key: cord- -qwhaesfk authors: hurley, walter l.; theil, peter k. title: perspectives on immunoglobulins in colostrum and milk date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: qwhaesfk immunoglobulins form an important component of the immunological activity found in milk and colostrum. they are central to the immunological link that occurs when the mother transfers passive immunity to the offspring. the mechanism of transfer varies among mammalian species. cattle provide a readily available immune rich colostrum and milk in large quantities, making those secretions important potential sources of immune products that may benefit humans. immune milk is a term used to describe a range of products of the bovine mammary gland that have been tested against several human diseases. the use of colostrum or milk as a source of immunoglobulins, whether intended for the neonate of the species producing the secretion or for a different species, can be viewed in the context of the types of immunoglobulins in the secretion, the mechanisms by which the immunoglobulins are secreted, and the mechanisms by which the neonate or adult consuming the milk then gains immunological benefit. the stability of immunoglobulins as they undergo processing in the milk, or undergo digestion in the intestine, is an additional consideration for evaluating the value of milk immunoglobulins. this review summarizes the fundamental knowledge of immunoglobulins found in colostrum, milk, and immune milk. the topic of immunoglobulins in milk immediately brings to mind the relationship between mother's milk, transfer of passive immunity from mother to neonate, and the immature immune system of the neonate. research in this field dates back to the late nineteenth century, however for many centuries herdsmen have capitalized on the linkage between maternal immune status and the immunological protection and development of the neonate [ , ] . immunoglobulins in mammary secretions come from several sources and represent a history of the antigen exposure of the mother and the response of her immune system. immunoglobulins are transported through the mammary epithelial cells by receptor-mediated mechanisms and transferred out of the mammary gland by milk ejection during suckling. the immunoglobulins then enter the environment of the gastrointestinal tract of the neonate. although that environment is primarily geared toward digestion to gain nutritional benefit, the immunoglobulins remain sufficiently stable to provide protective benefits for the neonate, either through uptake into the vascular system in the newborn of some species or through immunological function in the gastrointestinal tract. the immunoglobulins found in milk and the transfer of passive immunity from mother to neonate have been reviewed by many authors, with a partial listing referenced here [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the importance of homologous transfer of passive immunity between mother and neonate, there is considerable interest in the potential for heterologous transfer of passive immunity, such as immunoglobulins obtained from one species and utilized for passive immunity in another species. the ability to manipulate the immunological status of animals through vaccination against diseases that affect humans and the opportunity to harvest those immunoglobulins in the form of colostrum or milk has long been recognized [ , ] , and continues to be a topic of interest in both animal science and human medicine [ , , , [ ] [ ] [ ] . this review begins with a summary of some of the research on what has been termed -immune milk‖ and then discusses various aspects of immunoglobulins in mammary secretions (structure, function, concentration, sources, transport, species differences, and roles of immunoglobulins). finally, traits related to stability and processing methods for collecting milk immunoglobulins are reviewed. one intriguing application of our knowledge about bovine colostral and milk immunoglobulins comes through the opportunity to provide passive immunity against diseases in other species, especially in humans. the ability to direct the cow's immune system to produce antigen-specific antibodies that are secreted in colostrum and milk and may be used to provide protection against a specific disease continues to be an area of interest. for example, the widespread consumption of immune milk from cows inoculated against diseases such as avian influenza, sars, and other human respiratory diseases, has been suggested as a potential means of slowing outbreaks of the disease before they reach epidemic levels [ ] . a number of reviews have summarized and evaluated early attempts to develop and test the use of immune milk products to provide passive immune protection [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . several immune milk products are available commercially [ , , , , ] . safety issues associated with use of bovine immune milk products for human use have been discussed by others [ , [ ] [ ] [ ] . the discussion below provides some examples of immune milk products and their use against some animal and human diseases (sections . - . ). secretion of antibodies in breast milk from naturally immunized mothers can provide protection against enteric and other diseases in children [ ] . for example, elevated concentrations of antibodies specific for enteric pathogens, such as vibrio cholerae, in the mother's breast milk do not prevent colonization with the bacterium in the nursing child, but do seem to protect the infected child from developing diarrhea [ ] . breast feeding is associated with a reduced incidence of campylobacter diarrhea in young children compared with children that do not breast feed [ ] . in those children that are breast fed and do develop diarrhea, the human milk consumed may not contain iga antibodies specific for the common antigen of campylobacter [ ] , suggesting a degree of antigen specificity contained in the breast milk. the idea of immunizing the pregnant animal with the intent of controlling neonatal morbidity and mortality is well established [ ] . vaccination or natural immunization of the pregnant cow, ewe or sow against enterotoxigenic escherichia coli [ ] [ ] [ ] or intestinal viruses [ , ] , can provide a degree of protection for the newborn. as an example, while only limited protection against viral challenge occurred in calves fed once shortly after birth with a pooled colostrum from cows immunized against bovine rotavirus, a shorter duration of diarrhea was observed [ ] . on the other hand, calves fed milk supplements with low levels of a similar immune colostrum at each feeding for two weeks did have reduced virus sheading and reduced incidence of diarrhea [ ] . in primates, immunization of pregnant baboons with a rhesus rotavirus vaccine increased milk immunoglobulin and virus neutralizing titre [ ] . prenatal immunization of pregnant women with a single dose of meningococcal vaccine not only increased antigen-specific igg antibody in the infant's serum during the initial - months after birth, but antigen-specific iga in milk continued to be elevated at least up to months [ ]. as discussed in section , igg transfer to the offspring in humans occurs during late pregnancy and provides the initial systemic source of that immunoglobulin. infants consuming breast milk will primarily be consuming secretory iga (section ), which has significant protective activity in the intestine, as discussed in section . . the above examples of homologous transfer of passive immunity set the stage for considering the opportunities for heterologous passive transfer. immune milk products generally are some form of protein product derived from the colostrum and/or milk of dairy cattle. the cows typically are hyperimmunized against one or more antigens representing pathogens of bacterial or viral origin. crude preparations of the immunoglobulin from colostrum or milk may range from essentially no alteration of the immunoglobulin concentration in the product to partial immunoglobulin isolation or concentration in a whey protein concentrate. the primary immunoglobulin in cow colostrum and milk is igg, whereas the primary immunoglobulin in human milk is iga [ ] . nevertheless, bovine igg from colostrum or milk can be effective as a means of providing passive immunity to protect animals and humans from disease. the use of bovine colostral immunoglobulin preparations from immunized cows for disease protection of the neonate of other species has been demonstrated in swine [ ] , and experimental animal models such as mice [ , ] . there also are a number of examples of the use of bovine immune milk products in the treatment or prevention of human disease, especially in cases where the pathogen acts by way of the gastrointestinal tract. when considering these studies, it should always be kept in mind that the colostrum or milk preparations potentially contain other immune modulating substances than immunoglobulins, as discussed briefly below (section . ). the concept of using immune milk derived from hyperimmunized cows for treatment of human disease can be traced back to the s and earlier [ , ] . some of the early efforts in this field involved using immune milk products for treatment of rheumatoid arthritis and hay fever [ ] . immune milk preparations produced from milk from cows immunized with a heat-killed, lyophilized mixture of bacteria found to reside in the human gastrointestinal tract has been studied for the prevention and treatment of rheumatoid arthritis, high blood cholesterol, high blood pressure, and oral submucous fibrosis [ ] [ ] [ ] [ ] . on the other hand, most studies on the use of immune milk have examined the potential of immune milk for prevention and treatment of infectious diseases, particularly gastrointestinal disease. even milk that does not come form hyperimmunized cows may in some sense be regarded as immune milk. bovine anti-human rotavirus igg antibodies have been found in raw and pasteurized milk from cows that had not been specifically immunized against that virus [ ] . milk from non-immunized cows also has been found to contain measurable antigen-binding activity against several human pathogenic bacteria [ ] . several authors have tested the efficacy of immunoglobulin preparations with antibody activity against human rotavirus as a means of providing passive immunity to children. for example, children consuming a defatted colostrum preparation from cows immunized against a strain of human rotavirus had no improvement of symptoms when the infection was established (patients admitted to a hospital with rotavirus infection), however the preparation was effective in limiting diarrhea in children when consumed prior to the infection [ , ] . in another study, cessation of excretion of rotavirus in the stool of infants with acute rotavirus gastroenteritis was correlated with the presence of neutralizing activity in the stool after ingestion of a bovine whey protein concentrate from rotavirus-hyperimmunized cows [ ] , although there was not a significant decrease in duration of diarrhea in that study. other studies have found that treatment of children with hyperimmune bovine colostrum from cows immunized with human rotavirus serotypes reduces the duration and severity of diarrhea due to rotavirus [ ] , and can provide significant protection from rotavirus infection [ ] . enteropathogenic bacteria have also been the target for development of immune milk. over % of childrens' stools became negative for the e. coli strains used to hyperimmunize the cows that provided the source of immunoglobulin in a bovine colostrum/milk immunoglobulin concentrate consumed by children for days [ ] . interestingly, only one in nine children treated with the immunoglobulin concentrate, and having diarrhea that was associated with e. coli strains which were not used in the immunization of the cows, developed negative stools, underscoring the importance of the bacterial strain-specificity of the immune product. consumption of a hyperimmune immunoglobulin concentrate with a high antibody titer against a lipopolysaccharide isolated from shigella flexneri a also has been shown to provide protection against a challenge with the same strain [ ] . however, no difference in diarrhea or other symptoms in children with stools positive for s. dysenteriae was found whether treated with bovine colostrum from cows immunized against s. dysenteriae or with colostrum from cows not hyperimmunized [ ] . enterotoxigenic e. coli also is commonly associated with traveler's diarrhea. prophylaxis against this infection may be achieved by providing passive immunity with immune milk. a bovine whey protein concentrate from cows immunized with enterotoxigenic e. coli serotypes and consumed -times daily for seven days protected all of the adult volunteers from developing diarrhea after being challenged with an enterotoxigenic e. coli strain [ ] . in contrast, % of the volunteers who received control immunoglobulin concentrate prior to challenge developed diarrhea after the e. coli challenge. subsequent studies using igg isolated from bovine colostrum from cows hyperimmunized against specific e. coli colonization factor antigens also have shown protective effects in volunteers challenged with colonization factor antigen-bearing enterotoxigenic e. coli [ ] , however other studies by the same group did not demonstrate significant effects of similar milk immunoglobulin products [ ] . bovine colostrum concentrate preparations derived from cows that have not been hyperimmunized against specific antigens also may provide some benefit via passive immunization for some diseases. for example, a commercial product which is made from large standardized pools of colostrum collected from over cows has been used to treat a number of diseases [ , ] , including diarrhea caused by diarrheagenic e. coli [ ] . similar preparations from non-immunized cows may provide protection against bacterial toxins that are the cause of diarrhea in aids patients [ ] . these studies, along with the above mentioned study comparing colostrum preparations from cows immunized against s. dysenteriae or non-immunized cows [ ] , demonstrate that bovine colostrum contains significant antimicrobial properties as a result of natural exposure of the cows to antigens of pathogens that may afflict humans. another example of a potential use for bovine immunoglobulin preparations to control bacterial populations comes from studies on dental caries formation [ ] . the concept of prenatal immunization of the pregnant mother to protect the neonate against dental caries was demonstrated in rats [ ] . in applications to humans, bovine whey preparations of colostrum from cows immunized with caries-inducing bacterial strains (streptococcus mutans and streptococcus sobrinus), and containing over % immunoglobulin of which % was igg , has been used in several studies evaluating its effect on caries-producing bacteria. the colostral whey preparation reduced adherence of streptococcus mutans in vitro and caused aggregation of suspended bacteria [ ] , as well as inhibited glucose uptake by the test organism [ , ] . the whey preparation from hyperimmunized cows opsonized bacteria and enhanced in vitro phagocytosis of bacteria by human leukocytes [ ] . antibodies in the whey preparation remained functional when added to milk that had been treated via ultra-high temperature pasteurization or milk that was fermented to extend shelf-life [ ] . immune milk from cows hyperimmunized against seven streptococcus mutans strains reduced the recoverable bacterium in plaque samples from volunteers within seven days of initiation of mouth rinsing with the whey concentrate product [ ] . mouth rinsing with immune milk collected from cows immunized with a fusion protein representing two of the major factors implicated in oral colonization by streptococcus mutans inhibited recolonization of saliva and plaque by that organism [ , ] . immunodeficiency disorders often are associated with cryptosporidiosis, which can lead to chronic malabsorption and weight loss. in a case study of a child with congenital hypogammaglobulinemia, severe vomiting and diarrhea due to cryptosporidiosis, gastric infusion with hyperimmune bovine colostrum from cows immunized with cryptosporidium oocytes resolved the symptoms within a few days and oocyts were no longer found in stool samples after about eight days [ ] . similarly, in a child with aids who had severe diarrhea caused by cryptosporidiosis, administration of a commercial hyperimmune bovine colostrum preparation with anticryptosporidial activity improved the diarrhea and eliminated the parasite [ ] . in the cases where immune milk is collected from cows immunized against one or more pathogens, the immunization regimen occurs during the prepartum period of the cow. to put this in perspective relative to the lactation cycle of a cow, a brief reminder of that cycle may be helpful. depending on the management system used by a farm, most dairy cattle will have their first calf early in their third year, marking the start of their first lactation. the cow will be re-bred about two to three months into lactation. pregnancy is approximately days. at about months before expected calving date, or approximately months into lactation, milk removal is halted and the cow is given what is called a -dry‖ period. the mammary gland undergoes a process of involution during the early dry period where most residual milk components are broken down and resorbed [ ] . the mammary gland begins a redevelopment phase several weeks prior to calving. colostrum formation occurs in the days leading up to calving, coinciding with the early phase of lactogenesis (initiation of lactation). in the cow, lactogenesis begins shortly prior to calving and extends into the first few days postpartum. colostrum collected at the first milking of the cow after calving represents the accumulation of colostral products during the days leading up to parturition, including immunoglobulins which are at their highest concentration in the first milking. concentrations of immunoglobulins then decline rapidly in the subsequent several milkings [ ] . one application for immunization of pregnant or lactating animals comes from the arena of mastitis control in cattle. mastitis is the major disease in dairy cattle and most often is caused by intramammary infection [ , ] . vaccination of cattle against mastitis-causing pathogens has been an area of study for many years [ , ] . optimization of immunization schedules continues to be investigated [ ] . effective vaccines against mastitis-causing pathogens can increase antigen-specific immunoglobulins in the serum, which in turn can be increased in the mammary secretions. in the case of the j e. coli bacterin vaccine, the immunization also may be causing the mammary gland to become hyper-responsive to bacterial challenge [ ] , reminding us that enhancement of antigen-specific antibodies in the milk is not the only mechanism by which the vaccine may be having its effect. because the peripartum and early lactation periods are times of high susceptibility of the mammary gland to mastitis, many immunization schedules include prepartum immunizations during the dry period when milk is not removed and the mammary gland undergoes involution. it is also important to remember that cattle are generally immunosuppressed during the peripartum period [ , ] , potentially compromising the impact of immunizations administered just before or just after calving. coliform mastitis is one of the major types of mastitis in cattle [ ] . the more successful vaccination protocols for mastitis control have been with the j e. coli bacterin vaccine which is administered initially either just before or at the time of drying off [ , [ ] [ ] [ ] [ ] [ ] [ ] . these typically are followed by additional vaccine doses approximately mid-dry period. some protocols include an additional immunization within several days after calving [ , , , ] , while others also continue immunizations into the first three months of lactation [ , ] . attempts to vaccinate against other mastitis-causing pathogens have been met with more limited success. such vaccination protocols range from immunizations during the dry period [ ] , to peak lactation [ ] , and even late lactation [ ] . although most of the immunization protocols used in mastitis control administer the vaccine either intramuscularly or subcutaneously, intramammary immunization also can result in an increase in antigen-specific immunoglobulin in milk, as well as in the serum [ ] [ ] [ ] [ ] . a look across the immunization protocols used in studies to produce many immune milk products shows considerable variation, especially in the number and timing of immunizations. in those specifically collecting colostrum shortly after calving, multiple immunizations are administered during late pregnancy when the cow would be in the dry period [ ] [ ] [ ] , , , , , , , [ ] [ ] [ ] . mammary secretions then are collected either only at first milking [ ] , pooled from the first to milkings [ , , , ] , pooled from the first to days after calving [ , , , ] , or collected for longer periods into lactation [ ] . other studies have initiated immunizations during the late dry period and then continued vaccinating throughout lactation [ , , ], or only vaccinating during lactation [ ] . many of these studies used intramuscular or subcutaneous immunization, although some also have incorporated intramammary [ , , ] , or intravenous infusion [ ] . newer technologies for vaccine development and delivery may further enhance the production of immune milk products. immunization protocols that expose animals to specific antigens may enhance humoral immune responses in the mammary gland, including peptide-based vaccines [ ] , and dna-based vaccines [ , ] . delivery of antigen to the animal can also be achieved with antigen encapsulated in biodegradable microspheres [ ] , and with antigen-release devices [ ] . transgenic animals also have been used to produce antigens that then may be used to vaccinate animals against viral disease [ ] . the immunoglobulins, or antibodies, found in colostrum or milk are the same as those found in the blood or mucosal secretions. they are a family of proteins with a range of protective bioactivities. immunoglobulins are divided into several classes including igm, iga, igg, ige, and igd [ ] , and igg, iga and igm are the major immunoglobulin classes in mammary secretions. igm is the class that appears initially when an organism is exposed to an antigen for the first time (primary infection). igm has a low specificity and hence a lower potency in defeating the infection. iga is the major immunoglobulin class found in mucosal secretions and prevents mucosal infections by agglutinating microbes, whereas igg is the primary immunoglobulin class found in bovine colostrum and milk. several subclasses of igg exist, with igg and igg being the major immunoglobulins in serum. all monomeric immunoglobulins have the same basic molecular structure, being composed of two identical heavy chains and two identical light chains, with a total molecular mass of approximately kilodaltons (for details on immunoglobulin structure see [ , , ] ). both the heavy and light chains have constant regions and variable regions. heavy and light chains are linked together by disulfide bonds, resulting in the classic y-shape of the immunoglobulin molecule [ ] . the number and location of the disulfide bonds is dependent on the class of immunoglobulin. each immunoglobulin molecule has two antigen binding sites which comprise the antigen-binding fragment (fab). the fab includes the variable amino acid domain. at the other end of the molecule is the constant fragment (fc) which has a constant amino acid sequence among molecules of the same subclass and which confers the identity of an immunoglobulin as a particular subclass. the fc region of the molecule is the region that binds to fc receptors on various cell types. in the case of polymeric immunoglobulins, including the polymeric forms of iga and igm that are found in milk, the monomeric forms of the immunoglobulins are linked together through the covalent interaction with a joining (j) chain [ , ] . the result is a dimeric form of iga and a pentameric form of igm. binding of these immunoglobulins to the j chain also results in them having several special features, including: a high valency of antigen-binding sites, allowing them to agglutinate bacteria; limited complement-activating activity, which allows them to act in a noninflammatory manner; and a high affinity for the polymeric immunoglobulin receptor (pigr) that is responsible for transepithelial transport of iga and igm into mucosal secretions such as milk [ ] . the pigr and its relationship to the secretory component (sc) associated with secretory iga and secretory igm is discussed further below (section . ). the content of immunoglobulins in colostrum and milk is highly dependent on the animal species [ , ] . the same holds for the relative proportion of the immunoglobulin classes. these species differences are adaptations to the reproductive strategies of the animals and the degree of maturation of the offspring at birth. animal species may be divided into three classes [ ] : ( ) species where immunoglobulins are transferred mainly to the fetus via the placenta (humans and rabbits); ( ) species where offspring are born agammaglobulinemic and immunoglobulin transmission occurs via mammary secretions (ungulates such as horses, pigs, cows, and goats); and ( ) species where immunoglobulins are transferred both via placenta and mammary secretions (rats, mice and dogs). these adaptations have several consequences both for the composition of immunoglobulins in colostrum and milk, and for the role of colostrum. indeed, for animals like rats, mice, dogs and ungulates, uptake of colostrum of adequate quality and sufficient quantity is important for the offspring to boost the systemic immune function in the short term, whereas colostrum consumption in the human infant provides more protection for the gastrointestinal tract (see section . ). this is reflected in a lower total immunoglobulin content in human colostrum as compared to colostrum from the other species ( figure ) [ , , ] . human colostrum has a low content of igg ( %), and the igg required to provide systemic immunity is transferred across the placenta before birth. in contrast, colostral igg content in many other species is typically greater than % of the total immunoglobulin content (figure ). an additional consequence of different routes of immunoglobulin transmission relates to the changes in relative contents of immunoglobulins that occur in the transition from colostrum to milk within certain species (figure ). for example, the profile of immunoglobulins in human colostrum is similar to that found in milk, where the iga level is high in both colostrum and milk ( - % of total immunoglobulin). this is in contrast to the bovine mammary secretions where the high concentration of igg in colostrum declines rapidly with successive milkings. for animals like rats, mice, dogs and ungulates, the role of colostrum and milk immunoglobulins is to provide immune protection both systemically and for the gastrointestinal tract, which is reflected in large changes in the profile of immunoglobulins during the transition from colostrum to mature milk ( figure ). thus, for many species the proportion of iga increases between colostrum and milk. [ ] ; human and pig [ ] ; and horse [ ] . immunoglobulins found in mammary secretions arise from systemic and local sources. in the case of igg in milk, the major portion comes from the serum [ ] . while igg producing plasma cells may occur within the mammary tissue, their contribution to the igg in colostrum is minor compared with the igg derived from the serum. although limited paracellular passage of immunoglobulins may occur during inflammation (mastitis), uptake and transport of immunoglobulin across the mammary epithelial barrier is thought to occur primarily through an fc-receptor-mediated process [ , , [ ] [ ] [ ] . immunoglobulins are thought to bind to receptors at the basolateral surfaces of the mammary epithelial cell. these receptors are specific for the fc portion of the immunoglobulin molecule. the receptor-bound immunoglobulin is internalized via an endocytic mechanism [ ] , transported to the apical end of the cell and released into the alveolar lumen. recent studies have shed additional light on the details of this process [ ] . in the case of igg, the receptor responsible for transcytosis of igg into colostrum is referred to as fcrn, or the neonatal fc receptor, because it was initially identified in the neonatal rodent intestine as the receptor responsible for the specific uptake of maternal igg [ , ] . the fcrn also has been implicated in the trans-placental transport of igg in humans and other species [ ] [ ] [ ] , which may involve an endocytic and transcytotic process [ ] . since its initial discovery, fcrn has been described in many tissues [ ] . the receptor is a heterodimer composed of a membrane-bound α-chain similar to mhc class- molecules and a smaller mhc class- protein, β -microglobulin [ ] . binding of igg to fcrn is ph-dependent, with high affinity binding occurring at acidic ph, but only weak binding at neutral or basic ph [ ] . this observation suggests that igg taken up by the epithelial cells may bind to fcrn within an acidic environment in the endosomes. the precise mechanism of transport across the epithelial cell and release into the colostrum or milk remains to be demonstrated. the half life of igg in serum is typically longer ( - weeks) than that for iga or igm ( - days), and the half-life of igg is slightly longer than for igg [ ] . evidence suggests that igg has a higher affinity for fcrn than igg [ ] . in bovine colostrum, igg is many fold greater in concentration than igg [ ] , although they are of approximately equal concentrations in serum. it may be that the majority of the igg taken up by the mammary epithelial cell during colostrum formation is not passed on to the alveolar lumen, but rather is recycled back to the extracellular fluid. the fcrn is thought to have a major role in the recycling of igg in various tissues in the body [ ] [ ] [ ] . that is, igg that potentially may be lost through various tissues is recycled by the respective cells by binding to fcrn and recycled back to the blood or lymph. this is supported by studies of overexpression of fcrn in transgenic mice where there is an extension of the half-life of serum igg [ , ] , as well as a boosting of the overall humoral immune response of the mice [ ] . localization of fcrn in bovine, sheep and water buffalo mammary tissue indicates that the receptor is homogeneously distributed throughout the epithelial cells prior to parturition, but primarily localized at the apical surface of the mammary epithelial cells after parturition [ ] [ ] [ ] [ ] . while this type of observation corroborates the conclusion that fcrn plays an important role in igg transport during colostrum formation, at least in ruminant species, the precise meaning of this redistribution of fcrn staining in mammary cells remains to be determined. it is also interesting to note that the initial report of this distribution pattern in sheep mammary epithelium included the observation that the staining pattern became diffuse within the cells during mammary involution [ , ] . transport of igg also may increase transiently in mammary secretions during involution in cattle [ ] . hormonal and local factors have been implicated in the control of immunoglobulin transport during colostrum formation [ ] . haplotypes of the fcgrt gene, coding for the mhc class i α-chain of fcrn, are associated with serum concentrations of igg in neonatal beef calves [ ] and associated with igg concentrations in colostrum of dairy cows [ ] . haplotypes of the β -microglobulin gene (β m) also are associated with serum igg concentrations in newborn calves [ ] . in estimating mass transfer of igg into colostrum in dairy cattle, % of cows had mass transfer greater than one standard deviation above the mean, perhaps indicating a genetic or hormonal regulation of the variance of transport [ ] . clearly there is opportunity for genetic manipulation of igg transport in the mammary gland to enhance the concentrations of immunoglobulins in colostrum and milk. however, it should be remembered that serum igg concentrations in the periparturient cow are already decreased as a result of the extensive igg transport into the colostrum [ ] , and as indicated above, the cow is in an immunosuppressed state during the peripartum period [ , ] . the other major classes of immunoglobulins transported into colostrum and milk are iga and igm. immunoglobulin a is the major immunoglobulin in human colostrum and milk (figure ), however it is also present in milk of most other species. colostrum and milk iga and igm are found in the form of secretory iga, or siga, and sigm. much of these are produced by plasma cells in the mammary tissue. the plasma cells are part of the gut-associated lymphoid tissue (galt), the largest immune organ of an organism, which includes the peyer's patches, lymphoid and myeloid cells in the lamina propria and intraepithelial lymphocytes [ , ] . lymphocytes from the galt system migrate to the mammary gland and provide a direct link between the antigen exposure response in the mother's mucosal immune system, especially via the enteric mucosal immune system, and the secretory immunoglobulin repertoire of the mammary gland [ ] . this means that maternal colostrum and milk will contain antibodies specific for pathogens that may be encountered by the neonate's intestine and other mucosal tissues [ , , ] , providing a rationale for the observations summarized above that bovine colostrum from nonimmunized cows also may afford passive immune protection against human pathogens [ , ] . the immune connection between the galt and the mammary gland is of particular interest with respect to human milk where the major immunoglobulin is siga, which accounts for one of the key factors underlying the importance of breast feeding [ ] . the immune activation of galt in the human infant is delayed, and the milk siga and sigm provide the neonatal intestine a level of protection through their immune exclusion actions and their anti-inflammatory effects [ , ] . transepithelial transport of iga and igm across the mammary epithelial cells occurs via the polymeric immunoglobulin receptor (pigr) which is responsible for binding dimeric iga and pentameric igm in mucosal tissues [ , ] . the polymeric nature of iga and igm arises from their binding with the j-chain peptide [ ] . only iga or igm that contain the j chain have a high affinity for pigr [ , , ] . in fact, the j chain has been evolutionarily conserved within tetrapods to the point where human polymeric iga can bind to the pigr from the amphibian xenopus laevis [ ] . polymeric iga or igm bound to pigr is internalized and transported to the apical end of the mammary epithelial cell by an endocytic process. the pigr molecule is cleaved to release a receptor fragment, called secretory component (sc), which remains bound to the immunoglobulin molecule [ , ] . in the case of pigr receptor sites that are not occupied by immunoglobulin, the secretory component is still cleaved from the membrane-bound portion of pigr, resulting in release of free secretory component. the secretory component has protective effects of its own, potentially blocking epithelial adhesion of enterotoxigenic e. coli and neutralizing the effects of other pathogens [ ] . expression of pigr in the mammary gland is under control of hormones responsible for initiation of lactation [ ] . elevated transport of iga also may occur during mammary gland involution in cattle and persist longer into the involution process [ ] . part of the transfer of passive immunity story in mammals involves the timing and location of transfer of immunoglobulins from the mother to the offspring, while another part encompasses the fate and function of the immunoglobulins once in the neonate [ , , ] . in humans, intestinal transfer of maternal igg from colostrum is sparse in the neonate and their immune competency is assured by transfer via the placenta. in rats and mice, there is fcrn-mediated uptake of igg from the colostrum and milk in the neonate intestine. in ungulate species such as cattle, sheep, goats and pigs, the young are born essentially agammaglobulinemic and rely entirely on uptake of colostral immunoglobulins, especially igg, for systemic immune protection. the consumption of colostrum by the neonatal calf has significant effects on the gastrointestinal tract [ ] . the intestinal uptake in the immediate period after birth is transient and nonselective in species such as cattle, sheep, goats, swine and others. the intestinal cells become unable to absorb macromolecules within - h after birth probably as a result of developmental processes occurring in the enterocytes [ ] . the process whereby the intestinal cells gradually stop absorbing macromolecules is termed -closure‖. before closure, the enterocytes will nonselectively absorb large molecular weight proteins and other molecules [ ] . macromolecules so transported are released into the lamina propria and then are absorbed into the lymphatic or portal circulation. failure of passive transfer of immunity in these species is defined as occurring when a threshold concentration of igg is not reached before closure occurs, which in the calf corresponds to serum igg levels less than mg/ml [ ] . the maternal igg in the calf's blood gradually declines over the initial month after birth, and has a half-life of approximately days [ ] . milk siga is not taken up by the infant's intestinal mucosa [ , ] . in fact, gut closure in humans occurs before birth and little immunoglobulin is absorbed intact in the intestine after birth [ , ] . however, the presence of siga in the intestinal lumen is part of the protective function of the epithelial barrier in the intestine [ ] . milk siga in the intestine will bind bacteria, toxins and other macromolecules, limiting their ability to bind to intestinal cells and thereby be transported through the mucosa to the lamina propria to cause a systemic immune response [ ] . in adults of a pigr-deficient strain of mice, which do not transport siga into the intestinal lumen, there is an increased serum iga and igg that react with commensal organisms and food antigens [ ] . this may be occurring because siga is not being secreted into the intestinal lumen to participate in its role in immune exclusion (see section . ), and resulting in an increased uptake of food antigens and microbial antigens from the intestinal lumen which pass to the lamina propria and stimulate specific antibody responses [ ] . development of the galt system is dependent on microbial stimulation [ , ] . the microbe binding function of siga then modulates the early microbial colonization of the gastrointestinal tract and the interaction of those microbes with the developing neonatal immune system [ , , ] . from the discussion of immune milk products above it was clear that these products have protective effects on neonatal health, as well as infant and adult human health. the exact mechanisms by which immune milk products have their effects are less clear and deserve further investigation. below are summarized several perspectives to consider when evaluating the effects of immune milk products and the role of immunoglobulins in achieving those effects. it should be remembered that colostrum and milk not only contain immunoglobulins, but also contain a range of antimicrobial factors and factors that may impact the immune system [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these include the iron-binding antimicrobial protein lactoferrin, antibacterial enzyme lactoperoxidase, antibacterial and lytic enzyme lysozyme, oligosaccharides that function as analogues of microbial ligands on mucosal surfaces, antimicrobial heat stable peptides (defensins), and soluble cd . in addition, colostrum and milk contain leukocytes, including activated neutrophils, macrophages and lymphocytes. colostrum also contains cytokines and growth factors that may affect neonatal intestinal development, as well as intestinal immune responses to disease in adults [ , ] . the relative concentrations of these factors vary considerably among species. furthermore, colostrum provides a source of energy which may impact igg absorption in the neonate [ ] , and provide additional energy for an effective immune response. another point to consider is that, while most macromolecules are degraded by digestive enzymes, some portion of macromolecules is transported across the intestine intact, including proteins [ , ] . much of the immunoglobulin consumed in an immune milk can be expected to be partially or completely digested (discussed in section . ), however some portion of the immunoglobulin will remain intact or at least partially intact and capable of binding to an antigen. all colostrum and milk will contain some siga, even those collected from cattle. the siga present in these secretions may contribute to the protective effects of immune milk products. secretory iga is considered to be the primary immunoglobulin responsible for immune protection of mucosal surfaces such as the intestine [ ] . secretory iga and sigm, as polymeric forms of the respective immunoglobulins, are stabilized by their binding to sc. they have antimicrobial properties such as agglutination of microbes and neutralization of viruses, and noninflammatory extracellular and intracellular immune exclusion by inhibiting adherence and invasion of mucosal epithelia [ ] . the intracellular immune exclusion occurs when siga is being transcytosed by the enterocytes and comes into contact with viral particles within the endosomic system [ ] . secretory iga also neutralizes pathogens in the intestinal lumen [ ] . bacterial enterotoxins may be neutralized by binding siga and internalization into intestinal epithelial cells [ ] . in addition, iga has a major role in the immunosuppressive mechanisms in the intestine that inhibit proinflammatory responses to oral antigens, which is part of the oral tolerance mechanisms in the intestine [ ] . this suppression of the proinflammatory mechanisms is counterbalanced by systemic immune factors, including systemic igg, which may result in inflammation and tissue damage once an antigen crosses epithelia barrier to the lamina propria [ ] . after closure, any igg localized in the lamina propria, whether from systemic sources or from uptake from the intestinal lumen, could contribute to proinflammatory responses in the intestine [ ] . indeed, post-closure uptake of igg can occur via the fcrn receptor. fcrn has been identified in the human adult intestine [ , ] , consistent with the hypothesis that fcrn is involved in igg recycling (discussed in section . ). however, the transport of igg across the enterocyte seems to be bidirectional, lending support to the concept that igg in the intestine is involved in immune surveillance and defense of the mucosal lining [ ] [ ] [ ] . intestinal fcrn may deliver igg-antigen immune complexes to the lamina propria for immune processing [ , ] , thereby enhancing local mucosal immune response. on the other hand, functionally intact igg that remains in the intestinal lumen might be expected to bind antigens and participate in protection of the tissue through immune exclusion. the intestinal mucus layer does provide an important protective barrier in the interactions of the intestinal tissue with microbes [ ] . interestingly, an igg fc binding site has been identified in association with the intestinal mucus [ ] [ ] [ ] . this igg fc binding protein is distinct from the fcrn receptor. the fc binding protein may block passage of igg-antigen complexes to the enterocyte surface, thereby blocking their uptake and transport to the lamina propria, and perhaps allowing the complexes to be degraded in the intestinal lumen and excreted [ , ] . consumed colostrum also may impact immunological development of the neonate [ ] . these maternal antibodies may then inhibit infant responses to vaccine administration and impact development of the infant's immunity [ ] . in the case of dairy animals producing colostrum or milk immunoglobulins for human consumption, immunoglobulins are harvested at milking and undergo various types of processing whether it is to prolong the shelf-life of the milk, to concentrate or isolate the immunoglobulins from the mammary secretion, or to digest the milk in the intestine. through such processing, immunoglobulins are exposed to a number of conditions that may alter the structure and function of the protein. some of methods used to concentrate or isolate the immunoglobulins include steps that involve exposing the protein to heat, acid or pressure which may affect the conformation of the protein, and ultimately the immunological activity of the antibody. a range of methods have been used for isolation of immunoglobulins from colostrum or milk. these include traditional methods of ammonium sulfate precipitation and column chromatography [ , , , ] . affinity chromatographic methods used to isolate igg include lectins [ ] ; protein a or g chromatography [ , ] , and more recently, isolation with protein a/g immobilized electrospun polyethersulfone membranes [ ] ; metal chelate chromatography [ , ] ; and adsorption with polyanhydride microparticles [ ] . the range of detection and quantification methods for igg, most often analyzed by radial-immunodiffusion [ ] or enzyme-linked immunosorbant methods [ ] , are now expanding to include methods that detect multiple proteins, such as thermally addressed immunosorbant assays [ ] , and rapid methods that may be integrated into milking systems, such as surface plasmon resonance-based immunosensors [ ] . pepsin is a major proteolytic enzyme produced by the stomach. pepsin digestion of igg yields an f(ab') fragment that includes the two antigen-binding (fab) sites of the igg molecule [ , , , ] . intact immunoglobulin, f(ab') and other antibody formats are being exploited in development of antibody therapeutics [ ] . in the small intestine, immunoglobulins are further digested by pancreatic enzymes. one of them, trypsin, preferentially digests bovine igg over igm, whereas another enzyme, chymotrypsin, preferentially hydrolyzes igm over igg [ ] . bovine igg is more susceptible to hydrolysis by pepsin than igg , while igg is more susceptible to trypsin [ ] . immunoglobulins are relatively more resistant to gastrointestinal digestion than other milk or colostral proteins. upon ingestion and entry into the stomach, the caseins form a curd under the influence of the acidic environment and proteolytic activity. as a consequence, casein is retained in the stomach of the neonate longer than the whey proteins, including igg [ ] . in the intestine, the fate for the other major whey proteins is rapid digestion for α-lactalbumin, while β-lactoglobulin is more slowly digested. intestinal digestion of igg is among the slowest of the whey proteins and igg provides the smallest proportion of amino acids to the neonate relative to the other major whey proteins [ ] . in vitro incubations of iga and igg with small intestinal content of young lambs have shown that iga is more resistant towards digestion than is igg [ ] . in adult humans consuming a bovine whey protein concentrate, approximately % of igg and igm was detected by radial immunodiffusion from effluents from the jejunum, while % was detected in the ileum [ ] . these estimates of digestion of immunoglobulin compare with estimates of digestion of milk proteins in adult humans which are approximately % complete at the end of the jejunum and % complete by the end of the ileum [ ] , again underscoring the relative resistance of immunoglobulins to digestion in the gastrointestinal tract. detectable immunoglobulin in stool samples of infants fed the same immune product accounted for % of the ingested immunoglobulin [ ] . in adults fed a bovine immunoglobulin concentrate, fecal igg was typically less than % of ingested dose [ ] . detectable igg in the stool [ ] , or ileal effluent samples of adults [ ] , is not significantly increased by prior treatment with a proton pump inhibitor to reduce stomach acid production. however, encapsulation of the immunoglobulin product can significantly increase the igg detectable in the stool [ ] , although only low levels of igg are detectable in the ileum of adults ingesting encapsulated immunoglobulin [ ] . these studies suggest that degradation of immunoglobulins is occurring throughout the intestinal tract [ ] . the primary structure of the immunoglobulin found in intestinal effluents most likely is the immunoglobulin fab or f(ab') fragments found in their stool [ , ] , which nevertheless maintains its antigen-binding activity, as indicated by the correlation between appearance of the immunoglobulin and the virus-neutralizing activity observed in stool samples [ ] . in adults ingesting bovine anti-clostridium difficile immunoglobulins, toxin-neutralizing activity paralleled the bovine igg content in ileal effluent [ ] , and in stool samples [ ] . a pepsin-resistant form of bovine igg representing approximately % of colostral immunoglobulin has been isolated with a lectin that binds o-linked oligosaccharides [ ] , indicating that some proportion of igg in the gastrointestinal tract may remain intact. the ph of bovine mammary secretions transiently drops at calving (to approximately ph . ), then increases over several days to ph . to . [ ] , which is the ph characteristic of mature milk. therefore, bovine colostrum is slightly more acidic than mature milk. studies of isolated immunoglobulin stability over a ph range indicate that bovine igg isolated from milk is stable for several hours at °c when in ph - , however stability is significantly reduced at ph ≤ and ≥ [ , ] . the negative effect of ph on igg stability, even in the range of . - . , is enhanced under elevated temperature conditions [ , ] . the use of a multiple emulsion to encapsulate milk igg may increase stability of the protein against extreme acidic or alkali conditions, as well as against proteolytic degradation [ ] . however, emulsification by homogenization may reduce the residual igg content of the emulsion product [ ] , probably as a result of high shear forces [ ] . ultrasonic treatment of isolated igg also decreases residual igg content [ ] . immunoglobulins are thermolabile. exposure to temperatures of °c can reduce detectable isolated bovine igg by % in min, and by % at °c for s [ ] . heat exposure causes conformational changes in the igg molecule [ ] . antigen-binding activity of bovine igg also is reduced after heat treatment [ , ] . this is consistent with studies that suggest that the antigen-binding region of the immunoglobulin molecule is more thermolabile than the other regions of the molecule [ , ] . detectable igg in colostrum or colostral whey also are reduced by heat treatment, however at a slower rate than for isolated igg. thermal protectants such as sugars or glycerol can increase the stability of isolated igg to heat treatment [ , ] . many milk processing protocols include heat treatment of the colostrum, milk or whey. of the major immunoglobulin classes in bovine milk, igg is the most thermostable and igm is the least thermostable [ ] . commercial milk samples that have undergone a typical pasteurization process, including skim milk powder, can retain - % of the igg concentration compared with raw milk, while milk undergoing ultra-high temperature (uht) pasteurization contains little detectable igg [ , ] . nevertheless, antigen-specific igg in milk is relatively stable under typical conditions of pasteurization when compared with that in uht milk or cow milk-based infant formulas that undergo high-temperature processing [ , ] . flash-heat treatment of human breast milk, a method recommended by who to reduce vertical transmission of hiv in resource-poor regions, has minimal effects on milk iga and antimicrobial activity of the milk [ , ] . this method involves placing a jar of milk into a water bath, the water bath is heated to boiling, and then the jar of milk is removed and allowed to cool. the milk reaches a maximum temperature of - °c and is above °c for over min [ , ] . alternative methods of achieving microbial inactivation may offer a means of avoiding the impact of heat treatment on igg solutions. for example, high voltage pulsed electric fields have been used as a nonthermal processing method for pasteurization in various foods [ ] [ ] [ ] . pulsed electric field processing also generates heat, however temperature exposure of the fluid is less than °c, and total treatment time exposure is in milliseconds [ ] . that compares with more typical pasteurization process at about °c for min. microbial inactivation in bovine igg solutions as a result of pulsed electric fields did not change the secondary structure or the thermal stability of the secondary structure of the igg [ ] , and antigen-binding activity was unchanged [ ] . another emerging technology that may provide a nonthermal microbial inactivation treatment for milk uses exposure to pulsed ultraviolet light [ ] . high-pressure processing is another non-thermal method with the potential for inactivation of microbial and certain enzymes in food products, thereby extending shelf-life of the product [ ] . while the high-pressure process also generates heat during the treatment of the sample, lowering the initial temperature of the sample allows for control of the maximum temperature reached to be maintained within a desired range [ ] . to be effective in inactivating bacterial spores, high-pressure processing needs to be combined with moderate temperature treatment [ ] . moderate to extensive loss of immunoactivity of igg may occur depending on the conditions used for high-pressure processing of colostrum or other igg-containing fluids [ , ] . high-pressure processing also has been used for human breast milk with minimal effect on the milk iga [ ] . the issue of heating effects on immunoglobulin and colostrum also is important for control of various diseases that occur in cattle. collection and storage of colostrum from dairy cows shortly after calving has long been a common procedure. the stored colostrum then is fed to newborn calves to assure adequate uptake of igg for protection of the calf. several pathogens can be transmitted from cow to calf via colostrum or milk [ ] . colostrum may contain these pathogens as a result of shedding from the mammary gland, contamination of the colostrum after harvesting or improper storage of colostrum prior to feeding calves [ ] . one approach to allowing the neonate the benefits of colostrum from infected cows is collecting colostrum and batch pasteurization of pooled colostrum prior to feeding to the calves [ ] . volume of the batch of pooled colostrum that is pasteurized affects measurable igg concentrations in the colostrum, as well as igg serum concentrations attained in calves after feeding the colostrum [ ] . heat treatment of colostrum at °c for one to two hours does not alter measurable igg concentrations or viscosity of the colostrum, nor does that treatment affect antibody activity [ , ] . in addition, bacteria inoculated into colostrum prior to a heat treatment of °c for one hour are not detectable after the heat treatment [ ] . on-farm heat treatment of colostrum ( °c for one hour) results in higher concentrations of serum igg and greater apparent absorption efficiency of igg in new born calves consuming the treated colostrum than consumption of raw colostrum [ ] [ ] [ ] . colostrum and milk are rich sources of immunoglobulins. these secretions have developed through evolution to ensure homologous transfer of passive immunity from mother to offspring. the immunoglobulins that are passed from mother to her offspring, whether by transplacental transfer or by ingestion of colostrum and milk, can form an important link between the immunological experience of the mother and the immune capacity of the newborn. this immunological link also includes many immune factors that may be present in mammary secretions other than the immunoglobulins. the immunoglobulins in colostrum and milk also provide opportunities to harness their immunological function for the benefit of other animals, including humans. research has demonstrated that bovine colostrum and milk, whether or not they are from cows immunized against specific pathogens, provide a medium for the heterologous transfer of passive immunity, and may offer disease protection in a range of species. new technologies for enhancing efficacy of vaccination, enhancing stability and extending shelf-life of the immunoglobulin preparation while minimizing the impact of the processing, and extending the effectiveness of the immunoglobulin in the intestine, may enhance future use of colostrum and milk based on their potent immunological activity. while the mechanisms by which immunoglobulins are transferred from mother to neonate and their role in the neonate have become well documented, additional research is needed to clarify the mechanisms of action of the immunoglobulins derived from milk or colostrum when used in animals that are developmentally more mature. immunoglobulins and immunocytes in the mammary gland and its secretions immune components of colostrum and milk-a historical perspective immunoglobulins of the mammary secretions antibodies in milk bovine immunoglobulins: an augmented review mucosal immunity of the mammary gland and immunology of mother/newborn interrelation immunoglobulins of the mammary secretions antibodies in milk milk immunoglobulins and complement factors the immunological role of breast feeding the role of colostral antibodies in prevention of microbial infections the protective properties of milk and colostrum in non-human species colostrum and its benefits: a review immunoglobulins of the mammary secretions transfer of antibody via mother's milk. vaccine analysis of bovine immunoglobulin g in milk, colostrum and dietary supplements: a review immune components of bovine colostrum and milk the mucosal immune system and its integration with the mammary glands immune milk-a historical survey a review of the literature on some aspects of immune milk bovine milk antibodies for health bovine colostrum as a biologic in clinical medicine; a review-part i: biotechnological standards, pharmacodynamic and pharmacokinetic characteristics and principles of treatment bovine colostrum as a biologic in clinical medicine; a review-part ii: clinical studies bovine and human-derived passive immunization could help slow a future avian influenza pandemic vaccines and milk immunoglobulin concentrates for prevention of infectious diarrhea potential for immunological supplementation of foods passive protection against diarrhea disease passive and active protection against disorders of the gut passive immunity against human pathogens using bovine antibodies preventing infectious disease with passive immunization maternal antibodies, childhood infections, and autoimmune diseases regulation of immunoglobulin transfer into mammary secretions of ruminants allergenicity of orally administered preparations in food-allergic children effects of a milk-based bioactive micronutrient beverage on pain symptoms and activity of adults with osteoarthritis: a double-blind, placebo-controlled clinical evaluation analytical approach to determination of safety of milk ingredients from hyperimmunized cows protection against cholera in breast-fed children by antibodies in breast milk protection of breast-fed infants against campylobacter diarrhea by antibodies in human milk vaccines for preventing enterotoxigenic escherichia coli infections in farm animals immunity to escherichia coli in pigs: antibody secretion by the mammary gland after intramammary or intramuscular vaccination with e. coli vaccine the effect of oral immunization on the population of lymphocytes migrating to the mammary gland of the sow survey of immunoglobulin g content and antibody specificity in cow's milk from british columbia prevention of rotavirus infection by cow colostrum containing antibody against human rotavirus prevention of rotavirus infection by oral administration of cow colostrum containing antihumanrotavirus antibody use of bovine milk concentrate containing antibody to rotavirus to treat rotavirus gastroenteritis in infants hyperimmune cow colostrum reduces diarrhoea due to rotavirus: a double-blind, controlled clinical trial passive immunization of children with bovine colostrum containing antibodies to human rotavirus treatment of infantile e. coli gastroenteritis with specific bovine anti-e. coli milk immunoglobulins efficacy of bovine milk immunoglobulin concentrate in preventing illness after shigella flexneri challenge hyperimmune bovine colostrum in the treatment of shigellosis in children: a double-blind, randomized, controlled trial protection by milk immunoglobulin concentrate against oral challenge with enterotoxigenic escherichia coli milk immunoglobulin with specific activity against purified colonization factor antigens can protect against oral challenge with enterotoxigenic escherichia coli lack of prophylactic efficacy of an enteric-coated bovine hyperimmune milk product against enterotoxigenic escherichia coli challenge administered during a standard meal bovine colostrum ameliorates diarrhea in infection with diarrheagenic escherichia coli, shiga toxin-producing e. coli, and e. coli expressing intimin and hemolysin antibodies from colostrum in oral immunotherapy immunization against dental caries effective immunity to dental caries: passive transfer to rats of antibodies to streptococcus mutans elicits protection concentrated bovine colostral whey proteins from streptococcus mutans/strep. sorbinus immunized cows inhibit the adherence of strep. mutans and promote the aggregation of mutans streptococci generation of bovine immune colostrum against streptococcus mutans and streptococcus sobrinus and its effect on glucose uptake and extracellular polysaccharide formation by mutans streptococci combined inhibitory effect of bovine immune whey and peroxidase-generated hypothiocyanite against glucose uptake by streptococcus mutans effects of bovine immune and non-immune whey preparations on the composition and ph response of human dental plaque stability and activity of specific antibodies against streptococcus mutans and streptococcus sobrinus in bovine milk fermented with lactobacillus rhamnosus strain gg or treated at ultra-high temperature effect of immune bovine milk on streptococcus mutans in human dental plaque bovine milk antibodies against cell surface protein antigen pac-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of streptococcus mutans passive immunization with milk produced from an immunized cow prevents oral recolonization by streptococcus mutans remission of diarrhoea due to cryptosporidiosis in an immunodeficient child treated with hyperimmune bovine colostrum bovine colostrum immunoglobulin concentrate for cryptosporidiosis in aids mammary gland function during involution iga, igg , igg , igm and bsa secretion by the bovine mammary gland throughout lactation invited review: the role of contagious disease in udder health mastitis associate transcriptomic disruptions in cattle the immunophysiological basis for vaccinating ruminants against mastitis vaccines against bovine mastitis in the new zealand context: what is the best way forward? effect of changes in number of doses and anatomic location for administration of an escherichia coli bacterin on serum igg and igg concentrations in dairy cows potential mechanism of action of j vaccine in protection against bovine coliform mastitis effects of growth hormone, insulin-like growth factor-i, and cortisol on periparturient antibody response profiles of dairy cattle coliform mastitis effects of an escherichia coli j vaccine on mild clinical coliform mastitis efficacy of an escherichia coli j bacterin administered to primigravid heifers effects of adjuvants on safety and efficacy of an escherichia coli j bacterin effect of hyperimmunization with an escherichia coli j bacterin in adult lactating dairy cows milk and serum j -specific antibody responses, milk production change, and clinical effects following intramammary escherichia coli challenge for j vaccinate and control cows association of escherichia coli j -specific serum antibody responses with clinical mastitis outcome for j vaccinate and control dairy cattle study of the humoral immunological response after vaccination with a staphylococcus aureus biofilm-embedded bacterin in dairy cows: possible role of the exopolysaccharide specific antibody production in the protection from staphylococcus aureus induced mastitis efficacy of vaccination against staphylococcal mastitis: a review and new data immune responses to mycoplasma bovis vaccination and experimental infection in the bovine mammary gland immune response in the bovine mammary gland after intestinal, local, and systemic immunization the effect of intraperitoneal and intramammary immunization of sheep on the numbers of antibody-containing cells in the mammary gland, and antibody titres in blood serum and mammary secretions local vaccination with killed streptococcus uberis protects bovine mammary gland against experimental intramammary challenge with the homologous strain efficacy of immunization with ferric citrate receptor feca from escherichia coli on induced coliform mastitis anti-clostridium difficile bovine immunoglobulin concentrate inhibits cytotoxicity and enterotoxicity of c. difficile toxins bovine milk immunoglobulins for passive immunity to infantile rotavirus gastroenteritis production from dairy cows of semi-industrial quantities of milk-protein concentrate (mpc) containing efficacious anti-candida albicans iga antibodies method for treating inflammation using bovine milk induction of hiv- mpr - -specific iga and igg antibodies in caprine colostrum using a peptide-based vaccine immune responses to a dna/protein vaccination strategy against staphylococcus aureus induced mastitis in dairy cows mucosal genetic immunization against four adhesions protects against staphylococcus aureus-induced mastitis in mice immunization with staphylococcus aureus lysate incorporated into microspheres specific immune milk production of cows implanted with antigen-release devices highly protective e -csfv vaccine candidate produced in the mammary gland of adenoviral transduced goats immunoglobulins-basic considerations the structure of iga role of j chain in secretory immunoglobulin formation the total protein and immunoglobulin profile of equine colostrum and milk the immune system of the ruminant mammary gland and its role in the control of mastitis epithelial transcytosis of immunoglobulins identification and function of neonatal fc receptor in mammary gland of lactating mice fcrn-mediated antibody transport across epithelial cells revealed by electron tomography the neonatal fc receptor plays a crucial role in the metabolism of igg in livestock animals receptor-mediated transport of igg isolation and characterization of an fc receptor from neonatal rat small intestine human placental fc receptors and the transmission of antibodies from mother to fetus placental transport of immunoglobulin g. vaccine an interspecies comparison of placental antibody transfer: new insights into developmental toxicity testing of monoclonal antibodies endocytic and transcytotic processes in villus syncytiotrophoblast: role in nutrient transport to the human fetus an fc receptor structurally related to mhc class i antigens the protection receptor for igg catabolism is the β -microglobulin-containing neonatal intestinal transport receptor finally! the brambell receptor (fcrb) the role of the brambell receptor (fcrb) in liver: protection of endocytosed immunoglobulin g (igg) from catabolism in hepatocytes rather than transport of igg to bile position independent and copy-number-related expression of the neonatal fc receptor -chain in transgenic mice carrying a kb genomic fragment over-expression of the bovine fcrn in the mammary gland results in increased igg levels in both milk and serum of transgenic mice kacskovics, i. neonatal fcr overexpression boosts humoral immune responses in transgenic mice kasckovics, i. redistribution of the sheep neonatal fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs kasckovics, i. localization of the sheep fcrn in the mammary gland expression of the neonatal fc receptor (fcrn) in the bovine mammary gland expression and immunohistochemical localization of the neonatal fc receptor (fcrn) in the mammary gland of the egyptian water buffalo immunohistological localization of igg , iga and secretory component in the bovine mammary gland during involution association of bovine neonatal fc receptor -chain gene (fcgrt) haplotypes with serum igg concentration in newborn calves association of fcrn heavy chain encoding gene (fcgrt) polymorphisms with igg content in bovine colostrum beta- -microglobulin haplotypes in u.s. beef cattle and association with failure of passive transfer in newborn calves mass transfer of immunoglobulin g into colostrum production and turnover of igg and igg immunoglobulins in the bovine around parturition development and function of organized gut-associated lymphoid tissues iga plasma cell development mucosal immunity: integration between mother and the breast-fed infant immunoglobulin transport and the polymeric immunoglobulin receptor epithelial transport of iga by the polymeric immunoglobulin receptor the j chain is essential for polymeric ig receptor-mediated epithelial transport of iga secretory antibody formation: conserved binding interactions between j chain and polymeric ig receptor from humans and amphibians developmental expression of pigr gene in sheep mammary gland and hormonal regulation nutritional physiology of neonatal calves receptor mechanisms of the neonatal intestine and their relationship to immunoglobulin absorption and disease colostrum management for dairy calves absorption and endogenous production of immunoglobulins in calves iga and intestinal homeostasis biological functions of iga human milk: its components and their immunobiologic functions secretory antibodies reduce systemic antibody responses against the gastrointestinal commensal flora bovine neonatal immunology dairy products and the immune function in the elderly nutritional and physiological significance of human milk proteins growth factors and antimicrobial factors of bovine colostrum colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders protection of the neonate by the innate immune system of developing gut and of human milk biologically active breast milk proteins in association with very preterm delivery and stage of lactation regulation of intestinal epithelial function: a link between opportunities for macromolecular drug delivery and inflammatory bowel disease absorption of orally supplied immunoglobulins in neonatal piglets intestinal transport of some macromolecules in food macromolecular transport across the rabbit proximal and distal colon polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite giardia anti-inflammatory role for intracellular dimeric immunoglobulin a by neutralization of lipopolysaccharide in epithelial cells expression of the neonatal fc receptor, fcrn, on human intestinal epithelial cells bidirectional fcrn-dependent igg transport in a polarized human intestinal epithelial cell line immunoglobulin transport across polarized epithelia cells human neonatal fc receptor mediates transport of igg into luminal secretions for delivery of antigens to mucosal dendritic cells gut flora in health and disease identification of a unique igg fc binding site in human intestinal epithelium human iggfc binding protein (fcbp) in colonic epithelial cells exhibits mucin-like structure distribution and partial characterization of igg fc binding protein in various mucin producing cells and body fluids mechanisms by which maternal antibodies influence infant vaccine responses: review of hypotheses and definition of main determinants comparative studies of two types of bovine immunoglobulin g heavy chains isolation of bovine immunoglobulins resistant to peptic digestion: new perspectives in the prevention of failure in passive immunization of neonatal calves protein a/g chromatography purification of antibodies using affinity chromatography electrospun polyethersulfone affinity membrane: membrane preparation and performance evaluation selective concentration of bovine immunoglobulins and α-lactalbumin from acid whey using fecl separation of immunoglobulin and transferring from blood serum and plasma by metal chelate interaction chromatography protein adsorption on biodegradable polyanhydride microparticles immunochemical quantification of antigens by single radial immunodiffusion quantification of bovine igg in milk using enzyme-linked immunosorbant assay thermally addressed immunosorbent assay for multiplexed protein detections using phase change nanoparticles quantification of immunoglobulin g in bovine and caprine milk using a surface plasmon resonance-based immunosensor separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds physicochemical characteristics of proteolytic cleavage fragments of bovine colostral immunoglobulin g (igg ) potent antibody therapeutics by design the effect of trypsin and chymotrypsin on the bactericidal antibody activity of bovine colostrum proteolysis of bovine immunoglobulins colostrum protein digestion in newborn lambs n-labeled immunoglobulins from bovine colostrum are partially resistant to digestion in human intestine gastroileal nitrogen and electrolyte movements after bovine milk ingestion in humans survival of anti-colostridium difficile bovine immunoglobulin concentrate in the human gastrointestinal tract bovine immunoglobulin concentrate-colostridium difficile retains c. difficile toxin neutralizing activity after passage through the human stomach and small intestine mammary function during the nonlactating period: enzyme, lactose, protein concentrations, and ph of mammary secretions comparative studies in molecular stability of immunoglobulin g from different species effect of thermal protectants on the stability of bovine milk immunoglobulin g effect of ph on antigen-binding activity of igg from bovine colostrum upon heating specific activity against diarrheagenic bacteria in bovine immune milk and effect of ph on its antigen-binding activity upon heating efficiency and protective effect of encapsulation of milk immunoglobulin g in multiple emulsion temperature and ph dependence of immunoglobulin g conformation effect of heat treatment on the antigen-binding activity of anti-peroxidase immunoglobulins in bovine colostrum kinetic and thermodynamic parameters for heat denaturation of bovine milk igg, iga and igm thermal stability of bovine milk immunoglobulin g (igg) and the effect of added thermal protectants on the stability stability of bovine immunoglobulins to thermal treatment and processing effect of heat treatment on anti-rotavirus activity of bovine colostrum effect of flash-heat treatment on immunoglobulins in breastmilk effect of flash-heat treatment on antimicrobial activity of breastmilk pulsed electric field processing of beer: microbial, sensory, and quality analyses inactivation of e. coli in enriched soymilk using pulsed electric fields effects of pulsed electric fields on the activity of enzymes in aqueous solution effects of pulsed electric fields and thermal processing on the stability of bovine immunoglobulin g (igg) in enriched soymilk effects of pulsed electric fields and heat treatment on stability and secondary structure of bovine immunoglobulin g inactivation of staphylococcus aureus in milk using flow-through pulsed uv-light treatment system recommended laboratory practices for conducting high-pressure microbial inactivation experiments comparison of effects of high-pressure processing and heat treatment on immunoactivity of bovine milk immunoglobulin g in enriched soymilk under equivalent microbial inactivation levels compression heating influence of pressure transmitting fluids on bacteria inactivation during high pressure processing effect of heat and high-pressure treatments on microbiological quality and immunoglobulin g stability of caprine colostrum maintenance of breast milk immunoglobulin a after high-pressure processing heat treatment of bovine colostrum. i: effects of temperature on viscosity and immunoglobulin g level effect of on-farm commercial batch pasteurization of colostrum on colostrum and serum immunoglobulin concentrations in dairy calves effect of heat treatment of bovine colostrum on bacterial counts, viscosity, and immunoglobulin g concentration heat-treatment of bovine colostrum. ii: effects of heating duration on pathogen viability and immunoglobulin g effects of feeding heat-treated colostrum on passive transfer of immune and nutritional parameters in neonatal dairy calves feeding heat-treated colostrum or unheated colostrum with two different bacterial concentrations to neonatal dairy calves feeding heat-treated colostrum to neonatal dairy heifers: effects on growth characteristics and blood parameters key: cord- -r ii bu authors: butler, colin d.; corvalan, carlos f.; koren, hillel s. title: human health, well-being, and global ecological scenarios date: - - journal: ecosystems doi: . /s - - - sha: doc_id: cord_uid: r ii bu this article categorizes four kinds of adverse effects to human health caused by ecosystem change: direct, mediated, modulated, and systems failure. the effects are categorized on their scale, complexity, and lag-time. some but not all of these can be classified as resulting from reduced ecosystem services. the articles also explores the impacts that different socioeconomic–ecologic scenarios are likely to have on human health and how changes to human health may, in turn, influence the unfolding of four different plausible future scenarios. we provide examples to show that our categorization is a useful taxonomy for understanding the complex relationships between ecosystems and human well-being and for predicting how future ecosystem changes may affect human health. the interconnection between ecosystems and human activity is complex, important, and poorly understood. ecosystems support human health and well-being through their provisioning, regulating, cultural, and supporting services (butler and others ) . shortages of food, fiber, and other ecosystem products adversely affect human health via many direct and indirect pathways. the regulating functions of ecosystems that affect health include the purification of air and fresh water, the reduction of flooding and drought, and range limitation of certain vector-borne diseases. ecosystems also impact mental well-being through provision of cultural services, for example by providing totemic species and sacred groves, landscapes, and water bodies. these influence the aesthetic, recreational, educational, cultural, and spiritual aspects of the human experience. ecosystem changes have also altered the epidemiology of communicable and noncommunicable diseases, including through some pathways that would not be considered as arising from a reduced ecosystem regulating service. this article explores the impact that different plausible future scenarios (cumming and others ; raskin ) may have on health, and it also suggests how changes to health may feedback on and hence modify the course of the future. in doing so, we have categorized the effects of ecosystem change on human health into a useful taxonomy which can help predict which future ecosystem changes will impact human health and how. although positive scenarios are conceivable, in this article we are mainly concerned with how adverse ecosystem changes and reduced ecosystem services may harm future human health. until the very recent past, most human induced ecological changes have had favorable effects on human society and health. human health, judged by average life expectancy, has increased substantially, as has population size (riley, ; tuljapurkar and others ) . the reasons for these increases are well-known and include the mutually reinforcing and interacting elements of improved knowledge, technology, and social organization, including of public health (horiuchi ; szreter ) . a fundamental contributory factor has been the increased human capacity to modify ecosystems, for example, by increasing food supplies, by restricting populations of large carnivores, and by providing more fiber for fuel and shelter. this success is not unqualified. some adverse effects of our increased capacity to modify ecosystems on human health can already be seen. the transformation of ecosystems to provide certain benefits has reduced the scale and integrity of many ecosystems (pimentel and others ) . reduced ecosystem integrity decreases their ability to provide some ecosystem services, which can, in turn, have negative impacts on human health. this relationship is unlikely to be linear and may contain thresholds beyond which incremental loss of ecosystem services has a disproportionately negative effect on human-health and well-being. examples of the negative impacts of ecosystem change on human health abound. national life expectancy has fallen in several countries, including many parts of sub-saharan africa, haiti, russia, north korea, and ethiopia (farmer and others ; shkolnikov and others ; united nations population divisional ) . reduced ecosystem services may explain a part of these declines in national life expectancy and thus may be an underrecognized factor in the slowed rate of increase in global life expectancy. the problems of decreased provision of ecosystem services are often unequally distributed, with the majority of the burden falling on the poor. additionally, poor populations frequently lack the income and other means to substitute or partly compensate for reduced ecosystem services (for example, by boiling microbiologically contaminated water). in addition to the effect of ecosystem services on human health, human health itself influences access to critical ecosystem services and can modify the environmental impacts of human populations. for example, the aids epidemic in sub-saharan africa has reduced the provisioning ecosystem service of food supply (de waal and whiteside ) . the high prevalence of yellow fever and malaria delayed the construction of the panama canal, and sleeping sickness still limits human settlement and thereby affects human access to ecosystem services in parts of central africa (bhalla ) . a major challenge in this field is to apply real world data to conceptual models (miranda and others ) , to validate the models, and to develop approaches that can serve as a vehicle for generating hypothesis-driven research. although realization of these goals remains distant, the conceptual frameworks which will stimulate data acquisition and analysis in this field are developing (butler and others, ) . figure shows one such framework, linking natural and social systems with human wellbeing, of which health is an important component. here we introduce four categories of adverse effects on human health due to ecosystem change as a means to help understand the impacts of the different ecological scenarios on human health and well-being. in ascending order of scale, complexity, and lag-time, we call these adverse effects direct, mediated, modulated, and systems failure (see table ). we emphasize that, at their margins, these categories overlap because drivers may differ on temporal and spatial scales. figure graphically presents these concepts and provides a preliminary attempt to approximate the quantitative impact of the different categories. direct (adverse) health effects are manifested through the immediate impacts of the loss of a useful ecosystem service, such as the provision of sufficient food, clean water, fertile soil or the restriction of erosion and flooding. direct effects occur as the result of physical factors but do not include pathogens per se. miranda and others, ) climate change has recently been recognized as causing a substantial change in the lake tanganyika ecosystem. the fish catch has decreased due to a climate-related reduction in the nutrient supply (o'reilly and others ) . this reduction in ecosystem services places additional economic and nutritional stresses on an already poor and vulnerable human population. although data to measure the health effect of this reduced catch are unlikely to be available, the effect is probably adverse because it causes reduced income and reduced nutrition (verschuren ) . another example is the collapse of cod fishing in the north atlantic, which caused widespread unemployment, mental distress, and social dislocation, but little if any true under nutrition because the social mechanisms operating in canada were able to partially substitute for the lost provisioning services once supplied by the fishery. a third example of a direct health effect from a reduced ecosystem service is the disruption and physical injury caused by flooding. there is increasing recognition that floods are caused by the interaction of climatic and landuse changes (hellin and others, ; zhang and others, ) . there is also increasing evidence that mental and physical health is enhanced by contact with nature (friedman and thomas ) . reduction of the cultural services that ecosystems provide is likely to contribute to the already enormous burden of disease caused by impaired mental health. compared to direct effects, mediated effects have increased causal complexity and, in some cases, involve pathogens. some mediated effects have the potential for high rates of illness and death. there is also often a longer lag between the ecosystem change and the health outcome than for direct effects. however, by definition, mediated effects are insufficient in scale to cause the larger-scaled social collapse that we define as a modulated effect. many infectious and some chronic diseases fall in this category. the epidemiology of many communicable diseases is related to ecological factors. some major nonvector-borne diseases, including tuberculosis, measles, and influenza, are thought to have crossed into human populations because of close contact with domesticated animals (mcneil ; daszak and others, ; oxford and others, ) . changes to biodiversity may be associated with increased numbers of disease-transmitting insects. although contested, there are suggestions that malaria may also have become a significant human disease following the development of agriculture (pennisi ; joy and others ) . more recently, variant creutzfeld-jacob disease, nipah virus, and hendra virus illustrate novel infectious diseases that have entered human populations because of changed and more intensive animal feeding and farming practices (waltner-toews and lang ). the emergence of nipah virus may also have been related to bats fleeing from the intense drought and el niñ o-related fires in indonesia (epstein and others, ) . the long list of other infectious diseases related to ecosystem change (patz and others, ) includes schistosomiasis (li and others, ) , cholera (pascual and others, ) , and lyme disease (jones and others, ; blockstein ) . in many of these cases, the disease has emerged as a result of increased food-producing capacity of ecosystems-a provisioning ecosystem service-for example, by animal domestication, irrigation, dams, and other intensive farming practices. a tradeoff has been the unforeseen increase in the incidence and prevalence of many of these communicable diseases. some mediated health effects have also led to migration, while others have prevented the colonization of certain areas. for example, malaria has long restricted human settlement in lowland areas, including the terai in nepal, and many parts of equatorial africa. some chronic, noninfectious diseases can also be classified as mediated effects of ecosystem change, including allergies, asthma, and some forms of cancer and chronic lung disease. for example, lung cancer and pulmonary fibrosis have become particularly common in the region around the shrunken aral sea, as pesticide-contaminated dust from this human-made desert is inhaled (o'hara and others ). both long-distance dust transport and more localized air pollution are also related to ecosystem service change and have been linked with a number of diseases, including asthma and atopy (monteil ) . there is also increasing evidence that air pollution, often exacerbated by ecosystem change such as land clearing and fires, may aggravate heart disease (pyne ) . future ecosystem change, such as desertification, leading to a decrease in the ecosystem provisioning service of clean air, could thus alter the epidemiology of these diseases. these diseases are classified as mediated rather than direct because their connection to changed ecosystems is more complex than are direct effects. direct and mediated health effects are analogous to the direct and indirect health effects of climate change. in that classification, direct (adverse) health effects include phenomena such as heat stroke, while indirect effects include changes to certain vector-borne diseases because of altered patterns of temperature, humidity, and rainfall, and other effects secondary to extreme weather and adverse economic effects. it is possible that a novel emerging disease could escape from a remote ecosystem to enter the wider human population, as the plague and hiv probably did. however, at least in the case of hiv, its really major (modulated) impact depended on powerful social cofactors, including severe poverty, social practices and taboos, and poor governance (butler a (butler , b . the ecological factors that underlie the recent sars outbreak remain unclear (enserink and normile ) , but its origin and amplification in a region of china, characterized by extremely dense populations of humans and domesticated animals and by poor public health services (anonymous ) , is consistent with the view that human-dominated ecosystems today harbor more danger to population health than does the ''wild'' (oxford and others ) . a plausible example of this principle could be the widespread transmission of multi-or even omni drug-resistant tuberculosis emerging from a prison (tanne ; dye and others, ) . this scenario would have severe economic implications, especially for aviation and other industries perceived as increasing the probability of disease spread. we also identify a larger-scale, more lagged, and more causally complex adverse consequence of adverse ecosystem change, than either direct or mediated effects, which we call a modulated or tertiary effect (figure ). these effects include episodes of state failure, or of nascent or realized large-scale social and economic collapse. the role of environmental factors in the causation of large-scale conflicts, state failure, and social collapse is controversial (deudney ; gleick ; homer-dixon ; uvin ; cramer ) . we agree that causation for the phenomena is complex, but we assert that reduced ecosystem services and other adverse ecosystem changes are frequently a component of the causal webs that lead to these phenomena (butler and others ) . this may be of increasing significance in the near future as evidence accrues that ecosystems are being changed more frequently and at larger scales. there is compelling evidence that reduced ecosystem services were a causal factor for several large-scale social collapses and catastrophes, from both archeological sources (weiss and bradley ) and more recent history. two ancient cases are the collapse of the ancient mesopotamian and the mayan civilizations, contributed to, respectively, by increased salinity (jacobsen and adams ) and drought (haug and others ) . two more recent examples are the irish famine of the s and the rwandan genocide in . the irish famine was caused by the spread of a potato fungus (wilson ) interacting with a refusal by the british government to supply an effective substitute, such as famine relief (sen , pp - ) . the rwandan genocide also occurred as a result of the interaction of multiple factors, including poor governance, long-standing ethnic hatred, and rapid population growth. the violence was inflicted mainly by a large number of unemployed young men (mesquida and weiner ; potts ) , displaced from a livelihood in farming because of the shortage of fertile arable land, thus losing a key ecosystem service (andré and platteau ; butler- a) . in these examples adverse health effects are likely to be larger than those from mediated effects, although in some cases state failure may be limited to small populations, such as for the people of easter island or the norse in medieval greenland. inevitably, ecosystem service changes that contribute to modulated effects will be embedded in a mosaic of social, economic, and political cofactors. in turn, many of these cofactors are likely to have at least partial ecosystem change-dependent causation. depending on the knowledge, bias, and experience of the observer, the causal role of ecological factors in state failure may sometimes be underestimated, or even totally denied. for example, rotberg ( ) identifies the roots of state failure as based in ethnic, religious, linguistic, or other intercommunal enmity. he argues that state failure is ''man-made, not merely accidental nor-fundamentally-caused geographically, environmentally, or externally.'' we do not claim that reduced ecosystem services or other ecosystem changes that lead to adverse health effects are always a ''fundamental'' factor in state failure, but they are often important and usually identifiable. the enmity that rotberg refers to often arises over the distribution of diminishing per capita ecosystem services. there may be increasing recognition of this. for example, o'reilly and others ( ) concludes, in discussing the potential for further reduction in the ecosystem provisioning service of lake tanganyika, that ''the human implications of such subtle, but progressive, environmental changes are potentially dire in this densely populated region of the world, where large lakes are essential natural resources for regional economies.'' ecosystem services as a significant element in state failure may be underrecognized due to our tendency to discount the future possibility of thresholds or emergence. thresholds refer to sudden, nonlinear changes that result from a small increment and that are not intuitively predictable without prior experience (alley and others ; waldrop ; may ) . emergence refers to the new property that becomes apparent beyond the threshold. modulated and systems failure effects (described below) are emergent phenomena that become apparent when linked socioecological systems pass a threshold, caused by the interaction of numerous social, political, and ecological elements. we also describe an even larger scale phenomenon than state failure, as a result of coalescing, interacting modulated effects. we call this phenomenon ''systems failure.'' the increasing connections that insulate diverse human communities from scarcity also create large-scale vulnerabilities, magnifiable by feedbacks such as collapsed global trade, terrorism, technological breakdown, and radiating failure of institutions and governance. collapse could occur on a regional, continental, or even global scale. it is also possible, however, that a reverse state, ''systems success,'' could occur. large-scale epidemics exacerbated by chronic food insecurity, poor governance, and wide-scale conflict are plausible elements of this pathway. drug-resistant bacteria, in part driven by the excessive use of antibiotics in animal husbandry, could contribute to this, as could the emergence of new viruses. however, we stress that novel infectious agents are unlikely to lead to modulated or systems failure effects without significant cofactors. only modulated and systems failure effects are likely to be of sufficient scale to alter the unfolding of the various ecological and socioeconomic scenarios that are described elsewhere in this issue of ecosystems. cumming and others ( ) and raskin ( , this issue) review several socioecological scenarios that may unfold over this century. although all plausible futures are influenced by the same driving forces, these forces evolve in different ways in the different scenarios. demographic, economic, political, cultural, and social factors -including health -are codependent so that each factor will continually influence other factors. each scenario will influence and be influenced by ecological factors as well. just as in the past, the future world will contain a mixture of familiar and novel situations. scenarios seek to coalesce these myriad possibilities into a limited number of theme futures that have strongly plausible elements. although there are dozens of names for the existing environmental scenarios, most can be categorized into a surprisingly small group of core pathways, as described by cumming and others (this issue) . the names of the four scenarios considered in this article are market forces, reformed market, value change, and higher fences (see table ). it is difficult to succinctly describe these scenarios, but a number of axes can be identified along which they vary. for example, there is a spectrum identifiable between comparative economic deregulation (market forces) to a neo-keynesian model, here called reformed market. another spectrum can be identified between a concerted attempt to protect existing ecosystem services (the ''value change'' scenario) and a laissez faire approach to ecosystems and the nonliving environment, such as climate change (the ''market forces'' scenario). a third spectrum is identifiable between trade deregulation (''reformed market'') and continuing or even increased protectionism (''higher fences''). as well, the trend of global income distribution can be predicted from these scenarios, from a continuing increase (''higher fences'') to a marked decrease (''reformed market'' and ''value change''). the state of health for high-and low-income populations can be predicted, largely consequent to the anticipated change in income for each group. three of these scenarios, as very briefly described, are essentially optimistic, because they all assume an increase in income for high-and low-income populations. however, in the higher-fences world, it is conceivable that incomes will decline for populations that currently have a low income, and the increased global inequality in this scenario could exacerbate tensions between low and income populations. cumming and others ( ) examine the assumptions made about ecosystem resilience in each of four scenarios and find that the outcome of different scenarios is influenced by this resilience. table lists some key terms concerning health for both high-and low-income populations in these four scenarios. it also shows our estimation of the probability of changes in the ''health gap,'' that is, the gap between high-and low-income populations with respect to health. at present, for example, the burden of disease from diarrhea (of which nearly % can be attributed to unsafe water, poor sanitation, and hygiene) is over times higher in the least developed countries than in developed ones. the health gaps between high-and low-income populations have been systematically estimated using a measure called ''disability adjusted life years'' which accounts for total years of life lost because of disease and also for partial years of life lost because of disability (world health report ) . it is likely that in three of the four scenarios the health gap will decrease. this is because three of these scenarios assume gradual socioeconomic convergence between populations that are currently rich and poor. these scenarios postulate continued advances in science, technology, and the dissemination of information and expertise. the response to the sars outbreak illustrates the potential for a coordinated response to events that are perceived as sufficiently threatening. if convergence between rich and poor populations occurs, then coordinated responses to numerous health problems that continue to affect poor populations are likely. advances could improve new vaccines, attention to ''orphan'' diseases, and dis-tribution of the improved seeds and agricultural techniques needed to enhance food security. in these scenarios the health of high-income populations is also likely to improve, though not as rapidly as for low-income populations. in only one scenario -called ''barbarization'' (raskin, this issue) or ''higher fences'' (cumming and others, this issue) -is the health gap likely to increase. in this scenario, poor populations are increasingly ignored by the remaining population. food security of the poor is likely to further diminish, perhaps leading to positive feedbacks as malnutrition impairs cognitive development and further hinders education and the chance of skillful social and political responses. in this scenario the health of high-income populations is unlikely to be ideal: we identify obesity, diabetes, and anxiety as likely to increase, with their negative effects only partially countered by improved medical technology. modulated effects could sabotage even optimistic scenarios. table provides an estimation of the chance of systems failure, depending on assumptions concerning the resilience of ecosystems and the linked socioeconomic system. cumming and others ( ) have defined ecosystem resilience as the capacity to absorb anthropogenic impacts without the loss of essential structure or functions. we suggest that it is meaningful to assume that human populations are characterized by social resilience, which modifies their capacity to effectively deal with stress (carpenter and others ) . from a public health perspective, resilience may be defined as the capacity of society to respond to problems and challenges, over the short and long term, in ways that protect and advance public health, over the short and long term. affluence, education, social cooperation, technological capability, and flexibility are important determinants of the size of social buffers and human resilience. we suggest that systems failure effects are more likely to occur in the market-forces and higher-fences scenarios, because inequality between high-and low-income populations is likely to be the greatest in these scenarios. the most optimistic future for human health is likely to be if both ecological and social systems prove highly resilient. in this case both major ecosystem and social services are likely to be preserved even if ecosystem changes (currently perceived by many as adverse) continue to occur at a high rate. on the other hand, the chance of further modulated or even systems failure effects occurring is increased if ecosystem and social systems prove to be brittle. because the size of ecosystem service buffers is falling and the extent of ecosystem service resilience is uncertain, it is prudent to reduce fur- ther ecosystem damage as rapidly as possible. however, it is also prudent to conduct this in a way that minimizes harm to human health. continuing tradeoffs are likely to be required. for example, improving the health of populations in indonesia may require further clearing of forests to generate more income. but this process cannot be continued indefinitely. on the other hand, excessively strict protection of the surviving ecosystems could reduce the size of the socioeconomic buffer, thus perhaps increasing the longterm risk of a modulated effect. in reality, economic and social forces make an extremely rapid transition to full protection of surviving ecosystems unlikely, but it may be just as risky to delay protection in response to only short-term socioeconomic concerns. the causal relationship between ecosystem service change and impact upon human health remains incompletely understood. we have explored how ecosystem services impact human health and have proposed that adverse ecological changes can interact and feedback with dysfunctional social responses, leading to the development of states that we have termed mediated and systems failure. we have grouped the myriad possible interactions and cascading responses between ecosystem services and public health into four categories, a previously undescribed taxonomy which might help us better comprehend how ecosystem change and human health interact to affect the way the future unfolds. we believe that this is an important conceptual advance that will be useful for understanding the relationship between human health and changes in ecosystem services. continued analysis of cases studies, using both quantitative and qualitative methods, will advance our understanding of these relationships. several regions of the world, characterized by substantial ecological and social stresses, may be useful ''natural laboratories'' for this purpose, including sub-saharan africa, indonesia, and northern india. direct and mediated effects, although they may lead to some important changes to the path of the future, are unlikely to seriously compromise the development of regional or global civilization. despite occasional and localized setbacks, human health is likely to generally improve if future ecosystem changes result only in events such as occasional flooding, periodic disease outbreaks, and episodes of air pollution. on the other hand, modulated and systems failure effects, if they occur, have the power to alter the course of society in significant ways. if negative this will cause substantial harm to human health and well-being, and, by exacerbating poor governance, could also further erode ecosystem services. such events could derail even the most optimistic scenario. however, we do not deny the chance that positive systems effects could emerge, especially if ecosystem and social resilience remain high. current trends toward an increasingly large environmental footprint, further climate change, depletion of fossil fuels, and the erosion of existing ecological and social buffers are disturbing and unlikely to be sustainable (mcmichael ) . on the other hand, the increasing capacity to conceptualize, diagnose, and modify the global environment gives hope that humanity will self-organize in ways that can sustain both its social and ecological functions (crutzen ) . abrupt climate change land relations under unbearable stress: rwanda caught in the malthusian trap emerging stronger from the china crisis pan african group takes lead against the tsetse fly lyme disease and the passenger pigeon? entrapment: global ecological and/or local demographic? reflections upon reading the bmj's ''six billion day'' special issue hiv and aids, poverty, and causation ecosystems and human well-being. in: ecosystems and human well-being. a framework for assessment: a framework for assessment. millennium ecosystem assessment from metaphor to measurement: resilience of what to what? homo economicus goes to war: methodological individualism, rational choice and the political economy of war geology of mankind are existing global scenarios consistent with ecological feedbacks? infectious diseases of wildlife-threats to biodiversity and human health new variant famine: aids and food crisis in southern africa environment and security: muddled thinking erasing the world's slow stain: strategies to beat multidrug-resistant tuberculosis search for sars origins stalls conservation: our health depends on it [editorial] emerging diseases threaten conservation unjust embargo of aid for haiti pet ownership, social support, and one-year survival after acute myocardial infarction in the cardiac arrhythmia suppression trial (cast) environment and security: the clear connections climate and the collapse of maya civilization rainfall characteristics of hurricane mitch environmental scarcities and violent conflict: evidence from cases greater lifetime expectations salt and silt in ancient mesopotamian agriculture chain reactions linking acorns to gypsy moth outbreaks and lyme disease risk early origin and recent expansion of plasmodium falciparum epidemiology of schistosoma japonicum in china: morbidity and strategies for control in the dongting lake region how the biosphere is organized human frontiers, environments and disease: past patterns plagues and peoples human collective aggression: a behavioral ecology perspective policy concepts and applications dust clouds and spread of infection alternative projections of mortality and disability by cause - : global burden of disease study exposure to airborne dust contaminated with pesticide in the aral sea region climate change decreases aquatic ecosystem productivity of lake tanganyika world war i may have allowed the emergence of ''spanish'' influenza cholera dynamics and el niñ o-southern oscillation effects of environmental change on emerging parasitic diseases malaria's beginnings: on the heels of hoes? ecological integrity: integrating environment, conservation, and health. washington dc the population policy pendulum. needs to settle near the middle and acknowledge the importance of numbers small particles add up to big disease risk global scenarios: background review for the millennium ecosystem assessment rising life expectancy: a global history the new nature of nation-state failure development as freedom changes in life expectancy in russia in the s rapid economic growth and the four ds of disruption, deprivation, disease and death: public health lessons from th-century britain for st-century china? drug resistant tb is spreading worldwide a universal pattern of mortality decline in the g countries world population prospects: the revision. the demographic impact of hiv/aids tragedy in rwanda: the political ecology of conflict global change: the heat on lake tanganyika complexity. the emerging science at the edge of order and chaos a new conceptual base for food and agricultural policy: the emerging model of links between agriculture, food, health, environment and society what drives societal collapse? infectious disease: an ecological perspective world health organization china's forest policy for the st century we thank the millennium ecosystem assessment for providing many opportunities to discuss the main ideas expressed in this article with colleagues. we also thank professor d. crawford-brown and two anonymous reviewers for their comments and suggestions. key: cord- -fn zlutj authors: nan title: abstracts of the th annual meeting of the german society of clinical pharmacology and therapy: hannover, – september date: journal: eur j clin pharmacol doi: . /bf sha: doc_id: cord_uid: fn zlutj nan grapefruit juice may considerably increase the systemic bioavailability of drugs as felodipine and nifedipine. this food-drug interaction has potential practical importance because citrus juices are often consumed at breakfasttime when drugs are often taken. it is likely that a plant flavonoid in grapefruit juice, naringenin, is responsible for this effect (inhibition of cytochrome p- enzymes in the liver or in the small intestinal wall). ethinylestradiol (ee ), the estrogen of oral contraceptive steroides, shows a high first-pass-metabolism in vivo. therefore, the purpose of this study is to test the interaction between grapefi-uite juice and ee , the area under the serum concentration-time curve (auc _ h) ofee was determined in a group of young healthy women (n = ) on day + ofmenstruale cycle. to compare intraindividually, the volunteers were randomly allocated to two test days. the female volunteers took lag ee together with either ml of herb tea or with the same amount of grapefruit juice (content of naringenin mg/ ). furthermore, on the day of testing the women drank times ml of the corresponding fluid every three hours up to four times. the auc . h of ee amounts to . + . pg x mi- x h after the administration of the drug with grapefruit juice; that means % higher in comparison to + . pg x m - x h after concomitant intake of tea. also, the mean cmax-value increases to %, p _< . ( . + . pg x m - and . + . pg x m - , respectively). this result shows that the systemic bioavailability ofee increases after intake of the drug with grapefruit juice. the extent of this effect is lower than the extent of known interindividual variability. procarbazine is a tumourstafic agent widely used m hodgin's disease, non-hodgldn's lymphomas and mmours of brain and lung. procarbazine is an inactive prodrug which is converted by a cytochrome p mediated reaction to its active metabolites, in the first step to azoprocarbazine. the kinetics of both procarbazine and azoprocarbazine is not described in humans up to now. on turnout patients we have investigated the plasma kinetics of both procarbazine and azoprocarbazine after oral adminislxation of mg procarbazine in form of capsules and drink solution, respectively. a hplc method with uv-detection ( nrn) and detection limits of and ng/ml was developed for procarbazine and azoprocarbazine respectively. after both the capsules and drink solution the parent drug could be detected in plasma only for h. in contrast the tl/ of terminal elimination of azoprocarbazine was estimated in the range of , to , h with a mean of , h - + , h. the auc of procarbazine was less than % of that of azoprocarbazine. cma x values of azoprocarbazine were determined in the range of , to ,l gg/ml. in comparison to the drink solution we determined on the basis of the plasma levels of azoprocarbazine a bioavailability of the therapeutic used procarbazine capsules of , + , %. prostaglandin e (pge ) is used for the treatment of patients with peripheral arterial disease, and probably effective due to its vasodilator and antiplatelet effects. l-arginine is the precursor of endogenously synthesized nitric oxide (no). in healthy human subjects, larginine also induces peripheral vasodi]ation and inhibits platelet aggregation due to an increased no production. in the present study the influence of a single intravenous dose of l-arginine ( g, min) or pge ( p.g, min) on blood pressure, peripheral hemodynamics (femoral artery duplex sonography), and urinary no -and cgmp excretion rates was assessed in ten patients with peripheral arterial disease (fontaine iii -iv). blood flow in the femoral artery was significantly increased by l-arginine by % (p < . ), and by pge by % (p < . ). l-arginine more strongly decreased systolic and diastolic blood pressure than pge . plasma arginine concentration was increased -fold by l-arginine, but unaffected by pge . urinary excretion of no -increased by % after l-arginine (p < . ), and by % after pge (p = n.s.). urinary cgmp excretion increased by % after l-arginine and by % after pgei (each p = n.s.). we conclude that intravenous l-arginine decreases peripheral arterial resistance, resulting in enhanced blood flow and decreased blood pressure in patients with peripheral arterial disease. these effects were paralleled by increased urinary no -excretion, indicating that systemic no production was enhanced by the infusion. increased no -excretion may be a sum effect of no synthase substrate provision (l-arginine) and increased shear stress (pge and l-arginine). it is weli established that the endothelial edrf/no-mediated relaxing mechanism is impaired in atherosclerotic and in hypertensive arteries. recently it was suggested that primary pulmonary hypertension might be another disease in which the endothelial edrf/no pathway is disturbed. we tested the hypothesis that intravenous administration of l-arginine (l-arg), the physiological precursor of edrf/no, stimulates the production of no, subsequently increasing plasma cgmp levels and reducing systemic and / or pulmonary vasular resistance, in patients with coronary heart disease (chd; n = ) and with primary pulmonary hypertension (pph; n = ). l-arg ( g, min) or placebo (nac ) was infused in chd patients, and l-arg was infused in pph patients undergoing cardiac catheterization. mean aortic (pao) and pulmonary (ppul) arterial pressures were continuously monitored. cardiac output (co; by thermodilution), and total peripheral resistance (tpr) were measured before and during the infusions. plasma cgmp was determined by ria. in chd patients, pao decreased from . + . to . + . mm hg during l-arg (p< . ), whereas ppul was unchanged. tpr decreased from . -+ . to . + . dyne sec cm - during l-arg administration (p< . ). co significantly increased during l-arg (from . + . to . + . /min, p< . ). placebo did not significantiy influence any of the haemodynamic parameters, cgmp slightly increased by . + . % during l-arg, but slightly decreased during placebo (- . + . %)(p < . for l-arg vs. placebo). in pph patients, l-arg induced no significant change in pao, tpr, and co. mean ppul was . + . mm hg at the beginning of the study, but was only slightly reduced by l-arg to . + , mm hg (p = n.s.). plasma cgmp was not affected by l-arg in these patients. we conclude that l-arg stimulates no production and induces vasorelaxation in chd patients, but not in patients with primary pulmonary hypertension. thus, the molecular defects underlying the impaired no foimation may be different m both diseases. institutes of clinical pharmacology, *cardiology, and **pneumology, medical school, hannover, germany. the influence of submaximal exercise on the urinary excretion of , -dinor-pgflc, (the major urinary prostacyclin metabolite), , dinor-txb (the major urinary thromboxane a metabolite), and pge (originating from the kidney), and on platelet aggregation was assessed in untrained and endurance-trained male subjects before and after days of rag/day of aspirin. urinary , -dinor-txb excretion was significantly higher in the athletes at rest (p < . ). submaximal exercise increased urinary , -dinor- -keto-pgfl~ excretion without affecting , -dinor-txb or pge excretion or platelet aggregation. aspirin treatment induced an - % inhibition of platelet aggregation and , -dinor-txb excretion in both groups. however, urinary , -dinor- -keto-pgfl~ was inhibited by only % in the untrained, but by % in the trained group (p < . ). urinary pge was unaffected by aspirin in both groups, indicating that cyclooxygenase activity was not impaired by a systemic aspirin effect. after low dose aspirin administration, the same selective stimulatory effect of submaximal exercise on urinary , -dinor- -keto-pgfl~ excretion was noted in both groups as before. the ratio of , -dinor- -keto-pgfld , -dinor-txb was increased by exercise; this effect was potentiated by aspirin (p < . ). our results suggest that the stimulatory effect of submaximal exercise on prostacyclin production is not due to an enhanced prostacyclin endoperoxide shift from activated platelets to the endothelium, but rather the result of endothelial prostacyclin synthesis activation from endogenous precursors. mg/day of aspirin potentiates the favorable effect of submaximal exercise on endothelial prostacyclin production by selectively blocking platelet cyclooxygenase activity. institute of clinical pharmacology, medical school, hannover, germany. soluble guanylyl cyclases (gc-s) are heterodimeric hemeproteins consisting of two protein subunits ( kda, kda). the enzyme is activated by nitric oxide (no) and catalyzes the formation of the signal molecule "cgmp" (cyclic guanosine- 's'-monophosphate) from gtp. numerous physiological effects of cgmp are already very well characterized. however, detailed insights in the no-activation mechanism of this enzyme have been described to date only in a hypothetical model ( ). recently, this concept was supported by experimental data using sitedirected mutagenesis to create a no-insensitive soluble guanylyl cyclase mutant ( ). it is generally accepted that the prostethic heine-group plays a crucial role in the activation mechanism of this protein. nonetheless, some interesting questions with regard to structure and regulation of soluble guanylyl cyclases still need to be uncovered (e.g. activation with other free radicals, such as carbon monoxide). since this kind of studies is limited so far by isolating large quantities of a biologically active enzyme with conventional purification techniques, the recombinant protein was expressed in the baculovirus / insect cell system. we describe here the construction and characterization of recombinant baculoviruses, harboring the genes that encode both protein subunits of the soluble guanylyl cyclase. insect cells infected with these recombinant baculoviruses produce between - % (as related to total cell protein) of functional soluble guanylyl cyclase. positive infection was monitored as a change in morphology of the cells and by production of the respective recombinant viruses detected by polymerase-chain-reaction (pcr). so far examined, the recombinant enzyme exhibits similar physicochemical characteristics as the "natural" protein. exogenous addition of several heme analogues to the infected cells is able to either stimulate or inhibit the enzymatic activity of gc-s. we are confident to purify milligram amounts of the recombinant protein in the near future. pet studies of myocardial pharmacology have principally concerned the sympathetic nervous system and u'acers have been developed to probe the integrity of both pre-and post-synaptic sites. the sympathetic nervous system plays a crucial role in the control of heart rate and myocardial contractility as well in the conlrol of the coronary circulation. alterations of this system have been implicated in the pathophysiology of a number of .cardiac disorders, in particular, heart failure, ventricular arrhythmogenesis, coronary artery disease, idiopathic dilated and hypertrophic cardiomyopathy. several beta blockers have been labelled with carbon-ll for imaging by pet. the most promising of these is cgp which is a non-selective beta adrenoceptor anatagonist particularly suited for pet studies due to its high affinity and low lipophilicity, thus enabling the functional receptor pool on the cell surface to be studied. studies in our institution in a group of young healthy subjects have yielded bmax values of . _+ . pmol/g myocardium. these data are consistent with literature values of bmax for beta adrenoceptors in human ventricular myocardium determined by a variety of in vitro assays. a recent study in patients with hypertrophic cardiomyopathy has shown that myocardial beta adrenoceptor density is decreased by approximately - % relative to values in normal subjects. the decrease in receptor density occurs in both hypertrophied and nonhypertrophied portions of the left ventricle. these data are consistent with the hypothesis that sympathetic overdrive might be involved in the phenotypic expression of hypertrophic cardiomyopathy. a further decrease of myocardial beta adrenoceptor density (to levels well below _ - . pmol/g) has been observed in those patients with hypertrophic cardiomyopathy who procede to ventricular dilatation and heart failure. cyp a hydroxylates polycyclic aromatic hydrocarbons such as benzo(a)pyrene occurring e.g. in cigarette smoke. two hereditary mutations are discovered: ml, a t to c transition , bp downstream of exon ; m , located at position , in exon representing an a to g transition resulting an isoleucine to valine substitution in the heme-binding region. recently we could demonstrate in caucasians that carriers of the m -mutation possess an increased risk towards lung cancer (drakoulis et al clin.lnvestig. : , ) , whereas the ml-mutation shows no such association. the phasg-ii enzyme gstm catalyses the conjugation of glutathione to electrophilic compounds such as products of cyp ai. gstm is absent in . % of the caucasian population due to base deletions in exon and of the gene. we found no contrariety in the gstm distribution, including frequencies of type a (p.) and type b (v) among lung cancer patients (odds ratio = . , n = ; cancer res. : res. : , . lung cancer patients and reference patients were investigated for mutations of cypia and gstm by allele-specific pcr and rflp. a statistically significant higher risk for lung cancer among carriers of the m trait was found (odds ratio = . , p = . ). interestingly, amid lung cancer, m -alleles were less often linked to ml than in controls (odds ratio = . , %-confidence limits = . - . , p = . ). however, the frequency of cypia mutations did not differ among active and defective gstm types. consequently, we could not confirm in the caucasian population the synergistic effects of cypia mutations (especially m ) and deficient gstm as combined susceptibility factors for lung cancer as described among the japanese (cancer res. : , in healthy subjects the effect of gastrointestinal hormones like somatostatin and glucagon on splanchnic hemodynamics is not well defined due to the invasiveness of the direct measurement of e.g. portal vein (pv) wedged pressure. methods : now, we applied duplex sonography ( . m~z) and color coded flow mapping to compare the effects of ocreotide (i ~g sc), a long acting somatostatin agonist, and glucagon (i mg iv) on the hemodynamics of the pv, superior mesenteric artery (sma) and common hepatic artery (ha) in healthy volunteers ( g,i q; ± y; x ± sem). basal values of pv flow ( . ± . cm/s), pv flow volume ( ± ml/min), sma systolic (sf: ± cm/s) and diastolic flow (df: ± cm/s), sma pourcelot index (pi) ( . ± . ), ha sf ( ± cm/s) and df ( ± cm/s) and ha pi ( . ± . ) well agreed with previously reported results. within min ocreotide resulted in a decrease of sma sf (- ± %) sma df (- ± %), ha sf (- ± %) and ha df (- ± %). maximum drop of pv flow (- ± %) and flow volume (- ± %) occurred at min. all effects diminished at min. no significant change of vessel diameter and pi was seen. min following its application glucagon caused a highly variable, only short lasting increase of pv flow volume (+ ± %) and sma df (+ ± %). ha fd (+ ± %) showed a tendency to rise (ns). we conclude that in clinical pharmacology duplex sonography is a valuable aid for measuring effects of hormones and drugs on splanchnic hemodynamics. pectanginal pain and signs of silent myocardial ischemia frequently occur in hypertensives, even in the absence of coronary artery disease (cad) and/or left ventricular hypertrophy, probably due to a reduced coronary flow reserve. since the oxygen extraction of the heart is nearly maximal during rest, increases of oxygen demand cannot be balanced by increases of myocardial perfusion: to assess the frequency of ischemic type st-segment depressions in this patients and to determine the influence of heart rate (hr) and blood pressure (bp), simultaneous h hoher-and h ambulatory bp monitoring were performed in hypertensives (age - years, f, m) without cad before and after four weeks on therapy with the -blocker betaxolol. episodes of significant st-segment depressions (> . mv,> min) of a total length of min could be demonsu'ated in / patients ( %) without antihypertensive therapy_ systolic bp significantly increased from + . mmhg (mean + sd, p < . ) min before to a maximum of + . mmhg during the ischemic episodes, hr and rate-pressure product (rpp) increased from + . min -t and . + . mmhg x rain -t x to _+ . min-: and . + . mmhg x min - x (p < . ). the extent of st-segment depressions significantly correlated with hr and rpp (p < . ). drug therapy with - mg/d betaxolol for weeks significantly decreased mean hr, systolic' and diastolic bp (p < . ). ischemic episodes of a total length of min were recorded only in of hypertensives ( . %; p < . ; x -test). in conclusion, increases of hr and systolic bp seem to be the most important factors which induce myocardial ischemia in hypertensives without cad. as silent ischemia is a independent risk factor for sudden cardiac death and other cardiac events, specific antihypertensive therapy should not only be aimed to normalize blood pressure, but should also address reduction of ischemic episodes as demonstrated here. phosphodiesterase inhibitors exert their positive inotropic effects by inhibiting camp degradation and increasing the intracellular calcium concentration in cardiomyocytes. an identical phosphodiesterase type i[ has been demonstrated in platelets and vascular smooth muscle cells. we studied the influence ofpiroximone on platelet function in vitro and ex vivo and the hemodynaimc effects of a bolus application of piroximone in patients with severe heart failure (nyha iii-iv) using a swan -ganz-catheter. in order to study the influence ofpiroximone on platelet function in vitro, platelet rich plasma from healthy volunteers was incubated with piroximone ( - ~tmol/l) from minute to hottrs and aggregation was induced by addition of adp. for the ex vivo experiments platelet rich plasma was obtained from patients, who received piroximone in doses of . , . , . or . mg/kg bw. blood samples were drawn immediately before and , , , and minutes after bolus application. the adp-induced platelet aggregation was inhibited time-and dosedependently. the ic value for piroximoue in vitro amounted to + omol/ . in the ex vivo experiments the maximal inhibition of adp-induced aggregation was obtained in prp from patients who had received mg/kg bw piroximune minutes before. the admitdstration ofpiroximone resulted in a marked hemodynamic improvement with a dose-dependent increase in cardiac index and decreases in pulmonary artery pressure and resistance. to treat conditions associated with acute and chronic multiorgan dysfunction. studies indicate patients receive approximately ten drugs, on average during their icu stay, from several drug classes. commonly prescribed drugs include narcotics, sedatives, antibiotics, antiarrhythmics, antihypertensives, drugs for stress ulcer prophylaxis, diuretics, vasopressors, and inotropes. reports suggest surgical icu patients cost the hospital an average of $ , /patient in un-reimbursed costs under fixed-price reimbursement. furthermore, patients with the greatest drain in revenue received catecholamines, triple antibiotics, or antifungal agents. thrombolytics, antibiotics, plasma expanders, and benzodiazepines account for nearly twothirds of the cost of drugs prescribed in medical and surgical icus. agents with considerable economic impact include biotechnology drugs for sepsis. pharmacoeconomic data in icu patients suggest increased attention should be directed towards several areas, including patients with pneumonia, intraabdominal sepsis, nosocomial bloodstream infections, optimizing sedation and analgesic therapy, preventing persistent paralysis from neuromuscular blockers, preventing stress ulcers, treating hypotension, and providing optimal nutritional support. studies are needed to assess the impact of strategies to improve icu drug prescribing on length of stay and quality of life. if expensive drugs are shown to decrease the length of icu stay, then their added costs can have positive economic benefits to the health care system. the responses to min iv. infusions of the -and -adrenoceptor agonist isoprenalin (iso) and the -(and c~-) adrenoceptor agonist adrenalin (adr) at constant rates of ijg/min were evaluated noninvasively after pretreatment (pre-tr) with placebo (pl), mg of the -selective adrenoceptor antagonist talinolol (tal) and mg of the non-selective antagonist propranolol (pro) in healthy subjects. the following were analysed: heart rate (hr, bpm), pre-ejection time (pep, ms), ejection time (vet, ms), hr-corrected electromechanical systole (qs c, ms), impedance-cardiographic estimates of stroke volume (sv, ml), cardiac output (co, i/min) and peripheral resistance (tpr, dyn.s.cm - ) calculated from co and mean blood pressure (sbp and dbp according to auscultatory korotkoff-i and -iv sounds this indicates that ) about half the rise of hr and co and half the shortening of pep is -respectively ~ -determined, ) that predominant -adrenergic responses, whilst not affecting vet, take optimal benefit from the inodilatory enhancement of pump performance, ) that an additional -adrenergic stimulation is proportionally less efficient, as vet is dramatically shortened, thus blunting the gain in sv so that the rise in co relies substantially on the amplified increase of hr and ), vet is more sensitive than qs c in expressing additional -adrenoceptor agonism and ) prime systolic time intervals provide a less speculative and physiologically more meaningful represenation of cardiac pump dynamics than hr-corrected ones. zentrum flit kardiovaskul~re pharmakologie, mathildenstral e , mainz, brd a regression between blunting of ergometric rise of heart rate and l~ladrenoceptor occupancies in healthy man c. de mey, d. palm, k. breithaupt-grsgler, g.g. belz the hr-responses to supine bicycle ergometry ( min at appr. watt) were investigated at several time points after the administration of propranolol (pro: , , mg), carvedilol (car: . , , , mg), talinolol (tal: , , , mg), metoprolol (met: mg) and celipro-iol (cel: mg) to healthy man. the effects of the agents (= difference of the ergometric response for active drug and placebo) were analysed for both the end values (end) and the increments (inc) from resting values immediately before ergometry up to end. the effects were correlated with the %-~l-adrenoceptor occupancies estimated using a standard emax-model (sigmoidicity=l) from the concentrations of active substrate in plasma determined by i~l-adrenoceptor specific radioreceptor assay. the respective intercepts (i), slopes (s) and correlation coefficients (r) are detailed here below : inhibition of leukotrienes is a promising approach to the treatmer~t of several diseases because excess formation of these lipid mediators has been shown to play an important role in a wide range of pathophysiological conditions. since until recently we were not able to obtain specific drugs suppressing leukotriene biosynthesis or action for clinical practice, we started investigating the effects of putative natural modulators of leukotriene biosynthesis such as fish oil. healthy male volunteers were supplemented for days with fish oil providing mg eicosapentaenoic and docosahexaenoic acid per kg body weight and day. the urinary concentration of leukotriene e plus n-acetyl leukotriene e served as a measure for the endogenous leukotriene production, treatment resulted in a significant increase in the eicosapentaenoate concentration in red blood cell membranes. fish oil reduced the endogenous leukotriene generation in of the volunteers. the effect was associated with a decrease in urinary prostaglandin metabolites, determined as tetranorprostanedioic acid. in contrast to what was expected from published in vitro and ex vivo experiments, no endogenously generated cysteinyl leukotrienes of the series could be identified. the inhibitory effect of fish oil on the endogenous leukotriene generation was not synergistic to the effect of vitamin e, which also exhibited some suppressive activity. early clinical data on the effects of fish oil on teukotriene production in patients with allergy or rheumatoid arthritis are not yet conclusive. we conclude that fish oil exhibits some inhibitory activity on leukotriene production in vivo. the effectivity of fish oil may be attenuated by concomitant modulation of other mediator systems e.g. up-regulation of tumor necrosis factor production. • the number and affinity of platelet thromboxane (txa ) and prostacyclin (pgi )-receptors are regulated by several factors. we studied the influence of oral intake of acetylsalieylic acid (asa) on ex-vivo binding studies with human platelet membranes on the binding of the specific thromboxane a antagonist h-sq- and the pgi agunist h-l]oprost. the number of receptors (bmm) and the binding affinity (kd) were calculated using scatchard's plot analysis. in healthy male volunteers o significant difference was seen following intake of mg/d of asa for days (mean -+ sem): the potency of meloxicam (mel), a new anti-inflammatory drug (nsaid), in the rat is higher than that of well-known nsaids, in adjuvant arthrtitis rats, mel is a potent inhibitor of the local and the systemic signs of the disease. mel is also a potent inhibitor of pg-biosynthesis by leukocytes found in pleuritic exudate in rats. conversely, the effect of mel on pg-biosynthesis in isotated enzyme preparations from bull seminal vesicle in vitro, the effect on intragastric and intrarenal pg-biosynthesis and the influence on the txb - evel in rat serum is weak. in spite of the high antiinflammatory potency in the rat, mel shows a low gastrointestinal toxicity and nephrotoxicity in rats. -cyclooxygenase- (cox- ) has been recently identified as a isoenzyme of cyclooxygenase. nsaids are anti-inflammatory through inhibition of pg-biosynthesis by inducible cox- and are ulcerogenic and nephrotoxic through inhibition of the constitutive cox- . we have investigated the effects of mel and other nsaids on cox- of non stimulated and on cox- of lps-stimulated guine pig peritoneal macrophages. cells were cultured with and without lps for hrs together with the nsaid. arachidonic acid was then added for further mins, the medium removed and pge measured by ria. bimakalim, emd , is a new investigational k+-channel activator with vasod[lating properties. single pereral doses of . mg bimakalim, mg diltlazem, either alone or in combination, were investigated in healthy male supine volunteers ( to years of age) [n a placebo-controlled, periodbalanced, randemised, double-blind, way cross-over design. point estimates of the global effects of bimakalim [k] , di]tiazem [d] and their interaction [kxd, = in case of mere additivity] incl. % confidence intervals (ci) were analysed for systolic and diastolic blood pressure (sbp, dbp; mmhg), heart rate (hr; bpm), pq (ms), systolic time intervals (pep, qs c, lvetc; ms), cardiac output (co; i.min- ), total peripheral resistance (tpr; dyn.s.cm- ), heather index (hi; q.s- ); , h after dosing, *statistically significant at a= . : - to - - to - to - to . to . - . to , . to . * - . to , - . to - . * - . tol, - . to , - . to . - . to . - . to . - . to . - . to . - to - to -& to . - . to . afterload reduction and a drop in dbp occurred with bimakalim associated with a rise in hr and mild increase in cardiac performance, diltiazem (slightly) decreased afterload and bp with little (reflectory) accompanying changes and had a negative dromotropic effect. the combination caused additive effects. center for cardiovascular pharmacology, zekapha gmbh, mathildenstr. , mainz, germany. rheumatoid arthritis (ra) is characterized by an immunological mediated inflammatory reaction in affected joints. infiltration of granulocytes and monocytes is the pathophysiological hallmark within the initial phase of inflammation. these cells are able to synthesize leukotrienes. ltb is a potent chemotactic factor and therefore could be responsible for the influx of granulocytes from the circulation. cysteinyl leukotrienes ltc , d and e augment vascular permeability and are potent vasoconstrictors. ltb and cysteinyl leukotrienes have been detected in synovial fluid of patients with ra. however, these results are difficult to interprete, because the procedure is invasive and artificial synthesis cannot be excluded. we used a different, noninvasive approach by assessing the excretion of lte into urine. studies with hltc have demonstrated that lte is unchanged excreted into urine and is the major udnary metabolite of cysteinyl leukotrienes in man. udnary lte was isolated from an aliquot of a hour urine collection by solid phase extraction followed by hplc and quantitated by ria. nine patients were enrolled in the present study. all met the american college of rheumatology criteria for ra. patients were treated with nonsteroidal inflammatory drugs and disease modifying drugs. therapy with prednisolon was started after collection of the initial hour urine sample. disease activity was assessed by crp (mean + mg/l) and esr (mean _+ mm/hour platelet aggregation is mediated by the binding of an adhesive protein, fibrinogen, to a surface receptor, the platelet glycoprotein lib/ilia. gpiib/llla is one of a family of adhesion receptors, integrins, which consist of a ca++-dependent complex of two distinct protein subunits. under resting conditions, gpiib/llla has a low affinity for fibrinogen in solution. however, activation of platelets by most agonists, including thrombin, adp and thromboxane results in a conformational change in the receptor and the expression of a high affinity site for fibrinogen. binding of fibrinogen to platelets is a common end-point for all agonists and therefore is a potential target for the development of antiplatelet drugs. these have included chimeric, partially humanised antibodies ( e ), peptides and peptidomimetics that bind to the receptor and prevent fibrinogen binding. the peptides often include the sequence rgd, a sequence that is present in fibrinogen and is one of the ligand's binding sites. when administered in vivo, antagonists of gpiib/llla markedly suppress platelet aggregation in response to all known agonists, without altering platelet shape change, a marker of platelet activation. they also prolong the bleeding time in a dose and perhaps drug dependent manner, often to more than rain. in experimental models of arterial thrombosis, gpllb/llla antagonists have proved highly effective and are more potent than aspirin. studies in man have focused on coronary angioplasty, unstable angina and coronary thrombolysis and have given promising results. e given as a bolus and infusion combined with aspirin and heparin reduced the need for urgent revascularisation in patients undergoing high-risk angioplasty, although bleeding was more common. some compounds have shown oral bioavailability raising the possibility that these agents could be administered chronically. antagonists of the platelet gpiib/llla provide a novel and potent approach to antithrombotic therapy. drug databases on computers are commonly textfiles or consist of tables of generic-names or prices for example. until now pharmacokinetic data are not easily available for regular use, because searching parameters in a textfile is time consuming and personal intensive. on the other hand these pharmacokinetic data are the fundamental background of every dosage regimen and individual dosage adjustment. for many drugs elimination is dependent on the patients renal function. renal failure leads to accumulation, possibly up to toxic plasma concentrations. therefore, the decision was to build up a pharmacokinetic database. the aim is to achieve simplicity and effectiveness by using the basic rules. only three parameters are needed to describe the pharmacokinetics: clearance (ci), volume of distribution (vd) and half-life (t~). moreover, with two parameters the third can be calculated ancl'controlled by the equation: cl = vd * , / t½ according to the dettli-equation and the bayes' theorem estimation of individual pharmacokinetic parameters will be done by a computer program. the advantage is that the impact of therapeutic drug monitoring can be increased. using the population data and the bayesian approach, only one measurement of serum drug concentrations might be enough to achieve an individual dosage regimens (el desoky et al., ther drug monitor , : ) higher therapeutic security for the patient can be achieved. there is also a major pharmacoeconomic aspect: adapting drug dosage reduces costs (susanka et al., am j hosp pharm , : ) the basic database for future pharmacokinetic clinical desicions is going to be built up. the pharmacokinetic interactions with grape#uit juice reported for many drugs are attributed to the inhibition of cytochrome p enzymes by nanngenin, which is the aglycene of the bitter juice component nadngin. however, only circumstantial evidence exist that naringenin is indeed formed when grapefruit juice is ingested, and the lack of drug interaction when naringin solution is given instead of the juice is still unexplained. we investigated the pharmacokinetics of naringin, naringenin and its conjugated metabolites following ingestion of ml grapefruit juice per kg body weight, containing ijm naringin, in male and female healthy adults. urine was collected - , - , - , - , - , - , - and - hours alter juice intake. naringin and naringenin concentrations were measured by reversed phase hplc following extraction using ethyl acetate, with a limit of quantitation of nm. conjugated metabolites in urine were transformed by incubation with glucuronidase ( u/ml) / sulfatase ( u/ml) from abalone entrails for h at ph . and determined as parent compounds. additionally, naringin and naringenin concentrations were measured in plasma samples from grapefl'uit juice interaction studies conducted previously. neither naringin nor its conjugated products were detected in any of the samples. naringenin was not found in plasma. small amounts of nanngenin appeared in urine alter a median lag time of hours and reached up to . % of the dose (measured as nanngin). after treatment with glucuronidase / sulfatase, up to % of the dose was recovered in urine: the absence of naringin and its conjugates and the lag time observed for naringenin to appear in urine suggests that cleavage of the sugar moeity may be required before the flavonoid can be absorbed as the aglycone. naringenin itself undergoes rapid phase ii metabolism. whether the conjugated metabolite is a potent cytochrome p inhibitor is unknown but not probable. the pronounced variability of naringenin excretion provides a possible explanation for apparently contradictory results in grapefruit and/or naringin interaction studies. grapefruit juice increases the oral bioavailablity of almost any dihydropyridine tested, presumably due to inhibition of first-pass metabolism mediated by the cytochrome p isoform cyp a / . the mean extent of increase was up to threefold, observed for felodipine, and more pronounced drug effects were also reported. thus, a such interaction may be of considerable clinical relevance. no data are yet available for nimodipine. we conducted a randomized cross-over interaction study on the effects of concomitant intake of grapefruit juice on the pharmacokinetics of nimodipine and its metabolites m (pyridine analogue), m (demethylated) and m (pyridine analogue, demethylated). healthy young men ( smokers / nonsmokers) were included into the investigation. nimodipine was given as a single mg tablet (nimotop e) with either ml of water or ml of grapefruit juice (d~hler gmbh, darmstadt, mg/i naringin). concentrations ef nimodipine and its metabolites in plasma withdrawn up to hours p.ostdose were measured by gc-ecd, and model independent pharmacokinetic parameters were estimated. the study was handled as an equivalence problem, and anova based % confidence intervals were calculated for the test (=grapefruit period) to reference (= water period) ratios. the absence of a relevant interaction was assumed if the ci were within the . to . range: grapefruit juice was reported to inhibit the metabolism of a variety of drugs, including dihydropyridines, verapamil, terfenadine, cyclosporine, and caffeine. these drugs are metabolized mainly by the cytochrome p isoforms cyp a (caffeine and, in part, verapamil) and cyp a (others). theophylline has a therapeutic range of - mg/i and is also in part metabolized by cyp a . therefore, we conducted a randomized changeover interaction study on the effects of concomitant intake of grapefruit juice on the pharmacokinetics of theophylline. healthy young male nonsmokers were included. theophylline was given as a single dose of mg in solution (euphyllin e ), diluted by either ml of water or ml of grapefruit juice (d hler gmbhi darmstadt, mg/i nadngin). subsequently, additional fractionated . i of either juice or water were administered until hours postdose. theophylline concentrations in plasma withdrawn up to hours postdose were measured by hplc, and pharmacokinetics were estimated using compartment model independent methods. the study was handeled as an equivalence problem, and anova based % confidence intervals were calculated for the test (=grapefruit period) to reference (= water period) ratios (trnax: differences thus, no inhibitory effect of grapefruit juice on theophylline pharmacokinetics was observed. the lower contribution of cyp a to primary theophylline metabolism or differences in naringin and/or naringenin kinetics are possible explanations for the apparent contradiction between the effects of grapefruit juice on caffeine and on theophylline metabolism. the physical stability of erythromycin stearate film tablets was studied according to a factorial design with experimental variables temperature, relative humidity, and storage time. after one half year of storage at oc and % relative humidity, the fraction of dose released within min in a usp xxl paddle apparatus under standard conditions decreased from % for the reference stored at ambient temperature in intact blister packages to % for the stress-tested specimens. chemical degradation of the active ingredient did not become apparent before months of storage. under all other storage conditions, no effects of physical aging upon drug release were found. the bioequivalence of reference and stress-tested samples was studied in six healthy volunteers. the extent of relative bioavailability of the test product was markedly reduced (mean: . %, range: - %), mean absorption times of the test product were significantly prolonged. the results indicate that the product tested can undergo physical alterations upon storage under unfavourable conditions, and lose its therapeutic efficacy. it can be expected that this phenomenon is reduced by suitable packaging, but the magnitude of deterioration may cause concern. on the other hand, incomplete drug release is in this case easily detected by dissolution testing. whether similar correlations exist for other erythromycin formulations remains to be demonstrated. the efficacy of a drug therapy is considerably influenced by patient compliance. within clinical trials the effects of poor compliance on the interpretation of study results frequently leads to underestimating the efficacy of the treatment. in the evaluation of the "lipid research clinics primary coronary prevention trial" and the "helsinki heart study" special attention was focused on compliance with medication. the strong influence of compliance on clinical outcome and the dilutional effect of poor compliance on the efficacy of the respective drugs occured in both these trials. there are indirect (e.g. pill-count, patient interview) and direct methods (e.g. measurement of drugs, metabolites or chemical markers in body fluids) used to assess compliance with drug therapy. the indirect methods mentioned are commonly considered as unreliable. the direct methods can prove dose ingestion a short time before the sample is taken, however, they cannot show the time history of the drug use. an advanced method of measuring compliance is to use electronic devices. the integration of time/date-recording microcirculty into pharmaceutical packaging, so as to compile a time history of package use, provides real-time data as indicative of the time when dosing occurred. this method supports a precise, quantitative definition of "patient compliance" as: the extent to which the actual time history of dosing corresponds to the prescribed drug regimen. by taking real-time compliance data into account the results from clinical trials show not only clearer evaluations of drug efficacy and dese-reponse-relationship but also a better understanding of dose dependant adverse drug reactions. in the present study, we examined the usefulness of eroderm- and eroderm- . seventy five impotent men, to years old, participated in the present trial. the patients were classified into groups, patients each. the first group was treated by cream containing only co-dergocrine mesilate (eroderm- ), the second received a cream containing isosorbide dinitrate, isoxsuprine hcl and co-dergocrine mesilate (eroderm- ), while the third used a cream containing placebo. the cream was applied to penile shaft and gland / - hr before sexual stimulation and intercourse. the patients were asked to report their experience via questi'onnaire after one week. the results of treatment are as follows: seven patients ( %) who applied eroderm- indicated a full erection and successful intercourse. the use of eroderm- restored potency in patients ( %) of the second group. three men ( %) of psychogenic type reported overall satisfaction with placebo cream. treatment of impotence with eroderm cream was most successful in patients with psychogenic disorders which are often coincident with minor vascular or neurological disorders. fair results were reported by patients afflicted by moderate neurological disorders. except for one case of drug allergy following the use of eroderm- , no side effects were reported. we believe that eroderm cream has obvious advantages and may be a suitable treatment before the use of non-safe method as intracavernous medication. a new type of topically applied drugs (eroderm creams) for impotence is presented. eroderm creams contain vasoactive drugs. these drugs have ability to penetrate the penile cutaneous tissue and facilitate erection. in the present study, we examine the usefulness of eroderm- in the treatment of erectile dysfunction. eroderm- contains tiemonium methylsulfate, a.f. piperazine and jsosorbide dinitrate. a randomized, double blinded control trial on patients was performed. the etiology of impotence was investigated. all patients received eroderm- and placebo cream. the patients randomized into groups of . the first group received eroderm- on day and placebo cream on day , however, group two received placebo on day . the patients were advised to apply the cream on the penile shaft / - hr, before sexual stimulation and intercourse. the patients reported their experience via questionnaire. overall percent of patients demonstrated a response with eroderm- . the other responders reported a partial erection and tumescenous. three men ( %) reported a full crection and satisfied intercourse with either cream. these patients were psychogenic impotence. neither eroderm- nor placebo cream produced marked response in patients. four patients were venous leakage which were advised to use tourniquet at the base of penis after / hr. of cream application. only one of them indicated a good response. the highest activity proved to occur in psychogenic impotence. less rate of success was observed in patients with minor to moderate neurological and/or arterial disorders. no marked side effects were recorded. for these reasons eroderm- may be proposed as first line therapy of erectile dysfunction. control of cell proliferation is a basic homeostatic function in multicellular organisms. we studied the effects of some prostaglandins and leukotrienes and of their pharmacological inhibitors on cell proliferation in murine mast cells and mast cell lines, in a human promyelocytic cell line (hl- cells) and in burkitt's lymphoma cell lines. in addition, prostaglandin and leukotriene production was investigated in mast cells, representing putative endogenous sources of these lipid mediators. murine mast cells were derived from bone marrow of balb/c mice. proliferation of cells was estimated using a colorimetric assay (mtt-test). production of prostaglandin d (pgd ), pgj , delta- -pgj , leukotriene c (ltc ) and ltb by mast cells was determined by combined use of high performanceliquid chromatography and radioimmunoassay. pgd and its metabolites pgj and delta- -pgj exhibited significant antiproliferative effects in the micromolar range in mast cells, mast cell lines, hl- and burkitt's lymphoma cell lines whereas inhibition of cyclooxygenase by indomethacin was without major effects. ltc and ltb had a small stimulatory effect on cell proliferation in hl- cells. degradation and possibly induction of cell differentiation may have attenuated the actions of leukotrienes. the leukotriene biosynthesis inhibitors aa- and mk- reduced proliferation of hl- and lymphoma cells significantly but had no major effects on mast cell growth. on the other hand, mast cells stimulated with calcium ionophore produced pgd and its metabolites, as well as ltb and ltc in significant amounts. from our data we conclude that prostaglandins and leukotrienes may play an important role in the control of cell proliferation. we compared the pattern of drug expenditures of several hospitals in (size: to beds). a, b are university hospitals in the ,,old"and c,d,e are university hospitals in the ,,new" german countries, f is a community based institution in an ,,old" german country. main data source were lists comprising all drags according to their expenditures in a rank order up to %. items were classified into i) pharmaceutical products including immunoglobulines, ii) blood and -derived products (cell concentrates, human albumin, clotting factors) and iii) contrast media (x-ray). with regard to group i) the highest expenditures nccured in hospitals a and b whereas drug costs in c -e were / less and came to only % in hospital f. the main groups of drugs which together account for > % of these expenditures are shown in the table. ) products were about % up to % of group i and highest in hospitals a, b and e, but about / lower in hospitals c and d. these results suggest meaningful differences in the drug utilization between the old and new countries as well as betv,,een university institutions and community based hospitals. however, although all hospitals provide oncology and traumatology services and all university hospitals offer ntx, differences in other subspecialities e.g bone marrow and liver transplantation and treatment of patients with haemophilia must be considered, too. dr.medsebastian harder, dept c]inicai pharmacology, university hospital frankfurt, theodor stern kai , frankfurt/main frg m. hgnicka, r. spahr, m. feelisch, and r. gerzer organic nitrates like glyceryl trinitrate (gtn) act as prodrugs and release nitric oxide (no), which corresponds to the endogenously produced endothelium-derived relaxing factor. in the vascular tissue, no induces relaxation of smooth muscle cells, whereas in platelets it shows an antiaggregatory effect. both activities are mainly mediated via stimulation of soluble guanylyl cyclase (sgc) by no. in contrast to compounds which release no spontaneously, a membrane-associated biotransformation step is thought to be required for no release from organic nitrates. glutathione-s-transferasea and cytochrome p- enzymes have been shown to metabolize organic nitrates in the liver, but little is known as to whether these enzymes are involved in the metabolic conversion of organic nitrates in the vasculature. furthermore, it is still unclear whether or not platelets are capable of metabolizing organic nitrates to no. we isolated the microsomal fraction of bovine aorta in order to characterize the activities towards organic nitrates using the guanylyl cyclase reaction as an indirect and the oxyhemoglobin-technique as a direct measure for no liberation. gtn was metabolized to no by the microsomal fraction under aerobic conditions already in the absence of added cofactors. this activity was not influenced by the cytochrome p- inhibitors cimetidine and metyrapone. in contrast, the glutathione s-transferase substrate -chloro- , -dinitrobenzene and the glutathione s-transferase inhibitors sulfobromophthalein and ethacrynic acid did not affect no release, but potently inhibited sgc activity. blocking of microsomal thiol-groups resulted in a decreased no release from gtn. homogenates of human plateles isolated by thrombapheresis and stabilized by addition of mm n-acetylcysteine did not show no-release from gtn as determined by the stimulation of the platelet sgc even after addition of the possible cosubstrates glutathione and nadph. these data demonstrate ( ) that bovine aortic microsomes exhibit an organic nitrate metabolizing and no-releasing activity whose properties are clearly different from classical cytochrome p- enzymes and from glutathione s-transferases, and ( ) that human platelets itself are not capable of bioactivating organic nitrates and therefore require organic nitrate metabolism in the vessel wall for antiaggregation to occur. bioavailability of acesal ®, acesal ® extra, micristin ® (all mg acetylsalicylic acid -asa), and miniasal ® ( mg asa), opw oranienbufg, relative to respective listed references was studied in female and male healthy volunteers (age - y, weight - kg, height - cm). asa and salicylic acid (sa) were measured using an hplc method validated from ng/ml to pg/ml. extent of absorption was assessed by auc (bioequivalence range . - . ), rate by cr~/auc (bioequivalence range . - . ). geometric means and %-confidence limits of the ratios test/reference (multiplicative model) are shown in the acesal ® and micdstin ® were bioequivalent in rate and extent of absorption with the reference formulations. the fast liberating acesal ® extra was bioequivalent with respect to extent only. asa from miniasal ® was absorbed more slowly than from an asa solution (cm= ( %-range): - ng/ml and - ng/ml; t~ (min-max): . - . h and . - . h). asa from micdstin ® and the corresponding reference was absorbed more slowly than from acesal ® and acesal ® extra. this was accompanied by decreased aucasa (increase of first pass metabolism) and increased apparent trrz (absorption being rate limiting). all ratios of aucsa/aucasa after administration of mg asa were markedly higher than after mg asa. thus, the formation of salicyludc acid from sa might be capacity limited at doses of mg asa. in the study >>physicians' assessment of internal practice-conditions and regional health-services-conditions in accordance with ambulatory patient-management<< a sampie of primary care physicians -comprising gps and internists -provide data for continuons analyses of arnbulatory health care quality and structure. focussing on the physicians' drug prescription, the impacts of reform law (gesundheitsstralcturgesctz, gsg) upon primary care providers and their therapeutic decisions were examined in . four different surveys were carded out during the year, dealing with frequent patients' reasons for encounter in gps' offices. after a pretest was carried out, physicians reported on patient-physician-encounters, basing on mailed questionnaires. for every therapeutic change patients received, the reasons for the change were recorded (e.g, reform law, medical indication) and above the physicians' expectations towards three criteria to measure the quality: ) physicians' assessment of the patients' satisfaction, ) adverse drug effects, ) therapeutic benefit. according to therapeutic changes due to reform law (drag budgets, blacklist) it can be stated: ) therapeutic changes due to reform law were carried out with relevant frequency. ) the reform law was of different concern regarding the different reasons for encounter we investigate& ) the impacts' strangth of the legal control mechanisms differed among several groups of physicians: those who already have been liable to recourse before more often carried out therapeutic changes according to fixed drug budget. different multivariate logistic regression-models yield an estimation of the odds-ratio of about . ) therapeutic changes in accordance with the reform law having been carried out at the beginning of the year more often suffered from negative expectations towards the therapeutic quality then changes during the actual encounter, e.g. >>joint pains<c % decrease of the therapeutic benefit in contrast to %. during the year a decrease of negative expectations is noticable. ) patients overestimating the severity of their own health problem in comparison to the physician more often suffered from negative expectations towards the therapeutic change than other patients, e.g. concerning the therapeutic benefit ( % in contrast to %). generally speaking, firstly the reform law led to insecurities -particularly in the introduction phase -among susceptible subgroups of physicians and patients, regarding the four investigated reasons for encounter. secondly, there is evidence of a loss of quality in ambulatory health care -predominandy in terms of subjective criteria. these impacts considerably subsided within the field phase duration from may to november, . the presented analyses give evidence of the practicability of studies assessing effects of legally-initiated mechanisms to control health costs upon primary health care quality. postansehrifi: iseg, bissendorfer sm , d- hannover the cellular and biochemical mechanisms that lead to the longerm complicatiorts of diabetes mellitus are poorly understood. glycation is a multireaetion beginning with a condensation reaction between reducing sugars and reactive amino groups of proteins. the accumulation of the glycated products of various proteins may contribute to the development of the complications of diabetes and aging. in order to find suitable non-toxic glycation inhibitors we used sulfur deriva-tives (penicillamine, eaptopril and alpha-lipoic acid) and amino derivatives (aminoguani-dine and aminoguanidine propionic acid) and postulated the mechanisms involved.the inhibitou effect of the drags on the early (amadori product) and advanced glycation products (age). determined by affinity chromatography, deglycation method. i-iplc and fluorescence spectroscopy, differs markedly depending on the drag used and is greatest (up to %) with aminoguauidine and penicillamine ( mm) and smallest with alpha-lipoic acid. aminoguanidine propionic acid had no inhibitory effect on the glycation process. the ability of penicillamine to inhibit both the formation of amadori products and age was demonstrated by isolation of the adducts of penicillamine with ribose, glucose, glucosa-mine and p-tolylglucosamine, a standard for glycated protein. their structures were characterized using ir-and h-nmr and c-nmr spectroscopy. the oxidative deg-radation of glycated human serum albun{in in the presence of copper (ii), forming the carbox jmethyllysine, was inltibited by the sulfur derivatives. the in vitro formation of pentosidine, a crossliuking product was influenced by all drugs used however captopril, alpha-lipoic acid and aminoguanidine propionic acid do not condense with sugar derivatives.the mechanism by which captopril and alpha-lipoic acid inhibit the formation of age may be involve radical formation, where a spontaneus thiol/disul-fide interchange between drug and protein takes place. complications can appear at any time in diabetic patients.the cause of these complica-lions in some cases may be due glycation and/or oxidation. the clinical applications of substances which are able to prevent these events may have advantages. these results and the mechanism proposed show that the amino and sulfl~ydryl groups of drags reduce glycation and postglycation of proteins and thus have potential value in this regard. non-enzymatic glycation starts by covalent binding of single sugars to o~ or e amino groups of proteins. in subsequent reactions a number of early glycation products like schiff bases or amadori products, and of advanced glycation endproducts (ages) is formed. glycation is increased in a variety of proteins in diabetic patients [schleicher e ( ) diabetes und stoffwechsel : ] including human serum albumin (hsa), the red cell membrane, hemoglobin, collagen, laminin, immunoglobulin g, low-and high-density lipoproteins. the process is supposed to be a key mechanism in the pathogenesis of diabetic complications and in tisstie ageing [vlassara het al. ( ) lab invest : ] . inhibition of glycation may therefore be important for the prevention of late diabetic complications. we investigated the in vitro glycation of human serum albumin (hsa) and its inhibition by aminoguanidine, penicillamine, captopril and c~-lipoic acid ( ram). hsa ( g/l) was glycated by incubation with mm glucose in . m phosphate buffered saline, ph . , at °c for days under sterile conditions in the presence of . % natrium azide. the glycation rate was determined by two different methods: deglyeation -measures amadori products based on the colorimetry of -ketoglucose which is released from the glycated protein (ketoamine) the effect of aniseylated #asminogen streptokinase activator complex (apsac) on left ventricuiar (ll r) function ;,.,as investigated in ! patien~ with acute myocardial infarction (am!). a dose of q units apsac was given intraveneusiy to patien~ with ami within hours after onset of pain, patients underwent cardiac angiegraphy at minutes and at day . wail motion ,.~s measured by the global ejection fraction and by the centefline method in :'=he infarct and neninfar~ regions and expressed in units of sf~ndard deviations beiew normai, as defined in normai patient. group a with successfui ',th, rombo!ysis (timi and ) was compared with group b without successfui thrombolysis (timi and !). at minutes there vras no significant difference in giebal ejection fraction between both groups, by using the regiona! wa[i motion by the centerline method group a showed a significant improvement of lv function (p ~ . ). the benefit of successeli thrombeiysis was more marked in the peripheral infarct region than in the centrai infarct region. thus early repeffusion is an important factor in maintaining good lv function after ami. the re=gionai wai! motion in the infarct region seems to be more sensmve man me g!oba! eiection fraction to show ear!y differences in lv function after ami. the quinoline derivative bay x ((r)- -[ -(quinoliu- -yl-metho.,qc)phenyl]- cyclopentyl acetic acid) is a selective inhibitor of ltb -biosynthesis in various in vitro and in vivo models. it acts via binding to the integral nuclear membrane protein five-lipoxygenase activating protein (flap), which has been shown to be an essential component of the cellular leukotriene synthesis machinary. the function of flap is only poorly understood, but recently it has been shown that a radiolabeled photoaffinity-analog of arachidonic acid binds specifically to flap. consequently, a role of flap in the substrate transfer to -lipoxygenase ( -lox) has been suggested. using intact and fractionated human polymorphonuclear leukocytes (pmnl), we present data showin~ that exogenously added arachidonie acid diminishes dose-dependently binding of [ c]-bay x to its target. this effect correlates with the reduction of bay x mediated inhibition of ltb synthesis after stimulation of intact pmnl with tile calcium ionophore a in the presence of increasing extracellular arachidonic acid concentrations in addition, enhancing the release of intracellular arachidonic acid from phospholipids after activation of phospholipase a (pla ) with increasing a concentrations or after prolonged incubation of the cells with the ionophore results in a loss of bay x potency. to further study" the effect of arachidonic acid on the bay x mediated ltb synthesis inhibition, human pmnl were stimulated with paf, c a or fmlp in the presence of exogenous arachidouic acid. under such conditions, ltb synthesis is significantly enhanced in response to paf, c a or fmlp. which are only weak activators of cell-associated pea the effect of extracellular arachidonic acid is dosedependently inhibited by bay x . coincubation of the cells with arachidonic acid and paf, c a or fmlp, respectively, enhances synergistically the translocation of -lox from the cnosol to the membrane fraction. again, this effect is inhibited by bay x . in summary, using bay x , we provide evidence, that flap is involved in the utilization of the -lox substrate araehidouic acid from exogenous or endogenous sources for the agonist-induced leukotrieue biosynthesis. switzerland, but their effect on the expression and function of the multiple drug resistance gene product has not been studied in detail. we have therefore examined the effect of gm-csf and g-csf on the uptake and toxicty of vincristine with and without the chemosensitizers r-verapamil and norverapamil in the pglycoprotein-containing vincristine-resistant cell-line, hl- /vi,o. following a h pre-incubation with ng/ml g-csf or gm-csf, cells were washed and reincubated in rpmi medium with hvincristine and the modulators for hours. the uptake of hvincristine was increased up to -fold and this effect was equally marked when the vincristine-uptake was stimulated with the chemosensitizers in the range of . pg/ml to pg/ml. although cytokine-pretreatment increased the accumulation of vincristine, we could not show an increase in vincristine toxicity over h with the same concentrations of vincristine and chemosensitizers. it is concluded that the effects of cytokines on vincristine accumulation are compensated by a positive effect on cell-survival or cell-growth. department of clinical pharmacology, university clinic, frankfurt, theodor stern kai , frankfurt, germany. from the economic point of view people often tend only to regard the price of a product. savings for the health care system as a whole which could be gained by use of these drugs and which could more than compensate the high costs of medication are not considered. the cost-benefit-analysis enables a comprehensive analysis of the economic effectiveness of a pharmaceutical. the relevant cost factors such as the costs for in-and out-patient treatment depending on the therapeutic success and the inability to work are compared to the costs of medications. using the example of stroke prevention, it was possible to show that prophylaxis with an expensive but effective pharmaceutical is economically better than standard medication. according to the tass study ticlopidine can prevent at least more strokes per i treated patients than ass. on the basis of this study cost savings of up to dm , per i treated patients can be reached including higher costs of ticlopidine. projected onto i , patients up to dm , per year can be saved for the german health care system by the use of ticlopidine in secondary prophylaxis after tia. the naphthodianthrones hypericin (h) and pseudohypericin (ph) were proved to be elfectire in mild depressive disorders. recently they were discovered to exhibit high in vitro activity against enveloped viruses fostering clinical trials in infections with hiv or herpes viruses, for studying the pharmacokinetics of h and ph a solidified extract from st. john's wort (hypericum pefforatum l) was orally administered to healthy male volunteers in three single doses of , , and ~g of h and , , and p.g of ph, followed by a -days course of multiple dosing of p.g h and pg of ph tds. plasma concentrations were determined by hplc, the quantification limit was . #g/i (staffeldl et al., nervenheilkunde ( ) : ). single,dose kinetics, h showed a median lagtime of . (range: , - . ) h, which was significantly longer compared to , (range: . - , ) h of ph. h was eliminated with a median half-life of (range: - ) h, and in case of ph the hail-life ranged between . and (median: , ) h. median crux levels were . , . , . ~g/i for h and . , . , . pg/i for ph for the three single-dose levels given above, respectively. lowest dose crre, x and auc of h was lower then expected from medium and highesl dose, which was statistically significantly for crnax (p= . , anova; p= . , onferroni/dunn), indicating nonproportionality of pharmacokinetic response may exist in the lower dosing range. mul!iple-dose kinetics. during long-term dosing steady-state trough levels (ctreugh) of . (range: . - . ) ~g/i for h and . (range: -to. ) ~g/i for ph were reached after days for both. cmax after days of treatment were . (range: . c. kori-lindner, r. eberhardt the healthcare restructure act ( ) changed the evaluation of drugs from efficacy to cost effectiveness: therapeutic necessity, utility, purpose; medical progress and economic utility have to be considered. criterias and suroundings of pharmacooeconomic evaluations and studies according to the bealthcare restructure act are demonstrated by an example: "costs of nsaid-induced gastro-duodenal ulcers and prevention with miso~rostol". methods: cost-utility, cost-benefit, legal requirements, decision-model and decision -tree, basic calculation and the transfer to the socialsystem of the frg. premissions: rate of ulcers without prevention , %, with prevention , %; rate of compliance %, rate of hospitalisation % and duration of hospitalisation , days. rheumatic disorders have become a frequent reason for services m ambulatory care. therefore the use of drugs which are administered in rheumatic complaints is widespread. data of the sientific institute of statutory health insurances (arzneimittelindex ) demonstrate that the group of antirheumatics/analgetics held the first rank due to the number of prescriptions. with regard to the costs thts drug class were amongst the top three. apart from anurheumatics and analgetics different other drug classes are used for the treatment of rheumatic disorders for example corticoids. to estimate the total expenditure for drug utilization in out-patients suffer from musculoskeletal disorders data of the statutory health insurance dortmund (aok dortmund) were investigated. a % random sample of insurees (n= ) was analysed for diagnoses and drug therapy. the data were collected in a individual-related way for an observation period of one year ( ). the results demonstrate the high frequency of rheumatic disorders among out-patients: the -month prevalence of musculoskeletal disorders was about % in the studied population of whom one half suffered from back pain. approximately % of the patients were prescribed any drugs for treatment of rheumatic disorders. as might be expected nonsteroidal antiinflammatory drugs were used most frequently. the extensive use of topical products was a considerable factor of costs. more than one half of the patients received drugs for topical application. further details will be presented. diachronous data of statutory health insurences that have been gathered in a person-related way permit the estimation of drug expenditure in outpatients with regard to the several musculoskeletal disorders. misleading results of endocrine tests in patients suffering from liver diseases promted us to investigate the influence of liver function on the pharmacokinetics of dexamethasone (dex). it was the aim of this study to find out whether routine parameters of liver function may be usefull in roughly predicting the clearance of dexamethasone. methods : in patients liver function was rated on the basis of their plasma cholinesterase activity (che) and prothrombin time (pt). blood samples were withdrawn , , , , , , , and min following an iv bolus injection of mg of dexamethasone- -dihydrogenphosphate corresponding to , mg of dex for the determination of dex pharmacokinetics by ria. results : computer assisted best fit analysis ( compartment model) of the plasma dex decay did not show any influence of liver function on the distribution half-life (t½-a) and on the initial volume of distribution of dex, but a % increase in the terminal phase, "metabolic" half-life (t½-b) with decreasing liver function. while the t½-b increased from ± min in patients with a che activity > . ku/l to ± min in those with a che s . ku/l, the metabolic clearance rate (mcr) decreased from ± ml/min to iii ± ml/min. in patients on phenytoin the t½-b was reduced to <i min. che activity (r=- . ) and pt (r=- . ) were significantly (p< . ) correlated with the t½-b of dex. we conclude that the dosage of dexamethasone given for diagnostic or therapeutic purpose needs to be adjusted in patients on phenytoin therapy and in patients with liver disease causing a che s ku/l. etofenamate is one of the most frequently applied topical nsaids in germany. it can be regarded as a lipophilic prodrug of flufenamic acid, which is liberated after transdermal absorption and cleavage of the ester. we compared several topical nsaids in patients suffering from osteoarthrosis or rheumatoid arthritis with concomitant knee joint effusions. the products contained between and % of etofenamate and were administered according to the recommended dosage schedule. following multiple applications of defined doses we obtained synovial fluid and plasma specimen in order to measure the concentrations and to compare the bioavailabilily of several products. the analysis of synovial fluid and plasma samples was achieved by hplc. following the addition of other n-phenylanthranilic acid derivatives as internal standards the samples were extracted twice with a -fold volume of acetonitrile/methanol ( : ). the diluted extracts were concentrated with the use of preconditioned ods-columns and subsequently eluted with methanol. the separation of the products was perlormed on a reversed phase column with an elution medium of methanol/water/acetic acid ( : . : . ). the highest synovial fluid and plasma concentrations were measured after hours following the final administration of etofenamate. the concentrations reached average maximum values of . ng/ml of flufenanamic acid in plasma and . ng/ml in synovial fluid. the bioavailability could be estimated from the area under the concentration time interval between . and hours, during which samples were obtained. they served for the comparison of the topical products. from in vitro data of cyclooxygenase or lipoxygenase inhibition with n-phenylanthranilic acid derivatives it can be concluded, that the synovial fluid levels of etofenamate and its metabolites are unsufficient to produce effects relying on this mechanism. it can not be excluded, however, that other effects contribute to the clinical improvements observed by other investigators. in animal studies it has been shown to have excellent anti-arthritic efficacy and an improved tolerability profile over other nsaids. early preclinical studies have shown that mel preferentially inhibits cyclooxygenase- (cox- ). the aims of this three-week, parallel, multicentre trial were to assess the efficacy anct safety of two different doses of mel in comparison to placebo (pla) in patients with active ra. the specific question was, whether cox- inhibition relates to therapeutic efficacy and safety in the clinical'setting. methods: patients ( f, m, mean age years) were randomised (mel . mg= , mel mg = , pla= ) and analysed by an intention-to-treat analysis. all trial drugs were given orally once daily. results: patients ( %) in the pla group, patients ( %) in the . mg mel group and patients ( %) in the mg mel group discontinued the trial prematurely due to lack of efficacy or adverse events. mg mel was significantly superior (p< . ) to pla in of the primary endpoints: disease activity assessed by the investigator and by the patient and reduction of the tender/painful joint count. the difference between . mg mel and pla reached statistical significance in of the primary endpoints: disease activity assessed by the patient and reduction of the tender/painful joint count. in none of the primary endpoints a statistically significant difference between mg and . mg mel occured. global tolerance assessed by the patient and investigator at the last trial visit was similar in all three treatment groups. adverse events were slightly more frequent with meloxicam than with placebo. in none of the patients gastric or duodenal ulcers, perforations or bleeds were observed. conclusion: the efficacy of meloxicam . mg and mg per day was significantly superior to placebo in the treatment of patients with ra. there were no relevant differences regarding safety parameters between the meloxicam doses and placebo. the preferential cox- inhibition may be the reason for the favorable efficacy and safety profile of meloxicam. the major metabolites of morphine are morphine - -b-d-glucuronide (m g) and morphine - - -d-glucuronide (m g). in humans plasma aucs of m g and m g exceed those of morphine depending on form and route of application. m g and m g penetrate the blood-brain-barrier due to an unexpectedly high lipophilicity, m g is an antagonist able to provoke with&awl symptoms even in opioid naive animals. m g is an agonist markedly more potent than morphine. methods: pig brains were homogenized and the homogenate centrifuged to give a microsomal preparation free of nuclei and coarse debris. aliquots were incubated at °c in buffer (ph . ) containing . nm [ h]damgo. results: the ic for morphine, m g and m g were . nm, nm and . pm, respectively. m g and m g, respectively, shifted the ic for morphine markedly to the right. discussion: our results on the single substances confirm data from the literature indicating that m g and m g inhibit binding to the ~t-or less potent than morphine. from the fact that m g and m g shift the ic for morphine to the right we infer binding of m g and m g to the morphine binding site at the ~t-or. bypassing activity and producing cost of dm . ,-was not included in the above shown results. • blood products, coagulation factors and anttmierobial agents are the most expensive substances, however their use could be more restricted. • chemotherapy represents also a high-cost factor, but alternatives do not exist. [ntensiv care units are the most expensive wards consuming - % of the hospital budget, drug use reviews on sicu's of the university hospitals frankfurt ( beds) and jena ( beds) were compared after recording the indication related administration of pharmaceuticals and blood products over a months investigation period using notebook-pc's. all substances were included, wich produced % and % of total pharmacentical cost in the year before, respectively. in frankfurt and jena and patients were registered, causing total cost of dm . ,-and dm . . ,-and causing cost per patient and day of average dm ,and dm ,-, respectively. in jena most expensive diagnoses were severe trauma ( % of total cost; n= ), gastre-intestinal bleeding ( . %; n= ) and malignant growth ( . %; n= ). in frankfiart nigh cost diagnoses were liver transplantation ( % of total cost; n= l), severe trauma ( , %; n= ) and coronary bypass craring ( . %" n= ). although the patient populations were different, the percentage cost of the substance groups on total drug cost were the same in both sicu's: blced-products ± %, antibiotic and chemotherapeutics io, %, immuna-ghibulines , :t , % anyway, inside these groups drug usage patterns differed distinctly. for example, in jena the nigh-cost antibiotics were tobramycin, cefuroxim, amoxicillin and ambisome ® causing . %, . %, . % and % of total antibiotic cost, respectively. corresponding data for franlffurt are ambisome* . %, imipedem . %, cefotaxim , % and cefamandol . %. severe sepsis was the most abundant complication occuring in beth sicu's in patients and producing cost of dm . ,-in frankfurt and dm . ,-in jena. in sepsis blood products were the most expensive substances with . % (frankfurt) and . % (jena) followed by antibiotics with . % and . % and immuneghibulines with . % and . %, respectively conclusion: * recording the indication related drug administration drug usage patterns may be identified and targets for economical intervention may be found. • blood and blood-products must be included because they produce about % of total cost. • complications, especially sepsis, produce nigh cost, which should be included in the discussion with the health assurances concerning the payment system using diagnosis related groups. in a drug-utilisafion-review-group (durg) was founded by clinical pharmacologists. the task of this group is to identify drug usage patterns, to record indication related drug administration including cost and to analyse these data. as a result recommendations concerning rational and economical use of pharmaceuticals and blood-products were given (e.g. ht -receptor-antagonists, human albumin, g-csf the most expensive substances are blood-products ( %), antimicrobial agents ( %) and immunoglobulines ( %). in conclusion, at the universitiy hospital frankfurvmain the activities of the drug-utilisation-review-group seem to be successful and are at least inpart responsible for the reduction of drug-cost of more than dm mio last year. the aim of this study was to determine the extent and the time course of fenoterol induced tachycardia and hypokalemia during and after a continuous intravenous infusion with three different rates within the therapeutic range for tocolysis. furthermore we compared the point estimators and precision of parameters obtained with a standard two step approach and a population model. according to a randomized double blind multiple cross over design, healthy females received an intravenous infusion of either placebo or fenoterol ( . ijg/min, ijg/min, ljg/min) over hours. the plasma concentration of fenoterol, heart rate, blood pressure, plasma potassium and plasma glucose were monitored during and up to hours after the infusion. the plasma concentrations of fenoterol and potassium and the heart rate were fitted to an integrated pkpd model. both individual parameter estimates and population estimates were obtained with nonmem. both effects correlated best with the concentration in the central compartment. the relationship between heart rate and concentration was adequately described by an emax model. modelling of the dose dependent hypokalemic effect of fenoterol required an negative sigmoid emax model with an additional term allowing for passive diffusion of potassium from the intra-to the extracellular compartment. during the observation period, the concentration response relationship remained stable for both effects. as to be expected, the estimates of the population parameters were much more stable than those of the individual patients. it is concluded that the population method might be superior to the sts approach even in a data rich situation. volunteers received different controlled release formulations (a, b, c, containing mg is- -m ", ) in a randomised cross-over design. plasma samples were taken from to hours after application. one week wash-out phase was kept between investigations. is- -mn were determined in plasma using a gc-method. in vitro dissolution of a, ] , c was investigated by using the rotating bottle method according nf xiv. mean absorption time (mat) was calculated as the difference of mrt and ke - , the mean dissolution time (mtdiss) was calculated as proposed by brockmeier ( ). additionally half value duration (hvd) and plateau time opt) are reported. the difference of the dissolution profiles between the product is not reflected in the parameters mat, hvd and pt. although preparation c has the longest mtdiss it has the largest cmax value and shortest hvd. the origin of this apparent discrepancy between in vivo/in vitro profiles deserves further elucidation ( ) acta pharm. technol. ( ) calciumantagonists (ca) are lipophilic molecules which, however, have no common structural similarities. nevertheless, ca are characterized by the ability to attach to various receptors and binding places in cardiac and smooth muscle preventing calcium overload, necrosis and cellular death. in order to study the physiological effects of ca on cerebral cortex of normal and vit d calcified rats, i.v. infusion of ca have been applied. cerebral blood flow (via hydrogen clearance) and oxygen supply (via surface oxygen tension) have been measured in normal and sclerotic albino rats in order to differentiate cerebral and systemic effects. miniaturized surface oxygen and hydrogen electrodes were placed on top of the exposed cerebral cortex of ketamine~xylazine anesthetized rats. nifedipen, vcrapamil, diltiazem and flunarizine induced hypotension in normal and monckebcrg type sclerotic rats. nifedipen and verapamil showed comparable improvements in cbf and surface po , the initial hypotension recovered completely under nifedipen but not under verapamil. compared with vcrapamil, the hypotension was even more pronounced under flunarizine ( : %). nevertheless, in response to both ca cbf was improved to about %. however, diltiazem did not increase cbf due to hypotension. sclerotic rats showed a map-decrease from to mm hg, the po histogram was left-shifted. diltiazem did not improve the cortical supply of sclerotic animals. the influence of intracoronary enoximone at a dose of . mg/kg/lo min on global and regional wall motion, myocardial perfusion (contrast echo) and diastolic lv function during pacing-induced ischemia was investigated in pts. with significant lad stenoses. results: enoximone did not change either heart rate at "rest ( ± vs ± min -i) or blood pressure (lvsp: ± vs ± mmhg) . in the postpacing ischemic period, lvedp fell from a mean of . to . mmhg after enoximone (p< . ), dp/dtmax increased from to mmhg/s (p< . ) and regional ef from % to % (p< . ) with global ef remaining unchanged ( % vs %). st segment depression was reduced significantly from . to . mm (p< . ). enoximone induced an increase in myocardial perfusion by % (p< . ) in the stenosis-depending myocardial areas with shortening of the wash-out half-time of the echo contrast medium from a mean of to s (p< . ) . the isovolumetric relaxation (echo) was shortened by % (p< . ), the e wave by %, and dp/dtmin was increased by % (p< . treated with pivaloyl-pivaloyl-substituted - actamantibiotics belong to a new group of prodrugs with an improved bioavailability following oral administration. after metabolic cleavage the pivalic acid and the free - actam are eliminated separately. pivalic acid is subjected to enzymatic coupling with endogenous camitine leading to carnitinepivaloylesler, which can be found in large amounts in the urine. free camitine is reduced under the treatment which may lead to neurological side effects in patients with metabolic predisposition. we showed, that the pattern of other acylcarnitineesters excreted with the urine was not changed significantly after administration of a pivaloyl.substituted cephalosporine in healthy subjects. in order to compare the pattern of excreted carnitineesters we analyzed urine samples of pediatric patients treated with cefetamet-pivoxil. the analysis of carnitineesters in the urine was achieved by hplc following precolumn derivatization. as an internal standrad we used undecanoyl-carnitine aster, which does not occur in human plasma or urine. after the extraction using ion exchange columns the samples were incubated with p-bromo-phenacyl reagent. the mixture of derivatives was separated on a reversed phase column applying a gradient of different buffers. with this method the whole spectrum of carnitineesters can be quantitatively analyzed. the percentage of the pivalic acid ester eliminated with the urine in pediatric patients did not differ from that observed in healthy subjects, therefore an increased risk in this patient group cannot be inferred. institut for klinische pharmakologie, universit~.tsklinikum charit& humboldt-universit&t zu berlin, schumannstral~e , d- berlin; pharmacokinetics and bioavailability of bupranolol-l"rs and oral bupranolol in patients with impaired liver function compared to healthy volunteers k.-h. molz*, w. cawello**, g. haug-pihale*", r. bonn** w. hammes**, a. weil*** and g. popescu*** bupranolol is nearly completely absorbed after oral administration, but more than % of the parent compound are metabolized by first pass through the liver. it can be expected that transdermal application of the substance will bypass the high first pass metabolism resulting in higher plasma concentrations or lower therapeutic doses, respectively. a reduced capacity of metabolizing liver enzymes might mimic this effect. the study was performed to investigate if metabolisation by hepatic enzyme systems results in liver function dependent pharmacokinetics. special interest was to be given to possible differences between oral and transdermal application. in an open sequential single dose-study including patients with impaired liver function and age/sex/weight-matched healthy volunteers a bupranolol transdermal therapeutic system (containing mg/ h) and bupranolol tablets ( mg) were administered to each participant separated by a washout period of a week. phenotyping for mephenytoin and sparteine was done to exclude poor metabolizers. blood samples for the quantification of bupranolol plasma concentrations were drawn before and , , , , , , , , (before patch removal), , , , , , and hours after patch application as well as before tablet administration and . , , . , , , , , , , , , , , , and hours post dose. urine samples were collected in ('its) and (tablets) intervals after dosing. blood pressure and heart rate were measured before and , , , and hours after dosing, ecg's were recorded before and , and hours after dosing. the pharmacokinetic results indicate only minor differences between patients and healthy controls after patch applications. for example the main parameter auc( -~) were . ± . ng/ml.h (mean±sd) in healthy controls and . ± . ng/ml.h in patients. completely other situation was given in comparison of data from oral administration: auc( -=)decreased dramatically to . ± . ng/ml-h in healthy controls and increased to . ± . ng/ml.h in patients. vascular complications are a common fmding in patients with diabetes mellitus. it has been reported that elevated glucose impairs acetylcholine-but not nitruprusside-mediated relaxation of rabbit aorta in vitro suggesting an abnormal endothelium-mediated vasodilator mechanism (cohen, circulation ; (suppl. v): ). the purpose of the study was to investigate whether this phenomenon . implies general impairment of the nitric oxidemediated relaxation (or only specific interference with muscarinic receptors), and . can be also demonstrated for coronary arteries. porcine coronary arteries were cut in rings and were set up in organ baths for isometric tension recordings. rings were randomized and incubated with different glucose concentrations ( . , , , or . mm) for hours according to cohen and coworkers. rings were preconstricted with prostaglandin f ~t ( . - ~m); the nitric oxidemediated relaxation was induced by either substance p ( . - nm), bradykinin ( . - nm), or calciumionophor a ( - nm), the endothelium-independent relaxation by nitroglycerin ( - nm). in addition, the effects of glucose were studied on acetylcholine-( . - ~tm) and serotonin-( . - ~m) induced vascular responses. results: surprisingly, elevated glucose concentrations ( and mm) had neither an effect on the potency nor the efficacy of any of the agonists tested. it is concluded that this previously reported impairment of endothelium-mediated vasodilation of rabbit aorta by elevated glucose concentrations may not be important in other species or types of arteries. nitric oxide production may be induced by cytokine-mediated immune responses. we tested the hypothesis that graft rejection in cardiac transplanted patients is associated with an increased production of nitric oxide. the study group consisted of patients in whom one or several myocardial biopsies (total biopsies) were obtained to assess for graft rejection. at the day of biopsy, urine was collected for a time period of at least hours ( % isopropanol was added for antimicrobial activity). nitrate was measuerd in urine samples by gaschromatography. nitrate excretion was expressed as the quotient of nitrate and creatinine content (gmol nitrate/mmol creatinine). since nitric oxide is oxidized in biologic systems to nitrite and finally nitrate, it was assumed that nitrate excretion reflects total nitric oxide release. graft rejection was classified according to the hannover classification system (a /i= no/minor rejection, a -a =moderate/severe rejection). results: nitrate excretion showed a large interindividual variation ( to i.tmol nitrate/mmol creatinine). /£ tough not significant, the nitrate excretion tended to increase with the degree of rejection (a : + ; a : + ; a : + ; a / : + ; mean + se). in a subgroup of patients (n= ) who underwent several biopsies with and without rejection, moderate/severe rejection was significantly associated with increased nitrate excretion (a /ai: + vs. a -a : + , p< . ). conclusion: the urinary nitrate excretion is increased in patients with moderate/severe graft rejection. because of large variation in excretion, this marker may be not suitable for rejection diagnosis. the combination of thremboxane a~/prostaglandin endoperoxide (epo) receptor antagonism with thromboxane synthetase inhibition (trasi) is designed to both abolish the epos' prothrombotic role and direct epos from activated platelets to adjacent cells for the generation of antithrombotic prostaglandins. thus, we have investigated the effect of the potent trasi dt-tx on thrombus formation in models of vascular injury (of the media versus selective deendothelialization) in man ex vivo. healthy volunteers were treated with an oral dose of vehicle (placebo; n= ) or , , , mg; n= per dose). blood was taken before and , , and h after the ingestion, anticoagulated and allowed to flow over a thrombogenic cell-free subendothelial matrix (cfm) or a layer of human venous smooth muscle cells (smc) for min. the "en face" area of platelets/thrombi deposited was determined by microscopic morphometry. in the absence of cells of the vessel wall (cfm) the thrombus formation was inhibited by + % (+sd) after mg, +_ % after mg, + % after mg and +p % after mg dt-tx versus + % after placebo. the subclass of the large thrombi (= % of all platelets/thrombi but incorporating > % of the platelet mass) was much stronger affected by the dt-tx treatment: the mean area was reduced by +p % after rag, + % after mg, _+ % after mg, + % after mg and _+ % after mg dt-tx versus - + % after placebo. in the presence of cells of the vessel wall (smc) the overall thrombus formation was reduced by up to + % after only mg, + % after mg, +_ % after mg, _+ % after rag and -+ % after mg dt-tx versus +_ % after placebo. dt-tx , a molecule combining potent and specific th romboxane synthetase inhibition with prostaglandin endoperoxide/thromboxane a receptor antagonism, has been examined in healthy male subjects. collagen-induced platelet aggregation in platelet rich plasma prepared from venous blood was measu red photometrically before and up to hours after a single oral dose of , , , or mg dt-tx in a placebo-controlled, double-blind study. platelet aggregation was induced in the ex vivo samples by collagen in concentrations between . and p.g/ml to evaluate platelet aggregation in relation to the strength of the proaggregatory stimulus. the ecs , i.e. the concentration of collagen required for a half-maximal aggregatory response (defined as the maximal change of the optical density), was determined. in the placebo-treated control group, the mean ecso was + ng/ml collagen (+ se; n= ) before treatment. it then varied between + and +_ ng/ml collagen after treatment. the'ratio of the post-to the individual pre-treatment ecso values was . _+ . (n= ) at . h, . _+ . at lh, . _+ . at h, . - . at h, . + . at h and . + . at h. this indicates that the sensitivity of the platelets to collagen was not affected by the placebo treatment. oral treatment with dt-tx , however, strongly inhibited the aggregatory response of the platelets to collagen stimulation. the ecs -ratio was increased to a maximum of . the detection of endogenous opioids suggested the opinion that in case of the presence in the organism of a receptor for an exogenous substance there is probably a similar endogenous substance.the occurrence in the blood of persons, who were not treated with cardiac glycosides, of endogenous digoxin-like or ouabain-like [actors confirms that opinion. in our study we took up the research of other drug-like [actors in the blood serum of healthy people. in two hundered and twenty-five healthy volunteers (llom,ll f) non-smokers not receiving any treatment before or during the test and aged between ib and y(mean age y) the occurrence of drug-like [actors in blood serum was studied.the examinations were carried out with the use of the fluorescence-polarization-immunoassay (fpia)-tdabbott. th e presence of the following endogenous drug-like foctors in the blood serum was evaluated: quinidine,phenytoin, earbamazepine,theophylline, cyclosporineand gentamicin. the presence of endogenous phenytoin-like, theophyllinelike and cyclosporine-like [actors has been demonstrated. the drug-like [actors were not found in the case of quinidine ,carbamazepine and gentamicin. the phenytoin-like factor was found in , ~, theophylline-like [actor , ~ and cyclosporine-like [actor in , ~ of examined volunteers.the mean value of the drug-like [actors were as follow : phenytoin , ~ , pg/ml,theophylline , ~ o,ll pg/ml and cyclosporine , z , ng/ml. the supposition may be proponued that organism produces drug-like substances according to its needs. the acetylation and oxidation phenotypes were studied in healthy volunteers ( m, [) aged between ib and years (mean y) in the wielkopolska region in poland. the acetylation phenotype was studied with the use of sulphadimidine which was given in a dose of mg/kg b.w. per os.sulphadimidine was determined by a spectrophotometric method.the border value of m.r. was ~ in urine. the oxidation phenotype was studied with the use of sparteine which was given in a dose of , mg/kg b.w.per de. sparteine was determined by the gas chromatographic method in urine. if m~ was <hr these are values for intensive metabolism, if mr was , <mr( these are values for poor metabolism. the determinated phenotypes were bimodally distributed. our studies have short that healthy volunteers ( . ~) were slow and ( , ~) were fast metabolizers. intensive oxidation phenotype could be ascertained in volunteers ( , ~) and poor oxidation phenotype in volunteers ( , ~). these findings are comparable with the values typical for the caucasian population. the genes coding for rapid acety!ation were present in . % of the cases, genes coding for slow acetylation were detected in . %. the frequency of specific nat alleles were: r , . %; r , . %; sla, . %; slb, . %; slc, . %; s , . %; s , . %; and s , . %. there was no mutation found at nt. in cases ( . %) we have stated nat genoand phenotype concordance, but in cases ( . %), genotype and phenotype deviated from each other. seven individuals were genotyped as slas , one as slasle, and one as slas but were phenotypically rapid acetylators. one child was genotyped as r s a but proved a slow acetylator. we can confirm that in about % nat genotyping allows to properly predict nat phenotype. discrepancies may derive, i. a., from yet unknown mutations which we try to detect by dna-sequencing. several studies demonstrated that cardiac glycosides reduce diameter of epicardial coronary arteries. in patients (ffs) with coronary artery disease (cad) this vasoconstrictive side effect of glycosides may cause ischemic complications in the presence ot high-grade stenoses. this study evaluated quantitatively the effect of an i.v. injection of digoxin on diameter of epicardial coronary arteries in pts with cad. eleven grs (group ) were treated with aspirin mg/d, nine frs (group ) were treated with aspirin and rag nisoldipin hours prior to angiography. coronary angiograms were taken in identical projections before ( ) and , , and minutes after i.v. injection of . mg digoxin, at rain .ling of nitroglycerin (ntg) was given. the mean diameters of normal segments and the minimum diameters of stenoses were analysed with cms (medis, leiden). a total of normal and stenotic segments were analysed. figure shows the change of coronary diameter (% predrug-vahie; mean + se) for group i (left) and ii (right): ( -( -cyclohexylureido-phenoxy)- -hydroxy- -tert.-butylaminopropan; awd, dresden) is frequently used as a cardioselective g-adrenoceptor antagonist. talinolol metabolites in urine were detected by hplc and gc/ms with previously characterized reference compounds and were quantified by hplc with a normal phase silica column (oertel et al., br. j. clin. pharmacol. ( ) ) . less than one per cent of administered dose was recovered in urine as bydroxylated talinolol. other metabolites could be excluded. in serum after therapeutic doses talinolol metabolites were not traceable. however, in a suicidal patient with a parent compound serum concentration of ng/ml (manifold higher as after usual therapeutic dose) two hydroxy metabolites at concentrations near the detection limit ( ng/ml) were found. obviously only a small amount of the talinolol dose is metabolized in human. in this study the relation between structure, polarity and urinary recovery of the metabolites was examined. four different isomers of mono-hydroxylated talinolol were identified in urine of volunteers after a single dose of talinolol. eight isomers are possible: six hydroxy-cyclohexyl-compounds and two hydroxy-phenoxy-compounds. there was a low degree of specificity of biotransformation seen with respect to the point of attack on the cyelohexyl ring. hydroxylation of the phenyl ring could be excluded in human. using hplc methods very small quantities of reference compounds are sufficient to estimate and compare the polarity and hydrophily of similar compounds. long retention times in a normal phase system and short retention times in a reversed phase (rp) system are found for polar and hydropbilic compounds. the shortest retention times of all hydroxylated talinolol compounds in the normal phase system as well as the longest retention times in the rp system were found for the main metabolites -trans-and -cis-hydroxycyclohexyl-talinolol. accordingly, the main metabolites are the most polar and hydrophilic mono-hydroxylated talinolol isomers. lysine clonixinate is a non-steroidal antiinflarnmatory drug with marked analgesic effects. it is used for treatment of acute pain such as postoperative, dental or menstrual pain. we have determined the biosynthesis of thromboxane (tx)b z and prostaglandin (pg)f ~ in clotting whole blood ex vivo as well as collagen-induced platelet aggregation before and . , . (only txb and pgf ,~ biosynthesis), , . , , and hours after oral intake of mg lysine clonixinate. the results were compared with data obtained after oral intake of mg acetylsalicylic acid (asa). while both txb and pgf ,~ biosynthesis measured radioimmunologically were inhibited significantly (p< , and p< . , respectively) . hours, but not hours after lysine clonixinate, inhibition by asa was much greater and still highly significant (p< , ) after hours. the mean concentrations of lysine clonixinate and of asa inhibiting txb biosynthesis in vitro by % (ecs ) were . + . ~tm and . _+ . gm, respectively. aggregation of . ml aliquots of platelet-rich plasma induced by gg/ml collagen was inhibited by lysine clonixinate significantly (p< . ), but not completely for up to hours, while aggregation induced by gg/ml collagen was inhibited for up to hours only (p< . ). platelet aggregation induced by both collagen concentrations was inhibited by asa almost completely (p< . ) for at least hours. the ecs for in vitro inhibition of submaximal collageff-induced platelet aggregation was + btm for lysine clonixinate and + lam for asa. it remains to be investigated, whether other mechanisms than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate. the profile of side effects including only moderate inhibition of platelet function by lysine clonixinate as compared to asa might be an advantage with regard to its use as an analgesic. increased cell proliferation induced by ras-oncogenes is mediated by growth factors. growth factors bind to cell membrane surface receptors and activate tyrosine kinases leading to protein phosphorylation. this triggers posttranslational modification of the function of low molecular mass gtp binding ras proteins, e.g. p ras, including heterotrimetric g proteins. th~se proteins carry a c-terminal amino acid sequence, caax where c is cysteine, which is the target of isoprenylation by famesyl protein transferases (fpptases) and geranyl-geranyl protein transferases (ggptases). the amino acids of this sequence determine whether the ras protein is substrate for fptases or ggptases. this biochemical pathway is related to mevalonate metabolism, therefore, cholesterol biosynthesis shares several steps with isoprenylation. the active compounds include a series of isoprenoid inter-mediates, such as geranyl pyrophosphate (c- ), farnesyl pyrophosphate (fpp, c- ) and geranyl-geranyl pyrophosphate (ggpp, which are critical for cell proliferation. inhibitors of isoprenylation may (i) compete with fpp to its binding site on the alpha subunit of the enzyme, (ii) inhibit the zn protease of fptase beta subunit, and (iii) interfere with the binding of the caax peptide to its binding site on the beta subunit. the inhibitors include microbial products like manumycin, gliotoxin, acetylgliotoxin and pepticin-namins, fpp analogues, tetrapeptides and related peptidomimetics that may compete for the caax sequence, products of plant origin like limonene as well as synthetic compounds. all these compounds have been known to retard cell proliferation, and their effect can be abolished by mevalonic acid. inhibitors of hmg-coa reductase, such as lovastatin, which have been developed as antihypeflipidemic drugs, may also effective as anticancer agents. the recognition of the importance of mevalonate metabolism in cancerogenesis provides possibility to test simple chemical comp unds against tumor growth, and gives promise for pharmacological exploitation of inhibitors of isoprenylation and hmg-coa reductase inhibitors for cancer chemotherapy. dopamine (da) and epinine (epi, the active metabolite of ibopamine) are da-and ~-and ~-adrenergic receptor (r) agonists. to study whether da-r mediated effects can be separated from ~-and ~-ar effects we assessed in male volunteers effects of i.v. da and epi ( . ; . ; . and . ~g/kg/min for min each) on changes in serum prolactin (prl, da-r mediated), blood pressure (p) and heart rate (hr, ~-and ~-ar mediated), natriuresis (urinary excretion of sodium), diuresis (urine flow rate) and plasma noradrenaline (na) levels. in the . and . ~g doses da and epi did not affect psyst, pdiast and hr, but significantly decreased prl (da: from . ± . to . ± . ng/ml; epi: from . ±i.i to . ± . ng/ml, p<o. ). in the ~g dose da and epi increased psyst by mmhg, pdiast by and mmhg, and hr by and beats/min but further decreased prl. in addition, both da and epi significantiy increased diuresis and natriuresis; in contrast, only da, but not epi, dose-dependently increased plasma na ievels, an effect that was not inhibited by ~-ar antagonists. we conclude that in . and . ~g doses (plasma levels of - nmol/ ) oa and epl act only at da-r. in higher doses, however, da and epi can activate ~-and ~-ar whereby epi has a stronger ~-ar component than da. moreover, part of da-effects are indirect via release of endogenous na whereas epl-effects do obviously not include an indirect component. j. raderrnacher, r.mliller-bartels, f. barrels, h. baurngarten, k. thon, d. tsikas and j.c. fr ich, patients with essential hypertension were ireated with cicletanin ( rag/d) for weeks. compliance was verified by measuring urinary cicletanin excretion. before entering the study anti_hypertensive medication had been withdrawn for at least weeks (wash out phase). the study was preceeded by a placabo phase of week duration. blood pressure measurements and urine collections for measurements of , dinor- -keto-prostaglandin f a (a stable prostacyclin metabolite) were performed at the start and end of placebo phase, atter week of treatment with cicletanin and in monthly intervalls thereafter. cicletanin decreased systolic and diastolic blood pressure, the effect becoming significant at weeks. urinary excretion of the prostacyclin metabolite , dinor- keto pgfla.increased by %. hypertensive patients and control subjects (n= ; , dinor- -keto-pgfla: + pg/rnl] did not differ significantly in urinary excretion of , dinor- keto pgf ct. neither systolic nor diastolic blood pressure showed a significant correlation with , dinor- keto-pgf alpha excretion (systolic bp: r-- , , p-- , l; diastolic bp: r= , , p=l, ). these data show that cicletanin is effective in reducing blood pressure. increased prostacyclin production is probably not responsible for the observed reduction of blood pressure. in the past the realization of trials on the pharmacokinetics of nicergoline in human beings break down according to analytical difficulties. from animal studies with labelled compounds it is known, that nicergoline is rapidly absorbed from the gastrointestinal tract and two main metabolites (mdl, i-mmdl) can be detected.the mean elimination half-life for nicergoline is given with , h, for mdl - h and for i-mmdl with - h. the compound is excreted mainly by the kidney. due to known problems of the application of labelled drugs in human beings there are two approaches for the determination of these compounds: first the detection by monoclonal antibodies by using a ria-method, secondly a hplc-analysis. although there are promising in-vitro results of the immunological method, quantitative analyses of serum samples show deceptive results. the hplcassay reveales that in human serum samples after oral administration of nicergoline only the metabolites mdl and i-mmdl can be detected. further analytical development lower the limit of detection which should be i/i of c~x. with an improved hplc-method it will be demonstrated that only the measurement of the metabolite mdl is suitable to give sufficient results in pharmacokinetic trials and in studies on bioavailability with formulations of nicergoline. antiplatelet treatment with aspirin is well established as secondary prophylaxis after a transient ischemic attack or minor ischemic stroke, but the effect of aspirin treatment on the course of carotid atheroselerosis is unknown. we tried to investigate the effect of aspirin on the initial stages of carotid atherosc}erosis. patients were recruited from a prospective, randomized, double blind clinical trial to compare two doses of aspirin ( mg vs mg claity) with respect to restenoses after lower limb angjoplasty. of the patients admitted to the angioplasty trial, patients showing small carotid atheroma (< % lumen narrowing) were examined at entry and after year of aspirin treatment using a high-resolution ultrasound duplex system. disease progression and regression were defined by a change of maximal plaque area (as measured by longitudinal ultrasound sections) of more than standard deviations of the method. the change in plaque area was significantly different for the treatment groups: average plaque size remained'unchanged after treatment with mg aspirin daily but increased markedly after treatment with mg aspirin daily (p = . ). there were significantly more lesions in the -mg group showing progression than in the -mg group ( plaques [ %] vs plaques [ %], p = . ). ultrasonic disappearance of a lesion was observed only in the -mg group in cases ( soft plaques, ulcerative plaques, p = . ). the patients on mg aspirin who continued smoking during the study showed significantly more progression as compared to the non-smokers in the -mg group ( plaques [ %] vs plaques [ %], p = . ). the results of our study indicate that aspirin treatment slows carotid plaque growth in a dose-dependent fashion, with a dose of mg daily more efficient than mg a day. gentamicin is frequently applied in neonatal patients for the treatment of severe infections. the use is associated with high incidences of oto-and nephrotoxicity. ototoxicity occurs in - . % of the treated patients. it can be avoided, if the trough levels remain under p.g/ml between the administered doses. if the nephrotoxic effect, which occurs in - % of the treated patients, can also be reduced under these circumstances is not yet clear. recently it has been suggested to administer gentamicin only once daily in order reach the desired trough levels. this applies especially to children, in which the terminal half-life can be prolonged to approximately hours. this dosage regimen assures a sufficient effectivity due to the marked postantibiotic effect, but should reduce the incidence of the toxic effects. in order to test this hypothesis we perform a case-control study with neonatal children treated with gentamicin. the cases of nephrotoxicity during the previous months are identified from the hospital records. for these patients the plasma level time curves are obtained with the use of a population based pharmacokinetics programme, which considers the measured plasma concentrations and the administered doses. plasma concentrations were measured routinely in the process of therapeutic drug monitoring. from these curves the duration of times with concentrations lower than p.g/ml during the last days prior to the diagnosis are estimated. for the cases matched controls were identified according to age, age of gestation and other factors affecting the renal function e.g. comedication. different analyses are carried out according to the definition of exposure. the results wiij be discussed with regards to the modification of the dosage schedule for gentamicin and the application of population kinetics in order to reduce the adverse effects. economic evaluation of drug therapy is an important task of clinical pharmacology. drug costs amount to a relatively small percentage of total health care costs. some drugs may help to avoid more expensive therapeutic procedures. drugs with small therapeutic ratio need e therapeutic drug monitoring (tdm) to optimize therapeutic results and to avoid side effects. tdm may enhance the costs of drug therapy and the question arises, whether the additional costs are justified from the economic point of view. the aim of the study was to investigate the effectiveness of theophylline tdm in chronic obstructive pulmonary disease. methods: outpatients suffering from chronic obstructive pu[monary disease and receiving theophylline as a basic medication were included in this study. during the one-year observation period the parameters of pulmonary function were examined four times ( st, rd, th and th month) by the same physician and theophylline trough levels were estimated to individualize the dosage. the clinical outcome was assessed by intraindividual comparison. results: impaired parameters of pulmonary function were improved. hospital admissions were reduced. during the one-year observation period out of patients had hospital admissions with totally days. under the conditions of theophylline therapy without tdm during the year before of them had hospital admissions with days of stay in hospital. discussion: the results agree with a previous study on inpatients. there it was observed, that parameters of pulmonary function significantly were improved due to tdm-supported theophy[line therapy in comparison to a group receiving theophylline without tdm. despite the additional costs thaopylline tdm contributes not only to improved clinical outcome but also to decreased costs for the care of patients with chronic obstructive pulmonary disease. in cancer treatment arterial blood flow reduction by embolisation followed by intra-artedal chemotherapy may be advantageous by achieving high and prolonged drug concentrations in the tumor and lower systemic drug exposure. pharmacokinetics of mitomycin-c (mmc) were investigated in colorectal cancer patients (body weight: - kg) with liver metastases undergoing chemoembolisation. after hepatic artery branch catheterization and microspheres injection (polyvinylalcobel, mg mmc dissolved in ml physiological naci solution were infused in - rain, followed by ethiblock application. serum mmc-concentrations were determined from peripheral venous blood samples by reverse-phase hplcsystem with ultraviolet detection (c -column, elution: phosphate-buffer ( . m, ph: . )) : methanol, v:v= : , nm). pharmacokinetic parameters were computed using non-compartmental or two-compartment model analysis assuming linear kinetics (top fit . software). results: on the average, maximum serum concentrations were observed at tmax = . rain following the beginning of mmc-infusion and amounted to cmax = ng/ml (range: - ng/ml). mean steady state distribution volume was . i/kg with a central compartment volume of . i/kg. concentration-time curves could be descnbed by a distributional phase (t / = . min) followed by a terminal elimination phase (]' / = . min). mean total clearance amounted to . ml/min.kg. the area under the concentration time curve was pg.m - .min. conclusion: pharmacokinetic parameters for mmc-disposition in this study do not substantially differ from those reported for similar mmc-amounts administered as intravenous bolus. systemic mmc exposure does not seem to be reduced following the embolisation procedure used compared to intravenous application. pseudocholinesterase-activity (pche) is an important determinant of the elimination kinetics of drugs like mivacudum. therefore, the time course of pche was investigated in patients undergoing cardiosurgery with cardiopulmonary bypass (cpb) in normo-or hypothermia. anesthesia was induced and maintained with midazolam, propofol and fentanyl using mivacurium as a muscle relaxant. by starting cpb, i of pdming fluid were added to circulation volume. from collected blood samples, pche (u/i) was determined photometrically according to eiiman ( )(butyrylthiocholine, dithiobisnitrobenzoate, nm, ph= . , oc) . total plasma protein concentration (g%) was measured according to lowry, and the specific pche was calculated as the quotient of pche and protein concentration (u/g). serum hepadn-activity was determined in patients (berichreme heparin test kit). moreover, the influence of hepadn and aprotinin, both contained in the cpb pdming solution, on pche in vitro was examined using fresh serum from healthy volunteers (n= ). results: the induction of anesthesia had no significant influence on pche or protein concentration (p> . ). cpb induced a significant decrease of pche (- %)(p< . ) and protein concentration (- %)(p< . ) and a less pronounced numedcal reduction the specific pche (- %)(p> . ). the reduction of pche and protein concentration was not significantly affected by ending cpb (p> . ), and the values remained low over the remaining operation time. there was no significant difference in pche, measured at °c in vitro, or protein concentration between the normothermic and hypothermic group (p> . ). furthermore, there was no correlation between serum hepadn-activity and pche reduction. pche in the plasma of healthy volunteers was not significantly affected by either hepadn up to u/ml or apretinin up to u/ml (p> . ). conclusion: ( ) the concentration of the antitumor antibiotic mitomycin c (mmc), used in ophtalmic surgery for its antiproliferative effects, was measured in the aqueous humor of glaucoma patients undergoing trabeculectomy. sponges soaked with mmc-solution ( ul of mmc-solution . mg/ml: rag) were applied intraoperatively under the scleral flap for rain. to ul of aqueous humor were drawn with a needle min following the end of topical mmc-treatment. samples were assayed for mmc using a reverse-phase hplc-system with ultraviolet detection (c -column, elution: phosphate-buffer ( . m, ph: . ):methanol, v:v = : , nm). swabs were extracted in phosphatebuffer ( . m, ph: . ) before hplc-analysis. external calibration was used for mmc quantitetion. quantitation limit was ng/ml. in all aqueous humor samples mmc-concentration was below ng/ml. mmc in the swabs amounted to % of the mmc amount applied. conclusion: after intraoperetive topical application, mmc concentration in the aqueous humor of patients is very low. the substantial loss of mmc from the swabs used for the topical mmc-treatment suggests ( ) rapid systemic absorption of mmc and/or ( ) a loss through irngation of the operative field following topical mmc-application. institut fur pharmakologie und * klinik for augenheilkunde, universitcit k n, gleuelerstrasse , k n al a due to runaway costs of the national health service which are reflected as well in growing expenditures for drugs at the university hospital of jena investigation of indication related drug administration patterns becomes more and more interesting. this holds especially true for intensive care units (itu's) which are determined by similar high costs for technical equipment as for drugs ( ) although any economical considerations seem to be questionable due to ethical reasons ( ). over a month period indication related drug administrations of surgical itu's of the university hospital jena have been recorded and analyzed by using a pc-notebook. total expenditures for all included patients add up to dm . . regarding these drugs and blood products which caused % of total costs in . the leading substances ( antithrombin ill, human albumin %, prothrembine complex, ...) represent % of total costs including blood products, antibiotics and ig m endched intravenous immunglobine. therefore the indication of particulary these drugs became mere interesting for further investigations. already during the study actual discussions with the treating medical staff have been made leading to new developed therapy recommendations. providing same high standard of medical treatment a remarkable cost saving of some drugs by more cdtical and purposeful use could already be achieved as a first result. however, the results of the study underline impressivly the benefit of such investigations for improvement of drug treatment. the simple replacement of expensive drugs ( e.g. prothrembine complex ) by higher quantities of cheaper ones of the same indication group ( e.g. fresh frozen plasma ( )) does not necessarily mean less expenditures in all cases but may cause unsiderable side effects. ( ketokonazole is known to decrease pituitary acth secretion in vitro and inhibits adrenal ll-hydroxylase activity. to work out the clinical significance of both effects analysis of episodic secretion of acth, cortisol (f) and ll-deoxycortisol (df) was performed in patients with cushing's syndrome (cs) requiring adrenostatic therapy. methods : ketokonazole was started in ii patients with cs ( acth-secreting pituitary adenomas [cd], adrenal adenoma [aa] ). in of them ( cd, aa) blood samples were obtained for hours at i min intervals ( samples/patient) before and again under treatment (mean dose i mg/d, > weeks). hormone levels were measured by ria and secretion patterns analysed by means of pulsar, cluster and desade. patients were investigated only once because treatment was stopped due to side effects. results : the we conclude that the observed % increase of plasma acth and the % decrease of f/df ratio demonstrate that inhibition of adrenal li -hydroxylase activity is the primary mode of action of ketoconzole in vivo. even at high doses acth and f secretion patterns could not be normalized. the improvement of pain and swelling conditions by means of drugs is an important method of achieving an enhanced perioperative quality of life in cases of dentoalveolar surgery. in prospective, randomised, double-blind studies the influence of various concentrations of local anaesthetics and accompanying analgesic and antioedematons drugs was investigated in the case of osteotoimes. all of the studies were carded out according to a standardised study procedure. a comparison of the local anaesthetics articaine % mad articaine % (study ) demonstrated the superior effect of articaiue % with respect to onset relief on pain, period of effectiveness and ischaemia. recordings of the cheek swelling in the remaining studies were made both sonographically and with tape measurement, while the documentation of the pain was carried out by means of visual analogue scales on the day of operation and on the first and third post-operative days. tile perioperative, exculsive administration of x mg dexamethasone (study ) resulted in a significant reduction in the swelling ( %) while the exclusive administration of x mg lbuprofen (study ) was accompained by a marked decrease in pain ( %) but no significant reduction of swelling in comparison to the placebo group. the combination of x mg ibuprofen und mg methylprednisolone (study ) yielded a decrease in pain of . % and a reduction in swelling of %. a cdmparison between a mono-drug ibuprofen and a combination drug ass/paracetamol (study ) resulted in no significant difference in the reduction of swelling and pain and therefore highlighted no advantages for the combined drug. a mono-drug should therefore be given priority as an analgesic. the combinatton of ibuprofen und methylprednisolone offers the greatest reduction in pain and swelling. using the results of the randomised studies, a phased plan for a patietu-orietued, anti-inflammatory therapy to accompany dento-alveolar surgery is presented. in a placebo controlled study patients with congestive heart failure (nyka class ii) were treated orally for seven days with i mg ibopamine t.i.d, i subjects had a normal renal function (mean inulin clearance (gfr) ± , ml/min), i patients suffered from chronic renal insufficiency (gfr ± , ml/min; x ± sem). pharmacokinetic parameters of epinine, the maximum plasma concentration, the time to reach maximum plasma concentration and the area under the curve from to hours were unaltered in impaired renal function when measured on the first or on the seventh treatment day. however plasma concentrations in both groups were significantly higher on the first treatment day than after one week of ibopamine administration. in this context antipyrine clearance as a parameter of oxidative liver metabolism which might have been induced by ibopamine revealed no differences between placebo and ibopamine values. in conclusion kinetic and dynamic behaviour of ibopamine was not altered by impaired renal function. human protein c (hpc) is a vitamin k-dependent in the liver produced glycoprotein with anticoagulant properties. when active protein c splits the coagulation factors va and vuia by means of limited proteolysis (kisiel et al ) . its concentration in normal plasma is - lag/m[ i-ipc's biological importance became evident when a congenital protein c deficiency, which results in difficult recurrent thromboembolic diseases was discovered (griffin eta/ ) . the recognition of a congenital hpc deficiency, as wall as the connection between acquired protein c deficiency and the appearance of thromboembolic complications by means of highly accurate and sensitive ascertained methods is therefore of great practical importance for the clinic. murine monoclonal antibodies (moabs) against hpc were formed. antibody producing hybridomas were tested by an ,,indirect elisa" against soluble antigens. the plates were coated with purified hpc up to ng/ al. the peroxydase-system was used to identify antibodies the antibodies were tested with the remaining vitamin k-dependent proteins for cross-reactivity, as well as with hpc deficiency plasma for disturbances by other plasma proteins. the above described experiment represents a sensitive and specific method for measuring the hpc concentration with moabs. assessment of local drug absorption differences ("absorption window") in the human gastrointestinal tract is relevant for the development of prolonged release preparations and for the prediction of possible absorption changes by modification of gastrointestinal motility. current methods are either invasive and expensive (catheterization of the intestinum, hf-capsule method) or do not deliver the drug to a precisely defined localization. we evaluated the delay of drug release from tablets coated with methacrylic acid copolymer dissolving at different ph values as an alternative method. three coated preparations of caffeine tablets (onset of drug release in in vitro tests at ph . , . and . ) and an uncoated tablet (control) were given to six healthy male volunteers in a randomized order. caffeine was used because of its rapid and complete absorption and good tolerability. blood samples were drawn up to h postdose (coating ph . up to h postdose), and caffeine concentrations were measured by hplc. auc, time to reach measurable caffeine concentrations (tia~), tr, ax, cmax and mean absorption time (mat) values for coated preparations were compared to the reference tablet (mean + sd of n= ): the relative bioavailibility for the coated preparations did not differ from the reference, suggesting complete release of caffeine. all coatings delayed caffeine absorption onset. the tlag for the ph . preparation suggests that release started immediately after the tablet had left the stomach. the mean delay of . h for the ph . coating was highly reproducible and should reflect small intestine release. the ph . coating delayed absorption to the highest extent, however the drug was probably released before the colon was reached. there is evidence that nitric oxide (no) plays a role in cardiovascular disease like hypertension, myocardial ischemia and septic cardiomyopath.y. no stimulates the guanylyl cyclase leading to an increase m cgmp content we investigated by immunoblotting the expression of the inducible nitric oxide synthase (inos) in left ventricular myocardium from failing human hearts due to idiopathic dilative cardiomyopathy (idc, n= ), ischemic cardiomyopathy (icm, n= ), beeker muscular dystrophy (n= ) and sepsis (sh, n= ) compared to non-failing human hearts (nf, n= ). cytokine-stimulated mouse macrophages were used as positive controls sds-polyacrylamide gel electrophoresis ( . %) was perfomed with homogenates of left ventricular myocardium and mouse macrophages respectively. proteins were detected by enhanced chemiluminescence using a mouse monoclnal antibody raised against inos. furthermore, we measured the cgmp content in these hearts by radioimmunoassy. a band at about kda was observed in two out of three hearts from patients with sepsis and in stimulated mouse macrophage~ no inos-protein expression was detected in either non-failing human hearts (n= ) or failing human hearts due to idc, ihd or bmd. in ventricular tissue from patients with sepsis cgmp content was increased to % ( + fmol/mg ww, n= ) compared to non-failing hearts ( % or + . fmol/mg ww, n= ). in left ventricular tissue tissue from patients with heart failure due to idc, ihd and bmd cgmp content did not differ from that in non-failing hearts. it is concluded that an enhanced inos protein expression may play a role in endotoxin shock, but is unlikely to be involved in the pathophysiology of end-stage heart failure due to idc, ihd and bmd. (supported by the dfg.) nitric oxide (no) has been shown to be a major messenger molecule regulating blood vessel dilatation, platelet aggregation and serving as central and peripheral neurotransmitter; furthermore no is a crucial mediator of macrophage cytotoxicity. no production can be assessed reliably by determination of its main metabolites nitrite and nitrate in serum, reflecting no synthesis at the time of sampling, or in h urine, reflecting daily no synthesis. farrell et ai. (ann rheum dis ; : ) recently reported elevated serum levels of nitrite in patients with rheumatoid arthritis (ra). we report here total body nitrate production and the effect of prednisolone in patients with ra. nitrate excretion in h urines of patients with ra as defined by the revised criteria of the american rheumatism association was measured by gas chromatography at times: first before start of a antiinflammatory therapy with prednisolone, when the patients had high inflammatory activity as indicated by mean crp serum concentrations of + sd mg/i and elevated esr with a mean of ]: after hour. secondly - weeks after start of prednisolone therapy in a dosage of . mg/kg body weight, when the patients showed clinical and biochemical improvement (crp + mg/i, p< . , esg + , p< . , two-tailed, paired t-test). for comparison h urines from healthy volunteers were obtained. before start of predniselone therapy the urinary nitrate excretion in patients with ra (mean + sd p.mol/mmol creatinine) was more than twofold higher (p< . , twoaailed unpaired t-test) than in healthy volunteers ( + ~tmol/mmol creatinine). the urinary nitrate excretion decreased significantly (p< . , two-tailed, paired t-test) to + i.tmol/mmol creatinine under therapy with prednisolone, when inflammatory activity was reduced considerably. despite the decrease the urinary nitrate excretion was still twc, fold higher (p< . , two-tailed, unpaired t-test) in patients with ra than in the control group. our data suggest that the endogenous no production is enhanced in patients with ra. furthermore the results indicate that this elevated no synthesis could be reduced in accordance with suppression of systemic inflammation by prednisolone therapy. but now as ever the physicians are entitled to prescribe drugs which have to prepare in a pharmacy for a particular patient. little information is available on the frequency and patterns of these prescriptions. we had occasion to analyse the prescriptions of drugs which were prepared in pharmacies in north thuringia (east germany) from october to december at the expense of a large health insurance company (allgemeine ortskrankenkasse). the selected pharmacies are loealised in cities. we found prescriptions of drugs made up in pharmacies among a total number of reviewed drug prescriptions. this is . % of the total. most of these prescriptions were performed by dermatologists ( . %), general practitioners ( . %), paediatrists ( . %) and otolaryngologists ( . %). according to this, the most frequently prescribed groups of drugs were dermatics enteric eoated tablets with nag and nag acetylsalicylic acid (asa) have been developed wluch should avoid the known gastrointestinal adverse events by a controlled drug release mainly in the duodenum after having passed the stomach. a -way cross-over study in healthy male subjects, aged from - years, was conducted to investigate the pharmacokinetics, bioavailability, safety, and tolerance of asa and its metabolites salicylic acid and salicylurie acid following enteric coated tablets in comparison with plain tablets. asa and its metabolites were determined by a sensitive, specific, and validated hplc method. pharmacokinetic parameters were determined by non-compartreental analysis. bioequivalence was assessed by % confidence intervals. following the admimstration of enteric coated tablets, a delayed absorption can be observed for both the mg dose and the rag dose. this is likely due to a delayed release of the active substance from the enteric-coated tablets in the small intestine arer gastric passage. considering the mean residence times (mrt), there is a difference of at least . h following the enteric coated tablets compared to the plain tablets for asa and the two metabolites measured• this difference represents the sum of residence time in the stomach plus the time needed to destroy the coating of the tablet when it left the stomach• in general, the maximum observed concentrations of both enteric coated formulations occurred - h post dose. the pharmacokinetics of a novel immunoglobulin g (lgg) preparation (bt , biotest, dreieich, frg) have been determined in healthy, male anti-hbs-negative volunteers. for this preparation only plasma from hiv-, hbv-and hcv-negative donors was used, the quality control for the product was in accordance with the ec-guideline for virus removal and inactivation procedures. each volunteer received a single, intravenous infusion of ml bt containing g igg and anti-hbs > , iu. anti-hbs was used as a simply measurable and representative marker for the igg. blood samples for determination of anti-hbs (ausab eia, abbott, frg) were drawn before and directly after the infusion, after , , , and hours, on day , , , , , , , , , and . additionally, total protein, igg, iga, igm and c /c complement were measured and blood hematology and clinical chemistry parameters determined. the phar~gacokinetic parameters of anti-hbs were calculated using the topfit ~" pc program assuming a -compartment model. pharmacoeconomic evaluations (pe) describe the relationship between a certain health care input (costs) for a defined treatment and the clinical outcome of patients measured in common natural units (e.g. blood pressure reduction in mmhg), quality of life (qol) gained, lifes saved or even in money saved due to the improvement in patients functional status. this implies that the efficacy of a treatment has been measured and proven in clinical trials. in addition, in order to transfer data obtained in clinical trials to the clinical setting, an epidemiological database for diseases and eventually drug utiiization may be required. the evaluation of the efficacy depends on the disease to be treated or prevented and the mode of treatment. for acute, e.g. infectious diseases, the endpoint can be defined easily by the cure rate, but for pe the time (length of hospital stay) and other factors (e.g. no. of dally drug administrations) have to be considered. in the case of chronic diseases, e.g. hypertension or hypercholesterolaemia, surrogate endpoints (blood pressure or serum cholesterol reduction) and information on side effects may be acceptable for the approval, but cannot be used for a meaningful pe. the latter should include the endpoints of the disease, i.e. cardiovascular events (requiring hospitalisation and additional treatment) and mortality. furthermore, the qol has to be measured and considered for chronic treatment. several questionaires have been developed to measure the overall qol or the health related qol. especially the latter may be a more useful tool to detect mad quantify the impact of a treatment on qol. combining the clinical endpoint mortality and qol by using qalys (quality-adjusted lifeyears) may be a useful tool to determine the value and costs of a given drug treatment but cannot be applied to all treatments under all circumstances. sorbitol was used as a model substance to investigate the dynamics of the initial distribution process following bolus intravenous injection of drugs. to avoid a priori assumptions on the existence of well-mixed compartments data analysis was based upon the concept of residence time density in a recirculatory system regarding the pulmonary and systemic circulation as subsystems. the inverse gaussian distribution was used as an empirical model for the transit time distribution of sorbitol across the subsystems, distribution kinetics was evaluated by the relative dispersion of transit (circulation) times. the distribution volumes calculated from the mean transit times were compared with the modelindependent estimate of the steady-state volume of distribution. kinetic data and estimates of cardiac output were obtained from patients after percutaneous transluminal coronary angioplasty. each received a single . g iv bolus dose of sorbitol. arterial blood samples were collected over hours. while the disposition curve could be well fitted by a tri-exponential function the results indicate that distribution kinetics is also influenced by the transit time through the lungs, in contrast to the assumption of a wellmixed plasma pool underlying compartmental modelling. a karit@ "bu£ter" is used traditionally in west afr%can manding colture as a cosmetic to protect the skin against the.sun. gas chromatography was used to analyze the ingredients of karit@ butter from guinea. we found % palmitic acid, % stearic acid, % oleic acid and % linoleic acid and . % of other fatty acids with higher chain lengths like arachidonio acid. some of these are essential fatty aclds (vitamine f). furthermore karit@ contains vitamine a and d as well as triterpene alcohols and phytosterines. an original extract was used to prepare a skin cream. this preparation was tested in volunteers ( women, men; age - y.). the cream contained at least % karit@, glycerol, emulsifiers and no preservative agent except for sorbic acid. of the volunteers very well tolerated the cream and thought it effective. the skin became more tender and elastic. good results were obtained when the volunteers suffered from very dry skin. two of them who were known to be allergic against the most available skin creams had no problems in using our karit cream. pure karit@ butter was used for four months to treat an african infant with neurodermitis. after this time the symptoms had markedly improved whereas previous therapy trials with other usual topical medicaments had been unsuccessful. these pre-studies had shown that dermatologic preparations containing karit# may be a good alternative in the treatment of therapyreslstent skin diseases and may in some cases be able to replace eorticoid treatment. ) and a low molecular weight heparin preparation (fragmin ~, iu/kg bodyweight s.c.) on coagulation and platelet activation in vivo by measuring specific coagulation activation peptides [prothrombin fragment + (f + ), thrombin antithrombin iii complex (tat), -thromboglobulin (~-tg)] in bleeding time blood (activated state) and in venous blood (basal state). in bleeding time blood, r-hirudin and the heparin preparations significantly inhibited the formation of both tat and f + . however, the inhibitory effect of r-hirudin on f + generation was short-lived and weaker compared to ufh and lmwh and the tat/f + ratio was significantly lower after r-hirudin than both ufh and lmwh. thus, in vivo when the coagulation system is in an activated state r-hirudin exerts its anticoagulant effects predominantly by inhibiting thrombin (lla), whereas ufh and lmwh are directed against both xa and ila. a different mode of action of ufh and lmwh was not detectable. in venous blood, r-hirudin caused a moderate reduction of tat formation and an increase (at hour) rather than decrease of f + generation. formation of tat and f + was suppressed at various time points following both ufh and lmwh. there was no difference in the tat/f + ratio after r-h[rudin and heparin. thus, a predominant effect of rhirudin on ila (as found in bleeding time blood) was not detectable in venous blood. in bleeding time blood, r-hirudin (but neither ufh nor lmwh) significantly inhibited ~-tg release. in contrast, both ufh and lmwh caused an increase of ~-tg hours after hepadn application. our observation of reduction of platelet function after r-hirudin compared to delayed platelet activation following ufh and lmwh suggests an advantage of r-h[rudin over heparin, especially in those clinical situations (such as arterial thromboembolism) where enhanced platelet activity has been shown to be of particular importance. the human cytochrome p isoform cyp a determines the level of a variety of drugs metabolized by the enzyme, including caffeine (ca) and theophylline (th). more than compounds are potential or proven inhibitors of this enzyme. some of them were reported to be substrates or inhibitors to cyp a in vitro, ethers caused pharmacokinetic interactions with drugs metabolised by cyp a . we characterized a series of these compounds with.respect to their effect on cyp a in human liver microsomes in relation to-published pharmacokinetic interactions in vivo. cyp a activity in vitro was measured as ca -demethylation at the high affinity site in human liver microsomes, using rain incubation at °c with - jm caffeine, an nadph generating system, and inhibitor concentrations covering . orders of magnitude. apparent kr values were estimated using nonlinear regression analysis. for inhibitory effects on cyp a activity in vivo, the absorbed oral dose causing % reduction in ca or th clearance (edso) was estimated from all published interaction studies using the emax model. %)i followed by disinfectants ( . %)r ointments ( . %) and solutions ( . %) were the most frequent drug forms %) or german ( . %). our results show that even now drugs prepared trend analysis of the expenses at the various departments may be a basis for a ratio-hal and economic use of the drug budget. total drug expenses amounted to mill. dm in . s milldm ( %) were used in surgical departments with intensive care units (icu) (general surgery, kardiovascular surgery, neurosurgery, gynecology, anaesthesiology) of wtfich % are needed by the icu and % in the operating rooms. surgical departments without scu but similar patient numbers (ophthalmology, ent, orthopedics and urology) get only % of the budget ( % needed for the operating rooms). the medical departments spent s mill.dm of which icu needs only % whereas the oncology (oncu) and antiinfective units uses more than %• similar relation could be seen in the child hospital ( . milldm, %) where % were spent for icu and % for oncu. the departments of dermatology and neurology get %, the depart-merits of radiology, nuclear medicine and radiation therapy only % of the budget. antiinfective drugs (antibiotics, antimycotics, virustatics) are most expensive ( % of budget) followed by drugs used for radiological procedures ( %) sncreasing the knowledge about the costs of medical items and the rational and economical use may stop the overproportional increase of the drug budget the mostly used : ) and a -fold higher efficiency than the r-form the elimination of the talinolol enantiomers was studied in healthy volunteers (age: - years, body weight: - kg) given a single oral dose ( mg) or an intravenous infusion ( rag) of the racemi c drug. three volunteers were phenotypically poor metabolisers and nine were extensive metabolisers of the debrisoquine-type of hydroxylation. the r-and senantiomers of talinolol were analysed in urine by a hplc method after enantioselective derivatisation. the concentrations of the enantiomers within every sampling period as well as the amounts of s-and r-enantiomer this corresponds to a s/r-ratio of , + , . the mean total amount (= s-+ r-enantiomer) eliminated was on average % &the administered dose. after oral administration _+ % of the dose were eliminated within h. the amounts of talinolol enantiomers recovered were equally (senantiomer: _+ gg the ratios of s-to r-concentrations at every sampling interval and of every volunteer were assessed between , and , (mean: , after infusion and , after oral administration, respectively) medizinische fakult~t carl gustav cams, teelmische university, t, fiedlerstr nitric oxide (no), synthesized by the inducib]e form of no synthase, has been implicated as an important mediator of-specific and non-specific immune response, little is known about the in vivo synthesis or no in inflammatory joint diseases. therefore we have studied the excretion of the major urinary metabolite of no, nitrate, in rats with adjuvant arthritis, a well established model of polyarthritis in addition we assessed the urinary excretion of cyclic gmp, which is known to serve as second messenger for the vascular effects of no, synthesized by the constitutive form of no synthase, affecting blood vessels, plate]et aggregation and neurotransmission, in h urines of male sprague daw]ey rats at day after induction of adjuvant arthritis we measured nitrate excretion by gas chromatography and cyclic gmp by radioimmunoassay. for contro] we determined the same parameters in h urines of non-arthritic rats of the same strain and age, we found a significant (p < , two-tailed, unpaired t-test), more than -fo]d increase of urinary nitrate excretion in arthritic rats (mean ± sd pmo]/mmol creatinine) as compared to non arthritic rats ( _+ izmot/mmo] creatinine). urinary cyclic gmp excretion was slightly, but not significant]y lower in arthritic rats ( ± nmol/mmol creatinine) than in controls ( ± nmo]/mmo] creatinine).there were no major differences in food or water intake which cou]d account for these results. the increased urinary nitrate excretion accompanied by normal cyclic gmp excretion suggests that no production by the inducible form of no synthase is enhanced in rats with adjuvant arthritis institute of c]inica] pharmacology~ hannover medical school, d- hannover, germany and *research center gr@nentha] gmbh, zieg]erstr , d- aachen, germany background: pge has been shown to be efficacious in the treatment of critical leg ischemia. despite of an almost complete first pass metabolism in the lung the clinical effects of intraarterial and intravenous pge do not differ significantly. in addition, it is not fully understood which of the various pharmacological actions of pge is the main factor; by most authors, however, it is thought to be the increase of cutaneous and muscular blood flow. by means of [ - ]-h -pet, we studied muscular blood flow (mbf) of the leg in patients with peripheral arterial disease comparing intraarterial and intravenous pge . patients and methods: patients ( f, m; mean age y) with pad were studied, ( atherosclerosis, thromboangiitis obliterans). at the first day, pg pge were infused intraarterially within minutes; pet scanning of the lower leg was performed at minutes , und . at the following day, pg pge were infused intravenously within hours; pet scanning was performed at minutes , , and . results: in the infused leg the increase of mbf caused by intraarterial pge averaged + % at minute and _ % at minute ; in the not infused leg there was no effect. the increase rate in the infused leg was highly variable but did not correlate with sex, age, disease or clinical outcome. for intravenous pge the change of mbf at any time averaged almost %. conclusion: unlike intraarterial pge , intravenous pge does not increase the muscular blood flow of the leg. a comparable clinical effect provided, increase of muscular blood flow may not be considered the main way of action of pge in critical leg ischemia. eslrogen(er) and progesterone(pr) receptor status as well as lymph node involvement are important factors in predicting prognosis and sensitivity to hormone and chemotherapy in patients with breast cancer. prognostic relevance of ps -protein, egfr and cathepsin d is currently under debate. especially ps and egfr expression appears to provide additional information regarding the responsiveness of the tumour tissue to tamoxifen. the aim of the present study was to investigate the relationships between these parameters and established prognostic factors in breast cancer. in a prospective study ps and cathepsin d were assayed immunoradiometricauy in the tumour cytosol of patients, egfr was measured by elisa. relating the level of these factors to the lymph node involvement, menopausal status as well as turnout size, no significant association could be established. jn our findings er and pr are significantly correlated with the expression of ps but none is correlated with the cathepsin d status. egfr was shown to be inversely correlated with the content of er. a significant association between cathepsin d and ps could be established in patients with early recurrence. at a median follow-up of - months, recurrence was more common in patients with tumours having negative status for ps , independent of receptor status. in conclusion, because of the relative independence on the er and pr status and other prognostic factors and the influence on the recurrence behaviour, demonslrated here, and their role in promoting tumour dissemination and changing hormone therapy sensitivity, all three factors represent markers of prognostic relevance.deparlancnts of clinical pharmacology l, nuclear medicine and surgery ,pharmacoeconomic studies, conducted either separately from or together with clinical trials are increasing in both number and meaning. in a period of limited health care budgets, political and medical decision makers alike run the risk of accepting the results of such studies without critical reflection. careful evaluation of those studies by state-of-the-art methods is one way out of the trap. another could be to refer to ethical considerations. the problem in this context is, that the discussion concerning ethical aspects of pharmacoeconomic research, at least in europe, is just in its beginning. therefore, no widely accepted standards are available. but they are essential to answer four main questions: . who should perfom a pharmacoeconomic study? . which objectives should be considered? . what kind of study should be performed (e. g. cost-effectiveness, cost-utility, cost-benefit analysis)? . which consequences will be drawn from the results?based on the case study-orientated "moral cost-benefit model" (r. wilson, sci. tech. human values : - , ) , a three-step decision and evaluation model is proposed to handle bioethical problems in pharmacoeconomic studies: . moral risk analysis . moral risk assessment . moral risk management. possible practical consequences for decision making in research policy, study design and assessment of results are discussed. hirudin is the most potent known natural inhibitor of thrombin and is presently gaining popularity as an anticoagulant since recombinant forms have become available. the aim of the present study was to compare platelet aggregation, sensitivity to prostaglandin e (pge ) and thromboxane a (txa ) release in r-hirudinized and heparinized blood. platelet aggregation was measured turbidimetrically using a dual channel aggregometer (labor, germany) in blood samples of healthy volunteers anticoagulated with r-hirndin w (behring) and hepatin ( gg/mi blood each). aggregation was induced by arachidonic acid (aa; . , . and . ram) and adp ( . lam). pge in concentrations , and ng/ml was used. plasma txb content was measured by gas chromatography/mass spectrometry. this study showed a significantly lower a.a-induced platelet aggregation in r-hirudinized plasma. three minutes after the aggregation induction by . mm aa the plasma txb concentration was ng/ml in blood anticoagulated with rhimdin and . ng/ml in heparin-anticoagulated blood. the extent of the adp-induced aggregation was nearly the same in rhimdinized and heparinized plasma. platelet sensitivity to pge was significantly higher in r-hirudinized blood. thus, aa-induced platelet aggregation is significantly lower and sensitivity to pgei higher in r-himdin-anticoagulated blood in comparison with beparin-anticoagulated blood.university of tartu, puusepa str. , tartu ee , estonia anaemia has been reported in renal transplant (ntx) recipients treated with azathioprine (aza) and angiotensin converting enzyme-inhibitors (ace-i). an abnormal aza metabolism with increased -thioguanine nucleotide (tgn) levels in erythrocytes is a possible cause of severe megaloblastic anaemia (lennard et al, br j clin pharmaco ). methods: ntx patients receiving aza ( , _+ , mg/kg/d), prednisolone ( , + , mg/kg/d) and enalapril (ena) ( , + , mg/kg/d) for more than months were studied prospectively. blood samples were taken before and h after administration of aza on visits during ena treatment and weeks after ena had been replaced by other antihypertensives (x). tgn in erythrocytes, -mercaptopurin (mp) and -thiouric acid (tua) in h post dose plasma (p.) und h urine (u.) samples were analyzed by hplc using a mercurial cellulose resin for selective absorption of the thiol compounds. pharmacodynamic variables were hemoglobin (hb), erythropoietin (epo) and creatinine clearance (ci ace~,lcholine plays an important role in regulating various functions in the airway's. in human lung less is known about regional differences in cholinergic innervation and about receptor-mediated r%m.flation of acetylcholine release. in the present study the tissue content of endogenous acetylcholine and the release of newly-synthesized [~h]acetylcholine were measured in human lung human tissue was obtained at thoracotomy from patients with lung cancer moreover, in isolated rat tracheae with intact extrinsic vagal innervation possible effects of g__-adrenoceptor agonists on evoked ph]acctylcholine release were studied. endogenous acetylcholine was measured by hplc with ec-detection; evoked ph]acetylcholme release was measured after a preceding incubation of the tissue with [~h]choline. huma n large (main bronchi) and small (subsegmental bronchi) airways contained similar amounts of acetylcholine ( pmol/ mg), whereas significantly less acetylcholine was found in lung parenchym ( pmol/ mg). release of [ h]acetylcholine ,,,,'as evoked in human bronchi by transmural electrical stimulation (four s trains at hz). oxotremorine, an agonist at muscarine receptors, inhibited evoked [~hiacetylcholine release indicating the existence of neuronal inhibitor ' receptors on pulmona~ parasympathetic neurones. scopolamine shifted the oxotremorine curve to the right suggesting a competitive interaction (pa value: : slope &the schild plot not different from unity) however, a rather sluggish schdd plot was obtained for pirenzepine. scopolamine but not pirenzepine enhanced evoked [ h]acetylcholine release. the present experiments indicate a dense cholinergic innervation in human bronchi; release of aceu, lcholine appears to be controlled by facilitatory and inhibitou' nmscarinc receptors. in isolated, mucosa-intact rat tracheae isoprenaline ( nm) inhibited [~h]acetylcholine release evoked by preganglionic nerve stimulation isoprenaline was ineffective in mucosa-denuded tracheae or in the presence of indomethacin thus, adrenoceptor agonists appear to inhibit acetylcholine release in the airways by the liberation of inhibitoiy prostanoids from the mucosa. the occurrence of the non-enzymatic reactions between glucose and structural proteins is well known (vlassara h et al. ( ) lab invest : - ) . the reaction between proteins and fructose (i.e. fmctation), however, can also occur. like glucose-protein adducts the fructose analognes are able to form so-called advanced glycation endproducts (age). the inhibition of early and advanced products of fmctation may be ilnportant for the prevention of diabetic late complications (mcpherson jd et al. ( ) biochemistry : - . we investigated the in vitro fmctation of human serum albumin (hsa) and its inhibition by selected drugs. hsa was fmctated by incubation with mmol/ fructose in . i mol/l phosphate buffer, ph= . .,at ° c for days. the rate of fmctation was measured by the following methods: -a colorimetric method based on deglycatien of glycated, proteins by hydrazine (kobayashi k et ai.( ) bioi pharm bull : - ), -affinity chromatography with aminophenyl-boronate-agarose, -fluorescence measurement for the delermination of age we used aminoguanidine, pcnicillamine, captopril and alpha-lipoic acid( mmol/ ) to study the inhibition of hsa fmctation. after three weeks incubation the formation of early glycation products was inhibited by aminogalanidine ( %) and captopril ( %) whereas penicillamine and alpha-lipoic acid showed minimal inhibition. aminognanidine inhibited the formation of age by %, penicillamine by %, alpha-lipoic acid by % and captopril by %. these results may suggest a potential use of the investigated drags in the prevention of the formation of protein-fructose addncts. key: cord- - c mak authors: parker, philip d.; jerrim, john; anders, jake title: what effect did the global financial crisis have upon youth wellbeing? evidence from four australian cohorts date: - - journal: dev psychol doi: . /dev sha: doc_id: cord_uid: c mak recent research has suggested significant negative effects of the global financial crisis (gfc) on mental health and wellbeing. in this article, the authors suggest that the developmental period of late adolescence may be at particular risk of economic downturns. harmonizing longitudinal cohorts of australian youth (n = , ), we estimate the impact of the gfc on general and domain specific measures of wellbeing at age and . significant differences in wellbeing in most life domains were found, suggesting that wellbeing is susceptible to economic shocks. given that the gfc in australia was relatively mild, the finding of clear negative effects across ages is of international concern. the influence of macrolevel events and conditions on psychological variables is of central interest within the social sciences (fletcher, ) . in particular, there is growing interest in the influence of shifts in local and global economic conditions on personality (bianchi, ) , mental illness (sargent-cox, butterworth, & anstey, ) , and wellbeing (di tella, macculloch, & oswald, ; yang, ) . estimating the impact of such factors, however, has proven to be difficult. this is due to the use of cross-sectional designs that make it difficult to separate the influence of development (the degree to which there are changes in wellbeing that correspond to particular developmental stage) and period (cultural and economic conditions or events unique to a particular historical period ; fletcher, ; schoon, ; yang, ) . in this article, we took a multidisciplinary approach, using literature and methodological approaches from psychology, sociology, and economics to estimate the impact of the global financial crisis (gfc) on the multidomain wellbeing of australian youth. to do this we used four cohorts of the longitudinal study of australian youth (lsay) with wellbeing measured in domains. unlike previous research, we used longitudinal data from multiple birth-cohorts to estimate the effects of a unique and pervasive economic crisis. we also used wellbeing measured across most major life domains. this is in contrast to most research to date, which has focused on a single general domain. furthermore, we leveraged the unique opportunities afforded by the lsay to estimate these effects at two distinct ages ( and years of age). to do this we used statistical models, rarely used in previous research, to provide counterfactual estimates of the effect of the gfc (morgan & winship, ) . there has been growing interest in recent years of the effects of macrocontext (national or international conditions or events) on individual factors in psychology (fletcher, ) . however, the idea that dramatic changes in the global environment can have meaningful influence on individual psychology is not a new one. c. wright mills ( mills ( / ) laid the groundwork for this area of inquiry, stating "neither the life of an individual nor the history of a society can be understood without understanding both". in a pioneering study, glen elder's ( ) research on children growing up in the great depression prompted consideration of not only the influence of macrolevel conditions on progress and frustration in development, but also how such effects filter through to young people via links with local institutions, social ties, and family networks. elder ( ) noted effects of the great depression on social wellbeing, psychological health, and hope and optimism for the future; particularly among those who were younger and thus less cognitively developed. in addition, elder drew attention to the effect of economic downturns on populations of youth as a whole, in addition to those suffering abject and persistent deprivation (see elder & caspi, ) . thus, one needs to consider the effects of economic downturns on factors such as wellbeing across whole cohorts (jahoda, ) . recent research by di tella et al. ( ) found that a country's economic position has significant effects on wellbeing. indeed, di tella et al. indicated that rising unemployment that results from economic hardship has a critical effect not only on those who lose their job, but for the population as a whole. these effects were observed across a range of macroeconomic events including recessions, changes in gdp, inflation, and the relative generosity of the welfare system. schoon ( ) considered cohorts of british people born in , thus growing up in a golden age of economic stability and prosperity, and those born in , thus growing up in more economic vulnerable times. schoon reported that growing up in times of economic prosperity seems to be a protective factor against psychological distress and promotes wellbeing. conger, rueter, and conger ( ) , studying the effects of the severe economic downturn in the rural midwest of the united states found that economic distress affected young people's wellbeing via its impact upon parents' mood and parenting behavior. finally, forkel and silbereisen ( ) considered the effect of the reunification of germany on development. using a family stress model framework, they found that economic uncertainty had an effect on child wellbeing via parents depressed mood in the west, but less so in the more significantly altered society in the east. in relation to the gfc, a review by clark and heath ( ) found dips in trends in happiness and social wellbeing, including trust and experiences of prosocial behavior in the united kingdom and the united states. in australia, sargent-cox et al. ( ) focused on the influence of the gfc on australian seniors, suggesting that this group was at particular risk due to vulnerability in retirement savings as well as fear spread by the australian media. they also found significant increases in depression and anxiety. likewise the recent unicef innocenti report (fanjul, ) found that in of the oecd and non-oecd eu countries wellbeing decreased and experience of everyday stress increased from to . they attributed this impact as likely due to the gfc. taken together, the literature to date suggests three important considerations. first, changes in macrocontexts, and economic conditions in particular, can have meaningful impacts on wellbeing. second, these may have an impact upon everyone (i.e., those directly and indirectly affected). third, consideration of general wellbeing should be supplemented by consideration of domain specific measures within multiple life domains, given findings that social domains of life appear to be vulnerable to economic conditions. although elder ( ) focused on the effect of the great depression on youth, recent research has tended to focus on the elderly as a group of particular vulnerability. although the elderly were particularly exposed to the gfc (e.g., sargent-cox et al., ) , there are important reasons to also consider the develop-mental period ranging from the transition from high-school to the mid- s. here we explore the biological, social, and economic reasons for this. steinberg ( steinberg ( , has highlighted convincing biological, behavioral, and neurological evidence to extend the definition of adolescence up to mid- 's. steinberg's ( ) argument is both social, noting that youth are now becoming financially and socially independent at later ages, and biological, with evidence of continued and significant brain plasticity well into the mid- s. steinberg noted that this malleability means that young people are particularly vulnerable to toxic contexts that can lead to lifelong negative impacts. cummins ( ) likewise noted that wellbeing is particularly volatile during adolescence due to heightened biosocial change. this is consistent with the work of elder ( ) who noted that age was negatively related with impact of the great depression, hypothesizing that ongoing cognitive development meant that hardship had a more severe and long lasting impact. socially, not only is the post-high-school period defined by identity formation and uncertainty in social and occupational roles (arnett, ) but it is a period in which developmental transitions are both plentiful and of considerable importance to long-term status attainment (guo, parker, marsh, morin, ; parker, lüdtke, trautwein, & roberts, ; parker, thoemmes, duinveld, & salmela-aro, ) . the life span theory of control indicates that those making the transition from formal schooling to tertiary education or the labor market are particularly at risk of contextual events and influences (heckhausen, wrosch, & schulz, ; heckhausen & schulz, ; see also dietrich, parker, & salmela-aro, ) . such a period is defined by the convergence of developmental tasks from multiple life domains (educational, occupational, social, family, romantic, and values) and, as such, is one of the most critical developmental periods (zarrett & eccles, ) . from the perspective of life span theory of control (heckhausen & schulz, ) the particular danger of macroeconomic events, like the gfc, would be the potential to knock youth off a typical developmental track; delaying transitions, interfering with increasing independence from parents, and extending periods of career and educational uncertainty. for example, research on transition delays provides evidence that even a relatively short delay can have ongoing consequences for status attainment well into adulthood (see haase, heckhausen, & köller, ; heckhausen & tomasik, ; parker et al., ) . economically, not only is unemployment particularly high during this developmental period, but in australia, the united kingdom, and the united states the jump in unemployment levels during the gfc for those aged to was notably larger than for the working population as a whole; youth unemployment in australia jumped from . % to . %, whereas overall unemployment grew from % to almost %, in the period of to (our calculations are based on australian bureau of statistics data). as noted above, both unemployment and the risk of unemployment has a particularly detrimental effect on wellbeing (clark, georgellis, & sanfey, ) . the risk of unemployment can cause young people to make different choices about their educational and occupational plans than they otherwise would, which can put them at a distinct disadvantage when competing with their near age peers who entered this developmental period at a more economically advantageous time (see kahn, ) . finally, at the posthigh-school transition young people are increasing independence via entry into the labor market or tertiary education, yet they also remain strongly connected to parents (parker, lüdtke et al., ) . as such, the wellbeing of young people may suffer from both their own exposure to economic downturns but also that of their parents as suggested from a family stress model perspective (conger et al., ) . psychologists, economists, and sociologists have all been interested in the influence of both micro-and macrolevel conditions on wellbeing. a common thread across much of this research is general or aggregated wellbeing (e.g., life satisfaction). there is, in contrast, relatively little attention given to how such events might differentially affect multiple life domains. part of the reason is that it is difficult to determine how many and which life domains to cover. as cummins ( ) noted, if every human action is considered a life domain, true multidimensional measurement becomes impossible. derived from the work of cummins and colleagues, however, youth surveys of the australian population have covered between to life domains focusing on achievement, social life, community engagement, perspectives on the future, and living stan-dards. these domains are derived from empirical research on what most participants consider to be important and have been used over long periods of time, across countries, and age groups. this provides strong evidence of validity and utility of multiple dimensional measures of wellbeing in these areas (see cummins, ; tomyn, fuller tyszkiewicz, & cummins, , for a review). as cummins ( ) noted, there is value in a parsimonious multidomain approach, and the domains that are used here capture the domains that are relevant for the majority of young people (tomyn et al., ) . thus, taking a multidimensional perspective, we consider the degree to which there are differential impacts of events like the gfc on wellbeing measured in different domains. as noted above, there is some evidence to suggest that social wellbeing and optimism for the future is particularly at risk during economic hard times (clark & heath, ; elder, ; lau et al., ) , yet research in this area has been relatively limited in the number of domains explored. empirical research suggests economic conditions can lead to significant changes in wellbeing. this literature, however, has note. age is the average age at the first wave in the analysis. social class based on the erickson-goldthorpe-portocarero schema. three letter codes used for australian states. all figures use population weights. tended to use cross-sectional studies without the ability to follow individuals over time. here we make use of the unique opportunities afforded by the lsay datasets, which follow young people from four birth cohorts for up to years. the nature of the lsay data, four birth cohorts measured roughly three years apart, allows us to compare the influence of the gfc at two distinct ages in the post high-school transition period (i.e., age and ). as can be seen in table , the -year-old age group captures much of the movement of young people from high-school to tertiary education or the labor market. at age , young people appear to have mostly made this transition. the comparison of these age groups is opportunistic (i.e., due to the possibilities afforded by the data), however, and thus we have little evidence on which to assume the gfc would have differential effects. on this basis, we put forward the following hypotheses: hypothesis : the gfc will have a negative impact upon young people's wellbeing across the major domains of importance to late adolescents. we expect the influence of the gfc to differ by life domain, with particular impact on domains related to social life and long-term prospects. hypothesis : as existing research base is not yet large enough on which to make a strong hypothesis, we do not anticipate that there will be differences in the size of the effect of the gfc at age compared to . four cohorts of the lsay database were used. two of those cohorts did not go through the gfc during the time period covered in the study: birth cohorts (n ϭ , ; ages covered - ) and (n ϭ ; ages covered - ). two cohorts did experience the gfc during the study: birth cohorts at age (n ϭ , ; ages covered - ) and at age (n ϭ , ; ages covered - ). the cohorts are named after the modal birth year. the structure of the data is represented in figure . the and cohorts reflect representative samples of australian year nine students with wellbeing data collected years later. the and cohorts represent longitudinal extensions of the programme for international student assessment (pisa), a representative sample of -year-olds where wellbeing data was collected a year later. harmonization was based on modal grade in school rather than age in years. as a result there is a difference of several months in the average age of the cohorts for the waves of interest with the average age gradually increasing from to cohorts. this may be due in part to differences in how data was collected, but may also reflect a growing preference for later school intake ages by parents (see edwards, taylor, & fiorini, ) . population weighted demographics for each cohort can be found in table . all cohorts used a two-stage sampling procedure. the primary sampling unit was schools, selected with probability proportional to size. a random sample of students was then selected from within each school. weights are provided that aim to account for (a) particular design effect including the disproportionate sampling of schools and (b) participant attrition (marks & long, ) . thus the sample weights aim to provide unbiased estimates of the population consistently across the waves of the study. wellbeing. wellbeing was assessed using a measure similar to the personal wellbeing index (pwi) originally developed by cummins and colleagues (e.g., cummins, eckersley, pallant, van vugt, & misajon, ) . versions of this measure have been used in a number of large-scale panel studies in australia and beyond, including in all lsay cohorts. as such it provides a critical insight into historical trends in wellbeing of australian youth. there are domains covered by this instrument. two additional domains relating to the economy and the way in which the country is being run were excluded due to not being present at critical waves of the study. all variables begin with the stem "how happy are you with [domain]" (see below for suffixes), with response scales varying from (very happy) to (very unhappy). to aid interpretation, these answer points were reverse scored such that higher scores reflected greater happiness. an additional response point was included representing "can't say/don't know." this choice was selected by less than % of the sample on average and never more than % for any question in any wave. this response was coded as missing for the purposes of the current study. abbreviations will be used for the wellbeing variables (exact item suffix in brackets) as follows: general (your life as a whole), living (your standard of living), home (your life at home), future (your future prospects), career (your career prospects), work (the work you do, at study, at home or in a job), money (the money you get each week), leisure (what you do in your spare time), location (where you live), social (your social life), people (how you get on with people in general), and independence (your independence; being able to do what you want). gfc. the gfc is generally considered to have begun during . however, the impact on australia and the individuals in the study likely came later. sargent-cox et al. ( ) made the case that the impact of the gfc on australians, and particularly the psychological impact, should be dated to . we thus consider the gfc to have occurred when participants were aged in the cohort and for the cohort. marking the gfc at is both consistent with previous research, captures both the dramatic jump in unemployment levels that centered on this period and the zenith of media reporting on the gfc where there was a particular environment of heightened "panic, anxiety, and insecurity" (sargent-cox et al., , p. ). age-period-cohort effects. a long running concern in developmental psychology has been how to disentangle the effects of age, period, and cohort (see baltes & nesselroade, ; schaie & strother, ) . age effects are concerned with how old an individual is, cohort effects are concerned with the shared experiences of those who grow up in a similar historical context, whereas period effects are concerned with the impact of particular events that occur at a given time in history (see schoon, ; yang, ) . it is these period effects, and in particular changes in wellbeing that occurred after that are the focus of the current research. such research is limited by the requirement of having multiple cohorts of data that cover at least part of the life span. even when such data are available, there are concerns about identifying such effects given they are confounded (e.g., age ϭ period-cohort). to account for this we consider age as fixed (e.g., we only ever compare -year-olds to other -year-olds). second, we aim to minimize the influence of cohort by making statistical comparisons between cohorts who were born closest in time thus ensuring that they share much of the same historical context (see figure ). thus, when considering the influence of the gfc at age , we compare the cohort (as the exposed group) to the earlier cohort (as the nonexposed group). when considering the effect of the gfc at age , we compare only the cohort (as the exposed group) to the earlier cohort (as the nonexposed group; see figure ). counterfactual reasoning. in addition to concerns relating to isolating period effects, we were also concerned with providing estimates of the effect of the gfc that were as close to causal as the data would allow. to do this, we aimed to find counterfactual conditions that serve as an indication of what would have occurred to a variable of interest had a given event not occurred (morgan & winship, ) . put simply, in the case of the current research, we ask the question "what if the gfc never happened?" in the current research a birth cohort that experienced the gfc at a particular age serve as the exposed group (i.e., experienced the gfc at age or ) and the closest earlier cohort at the same age serves as the nonexposed group (i.e., did not experience the gfc at age or ). to increase our confidence that the control group acts as a sufficient counterfactual for the treatment group we used two approaches common in sociology and economics; namely a matching and a difference-in-differences (did) technique. propensity score matching. matching aims to find strategic subsamples of individuals in the exposed and nonexposed groups that either match participants across groups exactly on a small number of critical confounding variables, match approximately on a large number of confounding variables, or some combination of the two (morgan & winship, ) . in the current research we used a mixture of exact and approximate matching via a propensity score matching (psm) approach. here participants in the exposed and nonexposed groups were matched exactly on exogenous demographic variables (gender, state of residence, social class [erickson-goldthorpe-portocarero schema; ericson, goldthorpe, & portocarero, ] , and indigenous status) and postschool pathway variables (number of years of high-school completed, labor market status [employed, unemployed, not in labor market], and tertiary education status [enrolled, completed, dropped out, not relevant] measured at age for the -year-old comparison and for the -year-old comparison). participants were also propensity matched on age in days and all wellbeing variables up to the year prior to the gfc. the aim of psm is to create samples of exposed and nonexposed individuals who are similar (or balanced) on a wide range of potentially biasing covariates. initial analysis consisted of modeling the relationship between the covariates and presence in either the exposed or nonexposed groups. we used logistic regression to estimate the propensity score and, based on these scores, we used nearest neighbor matching with matches allowed when participants were within . of the standard deviation of the logit of the propensity score. as noted above, exact matching was used for several demographic, educational, and occupational status variables. one-to-one matching was used, without replacement (see stuart, ; thoemmes & kim, , for a review). propensity score estimation and matching were done with the matchit package in r (ho, imai, king, & stuart, ) and regression with clustered standard errors for school membership was conducted with the survey package (lumley, ) . hypotheses were tested using equation . here ␥ represented the effect of the wellbeing variable pre_y before the gfc (age for the year-old comparison and for the -year-old comparison), ␤ is the parameter of interest-the difference in y between the gfc exposed cohort (coded ) and control cohort (coded ). subscript j was the school that individual i was in at wave . importantly, psm allowed us to match participants on both grade in school and age in days, thus ensuring participants were similar in both biological and social developmental stage at the comparison point. did. as a robustness check, and to provide population estimates, we also adapted the logic of did to estimate the gfc influence across cohorts. a did approach estimates trends in a variable of interest in an exposed and nonexposed group. it assumes that both trends are essentially parallel and would remain so had an event of interest (e.g., the gfc) not occurred. a did approach estimates the shift from parallel trends at the exposure point (see figure ). the assumption is that this discontinuity in parallel trends provides an estimate of the effect of exposure to a given event (angrist & pischke, , provide a number of applied examples). typically this model is used to explore the potential effect of a treatment in two or more contemporaneous groups; one in which the treatment is present and one where it is not. for the gfc, however, young people either went through the historical period at a particular developmental stage or they did not. the multiple cohorts of lsay, however, allow us to extend the logic of the did approach to noncontemporaneous groups, given that the same measures were collected using the same survey collection procedure on participants of approximately the same age. as noted above, we thus make the assumption that cohort effects are negligible. following, angrist and pischke ( ) we fitted two sets of models. the first was a basic did model specified in equation : where ␤ is the first order estimate of cohort on the wellbeing variable y, ␥ is the first order estimate of the gfc and ␦ is the parameter of interest (i.e., whether there was a shift in trend for the gfc exposed cohort at the time of the gfc; see figure ). the subscripts t refer to individual observations at a given time wave, i refers to individual participants under which observations were nested, and j relates to the primary sampling unit which, in our case, was the school the individuals were in at the first wave of data collection. exploiting the fact that we had more than two waves of data, we also tested a model in which the assumption of common trends was partially relaxed. this second model was estimated using equation : in equation , and are included to relax the assumption of common trends, and allow for cohort specific linear trends. all other terms remain consistent with equation . missing data and survey design. as noted above the lsay database has a complex design. to account for this a series of weights were applied to ensure estimates were representative of the australian population. finally, even with the use of attrition weights there remains missing data holes where participants have failed to complete a particular item within a given wave. to account for this various complexities we (a) provide clustered standard errors for individual observations nested within participants who were themselves nested within schools; (b) apply sample and attrition weights; and (c) multiply impute wave specific missing data. imputation was achieved using a bootstrapped expectation maximization approach (honaker, king, & blackwell, ) . given that nonattrition related missing data was generally small (Ͻ %), five imputations was considered sufficient. the means and confidence intervals for each cohort were plotted in the following manner (see figure , e.g., plot). first, all cohorts were plotted on a single graph with solid lines representing observations that occurred before the expected impact of the gfc (i.e., - ). second, two close-up plots for each wellbeing domain were created, highlighting particular comparisons of interest. these close-up plots also provide insights into the comparisons of interest for the psm and did models. the first close-up compares the and cohorts at ages to . the second compares the and cohorts at ages to . given space restrictions, we provide an example plot for general wellbeing only (see figure ). however, all graphs, means and % confidence intervals, and an interactive graph are available from the paper website at https://pdparker.github.io/gfcweb/. microdata is available by application from the australian data archive (https://www.ada.edu .au/). a visual inspection of all the graphs suggested that the and cohorts had similar (or slightly higher) levels of wellbeing across domains than the earlier cohorts before the gfc. however, a relatively large gap emerges between the earlier and later cohorts, starting at age for the cohort and age for the cohort. thus, results were consistent with the hypothesis that the gfc had a negative impact on wellbeing. interestingly, there was some evidence of recovery in (ages for the cohort and for the cohort), where in many cases the wellbeing levels returned to those of the other cohorts before again diverging and growing progressively larger. finally, the first wave of the cohort was well below trend and may represent an outlier for consideration in later models. the close-up graphs provide strong evidence for the negative impact of the gfc with most of the relevant contrasts displaying overlapping confidence intervals in the years prior to the gfc before diverging. it was on this basis that we explored the hypotheses using psm and did models. two sets of psm models were estimated; one comparing the with the cohort at age and one comparing the to the cohort at age . matching was done exactly on gender, social class, state of residence, and indigenous status; as well as labor market status, university status, and number of years of high-school completed. propensity matching was done on age in days and all pre-gfc wellbeing variables. negative effects indicate that the gfc exposed cohort was lower on wellbeing than the comparison cohort (see table for results). matching suggested that the and cohorts were very similar with only % of the , assessed terms indicating a prematching difference of greater . of a standard deviation. after matching no term displayed a difference of greater than . standard deviation units. prematching the sample size was , . after matching this was only reduced to a balanced sample of , . table displays the differences in wellbeing at age for the and cohort controlling for pre-gfc levels. unsurprisingly, given the similarity between the two groups, matched and unmatched results were similar. in particular, the only factor that gfc exposure did not predict was satisfaction with money. furthermore, nine of the wellbeing domains had cohen's d differences greater than . . in order of effect-sizes these were social life, independence, general, living standards, career prospects, leisure time, future prospects, and home life. matching for the and cohorts revealed a greater prematching difference with % of the , assessed terms displaying a cohen's d differences of . or greater and % of terms greater than . . after matching, no term had a cohen's d difference greater than . . this matching resulted in a decline in sample size from , cases to a balanced sample of , . matching did result in a decline in the size of effects and the number that were statistically significant. however, eight out of wellbeing factors remained significant, and of those only three had effects sizes greater than . ; namely career prospects, home life, and people in general (in order of effect size). importantly, however, these results tended to be smaller than the comparison at age but generally not significantly so. indeed, z tests suggested only satisfaction with living standards, independence, and social life had significantly larger effects at age that . as a robustness check to the psm results we ran a series of did models. in this case two sets of models were estimated. first, we compared the cohort (who went through the gfc at age ) to the cohort. second, we compared the cohort (who experienced the gfc at age ) with the cohort. negative did estimates represent the disadvantage of the gfc exposed cohort over the comparison cohorts in terms of wellbeing. for the did at age , we found significant results for wellbeing variables when we assumed a common trend (satisfaction with money was not significant) and all were significant when we controlled for cohort specific trends. of these, nine had effect sizes larger that . . in order of effect size these were satisfaction with leisure time, social life, future prospects, independence, work, career prospects, home life, general, and people in general. interestingly, the gfc appeared to have a small positive effect on satisfaction with money. at age , results not controlling for trend were significant in all domains but only two domains when controlling for cohort specific trends (satisfaction with career prospects and work; see table ). as we noted above, the first time point for the cohort was considerably off trend and thus likely exerted considerable leverage on the linear trends. thus, we also estimated these models excluding the first wave. this resulted in seven out of significant results, with only career prospects having an effect size of the gfc greater than . . using z tests, the gfc had significantly larger effects for -year-olds than year-olds in terms of satisfaction with leisure time, where you live, social life, living standards, and future prospects (ordered in terms of size of difference). impact of the gfc on multidimensional wellbeing by taking advantage of the unique opportunities provided by the lsay data. we were able to overcome limitations in previous research via the use of multiple cohorts of longitudinal data to explore the influence of the gfc at two different ages in one general and domain specific measures of wellbeing. exploration of graphed means suggested significant divergence in wellbeing for the gfc cohorts in year to . of most concern, while there was evidence of recovery in in both the and cohorts, this gap reopened and grew larger. using the logic of psm and did models, these findings were also examined statistically. there was consistent evidence of a negative impact of the gfc in most domains at age with the exception of satisfaction with money; which was generally not significant and occasionally positive. the effect at age was more ambiguous, though generally suggested significant effects for over half the wellbeing domains. taken together, these results suggest significant though small differences at age than at age for wellbeing in at least three life domains. the current research across multiple models, using multiple comparisons, and across multiple domains suggested that the gfc did have a significant negative impact on the wellbeing of young people in australia. such a result is important, as the gfc had a much milder influence in australia than it did elsewhere. indeed, although youth unemployment jumped from . % to . % during the gfc in australia, it rose from under % to almost % in the united states (our calculation) during the same period. thus, although research in other countries is needed, it is likely that the results in countries such as the united states and the united kingdom, let alone greece, italy, ireland, and spain, was considerable. importantly, given our focus on the population as a whole, unmoderated by individual exposure, the effect sizes of above . standard deviation units, and often above . , were concerning given effects of unemployment of . (lucas, clark, georgellis, & diener, ) . this suggests that for particularly vulnerable groups, for example those who experienced the largest relative loss in status or income or became unemployed, the findings may have been considerably more dramatic. an interesting effect present in the trend plots for wellbeing was a drop in wellbeing in , consistent with our hypothesis, before a recovering during and then a step decline again from to . although we did not provide a hypothesis for this pattern, exploration of the unemployment rates provides a potential explanation (di tella et al. ( ) . in particular the pattern of decline and recovery is consistent across both wellbeing and unemployment. namely, unemployment rose sharply from to before recovering just as rapidly. from unemployment then increased steadily to levels worse than those at the initial impact of the gfc (see figure ). although it would be naïve to suggest that wellbeing naturally follows unemployment rates, it is fair to suggest that they do provide a proxy for general economic conditions in a country over a given time period. relatively little research has considered the differential effects of macro or micro contextual events on multiple domains of wellbeing. when such a comparison is made it is often done in relatively few domains. our research was one of the few to comprehensively test the impact of events like the gfc across a wide spectrum of youth's lives. previous research has suggested that social domains are particularly at risk. there was some evidence that this was the case with effects on satisfaction with social life, at age , and getting along with people in general at age being particularly affected. for both age groups, social domains, general life satisfaction, and satisfaction with career or future prospects appeared to be most strongly affected. such results are consistent with the developmental challenges these two groups face. in particular, these transition periods are primarily focused on the developmental tasks of developing new friendship networks and renegotiating existing relationships (tanner, ) . likewise, making appropriate transitions into higher education or the labor market are crucial during these age periods (dietrich et al., ) . importantly, these findings are also consistent with previous research on the gfc and the great depression where wellbeing in social domains and optimism for the future were particularly at risk (elder, ; clark & heath, ) . importantly, the findings suggest that the gfc had a significant impact across most life domains indicating that this event touched most aspects of young people's lives. importantly, the finding of small, nonsignificant results of the gfc on satisfaction with money suggests that results across domains were not merely a poisoned well effect (i.e., negative effects from one domain flooding through to all other domains). as such these findings indicate that economic hard times have a pervasive negative effect on the wellbeing of young people. although the type of domain effects across -and -year-olds were similar, a consistent finding was that effects were routinely smaller in the older age group. this difference, however, was only consistently significant in three cases; social life, independence, and living standards. these particular domains may be associated table standardized did estimates comparing and cohort (age ) with the many upheavals that occur during the post high-school transition (see dietrich et al., ) . as can be inferred from table , the gfc hit -year-olds at the end of high-school and in a period where most of the sample was establishing themselves in either university or the labor market. restructuring of old relationships and forming of new friendship circles after high-school is common during this period (see tanner, ) , which may explain why satisfaction with social life was affected more for -yearolds. likewise, during this transition young people are expected to considerably increase their independence from parents (parker, lüdtke et al., ) . although not the focus of this study, it may be that the gfc meant that -year-olds had less financial independence and were thus less able to establish greater independence either within the family home or by moving out. the older year-olds transitioned from high-school some years earlier and were thus able to at least begin the developmental tasks associated with restructure old and establish new relationships and gaining independence from parents during a more prosperous period. di tella et al. ( ) suggested that a payment of $ u.s. ($ u.s. in dollars; all conversions done using williamson, ) to the population in general may be sufficient to offset the effects of an economic recession on wellbeing. they do note, however, such a payment may not be sufficient for dramatic changes to economic conditions. the australian context provides a means of exploring this hypothesis given that the government provided payments of up to $ aud ($ u.s. in dollars) to % of the working age population and % of families (hyslop, ) . although not the main focus of the current research, satisfaction with money was the one domain to be largely unaffected by the gfc, suggesting a positive effect of the payment may have occurred. however, any potential effect of this payment appeared to be constrained to this domain only. there is some tension between the degree to which macroforces represent shared or qualitatively different experiences for different sectors of the community (elder, ) . here we focused on the population as a whole. although most research in psychology does focus on average treatment effects, exploring effects within particular strata is an important line for future research. this was difficult in the current case, however, where we had no data on individual exposure to the gfc, which would likely be the strongest moderator of any gfc effect (e.g., sargent-cox et al., ) . importantly, although we used rigorous designs by borrowing from the logic of did and psm regression in our research, the extent to which they represent causal effects is dependent on the degree to which the comparison cohorts represent true counterfactual counterparts to the gfc exposed cohorts. as we noted above we make the assumption that cohort effects are negligible. although we aimed to design our models as close to ceteris paribus comparisons as possible, readers should consider the potential biasing effect of birth cohort differences. finally, it should be noted that we used single-item measures for wellbeing in each life domain. multi-item measures would have allowed for latent variable modeling and thus a control for measurement error. the current article was concerned with whether the gfc had an effect on young people's wellbeing across multiple life domains. we focused on an age group that was undergoing a large number of developmental tasks at a critical period of life that has implications across the life span (dietrich et al., ) . we found that all domains were significantly affected in at least one case, with effect sizes often above . for those who were aged during the gfc. given that we were focused on a country in which the impact of the gfc was less sever than in the european union or the united states, these effects are of international concern. as conger et al. ( ) suggested, we cannot typically predict large-scale changes like the gfc; however, a better understanding of how such events impact young people is critical for marshaling an appropriate response. mastering metrics: the path from cause to effect emerging adulthood. a theory of development from the late teens through the twenties multivariate longitudinal and cross-sectional sequences for analyzing ontogentic and generational change: a methodological note entering adulthood in a recession tempers later narcissism scarring: the psychological impact of past unemployment hard times: the divisive tool of the economic slump the role of economic pressure in the lives of parents and their adolescents: the family stress model the domains of life satisfaction: an attempt to order chaos understanding the wellbeing of children and adolescents through homeostatic theory developing a national index of subjective wellbeing: the australian unity wellbeing index phase-adequate engagement at the post-school transition the macroeconomics of happiness who gets the "gift of time" in australia? exploring delayed primary school entry children of the great depression: social change in life experience economic stress in lives: developmental perspectives intergenerational class mobility in three western european societies: england, france and sweden children of the recession: the impact of the economic crisis on child wellbeing in rich countries does entering adulthood in a recession affect narcissism? robust evidence is still needed family economic hardship and depressed mood among young adolescents from former east and west germany achievement, motivation, and educational choices: a longitudinal study of expectancy and value using a multiplicative perspective goal engagement during the school-work transition: beneficial for all, particularly for girls a life-span theory of control get an apprenticeship before school is out: how german adolescents adjust vocational aspirations when getting close to a developmental deadline a motivational theory of life-span development matchit: nonparametric preprocessing for parametric causal inference amelia ii: a program for missing data the distributional effects of the australian cash bonus payments response to the global financial crisis economic recession and mental health: some conceptual issues the long-term labor market consequences of graduating from college in a bad economy the sars (severe acute respiratory syndrome) pandemic in hong kong: effects on the subjective wellbeing of elderly and younger people unemployment alters the set point for life satisfaction complex surveys: a guide to analysis using r. hoboken weighting the year cohort sample for differential response rates and sample attrition: technical paper no. . lsay technical reports the sociological imagination counterfactuals and causal inference personality and relationship quality during the transition from high school to early adulthood achievement, agency, gender, and socioeconomic background as predictors of postschool choices: a multicontext study i wish i had (not) taken a gap-year? the psychological and attainment outcomes of different post-school pathways the global financial crisis and psychological health in a sample of australian older adults: a longitudinal study the effect of time and cohort differences on the interpretation of age changes in cognitive behavior risk and resilience: adaptations in changing times should the science of adolescent brain development inform public policy? the influence of neuroscience on us supreme court decisions about adolescents' criminal culpability age of opportunity: lessons from the new science of adolescence matching methods for causal inference: a review and a look forward recentering during emerging adulthood: a critical turning point in life span human development a systematic review of propensity score methods in the social sciences the personal wellbeing index: psychometric equivalence for adults and school children seven ways to compute the relative value of a u.s. dollar amount, to present an age-period-cohort analysis. american sociological review the passage to adulthood: challenges of late adolescence key: cord- -bqwchhwk authors: persson, carl g. a. title: plasma exudation and asthma date: journal: lung doi: . /bf sha: doc_id: cord_uid: bqwchhwk several pieces of evidence support the view that exudation of plasma into the airway wall and into the airway lumen occurs in asthma. vascular leakage of plasma results from inflammatory mediator-induced separation of endothelial cells in postcapillary venules belonging to the tracheobronchial circulation. whereas proposed mediators of asthma induce reversible leakage, several antiasthma drugs exhibit antileakage effects in animals and humans. potential consequences of plasma exudation are many. mucosal/submucosal edema might contribute to airway hyperresponsiveness. plasma exudate in the airway lumen in asthma may contribute to sloughing of epithelium, impairment of mucociliary transport, narrowing of small airways, and mucus plug formation. exuded plasma may cause airway inflammation and constriction because of its content of powerful mediators, and chemoattractant factors and plasma proteins may condition the inflammatory cells abundant in asthmatic airways to release mediators in response to stimuli that otherwise would be innocuous to the cells. it is concluded that inflammatory stimulus-induced increase in macromolecular permeability of the tracheobronchial microvasculature and mucosa may be a significant pathogenetic mechanism in asthma and that the postcapillary venular endothelium and airway epithelium that regulate leakage of plasma are important effector cells in this disease. vascular leakage of plasma is a major sign of inflammation. this factor may deserve attention in asthma, a disease that has an important inflammatory component. however, the tracheobronchial venular endothelium, which regulates airway inflammatory plasma leakage, has not been considered an important effector cell of the lung. the occurrence of plasma leakage is nevertheless supported by findings of large amounts of plasma proteins in the sputum, mucus plugs, and in specific airway lavage fluid obtained from asthmatics [ , , , , , , ] . in addition, it has been observed in experimental animals that exposure of tracheobronchial mucosa to inflammatory mediators causes a rapid movement of large plasma solutes not only into the airway wall but also into the lumen [ , , ] . the physical and inflammatory effects of the plasma exudate and its content of protein-derived mediators would have a primary role in airway defense. plasma leakage in airways may be linked with many facets of the pathophysiology of airway diseases such as asthma and rhinitis [ , ] . vascular leakage of large molecules is an active process under physiological and pharmacologic control [ , , , , , , , , ] . this leakage is generally referred to as increased vascular permeability. this review discusses mechanisms and potential consequences of increased tracheobronchial microvascular permeability. tracheal and bronchial arteries carrying systemic blood nourish the walls of the airways and their accompanying nerves and vessels. species differences and variations within species exist for the origin and distribution of these arteries. capillary and precapillary anastomosis between the bronchial and pulmonary circulation has been demonstrated and there are different views as to how far the bronchial arteries travel in peripheral airways, but terminal bronchioles and alveoli may be supplied by these vessels [ , , , , ] . the bronchial circulation normally receives about % of the cardiac output [ ] and a large fraction of bronchial blood flow may go to the airway mucosa/submucosa [ ] . the bronchial veins from the first - generations of bronchi drain into the azygous veins and then into the right heart. the remaining bronchial blood drains into pulmonary capillaries and veins and enters the left heart [ , ] . different workers have demonstrated the presence of an extensive microvasculature in the trachea and bronchi [ , , , ] (fig. ) . on either side of the bronchial muscle layer there is an abundance of capillary-venular plexuses coupled to a relatively sparse arterial supply. both in the larger and the smaller bronchi the plexuses occupy the entire surface of the submucosal layer [ ] . a rich continuous subepithelial network of microvessels would regulate clearance, and possibly distribution (from large to small airways), of inflammatory mediators and inhaled drugs [ , ] . the profusion of the microvascular network of the airways may be illustrated by isolating the lung and perfusing it only through the pulmonary artery at normal pressures. the microvasculature of airways including the trachea will immediately be quite well perfused also, and this must have been accomplished by a retrograde microvascular flow along the airways [ ] . the superficial bronchial microcirculation also has a role in the temperature and moisture conditioning of inhaled air [ , ] . this function may be of particular relevance to asthmatic subjects who may respond with plasma exudation to inhalation of cold and dry air [ , ] . a role for plasma exudation in "dry-air-induced asthma" may be hypothesized for main reasons: ( ) in inflamed airways it is vessel fluid that humidifies incoming air whereas other sources are used under normal conditions [ , ] ; ( ) effective protection against this nonpharmacologic provocation is provided by drugs such as cromoglycate and glucocorticosteroids, which may have potent antileakage effects at airway endothelial-epithelial barriers [ , ] . alterations in bronchial blood flow will affect the delivery of plasma and white cells, the perfused surface area, and the microvascular hydrostatic pressure. the flow may be highl.y increased through tissue that is affected by inflamma-tion [ ] and the hydrostatic pressure increases in venules, from which exudation takes place in local hyperemia [ ] . the hydrostatic microvascular pressure is dependent not only on the arterial and venous pressures but also on the ratio of post-to premicrovascular resistance. vasodilation usually increase this ratio [ ] and thus promotes plasma exudation. it has long been recognized that once permeability is increased changes in blood flow may determine the degree of plasma exudation [ , ] . however, pronounced synergistic effects as demonstrated in the skin between flow-increasing and permeability-increasing mediators [ ] may not occur in airway mucosa/submucosa that has a high basal perfusion. the neural, hormonal, and pharmacologic regulation of tracheobronchial blood flow has many of the general characteristics of a systemic circulation [ , , , , ] . many agents including histamine, bradykinin, acetylcholine, substance p, vip, and prostaglandins have been demonstrated to increase tracheobronchial blood flow [ , , ] . under physiological conditions fluid equilibrium is maintained by a balance between the hydrostatic pressure in the capillary bed, which tends to drive fluid out of the vascular compartment, and a counteracting force of the osmotic pressure of plasma proteins. inflammation brings about dramatic changes in the transmural colloid osmotic pressure gradient. after excluding a number of factors (changes in the blood, increased microvascular pressure, changes in the surrounding tissue, etc.), julius cohnheim [ ] , more than years ago, concluded that the inflammatory extravasation of protein-rich fluid must be due to noxious stimuli acting directly on the microvascular wall to cause a molecular change resulting in increased permeability. cohnheim reported that the inflammatory exudate is concentrated and that this is due largely to its proteinaceous nature (differing from high-pressure edema fluid that is protein-poor) [ ] . somewhat reluctantly he could then make his reasoning fit with previous publications by julius arnold, who had shown that injected dyes always passed through the vessel wall between endothelial cells [ , ] . more recent ultrastructural, pathophysiological, and pharmacologic studies of systemic microvascular beds have shown that inflammatory mediatorinduced leakage of protein-rich plasma occurs in postcapillary venules (diameter - ~m) through large gaps (up to tzm) between endothelial cells [ , , , , , , , , , ] (fig. ). in the delayed inflammatory response to mild thermal burns and some other types of injurious stimuli an inflammatory exudate may come both from capillaries and venules [ , , ] , but no mediator has yet been demonstrated to produce capillary leakage [ , ] . the target cells for inflammatory mediators have thus been identified as venular endothelial cells. plasma escapes through the mediator-induced interendothelial gaps and filters through the endothelial basement membrane, which offers little hindrance to diffusion of plasma proteins [ ] . the concentration ratios of different proteins may be similar in blood plasma and in inflammatory exudate, indicating that there is a bulk flow of proteinaceous plasma out of leaky postcapillary venules [ , , ] . contraction of venular endothelial cells has been a favored mechanism to explain mediator-induced macromolecular leakage [ ] . this possibility is supported by the presence of actomyosin [ ] and bundles of fibrils that could form an endothelial contractile machinery [ ] . as in smooth muscle, endothelial contractility may be calcium-dependent. the contraction hypothesis is attractive because, as a corollary, the pronounced ability of endothelial cells to close mediator-induced gaps could be explained as a relaxation of these cells. another view on the mediator-induced deformation of endothelial cells has been discussed by zweifach [ ] . inflammatory leaks may be produced by effects on elements interlocking endothelial cells. a weakening of these forces may change endothelial cell shape and cause increased permeability. the attachment of these cells to the basement membrane is reported to be particularly loose in collecting venules [ ] , which would facilitate deformation there. also the surface material of the endothelial cells may be involved in macromolecular permeability [ ] . at sites of inflammation there may also be changes in the configuration of the filamentous gel making up the basement membrane, and the ground substance may be transformed from a gel to a sol state (shown by rapid dispersion of an injected colloid, which otherwise forms a distinct bleb only) [ ] . zweifach [ ] emphasized that in chronic diseases venules may be particularly sensitive due to defects in the collagenous and reticular fibers of the perivascular tissue that, together with the basement membrane, provide mechanical support for the vessel. since plasma exudation in theory can be causally linked with several facets of asthma pathology, it would be of interest to examine whether such differences exist between normal and asthmatic airway microvessels. recent ultrastructural examination of biopsies by laitinen and laitinen [ ] has demonstrated that subepithelial postcapillary venules have endothelial gaps in asthmatic but not in normal airways. new mediators are continually being discovered and characterized as factors of potential importance in asthma and other inflammatory diseases [ ] . irrespective of the chemical class of the mediator and whether it is applied extra-or intravascularly, the histologic and ultrastructural characteristics of the induced acute macromolecular leakage appear identical [ , , , t , ] . however, different vascular beds may differ in their sensitivity to individual mediators. not only are pulmonary microvessels quite resistant to histamine-type mediators [ , , , ] but also some systemic beds such as microvessels of rat intestinal mucosa may be resistant [ ] . the tracheobronchial microvasculature has generally responded in a sensitive way to leakage-inducing mediators [e.g., , ] . many proposed mediators of asthma are capable of inducing both bronchoconstriction and vascular leakage but may also differ in these effects. muscarinics are potent constrictors of airway smooth muscle but are without or almost without effects on microvascular permeability to macromolecules [ ] . histamine produces leakage in cat tracheal microvessels [ ] although it may relax rather than contract cat large airways [ , ] . similarly, paf-acether is a poor constrictor of guinea pig trachea but is effective in producing plasma exudate in this tissue [ , ] . due to their effect on vascular permeability, inflammatory mediators may in part produce bronchoconstriction through smooth muscle plasma-derived peptides. the acute allergen-induced response in guinea pigs may be associated with airway edema [ ] . although edema could not be demonstrated, vascular leakage of macromolecules into the airway wall and lumen was pronounced in an acute ige-driven anaphylactic reaction in the tracheal mucosa of anaesthetized guinea pigs [ , ] . extensive deposits of fibrin that would be secondary to a vascular leak were the most striking characteristic of ige-dependent late phase reactions in human skin, whereas cellular infiltration was not a consistent finding [ ] . chemical sensitizers such as plicatic acid and isocyanates (e.g., toluenediisocyanate [tdi]) are important inducers of occupational asthma. bronchial provocation with tdi causing both immediate and late phase responses is associated with significantly increased levels of albumin in bronchoalveolar lavage fluid [ ] . tdi is also a potent inducer of very prolonged (> h) vascular and epithelial macromolecular permeability in guinea pig trachea (erjefo_lt and persson, unpublished data). airway bronchial lavage in patients with asthma due to exposure to western red cedar (plicatic acid) showed a -fold higher albumin concentration in the lavage fluid than in normal subjects [ ] . the lavage was performed - h after provocation, when symptoms had subsided. preliminary observations with allergen and chemical sensitizers are thus compatible with the possibility that plasma exudate may contribute to latephase and sustained airway reactions. leakage of macromolecules is evident within ths of seconds after application of a directly acting inflammatory agent on systemic microvascular beds (including the tracheobronchial circulation). a maximum effect of mediators such as histamine, bradykinin, tachykinins, and leukotrienes is usually established within -• rain [ , , , , , , , , , ] . the response then declines quickly and normal low permeability is restored within a few minutes up to half an hour. during some time after the development of a response the affected tissue is partly refractory to further permeability effects [ , , , ] . tachyphylaxis or refractoriness is not absolute. prolonged vascular leakiness may be due to sequential effects of different mediators or intermittent release of mediators that avoid tachyphylaxis. since bradykinin did not exhibit tachyphylaxis in human skin responses [i , ], it may participate in delayed inflammatory reactions [ ] . menkin [ ] demonstrated that a cell-free plasma exudate injected into rabbit skin produced prominent vascular leakage of macromolecules. due to their distribution and activity, plasmaderived mediators may exert positive feedback effects on the venular wall and be responsible in part for maintaining high vascular permeability. the inflammatory breakdown of blood and tissue proteins will not only produce mediators but also increase the number of molecules and, hence, increase interstitial osmotic pressure, thus promoting transudation [ ] . miles and co-workers [ , , ] demonstrated interesting biphasic as well as sustained permeability responses to bacterial toxins injected into guinea pig skin. hence many types of injury may cause an immediate phase of leakiness that often is short-lasting; after - h a late phase of increased microvascular permeability follows that is much more sustained [ ] . we have recently observed that topical application of paf-acether on guinea pig tracheal mucosa in vivo produced both an acute [ i] and a late phase vascular leakiness h after provocation [ ] , which has not as yet been seen with other mediators. sticking of white cells to endothelium and their subsequent migration across the vascular wall characterize most inflammatory processes. as with protein leakage the leukocyte-endothelium interactions occur in postcapillary venules and the diapedesis is through endothelial intercellular junctions [ , , ] . however, inflammatory extravasation of leukocytes and macromolecular solutes is induced via different mechanisms and can occur separately: white cells apparently have a protein-tight seal during migration, and mediator-induced leakage of plasma can occur without any cellular escape [ , , , ] . tissue leukocytosis may be associated with delayed responses and a causal relationship between leukocytes and leakage has been suggested [ ] . however, neutropenia may not suppress the initial or the delayed permeability response [ , , ] , and mediators such as anaphylatoxins and paf-acether, which have been proposed to act through polymorphonuctear leukocytes, may have pronounced vascular leakage effects independent of leukocytes and platelets [ , ] . although participation of leukocytes is likely, their suggested pivotal role [ ] in the development of a sustained microvascular permeability in inflammation has not been proven. the mediators may come from many sources, the stationary and migrating inflammatory cells being the most widely explored. airway epithelial ceils may produce arachidonate mediators [ ] and vascular endothelial cells may also generate several of the inflammatory mediators, including paf acether and arachidonate products [e.g., ] . neuropeptides have been proposed as mediators of vascular permeability but substance p and other tachykinins may not be as effective as inflammatory agents in human airways as they are in the guinea pig [ ] . of particular relevance for the present discussion (i.e., the sequelae of microvascular leakage) must be the preformed and dormant protein mediators circulating in the bloodstream. huber and koessler [ ] included in their review the information that the serum of patient dying of asthma "was very toxic for animals, . c.c. causing death of a guinea pig." circulating kinins [ ] and esterase activity [ ] may be elevated and kininogen decreased [ ] in asthma. ciliary dyskinetic factors have been detected in asthmatic serum [ ] as have indices for activation of complement [ ] . hence, the plasma of asthmatic subjects may be particularly noxious. the plasma proteins leaking through the venular gaps may be activated by negative surface charges, proteases, and other factors during their transvascular passage and upon arrival in inflamed tissue [ , , , , , , , , ] . both in the airway wall and lumen proteins of the kinin, complement, clotting, and fibrinolysis systems may generate a variety of inflammatory, bronchoconstrictory, and chemoattractant mediators. bradykinin, which is a powerful provocateur in asthma [ , ] , is but of a plethora of plasma-derived mediators. gerberick et al. [ ] showed that rabbit alveolar macrophages were unable to release reactive oxygen intermediates unless they were conditioned by prolonged presence of plasma proteins. exuded plasma may thus, by direct actions on a variety of target cells and by recruitment and conditioning of inflammatory cells, be an amplifying factor that escalates and sustains the inflammatory process in asthmatic airways [ ] . the result of plasma leakage is generally thought of as edema, and edema is accepted as a characteristic sign of asthmatic airways [ , , , ] . this view agrees with general descriptions of inflammation of mucosal membranes and is supported by histologic preparations that show "edema spaces" in airways obtained from patients dying of asthma [ , ] . however, many workers who have done postmortem or biopsy examinations have not reported or been able to identify edema in asthmatic airways [ , , , , , , , , , ] . changes such as enlarged bronchial glands and increased thickness of the epithelial basement membrane and smooth muscle layer have been well documented [ , , ] , whereas edematous changes have not been easy to quantify. the relative lack of data on edema may be explained in part by movement of plasma exudate into the airway lumen. this possibility is supported by the abundant occurrence of plasma proteins in asthmatic airways [ , , , , , , ] and by observations in experimental animals. inflammatory mediators such as bradykinin, histamine, leukotrienes, tachykinins, paf-acether, and allergen applied to the tracheal mucosa of anesthetized guinea pigs produced acute extravasation of plasma and tracer macromolecules. edema could not be identified but extravasated large solutes were recovered in tracheal luminal fluid within a few minutes after provocation [ , [ ] [ ] [ ] ] . in contrast to tracheobronchial venules, the pulmonary microvessels are resistant to histamine-type mediators [ , , , ] . this aspect, together with abundant microvascular connections between the bronchopulmonary circulations, has stimulated a debate as to the relative importance of bronchial microvessels to fluid and protein exchange in pulmonary inflammation. in adult respiratory distress syndrome (ards) pulmonary edema and increased permeability to macromolecules in the pulmonary vessels and the alveolar wall are present [ ] , but bronchial venules may also leak plasma [ ] . wheezing is one of the symptoms of ards, and, as in bronchial asthma, survivors of ards may exhibit increased airway responsiveness to methacholine inhalation challenges [i ]. hence, it cannot be excluded that bronchovascular plasma exudation is one of the factors contributing to asthmalike symptoms in pulmonary inflammatory diseases. it has been calculated that small changes in mucosal thickness could have a profound influence on the tendency to airways closure as well as explain airway hyperresponsiveness to bronchoconstricting agents [ ] . slight edema of tissues between the bronchial muscle and the epithelium would thus only marginally reduce the baseline caliber of the airway lumen and be difficult to detect, but could cause abnormally large increases in resistance to airflow during bronchoconstriction. it has not yet been studied whether airway edema, similar to pulmonary edema [ , , ] , may lower the threshold for sensory receptor stimulation. in , fraenkel [ ] suggested that extensive epithelial shedding is a distinguishing characteristic of asthmatic airways. this proposal has been repeatedly substantiated [e.g., , ] and dunnill [ ] has suggested that mucosal edema and transepithelial passage of plasma exudate cause the shedding of epithelium. however, a significant volume of proteinaceous plasma can rapidly traverse the epithelial barrier without causing shedding [i ] . sustained inflam-mation and effects of potent and toxic cellular mediators such as eosinophilderived proteins [ ] may be required for significant shedding to occur. as discussed above several additional consequences of plasma exudation in the airways relate to the presence of plasma protein-derived mediators in the exudate that are capable of producing bronchoconstriction and inflammation. albumin is a normal constituent of tracheobronchial luminal liquid [ , , , ] . stockley et al. [ ] determined the relation between sputum/serum concentration ratios and stokes radius for selected proteins in chronic bronchitis. during stable noninfected conditions there was a significant negative correlation between the ratio and the protein size consistent with a passive diffusion of these proteins [ ] . during infection the ratio was increased more than -fold [ ] . this finding tallies with observations in upper airways: nasal washings obtained during viral rhinitis contain much elevated levels of serum proteins and kinin activity [ , , , ] . also in many noninfectious types of actue inflammation the epithelial permeability to macromolecules may increase dramatically. large amounts of charged macromolecules such as albumin and uncharged fluorescein-labeled dextran (mw , daltons) traversed vascular and epithelial barriers of guinea pig tracheas superfused with mediators or challenged with allergen [ , , ] . this highly increased permeability to inflammatory stimuli reversed spontaneously and was significantly prevented by drugs [ , ] . a pinocytotic transport mechanism would not suffice to bring about such a sudden transepithelial passage of a large volume of plasma [ , ] . perhaps intercellular junctions of tracheobronchial epithelium can be opened for macromolecular passage as in alveolar epithelium in high-permeability pulmonary edema [ , , ] . an ultrastructural study of inflamed guinea pig trachea has demonstrated that intraluminal horseradish peroxidase penetrates the wall between epithelial ceils [ ] . a series of observations of nasal liquid composition in atopic subjects challenged with allergen agrees with the notion that inflammatory stimuli induce a rapid, transient bulk flow of plasma macromolecules (and activation of peptide mediators) extravascularly and across the nasal epithelial lining [ , ] . it is of great interest that antiasthma drugs, notably glucocorticoids and xanthines, reduce this plasma leakage [ , , ] . mediator-induced increase in the permeability of the epithelial barrier can obviously be as dramatic as that across the venular endothelial lining. experiments indicate that the inflammatory plasma leakage does not require tissue destruction. instead, epithelial permeability to large molecules may be considered an active, reversible process that is under the control of mediators, hormones, and drugs. the subcellular~epithelial mechanisms involved in these permeability changes and details of intercellular pathways for leaking macromolecules remain unexplored. in recent years, airway mucosal or epithelial "permeability" has received widespread attention as a pathogenetic factor. what is usually measured in studies of this kind of "permeability," in particular in human subjects, is the rate of transfer of inhaled mtc-labeled diethylenetriamine penta-acetate (dtpa) into the blood. dtpa is a relatively small molecule (mw daltons) and its passage across airway epithelial-endothelial barriers may be regulated by mechanisms entirely different from those involved in leakage of large plasma proteins. hence neither vascular nor mucosal permeability to plasma macromolecules may be reflected in studies using dtpa. furthermore, lung clearance of inhaled dtpa may largely be across alveolar barriers. respiratory mucosal permeability determined with dtpa was not increased in asthma [ ] and, when it was increased, as in smokers, this did not correspond to increased airway reactivity [ ] . these observations may not be taken as evidence against a role for plasma exudation in asthmatic airways. many characteristics of asthmatic airways can be studied by analyzing the composition and pharmacologic effects of sputum [ ] . although their origin was not determined, anaphylatoxins have been identified in asthmatic sputa [ ] . asthmatic sputa have been demonstrated to produce smooth muscle contraction [ , ] and inhibition of ciliary motility [ ] . the factors responsible for these effects may well have come from the serum. they may be preformed mediators or mediators produced by activation of plasma proteins at exudation. using chemical analyses menders et al. [ ] confirmed the presence of plasma proteins in asthmatic sputa. ryley and brogan [ ] showed that the sol phase of asthmatic sputa contained large amounts of albumin and that glucocorticosteroid treatment reduced the plasma protein content along with improvement in lung function [ ] . in addition, they studied bronchitic sputa and concluded: this would imply that the sputum of the asthmatic patient had more in common with an inflammatory exudate than that of the chronic bronchitic.., this hypothesis is supported by the finding of a greater proportion of serum albumin and a greater variety of plasma proteins in the asthmatic as compared with the bronchitic sputum [ l. in a more quantitative study brogan et al. [ ] confirmed these findings and showed that levels of plasma proteins, but not secretory proteins, were elevated in asthmatic sputa. a high degree of plasma exudate in the airway lumen may also differentiate asthma from emphysema [ ] and cystic fibrosis [ ] . heilpern and rebuck [ ] not only demonstrated high levels of plasma proteins in asthmatic sputa, but also showed that cromoglycate normalized these levels. cromoglycate seems to share with several other antiasthma drugs antileakage effects directly on airway endothelial-epithelial barriers [ , ] . bronchoalveolar lavage is frequently performed in the clinical evaluation of lung diseases. it is used also in asthma but a relatively large contribution of alveolar liquids to the lavage fluid makes this technique less suitable for the identification of bronchial liquids. this point is illustrated in recent work by lam et al. [ ] . they found no difference in albumin levels between normals and asthmatics in large-volume bronchoalveolar lavage liquids. however, using a small volume lavage in a large bronchus they could demonstrate a -fold increase of albumin in asthmatic airways compared with controls [ ] . although many pieces of information are consistent with the pathophysiological importance of plasma and plasma-derived mediators in airway lumen, this subject has not been extensively reviewed. lord florey, who was experimentally acquainted with the possibility that a considerable amount of fluid may "come directly from the vessels" into the inflamed airway lumen [ ] , mentions only in passing, in his excellent chapter on inflammation of mucous membranes, that plasma exudate may seep through the mucosa [ ] . in julius cohnheim [ ] discussed how inflammation and plasma exudation might produce different results in different tissues and emphasized that in cavitary organs there may be a passage of exudate across the mucosal barriers. diseases with protein leakage into the gut have received attention due to the ensuing large fall in blood levels of plasma proteins [ ] . plasma exudate may escape into the gut lumen through a deranged mucosa and through an apparently intact mucosa [ ] . plasma albumin loss due to bronchial diseases has been suggested to occur [ , ] . for obvious reasons a luminal entry of plasma exudate would have rather more serious consequences for the lower airways than for the gastrointestinal tract. a rapid passage of exudate may increase the depth of the fluid layer in which the cilia beat, and hence cause marked inhibition of mucociliary transport [ ] . plasma exudate most likely participates in the formation of mucus plugs: fibrin formation would make the mucus firm and obstructive [ , ] ; albumin may interact with mucin to form viscous complexes [ ] ; plasma proteins may impede normal hydration of mucin [ ] . the plasma exudate may enter peripheral airways and compromise the surfactant activity, which in turn may lead to small airway narrowing [ ] . it is intriguing that different antiasthma drugs may prevent the mediator-induced microvascular leakage. drug-induced inhibition of vascular leakage can-not be expected to show an immediate reversal of edema because the rate of resolution of interstitial fluid is dependent on lymphatic drainage, which is a relatively slow process. however, if obstructive bronchial tone is dependent on a continuous supply of activated plasma protein mediators from leaky microvessels, an antileakage action might reverse the airway obstruction. it is of interest that protease inhibitors may prevent bronchoconstriction induced by various challenges in asthmatic subjects [ ] . in menkin [ ] found that adrenal cortex extract inhibited inflammatory vascular leakage. thirty-three years later leme and wilhelm [ ] demonstrated in rats that corticosteron prevents mediator-induced increase in vascular permeability and that this drug inhibits the enhanced venular responsiveness brought about by adrenalectomy. current developments include attempts to identify proteins induced by glucocorticoids. probably by binding with specific receptors followed by induction of anti-inflammatory proteins, glucocorticosteroids inhibit or reduce vascular leakage. in guinea pig airways glucocorticoids such as budesonide may reduce leakage of plasma across both endothelial and epithelial barriers [ ] . glucocorticoids have been shown to reduce plasma exudation in inflammatory airway diseases. ryley and brogan [ ] found a relationship between a lowering of the albumin concentration in sputa with steroid therapy and clinical improvement in an asthmatic subject. based on serial measurements of neuraminic acid concentration (indicator of bronchial secretion) in asthmatic sputa, keal [ ] inferred that the effect of steroid therapy "lies in the reduction of transudate rather than in any change in the bronchial mucosal gland secretion." moretti et al. [ ] studied patients with both reversible airways obstruction and bronchitis and showed that weeks' treatment with methylprednisolone brought about a dramatic reduction in the sputum concentration of albumin. stockley and associates [ , ] examined the effects of about week's therapy with prednisolone on sputum composition in patients with chronic obstructive bronchitis. the patients had no acute chest infections and had, therefore, relatively low levels of serum proteins in their sputa [ ] . still, after a few days of treatment a significant reduction was recorded in the ratio of sol-phase sputum concentration to serum concentration of albumin [ ] . an antiexudative effect would also reduce the entry of plasma proteins that have protective functions in the airway. the values of c~rantitrypsin followed the same pattern as those of albumin with steroid treatment [ ] . however, despite the reduced c~l-antitrypsin levels the inhibitory capacity of the sputum (evaluated on porcine pancreatic elastase) was increased, suggesting that the overall effect of glucocorticosteroids on the airway liquid proteinase-antiprotease balance may be beneficial [ ] . findings in nasal washing experiments lend further support to the theory of an antiexudative action of glucocorticosteroids. treatment for days with prednisolone significantly reduced clinical symptoms as well as amounts of albumin (kinins, tame-esterase activity, and histamine) in washings performed during nasal late reaction following challenge with allergen in allergic subjects [ ] . this study also produced the interesting information that generation of arachidonate products such as leukotrienes and prostaglandin d was not affected by the steroid treatment [i ] . attenuation of plasma exudation by glucocorticoids may contribute to the general efficacy of these drugs in asthma and to their potency in inhibiting latephase asthmatic responses and reducing airway hyperresponsiveness. antiasthma xanthines may be subdivided into adenosine-blockers such as theophylline and adenosine-nonblockers such as enprofylline [ , ] . these xanthines seem to share a number of potentially important pulmonary antiinflammatory effects [see ] . included among the anti-inflammatory actions is a vascular and epithelial antileakage effect that has been demonstrated in guinea pig airways [ , , , ] . it has also been shown that both enprofylline and theophylline prevent the development of pulmonary edema in guinea pigs inhaling histamine [ , ] . furthermore, antiasthmatic doses of theophylline reduced both symptoms and plasma exudation in human nasal mucosa provoked with different amounts of allergen [ , ] . cromoglycate also reduced macromolecular leakage across endothelial-epithelial barriers in guinea pigs. this action was not dependent on which mediator had induced the plasma leakage, nor was it due to a reduction of mucosal/submucosal blood flow [ , ] . the animal data may explain observations in asthmatic subjects reported by heilpern and rebuck [ ] years ago. they [ ] stated that their study • . . does not attempt to explain why sodium cromoglycate is also effective in non-allergic asthma. however, the evidence points to a previously unrecognized action of the drug, that of lowering albumin concentration in sputum to levels found in non-asthmatic patients. the significance of this finding awaits further study. the possibility that an anti-plasma-leakage action of cromoglycate is important and may compare favorably with other proposed mechanisms of action of the drug in asthma is discussed elsewhere [ ] . it has been widely held that the anti-inflammatory effects of sympathomimetic drugs reflect vasoconstriction and diminished blood flow to inflamed tissues. in addition, it has now been demonstrated that these drugs have a vascular antipermeability property. this is fl -adrenoceptor mediated and more than outweighs the slightly proinflammatory blood flow increasing effect produced by the/ z-receptor agonists [ , , ] . about years ago we demonstrated in guinea pigs that the fl -receptor agonist terbutaline given systemically or by the inhaled route effectively prevented the development of pulmonary edema induced by subsequent exposure to histamine [ ] . this protection might have been via effects on bronchial microvessels, because later studies have demonstrated that terbutaline reduces plasma leakage from the tracheobronchial microcirculation [ ] . fl -receptor mediated antipermeability effects on airway mucosa and microvessels { , ] in asthma remain to be examined. a pharmacologic mediator antagonist is by definition effective with some specificity only against type of mediator. thus, antihistamines, leukotriene antagonists, paf-acether antagonists, and tachykinin antagonists among others will specifically antagonize leakage produced by corresponding agonists. since the leakage-regulating endothelial cells of postcapillary venules harbor specific receptors for a large variety of inflammatory mediators, it cannot be expected that a single mediator antagonist alone could produce an acceptable antileakage response in inflammation. circulating plasma kinin in patients with bronchial asthma donnan mechanism of mucus hydration: effect of soluble proteins the physiological and pharmacological characteristics of the tracheal muscle a historical account of death from asthma biochemical mediators: release, chemistry, and function immunoelectrophoresis of nasal secretions collected during a common cold: observations which suggest a mechanism of seroimmunity in viral respiratory infections uber die kittsubstanz der endothelien quantitative studies of protein and water shifts during inflammation bronchial circulation in asthma role of tracheal and bronchial circulation in respiratory heat exchange interactions between histamine and bradykinin assessed by continuous recording of increased vascular permeability influx of kininogens into nasal secretions after antigen challenge of allergic individuals contractile proteins of endothelial cells, platelets and smooth muscle microvascular effects of anaphylatoxins c a and c a ~i albumin turnover and loss of protein into the sputum in chronic bronchitis arginine esterase activity of the plasma in different types of bronchial asthma airway mucosal permeability soluble proteins of bronchopulmonary secretions from patients with cystic fibrosis, asthma, and bronchitis vascular responses and their suppression: the role of endothelium the sequence of vascular events in early infective inflammation serum and sputum a macroglobulin in patients with chronic obstructive airways disease death in asthmatics intercellular cement and capillary permeability cohnheim j ( ) vorlesungen uber allgemeine pathologic i. august hirschwald modification of inspired air their discharge during spontaneous breathing and their stimulation by alloxan and pulmonary congestion ultrastructure of the prolonged vascular response induced by clostridium oedematiens toxin electron microscopic alterations at the alveolar level in pulmonary edema some properties of the iota-toxin of clostridium welchii including its action on capillary permeability ultrastructure of airways in children with asthma pulmonary and bronchial vascular systems antigen tracer studies and histologic observations in anaphylactic shock in the guinea-pig. ii kinin-like activity in nasal secretions of allergic patients bronchial secretions and mucociliary clearance sputum in bronchial asthma sputum and ciliary inhibition in asthma the pathology of asthma with special reference to changes in the bronchial mucosa the pathology of asthma a comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema respiratory mucosal permeability in asthma effects of adrenaline and terbutaline on mediator-increased vascular permeability in the cat trachea anti-asthma drugs attenuate inflammatory leakage of plasma into airway lumen bronchoalveolar neutrophilia during tdl-induccd late asthmatic reactions general pathology mucus secretion in the trachea zur pathologie des bronchialasthma relationships between pulmonary inflammation, plasma transudation, and oxygen metabolic secretion by alveolar macrophages effect of fibrin degradation products on microvascular permeability the role of the eosinophilic leukocyte in bronchial asthma the sticking and emigration of white blood cells in inflammation responses of skin blood vessels to bradykinin, histamine and -hydroxytryptamine biochemical study on sputum in asthma and emphysema plasma albumin loss due to bronchopathy spasm-producing substance in the sputum of patients with bronchial asthma effect of disodium cromoglycate (intal) on sputum protein composition the effect of bradykinin aerosol in guinea-pigs and in man the relation of ciliary insufficiency to death from asthma and other respiratory diseases the pathology of asthma protein permeability in the adult respiratory distress syndrome. loss of size selectivity of the alveolar epithelium a clinical and pathological study of fatal cases of status asthmaticus the pathology of bronchial asthma intravital and electron microscopic study of bradykinininduced vascular permeability changes using fitc-dextran as a tracer types of pulmonary microvascular injury acute inflammation bronchial-lumen und atemwiderstand effects of hemodynamic edema formation on peripheral vs. central airway mechanics protein-losing gastroenteropathy biochemistry and rheology of sputum in asthma increased airway mucosal permeability of smokers. relationship to airway reactivity bronchial circulation perfused via the pulmonary artery in guinea-pig isolated lungs is asthma also a vascular disease? effects of inflammatory and other mediators on airway vascular beds relationship between tracheal mucosal thickness and vascular resistance in dogs the effects of adrenalectomy and corticosterone on vascular permeability responses in the skin of the rat effect of bronchial lavage volume on cellular and protein recovery enhancement of the viscosity of mucin by serum albumin the stability of peripheral airways studies on inflammation i cellular and humoral mediators of pulmonary edema the site of leukocyte emigration during inflammation bronchial artery distribution in various mammals and in humans contribution of small vessel tone to the regulation of blood volume and formation of edema immunochemical studies of the asthmatic sputum effect of adrenal cortex extract on capillary permeability causes of death and pathologic findings in cases of bronchial asthma complement activation in asthma evaluated by the c d/c index large molecular substances as mediators of the inflammatory reaction enzyme-like globulins from serum reproducing the vascular phenomena of inflammation the activation of endogenous substances inducing pathological increases of capillary permeability the lung ciliary inhibition or destruction in tracheobronchial asthma effects of methylprednisolone on sputum biochemical components in asthmatic bronchitis inhibitory capacity of alpha~ antitrypsin in lung secretions: variability and the effect of drugs kinins and the kinin system as inflammatory mediators concentrations of immunoglobulins in nasal secretion from children with recurrent infections in the upper airways theophylline reduces the response to nasal challenge with antigen regulation of airway responsiveness and secretion: role of inflammation anaphylatoxins c a, c a and c a are produced by human alveolar macrophages in culture and are found in sputum of asthmatics solute permeability of the alveolar epithelium in alloxan edema in dogs the trachea and cuff-induced tracheal injury action of histamine on endothelial cells of guinea-pig isolated hepatic portal vein and its modification by indomethacin or removal of calcium mechanisms of blood vessel permeability derangement under the influence of permeability factors (histamine, serotonin, kinins) and inflammatory agents studies of reactivity and distribution of bronchial blood flow in sheep concentrations of gentamicin and carbenicillin in bronchial secretions role of plasma exudation in asthmatic airways overview of effects of theophylline cromoglicate, plasma exudation and asthma xanthines as airway antiinflammatory drugs bronchial microcirculation in vitro response to bronchodilator drugs inflammatory leakage of macromolecules from the vascular compartment into the tracheal lumen non-neural and neural regulation of airway microvascular leakage of macromolecules leakage of macromolecules from guinea pig tracheobronchial microcirculation. effects of allergen, leukotrienes, tachykinins, and antiasthma drugs the role of ¢l-receptor agonists in the inhibition of pulmonary edema airway microvascular and epithelial leakage of plasma induced by paf-acether and capsaicin vascular responses and their suppression: drugs interfering with venular permeability bronchial venular leakage during endotoxin shock effect of systemic glucocorticoid treatment on human nasal mediator release after antigen challenge paf-acether induced plasma exudation in rat skin is independent of platelets and neutrophils kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen protein composition of nasal secretion during respiratory virus infection the gradient of vascular permeability variation in the composition of sputum in chronic chest diseases on asthma: its pathology and treatment some histological changes in chronic bronchitis and asthma interaction of serum proteins with lung endothelial glycocalyx: its effect on endothelial permeability the activity of lung irritatant receptors during pneumothorax, hypertonea and pulmonary vascular congestion the late phase skin reaction: evidence for activation of the coagulation system in an ige-dependent reaction in man penetration of the respiratory epithelium of guinea-pigs following exposure to cigarette smoke in vivo and in vitro effect of bradykinin on bronchial motor tone in normal subjects and patients with airways obstruction long term follow-up and bronchial reactivity testing in survivors of the adult respiratory distress syndrome the microcirculation of the tracheal mucosa the role of some higher peptides in inflammation the inflammatory response a study of plasma proteins in the sol phase of sputum from patients with chronic bronchitis bradykinin and prostaglandin ex, e and f -induced macromolecular leakage in the hamster cheek pouch the effects of intermittent and continuous stimulation of microvessels in the cheek pouch of hamsters with histamine and bradykinin on the development of venular leaky sites mechanism of ionophore a induction of plasma protein leakage and of its inhibition by indomethacin chronic obstructive lung disease. a comparison between clinical roentgenologic, functional and morphologic criteria in chronic bronchitis, emphysema, asthma and bronchiectasis mucociliary function in bronchial asthma complement and chemotaxis control of vascular permeability by polymorphonuclear leukocytes in inflammation effect of corticosteroids on sputum sol-phase protease inhibitors in chronic obstructive pulmonary disease hageman factor and the contact activation system chemical mediators death from bronchial asthma vascular responses and their suppression: vasodilation and edema the role of polymorphonuclear leucocytes in acute inflammation in agranulocytic rats inflammation in agranulocytotic rats ciliary dyskinesia factors in cystic fibrosis and asthma the effect of synthetic leukotrienes on tracheal microvascular permeability pathophysiology of the blood vascular barrier microvascular aspects of tissue injury acknowledgment. i thank mrs lngrid erjeffilt for skillful co-work and my colleagues in lund for encouragement and helpful discussions. i thank mrs ingegerd k~illrn for expert secretarial assistance and mrs marianne persson and her colleagues for excellent library help. key: cord- -x kankxd authors: romero, cesar a.; orias, marcelo; weir, matthew r. title: novel raas agonists and antagonists: clinical applications and controversies date: - - journal: nat rev endocrinol doi: . /nrendo. . sha: doc_id: cord_uid: x kankxd the renin–angiotensin–aldosterone system (raas) regulates blood pressure homeostasis and vascular injury and repair responses. the raas was originally thought to be an endocrine system critically important in regulating blood pressure homeostasis. yet, important local forms of the raas have been described in many tissues, which are mostly independent of the systemic raas. these systems have been associated with diverse physiological functions, but also with inflammation, fibrosis and target-organ damage. pharmacological modulation of the raas has brought about important advances in preventing morbidity and mortality associated with cardiovascular disease. yet, traditional raas blockers such as angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) only reduce the risk of disease progression in patients with established cardiovascular or renal disease by ∼ % compared with other therapies. as more components of the raas are described, other potential therapeutic targets emerge, which could provide improved cardiovascular and renal protection beyond that provided by an ace inhibitor or arb. this review summarizes the present and future pharmacological manipulation of this important system. supplementary information: the online version of this article (doi: . /nrendo. . ) contains supplementary material, which is available to authorized users. more than seven decades ago, angiotensin, a poly peptide that is the product of the enzymatic activity of renin, was found in venous samples of ischaemic dog kidneys. in the s and s, two forms of angiotensin were reported: angiotensin- (ang i), which is amino acids long, and angiotensin- (ang ii), with amino acids. the latter form results from the metabolism of ang i by a dipeptidyl carboxypeptidase named angiotensinconverting enzyme (ace). the substrate of renin was found to be angiotensinogen, a serum globulin produced by the liver. also at this time, the role of ang ii in the regulation of aldosterone production by the adrenal cortex emerged. in the s, the main catalytic cascade of the reninangiotensin-aldosterone system (raas) was described ( figure ). plasma angiotensinogen is cleaved by renal renin, producing ang i, which is then converted to ang ii by endothelial ace, a process that occurs most extensively in lung tissue. ang ii was considered the most important raas mediator, with increased levels of ang ii being associated with vasoconstriction and increased blood pressure. ang ii binds to the type- ang ii receptor (at ) in a variety of tissues, including vascular smooth muscle and the adrenal grand, to mediate many mechanisms that lead to raised blood pressure. the stimulation of aldosterone production via the at receptor in the adrenal gland facilitates sodium retention by the kidney when aldosterone binds to the mineralocorticoid receptor. on the basis of the first experimental studies of ang ii, in which supraphysiological doses of this peptide were tested in dogs, the raas was related to the pathophysiology of hypertension mainly through the effects of this system on vascular constriction. however, more recent studies have helped clarify that blood pressure control by ang ii at physiological concentrations is more related to sodium and water handling than to arterial constriction. ang ii shifts the natriuresis-blood pressure curve, with increased levels of ang ii requiring increased blood pressure to eliminate the same quantity of sodium. part of this effect occurs via direct mediation by ang ii, which activates sodium transporters in the proximal tubules of the kidney. [ ] [ ] [ ] furthermore, ang ii, through binding to the at receptors, is the most potent regulator of aldosterone secretion in the adrenal cortex. , aldosterone binding to the mineralocorticoid receptor induces nongenomic (rapid) and genomic effects in many tissues, but mainly in the kidney, where it increases the epithelial expression of epithelial sodium channel (enac, also known as amiloride-sensitive sodium channel) at the level of the distal tubules, which favours the retention of sodium and water. blockade of the raas was a major breakthrough in the treatment of cardiovascular disease, lowering mortality and improving quality of life in patients with hyper tension, chronic kidney disease (ckd), myocardial infarction and heart failure. [ ] [ ] [ ] [ ] [ ] the classic raas antagonists target the angiotensinogen-angiotensin-at -aldosterone axis. however, as knowledge about the raas expands, the number of potential therapeutic targets in this system is increasing. in this review we the components of the raas are schematically represented in figure . renin is the rate-limiting step in ang ii production and is released by the juxta glomerular apparatus in the kidney in response to decreased renal perfusion pressure, low tubular salt load or sympathetic activation. renin is synthesized from prorenin, a pre-p roenzyme that is constitutively released, with plasma levels -fold higher than those of active renin. prorenin can be proteolytically activated in the kidney by neuroendocrine convertase (also known as prohormone convertase and proprotein convertase ) or cathepsin b and non-proteolytically (reversibly) in many tissues by the renin receptor (also known as renin/p rorenin receptor). these two types of activation require the propeptide to expose its active site, a cleft between two similar domains. the renin receptor also binds to mature renin, increasing its catalytic activity by - fold. the renin receptor can initiate ang ii-independent signal ling pathways, such as the mapk/erk pathway and pathways involving the zinc finger protein plzf. the effects of these signalling pathways are not clear and the renin receptor has not been shown to have a role in cardiovascular disease when the effects of ang ii are blocked. the secretion of renin is controlled by short-term and long-term negative feedback loops. short-term regulation is mediated directly by ang ii through binding to at , which induces inhibition of renin gene expression. the long-term negative feedback loop is an indirect consequence of the effects of increased ang ii levels: increased blood pressure, sympathetic inhibition of baroreceptor mechanisms and increased intratubular sodium levels. the classic effects of ang ii are mediated by the at receptor, which is present in practically all tissues (box ). blood pressure effects are modulated by vascular at receptors, whose activity produces vasoconstriction (rapid pressure effects) and by renal at receptors that mediate sodium and water reabsorption. many fibrotic and inflammatory effects of ang ii are also a consequence of at receptor activation. however, ang ii can also activate the type- ang ii receptor (at ), which is more sparsely expressed in body tissues than the at receptor, but which is upregulated in many cases of injury (for example, to the brain, heart and kidney). at is part of the protective arm of the raas, and, when activated, has antifibrotic and anti-inflammatory effects (box ). activation of the at receptor does not seem to have blood pressure effects. other non-classic raas components have been described in the past few years. a novel enzyme similar to ace was identified and called angiotensin-converting enzyme (encoded by ace and hereby referred to as ace ). this carboxypeptidase has great ang ii affinity and can transform this peptide to angiotensin - (hereby referred to as ang - ). ace can also convert ang i to angiotensin - (ang - ), which is then converted to ang - by ace. similarly, plasmatic endopeptidases can convert ang i to ang - . ang - binds to a g-protein-coupled receptor called proto-oncogene mas (encoded by mas and hereby referred to as mas ). this ace -ang - -mas axis has been reported to function as a counterbalance to the classic ace-ang ii-at axis, with opposite effects than those mediated by the classic axis (box ). another non-classic axis in the raas is the pathway mediated by angiotensin- (ang iv) and its receptor, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase, insulinresponsive aminopeptidase or irap). , this receptor, which was originally reported to colocalize with the glucose transporter glut- , has a role in the degradation of vaso pressin and oxytocin. ang iv inhibits the enzymatic activity of irap and knockout models and studies of non-peptide inhibitors suggest a role of irap in c ardiovascular disease, mainly in target-organ damage. [ ] [ ] [ ] the effects of irap inhibition are listed in box . beyond the original description of the endocrine roles of the raas, experimental studies have demonstrated that the system also has paracrine, autocrine and intracrine functions. different tissues (reproductive and digestive tissues, heart and brain) and cells (adipocytes and white blood cells) express raas components, which not only perform the classic raas function (fluid volume homeostasis), but are also involved in other physiological functions, such as ovulation, secretion of insulin by the pancreas, cognitive function, inflammation and cancer. , in addition, activation of local raass is strongly related to target-organ damage, mostly in the kidney and heart. four clinically established groups of drugs inhibit the raas: ace inhibitors, angiotensin receptor blockers (arbs, which block the at receptor), renin inhibitors and mineralocorticoid receptor blockers (box ). despite the important improvements achieved with these agents in slowing the progression of established cardiorenal disease, the ace inhibitors and the arbs only provide a % reduction in the relative risk of key points ■ renin-angiotensin-aldosterone system (raas) blockade with an angiotensin converting enzyme inhibitor or an angiotensin receptor blocker provides a % relative risk reduction for the progression of established cardiorenal disease compared with other non-raas blocking therapies ■ the raas is an endocrine, paracrine and autocrine system that regulates blood pressure homeostasis through effects on a variety of target organs, as well as having a role in the responses to vascular injury and repair ■ the raas is a complex system with a variety of sites suitable for pharmacological intervention ■ novel molecules that alter the production of various raas peptides or that alter receptor density, function or responsiveness to these peptides could have an important influence on haemodynamics and vascular structure and function www.nature.com/nrendo progression of cardiovascular disease when compared with non-raas blocking therapy. , , , moreover, no data have demonstrated beneficial effects of ace inhibitors or arbs in the primary prevention of either cardiac or kidney disease. therefore, much consideration has been given to how to improve upon raas inhibition with the classic, established drugs, by investigating new agonists and antagonists of the complex raas cascade. many theories about why traditional raas inhibition has not provided more clinical benefits have been proposed, but none of these theories has been validated clinically. in the early s, early clinical studies of the first oral ace inhibitor, captopril, demonstrated this agent has antihypertensive properties and improves the clinical status of patients with heart failure. ace inhibitors do not affect the levels of ace . even though the main mechanism of action of ace inhibitors is to limit the formation of ang ii, other activities, such as reducing the catabolism of other vasodilator oligopeptides (such as bradykinin) by ace, might have a role in the pharmacological effects of ace inhibitors. another tetrapeptide, n-acetyl-seryl-aspartyl-lysil-proline (ac-sdkp), whose levels are elevated in patients receiving ace inhibitors, could also be responsible for many of the antifibrotic and immune-beneficial effects of these agents. furthermore, ace inhibitors increase the levels of ang i, which shifts raas activity to the axis producing ang - . , some of the favourable pharmacological effects of ace inhibitors might be mediated though increased activity of the ace -ang - -mas axis. , currently, ten ace inhibitors are available in the usa and several more are available globally. the clinical effects of the various ace inhibitors are not markedly different, except for trandolapril and quinapril having a longer half-life and broader tissue distribution than the other agents owing to their liposolubility. many years of use have confirmed very good safety and tolerability profiles for these drugs, with cough ( - % of patients) and angioedema ( % of patients) being the most common adverse effects. this drug class is the first-line therapy for patients with hypertension, ckd and heart failure, [ ] [ ] [ ] with ace inhibitors successfully controlling blood pressure and reducing left-ventricular hypertrophy and all-cause mortality. the first oral arb, losartan, was made available in the s, and currently nine arbs exist for clinical use (box ). despite different pharmacokinetic characteristics, most arbs are used as once-daily drugs and have fewer adverse effects than ace inhibitors. in general, the indications for using arbs are the same as for using ace inhibitors; arbs are efficacious in hypertension - and heart failure, figure | components of the renin-angiotensin-aldosterone system and main classes of pharmacological activators and inhibitors of the system. prorenin can be activated proteolytically in the kidneys (by neuroendocrine convertase or cathepsin b) or nonproteolitically by the renin receptor in many tissues. circulating renin can also bind to the renin receptor, which increases its enzymatic activity. renin converts angiotensinogen to ang i, which can then enter three main pathways. these three axes, ace-ang ii-at -aldosterone, ace -ang - -mas and ang iv-irap, are highlighted. activation of the at receptor in the adrenal gland results in production of aldosterone, which can then bind to the mineralocorticoid receptor. abbreviations: ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; ang, angiotensin; arb, angiotensin receptor blocker; arn, angiotensin receptor-neprilysin; at , type- ang ii receptor; at , type- ang ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas , proto-oncogene mas; rh, recombinant human. protection. as with ace inhibitors, use of arbs increases the levels of ang ii as a substrate of ace and could have clinical benefits resulting from stimulation of the ace -ang - -mas axis. , in theory, combining ace inhibitors with arbs or with a renin inhibitor could have pharmacological benefits. however, no evidence of clinical benefits beyond those achieved with single-agent approaches was seen when combination therapy was used in patients with hypertension, coronary artery disease or ckd, and c ombination therapy could potentially be harmful. , mineralocorticoid receptor antagonists the mineralocorticoid receptor antagonist spironolactone was first used in the s as a potassium-sparing diuretic. since then, the systemic effects of aldosterone in hypertension, heart failure and target-organ damage became better known. randomized clinical trials confirmed the effectiveness of spironolactone to treat hypertension and showed that the drug has antifibrotic and anti-inflammatory properties. spironolactone binds to other receptors in addition to the mineralocorticoid receptor, such as androgen and progesterone receptors, which can have adverse effects such as gynaecomastia and decreased libido. in , a second mineralocorticoid receptor antagonist, eplerenone, was introduced. eplerenone has less affinity for sex hormone receptors than spironolactone; the drug has a nearly similar antihypertensive effectiveness to that of spironolactone, yet is less potent and has fewer adverse effects. , a third antagonist, canrenone, is available in some countries but scant clinical data on this drug exist. in addition to their use in primary hyperaldosteronism, mineralocorticoid receptor blockers are commonly used to treat ascites, heart failure and resistant hypertension. , direct renin inhibitors in , and after many attempts, the first oral direct renin inhibitor (dri), aliskiren, became available, replacing first-generation inhibitors of low bioavailability. aliskiren is an effective antihypertensive agent and dual therapy with aliskiren and an arb was tested in the altitude trial. however, in this trial, the combination of aliskiren and losartan did not show any benefits on cardiovascular or renal outcomes versus losartan alone, and dual therapy was associated with an increased rate of adverse events. improving the benefits of raas blockade beyond the effects of ace inhibitors or arbs will be challenging, but research in this area is abundant. several new nonpeptide drugs have been developed that modify raas activity (box ); however, most of them are at preclinical or early clinical stages. angiotensin-receptor-neprilysin inhibitors natriuretic peptides have an important role in sodium and water homeostasis, inducing natriuresis, inhibiting the hypothalamic-pituitary-adrenal system at all regulatory levels , and promoting vasodilatation; they also possess antiproliferative properties. , neprilysin is a vasopeptidase that metabolizes natriuretic peptides and other peptides, such as bradykinin. neprilysin inhibition increases the plasma levels of natriuretic peptides, but has modest effects in reducing blood pressure. however, combining neprilysin with a raas blocker is a powerful tool to increase natriuresis and vasodilation. the first therapeutic agent in this class was omapatrilat, a vasopeptidase that inhibits both neprilysin and ace. the cardiovascular effects of omapatrilat were encouraging, but a high rate of angioedema stopped clinical trials of this drug. the next generation of drugs in this group combines a natriuretic peptide inhibitor with an arb. lcz , the first agent of the angiotensinreceptor-neprilysin (arn) inhibitor class, combines a neprilysin inhibitor moiety with a valsartan moiety. this dual therapy decreases blood pressure in animals and healthy humans with low rates of adverse effects. this type of drug intervention is promising in the fields of h ypertension and heart failure. abbreviations: at , type- angiotensin ii receptor; at , type- angiotensin ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas , proto-oncogene mas; raas, renin-angiotensin-aldosterone system. a randomized clinical trial (rct) evaluated the effect of lcz in the treatment of hypertension. patients with mild to moderate hypertension (n = , ) received a single dose of , or mg of lcz , or , or mg of valsartan in a -week placebo-controlled trial. lcz showed a good safety profile and was s uperior to placebo and valsartan in reducing office blood pressure and pulse pressure. more than % of patients had their blood pressure controlled with the mg dose of lcz , whereas this proportion was only % in the group receiving mg of valsartan. in approximately patients per arm, ambulatory blood pressure monitoring was utilized. mean diastolic h blood pressure was not different between the groups receiving the two therapies, but mean systolic blood pressure was significantly reduced in the lcz group because of larger nocturnal decreases in blood pressure than those observed in the valsartan or placebo groups. the greater blood pressure reduction seen in patients receiving lcz when compared with patients in the other groups was not related to increased levels of the second messenger (cgmp) that mediates the actions of natriuretic peptides. a second rct in an asian population also confirmed the efficacy of lcz in treating hypertension. the parameter study is an ongoing study in elderly patients with wide pulse pressure, which evaluates the vascular effects of arn inhibition, including changes in arterial stiffness and central haemodynamics, and efficacy in reducing blood pressure. the trial will be finished in . the paramount study evaluated the effects of mg of lcz compared with mg of valsartan in almost patients with heart failure and preserved ejection fraction. even though the levels of n-terminal probnp (also known as b-type natriuretic peptide) were decreased and functional new york class of heart failure was improved at weeks of follow-up, no differences in echocardiographic parameters were observed. no changes were observed in the levels of plasma inflammation markers. surprisingly, proteinuria increased in the lcz group, an increase that was not observed in patients treated with lcz in the hypertension trials. this increase could be related to increased intraglomerular pressure related to increased levels of natriuretic peptides. the most important observations in this study were a mmhg decrease in systolic blood pressure and a mmhg decrease in diastolic blood pressure, which both persisted at weeks of follow-up in the lcz group, whereas the decreases in blood pressure seen in the valsartan group were . mmhg and . mmhg, respectively, over the same period. the paradigm-hf trial, in which therapy with mg of lcz was compared with therapy with mg of enalapril for the treatment of heart failure (with decreased ejection fraction), was stopped according to prespecified rules because lcz reduced the frequency of the primary outcome, a composite of death from cardiovascular causes or hospitalization for heart failure (hr . , p < . ). , blood pressure was signifi cantly lower in the lcz group than in the enalapril group. the lcz group had higher proportions of patients with hypertension and nonserious angioedema, but fewer patients with renal impairment and hyperkalaemia than the enalapril group. urinary levels of cgmp in the lcz group did not change during the trial. some post-hoc analyses have noted that the benefits of lcz in the treatment of heart failure do not correlate with the antihypertensive effects of the drug. arn inhibition could be a breakthrough in the hypertension field. the observed reductions in systolic nocturnal blood pressure and in pulse pressure might be an indication of a substantial effect of lcz on vascular function or on the central nervous system rather than an effect on natriuresis. nonsteroidal mineralocorticoid receptor antagonists (third-generation and fourth-generation compounds) have been developed with the aim of achieving cardiovascular benefits with fewer renal adverse effects than those of classic mineralocorticoid receptor antagonists (spironolactone, eplerenone and canrenone). abbreviations: ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; ang, angiotensin; arb, angiotensin receptor blocker; arn, angiotensin receptorneprilysin; at , type- angiotensin ii receptor; at , type- angiotensin ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas , proto-oncogene mas; raas, renin-angiotensin-aldosterone system. finerenone, previously called bay - , is a potent and promising mineralocorticoid receptor antag onist that conferred extensive cardiovascular and renal protection in rat models of hypertension and heart failure when compared with eplerenone. the arts trial assessed the efficacy and safety of this drug in patients with systolic heart failure and mild ckd. this phase ii study consisted of two parts. part a, which involved patients, evaluated the safety and tolerability of . , or mg daily doses of finerenone versus placebo. part b assessed the safety and efficacy of the drug in patients randomized in a : : : : : design to receive finerenone orally at doses of . , , or mg daily, or mg twice daily; placebo; or open-label oral spironolactone, which was given at an initial dose of mg daily and uptitra ted to mg daily if serum potassium concentrations allowed. finerenone decreased the levels of bnp and n-terminal probnp. the drug decreased albuminuria by a similar degree to spironolactone and was associated with lower incidence of hyperkalaemia and acute renal failure. more data are needed to assess the clinical effects of finerenone. the arts hf trial, which has been completed but has not been published yet, assessed the efficacy and safety of this drug when compared with eplerenone in patients with diabetes mellitus but without ckd or chronic left-ventricular dysfunction. another nonsteroidal drug, br- , has high potency and selectivity for the mineralocorticoid receptor but also blocks the l-type calcium channel, which might help lowering blood pressure. after binding to the mineralo corticoid receptor, br- triggers degradation of this receptor. no clinical trials of br- have been reported. pf- is a potent and selective nonsteroidal inhibitor of the mineralocorticoid receptor that has been tested in healthy humans , and patients with diabetic nephropathy, but was discontinued owing to adverse effects. sm- is another agent of this class that has potent activity as an antagonist of the mineralocorticoid receptor and, in hypertensive rats, diminishes blood pressure and reduces the urinary levels of inflammatory markers. sm- has not been tested in humans. aldosterone synthase inhibitors a different approach to avoid the deleterious effects of aldosterone is by inhibition of the expression of cyp b , the gene than encodes aldosterone synthase. inhibition at this level of the raas would block the effects that are mediated through the mineralocorticoid receptor and also those that are independent of this receptor. an additional potential benefit of this approach is the prevention of a reactive increase in aldosterone levels, which occurs when the receptor is blocked. the potential downside of this strategy is that aldosterone synthase inhibitors might also suppress cortisol release by inhibiting cytochrome p b , mitochondrial (also known as steroid β-hydroxylase, encoded by the cyp b gene), because the two enzymes have a high sequence homology. fad a is an isomer of fadrozole, an aromatase inhibitor. this drug decreased plasma and urinary aldosterone concentrations and improved cardiac and renal target-organ damage in several animal models. [ ] [ ] [ ] fad a has only a mild antihypertensive effect and, therefore, might be more clinically important in the treatment of heart failure than other approaches that have a more potent effect in lowering blood pressure. lci is an orally active and nonselective aldosterone synthase inhibitor that has been evaluated in human studies. in phase i studies, a . mg daily dose of lci was well tolerated and lowered plasma and urinary aldosterone levels without affecting cortisol secretion. at higher doses, however, cortisol secretion was diminished. phase ii studies demon strated that plasma and urinary aldosterone levels are decreased with lci therapy, but the raas is upregulated and slight hyponatraemia and increased serum potassium levels were reported. in a large study, patients were randomized to receive either lci . mg once daily (n = ), . mg once daily (n = ), . mg twice daily (n = ) or . mg once daily (n = ); eplerenone mg twice daily (n = ); or placebo (n = ) for weeks. lci intake was associated with a modest reduction in blood pressure that was lower than that achieved with eplerenone.when aldosterone synthase is inhibited by lci , cortisol levels remain normal, but -deoxycorticosterone levels increase, which means that the adrenocorticotropichormone-cortisol axis is stimulated by the inhibition of expression of the cyp b gene. the stimulation of this axis might be one of the reasons why the decrease in blood pressure is only modest even at high lci doses. lack of aldosterone synthase selectivity, a short halflife and lack of adequate antihypertensive efficacy in primary aldosteronism and resistant hypertension hinder the usefulness of lci in these areas. this agent, however, might be of value in the treatment of cushing disease. therefore, research is now set to develop second-generation aldosterone synthase inhibitors with better selectivity than lci , which would be associated with improved effects on reduction of blood pressure. n-(pyridin- -yl) benzamides are potential agents for future development in this drug class. , post-transcriptional regulation of cyp b and cyp b expression by dicer-dependent micrornas (mirnas) can affect secretion of aldosterone and cortisol in adrenocortical cells. therefore, adrenal mirnas might also be potential therapeutic targets. the rationale behind complete raas inhibition through blockade of the rate-limiting enzyme renin, and the expectation that this approach would more effectively prevent ang ii production than approaches targeted at other components of the raas is theoretically attractive. however, aliskiren is the only dri available. as add-on therapy to arbs, aliskiren did not improve renal or cardio vascular outcomes and was associated with more adverse effects than arb therapy alone. aliskiren has not been evaluated as initial or solo therapy, because other drugs have proven benefits when administered in this way. in the usa, the package insert of aliskiren contains a formal recommendation by the fda that the drug should not be used as dual therapy in association with arbs or ace inhibitors in patients with diabetes mellitus and renal disease. in , a new dri, act- , was shown to have an adequate safety profile in humans when administered once daily. act- inhibits plasma renin activity, while the levels of immunoreactive renin increase, a pattern typical of dris. however, the decrease in plasma renin activity is transient and is not dose-dependent at days. the mechanism under lying these observations is not clear, because the plasma levels of act- remain constant. the effects of this dri on blood pressure are controversial, as only one study showed a decrease in blood pressure when act- was used, and this effect was seen in the supine position, whereas other studies showed no differ ences between the effects of act- and placebo on blood pressure in healthy individuals and patients with hypertension. , more studies are necessary to clarify this inconsistency. the same pharmaceutical company that developed act- is developing another dri, act- . in early phase i and phase ii studies, act- has shown a good safety profile. not much information is yet a vailable about the antihypertensive efficacy of act- . for years, different agonist and antagonist peptides were used to study the function of the at receptor. the reported protective effects of this receptor were related to its antifibrotic and anti-inflammatory properties (box ). in , a selective non-peptide at agonist, compound , was introduced for research. most of the known effects of this agent have been described in different animal models. in rats with spontaneous hypertension, the drug reduces the amount of collagen in the extra cellular matrix and in vascular tissue in a bloodpressure-independent manner. compound has a similar effect in animal models of myocardial infarction, reducing scar formation and improving cardiac function. compound reduced proteinuria in the kidney of rat models of hypertension and kidney disease, and also decreased the levels of inflammatory markers in these models. , in the central nervous system, at receptor activation has neuroprotective effects and induces neuro regeneration. compound reproduces these effects, but has to be administered centrally to bypass the blood-brain barrier. however, systemic administration of compound might still have effects on neuroregeneration. compound does not have a net effect on blood pressure, which is similar to the situation seen with other at agonists. however, when the effects of the at receptor on vasoconstriction are blocked, the vasodilator properties of compound are revealed, and blood pressure decreases. , an at antagonist can reverse this effect. a study published in demonstrated that the increased urinary sodium excretion that is induced by compound is mainly due to effects on proximal tubular cells of the kidney, namely internalization of sodium/ hydrogen exchanger (nhe- ) and na + /k + atpase. continuous compound infusion recruited more at receptors to the proximal tubule brush border than an acute infusion. acute compound infusion did not change blood pressure, but in a -day experiment in which concomitant intrarenal ang ii infusion was administered, compound blocked the blood pressure effects of ang ii, increasing natriuresis. more studies are needed to clarify whether infusion time, time to achieve at receptor upregulation, or administration route can influence blood pressure responses to compound . even though no clinical trials are published yet, available evidence suggests that compound could modulate fluid retention, h ypertension and target-organ damage. the benefits of ace in vascular disease might be brought about by two different mechanisms. first, by participating in ang ii degradation, ace decreases the interaction of ang ii with at receptors, and second, by increasing ang - synthesis, ace induces vasodilation. the main function of ace has been proposed to be the counter balancing of the effects of the ace-ang ii-at -aldosterone axis. decreases in ace levels or increases in ace and ang ii levels induced damage in animal models of diabetic nephropathy or hyper tension. , in animal models, the human recombinant form of ace (rhace ) reduced progression of diabetic nephro pathy, decreased blood pressure (in models of hyper tension), and decreased cardiac fibrosis. , in , the pharmaco kinetic and pharmacodynamic characteristics of rhace were described in healthy humans. daily rhace intake decreased the levels of ang ii in a dose-dependent manner over h, with a transient increase in ang - levels, and more sustained increases in ang - degradation products, such as ang - . the authors of this study suggest that rhace is likely to reach the extravascular space, a hypothesis that is based on the large spread of rhace distribution they observed. this enzyme has a good safety profile and did not induce immunogenicity, even when repeated doses were administered. similarly to results reported in healthy animals, rhace did not affect blood pressure or heart rate in humans. thus, rhace is a promising drug for treatment of patients with intolerance to other drugs that target raas components, and also perhaps in acute illnesses, such as myocardial infarction or decompensated heart failure, in which the balance between ace and ace is disturbed. this drug is attractive in the renal field, because rhace might quickly reduce proteinuria, even in associ ation with an ace inhibitor. these two classes of drugs used together might have important protective effects mediated through substantial increases in ang - levels. we look forward to more human clinical trials in this area. aside from cardio vascular effects, some evidence exists that ace might facilitate recovery from acute lung injury, as rhace has been reported to ameliorate virus-mediated lung injury in mouse models. thus, rhace therapy in virus-mediated lung injury, especially influenza, in which ang ii levels are correlated with the degree of lung injury and with mortali ty, is potentially promising. similarly to ace administration, ang - binding to the mas receptor has shown beneficial effects in animal models; these effects are related to decreased fibrosis and inflammation and to blood pressure reduction by vaso dilation and enhancement of baroreflex sensitivity. more than years ago, the non-peptide compound ave showed similar effects to those of ang - in endo thelial cells. subsequent data demonstrated that ave and ang - compete for the same mas receptor in the kidney. this non-peptide mas receptor agonist has been studied in animals, in which it demon strated a capa city to decrease blood pressure, alone or in combination with renin inhibitors. ave was also demonstrated to attenuate acute and chronic renal injury in animal models. , in , ave was shown to improve penile erection through nitric oxide release in rats. ave has also been shown to ameliorate kidney inflammation, and, more interestingly, improve cell infiltration, cytokine release and histology in two rodent models of arthritis. unfortunately, no clinical trials have yet been performed to test this promising therapeutic approach in humans. the development of compounds that protect ang - from enzymatic degradation has also been the focus of interest. the combination of ang - with hydroxylpropyl-β-cyclodextrin protects ang - from degradation and acts as a long-lasting release system, enabling uptake of ang - in the colon. this drug combination has been tested in animal models of hypertension, in which it showed antihypertensive effects, and also had beneficial effects in models of diabetes mellitus and atherosclerotic inflammation. , phase i clinical trials are currently testing the safety profile of this combination therapy in humans (r. a. santos, personal communication). many oligopeptides result from ang ii degradation by endopeptidases. the hexapeptide ang - , known as ang iv, has been studied because it might have a role in protecting cognitive function. irap, the ang iv receptor, which is inhibited by ang iv, was identified in . irap colocalizes with the glucose transporter glut- and was originally thought to contribute to basal intracellular retention of the transporter. irap is linked to vasopressin and oxytocin degradation. a group of nonpeptide inhibitors of irap has been developed. hfi- is an irap-selective pyridinyl compound that enhances memory in rats. an australian group is working on the cardiovascular-protective effects of irap inhibition with hfi- in mice. preliminary data demonstrate that hfi- prevents cardiac and endothelial damage induced by ang ii, independent of blood pressure. hfi- also has anti-inflammatory properties. the association of irap with glut- is intriguing, and whether irap inhibitors could be used to improve insulin sensitivity remains an interesting opportunity. more studies are n ecessary to explore therapeutic uses for irap inhibitors. vaccines against the raas the high prevalence of hypertension in the world and the related cost of this burden have made the development of vaccines against the raas an attractive strategy, with the aim of achieving increased treatment compliance and public access to therapy, and a decrease in public-health costs. many attempts were made to generate vaccines against renin, ang i, ang ii and at . however, most of these attempts have been harmful or have not had clinical efficacy. an rct in which an anti-ang ii vaccine (cyt -angqb) was tested passed phase iia without safety problems, and showed a promising h reduction in blood pressure of . mmhg (systolic) and . mmhg (diastolic). however, subsequent studies could not confirm these results. novel strategies are currently in development, such as the association of ang ii as an epitope to a hepatitis a virus-like particle to generate an improved immune response. future studies are needed to explore the efficacy and safety of an i mmunological, chronic and irreversible blockade of the raas. although to improve upon the cardiovascular and renal protection offered by ace inhibitors and arbs will be diffi cult, novel drugs are being developed to shift the activity of the raas towards the protective cardiovascular arm (at and mas receptors). a role for raas blockade in other, non-cardiovascular pathologies (such as lung injury and cognitive disorders) is emerging. the combination of raas inhibition with other autacoid systems (endothelin blockade or natriuretic peptide stimulation) could improve the efficacy of traditional raas blockade and be the basis of specific tissue-targeted drug development. the substance causing renal hypertension the existence of two forms of hypertensin studies on angiotensinogen formation in a liver perfusion system stimulation of aldosterone secretion by angiotensin ii. a preliminary report mechanism of pulmonary conversion of angiotensin i to angiotensin ii in the dog historical perspective of the reninangiotensin system the role of angiotensin iistimulated renal tubular transport in hypertension renal proximal tubule angiotensin at a receptors regulate blood pressure chronic angiotensin ii infusion drives extensive aldosteroneindependent epithelial na + channel activation effect of angiotensin ii and of an angiotensin ii analogue (sar -ile -angiotensin ii) on blood pressure, plasma aldosterone and plasma renin activity in the dog expression of aldosterone synthase cytochrome p (p aldo) mrna in rat adrenal glomerulosa cells by angiotensin ii type receptor mechanisms of mineralocorticoid action effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. results of the survival and ventricular enlargement trial. the save investigators renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type diabetes congestive heart failure in renal transplant recipients: risk factors, outcomes, and relationship with ischemic heart disease effects of an angiotensinconverting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. the heart outcomes prevention evaluation study investigators effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy determinants of interindividual variation of renin and prorenin concentrations: evidence for a sexual dimorphism of (pro)renin levels in humans renin/prorenin receptors a novel signal transduction cascade involving direct physical interaction of the renin/prorenin receptor with the transcription factor promyelocytic zinc finger protein the (pro)renin receptor. a decade of research: what have we learned? the role of angiotensin ii in the feedback control of renin gene expression angiotensin ii and inflammation: the effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockade at receptor agonists: hypertension and beyond a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase update on the angiotensin converting enzyme -angiotensin ( - )-mas receptor axis: fetal programing, sex differences, and intracellular pathways angiotensin-converting enzyme , angiotensin-( - ) and mas: new players of the renin-angiotensin system circulating aminopeptidase activities in men and women with essential hypertension discovery of inhibitors of insulin-regulated aminopeptidase as cognitive enhancers angiotensin iv-evoked vasoprotection is conserved in advanced atheroma chronic angiotensin iv treatment reverses endothelial dysfunction in apoe-deficient mice at receptor/insulin regulated aminopeptidase inhibition protects against angiotensin ii-induced cardic fibrosis and vascular dysfunction role of angiotensin in ovarian follicular development and ovulation in mammals: a review of recent advances improved glucose-stimulated insulin secretion by selective intraislet inhibition of angiotensin ii type receptor expression in isolated islets of db/db mice physiology of local renin-angiotensin systems human t and natural killer cells possess a functional renin-angiotensin system: further mechanisms of angiotensin ii-induced inflammation prognostic impact of reninangiotensin system blockade in esophageal squamous cell carcinoma inhibition of renin-angiotensin system affects prognosis of advanced pancreatic cancer receiving gemcitabine inhibition of the renin-angiotensin system and target organ protection effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive heart failure. the solvd investigators sustained effectiveness of converting-enzyme inhibition in patients with severe congestive heart failure n-acetyl-seryl-aspartyl-lysylproline reduces cardiac collagen cross-linking and inflammation in angiotensin ii-induced hypertensive rats effects of captopril related to increased levels of prostacyclin and angiotensin-( - ) in essential hypertension guidelines for managing high blood pressure management of hypertension: summary of nice guidance an effective approach to high blood pressure control: a science advisory from the american heart association, the american college of cardiology, and the centers for disease control and prevention regression of left ventricular mass by antihypertensive treatment: a meta-analysis of randomized comparative studies angiotensin-converting enzyme inhibitors reduce mortality in hypertension: a meta-aalysis of radomized clinical trials of renin-angiotensin-aldosterone system inhibitors involving , patients a systematic comparison of the properties of clinically used angiotensin ii type receptor antagonists comparative risk for angioedema associaed with the se of drugs that target the renin-angiotensin-aldosterone system cardiovascular morbidity and mortality in the losartan intervention for endpoint reduction in hypertension study (life): a randomised trial against atenolol effects of the angiotensinreceptor blocker telmisartan on cardiovascular events in high-risk patients intolerant to angiotensin-converting enzyme inhibitors: a randomised controlled trial telmisartan, ramipril, or both in patients at high risk for vascular events effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: randomised trial -the losartan heart failure survival study elite ii effects of candesartan in patients with chronic heart failure and reduced left-ventricular systolic function taking angiotensin-converting-enzyme inhibitors: the charm-added trial angiotensin-receptor blockade versus converting-enzyme inhibition in type diabetes and nephropathy cardiorenal end points in a trial of aliskiren for type diabetes combined angiotensin inhibition for the treatment of diabetic nephropathy eplerenone in patients with systolic heart failure and mild symptoms eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction aldosterone receptor blockers spironolactone and canrenone: two multivalent drugs introduction to the revised american association for the study of liver diseases practice guideline management of adult patients with ascites due to cirrhosis the effect of spironolactone on morbidity and mortality in patients with severe heart failure. randomized aldactone evaluation study investigators low dose spironolactone reduces blood pressure in patients with resistant hypertension and type diabetes mellitus: a double blind randomized clinical trial efficacy of spironolactone therapy in patients with true resistant hypertension natriuretic peptides in the regulation of the hypothalamicpituitary-adrenal axis effects of natriuretic peptides upon hypothalamopituitary-adrenocortical system activity and anxiety behaviour molecular biology of the natriuretic peptide system: implications for physiology and hypertension biology of natriuretic peptides and their receptors blood-pressure reduction with lcz , a novel dual-acting inhibitor of the angiotensin ii receptor and neprilysin: a randomised, double-blind, placebo-controlled, active comparator study omapatrilat and enalapril in patients with hypertension: the omapatrilat cardiovascular treatment vs. enalapril (octave) trial pharmacokinetics and pharmacodynamics of lcz , a novel dualacting angiotensin receptor-neprilysin inhibitor (arni) efficacy and safety of lcz , a first-in-class angiotensin receptor neprilysin inhibitor, in asian patients with hypertension: a randomized, double-blind, placebo-controlled study rationale and study design of the prospective comparison of angiotensin receptor neprilysin inhibitor with angiotensin receptor blocker measuring arterial stiffness in the elderly (parameter) study the angiotensin receptor neprilysin inhibitor lcz in heart failure with preserved ejection fraction: a phase doubleblind randomised controlled trial atrial natriuretic peptide causes pre-glomerular vasodilatation and post-glomerular vasoconstriction in rat kidney angiotensin-neprilysin inhibition versus enalapril in heart failure baseline characteristics and treatment of patients in prospective comparison of arni with acei to determine impact on global mortality and morbidity in heart failure trial (paradigm-hf) independence of the blood pressure lowering effect and efficacy of the angiotensin receptor neprilysin inhibitor, lcz , in patients with heart failure with preserved ejection fraction: an analysis of the paramount trial discovery of bay - : a nonsteroidal antagonist of the mineralocorticoid receptor for the treatment of cardiorenal diseases finerenone, a novel selective nonsteroidal mineralocorticoid receptor antagonist protects from rat cardiorenal injury safety and tolerability of the novel non-steroidal mineralocorticoid receptor antagonist bay - in patients with chronic heart failure and mild or moderate chronic kidney disease: a randomized, double-blind trial a new mode of mineralocorticoid receptor antagonism by a potent and selective nonsteroidal molecule sm- , a novel selective and potent non-steroidal mineralocorticoid receptor antagonist with strong urinary na + excretion activity antihypertensive and cardiorenal protective effects of sm- , a novel mineralocorticoid receptor antagonist, in aldosterone/salt-treated rats investigation of aldosterone-synthase inhibition in rats aldosterone antagonism or synthase inhibition reduces end-organ damage induced by treatment with angiotensin and high salt aldosterone synthase inhibition improves cardiovascular function and structure in rats with heart failure: a comparison with spironolactone aldosterone synthase inhibition with lci : a proof-of-concept study in patients with primary aldosteronism the effects of aldosterone synthase inhibition on aldosterone and cortisol in patients with hypertension: a phase ii, randomized, double-blind, placebo-controlled, multicenter study effects of a novel aldosterone synthase inhibitor for treatment of primary hypertension: results of a randomized, double-blind, placebo-and active-controlled phase trial aldosterone synthase inhibition with lci : a proof-of-concept study in patients with primary aldosteronism lci , a potent β-hydroxylase inhibitor, normalizes urinary cortisol in patients with cushing's disease: results from a multicenter, proof-of-concept study n-(pyridin- -yl)benzamides as selective inhibitors of human aldosterone synthase (cyp b ) aldosterone synthase inhibition in humans microrna- is a novel regulator of aldosterone and cortisol production in the human adrenal cortex pharmacokinetics, pharmacodynamics, and tolerability of act- , a new direct renin inhibitor after multiple-ascending doses in healthy subjects entry-into-humans study with a new direct renin inhibitor effects of the renin inhibitor mk- (act- ) in patients with hypertension age and sex effects on the single-and multiple-dose safety and pharmacokinetics of the new renin inhibitor act- angiotensin type receptor agonist compound reduces vascular injury and myocardial fibrosis in stroke-prone spontaneously hypertensive rats angiotensin ii type receptor stimulation: a novel option of therapeutic interference with the renin-angiotensin system in myocardial infarction? stimulation of at receptor exerts beneficial effects in stroke-prone rats: focus on renal damage angiotensin at( ) receptor stimulation inhibits early renal inflammation in renovascular hypertension direct angiotensin at receptor stimulation using a novel at receptor agonist, compound , evokes neuroprotection in conscious hypertensive rats at -receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating bdnf expression at receptor activation induces natriuresis and lowers blood pressure angiotensinconverting enzyme : the first decade glomerular localization and expression of angiotensin-converting enzyme and angiotensin-converting enzyme: implications for albuminuria in diabetes transgenic angiotensinconverting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function human recombinant ace reduces the progression of diabetic nephropathy angiotensin-converting enzyme suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme in healthy human subjects angiotensin-converting enzyme protects from lethal avian influenza a h n infections angiotensin ii plasma levels are linked to disease severity and predict fatal outcomes in h n -infected patients ave , a nonpeptide mimic of the effects of angiotensin-( - ) on the endothelium nonpeptide ave is an angiotensin-( - ) receptor mas agonist in the mouse kidney effect of combination of renin inhibitor and masreceptor agonist in doca-salt-induced hypertension in rats renoprotective effects of ave , a nonpeptide mas receptor agonist, in experimental acute renal injury beneficial effects of the activation of the angiotensin-( - ) mas receptor in a murine model of adriamycininduced nepopathy ave , a non-peptide mas-receptor agonist, facilitates penile erection anti-inflammatory effects of the activation of the angiotensin-( - ) receptor, mas, in experimental models of arthritis an orally active angiotensin-( - ) inclusion compound and exercise training produce similar cardiovascular effects in spontaneously hypertensive rats treatment with angiotensin-( - ) reduces inflammation in carotid atherosclerotic plaques oral administration of angiotensin-( - ) ameliorates type diabetes in rats evidence that the angiotensin iv (at( )) receptor is the enzyme insulin-regulated aminopeptidase insulin-regulated aminopeptidase is a key regulator of glut trafficking by controlling the sorting of glut from endosomes to specialized insulin-regulated vesicles identification and characterization of a new cognitive enhancer based on inhibition of insulin-regulated aminopeptidase vaccine for hypertension: modulating the renin-angiotensin system effect of immunisation against angiotensin ii with cyt -angqb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase iia study construction, expression and immunogenicity of a novel anti-hypertension angiotensin ii vaccine based on hepatitis a virus-like particle author contributions c.a.r. researched data for the article. c.a.r. and m.r.w. wrote the article. all authors contributed substantially to discussion of content and reviewing and/or editing the manuscript before submission. key: cord- -y et pqg authors: dong, xiaoxv; fu, jing; yin, xingbin; yang, chunjing; zhang, xin; wang, wenping; du, xueying; wang, qingling; ni, jian title: cassiae semen: a review of its phytochemistry and pharmacology date: - - journal: mol med rep doi: . /mmr. . sha: doc_id: cord_uid: y et pqg cassiae semen (leguminosae), a well-known traditional chinese medicine, has been used for a number of centuries in areas of southeast asia, including korea, japan and china. the present review aims to provide updated and comprehensive information, on the botany, phytochemistry and pharmacology of cassiae semen. the available information on cassiae semen was collected using several different resources, including classic books on chinese herbal medicine and a number of scientific databases, including the china academic journals full-text database, pubmed, scifinder, the web of science and science direct. to date > chemical compounds have been isolated from cassiae semen, and the major components have been determined to be anthraquinones, naphthopyrones and volatile oil. the crude extracts and pure compounds of cassiae semen have been used as effective agents in preclinical and clinical practice due to their beneficial activities, including antihyperlipidemic, antidiabetic, neuroprotective, hepatoprotective, antibacterial, antioxidant and hypotensive activities. with the body of reported data, it has been suggested that cassiae semen has convincing medicinal potential. however, the pharmacological mechanisms of the main bioactive compounds and the association between structure and activity require further investigation. cassiae semen, also known as ʻjuemingziʼ in chinese, is the dry and mature seed of cassia obtusifolia l. or c. tora l., which belong to the cassia genus of leguminosae ( ) . it is cultivated in korea, japan and china, and is commonly consumed as a roasted tea ( , ) . in traditional chinese medicine, it has been used in treatments for hyperlipemia, diabetes mellitus, alzheimer's disease, acute liver injury, inflammation, photophobia, headache, dizziness and hypertension ( ) ( ) ( ) . phytochemical investigations have isolated and identified > compounds, including anthraquinones, naphthopyrones, volatile oils and sterols ( , ) . among these, anthraquinones are the primary functional components and possess a wide spectrum of pharmacological properties ( ) ( ) ( ) , including antihyperlipidemic, neuroprotective, hepatoprotective, antibacterial and antimutagenic activities ( ) ( ) ( ) . naphthopyrones, other primary components, exhibit antidiabetic ( , ) , antimicrobial ( ) , antiestrogenic ( ) , antiallergic ( ) and anthelmintic effects ( ) . at present, the pharmacopoeia of the people's republic of china recommends the use of chrysophanol and aurantio-obtusin as the indicator components, and the quality of cassiae semen is evaluated primarily by assessing the content of these two compounds ( ) . the purpose of the present review is to provide comprehensive information on the ethnobotany, phytochemistry and pharmacology of cassiae semen collated from previous studies, in order to facilitate the further study and application of cassiae semen, as well as generate a novel basis for the associations between structure and activity, and their molecular mechanisms of action. pairs and are glaucous, membranous, glabrous or pubescent, and have obcordate or obovate oblong morphology ( - cm long x . - . cm wide); the base is somewhat oblique, usually rounded and there are - pairs of main nerves. the petiolules are . - mm in length and their stipules are linear, pilose and caducous. it blossoms from july to september and produces fruit from september to october. flowers are usually in subsessile pairs in leaf axils, the pedicels are filiform and are - . cm in length. calyces are ovate, glabrous, membranous and comprised of five-parts; there are five petals, which are pale yellow, oblong, obtuse and the upper petal (standard) is two-lobed. the flowers have stamens, while the upper three are reduced to minute staminodes. the pods are slender, puberulous, subtetragonous, obliquely septate and, are ~ cm in length and - mm in width. however, the seeds of c. obtusifolia are a dark brown or green-brown, rhombohedral or short cylindrical, and are - mm in length and - mm in width. while c. tora seeds are a light brown, shiny, short cylindrical, and are - mm in length and - mm in width ( , ) . c. obtusifolia is cultivated in multiple provinces of china, including henan, hubei, shanxi, sichuan, zhejiang and anhui, and also other countries, including korea, india and japan. it is primarily distributed in moist and sunny places, in hillside shrubs and in the sandy soil of river banks ( ) . as a widely used traditional chinese medicine, there are some adulterants of this plant, including the seeds of c. occidentalis (leguminosae), c. sophera (leguminosae) and sesbania aculeata pers (papilionaceae) ( ) ( ) ( ) . to date, a number of methods have been developed to identify and distinguish these, including experiential identification, morphological identification, ultraviolet spectrophotometry, the thin layer chromatography method, high performance liquid chromatography (hplc), hplc-coupled with time-of-flight and ion trap mass spectrometry, and sds-page ( ) ( ) ( ) ( ) . among these methods, the hplc method is regarded as the most popular method for evaluating the quality and authenticity of cassiae semen. chrysophanol and aurantio-obtusin are used as the indicator compounds to characterize the quality of this plant and the minimum contents are defined as . and . %, respectively, in the pharmacopoeia of the people's republic of china ( ). a number of compounds, including anthraquinones, naphthopyrones, volatile oils and sterols, have been isolated from cassiae semen. anthraquinones and naphthopyrones, which exhibit multiple pharmacological activities, are considered the primary active ingredients of cassiae semen. all compounds isolated from cassiae semen are listed in table i, anthraquinones. cassiae semen contains structurally diverse and biologically active anthraquinones. thus far, ~ anthraquinones have been isolated and identified. the predominant anthraquinones are emodin-type anthraquinones, which include emodin, chrysophanol, physcion, aloe-emodin, rhein, obtusin, chryso-obtusin, aurantio-obtusin, obtusifolin, questin, -desmethylaurantio-obtusin, -desmethylobtusin, -desmethylchryso-obtusin, chrysophanol- , '-bianthrone, , -dihydroxyanthraquinone, -hydroxyemodin- -methylether, alaternin, , -dihydroxy- -methoxy- -methyl anthraquinone, -hydroxy- , -diformyl anthraquinone, chrysarobin, -o-methylchrysophanol, -o-methylemodin, , -dimethoxy- -hydroxy- -methyl- , -anthraquinone and l, , -trimethoxyl- , -dihydroxy- -methylanthraquinone (compounds - , respectively; fig. ); these have all been isolated from cassiae semen ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . there are also many combined anthraquinones (compounds - ; fig. ), which have been isolated from the seeds of c. obtusifolia or c. tora ( , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . naphthopyrones. naphthopyrones are the other characteristic components in cassiae semen. in , torachrysone, rubrofusarin and rubrofusarin- -o-β-d-gentiobioside were isolated from c. tora seeds (compounds - , respectively; fig. ) ( , ) . subsequently, toralactone, rubrofusarin triglucoside, cassiaside, nor-rubrofusarin gentiobioside, cassiaside b, cassiaside c, torachrysone apiglucoside, torachrysone gentiobioside, torachrysone tetraglucoside, cassiatoroside, demethylflavasperone gentiobioside and cassialactone gentiobioside were isolated from c. tora seeds (compounds - , respectively; fig. ) ( , , ( ) ( ) ( ) . in addition, cassiaside b and cassiaside c were identified in the seeds of c. obtusifolia ( ) . other naphthopyrones, including torosachrysone, isotoralactone, cassialactone, cassiaside b , cassiaside c , nor-rubrofusarin volatile oils. li ( ) extracted the volatile oils from cassiae semen by steam distillation, and subsequently identified components according to the gas chromatography/mass spectrometry analysis. among these peaks, the major volatile components were -octadecenoic acid (e) ( . %), n-hexadecanoic acid ( . %), , -anthracenedione, , -dihydroxy- -methyl ( . %), octadecanoic acid ( . %) and -octadecenoic acid methyl ester (z) ( . %) ( ) . other compounds. a range of other components have been isolated from cassiae semen, including malvalic acid, sterculic acid, mandelic acid, campesterol, aspidinol and , -dihydroxychromone (compounds - , respectively; fig. ) ( , , ) . in addition, the four f lavonoid compounds, chrysin, chrysin- -o-β-d-glucoside, galangin and cyanidenon, were also obtained and identified from cassiae semen (compounds - , respectively; fig. ) ( ). cassiae semen exerts a great variety of pharmacological activities due to its complex bioactive compounds. an overview of the pharmacological studies on cassiae semen is presented in detail in the following sections. antihyperlipidemic activity. in traditional chinese herbal medicine, cassiae semen is used for the prevention and alaternin- -o-β-d-glucopyranoside ( ) alaternin- -o-β-d-glucopyranoside ( ) aurantio-obtusin- -o-β-d-glucopyranoside ( ) chryso-obtusin- -o-β-d-glucopyranoside ( ) obtusifolin rubrofusarin ( ) rubrofusarin treatment of hyperlipidemia. several chinese herbal formulations containing cassiae semen is available in the chinese market for preventing the formation of atherosclerotic plaques ( ) . in certain asian countries, including china and korea, it is also commonly drunk as a roasted tea to reduce body weight ( , ) . previous studies using mice have evaluated the reductions in blood lipid contents induced by different cassiae semen extracts obtained through different methods, including supercritical fluid extraction, systematic solvents (petroleum ether, ethyl acetate, n-butanol, % ethanol and water) and ethanol precipitation following water extraction. the results revealed that the n-butanol and ethyl acetate extracts were the most effective ( , ) . in addition, the ethanol and aqueous extracts of cassiae emen significantly decreased the serum levels of total cholesterol (tc), triglyceride (tg) and low-density lipoprotein cholesterol (ldl-c), however, they increased the levels of high-density lipoprotein cholesterol (hdl-c) ( , , ) . similarly, he et al ( ) reported that treatment with the water extract form of c. obtusifolia seeds decreased the blood-lipid level by inhibiting cholesterol synthesis. cho et al ( ) demonstrated that soluble fibers from c. tora seeds markedly decreased liver tc and tg levels in rats fed with a high-cholesterol diet. the underlying mechanism may be mediated by increasing fecal bile acid excretion and downregulating the production of lipogenic enzymes ( ) . in addition, soluble fibers decreased the serum levels of tc, tg and ldl-c in patients with type ii diabetes without serious adverse effects ( ). liu et al ( ) revealed that the ethanol extract of cassiae semen upregulated the expression levels of peroxisome proliferator-activated receptor (ppar)-γ, sterol regulatory element-binding protein- c, hormone-sensitive lipase and triacylglycerol hydrolase, however, tumor necrosis factor receptor superfamily member was downregulated in adipose tissue. the anti-hyperlipidemia activity of cassiae semen is primarily due to its antioxidant components, such as anthraquinones and polysaccharides. there are a variety of bioactive anthraquinone components in cassiae semen, including chrysophanol, physcion, aurantio-obtusin, obtusifolin and emodin, which have been observed to decrease the levels of tc and tg ( , ) . previous studies have demonstrated that anthraquinones isolated from cassiae semen rubrofusarin triglucoside ( ) cassiaside ( ) nor-rubrofusarin gentiobioside ( ) cassiaside b ( ) cassiaside c ( ) torachrysone apiglucoside ( ) torachrysone gentiobioside ( ) torachrysone tetraglucoside ( ) cassiatoroside ( ) demethylflavasperone gentiobioside ( ) cassialactone gentiobioside ( ) torosachrysone ( ) isotoralactone ( ) cassialactone ( ) cassiaside ( , ) . these results were verified by a previous study, which applied an experimental hyperlipidemic rat model to investigate anthraquinone treatment ( and mg/kg, per os, for days). the tc, tg and ldl-c levels were significantly reduced in a dose-dependent manner, however, the levels of hdl-c increased. inhibition of cholesterol synthesis may be one of the underlying mechanisms involved in decreasing blood lipid levels ( ) . water-soluble polysaccharides (wsps) from cassiae semen markedly inhibited the activities of α-amylase and pancreatic lipase, however, protease activity increased. the results demonstrated that wsps had the ability to bind to bile acids and reduce the absorption of cholesterol, indicating that wsps may have potential as an effective herbal ingredient in functional food applications ( ) . a number of studies have demonstrated that cassiae semen exhibits anti-diabetic activity. a total of three naphthopyrone glucosides (compounds , and ) isolated from the butanol-soluble extract of cassia semen have been evaluated for their inhibitory activity on advanced glycation end products (ages) formation in vitro. the results revealed that these compounds possessed more potent inhibitory activity against ages compared with the aminoguanidine positive control ( ) . in addition, rubrofusarin- -o-βd-gentiobioside (compound ) and cassiaside (compound ) significantly inhibited the expression of transforming growth factor (tgf)- and extracellular matrix protein in glomerular mesangial cells cultured under diabetic conditions, suggesting that the active compounds in cassiae semen may be effective in the treatment of renal complications associated with diabetes ( ) . similarly, kim et al ( ) evaluated the preventive effects of the methanol extract of cassia semen ( mg/kg/day, for weeks) on the development of diabetic nephropathy in streptozotocin (stz)-induced diabetic rats. the results indicated that oral treatment with the cassia semen methanol extract inhibited the development of diabetic nephropathy by inhibiting ages accumulation, receptor for advanced glycosylation end product and cyclooxygenase- expression in the renal cortex of stz-diabetic rats ( , ) . in addition, zhu ( ) reported that the water extract of cassia semen exhibited protective activity against stz-induced renal fibrosis in diabetic rats. the underlying mechanisms may be associated with its ability to downregulate the expression of tgf-β , connective tissue growth factor and mothers against decapentaplegic homolog (smad ), as well as upregulating the protein expression of smad ( ) . neuroprotective activity. the ethanolic extract from the seeds of c. obtusifolia has been reported to have a neuroprotective effect in brain disease models. kim et al ( ) suggested that c. obtusifolia ( , or mg/kg) significantly attenuated scopolamine or transient bilateral common carotid artery occlusion ( vo)-induced memory impairment. these effects are mediated by the enhancement of the cholinergic nervous system via acetylcholinesterase inhibition in a dose-dependent manner [half maximal inhibitory concentration (ic )= . µg/ml] ( ). in addition, c. obtusifolia ( or mg/kg/day) exhibited a neuroprotective effect in a mouse transient global ischemia model due to its anti-inflammatory properties and the induced upregulated expression of phosphorylated cyclic amp response element binding protein and brain-derived neurotrophic factor ( ). drever et al ( ) demonstrated that treatment with c. obtusifolia ( . - µg/ml) significantly attenuated secondary calcium dysregulation and cell death induced by n-methyl-d-aspartate and -nitropropionic acid in mouse hippocampal cultures, and no significant effect on cell death was induced by incubation with naturally-secreted oligomers of amyloid (a)β. yi et al ( ) reported for the first time, that c. obtusifolia ( µg/ml) ameliorated the aβ-induced synaptic dysfunction model through anti-inflammatory and protein kinase b (akt)/glycogen synthase kinase- β pathways. the results suggested that the neuroprotective effect may be attributable to obtusifolin (compound ) and/or alaternin (compound ) ( ) . in a further experiment, c. obtusifolia ( . - µg/ml) inhibited cell damage against oxidopamine ( -ohda)-induced dopaminergic (da) neural toxicity in pc cells through an anti-oxidant and antimitochondrial-mediated apoptosis mechanism. in a mesencephalic da culture, c. obtusifolia ( . - µg/ml) protected the da cells against -ohda-and n-methyl- -phenylpyridinium iodide-induced in vivo ( ) function and attenuates mi/r-induced injury tc, total cholesterol; tg, triglyceride; ldl-c, low-density lipoprotein cholesterol; hdl-c, high-density lipoprotein cholesterol; pparγ, peroxisome proliferator-activated receptor; srebp- c, sterol regulatory element-binding protein- c; hsl, hormone-sensitive lipase; tgh, triacylglycerol hydrolase; fas, tumor necrosis factor receptor superfamily member ; ages, advanced glycation end products; tgf- , transforming growth factor- ; ecm, extracellular matrix; rage, receptor for advanced glycosylation end product; cox- , cyclooxygenase- ; ctgf, connective tissue growth factor; smad / , mothers against decapentaplegic homolog and ; ache, acetylcholinesterase; pcreb, phosphorylated cyclic amp response element binding protein; bdnf, brain-derived neurotrophic factor; da, dopaminergic; mda, malondialdehyde; mao, monoamine oxidase; sod, superoxide dismutase; ast, aspartate transaminase; alt, alanine transaminase; dpph, -diphenyl- -picrylhydrazyl; mi/r, myocardial ischemia and reperfusion. toxicities. in addition, c. obtusifolia ( mg/kg/day for days) significantly protected da neuronal degeneration in a -methyl- -phenyl- , , , -tetrahydropyridine-induced mouse parkinson's disease (pd) model by inhibiting the movement impairment and the loss of da neurons, indicating that c. obtusifolia may be a useful neuroprotective candidate for pd ( ) . in addition, protein and anthraquinone glucosides from cassia semen improved learning and memory capacity, inhibited the malondialdehyde (mda) and monoamine oxidase levels, and enhanced the level of superoxide dismutase (sod) in the cerebrum of senile mice ( ) . hepatoprotective activity. it was recorded in the compendium of materia medica that cassiae semen exhibited the functions of nourishing the liver and improving vision ( ) . in korea, the aqueous extract of c. tora l. seeds has been used for the protection of the liver. a weak anti-hepatotoxic activity in ccl -induced mice was observed when the drug was administered orally at a dose of mg/kg ( ) . the methanol extract of c. tora l. seeds exhibited significantly protective effects in primary cultured hepatocytes against ccl and toxicity. a total of two anthraquinone glycosides (compounds and ) and three naphthoypyrone glycosides (compounds , and ) from the methanol extract of c. tora seeds were the primary chemical constituents ( , ) . the preventative effects of cassiae semen on acute liver injury in mice induced by ccl have also been investigated ( , ) . when compared with the control group, varying concentrations of the aqueous extract of cassiae semen significantly increased the serum levels of sod, and decreased the serum levels of aspartate transaminase (ast), alanine transaminase (alt) and mda. these results indicate that cassiae semen is potentially beneficial in the treatment or prevention of hepatic damage ( , ) . in addition, the ethanol extract of cassiae semen has been observed to increase the serum levels of sod and to decrease the serum levels of tg, tc, mda, ast and alt ( ). total anthraquinones from cassiae semen exhibited a protective effect on alcohol-induced acute liver injury in mice by regulating fat metabolism, improving liver function and increasing the mrna and protein expression levels of ppar-γ ( , ) . antibacterial activity. antibacterial activity, an important effect of cassiae semen, has been comprehensively investigated. naphthalenes (compounds and ) and anthraquinones (compounds , and ) isolated from c. tora seeds exhibited significant antibacterial effects on four strains of methicillin-resistant staphylococcus aureus [minimal inhibitory concentration (mic) was - µg/ml] and a strain of methicillin-sensitive s. aureus. in addition, rhein (compound ) and torachrysone (compound ) from the seeds of c. tora exhibited antibacterial activity against escherichia coli k with mic values of and µg/ml, respectively ( ) . kim et al ( ) were the first to demonstrate that emodin (compound ) from c. tora seeds has a median lethal dose (lc ) value of . , . , . and . g/l against rhizoctonia solani, botrytis cinerea, phytophthora infestans and erysiphe graminis, respectively, and physcion (compound ) has an lc value of . , . , . , and . g/l against r. solani, b. cinerea, p. infestans and e. graminis, respectively. in addition, the lc value of rhein (compound ) is . , . , and . g/l against r. solani, b. cinerea and p. infestans, respectively ( ) . it has been reported that ethanol and aqueous extracts of c. obtusifolia seeds were inhibitory against helicobacter pylori strains (mic were and µg/ml, respectively) ( ). in addition, , -dihydroxyanthraquinone (compound ) isolated from c. obtusifolia seeds was revealed to inhibit the growth of clostridium perfringens and e. coli., indicating that this drug exhibited potent growth-inhibiting activities towards human intestinal bacteria ( ) . li et al ( ) demonstrated that the chloroform extract of the seeds of c. obtusifolia also exhibited different inhibitory activities against fusarium oxysporum and b. cinerea (ic values were . mg/ml and . mg/ml). antioxidant activity. the water extract of c. tora seeds accelerated the oxidation of deoxyribose induced by fe + -edta/h o and exhibited % inhibition of linoleic acid peroxidation at a concentration of . mg/ml. the underlying mechanisms of this may be mediated by reducing metal ions, scavenging hydroxyl radical and chelating ferrousion ( , ) . xv and hu ( ) demonstrated that the water extract of cassiae semen exhibited a potent ability to scavenge free oxygen radicals [ic values were mg/ml and µg/ml for hydroxyl radicals (oh -) and hydrogen peroxide (h o ), respectively]. wsp from cassiae semen ( . mg/ml) effectively inhibited superoxide radicals (o -) induced by pyrogallol autoxidation ( ) . the inhibitory effects of wsp on serum levels of mda were used to evaluate its antioxidation capabilities. the results demonstrated that wsp decreased mda serum levels with an ic value of . % ( ) . in another study, liu et al ( ) optimized the extraction conditions for wsp of cassiae semen (temperature ˚c, extraction time . h, solid-liquid ratio : ) and observed that wsp ( . µg/ml) had the ability to scavenge hydroxyl and superoxide radicals with scavenging rates of . and . %, respectively. in addition, ethyl acetate fraction and n-butanol fraction of cassiae semen were evaluated by dpph radical scavenging activity. the results revealed that the ethyl acetate fraction had a lower ic value of . g/ml, when compared with the value of . g/ml for n-butanol fraction. -desmethylaurantio-obtusin (compound ) exhibited good scavenging activity on dpph with an ic value of . ± . g/ml, while aurantio-obtusin- -oβ-d-glucopyranoside (compound ) and questin (compound ) exhibited moderate antioxidant activity, and their ic values were . ± . g/ml and . ± . g/ml, respectively. when compared with these results, chryso-obtusin (compound ) and aurantio-obtusin (compound ) demonstrated weaker antioxidant activity (ic > µg/ml) ( ) . the methanolic extract of c. tora seeds exhibited a high antioxidant activity on lipid peroxidation ( ) . similarly, in another study, yen et al ( ) demonstrated that the methanolic extract of c. tora seeds exerted a greater antioxidant activity than the other organic solvents (n-hexane and ethyl acetate). emodin was also revealed to be an antioxidative component ( ) . in addition, alaternin (compound ), cassiaside (compound ) and rubrofusarin- -o-β-d-gentiobioside (compound ) isolated from c. tora seeds exhibited good scavenging activity against dpph radicals with ic values of . , . and . µg/ml, respectively ( ). hypotensive activity. aqueous and ethanol extracts of cassiae semen have been reported to possess hypotensive effects ( ). koo et al ( ) reported that the water extract of c. tora seeds ( . , . , , , and mg/kg) consistently reduced arterial blood pressure in anesthetized rats. a potential reflex mechanism of this hypotensive action may involve a vagal reflex, which reciprocally inhibits the peripheral vasomotor tone via a reflex reduction in the sympathetic neural outflow to blood vessels ( ) . in addition, the media portion of the medullary reticular formation has been revealed to be directly involved in the hypotensive effect of c. tora seeds ( ) . furthermore, the ethanol extract of cassiae semen significantly decreased blood pressure in hypertensive rats by inhibiting receptor-controlled calcium channels on vessels and regulating the secretion of nitric oxide and inducible nitric oxide synthase ( ) . other activities. in addition to the pharmacological effects described above, cassiae semen and its ingredients have other pharmacological effects, including estrogenic, anti-allergic, antigenotoxic, anti-aggregatory, antimutagenic and cardioprotective effects. some of these effects are discussed briefly below. the estrogenic activity of c. obtusifolia seeds was evaluated by a recombinant yeast screening assay. the results revealed that % etoh extracts of this drug exhibited estrogenic relative potency [half maximal effective concentration (ec ) was . µg/ml) ( ) . cassiaside c (compound ) isolated from c. obtusifolia seeds exhibited a potent anti-allergic activity by inhibiting the histamine release from mast cells induced by antigen-antibody reaction ( ) . furthermore, gluco-aurantioobtusin (compound ) from c. obtusifolia seeds possessed potent inhibitory activities against arachidonic-acid-, adp-and collagen-induced platelet aggregations ( ). wu and yen ( ) demonstrated that the water extract of c. tora seeds exhibited potential antigenotoxic activities against the dietary mutagens glu-p- and trpp- in the ames test and the comet assay. the potential mechanisms may be associated with neutralization of the reactive intermediate of trp-p- and an antioxidant effect of the tested compounds ( ) . anthraquinone aglycones (compounds , and ) and naphthopyrone glycosides (compounds and ) from c. tora seeds exhibited significant antimutagenic activity in vitro. the mechanism associated with these compounds may be mediated via interactions with a microsomal activating system ( ) . fu et al ( ) reported that the water extract of cassiae semen ( mg/kg/day, for one week) effectively improved myocardial function, and attenuated myocardial ischemia and reperfusion-induced injury and apoptosis in diabetic animals, which is potentially attributable to the reduced plasma lipid levels and the triggered cell survival akt and extracellular signal-regulated kinases / signaling. in traditional chinese medicine, cassiae semen has long been used to clean the liver, brighten the eye, loosen the bowel to relieve constipation, and for the treatment of inflammation, photophobia, headaches, dizziness, hyperlipemia and alzheimer's disease. in addition, cassiae semen is commonly used in the composition of other herbs. although modern experiments have confirmed that this drug alone exhibits multiple pharmacological activities, it is important to investigate the molecular mechanisms of cassiae semen combined with other herbs based on traditional uses. a number of studies have investigated the effective constituents of cassiae semen from different batches and geographical areas. hplc-fingerprint chromatography is a common method to compare the differences ( - ). zhang et al ( ) developed a sensitive and reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (uhplc-esi-ms/ms) method to evaluate the quality of cassiae semen through simultaneous determination of components, providing a novel basis for the overall assessment of the quality of this plant. in addition, a novel nonaqueous capillary electrophoresis method was used for the analysis of aurantio-obtusin, emodin and rhein in cassiae semen with satisfactory results ( ) . yang et al ( ) was the first to simultaneously determine anthraquinones in rat plasma by uhplc-ms/ms following oral administration of cassiae semen extract. these results may support investigations into the bioactivity mechanism and clinical application of this drug ( ) . anthraquinones and naphthopyrones are considered to be the major constituents. therefore, characteristic compounds or a biological index should be established to evaluate the quality and ensure their clinical application is suitable. in the pharmacopoeia of the people's republic of china, chrysophanol and aurantio-obtusin are used as the indicator compounds to characterize the quality of cassiae semen with the minimum contents of . and . %, respectively ( ). a total of compounds including anthraquinones, naphthopyrones and volatile oil have been isolated and identified from cassiae semen (table i; figs. - ). it has also been suggested that certain efforts should be made to isolate and identify novel compounds from cassiae semen, in order to strengthen its pharmacological profile to develop it further as a candidate for novel drug developments in the future. pharmacological studies have revealed that cassiae semen possesses a variety of biological effects, including anti-hyperlipidemic, anti-diabetic, neuroprotective, hepatoprotective, antimicrobial, anti-oxidant and hypotensive activities ( , , , , ) . extracts and compounds responsible for the pharmacological properties have also been determined, as presented in table ii . although the pharmacological properties of certain traditional uses of cassiae semen have been validated, these studies were primarily conducted in vitro ( , , ) . therefore, the effects of these compounds require verification in vivo. in addition, the association between structure and activity, and the potential synergistic action exerted by the bioactive compounds requires further elucidation. it is anticipated that the comprehensive and current research on the pharmacological activities of extracts, as well as on active molecules isolated from cassiae semen, provided in this review will inspire novel strategies in therapeutics for curing a number of different ailments. medical science and technology press effects of cassia tora fiber supplement on serum lipids in korean diabetic patients quality evaluation of semen cassiae (cassia obtusifolia l.) by using ultra-high performance liquid chromatography coupled with mass spectrometry the advancement of the studies on the seeds of cassia obtusifolia the seed extract of cassia obtusifolia ameliorates learning and memory impairments induced by scopolamine or transient cerebral hypoperfusion in mice studies on the constituents of seeds of cassia obusifolia linn. the structures of two new anthraquinone glycosides two new glycosides from the seeds of cassia obtusifolia effect of polysaccharides of cassiae seeds on the intestinal microflora of piglets experiment study of total anthraquinone in cassiae semen on lipid peroxidation and ppar-gamma expression in liver tissues of rats with alcoholic fatty liver simultaneous determination of major components in cassiae semen obtusifoline by hplc chinese) extraction and identification of an antioxidative component from jue ming zi (cassia tora l hypolipidemic activity of seeds of cassia tora linn in vitro antimutagenic effects of anthraquinone aglycones and naphthopyrone glycosides from cassia tora naphthopyrone glucosides from the seeds of cassia tora with inhibitory activity on advanced glycation end products (ages) formation extract of cassiae semen and its major compound inhibit s b-induced tgf-beta and fibronectin expression in mouse glomerular mesangial cells phenolic constituents of cassia seeds and antibacterial effect of some naphthalenes and anthraquinones on methicillin-resistant staphylococcus aureus cassia tora: a phyto-pharmacological overview antiallergic agent from natural sources. structures and inhibitory effect of histamine release of naphthopyrone glycosides from seeds of cassia obtusifolia l in vitro anthelmintic activity of cassia tora editorial board of flora of china comparative identification of cassiae semen and sesbania aculeata pers comparative identification of semen cassiae and cassia sophera comparative identification of cassiae semen and cassia occidentalis l identification of cassia obtusifolia l. by tlc identification of cassia obtusifolia l., cassia tora. and cassia occidentalis l. by sds-polyacrylamide gelelectrophoresis quality evaluation of cassiae semen by both indicated component determination and hplc fingerprint components identification in cassiae semen by hplc-it-tof ms alaternin glucoside isomer from cassia tora progress in studies of active coustituents of anthraquinones and their biological activities from cassiae semen tumor promoting activity of , -dihydroxy- -methyl- -anthrone (chrysarobin) in female sencar mice study on anthraquinone constituents in the seed of cassia tora l studies on the chemical constituents from semen cassiae and the influence of processing studies on the constituents of the seeds of cassia obtusifolia l. i. the structure of obtusifolin studies on the constituents of seeds of cassia obusifolia l. ii. the structure of obtusin, chryso-obutsin and aurantio-obyusin studies on the constituents of the seeds of cassia obusifolia: the structures of three new anthraquinones study on anthraquinones constituents from semen cassiae isolation and identification of chemical constituents from seeds of cassia obtusifolia potential inhibitors of platelet aggregation from plant sources, v. anthraquinones from seeds of cassia obtusifolia and related compounds study on anthraquinone constituents in cassiae semen a new anthraquinone glycoside from the seed of cassia obtusifolia anthraquinone glycosides from the seeds of cassia tora a new anthraquinone glycoside from seeds of cassia obtusifolia studies on chemical constituents of roasted seeds of cassia obtusifolia glycosides from seeds of cassia obtusifolia anthraquinone constituents from seeds of cassia tora l chemical studies on the oriental plant drugs. xxi. the constituents of cassia tora l chemical studies on the oriental plant drugs. xx. the constituents of cassia tora l.( ). the structure of torachrysone studies on the constituents of the seeds of cassia tora l. ii.(on the purgative crude drugs. vii). the structure of the new naphthopyrone derivative, toralactone new antihepatotoxic naphthopyrone glycosides from the seeds of cassia tora a naphthalene glycoside from cassia tora studies on the constituents of the seeds of cassia obtusifolia l. the structures of two naphthopyrone glycosides studies on the constituents of the seeds of cassia obtusifolia: the structures of two new lactones, isotoralactone and cassialactone glycosides of roasted seeds of cassia obtusifolia two new glycosides from the genus of cassia comparison of volatile components of cassiae semen and semen seeds tea studies on chemical constituents of cassiae semen study on the chemical constituents, quality control and metabolism of cassia obtusifolia yishou jiangzhi (de-blood-lipid) tablets in the treatment of hyperlipidemia observation on cassia seed tea combined with walking exercise on weight loss in the elderly double intervention of cassia seed tea and sports on older women weight loss experimental study on the active situs of fetid cassia seed to reduce blood lipid and their dose-effect relation study on the effective part of reducing blood lipid in semen cassiae the effects of cassia obtusifolia seeds extracts on reducing blood lipid effects of ethanol extraction from cassiae semen on serum il- and tnf-α in hyperlipidemia rats study of the mechanism of cassia obtusifolia l in decreasing blood -lipid hypolipidemic effect of soluble fiber isolated from seeds of cassia tora linn. in rats fed a high-cholesterol diet effect of semen cassiae extracts on expression of lipogenesis genes in hyperlipidemia model mice study on the chemical constitutions of reducing blood lipid in cassiae semen the active ingredients of reducing blood lipid in semen cassia extraction and identification of an antioxidative component from jue ming zi (cassia tora l effects of protein and anthraquinone glucosides from cassia seed on serum lipid of hyperlipidemia rats effects of anthraquinones from cassia obtusifolia l. on cholesterol biosynthesis in cells composition, characteristics, and in-vitro physiological effects of the water-soluble polysaccharides from cassia seed extract of cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats effects of cs, a buoh-soluble fraction of cassiae semen methanolic extract, on cox- expression in renal cortex of stz-induced diabetic rats and cultured glomerular mesangial cells inhibitory effects of cassia seed on the renal fibrosis in diabetic rats the neuroprotective effects of the seeds of cassia obtusifolia on transient cerebral global ischemia in mice the seed extract of cassia obtusifolia offers neuroprotection to mouse hippocampal cultures cassia obtusifolia seed ameliorates amyloid β-induced synaptic dysfunction through anti-inflammatory and akt/gsk- β pathways cassiae semen, a seed of cassia obtusifolia, has neuroprotective effects in parkinson's disease models effect of protein and anthraquinone glucosides from semen cassiae on learning and memory capacity and related substances of senile mice induced by d-galactone jiangsu new medical college: encyclopedia of chinese materiamedica, i. shanghai science and technology press plants with liver protective activities (i) liver-protective and bowel-lubricating and defecation-promoting effects of crude and processed semen cassiae experimental study on protective effect of semen cassiae extract against acute liver injury study on the protective effect of cassiae semen extract against alcohol-induced acute liver injury in mice anthraquinones isolated from cassia tora (leguminosae) seed show an antifungal property against phytopathogenic fungi in vitro anti-helicobacter pylori action of chinese herbal medicines used to treat ulcer diseases growth responses of cassia obtusifolia toward human intestinal bacteria primary study on inhabiting of the extracts from cassia obtusifolia seeds against fusarium oxysporum and botrytis cinerea antioxidant effects of extracts from cassia tora l. prepared under different degrees of roasting on the oxidative damage to biomolecules antioxidant properties of water extracts from cassia tora l. in relation to the degree of roasting study on free radical scavenging capacity by cassia seed water extract in vitro study on purification and antioxidation of water-soluble polysaccharide isolated from semen cassia antioxidation study on water-soluble polysaccharide isolated from semen cassiae extraction of water-soluble polysaccharide and the antioxidant activity from cassiae semen target-guided separation of antioxidants from semen cassia via off-line two-dimensional high-speed counter-current chromatography combined with complexation and extrusion elution mode antioxidant activities of selected oriental herb extracts alatemin, cassiaside and rubrofusarin gentiobioside, radical scavenging principles from the seeds of cassia tora on , -diphenyl- -picrylhydrazyl (dpph) radical extraction of hypotensive principles from seeds of cassia tora a possible reflex mechanism of hypotensive action of extract from cassia tora seeds the involvement of medullary reticular formation in the hypotensive effect of extracts from seeds of cassia tora effects of cassiae semen extracts on vasorelaxation and its mechanisms in rat aorta in vitro estrogenic activities of chinese medicinal plants traditionally used for the management of menopausal symptoms antigenotoxic properties of cassia tea (cassia tora l.): mechanism of action and the influence of roasting process semen cassiae attenuates myocardial ischemia and reperfusion injury in high-fat diet streptozotocin-induced type diabetic rats hplc fingerprint of cassiae semen hplc fingerprint chromatogram of cassiae semen hplc fingerprint and chemical pattern recognition of cassiae semen study on fingerprint of cassiae semen, ultramicro powder and powder particle a novel nonaqueous capillary electrophoresis method for effective separation and simultaneous determination of aurantio-obtusin, emodin and rhein in semen cassiae and cassia seed tea simultaneous determination of seven anthraquinones in rat plasma by ultra high performance liquid chromatography-tandem mass spectrometry and pharmacokinetic study after oral administration of semen cassiae extract inhibitory activities of cassia tora and its anthraquinone constituents on angiotensin-converting enzyme potential hepatoprotective activity of ononitol monohydrate isolated from cassia tora l. on carbon tetrachloride induced hepatotoxicity in wistar rats traditional chinese medicine herbal extracts of cibotium barometz, gentiana scabra, dioscorea batatas, cassia tora and taxillus chinensis inhibit sars-cov replication the present review was financially supported by the collaborative innovation construction plan of beiji ng un iversit y of ch i nese medici ne (g ra nt no. -xtcx- ). key: cord- -al gxjw authors: calder, philip c. title: n− fatty acids, inflammation, and immunity— relevance to postsurgical and critically iii patients date: journal: lipids doi: . /s - - -z sha: doc_id: cord_uid: al gxjw excessive or inappropriate inflammation and immunosuppression are components of the response to surgery, trauma, injury, and infection in some individuals and these can lead, progressively, to sepsis and septic shock. the hyperinflammation is characterized by the production of inflammatory cytokines, arachidonic acid-derived eicosanoids, and other inflammatory mediators, while the immunosuppression is characterized by impairment of antigen presentation and of t helper cell type- responses. long-chain n− fa from fish oil decrease the production of inflammatory cytokines and eicosanoids. they act both directly (by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid metabolism) and indirectly (by altering the expression of inflammatory genes through effects on transcription factor activation). thus, long-chain n− fa are potentially useful anti-inflammatory agents and may be of benefit in patients at risk of developing sepsis. as such, an emerging application of n− fa is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. parenteral or enteral nutrition including n− fa appears to preserve immune function better than standard formulas and appears to partly prevent some aspects of the inflammatory response. studies to date are suggestive of clinical benefits from these approaches, especially in postsurgical patients. the systemic inflammatory response syndrome is the name given to the uncontrolled inflammatory response to insult or injury involving excessive production of inflammatory cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , and il- ( , ) . sepsis has been defined as "the systemic inflammatory response syndrome that occurs during infection" ( ) . sepsis is the leading cause of death in critically ill patients in western countries. using records from for state hospitals in the united states it was estimated that there were more than , cases of sepsis with a . % mortal-ity rate ( , deaths) and a total cost of almost us$ billion ( ) . death from septic shock is the result of multiple organ failures and represents the extreme end of a continuum of events of increasing severity and decreasing likelihood of survival ( , ; fig. ). the systemic inflammatory response syndrome, sepsis, and septic shock may together be termed as "septic syndromes." the involvement of inflammatory cytokines in septic syndromes has been long recognized and vervloet et al. ( ) wrote "these mediators [i.e., inflammatory cytokines] are largely, if not completely, responsible for the clinical signs and symptoms of the septic response to bacterial infection." in support of this idea, patients with sepsis were found to have markedly elevated circulating concentrations of tnf-α, tnf receptor , il- β, il- receptor antagonist (il- ra), and il- , and those patients with the highest concentrations were more likely to die ( ) ( ) ( ) ( ) . in addition, circulating white cells from septic patients exhibited high levels of activated nuclear factor kappa b (nfκb), a transcription factor that promotes the expression of numerous genes associated with inflammation, and again levels of activated nfκb were higher in those patients who went on to die ( ) . animal studies also support a role for inflammatory cytokines in the septic response. these studies have often used bacterial endotoxin (also called lipopolysaccharide) as a surrogate for infection, although endotoxin is a fragment of the gram-negative bacterial cell wall and not a viable organism. mice injected with endotoxin exhibit high circulating concentrations of tnf-α, il- β, il- , and il- , and survival of these animals can be improved by administering anti-cytokine antibodies ( , ) , cytokine receptor antagonists ( ) , or anti-inflammatory cytokines such as il- ( ) , or by knocking out the tnf-α receptor ( ) . despite this evidence, it is important to note that some studies report that many septic patients do not show detectable or elevated circulating concentrations of tnf-α or il- β ( ) ( ) ( ) ( ) . furthermore, it appears that inflammatory cytokines do play a beneficial role in sepsis. for example, in some animal models, blocking tnf-α increases mortality ( ) ( ) ( ) , while a tnf-α antagonist increased mortality in a clinical trial ( ) . thus, the situation regarding the pathological role of inflammatory cytokines in sepsis is unclear; it may be that a little is beneficial but that excess is harmful and that complete blocking negates the beneficial effects. another consideration is that there may be large between-individual differences in the generation of inflammatory cytokines, in the sensitivity to the harmful effects of these cytokines, and in the effects of blocking these cytokines. thus, there may be significant variation in the susceptibility of individuals to exhibit the systemic inflammatory response syndrome and to progress toward septic shock. this may partly relate to the extent and site of the initial injury, partly to the nature and site of the infection, if any, and partly to aspects of the patient's well-being prior to receiving the injury (e.g., nutritional state). it is now recognized that genetics may also play a role. in fact there are likely to be genetic variations in many aspects of the septic response to infection and injury. these most likely relate to adaptations of various population groups to withstand infection and injury in different ecological settings. in the context of this article, genetic variations in the propensity to produce inflammatory cytokines are of relevance. it is now recognized that there are single base variations in genes or in their promoter regions called single nucleotide polymorphisms or snps (pronounced "snips"). snps have been described for tnf-α, tnf-β, il- β, il- , il- , tnf receptors, il- receptors, il- ra, and for many other genes involved in the septic response ( ) . these snps are of functional significance since they partly determine the extent of expression of the gene once it is activated ( ) . thus tnf-α production by monocytes in response to endotoxin is higher in individuals who have a g rather than an a at - in the tnf-α gene promoter region ( ) . intriguingly, tnf-α production is also affected by a polymorphism in the tnf-β gene: tnf-α production by monocytes in response to endotoxin was higher if there was an a at + in the tnf-β gene than if there was a g ( ) . genotypes affecting tnf-α production appear to be of relevance with respect to sepsis mortality. for example, possession of a g at - in the tnf-α gene was found in % of patients with septic shock compared with % of controls, and among patients with septic shock this polymorphism was significantly more common among patients who died ( % vs. % among survivors) ( ) . in controlling for age, it was identified that, for the same clinical score, patients with a g at - of the tnf-α gene had a . -fold higher risk of death than those without a g ( ) . in another study, patients with sepsis who were homozygous for a at + in the tnfβ gene displayed significantly higher plasma tnf-α concentrations than heterozygotes or homozygotes for g, and they showed % mortality compared with % for heterozygotes and % for g homozygotes ( ) . in a more recent study, postoperative patients who were homozygous for a at + in the tnf-β gene had a . -fold higher risk of developing severe complications than heterozygotes ( ) . furthermore, among the patients who developed sepsis, those who were homozygous for a at + in the tnf-β gene were more likely to die ( vs. % for heterozygotes and % for homozygotes for g) ( ) . these findings raise the possibility of being able to identify patients at high risk of complications and mortality on the basis of genetic polymorphisms. although there has been much focus on the potential detrimental role of inflammatory cytokines in sepsis, other mediators including arachidonic acid-derived eicosanoids, reactive oxygen species, nitric oxide, and adhesion molecules are involved in the pathological processes that accompany critical illness. prostaglandin (pg) e is implicated in sepsis, burns, and critical illness ( , ) , while leukotriene (lt) b and oxidants released by neutrophils are involved in acute respiratory distress syndrome [see kollef and schuster ( ) ]. in addition to hyperinflammation, patients with sepsis also display immunosuppression ( ) ( ) ( ) . there are reports that septic patients have high circulating concentrations of the antiinflammatory cytokine il- and that these are strongly correlated with mortality ( , ) . note that this is contrary to the predicted effect of il- since this cytokine down-regulates tnf-α production and its early administration is protective in murine endotoxemia ( ) ( ) ( ) . however, the apparently harmful effect of il- may relate to the timing of its production. lymphocytes from patients with burns or trauma produce low levels of the t helper (th) -type cytokines [e.g., interferon (ifn)-γ] associated with host defense against bacteria and viruses but high levels of the th -and treg-type cytokines (il- , il- ) associated with inhibition of host defense against bacteria and viruses ( , ) . there also appears to be decreased monocyte expression of human leukocyte antigens (hla) ( ) ( ) ( ) ( ) , the proteins involved in antigen presentation to t cells, and this is associated with impaired ability of monocytes to stimulate t cells ( ) . interestingly, il- downregulates both th -type cytokine production and hla expression ( , ) , and this might be the origin of the harmful effect of this cytokine in septic patients. recent studies have revealed impaired proliferative or secretory functions of t cells from patients with sepsis, trauma, or burns ( , ) . the traditional view is that the immunosuppressed phase of septic syndromes lags behind the hyperinflammatory phase (fig. ) ; that is, initially sepsis is characterized by increased generation of inflammatory mediators (the systemic inflammatory response syndrome), but as it persists there is a shift toward an anti-inflammatory, immunosuppressed state sometimes called the compensatory anti-inflammatory response syndrome. however, some recent studies challenge this and suggest that the hyperinflammatory and immunosuppressed states coexist. some authors report that immunosuppression is present at the onset of sepsis ( , , ) , rather than being a later compensatory response. for example, tschaikowsky et al. ( ) identified that significantly decreased monocyte expression of hla-dr was evident at the onset of severe sepsis in postsurgical patients; in survivors there was some recovery of expression but in nonsurvivors there was a further decrease or even a permanent suppression of hla-dr expression. these authors identified that the timing of the peak of the systemic inflammatory reaction (identified as the time of maximum c-reactive protein concentration) coincided with the timing of the lowest monocyte expression of hla-dr. from this they concluded that decreases in monocyte hla-dr expression occur simultaneously with "signs of hyperinflammation" and as early as the onset of severe sepsis ( ) . thus, it appears that immune cells and cytokines have both detrimental and protective roles in patients as they move through the stages of sepsis. however, the traditional view that hyperinflammation precedes immunosuppression, as shown in figure , may be a simplification of the real situation, and this increases the challenge to finding interventions that might benefit high-risk patients. human immune and inflammatory cells are rich in polyunsaturated fa (pufa), especially arachidonic acid ( : n- ) [see calder ( ) ]. classically the influence of pufa on immunity and inflammation has been viewed as relating to their influence on eicosanoid generation ( ) ( ) ( ) ( ) . arachidonic acid is the principal substrate for cyclooxygenase (cox) and lipoxygenase (lox) enzymes giving rise to -series pg and thromboxanes (tx) or -hydroxyeicosatetraenoic acids (hete) and -series lt, respectively. these mediators have cell-and stimulus-specific sources and frequently have opposing effects (table ). for example, pge is produced mainly by monocytes, macrophages, and, to a lesser extent, neutrophils and inhibits the production of tnf-α and il- β [see miles et al. ( ) and references therein; , ] and il- ( , ) , while ltb is produced mainly by neutrophils, other granulocytes, and, to a lesser extent, monocytes and macrophages and increases the production of tnf-α and il- β [see rola-pleszczynski et al. ( ) and references therein]. thus, the overall physiological (or pathophysiological) outcome will depend upon the cells present, the nature of the stimulus, the timing of eicosanoid generation, the concentrations of different eicosanoids generated, and the sensitivity of target cells and tissues to the eicosanoids generated. recent studies have demonstrated that pge induces cox- in fibroblasts cells and so upregulates its own production ( ), induces production of il- by macrophages ( ) it is frequently considered that the effects of arachidonic acid are solely related to its role as an eicosanoid precursor. cell culture studies have shown that arachidonic acid acti- vates nfκb in a monocytic cell line ( ) , and induces tnf-α, il- α and il- β in osteoblasts ( ), il- in macrophages ( ) and osteoblasts ( ) , and cox- in fibroblasts ( ) , and it appears that these effects are exerted directly by arachidonic acid rather than by an eicosanoid metabolite. what is evident from these studies is that arachidonic acid may be able to regulate inflammatory mediator production in its own right and, if so, that it has effects that are sometimes the opposite of those of pge , for example, with respect to tnf-α production. a series of cell culture-based studies with human endothelial cells has suggested that another n- fa, linoleic acid ( : n- ), may also play a role in inflammation through activation of nfκb and increased production of tnf-α, il- , and other inflammatory mediators ( ) ( ) ( ) ( ) ( ) ( ) ( ) . increased consumption of long-chain n- pufa, usually as components of fish oil, by humans results in increased amounts of epa ( : n- ) and dha ( : n- ) in cells involved in immunity and inflammation [see calder ( ) ]. the incorporation of these fa from the diet into immune/inflammatory cells of humans is near-maximal within a few weeks ( , ) and occurs in a dose-dependent manner ( ) . incorporation of epa and dha into human cells is partly at the expense of arachidonic acid [see calder ( ) ], and the functional significance of this is that it decreases the amount of arachidonic acid available as a substrate for eicosanoid synthesis. thus, fish oil supplementation of the human diet has been shown to result in decreased production of pge ( - ), txb ( ), ltb and -hete ( , ) , and lte ( ) by inflammatory cells. however, the mechanism of the effect of long-chain n- fa on eicosanoid generation extends beyond simply decreasing the amount of arachidonic acid substrate. for example, epa competitively inhibits metabolism of arachidonic acid by cox ( - ) and -lox ( , ) . in vitro studies also report that dha can inhibit cox activity ( , ) but not that of -lox ( , ) . interestingly, however, both epa and dha suppressed cytokine-induction of cox- and -lox gene expression in cultured bovine chondrocytes and in human osteoarthritic cartilage explants ( , ) . by inhibiting cox and lox activities and by suppressing the up-regulation of the genes for these enzymes in response to inflammatory stimuli, long-chain n- fa act to oppose generation of eicosanoids from arachidonic acid. the final element of the effects of longchain n- fa on eicosanoid production is the ability of epa to act as a substrate for cox and lox enzymes, so giving rise to a different family of eicosanoids: the -series pg and tx, the series lt, and the hydroxyeicosapentaenoic acids (hepe). epa, which appears to be a good substrate for -lox ( , ) , is also a substrate for cox enzymes ( , ) . thus, fish oil supplementation of the human diet has been shown to result in increased production of ltb , lte , and -hepe by inflammatory cells ( ) ( ) ( ) , although generation of pge has been more difficult to demonstrate ( ) . the functional significance of this is that the mediators formed from epa are believed to be less potent than those formed from arachidonic acid. for example, ltb is -to -fold less potent as a neutrophil chemotactic agent than ltb ( , ) . recent studies have compared the effects of pge and pge on production of cytokines by cell lines and by human cells. bagga et al. ( ) reported that pge was a less potent inducer of cox- gene expression in fibroblasts and of il- production by macrophages. pge and pge had equivalent inhibitory effects upon production of tnf-α ( , ) and il- β ( ) by human mononuclear cells stimulated with endotoxin and upon production of ifn-γ production by mononuclear cells stimulated with mitogen ( , ) . however, il- production appeared to be less sensitive to pge than pge ( ) . studies using the isolated, perfused rabbit lung have identified contrasting effects of arachidonic acid-and epa-derived eicosanoids. infusion of escherichia coli hemolysin caused hypertension mediated by txb and increased vascular leakage mediated by -series lt ( ) . inclusion of arachidonic acid in the perfusate increased txb and -series lt generation, arterial pressure, and vascular leakage ( , ). in contrast, inclusion of epa in the perfusate decreased txb and -series lt generation, decreased arterial pressure and vascular leakage, and increased generation of txb and -series lt ( ) . perfusion with fish oil attenuated the hypertension induced by calcium ionophore ( ) . compared with soybean oil infusion, fish oil decreased the concentration of ltc by % and increased the concentration of ltc from barely detectable to very similar to that of ltc ( ) . in addition to long-chain n- fa modulating the generation of eicosanoids from arachidonic acid and to epa acting as substrate for the generation of alternative eicosanoids, recent studies have identified a novel group of mediators, termed e-series resolvins, formed from epa by cox- that appear to exert anti-inflammatory actions ( ) ( ) ( ) . in addition, dha-derived mediators termed d-series resolvins, docosatrienes, and neuroprotectins also produced by cox- have been identified, and these too appear to be anti-inflammatory ( ) ( ) ( ) . this is an exciting new area of n- fa and inflammatory mediators, and the implications for a variety of conditions may be of great importance. cell culture studies investigating the direct effects of arachidonic acid on inflammatory mediator production have also investigated effects of long-chain n- fa. epa did not activate nfκb in a monocytic cell line ( ), while epa and dha inhibited endotoxin-stimulated production of il- and il- by cultured human endothelial cells ( , ) . more recent studies showed that epa did not induce tnf-α, il- β, or il- α ( ) or il- ( ) in osteoblasts, and even countered the upregulating effect of arachidonic acid ( ) ; that epa and dha could totally abolish cytokine-induced up-regulation of tnf-α, il- α, and il- β in cultured bovine chondrocytes and in human osteoarthritic cartilage explants ( , ) ; and that epa or fish oil inhibited endotoxin-induced tnf-α production by monocytes ( ) ( ) ( ) ( ) . epa was also less potent than arachidonic acid in inducing cox- expression by fibroblasts and il- expression by macrophages ( ) . epa prevented nfκb activation by tnf-α in cultured pancreatic cells, an effect that involved decreased degradation of the in-hibitory subunit of nfκb (iκb), perhaps through decreased phosphorylation ( ) . similarly, epa or fish oil decreased endotoxin-induced activation of nfκb in human monocytes ( , , ) , and this was associated with decreased iκb phosphorylation ( , ) , perhaps due to decreased activation of mitogen-activated protein kinases ( ) . these observations suggest direct effects of long-chain n- fa on inflammatory gene expression via inhibition of activation of the transcription factor nfκb. animal feeding studies with fish oil support the observations made in cell culture with respect to the effects of long-chain n- fa on nfκb activation and inflammatory cytokine production. compared with feeding corn oil, fish oil lowered nfκb activation in endotoxin-activated murine spleen lymphocytes ( ) . feeding fish oil to mice decreased ex vivo production of tnf-α, il- β, and il- by endotoxin-stimulated macrophages and decreased circulating tnf-α, il- β, and il- concentrations in mice injected with endotoxin [sadeghi et al. ( ) and references therein]. several studies in humans involving supplementation of the diet with fish oil have demonstrated decreased production of tnf-α, il- β, and il- by endotoxin-stimulated monocytes or mononuclear cells (a mixture of lymphocytes and monocytes) ( ) ( ) ( ) ) . the study of caughey et al. ( ) reported a significant inverse correlation between the epa content of mononuclear cells and the ability of those cells to produce tnf-α and il- β in response to endotoxin. recent studies have confirmed the ability of dietary fish oil to decrease production of tnf-α ( ) and il- ( , ) by human mononuclear cells. furthermore, these studies provide for the first time information on the dose-response relationship between dietary intake of long-chain n- fa and production of these cytokines. it should be noted that there are also several studies that fail to show effects of dietary long-chain n- fa on production of inflammatory cytokines in humans [see calder ( ) for references]. it is not clear what the reason for this is, but the dose of n- fa used and other technical factors are likely to be contributing factors. one other factor that has recently been identified is polymorphisms in genes affecting cytokine production ( ) . it was found that the effect of dietary fish oil on cytokine production by human mononuclear cells was dependent on the nature of the - tnf-α and the + tnf-β polymorphisms. this study raises the possibility of being able to identify those who are more likely and those who are less likely to experience specific anti-inflammatory effects of fish oil. thus, examination of fa composition and of eicosanoid profiles, cell and tissue culture work, and animal and human feeding studies have revealed a range of anti-inflammatory actions of long-chain n- fa ( table ). these may be of benefit in sepsis, particularly during the "early" hyperinflammatory phase. the benefits of fish oil in animal models of experimental endotoxemia have been clearly demonstrated. for example, dietary fish oil or fish oil infused intravenously significantly enhanced survival of guinea pigs to intraperitoneal endotoxin compared with safflower oil ( , ) . dietary fish oil resulted in a decreased concentration of circulating postendotoxin eicosanoids (pge , txb , -keto-pgf α ) in rats and in decreased eicosanoid generation by alveolar macrophages ( , ) . furthermore, compared with dietary safflower oil, fish oil resulted in lower circulating tnf-α, il- β, and il- concentrations following endotoxin administration to mice ( ) . dietary fish oil also appears to decrease sensitivity to inflammatory cytokines ( , ) . fish oil decreased endotoxininduced metabolic perturbations in guinea pigs and rats ( , ) and improved heart and lung function and decreased lung edema in endotoxic rats ( , ( ) ( ) ( ) and pigs ( ) ( ) ( ) . in addition to effects on production of inflammatory eicosanoids and inflammatory cytokines, long-chain n- fa decreased generation of arachidonic acid-derived partial replacement of arachidonic acid in cell membrane phospholipids eicosanoids (many with inflammatory actions) inhibition of arachidonic acid metabolism by phospholipase a , cox, and -lox decreased induction of cox- , -lox, and -lox-activating protein however, it is the effects of lower amounts of long-chain n- fa that are of relevance to the patient setting. several studies in humans, typically providing long-chain n- fa as fish oil, and investigating aspects of cell-mediated immunity have been performed. phagocytic uptake of escherichia coli appears unaffected by dietary long-chain n- fa in humans ( ) ( ) ( ) ( ) . one study reported that fish oil decreased expression of hla-dp, -dq, and -dr on human monocytes ( ), suggesting impaired ability to present antigen, but there have been no studies attempting to confirm this finding. meydani et al. ( ) reported that fish oil providing . g epa plus dha per day decreased t-lymphocyte proliferation in older but not younger women. however, that study also reported increased oxidative stress in the older subjects ( ) , and it may be that the effect of n- fa was due to excessive lipid peroxidation. several other studies report no effect of various doses of longchain n- fa on lymphocyte proliferation ( , , ) , although there are studies reporting a decrease ( , ) . one recent study reported that long-chain n- fa caused a dosedependent increase in proliferation of t cells ( ) . it is noteworthy that the fish oil used was given in combination with an antioxidant mix. this might be important in terms of preventing excessive lipid peroxidation and so in determining the overall effect of n- fa. the study by meydani et al. ( ) also reported decreased production of il- in the older women, but this effect has not been confirmed by others in either older ( ) , young ( , ) , or mixed-age ( , ) subjects. a recent study reported a dose-dependent increase in ifn-γ production following n- fa supplementation as fish oil ( ) . that antioxidants were given in combination with fish oil may have been important in generating this finding. thus, the effects of long-chain n- fa on aspects of cellmediated immunity are rather unclear, although recent human studies suggest that adverse immune effects are not exerted at modest doses (see previous discussion for references) and that enhanced t-cell responses (proliferation and ifn-γ production) may occur at modest doses so long as antioxidants are also given ( ) . in terms of sepsis, the true test of immunocompetence occurs when live pathogens are administered. this is a different situation from using endotoxin that is not living and that therefore does not require a robust cell-mediated immune response to eliminate it. as indicated previously, it is clear that long-chain n- fa protect against the deleterious effects of endotoxin. however, the situation regarding live pathogens is much less clear. this is because animal studies, frequently using high intakes of n- fa, report opposing findings. infusion of fish oil into rats also receiving low-dose endotoxin decreased the number of viable bacteria in mesenteric lymph nodes and liver ( ) . fish oil did not decrease bacterial translocation across the gut, and so the authors concluded that fish oil must have improved bacterial killing. compared with linoleic acid-rich vegetable oils, fish oil fed to rats before exposure to live bacteria ( , ) resulted in increased survival, which was associated with decreased production of pge . more recently, infusion of fish oil after induction of sepsis by cecal ligation and puncture decreased mortality (and pge production) compared with vegetable oil ( ) . intragastric administration of fish oil into chow-fed rats before cecal ligation and puncture improved survival compared with saline or vegetable oil infusion ( ) . compared with vegetable oil feeding to mice, fish oil feeding increased survival to an intramuscular injection of klebsiella pneumoniae ( ) . the findings from these studies ( ) ( ) ( ) ( ) ( ) ( ) contrast with those reporting that fish oil feeding decreases the survival of mice to oral salmonella typhimurium ( ) and to intraperitoneal listeria monocytogenes ( ) , of guinea pigs to mycobacterium tuberculosis ( ) , and of neonatal rabbits to staphylococcus aureus ( ) . thus, animal studies do not provide a clear picture of the effect of high-dose fish oil on ability to survive an infectious challenge. there are few human studies that address exposure to long-chain n- fa and infection; most intervention studies performed to date have been too small and of too short duration to monitor infection as an outcome. however, it is worth noting that an epidemic of measles in greenland triggered by its introduction to a naive population by an infected danish sailor showed the same characteristics as previous epidemics in other naive populations ( ) . this suggests that the very n- fa-rich diet of the greenland inuits did not worsen their response to the virus and this could indicate that these fa do not increase infectious susceptibility in humans. surgery is typically accompanied by an inflammatory response that may be exaggerated in some patients, especially if the surgery is major. if the patient is exposed to pathogenic organisms and is unable to cope with these, then sepsis may develop. artificial nutrition is frequently used post-surgery and this may involve parenteral (i.e., intravenous) infusions, especially where the gastrointestinal tract is not fully functional (e.g., post-abdominal surgery). lipids are included in parenteral nutrition to provide an alternative source of calories to glucose and the lipid source used most frequently has been soybean oil, which is rich in the n- fa linoleic acid, although it also contains a proportion of α-linolenic acid ( : n- ). a meta-analysis of total parenteral nutrition suggested that inclusion of lipids might be detrimental (p = . for lipids vs. no lipids) ( ) , at least in very ill patients. it is not clear why this is, although a number of in vitro experiments have shown that soybean oil-based lipid emulsions can exert immunosuppressive effects [see calder et al. ( ) for references], which would clearly be detrimental in patients at risk of infection and sepsis. clinical trials provide conflicting evidence, some showing some immunosuppressive effects ( , ) and others not ( ) ( ) ( ) , at least in some patient groups. the concern about potential harm, the view of sepsis as a hyperinflammatory state followed by an immunosuppressed state (fig. ) , and the idea that n- fa might be "proinflammatory and immunosuppressive" has led to the development of alternative lipid emulsions for parenteral applications. emulsions using a mix of medium-chain triglycerides and soybean oil or based upon olive oil instead of soybean oil have been developed, but these will not be discussed here. however, of relevance to the present discussion is the development of emulsions that include fish oil as a partial replacement for soybean oil. several such emulsions have been tested in surgical patients. intravenous infusion of a lipid emulsion containing fish oil for d into patients who had undergone major abdominal surgery resulted in much higher ltc production by blood leukocytes stimulated ex vivo at d postoperation ( ) . in another study, patients who had undergone abdominal surgery received soybean oil or a mix of medium-chain triglycerides, soybean oil, and fish oil ( : : , by vol) for d post surgery ( ) . leukocytes from these patients produced more ltb and ltb isomers at postoperative days and . patients who had undergone major gastrointestinal surgery received a medium-chain triglyceride/soybean oil mix ( : , vol/vol) or a mix of medium-chain triglycerides, soybean oil, and fish oil ( : : , by vol) for d postsurgery ( ) . patients receiving fish oil got (days and ) and g (days , , and ) of long-chain n- fa per day. neutrophils from these patients produced less ltb and more ltb at postoperative days and . plasma tnf-α concentrations were lower in the fish oil group at day , while plasma il- concentrations were lower at day . the study did not report clinical outcomes. a more recent study infused a fish oil-rich formula on the day before abdominal surgery and on days to following abdominal surgery ( ) . on days and the patients also received standard total parenteral nutrition that included g of fat/d (n = ; n = in the control group). tnf-α production by endotoxin-stimulated whole blood tended to be lower at postoperative day in the fish oil group, but this was not significant. serum il- concentrations were significantly lower at days , , and in the fish oil group. monocyte expression of hla-dr was preserved in the fish oil group but declined at postsurgery days and in the control group. no differences in infection rates or mortality were observed. however, postoperative stay in intensive care tended to be shorter in the fish oil group ( . vs. . d) as did total hospital stay ( . vs. . days), although neither of these was a significant effect. postoperative stay on medical wards was significantly shorter in the fish oil group. another recent study compared the effects of lipid-free total parenteral nutrition or parenteral nutrition including % soybean oil or . % soybean oil plus . % fish oil for d after large bowel surgery ( ) . there were no differences between the groups with respect to the numbers of circulating lymphocytes, b cells, cd + cells, cd + cells, or natural killer cells before surgery or at days and postsurgery, although these were affected by surgery itself. there were no differences between groups with respect to t-lymphocyte proliferation, but il- production was increased in the fish oil group and the postsurgery decline in ifn-γ production was prevented by fish oil. these studies indicate that inclusion of fish oil in parenteral nutrition regimens for gastrointestinal surgical patients modulates generation of inflammatory eicosanoids ( ) ( ) ( ) and cytokines ( , ) and may help to counter the surgery-induced declines in antigen-presenting cell activity ( ) and t cell cytokine production ( ) . importantly, these studies do not reveal deleterious immunologic effects of fish oil infusion in these patients. furthermore, the only one of these fairly small studies to have examined hard end points like length of hospital stay suggests some clinical benefit from fish oil infusion in these patients ( ) . however, larger studies are required to evaluate the effects of this approach on complication rates, hospital stay, and mortality rate. a very recent report from a larger cohort of patients receiving parenteral nutrition postsurgery does indicate benefit of inclusion of fish oil in the regimen ( ) . patients received fish oil postoperatively (n = ) or controls received a : medium-chain triglyceride-soybean oil mix (n = ). there were no differences between the two groups with respect to the proportions of patients who died or developed wound infections or with respect to length of hospital stay. however, the proportion of patients who were readmitted to intensive care ( %) was significantly lower in the fish oil than in the control group ( %). a group of patients also received the fish oil-containing emulsion for d preoperatively (n = ). here there were a number of very significant benefits. this group showed a significantly decreased need for mechanical ventilation ( vs. % in the control group), a significantly shorter length of hospital stay ( vs. d) , significantly less need for readmission to intensive care ( vs. %), and a significantly lower mortality rate ( vs. %) ( ) . this study demonstrates a benefit from the inclusion of long-chain- fa in parenteral nutrition regimens used in abdominal surgery patients. however, it also demonstrates a much greater benefit if the fa are additionally provided before surgery, which, of course, is only possible in elective surgery. the greater benefit of preoperative infusion of longchain n- fa may relate to better incorporation of the fa into leukocytes and other tissues. enteral nutrition is an alternative form of artificial nutrition. it describes provision of nutrients directly into the gastrointestinal tract via a tube and is sometimes referred to as "tube feeding." enteral nutrition is used in patients with a functional gastrointestinal tract and is considered preferable to parenteral nutrition. the influence of enteral feeds including long-chain n- fa in their composition has been examined in surgical patients, generally in those who have undergone surgery to remove cancerous regions of the intestine. these studies have frequently used an enteral formula named impact ® (novartis, basel, switzerland), which contains arginine, long-chain n- fa, and nucleotides, each of which is lacking from control formulas. thus, any effects observed cannot be ascribed to a particular component of impact. the effect of impact on immunoinflammatory outcomes in surgical patients has been widely examined. daly et al. ( ) reported that impact results in time-dependent incorporation of epa into mononuclear cells and that this is associated with a timedependent decrease in pge production. studies have reported that impact increases phagocytosis by monocytes but not by neutrophils ( , ) , increases t-cell proliferation ( ) and cell-mediated immunity ( , ) , and decreases circulating concentrations of il- ( , ) . several of these studies report significantly improved clinical outcomes related to lower infection rate ( , , , ) and decreased length of hospital stay ( , , ) . studies of impact and similar enteral formulas investigating clinical outcomes in postsurgical patients have been subject to meta-analyses ( ) ( ) ( ) , which conclude that this approach to enteral nutrition significantly decreases infectious complications and length of hospital stay in elective surgery patients. it is possible that the modulation of inflammation and the improvements in immune function reported in these patients receiving impact contribute to the improved clinical outcomes. however, it is not possible to ascribe these benefits to long-chain n- fa. critically ill patients frequently require artificial support, depending upon the extent of organ damage or failure, and this will include nutritional support. the influence of enteral feeds including long-chain n- fa has been examined in critically ill patients; again, many of these studies have involved impact. a study in intensive care unit patients (a mix of trauma, sepsis, and major surgery patients) reported that impact resulted in higher t-cell proliferation at days and ( ), while a study of severe trauma patients reported greater hla-dr expression at day ( ) . these studies did not report improvements in clinical outcomes. studies of impact and similar enteral formulas investigating clinical outcomes in trauma and critically ill patients have been subject to metaanalysis ( ) ( ) ( ) . the most recent of these concluded that this approach to enteral nutrition decreases length of hospital stay but has no effect on infectious complications or mortality in critically ill patients ( ) . another trial performed in patients with moderate and severe acute respiratory distress syndrome used an enteral preparation that differed mainly in lipid source from the control ( ) . the control group of patients (n = ) received a formula in which the lipid source was % corn oil plus % soy lecithin. the experimental group (n = ) received a lipid source that was % canola oil, % medium-chain triglycerides, % borage oil, % fish oil, and % soy lecithin. the experimental formula also contained more vitamin c and vitamin e than the control and it contained β-carotene, taurine, and carnitine, which the control formula did not. patients receiving the experimental formula got about g of epa, g of dha, g of γ-linolenic acid, . g of vitamin c, iu of vitamin e, and . mg of β-carotene per day for d. by d the numbers of total leukocytes and of neutrophils in the alve-olar fluid declined significantly in the experimental group and were lower than in the control group. arterial oxygenation and gas exchange were improved in the experimental group. these patients had a significantly decreased requirement for supplemental oxygen, decreased time on ventilation support ( . vs. . d), and a shorter length of stay in intensive care ( . vs. . d) . total length of hospital stay tended to be shorter in the experimental group ( . vs. . d). significantly fewer patients in the experimental group developed new organ failure ( vs. %). the mortality rate was % in the experimental group and % in the control group, but this difference was not statistically significant. more recently, new data from this study have become available ( ) . patients receiving the experimental formula had significantly lower concentrations of il- in their alveolar fluid and tended to have lower concentrations of ltb and tnf-α. it is possible that the lower concentrations of ltb and il- , both of which are potent leukocyte chemoattractants, may have been responsible for the lower neutrophil infiltration reported in the experimental group, and indeed neutrophil counts were significantly associated with these concentrations ( ) . this study establishes that the experimental treatment decreases production of inflammatory mediators and infiltration of inflammatory leukocytes and that this can result in significant clinical improvement in extremely ill patients. because of the many differences in composition between the experimental and control formulas used it is not possible to ascribe the effects and benefits to any particular nutrient. however, the effects on ltb , il- and tnf-α concentrations are consistent with effects of long-chain n- fa reported elsewhere. recently, data from studies using parenteral nutrition with fish oil in sepsis patients have become available ( , ) . patients received a standard soybean oil-based emulsion or an emulsion containing fish oil for ( ) or ( ) d. blood leukocyte counts and serum c-reactive protein concentration tended to be lower, and production of ltb by stimulated neutrophils was significantly higher in patients receiving long-chain n- fa ( ). production of tnf-α, il- β, il- , il- , and il- by endotoxin-stimulated mononuclear cells did not increase during infusion of the fish oil-containing emulsion whereas production of the four proinflammatory cytokines was markedly elevated during the first d of soybean oil infusion ( ) . these studies establish that infusion of long-chain n- fa into patients with sepsis can modulate inflammatory mediator production and related inflammatory processes. however, the impact of this on hard clinical outcomes in these patients is not yet clear. in summary, long-chain n- pufa from fish oil decrease the production of inflammatory cytokines and eicosanoids. they act both directly, by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid metabolism, and indirectly, by altering the expression of inflammatory genes through effects on transcription factor activation. thus, long-chain n- pufa are potentially useful anti-inflammatory agents and may be of benefit in patients at risk of developing sepsis. an emerging application of n- pufa is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. parenteral or enteral nutrition including n- pufa appears to preserve immune function better than standard formulas and appears to partly prevent some aspects of the inflammatory response. studies to date are suggestive of clinical benefits from these approaches, especially in postsurgical patients. definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis strategies for the treatment of sepsis epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care has the mortality of septic shock changed with time? the epidemiology of the systemic inflammatory response syndrome derangements of coagulation and fibrinoloysis in critically ill patients with sepsis and septic shock tumor necrosis factor and interleukin- in the serum of children with severe infectious purpura procalcitonin and cytokine levels: relationship to organ failure and mortality in pediatric septic shock predictive value of nuclear factor κb activity and plasma cytokine levels in patients with sepsis passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin anti-cachectin/tnf monoclonal antibodies prevent septic shock during lethal bacteraemia a recombinant human receptor antagonist to interkeukin improves survival after lethal endotoxemia in mice interleukin- controls interferon-gamma and tumor necrosis factor production during experimental endotoxemia mice deficient for the kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to l. monocytogenes infection plasma tumor necrosis factor and mortality in critically ill septic patients persistently elevated soluble tumor necrosis factor receptor and interleukin- receptor antagonist levels in critically ill patients increased soluble interleukin- type ii receptor concentrations in postoperative patients and in patients with sepsis syndrome cytokine signalling-regulation of the immune response in normal and critically ill states anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia potential hazards of combination immunotherapy in the treatment of experimental septic shock tumor necrosis factor-dependent adhesions as a major protective mechanism early in septic peritonitis in mice treatment of septic shock with the tumor necrosis factor receptor:fc fusion protein gene polymorphisms, inflammatory diseases and cancer tumour necrosis factor (tnf) gene polymorphism influences tnf-alpha production in lipopolysaccharide (lps)-stimulated whole blood cell culture in healthy humans association of tumor necrosis factor (tnf) and class ii major histocompatibility complex alleles with the secretion of tnf-alpha and tnf-beta by human mononuclear cells: a possible link to insulin-dependent diabetes mellitus association of tnf , a tnf-α promoter polymorphism, with septic shock susceptibility and mortality a genetic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-α concentrations and outcome of patients with severe sepsis are postoperative complications genetically determined by tnf-β ncoi gene polymprophism? the role of prostaglandin e in immune suppression following injury ibuprofen restores cellular immunity and decreases susceptibility to sepsis following hemorrhage the acute respiratory distress syndrome delayed hypersensitivity: indicator of acquired failure of host defenses in sepsis and trauma the effects of injury on the adaptive immune response sepsis syndromes: understanding the role of innate and acquired immunity major injury leads to predominance of the t helper- lymphocyte phenotype and diminished interleukin- production associated with decreased resistance to infection anti-inflammatory cytokines interleukin reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia interleukin protects mice from lethal endotoxemia the cooperative effects of tnf-α and ifn-γ are determining factors in the ability of il- to protect mice from lethal endotoxemia monocyte hla-dr antigen expression characterises clinical outcome in the trauma patients changes in major histocompatibility complex class ii expression in monocytes and t cells of patients developing infection after surgery monocyte response to bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory cytokines during sepsis decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients dysregulation of in vitro cytokine production by monocytes during sepsis net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin- as a major functional deactivator of human monocytes selective defects of t lymphocyte function in patients with lethal intraabdominal infection relationships between t lymphocyte apoptosis and anergy following trauma sepsis after major visceral surgery is associated with sustained and interferon-γ-resistant defects of monocyte cytokine production coincidence of proand anti-inflammatory responses in the early phase of severe sepsis: longitudinal study of mononuclear histocompatibility leukocyte antigen-dr expression, procalcitonin, c-reactive protein, and changes in t-cell subsets in septic and postoperative patients n- polyunsaturated fa, inflammation and immunity: pouring oil on troubled waters or another fishy tale? dietary polyunsaturated fatty acids and eicosanoids: potential effects on the modulation of inflammatory and immune cells: an overview polyunsaturated fatty acids, inflammation and immunity dietary modification of inflammation with lipids n- polyunsaturated fatty acids and inflammation: from molecular biology to the clinic vitro effects of eicosanoids derived from different -carbon fatty acids on production of monocyte-derived cytokines in human whole blood cultures the modulatory effects of prostaglandin-e on cytokine production by human peripheral blood mononuclear cells are independent of the prostaglandin subtype prostaglandin e inhibits tnf production in murine bone marrow-derived dendritic cells prostaglandin e is a potent inhibitor of human interleukin production in murine bone marrow-derived dendritic cells differential regulation of cytokine and cytokine receptor genes by paf, ltb and pge differential effects of prostaglandin derived from ω- and ω- polyunsaturated fatty acids on cox- expression and il- secretion lipid mediator class switching during acute inflammation: signals in resolution endogenous anti-inflammatory mediators from arachidonate in human neutrophils lipoxin a analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis reduced inflammation and tissue damage in transgenic rabbits overexpressing -lipoxygenase and endogenous anti-inflammatory lipid mediators the inhibition of t-lymphocyte proliferation by fatty acids is via an eicosanoid-independent mechanism in vitro effects of eicosanoids derived from different -carbon fatty acids on t helper type and t helper type cytokine production in human whole-blood cultures nuclear factor κb is activated by arachidonic acid but not by eicosapentaenoic acid fatty acids and cytokine mrna expression in human osteoblastic cells: a specific effect of arachidonic acid arachidonic acid-induced il- expression is mediated by pkc-α activation in osteoblastic cells linoleic acid activates nuclear transcription factor-kappa b (nf-kappa b) and induces nf-kappa b-dependent transcription in cultured endothelial cells fatty acid-induced activation of vascular endothelial cells linoleic acid potentiates tnf-mediated oxidative stress, disruption of calcium homeostasis, and apoptosis of cultured vascular endothelial cells unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells effect of linoleic acid on endothelial cell inflammatory mediators linoleic acid-induced vcam- expression in human microvascular endothelial cells is mediated by the nf-kappa b-dependent pathway linoleic acid-stimulated vascular adhesion molecule- expression in endothelial cells depends on nuclear factor-κb activation the effects of short-and long-term supplementation with fish oil on the incorporation of n- polyunsaturated fatty acids into cells of the immune system in healthy volunteers encapsulated fish oil enriched in α-tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions the effect of low to moderate amounts of dietary fish oil on neutrophil lipid composition and function the effect of dietary supplementation with n- polyunsaturated fatty acids on the synthesis of interleukin- and tumor necrosis factor by mononuclear cells oral (n- ) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women the effect on human tumor necrosis factor a and fatty acids from vegetable oil or fish oil prostaglandin e production and t-cell function after fish-oil supplementation: response to antioxidant co-supplementation effects of dietary enrichment with eicosapentaenoic acid and docosahexaenoic acid on in vitro neutrophil and monocyte leukotriene generation and neutrophil function dietary ω- polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils n- fatty acids and cysteinyl-leukotriene formation in humans in vitro, ex vivo and in vivo manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach interaction between peroxidase and cyclooxygenase activities in prostaglandin-endoperoxide synthase eicosapentaenoic acid inhibits prostaglandin d generation by inhibiting cyclo-oxygenase- in cultured human mast cells effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of -lipoxygenase pathway products by ionophore-activated human neutrophils effect of docosahexaenoic acid (dha) on arachidonic acid metabolism and platelet function docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis n- fatty acids specifically modulate catabolic factors involved in articular cartilage degradation pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n- fatty acids fatty acid substrate specificities of human prostaglandin-endoperoxide h synthase- and - structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxidase h synthase- separation and quantification of pge following derivatization with panacyl bromide by high pressure liquid chromatography with fluorometric detection human neutrophil chemotactic and degranulating activities of leukotriene b (ltb ) derived from eicosapentaenoic acid characterization and biologic properties of , -dihydroxy derivatives of eicosapentaenoic acid, including leukotriene-b and the double lipoxygenase product synthesis of -series and -series leukotrienes in the lung microvasculature challenged with escherichia coli hemolysin: critical dependence on exogenous free fatty acid supply impact of arachidonic acid versus eicosapentaenoic acid on exotoxin-induced lung vascular leakage-relation to -series versus -series leukotriene generation alteration of n- fatty acid composition in lung tissue after short-term infusion of fish oil emulsion attenuates inflammatory vascular reaction anti-inflammatory lipid signals generated from dietary n- fatty acids via cyclooxygenase- and transcellular processing: a novel mechanism for nsaid and n- pufa therapeutic actions novel functional sets of lipid-derived mediators with anti-inflammatory actions generated from omega- fatty acids via cyclooxygenase -nonsteroidal antiinflammatory drugs and transcellular processing resolvins: a family of bioactive products of omega- fatty acid transformation circuits initiated by aspirin treatment that counter pro-inflammation signals novel docosatrienes and s-resolvins generated from docosahexaenoic acid in urine brain, human blood and glial cells: autocoids in anti-inflammation novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression neuroprotectin d : a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress the omega- fatty acid docosahexaenoate reduces cytokine-induced expression of proatherogenic and proinflammatory proteins in human endothelial cells docosahexaenoic and eicosapentaenoic acids inhibit in vitro human endothelial cell production of interleukin- fish oil decreases macrophage tumor necrosis factor gene transcription by altering the nfκb activity modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-α production by ω- fatty acid is associated with differential cyclooxygenase- protein expression and is independent of interleukin- nf-κb inhibition by ω- fatty acids modulates lps-stimulated macrophage tnf-α transcription eicosapentaenoic acid prevents lps-induced tnf-α expression by preventing nf-κb activation the anti-catabolic effects of n- fatty acids fish oil modulates macrophage p / mitogen-activated protein kinase activity induced by lipopolysaccharide fish oil suppressed cytokines and nuclear factor kappab induced by murine aids virus infection dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice n- pufa supplementation, monocyte pca expression and interleukin- production inhibition of tumour necrosis factor-α and interleukin- production by mononuclear cells following dietary fish-oil supplementation in healthy men and response to antioxidant co-supplementation comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects the ability of fish oil to suppress tumor necrosis factor-α production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor α production enhanced survival to endotoxin in guinea pigs fed iv fish oil emulsion endotoxin challenge after menhaden oil diet: effects on survival of guinea pigs effects of continuous tube feeding of dietary fat emulsions on eicosanoid production and on fatty acid composition during an acute septic shock in rats eicosapentaenoic acid reduces pulmonary edema in endotoxemic rats influence of butter and of corn, coconut and fish oils on the effects of recombinant human tumour necrosis factor-α in rats attenuation of the febrile response in guinea pigs by fish oil enriched diets diets enriched with n- fatty acids ameliorate lactic acidosis by improving endotoxin-induced tissue hypoperfusion in guinea pigs long-term feeding with structured lipid composed of medium-chain and n- fatty acids ameliorates endotoxic shock in guinea-pigs effects of eicosapentaenoic and γ-linolenic acids (dietary lipids) on pulmonary surfactant composition and function during porcine endotoxemia dietary fish oil and fish and borage oil suppress intrapulmonary proinflammatory eicosanoids biosynthesis and attenuate pulmonary neutrophil accumulation in endotoxic rats effects of eicosapentaenoic and gamma-linolenic acid on lung permeability and alveolar macrophage eicosanoid synthesis in endotoxic rats effects of a fish oil diet on pig's cardiopulmonary response to bacteremia effects of endotoxin on pigs prefed omega- vs omega- fatty acids-enriched diets select dietary fatty acids attenuate cardiopulmonary dysfunction during acute lung injury in pigs fatty acids and lymphocyte functions the effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man influence of dietary supplementation with long-chain n- or n- polyunsaturated fatty acids on blood inflammatory cell populations and functions and on plasma soluble adhesion molecules in healthy adults lack of effect of foods enriched with plant-or marine-derived n- fatty acids on human immune function the influence of different combinations of γ-linolenic acid, stearidonic acid and epa on immune function in healthy young male subjects fish oil supplementation inhibits the expression of major histocompatibility complex class ii molecules and adhesion molecules on human monocytes effect of long-term fish oil supplementation on vitamin e status and lipid peroxidation in women dietary supplementation with omega polyunsaturated fatty acids decreases mononuclear cell proliferation and interleukin beta content but not monokine secretion in healthy and insulin dependent diabetic individuals dietary supplementation with γ-linolenic acid or fish oil decreases t lymphocyte proliferation in healthy older humans fish oil-supplemented parenteral diets normalize splanchnic blood flow and improve killing of translocated bacteria in a low-dose endotoxin rat model dietary omega- fatty acids decrease mortality and kupffer cell prostaglandin e production in a rat model of chronic sepsis the fatty acid composition of maternal diet affects lung prostaglandin e levels and survival from group b streptococcal sepsis in neonatal rat pups parenteral supplementation with a fish oil emulsion prolongs survival and improves lymphocyte function during sepsis essential fatty acids influence survival in stress dietary fish oil supplementation in experimental gram negative infection and in cerebral malaria in mice fish oil decreases natural rresistance of mice to infection with salmonella typhimurium dietaey fish oil reduces survival and impairs bacterial clearance in c h/hen mice challenged with listeria monocytogenes influence of dietary (n- ) polyunsaturated fatty acids on leukotriene b and prostaglandin e synthesis and the time course of experimental tuberculosis in guinea pigs effect of dietary (n- ) and (n- ) fatty acids on in vivo pulmonary bacterial clearance by neonatal rabbits analysis of the incubation period for measles in the epidemic in greenland in using a variance components model total parenteral nutrition in the critically ill patient: a meta-analysis inhibition of lymphocyte proliferation in vitro by two lipid emulsions with different fatty acid compositions a prospective, randomized trial of intravenous fat emulsion administration in trauma victims requiring total parenteral nutrition influences of soybean oil emulsion on stress response and cell-mediated immune function in moderately or severely stressed patients effect of different types of total parenteral nutrition on t-lymphocyte subpopulations and nk cells effects of different lipid emulsions on lymphocyte function during total parenteral nutrition intravenous lipid dose and incidence of bacteremia and fungemia in patients undergoing bone marrow transplantation the effect of parenteral fish oil on leukocyte membrane fatty acid composition and leukotriene-synthesizing capacity in postoperative trauma impact of omega- fatty acid enriched tpn on leukotriene synthesis by leukocytes after major surgery influence of a total parenteral nutrition enriched with ω- fatty acids on leukotriene synthesis of peripheral leukocytes and systemic cytokine levels in patients with major surgery immunomodulation by perioperative administration of n- fatty acids impact of fish oil enriched total parenteral nutrition on dna synthesis, cytokine release and receptor expression by lymphocytes in the postoperative period perioperative administration of parenteral fish oil supplements in a routine clinical setting improves patient outcome after major abdominal surgery enteral nutrition during multimodality therapy in upper gastrointestinal cancer patients clinical outcome and immunology of postoperative arginine fatty acids, and nucleotide-enriched enteral feeding: a randomized prospective comparison with standard enteral and low calories/low fat iv solutions immune and nutritional effects of early enteral nutrition after major abdominal operations enteral nutrition with supplemental arginine, rna, and omega- fatty acids in patients after operation: immunologic, metabolic, and clinical outcome a prospective, randomised clinical trial on perioperative feeding with an arginine-, omega- fatty acid-, and rna-enriched enteral diet: effect on host response and nutritional status perioperative immunonutrition in patients undergoing cancer surgery enteral nutritional supplementation with key nutrients in patients with critical illness and cancer-a meta-analysis of randomized controlled clinical trials immunonutrition in the critically ill: a systematic review of clinical outcome should immunonutrition become routine in critically ill patients? a systematic review of the evidence effect of enteral nutrition on in vitro tests of immune function in icu patients: a preliminary report influence of arginine, omega- fatty acids and nucleotide-supplemented enteral support on systemic inflammatory response syndrome and multiple organ failure in patients after severe trauma and the enteral nutrition in ards study group ( ) effect of enteral feeding with eicosapentaenoic acid, γ-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome enteral nutrition with eicosapentaenoic acid, gamma-linolenic acid, and antioxidants reduces alveolar inflammatory mediators and protein influx in patients with acute respiratory distress syndrome ) ω- vs, ω- lipid emulsions exert differential influence on neutrophils in septic shock patients: impact on plasma fatty acids and lipid mediator generation parenteral nutrition with fish oil modulates cytokine response in patients with sepsis key: cord- -a obgg authors: canning, b.j.; spina, d. title: sensory nerves and airway irritability date: - - journal: sensory nerves doi: . / - - - - _ sha: doc_id: cord_uid: a obgg the lung, like many other organs, is innervated by a variety of sensory nerves and by nerves of the parasympathetic and sympathetic nervous systems that regulate the function of cells within the respiratory tract. activation of sensory nerves by both mechanical and chemical stimuli elicits a number of defensive reflexes, including cough, altered breathing pattern, and altered autonomic drive, which are important for normal lung homeostasis. however, diseases that afflict the lung are associated with altered reflexes, resulting in a variety of symptoms, including increased cough, dyspnea, airways obstruction, and bronchial hyperresponsiveness. this review summarizes the current knowledge concerning the physiological role of different sensory nerve subtypes that innervate the lung, the factors which lead to their activation, and pharmacological approaches that have been used to interrogate the function of these nerves. this information may potentially facilitate the identification of novel drug targets for the treatment of respiratory disorders such as cough, asthma, and chronic obstructive pulmonary disease. the primary function of the lung is gas exchange. the airways serve as a conduit for moving inspired air to the gas-exchanging regions of the lungs, and for expiration of co . airway and lung reflexes optimize lung capacity for gas exchange in response to a continually changing demand. airway reflexes also serve to preserve airway patency. these reflexes can become aberrant, however, and may worsen the symptoms of diseases such as asthma and chronic obstructive pulmonary disease (copd). multiple afferent nerve subtypes regulate these homeostatic and defensive reflexes, each subtype with unique physiological, anatomical, and pharmacological attributes. the properties of airway afferent nerve subtypes will be reviewed, as will their role in regulating bronchopulmonary reflexes and bronchial responsiveness. airway afferent nerve subtypes have been defined by their chemical and physical sensitivity, adaptation to mechanical stimulation, origin, myelination, conduction velocity, neurochemistry, basal activity, reflexes associated with their activation, and sites of termination in the airways, lungs, and brain stem. these various approaches to characterizing airway afferent nerves are hampered by their lack of specificity. but when used in combination, patterns of physiological and pharmacological attributes have emerged to help define at least four distinct subtypes of airway afferent nerves (canning et al. b ). slowly adapting receptors (sars) are the prototypical airway mechanoreceptors. the mechanical forces produced during breathing are the primary stimulus for sar activation, with sar activity increasing during inspiration and peaking prior to expiration (miserocchi and sant'ambrogio ; ho et al. ; schelegle and green ) . sars regulate the hering-breuer reflex, which terminates inspiration and initiates expiration when the lungs are adequately inflated (schelegle and green ) . sars thus play a primary role in regulating respiratory rate. sars can be differentiated from rapidly adapting receptors (rars) in some species on the basis of action potential conduction velocity, and in most species by their modest adaptation to sustained lung inflation (fig. ). sars may be differentially distributed in the airways of commonly studied mammalian species (schelegle and green ) . in cats, guinea pigs, and rats, few sars but many rar-like receptors and c-fibers can be found in the extrapulmonary airways. in dogs, sars may also be localized to the extrapulmonary airways (miserocchi and sant'ambrogio ; sant'ambrogio et al. ) . sars also differ from rars with respect to the reflexes they precipitate (see later). subtypes of sars have been described (miserocchi and sant'ambrogio ; schelegle and green ) . sars are generally unresponsive to chemical stimuli. with the exception of the small population of sars terminating in airway smooth muscle, this also includes an insensitivity to stimuli that initiate bronchospasm, pulmonary edema, pulmonary vascular congestion, or any stimulus that decreases lung compliance. the ion channels regulating the mechanical sensitivity of sars are also poorly defined. gadolinium ( mm applied repeatedly for min to sar receptive fields) slightly ( - %) reduced rabbit bronchial sar discharge to different levels of inflation pressures (ma et al. ) . other drugs reported to modify sar discharge include the voltage-sensitive k + -channel blocker -aminopyridine, the voltage-sensitive na + -channel opener veratradine, the na + -k + - cl À transporter inhibitor furosemide, sulfur dioxide, and the na + -k + -atpase inhibitor ouabain (davies et al. ; matsumoto et al. matsumoto et al. , matsumoto et al. , matsumoto et al. , matsumoto et al. , sudo et al. ; guardiola et al. ). an additional and unique physiological and pharmacological property of sars is their sensitivity to alveolar co concentrations (coleridge et al. ; fisher and sant'ambrogio ; green et al. ). as alveolar co increases, sar activity decreases. this contrasts sharply with bronchopulmonary c-fibers, which may be activated or at least sensitized by elevated alveolar co and/or decreases in extracellular ph (delpierre et al. ; lin et al. ) . the inhibitory effect of co on sars contributes in part to the hyperpnea associated with hypercapnea. the actions of co on sar excitability may occur secondary to effects on nerve terminal ph as evidenced by the preventive effect of the carbonic anhydrase inhibitor acetazolamide during co challenges (ravi ; matsumoto ; hempleman et al. ) . the expression of a carbonic anhydrase isozyme in sars has not been systematically evaluated. the term ''rapidly adapting receptor'' (rar) describes a subtype of airway and lung stretch receptor that are activated during the dynamic phase of lung inflation, but unlike sars become quiescent during static lung inflation (knowlton and larrabee ; widdicombe a) . often inappropriately, airway afferents that rapidly adapt to any stimulus are grouped into a broad and heterogeneous class, all of which are called ''rars.'' this has proven misleading (canning and chou fig. the characteristic features of airway and lung vagal afferent nerve subtypes are shown in these single-fiber recordings in the rat. c-fibers are generally unresponsive to mechanical stimulation, including the mechanical consequences of lung inflation and deflation, but are vigorously activated by capsaicin. the rapidly adapting stretch receptors (rars) and the slowly adapting stretch receptors (sars) are largely insensitive to capsaicin. both lung stretch receptor subtypes are responsive to lung inflation, with rar activity more prominent in species with higher respiratory rates. rars and sars are differentiated in part by their responses to sustained lung inflation. these vagal afferent nerve subtypes differentially regulate airway autonomic outflow, respiratory pattern, respiratory sensations, and cough. subtypes of each afferent class have been described and are found in all species thus far studied. (reproduced with permission from ho et al. ) ). in this review, the term ''rar'' refers to those intrapulmonary stretch receptors that rapidly adapt to sustained lung inflation (fig. ) . rars are considerably less active than sars during eupnea but more active than c-fibers. rars are also activated (either directly or indirectly) by a variety of mechanical stimuli in the lung, including airway smooth muscle contraction, pulmonary edema, decreased lung compliance, lung collapse, and negative airway luminal pressures (mills et al. ; armstrong and luck ; bergren ; ho et al. ; canning et al. ; canning and chou ) . rars are generally more responsive to chemical stimuli than sars, prompting use of the term ''irritant receptor'' to describe this airway afferent nerve subtype. what has been less clear, however, is whether the ability of these chemical stimuli to activate rars is through a direct action or secondary to a mechanical effect evoked in the lung. as mentioned above, rars are exquisitely sensitive to decreases in lung compliance (jonzon et al. ; yu et al. yu et al. , . accordingly, bronchoconstrictors such as serotonin, methacholine, histamine, and substance p and many other stimuli that initiate bronchospasm likely activate rars at least partly through their effects on lung mechanics (mills et al. ; mohammed et al. ; bergren ; canning et al. ; chou et al. ) . similar mechanisms may underlie the ability of pulmonary embolism and pulmonary vascular congestion to activate rars (mills et al. ; sellick and widdicombe ; ravi and kappagoda ; bonham et al. ) (fig. ) . but there are several reports suggesting a direct effect of autacoids such as atp, histamine, and serotonin on rars (vidruk et al. ; dixon et al. ; matsumoto and shimizu ; canning et al. ) . unlike sars and c-fibers, however, rars are largely unaffected by changes in alveolar co concentrations (sampson and vidruk ; ravi ) . as with sars, the ion channels regulating rar activation secondary to mechanical stimulation are poorly defined. gadolinium ( mm applied repeatedly for min to rar receptive fields) is reported to have a profound effect on rar excitation by lung inflation and pulmonary vascular congestion (ma et al. ) . given the distinctive accommodative responses of rars to sustained lung inflation, it seems possible that they express a unique set of ion channels that may be amenable to selective pharmacological modulation. this may prove useful in developing more selective drugs for treating respiratory disorders. with their defining physiological attribute of an axonal conduction velocity of ms À or less, bronchopulmonary c-fibers are the most readily identifiable vagal afferent nerve subtype innervating the airways. c-fibers can be activated by several chemical and mechanical stimuli, with responses depending upon the stimulus and the c-fiber subtype studied ricco et al. ; lee and pisarri ; undem et al. ) . the majority of c-fibers innervating the airways and lungs of all species are activated by the trpv receptor agonist capsaicin (figs. , ), a predictable observation, given the known expression patterns of trpv in afferent c-fibers throughout the body of most species (caterina et al. ) . but it is inappropriate to conclude from these data that responsiveness to capsaicin is the defining characteristic of airway c-fibers. c-fibers in dogs, rats, and mice that are not activated by lung capsaicin challenge have been described ho et al. ; kollarik et al. ) . moreover, perhaps secondary to the end organ effects associated with c-fiber activation (mucus secretion, vascular engorgement, airway smooth muscle contraction, altered respiratory pattern, and cough), other afferent nerve subtypes, especially intrapulmonary rars, can be activated by capsaicin challenge (mohammed et al. ; bergren ; morikawa et al. ) . a lack of responsiveness to mechanical stimulation and basal activity may also fail to differentiate c-fibers from other subtypes of bronchopulmonary afferent nerves. while c-fibers are generally less responsive to mechanical stimulation, they can be activated by punctate mechanical stimulation or lung inflation, and can have basal activity comparable to that of some rars fox et al. ; ricco et al. ; lee and pisarri ) . subtypes of bronchopulmonary c-fibers have been described. the coleridges defined c-fiber subtypes in dogs by their responsiveness to stimulants administered via the pulmonary or bronchial circulation . more recently, undem (kollarik et al. ; undem et al. ) described bronchopul-monary c-fiber subtypes in both guinea pigs and mice. in guinea pigs, c-fiber subtypes are differentiated on the basis of their ganglionic origin (nodose vs. jugular ganglia) and, by extension, their embryological origin, their sites of peripheral termination (extrapulmonary and intrapulmonary vs. exclusively intrapulmonary), expression of neurokinins, and responsiveness to adenosine, serotonin -ht , and atp receptor agonists chuaychoo et al. chuaychoo et al. , . these subtypes are known to have opposing effects on both cough and respiration, but both subtypes may initiate reflex bronchospasm upon activation (canning et al. a, b; chou et al. ; reynolds et al. ) (fig. ) . c-fibers arising from dorsal root ganglia also innervate the airways (martling ; kummer et al. ; dinh et al. ; oh et al. ; kwong et al. b) . their physiological properties and reflex actions have been only partially described. c-fibers are found throughout the airways and lungs of all species. in guinea pigs, the jugular-type c-fibers have been localized to both intrapulmonary and extrapulmonary airways, while the nodose-type c-fibers are found predominantly in the peripheral airways and lungs ). the extensively branched terminals of c-fibers in guinea pig and rat tracheae can be immunohistochemically labeled for the neuropeptides calcitonin gene-related peptide (cgrp), substance p, and neurokinin a (mcdonald et al. ; baluk et al. ; kummer et al. ; hunter and undem ; yamamoto et al. ). comparable structures can be fig. respiratory reflex effects evoked by histamine, adenosine, and capsaicin reveal the differential distribution of airway vagal afferent nerve subtypes and their distinct effects on respiratory pattern. histamine selectively activates intrapulmonary rars and initiates tachypnea. adenosine selectively activates pulmonary c-fibers and also initiates tachypnea. capsaicin activates both bronchial and pulmonary type c-fibers, initiating a profound slowing of respiration upon laryngeal challenge, tachypnea when capsaicin is inhaled (not shown), and both tachypnea and respiratory slowing following intravenous administration. (data adapted from chou et al. ) found in the airways of other species and in the peripheral airways of guinea pigs (dey et al. ; yamamoto et al. ; lamb and sparrow ; watanabe et al. ) . c-fiber terminals can also be found in the airway microvasculature and airway smooth muscle layer, and comprise at least a portion of paintal's j-receptors, suggesting peripheral/interstitial lung terminations (paintal ; mcdonald et al. ; baluk et al. ) . a myelinated (based on an axonal conduction velocity of ms À ) afferent nerve subtype with many shared physiological and pharmacological attributes of jugular c-fibers has also been described in guinea pigs (ricco et al. ) . these afferents have their cell bodies in the jugular ganglia and are activated by acid, hypertonic saline, bradykinin, and capsaicin. unlike jugular c-fibers, these capsaicin-sensitive ad-fibers terminate exclusively in the large airways (larynx, trachea, mainstem bronchi) and do not normally express the neuropeptide substance p (but can be labeled immunohistochemically for the structural protein neurofilament). trpv positive, substance p-negative nerve terminals have been described in the airway epithelium of guinea pigs and may correspond to this afferent subtype (watanabe et al. (watanabe et al. , . the existence of an ad afferent subpopulation expressing trpv in other species and their reflex effects in any species upon activation are unknown. in contrast to the indirect effects of autacoids and irritants thought to account for their activation of rars, there is molecular, immunohistochemical, and electrophysiological evidence to suggest that many mediators associated with airway inflammation act directly on bronchopulmonary c-fibers. stimuli known to activate airway and lung c-fibers include capsaicin and other trpv receptor ligands, acid, cationic proteins, bradykinin, thrombin, and other protease-activated receptor (par ) agonists, adenosine, -ht receptor agonists, nicotine, atp, prostanoids, and isoprostanes, and a variety of environmental irritants including acrolein, toluene diisocyanate, and ozone lee and pisarri ; undem et al. ; chuaychoo et al. chuaychoo et al. , nassenstein et al. ; taylor-clark et al. ). many of these stimuli work partly or entirely through gating of the ion channels trpv and trpa . pcr analyses confirm the expression of trpv and trpa , but also adenosine a , adenosine a , par , and multiple subunits of nicotinic receptors in bronchopulmonary c-fibers (chuaychoo et al. ; gu et al. ; kwong et al. a, b; nassenstein et al. ) . the responsiveness to such a variety of inflammatory mediators and environmental toxins and the reflexes initiated upon the activation of c-fibers lends credence to the notion that bronchopulmonary c-fibers are analogous to the nociceptors innervating somatic tissues. trpv -dependent signaling is not the same in all bronchopulmonary c-fibers and is at least suggestive of the differential expression of a ligand-transporting system in some c-fibers or perhaps unique gating mechanisms for trpv in the various bronchopulmonary c-fiber subtypes. olvanil and anandamide are reasonably effective and potent activators of intrapulmonary c-fibers in rats and in guinea pigs, but are minimally effective at evoking tracheal/bronchial c-fiber action potential discharge or tachykinin release from the peripheral terminals of bronchial and tracheal c-fibers (tucker et al. ; lin and lee ; kollarik and undem ; lee et al. ) . this inability to activate bronchial c-fibers is overcome with sustained incubation times, suggesting an impaired access to the intracellular binding site of trpv . conversely, cooling the terminals of pulmonary c-fibers rendered them considerably less responsive to olvanil and anandamide, but equally responsive to capsaicin. these data may predict the expression of an anandamidetransporting system in pulmonary c-fibers that is absent in bronchial and tracheal c-fibers (ligresti et al. ) . to date, however, no protein subserving this transporting function has been identified (glaser et al. ) . it is thus interesting that activation of trpv has been shown to promote the movement of extraordinarily large molecules from the extracellular to the intracellular space through the open trpv channel (meyers et al. ; binshtok et al. ). the hill coefficient for trpv activation is significantly different from unity, suggestive of cooperative binding properties (szallasi ; welch et al. ; . it seems possible that threshold trpv activation resulting in transient channel opening promotes additional agonist influx and further receptor activation. perhaps some subtle modification of trpv channel gating in c-fiber subtypes determines the ability of anandamide and olvanil to move through the open trpv channel. in addition to the autacoids listed above that activate bronchopulmonary c-fibers, many other mediators can sensitize them to subsequent activation. these include histamine via h receptors, cysteinyl leukotrienes via cyslt receptors, epinephrine via b receptors, and prostaglandin ep and tp receptor agonists (karla et al. ; morton , ; mcalexander et al. ; xiang et al. ; gu et al. ). prostaglandins also likely account for the sensitizing effects of protease-activated receptor (par ) agonists on bronchopulmonary c-fibers (gatti et al. ) . some mechanistic studies of these sensitizing effects have been carried out in patch-clamp analyses. other unique characteristics regulating airway c-fiber activation include sensitivity to changes in extracellular cl À and ca + concentrations, changes in airway surface liquid osmolarity, trpv -independent activation by acid (perhaps involving acid-sensing ion channels), and activation/ sensitization by co (delpierre et al. ; pisarri et al. ; fox et al. ; pedersen et al. ; kollarik and undem ; undem et al. ; lin et al. ; gu and lee ) . c-fiber-selective stimulants that readily initiate coughing in awake human subjects and in awake guinea pigs have consistently failed to initiate cough in anesthetized cats, dogs, or guinea pigs. on the basis of the studies of widdicombe ( a, b) published in and the results of vagal cooling studies in cats and dogs by tatar et al. ( tatar et al. ( , , it had become almost dogma that cough is initiated by activation of rars. but many well-known and even selective stimuli for rars, including a variety of bronchoconstrictors, negative airway luminal pressures, or inspiratory efforts against a closed glottis, have been consistently ineffective at evoking cough in either awake or anesthetized animals or humans. recent studies carried out in guinea pigs and a reappraisal of widdicombe's studies in cats suggest that a vagal afferent nerve subtype distinct from both c-fibers and rars plays an essential role in regulating the cough reflex in anesthetized guinea pigs and cats and likely in any species that has a well-defined cough reflex. these afferents have thus been called ''cough receptors'' (canning et al. (canning et al. , a canning and chou ) . cough receptors are differentiated from c-fibers and rars in guinea pigs by conduction velocity. with a conduction velocity of approximately ms À , these afferents conduct action potentials considerably faster than c-fibers ( ms À or less) but considerably slower than either rars or sars (more than ms À ). cough receptors are also differentiated from c-fibers and rars by mechanical sensitivity, being exquisitely sensitive to punctate mechanical stimulation ( - times more sensitive than c-fibers) but utterly insensitive to changes in airway luminal pressure or airway smooth muscle contraction, both of which activate rars. also unlike c-fibers, the cough receptors are insensitive to capsaicin and bradykinin (fig. ) . cough receptors are activated by acid but entirely through trpv -independent mechanisms (canning et al. (canning et al. , a . by combination of electrophysiological studies with intravital labeling methods, retrograde neuronal tracing, organotypic cultures, and immunohistochemistry, the peripheral terminals of cough receptors in the guinea pig trachea and bronchus have been identified (canning et al. a, b) . terminating between the epithelium and smooth muscle layers of the airways mucosa, the cough receptors assume a fig. electrophysiological characteristics of the extrapulmonary vagal afferent nerves regulating cough of guinea pigs. cough receptors and c-fibers are both activated by punctate mechanical stimulation and by acid, but the cough receptors are insensitive to capsaicin. capsaicin and other c-fiber-selective stimulants initiate coughing in awake animals and in awake human subjects, but have consistently failed to initiate coughing in anesthetized animals. in anesthetized guinea pigs, topical acid challenge of the tracheal mucusa initiates coughing, while topical capsaicin challenge does not evoke coughing. rather, capsaicin challenge in anesthetized guinea pigs evokes respiratory slowing and, occasionally, a profound apnea followed by gasping and a gradual recovery of a normal respiratory pattern. (reproduced with permission from canning et al. ) circumferential position in the extracellular matrix. branching is extensive at the terminals, with axons projecting from longitudinal nerve bundles through the smooth muscle layer. similar structures have been described in the airway mucosa of other species but their identity as ''cough receptors'' is unclear (larsell (larsell , gaylor ; yamamoto et al. ; yu ; de proost et al. ). immunohistochemistry confirms the selective expression of subtypes of na + -k + -atpase and na + -k + - cl À transporter in guinea pig cough receptors (canning et al. a, b; mcgovern , ) . more recently, tetrodotoxininsensitive na + channels have been localized to these cough receptors (kwong et al. a, b) . pharmacological analyses suggest that these regulators of ion flux and gradients, as well as cl À channels and voltage-sensitive k + channels, may be critical to the regulation of cough receptor responsiveness to chemical (acid) and punctate mechanical stimuli mcalexander and undem ; canning et al. a, b; mazzone and mcgovern ; canning ) . no other stimuli thus far studied, including a variety of autacoids and neurotransmitters and ion channel modulators, alter cough receptor excitability or the ability of acid or mechanical stimuli to initiate coughing in guinea pigs. parasympathetic nerves play a primary role in regulating airway smooth muscle tone and glandular secretion in the airways and also regulate pulmonary and bronchial vascular tone (canning ; wine ) . there are two anatomically, physiologically, and pharmacologically distinct parasympathetic pathways projecting to the airways with opposing effects on airway smooth muscle but synergistic effects on airway mucus secretion. parasympathetic-cholinergic nerves initiate airway smooth muscle contraction, pulmonary vascular dilatation, and mucus secretion upon activation, with acetylcholine acting in each target tissue via muscarinic m receptors. parasympathetic noncholinergic nerves also innervate the airways of most species, including humans. noncholinergic parasympathetic nerves utilize the peptide transmitter vasoactive intestinal peptide and related peptides (pituitary adenylate cyclase activating peptide, peptide histidine isoleucine, peptide histidine methionine) as well as the gaseous transmitter nitric oxide (formed from arginine by the neuronal isoform of nitric oxide synthase). upon activation, noncholinergic parasympathetic nerves evoke bronchodilatation, airway vascular dilatation, and mucus secretion. coincident activation of cholinergic and noncholinergic parasympathetic nerves may have synergistic effects on airway glandular secretion (choi et al. ; wine ) . airway and lung afferent nerve activation initiates myriad patterns of airway parasympathetic nerve responses (canning ) . at eupnea, basal parasympathetic tone appears to be necessarily dependent upon the ongoing activity of airway vagal afferent nerves, either rars or c-fibers (jammes and mei ; kesler and canning ) . with challenge, activation of bronchopulmonary c-fibers or rars increases airway cholinergic and noncholinergic parasympathetic nerve activity (fig. ) . activation of intrapulmonary stretch receptors (sars) by lung fig. reflex-evoked, airway parasympathetic nerve-dependent regulation of airway smooth muscle tone in guinea pigs in situ. (a) the c-fiber-selective stimulant bradykinin evokes reflex bronchospasm largely independent of any direct effects on airway smooth muscle. histamineevoked reflex bronchospasm occurs secondary to its direct effects on airway smooth muscle, which in turn activates intrapulmonary rars. evidence for the selective effects of bradykinin and histamine on c-fibers and rars, respectively, is apparent from the marked inhibition of bradykinin-evoked reflex bronchospasm by intravenous or intracerebroventricular administration of neurokinin receptor antagonists, which are without effect on histamine-evoked reflexes. neurokinins are selectively expressed by c-fibers in guinea pigs. (b) when rars and c-fibers are activated simultaneously, marked synergism is apparent. this synergistic effect of rar and c-fiber activation on airway parasympathetic tone may result from central convergence in the nucleus of the solitary tract of these afferent nerve subtypes. (c, d) the mean data for reflex bronchospasm and whole-lung-inflation pressures evoked by histamine, bradykinin, or the combination of histamine and bradykinin. (reproduced with permission from canning et al. and canning a, b) inflation or during the hyperpnea associated with exercise induces a withdrawal of parasympathetic cholinergic nerve activity and bronchodilatation, but has no effect on parasympathetic noncholinergic nerves. reflex regulation of airway parasympathetic nerves by vagal afferents may not be entirely unidirectional. secondary to the end-organ effects precipitated by parasympathetic nerve stimulation (e.g., mucus secretion, bronchospasm), action potential patterning in airway mechanoreceptors may change dramatically richardson et al. ) . this is especially true under conditions in which tidal volumes are held constant (e.g., mechanical ventilation). an increase in parasympathetic cholinergic tone will decrease airway volume and deadspace, resulting in an increase in end-inspiratory pressure with mechanical ventilation and an increase in alveolar stretch under any mode of static volume ventilation. the increase in alveolar distension will favor an increase in sar activation and a resulting withdrawal of cholinergic tone. in this way, airway afferent and efferent nerves may work in concert to establish a set point for airway parasympathetic tone (fisher and sant'ambrogio ; richardson et al. ; matsumoto ) . perhaps in copd, with alveolar destruction and increases in lung compliance, sar activation may be diminished, prompting the elevation in airway cholinergic tone observed in this disease (gross et al. ; canning ) . reflexes regulating noncholinergic airway parasympathetic nerves have been studied in guinea pigs, cats, and human subjects (szarek et al. ; ichinose et al. ichinose et al. , michoud et al. ; inoue et al. ; lammers et al. ; canning et al. ; kesler et al. ; mazzone and canning a) . unlike cholinergic contractions of the airway smooth muscle, which reach a near maximum within s and can reverse at the same rate, noncholinergic parasympathetic nerve mediated relaxations of airway smooth muscle are slow in both onset and reversal (chesrown et al. ; diamond and o'donnell ; irvin et al. ; matsumoto et al. ; lama et al. ; canning and undem ; canning et al. ; kesler et al. ; mazzone and canning a, b) . perhaps noncholinergic parasympathetic nerves function to restore or maintain airway patency during or at the conclusion of defensive reflexes (coburn and tomita ; canning et al. a) . consistent with this hypothesis, noncholinergic parasympathetic nerve activation is only modestly effective at preventing bronchospasm mediated reflexively or by direct actions on smooth muscle, but can gradually reverse an evoked contraction and modulate sustained cholinergic tone at eupnea (aizawa et al. (aizawa et al. , (aizawa et al. , bai et al. ; szarek et al. ; clerici et al. ; miura et al. ; inoue et al. ; matsumoto et al. ; canning et al. ; kesler et al. ) . sympathetic nerves innervate the airways and lungs of all species. in most species, including humans, sympathetic-adrenergic innervation of intrapulmonary airway smooth muscle is limited or nonexistent (canning ) . in all species, sympathetic aderenergic nerves have been found innervating the airway vascular smooth muscle. until recently, however, no study has directly addressed the reflex mechanisms controlling airway sympathetic nerve activity. we recently studied reflex regulation of airway sympathetic nerves innervating the trachealis of guinea pigs (oh et al. ) . the vagus nerves were cut bilaterally to limit the influence of airway parasympathetic nerves on smooth muscle tone. with the trachealis precontracted with histamine, capsaicin inhalation evoked a marked relaxation of the trachealis that was prevented by sympathetic denervation of the trachealis, propranolol, or dorsal rhizotomy (t -t ). retrograde tracing and electrophysiological analyses identified a population of capsaicin-sensitive spinal afferent nerves innervating the intrapulmonary airways and lungs. the majority of these spinal afferent nerves expressed substance p. not surprisingly, then, neurokinin receptor antagonists prevented the reflex-mediated relaxations evoked by capsaicin inhalation. interestingly, we found that the sympathetic reflexes evoked in the airways by capsaicin inhalation occurred without any coincident cardiovascular responses (oh et al. ). this adds further evidence against historical notions regarding sympathetic nerve function in homeostatic and defensive settings (morrison ; janig and habler ) . we also observed that stimulating the central cut ends of the vagus nerves evoked propranolol-sensitive relaxations of the trachealis (oh et al. ) . vagal afferents are known to regulate sympathetic outflow to multiple organs, including the airways (barman and gebber ; bachoo and polosa ; habler et al. ; huang et al. ) . in rats and in guinea pigs, bronchopulmonary c-fiber activation can also initiate an axon reflex, characterized by the peripheral release of neuropeptides that produce a variety of end-organ effects within the airways and lungs, including bronchospasm, mucus secretion, vascular engorgement, inflammatory cell recruitment, and plasma extravasation (barnes (barnes , canning et al. a, b) . the prominent role of the axon reflex in the response to a variety of experimental challenges in rats and guinea pigs prompted a nearly two decade effort to address the hypothesis that respiratory disorders such as asthma and copd were due in part to an axon reflex. this notion did not live up to its promise in rats and guinea pigs when evaluated in the human airways, in large part owing to the relative paucity of neuropeptidecontaining afferent nerve terminals in the airways and lungs of humans (hislop et al. ; howarth et al. ; chanez et al. ; lamb and sparrow ) . it is nevertheless possible that axonal reflexes regulate human airway function, but through the actions of transmitters (e.g., atp, glutamate) other than substance p, neurokinin a, and cgrp. the most effective stimulants of the axon reflex work through the gating of the ion channel trpv . capsaicin, for example, evokes a profound c-fiber discharge and an axon reflex, all of which are abolished when trpv gating is prevented. by contrast, bradykinin, which acts only partially through trpv gating on bronchopulmonary c-fibers, evokes little if any axon reflex (mizrahi et al. ; bramley et al. ; schlemper and calixto ) . other stimuli evoking an axon reflex include hypertonic saline, cold, dry air, par agonists, nicotine, immunosuppressants (cyclosporin a, fk ), and trpa receptor activation (lundberg et al. ; umeno et al. ; mapp et al. ; harrison et al. ; pedersen et al. ; yoshihara et al. ; carr et al. ; ricciardolo et al. ; andresen and saugstad ; taylor-clark et al. ) . a variety of stimuli have also been reported to inhibit the axon reflex through effects on the airway c-fiber terminal, including a adrenoceptor agonists, b adrenoceptor agonists, m-opioid receptor agonists, gaba b receptor agonists, nociceptin, neurotensin, galanin, serotonin (via -ht receptors), prostaglandin e , adenosine, phosphodiesterase type inhibitors, neuropeptide y, vasoactive intestinal peptide/ pituitary adenylate cyclase activating peptide, dopamine d receptor agonists, bradykinin channel openers, and histamine h receptor agonists (grundstrom et al. ; belvisi et al. belvisi et al. , giuliani et al. ; kamikawa ; matran et al. ; aikawa et al. ; stretton ; verleden et al. ; takahashi et al. ; undem et al. ; spina et al. ; fox et al. ; fischer et al. ; shah et al. ; birrell et al. ) . it is tempting to speculate that the ability of these agents to inhibit the action-potential-independent axon reflex predicts a peripheral site of action of these drugs on bronchopulmonary c-fiber activation. this seems unlikely. thus, prostaglandin e and adenosine both inhibit the axon reflex but activate and/or sensitize c-fibers to action potential formation (kamikawa and shimo ; aikawa et al. ; hong et al. ; ho et al. ) . the par agonist initiates an axon reflex but fails to initiate action potentials on airway c-fibers (carr et al. ) . removal of extracellular ca + reduces neuropeptide release from capsaicin-sensitive nerves, but enhances airway c-fiber excitability (hua et al. ; undem et al. ) . together, the data argue for an almost complete dissociation of the axon reflex from c-fiber action potential formation. changes in respiratory pattern attributable to airway afferent nerve activation have been studied extensively in animals (fig. ) . respiratory sensations such as dyspnea are less amenable to study in animals, but have been studied in human subjects. the classic triad of the pulmonary chemoreflex includes bradycardia and apnea followed by rapid shallow breathing (green and jackman ; lee et al. ) . both the apnea and the rapid shallow breathing depend upon pulmonary c-fiber activation (green and jackman ) . apnea/respiratory slowing can also be evoked by c-fiber activation in the extrapulmonary airways of anesthetized animals (palecek et al. ; chou et al. ) . in both animals and humans, activation of intrapulmonary c-fibers and rars can initiate tachypnea (mills et al. ; green and jackman ; chou et al. ) . in humans, the increase in respiratory rate evoked by pulmonary c-fiber activation with adenosine is accompanied by a sensation of dyspnea (burki et al. ) . dyspnea and ''breathlessness'' can be reduced by airway or vagus nerve anesthesia or transection (winning et al. ; davies et al. b; taguchi et al. ) . prostaglandin e worsens the sensation of dyspnea (taguchi et al. ). bradykinin, a selective stimulant for airway cfibers, reproduces the sensation of ''sore throat'' associated with upper respiratory tract infections (proud and kaplan ) . enhanced breaths (or sighs) become more frequent as airway lung compliance decreases. these have been attributed to the activation of rars and may serve to open closed airways during tidal breathing at rest or during bronchospasm dybas et al. ) . for good reason, much of the focus on respiratory sensations in disease has been directed to the activation of pulmonary c-fibers. but a role for sars in respiratory sensations should not be discounted. the accumulation of co in the alveoli would limit sar discharge, delaying inspiratory termination and thus prompting hyperpnea. in copd, with alveolar destruction, the lung stretch associated with a normal tidal volume may have limited stretching effects in the peripheral airways and thus may limit sar discharge, prompting a compensatory increase in end expiratory lung volume or an enhanced sensation of air hunger despite normal or nearnormal blood gases. the na-k + - cl transport inhibitor furosemide is reported to diminish air hunger sensation during breath hold, perhaps owing to an inhibition of rar discharge but an enhancement of sar discharge (nishino ; sudo et al. ) . the cough reflex is initiated by activation of the cough receptors and by activation of a c-fiber subtype innervating the large airways (canning and chou ). the role of c-fibers in cough has been the subject of considerable debate. the chemical stimuli most effective at activating bronchopulmonary c-fibers, including capsaicin, bradykinin, and acid, are similarly very effective at initiating cough in conscious human subjects and in conscious animals (forsberg et al. ; laude et al. ; karlsson and fuller ; jia et al. ; trevisani et al. ; dicpinigaitis ) . these stimuli work entirely or partly through trpv , and immunohistochemical and single-cell pcr confirms expression of trpv in airway c-fibers (myers et al. ; groneberg et al. ; watanabe et al. ; kwong et al. a, b) . prior capsaicin desensitization prevents citric acid induced coughing in awake guinea pigs, as does pretreatment with trpv receptor antagonists (forsberg et al. ; bolser et al. ; lalloo et al. ; trevisani et al. ; gatti et al. ; leung et al. ) . taken together, these and other observations argue strongly for a role of bronchopulmonary c-fibers in cough (canning et al. a, b) . but c-fiber-selective stimuli have consistently failed to evoke coughing in anesthetized animals (tatar et al. ; karlsson et al. ; tatar et al. ; canning et al. canning et al. , a . anesthesia has no effect on coughing evoked by mechanical or acid stimulation of the airway mucosa and does not prevent c-fiber activation or other c-fiber-dependent reflexes, and yet capsaicin and bradykinin do not evoke cough in anesthetized animals tatar et al. ; canning et al. a, b) . perhaps it should be expected that c-fiber-selective stimulants would fail to evoke coughing in anesthetized animals. airway and lung c-fibers share many characteristics with somatosensory nociceptors, and it is the objective of general anesthesia to prevent the sensations and reflexes associated with nociceptor activation. but while the effects of anesthesia on nociceptor signaling may explain the inability of c-fiber-selective stimulants to evoke coughing in anesthetized animals, anesthesia cannot account for the known acute inhibitory effects c-fiber activation may have on cough in anesthetized animals, or the inability of some cfiber stimuli to evoke coughing in conscious animals and in conscious human subjects (tatar et al. (tatar et al. , . we have recently addressed the hypothesis that c-fiber subtypes might account for these opposing effects on cough. subtypes have been described in several species kollarik et al. ; undem et al. ). in guinea pigs, airway vagal c-fiber subtypes can be differentiated by their ganglionic origin, distribution in the airways, and responsiveness to atp, adenosine, and serotonin -ht receptor agonists chuaychoo et al. chuaychoo et al. , . the ability of c-fiber activation to evoke coughing in awake guinea pigs is reasonably well established, and we also reported a facilitating effect of c-fiber activation on cough (mazzone et al. ; canning et al. a, b) . in these latter studies, capsaicin or bradykinin applied topically to the tracheal mucosa greatly enhanced sensitivity to subsequent tussive stimuli. on the basis of the location of these bradykinin and capsaicin challenges, c-fibers arising from the jugular ganglia likely promote coughing. by inference, then, we further speculated that nodose c-fiber activation might acutely inhibit coughing. consistent with this hypothesis, we found that selective activation of nodose c-fibers with adenosine or -methyl- -hydroxytryptamine did not evoke coughing but greatly reduced the ability of citric acid to evoke coughing in anesthetized animals. prior adenosine inhalation also inhibited capsaicin-induced coughing in conscious guinea pigs. the results of studies carried out in other species are at least consistent with the notion that c-fiber subtypes may have opposing effects on cough. in anesthetized dogs and cats, c-fiber activation by bradykinin, capsaicin, or phenyldiguanide (a -ht receptor agonist) does not induce cough but can inhibit cough (tatar et al. (tatar et al. , karlsson et al. ) . in rabbits, a species in which cough can be evoked by citric acid aerosol inhalation (consistent with a trpv -and c-fiber-dependent mechanism; tatar et al. , adcock et al. , it has also been reported that sulfur dioxide inhalation is acutely inhibitory for cough (hanacek et al. ) . sulfur dioxide is known to activate lung c-fibers (ho et al. ). adcock et al. ( ) speculated that the inhibitory effects of the compound rsd in cough induced in rabbits might be due to its ability to activate pulmonary c-fibers. humans readily cough to capsaicin and bradykinin challenge, but are refractory to serotonin and adenosine challenge (stone et al. ; burki et al. ) while intravenous capsaicin infusion is only minimally effective at evoking cough (winning et al. ). there is also a report of serotonin-mediated inhibition of cough in human subjects (stone et al. ) . a comparable inability of intravenously capsaicin to evoke coughing has been reported in studies using conscious nonhuman primates (deep et al. ). studies of airway reflexes in response to stimuli known to be selective for the various airway afferent nerve subtypes largely substantiate the accepted classification schemes for afferent nerves. implicit in the observation that afferent nerve subtypes subserve distinct reflex functions is that central termination sites of the various afferent nerve subpopulations must diverge to some extent, allowing for reflex specificity. from the little published evidence available, this notion would seem to be substantiated. most of the work on central terminations of airway sensory nerves has been carried out in cats and rats. bronchopulmonary c-fibers and rars terminate extensively and often bilaterally in the nucleus of the solitary tract (nts), particularly in the commissural and medial subnuclei (davies and kubin ; kalia and richter ; bonham and joad ; ezure et al. ; kubin et al. ; lipski et al. ; otake et al. ; mazzone and canning a, b; kubin et al. ) . sars terminate primarily ipsilateral to their vagal origin, rostral to obex in the lateral and interstitial subnuclei (kalia and richter ; davies et al. a,b; bonham and mccrimmon ; ezure et al. ; kubin et al. ) . no attempt at differentiating termination sites of rar, sar, or c-fiber subtypes has been described. in addition to the studies of sar, rar, and bronchopulmonary cfiber termination sites, some work has been done to identify the nts subnuclei regulating the cough reflex (gestreau et al. ; ohi et al. ; jakus et al. ) . electrophysiological and functional studies show evidence for bronchopulmonary afferent nerve convergence in the cns (takagi et al. ; paton ; silva-carvalho et al. ) . coincident activation of airway afferent nerve subtypes can have synergistic effects on airway reflexes, including reflex bronchospasm and cough (mazzone and canning a, b; mazzone et al. ) (fig. ) . such synergistic interactions may explain the association between extrapulmonary disorders (e.g., gastroesophageal reflux disease, allergic rhinitis) and cough. several studies have characterized the pharmacology of the primary central synapses for airway vagal afferent nerves and have revealed a prominent role for glutamate acting via non-nmda receptors (bonham et al. ; vardhan et al. ; karius et al. ; chianca and machado ; wilson et al. ; aylwin et al. ; ezure et al. ; haxhiu et al. ; mutolo et al. mutolo et al. , . notably, however, nmda receptor activation plays an essential role in the initiation of cough, explaining in part the ability of the antitussive agent dextromethorphan to prevent coughing in animals and in human subjects (canning et al. (canning et al. , a mutolo et al. ). other agents shown to act centrally in nts to regulate airway vagal reflexes include m-opioid receptor agonists (codeine, damgo), gaba b receptor agonists, sigma agonists, and trpv receptor agonists (mazzone and geraghty ; mazzone et al. ; ohi et al. ohi et al. , mutolo et al. mutolo et al. , . serotonin ( -ht) receptor antagonists have also been shown to act centrally to modulate airway reflexes, but their site of action has not been determined (bootle et al. ) . the tachykinins substance p and neurokinin a have been localized to airway afferent neurons, and tachykinin receptor antagonists have been shown to reduce or abolish coughing evoked in guinea pigs, dogs, rabbits, cats, and pigs bolser et al. ; moreaux et al. ; house et al. ; mutolo et al. ) . capsaicin microinjection in nts evokes respiratory reflexes in rats that are abolished by neurokinin receptor antagonists, while coughing evoked in rabbits and sensitization of cough induced in guinea pigs is markedly inhibited or abolished by nts microinjection of neurokinin receptor antagonists (mazzone and geraghty ; mazzone et al. ; mutolo et al. ) . a central site of action for neuroknin receptor antagonists in cough in cats and in guinea pigs has also been suggested (bolser et al. ) . reflex bronchospasm evoked by laryngeal capsaicin and by intravenous bradykinin in guinea pigs is also prevented by centrally acting neurokinin receptor antagonists (canning et al. ; mazzone and canning a, b) (fig. ) . neurokinin- receptor antagonists are also used clinically to treat emesis, a vagal reflex in humans that has many similarities to the cough reflex (hornby ; warr ) . it seems likely then that neurokinins released from the central terminals of airway afferent nerves may also modulate airway reflexes in humans and in other species. it is thus interesting and confusing that in electrophysiological recordings of nts neurons receiving synaptic input from airway afferent nerves, little evidence for an excitatory effect of neurokinins in otherwise healthy animals has been reported. indeed, in one study, exogenously administered substance p was found to act presynaptically to depress synaptic transmission in nts (sekizawa et al. ) . many of these studies involved recording from unidentified synapses or the synapses of rars or sars, which are unlikely to express substance p under normal conditions. but even in recordings in c-fiber relay neurons, synaptic transmission has been explained entirely by the actions of glutamate (wilson et al. ; mutoh et al. ) . this suggests that under the experimental conditions used for the electrophysiological recordings done to date, solitary tract stimulation is subthreshold in intensity, frequency, or duration for tachykinin release, the neurons selected for recording (i.e., neurons receiving monosynaptic input) are typically devoid of direct tachykinin input, or the process of tissue harvest and slice preparation effectively silences neurokinin-mediated effects in nts. a number of clinical features distinguish asthmatic subjects from other respiratory diseases and may be considered characteristic of this phenotype (avital et al. ) . these include an exacerbation of disease following exposure to b-adrenoceptor antagonists (bond et al. ) , an impairment in the ability to bronchodilate following deep inspiration (slats et al. ) , and their bronchoconstrictor sensitivity to a wide range of innocuous stimuli (cockcroft and davis ; van schoor et al. ) . it is well established that asthmatic subjects are invariably more responsive to a range of stimuli, as expressed by an increase in provocative concentration that induces a % fall in forced expiratory volume in s termed ''bronchial hyperresponsiveness'' (bhr). however, not only is there an increase in the sensitivity of the airways to a stimulus, but there is also an increase in the maximum degree of airway narrowing for a given dose of agonist (fig. ) . the importance of understanding the underlying mechanism contributing toward bhr is confirmed by a study showing that treating the underlying hyperresponsiveness leads to a better improvement in asthma symptoms (sont et al. ) . a number of mechanisms have been proposed to account for why asthmatic subjects are invariably more responsive to the external environment. these include an alteration in airway geometry due to an increase in airway smooth muscle thickness that would lead to a greater degree of airway narrowing for a given dose of agonist and/or perturbations in myosin-actin function resulting in a loss in the ability of smooth muscle to dilate in response to deep inspiration, thereby leading to enhanced bronchoconstrictor responses gil and lauzon ) ; the release of cytokines and growth factors from epithelial cells which stimulate mesenchymal cells and promote structural changes in the airways leading to airway remodeling, airway inflammation, and bhr (holgate ) ; and recruitment and activation of dendritic cells, t lymphocytes, and eosinophils whose cell-derived products trigger a cascade of events within the lung leading to epithelial cell damage, increased smooth muscle contractility, and airway remodeling (beier et al. ; hammad and lambrecht ; jacobsen et al. ; kallinich et al. ; lloyd and robinson ; rosenberg et al. ). these mechanisms are all thought to contribute toward bhr in asthma, are likely to be interrelated, and contribute to the overall expression of bhr. however, there is also good evidence for the contribution of airway sensory nerves in this phenomenon (spina and page ) that might be likened to allodynia and/or hyperalgesia, which are characteristic of pain syndromes (carr and undem ; . it is common for clinicians to use stimuli such as methacholine and histamine to induce bronchoconstriction because these agents are relatively convenient to use. however, although there is a separation in airways responsiveness to these agents between asthmatic subjects and healthy individuals, there is a considerable degree of overlap and it has been suggested that these agents may not be sensitive indicators of the asthma phenotype (avital et al. ; o'connor et al. ) . in contrast, asthmatic subjects invariably bronchoconstrict in response to the indirectacting stimuli described earlier, which provoke little if any response in otherwise healthy individuals or in subjects with other respiratory diseases (avital et al. ; van schoor et al. ) . asthmatic subjects bronchoconstrict in response to a number of physiological stimuli such as exercise, distilled water, cold air, and hypertonic saline which are otherwise refractory in healthy subjects. similarly, acidification, pollutants such as sulfur dioxide, and chemical substances, including adenosine, bradykinin, and neuropeptides, evoke bronchoconstriction in asthma but have little if any effect in fig. bronchial hyperresponsiveness (bhr) in asthma. it is convenient to measure changes in forced expiratory volume in s (fev ) to increasing doses of methacholine. in asthma, there is an increase in sensitivity (leftward position of the dose-response curve) often measured in terms of pc (dotted line) and reactivity (increase in slope) and in severe cases of the disease an inability to define the maximum degree value for airway narrowing compared with healthy subjects. however, bhr as measured by changes in fev to increasing doses of methacholine may not be a sensitive indicator of the asthma phenotype (see the text). an increase in bhr can occur during exacerbation of disease as observed naturally during the pollen season, in the case of an allergic asthmatic, or following the deliberate exposure to a relevant antigen (arrow). however, asthmatic subjects are invariably responsive to a wide range of physiological stimuli that are otherwise refractory in healthy subjects. an understanding of the mechanisms by which these stimuli induce bronchoconstriction suggests that sensitization of afferent pathways may underlie this phenomenon nondiseased individuals. these agents are commonly referred to as ''indirect-acting stimuli,'' since they do not appear to mediate bronchoconstriction by direct activation of airway smooth muscle. they are thought to elicit bronchospasm by activating a number of different cell types, including mast cells, vascular smooth muscle cells, and vascular endothelial cells, and/or airway nerves page , ; van schoor et al. ) . a number of studies which measured the generation of action potentials from individual afferent nerves using well-established electrophysiological techniques have shown that stimuli including sulfur dioxide, acidification, distilled water, bradykinin, neuropeptides, and adenosine can activate c-fiber and ad-fibers in vivo (table ) . it is therefore of interest that asthmatic subjects are sensitive to such stimuli, whereas healthy subjects are invariably unresponsive to these agents (van schoor et al. ) . this suggests that the mechanisms by which these stimuli provoke bronchoconstriction are upregulated in asthma and characteristic of this phenotype. furthermore, airways inflammation appears to be correlated better with bhr to indirect stimuli such as adenosine (van den berge et al. ) , bradykinin (polosa et al. ; roisman et al. ) , and hypertonic saline (sont et al. ) than it is to more direct acting stimuli such as methacholine. similarly, during an exacerbation of bhr following the deliberate exposure of an asthmatic subject to an environmental allergen (e.g., house dust mite) there is a preferential increase in bhr to an indirect-acting stimulus such as bradykinin compared with methacholine (berman et al. ) . on the other hand, a number of pharmacological drugs used to treat asthma, including nedocromil sodium and ipratropium bromide, suppress airways responsiveness to these indirect-acting stimuli, suggesting the likely involvement of neural reflexes (van schoor et al. ) . furthermore, it is now recognized that glucocorticosteroids preferentially suppress bhr to adenosine (ketchell et al. ; van den berge et al. ) and bradykinin (reynolds et al. ) compared with methacholine. it is also noted that there is often a wide variability in airway sensitivity to spasmogens in subjects with mild asthma and there is little or very poor correlation between airway sensitivity to indirect-acting bronchoconstrictor agents such as adenosine and sensitivity to direct-acting stimuli such as methacholine. also, bhr to an indirect-acting stimulus is greater during an exacerbation of asthma and lower following anti-inflammatory treatment compared with bhr to methacholine, which directly activates airway smooth muscle (o'connor et al. ; van schoor et al. ) . this apparent lack of correlation between bronchoconstrictor potency of these different types of stimuli suggest that alteration in the thickness of the airway wall (i.e., airway remodeling) alone cannot account for these discrepancies. if airway remodeling were responsible, then there would be a better correlation between bronchoconstrictor potency of indirect-acting and direct-acting stimuli. furthermore, inflammatory insults to the airway wall would cause a similar change in bhr to these different agents and, finally, there would be no preferential effect of drug treatment on bhr to different stimuli. together, the findings of clinical studies support the notion that inflammatory insults to the lung might increase the activity of neuronal pathways, thereby widdicombe ( a, b) , boushey et al. ( ) , ho et al. ( ) , matsumoto et al. ( ) , roberts et al. ( ) distilled water dog dog, guinea pig fox et al. ( ) , pisarri et al. ( ) bradykinin dog kaufman et al. ( ) guinea pig guinea pig bergren ( ) , fox et al. ( ) , ricco et al. ( ) mouse kollarik and undem ( ) neuropeptides rabbit rabbit, guinea pig bergren ( ) , bonham et al. ( ) , prabhakar et al. ( ) capsaicin cat, guinea pig cat, dog, guinea pig, rat, mouse armstrong and luck ( ) , bergren ( ) mohammed et al. ( , morikawa et al. ( ) coleridge and coleridge ( ) , dixon et al. ( ), fox et al. ( , ho et al. ( ) , jackson et al. ( ) , kollarik and undem ( ) , ricco et al. ( ) adenosine rat, guinea pig chuaychoo et al. ( ) , hong et al. ( ) , kwong et al. ( ) endotoxin rat rat lai et al. ( ) , ruan et al. ( ) rars rapidly adapting receptors resulting in heightened sensitivity of the lungs to these indirect-acting stimuli. furthermore, one cannot view bhr as a nonspecific phenomenon, that is to say, that asthmatics are hyperresponsive to all stimuli, but rather it is increasingly apparent that bhr is more heterogeneous than is widely appreciated (o'connor et al. ) and sensory nerves might be a common pathway through which bhr is manifested in respiratory diseases such as asthma. transient receptor potential (trp) channels are protein sensors for the perception of pain, taste, hearing, and smell and comprise at least six subfamilies (nilius et al. ) . one member of this superfamily (trpv ) is predominantly localized to small-diameter afferent neurons in dorsal and vagal sensory ganglia (szallasi and blumberg ) and activation by capsaicin gives rise to feelings of warmth, heat, and pain. the cloning and expression of trpv has increased our understanding of the role of this protein in neurogenic pain, but also with migraine, cough, irritable bladder disease, and gastrointestinal inflammation (cortright et al. ; geppetti and trevisani ; jia and lee ; kollarik et al. ; liddle ; ma and quirion ; okajima and harada ; storr ) . the role of these proteins in contributing to bhr has been investigated in a number of experimental models. it has long been recognized that capsaicin selectively activates a subpopulation of afferent nerves, the neuropeptide containing c-fibers. however, it is now well established that capsaicin may also target a subset of airway ad-fibers whose cell nuclei reside within the jugular but not nodose ganglion (myers et al. ) . the activation of both of these nerve types can lead to a number of physiological changes within the airways, including reflex bronchoconstriction, release of sensory neuropeptides, edema, cough, and submucosal gland secretion (de swert and joos ) . chronic treatment with capsaicin in various animal species leads to an impairment of somatosensory function as a consequence of depletion of sensory neuropeptide content, downregulation of trpv receptor expression, and/or destruction and loss of sensory nerves (watanabe et al. (watanabe et al. , . a consequence of chronic treatment with capsaicin upon neural function in the lung is an attenuation of bhr induced by a range of stimuli (table ) . thus, bhr induced by exposing nonallergic animals to lipid mediators, including platelet-activating factor and -hydroperoxyeicosatetraenoic acid, is attenuated. a similar observation was noted when bhr was elicited following exposure of nonallergic animals to lipopolysaccharide, ozone, citric acid, parainfluenza- virus, and poly(l-lysine) or exposure of allergic animals to inhaled antigens (table ). it has been concluded that the peripheral release of sensory neuropeptides per se was responsible for inducing bhr because depletion of sensory neuropeptides within the airways was a natural consequence of chronic treatment with capsaicin. indeed a variety of animal experimental data have provided a wealth of information concerning the potential role of tachykinins unlike in guinea pigs, rabbits, ferrets and humans, neurokinins released from capsaicin-sensitive nerves are bronchodilators in rats (and mice), which may explain in part the augmentation of bronchial responsiveness seen with capsaicin pretreatment in rats (manzini ; szarek et al. ) d bhr augmented at h but not at and h after lps challenge e data not analyzed statistically f treatment of neonates with capsaicin but measurement of bronchial hyperresponsiveness and inflammation performed in adult animals such as substance p, neurokinin a, and cgrp in altering both resident lung and inflammatory cells, thereby perpetuating the inflammatory process in the lung and contributing to this process and inducing bhr (de swert and joos ) . it is therefore surprising that neurokinin antagonists have thus far proved disappointing in clinical trials in asthma (de swert and joos ) . however, the rationale for the development of tachykinin antagonists was based on the assumption that local release of tachykinins from c-fibers was sufficient to perpetuate the inflammatory response. one can only conclude that other mediators released within the airways contribute to the inflammatory process, or the possibility that capsaicin is selective for c-fiber afferents in the airways needs to be revised since trpv may also be localized to non-c-fiber afferents, or is not necessarily colocalized with sensory neuropeptides (guo et al. ; myers et al. ; tominaga et al. ; watanabe et al. watanabe et al. , , and, therefore, sensory-neuropeptide-independent mechanisms may also operate in the lung. furthermore, most studies showing an importance for tachykinins in mediating bhr stem from studies conducted in the guinea pig, an animal rich in sensory neuropeptide innervation in the lung. in contrast, the rabbit is relatively resistant to the neuropeptide-depleting effects of capsaicin despite the inhibition of bhr induced by nonimmunological and immunological methods (herd et al. ; riccio et al. )and, therefore, mechanisms other than neuropeptide depletion must account for this phenomenon, and the peripheral release of sensory neuropeptides is not obligatory for the development of bhr. this may be one reason why, thus far, selective neurokinin antagonists have proved disappointing in the treatment of asthma (spina and page ) . studies using capsaicin have shown that the functional effect of neuropeptides on airway smooth muscle is species-dependent. rabbit, monkey, and human airways contract weakly on exposure to capsaicin in vitro (ellis et al. ; spina et al. ) , whereas guinea-pig airways are very sensitive (grundstrom et al. ) . these variable functional effects are superficially consistent with the differential localization of substance p and cgrp in the airways across species, as the occurrence of these neuropeptide-containing nerves in human and rabbit tends to be sparse (hislop et al. ; howarth et al. ; laitinen et al. ; lundberg et al. ) , whereas neuropeptides are found widely throughout the airways of guinea pigs (hua et al. ; nohr and weihe ; saria et al. ) . hyperalgesia induced by chemical and thermal stimuli is suppressed in trpv knockout mice, suggesting that this protein is an important transducer of pain (gunthorpe et al. ; julius and basbaum ) . it is therefore of interest that chronic treatment with capsaicin in humans can lead to a suppression of allodynia and hyperalgesia induced by intradermal injection of capsaicin (davis et al. ) and when applied topically to the nose, capsaicin reduces nasal hyperresponsiveness in allergic rhinitis patients (sanico et al. ) . a loss in trpv signaling might help explain the loss in bhr observed in various experimental animal models (spina and page ) . if the activation of trpv is important for the sensitization of primary afferent nerves in the lung, then it would seem reasonable to suggest that pharmacological antagonism of this protein might reduce bhr. unfortunately, very few studies have specifically addressed this question in the context of bhr. in one study, bhr was induced in the guinea pig by acute challenge with platelet-activating factor, which was inhibited following pretreatment with ruthenium red, a nonselective trp channel blocker (perretti and manzini ) . studies using trpv antagonists may require an additional control to rule out any possible bronchorelaxant capabilities that may confound any potential effect on bhr (skogvall et al. ). the effect of trpv antagonists on cellular recruitment has been investigated in a number of inflammatory models. the trpv antagonist n-( -chlorobenzyl)-n -( -hydroxy- -iodo- -methoxybenzyl) thiourea had no effect on neutrophil recruitment induced by injection of complete freund's adjuvant into the hindpaw of mice ). in contrast, capsazepine inhibited neutrophil recruitment to the lung induced by systemic administration of hydrogen sulfide donor (bhatia et al. ) and inhibited neutrophil recruitment in a model of colitis (kihara et al. ) and pancreatitis (hutter et al. ) . differences in the severity of the inflammatory models utilized in these studies may account for the lack of general consensus concerning the role of trpv in inflammatory cell recruitment; however, as indicated earlier, the findings of most chemical ablation studies using chronic treatment with capsaicin are consistent with the peripheral involvement of sensory neuropeptides in neutrophil recruitment to sites of inflammation (table ). there has also been a paucity of studies utilizing trpv gene deficient mice to study the role of this protein in bhr and inflammation. in one study, bhr in response to lipopolysaccharide challenge was significantly augmented in trpv knockout mice, and highlighted the existence of an anti-inflammatory substance (e.g., somatostatin) released from trpv -positive cells, which could act in a negative-feedback mechanism to limit the inflammatory response (helyes et al. ). however, these data are not consistent with those form chemical ablation studies showing that bhr to lipopolysaccharide is inhibited in the guinea pig (jarreau et al. ). this discrepancy may be due to the two different methods employed to ''impair'' trpv signaling. it could be envisaged that sensory nerves would be bombarded by multiple signals by a plethora of mediators released following the initial insult, resulting in the activation of various receptor proteins (e.g., bradykinin and ngf receptor, other trps) on primary afferent terminals. these signals would be processed at the level of the nts, but would also require the activation of trpv for a facilitated response (i.e., gain in function). however, the interpretation of these signals by the nts would be lost following chemical ablation with capsaicin, owing to destruction of the peripheral terminations of c-fibers in the airway, but these would be retained in trpv knockout mice with an intact afferent nervous system and therefore would still able to signal to the nts. alternatively, compensatory mechanisms during the development of trpv knockout mice might account for this anomaly. the implication is that impairment of sensory nerve function (e.g., via trpv desensitization) may be required instead of trpv antagonism to completely suppress bhr. in terms of the inflammatory response, it appears that neutrophil recruitment in joint inflammation was either unaffected (keeble et al. ) or augmented during inflammatory insults to the lung (helyes et al. ) and gastrointestinal tract (massa et al. ) . similarly, the amounts of tnf-a released within the extracellular space at sites of inflammation were either increased (clark et al. ) or unaffected (keeble et al. ) by gene deletion of trpv in different inflammatory models. hence, these murine models have been inconclusive concerning the role of trpv in mediating bhr and/or inflammation. the observation that activation of trpv may stimulate the release of an anti-inflammatory substance in the mouse also makes it difficult to elucidate the role of trpv in bhr and inflammation in this species (helyes et al. ). airway sensory nerves play an essential role in regulating airway and lung defensive and homeostatic reflexes. the afferent nerve subtypes regulating these reflexes have unique physiological and pharmacological attributes that are amenable to selective therapeutic interventions. there is extensive evidence to suggest that airway sensory nerves are dysregulated in disease. therapeutic strategies that target the excitability of airway sensory nerves at their central and peripheral terminations may provide symptom relief in conditions such as cough, asthma, and copd. rsd , a novel anti-tussive agent acting on airway sensory nerves tachykinin receptor antagonists and cough enhancement of the bronchial reactivity in immunized rats by neonatal treatment with capsaicin inhibitory actions of prostaglandin e on non-adrenergic non-cholinergic contraction in guinea-pig bronchi a possible role of a nonadrenergic inhibitory nervous system in airway hyperreactivity l-name-sensitive and -insensitive nonadrenergic noncholinergic relaxation of cat airway in vivo and in vitro role of nitric oxide released from inanc neurons in airway responsiveness in cats airway smooth muscle dynamics: a common pathway of airway obstruction in asthma effects of nicotine infusion on striatal glutamate and cortical non-protein-bound iron in hypoxic newborn piglets a comparative study of irritant and type j receptors in the cat adenosine, methacholine, and exercise challenges in children with asthma or paediatric chronic obstructive pulmonary disease nmda receptors contribute to primary visceral afferent transmission in the nucleus of the solitary tract properties of the inspiration-related activity of sympathetic preganglionic neurones of the cervical trunk in the cat the effects of parasympathectomy on serotonin-induced bronchoconstriction in the cat substance p-immunoreactive sensory axons in the rat respiratory tract: a quantitative study of their distribution and role in neurogenic inflammation basis for synchronization of sympathetic and phrenic nerve discharges asthma as an axon reflex neurogenic inflammation in the airways nf-kappa b: a pivotal role in asthma and a new target for therapy t-cell co-stimulatory molecules: novel targets for the treatment of allergic airway disease opioid modulation of non-cholinergic neural bronchoconstriction in guinea-pig in vivo modulation of non-adrenergic, non-cholinergic neural bronchoconstriction in guinea-pig airways via gabab-receptors sensory receptor activation by mediators of defense reflexes in guinea-pig lungs prostaglandin involvement in lung c-fiber activation by substance p in guinea pigs allergen-induced hyperresponsiveness to bradykinin is more pronounced than that to methacholine role of substance p in hydrogen sulfide-induced pulmonary inflammation in mice inhibition of nociceptors by trpv -mediated entry of impermeant sodium channel blockers effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and copd ruthenium red decreases capsaicin and citric acidinduced cough in guinea pigs central antitussive activity of the nk and nk tachykinin receptor antagonists, cp- , and sr , in the guinea-pig and cat getting to the heart of asthma: can ''beta blockers'' be useful to treat asthma? neurones in commissural nucleus tractus solitarii required for full expression of the pulmonary c fibre reflex in rat neurones in a discrete region of the nucleus tractus solitarius are required for the breuer-hering reflex in rat pulmonary stretch receptor afferents activate excitatory amino acid receptors in the nucleus tractus solitarii in rats substance p contributes to rapidly adapting receptor responses to pulmonary venous congestion in rabbits involvement of central -ht a receptors in the reflex activation of pulmonary vagal motoneurones by inhaled capsaicin in anaesthetized cats the response of laryngeal afferent fibres to mechanical and chemical stimuli the role of the epithelium in modulating the responses of guinea-pig trachea induced by bradykinin in vitro intravenous adenosine and dyspnea in humans reflex regulation of airway smooth muscle tone encoding of the cough reflex cough sensors. i. physiological and pharmacological properties of the afferent nerves regulating cough relaxant innervation of the guinea-pig trachealis: demonstration of capsaicin-sensitive and -insensitive vagal pathways multiple mechanisms of reflex bronchospasm in guinea pigs identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea-pigs mechanistic studies of acid-evoked coughing in anesthetized guinea pigs vagal afferent nerves regulating the cough reflex pharmacology of vagal afferent nerve activity in guinea pig airways trypsin-induced, neurokinin-mediated contraction of guinea pig bronchus the capsaicin receptor: a heat-activated ion channel in the pain pathway bronchial mucosal immunoreactivity of sensory neuropeptides in severe airway diseases in vivo demonstration of nonadrenergic inhibitory innervation of the guinea pig trachea microinjection of nmda antagonist into the nts of conscious rats blocks the bezold-jarisch reflex synergistic airway gland mucus secretion in response to vasoactive intestinal peptide and carbachol is lost in cystic fibrosis differential effects of airway afferent nerve subtypes on cough and respiration in anesthetized guinea pigs effect of -hydroxytryptamine on vagal c-fiber subtypes in guinea pig lungs evidence for both adenosine a and a a receptors activating single vagal sensory c-fibres in guinea pig lungs the transient receptor potential vanilloid (trpv ) receptor protects against the onset of sepsis after endotoxin nonadrenergic bronchodilation in adult and young guinea pigs evidence for nonadrenergic inhibitory nerves in the guinea pig trachealis muscle mechanisms of airway hyperresponsiveness impulse activity in afferent vagal c-fibres with endings in the intrapulmonary airways of dogs afferent vagal c fibre innervation of the lungs and airways and its functional significance effect of co on afferent vagal endings in the canine lung tracheal contraction and relaxation initiated by lung and somatic afferents in dogs trp channels and pain cationic protein-induced sensory nerve activation: role of substance p in airway hyperresponsiveness and plasma protein extravasation airway hyperreactivity induced by active cigarette smoke exposure in guinea-pigs: possible role of sensory neuropeptides projection of pulmonary rapidly adapting receptors to the medulla of the cat: an antidromic mapping study lung reflexes in rabbits during pulmonary stretch receptor block by sulphur dioxide pulmonary stretch receptor relay neurones of the cat: location and contralateral medullary projections extreme dyspnea from unilateral pulmonary venous obstruction. demonstration of a vagal mechanism and relief by right vagotomy cutaneous pretreatment with the capsaicin analog ne- prevents the pain to a burn and subsequent hyperalgesia role of vagal afferents in the reflex effects of capsaicin and lobeline in monkeys changes in activity of vagal bronchopulmonary c fibres by chemical and physical stimuli in the cat selective visualisation of sensory receptors in the smooth muscle layer of ex-vivo airway whole-mounts by styryl pyridinium dyes extending the understanding of sensory neuropeptides origin and colocalization of cgrp-and spreactive nerves in cat airway epithelium a nonadrenergic vagal inhibitory pathway to feline airways experimentally induced cough substance p expression in trpv and trka-positive dorsal root ganglion neurons innervating the mouse lung the effects of histamine, acetylcholine and -hydroxytryptamine on lung mechanics and irritant receptors in the dog the action of sodium cromoglycate on 'c' fibre endings in the dog lung deepbreath frequency in bronchoconstricted monkeys (macaca fascicularis) tachykinin-independent effects of capsaicin on smooth muscle in human isolated bronchi efferent projections of pulmonary rapidly adapting receptor relay neurons in the cat electrophysiological and pharmacological analysis of synaptic inputs to pulmonary rapidly adapting receptor relay neurons in the rat axonal projections of pulmonary slowly adapting receptor relay neurons in the rat nociceptin-induced inhibition of tachykinergic neurotransmission in guinea pig bronchus effects of inhaled co on airway stretch receptors in the newborn dog cough and bronchoconstriction mediated by capsaicin-sensitive sensory neurons in the guinea-pig an in vitro study of the properties of single vagal afferents innervating guinea-pig airways stimulation of guinea-pig tracheal afferent fibres by nonisosmotic and low-chloride stimuli and the effect of frusemide activation of large conductance potassium channels inhibits the afferent and efferent function of airway sensory nerves in the guinea pig protease-activated receptor- activation exaggerates trpv -mediated cough in guinea pigs the intrinsic nervous mechanism of the human lung activation and sensitisation of the vanilloid receptor: role in gastrointestinal inflammation and function differential brainstem fos-like immunoreactivity after laryngeal-induced coughing and its reduction by codeine smooth muscle molecular mechanics in airway hyperresponsiveness and asthma the tachykinin nk receptor antagonist sr inhibits citric acid-induced airway hyperresponsiveness in guinea-pigs modulatory action of galanin on responses due to antidromic activation of peripheral terminals of capsaicin-sensitive sensory nerves anandamide transport: a critical review pulmonary c-fibers evoke both apnea and tachypnea of pulmonary chemoreflex effect of pulmonary arterial pco on slowly adapting pulmonary stretch receptors increased expression of transient receptor potential vanilloid- in airway nerves of chronic cough cholinergic bronchomotor tone in copd. estimates of its amount in comparison with that in normal subjects prejunctional alpha adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission inhibition of the excitatory non-adrenergic, non-cholinergic neurotransmission in the guinea pig tracheo-bronchial tree mediated by alpha -adrenoceptors characterization of acid signaling in rat vagal pulmonary sensory neurons epinephrine enhances the sensitivity of rat vagal chemosensitive neurons: role of beta -adrenoceptor expression of neuronal nicotinic acetylcholine receptors in rat vagal pulmonary sensory neurons airway mechanoreceptor deactivation the diversity in the vanilloid (trpv) receptor family of ion channels immunocytochemical localization of the vanilloid receptor (vr ): relationship to neuropeptides, the p x purinoceptor and ib binding sites respiratory modulation in the activity of sympathetic neurones lung dendritic cell migration influence of lung stretch receptors on the cough reflex in rabbits stimulation of airway sensory nerves by cyclosporin a and fk in guinea-pig isolated bronchus the excitatory amino acid glutamate mediates reflexly increased tracheal blood flow and airway submucosal gland secretion role of transient receptor potential vanilloid receptors in endotoxininduced airway inflammation in the mouse benzolamide, acetazolamide, and signal transduction in avian intrapulmonary chemoreceptors capsaicin pretreatment prevents the development of antigen induced airway hyperresponsiveness in neonatally immunized rabbits immunohistochemical localization of peptide-containing nerves in human airways: age-related changes prostaglandin e( ) enhances chemical and mechanical sensitivities of pulmonary c fibers in the rat sensitivity of vagal afferent endings to chemical irritants in the rat lung the epithelium takes centre stage in asthma and atopic dermatitis activation of pulmonary c fibres by adenosine in anaesthetized rats: role of adenosine a receptors central neurocircuitry associated with emesis cough reflex in allergic dogs neuropeptide-containing nerves in endobronchial biopsies from asthmatic and nonasthmatic subjects release of calcitonin gene-related peptide and tachykinins from the rat trachea multiple tachykinins (neurokinin a, neuropeptide k and substance p) in capsaicin-sensitive sensory neurons in the guinea-pig fast ( hz and hz) and slow (respiratory) rhythms in cervical sympathetic nerve and unit discharges of the cat identification and substance p content of vagal afferent neurons innervating the epithelium of the guinea pig trachea transient receptor potential vanilloid (trpv- ) promotes neurogenic inflammation in the pancreas via activation of the neurokinin- receptor (nk- r) possible sensory receptor of nonadrenergic inhibitory nervous system nonadrenergic bronchodilation in normal subjects sensory receptors and reflex pathways of nonadrenergic inhibitory nervous system in feline airways possible roles of the peripheral vagal nerve in histamine-induced bronchoconstriction in guinea-pigs nonpurinergic nature and efficacy of nonadrenergic bronchodilation nedocromil sodium and sensory nerves in the dog lung eosinophils and asthma brainstem circuitry of tracheal-bronchial cough: c-fos study in anesthetized cats assessment of the pulmonary origin of bronchoconstrictor vagal tone neurophysiological analysis of target-related sympathetic pathwaysfrom animal to human: similarities and differences effects of capsaicin on the airway responses to inhaled endotoxin in the guinea pig role of trpv receptors in respiratory diseases anandamide induces cough in conscious guinea-pigs through vr receptors role of c fibers in physiological responses to ozone in rats rapidly adapting receptor activity in dogs is inversely related to lung compliance a unitary analysis of pulmonary volume receptors mechanisms of acid-induced activation of airway afferent nerve fibres in guinea-pig activation of bronchopulmonary vagal afferent nerves with bradykinin, acid and vanilloid receptor agonists in wild-type and trpv À/À mice capsaicin-sensitive and -insensitive vagal bronchopulmonary c-fibres in the mouse acid-sensitive vagal sensory pathways and cough an important role of tachykinins in ozoneinduced airway hyperresponsiveness the medullary projections of afferent bronchopulmonary c fibres in the cat as shown by antidromic mapping central pathways of pulmonary and lower airway vagal afferents the sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry role of pulmonary c fibres in adenosine-induced respiratory inhibition in anesthetized rats voltagegated sodium channels in nociceptive versus non-nociceptive nodose vagal sensory neurons innervating guinea pig lungs p x receptors differentiate placodal vs. neural crest c-fiber phenotypes innervating guinea pig lungs and esophagus improvement of symptoms of non-allergic chronic rhinitis by local treatment with capsaicin the involvement of hydroxyl radical and cyclooxygenase metabolites in the activation of lung vagal sensory receptors by circulatory endotoxin in rats immunohistochemical demonstration of substance p in the lower respiratory tract of the rabbit and not of man capsazepine inhibits cough induced by capsaicin and citric acid but not by hypertonic saline in guinea pigs the effects of electrical stimulation of myelinated and non-myelinated vagal motor fibres on airway tone in the rabbit and the cat three-dimensional mapping of sensory innervation with substance p in porcine bronchial mucosa: comparison with human airways capsaicin-induced bronchodilation in mild asthmatic subjects: possible role of nonadrenergic inhibitory system nerve termination in the lung of the rabbit the ganglia, plexuses and nerve-termination of the mammalian lung and pleura pulmonis a comparative study of the effects of citric acid, capsaicin and resiniferatoxin on the cough challenge in guinea-pig and man histamine enhances vagal pulmonary c-fiber responses to capsaicin and lung inflation pulmonary chemoreflex sensitivity is enhanced by prostaglandin e in anesthetized rats afferent properties and reflex functions of bronchopulmonary c-fibers effect of olvanil and anandamide on vagal c-fiber subtypes in guinea pig lung inhibition of citric acid-and capsaicin-induced cough by novel trpv- antagonist, v , in guinea-pig the role of transient receptor potential vanilloid (trpv ) channels in pancreatitis further evidence for the existence of a specific process for the membrane transport of anandamide stimulation of pulmonary vagal c-fibres by anandamide in anaesthetized rats: role of vanilloid type receptors stimulatory effect of co on vagal bronchopulmonary c-fiber afferents during airway inflammation identification of neurons receiving input from pulmonary rapidly adapting receptors in the cat allergen-induced airway remodelling involvement of tachykinins in endotoxininduced airway hyperresponsiveness airway hyperresponsiveness in a rat model of chronic bronchitis: role of c fibers respiratory tract inflammation during the induction of chronic bronchitis in rats: role of c-fibres cigarette smoke-induced airway oedema due to activation of capsaicin-sensitive vagal afferents and substance p release substance p-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man effects of gadolinium chloride on slowly adapting and rapidly adapting receptors of the rabbit lung inflammatory mediators modulating the transient receptor potential vanilloid receptor: therapeutic targets to treat inflammatory and neuropathic pain bronchodilatation by tachykinins and capsaicin in the mouse main bronchus bronchial smooth muscle responses evoked by toluene diisocyanate are inhibited by ruthenium red and by indomethacin role of substance p and neurokinin a in toluene diisocyanate-induced increased airway responsiveness in rabbits sensory nerves containing tachykinins and cgrp in the lower airways. functional implications for bronchoconstriction, vasodilatation and protein extravasation vanilloid receptor (trpv )-deficient mice show increased susceptibility to dinitrobenzene sulfonic acid induced colitis inhibition of cholinergic and non-adrenergic, noncholinergic bronchoconstriction in the guinea pig mediated by neuropeptide y and alpha -adrenoceptors and opiate receptors effects of vagal stimulation on slowly adapting pulmonary stretch receptors and lung mechanics in anesthetized rabbits effects of sustained constant artificial ventilation on rapidly adapting pulmonary stretch receptors and lung mechanics in rabbits effects of -hydroxytryptamine on rapidly adapting pulmonary stretch receptor activity in the rabbit effective sites by sympathetic beta-adrenergic and vagal nonadrenergic inhibitory stimulation in constricted airways inhibitory mechanism of co inhalation on slowly adapting pulmonary stretch receptors in the anesthetized rabbit effects of vagal and carotid chemoreceptor afferents on the frequency and pattern of spontaneous augmented breaths in rabbits excitatory mechanism of veratridine on slowly adapting pulmonary stretch receptors in anesthetized rabbits effects of potassium channel blockers on co -induced slowly adapting pulmonary stretch receptor inhibition effects of sodium and potassium channel blockers on hyperinflation-induced slowly adapting pulmonary stretch receptor stimulation in the rat the inhibitory effect of ouabain on the response of slowly adapting pulmonary stretch receptors to hyperinflation in the rabbit effect of ouabain on the afterhyperpolarization of slowly adapting pulmonary stretch receptors in the rat lung capsaicin inhibits airway hyperresponsiveness but not lipoxygenase activity or eosinophilia after repeated aerosolized antigen in guinea pigs evidence for differential reflex regulation of cholinergic and noncholinergic parasympathetic nerves innervating the airways synergistic interactions between airway afferent nerve subtypes mediating reflex bronchospasm in guinea pigs respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors na + Àk + À cl À cotransporters and cl À channels regulate citric acid cough in guinea pigs immunohistochemical characterization of nodose cough receptor neurons projecting to the trachea of guinea pigs synergistic interactions between airway afferent nerve subtypes regulating the cough reflex in guinea-pigs potassium channel blockade induces action potential generation in guinea-pig airway vagal afferent neurones inhibition of -lipoxygenase diminishes neurally evoked tachykinergic contraction of guinea pig isolated airway neurogenic inflammation in the rat trachea. ii. identity and distribution of nerves mediating the increase in vascular permeability lighting up the senses: fm - loading of sensory cells through nonselective ion channels reflex decrease of histamineinduced bronchoconstriction after laryngeal stimulation in humans activity of lung irritant receptors in pulmonary microembolism, anaphylaxis and drug-induced bronchoconstrictions epithelial irritant receptors in the lungs responses of pulmonary stretch receptors to static pressure inflations effect of nonadrenergic noncholinergic inhibitory nerve stimulation on the allergic reaction in cat airways effects of peptides and amines on isolated guinea pig tracheae as influenced by inhibitors of the metabolism of arachidonic acid effects of aerosol-applied capsaicin, histamine and prostaglandin e on airway sensory receptors of anaesthetized cats role of substance p and tachykinin receptor antagonists in citric acid-induced cough in pigs actions of moguisteine on cough and pulmonary rapidly adapting receptor activity in the guinea pig differential control of sympathetic outflow chronic passive cigarette smoke exposure augments bronchopulmonary c-fibre inputs to nucleus tractus solitarii neurones and reflex output in young guinea-pigs the role of excitatory amino acids and substance p in the mediation of the cough reflex within the nucleus tractus solitarii of the rabbit modulation of the cough reflex by antitussive agents within the caudal aspect of the nucleus tractus solitarii in the rabbit allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways expression and function of the ion channel trpa in vagal afferent nerves innervating mouse lungs transient receptor potential cation channels in disease physiological and pathophysiological implications of upper airway reflexes in humans tachykinin-, calcitonin gene-related peptide-, and protein gene product . -immunoreactive nerve fibers in alveolar walls of mammals selective airway responsiveness in asthma reflex regulation of airway sympathetic nerves in guinea-pigs functional and morphological organization of the nucleus tractus solitarius in the fictive cough reflex of guinea pigs codeine presynaptically inhibits the glutamatergic synaptic transmission in the nucleus tractus solitarius of the guinea pig regulation of inflammatory responses by sensory neurons: molecular mechanism(s) and possible therapeutic applications projections from the commissural subnucleus of the nucleus of the solitary tract: an anterograde tracing study in the cat vagal sensory receptors and their reflex effects reflex responses to capsaicin: intravenous, aerosol, and intratracheal administration pattern of cardiorespiratory afferent convergence to solitary tract neurons driven by pulmonary vagal c-fiber stimulation in the mouse selective stimulation of jugular ganglion afferent neurons in guinea pig airways by hypertonic saline activation of capsaicin-sensitive sensory fibers modulates pafinduced bronchial hyperresponsiveness in anesthetized guinea pigs vagal afferent and reflex responses to changes in surface osmolarity in lower airways of dogs sputum eosinophilia is more closely associated with airway responsiveness to bradykinin than methacholine in asthma role of the vagal afferents in substance p-induced respiratory responses in anaesthetized rabbits nasal provocation with bradykinin induces symptoms of rhinitis and a sore throat effect of carbon dioxide on the activity of slowly and rapidly adapting pulmonary stretch receptors in cats responses of pulmonary c-fibre and rapidly adapting receptor afferents to pulmonary congestion and edema in dogs airways hyper-responsiveness to bradykinin and methacholine: effects of inhaled fluticasone adenosine induces a cholinergic tracheal reflex contraction in guinea pigs in vivo via an adenosine a receptor-dependent mechanism presence and bronchomotor activity of protease-activated receptor- in guinea pig airways the effect of neonatal capsaicin on the development of bronchial hyperresponsiveness in allergic rabbits the effect of -hpete on airway responsiveness and pulmonary cell recruitment in rabbits interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways modulation of pulmonary stretch receptors and airway resistance by parasympathetic efferents role of sensory neuropeptides in piv- -infection-induced airway hyperresponsiveness in guinea pigs afferent vagal c-fibres are responsible for the reflex airway constriction and secretion evoked by pulmonary administration of so in dogs airway responsiveness to bradykinin is related to eosinophilic inflammation in asthma eosinophil trafficking in allergy and asthma sensory transduction of pulmonary reactive oxygen species by capsaicin-sensitive vagal lung afferent fibres in rats properties of 'irritant' receptors in canine lung hyperosmolar saline induces reflex nasal secretions, evincing neural hyperresponsiveness in allergic rhinitis lung mechanics and activity of slowly adapting airway stretch receptors evidence for substance p-immunoreactive spinal afferents that mediate bronchoconstriction an overview of the anatomy and physiology of slowly adapting pulmonary stretch receptors mechanisms underlying the contraction induced by bradykinin in the guinea pig epithelium-denuded trachea substance p presynaptically depresses the transmission of sensory input to bronchopulmonary neurons in the guinea pig nucleus tractus solitarii the activity of lung irritant receptors during pneumothorax, hyperpnoea and pulmonary vascular congestion nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway convergence properties of solitary tract neurons responsive to cardiac receptor stimulation in the anesthetized cat effects of capsazepine on human small airway responsiveness unravel a novel class of bronchorelaxants bronchial inflammation and airway responses to deep inspiration in asthma and chronic obstructive pulmonary disease the determinants of airway hyperresponsiveness to hypertonic saline in atopic asthma in vivo. relationship with subpopulations of peripheral blood leucocytes clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. the ampul study group airway sensory nerves in asthma -targets for therapy? pharmacology of airway irritability effect of capsaicin on paf-induced bronchial hyperresponsiveness and pulmonary cell accumulation in the rabbit regulation by phosphodiesterase isoenzymes of nonadrenergic non-cholinergic contraction in guinea-pig isolated main bronchus a comparison of sensory nerve function in human, guinea-pig, rabbit and marmoset airway effects of -hydroxytryptamine and -hydroxytryptophan infusion on the human cough reflex trpv in colitis: is it a good or a bad receptor? -a viewpoint modulation of neural bronchoconstrictor responses in the guinea pig respiratory tract by vasoactive intestinal peptide responses of tracheobronchial receptors to inhaled furosemide in anesthetized rats the vanilloid (capsaicin) receptor: receptor types and species differences vanilloid (capsaicin) receptors and mechanisms reflex activation of the nonadrenergic noncholinergic inhibitory nervous system in feline airways sensory nerve-and neuropeptidemediated relaxation responses in airways of sprague-dawley rats effects of bronchoconstriction and external resistive loading on the sensation of dyspnea prostaglandin e inhalation increases the sensation of dyspnea during exercise convergence of laryngeal afferents with different natures upon cat nts neurons modulation of neurotransmission in guinea-pig airways by galanin and the effect of a new antagonist galantide antinociceptive pharmacology of n-( -chlorobenzyl)-n -( -hydroxy- -iodo- -methoxybenzyl) thiourea, a high-affinity competitive antagonist of the transient receptor potential vanilloid receptor laryngeal and tracheobronchial cough in anesthetized dogs sensitivity of the cough reflex in awake guinea pigs, rats and rabbits lung c-fibre receptor activation and defensive reflexes in anaesthetized cats relative contributions of trpa and trpv channels in the activation of vagal bronchopulmonary c-fibres by the endogenous autacoid -oxononenal role of tachykinins in ozone-induced acute lung injury in guinea pigs tachykinins mediate the acute increase in airway responsiveness caused by toluene diisocyanate in guinea pigs the cloned capsaicin receptor integrates multiple pain-producing stimuli antitussive activity of iodo-resiniferatoxin in guinea pigs the endogenous cannabinoid agonist, anandamide stimulates sensory nerves in guinea-pig airways hypertonic saline increases vascular permeability in the rat trachea by producing neurogenic inflammation characterization of the vanilloid receptor antagonist iodoresiniferatoxin on the afferent and efferent function of vagal sensory c-fibers inhibition of neurally mediated nonadrenergic, noncholinergic contractions of guinea pig bronchus by isozyme-selective phosphodiesterase inhibitors physiology and plasticity of putative cough fibres in the guinea pig effect of extracellular calcium on excitability of guinea pig airway vagal afferent nerves corticosteroid-induced improvement in the pc of adenosine monophosphate is more closely associated with reduction in airway inflammation than improvement in the pc of methacholine indirect bronchial hyperresponsiveness in asthma: mechanisms, pharmacology and implications for clinical research effect of inhaled fluticasone on bronchial responsiveness to neurokinin a in asthma excitatory amino acid receptors in the nucleus tractus solitarius mediate the responses to the stimulation of cardio-pulmonary vagal afferent c fiber endings beta -adrenoceptor agonists inhibit nanc neural bronchoconstrictor responses in vitro capsaicinsensitive c-fiber-mediated protective responses in ozone inhalation in rats mechanisms by which histamine stimulates rapidly adapting receptors in dog lungs the neurokinin receptor antagonist aprepitant as an antiemetic for moderately emetogenic chemotherapy immunohistochemical localization of vanilloid receptor subtype (trpv ) in the guinea pig respiratory system immunohistochemical co-localization of transient receptor potential vanilloid (trpv) and sensory neuropeptides in the guinea-pig respiratory system the activation mechanism of rat vanilloid receptor by capsaicin involves the pore domain and differs from the activation by either acid or heat receptors in the trachea and bronchi of the cat respiratory reflexes from the trachea and bronchi of the cat non-nmda receptors transmit cardiopulmonary c fibre input in nucleus tractus solitarii in rats parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system the effect of airway anaesthesia on the control of breathing and the sensation of breathlessness in man respiratory and cardiovascular effects of central and peripheral intravenous injections of capsaicin in man: evidence for pulmonary chemosensitivity substance p released from intrinsic airway neurons contributes to ozone-enhanced airway hyperresponsiveness in ferret trachea involvement of thromboxane a( ) in airway mucous cells in asthma-related cough nerve endings in bronchi of the dog that react with antibodies against neurofilament protein morphological and quantitative study of the intrinsic nerve plexuses of the canine trachea as revealed by immunohistochemical staining of protein gene product distribution of trpv -and trpv -immunoreactive afferent nerve endings in rat trachea endogenous nitric oxide inhibits bronchoconstriction induced by cold-air inhalation in guinea pigs: role of kinins airway mechanosensors influence of lung stiffness on rapidly adapting receptors in rabbits and cats pulmonary rapidly adapting receptors reflexly increase airway secretion in dogs key: cord- -ad i wgj authors: nan title: th international congress on amino acids and proteins : vienna, austria, august – , date: journal: amino acids doi: . /s sha: doc_id: cord_uid: ad i wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from hcu patients were compared to controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control . ( . - . ) and for hcu . ( . - . ); p ϭ . ]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ . , p Ͻ . ). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. ( nmol/g; . %); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ ; - nmol/g; . - . %); rabbit ( nmol/ g; . %); pig (sus scrofa f. domestica, artiodactyla) ( nmol/ g; . %); bovine (bos primigenius f. taurus, artiodactyla) ( nmol/g; . %); seal (phoca vitulina, carnivora) ( nmol/g; . %), and rob (halichoerus grypus, carnivora) ( nmol/g; . %). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (Ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl- -thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere rp- e, ϫ mm, µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than , . the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by fer- d/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of adults ( males and females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: individuals ( %) belonged to the adult age group, persons ( %) to the mature and ( %) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over . . quantitative determination of free and bound -nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since -nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine -nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify -nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free -nitrotyrosine in rat plasma as well as bound -nitrotyrosine in tissue samples are reported. plasma samples were spiked with , , -d - -nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z ae for the analyte and m/z ae for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl n. -nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. -fold) by comparison with -nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r Ͼ . ) within concentration range . to . nm (equivalent to . - , fmol on column) and recoveries above %. inter-and intra-assay precision was determined below % over the concentration range . to . nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range . - . nm. this curve strongly correlated with a linear model (r Ͼ . ) and slope was . Ϯ . (mean Ϯ sd). basal level of -nitrotyrosine in rat plasma was measured to be within concentration range Ͻ lod to . nm. -nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl n at °c). resulting hydrolysates were analysed by lc-esi-ms/ms and nitrotyrosine was monitored in srm mode (m/z ae for the analyte and m/z ae for the internal standard). t. guszczynski , r. b. kapust , d. s. waugh , and t. d. copeland basic research laboratory, and macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv- . with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi ; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, g; soy peptone, g; yeast extract, g; dglucose, . g; nacl, g; dipotassium hydrogen phosphate, . g in l demineralized water adjusted to ph . . fermentation was conducted in nineteen -l shake flasks, each containing ml medium, for d at °c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water : : : ) yielding . g and . g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. ) and semipreparative hplc (nucleosil c- ; ϫ mm id; µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. . the -residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. h and c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of . the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful h and c signal assignment. a complete assignment of h and c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of h and c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe ϩ are converted to coloured products, with maximum wavelength at nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by ‰ δ n. in rats fed diets containing intrinsically labeled c casein or the corresponding amino acid mixture labeled with c leucine and n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta , m. yoshitake , h. yoshida , t. yoshitake , and m. yamaguchi faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, -hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of fluorogenic reagents, o-phthalaldehyde (with -mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality . to . mol·kg Ϫ using the "isoperibol" type calorimeter at . k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re ϩ . substances (glyh) . h o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re cl ] Ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re cl ] Ϫ in the substance (glyh) [re cl ]cl together with existence of quadruple re -re bond that is described first. in the substance [re (gaba) cl (h o)]cl . h o axial position of re ϩ fragment are substituted by ligands of different kind: h o and cl Ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p- -rp column allowed separation of caseins in min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of ml/min and a temperature of °c, using a mixture of two solvents. solvent a . % trifluoroacetic acid in water and solvent b was % acetonitrile- % water- . % trifluoroacetic acid. the effluent was monitored by a uv detector at nm. the determinations were performed in the linear range of . - . mg/ml for k-casein, . - . mg/ml for α-casein and . - . mg/ml for -casein. the detection limits were . , . and . mg/ml, respectively. the validity of the method was verified. the recoveries ranged from to % for cow's milk. the precision of the method was also evaluated, the % cv being less then . %. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine ( - µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to % seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by . -and . fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from % in freshwater to % in % seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for h in freshwater, and % seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec . . . ] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was , -fold with % yield. the molecular weight of the enzyme was estimated to be kda on sds-page and kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below % with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ , were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg ]-thf-γ exhibited higher restoring activity than that of our synthetic thf-γ . phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa ) was engineered to attain a pre-designed d structure and later modified by grafting short peptide fragments from human α interferon (aag ), and insulin molecules (aag ). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa ) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet- (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α -interferon fragments was studied in vitro using human fibroblasts cell line l- and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag ) acquired ability to stimulate glucose uptake by l- cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x m -/double knockout mice, transgenic for a single-chain hla-a - m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc and ba - . analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a binding peptides resulted in candidate peptides for immunogenicity screening. we have identified antigenic peptides of which peptides were found to be immunogenic in hhd mice. interestingly of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage- were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes , e. giralt , and d. andreu centro de investigação em química da universidade do porto, portugal department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of bases including the active site. the homology of the active site and the surrounding bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was . % and . %, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of amino acid residues. there was a high degree of homology in the sequence of amino acid residues (met ϳhis ) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments - : ksglkykk (g - ) and - khvvpnev vvqrlfqvkgrr (g - ), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol , -bisphosphate (pip ). the binding of peptides g - and g - to a cluster of four pip molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of , ps. the binding of the pip molecules to the peptides g - , g - showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip . during the binding some of the pip molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by -hydroxyphthalic anhydride ( hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus (hsv- ), bovine parainfluenza virus (pi- ) and porcine respiratory corona virus (prcv), the hp proteins were shown to be active against human herpes simplex virus only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with hp-albumin, hp--lactoglobulin and hplysozyme resulted in protection against hsv- infection whereas pre-incubation with hp-α-lactalbumin had no antiviral effect. however, all hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv- infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv- glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. ) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. , syrkin a. , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c. . . . , extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt- cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration - ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt- cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration mkm ( , mg/ml) not exerting toxical effect on mt- cells. the same effect is attained having used lo in doses - ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt- cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with - % ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y b) were cultured in eagle's minimum essential medium (mem) containing % serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. ) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k- as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was mm l-glutamine, mm taurine and . unit/ml ggt, ph , and -hr incubation of °c. forty-three mm gamma-glutamyltaurine was obtained and the yield was .%. gamma-glutamyltaurine was purified by dowex ϫ column and c column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex- t . mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and Ј, Ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with amino acid residues is first translated and then processed to a mature peptide with amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex- t , and expressed efficiently in e. coli bl as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: -deoxy-d-arabino-heptulonate- -phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by . , . , . and . folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional ( -d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant protein phosphatase. the two aliquots were then subjected to -d electrophoresis. the phosphoproteins could be mapped on the -d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately % of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately - % of the total proteins resolved by a large format d gel. to reduce the number of proteins to be identified the d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a -d gel, where about mg of protein amount has been resolved, , - , protein spots can be detected, using coomassie blue staining. the spots represent the products of only - different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis , j.-f. juranville , and l. suter genomics technologies, and drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, different gene products were detected. approximately % of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. ( ) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. ( ) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution d electrophoresis and detection of phosphorylated proteins labeled with p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. ( ) differential gene expression has been followed by using s met pulse chase labelling concurrently with endothelin stimulation. at least proteins showed significant changes in expression of d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by d gel electrophoresis and mass spectrometry d. gostick , s. cohen , p. young , b. karol , j. langridge , j. randell , t. slyker , and a. jacobson micromass, manchester, u.k. waters corporation, milford, massachusetts, and bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are possible sequences for a peptide containing any of the naturally occurring amino acids and having residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms ) is set to transmit only the fragment ion of interest. the ms quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms is not mass selective, operating in the rf only mode. the first data set at low energy ( ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of da (h po ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h po moiety is not observed, however a low mass immonium ion at m/z can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ p] inorganic phosphate. d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer , , m. berger , g. lubec , and m. fountoulakis f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland institute of cancer research, and department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha- chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately proteins were identified in the mitochondrial fraction, which were the products of different genes. about % of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about % were found in the microsomal fraction as well. of the proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec and m. fountoulakis department of neonatology, university of vienna, austria gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor -alpha , elongation factor , elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli , m. g. giuffrida , e. pessione , g. dellavalle , c. barello , e. griva , and c. giunta dipartimento di biologia animale e dell'uomo, università di torino, and csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller , r. wait , l. sironi , i. eberini , m. gemeiner , e. tremoli , and e. gianazza veterinärmedizinische universität, wien, austria imperial college school of medicine, hammersmith, london, u.k. universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional ( d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra , , s. fontana , p. cancemi , g. alaimo , and s. minafra , centre of experimental oncobiology, department of cell biology and development, and institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about % of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, -bc (minafra et al., br. j. cancer, , - , ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci , l. trabalzini , d. soldateschi , e. ferro , a. paffetti , and p. martelli dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses ( kbp): ad strain genome was completely sequenced, containing about open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: ) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of ) beta, early genes, needed for dna replication, and, finally ) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis ( d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad . the phenotypic d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the - th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe , k. landry , s. taurin , s. n. orlov , and a. v. pshezhetsky sainte-justine hospital research centre, and research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp family, a molecular chaperone grp , and so-called mortalin (grp ) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis ( d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c trap cartridge it is possible to pre-fractionate the peptides before separation on a c column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c cartridge whilst they are desalted. finally the peptides are eluted from the c pre-column, at nl/min, onto a um id ϫ cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of serum samples, immunofixation identified igg , igg , igg , and igg in , , , and cases, respectively. their light-chains were k in cases and λ in cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis ( -de) with various isoelectric focusing conditions as well as by -de ( -de of the proteins extracted from agarose after serum protein agarose electrophoresis). after -de or -de, the monoclonal γ-chains were not visualized in out cases, whatever the isoelectric focusing conditions that were tested. in cases, γ-chains were only detected using alkaline ph - gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p Ͻ . ) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ . , multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis ( -de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein (hsp ), hsp ry, heat shock cognate (hsc) , alpha crystallin b chain, glucose regulated protein (grp) and grp showed aberrant expressional patterns depending on brain region. hsp . , grp and t-complex (tcp- ) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin- -gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(Ϫ)epigallocatechin- -gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec . . . ), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately hivϩ patients; not yet on anti-viral treatment (cd count to ). methods: parameters measured included: cd count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within months, significant trends are noted. the cd count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; . /cmm and % confidence interval (c ), this was confirmed by the total lymphocyte court values. after months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla , i. giovannini , j. h. siegel , g. boldrini , and m. castagneto centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r ϭ . , p Ͻ . ). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ . ϩ . (pea) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (asp) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (logglu) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in patients with pd. vitamin e concentrations were also assessed in age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups ( . Ϯ . mmoli/l for pd patients and . Ϯ . mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli , institute of biological chemistry, university of urbino, italy department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers ( tocopherols and tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and out of main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than iu ( mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite , , -trimethyl- -(b-carboxyethyl)- hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari , s. mensi , g. mercurio , c. callà , l. colacicco , e. sacco , and s. lippa department of anaesthesiology and intensive care medicine, and department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients ( females, male; aged from to years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq ). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq and vitamin e showed a positive correlation with the plasma meth levels (p Ͻ . ; r ϭ . and p Ͻ . ; r ϭ . , respectively). since vitamin e and coq are mutually dependent-molecules, the administration of meth, essential substance for coq synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: ( ϩ ) ϩ ) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content - %), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing µg of the substance in ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in % and increased visual functions in % of the cases. in % of the patients visual acuity raised by . - . . electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich- (caspase- )/ ced- (caenorhabditis elegans death protease- )-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl- interacting mediator of cell death/bcl- related ovarian death gene) and p were significantly increased only in ad frontal cortex (p Ͻ . , in all cases). cerebellar bcl- levels were significantly increased in ad (p Ͻ . ) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar , m. brózik , r. tóbi , t. szabó , j. szakonyi , b. rojkovich , p. gergely , and f. hudecz research group of peptide chemistry hungarian academy of science, budapest, central laboratory of immunology, semmelweis university, budapest, and national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting - % of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( shqestrgrsrgrsgrsgs ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)- alpha and beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il -receptor antagonist (il- ra) is a protein structurally related to il- beta that effectively inhibits the proinflammatory effects of il- . a polymorphism in the Ј-flanking regulatory region at Ϫ of the il- alpha gene, which may cause an overexpression of il- alpha and a variable number tandem repeats (vntr) polymorphism in the il- ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il- beta gene have been reported to influence the protein production: one is located in the promoter region at position Ϫ and the other is in exon at position ϩ . to analyze the contribution of il- alpha, il- beta and il- ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in italian patients with clinically definite ms and healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h o /cl Ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b- proline and arginine residues, hydroxy- -aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl , svedberg units (s f ) - and ldl , s f - ) by gc-ms. paired knee synovial fluid and plasma samples were collected from subjects with ra. additionally, plasma samples were collected from healthy controls. the ldl hava content in plasma was not different between the groups (ra, . Ϯ . vs controls, . Ϯ . mol/mol apob- , p ϭ . ). the ldl hava content in plasma was significantly higher in ra ( . Ϯ . vs . Ϯ . mol/mol apob- , p ϭ . ). furthermore, synovial fluid ldl and ldl in ra contained elevated hava levels when compared with plasma concentrations (ldl syn , . Ϯ . mol/mol apob- (p Ͻ . ) and ldl syn , . Ϯ . mol/mol apob- (p Ͻ . )). results suggest that proline and arginine residues of apob- are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob- to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl were prone to direct oxidation of apob- . correlation between hava content in plasma and synovial fluid ldl and ldl in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis- -hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm ; : ) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg )-lys-chyp} to increase the % by wt of the analogue. rats were exposed to % o for da to induce ph. on da , and after da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at and da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da , rvp (mmhg) was Ϯ and hyp (µg/vessel) was Ϯ in at. rvp and hyp increased to Ϯ * and Ϯ *, respectively, in hypoxic animals (n ϭ ; *p Ͻ . vs. at). on da , rvps were at Ϯ , ht Ϯ *, hc Ϯ * †; hyps were at Ϯ , ht Ϯ *, hc Ϯ * † (n ϭ ; *p Ͻ . vs. at; †p Ͻ . vs. ht). from da to , rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p belonging to the cyp family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp e catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p homologous to cyp e . the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp s and by introducing new cyp s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens and s. beernaert pediatrician, metabolic diseases and dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over % of infants born to mothers with blood phenylalanine (phe) concentrations above µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, years old, who showed phe blood levels of - µmol/l under a strict diet (total protein content of . g/kg body weight/day with . g/kg natural proteins and . g/kg proteins provided by the aminoacid mixture pku (milupa, germany); , cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of . g/kg bw proteins and , cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to . - meq/l (ref. value . - . meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku , carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at c° for total time period minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p Ͻ . ) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over volatile components were separated, however only the most important component for caramel flavour were reported. maltol and hydroxymethyl- -furfural (hmf) and . h-pyran- -one, , dihydro- , -dihydroxy- -methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca ϩ -independent mechanism involving phosphorylation of ser by a phosphatidylinositol -kinase (pi -kinase) dependent pathway. we investigated the involvement of pi kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser in enos were increased by d-glucose ( mm, min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi -kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi -kinase dependent, ca ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered ( - times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku ( g ϭ adh , g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e components of the pyruvate, oxoglutarate and branched-chain -oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a -fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde- phosphate dehydrogenase (gapd) [e.c. . . . ] from the cytosolic domain of the membrane protein band (cdb ) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k ϭ . m Ϫ s Ϫ and . m Ϫ s Ϫ , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb and gapd. the cys in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form Ϫsno on, and decrease the affinity between, gapd and cdb . in intact rbc, both the no-donors -morpholino-sydnonimine (sin- ) and peroxynitrite (onoo Ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo Ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and Ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin- on gapd translocation. the results demonstrate that cdb and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than - days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of - days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about , - , ml of bile fluid, , - , ml pancreatic juice and , - , ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than days. if artificial nutrition is necessary for more than days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated patients with ppa and with mma in a period of months by weekly capillary blood punctures performed by the parents. age of the patients were from . until years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment ( - mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c /c -acylcarnitine quotient. mean value in mma and ppa patients showed a range of . - . (normal . ϩ/Ϫ . , n ϭ ), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with , , , tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci , m. sokoĺowska , m. zapaĺa , and l. wĺodek institute of pharmacology, polish academy of sciences, kraków, and institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland , , , -tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and -hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out h after a single dose of tiq ( mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs , r. steinfeld , a. kohlschütter , j. zschocke , and k. ullrich department of pediatrics, university of hamburg, and university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. . . . ) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor ( r)-l-erythro- , , , tetrahydrobiopterin (bh ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh . indeed, two such patients in our hospital were also sensitive to daily oral doses of - mg bh /kg. the subsequent molecular genetic analysis revealed that patient was homozygous for the widespread mutation y c and patient was compound heterozygous for the mutations a d and k n. it is striking, that all mutations are located distant from the known bh -binding site and thus, should not be associated with bh -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf ) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of % gypsy rna was achieved with about ng of the protein in ml of the reaction mixture. the binding constant was &times; m- , which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg ) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: ) tlc is employed when starting the investigation, ) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients ( in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo . we cloned each gene encoding ybbq and yhae into e. coli jm . both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of kda, whereas yhae was a tetramer (native molecular mass, kda). ybbq showed the maximum activity at ph . for the oxidation of d-serine. whereas optimum ph of yhae was ph . . they catalyzed oxidation of glycerate and -hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate -dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi , Á. bódi , l. gráf , a. perczel , a. patthy , and g. orosz department of organic chemistry, biochemistry, eötvös university, budapest, agricultural biotechnology centre, gödölló´, and research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd- family of canonical serine protease inhibitors. in these peptides, there is a p -p Ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p -p Ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p -p Ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p -p Ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara , t. nakagawa , and m. minami department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec . . . , mst), inactivation kinetics with -chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of -mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is . ϫ Ϫ min Ϫ and k is . mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin ( ) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of chemistry and pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit ( mg/kg) and oral administraton of melatonin ( mg/kg) at or h after anit injection. animals administered with melatonin at and h after anit injection were sacrificed and h, respectively, after the injection. liver injury appeared h after anit injection and developed at h. melatonin administered at h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g r, g d and r h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g r showed comparable specific activity to wtht, whereas g d and r h mutants exhibited remarkable reduction in specific activity. since homoallelism for g d mutation and heteroallelism for g r and r h mutation were identified in typical and atypical nkh, respectively, these results suggest that g and r h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: . native gel electrophoresis. . cross-linking of proteins using dibromoacetone. . radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with % ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat , s. nagata , h. misono , and k. packdibamrung department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified fold to homogeneity from aeromonas hydrophila. molecular mass of , daltons was estimated for alanine dehydrogenase by sephadex g- chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed polypeptide band with molecular mass of , daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at °c for h. optimum ph for reductive amination and oxidative deamination were . and . , respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were . , , . , and . mm, respectively. an elevation of apolipoprotein (apo) b- concentrations is a particular feature of several metabolic disorders, such as type diabetes (t d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b- of very low density lipoprotein subfractions (vldl , svedberg units (s f ) - and vldl , s f - ) kinetic studies were performed in subjects with t d, igt, fchl, and healthy controls using a tracer of either l-[ring- c ]-phenylalanine or l-[ , , - h ]leucine. these studies showed direct hepatic vldl apob- secretion to be increased in patients with t d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl apob- secretion. in all patients vldl catabolism is not essentially impaired. vldl apob- secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl apob- secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t d, igt, and fchl. vldl overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl and vldl secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg c) whose product was not inhibited by cysteine, and introduced satg c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda , e. suzuki , and t. kurata institute of environmental science for human life, and department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of . mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp , oatp and oatp . these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec . . . , pbgs), two zinc ions are coordinated by cys , cys and cys , and his and cys , respectively. the fomer zinc ion, closer to catalytic site lys , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep b cell, his was replaced with arg (h r). to elucidate the role of his in catalysis, the kinetic properties of wild type and h r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep b cell, respectively. each cdna encoding pbgs with Ј non-coding region was inserted into pet- b(ϩ) vector and then the constract was transformed into e. coli strain bl (de ). the cells were cultured in lb medium containing mg/ml ampicillin and µm zn ion for h at °c. after addition of mm isopropyl--d(Ϫ)-thiogalactopyranoside, cells were further cultured for h at °c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex , serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from - years and raised on pasture were used in the study with as control and as nutritionally treated. twenty four hours prior to capture the elk were offered either kg of a cereal grain based dietary supplement or kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # ). the amino acid content of the nutritional therapy product was minimally . g per kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus g per kg weight of leu and g/ kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels ( . nmol/l) compared to the untreated controls ( . nmol/l; p Ͻ . ). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl- produced µg/ml methionine in shake flasks with methionine yield at . g methionine/g glucose and specific methionine production at . mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt- produced µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte- produced . g/l methionine. the methionine yield was . g methionine/g glucose and its specific productivity was . g methionine/g dcw. clinical biochemistry, laboratory , luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and minutes after oral administration of mg of acetylcysteine. in the group of healthy persons the plasma glucose level feel by . % over the minute period. in the diabetics on the contrary, the plasma glucose level observed minutes after administration of acetylcysteine was . % higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of ml of distilled water ϩ . ml % glucose ϩ . ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by ml . % solution of acetylcysteine. in the glucagon group the distilled water was replaced by . % solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after minutes of incubation. a % diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a . % increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at , and % levels of fortification and the fortified yoghurt was analyzed fresh, and after and days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain ( . units/g) for minutes (tb) and rice bran hydrolysates with alcalase ( . units/g) for minutes (te) to yoghurt can ex-ceed - %, while fortification with sesame hydrolysed with papain ( . units/g) for minutes (td) and soybean hydrolysed with papain ( . units/g) for minutes (tc) can not reach up to %. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: ) controls; ) rats subjected to hbo for min once and immediately sacrificed; ) rats treated as group but for consecutive days and immediately sacrificed; ) rats treated as group but sacrificed h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups - , while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups - . this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker growing in pregnant rats which were distributed into six groups. three groups received normal diet ( % protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with % leucine ( % protein plus % leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, % of leucine) for days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ -pyrroline- carboxylate dehydrogenase, ec . . . ) in a girl aged months presenting with seizures and encephalopathy. l-∆ pyrroline- -carboxylic acid accumulates in this disorder and there is a - -fold increase in plasma proline. surprisingly, she also had vitamin b deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b coenzyme) was de-activated by l-∆ -pyrroline- -carboxylic acid. with high resolution h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph . and °c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline- -carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline- -carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young ( -month-old), middle-aged ( month-old) and old ( -month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b -b ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm and h. s. sharma department of pathology, university hospital, lund university, lund, and laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at °c for h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name ( mg/kg, i.p.) and l-nmma ( mg/kg, i.p.) min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk- , and cgp , respectively, were studied in both saline-pretreated and mptp-pretreated c bl/ mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk- distinguished from amantadine and cgp . in the study of long-term effect of nmda antagonists mk- was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer and a. fredriksson department of psychology, university of göteborg, and department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa ( mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk- , the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce , phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α -adrenoceptor antagonist, yohimbine, but not the α adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block , f. träber , s. flacke , f. jessen , ch. pohl , and h. h. schild department of radiology, department of psychiatry, and department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than spectroscopic data sets of patients with mnd and more than data sets of ad patients were acquired within the last years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin- . this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that -hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur and mglur ) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur in snpr projection neurons and mglur in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello , m. parodi , and m. balestrino centro di neurofisiologia cerebrale, cnr, genova and dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca ϩ into neurons. pretreatment of tissue with creatine ( mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower ( . mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s- . while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura- am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca ϩ ] i increase in response to k ϩ ( mm) depolarization was % higher than in wild-type. ω-agatoxin iva . µm decreased ca ϩ -influx in neocortical wild-type synaptosomes by %, and abolished differences between gsnϩ/ϩ and Ϫ/Ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was % higher and lesion size after mca occlusion was % higher than in wild-type. it is concluded that presynaptic ca ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ ) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv . Ϯ . , mtt . Ϯ . , ttp . Ϯ . , p Ͻ . ). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to µm glutamate (glu) for min, induce significant cell death in cerebellar granule cell cultures, h after treatment. in contrast, the combined treatment with µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to µm glu alone for min, glucose deprivation for min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by %, % and %, respectively. furthermore, we found that exposure to either µm glu, glucose deprivation or µm glu ϩ glucose deprivation decreased incorporation of [ h]cho into phosphocholine and increased the intracellular content of free [ h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf - .) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu receptor subtype by -methyl- -phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu / receptor by (Ϫ)- -oxa- aminobicyclo[ . . ]hexane- , -dicarboxylic acid (ly ) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep ( - µm), perfused through a microdialysis probe affected neither the basal nor the veratridine ( µm)-stimulated striatal da release. however, mpep given intraperitoneally ( mg/kg) diminished either the basal or the veratridine-evoked da release. ly ( - µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine ( ϫ mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep ( ϫ mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly may be a consequence of activation of striatal mglu / receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja , h. h. schild , and j. flammer department of radiology, university of bonn, germany university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that h of exposure to reduced glucose ( . mm instead of mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations ( mm instead of . mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid , -dihydroxy-l-phenylalanine (l-dopa) was first synthetized in . in , the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in , of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between and , showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in , the central antireserpine effect of d,l-dopa was described in mice and confirmed in with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years / , d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in , the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen , a. tasker , and j. zimmer anatomy and neurobiology, sdu-odense university, odense, denmark department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from -days old rats and grown on semipermeable membranes for - weeks before exposure. dom ( . - µm) was added to the medium, alone or together with the glutamate receptor antagonists ns- , nbqx or mk- , for hrs followed by hrs in normal medium. neuronal degeneration was monitored and ec values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at , , and hrs. the lowest ec values, obtained at hrs, were: ca ( µm), dentate granule cells (dg) and ca ab ( µm),ca c ( µm). protective effects of µm nbqx at hrs were seen against µm dom in dg, ca and ca c and against µm dom in ca and ca c. µm ns and mk only displayed protective effects together with nbqx. mk thus significantly increased the protective effect of µm nbqx in ca against µm dom in combination with µm nbqx and µm ns . we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur / and glur , and a decrease in the expression of mglur and its accompanying scaffolding proteins homer bc. this was accompanied by an increase in the capacity of mglur / receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur / receptors. in addition, there is reduced activity in the cystine/glutamate exchanger wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with -hydroxydopamine ( -ohda; :g icv), there was a % reduction in striatal tissue da content in adulthood, and a to fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa ( mg/kg i.p.) suppressed ho* formation. however, when glutamate ( mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns .) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk- . functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk- and flt- , will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased -fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk / ) were rapidly activated, which peaked within six hours of nmda treatment. erk / activation is completely blocked by mk- and partially blocked by k a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk / . as an extension of this work, we discovered a single nucleotide polymorphism in the human nr gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o •Ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to -nitrotyrosine ( -nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o •Ϫ /no • system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, -nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o to o •Ϫ and parent -nt. one-electron oxidation of -nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o •Ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and -nt. the nitration/denitration processes can be pathologic, but these also may play: ) a signal transduction role; ) a role of "blocker" for radical-radical reactions (scavenging of no • , no • and co •Ϫ by tyro • ); or ) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o •Ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than % of patients with type diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p was detected in heart of dm-rats. substantial changes in the methylation status of the p dependent p waf /cip -gene and the cyclin d -gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p -dependent activation of cardiac cell death. this pathway includes de novo methylation of the p -inducible p waf /cip -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor (igf- ). igf- is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf- is upregulated in areas of brain damage and the igf- system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf- is reduced. we recently observed a neuroprotective effect of topical application of igf- in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf- , as well, is known to be decreased. therefore, exogenous substitution of gh and igf- might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in -hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below % of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a , adrenergic a , opiod, and serotonergic b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype (mglur ), mpep ( . - mg/kg ip), or the agonist of group ii mglurs, ly ( - mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur , (rs) aida ( . - µg/ . µl), but not of the agonist of group ii mglurs, r, r-apdc ( . - µg/ . µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats ( - g) and male c bl/ mice ( - g). aida (group i mglurs antagonist) given i.v. in the dose of µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur antagonist) given i.p., was not effective in the despair test in rats. however, in doses of . , and mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep ( mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu receptors in the ca field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu receptors in the ca field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. .po a. . ) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t - segments resulted in profound edema formation and cell damage in the adjacent t and t segments at h. pretreatment with h- / ( mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t and t segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h- / . these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h- / has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in , creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound ( biological and synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p and cdc . transcriptional activity of p was shown by increased p dna binding activity and by the concomitant induction of the cdk inhibitor p , the cell cycle regulator gadd and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of -oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh increases in cultured cerebellar neurons or in ca pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after years of therapy % of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in -methyl- -phenyl- , , , tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg mice overexpressing human -amyloid precursor protein (huapp k n/m l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. , - , . recently, we also found that the overexpression of p protein, an activator of the kinase cdk , results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg mice at -months of age (i.e. - months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: ) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. , - ); ) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and ) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional ( -d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip ( , , , -tetrahydroizokasazolo, , -c pyridin- -ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult - week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of and mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at - hours and recovered at - hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/ mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: /sag/ ).] and in long-term ethanol-experienced rats only at a dose mg/ kg i.p. control group (cg) received saline ml/kg i.p. as can be seen in fig. and table the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. ) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at Ϫ mv. in a first survey, each of the solutes was briefly applied in a concentration of mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and -indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat ), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. p a .) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a -min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son ( %; p Ͻ . vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to %; p Ͻ . vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en - ). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets- and collagen α (vi) chain precursor, encoded on chromosome , was investigated in fetal brain by western blot and two-dimensional electrophoresis ( -de). western blot detected app and ets- that migrated at ϳ and kda, respectively. subsequent densitometric analysis of app and ets- immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α (vi) chain precursor, a protein resolved as a single spot on d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the d image master software revealed a significant decrease in fetal ds (p Ͻ . ) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd , apo- ), caspase- , bcl- and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase- , bcl- and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl- and annexins together with unaltered caspase- expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for h followed by h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff (dna fragmentation factor ), and flip (fadd like interleukin- -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf- (apoptosis protease activating factor- ), procaspase- and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase- and - were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n -( -iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, -nitroindazole ( -ni) and the no scavenger, -( -carboxyphenyl)- , , , tetramethylimidazole- -oxyl -oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by vessel occlusion for min. l-nio ( mg/kg), -ni ( mg/kg) and ptio ( mg/kg) were administered intraperitoncally min before ischemia. ptio, -ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio Ͼ -ni Ͼ l-nio. l-nio, but not -ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and -ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. -ni recovered in part ps amplitude min after ischemia. -ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase , m. tanaka , k. tujioka , e. hirano , and h. yokogoshi department of home economics, aichi university of education, kariya, aichi, and laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether -estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of wk old female rats: group . ovariectomized to reduce the level of plasma estradiol; group . ovariectomized and treated with estradiol; and group . sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif- (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that -f-phe- -oh-pro-arg-gly-trp-nh (inn ) is superior in all the statistical parameters used. in comparative testing inn was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn show that it is effective in over % of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn has a rapid onset of action, - days (vs. - weeks for currently marketed products) with sustained effects for months following to doses over - weeks. h. iwama , , a. umino , a. hashimoto , k. takahashi , n. yamamoto , and t. nishikawa , section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm genes (h and h ) , which are related with the metastatic potential of some tumors. although ndpk/ nm has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm -h in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional ( -d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm -h was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm -h upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d- -hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d- hydroxyglutarate (d- ), which is structurally similar to lglutamate (ϭ -amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d- induces excitotoxic neuronal damage. here we report that d- decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura- measurement. furthermore, fluorescence microscopy using dihydrorhodamine- revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d- . n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d- is an agonist at nmda receptors. patch-clamp investigation confirmed that d- activated recombinant nmda receptors in hek cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d- . we conclude that excitoxic mechanisms contribute to the neuropathology of d- hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c bl/ mice of different ages ( , - , and - months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e and z mrna density in naïve, ad lib-fed mice. correlations were found between changes in e subunit mrna and agonist binding and z mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e and e mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e and e and/or z subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices ( mm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of kϩ ( mm) or veratridine ( mm) minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( mm) perfused for minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( mm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( mm) increased the basal release of glutamate (p Ͻ . , n ϭ - ). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ h]cgp to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ h]cgp affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ h]cgp binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ h]cgp binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to days) with mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing mm k ϩ also exhibited multiple changes in nr splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding Ј and Ј splice variants. nr - and nr - (each containing exon ) were decreased, whereas nr - and nr - (each lacking exon ) were increased, accordingly. the predominant expression of nr -b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: Ϫ mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of Ϫ pa under control conditions. neurones treated with mm k ϩ showed a significantly diminished response to nmda (max. response: Ϫ pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of mm k ϩ on neuronal survival and maturation. (supported by bmbf grant gg / ) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp , hsp , and hsp . however, no investigation has been carried out on changes in level of apg- , a member of hsp family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp and apg- in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg- level was significantly decreased in the frontal cortex, hippocampus, and striatum days after kainate administration, while hsp level was increased in these regions following the administration. decreased level of apg- returned to the control levels in the three regions days after kainate administration. no significant changes were observed in levels of both hsp and apg- in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp , apg- expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide- ( - ) amide (glp- ) and its specific receptor (glp- r) have been found in the rat hypothalamus. in this study the actions of glp- and its related peptides, exendin- (glp- r agonist), exendin ( - ) (glp- r antagonist) and glp- ( - )amide (major glp- metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. i-glp- binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp- Ͼ exending- Ͼ exendin ( - ) Ͼ glp- ( - )amide. intracerebroventricular (icv) glp- ( nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas nmoles of exendin ( - ) was ineffective. exendin- produced a statistically significant reduction in the levels of trp, glu, and tyr. glp- ( - )amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin ( - ) or glp- ( - )amide blocked the effects of glp- on the levels of the amino acids. these data are consistent with exendin- being a glp- r agonist and exendin ( - ) being a specific glp- r antagonist. glp- ( - )amide, a primary metabolite of glp- , appears to act as an endogenous antagonist at the glp- r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck ( fmol) and/or cck antagonists ( fmol) were injected days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck ns; cck ) or a mixed cck b and cck a receptor agonist (cck s) were injected. in contrast, cck , a cck a receptor agonist or cck - and cck - were uneffective. furthermore, the ansiogenic effects of cck s were prevented by the previous ( min) administration of l , (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than % of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and -mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after days with maximum myoclonic activity ( jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by % (p Ͻ . ) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate ( . µ mol), aspartate ( . µ mol) or nmda ( - n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. -amino- -phosphonovaleric acid (ap ) and -amino- -phosphonopheptanoic acid (ap ) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like ( s, s)-acpd the agonist of group ii, and l-ap the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(Ϫ) and divalent cations in the na(ϩ)/cl(Ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat- (hgat- ) gaba transporter cdna. our results showed that cl(Ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat- (loo et al., j biol. chem. : - , ) . the cl(Ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat- transported cl(Ϫ) across the membrane with a stoichiometry of na(ϩ) : cl(Ϫ) : gaba, the transported cl(Ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(Ϫ)/cl(Ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat- is also modulated by divalent cations. the uptake of [ h]-gaba was inhibited significantly when both ca( ϩ) and mg( ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca( ϩ) and mg( ϩ) from the efflux buffer. the gaba transporter inhibitor skf a blocked the enhanced efflux, suggesting that the hgat- operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha subunit gene in knockout mice (ko). in the ca region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha containing gaba a receptors contribute to functional postsynaptic receptors on ca pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure ( hours) twice in week during weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon - mg/kg, mephebut - mg/kg, gammoxin - mg/kg, pyracetam - mg/kg. the treated males were mated with intact females during days. after the mate the treated males and more in days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased . % (p Յ . ) and . % (p Յ . ) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa . % (p Յ . ), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p Յ . ) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p Ն . ) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced . %. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino- -hydroxy- methyl- -isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk- (dizocilpine) . mg/kg sc. control rats received tap water only. behaviour of month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf , quinpirole, apomorphine, haloperidol). our results indicate that mk- applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo ) and mesencephalic trigeminal neurons (me ); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo and me during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo . furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer , m. dierssen , m. fountoulakis , and g. lubec department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap and the channel associated protein of synapse (chapsyn ) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on -dimensional gel, identified and quantified. we quantified members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins - and the collapsin response mediator protein- (crmp- ) in ds and control cortex samples. drp- and crmp- levels were comparable in the control and ds samples. evaluation of drp- , drp- and drp- revealed significantly decreased levels of of the spots assigned to drp- and increased levels of one spot assigned to drp- and increased drp- in ds brain. we conclude that as early as from the th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases , and activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases , , and activity in the caudate/putamen. zvad significantly decreased the levels of caspases , and but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap to the goldfish telencephalon in avoidance conditioning. in experiment , fish received no injection or microinjections of saline or various doses of ap to their telencephalon minutes before three semiweekly training sessions. fish were tested without injec-tions in session . a one-way anova with multiple comparisons on the test scores showed that ap produced anterograde amnesia in a dose-dependent manner. in experiment , fish received several training sessions and a microinjection of various doses of ap minutes before testing. the test scores showed that ap did not decrease avoidance responses, suggesting that microinjection of ap did not impair performance processes. in experiment , fish received microinjections of ap or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session . a one-way anova on the test scores showed that ap did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto and e. a. newsholme health science laboratory, tezukayama university, nara, japan department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan ( um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan ( mm/ min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average g) were made by tryptophan free feeding for weeks, and the rat's running time increased (Ͼ min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [ h]d-serine into the synaptosomal p fraction from the rat cerebral cortex. the cortical p fraction was able to accumulate [ h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of µm and a vmax value of pmol/mg protein/min. depletion of na ϩ and cl Ϫ ions remarkably decreased d-serine uptake into the cortical p fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo , p. j. lea , s. a. gaziola , a. p. pellegrino , and s. m. g. molina departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: -oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: ( ) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. ( ) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. ( ) the opaque- mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and -fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque- mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque- maize gene has been shown to regulate ak and lor activity. ( ) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. ( ) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl , ranging from . - mm, for up to h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as % of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: -fold), n-trimethylglycine ( -fold), histidine ( -fold), tryptophane ( , -fold), putrescine ( , -fold), spermine ( , -fold) and trigonelline ( -fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass ( %) as a result of growth inhibition ( %). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or -aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a to -fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a -fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in to -fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. Ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr ) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of Ϫ mv at ph . and involves a -electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of , bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova , m. zeh , o. kreft , s. maimann , h. hesse , and r. höfgen max-planck-institut für molekulare pflanzenphysiologie, potsdam, and institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque- mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during days of germination quantity of zn in primary leaves increased from , to , % and decreased in soil: in upper layer from , to , , midde layer from , to , , lower layer from , to , mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b , but these results can also be inaccurate. vitamin b deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b deficiency. twenty-seven patients (mean age - . years) with low serum b were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of . %- . % of neutrophils examined. there was a significant correlation of homocysteine (r ϭ . , p Ͻ . ) and mma (r ϭ . , p Ͻ . ) with serum b levels but no correlation of appendage number (r ϭ . ) with serum b . there was no correlation of appendage number with homocysteine (r ϭ . ) or mma (r ϭ . ). these results suggest that b -deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d ϭ placebo, d ϭ mg, d ϭ mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m ϭ min. after stress/before testdrink, m ϭ min.-, m ϭ min.-, m ϭ min.-, m ϭ min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha , theta (frontal) and decreasing beta relative-power earlier in recovery with d . these findings were related to relaxing effects. after ingestion of l-theanine alpha -, beta -power at occipital regions decreased faster (m ) to placebo recovery levels (m /m ). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus , j. gabryś , j. konecki , and j. shani department of pharmacology and department of histology and embriology, medical university of silesia, zabrze, poland department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin- or of some of its analogs was found to cause a selective inhibition, up to %, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type- somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type- receptor antagonist. a type- specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between and amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin- and vvhemorphin- , could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin- and mainly lvv-hemorphin- could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of days. noteworthy, continuation of treatment for aperiod of days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. ) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately % of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. ) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in % of the female patients. moreover, - % of these patients omplained about a distorted sensory perception of objects. ) young soccer players were observed during a period of months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany percent of amino acids in green tea leaves are represented by l-theanine ( -n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine ( , and mg) were administered to healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as , , , , and minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: . decreasing skin resistances after exercise could be established in each volunteer. . esg-activation levels before exercise could not even be reached again after a period of regeneration of / hours. . maximal electrodermal activity did not appear immediately after exercise, but after minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero and h. pitkänen , neuromuscular research center, department of biology of physical activity, university of jyväskylä, and rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment ten of them were given leucine ( mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly ( %) in the placebo group (p; n ϭ ) during weeks but not when leucine was taken. also total amino acids (taas) decreased strongly ( %) during weeks when dally protein intake was . g/kg body weight. in experiment the subjects (n ϭ ) carried out a single strength training seasion (sts) and consumed a drink containing leucine mg/kg body weight. following sts leucine in serum increased by % (ns) when leucine was taken but decreased strongly ( %) in p, in experiment the subjects (n ϭ ) underwent at days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ s with a recovery of s between the runs) until exhaustion. the subjects consumed drinks containing leucine ( mg/kg body weight) or placebo min before the test runs. following mare the concentration of leucine strongly increased by % whereas isoleucine ( %) and valine ( %) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment ) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of track and field adult male power athletes. in experiment eleven of them performed a -week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) ( %), branched chain amino acids (bcaas) ( %), essential amino acids (eaas) ( %) and leucine ( %) were observed following training with the daily protein intake of . g/kg body weight. in experiment eleven subjects performed a short run session (srs) of ϫ ϫ m with recoveries of s and s, and a long run session (lrs) of s runs with recoveries of s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by % in srs and by % in lrs. leucine decreased by % following srs but only by % (ns) following lrs. the peak blood lactate concentrations after srs and lrs were . Ϯ . mmol/l and . Ϯ . mmol/l, respectively. in experiment sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during minutes. the taas decreased significantly by %, bcaas by % and leucine by % following sts, while the peak blood lactate concentration was . Ϯ . mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of . g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have , , -and , , -trisubstituted pyridine skeletons both with three carboxylic acids and a mass of (c h n o ), identified as -( -amino- -carboxybutyl)- , -di-( -amino- -carboxypropyl)pyridine and -( -amino- -carboxybutyl)- , -di-( -amino- carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il- production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il- production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by % hr after the race (p Ͻ . ). there was an apparent decrease (only close to significance, p Ͻ . ) in il- production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was % higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response ( . %) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma ( . %). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il- production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il- production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward and l. m. castell departments of biochemistry, university catholique de louvain, belgium oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt and crt (a na ϩ and clЈ dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor . more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss , t. barthel , r. schnittker , k. e. geiss , w. falke , and l. r. juneja university of paderborn, germany taiyo kagaku co., yokkaichi, japan, isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ healthy students consumed drinks containing either , or mg l-theanine in randomized double-blind trials in the min - after a near maximal bicycle step test. measurements were done directly after exercise (m ) and (m ), (m ), (m ), (m ) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m (catecholamines) or m (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec . . . ), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from - kda. they contain different types of subunits, named b , b , b , a , a , a , ba , ranging from - kda. in the rabbit eye lens two -crystallin subunits ( b and b ), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b -and b -crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines ( , diaminooctane, , -diaminononane, , -diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni , d. bertoldi , , e. candioli , l. martinelli , and a. tassoni istituto agrario, san michele a/adige, and dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme ) is related to an intrinsic gtp-ase activity of type transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b , in transmethylation reaction, the significance of ,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b may be in direct relation with the formation of ,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase- and parp, a substrate of caspase- . the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca ϩ , but that putrescine is also capable of inducing influx of extracellular ca ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: / evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and / determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh ( . iu, s.c., saizen, serono, switzerland) was administered once daily for or days, to randomly selected rats on the second postoperative day. animals were sacrificed , and days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are . µm and µm, respectively. amino acid residues, cys , trp , glu , trp and tyr are strongly involved in both activities, and that glu , tyr , cys , cys , cys and glu are moderately involved in the activities. mutations of tyr , trp and trp mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys and tyr mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has % sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are µm and µm, respectively. it was found that two glutamate residues (glu and glu ) and four tyrosine residues (tyr , tyr , tyr and tyr ) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. . . . ) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg ) carrying the ttg gene sequence to c bl /n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor- a (eif a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif a activity in human epidermoid lung h cancer cells. we have found that h exposure to , iu/ml ifnα induces % growth inhibition and % apoptosis in h cells. moreover, ifnα induced a -fold increase of ttgase activity and expression that already occurred after h of exposure to the cytokine. this effect was paralleled by a . -fold enhance of ttgase mrnas. ifnα induced also a % increased eif a expression while an about % decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif a activity. adometdc deficient mice k. nishimura , f. nakatsu , , k. kashiwagi , h. ohno , t. saito , and k. igarashi graduate school of pharmaceutical sciences, chiba university, chiba, graduate school of medicine, chiba university, chiba, and cancer research institute, kanazawa university, kanazawa, japan the amd gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by % in amd ϩ/Ϫ mice, adometdc activity reduced only by % and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd Ϫ/Ϫ mice, we intercrossed amd ϩ/Ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd Ϫ/Ϫ mice from heterozygous intercrosses over. amd Ϫ/Ϫ embryos died early in development, between e . and e . days post coitum. in culture of blastocysts at e . , the shapes of all cell lines were normal, but amd Ϫ/Ϫ cells appeared to arrest the cell proliferation at day after the onset of cell culture. the arrest of amd Ϫ/Ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd Ϫ/Ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h o and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h o formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m ) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h o in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k ), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis( -aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis( -aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis( aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by °c by the addition of . mm of tetrakis( -aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis( -aminopropyl)ammonium prevents depurination most effectively. tetrakis( aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde -phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an -aa peptide bearing the simplest q n domain (q ), and polyamines of different size and shape as q-and nh -donor, respectively. in the present study we report that out of the lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm - , institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, -(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms ( , s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g- cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were . mm in the reaction mixture. bis(uracilyl)-analogs and -( -oxyethyl)ami-no- --d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ was stimulated . -fold and that of σ was stimulated . -fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ synthesis, whose translation is altered by polyamines. stimulation of σ synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( , , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than %. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol- -yl, isoxazol- -yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)- -amino- -( hydroxy- -methylisoxazol- -yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing h-pyran- -one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m ( ) tetrahedron : ). as a continuation of our investigation in this field we report here a detailed study of the transformation of h-pyran- -one derivatives with hydroxylamine ( , x ϭ o) and various hydrazines ( , x ϭ nr ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives . in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines also occured during the reaction course. some attempts to hydrogenate compounds towards α-amino acid derivaties by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin and were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position and were studied. in position several hydroxy-and serine related amino acids were incorporated, whereas in position the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser -endomorphin Ϫ . quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted h-pyran- -ones containing α, -didehydroamino acid unit in their structure as dienes in [ ϩ ]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of h-pyran- -ones upon the cycloaddition of acetylene derivatives the first intermediate formed ( ) still contains the carbon dioxide bridge. in many cases easily expels co and substituted benzene derivative is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co from the adduct there are two possible paths: so formed cyclohexa- , -diene ( ) is either aromatized into benzene derivative ( ) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts ( ) are formed. since co -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co -containing adducts ( ), while with alkenes we have isolated, depending on the structure pattern of the compound , all three types of products: aromatized , co containing and double adducts . especially the type is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc or orl . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc receptor and for full biological activities. noc( - )-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc( - )-nh , hoping that these derivatives retain the ability to bind lc receptor and are resistant against biological degradation: phe-nh-co--ala-noc( - )-nh phe--ala-nh-co-phe-noc( - )-nh phe-gly-nh-co--hphe-noc( - )-nh the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rЈ)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions Ñà ϩ or mg ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax and mgx where one metal ion binds together several (up to ) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca ϩ or mg ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to %). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose- p, the product of the reaction catalysed by glucosamine- p synthase is d-glucosamine -phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active from d-serine will be described and first results in the biological evaluation on glucosamine -phosphate synthase will be included. o. melnyk , d. bonnet , e. loing , l. bourel , and h. gras-masse -umr , -sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal - portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l- -(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a Јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur . as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly and its c epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[ , , ]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p , p and p Ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new Јdeoxynucleoside analogues modified at Ј-and Ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky and f. bordusa , max-planck-society, research unit "enzymology of protein folding", halle, and institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: ( ) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v protease (v protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev , a. k. dadayan , b. v. vaskovsky , and n. f. myasoedov institute of molecular genetics, russian academy of sciences, and shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ h] peptide analysis using h nmr spectroscopy was carried out, and the modified fragment [ h]actc - (met-glu-his-phe-gly-pro), with molar activity of ci/mmol and [ h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ amino-isobutyric acid) with molar activity of ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y , e. kontny , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il- trigger production of il- and il- also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il- and il- production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps ( h) in the presence of tau or tau-cl ( - µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il- and il- were inhibited by tau-cl with ic Ϸ µm and µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly ( . times) at low ( - µm) while reducing it (ic Ϸ µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (Ͼ µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl- ) to investigate the activation of a swelling sensitive [ h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, µm) increased transiently the rate coefficient for [ h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor -isobutyl- -methyl xanthine ( µm) increased the swelling activated efflux rate coefficient . times above the control level and the camp analogue dibutyryl-camp ( µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph Ͻ , is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: - °c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph of the aqueous solution decreases which correlates with a decrease of oxidation capacity of . % per day, probably originated by the elimination reaction r-ch -ncl ae r-chϭncl ϩ h ϩ ϩ cl Ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of mm ndct at ph and against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny , m. kurowska , j. kowalczewski , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for hours with rhpdgf or rhtnf-α. tau or tau-cl were added at - µm concentrations. cell proliferation was determined by incorporation of h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at µm concentration tau-cl inhibited (by Ϸ %) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p , p ) nor anti-apoptotic bcl- protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny , k. szczepań ska , j. kowalczewski , m. kurowska , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il- and il- by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil- . tau or tau-cl were added at - µm concentration. after . - h or h the dna binding activity of nfkb and ap- (emsa) and the expression of il- and il- mrnas (rt-pcr) was examined, respectively. results. il- raised nfkb and ap- activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il- Ͼ il- ) and the activity of transcription factors (nfkb Ͼ ap- ). conclusion. tau-cl inhibits transcription of il- and il- genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using h and c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within h, % of the total intracellular hypotaurine and . % of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, ) . ges, when administered to rats in their diet in the drinking water as a - . % solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within - weeks the levels of taurine reach their nadir ( - % of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in , koppel and associates (geggel et al., n. eng. j. med. : - , ) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. : - , . upon administering . % guanidinoethanesulfonic acid (ges) in the drinking water of rats for weeks, the taurine levels in cardiac tissue decline by %. however, the phosphorylation of a ϳ kdalton protein in the mitochondrial fraction of the heart tissue increased by % (j. mol. cell. cardiol. : - , ) . reversal of these effects could be accomplished by feeding the rat . % taurine in the drinking water for weeks. the ϳ kdalton protein was isolated by -dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ kdalton was pyruvate dehydrogenase (amino acids : - , ) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges ( %) for weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp ( ì) and radioactive atp for minutes. animals were also treated with taurine ( %) in their drinking water for - weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity Ϯ sem (normalized to mg protein) control Ϯ (n ϭ ) ges-treated Ϯ (n ϭ ) p Ͻ . (paired -test) control Ϯ (n ϭ ) taurine-treated Ϯ (n ϭ ) p Ͼ . control Ϯ (n ϭ ) ges followed by taurine Ϯ (n ϭ ) p Ͼ . these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume - % less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine ( - mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine ( mg/kg) also suppressed water intake. the taurine precursor hypotaurine ( - mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine ( - mg/kg ip), which suppressed ethanol intake and preference by approximately % without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi -kinase (phosphatidylinositol -kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl- . increases in the extracellular osmolality by either addition of mm taurine or mm mannitol to the incubation medium activates similar pathways. however, pi kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl- and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg human liver cells. the intracellular taurine content of hepg cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing nm angiotensin ii undergo hypertrophy, a process blocked by mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is : - . in the present study, we determined urinary sulfate and taurine in urine samples of healthy japanese women after h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, . Ϯ . ; free sulfate, . Ϯ . ; ester sulfate, . Ϯ . ; taurine, . Ϯ . ; urea, . Ϯ . . the ratio of total sulfate and taurine was : . . this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, . . this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi and h. oda laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine ( . - g/kg) were supplemented to hc for wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol α-hydroxylase (cyp a ) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp a gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ % of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ %), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ % in the presence of pdc and reduced in an equal degree (ϳ %) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no p a and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the and kda forms of mitogen-activated protein kinase (mapk) and the stressactivated kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p mapk. this kinase currently exists in four different isoforms which are p α, , γ and δ. the involvement of individual isoforms of p in enhancing the expression of carrier proteins for l-arginine will be discussed. gw is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. . bioorg. med. chem. lett., : , - ) . the systems associated with transport of this compound have been investigated using the macrophage cell line j . prior to each study, j cells were seeded in -well culture plates and allowed to adhere for h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution ( µl; °c) containing l-[ c]gw ( µciml Ϫ ) in the presence of either . mm or . - mm unlabelled substrate. in parallel studies transport ( µciml Ϫ , . mm) was monitored in the presence of mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of . mm of l-[ c]gw occurred in a time-dependent manner and was linear for up to min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of . mm and v max of . pmol·µg protein Ϫ min Ϫ . at mm, -methylaminoisobutyric acid (meaib), lalanine, l-valine and - -amino-bicyclo-( , , )-heptane- carboxylic acid (bch) caused little or no inhibition of l-[ c]gw ( . mm) uptake. in contrast, transport of l-[ c]gw was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, -diazo- -oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of . mm l-[ c]gw was inhibited in a concentration dependent manner by l-arginine (ki ϭ . mm), l-leucine (ki ϭ . ), don (ki ϭ . mm) and l-glutamine (ki ϭ . mm). taken together, these data suggest that gw may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw ; if so, the monophasic substrate kinetics imply that the two systems handle gw with similar affinity. other systems such as b ,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw is largely (ϳ %) na ϩ -independent. similarly, b ,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw does not interact with l-glutamine and thus unlikely to be involved in transport of gw , at least in j cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with - putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of h-l-alanine and h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and f hc) and seven light subunits are known. rbat and the light subunits b ,ϩ at and y ϩ lat are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b ,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat- , - a, - b, - and - ) with cat- a and - b being splice variants. in humans, only the "old" members of the family have been characterized (hcat- , - a and - b). hcat- and - b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat- a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat- a and hcat- b differ only in a region of amino acids. cat- , so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat- expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat- has recently been isolated, however, the transport activity of hcat- has not been characterized. when optimally aligned, the amino acid sequence of hcat- shows only about % identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat- , - (a or b) and - are about % identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat- and hcat- a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat- a compared to the high affinity cat-isoforms. to characterize the human cat- , we cloned a cdna encoding hcat- . when expressed in xenopus laevis oocytes, hcat- had a similar transport activity and affinity for l-arginine as hcat- or - b. hcat- mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat- is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat- also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat- , but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat- with the green fluorescent protein attached to the c-terminus. therefore, hcat- might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; . mg/ g b. wt. i. p.), sodium dichromate (cr; mg/ g b. wt. s. c.) and tl so (tl, mg/ g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in -(both sexes) and -day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times ( st day after cr in both age groups and in -day-old rats after cp and rd day after cp in adult rats; nd [ -day-old rats] and th - th day [ -day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious -day-old rats cr nephrotoxicity is not detectable after mg/ g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in -day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata , a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata and ata , have been cloned and functionally characterized. ata is expressed primarily in the brain and placenta whereas ata is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata . it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata cdna from a skeletal muscle cdna library using rat ata cdna as the probe. rat ata consists of amino acids and exhibits a high degree of homology in amino acid sequence to rat ata ( % identity) and rat ata ( % identity). interestingly, this new transporter also has a comparable degree of homology to sn and sn , the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata -expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k . for alanine (i.e., concentration of the amino acid yielding half-maximal current) is . Ϯ . mm and that the na ϩ : alanine stoichiometry is : . subsequently, we have cloned the human homolog of rat ata from a liver cell line (hepg ) cdna library. human ata also contains amino acids and shows % identity in amino acid sequence with rat ata . the sequence identity of human ata with human ata and human ata is % and %, respectively. the homology of human ata with human sn and sn is also similar ( % and % identity, respectively). the gene coding for human ata contains exons and is located on chromosome p . in the human, ata is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata for cationic amino acids is higher than for neutral amino acids. however, the human ata -mediated cationic amino acid transport is na ϩ -independent. in this respect, ata is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata and ata have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata also differs from ata and ata in substrate affinity. ata and ata interact with meaib with a k t of ϳ . mm whereas the affinity of ata for this model substrate is comparatively at least -fold lower (k t , ϳ mm). but, ata interacts with arginine with a k t value of . mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: ( ) cr, cp and tl were more nephrotoxic in -day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. ( ) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. ( ) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. ( ) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr -family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept that will be useful in further studies on structure and functions. starting from lys[z(no )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept . the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept with k i values as low as nm. this is the highest affinity of a ligand of pept ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept . ata is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ %) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic ϭ µm; ok hc , ic ϭ µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa ( µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b ,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b ,ϩ and lat , the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter protein (cat ). the regulation of translation of the cat mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif a. translation of the transporter mrna increases when eif a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif a are required for increased activity. we propose that eif a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some min following cd receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept , one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept is encoded by the pep- gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep- transporter in vivo, a c. elegans pep- mutant was constructed. the animals deficient in pep- show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep- gene in the mutant. the observed starved phenotype in pep- mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. , - , ) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a a , a b and a -adenosine receptor subtype, whereas negligible levels were detected for the a -receptor. adenosine ( µm, min) induced increases in l-arginine transport and no production were ca ϩ and camp independent and stimulated transport was abolished in cells depolarised with mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek / (pd , u ) but unaffected by inhibitors of pkc (calphosin c) and pi- kinase (ly ). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli , s. lord-fontaine , e. przybytkowski , and d. a. averill-bates department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr- gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h o and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c , at and °c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h o and aldehyde(s) relative to their drug-sensitive counterparts, auxb cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from -hydroxymethyl- methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts from the corresponding diol. treatment of with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams . activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi , m. a. s. di leo , s. caputo , n. gentiloni silveri , and g. ghirlanda department of pharmacology, university of sassari, and department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting % of diabetics and progressing to blindness in about %. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type and type diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic ( months) taurine ( % and %) supplementation in diabetic rats in comparison with vitamin e ( and ui). dietary taurine supplementation, for months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of -month duration, we found that cd, lp are significantly increased and they remained elevated for months. while, the na/k atpase is significantly decreased during the whole experimental time ( months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being % taurine more effective than % diets. the morphological examination reveals that in rats feed with % taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch (ch ) n xco iran applicated be ( mg/kg - days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p Ͻ . ; p Ͻ . ) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o Ϫ ), hydrogen peroxide production (h o ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o Ϫ , h o formation and mpo. taurine decreased o Ϫ and h o production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with -nitroso- -naphthol in the presence of nitric acid - -benzyo- -(alanyl)- -phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting -nitroso- -naphthol with -(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that , -benzo- phenoxazone derivative is not produced but a hydrogenated form of , -benzo- -phenoxazone which is probably , -benzo- -(α-hydroxy -methylaminopropyl)- -hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three ( ) breast cancer cell lines: mda-mb- , mda-n and hs- t. the % growth inhibitory (gi ) concentrations for these three cell lines were . ϫ Ϫ , . ϫ Ϫ and . ϫ Ϫ molar respectively. a. bocheva and t. pajpanova institute of molecular biology, bulgarian academy of sciences, and institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva , s. pancheva , and t. pajpanova institute of physiology, bulgarian academy of sciences, and institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh ) that was discovered in hypotalamus.the mif- exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a (txa ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue -ro-c h -coϳ, {where rϭh or ch co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at g for min at °c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp was used. the ic values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg , d-phe , d-trp , , leu ]sp (antagonist d) and the c-terminal analog [arg , d-trp , , mephe ]sp - (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp , glu(bu t ) ]sp - and [glp , glu(bu t ) ]sp - showed significant inhibition in the proliferation of the cancer cell lines hela and t d.in the present study the c-terminal analogs of sp [glp , d-trp , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) ]sp - ( ), [glp , trp , mephe , glu(bu t ) ]sp - ( ), [glp , mephe , d-trp , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) -oh]sp - ( ), [glp , d-trp , cys(acm) -oh]sp - ( ), [glp , d-trp , mephe , cys(acm) -oh]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , cys(acm) -oh]sp - ( ) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs - are peptide amides whereas the analogs - are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at Ј, Ј-positions j. velkov , i. stankova , a. ivanova , and a. tadjer department of chemistry, south-west university "neophit rilski", blagoevgrad, and department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives ( Ј, Ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, Ј, Ј-bis-o-n-α-benzyloxycarbonyl-valyl , Ј, Ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl, Ј, Јbis-o-n-α-benzyloxycarbonyl-phenylalanyl, Ј, Ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am ) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept substrate from a non-substrate?computer modelling of a large number of pept substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept , and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/ mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/ mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution ( . mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw human colon carcinoma cells and c rat glioma cells, stimulated human lymphocytes and macrophages. about ϫ cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). . mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately %, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f- -fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c- -methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c- ( min) in contrast to f- ( min). amino acid analogous labeled with f- like f- -fluoro-α-methyltyrosine (fmt), f- -fluoro-ethyltyrosine (fet), f- -fluoro-phenylanaline, f- -fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i- -iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i- allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo , , v. fiorito , a. pietrosanto , a. pepe , g. andria , and g. sebastio telethon foundation, rome, and department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim ) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim ), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the f heavy chain ( f hc) and a light chain represented by either the solute carrier family a, member (slc a ) or (slc a ). after excluding the f hc as the causative gene of lpi, we identified slc a as the lpi gene and characterized mutations in twenty-five patients from families ( italian, japanese, moroccan, greek, and pakistani; independent alleles) affected by lpi. thirty-two of the independent alleles ( . %) were characterized and fourteen mutations were identified. only five mutations (namely insatca, w x, delctct, ivs ϩ gaea, s r) were identified in more than one independent family. most mutations are located in the slc a coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc a gene in lpi, which, in addition to slc a , is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either f hc/ slc a or f hc/slc a , is still unknown. hypothetically, the activation of f hc/slc a in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n. cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal ( Ϯ vs . ϫ pmol/ cell/ min) and histaminestimulated ( Ϯ vs Ϯ pmol/ / min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of Ϫ Ϯ and Ϫ Ϯ mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions ( . m nacl, ph . ) were obtained in the temperature interval - c degrees. the measurements of the dielectric parameters were carried out at the frequency of . hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of - , - and - c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both and kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc - or pomc - with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three kda n-pomc fractions enhanced prl mrna levels by - %, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation ( . Ϯ . %) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all and kda isoforms activated the mc- and mc- receptor in cell lines in which these receptors were transfected. thus, att cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc- and mc- receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices ( µm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of k ϩ ( mm) or veratridine ( µm) minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( µm) perfused for minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( µm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( µm) increased the basal release of glutamate (p Ͻ . , n ϭ - ).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or na ϩ channels.glucagon-like peptide- ( - ) amide (glp ) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp on serotonin ( -ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp ( . µm) produced a significant decrease in levels of -ht by % after minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of hydroxyindolacetic acid ( -hiaa), the principal metabolite of -ht, and tryptophan the amino acid precursor of -ht, were also decreased significantly by % and % respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba ( %) and glu ( %) by glp was not significant.the results suggest that decreased synaptosomal levels of -ht and -hiaa caused by glp are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso treatment ( . Ϯ . vs control . Ϯ . µmol/mg prot.) and ( . Ϯ . vs control . Ϯ . nmol/mg protein p Ͻ . ) respectively. it was associated with significantly increased iron content ( . Ϯ . µg/mg prot. vs control . Ϯ . p Ͻ . ). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso and spermine, iron content was significantly decreased ( . Ϯ . p Ͻ . compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso treated only ( . Ϯ . ), but mda level didn't change ( . Ϯ . ). in addition, treatment with spermine alone resulted in increase of mda level ( . Ϯ . vs control p Ͻ . ), iron content didn't change ( . Ϯ . ), but carbonyl groups were decreased ( . Ϯ . vs control p Ͻ . ). feso treatment increased gsh level ( . Ϯ . nmol/mg prot. vs control Ϯ . ; p Ͻ . ) while in combination with spermine this increase was more profound ( . Ϯ . ; p Ͻ . vs control, p Ͻ . vs feso ). spermine alone produced similar increase of gsh level ( . Ϯ . , p Ͻ . vs control; p Ͼ . vs feso ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be ( mg/kg - days), second key: cord- -mcfnhscj authors: blecha, frank; charley, bernard title: rationale for using immunopotentiators in domestic food animals date: - - journal: adv vet sci comp med doi: . /b - - - - . - sha: doc_id: cord_uid: mcfnhscj nan to losses due to death, economic losses caused by bovine respiratory disease include reduced growth performance and increased treatment costs. these losses emphasize the need for alternative or comple mentary therapeutic approaches, such as immunomodulators, that may be well suited for the multifactorial etiology involved in the disease. in economical terms, mastitis is the most devastating disease affect ing dairy cows. in the united states, losses attributed to mastitis ap proach $ billion each year; % of this economic loss is due to a reduced milk yield as a result of subclinical mastitis (national mastitis council, ) . similarly, a french epidemiological survey found that mastitis was by far the most frequent pathology affecting dairy cows (barnouin et al., ) . vaccination against bacteria that cause intramammary infections has been attempted as a means of decreasing mastitis. however, even in studies that have shown beneficial effects of immuni zation against mastitis, vaccination did not prevent new intramam mary infections (pankey et al., ) . antibiotic therapy is used in the control of mastitis. however, because staphylococcus aureus mastitis responds poorly to antibiotic therapy and because of the problem of antibiotic residues in milk, the effectiveness of antibiotic therapy in mastitis prevention and treatment is limited. these specific examples emphasize the need to continue to search for more effective ways to minimize the impact of disease on animal pro duction. augmentation of the animal's immune response with the in tent of increasing resistance to disease-causing organisms should de crease the economic loss due to disease in food animal production. immunomodulation may provide an effective means of enhancing the ability of domestic food animals to withstand disease. when one considers the possibility of enhancing an animal's immune response, a question that must be addressed is whether specific or nonspecific immunomodulation is desired or required. specific immuno modulation involves the potentiation of the host's immune system toward a unique, specific antigen. vaccination programs are perhaps the best example of producing specific immunity in domestic food animals. nonspecific immunomodulation generally is an attempt to heighten immunologic capabilities at a time when an animal may be exposed to one or several pathogens and/or be immunocompromised. both of these concepts will be discussed further. the distinction between adjuvants and specific immunomodulators is blurred and may be only a matter of semantics. classical and new adjuvants offer the capability of enhancing specific immunity and are discussed in great detail in chapter of this volume. however, some substances that are not generally thought of as adjuvants, such as the interleukins and interferons, also induce a state of specific immuno modulation. for example, peripheral blood mononuclear cells from cat tle injected with recombinant bovine interleukin- display enhanced cytolytic capabilities against bovine herpesvirus-infected target cells (reddy et al., a) . however, protection against a bovine herpesvirus challenge was observed only in animals that received a vaccination against the virus in conjunction with injections of interleukin- . thus, in this case both nonspecific and specific immunomodulation was produced in cattle that were administered interleukin- , but only specific immunomodulation resulted in protection against a viral chal lenge. theoretically, the capability of potentiating the host's immune re sponse at a time when it might be immature, compromised, or overcome with pathogens should enhance the animal's ability to resist disease. this is the rationale for nonspecifically augmenting an animal's im mune response. nonspecific immunomodulation has potential in at least different conditions: ( ) during the neonatal period when the immune system may not be fully developed; ( ) during periods of stressinduced immunosuppression; and ( ) during virus-or bacteria-induced immunosuppression. because of a very efficient placental barrier, pig, horse, and ruminant fetuses are generally very well protected from in utero antigenic stim uli. therefore, although fully immunocompetent at birth, domestic food animal newborns differ from other mammalian neonates in being im munologically "virgin" (kim, ; salmon, ) and the development of totally effective immune defenses requires to weeks. during this critical neonatal period the young animal is highly susceptible to mi crobial infections. postnatal development of immune functions has been most exten sively studied in the pig (sterzl and silverstein, ; kim, ) . most immune parameters that have been studied appeared to be very low at birth and reached adult values at about month of age. thus, the percentage of Τ and Β lymphocytes in peripheral blood, as estimated by Ε-rosettes and anti-ig immunofluorescence techniques, was shown to increase from to % at birth to adult values by days of age (reyero et al., ) . a similar age-related increase has been described for serum concentrations of the third component of complement (c ) in pigs (tyler et al., ) . because of the high incidence and economic impact of respiratory and intestinal infections in young domestic animals, it is important to review studies related to the postnatal development of the mucosaassociated immune system in the pig. at birth, the intestinal, nasal, and tracheobronchial mucosa are devoid of plasma cells. plasma cells first appear in the respiratory tract at - days of age and reach adult values at - weeks of age. this postnatal development was described for cells containing iga as well as igm and igg (bradley et al., ) (chu et al., ; pabst et al., ) . inside the lung, residing at the air-tissue interface and directly exposed to inhaled microorganisms or air pollutants, the alveolar mac rophage functions as the primary defense against respiratory infections (hocking and golde, ) . functional properties of alveolar macro phages, including their immunological and antiinfectious features, have been studied in domestic food animals (khadom et al., ; charley, ) . rothlein et al. ( ) have studied the postnatal devel opment of alveolar macrophages in minnesota miniature swine. these researchers showed that lavage fluids from the lungs of newborn piglets were devoid of macrophages. however, within to days after birth, macrophages gradually appear inside the lung airspaces and adult values are reached at weeks of age. furthermore, macrophages col lected from piglets less than week old showed immature function, i.e., lower phagocytic capacity and enzyme content than adult cells. the postnatal development of lung macrophages appears to depend upon nonspecific antigenic stimulation since germ-free piglets have a much lower number of alveolar macrophages than specific-pathogen-free pig lets (rothlein et al., ) . additionally, alveolar macrophages from young piglets have been shown to be more permissive to pseudorabies virus, yielding higher virus progeny titers, than cells from older animals (iglesias et al., ) . a last example of an immune defect occurring during the neonatal period is given by studies on porcine natural killer (nk) cells. natural killer cell activity in the peripheral blood of newborn pigs is much lower (often undetectable) than the activity of adult cells. this nk cell defect has been observed regardless of the target cell system used: hu man tumor cells (huh et al., ) , virus-infected cells (cepica and derbyshire, a; yang and schultz, ), or porcine tumor b-cells (onizuka et al., ) . of particular interest are the observations that postnatal development of nk cells activity, which requires - weeks in specific-pathogen-free miniature swine (huh et al., ) and in conventionally reared large-white pigs , is de layed in germ-free miniature piglets (huh et al., ) . these data imply that microbial flora play a role in the maturation process of nk cell activity in neonates. , a) . this observation has led to the hypothesis that a nk cell defect could in part explain the great suscep tibility of piglets to coronavirus-induced transmissible gastroenteritis. indeed, adoptive transfer of adult pig leukocytes established functional nk cell activity in recipient piglets and reduced their susceptibility to a tgev challenge (cepica and derbyshire, b) . the examples described above illustrate the existence of several different immune defects (see table i ) in neonatal domestic food animals. this lower functional immune status during the neonatal period could explain some of the neonates' susceptibility to infectious diseases, especially intestinal infections. thus, the potential exists to increase the neonates' immune functions by using immunomodulators. a few studies have been conducted exploring means of enhancing the young animals' immune functions. for example, newborn piglets' nk cell activity was shown to be responsive in vitro to interferon (charley et al., ) , and in vivo to poly i : c (lesnick and derbyshire, ) or bacterial extracts (kim, ) . additionally, isoprinosine has been shown to enhance the immunocompromised immune status of artifi cially reared neonatal pigs (hennessy et al., ) . in the following chapters several immunomodulating strategies will be reviewed and should help to define possible immunotherapeutic approaches to en hance young domestic food animals' resistance to disease. (loan, ; filion et al., ) . the idea that stressed animals are more susceptible to disease generally relies on the assumption that alterations in immunocompetence have occurred (table ii) . indeed, some researchers have suggested that changes in immune function may be a useful indicator of stress in domestic food animals (kelley, ; siegel, ) . over the last decade several review articles have been written on the topic of stress and immunity in farm animals (kelley, (kelley, , (kelley, , (kelley, , observation reference blecha and minocha ( ) ; albani-vangili, ; siegel, ; blecha, a; griffin, ). if stress-induced changes in host immunity predisposes animals to disease, then methods of modulating the immune response in stressed animals should increase disease resistance (blecha, b) . when one attempts to intervene in an animal's response to a stressor, several different approaches can be envisioned (fig. ) . perhaps the best means of reducing the impact of stress on animal health is by providing a less stressful environment. however, deciding which envi ronment or management condition is the least stressful is not a simple or easy task (curtis et al., ; mcglone and hellman, ) . thus, several environments and management conditions have been evalu ated for their influence on immune function blecha et al., blecha et al., , blecha et al., , mcglone and blecha, ; minton etal, ) and for their impact on the physiology of the animal (dantzer and mormede, ) . another approach has been investigated as a method of reducing the influence of stress on susceptibility to disease: blocking the physiologic response to the stressor. the association between stress, neuroendo- crine responses, and alterations in immune function or disease sus ceptibility has been recognized for several years (munck et al., ; kelley, ; griffin, ) . when increased concentrations of glucocorticoids have been associated with lower immune responses, administration of drugs that block the synthesis of corticosterone, such as metyrapone, resulted in an abrogation of the stress-induced immu nosuppression (blecha et al., ) . recently, adrenal blocking chemicals (metyrapone and dichlorodiphenyldichlorethane) have been shown to increase the resistance of stressed chickens to viral and respi ratory infections (gross, ) . finally, when stress-induced immuno suppression has occurred, neurohormones, such as melatonin (maestroni et al., ) , immunomodulating drugs (hennessy et al., ; blecha, b; komori et al., ) , and cytokines (conlon et al., ) have been used to "up-regulate" or restore the immune response. it is likely that a combination of the approaches indicated above will pro vide the best means of reducing stress-induced disease problems in domestic food animals. c. pathogen-induced immunosuppression animals exposed to infectious disease often show depressed immune function. this is the case for several parasitic, bacterial, and viral infections. pathogenic bacteria have been shown to affect immune re sponsiveness of infected animals. thus, pasteurella hemolytica or hae mophilus pleur^pneumoniae, which both cause acute pneumonia in cattle and pigs, have been reported to exert toxic effects on lung macro phages and to alter macrophage phagocytic functions (markham and wilkie, ; bendixen et al., ) . during bacteria-induced mastitis, suppressed responses in lymphocyte proliferation and neutrophil phagocytic functions have been reported (nonnecke and harp, ; reddy et al., b) . immunosuppression of the host is also a frequent consequence of viral infections. several examples of virus-related im munosuppression are well documented in domestic food animals (table iii) , including viral diseases of great economic importance such as infectious bovine rhinotracheitis (bovine herpesvirus type- ) and pseudorabies, which cause severe pneumonia and death in cattle and pigs, respectively. as a consequence of virus-induced alteration of im mune function, animals become very susceptible to secondary bacterial infections. the detrimental effects of these virus-bacteria synergistic interactions are of particular importance in the case of respiratory infections. thus, following an initial viral multiplication in the lung, pathogenic bacteria proliferate, inducing the development of more se- in the production of domestic food animals several situations exist where disease decreases production efficiency. some of these diseases are exacerbated by a lowered or compromised immune response of the host. if immunomodulators can be used to augment immune function at critical periods during the production of food animals, such as the neonatal period, and prior to or during exposure to stressors or patho genic organisms, then the economic loss caused by infectious disease should be reduced. vere and acute lung lesions (jakab, ; babiuk et al., ) . if pathogen-induced immunosuppression can be moderated by immunomodulating substances, then the prospects for domestic food animals to withstand disease should be increased. stimulation of defense mecha nisms, especially lung immune defenses, will likely require activation of local lymphoid cells such as alveolar macrophages (charley, ) . targeting of immunomodulators to the critical organs will require special delivery systems, such as encapsulation in liposomes (fogler et al., ) , which should be considered in the field of domestic food animal immunoenhancement. stress e immunita. obiettivi vet the relative frequencies and distribution of immunoglobulin-bearing cells in the intestinal mucosa of neonatal and weaned pigs and their significance in the development of secretory immunity bovine respiratory disease: pathogenesis and control by interferon viral-bacterial synergistic interaction in respiratory disease enquêt e éco-pathologique continue toxicity of haemophilus pleuropneumoniae for porcine lung macrophages, peripheral blood monocytes, and testicular cells viral infections in domestic animals as models for studies of viral immunology and pathogenesis stress et immunité chez l'animal immunomodulation: a means of disease prevention in stressed live stock cold stress reduces the acquisition of colostral immunoglobulin in piglets suppressed lymphocyte blastogenic responses and enhanced in vitro growth of infectious bovine rhinotracheitis virus in stressed feeder calves adrenal involvement in the expression of delayed-type hypersensitivity to dnfb in stressed mice weaning pigs at an early age decreases cellular immunity shipping suppresses lymphocyte blasto genic responses in angus and brahman x angus feeder calves immunologic reactions of pigs regrouped at or near weaning decreased mononuclear cell re sponse to mitogens in artificially reared neonatal pigs influence of isoprinosine on bovine herpesvirus type- infection in cattle the respiratory tract immune system in the pig. i. distribution of immunoglobulin-containing cells in the respiratory tract mucosa effect of respiratory infections caused by bovine herpesvirus- or parainfluenza- virus on bovine alveolar macrophage functions inhibition of t-lymphocyte mitogenic responses and effects on cell func tions by bovine herpesvirus antibody-dependent and spontaneous cellmediated cytotoxicity against transmissible gastroenteritis virus infected cells by lymphocytes from sows, fetuses and neonatal piglets the effect of adoptive transfer of mononuclear leukocytes from an adult donor on spontaneous cell-mediated cytotocity and resistance to transmissible gastroenteritis in neonatal piglets le macrophage alvéolaire de porc: revue bibliographique effects of immunopotentiating agents on alveolar macrophage proper ties modifications des réactions immunitaires au cours de la peste porcine classique interferon-induced enhancement of newborn pig natural killing (nk) activity lymphoid tissues of the small intestine of swine from birth to one month of age the treatment of induced immune deficiency with interleukin- effects of sow-crate design on health and performance of sows and piglets stress in farm animals: a need for réévaluation new directions in bovine veterinary practice the possible role of stress in the induction of pneumonic pasteurellosis influence of isoprinosine on lymphocyte function in virus-infected feeder pigs in situ activation of murine macrophages by liposomes containing lymphokines bovine herpesvirus- and parainfluenza- virus interactions: clinical and immunological responses in calves stress and immunity: a unifying concept effect of social stress on the occurrence of marek's disease in chickens effect of adrenal blocking chemicals on viral and respiratory infec tions of chickens the effect of social isolation on resistance to some infectious diseases genetic and environmental effects on the response of chickens to avian adenovirus group ii infection air temperature selection guides for growingfinishing swine based on performance and carcass composition critère s biochmique s e t hématologique s d u stres s e t leur s relation s avec le s mécanisme s d e défens e reel immumoglobulin g, a and m antibody response in sow-reared and artificially reared pigs isoprinosine and levamisole immunomodulation in artificially reared neonatal pigs the pulmonary alveolar macrophage natural killing and antibody-dependent cellular cytotoxicity in specific-pathogen-free miniature swine and germ-free piglets. ii. ontogenic development of nk and adcc interactions of pseudorabies virus with swine alveolar macrophages i: virus replication viral-bacterial interactions in pulmonary infection stress and immune function: a bibliographic review immunobiology of domestic animals as affected by hot and cold weather stress et immunité des animaux domestiques. point vét immunological consequences of changing environmental stimuli cross-talk between the immune and endocrine systems whole blood leukocyte vs separated mononuclear cell blastogenesis in calves: timedependent changes after shipping bovine alveolar macrophages: a review developmental immunity in the piglet the effects of ok- on porcine nk and Κ cell system rapid effects of adrenocorticotropic hormone (acth) and restraint stressor on porcine lymphocyte function alleviation of depressed immunity caused by restraint-stress, by the immunomodulator, lobenzarit disodium (disodium -chloro- , '-iminodibenzoate) replication of transmissible gastroenteritis coronavirus (tgev) in swine alveolar macrophages activation of natural killer cells in newborn piglets by interferon induction bovine respiratory disease: a symposium role of the pineal gland in immunity. iii. melatonin antagonizes the immunosuppressive effect of acute stress via an opiatergic mechanism interaction between pasteurella hemolytica and bovine alveolar macrophages: cytotoxic effect on macrophages and impaired phagocytosis the welfare of sows and piglets: an examination of behavioral, immunological and productive traits in four management systems local and general effects on behavior and performance of two-and seven-week-old castrated and uncastrated piglets partial inhibition of the humoral immune response of pigs after early postnatal immunization fluctua ting ambient temperatures for weaned pigs: effects on growth performance and immu nological and endocrinological functions effect of transportation on blood serum composition, disease incidence, and production traits in young calves. influence of the journey duration physiological functions of glucocorticoids in stress and their relation to pharmacological actions the effects of road transportation on peripheral blood lymphocyte subpopulations, lymphocyte blastogenesis and neutro phil function in calves current concepts of bovine mastitis experimentelle untersuchungen zur wirkung einer chronischen aerogenen schadgasbelastung des saugferkels mit ammoniak unterschiedlicher konzentrationen. ii. die reaktion zellularer und humoraler infectionsabwehrmechanismen nh -exponieter saugferkel unter den bedingungen einer experimentallen pasteurella-multocidainfektion mit und ohne thermo-motorische belasturg effects of staphylococcus aureus on bovine mononuclear leukocyte proliferation and viability: modulation by phagocytic leuko cytes nonspecific cell-mediated cytotoxicity of peripheral blood lymphocytes derived from suckling piglets postnatal development and lymphocyte production of jejunal and ileal peyer's patches in normal and gnotobiotic pigs evaluation of protein a and commercial bacterin as vaccines against staphylococcus aureus mastitis by experimental challenge bovine recombinant interleukin- augments immunity and resistance to bovine herpesvirus infection bovine recombinant granulocyte-macrophage colony-stimulating factor aug ments functions of neutrophils from mastitic cows heat-and cold-stress suppresses in vivo and in vitro cellular immune responses of chickens development of peripheral Β and Τ lymphocytes in piglets suppression of neutrophil and lymphocyte function induced by a vaccinal strain of bovine viral diarrhea virus with and without the administration of acth development of alveolar macrophages in specific pathogen-free and germ-free minnesota miniature swine effect s o f ambien t temperature s o n induction o f transmissibl e gastroenteriti s i n feede r pigs immunologica l response s a s indicator s o f stress . world's losse s i n youn g calve s afte r transportation developmental aspects of immunity age-related variations in serum concentrations of the third component of complement in swine congenital infections with nonarbotogaviruses the effect of different climatic environment on metabolism and its relation to time of haemophilus pleurop neumonias infection in pigs physiologic concentrations of cortisol suppress cell-mediated immune events in the domestic pig ontogeny of natural killer cell activity and antibody dependent cell mediated cytotoxicity in pigs key: cord- - hf vyhz authors: abass, ahmed o; kamel, nancy n; khalifa, walaa h; gouda, g f; el-manylawi, m a f; mehaisen, gamal m k; mashaly, magdi m title: propolis supplementation attenuates the negative effects of oxidative stress induced by paraquat injection on productive performance and immune function in turkey poults date: - - journal: poult sci doi: . /ps/pex sha: doc_id: cord_uid: hf vyhz paraquat (pq) is used as a herbicide in agriculture and causes oxidative and inflammatory damage to animal tissues. the current study was conducted to investigate the positive effects of dietary propolis (pr), as a potent naturally produced antioxidant, on growth performance and immune function of turkey poults exposed to oxidative stress induced by pq injection. native male turkey poults (n = , -d-old) were randomly assigned into groups: poults received a basal diet with a daily subcutaneous pq injection of mg/kg bw for consecutive days (pq group), an experimental diet containing g/kg pr with a daily subcutaneous pq injection for days (pr+pq group), or received the experimental pr diet with a daily subcutaneous injection of . ml sterile saline for days (pr group); while the control poults received a basal diet with a daily subcutaneous saline injection for consecutive days (c group). the productive performance in the pq group showed a significant (p < . ) reduction in the weight gain (wg) and feed intake (fi), and impaired feed conversion ratio (fcr). propolis supplementation in the pr+pq group significantly ameliorated the pq effects on wg and fcr. turkey poults of the pq and pr+pq groups had a significant augmentation in the blood malondialdehyde (mda), tumor necrosis factor-α (tnfα), and corticosterone levels, and in contrast, a significant reduction in the triiodothyronine (t( )), when compared to the c group. while propolis significantly reduced the mda and corticosterone, and increased the t( ) levels in the pr+pq group compared to the pq group. furthermore, the dietary pr supplementation significantly limited the pq-suppressive effects on cell- and humoral-mediated immunity and lymphocyte proliferation of turkey poults. in addition, propolis supplementation in the pr and pr+pq groups markedly reversed the pq-induced dna fragmentation and heat shock protein (hsp ) over-expression in blood cells. it can be concluded that pr could improve turkey immunity and performance, particularly under inflammation and oxidative stress induced by pq exposure. oxidative stress develops when the generation of reactive oxygen species (ros) in a system exceeds the system's ability to neutralize and eliminate them. the imbalance can result either from a lack of antioxidant capacity caused by disturbance in production, distribution, or by an over-abundance of ros from an environmental or behavioral stressor (boelsterli, ) . ros could be produced by several agents and stres-that induced-oxidative stress in broiler chickens leads to proteolysis and gluconeogenesis (lin et al., ) , several pathologies incidence (fellenberg and speisky, ) , dna damage (huang et al., ) , and depression of immune function and growth performance with a high mortality rate (kamel et al., ; mehaisen et al., ) . depending on the severity of the oxidative harm, the consequence of these modulations can vary from modifying cell function to cell death, which negatively affects poultry flocks. paraquat (pq; , -dimethyl- , -bipyridium dichloride) is widely used in agriculture as a non-selective contact herbicide with redox activity. pq is known to exert its toxic effects via oxidative stress mechanisms (ray et al., ) . the potential mechanism of pq toxicity is the cyclic single-electron redox reaction that depletes cellular nicotinamide adenine dinucleotide phosphate (nadph) and generates superoxide anion. superoxide ions may form hydrogen peroxide and hydroxyl radicals; the latter being an extremely potent oxidant that may damage nucleic acids, proteins, and polysaccharides (tukmechi et al., ) . moreover, trace amounts of pq can be detected in more than crops such as corn, tomatoes, olives, field beans, and fruits (prasad et al., ) . pq effects in rodent models showed that prolonged exposure leads to accumulation and persistent damage in the brain, lung, and liver tissues (ortiz et al., ) . a study on paraquat toxicity in turkeys reported that all injected birds were affected at the dose of . mg/kg bw while the lethal dose was about mg/kg and mg/kg by intravenous and intraperitoneal injection, respectively (smalley, ) . propolis (pr) is an adhesive resinous material made by honey bees and it contains a variety of chemical compounds such as poly-phenols (flavonoid aglycones, phenolic acids and their esters, phenolic aldehydes, alcohols, and ketones), terpenoids, steroids, amino acids, and inorganic compounds (newairy and abdou, ) . flavonoid and phenolic compounds in propolis have been appeared to be capable of scavenging free radicals and thereby defending lipids and other compounds from being oxidized or destroyed during oxidative damage (seven et al., ). in addition, propolis is thought to be responsible for many biological and pharmacological activities including anticancer, antiinflammatory, anti-bacterial, antifungal, antiviral, antioxidant, hepato-protective, and immuno-stimulating activities (haščík et al., ) . many previous reports indicated that the inclusion of propolis in the poultry diet has a positive effect on the humoral immunity of laying hens (cetin et al., ; freitas et al., ) , on the hatchability and performance of quail chicks (aygun et al., ) , and on the hemoglobin concentrations and eosinophil count of blue-fronted parrots (silva et al., ) . based on pq actions, it is a good and welldocumented agent to induce oxidative stress when there is a need to provide a better understanding of this type of stress on poultry production. on the other hand, much research is focused on the ability of propolis to improve production performance, enhance immune function, and inhibit inflammatory response, which is very critical for poultry industry. however, it is not clear whether propolis supplementation could also reverse the inflammatory status and the negative effects of oxidative stress induced by paraquat treatment. thus, the current study was designed to investigate the effects of propolis supplementation on controlling the oxidative stress induced by paraquat injection to turkeys. the oxidative stress was determined as the level of mda, which is an important and significant biomarker of oxidative stress, in the blood. in addition, the inflammation status and immune function were evaluated in turkey poults after propolis supplementation with or without paraquat injection. furthermore, dna fragmentation test and stressed-related protein expression of heat shock protein (hsp ) were analyzed in the blood. in addition, growth performance of turkey birds was obtained under propolis supplementation in order to test whether it has the ability to reverse the negative effects of oxidative stress induced by paraquat. one hundred and twenty, -d-old, male baladi turkey poults were used in this investigation. baladi turkey is an egyptian native breed that is characterized by low growth performance and high immune response. the turkey poults were reared in an opened-house with feed and water ad libitum. a basal diet was formulated according to the recommendations of the regional center for food and feed in egypt to meet the local needs and was used as a control diet in the present study. the composition and the calculation analysis of the basal diet are presented in table . propolis was mixed into the basal diet to produce experimental diet containing . % propolis ( g propolis/kg diet). the experimental protocols were approved and carried out according to the regulation and guidelines set by cairo university ethics committee for the care and use of experimental animals in education and scientific research (cu-iacuc). propolis was collected from an apiary located at the faculty of agriculture, cairo university (giza province, egypt). the collected propolis was kept in a clean, dark bottle at • c until use in the experiment. phenolic acids and flavonoid contents were analyzed in a propolis sample using high-performance liquid chromatography (hplc). hplc was achieved on an agilent infinity hlpc series (agilent, santa clara, ca) equipped with quaternary pump, zorbax eclipse plus c column mm × . mm internal diameters, μm particle (agilent), operated at • c. the separation was achieved using a ternary linear elution gradient with hlpc grade water . % h po (v/v), methanol and acetonitrile. the injected volume was μl and the variable wavelength detector (vwd) was set at nm (ivanauskas et al., ) . the free radical scavenging activity of propolis samples was measured according to methods described by oktay et al. ( ) . briefly, the propolis was added to a solution of . mm of , diphenyl- -picryl-hydrazil (dpph) in methanol at different concentrations ( to μg/ml). the mixtures were shaken vigorously and allowed to stand at room temperature for min. then, the absorbance of reactions was measured using an automatic scanning spectrophotometer at nm. the chemical characteristics of propolis used in this experiment are presented in table . at d of age, turkey poults were randomly allocated into one of experimental groups (three pens per group × poults in each pen). the first group of poults served as a control, received the control diet and a single dose of . ml sterile saline per day for consecutive days through subcutaneous route (c group). the second group received the control diet and a single subcutaneously injected of paraquat (sigma aldrich, st. louis, mo), mg/kg body weight, per day for consecutive days (pq group). the third group received the experimental diet containing propolis and a daily subcutaneous injection of paraquat for a pe-riod of days (pr+pq group). the fourth group received the experimental propolis diet and a daily subcutaneous injection of saline for a period of days (pr group). at d of age, blood samples were collected from the brachial vein of the birds in each treatment using heparinized syringes and the physiological parameters were assayed ( samples from each pen for each parameter; n = ), including the mda and tumor necrosis factor-α (tnfα) levels in the peripheral blood mononuclear cells (pbmcs) isolated from blood, as well as the corticosterone and triiodothyronine (t ) hormone concentration in the plasma. the immune function of turkey poults in each group was determined by analyzing total white blood cells (wbcs), heterophil to lymphocyte (h/l) ratio, toe web swelling in response to phytohemagglutinin-p (pha-p) injection as an indicator for the cell-mediated immunity, antibody response to sheep red blood cells (anti-srbcs ab) injection as an indicator of the humoral-mediated immunity, and the stimulation index of peripheral tlymphocyte proliferation. furthermore, the dna fragmentation and blotting expression of hsp were analyzed in pbmcs isolated from blood samples in each group. in addition, the productive performance was obtained for each treatment group as will be mentioned later. the initial and final body weights were recorded individually at the beginning and at the end of the experiment ( and d of age). the entire weight gain was determined for each group. average daily feed intake was measured for each treatment group. the feed conversion ratio was calculated for each group. at d of age, blood samples from different birds (n = ) were collected from each treatment. pbmcs were isolated using histopaque- (sigma aldrich, st. louis, mo) as previously described . the cells were washed twice using roswell park memorial institute (rpmi) medium (gibco tm , thermo fisher scientific, waltham, ma), then re-suspended with pbs (ph . ), and the numbers were adjusted to be cells/ml. a mlcell suspension was centrifuged at , × g for min at • c. the pellets were collected and stored at − • c until processing. the cells were re-suspended with ml pbs, kept on ice for sec, and then sonicated for min. the homogenates were centrifuged at , × g for min at • c and supernatants were collected to determine mda and tnfα levels. the level of mda in the supernatant was determined by the thiobarbituric acid reaction method using a commercial assay (nanjing jiancheng bioengineering institute, nanjing, china). the level of tnfα in the supernatant was measured using chicken elisa commercial diagnostic kit (cat# wac- , wkea med supplies corp, changchun, china). the standard curves and calculations were performed following the kits protocol for each analysis. plasma corticosterone and t hormone assay at d of age, blood samples from each group were collected (n = ), centrifuged at , × g for min at • c, and then plasma was separated and stored at − • c. plasma corticosterone and t concentrations were determined in duplicates using chicken elisa kits (cat# mbs for corticosterone and cat# mbs for t ; mybiosource, san diego, ca). the intra-and inter-assay coefficient of variations was < % and < %, respectively for corticosterone, and was < % for t . the dynamic range of the assay was . to and . to ng/ml for corticosterone and t , respectively. total wbc count total leukocytes were performed manually in samples per group as described by gehad et al. ( ) . briefly, μl of brilliant cresyl blue dye was mixed with μl whole blood samples, and the total leukocytes were counted using a hemocytometer. h/l ratio h/l ratio was determined manually according to zhang et al. ( ) . in brief, blood smears ( slides per group) were fixed and stained using hema- (cat# - , , fisher scientific, pittsburg, pa). the differential leukocyte counts were performed for a total of leukocytes in each slide using light microscope and the h/l ratios were then calculated. toe web swelling the cell-mediated immune response was assessed using the swelling of toe web that induced by intradermal mitogen injection of pha-p. at d of age, the left foot of six turkey poults from each group was injected with μg of pha-p dissolved in . ml of sterile pbs buffer into the toe web between the third and fourth digits. the thickness of toe web was measured before injection and h after injection. the toe web swelling response was expressed in mm as the difference between the thickness before and after injection (loa et al., ) . anti-srbcs ab. the humoral-mediated immune response was assessed by evaluating the antibody production against srbcs. six turkey poults from each group were injected intravenously with ml of % saline suspension of srbcs at d of age. one wk following the injection (d ), blood samples was collected and the antibody production against srbcs was determined by microhemagglutination technique (loa et al., ) . antibody values were expressed as log of the reciprocal of the highest dilution where visible agglutination was observed. peripheral t-lymphocyte proliferation the pbmcs layer were carefully isolated from heparinized blood samples obtained from each group according to the method described by mehaisen et al. ( ) . after washing in rpmi culture medium, the viable lymphocytes were detected using trypan blue dye and plated in triplicate wells ( -well plate) at × cells per well. then, μl of concanavalin-a (sigma aldrich) at μg/ml was added to selected wells to induce the proliferation of t lymphocyte; while control wells received μl of rpmi- medium. after cells' incubation ( • c and % co for h), μl of mg/ml -[ , -dimethylthiazol]- , -diphenyltetrazolium bromide (sigma aldrich) was added to each well and incubated for another h. subsequently, μl of % sodium dodecyl sulfate dissolved in . m hcl solution was added to each well, then the plates were read using an automated elisa reader (model microplate reader, bio-rad laboratories, inc., hercules, ca) at nm. the stimulation index for t-lymphocyte proliferation was calculated as the optical density ratio of experimental group to blank control. the pbmcs were collected from blood samples ( samples for each group) as mentioned previously. the dna isolation was performed using the cells/tissue genomic dna extraction kit (cat# gk , generay biotech co., ltd., shanghai, china), according to the manufacturer's instructions. dna fragmentation was analyzed by % agarose gel (sigma-aldrich) electrophoresis for h at v. visualization of dna was analyzed by ethidium bromide fluorescence using a uvp ec imaging system (uvp inc., upland, ca) with visionworksls image acquisition and analysis software (version . . ; uvp inc., upland, ca). the expression of hsp in the collected pbmcs ( samples per group) was analyzed by western blot technique. the total protein ( μg) was loaded and separated on % polyacrylamide gel containing sodium dodecyl sulphate (sds-page). separated proteins were then transferred to poly-vinylidene difluoride membranes using a tank transfer for h at ma in trisglycine buffer containing % methanol. membranes were blocked with % skim milk for h and incubated overnight at • c with diluted primary anti-rabbit igg polyclonal antibody against hsp ( : , ; cell signaling technology, inc., danvers, ma), followed by a horse radish peroxidase conjugated secondary antibody against rabbit igg ( : , ; santa cruz biotechnology, inc., dallas, texas). to verify equal loading of samples, the membrane was incubated with monoclonal β-actin antibody ( : , ; santa cruz biotechnology, inc.), followed by a horse radish peroxidase conjugated goat anti-mouse igg ( : , ; santa cruz biotechnology, inc.). hsp was detected using the ecl chemiluminescence kit (ge healthcare life sciences, amersham place, little chalfont, buckinghamshire, uk). . ± . a . ± . b . ± . c a-d means within the same row with different letters are significantly different (p < . ). treatment groups: c, control group; pq, the group received a basal diet and a consecutive day injection of paraquat; pq+pr, the group received a consecutive day injection of paraquat and a diet supplemented with propolis; and pr, the group received a basal diet supplemented with propolis. treatment groups (n = number of birds per group): c, control group; pq, the group received a basal diet and a consecutive day injection of paraquat; pq+pr, the group received a consecutive day injection of paraquat and a diet supplemented with propolis; and pr, the group received a basal diet supplemented with propolis. a general linear model was performed using sas software (sas institute inc., ) to determine statistical differences between treatment groups (c, pq, pr+pq, and pr) for the productive traits, physiological parameters, and immunological parameters. significant treatment effects were detected by duncan's multiple range tests. results are expressed as lsm ± se and the significance level was set at p < . . the results reveal that turkey poults in the pq group had significantly (p < . ) reduced weight gain and feed intake compared to turkey poults in the c group (table ). in addition, the feed conversion ratio also in the pq group was impaired significantly (p < . ) compared to the c group. in contrast, the dietary propolis supplementation alleviated these reductions in weight gain and feed intake in the pr+pq group when compared to the pr group. furthermore, administration of propolis to the diets of the pr group significantly (p < . ) enhanced the weight gain compared to the c group. even though, the feed conversion ratio tended to be improved when propolis was supplemented to the diets of both pr+pq and pr groups; the differences were not significant (p > . ) when compared to the c group (table ) . the change in mda and tnfα levels in pbmcs as well as the plasma corticosterone and t concentra-tions after paraquat injection with or without dietary propolis supplementation in turkey poults are shown in table . compared with the control group, pq injection significantly (p < . ) increased the mda, tnfα, and the plasma corticosterone levels in both pq and pr+pq groups. however, adding propolis to the basal diet minimized (p < . ) the mda level and corticosterone concentration in the pr+pq group compared to the pq group. in contrast, the plasma t concentration was significantly (p < . ) lower in the pq group and higher in the pr group when compared to the c group. moreover, dietary propolis supplementation successfully alleviated the negative effect of pq injection and significantly (p < . ) raised the value of plasma t concentration in the pr+pq group, however, it remained lower than that of the c group (table ) . the effects of dietary propolis and paraquat injection on the immune function are presented in table . total wbcs were significantly (p < . ) lower in the pq group than in the c group. meanwhile, dietary propolis supplementation significantly (p < . ) increased the total wbcs in the pr group compared to the c group. not only that, but it also reversed the negative effect of pq injection by significantly (p < . ) raising the total wbcs in the pr+pq group as compared to the pq group. furthermore, the h/l ratio was significantly (p < . ) increased in response to oxidative stress induced by paraquat injection in the pq group compared to the c group. nevertheless, propolis supplementation significantly (p < . ) limited the increasing rate of h/l ratio due to pq injection in the pr+pq group compared to the c group (table ). in addition, oxidative stress induced by paraquat injection treatment groups (n = number of birds per group): c, control group; pq, the group received a basal diet and a consecutive day injection of paraquat; pq+pr, the group received a consecutive day injection of paraquat and a diet supplemented with propolis; and pr, the group received a basal diet supplemented with propolis. significantly (p < . ) reduced the toe web swelling in response to pha injection, antibody titers to srbcs, and t-lymphocyte proliferation in the pq group poults compared to the c group poults (table ). in contrast, adding propolis to the basal diet significantly (p < . ) alleviated the negative effect of paraquat injection on the previous immune parameters in the pr+pq group compared to the c group. moreover, supplementing the basal diet with propolis in the pr group significantly (p < . ) enhanced the t-lymphocyte proliferation compared to the c group (table ) . dna fragmentation test showed appearance of short dna fragments in response to pq injection ( figure ) . meanwhile, the dietary propolis supplementation to the turkey poults reduced the dna fragmentation in either the pr+pq or the pr group to a normal level similar to the control group. furthermore, hsp blotting level was influenced by the pq injection and dietary propolis supplementation (figure ). an overexpression of hsp was observed in the turkey poults of the pq group compared to the c group. however, data of hsp expression in both the pr and pr+pq groups show that propolis supplementation to the diets of turkey poults normalized the hsp expression to similar levels of the c group. paraquat is widely used as an herbicide in agriculture and may cause chronic health problems to animals and humans if it is absorbed through the skin or gastrointestinal and respiratory tracts (ortiz et al., ) . the deleterious effects of pq are mainly attributed to the extreme redox activity that, in turn, induces extensive damage for several organs and tissues, leading to high mortality rates in human (lin et al., ; kang et al., ) and animal populations (ray et al., ; tukmechi et al., ) . on the contrary, propolis, due to its antioxidation and anti-inflammation properties, has been recently used in poultry feeds as an alternative and practical way to alleviate deleterious effects of rearing under abnormal and stress conditions (seven et al., (seven et al., , . so far, the research regarding pq and pr effects on growth performance and immune function in animal science is very limited and the available discussion remains insufficient to understand such effects. in the present study, it was found that pq significantly suppressed weight gain and feed intake, and impaired feed conversion ratio by %, %, and %, respectively, compared to the control poults. a similar low growth performance was obtained by feeding paraquat to fish (babatunde and oladimeji, ) or rats (kimura et al., ) . the low performance after paraquat treatment is attributed to the oxidative stress that may have been due to active oxygen species formed by the action of paraquat. previous reports attributed the low performance in oxidative stressed-broilers to the reduction in associated metabolic and endocrine responses (lin et al., ) or the down-regulation of all sugar, peptide, and amino acid transporter genes in the small intestine (ebrahimi et al., ) . on the other hand, dietary propolis ameliorated the suppression of weight gain, feed intake, and feed conversion in turkey poults that were exposed to pq injection. furthermore, administration of propolis to the diets of the turkey poults without pq stress enhanced the weight gain by % compared to the control poults. this enhancement may probably be due to the ability of propolis to improve nutrients digestibility and absorption as a result of improving saccharase, amylase, and phosphatase activities (mutsaers et al., ) . additionally, shalmany and shivazad ( ) reported that the increase in feed intake could be linked to the palatable substances in propolis like resin, wax, honey, and vanillin. furthermore, kačániová et al. ( ) found that flavonoids in propolis have antibacterial activity and prevent the ability of pathogenic bacteria to attach to the intestinal epithelium (parkar et al., ) ; thus improving intestinal health, and consequently, enhance digestion and nutrient absorption (denli et al., ) . mda is one of the final products of lipid peroxidation and could be used as a direct indicator of oxidative stress induced by potential oxidants like paraquat in the present study. the significant increase in mda levels upon treatment with pq in turkey poults indicates the oxidative damages occurred to pbmcs. these results are consistent with the finding that various concentrations of pq increase the cellular peroxidation . when propolis was supplemented to the diets of turkey poults, a reduction in mda level was obtained in the pr+pq group compared to the pq group. previous studies reported that the supplemental antioxidants extracted from propolis, such as flavonoids and caffeic acid phenethyl ester, block ros production and protect cell membranes against oxidants (seven et al., ) . it was also suggested that polyphenols of propolis can reduce the negative effects of oxidative stress either by chelation of iron or by free radical trapping (thirugnanasampandan et al., ) . it was reported that tnfα is one of the cytokines involved in the early inflammatory phase of pq poisoning (amirshahrokhi, ) through the ros pathway. pq-induced ros accumulation could promote the production of tnfα to trigger the tissue injury; and reversely, increased tnfα concentration could promote further ros generation (edwards et al., ) . results of the present study demonstrate that tnfα concentration in the pbmc's was higher in the pq and pr+pq groups compared to the c group. the positive effect of propolis was not statistically significant in this study, however, it is well documented that propolis supplementation may attenuate the adverse effects of environmental stress and stress induced tissue damage by modulating the levels of cytokines such as tnfα (fitzpatrick et al., ; hu et al., ) . in addition to the increase in the tnfα concentration in the pbmc's, pq injection also induced a significant high plasma corticosterone concentration. this high corticosterone levels probably decreased feed intake and reduced intestinal absorptive surface area as reported by hu et al. ( ) . furthermore, the release of corticosterone and inflammatory cytokines exerted catabolic effects on proteins and lipids (siegel, ) . these findings may be responsible for the lower body weight gain in the pq poults as previously reported in the present study. in addition, the thyrotrophic [triiodothyronine (t ) and thyroxine (t )] axis is considered to be prerequisites for normal growth and development (decuypere et al., ) . it is shown in the current study that turkey poults of pq group had significant reduction in the plasma t concentration compared to the c group. steenland et al. ( ) reported an increased hypothyroidism in the people intoxicated with paraquat herbicide and found detectable levels of paraquat in their thyroid glands. goldner et al. ( ) observed thyroid adenomas in rats exposed to paraquat and suggested that the thyroid could be susceptible to the effects of paraquat. in contrast, the plasma t was significantly higher in the pr group than in the c group. the positive effects of propolis on t /t ratio was previously reported in broilers reared under heat stress condition (amen et al., ) , reflecting improvement in thyroid hormones secretions and higher conversion rate to the active form of thyroid hormone which is responsible for metabolism. the current study also demonstrates that dietary propolis supplementation significantly increased plasma t concentration in the pr+pq group compared to pq group which, consequently, alleviated the negative effect of paraquat injection on the growth performance of turkey poults. the immunological data obtained in the current work show that pq injection impaired the overall immune function of turkey poults. in pq group, the h/l ratio was increased, while the total wbcs, the toe web swelling in response to pha injection, antibody titers to srbcs, and t-lymphocyte proliferation were significantly decreased compared to the c group. these results are consistent with the data presented by riahi et al. ( ) who concluded that paraquat administration at high dose (intra-peritoneal injection of mg/kg bw for d) in mice has an inhibitory effect on the cell-mediated and humoral immunity; including higher neutrophil cells number, and lower hemagglutination antibody titer and lymphocyte proliferation. the increase in h/l ratio in the pq group may be due to the inflammation (dinis-oliveira et al., ) , which was represented by corticosterone and tnfα elevation in the same group. in a recent study, mehaisen et al. ( ) reported that corticosterone treatment has immunosuppressive effect in broiler chickens. in addition, free radicals are known to attack unsaturated fatty acid side chains of phospholipids, causing a substantial decrease of the membrane integrity and fluidity of immune cells (smith and heath, ) . it is possible that free oxygen radicals generated by paraquat could inhibit t cell proliferation via membrane lipids peroxidation (riahi et al., ) . in contrast, dietary propolis supplementation enhanced the total wbcs and t-lymphocyte proliferation in the pr group compared to the c group. not only that, but it also reversed the negative effect of paraquat injection on the immune function in the pr+pq group. these positive effects of pr and its ability to alleviate the pq effects on immunity may be due to the stimulation of intracellular antioxidants, which are important in lymphocyte activation and proliferation (annunziata and iorio, ) . paraquat was found to affect the animal cell through oxidative damage in cellular macromolecules including dna (lascano et al., ) . in the present study, the dna fragmentation test showed an appearance of short dna fragments in response to pq injection. the paraquat toxicity may be due to the excessive release of ros which, in turn, initiate lipid peroxidation, particularly in the polyunsaturated lipid compound of cell membranes (hayes and laws, ) . furthermore, the paraquat toxicity involves depletion of cellular reduction agent of nadph which is used as a cofactor for lipid and nucleic acid synthesis (kelner and bagnell, ) . these events induced by pq-oxidative stress may increase the dna damage at the cellular level (ribas et al., ) , and consequently, the dna fragmentation occurred in the pbmcs lead to the impaired immune function of the turkey poults that were injected with pq in the current study. on the other hand, hsp , which is classified as a constitutive protein synthesized to protect cells from stress (al-aqil et al., ) , was also analyzed in the pbmcs of turkey poults in the present study. the hsp was over blotted in the pq group compared with the c group. in accordance with our results, previous reports demonstrated higher expressions of hsp in rats (crum et al., ) and mice (nakanishi and yasumoto, ) that received pq compound in comparison with the controls. these findings show that pq administration appeared to activate oxidative degradation in the cell, and consequently, increased the level of oxidized proteins, which stimulate self-defense mechanism of the cell to increase the level of hsp . the dietary propolis supplementation to the turkey poults reduced the dna fragmentation and the over expression of hsp in the pr+pq and the pr groups to normal levels similar to the c group. phenolic acids and flavonoids are known as the basic compounds of propolis according to its quality and type (kurek-górecka et al., ) , and these compounds have a powerful antioxidative activity (gülçin, ) . it was demonstrated that such compounds inhibit the activity of participated enzymes in the ros creation such as camp phosphodiesterase, protein kinase c, ascorbic acid oxidase, lipoxygenase and na + /k + atpase (pietta, ) . moreover, the antioxidant compounds of propolis decrease the activity of xanthine oxidase, an oxidase of nadph, which is responsible for the appearance of superoxide anion radical (harborne and williams, ) . in addition, propolis could activate the cu/znsuperoxide dismutase, one of the most important antioxidant enzymes (de sá et al., ) . these antioxidative characteristics of propolis may participate in the mechanism of inhibiting the dna damage and hsp over-expression in pbmcs of turkey treated with pq, and then finally these birds can express a normal immune function. in conclusion, the pq injection in turkey poults induces an oxidative stress status and impairs the growth performance and immune function in treated poults. the negative effects of pq could be justified by the high levels of mda and tnfα in pbmc's, and the high corticosterone with low t concentrations in the plasma. furthermore, the dna fragmentation and hsp expression in the pbmcs explain the malfunction of these cells and the low immunity in the pq-treated poults. in contrast, the dietary propolis supplementation can alleviate the negative effects of pq-induced oxidative stress on the productive, physiological, and immunological parameters of turkey poults. the positive effects of antioxidative compounds in the propolis can decrease the mda, tnfα, corticosterone, dna fragments, and hsp levels. therefore, the addition of propolis at a rate of g/kg to the diet of turkey poults could be recommended as a potential nutritional strategy in order to improve their immunity and performance, especially under oxidative stress conditions by herbicides like paraquat. this study was funded by the general scientific research department at cairo university (gsrd-cu) under activities carried out by the project of rapid climate change in poultry cellular and molecular physiology (rcc-pcmp). ahmed o. abass (associate professor of poultry physiology, cairo university) was the principal investigator and research team leader of the project. the authors would like to thank abdel-rahman m. m. atta (professor of poultry immunology, cairo university) for his technical support during this study. the authors are very grateful to all the personnel from the poultry biotechnology lab and members of poultry services center at faculty of agriculture, cairo university, for their assistance in sample preparation and monitoring of birds throughout the experimental period. the effects of the hot, humid tropical climate and early age feed restriction on stress and fear responses, and performance in broiler chickens the effect of brazilian propolis on serum thyroid hormones in broilers reared under chronic heat stress. page p (abstract) in international poultry scientific forum anti-inflammatory effect of thalidomide in paraquat-induced pulmonary injury in mice the levels of glutathione and hemoglobin in sheep erythrocytes as a function of age effects of propolis on eggshell microbial activity, hatchability, and chick performance in japanese quail (coturnix coturnix japonica) eggs effects of paraquat on the growth and behaviour of oreochromis niloticus mechanistic toxicology: the molecular basis of how chemicals disrupt biological targets effects of diets containing different concentrations of propolis on hematological and immunological variables in laying hens neuroendocrine profile in a rat model of psychosocial stress: relation to oxidative stress heat shock protein responses to aging and proteotoxicity in the olfactory bulb effects of glucocorticoids on circulating concentrations of thyroxine (t ) and triiodothyronine (t ) and on peripheral monodeiodination in pre-and post-hatching chickens effect of dietary addition of turkish propolis on the growth performance, carcass characteristics and serum variables of quail (coturnix coturnix japonica). asian-australasian paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment effect of dietary lead on intestinal nutrient transporters mrna expression in broiler chickens antioxidants: their effects on broiler oxidative stress and its meat oxidative stability caffeic acid phenethyl ester, an inhibitor of nuclear factor-kappab, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats the effects of propolis on antibody production by laying hens the role of light program and melatonin on alleviation of inflammation induced by lipopolysaccharide injection in broiler chickens antioxidant activity of caffeic acid ( , -dihydroxycinnamic acid) upsides and downsides of reactive oxygen species for cancer: the roles of reactive oxygen species in tumorigenesis, prevention, and therapy advances in flavonoid research since the influence of propolis as supplement diet on broiler meat growth performance, carcass body weight, chemical composition and lipid oxidation stability handbook of pesticide toxicology biological markers of oxidative stress: applications to cardiovascular research and practice effect of corticosterone administration on small intestinal weight and expression of small intestinal nutrient transporter mrna of broiler chickens effects of ethanol and water extracts of propolis (bee glue) on acute inflammatory animal models heat stress impairs mitochondria functions and induces oxidative injury in broiler chickens evaluation of phenolic acids and phenylpropanoids in the crude drugs in vitro and in vivo antimicrobial activity of propolis on the microbiota from gastrointestinal tract of chickens depression of leukocyte protein synthesis, immune function and growth performance induced by high environmental temperature in broiler chickens absolute lymphocyte count as a predictor of mortality in emergency department patients with paraquat poisoning paraquat resistance associated with reduced nadph reductase in an energy-dependent paraquat-accumulating cell line protective effects of dietary nasunin on paraquat-induced oxidative stress in rats structure and antioxidant activity of polyphenols derived from propolis paraquat: an oxidative stress inducer. pages - in herbicides -properties, synthesis and control of weeds.hasaneen, mohammed nagib oxidative stress induced by corticosterone administration in broiler chickens (gallus gallus domesticus): . chronic exposure repeated pulse of methylprednisolone and cyclophosphamide with continuous dexamethasone therapy for patients with severe paraquat poisoning humoral and cellular immune responses in turkey poults infected with turkey coronavirus comprehensive growth performance, immune function, plasma biochemistry, gene expressions and cell death morphology responses to a daily corticosterone injection course in broiler chickens (r van den bos bee products (properties, processing and marketing) (marieke mutsaers induction after administering paraquat of heme oxygenase- and heat shock protein in the liver of senescence-accelerated mice effect of propolis consumption on hepatotoxicity and brain damage in male rats exposed to chlorpyrifos determination of in vitro antioxidant activity of fennel (foeniculum vulgare) seed extracts effects of antioxidant n-acetylcysteine against paraquat-induced oxidative stress in vital tissues of mice the potential influence of fruit polyphenols on colonic microflora and human gut health flavonoids as antioxidants toxicokinetics and toxicodynamics of paraquat accumulation in mouse brain effects of paraquat on antioxidant system in rats immunotoxicity of paraquat after subacute exposure to mice herbicide-induced dna damage in human lymphocytes evaluated by the single-cell gel electrophoresis (scge) assay. mutat brazilian propolis protects saccharomyces cerevisiae cells against oxidative stress the effects of propolis on biochemical parameters and activity of antioxidant enzymes in broilers exposed to lead-induced oxidative stress effects of propolis on selected blood indicators and antioxidant enzyme activities in broilers under heat stress the effect of diet propolis supplementation on ross broiler chicks performance action of brazilian propolis on hematological and serum biochemical parameters of bluefronted amazons (amazona aestiva, linnaeus, ) in captivity toxicity and hazard of the herbicide, paraquat, in turkeys paraquat lung: a reappraisal thyroid hormones and cytogenetic outcomes in backpack sprayers using ethylenebis(dithiocarbamate) (ebdc) fungicides in mexico analysis of chemical composition and bioactive property evaluation of indian propolis effect of acute and chronic toxicity of paraquat on immune system and growth performance in rainbow trout, oncorhynchus mykiss evaluation of models using corticosterone and adrenocorticotropin to induce conditions mimicking physiological stress in commercial broilers effects of corticosterone and dietary energy on immune function of broiler chickens antioxidant attenuation of ros-involved cytotoxicity induced by paraquat on hl- cells. health (irvine. calif) transport stress in broilers: i. blood metabolism, glycolytic potential, and meat quality key: cord- -ub p ngr authors: mollenhauer, hilton h.; james morré, d.; rowe, loyd d. title: alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity date: - - journal: nan doi: . / - ( ) -z sha: doc_id: cord_uid: ub p ngr monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. the outer surface of the ionophore-ion comples is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. the net effect for monensin is a trans-membrane exchange of sodium ions for protons. however, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself. one apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. this is accompanied by a disruption of trans golgi apparatus cisternae and of lysosome and acidic endosome function. at the same time, golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions. monensin effects on golgi apparatus are observed in cells from a wide range of plant and animal species. the action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. the effects of monensin are quite rapid in both animal and plant cells; i.e., changes in golgi apparatus may be observed after only – min of exposure. it is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of h(+) and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs. in the golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the golgi apparatus. in endocytosis, monensin does not prevent internalization. however, intracellular degradation of internalized ligands may be prevented. it is becoming clear that endocytosis involves both acidic and non-acidic compartments and that monensin inhibits those processes that normally occur in acidic compartments. thus, monensin, which is capable of collapsing na(+) and h(+) gradients, has gained wide-spread acceptance as a tool for studying golgi apparatus function and for localizing and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. among its advantages are the low concentrations at which inhibitions are produced ( . – . μm), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or atp levels) and a reversible action. because the affinity of monensin for na(+) is ten times that for k(+), its nearest competitor, monensin mediates primarily a na(+)-h(+) exchange. monensin has little tendency to bind calcium. not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed in cattle. the mechanisms by which monensin interact with coccidia and rumen microflora to achieved these benefits are reasonably well documented. however, the interactions between monensin and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensin poisoning are well known. equine species are particularly susceptible to monensin poisoning, and a common effect of monensin poisoning is vacuolization and/or swelling of mitochondria in striated muscle. other pathological injuries to striated muscle, spleen, lung, liver and kidney also have been noted. a consistent observation is cardiac myocyte degeneration as well as vacuolization. differences in cellular response resulting from exposure to monensin (i.e., golgi apparatus swelling in cultured cells, isolated tissues, and plants vs.mitochondrial swelling in animals fed monensin) suggest that myocardial damage is due either to a monensin metabolite or is a secondary response to some other derivation. however, as pointed out by bergen and bates [ ], the underlying mode of action of ionophores is on transmembrane ion fluxes which dissipate cation and proton gradients. consequently, some or all of the observed monensin effects in vivo in animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes. monensm, a na ÷ ionophore capable of collapsing na ÷ and h + gradients, has gamed wide-spread acceptance as a biochemical and biologacal investigative tool to study golgi apparatus function and to localize and identify the molecular pathways of subcellular vesicular traffic. among its advantages are the low concentrations at which lntubltions are produced ( . - . #m), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or atp levels), and a reversible action [ ] . the purpose of this review is to examine the mechanism of action and specificity of monensln in na+/h ÷ exchange and to attempt to reconcile tlus to the large body of structural and biochemical information on monensin toxicity derived from animal studies in , pressman and co-workers [ ] reported a class of anubiotics that induced alkali ion permeability in mitochondna and other membranous systems. these antibiotics functioned as lonophores (ion-carriers) to carry ions across hpid barriers as complexes soluble in the hpid phase of the membranes. the potential use of tonophores as probes of biological function, or as potential therapeutic agents, was recognized very early [ ] [ ] [ ] [ ] , but major economic importance was not forthcoming until the discovery of monensin in and the recognlnon of tts potential in the poultry mdustrty as a coccxdtostat [ ] subsequently, ~t was discovered that tonophores also could improve feed converston in rumtnants such as cattle [ , ] , thus adding further to their commercial value of the more than lonophores that have been reported [ ] , three, monensln, lasalocld and sahnomysin, have widespread commercial use of those licensed, monensin is probably used most widely. several monensins have been tdentified [ , ] _ monenstn a, and specfftcally the sodium salt of monensin a (hereafter simply designated as monensm) ts derived from streptomvces cmnamonensts [ ] , and a crude mycehal preparation (rumensm') containing about _ % monensin ts used commercially. the on spectfictty of monensm xs ag > na >> k > rb > cs > li > ca [ , ] with approximately a -fold selectlvry for sodtum over potassium [ ] and little tendency to bind calcmm [ ] i! . one of the original interests in ionophores was their percewed potential for directly modifying intracellular iomc gradients, pamcularly ca +, winch would lead, hopefully, to the development of useful pharmacologic agents or, alternatively, provide a tool for studying cellular functtons medmted by changes m ca + [ , ] . of parttcular interest were divalent xonophores such as x- a (normally considered a ca + ionophore -note, however, x- a complexes na ÷ and k + almost as well as ca +) winch have been shown to mduce contractaon of aortic strips and increase the rate of contractility of tsolated perfused rabbit heart [ , ] , and release ca + from energy-loaded vesicles derived from the sarcoplasrmc renculum of muscle [ ] [ ] [ ] x- a may also mcrease blood flow through coronary artenes and mcrease cardiac output [ ] . the emphasis on heart physiology stemmed from this organ's strong dependence on calcmm for proper functioning [ ] . indeed, subsequent studies of x- a used as a feed additive for poultry and cattle has shown that the heart ~s a primary target for lonophore toxicity [ ] . x- a affects many other cellular functions such as release of b~olog~cally active agents and the induction of sperm acrosome reactions of several species [ , ] . it was soon realized, however, that many of the ionotroplc effects of the divalent ionophores could be duplicated with even greater efficiency by monovalent ionophores such as monensm which complexes na + but almost no ca ÷ tins response apparently occurs because the movement of na ÷ into a cellular compartment by monensin fac~htates the entry of ca + by a na+-out/ca +-ln exchange [ , , , ] . thus, a ca ÷ shaft ts stall the pnmary factor medmtlng cellular responses although other factors may also play significant roles m monensm physiology for example, many tono-phores, either directly or as a result ol the lomc imbalance. may transport, promote uptake, or release effector substances such as serotonm, lustamme, prostaglandm and catacholamlne which, m turn. have profound effects on cellular function [ . ] slmdarly. monensm. through alteration of the ph of mtracellular compartments may lnhlbr the release and/or transport of numerous agents and. in so doing, perturbate cellular function in cardiac tissues, both posmve and negative motroplsm has been observed sequentially (the variable factor being time) m tissues following exposure to monensin [ ] concentrahon of monensln may. also. cause similar posttlve/neganve ,notroptc responses [ , . finally. some monensln mgested b}¢ an animal is metabolized to other tonophores, the properties of which are largely unknown thus, ionophores, in spite of many common characteristics, differ indtvtdually in their effects on cell~ moreover. cells may respond to both direct tonophore interaction as well as secondary effects that develop from the initial ionophore reactions. the latter is partlcularly likely in the whole antmal where metabolites with unknown properties are produced from lonophore breakdown and where changes m the products of one organ can affect the function of other organs_ monensin is an open chain molecule that is capable of ton complexation through a cyclic form stabilized by hydrogen bonding between the carboxyl and hydroxyl groups charge transfer bonding wltinn the cavity formed is responsible for on binding (fig ) _ because the affinity of monensln for na + is -ttmes that for k+. tts nearest competitor in biological systems. bergan and bates [ ] ) the aniomc form of the lonophore is stabilized by the polar environment characteristic to the surface of a membrane_ the ionophore is capable of ion pamng with a metal cation either at the terrmnal carboxyhc acid moiety or at other internal sites the binding of a cation initiates the formation of a hpophahc, cyclic catlon-lonophore complex that can diffuse through the interior of the blmolecular membrane structure_ after traversing the membrane, the complex is again subjected to a polar environment where the electrostatic forces that had stablhzed the complex are no longer greater than the unfavorable gibbs free energy change of cyclzzation the ionophore then releases its enclosed cation and reverts to the low energy acyclic conformation monensin, like other carboxyhc lonophores, binds metal ions through liganding s~tes such that the ions become centrally oriented ( fig. ) and masked from the extraceuular environment [ , , ] . the outer surface of the ionophore-lon complex is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes m nonpolar solvents [ , , ] in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse through the membranes from one aqueous membrane interface to the other [ , ] . once the ion traverses the membrane as a monensin-lon complex, the ion is released, and the monensin molecule picks up a proton to form an undissociated molecule which then retraverses the membrane to release the proton to the outside of the cell, vesicle, organelle, or other subcellular compartment [ ] (fig. ) . thus, the net effect for monensin is a trans-membrane exchange of monovalent ions for protons. the on transfer rates may be very high and can approach, or even exceed, normal enzyme diffusion rates [ ] , although the actual rate may be markedly altered depending on factors such as the concentration of k ÷ m the external medium and the type and concentration of permeant ion that accompariles the accumulated k + [ ] . however, effects of lonophores on cation transport and their distribution among different membrane-bounded compartments within the cell, will vary depending upon the physical and chemical properties of the different membranes. membrane fluidity, thickness, curvature, charge and orientation of polar head groups of phosphohpids, cholesterol content, and protein content, all influence solubihty, penetration, and expression of the lonophore [ ] . moreover, in asymmetric membranes (i.e., biological membranes), ionophores generally exhibit asymmetric transport properties [ ] . for example, kovac et al [ ] showed that both vahnomycm and nigericin (an lonophore similar to monensin) crossed the plasma membrane of saccharomyces cereotslae at a rather low rate but then were preferentially located, and active as ~onophores, m the tuner mltochondrlal membrane. thus, the physiological effects of monensln will depend on the membrane composition and functional characteristics of the different compartments involved. although mechanisms for on transfer through a blmolecular leaflet (membrane) have been proposed, questions still remain as to how this action is related to known effects of monensin on cell function and what relationships these may have, m turn, on blochermcal mechanisms leading to ammal toxaclty. while any consideration of ~onophore action must focus on the mechanism of lonophore interaction with biological membranes [ ] , the complexity of the process and the multiplicity of potentml pathways involved suggests that a single causal mechanism cannot explain both the cellular responses and the clinical expressions of toxic-]ty in animals monensin is cost-effective in increasing the yield of meat from both fowl and ruminants [ ] . in fowl, this increase m productivity is derived almost directly through the control of coccid]a that, if present, would adversely affect animal health [ ] in ruminants, increased product]vity appears to result from several factors, the most obvious being an increase in the effectiveness of feed utlhzation [ , , ] . these whole-animal effects (xe, systems effects) are well documented [ , [ ] [ ] [ ] and will be paraphrased only briefly here. the beneficial effects of monensm in cattle accrue, in part, through shifts m rumen rmcroflora population. for example, gram-positive bacteria (that are primarily acetate, butyrate, h , and formate producers) are inhibited by monensin; whereas, gram-negauve bacteria (many of which produce succinate) are less sensmve to monensm [ ] . the outer layer of the multflayered well of the gram-negatxve bacterium may contribute to this resistance by acting as a barrier to the penetration of [able i adapted from ledger and tanzer [ ] in ~e~retlon reduced secretion procollagen [ ] [ ] [ ] [ ] , fibronectm [ ] [ ] [ ] ] , proteoglycans [ , , ] , prolactln [ ] , alburmn [ ] , transfernn [ ] , promsuhn polypepudes [ ] , larmmn [ ] , a-amylase isoenzymes [ , ] , newly synthesized proteins [ ] , secretory proteins [ ] , proteins for fast axonal transport [ , ] , thyroxine-binding globuhn [ ] , acetylchohnesterase [ , ] , chononlc gonadotropin [ ] , phytohemagglutmm [ ] , very-low-density hpoprotem [ ] , maize rootcap polysaccharldes [ ] (see however, sticher and jones [ ] for lack of monensm effect), vesicular stomatltxs virus glycoprotein [ ] , extracellular matrix [ ] , type ii collagen [ , ] , reviews [ , , ] increased secreuon catecholarmne [ ] , catheps'n d [ ] _ defective processing pro-albumin to serum albumin [ ] , receptors for msuhn and somatatomedin c [ ] , pro-oplomelanocortm [ ] incomplete processing of ohgosacchandes (n-hnked and/or o-linked) myeloperoxldase [ ] , prenv glycoprotexn [ ] ; fibronectm [ ] , hcg subumts [ ] , blocked formation of complex ohgo-~acchandes [ ] , herpes simplex glycoprotelns [ ] , hla-dr-as-soclated mvanant chain [ ] , coronavlrus glycoprotem [ ] , review [ ] undersulphauon proteoglycans [ ] , glycosammoglycan chains [ ] , / -d-xyloside glycosammoglycans [ , ] in endocytosts and endosome actdzflcatton intubitlon of mternahzatlon arylsulfatase [ ] , lmmunoglobulln [ ] , a- -macroglobuhn [ ] , semlikl forest varus [ ] , horseradish peroxldase [ ] inhibition of dlssocmtion of mternahzed hgand asmloglycoprotems [ ] , asmlo-orosomucojd [ ] lnhlbxtlon of hgand transfer smb~s virus nuclear capsids to cytoplasm [ ] , epidermal growth factor, / -hexasamlmdase, low-density hpoprotein, lmmunoglobulin, and proteoglycans to lysosomes [ , , ] inhibition of acldfflcauon endocytlc vesicles [ ] [ ] [ ] , lysosomal and prelysosomal compartments [ ] , interference with semllka forest virus genome penetrauon [ ] , expression of diphtheria toxin [ ] ; recycling of ldl receptors [ ] , release of diphtheria toxin from endocyuc vesicles [ , ] lnhlbltton of mtracellular degradation proteoglycans [ ] , insuhn [ ] , lysosomal (methylarmne-sensxtlve) protem degradation [ ] inhibition of contraction of contractile vacuoles paramecmm aureha [ surface formation and growth altered secretion of cell surface molecules proteoglycan [ , , ] , type if collagen and/or procollagen [ , , ] ; fibronectin [ , , ] , lamlmn [ ] , mcorporauon of sulfaudes into myelin [ ] , incorporation of po protein and myehn basic proteins into myelin [ ] inhibition of scale morphogenesis scales of the green alga pyrarmmonas mconstans [ ] inhibition of cell spreading-cultured fibroblasts [ ] ; mesoderm ceils [ ] stimulation of receptor capping mouse t-lymphoma cells [ ] inhtbltlon of growth rye seedhngs [ ] , pelhu ] transport of molecule.~ recognition of independent secretory pathways acetylchohne receptor and acetylchohnesterase [ ] , membrane glycoprotems/ assembly of uukunlerm virus [ ] , ca +-dependent and ca +-mdependent secretion of a-amylase [ ] , proteoglycan and hyaluronate [ ] , prolactln [ ] ; galactosyl receptor [ ] maturation and/or transport of viral coat proteins vesicular stomatltis virus [ , ] , herpes sxmplex varus [ , ] , semhkj forest wrus [ ] , uukumerm wrus [ ] , alphavtrus [ ] , coronawrus glycoprotean [ ] , bovine herpes virus type glycoprotelns [ ] st~mulatlon of sugar and sugar nucleoude transport avian erythrocytes, isolated rat and mouse dmphragm muscle, and red cells [ , , ] ; mouse thymocytes [ ] redirection of secretory product plasmalemma to tonoplast [ ] inhibition of mtracellular transport: protein to rod outer segments [ ] , myeloperoxtdase [ ] , hcg subumts [ ] , accumulation of lammln [ ] ; gp glycoprotein [ ] , procollagen [ ] , fibronectm [ ] interactions wtth other toxm~ enhancement of toxaclty tlamuhn m swine [ ] , dlsulfide-hnked methotrexate-anti-transferrln receptor conjugate [ ] , specific cytotoxlcxty of a breast cancer-associated antigen lmmunotoxan m humans [ ] reduction of toxicity selenmm and vitarmn e [ ] monensm; although, a more direct influence revolving differenes in membrane energetlcs also has been implicated [ ] monensln also may decrease the degradation of dietary protein in the rumen and, thus, increase the amount of protein avaalable for dlgesuon and uptake in the small intestine [ ] . both a reduction in overall cell numbers m the rumen and a direct effect of monensln on bacterial protelnase and dearmnase activity have been suggested as contributing to this effect [ , ] . one of the first subcellular effects observed in relation to the topical apphcatlon of monensin was vacuolatlon of golgi apparatus clsternae [ ] . subsequently, xn vitro studies clearly demonstrated that monensin altered or inhibited numerous membrane-located phenomena (table i) . among these were the transfer of a -macroglobuhn from coated pits to receptosomes [ ] , recycling of low-density hpoprotem receptors [ ] , pinocytosis [ ] , transfer of product from endoplasmac reticulum to golgl apparatus [ ] , maturation and/or transport of viral coat proteins [ , , , , , , , ] , inhibition of transport of membrane proteins to rod outer segments [ ] , xnhibltion of cholesterol transport from the golgl apparatus to the mltochondnal site of steroidogenesis [ ] , blockage of phytohemagglutinln transport out of golgl apparatus and into protein bodies [ ] , inhibition of procollagen and fibronectm secretion from cultured human fibroblasts [ ] , inhibition of carbohydrate processing in cultured human flbroblasts [ ] , and inhibition of processing and cellular secretion [ , , , [ ] [ ] [ ] . additionally, cellular effects of monensm vary markedly depending upon the organism and the route of adrmnlstratlon. cultured cells, cells of tissue shces or explants, and plant organs that have received a topical exposure to monensin sufficient to inhibit growth or some cellular processes, usually show deviations in golgl apparatus structure and function. in contrast, ceils from animals poisoned by ingested monensin often exhibit gross mltochondrlal lesions without the corresponding golgl apparatus modifications. the reasons for these fundamental differences in cellular responses to monensin provide one focus for the present review and illustrate the many complexities surrounding the use of monensln as a probe specific to a single metabolic process. the primary functional unit of the golgi apparatus is a stack of membranous compartments (i e., the clsternae) each of which differs chemically, structurally, and functionally from the others [ ] [ ] [ ] [ ] . the number of cisternae per stack varies widely; although, in most animal cells and higher plant cells, there are about - cisternae per stack. each stack is polarized in the sense that product and membrane maturation appear to occur sequentially from a cls (fornung) face on one side to a trans (matunng) face on the other side [ ] . for simplicity, the stack may be divided into umts (measured from cis to trans faces) each of which represents a known set of functions. currently, there appear to be some - such units that make up each stack [ , ] . in reahty, however, it seems more likely that these changes in function occur gradually across the stack of clsternae rather than in discrete steps. there is, also, one or more membraneous structures (e.g., the trans golgl network [ ] or tgn and the partially coated retlculum [ ] or pcr) that he just off the trans poles of the stacks in plant cells, these structures appear to be derived from sloughed trans clsternae [ ] . tgn and pcr participate in the separation (i.e., sorting) of both secretion and endocytic products [ , [ ] [ ] [ ] [ ] and regulate the release of endocytosed substances through a ph-sensltlve mechamsm [ ] . the functions of these post-golgi apparatus structures are rapidly affected by monensin. several mechanisms for the movement of membrane and product through the golgi apparatus have been postulated. for example, movement may occur by sequential maturation of golgi apparatus elements (i.e., formation, maturation and loss of cxsternae) through the golgl apparatus stack [ ] . this would require the formation of new clsternae on one face of a golgl apparatus sack and commensurate loss of clsternae from the opposite face of the stack the source of these new clsternae is a special region of endoplasmic retlculum which gives rise to transition vesicles that move and condense on the forming (cls) face of the stack where they fuse together to form the new osternae [ ] [ ] [ ] product movement may also occur by shuttle vesicles at the peripheries of the clsternae that move proteins from one cisterna to the next [ ] . however, both direct (i e., nonvesicular) movement of substances into golgi apparatus cisternae and an endoplasmlc reticulum-mediated movement of product through the penpheral tubules of the clsternae must also be considered as viable options for the delivery and transfer of substances in and out of the golgl apparatus [ ] . the post-golgl apparatus structures appear to move membrane and product almost entirely via shuttle vesicles, many of which are coated [ ] [ ] [ ] monensin exerts its most profound effects on the trans cxsternae of the golgi apparatus stacks in those regions of the apparatus primarily associated with the final stages of secretory vesicle maturation and in post-golgl apparatus structures primarily associated with endocytosis and membrane/product sorting. because of its relative specificity, biologists have used monensln extensively as an inhibitor of trans golgl apparatus function. incorporation of radiolabeled [ s]methlonine into secreted lmmunoglobulin m molecules in monensintreated cells was reduced as was slalylatlon of immunoglobulin m and lymphoid cell surface glycoprotelns [ ] . these latter findings showed that the intracellular processing of n-asparagine-hnked oligosacchandes is altered in the presence of monensln with an effect primarily on those sugars (e.g., slalic acid, galactose, fucose) added late in the processing continuum [ ] flbronectin, secreted in human flbroblasts, was incompletely processed in the presence of monensln and exhibited a greater incorporation of mannose than did control protein molecules [ ] inhibition of fibronectln secretion in human melanoma also has been reported [ ] . similarly, when treated with monensln, rat astrocytes in primary culture accumulated lamimn, another matrix glycoprotem involved in cell adhesion [ ] . not only do the monovalent lonophores block transport and surface expression of several secretory glycoproteins in normal cell functioning [ , , ] and the transport of membrane glycoproteins or enveloped viruses [ , , , , , , , ] , they inhibit formation of cell surfaces including assembly of peripheral myelin [ , ] . in mouse thymocytes, monensin leads to ( stimulated incorporation of labeled sialyl-, galactosyl-, and n-acetyl glycosaminyl residues [ ] . this enhanced accumulation was not due to a &rect effect of monensin on glycosyltransferase activities but, rather, as a consequence of a greater entry and accumulation of labeled sugar nucleotides in the swollen clsternae_ galactosyl transferase itself was translocated through the golga apparatus at a slower rate with monensin_ however, the sialylatlon of the o- nked ohgosaccharides of the enzyme was unaffected by monensln treatment [ ] effects of monensin on glycosyltransferases also may be indirect. monensin has been reported to decrease galactosyltransferase activity m golgl apparatus of rat embryo fibroblasts [ ] although it had no effect on this activity in baby hamster kidney cells [ ] monensln is an especially useful inhibitor, since it blocks lntracellular transport of protein at the level of the golgl apparatus without directly affecting protein synthesis [ , ] the effect of monensin is considered to be on transport rather than on processing per se [ , ] one argument is that oligosaccharide processing of those glycoprotelns that reach the appropriate site occurs normally even xn monensin-treated cells [ , , , ] . however, these observations could be explained as well if processing of ollgosacchande chains of different secreted glycoprotelns occurred at different sites, only some of which were sensitive to monensln [ , ] . the ablhty of monensln to effectively 'freeze' processing of molecules at a particular stage had lead to its use in identifying transitory synthetic intermediates. examples include the insulin receptor where several polypeptlde precursors have been described [ ] , the intracellular accumulation of non-cleaved precursors of pituitary hormones that occur in the presence of monensin [ ] , and dissection of the pathway for secretion of gonadotropin by cultured human trophoblastic cells [ ] . in some instances, the effect of monensin may be to redirect, rather than block, the movement of golgl apparatus-derived product. for example, under normal conditions, proteins of developing seeds accumulate in a central vacuole which then partitions into smaller units of storage protein (i.e, the protein bodies). however, when treated with monensin, the golgi apparatus-derived transport of the protein vlclhn in pea cotyledon was redirected from the vacuole to the plasmalemma and the newly synthesized viclhn was released from the cotyledon cells to accumulate between the plasmalemma and cell wall [ ] monensin lnhibmon of golgi apparatus function ~s sufficiently well established [ , , , ] that the phenomenon is used widely as one criterion for verifying the passage of a biochemical entity through the golgi apparatus. thus, based on partial monensm inhibition, hammerschlag and co-workers [ , ] concluded that passage through the golgl apparatus was an obllgator~ step m the lntracellular routing of materials m ta,~t axonal transport_ bartalena and robblns [ ] showed that rnonensin ~mpeded the exit of thyroxin-binding globulin from the golgj apparatus w~thout affecting the terminal glycosylatlon of the protein yanagashito and hascail [ ] reported that monensin reduced and delayed transport of both secretory and membrane-associated forms of proteoglycans, suggesting passage through the golgl apparatus of rat ovarian granulosa cells m culture similarly, an involvement of the golgi apparatus m the transport of sulfatldes to myelin [ ] and phytohemaghitlnln to protein bodies m bean cotyledons [ ] were deduced from monensin mhlbmon fhckinger and co-workers [ ] using [ h]leucine, showed that all, or nearly all, of the protein secretory product of mouse epididymis principal cells pass through the golg~ apparatus in times approximately eqmvalent to those reported m other tissues. this transfer of product from golgi apparatus to the cell surface was largely blocked by monensin swelling of golgi apparatus cisternae observed in the electron microscope following fixation with glutaraldehyde is, perhaps, the most consistent visual in vitro demonstration of a monensin-mduced effect on a membranous cell compartment [ , , , , [ ] [ ] [ ] the swollen clsternae usually appear devoid of contents by electron microscopy (figs. and ) but an electron-dense substance may be precipitated through the osmium tetroxlde-zmc iodide reaction (unpubhshed data) although all clsternae of the golgi apparatus may swell in response to monensin ( fig. and a) , the major effect appears to be associated with the mature, or trans, parts of the golgi apparatus stacks (figs and b) [ , , ]_ gnffiths and co-workers [ ] showed that monensm inhibited the transport of viral membrane proteins from medml to trans golg~ apparatus cisternae, thus indicating a monensln block between medial and trans cisternae monensm also blocked tlamrmng of the high mannose bound to the viral membrane proteins and their conversion to complex ohgosacchartdes similarly, niemann and co-workers [ ] found that monensm blocked glycosylation of e glycoproteln of corona virus in infected mouse cells. srinlvas and co-workers [ ] reported failure to process simple endo-h-sensitive to complex endo-h-reslstant ohgosacchandes and reduced efficiency of cleavage of the prenv glycoprotein precursor to gp for evehne mouse cells infected with friend munne leukemia virus these findings indicate a block prior to entry into the golgl apparatus also, m cultured hepatoma cells, transport of vesicular stomatltis wrus (vsv) g protein was arrested prior to acquisition of endo-h resistance, suggesting a block early in the processing pathway [ ] . strous and co-workers [ ] showed that monensin affects primarily the galactosyltransferase-containlng c~sternae of the golgi apparatus based on studies of the metabohsm, localization, and biosynthesis of n-and o-linked oligosaccharides of galactosyltransferase in helz cells. the accumulation of incompletely processed glycoproteins indicates either an up-stream accumulation of secretory materials behind a golgl apparatus blockage by monensm [ ] or a monensin block near the exit site from endoplasmlc reticulum [ ] . monensin effects on golgi apparatus have been observed in a wide range of plant and animal species and appear to be a universal response to the topxcal applicatton of monensln. as pointed out above, monensln action is exerted on the trans half of the stacked clsternae (fig ) , often near the point of exit of secretory vesicles [ , , , , , , ] or, especially at low monensm concentrations or short exposure times, sometimes m the rmdreglon of the stacked cisternae [ , ] . intracellular transport may be blocked [ , , , , , ] , often wlthxn rmnutes after exposure to monensln [ , , , , ] . swollen units usually accumulate near the golgi apparatus [ , ] a monensm effect is quite rapid in both animal and plant cells; e, changes in golgi apparatus have been observed after only - rmn of treatment [ , , , ] . these early effects have been documented particularly well is suspension cultures of carrot (daucus carota l ) [ ] . when carrot cells were exposed to monensln at - m (which is approxamately the minimum concentration that will elicit a strong monensm response m plants), production of secretory vesicles ceased and, almost immediately, an increased number of clsternae in the dlctyosome stacks was observed. an average of one additional clsterna per stack was formed within the first - mln of monensin treatment and, in some experiments, a second clsterna was formed within about man. these effects occurred without significant swelling of cisternae. thereafter, vacuoles, representing intact swollen clsternae, began to accumulate in the cytoplasm at a rate of about one every - min (fig. ) . the mechanism postulated for this momentary increase of d~ctyosome clsternae was that monensln, acting on the trans pole of the dictyosome, blocked normal formation [ ] for details of procedure) the two samples differed onl~¢ m that the one dlustrated m {a) was adjacent to a 'natural' ( e_ uncut) surface of the hver lobe, whereat (b) was from a cell near the 'cut' surface of the tissue slice in both instances, come golgx apparatus (ga) clsternae were sv~ollen, however, m (a) swelling was progresse~e from cls to trans pole (direction of arrow) whereas m (b) swelling was t.onfined to the trans c~sterna note that m~tochondrm (m) were condensed of secretory vesicles but did not block the formation of new clsternae at the cis face of the apparatus however, as the trans clsternae began to swell, the swollen clsternae were eventually released as intact units that neither fragmented nor integrated (fused) with other cellular constituents (e.g., plasma membrane). with the scale producing green alga pyramimonas lnconstans, exposures of to several hours to monensln resulted m disorientation of the golg~ apparatus and disruption of scale morphogenesls [ ] . these effects were reversible more recent studies indicate that a similar pattern of swelling and accumulation of clsternae in the cytoplasm occurs in cultured animal cells [ ] . when h- hepatoma cells were treated for varying times with ~ to m monensin, one swollen clsterna per stack of clsternae was produced after - rain of treatment during tlus time, approximately one additional clsterna per stack was formed (fig. , inset) . as the clsternae veslculated, vacuoles began to appear m the cytoplasm these large swollen vacuoles were formed at the rate of one sin concentrations, the vacuoles were larger and appeared more rapidly than at low concentrations of monensln but the kinetics of vacuole formation were qualitatively similar. however, by h following treatment with - m momensin, all clsternae of the golgi apparatus appeared as vacuoles. the swollen trans compartments that accumulate in the golgl apparatus region with monensln inhibition may contain regmns that are clathrin coated; e.g., condensing secretory material (prolnsuhn) in pancreatic cells [ ] . the response of golgi apparatus of hver slices to monensin was qualitatively similar to that with hepatoma cells in culture [ ] . with liver shces, a fraction enriched in vacuoles was isolated and demonstrated to contain the trans golgl apparatus markers, galactosyltransferase, and thiarmne pyrophosphatase, in ratios sirmlar to those of golgl apparatus proper [ ] . in barley aleurone layers, the a-amylase and acid phosphatase activities that accumulated within aleurone cells following treatment with monensin, were localized in cellular components with buoyant densities intermediate between endoplasrmc reticulum and mitochondria and cosedimented with latent inoslne diphos-phatase activity, a putative golgi apparatus marker in plants [ ] . heupke and robinson [ ] reported a shift to higher density of golgi apparatus membranes from monensin-treated barley cells, a response no obvious from work with mammalian cells. the golgi apparatus clsternae that accumulated behind a monensin block in semlikl forest virus-infected bhk cells bound viral nucleocapsids, and the resulting increase in density permltted their separation by gradient centnfugatlon from other golgi apparatus elements [ ] . the effects of monensin on golgl apparatus, at least up to several hours of exposure, appear to be fully reversible [ , , , ] in carrot cells, normal secretory activity was resumed within nun after transfer of cells to a monensln-free medium; although, in these cells, the vacuoles formed during the monensln block, remained in the vicinity of the golgi apparatus for several hours or more, even after apparently normal secretory activity had resumed however, with the longer treatment times of several hours [ ] or days [ ] monensin apparently causes swelhng of golgi apparatus cisternae through a na+-in/h+-out exchange across the membranes leading to a net uptake of na ++ ci and entry of water [ , ] evidence in support of this concept was provided by studies with isolated chromaffin granules which lysed readily after brief exposure to monensln in na +-or k+-contalnlng isotomc media for swelling to occur, the membrane must normally be lmpermeant to cations as is known for the chromaffin granule_ the chromaffln granule membrane contains a h+-atpase which is electrogenexc and, in the presence of a permeant anion, acidifies the granule interior to ph _ thus, the operation of this pump in the presence of monensin drives net salt uptake [ ] . to test whether net salt uptake driven by the presence of a proton gradient also would explain the monensininduced swelling of golgi apparatus cisternae, wild-carrot cells in suspension culture were treated with drugs and inhlbltors known to interfere with proton gradients [ ] monensin-induced swelling of golgl apparatus in sjtu could be inhibited by the protonophore, carbonylcyanlde-p-trlfluoromethoxyphenylhydazone (fccp), but was only little affected by the inhibitor of lysosomal acidification, quercetln, or by the lysosomotropic amines, chloroquine, and ammonia. cyanide also dramatically decreased swelling, and arsenate (with prolonged treatments) reduced the number of swollen cisternae organic acids, by providing a readily permeable counterlon, promoted monensln-lnduced swelhng these data imply that the monensin-induced swelling of golgi apparatus cisternae involves a proton gradmnt at or near the mature poles of the golgi apparatus because monensin induces a " na+/h + exchange, and since the van't hoft factors for h + and na + are practically the same [ ] , the osmolanty of the cell content should not increase to cause swelhng without a net proton influx one explanation would be that the ph of golgi apparatus vesicles is highly regulated proton translocatlng atp hydrolyzing enzymes (h +-atpases) are associated with several components of cells that develop acidic intermrs such as endosomes, coated vesicles, lysosomes, and trans golgl apparatus clsternae [ ] . ewdence for the presence of an h +-atpase has been the demonstration of atp-dependent vesicle acxdlflatlon in golgl apparatus isolated from rodent liver [ ] [ ] [ ] , corn coleoptlles [ ] , and sycamore cells [ ] acldificatmn was demonstrated both by [lac]methylamlne uptake and by spectrophotometric assays of amd-quenched dye fluorescence (acridine orange, neutral red, or quinacrme) several lines of evidence confine the golgi apparatus h +-atpase to the trans cisternae. as emphasized in the preceding section, the early in sltu effects of monensm are frequently localized to the trans faces of the golgl apparatus the resultant swelling, whmh is proposed io be due to the accumulation of osmotically active ions in exchange for protons [ . ] , occurs predominantly m trans clsternae additionally, a basic congener o] dmltrophenol ( -( , -dlnitroanlhn o)- '-amlno-nmethyldlpropylamine, damp), which concentrates m acidic compartments as shown in fibroblasts by lmmunocytochemlstry is present onl) clsternae and vesicles associated with the trans faces of the golgl apparatus [ ] moreover, damp rapidly leaves these compartments when cells are incubated with monensm, thus further indicating that accumulation of damp is due to the acid ph some involvement of a low ph compartment is evidenced by the observatmn that some monensln effects on processing, ke, proteolytlc conversion of proalbumln [ ] , are mimicked by amines however, in promyelocytlc leukemia cells, processing of myeloperoxldase, while blocked by both monensln and chloroqulne, was not affected by nh~ cations, thus indicating that processmg is not necessarily influenced by ph-dependent mechanisms [ ] these results were interpreted as indicative of processing in golgl apparatus based on inhibition of transport by monensin and chloroquine rather than processing m lysosomes and other late, acidic compartments involving a ph-dependent mechanism [ confirmatmn of a trans location of the h ~-atpase has come from free-flow electrophoresis separations of golgl apparatus yielding cls, medial, and trans compartments m fractions of diffenng electrophoretic mobility [ , ] _ in these separatmns, proton pumping activity was found exclusively in the most electronegative fractions coming from the trans-most goigl apparatus re-gion_ gnfflng and ray [ ] have offered the suggestion that acldificatmn of clsternal lumlna may be part of an osmotic mechanism to compress and flatten the cisternae the latter, for example, might aid in the transfer of content into secretory vesmles. the inward pumping of protons would tend to favor the exit of na + and k + out of the clsternae furthermore, as the ph falls, those monovalent cations remaining would tend to combine with acidm groups of the golgx apparatus membranes, further reducing the osmolanty of the clsternal content relative to that of the external cytoplasm. thus, water would be driven osmotically out of the clsternae to both compress the clsternae, as seen along a pronounced cls to trans gradient for plant golgl apparatus [ ] , and, perhaps, to account for the condensation of secretory materials m condensing vacuoles and other trans golgi apparatus compartments (e.g, ref ). other cell compartments, including endocytic vesicles [ , ] , lysosomes [ , ] , multivesicular bodies [ ] , and coated vesicles [ ] [ ] [ ] have h+-atpases. all use h+-atpases to acidify their interiors and the enzymes responsible have been solubdized from lyso-somes and reconstituted into liposomes [ ] . vacuole (tonoplast) membranes [ ] , and possibly also plasma membranes of fungi [ ] , contain h÷-atpase. similarly, a gradient of acidification within the endocyuc pathway has been indicated from immuno-electron microscopy with protein a-colloidal gold and monospecific antibodies to the weak base pnmaquine [ ] . however, not all compartments with h+-atpases (e.g., cells, vacuoles, lysosomes, coated vesicles) swell in response to monensin. cell and/or vacuole swelhng may be hmlted due to the very large internal volumes revolved. the contracnon of contractile vacuoles of paramectum was inhibited reversibly by monensin and in a manner dependent upon the presence of na + [ ] but marked swelling was not observed. little is known about the swelhng response, ff any, of lysosomes, coated vesicles and other endocync compartments m response to monensin lntubition. their functions, however, are inhabited by monensin as will be emphasized in the section that follows. carboxyhc lonophores strongly inhibit proton uptake by photosynthenc preparations [ ] . in chloroplasts, swelling of thylakolds (inner membrane compartments) but not of the space between tuner and outer plastld membranes has been observed to result from monensln treatment [ ] . thylakold swelling, in contrast to swellmg of mitochondrial cristae and of golgl apparatus clsternae, was reduced upon mcubanon in darkness, again suggesting a relationship between swelling in the presence of monensln and the hght-driven proton gradient used for photophosphorylatlon [ ] mltochondrla have an outwardly directed energy-hnked proton pump and do not swell with monensln (rather, they tend to condense, see figs. , a and b) while the light-driven proton pump of chloroplasts and chromatophores, and that of the golgi apparatus pump, are directed reward causing the vesicles to swell. evidence for a secretory pathway bypassing the golga apparatus in the monensln-blocked cells is provided by kubo and pigeon [ ] who lnvesngated the effects of monensin on the synthesis and expression of membrane igm of a human lymphoblastold hne. they found altered processing of both /l, k chains and incomplete terminal glycosylations. yet, transport of the altered molecules was observed. that the aberrant processing did not influence markedly the membrane expression of the igm is consistent with a secretory pathway bypassing the golgl apparatus m monensin-blocked cells. slmdarly, a dual secretory pathway, only one part of which was suscepnble to monensin, was deduced from studies of a-amylase secretion m rice seed scutellum [ ] . in zea majze roots, monensln lnhib~ted secretion of aamylase but not polysaccharide slime [ ] . the blocked secretion that results m the intracellular accumulation of secretory products frequently is not absolute. some portion of the material synthesized is released from the monensm-mhabited cells and this material frequently exhibits an abnormal type of posttranslational modification. for example, those proteoglycans from chicken embryo chondrocytes secreted m the presence of monensln are vastly undersulfated [ , , , ] . thus, membrane and secreted molecules leaving the cell following a monensin block appear to have been denied the full range of processing enzymes they would normally encounter during transit through the cell however, whether the incompletely processed molecules bypass one or more parncular lntracelhilar compartments (eg, the golgi apparatus), or whether they pass through functionally incomplete compartments, remains to be determined. during maturation of uukunieml virus m baby hamster kidney cells, monensin appeared to mhibit a terminal step of virus assembly, but not the expression of virus membrane glycoproteins g and g at the cell surface. these findings suggest that both g and g could enter a functional transport pathway in the presence of monensm that bypasses the trans golgi apparatus compartment to become expressed at the cell surface [ ] . evidence for a golgi apparatus bypass has been presented m liver where secretory hpoproteins may move directly from endoplasnuc reticulum to the cell surface without direct golgl apparatus involvement [ , ] . at concentrations of - m or higher, monensin inhibited secretion of albumin, transferrln, and vsv proteins g and x destined for delivery to the cell surface to the same extent m rat hepatoma cells [ ] . this was taken as evidence that the same vesicles were used by all four proteins m their movement from golgl apparatus to the plasma membrane however, the time required to move from er to the golgi apparatus, based on sensitivity to endoglycosidase h, differed for secretory and membrane proteins. an even more striking observation was that following the monensm block, secretory proteins accumulated in an endo-h-sensitive form, whereas, membrane proteins were already endo-h-resistant. this strongly implies that membrane and secretory proteins are not m the same compartment initially and would support the concept of peripheral input of secretory proteins into the secretory vesicles of the golgi apparatus, at least in hver [ , , ] alonso and compans [ ] provided evidence for two distract pathways of glycoprotein transport in madin-darby canine kidney (mdck) cells only one of which was blocked by monensm however, in baby hamster kidney (bhk ) cells, both influenza virus and vsv maturation were sensitive to monensin. the vsv particles were synthesized m both mdck and bhk cells, but transport to the cell surface was blocked only in the mdck cells thus, there appear to be two distinct pathways of transport of glycoproteins to the plasma membrane in mdck cells, only one of which is blocked by monensln there is no information on the nature of the alternative transport vesicle that carries mfhienza virus to the cell surface of mdck cells if, in fact, a vesicle is involved. melroy and jones [ ] reported accumulation of normally secreted a-amylase within barley aleurone layers after monensln treatment. however, only isozyme was secreted normally whereas isozymes , and were not secreted also, in the perfused rat hver, monensin treatment has less of an effect on blliary secretion than on secretion of plasma proteins [ ] . sn-nllarly, in the transport of hla-dr a-and / chains [ ] , processing of n-linked carbohydrate chains to full endo-h resistance occurs however, with the associated i-chain, processing of both o-and n-linked carbohydrate chains ~s inhibited, and carbohydrate chains remain predornlnantly endo-h susceptible. here, the processing of membrane-associated proteins that occurs despite a monensln block may reside in intercalary clsternae that constitute the golgi apparatus region recently termed medial [ a ] there is now considerable evidence for monenslnsusceptible compartments in the endocytotic pathway. transfer of product to secondary lysosomes [ ] as well as virus penetration into cultured cells [ ] are impaired by monensm. stein and co-workers [ ] have shown that monensin blocks transfernn recycling by causing internahzed hgand to accumulate in the perinuclear regmn, primarily in multivesicular bodies, of the k cells used in the study. based on studies of hrp uptake in rat fibroblasts, wilcox and co-workers [ ] suggested that inhibition of endocytic events may be the consequence of an inhibition of membrane recycling within the cell rather than a direct effect of monensin at the cell surface. maxfleld [ ] reported that ~tm monensln resulted m an mcrease in internal ph of endocytic vesicles of cultured mouse fibroblasts from to above to account for its effects on receptor-mediated endocytosis similarly, marsh and co-workers [ ] concluded that the lnhlbmon of semhki forest virus penetration into cultured cells was the result of this increase in ph of endocyuc vacuoles and lysosomes above ph , the threshold for fusion activity of viral membranes monensln has also been shown to inhibit lysosomal degradation of protein by affecting lysosomal ph [ ] and to abolish aslaloglycoprotein degradation in cultures of rat hepatocytes through a ph shift in prelysosomal endocytlc vesicles [ ] . using digttal image analysis, tyco and co-workers [ ] showed that monensin raised the ph of endocytic vesicles in cultured human hepatoma cells and caused a hgand-lndependent loss o! receptors in other studies, monensln did not prevent lnternahzatlon of s-labeled proteoglycans by rat ovarian granulosa cells although their lntracellular degradation was completely inhibited [ ] . yet, degradation pathways involving proteolysls of both dermatin and heparln sulfate and limited endoglycosldlc cleavage of heparln sulfate continued_ these findings, while consistent with an involvement of both acidic and nonacldlc compartments, show that monensm inhibition is primarily on those processes that normally occur in acidic compartments such as endosomes or lysosomes by raising their ph. sirmlarly, with isolated hepatocytes, whittaker et al [ ] found no effect of monensin on insulin internalization but, rather, an impairment of ~ts degradation once lnternahzed. rustan and co-workers [ ] suggested that monensm inhibits both endo-and exo-cytosls by a similar mechanism, namely, disruption of proton gradients their conclusions were based on studies of rat hepatocytes in which monensm inhibited both secretion of very-lowdensity hpoprotelns, and binding and degradation of aslalofetuin both secretion and receptor binding were markedly decreased after only rmn of monensin treatment although no effect on protein synthesis was observed. however, secretion was more sensitive [o monensln than endocytosis, suggesting that monensin independently intubits endocytlc and secretory functions although the mechanisms may be similar. marnell et al [ ] explained the monensin block of the cytotoxic effect of &ptheria toxin on a sirmlar basis following endocytosls of the toxin, the toxin was assumed to penetrate the membrane of the endosome and enter the cytoplasm in response to an acid environment by neutralizing the ability of endosomes to acidify their interiors, monensin, like the lysosomotroplc amines, was able to block the low ph-dependent dissociation of receptor-hgand complexes and subsequent release of ligands either to the cytoplasm (viruses and toxins) or to lysosomes (endocytosed proteins such as ldl). this in turn would prevent recycling of receptors and membrane and eventually bring endocytosls to a halt due, not necessardy to an inhibition of the uptake processes per se, but perhaps, to blockage of an internal step very similar to that believed to be blocked at the goln apparatus. also consistent with sirmlar modes of monensin mhibition in processing both endocytlc and exocytic vesicles are findings that a single mutatmn in chinese hamster ovary cells impaired both golg¢ apparatus and endosomal functions in parallel included were the monensin sensitive steps of virus and toxin penetration from endosomes into the cytoplasm and of golgl apparatus-associated maturation of smbis virus [ ] . the alterations correlated with losses of atp-dependent vacuole acidification as if the atpase of endosome and golg~ apparatus shared a common genetically regulated subunit. ono et al. [ ] studied a monensln-reslstant mutant of mouse balb/ t cells which also proved to be a poor host for either vesicular stomatltis virus or semllkl forest virus multiplication. the mutant cells resistant to monensln, bound virus normally and contained acidic compartments. however, movement of virus from the cell surface to the endosome and lysosome compartments was extremely slow. thus, the ablhty of monensln to block processing of endocytic vesicles by making prelysosomal compartments less acidic, suggests a mechanism for perturbation of endocytosls based on its ionophorlc properties the mechanism could be slmxlar to the monensm-medlated exchange of monovalent alkah tons for protons that induces, by osmotic means, the observed swelhng of golgl apparatus clsternae. clearly, monensm does not interfere with the uptake and binding of particles at the cell surface. monensm is ineffective against activities that occur at the cell surface. hedm and thyberg [ ] showed that uptake of igg prebound to the cell surface was unaffected by monensln similar findings have been made in studies of receptor-medmted endocytosls of various other ligands [ , , ] . however, monensm may secondarily affect mternahzation through depletion of monensln-sens~tive receptor sites at the cell surface. this would occur if the cell surface receptors are recycled back into the cell and then blocked in the post-golgi region by monensin so that they could not return to the cell surface [ , , ] . thus, monensin inhibition of endocytlc events seems to be at the site of transfer from endocytlc vesicles to lysosomes [ , , , ] or, in monensin-sensltive endosomes, inhibition of the dissociation of ligand-receptor complexes [ ] most animal cells show a dose-related response to monensm that falls off rapidly at monensm concentrations less than - m consequently, most studies of monensln effects use monensin concentrations of - m or higher however, a few reports indicate a cellular response at monensm concentrations less than - m for example, receptor capping by lymphocytes is stimulated by low concentrations of monensin in the range - to - m and inhibited by monensm concentrations above - m [ ] cultured adrenal chromaffin [ ] and heart [ ] cells also are stimulated by low concentrations of monensln. though these effects occur at concentrations below the threshold effects for most golgl apparatus responses, they still presumably result from increased levels of cytoplasmic sodium due primarily to the ~onophore insertion at the plasma membrane fig (a) outer cap cells from a control (nontreated) maize root txp preserved by freeze-subsututlon [ ] . the form of dlctyosome (d) was normal and slrmlar to that following glutaraldehyde/osmlum tetroxade fixation (b) same except that root tip was treated for h with -s m monen~m before being preserved by freeze-substitution. the trans clsternae and/or secretory vesicles (arrowheads) were swollen, and mltochondna (m) were condensed (compare with mltochondna of a) w, cell wall, v, central vacuole swelling of golgl apparatus clsternae occurs in a wide range of plant and animal cells (see subsection iii-b ) and may be a universal response to monensln poisoning. however, in plants, and to a lesser extent animals, swelhng is influenced by the fixative used to preserve the cells specifically, morphological evidence of swelhng is less (in animal golgl apparatus) or nonexistent (in plant golgl apparatus) when the tissues are fixed in potassium permanganate as compared to fixation in ghitaraldehyde/osmium tetroxade [ ] . these effects could be due either to fixation artifacts or to differences between plant and animal golgl apparatus (e.g, ref_ ) this problem was evaluated by comparing the images of golgl apparatus preserved by various chemical fixatives as well as preservation by freezing and low temperature substitution in acetone and osmium tetroxade. presumably, the image following freeze sub-stitutlon would reflect the true ultrastructure more closely than the image following chemical fixation. the results of freeze substitution in both animal cells [ ] and maize root (figs a and b) , show swollen golgi apparatus c~sternae following monensln exposure in a pattern similar to that observed after glutaraldehyde/ osmium tetroxide fixation [ ] . however, using videoenhanced light microscopy and cultured bovine mammary epithelial cells, a marked swelling response to monensln was observed only after the addition of glutaraldehyde fixative to the monensln-incubated cells (morr , d j., mollenhauer, h h., spring, h., trendlenberg, m, morr , d m. and kartenbeck, j, unpubhshed data)_ thus, whether monensin-lnduced swelhng occurs m vlvo or is in response to aldehyde fixations remains an important questiion. no swelling was observed in golgl apparatus of protoplasts of carrot cells freshly prepared by digestion of cell walls when exposed to monensin even though such a response was obtained in the same cultures prior to wall dissolution [ ] . similarly, in thin slices of hver incubated in monensin, the golgl apparatus adjacent to cut edges of tissue slices showed a different swelling response than golgl apparatus adjacent to the uncut natural surfaces of the lobe (unpublished data) adjacent to a cut edge, fewer cisternae swelled and those that swelled were only in the most trans positions the basis for such differences is unknown but might, for example, indicate changes in cisternal proton pumping ability, or monensln uptake, in response to changes in the physiological state of the golgi apparatus brought about by the tissue excision the mechanism by which monensin interacts with coccldla and rumen rmcroflora is well documented [ , ] however, the interaction between monensm and the tissue of the host animal is less well understood even though the chnlcal manifestations of monensln poisoning are well known_ most striking are differences between the in wtro monensln effect ohserved in cultured plant and animal cells, and plants, and the in vwo effects observed m animals when used at recommended levels, either as a coccidlostat for poultry [ , ] or for cattle [ , , ] , monensin seldom causes poisoning nonetheless, misuse of the product, usually from improperly mixed or improperly distributed feed, may cause toxlcosis and death [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . horses, ponies and other equine species are particularly sensitive to monensln poisoning [ , . ] . the median lethal dosage (ldso) for the horse js - mg monensin per kg body weight compared to - mg per kg body weight for cattle and mg per kg body weight for poultry [ , , ] in mammals, the physical signs of monensln toxlcosls commonly include anorexia, diarrhea, depression, sweating, ataxm, palpitations of the heart, and sudden death following exercise [ , , , , [ ] [ ] [ ] [ ] stiffness of hindquarters and swollen gluteus muscles [ , ] , elevated pulse rate [ , , ] , and ecg abnormalities [ , , , ] also have been reported in fowl, the outstanding signs of monensln toxlcosls are drowsiness, excessive thirst, anorexia, depression and paralysis [ , , ] marked congestion in a variety of organs also has been noted [ ] severely poisoned birds may die in sternal recombency [ ] . routine clinical tests on serum from horses poisoned by monensm may show abnormally high values for blood urea nitrogen, total billrubin, creatlne kinase, lactate dehydrogenase and aspartate armnotransferase [ , , ] however, chnlcal manifestations are often variable makang interpretation and diagnosis difficult. moreover, serum levels of sodium, potassium, chlorine, calcium, phosphorus, and urea may remain at near-normal levels following monensin treatment [ ] _ in poisoned mammals and fowl, generalized congestion, hemorrhage, and macroscopic injury to striated muscle [ , , , ] , spleen [ , ] , lung [ ] , liver [ ] , and kidney [ , , , , ] have been noted. the most consistent macroscopic observation in ponies, cattle, pigs and fowl, has been cardiac myocyte degeneration and vacuollzatlon [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] in animals poisoned with relatively high doses of monensin, an initial condensation of heart mltochondria is often seen (fig ) however, with longer exposure times, some mitochondria swell and vacuolate with an almost total loss of matnx substance (fig ) _ intracnstae spaces generally remain unchanged with swelling being restricted to the mitochondrial matrix this is followed by loss or dilution of matnx components and a reduction in size of cnstae so that the rmtochondna appear as empty vacuoles with residual cristae typically, only some mitochondrla in a particular fiber become swollen and appear as vacuoles, and fig section of left ventricle from a rat treated wath a single mtraperitoneal mjecaon of mg monensm per kg body weight most rmtochondna (m) were either condensed or swollen in swollen rmtochondna, cnstae (arrowheads) were greatly reduced in extent but, otherwise, were of approxamately normal ttuckness thas dosage level (e_g, mg monensln per kg body wexght) is quite tugh for rats and often resulted m significant generahzed damage to the muscle fiber as dlustrated in the lower part of the rmcrograph however, rmtochondnal swelhng (but not condensauon) may occur as well at lower monensln dosages, even when no fiber damage can be identified ultrastructurally swelling and vacuohzatlon of mltochondna are progresslve with time whereas rmtochondnal condensation was dose-dependent and often occurred rapidly , a few were normal (arrowhead), but most were rmldly condensed swollen tmtochondna always appeared randomly distributed through a fiber although significant differences m rmtochondnal swelling were often noted between fibers (e g, see fig_ ) these are randomly distributed throughout the fiber (fig. ) . we have identified early stages of mitochondrial vacuolation and swelling in both the rat and pony (unpublished data), but these mitochondria were seldom plentiful, suggesting that transition to the vacuolated state, once started, was relatively rapid non-swollen mitochondria may appear condensed, especially after exposure to high levels of monensin ( fig. and ). granulation of mitochondna ( fig. ) was observed only occasionally and those granules that were present appeared similar to the tncalclum phosphate granules often associated with normal mitochondria [ , ] . however, whether monensln-lnduced granules contain ca ~ has not been determined. granules like these have been observed in lschermc and reperfused hearts and are considered indicative of calcium overload [ ] [ ] [ ] [ ] _ ca + overload may be a potential cause of cell death or cellular dysfunction in ischerma [ , [ ] [ ] [ ] [ ] [ ] [ ] although the effects are reversible if the cell is not too severely damaged [ , ] exaggerated matochondrial swelling has also been observed immediately following reperfuslon of hearts rendered lschemlc by occluding blood flow for a few minutes [ , ] whether excess mitochondrial ca + occurs as a result of monensin is not clear under appropriate condiuons, either monensin or na + in excess can block ca + accumulation and promote its release from both mitochondrla and sarcoplasrmc reticulum over a broad range of monensin and na + concentrations [ , , ] it is probable that the release of ca + by monensln can occur as the result of an increase in cytoplasmac na + from a monensin shuttle although monensin (at relatwely high concentrations) has been shown to release ca + directly m a na+-lndependent manner from cardiac sarcoplasmic retlculum [ ] in all probability, however, monensin is not present in myocytes at high concentrations since swelling of golgl apparatus cxsternae (a characteristic response to monensin) ~s never observed in these cells. the picture is further comphcated by the fact that ca + release patterns may vary according to type of cell (e g., white vs red muscle cells [ ] ) as well as the availability of extracellular ca + [ ] . although not related to this report, it may be of some interest to note that both mitochondrlal condensation and granulation are much more intense in x- a-treated animals (and cultured cells as well) than in comparable animals (or cells) treated with monensin [ ] _ the percentage of affected mltochondrla vaned markedly between muscle types and species of animal. analyses of striated muscle m ponies showed that there was a much greater ( -times) hkehhood of finding altered mltochondrla in heart tissues than m diaphragm or appendicular muscle [ ] a similar relationship existed in rats except that most swollen mitochondria were m the &aphragm [ ] . some antibioucs (e g, tiamuhn and avoparcm) may act synergistically with monensln to induce shifts in the distribution of swollen mltochondria [ ] and other cellular damage. differences in distribution patterns of swollen mltochondrla also were observed between red and white muscle fibers of the rat diaphragm [ ] . red and white fibers were dffferentmted structurally by size, mltochondrlal content, and z-band configuration [ , [ ] [ ] [ ] swollen matochondrm were present in all fiber types when the number of affected rmtochondna was small. however, when large numbers of swollen matochondrm were present, the dxsmbutlon pattern was heavily skewed toward the white muscle fibers (fig_ ). a differential effect of monensln was also noted by van vleet and co-workers [ ] who observed in swine severe damage m the diaphragm, vastus lateralls, sem~tendlnosus, triceps and intercostal muscles: moderate damage m longissimus lumborum muscle, and little (or minimal) damage in tongue. damage was greatest m muscles containing a high proportion of type fibers these distribution patterns, coupled with the characteristic form of the degenerating rmtochondrla, are not common observations and, therefore, may be strong indtcatots of monensin poisoning in animals_ swollen rmtochondrla have not been observed in any of the nonmuscle cells of the heart, diaphragm, or appendicular tissues or in hver, adrenal, or kidney cells thus, monensm adrmnistered to mammals in vlvo tends to induce rmtochondrial changes only m selected tissues and/or types of muscle fibers the mechanisms for these rmtochondrlal changes and reasons for the specificity are not known. these problems are compounded by the fact that matochondnal condensation, but not subsequent swelling and degeneration, occurs in cells exposed topically to monensin. whale mitochondnal aberrations are the primary morpholog~c indicators of monensin poisomng in striated muscle, other aspects of muscle ultrastructure may show deterioration [ ] or may remain relatively normal even with gross mltochondnal damage [ ] . generalized fiber and cell degeneration may occur following monensin poisoning [ , , ] . the extent of monensin-induced injuries appears to be time and dosage dependent. if death occurs shortly after monensin exposure, there may be httle or no recognizable evidence of pathologtcal change, at least in liver, kidney, and striated muscle. generalized necrosis appears to occur most often when monensln is administered over long periods of time. even with single doses of monensin, structural aberrations in striated muscle develop progressively over several days and then regress if the animal survives the imtial insult (unpublished data). we have not observed permanent injury in either striated muscle or liver of monensin-treated rats although such effects have been noted in other animals [ ] . many of the effects on striated muscle attributed to monensxn occur also with other lonophores irrespective of their ion specxficlties [ , ] this implies, again, that na + is not the direct cause of muscle perturbation but, rather, that the iomc imbalance resulting from the intracellular influx of na + triggers other cellular responses that lead to the observed perturbations. monensin is known to both inhibit and promote ca + accumulation in myocytes depending on the absence or availability, respectively, of external ca + stores [ , ] ; alter na + gradient-dependent ca + transfer through the basolateral plasma membranes of rat small intestine [ ] ; increase myocardial calcium activity [ ] , inhibit ca + accumulation by cardiac macrosomes or cause release of accumulated ca + stores [ ] ; and release ca + from macrosomes [ , ] . digitalis and other cardiac glycosides (which increase myocardial contractility) appear to act by altering intracellular na + concentration through inhibition of membrane-bound na +-, k+-actlvated adenosine triphosphatase which secondarily results in an increase in mtracellular ca + [see ] . calcium ionophores such as lasalocld and a have been suggested as potential probes for studying the effects of calcium imbalance on myocardial function [ ] . in retrospect, monensin affects the myocardlum in much the same way as do the calcium lonophores and, in some instances, to an even greater extent [ ] . these observations suggest that the xonotropic effects of monensln may be partially indirect; e g., through release of histamine and endogenous amines [ ] , or stimulation of synthesis and/or release of prostaglandln [ , , ] . thus, na + balance plays an indirect but critical role in modulating myocardial function however, a calcium-independent catecholamine depleting action of monensin in cultured rat pheochromocytoma cells [ ] and m bovine adrenal medullary cells and chromaffln granules [ ] suggests that monensln may also play a direct role in altering cellular function. similarly, sutko and co-wor~kers [ ] showed that both monensxn and mgencin produce their effects in guinea pig atria by direct action as well as by releasing catecholamines from tissue stores. differences in subcellular responses to monensin, between the whole ammal and isolated cells or organs, have been noted by us as well as by others [ ] . thus, swelling of golgl apparatus clsternae observed an cultured cells, tissue shces, and plant roots and stems, is not an aberration characteristic of the cells of animals poisoned by monensln. lack of golga apparatus welhng in animals infers that cells from monensin-poisoned ammals are bathed in body fluids containing less than - m monensln which is approximately the minimum effective dose of monensin that will cause sweulng of golgt apparatus clsternae in cultured animal cells. alternatively, lack of vacuolated and/or swollen mitochondna in cultured mammahan cells and plants generally imphes that the vacuolated and/or wollen rnttochondria observed in striated muscle from monensinpoisoned animals are secondary effects of monensin poisoning, perhaps caused by a metabohte of monensin [ , ] . alternatively, monensln could affect the synthesis and/or transport of humoral agents (e.g., catacholanunes) which, in turn, would alter muscle homeostasis and lead to the rmtochondnal aberrations observed in striated muscle. for plants, at concentrations of -s m, monensin was shown to inhibit gernunatlon and growth of ryegrass seedlings [ ] . the effects were primarily associated with poor root development and significant reduction of root mass as compared to controls leaf emergence and leaf mass was only slightly affected. at - m monensln, roots often did not emerge from the seed during germination and root mass of seedlings was often near zero. under these same conditions, shoot mass was reduced about % as compared to controls. monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of prinopally sodium ions for protons. the outer surface of the lonophore-ion complex as composed largely of nonpolar hydrocarbon, which tmparts a high solubility to the complexes in nonpolar solvents. in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. the net effect for monensin is a trans-membrane exchange of sodium ions for protons. however, the interaction of an ionophore with biologl-cal membranes, and its ionophorlc expression, is highly dependent on the blochemcial configuration of the membrane itself one apparent consequence of this exchange is the neutralization of acidic lntracellular compartments such as the trans golgi apparatus clsternae and associated elements, lysosomes, and certain endosomes. this is accompanied by a disruption of trans golgi apparatus clsternae and of lysosome and acidic endosome function. at the same time, golgl apparatus clsternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions monensin effects on golga apparatus are observed in cells from a wide range of plant and animal species the action of monensin is most often exerted on the trans half of the stacked clsternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensln concentrations or short exposure times, near the rmddle of the stacked cisternae. the effects of monensln are quite rapid in both animal and plant cells; l.e, changes in golgl apparatus may be observed after only - min of exposure it is implicit in these observations that the uptake of osmotically active cations is accompanied by a concornltant efflux of h + and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of clsternae observed in electron rmcrographs. in the golgi apparatus, late processing events such as terrmnal glycosylation and proteolytlc cleavages are most susceptible to inhibition by monensln. yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the golgi apparatus in endocytosls, monensln does not prevent internalization however, intracellular degradation of internalized ligands may be prevented. it is becormng clear that endocytosls involves both acidic and non-acidic compartments and that monensln inhibits those processes that normally occur in acidic compartments. thus, monensln, which is capable of collapsing na + and h + gradients, has gamed wide-spread acceptance as a tool for studying golgi apparatus function and for locahzlng and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. among its advantages are the low concentrations at which inhibitions are produced ( - . /~m), a minimum of troublesome side effects (e g., little or no change of protein synthesis or atp levels) and a reversible action. because the affinity of monensin for na + is ten times that for k +, its nearest competitor, monensxn mediates primarily a na+-h + exchange monensin has little tendency to bind calcium. not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed m cattle the mechanisms by which monensln interact with cocctdla and rumen rmcroflora to achieve these benefits are reasonably well documented. however, the interactions between monensm and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensln poisoning are well known equine species are particularly susceptible to monensm poisoning, and a common effect of monensln poisoning is vacuolizatton and/or swelling of rmtochondna m striated muscle other pathological injuries to striated muscle, spleen, lung, hver and kidney also have been noted a con-sistent observation is cardiac myocyte degeneration as well as vacuohzation differences m cellular response resulting from exposure to monensln (t e, golgi apparatus swelling in cultured cells, isolated tissues, and plants vs. mltochondrial swelling m animals fed monensln) suggest that myocardial damage is due either to a monensln metabohte or is a secondary response to some other derivation however, as pointed out by bergen and bates [ ] , the underlying mode of action of lonophores is on transmembrane ton fluxes which dissipate cation and proton gradients consequently, some or all of the observed monensin effects m vlvo m animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes. the role of membranes in metabohc regulation polyether antibiotics -naturally occurnng acid ionophores polyether antibiotics -naturally occurnng acid ionopbores polyether antlbloucs -naturally occurnng acid ionophores polyether antibiotics -naturally occumng acid ionophores (west-icy polyether antibiotics -naturally occurring acid ionophores the role of membanes m metabohc regulalaon polyether antlbtotlcs -naturally occurnng acid ionophores comp blochem phys- o protoplasma clba found symp ongm and continuity of cell organelles (relnert endocytosls (paston, i and wdhngham proc. natl acad th ann proc equine medacane and surgery current vetennary therapy food ammal practice (howard an atlas of fine structure calcium antagomsts and cardiovascular disease key: cord- -esnsa u authors: nan title: abstracts (th) tripartite meeting salzburg/austria, september – , date: journal: langenbecks arch chir doi: . /bf sha: doc_id: cord_uid: esnsa u nan the gastric secretion capacity increases during the first year after all types of vagotomy which is assumed to be important for the development of recurrent ulceration. however, the cause of this reestablishment of the preoperative gastric function is not adequately explained. the purpose of this study was to investigate the vagal innervation of the parietal cell mass in dogs / year after truncal vagotomy (tv), selective gastric vagotomy (sgv) and parietal cell vagotomy (pcv). material and methods: mongrel dogs supplied with a gastric fistula were used. the groups comprised dogs with tv, dogs with sgv and dogs with pcv. the acid secretion was measured before and one month and one year after vagotomy following insulin and pentagastrin stimulations. operative technique: a gastroduodenostomy was performed in addition to the gastric fistula operation in the dogs randomised for either tv or sgv. the vagotomy operations were performed after the prevagotomy secretion experiments. tv by a cm resection of the main truncs through a left side thoracotomy and sgv as described by amdrup [ ] . the pcv dogs had no gastroduodenostomy added but the technique at the lower esophagus was identical with the sgv and the dissection of the lesser curvature was extended cm distal of the sharp physiological and histologically determined borderline between the fundus and the antrum. about / year after the vagotomy the dogs were sacrificed by an acute experiment. the persistent or the regenerated vagal innervation of the parietal cell area was mapped out by neutral red excretion elicited by electrical stimulation of either the thoracic or the cervical vagal nerves [ ] . results: one year after vagotomy no increase in insulin response could be demonstrated in the tv and sgv groups, but a gradual increase to about % of prevagotomy output occured in all pcv dogs. reliable neutral red experiments were performed in tv, sgv and pcv dogs. the stimmulated neutral red excretion was scanty to the same extent at the cardia region in all three groups, but a distinct antralfundic border was outlined in all pcv dogs. the border was, however, only suggested in one of the sgv dogs. conclusion: pcv including denervation of cm distal of the antral-fundic border do not prevent a functional important reinnervation of the distal part of the parietal cell area. no important reinnervation seems to occur at the cardia region after any of the three vagotomy procedures. and insulin ( . ugkg - ) i.v. bolus; after uni-and then bilateral truncal vagotomy. each study was minutes and blood was taken at , , , and then min intervals. gastric acid was measured by autobiuret titration. plasma was stored at - °c until assayed for pp by ria using an antibody sensitive to fmol ml- . results are expressed as mean total increment _+ se. significance: p < . ". bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p < . ), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p < . ). bilateral and right hemi-vagotomy significantly inhibited pp release by bombesin ( < . ), however; only bilateral truncal vagotomy significantly inhibited pp release by insulin (p < . ). these results suggest that the measurement of pp release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released pp may specifically delineate the integrity of the right vagus. since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance. glucose (g) or oleate (o) empty more slowly from the stomach than . m nac (s). this chemoregulation may be achieved by resistance beyond the proximal stomach [ ] . we sougth to define the site of this resistance. expt. : gastric emptying of s and mm g was measured in dogs with intestinal cannulas cm distal to the pylorus while gastric pressure was maintained at , or cm h with a barostat. the dogs were studied either with an uninterrupted intestinal stream or with duodenal effluent diverted through a second barostat prior to its return downstream. since the latter ensured a constant pressure at the ligament of treitz, any observed effects on emptying could not be due to resistance further distally. expt. : emptying of s, g, and mm oleate emulsion was measurd in dogs without cannulas after control antroduodenal transection and after antrectomy while gastric pressure was controlled at , and cm h . emptying rates expressed as the average of the volumes emptied at the pressures. under both conditions in expt. emptying rose with pressure yet s emptied faster than g. thus a major site of chemoselective resistance to emptying of liquids lies proximal to the ligament of treitz. after antrectomy s and o emptied faster than before, however, the differences between saline and nutrients were maintained. resistance, therefore, does not reside in the antrum or pylorus and clearly the duodenum is implicated. gastric emptying of liquids may be abnormally rapid in du. this abnormality my be due to a selective loss of inhibition in response to acid since previous studies [ ] have demonstrated rapid emptying of acid solutions but normal emptying of glucose and fat. previous investigations, however, used meals of only one concentration, so differences between dus and normals (n) may have been minimized by selection of a supra-maximal inhibitory dose. furthermore intragastric ph was not maintained constant so emptying could have been slowed by higher rates of acidification in du. we therefore studied emptying of graded concentrations of citric acid, glucose and oleate in ml meals. gastric volumes were measured by the george technique in dus and n at rain intervals for min after ingestion. gastric ph was maintained constant by intragastric titration ( . for acid; . for nutrients). subjects received one type of meal on separate days and the doses were randomly administered. dose dependent inhibition of emptying in response to acid and nutrients occured in n and du, but emptying was faster in du (p< . ). with glucose and acid the difference occured within - min whereas with fat the difference occurred between - min. thus ( ) dus do not have a selective loss of inhibition of emptying in response to acid, and ( ) the mechanisms which control emptying of fat and which are disturbed in du differ from those which control emptying of glucose and acid. gross ra, isenberg ji, hogan d, samloff im ( ) the effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load and serum gastrin in duodenal ulcer and normal subjects. a means of reducing the requirement for postoperative nasogastric intubation would be advantageous. we report the effect of cimetidine on postoperative nasogastric aspirates. thirty patients undergoing elective abdominal aortic surgery or colonic resection were entered into a double blind randomised study, receiving cimetidine mg hourly or placebo (saline) by continuous intravenous infusion from the morning of the first postoperative day. volumes of hourly aspirates were recorded and ph and (h ÷) of samples measured. nasogastric tube removal accorded to standard clinical practice. it has been shown, that the duodenum has a better ability to resist acid than jejunum or ileum. this resistance was attributed to alkaline secretion (a.s.) of the mucosa, because pancreatic and bile secretion were excluded. recent in vitro studies demonstrated an energy dependent hco -transport (flemstroem am j physiol g : ). we were interested in studying the role of blood flow (bf) in the production of alkali in an in vivo preparation. min. three groups of animals were studied: i) controis with normovolemia for min. ii) normovolemia for min, thereafter min of shock induced by bleeding to mmhg mean arterial blood pressure. iii) min normovolemia and min vasopressin ( . i. u./kg/min) under normovolemia. in controls a small decrease in bfand a.s. as well was observed. both shock and vasopressin induced a significant reduction in a.s. and bf. the relation ofbf to a.s. was found to be exponential (y = . + . x- . x r= . ; p< . ). the reduction in a.s. during shock was much less, when shock induced acidosis was compensated by i.v.-infusion of hco ( . ,uequ/kg/min). conclusion: the alkaline secretion of the proximal duodenum is dependent on blood flow and the arterial [hco~. there has been considerable controversy as to the nature of the offending agent in the etiology in reflux oesophagitis in man. in rat studies, surgically induced reflux oesophagitis was shown to be correlated with the presence of active trypsin in the reflucting juice. reflux of bile and gastric juice with a ph of did not induce oesophagitis. even short periods of oesophagitis ( - weeks) showed an increasing amount ofpanmural fibrosis which even further increased after a reflux abolishing roux-y operation. the pattern of the collagen content of the full thikkness oesophageal wall was similar to that found in human reflux oesophagitis. the hypothesis that active trypsin is also in man responsible for reflux oesophagitis prompted to the following investigation. patients with typical gastrooesophageal reflux symptoms and control patients were endoscopically examined. on entering the stomach with the endoscope samples of gastric juice were taken for ph and active trypsin determinations. trypsin activity was determined with a kinetic method using s- (kabi vitrum b.v. amsterdam) as substrate. results: in all patients some trypsin could be detected in the gastric juice. in those patients with endoscopically erosive and/or ulcerative changes (n = ) the trypsin content was significantly elevated compared to the controls (/ < . wilcoxon). conclusions: the composition of gastric juice is determined by gastric secretion and duodenogastric reflux. duodenogastric reflux is increased in patients with symptoms of reflux oesophagitis [ ] . trypsin in gastric juice (ph . - ) will stable and partly active over prolonged periods [ ] . so from our study in man one may conclude that the high concentration of trypsin in the gastric juice in patients with reflux oesophagitis, may well be the etiological factor of the oesophagitis. disinfection, using artificial contamination of hands with escherichia coil and 'surgical' disinfection directed against the resident microflora of the hands. the authors who developed the procedure have reported unexpectedly high potency for n-propanol compared to iso-propanol, and, in turn, for iso-propanol compared to povidone-iodine and chlorhexidine preparations [ ] . however, using the same procedure we have been unable to find any significant differences in activity between these agents. we have identified several potential sources of error including the method of applying e. coliand its spontaneous loss of viability, the use of neutralizers before disinfection, the differing surfactant effects of the agents, and the absence both of a control untreated area and of a cross-over of disinfectants studied sequentially. in our parallel tests using an excision-sample technique [ ] which is considerably more sensitive than the dghm procedure, we have observed the following mean reductions in the counts of accessible bacteria: iodine in ethanol, %; povidone-iodine, %; chlorhexidine in ethanol, %; iso-propanol, the purpose of this study was to compare radiation injury in guinea pig small bowel ( ) devoid of contents ( ) containing bile ( ) containing pancreatic juice. one group was intact control animals not radiated. another was intact animals radiated. in group a roux y cholecystojejunostomy was constructed and the bile duct ligated. thus one limb contained bile, the other pancreatic juice. in group a blind roux y was constructed such that one limb was empty of contents and the other contained both bile and pancreatic juice. each animal was subjected to a single dose of rads via an abdominal port and sacrificed four days later. the severity of injury was judged by counting the number of surviving crypts per circumference. damage was further evaluated by histologic criteria -mucosal loss, edema, inflammation, hypervascularity and loss of mucus. the maximum histologlc grading score on this scale was . the microscopist was ,,blinded" as to the origin of each tissue section. for histologic grading all radiated groups were significantly different from control (p< . ) and from one another (/r< . ) except pancreatic juice vs. empty. intestine devoid of contents sustains a mild, but significant radiation injury. the presence of pancreatic juice enhances the damage. pure bile makes for yet more severe injury but still significantly less than normal whole intestinal contents which contain both bile and pancreatic juice. the main problem facing patients with ulcerative colitis after mucosal proctectomy and ileo-anal anastomosis is severe frequency of bowel action. our hypothesis was that either an artificial valve [ ] or reversed ileal loop might improve intestinal absorption and slow transit. we tested it in dogs after colectomy and low ileo-rectal anastomosis (ira). body weight, xylose absorption, serum albumin, folate, vitamin b , calcium, urea and electrolytes, full blood count, faecal weight and mouth to anus transit time [ ] were measured before operation. the dogs then randomly underwent either ira alone (control, c, n = ), ira with a cm reversed loop (ira + rl, n = ) or ira with an artifical valve (ira + v, n = ). body weight, haematological estimations and faecal chemistry were measured weekly for months post operatively. xylose absorption and transit time were measured a minimum of months after operation. body weight decreased significantly after each type of operation. there was no difference however, between (ira + rl) and c or (ira + v) and c. likewise, haematological and faecal measurements after both test operations did not differ significantly from c or from pre-op, measurements. the grosfeld valve prevented the reduction in transit time that occurred after the control operation, whereas the reversed loop did not. use of such a valve in combination with mucosal proctectomy might slow transit, but is unlikely to improve absorption. it seems worthy of further study. histologic grading___ sem crypt count-+-sem we have examined the effects of changes in intestinal blood flow induced by hypovolaemia, the mesenteric vasoconstrictors somatostatin and vasopressin and the mesenteric vasodilator prostaglandin e (pge ), on intestinal absorption, electrical activity and volume. in each of dogs a cm jejunal segment was isolated and serosal electrodes attached. in absorption experiments the segments were perfused at . ml/ rain with a solution of retool/ sodium and retool/ glucose. to measure segment volume, a solution of . g/ mannitol was perfused. this solution shows zero net absorption but does not alter electrical activity. intestinal motility and absolute volume were monitored by gamma camera. experiments were performed after a steady state was reached. absorption and transit time were measured using non-absorbable markers. electrical fast wave activity was reduced by (a) intravenous somatostatin . mcg/kg/h ( + . to _+ . ; mean_+sem/ rain) and abolished by (b) intravenous vasopressin . u/kg/h (p< . ). motility was inhibited and mean transit time was prolonged (a) . to . rain and (b) . to . rain (/r< . ). intestinal volume rose by (a) . ___ . ml and (b) . -+ . ml (p< , ). absorption was unchanged by somatostatin and fell with vasopressin ( . --- . to . -+ . ml/ min). acute blood loss sufficient to lower the central venous pressure by cm of water produced identical changes to somatostatin. pge produced no alteration in electrical activity or absorption. intestinal absorption is resistant to changes in intestinal blood flow whilst electrical activity is depressed and intestinal volume increased by mesentric vasoconstriction. the accepted views on the pathogenesis of amoebic lesions in complicated amoebic colitis are that amoebae produce a toxin resulting in cytolysis and necrosis. this concept may adversely affect the management of patients with complicated amoebic colitis by implying that once the amoebae are killed, the disease process is arrested and colonic perforation is unlikely. we present an alternative hypothesis that 'complicated amoebic colitis is the result of vascular compromise'. transmural disease is caused by amoebic invasion of vessels supplying a segment of the colon with subsequent thrombosis and ischaemic necrosis of the affected area. the ischaemic nature of the necrosis is suggested by its shape, and the demonstration of vascular thrombosis on dissection ofresected colons which have perforated. amoebic invasion of blood vessels can be demonstrated histologically. the ischaemic nature of the lesions can be confirmed by angiographic examination of the resected colon. vascular occlusion can be demonstrated pre-operatively with the use of selective mesenteric angiography, which clearly delineates the ischaemic segment of the colon. selective rnesenteric angiography in patients with fulminating amoebic colitis aided the preoperative diagnosis of colonic infarction before signs of visceral perforation had developed and permitted life saving surgical intervention. there is accurate correlation between angiographic and operative findings. in postdysenteric colitits associated with amoebic strictures of the colon, mesenteric angiography will demonstrate the ischaemic nature of the stricture and accurately define the extent of resection. after ca. the mean age at operation was and years respectively. anal canal length was . ___ . cm (mean + s.e.m.) after ia and . ___ . cm after ca. a resting tone of ___ cm water after ia and ----- cm water after cawas recorded. squeeze pressure was ___ cm water after ia and ----- cm water after ca. the recto-anal reflex was present in % of the ia patients and in % of the ca patients. a mean daily bowel frequency of . ___ . followed ia and +_ after ca. these findings show that after ia anal pressures are within the normal reference range for our laboratory [ ] . lower pressures were found after ca, probably due to the older age of the patients in this group [ ] . normal anal canal length, the integritiy of the striated sphincter and in most cases preservation of the recto-anal reflex contribute to satisfactory continence following peranal anastomoses. perineal descent is found in patients with idiopathic faecal incontinence (ifi) and patients presenting with symptoms of the descending perineum syndrome (dps), who have no incontinence. manometric, radiological and neurophysiological studies were performed in patients with ifi, all of whom leaked during rectal infusion of ml saline, patients with dps, who were continent of rectally infused saline, and matched controls. both patient groups exhibited similar degrees of perineal descent below the pubococcygeal line of straining (ifi, - . ___ . cm; dps, - . + . cm) compared with controis (- . __+ . cm;p< . in both cases), and similar prolongation of both the latency of the cutaneoanal reflex (ifi, ___ ms; dps, + ms; controls, + ms;/,< . in both cases), and motor unit potential duration (ifi, . ___ . ms; dps, . ___ . ms, control, . + . ms; p< . in both cases). moreover, both groups had an abnormal ano-rectal angle (/,< . ), though this was more obtuse in ifi than dps ( ___ ° vs ___ °;p< . ). however, while patients with ifi had lower sphincter pressures than normal (basal; ___ vs ___ cm water; / < . ; squeeze, ___ vs ___ cm water; / < . ), and required a lower rectal volume to inhibit sphincter tone for more than one minute ( + vs ___ ml; p< . l), these values were normal in patients with dps (basal; ___ cm water; squeeze, _+ cm water; rectal volume, ___ ml). these findings suggest that perineal descent and neuropathy are not necessarily associated with incontinence if sphincter pressures remain normal. aminoacids administered either enterally or intravenously stimulate gastric secretion in both dog and man. it has been suggested that absorption of amino acids from the gut into the circulation might contribute to the intestinal phase of gastric secretion. the mechanism of this stimulation by amino acids remains uncertain but in an earlier communication we reported the direct action on the parietal cell of phenylalanine, glutamine and alanine [ ] . in the present study using ( c) aminopyrine uptake as an index of dispersed parietal cell secretory function, the interaction between histamine and individual amino acids and the effect of the histamine h -receptor blocker ranitidine ( - molar) on these interactions have been examined. results (percentage of maximal stimulation produced by - molar histamine) results (percentage of maximal stimulation produced by - molar histamine) methionine glutamine alanine amino acids only ( - molar) amino acids and histamine ( - molar) - amino acids and ranitidine ( - molar) histamine and ranitidine -complete inhibition - . the response to the combination of amino acids and histamine was greater than the sum of the maximal responses to each of these agents alone. . the histamine h -receptor antagonist ranitidine completely inhibited the histamine stimulated response but only partially inhibited secretion stimulated by amino acids. we conclude that amino acids act directly on the parietal cell by a mechanism which is not solely dependent on histamine. sex related differences in gastric acid secretion have been documented (lilja et al, ) . it has been also reported that male rats have a significantly higher serum gastrin concentration than females and that oophorectomy increases serum gastrin to male levels (lichtenberger et al., ) . estimation of serum gastrin levels after administration of androgens has not been reported. aim. the aim of this study was to investigate the serum gastrin levels before and after oophorectomy and administration of estrogens and testosterone in female guinea pigs. method. groups of guinea pigs were used for this study, animals as controls, were given estrogens for weeks, were given testosterone for weeks and underwent oophorectomy. results. the mean serum (-+sem) gastrin value in controls was found + . pg/ml, after estrogens . -+ . pg/ml, after testosterone _+ . pg/ ml, after oophorectomy -+ . pg/ml. conclusion. it is concluded that estrogens decrease the serum gastrin, and that the increase of serum gastrin after administration of androgens and oophorectomy is statistically significant (/r< . ). the mechanism by which the ovarian hormones influence gastrin levels may be a sex-dependent change in the synthesis or secretion of gastrin. the reported inhibitory influence of estrogen on food consumption may be also the predominant factor in serum gastrin alterations. mean caloric intake was --+ kcal/day. moderate to severe dumping occurred in one patient. faecal fat was normal in while patients had greater than grams fat loss per day. weight loss was . _+ . % of recall weight. muscle mass measured by arm muscle circumference ( . _+ . cm) and creatinine heigth index ( ___ %) was normal for our patients but triceps skin fold was % less than normal ( . _+ . mm) indicating that in these patients fat stores were reduced. in six patients haemoglobin was less than grams per cent but serum iron, iron binding capacity and serum folate were normal. red cell folate was depressed in six patients whose haemoglobin values were normal. it is concluded that with this reconstruction total gastrectomy produces satisfactory digestive and "nutritional results. examination of factors affecting faecal fat loss and folate metabolism may help improve the nutritional status of these patients. in order to assess the optimal conditions for segmental pancreatic graft viability the following experiment was conducted. the duct obliterated splenic lobe of the canine pancreas was autotransplanted by end to side fashion to the femoral vessels (n = ). the graft was lodged in a subcutaneous pocket. a month later total pancreatectomy was completed. the dogs were supplemented with pancreatic enzymes (viokase). mean survival was ___ days. deaths were due to hyperglycemia when grafts lysed from local infection and thrombosis. in the second experimental group (n= ) the autografts were anastomosed to the iliac vessels and placed intraperitoneally followed by total pancreatectomy one month later. construction of a distal splenic arterio-venous fistula increased blood flow from . cc/min to cc/min. the pancreatic duct was obliterated with neoprene (n= ) silastic (n= ) or left open (n= ). three dogs died one month to four months due to graft fibrosis and failure. seven dogs had been followed from four months up to seven months when sacrificed. all were normoglycemic. mean k values for ivgtt were . . the normoglycemic dogs had mean plasma concentrations of amino acids similar to healthy controls. a two to threefold elevation was observed in pancreatectomized diabetic dogs for plasma leucine, isoleucine and valine. immunoreactivity of pancreatic polypeptide was measured by radioimmunoassay with an antibody raised against the human hormone by re. chance, lilly research laboratories. mean plasma levels rose form --+ s.e. pg/ml to over pg/ml following protein meal ( g ground lean beef/kg) in all normal control dogs (n= ), and to levels in the range of to over pg/ml in the same dogs following infusion ofbombesin ( pg/kg/ h) for min. mean basal levels were lower ( ___ . s.e. pg/ml) in dogs with autotransplants and did not increase significantly during any stimulatory test. the levels of pancreatic polypeptide did not distinguish between viable and failing grafts. in successful grafts histology showed fibrosis of the exocrine component. transmission electron microscopy revealed the presence of clusters of functional islet cells in the six-month implants. a and b cells were common. d cells were much less frequent. release of secretion granules into the perivascular connective tissue space was observed. the results indicate that vascularized segmental pancreatic graft comprising % to % of the pancreatic mass maintains normal carbohydrate and amino acid metabolism. graft survival was extended by intraperitoneal location and creation od distal splenic a-v fistula, but chronic graft fibrosis occurred regardless of the method of pancreatic duct treatment. the results further suggest the necessity of an intact vagal innervation for the physiological or pharmacological stimulation of pancreatic polypeptide release from thendocrine pancreas which is otherwise apparently functional. besides, these considerations rule out pancreatic polypeptide levels as a useful marker for evaluation of pancreas graft. polyisoprene ductal occlusion allows segmental pancreatic transplantation in man with satisfactory early islet cell function. however acute rejection remains a problem as the diagnosis, based on hyperglycaemia and glycosuria, represents a late manifestation of rejection. although llqndiumlabelled platelets have been used in the diagnosis of renal rejection [ ] , their use in pancreas transplants has not been studied. ~ in patients, autologous llqn-labelled platelets were injected on the second, sixth and tenth days following combined renal and pancreatic transplantation. graft radioactivity was expressed as the ratio of counts from the pancreas over a reference area on daily gamma images for days. one patient developed hyperglycaemia coinciding with renal rejection on day . over the previous h pancreas radioactivity had increased by %. in a further patient whose pancreas continued to function, a perigraft haematoma was recognised on the gamma image. the remaining patients had good pancreatic function and no xin-platelet accumulation: mean (_s.c. mean) pancreatic radioactivity was . + . on the third postoperative day and . _+ . at the end of study. these results demonstrate that ductal occlusion with polyisoprene does not cause significant platelet accumulation. hence min-platelets may potentially be used for the earlier diagnosis of pancreatic rejection. isograft models of pancreatic transplantation, methods involving closure of the duct system result in severe inflammatory changes and eventual fibrosis [ ] . these changes can be avoided by formal drainage of the duct into the bowel of urinary tract. inflammatory changes initiated by duct closure may contribute to and enhance allograft rejection. pancreas transplants were performed in rats using lewis (rt ) donors and streptozotocin-induced dia- betic da(rt a) recipients, using duct-ligation, open duct and ureteric duct drainage. cyclophosphamide produced significant prolongation ofnormoglycaemia in all groups and although there was a tendency towards longer function in the unligated groups there was not statistical difference (wilcoxon's unpaired test) between the methods of duct management. management of the pancreatic duct did not seem to have any immunological consequences for graft survival but septic complications, associated with the normoglycaemic deaths, were more common in immunosuppressed animals with draining ducts. between july , and march , combined pancreatic and kidney transplantation was performed in patients with type i diabetes who had all been on insulin therapy for at least years. age at the time of transplantation was to years. the pancreatic segment used for transplantation consisted of body and tail of the organ based on a vascular pedicle of the celiac axis and the splenic vein. imediately prior to intraperitoneal transplantation to the iliac vessels the ductal system was filled with to ml of prolamine, a rapidly solidifying alcoholic protein solution. simultaneous kidney transplantation was performed through a separate incision. five pancreas transplants were lost for non-immunological reasons. four of them never had any useful endocrine function, one graft was lost of venous thrombosis after hours of excellent function. in none of these patients did failure of the graft lead to any substantial complication. in the remaining patients initial graft function was excellent with plasma glucose normalizing within hours and subsequent normoglycemia on a regular diet without exogenous insulin administration. plasma glucose did not rise spontaneously during rejection episodes of the kidney and eleva-tions due antirejection treatment were promptly; reversed as high dose prednisolone was discontinued. in oral glucose tolerance test (ogtt) performed at to weeks in patients median glucose rose from a basal . mmol/l to . mmol/ and was back in normal range ( . mmol/ ) at h. basal insulin was - pmol/ and also returned to the normal range after h after a peak of - pmol/ at - rain. c-peptide showed a significant peaked rise over basal values ( , pmol/ ) in patients while all values were above pmol/l in the third. two of these patients have since rejected both organs. two tranplants still function very well after and months, respectively. in one of these patients the ogtt after months is even slightly better than the above -mentioned first test, and he shows a norreal insulin and c-peptide response. in the fourth patient an ivgtt performed at months and an ogtt at sixteen months were normal with insulin peaking at pmol/ and a c-peptide peak of pmol/ at min in the latter. -results at and months, respectively, will be presented. three problems persist in clinical organ preservation. these are failure of current systems to replenish the ischemically injured organ, to reliably extend the period of organ preservation and to definitely determine organ viability. previous studies have documented the value of electrochemical redox control in rejuvenation of the ischemically injured kidney during perfusion preservation. this study was undertaken to develop the cost effective technique applicable to current organ preservation systems, to test the reliability of redox measurement in prediction of organ viability and to determine the ability ofredox maintenance to safely extend the period of organ preservation. a disposable cell has been developed using reticulated vitreous carbon as an electrode which is driven by a potentiostat powered by a nine volt transistor battery. short term preservation studies using ischemically injured dog kidneys ( rain in-situ warm ischemia) were auto transplanted after h of pulsatile preservation to determine the optimum redox level of the hypothermic kidney. this was determined to be a - mv vs the standard calomel electrode. twenty-one adult mongrel dogs were equally divided into three groups. pulsatile perfusion preservation was extended to four days in group a with redox level monitored only. group b was treated with electrochemical reduction for four days and group c for six days. three of seven dogs survived in group a following auto transplantation and immediate contralateral nephrectomy. the kidneys of all survivors were able to bring perfusate redox potential under control and maintain this level throughout the preservation period. six of seven dogs in group b survived with a mean posttransplant peak serum creatinine of . rag/ ml. in group c four of seven kidneys supported life immediately. all redox controlled kidneys made copious amounts of urine. our data indicate that perfusate potential may be either monitored as a reliable index of organ viability or controlled to allow extended safe preservation. antithymocyte globulin (atg) has been shown to be an effective agent in combination with increased doses of steroids in reversing renal allograft rejection. since it is frequently undesirable to employ raised doses of steroids, the following study evaluated the effect of - i.v. daily doses of horse atg alone without additional steroids, on nd- th rejection episodes in recipients of cadaveric renal allografts ( rejections: biopsy-proven) detected within - mos. of transplantation. of rejection episodes, were successfully reversed, with return of serum creatinine to pre-rejection levels in episodes ( patients). three patients had primarily humoral rejections and returned to dialysis - mos. after treatment of the last rejection. levels of circulating horse immunoglobulins were obtained in all patients during and follwing administration of atg. recurrent rejections ( rd- th) following last treatment with atg ( - mos.) were seen in / patients. all patients had rapid immune eliminiation of atg ( / life - days) as compared to the patients who had no more than rejection episodes ( / life - days). atg without additional steroids is an effective agent for reversal of multiple renal allograft rejections which by biopsy are primarily cell-mediated. to be effective, heterologous atg must be given in adequate total doses and/or from appropriate heterologous source, to prevent rapid immune elimination by the recipient. the use of atg alone for treatment of recurrent allograft rejections is particularly recommended for its steroidsparing effect in treatment of multiple rejections and for those patients at high risk from steroid side effects. the significance of the monocyte crossmatch in lrd recipients of hla identical kidney grafts j. cerilli, l. brasile and s. rogers ohio state university hospitals, columbus, ohio, usa in a preliminary study from our transplant center, the presence of pre-formed antibody in recipient sera directed against monocytes from their respective living-related donors correlated with a poor clinical course. a poor prognosis for graft survival was found regardless of the hla match grade. to minimize the role of the hla system, only those living-related recipient/donor pairs who were hla identical at the a, b, c, d and dr antigen loci, and who exhibited severe immunological types of rejection were evaluated. due to the small numbers found in this category at any one center, this abstract represents an international study from different transplant centers. patients who met the criteria were studied for the presence of antibody directed against their respective donor's monocytes both pre-and posttransplant. in eighteen of these patients, cytotoxic antibody against their donor's monocytes was found in their pre-transplant sera. there was no detectable cytotoxic activity against their donor's t or b lymphocytes. two additional transplant recipients exhibited this antibody in post-transplant sera. again, no t or b lymphocyte cytotoxicity was detected. a control group of hla identically matched siblings who incurred no or minimal rejection demonstrated no anti-donor monocyte antibody. the results of this international study points towards a correlation between a high incidence of graft rejection and the presence of antibody directed against their respective donor's monocytes. therefore, in our view, the presence of anti-monocyte antibody to the prospective donor pre-transplant is a contraindication to transplantation. in patients with different liver diseases the reduced concentration of the peripheral blood t-cells and altered immune response were observed. the purpose of our studies was to investigate the mechanism of the immunological modification of the lymphoid system in the hepatic injury. studies were carried out in groups: ) lew rates were treated with dimethylnitrosamine (dmna), mg/kg b.w., i.v. for acute liver necrosis induction, ) cc ( . ml/ g b.w., i.v.) twice weekly, over weeks for chronic liver damage, ) ccl over weeks for liver cirrhosis (histologically examined). serum and thymus, spleen, mesenteric lymph nodes (ln) were removed and days after dmna treatment and and days after last cc injection. the total number of thymocytes and spleen cells was counted and the reactivity to pha and cona was measured. normal liver perfusate (lp) was prepared h after removing ( °c) by times perfusion in rain intervals (flow rate ml/g/min with ml/g of ringer). the effect oflp, sera, dmna and cc on the viability and pha response of normal thymocytes was tested in vitro. liver enzymes were evaluated, we found the decreased total number of thymocytes immediately after the stopping of medication (group : , ___ , %, group : , ___ %, group : less than % of control). proliferative response of the remaining cells in thymus to pha was much higher than normal thymocytes (group : ± % group : ___ %, group : nd). the total number of spleen cells was not changed but their response to cona was altered in the all groups and to pha only in group . pha response of ln-cells was decreased in the all groups. liver perfusate was cytotoxic ( ___ % of viable cells) and suppressive for pha response of thymocytes ( _+ % of control). the sera of rats with the hepatic damage showed an enhanced suppressive activity. cc and dmna did not have any effect on thymocyte in vitro. one month after last medication we observed the recovery of thymus involution (total i in the acute and partial in the chronic hepatic damage and cirrhosis). our results suggest that the liver origin cytotoxic and immunosuppressiv e factor(s) can be released from the damaged liver into the circulation (like to lp) and can destroy the thymus leading to the secondary changes in the other lymphoid organs. the grade of thymus alteration is dependent on the degree and the duration of hepatic damage and is reversable. the hepatic factor (s) showed the similar effect on the cortical population of thymocytes like the steroid immunosuppressants. liver grafts in the rat are in certain strain combinations not rejected and in this situation there is evidence for spontaneous donor specific tolerance [ ] . we have developed a model of auxiliary liver transplantation which would allow us to study the immunosuppressive properties apparently produced by a liver allograft. the portal vein is anastomosed to the left renal artery, the i.v.c. to the renal vein and the bile duct to the ureter. simultaneous kidney or heart allografts were performed. conclusions:auxiliary liver grafts are rejected. survival of heart or kidney grafts is not influenced by a simultaneous kidney or heart allograft. heart and kidney grafts are prolonged by simultaneous auxiliary liver grafts. [ ] . the role of suppressor cells in the initiation and propagation of malignant tumours in man, is less clearly defined. the present study, using in vitro mitogen assays (pha, cona, pwm) and various rosetting assays [ ] with specific monoclonal antibodies to lymphocytic helper (okt ) and suppressor (okt ) cells, has revealed the presence of such suppressor lymphocytes in women with clinically localised (breast and axilla) mammary carcinoma. lymplaocyte hyporeactivity to mitogens was found in % of lymphocyte preparations from blood and axillary lymph nodes of patients with breast cancer. in % of patients nodal lymphocytes were totally anergic. the most profound hyporeactivity, however, was detected in the lymphocyte subsets (< %) of specimens isolated from the breast carcinomas by collagenase digestion and sephadex g- column passage [ ] . the lymphocyte preparative techniques were not responsible for the low levels of responses detected. also, in situ prostaglandin synthesis and release did not appear to be involved in depressing lymphocyte reactivity [ ] . comparable percentages of suppressor cells (okt +) were detected within these different lymphocyte preparations. suppressor cells were not found in the lymphocyte preparations from the blood and lymph nodes of appropriate controls. from clinical and experimental studies it is known that blood transfusions may have immunosupressive as well as immunostimulating consequences. the effect of transfusions on graft survival has been extensively studied by our group in the bn to wag rat model. in this donor-host combination it was found that a donor specific pre-transplant blood transfusion could lead to a marked prolongation of heart and kidney graft survival, whereas the similar pretreatment resulted in accelerated rejection of bn skin allografts. this specific model was used to investigate the influence of a single bn transfusion on the growth of different syngeneic transplantable tumors in wag rats. the first tumor was a radiation induced basal cell carcinoma of the skin (t ), the second tumor was a chemically induced adenocarcinoma of the duodenum (t ). the antigenicity of both tumors was assessed in vivo, using classical challenge-protection experiments. it was observed that t i exhibited strong immunogenetic properties, whereas t was only weakly immunogenic. the doubling time oft i was . days, the doubling-time of the adenocarcinoma was days. intravenous inoculation of isolated t cells led to development of lung nodules which could be counted after days. wag rats were injected i.e. with ml of bn blood or syngeneic blood (controls) at - days before tumor challenge. t l was given in two different ways: a) sc. implantation of+ x mm pieces, b) i.e. injection of isolated tumor cells. t was implanted sc. only. each experimental group consisted of animals. for t it was found that allogeneic blood transfusions caused a slight (but not significant) inhibition of subcutaneous tumor growth. however, in the t lung-metastasis model it was observed that a single bn blood transfusion led to a % reduction of nodules, counted at weeks after inoculation. this reduction in number and size of nodules was highly significant (p< . ). for tumor t , the bn blood transfusions evoked a strong inhibitory effect on the growth of the sc. implanted tumor. at weeks after implantation all tumors in the control group had grown to a diameter of - rnm (average diameter . ram). in the group pretreated with bn blood, only of tumors were palpable at that time (average diamter mm). for t it was further investigated whether a single bn transfusion, given one week after i.e. tumor cell inoculation, would have any influence on tumor growth. no significant effect on number or size of the lung nodules could be noticed, if anything, the transfusion appeared to have a stimulatory effect. the results indicate that allogeneic transfusions can lead to a substantial modification of tumor growth, depending on tumor type and site of implantation. this observation may have important clinical implications. we report here the serial study of circulating immune complexes (cic) in two human tumor systems, colorectal cancer and gestational trophoblastic neoplasia (gtn). cic were assayed by antigen nonspecific insolubilization induced by . % polyethylene glycol (peg) and monitored as a od o changes by spectrophotometry. all of the serially studied colorectal cancer patients presented with elevated cic levels (mean = + zt od ) as compared to our standard cic level for pooled normal human sera ( --+_ aod , p< . ). initial values in these patients range from to a od with no correlation to tumor load, site of presentation, or subsequent clinical course. in / patients who underwent ,,curative" resection of primary or metastatic colorectal cancers, serial cic elevations occured only when antigen excess (measured by simultaneous carci-noembryonic antigen [cea] assay) decreased. immunoglobulin components of fractionated cic showed predominantly iga subclass. in gtn patients followed with serial cic and simultaneous human chorionic gonadotropin (hcg) assay, only those patients documented to enter hcg remission after molar evacuation showed significant elevation of cic. chromatographic fractionation of peak cic in one such patient defined three irnmunoglobulin containing fractions showing immunoreactivity to one of four paternal hla haplotypes (aw ). one ,,antigen" fraction (< . mw) from this complex completely inhibited reference anti-aw binding. as in the colorectal cancer patients, these data show that cic rise only when antigen excess decreases (reflected in the gtn patients by hcg normalization). in addition, some gtn patients may react to immunogenic paternal hla haplotypes as part of their response to molar pregnancy. dfmo, an enzyme-activated irreversible inhibitor of ornithine decarboxylase (odc), reduces tumor polyamine levels, inhibits growth of emt sarcomas and hepatomas in experimental animals, and induces remission in human leukemia. a renal adenocarcinoma (ra) cell suspension, or a mm segment ofwilms' (wm) tumor was transplanted intrarenally into balb/c mice (n-- ) or subcutaneously into wistar-furth rats (n= ) respectively. dfmo ( %) in drinking water was administered to half the animals in each group throughout the experiment. at days ra tumors in dfmo-fed mice weighed % less than tumors in control animals (p< . ); wm tumor weight at days was not affected by dfmo feeding. the mean number of lung metastases in dfmo-fed r.a-bearing mice was . and in ra-bearing control mice was . (p< . ). dfmo caused - % inactivation of tumor odc, reduced ra putrescine levels by /o (p< . ), reduced wm putrescine levels by % (d < . ), reduced wm spermidine levels by % (p< . ) and increased wm spermine levels by % (p< . ). dfmo feeding did not alter dna content of ra or wm tumors. although final carcass weight was similar in all animals, dfmo feeding progressively reduced total body weight (tbw) of mice, but not rats, until at day the tbw of dfmo-fed mice was . % less than tumor-bearing control mice (p< . ). dfmo-fed mice bearing ra tumors survived . -t- . days longer than control mice (p< . ). reduction of polyamine levels in wilms' tumors does not affect tumor growth. lowering of renal ade-nocarcinoma putrescine levels by continuous feeding of dfmo to tumor-bearing animals decreases tumor growth, reduces lung metastases, and increases host survival. we have previously demonstrated that vagal nerve stimulation releases -ht into the lumen of the feline gut. this study was initiated to: . determine if substance p (sp) and motilin (mt), other enterochromaffin cell products, are released simultaneously, . to evaluate if this release is under cholinergic or adrenergic control. in cats, cm isolated in situ segments of proximal jejunum were perfused with saline at ml/min ( ~c). perfusate samples were evaluated at rain intervals and concentrations of -ht, sp, motilin were measured by ria's developed in our laboratory. after two -min basal periods, the supradiaphragmatic sectioned vagus nerves were stimulated electrically ( v, m s, hz). the output from the loop in ng/ min ( -ht) and pg/ rain (sp and mt) was: introduced into the jejunum of conscious dogs through an external small bowel fistula. the gut was perfused at ml min- with a physiological electrolyte solution containing the non-absorbable marker polyethylene glycol (mol. wt. ; g - ); water and electrolyte absorption and transit time (tt) were measured during intravenous (i.v.) administration of each peptide, and during preceding and succeeding i.v. control infusions of . m naci. separate studies showed jejunal absorption and tt to be constant over prolonged periods during i.v. nac administration. bombesin ( pmol kg-lmin- ) and neurotensin ( pmol kg-~min ) significantly reduced jejunal water absorption; bombesin and enkephalin ( . nmol kag- rain- ) significantly prolonged tt; and enkephalin encreased water absorption (/ < . in all cases). measurement of plasma neurotensin during i.v. infusion indicated that physiological blood levels were not exceeded during these studies. conclusion: a number of peptides may be involved in the regulation of small bowel function. the effect of neurotensin on jejunal water transport provides a possible mechanism linking raised blood neurotensin levels with intestinal intraluminal fluid accumulation in the dumping syndrome. in order to establish whether this release was under adrenergic control, cats had cervical ganglionectomies. using the same electrical paramenters, stimulation of the cut cervical vagus nerves resulted in identical -ht responses as above. in additional cats atropine administration (lmg/kg iv) totally abolished the -ht responses to vagal nerve stimulation. the paralled release of -ht, sp, and mt following vagal nerve stimulation, strongly suggest that the ec cell is the source of these luminal hormones. this release appears to be under cholinergic control. since diabetes mellitus is markedly improved immediately after jejunoileal bypass before significant weight loss, but only gradually and often incompletely changed after gastric bypass, it seemed appropriate to investigate the effects of intestinal exclusion on experimental diabetes. studies were performed on alloxan diabetic sprague-dawley rats. two days after jejunal exclusion (je) in rats (resection of proximal / of small intestine), fasting blood sugars (fbs) decreased , from to mg/dl, and averaged mg/dl at weeks. after ileal exclusion (ie) in rats (resection of distal / of small intestine), fbs fell % in days, from to mg/dl, but increased % above preoperative levels to mg/ dl at weeks. sham operated rats responded similarly to ie rats. after alloxan and operations, all groups lost weight, but only je rats began to increase weight at a normal rate. increased water intake, polyuria ( ml/ h), and glycosuria ( rag/all), were present in ie rats; je rats were normal (urinary output < ml/ h, and urinary glucose mg/dl). oral glucose tolerance tests (gtt) were extremely abnormal in ie rats, similar to those in non-operated alloxan rats, while gtt curves in je rats were similar to normal animals, with some elevation at , and min. serum insulin levels remained low in all alloxan treated rats after jejunal exclusion. possible mechanisms, currently under study, relate to carbohydrate malabsorption and changes in enteric chemical mediators. the purpose of the present study was to investigate the changes in somatostatin release and somatostatin-containing cells of the pancreas and stomach of the streptozotocin (stz) -induced diabetic rat after the amelioration of diabetes by whole pancreatic transplantation. highly inbred lewis rats were divided into three groups: ( ) normal rats, ( ) stzinduced diabetic rats and ( ) transplanted rats. diabetes was induced by the administration of stz ( mg/kg). on the seventh day after stz treatment, pancreatic transplantation was performed. four weeks after the transplantation, in vivo and in vitro, studies were performed. pancreatic d cells and gastric somatostatin-containing cells were stained with antibody enzyme method. studies in vivo showed marked improvement of the impaired arginine-induced insulin release by the transplantation. studies in vitro employing isolated perfused rat pancreas and stomach revealed following results: mean basal pancreatic somatostatin release in normal, diabetic and transplanted rats were ___ , -t- , and __+ pg/ml, respectively. total amount of pancreatic somatostatin release in each group during arginine stimulation ( . . mm) were --+ , ___ , and -+ pg/ min, respectively. significantly higher somatostatin release was obtained from the diabetic pancreas, which was, however, reduced to normal after the whole pancreatic transplantation. on the other hand, insulin release from the diabetic pancreas was severly impaired and pancreatic transplantation had not effect on insulin release from the host pancreas in the transplanted rats. as to the glucagon release, there was not significant difference among them. mean basal gastric somatostatin release in normal, diabetic and transplanted rats were -t- , ___ , and + pg/ml, respectively. there was no significant difference between normal and diabetic rats, though the significant decreased value was obtained in the transplanted rats. (vs. normal;/ < . , and diabetes; / < . ). on the other hand, glucagon-stimulated peak values in these groups were _ , ___ , and + pg/ml, respectively. glucagon-stimulated gastric somatostatin release in diabetic rats was significantly increased, but reduced to normal value by pancreatic transplantation. also, a number of pancreatic d cells and gastric somatostatin-containing cells were markedly increased on the diabetic rats. on the other hand, a number of these cells in the transplanted rats were descreased to normal levels. in summary, enhanced pancreatic and gastric somatostatin release and cells in the diabetic rats were both normalized after the amelioration of diabetes by the whole pancreatic transplantation. from these results, it is suggested that pancreatic and gastric somatostation are regulated by circulation and/or metablic of nutrients. doppler velocity recordings are widely used for the non-invasive diagnosis of carotid arterial disease. although detailed analysis of carotid doppler spectral information has been suggested as a method for improving diagnostic sensitivity, the accuracy of the relationship between the doppler recording and the true instantaneous velocity profile has not been established. the purpose of this study is to determine if a cw doppler velocitymeter can accurately transduce the true instantaneous blood flow velocity information. methods: a pulsatile flow model has been constructed in which it is possible to record the instantaneous doppler spectrum and simultaneously photograph and measure the true velocity profile. a computer controlled pump generates a carotid waveform in tubes without stenoses and with, symmetrical stenoses. flow is visualized using the photochromic dye tracer technique. a short burst of uv light from a laser is passed across the tube. a narrow band of the fluid turns blue, its movement is photographed, and the instantaneous velocity profile determined every msec throughout the pulse cycle. at the same time, the instantaneous doppler spectral information is recorded by a frequency analyzer. the results follow. for pulsatile laminar flow, the doppler spectrum correctly recorded the true velocity spectrum, including the instant/~neous maximum velocity and mean velocity. for disturbed flow, it was not possible to show the same direct relationship between the doppler spectral recordings and the blood flow velocity. in conclusion doppler velocitymeters accurately transduce velocity information when flow is laminar but when flow is disturbed there is not a direct relationship between doppler recordings and the true velocity profile. consequently, one should be cautious in attempting to relate doppler measurements of disturbed flow directly to the true changes in the velocity pattern. early failure of arterial reconstruction may originate in poor patient selection. in aorto-iliac stenosis (ais) selection for operation relies upon clinical examination of the femoral pulse and radiology. since single plane arteriography is inadequate for accurate definition ofiliac stenosis [ , ] , this paper compares clinical examination, doppler ankle systolic pressure (aspi) and femoral signal analysis (laplace transform damping, ltd, and pulsatility index, pi) [ ] with biplanar contrast studies. i at biplanar angiography of ischaemic lower limbs had ais with diameter reduction from - %, of the remainder, were normal (< % stenosis) and were ,,occluded" ('_-- / ). nearly two thirds ( %) of limbs with a clinically normal femoral pulse had identifiable arteriographic stenosis (~-__. / ), upstream abnormality was predicted incorrectly in % and the overall accuracy of clinical examination was %, both for detecting stenosis and predicting its severtiy. aspi (median . ; % confidence ,limits . - . ) and pi ( . ; . - . ) although correlated with stenosis (aspir = . ; p = . , pir = . ; p= . variance analysis for linear regression), did not aid the clinician further (accuracy %). however ltd ( . ; . - . ) was well correlated (r= . , p= . ) and did improve assessment ofiliac stenosis (accuracy %). the need for biplanar arteriography is reiterated and its use with doppler signal analysis should improve the evaluation of aorto-iliac disease. in the absence ofa non-invasive method for estimating volume flow in an individual artery, local blood pressure measurement has proved, with certain limitations, useful in assessing the cardiovascular system. now ultrasound technology has progressed to enable blood flow in an artery to be measured noninvasively. we report the results o four evaluation ofa mhz, computerized, channel, pulsed doppler vessel imaging and flow measuring instrument in in-vivo experiments. computed blood flow was compared to actual blood flow (calculated by timed collection) in anaesthetised dogs. correlation between computed and actual blood flow was stronger in the larger abdominal aorta than in the smaller common carotid arteries. from the regression plot, the coefficients of determination, p, were: . (exposed aorta scans); . (transcutaneous carotid scans); and . (exposed carotid scans). stepwise regression analysis showed the computed flow values to be independent of probevessel angle, depth and lumen diameter for vessels greater than . mm in diameter. these results suggest that this pulsed doppler instrument has the versatility and accuracy essential for diagnostic flow measurements in the main conducting arteries of the neck and limbs and in vascular bypass grafts. in the assessment of patients undergoing carotid artery surgery, many laboratory methods are available in addition to angiography. in a series of patients experience has been gained with the use of eeg, tc m isotope scanning, opg, ct scanning and doppler. in a year follow up over % of patients had a satisfactory outcome. an early mortality of % in the beginning of the series has been eliminated due to improved selection. in this report the application of multi-gated pulsed doppler techniques is reported. this allows a non invasive measurement of mean volume flow in the common carotid artery with a method reproducibility of + %. mean volume flow in undiseased arteries ( subjects mean age years) was found to be ± (s.d.) ml/min. from this a lower range for normal flow of ml per minute ( x s.d.) was selected. patients were investigated before surgery and a follow up examination was performed at a mean interval of v months post operatively. groups were defined. group a > ml/min; group b - ml/min; group c < ml/min, of arteries in group a before surgery, remained in the group and dropped to group b. in group b, of arteries, go to group a, remain and dropped to group c. in group c, out of arteries moved to group a and to group b. thus of arteries with below normal volume flow before surgery, were returned to normal range and further improved. remain unchanged and disimproved. of the arteries examined ;/ months after surgery, are in the normal range and a further improved. remain unchanged and disimproved. of the arteries examined / months after surgery, are in the normal range in flow values and a further remain unchanged. the non-invasive and isotope techniques have a valuable and practical application in assessment of carotid artery surgery. timing is influenced by the finding fo infarction on ct or isotope scanning. doppler techniques are useful not only in defining severity of diseas and sub-radiological plaques, but valuable flow information can be obtained by pulsed doppler pre and post operatively. this may help in identifying patients who need further medical or surgical treatment. stepwise logistic regression-amodel for predicing success of femorai-popliteal bypass grafts the objectives of this study were to identify the preoperative factors that influenced postoperative patency of femoral-popliteal grafts and to develop a model that could be used prospectively to determine the probability of successful outcome. data base material consisting of history, physical examination, laboratory data, angiography, and operative findings in patients undergoing femoral-popliteal bypass grafting was entered into a computer programmed for stepwise logistic regression analysis. the computer identified and ranked factors that influenced outcome. the top five factors (other than technical problems) included quantity of runoff, previous ipsilateral femoral-popliteal bypass, preoperative prediction of potential amputation level, concurrent proximal vascular reconstruction, and the location for distal graft anastomosis. having established the computer data base, it is now possible to enter information from new patients into the computer which will weigh all factors and indicate the likelihood of surgical success. in addition, tables can be generated which will look at simple combinations of variables to predict patency. for example, in a patient about to undergo a primary femoral-popliteal bypass with no anticipated technical problems, the likelihood of success as a function of runoff and preoperative amputation level is as follows: irreversibility of shock and ischemic injury is generally considered a consequence of extensive cellular injury. to study the role of intravascular coagulation in shock, rats were bled to a mean arterial pressure of mm hg for hrs or % uptake of shed blood, whichever occured first. return of shed blood with these data provide the patient and the surgeon with a quantitative prediction for success and permit an informed decision when considering therapeutic alternatives. potential cytoprotection by heparin was studied by similarly bleeding additional rats; controls were only cannulated. twenty pairs were heparinized (h); were not (nh). paired-bled and control rats were sacrificed following hemorrhage, liver (l) and kidney (k) mitochondria were isolated, and the isolates were studied by the polarographic technic with glutamate and succinate to determine the respiratory control index (rci) as a measure of cellular injury. results were: an equal volume of isotonic saline resulted in a % survival; % uptake of blood during shock allowed prediction of survival. an additional rats were then randomized (coin toss) to heparinization versus no heparin prior to shock, and were similarly bled and resuscitated. significantly improved survival (p< . ) was seen in heparinized ( / ; / ) versus nonheparinized rats ( / ; %). uncoupling and inhibition of mitochondrail function were noted in both h and nh rats with rci being significantly reduced from control. however, there was no difference between h and nh compared to each other. heparin does not provide cytoprotection during shock; improved survival with heparin may rather be a consequence of improved reperfusion of tissues following the shock episode. fl-endorphin (b-end) has been postulated to play a role in the pathogenesis of shock because the opiate "antagonist naloxone improves the macrohemodynamics in various shock models [ ] . however, plasma levels ofopioid peptides have not been determined as yet. the aime of our study was to measure the plasma concentrations of various peptides and to evaluate the influence of naloxone particularly on the plasma concentration of r-end. in anesthetized foxhounds, the adrenolumbar vein was cannulated and hemorrhagic shock (map = mm hg for h) was induced according to wiggerstechnique. the plasma levels offl-end, methioninenkephalin (m-enk), and leucine-enkephaline (l-enk) were simultaneously determined in c.v. and/ or adrenal venous blood by a specific ria. crossreactivity of r-end withfl-lipotropin was about %. the enk-antibodies crossreacted with less than %. five dogs received an i.v. bolus of naloxone ( mg/ kg) and a subsequent naloxone infusion of mg/kg/ h after h of hypovolemia. eleven dogs served as control and received equivalent volumes ( mg/kg per h) of ringer solution. hemorrhage resulted in a sharp rise of central venous plasma levels particularly of m-enk and l-enic this effect was even more pronounced in the adrenal's effluent system.fl-endorphin levels remain elevated whereas the enk secretion began to decrease h after hemorrhage. naloxone treatment inhibited any spontaneous fall of adrenal enkephalin release during the shock phase and the values remained elevated - fold. volume substitution with autologous blood resulted in a normalization of all peptide levels. these data demonstrate that hemorrhagic shock will cause stimulation of endogenous opioid peptides. the high levels of enkephalins in the adrenolumbar vein indicate that the adrenal gland is the main source of these peptides in the circulation. in addition toil-end, the enk seem to play a role in the pathogenesis of shock as well. at our present state of knowledge, however, it is difficult to design a coherent concept of mechanisms involved. this shows that cp treated cells bound nearly as much [ t]-acth analog as control cells but there was very little specific binding to sp treated cells. low concentrations of acth effectively displaced the acth analog whereas exposure of adrenocortical cells to sp resulted in a significant decrease in acth receptors. this suggests that sp has a factor(s) that binds to acth receptors of adrenocortical ceils which may adversely affect the stress response of shock. f- has been proposed as superior to other asanguinous fluids due to increased oxygen carrying capacity. evaluation to date has been largely uncontrolled and at extremes of hemodilution (hct. < %) rarely seen clinically. near infrared spectrophotometric monitoring of brain cytochrome a, a redox state, a sensitive indicator ofintramitochondrial oxygen availability, offers a unique opportunity to contrast f- with balanced salt-albumin (bsa) and whole blood saline (wbs) as a resuscitative regimen in a clinically relevant model. fifteen rats were subjected to minutes of hypoxia (fio = , %) and hemorrhagic hypotension (map = mmhc), then randomly allocated to one of three groups and resuscitated by fio = % and infusion of either f- , bsa, or wbs. cytochrome a, a redox state was monitored continuously at run. thirty additional rats were sacrificed at baseline, end shock and and minutes post resuscitation for cerebral cortical atp and lactate assay. despite hematocrits as low as % in the bsa and f- groups, there were no significant differences / < . between groups in the parameters of oxygen sufficiency; atp, lactate, and cytochrome a, a redox state. we assume differences in cardiac output compensated for differences in arterial oxygen content. on this basis we suggest perfluorochemical utilization should be limited to situations in which hematocrits are < % and when cardiac reserve is limited. metabolites of the prostaglandin endoperoxide h (pgh ) affect both vascular tone and platelet aggregation and thereby may influence blood flow. we, therefore, determined the metabolites formed from pgh by microsomes isolated from human saphenous vein used for aortocoronary bypass surgery. in the absence of reduced glutathione (gsh), the enzymatic metabolism of c-pgh produced only prostacyclin (pgi ) as measured by the formation of its stable breakdown product -keto-pgfla. the amount of pgi formed varied from - % of the substrate depending upon the microsomal protein and pgh concentrations. in addition, the nonenzymatic breakdown of pgh resulted in the formation of pgf a, pge , pgd and heptadecatrienoic acid (hht). there was no formation of thromboxane a (txa ) as measured by the absence of its stable breakdown product txb . in the presence ofgsh, a required cofactor for microsomal pge isomerase activity, the formation of pge was augmented fold (up to % of the substrate) indicating enzymatic for-mation of pge . the gsh either did not alter or augmented (less than fold) the formation of pgi . the increased formation of pge in the presence of gsh was at the expense of decreased nonenzymatic breakdown of pgh to pgf a, pgd and hht. these data suggest that prostacyclin synthetase activity may serve to protect the vessel graft from platelet aggregation and/or vessel spasms and may possibly serve as an indicator of graft viability. thrombosis is a frequent cause of early arterial bypass graft failure and platelets are known to be major determinants ofthrombus formation on arterial surfaces. pgi and flbriolytic activators from the vascular wall counteract intravascular thrombosis. the aim of this work was to study the effect of arterial grafting on the aforementioned mechanisms. cm lengths of tanned human umbilical vein grafts (huvg) with an internal diameter of mm were interposed end-to-end in the carotid arteries (c.a.) and jugular veins (j.v.) of sheep. placed in the c.a.'s, grafts with restricted flow ( cc/min) were removed on the th postoperative day (group i); grafts with unrestricted flow ( ___ cc/min after placement) were taken out days later (group ii) and grafts placed in the j.v.'s were removed days after surgery (group iii). upon removal, the grafts were checked for patency and sections from the proximal and distal anastomoses and midgraft were obtained for determination of pgi production (ria) fibrinolysis activators activity (histochemical method) and for light and scanning electron microscopy. sections from the femoral arteries were also obtained. the results of pgi generation are expressed in ng/ml/cm% all grafts showed fibrinolytic activity in the adventitia but grafts in group ii also showed fibrinolytic activity in the intima. early neointimal fibrous hyperplasia (nfh) characterized by proliferation of smooth muscle cells was present in group ii. the, occluded grafts showed organizing thrombus material and inflammatory cells and the patent ones showed fibrin and scattered inflammatory cells. in groups i and iii, sem revealed numerous platelets and rbc's incorporated into a proteinaceous material overlying the anastomoses and in some specimens obvious thrombus material was present. in group ii, the anastomotic areas were covered with large endothelial cells, nonetheless, some areas were denuded and small thrombi were occasionally noticed. in conclusion: . anastomotic sites create a strong stimulus for thrombus formation despite a high production of pgi . this suggests that antithrombotic therapy may be necessary to prevent early failures. . huvg develop the capacity to produce pgi and fibrinolytic activators and . although the etiology of nfh remains obscure, the decreased levels of pgi in group ii suggest that exhaustion of pgi generation from the endothelium might occur leading to proliferation of smooth muscle cells (nfh). these cells will in turn supply pgi if a persistent stimulus exists. permeability of intestinal capillaries to fibrinolytic products d. manwaring and p. william curreri department of surgery, university of south alabama college of medicine, usa fibrin/fibrinogen degradation product d (fdp-d) is significantly elevated in the serum of patients after trauma or sepsis. purified fdp-d infused into nontraumatized rabbits precipitates thrombocytopenia, complement depletion, pulmonary dysfunction and increased permeability of lung capillaries to i s-albu -composite fibrin plate assay size oflytic zones (mmx) after h incubation at °c rain. in order to determin of products of fibrinolysis alter fluid filtration or permeability, either purified fdp-d or fdp-e were tested in an isolated, autoperfused cat ileum preparation. steady-state lymphatic: plasma protein concentration ratio (cl/cp) and lymph flows (ql) were measured at a venous outflow pressure of mmhg. data was analyzed for each animal group by the paired student t test for ql, cl/cp and protein clearance (ql x cl/cp). in cats which received fdp-d (n= ), ql and clearance increased five-fold (p< . ), but cl/cpwas not altered, which suggests a permeability change. ileal mucosal biopsies prepared for histology had villi that were de-epithelialized and platelet clots in blood vessels. fdp-e (n= ) provoked a slight increase in ql (p< . ), but not in cl/cp or clearance. histology was normal. (fdp-e causes no pathological lung change in awake rabbits). fdp-d may contribute to various organ pathologies after trauma. the effect of aspirin on the fibrinolytic activity of viable granulocytes r.c. franz, w.j.c. goetzee, b. rotunno and r. anderson department of surgery, university of pretoria, rsa although several influential authors have suggested that low dose ( rag) acetyl salicylic acid (asa) represents a balanced daily antithrombotic regimen probably by both inactivating thromboxane a production and enhancing prostacyclin synthesis little is known about the effect of aspirin on the fibrinolytic activity of live granulocytes [ ] . the present study was designed to evaluate this effect in vivo. methods: granulocytes from fasting samples of heparinized venous blood taken from male volunteers were separated from monomuclear cells and platelets by density gradient centrifugation (ficoll: sodium metrizoate). viable granulocyte suspensions and plasma samples were placed as drops on a coin- before aspirin after aspirin p = [ ] . the experiment was repeated h after each subject had ingested . g of aspirin. the results are summarized in table . . there appears to be a significant increase in granulocyte fibrinolytic activity h after the ingestion of .sg of aspirin. . this increment is insufficient to overcome the resting inhibitor potential of plasma on granulocyte fibrinolysis. . aspirin does not evoke a significant increase in plasminogen activator-(urokinase) induced flbrinolysis in platelet free plasma or in the combined system of granulocyte-plasma mictures. . the optimal dosage of aspirin as a fibrinolytic agent requires further study. the terminal vascular bed of malignant tumors is characterized by a lack of organization, differentiation and sufficient developement of nutritional capillaries. as a result, malignant tumors reveal consistently small regions of low or even no perfusion. pre-vious data in a melanoma indicate that due to the rarefication of capillaries, the full impact of tumor treatment ±st diminished by an elevated microvascu lar resistance, which could significantly affect the impact of tumor therapy. since the improvement of the blood's fluidity has been shown as one therapeutic modality to increase significantly the capillary blood flow, it was assumed that this measure might enhance the accessibility of tumor tissue for bloodborne drugs. this study was aimed to investigate the effects of the improvement,of microcirculatory flow on tumor growth and tissue oxygenation. moreover, the response of the melanoma to chemotherapy was evaluated when isovolemic hemodilution was employed in conjunction with chemotherapy. a transparent chamber technique, intravital microscopy, a platinum multiwire electrode (local po measurement) and quantitative television image analysis (capillary blood cell velocity and diameter) were employed to study the microvasculature in the amelanotic melanoma a-mel- of hamsters in the event of hemodilution without and in conjunction with chemotherapy ( mg/m dtic, dimethyl-triazeno-imidazol-carboxamid). permanent indwelling catheters in carotid artery and jugular vein served for measuring systemicpressures, heart rate, for withdrawing blood and the infusion of dtic and/or dextran . after inoculation of x cells of the amelanotic hamster melanoma a-mel- into s.c. tissue in the preparation, this tumor re~ched a diameter of approx mm within five days. the reduction of systemic hematocrit from . to . ( . ± . ml blood vs dextran , animals) at a tumor diameter of mm increased the growth rate of the melanoma by about % while enhancing significantly the volume flow through capillaries and the mean local po . table control hemodilution capillary velocity (ram/s) . _ . . ± . capillary blood flow (ml/min x - ) . ± . . ± . mean local po (mmhg) . ( - ) . ( - ) the frequency distribution of local po on the tumor's surface showed a distinct shift toward higher po values with still some hypoxic regions remaining. intravital microscopy, however, revealed petechial bleeding and localized, interstitial edema which compressed a small number of capillaries. by contrast, the tumor's diameter remained at app. mm for a period of ten days with chemotherapy alone ( animals). in one of the animals, a complete stop in the melanoma microcirculation was seen within four hours after infusion of dtic followed by a significant decrease of tumor diameter. when chemotherapy was initiated in hemodiluted animals, neither retardation of tumor developement nor vascular obstruction was observed ( animals). conclusion: capillary blood flow of the melanoma can be enhanced by hemodilution thus diminishing tissue hypoxia. this measure, however, was associat-ed with an increase in melanoma diameter of %. at the present, we investigate whether, in hemodiluted animals, a reduction of tumor size can be obtained with a higher dose of dtic. ). however, the etiology of stress hyperglucagonemia in the immobilized rat is only poorly defined. since during restraint stress, catecholamines (ca) are elevated and stimulation ofglucagon by ca is accepted (woods sc d jr [ ] physiol rev. : ), we decided to study by surgical means the the relative contribution to glucagonemia of different sources of ca, i.e. peripheral sympathetic nervous system and adrenal medulla. methods: male sprague-dawley gastric fistula rats (n= ), weight approx. g, were subjected to either sham-op or various sympathectomies [microsurgical splanchnicotomy = s-sx; chemical sympathectomy = c-sx ( mg/kg -oh-dopamine ip two days prior to the experiment); adrenal demedullation = amx; combinations: s-sx + amx; c-sx + amx]. gastric acid secretory trials (duration h), preceded by a h fasting period were carried out - days following the operation. at the start of the experiment an intraperitoneal polyethylene tube, was inserted into the abdominal cavity of the rats, allowing a constant infusion of physiological saline ( ml/ kg/h). in addition, stress was performed by pairwise restraint of the extremities and small electric shock waves applied by a tail electrode. at the end of the experiment, blood was drawn from the vena portae and the abdominal aorta for plasma and serum. hormones (glucagon, insulin) were measured by radioimmunoassay, glucose enzymatically, volume was read to the next . ml, acidity by microtitration. results (see table) : volume and acidity are not changed by the various sympathectomies, when compared to the sham group. the same is true for acid secretion, except in s-sx + amx, where it is elevated. glucagon in peripheral plasma is elevated in c-sx, amx and c-sx + amx. in the portal vein, glucagon is dccreased in s-sx + amx ( ___ pg/ ml) and elevated in c-sx + amx ( ___ pg/ml) when compared with sham rats ( --. pg/ml). the portal/aortal glucagon ratio is significantly decreased only in c-sx and amx ( . --+ . , . --. . , resp.) when compared with sham ( . --_ . ). insulin is increased only in amx, glucose decreased in amx, s-sx + amx and c-sx + amx, insulin and glucose are unchanged in the other groups. conclusion: . stress hyperglucagonemia in the rat is confirmed (levels during zero stress - pg/ml) and also the rise in insulin following removal ofadrenomedullary ca (but not other sympathectomy); . the blood glucose fall (amx; s-sx + amx; c-sx + amx) is not uniformly paralleled by hyperglucagonemia, but in the case of amx it may be secondary to relative stress hypoglycemia owing to removal of adrenal medullary ca or reactive insulin release; . mechanism underlying the increased systemic glucagon despite partial (c-sx; amx) or total (c-sx + amx) sympathectomy are yet unknown. . during stress the enterogastrone component of hyperglucagonemia may be of minor importance. evlw showed good agreement with gravimetric lung water determinations. significant lung water accumulation was produced by pressure elevations over mmhg. reductions in plasma oncotic pressure significantly increased transvascular fluxes at each level of pressure elevation. however, fluid accumulation was not significantly greater during hypoproteinemia. we conclude that a - % reduction in plasma oncotic pressure does not contribute to increased high pressure edema because the lymphatic safety factor is augmented. this phenomenon may explain the well tolerated state of hypoproteinemia in patients after hemorrhagic shock. computerized gamma scintigraphy is a new technique for the analysis of albumin flux in the acute respiratory distress syndrome (ards). the objectives of this study were to obtain normal control values and to determine the method's validity in patients with cardiogenic vs. permeability pulmonary edema. methods: following mci mtechnetium-human serum albumin, lung :heart radioactivity ratios were determined. this ratio remains constant unless there is a leak o falbumin, when a rising ratio is seen, called the ,,slope index" (si). si's were determined in control individuals who had :> % left ventricular ej ection fraction and < s pulmonary circulation. thirtythree studies were obtained in patients using a portable gamma camera. fourteen patients had clinical evidence of ards. results: studies were considered positive if the si was s.d. > control mean ( - . ___ . x - units/ min). among positive studies, all had diffuse air space disease on chest radiographs. their average pulmonary capillary wedge pressure (pcwp) was . _+ . mmhg. the average artertial: alveolar oxygen tension ratio (a/a)o was . +-- . on . cm h peep, which were both significantly (p< . ) different from patients with normal si's. positive si's were present from hours to days following the apparent onset of ards in patients. recovery of gas exchange was associated with normal si's on repeat studies in patients. of patients with cardiogenic pulmonary edema, had negative studies ( - mmhg pcwp) and i a positive study ( mmhg pcwp). conclusion: gamma scintigraphy was a sensitive, non-invasive tool for the detection of a pathological increase in pulmonary protein flux, which was usually normal in cardiogenic pulmonary edema. positive scintigraphy was associated with significantly impaired gas exchange. the method documented that the leak of albumin in ards may last for days but resolves with recovery. cancellous bone thermocoagulation ph. dumontier, r. benichoux and a. vidrequin institut de recheres chirurgicales -c.h.u. de brabois vandoeuvre les nancy cedex, france electrocoagulation can not stop bleeding from the cancellous part of a sectioned bone. therefore we tested the efficiency ofthermocoagulation by hot air. the hot air generator delivers a flow, of non illtercd air, at - /min at a fixed temperature of °c measured at the exit of a mm diameter pipe and °c at the site of bleeding. the generator sustains usual steam sterilization. dogs were operated on both patellae, femoral short segments and iliac crests giving different site of cancellous bone. in vitro sterility studies: the conduit of hot air was applied at various distances, from cm to meters, above a petri plate containing culture material. thus the turbulence in the atmosphere around the zone of thermocoagulation has been bacteriologically controlled and a particle counter used. in vivo thermocoagulation: three sites of cancellous bone were used in anesthetized dogs, using a sterile procedure: the iliac crest, a small segment of the femoral bone and the divided patella. . five iliac crests were divided and bleeding measured after thermocoagulation. . the segmental femoral resections were thermocoagulated. . the patellae were vertically divided and each section submitted separately either to thermo or electrocoagulation. the pipe of thermo- coagulation was directed to the bleeding surface at a cm distane, sweeping it during to seconds. the bleeding was compared by photography and the two fragments were approximated by a wire synthesis to provide a bone fusion. in few cases both sides were thermocoagulated. results: in vitro: no contamination was found in the atmosphere, up to a distance of meters. there was a significant decrease of particles around the operating site ( % less, at cm and % less, at meters). in vivo: the bleeding was weighted around % less than with conventional coagulation. thermocoagulation did not delay or disturb the healing of the patella after wire synthesis. the in vitro nucleation time of cholesterol crystals from gallbladder bile of patients with gallstones is more rapid than that from normal persons, (holan rt [ ] gastroenterology : ). this study determined whether this was due to a gallbladder or liver defect and wether the defect was the addition of a nucleating factor or the deletion of an antinucleating factor. hepatic and gallbladder bile were gathered at surgery in stone patients and gallbladder bile in patients with a normal biliary tract. after ultracentrifugation, the isotropic phase was observed daily by polarizing microscopy until cholesterol crystals appeared. in gallstone patients, the nucleation time of gallbladder bile was significantly more rapid, . days+ . sem, than that of hepatic bile . + . days, although hepatic bile was significantly more saturated with cholesterol [cholesterol saturation index (csi), . _+ . ], than gallbladder bile, (csi, . _+ . ). thus the characteristic short nucleation time of stone formers is due to an alteration in bile after it enters the gallbladder. to determine whether the gallbladder defect was due to addition of a nucleating factor or the deletion of an antinucleating factor, isotropic phases of normal gallbladder bile and that from stone formers were mixed and nucleation time determined. mixtures of up to % normal bile had pathological nucleation times demonstrating that the defect is the addition of a nucleating factor by the gallbladder, and that this factor is potent. the rate of formation ofgaustone precursor crystals in bile, although faster in gallstone patients than in controls, is unrelated to the degree of cholesterol supersaturation [ ] , implying that other factors are involved. two competing factors seem likely; (a) secondary seed crystals in bile may trigger and accelerate gallstone crystal formation from supersaturated solution; (b) "poisons" in bile may retard or inhibit crystal growth. because of the complexity of bile itself, experiments were performed in highly purified mixtures of bile salt, lecithin and cholesterol, in concentrations closely resembling those of gallbladder bile. (a) lipid solutions were seeded with calcium carbonate, hydroxy-apatite, calcium bilirubinate and biliary mucus, all of which are found in gallstones [ ] . cholesterol crystal formation was significantly faster in_ the presence of all of the seed compounds tested (x= . /~g ml-lh- ) than in unseeded controls ( . /ag ml-~h- ) (/ < . ). (b) substances with "crystal-poisoning" properties included heparin, chondroitin sulphate and bile salt. these and changes in ph altered the quantitiy ( - % decrease) and rate of calcium carbonate and calcium phosphate crystal formation. we suggest that gallstone precursor crystal formation may be affected by a subtle balance between crystal seeding and crystal growth-inhibition, both due to the presence of other compounds in bile. at the last tripartite meeting we reported experimental data on a new method to destroy concrements of the kidney in situ by shockwaves allowing for spontaneous excretion via the urinary tract [ ] . the shockwaves are generated externally by underwater discharge of a condensor with sparking electrodes which are localized in a focus o fan elliptic cavity. for treatment the renal concrement must be positioned exactly in the second, virtual focus opposite to the elliptic cavity. since then, altogether patients were subjected to this form of treatment in our institute by the colleagues of the department of urology at the university of munich. % of the patients got rid of their concrements within a few days, in . % small remnants remained in the renal pelvis, and in . % ( cases) additional surgery became necessary. meanwhile this technique is employed on a routine basis in the department of urology of the university of munich [ ] . the experimental as well as clinical results were considered encouraging enough to extend the technique for the treatment of biliary concrements. for this purpose, human gallbladder concrements of different composition (bilirubin, cholesterol) were implanted into the gallbladder of dogs for exposure to shockwave treatment after wound healing. under in vitro-conditions the biliary concrements could be crushed into any size desired, irrespective of their composition, while only in out of experiments this was accomplished under in vivo-conditions. blockage of the biliaiy duct after shockwave exposure was never observed. concrements which were experimentally implanted into the bile duct could be visualized without difficulties by contrast medium. here, destruction by shockwaves was accomplished as well. currently experiments are conducted to dissolve remnants of biliary concrements after treatment by administration of desoxycholic acid. precise positioning of the gallbladder concrements in the second virtual focus is a problem which has not been satisfactorily solved so far, because the concrements cannot be visualized by conventional x-ray techniques. alternatively it is attempted to employ ultrasound, or visualization by retrograde injection of xray contrast medium through a catheter. in experimental animals, we leave a t-formed drain in the gallbladder for injection of contrast medium. in animal experiments conducted so far, histological or clinical evidence for tissue damage has not been obtained, as is the case with shockwave treatment of kidney stones. we are convinced that treatment of biliary concrements by shockwave exposure can be employed under clinical conditions in the near future. exploration of the common bile duct (cbd) for calculi, particularly in the presence of obstructive jaundice, is a procedure with considerable mortality and morbidity. to avoid the problem of retained stones, choledochoduodenostomy and transduodenal sphincteroplasty have been recommended, but have their own complications. this morbidity might be reduced by removal of cbd calculi prior to surgery. endoscopic sphincterotomy (es) allows this. a review of cases of es performed for calculi indicated that this was a safe (complications % no deaths) and reliable procedure ( % success rate). a study was conducted of patients with known cbd stones who had either preliminary es followed by operation at a later date (group i) or operation alone (group ii). this study showed a lower morbidity in group i. a prospective randomised controlled study has begun on the basis of these findings and the data from both studies are shown in the table. these results suggest that pre-operative endoscopic sphincterotomy my reduce the morbidity of cbd stones. group ii n = two controversies regarding the physiology of the biliary sphincter (bs) concern its functional independence from the duodenum [ ] and those aspects of its acitivity which control bile flow [ ] . the rabbit was chosen as the experimental animal as it has an easily identifiable sphincter. during anaesthesia induced by intravenous pentobarbital sodium, recordings of the electrical and mechanical activity of the bs and duodenum were made from (a) starved, (b) fed and (c) starved animals during administration of cholecystokinin, pentagastrin, secretin and glucagon. spike complexes (sc) were ordinarily associated with mechanical acitivity of the sphincter and duode-num. of . sphincter sc, recorded in animals, ( %) were not associated with duodenal acitivity, whereas . of ( . %) duodenal sc were accompanied by synchronous bs activity. this supports the hypothesis that the rabbit's bs can contract independently of the duodenum but that duodenal contraction is usually accompanied by simultaneous contraction of the bs. sphincter scs correspond to its phasic acitivity. food and cholecystokinin increased the number of sc without altering the baseline pressure of the perfused common bile duct. pentagastrin produced a transitory increase in sphincter activity whereas :secretin and glucagon were without effect. phasic activity of the spincter may influence bile flow through the choledochoduodenal junction. natural blood coagulation finally results in the formation of fibrin, which is one of the most important components of hemostasis in the human organism and thus provides the basis of all reparative procedures that are part of wound healing. it stands to reason to utilize the properties of fibrin for hemostasis during surgery and for joining severed tissue. first attempts of this kind were made at the beginning of this century. but only after greater insight had been gained into the coagulation proc- ess and the manufacturing techniques of blood derivatives had become more sophisticated, the essential breakthrough was made. a biological adhesive system has been developed, which consists of highly concentrated fibrinogen, thrombin and clotting factor xiii. this tissue sealant is completely resorbable and of high adhesive property. further advantages are elasticity of consistence and excellent tissue compatibility. after extensive animal experimentation, first clinical experience was made in . in the meantime the fibrin-adhesive-system (fas) has been introduced into numerous surgical disciplines with excellent results. the outstanding properties are: atraumatic tissue synthesis; enhancement of fibroblast proliferation and promotion of rapid wound healing; obtaining of local hemostasis by sealing bleeding surfaces, which is of special importance in the treatment of patients suffering from hemophilia or during operations under heparinization. the authors experience in using the fas within the last years is reported and a review over indications, techniques and advantages of this method is given. bile salts have been shown to enhance the stability and prolong the activity of intraluminal pancreatic enzymes and may therefore influence the effects of impaired exocrine secretion in patients with pancreatitis [ ] . individual bile salts in the peak min collection of duodenal fluid following cck/secretin administration have been quantitated by high performance liquid chromatography in patients without pancreatic or hepatic impairment (group c), patients with acute pancreatitis (group ap) and patients with chronic pancreatitis (group cp) all with functioning gallbladders, and patients with gallstone related acute pancreatitis (group gs). the peak total bile salt output in moles and the trihydroxy: dihydroxy (tri:di), primary:secondary (p:s) and glycine:taurine (g:t) bile salt ratios are shown below (mean___ sem). the duodenal aspirates contained detectable amounts of taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate but not lithocholate or free bile salts. the low total bile salt output in groups cp and gs were due to decreased levels of all the individual bile salts. although the bile salt pattern in groups c and ap were similar, the relative proportions of trihydfoxy and secondary bile salts were higher in groups cp and gs respectively. these results indicate that patients with chronic pancreatitis without obvious large bile duct obstruction have an impaired bile salt output into the duodenum and this may exacerbate the effects of pancreatic exocrine insufficiency. an elevated amylase creatinine clearance ratio (accr) was considered a specific test for the diagnosis of acute pancreatitis (ap). however, it has been found elevated in other diseases as well as after surgery. the aim of this study was to evaluate prospectively the accr levels in patients with ap and in several groups of surgical patients. we studied subjects divided into groups: group a: acute pancreatitis (n= ). group b: patients undergoing biliary tract surgery (n= ). group c: peptid ulcer patients undergoing gastric surgery (n= ). group e: patients undergoing cardiac surgery under extracorporeal circulation (n = ). group f: control group of healthy subjects (n = ). the accr was determinated using the levitt method. in the surgical groups the accr was measured before and after the operation. amylase was determinated by the phadebas amylase test. ap was diagnosed on the basis of both clinical and radiological findings and the presence of high serum amylase levels. this diagnosis was confirmed through laparotomy in cases ( %). the accr was . + . % (mean___ sd) in group f, control group, and . + . % in group a, acute pancreatitits p< . . accr values below % were found in cases of acute pancreatitis ( %). among those patients whose ap diagnosis was confirmed through surgery the accr was . + . % (mean_+sd), higher than in the rest of the ap patients, . ± . % (p< , ). in groups b,c,d and e (surgical groups) the accr before the operation was . ± . %, . ± . %, . + . % and . + . % (mean___ sd), respectively. after the operation it was: . + . %, . + %, . ± . % and . + . %, respectively. on the average, we found an increase in the accr levels after the operation in the biliary tract group (p< . ), but not in the other surgical groups. in patients ( %), the accr after operation was above the upper limit of normal. none of these patients had symptoms compatible with clinical pancreatitis. in conclusion: . the accr increase in acute pancreatitis (sensitivity: o/ ) . the average accr increase after biliary tract surgery, but not after either gastric or thyroid surgery or after cardiac surgery under extracorporeal circulation. however, it is possible to find isolated cases with high accr after any type of surgery without any symptoms of pancreatitis, suggesting that an increase of accr may be an unspecific finding in postoperative patients which require further investigations. out of patients with acute pancreatitis developed a fulminant type. were males and females, mean age years (range - years). all patients were primarily treated by peritoneal lavage applied at laparotomy. indication for laparotomy was sudden deterioration with ( ) or without ( ) organ failure. a necrotic or hemorrhagic pancreas was found in every patient. the pancreas was exposed and soft and large catheters were placed close to the pancreas. mean duration of lavage (ll/h) was days. due to secondary deterioration a pancreas resection was performed - days later in nine patients. patients with acute fulminant pancreatitis died, a mortality of . %. all patients with the mild type of acute pancreatitis survived, thus the overall mortality was . %. none treated by peritoneal lavage only developed diabetes mellitus, whereas out of surviving patients with an additional pancreas resection had this complication. patients with acute fulminant pancreatitis displayed or more ranson criteria [ ] and six died. however, no less than of those with a mild type of acute pancreatitis fulfilled or more of these criteria and they all survived. a laparotomy -not a laboratory test -is necessary to confirm the diagnosis of acute fulminant pancreatitis. the indication for laparotomy is mainly clinical and therefore such a patient should preferably be handled by surgeons or physicians experienced in this disease. after confirmation of a correct diagnosis peritoneal lavage is one of the methods by which the mortality of acute fulminant pancreatits -which by conservative means is - % -can be reduced. coincidences ofhyperparathyroidism and pancreatitis have been given up by different author varying between % and %. frequently a causal relationship has been defended. recently, however, any causality has been queried [ ] . we analysed our own series of patients with surgically and histopathologically confirmed primary hyperparathyroidism (phpt) (n= ) and found a coincidence with a coexisting or prior pancreatitis of . % (n = ). from this a causal relationship cannot be concluded. however, out of these patients (i.e. . %) had an acute onset or exacerbation of a pancreatitis immediately following the parathyroidectomy, which is strikingly more than one would expect after an operation without any anatomical relation to the pancreas (< . %) [ ] . none of these patients had another cause of the pancreatitis such as cholelithiasis or alcohol abuse. hence a causal relationship cannot be excluded. it is imaginable that excessive amounts of parathyroid hormone are released during the surgical manipulation of the pathologically altered parathyroids. the postoperative pancreatitis may be caused by the acute elevation of parathyroid hormone levels in the presence of hypercalcemia. in our series the parathyroidectomy was combined with a partial or total thyroidectomy in patients. in another patients a thyroid operation was performed prior to the parathyroidectomy. although calcitonin has been employed as a possible therapy in patients with acute pancreatitis (ap), the normal levels of this substance in ap are not well documented and have not been correlated with pth. furthermore no definitive role in human calcium homeostasis is accepted for calcitonin, whereas high pth levels have previously been correlated with hypocalcaemia [ ] . in patients with ap the mean serum calcitonin on admission was ng/ , and the peak level mean lag/ (upper limit of normal ng/ ). calcitonin levels were higher in severe than mild ap, and of patients with levels > ng/ , were objectively graded as severe ap [ ] . in the hypocalcaemic patients, with corrected serum calcium < . mmol/ all recorded calcitonin levels > ng/ and had elevated pth. nine of the patients had significantly elevated pth ('> rig/l) and of these only did not have an associated elevation in calcitonin. the high calcitonin levels in patients with ap suggest that supplementary calcitonin intended to inhibit pancreatic secretion is unnecessary. at present it is merely speculative to suggest a role for calcitonin in ap but intriguing to report a tendency to parallel the elevations of pth. nuclide labelled microspheres were used to measure pancreatic and other visceral blood flow in two groups of conscious dogs before and after intravenous alcohol infusion. blood alcohol concentrations at the time of blood flow measurements were similar to those encountered in intoxicated humans. thus dogs (groups a) were given a somewhat low dose of alcohol to produce a mean blood alcohol level of . gm d - , while dogs (group b), received substantially greater doses of alcohol, and reached a mean blood alcohol of . gm dl- . wilcoxon matched pairs signed rank test confirmed a biphasic, concentration-related, response of pancreatic blood flow after alcohol infusion; no such response was found in blood flow to other viscera. moderate alcohol levels (group a) were associated with a decrease in pancreatic blood flow (p< . ), while high blood alcohol concentrations resulted in increased pancreatic blood flow (p'< . ). colonic blood flow increased in both groups a and b, but blood flow to the gallbladder, small intestine and the parotid gland increased in group b only. gastric, duodenal, renal, hepatic (arterial) and cerebral perfusion did not change. in addition, direct observations of the surface of the pancreas showed occasional haemorrhagic areas and mottling. these findlings however could not be confirmed by objective attempts to measure blood flow in such discrete areas in conclusion pancreatic blood flow shows a biphasic, concentration-related, response shortly after intoxication. this response appears to be peculiar to the pancreas and does not occur in other viscera. we recently showed that acute ethanol (e) and/or aspirin (a) ingestion increased the permeability of the pancreatic duct to large molecules, this suggested that pancreatic enzymes might leak from the duct into the parenchyma, causing pancreatic disease. this is a new concept in the study ot he pathophysiology of this organ (to be presented at aga, may, , plenary session). in the present experiments wie studied the effects of chronic e/a ingestion on pancreatic function in dogs. methods: dogs were fitted with duodenal and gastric cannulas. after recovery, baseline secretory sutdies were performed by cannulating the pancreatic duct and collecting pancreatic juice during secretin infusion ( . (submax) or . u/kg-hr. (maximal) iv). at least studies were performed in each dog at each dose. after baseline studies, dogs (group e) were given daily intragastric ethanol ( gm/kg-day). dogs (group a/e) were given e and a ( mg/kgday). after - weeks, secretory studies were repeated. results: all dogs gained weight (x = . kg). the pancreases appeared normal by light microscopy. drug treatment increased volume and hco output by and %, respectively, in group e, submax secretin, but decreased them by and % in group a/e animals. (p= . vs. predrug values and group e vs. a/e values). drug treatment decreased volume and hco output in both groups by - °/c (p= . ) after maximal secretin stimulus. (manova test for all statistical analyses). conclusions: consumption of e alone increased volume and hco output after submaximal and decreased them after a maximal secretin stimulus. this confirms the work of sarles. consumption of e and a reduced volume an i-ico output after secretin at both doses. thus chronic a ingestion further impaired pancreatic function in these animals. since only a small proportion of chronic alcoholics develop clinically significant pancreatic disease, an aggravating "cofactor" may be operating in this group. chronic asa ingestion, not uncommon in alcoholics, may represent such a cofactor. the development of diabetes mellitus in pancreatic cancer is well known but the standard oral glucose tolerance test is not recognised as a useful diagnostic indicator. glucose homeostasis, insulin and c-peptide secretion in response to intravenous glucagon were studied prospectively in patients with suspected pancreatic cancer and assessed as to their diagnostic value. fasting patients were given glucagon, m.g., i.v., and serial measurements of blood glucose, plasma insulin and c-peptide concentrations made for min. subsequently it was shown that patients had pancreatic cancer and the remainder constituted a control group. there was not significant difference in the rise in blood glucose between the groups after glucagon. the mean plasma insulin concentrations rose rapidly in both groups peaking between and rain but the values were sginificantly lower in the pancreatic cancer group (p< . at min: p< . at min: p< . at min). a similar pattern was observed with c-peptide. in patients with obstructive jaundice the plasma insulin response was a better discriminator of pancreatic cancer. we conclude that abnormal pancreatic beta cell funktion exists in patients with pancreatic carcinoma, detectable before any change in glucose homeostasis, particularly in patients with obstructive jaundice. the glucagon stimulation test may have a useful role in the diagnosis of pancreatic cancer. t-suppressor cells (t~) have previously been implicated as mediators of graft surival in baboons tolerant to their renal grafts [ ] . in addition, it seems that t-helper cells (th) are also affected by totallymphoid irradiation in that they are unable to provide help in mitogen-induced t-cell responses or pokeweed mitogen induced immunoglobulin synthesis in bcells. in this study the evolution of t~ and th is followed in baboons undergoing graft rejection at different rates. peripheral blood was collected from the animals at weekly intervals after renal transplantation, defibrinated and the lymphoid cells isolated by flotation on ficol hypaque. the t-lymphocyte fraction was purified by filtration through a column packed with nylon wool. th, ts and total t-cells were enumerated using monoclonal antibodies okt , and respectively (ortho). the th/ts rations reported were taken immediately before a rapid rise in serum creatinine occurred. in longlived baboons who maintained normal creatinine values, a mean of the ratios over the last month was used. b rats are produced by sublethal ( rad) x-irradiation of thymectomized animals, reconstituted with bone marrow from syngeneic, thymectomized, thoracic duct drained donors. in these lew rats, (lew x bn)f cardiac allografts survice indefinitely (> days); unmodified lew rats acutely reject such allografts ( -+ days). in this study, we have tried to restore the processes of acute rejection in b recipients. graft survival appeared independent of blocking factors or suppressor cells, as transfer of serum or lymphocytes from b recipients into syngeneic normal animals failed to increase survival of test allografts, placed subsequently. similarly, immunogenicity of long surviving grafts was unchanged; such grafts functioning > days and retransplanted into normal animals were rejected acutely. adoptive transfer of unseparated spleen cells (sl) from nonimmune syngeneic animals produced slow rejection ( --+ days) in b rats; sensitized slwas somewhat more effective ( _+ day). transfer of x syngeneic peritoneal exudate (pe) cells plus sensitized sl caused acute rejection in % orb recipients ( _ days), the remainder experiencing rejection at c weeks. pe cells harvested from rats injected ip with thioglycollate days previously, were primarily macrophages/adherent cells, as the cells used were that fraction sticking to plastic dishes and removed with lidocaine (purity > %). in vitro, b rat macrophages were abnormal, having only % of capacity of normal macrophages to promote production of interleukin (il ) when co-cultured with purified t lymphocytes. however, b recipients experienced acute graft rejection ( -+ days) after transfer of sensitized sl plus semipurified il , thus bypassing the above defect. addition ofll to the t cell (purity > °/ ) equivalent of sl (purified over degalan bead columns coated with rabbit antirat lgg, nonadherent fraction) failed to reestablish acute rejection ( -+ days), while further addition of b lymphocytes (degalan bead adherent fraction, purity > %) or macrophages was uninfluential ( ___ days and ___ days, respectively). transfer of il- alone never produced rejection. acute rejection can be re-established, however, by increasing the number oft lymphocytes ( , transferred concomitantly with il ). thus, the state of unresponsiveness in b rats can be reversed in vivo by adoptive transfer of particular cellular elements in the presence of growth factors; increased graft survival seems dependent ultimately upon il- production by sensitized t cells, presumably t helper cells. the relative inability of b rat macrophages to promote production of il- by t cells may be primarily responsible for the immunological deficit of the b rat. one of the most intriguing findings ofcyclosporin a (cya) immunosuppression is that in some species a short course of treatment will produce very prolonged allograft survival. we have tested the ability of cya to prolong the survival ofvascularized heart, kidney and pancreas allografts by direct comparison in a da (rt a) to lew (rt ~) rat allograft model. accessory abdominal heart and orthotopic left kidney transplantation were performed using standard microsurgical techniques. in renal transplantation the left kidney was removed at the time of transplantation, the remaining right kidney days thereaf- ter. streptozotocin-diabetic animals received ductligated pancreas whole organ grafts isolated on the portal vein and a segment of the aorta giving offthe coeliac axis and the superior mesenteric artery. rejection was taken as complete stop of palpable pulsations in heart transplantation, the day of death in renal transplantation and recurrance of hyperglycemia above mmol/ in pancreas transplantation, respectively. cyclosporin a mg/kg body weight, dissolved in olive oil, was administered intramuscularly for days starting with the day of transplantation. in all instances functional demonstration of rejection was confirmed by histological examination. cyclosporin a is effective to prolong the survival of vascularized heart, kidney and pancreas allografts. while cya is administered none of the grafts has been rejected. however, following withdrawal of the drug pancreas grafts are rejected within days and heart grafts within days. none of the kidney grafts has been rejected so far. the differential susceptibility of vascularized heart, kidney and pancreas allografts to cya immunosuppression may be caused by differences in immunogenicity due to organ specific alloantigens or a differential representation of spezialized antigen presenting cells. it may also reflect different patterns of rejection of the various organs. during cya administration all rejection processes are effectively suppressed. in the maintaince phase after withdrawal of cya such immune responses may prevail and ultimately lead to rejection of pancreas and to a lesser degree of heart allografts. the venous allograft still remains an attractive alternative for the reconstruction of small caliber vessels. however, when the venous graft is introduced to a non-histocompatible host, rejection and early occlusion is the rule. this study evaluates the use of cyclosporin a (cya) as a graft pretreatment, or systemic immunosuppressant for venous allografts. in addi-tion, cryopreservation techniques for pretreated venous allografts was investigated. adult mongrel dogs, weighing between and kg, were used as recipients for donor jugular vein segments ( - cm) which had been excised and flushed with cc of plasma protein fraction (ppf) at °c. these venous allografts were anastamosed end-to-end into a carotid artery of the recipients. the animals were divided into five groups as follows: group i (n= ) received untreated venous allografts without subsequent immunosuppression, group ii (n = ) was the same as group i with minimal immunosuppression (azathioprine . mg/kg/day). in group iii (n= ) the animals were transplanted with venous grafts stored in cc of plasma protein fraction (ppf) containing cy a ( mg/ ) at ° c for hours, immunosuppression was as in group ii. in group v (n= ) the animals received allografts that had been cryopreserved in a % dmso solution at - ° c for - days and then the animals had azathioprine as in group ii. in group v (n= ) venous allografts recipients were treated with systemic cya ( mg/kg/day x weeks, followed by mg/kg/day x weeks) as the only immunosuppression. the patency of the allografts was evaluated at , , , and weeks post transplantation. patency results at one month showed that azathioprine alone failed to improve the patency rate (gr. i and ii = % patency). cya graft pretreatment, however, significantly improved the one month patency (gr. iii = %). in addition, cryopreservation appeared to enhance the graft pretreatment effect of cya (gr. iv; one month patency = . %) of the allografts. finally, systemic cya proved very effective in preventing rejection and occlusion (gr. v; one month patency = %). grafts that remained patent for a initial critical period of - weeks, all showed long term patency. the effect of cya in preventing graft rejection was further documented by histiological studies of the allografts which showed a marked cellular infiltration and degenerative changes in all the grafts of the control group as compared to minimal or no cell infiltration in the patent grafts of the treatment groups. in summary, it appears that cya used as a graft pretreatment with minimal immunosuppression of the recipient, in conjunction with cryopreservation or given systemically as the sole immunosuppressant can significantly improve the survival of venous allografts. in our previous reports, it was shown that isolated hepatocytes transplanted into the splenic parenchyma of syngeneic rats, proliferated markedly and recomposed the hepatic tissue. this experimental system provided a new model to elucidate the mechanism of hepatic regeneration which could not be obtained in in vitro cell culture experiments. in the present paper, fetal hepatic tissue instead of isolated adult rat hepatocytes were transplanted into the rat spleen. we document briefly long-term morphological observations on the transplanted fetal hepatic tissue with special reference to proliferation of the hepatocytes and bile ducts. materials andmethods:wistar rats were mated in our laboratory for a fetal liver source. gestation day was when a plug or sperm were observed in the vaginal smear. fetuses used were of to days gestation. about ten fetal livers which were obtained from one maternal rat, were minced with scissors. the liver fragments were washed three times with saline solution. transplantation was carried out by direct injection into the spleens of syngeneic adult rats using a gauge needle. half of the liver fragments obtained from one maternal rat were innoculated into the spleen of one animal. a total of approximately rats with transplanted liver fragments were killed , , , and days and then every two to three months until one year after transplantation. the spleens removed were stained by h.e., pas and silver nitrate for histological examination. results: fetal livers exhibited no lobular architecture or hepatic cord structure. the very sparse cytoplasm of the hepatocytes and many hemopoetic cells among the hepatocytes were characteristically found only in the fetuses. one week after transplantation, the survived hepatocytes revealed almost the same morphological features as in fetal liver except for the presence of several proliferated bile ducts around the hepatocytes. two weeks later, the hepatocytes formed apparent hepatic cord structures and the extoplasm of each hepatocyte increased abunduntly and became acidophilic as seen in normal neonatal hepatocytes. hemopoetic cells disappeared. four weeks later, hepatocytes began to proliferate sporadically among the markedly proliferated bile ducts, groups of survived hepatocytes with cord structure were very similar to a neonatal liver except for the lack of the glisson's area. two or three months later, proliferation of the hepatocytes became prominent. there seemed to be no interrelationship between proliferated hepatocytes and bile ducts. one year after transplantation, a white nodule was observed on the spleen macroscopically and it consisted of numerous bile ducts and hepatocytes with or without cord structure on histology. summary: . fetal hepatocytes transplanted into the spleen, differentiated to almost normal neonatal hepatocytes two weeks after transplantation. . hepatocytes began to proliferate about weeks after transplantation. . three days after transplantation, proliferation of bile ducts was already observed independent of the transplanted hepatic tissue. . when comparing the difference in proliferation between fetal hepatic tissue and isolated hepatocyte transplantation, marked proliferation of the bile ducts in fetal hepatic tissue was observed and fetal hepatocytes proliferated more rapidly, while there were no proliferated bile ducts in isolated hepatocyte transplantation. pretransplant splenectomy (sx) has been of disputed benefit since its introduction two decades ago. of patients with first cadaver transplants treated at our institution between dec. and dec. have had pretransplant sx. at six monts, sx patients had % better kidney survival, but this benefit was lost shortly after year and by and years was % and % worse in sx patients. patient survival for sx and no sx was identical for the first year but was % and % worse by and years respectively in sx patients. thus, the early improvement in kidney survival was more than offset by a late high mortality. a rational basis for selecting patients who might benefit most from pretansplant splenectomy is urgently needed. since july, , patients ages - have received first cadaver transplants after having been tested for reactivity to dncb. nine of dncb negative patients had splenectomy as did of dncb positive patients. kidney survival at year for dncb negative patients without sx was %; for dncb negative with sx, %; for dncb positive without sx, %; for dncb positive with sx, %. rejection was the sole cause for kidneyloss in dncb positive patients without sx. however, of dncb negative patients with splenectomy died, primarily of septic complications. since survival of sx patients has been % compared to % in non sx patients (p< . ). sx appears to be beneficial in dncb positive patients but has an adverse effect in dncb negative patients because of an increased susceptibility to fatal infections. prior blood transfusion improves renal graft survival [ ] . plasma from uraemic patients suppresses the in vitro responses of normal lymphocytes to antigen (plasma suppressive activity, psa) and this effect is mainly attributable to the plasma protein macroglobulin (a m) [ ] . the aims of the present study were: a) to identify changes in psa and a m concentration in uraemic subjects following primary blood transfusion. b) to correlate the psa of transfused renal transplant recipients with subsequent graft survival. a) ten potential transplant recipients were studied before and after their first blood transfusion. following blood transfusions the psa increased significantly (p< . ) reaching a maximum at two months. there was no significant change in the plasma a m concentration over the same period. b) the plasma of consecutive chronic renal failure patients was tested for psa prior to renal transplantation and before institution of immunosuppressive therapy. all but two patients had received previous blood transfusions. after transplantation patients were followed for a minimum of months and a maximum of months. grafts failed for non-immunological reasons and were excluded from the study group. patients were divided into two groups according to the degree of suppressive activity of their plasma. a volume of /t , producing a % inhibition of normal lymphocytes was used as a treshold to differentiate those with a high or low suppressive activity. graft survival in the first three months was significantly better, % (/ < . ) for those recipients with a high psa as compared to % for those with a low psa. we conclude that blood transfusion causes a significant increase in psa although not a m concentration and that patients with high psa have a better graft survival. the effect of in vitro steroid on antibody dependent cellular cytotoxicity (adcc) was studied in patients awaiting renal allotransplantation and the results were correlated with transplant outcome. recipients of primary cadaveric allografts were classified as steroidsensitive or steroid-resistant from the degree of adcc suppression induced in vitro by methylprednisolone, patients being steroid-sensitive and steroid-resistant. following transplantation patients received azathioprine and prednisone, and rejection crises were treated with bolus doses of methyl-prednisolone. graft failure occured in of the steroid-sensitive patients, and in of the steroid-resistant patients. the observed one year graft survival rate was . % for the whole group, . % for the patients with steroid-sensitive adcc and . % for those with steroid-resistant adcc, the difference between the two groups being highly significant (xz= . ). a high incidence of early graft failure was seen in steroid-resistant adcc patients, . % of grafts being lost in the three months after transplantation, as compared with only of graft failures in the steroid-sensitive adcc group in the same period. analysis of hla-a, hla-b and hla-dr incompatibilities showed no significant difference between the groups, and since all patients had received deliberate pregraft blood transfusion, the difference in survival rates between the two groups appears to be independent of these two variables. these findings confirm our preliminary observation that pregraft assay of adcc response to in vitro steroids identifies those patients who are unlikely to respond to steroid therapy in the treatment of rejection, and in whom alternative forms of therapy may be appropriate. post-operative dxt, whilst not influencing survival, protected patients from loco-regional recurrence < . , hazard ratio (hr) = . ). interestingly it was found to be most effective against axiallary node recurrence (p< . , hr = . ), reasonably effective against chest wall recurrence (/ < . , hr= . ) but conferred no protection against supraclavicular node recurrence (hr = . ) in spite of a supraclavicular field being routinely employed in the radiotherapy technique. with such large numbers involved, this trial has facilitated the study of the prognostic significance of sub-groups of patients with different patterns oflocoregional recurrence as first evidence of treatment failure (see table) . of those patients developing loco-regional recurrence who have since died ( out of in wp group; out of in dxt group) % in the wp group and % in the dxt group did so with evidence of persistent loco-reglonal disease. however, the incidence of uncontrolled local disease at death was higher in the wp group overall. stress as well as dietary fatty acids have been shown to prolong allograft survival in rats [ ] . poly unsaturated fatty acids (linoleic acid, arachnoidic acid) have been reported to depress immune response [ ] . depressed immune response was suggested to correlate with a higher incidence of spontaneous tumor [ ] as well as with an increased growth rate of inoculated tumors [ ] . the objective of this study was to elucidate the effect of two environmental factors i.e. chronic stress (change in light/dark pattern) and diets low and high in linoleic acid on immune response and growth of transplantable tumors in bn rats. immune response: four experimental groups (n > ) were used in immune response studies. group i: high linoleic acid dietl; group i : low linoleic diet , group iii: l/d shift weekly, normal diet and group iv: controls on normal diet, normal lighting. seven weeks after the start of the experiment the immune response was measured. the results showed that corticosterone levels were slightly increased in all experimental groups, although only the high linoleic group showed statistic significant difference with the control group. cellular immune response (con a stimulation and popliteal node assay) was decreased in all experimental groups compaired to controls. transplantable tumors: x leukemia cells were injected i.v. and pieces of mm of an spontaneous adrenal cortical carcinoma, a urethral squamous cell carcinoma and a round cell cervix sarcoma were implanted subcutaneously. all tumors were inoculated in groups of animals each. spleen weight as a measure of leukemia growth was high in the control group and low in the experimental group. the same pattern was seen in the growth of the subcutaneously implanted adrenal cortical carcinomas. both the urethral squamous cell carcinoma and the round cell cervix sarcoma, being non-immunogenic, did not show any difference in growth. so far, it can be concluded, that the immunosuppression as induced by mild chronic stress or dietary fatty acids does not lead to enhanced tumor growth. in contrary, the results of both leukemia and adrenal cortical carcinoma show a possible reserve effect. little is known of the derivation or content of human breast cysts. recent reports have shown wide variations in the content of steroid hormones, particularly dehydroepiandrosterone sulphate (dhas) [ , ] . no explanation for this is apparent. to confirm the large variation in dhas concentrations and to further define the contents of cyst fluids, cysts from patients have been analysed for dhas, sodium and potassium. dhas concentrations ranged from . - pmol/ . both sodium and potassium content also varied widely (sodium - pmol/ and potassium - ~umol/ ). there was a significant direct correlation between the content of potassium and dhas in cyst fluid (p< . ) and a significant negative correlation with sodium content (/ < . ). three separate subpopulations of cysts could be identified according to the sodium and potassium content and these were, predominantly potassium cysts ( ), predominantly sodium cysts ( ) and mixed cationic cysts ( ). the median dhas concentration of the potassium cysts was pmol/ similar to the levels found in human breast secretions [ ]. in contrast the median concentration of dhas in the predominantly sodium cysts was pmol/ and significantly different (p< . ), with many of these cysts having dhas concentrations in the same range as those found in plasma. the remaining mixed cysts had a median dhas concentration intermediate between the two main groups. it may be that the variation in cationic content and dhas concentration in these two major subpopula-tions of human breast cysts represents either, derivation from two different sources, namely breast secretions and plasma or marked differences in the secretory activity of the epithelium lining these two groups of cysts. there is no uniform agreement on the correct management of patients with invasive lobular carcinoma (ilc). it is widely considered that in ilc there is an increased risk of developing a contra-lateral carcinoma and the major controversy surrounds the management of the second breast. the survival of patients with ilc was significantly better than that of idc fp<: . ). six patients had bilateral carcinomata at diagnosis and a further developed a contra-lateral carcinoma during the period of follow-up ( to years). survival data showed poor survival for patients with simultaneous bilateral disease, but no difference in survival for patients with metachronous bilateral or unilateral disease. this suggests that the later development of a second carcinoma does not necessarily reduce the probability of survival for patients with ilc. the major factor predicting patients at risk of developing a contralateral carcinoma was histologi- cal type. of patients with a particular histological pattern of ilc [ ] with a classical pattern of spread but showing nuclear pleomorphism and cellular cohesion, developed a contralateral carcinome, compared with a further in the remaining patients (p< . ). if bilateral mastectomy is justified it ought to be restricted to patients with this histological type of ilc. both the anti-oestrogen tamoxifen and cyclical combined chemotherapy will provide significant palliation in advanced breast cancer. the optimal use of these agents requires further evaluation and thus this trial was designed to compare a combination ofcytotoxic therapy and tamoxifen, against cytotoxics alone in patients with advanced breast cancer. post-menopausal patients presenting with metastatic breast cancer, locally advanced cancer extending beyond the breast and regional nodes, or with tumor recurrence following primary local treatment were allocated to the treatment arms via sequential manner. doxorubicin, cyclophosphamide, -fluouracil, and vincristine were given intravenously once every weeks. tamoxifen was prescribed in a dose of mg. b.d. on failure or relapse from one of the single modality arms, a crossover of those arms occurred. the combination consisted of both the above therapies. assessment of therapies was made in terms of objective response (uicc criteria), duration of response, and survival. we have previously reported that the combination results in a significantly greater response rate [ ] . as a result of stenosis reducing flow or by platelet embolisation [ ] . as neither aniography nor ultrasound can identify thrombotic activity we have evaluated gamma camera neck imaging using n indium platelets. labelled platelets on endarterectomy specimens were also measured and the activities found were then examined in a theoretical model. twentyfive patients with tia received rain platelets and sequential gamma images were interpreted by two observers, carotid endarterectomy in patients allowed measurement of specimen radioactivities. angiography and doppler spectral analysis [ ] were also performed. all endarterectomy specimens contained labelled platelet deposits with the most active equivalent to platelets from . ml of blood. this activity level was at the threshold of resolution in the theoretical model. both observers agreed that of the carotid bifurcations showed platelet accumulation on imaging. of the atheromatous ulcers demonstrated by angiography were visualised, but only of stenoses greater than per cent were detectable~ since ultrasound identified all stenoses only one angiographically diseased carotid was not detected by combining doppler and platelet imaging. diseased carotids accumulate rain platelets with the more thrombogenic ulcerated plaques identified more frequently than stenoses. long term follow-up is required to establish the clinical relevance of platelet deposition. major problem in vascular endoscopy is the existence of blood which prevents clear visualization. we devised a new technique using a combination of balloon catheter and slender fiberoptic endoscope, by which clear visualization was obtained experimentally and clinically. three to four pairs of orifices of intercostal arteries were also visualized in one visual field. in some dogs, acute aortic dissection was experimentally created by means of blanton's method. the entry, which was located at the descending aorta just distal to the left subclavian artery, was clearly identified. complete occlusion of blood flow and clear visualization could be obtained when balloon pressure exceeded systemic blood pressure. clinical study: in six patients requiring major vascular reconstruction of the aorta (abdominal aneurysm , leriche's syndrome , dissecting aneurysm ), vascular endoscopy was performed intraoperatively. in five patients, balloon catheter was introduced through the one of the limbs of y graft after proximal anastomosis. in each case, orifices of the major abdominal aortic branches were clearly observed. irregular orifices and atheromatous plaque of the aortic intima which were not expected from aortogram, were also identified in all patients. intimal tears by vascular claps were more extensive than expected and anastomotic suture lines were able to be checked from inside. in a case of dissecting aneurysm, balloon catheter was advanced through the mm graft which was sutured to the common femoral artery with finding the entry just above the left renal artery. using fiberoptic endoscope and balloon catheter was useful to observe orifices of the major aortic branches, unexpected intimal tears by vascular clamps and atheromatus plaques. it was particularly usbful to check the anastomotic suture line from inside of the aorta and to identify the exact location of the entry in dissecting aneurysm. vascular endoscopy could be one of the invaluable methods to examine, diagnose and treat the patients requiring aortic, caval and other major vascular surgery. ( ) produced endothelial injury and a local increase in shear stress in cynomolgus monkeys by suture plicating and constricting the aorta and then feeding an atherogenic diet for months. our findings reveal that carotid plaques localize on the outer wall of the internal carotid (plaque thickness . --+ . mm) which is an area of low flow velocity ( - ___ cm/s at re ) and shear stress ( -+ dynes/cm ) and not at the flow divider (thickness . ___ . mm, p< . ) which is an area of high flow velocity ( --- cm/s) and shear ( -+ dynes/cm ). distal to the carotid bulb, velocity and shear increased on the outer wall and little or no plaque was observed. in experimental coarctations, no endothelial damage was observed (sem and tem) within the high-shear coarct channel and the channel was noted to be free ofatherosclerotic plaque despite the development of extensive diet-induced lesions proximally and distally. thus, high flow velocity and shear stress do not appear to produce endothelial damage in vivo. in addition, plaques were minimal in high shear areas in the human carotid bifurcation and high shear appears to have an inhibitory effect on experimental plaque formation. these data contradict previous investigations implicating high shear stress in plaque pathogenesis. in contrast, host aortic endothelium (ae) fails to cover large vp by pannus ingrowth even over much longer times. to see if iaes succeeds because of inherent differences in growth potential between ae and ve, we used ae to seed cm x mm diameter dacron velour infrarenal vp in dogs. an average of x cells obtained by trypsin/collagenase digestion of the bypassed aortic segment was used to seed each vp by a step preclotting method. the identity of ae was confirmed by stains for factor viii antigen. viability of seeded ae was verified by growth of subaliquots in tissue culture. six weeks after surgery central segments of aeseeded (n = ) and control unseeded (n = ) vp were compared by light and scanning electron microscopy using an endothelial coverage score range of - (for fibrin/platelet thrombi) to + (for confluent endothelial coverage). ae-seeded vp had a score of+ . ___ . (mean___ sd) versus. - . ___ . for controls (p< . ). in addition to endothelial coverage, the subluminal smooth muscle and intramural vasa vasorum previously reported in ve-seeded vp were also seen in ae-seeded vp. since ae and ve seeding give identical results, the success of iaes with ve cannot be due to inherent biological differences in mitotic potential between ae and ve. iaes must instead achieve additional endothelial growth either through a) the action of the proteolytic enzymes used for cell harvest or b) mitogenic stimuli to nonconfluent cells at the edges of seeded cell clusters on the vp. further improvement of the efficiency of iaes to allow use of less harvested vein per cm of vp should come from enhancing one or both of these effects. pyrolytic carbon is a crystalline form of carbon that has been extensively used in the construction of cardiac and bone prostheses. since it has also been suggested that pyrolytic carbon will prevent thrombosis from occuring in vascular prostheses, the aim of the present study performed in dogs was to test the immediate blood compatibility of this material and to evaluate its biocompatibility when inserted as vascular substitute. after pryolysis of a gazeous hydrocarbon, the carbone crystalite was deposited on a knitted textile surface or tube. its surface examined by scanning electron microscopy (sem) was rough and porous to a depth of p. this material was tested °) for immediate hemocompatibility as inserts within the vascular lumen (aorta and inferior vena cava). the specimens were examined sequentially by sem and histology at , , , s and min after reestablishment of the blood flow, ° ) for long term biocompatibility as vascular cylinders ( mm id) inserted either in the aorta or inferior vena cava or as intraatrial (left or right) implants. patency of vascular cylinders was tested during postoperative month by doppler ultrasound investigations, specimens were examined by histology, electron microscopy (scanning transmission) at , and days following implantation. satellite lymph nodes were examined by histology. already s after establishement of the blood flow, platelet adhesion and limited fibrin mesh with few erythrocytes developed on the material. platelet aggregates of limited extent were only observed on intravenous implants. plasmatic protein deposition, an early event on polymeric vascular material was not observed. after s a fibrino-erythrocytic membrane recovers completely the material. except in the case of intravenous insert, no thrombosis developed at the contact of intraarterial or intracardiac implant. after days it was completely recovered by a - fibrocellular layer consisting of large myofibroblasts with microfilaments, newly synthetized collagen and elastin. the blood interface was of fibrous nature. at one month by sem, endotheliallike cells developed in a mosaic-like pattern, characterized by transmission e.m., by microvillous projections, numerous pinocytic vesicles and intercellular tight junctions. this endothelial-like cell lining was complete months after implantation. their immunocytochemical properties are now under investigation using specific anti-dog factor viii-rag sera. although preliminary, the present results suggest that among the numerous vascular biomaterials tested, pyrolytic carbon may represent a unique feature of rapid cell development and differentiation of endothelial lining at the blood material interface. department of connective tissue biology, institute of anatomy, university of aarhus, aarhus, denmark in the surgical clinic a significant number of patients report that their incision wound has burst, even though the scar appears to be intact. by mechanical testing of strips from skin wounds we have noticed a breaking pattern, in which the deepest layer of the wound ruptures earlier than the superficial part. therefore, we have investigated the strength and extensibility of rat skin wounds at different levels (superficial-deep) of the epidermis ( . mm), dermis ( . - . mm) and m. panniculus carneous ( . - . mm), average thickness is indicated. , and day old standardized skin wounds from the dorsal region of rats have been used. strips were punched out at right angle to the wound line and mounted in a materials testing machine. the strips were stretched until rupture and load-strain curves registered continuously. simultaneously, the strips were transluminated and the breaking pattern was studied by taking photographs of the wound specimens during the mechanical testing ( - photographs of each specimen). the photographs were marked on the load-strain curve by means of a connection between camera and x-y-recorder. from the load-strain curves the maximum load and the failure energy were calculated. the breaking patterns of , and day old wounds were found to be similar. the deep part of the wounds ruptured first. the force required to break the deep part was less than that required to break the superficial part of the wounds. the musculus panniculus carneous was very extensible and did not break. however, it possessed only minimal strength. quantitative measurements of the strength of the combined superficial-deep layers were performed on mm wound strips. specimens contained the superficial . , . and . mm of the wound area and were produced by cutting off the deep layer parallel to the skin surface. specimens containing the total wound area down to the musculus panniculus carneous were produced by cutting off the muscular tissue. these specimens were mechanically tested as described above. the present studies demonstrate the mechanical inhomogeneity of incision wounds. a new method for testing the mechanical properties of the tissue of incision wounds at various levels (superficial-deep) is presented. the superficial layer of an incision wound contributes a major part of the strength of the wound and is more extensible than the deep layers. these results may explain the clinical observations. the effect on wound healing of different kinds of vitamins is worth investigating, since the efficiency of vitamin c has been dearly demonstrated. the possible action of vitamin bs-whose trophic effect on skin is well known -has been experimentally studied on skin and aponeurosis healing after a standard laparotomy. materials and methods: experiments were carried out on five months old rabbits which were randomly divided into three groups: in group i, animals served as controls ( animals in sequence of days from the th to the th post-operative day), group ii, animals injected with vit b ( mg/kg of body weight/ h) and group iii, animals injected with a placebo ( animals in sequence of days for each group). in each case four samples were tested of skin and aponeurosis for determinating tensile strength, directly recorded with an original technic [ ] : this new apparatus allowed us to obtain simultaneously two dynamic parameters, the healing tensile strength and stretching of the scar. results: . no significant difference was found between controls (group i) and the placebo group (group iii) both for resistance of skin and the aponeurosis. . as far as vitamin b treated animals were concerned (group ii) there was no significant difference regarding skin resistance when compared with the other two groups. . inversely aponeurosis resistance become significantly greater when measured on the th (p< . ), th (/r< . ) and th (p< . ) post-operative day. in mongrel dogs ( x cm cranial based rectus abdominis) mc and corresponding rp flaps were raised. in group i ( dogs) skin bf was determined from the clearance curve for ~ xenon injected intradermally and measured with a computer-linked gamma camera. in group ii ( dogs) subcutaneous pro was determined by a recently developed method using a silastic tonometer, subcutaneously implanted. the pro inside the tonometer was measured in infused saline, by a platinum oxygen needle electrode and a silver/silver chloride reference electrode. b f and pto were measured before and after the flaps were raised and on postoperative days (pod) , , and . pto were taken at various inspiratoric oxygen levels (f~o ) ranging from % (air) to % oxygen. intact areas lateral to the flaps and in flap regions prior to surgery served as controls. immediately after surgery bf in the mc increased while in the rp flaps was %, % and % of the flow in the mc flaps, in lateral intact area and in the preoperative areas (p< . ). during pod - bf in the rp flaps increased to the preoperative level, but not to the increased levels found in the mc flaps and the lateral intact areas. by pod there were no differences in bf between the two types of flaps and the lateral areas, but all were higher than corresponding preoperative values (/'< . ). tissue oxygen tension showed a dramatic fall pod , and in the rp flaps for all fio , and for all days the values were lower than the preoperative level (p< . ). one rp developed pod distal necrosis and the pro was then even with a fio of %. the mc flap showed an increased pro on the operative day but at pod the values were slightly lower than the preoperative level, but pod , and the values for all fio were higher than for rp flaps (p< , ). at pod the pro reached preoperative level for rp as well as mc flaps. lateral intact areas showed comparable changes to that observed in the mc flaps. it is concluded that the mc flap demonstrates superior bf as well as pro when compared to the rp flap. early postoperative pro in the distal part of the rp flaps is critically low despite of increasing f~o to % and increasing bf. differences in bf and pto may be the biologic factors responsible for the superior healing characteristics of the mc flap. ( ) atp~adp + pi (inorganic phosphate) ( ) pcr + adp~atp the net result ofreaotjoxas and is a fall in pcr and a rise in pi while atp ~'emains relatively constant. all of the phosphorus metabolites are easily measured in gastrocnemiaas muscle using pnmr spectroscopy. normal volunteers and patients with angiographically documented arterial occlusions were studied in a / " bore oxford research systems tmr- spectrometer at rest and after exercising each limb separately. normal resting values ofpcr/p iwere > and the nmr index = p/(p~+pcr) was . _+ . (s.d.). limbs with femoral arterial occlusions whose ankle systolic pressure index was < . had nmr index which was significantly elevated above norreals ( . + . p< . ) indicating a failure of metabolic compensation for reduced bloodflow and oxygen delivery, although atp concentration was norreal. exercise produced a five-fold rise in nmr index in both normal and diseased legs. spectra were taken over one minute intervals during the recovery period and in normal limbs returned to resting values within rain. the recovery period was considerably slower in the diseased limbs indicating abnormal mitochondrial oxygen delivery and impaired mitochondrial formation of atp. these data demonstrate the feasibility of using pnmr to non-invasively probe the biochemical abnormalities of energy metabolism in patients with peripheral vascular disease. the incidence of urinary calculous disease (ucd) in the south african black population is very low in comparison with the white population group. no biochemical differences in serum nor urine account for this discrepancy and no other measurable parameters have demonstrated any difference between the two groups. urinary particulate activity measurements have demonstrated differences between normal persons and those with ucd who are otherwise biochemically similar, and it would therefore seem rational to expect such measurements to demonstrate differences between the two population groups. urinary particulate activity was measured in the urin of normal whites and normal blacks, the two groups being matched for age, height and weight, and monitored under normal dietary hydrational and environmental conditions. the three parameters of particulate nucleation, growth and aggregation were measured and the two groups compared. particulate nucleation demonstrated the most significant contrast between the two groups with the production of new particles through nucleation being far greater in the white group than that which occurred in the black group (p< . ). particulate growth occurred at similar rates in the two groups although at slightly higher rates in the white group. particulate aggregation occurred at a greater rate in the white group but the difference between the two groups was not statistically significant. the differences between the two groups are shown to occur as a consequence of differing rates of particulate nucleation although the rates of particulate growth and aggregation are parallel. whilst the factors responsible for the low nucleation rate in black person remain unknown their effect can now be measured quantitatively through the parameters of urinary particulate activity. blood levels of ketone bodies appear to determine skeletal muscle amino acid release; high levels conserve protein and attenuate gluconeogenesis. starvation indt/ced ketosis is suppressed by infection [ ] . to determine if the relative hypoketonaemia following sepsi s in turn contributes to increased glucogenesis, arterial substrates and glucose production (constant infusion - h(n)-glucose) were measured before and after infusion of na-dl-./ -hydroxybutyrate (/ oh) to raise levels three-to fourfold in fed (n= ), in fasted (n = ) and in fasted-infected (n= ) animals. in fasted-infected animals before infusion ketosis did not occur (/ oh . ± . mm/ fasted; . ± . fasted-infected) and basal glucose turnover was increased ( . ± . /tm/kg/min fasted; . ± . fastedinfected). with infusion of glucose and alanine concentrations decreased as expected in fed and fasted animals but not in fasted-infected (glucose . ± . ram/ befor; . + . mm/ after). glucose production also fell significantly in the fed ( . ± . /~m/kg/min before; . ± . after) and fasted ( . ± . v. . ± . ) groups but was unaffected by infusion in the fasted-infected group ( . +- . v. . + . ). the accelerated rate of gluconeogenesis in infection is thus not a consequence of hypoketonaemia. the usual reciprocal relationship between glucose and ketone utilisation during feeding and fasting has not been demonstrated~in sepsis. preliminary experiments in a hindlimb model support the hypothesis that during infection amino acid release from muscle is not affected by ketone levels. we have developed a technique for measuring the total body carbon of the living subject which is suitable for measuring the critically-ill as well as the ambulatory patient. by combining this measurement with that of total body nitrogen and calcium [ ] an estimate of total body fat is derived. measurement at the beginning and end of a given period enables the changes in total body protein and fat to be obtained, as well as the patient's energy expenditure if energy intake is also known. the method is a radiation technique. the supine patient is irradiated laterally with a horizontal beam of fast neutrons and the resulting gamma rays from the body are detected by a radiation detector placed unterneath the subject. the nuclear reaction employed is the inelastic scattering of fast neutrons by the carbon nuclei of the body with the emitted gamma rays having an energy of . mevi in the initial application, measures of total body fat obtained using the technique were compared with those derived from skinfold thicknesses in six volunteers: there was no significant differences between the two measurements, (see table) . the method is being employed in studying the changes in total body protein and fat, and the energy requirements of surgical patients receiving nutrition. in order to investigate the mechanism of this effect, normal monocytes were incubated at °c for min (with intralipid /a/ml) and their function assessed by three different techniques (chemotaxis, phagocytosis and chemiluminescence). all three methods showed impairment of function following exposure to intralipid. in order to try and prevent this potentially damaging effect, heparin was added to the various in vitro tests and found to cause marked impairment of phagocytosis. (p< . ) to assess its effect in vivo, volunteers were given , units of subcutaneous heparin h prior to intravenous intralipid (as above). although the use of heparin did not affect either immunological function, it completely prevented the fall of monocyte chemotaxis following intralipid alone. these findings suggest that monocyte function may be impaired by the presence ofintracellular lipid particles. the use of s.c. heparin may help to alleviate this problem and could, therefore, be beneficial to ill and often septic patients requiring intravenous nutrition. to investigate the effect of elevated glucocorticoids of stress and trauma on peripheral glutamine metabolism, . mg/kg bw dexamethasone was injected daily intramuscularely in adult mongroel dogs over a period of weeks. at least weeks prior to the experiments catheters were placed into the animal's abdominal aorta ( ) and caval vein ( ) in order to measure a-v differences and hindquarter blood flow. during dexamethasone treatment nitrogen balances were negative, - . - - g n per day, whereas slightly positive n-balances were observed during the control period ( . +__ . g n/day). muscle glutarnine concentrations declined constantly from . + . mmol/ intracellular water to . - - . by % within two weeks. whole blood arterial and venous plasma concentrations remained constant. to test the hypothesis of increased peripheral glutamine utilisation or decreased glutamine formation, the activities of glutaminase and glutamine synthetase were measured in a muscle homogenate obtained before and days after dexamethasone treatment. both enzyme activities were found to be unchanged. hindquarter glutamine efflux increased from . + . pmol/min in the control state to . + . during dexamethasone treatment indicating a fold muscle glutamine output. this increased glutamine output was enirely due to increased a-v differences and despite decreased hindquaarter blood flow during dexamethasone. it is concluded that dexamethasone reproduces the metabolic response of trauma and sepsis in terms of negative nitrogen balance and muscle glutamine depletion. muscle glutamine is shifted from peripheral tissues to visceral organs with muscle compensating for visceral demands rather than skeletal muscle being the primary target of corticoid action. it has been suggested that there is abnormal glucose utilisation in malnourished patients and that this may explain the adverse clinical sequelae of high rates of glucose infusion during intravenous feeding. we have investigated the hypothesis that there is a depression of the key enzymes of glucose oxidation in the muscle of malnourished patients which is due to an alteration of muscle fibre type proportions. malnourished patients (p) ( m, f + yrs) our results demonstrate that there is a positive correlation between preoperative cp and stage of cancer ' = . +- . x; r= . ;/ < . ). nevertheless before surgery there is no difference between cp values in the two groups considered (g.c.= . ___ . mg %;p.u. = . ---- . mg%), but after surgical trauma cp presents a positive response in patients with p.u. (mean increase + . %), whereas it acts as a negative ap protein in g.c. patients (mean decrease - / ) (p< . ). moreover malnourished g.c. patients present a reduction of cp values ( - %) which is greater than g.c. patients with albumin > . g% ( - . %); this difference is not statistically significant. cancer patients undergoing palliative (n= ) or radical surgical procedure (n= ) show parallel decrease of preoperative cp ( - %), the first group presenting higher preoperative values in relation to the tumor diffusion. in conclusion our results demonstrate that cp is not only a positive ap protein, but in some circumstances it may act as a negative pa protein depending on the underlying disease and the preoperative nutritional status. in this study the free aa concentration in liver tissue of non septic patients (cholecystectomy) were compared with those of septic patients (abdominal sepsis). the liver specimens were taken intraoperatively..the nature and possible risk involved in this study were explained to the patients and their consent obtained. the data presented in this abstract are part of a metabolic screening program of septic patients including the determination of aa (plasma, muscle), hormones (insulin, glucagon, cortisol), nutritional parameters (prealbumin, retinol-binding protein, transferrin), and of energy metabolism (atp, adp, glucose, free fatty acids). for the determination of the free aa the intra-and extracellular water content (chlorid method) and of fat content of the liver specimen were analysed. a membrane potential o f - mv was assumed. the aa analysis were performed with an automatic aa analyser (kontron, svitzerland) by means of an ionexchange resin (durrum dc- ) and a lithium buffer system (durrum-pico buffers). conclusions: . this study reveals decreased concentrations of nearly all aa in liver tissue of septic patients (exception: phenylalanine, tyrosine, cystathionine). . the significantly decreased concentrations of the gluconeogenetic aa (thr, ser, ala) indicate that the gluconeogenetic capacity of the liver is not exhausted through an increased uptake of those aa as shown earlier by wilmore et al. [ ] . an increased administration of gluconeogenetic and basic aa (lys, his) may normalise the aa pattern in the liver of septic patients. the liver is being increasingly recognized as a critical organ in postoperative multiple organ failure. the principle factors precipitating postoperative multiple organ failure were sepsis, hypotension and injury to the liver. previous studies from our laboratory have shown that hepatic failure, which has a high mortality rate, is linked to the marked decrease in energy charge. in order to evaluate the possible presence of metabolic blocks, the changes in the ratio of acetoacetate to fl-hydroxybutyrate (ketone body ratio), which reflects the hepatic mitochondrial redox potential, were analyzed in relation to energy charge in hepatectomized, jaundiced, hemorrhagic-shokked and septic animals, as well as patients with postoperative multiple organ failure. experimental: . in hepatectomized rabbits, mitochondrial phosphorylative activity increased to % of the control and the energy charge level decreased from the normal level of . to . at h after hepatectomy (/ < . ). afterward, these values returned to preoperative levels within a week. the ketone body ratio in arterial blood was positively correlated with hepatic energy charge (r= . , p~ . ). . in jaundiced rabbits, the hepatic energy charge decreased rapidly after the bile duct ligation along with the decrease of mitochondrial pbospborylative activity. the hepatic energy charge fell from . to . at h postoperatively with a maximum incidence of mortality. moreover, changes in the blood ketone body ratio were positively correlated with the hepatic energy charge (r= . ,/~ . ). the decrease in the blood ketone body ratio was attributed to the restricted mitochondrial reoxidation of nadh due to an inhibition of oxidative phosphorylation. . in hemorrhagic-shocked rabbit with a mean arterial blood pressure of mmhg, the changes in the blood ketone body ratio were correlated with hepatic energy charge (r= . , p< . ). . in septic pigs subjected to the ligation and perforation of the cecum, the hepatic energy charge level decreased gradually from . to . and the mitochondrial phosphorylative activity was enhanced to % of controls in the hyperdynamic state. in the hypodynamic state, the hepatic energy charge level fell drastically . concomitant with the decrease in mitochondrial phosphorylative activity and blood ketone body ratio. from these results, the blood ketone body ratio may be regarded as a reliable indicator for assessing the degree of decreased energy charge. clinical: changes in the blood ketone body ratio were measured in patients who underwent major surgery such as hepatectomy. these patients were classified into groups according to the postoperative changes in blood ketone body ratio: group a without decrease to below . , group b with transient decrease to . , group c with progressive decrease to below . and group d with terminal decrease to below . . all group a and b patients tolerated the operation well. by contrast, the group c patients showed multiple organ failure with % mortality rate, which involved pulmonary failure ( %), hepatic failure ( %), gastrointestinal bleeding ( %), renal failure ( / ), cerebral failure ( %) and coagulopathy ( %). all patients who transitioned to the terminal stage of group d died of cardiogenic decompensation. in patients of group c, the decreased blood ketone body ratio was restored with the amelioration of clinical symptoms after ex vivo pig or baboon liver crosshemodialysis and patients of them were later discharged. evidence presented indicates that the decreased blood ketone body ratio has a direct bearing on multiple organ failure. conclusion: sepsis, hypotension or injury to the liver are a metabolic burden to the liver mitochondria which can result in mitochondrial impairment leading to a marked decrease in hepatic energy charge. such impairment ultimately leads to multiple organ failure as a result of the critically decreased energy and substrate store and the reduced protein synthesis relative to demand in the various organs. in interferon-treated cells, the '- 'a synthetase, activated by double stranded rna, polymerizes atp into a series of oligonucleotides characterized by '- ' phosphodiester bonds and collectively designated '- 'a. these activate an endoribonuclease which cleaves rna. other regulatory functions of this enzyme may be expected because of its wide occurence in mammalian cells (untreated with interferon), where its activity appears, in vitro, to be dependent on the growth conditions, hormone responses regenerating liver after partial hepatectomy is often used as a model for the study of control growth and cell proliferation in vivo. in order to evaluate the role of the '- 'a synthetase in the processes leading to initiation of cell division, we measured this enzymatic activity in the rat liver during the first h after partial hepatectomy. partial hepatectomy ( rats) was performed under neuroleptanalgesia by removing the median and the left lateral lobes of the liver according to the method of higgins and anderson. control animals ( rats) were subjected to a sham operation. after selected time intervals, the animals were sacrificed and the enzymatic activity in the regenerated liver was measured. the '- 'a synthetase activity present in the two first removed lobes was defined as %. we observe a very rapid decrease of enzymatic activity which reaches % already h after partial hepatectomy. the lower level of enzymatic activity ( %) is measured between and h after partial hepatectomy. this minimum is followed by a slow restoration of the activity (at h: %). during this early phase of liver regeneration, a maximal incorporation of tritiated thymidine in dna takes place h after surgery. so well differentiated liver cells have elevated levels of '- 'a synthetase. but, after partial hepatec-tomy, the '- 'a synthetase activity decreases dramatically before the first wave of cell mitosis. these observations clearly illustrate the relationship between '- 'a synthetase activity and the growth status. moreover, this drop of enzymatic activity may be a trigger for the initiation of cell division. the primary event or events setting in motion the process of liver regeneration after partial hepatectomy (ph) remain unsettled. regarding the so called hepatotrophic factors, i.e. insulin, glucagon and recently egf, present evidence suggests that they would play mainly a promoting rather than a initiating role. early changes such as glycogen breakdown, fat infiltration and changes in adenine nucleotides and mitochondrial phosphorylative activity are usually, at least the first two, ascribed to metabolic overload of the remaining liver (bucher et al ( ) johns hopkins med, j, : ). so far, however, little attention has been paid to a possible involvement of this phenomenon in the initiation of liver regeneration. attempts were therefore made to modify metabolic overload through early changes in energy metabolism in order to study their influence on the pattern of dna synthesis. fed or h fffsting male wistar rats weighing + g were examined after ph at , , , , and h for adenine nucleotides, oxidative phosphorylation and dna synthesis, based on the rate of~h thymidine incorporation. fasting animals received continuous infusion of % dextrose for h at the rate of . ml/ g/h. in addition to the above mentionned parameters hepatic glycogen and fatty acids were measured. within h partial hepatectomy caused a decrease in hepatic atp which was maximal at h (from . ___ . to . + . /~ moles/g p< . ); in energy charge (atp + . adp/atp + adp + amp) from . -+ . to . ___ . (p< . ) and increase in phosphorylation potential which was maximal at h (from + to _ p< . ). dna synthesis began at h reaching a peak by h. glucose infusion to ph rats suppressed the decrease of hepatic atp, . ___ . / mole/g at h vs . _ . in the control group, prevented glycogen depletion (histochemical estimation) and the increase in fatty acids (two folds increase in ffa and triglycerides (tg) at h vs folds in ffa and folds in tg in the control group),with little effect on mitochondrial activity. the initiation of dna synthesis was delayed and the whole pattern was considerably modified. cessation of glucose infusion restored the usual rate of h thymidine incorporation after a late fall of hepatic atp. in conclusion, glucose infusion was shown by one of us to modify the hormonal response to ph, but insulin and glucagon administration to glucose treated animals failed to normalize the pattern of dna synthesis. it is suggested that metabolic overload as estimated on the basis of early changes in energy matabolism may account for one of the events involved in the initiation of dna synthesis after ph. infusion on liver cell regeneration after partial hepatectomy in the rat b. de hemptinne, j.f. ngala and l. lambotte university of louvain, laboratory of experimental surgery ucl , brussels, belgium after partial hepatectomy (ph) the portal and the peripheral blood serum concentration of immunoreactive insulin suffers a drastic fall and levels ofglucagon show a rapid increase which is maximal h after the liver resection. as these changes appear closely correlated to the blood glucose levels which show a % decrease at h and progressive restoration towards normal values up to h, attempts have been made to alter the insulin/glucagon ratio by glucose infusion after ph and study its relation to liver regeneration. the purpose of this work is thus to determine after ph and hypertonic ( %) glucose infusion: . the effect of glucose on insulin and glucagon blood levels over h; . the repercussion of the insulin/glucagon modified ratio on dna synthesis; . the possible improvement of dna synthesis by extensive glucagon infusion. male fisher rats underwent a standard % ph. a % glucose solution or isotonic salin was infused at a constant rate of . ml/h through a cannula placed in the iliac vein. the rats were sacrified at , , , , and h. ( h) thymidyne ( .. /~ci/mmol) was injected h prior to sacrifice and the liver caudate lobes removed for analysis of ( h) thymidine uptake into nuclear dna. blood samples were withdrawn from the portal vein, the inferior vena cava and the aorta for glucose, insulin and glucagon assays. compared to the salin treated group, the infusion of glucose while keeping a normal steady blood glycemia was responsible of a marked increase of insulin ( . --+ . ng vs . _+ . ng,/ < . ) and decrease of glucagon ( . -+ . ng vs . + . ng, p< . ), with a major switch of the insulin/glucagon ratio at h after ph (from . to . ). dna synthesis started at h in both series, but was very significantly impaired at h in the glucose infused group ( -+ cpm/mg dna vs + cpm/mg dna, p< . ). infusion of increasing doses of glucagon (from . to mg/kg/day could not restore the impaired dna synthesis. only a slight improvement was recorded at . mg/kg/day as the insulin/glucagon ratio tended to approach that of the control group. fractionation of various doses of glucagon over the h perfusion time, in such a way that the changes in concentration of glucagon after partial hepatectomy was imitated, remained unsuccessful to improve thymidine incorporation. in conclusion: . the infusion of hypertonic glucose which impairs dna synthesis after ph, modifies markedly the insulin and glucagon secretion. . if a specific insulin/glucagon ratio after ph is important to sustain normal regeneration, its modification does not seem to be the major factor contributing to the blunted dna synthesis response in the hypertonic glucose infusion model. operative mortality of emergency shunt operations or esophageal transection during acute hemorrhage from ruptured esophageal varices in cirrhotic patients (child's category c) has been intolerably high. hence, the emergency operations in these patients should be avoided when the bleeding could be stopped by non-operative measures. when the emergency operation eventually becomes inevitable, the operative procedures should be simple and of short duration. in , we have introduced the endoscopic balloon tamponade method for the management of esophageal variceal bleeding. the new balloon tube used in this method has essentially the similar structure as the sengstaken-blakemore tube, but is made of translucent plastic materials, and has a larger internal diameter so that a small caliber optic fiberscope (ex. bronchofiberscope) can be passed through the tube. by this method, the endoscopic observation of the esophagus is possible through the translucent balloon tube during tamponade of the ruptured varices. it is possible to know directly whether the bleeding from varices has been successfully stopped or not, which seems to be an advantage over the blind tamponade method using the sengstaken-blakemore tube. this endoscopic tamponade method has been used for emergency hemostasis of acute bleeding from ruptured esophageal varices in patients. the mean initial tamponade pressure by the esophageal balloon necessary to stop bleeding was _+ mrnhg and the average duration of tamponade was h. the location of the ruptured v~ices observed by this method was in the lower esophagus within cm of the esophagocardiac junction in % of the cases. the bleeding has been stopped in occasions ( . %) by this method. no significant complications other than atelectasis in patients have been observed. in patients, the bleeding was initially stopped by the new translucent balloon tube, but recurred within h after decompression of the esophageal balloon. tamponade was repeated for several times since these patients were in the category c of the child's classification, however, the emergency operation eventually became necessary. transthoracic esophageal transection was performed using autosu-ture apparatus, eea, in these patients. one patient died of liver failure in the th postoperative day, but others survived the operation and recovered. the use of eea in transthoracic esophageal transection simplifies the esophageal anastomosis, and shortens the duration of operation by rain. we have so far used the autosuture apparatus, eea ( mm cartridge) in elective esophageal transection of patients. neither anastomotic bleeding or insufficiency has been encountered. acute variceal bleeding in category c patients should be treated initially by endoscopic balloon tamponade, when emergency operation is inevitable, transthoracic esophageal transection using eea is the operation of choice. arterialisation of the portal vein in conjunction with an end-to-side portacaval shunt has been proposed as a method of improving survival following shunting [ ] . however, there is little experimental evidence to support this suggestion and so we have examined the effects of arterialisation of the portal stump in cirrhotic rats. rats with dimenthylnitrosamine-induced cirrhosis were used in this study. of the rats received an end-to-side portacaval shunt, had a portacaval shunt and the portal stump arterialised with the left gastric artery, eight were sham-operated. liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before and after shunting. three weeks after surgery lbf and whvp measurements were repeated, the animals bled and the liver removed and weighed. an end-to-side portacaval shunt led to an immediate fall in lbf ( . _+ . to . _+ . mls/min/ g-~) and whvp ( . _+ . to . -+ . mmhg). however, if the portal stump was arterialised lbf ( . -+ . to . + . ) and whvp ( . _+ . to . _+ . ) did not change significantly. no further changes in lbf and whvp were observed three weeks after operation in any group of animals. arterialisation of the portal stump prevented the loss in body weight, loss in liver weight deterioration of liver function tests and hyperammonemia observed in animals with a portacaval shunt alone. these findings suggest that arterialisation of the portal stump may prevent some of the deleterious effects after shunting. departments of surgery and pathology, university of virginia hospital, charlottesville, virginia , usa we have hadexperience with operative restoration ofhepatopedal portal blood flow in five patients intolerant of total splanchnic shunting. hepatopedal flow was reestablished by takedown of the total shunt and construction of a selective, distal splenorenal shunt, or by isolation and arterialization of the hepatic limb of the shunted portal vein. in two patients, shunt revision was undertaken electively for chronic encephalopathy, unresponsive to low protein diet, intestinal antibiosis and oral lactulose. each individual had been hospitalized more than eight times for encephalopathy or coma. nine and months postoperatively, both patients have had no encephalopathy on unrestricted protein intake, and work. actively as homemakers. serial liver needle biopsies have shown bilobed nuclei and enhanced mitotic activity suggesting hepatocyte regeneration. in three patients, shunt conversion or arterialization was undertaken in desperate circumstances, characterized by liver failure (bilirubin > mg/dl, albumin < . girl, prothrombin time > " s)~ coma and respirator dependency. although two patients showed immediate, marked improvement in mentation, all three died of intraabdominal hemorrhage in the first few postoperative days in spite of prolonged attempts to achieve hemostasis and maximum blood product support. three conclusions can be drawn from this limited experience: . total shunt procedures which are anatomically suitable for subsequent conversion to a selective configuration or for hepatic limb arterialization should be favored over those not offering such potential; . at a time of election, restoration of hepatopedal portal flow can be accomplished in patients with side-to-side portacaval or hemodynamically equivalent shunts with considerable benefit; and . similar procedures in patients with fulminant liver failure are unlikely to succeed. peritoneal-venous (leveen) shunts are associated with a significant incidence of disseminated intravascular coagulation (d.i.c.). this study identifies a site of origin, a pathogenic mechanism, and the hemostatic pathway which accounts for the thrombogenicity of human ascites. ml of peritoneal fluid were removed percutaneously from individuals with malignant and cirrhotic ascites. ficoll-hypaque column chromatography and ultracentrifugation were utilized to prepare four fractions: cellular; a low speed cell-free fluid; a high speed supernatant; and the precipitate from the high speed centrifugation. the cellular fraction from both types demonstrated an ability to shorten a one stage clotting time by % relative to saline and endotoxin controls. similarly, low speed cell-free fluid shortened the clotting time of pooled normal plasma by %; was also effective in factor viii (required for intrinsic pathway of coagulation) deficient plasma; but had no effect on factor vii (required for extrinsic pathway of coagulation) deficient plasma or platelet aggregation and release. the high speed supernatant was demonstrably less thrombogenic. the resuspended precipitate shortened the clotting time of pooled normal plasman by % and of factor viii deficient plasma from infinity to s. in contrast, this material was ineffective on factor vii deficient plasma. we conclude that the thrombotic potential of human ascites derives from peritoneal cells, either leucocytes or malignant cells. consistently, the thrombotic factor exists in suspension and is thromboplastin-like in its behavior, operating through the extrinsic pathway of coagulation. thus, a site of origin and a pathway of activity for the thrombotic agent in human ascites is identified. the effect of somatostatin in hepatic haemodynamics in the cirrhotic rat s. jenkins, p. devitt and r. shields department of surgery, university of liverpool, liverpool, u.k. although somatostatin has been suggested as an alternative treatment to vasopressin in the emergency control ofbleeding oesophageal varices [ ] , recent studies on its effect on wedged hepatic venous pressure in cirrhotic patients have provided conflicting results [ , ] . therefore we have examined the effect of somatostatin on hepatic heamodynamics in cirrhotic rats. rats with dimethylnitrosamine-induced cirrhosis received a bolus injection of , or pg/kg body weight of somatostatin followed by a min infusion of either , or pg/h/kg body weight. control rats received saline only. portal venous flow (pvp) portal pressure (pp), liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before, during and after somatostatin infusion. at the lowest rate of somatostatin administration (bolus injection of pg/kg body weight followed by an infusion of pg/h/kg body weight)there was a rapid decrease in pp ( . + . to . --+ . mmhg), whvp ( . ___ . to . --- . mmhg) and pvf ( . ___ . to . ___ . ml/min). rain after the start of the infusion pp, whvp and pvf were still signaflcantly lower than preinfusion levels. at higher rates of somatostatin infusion there was no furhter decrease in pp, whvp and pvf. lbf was significantly reduced at all the doses of somatostatin. the highest rate of infusion of somatostatin ( pg) produced a significantly greater reduction in lbf than either or pg. these data suggest that somatostatin may have a role in the management of portal hypertension, but that higher doses may have a detrimental effect on lbf. er positive breast cancers preferentially metastasize to bone ( ) prostaglandin e (pgez) is synthesized by breast cancers and potentiates bone resorption in vitro ( ) . this study investigates the relationship between er status and pge synthesis in breast cancer cells. pge was measured by radioimmunoassay in primary breast cancers: a) after ethanol inhibition of further prostaglandin (pg) production -,,basal pg" b) after stimulating pg production with excess arachidonic acid -,,total pg". ,,total" minus ,,basal" = ,,synthesized pg". in order to relate pg production to breast cancer cells, measured pge values have been corrected for the epithelial cellularity of each tumour. pge x corrected pge = actual (%) cellularity cellularity was evaluated by a proportional count of cancer cells expressed as a percentage against non cellular material in all fields of - histological sections at x magnification. we conclude that er positive tumour cells synthesize greater amonts pge than er negative cells. this may account for the greater tendency of er positive cancers to recur in bone. oestrogen receptor activity (er) is of prognostic value in breast cancer [ ] . however, the chosen cutoff between er-negative and -positive is arbitrary and likely to affect its prognostic value. in patients with invasive breast cancer treated by mastectomy, tumour er was determined [ ] and the patients were followed up until first recurrence. using a computer program to perform repeated logrank analysis, the effect of varying the cut-off on prognostic value of erwas studied between and fmol/mg protein. er levels were higher in postmenopausal patients ( - , median , n= ) than in pre-menopausal patients ( - , median , n= ). for the whole group, optimal prognostic discrimination was achieved with a cut-off fmol/mg protein. in premenopausal patients, the effect of varying cut-off was complex, leading to an optimum of fmol/mg protein, whilst in post-menopausal patients the optimum was fmol/mg protein. these findings were unexpected and may relate to the relationship of er to tumour cellularity [ ] . it is concluded that: . the failure of some centres to relate er to prognosis may be due to the inappropriate cut-off point chosen; . in our patients, the optimal cut-off was fmol, which agrees with the level suggested by the british breast group [ ], though it differed between pre-and post-menopausal patients; . optimal cut-off for prognosis may differ from that for predicting response to endocrine therapy. previous studies have reported that the use of adjuvant chemotherapy improves relapse free survival following mastectomy. the effect on overall survival is less certain. patients with histologically confirmed axillary node metastases were randomised to receive postoperative radiotherapy (rt), chemoterapy (cmf) or radiotherapy plus chemotherapy (rt + cmf). patients have now been followed for between months and years. patients initially treated with rt received chemotherapy on failure where possible. of patients receiving rt alone, have developed distant recurrence and have died. of receiving cmf alone have developed distant recurrence and have died (see table) . although the use of adjuvant chemotherapy significantly prolongs the relaps free interval there is no corresponding improvement in overall survival. a sample of patients with histologically proven prostatic carcinoma underwent transrectal find needle aspiration at six month intervals, for a mean follow-up of months, as an out-patient procedure, without significant side effects. patients were followed from time of initial diagnosis. the others had been on treatment for a mean period of months before the study commenced. in of the new patients, cytology was positive at the time of histological diagnosis, and correlated with the histological grade. there were no false positives. repeat cytology on patients after months showed positive and in of these the disease was progressing, and in one it was stable. of the who were negative, were stable and one progressing. of the patients already on therapy had positive cytology at their initial aspiration - showing progressive disease and being stable. patients whose cytology was initially negative became positive months later and all had disease progression at this time_ patients had negative cytology on or more occasions and in only cases was there evidence of disease progression. cytology showed evidence of squamous metaplasia on follow-up in of the patients whose condition was stable. the prognosis for all patients was assessed on the basis of gleeson's score and aspiration biopsy has been shown to be a safe and useful procedure for monitoring disease activity and response to treatment. the optimum method of restoring the ability to swallow in patients with unresectable carcinoma of the oesophagus remains controversial. this study evaluates the palliative potential of pulsion intubation versus retrostemal gastric bypass of the excluded oesophagus in unresectable carcinoma of the upper thoracic segment ( - cm from the incisor teeth). patients and methods: patients were prospectively randomised for treatment by pulsion intubation ( patients) or gastric bypass ( patients). non-resectability was indicated by ( ) tumour lengths greater than cm, ( ) tracheal or bronchial invasion, ( ) disrant dissemination, ( ) mediastinal invasion. the operative mortality, morbidity, palliation of dysphagia and postoperative nutritional status was compared in the two groups. results: mortality: intubation resulted in deaths, a mortality rate of , %. gastric bypass resulted in deaths, a mortality rate of , %. complications: intubation was complicated by respiratory infection ( ), tube migration ( ), respiratory obstruction ( ), oesophageal perforation ( ), bleeding ( ). gastric bypass was complicated by chest infection ( ), pneumothorax ( ), wound infection ( ), subphrenic abscess ( ), anastomotic leak ( ), pulmonary embolism ( ), purulent neck discharge ( ). ability to swallow: palliation of dysphagia was achieved in % of patients following intubation and % of patients following bypass. postoperative nutritional status: improvement in nutritional status was more rapid following intubation. conclusion: pulsion intubation is the preferred palliative procedure because of fewer complications and lesser degree of postoperative catabolism. the current technique for investigating the response of vascular prosthetic materials to infection is by challenge with a sub-lethal dose of bacteria, usually an intravenous infusion of organisms in an animal model. this large bacterial innoculum, however, obscures any difference in the infectibility of prostheses that may be inherent in the material, its incorporation into host tissues, or its resistance to infection. we have developed a sensitive method for determining the susceptibility to infection of vascular prostheses based on calculation of the number of bacteria required to infect a specific prosthesis in % of trials (ids ). following implantation of the prosthesis to be tested in the canine infra-renal aorta, a dose-response curve was generated by the intravenous injection of known innocula of s. aureus (at log intervals from to bacteria). at six weeks the prosthesis was harvested and cultured to document infection with s. aureus. a characteristic sigmoid curve resulted from which the ids was determined. to test this method, a comparison was made between commercial human umbilical vein grafts (huvg) and huvg impregnated with silver sulfadiazine in dogs. although both prostheses were infected by the standard innoculum, a greater than tenfold increase in the resistance to infection by the treated huvg (< to ) was demonstrated in the dose-response curves. since the number of bacteria in a postimplant bacteremia rarely exceeds organisms/ml, such differences in infectibility are clinically significant. ids determination provides a sensitive, reproducible method for quantitating resistance to infection in vascular protheses. prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. it is accepted that infection arises from operative contamination, bacteraemic seeding and abscesses or viscus eroding into the graft. this study investigates the role played by distal sepsis on groin grafts. ten dogs had a specific strain of staphyloccocus aureus inoculated onto a surgical wound in the right foot pad. five days later interposition mm dacron grafts were implanted into the ipsilateral and contralateral groin in continuity with the superficial femoral artery. ten days following this the grafts were removed for bacteriological and histological examination. blood cultures and lymphnode cultures were also taken at this time. in seven dogs the specific staph, aureus, was grown from both grafts. two dogs failed to grow the specific staph, aureus from either graft. these results are significant at the % level using fischer's exact test. blood cultures grew staph, aureus from only one dog. ipsilateral lymphnode cultures yielded the specific staph, aureus in seven dogs. we believe that distal sepsis plays an important role in the estabishment of graft sepsis. the mechanism of spread would appear to be via the lymphatics. the influence of tumor growth on wound healing p.w. de graaf and a. zwaveling laboratory experimental surgery, state university, leyden, the netherlands leakage of a colonic anastomosis is a complication each surgeon fears, because it usually leads to a prolonged hospital stay and is accompanied by a high mortality. carcinoma as an indication for operation and preoperative malnutrition are considered to be high risk factors. the subject of this study was to measure the influence of malignant tumor growth at a distant site on woundhealing in colon and skin, and to find a correlation between the influence of the nutritional state of the experimental animal, and the influence of malignant tumorgrowth on woundhealing. we also tried to find ways to compensate the possible unfavourable influence a malignant tumor might have on woundhealing. the study was performed in rats, the tumor used was a rhabdomyosarcoma, transplanted subcutaneously in the rats right flank. parameters for woundhealing were tensile strength of the skin-incision, and bursting pressure of a colonic anastomosis. six groups of rats were studied, each rat undergoing a standardized colonic operation after two or four weeks. the groups consisted of: control animals ( ); tumorbearing animals ( ), without tumor removal; malnutrltioned animals ( ); tumorbearing animals receiving intravenous hyperalimentation ( ), without tumor removal; animals undergoing tumor removal and colonic operation in one session ( ); animals undergoing the colonic operation two weeks after tumor removal ( ). woundhealing was negatively influenced in an early stage of tumorgrowth (f< . ). four weeks of tumor growth did not impair woundhealing to a greater degree than two weeks. laboratory determinations showed no deviations. malnutrition, leading to less weight gain than in control and tumor bearing animals took much longer to influence woundhealing than tumor pearing. this is in accordance with publications by irvin and hunt ( ), devereux et al ( ) and garattini and guaitani ( ) , which led us to the conclusion that the negative effect of tumor bearing on woundhealing was not solely the result of anorexia. hyperalimentation in tumorbearing rats resulted in undisturbed woundhealing, despite the fact that hyperalimentation as practised by us (with amino-acids and carbohydrates) stimulated tumorgrowth. we concluded from these experiments that tumorgrowth caused a metabolic chaos in the animal, which in turn led to a disturbance in woundhealing. intravenous hyperalimentation could apparently compensate this. woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in two sessions (p< . ). irvin an hunt ( ) have demonstrated however that extraabdominal trauma had no influence on colonic healing. this led to the conclusion that the diminished woundhealing found in rats operated in one session was an effect of the tumor. obviously this influence is not instantly removed with tumor excision. this study offers a theoretical foundation for the clinical observation that in operations for colonic carcinoma with a substantial operative trauma, the anastomosis needs extra protection and/or needs to be performed in a second session. the aorta of the blotchy mouse undergoes dilatation during adult life, resulting in the formation of fusiform aortic aneurysms. the mutation is x-linked, so only the males are affected. we have found a shift in the fluorescent spectrum of insoluble, hydrolyzed, dermal collagen in these mice; suggesting the presence of an abnormal crosslinkage compound. the purpose of this report is to describe additional studies carried out on the soluble collagen fraction in these animals, which lend support to the hypothesis of a crosslinkage abnormality. dermal collagen was prepared from one-monthold mutant mice and their normal male littermates by sequential extractions in salt solution, acetic acid, and urea. amino acid analyis and sds gel electrophoreses of the salt-soluble fractions revealed similar profiles of amino acids and collagen-chain types, suggesting that there is no abnormality of the basic building blocks of collagen in the mutants. fluorescent spectroscopy of the acid hydrolysates of these fractions also demonstrated similar absorption and emission peaks. however, reduction with sodium porohydride abolished fluorescence in the collagen fraction from the mutants. results ( emission m a x ± sem): normal blotchy before reduction . ± . . ----- . after reduction . + . none sodium borohydride is used experimentally to stabilize labile collagen cross-linkages. the present studies indicate that the salt-soluble collagen from the blotchy mice is borohydride-reactive and is thus chemically ,,unstable". these studies suggest a rational for developing probes based on these fluorescent croperties of investigate collagen from human subjects affected with aneurysms. one third of testes are removed after torsion ( , ) because of delay in presentation and diagnosis. in a retrospective study of patients with torsion our salvage rate was %. nineteen percent were misdiagnosed as epididymitis by a junior doctor and % by a surgical registrar. doctors could not distinguish between torsion of the testis and its appendages. in the literature, eight factors have been reported to be of discriminant value (table ) . a computer program, using these parameters and bayes' theorem, was written to calculate the most likely diagnosis. data from the patients with torsion and patients with acute epididymitis were analysed by this program ( table ) . the program correctly diagnosed all cases of epididymitis. of patients with torsion of the testis . in this study we determined the critical ischemic time for the development of an arrhythmogenic potential. egg and bipolar electrograms were recorded from the his bundle, ventricular endocardium and epicardium in twelve anesthetized dogs. short bursts ofventricular pacing ( beats; - beats/min) were used to induce ventricular arrhythmias before and after lidocaine administration ( , , mg/kg). previously, lidocaine has been shown to exacerbate conduction delay in ischemic myocardium leading to reentrant ventricular arrhythmias. in the control state, ventricular pacing with or without lidocaine induced either no response or single or repetitive ventricular responses. sustained ventricular tachycardia was never evoked in the control studies. in six dogs, after min of left anterior descending coronary artery ligation and reperfusion, ventricular pacing with and without lidocaine produced sustained ventricular tachycardia in one animal. in another six dogs, after rain ofligation and reperfusion, one dog showed sustained vertricular tachycardia in response to ventricular pacing alone. the other showed sustaine~l vertricular tachycardia after ventricular pacing with lidocaine. sustained ventricular tachycardia averaged /min and degenerated into ventricular fibrillation within - rain. in conclusion - min of ischemia appears to be the critical period for development of malignant arrhythmogenic potential in the canine heart. administration of lidocaine during this critical period enhances the inducibility of cardiac arrhythmias, which may have clinical relevance in the management of acute myocardial infarction. untreated coronary artery air embolism in the experiments without ecc (group i) resulted in a transmural myocardial ischemia with a mortality rate of % in contrast there were no death in the experiment when extracorporeal circulation was used (group ii to v). the best therapeutic effect with regard to the reversibility of temporary myocard ischemia following coronary artery air embolism was achieved by increasc of the postembolic perfusion pressure (group v), a finding being significantly different (p< . ) to groups i through iv. also volume depletion of the left ventricle on ecc (group ii) resulted in a faster regression of the left ventricular hypothermic area compared to group i (p<: . ). the application of dipyridamole (group iii) and st.thomas cardioplegic solution (group iv) was not able to produce a significant therapeutic effect. the increase of perfusion pressure following coronary artery air embolism has demonstrated to be the most effective procedure to achieve reversibility of myocardial ischemia. to transfer these experimental findings into clinical application is suggested because of its practicability and convenience. transmural biopsies were obtained before and , , , , , min after acc for biochemical and structural analysis. myocardial temperature (t) ph (mph), and pco ) were measured continously with a thermistor, a new ph electrode, and a mass spectrometer respectively. in group i (n= ) acc was under normothermia. in group ii mean t was reduced to °c by the administration of cold potassium cardioplegia immediately after acc and consecutively every min. mph at the end of acc reached . -t- . group i) and . ___ . (group ii) (/ < . ); pmco rose from ___ to _+ mmhg in group i and did not change in group ii. tissue content of atp decreases by % group i) and by °/ (group ii) (f< . ); tissue creatine phosphate fell by % (group i) and by % (group ii) (p< . ). changes in atp and creatine phosphate as well as in tissue glucose and glycogen related ton concomitant changes in ph. the degree of ischemic damage assessed histologically by a mean ischemic score was . +-- . in group i and . _+ . in grup ii (p< . ). irreversible structural demage assessed by electron microscopy occurred in group i - min after acc and was associated with a mph below . . no such damage was observed in group ii. we conclude: . irreversible damage during normothermic arrest occurs considerably later than has been reported for regional ischemia in the beating heart; . during h of acc cold potassium cardioplegia does not completely protect against id when mean t is °c; and . on-line oaeasurement of mph reflects the inadequacy of' mp more accurately than do either pmco or t and thus, provides a useful potential method for intraoperative monitoring of mp. in rabbits, infusion cardioplegia was installed at , , and °c respectively. the infusate contained na , k , , , or , ca , or mmol/ , and varying amounts of glucose. measured osmolality varied from to mosmol/kg. the hearts were excised at the end of a rain infusion period; one half was immediately frozen, the other half was incubated for varying periods of time. specimens of left ventricular myocardium were analyzed for metabolite and electrolyte contents. at termination of the cardiplegic perfusion, total adenine nucleotides (tan) and total creatine (tcr) were within control ranges under all conditions. however, atp and ecp = (atp + . adp)/tan as well as creatine phosphate (crp) and the ratio crp/tcr decreased significantly with increasing dosages of k and ca and higher temperatures. there were corresponding increases in adp, amp, and free creatine. at °c, there were no such changes except in hearts perfused with retool/ k and mmol/ ca. on the contrary, crp and the ratio crp/ tcr were elevated above the control values in hearts perfused with ca-free solutions containing between and mmol/ k. the decrease in atp served as the parameter of the ischemia-induced metabolic alterations and the energy deficit developing during cardiac arrest. at °c, it averaged . and . /zmol/g (p< . ) after min of arrest induced by and mmol/l k respectively. ca, or retool/ respectively, significantly increased the atp-decay to . and more than . pmol/g/lo min respectively (/ < . and p< . ). hypothermia of °c reduced the rate of metabolic deterioration and suspended the influence of the k-dosage. however, the ca-effect was still visible: induction of cardiac arrest by and mmol/l k without/with ca decreased atp by . / . pmol/g (p< . ) and . / . pmol/g (n.s.) respectively within rain. at °c, the rate of metabolic alterations was further reduced. the effects of k-dosage and of ca were completely abolished. the atp-decreased . pmol/g during min of arrest. although higher concentrated k-solutions for infusion cardioplegia do not enhance the ischemiainduced myocardial metabolic alterations in deep hypothermia, they are not recommended for practi-despite the advantages of cbk carrdioplegia, the severely impaired myocardium and/or long ischemia time continue to be a challenge. because of the association of ca ~ with cell injury and death the use of ca ~ channel blockers is logical. investigation of cbd revealied no advantages over cbk. the combination of k and d is appropriate because of their different mechanisms of action. ten dogs had one hour of myocardial ischemia (mi) with topical ice (temp ___ °c) after coronary perfusion with ml cb ( ___ l___c) containing k, meq/l and d, pg/kg. eight dogs had two hours of mi after perfusion with ml cb containing k, meq/l and d, /zg/kg. six dogs received the same treatment as the previous group except that d was increased to /zg/kg for all four perfusions. baseline studies were repeated after min ofreperfusion without the use of ca ~ or inotropic agents. heart rate, peak systolic pressure, vc¢, v~,~x, peak + dp/dt, peak -dp/dt, dp/dt over common peak isovolumic pressure, left ventricular compliance, stiffness and elasticity and great water were unchanged from control. coronary vascular resistance was unchanged in groups one and two but declined in group . creatine phosphate returned to control but atp, adp and the adenosine pool were depressed. ultrastructure was well preserved. in / dogs defibrillation was not required whereas / dogs with cbk and / with cbd required defibrillation. these data suggest that d is a worthwhile addition to cbk. early one-stage repair of some congenital vascular anomalies might be accompleshed readily if the material used for grafts grew along with the reconstructed tissues. we have evaluated the capacity of tubular grafts constructed from anterior rectus sheath to serve as growing segmental replacements of the descending thoracic aorta of puppies (age weeks). grafts measuring cm in length were placed in animals; were implanted with attention to tissue preservation (live grafts) while received grafts subjected to prior freezing and thawing in order to kill the donor tissue cells (devitalized grafts). grafts were measured initially and at sacrifice , or months later. each months animals with live grafts and with devitalized grafts were sacrificed. no aneurysmal dilatations or grafts ruptures were observed in any of the specimens. by months, the live grafts had increased . - - % in length and . ___ % in diameter, while length and diameter of the devitalized grafts showed minimal growth ( . ___ /o and . ___ o/o respectively). during the same period the length of the thoracic aorta increased . ___ % in the live graft group and . ___ % in the devitalized graft group. grafts were lined by endothelium and organized into transmural zones. the thickness of each zone in the live grafts remained static from to months, whereas thickness of the middle and outer zones in the devitalized grafts increased to fold. in living grafts the layers consisted principally of newly formed fibrocellular tissue; the rectus sheath was reduced to a atrophic fibrous band. by contrast, the rectus sheath remained prominent in the devitalized graft specimens. cellularity (cells/field) of the live grafts was much greater than that of the devitalized grafts (ratio . ___ : . ----- ). a newly formed, fibrocellular layered structure replaces the rectus sheath in the live grafts. these findings indicate that rectus sheath grafts are capable of growth in the young animal, providing that at implantation the tissue is well preserved. it is well known that durability of valvular bioprostheses (vbp) depends mainly on the structure of constituent biomaterial and long-term preservation of the collagen network. to our knowledge, no studies on ultrastructure of tissues currently used for vbp has been reported so far after a long time of chemical preservation. the presentation of a new anysotropic tissue, xenogenic cervical duramater (xcd), with a collagen tridimensional network, and a comparative study of scanning (sem) and transmission (tem) electron microscopy of bovine pericardium (bp), human duramater (hd) and procine aortic-cusp (pao) represent the purpose of the present communication. samples of the tissues were obtained in semisterile fashion, rinsed and fixed in karnovsky medium at °c for h. materials were then minced on paraphine plates and washed with . m cachodylate buffer for h and dehydrated in increasing concentrations of ethanol ( %- %) and intermediate exposure to % uranyl acetate for the in-bloc contrasts. were conducted, and results are exposed in table . as a general rule, histologic evaluation was by far, more satisfactory for tissues preserved with . % glutaraldehyde (xcd, pap) than those with % glycerol (hd) and % formaldehyde (bp) in combination. it is concluded that the new anysotropic tissue herein presented, xcd, can be appropriately preserved with . % glutaraldehyde as collagen preservation is concerned, and it's anysotropic and ultrastructural features, maintained for as long as months as assessed by sem and tem. albeit, clinical use of this new biomaterial is subjected to further experimental and animal trials. tetralogy of fallot and absent pulmonary valve (tapv) is associated with massively dilated pulmonary arteries which cause tracheobronchial compression in the newborn and heart failure and cyanosis in older patients. corrective operations have been attended by mortality rates exceeding % due to pulmonary insufficiency causing right heart failure (rhf) and pulmonary complications. pulmonic valve insertion (pvi) with complete repair has resulted in improved survival. the purpose of this paper is to assess the results of total correction and pvi in ten patients. during the last five years, patients with tetralogy were corrected. of these, ten patients (ages days to years) had absent pulmonary valve. one patient ( days) present with severe rhf and pul-monary insufficiency and nine patients presented with mild rhf and cyanosis. chest roentgenographs showed increased cardiothoracic ratio and pulmonary prominence in all. arteriography revealed massively enlarged pulmonary arteries with a mean right pulmonary artery to aorta size ratio of . to . associated pulmonic stenosis and insufficiency was present in all. seven patients underwent closure of ventricular septal defect (vsd) and pvi. of these, three had pvi ( tissue and prosthetic) with outflow patch and four had right ventricle to pulmonary artery (rv-pa) tissue valved conduits. two patients had repair without pvi, and one had repair with a monocusp pericardial valve patch. nine patients have done well with no episodes of thromboembolism or infection. death occurred in a day old infant who had vsd closure and relief of pulmonic stenosis. pulmonary valve insertion seems indicated in these patients at it lowers peak pulmonary artery pressure and thus reduces compression effects on the trachea and bronchi. as well, when pvi was used right heart failure was not noted postoperatively. although in experimental acute myocardial ischemia intraaortic balloon pumping (iabp) appears to increase regional contractility of ischemic segments of the left ventricle by increasing the collateral flow, evidence for this effect of iabp in patients with refreactory myocardial ischemia is not available. this study was done to analyze the effect of labp on global and segmental left ventricular performance in patients with refractory, acute myocardial ischemia using gated blood pool scanning with tc- albium tracer. eight patients were studied on-and-off iabp prior to and following coronary bypass surgery. left ventricular (lv) wall motion analysis was undertaken and both cardiac output (co) and left ventricular filling pressure (lvfp) were determined from thermistor-tipped pulmonary artery catheters. when placed on iabp, mean preoperative global ejection fraction (gef) increased ( . - . (off) to . - . (on); p< . ) abut no changes in co or lvfp were observed. when comparisons were made praend postoperatively, co increased after operation (p< . ), due chiefly to an increase in heart rate ( - to - beats/rain; p< . ), but gef was unchanged whether or not iabp was on. only segmental wall motion analysis pre-and postoperatively revealed that iabp increased regional ejection fraction (ref) and contraction velocity by % (mean) in the angiographically determined regions of myocardial ischemia and these regional changes were maximal during the last one-third of the lv contraction cycle. it appears that ref is a more sensitive indicator of changes in lv performance than co and gef in these patients. further, this study indicates that iabp does increase the regional wall contracility in the ischemic areas of the myocardium in man. the use of aspirin (asa) to inhibit platelet thromboxane synthesis (tbx ) in patients undergoing coronary artery bypass grafting (cabg) has been advocated to reduce the potential for graft occlusion. however, asa in conventional doses also inhibits the venous synthesis of prostacyclin (pgi ) a potent anti-thrombotic factor, and may contribute to excessive surgical bleeding. to investigate the relationship between asa dose, blood loss and inhibition of venous synthesis of pgi , patients undergoing cabg were studied. control patients (no asa) were compared to those receiving or mg. asa as a single dose - hours prior to surgery. production of pgi by saphenous vein specimens removed at the time of surgery was measured by radioimmunoassay (ria) of -keto pgf~a following incubation with um na arachidonate. blood loss was assessed by chest tube drainage and transfusion requirement in the perioperative period. serum tbx was measured by ria following asa ingestion. transfusion (units), drainage (cc), vein pgi (pg/mg tissue) and serum tbx (ng/ml) were (mean -sem): therefore, asa mg or mg did not increase blood loss during cabg. however, asa mg, significantly reduced saphenous vein pgi synthesis (/ < . ) while the lower dose asa mg spared the production of pgi . asa is both doses inhibited tbx (p< . ) and blocked platelet aggregation. low dose asa deserves further investigation as an anti-thrombotic agent since it does not appear to increase operative bleeding or significantly inhibit venous pgi production. transfusion postoperatively, igg increased progressively in patients in group a, reaching preoperative levels on the th or th day, whereas in patients in group b, igg remained at lower than preoperative levels during the first week. the number of patients with abnormally low lavels of igg during the whole po period was significantly higher in group b (p"~. ). no significant differences were found in the others studied variables. one patient in group a had infection. three patients in group b had infections. conclusion: high doses of fab igg administered during ohs and the first po days are effective in lessening the po decrease oflgg and thus may be beneficial in preventing po infection. a water insoluble but very hygroscopic polyvinyl alcohol powder, zy- ", is capable of significantly stimulating the cicatrization of open, contaminated skin wounds in rats. when it is compared to other substances in clinical use such as powders containing antibiotics, antiseptics, amino-acids, enzymes or a dextranomer, no other agent tested was capable of producing a similar beneficial effect. these excellent experimental result justified pilot clinical trials on patients: varicose vein ulcers, atherosclerotic leg ulcers, infected traumatic wounds and infected postoperative wounds. whether in rats or in patients, zy- powder removed secretion, bacteria and tissue debris; it produced an early and increased proliferation of fibroblasts with the appearance of dense granulation tissue; it reduced wound size after less than three applications permitting very early grafting of the wound of eventually, cicatrization advanced to complete epithellzation. regardless of whether the treatments were applied daily in the hospital bed or every other day in outpatients, there were not complaints of unpleasant sensations such as itching, burning or pain. few studies have dealt with long term healing in stomach and none is available for duodenum. this study evaluates morphology and development of mechanical strength and collagen concentration in and adjacent to wounds after to ]g days of healing. incisions were made in the non-glandular (rumen) and in the glandular oxyntic (corpus) part of the stomach and in duodenum of male wistar rats, intact rat served as controls. the wounds were dosed with - polypropylene sutures using a single sutur technique. after , and days of healing complete load deformation curves were determined on strips perpendicular to the incision line after the sutures were cut. collagen distribution was measured as hydroxyproline in strips parallel to the incision line. wound tissue continued to gain strength up to days of healing (breaking energy significantly increased in all tissues from to days of healing). no such increase was found for wounds tested togetherwith adjacent tissue, because the "point of maximum weakness" had moved laterally from the incision line with healing time. wound collagen concentration increased in corpus and duodenum between and days of healing, but was unchanged between and days. the biochemical active zone around the incision line was unchanged - mm on each side from day to . conclusion: while the wound tissue itself continues to gain strength during the days ofheaiing studied, the increase seen after days does hot,enhance the functional properties of the organ as a'whole. the "point of maximum weakness" has at that time moved to the borderline of the biochemically active zone, which lies outside the tissue area enclosed by sutures. myocutaneous flaps based on either the inferior or superior epigastric artery may be used to cover extensive soft tissue defects from the mid thigh to the clavicle. we report our experience with rectus abdominis flaps, based on the superior epigastric artery and based on the ingerior epigastric artery. the flap is easily elevated, no skin loss has occurred and primary closure of the donor defect has been achieved in every case. in a follow-up period of between month and months no patient has developed an incisional hernia. the flaps have been used to cover extensive chest wall resection for recurrent carcinoma of the breast ( patients), extensive radionecrosis of the chest wall ( case) as part of a primary breast reconstructive procedure ( patients) and to replace soft tissue overlying the femoral vessels after radical dissection of the groin for metastatic tumour ( patients). of the patients undergoing primary reconstruction required additional subpectoral prosthesis but in adequate bulk was provided by the flap itself. primary healing occurred in all cases. this easily raised and reliable flap will shorten hospital stay after major ablative surgery for recurrent disease and provides a useful addition to methods of breast reconstruction. a. watson department of surgery, royal lancester in girmary, lancaster, u.k. occult gastro-intenstinal bleeding which defies readiological and endoscopic diagnosis provides one of the greatest diagnostic challenges in surgery. lesions producing this clinical situation are often very small by definition and not infrequently mucosal angiodysplastic lesions, localisation of which may be extremely difficult pre-operatively and indeed at laparotomy. various methods have been described in an attempt to improve pre-operative localisation of such lesions. string tests an dthe detection of sicr labelled red cells in the faeces are notoriously inaccurate. arterioghraphy is invasive and usually necessitates a bleeding rate in excess of ml per day. scintiscanning after red cell tagging with mtc gastro-intestinal blood loss, but localisation is difficult. a method oflocalisation of such lesions using slcr labelled red cells and intestinal intubation is described. after labelling of the red cells, a miller-abbot intestinal tube with the balloon inflated and rendered radio-opaque is passed and samples of gastrointestinal secretions aspirated at each cm during passage down the gastro-intestinal tract. samples are counted on a well scintillator counter and when a sample of high radioactivity is obtained, localisation is assessed both by the length of tube passed and by instilling dilute barium down the miller-abbot tube underfluoroscopic control. this method has been used successfully in five patients which were subsequently treated surgically with localised resection resulting in cessation of bleeding. case histories together with photographs of the method and respected specimens will be presented in poster form. postoperative sepsis is tlae most frequent complication of surgery and is the commponest cause ofprolungation of hospital stay. purpose of the study is to prospectively evaluate incidence predisposing factors, bacteriology and costs of postoperative infections. consecutive surgical patients admitted to our institute from may to july were studied. patients undergoing minor surgical procedures (wound less than cm) were excluded from the study. patients were evaluated daily during hospital stay for onset of infection and results recorded in a data sheet. hemocultures in septic patients and free plasma, activated partial thromboplastin time, prothrombin time, and thrombin time, ristocetin test for soluble monomer complexes and fibrin degradation products (fdp, welco test) euglobulin lysis time (elt) and platelet counts. conclusion: . thrombocytopenia is not a typical feature of boomslang coagulopathy. . the demonstration of soluble monomer complexes is blood samples (positive ristocetin test) appears to be an early and consistent indication of intravascular coagulation. . despite unequivocal evidence of advanced consumption, platelet function seems to be maintained, thus preventing complete heamostatic failure. . support for this view is found in the clinical observation that bleeding is essentially mild and late in onset. . thrombelastographic hyperretractility the "spinning top" appearance is a constant feature and is probably mediated via the platelets. . although d. (vpus venom is extremely potent and often fatal, the antivenom is rapidly effective despite advanced consumption. this mechanism which may involve the platelet release reaction has not been fully elucidated and deserves detailed study. in most cases artificial ventricles are driven pneumatically with the advantage of easy handling and the disadvantages of regulation problems, high weight and volume of the driving units and the danger of air embolism in the case of membrane rupture. the driving unit developed at innsbruck universiy consists of a microprocessor-controlled electromagnet driving a pump that functions as a safety chamber (sk). force transmission to the artificial ventricle (ellipsoid heart-eh) is effected hydraulically. thus there is a fixed connection between armature stroke and membrane movement in the eh. the armature position is measured by the microprocessor by means of a position sensor with programmable switch-over points determining the change from systole to diastole so that idling and beat volumes, respectively, of the eh can be programmed. the sk is a newly designed double rolling membrane pump for pressure and suction with very low compliance. pressure and suction are determined by the armature force which is proportional to the microprocessor-controlled current in the solenoids. thus a very positive control of the pumping action is possible. in mock circulation experiments with the hydraulic safety driving unit the cardiac output (co) was between /min and /min at beat frequencies of to bpm. with constant piston-stroke a direct relation between frequency and co was observed. the influence of preload (starling's law) and afterload decreases with increasing armature force (decreasing periods of systole and diastole, respectively) which is due to friction in the hydraulic transmission system. improvements in the geometry of the next system should reduce these losses. in vivo experiments confirmed the hemodynamic efficiency of the system. applied as lvad the system proved to relieve the beating heart considerably. in the case of heart fibrilation the circulation could be maintained by the safety driving unit. the variable height differences between driving unit and eh as they occur in long-term experiments (animal lying or standing) affect only the ratio systole/diastole periods with the co remaining constant. mechanically assisted circulation is indicated in patients with cardiac failure after open heart operations. the clinical experiments show that left ventricular assist devices are capable only in a few cases to maintain full circulation. the limiting factor is the additional right heart failure syndrome. in patients with postoperative cardiac failure, a distinction must be made between isolated left heart failure and total heart failure. in patients with total heart failurebased on diffuse coronary sclerosis or on an increased pulmonary resistance -left ventricular assistance alone is not effective and right ventricular support is desired. therefore, a simple, quick and safe biventricular assist device is necessary. with the biventricular bypass it is possible to maintain complete circulation in cases of cardiac insufficiency. for a successful outcome in patients with low cardiac output after cardiopulmonary bypass, the e-bvad was evaluated in animal experiments (with calves in acute and survival experiments) in cardiac failure situations. the cannulas for the inflow are put into the left and right ventricle, the outflow tracts into the descending aorta and the pulmonary artery. both parts are connected with the ellipsoid hearts. with the e-bvad it is possible to have a replacement of the heart -a total heart assistance similar to the total artificial heart. in cases of clinical emergency this device can be recommended because of the satisfactory hemodynamic effects achieved and the small degree of traumatic hemolysis ( , mg% free hemoglobin). it represents an easy and quick implantable system for total functional heart replacement. the realtionship between acute pancreatitis (ap) and the enzyme and hormone level in blood and gastric and duodenal juices is the factor that had driven us to do this experiment that would complete the study of the physiopathology of this illness. material and methods. we used eight -year-old dogs. a) production of two antiperistaltic fistula in the mann and bullman way; one gastric and the other duodenal (first part). . blockage of the gastric and duodenal compartments: by the gastric fstula we introduced one foley's bucket with closed point and inflatable globe to block the pylorus. by means of the duodenal fistula we introduced another foley's bucket into the duodenum. once inflated, the ball blocks the first duodenal portion. . juice extraction: by adjacent openings in the gastric bucket blocking the pylorum, we extracted the gastric juice separately. with another thin foley's bucket, that was introduced by the duodenal fistula near the already blocked one, to block the third part of duodenum when the globes inflates. b) production of ap: days after the first surgical operation, by the morandeira method with intraparenchymal injection of a mixture of autologous bile and olive oil. results. one of the animals died on the fifth day after the first operation. + no animal died spontaneously after the second operation. they were killed on the th day. in each one acute necrotizing pancreatitis could be verified. + the radiograph showed that the gastric and duodenal compartments were sealed. -it was easy to separately extract blood and gastric and duodenal juices. conclusion. we believe that this method is complete, simple, with good results and very useful for the study of the physiopathology of ap. liver samples were taken form patients operated for cholelithiasis and common bile duct obstruction by gallstones or pancreatic tumour. early evident histoenzymatic deficiency was found even without jaundice or liver structural lesions. in the bilio-obstructive jaundice, the histochemical methods revealed more marked liver impairment, as compared to the histological picture. the degree of the enzymatic depression could explain the occurence of the postoperative liver failure in some patients. experimentally, the effect of common bile duct ligation was studied in rats. group i = healthy controls. group ii = aspartate ( ml % sol.) was injected i.p. in rats daily, for a week. group iii = the common bile duct was ligated under other anes-thesia, in rats h before killing, and group iv=in another rats days before killing. group iv =in rats aspartate was administered h before an twice after choledocus ligation, and group vi = in another rats aspartate was injected daily for a week after surgery. the liver slices frozen in liquid nitrogen were cut in a slee-type cryostat and the histochemical reactions were performed according to arnold, chayen-bitensky and lojda-gossrau-schiebler's methods. the biochemical reactions were performed using merckotests and merz-dade test-kits. after h, and still more after days, very severe structural, histochemical and enzymatic lesions developed. the liver cell glycogen and rna, as well as the "marker" enzymes of infrastructures markedly diminished: nach -tetrazolium reductase - . %, glucose- -phosphatase - . %, alphanaphthylacetat esterase - . %, atp-ase - . %, '-nucleotidase - . %, pas-reaction - . %, mgp-staining - . %. aspartate treatment seems to exert a protecting effect against the noxious action of retained bilirubin and conjugated bile salts upon the liver cells: nadh -tetrazolium reductase + . %, gsp-ase + . %, esterase + . %, atp-ase + . %, 'n-ase + . %, pas + . %, mgp + . % and lipids - . %. but it does not influence the biochemical signs ofcholestasis: bilirubinemia, alkaline phosphatase, leucinearylamidase, gamma-glutamyltransferase, lactate dehydrogenase. aspartate prevented partially but significantly only the increase of cytolysis enzymes: asat - . % and alat - . %. the histochemical and enzymatic results are in agreement with the severity ofmorphologic changes. therefore, aspartate treatment might be adequate for the preoperative improvement of the liver function in cases of obstructive-jaundice, in order to reduce the incidence of liver failure, without influencing cholestasis. oral glucose tolerance tests (ogtt) y. yamoaka, a. sugitani, ic kimura, ic ozawa and y. tobe department of surgery, kyoto university medical school, sakyo-ku, kyoto, japan two patients with cholecystographic evidence of septate gallbladder underwent dynamic hepatobiliary radionuclide scanning with mtc-ehida. when a steady state of emission had been reached from both gallbladder lobes, cholecystokinin was infused incrementally in four doses ( . , . , . and . ivy-dog-units kg-lmin - ) and counts from the gallbladder analysed by computer. in each an obvious functional difference was revealed between the lobes: both distal lobes ceased emptying abruptly during the third hormone infusion whereas the proximal lobes continued to empty predictably [ ] . in one case, histology revealed the septum to be a well-developed smooth muscle ring with cholecystitis glandularis proliferans confined to the distal lobe; the other case awaits surgery. the cause of the difference in response was probably contraction of the muscle in the septum acting as a sphincter. this study provides indirect confirmation that pain in septate gallbladder is due to septum contraction and raised distal intralobe pressure. it is known that the gallbladder stone passes into the common bile duct. furthermore, common bile duct stones pass to the duodenum. these gallstone movements were analysed in cases during the past six years. the phenomenon in which gallbladder stones pass through the cystic duct down to the common bile duct are seen in cases where stones are small and numerous, the cystic duct is wide and connected with the common bile duct angularly or in parallel. in . % of our cases common bile duct stones passed into the duodenum. there were neither inflammatory stenosis of the terminal choledouchous, high grade pappilitis nor severe obstruction of the common bile duct in these cases. it appears that the movement of gall stone is related to size and number of stone and morphological variations in the biliary system. the study was conducted in order to investigate whether intraoperative mano~etry of the bile ducts could be of additional valtie in diagnosis of afflictions of biliary tract or of the papilla. in contrast to current methods of water manometry it seemed important to the authors to use a simple and safe method which could deliver precise results. method: electromanometrics studies were carried out with a cannula placed in the common duct. three manometric parameters were obtained for evaluation. . resting pressure (rp); . pressure increase after injection of ml saline (lml/s) (pi); . time in seconds needed for return of initial pressure (bile flow) (tr). for pressure measurements a pressure transducer with a recorder was used. results: (table) manometry results in patients with calculi in the common duct or with organic stenosis of the papillawere significantly changed as compared with normal findings in the bile ducts. false positive results (proven by cholangiography and biopsies of the papilla) were found in . % of cases, false negatives in % % of all patients with patho-histological changes in the papilla had pathological bile pres-. sures. although false positive and negative results were mainly caused by methodical errors, the described method delivers a high percentage of correct results in agreement with the final diagnosis. it is avery sensitive method in indicating impaired and reduced bile flow and discovers early pathological changes in the papilla as proven by histological examinations. a functional anhepatic state in experimental animals for biochemical studies and as a model for treatment of acute liver failure can be achieved by different surgical procedures like portocaval shunt and complete arterial devascularization or by replacement of the liver with vascular prosthesis and protocaval shunt. we developed a simple hepatectomy procedure using t/ inch silastic tubing, carbon-y-connector and specially designed "vascular spirales" to restore normal portal and caval blood flow. this model was used for auxiliary liver perfusion studies in heparinized animals; the operation takes min, requires no surgical skill, vascular occlusion is less than min, no signs ofsplanchic hypertension are present and blood loss in fully heparinized animals over h is insignificant. one of the main problems in atypical and anatomical liver resections consists of achieving appropriate hemostasis. also because bile capillaries are opened when liver tissue is separated there is the danger of infection and subphrenic or parahepatic abscesses. the authors have used the human fibrinogen clue to seal the surface of the resected liver in cases ( hemihepatectomies and resections). even in anatomical resections exists, in the majority of cases, a diffuse bleeding or oozing of the resected area. this can be completely controlled by sealing the surface with the fihrinogen adhesive. also one of the main advantages of the fibrinogen consists of having the possibility to avoid the insertion of numerous tubes for drainage. all our cases were drained by a single silicon tube. the postoperative courses were uneventful in all cases no major complications were observed. liver regeneration is thought to be stimulated by changes in portal blood constituents, or flow or by a substance in the hepatocytes. rice et al. have documented increases in total hepatic perfusion in rats during liver regeneration. this study has measured the portal and arterial components sequentially in large animals. young pigs ( - weeks) were subjected to shamoperation or % partial hepatectomy (ph). blood flow in the portal vein or hepatic artery were measured pre-operatively and at intervals of h postoperatively using cuff probes of an s.e.m. electromagnetic flowmeter; these were positioned daily under light anaesthesia. systemic arterial and portal venous pressures were measured via indwelling catheters. previous studies have shown that the peak of regenerative response occurs in pigs to days after ph. the mean pre-operative total liver blood flow increased from . + . (s.e.m.) ml.g-lmin- to a peak of + % on day . thereafter flow declined towards normal by day . the pre-operative portal component was ___ %; this increased to + . % on day and returned to normal level within to days. it seems that both total hepatic flow and the portal component increase markedly just preceding the peak of regeneration. this response may stimulate regeneration, or merely accompany it, or may be proyoked by similar stimulators. the analysis of individual bile acids is relevant to several clinical investigations although established techniques have a number of disadvantages. to overcome these we have developed a simple hplc method using a waters associates rcm radial compression module with a radial-pak c , ,u, mm id, reverse-phase cartridge (column). bile acids were eluted from the column at a flow of . ml min- with a mobile phase of methanol:water ( : v/v) containing . % (v/v) acetic acid and adjusted to ph . with m naoh. a mixture of standards of cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids and their glycine and taurine conjugates were resolved, while the conjugates alone were completely separated in under rain. the technique has been applied to the study of human bile from the common duct, gallbladder, tube and duodenal fluid, and serum from patients with hepatobiliary disorders. bile acids in these samples were rapidly extracted using a sep-pak c cartridge before being applied ( /a to pl) to the hplc system, although bile could be analysed directly without extraction. quantitation (~mol ml- sample) of separated bile acids was achieved by comparison with standards using an on-line integrating computer and less than nmol could be detected using a refractive index detector. acute hepatic failure induced by total liver devascularization in pigs -amino acid uptake by combined charcoal -resin hemoperfusion support system where losses averaged %. jib patients had progressively greater losses with increasing preoperative weight (/ lbs- %, - lbs- %, - lbs- %, - lbs- %, lbs- %). weight loss in patients dbs was statistically and clinically greater with jib. . diabetics, especially insulin-dependent, were rapidly cured after jib. normal plasma glucoses, serum insulins, and oral glucose tolerance curves were usually seen within l postoperative mouth, unrelated to weight loss. all patients requiring insulin or oral medication preoperatively could discontinue mediation usuallywithin a weekpostoperatively. improvement after gb was gradual, appeared related to weight loss, and was often incomplete. . hypercholesterolemic patients had % decreases, from to mg/dl, after jib, but only % decreases after gb. all serum cholesterols were normal after jib, but % remained elevated after gb. because of complications after jib, some needed reoperation and conversion to gb. fortunately, most benefits were retained after conversion to gb. we suggest considering jib for "superobese", diabetic and hypercholesterolemic patients. in the past years jenunoileal bypass procedures were performed. the most common complications and side effects were frequent abdominal pain, or discomfort and flatulence in almost half of the cases. in addition kidney stones, blind loop invagination, electrolyte and liver dysfunction problems could be observed. the over all complication rate was %, the postoperative mortality rate %. to eliminate the blind loop of the small intestine, to maintain enterohepatic circulation of bile, and to diminish undesirable side effects in the conventional jejunoileal bypass, biliointestinal bypass was introduced in . twenty patients with a mean weight of kg were subjected to primary biliointestinal bypass within the last two and a half years. three additional patients had secondary biliointestinal bypass due to side effects, especially diarrhea and flatulence, of jejunoileal bypass, performed two to four years previously. the surgical procedure entailed establishment of an end-to-side jejunoileostomy. cm of the jejunum and cm of the ileum were left in the continuity. the blind loop of the jejunum was anastomosed to a functioning gallbladder. so far the above mentioned complications of conventional jejunoileal bypass could be remarkebly diminished. there have been no deaths in this material and no metabolic side effects. frequent diarrhea is avoided by reduced bile spill-over to the colon. the weight reduction has been satisfactory. in summary: due to a lesser complication rate biliointestinal bypass seems to be superior to the simple jejunoileostomy in the treatment of morbid obesity. hyperplasia or neoplasia g.w. geelhoed, c.j. schaeffer and p. daudu department of surgery, george washington university medical center, washington, usa patients with various thyroid disorders who were undergoing thyroid operation were studied for the presence of absence of estradiol and progesteronebinding proteins in thyroid tissue. steroid receptor assays were carried out using similar techniques and standards as those routinely employed for study of breast cancer specimens. quantitative data were collected by coded specimen number by an observer unaware of the patients' clinical diagnoses, and diagnostic correltions were drawn following de-coding. there was no correlation with age or sex and the presence or quantitative value of steroid-binding site. specimens with the histologic diagnosis of follicular adenoma had estradiol binding sites, and had them at high levels ( . to . ) femtomoles/mg cystosol protein). patients with the histologic diagnosis of thyroid hyperplasia had marginally positive estradiol-binding and lacked progesterone binding. patients with adenocarcinoma of the thyroid exhibited neither estradiol nor progesterone-binding in significant quantity. presence or absence of steroid binding sites in thyroid tissue appears independent of sex or age, but correlates with benign neoplasia of the thyroid, suggesting a possible etiologic association for thyroid adenomas. patients with esophageal cancer underwent resection and primary reconstruction of the esophagus by posterior invagination esophagogastrostomy which we devised. the anastomosis was made in the cervical level in cases and in the left thoracic cavity in cases. functions of stomach placed in the posterior mediastinum were examined in patients surviving more than years and in patients surviving less than years. within one year postoperatively, the absorption ofvitamine b decreased markedly. one and a half year after operation, however, the secretion of castle's instrinsic factor recovered and it showed normal values in patients surviving over years. in secretion of gastric acid by tetragastrin, both total acidity and free hydrochloric acid increased in the progress of the postoperative period and showed normal values in all patients surviving over years. this fact was also confirmed by endoscopic observation of color development by congo-red in the gastric mucosa; its coloring area was scattered in patients surviving years, but extended to the whole surface in patients surviving over years. roentgenographycally in head-down position, the surgically created fornix with a sharp angle of his was effective in preventing gastroesophageal reflux completely. the strain combination of donor and recipients. the nature of the mechanisms determing the different fate of allografts remains obscure and all interpretations of the above findings must be speculative. however, the long-term acceptance of hepatic allografts in the rat appears to be similar to that in other species and emphasizes the role of the liver as an immunologically favoured organ. footnote: part of the work (intrahepatic bile duct proliferation!) contained in this abstract will be presented at the xvii essr meeting . peoples' friendship university, tamojenii pr. , department of operative surgery, moscow, ussr a transplanted kidney, besides tissue incompatibility reaction, is influenced by non-specific injuring factors, including decentralisation. for normal functioning of a kidney transplant over a long period of time we carried out an experimental study. the purpose of the study was the investigation of operative reinnervation possibilities and its influence upon the functional state of a transplanted kidney. for the kidney reinnervation kirpatovski's method for suturing perivascular fascial tissue flaps of anastomosed vessels with branches of vegetative plexus nervosus on them was used. experiments were carried out on mongrel dogs, both male and female. in the first group of experiments a kidney was transplanted into the pelvis while the opposite kidney was preserved or ablated. in the second group the kidney autotransplanted into pelvis was reirmerved by the described method with preserving the contralateral kidney in part of the animals and ablating the kidney in the rest of them. the third, fourth and fifth were control groups. in the third group the kidney was denerved, in the fourth it was denerved and reinnerved by the mentioned method, in the fifth group unilateral nephrectomy was performed. after the operation the animals, in different periods of time (from days to years), were studied by isotopic renography and by scanning kidney's by jbl-hippuran and neohydrin-hg ° intravenous injections. results of the isotopic renography were estimated qualitatively and quantitatively by universal renographic index (hirakawa-corcoran, ). results of the experiments indicate that autotransplantation and denervation of a kidney without its reinnervation were followed by lowering of a kidney vascularisation and its secretory and excretory functions. during the first or months after the operation a tendency to improvement of the kidney function took place; later those functions gradually oppressed and the renographic index lowered down to . +__ . , . +--. . (p< . ) respectively according to the groups. after operative reirmervation of a kidney autotransplant and denervation of a kidney without its transplantation gradual improvement of separate functions of the kidney took place: in or months the kidney circulation was restored; in or , and in or months respectively, secretory and excretory functions of the kidney restored; renographic index reached its norm after or months and remained stable during years (according to the groups . +___ . , . - - . , p> . respectively). thus, the operative reirmervation of a kidney prevented depression of its functions and provided its normal functioning during a long period of time. introduction of fine needle aspiration cytology in renal transplantation gives the possibility not only to distinguish acute tabular necrosis, arterial and venous obstruction and viral infection from acute rejection, fnc also seems to allow judgement of the effectiveness of various types ofimmunosuppressive therapy during rejection episodes. method: after sterile fine needle biopsy the cytological evaluation of the aspirate was performed on cytocentrifuged smears after staining the cells by the method of may-grtinwald-giemsa. thus normal and "activated" mononuclear cell populations as well as parenchymal cells such as tabular and endothelial cells and their pathological changes could easily be recognized. the white blood cell types in fnc were compared with those of the peripheral blood. the difference in number due to the graft invading cells was expressed as "increment". the findings of fnc were correlated with clinical signs and serum creatinine values. results: fnc allows to judge the in situ inflammation in the rejecting kidneys within h. non-rejection grafts show cell counts comparable to peripheral blood. with onset of rejection (grade ) t-and bblasts and monocytes appear in the graft. with sever-ity of the rejection (grade ) monocytes and lymphocytes as well as blood cells increase in numbers. acute rejection (grade ) is heralded by high numbers of monocytes and macrophages. acute tubular necrosis (atn) and virus infections can be distinguished from rejection episodes to be treated. in all cortisone and azathioprine resistant cases where alg was used in order to suppress inflammation during rejection episodes it reduced not only the peripheral white blood cells, but also within h after start of alg therapy the number of in situ cells. beside reduction of inflammatory cells typical changes in shape of the nucleus and density of chromatine in up to % of the lymphocytes and granulocytes could be detected. these changes look closest to what has been described as apoptoses. conclusion: cortisone and azathioprine resistant rejection episodes could be monitored within - h using fine needle aspiration cytology. this method is safe, cheep, and it provides good information about the in situ situation of the graft, it allows to distinguish acute rejection from atn and other situations where higher immuno-suppressive therapy, especially with cortisone, could only be harmful for the patient. criteria of the border of normothermic ischemic tolerance in dog kidneys are first normal pah-and exogenic creatinine-clearances compared with unilateral nephrectomized dogs in neuroleptic analgesia under excessive water and sodium load and second perfectly normal kidneys after two weeks in pathological examination. the protective solution htk ® by bretschneider has a superior buffering capacity compared to euro-collins-solution ® , which, by itself, enhances anaerobic glycolysis by substrate stimulation (glucose) especially between ° and °c. without preservation in normothermia oligo-anuric arf is observed at the border of ischemic tolerance ( - min; °c). the obligo-anuric arf is dependend on ischemia which will result in necrosis of the kidney after rain and °c ischemic temperature. the border ofischemic tolerance corresponds to an intrarenal ph of less than . (outer stripe of medulla), and medullary lactate levels of to pmol/gdw. substrate stimulation of anaerobic glycolysis limits the effectiveness of euro-collins-solution ® above °c earlier than anaerobic glycolysis in pure ischemia. after warm ischemia (>--__.. °c; min) all kidneys protected with euro-collins-solution ® show anuric arf, whereas all htk®-protected kidneys ( to min of warm ischemia; °c) are having polyuric arf. under these conditions, renal ph will not increase above nmol/ h+-concentra -tion. the border of ischemic tolerance for htk ®protection ofkidneys is rain- °c and seems to be limited by a transitory secondary postischemic oliguria ( - h). in summary: intrarenal anaerobic glycolysis limits tolerance of pure warm ischemia by inducing anuric arf. in contrast polyuric renal failure is observed at the border ofischemic tolerance by using the protective procedure with the htk®-solution mentioned above. at least % of unrelated pigs respond to liver allografting with a rejection episode which they overcome without immunosuppression; kidney transplants are rejected uniformly within to days. in this study, mlc responses were measured at weekly intervals in such animals. non-litter mate pigs were subjected to autograft, exchange liver allograft, or exchange renal allograft. blood from mlc responses were taken from unaesthetised pigs before and at weekly intervals after operemained the same for each pair of transplants. in of pairs of liver allografts, mlc responses were depressed up to fold for weeks post-operatively. in all these pigs, the mlc responses were initially high. in pairs where the mlc response was initially low, there was no change after the transplant, and in one pair with a low pre-operative response, there was a marked increase post-operatively. in pairs of kidney allografted pigs, there was a similar depression in the single post-operative sample available. in liver autografted pigs, there was a slight post-operative rise in mlc response. it appears that liver and kidney transplantation but not hepatic autografting, markedly depress sequential post-operative mlc responses. the process of ischaemic kidney degeneration was estimated by measuring the adenine nucleotide (atp)levels and the effect of inosine and naftidrofuryl (praxilene) was assessed. min ischaemia was induced on both dissected kidneys of wistar male rats. then, either both clamps were removed and the right kidney was excised after i rain reperfusion the role of mononuclear phagocytes in various disease states has been extensively studied by phagocytosis, fc and complement receptor sites, and enzyme content. chemotaxis of peripheral blood mononuclear phagocytes, however, has not been studied to any great extent. we have therefore investigated a method for quantifying chemotaxis by mononuclear phagocytes. mononuclear phagocytes were purified from peripheral blood by centrifugation over ficoll-hypaque and a discontinuous percol gradient. chemotaxis was quantified by the 'under-agarose method'. mononuclear phagocytes were allowed to migrate towards the chemotactic agent zymosan activated serum (zas), and the control non-chemotactic agent eagles medium. the distance of cell migration was measured after hours incubation at °c in % co with the aid of a microscope eyepiece graftcule. using non-sepcific esterase staining the purity of mononuclear phagocytes obtained was %___ % and the viability by trypan blue dye exclusion was at least % preliminary results have shown selective migration by purified human peripheral mononuclear phagocytes towards zas. this method may therefore represent a means of investigating an important role of these cells in disease states. the intrathoracic pressure rises when a person exhales into a manometer to such an extent that finally the venous blood-flow to the heart stops. it is supposed that this venous stop flow pressure (vsfp), measured by an ultrasound device, is equal to the central venous pressure. in a prospective clinical trial by two independant examiners in % of the cases (n= ) the correlation was within cm h . therefore, the central venous pressure (cvp) can be measured non-invasively with an ultrasound device. we have previously reported that opiate receptor blockade with naloxone (nal) significantly improves cardiovascular function and survival in canine hemorrhagic shock [ , ] . since species differences do exist, we investigated the effects of nal in cynomulgus monkeys anesthetized with n /o . blood was withdrawn to achieve a mean arterial pressure (map) of mmhg (t--- ) which was maintained until t= h when the reservoir was clamped and the animals treated with either nal at mg/kg bolus plus mg/kg.h infusion i.v. or . % nac in equivalent volumes. there were no significant differences between nal (n= ) and con (n= ) in map, left ventricular contractility (lv dp/dt max, mmhg. vs), and survival; the nal animals, however, were acidotic (pha . ----- . ) and colder ( . - . °c) than con ( . +__ . , . -+- . ). map responses to nal were proportional to ph a complications of vascular bypass grafts, especiallyin the femoro-popliteal region are common. the most frequent of these by far is occlusion. other late complications iclude leaking and false aneurysm formation at the anastomotic site, dilatation of the graft material and stenosis ofanastomotic sites. the most commonly employed non-invasive studies render little information of value in the diagnosis of these complications. we have recently undertaken a study to evaluate arterial grafts at various intervals after implantation with the use of a duplex ultrasonic scanner. imaging of graft material with this system ist excellent and patency of the lumen can easily be established. graft diameter can be measured and accumulation of material within the lumen can be measured and quantitated. although anastomoses are not always clearly visualised they are often seen satisfactorily for diagnostic purposes. graft dilatation, false aneurysm formation and occlusions can be accurately diagnosed with this method. we have also studied the behaviour of femoro-popliteal grafts across the knee joint using the duplex scanner. as a non-invasive technique scanning with a duplex system has many advantages enabling frequent investigations and therefore early recognition of complications where synthetic material vascular grafts have been used for arterial bypass. an experimental model for dissecting aortic aneurysm has been designed to obtain much better understanding of the disease, leading to more effective methods of surgical treatment. dissection of the descending thoracic aorta was successfully performed by modified blanton's prodedure in % of more than mongrel dogs. fals lumens ruptured distally into true lumens to form a doublebarreled aorta in % of the dogs. the method of surgical treatment performed were ( ) closure of entry by direct suture, ( ) closure of entry by inserting dacron vascular prosthesis with a stainless steel ring, and ( ) bypass grafting with vascular prosthesis and ligation of the thoracic aorta. in dogs treated by methods ( ) or ( ) at the time of performing the dissection, cine angiography revealed that false lumens hat thrombosed within month and dissection was found to have completely healed on autopsy. in chronic cases treated by methods ( ) or ( ), false lumens were all patent with various degrees of formation of mural thrombi at observation of to months after surgical treatment. these results indicate that closure of entry for acute dissecting aneurysm should be a curative procedure. studies are now continuing on chronic dissecting aneurysm. the lack of controlled trials not rarely resulted in the incorrect acceptance o fan ineffective operation as an effective one igation of internal mammary arteries for relief of angina pectoris, for example). this failure also explains the non-stop controversies over the appropriate operation (and whether the question operate at all) for various myocardial revascularization procedures, over the thymectomy for myasthenia gravis, the limited vs radical mastectomy for breast cancer etc. several clinicians and surgeons assert that controlled clinical trials for new operations are unrealistic and naive because of some important differences between operations and drugs. compared with medical trials surgical ones are often really more difficult to be carded out. they are subject to more restrictions, ethical, statistical and practical, and may require longer or even remarkably long times. in any case, patients must nevertheless be sheltered from harmful and ineffective surgical operations since luckily not all the undoubted difficulties are unsurmountable, and prospective controlled trials of surgery, including random allocation of patients, are quite feasible. comparison between surgical and non-surgical treatment is a really particularly suitable field for these studies. comparison of effectiveness of two operations of the same type or of different types can also be carried out, but only on certain conditions and using suitable and appropriate expedients. also the doubleblind approach may be feasible, although remarkable restrictions and adequate cooperation are necessary. acceptance in trials of surgery as placebo is objectively very difficult, and obviously it has to be considered only when an effective operation for the considered disease does not exist. the ethical sound of this delicate aspect of clinical research can be minimized enough only if ( ) there are substantial doubts about the effectiveness of the intended operation and ( ) requested placebo-surgery is of very slight importance. posters for scientific meetings mary evans and a.v. pollock scarborough hospital, north yorkshire y ql , u.k. the display of information in posters antecedes even the,art of writing. they have been used to advertise, to educate and to inform and their function is to disseminate information to a wide audience quickly, simply and effectively. in medicine they have been used since the thirteenth century for the promotion of health education. in recent years poster sessions have been introduced into scientific meetings as an alternative to the spoken paper for the presentation of original work. following this experimental procedure hepatic coma supervenes in - h and animals die after ca. h. five male pigs, aged - months, weighing - kg were studied ca. h after operation. the detoxifying system consisted of activated charcoal (norit ¢ (lmm x ram) and ionexchange resin (dowex lx ), subsequently inserted in an extracorporeal circulation. plasma amino acids were determined serially ( , , , rain) in and out the detoxifying system. plasma extraction of individual amino acids (means_ se in/zmol/min) is reported in the table a remarkable extraction was present in the first rain and chiefly involved aromatic amino acids and tryptophan which did not further increase. during hemoperfusion, the molar ratios between neutral amino acids improved. the ratio of branched-chain to aromatic amino acids increased from late hepatic effects of small intestinal bypass (sibp) for obesity plasmafl-endorphin (/ -end, pg/ml, by ria) was related to t:fl-end = (t- ) + , r= . , p< . . when acid-base balance and temperature were maintained, nal (n= ) significantly (*p< . ) improved map and lv dp pg/ml in nal and + pg/ml in con). r-end is released in and contributes to the cardiovascular depression of primate hemorrhagic shock. nal reverses this depression and improves survival but only when arterial ph and core temperature samples of exudates at site of infection were taken wherever possible for aerobic and anaerobic cultures. in following infections were recorded: wound, respiratory tract, thrombophlebitis, indwelling intravenous catheter, unidentified origin fever (> °c lasting more than days) (fuo) and miscellaneous. incidences: patients ( %) had postoperative septic complications. wound infection was recorded in patients ( . %), respiratory tract infection in ( %), urinary tract infection in ( %), fuo in ( . %) thrombophlebitis in ( . %) and miscellaneous infections in ( . o/ ). predisposing factors: wound infections were / ( . %) in clean operations, / ( . %) in potentially contaminated, / ( %) in contaminated and / ( . o/ ) in dirty.wound infections were diagnosed later (mean th day) in clean than in dirty operations (mean th day) (p< . ). a statistically significant correlation was found between wound infection and leght of preoperative hospital stay: from . % in patients operated on within - days to . % for patients operated after days or more (p< . ). no correlation was found between wound infection and age. urinary infections were more frequent when the patients were catheterized at least once in the postoperative period ( % vs %). a statistically significant correlation was found between the incidence of respiratory infections and duration of anaesthesia ( . %----< min, / > < min, / > min;/~ . ). bacterology: out of cultures gave positive results ( %): aerobes were isolated in samples ( / ); mixed aerobes and anaerobe in ( . o/ ); anaerobes in ( . %). bacteroides fr. was the commonest isolated anaerobe in all types of sample except hemocultures were propionibacterium aches was the most frequent. a statistically significant correlation was found between the incidence of recvery of anaerobes and intraoperative contamination ( . % in clean operations, . % in potentially contaminated, . % in contaminated and . % in dirty; p< . ).in wound infections the most frequent aerobes were staphylococcus in clean and e. coli in contaiminated operations. costs: mean postoperative hospital stay of patients with septic complications was days, whereas patients with no postoperative sepsis were discharged after an average of days (p< . ). mean daily cost in our hospital was extimated $ ; accordingly, the mean postoperative hospital stay of patients with sepsis costs $ vs $ of that o~ the patients without postoperative infections. overwhelming postsplenectomy sepsis/infection has been accepted the highest risk with more than percent mortality due to pneumococcus species. however, e. coli, pseudomonas and staph. aureus have been reported a deadful threat to the splenectomized individual also. pneumococcal challenge after splenectomy in animal experiment and after partial salvage has been well examined. staph. aureus-challenge has not been tried after splenic repair, so far. material and method: a total of nmri-mice ( - g) have been subjected to: sham-operation, splenectomy, peritoneal splenosis, controls as non-operated group.the technique was similar to the one performed in rabbits, prior reported. weeks postoperatively, the mice were exposed to intraperitoneal injection of staph.aureus concentrations of °c/ml down to c/ml. the peritoneal cavity of mice has a high resistance to staph.aureus, as only no. proved lethal. results: whereas the macroscopic view post mortem seemed promissing, showing almost total salvage of the splenic particles weeks postoperatively bacterial challenge with staph.aureus was contradictory: x s c/ml was survived by all splenectomized mice, by only percent of the splenosis micecand by none of the sham and nonoperated mice. due to the small number of only mice within this mice-pathologenic staph. aureus species, we may not draw definite conclusions. as for one, the intraperitoneal application may not be the natural way of septicemia. on the other hand, splenic remnants may not be as effective in the protection of staph.aureus-sepsis as in a pneumococcal challenge. therefore, further studies with staph.aureus species in other animals and by other application route will be needed! studies of the coagulant effects of boomslang (dispholidus typus) venom have indicated that the coagulant effect was mainly due to its ability to activate prothrombin. it also activates prethrombin i, factor x and possible factor ix as well [ ] . although boomslang envenomation is said to represent a classic model of disseminated intravascular coagulation, certain features are worthy of critical thought. the coagulation profile of a nine year old lad, who was admitted to the h.f. verwoerd-hospital, h after a reputed boomslang bite, provided the impetus to explore the in vivo effect ofcurde. d. typusvenom on the thrombelastographic and other haemostatic changes in the chacma beboon. methods: . mg, . rag and . mg respectively of crude. d. typus venom were injected subcutaneously in adult baboons. serial determinations of the flow parameters were done over - days. thrombelastography on whole blood an platelet this report presents the ten year survey of liver mitochondria analized in patients. . two mechanisms were of major importance in the augmentation of mitochondrial ability to synthesize atp: a) the enhancement of atp-generating capacity per unit of respiratory assemblies and b) the increase in respiratory enzyme contents. those compensatory mechanisms were functional within a certain range of contents in cytochrome a ( . - . nmol/mg protein, normal; . ). hepatic insufficiency was observed in patients with cytochrome a contents less than . or more than . . . in all patients with cytochrome a of . - . nmol/mg, the blood glucose level after an oral glucose load ( g) returned toward normal within h (parabolic pattern). in patients with cytochrome a of more than . , the blood glucose level did not return toward normal within h (linear pattern). parabolic and linear patterns were intermingled in the patients with cytochrome a form . to . .in this report, we will emphasize the importance of preoperative ogtt and measurement of cytochrome a contents during operation for the prediction of operative prognosis, better than any other currently used index of liver function, in the patients receiving major surgery such as hepatectomy or operation for esophageal varices in severe cirrhosis. the reticulo-endothelial system (res) has largely been ignored in studies of liver regeneration. in this study, the res was "blocked" with colloidal carbon, or was stimulated with olive oil or with glucan. indices of liver regeneration were the thymidine kinase acitivtiy (tk) and the number of mitotic figures (mi) in liver biopsies. fibronectin was measured as a putative index of kupffer cell function.four animals in each group were sacrified at , , , , , and h after sham-operation or % partial hepatectomy (ph). group : carbon suspension (pelikan ink, wagner) mg/ g rat single injection pre-operative; group : olive oil ( % in % dextrose water + . % tween ) . ml/ g rat single injection pre-operative; group : as group given at intervals of h; group : glucan . rag/ g rat pre-op, and at intervals of h.results: glucan or a single dose of oil increased tk in ph and sham-operated rats but did not influence m.i. carbon inhibited mi but did not influence tk. fibronectin levels were increased in sham-operated and ph rats after daily oil or glucan.in conclusion, no administration enhanced both tk and mi but stimulation of the res with glucan or oil did increase fibronectin levels in both sham and ph rats. it appears that the res does not have a role in liver regeneration as assessed above, but fibronectin appears to have been an indicator of res activity. anatomical abnormalities of the gallbladder include multiseptate and bilobed organs, and the more common transverse septate variant. patients with the latter type often have typical biliary symptoms which are thought to be caused initially by raised intracystic pressure and subsequently by inflammation and calculi formation in the distal lobe [ ] .this study evaluate~ the long term effects of sibp on hepatic lipid and fibrous tissue accumulation. liver was obtained at the time of abdominal operation from normal weight patients, morbidly obese patients (mean body weight + kg) and patients or more years (mean -+ months) postoperative from sibp. from wedge liver biopsies hepatic total lipid, triglyceride, cholesterol, phospholipid and protein contents were determined as well as the activities of acetyl coa carboxylase and fatty acid synthetase. histologic evaluation of needle biopsies of the liver was used to quantitatively estimate the amount of hepatic steatosis and fibrosis present.there was also a significant histopathologic increase in hepatic fibrosis present in the liver tissue from sibp patients when compared to hepatic fibrosis in liver from obese patients as well as when compared to liver specimens obtained from the sibp patients at the time of their sibp. the effect of reversal of the sibp on hepatic fat content and fibrosis was determined by transcutaneous liver biopsy - mos. following take down of sibp. post-reversal histologic quantitation of hepatic fibrosis and inflammation was significantly decreased from pre-reversal values and the improvement was greater in patients who lost weight. patients year after sibp have persistent hepatic steatosis with hepatic fibrosis and inflammation. improvement in these parameters may be anticipated following reconstruction of the intestinal tract particularly if weight loss is maintained. the aim of this paper is to study the metabolic effects of gastric bypass in dogs. we used dogs weighing between and kg. the animals were divided into three groups: a) five dogs without surgical operation (control); b) six dogs with a fundoj ejunal bypass, and c) six dogs with a gastroplasty. in group b and c the exclusion was / of the stomach. in all of the animals and groups were determined: cholesterol , live r function tests, calcium, magnesium-and electrolyte levels, the body weight evolution and the apparent-digestibility coefficient (adc) of fat, protein, minerals and glucosides for a type of diet. the composition of this diet in dry substance was: fat . %, protein . %, glucosides . % and minerals . %. these analyses were determined five times during the twelve months of control. the animals were kept in individual metabolism cages which allow feces and urine to be gathered separately as well as the food consumed to be controlled. the cages were housed in a room thermoregulated at °c± . the cholesterol, calcium, magnesium and electrolyte levels were normal during the twelve months of control in the three groups. the liver function tests were normal in all groups with the exception of serum aspartate transaminase initially deteriored in group b. the body weight evolution was significantly diminished in groups b and c compared with the control group (p< . and p< . ). the results of adc expressed in % ± sd were.by roviding a functional gastric capacity of less than ml and consequently a forced reduction in food intake the gastric bypass produces an effective loss of weigth. our results suggest that the gastric bypass can be considered as an effective and safe alternative to intestinal bypass for treatment of morbidly obese subjects who have failed nonsurgical treatment; however, a great and deeper research is necessary to discover the possible side effects of gastric bypass surgery; then its ultimate benefits will be fully understood. there was no operative mortality. the percent of the excess weight lost following the two operations is shown and compared with weight lost following gastric bypass. it is concluded that: . complications occured more often following gastroplasty, . % compared with . % of the patients treated by gastrogastrostomy. . the amount of excess weith lost was greater after gastroplasty, a mean of % for patients followed for two years, than after gastrogastrostomy, a mean of % for patients followed for two years. . because of the incidence of stomal obstruction requiring reoperation following gastroplasty, . % compared with . % after gastrogastrostomy, is our preferred operation. . to improve weight loss after gastrogastrostomy the ta- is now used instead of the ta- stapling instrument. the proximal gastric pouch is reduced in size to - ml and the stoma made smaller to ram. an artifical esophagus was made of silicone rubber tube covered with a dacron mesh. a segment of thoracic esophagus of dogs was replaced with this graft using three different types ofamastomosis, i.e., overlayer end-to-end anastomosis, two layer end-toend anastomosis both using a flanged tube, and monolayer end-to-end anastomosis with noflange tube. seven of dogs ( %) survived more than months without complications, and of them more than years. in of of the prolonged survivors, extrusion of the graft was recognized in the rd to th month after operation. esophageal stenosis increased slightly up to the th month after extrusion of the graft, but it did not further advance until sacrifice. in these dogs, mucosal regeneration of the neoesophagus was complete with muscle layers and mucous glands in the submucosa recognized microscopically. proximal esophagus from the replaced portion was apparently dilatated more than that of the distal portion. there was no definite difference between the anastomotic techniques with regard to complication or prognosis. these results suggest a possibility for clinical trials. paper withdrawn attempts to reduce nephrovascular hypertension by surgical techniques deviating renal venous blood and renin directly to the hepatic filter are at present discouraging. experimental data with portocaval transposition are contradictory, due mostly to the use of heterogeneous biological models (mongrel dogs) and collateral circulation developement. renal to portal vein end-to-side (ets) and end-to-end (ete) anastomoses in the rat were therefore tested as possible experimental models. we observed that even after right kidney decapsulation, collateral vein circulation develops through the periureteral plexus, particularly after ets shunts, one month after surgery. nevertheless, collateral cirulation in the male rat can be prevented by ligature and cutting offthis plexus near the kidney. in the female rat instead, collateral circulation is still possible. in fact, newly formed pericapsular veins join the right kidney to the right ovaric vein and therefore preventive ligature and eradication of these vessels is also necessary. in conclusion, both models (ete-ets shunt) appear feasible in the rat, and reliable for studies dealing with nephrovascular hypertension and hepatic metabolism of renin. the handling of the exocrine system is one of the main problems inpancreas transplantation. one trial to overcome this problem consists of the occlusion of the pancreatic duct system. a the theoretical disadvantage is the induction of a secretion oedema which may lead to blood flow disturbance followed by venous thrombosis. the aim of the present experiments was to study the effect of an anti-oedematous drug (cumarin and rutin sulphate (venalot ®) on the blood flow of autologous segmental pancreatic transplantation was tested by chosing the cervical region of the dog as the site of grafting. material and methods: dogs weighting - kg were used. these dogs were divided into two groups (control group, n= ; treated group, n= , venalot ® dose: rag, cumarin/kg/b.w.). the body and the left limb of the pancreas was removed, perfusion with eurocollins was started wia the splenic artery and the duct system was injected with prolamine. a distal a. v. fistula of the splenic vessels was performed. this conditioned graft was transplanted at the neck of the same dog, performing the anastomoses between the carotid and the splenic vessels. the blood flow of the pancreatic graft was measured with radioactively labelled microsperes of /~m immediately after, as well as days after grafting. the developing oedema of the graft was estimated by weight gain, histological and electron microscopic investigation. residual exocrine function of the graft was measured by determining the serum lipase and amylase levels.results: the autotransplantation of an segmental pancreatic graft to the neck of a dog is a feasable and technically easy surgical procedure, without major venalot -treated dogs local complications. in the ® there was a higher weight increase of the graft in comparison to the controls. as tested so far there was no difference in the behaviour of the i~lood flow of the grafts in both experimental groups. there was a significant increase of serum amylase and lipase values in the venalot®-trea'ted group. conclusion: the technique of cervical segmental pancreatic transplantation in dogs is recommended in cases where immunological monitoring (aspiration cytology, biopsies) is the aim of the study (good access to needles). the technique of microspheres injection is a useful method for the examination of the blood flow in segmental pancreatic grafts in dogs. the prolamine-induced secretion-oedema of ductoccluded pancreatic grafts is resistant to venalot ®treatment. graft pretreatment has been used in various organs to prolong auograft survival. we have recently demonstrated that graft pretreatment of canine renal allografts with cyclosporin a (ca a) led to improved animal and graft survival. our present study assesses the effect ofcy a as a graft pretreatment on pancreatic islet cell allografts. pancreases were removed from unrelated donor mongrel dogs and placed in iced saline ( °c) after the collagenase digestion. the tissue fragments were washed once more and then another mg cy a was added to the preparation prior to intrasplenic injection of the islet cell allografts. three groups were studied: group i (n= ) served as pancreatectomized non-transplanted controls, group ii (n = ) received a non-pretreated islet cell allograft after total pancreatectomy and group iii (n= ) received a cy a graft pretreated islet cell transplant after total pancreatectomy. all animals were given minimal immunosuppression with azathioprine ( rng/kg/day x , followed by , mg/kg/ day). blood glucose values were monitored to determine engraftment and subsequent rejection. hyperglycemia was considered when plasma glucose values rose above mg/dl. all pancreatectomized controls (group i) became hyperglycemic by the first post-operative day. non-pretreated islet cell allografts in group ii had variable function and became hyperglycemic between one and nine days after transplantation. only five of ten animals in group iii, receiving cy a pretreated islet cells, became hyperglycemic levels greater than days and one died of unknown causes immediately after transplantation. the following table presents the animal survival data for the day follow-up period.although the exact mechanism of action is not known, this study indicates that cy a graft pretreatmerit can be beneficial in prolonging pancreatic islet cell allograft survival, further studies will optimize the use ofcy a in this application and hopefully contribute to the improvement of pancreatic islet cell transplantation. grafting of vascularised organs has become a standard procedure in surgical research. however, only few data of the fate oforthotopicliver transplantation in the rat are available, probably because of the difficult ~iargery involved. this communication gives an account of the outcome of liver allografts and liver isografts in four inbred strains of rat. the following groups of allo and isografts are formed; the survival time is given in days postoperatively.the technical details of the operative procedure of orthotopic liver transplantation in the rat are described elsewhere (eur surg res : ( ). the distribution of death among allallograft groups show blocks of survivors. the first block includes animals surviving up to days. acute rejection and infection are the diagnosed causes of death in this compartment. the second block includes animals surviving up to days. in the grafts, which belong to this compartment a intrahepatic bile duct proliferation is a frequent and dominant histological feature. because of the development of identical lesions in isograft surviving the same periode and the induction of this lesions by common bile duct ligation experiments, chronic rejection is excludet as the cause of death. the third block includes all so called long-term survivors, in which the structure of the grafts are remarkably well preserved and very few abnormalities are present. the occurence of fatal acute rejection and long-term survival in each allograft group demonstrates, that the fate of the orthotopically transplanted livers in not dependent upon key: cord- -biop gyd authors: ali, muhammad; khan, tariq; fatima, kaneez; ali, qurat ul ain; ovais, muhammad; khalil, ali talha; ullah, ikram; raza, abida; shinwari, zabta khan; idrees, muhammad title: selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions date: - - journal: phytother res doi: . /ptr. sha: doc_id: cord_uid: biop gyd insight into the hepatoprotective effects of medicinally important plants is important, both for physicians and researchers. main reasons for the use of herbal medicine include their lesser cost compared with conventional drugs, lesser undesirable drug reactions and thus high safety, and reduced side effects. the present review focuses on the composition, pharmacology, and results of experimental trials of selected medicinal plants: silybum marianum (l.) gaertn., glycyrrhiza glabra, phyllanthus amarus schumach. & thonn., salvia miltiorrhiza bunge., astragalus membranaceus (fisch.) bunge, capparis spinosa (l.), cichorium intybus (l.), solanum nigrum (l.), sapindus mukorossi gaertn., ginkgo biloba (l.), woodfordia fruticosa (l.) kurz, vitex trifolia (l.), schisandra chinensis (turcz.) baill., cuscuta chinensis (lam.), lycium barbarum, angelica sinensis (oliv.) diels, and litsea coreana (h. lev.). the probable modes of action of these plants include immunomodulation, stimulation of hepatic dna synthesis, simulation of superoxide dismutase and glutathione reductase to inhibit oxidation in hepatocytes, reduction of intracellular reactive oxygen species by enhancing levels of antioxidants, suppression of ethanol‐induced lipid accumulation, inhibition of nucleic acid polymerases to downregulate viral mrna transcription and translation, free radical scavenging and reduction of hepatic fibrosis by decreasing the levels of transforming growth factor beta‐ , and collagen synthesis in hepatic cells. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. liver disorders have been classified in the high priority areas of health care. according to an estimate by the world health organization, approximately million people of the world are suffering from a severe form of liver disorders, that is, chronic hepatitis (al-asmari et al., ) . medicine of herbal origin may serve as a feasible therapy for the prevailing liver problems because of their safety, easier availability, cost effectiveness, and environment friendliness (izzo, hoon-kim, radhakrishnan, & williamson, ) . medicinal plants have acquired importance in healthcare system throughout the world for their proven and effective therapeutic properties (helmstädter & staiger, ). an estimated % of the world's population is relying on medicines that contain compounds of herbal origin (ekor, ) . the international union for conservation of nature has suggested that approximately , to , flowering plants are used for medicinal purposes (chen, li, ren, & hu, ) . many factors regarding these medicines are important. herbal medicines are claimed to both treat and prevent diseases, which adds to a deep belief that these abbreviations: alt, alanine aminotransaminase; asp, angelica sinensis polysaccharides; ast, aspartate transaminase; egf, epidermal growth factor; hbv, hepatitis b virus; lbps, lycium barbarum polysaccharides; wf , woodfordia fruticosa flower extract. treatments are safe because they are "natural and gentle" and therefore, a harmless alternative to the conventional medicine. moreover, the latter may sometimes cause disappointing results and undesirable side effects in patients (izzo et al., ) . in addition, the less expensive herbal products are often not subject to strict regulations and medication prescribed by a physician or other qualified practitioners (hunter & hegele, ) . although medicinal plants have been used globally, their wider usage is limited to a few countries like japan, india, china, pakistan, thailand, iran, and some african countries (bahmani et al., ; iwu, ; li, ; sivasankari, anandharaj, & gunasekaran, ) . other countries are also encouraging the use of plant-based medicinal products in their healthcare systems. for example, natural health product regulations of canada for the plant-based product in healthcare encourages usage of modern technology and evidencebased scientific support towards promoting medicinal plants and the associated products (tomlinson & akerele, ) . a major concern of scientists investigating herbal treatments is that the chemical composition of the plants contributing to their biological effects is mostly undetermined (ling et al., ). herbs and herbal medicines have been used for the treatment of liver diseases for a long time (dhiman & chawla, ) . there are many herbs having ingredients that are potential sources of medicine for the treatment of liver diseases having various modes of actions and bioactivities (babu, bhuvaneswar, sandeep, ramaiah, & rajendra, ; gnanadesigan, ravikumar, & anand, ; pereira, barros, & ferreira, ) . however, several of them are well-studied for their bioactive components and the mechanism of hepatoprotective activity. in the current review, we have selected some of these compounds for which elaborate detail about hepatoxicity is available in literature in the form of either in vivo studies, study into biochemical parameters and bioactive compounds. this article highlights the possible ways of inducing hepatoxicity in mice models and encompasses the mechanisms in which certain medicinally important plants perform their hepatoprotective activity. the article further aims to summarize studies conducted on the composition, pharmacology, and nature of the selected plants in the light of possible mechanism deduced from experimental trials. a thorough search was conducted on the electronic literature databases, google scholar, pubmed, scopus, and web of science. literature was retrieved using the key words and phrases "hepatitis c", "hepatoprotective activity", "mechanism of action", "medicinal plants", "herbal", and "treatment". about relevant articles were extracted after a narrow search for a combination of the keywords and subsequent analysis per the inclusion criteria. there were two sets of criteria applied to articles for inclusion in this manuscript. according to the first, "general criteria", articles selected for this manuscript were those which (a) reported plants and their parts that were traditionally applied to hepatitis and liver disorders and any other type of hepatoprotective activity; (b) reported extract or pure compounds important for their hepatoprotective role; and (c) attempted to explain the mechanism of hepatoprotective action of these plants. the nd criteria were used for selecting those plants that are discussed in detail (shown in figure ). for this purpose, seventeen plants were selected for which recent articles were available that (a) studied in vitro and in vivo hepatoprotective activities of herbal products, (b) reported active compounds from the plant, and (c) described the mechanism of action herbal hepatoprotective products. the plants described in detail were selected if literature available for them fulfilled at least two of the above -point criteria. each of the selected plants was discussed; mainly focusing its hepatoprotective activities, active compounds, and possible mechanism of action. in addition, featured hepatoprotective herbal combinations have been deliberated. toxicity and quality control issues associated with these herbs/herbal products have been debated. two of the authors independently reviewed all the full-text articles obtained during the electronic search. data from the eligible articles were extracted; all the disagreements were discussed and were referred to a third reviewer (one of the author) for a final decision. all the data were extracted in two tables (tables and ) , and the mechanisms of action were explained in respective subheadings and demonstrated through four different figures (figures - , and ). the figures were constructed in chembiodraw ultra (version . ) software package. furthermore, the plant database "plant list" was used for the taxonomic categorization of all the documented plant species (theplantlist, ) . the prerequisite to screen/study any medicinal compound for its hepatoprotective activity is to develop a model (animal model or cell culture model) in which hepatic injury is induced (salehi, karegar-borzi, karimi, & rahimi, ) . several studies have manipulated mice models to induce hepatotoxicity and then treat those induced liver diseases using herbs and herbal products (figure ). this approach provides insight into how hepatitis and other liver diseases are caused. however, for many plants, their mechanism of action against hepatotoxic agents is not well-documented. the common prototype applied for hepatoprotective drug screening is the carbon tetrachloride (ccl ) induced hepatic injury (rodrigues et al., ) . as ccl have been reported for its damaging effects on the liver because on metabolism by p , it produces free radicals (johnston & kroening, ) . these free radicals cause lipid peroxidation by binding to dna, proteins, or lipids (yasuda, izumi, shimada, kobayakawa, & nakanishi, ) . the degree of hepatic injury is evaluated by the higher level of biochemical parameters that is ascribed to the production of trichloromethyl free radicals which eventually causes lipids peroxidation present in cellular (dusheiko, ) calotropis procera (aiton) dryand. crude hydro-ethanol solution extract prevents of the depletion of gsh levels. c. procera contains flavonoids thus it also performs the antioxidant activity (mcomish et al., ) clerodendrum abilioi r. fern. ethanol extract decreased the serum enzyme alt, ast, alp, tgl, and total cholesterol and considerably increased the glutathione level (chamberlain, adams, saeed, simmonds, & elliott, ) ficus carica l. leaves crude petroleum ether extract reduction in the levels of alt and ast. the petroleum ether extract of ficus leaves repair the damaged liver cell (gond & khadabadi, ) glycyrrhiza uralensis -glycyrrhizin glycyrrhizin administered in plc/prf/ cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes (sato et al., ) momordica dioica roxb. ex willd. alkaloids, phenolic compounds, glycosides, flavonoids oral administration of the extract significantly normalized and restored the elevated serum enzymatic levels of ast, alt, salp, and total bilirubin. its hepatoprotective activity is due to the antioxidant and free radical scavenging activity. (surai, ) nelumbo nucifera gaertn. leaves catechin glycoside, myricitrin- -o-glucoside, hyperin, isoquercitrin, quercetin- -o-rhamnoside, astragalin lotus leaf extract possess significant hepatoprotective and antioxidant activity in ccl -induced toxicity rat model. free radicalscavenging and antioxidant activity due to the presence of some flavonoids and phenolic compounds results in the hepatoprotective activity. (theplantlist, ) paeonia lactiflora pall. and a. membranaceus (fisch.) bunge. -progression of ccl -induced hepatic fibrosis was inhibited in rates by decreasing the level of tumor growth factor-β and inhibit collagen synthesis (sun et al., ) s. miltiorrhiza bunge. roots -s. miltiorrhiza could reverse the ccl -induced fibrosis treatment by decreasing the levels of transforming growth factor-β , procollagens i and iii, and metalloproteinase- and decreasing the levels of metalloproteinase- in liver of the affected rates (wasser et al., ) s. miltiorrhiza bunge. roots s. miltiorrhiza polysaccharides protects liver against immunological injury by adjusting the levels of alanine aminotransferase, aspartate aminotransferase, nitric oxide, tumor necrosis factor and interleukin- (zein et al., ) solanum nigrum l. total decoction crude aqueous extract inhibited thioacetamide-induced collagen (α ) and transforming growth factor-β mrna levels in the liver of mice with thioacetamide-induced liver fibrosis (hsieh et al., ) s. nigrum l. and cichorium intybus l. crude plant extract protect dna against oxidative damage in the reaction mixture containing calf thymus dna and free radical generating system (sultana et al., ) (continues) membrane (chen, yu et al., ) . figure shows the different strategies applied for studying the in vivo effects of induced hepatotoxicity in mice models. the leaves are marked by distinct white "milky" veins that give the plant its common name (theplantlist, ) . historically, s. marianum was used medicinally to treat disorders of the gallbladder, spleen, and liver, but the most important medicinal application of s. marianum is its use as a hepatoprotective herbal treatment and as supportive treatment for chronic inflammatory liver disorders such as hepatitis, cirrhosis, fatty infiltration, and some other forms of liver damages due to toxic chemicals, poisonous mushrooms, and alcohol (freitag et al., ) . the most important component extracted from s. marianum is silymarin (lu, lu, chen, zhang, & wu, ; wu, wang, & que, ) , which is used to treat a variety of liver disorders, including chronic and acute viral or drug/toxin-induced hepatitis, alcoholic liver disease, and liver cirrhosis (lu et al., ) . silymarin is a combination of different ingredients with silibinin as the most active among them (surai, ) . silymarin has been approved for clinical studies in treating the hepatitis c virus infection (ferenci et al., ) . there are many studies on the mechanism of hepatoprotective effects of silymarin. recently, tunca et al. ( ) showed that silymarin has a protective action on pyridine-induced hepatic injury in syrian hamsters. the study concluded that it decreases the metabolic activation of pyridine (by decreasing the cytochromes p a protein concentration) and control the elevation of inducible nitric oxide synthase expression. all these factors play a protective role in liver injury. in another study, farghali, kamenikova, hynie, and kmonickova ( ) concluded that in addition to inhibition of lipid peroxidation, the hepatoprotective activity against thioacetamide-induced hepatotoxicity (khatri, garg, & agrawal, ) tephrosia purpurea (l.) pers. decreased serum aspartate aminotransaminase ( % and %), alanine aminotransaminase ( % and %), gamma glutamyl transpeptidase ( % and %), alkaline phosphatase ( % and %), total bilirubin ( % and %), and liver mda levels ( % and %), and significant improvement in liver glutathione ( % and %) when compared with thioacetamide-damaged rats. (hosseinzadeh & nassiri-asl, ) vitex negundo l. administration of ethanol solution extract of vitex leaf caused a significant decrease in tb, ast, alt, and alp levels in rats. (abdulkarim et al., ) zanthoxylum armatum dc. bark berberine elevated serum enzymatic levels of serum transaminases, alkaline phosphatase. total bilirubin was considerably restored to a normal level. (cha et al., ) note. gpx = glutathione peroxidase; mda = malondialdehyde; ast = aspartate transaminase; alt = alanine aminotransaminase; ccl = carbon tetrachloride; sod = superoxide dismutase; gsh = glutathione; tb = total bilirubin; alp = alkaline phosphatase hbsag = hepatitis b surface antigen; tgl = triglyceride lipase. inhibition of the increased intracellular ca i plays a critical role in the hepatoprotective effect of silymarin. although, upadhyay, kumar, and singh ( ) showed that silymarin restores the changes in the expression and activity of cytochrome p (cyp) enzymes (cyp a , cyp a , and cyp e ), glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. glycyrrhizin administered in plc/prf/ cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes glycyrrhizin administered intraperitoneally inhibits the lipopolysaccharide-and d-galactosamine-induced liver injury by preventing inflammatory responses and il- production in mice glycyrrhizin inhibited anti-fas antibody-induced hepatitis in mice by acting upstream of cpp -like protease administration of glycyrrhizin or glycyrrhetinic acid, significantly suppressed α (i) collagen gene promoter activation and progression of liver fibrosis induced by repeated ccl injections in transgenic mice (liew, erali, page, hillyard, & wittwer, ; martell et al., ; ogata, alter, miller, & purcell, ; sato et al., ) phyllanthin phyllanthus amarus schum. et thonn. phyllanthin help in restoration of antioxidant potential of rat hepatocytes, level of gsh, and sod and gr activities reduced by ethanol (chirdchupunseree & pramyothin, ) p-methoxy benzoic acid capparis spinosa l. the compound alleviated the enzyme levels increased as result of administration of ccl , and pcl (gadgoli & mishra, ) silymarin silybum marianum (l.) gaertn. silymarin attenuated the rifampicinand/or pyrogallol-induced hepatotoxicity by restoring the alterations in the expression and activity of cyp a and cyp e , glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. silymarin suppresses n-nitrosodiethylamine induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals (farghali et al., ; upadhyay et al., ) note. hbsag = hepatitis b surface antigen; cpp = -kda putative cysteine protease; ccl = carbon tetrachloride; gsh = glutathione, sod = superoxide dismutase; gr = glutathione reductase; cyp = cytochrome p ; pcl = paracetamol. g. glabra is a member of the glycyrrhiza genus (isbrucker & burdock, ) , an ancient genus that contains the most commonly used herbs in chinese traditional medicine (hosseinzadeh & nassiri-asl, ) . glycyrrhiza species are considered among the most important herbaceous plants for a diverse array of pharmacological activities (hosseinzadeh & nassiri-asl, ) . chemical structures of ( ) cryptotanshinone, ( ) phyllanthin, ( ) quercetin, ( ) glycyrrhizin, ( ) silymarin, and ( ) p-methoxybenzoic acid, also known as p-anisic acid. all the images were adopted from ncbi-pubchem with the compound ids; , , , , , and , respectively (pubchem, ) (mao et al., ) . one of the bioactive compounds from p. amarus is phyllanthin, which is a lignan compound and is traditionally applied in the treatment of many liver diseases (hanh, sinchaipanid, & mitrevej, ) . it was shown to have hepatoprotective effects on ethanol-induced oxidative damage in primary culture of rat hepatocytes through its antioxidant activity especially the activities of superoxide dismutase (sod) and glutathione reductase (chirdchupunseree & pramyothin, ) . previously, naaz, javed, and abdin ( ) . | c. intybus l. c. intybus l., commonly known as chicory, belongs to the lactuceae family and is typical mediterranean plant indigenous to western asia, europe, north america, and egypt, which varies in perianth color from white, red to blue (norbaek, nielsen, & kondo, . | s. nigrum l. s. nigrum l., commonly known as "black nightshade", is a species in the family solanaceae ( ccl -induced hepatic necrosis. this hepatoprotective effect might be due to its adjustment of antioxidant activity, detoxification enzymes, and its free radical scavenger effects. in another study, hsieh, fang, and lina ( ) induced liver fibrosis by administering thioacetamide in mice and treated them with distilled water and s. nigrum extract via oral administration for weeks. this treatment alleviated the hepatic hydroxyproline and α-smooth muscle actin protein levels in mice and inhibited thioacetamideinduced collagen and transforming growth factor-β mrna levels in the liver. histological examination of liver also confirmed that this extract reduced the degree of fibrosis caused by thioacetamide treatment which is the probable reason for the reduction of hepatic fibrosis. . | s. mukorossi gaertn. s. mukorossi gaertn., commonly known as ritha or aritha, is abundantly found in india. its fruit is reported to have expectorant, purgative, antidotal, and abortifacient effects. additionally, it is used in epilepsy, extreme salivation, and chlorosis (suhagia, rathod, & sindhu, ) . the saponins extracted from this plant are spermicidal (in vitro) and due to this property, it has been used in contraceptive cream (rastogi & mb, ) . pharmacological studies of s. mukorossi have shown their potential effect as hepatoprotective agents (upadhyay & singh, ) . to assess the hepatoprotective activity of the s. mukorossi, wistar male rats were treated with ccl . administration of ccl to normal rats increased the serum levels of alt, ast, alp, and bilirubin. these enzymes eventually cause damage to the hepatic cells. the ccl -treated liver cells cultured on petri plates were treated with the extracts of s. mukorossi and were reported to alleviate the levels of these enzymes. when histopathological studies of the ccl -treated rats were performed, they showed that it also causes the demolition of architectural configuration of target cells. however, rats that were treated with s. mukorossi presented normal lobular structural design, which shows its reparative properties and thus its hepatoprotective effects. . | g. biloba l. g. biloba l. belongs to family ginkgoaceae (theplantlist, ) . it is one of the significant herbs of the chinese traditional medicine. g. biloba leaf extract has been reported to have therapeutic activities against age-related memory deficit problems, including alzheimer's and dementia; cardioprotective, antiasthmatic, antidiabetic, hepatoprotective, photoprotective effects, dna repair mechanism, antioxidant, and antiinflammatory activities (mohanta, tamboli, & zubaidha, ) . g. biloba has been associated with a strong hepatoprotective activity through numerous studies (parimoo et al., ) . g. biloba amplifies cellular antioxidant protection system consisting of glutathione peroxidase, glutathione s-transferase, glutathione reductase, nonprotein thiols, catalase, and antioxidant enzymes (sod). the binding of an individual part of herbal tracks to that of phosphatidylcholine produces phytosome having better efficacy compared with traditional herbal extracts (naik, pilgaonkar, & panda, ) . rifampicin is an antibiotic widely used in tuberculosis chemotherapy. it has been reported to cause hepatoxicity, the reason of which is unknown as it is always given in combined form with other antibiotics such as isoniazid and ethambutol. wistar albino rats were treated with rifampicin that caused hepatoxicity in them. their blood samples were taken, and assays of their blood samples were performed to know the levels of sgpt, sgot, and alp. the elevated levels of sgot, sgpt, and alp show liver damage as these enzymes escape from the liver into the blood in case of liver damage. with parallel treatment through ginkoselect phytosome® and the standard drug silymarin, the markers enzymes levels in serum were nearly at a normal level or marginally elevated. this suggests the hepatoprotective quality of g. biloba plant. it also elevates total protein levels and albumin, which shows its hepa- with activities against chronic hepatic fibrosis (nitha, prabha, ansil, & latha, ) . it also shows antiinflammatory, antibiotic, antileprosy, and antihelminthic properties (arya et al., ; shoaib et al., ; syed & khan, ) . in an experiment designed to check the hepatoprotective effect of w. fruticosa flower extract (wf ), albino wistar rats were administered with ccl which resulted in the increased level of alp, ast, alt, and lactate dehydrogenase. these enzymes leak from serum into the blood. thus, ccl damage causes loss of enzymes which are responsible for drug metabolism (chandan et al., ) . these rats were administered with wf . the extract reversed the elevated lipid peroxidation and regulated the liver glucose- -phosphate and gsh levels. these results are in line with former information for other hepatoprotective agents . . | v. trifolia l. v. trifolia l., known generally as chaste tree, is a high-value medicinal plant that belongs to the family verbenaceae. its leaves are effective as plaster against pains, infections, and fever. its fruits are used in curing amenorrhea, and the flowers are effective against fever (chan, baba, chan, kainuma, & tangah, ) . the active constituents of this plant are essential oil (kvasnicka, biba, sevcik, voldrich, & kratka, ) , viterifolins, and diterpenes. it also possesses some important pharmacological qualities, that is, antipyretic (rani & sharma, ) , antibacterial (lawitz et al., ) , antiallergic, and antiasthmatic properties (lawitz & gane, ) . medical practitioners use this plant in the treatment of acute jaundice. however, literature study suggested that this plant is not well screened for its hepatoprotective activity. nonetheless, the tribal groups of western ghats use this plant leaf extracts in treating jaundice, and these results give some scientific evidence of hepatoprotective activity. . | s. chinensis (turcz.) baill s. chinensis (turcz.) baill is widely used in traditional and modern chinese medicine for the treatment of many disorders including insomnia, respiratory failure, and weakness. moreover, mental health improving ability along with fatigue reduction property is also validated for s. chinensis in russian medicine (szopa, ekiert, & ekiert, ) . in general, dibenzocyclooctadiene lignans found in s. chinensis are known to exhibit potent hepatoprotective activity (zheng et al., ) . in one of the study of individual lignin, gomisin a was found responsible for the acceleration of hepatocytes proliferation and increase hepatic flow (panossian & wikman, ) . furthermore, elevation of mitochondrial glutathione concentration was found to be linked with γ-schisandrin hepatoprotective mechanism. the increase in vitamin c concentration in the liver of test animals upon treatment with γ-schisandrin also validates its hepatoprotective ability. another individual lignin, schisandrin b was also found to counter oxidative harm to liver tissues (thandavarayan et al., ; xin et al., ) . in one scientific study, the hepatoprotective mechanism against acetaminophen-induced liver injury of six schisandra lignans (deoxyschisandrin, schisantherin a, schisandrin b, gomisin a, schisandrin c, and schisandrin) was elucidated. the hepatoprotective ability of these lignins was found to be associated with inhibition of cytochrome-mediated bioactivation (jiang et al., ) . furthermore, another mechanistic study investigated the hepatoprotective effect of schisandra polysaccharide in nonalcoholic fatty liver disease mice models. the results demonstrate potential down regulation of hepatic lipogenesis genes and lxrα/srebp- c/ fas/acc and srebp- /hmgcr signaling pathways in the liver (wang, song et al., ) . . | c. chinensis lam. c. chinensis lam. also known as chinese dodder is a parasitic plant having diverse traditional medicinal uses as a tonic, sex enhancer, and abortion preventer (zheng, dong, & she, ) . studies also have scientifically validated the hepatoprotective activity of c. chinensis (donnapee et al., ) . yen, wu, lin, and lin ( ) chinensis seeds ethanol solution extract was found to be more effective in rats with acetaminophen-induced hepatotoxicity (yen, wu, lin, cham, & lin, ) . the mechanism of hepatoprotective potential as demonstrated by ethanol solution extract of c. chinensis is proposed to be the elevated activities of antioxidant enzymes. . | l. barbarum l. l. barbarum l. berries are very famous in traditional chinese medicine for the treatment of inflammation, cancer, eye disorders, throat infection, and anemia. the use of these berries has been validated as food and also has gained great importance due to its significant antioxidant potential (cheng et al., ) . the major active components of l. barbarum berries are l. . | a. sinensis (oliv.) diels a. sinensis (oliv.) diels is reported in chinese herbal medicine for the treatment of cardiovascular disease, anemia, and hepatic disorders (bunel, antoine, nortier, duez, & stévigny, ) . the a. sinensis polysaccharides (asp) extracted from a. sinensis roots having the average molecular weight of , da is regarded as a potential active component of a. sinensis that exhibits a wide range of pharmacognostic properties (hsu, tsai, & tsai, ) . the hepatoprotective potential of asp in ccl -induced liver injury and via using ischemia/reperfusion rat is widely established (zhang et al., ) . wang, wen, li, zhang, & yang ( ) the number of hepatoprotective products from plants is ever increasing. in addition to the hepatoprotective role of a general class of phenolics and flavonoids, many studies have defined specific compounds for their preferable role in hepatitis and other liver disorders (shehab et al., ) . some of the important plants and their products are highlighted in figure . among the many different compounds, few are distinguished for their promising role in liver inflammation. we have, therefore, selected six important compounds for their hepatoprotective role (table ) . quercetin, for instance, a major flavonol commonly found most of the plants, is a potent hepatoprotective agent. the first basis of quercetin-based potency has been attributed to the antioxidant activity of this compound. one of the specific mechanisms of quercetin supplementation was established through ethanol-induced cytotoxicity which affects the activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. for instance, quercetin supplementation restored the glutathione reductase activity which was affected by ethanol that in turn reduced the glutathione content of liver. quercetin supplementation has also been attributed to hepatoprotection against metals, pesticides, drugs, toxins, and viruses (miltonprabu et al., ) . rhein ( , -dihydroxyanthraquinone- -carboxylic acid) is another important hepatoprotective compound extensively found in medicinal herbs, such as rheum palmatum l., cassia tora l., polygonum multiflorum thunb., and aloe barbadensis miller. the mechanism of action through which rhein acts has been described as modulation of cyp enzymes in rat liver, attenuation of total cholesterol and triglyceride levels in serum, and amelioration of glucose and lipid metabolism. similarly, rhein downregulated the levels of serum alt, hyaluronic acid, procollagen type iii, and liver malondialdehyde and inhibited the expression of transforming growth factor beta and alpha-smooth muscle actin in tetrachloride/ethanol-induced liver fibrosis rats . similarly, flavonoids, lignans, terpenoids, and steroids from vitex negundo l. have also been shown to demonstrate hepatoprotective activities (zheng et al., ) . extracts containing these compounds have been shown to improve biochemical and functional parameters and thus alleviate ccl -induced damage in liver rats. negundoside, for instance, which is a glycoside, has been demonstrated to reduce calcium-mediated toxicity and ccl -induced oxidative stress through regulation of calcium homeostasis and decreasing the production of ros and lipid peroxidation (zheng et al., ) . table with their structures given in figure . the present literature is not sufficient to assess the safety of most of the hepatoprotective and liver regenerative herbs and herbal products, as most of the studies focus on their antihepatotoxic effects only. however, some previous experiments on rats show that no adverse effects were observed by administering intraperitoneal injection of the a. membranaceus extracts at . g/kg for days, whereas large doses ( g/kg) of a. membranaceus root extracts resulted in mutagenicity in mice when injected directly into the stomach lining ). in another study, sato et al. ( ) observed no significant toxicity of various concentrations of glycyrrhizin on plc/prf/ cells in vitro. gadgoli and mishra ( ) reported that p-methoxy benzoic acid extracted from c. spinosa was nontoxic at mg/ml when applied to rat hepatocytes in vitro. this supports the claims made in the traditional system of medicine. similarly, silymarin is shown to have a lack of toxicity and side effects even at high doses (upadhyay et al., ) . isbrucker and burdock ( ) reported that no-observed-effect levels for purified glycyrrhizin are in the range of - mg/ kg/day and concluded that current levels of consumption of licorice extract products and glycyrrhizinate are safe. although, several herbals show potential activity for the treatment of acute and chronic liver diseases premarketing drug-testing, and pharmacovigilance is needed as with any other drug. so far, herbals to treat chronic liver diseases should not be recommended outside clinical trials as the evidence supporting its use is insufficient (stickel, patsenker, & schuppan, ) , and publications relevant to the cytotoxicity of medicinal plants should be encouraged (mukhtar et al., ) . moreover, there are issues like approval of the plant products/extracts as a drug from regulatory agencies such as the food and drug administration or any other equivalent agencies. extensive literature survey of hepatoprotective plants clearly indicates that herbal drugs have an enormous potential for the treatment of liver diseases. in this article, we reviewed the scientific merit of selected plants studied for their hepatoprotective mechanism of action. the major hepatoprotective mechanism identified by the majority of the studies is through combating the oxidative stress that damages the liver. we have summarized the effect of extracts and compounds from different herbs on liver injury considering changes in their biochemical parameters. we also presented the possible data available in the literature for different plants regarding their phytochemical constituents. we, therefore, conclude that herbs and herbal preparations are among the most important sources of hepatoprotective and liver regeneration medicines. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. moreover, a combination of the traditional herbal medicines with the modern and conventional medicine may be one of the best options for the treatment of liver disorders and other diseases and infections, soon. the importance of medicinal plants can be determined from world health organization's estimates, which states that up to % of the world's population fulfill their healthcare needs from medicinal plants (mukhtar et al., ) . there has been a significant rise in using overthe-counter medicinal plant products containing powerful medicinal drugs and are believed to have to produce progressive effects with reduced side effects. however, therapeutic failures or adverse effects have been observed in many cases as pharmacological mechanisms of the herbal mixtures/preparations are not well-studied. the most important concern involving the use of medicinal plants is to identify and standardize the exact method of preparation of an extract, identification of active ingredients and details of administration (yip & kwan, ) . in this relationship, the screening and characterization of other undiscovered herbal products in traditional medicine is needed. the integration of the therapeutic use of traditional chinese medicinal knowledge with the synthetic and traditional oriental medicinal knowledge is a key area of research (cho & leung, ) . however, medicinal plants cultivated in different geographical regions are believed to differ in therapeutic effects in different diseases and infections. for example, a. membranaceus used in chinese traditional medicine, from certain locality contains more favorable trace elements and fewer harmful trace elements than those from other localities. in this context, the use of new therapeutic strategies based on natural plants may be useful to provide minimal toxicity, higher effectiveness, and a wider therapeutic background for effective manipulation than existing pharmaceutical products (panico et al., ) . authors declare that they have no competing interests. animals not applicable. the current article is a review article and does not contain any studies with human participants performed by any of the authors. hepatitis c virus genotypes and hepatitis g virus in hemodialysis patients from syria: identification of two novel hepatitis c virus subtypes hepatoprotective activity of two plants belonging to the apiaceae and the euphorbiaceae family a review of hepatoprotective plants used in saudi traditional medicine. evidence-based complementary and alternative medicine: ecam a systematic review of treatment response rates in pakistani hepatitis c virus patients; current prospects and future challenges in vivo hepatoprotective activity of the aqueous extract of artemisia absinthium l. against chemically and immunologically induced liver injuries in mice effect of ethanol extract of flowers of vitex trifolia linn. on ccl induced hepatic injury in rats extract of woodfordia fruticosa flowers ameliorates hyperglycemia, oxidative stress and improves beta-cell function in streptozotocin-nicotinamide induced diabetic rats hepatoprotective role of ricinus communis leaf extract against d-galactosamine induced acute hepatitis in albino rats a review of the health effects and uses of drugs of plant licorice (glycyrrhiza glabra l.) in iran nephroprotective effects of ferulic acid, z-ligustilide and e-ligustilide isolated from angelica sinensis against cisplatin toxicity in vitro use of a signature nucleotide sequence of hepatitis c virus for detection of viral rna in human serum and plasma complete nucleotide sequence of a type hepatitis c virus variant, the predominant genotype in the middle east medicinal plants of sandy shores: a short review on vitex trifolia l. and ipomoea pes-caprae hepatoprotective activity of woodfordia fruticosa kurz flowers against carbon tetrachloride induced hepatotoxicity conservation and sustainable use of medicinal plants: problems, progress, and prospects efficiency of transcellular transport and efflux of flavonoids with different glycosidic units from flavonoids of litsea coreana l. in a mdck epithelial cell monolayer model global prevalence of preexisting hcv variants resistant to direct-acting antiviral agents (daas): mining the genbank hcv genome data the effect of lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats an evidence-based update on the pharmacological activities and possible molecular targets of lycium barbarum polysaccharides. drug design protective activity of phyllanthin in ethanol-treated primary culture of rat hepatocytes in vitro and in vivo immunomodulating and immunorestorative effects of astragalus membranaceus herbal products: benefits, limits, and applications in chronic liver disease herbal medicines for liver diseases cuscuta chinensis lam.: a systematic review on ethnopharmacology, phytochemistry and pharmacology of an important traditional herbal medicine summary: antiviral treatment of hepatitis c virus the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety hepatoprotective efficacy of cichorium intybus l. extract against carbon tetrachloride-induced liver damage in rats silymarin effects on intracellular calcuim and cytotoxicity: a study in perfused rat hepatocytes after oxidative stress injury silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy hepatoprotective effect of silymarin (silybum marianum) on hepatotoxicity induced by acetaminophen in spontaneously hypertensive rats antihepatotoxic activity of p-methoxy benzoic acid from capparis spinosa hepatoprotective activity of ceriops decandra (griff.) ding hou mangrove plant against ccl induced liver damage hepatoprotective activity of ficus carica leaf extract on rifampicin-induced hepatic damage in rats ameliorating effects of compounds derived from salvia miltiorrhiza root extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion physicochemical characterization of phyllanthin from traditional use of medicinal agents: a valid source of evidence a biomedical investigation of the hepatoprotective effect of radix salviae miltiorrhizae and network pharmacology-based prediction of the active compounds and molecular targets pharmacological effects of glycyrrhiza spp. and its bioactive constituents: update and review inhibitory effect of solanum nigrum on thioacetamide-induced liver fibrosis in mice inhibitory effect of angelica sinensis extract in the presence of -hydroxypropyl-β-cyclodextrin functional foods and dietary supplements for the management of dyslipidaemia the efficacy of liv- on liver cirrhotic patients: a randomized, double-blind, placebo-controlled first approach. phytomedicin: international journal of phytotherapy and phytopharmacology a review on hepatoprotective and immunomodulatory herbal plants risk and safety assessment on the consumption of licorice root (glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin handbook of african medicinal plants a critical approach to evaluating clinical efficacy, adverse events and drug interactions of herbal remedies a review on phytochemical and pharmacological properties of litsea coreana hepato-protective effects of six schisandra lignans on acetaminopheninduced liver injury are partially associated with the inhibition of cyp-mediated bioactivation comparison of the nutritional value and biological activities of the acetone, methanol and water extracts of the leaves of solanum nigrum and leonotis leonorus mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes evaluation of hepatoprotective activity of aerial parts of tephrosia purpurea l. and stem bark of tecomella undulata in vivo glycyrrhizin accelerates liver regeneration and rapidly lowers serum transaminase activities in % partially hepatectomized rats in vitro antioxidant activities of methanol extracts of five phyllanthus species from india. lwt -food science and technology analysis of the active components of silymarin hepatoprotective effects of chinese medicinal herbs: a focus on antiinflammatory and anti-oxidative activities a protein with antiproliferative, antifungal and hiv- reverse transcriptase inhibitory activities from caper (capparis spinosa) seeds sofosbuvir for previously untreated chronic hepatitis c infection simeprevir plus sofosbuvir, with or without ribavirin, to treat chronic infection with hepatitis c virus genotype in non-responders to pegylated interferon and ribavirin and treatment-naive patients: the cosmos randomised study aqueous extract of solanum nigrum inhibit growth of cervical carcinoma (u ) via modulating immune response of tumor bearing mice and inducing apoptosis of tumor cells chinese & related north american herbs: phytopharmacology & therapeutic values a review of recent research progress on the astragalus genus hepatitis c genotyping by denaturing high-performance liquid chromatography hepatoprotective effects of solanum nigrum linn extract against ccl( )-induced oxidative damage in rats a pharmaceutical preparation of salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin ii-stimulated collagen synthesis in fibroblasts hepatoprotective effects of solanum nigrum against ethanol-induced injury in primary hepatocytes and mice with analysis of glutathione s-transferase a synchronized and sustained release of multiple components in silymarin from erodible glyceryl monostearate matrix system hepatoprotective activity of vitex trifolia against carbon tetrachloride-induced hepatic damage dual effects of lipophilic extract of salvia miltiorrhiza (danshen) on catecholamine secretion in cultured bovine adrenal medullary cells the genus phyllanthus: an ethnopharmacological, phytochemical, and pharmacological review hepatitis c virus (hcv) circulates as a population of different but closely related genomes: quasispecies nature of hcv genome distribution geographical distribution of hepatitis c virus genotypes in blood donors: an international collaborative survey hepatoprotective effect of quercetin: from chemistry to medicine phytochemical and medicinal importance of ginkgo biloba l antiviral potentials of medicinal plants hepatoprotective effect of ethanolic extract of phyllanthus amarus schum. et thonn. on aflatoxin b -induced liver damage in mice pharmacological effects of capparis spinosa l neuropharmacological evaluation of ginkgo biloba phytosomes in rodents methanolic extract of woodfordia fruticosa kurz flowers ameliorates carbon tetrachlorideinduced chronic hepatic fibrosis in rats anthocyanins from flowers of cichorium intybus nucleotide sequence and mutation rate of the h strain of hepatitis c virus. proceedings of the national academy of sciences of the united states of america protective effect of capparis spinosa on chondrocytes pharmacology of schisandra chinensis bail.: an overview of russian research and uses in medicine hepatoprotective effect of ginkgo biloba leaf extract on lantadenes-induced hepatotoxicity in guinea pigs preventive effects of a purified extract isolated from salvia miltiorrhiza enriched with tanshinone i, tanshinone iia and cryptotanshinone on hepatocyte injury in vitro and in vivo phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review a review: the pharmacology of isoliquiritigenin extraction, identification, fractionation and isolation of phenolic compounds in plants with hepatoprotective effects neuropharmacological activity of solanum nigrum fruit hepatitis c virus (hcv) genotypes distribution: an epidemiological up-date in europe cytoprotective role of solanum nigrum against gentamicin-induced kidney cell (vero cells) damage in vitro national center for biotechnology information. pubchem compound database glycyrrhizin alleviates experimental allergic asthma in mice the genus vitex: a review compendium of indian medicinal plants potential anti-inflammatory effects of artemisia gorgonum on rat liver injury induced by ccl -erratum. microscopy and microanalysis: the official journal of microscopy society of america flavour profile of capers (capparis spinosa l.) from the eolian archipelago by hs-spme/gc-ms medicinal plants for management of gastroesophageal reflux disease: a review of animal and human studies therapeutic basis of glycyrrhizin on chronic hepatitis b impact of phenolic composition on hepatoprotective and antioxidant effects of four desert medicinal plants scientific investigation of crude alkaloids from medicinal plants for the management of pain an ethnobotanical study of indigenous knowledge on medicinal plants used by the village peoples of thoppampatti ripe fruit of solanum nigrum l. inhibits cell growth and induces apoptosis in mcf- cells herbal hepatotoxicity cichorium intybus: traditional uses, phytochemistry, pharmacology, and toxicology sapindus mukorossi (areetha): an overview evaluation of hepatoprotective effects of helminthostachys zeylanica (l.) hook against carbon tetrachloride-induced liver damage in wistar rats crude extracts of hepatoprotective plants, solanum nigrum and cichorium intybus inhibit free radical-mediated dna damage effects of purified herbal extract of salvia miltiorrhiza on ischemic rat myocardium after acute myocardial infarction effects and mechanisms of extract from paeonia lactiflora and astragalus membranaceus on liver fibrosis induced by carbon tetrachloride in rats silymarin as a natural antioxidant: an overview of the current evidence and perspectives chromatographic profiling of ellagic acid in woodfordia fruticosa flowers and their gastroprotective potential in ethanol-induced ulcers in rats current knowledge of schisandra chinensis (turcz.) baill.(chinese magnolia vine) as a medicinal plant species: a review on the bioactive components, pharmacological properties, analytical and biotechnological studies differences in the hepatitis c virus genotypes in different countries schisandrin b prevents doxorubicin induced cardiac dysfunction by modulation of dna damage, oxidative stress and inflammation through inhibition of mapk/p signaling the plant list version . . published on the internet medicinal plants: their role in health and biodiversity pyridine induction of cytochrome p a , inos and metallothionein in syrian hamsters and protective effects of silymarin pharmacological effects of sapindus mukorossi effect of silymarin on pyrogallol-and rifampicin-induced hepatotoxicity in mouse effects of an extract from phyllanthus niruri on hepatitis b and woodchuck hepatitis viruses: in vitro and in vivo studies in vitro plantlets from alginate-encapsulated shoot tips of solanum nigrum l protection of lethal toxicity of endotoxin by salvia miltiorrhiza bunge is via reduction in tumor necrosis factor alpha release and liver injury angelica sinensis polysaccharide attenuates concanavalin a-induced liver injury in mice ethnobotany, phytochemistry, and pharmacology of the genus litsea: an update schisandra polysaccharide inhibits hepatic lipid accumulation by downregulating expression of srebps in nafld mice salvia miltiorrhiza reduces experimentally-induced hepatic fibrosis in rats enhanced bioavailability of silymarin by self-microemulsifying drug delivery system effects of salvianolic acid a on oxidative stress and liver injury induced by carbon tetrachloride in rats lycium barbarum polysaccharide attenuates alcoholic cellular injury through txnip-nlrp inflammasome pathway schisandrin b attenuates the inflammatory response, oxidative stress and apoptosis induced by traumatic spinal cord injury via inhibition of p signaling in adult rats the protective effect of tinoridine against carbon tetrachloride hepatotoxicity nanoparticles formulation of cuscuta chinensis prevents acetaminopheninduced hepatotoxicity in rats hepatoprotective and antioxidant effects of cuscuta chinensis against acetaminopheninduced hepatotoxicity in rats salvia miltiorrhiza bunge and its active component cryptotanshinone protects primary cultured rat hepatocytes from acute ethanol-induced cytotoxicity and fatty infiltration molecular identification of astragalus membranaceus at the species and locality levels hepatitis c virus genotypes in the united states: epidemiology, pathogenicity, and response to interferon therapy. collaborative study group systematic review of the renal protective effect of astragalus membranaceus (root) on diabetic nephropathy in animal models extraction, chemical analysis of angelica sinensis polysaccharides and antioxidant activity of the polysaccharides in ischemia-reperfusion rats levistilide a inhibits angiogenesis in liver fibrosis via vascular endothelial growth factor signaling pathway phytochemical and pharmacological profile of vitex negundo modern study of traditional chinese medicine schisantherin a protects against liver ischemia-reperfusion injury via inhibition of mitogen-activated protein kinase pathway selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions key: cord- -e kuwf authors: nan title: opinion of the scientific panel on animal health and welfare (ahaw) on a request from the commission related with the risks of poor welfare in intensive calf farming systems date: - - journal: efsa j doi: . /j.efsa. . sha: doc_id: cord_uid: e kuwf nan summary efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission asked efsa to update the findings of the scientific veterinary committee (animal welfare section) report, on the welfare of calves of november , in the light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. in this report a risk assessment was made and the relevant conclusions and recommendations are forming the scientific opinion by the ahaw panel. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economical considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update o the previous svc report with the exception of economical aspects which are outside of the mandate for this report. the various factors potentially affecting calves' health and welfare, already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently systematically determined whether they constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. in line with the terms of reference the working group restricted itself to (in essence a qualitative) risk assessment although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport and slaughter, this report does not consider animal health and welfare aspects of calves during transport and slaughter but such information can be found in a recently issued comprehensive report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, ) and in the efsa report "welfare aspects of animal stunning and killing methods" (ahaw / ). in relation with the food safety aspects, main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, the main risk factors contributing to increased prevalence/levels of the above foodborne pathogens, as well as generic principles for the risk reductions are known. the latter are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures (e.g. gfp-ghp). in general, the conclusions made in the previous svc report remain. however, recent research has provided for some additional conclusions. the risk analysis is presented in the tables of annex . the graphics in this table are not intented to represent numerical relationships but rather qualitative relations. in some instances the exposure could not be estimated due to lack of data, in which cases the risks where labelled "exposure data not available". the following major and minor risks for poor animal health and welfare have been identified for one or several of the various husbandry systems considered: the hazards of iron deficiency and insufficient floor space are considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet are considered to be moderately serious. for these hazards, there is no consensus on the exposure of calves mainly due to lack of data and that is why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding castration and dehorning (and disbudding) without anaesthetic drugs, there is a variation in relation to national legislation why the risk of poor welfare in relation to castration and dehorning has a wide range between countries. council directive / /eec laying down minimum standards for the protection of calves as amended by council directive / /ec requires the commission to submit to the council a report, based on a scientific opinion, on intensive calf farming systems which comply with the requirements of the wellbeing of calves from the pathological, zootechnical, physiological and behavioural points of view. the commission's report will be drawn up also taking into account socio-economic implications of different calf farming systems. it should be noted that the scientific veterinary committee (animal welfare section) adopted a report on the welfare of calves on november which should serve as background to the commission's request and preparation of the new efsa scientific opinion. in particular the commission requires efsa to consider the need to update the findings of the scientific veterinary committee's opinion in light of the availability of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission requires efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. the mandate outlined above was accepted by the panel on animal health and welfare (ahaw) at the plenary meeting, on / march . it was decided to establish a working group of ahaw experts (wg) chaired by one panel member. therefore the plenary entrusted a scientific report and risk assessment to a working group under the chairmanship of prof. bo algers. the members of the working group are listed at the end of this report. the scientific report is considered for the discussion to establish a risk assessment and the relevant conclusions and recommendations forming the scientific opinion by the ahaw panel. according to the mandate of efsa, ethical, socio-economic, cultural and religious aspects are outside the scope of this scientific opinion. . . the working group set out to produce a document in which the various factors potentially affecting calves' health and welfare [already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently to systematically determine whether these factors constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. it should be noted, however, that this does not imply that a hazard that has a serious effect on just a few animals should not be dealt with by managers on farm level as the suffering imposed on some animals constitute a major welfare problem for those individuals. in line with the terms of reference the working group restricted itself to (in essence qualitative) risk assessment, i.e. only one of three elements essential to risk analysis a risk assessment approach was followed, similar to the one generally adopted when assessing microbiological risks, i.e. along the lines suggested at the nd session of the codex alimentarius commission (cac, ) . incidentally, these guidelines have been characterized by the cac as 'interim' because they are subject to modifications in the light of developments in the science of risk analysis and as a result of efforts to harmonize definitions across various disciplines. cac's guidelines are in essence exclusively formulated for the purpose of assessing risks related to microbiological, chemical or physical agents of serious concern to public health. consequently -considering their disciplinary focus -the working group had to adapt the cac definitions to serve their purpose. these adapted definitions, have, in alphabethical order, been included in chapter (see risk analysis terminology) the objectives of this report are to review and report recent scientific literature on the welfare including the health of intensively reared calves, to report on recent findings as an update to the scientific veterinary committee's previous report, to make a qualitative risk assessment concerning the welfare of intensively kept calves. where relevant, food safety implications of different farming systems are also considered. the report is structured in five major parts. the first three follow the scientific veterinary committee's previous report "on the welfare of calves" with introductory chapters - on background, measurements and needs in relation to calf welfare, chapter describing housing, diet and management and chapter describing comparison of systems and factors. in chapter common disease and use of antibiotics is described. the other two parts involve aspects of meat quality and food safety (chapter ) and the risk assessment (chapters ). conclusions and recommendations from the previous svc document together with updated conclusions derived from recent research findings are presented in chapter . effect of transport and slaughter on calves' health and welfare although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport, this report does not consider animal health and welfare aspects of calves during transport because there is already a comprehensive recent report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, . the report takes into account all aspects related with transport that could affect the health and welfare of cattle and calves, including the direct effects of transport on the animals and the effects of transport on disease transmission. the loading methods and handling facilities for cattle, the floor space allowance, the relationships of stocking and the density requirements, the vehicle design, space requirements and ventilation for cattle transporters (see also the ahaw scientific opinion related to standards for the microclimate inside animal road transport vehicles, efsa-q- - ), the behaviour of cattle during road transport, the road conditions, long distance transport and the travel times are also reviewed. recommendations for all these aspects are also given in that report. feeding and housing systems, weaning strategies and quality of solid and liquid feed . . . feeding systems and weaning strategies recommendations without a fully functional rumen, calves will be unable to utilise nutrients provided in the post-weaning dry feed diet. attention must paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. the provision of solid feeds with adequate content and balance to veal calves is a prerequisite for the development of a healthy and functional rumen, the prevention of abnormal oral behaviours, and the stimulation of normal rumination activity. although some solid feeds may exacerbate problems with abomasal ulcers in milk-fed veal calves, properly balanced rations seem to moderate this effect. nutritional factors are clearly involved in the etiology of abomasal ulcers in veal calves. important elements include the consumption of large quantities of milk replacer and the interaction between a milk replacer diet and the provision of roughage. if vegetable proteins are not properly treated, milk replacers may cause hypersensitivity reactions in the gut, which may compromise calf welfare. it is recommended that solid feeds provided to veal calves, in addition to milk replacer, are adequately balanced in terms of the amount of fibrous material, which will promote rumination, and other components such as proteins and carbohydrates, which stimulate rumen development and support a healthy function of the digestive system. since milk replacer formulations are frequently changing, it is recommended to carefully and consistently examine allergenic properties and other possibly detrimental effects of all milk replacers before they are used on a large scale. if the concentration of haemoglobin in the blood of calves drops below . mmol l - , the ability of the calf to be normally active as well as lymphocyte count and immune system function are substantially impaired, and there is reduced growth rate. below . mmol l - , veal calves exhibit a number of adaptations to iron deficiency, including elevated heart rate, elevated urinary noradrenaline and alterered reactivity of the hpa axis. there is a lack of data on the variability in groups of calves. hence, when haemoblogin levels are found to be below . mmol l - in groups of young veal calves, it is field practice to give supplementary iron. for older calves, including those in the last four weeks before slaughter, efficient production is possible in individual calves whose haemoglobin concentration is above . mmol l - . if the concentration of haemoglobin in blood is not checked at all, there is a high risk of anaemia that is associated with poor welfare, for all calves fed a diet with a very low iron content. anaemia can be identified and quantified adequately if checks are carried out on veal production calves of - weeks, for example, when the calves are brought into a unit, between - weeks of fattening, and during the last four weeks before slaughter. if the concentration of haemoglobin in the blood of a group of calves during the last four weeks before slaughter is a mean of . mmol l - , some calves may have a concentration substantially lower than the group-mean, and hence their welfare may be poor. in order to avoid anaemia levels that are associated with poor welfare because normal activity is difficult or not possible and other functions are impaired, it is advisable that diets should be provided that result in blood haemoglobin concentrations of at least . mmol l - throughout the life of the calf. in order to avoid serious impairment of immune system function and hence poor welfare, no individual calf should have a blood haemoglobin concentration lower than . mmol l - . in most cases this is achieved by adjusting the concentration of iron in the diet and having an adequate checking system so that the above condition is avoided. other treatment may be needed for calves with clinical conditions which cause anaemia but which are not related to diet. since the lowest haemoglobin concentrations in the blood of veal calves are usually reached during the last four weeks before slaughter, these blood concentrations should be checked at this time. such controls would help to see if measures are necessary to be taken or not. a checking system using a mean level, but whose aim is to avoid the risk of a low haemoglobin concentration in any individual lower than . mmol l - would have to use a mean substantially higher than , mmol l - , probably mmol l - , and an appropriate sample size. in order to avoid poor welfare associated with anaemia, as explained in the conclusions (above), measurements of average blood haemoglobin concentration are not a satisfactory means of avoiding poor welfare but the use of a minimum level of . mmol l - for individual calves would achieve this. there is a lack of data on the haemoglobin levels and variation in groups in slaughtering calves. to gain more information as a basis for further actions and recommendations, it is advisable to perform sampling of calves at slaughter, by checking the haemoglobin level on a random basis in groups of calves. space and pen design recommendations space should be enough to allow animals to fulfill their needs for social behaviour, lying and grooming. as the pen shape affects the use of space by animals, pens should be rectangular rather than square and pen space should be divided into different usable areas. as the floor type affects the resting and lying postures of calves it should be comfortable. wet floors should be avoided due to thermal and resting problems. degree of social contact conclusions group housing can help calves to acquire social skills. some experience of mixing is important as calves that have been reared for a while in groups dominate calves that have always been in individual crates. when calves are mixed together in the first few days of life, and then kept for some weeks in a social group, there may be poor welfare because of the following risks: . especially when individuals are provided with inadequate access to teats and roughage in the diet, cross-sucking and other abnormal sucking behaviour may occur. . some individuals may be unaccustomed to the food access method, for example they may have only received food via a teat, and may find it difficult to drink from a bucket. . calves coming from different buildings, perhaps from different farms, may carry different pathogens and hence there is a risk of disease spread in all the calves that are put in the same airspace or are otherwise exposed to the pathogens. since calves are social animals, they should be kept in social groups wherever possible. these groups should be stable with no mixing or not more than one mixing. it is advisable for calves in the first two weeks of life not to be mixed with other animals. if calves from different buildings, perhaps different farms, are to be mixed in a pen or are to be put in different pens in the same airspace, quarantining animals for - weeks can reduce disease in the calves and hence prevent poor welfare. although cross-sucking can sometimes be minimised by provision of teats, water and roughage, if this is not possible, mixing into groups could be delayed for three to four weeks. calves fed by various means may require careful supervision after being put into groups in order that they learn how to feed effectively. temperature, ventilation and air hygiene calf rearing causes significant emissions of substances such as nitrate, phosphate, heavy metals and possibly antibiotics in manure and liquid effluents. in addition, there are odours, gases, dusts, micro-organisms and endotoxins in the exhaust air from animal houses. also in the handling of manure in storage and during application of manure and during grazing. these effluents can have distinct impacts on air, water, soil, biodiversity in plants, forest decay and also on animals and including humans. calf houses possess a high potential for emissions of ammonia and other gases. dust, endotoxins and micro-organisms are emitted in lower amounts than from pig or poultry production. odour, bioaerosols, ammonia, nitrogen, phosphorous and heavy metals may either have a local or a regional impact. gases such as methane and nitrous oxide contribute to global warming. respiratory disorders are the second largest reason for morbility and mortality in calf rearing. the most important causes are environmental conditions such as hygiene, management and the physical, chemical and biological factors in the environment. ventilation plays a decisive role in reducing the incidence of respiratory disease. temperatures below ºc can compromise lung function. ammonia concentrations of more than ppm seem to increase respiratory infections. relative humidity of more than % bear the risk of increased heat dissipation and can help bacteria to survive in airborne state. air velocities close to the animals of more than . m/s can significantly increase respiratory sounds in calves. sufficient air space in confined buildings can help to reduce the concentration of airborne bacteria. calf houses contain relatively high amounts of endotoxins ( eu) (eu: endotoxin unit, see scientific report, www.efsa.eu.int) there is concern that antibiotic residues may contribute to the development of bacterial resistance. local and regional environmental problems are enhanced by high animal densities and insufficient distances between farms and residential areas. the exact quantitative contribution of calf rearing to environmental pollution and its impact on water, air, soil vegetation and nearby residents is not yet well understood. when housing systems are compared, although dust emission levels will seldom pose problems for the health of calves, ammonia emission levels may be high enough to exacerbate calf disease, especially when calves are kept in slatted floor units. the development of low emission production systems should be encouraged including mitigation techniques, e.g. biofilters, bioscrubbers, covered manure pits and shallow manure application. in particular there is need to reduce ammonia emissions from slatted floor units or to reduce the usage of such systems. adequate and efficient feeding regimes are required with minimal wastage of nitrogen and phosphorous and limited use of growth promoters and drugs. there is an urgent need for cooperative research to design appropriate ventilation systems to improve health and welfare of calves kept in confined rearing conditions. temperatures for young calves should range between and ºc. ammonia concentrations should be kept as low as possible preferably not more than ppm. housing and management should aim a reducing dust, bacteria and endotoxin concentrations in the animal house air. minimum ventilation rates of m per kg live weight should be applied. human-animal relationships recommendation stockpersons should be appropriately trained so that they have sufficient skills in rearing calves. they should have a positive attitude towards animals and work with them in order to minimise stress and to maintain a high quality of health control. rough contact (e.g. use of painful device such as an electric prod, loud noises) should be avoided and gentle contacts (e.g. talking softly, stroking, offering food) should be encouraged. this sort of contact is of particular importance for calves in groups or with their dam that tend not to approach humans readily. dehorning and castration if cattle are to be dehorned, it is recommend to disbud young cattle rather than to dehorn older ones. disbudding by cautery is recommended over other methods. local anaesthesia (e.g. - ml lidocaïne or lignocaïne % around the corneal nerve) and analgesia with an nsaid (e.g. ml flunixin meglumine or - . mg ketoprofen % / kg body weight) should be given - min before disbudding. if cattle are to be castrated, it is recommended to castrate calves as early as possible (no later than . mo and preferably at wk of age), to use the burdizzo method, and to provide appropriate anaesthesia and analgesia (e.g. ml lignocaine % in each testicle through the distal pole and mg ketoprofen % / kg body weight injected intravenously both min before castration). prevention of typical calf diseases in the first months of life such as diarrhoea and enzootic bronchopneumonia requires a systematic approach by improving management and housing conditions, specifically the preparation of the cow, hygiene of the calving environment, including dry clean bedding and high air quality, immediate supply with maternal antibodies, no mixing with older animals and careful attention and a rapid response to any sign indicating disease. main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens on farms depend on the status and the inter-relationship of different contributing factors that are inherently highly variable. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, generic principles for risk reductions for the main foodborne pathogens at calf farm level are known and are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures based on gfp-ghp. for quantitative food safety risk categorization of farming systems individually, and/or their related ranking, further scientific information is needed. accordingly, related research should be encouraged. the conclusions of the scientific veterinary committee report on the welfare of calves are presented in table below together with additions relevant in the light of this update of the svc report. the best conditions for young rearing calves involve leaving the calf with the mother in a circumstance where the calf can suckle and can subsequently graze and interact with other calves.c agreed where the calf will be separated from its mother at an early age, evidence suggests that it is normally beneficial for the calf if the mother is allowed to lick the calf thoroughly for a few hours after birth.c agreed r whenever possible, cows should be given the opportunity to lick the calf during at least three hours after parturition. it is important that the calf should receive sufficient colostrum within the first six hours of life and as soon as possible after birth, in conditions which facilitate antibody absorption, preferably by suckling from the mother, so as to ensure adequate immunoglobulin levels in the blood. r where necessary, suckling assistance or additional colostrum should be provided for calves left to suckle from the dam.r agreed agreed calves need resources and stimuli which are normally provided by their mothers. all calves should be given adequate food and water, appropriate conditions of temperature and humidity, adequate opportunities to exercise, good lying conditions, appropriate stimuli for sucking during the first few weeks of life and social contacts with other calves from one week of age onwards. specific aspects of housing and management which fulfill these conditions are detailed. agreed young calves reared without their mothers should receive considerate human contact, preferably from the same stockperson throughout the growing period.r agreed stockpersons should be appropriately trained so that they have sufficient skill in the rearing of calves. they should have positive attitudes towards animals and to working with them in order to handle them while minimising stress and to maintain a high quality of health control. rough contacts (e.g. use of a painful device such as an electric prod, or loud noises) should be avoided and gentle contacts (e.g. talking softly, petting, offering food) should be encouraged. these contacts are of particular importance for calves in groups or with their dam that may tend not to approach humans easily. where calves cannot be kept with their mother, the system where welfare is best is in groups with a bedded area and an adequate space allowance available to them.c agreed. r see below. the welfare of calves is very poor when they are kept in small individual pens with insufficient room for comfortable lying, no direct social contact and no bedding or other material to manipulate. c agreed r as the floor affects the resting and lying posture of calves they should be useful to have a comfortable floor. wet floors should be avoided due to thermal and resting problems. tethering always causes problems for calves. calves housed in groups should not be tethered except for periods of not more than one hour at the time of the feeding of milk or milk substitute. individually housed calves should not be tethered. r calves are vulnerable to respiratory and gastro-intestinal disease and welfare is poor in diseased animals. better husbandry is needed to minimize disease in group housing conditions but results that are as good as those from individual housing can be obtained. c every calf should be able to groom itself properly, turn around, stand up and lie down normally and lie with its legs stretched out if it wishes to do so. r in order to provide an environment which is adequate for exercise ,exploration and free social interaction, calves should be kept in groups. calves should never be kept at too high stocking density. the following requirements are based on evidence of increasingly poor welfare as space allowance decreases. the space allowance should provide, especially for allowing resting postures, an area for each calf of at least (its height at the withers) x (its body length from the tip to its nose when standing normally to the caudal edge of the tuber ischii or pin bone x . ). the length measurements takes account of the forward and backward movements involved in standing up and lying down. this calculation takes account of differences in size amog breeds and with age. as a guideline, for holstein calves this area is . m at weeks, . m at weeks and . m at weeks. r r since calves are social animals, they should be kept in social groups wherever possible. these groups should be stable with no mixing or not more than one mixing. it is advisable for calves in the first two weeks of life not to be mixed with other animals. c when calves are mixed together in the first few days of life, and then kept for some weeks in a social group, there may be poor welfare because of the following risks: -especially when individuals are provided with inadequate access to teats and roughage in the diet, cross-sucking and other abnormal sucking behaviour may occur. -some individuals may be unaccustomed to the food access method, for example they may have only received food via a teat, and may find it difficult to drink from a bucket. -calves coming from different buildings, perhaps from different farms, may carry different pathogens and hence there is a risk of disease spread in all the calves that are put in the same airspace or are otherwise exposed to the pathogens. r if calves from different buildings, perhaps different farms, are to be mixed in a pen or are to be put in different pens in the same airspace a quarantine situation should be used in order to reduce disease in the calves and hence prevent poor welfare. for a given space allowance per calf, increasing group size results in a larger total area and hence better possibilities for exercise, social interaction and improved environmental complexity. c larger groups are preferred because of the better possibilities for providing an adequate environment but there are limits to the numbers of animals which should be in one building section and risks associated with mixing of calves from different sources should be considered. r agreed r the space provided for calves should be enough to allow animals to fulfill their needs for social behaviour, lying and grooming. space allowance per animal should be greater for groups of - animals and for feeding systems, and pen shapes or flooring materials that necessitate extra space availability. if the preferred system, group housing, is not possible then individual pens whose width is at least the height of the calf at the withers and whose length is at least the length of the calf from the tip of its nose when standing normally to the caudal edge of the tuber ischii or pin bone x . should be used. this space requirement is calculated on the basis of the space required for normal agreed r as the pen shape affects the use of space by animals, pens should maximize the perimeter and pen space should be divided into different usable areas. movements and evidence of increasingly poor welfare r appropriate bedding for example straw is recommended. bedding must be changed at appropriate intervals and every calf should have access to a dry lying area. slatted floors must not be slippery and must not be a cause of tail tip necrosis. r agreed see above buildings should be adequately ventilated taking into account of the number of animals present and the external conditions. the air space in the building should be m per calf up to weeks of age and an amount of air space which increases with age is needed for older calves.r agreed c -calf rearing causes significant emissions such as nitrate, phosphate, heavy metals and possibly antibiotics in manure and liquid effluents as well as odour, gases, dusts, micro-organisms and endotoxins in the exhaust air from animal houses, from manure storage facilities, during application of manure and during grazing. -these effluents can have distinct impacts on air, water, soil, and thus also on animals. -calf houses possess a high potential for emissions of ammonia and other gases. dust, endotoxins and micro-organisms are emitted in lower amounts than from pig or poultry production. -respiratory disorders are the second largest reason for morbidity and mortality in calf rearing. the most important reason are environmental conditions such as hygiene, management and the physical, chemical and biological factors of the aerial environment. -ventilation plays a decicive role in reducing the incidence of respiratory diseases. temperatures below c can compromise lung function. -ammonia concentrations of more than ppm seem to increase respiratory affections. relative humidity of more than % bear the risk of increased heat dissipation and can help bacteria to survive in airborne state. -air velocities close to the animals of more than . m/s can increase respiratory sounds in calves significantly. -sufficient air space in confined buildings can help to reduce the concentration of airborne bacteria. -calf houses contain relatively high amounts of endotoxins. -there is concern that antibiotic residues may contribute to the development of bacterial resistance. -environmental problems in calf houses are enhanced by high animal densities, insufficient distances between farms. -when housing systems are compared, although dust emission levels will seldom pose problems for the health of calves, ammonia emission levels may be higher enough to exacerbate calf disease, especially in slatted floor units. r -the development of low emission production systems should be encouraged including mitigation techniques, e.g. biofilters, bioscrubbers, covered manure pits and shallow manure application. in particular there is need to reduce ammonia emissions from slatted floor units or to reduce the usage of such systems. -adequate and efficient feeding regimes are required with minimal wastage of nitrogen and phosphorous and limited use of growth promoters and drugs. -there is an urgent need for cooperative research to design appropriate ventilation systems to improve health and welfare of calves kept in confind rearing conditions. -temperatures for young calves should range between and c. -ammonia concentrations should be kept as low as possible, preferably not more than ppm. -housing design and management procedures should aim to reduce dust, bacteria and endotoxin concentrations in the animal house air. -minimum ventilation rates of c per kg live weight should be applied. calves which lack specific nutrients, including iron, which are given poorly balanced diet, and which are not provided with adequate roughage in the diet after four weeks of age can have serious health problems, can show serious abnormalities of behaviour, and can have substantial abnormalities in gut development. c every calf should receive a properly balanced diet with adequate nutrients.r agreed r it is recommended that solids feeds provided to veal calves, in addition to milk replacer, are adequately balanced in terms of the amount of fibrous material, which will promote rumination, and other components such as proteins and carbohydrates, which stimulate rumen development and support a healthy function of the digestive system. c if the concentration of haemoglobin in the blood of calves drops below . mmol l - , the ability of the calf to be normally active as well as the lymphocyte count and immune system function are substantially impaired, and there is reduced growth rate. below . mmol l - , veal calves exhibit a number of adaptations to iron deficiency, including elevated heart rate, elevated urinary noradrenaline and alterered reactivity of the hpa axis. hence it is normal practice to identify young veal production calves with less than . mmol l - haemoglobin in plasma and to provide supplementary iron in addition to that normally included in the diet. for older calves, including those in the last four weeks before slaughter, efficient production is possible in individual calves whose haemoglobin concentration is above . mmol l - . if the concentration of haemoglobin in blood is not checked at all, there is a high risk of anaemia that is associated with poor welfare, for all calves fed a diet with very low iron content. anaemia can be identified and quantified adequately if checks are carried out on veal production calves of - weeks, for example, when the calves are brought into a unit, between - weeks of fattening, and during the last four weeks before slaughter. if the concentration of haemoglobin in the blood of a group calves during the last four weeks before slaughter is a mean of . mmol l - , some calves may have a concentration substantially lower than the group-mean, and hence their welfare may be poor. r in order to avoid anaemia levels that are associated with poor welfare because normal activity is difficult or not possible and other functions are impaired, it is advisable that diets should be provided that result in blood haemoglobin concentrations of at least . mmol l - throughout the life of the calf. in order to avoid serious impairment of immune system function and hence poor welfare, no individual calf should have a blood haemoglobin concentration lower than . mmol l - . in most cases this is achieved by adjusting the concentration of iron in the diet and having an adequate checking system so that the above condition is avoided. other treatment may be needed for calves with clinical conditions which cause anaemia but which are not related to diet, r since the lowest haemoglobin concentrations in the blood of veal calves are usually reached during the last four weeks before slaughter, these blood concentrations should be checked at this time. such controls would help to see if measures are necessary to be taken or not. a checking system using a mean level, but whose aim is to avoid the risk of a low haemoglobin concentration in any individual lower than . mmol l- , would have to use a mean substantially higher than , mmol l- , probably mmol l- . in order to avoid poor welfare associated with anaemia, as explained in the conclusions (above), measurements of average blood haemoglobin concentration are not a satisfactory means of avoiding poor welfare but the use of a minimum level of . mmol l - for individual calves would achieve this. some non-milk proteins are inappropriate for use in a milk substitute fed to calves because they produce allergenic reactions. some carbohydrates cannot be easily or properly digested by calves and they may cause digestive upset. no milk substitute should be fed to calves unless it can be easily digested and does not cause harmful reactions in the calves. r acidification of milk can reduce the incidence of diarrhoea, but any forms of acidified milk which are unpalatable to calves or which harm the calves should not be used. r every calf should be fed fermentable material, appropriate in quality and sufficient in quantity to maintain the microbial flora of the gut and sufficient fibre to stimulate the development of villi in the rumen. roughage, in which half of the fibre should be at least mm in length, should be fed to calves. they should receive a minimum of g of roughage per day from to weeks of age, increasing to g per day from to weeks of age but it would be better if these amounts would be doubled. the development of the rumen should be checked by investigating villi development in a proportion of calf guts after slaughter. r agreed r without a fully functional rumen, calves will be unable to utilise nutrients provided in the post-weaning dry feed diet. attention should paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. there are clear signs of increased disease susceptibility and immunosuppression in calves up to weeks of age, whose blood haemoglobin concentration is below . mmol/liter. however, in some studies the antibiotic treatment was not higher in calves whose haemoglobin was near to mmol/litre than in calves whose level was near to mmol/litreat weeks of age. studies of exercise in anaemic calves show that there can be problems during exercise at a level of , mmol/litre. c all calves should be fed in such a agreed see way that their haemoglobin level does not fall below a minimum of . mmol/litre. r where calves are fed a diet which is lower in iron than mg/kg, an adequate sample of animals should be checked at and weeks of age in order to find out whether the blood haemoglobin concentration is too low. r agreed see young calves have a very strong preference to suck a teat or teat-like object. it is preferable for calves to be fed milk or milk substitute from a teat during the first four weeks of life. calf welfare is improved if a non-nutritive teat is provided during the first four weeks of life especially if they are not fed from a teat. c when young group-housed calves are fed milk or milk substitute, the social facilitation effects of having a group of teats close together are beneficial. it is also advisable for several teats to be provided in groups of older calves. transponder controlled feeder systems have been found to work well. c the feeding to calves of large quantities of milk or milk substitute in a single daily meal can cause digestive problems. hence when calves are fed more than % of body weight in milk or milk substitute each day, this should be fed in at least two meals per day. r calves fed ad libitum, or close to this level should not be weaned off milk or milk replacer until they are consuming a minimum of g of concentrates per head per day in the week prior to weaning. where calves are fed restricted quantities of milk or milk replacer before weaning they should not be weaned until they are consuming a minimum of g of concentrates per head per day in the week prior to weaning. r calves which are diseased and calves which are in hot conditions often need to drink water as well as milk or milk substitute and all calves drink water if it is available. the provision of milk or milk substitute is not an adequate alternative for provision of water. hence calves should be provided daily with water to drink. it is recommended that drinkers be provided in all pens. r agreed r prevention of typical calf diseases in the first months of life such as diarrhoea and enzootic bronchopneumonia requires a systematic approach by improving management and housing conditions, specifically the preparation of the cow, hygiene of the calving environment, including dry clean bedding and high air quality, immediate supply with maternal antibodies, no mixing with older animals and careful attention and early reaction of all signs of any beginning diseases. dehorning calves between - weeks by cauterisation with adequate anaesthesia and analgesia (no precision given) castrate calves at months with adequate anaesthesia and analgesia (no precision given) r dehorning: if cattle are to be dehorned, it is recommended to disbud young cattle rather than to dehorn older ones. disbudding by cauterisation is recommended over other methods. local anaesthesia (e.g. - ml lidocaïne or lignocaïne % around the corneal nerve) and analgesia with a non steroidal anti-inflammatory drug ( ml flunixin meglumine or - . mg ketoprofen % / kg body weight) shall be performed - min before disbudding. r castration:if cattle are to be castrated, it is recommended to castrate calves as early as possible (no later than . mo and preferably at wk of age), to use the burdizzo method, and to provide appropriate anaesthesia and analgesia (e.g. ml lignocaine % in each testicle through the distal pole and mg ketoprofen % / kg body weight injected intravenously both min before castration). -main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. -the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens on farms depend on the status and the inter-relationship of different contributing factors that are inherently highly variable. -present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. -nevertheless, generic principles for risk reductions for the main foodborne pathogens at calf farm level are known and are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures based on gfp-ghp recommendations for future research . it is recommended that future research should be conducted within the following areas: -hemoglobin levels and iron deficiences of veal calves aged - weeks. -the monitoring of haemoglobin in groups of calves using representative samples -exposure to allergenic proteins -solid and liquid food balance. exposure to too rich diets and changes in feed composition. -space requirements -health monitoring systems and the effect of such on clinical health in calves -infection transmission (respiratory and digestive diseases) due to direct contact between calves in relation to social benefits of mixing -pain relief when disbudding, dehorning and castrating calves -design of appropriate ventilation systems for calves in confined rearing conditions -health and environmental effects of feeding minerals as antimicrobial agents -for quantitative food safety risk categorization of farming systems individually, and/or their related ranking, further scientific information is needed. accordingly, related research should be encouraged. references used in this scientific opinion are available and listed in the scientific report published at the efsa web (www.efsa.eu.int). the ahaw panel wishes to thank the members of the working group chaired by panel member summary efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission asked efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. in this report a risk assessment was made and the relevant conclusions and recommendations are forming the scientific opinion by the ahaw panel. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economical considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update o the previous svc report with the exception of economical aspects which are outside of the mandate for this report. the various factors potentially affecting calves' health and welfare, already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently systematically determined whether they constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. in line with the terms of reference the working group restricted itself to (in essence a qualitative) risk assessment although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport and slaughter, this report does not consider animal health and welfare aspects of calves during transport and slaughter but such information can be found in a recently issued comprehensive report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, ) and in the efsa report "welfare aspects of animal stunning and killing methods" (efsa, b) . in relation with the food safety aspects, main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, the main risk factors contributing to increased prevalence/levels of the above foodborne pathogens, as well as generic principles for the risk reductions are known. the latter are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures (e.g. gfp-ghp). in general, the conclusions made in the previous svc report remain. however, recent research has provided for some additional conclusions. the risk analysis is presented in the tables of annex . the graphics in this table are not intented to represent numerical relationships but rather qualitative relations. in some instances the exposure could not be estimated due to lack of data, in which cases the risks where labelled "exposure data not available". the following major and minor risks for poor animal health and welfare have been identified for one or several of the various husbandry systems considered: the hazards of iron deficiency and insufficient floor space are considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet are considered to be moderately serious. for these hazards, there is no consensus on the exposure of calves mainly due to lack of data and that is why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding castration and dehorning (and disbudding) without anaesthetic drugs, there is a variation in relation to national legislation why the risk of poor welfare in relation to castration and dehorning has a wide range between countries. tables which clarify the risk assessment have been included in annex . calf a calf is a young bovine which is significantly younger and smaller in size than an adult of the same species and breed and which is not reproductively active. there is a gradual transition from a newborn animal, dependent on milk, to an animal with many adult characteristics. few people would use the term calf for domestic cattle of - months whilst most would call an animal of months or somewhat older a calf. in this report, calf is used for animals of up to months of age. however, in deciding on the end of the calf stage, any definition based on age or weight is arbitrary. the term calf is not normally restricted to animals that are unweaned or monogastric rather than having some degree of development of the rumen for its specialist function. the removal of the horn bud or the actual horn depending on the breed and the age of the animal. endotoxin unit (eu) endotoxin activity of . ng of reference endotoxin standard, ec- or eu/ng (fda). to convert from eu's into ng, the conversion is eu/ng. a process where water bodies receive excess nutrients that stimulate excessive plant growth (i.e. water pollution). intensively reared calf a calf which is not kept extensively at pasture. according to the council of europe european convention for the protection of animals kept for farming purposes, (chapter i, article ), "modern intensive animal farming systems are systems in which mainly technical facilities are used that are primarily operated automatically and in which the animals depend on the care of and supply from the farmer". nsaid non-steroidal anti-inflammatory drug. the process by which a mother mammal allows a young animal to obtain milk from its teats. odds ratio (or) the odds ratio is a measure of effect size particularly important in bayesian statistics and logistic regression. infection of the navel. meat produced from animals slaughtered at - weeks of age and supplied with roughage from at least months of age onwards. there is not any classification system for veal carcasses agreed across the eu. the only existing classification system would rather relate to a general beef carcass classification system, which comprises the following categories: however, these categories are valid for cattle having a live weight of more than kg. consequently, some member states have issued their own national schemes for veal carcass classification. in trade, there is agreement between importing and exporting countries that veal originates from calves which were fed predominantly milk replacers, and which displays a light colour. the age limit is around months. some countries such as the netherlands market meat of animals of the age of to months, as pink veal. the eu subsidies scheme represents an important incentive for pink veal production. the determination of the relationship between the magnitude of exposure of calves to a certain hazards and the severity and frequency of associated adverse effects on calf welfare. the quantitative and qualitative evaluation of the likelihood of hazards to welfare occurring in a given calf population. any factor, occurring from birth to slaughter, with the potential to cause an adverse effect on calf welfare. the qualitative and quantitative evaluation of the nature of the adverse effects associated with the hazard. considering the scope of the exercise of the working group the concerns relate exclusively to calf welfare. the identification of any factor, from birth to slaughter, capable of causing adverse effects on calf welfare. a function of the probability of an adverse effect and the severity of that effect, consequent to a hazard for calf welfare. the process of determining the qualitative or quantitative estimation, including attendant uncertainties, of the probability of occurrence and severity of known or potential adverse effects on welfare in a given calf population based on hazard identification, hazard characterisation, and exposure assessment. unaltered remain the following cac (codex alimentarius commission) definitions (note: for completeness all definitions used by cac -while not necessarily used in this document -have been included): a risk assessment that provides numerical expressions of risk and an indication of the attendant uncertainties (stated in the expert consultation definition on risk analysis). a risk assessment based on data which, while forming an inadequate basis for numerical risk estimations, nevertheless, when conditioned by prior expert knowledge and identification of attendant uncertainties, permits risk ranking or separation into descriptive categories of risk. a process consisting of three components: risk assessment, risk management and risk communication. a scientifically based process consisting of the following steps: i) hazard identification, ii) hazard characterisation, iii) exposure assessment and iv) risk characterisation. the interactive exchange of information and opinions concerning the risk and risk management among risk assessors, risk managers, consumers and other interested parties. output of risk characterisation. the process of weighing policy alternatives in the light of the results of risk assessment and, if required, selecting and implementing appropriate control options (i.e. prevention, elimination, or reduction of hazards and /or minimization of risks) options, including regulatory measures. a method to examine the behaviour of a model by measuring the variation in its outputs resulting from changes to its inputs. characteristics of a process where the rationale, the logic of development, constraints, assumptions, value judgements, decisions, limitations and uncertainties of the expressed determination are fully and systematically stated, documented, and accessible for review. a method used to estimate the uncertainty associated with model inputs, assumptions and structure/form. the process by which a young mammal obtains milk from the teat of its mother or another lactating female by sucking. the term veal refers to the meat produced from calves, principally those of the species bos taurus and bos indicus. there are several meat products from calves. they are generally distinguished by their colour: "pale" or "white" veal is generally produced from an animal under months of age and fed mostly milk or milk replacer; "pink" veal is generally produced from an animal of up to months fed larger amounts of solid foods and possibly weaned. meat from calves of - months is called young beef. weaning, weaned in mammals, weaning is a gradual process during which the young animal receives less and less milk from its dam and consumes more and more solid food. it is accompanied by changes in the dam-offspring relation. in farming, calves are often separated from their dams soon after birth and receive milk (or milk replacer) from humans or a machine. although separated from the dam, calves are considered as un-weaned as long as they are fed milk. suckler calves are left with their dam for some months and are generally weaned some time before the next calving by separating them suddenly from the dam. calves normally commence eating solid food at - weeks, although some start earlier, and they eat enough solid food for development of a functional rumen to start by about weeks of age. a weaned animal is one that no longer needs to suckle and so does not consume milk in any significant quantity indicating that the weaning process has finished. council directive / /eec laying down minimum standards for the protection of calves as amended by council directive / /ec requires the commission to submit to the council a report, based on a scientific opinion, on intensive calf farming systems which comply with the requirements of the wellbeing of calves from the pathological, zootechnical, physiological and behavioural points of view. the commission's report will be drawn up also taking into account socio-economic implications of different calf farming systems. it should be noted that the scientific veterinary committee (animal welfare section) adopted a report on the welfare of calves on november (svc, ) which should serve as background to the commission's request and preparation of the new efsa scientific opinion. in particular the commission requires efsa to consider the need to update the findings of the scientific veterinary committee's opinion in light of the availability of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission requires efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. the mandate outlined above was accepted by the panel on animal health and welfare (ahaw) at the plenary meeting, on / march . it was decided to establish a working group of ahaw experts (wg) chaired by one panel member. therefore the plenary entrusted a scientific report and risk assessment to a working group under the chairmanship of prof. bo algers. the members of the working group are listed at the end of this report. this report is considered for the discussion to establish a risk assessment and the relevant conclusions and recommendations forming the scientific opinion by the ahaw panel. according to the mandate of efsa, ethical, socio-economic, cultural and religious aspects are outside the scope of this scientific opinion. in , the scientific veterinary committee of the european commission published the report on the welfare of calves. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economic considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update of the previous svc report with the exception of economic aspects which are out of the mandate for this report. this report represents an update of the previous svc report ( ) with a risk assessment perspective. factors which are important for calf welfare include housing (space and pen design, flooring and bedding material, temperature, ventilation and air hygiene), feeding (liquid feed, concentrates, roughage) and management (grouping, weaning, human-animal relations). the measures used to assess welfare include behavioural and physiological measures, patho-physiological measures and clinical signs as well as production measures. as explained in the glossary, in this report young bovines are called calves up to a maximum of eight months of age and veal is the meat of a calf. countries with substantial production of veal are france, italy, the netherlands, belgium, spain and germany. significant veal production exists also in portugal, austria and denmark, the production of white veal, from calves that have been fed predominantly milk replacer and which has a light colour, takes place largely in france, the netherlands, belgium and italy. the eu subsidies scheme represents an important incentive for pink veal production. most calves produced for further rearing are in france, germany, uk, ireland and italy. the ways of keeping calves vary considerably from country to country and between breeds. most dairy calves are separated from their dam at birth and artificially fed whereas calves from beef breeds generally suckle their dam. according to eu statistics, in in the eu ( ) , , calves were reared for slaughter (table ) and , , calves were reared for other reasons than slaughter (table ). in total (table ) , tonnes of calf meat were produced in eu ( ) which probably implies that about , tonnes were produced in eu ( ) during . human consumption of meat from calves decreased slightly from to in eu ( ) ( table ). : item and element selected in the eurostat database to make the query and extract the data the working group set out to produce a document in which the various factors potentially affecting calves' health and welfare [already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently to systematically determine whether these factors constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. it should be noted, however, that this does not imply that a hazard that has a serious effect on just a few animals should not be dealt with by managers on farm level as the suffering imposed on some animals constitute a major welfare problem for those individuals. in line with the terms of reference the working group restricted itself to (in essence qualitative) risk assessment, i.e. only one of three elements essential to risk analysis a risk assessment approach was followed, similar to the one generally adopted when assessing microbiological risks, i.e. along the lines suggested at the nd session of the codex alimentarius commission (cac, ) . incidentally, these guidelines have been characterized by the cac as 'interim' because they are subject to modifications in the light of developments in the science of risk analysis and as a result of efforts to harmonize definitions across various disciplines. cac's guidelines are in essence exclusively formulated for the purpose of assessing risks related to microbiological, chemical or physical agents of serious concern to public health. consequently -considering their disciplinary focus -the working group had to adapt the cac definitions to serve their purpose. these adapted definitions, have, in alphabethical order, been included in chapter (see risk analysis terminology). the objectives of this report are to review and report recent scientific literature on the welfare including the health of intensively reared calves, to report on recent findings as an update to the scientific veterinary committee's previous report, to make a qualitative risk assessment concerning the welfare of intensively kept calves. where relevant, food safety implications of different farming systems are also considered. the report is structured in five major parts. the first three follow the scientific veterinary committee's previous report "on the welfare of calves" with introductory chapters - on background, measurements and needs in relation to calf welfare, chapter describing housing, diet and management and chapter describing comparison of systems and factors. in chapter common disease and use of antibiotics is described. the other two parts involve aspects of meat quality and food safety (chapter ) and the risk assessment (chapter ). conclusions and recommendations from the previous svc document together with updated conclusions derived from recent research findings are presented in the scientific opinion (www.efsa.eu.int). effect of transport and slaughter on calves' health and welfare although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport, this report does not consider animal health and welfare aspects of calves during transport because there is already a comprehensive recent report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, . the report takes into account all aspects related with transport that could affect the health and welfare of cattle and calves, including the direct effects of transport on the animals and the effects of transport on disease transmission. the loading methods and handling facilities for cattle, the floor space allowance, the relationships of stocking and the density requirements, the vehicle design, space requirements and ventilation for cattle transporters (see also the ahaw scientific opinion related to standards for the microclimate inside animal road transport vehicles; efsa, ), the behaviour of cattle during road transport, the road conditions, long distance transport and the travel times are also reviewed. recommendations for all these aspects are also given in that report. the following general requirements in relation to animal welfare were annexed as a protocol to the eu treaty of amsterdam in : "in formulating and implementing the community's agriculture, fisheries, transport, and internal market policies, the community and the member states shall pay full regard to the welfare requirements of animals, while respecting the legislative provisions and customs of member states relating to religious rites, cultural traditions and regional heritage." in the introduction to the proposed eu constitution, the following extended wording is included: "in formulating and implementing the european union's agriculture, fisheries, transport, internal market, research and technological development and space policies, the union and the member states shall pay full regard to the welfare requirements of animals, as sentient beings, while respecting the legislative provisions and customs of member states relating to religious rites, cultural traditions and regional heritage." this wording reflects the ethical concerns of the public about the quality of life of the animals. it also takes into account customs and cultural traditions. farm animals are subject to human imposed constraints and for a very long time the choice of techniques has been based primarily on the efficiency of production systems for the provision of food. however it is an increasingly held public view that we should protect these animals against mistreatment and poor welfare. in order to promote good welfare and avoid suffering, a wide range of needs must be fulfilled. these needs may require the animal to obtain resources, receive stimuli or express particular behaviours (hughes and duncan, ; jensen and toates, ; vestergaard, ) . to be useful in a scientific context, the concept of welfare has to be defined in such a way that it can be scientifically assessed. this also facilitates its use in legislation and in discussions amongst farmers and consumers. welfare is clearly a characteristic of an individual animal and is concerned with the effects of all aspects of its genotype and environment on the individual (duncan, ) . broom ( ) defines it as follows: the welfare of an animal is its state as regards its attempts to cope with its environment. welfare therefore includes the extent of failure to cope, which may lead to disease and injury, but also ease of coping or difficulty in coping. furthermore, welfare includes pleasurable mental states and unpleasant states such as pain, fear and frustration (duncan, ; fraser and duncan, ) . feelings are a part of many mechanisms for attempting to cope with good and bad aspects of life and most feelings must have evolved because of their beneficial effects (broom, ) . although feelings cannot be measured directly, their existence may be deduced from measures of physiology, behaviour, pathological conditions, etc. feelings cannot be directly measured and therefore care is necessary to avoid uncritical anthropomorphic interpretations (morton et al., ) . good welfare can occur provided the individual is able to adapt to or cope with the constraints to which it is exposed. hence, welfare varies from very poor to very good and can be scientifically assessed. measures which are relevant to animal welfare during housing, i.e. largely longterm problems, are described by broom and johnson ( ) and by broom ( broom ( , a . production criteria have a place in welfare assessment. however, although failure to grow, reproduce etc. often indicates poor welfare, high levels of production do not necessarily indicate good welfare. physiological measurements can be useful indicators of poor welfare. for instance, increased heart-rate, adrenal activity, or adrenal activity following acth challenge, or reduced heart-rate variability, or immunological response following a challenge, can all indicate that welfare is poorer than in individuals which do not show such changes. the impaired immune system function and some of the physiological changes can indicate the pre-pathological state (moberg, ) . in interpreting physiological measurements such as heart rate and adrenal activity it is important to take account of the environmental and metabolic context, including activity level. behavioural measures are also of particular value in welfare assessment (wiepkema, ) . the fact that an animal avoids an object or event, strongly gives information about its feelings and hence about its welfare (rushen, ) . the stronger the avoidance the worse the welfare whilst the object is present or the event is occurring. an individual, whom is completely unable to adopt a preferred lying posture despite repeated attempts will be assessed as having poorer welfare than one which can adopt the preferred posture. other abnormal behaviour which includes excessively aggressive behaviour and stereotypes, such as tongue-rolling in calves, indicates that the perpetrator's welfare is poor. very often abnormal activities derive from activities that cannot be expressed but for which the animal is motivated. for example, calves deprived of solid foods and hence lacking the possibility of nutritive biting, develop non-nutritive biting. whether physiological or behavioural measures indicate that coping is difficult or that the individual is not coping, the measure indicates poor welfare. studies of the brain inform us about the cognitive ability of animals and they can also tell us how an individual is likely to be perceiving, attending to, evaluating, coping with, enjoying, or disturbed by its environment so can give direct information about welfare (broom and zanella, ) . in studies of welfare, we are especially interested in how an individual feels. as this depends upon highlevel brain processing, we have to investigate brain function. abnormal behaviour and preferred social, sexual and parental situations may have brain correlates. brain measures can sometimes explain the nature and magnitude of effects on welfare. the word "health", like "welfare", can be qualified by "good" or "poor" and varies over a range. however, health refers to the state of body systems, including those in the brain, which combat pathogens, tissue damage or physiological disorder (broom and kirkden, ; broom, ) . welfare is a broader term than health, covering all aspects of coping with the environment and taking account of a wider range of feelings and other coping mechanisms than those associated with physical or mental disorders. disease, implying that there is some pathology, rather than just pathogen presence, always has some adverse effect on welfare (broom and corke, ). the pain system and responses to pain are part of the repertoire used by animals to help them to cope with adversity during life. pain is clearly an important part of poor welfare (broom, b) . however, prey species such as young cattle and sheep may show no behavioural response to a significant degree of injury (broom and johnson, ) . in some situations responses to a wound may not occur because endogenous opioids which act as analgesics are released. however, there are many occasions in humans and other species when suppression of pain by endogenous opioids does not occur (melzack et al., ) . studies of the brain inform us about the cognitive ability of animals and they can also tell us how an individual is likely to be perceiving, attending to, evaluating, coping with, enjoying, or disturbed by its environment so can give direct information about welfare (broom and zanella, ) . in studies of welfare, we are especially interested in how an individual feels. as this depends upon high-level brain processing, we have to investigate brain function. abnormal behaviour and preferred social, sexual and parental situations may have brain correlates. brain measures can sometimes explain the nature and magnitude of effects on welfare. the majority of indicators of good welfare which we can use are obtained by studies demonstrating positive preferences by animals (dawkins, ) . methods of assessing the strengths of positive and negative preferences have become much more sophisticated in recent years. the price which an animal will pay for resources, or pay to avoid a situation, may be, for example, a weight lifted or the amount of energy required to press a plate on numerous occasions. the demand for the resource, i.e. the amount of an action which enables the resource to be obtained, at each of several prices can be measured experimentally. this is best done in studies where the income available, in the form of time or energy, is controlled in relation to the price paid for the resource. when demand is plotted against price, a demand curve is produced. in some studies, the slope of this demand curve has been measured to indicate price elasticity of demand but in recent studies (kirkden et al., ) it has become clear that the area under the demand curve up to a particular point, the consumer surplus, is the best measure of strength of preference. once we know what animals strongly prefer, or strongly avoid, we can use this information to identify situations which are unlikely to fulfil the needs of animals and to design better housing conditions and management methods (fraser and matthews, ) . however, as pointed out by duncan ( duncan ( , , all data from preference studies must be interpreted taking account of the possibilities that, firstly, an individual may show a positive preference for something in the shortterm which results in its poor welfare in the long-term, and secondly, that a preference in a simplified experimental environment needs to be related to the individual's priorities in the more complicated real world. each assessment of welfare will pertain to single individual and to a particular time range. in the overall assessment of the impact of a condition or treatment on an individual, a very brief period of a certain degree of good or poor welfare is not the same as a prolonged period. however, a simple multiplicative function of maximum degree and duration is often not sufficient. if there is a net effect of poor welfare and everything is plotted against time, the best overall assessment of welfare is the area under the curve thus produced (broom, c) . . the needs and functioning of calves . . the concept of needs in assessing the needs and functioning of calves, many different approaches can be taken. one is to study, at a fundamental level, the physiology and behaviour of cattle and the ways in which they have evolved, in order to try to understand their causation and function. needs are in the brain but may be fulfilled by obtaining resources, physiological change, or carrying out a behaviour. in order to conclude that a need exists to show certain behaviour, it is necessary to demonstrate that the calves used in modern production systems are strongly motivated to show the behaviour and that, if the need is not provided for, there are signs of poor welfare such as abnormal behaviour or physiology or pathological effects (see chapter ). where the housing design allows the animals to show the behaviour that they need to show, this will promote the avoidance of poor welfare. a need is a requirement, which is a consequence of the biology of the animal, to obtain a particular resource or respond to a particular environmental or bodily stimulus. an animal may have a need that results in the existence at all times of mechanisms within the brain and abilities to perceive stimuli and respond appropriately. however, this does not mean that every individual at all times needs to carry out the response. for example, a calf has a need to avoid attack by a predator but it does not need to carry out anti-predator behaviour if no individual perceived as a predator is present. there are some needs which require urgent fulfilment, otherwise the body functioning will be impaired and in the medium or long term, the animal may suffer. for example, an adequate amount of an essential nutrient or avoidance of exposure to a serious disease. there are other needs which, if not fulfilled lead to frustration and excessive activities in an attempt to fulfil the need. the resulting poor welfare may be extreme and prolonged. needs to avoid predation and other danger mean that animals have a negative experience in some situations. close human presence and handling of animals may elicit physiological and behavioural anti-predator responses. the avoidance of such situations can also be considered as a need. calves require space to perform activities such as resting, feeding, exploring, interacting and escaping from perceived danger. to assess what risks of poor welfare are involved when the housing circumstances do not allow certain activities, it can be helpful to consider why the calves are intrinsically motivated to perform the activities. the selection criteria applied to modern cattle genotypes have resulted in changes in morphological phenotype. although these have not altered the categories of needs of calves, they may have altered rates of growth and energy partitioning so that the timing of problems and the probability that they will arise may be changed. the overall need of calves is to maintain bodily integrity while growing and preparing for adult life. in order to do this, calves have a series of needs that are relevant to the housing and management conditions imposed upon them by humans. the needs of calves are described in detail by broom ( broom ( , . in listing needs and in later consideration of how to provide for them, it is assumed that extreme human actions, such deliberately creating a large wound or infecting an animal with a dangerous pathogen, will not occur. the list of needs is not in order of importance. some of the needs mentioned here are discussed at greater length in the previous report. . . . to breathe calves need air that has sufficient oxygen and a low level of noxious gases in it. calves may be adversely affected by some of the gaseous products of the breakdown of animal faeces and they show preferences that help them to avoid any harm that they may cause. calves need to rest and sleep in order to recuperate and avoid danger. they need to use several postures which include one in which they rest the head on the legs and another in which the legs are fully stretched out (de wilt, ; smits, , ) . sleep disruption may occur if comfortable lying positions cannot be adopted or if there is disturbance to lying animals because they are trodden on or otherwise disturbed by other calves. exercise is needed for normal bone and muscle development. calves choose to walk at intervals if they can, show considerable activity when released from a small pen and have locomotor problems if confined in a small pen for a long period (warnick et al., ; dellmaier et al., ; trunkfield et al., ) . calves living in natural conditions would be very vulnerable to predation when young. as a consequence, the biological functioning of calves is strongly adapted to maximise the chance of recognition of danger and escape from it. calves respond to sudden events and approaches by humans or other animals perceived to be potentially dangerous with substantial sympathetic nervous system and hypothalamic-pituitary-adrenocortical (hpa) changes. these physiological changes are followed by rapid and often vigorous behavioural responses. fear is a major factor in the life of calves and has a great effect on their welfare. . . . to feed and drink . . . . sucking the calf needs to attempt to obtain nutrients at a very early stage after birth and shows behavioural responses that maximise this chance. as a consequence, from an early age, calves have a very strong need to show sucking behaviour and if a calf is not obtaining milk from a real or artificial teat, it sucks other objects (broom, (broom, , metz, ; hammell et al., ; jung and lidfors, ) . the need of the calf is not just to have the colostrum or milk in the gut but also to carry out the sucking behaviour on a suitable object (jensen, ) . further, the sucking is of importance for the release of gastrointestinal hormones. it has been shown in calves that oxytocin is released during milk ingestion. the amount released, however, was less in calves drinking their milk from a bucket compared with calves suckling the dam (samuelsson, ) . peripheral oxytocin stimulates the release of glucagon from the pancreas whereas central oxytocin increases hunger and the release of gastrointestinal hormones promoting growth (stock et al., ; björkstrand, ) . in the early days after birth, calves are motivated to suck and obtain milk. however, calves also have a need to obtain sufficient water and will drink water even when fed milk. if the temperature is high, calves will drink water if it is available and sick calves will also choose to drink water. if water is not available, over-heated calves and sick calves may become dehydrated. sick calves may become dehydrated even when water is offered. calves with acidosis with or without diarrhoea often lose their suckling reflex. this may also happen in calves with hypoglycaemia and septicaemia (berthold, pers. com). after the first few weeks of life, calves attempt to start ruminating. if they have received no solid material in their diet, calves still try to ruminate but cannot show the full rumination behaviour. in addition to the need to suck when young, calves need to manipulate material with their mouths. they try to do this whether or not they have access to solid material and they will seek out solid material that they subsequently manipulate. (van putten and elshof, ; webster, ; webster et al., ) . calves eat solid food better when water is offered simultaneously. certain rapidly digestible carbohydrates are necessary for the development of ruminal papillae with associated physiological development and fibrous roughage helps the anatomical development of the rumen. so it is clear that calves need appropriate solid food in their diet after the first few weeks of life; first, food that is digested rapidly and provide fatty acids; then fibrous foods. rumen development is enhanced when calves are fed with concentrates, water and roughage such as hay. . . . to explore exploration is important as a means of preparing for the avoidance of danger and is a behaviour shown by all calves (kiley worthington and de la plain, ; fraser and broom, ) . exploration is also valuable for establishing where food sources are located. calves need to explore and it may be that higher levels of stereotypes (dannemann et al., ) and fearfulness (webster and saville, ) in poorly lit buildings or otherwise inadequate conditions are a consequence of inability to explore. . . . to have social contact . . . . maternal contact the needs of young calves are met most effectively by the presence and actions of their mothers. in the absence of their mothers, calves associate with other calves if possible and they show much social behaviour. the need to show full social interaction with other calves is evident from calf preferences and from the adverse effects on calves of social isolation (broom and leaver, ; dantzer et al., ; friend et al., ; lidfors, ) . to minimise disease during the first few hours of life, the vigorous attempts of the calf to find a teat and suckle should result in obtaining colostrum from the mother. this colostrum includes immunoglobulins that provide passive protection against infectious agents. hence the needs of the calf have an evident function that is not just nutritional. calves also show preferences to avoid grazing close to faeces. they also react to some insects of a type which may transmit disease. if infected with pathogens or parasites, calves will show sickness behaviour that tends to minimise the adverse effects of disease (broom and kirkden, ) . young calves, less than four weeks of age, are not well adapted to cope with stressful events such as handling and transport, often suffering very high rates of mortality and the younger the calves are, the higher their mortality (staples and haugse, ; mormède et al., ) many succumbing to pneumonia or scouring, within four weeks of arrival at the rearing unit (staples and haugse, ). an inability to mount an effective glucocorticoid response, which is adaptive in the short term, may be a contributing factor to the high levels of morbidity and mortality which occur in young calves (knowles et al., ) as may neutrophilia (simensen et al., ; kegley et al., ) , lymphopaenia (murata et al., ) and suppression of the cell mediated immune response (kelley et al., ; mackenzie et al., ) . to groom grooming behaviour is important as a means of minimising disease and parasitism and calves make considerable efforts to groom themselves thoroughly (fraser and broom, ). calves need to be able to groom their whole bodies effectively. to thermoregulation calves need to maintain their body temperature within a tolerable range. they do this by means of a variety of behavioural and physiological mechanisms. . . . . selection of location when calves are over-heated, or when they detect that they are likely to become over-heated, they move to locations that are cooler. if no such movement is possible, the calf may become disturbed, thus exacerbating the problem and other changes in behaviour and physiology will be employed. responses to a temperature that is too low will also involve location change if possible. . . . . body position over-heated, or potentially over-heated, calves adopt positions that maximise the surface area from which heat can be lost. such positions often involve stretching out the legs laterally if lying and avoiding contact with other calves and with insulating materials. if too cold, calves fold the legs and lie in a posture that minimises surface area. . . . . water drinking over-heated calves will attempt to drink in order to increase the efficiency of methods of cooling themselves. to avoid harmful chemical agents calves need to avoid ingesting toxic substances and to react appropriately if harmful chemical agents are detected within their bodies. . . . to avoid pain calves need to avoid any environmental impact or pathological condition that causes pain. the text in this section refers to current situation in eu countries. calf housing in other countries may be different. replacement dairy calves . . . diet brief description of the diet of replacement heifer calves. this has not really changed since the report. following birth, calves receive (or should receive) colostrum and are than reared with whole milk or milk replacer. calves are weaned; weaning ages and weaning strategies may differ according to region or country. briefly mention current weaning strategies. calves receive starter and, for example, hay and maize silage to promote rumen development. according to the latest eu regulation on the housing of calves (council directive eu / /ec), group housing is compulsory for calves older than weeks, unless there is any need for isolation certified by a veterinarian. individual housing of rearing calves younger than weeks, is quite common in the european dairy industry. below, the most important housing systems for replacement heifer calves are briefly listed. . . . . hutches: partially closed, outside area hutches are made of plywood, plastic or fibre glass. if hutches are made from a synthetic opaque material, this prevents the greenhouse effect inside the hutch and reduces heat stress. if reflective material is used (light coloured), the sun rays are reflected which reduces the risk for overheating. the size of hutches may vary from . - . m width and . - . m length. a layer of sand, e.g. cm gravel or crushed stone can be placed under the calf hutch. litter may be provided preferably as straw, as it provides the warmest surface temperature (panivivat et al., ) , but also wood shavings, sawdust or newspapers are used and the layer should be thick enough to provide a comfortable and dry bed. calf hutches provide three different environments, as the inside is dry and protected from the weather and outside the calf is able to get limited exercise and sunlight. the calf can be also position itself half in and half out, getting sunlight and being protected from wind. hutches should be placed where they catch the most sunlight and avoiding hot, windy and wet locations. nevertheless, during hot summer conditions hutches should be placed in a shady area to avoid overheating. in the rear wall, a hole that can be closed provides better air ventilation within the hut in warm weather. in the hutches, the calf can be kept using wire panels in a building with an outdoor run, preferably of more than . m , enabling some contact to other calves. calves can also be fed outside using a milk bucket support, a dry feed recipient support and a hay rack. other hutch types locate feed and water pails inside the hutch. individual pens are situated in a roofed building. the area should be wellventilated so that the air is dry and fresh, but draught has to be avoided. separation from adult cows is advantageous with respect to disease prevention. pens are either made from hard material with concrete walls or dismountable with three solid sides (i.e. plywood) and an open front (see figure ). walls have to be perforated according to council directive eu / /ec in holdings with more than five calves, which allow at least limited social contact with other calves, one of the key needs of calves. the open front gets fresh air to the calf and makes them easier to feed through a bucket support provided on the front. hardwood is normally used for the floor, which is covered with a litter that is thick enough, dry and clean. totally slatted floors are in use also, made of wood, plastic or metal, but require more care for air temperature. the . - . m x . - . m pen can be put mm above the ground allowing for draining and the removal of urine. dismountable individual pens should be designed in such a way that they can be taken apart and stored when they are not needed, and also easily cleaned with a skid-steer loader or small bucket tractor. in case of cold weather, a plywood cover can be placed over the rear portion of the pen to preserve heat produced by the calf. in hot weather, a removable panel at the rear of the shelter can be opened to provide additional air exchange. collective hutches may house a group of between and calves. the hutches are made of synthetic materials or wood. the inside of the hutch is provided with litter and some hay may be put in a rack. roughage is distributed at a feeding barrier and anti-freeze drinking devices are needed if freezing temperatures may occur. with collective hutches fastened on concrete, a good outdoor run has a non-slippery surface. manure and bedding have to be removed manually or the collective hutch has to move over a few metres distance by means of a tractor and guide-blocks. as for the individual hutches, the location has to be chosen carefully to avoid overheating during summer and provide protection from wind and rain entering the hutch during cold seasons, but give as much sunlight as possible. when sufficient straw and proper ventilation is provided these are the most suitable facilities for young replacement heifer calves. if the calves stay there for several months it is necessary to provide a passage on slippery free concrete. if the floor of this passage is quite rough this will prevent slipping. the concrete floor may be replaced by a slatted floor provided that the spacing between slats agrees with the age of the animals. the lying area can be built in different ways and littered with different materials. in the deep litter system, the dung is removed at regular intervals from every few weeks to twice per year. . . . . group pens inside another common system for group housing of replacement heifer calves is group housing inside, in straw littered pens usually with - calves per pen. calves may enter such group housing already after weeks of individual housing. the regulatory change with regard to calf housing together with a general trend towards larger dairy farms has increased the interest in group housing systems for rearing calves during the milk feeding period (hepola, ; jensen, ) . in addition to systems with small groups of calves ( - animals per group) kept on straw and usually bucket-fed, calves are increasingly kept in larger groups ( up to about calves) with computer-controlled automatic milk feeders. an automatic milk feeder may contain two milking dispensers, and each milking dispenser can be used for about calves. to prevent hierarchic and health problems within the group, calves are grouped with a limited age difference between the animals. calves receive milk replacer according to their needs or ad libitum. when calves are fed according to their needs, a radio-frequency electronic identifier can be used, with a transponder inserted in the collar, in an ear tag, injected under the skin or inside a ruminal bolus swallowed by the animal. the diet of the vast majority of veal calves in the european union is determined by the market demand for "white meat", i.e. meat with low myoglobin content. the production of white veal meat comes from a tradition of fattening calves thanks to a diet based on milk, which is naturally poor in iron, and slaughtering the animals when they are young. nowadays most veal calves are fed milk replacers that contains a variable proportion of milk powder and which iron content is maintained at a low level. this results in relatively low blood haemoglobin levels. an average blood haemoglobin level at slaughter between . and . mmol/l is compatible with an acceptable meat colour. as haemoglobin levels increase, the number of animals whose meat is darker in colour increases. in order to prevent calves from having haemoglobin levels that are too low, early in the production phase, the iron supply in the milk replacer fed during the first - weeks of the fattening period (starter) is usually about ppm, whereas iron supply in the milk replacer fed during the remainder of the fattening period (fattener) is ppm. moreover, blood haemoglobin levels are generally monitored, most intensively upon arrival at the fattening unit, and calves with levels below age-dependent thresholds are treated with iron, either individually or group-wise. thus, blood haemoglobin levels usually gradually decline across the fattening period, and the lowest average levels are supposed to be reached during the last four weeks prior to slaughter. some veal calves are still fed raw milk. in case of dairy breeds, the cows are generally milked and the milk is given to calves in buckets. in case of beef breeds, the calves are led twice a day for suckling their dam or another cow. according to the latest amendment to the annex of council directive / /eec (commission decision / /ec) calves should receive sufficient iron to ensure an average blood haemoglobin of at least . mmol/l, and calves over two weeks old should be provided daily with some fibrous feed which should increase from to a minimum of grams per day from the beginning to the end of the fattening period. the main types of solid feed given to veal calves differ somewhat between the veal producing countries in europe. in france and italy solid feeds for veal calves usually consist of chopped straw or pelleted dry feed consisting of both fibrous (e.g. straw) and concentrate-like (e.g. cereal) materials. in the netherlands, maize silage is a popular roughage source for white veal calves, provided that the iron content is not too high (an upper limit of - pp/kg dry matter is generally imposed). maize silage is usually fed in relatively high amounts, with maximum daily amounts of up to . kg ( gr dry matter)/calf/day. other feeds used in the veal industry include chopped straw and rolled barley. white veal calves are fattened for approximately weeks in italy and the netherlands, and for - weeks in france. besides the production of white veal meat, several systems exist across europe that lead to the production of so-called "pink veal meat". the main differences from the more conventional production of white veal are that the calves are reared for a longer period and they receive higher amounts of solid foods. as a consequence the muscles have a higher content of myoglobin, hence the darker colour of the meat. in france, the calves are most often from suckler beef breeds; they are reared with their dam and may be weaned before the end of rearing. in the netherlands, pink veal meat is generally produced from calves of dairy breeds. pink veal calves are weaned at - weeks of age. after weaning, they receive a diet of ad lib roughage (frequently maize silage) and by-products. pink veal calves are not restricted with regard to dietary iron supply and, consequently, develop normal haemoglobin levels and the associated "red" (pink) meat colour. the age at slaughter can vary from calves of - months to young bred animals of - months with the slaughter age of individuals depending on the production rate. these products are labelled to help consumers to distinguish them from white veal meat. in line with the latest eu regulation (council directive eu / /ec), individual housing of veal calves has been officially abolished in the european union. already in the s extensive studies were initiated with the aim to develop a practically feasible husbandry system for group housing of veal calves. at present the systems involve both large and smaller groups. housing of calves is in groups of - animals, with a slight trend towards larger group sizes ( - calves per group). the floor can be bedded with straw or wood shavings but is more commonly made of wooden slats. wooden slats require less labour and straw or woodshavings easily become dirty and wet. calves are kept in individual pens, sometimes called "baby-boxes" for a period of - weeks upon arrival at the fattening unit to prevent overt preputial sucking thereafter and to be able to monitor more closely the health of calves. baby boxes are usually made of galvanised or wooden partitions placed inside the group pen. in these boxes, calves are bucket-fed individually. after - weeks, these temporary partitions are removed and calves are free to move around in the pen. calves are fed milk replacer in a trough or in individual buckets. a crucial management procedure associated with trough feeding is the regular re-grouping of calves, to maintain homogeneous groups in terms of calf weight and particularly drinking speed throughout fattening. experimental work confirmed the feasibility of this procedure in that calves could be repeatedly regrouped without effects on their health, growth rate and a number of physiological measures of stress . in this latter study, aggression between calves was rare, and calves seemed to habituate to repeated mixing. individual calves not thriving on milk replacer because of drinking problems, are provided with floating teats or with a teat-bucket. veal calves are sometimes kept in pairs. this type of housing results in less availability of space for movement and social opportunities than in larger groups of calves but is reported to have no disadvantages in health, weight gain and the occurrence of cross-sucking (chua et al., ) . suckling veal calves are generally accomodated in small groups. as in the rearing of dairy calves, automatic feeding systems have been extended to veal production systems, particularly since increasingly sophisticated computer technology is becoming available for sensor-aided recognition of individual animals, and to control feeding times and intake. calves are usually housed in large groups ( - calves) and receive milk replacer via an automatic feeding machine. with such feeders, calves suck to obtain their milk. the floor generally consists of wooden slats, or concrete in combination with wooden slats. some veal calves are kept on straw bedded floors, or have access to rubber mats or concrete covered by rubber. calf rearing and animal environmental pollution . . . general introduction in the report there was a short chapter on calf production and environmental pollution referring to gases (ammonia, nitrous oxide, carbon dioxide). manure resulting from calf production was seen as a fertiliser only. this chapter briefly describes in a condensed way the impact of modern animal calf production affects the environment of the animals. modern animal production is a source of solid, liquid and gaseous emissions which i.a. can be harmful to the animals. solid and liquid manure and waste water contain nitrogen and phophorus which are the most important plant nutrients, but are harmful when applied to agricultural land in excess amounts thereby leading to pollution of ground water by nitrates, surface water with phosphorous causing eutrophication and soil with heavy metals such as zinc and copper which are used as growth promotors in the feed stuff, all of which can affect the animals if returned to them. a third group of potentially hazardous effluents are drug residues, such as antibiotics, which may be present in the excreta of farm animals after medical treatment and which are passed to the environment during grazing or spreading of animal manure where they may conceivably contribute to the formation of antibiotic resistance in certain strains of bacteria. the same risk arises when sludge and waste water from sewage plants containing residues of antibiotics and other drugs from human consumption are discharged as fertiliser in the soil and water body of agricultural land. the most important aerial pollutants from calf rearing systems are odours, some gases, dust, micro-organisms and endotoxins, together also addressed as bioaerosols (seedorf and hartung, ) , which are emitted by way of the exhaust air into the environment from buildings and during manure storage, handling and disposal. aerial pollutants can give cause for concern for several reasons. e.g. an animal's respiratory health may be compromised by these pollutants. in fattening units, up to % of all calves may show signs of pneumonia, pleuritis or other respiratory disease within the first three weeks of housing when the calves come together from different herds (see chapter on temperature, ventilation and air hygiene). the travel distance of viable bacteria from animal houses via the air is presently estimated at m (müller and wieser, ) downwind why there is a possible transmission between animal houses. very little is known about the distribution characteristics of dust particles, endotoxins, fungi and their spores, in the air surrounding animal houses. recent investigations showed dispersion of staphylococcae sp. (bioaerosols) up to m (schulz et al., ) from a broiler barn. the contribution of calf production is presently unknown. it is estimated that calves produce about . kg fresh manure and . kg slurry per animal and day. this is a share of . % in the total amount of fresh manure produced in cattle farming (richter et al., ) . manure suspected to contain pathogens such as salmonella should be stored for at least months without adding or removing material and subsequently applied to arable land where it is ploughed in, or it is disinfected before any further use. the second area of concern is the emission compounds such as gases, odours, dust, micro-organisms and other compounds like endotoxins which are regularly present in calf house air where they can cause or exacerbate respiratory disorders in animal and work force. the quantities emitted from calf houses are summarised in seedorf et al. ( b) there are considerable emission amounts from calf husbandry. the emissions of micro-organisms are higher than from dairy or beef barns but distinctly lower than from pig or poultry production (seedorf et al., a) . the same is true for endotoxins which are one log lower in cow barns but distinctly higher in pig and poultry houses. the dust emissions can be times higher in piggeries and times higher in broiler barns . the ammonia concentration is usually lower than in piggeries or laying hen houses. however this depends greatly on the housing and manure management system. in a us study johnson et al. ( ) reported that cow-calf, stocker and feedlot phases contribute considerable amounts of nitrous oxide and methane to the emissions from cattle production. . comparison of systems and factors . . feeding and housing systems, weaning strategies and quality of solid and liquid feed . . . feeding systems the main potential problems associated with the housing of calves in large groups with automatic feeders include: cross sucking, i.e., non-nutritive sucking of parts of another calf's body (in particular the ears, mouth, navel, udder-base and, in case of bull calves, the scrotum and prepuce) (plath et al., ; bokkers and koene, ; jensen, ) , competition for access to the feeder (jensen, ; , and health problems, in particular a high incidence of respiratory disease (maatje et al., ; plath et al., ; svensson et al., svensson et al., , hepola, ; engelbrecht pedersen et al., ) . a number of factors have been identified that are likely associated with some of these problems, although conflicting results have been reported. cross-sucking is linked with the sucking motivation of calves and, hence, measures to reduce the motivation of calves for non-nutritive sucking may reduce the occurrence of cross-sucking (de passillé, ). an increased milk allowance also reduced non-nutritive sucking on a teat as well as cross-sucking in group-housed calves in one experiment (jung and lidfors, ), but did not affect cross-sucking in another (jensen and holm, ) . reducing the milk flow rate decreased nonnutritive sucking on a teat in individually housed calves (haley et al., ) , but failed to influence cross-sucking in group-housed ones (jung and lidfors, ; jensen and holm, ) . alternatively, it has been suggested that hunger may also control the level of non-nutritive sucking and possibly cross-sucking (jensen, ) . this idea is consistent with the observations that the duration of unrewarded visits to an automatic feeding station increased during gradual weaning (jensen and holm, ) , and that under practical farm conditions the frequency of cross-sucking among dairy calves around weaning is increased with decreasing availability or energy density of solid feeds (keil et al., ; keil and langhans, ) . in contrast to other calves, white veal calves are not weaned, receive large amounts of milk replacer and usually obtain only restricted amounts of solid food. these additional factors may also affect and perhaps exacerbate crosssucking in systems with an automatic feeder (jensen, ) . results by veissier et al. ( ) showing that bucket-fed group-housed veal calves show less cross-sucking than those fed by an automatic feeder again seem to implicate factors other than sucking motivation per se in the development and expression of cross-sucking. on the other hand, rearing calves in large groups with an automatic feeder allows more interactions between calves and offers calves the possibility to suck milk. competition for access to an automatic milk feeder was increased in groups of or calves in comparison with groups of or , respectively (herrmann and knierim, ; jensen, ) , and under dietary conditions of relatively low milk allowance and reduced milk flow rate (jensen and holm, ) . protecting calves from displacement at the feeder may also be accomplished by fitting a closed feeding stall to the station (weber and wechsler, ) . in comparison with the usual setup, this modification increased the duration of visits to the feeder as well as the duration of non-nutritive sucking on the teat after milk ingestion, and significantly reduced the frequency of cross-sucking within minutes after milk ingestion. however, the incidence of cross-sucking performed without prior milk ingestion was not affected by the design of the feeder (weber and wechsler, ) . in a recent comprehensive review, jensen ( ) observes that there is a lack of knowledge on the effect of different weaning methods on cross-sucking. she also concludes that future research should focus on preventive measures to reduce cross-sucking and problems with aggression in automatically fed calves, including the establishment of appropriate numbers of calves per feeder. the apparent increase in health problems of calves kept in large groups with automatic feeders might be related to group size rather than to feeding system. a comparison of two different group sizes of calves fed by an automatic milk feeder showed that calves housed in groups of - had a higher incidence of respiratory illness and grew less than calves housed in groups of - (svensson and liberg, ) . similarly, placement of preweaning heifer calves in groups of or more was associated with high calf mortality in a large scale epidemiological survey (losinger and heinrichs, ) . interestingly, in a study by kung et al. ( ) , group-housed calves fed by an automatic feeding system for milk supply had fewer days of medication than those kept individually in separate calf hutches. these authors also emphasize the importance of good management and frequent observations of calves as an integral part of a successful rearing program. likewise, howard ( ) specifically links good and correct management practices with the prevention of disease and successful group housing of dairy calves. natural weaning in cattle takes place when young animals are around - months of age. depending on productive system, weaning can usually occur between and months of age. dairy calves are usually reared away from their dams and they are given milk or milk replacer until weaning at to weeks of age. however holstein calves can be weaned at to weeks of age (early weaning). beef calves are usually weaned at to months of age depending on season of birth. early weaning of beef calves may be considered as a management practice in poor climate conditions and where forage quality is poor later in the grazing season. several studies have shown that it is possible to wean calves at very young ages based on concentrate intake (svc, ) . however, regardless of the productive system, weaning is effective and does not cause health and welfare problems to calves when it occurs as a smooth transition from an immature to mature ruminant with an adequate size and development of the reticulo-rumen for efficient utilisation of dry and forage based diets. at birth, the reticulum, rumen, and omasum of the calf are undeveloped, nonfunctional and small in size compared to the abomasum and rumen remains underdeveloped during the first - months of age. calves being ruminant animals require a physically and functionally developed rumen to consume forages and dry feeds. however, the rumen will remain undeveloped if diet requirements for rumen development are not provided. solid feed intake stimulates rumen microbial proliferation and production of microbial end products, volatile fatty acids, which initiate rumen epithelial development . solid feeds are preferentially directed to the reticulo-rumen for digestion, however they differ in efficacy to stimulate rumen development. recent studies have shown that addition of yeast culture ( %) increased calf grain intake, but did not affect rumen development in young calves ; while papillae length and rumen wall thickness were significantly greater in week old calves fed calf starters containing steamflaked corn over those fed dry-rolled and whole corn when these corn supplements made up % of the calf starter showing that the type of grain processing can influence rumen development in young calves. forages seem to be the primary stimulators of rumen muscularization development and increased rumen volume (zitnan et al., ) . large particle size, high effective fibre content, and increased bulk of forages or high fibre sources physically increase rumen wall stimulation, subsequently increasing rumen motility, muscularization, and volume coverdale et al., ) . besides, solid feeds other than forages or bulky feedstuffs can be effective in influencing rumen capacity and muscularization. coarsely or moderately ground concentrate diets have been shown to increase rumen capacity and muscularization more than finely ground or pelleted concentrate diets, indicating that extent of processing and/or concentrate particle size affects the ability of concentrates to stimulate rumen capacity and muscularization (beharka et al., ; greenwood et al., ) . therefore, it seems that concentrate diets with increased particle size may be the most desirable feedstuff for overall rumen development, due to their ability to stimulate epithelial development, rumen capacity, and rumen muscularization . calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. but, as pointed out from recent research attention must paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. early weaning systems should not be used if the animals are in a negative energy balance. . . . quality of solid and liquid feed . . . . solid feed: concentrates and roughage traditionally, veal calves were fattened on a diet consisting exclusively of milk replacer. calves fed in this manner show a number of welfare problems (reviewed in the previous report), including abnormal behaviours and disease associated with lack of rumen development. to better safeguard the welfare of calves, provision of (some) solid feed to veal calves has become compulsory according to the latest amendment to the annex of council directive / /eec (commission decision / /ec). however, provision of roughage to veal calves fed a regular milk replacer diet, has clearly been demonstrated to increase the incidence of abomasal ulcers, in particular in the pyloric part (which connects to the duodenum) (wensink et al., ; welchman and baust, ; breukink et al., ) . thus, recent studies have largely focussed concurrently on the effects of provision of roughage on calf behaviour, abomasal lesions and rumen development, in an attempt to identify feeds that may benefit veal calf welfare without compromising abomasal integrity. in a comprehensive eu-funded project, a range of different types of roughage/solid feeds (straw, maize silage, maize cob silage, rolled barley and beet pulp) in different amounts ( versus gr dry matter) and of different particle sizes and physical characteristics (i.e., chopped versus ground, dried versus fresh, un-pelleted versus pelleted) were given to veal calves in addition to milk replacer in large-scale multifactorial trials (chain management of veal calf welfare, ; cozzi et al., ; mattiello et al., ) . control treatments consisted of milk replacer only, and milk replacer with ad lib access to hay. another control group consisted of bull calves reared in a similar way to normal dairy calves, i.e. the animals received ad libitum hay and concentrates and were weaned at weeks of age. in comparison with milk replacer only, those types of roughage that were richest in fibrous material, i.e. straw (regardless of amount and physical structure) and hay, significantly reduced the level of abnormal oral behaviours (composed of tongue rolling, tongue playing and compulsive biting/sucking of substrates), and concomitantly increased the level of rumination. weaned calves exhibited no abnormal oral behaviours. higher levels of rumination in veal calves as a function of the fibre content of the solid feed were also reported by morrisse et al. ( morrisse et al. ( , . in line with these findings, veissier et al. ( ) , observed reduced levels of biting at substrate and more chewing behaviour in veal calves provided with straw compared with un-supplemented controls. previously, it has been suggested that a sucking deficit causes abnormal oral behaviours in calves (sambraus, ) . more recent data, however, clearly identify the lack of appropriate roughage as a major determinant of abnormal oral behaviours in veal calves. correspondingly, bokkers and koene ( ) found no differences in abnormal oral behaviours between group-housed veal calves fed either by bucket or by an automatic feeder. results obtained in veal calves are also fully consistent with data in cows (redbo et al., ; redbo and nordblad, ) and other ruminants such as giraffes (baxter and plowman, ) , which all link increased levels of abnormal oral behaviours with feeds poor in fibre. in agreement with previous data, most roughages provided to milk-fed veal calves significantly increased the incidence of abomasal lesions, particularly ulcers in the pyloric region, in comparison with the feeding condition without additional roughage (chain management of veal calf welfare, ; . incidences of abomasal ulcers (expressed as the percentages of calves with one or more lesions) among weaned bull calves, calves fed milk only, and veal calves given supplemental roughages were , between - , and between - %, respectively. this suggests that the interaction between roughage and a milk replacer diet rather than roughage per se, is involved in the etiology of abomasal ulcers in veal calves. these findings support the hypothesis that pyloric ulcers in milk-fed veal calves may be caused by local ischaemia followed by focal necrosis as a consequence of strong contractions of the pyloric wall when large volumes of milk are consumed. provision of roughage, in turn, would then exacerbate an existing problem in that roughage particles exert a mechanically abrasive effect on a sensitive abomasal mucosa, and delay the healing of any lesions already present (unshelm et al., ; dämmrich, ; krauser, ; welchman and baust, ; breukink et al., ) . this explanation may also fit the observations that veal calves fed either hay or a combination of concentrates and straw exhibited similar incidences of abomasal ulcers to those fed milk replacer only (chain management of veal calf welfare, ; veissier et al., ) . these roughages represent more balanced feeds, accompanied by better rumen fermentation. this may have improved ruminal digestion of fibres, thereby preventing sharp undigested particles entering the abomasum. other factors proposed or examined in relation to the pathogenesis of abomasal ulcers in calves include stress, infection with bacteria, trace mineral deficiencies, and prolonged periods of severe abomasal acidity (lourens et al., ; mills et al., ; jelinski et al., ; de groote et al., ; palmer et al., ; ahmed et al., ahmed et al., , ahmed et al., , . however, so far none of these factors have been convincingly related to abomasal ulcers in veal calves. calves fed milk only, showed a high incidence of ruminal hairballs. in different experiments between - % of milk-fed veal calves had hairballs (chain management of veal calf welfare, ; cozzi et al., ) . feeding roughage gave a profound reduction of hairballs; depending on the type of roughage the incidence varied between - %. similarly, morisse et al. ( ) reported a marked reduction of ruminal hairballs in calves fed pelleted straw and cereals. this reduction was thought to result from a continuous elimination of ingested hair by improved ruminal motility. however, it may well be that abnormally high self-licking behaviour is reduced when roughage is provided. it is suggested that further optimising the composition of roughage in terms of adequate rumen development and rumen function, may eventually result in feeds that promote rumination and reduce abnormal oral behaviours without damaging the digestive apparatus (chain management of veal calf welfare, ; morisse, ; mattiello et al., ) for all newborn calves, receiving an adequate amount of high quality colostrum is essential for their health and survival. in comparison with mature milk, colostrum contains greater concentrations of total solids and of fat, protein, vitamins and minerals. most importantly, colostrum provides the calf with immunoglobulins (igg), which are vital for its early immune protection. in addition, colostrum contains a range of other non-nutrient and bioactive components including various types of cells, peptide hormones, hormone releasing factors, growth factors, cytokines and other bioactive peptides, oligosaccharides and steroid hormones. these factors modulate the microbial population in the gastrointestinal tract, have profound effects on the gastrointestinal tract itself (e.g. cell proliferation, migration, differentiation; protein synthesis and degredation; digestion, absorption, motility; immune system development and function), and in part exert systemic effects outside the gastrointestinal tract on metabolism and endocrine systems, vascular tone and hemostasis, activity and behaviour, and systemic growth (waterman, ; blum, ) . the highest quality colostrum, or true colostrum, is obtained from the very first milking after parturition. thus, provision of first colostrum to newborn calves is one critical factor for successful calf rearing. the timing of provision of colostrum is also crucial since the ability of the calf's small intestine to absorb large proteins such as igg decreases rapidly following birth. consumption of sufficient colostrum within the first h of life is needed not only for an adequate immune status but also to produce the additional important and favourable effects on metabolic and endocrine traits, and on vitality. finally, colostrum should be regularly provided for a sufficient length of time, preferably for the first three days after birth (hadorn et al., ; waterman et al., ; rauprich et al, ) . although the importance of colostrum for calf health and survival is generally recognized, actual practices in calf rearing do not always favour adequate colostrum intake in newborn calves, and may therefore pose a risk for their welfare. after the period of colostrum feeding, calves can be switched to whole milk or a high quality milk replacer. in the case of rearing calves, both sources of liquid feed are used, although the majority of dairy calves are currently reared on a milk replacer diet. milk replacers are usually less costly than saleable whole milk, and the feeding of raw waste milk may pose several health and contamination risks, including the transfer of infectious diseases to the calf, and problems with antibiotic residues or overdoses (wray et al., ; selim and cullor, ; waltz et al., ) . at present, good quality milk replacers may provide comparable performance to whole milk. however, pasteurization of waste milk prior to feeding it to calves may also represent an effective and viable alternative for minimizing health risks (stabel et al., ) . results from a recent clinical survey by godden et al. ( ) even suggested that dairy calves fed pasteurized waste milk have a higher growth rate and lower morbidity and mortality rates than do calves fed conventional milk replacer. with the exception of production systems involving suckler cows, veal calves are generally fattened on milk replacer diets. over time, formulations of commercially available milk replacers for veal calves (as well as those for dairy calves) have become more and more sophisticated. at the same time, economic pressures continuously prompt the industry to reduce feeding costs and to consider alternative components and raw materials. originally, proteins in milk replacers were milk-based, and skim milk powder constituted the major protein source. subsequently, milk replacers based on whey powder became available. approximately during the last two decades, attempts have been made to replace animal-based proteins in milk replacers by vegetable proteins, mainly from soybean and wheat and, to a lesser extent, pea and potato. initially, some of these attempts met with little success because of health problems in the calves. for example, compared to calves fed diets based on skim milk powder, calves fed milk replacers containing heated soybean flour developed severe immunemediated gut hypersensitivity reactions characterized by partial atrophy of the small intestinal villi, malabsorption, diarrhoea, and large infiltrations of the small intestine by immune cells, accompanied by the presence of high antibody titres against soy antigens in plasma and intestinal mucous secretions (lalles et al., a (lalles et al., , b (lalles et al., , (lalles et al., , dreau et al., ; dreau and lalles, ) . however, the nutritional utilization of vegetable proteins can be improved by a variety of technological treatments including, for example, heating, protein hydrolysis, and ethanol extraction. such treatments reduce anti-nutritional factors and antigenic activity, and increase protein digestibility by denaturing three-dimensional structures (lalles et al., c (lalles et al., , d . at present a number of processed plant proteins are successfully applied in combination with milkbased protein sources in milk replacers for (veal) calves, including hydrolysed soy protein isolate and hydrolysed wheat gluten. recent research in the area of plant proteins in milk replacer formulas is focussed on understanding mechanisms underlying the flow of proteins in duodenal digesta, and the interaction of dietary peptides with the gut, in particular at the level of the mucus layer (montagne et al., (montagne et al., , (montagne et al., , . results of this type of work may further enhance the use of plant proteins in milk replacers for calves. in addition to an enhanced risk for gut problems, low quality milk replacers may also cause dysfunction of the oesophageal groove reflex, which may result in ruminal acidosis. in this respect, temperature is also an important quality feature; a too cold drinking temperature of the milk replacer attenuates the oesophageal groove reflex (gentile, ) . if vegetable proteins are not properly treated, milk replacers may cause hypersensitivity reactions in the gut, which may compromise calf welfare. low iron dietary supply is a prerequisite for the production of white veal. the blood haemoglobin level in veal calves towards the end of fattening (between . and . mmol/l), is generally considered a threshold below which iron deficiency anaemia occurs (bremner, ; sprietsma, a, b; postema, ; lindt and blum, a) , although some authors have argued that this level is already below a critical value (welchman et al., ) . when calves were forced to walk on a treadmill, those with a mean haemoglobin level of . mmol/l consumed more oxygen and exhibited higher cortisol levels after walking than calves whose haemoglobin level was . . or . mmol/l (piguet et al., ) . on the other hand, blood lactate after transport was not significantly different between groups of calves with average haemoglobin levels of . and . . mmol/l, respectively (lindt and blum, b) . there is a large body of evidence showing that iron deficiency anaemia may compromise immunocompetence, in particular cellular immune function, in a range of species including laboratory rodents and humans (dallman, ; dhur et al., ; galan et al., ; latunde-dada and young, ; ahluwalia et al., ) . in human children, iron-deficiency states have been epidemiologically associated with increased morbidity due to respiratory infection and diarrhoea (keusch, ; de silva et al., ; levy et al., ) . this justifies the question of whether dietary iron supply and associated haemoglobin levels are sufficient to guarantee adequate health in white veal calves. previous results concerning the relationship between clinical health and anaemia in veal calves are scarce, and were inconclusive. using very small numbers of calves, möllerberg and moreno-lopez ( ) found no difference between iron anaemic and normal calves in the clinical response to infection with an attenuated parainfluenza- virus strain, whereas sárközy et al. ( ) reported a depressed immune response as reflected in significantly lower antibody levels in anaemic calves compared with controls following inoculation with a live adenovirus. in a study by gygax et al. ( ) , cellular immune function was depressed, and disease incidence, especially of respiratory infections, was increased in calves fed low amounts of iron. however, in this particular study, haemoglobin levels dropped considerably below the value of . mmol/l. a more recent study (van reenen et al., ) , therefore, aimed to examine immunocompetence in a bovine herpes viral (bhv ) infection model in white veal calves with blood haemoglobin levels maintained at all times above or just at . mmol/l. calves daily supplemented with extra iron exhibited normal haemoglobin levels across the entire experiment (average approximately . mmol/l), whereas white veal calves had average haemoglobin levels at the time of bhv infection and at slaughter of approximately . and . mmol/l, respectively. dietary iron supply did not affect the reactions of calves to bhv infection (clinical signs, viral excretion in nasal fluid, antibody reponse), white blood cell and lymphocyte counts, and growth rate. by contrast, in comparison with calves with high haemoglobin levels, white veal calves exhibited a higher heart rate during milk intake, had consistently elevated levels of urinary noradrenaline, and showed enhanced plasma acth and reduced plasma cortisol responses in a number of hpa axis reactivity tests. these latter findings concur with increased heart rate and catecholamines in urine, and altered responsiveness of the hpa axis in iron-deficient or anaemic humans and laboratory rodents (voorhess et al., ; dillman et al., ; dallman et al., ; groeneveld et al., ; saad et al., ) . these physiological changes are part of an elaborate adaptive response to iron deficiency (beard, ; rosenzweig and volpe, ) , which also involves alterations in glucose metabolism (blum and hammon, ) . veal calves with blood haemoglobin levels clearly below . mmol/l demonstrated reduced growth rates as well as a large depression in white blood cell and lymphocyte counts (reece and hotchkiss, ; gygax et al., ) . thus, it is suggested that maintaining blood haemoglobin in individual veal calves over . mmol/l induces a number of physiological adaptations which seem universal for iron-deficient mammals in general, but do not harmfully compromise biological capacities in terms of growth and immunocompetence. in actual practice, however, the haemoglobin threshold of . mmol/l is currently considered at the group rather than at the individual level. for example, an average haemoglobin level of . mmol in a group of finished veal calves is assumed to be exactly at the lower threshold value. however, depending on the variation between individuals, if a group of calves has an average haemoglobin level of . mmol/l, then some individuals within that group may have levels well below this lower threshold value. in fact, based on an analysis of the variation between calves in blood haemoglobin levels, it has been argued that the haemoglobin threshold for anaemia of a group of veal calves should be higher than that of an individual calf, i.e. an average level of . rather than mmol/l (van hellemond and sprietsma, a) . in order to prevent anaemia during fattening, blood haemoglobin levels are monitored to some extent in white veal calves, and animals are treated with supplemental iron according to age-dependent haemoglobin thresholds. however, systematic monitoring generally occurs only on two occasions: within the first - weeks upon arrival at the fattening unit, in all animals, and between - weeks of fattening, in a sample of calves. outside these instants, individual calves may receive iron supplementation in the presence of clinical signs of iron deficiency. but once clinical signs are apparent, haemoglobin levels are usually well below . mmol l - (blaxter et al., ; bremner et al., ) . since blood haemoglobin levels are not routinely monitored in veal calves beyond the th week of fattening, there is a likelihood of too low haemoglobin levels occurring in part of the animals, in particular towards the end of fattening, when low haemoglobin levels are most likely to occur. general housing calves kept indoors are housed in an environment where several important factors interact such as space, pen design, social contacts, flooring and bedding material as well as climate. in experimental studies, usually one or a few of these factors are varied and the others controlled for. however in larger epidemiological studies many of these factors vary and their interaction can be measured. in a study of heifer calves in swedish dairy herds the effect of draught, cleanliness of the animals, hygiene level of the farm, placing of the calf pens, nature of the pen walls, air volume per animal, management factors such as status of the caretaker and feeding routines was evaluated by means of a two-level variance component logistic model. the placing of calf pens along an outer wall was significantly associated with the risk of diarrhoea (odds ratio (or): . , p< . ), the risk for respiratory disease was significantly associated with an ammonia concentration below ppm (or: . , p< . ) while the or for moderately to severely increased respiratory sounds was significantly associated with draught (or: . , p< . ) (lundborg et al., ) . odds ratios for respiratory disease were increased in calves housed in large-group pens with an automatic milk-feeding system (or: , ) . the report highlights that the housing systems of calves and the available space affect the development and determine which behaviours the animals are able to perform. the report (svc, ) recommends the minimum space for both single crate and group pen and it points out how lack of space can affect health and welfare of reared calves (maatje and verhoeff, ; dantzer et al., ; friend et al., ) . the report also suggests that shape of the pen can be important to the animal. recent studies confirmed that the space available can affect both behavioural and physiological traits and productive performances of cattle. however, the majority of them compare behaviour, production or other indicators of calves reared in individual crates versus group pens (vessier et al., ; andrighetto et al., ; jensen, ; verga et al., ; cozzi et al., ; bokkers and koene, ) or tethered or single pen (terosky et al., ; wilson et al., ) which were already discussed in chapter . little research has been done to directly compare behavioural and physiological indicators of welfare in calves reared in pens of various space allowances. in dairy calves it has been shown that spatial environment stimulated play: calves in small group pens performed less locomotory play that the ones kept in larger pens (jensen et al., ; jensen and kyhn, ) . it has been reported in a preliminary study that dairy calves kept, from birth to month of life in larger stalls ( . m x . m) showed a higher percentage of lying behaviour and grooming than calves kept in smaller stalls ( . m x . m); besides, lymphocyte proliferation was significantly higher in calves reared in large stalls (ferrante el al., ) . it is known that cattle prefer to use the perimeter of pens rather than the central area (stricklin et al., ; hinch et al., ; fraser and broom, ) . the ratio between the number of corners in the pen and number of animals seems to influence the individual space, the space that calves try to keep to other calves, as showed by simulation models (stricklin et al., ) . therefore pen shapes maximising the perimeter to area ratio might be preferable for cattle (jóhannesson and sørensen, ) . for this reason it has been pointed out that measurements such as pen perimeter, the number of corners and the diagonal distance of the pen could be important for dairy cattle (jóhannesson and sørensen, ) . however there is a lack of knowledge on this topic on calves. in a study on veal calves most of the animals lying next to the wall, the quieter and drier part of the pen, stood more on the side of the far pen and eliminated in the feeding area (stefanowska et al., ) . calves kept in a large group ( animals) and fed using an automatic milk replacer showed an elevated use of the area around the partition of the pen and they spent little time in the centre of the area. (morita et al., ) , this use of the pen space could lead to a pen design functionally divided into a walking and feeding area and a lying area. the report concludes that slatted floors must not be slippery, it also recommends appropriate bedding, for example straw, and that every calf should have access to a dry lying area. the report highlights that housing and management conditions can affect the posture adopted when lying and resting in calves. . . . recent findings regarding importance of floor and bedding materials slatted floors have been used for many years as convenient for intensive housing for beef cattle but concerns have been expressed about their effects on animal welfare (scahaw, ) . the type of surface not only affects the movements of getting up and lying down, lying and resting behaviour of the fattening animals but also other behavioural traits and physiological indicators of stress (scahaw, ) . moreover when cattle can choose between different floor types they prefer deep litter to slatted floor especially for resting. many studies were conducted in order to analyse the floor comfort in the lying area in dairy and in beef cattle (for a list of references see tuyttens, ; scahaw, ) . the group pens for veal calves do not have separate lying areas and therefore the animals spend all their time on the same surface. if the floor is too hard for lying or too slippery, discomfort, distress and injury may result. a suitable floor is very important for calves as adequate rest is essential for the good welfare of young growing animals, moreover a positive correlation between the amount of rest and growth rates has been observed for growing cattle (mogensen et al., ; hanninen et al., ) . adequate resting is important both for sleep and temperature regulation. veal calves are often housed on slatted floors, commonly made of hardwood, a product that is controversial because it often comes from unsustainable forestry in tropical countries (stefanowska et al., ) , or on concrete floors due to the fact that bedding material is costly and requires more labour and can cause problems in manure handling systems. wooden slatted floors can absorb liquid from manure and a wet surface is not comfortable for moving and lying (verga et al., ) . even if straw bedding provides better floor comfort to animals than slatted or concrete floors, suitable alternatives to reduce or eliminate the use of straw bedding are available for cattle (tuyttens, ) . recent studies have investigated the effect of the texture (how soft) and the thermal properties of floor on lying postures and resting behaviour of calves. in cool or drafty floors calves spent less time resting on the side and rest curled up in order to conserve heat (hanninen et al., ) . in contrast with adult dairy cows which rested longer and lay down more frequently on softer floors, there was no effect of type of floor (concrete floor or rubber mats) on resting behaviour of dairy calves (hanninen et al., ) . in another experiment where veal calves could choose to use a hardwood slatted floor surface or a synthetic rubber coated floor surface the calves preferred the wooden floor for lying (stefanowska et al., ) . moreover the animals rested in the drier part of the pen (stefanowska et al., ) . from these studies it seems that the texture of the floor is not as important to calves as to older animals, whereas thermal comfort seems to affect lying and lying postures. panivivat et al. ( ) investigated growth performance and health of dairy calves bedded with five different types of materials (granite fines, sand, rice hulls, long wheat straw, wood shavings) for days during august to october from birth. overall average daily gain and dry matter intake of calves did not differ with bedding type, although during week , calves housed on rice hulls had the greatest dry matter intake and those housed on wood shavings had the lowest. during week , calves housed on granite fines and sand were treated more often for scours, and calves housed on long wheat straw received the fewest antibiotic treatments (week by bedding material interaction). granite fines formed a harder surface than other bedding, and calves housed on granite fines scored the dirtiest. long wheat straw had the warmest surface temperature, and rice hulls and wood shavings were warmer than granite fines and sand. serum cortisol, alpha ( )-acid glycoprotein, immunoglobulin g concentrations, and the neutrophil:lymphocyte ratio were not affected by bedding type. on day , coliform counts were greatest in rice hulls. after use, coliform counts were greatest in long wheat straw (week by bedding material interaction). growth rates of calves bedded for d with bedding types did not differ; however, the number of antibiotic treatments given for scours was greatest on granite fines and sand; coliform counts in the bedding were highest in rice hulls before use and in long wheat straw after days of use. degree of social contact the report recommends that calves are cared for by their dam after birth so that they are licked and receive colostrum and that calves are not deprived of social contact, especially with other calves because ) calves for social contacts; ) calves isolated from other calves express more abnormal activities (e.g. excessive grooming, tongue rolling), are hyper-reactive to external stimuli and their subsequent social behaviour is impaired; and ) in combination with restricted space or lack of straw, individual housing induces a chronic stress state as assessed through enhanced responses to an acth challenge. . . . recent findings regarding contacts with the dam the bond between dam and calf is likely to develop very soon after birth: calves separated from their dam at h can recognise the vocalizations of their own dam one day later (marchant-forde et al., ) in their review about early separation between dairy cows and calves, flower and weary ( ) conclude that, on the one hand, behavioural reactions of cows and calves to separation increase with increased contacts but, on the other hand, health and future productivity (weight gain for the calf, milk production for the cow) are improved when the two animals have spent more time together. calves reared by their dam do not develop cross-sucking while artificially reared calves do so (margerison et al., ) . the provision of milk through a teat, a long milk meal, and the possibility to suck a dry teat can decrease non-nutritive sucking in artificially reared calves but do not abolish it (review by jensen, ; lidfors and isberg, ; veissier et al., ) . the presence of adult cows other than the dam do not help calves to get accustomed to new rearing conditions, as observed by schwartkorf-genswein et al. ( ) for calves submitted to feedlot conditions. . . . recent findings regarding contacts with other calves recent studies confirmed that calves are motivated for social contact. such a motivation was shown using operant conditioning by holm et al. ( ) ; furthermore calves that are housed individually engage in more contacts with their neighbours than calves housed in pairs (raussi et al., ) . the presence of a companion can reduce emotional responses of calves. this, for instance, is the case when group housed calves are exposed to a novel situation like a novel object (boivin et al., ) , a novel arena (jensen et al., ) , a sudden event (veissier et al., ) , or a lorry (lensink et al., ) . humans are not a good substitute for social contacts. individually housed calves interact more with their neighbours compared with pair-housed calves, even when they receive additional contacts from the stockperson (e.g. stroking, letting suck fingers, speaking softly) (raussi et al., ) . (see section on humananimal relationships). . . . comparison between individual housing vs. group housing individual housing can be stressful to calves as measured by adrenal responses to acth (raussi et al., ) . group housed calves are generally more active than individually housed calves as far as gross activity is concerned (more time spent moving or eating, less time spent idling or lying) (babu et al., ; raussi et al., ) . group housing can benefit production: xiccato et al. ( ) found that calves housed in fours put on more weight than calves tethered in individual crates. however, this seems not to be the case when the calves are not tethered in individual crates (veissier et al., ) . group housed calves are less easy to handle. human contact is thus essential for them to become accustomed to humans and to react less to handling (lensink et al., ; mogensen et al., ) . group housing can help calves acquiring social skills (boe and faerevik, ) . some experience of mixing is of particular importance: calves that have been reared for a while in a group dominate calves that have always been in individual crates (veissier et al., ) . by contrast, it is not clear whether repeated mixing would be beneficial or harmful to calves veissier et al., ) . recent research (e.g. svenson et al., and svensson and liberg, ) suggests that transfer from individual pen to group-housing during the second week of life is disadvantageous for health reasons (see chapter ) and, that a delay in mixing until the calf is weeks old may be preferable. additional research seems necessary to establish what mixing age would be preferable from a health and welfare perspective. . . temperature, ventilation and air hygiene the importance of the aerial environment inside a calf house for the health status of the animals was stressed in the report, and it still seems to be one of the major factors which cause morbidity and mortality (svensson et al., ) . bioaerosols (micro-organisms, dust), low air temperatures together with high air humidity, gases such as ammonia, draught, insufficient air space and poor ventilation form a complex environmental situation which can be detrimental particularly for the respiratory health of young calves (lundborg et al. ; svc, ) . . . . temperature and relative humidity a healthy calf consuming a sufficient amount of feed has a wide zone of thermal neutrality. there is no difference in the performance of healthy, normal eating calves at temperatures ranging between ºc and ºc provided it is dry and not exposed to draughts. above ºc conditions in confined calf houses can start to become uncomfortable. moran ( ) suggests that the ideal temperature and relative humidity for calves are ºc and %, respectively. however, there is a large number of influencing factors to consider which can alter the situation for a calf substantially. lower critical temperatures for a calf in calm air and full feed are different whether it stands (- ºc) or is lying on dry concrete (- ºc) or on dry straw (- ºc) (thickett et al. ). the younger the animal the higher is its demand for the thermal environment. by week of age, the lower critical temperature in still air is approximately ºc (webster, ) . this temperature can be significantly changed by draught, wet coat and feeding level. young calves start to shiver at ºc when they are exposed to draught even if their coat is dry and they are fed sufficient feed. when fed on maintenance only level shivering starts at ºc. if their coat is wet and they are exposed to draught shivering starts at ºc when on full feed and ºc when fed on a low level (moran, ) . no signs of shivering are observed at ºc when the coat is dry with no draught and feeding is at a normal level. cold stress in calves can be prevented by providing dry lying areas, appropriate feeding and draught free ventilation. dry bedding such as straw significantly improves thermal comfort for the lying calf. in summer situations reduced feeding (but with sufficient water supply!) or feeding calves in the cooler evening or at a reduced animal density per pen and increased ventilation rates can help to lower heat stress. heat stress can also be reduced through constructing sheds with insulated roofs and well ventilated walls. calf houses with a solid wall construction and a high capacity to store energy combined with an efficient ventilation system can also contribute to create comfortable environmental temperatures for young calves kept indoors all year (din , ) . the preferred environmental temperature for calves is not fixed, it largely depends on management and other environmental factors such as wind speed and humidity of the air. the generally accepted range of relative humidity for calf barns is between and % with an optimum around % which is not too humid to dissipate excess heat and not too dry to dry out the respiratory pathways predisposing the mucous membranes to infectious and noxious agents present in the inhaled air. air humidities of more than % can lead to condensation on the walls and ceiling increasing the risk of wetting the animals by water dripping off these surfaces. high relative humidities can also impair the insulation properties of the walls increasing heat losses. cold and humid air at high velocities can considerably increase the heat loss of animals. lundborg et al. ( ) showed that draughts greater than . m/s measured close to the animal, significantly increased the odds ratio for moderate to severe respiratory sounds. the higher the humidity the higher the risk of wet skin and cooling and shivering. high air humidity increases the probability that bacteria survive in an airborne state and are transmitted between animals in the same pen and between animal pens (wathes, ) . there are existing numerous reports from s onwards of the survival of bacteria and viruses employing simple regression models to describe the loss of viability of microbes over time. however, these models lack an insight into biophysical and biochemical mechanisms of cell and virus death. as long as we do not understand these mechanisms, the measures to reduce the air pollutants are limited to either increased ventilation or increased air space. the smaller the air space per animal the more sophisticated the ventilation system must be. the influence of air space was demonstrated by wathes ( ) showing that doubling of the air space in a calf barn from to m³ per calf had the same effect on the concentration of airborne bacteria as a six fold increase in ventilation rate (air exchange rate). an air space of to m³ per calf was recommended by hilliger ( ) from experience. . . . air quality aerial pollutants in confined animal houses are widely recognised as detrimental for respiratory health. primary and opportunistic microbial pathogens may cause directly infectious and allergic diseases in farm animals, and chronic exposure to some types of aerial pollutants may exacerbate multi-factorial environmental diseases, such as enzootic bronchopneumonia. the factors can be inadequate environmental conditions, e.g. too low temperatures, high ammonia concentrations and poor ventilation resulting in low air quality. poor air and surface hygiene in calf buildings are nearly always associated with intensive systems of husbandry, poor standards of management and high stocking densities (wathes ) . the most common aerial pollutants in calf housing are summarised in table . . gases such as ammonia (nh ), hydrogen sulphide (h s), carbon dioxide (co ), and more than hundred trace gases form an airborne mixture of bioaerosols composed of about % organic compounds and can also contain endotoxin, antibiotic residues and further trace components. significantly high amounts of endotoxins were found in calf house air while bacteria and dust are relatively low compared with pig and poultry houses and suggest that a high number of gram-negative bacteria are present in the air. the average concentration of ng/m³ endotoxin given in table . represents about eu (endotoxin units) according to the new nomenclature. it seems rather high in comparison to a formerly proposed occupational threshold of eu for humans at the work place (rylander and jacobs, ) . it can be assumed that high endotoxin concentrations in calf house air may substantially contribute together with the other bioaerosol compounds and the physical environment to the high level of respiratory disorders in young calves up to days (assie et al., ) . in general, there is little detailed knowledge on the specific composition of bioaerosols in calf keeping systems and which factors cause respiratory diseases. assie et al. ( ) found e.g. a tendency to higher repiratory disorders in non-weaned calves reared in loose-housing yards compared with tied-cow stalls. the highest incidence rates of cases were observed between november and january, while daily meteorological conditions obviously did not influence incidence rates. one of the most detrimental gases in calf barns is ammonia which is formed by bacterial degradation of nitrogen containing compounds in urine and faeces. it is the most widespread naturally occurring alkaline gas in the atmosphere and a strong irritant in animals and humans. concentrations in the air of more than ppm can impair the proper functioning of the mucous membranes of the respiratory tract and predispose to infection. in a recent study lundborg et al. ( ) found that the risk for respiratory disease was significantly associated with an ammonia concentration below ppm (or: . ; p< . ). high concentrations of hydrogen suphide can be released in high amounts from stored liquid calf manure when it is stirred up before removal from the slurry pit of the barn. concentrations of about ppm are acute fatal. the composition of the inhalable and respirable particles in animal houses is associated with compounds such as dried dung and urine, skin dander and undigested feed. the majority of bacteria found in shed airspace have been identified as gram-positive organisms, with staphylococci spp. predominating (cargill et al., ) . a survey by heinrichs et al. ( ) showed the importance of good ventilation which removes dust and other respirable particles as well as noxious gases and is essential for calf health. adequate ventilation is seen as vital to help to reduce the incidence of respiratory disease. air inlets should be above calf height and the outlet at least . m above the inlet (howard, ) . however, heating and ventilation in combination with an air filtration system significantly improved the environment in a calf house but did not completely eliminate pneumonia (bantle et al., ) . this may have to do with other factors such as ambient temperature. in a recent study by reinhold and elmer ( ) some compromise in lung function (compared with controls) was seen in calves exposed to an ambient temperature of ºc. . . . . light in the past, veal calves were often kept in dark to reduce muscle activity but the requirements for light have increased over the last years from to lux (bogner and grauvogel, ) , to over lux (irps, ) to lux for at least h and according to daylight circadian rhythm. (tierschutz-nutztierhaltungsverordnung germany, ) . there is wide agreement that calves need light for orientation in their boxes or pens and for social contact. a precise threshold has not been determined. there is a need for air movement around calves to supply fresh air and to remove excessive heat, moisture and air pollutants (gases, dust, microorganisms). good ventilation systems provide this exchange. however, high air speeds close to the animals can lead to draughts and should be avoided. draughts happen when part of an animal is hit by an air stream with a higher velocity than the ambient air movement and which has a substantially lower temperature than the surrounding air, causing a feeling of cold and physiological reaction in that particular part of the body. draught can lead to poor welfare and disease when it continues and the animal cannot escape, e.g. when it is tethered. it is generally recommended that wind speeds around animals should be between . m/s in winter (least value) and about . m/s in summer. these values strongly depend on relative humidity and temperature of the air and whether the skin of the animals is dry or wet, full fleece or shorn. in confined buildings this complex relationship between the various factors is strongly influenced by the ventilation system and the ventilation rate which is necessary for the number of animals kept in the animal house. it seem useful to develop a more comprehensive model for the interaction of the different air quality compounds and the air exchange rate to improve our understanding of the welfare and health impacts arising from the air environment. . . . ventilation ventilation plays an essential role in improving air quality in calf barns. this applies to free ventilated and forced ventilated houses. calculations of ventilation rates are usually based on heat removal in summer and moisture removal in winter, and give some guideline temperatures and humidities of the air which should not be exceeded (e.g. cigr, ; din , ) . ventilation rates in calf barns can only be calculated satisfactorily for confined buildings. minimum ventilation rates around m³ per kg live weight should be sufficient to keep the air quality within acceptable limits if the air distribution system ensures an even air exchange in all parts of the building. such guidelines (cigr) and norms (din , ) cannot guarantee healthy calves but they can substantially help in designing confined calf houses. it seems useful to standardise the air quality and ventilation requirements for confined calf houses in europe in order to reduce respiratory disorders, suffering of the animals and economic losses. the report highlights two aspects of human-animal relationships: the skills and motivations of caretakers to raise healthy calves, which are of particular importance for indoor calves and in large groups and are linked to the health status of calves; the physical contacts between caretakers and calves to improve subsequent reactions of calves to humans. it recommends careful monitoring (by the same person throughout rearing) and careful handling to habituate calves to human contacts. recent studies confirmed that the stockpersons have a great impact on both the productivity (e.g. growth) and the welfare of farm animals (stress responses, fear reactions during handling) (boivin et al., ; reviewed by hemsworth and coleman, ) . the effect of stockmanship is two-fold: good stockmanship leads to healthy animals and less stressful human-animal interactions. stockman skills are associated with positive attitudes towards work and towards animals. in calf production, a better health status is observed on farms where the caretaker (also the owner) believes that calves are sensitive animals and he/she has a positive attitude towards farming tasks in calf production (lensink et al., b) contacts given by stockpersons to animals depend also on human attitudes. stockmen that are positive towards gentle contacts with calves (e.g. stroking, talking) are more likely to provide calves with such contacts (lensink et al., a) . it is not only the duration of contact but also its nature that plays a role. gentle contacts (e.g. stroking, talking, letting a calf suck fingers, offering food) lead to calves approaching humans as they have less fear of handling (boivin et al., ; jago et al., ; lensink et al., b) . whereas rough contacts (e.g. hitting with a stick, use of nose tongs or an electric prod) lead to fear reactions in presence of humans (rushen et al., ) . the electric prod seems particularly stressful to calves (croney et al., ) and noises (metal clanging, shouts by humans) will also increase stress during handling (waynert et al., ) . during transport to slaughter, less fear responses to handling (e.g. due to regular previous experience of gentle contact) not only improves the welfare of calves but also improves meat quality (lower ph and lighter colour) (lensink et al., c; lensink et al., a) . cattle are able to distinguish between familiar and unfamiliar persons (rybarczyk et al., ; taylor and davis, ) . among familiar persons, they distinguish between those who have been rough with them and those who have been gentle (e.g. stroking, brushing, and offering food) (de passillé et al., ) . compared with individually housed calves, calves housed in groups tend not to approach humans and are more difficult to handle (lensink et al., b) . the presence of the dam can lower the effectiveness of gentle contacts with animals (boivin et al., ) . contact early after birth can be more effective that contact provided later; however, regular contact is necessary to maintain a lower level of fear responses to humans (boivin et al., ) . the report recommended: to dehorn calves between - weeks by cauterisation with adequate anaesthesia and analgesia (no details given) to castrate calves at months with adequate anaesthesia and analgesia (no details given) . . . dehorning dehorning means the removal of horns while disbudding (on young animals) corresponds to the removal of horn buds. disbudding can be performed by cautery, or by rubbing or covering the horn buds with a chemical (naoh, koh or colloidon), or by amputation with a specifically designed sharp tool, a scoop. recent publications confirmed that disbudding and dehorning are painful to cattle (stafford and mellor, ) . the existence of pain is deduced from observations of an increase in blood cortisol for several hours after dehorning and from specific pain related behaviour: head shaking, ear flicking (faulkner and weary, ) disbudding without anaesthesia or analgesia is painful to calves, even when young, and dehorning with a wire-saw is painful to cows even if anaesthesia is carried out (taschke and folsch, ) . disbudding by cautery (hot iron, electric tool) and chemical disbudding (naoh) are less painful than disbudding with a scoop (stilwell g. et al., a; stilwell g. et al., b; sylvester et al., ) . local anesthesia ( - ml lidocaine or lignocaine % around the corneal nerve - min before disbudding) can abolish the pain that immediately follows cautery and largely diminishes the pain caused by disbudding by other methods; the effects last for the few hours and when the nerve block has lost its effect, pain ensues stilwell et al., b; sutherland et al., b) . local anesthesia plus analgesia with a non-steroidal anti-inflammatory drug (nsaid)(e.g. ml flumixin meglubine or - . mg/kg ketoprofen ( %) min before disbudding) abolish pain caused by cautery but only reduces it in the case of disbudding with a scoop (unless it is followed by cautery) (sylvester et al., ; faulkner and weary, ; sutherland et al., a; stilwell et al., b) . in their review, stafford and mellor ( ) concluded that cautery is the less painful method for disbudding and that optimal pain relief is obtained with xylazine sedation, local anaesthesia and analgesia with a non-steroidal antiinflammatory drug. the report recommend that when cattle are to be castrated this should be done at around months of age and under appropriate anaesthesia and analgesia. methods to castrate cattle are: clamping (generally with a burdizzo clamp), constriction of the blood supply with a rubber ring, and surgical removal (cutting of the scrotum then traction on the testes and spermatic cords or cutting across the spermatic cross). calves are castrated as early as week up to over months (see review of practices used in uk by kent et al., ) . castration is painful whatever the method used and whatever the age of the calf (molony et al., ; robertson et al., ) . acute pain is deduced from the observation of increases in blood cortisol and abnormal postures (immobility), and behaviours such as foot stamping and kicking. chronic pain is deduced from the observation of activities targeting at the site of castration (e.g. licking, head turning, alternate lifting of the hind legs, and slow movements of the tail) as well as abnormal standing. burdizzo clamping and surgery induce acute pain for at least h (molony et al., ; obritzhauser et al., ; robertson et al., ) . burdizzo is less painful than surgery but may also cause pain for longer (at least h) due to scrotal inflammation (stilwell, pers. comm.) . castration with a rubber ring causes both acute and chronic pain for at least . mo (molony et al., ) castration is less painful for wk old calves than for - wk old calves (robertson et al., ) and it is less painful at . months than at older ages (ting et al., ) . local anesthesia ( ml lignocaine % into each testicle through the distal pole) abolishes the acute pain associated with castration by a ring or a band (stafford k.j. et al., ) . it reduces but does not abolish acute pain associated with castration by surgery or clamping (fisher et al., ; stafford et al., ) . analgesia with a nsaid drug (e.g. ketoprofen % mg/kg body weight) reduces the pain associated with clamping (ting et al., ) . some analgesics (e.g. caprofen, , mg/kg body weight) are effective for longer than h and are thus more likely to provide more effective pain relief (stilwell, comm. pers.) . local anaesthesia plus analgesia appears to eliminate the acute pain due to castration by surgery or clamping . the most important diseases in young calves are diarrhoea and respiratory disease (olsson et al., ; sivula et al., ; virtala et al., a , donovan et al., lundborg, ) . a prospective study was carried out on randomly selected beef herds in the midi-pyrenees region in france (bendali et al., ) . the objective was to describe diarrhoea and mortality in beef calves from birth to days of age. calves ( , ) were followed from december to april , and a total of visits allowed records of herd management practices, individual data and environmental conditions to be collected. the incidence rate for diarrhoea during the neonatal period was . %, and varied markedly between herds. eighteen herds did not suffer from diarrhoea, while five herds had an incidence of more than %. results indicate that % of diarrhoea appears during the first week and only % after the second week of life. the greatest risk of diarrhoea for a calf was during the first and second weeks of life ( . and . times, respectively). the month of birth was also significantly associated with morbility, the highest incidence was observed in december and march ( . % and . %, respectively) . the global mortality rate was . % and was two-times higher in december than in other months. forty per cent of herds did not exhibit mortality, and % had mortality rates greater than %. in a study of calf health in cow-calf herds in switzerland, busato et al. ( ) found that of calves included in the study % of the calves had been treated by a veterinarian. of those, % of the treatments were given because of diarrhoea and % because of respiratory disease. another swiss study (frei et al., ) showed that in swiss dairy herds, the incidence density (id) per animal-years of diarrhoea, omphalitis (infection of the navel), respiratory diseases and other diseases were . , . , . and . respectively. in a study of nine herds and calves with swedish red and whites, swedish holsteins and some crossbreeds the effect of group size on health was studied (svensson and liberg, ) . after transfer to group pens (at - days of age) . % of the calves had diarrhoea, . % had omphalophlebitis/umbilical abscess, . % had a clinical respiratory-tract disease and . % had weak calf syndrome. of all calves, in . % there was associated general condition impairment. in . % of the diarrhoea cases antibiotics were used as treatment and of the clinical respiratory-tract cases % were treated with antibiotics. several factors have been associated with an increased risk of infectious disease during the first days of life, particularly factors affecting serum immunoglobulin concentration. in a study of dairy herds in south-west sweden, svensson et al. ( ) clinically monitored the health of heifer calves from birth until days of age. % of the calves developed one or more diseases during this period. most of the diarrhoea cases were mild ( %) whereas of the cases of respiratory disease % were severe. the total morbidity was . cases per calf-month at risk and the incidence rates of arthritis, diarrhoea, omphalophlebitis, respiratory disease and ringworm were . , . , . , . and . cases per calf-month at risk respectively. odds ratios were calculated for severe diarrhoea in calves born in the summer (or: . ) and receiving colostrums through suckling instead of a bucket or nipple (or: . ). it has been shown that calves left with their mothers have a delayed/longer time to ingest colostrum and often fail to ingest adequate volumes (rajala and castrén, ) . svensson and liberg ( ) found that the health status of the mother cow - days before calving, length of dry period, retained placenta and somatic cell count were predisposing risk factors for respiratory disease in the calf. svensson et al. ( ) were also able to demonstrate that the odds ratios for respiratory disease and increased respiratory sounds were increased in calves housed in large group pens with an automatic milk-feeding system (or: . and . ). similar results have been reported by maatje et al. ( ) and plath ( ) . there was a decreased odds ratio for respiratory disease if calving was supervised (or: . ) . if birth was taking place in individual maternity pens or in tie stalls instead of in cubicle or group maternity pen, the odds ratio for increased respiratory sounds was . or . respectively. % of the diarrhoea cases were treated with antibiotics whereas % of the respiratory cases were treated using antibiotics. in another study of nine farms, svensson and liberg ( ) found that in pens for six to nine calves there was a significantly reduced risk of clinical respiratory tract disease (or: . - . ) compared with pens with - calves and there was also an association with the age at transfer to the group pen. the risk of diarrhoea was not affected by housing the calves in differently sized groups. however, calves housed in large sized groups grew significantly less quickly (approximately g/day) than calves housed in groups of six to nine. serological responses to respiratory viruses (e.g. bovine respiratory syncytical virus, parainfluenza virus, corona virus and viral diarrhoea virus) showing that animals within a herd are usually either all seropositive or all seronegative, indicate that infections spread to all calves in the herd when introduced or activated (hägglund et al., in press) and hence that aerosol is an important means for the spread of viruses. however, an infected animal is not equivalent to a diseased animal. it has been shown that calves housed in adjacent pens can maintain quite different levels of disease. svensson and liberg ( ) reported that calves in a group of had a significantly higher incidence of clinical respiratory disease that calves in an adjacent pen kept in groups of . engelbrecht ( ) reported calves transferred to group pens in a batchwise manner had significantly higher prevalence of diarrhoea and respiratory disease than calves in adjacent pens that were transferred to and from the group pen continuously. in both studies calves had no direct contact with calves in adjacent pens. these results indicate an important role of direct contact for the transmission of respiratory disease and hence the importance in disease control of decreasing direct contact between calves within the same building by means of solid walls. svensson and liberg ( ) also reported that the age at transfer from single pens to group pens was associated with the risk of respiratory disease, indicating that delaying transfer to after two weeks of age might be preferable for health reasons. enteritis is the most common disease in calves less than a month old (virtala et al., b; wells et al., ; radositis et al., ) . diarrhoea is caused by dietary factors or caused by infections due to viruses, bacteria or parasites. routines in distributing colostrum to the calf are crucial for transferring immunoglobins to the calf and to obtain a good health. (rajala and castrén, ; liberg and carlsson, ) . enteritis is clinically recognized by the observation of faeces with a looser consistency than normal. colour as well as smell of the faeces might be affected. diseased animals exhibit fever and may be inactive usually as a result of dehydration and possibly acidosis (radositis et al., ) . usually it is not possible to differentiate between different agents causing the diarrhoea by clinical findings. rotavirus is worldwide a major cause of diarrhoea and it is an often detected agent among young calves with enteritis (e.g. björkman et al., ) . rotavirus affects calves usually between and weeks of age and the diarrhoea can vary from very mild to lethal (de leeuw et al., ) . the virus is excreted through faeces of infected animals and is very resistant for several months, that is why cleaning of pens is necessary to break the infectious path (saif and theil, ) . bovine coronavirus is most commonly seen in calves at about week of age (fenner et al., ) . escherichia coli k + may cause diarrhoea in young calves although it is a part of the normal intestinal flora. poor routines for transferring colostrum to the calf, stress etc. might trigger a diarrhoea outbreak (wray and thomlinson, ) . severity of the disease may vary but with a high proportion of mortality (radositis et al., ) . only amoxicillin is recommended for the treatment of diarrhoea caused by e. coli bacteria associated by systemic illness. in calves with diarrhoea and no systemic illness (normal appetite for milk, no fever), the health of the calves should be monitored carefully and no antibiotics should be administered (constable, ) . bovine viral diarrhoea may occur at any age. the infection causes an immunosuppression in infected animals which may lead to infections with other intestinal or respiratory pathogens (elvander et al., ; de verdier klingenberg, ) and it may increase the mortality rate in the herd (ersböll et al., ) . salmonella spp, mainly s dublin and s typhimurium can affect calves usually between and weeks of age. the pathogen is introduced into the herd via infected feed, water, pastures, cattle or humans or via other animals entering the herd. calves are infected orally and clinical signs are fever and bloody diarrhoea (carter and chengappa, ) . clostridial infections in the gastrointestinal tract are sometimes a problem in calves. usually, the calf, less than days of age, develops haemorrhagic, necrotic enteritis and enterotoxemia, often associated with clinical abdominal pain. affected calves exhibit tympany, hemorrhagic abomasitis and ulcerations in abomasum (songer and miskimins, ) . as yet, relatively little is known about the etiology aside of the participation of c. perfringens type a. overfeeding or feeding which decreases gut motility is suggested to contribute to the occurrence of the disease (songer and miskimins, ) eimeria spp. are frequently present in cattle in europe (bürger, ) . predominantly e. ellipsoidalis was found in housed calves in east germany (hiepe et al., ) and the distribution may differ from country to country. svensson ( ) found a predominance of e. alabamensis in swedish dairy calves. clinically, signs are rarely seen but diarrhoea can occur usually as a result of exposure at the first grazing season in areas contaminated with oocysts (svensson, ) . there is evidence that infection rates have increased since the prohibition of tethering (berthold, pers. com.). emeria bovis and emeria zuernii are other intracellular protozoan parasites belonging to the same group and with a worldwide distribution (urquhart et al., ) . it is often seen in calves between - months of age (holliman, ) . the disease is triggered by stress such as very cold or hot climate (urquhart et al., ) . cryptosporidial infection in calves less than days old is significantly associated with the risk of infection in the dairy herd. the risk increases when animals are grouped together and when hygiene and management practices are deficient (attwill et al., ; mohammed et al., ) . factors associated with a decreased risk of infection in preweaning calves were shown to be use of ventilation in calf rearing areas, daily addition of bedding, feeding of milk replacer, daily disposal and cleaning of bedding and use of antibiotics. in addition, postweaning moving of animals was also associated with a decreased risk of infection with c. parvum (mohammed et al., ) . perryman et al. ( ) showed that with appropriate supply of immune colostrum, diarrhoea can be prevented. two species are distinguished: c. parvum and c. andersoni, although only c. parvum has been shown to be associated with diarrhoea . cryptosporidia parvum is an intracellular protozoan parasite belonging to coccidiae. in the uk c. parvum has been considered to be one of the most common agents in neonatal diarrhoea in calves (reynolds et al., ) . in denmark it was found mixed with other enteropathogens in % of diseased calves (krogh and henriksen, ) . in two recent swedish studies it was found in % and % respectively of calves with diarrhoea (de verdier klingenberg and svensson, ; björkman et al., ) . the most common form of respiratory disease affecting young calves is enzootic pneumonia (ames, ; radositis et al., ) . it is considered to be a multifactorial disease with causative agents, individuals and environmental factors as important components (ames, ) . enzootic pneumonia usually affects calves between and months of age (radositis et al., ) . the signs usually found are fever, nasal discharge, coughing and increased respiratory sounds when lung auscultation is performed. secondary bacterial infections may occur which might increase the fever. diagnosis of etiological factors may be achieved from serological examinations, viral examinations from nasal discharge or at autopsy. bovine respiratory syncytical virus (brsv) is a worldwide present agent with seasonal peaks during autumn and winter (baker and frey, ) . the virus is thought to be transmitted from infected animals, by transmission of humans or by airborne transmission (van der pohl et al., ; elvander, ) . morbidity can be high but mortality is usually low . another virus with a milder course of disease is para-influenza- virus but the virus can cause immunosuppression predisposing to secondary bacterial infections (adair et al., ) . the most common bacterial pathogens in calves with respiratory disease are pasteurella multocida and manheimia hemolytica (mosier, ; bengtsson and viring, ) . these agents are usually found in the bovine nasopharynx and may, as a result of viral disease proliferate and colonise the lungs of the calf (kiorpes et al., ) . haemophilus somnus was shown to be commonly present in danish calves (tegtmeier et al., ) where no such agents were found in swedish calves (bengtsson and viring, ) . arcanobacterium pyogenes and staphylococcus aureus (carter and chengappa, ) as well as mycoplasma spp. (ames, ) are other agents found in immune depressed calves with other infections. infections may occur in the umbilical cord (radositis et al., ) of newborn calves. various bacteria are found and through a bacteraemia infection may spread to to joints, meninges and internal organs (radositis et al., ) . omphalitis is painful in response to palpation of the umbilicus. arthritis is often secondary to an umbilical infection and usually affects the calf during its first month resulting in warm and swollen joints, fever and lameness (radositis et al., ) . the effect of environmental factors on the risk of diarrhoea and respiratory disease was studied by lundborg et al. ( ) in the same farms and calves as previously reported by svensson et al. ( ) . they found that the placing of calf pens along an outer wall was associated with a significantly higher risk of diarrhoea (or: . ). an ammonia level below ppm was significantly associated with the risk of respiratory disease (or: . ) but variations of ammonia levels were low, while the odds ratio for increased respiratory sounds was associated with a bvdv infection in the herd (or: . ) and draught (or: . ). absence of draught was associated with the risk for infectious diseases other than diarrhoea and respiratory disease (or: . ), a finding which could not be explained by the authors. an increased calf mortality rate in herds with a bvdv infection has also been reported by ersböll et al. ( ) and the eradication of bvdv infection in a dairy herd has been demonstrated to decrease the incidence of calf diarrhoea (de verdier klingenberg et al., ) . typically, clinical experience is that the incidence and prevalence of infectious respiratory disease is much higher in rearing systems where the calves have been bought and transported from several farms where they were born to a specific rearing farm than if they are reared on the farm they were born on. calves reared indoors commonly develop complex respiratory diseases. bergmann ( ) reported that % of calves of a large fattening unit with several thousand calves suffered from bronchopneumonia within the first six month of their life. similar figures were reported from herrmann ( ) with a prevalence of to %, lämke et al. ( ) % and busato et al. ( ) %. the disease seems to be continuously present and does not come or go in form of isolated outbreaks. therefore kielstein et al. ( ) called it enzootic pneumonia. it is a typical multifactorial disease caused by a variety of different types of micro-organisms which are always present but becoming a nuisance only when additional factors contribute (grunert, ) . the most prominent reasons for losses of calves in the first weeks of life are respiratory and digestive disorders (katikaridis ; girnus, ) . losses can reach to per cent in the first six months of rearing (berchtold et al. and others). estimations show that the financial losses are reaching million €/year in germany (biewer, ) . this sum does not cover the costs of veterinary treatment and reduced growth rates of the calves. there are several epidemiological studies on the different diseases in calves in the first couple of weeks of life (katikaridis, ; biewer, ; girnus , svenson et al., . heinrichs ( ) reported that % to % of , fallen calves coming for post-mortem dissection showed digestive disorders. calves weeks of age died predominantly of respiratory diseases ( % to %). an investigation of , calves less than months shows that enzootic bronchopneumonia can already start with the age of weeks (buhr, ) . the calves were not older than months. % displayed abomasum enteritis. % of the animals suffered from pneumo-abomasal enteritis. only . % of the fallen animals suffered from a distinct pneumonia. an epidemiological survey of calf losses in free range and suckling cow herds showed that the percentage of calf losses is increased with herd size. % of herds with less than suckling cows had a calf mortality of less than %. in herds with more than cows, these were % of all investigated farms, calf losses were higher than % (laiblin and metzner, ) . main disorders were again diseases of the digestive tract ( %) and respiratory tract ( %). the authors calculated that the disease risk for calves born from cows that were housed during the calving season was . times higher compared with cows kept the whole year on free-range. the epidemiological data from the vast majority of investigations suggest considerable differences in morbidity and mortality of calves among different farms. this implies that the management and housing conditions greatly influence heath, welfare and survival of calves in the first months of their life. the situation was not substantially improved by vaccination of cows against a cocktail of infectious agents causing diarrhoea. there are no experimental studies available to indicate whether or not there is any advantage to calf welfare of preventing individuals in separate pens from social contact as opposed to a disadvantage to calf welfare of greater spread of disease with housing where such social contact is possible. in general, disease spread occurs in buildings with continous air space and contact is not a clearly identified factor. however, recent results indicate an important role of direct contact for the transmission of respiratory disease and hence the importance in disease control of decreasing direct contact between calves within the same building by means of solid walls (see chapter / / ). antibiotic resistance although the use of antibiotics as growth promoters is being progressively restricted through eu regulations, they are still used in large quantities in calf rearing for both prophylactic and therapeutic purposes. in those instances where calves are not reared on site but transported to other locations and mixed in groups, the incidence of clinical illness is high and the use of antibiotics is frequent. in a study of antibiotic resistance, berge et al. ( ) found high levels of multiple resistance in calf commensal faecal escherichia coli both on farms with calf production and on dairy farms. the investigators found that escherichia coli from calves on dedicated calf-rearing facilities was more likely to be multiple-resistant than e. coli from dairy-reared calves (or: . ) (berge et al., a) . in her phd thesis, berge ( ) showed that both prophylactic use of antibiotics in milk replacer and individual antibiotic therapy increased the resistance of faecal e. coli in calves. e. coli isolates from calf ranches were the most resistant, with in order of decreasing levels, isolates from feedlots, dairies and beef cow-calf farms. on organic dairies fewer resistant e. coli isolates were found in comparison with conventional dairies. e. coli isolates from beef cow-calf farms were less resistant if the farms were on remote locations compared to those on locations close with dairy intense areas. the use of antibiotics to treat clinical illness will increase the welfare of the animal given that the drug has a beneficial clinical effect. however, the frequent use of antibiotics results in increasing resistance in bacteria such as e. coli and thus poses a threat to the welfare of calves in a longer perspective as well as to man (aarestrup and wegener, ) . in a clinical trial on a calf ranch in california, it was shown that the most important factor for decreasing morbidity and mortality was to ensure adequate passive transfer through colostrum (berge et al., c) . thereafter, the ability to use antibiotics for clinical treatment of disease was important to decrease morbidity and mortality. the use of antibiotics in the milk replacer had a minor protective effect on calf health. the authors concluded that in order to minimize prophylactic use of antibiotics, adequate passive transfer of colostrum needs to be assured. furthermore measures need to be taken to optimize nutrition, decrease environmental stress and pathogen load on the farms. in the same study, the antibiotic resistance patterns of the commensal faecal e. coli of calves receiving antibiotics in the milk-replacer, antibiotics for clinical disease, and no antibiotic therapy were compared (berge et al., ) . the study showed the emergence of highly multiple resistant e. coli in the calves receiving antibiotics in the milk-replacer. the commensal faecal e. coli were predominantly resistant to at least of antibiotics tested. the resistance covered the antibiotics available for clinical therapy. antibiotic treatments for clinical disease resulted in a transitory shift to more resistant faecal e. coli, but the effect was not detectable approximately days post-treatment. the effect of clinical therapy with antibiotics was similarly assessed in steers in south dakota. in a feedlot study a single dose injectable florfenicol to steers resulted in transitory shifts to increasing levels of multiple resistant e. coli in the faeces. the e. coli from the treated steers were not only more resistant to chloramphenicol (same antibiotic group as florfenicol), but were increasingly resistant to several other antibiotics in other antibiotic classes. (berge et al., b) in dairy cattle it has been estimated (kelton et al., ) that between and % of all lactations include a mastitis infection. most of these cases are treated with antibiotics. milk must be withheld from sale during the treatment and for the compulsory withdrawal period. such "waste milk" is often fed to calves as it is the most economical alternative from the farmer's perspective. earlier studies have previously given various results on how antibiotic resistance develops as a result of the use of this procedure. recently, a controlled, multiple-dose experiment by langford et al. ( ) found an increasing resistance of gut bacteria to antibiotics with increasing concentrations of penicillin in milk fed to dairy calves. in a multi-farm study in california (berge et al., ) including dairies, no association was found between increasing levels of antibiotic resistance in calf faecal e. coli and the consumption of waste milk. it should, however, be noted that mastitis in these dairies are predominantly treated with intra-mammary antibiotics (cephalosporins) and injectable antibiotics are rarely used for mastitis treatments (berge, non-published data) . the use of rearing systems for calves that increase the incidence of disease and thus the use of antibiotics for either preventive or clinical purposes should be avoided. further, there is a risk that the use of "waste milk" for calves will increase antibiotic resistance in gut bacteria in calves. . food safety aspects of calf farming foodborne hazards that can be present on calf farms include biological and chemical hazards. biological hazards associated with calf farming include following main examples: a) bacterial foodborne pathogens salmonella spp., human pathogenic vtec (hp-vtec), thermophilic campylobacter spp., and mycobacterium bovis; and b) parasitic foodborne pathogens tania saginata cysticercus and cryptosporidium/giardia. on-farm control of chemical foodborne hazards is outside the scope of this chapter and will not be considered. faecal shedding of foodborne pathogens can occur in calves and adult bovines without symptoms of disease; but the shedding pattern may differ between the two age categories. in the conventionally reared animal the intestinal tract becomes colonised from birth by combinations of bacterial species until the characteristic and complex flora in the adult animal is achieved. in the early stages of the process infections with bacterial pathogens are common. once the indigenous flora is established it resists colonization by pathogens and other 'foreign' strains by competitive exclusion . the gut flora of the bovine species changes with ruminal development and the population of faecal coliforms of the adult differ markedly, both qualitatively and quantitatively, from that of the young; particularly that of veal calves fed milk replacer (smith, some early studies (gronstol et al., a; gronstol et al., b) , based on experimental salmonella infection of calves, described virulence, spread of infection and the effects of stress on the carrier status. hinton et al. ( hinton et al. ( , a hinton et al. ( , b determined the incidence of salmonella typhimurium (dt and dt ) excretion by veal calves fed milk replacer and report that, while initially low on in-take at around days of age, its incidence rose to a peak by days. the level of salmonella contamination of the environment also affects the incidence of infection in housed animals (hinton et al., a (hinton et al., , b . provided calves are reared in separate fattening units and slaughtered on separate slaughterlines the incidence of salmonellae in calves can be maintained at a very low level (guinée et al., ) . during meat inspection clinical salmonellosis is sometimes diagnosed in a herd of veal calves; however, the prevalences are usually of the order of magnitude of per thousand and the strains isolated are generally restricted to salmonella typhimurium (occasionally) and more commonly, salmonella dublin (up to %). although salmonellae may not be isolated from faeces, significant proportions of calves slaughtered commercially ( . - . %) have contamination involving hepatic lymph nodes, liver, mesenteric lymph nodes and, because of cross contamination, they may ultimately also be isolated from the carcass surface (nazer and osborne, ; wray and sojka, ) . studies conducted in the netherlands in the late 's indicate that microorganisms may be released from lymph nodes through transport stress and may appear in the faeces. this results in young veal calves being cross-infected in transit and at markets; however, in dutch studies faecal samples from no more than . % of the animals were found to contain salmonellae on arrival at the fattening units (van klink and smulders, ) . moreover, within weeks of arrival, faeces samples become negative again (van zijderveld et al., ) . subsequent studies by the same workers (van zijderveld et al., unpublished, cited by van klink and smulders, ) indicate that faecal samples from calves which had survived clinical salmonellosis also become culture-negative, albeit only after weeks. these findings suggest that, provided stressful transport conditions are avoided and sufficient hygienic care is taken to avoid cross infection during transport to the abattoir, the extent of introduction of salmonellae to the veal slaughterline is indeed extremely low. this is substantiated by repeated failure to isolate salmonellae from carcass surfaces of veal calf populations, and from their livers and offal meats (van klink and smulders, ) . as with other bacterial foodborne pathogens, antimicrobial resistance in salmonella shed by calves represents an additional food safety risk. numerous studies have shown that use of antimicrobials in food producing animals selects for resistance in non-typhoid salmonella spp. and that such variants have been spread to humans (who, ; walker et al., ; fey et al., ) . in general, antimicrobial resistance in s. typhimurium isolates from bovines in the eu was widespread in , with highest prevalence of resistance to ampicilin, sulfonamide, tetracycline and streptomycin (efsa zoonosis report, b), but the data does not relate specifically to calves. vtec is a group of e. coli that produces one or more verocytotoxins (vt) also known as shiga toxins (stx), but not all members of this group cause foodborne disease in humans. in the opinion on verotoxigenic e. coli (vtec) in foodstuffs (scvph, c) , vtec that have been associated with causing human disease were referred to as human pathogenic vtec (hp-vtec). foods of bovine origin (e.g. beef, milk) have been implicated in a number of foodborne outbreaks caused by hp-vtec (borczyck et al., ; chapman et al., ; martin et al., ; pennington, ; scvph, a) ; these include several serotypes (e.g. o , o , o , o and o ). when adult cattle were inoculated with vtec o , they showed no outward signs of infection and the organism was cleared from the gastrointestinal tract within two weeks (wray et al., ) . the organism seems to be a constituent of their naturally-occurring microflora, and longitudinal studies show most cattle occasionally carry e. coli o in their faeces (hancock et al., ; lahti, ) . however, the prevalence of infection with hp-vtec of, and the shedding patterns in, cattle can vary due to variable factors including age, immunocompetence status, husbandry conditions, season and geographical areas. prevalence of vtec o is usually higher in younger animals (synge, ; cray and moon, ; hancock et al., ; mechie et al., ; van donkersgoed et al., ) . in calves, the prevalence of e. coli o :h can range from zero to . % prior to weaning, and often increases after weaning (bonardi et al., ; laegried et al., ) . calves normally show no, or little, sign of infection, perhaps only some excess faecal mucus (myers et al., ; synge and hopkins, ; brown et al., ; richards et al., ; wray et al., ) ; the shedding rate can fall rapidly in the first two weeks after inoculation and continue intermittently for several weeks. in the first three months of life on contaminated farms, faecal prevalence can increase from around % to %, possibly due to the decline in maternal immunity (busato et al., ) . fasting showed little effect on the carriage and excretion of e. coli o in calves (harmon et al., ) . less information is available on non-o hp-vtec in calves that have potential to cause enterohaemorrhagic disease in humans, so establishing indicators for virulence and clarifying the epidemiology of such serotypes is needed. thermophilic campylobacter spp. according to the biohazard scientific report on campylobacter in animals and foodstuffs (efsa, a) , the most important species of campylobacter are the thermophilic species c. jejuni ssp. jejuni, c. coli and c. lari; other species which are known to cause human illness are c. upsaliensis, c. fetus ssp. fetus and c. jejuni ssp. doylii. the most common species recovered in human disease is c. jejuni. campylobacter spp. can be found throughout the intestine of cattle, but the level of the organism in the rumen is significantly lower than that found in the small intestine and in faeces (stanley et al., ) . the class of cattle also has an effect on the level of campylobacter spp. found in the faeces; faeces may contain around cfu/g in calves, around cfu/g in beef cattle and around cfu/g in adult dairy cattle. campylobacter spp. are more often found in the faeces and intestine than in the rumen, while campylobacter jejuni prevalence is much less than that of campylobacter hyointestinalis (ataby and corry, ; grau, ) . campylobacter jejuni has been found in calves in % of beef farms, campylobacter coli in % and campylobacter hyointestinalis in % (busato et al., ) . within the herds, zero to over % of the calves may be excreting campylobacter spp.; % of the calves may be positive for campylobacter spp. in herds without evidence of diarrhoeal disease (myers et al., ) . in this study, % of the isolates were c. jejuni. in a study of veal calves at slaughter, c. jejuni was found in % of calf rumen samples (in low numbers; < /g) and in % of calf faecal samples (grau, ) , whilst c. hyointestinalis was found in % of calf rumen samples (in numbers > /g) and in % of faecal samples. the coats of the calves were also contaminated, as % were positive for c. jejuni and % for c. hyointestinalis. as with other bacterial foodborne pathogens, antimicrobial resistance in thermophilic campylobacter spp. shed by calves represents an additional food safety risk. in , although the total number of isolates was relatively small and the data are not related specifically to calves, some eu member states reported relatively high prevalence of resistance to quinolones, fluoroquinolones and tetracyclines in campylobacter spp. including c. jejuni from bovines (efsa zoonosis report, ) which can be an emerging public health concern. . . mycobacterium bovis efsa a, opinion on the risk assessment of a revised inspection of slaughter animals in areas with low prevalence of cysticercus); therefore, the parasite will not be further considered here. cryptosporidium parvum and giardia duodenalis are protozoan parasites that have caused disease in humans primarily via contaminated water or foods (e.g. salads), but also via chicken salad and milk drinks. high prevalences of cryptosporidium and giardia in veal calves (the age group - weeks) have been reported (van der giessen et al., ) . however, in this study, all isolates from the former group belonged to the pathogenic cryptosporidium parvum genotype , whilst only few isolates from the latter group showed similarities with giardia isolates from humans. other authors also reported presence of these protozoan parasites, cryptosporidium (de visser et al., ) and giardia (trullard ; mcdonough et al., ) in veal calves. risk evaluation and principles of food safety assurance at calf farm level the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens at calf farms depend on a large number of risk factors that are inherently variable even at single-farm level. the complexity of the problem is further exacerbated by the existence of a number of different farming systems for veal calves in the eu; and even within each of the main farming categories (e.g. intensive vs extensive) a large number of "epidemiological" subcategories exists that differ with respect to one or more risk factors. therefore, presently, both knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative categorization of different types of calf farms and/or their quantitative comparison/ranking with respect to main foodborne pathogens. nevertheless, the role of some main factors contributing to an increase in prevalence and/or in levels of foodborne pathogens in food animals on farms (including calves) are reasonably well understood, as are the generic principles of their control. they are indicated in a condensed form in table . . it is logical that calves from farming systems in which fewer of the contributing factors exist and where the controls are more complete/efficient will represent lower foodborne pathogen-risk than calves from farming systems having opposite contributing factors-controls situation. therefore, future food safety risk categorization of individual farming system, or related between-systems comparisons, would be dependent upon obtaining and analysing quantitative information on: a) status/levels of contributing factors; b) status/levels of hazards of main concern; and c) existence and effectiveness of their controls. increased "importation" and spread of pathogens via animals "asymptomatic excretors" animal supply only from known, epidemiologically "equivalent" sources; "all in-all out" system presence of animal diseases spread of zoonotic agents in calves global disease control programmes; heard health plans *good farming practice-good hygiene practice . risk assessment . . introduction to risk assessment approach when the ahaw panel of efsa was confronted with the tasks of updating the report (svc, ) , the working group members were asked to make it on the basis of a risk assessment , and particularly to consider the possible effects on the calf and, where relevant, on food safety. it appeared entirely feasible for the working group members to follow this part of a risk analysis approach where risks were defined as those concerning the welfare of calves. the risk of concern in this report is that the welfare of the calves will be poor. this may involve an increased risk of injury, of disease, of negative feelings or of failure to cope. the time span of such poor welfare might vary from short to long and severity can vary from low to high. a member experienced in risk assessment procedures was included in the working group from the start. initially, the procedure adopted for the risk assessment was identified and presented to the participants of the whole working group. when identifying the hazards, it has been assumed that the managers of the farm and animal keepers have a basic knowledge, that they have undergone training, that they are aware that the particular constraints on the farm do not hamper their work (e.g. lack of facilities on a farm). however, it is pointed out that under practical conditions hazards may interact, e.g. inadequate air flow may interact with poor air quality, inadequate clinical health monitoring may interact with inadequate haemoglobin monitoring, etc. the identification of hazards and consequential risks to welfare, as well as the risk assessment approach, were agreed by the working group. steps of the risk assessment a. multidisciplinary approach the expert working group it was selected on the basis of having expertise in animal science, ethology, veterinary medicine, risk assessment and food safety. b. listing of potential hazards, hazard characterization and exposure assessment the first step was to describe the needs of calves (see chapter and listed below). then, hazards that might compromise those needs were identified (table . ) and related to each specific need (table . ). the hazards were characterized in relation to the impact they have on the animal. the exposure to the hazard might vary between different rearing systems. for this purpose a set of different rearing categories was developed (table . ) as well as scoring categories for the hazard characterisation, exposure assessment and risk evaluation (table . ). b.a. small groups, bucket fed (not suckling) + solid foods, weaned at - months b.b. groups with an automatic feeding system (not suckling) + solid foods, weaned at - months b.c. feed lots (high density groups within outside pens) b.d. hutches outside, bucket fed (not suckling) + solid foods, weaned at - months beef calves c.a. suckler calves in small groups kept inside, led twice a day to the dam for suckling up to - months the hazards were identified and characterized, as well as, an estimate of the probable exposure. however, to ensure that these estimates of exposure correspond with current practice in various european calf production systems, a group of veterinarians, experts in clinical practice in calf production, named the "consultation group", was identified. criteria for invitation were the following; predominantly engaged in clinical practice; extensive clinical experience in calf medicine; and covering various geographical areas where calf production is significant in the eu. another important criterion was that the consultant should not be affiliated with the calf production industry. in total veterinarians accepted an invitation to assist in the exposure assessment. the experience of the individuals covered the various husbandry systems and important veal producing countries in europe. the consultation group prompted that for the exposure assessment, a quintile distribution (i.e. five classes of % increments) of exposure classes be adopted. in some instances the estimates of the wg and the consultation group on exposure did not agree in which case the opinion of the consultation group was interpreted to represent the factual situation. in other instances the exposure could not be estimated due to lack of data, in which cases the risks were labelled "uncertain". for hazards characterized as moderately to very serious, this uncertainty is highlighted. table in annex show the agreed scoring between the wg members and the "consultation group" for the hazard characterisation, exposure assessment and risk evaluation. inadequate colostrum intake -quantity b) inadequate colostrum intake -quality c) inadequate colostrum intake -duration ) iron deficiency resulting in haemoglobin levels below . mmol/l ) deficiency of other minerals (cu, se) ) insufficient access to water (not milk) (especially during warm season) ) allergenic proteins ) insufficient appropriately balanced solid food ) overfeeding (too rich diet) ) underfeeding ) too low temperature of milk or milk replacer ) exposure to excessively contaminated feed that results in pathology ) no access to natural teat or artificial teat housing ) high humidity and too high or low a temperature ) indoor draughts ) inadequate ventilation, inappropriate airflow, and air distribution within the house, airspeed, temperature ) poor air quality (ammonia, bio-aerosols and dust)) ) poor air quality ( major risk ; is considered not applicable c. assessment of whether hazards pose risks (substantiation by scientific evidence) as a consequence of the hazard characterisation and exposure assessment, the risk for poor animal welfare and health was assessed by integrating the hazard character with exposure according to the table . below. the risk was assessed as "major" if the hazard was judged to have a very serious effect and the exposure was frequent or very frequent or if the hazard was serious and exposure was very frequent. the risk was assessed to be "minor" if the hazard was very serious and exposure was rare, if hazard was moderately serious and exposure was moderately frequent or if hazard was adverse and exposure was very frequent. the hazards of iron deficiency and insufficient floor space is considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet is considered to be moderately serious. for these hazards, there is not enough information on the exposure of calves mainly due to lack of data why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding the hazard ) castration and dehorning without anesthetic and analgesic drugs, there is a variation in relation to national legislation as to why the risk of poor welfare in relation to castration and dehorning is widely different between countries. further, there is a variation in the use of analgesia during the time after the surgery is carried out which also affects the welfare of the calf. the ahaw panel wishes to thank the members of the working group chaired by panel member the needs and functioning of calves the needs of calves hutches: partially closed, outside area individual pens: open front structure straw yard with bedded lying area . . . group pens with automatic milk feeder . calf rearing and animal environmental pollution . . the quantitative share of calf production in the pollution problem . calf diseases and use of antibiotics human pathogenic-verotoxigenic escherichia coli (hp-vtec) on calf farms risk evaluation and principles of food safety assurance at calf farm level listing of potential hazards, hazard characterization and exposure assessment assessment of whether hazards pose risks (substantiation by scientific evidence) annex . hazard characterisation and exposure assessment the effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in campylobacter and escherichia coli cytotoxic interactions between bovine parainfluenza type virus and bovine alveolar macrophages immune function is impaired in iron-deficient, homebound, older women effect of orally administered cimetidine and ranitidine on abomasal luminal ph in clinically normal milk-fed calves effect of feeding frequency and route of administration on abomasal luminal ph in dairy calves fed milk replacer effect of orally administered omeprazole on abomasal luminal ph in dairy calves fed milk replacer dairy calf pneumonia. the disease and its impact effect of type of housing on veal calf growth performance, behavior and meat quality incidence of respiratory disorders during housing in non-weaned charolais calves in cow-calf farms of pays de la loire (western france) age, geographic, and temporal distribution of fecal shedding of cryptosporidium parvum oocysts in cow-calf herds effect of individual versus group rearing on ethological and physiological responses of crossbred calves bovine respiratory syncytical virus bovine respiratory syncytical virus ventilation system to reduce pneumonia in a warm calf house: a case study the effect of increasing dietary fibre on feeding, rumination and oral sterotypies in captive giraffes (giraffa camelopardalis) neuroendocrine alterations in iron deficiency effects of form of the diet on anatomical, microbial, and fermentative development of the rumen of neonatal calves pattern of diarrhoea in newborn beef calves in south-west france respiratory infections -project, panorama and treatment strategies kälberkrankheiten spatial, temporal and management-specific factors influencing antibiotic resistance and carbohydrate fermentation patterns in bovine enteric escherichia coli and the clinical consequences of limiting antibiotic use in pre-weaned calves assessing antibiotic resistance in fecal escherichia coli in young calves using cluster analysis techniques animal and farm influences on the dynamics of antibiotic resistance in faecal escherichia coli in young dairy calves assessing the effect of a single dose florfenicol treatment in feedlot cattle on the antimicrobial resistance patterns in fecal escherichia coli a clinical trial evaluating prophylactic and therapeutic antibiotic use on health and performance of calves a field trial evaluating the influence of prophylactic and therapeutic antibiotic administration on antibiotic resistance in fecal escherichia coli in dairy calves cryptosporidium parvum, giardia intestinalis and other enteric pathogens in swedish dairy calves role of oxytocin in glucose homeostasis and weight gain iron-deficiency anaemia in calves endocrine and metabolic aspects in milk-fed calves nutritional physiology of neonatal calves grouping and social preferences in calves, heifers and cows emotional reactivity affected by chronic stress: an experimental approach in calves submitted to environmental instability is gentling by people rewarding for beef calves? early contact with peers or a stockperson influences later emotivity and social behaviour of dairy calves the maternal environment limits the effect of early human nursing and contact on lambs' socialisation to the stockperson stockmanship and farm animal welfare activity, oral behaviour and slaughter data as welfare indicators in veal calves: a comparison of three housing systems isolation of verocytotoxin-producing escherichia coli o :h from cattle at slaughter in italy bovine reservoir for verotoxin-producing escherichia coli o :h . the lancet(i anaemia and veal calf production abomasal ulcers in veal calves: pathogenesis and prevention husbandry methods leading to inadequate social and maternal behaviour in cattle indicators of poor welfare needs and welfare of housed calves. in: new trends in veal calf production scientific research on veal calf welfare welfare, stress and the evolution of feelings welfare assessment and problem areas during handling and transport the use of the concept animal welfare in european conventions, regulations and directives. food chain evolution of pain coping, stress and welfare welfare behaviour and welfare in relation to pathology the effects of group-rearing or partial isolation on later social behaviour of calves approaching questions of stress and welfare effects of disease on farm animal welfare bem-estar animal: conceito e questões relacionadas -revisão brain measures which tell us about animal welfare experimental escherichia coli o :h carriage in calves untersuchung von todesursachen bei kälbern in schleswig-holstein in den jahren eimeria-infektionen beim rind calf health in cow-calf herds in swotzerland prevalence and infection risks of zoonotic enteropathogenic bacteria in swiss cow-calf farms principles and guidelines for the conduct of microbiological risk assessment. document cac/gl the influence of air quality on production increases associated with all-in/all-out management essentials of veterinary bacteriology and cattle as a possible source of verocytotoxin-producing escherichia coli o infections in man effects of pair versus individual housing on the behavior and performance of dairy calves antimicrobial use in the treatment of calf diarrhoea effects of various levels of forage and form of diet on rumen development and growth in calves somministrazione di un mangime solido a vitelli a carne bianca stabulati in gabbia individuale the provision of solid feeds to veal calves: i. growth performance, forestomach development, and carcass and meat quality experimental infection of calves and adult cattle with escherichia coli o :h effects of handling aids on calf behavior iron deficiency and the immune response the pituitary-adrenal response to stress in the iron-deficient rat indicators relevant to animal welfare: a seminar in the c.e.c. programme of coordination of research on animal welfare the behaviour of calves under four levels of lighting the effect of different housing conditions on behavioural and adrenocortical reactions in veal calves from an animal's point of view: motivation, fitness, and animal welfare phylogenetic characterization of 'candidatus helicobacter bovis', a new gastric helicobacter in cattle rotavirus infections in calves in dairy herds sucking motivation and related problems in calves dairy calves discrimination of people based on previous handling iron supplementation improves iron status and reduces morbidity in children with or without upper respiratory tract infections: a randomized controlled study in colombo, sri lanka enhancement of clinical signs in experimentally rotavirus infected calves by combined viral infections group a rotavirus as a cause of neonatal calf enteritis in sweden incidence of diarrhea among calves after strict closure and eradication of bovine viral diarrhea virus infection in a dairy herd enteric infections in veal calves: a longitudinal study on four veal calf units behaviour and welfare of veal calves in relation to husbandry systems comparison of four methods of calf confinement. ii report of the scientific committee on animal health and animal welfare. health and consumer protection, directorate-general, directorate c -scientific opinions iron status, immune capacity and resistance to infections planungs-und berechnungsgrundlagen für geschlossene zwangsbelüftete ställe (thermal insulation for closed livestock buildings. thermal insulation and ventilation forced. part : principles for planning and design for closed ventilated livestock buildings). deutsches institut für normung e assosiations between passive immunity and morbidity and mortality in dairy heifers in florida, usa contribution to the study of gut hypersensitivity reactions to soybean proteins in preruminant calves and early-weaned piglets igg, iga, igg and igg specific responses in blood and gut secretion of calves fed soyabean products the interpretation of preference tests in animal behaviour animal right-animal welfare. a scientist's assessment measuring preferences and the strength of preference animal welfare as defined in terms of feelings. acta agriculturae scandinavica section a opinion of the scientific panel on biological hazards of the european food safety authority on standards for the microclimate inside animal transport road vehicles opinion of the scientific panel on biological hazards of the european food safety authority on "the risk assessment of a revised inspection of slaughter animals in areas with low prevalence of cysticercus welfare aspects of the main systems of stunning and killing the main commercial species of animals opinion of the scientific panel on biological hazards of the european food safety authority on "campylobacter in animals and foodstuffs the community summary report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance in the european union in opinion of the scientific panel on biological hazards of the european food safety authority on "an assessment of the public and animal health risks associated with the adoption of a visual inspection system in veal calves raised in a member state (or part of a member state) considered free of tuberculosis an experimental study of a concurrent primary infection with bovine respiratory syncytical virus (brsv) and bovine viral diarrhoea virus (bvdv) in calves a study of bovine respiratory syncytical virus infections in swedish cattle performance and health of group-housed dairy calves fed milk automatically versus manually virkning av forskellige insaettelsestrategier på tillvaekst og sundhed hos gruppeopstaldede kalve increased mortality among calves in danish cattle herds during bovine virus diarrhoea infection reducing pain after dehorning in dairy calves veterinary virology the effect of the size of individual crates on the behavioural and immune reactions of dairy calves ceftriaxone-resistant salmonella infection acquired by a child from cattle effect of castration method and the provision of local anesthesia on plasma cortisol, scrotal circumference, growth, and feed intake of bull calves farm animal behaviour and welfare farm animal behaviour and welfare preference and motivation testing pleasures, "pains" and animal welfare: towards a natural history of affect the effects of early separation on the dairy cow and calf the production system and disease incidence in a national random longitudinal study of swiss dairy herds comparison of four methods of calf confinement. i interleukin production in iron-deficient children rumional acidosis in milk fed calves. large animal veterinary rounds inzidenz und verlauf von neugeborenendurchfall bei kälbern in einem praxisgebiet in oberbayern economic analysis of feeding pasteurized nonsaleable milk versus conventional milk replacer to dairy calves campylobacter jejuni and campylobacter hyointestinalis in the intestinal tract and on the carcasses of calves and cattle a new method of measuring diet abrasion and its effect on the development of the forestomach urinary catecholamines in iron-deficient rats at rest and following surgical stress experimental salmonella infection of calves. . the effect of stress factors on the carrier state experimental salmonella infection of calves. . virulence and the spread of infection concentrations and emissions of ammonia in livestock buildings in northern europe zur Äthiologie und prophylaxe der perinatalen mortalität beim kalb collegium veterinarium xxiv salmonella bij gezonde runderen en kalveren in nederland immune functions of veal calves fed low amounts of iron delaying colostrum intake by one day has important effects on metabolic traits and on gastrointestinal and metabolic hormones in neonatal calves dynamics of virus infections involved in the bovine respiratory disease complex in swedish dairy herds. the vet effects of resistance to milk flow and the provision of hay on non-nutritive sucking by dairy calves sucking behaviour of dairy calves fed milk ad libitum by bucket or teat a longitudinal study of escherichia coli o in fourteen cattle herds resting behaviour, growth and diarrhoea incidence rate of young dairy calves housed individually or in groups in warm or cold building the effect of flooring type and social grouping on the rest and growth of dairy calves fecal shedding and rumen growth of escherichia coli o :h in fasted calves feeding the newborn dairy calf calf and heifer rearing : principles of rearing the modern heifer from calf to calving the national dairy heifer evaluation project: a profile of heifer management practices in the united states human-livestock interactions: the stockperson and the productivity and welfare of intensively farmeed the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves effects of confinement on rebound of locomotor behaviour of calves and heifers, and the spatial preferences of calves the effects of feeding method, milk allowance and social factors on milk feeding behaviour and cross-sucking in group housed dairy calves computer-controlled milk feeding of dairy calves: the effects of number of calves per feeder and number of milk portions on use of feeder and social behaviour who needs 'behavioural needs'? motivational aspects of the needs of animals play behaviour in group-housed dairy calves, the effect of space allowance the effect of milk flow rate and milk allowance on feeding related behaviour in dairy calves fed by computer controlled milk feeders effect of single versus group housing and space allowance on responses of calves during open-field tests play behaviuor in domestic calves kept in pens: the effect of social contact and space allowance evaluation of welfare indicators for social environment in cattle effect of amount of milk, milk flow and access to a rubber teat on cross-sucking and non-nutritive sucking in dairy calves epidemiologische erhebungen zur kälberdiarrhoe in einem praxisgebiet in oberbayern effect of shipping and chromium supplementation on performance, immune response, and disease resistance of steers the development of intersucking in dairy calves around weaning factors associated with intersucking in swiss dairy heifers whole blood leukocytes vs separated mononuclear cell blastogenesis in calves. time-dependent changes after shipping recommendations for recording and calculating the incidence of selected clinical diseases in dairy cattle castration of calves: a study of methods used by farmers in the united kingdom onderzoek naar de uit ethologisch oogpunt minimaal gewenstle boxmaten voor vleeskalveren met een gewicht van tot kg spatial requirements of individually housed veal calves of to kg nutritional effects on response of children in developing countries to respiratory tract pathogens: implications for vaccine development problems on the etiological importance of pasteurella, mycoplasma and parainfluenza- -virus in enzootic pneumonia of calves the behaviour of beef suckler cattle enzootic pneumonia in calves: clinical and morphological features theoretical comparison of the consumer surplus and elasticities of demand as measures of motivational strength effects on calves less than one month old of feeding or not feeding tham during road transport of up to hours untersuchungen zur pathogenese der pylorusulzera beim mestkalb bovine cryptosporidiosis in denmark. ii. cryptosporidia associated with neonatal calf diarrhoea an evaluation of two management systems for rearing calves fed milk replacer prevalence of escherichia coli o :h in range beef calves at weaning cattle and reindeer as possible sources of escherichia coli o infection in humans aktuelle probleme der tierärztlichen betreuung von mutterkuhherden mother-young behaviour in cattle prevention of microbial contamination of red meat in the ante mortem phase; epidemiological aspects small intestinal motility disorders in preruminant calves chronically fed a diet based on antigenic soya : characterization and possible mediators influence of soya antigen levels in milk replacers on the disruption of intestinal motility patterns in calves sensitive to soya systemic and local gutspecific antibody responses in preruminant calves sensitive to soya feeding heated soyabean flour increases the density of b and t lymphocytes in the small intestine of calves hydrolysed soy protein isolate sustains high nutritional performance in veal calves antigenicity and digestive utilization of four soya products by the preruminant calves antibiotic resistance in gut bacteria from dairy calves: a dose response to the level of antibiotics fed in milk iron deficiency and immune responses the relationship between farmers' attitude and behaviour towards calves, and productivity of veal units reducing veal calves' reactivity to people by providing additional human contact the influence of farmers' behavior towards calves on animals' responses to transport and quality of veal meat the farmers' influence on calves' behaviour, health and production of a veal unit the impact of gentle contacts on ease of handling, welfare, and growth of calves and quality of veal meat reactions of calves to handling depend on housing condition and previous experience with humans effects of supplemental yeast culture on rumen development, growth characteristics, and blood parameters in neonatal dairy calves effects of corn processing on growth characteristics, rumen development, and rumen parameters in neonatal dairy calves anemia as a risk factor for infectious diseases in infants and toddlers: results from a prospective study colostrum in swedish dairy cows: quality and effects on passive immunity in the calves cross-sucking in group-housed dairy calves before and after weaning off milk intersucking in dairy cattle--review and questionnaire cryptosporidium andersoni n. sp. (apicomplexa: cryptosporidiidae) from cattle, bos taurus occurrence of iron deficiency in growing cattle growth performance, haematological traits, meat variables, and effects of treadmill and transport stress in veal calves supplied different anounts of iron prevention of microbial contamination of red meat in the ante mortem phase; epidemiological aspects management practices associates with high mortality among preweaned dairy heifers the gastric mucosal barrier and abomasal ulcers in veal calves housing, management and health in swedish dairy calves. doctoral thesis herd-level risk factors for infectious diseases in swedish dairy calves aged - days automated feeding of milk replacer and health control of group-hiused veal calves automated feeding of milk replacer and health control of group-housed veal calves effect of transportation and weaning on humoral immune responses of calves responses of dairy cows and calves to each other's vocalisations after early separation cross-sucking and other oral behaviours in calves, and their relation to cow suckling and food provision isolation of escherichia coli o :h from dairy cattle associated with two cases of haemolytic uraemic syndrome the provision of solid feeds to veal calves: ii. behavior, physiology, and abomasal damage enteric pathogens in intensively reared veal calves a fifteen month study of escherichia coli o :h in a dairy herd a comparison of catecholamine and cortisol responses of young lambs and calves to painful husbandry procedures acute pain in an emergency clinic regulation of sucking behaviour of calves laboratory findings associated with abomasal ulcers/tympany in range calves biological response to stress: key to assessment of animal well-being? association between resting behaviour and live weight gain in dairy heifers housed in pens with different space allowance and floor type long-term effect of housing method during the first three months of life on human-animal relationship in female dairy cattle risk factors associated with cryptosporidium parvum infection in dairy cattle in southeastern new york state the response of normal and iron anemic calves to nasal infection with an attenuated strain parainfluenza- virus assessment of acute and chronic pain after different methods of castration of calves intestinal digestion of dietary and endogenous proteins along the small intestine of calves fed soybean or potato influence of dietary protein level and source on the course of protein digestion along the small intestine of the veal calf effect of diet on mucin kinetics and composition: nutrition and health implications calf rearing a practical guide, natural resource and environment influence of dry feed supplements on different parameters of welfare in veal calves the effect of four fibrous feed supplementations on different welfare traits in veal calves behavioural investigation of group rearing calves in automatic milk replacer feeding system effect of transportation on blood serum composition, disease incidence, and production traits in young calves. influence of the journey duration critical anthropomorphism, animal suffering and the ecological context bacterial pneumonia dust and microbiol emissions from animal production influence of truck transportation of calves on their cellular immune function prevalence of enteric pathogens in the feces of healthy beef calves salmonella infection and contamination of veal calves: a slaughterhouse survey comparison of two castration methods in cattle: plasma cortisol levels, leukocyte count and behavioral changes calf diseases and mortality in swedish dairy herds abomasal ulcers in captive white-tailed deer (odocoileus virginianus) growth performance and health of dairy calves bedded with different types of materials factors involved in recent outbreaks of escherichia coli o :h in scotland and recommendations for its control protection of calves against cryptosporidiosis with immune bovine colostrum induced by a cryptosporidium parvum recombinant protein treadmill exercise of calves with different iron supply, husbandry, and work load beurteilung verschiedener tränketechniken und betreuungsmassnahmen hinsichtlich ihrer auswirkungen auf die oralen aktivitäten, den gesundheitszustand und die mastleitung über zwei bis acht wochen alter mastkälber in gruppenhaltung. dissertation. institut für tierhygiene und tierschutz der tierärztlichen hochschule hannover group housing for two to eight week old fattening calves veterinary and zootechnical aspects of veal production veterinary medicine. a textbook of the diseases of cattle, sheep, pigs, goats and horses serum immunoglobulin concentrations and health of dairy calves in two management systems from birth to weeks of age influence of feeding different amounts of first colostrum on metabolic, endocrine, and health status and on growth performance in neonatal calves the impact of social and human contacts on calves' behaviour and stress responses stereotypies om heifers are affected by feeding regime feeding level and oral stereotypies in dairy cows blood studies and performance among calves reared by different methods consequences of short term fluctuations of the environmental temperatures in calves--part : effects on the health status of animals within three weeks after exposure microbiology of calf diarrhea in southern britain studies on the presence of verocytotoxic escherichia coli o in bovine faeces submitted for diagnostic purposes in england and wales and on beef carcases in abattoirs in the united kingdom effect of different methods of castration on behaviour and plasma cortisol in calves of three ages iron, thermoregulation, metabolic rate the validity of behavioural measures of aversion: a review fear of people by cows and effects on milk yield, behavior, and heart rate at milking recognition of people by dairy calves using colour of clothing endotoxins in the environment: a criteria document reduced cortisol secretions in patients with iron deficiency viral diarrhea in man and animals mouth-based anomalous syndromes the influence of management routines on endocrine systems involved in the control of lactation in dairy cattle immune response in anemic calves the welfare of cattle kept for beef production. directorate general health and consumer protection, directorate c -scientific health opinions, unit c -management of scientific committees staphylococci as an indicator for bacterial emissions from a broiler house the use of trainer cows to reduce stress in newly arrived feedlot calves scvph (scientific committee on veterinary measures relating to public health) scvph (scientific committee on veterinary measures relating to public health) scvph (scientific committee on veterinary measures relating to public health) bioaerosole in und aus der landwirtschaftlichen nutztierhaltung (vorläufiger titel). kuratorium für technik und bauwesen in der landwirtschaft (ktbl, hrsg.) concentrations and emissions of airborne endotoxins and microorganisms in livestock buildings in northern europe a survey of ventilation rates in livestock buildings in northern number of viable bacteria and presumptive antibiotic residues in milk fed to calves on commercial dairies effects of transportations, a high lactose diet and acth injections on the white blood cell count, serum cortisol and immunoglobulin g in young calves descriptive epidemiology of morbidity and mortality in minnesota dairy heifer calves the development of the bacterial flora of the faeces of animals and man: the changes that occur during aging clostridial abomasitis in calves: case report and review of the literature destruction of mycobacterium paratuberculosis, salmonella spp., and mycoplasma spp effects of local anaesthesia or local anaesthesia plus a non-steroidal anti-inflammatory drug on the acute cortisol response of calves to five different methods of castration dehorning and disbudding distress and its alleviation in calves seasonal variation of thermophilic campylobacters in lambs at slaughter losses in young calves after transportation reaction of claves to two flooring materials offered simultaneously in one pen effects of three different methods of dehorning on cortisol levels and behaviour of calves evaluation of the effect of local anaesthesia and local anaesthesia associated with analgesia on the levels of cortisol after hot-iron, chemical or scoop dehorning effects of in vivo release of insulin and glucagon studied by microdialysis in rat pancreas and audiographic evidence for ( h)-oxytocin binding sites within the islets of langerhans some theoretical and observed relationships of fixed and portable spacing behavior of animals selfish animats and robot ethology: using artificial animals to investigate social and spatial behavior effect of local anaesthetic combined with wound cauterisation on the cortisol response to dehorning in calves cortisol responses to dehorning of calves given a -h local anaesthetic regimen plus phenylbutazone, ketoprofen, or adrenocorticotropic hormone prior to dehorning scientific veterinary committee, animal welfare section. directorate generale for agriculture, vi/bii. . adopted the effect of group size on health and growth rate of swedish dairy calves housed in pens with automatic milk-feeders bovine coccidiosis with special reference to eimeria alabamensis infections in grazing calves morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases health status of dairy calves kept in individual pens or in group pens with or without automatic milk feeder acute cortisol responses of calves to scoop dehorning using local anaesthesia and/or cautery of the wound verotoxigenic escherichia coli o in scottish calves verocytotoxin-producing escherichia coli: a veterinary view concentrations and emissions of airborne dust in livestock buildings in northern europe ethological, physiological and histological aspects of pain and stress in cattle when being dehorned individual humans as discriminative stimuli for cattle (bos taurus) pathological and microbiological studies on pneumonic lungs from danish calves effects of individual housing design and size on special-fed holstein veal calf growth performance, hematology, and carcass characteristics calf rearing verordnung zum schutz landwirtschaftlicher nutztiere und anderer zur erzeugung tierischer produkte gehaltener tiere bei ihrer haltung (tierschutz-nutztierhaltungsverordnung -tierschnutztv)*) vom geändert durch die erste verordnung zur Änderung der tierschutz -nutztierhaltungsverordnung vom effects of age of holstein-friesian calves on plasma cortisol, acute-phase proteins, immunological function, scrotal measurements and growth in response to burdizzo castration effect of ketoprofen, lidocaine local anesthesia, and combined xylazine and lidocaine caudal epidural anesthesia during castration of beef cattle on stress responses, immunity, growth, and behavior etude de la prévalence de l'infection des veaux par giardia duodenalis en pays de la loire effects of housing on responses of veal calves to handling and transport. in new trends in veal calf production the importance of straw for pig and cattle welfare: a review behavioural and physiological studies on rearing and veal calves molecular epidemiology of cryptosporidium parvum and giardia duodenalis to study the potential for zoonotic transmission dynamics of bovine respiratory syncytical virus: a longitudinal epidemiological study in dairy herds the prevalence of verotoxins, escherichia coli o :h and salmonella in the feces and rumen of cattle at processing haemoglobin and haematocrit levels in veal calves is anaemia of veal calves normochromic or hypochromic a comparison of different enrichment media for the isolation of salmonella dublin from livers, kidneys and muscles of salmonella-positive veal calves zusatzfuttering von stroh an mastkälber the effect of housing system and dietary iron supply on stress physiological measures and resistance against an experimental infection with bovine herpesvirus (bhv ) in veal calves enkele epidemiologische aspecten van salmonellose bij mestkalveren in nederland calves' responses to repeated social regrouping and relocation does nutritive and non-nutritive sucking reduce other oral behaviors and stimulate rest in calves nonnutritive oral activities and stress responses of veal calves in relation to feeding and housing conditions providing social contacts and objects for nibbling moderates reactivity and oral behavior in veal calves the effects of rearing in individual crates on subsequent social behaviour of veal calves somministrazione di un mangime solido a vitelli a carne bianca stabulati in gabbia individuale o in box di gruppo. . parametri comportamentali, fisiologici e patologici behaviour and performance of veal calves under different stabling conditions assessing animal welfare: the significance of causal studies of behaviour at the motivational level epidemiologic and pathological characteristics of respiratory tract disease in dairy heifers during the first three months of life morbidity from nonrespiratory diseases and mortality in dairy heifers during their first months of life iron deficiency anemia and increased urinary norepinephrine excretion decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant salmonella typhimurium dt otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis effects of group, individual, and isolated rearing of calves on weight gain and behavior colostrum -the beginning of a successful calf raising program. dairy feed facts air and surface hygiene the response of beef cattle to noise during handling reduction of cross-sucking in calves by the use of a modified automatic teat feeder calf husbandry, health and welfare rearing of veal calves the effect of different rearing systems on the development of calf behaviour a survey of abomasal ulceration in veal calves haematology of veal calves reared in different husbandry systems and the assessment of iron deficiency health status of preweaned dairy heifers in the united states the effect of feeding pellets of different types of roughage on the incidence of lesions in the abomasum of veal calves st joint fao/oie/who expert workshop on non-human antimicrobial usage and antimicrobial resistance: scientific assessment on the significance of ethological criteria for the assessment of animal welfare effects of individual housing design and size on behaviour and stress indicators of special-fed holstein veal calves feeding antibiotic-contaminated waste milk to calves--effects on physical performance and antibiotic sensitivity of gut flora reviews of the progress of dairy science: bovine salmonellosis factors influencing occurrence of colibacillosis in calves natural and experimental infection of normal cattle with escherichia coli o rearing veal calves with respect to animal welfare: effects of group housing and solid influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves the members of the working group which authored this report were: animals. cab international, oxon/new-york pp.hepola, h., . milk feeding systems for dairy calves in groups: effects on feed intake, growth and health. applied animal behaviour science : - .herrmann, j., knierim, u., hinton, m., linton, a.h., . antibacterial drug resistance among escherichia coli isolated from calves fed on a milk substitute diet, vet. rec., , - .hinton, m., ali, e. a., allen, v., linton, a. h., . the excretion of salmonella typhimurium in the faeces of calves fed on a milk substitute diet. j. hyg. camb. , - .hinton, m., hedges, a. j., linton, a. h., a . the ecology of escherichia coli in calves fed on a milk substitute diet. j. appl. bacteriol. , - .hinton, m., linton, a. h., hedges, a. j., b . the ecology of escherichia coli in calves reared as dairy-cow replacers. j. appl. bacteriol. , - .hinton, m., rixson, p. d., allen, v., linton, a. h., a . the persistence of drugresistant escherichia coli strains in the majority faecal flora of calves, j. hyg. camb., , - .hinton, m., suleyman, i. o., allen, v., linton, a. h., b . further observations on the excretion of salmonella in the faeces of calves fed milk substitute, j. hyg. camb., , - .holliman, a., . overview of coccidiosis -recent observations. cattle practice ; - .holm, l., jensen, m. b., jeppesen, l. l., key: cord- -yzv e l authors: kaye, alan david; chernobylsky, david j.; thakur, pankaj; siddaiah, harish; kaye, rachel j.; eng, lauren k.; harbell, monica w.; lajaunie, jared; cornett, elyse m. title: dexmedetomidine in enhanced recovery after surgery (eras) protocols for postoperative pain date: - - journal: curr pain headache rep doi: . /s - - -z sha: doc_id: cord_uid: yzv e l purpose of review: effective acute pain management has evolved considerably in recent years and is a primary area of focus in attempts to defend against the opioid epidemic. persistent postsurgical pain (ppp) has an incidence of up to – % and has negative outcome of quality of life and negative burden on individuals, family, and society. the american society of anesthesiologists (asa) guidelines states that enhanced recovery after surgery (eras) forms an integral part of perioperative surgical home (psh) and is now recommended to use a multimodal opioid-sparing approach for management of postoperative pain. as such, dexmedetomidine is now being used as part of eras protocols along with regional nerve blocks and other medications, to create a satisfactory postoperative outcome with reduced opioid consumption in the post anesthesia care unit (pacu). recent findings: dexmedetomidine, a selective alpha( ) agonist, possesses analgesic effects and has a different mechanism of action when compared with opioids. when dexmedetomidine is initiated at the end of a procedure, it has a better hemodynamic stability and pain response than ropivacaine. dexmedetomidine can be used as an adjuvant in epidurals with local anesthetic sparing effects. its use during nerve blocks results in reduced postoperative pain. also, local infiltration of iv dexmedetomidine is associated with earlier discharge from pacu. summary: perioperative use of dexmedetomidine has significantly improved postoperative outcomes when used as part of eras protocols. an in-depth review of the use of dexmedetomidine in eras protocols is presented for clinical anesthesiologists. enhanced recovery after surgery (eras) protocols are a series of perioperative care protocols that promote early postsurgical recovery [ ] . the main aspects of eras involve decreasing postoperative stress and protecting organ function, perioperatively. this is accomplished via pre and post-operative counseling, enhanced nutritional support, early mobilization and standard utilization of anesthetics and analgesics. the significance of eras protocols is evident through a review of the literature, which demonstrates that they improve patient outcomes and have advanced patient care. as such, eras protocols have a distinct place in modern medical care, which has added to the continued implementation of such protocols nationwide. and while the role of eras protocols have been demonstrated, a significant area of improvement with context to postoperative pain management needs to be addressed, so that patients can benefit postoperatively through implementation of these protocols. doing so would maximize the benefits for patients that are receiving care through the implementation of eras protocols [ ] . enhancing the management of persistent perioperative pain would translate into decreased time in the postanesthesia recovery unit (pacu), reduced opioid consumption, earlier hospital discharge, and improvement in patient satisfaction. a key strategy for enhancing persistent perioperative pain management and reducing pacu time includes incorporating a multimodal analgesia approach and reducing opioid use [ ••, ••] . dexmedetomidine, therefore, is a crucial component of the multimodal analgesia approach. a large collection of studies have demonstrated the efficacy of dexmedetomidine to be superior to other agents in the perioperative setting [ - , ••, , ••, - ] . for example, dexmedetomidine has superior pain control in several types of laparoscopic procedures, such as bariatric and gynecological procedures, and opensurgery approaches, such as colorectal, major spine, and abdominal hysterectomy surgeries. patients report higher satisfaction scores after dexmedetomidine, which is likely related to decreased pain and decreased postoperative nausea and vomiting (ponv). for these reasons, many providers opt for using dexmedetomidine as part of multimodal therapy. this manuscript will comprehensively discuss dexmedetomidine with regards to postoperative pain management, eras protocols, pharmacokinetics, pharmacodynamics, cardiovascular and respiratory effects, toxicity, drug interactions, abuse and dependence. this review will also include a discussion of clinical considerations, incorporating an extensive review of the positive and negative studies, will follow the reporting of drug formulation, and dosing regimens. dexmedetomidine is a potent, versatile, and highly selective short-acting alpha- agonist with sedative, anxiolytic, perioperative sympatholytic, and hypnotic effects [ ] . dexmedetomidine is highly selective alpha- agonist similar to clonidine. with regards to alpha :alpha receptor specificity; clonidine has alpha :alpha specificity of : ; where as dexmedetomidine has specificity of : . in , the fda approved dexmedetomidine use for procedural sedation and for sedation in non-intubated patients. it also has analgesic, anesthetic-sparing, and sympatholytic properties [ ] . dexmedetomidine has the ideal characteristics of a sedative for icu use, with predictable sedation and hemodynamic stability and is easy to titrate [ ] . i n t h e u s a , d e x m e d e t o m i d i n e i s a v a i l a b l e a s dexmedetomidine hydrochloride, equivalent to μgm/ml. this formulation needs to be diluted to μgm/ml. prediluted solutions containing μgm/ml in sodium chloride . % are also available. for icu sedation, a loading dose of μgm/ml over min is infused followed by maintenance dose of . to . μgm/h; adjusted to desired level of sedation and avoiding any hypotensive effects. similar dosing regimen of μgm/ml over min is infused followed by maintenance dose of . μgm/h for procedural sedation [ ] . dexmedetomidine is highly selective for alpha- : alpha- in ratio of : [ ] . as previously mentioned, it is a potent alpha- adrenergic receptor agonist, yet the molecular mechanism is not fully clear. it is likely due to activation of inhibitory g proteins and the nitric oxide cgmp pathway. dexmedetomidine produces an agonist affect after binding to g protein-coupled receptors which have three subtypes (alpha- a, alpha- b, and alpha- c). alpha- a and alpha c receptors are primarily found on cns and alpha- b are found on vascular smooth vessels. dexmedetomidine binds selectively to alpha- a receptors via all three receptors, which inhibits adenyl cyclase reducing levels of adenosine monophosphate and leading to hyperpolarization of noradrenergic neurons. this leads to the suppression of nerve conduction by inhibiting calcium entry required for neurotransmitter vesicle fusion. this negative feedback back loop leads to attenuation of sympathetic response and decreases both heart rate and blood pressure [ ] . at the alpha- receptor, dexmedetomidine, causes inhibition of norepinephrine release from presynaptic neurons, produces centrally induced sedation via alpha- receptors in the locus ceruleus and centrally mediated pain modification via the dorsal horn [ ] . dexmedetomidine is a highly lipophilic drug and follows the two-compartment model, following rapid distribution and redistribution. the volume of distribution and clearance seems to be affected by patient bmi, hepatic function, plasma albumin binding, and cardiac output. although it was initially approved for only iv use, it is now also available in both intranasal and oral preparations. dexmedetomidine is well absorbed via intranasal and buccal route and these routes could be used for uncooperative pediatric or adult patients. following intravenous administration, dexmedetomidine undergoes rapid distribution with distribution half time (t / ) of min followed by terminal half-time (t / ) of h. the onset of action after an iv loading dose is usually - min and it peaks effect in to min. intranasal route has onset of action in min with peak effect in to min. there is no difference in the pharmacokinetic profile of either males or females, and both have similar protein binding [ ] . after the oral administration, dexmedetomidine undergoes extensive first pass metabolism and has a poor bioavailability of only % [ , ] . dexmedetomidine is highly protein-bound. almost % is bound to albumin and alpha- -glycoprotein with a wide volume of distribution and is known to readily cross blood brain and placental barriers. adult volume of distribution is around . to . l/kg. infant and children less than years of age and patients with low albumin have larger volume of distribution [ ] . biotransformation is the primary route of elimination for dexmedetomidine with less than % excreted unchanged (of which % is excreted really and % via stool). dexmedetomidine has hepatic extraction ratio of . [ ] . uridine ′-diphospho-glucuronosyltransferase also known as ugt b and ugt a are responsible for metabolizing dexmedetomidine. up to % of metabolism is via hydroxylation by cyp a in the liver microsomes [ ] . large volume of distribution and increased elimination half-life was seen in patients with hepatic impairment, due to higher unbound frac- renal impairment does not affect the dexmedetomidine dosing, but sedative effects may last longer. this could be due to higher unbound drug [ ] . dexmedetomidine clearance ranges from . to . l/min in healthy adults [ ] . low albumin levels, especially in the icu patients, can have an effect on clearance. however, the effect of low albumin could be marginal, since for compounds with high extraction ratios, blood flow and not plasma protein effects the clearance of drug. decreased cardiac output also has impact and leads to reduced dexmedetomidine clearance [ ] . nevertheless, low albumin levels, end organ failure, and hemodynamic changes all are factors to be considered in an icu patient that can affect the pharmacokinetics of dexmedetomidine. alpha- receptor polymorphisms and ethnicity are other factors that can affect the pharmacokinetics of the drug [ ] . to achieve a specific blood plasma concentration, a targetcontrolling infusion protocol has also been suggested to maintain the richmond agitation-sedation scale between zero and minus three (− ). factors like height, total body weight, and serum albumin levels can affect achieving the steady-state concentration. dexmedetomidine exhibits linear kinetics between the dose range of . to . μg/kg/h and it is % bound in steady state [ ] . the context sensitive half time (t / ) of dexmedetomidine can range from min after a -min infusion [ ] . context sensitive time half time (t / ) can be over h after a prolonged -h infusion. clearance of dexmedetomidine matures with age reaching maturity at year of age and is reduced by % after a cardiac surgery [ ] . as mentioned, dexmedetomidine exerts its sedative and hypnotic effects by action on central presynaptic and postsynaptic alpha- receptors. these effects are concentration-dependent between . and . ng/ml. postsynaptic alpha- binding in locus cereus and spinal cord leads to sedation and analgesia. its affinity to alpha- receptor leads to vagolytic effects leading to bradycardia and vasodilation [ ] . dexmedetomidine is known to mimic natural sleep while maintaining a normal physiological sleep and wake cycle. the patient remains arousable which decreases the risk of delirium. respiratory depression is rare when used in dosages between . and . μg/kg/h. dexmedetomidine suppresses pain transmission, likely through inhibiting the release of neurotransmitters such as substance p, which may alter the perception of pain [ ] . dexmedetomidine is approved for only h of icu use. studies have shown an acceptable safety profile for use up to days in the icu patients. dexmedetomidine also reduces mechanical ventilation and length of icu stay by % and %, respectively [ ] . the most frequently observed side effects include hemodynamic alterations such as hypertension, bradycardia, and hypotension due to postsynaptic alpha- receptor activation. others include bradycardia, dry mouth, and nausea. additional reported side effects include fever, muscle weakness, bronchospasm (especially in asthmatic patients), respiratory depression, conduction abnormalities, arrhythmia, a-v block, tachycardia, syncope, neuropathy, paresthesia, potassium abnormality with ekg changes, lactic acidosis, and elevated glucose levels [ ] . tachyphylaxis can also occur if given more than h as intravenous infusion. dexmedetomidine has a biphasic hemodynamic response. a bolus of high-dose dexmedetomidine can result in tachycardia and elevated blood pressure, whereas a low-dose bolus can decrease blood pressure and decrease cardiac output but preserve stroke output. this is due to alpha- -mediated vasoconstriction, which eventually leads to baroreceptor-mediated bradycardia and increased vagal activity, resulting in hypotension. dexmedetomidine also results in a reduction of circulating catecholamines, due to its sympatholytic effects [ ] . dexmedetomidine loses its alpha- receptor agonism if infused as bolus via rapid infusion, leading to increase in blood pressure and low heart rate that eventually normalizes in min. this effect is primarily mediated via central alpha- a receptors. hypertension can also be observed because of activation of alpha- b receptors. therefore, extreme care must be taken when using dexmedetomidine in patients who are volume-depleted and have underlying cardiac issues. high doses of dexmedetomidine can result in pulmonary hypertension and can be a limiting factor for its use in patients with underlying cardiac disease. at low plasma concentrations of . ng/ml, respiratory drive and the ventilatory response of co are preserved. with increasing doses, there is a slight fall in tidal volume. even at supra-therapeutic concentrations of . ng/ml, respiratory drive is unaffected [ ] . hypercapnic ventilatory response has been observed with dexmedetomidine use and this decreases with age but can lead to respiratory depression, especially in the elderly population in conjunction with other hypnotics or opioids that depresses respiratory drive. therefore, it has been approved for icu sedation only, with continuous cardiac and respiratory monitoring. however, the overall effects of dexmedetomidine on the respiratory system when combined with other anesthetic drugs are minimal. dexmedetomidine also demonstrates protective effects by attenuating oxidative stress from acute lung injury by inhibiting the generation of ros (reactive oxidative species). this is in part due to its activation of alpha- adrenoreceptor effects, which promotes cell survival and proliferation of lung alveolar epithelial cell in acute lung injury (ali). therefore, dexmedetomidine has established itself as sedative of choice in patients with ali [ ] . side effects in the elderly can be more pronounced, especially hemodynamic side effects. hypotension can result if a loading dose of more than . μm/kg is used. it is recommended to be cautious when using dexmedetomidine in the elderly due to a higher incidence of hypotension and bradycardia, as they often have numerous comorbidities [ ] . therefore, a continuous monitoring of pulse oximetry and ekg is recommended for continuous infusion, especially in patients with cardiac ejection fraction less than %, or with other underlying cardiac comorbidity. monitoring respiration in an obese patient is important. they are more likely to have obstructive sleep apnea and when dexmedetomidine is used in conjunction with other opioids, this may compound the problem. beneficial effects with use of dexmedetomidine include less use of volatile gas, less opioids requirements with better pain control, and less antiemetic requirements. no human studies are available with use of dexmedetomidine and drug dependence, but clonidine-like withdrawal symptoms have been noticed. animal studies following abrupt withdrawal of dexmedetomidine have shown symptoms of nervousness, headache, and agitation. elevation of blood pressure and catecholamine levels along with elevated plasma catecholamines is also seen. a recent study showed that use of dexmedetomidine in patients with substance abuse had shorter icu and ventilator-dependent days [ ] . the patients with substance abuse also had beneficial effect from anxiolytic and analgesic effects of dexmedetomidine without respiratory depression. hence, it may be a sedative of choice in patients with substance abuse. although the mechanism of dexmedetomidine in attenuating withdrawal symptom of opioids is poorly understood, it is hypothesized that use of strong opioids like heroin leads to a hyperadrenergic state. thus, use of alpha- agonists decreases the sympathetic outflow and counteracts the physiological effects, but the clear mechanism of dexmedetomidine effects is still unclear. further studies may be warranted in formulation of icu treatment protocol for dexmedetomidine for use as part of substance abuse withdrawal regimen protocol [ ] . when dexmedetomidine is used in co-administration with other anesthetics, sedatives, and opioids, this may synergistically enhance their effects. similarly, caution must be exercised when using vasodilators or negative chronotropic a g e n t s , s i n c e d e x m e d e t o m i d i n e m a y e n h a n c e cardiodepressant side effects. vigilance should be used in patients who have greater than % decrease in heart rate from their baseline, because these patients are predisposed to severe bradycardia that could progress to pulseless electrical activity [ ] . a rapid bolus could result in fatality; therefore, caution must be exercised even in healthy adults with high vagal tones. no meaningful side effects are seen when dexmedetomidine is used with neuromuscular blocking agents. caution should also be exercised in patients receiving dexmedetomidine beyond h as this has been associated with both tachyphylaxis and tolerance. there are no studies involving dexmedetomidine and pregnancy. nevertheless, it has been suggested that a lactating woman who received dexmedetomidine may pump and discard breast milk for next h. dexmedetomidine can cause significant nerve damage in diabetic rats when used for local nerve blocks [ ] . although this produced significant motor and sensory blockade but also warranting the caution to be used in patients with peripheral neuropathy while using precedes in combination with local anesthetics [ ] . similarly, in another study when dexmedetomidine was used as an adjuvant for a nerve block, it not only prolonged nerve block duration but increased systemic side effects [ ] . overdosage of dexmedetomidine primary leads to cardiodepressant side effects that may require supportive therapy. dexmedetomidine has been extensively studied in pediatric intensive care patients, pediatric cardiac, and general surgery patients. its sympatholytic effects are generally beneficial for patients undergoing cardiac procedures [ ] . the recommended adult dose may also be given in children, given as a loading dose of . to μg/kg/h over min and a maintenance does of . to . μg/kg/h to achieve a cook scale between and points [ ] . dexmedetomidine clearance is about % in newborns that eventually matures to adult levels by the end of new neonatal age. neonates have larger volume of distribution with increased elimination half-life due to liver immaturity and lower albumin levels [ ] . also, a higher concentration of dexmedetomidine is found in neonatal brains due to immature blood-brain barrier. at lower doses, no cardiopulmonary side effects occur but hypothermia and bradycardia have been reported with use of dexmedetomidine in neonates [ ] . in older children, dexmedetomidine is well tolerated and efficacy similar to that seen in adults. analgesia with non-opioids analgesics like dexmedetomidine are being used as part of an eras protocol intraoperatively, along with regional nerve blocks to attain a satisfactory postoperative outcome with reduced requirements of opioids in pacu in the pediatric population. with more requests for procedural sedation for diagnostics procedures like mri, ambulatory center procedures, dexmedetomidine has become an attractive option for non-iv route of sedation especially buccal administration at least min before the elected time in a dose to μg/kg. this provides adequate sedation in approximately % of patients with a failure rate of % requiring other modes of sedation [ ] . delirium is an acute confusional state wherein the patient's cognitive functioning is impaired with inability to process awareness of environment and attention. most patients transition to normal consciousness smoothly, after the anesthetic agents are disconnected at the end of a surgical procedure. a select few patients may end up having emergence delirium with the risk being higher in pediatric age groups and the elderly population. the incidence has been reported up to % in pediatric age group and this can increase risk of postoperative respiratory depression and airway obstruction [ ] . amongst the numerous agents available, dexmedetomidine has found to be beneficial, especially in sevoflurane-induced e m e r g e n c e d e l i r i u m . a t a d o s e o f . μ g / k g , dexmedetomidine is beneficial in reducing the incidence of emergence delirium and negative postoperative behavioral changes (npobc). caution must be exercised with vigilant cardiopulmonary monitoring when dexmedetomidine is administrated after induction of anesthesia. the data on negative postoperative behavioral changes is limited, but up to % children undergoing surgical procedures under general anesthesia manifest some kind of behavioral symptoms including but not limited to inconsolable crying, irritability, feeding and sleeping issues, temper tantrums, and nightmares that could manifest anywhere from postoperative day to to a week or later after discharge. such symptoms have been prone to be decreased and limited by the use of dexmedetomidine when quantified by pediatric anesthesia emergence delirium (paed) scale [ ] . although the incidence of emergence agitation is not as high as emergence delirium in the pediatric age group, it has been found to be in ranges of~ % when using sevoflurane alone versus when dexmedetomidine is used in addition to sevoflurane with incidence of only % when quantified on riker sedation agitation scale. although a recent published study did show that with dexmedetomidine, infusion leads to decreased norepinephrine and epinephrine, suggesting that dexmedetomidine's effects are primarily by reducing catecholamines and not via anti-inflammatory effects [ ] . dexmedetomidine can also decrease the occurrence of emergence delirium, especially in the pediatric population. it has been found that patients treated with dexmedetomidine had reduced incidence of emergence delirium when compared with midazolam. even with lorazepam, dexmedetomidine had lower incidence of emergence delirium. therefore, dexmedetomidine could be used prophylactically or in an emergent setting for the prevention or control of emergence delirium [ ] . for patients at risk, a dose of . mcg/kg of dexmedetomidine may be slowly injected intravenously and to treat patients emergently, a dose of . mcg/kg of dexmedetomidine may be used [ ] . atipamezole is highly selective alpha- antagonist approved only for use in veterinary medicine. further research and clinical studies are warranted before it is approved for use in humans. atipamezole rapidly reverses both sedative and sympathetic effects of dexmedetomidine. higher doses of atipamezole - μg/kg can quickly reverse side effects of iv dexmedetomidine. numerous meta-analyses demonstrate the effectiveness of dexmedetomidine for postoperative pain control [ ••, - , ••, ••, ]. in a meta-analysis of patients, dexmedetomidine reduced opioid consumption by % at h postoperatively [ ] . dexmedetomidine has a stronger analgesic effect than clonidine and acetaminophen, but weaker than ketamine or non-steroidal anti-inflammatory drugs [ ] . this not only makes dexmedetomidine an attractive agent for eras, but also for chronic pain patients [ ] . in a meta-analysis, although dexmedetomidine reduced pain intensity, opioid consumption, and postoperative nausea and vomiting (ponv), it had no effect on recovery time [ ] . notably, in a cochrane review of dexmedetomidine use in abdominal surgery, there was too much heterogeneity of the data for meta-analysis [ ] . dexmedetomidine is an effective analgesic for pediatric patients and has the added benefits of reducing anxiety and emergence agitation [ ••, [ ] [ ] [ ] . there is variability in the timing of dexmedetomidine administration without consensus on the optimal time for administration. when given preoperatively, a single mcg/kg dose of dexmedetomidine given min prior to induction has been shown to reduce postoperative opioid use [ ] . multiple studies demonstrate the effectiveness of intraoperative dexmedetomidine and it has also been shown to be superior to intraoperative remifentanil, providing better postoperative analgesia with fewer side effects [ ] [ ] [ ] [ ] [ ] . in two recent metaa n a l y s e s , p a t i e n t s t h a t r e c e i v e d p o s t o p e r a t i v e dexmedetomidine infusion and iv opioid patient-controlled analgesia (pca) had lower postoperative pain scores and lower opioid consumption in the first h postoperatively with decreased ponv and pruritis compared with those with pca alone [ , ] . one randomized controlled trial comparing dexmedetomidine pca with fentanyl pca for postoperative pain control found that although there was no significant difference in vas pain score at h postoperatively, the patients with the dexmedetomidine pca had higher patient satisfaction with pain control, faster return of bsowel function, and a lower incidence of ponv [ ] . as most eras pathways do not utilize iv opioid pcas, there may be a limited role for dexmedetomidine pca. however, it may be an attractive alternative to opioid pca in patients that require pca despite other multimodal analgesics. another potential use for dexmedetomidine perioperatively for postoperative pain is as an adjunct for regional anesthesia [ , ] . regional anesthesia is an important modality for minimizing opioid use as part of an eras multimodal analgesic regimen. the addition of dexmedetomidine to neuraxial analgesia, iv regional anesthesia, and peripheral nerve blocks [ , ] can hasten block onset, prolongs duration, and reduces opioid use [ , ] . in a meta-analysis of over patients, the addition of dexmedetomidine to brachial plexus blocks led to faster block onset, longer block duration, improved analgesia, and reduced postoperative morphine requirements by . mg [ ] . while dexmedetomidine is effective as an adjunct to regional anesthesia, it is not as effective as dexamethasone and carries a higher risk of hypotension and sedation compared with dexamethasone [ ] . this may limit its widespread use as an adjunct to regional anesthetic eras pathways and may support its preferential use as an intravenous infusion. the vast majority of dexmedetomidine studies for postoperative pain control report intravenous administration of dexmedetomidine; however, there is heterogeneity of the doses given and the optimal dose is unknown. table lists dexmedetomidine doses which have been described in the literature for various routes of administration [ , ••, , , , , , , , ] . of note, a loading dose of iv dexmedetomidine may not be necessary. in two recent randomized studies, there was no difference in -h opioid consumption between those who received a loading dose of mcg/kg dexmedetomidine immediately before induction, followed by . mcg/kg/h infusion and those who only received dexmedetomidine infusion [ , ] . a bolus of ≥ . mcg/kg/h in pediatric patients is sufficient to decrease postoperative pain scores and opioid requirements, even without a continuous infusion [ ••] . while intravenous administration is the most widely used, other routes of administration of dexmedetomidine have been described, including transdermal, intramuscular, oral, buccal, and intranasal, not to mention its use as an adjunct for regional anesthesia [ ] . these alternate routes of administration could potentially be important particularly for patients with limited or difficult iv access, including pediatric and autistic patients, and could be ideal for chronic pain patients to use on an outpatient basis. the bioavailability and onset of action can differ significantly depending on the route of administration. the mean absolute bioavailability for oral, intranasal, buccal, and intramuscular administration is %, - % [ , ] , %, and - % respectively [ ] [ ] [ ] . there is high interindividual variability in dexmedetomidine pharmacokinetics, which is influenced by body size, liver function, cardiac output, and albumin levels [ ] . given the low bioavailability of oral dexmedetomidine, it is not surprising that oral dexmedetomidine ( mcg/kg) provides inferior pain relief to oral ketamine ( mcg/kg) in burn patients [ ] . a newly developed orally disintegrating tablet has . % release of the drug after min, which has potential for postoperative eras use [ ] . with - . mcg/kg intranasal or transmucosal dexmedetomidine, patients have a similar sedation and anxiolytic effect as midazolam, but less postoperative pain and less sympathetic stimulation [ ] . this route has a slower onset than intravenous with a peak plasma concentration is reached by min [ ] . although buccal dexmedetomidine ( . mcg/kg) and intramuscular dexmedetomidine ( . mcg/kg) provide equal sedation and anxiolysis, buccal dexmedetomidine results in better analgesia t h a n i n t r a m u s c u l a r [ ] . a f t e r i n t r a m u s c u l a r dexmedetomidine, there is a large range in time to peak concentration ranging from min to as high as . h, and an elimination half-life of . - . h [ , ] . the addition of dexmedetomidine has been described as an adjunct for neuraxial analgesia, iv regional anesthesia, and peripheral nerve blockade [ ] . one mcg/kg dose of dexmedetomidine has been described for lumbar epidural and caudal analgesia; mcg dose has been described for intrathecal use. as an adjunct for peripheral nerve blockade, most studies use mcg/kg dexmedetomidine in peripheral nerve blockade [ ] . although in a meta-analysis of dexmedetomidine use in brachial plexus blockade, − mcg was recommended to maximize sensory block duration while minimizing risk of bradycardia and hypotension [ ] . there seems to be little evidence of adverse events with alternate routes of dexmedetomidine administration; however, there is evidence of neurotoxicity with mcg/kg epidurally in an animal model [ ] . as mentioned, dexmedetomidine has gained popularity in large part related to its ability to reduce reliance on opioids in postsurgical analgesia. shariffuddin et al. demonstrated in a double-blinded, randomized controlled study that a single preoperative dose of dexmedetomedine . μg kg − in patients undergoing either ureteroscopy or ureteric stenting resulted in a clinically significant reduction of anesthetic and opioid use both intraoperatively and postoperatively. they reported a reduction of the mac of sevoflurane ( . ( . ) vs. . ( . ), p = [ , ] . - mcg/kg . - . mcg/kg/h iv pca (with opioid) [ , ] . - mcg or . mcg/kg . - . mcg/kg/h basal rate or . - mcg/h - -min lockout iv pca (as sole agent) [ ••] . mcg . mcg/kg/h -min lockout oral [ ] m c g / k g intranasal [ , ] - mcg/kg buccal [ ] . mcg/kg intramuscular [ ] . mcg/kg iv, intravenous; pca, patient-controlled analgesia; min, minute . ) needed to achieve adequate sedation, as well as a % reduction of pain immediately postop with further reduction lasting until pod [ ] . panchgar et al. had similar results in laparoscopic surgeries with a loading dose of μg/kg body weight and then a maintenance dose of . μg/kg/h for the remainder of the procedure. time to rescue analgesia postoperatively was min in the control group vs. min in the dexmedetomidine group. the total -h analgesic need was also significantly less for the dexmedetomidine group ( mg) vs. the ns control group ( mg) [ ] . a recent meta-analysis involving studies and patients showed that dexmedetomidine used in conjunction with opioids in patient-controlled analgesia resulted in lower overall opioid utilization with no increase in adverse reactions [ ] . dexmedetomidine has been paired with propofol to achieve opioid-free total intravenous anesthesia in gynecologic laparoscopy. it demonstrated improved pain scores, delayed rescue analgesia, and decreased total rescue analgesic dose [ ] . the effects of dexmedetomidine on local anesthetic and nerve blocks have also been studied. dexmedetomidine significantly prolonged postoperative analgesia in children undergoing ilioinguinal/iliohypogastic nerve block for hernia repair [ ] . it had better analgesia and fewer adverse reactions than fentanyl when added to bupivacaine for epidural anesthesia [ ] . it similarly outperformed fentanyl when combined with ropivacaine administered intraperitoneally to control pain following laparoscopic cholecystectomy [ ] . other studies have shown that dexmedetomidine can significantly reduce the incidence of postoperative nausea [ ] . song et al. demonstrated that within a high-risk group, dexmedetomidine administered min before the completion of surgery reduced the frequency and severity of nausea [ ] . postoperative delirium is another complication that could potentially benefit from the addition of dexmedetomidine. one study showed that there was a reduction in both the incidence and severity of delirium in pod - in patients undergoing pulmonary resection due to lung carcinoma [ ] . other trials demonstrated that dexmedetomidine may decrease negative postoperative behavioral changes and agitation in pediatric patients without excessive sedation or other negative side effects [ , ] . the major side effect noted in many of these studies was hemodynamic instability in the form of bradycardia and hypotension [ , , ] . these changes, although statistically significant, were well tolerated by most of study participants. some studies suggest that this is beneficial in that it curtails hemodynamic stress response generated by the trauma of surgery [ ] . still other studies did not find dexmedetomidine to be superior to other drugs. one such study found that fentanyl p r o v i d e d l o n g e r p o s t o p e r a t i v e a n a l g e s i a t h a n dexmedetomidine when added to lidocaine in women undergoing spinal epidural during elective c-section, although it did have a higher incidence of nausea and vomiting [ ] (see table ). enhanced recovery after surgery is an approach to patient care that focuses on optimizing the postoperative period. this includes implementing protocols meant to reduce postoperative complications, patient discomfort, and length of hospital stay. dexmedetomidine is a highly selective a -adrenergic agonist, which has become a valuable addition to the multimodal approach to anesthesia. its sedative, anxiolytic, and analgesic properties are useful in potentiating postoperative analgesia. these features make it a useful adjuvant to the anesthesia protocol, especially in the context of enhanced recovery after surgery. dexmedetomidine acts in the locus ceruleus and spinal cord, inhibiting presynaptic release of norepinephrine. this results in sedation, analgesia, and a centrally mediated sympatholytic effect [ ] . use of dexmedetomidine has been shown to reduce the anesthetic and opioid requirements both intraoperatively and postoperatively. dexmedetomidine has also been shown to reduce the incidence of postoperative nausea, vomiting, delirium, and agitation with minimal effect on respiratory drive [ ] . these features make it a valuable tool in achieving the goals of enhanced recovery after surgery. many of its on and off label uses have been studied. it has been used for sedation in the icu, as an adjuvant for epidural and peripheral nerve block, and for preprocedure anxiolysis [ ] . more research is warranted to better understand dexmedetomidine's far reaching applications. the main adverse event healthcare workers should be cautious of when administering dexmedetomidine is hemodynamic instability, namely bradycardia, hypotension, and hypertension. song et al. found that patients who received dexmedetomidine intraoperatively had almost a -fold increase in bradycardia compared with the control group [ ] . meanwhile, shariffudin et al. found a significant decrease in the systolic blood pressure at the -min mark after infusion. this phenomena disappeared by the -min mark and did not return for the remainder of the case [ ] . it appears that these episodes of hemodynamic disturbance are associated with the use of a loading dose or fast initial infusion rates. one way to mitigate this is to forgo a loading bolus and instead utilize a slower basal infusion rate. although these hemodynamic changes do not cause issue for the majority of patients, healthcare providers should use caution when administering dexmedetomidine to patients who are less able to tolerate bradycardia. these might include patients with cardiac conduction abnormalities, those taking medications that alter cardiac conduction, and the elderly [ ] . additionally, care should be taken to adjust the dosing in patients with hepatic impairment, as dexmedetomidine is predominantly metabolized in the liver [ ] . although dexmedetomidine has proven to be a relatively safe drug, the lack of a reversal agent is an issue. atipamezole is a synthetic a -antagonist that has been shown to reverse the actions of dexmedetomidine; however, it is currently only approved for use in dogs. more studies are needed to assess the effectiveness and safety of atipamezole in humans. g a i n i n g a p p r o v a l f o r h u m a n u s e w o u l d m a k e dexmedetomidine an even more attractive option [ ] . there is currently a paucity of data addressing the potential neuroprotective, cardioprotective, and renoprotective effects of dexmedetomidine. the trials that have been conducted are mostly animal models, but have been encouraging enough to merit future research in humans [ ] . conflict of interest david chernobylsky, pankaj thakur, harish siddaiah, rachel kaye, lauren eng, monica harbell, jared lajaunie, and elyse cornett declare no conflict of interest. alan kaye is a section editor for current headache and pain reports. he has not been involved in the editorial handling of this manuscript. dr. kaye is also a speaker for merck. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. enhanced recovery after surgery (eras) protocols: time to change practice? asra news -review of dexmedetomidine (precedex) for acute pain and analgesia -american society of regional anesthesia and pain medicine multimodal analgesia for controlling acute postoperative pain multimodal approaches to analgesia in enhanced recovery after surgery pathways dexmedetomidine in anaesthesia the effects of dexmedetomidine and remifentanil on hemodynamic stability and analgesic requirement after craniotomy the effect of dexmedetomidine on postoperative opioid consumption and pain after major spine surgery effect of dexmedetomidine alone for intravenous patient-controlled analgesia after gynecological laparoscopic surgery efficacy of intraoperative dexmedetomidine compared with placebo for postoperative pain management: a meta-analysis of published studies dexmedetomidine versus remifentanil in postoperative pain control after spinal surgery: a randomized controlled study facilitatory effects of perineural dexmedetomidine on neuraxial and peripheral nerve block: a systematic review and meta-analysis dexmedetomidine: new avenues dexmedetomidine infusion during laparoscopic bariatric surgery: the effect on recovery outcome variables clinical pharmacokinetics and pharmacodynamics of dexmedetomidine dexmedetomidine in current anaesthesia practicea review publications private limited alpha- adrenergic receptor agonists: a review of current clinical applications clinical pharmacokinetics and pharmacodynamics of dexmedetomidine prolonged dexmedetomidine infusion and drug withdrawal in critically ill children dexmedetomidine attenuates oxidative stress induced lung alveolar epithelial cell apoptosis in vitro dexmedetomidine: sedative of choice in substance abuse dexmedetomidine infusion to facilitate opioid detoxification and withdrawal in a patient with chronic opioid abuse dexmedetomidine-associated bradycardia progressing to pulseless electrical activity: case report and review of the literature dexmedetomidine enhances ropivacaine-induced sciatic nerve injury in diabetic rats prolonged duration local anesthesia using liposomal bupivacaine combined with liposomal dexamethasone and dexmedetomidine safety and efficacy of buccal dexmedetomidine for mri sedation in school-aged children dexmedetomidine for the prevention of emergence delirium and postoperative behavioral changes in pediatric patients with sevoflurane anesthesia: a double-blind, randomized trial intraoperative use of dexmedetomidine for the prevention of emergence agitation and postoperative delirium in thoracic surgery: a randomized-controlled trial effect of perioperative systemic α agonists on postoperative morphine consumption and pain intensity: systematic review and meta-analysis of randomized controlled trials efficacy of intraoperative dexmedetomidine compared with placebo for surgery in adults: a meta-analysis of published studies dexmedetomidine for craniotomy under general anesthesia: a systematic review and meta-analysis of randomized clinical trials perioperative analgesic profile of dexmedetomidine infusions in morbidly obese undergoing bariatric surgery: a meta-analysis and trial sequential analysis dexmedetomidine as a sedative and analgesic adjuvant in spine surgery: a systematic review and meta-analysis of randomized controlled trials dexmedetomidine reduces perioperative opioid consumption and postoperative pain intensity in neurosurgery medicine (baltimore). ; :e . excellent study of dexmedetomidine reducing opioid comsumption postoperatively effect of intravenous dexmedetomidine during general anesthesia on acute postoperative pain in adults optimizing perioperative analgesia for the complex pain patient: medical and interventional strategies perioperative dexmedetomidine for acute pain after abdominal surgery in adults effects of preanesthetic administration of midazolam, clonidine, or dexmedetomidine on postoperative pain and anxiety in children effect of dexmedetomidine on preventing postoperative agitation in children: a meta-analysis evidence for the efficacy of systemic opioid-sparing analgesics in pediatric surgical populations the effect of pre-anaesthetic administration of intravenous dexmedetomidine on postoperative pain in patients receiving patient-controlled morphine intraoperative infusion of dexmedetomidine reduces perioperative analgesic requirements effects of intravenous lidocaine, dexmedetomidine and their combination on postoperative pain and bowel function recovery after abdominal hysterectomy the efficacy of ultrasound-guided type ii pectoral nerve blocks in perioperative pain management for immediate reconstruction after modified radical mastectomy. a prospective, randomized study comparison of the pro-postoperative analgesia of intraoperative dexmedetomidine with and without loading dose following general anesthesia intra-operative analgesia with remifentanil vs. dexmedetomidine: a systematic review and meta-analysis with trial sequential analysis dexmedetomidine and sufentanil combination versus sufentanil alone for postoperative intravenous patient-controlled analgesia: a systematic review and meta-analysis of randomized controlled trials optimization of postoperative intravenous patient-controlled analgesia with opioiddexmedetomidine combinations: an updated meta-analysis with trial sequential analysis of randomized controlled trials adjuvant agents in regional anesthesia in the ambulatory setting. curr pain headache rep dexmedetomidine in perioperative acute pain management: a non-opioid adjuvant analgesic dexmedetomidine as an adjuvant to local anesthetics in transversus abdominis plane block evidence basis for using perineural dexmedetomidine to enhance the quality of brachial plexus nerve blocks: a systematic review and meta-analysis of randomized controlled trials efficacy and safety of dexmedetomidine as an adjuvant in epidural analgesia and anesthesia: a systematic review and meta-analysis of randomized controlled trials dexamethasone is superior to dexmedetomidine as a perineural adjunct for supraclavicular brachial plexus block safety and sedative effect of intranasal dexmedetomidine in mandibular third molar surgery: a systematic review and meta-analysis. drug des dev ther formulation optimization and assessment of dexamethasone orally disintegrating tablets using box-behnken design dexmedetomidine added to an opioid-based analgesic regimen for the prevention of postoperative nausea and vomiting in highly susceptible patients: a randomised controlled trial perioperative dexmedetomidine reduces delirium in elderly patients after lung cancer surgery opioid-free total intravenous anesthesia improves postoperative quality of recovery after ambulatory gynecologic laparoscopy comparison of the morphinesparing effect of intraoperative dexmedetomidine with and without loading dose following general anesthesia in multiple-fracture patients mechanism-based population pharmacokinetic and pharmacodynamic modeling of intravenous and intranasal dexmedetomidine in healthy subjects bioavailability of dexmedetomidine after intranasal administration bioavailability of dexmedetomidine after extravascular doses in healthy subjects oral ketamine and dexmedetomidine in adults' burns wound dressing-a randomized double blind cross over study comparison of buccal and intramuscular dexmedetomidine premedication for arthroscopic knee surgery the pharmacokinetics and hemodynamic effects of intravenous and intramuscular dexmedetomidine hydrochloride in adult human volunteers pharmacodynamics and pharmacokinetics of intramuscular dexmedetomidine comparison of dexmedetomidine and fentanyl as local anesthetic adjuvants in spinal anesthesia: a systematic review and meta-analysis of randomized controlled trials effect of singledose dexmedetomidine on postoperative recovery after ambulatory ureteroscopy and ureteric stenting: a double blind randomized controlled study the effectiveness of intravenous dexmedetomidine on perioperative hemodynamics, analgesic requirement, and side effects profile in patients undergoing laparoscopic surgery under general anesthesia effect of dexmedetomidine as an adjuvant to ropivacaine in ilioinguinaliliohypogastric nerve blocks for inguinal hernia repair in pediatric patients: a randomized, double-blind, control trial dexmedetomidine versus fentanyl added to bupivacaine for epidural analgesia in combination with general anesthesia for elective lumbar disc operations: a prospective, randomized double-blinded study intraperitoneal ropivacaine with dexmedetomidine or fentanyl for postoperative analgesia following laparoscopic cholecystectomy: a comparative randomized trial effect of single-dose dexmedetomidine on intraoperative hemodynamics and postoperative recovery during pediatric adenotonsillectomy comparison of dexmedetomidine and fentanyl as an adjuvant to lidocaine % for spinal anesthesia in women candidate for elective caesarean. open access the comparison of dexmedetomidine and midazolam premedication on postoperative anxiety in children for hernia repair surgery: a randomized controlled trial postoperative pain control after the use of dexmedetomidine and propofol to sedate patients undergoing ankle surgery under spinal anesthesia: a randomized controlled trial dexmedetomidine and general anesthesia: a narrative literature review of its major indications for use in adults undergoing non-cardiac surgery dexmedetomidine: review, update, and future considerations of paediatric perioperative and periprocedural applications and limitations dexmedetomidine: present and future directions publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - ht cqhu authors: smith, silas w. title: drugs and pharmaceuticals: management of intoxication and antidotes date: - - journal: molecular, clinical and environmental toxicology doi: . / - - - - _ sha: doc_id: cord_uid: ht cqhu the treatment of patients poisoned with drugs and pharmaceuticals can be quite challenging. diverse exposure circumstances, varied clinical presentations, unique patient-specific factors, and inconsistent diagnostic and therapeutic infrastructure support, coupled with relatively few definitive antidotes, may complicate evaluation and management. the historical approach to poisoned patients (patient arousal, toxin elimination, and toxin identification) has given way to rigorous attention to the fundamental aspects of basic life suppport — airway management, oxygenation and ventilation, circulatory competence, thermoregulation, and substrate availability. selected patients may benefit from methods to alter toxin pharmacokinetics to minimize systemic, target organ, or tissue compartment exposure (either by decreasing absorption or increasing elimination). these may include syrup of ipecac, orogastric lavage, activated single- or multi-dose charcoal, whole bowel irrigation, endoscopy and surgery, urinary alkalinization, saline diuresis, or extracorporeal methods (hemodialysis, charcoal hemoperfusion, continuous venovenous hemofiltration, and exchange transfusion). pharmaceutical adjuncts and antidotes may be useful in toxicant-induced hyperthermias. in the context of analgesic, anti-inflammatory, anticholinergic, anticonvulsant, antihyperglycemic, antimicrobial, antineoplastic, cardiovascular, opioid, or sedative-hypnotic agents overdose, n-acetylcysteine, physostigmine, l-carnitine, dextrose, octreotide, pyridoxine, dexrazoxane, leucovorin, glucarpidase, atropine, calcium, digoxin-specific antibody fragments, glucagon, high-dose insulin euglycemia therapy, lipid emulsion, magnesium, sodium bicarbonate, naloxone, and flumazenil are specifically reviewed. in summary, patients generally benefit from aggressive support of vital functions, careful history and physical examination, specific laboratory analyses, a thoughtful consideration of the risks and benefits of decontamination and enhanced elimination, and the use of specific antidotes where warranted. data supporting antidotes effectiveness vary considerably. clinicians are encouraged to utilize consultation with regional poison centers or those with toxicology training to assist with diagnosis, management, and administration of antidotes, particularly in unfamiliar cases. the challenges to effective evaluation and management of a patient poisoned by drugs and pharmaceuticals are diverse. the circumstances surrounding exposure are often incompletely accessible. poisoning signs or symptoms may be subtle or delayed. patient-specific factors -pharmacogenetics and unique susceptibilities, drug-drug interactions, cultural or geographic practices, and underlying comorbidities -may complicate presentation, response to treatment, and outcome. polypharmacy or mixed exposures may confuse the clinical presentation. compared to the near-inexhaustible list of products and possible combinations, few specific antidotes exist. the toxicological profiles of newly intro-organ injury. specific management of toxicant-induced hyperthermias follows later. hypothermia may require active or passive rewarming techniques. clinical hypoglycemia, which implies neuroglycopenia, must be rapidly reversed with administration of . - . g/kg of age-appropriate dextrose-containing solutions (d in adults, d in children, and d in neonates). benzodiazepines (e.g., diazepam, midazolam, and lorazepam) are generally well tolerated and are first line agents for drug-and withdrawal-induced seizures and agitation. persistent or refractory seizures should prompt consideration of empiric administration of pyridoxine and barbiturates (phenobarbital, pentobarbital), propofol, or ultimately, general anesthesia. coincident endotracheal intubation may be required. phenytoin and non-barbiturate anticonvulsants are typically ineffective or harmful in toxin-induced seizures [ , ] . altered mental status should also prompt parenteral administration of mg thiamine hydrochloride. alcohol-dependent patients without clinically apparent wernicke's encephalopathy may require at least mg of parenteral thiamine to improve neurological symptoms; overt wernicke's encephalopathy necessitates a minimum of mg thiamine hydrochloride three times daily for - days [ ] . naloxone use is considered in a separate section. toxidromes (toxic syndromes) are characteristic signs and symptoms that correlate with exposure to certain xenobiotics. identifying toxidromes suggests the etiology of the patient's condition and helps guide management. "classic" class-effect toxidromes include anticholinergic, cholinergic, sedative-hypnotic, sedative-hypnotic withdrawal, opioid, and opioid withdrawal. these should be actively sought and managed if identified. while the patient is being stabilized, diagnostic investigations including a complete and thorough history and physical examination, laboratory analyses, and radiological studies may be undertaken to further characterize the exposure and effect. for significantly compromised patients, a typical "chemistry panel" (providing electrolytes, blood urea nitrogen, creatinine, and indirectly the anion gap), a complete blood count, arterial (or venous) blood gas, and lactate are reasonable studies. urine or serum ketones may be required to determine the etiology of acidemia. female patients benefit from an assessment of pregnancy status. it is useful to determine a serum acetaminophen concentration in suicidal patients or those with altered consciousness, as patients with significant acetaminophen poisoning may present without a toxidrome. serum acetaminophen is detectable in - % of patients without a reported history of ingestion; treatable concentrations are found slightly less frequently [ , ] . toxin-specific studies and other serum determinations are often not rapidly returned and should be obtained only if suggested by the history, physical examination, or bedside testing. urine drug screening (uds) is of minimal use in the acute management of intoxication. results are not typically returned for hours; a reported "positive" substance may not be the proximate cause of the presenting condition (as the measured metabolites may persist in urine for days to weeks); and the uds lacks sensitivity and specificity (particularly for opioids, benzodiazepines and other sedative-hypnotics, and amphetamines). selected patients may benefit from methods to alter toxin pharmacokinetics -limiting exposure. a discussion of these modalities and their risks and benefits occurs in the following section. ultimately, patients will require disposition depending of severity of presentation and anticipated sequelae, which may range from admission to intensive care units, cardiac monitoring (telemetry) units, ward beds, continued emergency department evaluation, to discharge. a psychiatric assessment and social assessment, when appropriate, should precede release from medical care. appropriate and early consultation with medical toxicologists or regional poison centers may also assist with diagnosis and management. in the u.s., this has been simplified by a uniform telephone number ( . . . ) for regional poison center consultation. the international programme on chemical safety (ipcs) maintains a world directory of poison centers (http://www.who.int/ipcs/poisons/centre/directory/en/). adjuncts to alter toxicant pharmacokinetics aim to minimize systemic exposure (either by decreasing absorption or increasing elimination) or to minimize exposure of a target organ or tissue compartment. in practice, this is achieved by expulsion or removal from the upper gastrointestinal tract (induced emesis, gastric lavage, or endoscopy); intraluminal binding to adsorptive materials (activated charcoal); or increasing intestinal transit time (cathartics and whole bowel irrigation). endogenous elimination may be improved by more effective urinary clearance (urinary alkalinization and forced diuresis), improved hepatobiliary clearance, or "gut dialysis" with multiple-dose activated charcoal. rarely, hepatic metabolism is altered to preclude ultimate toxicant formation (e.g., cimetidine to mitigate production of dapsone's methemoglobinemia inducing metabolite). exogenous clearance utilizes hemodialysis, charcoal hemoperfusion, continuous renal replacement therapies, and exchange transfusion. all the adjuncts attempt to shift where a patient lies upon a particular dose-response curve (fig. ) . drug recovery following gastrointestinal emptying techniques has been inconsistent; human studies attempting to demonstrate a survival benefit of any decontamination modality are inconclusive. randomized trials in which a control group might not receive any decontamination could be considered unethical; volunteer studies using sublethal doses of xenobiotic cannot show mortality benefit. as might be anticipated from the fact that supportive care suffices for the majority of poisoned patients, a typical study of routine administration of charcoal following oral overdose of primarily benzodiazepines, acetaminophen, and selective serotonin reuptake inhibitors could not demonstrate benefit [ , , ] . past studies have suffered from significant exclusions. recommendations are based both on theoretical grounds (animal and in vitro studies demonstrating lower peak serum concentration or faster serum drugs and pharmaceuticals: management of intoxication and antidotes clearance) and human studies with surrogate endpoints such as marker studies or area under the curve of plasma concentration versus time (auc) improvement. aggressive detoxification may be required for certain lethal toxins for which few antidotal options exist. most gastric emptying techniques are thought to be relatively ineffective beyond hour. these constraints diminish possible benefit. for example, the median time from ingestion to arrival at a health care facility is on the order of hours, and only about % of patients can be lavaged within the idealized -hour time frame [ ] . although in ideal situations (patients presenting early to experienced health care providers with readily available ipecac syrup) pill retrieval averages - %, ipecac's benefits can be completely negated when administration is delayed as briefly as min [ ] [ ] [ ] [ ] . when orogastric lavage is performed by experienced providers within min of ingestion, clinical manifestations of ingested xenobiotics have been prevented [ ] . practically, efficacy of tablet retrieval rates reduces to % in some cases and improvements in auc vary from zero to % (averaging ~ %) [ , [ ] [ ] [ ] . similarly, restricting activated charcoal (ac) administration to patients presenting to health care within the first hour post ingestion would exclude up to % of poisoned patients from the potential benefits of ac when administered beyond an hour [ , ] . earlier administration of ac is more efficacious [ ] . however, home and prehospital use of ac decreases the time to treatment, but has not improved clinical outcomes [ ] . drugs with opioid or anticholinergic properties that decrease peristalsis or particularly large ingestions, which independently decrease intestinal motility, may modify decision making in delayed presentations [ , ] . figure . adjuncts to alter toxicant pharmacokinetics attempt to shift where a patient lies upon a particular (idealized) dose-response curve. risk will likely outweigh benefit if the patient begins at point a (negligible morbidity and mortality) and systemic exposure is reduced to b. this is the case for many drug poisonings which are managed effectively by supportive care alone. decontamination might provide significant benefit if the patient lies upon the steep aspect of the curve [reduction from c to d -the same fixed amount as from a to b (although a percentage reduction could also be envisaged)]. with overwhelming overdose (point e), despite decontamination, benefit would be unlikely (point f). independent of side effects, the efficacy of one modality over another or combination therapy is debated. some studies rate ipecac syrup more efficacious than orogastric lavage, but most studies have found little or no difference, and neither has been shown to be more effective than spontaneous emesis [ , , , ] . ac has demonstrated ~ % better reductions in auc than ipecac, which may improve or worsen its efficacy [ , , ] . gastric lavage adds no benefit to ac, except for the most critically ill patients [ , , ] . compared directly, ac has better impact than lavage on auc and clinical effect [ , , ] . data are equivocal regarding whole bowel irrigation's ability to function similar to multiple-dose ac (mdac) as a medium for "gut dialysis" [ , ] . syrup of ipecac is obtained from a root extract of the amazonian flowering plant psychotria ipecacuanha [ ] . its active alkaloid components, cephaeline and emetine, induce emesis via local irritation and central stimulation of -hydroxytryptamine (serotonin) -ht receptors [ ] . following appropriate dose ( ml for infants, - ml for children under , and ml otherwise), roughly % of patients have a first episode of emesis within min [ , ] . patients average three episodes in min [ ] . however, since ipecac's removal from most homes, the median time to administration in the acute care setting is delayed on the order of an hour, with only one-third of patients successfully vomiting within the first hour post ingestion [ ] . indications for ipecac are limited. a routinely cited example is a patient known to have taken multiple lithium tablets, which do not bind ac and may not fit through a lavage tube, who presents early to health care [ ] . the american academy of pediatrics no longer recommends ipecac syrup for home use; ipecac use does not impact outcomes or decrease utilization of emergency services [ , ] . ipecac may or may not have a role in other rare ingestions that mandate gastrointestinal decontamination, but are not amenable to orogastric lavage, ac, whole bowel irrigation, or an antidote; the patient must present alert and early (< min post ingestion) to medical care [ ] . unsurprisingly, ipecac's most common side effect is persistent emesis. as many as eight emetic episodes occurring more than min after ipecac administration have been reported [ ] . this impairs administration of oral therapeutic agents, as induced emesis can last up to several hours [ ] . prolonged vomiting associated with induced sedation or absent airway reflexes increases the risk of aspiration bronchospasm, pneumonitis, and pneumonia [ , ] . other life-threatening side effects have been reported, including bradycardia, cns depression, mallory-weiss esophageal tears, pneumomediastinum, pneumoretroperitoneum, and intracranial hemorrhage [ ] . emesis of caustics reexposes damaged esophageal mucosa to the caustic agent. analogous pulmonary aspiration concerns accompany induced emesis of hydrocarbons. orogastric lavage is performed via a large bore orogastric tube (adults, [ ] [ ] [ ] [ ] [ ] french; children, - french) with fenestrae large enough to accommodate whole tablets [ ] . serial -ml aliquots ( - ml in pediatric patients) of normal saline or lactated ringer's solution are administered and suctioned until retrieved liquid is clear. orogastric lavage can be expected to have its best risk-to-benefit ratio when patients present early enough to have a significant gastric burden, and when severe toxicological effects are manifest or expected to become manifest [ , ] . because advancement of stomach contents does occur despite proper left lateral decubitus positioning [ ] , ac (see below) is sometimes provided prior to crystalline lavage [ , ] . introduction of a large, relatively rigid tube requires a cooperative patient with a protected airway (typically an endotracheal tube if the patient is ill enough to warrant gastric lavage). orogastric lavage risks hypoxia, dysrhythmia, laryngospasm, hypothermia, gastrointestinal or pharyngeal traumatic laceration or perforation, fluid and electrolyte abnormalities, and vomiting with subsequent aspiration pneumonia [ , , ] . ac is a convoluted macromolecule created via pyrolysis of carbonaceous material and subsequently "activated" with steam to further increase surface area [ ] . the multiple pores of various size on the surface of each macromolecule of ac account for its high adsorptive affinity for a multitude of xenobiotics -particularly chemical species that are nonionized, aromatic, and/or branched [ , , ] . maximal xenobiotic binding occurs in - min [ ] . ac decreases auc by as much as %, seems to improve clinical outcomes for critically ill patients, and may benefit in certain poisonings such as acetaminophen [ , , , ] . it also increases the rate of endogenous clearance of drugs with long half-lives and some degree of entero-enteric or enterohepatic circulation [ , , ] . those findings suggested the use of mdac as a "gut dialysis" for toxins with slow pharmacokinetics [ , ] . a meta-analysis of volunteer studies demonstrated increased clearance of xenobiotics with longer half-lives, but not necessarily improved clinical outcome [ , ] . mdac has enhanced amitriptyline, carbamazepine, dapsone, dextromethorphan, phenobarbital, phenytoin, quinine, and theophylline elimination, although without definitive clinical benefit in controlled trials [ , , ] . two studies provided conflicting results for survival benefit of mdac for yellow oleander poisoning [ , ] . the fraction of unbound xenobiotic decreases as the charcoal-to-toxin ratio increases from . : up to : , although the yield curve levels off near : [ , ] . in theory, the dose of ac administered to a poisoned patient would be ten times the mass of ingested xenobiotic, but those values are unknown in most clinical situations [ ] . ac is practically dosed based on the patient's weight ( g/kg), which can be divided into multiple smaller doses to be administered every - hours [ ] . although optimum dosing is unclear, mdac is administered hourly, every hours, or every hours at a dose equivalent to . g/hour [ ] . pediatric charcoal doses are lower due to generally smaller ingestions and gut capacity. the total dose administered is the major determinant of efficacy particularly for larger overdoses, and can be administered continuously [ ] . emesis occurs in up to % of patients receiving ac; patients receiving ac via nasogastric tube or who vomited previously are at greater risk for emesis [ , ] . rarer complications include aspiration and intestinal obstruction or perforation [ , , , ] . aspirated ac may produce bronchiolitis obliterans, acute respiratory distress syndrome (ards), and death [ ] . ac adheres to mucosa and obscures endoscopy; mineral acids and bases will not adhere to charcoal. ac poorly adsorbs short chain alcohols and metals such as iron, lead, and lithium [ ] . ac administration requires an intact mental status or protected airway. flavoring agents increase the palatability of ac for volunteers, but poisoned patients do not show increased compliance/tolerance with flavored ac [ ] . cathartics induce watery evacuation of bowel within a few hours. hyperosmotic cathartic agents such as sorbitol are non-absorbed, osmotically active substances that draw water into the lumen, where increased intestinal volume and pressure promote peristalsis. so-called "saline" cathartic agents such as magnesium salts also directly stimulate smooth muscle to induce peristalsis [ ] . cathartics alone are not recommended for ingested poisons [ ] . cathartics have many adverse effects, including volume depletion, hypernatremia, hypermagnesemia, hyperphosphatemia, hypocalcemia, metabolic alkalosis, pain, nausea, emesis, and flatus [ , ] . sorbitol or laxatives are sometimes used in conjunction with the first dose of ac. while theoretically beneficial -minimizing the possible constipation of ac or promptly delivering ac to the duodenum, they do not increase the efficacy of ac [ , , ] . sorbitol is implicated in the fluid/electrolyte changes that occur with mdac: hypermagnesemia, hypernatremia, and volume depletion [ , , ] . repetitive cathartic doses have been associated with rectal prolapse and death [ , ] . whole bowel irrigation (wbi) employs polyethylene glycol (peg), a large, non-absorbable organic polymer and an electrolyte lavage solution (els) is-drugs and pharmaceuticals: management of intoxication and antidotes osmotic to serum. large peg-els volumes are introduced into the alimentary canal with less risk for fluid and electrolyte shifts caused by traditional cathartics. peg-els provides non-viscous bulk for rapid transit of material in a normally functioning gastrointestinal tract. wbi should induce evacuation within min, but requires hours on average for complete effect. reported improvements in auc are modest given the more rapid absorption time for most pharmaceuticals [ ] . however, reduction in auc can be as high as % with poorly absorbed products or modified release preparations [ ] . wbi might be considered for slowly absorbed significant ingestions such as iron, lead, and lithium, as well as modified-release preparations of β-adrenergic antagonists, bupropion, calcium-channel antagonists, carbamazepine, and theophylline [ ] [ ] [ ] [ ] . wbi is also employed to rid patients of enterally transported illicit substances which produce toxicity upon packet rupture or leakage (e.g., cocaine, heroin, and methamphetamine) [ ] . standard dosing protocols are . - l/h ( ml/kg per h) enterally until rectal effluent is clear [ ] . at this point, intestinal contents are assumed to have been displaced, although this is not always true [ , ] . nasogastric tube placement is generally required to sustain compliance with the large volume requirements, and pretreatment with an antiemetic is prudent [ ] . wbi may produce nausea, vomiting, cramping, and flatus. peg-els for colonoscopy has precipitated colonic perforation [ ] . unintentional bronchial administration of peg-els can produce acute lung injury [ ] . ileus, obstruction, perforation or threatened perforation should preclude wbi; a protected airway is required. desorption of toxins from ac by peg has been demonstrated in vitro and in vivo [ , ] . support for endoscopic therapy consists of limited case reports of retrieval in ingestions of cocaine packets, lead pellets, and medication such as sustained release calcium channel antagonists, clomipramine, iron, and meprobamate [ ] [ ] [ ] [ ] [ ] . the procedure might be warranted for certain ingestions or cases of pharmacobezoar formation of toxic substances. complications include perforation, aspiration, hemorrhage, and anesthetic-associated hemodynamic changes. when endoscopy fails, surgery may be required for definitive removal [ , ] . surgery may be required in patients with enterally transported illicit substances either due to failure of passage (with or without wbi), obstruction, or severe toxicity upon packet rupture or leakage [ , ] . weak acids in an alkaline environment exist predominantly in ionized form. biological membranes are relatively impermeable to these charged molecules. alkaline serum thus inhibits the diffusion of acidic toxins (low pk a ) across cellular membranes. similarly, an alkaline urinary ph promotes renal sequestration (or "ion-trapping") of acidic species from the systemic circulation. the relative intolerance of biological systems to acidosis limits the effectiveness of converse urinary acidification (via ascorbic acid or diluted hcl solutions) for renal sequestration of weak bases. critically ill patients may have reduced drug clearances due to decreased hepatic and renal perfusion, and thus interventions that increase clearance/ elimination have the potential to significantly reduce toxicity [ ] . alkalinization improves renal elimination of chlorpropamide, , -dichlorophenoxyacetic acid, diflunisal, fluoride, mecoprop, methotrexate, phenobarbital, and salicylate [ ] . urine alkalinization is considered first line therapy in patients with moderate salicylism who do not meet hemodialysis indications. dosing of - meq/kg of . - . % bicarbonate provided over - min is followed by "normal" bicarbonate infused at double the standard rate of i.v. fluid maintenance. the "normal" bicarbonate solution is prepared by adding three ampules of sodium bicarbonate (totaling - meq) in l % dextrose in water (d w). the rate is titrated to maintain an alkaline urinary ph, without exceeding a serum ph of . [ ] . alkalemia decreases ionized calcium. volume overload may occur, particularly in patients with congestive heart failure, acute renal failure, or end-stage liver disease. bicarbonate treatment induces hypokalemia. as the proximal renal tubular cells conserve serum potassium by exchanging protons for urinary potassium, this defeats urinary alkalinization. therefore, maintaining a normal serum potassium, with frequent monitoring and supplemental administration and/or inclusion in the bicarbonate solution, are important components of urine alkalinization. saline diuresis is utilized to improve excretion and minimize toxicity of overdose of ions such as magnesium, calcium, and lithium in patients who do not meet hemodialysis indications [ ] [ ] [ ] . hypermagnesemia may occur with excessive antacid use, gargling or ingesting magnesium sulfate compounds, and iatrogenic error [ , ] . hypercalcemia can result from excess calcium (in antacid tablets) or vitamin d ingestion or parenteral administration [ , ] . renal lithium toxicity presumably results from cytotoxic accumulation of lithium entering via the apical epithelial sodium channel [ ] . ensuing nephrogenic diabetes insipidus, characterized by increased water and sodium diuresis, can result in dehydration, hyperchloremic metabolic acidosis, and renal tubular acidosis. in volume depletion, activation of the reninangiotensin-aldosterone axis leads to active resorption of sodium, and thus lithium, from the distal convoluted tubules. therefore, adequate volume repletion with saline is prerequisite for effective renal elimination of lithium. boluses of . % sodium chloride are administered until the patient is clinically euvolemic. saline infusion is then provided at . - times a standard maintenance rate. throughout treatment renal function, urine output, and electrolytes are monitored. congestive heart failure, renal failure, or end-stage liver disease moderate volume administration and make saline diuresis less attractive than hemodialysis in significant ingestions. loop diuretics such as furosemide inhibit sodium resorption in the proximal convoluted tubules, and would theoretically promote elimination of lithium as natriuretics. however, these effects are countered by the action of the renin-angiotensin-aldosterone axis on the distal convoluted tubules, and diuretics do not seem to improve outcomes in lithium overdose or radiographic contrast exposure [ , ] . in hemodialysis (hd) the patient's blood is pumped through a circuit that includes a cartridge consisting of thousands of semi-permeable, membranelined capillary tubes. the blood traverses the cartridge counter-current to a circulating buffered salt solution (a.k.a. dialysate) before returning to the patient's venous circulation. diffusible molecules flow down their electrochemical gradient from the serum to the dialysate. hemoperfusion (hp) employs a similar circuit, but the cartridge is enveloped with ac (rather than a circulating dialysate) to adsorb xenobiotics regardless of plasma protein binding, and leave serum electrolytes largely unchanged. continuous arteriovenous or venovenous hemofiltration (cavh or cvvh) employ lower pressures and flow rates than hd over longer sessions for patients unable to tolerate hd or to remove xenobiotics with slow tissue redistribution [ , ] . peritoneal dialysis (pd) is ineffective in poisoning management, given its inherently slow kinetics and the availability of hd [ ] . extracorporeal therapies may be warranted when criteria are met for both the xenobiotic and the patient [ ] . favorable dialyzable toxin properties include low volume of distribution (v d ), relatively low molecular weight, and poor serum protein binding (or binding that worsens in overdose, as is the case for salicylate and valproate) [ ] . patient characteristics suggesting extracorporeal therapy include signs or symptoms of significant end organ toxicity; impaired elimination secondary to baseline comorbidities or critical illness-induced hypoperfusion; inability to tolerate or refractory to antidotal strategies (such as bicarbonate or saline); inadequate response to supportive care measures; concurrent electrolyte derangements (e.g., metformin-associated lactic acidosis); or serum drug concentrations historically associated with severe outcome [ ] . traditionally, charcoal hp was used for xenobiotics significantly bound to plasma proteins, but its use is declining while (high-flux membrane) hd increases. methanol, ethylene glycol, salicylates, lithium, halides, theophylline, and metformin-associated lactic acidosis are commonly treated with dialysis [ ] . hd is used for valproate and carbamazepine poisoning; however, in the absence of high-flux dialysis membranes, the characteristics of charcoal hp may more appropriately address the larger v d and protein binding [ ] . common side effects of extracorporeal elimination include hypotension, bleeding, and infection. enhanced clearance of therapeutic medications and antidotes (e.g., antibiotics, fomepizole, n-acetylcysteine, water-soluble vitamins) may occur. the need for dialysis must be anticipated early; several hours of preparation time may be required to secure vascular access, equipment, and personnel. exchange transfusion is a total blood volume exchange administered in small aliquots. serial frequent phlebotomy of a small amount of circulating blood occurs with simultaneous transfusion of equivalent donor blood. this process is repeated until two to four vascular volumes have been exchanged. while the procedure is very rarely used for toxin removal, exchange transfusion is more familiar to clinicians treating severe hemolytic diseases of the newborn, hyperbilirubinemia without hemolysis, and sickle cell crisis. exchange transfusion removes xenobiotics that are large or bound to plasma proteins, such as thyroxine, iron, or theophylline [ , ] . for lifethreatening ingestions, exchange transfusion is a viable option for neonates and infants whose immature vasculature cannot tolerate extracorporeal elimination modalities or in institutions lacking pediatric dialysis capacity. exchange transfusion has been successfully employed in pediatric iron, isoniazid, phenobarbital, salicylate, theophylline, and vincristine overdose [ ] [ ] [ ] [ ] [ ] [ ] . it has also been suggested for refractory drug-induced methemoglobinemia [ ] . whole blood exchange was utilized in an adult with a fold cyclosporine dosing error [ ] . anticipated complications arise from vascular access, bleeding, hypoglycemia, hypotension, and blood product administration (immune-mediated reactions, blood incompatibility, and infections). several hyperthermic syndromes are caused by xenobiotics. these are generally spectrum disorders, whose features may overlap with other conditions such as cns infection, agitated delirium, and sepsis. malignant hyperthermia (mh) occurs in patients with an autosomal-dominant defect in genes encoding the skeletal muscle ryanodine receptor (ryr- ) or the voltage-gated calcium channel (cav . ) who are exposed to volatile anesthetics or depolarizing muscle relaxants (succinylcholine) [ ] . hypomagnesemia may increase the probability and possibly severity of an mh event [ ] . the subsequent rapid increase in myoplasmic calcium concentration increases muscle metabolism and heat production and produces muscle contractures and hyperthermia. neuroleptic malignant syndrome (nms) is characterized by high fever, autonomic instability, altered mental status, and muscle rigidity. potent antipsychotics (neuroleptics) such as haloperidol and other medications (metoclopramide, droperidol, and promethazine) with significant dopamine antagonism, as well as abrupt cessation of dopaminergic agents such as those used in parkinsonism, can precipitate this life-threatening syndrome [ ] . nms typically develops over several days and is characterized by ''lead-pipe'' rigidity [ ] . drugs that impair serotonin breakdown or re-uptake, those that act as serotonin precursors or enhance its release, or those that are serotonin agonists may lead to serotonin syndrome. like nms, serotonin syndrome is a spectrum disorder for which various signs and symptoms have been proposed to establish diagnosis (e.g., sternbach and hunter criteria) [ , ] . in its most severe form it consists of high fever, autonomic instability, altered mental status, and may have associated diaphoresis, shivering, tremor, diarrhea, or spontaneous clonus. in serotonin syndrome, onset of symptoms is usually rapid, with % of patients with the serotonin syndrome presenting within hours of drug exposure, and tremor and hyperreflexia predominant in the lower extremities may be a prominent feature [ ] . sympathomimetic-associated hyperthermia, seen with acute intoxication with cocaine, amphetamines, substituted amphetamines, and phencyclidine, may be clinically indistinguishable from serotonin syndrome [ ] . additionally, the agitated delirium engendered by these agents may be difficult to distinguish from that induced by hyperthermia itself. patients with anticholinergic-associated hyperthermia will generally present with a compatible "toxidrome" -agitation; mydriasis; dry, hot, and erythematous skin; hypoactive bowel sounds; and urinary retention. while rare, thyrotoxicosis factitia, the ingestion of excess thyroid hormones due to inadvertent intake (pharmaceutical or food contamination), misuse (dieting), or significant intentional ingestion may produce hyperthermia [ , ] . hyperthermia may accompany toxicity with agents that uncouple oxidative phosphorylation (e.g., salicylates, dinitrophenol, pentachlorophenol) [ ] . multiple medications can also complicate or contribute to environmental hyperthermia. several reviews and epidemiological data from major heat waves have demonstrated that anticholinergics, antiepileptics, antihistamines, antihypertensives in general and diuretics in particular, antipsychotics, and others contribute to excess morbidity and mortality [ , ] . conversely, exogenous heat stress can increase mortality from specific xenobiotics. in an urban setting at ambient temperatures above . °c, the mean daily number of fatal cocaine overdoses increased markedly [ ] . regardless of the cause for the hyperthermic syndrome, cessation of any possible offending or contributing agents and rapid cooling is critical. the degree of hyperthermia produced correlates with death and neurotoxicity in animal models, and temperature normalizing intervention is critically impor-tant in attenuating cns injury and mortality [ ] . studies from the chicago and france heat waves show that this is rarely done in a timely manner (if at all) in cases of environmental hyperthermia, with devastating results [ , ] . the benefits of rapid cooling by ice water immersion were demonstrated over years ago [ ] . a large review concluded that cooling methods based on evaporative heat loss are less efficient than immersion in ice water in dissipating heat [ ] . additional studies demonstrate that cooling rates of up to . - . °c/min can be achieved with immersion, two to three times that of evaporation [ , ] . regardless of the method used, effectiveness should be repeatedly assessed. sedation with benzodiazepines and rigorous supportive care are necessary adjuncts in significant cases. this is primarily accomplished with titrated doses of benzodiazepines to inhibit muscle rigidity and control agitation. animal models have demonstrated the benefit of benzodiazepines in prolonging survival, preventing seizure, and attenuating agitation in the toxicological hyperthermias [ , ] . phenytoin is ineffective in animal models [ ] . phenothiazines and butyrophenones, while reported, may have delayed onset and compromise mental status, lower seizure threshold, impair heat dissipation, and worsen hypotension [ ] . neuromuscular paralysis may be required to limit further heat generation in cases of nms, serotonin syndrome, and sympathomimetic-associated hyperthermia. as the pathophysiology of mh is beyond the neuromuscular junction, paralytics are unlikely to provide benefit. rapid i.v. administration of dantrolene, a direct-acting skeletal muscle relaxant, is the only drug proven effective for prevention and treatment of mh. dantrolene disrupts the pathogenic excitation-contraction coupling by acting at ryr- to suppress depolarizationinduced sarcoplasmic reticulum calcium release and normalize the voltage dependence of contractile activation [ ] . reversal of increased myotube sensitivity may also play a role [ ] . intravenous - mg/kg dantrolene is repeated until symptoms are controlled or mg/kg (or more) has been administered. following initial treatment, - mg/kg i.v. or per os is given every hours for - hours to prevent recurrence. dantrolene is packaged in vials containing mg dantrolene sodium; thus, multiple vials are needed for treatment of adult patients. a large review of nms cases did not suggest a beneficial role for dantrolene, although one case-controlled analysis found benefit [ , ] . bromocriptine, a dopamine agonist, has been used (off-label) to treat nms at doses ranging from to mg every hours [ ] . common side effects include hypotension, dyskinesia, erythromelalgia, and hallucinations. cyproheptadine, developed as an antihistamine, additionally antagonizes -ht receptors. cyproheptadine for serotonin syndrome (off-label) is initially used in a dose range of - mg, followed by mg every hours for persistent symptoms; upon symptom control, mg maintenance dosing is provided every hours [ ] . the tablet form necessitates administration orally or crushed via nasogastric tube. n-acetylcysteine (nac) provides an effective means of prevention and treatment of acetaminophen (n-acetyl-p-aminophenol, apap; paracetamol)induced hepatotoxicity. nac is also employed to preclude radiographic contrast-induced nephropathy [ ] . the ultimate toxicant of apap, n-acetyl-pbenzoquinone imine (napqi) generated primarily by cyp e and cyp a , depletes glutathione (gsh), binds intracellular components, and, through an incompletely understood process, produces hepatic injury, centrilobular necrosis, or hepatic failure [ , ] . nac works by multiple mechanisms. it augments apap sulfation to a nontoxic metabolite, it acts as a glutathione precursor or glutathione substitute to detoxify napqi, and possibly reverses napqi oxidation [ , ] . nac provides substantial benefit even in cases of delayed presentation following overdose [ ] . extra-hepatic benefits of nac include improving cardiac index and systemic mean oxygen delivery despite decreasing systemic vascular resistance [ ] . in a range of hepatic disorders, nac improved baseline oxygen delivery, oxygen consumption, and dye clearance in a majority of patients [ ] . liver blood flow and cardiac index improved in septic shock patients provided nac [ ] . only l-nac is beneficial. animal experiments demonstrate that the l-isomer, derived from physiological l-cysteine, prevents hepatotoxicity and provides prolonged elevations of hepatic glutathione [ ] . nonphysiological d-nac cannot increase glutathione stores or prevent hepatotoxicity, despite increasing acetaminophen sulfation [ ] . according to rumack [ ] , the oral nac dosing strategy was reached by estimating the absorption and turnover rate of glutathione at mg/kg per h and an fda safety factor of , to yield mg/kg every hours [ (mg/kg per h) × (h) × (safety factor) = ≈ mg/kg every h]. there were several assumptions as to "normal" hepatic glutathione levels and apap to napqi conversion. a mg/kg loading dose was added to provide an early high hepatic dose. the -hour duration of oral therapy was based on previous observations of multiple patients with prolonged apap half-lives and a desire to implement a protocol that would accommodate those with half-lives longer than hours (anticipating disappearance after five half-lives). while many have suggested that the -hour oral course is excessive, particularly after apap has disappeared from the serum, the optimal duration of therapy is unclear. studies assessing a shortened or "patient-tailored" approach have been small or methodologically limited [ , ] . the rumack-matthew nomogram guides initiation of nac therapy in single acute ingestions. the "treatment line" is anchored at an apap serum concentration of either μg/ml (" line") or μg/ml (" line") at hours post ingestion and decreased by % every hours. the slope of the treatment line does not reflect apap kinetics. the " line" is utilized in all patients in the u.s. and australia; in the u.k. and elsewhere the " line" is employed, with a " line" modification for an array of individuals deemed at "high-risk": ethanol tolerant, those at risk for glutathione depletion (malnutrition, hiv, eating disorders, cystic fibrosis), pregnancy, and those prescribed enzyme-inducing drugs (carbamazepine, phenytoin, phenobarbitone rifampacin, isoniazid, etc.) [ , ] . the u.s. multicenter study substantiated the safety and efficacy of its approach [ ] . proponents of the " line" point to the fact that . - . % of patients above the " line" but below the " line" developed biochemical hepatotoxicity (aspartate aminotransferase, ast > iu/l at any time during their course) in the u.s. multicenter trial and that patient deaths have occurred in untreated patients "between the lines" [ , ] . in patients presenting near hours after ingestion, or if a level is not available before hours post ingestion, nac is begun while awaiting apap results and then continued or stopped once the results are available and have been plotted on the nomogram. if the time of ingestion is unknown or more than hours has passed, nac is administered. when apap concentration and transaminase results are obtained, if transaminases are elevated or if measurable apap exists, a full course of treatment is provided. with normal aminotransferases and without detectable apap, treatment is not required. concentrations obtained less than hours post ingestion are not useful except to completely exclude ingestion (i.e., it is useful only if the apap concentration is undetectable). ongoing absorption may place individuals above the line at hours, or metabolism or charcoal administration may result in a patient falling below the nomogram at hours. in cases of chronic ingestion (> . g/day in adult), laboratory evaluation and treatment are provided as for an unknown time of ingestion. with elevated transaminases or measurable apap, nac is provided. oral nac is cheap and familiar to clinicians. it has minimal side effects (other than vomiting and odor) and is preferred in patients with bronchospastic disease. its use can become problematic in cases where oral delivery is compromised, e.g., in patients with depressed mental status, significant vomiting, or impaired gastric motility. use of an anti-emetic is encouraged. intravenous nac appears to be similarly efficacious to oral nac and eliminates many delivery issues. it has a much shorter therapy course ( hours), expediting medical and psychiatric disposition. it avoids first pass metabolism in cases where the liver is not the only target or interest, such as those with cerebral edema or pregnancy. while i.v. nac is slightly more expensive, total hospital charges may be less due to decreased treatment time. histamine-mediated anaphylactoid reactions are more commonly seen with rapid i.v. loading and in patients with lower apap levels [ ] . mild reactions have been treated by slowing the infusion rate and providing i.v. diphenhydramine, although this might alter nac and apap kinetics. dosing complexity - mg/kg in ml of % dextrose over hour, followed by mg/kg in ml of % dextrose over hours ( . mg/kg per h), and then mg/kg in ml of % dextrose over hours ( . mg/kg per h) -yields frequent administration errors [ ] . the supplied % solution was too concentrated for children, and dilution according to adult guidelines resulted in excess free water, and cases of hyponatremia and seizures [ ] . the current u.s. package prescribing information (http://www.acetadote.net/pi_acetadote_revised_apr .pdf) and dosage calculator website (http://www.acetadote.net/dosecalc.shtml) provide dosing and administration guidelines in patients of less than kg. in a study limited by different comparison groups, data acquisition methodology, treatment location and several other factors, -hour only i.v. nac was favored in patients with early presentation (< hours), whereas late presentation favored oral -hour nac [ ] . however, continuous i.v. infusion in delayed presentations with apap-induced fulminant hepatic failure showed clear benefit in a prospective study [ ] . whatever the route, prior to cessation of nac therapy, negative apap concentrations and normal transaminases must be ensured, particularly in cases of massive ingestion; hepatotoxicity may follow premature cessation of therapy [ , ] . the -hour maintenance dose is continued in patients receiving i.v. nac until apap is undetectable and transaminases are normal (or at baseline). experimental evidence and human case reports demonstrate both delayed absorption, delayed increase following initial decline, and "crossing the nomogram" with extended-relief, opioid-or anticholinergic-containing apap products, or co-ingestants [ , ] . in cases of hepatic failure, i.v. nac is continued until resolution, transplant, or death. historically, physostigmine (eserine), a reversible carbamate inhibitor derived from the seed (calabar bean) of the vine physostigma venenosum balfour, was used in the ancient trial by ordeal [ ] . medicinal use of physostigmine was first reported in to reverse severe atropine poisoning [ ] . naturally available (-)-physostigmine is over times more effective in inhibiting acetylcholinesterase and butylcholinesterase in tissue, erythrocytes, and serum in humans and animal models than its stereoisomer [ , ] . this activity depends upon interactions within the hydrophobic pocket of the acetylcholinesterase active center, which is distinct from the catalytic site [ ] . additionally, physostigmine binds nicotinic receptors close to, but distinct from, the acetylcholine binding site on the α-subunit [ ] . at low doses, physostigmine functions as an ineffective nicotinic receptor agonist, while at higher doses it produces marked channel blockade. physostigmine's nonspecific analeptic properties [ ] are no longer considered useful in overdose, given the clear benefits of supportive care. indiscriminate use of physostigmine and an incomplete understanding of the pathophysiology of tricyclic antidepressant (tca) poisoning was associated with bradydysrhythmias including asystole, seizure, and several deaths [ , ] . in animal models, physostigmine is ineffective in attenuating tcainduced seizures [ ] . it failed to abolish dysrhythmias, decreased blood pressure, and at high doses enhanced tca toxicity [ ] . physostigmine is currently recommended as a diagnostic and therapeutic agent for antimuscarinic poisoning [ ] . patients should have clear peripheral or central manifestations of the anticholinergic toxidrome. as a tertiary amine, physostigmine can cross the blood-brain barrier to reverse the central effects. an ecg should exclude sodium or potassium channel blockade (qrs or qt prolongation). excessive physostigmine will produce a cholinergic syndrome, with muscarinic and nicotinic effects. as the adverse effects of bradycardia and bronchorrhea can produce significant morbidity, continuous cardiac monitoring and immediate access to atropine are recommended during physostigmine administration. physostigmine, - mg in adults and . mg/kg (maximum . mg) in children is infused slowly over at least min [ ] . repeat doses every - min can be provided if an adequate response does not occur and adverse effects are absent. re-bolusing may be required in the setting of antimuscarinics with a prolonged duration of action. the anticonvulsants include carbamazepine, ethosuximide, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, tiagabine, topiramate, valproic acid (vpa), vigabatrin, and zonisamide. these drugs enjoy widespread approved and offlabel use for additional conditions, e.g., fibromyalgia (pregabalin); neuropathy and neuropathic pain (carbamazepine, gabapentin, lamotrigine, levetiracetam, and pregabalin); panic disorder (tiagabine); migraine prophylaxis and treatment of obesity, ethanol dependence, and depression (topiramate); and bipolar disorder (carbamazepine, lamotrigine, and vpa). treatment of anticonvulsant overdose is largely supportive, with particular attention to the cns-depressant and cardiovascular effects of some of these agents. l-(r)-carnitine exists as the sole specific antidote in this class for significant vpa (di-n-dipropylacetic acid, -propylpentanoic acid) poisoning. patients with drug-associated mitochondrial toxicity (particularly from nucleoside analogs) and anthracycline cardiotoxicity might also benefit from its administration [ , ] . the anticonvulsant properties of vpa derive from its ability to increase γ-aminobutyric acid (gaba) availability via inhibition of gaba transaminase and succinic semialdehyde dehydrogenase, to attenuate n-methyl-daspartate (nmda)-type glutamate receptor excitatory effects, and to slow the rate of recovery from sodium channel inactivation [ ] [ ] [ ] . additionally, vpa appears to affect inositol levels similar to lithium. therapeutic concen-trations are - mg/l. potentially toxic concentrations are greater than mg/l. oral absorption of vpa is excellent [ ] . peak plasma concentrations are generally seen in - hours, although this may be markedly delayed by overdose, enteric coating, or meals [ ] . manifestations of significant vpa toxicity include cns effects (lethargy, seizure, coma, cerebral edema), respiratory depression, metabolic derangement (hypernatremia, hyperammonemia, hypocalcemia, metabolic acidosis, carnitine deficiency), gastrointestinal effects (nausea, vomiting, and abdominal pain), pancytopenia, pancreatitis, and hepatotoxicity [ , ] . valproate toxicity is seen both in intentional acute overdose and in those on chronic therapy, either without adequate carnitine supplementation or on complex regimes. cells attempt to metabolize the vpa that is not directly excreted or glucuronidated in a manner similar to other fatty acids (fig. ) . thus, vpa is conjugated with coenzyme a (coa). carnitine enters via an atp-dependent transporter. vpa is then transferred to carnitine, the normal mechanism for fatty acids entry into the mitochondrion. however, vpa-carnitine both inhibits the carnitine transporter and also diffuses out of the cell to be lost via renal excretion [ ] . renal resorption of carnitine is also impaired [ ] . these factors contribute to intracellular carnitine depletion. once vpa-carnitine is shuttled into the mitochondrion, it is reattached to coa. it then undergoes β-oxidation, in an attempt to generate -carbon molecules for entry into the krebs cycle. the -en-vpa-coa product is neurotoxic with a prolonged half-life. the terminal -keto-vpa product traps coa, leading to its mitochondrial depletion. decreased mitochondrial coa yields decreased atp production, diminishing usable cellular energy currency and further limiting carnitine entry into the cell (via an atp-dependent carnitine transporter). once carnitine is depleted, normal fatty acid metabolism cannot occur [ ] . fatty acid build up is thought to underlie the reye's-like steatohepatitis, which can be seen in toxicity [ ] . coa is also needed to make n-acetylglutamate, an activator of carbamoylphosphate synthetase i (cps i), a critical enzyme in the urea cycle. when its effectiveness is limited due to inadequate activator, ammonia cannot be incorporated, and consequently, its concentrations increase. furthermore, as coa is depleted, β-oxidation shifts to omega (ω), or terminal carbon oxidation. this creates (among others) the hepatotoxic -en-vpa product. -en-vpa additionally inhibits cps i, further preventing nitrogen elimination and contributing to hyperammonemia. l-carnitine (levocarnitine) supplementation has been recommended to reverse the adverse metabolic effects of vpa in cases of vpa-induced hepatotoxicity, vpa overdose, and primary carnitine-transporter defects [ , ] . hyperammonemia and serum and muscle carnitine deficiency are well described in patients chronically taking vpa [ ] [ ] [ ] . several studies and case reports demonstrate that carnitine supplementation reverses clinical symptoms, hypocarnitinemia, hyperammonemia, and vpa half-life prolongation in patients with toxicity due to chronic administration [ ] [ ] [ ] . in patients with acute vpa overdose, limited clinical and laboratory data derived from case reports also suggest that reversal of metabolic derangements and improvement in clinical symptoms occurs when carnitine is provided [ ] [ ] [ ] . a single large retrospective analysis showed a significant survival benefit with i.v. carnitine supplementation (with vpa cessation) in patients with valproate-induced hepatotoxicity [ ] . l-carnitine dosing for cases of overdose is not currently evidence based. an oral or i.v. dose of mg/kg per day, divided and given every hours (maximum daily dose g), is provided to those patients with acute overdose and [ , , , , ] . asymptomatic hyperammonemia or hepatotoxicity in the absence of cns depression or metabolic derangement [ ] . symptomatic patients with hyperammonemia or symptomatic hepatotoxicity should receive mg/kg l-carnitine i.v. over min (maximum g), followed by mg/kg every hours over - min until clinical improvement occurs [ , ] . others have supplemented at the higher dosing strategy when vpa concentrations exceed mg/l [ ] . in addition, given the decrease in protein binding that occurs, hemodialysis or hemoperfusion is recommended for patients with vpa concentrations exceeding - mg/l or with severe clinical symptoms [ ] . l-carnitine is generally well tolerated. side effects associated with carnitine supplementation are nausea, abdominal discomfort, dose-related diarrhea, and fishy body odor [ ] . a small retrospective chart review found no adverse effects or allergic reactions in vpa overdose patients administered carnitine [ ] . the current l-carnitine package inserts have no warnings or contraindications, but note that seizures have been reported to occur in patients, with or without pre-existing seizure activity, who received either oral or i.v. l-carnitine [ ] . up to mg/kg per day for days has been provided without complications [ ] . the d-isomer and the racemate (d,l-carnitine) are contraindicated. historic use of racemic d,l-carnitine was associated with myasthenialike syndromes and cardiac dysrhythmias, which disappeared after l-carnitine administration [ ] . d-carnitine also competitively depletes cardiac and skeletal muscles and kidneys of l-carnitine [ ] . dextrose dextrose (d-glucose) is indicated to rapidly reverse organic or toxin-induced hypoglycemia (e.g., from sulfonylureas, insulin, ethanol, salicylates, β-adrenergic antagonists, quinolines, pentamidine, ritodrine, and disopyramide) [ , ] . hypoglycemia onset may be significantly delayed with certain agents (e.g., long-acting insulin or sulfonylureas). limited cns glycogen stores (in astrocytes) and the inability to acutely use free fatty acids make the cns particularly vulnerable to hypoglycemia [ ] . patients (and providers) may be unaware of hypoglycemia in the absence of objective testing; both the counter-regulatory autonomic response and overt neurological deficit may be absent [ , ] . additionally, significant neuroglycopenia and hypoglycemia-associated delirium (particularly in salicylism) may occur despite a "normal" peripheral blood glucose [ ] . a wide range of clinical presentations have been described, including diaphoresis, nausea, tachycardia, tremor, hypothermia, focal neurological deficits, and cns agitation, confusion, or depression. these are generally reversible upon prompt treatment. untreated hypoglycemia may result in seizure, coma, and death. hypoglycemic seizures increase cerebral metabolic rate, contribute to atp depletion, and produce irre-versible brain damage [ , ] . for these reasons, when bedside testing is unavailable, a risk-benefit calculation has generally favored empiric dextrose administration in the absence of a very clear alternative history or explanation for altered mental status. following a determination of absolute or relative hypoglycemia, . - . g/kg i.v of age-appropriate dextrose containing solutions should be provided immediately -d w ( g/ ml) in adults, d w ( g/ ml) in children, and d w ( g/ ml) in neonates. frequent re-evaluation of response to therapy is required. glucose uptake and distribution, hyperglycemia-induced insulin secretion in those with a competent pancreas, and ongoing toxin exposure may cause recurrent hypoglycemia and necessitate repeat dosing. feeding, which provides significantly more calories than each ml ampule of d w ( kcal according to one manufacturer [ ] ), should be commenced as soon as practicable. while d w "maintenance" solutions may be subsequently required, at an infusion rate of ml/h, this concentration only provides kcal per hour. continuous infusion of more concentrated solutions (e.g., d w) requires a central venous catheter for administration. only the d-isomer is clinically useful. most glucose transporters (gluts) and the specific transporter required for facilitated diffusion of glucose across the blood-brain barrier, glut (slc a ), have a high affinity for d-glucose and negligible affinity for l-glucose [ , ] . d-glucose is also generally favored over other d-glucose epimers such as d-mannose or d-galactose. d w is hypertonic and may cause phlebitis or thrombosis at the site of injection. extravasations of solutions containing as low as % dextrose have caused significant tissue injury and necrosis, particularly in young children [ ] . pseudoagglutination of red blood cells may occur if concentrated dextrose solutions without electrolytes are administered simultaneously with blood through the same infusion set [ ] . hypertonic dextrose administration may also induce generally clinically irrelevant hypophosphatemia [ ] . octreotide acetate, a synthetic somatostatin analogue, is now favored in cases of refractory hypoglycemia due to sulfonylureas or quinine. it is fda approved for treatment of acromegaly, carcinoid tumors, and vasoactive intestinal peptide tumors [ ] . it is a more potent inhibitor of insulin secretion than the natural hormone [ ] . in pancreatic β-islet cells, atp generated from glucose uptake and subsequent metabolism normally induces closure of the atp-dependent potassium channel by binding to its pore subunit (fig. ) . sulfonylureas similarly induce channel closure after binding to a regulatory (sur ) subunit. increased intracellular potassium triggers calcium entry through voltage-dependent calcium channels, leading to increased cytosolic calcium and insulin exocytosis [ , ] . additionally, atp contributes to insulin vesicles movement and provides a substrate for protein kinase a (pka)-mediated phosphorylation. octreotide binds to the somatostatin receptor (primarily sstr ) [ ] . the subsequent effects continue to be explored and include inhibitory calcium channel effects, inhibition of adenylyl cyclase, and dephosphorylation of specific proteins required for movement and/or docking of vesicles [ , , ] . octreotide effectively suppresses endogenous insulin release in controlled studies in diabetics and in cases of sulfonylurea overdose, but does not (and would not be expected to have) an effect on exogenously administered insulin [ ] [ ] [ ] . several factors support octreotide usage following failure of initial dextrose administration and feeding. bolused dextrose may produce hyperglycemia and thus subsequently stimulate an exaggerated insulin response, particularly when figure . pancreatic β-islet cell mechanisms of insulin release and octreotide action (see text for details). enzymes and substances: ac, adenylyl cyclase; atp, adenosine triphosphate; camp, cyclic adenosine monophosphate; glut , glucose transporter ; pka, protein kinase a; sfu, sulfonylurea; sstr, somatostatin receptor. symbols: ⊕, agonism or co-factor; ⊗, antagonism. data used can be found in [ ] [ ] [ ] [ ] . sulfonylureas persist. this contributes to recurrent (sometimes more significant) hypoglycemia. a vicious cycle of serum glucose concentrations is described in case reports and controlled trials following dextrose administration after sulfonylurea exposure [ ] [ ] [ ] . additionally, as has been demonstrated, classic neuroglycopenic symptoms may not be present, and patients may need to be admitted during periods when circadian sleep patterns would complicate assessment. octreotide administration also obviates the concern of excess water administration in pediatric patients receiving i.v. dextrose solutions. relatively few trials are available to judge the efficacy of octreotide for sulfonylurea-induced hypoglycemia. in one study, glipizide was used to induce induced hypoglycemia ( mg/dl) in eight healthy volunteers, who were then resuscitated with dextrose infusion, diazoxide, or octreotide [ ] . dextrose requirements were markedly less in patients provided octreotide and hypoglycemic events were markedly attenuated after all therapies were stopped. one retrospective chart review of nine patients demonstrated that octreotide significantly reduced the number of recurrent hypoglycemic events and dextrose requirement [ ] . one prospective randomized controlled trial in poisoned patients, despite a failure to control for carbohydrate intake and having an unusual dosing strategy (a single octreotide μg dose subcutaneously), demonstrated consistently higher glucose values for the duration for which octreotide would be expected to be effective ( - hours) [ ] . controlled animal studies with - μg octreotide demonstrated a similar decrease in hypoglycemic events [ ] . the remainder of human clinical experience of the effectiveness of octreotide in sulfonylurea overdose comes from abstracts, case reports, and case series (e.g., [ , , [ ] [ ] [ ] ). pediatric experience in sulfonylurea overdose comes only in the form of limited abstracts and case reports in children aged months to years [ , [ ] [ ] [ ] . however, octreotide has been used for prolonged periods to treat persistent hyperinsulinemic hypoglycemia of infancy [ , ] . two human studies examined the effectiveness of octreotide in quinineinduced hypoglycemia. in one study of nine healthy volunteers, μg/hour octreotide as a continuous i.v. infusion abolished quinine-induced insulin release [ ] . the authors reported resolution of hypoglycemia in an additional patient being treated with quinine for plasmodium falciparum malaria. a subsequent study in eight patients with p. falciparum malaria confirmed octreotide suppression of quinine-induced hyperinsulinemia [ ] . optimal dosing of octreotide has not been definitively determined. initial doses of - μg subcutaneously in adults have been reported, although μg every - hours is commonly provided [ , ] . in children, an initial dose of . - . μg/kg is used, although up to . μg/kg (or more) has been reported [ , ] . peak serum concentrations are achieved within min after subcutaneous administration and within min after a short ( min) i.v. infusion [ ] . the elimination half-life (by either route of administration) is approximately . hours. in patients with severe renal impairment (which may have contributed to sulfonylurea-induced hypoglycemia in the first place), the plasma clearance is reduced by % [ ] . the subcutaneous route is recommended due to longer duration of effect, as i.v. administration has resulted in treatment failure [ ] . side effects are generally minimal. octreotide does inhibit gallbladder contractility and decreases bile secretion in normal volunteers [ ] . when octreotide has been used to reverse sulfonylurea-induced hypoglycemia, bradycardia, hypokalemia, anaphylactoid reaction, and hypertension and apnea have been reported [ , ] . other adverse events include nausea, abdominal cramps, diarrhea, fat malabsorption and flatulence [ ] . octreotide also suppresses glucagon release, although hypoglycemia has been a concern only in patients on long-term therapy for organic hyperinsulinemia [ ] . glucagon is not generally recommended to correct hypoglycemia. glycogen stores are frequently depleted by the time toxin-induced hypoglycemia manifests; glucagon's half-life (less than min) is inadequate given the prolonged duration of the effect of sulfonylureas; and glucagon may exacerbate hyperinsulinemia [ ] . diazoxide, an antihypertensive agent, which reduces insulin release by opening the atp-dependent potassium channel, is now of historical interest due to associated hypotension, reflex tachycardia, nausea and vomiting, and fluid retention [ , ] . since its introduction in , isoniazid (inh, isonicotinic hydrazide, pyridine- -carbohydrazide) has remained a mainstay for treatment and prophylaxis of mycobacterial infections [ ] . the adult single tablet, mg daily dose ( . mg/kg in a kg individual) targets a peak plasma concentration of - μg/ml [ ] . acute inh toxicity may occur following ingestion of mg/kg inh; it is common above - mg/kg [ ] . the relatively narrow therapeutic window poses a significant risk for those with suicidal intent and for those who ingest extra pills to "catch up" after a brief period of incomplete compliance [ ] . historically, death rates of % were reported [ ] . seizures refractory to typical therapy, severe metabolic lactic acidosis, and coma may occur as early as min post ingestion due to the rapid and nearly complete absorption of inh from the gastrointestinal tract. seizures may occur at lower doses in those with pre-existing susceptibility. associated respiratory failure, hypotension, and rhabdomyolysis may ensue. in patients provided . - . g ( - mg/kg) inh due to medication error, all experienced nausea or vomiting, vertigo, and coma within min to hours after ingestion [ ] . abnormal generalized discharges as sharp and slow waves were seen on eeg in all patients. chronic inh toxicity may present with nausea, vomiting, hepatitis, hemolytic anemia, and neurological findings (restlessness, neuropathy, cerebellar findings, and psychosis). the acute clinical effects are a product of the multiple biochemical actions of inh, which lead to pyridoxine depletion and subsequent neuronal hyperexcitability (fig. ) [ , [ ] [ ] [ ] . inh hydrazones inhibit pyridoxine phosphokinase, which activates pyridoxine. inh hydrazines and hydrazides inactivate active pyridoxal -phosphate. inh metabolites also complex with pyridoxal -phosphate, leading to increased urinary elimination. glutamic acid decarboxylase (gad) and gaba transaminase (gaba-t) both require pyridoxal -phosphate as a co-factor. inhibition of gad exceeds that of gaba-t [ ] . the resulting gaba depletion and loss of neuronal inhibition is thought to underlie seizure activity. metabolic acidosis may be profound -survival has been reported with a ph of . [ ] . seizure-associated lactate generation is substantial; inh-induced metabolic acidosis does not develop in paralyzed dogs (despite eeg evidence of seizure) [ ] . importantly, merely correcting the acidosis (e.g., by bicarbonate) does not prevent additional seizures or terminate inh toxicity [ , ] . inh also impairs lactate conversion to pyruvate (fig. ) . increased metabolism of fatty acids due to impaired glucose metabolism with hyperglycemia and ketonuria has been reported [ , ] . inh also impairs cellular reduction-oxidation capacity via competitive inhibition of nad [ , ] . pyridoxine deficiency also appears to play a role in inh-induced mental status changes (coma and lethargy) [ , , ] . appropriately dosed pyridoxine (vitamin b ) has been the mainstay of antidotal therapy for inh intoxication since the early reports of benefit versus his- figure . mechanisms of isoniazid (inh) toxicity (see text for details). enzymes (italicized): gaba-t, gaba transaminase; gad, glutamic acid decarboxylase; got, glutamic-oxaloacetic transaminase; ldh, lactate dehydrogenase; ppk, pyridoxine phosphokinase; and sr, serine racemase. substances: gaba, γ-aminobutyric acid; and ssa, succinic semi-aldehyde. symbols: ⊕, agonism or co-factor; ⊗, antagonism. data used can be located in [ , [ ] [ ] [ ] ] . drugs and pharmaceuticals: management of intoxication and antidotes torical controls [ ] . exogenous vitamin b provides the necessary precursor for the co-factor for gaba regeneration. clinical experience with pyridoxine comes from case series, case reports, and animal data [ , , , , [ ] [ ] [ ] . clinical trials are absent due to ethical considerations. vitamin b (as pyridoxine hydrochloride) is provided on a gram per gram basis for each gram of inh ingested, to a maximum of g or mg/kg (the empiric dose in ingestions of unknown quantity) [ , , ] . a repeat dose can be provided if necessary. due to the large amount of pyridoxine required, inadequate stocking and depletion of institutional and entire regional supplies have been widely reported [ , , ] . in the convulsing patient, pyridoxine is administered i.v. at . g/min ( g maximum) until seizure termination, with the remainder over - hours. pediatric dosing should not exceed mg/kg ( g maximum). large doses of pyridoxine have been safely administered; however, sensory neuropathy may occur with massive acute doses (> g) or chronic large daily doses [ ] . co-administration of benzodiazepines is synergistic in controlling seizures [ , ] . massive inh ingestion may require additional sedative hypnotics or anesthetic agents to suppress seizures [ ] . inh is dialyzable, and hemodialysis has been used successfully in cases refractory to antidotal treatment, in those with extremely high plasma inh concentrations, and in patients with renal failure [ , ] . pyridoxine also appears to rapidly reverse the impaired consciousness seen in inh overdose [ , ] . the cns excitatory neurotransmitters include glutamate and d-serine, which with glutamate is a co-agonist of the nmda receptor [ ] . examination of the metabolic pathways affected by pyridoxal -phosphate depletion (fig. ) suggests that inadequate stores of these neurotransmitters (due to inadequate co-factors for glutamic-oxaloacetic transaminase and serine racemase) might be contributory, in addition to general substrate or catecholamine deficiency. pyridoxine therapy is also recommended for poisoning through other hydrazines or hydrazine precursors (e.g., gyrometra mushrooms, monomethylhydrazine, and unsymmetrical dimethylhydrazine fuel). pyridoxine is effective in treating the chronic inh-associated neuropathy, particularly in patients with renal failure. doses of - mg pyridoxine/day have typically been used in the chronic setting [ ] . pyridoxine has no effect in prevention or treatment of inh-associated hepatic injury. antineoplastic agents are used for the treatment of a variety of benign and malignant neoplasms. some antineoplastic agents (such as the antifolates) have an expanded spectrum that includes use in rheumatology, dermatology, and obstetrics and gynecology. toxicity may be due to the agent itself or delivery of the agent to an unintended target (e.g., extravasation). several antidotes are used in a prophylactic fashion or on chronic basis. amifostine (wr- ) -which is dephosphorylated by alkaline phosphatase to an activated, protective thiol form -is approved to decrease toxicity associated with radiotherapy and renal injury associated with cisplatin [ ] . it has also been used to reduce chemotherapy-induced neutropenia; genitourinary injury associated with cyclophosphamide; and transfusion requirements, gastrointestinal and hepatic toxicity in pediatric patients [ , ] . cyclophosphamide and ifosfamide induce bladder toxicity (hemorrhagic cystitis) via their metabolite acrolein. mesna ( -mercaptoethane sulfonate), a thiol agent that complexes with and inactivates acrolein, is provided orally or i.v. as prophylaxis [ ] . diethyldithiocarbamate (ddtc), the major metabolite of disulfiram, is an investigational agent for prevention of neuropathy from cisplatin and its analogs; it increased nephrotoxicity in one study [ ] . granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage colony-stimulating factor (gm-csf), erythropoietin (hemopoietin) and its derivatives, oprelvekin (recombinant interleukin- ), and other stimulating factors are employed as adjuvants to reconstitute various hematopoietic lines damaged by chemotherapy and radiation [ , ] . palifermin (recombinant truncated human keratinocyte growth factor) is used to prevent severe mucositis in patients receiving stem-cell transplantation with a total body irradiation conditioning regimen [ ] . the remaining section focuses on antineoplastic antidotes used in the acute setting. a dreaded complication of administration of vesicant chemotherapeutic agents is extravasation. risk factors for extravasation include small, fragile, or sclerosed veins, obesity, comorbid conditions (diabetes, circulatory disorders, impaired sensory perception), use of rigid i.v. catheters, and clinicians' lack of knowledge and skills [ ] . redness, burning pain, and swelling may portend later blistering, ulceration, and necrosis. dexrazoxane is u.s. fda approved for treatment of extravasation resulting from i.v. anthracycline chemotherapy, to diminish tissue damage and the need for surgical excision of necrotic tissue [ ] . clinical efficacy data comes from two simultaneously reported openlabel, single-arm, prospective multicenter studies in which only out of patients with biopsy-proven extravasation required surgical debridement [ ] . additional instances of successful dexrazoxane treatment of anthracycline extravasation are provided as case reports ( [ ] and others). dexrazoxane is provided once daily for consecutive days, with the first infusion initiated as soon as possible. daily doses are as follows: day , mg/m (maximum mg); day , mg/m (maximum mg); day , mg/m (maximum mg) [ ] . the dose is reduced by % in patients with creatinine clearance of less than ml/min. in mice, efficacy rapidly decreased when dexrazoxane was provided beyond hours after extravasation [ ] . dexrazoxane's mechanism of action appears to involve reversible inhibition of topoisomerase ii and inhibition by its metabolite, an ethylenediamintetraacetic acid (edta) analogue, of free radical formation via iron removal from the iron-doxorubicin complex [ ] . topoisomerase ii-independent effects have also been described [ ] . in contrast, some authors have encouraged the nonconcurrent, off-label use of topical dimethyl sulfoxide (dmso) for anthracycline extravasation because of the risk of infection, neutropenia, and thrombocytopenia associated with dexrazoxane [ ] . dexrazoxane is also used prophylactically to limit anthracycline-associated cardiomyopathy [ ] . in , methotrexate (mtx) joined the oncological armamentarium for leukemia [ ] . mtx treatment of solid cancers was reported in , and it gained fda approval for psoriasis in [ , ] . mtx is now used intramuscularly, intrathecally, i.v., and orally for a range of dermatological, rheumatological, obstetric, and gynecological conditions. the dose ranges from . - mg orally once weekly for psoriasis or rheumatoid arthritis to - g/m or more for osteosarcoma, leukemia, and lymphoma [ ] [ ] [ ] . mtx poisoning may result from intentional overdose; unintentional ingestion, prescription, dispensing, administration, and patient errors; or renal insufficiency leading to persistent mtx in patients receiving high-dose chemotherapy regimens [ , ] . mtx antagonizes folate metabolism (and rapidly proliferating cells) via multiple mechanisms. dihydrofolate reductase inhibition by mtx and its polyglutamated metabolites ensures that neither dihydrofolate nor active tetrahydrofolate can be generated from folate, nor can existing dihydrofolate be recycled. thymidylate synthase inhibition compromises thymidine synthesis. purine ring synthesis is impaired by inhibition of the participating enzymes amidophospho-ribosyltransferase (ppat) and -aminoimidazole- -carboxamide ribonucleotide transformylase (aicart) [ , ] . maintenance of brisk urinary elimination with i.v. hydration and urinary alkalinization are standard therapies for patients receiving mtx. mtx is ten times more soluble in alkalinized urine (i.e., ph . ) than at ph . [ ] . folate (folic acid) is an ineffective therapy for mtx poisoning. while folate will inhibit renal resorption of mtx, persistent dihydrofolate reductase inhibition by mtx inhibits folate's activation. leucovorin (folinic acid, -formyltetrahydrofolic acid, citrovorum factor) sustains the folate cycle by bypassing the blocked dihydrofolate reductase pathways. addition of leucovorin "rescue" permitted the administration of very-high-dose mtx chemotherapy [ ] . however, in patients receiving mtx chemotherapy, -hour mtx concentrations greater than × - m ( μmol/l), -hour concentrations greater than × - m ( μmol/l), or -hour concentrations greater than × - m ( . μmol/l, nm), or those with evidence of renal dysfunction are considered at high risk for toxicity [ ] . in the setting of mtx persistence or toxicity, leucovorin i.v. doses are increased to mg/m or mg/m every hours according to established nomograms; doses and as high as g/day have been used [ , ] . leucovorin therapy continues until mtx concentration are less than . × - - . × - m ( . - . μmol/l, - nm) [ ] . however, adequate leucovorin concentrations cannot be achieved for competitive reversal of mtx toxicity when mtx concentrations are persistently above - μmol/l; other antidotal strategies are then considered [ ] . treatment of patients ingesting mtx should not be delayed pending mtx concentrations. inhibition of dna synthesis is nearly complete when mtx plasma concentrations are greater than × - m ( . μmol/l, nmol/l) [ ] . therefore, leucovorin is provided until mtx concentrations are less than × - m in patients receiving mtx for non-oncological indications or in patients not receiving mtx therapeutically [ ] . only leucovorin's s-form [levoleucovorin, ( s)-leucovorin] is active and rapidly metabolized to usable, reduced folates; the inactive isomer is slowly eliminated by renal excretion during i.v. administration [ ] . leucovorin was available in the u.s. only as a racemate until , when levoleucovorin received fda approval. levoleucovorin at one-half of the usual racemic dose (as it is entirely active) appears to provide similar rescue therapy in high-dose mtx chemotherapy [ ] . oral rescue is not routinely recommended as leucovorin's bioavailability is poor above mg due to saturation of active intestinal transport [ ] . the calcium content of leucovorin ( . meq calcium/mg leucovorin) mandates that infusion should not exceed mg/min. intrathecal administration of leucovorin is contraindicated, as death may result [ ] . glucarpidase (carboxypeptidase g , cpdg ) is undergoing evaluation as an additional antidote for mtx toxicity. u.s. or european marketing approval for glucarpidase has not been granted at the time of writing. competitive and complete reversal of mtx toxicity by leucovorin may not be possible at mtx concentrations above μmol/l (and perhaps even lower) [ , , ] . patients with systemic mtx toxicity (significant mucositis, gastrointestinal distress, myelosuppression, hepatitis, or neurotoxicity), persistent serum mtx, and renal impairment following high-dose mtx have been considered for glucarpidase therapy in addition to leucovorin. recommendations for glucarpidase above certain mtx concentrations have varied by malignancy, degree of renal impairment, initial mtx dose, and serum mtx concentration (e.g., clinical trials nct , nct , and [ , [ ] [ ] [ ] ). purification of "carboxypeptidase g", a pseudomonad zinc-dependent enzyme capable of mtx cleavage, was reported in [ ] . its antidotal potential was suggested in . in mice injected with lethal mtx doses, carboxypeptidase g rapidly decreased mtx concentrations and improved survival [ ] . cpdg selectively eliminated systemic mtx in patients treated with high dosages targeting cns malignancy, and rescued a patient receiving mtx with renal failure in [ , ] . after the original enzyme source of cpdg was lost, a revived recombinant cpdg product demonstrated success in both i.v. and intrathecal rescue of mtx overdose in non-human primates [ ] [ ] [ ] . successful use in multiple case reports and human trials in adult and pediatric patients with i.v. and intrathecal mtx overdose emerged [ , , , , [ ] [ ] [ ] . glucarpidase is a dimerized protein with two domains -a zinc-dependent catalytic domain that removes c-terminal glutamate residues of folate and folate analogues and a β-sheet interaction site [ ] . glucarpidase splits mtx and its -hydroxy-mtx metabolite into inactive -{[ , -diamino- -(pteridinyl)methyl]-methylamino}-benzoic acid (dampa) and hydroxy-dampa plus glutamate [ , ] . mtx serum concentrations decline by - % within minutes after glucarpidase [ , , , , , ] . intracellular, intraluminal (gastrointestinal tract) and intracerebral mtx is unaffected, creating the potential for rebound concentrations and persistent cytotoxicity [ , , , [ ] [ ] [ ] . leucovorin therapy must continue after carboxypeptidase administration. dampa's poor urinary solubility also requires ongoing alkalinization and saline diuresis to prevent renal precipitation [ , ] . anti-glucarpidase antibodies have been detected in patients receiving glucarpidase, although patients have been successfully treated with additional doses of glucarpidase for persistently elevated mtx concentrations [ , , , , , , ] . hplc must be used to determine actual mtx concentrations after glucarpidase as both mtx metabolites, -hydroxy-mtx and dampa, interfere with immunoassay techniques [ ] . glucarpidase has an affinity for mtx approximately -to -fold higher than it does for leucovorin; however, its affinity for folate and -methyltetrahydrofolate are similar [ , ] . glucarpidase eliminates active levo-( s)-leucovorin about % faster than nonphysiological dextro-( r)-leucovorin [ ] . a study to address the clinical consequence is ongoing. because of the stereoselective destruction of active leucovorin and its metabolites, many protocols attempt to separate leucovorin administration from glucarpidase administration by - hours. administration of glucarpidase more proximate to leucovorin administration, and which antidote to provide should glucarpidase become available at a leucovorin dosing interval, requires a thoughtful benefit-risk assessment. countryspecific information on obtaining glucarpidase, institutional review board protocol, and consent issues have been made available online (www.btgplc.com/ btgpipeline/ /voraxaze.html; and www.fda.gov/cder/cancer/singleind. htm). cardiovascular pharmaceuticals comprise a wide variety of agents including anti-dysrhythmics, β-adrenergic antagonists (β-blockers, bbs), angiotensin antagonists, calcium channel antagonists (ccbs), cardioactive glycosides, and imidazoline derivatives. overdose of these agents alone or in combination can generate potentially lethal combinations of impaired conduction, dysrhythmia, vasodilatation, and negative inotropy. management of severe cases may necessitate diagnostic adjuncts such as echocardiography and right heart catheterization (swan-ganz measurements). in cases refractory to routine supportive care, vigorous gastrointestinal decontamination, and pharmacological intervention, aggressive measures including cardiac pacing, intra-aortic balloon counter-pulsation, or extracorporeal circulation (cardiopulmonary bypass) may be required until toxin elimination can be achieved [ ] . cardiac pacing may improve heart rate without increasing cardiac output if inotropy is compromised. use of naloxone in the management of overdose of clonidine and angiotensin receptor antagonists and angiotensin converting enzyme inhibitors is provided in the opioid antagonists section. strategies to mitigate the anticoagulant toxicity of vitamin k antagonists (i.e., coumadin) including exogenous oral or i.v. vitamin k, fresh frozen plasma, prothrombin concentrates, and recombinant factor vii are detailed in the american college of chest physicians evidence-based clinical practice guidelines [ ] . the guidelines also address protamine sulfate for reversal of heparin anticoagulation and use of nonheparin anticoagulants for treatment and prevention of heparininduced thrombocytopenia [ , ] . atropine (d,l-hyoscyamine) is familiar to clinicians due to its use in several advanced cardiac life support (acls) algorithms [ ] . atropine is a centralacting, competitive antagonist of muscarinic acetylcholine receptors (m -m ) [ ] . it is used to counteract bradycardia from bbs, ccbs, cardioactive glycosides, and clonidine. atropine increases basal heart rate; it does not affect the basal force of contraction [ ] . positive chronotropy alone may not produce systemic benefit in severe poisoning, and conduction system poisoning may limit responsiveness to atropine [ ] . for symptomatic bradycardia, atropine . - . mg (pediatric dose: . mg/kg) i.v. is provided every - min to a maximum dose of mg. paradoxical parasympathetic response may occur during slow infusions or doses less then . mg ( . mg minimum in children) [ ] . in slowing gastrointestinal motility, atropine may impair decontamination with wbi or ac. ccbs antagonize l-type calcium channels, slowing entry of calcium ions during myocyte depolarization; however, intracellular calcium release is not directly affected. this disrupts calcium-mediated excitation-contraction coupling, action potential generation and conduction, and vascular smooth muscle tone [ ] . exogenous i.v. calcium is indicated in cases of ccb and bb toxi-city [ ] . in animal models, calcium salts reverse ccb-induced deficits in contractility, blood pressure, and cardiac output [ ] . multiple uncontrolled cases reports document the effectiveness of calcium salts; however, interpretation of effectiveness is complicated by the co-administration of other modalities. some authors advocate aggressive high-dose calcium therapy, providing large amounts of calcium without apparent ill effect [ ] . this approach does carry a risk of death from hypercalcemia [reported concentration, . mg/dl ( . mmol/l) after g calcium] [ ] . others recommend a bolus dose followed by continuous infusion to maintain physiological calcium levels [ ] . peripheral administration as calcium gluconate decreases the risk of extravasation and tissue necrosis. a standard container of ml of % calcium gluconate provide . meq ( mg) elemental ca + ; ml of % calcium chloride ( g total cacl ) yields . meq elemental ca + [ ] . a suggested approach is to initially administer a . ml/kg ( . meq/kg) bolus of % calcium gluconate ( . ml/kg % cacl ) over - min [ , ] . empirically, this is roughly one vial ( g) of % cacl or three vials ( g) of % calcium gluconate i.v. the bolus may be repeated several times. due to bolus dissipation, most patients are placed on an infusion of % calcium gluconate at . - . ml/kg per hour ( . - . meq/kg per hour) or . - . ml/kg per hour [ , ] . serum phosphate, calcium, and hydration status should be closely monitored. calcium administration for hyperkalemia has been generally contraindicated in cases of cardioactive glycoside toxicity, out of concern for dysrhythmias or systolic arrest (also known as "stone heart") [ ] . while more recent studies have challenged this assertion, it is advisable to withhold calcium until the definitive cardiac glycoside antidote, digoxinspecific fab fragments, has been provided [ ] . digoxin and cardioactive glycosides inhibit the cardiac sodium-potassium atpase. the subsequent accumulation of sodium in the cytoplasm dissipates the driving force for calcium expulsion via the sodium-calcium exchanger. increased intracellular calcium enhances actin-myosin coupling, myocyte contraction, and inotropy. in overdose, the excess calcium may result in membrane hyperexcitability and delayed after-depolarizations. increased vagal tone decreases conduction through the av node. the combination of increased automaticity and vagotonicity may yield lethal ventricular escape rhythms. digoxin-specific antibody fragments bind free digoxin in serum to decrease digoxin serum concentrations to undetectable levels within minutes [ ] . successful reversal of digoxin toxicity with digoxin-specific fab was first reported in [ ] . the results of a prospective multicenter study demonstrated significant effectiveness in reversing life-threatening digitalis toxicity, and more recent studies confirm ongoing fab fragment utility [ , ] . digoxin-specific fab were also shown to be effective in children [ ] . digoxin-specific fab are produced from purified ovine-derived immunoglobulin g. cleaving the fc antibody portion significantly improves renal excretion of the complex, decreases immunogenicity, and facilitates diffusion of remaining free fab into tissue [ ] . reflecting digoxin redistribution from target organs of toxicity, the initial response to digoxin-specific fab was min ( - min), and complete reversal of systemic toxicity occurred on average by min ( - min) [ ] . indications for therapy include life-threatening or progressive dysrhythmia or shock; potassium greater than . meq/l (acute poisoning); chronic poisoning with other end-organ manifestations such as altered mental status, significant gastrointestinal symptoms or renal impairment; or serum digoxin concentration > ng/ml or greater than ng/ml beyond hours after ingestion. hyperkalemia is rapidly reversed by digoxin-specific fab [ ] . one vial neutralizes approximately . mg of digoxin (or digitoxin). dosing is based either on amount ingested [number of vials = amount ingested (in mg) × . (oral bioavailability) / . ], or a serum concentration [number of vials = serum digoxin concentration (ng/ml) × patient weight (kg) / ]. the number of vials is rounded up and administered i.v. over min. empiric therapy is - vials for adult or pediatric patients in acute poisoning or - vials ( - vials in children) in chronic poisoning. partial reversal is recommended by some authors [ ] , but is not common u.s. practice due in part to concern for recrudescent toxicity with inadequate therapy [ ] . following treatment, free digoxin concentrations may rebound upwards within - hours, most likely reflecting tissue redistribution into the vascular space [ ] . this provides a measure of protection against development of significant congestive heart failure (chf) in patients dependent upon digoxin for inotropy, although exacerbation of chf may occur [ ] . clinically significant late rebound of digoxin concentrations and toxicity have occurred in patients with marked renal dysfunction [ ] . immunogenicity from repeat digoxin-specific fab has generally not been significant, although allergic reactions have been infrequently reported with administration [ ] . digoxin-specific fab has been used clinically or experimentally to treat poisoning by other cardiac glycosides -yellow oleander (thevetia peruviana), nerium oleander, chan su and "love stone" (extract of the bufo bufo gargarizans toad) [ , ] . higher dosing may be required due to poor binding affinity. bbs competitively antagonize catecholamine effects at cardiac β-receptors, leading to decreased inotropy and slowed conduction through the av node. bradycardia, conduction delay, hypotension, and decreased cardiac output may accompany significant poisoning. bb interference with gluconeogenesis and glycogenolysis may lead to hypoglycemia, as well as blunt the catecholamine response that is important in its recognition. glucagon, a -amino acid peptide hormone secreted by pancreatic α-cells, counteracts hypoglycemia and the actions of insulin; regulates gastrointestinal motility; and mediates the rate of renal filtration, urea excretion, and water resorption [ ] . the current glucagon product is now produced in non-pathogenic e. coli by recombinant techniques [ ] . myocardial binding occurs at a distinct glucagon receptor (gcgr) coupled with the β-agonist binding site. antidotal (off-label) use of glucagon thus bypasses β-receptor blockade to directly induce g-protein-mediated stimulation of adenylate cyclase to convert atp to camp [ ] . camp, in turn, activates protein kinase a (pka), which promotes the phosphorylation and opening of dormant l-type calcium channels to improve calcium-dependent excitation-contraction coupling [ ] . another proposed mechanism is c-terminal cleavage of glucagon to miniglucagon, which has a direct effect on sarcoplasmic reticulum calcium stores via arachidonic acid [ ] . in human volunteers evaluated by cardiac catheterization, glucagon increased heart rate, cardiac index, and mean atrial pressure, but not left ventricular end-diastolic pressure (edp) or systemic vascular resistance (svr) [ ] . clinical experience in overdose consists primarily of case reports [ , ] . due to the complex nature of overdose, glucagon is often used in combination with other agents in severe bb overdose. additionally, several ex vivo experiments, controlled animal studies, and uncontrolled case reports have demonstrated that glucagon can be beneficial in ccb exposure [ ] [ ] [ ] . the recommended initial bolus dose of glucagon is - μg/kg, which may be repeated after - min [ ] . a continuous infusion corresponding to the total effective bolus reversal dose is then provided per hour (e.g., if clinical response was observed following administration of mg, mg, and finally mg, the hourly infusion would be mg/hour). the effects of glucagon administered i.v. begin within - min, peak at - min and last for approximately min [ ] . nausea and vomiting are common and should be anticipated. this may complicate management of patients with depressed mental status or airway concerns. flushing, transient hyperglycemia, and smooth muscle relaxation, and ileus may also occur. since ccbs antagonize the l-type calcium channel in pancreatic islet cells, a subsequent decreased insulin production can produce hyperglycemia [ ] . animals poisoned by ccbs have impaired myocardial fatty acid uptake (leaving them dependent upon carbohydrate metabolism), impaired uptake of glucose, and myocardial insulin resistance [ , ] . in humans, intracoronary verapamil increased glucose release and altered myocardial lactate use from consumption to release [ ] . decades ago, glucose-insulin-potassium (gik) was proposed as adjuvant therapy for acute myocardial infarction, with the intent of suppressing uptake of free fatty acids, improve myocardial energy production, and stabilize intracellular potassium [ ] . randomized trials of gik therapy in patients with acute myocardial infarction (ami) have not shown benefit, although the insulin doses tend to be low (in general, ≤ . u/kg) [ ] . experience in the surgical literature in cases where much higher insulin doses have been used has been somewhat different [ ] . patients undergoing aortic valve replacement and coronary artery bypass who received high-dose insulin at unit/kg per hour demonstrated more rapid lactate clearance, lower glucose, lower dobutamine requirements, a trend for improved cardiac indices, and potential anti-inflammatory benefit (lower c-reactive protein and free fatty acid levels) [ ] . insulin doses of . units/kg were tolerated without excess increase of insulin-induced potassium elimination [ ] . in combination with dopamine, insulin units/kg was used to significantly augment cardiac output and decrease systemic vascular resistance in post-coronary artery bypass graft (cabg) patients without generating excess in oxygen demand [ ] . additional benefits of high-dose insulin included overcoming insulin resistance, increased expression of glucose transporters, and improved turnover of sodium-potassium-atpases [ ] . the basis for high-dose insulin euglycemia therapy (hiet) (off-label) in overdose has been explored in a series of animal models of ccb and bb toxicity [ , , [ ] [ ] [ ] . hiet increased myocardial lactate uptake and improved systolic and diastolic heart function. insulin outperformed epinephrine and glucagon [ ] [ ] [ ] . multiple human cases of successful management of ccb overdose with hiet have been described [ , ] . because the beneficial cardiovascular effects of hiet are not seen for - min after initiation, it must be considered early, before patients become unsalvageable [ ] . a proposed dosing scheme includes a bolus dose of regular insulin of . units/kg, followed by an infusion of . - . units/kg per hour, titrated upwards as necessary [ ] . a dextrose bolus is also provided unless significant hyperglycemia exists, followed by an infusion of . - . g/kg per hour to maintain blood glucose between and mg/dl. persistent physician reticence to utilizing the high-dose insulin out of concern for excess hypoglycemia presents an obstacle for implementation of adequate hiet [ ] . this ignores a body of physiological data that demonstrate that the insulin transport follows saturation kinetics [ , ] . alternatively, it has also been demonstrated that insulin-stimulated glucose clearance reaches a maximum in both lean and obese subjects [ ] . taken together, this suggests that, from a therapeutic standpoint, since insulin effect via insulin receptors appears saturable, additional mechanisms must be at work. the effects of hiet may include counteracting ccb-mediated insulin impairment or shockinduced hyperglycemia, improving myocardial substrate utilization, and improving myocardial metabolism [ ] . from an adverse-effects standpoint, once adequate and ongoing glucose has been provided, hypoglycemia should not present an excessive risk [ ] , although frequent serum glucose and potassium evaluation are obvious components of hiet therapy. due to the high dosing, insulin may persist after the infusion cessation and necessitate ongoing supplemental dextrose beyond insulin infusion. as hypokalemia is an intracellular result of shift, it is supplemented cautiously. during administration of local anesthetics, severe toxicity may result from systemic absorption or unintended intravascular administration. loss of consciousness, dysrhythmia, cardiovascular collapse, seizures, and lactate-associated acidemia may rapidly ensue [ ] . furthermore, in animal models, for some of the local anesthetics (bupivacaine, levobupivacaine, and ropivacaine), treatment with "standard" advanced cardiac life support (acls) drugs such as epinephrine may precipitate ventricular dysrhythmia [ ] . following a serendipitous observation that pretreatment with a lipid emulsion altered the dose-response to bupivacaine-induced asystole, murine and canine studies provided evidence of survival benefit with lipids in bupivacaine toxicity [ , ] . case reports of successful resuscitation of patients severely affected by bupivacaine, levobupivacaine, mepivacaine, prilocaine and ropivacaine (alone or in combination) followed [ , [ ] [ ] [ ] . pediatric experience is limited to a case of successful resuscitation following lidocaine/ropivacaine toxicity from a posterior lumbar plexus block [ ] . lipid therapy has been successfully applied in human bupropion toxicity and combined quetiapine and sertraline overdose [ , ] . animal models have suggested a possible benefit in clomipramine, propranolol, thiopentone, and verapamil poisoning [ ] [ ] [ ] [ ] . an understanding of lipid's mechanism of action is incomplete. it may act as a "circulating lipid sink" in which excess lipophilic drug may dissolve; modulate intracellular processes; or provide an alternative myocardial energy supply [ ] . presumably due to central sympathetic activation, human volunteers given a -hour lipid emulsion ( %) infusion had increased systemic vascular resistance, blood pressure, muscle sympathetic nerve activity, and concentrations of insulin and aldosterone, without increased cardiac output [ ] . lipid emulsion increased inotropy in both spontaneously beating and paced isolated rat hearts poisoned with levobupivacaine [ ] . dosing guidelines for the off-label use of lipid emulsion in resuscitation are provisional, as optimal bolus and continuous infusion therapy and timing are still being explored. the association of anaesthetists of great britain and ireland recommends an i.v. bolus of . ml/kg intralipid ® ( %) over min, which may be repeated twice at -min intervals if an adequate circulation has not been restored [ ] . following the initial bolus, an infusion is commenced at . ml/kg per min (which may be increased to . ml/kg per min in inadequate circulation). propofol is an inadequate substitute [ , ] . ongoing lipid therapy may be required as recrudescence may occur [ ] . hyperamylasemia may be anticipated. additional concerns include pancreatitis, allergic reactions, acute myocardial infarction, fat embolism, and altered coagulation [ ] . in lapine and porcine models of asphyxial cardiac collapse (pulseless electrical activity or arrest), lipid emulsion was markedly ineffective [ , ] . in vitro, lipid affinity for both bupivacaine and ropivacaine is also adversely affected by low ph (by a factor of . in a ph drop from . to . ) [ ] . these data suggest that ventilatory status must be aggressively addressed early in toxicity. due to their physicochemical characteristics and structure, many non-antiarrhythmic drugs are able to antagonize or alter expression of the myocardial potassium delayed rectifier channel (herg, kcnh , lqt ). with channel block, potassium efflux is compromised, and the repolarizing cardiac i kr current is impaired. the surface ecg reflects this as qt prolongation. age, female gender, comorbidities such as structural heart disease, electrolyte disturbances such as hypokalemia, and heart rate (bradycardia) may provide additional risk. certain antibiotics, antihistamines, antipsychotics, antidepressants, and methadone are prone to induce qt prolongation. qt prolongation is associated with torsade de pointes, a polymorphic ventricular arrhythmia that can degenerate into ventricular fibrillation, cardiac arrest and sudden death [ ] . if significant qt prolongation (qtc > ms) is detected, administration of - g magnesium sulfate i.v. (pediatric dose, - mg/kg) over to min (depending on urgency of presentation), followed by an infusion of - mg/min is suggested [ ] . rapid infusion may cause hypotension, and magnesium should be administered cautiously in renal failure. a second bolus can be provided - min later [ ] . magnesium sulfate i.v. is effective in arrhythmias occurring due to early or delayed depolarization-induced triggered activity [ ] . acceleration of the heart rate with isoproterenol or transvenous pacing (overdrive pacing) may be needed to preclude recurrence of torsade de pointes while correction of underlying risk factors (hypokalemia and hypocalcemia) ensues. immediate non-synchronized defibrillation is required for unstable polymorphic ventricular tachycardia or ventricular fibrillation. severe cardiovascular toxicity may result from blockade of cardiac sodium channels by tricyclic antidepressants (tcas) -leading to conduction delays, dysrhythmias, and myocardial depression. tcas adversely affect maximum upstroke velocity (v max ), which approximates the magnitude of sodium entry [ ] . the sodium channel blockade displays rate dependence. at slow rates the tca has time to disassociate, allowing channel recovery. at faster rates, block progressively worsens. given the anticholinergic effects of tcas that speed the heart rate, this is a significant concern. however, attempts to decrease heart rate with propranolol produced hypotension and lethality in canine studies [ , ] . with progressive sodium channel block, ventricular impulse propagation becomes delayed. sodium channel blockade manifests on the surface ecg as qrs widening. a qrs equal or greater than ms is a significant predictor of seizure; a qrs ≥ ms predicts ventricular dysrhythmia [ ] . the right bundle branch has a relatively longer refractory period, and it is affected disproportionately by impaired intraventricular conduction delay. rightward terminal axis shift or outright bundle branch block may be present [ ] . these rightward terminal forces may also produce terminal r waves in leftwarddirected leads [ ] . acidemia secondary to hypoperfusion or seizure may generate progressively worsened block. in an acidemic environment, free tca concentrations increase as binding to α- acid glycoprotein decreases, the tca ionized fraction increases, and sodium channel blockade worsens [ ] . seizures are severe and consequential, leading to qrs widening and hypotension [ ] . administration of sodium bicarbonate improves v max and action potential amplitude by increasing extracellular ph and sodium concentration [ ] . consequentially, compromised myocardial inotropy, conduction aberrations, and dysrhythmia are reversed. several animal studies have demonstrated these beneficial effects [ , ] . both the sodium and alkalemia induced by sodium bicarbonate improve cardiac performance [ ] . the enhanced inotropy with sodium bicarbonate is independent of and additive to vasopressor treatment [ ] . hyperventilation-induced alkalinization similarly narrows the qrs [ ] . sodium bicarbonate outperformed hyperventilation in a swine model, although hypertonic saline was superior to both [ ] . this approach has been reported clinically [ ] . while sodium bicarbonate is recommended for qrs widening in tca evidence-based consensus guidelines for out-ofhospital management, actual human studies are not as extensive as one might suspect [ , ] . initially, hypertonic sodium bicarbonate - meq/kg i.v. is provided, preferably with continued ecg monitoring of the qrs. institutions usually stock either an . % solution ( meq/ml sodium and bicarbonate ions) or a . % solution ( . meq/ml sodium and bicarbonate ions). rarely, a % solution may be encountered ( . meq/ml). a "standard" -ml ampule of . % or . % solutions would deliver or . meq of nahco . several boluses may be required, either initially or as the bolus effect declines due to redistribution [ ] . ongoing alkalinization should be provided as discussed previously, with a goal of serum ph . . if sodium bicarbonate administration is problematic due to fluid load, hyperventilation and/or hypertonic saline may be required [ , ] . due to mechanistic similarities, sodium bicarbonate has been recommended for qrs widening seen in poisoning by vaughn-williams class ia and ic antidysrhythmics, cocaine, diphenhydramine, carbamazepine, and propoxy-phene [ ] [ ] [ ] [ ] . treatment of bupropion-induced qrs widening with sodium bicarbonate has met with both success and failure [ ] . sodium bicarbonate has also been suggested to treat qrs widening from venlafaxine; similar effects seen with lamotrigine might also be amenable [ , ] . sodium bicarbonate therapy may have a role in taxus species (yew berry) toxicity [ ] . treatment of amantadine-induced qrs widening with sodium bicarbonate may be complicated by concurrent profound hypokalemia [ ] . naloxone is a competitive opioid antagonist at all receptor subtypes [ ] . it can prevent or reverse the effects of opioids, notably cns and respiratory depression. massive doses of naloxone ( . mg/kg with . mg/kg per hour infusion) have been administered safely in non-opioid tolerant individuals suffering from spinal cord injury [ ] . however, indiscriminate use of naloxone in opioid-tolerant individuals can precipitate acute opioid withdrawal, with attendant acute lung injury, seizure, hypertension, or cardiac dysrhythmia [ ] . these are likely associated with the abrupt, significant, and sustained increases in plasma catecholamine concentrations (epinephrine and norepinephrine) that accompany narcotic reversal, particularly in the setting of hypercapnia [ ] . withdrawal-induced vomiting may compromise the airway in patients with concomitant sedative-hypnotic ingestion. precipitated withdrawal-associated agitation and violent behavior may require chemical restraint, leading to a vicious cycle of compromised cns and cardiopulmonary function as naloxone wears off. self-release and relapse following naloxone administration is also a concern in opioids with prolonged duration of effect (methadone, controlled-release oxycodone hydrochloride, etc.). naloxone is no longer recommended as the initial resuscitation of newborns with respiratory depression in the delivery room; precipitation of acute neonatal opioid withdrawal may produce severe consequences [ ] . sudden cardiac arrest has occurred in preterm neonates given naloxone to reverse opioid overdose [ ] . naloxone is utilized in those individuals with clear evidence of the opioid toxidrome. those with a respiratory rate ≤ or hypopnea are likely to benefit [ ] . the goal of therapy is titration to adequate ventilatory status without withdrawal. after normocapnia is achieved by supported ventilation, this can be done with i.v. administration of . - . mg initially (e.g., ml of . mg naloxone in ml diluent or mg naloxone in ml diluent). due to rapid onset, effectiveness can be assessed, and if required, the dose can be titrated upwards incrementally to . mg, mg, or even mg. patients without response to mg naloxone are unlikely to have opioid-induced respiratory depression. nonopioid-dependent adults are administered . - mg i.v. pediatric dosing for infants and children from birth to years of age or less than kg body weight is . mg/kg; children older than years of age or weighing more than kg are provided mg [ ] . for longer acting opioids, following adequate initial opioid antagonism, two-thirds of the initial naloxone reversal bolus is provided as a continuous i.v. infusion [ ] . naloxone can successfully antagonize buprenorphine overdose in children, although prolonged therapy and monitoring may be required [ ] . higher doses may be required due to reverse buprenorphine effects because of its high affinity for opioid receptors [ ] . naloxone has also been used to reverse clonidine toxicity, although this is not always the case [ ] . it has been postulated that patients with higher hyperadrenergic tone (who have higher concentrations of endogenous opioids) or those in whom clonidine induces more endogenous opioid release may respond best to naloxone [ ] . mental status, blood pressure, and heart rate may respond differently. naloxone has been employed in angiotensin converting enzyme inhibitor overdose. one author reported that a . mg bolus of naloxone followed by repeat mg bolus reversed hypotension due to overdose with mg captopril [ ] . naloxone has been ineffective in reversing hypotension in other cases complicated by co-ingestants [ ] . the mechanism may relate to antagonism of endogenous opioids [ ] . co-administration of . mg/kg naloxone mitigated captopril-related decreases in systolic and diastolic blood pressure in healthy volunteers [ ] . a placebo-controlled study of healthy men found that naloxone pretreatment with mg followed by . mg/hour infusion precluded systolic blood pressure decrease induced by captopril ( mg) [ ] . under different experimental conditions [naloxone, . mg bolus and a -hour continuous infusion ( . mg/hour), and captopril ( mg)], no difference was observed [ ] . analogous to naloxone antagonism at opioid receptors, flumazenil competitively antagonizes benzodiazepine receptors -allosteric sites located at the macromolecular gaba a receptor complex, which regulate chloride ion flux within the associated ion channel [ ] . flumazenil reverses the sedative, psychomotor, and amnestic effects of benzodiazepines [ ] . flumazenil's effectiveness depends upon the number of benzodiazepine receptors that can be occupied according to the mass-action law, the affinity of a particular benzodiazepine for the receptor, and the free benzodiazepine concentration near the receptor [ ] . in contrast, antagonism of benzodiazepine-induced respiratory depression is inconsistent, and acute tolerance may develop to large doses [ ] [ ] [ ] . flumazenil administration can reverse bispectral index (bis) depression and permit earlier emergence from anesthesia in patients provided non-benzodiazepine anesthesia (propofol/remifentanil) [ ] . postulated mechanisms included intrinsic cns stimulant activity or antagonism of endogenous benzodiazepine-like ligands (endozepines). under certain experimental conditions, flumazenil may also demonstrate partial agonist or even inverse agonist activity [ , ] . the appropriate utilization of flumazenil as an antidote in patients with benzodiazepine overdose is still a matter of debate. patients who ingest benzodiazepines alone or in combination generally have acceptable outcomes with supportive care alone. proponents argue that awakening is therapeutic and diagnostic, obviates requirements for investigatory procedures, and limits complications of sedation. opponents point to the low risk of mortality with benzodiazepine ingestion, frequent co-ingestants for which flumazenil is ineffective or contraindicated, relapse, and risks of reversal. while flumazenil can be administered safely, indiscriminate flumazenil administration may produce an acute withdrawal syndrome in benzodiazepine-dependent patients, seizures, dysrhythmias, vomiting, and agitation [ ] [ ] [ ] [ ] . flumazenil is not recommended in cases complicated by co-ingestants capable of inducing seizures or dysrhythmias (e.g., bupropion, carbamazepine, chloral hydrate, chlorinated hydrocarbons, chloroquine, cocaine, cyclic antidepressants, cyclosporine, isoniazid, lithium, methylxanthines, monoamine oxidase inhibitors, phenothiazines, and propoxyphene) [ , , ] . as might be anticipated with an antidote of lesser half-life than many benzodiazepines, clinical condition may deteriorate following initial improvement, mandating ongoing monitoring. in one study, patients with primarily benzodiazepines ingestion remained awake for ± min following flumazenil; this was markedly decreased to ± min with co-ingestants [ ] . this may be problematic in patients who, once aroused, demand release from medical care. after excluding co-ingestants of concern, vital sign abnormalities, and an aberrant ecg, and considering the risk-benefit ratio, flumazenil is administered slowly i.v., titrated to clinical effect ( . mg/min, max ≤ mg) [ ] . offlabel continuous infusions of . - . mg/hour have been provided to preclude relapse. patients poisoned by pharmaceuticals present many challenges to the treating clinicians. they generally benefit from aggressive support of vital functions, a careful history and physical examination, specific laboratory analyses, and a thoughtful consideration of the risks and benefits of decontamination and enhanced elimination. data on the effectiveness of certain antidotes ranges from isolated case reports to robust clinical trials. clinicians are encouraged to liberally utilize consultation with regional poison centers or those with toxicology training to assist with diagnosis, management, and administration of antidotes, particularly in unfamiliar cases. no outside funding or support was received. the author has no financial interest in any products mentioned or the companies that produce them. use of trade names is for identification purposes only and does not constitute endorsement by the author, the nyu school of medicine, the new york city poison control center, or the new york city department of health and mental hygiene. within the medical literature, pharmaceuticals, pharmaceutical combinations, and other products are used offlabel as antidotal therapies; off-label uses are referred to in this review. this is for discussion purposes only and does not constitute endorsement of off-label use by the author, the nyu school of medicine, the new york city poison control center, or the new york city department of health and mental hygiene. as medicine is an ever-changing science, readers are encouraged to confirm the information contained in this review -by consulting product and safety information sheets, regional poison centers, those with toxicological expertise, and other resources -particularly in the case of new or infrequently used drugs. analeptic use in clinical toxicology: a historical appraisal treatment of choice in barbiturate poisoning; series of twenty-nine cases of barbiturate poisoning treated with pentylenetetrazole (metrazol) and supportive therapy a study of the anti-barbiturate effects of bemegride respiratory stimulant effects of ethamivan and picrotoxin the case against the use of respiratory stimulants a double blind comparison of doxapram, ethamivan and methylphenidate nagd regimen for the coma of drug-related overdose physostigmine in coma due to drug overdose physostigmine for γ-hydroxybutyrate coma: inefficacy, adverse events, and review the poisoned patient with altered consciousness. controversies in the use of a 'coma cocktail anonymous ( ) practical pharmacology. xxiv management of oil of citronella poisoning emetics, cathartics, and gastric lavage fullers earth; its absorptive power, and its antidotal value for alkaloids management of acute childhood poisoning therapeutic trends in the treatment of barbiturate poisoning. the scandinavian method patients with acute poisoning seen in a general medical unit ( - ) antagonism of imipramine poisoning by anticonvulsants in the rat phenytoin prophylaxis of cardiotoxicity in experimental amitriptyline poisoning wernicke's encephalopathy: new clinical settings and recent advances in diagnosis and management acetaminophen and salicylate serum levels in patients with suicidal ingestion or altered mental status value of rapid screening for acetaminophen in all patients with intentional drug overdose a randomized clinical trial of activated charcoal for the routine management of oral drug overdose how feasible is it to conform to the european guidelines on administration of activated charcoal within one hour of an overdose? position statement: ipecac syrup gastric emptying procedures in the self-poisoned patient: are we forcing gastric content beyond the pylorus? residual gastric content after gastric lavage and ipecacuanhainduced emesis in self-poisoned patients: an endoscopic study efficacy of ipecac during the first hour after drug ingestion in human volunteers gastric decontamination performed min after the ingestion of temazepam, verapamil and moclobemide: charcoal is superior to lavage gastric lavage for liquid poisons efficacy of ipecac-induced emesis, orogastric lavage, and activated charcoal for acute drug overdose position paper: gastric lavage efficacy of superactivated charcoal administered late ( hours) after acetaminophen overdose activated charcoal alone or after gastric lavage: a simulated large paracetamol intoxication out-of-hospital administration of activated charcoal by emergency medical services prolonged gastric emptying half-time and gastric hypomotility after drug overdose effect of anticholinergic drugs on the efficacy of activated charcoal efficacy of gastric emptying: gastric lavage versus emesis induced with ipecac superiority of activated charcoal alone compared with ipecac and activated charcoal in the treatment of acute toxic ingestions management of acutely poisoned patients without gastric emptying assessment of the efficacy of activated charcoal following gastric lavage in acute drug emergencies gastric emptying in acute overdose: a prospective randomised controlled trial comparison of three methods of gut decontamination in tricyclic antidepressant overdose whole-bowel irrigation versus activated charcoal in sorbitol for the ingestion of modified-release pharmaceuticals multiple-dose charcoal and whole-bowel irrigation do drugs and pharmaceuticals: management of intoxication and antidotes not increase clearance of absorbed salicylate ipecacuanha: the south american vomiting root toxicology of ipecac: a review effectiveness of -ml versus -ml doses of syrup of ipecac in children urinary excretion of ipecac alkaloids in human volunteers position paper: ipecac syrup evaluation of the time frame for home ipecac syrup use when not kept in the home poison treatment in the home home syrup of ipecac use does not reduce emergency department use or improve outcome a comparison of ipecac syrup and apomorphine in the immediate treatment of ingestion of poisons the frequency of complications associated with the use of multiple-dose activated charcoal esophageal laceration and charcoal mediastinum complicating gastric lavage severe hypernatremia secondary to gastric lavage advantage of high-surface-area charcoal for gastrointestinal decontamination in a human acetaminophen ingestion model position paper: single-dose activated charcoal clinical pharmacokinetics of oral activated charcoal in acute intoxications efficacy of activated charcoal administered more than four hours after acetaminophen overdose activated charcoal reduces the need for n-acetylcysteine treatment after acetaminophen (paracetamol) overdose acceleration of the body clearance of phenobarbital by oral activated charcoal enhancement of theophylline clearance by oral activated charcoal gastrointestinal clearance of drugs with activated charcoal european association of poisons centres and clinical toxicologists ( ) position statement and practice guidelines on the use of multi-dose activated charcoal in the treatment of acute poisoning physicochemical characteristics of drugs and response to repeatdose activated charcoal randomized study of the treatment of phenobarbital overdose with repeated doses of activated charcoal the treatment of tricyclic antidepressant overdose with repeated charcoal multiple-dose activated charcoal for treatment of yellow oleander poisoning: a single-blind, randomised, placebo-controlled trial multiple-dose activated charcoal in acute self-poisoning: a randomised controlled trial effect of charcoal-drug ratio on antidotal efficacy of oral activated charcoal in man the effect of food and ice cream on the adsorption capacity of paracetamol to high surface activated charcoal: in vitro studies role of repeated doses of oral activated charcoal in the treatment of acute intoxications aspiration of activated charcoal and gastric contents risk factors for emesis after therapeutic use of activated charcoal in acutely poisoned children gastrointestinal tract perforation with charcoal peritoneum complicating orogastric intubation and lavage gastrointestinal obstruction associated with multiple-dose activated charcoal fatal pulmonary aspiration of oral activated charcoal activated charcoal and the absorption of ferrous sulfate in rats treatment of acetaminophen ingestion with a superactivated charcoal-cola mixture effects of emetic and cathartic agents on the gastrointestinal tract and the treatment of toxic ingestion position statement: cathartics severe hypermagnesemia due to multiple-dose cathartic therapy electrolyte disorders following oral sodium phosphate administration for bowel cleansing in elderly patients sorbitol catharsis does not enhance efficacy of charcoal in a simulated acetaminophen overdose the effects of charcoal and sorbitol (alone and in combination) on plasma theophylline concentrations after a sustained-release formulation hypernatremia due to repeated doses of charcoal-sorbitol fatal poisoning from sodium phosphate enema. case report and experimental study rectal prolapse after oral cathartics effect of whole bowel irrigation on the pharmacokinetics of an acetaminophen formulation and progression of radiopaque markers through the gastrointestinal tract position statement: whole bowel irrigation activated charcoal alone and followed by whole-bowel irrigation in preventing the absorption of sustained-release drugs use of whole bowel irrigation in an infant following iron overdose whole-bowel irrigation as a treatment for acute lithium overdose whole bowel irrigation in an acute oral lead intoxication parachuting" meth: a novel delivery method for methamphetamine and delayed-onset toxicity from "body stuffing drugs and pharmaceuticals: management of intoxication and antidotes efficiency of whole bowel irrigation with and without metoclopramide pretreatment colonic perforation with volume laxatives life-threatening respiratory failure following accidental infusion of polyethylene glycol electrolyte solution into the lung theophylline desorption from activated charcoal caused by whole bowel irrigation solution endoscopic removal of iron bezoar following acute overdose endoscopic removal of slow release clomipramine bezoars in two cases of acute poisoning bezoar formation requiring endoscopic removal after intentional overdose of extended-release nifedipine endoscopic removal of a cocaine packet from the stomach acute pediatric lead poisoning: combined whole bowel irrigation, succimer therapy, and endoscopic removal of ingested lead pellets gastrotomy -a surgical approach to iron overdose radiopacity of clomipramine conglomerations and unsuccessful endoscopy: report of cases prognosis of cocaine body-packers surgical treatment in cocaine body packers and body pushers the altered pharmacokinetics and pharmacodynamics of drugs commonly used in critically ill patients position paper on urine alkalinization acute hypermagnesemia: a rare complication of antacid administration after bone marrow transplantation methods used to decrease lithium absorption or enhance elimination hypercalcemic crisis in pregnancy associated with excessive ingestion of calcium carbonate antacid (milk-alkali syndrome): successful treatment with hemodialysis hypermagnesemia-induced fatality following epsom salt gargles excessive calcium ingestion leading to milk-alkali syndrome pamidronate treatment of hypercalcemia caused by vitamin d toxicity lithium nephrotoxicity revisited forced euvolemic ciuresis with mannitol and furosemide for prevention of contrast-induced nephropathy in patients with ckd undergoing coronary angiography: a randomized controlled trial lithium poisoning: pharmacokinetics and clearance during different therapeutic measures treatment of life-threatening lithium toxicity with high-volume continuous venovenous hemofiltration clinical pharmacokinetics during continuous haemofiltration treatment of acute leukemia with amethopterin ( -amino, -methyl pteroyl glutamic acid) principles and techniques applied to enhance elimination successful treatment of valproic acid overdose with hemodialysis hemodialysis in the treatment of lactic acidosis in diabetics treated by metformin: a study of metformin elimination hemodialysis is as effective as hemoperfusion for drug removal in carbamazepine poisoning severe iron intoxication treated with exchange transfusion exchange transfusion in the treatment of severe theophylline poisoning exchange transfusion in severe infant salicylism acute poisoning with isoniazid treated by exchange transfusion exchange transfusion treatment in a newborn with phenobarbital intoxication vincristine overdose: experience with patients drug-induced methaemoglobinaemia successful whole blood exchange by apheresis in a patient with acute cyclosporine intoxication without long-term sequelae therapeutic approaches to ion channel diseases halothane modulation of skeletal muscle ryanodine receptors: dependence on ca + , mg + , and atp medical complications of psychiatric treatment the hunter serotonin toxicity criteria: simple and accurate diagnostic decision rules for serotonin toxicity the serotonin syndrome the serotonin syndrome toxin-induced hyperthermic syndromes a thyrotoxicosis outbreak due to dietary pills in paris thyrotoxicosis factitia in a post-aortocoronary bypass patient pediatric fatality following ingestion of dinitrophenol: postmortem identification of a "dietary supplement shortand long-term outcomes of heatstroke following the heat wave in lyon, france near-fatal heat stroke during the heat wave in chicago ambient temperature and mortality from unintentional cocaine overdose further studies of the role of hyperthermia in methamphetamine neurotoxicity heat stroke: clinical and chemical observations on cases cooling and hemodynamic management in heatstroke: practical recommendations whole-body cooling of hyperthermic runners: comparison of two field therapies emergency treatment of exertional heatstroke and comparison of whole body cooling techniques diazepam and chlormethiazole attenuate the development of hyperthermia in an animal model of the serotonin syndrome antagonism of cocaine, amphetamine, and methamphetamine toxicity effects of diltiazem on hyperthermia induced seizures in the rat pup dantrolene stabilizes domain interactions within the ryanodine receptor enhanced excitation-coupled calcium entry in myotubes expressing malignant hyperthermia mutation r c is attenuated by dantrolene managing an effective treatment for neuroleptic malignant syndrome drug treatment of the neuroleptic malignant syndrome contrast medium-induced nephropathy: strategies for prevention acetaminophen hepatotoxicity: the first years acetaminophen bioactivation by human cytochrome p enzymes and animal microsomes acetylcysteine for acetaminophen poisoning mechanism of action of n-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo intravenous acetylcysteine in paracetamol induced fulminant hepatic failure: a prospective controlled trial improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure n-acetylcysteine improves indocyanine green extraction and oxygen transport during hepatic dysfunction n-acetylcysteine increases liver blood flow and improves liver function in septic shock patients: results of a prospective, randomized, double-blind study selective effects of n-acetylcysteine stereoisomers on hepatic glutathione and plasma sulfate in mice dissociation of increased sulfation from sulfate replenishment and hepatoprotection in acetaminophen-poisoned mice by n-acetylcysteine stereoisomers a patient-tailored n-acetylcysteine protocol for acute acetaminophen intoxication a prospective evaluation of shortened course oral n-acetylcysteine for the treatment of acute acetaminophen poisoning guidelines for the management of paracetamol poisoning in australia and new zealand -explanation and elaboration efficacy of oral n-acetylcysteine in the treatment of acetaminophen overdose. analysis of the national multicenter study ( to ) deaths from low dose paracetamol poisoning acetaminophen misconceptions risk factors and mechanisms of anaphylactoid reactions to acetylcysteine in acetaminophen overdose frequency of medication errors with intravenous acetylcysteine for acetaminophen overdose dilution of intravenous n-acetylcysteine as a cause of hyponatremia comparison of the -hour intravenous and -hour oral acetylcysteine protocols for the treatment of acute acetaminophen poisoning hepatotoxicity despite early administration of intravenous n-acetylcysteine for acute acetaminophen overdose acetaminophen overdose with altered acetaminophen pharmacokinetics and hepatotoxicity associated with premature cessation of intravenous n-acetylcysteine therapy pharmacokinetic effects of diphenhydramine or oxycodone in simulated acetaminophen overdose tylenol extended relief overdose on the characters, actions, and therapeutic uses of the bean of calabar the first use of physostigmine in the treatment of atropine poisoning. a translation of kleinwachter's paper entitled 'observations on the effect of calabar bean extract as an antidote to atropine poisoning comparative inhibitory effects of various physostigmine analogs against acetyl-and butyrylcholinesterases inhibition of acetylcholinesterase from electric eel by (-)-and (+)-physostigmine and related compounds accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions unconventional ligands and modulators of nicotinic receptors asystole complicating physostigmine treatment of tricyclic antidepressant overdose assessing physostigmine's contraindication in cyclic antidepressant ingestions mechanism of imipramine-induced seizures in amygdala-kindled rats failure of physostigmine in intoxications with tricyclic antidepres-drugs and pharmaceuticals: management of intoxication and antidotes sants in rats a comparison of physostigmine and benzodiazepines for the treatment of anticholinergic poisoning physostigmine: is there a role for this antidote in pediatric poisonings? detecting life-threatening lactic acidosis related to nucleosideanalog treatment of human immunodeficiency virus-infected patients, and treatment with l-carnitine supplementation with l-carnitine does not reduce the efficacy of epirubicin treatment in breast cancer cells science review: carnitine in the treatment of valproic acid-induced toxicity -what is the evidence? case files of the children's hospital of michigan regional poison control center: the use of carnitine for the management of acute valproic acid toxicity basic pharmacology of valproate: a review after years of clinical use for the treatment of epilepsy valproic acid metabolism and its effects on mitochondrial fatty acid oxidation: a review delayed valproic acid toxicity: a retrospective case series acute valproate poisoning: pharmacokinetics, alteration in fatty acid metabolism, and changes during therapy valproic acid toxicity: overview and management carnitine disposition before and during valproate therapy in patients with epilepsy involvement of recognition and interaction of carnitine transporter in the decrease of l-carnitine concentration induced by pivalic acid and valproic acid acute valproate-associated microvesicular steatosis: could the [ c]methionine breath test be useful to assess liver mitochondrial function? carnitine as an antidote for acute valproate toxicity in children l-carnitine supplementation in childhood epilepsy: current perspectives serum and muscle carnitine levels in epileptic children receiving sodium valproate vigabatrin, lamotrigine, topiramate and serum carnitine levels carnitine deficiency and hyperammonemia in children receiving valproic acid with and without other anticonvulsant drugs the effect of carnitine supplementation in valproateinduced hyperammonaemia diet-and valproate-induced transient hyperammonemia: effect of l-carnitine carnitine deficiency during valproic acid treatment valproic acid overdose and l-carnitine therapy a case of valproateassociated hepatotoxicity treated with l-carnitine valproate-associated hyperammonemic encephalopathy effect of l-carnitine treatment for valproate-induced hepatotoxicity the role of carnitine supplementation during valproic acid therapy valproic acid overdoses: a retrospective study comparing serum drug levels and the incidence of adverse outcomes l-carnitine was safely administered in the setting of valproate toxicity carni-tor ® (levocarnitine) oral solution ( g per ml multidose). carnitor ® sf (levocarnitine) sugar-free oral solution ( g per ml multidose) a case of hemoperfusion and l-carnitine management in valproic acid overdose emg changes in chronically dialyzed uraemic subjects undergoing d,l-carnitine treatment enhancement of activities relative to fatty acid oxidation in the liver of rats depleted of l-carnitine by d-carnitine and a γ-butyrobetaine hydroxylase inhibitor drug-induced hypoglycemia. a review of cases drug-induced disorders of glucose metabolism. mechanisms and management astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter brain glucose uptake and unawareness of hypoglycemia in patients with insulin-dependent diabetes mellitus neuroglycopenia and adrenergic responses to hypoglycaemia: insights from a local epidemic of serendipitous massive overdose of glibenclamide reversal of salicylate-induced euglycemic delirium with dextrose hypoglycemic seizures during transient hypoglycemia exacerbate hippocampal dysfunction the distribution of hypoglycemic brain damage concentrated dextrose for intravenous administration docking studies show that d-glucose and quercetin slide through the transporter glut peripheral intravenous infiltration necrosis the effect of grams i.v. glucose on serum inorganic phosphate levels sandostatin ® octreotide acetate injection prescribing information pharmacological agents that directly modulate insulin secretion oral hypoglycemic agents drugs and pharmaceuticals: management of intoxication and antidotes inhibition of insulin secretion as a new drug target in the treatment of metabolic disorders molecular signaling of somatostatin receptors effect of octreotide on insulin requirement, hepatic glucose production, growth hormone, glucagon and c-peptide levels in type diabetic patients with chronic renal failure or normal renal function octreotide for pediatric sulfonylurea poisoning a diabetic woman with worsening heart failure, hunger, and tremor effectiveness of octreotide in a case of refractory sulfonylureainduced hypoglycemia octreotide reverses hyperinsulinemia and prevents hypoglycemia induced by sulfonylurea overdoses octreotide: an antidote for sulfonylureainduced hypoglycemia comparison of octreotide and standard therapy versus standard therapy alone for the treatment of sulfonylurea-induced hypoglycemia the effectiveness of various doses of octreotide for sulfonylurea-induced hypoglycemia after overdose successful treatment of sulfonylureainduced prolonged hypoglycemia with use of octreotide octreotide for sulfonylurea-induced hypoglycemia following overdose anaphylactoid reaction to octreotide case files of the medical toxicology fellowship training program at the children's hospital of philadelphia: a pediatric exploratory sulfonylurea ingestion bradycardia and hyperkalemia associated with octreotide administration octreotide in children with hypoglycaemia due to sulfonylurea ingestion laparoscopic pancreatectomy for persistent hyperinsulinemic hypoglycemia of infancy octreotide therapy for persistent hyperinsulinemic hypoglycemia of infancy effectiveness of sms - , a synthetic, long-acting somatostatin analogue, in treatment of quinine-induced hyperinsulinaemia hypoglycaemia and counterregulatory hormone responses in severe falciparum malaria: treatment with sandostatin bench-to-bedside review: antidotal treatment of sulfonylurea-induced hypoglycaemia with octreotide somatostatin and somatostatin analogues: pharmacokinetics and pharmacodynamic effects glyburide sold as "street steroid" causes hypoglycemia complicated by inappropriate iv administration of octreotide long-term non-surgical therapy of severe persistent congenital hyperinsulinism with glucagon isoniazid-associated hepatitis. report of an outbreak therapeutic drug monitoring in the treatment of tuberculosis pyridoxine in clinical toxicology: a review acute isoniazid poisoning accidental isoniazid poisoning -a report the origin and turnover of d-serine in brain glutamatergic regulation of serine racemase via reversal of pip inhibition the effect on gaba metabolism in brain of isonicotinic acid hydrazide and pyridoxine as a fuction of time after administration profound acidosis caused by isoniazid ingestion convulsions as the etiology of lactic acidosis in acute isoniazid toxicity in dogs single high-dose pyridoxine treatment for isoniazid overdose isoniazid self-poisoning fitzpatrick's dermatology in general medicine acute isoniazid poisoning in childhood reversal of prolonged isoniazid-induced coma by pyridoxine acute isoniazid neurotoxicity in an urban hospital isoniazid overdose: four case reports and review of the literature evaluation of diazepam and pyridoxine as antidotes to isoniazid intoxication in rats and dogs acute isoniazid toxicity and the need for adequate pyridoxine supplies isoniazid toxicity: a survey of pyridoxine availability sensory neuropathy from pyridoxine abuse. a new megavitamin syndrome treatment of refractory seizures in massive isoniazid overdose clinical practice guideline update: use of chemotherapy and radiation therapy protectants cytoprotective effects of amifostine in the treatment of childhood malignancies amifostine and glutathione prevent ifosfamide-and acrolein-induced hemorrhagic cystitis drugs and pharmaceuticals: management of intoxication and antidotes randomized placebo-controlled multicenter evaluation of diethyldithiocarbamate for chemoprotection against cisplatin-induced toxicities myeloid growth factors neumega ® [nu-meg<a] (oprelvekin) european oncology nursing society extravasation guidelines distributed by integrated commercialization solutions. manufactured by ben venue laboratories inc., and hameln pharmaceuticals gmbh for topotarget a/s. marketed by treatment of anthracycline extravasation with savene (dexrazoxane): results from two prospective clinical multicentre studies treatment of anthracycline extravasation with dexrazoxane -clinical experience dexrazoxane (totect): fda review and approval for the treatment of accidental extravasation following intravenous anthracycline chemotherapy management of anthracycline extravasation injuries topoisomerase iiα-dependent and -independent apoptotic effects of dexrazoxane and doxorubicin anthracycline extravasation: continue using dimethyl sulfoxide the response of acute leukemia to the administration of the folic acid antagonists, aminopterin and a-methopterin effect of methotrexate therapy upon choriocarcinoma and chorioadenoma immunosuppressant and cytotoxic drugs: unapproved uses or indications methotrexate in psoriasis: consensus conference the use of methotrexate in rheumatoid arthritis carboxypeptidase g rescue in patients with methotrexate intoxication and renal failure iatrogenically-related, fatal methotrexate intoxication: a series of four cases high-dose methotrexate-induced nephrotoxicity in patients with osteosarcoma polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis mechanisms of action of methotrexate theoretically required urinary flow during high-dose methotrexate infusion methotrexate: clinical pharmacology, current status and therapeutic guidelines high-dose methotrexate with citrovorum factor rescue: predictive value of serum methotrexate concentrations and corrective measures to avert toxicity high-dose leucovorin as sole therapy for methotrexate toxicity threshold methotrexate concentration for in vivo inhibition of dna synthesis in normal and tumorous target tissues clinical pharmacokinetics of high-dose leucovorin calcium after intravenous and oral administration safety and efficacy of l-leucovorin rescue following high-dose methotrexate for osteosarcoma intrathecal leucovorin after intrathecal methotrexate overdose carboxypeptidase-g rescue in cancer patients with delayed methotrexate elimination after high-dose methotrexate therapy the reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides glucarpidase (carboxypeptidase g ) intervention in adult and elderly cancer patients with renal dysfunction and delayed methotrexate elimination after high-dose methotrexate therapy treatment of accidental intrathecal methotrexate overdose with intrathecal carboxypeptidase g understanding and managing methotrexate nephrotoxicity carboxypeptidase g: purification and properties enzymatic cleavage of methotrexate provides a method for prevention of drug toxicity comparative effects of citrovorum factor and carboxypeptidase g on cerebrospinal fluid-methotrexate pharmacokinetics hemodialysis and enzymatic cleavage of methotrexate in man methotrexate pharmacokinetics following administration of recombinant carboxypeptidase-g in rhesus monkeys rescue of experimental intrathecal methotrexate overdose with carboxypeptidase-g successful carboxypeptidase g rescue in delayed methotrexate elimination due to renal failure successful treatment of intrathecal methotrexate overdose by using ventriculolumbar perfusion and intrathecal instillation of carboxypeptidase g optimal management of methotrexate intoxication in a child with osteosarcoma drugs and pharmaceuticals: management of intoxication and antidotes crystal structure of carboxypeptidase g , a bacterial enzyme with applications in cancer therapy pharmacokinetics and metabolism of the methotrexate metabolite , -diamino-n -methylpteroic acid carboxypeptidase-g rescue in a patient with high dose methotrexate-induced nephrotoxicity carboxypeptidase-g , thymidine, and leucovorin rescue in cancer patients with methotrexate-induced renal dysfunction favorable outcome in excessive methotrexate (mtx) intoxication after high-dose (hd) mtx therapy by early use of carboxypeptidase g (cpg ) carboxypeptidase g rescue after high-dose methotrexate comparative effects of citrovorum factor and carboxypeptidase g on cerebrospinal fluid-methotrexate pharmacokinetics treatment of accidental intrathecal methotrexate overdose with intrathecal carboxypeptidase g interactions of carboxypeptidase g with s-leucovorin and r-leucovorin in vitro: implications for the application in case of methotrexate intoxications interference of -hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays pre-authorisation evaluation of medicines for human use. withdrawal assessment report for voraxaze. emea/chmp/ / purification and properties of carboxypeptidase g from pseudomonas sp. strain rs- . use of a novel triazine dye affinity method tox-acls: toxicologic-oriented advanced cardiac life support pharmacology and management of the vitamin k antagonists: american college of chest physicians evidence-based clinical practice guidelines treatment and prevention of heparininduced thrombocytopenia: american college of chest physicians evidence-based clinical practice guidelines pharmacotherapy considerations in advanced cardiac life support muscarinic receptors in the mammalian heart calcium channel blocking drug overdose: an australian series management of β-adrenergic blocker and calcium channel antagonist toxicity a fatal case of iatrogenic hypercalcemia after calcium channel blocker overdose management of calcium channel antagonist overdose calcium gluconate injection, usp % assessment of the synergistic relationship between serum calcium and digitalis case files of the medical toxicology fellowship of the california poison control system-san francisco: calcium plus digoxin-more taboo than toxic treatment of severely digitalis-toxic patients with digoxin-specific antibody fragments reversal of advanced digoxin intoxication with fab fragments of digoxin-specific antibodies treatment of cases of life-threatening digitalis intoxication with digoxin-specific fab antibody fragments. final report of a multicenter study digoxin-specific fab fragments as single first-line therapy in digitalis poisoning the use of digoxin-specific fab fragments for severe digitalis intoxication in children review of clinical experience with digoxin immune fab (ovine) digoxin-specific antibody fragments: how much and when? pharmacokinetic aspects of digoxin-specific fab therapy in the management of digitalis toxicity late rebound digoxin toxicity after digoxin-specific antibody fab fragments therapy in anuric patient recurrent digoxin overdose and treatment with digoxin-specific fab antibody fragments successful treatment of oleander intoxication (cardiac glycosides) with digoxin-specific fab antibody fragments in a -year-old child: case report and review of literature efficacy of digoxin specific fab fragments (digibind) in the treatment of toad venom poisoning benefits and limitations of reducing glucagon action for the treatment of type diabetes information for the physician. glucagon for injection (rdna origin) treatment of poisoning caused by β-adrenergic and calcium-channel blockers arachidonic acid drives mini-glucagon action in cardiac cells cardiovascular effects of glucagon in man towards evidence based emergency medicine: best bets from the manchester royal infirmary. glucagon for the treatment of symptomatic β blocker overdose glucagon use in symptomatic β blocker overdose treatment of verapamil overdose with glucagon in dogs amelioration of nifedipine poisoning associated with glucagon therapy glucagon antagonism of calcium channel blockerinduced myocardial dysfunction myocardial metabolism dur-drugs and pharmaceuticals: management of intoxication and antidotes ing graded intraportal verapamil infusion in awake dogs beneficial myocardial metabolic effects of insulin during verapamil toxicity in the anesthetized canine myocardial lactate release after intracoronary verapamil application in humans: acute effects of intracoronary verapamil on systemic and coronary hemodynamics, myocardial metabolism, and norepinephrine levels effect of glucose-insulinpotassium infusion on mortality in critical care settings: a systematic review and meta-analysis forty years of glucose-insulin-potassium (gik) in cardiac surgery: a review of randomized, controlled trials anti-inflammatory effect of high-dose insulin treatment after urgent coronary revascularization surgery therapy with insulin in cardiac surgery: controversies and possible solutions high-dose insulin improves the efficacy of dopamine early after cardiac surgery. a study of myocardial performance and oxygen consumption insulin improves heart function and metabolism during non-ischemic cardiogenic shock in awake canines insulin improves survival in a canine model of acute β-blocker toxicity insulin versus vasopressin and epinephrine to treat β-blocker toxicity relative safety of hyperinsulinaemia/euglycaemia therapy in the management of calcium channel blocker overdose: a prospective observational study tarka(r) (trandolapril/verapamil hydrochloride extended-release) overdose the effect of insulin dose on the measurement of insulin sensitivity by the minimal model technique. evidence for saturable insulin transport in humans interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals dose-response characteristics of insulin action on glucose metabolism: a non-steady-state approach levobupivacaine-induced seizures and cardiovascular collapse treated with intralipid cardiac resuscitation after incremental overdosage with lidocaine, bupivacaine, levobupivacaine, and ropivacaine in anesthetized dogs lipid emulsion infusion rescues dogs from bupivacaine-induced cardiac toxicity resuscitation with lipid versus epinephrine in a rat model of bupivacaine overdose intravenous lipid infusion in the successful resuscitation of local anesthetic-induced cardiovascular collapse after supraclavicular brachial plexus block successful resuscitation of a patient with ropivacaineinduced asystole after axillary plexus block using lipid infusion reversal of central nervous system and cardiac toxicity after local anesthetic intoxication by lipid emulsion injection successful resuscitation after ropivacaine and lidocaine-induced ventricular arrhythmia following posterior lumbar plexus block in a child early treatment of a quetiapine and sertraline overdose with intralipid use of lipid emulsion in the resuscitation of a patient with prolonged cardiovascular collapse after overdose of bupropion and lamotrigine intralipid outperforms sodium bicarbonate in a rabbit model of clomipramine toxicity intralipid infusion ameliorates propranolol-induced hypotension in rabbits hemodynamic effects of intravenous fat emulsion in an animal model of severe verapamil toxicity resuscitated with atropine, calcium, and saline intralipid ameliorates thiopentone induced respiratory depression in rats: investigative pilot study non-esterified fatty acids increase arterial pressure via central sympathetic activation in humans the effects of lipid infusion on myocardial function and bioenergetics in l-bupivacaine toxicity in the isolated rat heart the association of anaesthetists of great britain & ireland ( ) guidelines for the management of severe local anaesthetic toxicity intravenous lipid emulsion for local anesthetic toxicity: a review of the literature recurrence of cardiotoxicity after lipid rescue from bupivacaine-induced cardiac arrest intralipid infusion diminishes return of spontaneous circulation after hypoxic cardiac arrest in rabbits a comparison of the combination of epinephrine and vasopressin with lipid emulsion in a porcine model of asphyxial cardiac arrest after intravenous injection of bupivacaine binding of long-lasting local anesthetics to lipid emulsions in-hospital cardiac arrest is associated with use of non-antiarrhythmic qtc-prolonging drugs current concepts in the mechanisms and management of drug-induced qt prolongation and torsade de pointes novel therapeutics for treatment of long-qt syndrome and torsade de pointes mechanism of reversal of toxic effects of amitriptyline on cardiac purkinje fibers by sodium bicarbonate experimental amitriptyline intoxication: treatment of cardiac toxicity with sodium bicarbonate tricyclic antidepressant overdosage: experimental studies on the management of circulatory complications value of the qrs duration versus the serum drug level in predicting seizures and ventricular arrhythmias after an acute overdose of tricyclic antidepressants electrocardiographic criteria for tricyclic antidepressant cardiotoxicity drugs and pharmaceuticals: management of intoxication and antidotes ecg lead avr versus qrs interval in predicting seizures and arrhythmias in acute tricyclic antidepressant toxicity effects of high dose α- -acid glycoprotein on desipramine toxicity in rats cardiovascular repercussions of seizures during cyclic antidepressant poisoning epinephrine and sodium bicarbonate independently and additively increase survival in experimental amitriptyline poisoning improvement of cardiac conduction after hyperventilation in tricyclic antidepressant overdose experimental tricyclic antidepressant toxicity: a randomized, controlled comparison of hypertonic saline solution, sodium bicarbonate, and hyperventilation reversal of severe tricyclic antidepressant-induced cardiotoxicity with intravenous hypertonic saline solution sodium bicarbonate treatment for tricyclic antidepressant arrhythmias in children tricyclic antidepressant poisoning: an evidence-based consensus guideline for out-of-hospital management diphenhydramine-induced wide complex dysrhythmia responds to treatment with sodium bicarbonate propoxyphene-induced wide qrs complex dysrhythmia responsive to sodium bicarbonate -a case report case files of the medical toxicology fellowship at the toxikon consortium in chicago: cocaine-associated wide-complex dysrhythmias and cardiac arrest -treatment nuances and controversies acute carbamazepine toxicity associated with a widened qrs interval treated with intravenous sodium bicarbonate bupropion-associated qrs prolongation unresponsive to sodium bicarbonate therapy a fatal case of venlafaxine overdose self-poisoning with lamotrigine management of isolated yew berry toxicity with sodium bicarbonate: a case report in treatment efficacy cardiotoxicity after massive amantadine overdose naloxone in opioid poisoning: walking the tightrope a randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. results of the second national acute spinal cord injury study adverse events after naloxone treatment of episodes of suspected acute opioid overdose narcotic reversal in hypercapnic dogs: comparison of naloxone and nalbuphine the ilcor consensus on science with treatment recommendations for pediatric and neonatal patients: neonatal resuscitation cardiac arrest following naloxone in an extremely preterm neonate the empiric use of naloxone in patients with altered mental status: a reappraisal a dosing nomogram for continuous infusion intravenous naloxone adverse effects in children after unintentional buprenorphine exposure naloxone reversal of buprenorphine-induced respiratory depression accidental clonidine patch ingestion in a child clonidine toxicity revisited naloxone reversal of hypotension due to captopril overdose profound prolonged hypotension following captopril overdose attenuation of the antihypertensive effect of captopril by the opioid receptor antagonist naloxone effect of naloxone on the actions of captopril naloxone does not modify the antihypertensive effect of captopril in essential hypertensive patients molecular structure and stereoelectronic properties of sarmazenil -a weak inverse agonist at the omega modulatory sites (benzodiazepine receptors): comparison with bretazenil and flumazenil flumazenil: us clinical pharmacology studies clinical pharmacology of flumazenil sedative and ventilatory effects of midazolam infusion: effect of flumazenil reversal the effects of large-dose flumazenil on midazolam-induced ventilatory depression effect of flumazenil on benzodiazepineinduced respiratory depression effect of flumazenil on bispectral index monitoring in unpremedicated patients electrophysiology of benzodiazepine receptor ligands: multiple mechanisms and sites of action characterization of the analgesic effects of the benzodiazepine antagonist, ro - empiric use of flumazenil in comatose patients: limited applicability of criteria to define low risk use of flumazenil in the treatment of drug overdose: a double-blind and open clinical study in patients flumazenil and seizures: analysis of cases treatment of benzodiazepine overdose with flumazenil. the flumazenil in benzodiazepine intoxication multicenter study group a risk-benefit assessment of flumazenil in the management of benzodiazepine overdose valproic acid and metabolites: pharmacological and toxicological studies the author is indebted to lewis s. nelson, md, facep, facmt for manuscript review. key: cord- - e vo xr authors: lang-illievich, kordula; winter, raimund; rumpold-seitlinger, gudrun; schicho, kurt; dorn, christian; klivinyi, christoph; bornemann-cimenti, helmar title: the effect of low-level light therapy on capsaicin-induced peripheral and central sensitization in healthy volunteers: a double-blinded, randomized, sham-controlled trial date: - - journal: pain ther doi: . /s - - - sha: doc_id: cord_uid: e vo xr introduction: several clinical trials have demonstrated that low-level light therapy (lllt), a method of photobiomodulation, is an effective analgetic treatment. however, the mechanism of action has not yet been finally clarified. in particular, unanswered questions include whether it only affects peripheral or whether it also affects the spinal or supraspinal level. this study aimed to evaluate the effect of low-level light therapy on primary and secondary hyperalgesia in a human pain model. methods: this study was planned as a randomized, sham-controlled, and double-blinded trial with repeated measures within subject design. capsaicin was applied on both forearms of ten healthy volunteers to induce peripheral and central sensitization. one forearm was treated with low-level light therapy; the other served as sham control. results: low-level light therapy significantly increased the mechanical pain threshold, heat pain threshold, and decreased pain intensity. conclusions: our data indicate that low-level light therapy is effective at reducing the heat and mechanical pain threshold in a human pain model, pointing to a significant modulating effect on peripheral and central sensitization. these effects—especially in the absence of reported side effects—make low-level light therapy a promising tool in pain management. the application of low-level light therapy to treat chronic pain should be considered for further clinical trials. low-level light therapy (lllt), a method of photobiomodulation, is well established in various fields of medicine. in general, lllt is the clinical application of light with wavelengths usually in the range of to nm and a typical power density from mwcm - to wcm - [ ] . indications for lllt are the improvement of wound healing and tissue regeneration, scarring, and perfusion, as well as pain therapy. in the last few decades, the phenomenon of sensitization has been increasingly studied for its fundamental role in pain medicine [ , ] . sensitization is generally regarded as a physiological, adaptive, and self-limiting process caused by nociceptive stimuli to facilitate healing of an injury. however, prolonged sensitization is maladaptive and represents a major contribution to the development of pain chronification. systematically, sensitization can be grouped into central and peripheral mechanisms. peripheral sensitization causes the so-called primary hyperalgesia at the nociceptor level due to an excessive stimulus response by decreasing the threshold for nociception in the peripheral nervous system [ ] . the heat pain threshold (hpt) is generally regarded as a marker of primary hyperalgesia [ ] . in contrast, central sensitization causes secondary hyperalgesia, which is quantified by the mechanical pain threshold (mpt). central sensitization is determined by the neuroplastic changes occurring at the spinal or supraspinal level [ ] . the distinction between these two mechanisms is relevant because peripheral and central sensitization responds to different therapeutic approaches [ ] . clinically, the two are likely to coexist in the majority of patients experiencing pain; in pain chronification, however, the central processes reach major relevance [ ] . they play a pivotal role in the development and maintenance of various chronic pain conditions [ ] . for example, central sensitization contributes to the transition process from acute to chronic pain and the development of persistent postoperative pain [ ] . despite convincing results in some clinical trials [ ] [ ] [ ] [ ] [ ] , the mechanism of action by which lllt relieves pain has not yet been clarified. in particular, unanswered questions include whether lllt only takes effect peripherally or whether it also effects the neuroplastic changes on the spinal or supraspinal level. to provide an answer to these questions, we use a human pain model developed to mimic different aspects of clinical pain and to study pain mechanisms in order to examine whether peripheral or central mechanisms contribute to the analgetic effect of lllt. this study was designed as a randomized, shamcontrolled, and double-blinded trial with repeated measures within subject design at the medical university of graz, austria. after receiving approval from the ethics committee (ek - ex / ), healthy male and female subjects aged - willing to participate were included. exclusion criteria were defined as: known allergies or hypersensitivity to capsaicin, history of skin diseases, neurological diseases, diabetes mellitus, pregnancy, and consummation of analgetic or psychotropic drugs taken within month before study participation. those eligible were informed about the study details by the principal investigator and, after giving written consent, were included in the study. during the first visit, demographic information, including fitzpatrick classification of skin color, was obtained from all patients [ ] . no previously published data were available as a source for an a-priori sample-size calculation. thus, we powered the study to meet the following parameters: the significance level of . and beta was . . in a two-sided paired t test, a sample of ten participants is suitable for detecting an effect size of c . the trial took place in a quiet room with an average temperature of * °c over the whole duration of the capsaicin application and irradiation procedure. probands were situated in the room for at least min to allow acclimatization. we used a human pain model capable of inducing the central and peripheral mechanisms. capsaicin application rapidly produces local neurogenic inflammation (characterized by edema and erythema) when locally administered to human skin by stimulating the trpv receptors on dermal sensory nerve endings [ , [ ] [ ] [ ] [ ] [ ] . in this pain model, heat hyperalgesia (i.e., the increased sensitivity to heat stimulation at the site of application of capsaicin) has been found to be due to peripheral sensitization, whereas mechanical hyperalgesia (i.e., increased sensitivity to mechanical stimulation beyond the area of capsaicin) is due to central sensitization [ ] . before the capsaicin application, skin temperature was measured using an infrared thermometer. in the case of skin temperature below °c, the area was covered by a blanket until the required skin temperature was reached. a mm mm cutaneous capsaicin patch % (qutenza, grünenthal, aachen, germany) with mg of capsaicin per cm (corresponding to . mg of capsaicin in total) was applied in the volar side in the middle of both distal forearms of the participants. the patches were loosely fixed with bandages. after min, the patches were removed, and the exposed skin areas were treated with the appropriate cleansing gel (supplied with qutenza and intended for removal) according to the manufacturer's recommendations. during the capsaicin application, patients were randomized to determine which arm was treated with lllt. the other arm received the same treatment with an unactivated device and served as sham-control. randomization was carried out by a coin toss (left or right) by a nurse, who had no further involvement in the study except for the irradiation procedure. the observer was not present during the randomization and intervention. the nurse was instructed not to share any information about the allocation of the side of irradiation with the volunteers and observers. the volunteers were blinded with opaque goggles. in addition, they wore earplugs to prevent them from hearing the ventilator noise of the lllt system. immediately after removing the capsaicin patch, the two forearms were positioned in the same way on a base divided by an opaque screen to avoid scattered radiation (fig. ) . the nurse, who was also responsible for the randomization, positioned two identical lllt devices (repuls , repuls lichtmedizintechnik gmbh, vienna, austria) simultaneously with a cmdistance ring in direct contact with the skin. according to the randomization procedure, one lllt device remained turned off. the other forearm was irradiated with pulsating and cold red light of nm. the intensity was mwcm - , which corresponds to a power density of mw. the pulse frequency was set to . hz. a duration of min was chosen based on the manufacture's recommendations. measurement was performed directly after intervention. pain intensity was quantified using a numeric rating scale (nrs - ) immediately after intervention on the irradiated and non-treated forearms. assessment of the sensory thresholds was based on the specifications of the german research association for neuropathic pain [ , ] . hpt was assessed on both the lllt irradiated forearm and non-treated forearm (= control). it was evaluated using a computer-controlled peltier element, the tsa-ii quantitative neu-rosensory analyzer (medoc ltd., ramat yishai, israel), with a mm mm thermode. as heat hyperalgesia is only present in the area of primary hyperalgesia [ ] , the probe of the tsa was exactly positioned to match the marked edges of the capsaicin patch. the baseline temperature of the probe was °c. the ramp for the temperature increase of the probe was set to °c/s. the volunteers were instructed to press a button as soon as they felt a sensation that was not only warm but also accompanied by a burning, stinging, drilling, or dragging feeling. mpt was assessed on both sides cm proximal to the marked area using pinpricks with a calibrated force of , , , , , , and mn and a flat contact area . mm in diameter (mrc systems gmbh, heidelberg, germany). the pinpricks were applied with a stepwise which increased force. the volunteers were instructed to indicate the force where they first perceived a sharp sensation. they were then asked about any side effects at the end of the irradiation and the end of the measurement. the data were analyzed for normality by the shapiro-wilk test. accordingly, a comparison of the sides was calculated using a paired t test or wilcoxon signed-rank test, as appropriate. the significance level a was set to . . to quantify the magnitude of the effect size, cohen's d for repeated measurement was calculated with their corresponding % confidence interval ( % ci). the effect sizes were summarized in three categories: small ( . \ d \ . ), medium ( . \ d \ . ), and large (d [ . ) [ ] . the force measurements of the mechanical pain sensitivity were transformed into decadic logarithms. before the logarithmic transformation, a constant of . was added to all zero and non-zero raw data in order to avoid a loss of zero values [ ] . the data were summarized as mean and standard deviations. all analyses were conducted with ncss [ncss statistical software ( ); ncss, llc. kaysville, ut, usa]. data analyses were performed by an independent researcher blinded to the group assignment. height (cm) . ± . this manuscript was prepared according to the consort guidelines. all procedures were in accordance with the helsinki declaration. in july and august , seven females and three males were assessed for eligibility and included in the trial. patient demographics are presented in table . the age of the probands ranged from to years. eight subjects were right-handed and two were left-handed. all participants were caucasian and had dark white skin, according to skin type of the fitzpatrick classification. the treatment side was randomly allocated to the right side in five subjects and to the left side in five subjects. skin temperature was in the range of ± °c before the capsaicin application for all probands without the need for external warming. a schematic diagram of the time-course of the study is presented in fig. all the participants completed the study. there were no unexpected side effects reported (fig. ). our study shows that lllt reduces pain intensity, as well as both peripheral and central sensitization in a human pain model. a few clinical trials have demonstrated that lllt alleviates pain. most of these studies were carried out in postoperative scenarios. lllt was shown to be effective in decreasing mean pain intensity in patients undergoing hip arthroplasty [ ] , coronary bypass surgery [ ] , and orthodontic tooth movement [ ] . likewise, in a non-surgical acute pain setting, pre-treatment with lllt before radiotherapy in breast cancer patients had a dual beneficial effect in reducing the severity of radiodermatitis and ameliorating pain intensity due to radiation-induced skin toxicity. there are very few studies that concentrate on lllt and chronic pain. in patients with nonspecific chronic knee pain, treatment with lllt decreased pain and improved quality of life [ ] . the results of our trial are in line with the findings of the above-mentioned small number of clinical studies. the pain intensity our probands experienced on the irradiated forearm was reduced by nearly one-quarter compared with the intensity on the sham-treated forearm, representing a medium effect size. proposed mechanisms of analgetic action include local release of serotonin, endorphins, and anti-inflammatory effects [ ] . additionally, activation of peripheral opioid receptors may contribute [ ] . our data have shown that hpt experienced a statistically significant increase in the lllttreated forearm compared with the sham-treated forearm. this corresponds to a reduction in peripheral sensitization. the intracellular mechanisms of sensitization of nociceptors-the structure where the peripheral sensitization mainly takes place-are not yet fully understood. the complex processes of immunology, however, which have recently been well studied, should attract particular attention. several studies have shown that pronociceptive cytokines like tumor necrosis factor a (tnf-a), il- b, and il- , as well as antinociceptive cytokines like il- contribute to balanced nociceptor sensitivity. lllt contributes to a decrease in the pro-inflammatory cytokines il- b and il- as well as an increase in the anti-inflammatory cytokine il- [ , ] . in clinical trials, tnf-a and il- showed decreased serum levels after hip arthroplasties were treated with lllt [ , ] . the effect of lllt on pro-inflammatory tnf-a was studied in a sciatic nerve crush model in mice, but the results were controversial [ , ] . the release of opioid peptides by immune cells is another peripheral endogenous antinociceptive mechanism involved in counteracting inflammatory hyperalgesia [ ] . in this context, the lllt-related activation of peripheral opioid receptors, which involve the recruitment of opioid-containing leukocytes, are certainly of great relevance [ ] . mechanical pain sensitivity differed significantly between the irradiated and non-irradiated forearms, pointing to an influence of lllt on central sensitization. although these results may appear counterintuitive at first glance, there are some possible explanations for how changes at the spinal and supraspinal level can be caused by topical treatment. however, as our study was not designed to elucidate the underlying mechanisms, we can only speculate on the potential explanations for our observed antihyperalgetic effect. nitric oxide (no) is regarded as an important element in central sensitization of nociceptive posterior horn neurons [ ] [ ] [ ] . there are references that show the involvement of no in cellular mechanisms of low-level light therapy [ , ] . glutamergic calcium conducting ion channels, in particular, nmda receptor channels, as well as the activation of trpv receptors, are essential for central sensitization processes (i.e., long-term potentiation) [ ] . pigatto et al. demonstrated in a very recent study the involvement of both trpv and glutamate on the analgetic effect of lllt [ ] . one could therefore speculate that these mechanisms may also represent a potential rational for our findings. peripheral and central sensitization are two pivotal mechanisms in our current understanding of pain physiology. while peripheral sensitization is often linked to inflammation, which is a typical component of various acute pain scenarios, central sensitization is regarded as a key domain in the chronification of pain [ , ] . previous clinical studies on the effectiveness of lllt have been conducted primarily for the treatment of acute pain [ , , , ] . these convincing results can be explained by the effect on peripheral sensitization, which we showed in the present study. our results provide the mechanistic background of the effect of lllt on acute pain disorders. what surprised us was the central effect of lllt. the effect size of these results was even larger than the effect on pain intensity. until now, the application of lllt in chronic pain scenarios has only been evaluated in a single clinical trial, which suggested it as an effective treatment option [ ] . our findings encourage further clinical trials on chronic pain. the present study has limitations that need to be addressed. first, the duration of the lllt application was chosen arbitrarily. as no previously published evidence was available, we followed the manufacturer's recommendations. another limitation is that we applied only a single lllt treatment. repeating the lllt therapy several times would not have been reasonable due to the temporal dynamics of the chosen pain model [ ] . however, even our single treatment resulted in medium to strong effects. evaluation of repeated applications in a prolonged human pain model would thus be theoretically interesting but would raise major ethical issues. therefore, the effectiveness of different treatment durations should be further studied in clinical scenarios related to chronic pain. another interesting parameter would have been the size of the flare area, which occurs as a result of neurogenic inflammation. unfortunately, we did not have access to the necessary measurement systems for a valid assessment, such as a laser doppler flowmeter. finally, we have to emphasize that this study was not designed for elucidating background mechanisms but to assign the analgetic effect to clinically relevant modes of action. all assumptions on potential underlying causes and contributors are speculative. our data indicate that lllt is effective in reducing hpt and mpt in a human pain model, pointing to a significant modulating effect on peripheral and central sensitization. these effects-especially in the absence of reported side effects-make lllt a promising tool in pain management. the application of lllt to treat chronic pain should be considered for further clinical trials. funding. this study has been carried out in the course of a research project supported by the austrian research promotion agency ffg, project no. . dr. kurt schicho has proposed and managed the parts of this project belonging to the field of biomedical engineering. authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. authorship contributions. kli: design of the study, data acquisition, preparing and approving the manuscript. rw: design of the study, analyzing the data, drafting and approving the manuscript. grs: design of the study, data interpretation, drafting and approving the manuscript. ks: design of the study, data acquisition, drafting and approving the manuscript. cd: design of the study, data acquisition, drafting and approving the manuscript. ck: design of the study, data acquisition, drafting and approving the manuscript. hbc: design of the study, data interpretation, revised and approving the manuscript. disclosures. kordula lang-illievich, raimund winter, gudrun rumpold-seitlinger, kurt schicho, christian dorn, christoph klivinyi, and helmar bornemann-cimenti have nothing to disclose. compliance with ethics guidelines. the study protocol was approved by the ethics committee of the medical university of graz, austria (ek - ex / ). the study was carried out in accordance with the helsinki declaration. all participants provided informed consent. data availability. the datasets analyzed during the current study are available from the corresponding author on reasonable request. open access. this article is licensed under a creative commons attribution-noncommercial . international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/bync/ . /. proposed mechanisms of photobiomodulation or low-level light therapy nociceptor sensitization in pain pathogenesis central sensitization: implications for the diagnosis and treatment of pain neuropathic pain and neuroplasticity in functional imaging studies pathophysiological mechanisms of neuropathic pain: comparison of sensory phenotypes in patients and human surrogate pain models pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain peripheral and central mechanisms of cutaneous hyperalgesia the area of secondary hyperalgesia following heat stimulation in healthy male volunteers: inter-and intraindividual variance and reproducibility assessment and manifestation of central sensitisation across different chronic pain conditions the effect of two phototherapy protocols on pain control in orthodontic procedure-a preliminary clinical study photobiomodulation therapy (pbmt) on acute pain and inflammation in patients who underwent total hip arthroplasty-a randomized, triple-blind, placebo-controlled clinical trial adjunctive use of combination of super-pulsed laser and light-emitting diodes phototherapy on nonspecific knee pain: double-blinded randomized placebo-controlled trial lowlevel laser and light-emitting diode therapy for pain control in hyperglycemic and normoglycemic patients who underwent coronary bypass surgery with internal mammary artery grafts: a randomized, double-blind study with follow-up photobiomodulation therapy for the management of radiation-induced dermatitis ultraviolet-induced pigmentary changes: benefits and hazards neurogenic hyperalgesia: the search for the primary cutaneous afferent fibers that contribute to capsaicin-induced pain and hyperalgesia dynamic and static components of mechanical hyperalgesia in human hairy skin dorsal root reflexes and cutaneous neurogenic inflammation after intradermal injection of capsaicin in rats a data science approach to the selection of most informative readouts of the human intradermal capsaicin pain model to assess pregabalin effects preclinical assessment of inflammatory pain does electroacupuncture have different effects on peripheral and central sensitization in humans: a randomized controlled study qst-quantitative sensorische testung-handlungsanweisung für den untersucher quantitative sensory testing in the german research network on neuropathic pain (dfns): standardized protocol and reference values secondary hyperalgesia to mechanical but not heat stimuli following a capsaicin injection in hairy skin effect size estimates: current use, calculations, and interpretation the use of transformations light-emitting diode therapy induces analgesia in a mouse model of postoperative pain through activation of peripheral opioid receptors and the larginine/nitric oxide pathway effect of lowlevel laser therapy ( nm) on acute inflammation induced by tenotomy of achilles tendon in rats lightemitting diode therapy reduces persistent inflammatory pain: role of interleukin and antioxidant enzymes low-level red led light inhibits hyperkeratinization and inflammation induced by unsaturated fatty acid in an in vitro model mimicking acne lightemitting diode therapy induces analgesia and decreases spinal cord and sciatic nerve tumour necrosis factor-a levels after sciatic nerve crush in mice tissue monocytes/macrophages in inflammation: hyperalgesia versus opioid-mediated peripheral antinociception inhibition of inducible nitric oxide synthase in persistent pain the possible role of the no-cgmp pathway in nociception: different spinal and supraspinal action of enzyme blockers on rat dorsal horn neurones cgmp produced by no-sensitive guanylyl cyclase essentially contributes to inflammatory and neuropathic pain by using targets different from cgmp-dependent protein kinase i cellular effects of low-power laser therapy can be mediated by nitric oxide pretreatment with light-emitting diode therapy reduces ischemic brain injury in mice through endothelial nitric oxide synthase-dependent mechanisms photobiomodulation therapy reduces acute pain and inflammation in mice age-related differences in the time course of capsaicin-induced hyperalgesia involvement of the proinflammatory cytokines tumor necrosis factor-alpha, il- beta, and il- but not il- in the development of heat hyperalgesia: effects on heat-evoked calcitonin gene-related peptide release from rat skin anti-inflammatory interleukin- therapy in cci neuropathy decreases thermal hyperalgesia, macrophage recruitment, and endoneurial tnf-alpha expression we thank the participants of the study and the team of the interdisciplinary pain clinic graz. key: cord- -ampqjbqw authors: chacón‐labella, julia; garcía palacios, pablo; matesanz, silvia; schöb, christian; milla, rubén title: plant domestication disrupts biodiversity effects across major crop types date: - - journal: ecol lett doi: . /ele. sha: doc_id: cord_uid: ampqjbqw plant diversity fosters productivity in natural ecosystems. biodiversity effects might increase agricultural yields at no cost in additional inputs. however, the effects of diversity on crop assemblages are inconsistent, probably because crops and wild plants differ in a range of traits relevant to plant–plant interactions. we tested whether domestication has changed the potential of crop mixtures to over‐yield by comparing the performance and traits of major crop species and those of their wild progenitors under varying levels of diversity. we found stronger biodiversity effects in mixtures of wild progenitors, due to larger selection effects. variation in selection effects was partly explained by within‐mixture differences in leaf size. our results indicate that domestication might disrupt the ability of crops to benefit from diverse neighbourhoods via reduced trait variance. these results highlight potential limitations of current crop mixtures to over‐yield and the potential of breeding to re‐establish variance and increase mixture performance. global agricultural production needs to rise by - % to accommodate the projected demands for food by (tilman et al. ). this constitutes a major challenge, in particular if it is to be met without substantial expansion of croplands and reduced usage of synthetic fertilisers and pesticides (foley et al. ; gurr et al. ; wan et al. ) . therefore, farming practices that increase crop production in a sustainable way are needed. the promotion and management of biodiversity effects, that is, higher productivity of species mixtures than expected from their respective monocultures, might contribute to address this challenge (brooker et al. ; isbell et al. ; martin-guay et al. ) . however, while biodiversity effects have been solidly demonstrated in natural ecosystems (loreau & hector ; tilman et al. ; hooper et al. ; cardinale et al. ) , the literature reports contrasting responses in mixtures of arable crops, ranging from positive (kiaer et al. ; lin ; reiss & drinkwater ) to negative (pimentel et al. ; letourneau et al. ; snapp et al. ) , with considerable variation between years, locations and crop species (fridley ; hauggaard-nielsen et al. ; smith et al. ; snapp et al. ) . here, we argue that evolution of plant phenotypes after domestication might partly account for such discrepancies between the behaviour of wild and crop plants in mixtures. experiments in agricultural and natural settings use different experimental setups, which might explain part of the disparities (brooker et al. ) . for instance, biodiversity-ecosystem functioning (bef) experiments use regular spacing among plants and keep fixed plantation densities (trenbath ; schmid & hector ; bruelheide et al. ; schmid et al. ) . experiments with crops use more variable spatial arrangements (e.g. intercropping patterns, mal ezieux et al. ), and generally very different metrics (land equivalent ratios vs. additive partitioning of biodiversity effects; adetiloye & ezedinma ; loreau & hector ). therefore, different methods might result in discordant biodiversity effects. another cause of discrepancy might be the values of the traits of the plant species and genotypes involved in the mixtures. crop phenotypes are different from those of their wild progenitors (meyer et al., ) or other wild plants (milla et al. ) . herbaceous crops are generally larger seeded than their wild relatives, have lost seed dormancy and dispersal mechanisms, and show erect and compact growth habits (harlan et al. ; meyer et al., ) . further traits that might have changed after crop domestication can be relevant for plant performance in mixtures. for example, increased plant canopy height and leaf area after domestication (milla et al. ; suggest that crops are stronger competitors for light than their wild progenitors. additionally, genetic bottlenecks (due to selection processes by early farmers) and crop breeding for maximum monoculture yield have substantially reduced the genetic diversity of crops (tanksley & mccouch ; doebley et al. ; zeder ) . this reduction in genetic diversity might lead to increased phenotypic uniformity, that is, constant phenotypes in different environments (bloomfield et al. ) . genetic variability and phenotypic plasticity trigger resource use complementarity in mixtures (mal ezieux ; zeller et al. ; zuppinger-dingley et al. ; prieto et al. ) . indeed, a recent study employing genetically divergent arabidopsis accessions suggests that biodiversity effects can be partly tractable in the genotype (i.e. allelic mixtures at locus chr @ . were more productive in terms of biomass than the most productive monoallelic community; wuest & niklaus ) . thus, if domestication and further breeding have reduced genetic diversity and increased constancy of phenotypes, achieving higher productivity in crop mixtures through increased niche complementarity might be compromised. in addition, recent evolution under monoculture might also have diminished the ability of crops to perform in mixtures. current genotypes of major arable crops were selected to maximise yields in monospecific stands (weiner et al. ) , which entailed adaptations to perform in an intraspecific neighbourhood (e.g. high phenological synchrony). in contrast, wild plants composing the natural and seminatural grasslands typical of bef experiments, as well as crop wild progenitors, have evolved in mixed plant communities for their entire evolutionary history. local adaptations to a diverse neighbourhood can evolve over short-time periods and differently depending on the biotic (zuppinger-dingley et al. ) and abiotic conditions (i.e. elevated co ; kleynhans et al. ) . indeed, competitive hierarchies and further adaptations through niche differentiation or facilitation (which are the mechanisms underlying biodiversity effects) are more likely to evolve in diverse communities than in monospecific stands (sch€ ob et al. ) . a recent study found that biodiversity effects in mixtures of genotypes with a recent history of coexistence were higher than in mixtures of genotypes with a monoculture history (zuppinger-dingley et al. ) . importantly, in a short period of years of coselection history in mixtures, plants evolved adaptations to improve over-yielding (zuppinger-dingley et al. ) . given that most crop species have been selected under monoculture conditions over centuries, the results of zuppinger-dingley et al. ( ) suggest that crop plants might be less suited to over-yielding in mixtures, and might benefit less from biodiversity effects than their wild progenitors. the primary goal of this study was to investigate how domestication has affected the ability of annual crops to deliver biodiversity effects. specifically, we compared the performance and functional trait diversity of assemblages of annual crops and of their wild progenitors, set to grow in mesocosms of varying levels of diversity. we hypothesised that: ( ) crop species will show lower biodiversity effects than their wild progenitors, and ( ) weak biodiversity effects in crop mixtures will be driven by increased similarity in functional traits within assemblages. to test whether domestication has affected the ability of annual crops to perform in mixtures, we conducted a biodiversity-ecosystem functioning (bef) experiment with annual crop species. we selected eight crops (two forbs, two c grasses, two c grasses, and two legumes), and gathered seeds of a domesticated and a wild progenitor accession of each (table ) . we grew and harvested each species in monoculture and in three types of mixtures, separately for wild progenitor and domesticated accessions. to calculate biodiversity effects, we followed the additive partitioning method of loreau & hector , and partitioned net biodiversity effects into complementarity and selection effects. additionally, we measured a suite of plant traits (height, leaf area, leaf mass per area, and leaf mass fraction) that influence growth and plant-plant interactions in mixtures prieto et al. ) . we calculated within-mixture trait differences (i.e. ratio between absolute differences and trait means) to address whether differences in the ability to over-yield are modulated by phenotypic diversity within assemblages. to include the most prominent functional groups in annual croplands, we used two species of each of the major functional groups of annual crops: c grasses, c grasses, legumes, and forbs (table ) . plants were grown in l pots with a fixed density of four individuals across all treatments. individuals of the accessions were grown in monocultures and in mixtures. each type of mixture included all possible combinations of species and functional groups in the experiment, separately for wild progenitor and domesticated accessions. we built three types of mixtures: one type of within-functional group polycultures and two types of among-functional group polycultures ( fig. ) . within-functional group polycultures were composed of two individuals of two different species of a given functional group. the among-functional group polycultures consisted either of a mix of two different species of two different functional groups, or a mix of four different species, belonging each to one of the four functional groups. we implemented four levels of increasing diversity: monocultures ( combinations, pots), within-functional group two species mixtures (two species of the same functional group; combinations; pots), among-functional group -species mixtures (two species from two different functional groups; combinations; pots), and among-functional group -species mixtures (four species of four different functional groups; combinations; pots). experimental design, treatments, and species combinations within each treatment are summarised in table s . detailed information about the assignment of wild progenitors, or seed accession identifiers and seed donors can be found in table s . in may , seeds of each accession were germinated in small individual pots ( cm) filled with commercial topsoil (klasmann; traysubstrate ). once germinated, and as soon as transplanting was feasible without compromising plant establishment, randomly selected seedlings were transplanted to the experimental pots ( . . cm, l). the pots were filled with conventional, washed river sand and supplemented with slow-release fertiliser ( gdm - ; basacote plus m; compo, barcelona, spain; see fertiliser components on table s ). the amount of fertiliser was set to grow plants under high-nutrient availability conditions (day et al. ) . all pots were randomly located on three greenhouse benches at universidad rey juan carlos, mostoles-madrid, central spain ( ° ´ ″ n, ° ´ ″ w), and received full sun (mean midday photosynthetically active radiation [par] c. µmol m À Ás À , measured in three consecutive clear days of the experiment; li-cor, lincoln, ne, usa). watering was applied regularly via sprinklers to maintain the pots at - % of their field water holding capacity. pots were regularly weeded to keep the species composition as designed. replacement of dead seedlings took place during the first days of the experiment, by seedlings of the same age. after that period, all pots lacking one or more individuals were discarded. the experiment ran until reproductive development was observed in the earliest flowering accessions, which set the harvest date of the experiment. pesticides were not needed during the experiment. details on the number of replicates per treatment and domestication status can be found in table s . to explore how the variation in functional traits within pots (character displacement) underlies biodiversity effects, we measured plant height (h), leaf area (la), leaf mass per area (lma) and leaf mass fraction (lmf). plants that hold more than optimal h or la are competitively superior in intercepting light and generally displace shorter and smaller-leaved species (westoby et al. ; anten ) . high variation in h and/or la is often associated with increased productivity via selection effects (cadotte ) . lmf is directly and lma is inversely related to plant relative growth rate (rgr; reich et al. ; poorter & garnier ) . variation in lma in stands is related to different light-use strategies and increased productivity via complementarity (roscher et al. ; cadotte ) . lmf is the proportion of biomass allocated to leaves. at high densities, variation in lmf would indicate different strategies to capture light and other resources (poorter et al. ) . measurements of all traits were performed at the individual level. plant height (h; cm) was measured just before harvest, when reproductive development started in the earliest flowering accession ( weeks after transplanting into the experimental pots). aboveground biomass was harvested at ground level separately for each individual in the experiment, separated into leaves and other organs, and oven dried at °c for days prior to weighting. leaf area was measured as the lamina area (la, in mm ) of a representative leaf by scanning a fresh leaf immediately after plant harvest, and its dry mass was used to calculate leaf mass per area (lma, in g mm À ). leaf mass fraction (lmf, unitless) was measured as the ratio of the total leaf mass to the total dry mass of each individual plant. to calculate the relative differences in height, leaf area, lma and lmf within pots, we calculated all the absolute differences between each two individuals of a pot divided by the mean of the two, and then calculating the mean of all the values. biodiversity effects are based on the differences between the observed yields of each species in mixtures compared to monocultures, and may be partitioned into complementarity effects (ces) and selection effects (ses) following the approach of loreau & hector . ces measure if species' yields in a mixture are on average higher or lower than expected by the relative yields of the component species in monoculture. positive ces are indicative of niche partitioning and facilitative interactions resulting in an increase in total resource use, while negative complementarity effects indicate competitive interactions among plants resulting from either chemical or physical interferences (loreau & hector ) . ses are measured as the covariance between the deviation from the expected relative yield of a species in a mixture and the yield of the same species in monoculture. ses occur when the change in the relative yield of a species in a mixture is a function of its yield in monoculture (i.e. a species with high or low yield in a monoculture yields disproportionately more, or less, respectively, in mixture). net effects (nes) are the sum of ces and ses. all three effects can be either positive or negative. ne has an expected value of zero under the null hypothesis of no biodiversity effects (see the supporting information for calculation details). biodiversity effects in crops vs. wild progenitors and across diversity levels we compared the performance of wild and domesticated plant species mixtures using mixed effects models with residual maximum likelihood estimation. significance tests were based on approximate f-tests. biodiversity effects (nes, ces and ses; g/ pot) were the response variables in the models. absolute values of nes, ces and ses were square root-or log-transformed to meet assumptions of homoscedasticity, and after that, their original signs put back to the transformed values for analyses, which allowed maintaining the direction of the effects (loreau & hector ) . the fixed terms of the model were, in this order, bench (three levels), domestication status (dom: crop vs. wild), species richness (spr: two vs. four species), functional group richness (fgr: one vs. two functional groups), functional group combination (fg comb: eleven levels), and their interactions. not all possible combinations between functional group richness and species richness existed. several of the combinations are impossible (e.g. four functional groups and one species) while others are possible but were not included in the design (e.g. one functional group and four species, or two functional groups and two species). to deal with such a non-fully factorial design, we used a nested structure of the fixed-effect terms. thus, fgr (one vs. two functional groups) was nested within spr (two vs. four species), in such a way that the factor fgr only applies to one level of spr, that is, two species. a total of biodiversity effects were calculated in mixture pots (more details in table s ). species combination was used as a random intercept term. no outliers were excluded from the analyses. the full model is shown in table . to test whether the results were consistent across functional groups, we split the data into four smaller, overlapping, data sets, by the presence of each functional group (legumes, forbs, c grasses or c grasses) in the pots. then, we performed the analyses for each of these new data sets and tested the effects of the different predictors on biodiversity effects separately per functional group. to test whether character displacement within pots is the underlying mechanism of biodiversity effects, we tested whether the relative differences in traits follow the same significant direction as ce or se does for crops and wilds. for this purpose, we used the mean relative differences for a given trait in mixture pots as a response variable, using the same mixed model as the one employed for explaining the biodiversity effects (ne, ce and se) in crops vs. wilds progenitors. the full model is presented in table s . to directly test the association between biodiversity effects and the differences in particular functional traits, we ran mixed models using the biodiversity effects (ne, ce and se) as response variable, but adding the mean differences per trait as an explanatory variable. this model is presented in table s . when the variation in a given functional trait correlated with ces or ses, we explored these specific relationships further using standardised major axis (sma) regressions (warton et al. ) . first, we fitted sma regressions separately for crops and for wild progenitor pots and tested for a common slope using a bartlett-corrected likelihood ratio test (warton & weber ) . if the assumption of a common slope was justified, a wald test was performed to identify significant differences in elevation and shifts along the common axis between crops and wild progenitors. all statistical analyses were conducted using r version . . (r core team ). mixed models were fitted using the function lmer from package lme (bates et al. ) . significance of the fixed factors was assessed with type i anova tests from the package lmertest (kuznestsova et al. ) . we estimated conditional (variance explained by fixed and random factors) and marginal r (variance explained by fixed factors alone) using the function r.squaredglmm from the mumin package (barton ) . sma analyses were performed using the smart library in r (warton et al. ) . plots were produced using ggplot (wickham ) . net biodiversity effects were significantly higher in mixtures of wild progenitors than in the mixtures of their domesticated counterparts ( fig. a ; table ; p = . ). differences in nes between domestication statuses were largely driven by stronger positive ses in the mixtures of wild progenitors compared with the mixtures of crops ( fig. a ; table ; p = . ). biomass was % higher, on average, in the mixtures of wild progenitors compared to the biomass expected from monocultures (fig. s ) . the effects of plant domestication status on se were consistent across the four functional groups evaluated ( fig. s ; table s ). complementarity effects were also generally positive, but similar among domestication statuses (fig. a) . increasing diversity from two to four species or from one to two functional groups had no significant effects on nes ( fig. b and c) . the effects of species richness (p = . ) and functional group richness (p = . ) on se were positive in both mixtures of crops and wild progenitors (fig. f , g; table ). although ce decreased with increasing levels of species richness (fig. d) , this negative effect of species richness on ce was not significant. conversely, functional group richness increased ce in crop mixtures but decreased in mixtures of wild progenitors (interaction between domestication status and functional group richness; p = . ; table ). this trend was stronger in the pots containing c grasses ( fig. s b for c grasses; table s ; p = . ). character displacement within mixtures varied between domesticated crops and their wild progenitors in a trait-specific manner. crop mixtures showed lower variation in leaf area than their wild progenitors ( fig. b ; p < . ), and higher variation in lma and lmf (p = . and p < . respectively; fig. s ; table s ). variation in plant height was similar in pots of wilds and domesticated crops (p = . ; figure s ; table s ). selection effects correlated positively with the relative differences in leaf area (p = . ; r m ¼ : ; r c ¼ : ; fig. a ; figure s ; table s ). therefore, given that wild mixtures showed higher variation in leaf area than crops, and that such variation correlated with ses, we explored this relationship further using sma regression (warton et al. ) . ses were significantly correlated with leaf area differences across domestication statuses (p < . , slope = ) and individually for both samples. sma slopes were significantly different from zero (domestic crops: p < . , slope = . , wild progenitors: p < . , slope = . ), homogeneous among domesticated and wild progenitors (p> . ), and with a significant shift along the common slope (p < . ). thus, sma analyses support the results ( ) that ses are partly explained by differences in leaf area within pots, and ( ) that pots of wild progenitors have more diverse leaf area scores compared to domesticated crops (see fig. ). additionally, sma analyses show that diversity in leaf area within pots affects ses in a similar way and intensity (i.e. slope) in domesticated and wild assemblages, though at different positions along the difference-in-leaf-area range. our results show that diversity promotes productivity also in arable crops, but suggest that through the course of plant domestication crop varieties have lost part of their capability of delivering biodiversity effects. we found greater biodiversity effects in wild progenitors' assemblages, compared to their crop counterparts, and provide evidence that crops might have lost their ability to benefit from biodiversity effects via reduced functional trait variation within mixtures. interestingly, the stronger biodiversity effects shown by wild progenitors are attributable to stronger selection effects, which in turn were primarily associated with larger differences in leaf size. positive selection effects occur when species with higher than average biomass in monoculture yield higher than figure biodiversity effects across domestication statuses and diversity levels. (a) biodiversity effects (net, complementarity and selection effects) in the mixtures of domesticated crops and wild progenitors. biodiversity effects were assessed by additive partitioning of net effects, and decomposed into complementarity and selection effects (n = ). (b-g) biodiversity effects split into net, complementarity and selection effects of crops and their wild progenitors, grouped per increasing species richness (red) and functional group richness (blue). values are means ae standard error of the mean (sem) calculated from raw data. these data are shown separately per functional group (legumes, forbs, c or c grasses) in the supporting information (figures s , s and s ) expected in mixture. functional differences within assemblages are relevant to plant-plant interactions and underlie biodiversity effects, either because they promote a complementary use of resources or because they trigger selection effects (fox ; mal ezieux et al. ; zuppinger-dingley et al. ; prieto et al. ) . however, functional differences are different between crops and wild plants (meyer et al., ; milla et al. ). thus, it is possible that some trait values, or combinations of trait values, necessary for enhancing yield in mixtures are absent or downplayed in current crop varieties, as shown here for leaf area. the consequences for fostering crop yields in a sustainable way are wide, as crops might carry an evolutionary legacy that impacts the capacity of crop mixtures to over-yield. although wild progenitors showed higher net biodiversity effects than crops, we show that both domestication statuses over-yielded in mixtures. our results for arable crops are in line with diversity-productivity relationships reported for other types of plant communities (loreau & hector ; tilman et al. ; hooper et al. ; cardinale et al. ) , and concur with previous studies reporting positive biodiversity effects in crop mixtures (li et al. (li et al. , (li et al. , kiaer et al. ; lin ; davis et al. ; reiss & drinkwater ; but see pimentel et al. ; letourneau et al. ; snapp et al. ) . our study expands the current knowledge for arable crops by using a bef experiment approach, which specifically tests the role of increasing diversity, thereby ruling out the confounding effects of disparate spatial scales (malezieux et al. ; brooker et al. ) , compositional differences (no balanced combination of functional types or gradients of diversity; mal ezieux et al. ) and temporal scales (smith et al. ) of agricultural trials. biodiversity can promote over-yielding through complementarity and/or selection effects (fox ; barot et al. ), figure relative differences in leaf area between domestication statuses and their relationships to selection effects. (a) relationship between selection effects and the relative differences between species within mixtures pots. the solid line shows the slope of a mixed effects model. the grey area shows the % confidence intervals of the fit line. data points are observed scores for crops (hollow dots) and wild progenitors (yellow). the marginal density plots for the selection effects and the relative differences in leaf area are shown split for crops (hollow area) and wild progenitors (yellow area). (b) relative differences of leaf area within pots, for each domestication status in mixture assemblages. values are means ae standard error of the mean, calculated from raw data. significance of the regression slope and significant differences between domestication statuses at the inset are indicated by asterisks (***p < . ; **p < . ; *p < . ). the same relationships for the relative differences in height, leaf mass fraction (lmf) and leaf mass area (lma), and the selection effect are provided in supporting information ( figure s ). whose relative relevance varies among study cases and duration of experiments (fargione et al. ). in our experiment, ses were much stronger than ces. selection effects are frequently disregarded as relevant contributors to over-yielding because they might signal strong competitive hierarchies, and thus suppressed species and preclusion to achieve transgressive over-yielding (loreau ) . however, the fact that productive species over-yield disproportionately does not necessarily come at the cost of other species under-yielding. in our experiment, the patterns of relative yields of individual species suggest that dominance was not a relevant mechanism triggering ses (see fig. s ), which highlights that ses are useful drivers of biodiversity effects and should not be overlooked in crop mixtures. interestingly, ses were stronger in assemblages of wild progenitors than in assemblages of crops, consistently across species and functional group richness levels. moreover, dissimilarity in leaf areas, which were larger in mixtures of wilds, partly accounted for the lower capacity of crops to over-yield in mixtures. there is general consensus that functional differences between co-occurring species underlie the link between diversity and productivity (cardinale et al. ). this link strengthens over time, due to adaptations through natural selection (zuppinger-dingley et al. ) . thus, given time, populations of diverse mixtures are expected to be better adapted to growing with interspecific partners (dawson & goldringer ) . for example, allard & adams found that mixtures formed by varieties of barley selected as pure lines were less productive compared to mixtures of barley lines cultivated together for many generations. this is consistent with grassland bef experiments showing that species with a co-selection history in mixtures develop functional trait divergence and increase biodiversity effects (both complementarity and selection effects; zuppinger-dingley et al. ) . indeed, frequent biotic interactions have been documented to impose selection on plasticity in functional traits and result in functional divergence between neighbouring plants in experimental (lipowsky et al. ) and natural grasslands (abakumova et al. ) . our results concur with these findings and provide evidence that crop breeding, as a selection process that generally occurs in monospecific stands, might have disrupted the ability of crops to develop strong biodiversity effects in diverse environments. these differences between crop plants and wild progenitors might arise either from directional selection in crops, or from reduced plasticity after domestication in certain traits , which remains to be investigated. in addition, arable cropping systems might pose limitations to co-evolutionary adaptations that promote complementarity (zuppinger-dingley et al. ). ces generally increases over time (fargione et al. ; lipowsky et al. ; reich ; zuppinger-dingley et al. ) , which indicates co-adaptation of the genotypes at play (zuppinger-dingley et al. ) , and might facilitate transgressive over-yielding in the long term (cardinale et al. ). since intercrops are rarely re-seeded on-farm in arable systems, which was mirrored in our experiment by using seeds with no previous history of coexistence, such opportunities for co-adaptation are precluded. we speculate that the ability of domesticated crops to adapt to local neighbourhoods would be hampered, either because they exhibit lower genetic variation and evolutionary potential (doebley et al. ; miller & gross ; zhang et al. ) and/or lower potential for plasticity due to an evolutionary history of coexistence in monocultures (abakumova et al. ) . in other words, mixing pure varieties that exhibit local adaptation is probably significantly advantageous in terms of monoculture yield and other ecosystem services, but not necessarily optimal for the performance in mixtures. thus, we suggest identifying traits, or trait combinations, that make mixtures of wild progenitors more productive, and bringing back the genes underlying those phenotypes to current varieties (zhang et al. ; wuest & niklaus ) . alternatively, instead of focusing on single beneficial traits, breeding for mixing ability can be oriented to increase genetic diversity and/or plasticity within crop varieties. such strategies can be reinforced with classical breeding on standing variation within crops, and with multi-generation programs that allow de novo adaptations to multi-specific settings. finally, it is important to acknowledge that some specificity of our set up and study system call for caution when generalising our findings, and might explain some discrepancies with preceding literature. for instance, it is remarkable that ces in our experiment, even if positive, were generally weak. previous studies on intercropping suggest that ces can be quite large (li et al. (li et al. , (li et al. , davis et al. ) . free-rooting intercrop experiments allow root growth complementarity, and displacement in phenological traits, more easily than in greenhouse settings (li et al. (li et al. , . other benefits of diversification, like increases in soil biodiversity or in soil carbon storage, are better captured in the long term and at larger spatial scales (davis et al. ) . additionally, in spite of our efforts to include diverse crops from diverse functional types, our set up was limited to eight crop species, and ignored varietal and geographical diversity within the gene pool of each crop. clearly, further studies addressing more crop species, and wider genetic, varietal and domestication status diversities within species, are needed. in this sense, to test if genotypes that are more closely related to their wild progenitors also show stronger biodiversity effects a set up that includes multiple stages along a sequence of domestication of a given species would be suitable (see e.g. roucou et al., ) . such an approach would reveal if a true 'dose-response' relationship exists between biodiversity effects and domestication. our bef experiment on eight arable crops revealed that domestication has reduced the ability of domesticated crops to benefit from biodiversity effects. we have shown that mixtures of wild progenitors attain higher biodiversity effects than mixtures of crops, driven by larger selection effects that are associated to larger differences in leaf area within assemblages. our analysis showed that mixing annual crops might bring yield benefits if the composing species exhibit contrasting values of key functional traits. since crop plants have a recent history of selection under monoculture, we argue that crop breeding might have hindered the ability of crop plants to perform in interspecific neighbourhoods through reduced genetic variance or plasticity in key traits. it will be interesting to explore to what extent the different functional divergences found within crops vs. wild mixtures arise from a directional selection of certain phenotypes or from a reduced plasticity in certain traits in crops. moreover, as the adaptations to diverse coexistence environments are expected to increase over time, we propose breeding strategies including multiple generation programs that permit local adaptations to arise. thus, further breeding programs should re-establish genetic diversity and plasticity to increase the performance of mixtures. provided that implementing genes from wild progenitors has proved useful for multiple breeding purposes (zhang et al. ; wuest & niklaus ) , and that we show here that wild progenitors might also have useful traits for coexistence in mixtures, we recommend a focus on wild relatives to breed crops for optimal polycultures. the authors acknowledge the funding from mineco grants cgl - -r and pcin- - . we are also grateful to carlos ingala, marina ramos-muñoz and mario blanco-s anchez for assistance in data gathering, and to three anonymous reviewers for their critical revisions of the manuscript. j.c-l. acknowledges the agricultural ecology group at eth zurich, and the juan de la cierva formaci on program (fjci- - ) for the financial support. cs was supported by the swiss national science foundation (pp p - ). p.g.p. acknowledges the spanish ministry of economy and competitiveness for financial support via the juan de la cierva incorporaci on program (ijci- - ) . sm was supported by the juan de la cierva and ram on y cajal programs. authorship rm originally formulated the idea and designed the experiment. rm, pgp and sm conducted the experiment. jcl analysed the data and wrote the manuscript with inputs from all other authors. all authors provided technical assistance and contributed substantially to revisions. data supporting the results are available from the dryad digital repository: https://doi.org/ . /dryad. q p b ). plasticity in plant functional traits is shaped by variability in neighbourhood species composition a land equivalent coefficient (lec) concept for the evaluation of competitive and productive interactions in simple to complex crop mixtures population studies in predominantly self-pollinating species xiii. intergenotypic competition and population structure in barley and wheat optimal photosynthetic characteristics of individual plants in vegetation stands and implications for species coexistence designing mixtures of varieties for multifunctional agriculture with the help of ecology. a review mumin: multi-modal inference. model selection and model averaging based on information criteria (aicc and alike) fitting linear mixed-effects models using lme sustainable harvest: managing plasticity for resilient crops improving intercropping : a synthesis of research in agronomy, plant physiology and ecology designing forest biodiversity experiments: general considerations illustrated by a new large experiment in subtropical china functional traits explain ecosystem function through opposing mechanisms impacts of plant diversity on biomass production increase through time because of species complementarity the functional role of producer diversity in ecosystems increasing cropping system diversity balances productivity, profitability and environmental health breeding for genetically diverse populations: variety mixtures and evolutionary populations the effects of spatial pattern of nutrient supply on yield, structure and mortality in plant populations the molecular genetics of crop domestication from selection to complementarity: shifts in the causes of biodiversity-productivity relationships in a long-term biodiversity experiment solutions for a cultivated planet interpreting the 'selection effect' of biodiversity on ecosystem function resource availability dominates and alters the relationship between species diversity and ecosystem productivity in experimental plant communities multi-country evidence that crop diversi fi cation promotes ecological intensi fi cation of agriculture comparative evolution of cereals grain legume -cereal intercropping: the practical application of diversity, competition and facilitation in arable and organic cropping systems effects of biodiversity on ecosystem functioning: a consensus of current knowledge benefits of increasing plant diversity in sustainable agroecosystems grain yield increase in cereal variety mixtures: a meta-analysis of field trials adaptation to elevated co in different biodiversity contexts lmertest package: tests in linear mixed effects does plant diversity benefit agroecosystems ? a synthetic review interspecific complementary and competitive interactions between intercropped maize and faba bean root distribution and interactions between intercropped species diversity enhances agricultural productivity via rhizosphere phosphorus facilitation on phosphorus-deficient soils plant diversity and overyielding : insights from belowground facilitation of intercropping in agriculture resilience in agriculture through crop diversification: adaptive management for environmental change selection for monoculture and mixture genotypes in a biodiversity experiment diversity in plant breeding: a new conceptual framework biodiversity and ecosystem functioning: recent theoretical advances partitioning selection and complementarity in biodiversity experiments designing cropping systems from nature mixing plant species in cropping systems: concepts, tools and models. a review the new green revolution: sustainable intensification of agriculture by intercropping differential plasticity to water and nutrients between crops and their wild progenitors tansley review patterns and processes in crop domestication: an historical review and quantitative analysis of global food crops growing larger with domestication: a matter of physiology, morphology or growing larger with domestication: a matter of physiology shifts and disruptions in resource-use trait syndromes during the evolution of herbaceous crops phylogenetic patterns and phenotypic profiles of the species of plants and mammals farmed for food from forest to field: perennial fruit crop domestication environmental, energetic, and economic comparisons of organic and conventional farming systems ecological significance of inherent variation in relative growth rate and its components tansley review biomass allocation to leaves, stems and roots : meta-analyses of interspecific variation and environmental control complementary effects of species and genetic diversity on productivity and stability of sown grasslands impacts of biodiversity loss escalate. science ( -) from tropics to tundra: global convergence in plant functioning cultivar mixtures: a metaanalysis of the effect of intraspecific diversity on crop yield: a contrasting effects of intraspecific trait variation on trait-based niches and performance of legumes in plant mixtures shifts in plant functional strategies over the course of wheat domestication the value of biodiversity experiments a guide to analyzing biodiversity experiments evolution of facilitation requires diverse communities effects of crop diversity on agroecosystem function: crop yield response management intensity -not biodiversity -the driver of ecosystem services in a long-term row crop experiment seed banks and molecular maps: unlocking genetic potential from the diversity and productivity in a long-term grassland experiment global food demand and the sustainable intensi fi cation of agriculture biomass productivity of mixtures increasing plant diversity with border crops reduces insecticide use and increases crop yield in urban agriculture common slope tests for bivariate errors-in-variables models smatr -an r package for estimation and inference about allometric lines evolutionary agroecology: individual fitness and population yield in wheat (triticum aestivum) plant ecological strategies : some leading dimensions of variation between plant ecological strategies: some leading dimensions of variation between species ggplot : elegant graphics for data analysis a plant biodiversity effect resolved to a single chromosomal region central questions in the domestication of plants and animals mixtures of genetically modified wheat lines outperform monocultures back into the wild -apply untapped genetic diversity of wild relatives for crop improvement selection for niche differentiation in plant communities increases biodiversity effects additional supporting information may be found online in the supporting information section at the end of the article. key: cord- - ckuya w authors: ninfali, paolino; antonelli, antonella; magnani, mauro; scarpa, emanuele salvatore title: antiviral properties of flavonoids and delivery strategies date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: ckuya w this review summarizes the latest advancements in phytochemicals as functional antiviral agents. we focused on flavonoids, like apigenin, vitexin, quercetin, rutin and naringenin, which have shown a wide range of biological effects including antiviral activities. the molecular mechanisms of their antiviral effects mainly consist in the inhibition of viral neuraminidase, proteases and dna/rna polymerases, as well as in the modification of various viral proteins. mixtures of different flavonoids or combination of flavonoids with antiviral synthetic drugs provide an enhancement of their antiviral effects. recent strategies in drug delivery significantly contribute to overcoming the low bioavailability of flavonoids. frequent viral infections worldwide have led to the need for new effective antiviral agents, which can be identified among the various phytochemicals. in this light, screening the antiviral activities of a cocktail of flavonoids would be advantageous in order to prevent viral infections and improve current antiviral therapies. in the past two decades, studies conducted in our laboratory focused on the antioxidants present in vegetable foods and on their capacity to reduce the adverse physiological effects of the oxygen free radicals [ , ] . the research was also aimed at the technologies of food transformation in order to preserve, as much as possible, the antioxidants in the final products [ ] . antioxidants are mainly represented in nature by the liposoluble vitamins e and a, β-carotene, hydro-soluble vitamin c and a wide range of amphipathic molecules, broadly termed phenolic compounds [ ] . these compounds are divided into several sub-classes, including phenolic acids and analogues, stilbenes, flavonoids and their analogues. flavonoids possess important health protective effects, including anti-inflammatory, anticancer and antiviral properties [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there are in nature more than flavonoids, which have been structurally identified and divided in classes: flavones (e.g., apigenin), flavanols (e.g., quercetin), flavins (e.g., epigallocatechin- -gallate), isoflavones (e.g., genistein) and anthocyanidins (e.g., cyanidin). flavonoids occur in their free or conjugated form or are often esterified with one or two sugars with o-glycosidic or c-glycosidic bonds [ ] . in the past decade we purified and studied the biological effects of a group of the flavonoid c-glycosides derived from apigenin, namely vitexin, vitexin- -o-rhamnoside and vitexin- -o-xyloside ( figure ). the interest in the antiviral activity of natural flavonoids has increased in the last decade because of the frequency of viral infections, particularly influenza infections, which affect several million patients annually [ ] . while vaccination is the primary strategy for influenza prevention, there are scenarios for which vaccination is not possible and antiviral molecules represent an important sanitary presidium. synthetic antiviral drugs often show limited efficacy and serious adverse effects [ ] , whereas herbal extracts, known for their antiviral properties with no or mild side effects, may be a viable alternative for treating various viral diseases [ ] . viruses consist of nucleic acid (dna or rna) enclosed in a protein structure, called capsid, which can be surrounded by a lipid membrane named the envelope. the infective unit of the virus is called the virion and this parasite can replicate only inside host cells, hijacking the molecular machinery and controlling dna replication, rna transcription and the protein translation processes. viruses attack the host cells through adsorption to receptors specific for each type of target cells, penetrating through the cell membrane, then the genetic material of the virus is liberated to replicate its own genome, produce new viral proteins and obtain new virions [ ] . in , the antiviral effects of various phytochemicals were reviewed by kapoor et al. [ ] , taking into consideration many categories of compounds, ranging from flavonoids to saponins and lignans. in the same year, another group published a review focused specifically on the antiviral effects of the six classes of flavonoids [ ] . in , an interesting review regarding the methods for delivery of phytochemicals to increase their bioavailability in human tissues has been published [ ] . in this review, we first summarize flavonoids along with their class and plant sources, with particular attention to apigenin, vitexin and its derivatives (table ) . we then discuss the antiviral action mechanisms of flavonoids, their combinations with conventional antiviral drugs in multitarget cocktails, and the delivery strategies used to increase their bioavailability and antiviral efficacy. the interest in the antiviral activity of natural flavonoids has increased in the last decade because of the frequency of viral infections, particularly influenza infections, which affect several million patients annually [ ] . while vaccination is the primary strategy for influenza prevention, there are scenarios for which vaccination is not possible and antiviral molecules represent an important sanitary presidium. synthetic antiviral drugs often show limited efficacy and serious adverse effects [ ] , whereas herbal extracts, known for their antiviral properties with no or mild side effects, may be a viable alternative for treating various viral diseases [ ] . viruses consist of nucleic acid (dna or rna) enclosed in a protein structure, called capsid, which can be surrounded by a lipid membrane named the envelope. the infective unit of the virus is called the virion and this parasite can replicate only inside host cells, hijacking the molecular machinery and controlling dna replication, rna transcription and the protein translation processes. viruses attack the host cells through adsorption to receptors specific for each type of target cells, penetrating through the cell membrane, then the genetic material of the virus is liberated to replicate its own genome, produce new viral proteins and obtain new virions [ ] . in , the antiviral effects of various phytochemicals were reviewed by kapoor et al. [ ] , taking into consideration many categories of compounds, ranging from flavonoids to saponins and lignans. in the same year, another group published a review focused specifically on the antiviral effects of the six classes of flavonoids [ ] . in , an interesting review regarding the methods for delivery of phytochemicals to increase their bioavailability in human tissues has been published [ ] . in this review, we first summarize flavonoids along with their class and plant sources, with particular attention to apigenin, vitexin and its derivatives (table ) . we then discuss the antiviral action mechanisms of flavonoids, their combinations with conventional antiviral drugs in multi-target cocktails, and the delivery strategies used to increase their bioavailability and antiviral efficacy. the most common flow chart for studies regarding the antiviral activity of phytochemicals is focused on the immediate testing of the whole natural extract, by dividing the polar from the non-polar constituents. after that, fractions with remarkable activity in in vitro antiviral assays, such as cytophatic effect, neutralization assay, hemagglutination, viral enzyme inhibition, or virion number reduction assay, are further fractionated through chromatographic techniques in order to purify the active phytochemicals. effective compounds are then used on virus-infected animals or in human clinical trials in order to assess their effective antiviral properties [ ] . the complete procedure, when interfaced with chemical libraries, represents the basis for high throughput screening assays [ ] . the parameter used for assessing antiviral efficiency of drugs and phytochemicals is represented by % inhibitory concentrations (ic ) or by % effective concentration (ec ). table shows the main flavonoids studied for antiviral activity along with their investigated mechanisms of action. in the first section, we point out the studies focused on apigenin, vitexin and their derivatives, which were found active against many viruses like human hepatitis c virus (hcv), herpes simplex virus- (hsv- ), human hepatitis a and b viruses (hav; hbv), rhesus rotavirus (rrv) and influenza virus. the natural extract of eclipta alba (asteraceae) was shown to be able to inhibit the hcv replicase in a cell culture system, which resulted in reduced hcv rna titer and translation level of viral proteins [ ] . through bioassay-based fractionations of the natural extract, the authors identified two flavonoid compounds: apigenin and luteolin, which, tested individually, exhibited a dose-dependent inhibition of hcv replicase in vitro [ ] . quercetin, extracted from embelia ribes (mirsinaceae), exhibited antiviral effects against hcv, exerted through activity inhibition of the viral protease non-structural protein (ns ), leading to a decrease in hcv replication [ ] . furthermore, the flavanol quercetagetin was found to inhibit hcv rna-dependent rna polymerase (rdrp) through inhibition of rna binding to the viral polymerase, a mechanism associated with broad genotypic activity against several hcv strains and a high barrier to resistance mechanisms [ ] . luteolin and quercetin showed anti-hcv effects in hepatoma huh- cells transfected with non-structural protein b (ns b) cloned gene vector, that codifies for the ns b polymerase of hcv virus [ ] . naringenin and quercetin possess antiviral activity against hcv, but naringenin showed stronger inhibition of virion assembly, whereas quercetin inhibited viral translation by blocking non-structural protein a (ns a) and internal ribosome entry site (ires)-mediated translation, as well as heat shock protein (hsp ) induction [ ] . bioinformatics tools were also used to study the interaction of various phytochemicals with viral proteins that possess pivotal roles in the production of new virions and in the infection of the host cells. this approach may be a useful premise for deeper investigation regarding flavonoids which have provided interesting evidence of interactions with viral proteins. for instance, naringenin, diosmetin, apigenin and luteolin were all able to bind to the ns b protein of hcv with higher affinity when compared with the antiviral drug sofosbuvir, inhibiting the activity of this viral enzyme [ ] . epigallocatechin- -gallate (egcg), the principal tea derived catechin, efficiently inhibited cell culture-derived hbv entry into hepatocellular cell lines, independent of the hbv genotypes, through a mechanism that involves the clathrin-dependent endocytosis of the hbv receptor sodium taurocholate co-transporting polypeptide (ntcp) from the plasma membrane, followed by its protein degradation [ ] . the extract obtained from erythrina speciosa (fabaceae) exerted antiviral effects against hav-h viruses mainly due to vitexin which, isolated from the extract, exhibited an antiviral activity against this virus with ec = . ± . µm [ ] . the authors showed that the flavone vitexin can interact with the binding pocket of hav c proteinase, inhibiting this enzyme [ ] . hcv virions inhibition of virion assembly vitexin, extracted from the plant flos trollii, (caryophyllaceae), exerted its anti-h n influenza virus effects through partially down-regulating toll-like receptor (tlr ) and toll-like receptor (tlr ) pathways and up-regulating toll-like receptor (tlr ) molecular pathway [ ] . tlrs are transmembrane glycoproteins, which are privileged targets of several viruses and can be activated by endogenous molecules in the context of inflammation. tlr activation produces pro-inflammatory cytokines through nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) signaling pathway or through interferon regulatory factor (irf ) and interferon regulatory factor (irf ). some viruses enter the host cells by binding tlr , but after their entrance the viruses are able to inhibit cytokine production, thus impairing the immune response. phytochemicals able to decrease the binding between tlrs and virus particles can slow the infective process. interestingly, virus infection can lead to an induction of the inflammation process, characterized by excessive production of nitric oxide (no), interleukin- (il- ), interleukin- (il- ) and tumor necrosis factor-α (tnf-α). it was shown that various flavonoids enhance the production of interferon-β (ifn-β) in order to counteract the viral infections [ ] . baicalin, baicalein, wogonin, chrysin and oroxylin a, isolated from scutellaria baicalensis, showed anti-h n activities, with ic values of . , . , . , . and . µm, respectively, and were all more potent than the conventional antiviral drug oseltamivir phosphate, which had an ic of . µm [ ] . these flavonoids increased the transcriptional activity of nuclear factor erythroid -related factor (nrf ), the master regulator of the antioxidant responses, whose activation is related to the antiviral effects of scutellaria baicalensis [ ] . the natural extract of tetrastigma hemsleyanum (vitaceae) contains many flavonoids, including vitexin, vitexin- -o-rhamnoside, isorhamnetin, rutin, kaempferol, astragalin, quercitrin, quercetin and iso-quercetin, which were shown to be able to exert anti-influenza virus activity, with different efficiency, through the reduction of the number of plaques induced by the influenza virus in infected madin-darby canine kidney (mdck) cells [ ] . similarly, the flavonoid quercetin- -rhamnoside, extracted from houttuynia cordata (saururaceae), exerted anti-influenza a/ws/ activity reducing virus-mediated cytopathic effects, directly interacting with virus particles [ ] . furthermore, the same authors showed that quercetin- -rhamnoside exerted anti-influenza virus activity in mice, when used at . mg/kg/day for six days after influenza virus infection [ ] . flavonoids sanggenon o, egcg and chamaejasmin were all potentially able to inhibit dengue virus replication by blocking the asn- glycosylation site of the viral protein non structural protein (ns ) [ ] . baicalin, a flavonoid derived from scutellaria baicalensis (lamiaceae), exhibited viricidal activity against dengue virus- (denv- ) extracellular virions with ic = . ± . µm, exerted an anti-adsorption effect with ic = . ± . µm and also inhibited virus replication with ic = . ± . µm [ ] . studies of the antiviral effects of the flavonoids fisetin, quercetagetin, and pinocembrin showed that fisetin can inhibit the replication molecular machineries of dengue virus and enterovirus a [ ] . furthermore, other antiviral mechanisms of the pinocembrin consist in targeting the molecular machinery used by the zika virus to replicate its own genome inside the host cells [ ] . this flavanone acts on the post-entry processes of zika virus replication cycle through the inhibition of both viral rna production and synthesis of envelope proteins [ ] . interestingly, the plant moringa oleifera lam (moringaceae) provides a rich and rare combination of several phytochemicals, including the flavonoids quercetin and kaempferol, and its leaves extract can be applied as a prophylactic or therapeutic anti-hsv- medicine [ ] . the extract obtained from moringa oleifera lam remarkably reduced the plaque formation induced by wt hsv, thymidine kinase-deficient hsv and phosphonoacetate-resistant hsv strains [ ] . furthermore, the extract obtained from erythrina speciosa possessed antiviral properties against the hsv- virus, mainly due to vitexin which exhibited an antiviral activity with ec = . ± . µm, exerted through the interaction of this flavone with the binding pocket of hsv- thymidine kinase [ ] . vitexin and luteolin from aspalathus linearis (fabaceae) showed antiviral activity against rrv with ic of µm and µm , respectively; interestingly, apigenin- -o-glucoside from melissa officinalis (labiateae) inhibited viral growth, with an ic of µm, through the reduction of the number of rrv-induced plaques in infected ma cells [ ] . tangeretin and nobiletin, two polymethoxyflavones extracted from citrus reticulate "chachi", possess anti-rsv properties. tangeretin exhibited a dose-dependent inhibition of rsv-induced plaque formation on hep- cells, through inhibition of rsv entry into host cells and viral replication. furthermore, tangeretin decreased the levels of rsv phosphoprotein (p protein), which is associated with the viral genome to form the holo-nucleocapsid. the extent of the antiviral effect of this phytochemical was comparable to the conventional antiviral drug ribavirin [ ] . the knowledge of the molecular mechanisms of virus infection and phytochemical antiviral actions is fundamental in planning an effective therapeutic approach. the main mechanisms involved in the antiviral effects of phytochemicals are focused on the targeting of viral enzyme activities. many natural molecules target the catalytic activity of the influenza virus neuraminidase, also called sialidase. this enzyme is a glycoside hydrolase that cleaves the glycosidic linkages of sialic acids ( figure ). various phytochemicals inhibit the activity of viral sialidases, hampering the release of new virions from the infected cells and preventing new infections [ ] . another enzyme with a pivotal role in influenza a virus infection is rdrp, which is composed of three subunits: polymerase acidic subunit (pa), protein binding subunit (pb ) and protein binding subunit (pb ). the enzyme synthesizes viral mrnas using short capped primers derived from host cellular mrnas, which are cut by the viral endonuclease. the n-terminal domain of the pa subunit contains a typical endonuclease active site and harbors the rna/dna endonuclease activity, which is essential for viral growth [ ] . the knowledge of the molecular mechanisms of virus infection and phytochemical antiviral actions is fundamental in planning an effective therapeutic approach. the main mechanisms involved in the antiviral effects of phytochemicals are focused on the targeting of viral enzyme activities. many natural molecules target the catalytic activity of the influenza virus neuraminidase, also called sialidase. this enzyme is a glycoside hydrolase that cleaves the glycosidic linkages of sialic acids (figure ). various phytochemicals inhibit the activity of viral sialidases, hampering the release of new virions from the infected cells and preventing new infections [ ] . another enzyme with a pivotal role in influenza a virus infection is rdrp, which is composed of three subunits: polymerase acidic subunit (pa), protein binding subunit (pb ) and protein binding subunit (pb ). the enzyme synthesizes viral mrnas using short capped primers derived from host cellular mrnas, which are cut by the viral endonuclease. the n-terminal domain of the pa subunit contains a typical endonuclease active site and harbors the rna/dna endonuclease activity, which is essential for viral growth [ ] . enzymes involved in the hcv virus replication, like the protease ns and the polymerase ns b can also be efficiently inhibited or modulated by phytochemicals ( figure ). ns is a hcv nonstructural protein, which acts as a serine protease. its n-terminal domain can interact with the viral non structural protein (ns ), while the c-terminal domain acts both as helicase and nucleoside triphosphatase. enzymes involved in the hcv virus replication, like the protease ns and the polymerase ns b can also be efficiently inhibited or modulated by phytochemicals ( figure ). ns is a hcv nonstructural protein, which acts as a serine protease. its n-terminal domain can interact with the viral non structural protein (ns ), while the c-terminal domain acts both as helicase and nucleoside triphosphatase. the ns b protein is rdrp with a pivotal role in replicating hcv's viral rna by using the viral ssrna+ as a template to catalyze the polymerization of ribo-nucleoside triphosphates during replication of the viral genome. interestingly, quercetin, apigenin and luteolin effectively inhibit ns b polymerase activity [ ] . when phytochemicals have been combined among them or with synthetic antiviral drugs, synergistic therapeutic effects were often evidenced. overall, when synergy occurred, it was often justified by the fact that the different molecules target different steps in the the ns b protein is rdrp with a pivotal role in replicating hcv's viral rna by using the viral ssrna+ as a template to catalyze the polymerization of ribo-nucleoside triphosphates during replication of the viral genome. interestingly, quercetin, apigenin and luteolin effectively inhibit ns b polymerase activity [ ] . when phytochemicals have been combined among them or with synthetic antiviral drugs, synergistic therapeutic effects were often evidenced. overall, when synergy occurred, it was often justified by the fact that the different molecules target different steps in the molecular mechanisms of viral infection, and the final antiviral effect results therefore potentiated. naringenin is a flavanone, which exhibits anti-hcv activity by blocking the assembly of hcv particles [ ] . the phytochemical quercetin exerted anti-hcv activity by reducing internal ribosome entry site-(ires-)mediated translation and also inhibiting the viral non-structural protein ns a as well as the protease ns [ ] . when naringenin and quercetin were used together they suppressed hcv rna replication and inhibited viral replication to a higher extent when compared with the phytochemicals used individually, thus demonstrating a synergistic effect [ ] . ladanein, a flavone isolated from marrubium peregrinum l. (lamiaceae), exploited antiviral effects through the inhibition of the post-attachment entry step of hcv virions, with an ic of . µm. ladanein, in combination with cyclosporine, showed a remarkable synergistic antiviral effect against various hcv genotypes [ ] . the natural extracts from nymphaea alba l. (nymphaeaceae), containing iso-quercetin, hyperoside, quercetin, reynoutrin, apigenin and isokaempferide, showed anti-hcv activity, suppressing hcv ns gene expression in the transfected huh- cell line and inhibiting the viral genotype a replication. furthermore, the combination of nymphaea alba l. extract with the conventional drug boceprevir displayed synergistic effects for inhibition of hcv replication in a docking bioinformatics model [ ] . an antiviral role of phytochemicals was also linked to the receptors recognized by viruses for their endocytosis, such as the membrane receptor ntcp. this protein has a mass of kda and is involved in the transport of bile salt molecules, steroid hormones, thyroid hormones and xenobiotics. ntcp is required for the entry in hepatocytes of both hbv and human hepatitis d virus (hdv). in fact, the virus-receptor interaction leads to the clathrin-dependent endocytosis of hbv virions, which can replicate inside the host cells [ ] . egcg, used at the dose of µm, induced clathrin-dependent endocytosis of ntcp from the plasma membrane, leading to its degradation and inhibiting the entry of hbv virus into immortalized human primary hepatocytes (figure ). two hiv enzymes address pivotal roles in virus replication and virion production: hiv reverse transcriptase and the homodimer of hiv protease (figure ). hiv reverse transcriptase is an rna-dependent dna polymerase (rddp) and builds ssdna based on an rna template in its polymerase active site; the enzyme also cleaves the original rna template into pieces through its nuclease active site and finally it polymerizes a second dna strand to form the final dsdna, which is integrated in the host cell genome. interestingly, it was shown that egcg suppressed hiv- infection in hela-cd -ltr-β-gal cells, with a ec of . µm, by acting as a non-nucleoside hiv reverse transcriptase inhibitor [ ] . furthermore, it was demonstrated that the flavonoids myricetin- -rhamnoside and myricetin- -( -rhamnosylgalactoside) possessed antiviral activity in vitro against hiv with ec of µm and µm, respectively [ ] . this antiviral effect was exerted through the inhibition of hiv reverse transcriptase with ic of . µm for myricetin- -rhamnoside and of . µm for myricetin- -( -rhamnosylgalactoside) [ ] . hiv protease is a retroviral aspartyl protease, which cleaves newly synthesized viral polyproteins (namely gag-pol) at nine cleavage sites to create the mature protein components of the virion. mature hiv- protease exists as a kda homodimer, each one containing an asp amino acid, which acts as the catalytic residues are able to hydrolyze peptide bonds on the gag-pol polyproteins into fully functional viral proteins, like reverse transcriptase and integrase. kehinde et al. ( ) [ ] showed that the phytochemicals kaempferol- -o-glucoside and egcg were able to interact with hiv- protease, showing pronounced structural evidence as potential hiv- protease inhibitors ( figure ) . interestingly, phytochemicals can also be used to reduce the extrusion of antiviral drugs from infected cells. in fact, apigenin, fisetin and luteolin were able to slow down the elimination of the antiviral drugs atazanavir, lopinavir, darunavir and saquinavir, which target the viral protease of hiv- [ ] . dependent dna polymerase (rddp) and builds ssdna based on an rna template in its polymerase active site; the enzyme also cleaves the original rna template into pieces through its nuclease active site and finally it polymerizes a second dna strand to form the final dsdna, which is integrated in the host cell genome. interestingly, it was shown that egcg suppressed hiv- infection in hela-cd -ltr-β-gal cells, with a ec of . µm, by acting as a non-nucleoside hiv reverse transcriptase inhibitor [ ] . furthermore, it was demonstrated that the flavonoids myricetin- -rhamnoside and myricetin- -( -rhamnosylgalactoside) possessed antiviral activity in vitro against hiv with ec of µm and µm, respectively [ ] . this antiviral effect was exerted through the inhibition of hiv reverse transcriptase with ic of . µm for myricetin- -rhamnoside and of . µm for myricetin- -( -rhamnosylgalactoside) [ ] . hiv protease is a retroviral aspartyl protease, which cleaves newly synthesized viral polyproteins (namely gag-pol) at nine cleavage sites to create the mature protein components of the antiviral activity of flavonoids may be also due to the modulation of host cell enzymes used by the virus to take advantage for infection, such as the enzymes with a pivotal role in the production of radical oxygen species (ros), utilized to increase the number of virions. regarding hsv- , which persists in the host in sensory neurons in latency, the enzyme nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox) family is a useful source of ros, which can be used to reactivate the viral infection under oxidative stress conditions [ , ] . interestingly, delphinidin- -rutinoside obtained from extracts of solanum melongena l. (solanaceae), inhibited hsv- replication through the reduction of nox protein levels, when added for h after viral adsorption on vero cells [ ] . the extract obtained from veronica persica poir (plantaginaceae) possessed a dose-dependent inhibitory activity against the herpes simplex virus strains hsv- and hsv- and a prominent synergistic activity in combination with acyclovir in anti-hsv therapy, exerted through a reduction of the percentage of plaque numbers of both hsv- and hsv- in the infected cero cells [ ] . the natural extract of disticella elongata (bignoniaceae) exhibited antiviral effects against denv- virus and this effect was mainly due to pectolinarin and acacetin- -o-rutinoside [ ] . when the two flavonoids pectolinarin and acacetin- -o-rutinoside were used in combination, the antiviral effect was eight times more potent against denv- virus (with ec = . ± . µm) than the flavonoid pectolinarin used alone (with ec = . ± . µm). the selectivity index of the combination (si = ) was remarkably higher than the si of the isolated pectolinarin (si = . ), indicating that the phytochemical mixture specifically inhibited viral growth, with negligible effects on the vitality of the cells infected by denv- virus. the ethanol extract obtained from the leaves of disticella elongata showed antioxidant activities; therefore, it could detoxify cell damaging free radicals present in denv- viral infections [ ] . the low water solubility and low bioavailability of natural flavonoids represent the major limit for their use in the nutraceutical sector. many delivery strategies have been used by researchers for increasing flavonoid bioavailability following oral consumption [ ] , including: micelles, nanoparticles, microspheres, crystals, dendrimers, the self-micro-emulsifying drug delivery system (smdds) and the self-nanoemulsifying drug delivery systems (snedds), as recently reviewed [ , ] . for instance, it was shown that the catechin and egcg-loaded chitosan nanoparticles led to a higher rate of intestinal absorption of the two phytochemicals [ ] . interestingly, in chitosan particles the flavonoids maintain their antioxidant activity and can exploit their antioxidant effects in the blood stream [ ] . the loading of myricetin into a cationic polymeric nanoparticle carrier with a cationic corona and hydrophobic core was investigated in order to improve the clinical relevance of this natural flavonoid by increasing its solubility [ ] . smdds has been used to overcome the problem of low bioavailability of hydrophobic molecules as, in the intestinal lumen, the oil-in-water microemulsions containing phytochemicals may be efficiently formed with a consequent increase of the intestinal absorption of the flavonoid [ ] . interestingly, puerarin, an isoflavone isolated from the root of pueraria lobata, exhibited . fold higher bioavailability when prepared using the smdds technique [ ] . furthermore, the synthesis of silver nanoparticles linked with phytochemicals and their use for antimicrobial and antiviral treatments have been described, highlighting the various molecular mechanisms that lead to the phytochemical-mediated inhibition of viral replication [ ] . poly (d,l-lactide) (pla) nanoparticles and polymeric micelles contributed to a more sustainable and efficient release of flavonoids characterized by a poor bioavailability, like quercetin and apigenin [ , ] . quercetin was successfully encapsulated on the most uniformly distributed type of pla- nanoparticle, synthesized using lonicera japonica leaf extract, showing that these nanoparticles allowed a slow release of quercetin [ ] . this nanoparticle approach paves the way for encapsulating drugs, small molecules, nutraceuticals and other bioactive ingredients to obtain safer cellular uptake, improved biodistribution, specific targeted delivery and enhancement of the therapeutic antiviral efficacy of encapsulated drugs and phytochemicals [ ] . the increase in antiviral efficacy and bioavailability of flavonoids may be attained through their encapsulation into red blood cells (rbcs), as has occurred for other type of antiviral drugs and molecules, such as fludarabine (figure ) [ ] , vincristine and vinblastine [ , ] . the idea of using rbcs as delivery system for flavonoids takes its advantage from the favorable characteristics of these cells. they have a long life-span of about days and have a widespread circulation throughout the body, and hence can be used as drug reservoirs, enabling them to facilitate sustained drug release. moreover, rbcs protect encapsulated drugs and molecules from degradation; they are completely biodegradable and show no or only minor immunogenic responses. interestingly, the rbcs were used to determine cellular antioxidant activity of some flavonoids, specifically vitexin, vitexin- -o-xyloside and vitexin- -o-rhamnoside, with the aim of predicting their bioavailability [ ] . moreover, it was demonstrated that flavonoids could have beneficial effects in preventing oxidative damage of erythrocyte membrane [ , ] . some authors have also evidenced the possibility that human rbcs play a pivotal role in the distribution and bioavailability of circulating flavonoids such as quercetin [ ] . furthermore, it was shown that drug-loaded rbcs can be modified in order to increase their macrophage-mediated phagocytosis by inducing band clustering and opsonization through the complementary and autologous iggs [ ] . in future perspective, this approach could be considered in order to possibly improve the antiviral activity of some flavonoids, like baicalin, that was able, like fludarabine [ ] , to act against hiv- chronic infection of human monocytes and macrophages, inhibiting the fusion of hiv virus envelope proteins with these cells [ ] . although polymeric nanoparticles, liposomes, dendrimers, micelles and inorganic nanoparticles have been widely accepted as drug delivery systems, they show toxicity and a short lifetime, thus making them relatively disadvantageous when compared with natural cell carriers, such as rbcs. for this reason, some authors in recent years have tried to mimic the erythrocyte cell membrane to produce biocompatible nanocarriers in order to decrease their toxicity and to prolong their survival in blood circulation [ ] [ ] [ ] . rbcs, which are biodegradable and non-immunogenic, can be used as a valuable carrier system with a lifespan that is remarkably prolonged and controllable when compared to synthetic carriers. several approaches have been developed to load peptides, drugs and molecules into rbcs or to attach these agents onto rbcs' outer membrane surface by either chemical or physical methods [ ] and the possibility of loading drugs into autologous rbcs prior to their transfusion into patients has been studied in small animal models and primates, as well as in clinical studies of human patients [ ] [ ] [ ] [ ] . the topic of phytochemical encapsulation in rbcs remains open for further studies, but we believe that flavonoid derivatives and nanoparticles able to bind flavonoids could be successfully considered for this application in the near future. diet and life-style play important roles in the defense against the attacks of viruses. the relationship between diet and the immune system involves the microbiota, i.e., the ecological community of commensals and potentially pathogenic microbes and symbionts that live in the gut [ ] . the diet modulates the microbiota composition, leading to an increase or a decrease in immune defenses [ ] . the mediterranean diet (and in general diets rich in fruits, vegetables and herbs) maintains gut microbiota homeostasis and provides protection against microbes and viruses [ ] . the cross-talk between microbiota and immunity is supported by the aryl hydrocarbon receptor (ahr), which is ubiquitous, but mainly present in the cytoplasm of immune cells [ ] . it was demonstrated that ahr binds different ligands, namely metabolites, pollutants and pathogenic molecules, and after this interaction it translocates into the nucleus, where it induces specific transcription programs and modulates the defensive functions of both t and b cells, dendritic cells and monocytes [ ] . interestingly, it was shown that flavonoids can bind ahr [ ] . on this basis, people eating vegetables, all of which contain flavonoids to a different extent, would strengthen their immune system [ ] . this strengthening is a general effect that occurs with many nutrients, but there are more specific antiviral effects attributable to the flavonoids treated in this review. flavonoids, like apigenin, vitexin, quercetin, rutin and naringenin, show a wide range of antiviral effects (table ) , because they are able to target different pathways of viral infections. it is therefore possible to increase the chances of blocking viral replication using mixtures of flavonoids with synergistic antiviral effects, because of the pleiotropic effects of their combination. an important question that arises is focused on the reasonable concentration range of flavonoids that should be used for an effective antiviral therapy, which is hard to be reached by diet alone. recent experiments performed in in vivo studies demonstrated the protective efficacy of various flavonoids, tested in the range - mg/kg body weight per day, in newborn mice challenged with a lethal dose of the enterovirus [ ] . interestingly, apigenin ( mg/kg), luteolin ( mg/kg) and quercetin ( mg/kg) conferred survival protection of . %, . % and . %, respectively, from the lethal enterovirus infection; moreover, isorhamnetin provided the highest survival protection of % at a dose of mg/kg. the authors hypothesized that the differences in concentrations are due to different times of absorption and renal clearance of these flavonoids [ ] . the flavanol isorhamnetin was studied also by dayem et al. [ ] , who showed that oral administration of this flavonoid at mg/kg/day for five days in mice infected with the influenza a virus significantly decreased lung virus titer by two-fold, increased the survival rate (which ranged from % to %) and decreased mice body weight loss by %. these authors hypothesized that the methyl group of the b ring of isorhamnetin may contribute to its strong antiviral potency against influenza a virus [ ] . guo et al. [ ] focused their in vivo studies on the flavone wogonin, showing that, in human hbv-transgenic mice, this flavonoid administered once a day at , and mg/kg reduced plasma hbsag level and immunohistological staining of the liver confirming the hbsag reduction exerted by wogonin [ ] . the potentiality of flavonoid bioactivity in vivo depends on the extent of their absorption after ingestion and their ability for distribution in various target tissues. in this light, liu et al. [ ] developed a quercetin-loaded cationic nanostructure lipid carrier (q-cnlc), which increased the in vivo bioavailability of this flavonoid. this quercetin-nanostructure complex benefits from its strong interactions with negatively charged intestinal mucosa, which could increase its residence in the gastrointestinal tract. moreover, the authors showed an entrapment efficiency of quercetin of . % and a slower release of this flavonoid from the q-clnc, when compared with the release profile of a simple quercetin solution, indicating that q-clnc exhibited a sustained and controlled release of this flavanol, in order to maintain its effective therapeutic concentration [ ] . in addition, the same authors performed in vivo tissue distribution studies in c bl/ j mice, comparing treatment with mg/kg of orally administered quercetin solution with the administration of mg/kg of q-cnlc, showing that the relative quercetin uptake from q-cnlc was . fold higher in lungs, . fold higher in liver and . fold higher in kidneys. on the contrary, the relative quercetin uptake from q-cnlc was lower in spleen, heart and brain, when compared with the quercetin solution [ ] . these results indicate that the most suitable delivery strategy should be chosen in order to target a specific organ with a particular flavonoid-nanostructure complex for future clinical applications. furthermore, the safety of the used phytochemical should also be considered, as has already been done for hydroxytyrosol, which is quickly absorbed and eliminated by the kidneys in either free or conjugated forms. hydroxytyrosol has been considered safe at mg/kg/day by the european food safety authority (efsa) panel [ ] . in this light, we think that, for a kg person, a flavonoid range between . - . g/day should be proposed. these values are close to the daily polyphenol intake in humans, calculated in various diet surveys [ ] , such as the supplementation en vitamines et mineraux antioxydants (su.vi.max) study, which ranked the polyphenol intake at . ± . g/day [ ] . accordingly, in another dietary intervention trial aimed at improving cognition in older adults, a total of . g/day of flavonoids was applied [ ] . indeed, based on these results, we think that . - . g/day of flavonoids could be a reasonable concentration range in order to start preliminary experiments, focused on assessing the in vivo antiviral effects of flavonoids. concerning the combination of flavonoids in an antiviral cocktail, each phytochemical may be used initially at a concentration of about . g/day with the aim of reaching an intake of g/day of antiviral flavonoids. in the case of antiviral synergistic effects [ , ] or increase of the absorption of one specific flavonoid exerted by other phytochemicals [ ] , the individual flavonoid concentrations can be modulated, according to the extent of the effect. it should also be emphasized that a significant antiviral effect is linked to the type of flavonoid mixture, the delivery system used, the pharmacokinetic pattern, the number of targets involved and the number of required daily doses. once these aspects have been defined, the more suitable regimen of administration consists of starting with the lowest effective concentration for a fixed time and proceeding with increasing doses of the antiviral flavonoids. monitoring the markers of antiviral efficacy and any side effects should also be considered in order to evaluate the risks-benefits pattern. overall, our review shows that many flavonoids exhibit antiviral activity and could offer a promising alternative for prevention of and therapy for viral infections. flavonoids are present in many vegetables and the first protection for the immune system resides in the ability to seek foods rich in bioactive nutrients. education programs for a healthy diet should be implemented during the outbreaks of viral infections [ ] . in fact, a diet rich in vegetables activates the ahr in the gut for maintenance of microbiota homeostasis, which in turn regulates the immune system. in the critical periods of viral infections, oral dietary supplementation with nutraceutical preparations based on combinations of flavonoids can be useful in order to inhibit different steps of the viral infective cycle. molecular mechanisms underlying the antiviral effects of flavonoids, herein described, mainly focus on the inhibition of viral enzyme activities: neuraminidases, dna/rna polymerases and proteases. therefore, a cocktail of flavonoids, selected for their efficacy in the inhibition of different viral enzymes, could be associated with elevated immune response and offer a promising option for antiviral therapies. this option acquires great importance considering that the viral genome frequently mutates, due to the lack of proof-reading activity of most of the viral polymerases. these mutations could hamper the efficacy of antiviral synthetic drugs. on the contrary, antiviral flavonoids, as well as the combination of synthetic antiviral drugs with flavonoids, would enhance therapeutic strategies by targeting the multiple signaling pathways involved in the viral infections [ ] . the active concentration of the flavonoids should be investigated, considering the pharmacokinetic studies available in the literature and the synergistic effects of the specific flavonoid combinations [ ] [ ] [ ] . the scarce intestinal absorption and bioavailability of flavonoids, when given through food or in pills, may be enhanced by the use of new drug delivery strategies [ ] . in fact, since flavonoids have some drawbacks after oral administration such as low stability, bioavailability and bio-efficacy, researchers are developing biocompatible nanomaterial synthesis as novel delivery systems (including nanospheres, nano-capsules, micro and nano-emulsions, micelles, solid lipid nanoparticles and capsules), for overcoming the delivery challenges of flavonoids in the biomedical sector. phytochemical-nanomaterial complexes can represent innovative drug delivery strategies (alongside those already known) for new antiviral therapies against the seven baltimore virus classes [ ] . interestingly, three patents regarding the antiviral effects of flavonoids (us , , ; ep ; us , , ) have been already assigned to the korea research institute of bioscience and biotechnology and advanced life sciences inc. [ ] . however, an important step that must be achieved is to obtain the authorization of novel food including flavonoids with antiviral properties, following regulation eu / [ ] . nowadays, this authorization has been given only to hydroxytyrosol [ ] and few other nutrients. if the antiviral efficacy and safety of flavonoids and their mixtures can be clearly demonstrated in vivo, it would be possible to obtain european food safety authority (efsa) authorization of novel foods, in order to provide new natural tools for preventing and facing outbreaks of viral infections. indeed, when flavonoids are administered through nano-sized delivery systems, they show increased stability and bioavailability with an enhanced and prolonged activity. however, the in vivo behavior and the antiviral actions of these nano-delivery systems are still under experimental evaluation. interferon regulatory factor irf interferon polyphenols and antioxidant capacity of vegetables under fresh and frozen conditions antioxidant capacity of extra-virgin olive oils antioxidant capacity of vegetables, spices and dressings relevant to nutrition effect of flavonoids on human health: old subjects but new challenges vitexin -o-xyloside, raphasatin and (-) eepigallocatechin- -gallate synergistically affect cell growth and apoptosis of colon cancer cells betalains increase vitexin- -o-xyloside cytotoxicity in caco- cancer cells antiproliferative activity of vitexin- -o-xyloside and avenanthramides on caco- and hepg cancer cells occurs through apoptosis induction and reduction of pro-survival mechanisms natural and synthetic avenathramides activate caspases , , and downregulate htert, mdr and cox- genes in caco- and hep b cancer cells a combination of moringin and avenanthramide f inhibits the proliferation of hep b liver cancer cells inducing intrinsic and extrinsic apoptosis flavonoids from beta vulgaris cicla and betalains from beta vulgais rubra: antioxidant, anticancer, antiinflammatory activities-a review characterization and biological activity of the main flaonoids from swiss chard (beta vulgaris subspecies cycla) oseltamivir resistance-disabling our influenza defenses twenty years of research into medicinal plants: results and perspectives combination of western medicine and chinese traditional patent medicine in treating a family case of covid- in wuhan. front antiviral phytochemicals: an overview flavonoids: promising natural compounds against viral infections antiviral effects of phytochemicals from medicinal plants: applications and drug delivery strategies antiviral activity of disticella elongata (vahl) urb. (bignoniaceae), a potentially useful source of anti-dengue drugs from the state of minas gerais identification and evaluation of anti hepatitis c virus phytochemicals from eclipta alba an evaluation of the inhibitory effects against rotavirus infection of edible plant extracts qualitative and quantitative analysis for the chemical constituents of tetrastigma hemsleyanum diels et gilg using ultra-high performance liquid chromatography/hybrid quadrupole-orbitrap mass spectrometry and preliminary screening for anti-influenza virus components anti-h n virus, cytotoxic and nrf activation activities of chemical constituents from scutellaria baicalensis molecular docking based screening of plant flavonoids as dengue ns inhibitors a polyphenol rich extract from solanum melongena l. dr peel exhibits antioxidant properties and anti-herpes simplex virus type pharmacoinformatics approach for investigation of alternative potential hepatitis c virus nonstructural protein b inhibitors epigallocatechin- -gallate inhibits entry of hepatitis b virus into hepatocytes the pharmacokinetic properties of hiv- protease inhibitors: a computational perspective on herbal phytochemicals anti-hepatitis c virus activity and synergistic effect of nymphaea alba extracts and bioactive constituents in liver infected cells moringa oleifera: a food plant with multiple medicinal uses a plant-derived flavonoid inhibits entry of all hcv genotypes into human hepatocytes the role of the glycosyl moiety of myricetin derivatives in anti-hiv- activity in vitro antiviral activity of polymethoxylated flavones from guangchenpi, the edible and medicinal pericarps of citrus reticulata "chachi antiviral activity of pinocembrin against zika virus replication characterization and evaluation of self-microemulsifying sustained-release pellet formulation of puerarin for oral delivery phytochemicals as antiviral agents: recent updates suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns protease activity inhibitory effects of quercetin -rhamnoside on influenza a virus replication anti-influenza a virus mechanism of three representative compounds from flos trollii via tlrs signaling pathways inhibition of rna binding to hepatitis c virus rna-dependent rna polymerase: a new mechanism for antiviral intervention therapeutic potential of taraxacum officinale against hcv ns b polymerase: in-vitro and in silico study divergent antiviral effects of bioflavonoids on the hepatitis c virus life cycle breaking down the barriers to a natural antiviral agent: antiviral activity and molecular docking of erythrina speciosa extract, fractions, and the major compound epigallocatechin gallate inhibits the hiv reverse transcription step baicalin, a metabolite of baicalein with antiviral activity against dengue virus quercetin- -rhamnoside exerts antiinfluenza a virus activity in mice virology blog: about viruses and viral disease inhibition of sialidase activity as a therapeutic approach anti-influenza activity of marchantins, macrocyclic bisbibenxyls contained in liverworts a sequence data resource for hepatitis b virus corriere nazionale: i virus che spaventano la scienza infectious agents and neurodegeneration influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by nox -derived ros antiviral activity of veronica persica poir. on herpes virus infection nanoparticulate delivery systems for antiviral drugs chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (+)-catechin and (-)-epigallocatechin gallate orac of chitosan and its derivatives. food hydrocoll electrostatic interactions enable nanoparticle delivery of the flavonoid myricetin development of self-microemulsifying drug delivery system (smedds) for oral bioavailability enhancement of simvastatin in beagle dogs phytochemical-assisted synthetic approaches for silver nanoparticles antimicrobial applications: a review development of biodegradble nanoparticles for delivery of quercetin preparation and in vitro evaluation of apigenin-loaded polymeric micelles plant extract synthesized pla nanoparticles for controlled and sustained release of quercetin: a green approach domínguez-perles, r. nanoparticles and controlled delivery for bioactive compounds: outlining challenges for new "smart-foods" for health drug-loaded red blood cell-mediated clearance of hiv- macrophage reservoir by selective inhibition of stat expression the terpene-indole alkaloids loaded erythrocytes as a drug carrier: design and assessment study of desorbtion and exemption of terpeno-indole alkaloids of vinkristin and vinblastin from erythrocitary cell carriers the cellular antioxidant activity in red blood cells (caa-rbc): a new approach to bioavailability and synergy of phytochemicals and botanical extracts protective effect of flavonoids against red blood cell hemolysis by free radicals inhibition of free radical initiated peroxidation of human erythrocyte ghosts by flavonols and their glycosides human red blood cell sas a natural flavonoid reservoir drug delivery through phagocytosis of red blood cells flavonoids as antiviral agents for enterovirus a (ev-a ) red blood cell membrane-camouflaged nanoparticles: a novel drug delivery system for antitumor application red blood cell-mimicking synthetic biomaterial particles using mechanobiological mimicry of red blood cells to extend circulation times of hydrogel microparticles engineering erythrocytes for the modulation of drugs' and contrasting agents' pharmacokinetics and biodistribution red blood cell-based delivery of drugs and nanomaterials for therapeutic and diagnostic applications delivery of drugs bound to erythrocytes: new avenues for an old intravascular carrier observation of erythrocyte dynamics in the retinal capillaries and choriocapillaris using icg-loaded erythrocyte ghost cells erythrocytes as carriers for drug delivery in blood transfusion and beyond the potential impact of gut microbiota on your health: current status and future challenges. asian pac effect of fruit and vegetable consumption on immune function in older people: a randomized controlled trial polyphenols and tryptophan metabolites activate the aryl hydrocarbon receptor in an in vitro model of colonic fermentation antiviral efficacy of flavonoids against enterovirus infection in vitro and in newborn mice antiviral effect of methylated flavonol isorhamnetin against influenza anti-hepatitis b virus activity of wogonin in vitro and in vivo characterization and biodistribution in vivo of quercetin-loaded cationic nanostructured lipid carriers ec regulation / . safety of hydroxytyrosol as a novel food pursuant to regulation (ec) no / systematic review on polyphenol intake and health outcomes: is there sufficient evidence to define a health-promoting polyphenol-rich dietary pattern? nutrients dietary intake of polyphenols in french adults enhancing dentate gyrus function with dietary flavanols improves cognition in older adults enhancement of flavonoid ability to cross the blood-brain barrier of rats by co-administration with α-tocopherol the food systems in the era of the coronavirus (covid- ) pandemic crisis. foods viral mutation rates flavonoids loaded in nanocarriers: an opportunity to increase oral bioavailability and bioefficacy nanomaterials designed for antiviral drug delivery transport across biological barriers of the european parliament and of the council and repealing regulation (ec) no / of the european parliament and of the council and commission regulation (ec) no this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors want to thank university of urbino carlo bo for financial support. the authors declare no conflict of interest. the funders had no role in the writing of the manuscript or in the decision to publish this manuscript. key: cord- -bcw f b authors: nan title: abstracts: th ebsa european biophysics congress, august rd– th , budapest, hungary date: - - journal: eur biophys j doi: . /s - - -z sha: doc_id: cord_uid: bcw f b nan o- structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md , usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr , ). together with the light driven clpump halorhodopsin chr is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o- molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station , ecole polytechnique fé dé rale, ch- lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over percent for single junction and percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi- d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale ( - nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier ] - nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier ] meta-stable - nm raft domains that can be turned into stable * - -nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier ]) and facilitated by raft-lipid interactions. [tier ] protein complexes of various sizes ( - nm) and lifetimes. i will also talk about how domains of tiers and are coupled to the membrane partitioning (tier ). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh and daam-fh -fh fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh -fh fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh -fh also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh -fh , remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = , , , , ). d o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about % of the nitroxides in the upper part of the chain (n = , ) are h-bonded by a single water molecule and this fraction reduces to % at the chain terminus (n = , ). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about % to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both -and -sasl in the protein cavity. the temperature dependence of the librations is more marked for -sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol- , -bisphosphate (pip ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf , hela and g tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps . h o) on the malignant cell lines (g , hela, mcf ) irradiated with the jcm - doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps . h o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p- nmr analysis of the respiratory syncytial virus m - protein structure and of its interaction with some of its targets c. sizun the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m - acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m - shares structural similarity with vp , a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m - might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger , , ingo mey and claudia steinem institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs - ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol , bisphosphate (pip ) in the membrane, followed by phosphorylation of the threonine residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b- (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp- - , vega- - . modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. these processes are involved in the pathogenesis of aging and diabetes. non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [ ] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [ ] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, rue becquerel strasbourg, france. cnrs, umr , strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches Ícaro p caruso human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin- -rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration ( lm) constant and stoichiometrically varying the rutin concentration ( - lm) . the emission spectra were obtained in the range of to nm, with the excitation wavelength at nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were . and . m - at and k, respectively. from the modified stern-volmer association constants . (at k) and . m - (at k) were calculated the thermodynamic parameters dh = . kj mol - , dg k = - . kj mol - and dg k = - . kj mol - , and ds = . kj mol - k - . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites . and . , and binding constant . m - and . m - at and k, respectively. the efficient quenching of the trp fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = . nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b lyp functional with - g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about . ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code ao ) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp ) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin - on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's greatest discoveries, ) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph . a : stoichiometry of complexation was detected for both complexes, with association constants of m - and m - for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by mm ttc increased % upon complexation. supported by (fapesp # / - , - ) brazil. p- itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas , dominique burnouf , eric ennifar , sondes guedich , guillaume bec , guillaume hoffmann isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv- reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: ( ) the binding of the tpp ligand and ( ) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least times lower than gfp. this difference is due to an intrinsic mutation of cfp (n i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w (po --bound) and w (non-po --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w . the water h-bond imbalance of - kj mol - is equalized at little enthalpic cost upon formation of a contiguous water network (at - h o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w -dna interactions by additional w -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial -mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh domains of cd ap protein to study interaction with their ligand i.f. herranz-trillo , j.l. ortega-roldan , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin /cms family of adaptor proteins. in this family all three n-terminal sh domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh domains, suggesting an overlapping specificities in binding. cd associated protein (cd ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd ap-sh a/ sh b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over - million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the : and : stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n . microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu + ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu + ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of , , , mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu + complexes in aqueous solution at two ph values, . and . , the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu + complexes. the formation of amb-cu + complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n . membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin- , however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the xhis-tagged protein was immobilised via specific binding to a cu + terminated sensor surface, with a change in frequency indicating approximately % coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix , *, zeger debyser , johan hofkens and yves engelborghs laboratory for biomolecular dynamics, university of leuven, belgium, laboratory for molecular virology and gene therapy, university of leuven, belgium, laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p has well characterized protein and chromatin interacting regions. we studied ledgf/p in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p and the hiv- integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b or c , play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b and c into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [ ] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [ ] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [ , ] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [ ] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t and its distant relative coliphage rb encode co-chaperon proteins (respectively gp and coco) that can replace groes in the chaperonin complex. gp is also required in the folding of the major capsid protein of the phage. prd is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd . we have found evidence that prd protein p can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, svobody sq., kharkiv, , ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin ( mol %) and cholesterol ( mol %). to achieve this purpose, two fluorescent probes with different bilayer location, , -diphenyl- , , -hexatriene (dph) distributing in membrane hydrocarbon core and -lauroyl- -dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range lm [ k d [ nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper , , d. spridon , l.j. van ijzendoorn , m.w.j. prins , eindhoven university of technology, department of applied physics, eindhoven, the netherlands, dutch polymer institute, eindhoven, the netherlands, philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. , p. , ). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a - kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin- (twf ) on the monomeric actin. we determined the affinity of twf to the atp-actin monomer with fluorescence anisotropy measurement (k d = . lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf . in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf . we determined the thermodynamic properties of twf and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe o np was studied in female wistar rats i.v. administered either placebo ( % v/v rat serum in . % nacl), suspension of tio np (positive control, bimodal / nm distribution), or fe o np (bimodal / nm distribution) in doses of . , . or . mg/kg. rats were sacrificed h, -, -and -days after np injection (n= - /each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered mg fe o /kg. plasma mg levels rose in this group and weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe o np. study was supported by fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: . biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in % of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. detailed information on the mechanism and the thermodynamics of the reaction was revealed: first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h po hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin- (gal- ) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal- . our goals are two-fold: ( ) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and ( ) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away -phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (* %) is constituted by amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab( - ) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab( - ) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag , a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of °c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak , b. ró _ zycka-roszak , e. woźniak , r. skrzela , k.a. wilk department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = , , , ) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n . every year over million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ ] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [ ] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [ ] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [ ] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu - -bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse , gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse , matteo castronovo , bojana lucic , alessandro vindigni , giacinto scoles and loredana casalis sincrotrone trieste s.c.p.a., trieste, italy, temple university, philadelphia, usa, protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm . the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt . in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp ) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at °c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween , tween , and pluronic f . znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of . and . mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph . ) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f , to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. , in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano ( ) ( ): , bano et al. nano lett ( ) ( ): - ). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the ( - ) and ( - ) segments of crotamine, a toxin from crotalus durissus terrificus venom [ ] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol : ) and anionic (popg; popc:popg : ) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p * - ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r - , a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r - depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r - to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats and that are involved in the interaction with anionic suvs. results indicate that dys r - constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp- function by occupying a binding site near the active center of the enzyme (bika di et al. ). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp- (cdmmp- ) was expressed in e.coli bl (de ) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp- activity, however, determination of ic % or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp- in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of to da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [ ] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc [ ] in liposomal and planar bilayer systems was possible. abcc -containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc for immobilization via ni + and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met and his residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met bond. this allows the binding of others endogenous ligands forming alternative low spin species , or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h q, h n, and the double one h q/h n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m , to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of . gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin -acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin complex, and study a possible side-binding effect of the leiomodin . the cardiac leiomodin of rattus norvegicus is a kda molecular weight protein, which contains a kda n-terminal, a kda leucin reach repeat (lrr) and a kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin concentration. the nucleator activity of leiomo-din was ionic strength dependent. the data also confirmed that leiomodin is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin . these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc- ( , ', , '-tetrachloro- , ', , '-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh -da ( ', 'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, - , japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri( -n-methylpyridyl)-mono-( -carboxyphenyl)porphyrin and bi-cationic meso- , -bis( -n-methylpyridyl)- , di-( -carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t phage. the wide nucleotide specificity of -phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay , j. vincze , e. balogh hheif, budapest, hungary, nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [ ] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [ , ] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [ , ] . the initially formed cpps show hydrodynamic radii in the range of nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of - nm [ ] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [ ] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna ' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst ) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles ) to detect the expression/activation of critical cell signaling proteins at the single cell level; ) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and ) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b -microlgobulin (b -m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in , cells from localized pca tissue areas (gleason and ) of patients with known metastatic status. the overall median % triple positive for b -m + /pcreb + /ar + cells was . %. the median triple positive for the samples with metastatic potential was % compared with those without metastatic potential (median = %); p = . by a wilcoxon rank sum test. the results were confirmed in pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl- were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o- optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp s and vsfp s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding % dr/r in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr ) slowing with depolarisation and chief (chr /chr hybrid) in contrast, accelerating. new chr point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm ncrna in a sknbe -derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm at its basal level (mock cells) or at . fold higher levels (s cells). the two cell lines shared very similar pools of functional k channels, but s cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s rapid application of gaba elicited a current with a ec = . lm; this current was antagonized by bicuculline ( lm) and potentiated by zaleplon (ec = nm). in mock cells, real time pcr evidenced a high level of gaba a a subunit, while in s cells a significant expression of a and a was detected, whereas a mrna was downregulated by %, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of lm to lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin- (chr ), the panel of excitatory opsins now includes an array of chr variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o- subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav . subunit in pc proximal and distal dendrites, where their density is -fold lower than that found in aiss. a gradual decrease in nav . density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv . voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav . channels, the density of kv . first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca + redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca + response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca + homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca + ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na + concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca + -homeostasis since bz-induced decrease of: in striated muscle ca + release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca + homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca + release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca + release events on images recorded from fibres loaded with fluorescent ca + indicators. to elucidate the effects of ptdinsp-s on ca + release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins p, ptdins p, and ptdins( , )p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca + release events. supported by the hungarian national science fund (otka ), té t. calcium sparks elicited by mmol/l caffeine and by a depolarization to - mv were recorded at high time resolution on both x-y ( frames/s) and line-scan images ( lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, . and . % of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f ; . ± . vs. . ± . ; n = vs. ; mean ± sem, p \ . ) and the full width at half maximum (in lm; parallel with fiber axis: . ± . vs. . ± . ; perpendicular to fiber axis: . ± . vs. . ± . ) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the , dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc . the activation of nfatc dependent genes is consistent with an increase of the il secretion from primary coltures human myotubes from ccd patients and with fibre type predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p- ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew , paula pescador , matthias schade , julian appelfeller , jü rgen liebscher , oliver seitz , daniel huster , andreas herrmann , and anna arbuzova humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, humboldt universitä t zu berlin, institute of chemistry, berlin, germany, universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of nm and - lm and a length of - lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of t -mdr cells. these cell surface transporters have a uic conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least -fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il- is a multifunctional cytokine with pleiotropic effects on t cells. the il- r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il- and - , important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il- and il- r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il- signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il- r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il- r with lipid rafts as well as with the il- r/mhc superclusters was determined by clsm. molecular scale interactions of il- ra with il- r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il- r signaling was assessed by following the effect of cholesterol depletion on il- induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a -mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp and trp ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad , india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij and brij , at °c and °c, and compared them to drms obtained with triton x- (tx ). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx while pe content was decreased in tx drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin- and stomatin) were present in all drms. contrary to tx drms, analysis of electron paramagnetic resonance spectra (with -doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to %, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of - nm diameter and - nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of - ) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg , gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [ nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca + controlled all-or-none like recruitment of synaptotagmin- c ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin- (syt) is the major ca + sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca +-mediated membrane recruitment of the cytosolic c ab domain. the ca +-dependent recruitment of syts c ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c ab and different c ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca +] . this ca + controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t= °c; ph . ). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd , mediating ,,dynamic'' adhesion in cell rolling and icam- , mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd , icam- and the mhci is investigated on the nm-and lmdistance levels on ls t colon carcinoma cells in two different conditions of lymphocyte homing: ( ) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam- and down-regulating that of cd . ( ) crosslinking of cd and icam- representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd and icam- molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann , hetvi gandhi , remigiusz worch , , robert weinmeister , christian bö kel , and petra schwille biophysics research group, technische universitä t dresden, germany, crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only such kinases in humans (jak , , and tyk ), which associate to different cytokine receptor chains. here we study the dynamics of gfp-tagged jak and jak in epithelial cells with fluorescence correlation spectroscopy (fcs). jak and jak behave differently in various aspects: in the absence of receptors, jak still binds the membrane, whereas jak diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak to be ± nm. the association of jak with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak diffuses as a monomer in the rather small cytoplasmic pool, whereas jak diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide (hnp ) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp upon membrane binding/insertion were followed, showing that hnp have quite distinct preferences for mammalian and fungal membrane model systems. hnp showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp membrane interaction process were given by fluorescence quenching measurements using acrylamide, -doxylstearic acid ( ns) or ( ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads ( or nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly % of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the -hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. image gauge v . . the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, stanford street, london se nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb ) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from nm at temperatures below lipids phase transition to nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin- includes a short sequence containing a crac motif (v tkywfyr ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav- (v -l ) nonapeptide encompassing this motif and including the first residue of cav- hydrophobic domain (l ), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., , eur. biophys. j., , - ). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/ / . our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-( -betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of °c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at °c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at °c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica , artur wrona and olga hrydziuszko institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data ( % decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding %) we have shown that the pore radius can measure up to . nm (for a nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to . nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat and stat ) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat and stat in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat and stat . strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [ ] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol ( : : ) with or mol % of ganglioside gm , which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au ( ), although surface modification with -mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [ , ] . in this presentation, we report a detailed mechanism by which ab-( - ) fibrillizes in raft-like lipid bilayers composed of gm /cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e , e , and p ) and non-structural ones (ns , ns , ns a, ns b, ns a and ns b), the latter associated with the membrane originated from the er in the emerging virus. the ns b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns b, a detailed structure determination of this protein is very difficult. the ns b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns b is constituted by two a helices, h (approximately from amino acid to ) and h (approximately from amino acid to ), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu - -bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. , ( )). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c : ceramide) or no (c : ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a : popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability ( - ). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), ( ) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties ( ) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf w. s. aureus lipid extracts from cells grown at ph . and . , studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was * - Å less than ph . . with peptide, bilayer separations of ph . and . extracts were reduced by * Å and * Å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of / of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs Á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr residues and the pi -kinase / akt pathway specific tyr residues in nonconfluent cultures. tyr residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, , rue du loess, strasbourg cedex, f- , france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin- and - receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il- / r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft . t lymphoma cells by increasing the ec. k+ level or by blocking kv . voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il- / r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il- -induced receptor activity, as monitored by measuring stat -phosphorylation, increased upon depolarization, whereas il- induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il- r and il- r with mhc i as well as between il- r and il- r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il- /il- induced phosphorylation of stat transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il- binding and the formation of the receptor complex (il- r alpha -il- r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il- r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin- (il- ) is an important cytokine involved in adaptive immunity. il- binds with high affinity the singlepass transmembrane receptor il- ra. the occupied complex, il- /il- ra then engages either il- rc or il- ra , to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il- ra and il- ra . in contrast, type i complexes containing il- rc could not be observed. however, ectopic expression of gfp-tagged il- rc/jak induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea and rab gtpases. the il- ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il- r heterodimer may require internalization and that early endosomes serve as a platform for il- signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) ( ). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses ( - ). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a -residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the -residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm gangliosides. gm takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup (sup nm) on the mobility of gm on the plasmamembrane of living cells. preformed sup nm aggregates were incubated with cells and gm molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm were then tracked. the mobility of gm was found to decrease dramatically in the presence of sup nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm might imply a consequent loss of function of gm , thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form to . and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega , and , cex sas nanofluid and by esf project no. . understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from to lm x lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe o nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe o . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega , and , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e , e or e , of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including - - proteins. - - proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of - - (eta) with as in lbs. herein we show how - - (eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, - - (eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since - - (eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain ( - ) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m '' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta ( - ) we present an experimental study on the fibril formation of ab( - ) peptide at ph . . the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin - was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin - (amyl - ), reverse amylin - (rev-amyl - ) and scrambled amylin version scr-amyl - . the results show that for amylin - peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [ ] . md show that the x revamyl- - stack has looser selfassembly than the x amyl- - stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [ ] . the results of md show that x amyl- - could have a turn, which consists with ftir data [ ] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [ ] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor (a -lz). first, a -lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a -lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a -lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a -lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble -subunits and -subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega , , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca + pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause % leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m , is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab and ab induce aggregation and toxicity of ab while the behavior of ab is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept and smsept , which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept was able to hydrolyze gtp, whereas smsept was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of °c for smsept and °c for smsept . these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after -st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after -st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients hours after -st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from to bar), and small angle x-ray scattering (saxs) changing the temperature (between - °c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d = . - lm /s corresponding to small oligomeric forms, whereas the slow component with d = . - . lm /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h . the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § - - and - - ) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p -dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm and mcm . furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu , and its mammalian counterpart, gon l. preliminary data showed that udu and gon l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu and gon l can bind to mcm and mcm and they are localized in the nucleus. these data suggest that udu and gon l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, nanyang drive, , singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of -nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position of the histone h . exceptional importance of the h lys acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [ ] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm - ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [ ] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from cm - to cm - . ir spectra show that in ph . - . interval carbonyl (c=o) band at - cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph . significantly decreases band intensity at cm - due to unbounded c =o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in - cm - frequency interval, corresponding to o-h group in-plane bending vibration ( - cm - ). as for acidic conditions, it was observed that under the extreme ph (* . ) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at cm - and cm - corresponding to out-of-plan deformation of nh groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb / also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb , hmgb-domain recombinant proteins and linker histone h . we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb / regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h . based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy -dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh . ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm. plasmid effect on the repair of dna j. vincze , i, francia , g. vincze-tiszay hheif, budapest, hungary, univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm. plasmid on repair of single strand breaks in dna induced by cogamma irradiation in e.coli k ab (wild type) and its different rec mutant cells. the pkm. resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm. , the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm. r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k , k ), derived from the parental hybrid peptide ca( - )m( - ), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [ ] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca( - )m( - ) [ ] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg ( : ) and pope:popg( : ) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: ( ) dioleoylphosphatidylcholine (dopc); ( ) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and , -bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw together with a longrange component of the correlation energy [ ] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some % to % of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter (ctr ), which is the main copper transporter in the human cell, and the kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca + and predicts the blockade of alkali metal ion currents by micromolar ca +. conductance patterns observed in varied mixtures containing na+ and li+, or ba + and ca +, are predicted. ca + is substantially more potent in blocking na+ current than ba +. in apparent contrast to experiments sing buffered ca + solutions, the predicted potency of ca + in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca + for the pore. these experiments depend on the problematic estimation of ca + activity in solutions buffered for ca + and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca + versus na+ is always larger than the dynamical selectivity because ca + ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations . the shift of vibrational fingerprints in the - cm - frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis and compared with the present and previous experimental data . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [ ] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au( ) surfaces by means of the dft [ ] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([ %) differ only at the th position, k (mb-i) and e (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v g (jmr) and d h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng and suet-mien tan biophysics group, a*star institute of high performance computing, and, school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb tm that is absent in the better-studied platelet integrin aiibb [ ] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb within the simulation timescale as compared to its 'lossof-function' b t g mutant or aiibb [ ] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb , suggestive of tighter packing. the likely conservation of this polar interaction across the b integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [ ] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [ ] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [ ] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at to k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm -dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm [ ] [ ] [ ] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm . we have found that the allosteric effect in crm -dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm . light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin- (chr ) and channelrhodopsin- (chr ) , which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels . the n-terminal putative seven-transmembrane region of chr was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr) . as for the majority of membrane proteins, there is no d-structural data for chr available yet. here we present homology models of chr using two high-resolution x-ray template structures of br ( qhj ) respectively asr ( xio ) in order to get structural and functional insights into chr . with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs . . . comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr . here we show that the br-based homology model is a reliable model of chr exhibiting structural features already found experimen-tally . our br-based homology model of chr allows predictions of putative crucial residues within chr . so we proposed several mutations within the chr sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [ ] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [ ] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd- , a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter and its surface nanomodifications . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest , . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio surface . [ ] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin (hsaqp ) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp tetramer (pdb code d s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp tetramer. elucidating the modular structure of the protein g c fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to membrane systems spanning over to us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to * a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than compounds (us nci protocol (dtp) based on obtained directed library over new compounds, among them compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [ , , ] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [ ] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [ ] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [ ] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted ( °respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale ( - ns), however, this process may be affected by unfavorable energy barriers (from to kj mol - ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p y and p y . to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p y is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp , member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp with nd of his , which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a % w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated * such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by - %) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo u and m a . in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [ ] [ ] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [ ] [ ] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [ ] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [ ] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex ( b q) [ ] . ns md trajectories (charmm force field) were analyzed and roles of ca + and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [ ] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [ ] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechan- hiv- protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via all-atom explicit solvent molecular dynamics simulation runs of ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [ ] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [ ] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [ ] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [ ] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [ ] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [ ] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [ ] [ ] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type (hiv- ) gp and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv- entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv- integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p [ ] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [ , ] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv- integrase and block the interaction with ledgf/p . the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p and hiv- integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [ ] currently the most potent inhibitors show an in vivo ic of nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [ ] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level , . this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy - . despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [ ] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [ ] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [ ] , light exposure minimization and object tracking [ ] , or super-resolution strategies. the features and operation of the rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional ( d) volume with a video-rate scanning. we present d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than frames per second lateral scanning with fast refocusing enables the d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz , , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [ ] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated , lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [ ] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [ ] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl- protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u- mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy ( pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di- -anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye ( lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation ( min.; mw/mm ) of dye loaded tissue had no statistically significant effect on apd or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd (by %) and conduction velocity ( %). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m ) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with % saccharose, r and r with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at hours and after days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * - lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution ls) and a pixel array of spads. the spad array has a full frame time resolution of - ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional ( d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution ( . - Å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the . to Å resolution range. a recent example is our Å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex , two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at - Å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all species we investigated ( mammals, fungi, plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra . one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\ ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (* nm) as the load is increased, until it suddenly vanishes as the isometric force is reached ( . ± . pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for * % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle * residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a b ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz , pá l petrovszki , zoltá n kó ta the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at . hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the degrees rotor steps, meaning that the rotation rate of the rotor is around hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [ ] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [ ] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [ ] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [ ] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp of the c subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published in march , and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range . - pn force and - , nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin- family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin- function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin motility is exceptional in the kinesin- family. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of amino acids in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin- kip and of the spindle-organizing protein ase affected cin velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large ( nm) and one small ( nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferÒ and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # - - -a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph . and °c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg + ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* %) and b-sheet (* %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix ). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position , which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop '' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop '' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: ( ) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the human members (hspb - ), this study focused on hspb (hsp involved in some cancers), hspb (lens specific), hspb (lens, muscle, heart, lung) and hspb -r g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [ , ] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [ fe- s] cluster [ , ] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with , and kcal/mol at , and °c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with , and kcal/mol and , and °c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from to mol %), the first transition was shifted to a lower temperature value around °c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type (mfe- ) from different organisms. mfe- is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe- has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe- . mutations in the mfe- -coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe- protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura , - warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov , marko sustarsic , arnold j.m. driessen groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa /alb chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [ ] [ ] . the t ss consists of multiple copies of * different proteins (totalling * mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase (dgat ) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat binding sites (sit and sit ) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit showed to bind more strongly in nonpolar membrane models, while sit was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a nm blue shift in fluorescence emission. the binding of sit and sit peptides to negative liposome gave dissociation constants (k d ) of and . lm, respectively, and a leakage action -fold higher to sit . the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [ ] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp . this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s and s leads to the binding of the - - protein. the - - proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the - - protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the - - :pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by - - binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs at . Å resolution was solved. the stoichiometry of - - f /rgs protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between - - f and rgs protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build d model of - - f /rgs protein complex. our model revealed new details on architecture of complex formed by - - proteins. to date all known structure of - - proteins complexes suggests that the ligand is docked in the central channel of - - protein. our results indicate that the rgs domain of rgs protein is located outside the central channel of - - f protein interacting with less-conserved residues of - - f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca + ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n -( -carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas and pah genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas and pah using biophysical techniques. pah and ceas genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah and ceas were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to °c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at °c. moreover ceas and pah showed to be stable over a wide ph range (ph . - . ). we are currently working on improving ceas crystals which are a promising step towards the elucidation of the ceas structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from to nm, last for min for three scans with a nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g and later as a gap junction-like feeding tube through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha l glyr are clustered, while the alpha k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [ ] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [ ] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution d imaging and d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of nm in the plane and nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol , -bisphosphate (pi( , )p ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi( , )p in synaptic active zones is important for synaptic transmission. however, pi( , )p does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi( , )p is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi( , )p clusters and on synaptic protein-pi( , )p interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about % of the chromosome to be transferred into the forespore. the remaining (* mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * lm /s during nuclear cycles - , both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor (tlr ) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr organisation is required for the signalling process. macrophages natively express tlr at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [ ] , reversible on-off qd photoswitching [ ] , and ph sensing [ ] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [ ] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [ ] [ ] [ ] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [ ] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [ ] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [ ] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [ ] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the or amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than * nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to * nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [ ] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [ ] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a ). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected time-lapse images (time intervals min) of dual-marked a cells under three different light intensities ( . , . and . mw/cm ) and a variety of pulse lengths ( . , . , , . and ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than . mw/cm together with pulse durations longer than ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [ ] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [ ] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [ ] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [ ] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [ ] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb . fluorescence intensity f , f , f and f was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the and hours old seedlings, and bgf intensity decreased of hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße , , germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto was coupled to the halotag ligand (htl) and fast reaction of htl-atto with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for : labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than g proteincoupled receptors (gpcrs); overall, - % of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of * ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk ( ) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph , the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph , repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants ( ) ( ) ( ) ( ) . fss with a polymeric hydrophilic head proved to be mild towards mps ( ) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods ( ) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans ( ). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * k the dynamic response observed in the pico-to nano-second time regime is driven by ch dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [ ] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [ ] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [ ] , preferential exclusion [ ] and water replacement [ ] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of , -dipalmitoyl-phosphatidylcholine (dppc) in d o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & . - . . under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [ , ] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [ ] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [ ] . in recent papers [ , ] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [ , ] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa , , mikio kataoka , japan atomic energy agency, tokai, japan, juelich centre for neutron science, forschungszentrum juelich gmbh, nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, É va juhá sz, jó zsef tímá r, balá zs heged} us nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within hours and the -hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the -hour migration distance ranged from to micron and from to micron in mesothelioma and melanoma cells, respectively. the lowest -hour cell division probability was found to be . in both the melanoma and mesothelioma series while the highest proliferation activity reached . and . in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r= , ; p= , ) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n ba titin isoform as compared to short n b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße , gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p . paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp / protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp / machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal and tip in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea , the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin- motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider , marco tarantola , and andreas janshoff institute of physical chemistry, georg-august-university, gö ttingen, germany, max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor (tgf-b ) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a and mda-mb was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [ ] to fission yeast cells [ ] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea travels at the tips of growing microtubules and is delivered to the cell ends [ ] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain isoform (tmbr- ) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr- to actin was* lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by % in the presence of tmbr- . the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr- seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [ ] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a , um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [ ] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf- , associated to a low metastatic potential showed tether stiffness of pn/um in average. the second cell line, mda-mb , poorly differentiated with a high metastatic potential had a tether stiffness of pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p ) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt and histone deacetylase- , function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. we extensively characterize important liposomal properties, finding diameters between and lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp / complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal nm and br ) on the activity of mdia formin and arp / complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia -, and arp / complexmediated actin assembly. the activity of the arp / complex is inhibited by sktm and tm nm , while tmbr does not have any effect. all three tm isoforms inhibited the activity of mdia . these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with , and lg/ml of collagen type i for h and h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf insect cells) was implemented. initial expression levels were determined by either partial purification using ni ? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain - % a-helices and - % b-sheets in agreement with estimates based on homology with the ca atpase serca . we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v . membrane distribution jimé nez-garduño am , , pardo la , ortega a , stü hmer w unam, mexico-city, mexico, mpi-em, gö ttingen, germany the potassium channel k v . is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v . has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v . in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv . associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv . fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v . current density. we suggest that k v . coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [ ] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , Ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v . , ca v . and ca v . channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v . channels on-gating is shifted to more positive membrane potentials by about mv compare to ca v . and ca v . channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v . and ca v . channels compare to ca v . channels. further, more charge is moved per unit of macroscopic current amplitude in ca v . channels compare to ca v . and ca v . channels. supported by apvv- - and vega / / . the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i e of the adult rat muscle-type sodium channel (rna v . ) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i e was expressed in tsa cells and studied by means of the patch-clamp technique. when applied from the extracellular side lm qx stabilized the slow but not the fast inactivated state in i e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger isoform (nhe ), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe . we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe , these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida , - wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v . ) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v . channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w . w c, w c and w a at the outer tryptophan-rich lip, as well as d c, e c, k c and a c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a -fold inhibition loss was observed, tryptophan is crucial for the reversible blocking effects of pq. as a result, w c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm , tm ) a-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm inner helices are crossed each other near the cytoplasmic side and leu and val in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe in tm outer helices was dragged by lipids, leading to an opening of mscl. thus phe was concluded to be the major tension sensor. during opening, tm inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of * to * kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed to of * - nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length * kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length * kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was * . indicating % colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. %) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn . electrophysiological investigation of the hvdac ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. , d- gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel (hvdac ) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of - %. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ± mv and evaluation of the resulting currents. the hvdac was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y and f were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y a. in this study we provide a structural interpretation for this observation. md simulations on the y a mutant suggest side chain rearrangements of f located one helical turn below y . loss of p-p stacking induces reorientation of f from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y insensitive drugs can still favorably interact with the reoriented f aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s ) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa is very weak (apparent number of gating charges * . versus in k ? channels) and its putative voltage-sensing domain most likely lies outside the s because trpa completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa . in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p- action of double stress on photosystem aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd ? and co ? ) on activity of ps in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd ? ( - m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn -cluster or y z on donor side of ps , is inhibited stronger than at action of only co ? . the steadystate level at cd ? treatment is remain stable, while at co ? action it is increased. simultaneous action of cd ? and photoinhibitory illumination ( lmol photons m - s - ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co ? and excess light. result indicates that damage sites of action cd ? and co ? are donor and acceptor side of ps , accordingly. we assume that binding site of cd ? is y z or camn -cluster, one of the recombination partners with p ? on the donor side of ps . thereby, action of cd ions on donor side of ps leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos , z. gingl and p. maró ti department of medical physics and informatics, university of szeged, hungary, department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up with better time resolution ( ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: ) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, ) electron transfer in the reaction center, cytochrome bc complex and in between obtained from relaxation of the fluorescence and ) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at and nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [ ] and photoprotection [ ] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s state of a carotenoid [ ] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [ ] reveal that each chlorophyll is surrounded by or molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [ ] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [ ] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being : for the rfpcp and : for the hspcp [ ] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( o ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative o sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße - , d- mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q type or qequenching), an additional quenching center responsible for qi type (identical to q center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp and cp we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [ , ] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [ , , ] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence ( ). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae ( ) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated ( ). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s , s). the ratio k k sv • s /s was suggested as a measure of the functional domain size. values in the range of - were found for lhcii macroaggregates and - for native thylakoid membranes, corresponding to domain sizes of - chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r- and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from ls to s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc complex of the bacterium. in the shorter (\ ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r- were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at nm was divided into a ns and a ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. ( ) febs lett. , - ] to spectrally silent conformational changes. the nature of the ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker , szilvia z. tó th , lá szló ková cs , alfred r. holzwarth and gy} oz} o garab institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, * - % of the variable fluorescence intensity relaxes within ms in darkness and can be re-induced within ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, * % of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below - °c), where the q a -/s recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne , edward o'reilly , cecile blanchard , alexandra olaya-castro and james n. sturgis lism, cnrs and aix-marseille university, marseille, france, department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh , lh and lh . here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo , g. bencsik , g. kozak , cs. visy , z. gingl , k. hernadi , k. nagy , gy. varo and l. nagy departments of medical physics and informatics, physical chemistry and materials science, technical informatics and of, applied and environmental chemistry, university of szeged, hungary, institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th , , herbert van amerongen , , gy} oz} o garab , lá szló ková cs institute of plant biology, biological research center, hungarian academy of sciences, hungary, wageningen university, laboratory of biophysics, wageningen, the netherlands, microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp , kocp , kopsbw, kopsbx, dgd ), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan , hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. /c, h- budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl , k.yoshihara university of warmia and mazury, department of physics and biophysics, olsztyn, poland, suntory institute for bioorganic research, wakayamadai, osaka , japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with -fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h o although the reaction can occur in the presence of oxygen with lower rate [ , ] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h o as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch ) -cooh/-s-(ch ) -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h o or d o as solvent, under the variable temperature ( - k) or pressure ( - mpa) conditions, revealing the overall reorganization free energy of . ± . ev, activation volume of - . ± . cm mol - and inverse solvent kinetic isotope effect of . ± . ( °c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode ), or attached to sams through the electrostatic interaction (mode ), or specific ''wiring'' (mode ). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode ). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to -cis, -anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp , proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp to the schiff base, and reprotonation of asp from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [ ] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl to asn (l dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between cm - and cm - to histidine vibrations. these bands show the protonation of a histidine, most likely hisl , during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [ ] [ ] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [ ] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [ ] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [ ] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, -(n-ethyl-n-isopropyl)-amiloride (eipa) [ ] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [ ] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [ ] [ ] . we will present results of the first computational studies of nor (both cnor and qnor types) [ ] [ ] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [ ] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for c-type cytochromes ( ). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions ( ) ( ) ( ) . a putative outer-membrane cytochrome, omcf, is crucial for fe ? and u ? reduction and also for microbial electricity production ( ). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c of photosynthetic algae and cyanobacteria ( ). the structure of oxidized omcf was determined ( ) being the first example of a cytochrome c -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( n and c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( ( )). years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… ( )). in spite of superimposable d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers ( )…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie- and rhodobacter ferroxidans sw both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie- the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for . h, h and h. after . h, aox activity was increased only in specimens incubated in anoxic conditions ( . %). after h, increase in aox activity was significant in both hypoxic and anoxic specimens ( . % and . %, respectively), with even greater increase after h, . % for hypoxic and . % for specimens in anoxic conditions. mycelia treated for h was then oxygenated for minutes. this induced decrease in aox activity of % in anoxic and even . % in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f f -atpase on hydrogenase activity during glycerol fermentation k. trchounian , , g. sawers , a. trchounian department of biophysics, yerevan state university, yerevan, armenia, institute for microbiology, martin-luther university halle-wittenberg, haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd- , hyd- and hyd- have been well characterized. hyd- has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph . [ ] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f f -atpase. indeed, at ph . in an e. coli mutant (dk ) lacking f f overall hyd-activity was reduced to approximately % of the wild type activity; hyd- , but not hyd- , was detected in an in-gel activity assay. f f is therefore suggested to be required for hyd- activity. at ph . in glycerol medium hyd-activity in dk was * % of wild type activity and hyd- and hyd- exhibited only weak activity. this indicated a significant f f contribution towards hyd-activity as ph decreased. furthermore, at ph . hydactivity was negligible and only a very weak activity band corresponding to hyd- could be observed. these results suggest that f f -atpase is essential for hydrogenase activity during glycerol fermentation at ph . . taken together, the results suggest an interdependence between hyd- , hyd- and f f -atpase activity. [ ion channels and transporters control many facets of cancer cell biology and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology , . we have contributed to this field showing that k v . (herg ) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in ). the herg dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia and colorectal cancer , herg overexpression confers a higher malignancy to neoplastic cells. moreover, herg blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg blockers overcame chemoresistance in acute leukemias as well as reduced gi cancer growth, angiogenesis and metastatic spread . the overall message emerging from our data is that the herg protein represents a novel biomarker and drug target in oncology. up to now, herg was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg blockers that exhibit no cardiotoxicity and are more specific for the herg channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl. k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv . gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\ . mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv . gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd and anti-cd stimulation. our results indicate that the biophysical parameters of kv . gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd and anti-cd exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim ) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b -subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using -d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p ). it has been over years since sensory neuron-specific sodium channel na v . was identified. since then na v . has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v . in unmyelinated primary sensory neurons. na v . was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v . in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v . cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd complex and cd . we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv . (tea or anuroctoxin), ikca (tram- ), or the crac channel ( -apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc a ) are associated with several diseases including cancer. however, data about napi b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc a gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc a gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc a in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc a gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in , in and in spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h o and d o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf- breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for , , , and hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca ? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k p channels (twik channels) and k p channels located at the plasma membrane (trek / channels). living at the edge: volume the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca ?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca ? by extracellular ca ? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than %w tested using ll of mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd , ubq, a-, b-casein, and wt, a t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's d orientation. therefore, several techniques have been proposed in order to determine the full d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [ , , ] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in d orientation of single molecules down to a nanosecond timescales. the d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a - % higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from . (physiological) to . (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. , kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic f-labeled amino acid f-phg. using f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. : ]. here, we present novel analogs of gs with different f-prolines in the b-turn region, and with cf -bpg in place of leu. based on these f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h n /victoria/ / influenza virus strain has been submitted to various bpl inactivation conditions (from lm to mm). cell infection ability was progressively reduced and entirely abolished at mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph on gm liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, -hydroxylated fatty acid. minerval ( -hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of -hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, -hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at °c in the presence of triton x- . the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of -hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied -hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c -hpc, in mixtures of popc with and mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional ( d) counterpart of the classical stern-volmer analysis for three-dimensional ( d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of nh and oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = , , , ) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l and a . for cytotoxicity test, the cells were seeded in -well plates ml of Á cells/ml in the culture medium eagle'a or dulbecco with % calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately % of cells was taken as tccd . this work was supported by grant n n . membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre , d. arrais , j. martins , ibb -cbme, faro, portugal, dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a- , glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until . m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above . m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in , usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [ , ] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di( : ), mixed ( : )( : ), and methylated di( : - me). in order to minimize the effect of headgroup electrostatics, measurements where performed in m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from . seconds in the dope lipid to seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n and n n . many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, : - , ( )). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., , , ( )) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after days and reached % conversion in days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz , halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani , , alessandra filabozzi and giovanna fragneto dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d (small momentum-transfer diffractometer) and d (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h- budapest pazmany p. stny. /a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. ( )). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor -prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr and ccr mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain (tmd ) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr and ccr , coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr and ccr that is responsible for cholesterol requirement for cxcr and ccr conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr -using and ccr -using hiv strains. putative crac sequences involve residues v /l -y -k in cxcr and l /v /v -y -k in ccr . in cxcr , crac motif is highly conserved across chordata species, whereas in ccr , crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t and t data showed that there are two reorientation types present for every protein solutions below °c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from to residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) , . in the present work, al ? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a c and a c. the average lifetime of donor in the presence of acceptor increases with the increase of al ? concentration, indicating as adopts a more extended conformation upon al ? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al ? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from ± lm /s as monomer state to ± lm /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly % of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [ ] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [ ] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [ ] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [ , ] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase d structure. measuring hsqcs peaks intensities up to bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the residues of each mutant. so-fast hmqc experiments were also performed to measure folding and unfolding rates from bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i ) and a -residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of - gpa and - °c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p , the tumor suppressor p , and the adenovirus e a oncoprotein. the globular domains of cbp/p are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p . many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p -mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, Á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t - ., budapest, h- , hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around %. this transition is characterized by a scale-free power-law cluster size distribution with an exponent . . we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a well- we focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [ , with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes ( ), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of , and kda which were bound cgmp more intensively. after d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [ ] . helix specific interactions also cause tilt, in one direction, between two dna fragments [ ] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [ ] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [ ] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [ ] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn- and - using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [ ] [ ] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n , the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, ). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science ). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a or a -like subunit [ ] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [ ] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from . % to . %) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb , which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein (nsp ) and non-structural protein (nsp ). this hexadecameric nsp -nsp complex is a hollow, cylinder-like structure assembled from eight copies of nsp and held together by eight nsp molecules [ , ] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp -nsp form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks- with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of * - monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used nm diameter aunp loaded with rhodamine g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. nm and nm diameter aunp loaded with doxorubicin were incubated at different time points with a cell line (human adenocarcinomic alveolar basal epithelial cells). only nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\ nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with - lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of ', '-dichlorofluorescin, the effect being higher for the fenton-modified dnps after -h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [ ] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation ( - lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [ ] . upon illumination, the chromophore retinal isomerizes around the c -c double bond [ ] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [ ] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [ ] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [ ] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan ) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [ , ] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln- , p-c-met and mcl- , which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: ) fixed xenograft tissues from an established ltl castration resistance human prostate cancer or crpc model; and ) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in ) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and ) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) are already quite popular among many scientific and technological disciplines . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les- , , which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les- containing isatine fragment, activated caspase- involved in receptor-mediated apoptosis, while les- possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase- , and les- which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les- and les- were combined into one molecule. in vitro studies showed that such hybrid molecule called les- possessed times higher anticancer potential (ic = lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from ± nm till ± hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe o ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str , , berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection ( nm) vs neurite indentation ( - nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. , d- gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr is also expressed by tumor cells. first experiments show that activation of tlr displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe o -nanoparticles (nps) to stimulate tlr in a cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe o -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [ ] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [ ] . three types of colloids were prepared at various temperatures ( , and °c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of - nm. for agnps synthesized at °c, for example, a bimodal size distribution was observed (about and nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used -mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: ) quality and quantity of structural blocks of nc, and size unimodality; ) branching at specific sites in polymer chain of nc; ) providing nc with reactive chemical groups; ) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: ) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; ) dna for transfection of mammalian, yeast and bacterial cells; ) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants # , # , # . in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\ nm) is a very promising candidate [ ] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [ ] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between and nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as lm. it is generally accepted, that h s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h o . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h o . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a % delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible fold drop of br mean resistance over * cycles of interspersed illuminations at the same gold-br-gold junction when k[ nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at nm were used along with synchronized ultrafast laser pulses at nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, nm, fs, hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within * min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si- ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b f cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b f murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& . s/m), f co of control and gt-cells was found to range from to khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation , inverse emulsion techniques and more recently microfluidic techniques such as double emulsion or jetting method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than %. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range - lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d domain of flagellin subunits, comprising residues - , protrudes at the outer surface of flagellar filaments. the d domain has no significant role in the construction of the filament structure. thus, replacement of d may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g pamam dendrimer was modified with anti-tag nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. dept of chemistry, faculty of science, national university of singapore, singapore, dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll and ficoll is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of to mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, -cell and -cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of -cell and -cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly- hydroxybutyrate (phb) [ ] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at * cm - to that of cell proteins (amide ii band at * cm - ), showing a. brasilense to be able to produce phb up to over % of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d is not essential for filament formation. the concept in this project is to replace the d domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table ) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck , nk , nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated fe ions of differing energies swift heavy ion irradiation of srtio under grazing incidence'' proc. natl. acad. sci. usa forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta -adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin , , viktor foltin , , erich gornik , rumen stainov , , tanya zlateva nü rnberg. icsd e.v. postfach (pob) , d- mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. / a/jan stretch ( - mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins , . acknowledgments this work was supported by project grant ptdc/qui/ / (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: . enlargement of executive committee by a. honorary & presidents (permanent - : moral support & - fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). . implication of interdisciplinary topics to esba/iupab congressprogrammes, . also for biophysical journals. . organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. . co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals .- . could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin , , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon , zsolt má rtonfalvi , pasquale bianco , beá ta vé rtessy , mikló s kellermayer micro-viscosimeter generated and manipulated by light andrá s buzá s , lá szló oroszi , ló rá nd kelemen , pá l ormos temesvá ri krt proc. natl. acad. sci. usa neural signal recordings with a novel multisite silicon probe gergely má rton , anita pongrá cz , lá szló grand , , É va vá zsonyi pé ter pá zmá ny catholic university, faculty of information technology, h- , /a prá ter st multiscale pattern fabrication for life-science applications francesco valle , beatrice chelli , michele bianchi , eva bystrenova , marianna barbalinardo , arian shehu , tobias cramer , mauro murgia , giulia foschi miroslava kuricova , jana tulinska , aurelia liskova , eva neubauerova , maria dusinska , , ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci , jelena ban , elisabetta ruaro , massimo tormen , marco lazzarino , and vincent torre light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: ) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). ) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from . to . and the photochemical rate constant from Á s - to Á s - with an apparent halftime of about - hours after change from anaerobic to aerobic atmosphere. ) the electrogenic signal (absorption change at nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi , m. bokor , p. kamasa , p. tompa , k. tompa research institute for solid state physics and optics, hungarian academy of sciences, pob. , budapest,hungary, institute of enzymology, biological research center, hungarian academy of sciences, pob. , budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at . mhz frequency in the - °c to ? °c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below °c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers and the freely-orbiting magnetic tweezers . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench , . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then , % and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . , thus, notable variations in the rna binding specificity was observed for the divalent ions of mg ? , ca ? and mn ? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m g modification together with the principal stabilizing m a modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the -base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with -base-long indifferent gene segments for use as molecular handles, was cloned into a pet b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of - pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at * , , nm, corresponding to * , , bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert , structural biology brussels, vrije universiteit brussel, pleinlaan , brussel, belgium, department of structural biology, vib, pleinlaan , brussel, belgiumin , scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments ( kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse , osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar and ifnar . detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar and ifnar with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar / in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o- self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse , osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar and ifnar based on mvqd-trisnta were realized on live cell [ ] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u a cells stably expressing ifnar at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat , we observed the dynamic coming-and-going contact between the microcompartments of ifnar and stat .a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the . - hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph . to . . forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted . ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as . these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann , christoph schweiger , feng zhang , wolfgang. j. parak , thomas kissel ,# , pilar rivera_gil ,# biophotonics, institute of physics, philipps university of marburg, pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe o ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe o nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than % of all drugs and are encoded by about % of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) , to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin- will be presented . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is lm wide, lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [ ] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy y, n , ne- c) achieving an extremely accurate guidance [ ] . local surface topography is also known to influence the cell fate [ ] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [ , ] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio nps in concentrations: . ; and lg/cm for h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for h was suppressed significantly (p. , p. ). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p. ). no alteration was found in lymphocyte proliferation treated in vitro with tio nps for h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from to nm. on higher pillar neuronal differentiation reaches * % hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord- - jx t ol authors: palloni, alberto; mceniry, mary; huangfu, yiyue; beltran-sanchez, hiram title: impacts of the flu on survivors' nutritional status: a double quasi-natural experiment date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jx t ol robust empirical evidence supports the idea that embryonic and, more generally, intrauterine disruptions induced by the -flu pandemic had long-term consequences on adult health status and other conditions. in this paper we assess the -flu long-term effects not just of in utero exposure but also during infancy and early childhood. a unique set of events that took place in puerto rico during – generated conditions of a “double quasi-natural experiment”. we exploit these conditions to empirically identify effects of exposure to the flu pandemic and those of the devastation left by an earthquake-tsunami that struck the island in . because the earthquake-tsunami affected mostly the western coast of the island whereas early (in utero and postnatal) exposure to the flu was restricted to those born in the interval – , we use geographic variation to identify the effects of the quake and timing of birth variation to identify those of the flu. we benefit from availability of information on markers of nutritional status in a nationally representative sample of individuals aged and older in . we make two contributions. first, unlike most fetal-origins research that singles out early nutritional status as a determinant of adult health, we hypothesize that the flu damaged the nutritional status of adult survivors who, at the time of the flu, were in utero or infants. second, we target markers of nutritional status largely set when the adult survivors were infants and young children. estimates of effects of the pandemic are quite large mostly among females and those who were exposed to the earthquake-tsunami. impacts of the flu in areas less affected by the earthquake are smaller but do vary by area flu severity. these findings constitute empirical evidence supporting the conjecture that effects of the flu and/or the earthquake are associated not just with disruption experienced during the fetal period but also postnatally. the spanish flu virus of - is an example of a perfect storm: like hiv and unlike seasonal influenza, it was highly lethal but, unlike hiv and like other influenza, it was rapidly and efficiently spread [ ] [ ] [ ] [ ] [ ] . the combination of these two traits made the pandemic one of the deadliest in human history [ , ] . as in the rest of the world, the a/h n influenza in puerto rico was characterized by its unique temporal sequence, peculiar age pattern, and case morbidity and lethality [ ] [ ] [ ] . jointly, the age pattern of incidence and morbidity and mortality levels, created unfavorable conditions for all but especially for women of childbearing ages and those who were pregnant at the time or who had recently given birth [ , ] . these conditions may have compromised not just fetal growth but also infant and young children' health, both highly dependent on maternal health status and parental care. as if the onslaught of the flu had not been enough, on october of , precisely when the pandemic was gathering force during its second, most lethal wave, a strong earthquake (the san fermin earthquake) struck the western part of the island. this was immediately followed by a tsunami, two major aftershocks in a two-month interval following the earthquake, and multiple smaller ones spread over the subsequent year or so [ ] . research on the lasting effects of the pandemic are strengthened by the fact that the event can be considered a quasi-experiment: it was unexpected, difficult to avoid and, in most cases, there were no contemporaneous exogenous events that could have produced similar outcomes [ , ] . the puerto rican earthquake was also unexpected, hard to avoid in areas struck by it and, asides from the flu pandemic, unaccompanied by other major events that could have scarred even more an already vulnerable population. thus, in one stroke, an unlikely combination of two events handed us conditions of a unique double quasi experiment. this paper departs somewhat from others on effects of flu pandemic. first, it seeks to shed light on a rather unexplored dimension of the pandemic, namely, its effects on markers of nutritional status of individuals who were exposed to it in utero or during infancy. with the exception of one study [ ] , we know of no other attempt to investigate such an association. analyses of impacts on the nutritional status of flu survivors require a focus on mechanisms that could disturb physiological growth and developmental processes during infancy, early childhood and even early adolescence, not just those that operate in utero. it is known that embryonic and, more generally, intrauterine disruptions influence neural development (brain tissue), metabolic balance (pancreas, liver), nephron growth (kidneys and blood pressure regulation) or lung and heart functioning [ ] [ ] [ ] [ ] . in addition, embryonic and fetal development is also about growth of cartilage, bone, and muscle tissue, all of which are implicated in subsequent postnatal physical development [ ] . furthermore, impairment of growth processes that occur during the fetal period can be aggravated if postnatal conditions deteriorate. thus, fetal growth could be compromised when pregnant mothers experience illnesses and are exposed to extreme stress. in addition to these pre-natal mechanisms, the flu and the earthquake separately and jointly must have triggered conditions that, in all likelihood, disrupted processes of growth and development that take place during the first year of life and even later, during early childhood. with the benefit of hindsight that the covid pandemic permits, we know that this must also have been the case in as infant and child malnutrition in low-to middle -income countries today are expected to be severely affected with lasting future consequences [ ] . the social and economic aftermath of the flu must have borne close resemblance to the devastation being caused by covid and the interventions implemented to halt the spread of the virus: sharp declines in household income, increases in poverty and destitution, lack of access to social, health and other protective services, and massive disruption of food supplies and other resources. but there is more: when, due to illnesses or death, mothers cannot breastfeed normally, are unable to provide adequate maternal care, proper nutrition, grooming, and hygiene, early growth and development could go astray. furthermore, when infants and children are exposed to subsequent, lasting adverse environmental and material conditions, catch-up growth may be a non-starter [ , ] . if, in addition, an earthquake-tsunami struck some areas, the destruction there could only have been made worse. for these reasons, it is important to assess not just the flu 's impacts of in in-utero exposure, but also those generated by adverse postnatal conditions. second, we build the case on a unique quasi-experimental research design, a product of the occurrence of two simultaneous events, one involving timing of exposure (to the flu and the earthquake) and the other geography of exposure (areas with different flu severity and differentially affected by the earthquake-tsunami). we aim to show that the flu pandemic and the earthquake-tsunami combine to generate impacts that neither of these events could have produced separately and are strongly associated with both gestational and postnatal exposures. human physical growth depends on early embryonic and fetal events, maternal exposures (including stressors), maternal health status, and parental effects, including maternal capacity to nourish during the fetal and postnatal stages [ ] [ ] [ ] . of particular importance is the length and intensity of breastfeeding [ ] [ ] [ ] , protection from infections and parasitic diseases [ ] , recovery from illness [ , ] and reduction of environmental stressors [ ] . these parental effects are strongly associated with maternal (and paternal) health status, household (family) environments and access to resources. embryonic and fetal growth. by and large, fetal nutrition depends on maternal diet and placental capacity to deliver nutrients (including oxygen, fat, proteins, hormones, scfa) [ ] . it is well-known that maternal nutritional status influences the entire process of fetal development and can have strong impacts of the infant's subsequent growth [ ] . it is also known that poor maternal health status can derail the normal course of a pregnancy and complicate delivery. in particular, maternal infections during pregnancy could compromise normal fetal development and the ultimate effects depend on both the timing of infections, their intensity, and duration. these effects are associated with inflammatory responses induced by the infections themselves. in addition to the potentially fetal organogenic damage associated with the flurelated cytokine storms [ , ] , bouts of hyperthemia induced by the inflammation can also lead to deleterious outcomes, including miscarriages, premature labor, stillbirths, congenital anomalies, and growth restrictions [ ] [ ] [ ] . the latter are a result of irregularities of the physiology of bone and muscle tissues formation. as is known, bone develops from embryonic mesoderm and proceeds by ossification of cartilage tissue formed from mesenchyme. in addition to bone formation, hyperthermia can also affect limb myogenesis as it disrupts and delays the involvement of several crucial regulatory factors. jointly, dysregulation of bone and muscle tissue formation can compromise normal physical growth [ ] . early and late infant development. because of mother's milk properties, intensity and length of breastfeeding is of crucial importance for an infant's early growth, particularly during the first months of life [ , ] . aside from its beneficial nutritional properties [ ] , breastmilk contains important compounds that strengthen infants' immune response and act as a shield to reduce risks of viral, bacterial, and parasitic diseases [ ] . most infant infections and parasitic diseases reduce appetite, limit food intake and/or impair the child's nutrient absorption capabilities [ ] . thus, the combination of illnesses and breastfeeding interruption, cessation, or irregularities during the first months can compromise not only the quality and quantity of nutrients available for early growth but also reduce absorption and metabolization of those available [ ] . these disruptions compromise the ability of an organism to satisfy energetic demands to sustain rapid cell division and organ growth and formation during critical periods [ ] . although early growth faltering can be offset by subsequent catch-up growth phase, this will not take place in the absence of material conditions that can sustain rapid growth and maturation [ , ] . in populations with widespread poverty and vulnerable maternal health status, the process of catch-up growth may never get off the ground and children who could have benefitted from it will fail to attain physical growth milestones [ ] . gender differentials in early growth and development. there are important differences between female and male physical growth and development trajectories [ ] . should one expect that responses to insults are also different? why should this be the case? there are three sets of factors that can cause gender differentials, one promoting stronger effects among females and the other generating stronger effects among males. first, male embryos are known to be more vulnerable than female embryos [ ] . this implies that an early sieve operates to remove some of the vulnerable male embryos even in the absence of exogenous shocks. if, in addition, conditions under which growth and development take place turn negative during critical periods, male embryos and fetuses that could have experienced more serious post-natal developmental problems will never be born. furthermore, it is well-known that male infants experience higher mortality than female infants [ ] . excess infant mortality can operate as a second sieve disproportionally selecting out more frail male births. thus, it is quite possible that selection forces alone result in stronger effects among females who are, on average, subject to weaker early selection pressures. however, a second set of factors could amplify effects among males and attenuate them among females. in many low-to middle-income countries, in the past and even more recently, there was pervasive male child preferences that, despite not resulting in outright child neglect, biased the flow of scarce parental resources toward male children [ , ] . it is plausible to conjecture that under added sources of stress induced by exogenous shocks, male child preferences could have more dire consequences among females than during normal periods, particularly among girls who are more vulnerable to begin with. this is a source of selection that should attenuate effects of early insults among females since those who survive them are of lower than average frailty. finally, an alternative explanation that does not rely on selection arguments has to do with gender differentials in growth and development. it has been known for quite some time [ ] that male physical growth stretches over a longer period of time than female physical growth. this increases males' window of opportunity for replenishment and recovery from past interruptions of development and growth. if this is so, males who survive to adulthood could have benefited from these added opportunities and be less likely to carry through life scars associated with very early insults. given the nature of our data, it is impossible to sort out between these various forces that combine to produce observed gender differential in responses to the flu and earthquake. long lasting effects of the flu. the above considerations lead us to hypothesize that exposure to the flu during two critical periods, e.g in utero and/or during infancy, must have had non-negligible influences on early nutritional status and should be reflected in poor adult markers of physical growth. by the same token, exposure to stresses and material deprivation brought about by the earthquake-tsunami could have disrupted embryonic, fetal and postnatal growth and, as consequence, facilitated growth faltering and attainment of substandard markers of physical growth. furthermore, as did happen in other populations, the effects of the flu are likely to be stronger among those who experienced the pandemic in areas more severely affected by it [ , ] . finally, both in utero and postnatal vulnerability to the flu was likely augmented by conditions associated with the earthquake [ ] . if so, we should find that the impact of the flu among individuals exposed to the pandemic in utero or during the first year of life) and individuals exposed later in childhood or adolescence) is larger among those born in areas struck by the earthquake-tsunami (e.g. exposed to the earthquake) than among those born elsewhere in the island. to test these hypotheses, we use a study design that relies on geographic identification of local areas (municipio of birth) classified by flu and earthquake severity. we use dates of birth to assess exposure during critical periods. table is a representation of the design. we use prehco (puerto rican elderly health conditions) data base [ ] . prehco is a twowave panel of the non-institutionalized puerto rican population aged table . puerto rican birth cohorts born before , were exposed to mortality experiences similar to those in latin american countries. their infant and early child mortality levels correspond to life tables with life expectancies at birth not exceeding years and are in the range of - per , births. up until and during their late adolescence and early adulthood, these birth cohorts' mortality conditions are embedded in life tables with life expectancy at birth of around - years. mortality improvements relative to early infancy and early childhood largely originate in eradication of parasitic and vector born infectious diseases (period - ) as well as to increasing standards of living that marked the period - . finally, their post- mortality experience is heavily influenced by the introduction of (ii) the severity of the earthquake is gauged by distance to epicenter and we classify municipios as affected (high severity of earthquake) and non-affected (low severity of earthquake) according to distance from epicenter (see methods section). (iii) the exposure contrast between cells a-d, on one hand, and cells e-h, on the other, is associated with a cohort' s timing of birth and the conditions to which they were exposed during two critical period (in utero and infancy). birth cohorts belonging to these cells are at risk of developing growth problems whereas those belonging to cells e-h are not. the contrast between cells a and b, on one hand, and c and d, on the other, is associated with severity of the event, namely, severity of the flu and "severity" of earthquake (municipios that suffer the earthquake vs municipios that were less affected or not affected at all). there are also contrasts between birth cohorts that belong to mixtures of municipios of type a or b and c or d. thus, those exposed individuals belonging to cohorts born in municipios that are simultaneously of type b and d are expected to suffer the most. we expect no such contrasts across birth cohorts born in municipios of type e-h. https://doi.org/ . /journal.pone. .t chemotherapy (antibiotics, sulfa) and vaccinations as well as by increased prevalence of modern chronic illnesses, including type ii diabetes, heart disease and cancer [ ] life expectancy at birth between the years and , the period during which prehco was fielded, was of the order of - . these cohorts' life expectancy at ages is of the order of - years. in summary, the birth cohorts we study experienced highly heterogeneous mortality conditions throughout their lifetime, ranging from severe during their early childhood years to mild and lenient in later life. flu exposure. in contrast to other studies of the flu, we identify a wider period during which exposure is assumed to have taken place and, in addition to the fetal period, include time intervals during which post-natal care may have been disrupted as a consequence of the epidemic and/or the earthquake. if, as it is most likely, the post-natal mechanisms are also relevant for outcomes other than physical growth markers (adult health, mortality, cognition, educational attainment, etc. . .), studies that ignore them will underestimate the total effects when using a restrictive definition of exposure for some "treated" cases will be assumed to be "controls". to capture the extended exposure including gestational and post-natal exposures we define a dummy variable attaining the value if an individual's birth is reported to have taken place during or during the first six months of (see appendix for alternative definitions of exposure). this indicator is a compromise between preservation of the ability to assign effects to fetal and post-natal exposure according to timing and duration and sample size constraints. flu severity. because we lack information on the incidence and case fatality of the pandemic in puerto rico, we follow past research and create a proxy indicator of flu severity using the excess total mortality registered during the flu period [ ] . to construct an index of severity we consider total mortality during the two years period - for each of the municipios, the smallest administrative units in puerto rico, estimate expected deaths using age-specific mortality rates during in the us, and then compute the ratio of mortality rate observed in a municipio to the observed rate [ ] . the resulting quantity is an indirectly standardized mortality ratio, a conventional index computed when information of age specific death rates is absent. we classify as high severity all municipios above the th centile of the severity index distribution. those between the th and th centiles are classified as intermediate severity and the remaining as low severity (see s fig) . earthquake-tsunami exposure. exposure to earthquake-tsunami is assessed according to the timing of birth of a cohort and municipio of birth. we consider that a birth cohort's early conditions may have been affected by the earthquake if the timing of birth falls within the same window defined as critical for the flu (see above). thus, to capture extended exposure including gestational and post-natal exposures we use the same dummy defined to flag flu exposure. earthquake severity. we classify municipios of birth into three groups depending on the severity of the earthquake: (i) most severe (all municipios in the west coast of puerto rico, (ii) mild (all municipios to the east and immediately adjacent to those classified as severe and (iii) not severe, (the remaining municipios) (see s e table for municipio membership by classes in what follows we use a / dummy variable to flag municipios in group (i). municipio poverty levels. to assess poverty levels in municipios of birth we adopt the classification constructed by clark [ ] . municipios were grouped into three classes according to their population size, assessed value, and government income. a total of municipios are plos one either in the wealthiest or an intermediate class and the remaining ones are in the poorest category. in this paper we use a / binary indicator to contrast the poorest and the remaining municipios. when using this indicator in models with no fixed effects we verify that the indicator behaves as expected, namely, it is strongly and negatively correlated with both knee height and height. the models we discuss in this paper, however, only rely on a fixed effects formulation and the impact on municipio of birth poverty levels is absorbed by the fixed effects. thus, an important set of confounders associated with early poverty are neutralized in the fixed effect model. markers of early nutritional status: knee height and adjusted height. we use pre-hco's anthropometry module for the assessment of height and knee height [ ] . to attenuate biases due to skeletal compression, we adjust height measures using estimates of compression by gender and age observed in a sample of individuals who were followed for a long period of time (see s fig) [ ] . the magnitude of the adjustments is considerable and, if anything, they will lead to overcorrection and downward biases of effects on height. on the other hand, knee height is also a marker of early nutritional status and is unaffected by skeletal compression. other outcomes are frequently studied in the literature on the pandemic. among them is bmi. we do not examine these here since our interest is on markers of early nutritional status and neither bmi nor any of many indicators available in the survey are suitable. we use ols regressions with municipio fixed effects and treat adjusted height and knee height as continuous variables. we also estimated sur, models to account for correlation between knee height and height. results from these models are available upon request. although there are some differences between sur and ols estimates (standard errors of regression coefficients), the differences are minor and do not affect inferences. we also estimated biprobit models using binary indicators for knee height and height. the drawback of these models is that they depend on arbitrary cut points. in all cases we use our preferred measures of exposures, namely, exposure = to flag birth cohorts born during the critical period defined before and to distinguish them from birth cohorts born before and after such critical period (exposure = ). we consider severity of flu using two alternative indicators. the first is a single dummy variable (severity_flu) that distinguishes between municipios where the flu is considered to be severe from those in which it was mild. the second consists of two dummies that identify municipios with severe and intermediate severity. a dummy variable, severity_earth, serves to distinguish municipios which were severely affected by the earthquake from all others. in addition, the model contains first, second and third order interaction effects. the specification for outcome k (k = for knee height and k = for height) is where z ki is the outcome k for individual i. c i is a vector of control variables that includes years of education, year of birth, and a dummy variable for gender. this regression formulation is a difference-in-difference model that seeks to identify (i) differences in the impact of the flu by timing of exposure between high and low flu severity areas and (ii) differences in the impact of the earthquake by timing of exposure between areas affected by high and low severity of the earthquake-tsunami and, finally, differences in the effect of exposure between areas affected by both high severity of flu an earthquake and those only impacted by one of these. exposure i is a / variable for exposure to flu/earthquake. severity_flu i is a / variable for flu severity in the municipio of birth; in some models we used a more fine-tuned classification and employed two dummies to distinguish municipios with high, moderate(intermediate) and low severity. severity_earth i is a / variable for earthquake severity in the municipio of birth, and ε ki is an idiosyncratic error. in turn, the parameters for the equation of outcome j are a constant, α k , and a vector of effects associated with controls, β k . the effect of exposure, γ k , reflects the combined impacts of flu and earthquake exposure for cohorts born during the critical period. the parameter θ k measures the difference of effects of flu exposure between those born in high and low severity municipios whereas the difference of effects of exposure between those born in municipios affected by the earthquake and those born in the remaining municipios is, δ k. finally, μ k is a measure of the excess impact of exposure during the critical period among individuals born in municipios with high severity flu and affected by the earthquake relative to those also exposed but in areas of low flu severity and not affected by the earthquake. to assess differentials by gender we also introduce interaction terms involving the / binary variable for gender (female = for females), timing of exposure, and flu and earthquake severity. a few remarks are important. first, our models do not include a control for age as there is no relation between markers of nutritional status and age. second, additional controls were tried but discarded since they did not change results. third, all our initial models included a linear term for birth year to account for secular trends in height and knee height as well as to purge out effects of other time variant factors related to birth cohorts. since estimates from models that did include these covariates are no different from those that did not, we only discuss results from the latter. fourth, we estimate models with municipio fixed effects. this implies that the main effects of earthquake and flu severity, as well as other variables at the municipio level, are absorbed by the fixed effects. finally, we estimate models with adjusted variance-covariance matrices of errors to relax assumption of independence of observations and requiring only. model justification and interpretation. estimates from the above model can be interpreted causally only if some conditions are satisfied. first, other than the earthquake and flu, there are no concurrent phenomena that could influence outcomes. second, the occurrence of earthquake and flu are uncorrelated. third, unmeasured cohort conditions that influence the outcomes of interest vary continuously between cohorts. finally, selection due to differential survival among those exposed and non-exposed is minor. the first condition is likely to be met since, to our knowledge at least, other than being very marginally affected by military mobilization and disruptions caused by wwi, puerto rico was not affected by other simultaneous large-scale disruption. the second condition is also likely to be met. however, our assessment of flu severity relies on estimated excess deaths during the flu period. because the earthquake also contributed to excess deaths, at least in areas severely affected by it, the severity indicator could introduce a correlation between the two events. since in most of our analysis we rely on a coarse binary indicator for severity, this correlation is unlikely to influence results. the third condition refers to the possibility than unmeasured conditions associated with the nutritional status of cohorts born at the time of the flu and earthquake, changed abruptly as a consequence of these shocks themselves and/or some concurrent event. for example, if draftees to wwi armies were disproportionately drawn from middle income classes, the composition of births by class origin during that period suddenly changed. since social class of origin is strongly associated with nutritional status, these birth cohorts would be composed by individuals who are at higher risk of manifesting deficits in adult markers, independently of the flu and the earthquake. but, the population of puerto rican wwi draftees from middle to high classes in us armies must have been insignificant since the total number of puerto rican recruits was in the hundreds, not thousands. the final condition is that there is no selection due to differential survival of individual exposed and non-exposed to exogenous shocks. because conditions that determine poor early nutritional status also increase child and adult mortality risks, it is quite likely that selection in our sample of older adults will induce to downward biases on estimates of effects of exposure to flu and earthquake. in s file we provide a rough assessment of the potential magnitude of these biases. we first discuss results from baseline models for knee height and height that only include exposure during the critical period (and years of education as a control variable). we then examine whether there are differences in the effects of exposure across areas with contrasting flu severity. this is followed by a review of models that also include a dummy variable for earthquake and higher order interaction effects. these models enable us to assess whether the pandemic and the earthquake combine to induce impacts of larger magnitude than either event separately. individual regression coefficients are displayed in tables - (table is a compact summarizes of estimates of total (net) effects.) the flu: models for knee height and height including exposure during critical period and flu severity baseline models. table displays estimates of coefficients for models with knee height and height as dependent variables, control variables, and the additive and interaction effect of gender. as assessed by the overall value of r-square, the model for height fits much better than the one for knee height (. vs . ). the reason for this is that gender is a much stronger predictor of height than of knee height. in any case, the goodness of fit of these models is quite good for this kind of individual level data. note that the coefficients for the single control variable, years of education, is very large and in the expected direction. this result is reassuring because it confirms that educational attainment reflects early conditions that influence markers of nutritional status. note that because individual adult educational attainment itself could be influenced by the pandemic, the relation between education and markers of nutritional status could also be spurious. second, the effects of exposure during the critical period (exposure) are in the expected direction although it is only statistically significant in the model for knee height where the magnitude of the coefficient is close to twice the standard error. the reduction in knee height among those exposed during the critical window is about . cms or percent of the mean value in the sample and the corresponding regression coefficient is marginally significant (- . (. )). the magnitude of this effect is slightly smaller than a third of the total effect of gender (- . ), the covariate that has the largest effect in the model. it should be kept in mind that this estimate mixes the impact of exposure to both flu and earthquake. the total reduction in knee height among exposed females is . (- . +(-. )) and an f-test reveals this is significant at the . level. third, the model for height reveals a gender differential that penalizes females: the reduction in height among exposed females is of the order of . cms, about percent of the mean value in the sample, with a coefficient about twice its standard error. an f-test confirms that the total effect exposure on females' height is different from (pr f o > f � = . is . ). thus, exposure during critical period leads to losses of knee height and height but gender differentials in these losses are only manifested in adjusted height. models with flu severity. table displays only estimates of parameters of interest from models that include the interaction term between exposure and flu severity as well as those associated with the gender variable. as in the previous case, the model does not control for earthquake and the effect associated with the variable exposure absorbs both the impact of the flu and the earthquake. three results stand out. the additive effect of exposure on knee height is properly signed and slightly larger than in the baseline model (- . (. )) whereas the added impact on females is close to zero (. ( . )). the effect of exposure is larger in the hardest hit regions and leads to a decrease of about . cms but the regression coefficient does not pass a standard statistical test (-. ( . ). third, the impact among exposed females in high severity area is weak (-. ( . )) and statistically insignificant. the model for height leads to somewhat different inferences, however. as in the previous case the additive effect of exposure is small but, unlike the model for knee height, there is an important gender differential as exposed females lose about cms (about percent of the mean) and the regression coefficient estimate is more than twice its standard error (- . ( . )). in fact, an f-test reveals that the total impact among females (the additive effect of exposure and the interaction between exposure and gender) is statistically different from (pr f o > f � = . is . ). however, there is no evidence of a contrast between impact of exposure in the total population (- . ( . )) nor among females ( . ( . )). fig a and b display box-plots of predicted values of knee height and height for exposed and non-exposed individuals born in high and low flu severity municipios using coefficients from table . the gradient between exposed and non-exposed and between those born in different flu severity municipios is apparent for knee height and of the order of to cms. the contrasts are in the expected direction for height but are of smaller magnitudes. because we find important differentials by gender, we also estimated a model with the female subsample only. although we lose some statistical power, we gain somewhat by reducing the confounding impact of heterogeneity of effects by gender. furthermore, to fine-tune the indicator for flu severity we use all three classes of severity (small, moderate and severe). table displays the new models' estimates. these suggest somewhat stronger inferences than allowed by the previous model. in fact, although the additive effects of exposure on knee height is correctly signed but small, the interaction effect associated with the most severely affected municipios are quite large and more than twice their standard errors (- . (. )). exposed females in areas of high severity lost a total of nearly . cms in knee height or about percent of the mean in the sample. the f-test indicates that the total effect of flu exposure is statistically significant (prf o >f � = . is . ). the results for height are even stronger: exposed females in high severity areas lost a total of . cms (- . + (- . )) or about percent of the mean in the sample and both the additive and interaction coefficients are statistically significant. as in the case of knee height, the f test for the total impact on height confirms that the overall impact is important (prf o > f � = . is . ). fig a and b display plots of predicted values of knee height and height by flu severity using coefficients from table . the gradients are sharp and suggest losses of about . to cms of knee height and to cms of height among those exposed in high flu severity areas (relative to those born in low flu severity areas). contrasts are large (between and cms of knee height and and cms in height) between those exposed and not exposed. the observed patterns identified above can be summarized as follows: had we been interested only on the impact of the flu and ignored the concurrent earthquake-tsunami, we would have stopped the analysis here. our conclusion would have been that females bore the brunt of the flu pandemic and that those born in municipios hardest hit by the flu experienced damage in both phenotypes of interest as they lose as much as to cms of knee height and - cms of height. however, during the period that the pandemic prevailed in the island, and coinciding with its peak strength, a strong earthquake-tsunami castigated the west costal and proximate interior region of the island. it is then possible that inferences from models that ignore the earthquake attribute to the flu (in high and low severity areas) impacts that might be attributable to the earthquake. this is an example where the joint occurrence of two exogenous shocks, the flu and earthquake, violates one of the prerequisites needed to justify treating the pandemic as a quasi-natural experiment. the only escape from this trap is to estimate models that account for earthquake severity. could the estimated influenza effects be confounded with impacts of the earthquake? were the effects of the flu magnified in areas affected by the earthquake or those of the earthquake magnified in areas of high flu severity? to answer these questions, we estimated models including the additive and interactive effects of the earthquake. we seek to determine whether (i) there are independent effects of the flu and the earthquake and their relative magnitudes, (ii) there are important synergies between the two events, and (iii) females were singularly affected. table shows results of the full models that include exposure, flu severity, and earthquake severity as well as interaction effects associated with gender. because the introduction of a new dummy for earthquake and associated interaction effects leads to substantial losses of observations in some cells of the data, we return to use the binary variable for severity of earthquake as identified in table. the results for knee height are somewhat mixed but they shed light on an intriguing story and are summarized below. the results for height, on the other hand, are weaker although they too reveal important regularities. knee height. does exposure during critical windows make a difference? the additive effect of exposure in the model for knee height is properly signed but significant only at . level (-. (. )). in contrast, exposure during critical windows among individuals born in municipios more severely affected by the earthquake translates into sizeable and significant effects independently of gender. the estimate of the corresponding regression coefficient implies knee height reductions of the order of . cms and the associated regression coefficient is significant at the . level. however, exposure in high flu severity areas does not appear to lead to important reductions in knee height as the estimated effect is negative, as expected, but does not attain statistical significance (- . ( . )). similarly, exposure during critical periods in municipios of high flu severity and also affected by the earthquake does not result in particularly large negative impact. in fact, the opposite appears to be the case as the additive effect of exposure during critical periods in areas affected by the earthquake and with high flu severity is positive albeit with a large standard error ( . ( . )). the final problem we investigate is whether the above patterns are different for females. exposed females in the general population are not affected ( . ( . )). nor are those exposed and born in earthquake municipios ( . ( . )) or exposed and born in high flu severity areas ( . ) ). however, exposed females born in areas struck by the earthquake and of high flu severity are singularly affected. in fact, we find a sizeable and significant impact among females exposed during the critical period who were born on earthquake and high flu severity areas. the estimate of effects for these females implies a reduction of knee height of about - . cms, a figure that is statistically significant despite a large standard error. because the additive effect of exposure in areas of high flu severity and affected by the earthquake implies an average increase in knee height of about . , albeit with a large standard error ( . ), the total effect among females is negative and leads to a loss of . cms. to check the importance of these effects we employ two f-tests. the first corresponds to the null hypothesis that the magnitude of the overall effects of the earthquake and the flu among females is zero, that is, the sum of the effects females' experience by virtue of being exposed during critical periods, being born in areas affected by the earthquake, and with high flu severity is not significantly different from . the observed total or net impact ( . ) yields an f = . (prf o > . is . ). the second test seeks to verify the null hypothesis that the difference between effects of females exposed during critical windows and those not exposed is . the observed value of the test statistic is equal to - . among females exposed and -. among those not exposed. the difference translate into an f statistic ( . ) that would be observed in a population where the difference is zero with a probability of less that . . these two tests offer empirical evidence confirming that females whose exposure during critical windows took place in areas of high flu severity and affected by the earthquake are most damaged by these events. fig a and b are box plots of predicted values of knee height for selected categories of exposed and non-exposed individuals who experienced combinations of the worst conditions. although there is important within-category variability (particularly in the worst one, namely, among individuals exposed in high earthquake and flu severity areas) the between category gradients are in accordance with the conjectures. height. the results for height are weaker than those for knee height even for females. exposure during critical periods by itself does not influence height (. ( . )) nor does it whether it is combined with the earthquake (. ( . )) or high flu severity areas (- . ( . )) or both ( . ( . )). the last two regression coefficients are large but very noisy, possibly due to small number of cases. as was the case for knee height, exposed females are more affected as they lose . cms and the corresponding regression coefficient is marginally significant. females who were exposed in earthquake areas experience reductions in height but the estimate is too noisy (- . ( . )). the effect on females who were exposed in high severity areas appear to gain in height but, here again, the regression coefficient is subject to quite a bit of uncertainty (or in high flu severity municipios ( . ( . ))). finally, and unlike the case of knee height, height reduction among exposed females born in earthquake and high severity areas is large but only half its standard error (- . ( . )). as in the case of knee height, however, the total impact on exposed females is equivalent to a total loss of about . cms, and statistically significant (prf o >f = . ). the difference between impacts on females exposed and those not exposed is . cms (prf o > f = . ) the above results suggest four inferences. first, the flu pandemic alone had important impacts on knee height and height but only among females born in high severity flu areas. second, the earthquake-tsunami and the flu pandemic combined to induce damage but mostly on females' knee height and somewhat less on females' height. the net effects on knee height are sizeable, translate into reductions of the order of . cms or about percent of the mean value of knee height in the sample, and are statistically significant from . the net effects on height are also statistically significant and equivalent to losses of cms representing about percent of the mean value in the sample. the total effects of female exposure on knee height are two to three times larger among those who were born in municipios with high flu severity and affected by the earthquake. table is a synthetic representation of key results for the female subpopulation. the table includes a brief summary of main findings (columns and ) and estimates of total effects and associated f-statistics (columns and ). yellow cells represent findings consistent with initial conjectures. green cells are associated with weaker evidence and grey cells with little or no empirical support. an examination of yellow cells suggests that female exposure during the critical period led to total losses in knee height and height of the order . and . cms respectively (first row of and last two columns of table). exposure within high flu severity areas increases these losses to . a . cms (fifth row and last two columns of the table). finally, when exposure takes place in areas hit hard by both the earthquake and the flu losses increase to about . and . cms respectively (next to last row and last two columns). a cautionary note on interpretation. the impacts in table are not trivial and, given the definition of exposure we use here, they are consistent with the idea that it is the combination of perturbations during the fetal and the postnatal period that mediate the impacts of the two external shocks that struck this population. these effects are larger than those one gets when using an exposure variable that constrains the critical window to the fetal period only. but, how strongly can we lean on this interpretation of findings? although the empirical analysis presented above is suggestive and consistent with the initial conjecture there are alternative interpretations. human markers of physical growth and development, other than those assessed at birth, are shaped by a multiplicity of factors, from genetic make-up to pre-pregnancy maternal health status to exposures in utero, to conditions encountered in infancy, early childhood and adolescence, and to adult and older adult experiences. even though the two markers of nutritional status we use here, height and knee height, measure phenotypes whose growth and development dynamics are subject to important constraints that are set early in life, they are also shaped by conditions experienced after the narrow window we flagged here as critical. furthermore, these conditions are directly or indirectly related to the two exogenous events of interest. fig is a rendition of the main relations involved. the figure is designed to emphasize that the way to secure evidence supporting a strong interpretation that attributes effects of external events on adverse early conditions only is to control for "other factors" also influenced by the events. although the effects of the latter could very well be accounted for by individual measures (education) and contextual indicators (municipio poverty index), all of which are included in our models, they may be insufficient to purge effects represented by the path in the lower part of fig . childhood stunting induced by a protracted period of poverty following the earthquake may derail physical growth permanently. if so, the effects we estimate cannot be solely imputed to exposure during the early critical window we focus on here. but, by the same token, catch-up growth in early adolescence, years after the event of interest took place, could conceal effects of exposures in the critical window. these two sets of effects will offset each other, and we will not be able to rank them in importance. for this reason, we argue that it is inappropriate to downplay the mediating role of early conditions. the two markers of physical growth, but less so knee height than height, are influenced by heterogeneous conditions that are experienced in very different windows of time during fetal development (particularly during the last three months of pregnancy), infancy, early childhood and adolescence. because they involve biological processes that unfold at different stages, they are not perfectly correlated. in fact, the correlation between these two measures ranges from . to . [ ] [ ] [ ] . also, it is known that birthweight is correlated with adult height and that this association is partially explained by intrauterine conditions and genetic factors [ ] . although we know of no study confirming this, it must be the case that knee height is also highly correlated with birth weight and, as height, is influenced by very early conditions, e.g. before birth, as well. to capture these processes a more complex model is needed, one that includes measures of exposure in multiple critical periods thus allowing room for an assessment of time-varying effects on single metrics of nutritional status as well as effects heterogeneity across metrics. we conclude that even if we cannot attribute in toto the estimated effects to mediating paths only involving very early conditions, the evidence gathered suggests that these conditions are important contributors to markers of nutritional status. robustness of findings: systematic errors, small sample sizes, the role of chance and selection. although estimates of effects on knee height are, by conventional standards, "statistically significant" (at levels p < . or less), we should provide additional empirical evidence to support our inferences. we do this for three related reasons. the first is that the estimates could be contaminated by systematic measurement of errors. the second is that they are based on a small sample from which the model we estimate demand quite a lot. the third is that the results may be due to chance. systematic errors. we conducted two tests to assess possibility of systematic underestimation of knee height among those exposed to the flu and born in areas impacted by the earthquake. first, the distribution of knee height shows no deviant extreme cases and the smallest values in earthquake areas are within . of a standard deviation. second, in a more radical test we ignored the lowest values of knee height and re-estimated models. to turn estimated effects from worthy of note (p < [. -. ]) to mundane (p>. ) we need to exclude observations of knee height below the first quartile of the distribution, a rather implausible surgery. the role of chance. most epidemiological and population health research highlights findings on the basis of classic-fisher criteria, that is, based on a priori chosen significance level (say α < . in a two-tailed test). the analysis we carried out was couched on this model as we highlighted results that would pass standard statistical tests with α < . in two-tailed test. we are saying nothing new when we point out that this type of criterion can be misleading. this is of concern as extreme values of a statistic can be obtained just by chance and are more likely to occur with small samples and demanding models such as ours. to assess this possibility, we pursue two routes: (a) perform a permutation test [ ] and (b) compute bounds for false discovery rates [ ] . (a) permutation test: we implement permutation tests on estimates of effects obtained in the most complex model that includes third order interaction effects (female exposure x severity x earthquake). the objective is to add some strength to inferences drawn from conventional hypothesis testing, namely, that the effects on knee height (and height) of the size we observe occur with small probabilities, ideally below . . this exercise suggests that in a permutation repeated times the coefficient of the most extreme of exposure (in high flu severity and earthquake) would be lower than what we observe between and percent of the time. this is a bit less stringent and provides weaker support than the conventional test statistic (see s fig) . (b) false discovery rate: this is the conditional probability that if the null hypothesis is rejected, it is erroneously rejected. this quantity is usually quite different from the conventional α as it is a function of α, power, and the true magnitude of effects. alternative values of these parameters lead to the graph s fig. given our p-values ([. -. ]) and approximate power (. -. ), we conclude that the probability of uncovering effects only by chance is between . and . , hardly a comforting range but quite common in clinical and population studies [ ] . selection effects. it is highly likely that the sample of survivors to age is highly selected and that selection could have been a function of nutritional status and, therefore, a partial result of the events of interest. prehco is not unlike other survey of elderly people, all of which confront this problem. we argued before that these biases are in all likelihood attenuating estimates of effects. in s file we develop simple expressions to evaluate the potential magnitude of these biases and conclude that, under conditions prevailing in puerto rico, we might be underestimating effects by as much as to percent. is the magnitude of the flu effects relevant? while empirical findings confirm that flu exposure, in the broadest sense defined here, is associated with markers of early nutritional status, it is unclear whether their magnitude is substantively meaningful. to shed some light on this issue we proceed indirectly and compare predicted changes in individual stature associated with flu exposure with changes in stature throughout the period of mortality decline in western europe. because knee height appears to be the metric most sensitive to exposure, an ideal test would have been to use the estimated changes in knee height we observe here as a result of exposure with those experienced by other populations. because there are no historical records of knee height, we cannot perform this test. however, we can exploit the fact that knee height is moderately associated with adjusted height (r-squared~. in our sample) and still draw tentative inferences by predicting changes in height from estimated changes in knee height. we find that the reduction in height implied by the estimated reduction in knee height due to flu exposure (between . and . cms for an average of . of the sample's knee height standard deviation) is associated with a proportionate adjusted height reduction of about . (based on a log-log regression of adjusted height on knee height). note that it took forty years, between and , for the mean height of the dutch population to experience proportionate gains that are half as large as the losses induced by the flu and earthquake. a similar inference follows from a comparative assessment of the average reduction in height associated with exposure during critical periods in high flu and earthquake severity areas. as an added piece of evidence consider this: an increase of cm in knee height in our sample translates into a decrease in mortality risks above age of the order of %. this, in turn, translates into an increase in life expectancy at that age from . to . , about percent (calculated using life tables for females in model west of the coale-demeny female life table system using levels and ). is the evidence retrieved here strong enough to support the argument the impact of the flu and earthquake may operate outside the narrow window of fetal exposure? we suggested that past research on the long-run effects of the influenza may be somewhat limited by a preoccupation with fetal exposure. this is justified since there is strong evidence supporting the idea that embryonic and, more generally, intrauterine disruptions are influential. however, fetal development is also about growth of cartilage, bone and muscle tissue, all of which are implicated in subsequent postnatal physical development. furthermore, impairments in the fetal period can only be aggravated if post-natal conditions are unfavorable, as may happen as a consequence of maternal or paternal illness. this justifies our claim that the flu pandemic could have also perturbed the post-natal period and through both, fetal and postnatal exposures, affected children's nutritional status. our estimates, particularly those for females born in high severity areas and/or in areas impacted by the earthquake-tsunami, are large, statistically "relevant", and robust to checks. this evidence does not imply that fetal exposure is irrelevant but that it, together with postnatal conditions, may combine in a highly poisonous cocktail that impedes attainment of physical growth landmarks. the paper has shortcomings. first, an ideal test of our hypotheses requires to contrast effects in multiple windows of exposure (fetal, infancy, and early adolescence). although we did define alternative critical windows (see s -s tables) model estimation using more than one critical window became quite difficult due data sparsity. second, while both nutritional status markers are sensitive to the exogenous shocks, one is more so than the other. unlike knee height, adjusted height is more likely to be influenced by measurement errors and by events that take place in windows of time more removed from infancy and early childhood. it is difficult to tell whether the differential responses of these markers is as a result of errors or due to real differences in physiological processes that underpin development of different parts of the human body. third, the sample is small and vulnerable to produce effects where there are weak ones. unlike other research, we are not dealing with observations in the tens of thousands but with an effective sample size orders of magnitude below that. buffering against this possibility with permutation tests and computations of false discovery rates can only suggest but not prove that our results are immune to false discovery. fourth, for the most part our results only offer support for effects on females, not on the entire population. although we propose conjectures that could explain gender differentials, we have no way of testing them with the data at hand. finally, and on a different note, puerto rico is a tiny dot in a world map, with a population size that has always been, then and now, an infinitesimal fraction of the total world population. why would anybody bother with all of this? it turns out that the unlikely collusion of two simultaneous natural disasters and the accidental availability of empirical records of survivors, generates a unique opportunity to identify effects of broadly defined early exposures to shocks. in particular, we uncovered some evidence suggesting that past research on the impacts of the flu pandemic may have missed something important: the influence of the combined disruption of fetal and postnatal life on the ultimate fate of subsequent physical growth. we are not so much trumpeting a new finding as we are identifying a relation that deserves a second look in future research on the pandemic or, importantly, on the longlasting effects of the covid pandemic. supporting information s the long-lasting influenza: the impact of fetal stress during the influenza pandemic on socioeconomic attainment and health in sweden from two mutations, an important clue about the spanish flu death patterns during the influenza pandemic in chile estimating reproduction numbers for the - and - influenza pandemics in the city of madrid effects of the spanish influenza pandemic of - on later life mortality of norwegian cohorts born about mortality patterns associated with the influenza pandemic in mexico: evidence for a spring herald wave and lack of preexisting immunity in older populations updating the accounts: global mortality of the - "spanish" influenza pandemic the long-lasting influenza: the impact of fetal stress during the influenza pandemic on socioeconomic attainment and health in sweden the long-term consequences of the global influenza pandemic: a systematic analysis of ipums international census data sets spanish flu and early th-century expansion of a coronary heart disease-prone subpopulation is the influenza pandemic over? long-term effects of in utero influenza exposure in the post- lingering prenatal effects of the influenza pandemic on cardiovascular disease government printing office fetal origins of adult diease: strength of effects and biological basis mothers, babies, and health in later life the fetal matrix: evolution, development and disease: cambridge development and evolution: cambridge impacts of covid- on childhood malnutrition and nutrition-related mortality. the lancet short stature associated with intrauterine growth retardation: final height of untreated and growth hormone-treated children patterns of catch-up growth the develpmental origins of health and disease: cambridge the developmental origins of health and diseases (dohad) concept: past, present, and future london the intrauterine and early postnatal origins of cardiovascular disease and chronic bronchitis the nature of child malnutrition and its long-term implications community and program influences on the mortality of malaysian infants effects of inter-birth intervals and breastfeeding on infant and early childhood mortality synergism of nutrition, infection, and immunity: an overview resistance and life expectancy: disease and death during the economic development of japan an analytical framework for the study of child survival in developing countries. population and development review maternal care, gene expression, and the transmission of individual differencesin stress reactivity across generations building muscle: molecular regulation of myogenesis. cold spring harbor perspective in biology maternal nutrition and fetal development the cytokine storm of severe influenza and development of immunomodulatory therapy viral infections during pregnancy fever in pregnancy and its maternal and fetal outcomes pax induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors the optimal duration of exclusive breastfeeding: a systematic review human milk composition: nutritents and bioactive factors the immunological components of human milk and their effect on immune developmenty in infants nutrition and health from womb to tomb the interaction between nutrition and infection: world health organization the developing human: clinically oriented embryology ( th ed): philadelphia: sanuders maternal and child undernutrition: global and regional exposures and health consequences long-run consequences of exposure to natural disasters boys live dangerously in the womb regional model life tables and stable populations the care of children: the influence of medical innovation and medical institutions on infant mortality - diffusion of ideas about personal hygiene and contamination in poor countries: evidence from guatemala. social science & medicine sex differences in human development inter-university consortium for political and social research [distributor adult mortality in latin america and the caribbean observations on mortality during the influenza pandemic porto rico and its problems prehco protocol: an exercise to examine interviewer measurement error for anthropometric measures. unpublished report decline of height with age in adults in a general population sample: estimating maximum height and distinguishing birth cohort effects from actual loss of stature with aging stature estimation in critically ill patients equations for predicting stature in white and black elderly individuals stature predictions equations for elderly hispanics in latin america by sex and ethnic background associations between birth size and later height from infancy through adulthood: an individual based pooled analysis of twin cohorts participating in the coda twins project the design of experiments an investigation of the false discovery rate and the misinterpretation of p values we thank berty lumey for very helpful comments to the first draft of the manuscript. conceptualization: alberto palloni. key: cord- -dzm xi authors: filardi, tiziana; varì, rosaria; ferretti, elisabetta; zicari, alessandra; morano, susanna; santangelo, carmela title: curcumin: could this compound be useful in pregnancy and pregnancy-related complications? date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: dzm xi curcumin, the main polyphenol contained in turmeric root (curcuma longa), has played a significant role in medicine for centuries. the growing interest in plant-derived substances has led to increased consumption of them also in pregnancy. the pleiotropic and multi-targeting actions of curcumin have made it very attractive as a health-promoting compound. in spite of the beneficial effects observed in various chronic diseases in humans, limited and fragmentary information is currently available about curcumin’s effects on pregnancy and pregnancy-related complications. it is known that immune-metabolic alterations occurring during pregnancy have consequences on both maternal and fetal tissues, leading to short- and long-term complications. the reported anti-inflammatory, antioxidant, antitoxicant, neuroprotective, immunomodulatory, antiapoptotic, antiangiogenic, anti-hypertensive, and antidiabetic properties of curcumin appear to be encouraging, not only for the management of pregnancy-related disorders, including gestational diabetes mellitus (gdm), preeclampsia (pe), depression, preterm birth, and fetal growth disorders but also to contrast damage induced by natural and chemical toxic agents. the current review summarizes the latest data, mostly obtained from animal models and in vitro studies, on the impact of curcumin on the molecular mechanisms involved in pregnancy pathophysiology, with the aim to shed light on the possible beneficial and/or adverse effects of curcumin on pregnancy outcomes. maternal nutrition is an essential and modifiable environmental factor that deeply influences maternal and offspring health in the short and long-term [ ] [ ] [ ] [ ] [ ] [ ] . genetics, nutrition, and other environmental factors significantly contribute to the physiological immune and metabolic modifications occurring in pregnancy, to favor maternal adaptation to the growing and developing fetus. maternal malnutrition adversely affects these dynamic processes by acting on the mechanisms related to the nutritional programming, including nutrition sensing signals, epigenetic regulation, gut microbiome, as well as on the nutrient-nutrient and nutrient-drug interactions, modulating maternal and fetal genes in a sex-specific manner [ , [ ] [ ] [ ] [ ] . bw/day of curcumin as a food additive [ ] . however, despite its potential therapeutic benefits, curcumin is poorly bioavailable due to its rapid metabolism, and the small portion of substance that is absorbed is extensively bio-transformed into its water-soluble metabolites, glucuronides, and sulfates [ ] . therefore, several strategies have been developed to enhance its bioavailability and efficacy, to increase oral and gastro-intestinal absorption, and to reduce the clearance from the body [ ] [ ] [ ] . for this purpose, taking into consideration that curcumin is fat-soluble, several delivery systems have been developed to obtain a number of formulations by mixing curcumin with different materials, including adjuvants, such as piperine [ , ] . micelles, liposomes, phospholipid complexes, phytosomes, emulsions, microemulsions, nano-emulsions, solid lipid nanoparticles, nanostructured lipid carriers, biopolymer nanoparticles, and microgels represent different and recent technical approaches to encapsulate curcumin [ ] [ ] [ ] , although further studies are needed to evaluate their effectiveness and safety as potential health-promoting compounds in humans. it is well known that dynamic changes in insulin sensitivity take place during healthy pregnancy to allow adequate supply to the growing fetus [ ] . in pregnancy, several players, including hormones, cytokines, and metabolic factors, contribute to the development of insulin resistance through complex mechanisms, not yet completely understood [ , ] . maternal obesity, related to unhealthy diet and lifestyle, can negatively affect insulin sensitivity leading to the development of gdm and type diabetes (t d), with serious short and long-term health consequences for both the mother and the offspring [ , ] . recent evidence emphasized the anti-hyperglycemic activity of curcumin, both in animals and humans [ ] . specifically, this compound had the capability to improve glucose uptake, insulin sensitivity, and pancreatic β-cell function, as well as liver and kidney function, and to reduce glucose and lipid levels, oxidative stress, and inflammation [ ] , by interacting with almost all the players involved in these processes, as demonstrated in in vitro studies [ , ] . as regards human studies, the effects of curcumin supplementation have been evaluated in several randomized controlled trials. a recent intervention study showed that mg/day curcumin supplementation ( mg capsules: mg of curcumin, mg of demethoxycurcumin, and mg of bisdemethoxycurcumin) for weeks reduced triglycerides (tg) and c-reactive protein (crp), and increased adiponectin levels [ ] , whereas mg/day curcumin co-administered with piperine mg/day for three months was able to reduce blood glucose, c-peptide, glycated hemoglobin (hba c), alanine aminotransferase (alt) and aspartate aminotransferase (ast), in patients with t d [ ] . another study showed that the daily ingestion of mg turmeric powder for eight weeks resulted in a reduction in body weight, low density lipoprotein-cholesterol (ldl-c), and tg levels, with no significant effects on glycemia, crp, and hba c, in hyperlipidemic t d patients [ ] . in obese women with polycystic ovary syndrome (pcos), mg/day curcumin supplementation ( mg twice daily: - % curcumin, - % demethoxycurcumin and . - . % bisdemethoxycurcumin) for six weeks improved serum insulin and the quantitative insulin sensitivity check index [ ] . a recent meta-analysis reported that curcumin intake was associated with reduced body mass index (bmi), body weight, body fat, leptin value, and increased adiponectin levels in patients with metabolic syndrome and related disorders [ ] . overall, the dosage and duration of curcumin supplementation appear to differently modulate glucose metabolism in humans. a recent promising approach to treat hyperglycemia consists of combining the effects of curcumin and the ongoing antidiabetic agents, as observed in diabetic rats treated with a combination of curcumin and metformin. specifically, this association improved hyperglycemia, dyslipidemia, and oxidative stress, increasing the activity of the antioxidant enzyme paraoxonase (pon ), in diabetic rats [ ] . dietary bioactive compounds might have beneficial effects on gdm [ , ] . in particular, curcumin appeared to improve gdm and gdm-related complications in a recent study in a mouse model. specifically, c bl/ksjdb/+ diabetic pregnant mice were supplemented with different curcumin dosages: mg/kg and mg/kg/day, from gestational day zero (gd ) to gd . results showed that mg/kg curcumin significantly reduced blood glucose and insulin levels, increased hepatic glycogen content, and improved oxidative stress by reducing thiobarbituric acid reactive substance (tbars) and increasing glutathione (gsh) levels, superoxide dismutase (sod), and catalase (cat) activities in the liver of diabetic pregnant mice at gestational day . the reduced ' adenosine monophosphate-activated protein kinase (ampk) and increased histone deacetylase hdac activities observed in gdm liver were reverted by curcumin treatment. furthermore, curcumin positively influenced the offspring of mothers with gdm, restoring litter size and birth weight, and inducing the reduction of glucose- -phosphatase (g pase) expression and activity in the liver [ ] ( table ) . congenital birth defects, including neural tube defects (ntd), occur more often in the offspring of diabetic mothers. in a recent study, mouse embryos (at e . of development) were cultured for h with mg/dl glucose, in the absence or presence of curcumin ( and µm). remarkably, µm curcumin was able to reduce the rate of embryos with ntd induced by high glucose. curcumin reduced high glucose-induced oxidative and nitrosative stress [i.e., decreased -hydroxynonenal ( -hne), nitrotyrosine levels, and lipid hydroperoxide (lpo)], as well as endoplasmic reticulum (er) stress (i.e., decreased expression of er-markers stress such as phosphorylated protein kinase-like endoplasmic reticulum kinase (p-perk), phosphorylated inositol-requiring protein- α (p-ire α), phosphorylated eukaryotic initiation factor α (p-eif α), c/ebp-homologous protein (chop), binding immunoglobulin protein (bip), and x-box binding protein (xbp ). moreover, µm curcumin inhibited the cleavage of pro-apoptotic caspases (i.e., casp- and - ) [ ] . although the results from preclinical studies are overall promising, further research is needed to better understand the molecular mechanisms underlying diabetic complications, as well as the pharmacodynamics and pharmacokinetics of curcumin in pregnancy, to conceivably employ this compound as a therapeutic agent for human pregnancy complications. critical changes in the cardiovascular system occur in physiological pregnancy, to ensure maternal and fetal adaptation to the increased metabolic demand and to guarantee adequate uteroplacental circulation for fetal growth. a healthy pregnancy is hallmarked by systemic vasodilatation, significantly related to the high levels of estrogen and progesterone. cardiac output and heart rate rise during gestation and the activation of the renin-angiotensin-aldosterone system leads to a significant increase in total blood volume. alterations in these processes are associated with maternal and fetal morbidity and mortality [ ] . obesity, older maternal age, and diabetes mellitus increase the risk of cardiovascular diseases in pregnancy ( - %), with a higher prevalence when including hypertensive disorders-chronic hypertension, pregnancy-induced hypertension, pre-eclampsia, and hellp syndrome (hemolysis, elevated liver enzymes, and low platelet count) [ ] . considering the anti-inflammatory, antioxidant, and antiangiogenic activities observed in several studies, curcumin is a potential therapeutic compound in cardiovascular disorders [ ] . preeclampsia (pe) is a systemic syndrome characterized by hypertension and proteinuria, which begins after weeks of gestation; it occurs in - % of pregnancies, and it is a leading cause of maternal and fetal morbidity and mortality [ ] . although the pathophysiology of pe remains to be elucidated, alterations in maternal vascular physiology have been described, leading to a generalized vasoconstrictive state, systemic oxidative stress, inflammation, and endothelial cell dysfunction, with severe adverse effects on the placenta, one of the major organs that develops after conception [ , ] . strategies to reverse or arrest the pathological processes of pe are aimed at reducing excessive inflammatory response, micro-emboli formation, and vasoconstriction by using specific drugs or natural products [ ] . for this purpose, studies in animal models have been performed. it has been observed that in lipopolysaccharides (lps)-treated pregnant rats to create a pe model (lps . µg/kg on gestational day ), the administration of curcumin ( . mg/kg, from gd to gd ) improved hypertension, proteinuria, and renal damage, and reduced serum levels of il- and monocyte chemoattractant protein- (mcp- ). curcumin treatment ameliorated inadequate trophoblast invasion and spiral artery remodeling, significant histopathological alterations observed in pe. analysis of placental tissue showed that curcumin administration decreased the lps-induced expression of the inflammatory molecules toll-like receptor (tlr)- , il- , and the proinflammatory transcription factor nf-kb. according to the obtained data, the authors hypothesized that curcumin may positively modulate the cascade of different signaling pathways involved in pe development [ ] . similar results were obtained in a mouse model of lps-induced pe. in this study, in addition to blood pressure and proteinuria reduction, curcumin increased the number of live pups, fetal and placental weight, and decreased fetal desorption. these effects were associated with the inhibition of placental expression of tnf-α, il- β, il- cytokines, and mcp- and mip- chemokines, and with a reduction in macrophage infiltration. the reduced inflammatory status was accompanied by increased activation of the serine/threonine-specific protein kinase akt, involved in cellular proliferation [ ] . neo-vascularization is a critical event mediated by several angiogenic factors-including the vascular endothelial growth factor (vegf), fibroblast growth factors (fgfs), matrix metalloproteinases (mmps)-and inflammatory factors such as cyclooxygenase (cox)- and nf-kb, occurring not only in tumor progression but also in early placentation [ , ] . curcumin appears to modulate the above-mentioned factors, influencing vessel formation by acting either as a proangiogenic or as an antiangiogenic molecule, depending on the concentration and the cell type [ ] . a recent study investigated the effect of curcumin in htr /svneo trophoblasts cells, a model of the human first-trimester placenta. incubation with curcumin at low concentration ( - µm for h) stimulated (i) proliferation with concomitant activation of akt, (ii) tube formation of placental trophoblast htr /svneo cells, (iii) and increased the expression of the proangiogenic factors vegf, vegfr , and fabp . in addition, curcumin treatment strongly increased the mrna and protein expression of hla-g, involved in the immune regulation during trophoblast invasion; and mrna expression of a relevant number of genes related to the notch-signaling pathway, which regulates angiogenesis. the authors examined the promoter methylation of genes involved in metabolic and oxidative stress and observed that curcumin induced hypomethylation in genes involved in the protection against oxidative stress and dna damage. altogether these data indicate that curcumin is able to promote angiogenesis and to activate protective pathways in the first trimester of pregnancy, and supports the development of the placental trophoblast [ ] . moreover, htr /svneo trophoblast cells were used to evaluate the protective effects of curcumin against oxidative stress induced by h o ( µm for h). results showed that pretreatment with curcumin ( µm for h) increased cell viability, upregulated the activities of the antioxidant enzymes cat and glutathione peroxidase (gsh-px), reduced the h o -induced ros accumulation and the apoptotic rate. at molecular levels, these data were associated with an increased nuclear translocation of the antioxidant transcription factor nrf , and reduced expression of cleaved-caspase [ ] . the anti-inflammatory activity of curcumin has been also observed in vitro in human gestational tissues treated with lps. specifically, incubation with curcumin ( µm for h) reduced il- release, and il- and il- mrna expression induced by lps, in both placenta and fetal membranes. moreover, curcumin decreased placental cox- mrna expression, prostaglandin pge and pgf a release, and the expression and activity of the matrix-degrading enzyme mmp- , in association with reduced activation of nf-kb [ ] . although several clinical trials emphasized the benefits of curcumin in different pathological contexts [ , , , ] , there are few data on curcumin supplementation in human pregnancy. recently, a double-blind randomized clinical trial involving pregnant women with preeclampsia was conducted to evaluate the possible effect of curcumin on the expression of cox- and il- , thought to have a role in the pathogenesis of pe. the enrolled patients were randomized to receive either curcumin mg/d (n = ) or placebo (n = ) [ ] . the authors analyzed the circulating levels of il- and cox- , at t , min after curcumin ingestion, and h after delivery. results showed that curcumin did not modify the expression of the analyzed molecules at any tested time. the authors hypothesized that the absence of effect might be due to the low dose of curcumin, taking into account that in non-pregnant subjects doses can reach more than g/day [ ] . during the antenatal and postpartum periods, women are particularly prone to develop mental disorders, including depression. postpartum depression (ppd) occurs in - % of women, leading to significant health consequences for both mother and offspring [ ] . this condition has been largely underestimated and understudied so far. hence, its prevalence is supposed to be higher, conceivably reaching % of women. symptoms of depression begin during pregnancy in about % of women and numerous environmental, genetic, biochemical, and epigenetic factors likely contribute to the onset of ppd [ ] [ ] [ ] , although the exact mechanisms responsible for this condition are not yet completely known. several pharmacological and psychological approaches are currently adopted to treat ppd, even though complementary and alternative medicine have also been taken into consideration. increasing data have suggested the neuroprotective roles of a healthy diet, rich in fruit and vegetables, highlighting its positive influence on mental health [ ] . on the contrary, an unhealthy dietary pattern increases the risk of systemic low-grade inflammation and neuroinflammation, known to be associated with ppd [ ] . the neuroprotective and antidepressant benefits of curcumin have been known for a long time [ ] [ ] [ ] . several preclinical studies have suggested potential positive effects of curcumin in treating neurological disorders, such as alzheimer's disease, parkinson's disease, multiple sclerosis, migraine, epilepsy, brain and spinal cord injury, and depression [ , , ] . lopresti and colleagues have investigated the effects of curcumin on depression outcomes in humans. they observed that eight-week curcumin supplementation ( mg twice a day) in subjects with major depressive disorder (mdd) was effective in reducing depressive and anxiety symptoms, as demonstrated by the reduction in total depressive symptoms (total ids score), mood/cognitive depressive symptoms (idsm), arousal-related symptoms (idsa), and trait anxiety (stait) [ ] . this supplementation resulted in an increase in urinary levels of both the arachidonic acid metabolite thromboxane b (tbx-b ) and the neuropeptide substance p (sub-p), potentially involved in depression mechanisms. moreover, although curcumin did not modify plasma levels of endothelin- and leptin, a greater antidepressant benefit was observed in subjects with the highest baseline levels of these molecules. the authors hypothesized that curcumin might act by increasing endothelin and leptin receptor activities [ ] . similarly, in another trial, mg/day curcumin ingestion for six weeks or the administration of the antidepressant drug fluoxetine showed comparable efficacy in subjects with mdd [ ] . a recent meta-analysis provided relevant information about curcumin use in depression. specifically, this analysis revealed that curcumin administration (i) appears to be more effective in reducing depression symptoms at a higher dosage ( g/day) and for six weeks or more; (ii) can enhance the action of antidepressants; and (iii) has more effects on subjects with major depression and without other comorbidities [ ] . these results indicate the need for further study to better comprehend the mechanisms of action of curcumin in depression treatment. data obtained from animal and in vitro studies have indicated that curcumin might exert antidepressant activity by acting on different signaling pathways involved in mental disorders. specifically, this compound is able to ameliorate the hypothalamic-pituitary-adrenal (hpa) axis disturbances [ ] . curcumin can influence the unbalanced release of monoamine neurotransmitterssuch as serotonin ( -ht), dopamine (da), noradrenaline, and glutamate-the expression of monoamine oxidase (mao), the expression of neurotrophic factors such as brain-derived neurotrophic factor (bdnf) and neurogenesis, as well as the dysregulated immune system function and oxidative and nitrosative stress. thus, curcumin appears to promote neurogenesis and inhibit neuronal cell apoptosis [ , , , ] . despite the consistent evidence of efficacy and safety of curcumin treatment in other pathological conditions, to date, data on its effects on depression in pregnancy are completely lacking. however, in the last years, there has been a growing awareness of the possible role of anti-inflammatory micronutrients in improving ppd symptoms [ ] . according to the theory of the fetal origin of adult diseases (foad) hypothesized by david barker, the intrauterine environment has a relevant role in fetal growth and development and influences disease susceptibility in the offspring in the short and long term [ ] . the physiological processes of pregnancy require immune and metabolic modifications to accommodate the growing fetus; maternal malnutrition negatively influences this dynamic equilibrium, leading to tissue-specific impairment, with serious adverse outcomes for both mother and child [ , ] . taking into consideration the importance of nutrition in human development, there is a need for better understanding the nutritional programming and the related mechanisms and players acting during pregnancy. the placenta has the fundamental role of transferring nutrients to the fetus, and alterations in placental function have severe effects on fetal growth. placental insufficiency is the most common cause of fetal growth restriction (fgr), a serious condition that affects - % of all newborns [ ] . although the pathophysiology of fgr is not completely known, excessive oxidative stress and inflammation, as well as the activation of a complex network of several signaling pathways, appear to be involved [ , ] . the antioxidant and anti-inflammatory effects exerted by curcumin on the placenta [ ] were confirmed in a mouse model of fgr fed with a low-protein (lp) diet [ ] . the authors showed that maternal supplementation with curcumin ( mg/kg day, from . to . gd) induced a potent antioxidant response in lp-fed pregnant mice; specifically, curcumin (i) increased gsh-px activity, nfr mrna expression, and the blood sinusoids area; (ii) reduced malondialdehyde (mda) content and apoptosis in the placenta, leading to increased placental efficiency; and (iii) elevated the expression of the antioxidant genes sod , sod , and cat, and protein expression of nrf and eme oxygenase- (ho- ) in the liver. overall, curcumin supplementation during pregnancy was able to revert tissue damage and contrast the decrease in fetal weight induced by a lp diet [ ] . curcumin appeared to improve birth weight, inflammation, and oxidative damage also in fgr newborn rats. indeed, fgr rats supplemented with mg/kg curcumin (at six weeks of age for six weeks) displayed reduced levels of the inflammatory cytokines tnf-α, il- β, and il- , reduced activity of ast, alt, and mda enzymes, and increased gpx and gsh activity in serum. antioxidant defense in the liver was significantly improved as well. the attenuation of the inflammatory status induced by curcumin was associated with (i) reduced activation of nf-kb and jak ; (ii) increased expression of the antioxidant genes (nqo , hmox , gst, gpx , and sod ), and activation of their regulatory transcription factor nfr , in the liver [ ] . successively, the same authors investigated the effects of curcumin on insulin resistance (ir) and hepatic lipid accumulation in fgr newborn rats. specifically, supplementation with mg/kg curcumin (at six weeks of age for six weeks) attenuated ir by reducing serum insulin, glycemia, and homeostasis model assessment of insulin resistance (homa-ir). furthermore, in the liver, curcumin diminished total cholesterol, tg, and non-esterified fatty acids (nefa); increased glycogen concentration and induced the activation of lipolytic enzymes, together with a reduction in irs- and akt phosphorylation, a decrease in cd , srebp- , and fasn expression, and an increase in pparα levels. overall, these data showed that curcumin could improve ir and lipid accumulation in the liver by regulating insulin signaling pathways, and promoting lipolysis and fatty acid oxidation in fgr rats [ ] . of note, curcumin alleviated also jejunum damage in fgr growing pigs. indeed, the addition of mg/kg curcumin to diet improved antioxidant defense (i.e., increased sod and decreased mda activity), immune-related gene expression (reduced mrna of tnfα, il- , and ifnγ, and increased il- ), and decreased apoptotic genes, such as caspase and bax in the jejunum. moreover, curcumin supplementation increased mrna expression of the tight junction-related gene ocln [ ] . preterm birth (ptb) is a pregnancy complication that affects about % of births worldwide and is associated with increased maternal and neonate morbidity and mortality [ ] . an altered inflammatory status appears to be associated with ptb. thus the anti-inflammatory activity of curcumin has been evaluated in a mouse model of ptb, obtained through lps injection in the abdominal cavity [ ] . the injection of mg/kg curcumin into the abdominal cavity, one day before (preventative group) or one day after (treatment group) lps treatment, significantly reduced serum levels of tnf-α, il- , and mda, and increased sod levels, in both the experimental conditions, in pregnant mice. the staining intensity of nf-κb p showed that curcumin was able to reduce the lps-induced expression of this inflammatory transcription factor in placental tissue both in the preventative and in the treatment group [ ] . besides maternal nutrition, many other factors, including exposure to chemical and natural toxic agents, drugs, alcohol, smoking, and maternal stress influence fetal growth and development [ ] . among the myriad of properties, curcumin appears to be able to reduce toxicity induced by several environmental agents in different organs and tissues, including the brain and liver [ ] . bisphenol-a (bpa) is a chemical substance adapted to produce plastic. it has been considered an endocrine disruptor by the european chemicals agency (echa ) [ ] due to its estrogenic activity. bpa exposure in pregnancy is associated with negative outcomes, including impaired fetal growth and childhood adiposity [ ] . remarkably, this synthetic compound affected the processes of neurogenesis in the hippocampus of the developing rat brain, and curcumin treatment showed neuroprotective activity by reverting bpa-induced effects. specifically, pups from a pregnant rat receiving bpa ( µg/kg body weight/day from gd to pnd ) were treated with curcumin ( mg/kg body weight/day from pnd to pnd ). the authors performed accurate experiments on embryo and pup brains and examined the expression of genes and pathways involved in neurogenesis. they observed that curcumin attenuated the bpa-induced reduction in neuronal stem cells (nsc) proliferation and differentiation. at molecular levels, the improvement in neurogenesis was associated with the enhanced expression of the proneural transcription factors neurogenin and neurod , the reduced expression of the proapoptotic molecule bax, the increased expression of the antiapoptotic molecule bcl- , and the activation of wnt/βcatenin signaling that regulates nsc proliferation and differentiation. of note, the benefits of curcumin resulted in improved learning and memory in bpa-treated pups [ ] . mercury (hg) is a widely diffused toxic heavy metal that occurs naturally in three forms, namely metallic hg, organic hg, and inorganic hg. human exposure to hg occurs mainly through the environment (e.g., mercury-contaminated sea fish, dental amalgam). of note, occupation (e.g., mining) is another important source of exposure for humans and is associated with possible multi-organ toxicity [ ] . as for the influence of hg on neurodevelopment, a cross-sectional study, involving healthy saudi mothers and their infants (age - months), showed an association between hg exposure and neurodevelopmental delay, with possible negative effects persisting also in adulthood [ ] . interestingly, curcumin appeared to mitigate hg toxicity in animal models [ ] . specifically, pregnant mice were exposed (from gd to pnd) to ppm mercuric chloride (hgcl ) in the presence or absence of and ppm curcumin. hg exposure induced serious damage to the development of neuromotors, and increased anxiety behavior in pups. curcumin administration improved neurodevelopment and reduced anxiety, by restoring the levels of neurotransmitters da, -ht, and acetylcholinesterase (ache), and of the antioxidant gsh, decreased by hg exposure, in forebrain pups [ ] . moreover, by using the same experimental conditions, the authors analyzed changes in body weight, sexual behavior, and fertility in male and female pups. the obtained data showed that curcumin counteracted the perinatal effects of hg exposure by increasing (i) body weight, liver and brain weight in male and female pups; (ii) epididymis, seminal vesicle, testis weight in males; and (iii) ovary weight in females; also sexual behavior was improved in both sexes. moreover, curcumin increased testosterone and fsh levels, and sperm motility in males, as well as fsh, lh, and progesterone in females, reduced by hg exposure [ ] . lead (pb) is a heavy metal widely spread in the environment. it is extremely dangerous for both animals and humans. lead exposure occurs mainly through food and water contamination, and air pollution. lead can cross the placental and blood-brain barrier, inducing neurotoxicity. curcumin exerted neuroprotective effects contrasting lead-induced damage in rats. the concomitant exposure of rat mothers to pb ( g/l) and curcumin ( g/kg) during pregnancy and lactation resulted in the recovery of the pb-induced altered sensorimotor functions in neonatal rats. pb neurotoxicity produced alterations in locomotor neuronal network development and curcumin treatment reversed these anomalies, allowing normal locomotor behavior. these findings indicate that curcumin has the capability to prevent central nervous system dysfunction induced by lead during the earlier stages of development [ ] . celecoxib is a selective inhibitor of cox- that is able to reduce pain and inflammation caused by several inflammatory conditions [ ] . since recent data have shown that the inhibition of cox- reduced adult neural cell proliferation and differentiation [ ] , wang et al., investigated the neuroprotective action of curcumin on fetal brain development in pregnant mice treated with celecoxib [ ] . specifically, pregnant mice were pretreated with curcumin ( nmol/kg body weight) from embryonic day (e) . to e . , and then with celecoxib ( mg/kg body weight) from e . to e . . results showed that curcumin counteracted the celecoxib-induced inhibition of neurogenesis in the fetal frontal cortex, by increasing proliferation and cyclin d expression in neural progenitor cells, and by activating wnt/βcatenin signaling (i.e., decreased expression of glycogen synthase kinase beta (gsk- β), and increased expression of βcatenin) [ ] . valproic acid (vpa), a branched short-chain fatty acid, is an antiepileptic agent that has been associated with congenital malformations, including alterations in fetal brain development, and consequent intellectual disabilities and autistic spectrum disorders in the offspring [ ] . curcumin appears to attenuate the vpa-induced brain damage, as observed in a rodent model of autism. neonatal rats, born to mothers treated with vpa from . gestational day, received a single dose of curcumin ( g/kg day), and their brains were analyzed days after birth. curcumin was able to ameliorate body and brain weight, and the altered expression of il- , ifn-γ, gsh, cyp , in the brain of vpa-exposed pups [ ] . prenatal alcohol exposure (pae) has dramatic effects on fetal growth and development (fetal alcohol spectrum disorders: fasd) and is responsible for neurodevelopmental disorders (i.e., neurocognitive and behavioral deficits, and increased susceptibility to mental health disorders) and birth defects (growth deficits and physical abnormalities). pae induces chromosomal rearrangements and epigenetic alterations, therefore leading to altered gene-environment interactions that are responsible for alcohol-induced disorders [ ] . curcumin ( mg/kg body weight), administered during the peri-adolescence period (pnd - ), appeared to counteract fetal brain damage induced by prenatal and lactational alcohol exposure (plae; % (v/v) alcohol solution) in mice. the authors showed that curcumin improved anxiety and memory deficits caused by plae, and these improvements were associated with reduced microglia activation and astrogliosis. at molecular levels, curcumin reduced protein expression of il- , tnf-α, and nf-kb. these data showed that curcumin may act against cognitive deficits and neuroinflammation induced by alcohol exposure in pregnancy [ ] . curcumin can counteract the deleterious effects of pae on cardiac development, as demonstrated in a mouse model. pregnant mice were daily exposed to ethanol ( % v/v in saline) between embryonic days . to . ; at embryonic day . , mice were euthanized and embryonic hearts were removed. results showed that pae treatment increased apoptosis in pup hearts; this finding was associated with higher levels of caspase- and - mrna expression, and reduced bcl- mrna expression, due to a different modulation of histone h k acetylation near the promoter regions of caspase- , caspase- (hyperacetylation), and bcl- (hypoacetylation). in vitro, curcumin ( µm for h) treatment abolished apoptosis and reverted the expression of caspases and bcl- , induced by alcohol ( mm), in cardiac progenitor cells. these results highlighted the capability of curcumin to prevent congenital heart diseases induced by pae in pregnancy, by acting as an epigenetic modulator [ ] . embryonic development is a complex process that is finely regulated and highly susceptible to environmental influences. therefore, it is reasonable to hypothesize that the anti-inflammatory, antioxidative, antiproliferative, and antiangiogenic properties of curcumin could interfere with the blastocyst stage, implantation and post-implantation development of embryos [ ] . chen and colleagues evaluated the possible embryotoxicity of curcumin in mouse blastocysts both in vitro and in vivo. they observed that curcumin ( µm for h) induced apoptosis in mouse blastocysts, and reduced implantation rate and development, in vitro. then, embryos treated with curcumin were transferred in vivo; results confirmed a significant reduction in implantation ratio, and, among the implanted embryos, a higher rate of failure to develop normally. the authors evaluated the possible mechanisms responsible for these effects and found that curcumin-induced apoptosis was associated with the modulation of pro-and anti-apoptotic molecules (i.e., increased bax and reduced bcl- expression), ros generation, and caspase- activation [ ] . additionally, the same authors showed that curcumin ( µm) adversely affected oocytes maturation, in vitro. this effect resulted in a reduced ability of oocytes to be fertilized, increased blastocyst apoptosis, and reduced blastocyst implantation ratio and development. these results were confirmed in oocytes collected from female mice after feeding them with curcumin supplementation ( µm) for four days [ ] . another in vitro study highlighted that the degree of damage induced by curcumin ( , , or µm curcumin for h) on mouse blastocyst at the implantation stage and during the early post-implantation stage is dose-dependent. specifically, µm and µm curcumin inhibited cell proliferation of the blastocyst but increased the formation of trophoblastic giant cells, whereas µm curcumin exposure was lethal to all blastocysts, and induced severe damage to the implanted blastocysts [ ] . further evidence on these effects comes from a recent study in zebrafish. the exposure of zebrafish embryos and larvae to different concentrations of curcuma longa extract ( . , . , . , . , . , and . µg/ml) at different hours post fertilization (hpf: , , , , h) showed that a dosage above . µg/ml had toxic effects, and a dosage of . µg/ml increased embryo mortality and induced morphological deformities in larvae [ ] . despite the potential benefits of curcumin described in different pathological conditions, all these data indicate that dosage and time of exposure throughout pregnancy should be carefully evaluated to avoid serious damage to embryo development. the use of the natural product curcumin to treat medical conditions is spreading around the world. there is an increasing public interest in the potential health benefits of this compound, as evidenced by the large number of currently available curcumin formulations, aimed at increasing its bioavailability and efficacy, and by the considerable number of scientific papers published over the last years. this review has drawn attention towards the effects of curcumin on pregnancy and pregnancy complications, considering that during gestation, mother and fetus undergo significant (patho-) physiological changes. almost all data emphasizing the numerous biological activities of curcumin have been obtained from pregnant rodents and in vitro studies. curcumin appeared to ameliorate diabetes in a gdm mouse model, as well as pe in a pe rat model, and was found to be neuroprotective against environmental toxic agents. the antidepressant activity of curcumin has also been tested in humans. however, to date, studies on the possible beneficial effects of curcumin on ppd, a largely underestimated and understudied condition, are completely lacking. as regards fetal growth and development, curcumin counteracted the modifications associated with fgr and ptb in rodent models but negatively affected blastocyst stage, implantation and post-implantation embryo development in healthy animals. altogether, these results indicate that the use of curcumin in pregnancy must be carefully evaluated. the growing use of curcumin as self-medication along with the misleading perception that "natural" is the equivalent of "safe" are additional issues of concern. further studies are needed to clarify whether pregnancy might benefit from curcumin's properties; for this purpose, the collaboration between multidisciplinary scientific teams is essential to provide a holistic view of the complex networks between natural products and human physiology. systems biology and the recently developed network pharmacology represent new strategies to better comprehend the mechanisms underlying curcumin activities in the human body. maternal nutrition and birth outcomes dietary interventions for healthy pregnant women: a systematic review of tools to promote a healthy antenatal dietary intake tain the good, the bad, and the ugly of pregnancy nutrients and developmental programming of adult disease management of reproduction and pregnancy complications in maternal obesity: which role for dietary polyphenols? could gestational diabetes mellitus be managed through dietary bioactive compounds? current knowledge and future perspectives maternal obesity, inflammation, and developmental programming non-coding rna: role in gestational diabetes pathophysiology and complications epigenetics and human reproduction: the primary prevention of the noncommunicable diseases genes and diet in the prevention of chronic diseases in future generations herbs and spices-biomarkers of intake based on human intervention studies-a systematic review curcumin: a review of its effects on human health antidotal or protective effects of curcuma longa (turmeric) and its active ingredient, curcumin, against natural and chemical toxicities: a review cellular and molecular mechanisms of curcumin in prevention and treatment of disease curcumin ameliorates gestational diabetes in mice partly through activating ampk curcumin stimulates angiogenesis through vegf and expression of hla-g in first-trimester human placental trophoblasts maternal curcumin supplementation ameliorates placental function and fetal growth in mice with intrauterine growth retardation curcumin's nanomedicine formulations for therapeutic application in neurological diseases rationale for natural neurosteroid-based interventions for postpartum depression phytochemical evaluation, embryotoxicity, and teratogenic effects of curcuma longa extract on zebrafish (danio rerio). evid.-based complementary altern curcumin as "curecumin": from kitchen to clinic high-performance thin layer chromatographic method for quantitative determination of curcuminoids in curcuma longa germplasm curcumin: a modulator of inflammatory signaling pathways in the immune system pharmacokinetics, pharmacodynamics, and pkpd modeling of curcumin in regulating antioxidant and epigenetic gene expression in healthy human volunteers curcumin as an alternative epigenetic modulator: mechanism of action and potential effects mutual two-way interactions of curcumin and gut microbiota potential effects of curcumin in the treatment of covid- infection food & drug administration. gras notice inventory scientific opinion on the re-evaluation of curcumin (e ) as a food additive formulations of curcumin nanoparticles for brain diseases phytosomal curcumin: a review of pharmacokinetic, experimental and clinical studies highly bioavailable forms of curcumin and promising avenues for curcumin-based research and application: a review curcumin delivery mediated by bio-based nanoparticles: a review determinants of maternal insulin resistance during pregnancy: an updated overview gestational diabetes mellitus: the impact of carbohydrate quality in diet cross-talk between fetal membranes and visceral adipose tissue involves hmgb -rage and vip-vpac pathways in human gestational diabetes mellitus the risk stratification of adverse neonatal outcomes in women with gestational diabetes (strong) study impact of risk factors for gestational diabetes (gdm) on pregnancy outcomes in women with gdm curcumin and type diabetes mellitus: prevention and treatment antidiabetic properties of curcumin ii: evidence from in vivo studies the effects of curcumin supplementation on high-sensitivity c-reactive protein, serum adiponectin, and lipid profile in patients with type diabetes: a randomized, double-blind, placebo-controlled trial effects of curcuminoids plus piperine on glycemic, hepatic and inflammatory biomarkers in patients with type diabetes mellitus: a randomized double-blind placebo-controlled trial effect of turmeric on glycemic status, lipid profile, hs-crp, and total antioxidant capacity in hyperlipidemic type diabetes mellitus patients the effects of curcumin supplementation on glycemic status, lipid profile and hs-crp levels in overweight/obese women with polycystic ovary syndrome: a randomized, double-blind, placebo-controlled clinical trial the effects of curcumin on weight loss among patients with metabolic syndrome and related disorders: a systematic review and meta-analysis of randomized controlled trials curcumin combined with metformin decreases glycemia and dyslipidemia, and increases paraoxonase activity in diabetic rats epigenetics and gestational diabetes: consequences in mother and child curcumin ameliorates high glucose-induced neural tube defects by suppressing cellular stress and apoptosis curcumin improves lps-induced preeclampsia-like phenotype in rat by inhibiting the tlr signaling pathway curcumin inhibits placental inflammation to ameliorate lps-induced adverse pregnancy outcomes in mice via upregulation of phosphorylated akt curcumin protects human trophoblast htr /svneo cells from h o -induced oxidative stress by activating nrf signaling pathway dietary phytophenols curcumin, naringenin and apigenin reduce infection-induced inflammatory and contractile pathways in human placenta curcumin's effect on cox- and il- serum in preeclampsia's patient undergo sectio caesarea with spinal anesthesia. open access maced dietary curcumin supplementation attenuates inflammation, hepatic injury and oxidative damage in a rat model of intra-uterine growth retardation curcumin attenuates insulin resistance and hepatic lipid accumulation in a rat model of intra-uterine growth restriction through insulin signalling pathway and sterol regulatory element binding proteins effect of curcumin on expressions of nf-κbp , tnf-α and il- in placental tissue of premature birth of infected mice. asian pac correction to: bisphenol-a mediated inhibition of hippocampal neurogenesis attenuated by curcumin via canonical wnt pathway neurobehavioral protective properties of curcumin against the mercury chloride treated mice offspring curcumin palliative effects on sexual behavior, fertility and reproductive hormones disorders in mercuric chloride intoxicated mice offspring disturbed sensorimotor and electrophysiological patterns in lead intoxicated rats during development are restored by curcumin i celecoxib-induced inhibition of neurogenesis in fetal frontal cortex is attenuated by curcumin via wnt/β-catenin pathway postnatal treatment using curcumin supplements to amend the damage in vpa-induced rodent models of autism curcumin treatment attenuates alcohol-induced alterations in a mouse model of inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis hazardous effects of curcumin on mouse embryonic development through a mitochondria-dependent apoptotic signaling pathway injurious effects of curcumin on maturation of mouse oocytes, fertilization and fetal development via apoptosis effect of curcumin on in vitro early post-implantation stages of mouse embryo development pregnancy and cardiovascular disease a systemic review on the antioxidant and anti-inflammatory effects of resveratrol, curcumin, and dietary nitric oxide supplementation on human cardiovascular health analysis of causes of maternal death: a systematic review disruption of placental homeostasis leads to preeclampsia the hmgb /rage pro-inflammatory axis in the human placenta: modulating effect of low molecular weight heparin preeclampsia: a review of the pathogenesis and possible management strategies based on its pathophysiological derangements extravillous trophoblast invasion in placenta accreta is associated with differential local expression of angiogenic and growth factors: a cross-sectional study effects of curcumin on vessel formation insight into the pro-and antiangiogenesis of curcumin. evid.-based complementary altern curcumin, the golden nutraceutical: multitargeting for multiple chronic diseases pathophysiological mechanisms implicated in postpartum depression antenatal depression and its association with adverse birth outcomes in low and middle-income countries: a systematic review and meta-analysis maternal depression and inflammation during pregnancy fruit and vegetable intake and mental health in adults: a systematic review multiple antidepressant potential modes of action of curcumin: a review of its anti-inflammatory, monoaminergic, antioxidant, immune-modulating and neuroprotective effects linking what we eat to our mood: a review of diet, dietary antioxidants, and depression curcumin for depression: a meta-analysis the role of curcumin administration in patients with major depressive disorder: mini meta-analysis of clinical trials benefits of curcumin in brain disorders curcumin for the treatment of major depression: a randomised, double-blind, placebo controlled study curcumin and major depression: a randomised, double-blind, placebo-controlled trial investigating the potential of peripheral biomarkers to predict treatment response and antidepressant mechanisms of change efficacy and safety of curcumin in major depressive disorder: a randomized controlled trial curcumin reverses the effects of chronic stress on behavior, the hpa axis, bdnf expression and phosphorylation of creb the influence of antidepressants on the immune system curcumin in antidepressant treatments: an overview of potential mechanisms, pre-clinical/ clinical trials and ongoing challenges developmental origins of adult health and disease fetal growth restriction-diagnostic work-up, management and delivery coordinated and highly regulated changes in placental signaling and nutrient transport capacity in iugr curcumin alleviates iugr jejunum damage by increasing antioxidant capacity through nrf /keap pathway in growing pigs global burden of preterm birth developmental programming of fetal growth and development msc unanimously agrees that bisphenol a is an endocrine disruptor bisphenol a and phthalates in diet: an emerging link with pregnancy complications medicinal plants and natural products can play a significant role in mitigation of mercury toxicity mercury (hg) exposure and its effects on saudi breastfed infant's neurodevelopment comparison of pharmacological and genetic inhibition of cyclooxygenase- : effects on adult neurogenesis in the hippocampal dentate gyrus adverse effects of prenatal and early postnatal exposure to antiepileptic drugs: validation from clinical and basic researches fetal alcohol spectrum disorders: genetic and epigenetic mechanisms author contributions: conceptualization, c.s. and s.m.; pubmed search, t.f. and r.v.; writing-draft preparation, t.f. and c.s.; writing-review and editing, s.m., e.f. and a.z.; supervision and critical revision s.m., r.v. and c.s. all authors have read and agreed to the published version of the manuscript.funding: this review received no external funding. the authors declare no conflict of interest. key: cord- - jfr wl authors: nelson, randy j.; demas, gregory e.; klein, sabra l.; kriegsfeld, lance j. title: minireview the influence of season, photoperiod, and pineal melatonin on immune function date: - - journal: j pineal res doi: . /j. - x. .tb .x sha: doc_id: cord_uid: jfr wl abstract: in addition to the well‐documented seasonal cycles of mating and birth, there are also significant seasonal cycles of illness and death among many animal populations. challenging winter conditions (i.e., low ambient temperature and decreased food availability) can directly induce death via hypothermia, starvation, or shock. coping with these challenges can also indirectly increase morbidity and mortality by increasing glucocorticoid secretion, which can compromise immune function. many environmental challenges are recurrent and thus predictable; animals could enhance survival, and presumably increase fitness, if they could anticipate immunologically challenging conditions in order to cope with these seasonal threats to health. the annual cycle of changing photoperiod provides an accurate indicator of time of year and thus allows immunological adjustments prior to the deterioration of conditions. pineal melatonin codes day length information. short day lengths enhance several aspects of immune function in laboratory studies, and melatonin appears to mediate many of the enhanced immunological effects of photoperiod. generally, field studies report compromised immune function during the short days of autumn and winter. the conflict between laboratory and field data is addressed with a multifactor approach. the evidence for seasonal fluctuations in lymphatic tissue size and structure, as well as immune function and disease processes, is reviewed. the role of pineal melatonin and the hormones regulated by melatonin is discussed from an evolutionary and adaptive functional perspective. finally, the clinical significance of seasonal fluctuations in immune function is presented. taken together, it appears that seasonal fluctuations in immune parameters, mediated by melatonin, could have profound effects on the etiology and progression of diseases in humans and nonhuman animals. an adaptive functional perspective is critical to gain insights into the interaction among melatonin, immune function, and disease processes. seasonal breeding is a salient component of the life history strategies of most animals. although seasonal breeding is the primary seasonal phenomenon studied, it is only one component of a web of complex seasonal adjustments that permit individuals to maintain a positive energy balance despite fluctuating ambient temperature, food availability, and other challenging environmental conditions [re-viewed in bronson, ; moffatt et al., . individuals use photoperiodic information to initiate or terminate specific seasonal adaptations, including reproduction, in order to maintain a positive energy balance [reviewed in bartness and goldman, ; heldmaier et al., ; saarcla and reiter, . the annual cycle of changing photoperiod can be used by nontropical animals as a very precise temporal cue for the time of year. ambient pho-toperiodic information is transduced by the pineal gland into a melatonin signal. the secretory pattern of melatonin allows individuals to ascertain the time of year and thus anticipate predictable seasonal environmental changes [reviewed in bartness and goldman, ; reiter, . these seasonal adaptations ultimately enhance survival and presumably increase fitness [bronson, . although maintenance of a positive energy balance is critical for survival and reproductive success [reviewed in bronson and heideman, ; nelson et al., , other threats to survival must also be met in order for individuals to increase their fitness. they must avoid predators and potentially dangerous interactions with conspecific competitors, as well as avoid succumbing to disease. immunological resistance requires energy. in fact, the cascade of cellular events during the acute phase immune response and inflammation, and the elevation of body temperature in response to cytokine activation, presumably requires substantial energy, although precise quantification is lacking [henken and brandsma, : maier et al., . cytokine activation elevates body temperature and the energy requirements of inflammation and acute phase immune responses may increase metabolic rates >lo% per degree of body temperature elevation [reviewed in maier et al., . thus, a general energy deficit can increase the risk of infection and death because insufficient energy reserves may be available to sustain immunity. stress can also compromise immune function [see ader and cohen, ; dunn, ; o'leary, for reviews]. prolonged or severe food shortages may evoke secretion of glucocorticoid hormones [nakano et al., ; jose and good, ; glucocorticosteroids actively compromise aspects of immune function [kelley, ; munck and guyer, ; maier et al., ; and see below] . many other conditions perceived as stressful, such as reduced food availability, low ambient temperatures, overcrowding, lack of shelter, or increased predator pressure, can recur seasonally leading to seasonal fluctuations in immune function among individuals, and seasonal changes in population-wide disease and death rates [lochmiller et al., . a dynamic relationship exists between longevity and reproductive fitness [stearns, . in addition to the well-established seasonal cycles of mating and birth, there are also seasonal cycles of illness and death among many populations of animals [e.g., bradley et al., ; lochmiller et al., ; mcdonald et al., ; mihok et al., . because many stressful environmental conditions are somewhat recurrent, we hypothesize that animals have evolved mechanisms to combat seasonal stress-induced reductions in immune function. from an evolutionary and ecological perspective, it is reasonable to expect that animals have evolved the ability to forecast recurrent conditions associated with immunosuppression and bolster immune function in advance of these challenging conditions in order to maximize survival. the working hypothesis of this review is that individuals use photoperiodic information to bolster immune function in anticipation of challenging energetic conditions that may otherwise compromise immune function. all laboratory studies of photoperiodic effects on immune function have reported enhanced immune function in short day lengths ( table ) . although many field studies support this hypothesis, with data suggesting enhanced immune function and decreased disease prevalence during the winter as compared to the summer, a substantial number of studies have reported the opposite pattern of results (table ) ; i.e., immune function is lowest during short days. these conflicting results can be resolved by considering additional environmental factors, not usually manipulated in laboratory studies. for example, winter-associated stressors (e.g., restricted food and low ambient temperatures) appear to counteract short day enhancement of immune function in the lab [reviewed in demas and nelson, ) . thus, we predict enhanced immune function should be observed during mild winters, whereas compromised immune function should be expected during challenging winters. long-term field studies are required to test this hypothesis. evidence will be presented that pineal melatonin plays a critical role, both directly and indirectly through its effects on other hormones, in mediating photoperiodic modulation of immune function. although the effects of melatonin on immunity are well-established [see giordano et al., ; pioli et al., ; maestroni, ; caroleo et al., ; guerrero and reiter, for recent reviews], our goal in this review is to provide an ecological context for the effects of melatonin upon immune function, and to suggest why this phenomenon might be adaptive and functional, rather than merely a physiological oddity. knowledge of the adaptive and functional significance of seasonal fluctuations in immune function may help to provide an improved understanding of the possibilities, as well as the constraints, of melatonin immunotherapy. seasonal cycles in the development, regression, and regeneration of the thymus, spleen, and bursa of fabricius have been described in a wide variety of vertebrate species [brainard et al., ; ; wurtman et al., vriend and lauber, brainard, vaughan et al., mahmoud et al., blom et al., blom et al., blom et al., dobrowolska & gromadzka-ostrowska, champney and mcmurray, zapata et al., . in common with seasonal fluctuations in reproductive organ mass, seasonal changes in lymphatic organ size were presumed to reflect changing organ function. regression of the thymus, bursa, and spleen after puberty and the obvious link among these organs to seasonal changes in reproductive function, prompted many early hypotheses suggesting that these lymphatic organs regulated or influenced breeding [aimc, ; riddle, . for example, the avian thymus was originally suggested to provide the "egg envelope" [riddle, . other investigators hypothesized that the thymus was somehow involved with the onset of puberty because it was noted that castration "caused" hypertrophy of the thymus [hammar, ; gregoire, . the thymus and pineal gland have been functionally linked since early in this century. for example, the thymus and pineal gland were reported to function together to enhance somatic growth and development [berman, . treatment with pineal extracts increased thymic mass and induced lymphoidal cell hyperplasia [milcu and pitis, . furthermore, perinatal pinealectomy caused thymic regression [devecerski, . the discovery that the thymus, bursa, and spleen are major components newson, shivatcheva & hadjioloff, newson, shiffrine et al., lochmiller et al.. sealander, stone, aim& hussein, et a ., el ridi et. al., hussein, et al., of the immune system, and the subsequent pursuit of molecular analyses of immune function have ignored, until very recently, the molar relationship, and possible bidirectional interactions, between immune function and the reproductive system [maier et al., . the topic of seasonal variation in the immune systems of poikilothermic animals has recently been reviewed [zapata et al., . there seems to be no consistent seasonal pattern of immune responsiveness in poikilotherms; however, steroid hormones profoundly affect immune function in these animals and seasonal fluctuations in immune function have been linked in some cases to interactions among different steroid hormones [zapata et al., . the role of melatonin in mediating these effects in poikliotherms is largely unknown. in the field, seasonal changes in lymphatic tissue have been thoroughly investigated in birds, but much less so in mammals. avian splenic and thymic sizes are minimal when the gonads undergo vernal recrudescence [e.g., krause, ; riddle, ; oakeson, ; hohn, ; f h g e and silverin, ; john, . mallard ducks (anus plutyrhynchos), in common with other homeothermic vertebrates, undergo thymic involution at puberty [hohn, . a pronounced regeneration of thymic tissue has been observed at the end of each breeding season in the middle of summer. in both male and female adult mallards, however, thymic tissue regresses again prior to the autumnal migration [hohn, . the physiological stress associated with migration and breeding was considered to be incompatible with full thymic size and function [hohn, . similar observations have been made for house sparrows (passer domesticus) and robins (turdus migrutorius) [hohn, . the spleen of white-crowned sparrows (zonotrichia leucophrys garnbelii and . . nuttulli) also regresses at the beginning of their breeding season. this splenic regression cannot be attributed to the "stress of migration" because both migratory and nonmigratory populations displayed similar seasonal patterns of splenic size [oakeson, ; . splenic size (corrected for lean body mass) was lowest prior to breeding and highest at the end of the breeding season in white-crowned sparrows. similarly, migratory pied flycatchers (ficedulu hypoleucu) in sweden also displayed a seasonal cycle of splenic development. splenic regression was observed at the onset of the vernal breeding season; subsequent splenic development was exhibited by the adults during incubation and feeding of the hatchlings [fange and silverin, . the adaptive significance of the development of the spleen prior to the autumnal migration has been suggested to reflect enhancement of immune function, particularly of the young birds after hatching, in advance of winter [fange and silverin, . one parsimonious proximate explanation for the seasonal pattern of lymphatic organ development among birds is that the high gonadal steroid levels associated with breeding are incompatible with highly developed lymphatic tissue. the role of melatonin in mediating seasonal fluctuations in avian immune function has not been examined. the proximate explanation that high gonadal steroid levels are associated with low lymphatic organ weights might also account for some of the data concerning seasonal fluctuations in mammalian lymphatic organ size. for example, mean splenic reticular cell counts varied seasonally in red-backed mice (clethrionomys rutilus), with the main peak observed in early winter and lesser peaks observed in late winter and midsummer [sealander and bickerstaff, . thymus weights were largest in february and spleen weights were largest in september and october; the lowest weights for both organs occurred in july [sealander and bickerstaff, . similarly, thymus masses of pine voles (microtus pinetorurn) were highest in early autumn when reproductive organ masses were declining [valentine and kirkpatrick, . adult and subadult cotton rats (sigrnodon hispidus) display a seasonal cycle of thymic development and regression; thymic masses were depressed during the summer and were maximal during the winter of some, but not all, years [lochmiller et al., . peak splenic masses and peak number of splenocytes were recorded in autumn and late winter, respectively in cotton rats. seasonal changes in lymphatic tissue have also been noted in hibernating mammals. the spleen and gut-associated lymphoid tissues of both hibernating and non-hibernating european ground squirrels (citellus citeflus l.) were examined and a circannual rhythm in the morphology of the splenic lymphoid tissue, as well as the lamina propria of the mucosa, and peyer 's patches was reported [shivatcheva and hadjioloff, a; bl . these lymphatic tissues regressed in the autumn in both hibernating and nonhibernating squirrels, but regression was reported to be more complete in hibernating animals. notably, proliferation and hypertrophy of splenic and gut-associated lymphoid tissues were observed in squirrels prior to arousal in the spring [shivatcheva and hadjioloff, a; bl . the physiological effects of torpor and hibernation on immune function remain unspecified. although suggestive, seasonal changes in lymphatic tissue do not directly inform about alterations in immune function, per se. in the following section, the literature on seasonal . also, infected chickens significantly increased shedding of laryngotracheitis virus after egg laying had commenced [hughes et al., . seasonal changes in mammalian immune function and disease prevalence have also been reported. for instance, seasonal variation exists in the ability of bank voles (clethrionomys glareous) to infect larval ticks with lyme disease (borrelia burgdorferi) [talleklint et al., . although larval tick infestations of voles were highest in june and july, nearly % of borrelia infections occurred during august and september. virtually no infections occurred during the winter. whether these data reflect a seasonal alteration in the immune function of the host or reflect the latency to infection from year to year requires further study. lymphocyte proliferation in response to the mitogens, concanavalin a (con a) and an extract of pokeweed (phytolacca americana) (pwm) was assessed in cotton rats (s. hispidus) [lochmiller et al., . in addition to elevated humoral responses, cotton rats trapped in february also displayed elevated lympho-proliferative responses to con a and pokeweed coinciding with increased numbers of total splenocytes harvested [lochmiller et al., (table ) . total white blood cell (wbc) numbers reached minimum values in december , july , february , and february . the highest numbers of plaque-forming cells (pfc) were recorded in december and february . in another study of rodents, richardson's ground squirrels (citellus richardsoni richardsoni) were trapped during the spring and summer and maintained in natural photoperiods at - °c [sidky et al., . five days after immunization with sheep red blood cells (srbc), the animals were bled and their spleens removed. antibody response to srbc decreased significantly during the winter, reaching the lowest level in january. spleen cell suspensions were tested for the presence of hemoly sin-forming cells by a modification of the pfc assay. pfcs decreased significantly during the winter, reaching the lowest levels in january. however, the number of nucleated cells per spleen increased during the winter, reaching a maximum in january ( % of may values). the fact that these squirrels normally hibernate through the winter may explain the lack of winter enhancement of immune function. again, the effects of hibernation on immune function are virtually unknown. a study of cattle in the southern hemisphere revealed seasonal variation in naturally occurring antibody production against the antigen, substance j, a compound detected on the erythrocytes of some cattle [stone, . blood samples were drawn from cattle monthly and added to culture plates con-changes in immune function and disease prevalence is briefly reviewed. lymphatic organ development is suppressed among birds when gonadal steroid levels are elevated. breeding coincides with an increased prevalence of some avian diseases, and this increased disease rate apparently reflects reduced immune function [john, . numerous studies have demonstrated a seasonal change in parasite and pathogen prevalence [lord, ; descbteaux and mihok, ; lochmiller et al., . in some cases, this seasonal variation seems to reflect seasonal changes in the prevalence of the vector. for example, autumnal epidemics of typhus (rickettsia prowazekii) in wild flying squirrels (glaucomys volans) correspond closely to the maximal numbers of the arthropod vectors, viz, fleas and lice [sonenshine et al., . the overwhelming evidence, however, indicates that seasonal fluctuations in disease and death rates reflect a seasonal change in the immune function of the host, rather than seasonal changes in the parasite or pathogen [john, . for example, house sparrows (passer domesticus) infected with avian malaria (plasmodium relictum) display a relapse of symptoms occurring synchronously throughout a population of infected birds and coincident with the onset of vernal breeding activities [applegate and beaudoin, . gonadotropin treatment, either alone or in combination with corticosterone, stimulated gonadal development, but did not change the timing of incidence of the relapse. these results suggest that gonadal steroids are not involved in the prevalence of avian malaria, but that another factor associated with breeding affects susceptibility to this disease. the interaction between steroid hormones and immune function is described more fully below. ducks infected with leucocytozoon, a parasite related to avian malaria, also display a vernal relapse of symptoms [chernin, . when day lengths were experimentally increased during the winter, the relapse could be phase-advanced. malaria among humans has also been reported to show increased vernal relapses, but these relapses have been considered to be due to a fixed interval of disease progression following autumnal infections [coatney and cooper, ; also see below]. suppression of immune function during breeding has also been reported for birds with viral infections. for example, homing pigeons (columba livia) maintained in semi-natural conditions and latently infected with pigeon herpes virus displayed an increased rate of viral shedding during breeding [vindervogel et al., taining substance j. low antibody titers were present in january (summer), with levels rising thereafter to peak levels in august (winter). after this peak, levels began to drop, again returning to a minimum in january. similarly, the rate of seropositive responses in cattle to borrelia burgdorferi varied seasonally with the population infection incidence highest during the summer (up to . %) and lowest during the winter ( % in january) [isogai et al., . the seasonal occurrence of the equid herpes virus- (ehv- ) in foals was studied in australia [gilkerson et al., . nasal swabs were obtained once a month for a year in order to detect the presence of ehv- antibodies. twenty-six foals were ehv- positive, and all of these seropositive animals were discovered in the summer months of january, february, and march ( in january and march, in february) [gilkerson et al., . no seropositive animals were detected in the winter months. outbreaks of european brown hare syndrome (ebhs) displayed a strong seasonal fluctuation among lepus europeus in sweden with peak occurrence observed during the winter . similarly, rabbit viral hemorrhagic disease (vhd) exhibited a peak incidence during the winter. again, the extent to which these animals were engaged in winter breeding was not reported. outbred male and female beagle dogs ( days of age) maintained in open colonies were examined to assess seasonal changes in immune function [shifrine et al., al . whole blood lymphocyte proliferation tests were conducted by adding either phytohemagglutinin (pha) or con a to the monthly samples. the results of the lymphocyte proliferation tests demonstrated a peak in june/july and a trough observed in january. the reproductive status of these dogs was not described. in a follow-up study, blood samples were taken from beagles at various times throughout the year [shifrine et al., bl . another lymphocyte proliferation test was conducted on the samples. greatest lymphocyte proliferation in response to both mitogens occurred during the summer, but the peak for samples incubated with pha was phased-advanced several weeks as compared to samples incubated with con a. in summary, immune function in birds and some mammals appears to be generally compromised, and diseases are more prevalent, during the breeding season. although there are data from many sources indicating seasonal changes in lymphatic tissue size and morphology, as well as immune function, there is significant variation among different populations of animals. because so many factors can influence steroid hormone levels and these factors vary across populations, field studies are difficult to compare. determining the causative agents and understanding the additive effects of these agents on the immune system requires laboratory studies in which one or more factors are altered in an otherwise stable and controlled environment. when only photoperiod has been experimentally manipulated in a controlled environment, the results clearly indicate that short days are coincident with elevated lymphatic organ mass and immune function. these studies are reviewed in the following section. laboratory strains of rats (rattus norvegicus) are traditionally considered to be reproductively nonresponsive to photoperiodic information [nelson et al., . nevertheless, maintaining adult wistar male rats in constant dark (dd) for weeks increased thymic mass by % over rats maintained in an ld : photoperiod; most of the increase was observed in the lymphatic tissue within the thymic medulla [mahmoud et al., ) . the number of thymocytes also increased in dd animals. rats maintained for weeks in constant bright light (ll) decreased thymic mass to % of values of ld : rats; the reduction in total volume represented mainly reductions in the thymic cortex [mahmoud et al., . because photoperiod does not affect steroid hormones in male rats [nelson et al., , these data strongly suggest that melatonin acts directly upon immune function [mahmoud et al., . previous studies on rats have indicated slight photoperiod-induced changes in splenic weight [wurtman and weisel, . laboratory strains of house mice (mus musculus) also display seasonal rhythms of immune function despite insignificant reproductive response to photoperiod. for instance, young c bl/ mice (mus musculus) were maintained in an ld : photoperiod [brock, ; splenic lymphocytes were stimulated with mitogens and viable and non-viable lymphocytes were counted throughout the year. peak responses in t and b lymphocyte populations were - times higher in march-april and february-march than in either of the two previous decembers. summer comparisons were not reported. again, these animals, like laboratory strains of rats, typically are reproductively nonresponsive to photoperiod [nelson, . differences in seasonal patterns have been reported between strains of mus. the maximal numbers of splenic pfc to srbc injection occurred in spring for cdl females and in summer for b c fi mice [ratajczak et al., . short day lengths appear more effective at me-elevated splenic mass [vaughan et al., . elevated splenic mass could be prevented in short-day hamsters by pinealectomy [vaughan et al., . importantly, melatonin mediates immune function [maestroni, . in virtually all cases examined, melatonin enhanced humoral and cell-mediated immunity [maestroni, ; guerrero and reiter, . melatonin treatment of both normal and immunocompromised house mice elevated in vitro and in vivo antibody responses [caroleo et al., ; maestroni, . impaired t-helper cell activity in immunocompromised mice is restored by melatonin treatment [caroleo et al., . antigen presentation by splenic macrophages to t cells is also enhanced by melatonin; furthermore, this enhancement is coincident with an increase in major histocompatibility (mhc) class i molecules, as well as interleukin ( l)- and tumor necrosis factor (tnf,) production [pioli et al., . murine antibody-dependent cellular cytotoxicity (adcc) is reduced in adult mice that were pinealectomized prior to days of age [vermeulen et al., . adcc is a lytic process that occurs when lymphocytes bind to specific antibody-coated target cells through receptors for the fc portion of the ig molecule expressed on their membrane. the impairment in adcc appears peripubertally, around days of age, suggesting an involvement of sex steroid hormones [vermeulen et al., . pinealectomy also ameliorates collagen -induced arthritis in mice [hansson et al., , as well as inhibits humoral immune function and suppresses bone marrow progenitors for granulocytes and macrophages [kuci et al., . additionally, natural killer (nk) cell activity and il- production are reduced in mice after pinealectomy [del gobbo et al., . as predicted [maestroni, ; guerrero and reiter, , melatonin receptors have been isolated on circulating lymphocytes [calvo et al., ; pang and pang, ; pang et al., ; poon and pang, ; liu and pang, , as well as on thymocytes and splenocytes [lopez-gonzales et al., ; martin-cacao et al., ; rafii-el-idrissi et al., . the melatonin receptors on lymphatic tissue appear similar in kd values to melatonin receptors localized in rat and hamster brains, and also seem to be coupled to g-protein(s) [calvo et al., . melatonin partially inhibits cyclic amp production in human lymphocytes, but only at pharmacological doses [rafii-el-idrissi et al., . the circadian synthesis and release of melatonin modulates antibody response and also alters tumorigenesis [see blask, . at the normal cellular level, melatonin is believed to affect antimitotic processes as well as cytotoxic activity [boucek and alvarez, ; poffenbarger and fuller, ; win-diating immune function in individuals with robust reproductive responses to photoperiod. for instance, splenic weights of deer mice (peromyscus maniculatus) [vriend and lauber, , and syrian hamsters (mesocricetus auratus) [brainard, were reduced in short days. splenic masses, total splenic lymphocyte numbers, and macrophage counts were significantly higher in hamsters exposed to short days, as compared to animals exposed to long photoperiods [brainard et al., ; . however, photoperiod did not affect thymic weight or antibody production in hamsters [brainard et al., . photoperiodic influences on lymphocyte number and total white blood cell count have been reported for deer mice [blom et al., . animals maintained in short day lengths (ld : ) possessed more white blood cells than animals maintained in long day lengths (ld : ); neutrophil numbers were unaffected by day length in adult female mice. more recently, deer mice maintained in short days displayed faster healing rates than long day mice [nelson and blom, . short day lengths appear to bolster immune function (table ) . one likely physiological mechanism by which photoperiod affects immune function is via alterations in the pattern of melatonin secretion. importantly, lymphatic cells of both birds and mammals possess melatonin receptors [reviewed in calvo et al., . in vivo melatonin treatment bolsters immune function. the pattern of melatonin release induced by short days affects the secretion of other hormones. the precise mechanisms through which photoperiod interacts with the endocrine system and exerts influences on the immune system are not known. the presence of receptors for both androgens in the thymus and for estrogens in the cytosol of circulating lymphocytes might explain why these steroid hormones play an important role in the mediation of immune function [grossman, ; hall and goldstein, . prolactin is another hormone that is profoundly affected by day length and also affects immune function. thus, photoperiodic effects on immune function may reflect photoperiod-mediated changes in blood concentrations of prolactin. consequently, the possibility that melatonin might act both directly and indirectly on the immune system is strong. the effects of these specific endocrine interactions upon immunity are reviewed in the following sections. the pineal gland and the primary secretory pineal product, melatonin, can affect lymphatic tissue sizes. for example, exposure of male and female hamster to short days or daily afternoon melatonin injections ston et al., . when the synthesis of endogenous melatonin is blocked, antibody production is depressed [maestroni and pierpaoli, ; maestroni, et al., . in contrast, transplantation immunity is not affected by pinealectomy [maestroni and pierpaoli, ; maestroni, et al., . pharmacological and surgical pinealectomy also modulate other immune parameters including plaque-forming cells and blastogenic responses of splenocytes and thymocytes to various mitogens [becker et al., ; kuci et al., . furthermore, elimination of melatonin synthesis by pinealectomy profoundly decreased the proliferation of bone marrow progenitors for granulocytes and macrophages (cfu-mg); disruption of the night-time peak of melatonin completely abolished cfu-mg proliferation [kuci et al., . whenever examined, compromised immune function caused by pinealectomy could be ameliorated by melatonin replacement therapy [maestroni, . the effects of melatonin on immune function appear to be related to seasonal changes in tissue sensitivity to this indoleamine. for example, in balb/c mice melatonin injections enhanced adcc in response to chicken red blood cells (crbc) when given during the summer [giordano et al., . melatonin treatment during the winter failed to enhance adcc. melatonin is important in many disease processes, especially cancer [blask, ; maestroni, ; guerrero and reiter, ; giordano et al., ; pioli et al., ; caroleo et al., ; nelson and demas, . the overwhelming majority of studies indicate that melatonin is an oncostatic hormone. a number of treatments for cancer now incorporate melatonin as part of the immunomodulatory therapy [e.g., see barni et ai., ; neri et al., ; lissoni et al., ; . in summary, melatonin appears to enhance immune function in most cases. in common with reproductive responses mediated by melatonin, there may be a temporal component to the biological actions of this indoleamine. most studies of melatonin effects on immune function have used animals that are not particularly responsive to this hormone (e.g., laboratory rodent strains) and may have overlooked the temporal components of melatonin influences. again, sustained release of melatonin is higher in short, as compared to long days. short-day induced changes in melatonin secretion evoke a cascade of other endocrine changes. notably, steroid hormone and prolactin secretion declines dramatically in short days. short days, or timed melatonin treatments, elicit gonadal regression in many species. gonadal regres- sion in males is coincident with reduced circulating levels of testosterone. testosterone generally suppresses immune function. castration of adult male rodents results in increased humoral and cell-mediated immunity, as well as increased lymphatic organ size, including thymic, splenic, and lymph nodal masses [schuurs and verheul, . castration of male rodents leads to immune parameters that are similar, but not equivalent to females [grossman, ; this suggests that some of the sex difference in immune function is organized prior to puberty. treatment of adult castrated males with physiological doses of testosterone restores (i.e., compromises) immune function to pre-castration levels [schuurs and verheul, ; grossman, . testosterone treatment of castrated or intact male rats or mice significantly suppresses humoral and cell-mediated immunity, as well as thymic mass [schuur and verheul, ; grossman, . androgen receptors have been identified in thymic tissues, particularly in the epithelial, lymphatic portion of the thymus [mccruden and stimson, ; sasson and mayer, . because no androgen receptors have been identified on circulating lymphocytes, androgenic effects on lymphocytes may be indirect or act through aromatization of androgens to estrogens [mccruden and stimson, . because blood androgen levels decrease in short days, this photoperiodic treatment is similar to a functional castration. thus, enhancement of immune function could be due to removal of the immunosuppressive effects of androgens in shortday animals. dehydroepiandrosterone (dhea) is a weak androgen that is produced primarily in the adrenal cortex. dhea serves as an intermediate in the production of androstenedione from a-hydroxyprogesterone. in addition to its role in the steroid biosynthesis pathway, a number of recent reports suggest that dhea may have potent physiological effects on immune function [casson et al., ; morales et al., . dhea acts as an antiglucocorticoid, and enhances il- production and cytotoxicity of activated t cells in mice and humans [suzuki et al., . dhea also increases immunological protection against a herpes virus type encephalitis, and also protects against systemic coxsackievirus b infection [loria and padgett, . dhea-treatment also prevented the reduction in humoral and cellular immune function usually observed after thermal injury [araneo et al., . the role of dhea in seasonal fluctuations in immune function requires investigation. in contrast to the pattern of androgen receptor localization, estrogen receptors have been localized in ratory burst and phagocytosis of peritoneal macrophages from both young and old mice [chen and johnson, . prolactin induces resting lymphocytes to divide, and can also affect the magnitude of their response to polyclonal stimuli. prolactin also influences the effector phase of the immune response, including increased response of nk cells, t-cells, and b-cells to mitogenic signals [matera et al., . membrane-bound prolactin receptors have been discovered on lymphocytes [reber, ; bernton et al., ; . furthermore, prolactinlike substances have been identified in mouse splenocytes and human b-lymphoblastoid cell lines [sabharwal et al., ; reber, . cyclosporin a directly competes with prolactin for binding of the lymphatic receptors. it has been proposed that the immunocompromising effects of cyclosporin a may result from interference with a prolactin-like autocrine growth factor for lympho-proliferation [sabharwal et al., ; reber, . the cytosol of circulating lymphocytes [danel et a!., ; grossman, , cd + cells [cohen et al., ; stimson, , and thymic cells [danel et al., ; nilsson et al., ; weusten et al., . physiological treatments with estrogen or the estrogen receptor antagonists, tamoxifen or fc- a, enhance pokeweed mitogen (pwm)-induced immunoglobulin synthesis of b-lymphocytes [paavonen and anderson, ; sthoeger et al., . treatment of intact male or gonadectomized male or female mice and rats with physiological or supraphysiological doses of estrogens increases antibody responses to a variety of t-dependent and tindependent antigens [inman, ; myers and peterson, ; brick et al., . cyclic exposures to pharmacological doses of estrogens are more effective in boosting antibody formation than chronic exposure to pharmacological estrogen doses [schuurs and verheul, . however, prolonged pharmacological doses of estrogens may also suppress cell-mediated immunity [grossman, ; kuhl et al., . taken together, the effects of physiological doses of estrogen appear to enhance immune function. blood estrogen (and androgen) levels are low in short-day females. thus, enhancement of winter immune function is unlikely to involve photoperiod-mediated changes in blood levels of estrogens. the proximate effects of estrogens probably account for the superiority of female immune function as compared to males [grossman, ; schuurs and verheul, . exposure to short day lengths reduces blood prolactin levels in every mammalian species thus far examined [goldman and nelson, . treatment with melatonin in ways that mimic release patterns associated with short day lengths also suppresses blood prolactin titers [goldman, ; bittman, . prolactin has pronounced effects upon immune function in a variety of species [reviewed by bernton et al., ; ; reber, ; arkins et al., ; matera et a]., ; castanon et al., . generally, prolactin maintains or enhances normal immunological activities, but there are also examples of prolactin compromising immune function, particularly at high or low circulating levels [reber, . because exposure to short day lengths suppresses circulating prolactin levels, this hormone is a possible candidate for mediating some of the reported seasonal changes in immune function. hypophysectomy of rats results in compromised humoral and cell-mediated immunity; immune function can be restored by prolactin replacement therapy [reber, . prolactin elevates the respi- many interactions between glucocorticoids and immune cell function have been reported in relation to environmental stress [reviewed in nakono et al., . however, the mechanisms underlying seasonal changes in stress hormones and immune function have not been elucidated. adrenocortical hormones, especially glucocorticoids, suppress immune function in both humans and nonhuman animals [baxter, ; clamin, ; hauger, ; ader and cohen, ; black, . glucocorticoids are released in response to stressful stimuli, and can compromise cellular and humoral immune function [berczi, ; levi et al., . adrenalectomy enhances lymphatic organ masses and bcell activities [del rey et al., . the precise mechanisms by which the immune system is affected by the hypothalamo-hypophysial-adrenocortical axis are unknown, but probably involve cytokine release rates from activated immunological cells [besedovsky et al., ; besedovsky et al., ; besedovsky and del rey, . regardless of mechanism, substantial evidence links glucocorticoids with suppressed immune function. recently, a direct link between melatonin and glucocorticoid biology has been established. generally, melatonin enhances immune function, whereas glucocorticoids compromise immune function [gupta, ; maestroni et al., ; maestroni, ; maier et al., . melatonin treatment can ameliorate the immunocompromising effects of glucocorticoids [maestroni et al., ; aoyama et al., ; . cortisol treatment of ducklings reduced the number of thymic melatonin receptors [poon et al., . similarly, chronic melatonin treatment decreased the density of thymic glucocorticoid and progestin receptors in rats [persengiev et al., . previous studies have demonstrated that environmental stressors elevate blood glucocorticoid levels and that high glucocorticoid levels suppress immune function [baxter, ; clamin, ; hauger, ; ader and cohen, ; black, ; fauci, ; kawate et al., ; besedovsky and del rey, . for example, low ambient temperatures are often perceived as stressful, and can potentially depress immune function [e.g., clamin, ; macmurray et al., ; monjan, . winter survival in small animals is hypothesized to require a positive balance between short-day enhanced immune status and glucocorticoid-induced immunosuppression [demas and nelson, . this immunosuppression may be due to many factors, including overcrowding, increased competition for scarce resources, low temperatures, reduced food availability, increased predator pressure, or lack of shelter. each of these potential stressors may cause high blood concentrations of glucocorticoids. winter breeding with its concomitant elevation in sex steroid hormones may also cause immunocompromise [e.g., tang et al., ; lochmiller et al., . presumably, winter breeding occurs when other environmental stressors such as temperature and food availability are not severe. the balance of enhanced immune function (i.e., to the point where autoimmune disease becomes a danger) against stress-induced immunosuppression (i.e., to the point where opportunistic pathogens and parasites overwhelm the host) must be met for animals to survive and become reproductively successful. thus, the mediation of reproductive function and immune function will likely be intertwined [besedovsky and del rey, . although stress generally results in compromised immune function, the degree of immunological response to stress varies seasonally. for example, rat hemagglutination titer response to srbc was suppressed in response to electric-shock stress compared to control animals during the winter. in the summer, however, shock-stressed rats displayed enhanced antibody response to srbc relative to control animals [amat and torres, . recently, the interaction between photoperiod and temperature was examined on antibody levels and splenic mass in male deer mice [demas and nelson, . animals were maintained in ld or ld : photoperiods in either " or °c temperatures. serum igg levels were elevated in shortday mice maintained at normal room temperature as compared to long-day animals (fig. i) levels; mice exposed to short days and low temperatures had igg levels comparable to long-day mice maintained at °c. in other words, short days elevated igg levels over long days. low temperatures caused a significant reduction in igg levels. the net effect of short-day enhancement and low temperature reduction of igg levels is no appreciable difference from baseline (i.e., long-day mice kept at °c). this adaptive system may help animals cope with seasonal stressors and ultimately increase reproductive fitness. the possibility that photoperiod affects human reproductive function remains open [ see bronson, . similarly, the possibility that photoperiod affects human immune function is largely unexplored. many human diseases show remarkable seasonal fluctuations (table ) . malaria is a common seasonal disease among humans residing in the tropics [theander et al., . there are a number of reports of seasonal changes in immune function associated with malaria. as noted previously, malaria has been reported to show increased vernal relapses in humans. typically, these relapses have been at- cornelius, hall, miller, cavallaro and monto, hamre and beem, hendley et al., beal, carpenter and gardner, glezen et al., kapikian et al., chougnet et al., douglas et al., biller et al., dunnigan and harland, biller et al., blom and dahlquist, winter ownby et al., spring-early summer chleboun and gray, cohen et al., kirkham et al., summer sankila et al., spring-summer jacobsen and janerich, kirkham et al., mason et al., fall-winter hostmark et al., afor cases speculated to be caused by a respiratory virus. tributed to a fixed interval of disease progression following autumnal (onset of the wet season) infections [coatney and cooper, . antigen-induced cellular immune responses to plasmodium falciparum are compromised during acute malaria onset [abu-zeid et al., ; chougnet et al., ; theander et al., . lymphocyte proliferation responses (against non-malaria antigens) of healthy individuals were also compromised during the malaria transmission season [theander et al., . this suggests that immune function might be suppressed during the time of plasmodium falciparum infections. as is the case for nonhuman animals, the extent to which the seasonal changes in human disease and death rates reflect changes in the host or in the pathogen requires further study in most cases. the interaction among melatonin deficiencies, immune function, and disease processes may be profound. there is evidence from several sources that day length may affect human immune function and disease prevalence. individuals suffering from seasonal affective disorder (sad) often exhibit aberrations in their immune cell counts, especially during their winter-depression [rosen et al., . for example, some patients with sad display aberrant lymphocyte proliferation in response to mitogenic stimulation [skwerer et al., . treatment of the sad symptoms with bright illumination ameliorates these immunological abnormalities [skwerer et al., . total number of circulating nk cells was reduced among sad patients in another study [kasper et al., ; the reduction was inversely related to the score attained on a test of depression. after bright light therapy, the symptoms of depression ameliorated and nk cell numbers increased. furthermore, lymphocyte proliferation in response to con a and pwm improved after phototherapy [kasper et al., . thus, these studies indicate that immune function is significantly compromised in the winter among patients who suffer from sad [rosen et al., . another observation that is consistent with a photoperiodic influence on human immune function is the association between latitude and multiple sclerosis (ms) [davenport, ; limburge, ; kurtzke, ; . the prevalence of ms increases at higher latitudes, both north and south [reviewed in rosen et al., . a consistent correlate with ms is the amount of december solar radiation (i.e., high numbers of sunny hours in december are associated with low numbers of ms cases in the region) [acheson et al., . seasonal changes in human immune function have also been established in healthy people. accordingly, both measurements of cellular and humoral immunity display seasonal variation. for example, the percentage of viable b and t cells was significantly elevated in winter subjects compared to those tested in the summer [macmurray et al., . in another study, a group of six blood donors was tested, and absolute values and percentage of b and t-cells in peripheral blood were examined over the course of year [bratescu and teodorescu, . although the total number of lymphocytes and leukocytes did not vary throughout the year, the proportion of b cells to t cells was nearly doubled during the winter months compared to the summer months [bratescu and teodorescu, . seasonal differences in mitotic activity of normal human peripheral blood lymphocytes have also been examined [boctor et al., . in healthy males and females, increased proliferative responses of peripheral blood lymphocytes to con a and pha were observed in the summer months compared to the winter months [boctor et al., . observations of humoral immunity in humans have yielded conflicting results. in one study, blood samples of patients from five different va hospitals had significantly higher igg levels in the winter samples compared to those of summer for four out of five hospitals [macmurray et al., . conversely, examination of seasonal variation in a variety of serum proteins from adult and children outpatients, revealed that concentrations of igg were greater during the summer months as compared to the winter months [lyngbye and krflll, . iga and igm levels did not differ significantly across seasons in these studies [lyngbye and krflll, ; macmurray et al., . in another study of healthy adults and children, no significant seasonal changes were observed on serum igg, igm, or iga levels [stoop et al., . similarly, serum sampled over a month period from healthy adults and children, revealed no seasonal changes in immunoglobulin concentrations, though serum igm showed the greatest variability in the fall-winter period [nordby and cassidy, . taken together, the seasonal, photoperiodic, and pineal melatonin studies suggest that melatonin enhances immune function. although progress is being made to determine the physiological mechanisms underlying the effects of melatonin on immune function, new insights may be gained by understanding the adaptive significance of melatonin effects on immune function. answers at this level of analysis might guide questions on the proximate, physiological level of analysis. similar to humans, laboratory strains of rats and mice are traditionally unresponsive to melatonin. these laboratory species may be useful for understanding the effects of melatonin on human immune function. alternatively, these artificially selected species, especially albino strains, may present limitations on our understanding [e.g., turek et al., ; vollrath et al., ; champney et al., ; olcese and reuss, ; webb et al., ; lynch et al., . the effects of timed infusions of melatonin that mimic naturally occurring patterns of endogenous secretion are also required to understand melatonin-immunity interactions. melatonin may enhance immune function to help the individual cope with seasonal stressors that would otherwise compromise immune function to critical levels. fluctuating immune function may represent adaptations that have evolved to increase the odds of surviving changes in energy availability. this review systematically examined the interaction of melatonin, photoperiod, and immune function in an ecologically-relevant manner. the clinical implications of seasonal fluctuations in immune function may be significant in forecasting and treating human diseases. preparation of this review and funding of unpublished experimental data were supported by usphs grants hd and ca . we thank drs. thomas hahn and courtney devries for reading the manuscript. stress and mortality in a small marsupial neuroimmunology: modulation of the hamster immune system by photoperiod effect of light irradiance and wavelength on the syrian hamster reproductive system neuroendocrine regulation of immune parameters circannual variations in the b cell/ t cell ratio in normal human peripheral blood hormonal modulation to thymus-independent and thymus-dependent antigens in autoimmune nzb/w mice seasonal rhythmicity in lymphocyte blastogenic responses of mice persist in a constant environment mammalian reproductive biology seasonal variation in human reproduction: environmental factors ) immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells doria ( ) melatonin as immunomodulator in immunodeficient mice environmental findings and sudden infant death syndrome oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women prolactin as a link between behavioral and immune differences between the roman rat lines monto ( ) community-wide outbreak of infection with a e-like coronavirus in tecumseh, michigan hormonal modulation of pineal melatonin synthesis in rats and syrian hamsters: effects of streptozotocin-induced diabetes and insulin injections in vivo activation of macrophages by prolactin from young and aging mice the relapse phenomenon in leucocytozoan infections of the domestic duck tancini ( ) a randomized study of low-dose subcutaneous interleukin- plus melatonin versus supportive care alone in metastatic colorectal cancer patients progressing under -fluorouracil and folates mammalian pineal melatonin: a clock for all seasons forsham ( ) the effects of glucorticoids some epidemiological factors about sudden infant death syndrome (sids) in south australia circadian variations in the immunomodulatory role of the pineal gland the influence of the pituitary-adrenal axis on the immune system the glands regulating personality prolactin and prolactin secretagogues reverse immunosuppression in mice treated with cycteamine, gluccocorticoids, or cyclosporin-a feed-back interactions between immunological cells and the hypothalamus-pituitaryadrenal axis lymphokine-containing supernatants from con a-stimulated cells increase corticosterone blood level immunoregulatory feedback between interleukin-i and glucocorticoid hormones seasonal variation of stroke-does it exist central nervous system-immune system interactions: psychoneuroendocrinology of stress and its immune consequences the pineal: an oncostatic gland? in: the pineal gland dahlquist ( ) epidemiological aspects of the natural history of childhood diabetes immune function in deer mice: developmental and photoperiodic effects seasonal differences in the rhythmicity of human male and female lymphocyte blastogenic responses -hydroxytryptamine: a cytospecific growth mediator of cultured fibroblasts the profile of breast cancer in western australia ) humoral and cell-mediated immune responses to the plasmodium falciparum antigens pf iresa and cs protein: seasonal variations in a population recently reexposed to endemic malaria corticosteroids and lymphoid cells recrudescence and relapse in the vivax malaria seasonality in the occurrence of breast cancer seasonality of gonorrhea in the united states specific estrogen binding sites in human lymphoid cells and thymic cells multiple sclerosis from east and point of geographic distribution and race photoperiod and temperature interact to affect immune parameters in male deer mice (p eromyscus manicutatus) serologic study on the prevalence of murine viruses in a population of wild meadow voles (microtus pennsylvanicus) age and androgen-related changes in morphological parameters, haematological indices and serum protein fraction in common vole (microtus arvalis pall) growing in different photoperiods seasonal, regional and secular variations of cardiovascular and cerebrovascular mortality in new zealand psychoneuroimmunology for the psychoneuroendocrinologist: a review of animal studies of nervous system-immune system interactions effect of seasonal variations on the immume system of the snake psammophis schokar silverin ( ) variation of lymphoid activity in the spleen of a migratory bird, the pied flycatcher ) corticosteroids and circulatory lymphocytes epidemiology and diagnosis of the european brown hare syndrome in scandinavian countries: a review ) epidemiological investigation of equid herpesvirus- (ehv- ) excretion assessed by nasal swabs taken from thoroughbred foals seasonal variations in antibody cellular cytotoxicity regulation by melatonin downs ( ) mortality and influenza nistico ( ) pinealectomy inhibits interleukin- production and natural killer cell activity in mice the physiology of melatonin in mammals melatonin and seasonality in mammals sur le mechanisme de i'hypertrophie thymique declanchee par la castration regulation of the immune system by sex steroids interactions between the gonadal steroids and the immune system a brief survey of pineal gland-immune system interrelationships the pineal gland: its immunomodulatory role respiratory syncytial virus die menschenthymus in gezundheit und krakheit. i das organ unter anomale verhaltniss. zeitung mikroskopanatomie forschung beem ( ) virologic studies of acute respiratory disease in young adults. v. coronavirus e infections during six years of surveillance pinealectomy ameliorates collagen -induced arthritis in mice aguilera ( ) corticotropin releasing factor receptors and pituitary adrenal responses during immobilization stress ) photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation coronavirus infections in working adults. eight-year study with e and oc the effect of environmental temperature on immune response and metabolism of the young chicken. . effect of the immune response to sheep red blood cells on energy metabolism seasonal cyclical changes in the thymus of the mallard seasonal recrudescence of the thymus in adult birds seasonal variations of symptoms and occurrence of human bladder carcinomas. scand effects of certain stress factors on the re-excretion of infectious laryngotracheitis virus from latently infected carrier birds a randomized study of immunotherapy with low-dose subcutaneous interleukin- plus melatonin vs chemotherapy with cisplatin and etoposide as first-line therapy for advanced non-small cell lung cancer ]iodomelatonin binding sites in the bursa of fabricus of birds: binding characteristics, subcellular distribution, diurnal variations and age studies temporal variation in humoral and cell-mediated immune response in a sigmodon hispidus population interaction of melatonin with human lymphocytes: evidence for binding sites coupled to potentiation of cyclic amp stimulated vasoactive intestinal peptide and activation of cyclic gmp ) seasonal reproduction of vampire bats and its relation to seasonality of bovine rabies mobilization of cutaneous immunity for systematic protection against infections light intensities required to suppress nocturnal melatonin secretion in albino and pigmented rats ) quantitative immunoelectrophoresis of proteins in serum from a normal population: season-, age-, and sex-related variations sex hormones and immune function endocrine changes in dasyurid marsupials with differing mortality patterns circannual changes in immune function the immunoendocrine role of melatonin role of the pineal gland in immunity. circadian synthesis and release of melatonin modulates the antibody response and antagonizes the immunosuppressive effect of corticosterone pharmacologic control of the hormonally mediated immune response continuous darkness and continuous light induced structural changes in the rat thymus psychoneuroimmunology: the interface between behavior, brain, and immunity ) binding of -[' ]melatonin by rat thymus membranes during postnatal development seasonal variation in breast cancer detection: correlation with tumour progesterone receptor status effect of seasonal variation on immune system of the lizard. scincus scincus immunologic sex differences and the female preponderance in systemic lupus erythematosus. arthritis rheum enokidani ( ) seroepidemiological survey for antibody to borrelia burdorferi in cows seasonal variation in the diagnosis of breast cancer the avian spleen: a neglected organ quantitative effects of nutritionally essential amino acid deficiencies upon immune responses to tumors in mice human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children immunological correlates of seasonal fluctuations in mood and behavior and their relationship to phototherapy studies on the bioperiodicity of the immune response. ii. covariations of murine t and b cells and a role of corticosteroids ) immunological consequences of changing environmental stimuli seasonality and breast cancer die milz circadian variations of the immunomodulatory role of the pineal gland the effect of sex steroids and hormonal contraceptives upon thymus and spleen of intact female rats a reassessment of the distribution of multiple sclerosis geographic distribution of multiple sclerosis: an update with special reference to europe and the mediterranean region circadian rhythms in circulating t lymphocyte subtypes and plasma testosterone, total and free cortisol in five healthy men the geographic distribution of multiple sclerosis and its estimated prevalence in the united states lewinski ( ) immunoendocrine therapy with low-dose subcutaneous interleukin- plus melatonin of locally advanced or metastatic endocrine tumors oberholtzer ( ) modulatory effect of prolactin on the resting and mitogeninduced activity of t, b, and nk lymphocytes anemia at the onset of winter in the meadow vole (microtus pennsylvanicus) pitis ( ) contributions l'etude de la correlation thymo-epiphysaire respiratory syncytial virus and the use of ribavirin winter adaptations of male deer mice and prairie voles that vary in reproductive responsiveness to photoperiod stress and immunologic competence: studies in animals effects of replacement dose of dehydroepiandrosterone in men and women of advancing age estradiol induced alterations of the immune system. i. enhancement of igm production significance of increased secretion of glucocorticoids in mice and rats injected with bacterial endotoxin photoperiodic responsiveness in house mice mechanisms of seasonal cycles of behavior photoperiodic effects on tumor development and immune function seasonal changes in immune function photoperiodic effects on reproductive function in male rats grechi ( ) modulation of human lymphoblastoid interferon activity by melatonin in metastatic renal cell carcinoma. a phase i study seasonal differences in reticulocyte count, hemoglobin levels and spleen weight in wild voles specific binding of -beta estradiol in the human thymus seasonal effect on the variability of summer immunoglobulin levels cyclic changes in liver and spleen weight in migratory whitecrowned spmws stress, emotion, and human immune function magnetic field effects on pineal gland melatonin synthesis: comparative studies on albino and pigmented rodents seasonal variation in tumor size at diagnosis and immunologic responses in human breast cancer the oestrogen antagonists, tamoxifen and fc- a, display oestrogen like effects on human lymphocyte functions in vitro pang ( ) [ ]iodomelatonin binding sites in the lung and heart: a link between the photoperiodic signal, melatonin, and the cardiopulminary system pang ( ) high affinity specific binding of -[ ]iodomelatonin by spleen membrane preparations of chicken velev ( ) melatonin effects on thymus steroid receptors in the course of primary antibody responses: significance of circulating gluccocorticoid levels melatonin increases antigen presentation and amplifies specific and nonspecific signals for t-cell proliferation is melatonin a microtubule inhibitor? evidence for a direct action of melatonin on the immune system cortisol decreases [' ] iodomelatonin binding sites in the duck thymus pang ( ) [ ]iodomelatonin binding sites in spleens of guinea pigs specific binding of -[' ]iodomelatonin by rat splenocytes: characterization and its role on regulation of cyclic amp production evidence for genetic basis of seasonal differences in antibody formation between two mouse strains prolactin and immunomodualtion melatonin: the chemical expression of darkness - . riddle, . ( ) studies on the physiology of reproduction in birds. xix. a hitherto unknown function of the thymus tiple sclerosis and latitude: a new perspective on an old association function of melatonin in thermoregulatory processes malarkey ( ) prolactin synthesized and secreted by human peripheral blood mononuclear cells: an autocrine growth factor for lymphoproliferation muivariation in the capacity of the bank vole to infect larval ticks (acari: ixodidue) with lyme's disease spirochete, borrelia burgdorferi interaction of cold and starvation in the regulation of plasma corticosterone levels in the male rat reduced cellular immune reactivity in healthy individuals during the malaria transmission season differential effects of melatonin on the testes of photoperiodic and nonphotoperiodic rodents ) seasonal changes in reproductive and related organs in the pine vole, microrus pinetorum, in southwestern virginia splenic hypertrophy and extrameduallry hematopoiesis induced in male syrian hamsters by short photoperiod or melatonin injections and reversed by melatonin pellets or pinealectomy neonatal pinealectomy impairs murine antibody-dependent cellular cytotoxity observation of pigeon herpesvirus re-excretion during the reproduction period in conventionally reared homing pigeons serotonin and melatonin contents in the pineal glands from different stocks and strains of laboratory rats effects of light intensity, wavelength and quanta on gonads and spleen of the deer mouse photoreceptor damage and eye pigmentation: influence on the sensitivity of rat pineal n-acetyltransferase activity and melatonin levels to light at night. neuroendocrinology margulis (i ) melatonin: cellular effects on live stensors correlated with the inhibition of choline-binding on microtubule protein environmental lighting and neuroendocrine function: relationship between spectrum of light source and gonadal growth torroba ( ) seasonal variations in the immune system of lower vertebrates does the month of diagnosis affect survival of cancer patients? antiglucocorticoid activity of androgens in rat thymus lymphocytes effects of gender and sex steroids on the immune response seasonal changes in reticulocyte number and in relative weights of the spleen, thymus, and kidneys in the northern red-backed mouse seasonal variations in lectin-induced lymphocyte transformation in beagle dogs seasonal variation in cell mediated immunity of clinically normal dogs seasonal involution of gut-associated lymphoid tissue of the european ground squirrel seasonal variations of the immune response of ground squirrels kept at - °c. can neurobiology of seasonal affective disorder and phototherapy elisberg ( i ) epizootiology of epidemic typhus (rickettsi prowazekii) in flying squirrels life-history tactics: a review of the ideas regulation of the immune response by sex hormones. i. in vitro effects of estradiol and testosterone on pokeweed mitogeninduced human b cell differentiation oestrogen and human t lymphocytes: presence of specific receptors in the t-suppressor/cytotoxic subset the j substance of cattle . seasonal variation of the naturally occurring isoantibodies for the j substance serum immunoglobulin levels in healthy children and adults dehydroepiandrosterone enhances il production and cytotoxic effector function of human t cells key: cord- -xgikbdns authors: Ühlein, e. title: Übersicht Über neue ernährungswissenschaftliche publikationen date: - - journal: z ernahrungswiss doi: . /bf sha: doc_id: cord_uid: xgikbdns nan shoe~taxer, w. c., yanof, h. m., tui~k, l. n., u. wilson, t.h.: glucose and fructose absorption in the unanesthetized dog. gastroenterology [ ] nr. , s. ff . ( s.) . * sieaei~, p. s., u. correia, iv] . j. : speed of resumption of eating following distraction in relation to number of hours food-deprivation. physiol. ree. [ ] nr. i, s. ff. ( s.) . si~eth, r. n., u. christie, it. j. : studies on the absorption of insulin from the gastrointestinal tract of the rabbit. diabetes [ ] nr. , s. ff. ( s.) . *st~oh~.x~r, g.: ~iessung der ksrperradioaktivitiit zur bestimmung der eisenresorption beim mensehen. nuclear med., suppl. [ ] s. ] . tallis, g. ] ~., moore, ~. w., u. gream, b. d. : specific gravity of live sheep. nature [ ] nr. , s. . tanaka, s., nu-kasawa, a., yoshikawa, h., u. yoneyama, y. : protein nutrition and radiation damage in mice. nature [ ] nr. , s. / . tho~tson, a. m., u. billv, wlcz, w. z. : nutritional status, maternal physique, and repro- ductive efficiency. prec. i~utrition see. [ ] nr. , s. / . toothill, j. : the effect of certain dietary factors on the apparent absorption of magnesium by the rat. brit. j. nutrition [ ] nr. , s. . *trail, j. c. m. : upgrading the indigenous poultry of uganda. i.: the growth rates and feed conversion from hatching to maturity of indigenous poultry crossed with four imported breeds. j. agrie. sei. {} [ ] nr. , s. ft. ( s.) . van i~iekerk, b. d. h., i~eid, j. t., be~s~uoun, a., u. paladines, . l.: urinary ereatinine as an index of body composition. j. nutrition [ ] nr. , s. / . voigtl~ndeg, h. : der rhythmus des brotessens. lebensmittel u. ern~hrung [ ] nr. , s. ] . wadsworth, g. r. : nutrition surveys: clinical signs and biochemical measurements. prec. l~utrition see. [ ] nr. , s. ] . watson, w. c., gordon, r. s., kx~en, a., u. jove~, a. : the absorption and excretion of castor oil in man. ft. pharmacy pharmacol. [ ] nr. , s. / . wkeel~r, r. r. : evaluation of various indicator techniques in estimating forage intake and digestibility by range cattle. diss. abstr. [ ] nr. , . williams, w. p., dawxs, r. e., u. couch, j. r. : the utilization of earotenoids by the hen and chick. poultry sei. [ ] nr. , s. ft. ( s.) . wolford, j. i-i., ringer, ~. k., col~_ai% t. h., u. zrsdel, h. c. : individual feed consumption of turkey breeder hens and the correlation of feed intake, body weight, and egg production. poultry sci. [ ] nr. , s. ff. ( s.) . youl~o, g. m., tensuan, r. s., saimt, f., u. hol~es, f. : estimating body fat of normal young woman. visualizing fat pads by soft-tissue x-rays. j. amer. dietetic assoc. [ ] nr. , s. ff . ( s.) . zuck~r, t. f., u. ziicker, l. hi. : fat accretion and growth in the rat. j. nutrition [ ] nr. , s. / . intermediary metabolism c - kohlenhydrate -garbohydrates +n. n. : enzymatic defects in the hereditary fructosurias. nutrition i~ev. [ ] i~r. , s. / . *agosi~, hi., scar~elli, n., di~amarca, iv] . l., u. arav~i~a, l. : intermediary carbohydrate metabolism in triatoma infestans (insecta: hemiptera). ii. : the metabolism of c-glucose in triatoma infestans nymphs and the effect of ddt. comp. bioehem. physiol. [ ] nr. , s. ff . ( s.) . baens, g. s., lv-~deen, e., u. cornblxth, m. : studies of carbohydrate metabolism in the newborn infant. vi. : levels of glucose in blood in premature infants. pediatrics [ ] nr. , s. ff . ( s.) . * b~akeb, j., u. mapson, l. w. : studies in the respiratory and carbohydrate metabolism of plant tissues. xil : l~tlrther studies of the formation of co~ and the changes in lactate, alcohol, sucrose, pyruvate, and cr in potato tubers in nitrogen and in air following anaerobic conditions. prec. royal soc. b. [ ] nr. , s. ff. ( s.) . *bean, r. c., porter, g. g., u. bur, b. k.: carbohydrate metabolism of avocado. il: formation of sugars during short periods of photosynthesis. plant physiol. [ ] nr. , s. ff. ( s.) . b~gy~, e. n.: quantitative aspects of glucose metabolism in pregnant and nonpregnant sheep. amer. j. physiol. [ ] nr. i, s . / . b~-~t~au~, p. l.: effect of temperature on the fate of certain sugars in the gut of oxycarenus hyalinipennis costa (heteroptera: lygaeidae). naturwissenschaften [ ] nr. , s. . * broit~x~, s. a., u. zamchec~, n. : utilization and excretion of d-xylose in thyroxinetreated rats. j. l~bor. elin. med. [ ] nr. , s. ff. ( s.) . davidsoi% e. a., u. small, w. : metabolism in vivo of connective tissue mncopolysaccharide. i. : cimndroitin sulfate c and keratosulfate of nucleus pulposus. biochimica biophysiea aeta [ ] nr. , w. : metabolism in vivo of connective tissue mucopolysaccharide. il : chondroitin sulfate b and hyaluronic acid of skin. biochimica biophysica acta [ ] nr. , s. / . --u. s~a~l, w.: metabolism in rive of connective tissue mucopolysaccharide. iil: chondroitin sulfate and keratosulfate of cartilage. bioehimica biophysica aeta [ ] nr. , s. / . dea~, j. m.: a comparative study of carbohydrate metabolism in fish as affccted by temperature and exercise. diss. abstr. [ ] nr. , - ( s.) . de ] ode, t~,. c., steele, r., altszuler, n., du~, a., u. bishor, j. s. : effects of insulin on hepatic glucose metabolism and glucose utilization by tissues. diabetes [ ] l~t. , s. ft . ( s.) . *edel)ian, j., recaldi~, d. a. c. l., u. dic~erson, a. g. : the metabolism of fructose polymers in plants. . : the activity of f-fruetosylsucrose]sucrose transfruetosylase in living tissue of helianthus tuberosis l. bull. res. council israel a [ ] nr. , s. ff . ( s.) . * farooqi, m. i. h., u. karl, k. n.: potassium nitrate and carbohydrate contents of argemone mexieana l. at different stages of its growth. current sci. [ ] nr. , s. ft . (i s.) . * geissb#~ler, f., u. fav~oe~, p. : ] ~ffet des rayons x sur le m tabolisme du glucose et des acides gras chez la souris m~le. helvetica physiologica et pharmacologica acta [ ] nr. , s. c ff. ( s.) . gra~cr, b. r. : an investigation of the mechanism of absorption of sugars by plant cells. diss. abstr. [ ] nr. , - ( s.) . * gayeos~i, . d., t~ay~r, w. r., gayboskl w. a., gabaielson, i. w., u. sl'mo, h. m. : a defect in disaceharide metabolism after gastrojejunostomy. :new engl. j. med. [ ] nr. , s. s/s . * hxtch, m. d., u. glasziou, k. [ ] nr. , s. ff. ( s.) . heald, p. j. : the mctabolism of carbohydrate by liver of the domestic fowl. biochem. j. [ ] nr. , s. / . h~sch~l, m. j., i-lrr,tj, w. b., u. poster, j. w. g. : carbohydrate digestion in the small intestine of the young steer. prec. nutrition soc. [ ] nr. , s. v/vi. heb~h~d~z, a., u. sols, a. : transport and phosphorylation of sugars in adipose tissue. biochem. j. [ ] nr. , s. / . h, , .z, h., eric, , , c., u. glaubitt, d. : veriinderung yon zelldichte und polysaccharidstoffwechsel im alternden bindegewebe. klin. wschr. [ ] nr. , s. / . holt, p. r., h&essler, h. a., u. issv, lbacher, k [ ] nr. , s. ff. ( s.) . je ni~os, a. c., u. morton, r. k. : changes in carbohydrate, protein, ni~ogenous compounds of developing wheat grain. australian j. biol. sei. [ ] , s. ff. ( s.) . * kok, b., hoch, g., u. coorv, l~, b .: sensitization of chloroplast reactions. i. : sensitization of reduction and oxidation of cytochrome c by chloroplasts. plant physiol. [ ] nr. , s. ff. ( s.) . *levin, :b., berholzer, v. g., slqodorass, g. j. a. i., stimm~er, l., u. wilmers, m. j. : fruetosaemia. an inborn error of fructose metabolism. arch. disease childhood [ ] nr. , s. ff. ( s.) . lindquist, b., u. m_eeuwlsse, g. w. : intestinal transport of monosaccharides in generalized and selective malabsorption. acta paediatrica suppl. , s. ll ff. ( s.) . lui~dholm, l., u. i~ottme-lundholi~i, e r, j. a. : i. : glucose tolerance in swine as related to post-mortem muscle characteristics. ii. : the relationship of niacin and niacin coenzymes with various post-mortem properties of porcine muscle. diss. abstr. [ ] nr. , - ( s.) . *ivir/~z, m. : carbohydrate metabolism following traumatization in the noble-collip drum and shock due to burns in rats. physiologica bohemoslovenica [ ] nr. , s. ft. ( s.) . * murder, r., u. smith, f. h.: abnormal carbohydrate metabolism in pancreatic carcinoma. i~ied. clinics north americ~ [ ] nr. , s. ff. ( s.) . *randle, p. ft., garland, p. b., hales, c. n., u. newsholme, e. a.: the glucose fattyacid cycle. lancet [ ] nr. , s. ff. ( s.) . rossi, f., z~ri, m., u. g] av.enbau~, a. l.: evidence for the existence of ~he hexose monophosphate pathway for glucose metabolism in the normal and denervated skeletal muscle of rats. biochcm. j. [ ] nr. , ] . *ruiteb, j., wei-i~bei~o, f., u. morrisoi~, a. : the stability of glucose in serum. clinical chem. [ ] bit. , s. ff. ( s.) . $sac]ter, j. a., ]: l~tch, ~r d., u. glasziou, k. t [ ] nr. , s. ff. ( s.) . *scm~ass~k, h.: metabolite des kohlenhydratstoffwechsels der isoliert pcrfundierten rattcnleber. biochem. z. [ ] iqr. , s. ff. ( s.) . *sch~reiu, c. : relation between fructose content of semen and fertility in man~ j. reproduction fertility [ ] i~ir. , s. ff. ( s.) . * schlubac~, it. h., u. g~em~, m neptune, e. ~., u. sudduth, h. c. -" toxic effects of oxygen at hight pressure on the metabolism of d-glucose by dispersion of rat brain. biochem. j. [ ] nr. , s. / . *waaei~, h. g.: pathways of utilization of ( - c) glucose and ( -x c) glucose in slices of peas. j. exper. botany [ ] nr. , s. ff. ( s.) . weic-~r, h., schs~r~cl, h., u. re~schle~, h. e.: ~ber physiologische glucose-ansscheidung im urin yon stoffwechsel-gesunden. klin. wschr. [ ] nr. , s. . w~ovse, s., fr~ed~ia~r~, b., u. reicm~d, g. : effects of insulin on hepatic glucose production and utilization. diabetes [ ] nr. i, s. lff i~ou~, l~i., u. kum~e~ow, f. a. : fatty acid composition of lymph lipids from rats fed fresh and thermally oxidized fats. j. dairy sci. [ ] i~r. , s. ff. ( s.) . * blou~rr, a. w., u. co~, m.: tissue lipid pattern in a case of xanthoma disseminatum. arch. intern. ivied. lu [ ] nr. , s. ] . bortz, w., anr~m~, s., u. c~o~, i. l.: localization of the block in lipogenesis resulting from feeding fat. j. biol. chem. [ ] nr. , s. / . bronsert, u., hartmani~', f., u. mitzkat, ii.-j.: die wirkung yon milchs~ure im fettstoffwechsel der leber bei experimenteller fettlcber der ratte. naturwisscnschaften [ ] i~r. , s. . br w~, m. l. : effect of a low dietary level of three types of fat on reproductive performance and tissue lipid content of the vitamin b -deficicnt female rat. j. nutrition [ ] nr. , s. . *busfm~k, e. r., tho~rrson, r. h., u. weedo~, g. d. : metabolic response to cold air in men and women in relation to total body fat content. j. appl. physiol. [ ] nr. of mast cells to fat transport. ann. new york acad. sci. [ ] ar~. , s. ff. ( s.) . katorsx~, b. a.: the influence of growth hormone on fat and protein metabolism. diss. abstr. [ ] nr. , -- ( s. amer. j. clinical nutrition [ ] nr. , s. ff. ( s.) . mxmo~, j. e.: the influence of diet and age on lipid metabolism of chickens. diss. abstr. [ ] nr. , - ( s.) . *me, i, f. : a study on the lipid metabolism in the patients with atherosclerosis especially on the cholesterol in serum lipoprotein fl-fraction. japan. heart j. [ ] nr. brown, l. d., grev~es, %. m., u. duncan, c. w. : effect of protein level in milk replacers on growth and protein metabolism of dairy calves. j. dairy sci. [ ] nr. , s. ff. ( s.) . *ma~i)elst~, j. : protein turnover and its function in the economy of the cell. arm. new york acad. sci. [ ] nr. , s. ff. ( s.) . mast~as, c. j. : nucleic acids and protein stores in the merino sheep. australian j. biol. sci. [ ] nr. , s. ff. ( s.) . noack, r.: zur biochemischen funktion des proteins als grundlage der bedarfsaormen. ernehrungsforschung [ ] nr. , s. / . *par.r. anin, a. u : le m tabolisme pro~ique dans le systems nel-ceux. studii si cercet~ri de biochimie [ ] nr. , s. ft. ( s.) . roouski, j., ~i[akowska, k., i~sn~, j., stankowska, a., hryniewiecki, l., u [ ] nr . [ ] . suppl. to nr. , s. ff. ( s.) . co~solazio, c. f., ~ixtous~, l. ., nv. lson, r. a., ~[a~drng, r. s., u. can" a~, j. e.: excretion of sodium, potassium, magnesium, and iron in human sweat and the relation of each to balance and requirements. j. nutrition [ ] nr . goodwin, a. f., u sci. [ nr. , s. ff. ( s.) . *gerlinger, p., much, j.-v., u. clukvert, j.: note pr liminaire sur raction du eyclophosphamide (endoxan) sur le d veloppement de l'embryon. c. r. acad. sci. [ nr. , s. ff. ( s. [ nr. , - ( s.) . nelson, f. e., jensen, l. s., u *caruzzo, c., tartara, d., pagano, p. g., pellegrini, a., costanzo, f., u [ ] nr. , -- ( s.) . [ ] nr. , s. ll ff. ( s.) . gal/~bos, j. t., asada, m., u. s~ks, j. z. : the effect of intravenous ethanol on serum enzymes in patients with normal or diseased liver. gastroenterology [ ] nr. , s~ ff. ( s.) . gall, c., u. weissw~.~le~, p. : un~ersuchungen fiber die labf~higkeit der milch und ihre beziehung zur mineralsteff-fiitterung der kfihe. milchwissenschaft [ ] nr. , s. ff. (? s.) . zitat: dt. lebensmittel-rdsch. [ ] nr. , s. . gasic, g., u. morrison, a.b.: mucopolysaccharides of renal collecting tubule ceils in potassium deficient rats. prec. soc. exper. biol. med. [ ] l~r. , s. ff. ( s.) . gear~-, t. f. : oak wilt development and its reduction by growth regulators. i. production and activity of oak wilt fungus pectinase, cellulase, and auxin. ii. effect of halogenated benzoic acids on oak trees, the oak wilt diseases, and the oak wilt fungus. diss. abstr. [ ] nr. , -- ( s.) . *g~l~,~cs~,r, f., g~ti, t., gy~g~, k., u. s s, j.: effec~ of cardiopathogenic diet on the thiopental anaesthesia. acts physiologica aead. sci. hung. suppl. nr. , s. . gar~'ls, j., piazu~lo, e., u [ ] nr. , -- ( s.) . ikir~a, h., u. t~, k. v. : activity of gibberellin 'd' on the germination of photosensitive lettuce seeds. nature ] nr. , s. / die wirkung des glucagons auf den blutzuckerspiegel in abh~ngigkeit yore alter. z. aiternsforsch. [ ] nr. , s. / . *loosli, j. k. : primary signs of nutritional deficiencies of laboratory animals. j. amer. veter, reed. assoc. [ ] nr. , s. ft. ( s.) . lowrey, r. s., pond, w. g., lo sli, j. k. u. barnes, r. h.: effect of dietary protein and fat on growth, protein utilization, and carcass composition of pigs fed purified diets. j. animal sci. [ ] nr. , s. ft. ( s.) . lvnd, c. c., u. ) [ ] nr. , s. ff. ( .) . nr~cleod, l. b. : effect of liming and potassium fertilization on soil solution and on yield and composition of alfalfa and orchard grass mixtures. dlss. abs~r. [ ] nr. , -- ( s.) . ~ds~n, k. ., u. edmonds, e. j. : prolonged effect on caries of short-term feeding of rice hulls to cotton rats. j. dental res. [ ] nr. , s. ff. ( s.) . m~a~.~, a. c. : biological responses of young rats fed diets containing genistin and genistein. j. nutrition [ ] nr. , s, ] . ~_ajaj, a.s., dnc~g, j.s., azzam, s.a., u. da_~by, w. j.: vitamin e responsive megaloblastic anemia in infants with protein-calorie malnutrition. amcr. j. clinical nutrition [ ] nr. , s. ft. ( s.) . v~jcm~owicz, e., u. quxstel, j. h. : effects of aliphatic alcohols on the metabolism of glucose and fructose in rat liver slices. canad. j. bioehem. physiol. [ ] nr. , s. ff. ( s.) . m_al~otra, o. p., n~a-wl)ov, a.v., r~ber, e. f., u. norton, h . w., effects of rat strain, stilbestrol, and testosterone on the occurrence of hemorrhagic diathesis in rats fed a ration containing irradiated beef. j. nutrition [ ] nr. , s. ff. ( ,) . --, u. r~ber, e. f. : effect of methionine and age of rat on the occurrence of hemorrhagic diathcsis in rats fed a ration containing irradiated beef. j. nutrition [ ] nr. , *misga, u. k. : effect of corn oil feeding on the lipids of dog bile. indian j. exper. biol. [ ] nr. , s. / . * miyao, m., tsvn•isnz, m., nagano, k., u. ttosogi, t.: experimental studies on the digestibility and absorbability of milk proteins. . effects of carbohydrate addition on the digestibility and absorbability of cow's milk proteins. tokushima j. exper. med. [ ] nr , nr. , s. ff. ( s.) . nagra, c. l., brerrenbach, r. p., u. meyer, ~. k. "-influence of hormones on food intake and lipid deposition in castrated pheasants. poultry sci. [ ] nr. , s. ff. ( s.) . *+na~:ler, w. g.: the significance of calcium ions in cardiac excitation and concentration. amer. heart j. [ ] nr l~, b. l., u. re(~a~, w. s., u. dobozy, a.: effect of vitamin a on the nucleic acid metabolism of rats. acts biologics acad. sci. hungariae , suppl. , s. . ~itz, k., u. loeser, a. : uber den einflufl appetithemmender substanzen auf das fettgewebe. klin. wschr. [ ] nr. , s. . * rstadius, k., nordstrom, g., u. lannek, n. : combined therapy with vitamin e and selenite in experimental nutritional muscular dystrophy of pigs. cornell veterinarian [ ] nr. , s. ft. ( s.) . ern~hrung und therapie. med. u. ern~hrung [ ] nr. , s. / . *--pflanzliche polysaccharide zur steigerung der kiirpereigenen abwehr. ivied. welt , nr. , s. pxrkrnson, t. m., u. lso~r, j. a. : inhibitory effects of bile acids on the uptake, metabolism, and transpor~ of water-soluble substances in the small intestine of the rat. life sci. , nr. , s. ] . *pxl~o% j.-l., u. mord~l~t-dx~rin~, )i..' action du potassium et du calcium sur l'histaminopexie s~rique. recherches sur le cobaye, le rat et l'homme. j. physiol. [ ] nr. , s. ff. ( s.) . * patek, a. j., de fr tsc] t, n. m., kendall, f. e., n. hirsch, r. l.: corn and coconut effects in dietary cirrhosis of rats. arch. pathology [ ] nr. , s. ff. ( s.) . patil, s. s.: the relation of ehlorogenic acid and total free phenols in potato plants to resistance to infection by verticillum alboatrum. diss. abstr. [ ] nr. , -- ( s.) . petuet,y, f.: diskussionsbemcrkungen zu rcferaten fiber sauermilchprodukte auf einer vortragsveranstaltung der gesellsehaft fiir erniihrungsbiologie e.v., miinchen, . juni . milchwissensehaft [ nr. , s. ] . pfei~ter, ( . j., gass, g. h., u. schw~rz, ( . s.: reduction by chlorpromazine of ulcem due to acute starvation in mice. nature [ ] nr. , s. . prrrllros, a. w., newc m~, tl r., u. sha.wklrs, d. r.: long-term rat feeding studies on irradiated chicken stew and irradiated cabbage. toxicol. appl. pharmacol. [ ] nr. , s. ] . prrmt~s, p. h., sutti~, j. w., u. ze~rowski, e. j. : effects of dietary sodium fluoride on dairy cows. vii.: recovery from fluoride ingestion. j. dairy sei. [ ] l~r. , s. ff. ( s. browi~, t. h., u. levee, r. v.: the effect of milk intake on nematode infestation of the lamb. prec. nutrition oc. [ ] nr. , s. / . * srnwrvasan, m., n~o~bhusha_~a~, a., u. sm~rrvas~, k. s.: changes in serum inorganic phosphate following ingestion of protein. curr. ci. [ ] nr. , s. . stallwoth, h. : some effects of . -dichlorophen-oxyacetie acid on sweet corn (zea mays rugosa l.) with emphasis on yield, tillering, root development, and exudation of electrolytes from roots and stems. diss. abstr. [ ] nr. , --- ( s.) . starcher, b., u. i~.ratzer, f~ h.: effect of zinc on bone alkaline phosphatase in turkey poults. j. nutrition [ ] nr. , s. / . stei~-~off, d., u. ~rqu~lrdt, p. : kombination yon kaliumpyrosulfit und ~xthy]alkohol ira tr~nkungsversuch an ratten. arzneimittelforschung [ ] nr. , s. / . stevermer, e. j. : influences of level of nutrition of the boar and of ionic environment of the spermatozoa on the properties of boar semen. diss. abstr. [ ] nr. , -- ( s.) . stn~p~., f., u. c~warz, k.: incorporation of valine-l-x*c into serum and tissue proteins of rats fed torula yeast diets. j. nutrition [ ] nr. , s. / . ton~., d. b., u. co~or, w. e. : the prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients. diabetes [ ] nr. , . ft. ( s.) . *stormont, j. •., u. waterhouse, c. : effect of variations in previous diet on fasting plasma lipids. j. labor. clinical med. [ ] nr. , s. ff. ( s.) . * stuart, a. e., u vity of the heart extract of rats. medlcina exporimentalis [ ] nr. , s. ff. ( s.) . szepsenwol, j.: carcinogenic effect of egg white egg yolk and lipids in mice. prec. soc. exper. biol. med. [ ] nr. , s. ff. ( s.) . *t~cs, l, u. ny~i, i.: effect of saline infusion, acth infusion, and blood transfusion on the hormone excretion of patients with hypereme~is. act~ medics aead. sei. hun. garicae [ ] nr. , s. ff. ( s.) . tanner, j. w.: an external effect of inorganic nitrogen on nodulation. diss. abstr. [ ] nr. , -- ( s.) . --, u [ ] nr. , s. ff. ( s.) . *t~i~er, l., m~az, m., u. cs:menafiov , m. : the effect of glucose and glucose together with insulin on the resistance of fasted rats to trauma in the noble-collip drum. physiologia bohemoslovenica [ ] nr. , s. ff. ( .) . ude~iend, s. : factors in amino acid metabolism which can influence the central nervous systems. amer. j. clinical nutrition [ ] nr. , s. ff. ( . .) vahouny, g. v., moede, a., silver, b., n . treadw~ll, c. r. : nutrition studies in the cold. iv. effect of cold environment on experimental atherosclerosis in the rabbit. j. nutrition [ ] nr. , s. / . van p lsum, j. f., olsen, b., taylor, d., rozyc'ki, t., u voss, r. d.: yield and foliar composition of corn as affected by fertilizer rates and environmental factors. i)iss. abstr. [ ] nr. , -- ( s.) . *vyval,ro, i. g. : the effect of gibberellin on the transformation of substances in germinating corn seeds. i)oldady akad. nauk sssr [russ.] [ ] nr. , s. ff. ( s.) . wao~ei~, g. r., cl~mk, a. j., hays, v. w., u amer. j. clinical nutrition [ ] lgr. , s. ff. ( s.) . the assessment of marginal protein malnutrition. prec. nutrition soc. [ ] mr. , s. ] . --, u. stnp~, j. m. l.: the free ly~ine and amino nitrogen content of liver, muscle, and serum in normal and protein-depleted rats. prec. nutrition soc. [ ] mr. i, s . viii/ix. *watson, w. c.: the morphology and lipid composition of the erythrocytes in normal and essential-fatty-acid-deflcient rats. bri . j. haematology [ ] lgr. , s. ff. ( s.) . waite, r., u. : blackburn, p. s. : the relationship between milk yield, composition and tissue damage in a case of subclinical masticis. j. dairy res. [ ] , nr. , s. ff. ( s.) . *n. n.: paraty ~hoid fever from frozen chinese eggs. brit. reed. j. . ( s.). *n. n. : radioactivity and human diet. chem. ind. , nr. , . . ~q. ~.: eh~digungen dutch konservlerungsmittel bei zitrusfrfichten? dr. reed. wschr. [ ] nr. , s. . *n. n. : salt-poisoning in infancy. lancet [ ] l~r. , s. . n.n.: kouoquium des arbeitskrcises hamburg der gdch-fachgruppe lebensmittelchemic und gerichtliche chemic am . januar . lebensmittelchem. u. gerichtl. chem. [ nr. , s. . *n. n. : smoking and heath disease. new england j. med. [ ] nr. , s. . +hi. n. : toxic components of lathyrus peas. nutrition rev. [ ] nr. , s. / . +n. n. : cariogenic ability of different diets. nutrition roy. [ ] nr. , s. ] . +n. ~.: aminoaeiduria in lead intoxication. nutrition rev. [ ] nr. , s. / . +n. n. : pyridoxine and dental caries. human studies. nutrition rev. [ ] nr. , s. . +n. n.: pyridoxine and dental caries. animal studies. i~utrition rev. [ ] nr. , s. . +hi. n. : rcporb: the prophylactic requirement and the toxicity of vitamin d. pediatrics [ ] nr. , s. ff. (? s.). axrkroa, a.: caesium- from fall-out in human milk. nature [ ] nr. , s. ff. ( s.) . *allcroft, r., u. car~agha~, r. b. a. : groundnut toxicity: an examination for toxin in human food products from animals fed toxic groundnut meal. veteri. rec. [ ] nr. , s. ff. ( s.) . *--, l~wis, g., u. hill, k. ~.: groundnut toxicity in cattle: experimental poisoning of calves and a report on clinical effects in older ca~tle. ve~er. rcc, [ ] nr, , s. ff. ( s. nr. , , nr. , , , , nr. , nr. , influence of calcium and ouabai bain upon potassium influx in human erythrocytes the enzymatic assimilation of nitrate in the tomato plant translocation of '~p, i~n, and ~c in plants einige neue gesiehtspunktr zum caleiumstoffwechsel dis~ibution of water, sodium, and potassium in resting and stimulated mammalian muscle. canad the influence of vitamin bi, on calcium ('sca) metabolism of maxillodental tissues kidney, water, and electrolyte metabolism intermediiirer elektroly~toffwechsel und zellgrenzfl~chenphysiologie im theoretischen zusammenhang mit der krebsentstehtmg. tell i dcr einflul~ yon vollkornbrot auf den calcium-stoffwechsel bei schulkindern recherches sur le m tabolisme du soufre. x. : la non- quivalence de la eystine et de la eyst ine dans la couvcrture des besoins sufr~s du rat adulte potassium-magnesium antagonism in soils and crops low serum iron levels in obese adolescents metal content of human organs studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to weeks of age iron absorption and excretion in experimental iron deficiency the measurement of exchangeable magnesium in dogs the copper metabolism of warmblooded animals with special reference to the rabbit and the sheep comparative studies of the metabolism of strontium and barium in the rat the utilization of iron in erythropoiesis binding of strontium in blood ~iolybdenum, copper, and zinc contents of mouse liver and sarcoma treated with molybdenum compounds biochemical effects of zinc deficiency in tomato plants excretion of sodium, potassium, magnesium, and iron in human sweat and the relation of each to balance and requirements turnover rate of zinc in the body as determined by the study of szn in rats a study of the iron absorption in mice as modified by various agents funktionsteste des radiojodstoffwechselstudiums und ihre bedeutung in der diagnostik der sehilddrfisenerkrankungen. ~rztl. laboratorium the fate of radioiodine after parcnteral administration a possible humeral regulator of iron absorption beitrag zur kl~rung der ursachen der anreicherung yon caesinm- im rganismus blood-and serum-level of watersoluble vitamins in man and animals significados metabslicos do ~cido ~-lip ico. . o ~cido r e o metabolismo do ferro observations on a magnesium-fluoride interrelationsip in chicks prevention of ,meat anemia" in mice by copper and calcium iron metabolism in experimental pyridoxine deficiency aluminium in soils and plants on the coastlands of british guinea physiology of adolescence. ii. : nutrition -basal oxygen consumption -energy expenditure and balance -nitrogen metabolism -calcium metabolism -iron metabolism -red cell mass and hemoglobin dietary strontium and calcium, and deposition of sr and asca in the bones of rats -~iengenelementansatz wachsender sehweine bei unterschicdiichen cuso -zulagen differences in copper retention in two strains of chickens untersuchungen fiber anreicherung und verteilung yon rubidium in gerstenkcimpflanzen [ ] nr. , s. / . *+lv~ointyr~, i.: an outline of magnesium metabolism in health and disease. a review uptake by the root and subsequent distribution within the potato plant of strontium- leached from the foliage nor zinkstoffwechsel in der schwangerschaft foetal metabolism of caesium- in the rat magnesium metabolism of chickens zinc metabolism in patients with the syndrome of iron deficiency anemia hepatospenomegaly dwarfism, and hypogonadism. labor. clinical mcd c~isium- trod kalium in menschlichen organen und in der milch / l~ole of the genotype in controlling accumulation of strontium- by plants copper and zinc interrelationships in the pig effect of chromium, cadmium, and other trace metals on the growth and survival of mice studies in the metabolism of zinc. iv. some observations on the urinary zine-porphyrin relationship in non-porphyries and in a patient with aeutm intermittent porphyria aastrontium balances in man studies on zinc metabolism. ii.: effect of the diabetic state on zinc metabolism: an experimental aspect effect of diabetic state on zinc metabolism: a clini. cal aspect copper metabolism and the liver iron metabolism and the liver with particular reference to the pathogcnesis of haemachromatosis studies on iron metabolism uber den eisenstoffwechsel. (bemerkung zu t. su~di~, miinchener reed yifinchener reed. wschr. [ ] nr. , s. . c - wirkstoffe -biocatalysts +n. n. : vitamin a in human livers. nutrition biological half.llfe of vitamin b~ in plasma hypervitaminosis a and mast cells. a study of the interrelationship of mast cells and vitamin a in vivo and in vitro evidence concerning the human requirement for vitamin bi~. use of the whole body counter for determination of absorption of vitamin blz vitamin c in plasma and leucocy~s of smokers and non-smokers some factors affecting the absorption of vitamins the determination of vitamin a in animal tissues and its presence in the liver of the vitamin a deficient rat metabolic activities of vitamin a and related compounds in animals. i.: role of vitamin a in intestinal muscular contraction zum vitamin-b--haushalt dcr ratte bei sorbitfiitterung contribution a l'~tude do la relation entre la vitamine big et la glande thyrolde effects of deficiencies of certain b vitamins and ascorbic acid on absorption of vitamin blv amer ascorbic acid metabolism in plants. ii. : biosynthesis dietary and thyroid interrelationships affecting vitamin a status of feedlot beef cattle die wirkung gesteigerter kupferzufuhr auf den vitamin-c-haushalt vom meerschweinchen bei parental zugeffihrter ascorbins~ure kritische auswe~ung der naeh erschienenen arbeiten fiber gebundene ascorbins~ure im tierischen gewebe metabolism and biological activity of vitamin a acid in the chick biochemical studies of vitamin metabolism in poultry urine. ii. : on the excretion of thiamine in poultry urine after subcutaneous and oral administrations of some thiamine derivatives untersuchungen fiber die speiehcrung und fiber die ansscheidung yon vitamin a nach ungeniigender vitamin-a-versorgung bei legenden hfihnern studies on metabolism of vitamin a. .: the biological acticity of vitamin a acid in rats vitamin a and cholesterol absorption in the chicken studies on the interaction of vitamin bi~, intrinsic factor, and receptors. il the possible absorption of intrinsic factor human growth hormone in infant malnutrition macro-and micromethods for the determination of serum vitamin a using trifluoroacetic acid the activation of sulphate by extracts of cornea and colonic mucosa from normal and vitamin a-deficient animals the importance of blood as a pool of vitamin d studies on metabolism of vitamin a. .: enzymic synthesis ~nd hydrolysis of phenolic sulphates in vitamin-a-deficient rats human metabolism of l-ascorbie acid and erythorbic acid tissue distribution and storage forms of vitamin b~, injected and orally administered to the dog relation between vitamin a, tocopherol, and cholesterol serum levels in the elderly interrelationships of vitamin bi~, folio held, and ascorbie acid in the megaloblastie anemias zum wirkungsmechanismns des vitamins e. helvetica physiologica et pharmacologica effect of some physiologic factors on the absorption of vitamin bi~ in rats transport of dietary nitrogen mitochondrial fatty acids of fish and fish-eating birds the biosynthesis of fatty acids influence of age and dietary protein on cerbain free amino acids in chick blood plasma nitrogen metabolism in coldexposed rats ~ber des vermehrte auftreten yon fettsiiuren mit bis c-atomen in den depotfetten siiugender ratteu und den ubergang der linolsi~ure yon den mfittern auf die jungen die bildung yon antiksrpcrn gegen verschicdene kuhmilehproteine bei neugeborenen, kindern, erwachsenen und graviden the myocardial arterio-venous differences of free amino acids and of free fatty acids in healthy individuals, patients with diabetes and with essential hypercholesterolemia urinary amino acids on phenylalanine-tyrosine-supplemented diets difference in the metabolic fate of acetate and ethanol fed to higher plant tissues iiemodynamic relationships of anaerobic metabolism and plasma free fatty acids during prolonged, strenuous exercise in trained and untrained subjects effects of palmitate on the metabolism of leukooytes from-guinea pig exudate the dynamics of plasma free fatty acid metabolism during exercise ~ber die retention, den abbau und die ausseheidung yon -thion-tetrahydro-l. . -thiadiazinen transport systems for amino acids effects of protein intake and cold exposure on selected liver enzymes associated with amino acid metabolism quantitative studies on tryptophan metabolism in the pyridoxine-deficient rat effect of desoxypyridoxine-induced vitamin b~ deficiency on polyunsaturated fatty acid metabolism in human beings studies on the wheat plants using carbon- compounds. xix.: observations on the metabolism of lysine- c. canad genetic defects of amino acid metabolism. pediatric clinics north america metabolism of tryptophan in diabetes mellitus the two-carbon chain in metabolism der einfluss yon vitamin a auf den citronens~urestoffwechsel ~)ber phenolspeicherung und phenolabbau in wasserpflanzen. naturwissensehaften effect of physical exercise on nitrogen balance in obese subjects metabolism of nitrogen compounds in the rumen of ruminants. izvest. acad der intermedli~r-stoffwechsel s~ffweehsel der carotine im hiihnerembryo nucleic acid metabolism of germinating corn seedlings carbon metabolism of ~ c-labelled amino acids in wheat leaves. ii.: serine and its role in glycine metabolism bovine metabolism of insecticides. the metabolism of ~evin in dairy cows stoffweehsel der carotine. wiss. veroff. dr. oes. ern~hrnng metabolism of labelled linolcic-l-tac acid in the sheep rnmen nonessential nitrogen supplements and essential amino acid requirements. nutrition rev vitamin c requirements of man re-examined. new values based on previously unrecognized exhalatory excretory pathway of ascorbie acid studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to weeks of age water requirements of men as related to salt intake evidence for a high zinc requirement at the onset of egg production effect of lysine and glyeine upon arginine requirement of guinea-pigs the cobalt requirement of subterranean clover in the field fluid and electrolyte, requirements of newborn infants with intestinal obstruction the requirement and availability of dietary iron for young pigs studies on the protein and methionine requirements of young bobwhite quail and young ringnecked pheasants phenylalanine requirement of women consuming a minimal tyrosine diet and the sparing effect of tyrosine on the phenylalanine requirement effects of starvation on the cardiovascular system of the chicken calcium and phosphorus requiremeats of finishing broilers using phosphorus sources of low and high availability water intake of normal children sex differences in the ~ oeopherol requirement of rats as shown by the haemolysis test further studies on protein and energy requirements of chicks selected for high and iow body weight smoking and blood dotting dental caries and trace elements a statement approved by the board of directors of the canadian heart foundation the question of fats. il : fats and disease moldy peanuts and liver cancers vitamin c and healing of wounds berieht fiber die vortragstagung des fachverbandes lehensmittelchemie der chemisehen gesellschaft in der d])r veto . his diet and human depot fat ethanol and plasma free fatty acid in man dietary water and protein efficiency in rats. nutrition rev. [ ] nr. , s. / . +n. n. : nature of the coagulation defect in rats fed diets producing thrombosis or experimental atheroselerosis factors affecting growth depression by raw soybeans bones in undernourished animals. nutrition rev. [ ] nr. , s. . +n. n. : vitamin e and the etiology of muscular dystrophy in the rabbit riboflavin eoenzymes and congenital malformations the thyroid gland in infant malnutrition. nutrition rev. [ ] nr. , s. . +n. n. : a proposed mechanism for the effect of fats on serum cholesterol effect of varying levels of dietary protein on synthesis and excretion of urea dietary phosphates and dental caries folic acid restriction and cancer inhibition changes induced by lipoie acid in normal rat liver vitamin b deficiency and tryptophan metabolism effect of ubichromenol on development of encephmomalacia in vitamin e deficient chicks leucine-induced hypoglycemia nutritional muscular dystrophy in lambs. nutrition rev. [ ] nr. , s. / . +n. n.: amino acid imbalance in cold-exposed rats. nutrition rev. [ ] nr. , s. . +n. n.: milk and athletic performance effect of vitamin a deficiency on the ubiquinone content of rat liver idiopatjaie stea~orrhea gastrointestinal protein loss in iron-deficiency anemia nutritional cirrhosis of the liver. nutrition rev. [ ] nr. , s. / . +n. n. : exercise and heart disease calcium deficiency in the etiology of osteoporosis the relation of dietary fat to the fatty acids in the intestinal wall clot-strength and elot-lysis in rats fed hyperlipemic diets effect of potassium iodide and duodenal powder on the growth and organ weights of goitrogen-fed rats ver~nderungen im stoffwechsel und wachsturn junger tomatenpflanzen nach giberillins~,urebehandiung effect of restricted feeding during the growing period on reproductive performance of large type white turkeys keys, a. l glucose, sucrose, and lactose in the diet and blood lipids in man ambient temperature and survival on a protein-deficient diet some considerations of changes in total body composition in relation to nutritional status the effect of variations in the energy and protein levels of the ration upon performance in the pig studies in choline deficiency. fate of injected - ~c-pal. mitio acid and fatty acid spectra in fasting and refed rats bartou i~ vitamin a requirements of chicks at moderately elevated temperature influence of age and dietary protein on eer~ain free amino acids in chick blood plasma the effects of nicotine on weight increment, activity, food intake, and water intake in weanllng albino rats effect of pyridoxine deficiency upon delayed hypersensitivity in guinea-pigs vitamin a deficiency in chickens ccrebellar encephalomalacia produced by diets deficient in toeopherol vitamin b deficiency in indian infantm plasma liplds in scurvy: effect of ascorblc acid supplement and insulin treatment effect of gibberellin on the variations of the growth-point in winter wheat uptake of dinitrophenol and its effect on transpiration. calcium accumulation in barley seedlings l~[ethylmalonate excretion in vitamin b~ deficiency reduction of plama cholesterol levels in atherosclerosis by diet and drug treatment. australasian ann effects of reduced dietary intake on the activities of various enzymes in the livers and kidneys of growing male rats the effect of feeding d-methionine on the d-amino acid oxidase activity of chick tissues l~etabolic effects of dietary protein level in cold-exposed rats relative effects of rapeseed oil and corn oil on rats subjected to aclrenalectomy, cold, or pyridoxine deprivation metabolic effects of dietary protein level with caloric restriction in coldexposed rats. canad metabolic effects of three dietary protein levels fed isocalorically to coldexposed rats. caned die nolle versehiedener fette im eiweis des organismus. nahrung the bursa of fabricius and xanthine oxidase activity of liver and kidney following dietary supplementation of iodina~d casein to chickens effects of linolsate and dietary fat level on plasma and liver cholesterol and vascular lesions of the cholesterol-fed rat a comparative study of the effect of bile acids and cholesterol on cholesterol metabolism in the mouse, rat, hamster, and guineapig the effects of ruminal and plasma sodium concentrations on the sodium appetite of sheep effect of level and sequence of feeding and breed on ovulation rate, embryo survival, and fetal growth in the mature ewe inhibitory effects of carbohydrates on histamine release and mast cell disruption by dextran fett-s~urestoffwechsel bci the effect of calcium infusions, parathyroid hormone, and vitamin i) on renal clearance of calcium nutritional supplementation during pregnancy effect of level of dietary protein with and without added cholesterol on plasma cholesterol levels in man die schilddrfisenfunktion bei enteralem eiweiflverlust effects of pelleting and varying grain intakes on milk yield and composition fatty acid composition of lipids of serum and aorta in the chicken on different diets rat intestinal suerase. ii.: the effects of rat age and sex and of diet on suerase activity the effect of selenium administration on the growth and health of sheep on scottish farns die wirkung yon vitamin b bei leukopenien effect of energy source and level of alfalfa pellets on growth and tissue hpids of beef calves effect of magnesium deficiency on mast cells and urinary histamine in rats histamine-liberating effect of magnesium deficiency in the rat zur wirkung des wassers bei der seitenwurzelbildung an luftwurzeln influence of the aqueous potato extract and its fractions on growth and spore formation of the b. pumilus and the production of the antibiotic tetaine influence of mineral nutrition on the resistance of peach tree to fusicoceum amygdali de la croix the effect of dietary protein on the course of various infections in the chick bifidus intestinal flora in infants fed on mamysan b. acta paediatriea effect of food fats on concentration of ketone bodies and citric acid level in blood and tissues is there a hemostatie effect of peanuts in hemophilioid disorders? milk allergy in infancy dental effects of fluoridation of water with particular reference to a study in the united kingdom influence of previous feeding with a high-fat diet on liver steatosis produced by acute starvation of growth hormone in mice effect of amino acid imbalance on nitrogen retention. ii.-interrelationships between methionine, valine, isoleucine, and threonine as supplements to corn protein for dogs supplementation of cereal proteins with amino acids. v. : effect of supplementation lime-treated corn with diffe. rent levels of lysine, tryptophane, and isoleueine of the nitrogen retention of young children the interrelation of nutrient supply, leaf nutrient content, and vegetative growth of ilex crenata gastric content of fasted primates. a survey serum cholesterol in vitamin c deficiency in man the effect of carbohydrates on the production of staphylococcal pigment effect of a low dietary level of three types of fat on reproductive performance and tissue lipid content of the vitamin b -defieient female rat effect of magnesium deficiency on synthesis of hear~ and liver mit~chondria phospholipids ~ber den einflub l~nger dauernder ksrperlicher inaktivit~t auf die blutzuckerkurve nach oraler glucosebelasttmg. helvetica medica acts action of trace elements on the metabolism of fluoride zur frage des einflusses yon kondensmilch und einer protein-valerina-polyphosphat-komplexverbindung auf die kreislaufwirktmg des kaffees beim menschen modifications de la croissance de la plantule de lapin blanc (lupinns albns l.) provoqu es par une diminution exp rimentale des r serves influence of the dietary protein level on the magnesium requirement effects of high levels of copper and chlorotetracycline on performance of pigs effect of dietary calcium lactate and lactitic acid on faecal escherichia coli counts in pigs die entwickhing von calcium-mangelsymptomen. z. pflanzenern~hrung, diingung, bodenkde. [ ] nr. , s. / . --calcium-mangelsymptome an hsheren pfianzen copper deficiency in relation to swayback in sheep. i. : effect of molybdate and sulphate supplements during pregnancy long-term, low fat, low protein diets and their effect on normal trappist subjects further studies of the influence of diet on radiosensitivity of guinea-pigs, with special reference to broccoli and alfalfa effects of the infusions of ammonia, amides, and amino acids on excretion of ammonia answirlmngen langfristig fettreicher ern~ihrung auf das plasma-cholesterin the relationship of dietary energy level and density to the growth response of chicks to fats influences of dietary carbohydrate-fat combinations on various functions associated with glycosis and lipogenesis in rats. i. effects of substituting sucrose for rice starch with unsaturated and with saturated fat compensatory carcass growth in steers following protein and energy restriction fatty acid composition and glyceride structure in rats fed rapeseed oil or corn oil. canad influence of selective and nonselective hydrogenation of rapeseed oil on carcass fat of rats. canad studies in serum lipids. with special reference to spontaneous variations and the effect of short-term dietary changes experimentelle untersuchungen zur wirkung yon kaffeefett evaluation of the effect of breed on vitamin be requirements of chicks -iigh salt content of western infant's diet: possible relationship to hypertension in the adult the effect on the serum cholesterol levels of the consumption of a special dietary fat with a high content of unsaturated fatty acids in elderly people effect of dilution of the diet with an indigestible filler on feed intake in the mouse effect of tea and its tannins upon capillary resistance of guinea-pigs food input and energy extraction efficiency in carassins auratns effect of calcium and magnesium upon digestibility of a ration containing corn oil by lambs effects of calorie restriction during the growing period on the performance of egg-type replacement stock effects of insulin on hepatic glucose metabolism and glucose utilization by tissues cellulase and polygalacturonase in tomato fruits and the effect of calcium on fruit cracking effect of nutritional muscular dystrophy and of starvation on amino acid penetration in rabbit tissues plasma protein synthesis in nutritional muscular dystrophy inulin and sucrose distribution in tissues of vitamin e-deficient and control rabbits protein-bound dyes in the serum and liver of rats fed aminoazo dyes vanadium. excretion, toxicity, lipid effect in man the influence of vitamin a status on the proteoly~ic activity of lysosomes from the livers and kidneys of rats disauxie metainfettive e da malnutrizione induced drinking in dogs: comparative effects of hypertonic sodium chloride and sorbitol the influence of early nutrition on brain cholesterol accumulation during growth changes in composition of the saliva of cows on grazing heavily fertilized grass. res. veter changes in composition of the saliva of sheep on feeding heavily fertilized grass efect of varying alfalfa: barley ratios on energy intake and volatile fatty acid production by sheep the influence of calcium on the secretory response of the submaxillary gland to acetyi-choline or to noradrenaline clinical dentistry and fluoride food allergy as a cause of abdominal pain the effect of various dietary levels of ddt on liver function, cell morphology, and ddt storage in the rhesus monkey effect of natural and purified diets on survival of x-irradiated mice effect of autoclaving and ])'sine supplementation of skimmilk-powder diets on growth and caries in rats effects of alcohol intake on subjective and objective variables over a five-hour period nitrogen metabolism of african cattle fed diets with an adequate energy feeding value of fl-caroteno following treatment with n~o~ the relationship of the quantity and quality of dietary fats to serum cholesterol levels in men of different ages and weights effects on girls of greater intake of milk, fruits, and vegetables effect of antioxidant, protein, and energy on vitamin a and feed utilization in steers the growth-maintaining activity of ascorbic acid the effects of an induced pyridoxine and pantothenie acid deficiency on excretions of oxalic and xanthurenic acids in the urine influence of lactose and dried skim milk upon the magnesium deficiency syndrome in the dog. i.: growth and biochem chronic malnutrition in turkey. v. study on serum fatty acids in malnourished children the effect of nutrition conditions on the growth of and nitrogen accumalation by fodder beans when sown together with indian corn effects of a diet high in polyunsaturated fat on the plasmalipids of normal young females citrate and action of vitamin d on calcium and phosphorus metabolism beeinflussung der sportliehen lelstungsf/~higkeit durch eine geeignete er-nehrung effect of dietary erotic acid on liver proteins effect of barbiturie acid and ehlortetraeyeline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks effects of feeding low levels of dimethoate on milk and whole blood eholinesterase activity of dairy cattle changes in serum lipoproteins after a large fat meal in normal individuals and in patients with isehemic hear~ disease the relationship of specific nutrient deficiencies ~) antibody response in swine. i. : vitamin a. ii. : pantothenie acid, pyridoxine or riboflavin. i)iss. abstr relationship of specific nutrient deficiencies to antibody production on swine. i. : vitamin a relationship of specific nutrient deficiencies to antibody production in swine. il : pantothenic acid, pyridoxine or riboflavin histechemistry of dietary cardiac lesions effect of various levels of fluorine, stilbestrol, and oxytetraeycline, in the fattening ration of lambs uptake of copper and its physiological effects on chlorella vulgarls the effects of a small dose of ethyl alcohol on certain basic components of human physical performance. i. the effect on cardiac rate during muscular work. arch. internat some effects of feeding stilbestrol, chlortetracycline and penicillin with alfalfa soilage on steer performance and carcass quality experimental induction of ciguatera toxicity in fish $hrough diet effects of potassium fertilizer, age of ewe, and small magnesium supplementation on blood magnesium and calcium levels of lactating ewes the influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep un~emuchungen fiber den umsatz wachsender schweine ab geburt. . mitt eczema and cow's milk. brit. med. j. , nr. , s. . isaacso~, a. : the effects of zinc on responses of frog skeletal muscle effects of zinc on responses of skeletal muscle effect of diet on work metabolism carbohydrate-phosphorus metabolism in the skeletal muscles of epinephrectomized animals durlng treatment with cortisone and vitamins c and p. ukrainskii biokhimichnii zhurnal composition of dietary fat and the accumulation of liver lipid in the choline-deficlent rat nutrition and palatability the incidence of protein-calorie malnutrition of early childhood theories on the mode of action of fluoride in reducing dental decay saccharase deficiency, familial entailing intolerance ~ cane sugar. acts paediatrica raw and heat-treated soybeans for growing-finishing swine and their effect on fat firmness effect of the administration of isoniazid and a diet low in vitamin be on urinary excretion of oxalic acid dietary and thyroid interrelationships affecting vitamin a status of feedlot beef cattle ration effects on rumen acids, ketogenesis, and milk composition. i.: unrestricted roughage feeding the effect of supplements of groundnut flour or groundnut protein isolate fortifed with calcium salts and vitamins or of sklmmilk powder on the digestibility coefficient, biological value and net utilization of the proteins of poor indian diets given to undernourished children a comparison of skin-testing with natural foods and commercial extracts die wirkung yon hungern auf den ammoniakgehalt und das ph der pansenfi(issigkeit sowic auf die harnstoff-, cholesterin-und zuckerkonzentration im blu~. acts veterinaria acad increase of plant virus infection by magnesium in the presence of phosphate effect of intramuscular injection of magnesium sulphate solution on the growth rate and serum composition in rats diskussionsbemerkungen zu referaten fiber sauermilchprodukte auf einer vortragsveranstaltung der gesellschaft fiir eru~ effect of massive sodium bicarbonate infusion on renal function excessive insulin response to glucose in obese subjects as measured by immunoehemical assay die wirkung gesteigerter kupferzufuhr auf den vitamin-c-hanshalt yon meerschweinehen bci paren~ral zugef'dhrter ascorbinsrure the influence of growth hormone on fat and protein metabolism. dies. abs~r. [ ] nr phenolearbons~iuren in mensehlichen nahrungsprodukten. zum vorkommen yon phenolcarbons~uren in mensehlichen nahrungsprodukten und ihr einflul~ auf den intermedit~ren stoffwechsel influence of pregnancy and an oxidized lipid diet on the fatty acid composition of blood and tissue an experimental approach to the mechanisms of weight loss. ii. a comparison of effects of thyroxine, fat-mobilizing substance (fms) and food deprivation in achieving weight loss in mice fat accumulation in the regenerating media of arteries in rats given an atherogenic diet the effect of nutrition on the growth of faseiola hepatica in its snail host the effects of specific viruses, virus complexes, and nitrogen nutrition on the growth, flowering, and mineral composition os strawberry plants body weight and food intake as initiating factors for puberty in the rat the role of catecholamines in the free fatty acid response to cigarette smoking die renale steroidausseheidung bei experimentellem vitamin-e-mangel peculiar features of respiration and redox processes in the rice plants grown under different nutritional conditions der bohnenkaffee und die migrrne repeatability of litter size and weight in the laboratory rat as affected by selection and plane of nutrition wirkungen yon muskelextrakt auf den stoffwechsel. arzneimittelforschung is [ ] nr. , s. / einflul] der silageftitterung auf die qualit~t der butter einflul] der silageftitterung auf die qualit~t yon milch und milchproduk. ten. . mitt.: einflul] von silagefiitterung auf die organoleptischen eigenschaften dcr milch effect of protein intake and cold exposure on selected liver enzymes associated with amino acid metabolism body iron levels and hematologic fin. dings during excess methionine feeding der einttul~ des kulturmediums auf die bildung von streptolysis s durch ruhcnde zellen influence of polyphosphates in chilling water on quality of poultry meat influence of breed-type, feed level, and sex on characteristics of the lamb carcass, and some relationships among live animal and carcass measurements the toxic action of phenothiazine and some disturbances of intermediary metabolism in undernourished sheep efficiency of feed use in beef cattle uber die wirkung der nalmmgsfette auf die blutlipoide, teil i. ernahrungs-umsehau [ ] nr. , s. / . --~ber die wirkung der nahrungsfette auf die blutlipide fette und blutgerinnung. bibliothcca hacmatologiea effect of heavy cigarette smoking on postprandial triglycerides, free fatty acids, and cholesterol role of calcium in fibrin formation glucose- -phosphate dehydrogcnase and aldolase in lenses of lactose-fed rats -effect of riboflavin, choline, pantothenic acid and vitamin a on the excretion of sodium in urine of rats the effect of waterwashed rice in the diet on the growth, excretion of sodium in urine and blood pressure of rats effect of aseorbic acid, vitamin b, and milk on the dark adaption effect of single deficiency of vitamin a, thiamine der einflub yon vitaminen auf die psyehomotorisehe leistungsi'~higkcit beim menschen. naunyn-schmiederberg's arch. exper feeding response of adult tribolium to carbohydrates in relation to their utilization nutritional secondary hyper hieronymi: influence of nutritional conditions on the cellular rna metabolism in rive and in vitro diffusible auxin increase in a rosette plant treated with gibberellin transamination in muscular dystrophy and the effect of exogenous glutamate: a study on vitamin e deficient rabbits, and mice with hereditary dystrophy. canad effect of auxin on the emergence of lateral roots in p. mungo seedlings the effect of nutritive status on oestrus, ovulation, and graafian follicles in merino ewes biologische wirkungcn autoxydierter, epoxydierter und bestrahlter fette s~ure-basen-gleichgewicht mad chronische acidogene und alkalogene eruehrung effect of protein level in milk replacers on growth and protein metabolism of dairy calves effect of sodium bicarbonate in the drinking water of ruminants on the digestibility of a pelleted complete ration sucrose diet and bfliary chelate excretion in rats: with note on procedure for chelate determination eirdlub sauerer milcherzeugnisse auf die darmflora untersuchungen fiber die speicherung und die ausscheidung yon vitamin a nach ungenfigender vitamin-a-versorgung bei legenden hiihnern the effects of low magnesium intake on lactating ewes effect of vitamin a on the content of pyridinnucleotides, pyrovic, and lactic acid and on anaerobic phosphorylation. ukrainskii biokkimichnii zhurnal the pyridine nucteotides. a study of a method of measurement. a study of the alterations in rat fiver under the conditions of diabetes and starvation. a preliminary study of various marine fish tissues with the emphasis on the islet of langerhans iron uptake-transport of soybeans as influenced by other cations hshe und zeitpm~kt der dfingung yon sommerweizen mit chlorcholinchlorid zur vcrkiirzung der halmlange nutritional significance of soluble nitrogen in dietary proteins for ruminants effects of long-term feeding of vegetable fats on atherosclerosis effects of feeding various mile, corn, and protein levels on laying performance of egg production stock some observations on the influence of a magnesiumdeficient diet of rats, with special reference to renal concentrating ability effect of gibberellic acid on flowering of apple trees the effects of dietary fat and energy levels on the performance of caged laying birds motivational producing properties of the feeding system of the rat hypothalamus the influence of diet and age on lipid metabolism of chickens effect of age on the response of chickens to dietary protein and fat absorption and excretion of biotin after feeding minced liver in achlorhydria and after partial gastrectomy variations de la calcsmie du chien normal apr~s ingestion de cholesterol eristallis~ dans l' thanol ou dans rhexane changes in activity of rat epididymal adipose tissue in vitro due to time elapsed since last feeding differences in rat strain response to three diets of different compositions l'alcoolisme ~ l'hspital psychiatrique de colsou (martinique). ann. m~dico-psychologiques nitrogen, lipid, glycogen, and deoxyribonu. eleie acid content of human liver. the effect of brief starvation and intravenous administration of glucose some metabolic and nutritional factors affecting the survival of erythrocytes erythrocyte survival of rats deficient in vitamin e or vitamin b . j. nutrition [ ] nr. , s. / nutrition and lactation the effect of administering sodium chloride, sodium bicarbonate, and potassium bicarbonate to newly born piglets strontinm- and calcium in milk of miniature swine further studies on cariostatic effect of organic and inorganic phosphates urinary excretion of magnesium in man following the ingestion of ethanol the effects of magnesium compounds and of fertilizers on the mineral composition of herbage and on the incidence of hypomagnesaemia in dairy cows behavioral, dietary, and autonomic effects of ehlordiazepoxide in the rat the effect of a high and low sodium diet in a patient with familial periodic paralysis the effect of quaternary ammonium anion exchange resin on plasma and egg yolk cholesterol in the laying hen vitamin e deficiency and ion transpor~ in rat liver slices effect of level of nitrogen on growth and reproductive physiology of young buus and rams influence of low protein rations on growth and semen characteristics of young beef bulls, if treatment of nutritional cirrhosis in rats with choline and methionine; with special reference to fibrogenesis and fibroelasia probleme der beurteilung yon sauermilcherzeugnissen. milchwissen. schaft [ ] nr. , s. / . --antwort auf die diskussionsbemerkungen auf einer vortragstagung der gesellschaft ffir ern~hrungsbiologie e. v., miinehen, am response of early-weaned pigs to variations in dietary calcium level with and without lactose effect of low calcium diet on bone crysta]linity and skeletal uptake of sca in rats response of rural guatemalan indian children with hypocholesterolemia to increased crystalline cholesterol intake source of plasma free fatty acids in dogs receiving fat emulsion and heparin alcoholic intoxication in the newborn infant. bril mcd dental caries of rats fed a rice diet and modifications a study of zinc deficiency in the dairy calf effects of different levels of zinc and phosphorus on the growth of subterranean clover (trifolium subterraneum l) lrber den einflu yon fluor auf den wassergehalt des knochens gegevens ovvr vitamine b,-deficientie, -behoefto en -voorziening the liquefying action of pancreatic, cereal, fungal, and bacterial co-amylases ern~ihrung und endokrines system . mitt.: der einfiul der erniihrung auf die schilddriise the effect of saline water on kidney tubular function and electrolyte excretion in sheep zinc and iron deficiencies in male subjects with dwarfism and hypogonadism but without ancylostomiasis, schistosomiasis or severe anemia die yextriiglichkeit yon xyli~ beim diabetiker einflni der laevulose auf die fu~ionsbrelte. sport~tl. praxis der einflul~ yon vitamin a auf den zitronensi~urestoffwechsel studies on the growth-promoting value and digestibility of passion fruit seed oil ration effects on drylot steer feeding patterns dextrothyroxine on metabolism of cholesterol some effects of feeding lactates to dairy cows copper deficiency problems in south-east scotland bone changes in iron deficiency anaemia a preliminary report on nucleic acid levels in mineral deficient plants metabolism of histidine in protein malnutrition ttypoglycemie effect of l-leucine during periods of endogenous hyperinsulinism nutritional studies with the guinea-pig. viii. : effect of different proteins, with and without amino acid supplements, on growth some effects of sulphur-containing amino acids on the growth and composition of wool effects of hunger and vi value on vi pacing potency of conditioned reinforcem based on food and on food and punishment sfibwaren und karies in theorie und praxis effect of magnesium on the changes of myocardial potassium confent untersuchungen fiber den einflub oral verabreiehten oxytetraeyclins auf leberlipide und serumeholesterin der weil~en ratte further studies on manganese nutrition of tobacco in relation to virus infection and synthesis aminobutyric acid (),-aba)-vitamin be relationships in the brain serum lipids and diet: a comparison between three population groups with low, medium, and high fat intake effects of light and gibberellin on elongation of intact wheat coleoptiles experimental magnesium deficiency in the cow thyroid function in chickens and rats: effect of iodine content of the diet and hypophysectomy on iodine metabolism in white leghorns cockerels and long-evans rats kuhmilehallergie beim sgugling und a rapid method for assessing drug inhibition of feeding behavior variation in, and the effects of vitamins on vertieillinm albo-atrnm influence of high level vitamin a supplement on semen characteristics and blood composition of breeding bulls influence of diet on viral hepatitis der einflul] der stiekstoffdfingung auf die zusammcnsetzung yon karf~ffeleiweib insulin response to fructose and galactose the effect of excess vitamin a on the oxygen consumption of young female rats effect of dietary amino acid pattern on plasma ~mino acid pattern and food intake gibbere/lln: effect on diffusible auxin in fruit development effect of intravenous versus oral fat administration in fat-deiicient dogs plasma vitamin bi~ levels in some nutritional deficiency states nutritional factors influencing the conversion of tryptophan to niacin pancreatic hypertrophy and chick growth inhibition by soybean fractions devoid of trypsin inhibitor production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens effect of palm jaggeries on the growth and blood and liver composition of albino rats kept on rice and jowa effects of polyphosphates on water uptake, moisture retention, and cooking loss in broilers untersuchungen fiber den ern~hrungsphysiologischen wer~ yon kasein entgegnung zu diskussionsbemerkungen auf einer vortragsveranstaltung der gesellschaft for ern~ihrungsbiologie e. v., mfinchen, am . juni die erg~nzungswirkung yon dl-methionin allein oder in kombination mit l-lysin beim wachsenden schwein der einflul~ der nahnmg auf den kauapparat der einflu der nahrung auf den kauapparat. teil il changes in bone mass and density in living rats during the manipulation of calcium intake effect of chromium, cadmium, and other trace metals on the growth and survival of mice a study of fermentation in the production of mahewu, an indigenous sour maize beverage of southern africa the vitamin b~ deficiency syndrome in human infancy, biochemical and clinical observations lipids in chick urine: the influence of dietary rapeseed oil effects of dietary nitri~ on the chick" growth, liver vitamin a stores, and thyroid weight influence of radioactive sodium- on higher nervous activity of dogs, when chronically administered into the organism in comparatively small doses the change of erythroeyte blood composition in persons with prolonged complete alimentary starvation (without limiting the water intake) and subsequent feeding dental abnormalities in rats attributable to protein deficiency during reproduction the effect of environment, and nutrition of pathogen and host, in the damping off of seedlings by rhizoctonia solani effect of dietary protein on fructose, citric acid, and -nucleotldase activity in the semen of bulls the effect of fluorine on dairy cattle. v. : fluorine in the urine as an estimator of fluorine intake some effects of diet and therapy on the survival and metabolism of adrenalectomized rats effect of methonine and choline deficiency on liver choline oxidase activity in young rats untersuchungen fiber die wasserlssliehen hemmstoffe aus dem chnittholz der weinrebe (vi~is vinifera l.). i. zur wirkung der hemmstoffe auf die keimlmg mad entwicklung yon rebs~mlingen nutrition and palatability. lancek the effect of feeding fluoride on some enzymes of bovine tissues diet and histamine in the rmninant effect of food reflexes on cholinestera~e activity of cortical tissue. federation essential fatty acid deficiency and rat liver homogenate oxidations the effect of vitamin and antibiotic injections on early turkey poult growth and mortality alimentary production of gallstones in hamsters. . studies with rice starch diets with and without antibiotics nitrogen studies with apple and cranberry the influence of diet on the quality of faecal fat in patients with and without steatorrhoea uber die unterschiediiche beeinflussung des tryptophansteffwechsels dutch vitamin b -mangel in der ratte. hoppe-seyler's influence of vitamin b and its coenzyme upon incorporation in rive of amino acids into tissue proteins in rats relativer vitamin b -mangel hei erkrankungen der schilddriise strukturanomalien der z/ihne bei vitamin d-mangel-raehitis und der vitamin d-resistenten renalen rachitis the effect of fluoride on bone effects of insulin on hepatic glucose production and utilization prevention by hydrocortisone of changes in connective tissue induced by an excess of vitamin a acid in amphibia acute hypervitaminosis a in guinea-pigs. i. : effect on acid hydrolases der einfiu~ yon vollkornbrot auf den calciumstoffwechsel bei schulkindern effect of feeding milk replacers with varying amounts of f~ for hothonsc lamb production egg yolk and serum cholesterol levels: importance of dietary cholesterol intake effect of protein intake on glutamic dehydrogenase and amino acid desruination in rive observations in experimental magnesium depletion effect of gibbercluc acid on growth, gibberellin content, and chlorophyll content of leaves of potato ~ physiological factors influencing growth, reproduction, and production of well-fed dairy heifers. i. age at first breeding. ii. feeding of diethytstilbestrol tm~ influence of diet on the development of parotid salivation and the rumen of the lamb bericht fiber den wissenschaftlichen kongreb der deutschen geseuschaft fiir erniihrung influence of variations in dietary calcium: phosphorus ratio on performance and blood constituents of calves the lack of a consistent chick growth response to norwegian kelp meal some effects of kinctin on the growth and flowering of intact green plants incorporation of [, p] orthophosphate into phospholipids of frog tissues during feeding and stmrvation growth-modifying and antimetabolite effects of amino acids in chrysanthemum a study of techniques used by advertisers in dealing with weight control. a national health problem lipid excretion. .: examination of faecal lipids of rats injected intravenously with serum lipoprotcin containing ~ac-labelled cholesterol effect of diet and diabetes on plasma glucose, fatty acid, and insulin effect of cigaret smoking during pregnancy: study of cases. obstetrics gynecology respiration and phosphorylation in live homogenates from rats exposed to hypoxia and food restriction the influence of mi]l~ fat depressing rations on the yield and composition of bovine milk phosphatides and cholesterol in the rat bed: effects of growth, diet, and age the effect of plant nutrients and antagonistic microorganisms on the damp. ing-off of cotton seedlings caused by rhizoctonia solani kurus l~utrition of gram-negative anaerobic bacilli. nutrition rev. effects of glucose on the production by escherichia coli of hydrogen sulphide from cysteine. j. general iylierobiol. enumeration of psyehrophilie microorganisms vitamin requirements of three pathogenic fungi calorie requirements of rat intestinal microorganisms specificity of the salt requirement of halobacterium cutirubrum influence of the aqueous potato extract and its fractions on growth and spore formation of the b. pumilus and the production of the antibiotic tetaine the relationship between hormonal activity and sugar metabolism in protoparce scxta (joka~sen) and blabcrus craniifer bur~ieister apparent incorporation of ammonia and amino acid carbon during growth of selected species of ruminal bacteria l~ber die wirkung anorganischer st~ube auf das wachstum yon mikroorganismen effect of dietary calcium lactate and lactic acid of faecal eseherichia coli counts in pigs uber den einflul des n~ihrsubstrats auf die hemmung des bakterienwachstums durch cyanid autoradiographic studies of the differential incorporation of glycine, and purine and pyrimidine ribosides by paramecium aurelia correlation between the essential amino-acid requirements of staphylococcus aureus, their phage types, and antibiotic patterns nutrition and metabolism of marine bacteria. xii.: ion activation of adenosine triphosphat~se in membranes of marine bacterial cells carbon dioxide fixation in bacillus anthracis bacterial growth under conditions of limited nutrition interrelationship between temperature and sodium chloride on growth of lactic acid bacteria isolated from meat-curing brines morphological variation and nutrition of a new monoeentric marine fungns feeding stimulants required by a polyphagons insect, schlstocera gregaria vitamin requirements of root nodule bacteria phcnotypic, genotypic, and chemical changes in starving populations of aerobacter aerogenes studies on the d-amino acids. ii.: utilization of d-amino acids by lactic acid bacteria role and formation of the acid phosphatase in yeast der einflub yon co~-partia]druck und glucose-konzentration auf wachsturn und stiekstoffbindung yon azotobacter chroococcum bei~ inorganic polyphosphate metabolism in chlorobium thiosulfatophilum effects of molybdenum, vanadium, tungsten, and cobalt on growth of rhizobia and their hosts nutrition of leptospira pomona. ii.: fatty acid requirements sterilization by beta-propiolactone of solid nutrient media for eultivation of moulds the digestion of natural food protein by the elearnose skate raja eglanteria (bose.) utilization of amino acids during metabolism in escheriehia coil the effect of nutrition on the growth of fasciola hepatica in its snail host cobamide coenzyme contents of soybean nodules and nitrogen fixing bacteria in relation to physio]ogical conditions determination of carbohydrate metabolism of marine bacteria the amino acid requirements of various types of shigella mushroom culture. factors affecting the growth of morel mushroom myecelium in submerged culture lebensmittelzusatzstoffe und mutagene wirkung. vii. : priifung einiger xanthen-farbs~offe auf mutagene wirkung an escherichia coll the biological control of glycogen metabolism in agrobaeterium tumefaeiens the maintenance requirement of escheriehia coll methionine requirement for growth of a species of mieroeoecus ~iorphogenesis and nutrition in the memnionella-stachybotrys group of fungi viable organisms from feces and food-s~uffs from early antarctic expeditions the metabolism of yeas~ sporulation. v. : stimulation and inhibition of sporulation and growth by nitrogen compounds the effect of lipids on citric acid production by an aspergillns niger mutant relationship between deuterium inhibition of growth and glucose catabolism in saecharomyees cerevisiae function of trehalose in baker's yeast (saccharomyces cerevisiae). arch. biochem preparation and lyophilization of colicine suspensions. i. production of eolicines in liquid nutrient media lvber den einflub der kulturbedingungen auf die stramenempfmdliehkeit der glueoseoxydation in baeterium cadaveris nutritional requirements and metabolism of myeoplasma laid-lawil j. gen. microbiol. die wirkung subletaler konzentrationen yon sorbinsi~ure auf escherichia coli und aspergillus niger the genetic analysis of carbohydrate utilization in aspergillus nidulans the amino acid nutrition of respiration deficient and respiration competent saecharomyces. a. van leewenhoek nutritional studies of some membem of mucorales. iv.: . sugars, amino-, and organic acids of the myceaium selektivn~hrboden fiir staphylokokken effects of certain amino acids in anthranilate production in neurospora crassa studies on the polysaccharide-fermenting lactic acid bacteria. in. : nutritional requirements and the existence of fermentation promoting factors for sucrose and inulin the catabolism of proteins and nucleic acids in starved aerobacter aerogenes aerobic fermentation and the depletion of the amino acid pool in yeast cells influence of hydrogen ion concentrations on the utilization of sodium nitrite by fungi oxidative metabolism of glucose in leaf tissues infected with tobacco mosaic virus differential effects of amino acid deficiencies on bacterial cytochemistry nutritional requirements of an aspergiuus niger mutant for citric acid production culture de tissus d'insectes ~, l'aide d'extrait d'embryon de poulet en l'absenee d' h molymphe. c. r. acad utilization of some carbon and nitrogen sources by pseudomorms fluorescens on the selection of microorganisms for use in bacterial fertilizers in vitro and -rive uptake of carbon- labelled alanine and glucose by ascaridia galli, parasitic nematode of chickens growth of psychrophiles. i. : lipid changes in relation to growgh-temperature reductions vitamin requirements of listeria monoeytogenes parasitism and nutrition of gonatobo~rys simplex the effect of alkali metals on the growth of staphylococcus pyogenes the uptake of potassium and rubidium by staphylococcus pyogenes metabolism of nucleic acids and of nucleotides in the course of synchronous development of azotobacter vihelandii studies on the biotin-oleie acid requirements of a lactobaeillus plantarum variant isolated from chick feces unusual response to iron-dextran. brit. *ned. j. , nr. , s. . +in'. n.: tissue trypsin and trypsinogen levels in pancreatitis skeletal development of suckling kittens rate of liver regeneration atherogenesis in the monkey the significance of serum triglyeerides anaemia and parasitism in man physiological mechanisms in nutritionally-induced differences in ovarian activity of mature ewes bone development in suckled pigs production performance of artificiauy and non~r~ifically sired herd-mates in wisconsin search for an unidentified nutrient in mammalian liver. part i.: growth studies with various ox liver preparations proline control of the feeding reaction of cordylo-phora relationship between longevity and production in holstein-friesian cattle circadian adrenal cycle in c mice kept without food and water for a day and a half metabohc pmduc~s form labelled ethanol. iv. : disappearance of ethanol-carbon from morphological fractions and lipids of rat tissues acetate utilization by maize roots vajda, ] . : i~c~sll-rcs of body fat and hydration in adolescent boys some characteristics of a proteolytic enzyme system of pseudomonas fluorescens some metabolic relationships between host and parasite with particular reference to the eimcriae of domestic poultry nucleotide degradation in the muscle of iced haddock (gadns aeglefinns), lemon sole (pleuronectes microcephalus), and plaice (pleuronectes platessa) effect of starvation and -mcthylprednisolone (m_edrol) on the acid phosphatase of rat liver and muscle metabolic patterns in preadolescent children. vii. : intake of niacin and tryptophan and excretion of niacin or tryptophan metabolites biochemical changes in fish muscle during rigor morris studies on ornithine synthesis in relation to benzoic acid excretion in the domestic fowl effect of manual total collection of feces upon nutrient digestibilities polarographie studies on storage of meats. xxii. : influence of proteolytic enzymes on the polarographie wave of beef protein solutions post-mortem changes in chilled and frozen muscle genetic-nutritional interactiions as affecting the early growth rate of chickens effect of unequal milking intervals on lactation milk, milk fat, and total solids production of cows changes with age in glutamic oxalacetic transaminase activity of sonically oscillated tureen juice compared to total steam volatile fatty acids in calves fed different roughages catecholamine metabolism and some functions of the nervous system a study of some of the conditions affecting the rate of excretion and stability of creatinine in sheep urine changes in feeding behavior after intracerebral injections in the rat kanzcrogene substanzen in wasser und boden food additives and contaminants and cancer milk allergy in infancy food poisoning due to salmonella cnteritidis vat the mineral element content of spring pasture in relation to the occurrence of grass tetany and hypomagnesaemia in dairy cows insecticide residues in meat. residues in body tissues of livestock sprayed with sevin or given sevin in the diet over de giftigheid van solanum-alkaloidcn toxic products in groundnuts zur beziehung zwischen lipidcn hepatotoxicity of foods: a consideration of the hepatotoxicity of a few phanerogams and eryptogams. their possible influence in the pathogenesis of cirrhosis and hepatoma food-poisoning potential of the enterococei vanadium. excretion, toxicity, lipid effect in man an institutional food-poisoning outbreak examination of market milk of novokuznctsk for brucellosis an outbreak of food poisoning in a mental hospital food allergy as a cause of abdominal pain radioactivity in the diet the effect of microbial contamination on the requirement of chicks for certain nutrients the acute oral toxicity of cottonseed pigment glands and intraglandular pigments sur l'absorption du edsium radioactif par rorge. c. r. hebd. s ances aead die experimentelle alimentiire lebernekrose a]s empfindlicher indikator bei thermiseher belastung der milch. uber die magermilehtroekntmg the comparative toxicity of ethylene dibromide when fed as fumigated grain and when administered in single daily doses repository polyvalent insect antigen treatment for patients sensitive to hymenoptera. a clinical evaluation precursors of carcinogenic hydrocarbons in tobacco smoke toxin production in naturally separated liquid and solid components in preparations of heated surface-ripened cheese inoculated with clostridium botulinum allergieversuehe am tier zur ~tiologie der sogenannten margarinekrankheit. dr. reed. wschr. [ ] nr. , s. / . --, allergenwirkung oder immunologisohe adjuvanswirkung in der ~tiologie der sogenarmten margarinekrankheit radium- in human diet and bone miodine in the thyroids of north american deer experimental groundnut poisoning in pigs cholesterol as carcinogen safety factors in water fluoridation based on toxicology of fluorides entelo epidemiologische gegevens over her ,planta-exantheen" te rotterdam, verkregen door enquetc-onderzoek planta-~x~ntheem" epidemie te rotterdam in de m~nden ~ugnstu~ en september salmonella-verontreiniging van plantaardige grondstoffen veer voedingsmiddelen van mens en dier increase of strontium- and caesium- sodium fluoride intoxication salmonellosis in the netherlands zwei seltene salmonellenfunde untersuehungen fiber die chronisehe toxizit~t der ascorbins~ure bei der ratte captan in green vegetables rfickst~nde yon pflanzenschutzmitteln, insektiziden mid dergleichen in der nahrung und ihre bedeutung ffir die gesundheit der gehalt der milch an j, ,~co ' u ba _{_ la in der deutschen )iileh in der zeit yon langfristige nutritive anwendung yon antibiotika in der tierern~hrung im hinblick auf die menschliche gesundheit mi~ besonderer beriicksichtigung yon chlortetrazyklin a milk-borne outbreak of food poisoning due to salmonella heidelberg ergebnisse yon schwebversuehen an farbstoffen zur farbmattierung yon tabakwaren nutritional secondary hyperparathyroidism of the cat insecticide residues in fat. a screening method for chlorinated pesticide residues in fat without cleanup untersuehungen fiber die quantitative verteilung radioaktiver falloutprodukte in milch too many vitamins radios~ron$ium removal from milk. determination of apparent equilibrium constants of the exchange reactions of sodium, potassium, calcium, and magnesium with amberlite ir- ern~hrungsphysiologische eigenschaften der margarine. fette, seffen, anstrichmittel smoking and cancer: retrospective studies and epidemlologieal evalution beobachtungen fiber den verlauf der alkoholkrankheit am krankengut einer heilanstalt die verschmutzung yon trinkwasser dutch i)etergentien grain fumigant residues. occurrence of bromides in the milk of cows fed sodium bromide and grain fumigated with methylbromide insecticide persistence. the disappearance of endrin residues on cabbage lebensm ittelchem u. gerichtl reproductive performance of female miniature swine ingesting strontium- daily toxicity of nitrate nitrogen to cattle methods of residues of phosphated insecticides and miticides in foods on bacillary excretion in food toxinfections of salmonella etiology relationships between the deposition of strontium- and the contamination of milk in the united kingdom staphylococci in cottage cheese is~cs and potassium in people and diet. -a study of finnish lapps effect of treatment of seeds with -chloroethanol on the resistance to boron toxicity in wheat seedlings desferrioxamin, eine neue das eisen bindende und eliminierende substanz zur behandlung der rim~rcn und sckund~ren i-i~mochromatose akuter eisenvergiftungen toxic products in groundnuts smoking, arteriosclerosis, and age the incidence of milk sensitivity and the development of allergy in infants einflul] yon fluor und jod auf den stoffwechsel, insbesondere auf die schilddrtise quelques exemples illnstrant la valour et l'utllit des m~thodes de lysotypie clans certaines salmonelloses humaines d'origine alimentaire food poisoning caused by panthogenic halophilic bacteria (pseudomonas enteritis txkikawa): %ep rt of four autopsy cases procaine penicillin g in milk following intramuscular injections comparative subacute toxicity for rabbits of citric, fumaric, and tartaric acids distribution of pesticides in fermentation products obtained from artificially fortified grape musts mercurial fungicidal seed protectant toxic for sheep and chickens the problem of salmonella food poisoning dietary factors in the pathogenesis and treatment of cirrhosis of the liver. med. clinics of north america an outbreak of salmonella food poisoning in l~ehmadabad town, kaira i)istxiet, gnjaxat ~a~e la tossinfezionl alimentarl da salmonella nell' spedale maggiore di milano dal al water intoxication due to oxytocin: reporb of a case c~sium- und kalium in menschlichen organen und in der milch / caesium- in dried milk produets in relation to phytoellmatic zones smoking and oral cancer occurrence of hepatomas in rats fed diets containing peanut meal as a major source of protein nachweis yon mangan- und kobalt- in pflanzen als fo]ge russischer kernwaffenvcrsuche e-ruhr-epidemie durch speiseeis bcricht fiber eine arbeitstagung bei der internationalen atomenergic-behsrde in wien vom . bis . i)ezember . i)t. lebensmittel-rdsch the development of microbiological standards for foods. j. milk food technol a case of breslau salmoneliosis caused by eating chicken internationales rundgespr~ch fiber lebensmittelchemische probleme in wiesbaden und eltvllle a. rh. ( vortragsreferate) staphylococcal infection of raw milk as a cause of food poisoning. monthly bull. ministry health pub carcinogenic effect of egg white, egg yolk, and lipids in mice eczema and cow's milk exitus letalis nach lebensmittelvergiftung dutch bacillus cereus repression of staphylococcus aureus by food bacteria. ii. : causes of inhibition a further report on the radioactive contamination of milk and milk products in japan. determination of strontlum- and cesium- concentrations in milk powder in japan concerning sporadic salmonelloses insecticide residues. extraction and cleanup studies for parathion residues on leafy vegetables salmonellosis epidemiology zur frage der deponierung yon nutrltiven allergenen im organismus. allergic, _~sthma allergic children with various symptoms caused by cows' milk messungen der umweltsradioaktivit~t und der radioak-tivit~t yon lebensmitteln im jahre ein cxperimenteller bcitrag zur tabakrauehkanzerogenese. dr. reed. wschr. [ ] nr. . u n. : contamination of leaves by radio active fall-out insecticide residues in milk and meat. residues in butterfat and body fat of dairy cows fed at two levels of kelthane ( . and insecticide residues in milk. residues in milk from dairy cows fed low levels of toxaphene in their daily rations tier-und pfhnzenerniihrnug _anlrnal and plant nutrition summary of ,tropical crops soil, analysis, and its relation to plant composition and growth fertilisers and plant nutrients ulcers in swine tnfluence of chelating agents on the concentration of some nutrients for plants growing in soil under acid and under alkaline conditions nutritional evaluation of permanent pastures with dairy cattle in louisiana the herbage intake of eattle grazing lucerne and cocksfoot pastures terminology and methods for feeding and weighing animals the effect of feeding on evaporative heat loss and body temperature in zebu and jersey heifers studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to weeks of age some factors affecting iron uptake by strawberry plants feed consumption during lactation and involution in sprague-dawley-rolfsmeycr rats the effect of variations in the energy and protein levels of the ration upon performance in the pig use of barley in high-efficiency broiler rations. . poultry sci tierarzncimittcl und aufzuchtmittel in der landwirtschaftlichen praxis. gesund. heitliche erwggungen zum schutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmitteln in der landwirtschaftlichcn praxis, tell ii mechanism for movement of plant nutrients from soil and fertilizer to plant root growth rate of the tea leaf as determined by shade and nutrients. empire j. exper note on induction of flowering in ~railing shoots of clones of saccharum spontaneum effect of level and sequence of feeding and breed on ovulation rate, embryo survival, and fetal growth in the mature ewe evidence for a high zinc requirement at the onset of egg production aufnahme und wirkung des mikronghrstoffs knpfer in ionogencr und ehelatisierter form bei gerste effects of pelleting and varying grain intakes on milk yield and composition the relationship of gibberellic acid to flower initiation in column stock, math~ela incana the effect of selenium administration on the growth and health of sheep on scottish farms the horsebean (vicia faba l.) as a vegetable protein concentrate in chick diets size and feeding of different types of fishes the influence of age of chicks on their sensitivity to raw soybean oil meal influence of the mineral nutrition on the resistance of peach trees to fusicoceum amygdali de la croix granular fertilizer. influence of associated salts on plant response to dicalcium phosphate a comparison of feeding growing pigs once or twice daily the interrelation of nutrient supply, leaf nutrient content, and vegetative growth of ilcx crenata 'green island' effect of rationing grass on the growth rate of dairy heifers and on output per acre, with a note on its significance in experimental design experiments on the nutrition of the dairy heifer. iv.: protein requirements of -year-old heifers grass silage vs. hay for lactating dairy cows hay vs. silage for two to six months old dairy calves weaned at or days effects of high levels of copper and ehlortetracycline on performance of pigs seedlings resistance of corn to leaf feeding of the european corn borer ostrina nubflalis ease of hydrolysis of the hemieeiluloses of forage plants in relation to digestibility bodenkde. [ ] mr. , s. / . --caleium-mangelsymptome an h heren pflanzen effects of frequency of feeding on urea utilization and growth charae%oristics in dairy heifers factors affecting the voluntary intake of foods by cows. . : a preliminary experiment with ground, pelleted hay the relationship of dietary energy level and density to the growth response of chicks to fats salt tolerances of pinus thunbergii compensatory carcass growth in steers following protein and energy restriction a guide to production, care, and use of laboratory animals. federation prec. estimation of essential fatty acid intake in swine automatic dispensing at frequent regular intervals of liquid diet for piglets chelation in nutrition. chelates and the trace element nutrition of corn der einflul yon humuss~ure auf wachstum und ver~inderungen des freien zuekergehaltes bci winterweizenpflanzen, die im dtmkeln kultiviert wurden a comparison of the growth of chicks in the gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin an evaluation of weed competition and the effects of weed extracts and leachates on the development of field corn (zea mays l.) and oats (arena sativa l digestibility of rations containing different sources of supplementary protein by young pigs the effects of urea supplements on the utillzation of straw plus molasses diets by sheep production performance of artificially and nonartifieiallysired herd-mates inwiseonsin dietary phosphorus for laying hens tolerance to acid soil conditions in barley effect of calcium and magnesium upon digestibility of a ration containing corn oil by lambs effects of caloric restriction during the growing period on the performance of egg-type replacement stock untersuchungen fiber die verwertung yon calcium-und phosphorsalzen aus fisehgr~itenmehl einigcr frischfische und fischkonserven bei der verffitt~rung a nut~rient requirement for optimum water absorption by intact root systems preparation of feed for animal nutrition experiments responses of winter wheat to nitrogen and soil nitrogen status studies on calcium requirements of broilers znm problem dcr nahrungspflanzenwabl der aphiden some factors affecting leaf development and longevity and the subsequent yield of corn grain efficiency of energy utilization by young cattle ingesting diets of hay, silage, and hay supplemented with lactic acid the effects of a plant steroid on body weight and feed efficiency of broilers feeding troughs for guinea-pigs beitrag zur ]~ackfruchtmast mit schweinen unter besondercr beriicksichtigung des n~hrstoffgehaltes der beifuttermischungen und der the feeding of thyroprotein to lactating sows zur planung, durchfiihrnng und auswertung yon schweinemastvcrsuchen bei gruppenfiitterung the influence of barbituric acid derivatives on the development of plant roots and root hairs factors affecting the voluntary intake of food by cows. .: the relationship between the voluntary intake of food, the amount of digesta in the retieulo-rumen, and the rate of disappearance of digesta from the alimentary tract with diets of hay, dried grass or concentrates artificial food for oak-silkworm raising the comparative toxicity of ethylene dibromide when fed as fumigated grain and when administered in ingle daily do~ ~ gibberellin at the vineyard oak wilt development and its reduction by growth regulators. i. production and activity of oak wilt fungus pectinase, ecmulase, and auxin. ii. effect of halogenated benzoic acids on oak trees, the oak wilt diseases, and the oak wilt fungus regulating nutrient intake in laying hens with diets fed ad libitum some effects of different soils on composition and growth of sugar beet production of fodder yeast from barley. i. preliminary studies on the use of the waldhof fermenter development and nutrition of new species of thranstochytrium studies on the effect of frequency of feeding upon the biology of a rabbit-adapted strain of pediculns humanus the influence of previous vitamin k nutrition on thromboplastie activity of brain extract the effect of nutrition conditions on the growth of and nitrogen accumulation by fodder beans when sown together with indian corn. i)oklady akad. nauk sssr dutch phenylbors~ure induzierte fragen der resistenzsteigerung in der modernen gefliigelhaltung chelation in nutrition. metal chelates in plant nutrition beziehungen zwischen dcm kupfergehalt und dem zeitlichen auftreten yon kupfermangelsymptomen an hafer in wasserkultur mit kleincn bodenmengen increased tolerance of bean plants to soil drought by means of growth-retarding substances effect of monocaleium and diammonium phosphates on crop yield, and their influence on soil solution movement and characteristics when associated with different salts effect of barbituric acid and ehlortetraeyeline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks effects of feeding low levels of dimethoate on milk and on whole blood cholinesterase activity of dairy cattle die ziichtung von fleischschweinen und die folgeerscheinungen, die sich besenders im hinblick auf die qualit~t yon fleiseh und fett ergeben the relationship of specific nutrient deficiencies to antibody response in swine. i.: vitamin a. ii.: pantothenie acid, pyridoxine or riboflavin further studies of diet composition on egg weight effect of various levels of fluorine, stilbestrol, and oxytetraeycline, in the fattening ration of lambs studies on the properties of l~ew zealand butterfat. viii. the fatty acid composition of the milk fat of cows grazing on ryegrass at two stages of maturity and the composition of the ryegrass lipids soil potassium and the growth of vegetable seedlings artificial feed for silkworm, bombyx mori some effects of feeding stilbestrol, ehlortetracyeline, and penicillin with alfalfa soflage on steer performance and carcass quality food agric. [ ] nr. , s. / . --, mineral supplements for sheep the influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep untersuehungen fiber den umsatz waehsender sehweine ab geburt. . mitt growth of edible emorophyllous plant tissues in vitro chelates in agriculture. metal chelation by glucose-ammonia derivatives economic analysis of high-level grain feeding for dairy cows evaluation of the dacron bag technique as a method for measuring cellulose digestibility and rate of forage digestion n~ihrlssungen fiir zuckerriiben in wasser-und sandkultur activity of gibbereuin:'d' on the germination of photosensitive lettuce seeds raw and heat-treated soybeans for growing-finishing swine, and their effect on fat firmness ration effects on tureen acids, ketogenesis, and milk composition. i.: unrestricted roughage feeding a new growth stimulant, ~ growth hormone the effects of specific viruses, virus complexes, and nitrogen nutrition on the growth, flowering, and mineral composition of strawberry plants peculiar feature of respiration and redox processes in the rice plants grown under different nutritional conditions einflul] der silagefiitterung auf die qualit~t yon milch und milchprodukten. . mitt. : einflul] der silagcfiitterung auf die organoleptischen eigenschaften dcr milch effect of grinding and pelleting on the utilization of coastel bermuda grass hay by dairy heifers langfristige nutritive anwendung yon antibiotika in der tierernlihrung im hinblick auf die menschliche gemmdheit mit besonderer beriicksichtigung yon chlor-te~azyklin mode of action of growth retarding chemicals yield of sugarcane in louis-ana as influenced by soil moisture status and climate diss effect of auxin on the emergence of lateral roots in p. mungo seedlings compound mouse diets a semipurified caries-test diet for rats present status of feeding antibiotics to htctating dairy cows effect of sodium bicarbonate in the drinking water of ruminants on the digestibility of a pelleted complete ration semi-purified diets for sheep effect of vacuum-drying, freeze-drying, and storage environment on the viability of pea pollen. ii. : effect of boron, sucrose, and agar on the germination of pea pollen hshe und zeitpunkt der diingung yon sommerweizen mit chlorcholinchlorid zur verkiirzung der halml~nge nutritional signifieance of soluble nitrogen in dietary proteins for ruminants primary signs of nutritional deficiencies of laboratory animals ]~ffe[~b of dlctary pro~in and fat on growth, protein utilization, and carcass composition of pigs fed purified diets trans-fetts~iuregehalt yon schweineschmalz nach fiitterung yon sehweinen mit rindertalghaltigem kraftfutter. (ein beitrag zur quantitativen infrarotspektroskopischen bestimmung yon trans-fetts~uren in fetten effect of liming and potassium fertilization on soil solution and on yield and composition of alfaffa and orchard grass mixtures effects of feeding various milo, corn, and protein levels on laying performance of egg production stock effect of gibberellie acid on flowering of apple trees the effects of dietary fat and energy ]evels on the performance of caged laying birds effect of age on the response of chickens to dietary protein and fat chemical control of flowering. concentration of a floral-inducing entity from plant extracts strontium- and calcium in milk of miniature swine studies on the properties of newzealand butterfat. vii. effect of the stage of maturity of ryegrass fed to cows on the characteristics of butterfat and its carotene and vitamin a contents new radioactive tests show how termites feed mechanisms regulating the feeding rate of daphni~ magna straus influence of low protein rations on growth and semen characteristics of young beef bulls a study of zinc deficiency in the dairy call effects of different levels of zinc and phosphorus on the growth of subterranean clover (trifolium subterraneum l.). australian j. agrie absorption, translocation, exudation, and metabolism of plant growth-regulating substances in relation to residues the effect of the performance of growing pigs of the level of meal fed in conjunction with an unrestricted supply of whey increase in yield of legumes by fer~iliser mixture with lime chemically defined medium for growth of animal cells in suspension dis sieherung der eiweiljwrsotgung in dor l~ndwirt influences of previous calcium and phosphorns intake and plant phosphorus on the requirement of developing turkeys for calcium and phosphorus relationship between isotopicauy exchangeable calcium and absorption by plants effect of adding buffers to all-concentrate rations on fcedlot performance of steers, ration digestibility, and intrarumen environment lysine supplementation of corn -and barley-base diets for growing-finishing swine the effect of gibbereliin on the germination of seeds of arboreal plants effect of physical state of coastal bermuda grass hay on passage through digestive tract of dairy heifers nitrate reduction and carotene stability. effect of nitrate and some of its reduction products on carotene stability, d. agric. food chem chemical preparations for plant protection untersuchungen fiber die ksrperzusammensetzung und den stoffansatz waehsender mastschweine und ihre beeinflussung dutch die erniihrung. . mitt the cobalt requirement of sub-~erranean clover in the field comparing mile and corn in broiler diets on an equivalent nutrient intake basis effect of mineral nutrition on the invasion and response of turnip tissue to plasmodiophora brassicae wor the relation of chlorogenic acid and total free phenols in potato plants to resistance to infection by verfieillium alboafxum nitrogen and potassium as variables influencing soluble nitrogen and organic acid accumulation in soybean (glyoine max). di~s. abstr. bett~r british beef and barley feed. veter effect of nitrogen fertilization upon yield and digestibility of aftermath timothy forages fed to dairy heifers ration effects on dltlot steer feeding patterns effects on zea mays seedlings of a strontium replacement for calcium in nutrient media evaluation of albumen quality in a poultry breeding program nutritional studies with the guinea-pig. viii. : effect of different proteins, with and without amino acid supplements, on growth some effects of heredity and environment on appetite in dairy animals further studics on manganese nutrition of tobacco in relation to virus infection and synthesis amillo acid supplementation of pig diets chelation as a basic biological mechanism der einflu~ der stiekstoffdiingung auf die znsammensetzung yon kartoffeleiweiil z studies on photosynthesis. i. : biosynthesis of sucrose from glycolate. par~ ii. : bicarbonate utilization by washed algae production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens alteration of post-mortem changes in porcine muscle by preslaughter heat treatment and diet modification chelation in nutrition. soft microorganisms and soil chelation. the pedogenie action of lichens and lichen acids die ergiinzungswirkung yon dl-•ethionin allein oder in kombination mit l-lysin beim wachsenden schwein untersuchungen iibcr den einflub unterschiedlicher wasservemorgung auf ertr~ge, gehalte an ~therischem , trenspirationsquotienten, biattgrsl~en und relative ~)ldriisendichtsn bei einigen arten aus der familie der labiaten. . teil gehalte an iitherischem , transpirationsquotienten, blattgrsflen und relative -driisendichten bei einigen arten aus dcr familie der labiaten. iii.: blattgrsflen, relative driisendichten, anzahl am haupttricb inseriertcr blattpaare und internodien. liingen cadmium: uptake by vegetables from supcrphosphate in soil studies on the protein and methionine requirements of young bobwhite quail and young ringnecked pheasants chelation in nutrition. evidence for natural chelates which aid in the utilization of zinc by chicks selective fertilization of apple-trees some soils and fertilizer relationships of the cavendish banana (muss cavendlshl lambert) on three different soils in costa rice soil organic phosphorus and the phosphorus nutrition of plants the effect of heat treatment on the nutritive value of milk for the young calf. . : a comparison of spray-dried skim milks prepared with different preheating treatments and roller.dried skim milk, and the effect of chlortetracyclinc supplementation of the spray-dried skim milks the effect of heat, ~reatmen~ on the nutritive value of milk for the young calf. . :the effect of the addition of calcium a biological assay for metabolizable energy in poultry feed ingredients together with findings which demonstrate some of the problems associated with the evaluation of fats feed additives in livestock rations: part i. : urea in dairy rations. part.i/: use of thyroprotein in cattle nutrition diet and histamine in the ruminant synthetic ion-exchange resins as a medium for plant growth nutrition of vibrio fetus theoretical basis of unicellula algae cultivation amino acid supplementation of barley diets for growing swine some effects of . -dichiorophen-oxyacetic acid on swect corn (zea ]~ays rugosa l.) with emphasis on yield, tillering, root development, and exudation of electrolytes from roots and stems. i)iss. abstr feeding and management of broiler strain breeder hens relationships among seven elements in the nutrition of corn in sand culture an external effect of inorganic nitrogen on nodulation influence of enzyme supplements in lamb fattening rations gravenstein and jonathan apples produced with giberellle acid the role of carotene in the dairy cow. wiss. ver ff. dr. ges. ern~hrung vitamin a-wirksamkeit der carotine bei versehiedenen tierarten. wiss. ver- ff. dt. ges. eru~hrung upgrading the indigenous poultry of uganda. i. : the growth rates and feed conversion from hatching to maturity of indigenous poultry crossed with four imported breeds effect of different kinds of litter on growth and feed efficiency in chick rearing investigation of the mineral nutrition of datura innoxia the effect of flooding in the availability of phosphorus and on the growth of rice nutrition of the boll weevil larva ascorbic acid in the nutrition of plant-feeding insects effects on the s~maeh worm, i-iaemonehus contortus, of feeding lambs natural versus semipuriiicd diets yield and foliar composition of corn as affected by fertilizer rates and environmental factors. i)iss. abstr. ~ [ ] nr praktische erfahrm]gen in der carotinoidversorgung yon vsgeln effect of protein-energy relationship on the performance and carcass quality of growing swine the use of quarter samples in the assessment of the effects of feeding treatments on milk composition * calcium and phosphorus requirements of finishing broilers using phosphorus sources of low and high availability amino acid supplementation of peanut meal diets for broiler chicks the effects of feeding various levels of vitamin a on chicks with cecal coccidiosis chelation in nutrition. review of chelation in plant nutrition water use by irrigated arabia coffee in the failure of certain dietary ingredients to affect the incidence of blood spots in chicken eggs dcr einflul~ der anbauverh~ltnisse auf die eigensehaften der kartoffelknolle und der st~rke effect of feeding milk replacers with varying amounts of fat for hothouse lamb production l~hysiologieal factors influeneelng growth, reproduction, and production of wcll-fed dairy heifers. i. ago at first breeding. ii. feeding of diethylstflbestrol results of an experiment ot rothamsted testing farmyard manure and n, p, and k fertilizers on five arable crops. i. : yields results of an experiment at rothamsted testing farmyard manure and n, p, and k fertilizers on five arable crops. ii.: nutrients removed by crops the utilization of carotenoids by the hen and chick some effects of potassium and lime on the relation between phosphorus in soil and plant, with particular reference to glasshouse tomatoes, carnations, and winter lettuce the lack of a consistent chick growth response to norwegian kelp meal further studies on protein and energy requirements of chicks selected for high and low body weight some effects of kinetin on the growth and flowering of intact green plants individual feed consumption of turkey breeder hens and the correlation of feed intake, bocly weight, and egg production crop analysis technique for studying the food habits and preferences of chickens on range supplemental value of turkey protein for wheat herbicides and plant growth regulators preparation of purified ration for chick. parg iv. : preparation of crystalline amino acid diet evaluation of algae as a food for human diets the influence of milk fat depressing rations on the yield and composition of bovine milk the effect of plant nutrients and antagonistic microorganisms on the damping-off of cotton seedlings caused by rhizoetonia solani kukn ki;-;sehe ern~ihrung und di~tetlk clinical nutrition and dietetics a statement approved by the board of directors of the canadian heath foundation radioactivity and human diet probleme der ern~hrung durch gefrierkost. sympomum der d utschen gcgell~ehaft ffir ernghrung veto . bis . mgrz klinische ernghrungslehre" und wissenschaftlicher kongreb der deutschen gesellschaft flit ern~hrung an der johannes-gutenberg-universit~t mainz veto . bis wissenschaftlicher kongreb der deutsehen gesellschaft ftir ern~hrung an der johannes-gutenberg-universit~t mainz am . und ernghrung nnd digit" . deutseher kongreb ffir ~irztliehe fortbildung in berlin veto . bis arbeitstagung fiber klinisebe ernghrungslehre. ern~ foods of the future (forts.). problems in space foods and nutrition. foods for extended space travel and habitation the question of fats. ii.: fats and disease behandlung fettbedingter gerinnungsstgrungen mit lipostabil sugar and dental caries obesity and sugar addiction hunger and malnutrition lancet nutrition and general practice bericht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellschaft in der ddr vom arbeitstagung fiber kommission ffir volksern~hrung, lebensmittelgesetzgebung und -kontrolle (eek) zu yi~inden des eidg the national diet-heart study low fat diet in familial mediterranean fever the thyroid gland in infant malnutrition evaluation of fao amino acid reference pattern studies on the physiology of nutrition in surinam rickets in southern israel diet and heart disease maiskeims in ernt~hrung und ditltetik apha conference report safe and nutritious food supply malnutrition and disease expert committee on medical assessment of nutritional status protein malnutrition the fat tolerance curves of patients with hyperllpidcmia and athcrosclcrosis die lactose im rahmen der ernt~hrung effect of environment on nutritional status zur theorie und praxis der zuckerkrankheit. wiener z dietetically induced experimental flous of rats physikalisch-diiitetische therapie yon hautkrankheiten. arch. phys. therapie err~hrungsforschung [ / ] :nr. , s. / . +bo rtiw~l, p. w. : milk-borne disease consumers' reactions to instand foods de voeding van woonwagenbcwoners experimental investigations on nutrition and human behavior. a post-script. amer di~tetische therapie der chronischen herzinsuffizienz construction and validation of the food attitude scale why we have a safe and wholesome food supply use of food in a psychiatric setting stature and nutrition in cystinuria and hartnup disease ])as endokrinologisehe syndrom des proteinmangels urinary excretion of . -dlhydroxyphenylalanine (dopa) in two children of short stature with malnutrition current problems affecting consumption of milk and indnstry's response to them preparedness for emergency feeding fluoridation and public relations dieetprodukten in vlaanderen incorporation of labelled glycine into erythrocyto glutathione of rabbits; effect of nutritional muscular dystrophy hot wcreldvoedselvraagstuk sniker -glycogcen -tandbederf dietary in take in patients with arthritis and other chronic diseases a clinical trial of iron-fortified bread effects of freater intake of milk, fruits, and vegetables joint fao/w/to expert committee on nutrition" fiber eine sitzung in genf vom . his serum cholesterol in a military population. its relation to obesity and the military diet ). ~z~, a. c. some nutritional problems of older age groups neuere biochemisehe untersuchungen zur diagnostik und therapie yon b-vitamin-mangelzust~nden call-harvard nutrition project. iii.: the erythroid atrophy of severe protein deficiency in monkeys cali-harvard nutrition projeet. ii. : the erythroid a~rophy of kwashiorkor and marasmus zur hshe des erwiinsehten fottverbrauehs ern~hrung des sportlers voeding moderne ern~hrungsbedarfsnormen. i. mitt. z. ges. hyg. [ ] nr. , s. . *--~ioderne ern~hrungsbedarfsnormen. . mit~.: z. ges. hyg. a comprehensive home-care program for the chronically ill fat-modified foods for serum cholesterol reduction besonderheiten der ern~hrung alter menschen chronic malnutrition in turkey. v. studies on serum fatty acids in malnourished children prevention of ,meat anemia" in mice by copper and calcium beeinflussung der sportlichen leistungsfdhigkeit dutch eine geeignete er-n~ihrung physiology of adolescence. ii, l~u-trition -basal oxygen consumption -energy expenditure and balance -nitrogen metabolism -calcium metabolism -iron metabolism -red cell mass and hemoglobin ern~hrungsproblemc bei chirurgisehcn kranken. wiss. versff. dr. ges. ern~hrung moderne vitamin b~-therapie: oral, rektal oder parenteral ? a palatable diet for producing experimental folate deficiency in man smoking in hospital was ist hungern und was heibt the cultivation of tflapia. this prolific fish as a fine source of proteinrich food in underdeveloped areas pyridoxine supplementation during pregnancy. clinical and laboratory observations on japanese foods nutritional sequelae of ga~trio surgery koehsalzarme kost und nierenerkrankungen theoretische und praktische grundiagen der ernilhrung in der fett in der diabeteskost foods or supplements? g zur hygiene und ,di~tetik des rauehens zur dis behandlung der uremic anaphylactie shock of the lungs triggered by mieroaspiration of cows' milk: a form of sudden unexpected death in early infancy the food service industry and its relation to the control of foodborne illness die konservative therapie des peptischen gesehwiirs gezondheid op reis addictive aspeeta in heavy cigarette smoking die ern~hrung im rahmen de~ heilvers im kurort the diet in renal faiiure is the rationale for gaetrointestinal diet therapy sound? familie-beruf-ern~hrung die dfiitbehandiung der leberkrankheiten. i)t. reed vom hunger his zum ~beritul -weltweite ern~hrungsprobleme die bedeutung der vitamine in der t~tglichen erniihrung s~ure-und baseniibersehiissige naln'ung. therapiewoehe [ ] nr. , s. / . --ss und chronische acidogene und alkalogene erns z. em~hrungswiss industrial lunches and public health the assessment of nutritional status in man: chairman's opening remarks therapie der essentiellen hypertonie speeifieke voedings-en voorlichtingsproblemen in tropische landen wie kann man unsere kos~ und unsere kostgewohnheiten beeinflussen? neue konzeptionen in der wasser-und salzsubstitution some aspects of the relation of nutrition and pregnancy is coronary heart disease preventable? world hunger demineralization of whey. use of its protein in infant feeding elaidinized olive oil and cholesterol atherosclerosis soziatrischo aspekte dee genul)-und arz~aeimittelkonsums verwendung yon htilsenfriichten in der diabetiker-di~t sanitation and dishes sanitation and dishes. aspects old and new. part ii smoking, arteriosclerosis, and age neue weg~ zur erni~hrungsphysiologi~chen aufwertung yon getreide-erzeugnissen diphyliobothrium latum and human nutrition, with particular reference to vitamin bli deficiency milk and diverticulosis dietary factors in the pathogenesis and treatment of cirrhosis of the liver. ivied. clinies north america zur frage der quanti~tiven charakteristik der ern~hrung der berufst probleme der gemeinschaftsverpflegung aus der sieht des ern~hrungsphysio-logen gegevens over vitamine b,-deficientie, -behoefte en -voorziening use of government-donated foods in a rural community fluoride, teeth, and the analyst eiweil bedarfsnormen im rahmen unserer ern~hrungsrichts~tze physiologic discomforts in navy protective shelter tests di~tvorschl~ige : akute nierenentziindtmg siii]waren und karies in theorie und praxis ernehrtmgsberatung im krankenhaus use of a low-sodium formula as an improved karell diet, with emphasis upon the outpatient management of heart failure and lymphedema pflanzliches eiweil~ fiir die erniihrung des menschen influence of diet on viral hepatitis influence of siblings on student smoking patterns voeding yon leerlingen van een lagere technische school. ii. calorie~n-en nutri~ntenwaarde early use of circulating blood volume, weights, and normal diet in acute renal failure fette in tier nahrung. dr. recd. wschr. [ ] nr. , s. / . *scm~-idt-b~bach, a.: ~j~ber die di~tetik der sauermilch begriff und anfgabe di~tetischer lebensmittel zur ursache yon geschmackskalamit~ten in trinkwassertalsperren psychologische motive im wandel des brotverzehrs improving levels of nutrition through better food practices the vitamin b~ deficiency syndrome in hun~n infancy. biochemical and clinical observations treatmen~ of ,refractory obesry" with ,formula die~ nr. , s. ff. ( s.). elwzea, c. c.: morphologie constitution and smoking prediction the outcome for obese dieters symposium fiber probleme der ern~hrung durch gefrierkost in karlsruhe yore coordination of long-term care of :pku children die di~t bei diabetes meuitus a nutritional supplement (nutrament) for elderly patients dietary intake of five groups of subjects. -hr. recall diets vs. dietary patterns the prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients stand und perspektiven der eiwei~versorgung. zur zielsetzung des verhandiungsthemas de voeding van rijswerkers signification des standards calorieo-azotes utilists en france erg~nzungen veto standpunkt des lebensmittelehemikers zu (dem beitrag) official acceptance of homogenized milk in the united states advances in nutrition and dietetics nutrition of naval recruits during a shelter habitability study hot ombuigen van voedingsgewoonten de voeding van schippemkinderen san boord en in de internaten voeding [ ] iqr. , s. / . --le traitement de rinsuffisance rtnale her zoutarme-eiwitarme dicer ein beitrag zur allgemeinen el problematik, ausgehend yon einer anorexia nervosa rroblem~ in nutrltlon~l supplementation an ~mri~hnl~ut detection of nutritional imbalances theorie und praxis der schwangerenern~hrung die pharmakologisehe beeinflussung yon hunger mad s~ttigung problems in the evalutaion of nutritional status in chronic illness europ~ische di~ttagung in amstercl~m ( . bls entwieklung des brot-und gctreidevcrzehrs in der neuercn zeit nutrition and palatability coehac dis~tse. -biochemical and technological aspects die di~itetik der :fettsucht kinderemll}~mg nutrition of infants and children report on infant feeding childhood nutrition in lapland. :nutrition rev the thyroid gland in infant malnutrition. nutrition rev. [ ] nr. , s. . +n. n.: physical activity of obese girls appraisal of nutritional adequacy of infant formulas used as cow milk substitutes isomaltose intolerance causing decreased ability to utilize dietary starch passagere hypereh]or~mische azidose bei zwei ausgetragenen s~uglingen wghrend s~uremflch-em~hrung ober erfahrungen in der frfihgeborenonaufzucht mit einer neuen bedarfsangepa ten friilmahrung nutritional defects in adolescence g p intravenous glucose tolerance in the normal newborn infant: the effects of a double dose of glucose and insulin ! attitudes towards physical ac. tivity, food, and family in obese and nonobese adolescent girls urinary excretion of . -dihydroxyphenylalanine (dopa) in two children of short stature with malnutrition high salt content of western infant's diet: possible relationship ~o hypertension in the adult vitamin e to premature infants des enzym yon ~'leming (lysozym) und seine bedeutung flit die si~ugllngsern~ihrung. ann investigation on the relation of between-meal eating and dental caries of sixth-year molars in school children studies in infantile malnutrition. i.: nature of the problem in peru chronic malnutrition in turkey. v. studies on serum fatty acids in malnourished children emi~hrnng mad faekalc lysozymaktivitiit beim s~iugiing role of linoleio acid in infant nutrition factors related to the eating behavior and dietary adequacy of girls to years of age. dies. abstr. des vitamin c im jugendalter. ii. mitt.: uber die wirkung yon natiirlichem und synthctisehemvitamin c bei l~ngeren zugaben the incidence of protein-calorie malnutrition of early childhood ein besonderer znsammenhang zwischen dem bedarf an nahrungsfett und dem stoffwechsel in den ersten lebensjahren the effect of supplements of groundnut flour or groundnut prorein isolate fortified with calcium salts and vitamins or of skim-milk powder on the digestibility coefficient, biological value, and net utilization of the proteins of poor indian diets given to undernourished children lysine fortifications of wheat bread fed to haitian school children lrber den -stunden-rhythmus der kalorienerzeugung bei friihgeborenen beitriige zur frage der spezifisch-dyrmmi~ehen wlrkung auf grund yon glykokoll-belastungen bei friihgeborenen. acta paediatriea acad carotine in tier stiuglingserni~hrung. wiss. versff. d$. ges. ernkhrung liver and depot lipids in children on normal and high carbohydrate diets response of rural guatemalan indian children with hypocholesterolemia to increased crystalline cholesterol intake feeding value of soy milks for premature infants early feeding and birth difficulties in childhood schizophrenia. a brief study zusammenh~nge zwischen stoffwechsel und fl~issigkeitsbedarf beim s~ug-ling kuhmilchauergie beim s~ugling und ,cot death". die unspezifische kumulative sensibilisicrung malnutrition and the health of children practical aspects of infant feeding breast-feeding, weaning, ~nd acculturation appetithemmer in der ]~ehandlung der fettsucht bei kindern. miinchener med the effect of different amounts of vitamin d on growth and serum levels of calcium, inorganic phosphorus, and alkaline phosphatase in premature infants partition of urinary nitrogen in children with kwashiorkor treated with animal and vegetable proteins der einflud yon voukornbrot auf den caleiumstoffweehsel bei schulkindern ist eine rektale vitamin blz-behandlung vertretbar? dr ethyl alcohol in the pathogenesis of gout c|e~rance of infused fat emulsion in diabetic dogs praktische durehffihrung der parentcralon ern~hrung die parcntcrale ern~hrung chirurgischor paticntcn. wiss. ver ff. dr. ges. em~hrung verwertung intravenss verabfolgter aminos~,urengemische. wiss. versff. dr. ges die erkcnnung yon fe~-transportstsrungen und ihre bedeutung ftir die intra-ven se fettzufuhr intravenous glucose tolerance in the normal newborn infant: the effect of a double dose of glucose and insulin new intravenous fat emulsion indikationen und kontraindikationen der intraven sen fettzufuhr in der chirurgie anwendung intraven s gegebener aminos~urengemische in dcr p~diatrie ymtravensse fettinfusionen. wiss. versff. dt. ges. ern~rung ~qotwendigkcit und erfolge der parenteralen mad sonder-ern~hrung moderne vitamin blz-therapie: oral, rektal oder p~renteral? mcd anwendung intravenss gegebener aminos~urengemische in der gyn~kologie und geburt~hilfe aminos~ureninfusionen. schweiz. reed. wsehr. klinische anwendung mad erfahrungen bei der verabreiehung intravensser fettemulsionen an ehirurgischen patienten diskussionsbemerkung zum thema: die parenteralo ern~hrung ~tude exp~rimentale de la tolerance d'une solution de graisse vsgstale d'administration intraveineuse ern~hrungsphysiologische grundlagen der parenteralen ern~hrung erfahrungen mit der parenteralen ern~hrung mittels fettinfusionen. helvetica chirurgica aeta nitrogen, lipid, glycogen, and deoxyribonucleic acid content of human liver. the effec~ of brief starvation and intravenous administration of glucose techn~ und indikationen der parenteralen ern~hrung des neugeborenen die praktische organisation der klinisehen infusionstherapie mit zuckerund elektrolytlt)sungen. l ~ed untersuchungen und bcobachtungen fiber intravensse fettinfusionen in der inneren klinik. wiss. versff. d$. ges. ern~hrung l'alimentation parenttrale, mulsions lipidiques. (a suivre) ann intravcnsse ern~hrungstherapie mit fettemulsionen parenteral-und sondeneru~hrte patienten zur rekt~len kaliumsubstitufion parenteraie ern~hrtmg mit fettemulsionen konservierung mad zubereitung yon lebens-und futtermitteln nutritional hygiene, preservation and preparation of foodstuffs and feeds n. n.: vortragsveranstaltung ,fleischhygieno" der th seminar on the use of radioisotopes in nutrition science and of ionising radiation in food technology. strasbourg, st - th october progress of food irradia~on work and programmes in o.e.c.d. member countries ( berichte) safe heat processing of canned cured meats with regard to bacterial spores the role of food science i and technology on the freeze dehydration of foods public health aspects of handling animal products in the txopics fack)rs affecting bacfcrial spoilage of animal products at elevated temperatures. food technol sterilized concentrated millr. food teehnol. [ ] nr. , s. / , . n.n.: foods of the future. now opportunities for flavor modification unrestricted approval for irradiated bacon -a sanitary standards for multiple-use rubber and rubber-like materials used as product contact surfaces in dairy equipment -a sanitary standards for batch and continuous freezers for ice cream, ices, and similarly-frozen dairy foods bericht fiber die vortrag~tagung des fachverbandes lebensmittelchemio der chemischen gesellsch~ft in der ddr vom . bis lebensmittelchem. u. geriehtl. chem. fluoridation in great britain die enzymatische phycinspaltung in geschrotetem getreide in abh~ingigkeit yon der relativen lufffeuchtigkeit the effect of certain antioxidants during freezer storage of pork chops and sausage the mechanics of treating hatching eggs for disease prevention beitrag zur sfil~gerinnung yon kakaotrunk jodophore als desinfektionsmittel in der milchwirtschaft. milchwissenschaft [ ] nr. , s. ff. (? s.). zitat: dr. lebensmittel tierarzneimittel und anfzuchtmittcl in der landwirtschaftllehen praxis. gesundheitliche erwrgungen znm sehutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmitteln in der landwirtschaftlichen praxis n~ihrwertminderung dureh zubereitung dcr nahrung suue modifieazioni della flora mierobiea dei mollusohi eduli par effetto di eonservazione impropria verlinderungen des inhaltes yon dosenkonserven w~hrend lgngerer lagerung digtbrote aus der sieht ihrer praktischen gestmtung. wiss. versff. dr. ges. ern~hrung l erniihrungshygienische untersuchungen in kindergarten an budapester kindern im alter yon bis jahren. z. ges. hyg. die eignung der bakteriologischen untersuehung yon kannenmilchproben als grundlage eines eutergesundheitsdienstes adequacy of cooking procedures for the destruction of salmonellae zur revitaminierung des mehles bzw. brotes. wiss. versff. dr. ges. er-n~hrung conventionele verwarningsmethoden beitrag zur kenntnis der wechselwirkungen zwischen proteinen und poly. phenolcn der kakaobohnen wtihrend der fermentation nihydrazone feed medication ag~ins~ ar~iiieiaily induced escheriehia eoli air-sac infection foam-mat dried orange juice. i. time-temperature drying studies lebensmittelhygicnische probleme bei der herstellung yon gemeinschaftsverflpegung. . mitt. : z. ges. hyg. lebensmittelhygienische probleme bci der herstellung yon gemeinschaftsverpflegung. . mitt.: z. ges. ttyg. untersuchungen fiber die temperaturvorg~nge im innern yon lebensmittein w~hrend ihrer thermischen zubereitung, erl~uter~ am kochen yon kartoffelklsl~en. z. ges. hyg. zur gewinnung yon niederverestertem pektin aus toehnisehen apfelpektinextrakten mit ammoniak: einflul] der entesterungsbedingungen auf das geliervermsgen hygienisohe beurteilung einer dutch clostridlum verursaehten massen. lebensmittelvergiftung [ungar neuartige teehnik der lebensmlttelverpaekung filr ge. schmaeks-aromastoffe und andere artikel bei ~berdruek studies with a natural source of xanthophylls for the pigmentation of egg yolks and skin of poultry die problematik der tuberkulosebeurteilung in der sehlachttier-und fleischuntersuchung freezing rate of beef as affected by moisture, fat, and wrapping materials ~ber mit komblnier~en konservierungsmitteln hergestellte konfitiiren und consumers' reactions to instant foods. food teclmol effect of supplementing lime-~reated corn with different levels of lysine, tryptophane, and isoleueine on the nitrogen retention of young children effect of freezing on autoxidation of oxymyoglobin solutions the control of gloecsporium album rot of stored apples by orchard sprays which reduce sporulation of wood infections bakteriologische befunde bei der spelseeisuntersuchung im sommer the microbiology of vacuum packed sliced bacon the stability of canned foods in long erm storage the effect of proofing and baling on concentrations of organic acids, carbonyl compounds, and alcohols in bread doughs prepared from pre-ferments nutrients in raw vs. cooked globe artichokes effect of gamma-radiation, chemical, and packaging treatments on refrigerated life of strawberries and sweet cherries. food teehnol beeinflussung der wirkung yon kaffeeinhaltsstoffen dutch be-s~immte behandiungsverfahren der l~hbohne. (eine tierexperimentell-toxikologische studie influence of surface pasteurization and ehlortetraeycline on bacterial incidence on fryers the hydrolysis of grass hemicelluloses during ensilage post-harvest storage studies with selected fruits the science of food technology in venezuela beitrag zur aufbewahrung von sti~rkesirup in verzinkten ge-f~ben inactivation-rate studies on a radiation.resistant spoilage microorganism. ii. : thermal inactivation rates in beef the oceurrence and growth of staphylococci on packed bacon, with special reference to staphylococcus aureus zur verhiitung yon lebensmittelinfektionen in grobklichenanlagen dutch desinfektionsmabnahmen. ~rztl the influence of selected bacteria upon the flavor of a precooked frozen poultry product flour maturing and bleaching with aeyclie acetone peroxides effect of processing conditions on dry-heat expansion of bulgar wheat zur vcrwendung von pentachlornitrobenzol bei der lagerung yon kohl. dr. lebensmittel-rdsch. [ ] nr. , s. los mati~res f cales des pores et les selles des ouvriers d'aba~toir constituent une source permanente de diss mination des salmonella studies on cooking fats and oils aspect sanitaire et l~gal aetuel des aliments conserv&s. rev. d'hyg over de betekenis van postduiven als besmettingsbron van levensmiddelen met salmonella-kiemen. ti]dschr. v. diergeneesk over bet voorkomen van salmonella-kiemen bij slagerijen. tijdschr. v. diergeneesk ursache und entstehung yon brotfehlern les salmonella des oeufs et ovoproduite frangais eg trangers the microstructure of baked products and doughs. food technol tomsto powder by foam-mat drying zitat: dt. lebensmittel-rdsch. [ ] nr. , s. . --die verwendung yon gefrosteter saline zur butterherstellung. teil iv. die ergebnisse der in d~nemark, frankreich und in den l~iederlanden durchgefiihrten praktischen versuche und ihre bedeutung ffir die in der deutschen molkereiwirtschaft erfolgendo verwendung yon gefrosteter sahne bei der butterherstellung tenderne~ of the turkey meat as influenced by pre-cooling before proce~ing and hand masv~ging vortragsmaterialien flit die ern~hrungsproplldeutik. (erlruterungen zu insgesamt groben sehautafeln.) . mitt. : behandlung der tafeln iv bis vi. ernahrungsforschung die unterschiedliehe problematik und ihre konsequcnzen bei der bekrmplung der rinder-und schwefnefinnen salmonellae from flies in a mexican slaughterhouse factors affecting quality of pies prepared from frozen bulkpack red sour pitted cherries zur bedeutung antimikrobiellcr stoffc in der nahrung modified equipment for pasteurizing and deodorizing market milk and for pasteurizing, deodorizing, and slightly concentrating cheese milk adhesion of coatings on frozen fried chicken oob~age eheeae problems in production and sanitation. publle health aspects ~ber den einflub yon licht, auerstoff und tempera~ur auf die hal~barkoib yon verpaektem emmentaler ks in scheiben aktuelle notwendigkeiten -gesetzliche m glichkeiten zur fischkfihlung in eis und seewasser techniques de recherche des salmonella dans les oeufs frais et de conserve food hygiene on board ship safety factors in water fluoridation based on toxicology of fluorides the effect of oiling before and after cleaning in maintaining the albumen condition of shell eggs lebensmi~t~l-aerosole. fette preservation of the natural color in processed sweetpotato products. i.: flakes. food technol temporary inhibition of fermentation in apple juice preservatives and artificial sweeteners the mechanism of the development of rancidity in frozen fresh pork sausage and practicable methods for its inhibition die entwicldung der trinkwasseriinoridierung in den usa microbiological principles in prcpaeking meats st~ndard-kapazit~tstest ffir die bestimmung der desinfektionswirkung yon desinfektionsmitteln in der milchwirtsehaft. internationaler standard fil/ii)f - die anwendung einiger arteu, bzw. st~mme, yon propions~urebakterien zur herstellung bestimmter k~scsorten mit hohem vitamin b -gehalt the extraction of pectins from apple marc preparations zur hygiene und ,di~tetik des rauchens studies on control of respiration of mcintosh apples by packaging methods. food teehnol effects of ingredients used in condermed and frozen dairy products on thermal resistance of potentially pathogenic staphylococci der frisehkllse und seine verpackung studies on the viscosity of mayonnaise. ii.: the influence of addition of vinegar on the vi~co~isy of mayonnaise e~'ec~ of chemical v~lditives on the spreading quality of butter. ii. laboratory and plant churnings studies on browning mechanisms of fruit juice products. i.: changes in chemical composition which accompany browning of commercial concentrated lemon juice during storage ergebnisse der dlg-qualit~tspriifung fiir speiseeis. dr. molkerei-ztg beeinttnssung versehiedenartig verpaekter lebensmittel dureh desinfektion mit formaldehyd grunds~itzliches zur stabilisierung und solubilisierung yon carotin und carotinoidpr~paraten riickst~nde yon pflanzensehutzmitteln, insektiziden und dergleichen in der nahrung und ihre bedeutung fiir die gesundheit absehliebende stellungnahme lebensmittel-rdsch langfristige nutritive anwendung yon antibiotilm in der tierern~hrung im hinbliek auf die menschliehe gesundheit mit besonderer beriieksiehtigung yon chiortetrazyklin nutritional studies on the utilization of distiller's stillage. part l: insolubles of me]lasses-butanol distiller's stillage auswertung der dlg-priifung fiir frischk~ise in verbraueherpaekungen zu den fermentativen eigensehaften der milchs~urebakterien (,laetobacillus meijerinek"), zugleieh ein beitrag zur vermeidung yon fehlfabrikaten bei roh-nnd briihwurst. arch. lebensmittel-hyg microbiological aspects of one-trip glass bottles as used by the carbonated beverage industry de bereiding van bouillon aktnelle milchhygienische aufgaben und zicle des organisation der ~berwachung der umweltradioaktivit~t unter besonderer beriieksichtigung der l~berwaehung des gehaltes yon lebensmitteln an radioaktiven stoffen. dr. lebensmittel bakteriologie der auermilcherzeugnisse l~ber die italtbarkeit yon lebensmittelkonserven preparation of aeid-modifid flour for tub sizing radiostrontium removal from milk. determination of apparent equilibrium constants of the exchange reactions of sodium, potassium, calcium, and magnesium wish amberlite ir- probleme der vitaminierung yon brot the effect of several operational variables on the rate of freeze-drying of beef studies on beef quality. x. effect of temperature, freezing, frozen s~orage, thawing, and p~ on the rate of hypoxanthine production. div. food preservation techn retardation of gelation in high temperature-short-time sterile milk concentrates with polyphosphates nonenzymatic bread browning and flavor. changes in amino acids and formation of earbonyl compounds during baking ttinweise fiir konservierende wirkungen synthetiseher senfslbildner naeh versuehen an fisehen neuo wege zur herstellung haltbarer fisch-pr~iserven behavior of ethylene dibromide, methyl bromide, and their mixtures. i. : in columns of grains and milled materials der einflui~ des wksserns auf die kartoffel irradiation of fruits and vegetables in india effect of storage in nitrogen on the soluble sugar and dry matter contents of ryegrass drying of seaweeds and other plants. v. throughcirculation drying of asophyllum nodosnm in a semi-continuous dryer niacin, thiamin, and riboflavin in fresh and cooked pale, soft, watery versus dark, firm, dry pork muscle nouvelles observations concernant la survie des salmonellae clans les fromages pyroearhonie acid diethyl ester as a potential food preservative the effect of phosphates on moisture absorption, retention, and cooking losses of broiler carcasses gur hygienisehen beur~ilung der trinkwasserverh~ltnisse des oberon vogtlandes -eine hydrobiologische s~udie. z. ges. hyg. studies on preserving quality in market eggs rapid detection of faecal coliform bacteria in the food processing plant. j. milk food technol the relationship between the loss of water and carbon dioxide from eggs and the effect upon albumen quality plastic pacckaging of eggs. study on improvement of digestibility of milk protein. i.: the effect of heating, adjustment of activity of calcium ion, addition of whey protein, homogenization, and elimination of coarse casein micelle from milk by ultracentrifuge on the digestibility of milk especially on the coagulability of it part iii.; the digebtibility of slightly hydrolized milk with proteinase and the preparation of rnill~ which has same eoagulability as human milk. g. agrie, chem. see study on improvement of digestibility of milk protein. part iv. : the nature of coagulation of casein of milk preparation which has same coagulability as human milk the influence of added microorganism on the quality of margarine. i. : the influence of mold inoculation die ilberung yon tafelw/~ssern. dr. lebermmittel-rdsch technological aspects of the radiation pasteurization of foods rapid hydration of dried fruits. food technol untersuchungen fiber polygalakturonase-enzyme aus sehimmelpilzen. . mitt.: eigenschaften der polygalakturonasen aus schimmclpilzen role of individual phospholipids as antioxidants association of veterinary food hygienists symposium on the marketing, transport, and slaughter of calves. iil: scientific aspects. ve~cr ricerche sulla resistenza della brucella abor~us helle salsicce. riv pr senee des salmonelles dans les viandes. donn es frangaises et tran-gbres biochemisehe vorg~nge inl fleisch bei der lagerung einflul des r stgrades yon kaffee auf die extinktion w~briger extrakte und die menge der trockensubstanz die herstellung yon quark und weillk~e unter ansnfitzung ss eiweibstoffe der milch vcrluste yon vitamin b~ und c beim kochen und turmkoehen yon gemfise effect of chilled storage on the frozen storage life of whiting salmonellenfunde in einer importsendung amerikanischer tiefgefrierhiihner. arch. lebensmittel-hyg iron sulfide blackening in canned protein foods: oxidation and reduction mechanisms in relation to sulfur and iron raft research on food preservation by irradiation in poland no~: gas chromatography of chicken and turkey volatiles: the effect of temperature, oxygen, and type of tissue on composition of the volatile fraction l'inaetivation dens l'eau de meret l'eau d'alimentation de eertains entdrovirus de voedingswaarde van aardappelen van versehiuende re, sen en de invloed daarop van bemesting en bewaring effecb of l-arab]nose and d-xylose on dough fermentation and crust browning gelation of egg yolk corn carotenoids: effects of temperature and moisture on losses during storage salmonellenfunde in einer importsendung amerikanischer tiefgefriorhiihner. arch. lebensmittel-ttyg bacteriological examination of unbottled soft drink ~berbliek fiber kunststoff-folien und -kombinatlonen ale verpackungsmaterial in der mflchwirtschaft pigmentierung des eidottem bei gettiigel. wiss. versff. dr. ges. eruiilu'ung s~ beltrag zur bedeutung wasserlsslieher hochmolekularer kohlenhydrate f'tir die verkleisterung der st~irke einfiul ehemischer verbindungen auf die antimikrobielle konservierungs-~toffwirkung. . mitt.: einflub verschiedener stoffgruppen auf die konservierungswirkung gegen aspergillus niger a quantitative s~udy of changes in dried skim-milk and lactose cnscin in the 'dry' state during storage the role of the major sugars of potatoes in ~he browning roa tion during chipping probleme der zuverl~sigkeit yon kunststoffen zur lebensmittelverpackung in europi~ischer sicht bakteriologiseh-hygienisehe beur~ilung yon speiseeis weizenkeime ale wertvoller rohstoff-einige ~ragestellungen und probleme probleme der frischhaltung und haltbarmachung yon brot end backwaren the effect of selected polymers upon the albumen quality of eggs after storage for short periods preparation and quality evaluation of processed fruits and fruit products with sucrose and synthetic sweeteners the microflora within the tissue of fruits and vegetables changes in carbohydrate and phosphorus content of potato tubers during storage in nitrogen preparation of "natural" cow-milk fat globules; preliminary investigation of materials adsorbed at their surfaces lethal doses of gamma radiation of some fruit spoilage microorganisms alteration of post-mortem changes in porcine muscle by preslaughter heat treatment and diet modification ober die msglichkeiten end grenzen eines effects of polyphosphates on water uptake, moisture retention, and cooking loss in broilers flavors imparted to dairy products by phenol deriva aromatisehe crackproduktc yon sterinen. (i). z. ern~hrungs-wiss dose requirements for the radiation sterilization of food berichb fiber eine arbeitstagung bei der internationalen atomenergie-beh rde in wien vom zur bok~mpfung d r rinderfinne zum einfiu]~ handelsfiblicher, in lebensmittelbetrieben gebr~uch-]icher desinfektionsmittel auf lactobakterien; zugleich ein beitrag zur desinfektion in der marinadenindustrie einflu~ chemischer umsetzungen bei trockenen lebensmittelgemischen in hinsich~ auf die lagerfestigkeit. vi. mitt. : lebensmittelgemische mit troekenmagermileh als hauptkomponente. z. lebensmittel-untersuchung u die technologie yon sauren milcherzeugnissen, insbesondere der sauermilcharten und sauerrahmarten effects of several edible coatings on poultry meat quality how to control insects in stored foods. part die antibiotika und die ans ihrer anwendung fiir die ~iilehwirtsehaft sich ergebenden probleme zur haltbarkeitsverli~ngerung empfindlicher l~ahrungs-und genuflmittel dureh abpaeken unter vakuum. fette, seifcn the diffusion of hydrogen through tinplate containers packed with grapefruit juice effect of ice cream stabilizem on the freezing characteristics of various aqueous systems ist die infektion mit trichinen aus amtlich untersuchtem schweinefieisch im liehte der mathematisehen analyse der bestimmungen der fleischbcschau yon schweinefleisch msglieh? hygiene in milk production, processing, and distribution effect of pre-eooling eggs and cartons upon quality after storage a biological after-effect in radiation-processed chicken muscle accounting for farm tank milk factors related to the flavor stability during storage of foam-dried whole milk. iii. effect of antioxidants untersuchungen zur hygienischen beurteihing yon ~ietallverunreinlgungen in lvben~mitteln the effect of bleed time prior to scald and refrigerated storage upon bacterial counts in the axillary diverticula of the interclavicular air sac of chickens techniques de recherche des salmonella darts les viandes the serotypes of salmonella isolated from foods carotinverluste beider zubereitung der nahrung. wiss. vcrsff. dr. gcs. er-n~hrung stability of ascorbie acid in a liquid multivitamin emulsion containing sodium fluoride the effect of storage time and holding temperature on egg interior quality in uganda uber den einflul versehiedener fangverfahren auf die qualit~t und lagerreserve der fische zerst~ubungstrocknung yon tomatenkonzentraten. dr. lebensmittel radiation pasteurization of fresh fruits and vegetables a bacteriological survey of certain processed meat~. part l population studies at packet and retail levels association of veterinary food hygienists symposium on the marketing, transport, and slaughter of calves. l : marketing and slaughter die kombinierte verarbeitung yon kartoffeln auf st~rke und alkohol (fortschrittsbericht) usaec program in radiation research preservation of certain fish and fruits biochemical and quality changes in chicken meat during storage at above-freezing temperatures inleidend onderzoek naar strnctuurveranderingen die ontstaan bi] verhitten van plantaardige produkten veranderingen van hot vetgehalte bij de bereiding van vlces a study on the relationship between the factors influencing the time of cheese salting maple sirup. xxi. : the effec~ of temperature and formaldehyde on the growth of pseudomonas geniculata in maple sap vacuum-tempering corn for dry. milling organoleptische eigenschappen, thiamine-en ascorbinezuurgehalte van enige week-en diepvriesgroenten growth of psyehrophiles. il : growth of poultry meat spoilage bacteria and some effects of chlortctracycline tests of corn stored four years in a commercial bin association of veterinary food hygienists symposium on the marketing, transport and slaughter of calves. ii. : the slaughter and inspection of calves. veter the effect of processing conditions upon the nutritional quality of vegetable oils berieht fiber den wisscnsehaftlichen kongrel der dcutschen gesellschaft ftir ern~hrung ( vortragsrcferate) an objective measurement of the freshness of ready-to-cook broilers the fieldman's responsibilities in milk quality and procurement studies on the bacteria found in the wine during its making. i.: multiplication of bacteria in wine and male-lactic fermentation hydrophilic colloids as additives in white layer cakes the acceptability of cooked poultry protected by an edible acetylated monoglyeeride coating during fresh and frozen storage istes zu vertreten, dal das fleiseh sehwachfinniger rinder auch in gebriitetem zust~nd eingcfrorcn wird? arch. lebensmittel-hyg =[efenlnfit.ierte kondensmilch als ursache yon fehlfabrikation bei schokolade. arch. lebensmittel.hyg. [ ] nr. , s. . m, ober die bedeutung aerober sporenbildner als bombageerreger yon wfimtehenkonscreen. arch. lebensmittel-hyg aluminiumfolio zur verpaekung tiefgekiihlter und gefriergetrockneter lebcnsmittel. fette fiber die milcldtuorierung. bull. schwciz. akad. reed. wiss vitamin stability in diets sterilized for germfree animal~ die trinkwasserversorgung ernliln-ungsstatistlk nutritional statistics african nutrition problems der vcrbrauch yon alkoholisehen getr~,nken in sterreich childhood nutrition in lapland overweight children in stockholm iron deficiency in the finnish population vitamin b deficiency in indian infants the indices of nutritional change in great britain dietary values from a h recall compared to a -day survey on elderly people her vaststellen van de voedingstoestand van sen bevolldng in de tropen en subtropen dental effects of fluoridation of water with particular reference to a study in the united kingdom de voedlng van woonwagenbewoncrs nutritional attitudes of some london housewives de vocdingsgewoonten van bcjaarden in amsterdam community studies of drinking behavior fats and carbohydrates as factors on atherosclerosis and diabetes in yemenite jews nutritional beliefs among a low-income urban population algemene gezondimidsaspccten van de vocding in de ontwikkelingsgcbieden a comparative study of the nutritional adequacy of the morning intake of women clerical workers and women factory workers serum cholesterol in a military population. its relation to obesity and the military diet nutrient intakes of healthy older women analysis of the structures of food consumption by groups in japan thiamin (vitamin b,)-untercru~hrung in deutschland? lvied vortragsmaterialien ftir die ern~hrungsprops (erli~uterungen zu insgesamt groben sehautafeln.) . mitt.: behandlung der tafein iv bis vi. erns [ ] nr. , s. zur ern~ihrungssituation in arbeitexfamilien aus verschiedcnen bezirken der ddr. . mitt. : ern~hrungssoziologischc answertung der lebensmittelverzehrungen in itaushaltungen mit erwachscnen und kindern~ stand studies in infantile malnutrition. i. : nature of the problem in peru zur ~ethodik yon ern~ihrungserhebungen bei der gemeinschaftsverpflegung weight changes in relation to birthweight of papuan, indonesian, and chinese children during the first two weeks of life numbers of tasters required to determine consumer preferences for fruit drinks onderzoek naar de menupatronen in de noordoostpolder smoking habits of medical and non-medical university staff i**cs and potassium in people and diet. -a study of finnish lapps. ann. acad. scientiarum fennicae a ~berbliek fiber die radioaktivit~t der in sterreich im jahre konsumierten lebensmittel diet and plasma cholesterol in bank men der mengenmi~bige getr~inkeverbrauch je einwohner im bundesgebiet predictors of human food consumption use of goverument.donated foods in a rural community bericht fiber die durum-und teigwarentagung der arbcitsgemeinschaft der oetreldeforschung e. v. vorn . bis . miirz voeding van leerlingen van con lagere teehni~eho school. ii. calorie~in-en nutriiintenwaarde nutritional deficiencies in developing countries dietary survey in surinam vitamine a-tekor~en op de aarde schwierigkeiten beim erreichen einer vollwertigcn ern~ihrung in ausgew~hl-ten vcrbrauchergruppen die entwicklung des brot-und getreideverzehrs in der neueren zeit. wiss. ver ff. dr. ges. ern~hrung n~rwert und zusammensetzung yon lebens. und futtermltteln nutritive values, composition of food~tuffe and f physical chemistry of ice cream vitamin e in human nutrition appraisal of nutritional adequacy of infant formulas used as cow milk substitutes enkele gegevens betreffende de calorisehe waarde van klsine hepjes, gerechtcn en maaltijden the public health aspects of the use of antibiotics in food and foodstuffs. report of an expert committee niihrwertminderung dutch zubereitung der nahrung metals and other elements in foods die farbe der nahrungsmittel in anthropologischer sicht the enzymatic destruction of carotene and carotenoids foodstuff flavors. some factors affecting the flavor of sodium caseinate chemical and radiochemical composition of the rongelapese diet the organic constituents of food. i. : lettuce the influence of dehydration of foods on the digestibility and the biological value of the protein a stfidy of two methods of assessing vitamin b nutriture mincraisalze mad spurenelemente in der nahrung distribution of the bound form of nicotinic ac|d in natural material~ the distribution of c~rotenoids in nature and their biological significance zur bedeutung antimikrobieller stoffe in der mahrung die konsistenz yon margarine mad fetten an improved nutrient solution for diploid chinese hamster and human cell lines extraneous materials in foods and drugs the composition of food flavors studies on pantothenic acid intake. i. pantothenic acid content in japanese foods haben wir mangel an essentiellen fettsiiurcn? phonolcarbons~uren in menschlichen nahrungsprodukten. zum vorkommen yon phenolcarbons~uren in menschlichen nahrungsprodukten und ihr einttud auf den intermedi~ren sboffwechscl drugs in feeds relationship between the sulphur/nitrogen ratio and the protein value of diets gums in foods zur definition der begriffe ,aroma" und begriff und aufgabe dii~tetischer lebensmittel valettr vitaminique des carot nes pour rhomme. wiss. versff. dr. ges. ern~hrung internationales rundgespriich fiber lebensmittelchemische probleme in wiesbaden und eltville a. rh. ( vortragsreferate) consumer awareness of texture and other food attributes organisehe und organisierte substanz in der lebensmittelchemie frost resistivity of fruit plants californ'ia association of chemistry teachers : inorganic nutrients in the sea fluoride in food antibiotics in feeds and other products planktorm as foods relation between color of cranberries and color and stability of sauce berieht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellsehaf$ in der ddr vom . bis bulletin on tobacco evaluation of algae as a food for human diet~ digestibility of high-amylose corn starch. nutrition red. [ ] nr. , s. / . ~. n. : comparative evaluation of corn mesa and steam-processed whole corn flours the major anthocyanin pigments of vitis vinifera varieties flame tokay, emperor, and red ~alaga peonidin- -monoglucoside in vinifera grapes formation and distribution of amylosc and amylopectin in the starch granule nutrient in seeds. amino composition of some seeds sugar levels in fruits of the lowbush blueberry estimated at four physiological ages the relation of pectic substances to firmness of processed sweet potatoes (ipomoea berates) processed vegetable produc~s the protein composition of different flours and its relationship to nitrogen content and baking performance relationship between 'antitryptic factors' of some plant protein feeds and products of proteolysis precipitable by trichloroacetic acid. g. sci. food agric a thermostable haemolytic factor in soybeans foam-mat dried orange juice. i. time-temperature drying studies bound" growth inhibitor in raw soybean meal leaf analysis as a guide to the nutrition of fruit crops. ii. : distribution of total n, p, k, ca and mg in the laminae and petioles of raspberry (rubns idaeus l.) as influenced by soil treatments nutritive value of pumpkin seed. essential amino acid content and protein value of pumpkin seed (cueur bi~a farinoaa) effect of cooking and of amino acid supplementation on the nutritive value of black beans (phaseolns vulgaris l.) supplementation of cereal proteins with amino acids. iv.: lysine supplementation of wheat flour fed to young children at different levels of protein intake in the presence and absence of other amino acids uber den chemischen total ascorbic acid in potatoes. raw, fresh, mashed, and reeonstituted flakes moisture contents of hard red winter wheat as determined by meters and by oven drying, and influence of small differences in moisture content upon subsequent deterioration of the grain in storage rheological studies with canned tomato juice the chemical composition of maple sugar sand soluble carbohydrate content of varieties of te~raploid ryegrass natiirlicher gehalt und stabilit~t yon carotine und carotinoiden in citrnss~ften ~ber wein und weinuntersuchungen fatty acids and other lipids in mayonnaise cyclic fatty acid yields from linseed oil factors ~ffecting enzymatic solubilization of beef proteins weibulls original ring, en ny medeltidig varvetesort. (a new medium early variety of spring wheat, weibull's ring.) agri hortique genetiea ober die askorbins~uresynthese in zerschnittene kartoffeln die bestimmung der amylaseaktivit~t und einige studicn fiber amylaseaktivit~t in gekeimtem roggen effect of processing conditions on dry-heat expansion of ~ulgar wheat oils, fats, and waxes ~.~ber das der johannisbro~.kerne. fetto, seifen fruit and fruit products uber die variabili~it einiger eigcnschaften der kartoffelstilrke in abhiingigkcit yon witterung a short.term effect of weather on malie acid in pineapple fruit the specific surface of flour and starch granules in a hard winter wheat flour and in its five subsieve-size fractions oranges and lemons factors affecting quality of pies prepared from frozen bulk-pack red sour pitted cherries studies on the consish~ncy of thiamin and protein contents of pure.bred strains of rice a comparison of the nutritional value of protein from several soybean frac ions zum vitamin-und aminos~uregehal~ yon maisquellwasser dark discoloration of canned all-green asparagus. i. chemistry and related factors enzymatic enhancement of flavor peetinestcrase in normal and abnormal tomato fruit storage effects on winter squashes. varietal differences and storage changes in the ascorbie acid content of six varieties of winter squashes grape pigments. concord grape pigments corn meal as a source of ribonuclease banana odor components. volatile components of bananas. part i. isolation of an odor concentrate. part il separation and identification lipids of algae. ill. : the components of unsaponifable matter of the algae chlore]la volatile esters of bartlett pear. ii studies on the nutritive value of raw and cooked soybeans for growing rats and swine and their effect on fat firmness characterization of fruit juices by acid profiles nitrate content of beets, collards, turnip greens lysine fortifications of wheat bread fed to haitian school children studies on the flavor of green tea. par~ iv. : dimethyl sulfide and its preeursor funktionelle eigenschaften yon lehens-mittclst~rken ~)ber das vorkommen yon xylit im speisepi]z champignon ein beitrag zur znsammensetzung yon apfeisinens~ften aus spanischcn und l~iarokko-apfelsinen an examination of the free amino acids of the common onion (allium cepa) a trypsin inhibitor in wheat flour the protein quality, digestibility, and composition of algae, chlorella chemical and color changes in canned apple sauce digestibility of the a-cellulose and pentosan components of the cellulosic mieelle of rescue and alfalfa safeness and serva. bility of meringued pie alcoholic beverages diurnal-nocturnal changes in the starch of tobacco leaves sugar and sugar products modification of flour proteins by dough mixing: effects of suffhydryl-blocking and oxidizing agents ~ber das haferprotein. z. lebensmittel-untersuchung u sodium and potassium in wines and distilled spirits non-volatile organic acids of the dwarf cavendish (chinese) variety of banana~ biological evaluation of soybean meal and cottonseed meal by amino acid digestibility and protein efficiency ratio studies. oiss. abstr. [ ] l~r oxidation-reduction potentials of sak~f and synthetic sak . xi.: on the relationship of the various fermenting processes of sak~ to oxidereduction potentials and indicator time test (i.t.t.) values of sak mash neue wege zur ern~hrungsphysiologischen aufwcrtung von getreideerzeugnissen cereal products ascorbie acid in dehydrated po~es die eiweibqualit~t yon ,getoastetem" (dampferhitztem) und ungetoastetem sojaextraktionssehrot color studies on processed dried fruits studies on the basic amino acid of the soy sauces and the seasoning liquids. ii.: the quantitative changes of l-arginine in the process of soy sauces brewing studies on the flavorons substances in soy sauce. xxii. : the differences between the soy sauce made from soy bean and wheat and that made from defatted soy bean and wheat feeding value of soy milks for premature infants flavors and non-alcoholic beverages fat content and fatty acids in some commercial mixes for baked products sensory examination of four organic acids added f~ wine ber die inhaltsstoffe der robktmtanie und versuehe zu ihrcr gehaltsbestimmung. diss. univ. hamburg, . malt beverages, sirups, extracts, and brewing materials vitamin b and niacin in potatoes. retention after storage and cooking chemical investigation of some wild indian legumes zusatzstoffe der margarine. forte componen~ glyeerides of an indian fresh-water fish fat einflub des rsstgrades yon kaffce auf die extinktion w~$riger extrakte und die v[enge der trockensubstanz gaschromatographische untersuehungen yon fusclslen aus vcrsehiedchen g~irprodukten. . mitt.: problemsteilung und literaturfibemieht de voedingswaarde van aardappelen van versehillende rassen en de invloed daarop van bcmesting en bewaring eleetrophoretic separation of beet pigments studies on the growth-promoting value and digestibility of passion fruit seed oil polycyclisehe und aliphatisehe kohlenwasserstoffe dc~ tabakrauehes carotenoid, oil, and tocopherol content of corn inbreds location and possible role of esterified phosphorus in starch fractions untersuchungen fiber die chemischen u beim a]tern yon r stkaffee. . mitt.: gaschromatographische analyse der leichtflfichtigen aromabestandteile nature, origin, and prevention of hydrogen sulphide aroma in wines the magnesium contents of soil and crops versuehe zur ehemischen differenzierung der eiweibst~ffe des weizens und roggens lemon juice composition. iii.: characterization of california-arizona lemon juice by use of a multiple regression analysis weizenkeime als wertvoller rohstoff -einige fragestellungen und probleme isolation of gram quantities of a rhamnoglucoside of apigenin from grapefruit determination of distribution of water in wheat grains by interference microscopy preparation and quality evaluation of processed fruits and fruit products with sucrose and synthetic sweeteners changes in carbohydrate and phosphorus content of potato tubers during storage in nitrogen chemical composition of some natural and processed orange juices effects of various factors in the candy test zum stand der kenntnlsse fiber die v{eclmelwir-kung zwischen nativer sts und wasser some volatile compounds from cooked potatoes firmness of canned apple slices as affected by maturity and steam-blanch temperature nutritive values of ten samples of western canadian grains proteins of wheat and flour. the separation and purification of the pyrophosphate-soluble proteins of wheat flour by chromatography on dear-cellulose changes in quality and composition produced in wine by s~ gamma irradiation citrus essential oils. iii. evaluation of silician natural lemon oils mineral analysis of plant tissues a. : nature of colloids in clarified cane juices studies on amino acid content of rice. i. : amino acid composition of polished rice glutelln estimated by beckman amino acid analyzer yliichtige carbonylverbindungen in honig neue aminos~iuren in h heren pflanzen studium der wirkung der gammastrahlung auf die l-ascorbins~iure flour liplds and oxidation of sulfhydryl groups in dough zur kenntnls eincr weiteren in der sti~rke vorkommenden kohlenhydrat-komponente. ern~ihrungsforschung lemon juice composition. ii. : characterization of california.arizona lemon juice by its polyphenolic content lemon juice composition. i.: characterization of california-arizona lemon juice by its total amino acid and -malic acid content safflower amino acids amino acid composition of safflower kernels, kernel protein, and hulls, and solubility of kernel nitrogen citrus fruit enzymes. i. : ascorbie acid oxidase in oranges untersuehungen fiber die verf~rbung gekochter kartoffeln an den sorten des kulturlrartoffelsortiments ides instituts ffir pflanzenziiehtung groi~-liisewitz nutritive value of red kidney beans (phaseolns vulgaris) for chicks instability in potable spirits. ii.: rum and brandy effect of size classification and maturity on the protein content of alaska and perfection peas ma~tr~, d. c.: ascorbie acid retention and color of strawberries as related to low-level irradiation and storage time historical aoac data on four fema samples of vanilla extract the acid-extracted pentosan content of wheat as a measure of milling quality of pacific northwest wheats petroleum ether extraetables in tobacco isolation, origin, and synthesis of a bread flavor constituent the protein composition of airclassifled flour fractions * studies on the flavor of green tea. v. : examination of the essential oil of the tea-leaves by gas lipid chromatography nutritive value of starches. iv.; comparison of digestibility of natural starches estimated by a new procedure o c lebensmittel tierischen ursprungs _foodstu~$ o/animal origin n.n.: gdch-faehgruppe ,lebensmib~el. und gerichtliche chemie bericht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellsehaft in der ddr yore . bis a taxometric study of the propionic acid bacteria of dairy origin comparisons of the caseins of buffalo's and cow's milk matiirlicher gehalt und stabilit~t der carotinoide in fetten und milchprodukten. wiss. versff. dr. ges. ernhhrung antibiotics in milk vitamin a and d enrichment of nonfat dry milk some characteristics of yolk solids affecting their performance in cake doughnuts. i. effects of yolk type, level, and contamination with white proteine des eidotters post-mortem changes in the muscles oflandrace pigs t~ber den fettgehalt yon flcischkonserven. dr. lebensmittel der einflul~ der verfahrenstechnik auf die ks jahreszeitliche einfliisse alff den gehalt der fischmuskulatur an freien ami-nos~iuren u. deren bedeutung fiir die qualitiit fangtechnik und fisch-qualit~t. fette spcei~c distribution of fatty acids in marine lipids a comparison of pigs slaughtered at three diffcrcn~ weights. i. : carcass quality and performance a comparison of pigs slaughtered at three different~ weights. ii. : association between dissection results, various measuremen~ and visual assessments a note on the effect of heat on the colour of goat's milk chemical and nutritional changes in stored herring meal. .: nutritional significance of oxidation of the oil relation of pork muscle quality factors to zinc con~ent and other properties the chemical nature of the characteristic flavor of cultured buttermilk occurence of vaniuin in hea~ed milks the electrophoretie properties of the proteins in cottage cheese curd the influence of post-mortem glycolysis on poultry tenderness seasonal variations in cod liver oil isolation and characterization of the flavor components of rancid pork some problems in the evaluation of egg albumen quality quality evaluation studies of fish and shellfish from certain northern european waters indices for lamb carcass composition subjective and objective evaluations of prefabricated cuts of beef ils, fats, and waxes die bildung yon eiskristallen in diinnen milchschichten. milchwissensehaft the incidence of bacteria in cheese milk and cheddar cheese and their association with flavour body composition of market weight pigs some factors affecting tenderness of turkey meat ovine bioenergetics and nutritional efficiency, with special reference go forage utilization die ziichtung yon fleisehsehweinen und die folgeerseheinungen, die sich insbesondere im hinbliek auf die qualit~t yon fleisch und fett ergeben. arch. lebensmitgel-ityg n the structure of highly unsaturated fatty acids of fish oils by high resolution nuclear magnetic resonance spectral analysis the fatty acid composition of the milk fat of cows grazing on ryegrass at two stages of maturity and the composition of the ryegrass hpids effect of intrauterine infusion of penicillin-streptomycin and furacin and vaginal deposition of furacin on chemical residues tevei~ in millr beziehungen zwisehen l-aseorbinsaure und milch comparison of chemical and organoleptic data obtained on thawed and unthawed frozen cod, haddock, and perch fillets die ~ m~uosfiurenzusammensetzung der ziegenmflch und des ziegenmileh-caseins food flavors and odors. meat flavor: lamb dairy products de ehemische samenstelling van visen visprodukten chemical studies on the herring (clupen harengus). vii.: collagen and cohesiveness in heat-processed herring and observations on a seasonal variation in collagen content growth and pro~eolycie activity of pseudomonas fluoresccns in eggs and egg products the fatty acid composition of some perirenal and subcutaneous beef depot fats inactivation of peroxidase in milk by homogenization a study of the "cured meat" color producing reaction and the effects of some curing adjuncts t~yoer den fettgchalt yon br'tihwiirsten stress effects, eareass composition, and carcass quality in lambs effect of chemical additives on the spreading quality of butter. ii. laboratory and plant ehurnings preliminary studies on protein and moisture relationship in fresh and proeessed hams die zusammensetzung des kuhmilchfettes in abh~ngig-keit yon der ffitterung. fette chemical characterization of off-flavors in concentrated and nonfat dry milk zur ausseheidung yon xanthindehydrase mid molybdiin in der kuhmilch copper distribution in milk during early lactation die physikaliseh-ehemischen ursaehen der hitzestabilitet yon mileheiweil]stoffen. milehwissenscha sobn~a: ~oer einige verenderungen in den caseinfraktionen normaler und anomaler milch (colostralmilch und milch an lviastitis erkrankter kiihe). milchwissensehaft some characteristics of yolk solids affecting their performance in cake doughnuts. ii. variability in commercial yolk solids zum problem des znsammenhanges zwisehen der konsistenz und der physikalischen struktur der butter. fette relationships between milk fat acidity, short-chain fatty acids, and rancid flavors in milk induced and natural inhibitory behavior of milk and significance to antibiotics disc assay testing. j. dairy sei. [ ] nr. , s. ff. ( s.). --some distribution patterns of cottage cheese particles and conditions contributing to curd shattering natural inhibitory eharacteristies of some irish manufacturing milks thermophylie aetinomycetes in milk and dairy products. mikrobiologiya the free fatty acids of purdue swiss-type eheese die zusammensetzung yon siilzen lind ihre beurteilung im regierungsbezirk diisseldorf not~ on tyrosine production in frozen stored liver studies on the muscles of meat animals. hi. : comparative composition of various muscles in pigs of the three weight groups studies on beef quality. x. effect of temperature, freezing, frozen storage, thawing, and pe on the rate of hypoxanthine production. die. food preservation t~chn increased iodine in milk as a countermeasure for ~liodine time-temperature studies of baked, loaves. meat, fish, and poultry vergleichende untersuehungon an butter und einem butter~hnli-chen umgeesterten fort. forte, seifen, anstrichmittel zur beziehung zwisehen fettgehalt und wassergehalt bei ungewa~ohener s/il~rahmbutter (fritzbutter) variation of ovine fat composition within the carcass studies on the properties of new zealand butterfat. vii. effect of the stage of maturity of ryegrass fed to cows on the characteristics of butterfat and its carotene and vitamin a contents studies on turkey body composition. . poultry sci thermal conductivity of beef studies on turkey body composition. free fatty acid, tyrosine, and ~ changes during ripening of blue cheese made from variously treated milks l : factors influencing the nutritional value of fish flour. il: availability of lysine and sulphur amino acids. canad characterization of flavor compounds isolated from evaporated milk effect of egg yolk size on yolk cholesterol concentration ~tudo immuno~lectrophor~tiquo du lair dans los divers types de mammites. i. milchwissenschaft rdsultats hemmstoffe in der anlieferungsmflch und methoden zu ihrem nachweia eiweibverenderungen gefriergetroekneter muskulatur meat and meat products effect of high doses of vitamin a palmitate on vitamin a aldehyde, esters, and alcohol and carotenoid contents of hen's eggs. brig. j. nutrition [ ] nr. , s. / . --the amounts of vitamin a aldehyde, esters, and alcohol and of earotenoids in hen's eggs and in day-old chicks nutritive value of marine oils i. : ]~lenhaden oil at varying oxidation levels, with and without antioxidants in rat diets a quantitative study of changes in dried skim-milk and lactose-casein in the 'dry' state during storage physico chemical characteristics of canadian milk fat. unsaturated fatty acids collagen content and its relation t~ tenderness of connective tissue in two beef muscles ~ber eine braune verf~rbung yon mariniertem hering. dr. lebensmitbel versuche fiber den sonnenlichtgesehmaek in mit ass markierter milch effect of unequal milking intervals on lactation milk, milk fat, and total solids production of cows wei~re untersuehungen fiber die nitratreduktase verschiedener an der reifung yon rohwurst beteiligter mlkroorganismen nutritive value of leg of lamb roasts. l~oisture, energy, protein, fat, and iodine values ~ber einige gul~sigkai~s~renzen bei konsistenzfehiern yon butter influence of linoleic acid content of milk lipids on oxidation of milk and milk fat fish hydrolysates-iii.: influence of degree of hydrolysis on nutritive value einige beobaehtungen fiber die bildung des durch lieht verursaehten oxydationsgesehmackes s.). zitat: dt. lebensmittel comminuted meat emulsions: factors affecting meat proteins as emulsion stabilizers factors related to the flavor stability during storage of foam-dried whole milk. iil effect of antioxidants upgrading the indigenous poultry of uganda. hi. : shell and egg interior quality relation between carcass composition and live weight of sheep diethylstilbestrol occurence in eggs of subcutaneously injected hens biochemical and quality changes in chicken meat during storage a~ above-freezing temperatures investigations on the allerged goitrogenie properties of milk fish and other marine products bioehemieal properties of pork muscle in relation to curing potassium content of dried milk vergleiehende histometrische untersuehungen fiber den kollagengehalt yon brfihwfirsten a comparison of the volatile compounds of fresh and decomposed cream by gas chromatography studies on the volatile carbonyl compounds in ladino clover and their influence on the flavor of milk aut~xidation of fish oils. ii.: changes in the carbonyl distribution of autoxidizing salmon oils the public health aspects of the use of antibiotics in food and feedstuffs. repor~ of an expert committee composition and digestibility of corn silage as affected by fertilizer rate and plant population factors influencing the nitrate content of forage the component sugars and rate of hydrolysis of forage hemicelhflose as related to digestibility digestibility trials on forages in trinidad and their use in the prediction of nutritive value acetyl-(para-nitrophenyl)-sulfanilamide in feeds distribution of major and trace elements in some common pasture species -dimcthyl -( , , -trichlorophcnyl)phosphorothioate) in feeds feeding value of low-moisture alfalfa silage from conventional silos ovine bioenergeties and nutritional efficiency, with special reference to forage utilization a system for naming and describing feeds, energy terminology, and the use of such information in calculating diets supplemental methionine in a sixteen percent protein diet for laying chickens l : organic arsenicals in feeds cellulose degradation by enzymes added to ensiled forages influence of corn distillers dried grains with sohbles on the feeding value of wheat silage zinc content of certain feeds, associated materials, and water der natiirliche gehalt und die stabilit~it yon carotin und carotinoiden in lieu und silage forage digestibility. benzene-ethanol extracts of forage and faeces as indicators of digestibility the prediction of the metabolizable energy content of poultry fcedingstuffs from a knowledge of their chemical composition factors affecting the metabolizable energy content of poultry feeds. facters affeeting the metsbohzablo energy content of poultry feeds unidentified chick growth factor in fish solubles diet and histamine in the ruminant. occurrence of histamine in silage ethopabate in feeds die isolierung yon apocarotinalen aus luzernemehl isoflavone contents of red and subterranean clovers metabolizable energy of some oil seed meals and some unusual feedstuffs ii methoden der untersuchung yon lebens-und futtermitteln techniques analysis of foodstuffs and feeds n.n.: gdch-faehgruppe analysis of foods by neutronaetivation techniques continuous measurement of dissolved solids in food processes by critical-angle refractometry berieht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellschaft in der ddr vom . bis a rapid test for anionic detergents in drinking water fortschritte in der lebensmittelehemio dureh moderne analysenmethoden direct potcntiometrio determination of chloride in cheese modification of the polarimetrie starch determination on hig-hamyloso corn ~iethoden der ]~berwachung des wassers auf radioaktiviuit. btmdes prediction of quality in protein concentrates by laboratory procedures involving determination of soluble nitrogen direct chromatographic analysis of milk die eignung der bakteriologischen untersuehung yon kannenmilchproben als grundlage eines eu~ergesundheitsdienstes. arch. lebensmittel-i~yg nachweis fremder carotinoide in rangensiiften mittels diinnschichtchromatographie. i)t. lebensmit~el-l~dseh determination of phosphate composition of stock food calcium phosphate. . assoe. off. agric. chemists molybdenum in plants and animals. determination of molybdenum in biological materims with dithiol control of copper interference the determination of dissolved oxygen in canned drinks using a vibrating mercury-plated platinum electrode determination of chromium and lead in periodic acid solution and dialdehyde starch the utilization of infrared and ultraviolet spectrometric procedures for assay of pesticide residues ultramicro determination of potassium and sodium in biologic fluids gum quautativen naehweis des pektins und der alginsiiure ein schliffapparat zur abtrennung yon flfichtigcn stoffen mittel~ wasscrdampfdcstillation ~tude par chromatographic on phase gazeuse des acidea gras du beurre fabriqu en italic et clans d'autres pays. application ~, la recherche des falsifications clans le bcurre commercim. ann. falsiticatiorm expertise chim note on the determination of caffeine in coffee a comparison of the press method with taste-panel and shear measurements of tenderness in beef and lamb muscles zur diehtebestimmung der milch mit der neuen milehspindel. lebensmittelchem, u. geriehtl. chem. [ ] nr. , s. / . --aufschlu -.i~thercxtrakt und titrationswert bei der teigwarenunbersuehung als beispiel einer dynamisehen lebensmittelanalyse quantitative measures of carcass composition and qualitative evaluations fruit preservatives analysis. determination of calcium in cherry brines by versenat~ titration: elimination of anthocyanin interference by means of carbonyl reagents, g. agric. food chem allgemeine prinzipien der analytik yon carotinen und carotinoiden zur bestimmung yon ethoxyquin kolorimctrische bestimmung yon dipterex-riickst~nden yon lebensmittein the direct determination of shear stress-shear rate behavior of foods on the presence of a yield stress methods for evaluating bhe feeding quality of meat-andbone meals a paper chromatographic method for determination of vanillin and ethyl vanillin in vanilla flavorings ~drber die anwendung der papierchromatographisehen analyse auf dem fettgebiet. . mitt.: uber ls~iurereiehe samenole. ermittlung der konsti~uierenden fettsiiuren der samensie yon margosa (azadiraehta indies), cashewkern (anacardicum oecidentale) und putranjiva roxburghli. nahrung die jodzahl des riickenspeeks im verhiiltnis zu der qualit~t des futterfettes, dem alter der schweine und dem fettungsgrad bei sehweinen der ditnisehen landrasse determination of parathion, methyl parathion, epn, and their oxons in some fruit and vegetable crops an improved chromatographic method for determining trace elements in foodstuffs determination of guthion residues on fruits techniques used in meat flavor research the analysis of edible oils contaminated with synthetic ester lubricants colorimetrische bestimmtmg yon nitrat und nitrit in biologisehem material confrontation de quelques proe~d s de dosage iodometrique de l'anhydride sulfureux dans les vins zur anwendung der massenspektroskopie zur strukturermittlung yon naturstoffen, mit besonderer berfieksiehtigung der lebensmittelanalytik z lebensmittel cellulose solubility as an estimate of cellulose digestibillty and nutritive value of grasses the -thiobarbiturie acid reagent for determination of oxidative rancidity in fish oils bet die eignung yon daphnia magna zur ermittlung yon riieksti~nden auf frisehem bst und gemfise bemcrkung zum diinnschiehtchromatographischen kakaoschalennaehweis nach the determination of vitamin a in animal tissues and its presence in the liver of the vitamin a-deficient rat elution column preparation of leaf sample for flame photometry. ii. : determination of calcium in tobacco enzymatic-ultraviolet method for determination of uric acid in flour die bestimmung der amyiaseaktivit~t und einige studien fiber amylaseaktivit~it in gekeimtem roggen identification and determination of ascorbic acid (vitamin c) with janus green and its localisation in mitochondria cho]estehnbestimmung im kleinen laboratorlum chick edema factor. iii.: application of mieroeoulometric gas chromatography to detection of chick edema factor in fats or fatty acids bioassay of chick edema factor. collaborative study determination of nih te and nitrate in meat products the measurement of the surface areas of milk powders by a permeability procedure sedimentbeurteilung und sehaim-~iastitis-test als sortierverfahren zur ermittlung yon sekretionsstsrungen bei der ~iassenuntersuchung yon milchproben aoac methods for nutritional adjuncts die ermittlung der ribonucleins~ure im pflanzenmaterial beitrag zur analytischen beurteilung des frischezustandes der pharmazeutisch verwendeten e und fe~te sehnellbestimmung yon kupfer in fe~n applications of oscillographic polarography to the determination of organophosphorus pesticides. ii. : a rapid screening procedure for the determination of parathion in some t~its and vegetables zur standardisierung der vitaminb~-bestimmung in getreide und getreideprodukten eine mikromethode zur bestimmung des fettgehaltes der milch kleiner laboratoriumstiere the determination of organophosphato pesticides and their residues by paper chromatography die l~fikrochemie beim studium yon nahrung und erni~hrung aearicide residues. an improved method for kelthane residue analysis with applications for determination of residues in milk collaborative study of the determination of ethoxyquin in feeds ~rber den naehweis yon quellstoffcn in fleischwaren und m gliche stsrungen durch andere polysaccharide ersatz manueller labormethoden der l sungsspektralanalyse durch den automaten determination of total acids in wines: american society of enologists determination of aldehydes in wines and spirit~ by the direct bisulilte method ~fber einen vereinfachten nachweis des vitamin b mit poteriochromonas slipitata extraction of nys~tin in animal feeds for microbiological analysis zur quantitativen bestimmung der sorbinsi~ure mit dem thiobarbitur-s~urereagenz cottage cheese problems in production and sanitation. quality control in cottage cheese sitzung des arbeitskreises berlin der gdch-fachgruppe lebensmittelchemie und gerichtliche chemie am . . in ]~erlin-dahlem ( vortragsreferate) ein neues elektronisches sctmeuverfahren zur ermittlung der ~risehe yon seefischen fatty acids of lard. a. identification by gas.liquid chromatography oxydative abbauprodukte der l-aseorbins~ure. . mitt. : papierchromatographischer l~achweis analysis of orange juice for total earotenoids, carotenes, and added betacarotene. food technol. [ ] nr. , s. / . u. r~ck~, a. a. : refractometrio measurement of soluble solids in orange juice analysis of iron chelates in plant extracts. il: ferric ethylenediamine'bis'(~ acid) determination of n-aeetylglucosamine- -phosphate and n-aeetylglucosamine in milk arsenic in foods: collaborative comparison of the aminemolybdenum blue and the silver diethyldithiocarbamate methods improved method for testing macaroni products neuere beitri~ge zur chemic der st~rkefraktionen. . mitt.: die mikro-ameisens~urebestimmung bei der perjodat- xydatonsbestimmungsmethode yon stiirke recherche des falsifications dans les extraits de vanille. ann. falsifications expertise chim bestimmung des gesamtstckstoffgehaltes yon milch nach der kjwld~t:l-methode. internationaler standard fil vorteile und grenzen des einsatzes yon markiertem phosphat bei untersuchungen am hiihnerei paper chromatography of carotene and carotenoids determinaton of sevin insecticide residues in fruits and vegetables insecticide residues in meat and eggs. determinaton of sevin insecticide and its metabolites in poultry tissues and eggs bcstimmung der jodzahl yon fetten und en mittels n-bromsuccinimid. . mitt.: l~ber eine !~i gliehkeit zur bestimmung der gesamtjodzahl yon el~iostearins~ure und holz . nahrung [ ] nr. , s. / . kxrr:~e~r, m. a. : ~ ber die anwendbarkeit des thiobarbi~urs~iuretestes boi der untersuchung yon milchprodukten application of gas chromatography to the measurement of gas permeability of packaging materials a sensitive method for quantitative microdetermination of lipids comparison of chemical and microbiological methods for the determination of procaine penicillin in prcmixes and mixed feeds electrophoretc analysis of flour proteins from various varieties of wheat katalytische methode zur bestmmung kleinster manganmengen in lebensmitteln am beispiel der milch eine methode zur papierchromatographischen qualits yon silagen comparison of methods of measuring potassium in pork and lamb and prediction of their composition from sodium and potassium electron capture gas chromatography for determination of ddt in butter and some vegetable oils eine methode zum schnelinachweis yon nitriten in yleiseh-und wurstwaren. arch. lebensmittel-hyg determination ofglyodin residues on pears and peaches eleetrophoretic analysis of flour proteins erstes internationales symposium fiber methoden zur analyse yon lebensmitteln in bordeaux-tatence (frankreich) veto . bis . oktober on the problem of luminescence technique of protein definition in milk beitr~ge zur aminos/iuren-bestimmung in biologischem material aktuelle fragen der lebensmitteluntersuehung, insbesondere der histologischen wurstanalyse untersuchungen yon importiertem tiefgefrorenem tiasenfleisch argentinischer herkunft fish hydrolysates. iv.: microbiological evaluation untersuehungon zum naehweis yon emulgatoren in lebensmit~eln. . mitteilung. forte use of a slice-tenderness evaluation device with pork studies on improvements in quantitative paper chromatography of amino acids in foods. i. : research on procedure of development studies on improvements in quantitative paper chromatography of amino acids in foods. ii.: selection of solvent systems determination of the equilibrium relative humidity of foods untersuchungen fiber die methodik der quantitativen bestimmung yon heizolen und flfissigen treibstoffen im wasser determination of ~-lactalbumin in complex systems hydrogen sulphide in cheddar cheese; its estimation and possible contribution to flavour ascorbic acid measurement. polarographic determination of total ascorbic acid in foods trans-fettsguregehalt yon sehweineschmalz nach fiitterung von schweinen mit rindertalghaltigem kraftfutter. (ein beitrag zur quantitativen infrarotspektroskopischen bestimmung yon trans-fetts~uren in fetten the importance of starch on the microscopic identification of cereal grains in feeds insecticide residues. chromatographic identification of some organophosphate insecticides in the presence of plant extracts chemical and biological estimation of the carotene content in fresh and processed italian apricots sialir acid as an index of the u-casein content of bovine skimmilk foodstuffs analysis. nonvolatile acids of blueberries le dosage de la matibre grasse dans les fromages. ]~tude critique de la m thode en usage au laboratoire municipal la d termination de residus d'insecticides et de fongieides par la m thode polarographique fusel oil determination by gas.liquid chromatography * die brabanter mastitis-reaktion, ein neues verfahren zur ermittlung yon sekretionsstsrungen des euters dutch die kannenmilchuntersuchung uber eine enzymatisehe _&pfels~urebestimmung in wein und traubensaft direct microscopic technique to detect viable yeast cells in pasteurized orange drink die enzymatisehe bestimmung der glucose und saecharose und ibre anwendung in der lebensmittelanalyse a modified zirconium-alizarin method for determining fluoride in natural waters paper chromatography of some cholesterol derivatives determination of moisture by nuclear magnetic resonance and oven methods in wheat, flour, doughs, and dried fruits dye binding by soybean and fish meal as an index of quality the absorptiometrie determination of silicon in water: part l: formation, stability, and reduction of cr and fl-molybdosilicie acids column chromatography of soybean whey proteins enzymatic determination of carbon dioxide in lightly carbonated wines. collaborative study nachweis yon biiffelmilch als verf/flschungsmittel in kuhmilch durch serologisehe methoden photometric determination of phosphate in wines pertinent references to analytical lipid methods published recently, ft spectrophotometric estimation of nucleic acid of plant leaves hemmstoffe in der aalieferungsmilch und methoden zu ihrem nachweis determination of potassium in tobacco determination of chlorides in tebacco phospholipase c determination by egg yolk turbidimetry oher die inhaltsstoffe der rol]kastanie und versuche zu ihrer gehaltsbestimmung mierobiologlcal method for assaying nystatin in animal feeds / ] nr. , s. / . --gaschromatographische untersuchungen yon fuselslen aus versehiedenen g~rproduk-ten. . mitt.: methodik der fusel bestimmung lactose activity measurements. evaluation of laetase preparations for use in breadmaking comparison of methods for determination of lysino in cereals direct determination of calcium in plants, soils, and milk by means of a flame photometer colorimetrie determination of urea in feeds kolorimetrische bestimmung des dihydrox-yacetons fluoride, teeth, and the analyst the quantitative micro-determination of biphenyl in citrus fruit kolorimetrisehes verfahren zur gleichzcitigen bestimmung der weinsiiure und milchsiiurc in wein und most estimation of extra-cellular starch of dehydrated potatoes versuehe zur chromatographischen trennung yon kohlenhydra~en und eiweil en aus dem w~ibrigen extrakt yon roggenvollkornmehl quantitative determination of the amino acid content of rumen fluid from twin steers fed soybean oil meal or urea vemnche zur chemischen differenzierung der eiwei•stoffe des weizens und roggens aromastoffe des brotes. versuch einer auswertung chemiseher gesehacksanalysen mit hilfe des schwellenwertes zur trennung yon sacchariden an kohle]celit~-s~ulen. ern~hrungs-fomchung untersuehungen zur bestimmung der lsslichkeit yon milchpulver * selection of a medium for the isolation and enumeration of enterocoeei in dairy products colorimetric determination of amino nitrogen in corn syrups stxogene und versuche zu ihrem nachweis in gefliigelflelsch. dt. lebensmittel ein beitr~g bert. die verwendung der anatysen-q~arzlampe zu friihzeitiger erkennung der l~anzigkeit colorimetrlsche methode zur bestimmung yon -monoglyceriden in eiskrem determination of fusel oil in distilled spirits zu den m glichen fehlerquellcn bei der histometrischen ermittlung des kollagen-, bzw. gelatine-gehaltes bei briihwiirsten studium der uv-spektren der auf hshere tempcraturen erhitzten e measuring of oil-binding characteristics of flour naehweis der konservierungsmittel mit hilfe cl~r papierehromatographie identification of ch]orogenic acid in castor bean and oranges. canad evaluation of forages in the laboratory. iii.: comparison of various methods for predicting silage digestibility feed microscopy essential oils. determination of botanical and geographical origin of spearmint oils by gas chromatographic and ultraviolet analysis fluorometric determination of chlortetracycline in premixes dfinnschicht-chromatographie yon carotin-und carotinoidgemischen measurement of the sub-sieve particle size distribution of flour chemisehe bestimmung der riickst~nde yon parathion, malathion und diazinon auf blumenkohl, kohlrabi, bohnen und gurken determination of cadmium anthranilate in feeds nachweis yon penicillin und anderen antibiotika in milch microbiological evaluation of protein quality with tetrahymenapyriformis w. .: a simplified assay procedure peanut lipoprotein. ii. : analysis in foods by gas chromatography beitrag zum papierchromatographisehen und spektrophotometrisehen i~achweis fettlsslieher synthetiseher farbstoffe in lebensmitteln und kosmetika elektroehemische sauerstoffbestimmung in olefinischen fetich bestimmung der gesamten sehwefligen s~ure in getr~nken the modified whiteside test. recommended procedures for bulk or blended milk deliveries die bestimmung yon mileheiweib in fleiseherzeugnisscn. dr. lebensmittel zur bestimmung des veresterungsgrades yon pektin a quantitative fluorometrie method for the determination of serpasil (reserpine) in feeds at the micro level fluorimetric mierodetermination of carbohydrates zur l~iethodik der klebrigkeitsbestimmung yon brot frage der papierchromatographischen untersuchung von amylopektin und amylose chemical determination of diethylstilbestrol residues in the tissues of treated chickens gas chromatographic identification of components in m~ple sirup fl~vor extract dark discoloration of canned all-green asparagus. ii. development of a new tin plate for its control thermal conductivity and density of chicken breast mnsele and skim beitrag zur bestimmung der fluoreszenz in spriten microbiological determination of alanine in proteins and foods collaborative study of the method for counting microorganisms in maple sirup glass fiber paper strip charring. a rapid and simple method for monitoring column chromatography of lipids verbesserte mcthode zum serienm~bigcn quantitativen nachweis yon insektizidrficksti~nden bei bst und gemfise use of the shear press in determining fibronsness of raw and canned green asparagus betrachtungen fiber verschiedene methodcn zur bestimmnng des bindegewebeanteiles in rohem fleisch und fleischwaren. arch. lebensmittel-hyg the determination of citric acid in milk and milk sera formaldehyde in maple sirup: an adaption of the nash method polarographische bestimmung dcr ascorbinsiiure und des gesamt-vitamin c untersuchungcn fiber die mbglichkeit zur objektiven beurteilung der organoleptischen eigensehaften yon kokosraspeln analysis of the flavor and aroma constituents of florida orange juices by gas chromatography assay for cyzine in finished feeds dfinnsehicht-ehromatographische trennnngen yon synthetischen lebensmitteffarbstoffen auf ceuulose-schichten evaluation of quantitative methods of determining peroxidase in vegetables. i.: the indophenol and the o-phenylenediamine methods ein beitrag zur untersuchung thermisch oxydierter fette. dt. lehensmittel a more accurate method for determination of caffeine in decaffeinated coffee bestimmung yon vitamin c in friichten, fruchtsiiften, gemiise und konserven naeh der methode naeh tillma~s unter ausschaltung reduzierender stoffe lebensmittelrecht und lebensmitteliiberwaehung nutritional laws and nutritional control briihwiirst~ einfaeher qualit~t, die zu einem kaliber unt~r mm in den verkehr gebracht werden, sind i.s. des w nr. lmg irreffihrend aufgemaeht new regulations under the food and drugs act lebcnsmittel-rdseh orb der kenntlichmaehung fremder stoffe probleme des geltenden lebensmittelrechts glasurmittel ftir r stkaffee beschr~nkt zugelassen food technol. [ ] nr. , s. . n. iv. : revised u. k. preservatives regulations. food teehnol ausschub fiir lebeusmittelrechtliche fragen der fachgruppe lebensmittelehemio und gerichtliche chemic in der geseuschaft dcutscher chemiker vi. t~tigkeitsberieht der eidg. kommission fiir volksern~hrung, lebensmittelgesetzgebung und -kontrolle (eek) zu h~nden des eidg. departementes des i_nnern, umfassend die jahre federal food, drug, and cosmetic act, as amended. selected u. s. government publ. nr. ; h. catalog i~o verkauf yon frikadellen mit brotzusatz in gastwirtsehaften tierarzneimittel und aufzuchtmittel in der landwirtschaftlichen praxis. gesundheitliche erw~gungen zum schutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmit~ln in der landwirtschaftliehen praxis. teil hi der amtsarzt und das neue lebensmitt~lgesetz. off ist der beschlub des bundesgerichtshofes vom . . - stl~ -geeignet, den vertrieb yon hackfleiseh befriedigend zu regeln ? arch. lebensmittel-hyg zur beurteilung des saccharingehaltes yon meerrettiehzubereitungen. dt. lebensmittel zum begriff ,mai]gebend" in w a abs. des lebensmittelgesetzes aspect sanitaire et ldgal actuel des aliments conservds verbrauchererwaxtung und lebensmitte]kontrolle bei fleiseherzeugnissen bei ger~ueherten fleischwaren. sehwarz-w~lder speck, schwarzw~lder sehinkenspeck, sehwarzw~lder sehinken. arch. lebensmittel-ttyg kampf der wasserverseuchung. aktuelle notwendigkeiten -gesetzliehe ~/isgliehkeiten. ~iiinchener reed. wschr. lebensmittelreehtliehe stellung yon carotin und carotinoiden in der schweiz die lebensmittelgesetzgebung sterreiehs, der sehweiz und der bundesrepublik deutschland. eine vergleiehende untersuchung arzneimittel-lebensmittel dr. lebensmittel-rdseh erwiderung des autors (zur stellungnahme yon f. nitzsc~, d~. lebensmittel-rdseh. [ ] nr. , s. ). i)t. lebensmittel das lebensmittelgesetz und seine auswirkung auf die gummiindtlstrie codex alimentarius austriacus absehliel]ende stellungnahme der ort der kenntlichmachtmg fremder stoffe. dr. lebensmittel-rdsch _rreffihrende bezeiehnung und angaben bei limonade aus mineralamem tafelwasser. dr. lebensmittel zur herkunftsbezeichnung yon lebensmitteln. dt. lebensmlttel aktuelles zur lebensmitteliiberwachung einige beispiele fiir die auswirkung der neuen lebensmittelrechtliehen vorschriften auf erniihrungs-und landwirtschaft auf dem wege zum einheitliehen lebensmittel-rech~ zur ~r yon b. rsssl~: welehe anforderungen sind an alkoholhaltige siigwaren zu stellen? dr die silbertmg yon tafelwiissern mokka-kaffee" auch im handel mit kaffeebohnen keine tterkunftsbezeiehnung [ ] nr. , s. . --zuliissigkeit trod grenzen bildlicher darstellungen yon fleiseh und ylelscherzeugnissen auf paclmngen yon suppen in trockener form. db. lebensmit~el-l~lsch bedeutung und beur~eitung yon galtstreptokokken in vorzugsmileh. arch. lebensmittel-hyg auf clipverschliissen zul~ssig ist die infektion mit trichinen aus amtlich untersuehtem schwelnefleiseh im lichte der mathematischen analyse der bestimmungen der fleischbesehau yon schweinefleiseh msglich? arch. lebensmittel-hyg intema~ionales rundgespr~ch fiber lebensmitte]ehemische probleme in wiesbaden und eltville a. rh. z. lebensmittel-untersuch. u. -forschung. [ ] nr. , s. . --kommission zur priifung fremder stoffe bei lebensmitteln (fremdstoff-kommission) der deutsehen forsehungsgemeinsehaft entwicklung des lebensmittelrechts im nationalen und internationalen bereich entwicklung des lebensmittelrechts im nationalen und inter~ationalen bereich entwicklung des lebensmittelrechts im nationalen und internationalen bereich de warenwet en de hierop berustendc besluiten zum entwurf einer harmonisierung der lebensmittelrechtlichen bestimmungen fiber konservierungsstoffe im gebiet der europaischen wirtschaftsgemeinschaft. er lebensmittelrechtliche stellung yon carotinen und carotinoiden in der bundesrepublik deutschland die instrumente der lebensmittelfiberwachung in osterreich sterreichischer standpunkt zur l~rage der f~rbung yon lebensmitteln mit carotinen und carotinolden. wiss. ver ff. dr. ges. ernkhrung lst es zu vcrtreten, dab das fleisch schwacbfinniger rinder auch in gebr~tetem zustand eingefroren wird? z lrch. lebensmittel-iiyg richtlinien fiber die zulassung yon gegeusachverst~ndigen zur untersuchung yon lebensmittel-gegenproben die ]ebensmittelrechtliche bedeutung yon bildiichen darstellmlgen auf verpackungen diverse problems (education, documentation associations, terminology etc the nutritional education of the food technologist proposed formation of a food engineering panel within the food group studium der hauswirtschafts-und ern~hrungswissenschaften. ern~hrungswirt-sehaft l an information service for the american food industry st international congress of food science and technology. symposium on education and training technically trained people for developing countries lft paces the food frontier un committee on food additives international standardization of fruit and vegetables. food technol terminology and methods for feeding and weighing animals food additives and food standards beitriige zur durehffihrung der umweltsradloaktivitiits- berwaehung training dairy personnel in denmark training opportunities for the sanitarian-specialized in-service training r die benennungen honigkueheniihnlicher oebiieke als lobkuehen und lebzelten sowie als biber und bibenzelten. dt. lebensmittel nutrition and dietetics for the medical student problem of keeping dairy plants supplied with the foreman-type employee literaturdokumentation fiir hochschulassistenf~n a system for naming and describing feeds, energy terminology, and the use of such information in calculating diets a brief sketch of veterinary periodical literature in great britain before the foundation of the veterinary record a conference on nutrition teaching in medical schools the food service industry and its relation to the control of foodborne illness research and educational progress in nutrition informationsm gliehkeiten fiir tieriirzte auf dem measuring readability of health education literature the dai~y literature problem the central food technological research institute the education and function of the nutritionist training in food service for nursing homes. l: tools for evaluation training in food service for nursing homes. iii.: observations on management of twelve units pennsylvania takes a look at nutrition in the orthopedic program zur definition der begriffe ,aroma" und l~ber die notwendigkeit einer priifung for beamtete sehlaehthofleiter. arch. lebensmittel-hyg organisation der dokumentation in einem forschungsinstitut (bundes. forsehungsanstalt f'tir lebensmittelfrischhaltung in karlsruhe). dt. lebensmittel-rdsch on changing the name of our assoziation. for a name change pporbunities in nutrition education questionnaires to identify nursing homes most in need of dietary counsel. publ. health irep lebensmittelwissenschafttiche institute in bulgarien zur stellung des psychiaters in der alkoholfrage naas nutrition chemists. vcter on changing the name of our association. against a name changing begriffe) in sprachen: deutseh, russiseh, polnisch, tsehechisch, slowakisch, ungariseh, bulgariseh, serbokroatiseh, rum~nisch, englisch the role of nutrition in the teaching of medicine key: cord- - yniw t authors: ben-chetrit, eldad title: colchicine date: - - journal: textbook of autoinflammation doi: . / - - - - _ sha: doc_id: cord_uid: yniw t colchicine is an alkaloid which was originally extracted from bulbs of a plant called colchicum autumnale (meadow saffron). its active pharmacological component was isolated in and in the active ingredient was purified and named colchicine. it consists of three hexameric rings termed a, b, and c. it was first recommended for the treatment of gout by alexander of tralles in the sixth century ad. later it has been employed for suggested and approved indications including primary biliary cirrhosis (pbc), alcohol induced hepatitis, psoriasis, behçet disease, sweet syndrome, scleroderma, sarcoidosis and amyloidosis. perhaps the most effective results have been obtained in the prophylaxis of familial mediterranean fever (fmf). colchicine is absorbed in the jejunum and ileum and is trapped in the body tissues. it is metabolized in the liver and the intestine by cytochrome p (cyp) a and p-glycoprotein (pgy) . colchicine is excreted mainly by the biliary system, intestines and the kidneys. it has a narrow therapeutic range, but with normal liver and kidney functions is relatively safe and can be used during pregnancy, nursing and in infants. the main mechanism of action of colchicine is probably through interaction with microtubules affecting leukocyte chemotaxis, thereby suppressing inflammation. the blood level of colchicine may be affected by concomitant drug administration and therefore, caution should be exercised when such medications are added. colchicine is an alkaloid which has been used for centuries for treating gout [ ] . the source of its name is colchis, a kingdom on the black sea in western georgia. its history is related to medea who was the daughter of aeetes, ruler of colchis. aeetes kept the "golden fleece" under heavy guard. his daughter, medea used to "harvest grasses and to extract harmful juices squeezed from twisted roots" [ ] . among the most potent products squeezed from these bulb-corms was the juice of colchicum autumnale, the yellow crocus of colchis. on his quest to fetch the "golden fleece" from colchis, jason met medea and fell in love with her. the princess used her potions (most of which consisted of concentrated colchicine) to help jason poison the warriors and dragons that stood guard over the fleece. the use of the bulb-like corms of colchicum for gout traces back to a.d., as the "hermodactyl (iris)" recommended by the byzantine physician, alexander of tralles (today-aydin in turkey) [ ] . while the greeks and romans knew about the use of colchicine for gout, the drug wasn't available in pure form until the late nineteenth century. the active pharmacological component of the plant, colchicum, was isolated in and in , p.l. geiger purified the active ingredient, which he named colchicine [ ] . in the nineteenth century, alfred garrod, dyce duckworth, and many others, reached a consensus that colchicine was relatively specific for the treatment of gout [ ] . pernice, an italian pathologist, found that when therapeutic doses of colchicine were given to experimental animals, lesions were produced in the nuclei of gastric and intestinal cells as these cells were arrested in metaphase [ ] . in , ed taylor and gary borisy used tritiated colchicine to identify the target of colchicine in dividing and non-dividing cells [ ] . the protein they identified was the dimeric building block of microtubules, subsequently given the name "tubulin" by mohri [ ] . we now know that the traffic of intracellular materials is carried over tracks formed by microtubules [ ] . in the last years, colchicine has been employed for an increasing number of diseases, in addition to gout, including familial mediterranean fever (fmf), idiopathic recurrent pericarditis, behçet disease, sweet syndrome, systemic sclerosis, amyloidosis and hepatic cirrhosis [ ] (table . ). in acute gout, colchicine is effective in alleviating the acute attack and as a prophylactic medication. in behçet disease, colchicine is effective mainly in the treatment of mucosal ulcers, especially in female genitalia. in systemic sclerosis, colchicine may decrease the stiffness of the skin whereas in amyloidosis it may result in regression of amyloid deposition loci where serum amyloid a (saa) fibers are deposited [ , ] . since , colchicine has gained popularity as being the main effective remedy for fmf [ ] . in this disease, colchicine prevents the occurrence of the acute inflammatory episodes and the development of amyloidosis. however, colchicine is not effective in controlling acute attacks when administered once they occur. • colchicine is an alkaloid which is absorbed in the jejunum and ileum and is trapped in the body tissues • colchicine is metabolized in the liver and the intestine by cytochrome p (cyp) a and p-glycoprotein (pgy) • colchicine is excreted mainly by the biliary system, intestine and the kidneys • the half-life of colchicine is between and h following oral ingestion • caution should be exercised in patients with liver or kidney disease or damage colchicine is a tricyclic, lipid-soluble alkaloid with the chemical formula; n- ( , , , , tetrahydro-l, , , , tetramethoxy- oxobenzo[a] hep-tain- -yl) acetamide n ( fig. . ). pharmacokinetic studies of colchicine are limited, and their results somewhat contradictory because of the different methods used for its measurement. previously, most studies employed thin-layer chromatography, whereas recently a more sensitive radioimmunoassay and high performance liquid chromatography with mass spectrometry (hplc-ms) has been used [ ] . colchicine can be administered by mouth as . , . and in some localities mg tablets. until recently an intravenous (iv) formula was also available. however, in , the united states food and drug administration (fda) withdrew approval following several cases of fatal toxicity [ ] . formerly, it was thought that colchicine is almost completely absorbed after oral administration. however, recent studies have shown that its bioavailability ranged from to % [ ] . the exact site of absorption is unknown, but the drug seems to reach the jejunum and ileum because dysfunction of these bowel regions is common in cases of chronic colchicine overdose [ ] . the plasma membrane-localized multidrug transporter p-glycoprotein (pgy ) [also known as atp-binding cassette subfamily b member (abcb ), multiple drug resistant (mdr ), and cd ] transports multiple classes of substrates across cell membranes [ , ] . altered intestinal pgy expression can change the absorption of drugs transported by this peptide, including colchicine. therefore, pgy- is critical in the regulation of its bioavailability and plasma concentrations, which can lead to sub-optimal therapeutic effects or, alternatively, to drug toxicity [ , ] . also, pgy clearly participates in the removal of drug metabolites through bile, the intestinal lumen, and urine [ ] [ ] [ ] [ ] . colchicine is predominantly eliminated by biliary excretion and through the stool, with gastrointestinal tract lining cell turnover playing a variable role in this process [ , , ] . normally, a lesser, but significant role in colchicine metabolism (~ %) is played by enteric and hepatic cytochrome p a (cyp a ), which catalyzes the demethylation of colchicine to and demethtylcolchicine, which are inactive metabolites [ ] . renal elimination has been estimated to be responsible for - % of drug disposition in normal subjects. however, cyp a and renal disposition of colchicine can be significantly impacted by certain drug-drug interactions that affect pgy- (abcb ), as well as hepatobiliary dysfunction and aging [ , ] . the bioavailability of oral colchicine tablets is comparable in the young and elderly, but colchicine pharmacokinetics differ markedly, with volume of distribution at steady state (vss) and total body clearance significantly reduced in the elderly, and the plasma cmax significantly higher at equivalent colchicine doses [ ] . over the last decade, several single nucleotide polymorphisms of pgy (abcb ) were identified with the potential to influence expression and quantitative transporter function [ ] . for instance, - % of patients diagnosed with fmf do not respond to treatment with colchicine. compared with non-responders (who usually have more severe disease), treatment responders had a twofold greater concentration of colchicine in mononuclear cells, which was attributed to a potential genetic effect independent of mefv mutations [ , ] . specifically, the distribution of abcb c and t-genotypes was significantly different between colchicine-responders and non-responders, with the c allele significantly increasing the risk of resistance to colchicine, whereas in patients with the t genotype a decreased colchicine dose was needed to obtain an adequate response [ ] . wallace and ertel [ ] were pioneers in studying the pharmacokinetics of colchicine. they found that after iv administration of mg of the drug to healthy volunteers, the peak plasma concentration averaged . + . μg/ ml. after iv administration of . mg of colchicine, a rapid drop of plasma levels occurred during the first min, followed by a logarithmic decline [ ] . the rapidity of colchicine disappearance from the plasma and its persistent excretion days after drug ingestion suggest that it is trapped in body tissues for a prolonged period. indeed, the [ ] . an example for such binding is the detection of colchicine in leukocytes days after a single iv dose [ ] . furthermore, it was shown that in plasma % of the colchicine is bound to albumin [ ] . after oral administration of colchicine, the tmax (the time needed to reach peak plasma levels) is - h [ ] . however, maximal anti-inflammatory effects develop over - h based upon intra-leukocytes accumulation of the drug [ ] . initial studies reported terminal elimination half-life time (t / ) from min (after iv administration) to h (after oral ingestion) [ ] . in an additional study, the mean t / was . h, similar to findings in patients with fmf without renal or liver disease (t / of about ± h) [ , ] . in a study in which we evaluated the pharmacokinetics of colchicine in patients with fmf with and without renal or liver disease, we found that colchicine clearance was significantly impaired in those with kidney or liver failure [ ] . the t / of colchicine in patients with severe renal failure was two-to threefold longer and in a patient with both renal failure and cirrhosis tenfold longer than in patients with normal renal and liver function. leighton et al. [ ] reported similar results in patients with liver cirrhosis. these findings suggest that patients with either liver or renal disease should be closely monitored even when treated with conventional doses of colchicine. • colchicine binds non-polymerized tubulin forming a tubulin-colchicine complex • heterodimers of αand β-tubulin form microtubules that can elongate and contract as filaments • colchicine, can bind the tubulin molecule and inhibit its polymerization into microtubules in vitro • since microtubules are involved in cell division, in signal transduction, regulation of older studies claimed that most of the effects of colchicine at the cellular level are attributed to its interaction with tubulin, the main building block of microtubules [ ] . colchicine binds in an equimolar and poorly reversible manner to soluble non-polymerized tubulin with high activation energy, forming a tubulin-colchicine complex [ , , ] . heterodimers of α-and β-tubulin form dynamic polymers termed microtubules that can elongate and contract as filaments, to change the structure and function of the cytoskeleton, exemplified by the interphase microtubule network and the mitotic spindle. microtubules are involved not only in cell division, but also in signal transduction, regulation of gene expression, migration, and secretion [ ] . microtubules are widely distributed in organelles present in nerve cells, ciliated cells, leukocytes, and sperm tails. it was shown that tropolone methyl ester, which is a precise analog of the ring c of colchicine, can bind the tubulin molecule and inhibit its polymerization into microtubules in vitro. furthermore, mescaline, which is an analog of the methoxyphenyl moiety of ring a of colchicine, also may inhibit microtubular assembly [ ] . these data suggest that the action of colchicine is dependent on its two rings which bind microtubules, inhibiting the movement of intracellular granules, thereby disturbing the secretion of various components to the cell exterior. colchicine has an inhibitory effect on several leukocyte functions such as adhesiveness, ameboid motility, mobilization and degranulation of lysosomes [ ] . however, the most potent effect of colchicine is on leukocyte chemotaxis [ ] . of all the effects of colchicine, only the inhibition of chemotaxis was shown to occur at concentrations as low as × − mol/l. a higher dose is necessary to inhibit other effects. it was suggested that the primary anti-inflammatory effect of colchicine is derived from its potent inhibitory effect on leukocytes chemotaxis [ ] . however, additional studies have shown that colchicine decreases the expression of adhesion molecules on neutrophil membranes, leading to significant inhibition in migration and interaction with endothelial cells [ ] . other investigators have shown that colchicine may modulate cytokine production by polymorphonuclear cells. several studies have shown a relatively high concentration of colchicine in leukocytes explaining its potential effect on these cells [ , ] . however, the cause for the special "affinity" of colchicine to leukocytes was unclear. we and other investigators, have shown that granulocytes have low activity of the pgy efflux pump and therefore when colchicine enters these cells it accumulates in their cytoplasm [ , ] . conversely, lymphocytes and mononuclear cells have a higher activity level of pgy . therefore, some of the colchicine entering these cells is effluxed, explaining why the level of colchicine in granulocytes exceeds that of lymphocytes and monocytes. mechanisms of colchicine several mechanisms have been ascribed to the therapeutic action of colchicine (table . ). bessis and gorius discovered that colchicine disrupts microtubules in a dose-dependent fashion [ ] . colchicine does not enhance microtubule dissolution but abrogates the process of microtu-bule self-assembly by forming tubulin-colchicine complexes [ , ] . colchicine reduces the generation of tumor necrosis factor (tnf)-α by macrophages and its receptors on endothelial cells [ , ] . colchicine also has been shown to interfere with the interaction of neutrophils and the vascular endothelium by abrogating their binding to adhesion molecules. colchicine abrogates the e-selectin-mediated adhesiveness of the cytokinestimulated vascular endothelium to neutrophils. it also alters the distribution of the adhesion molecules on the surface of endothelial cells and neutrophils, significantly reducing their interaction [ ] . in addition, at high concentrations colchicine suppresses phospholipase a activation, lysosomal enzyme release and phagocytosis [ ] . conversely, colchicine does not exert its anti-inflammatory effect through inhibition of cyclooxygenases [ ] . in , the gene likely to cause fmf was isolated and cloned [ , ] . following the isolation of the mefv gene and the finding that it is fully expressed in neutrophils, a question was raised regarding the possibility of a direct effect of col- abrogates the microtubule self-assembly by forming complex with tubulin reduces the generation of tumor necrosis factor (tnf)-α by macrophages abrogates e-selectin-mediated adhesiveness of endothelium to neutrophils alters distribution of adhesion molecules on endothelial cells may suppress phospholipase a activation (at high concentrations) may inhibit lysosomal enzyme release, and phagocytosis raises mefv gene expression in a cell line of peritoneal fibroblasts may modulate the expression of numerous genes in endothelial cells suppresses the activation of caspase- may inhibit superoxide production by neutrophils leads to release of guanine nucleotide exchange factor (gef)-h factor thereby activating rat sarcoma homolog gene family, member a (rhoa) reduces neutrophils elasticity and relaxation inhibiting chemotaxis inhibits liver fibrosis by inducing stellate cell apoptosis inhibits anti-transforming growth factor (tgf)-β activity chicine on the gene or its protein. in a study where we tested this hypothesis, we showed that colchicine did not up-regulate the expression of mefv gene in neutrophils [ ] . however, colchicine increased the mefv gene expression in a primary cell line of peritoneal fibroblasts. the exact significance of this finding is unknown. nevertheless, it should be borne in mind that peritoneal cells comprise an important target in the acute attack of fmf (peritonitis), suggesting a potential local effect of colchicine. a few studies suggested that colchicine modulation of pyrin expression and interaction with pyrin in the cytosol contributed to its efficacy in fmf [ , ] . at relatively high concentrations, colchicine also modulates the expression of numerous genes in cultured endothelial cells, with a significant delay in the onset of action [ ] . this may explain the observation that initiation of colchicine treatment during acute attacks of fmf does not effectively terminate them. colchicine has recently been shown to suppress the activation of caspase- , the enzymatic component of the nucleotide-binding oligomerization domain (nod) receptor family pyrin (nlrp ) inflammasome. caspase- suppression blocks conversion of pro-interleukin (il)- β to active il- β, leading to secondary reduction in cytokines such as tnf-α and il- . the effect of colchicine on this process may be upstream of the inflammasome, rather than a direct effect [ ] . to date, inflammasome inhibition has been assessed at colchicine concentrations to -fold higher than that achieved in the serum. whether colchicine inhibits caspase- at physiologic concentrations, or whether colchicine accumulation in leukocytes is sufficient to block the inflammasome, remains to be determined. another mechanism by which colchicine may suppress inflammation is by inhibition of superoxide production by neutrophils. chia et al. demonstrated that colchicine inhibits monosodium urate (msu)-induced superoxide production by murine peritoneal macrophages in vivo at doses times lower than that required to inhibit neutrophil infiltration [ ] . this suggests that superoxide anion production is more sensitive to suppression by colchicine than microtubule formation involved in cell migration. attacks of fmf? • rat sarcoma homolog gene family, member a (rho a) protein is a peptide which controls the action of gtpases thereby affects tubulin dynamics • pyrin is a specific immune sensor (pattern recognition receptor-prr) for bacterial modifications of rho and gtpases • activation of rhoa inhibits pyrin activity while inactivation of rhoa causes over activation of pyrin resulting in increased production of interleukin (il)- , thereby enhancing inflammation • colchicine may activate rhoa by guanine nucleotide exchange factor (gef)-h , thereby suppressing pyrin activity and inflammation • colchicine also disrupts microtubules structure reducing neutrophils membrane elasticity and relaxation, thereby preventing their extravasation from the blood vessels to the inflammatory site to understand this process, we first need to explain the cellular role of pyrin. pyrin is the protein encoded by the mefv gene, which is mutated in fmf (see chap. ). rat sarcoma homolog gene family, member a (rho a) protein is an intra-cellular peptide which controls the action of gtpases. gtpases are important enzymes in regulation of actin and tubulin dynamics. actintubulin interaction has a major role in the motility and chemotaxis of neutrophils [ , [ ] [ ] [ ] . bacterial toxins such as that of clostridium may modify the effect of rho on gtpases, thereby inhibiting actin activity and leukocytes chemotaxis. xu et al. found that pyrin is a specific immune sensor (pattern recognition receptor-prr) for bacterial modifications of rho and gtpases [ ] . pyrin does not directly recognize rho modification, but probably senses events downstream of rho modification. xu et al. showed that activation of rhoa inhibits pyrin activity while inactivation of rhoa causes over activation of pyrin resulting in increased production of il- , thereby enhancing inflammation. clostridium toxin inactivates rhoa thereby leading to less production or recruitment of protein kinases (pkns) and thereby less phosphorylation of pyrin [ ] . ** peptides (**the name reflect the migration pattern on d deae gel of these acidic proteins family) are group of proteins which interact with phosphorylated pyrin [ ] . decreased phosphorylation of pyrin leads to less interaction and reduced binding by peptides. this increases the amount of free pyrin which can recruit apoptosis-associated speck-like protein containing a card (asc) and procaspase − to construct the pyrin inflammasome, leading to increased secretion of il- β and an enhanced inflammatory process (fig. . ) . colchicine may also suppress inflammation in fmf by the following mechanism. it binds to tubulin and depolymerizes microtubules, resulting in the release of the rhoa activator, guanine nucleotide exchange factor (gef)-h , which is inactive when bound to microtubules [ , ] . thus, colchicine indirectly activates rhoa. activation of this protein results in enhancement of pyrin phosphorylation, thereby higher binding by the pep-tides and hence, less free pyrin is available for constructing the inflammasome, required for secreting inflammatory cytokines, including il- . in a recent study, we looked more closely to the mechanistic features of the effect of colchicine on the neutrophil membrane [ ] . we found that colchicine disrupts the microtubules structure and reduces neutrophil elasticity and relaxation, thereby preventing their extravasation from the blood vessels to the site of inflammation. this may be the final and most effective step in inhibiting chemotaxis. colchicine has anti-fibrotic effects. in a rat model of hypertensive chronic kidney disease, colchicine inhibited renal fibrosis via inhibition of rhoa signaling and infiltration of inflammatory cells [ ] . in another rat model, colchicine inhibited liver fibrosis by inhibiting the activation of hepatic stellate cells and inducing stellate cell apoptosis [ ] . in an encapsulating peritoneal sclerosis model, colchicine inhibited anti-transforming growth factor (tgf)-β activity [ ] . • colchicine is a relatively safe medication but has a narrow therapeutic window • the metabolism of colchicine is affected by the functions of cytochrome p (cyp) a , p-glycoprotein (pgy) , the liver and the kidneys and by additional medications taken concomitantly colchicine is a relatively safe medication but has a narrow therapeutic window [ , ] . colchicine is well tolerated when taken in age dependent doses that are less than mg/day in children (up to year old) and mg/day in adults with normal liver and kidney function, when not taking concomitant drugs that may affect its pharmacokinetics. nevertheless, therapeutic oral doses of colchicine ( - mg/day), may cause cramping, abdominal pain, hyperperistalsis, diarrhea and vomiting. these effects are usually mild and transient. when abdominal cramps persist, lowering colchicine dose may be effective. when diarrhea persists dividing the dose of colchicine (twice daily) may be helpful. in resistant cases, a short course of anti-diarrhea medications such as loperamide may be beneficial [ ] . decreasing use of lactose containing products or using lactase prior to their consumption may also reduce symptoms. leukopenia is a very rare adverse event in therapeutic dose and neuromuscular complications may occur when renal functions are impaired or with use of concomitant drugs such as clarithromycin. colchicine overdose may lead to a cholera-like syndrome associated with dehydration, shock, multiple organ failure, alopecia, disseminated intravascular coagulation (dic), seizures, coma and death [ ] . colchicine doses of . - . mg/kg are highly toxic, and doses of more than . mg/kg are typically lethal [ ] . cumulative doses of colchicine causing toxicity when administered intravenously were mg given over days, mg in days and even mg given within days. the lowest reported oral dose causing lethal colchicine toxicity was mg given over days to a -year old male [ ] . the course of colchicine intoxication can be divided into three stages, with overlap between the stages. in the first stage, gastrointestinal symptoms dominate. there may be excessive fluid loss through diarrhea, leading to volume depletion and dehydration. this stage develops within - h following ingestion of the drug. the second stage is dominated by multi-organ failure which may include: bone marrow failure, renal insufficiency, adult respiratory distress syndrome (ards), arrhythmias, disseminated intravascular coagulation (dic), neuromuscular disturbances and alopecia. this stage develops over - days. patients surviving this stage may enter the third stage which is characterized by bone marrow recovery and rebound leukocytosis, resolution of organ failure and regrowth of hair. clinical management of colchicine intoxication is basically supportive. in a single case, treatment with f(ab) fragments of anti-colchicine antibodies was successful [ ] . unfortunately, these antibodies, raised in goats, are not currently available. one of the problems of managing colchicine overdose is that it is not dialyzable using regular dialysis membranes. recently, new high flux polysulfone membranes have been introduced to improve dialysis [ ] . many medications and substrates which were non-dialyzable with older membranes are now dialyzable. colchicine is one of the medications of which steady state levels were reduced when given to patients with fmf on high flux dialysis [ ] . however, our study showed that the rate of excretion of colchicine by these membranes is far less than the rate needed for effective treatment of colchicine intoxication. drug-drug interactions have increasingly become apparent as a cause of colchicine toxicity in patients treated with "standard" daily prophylactic regimens. abcb (pgy ) can undergo conformational changes with expression modulation thereby promoting clinically significant colchicine drug-drug interactions [ , , , ] . cyclosporine is a prime example of a drug that can inhibit or modulate the abcb transporter [ ] . potentiation of colchicine neuromyopathy can occur within weeks of commencement of cyclosporine therapy. this complication is often associated with cyclosporine nephropathy and a decline in the glomerular filtration rate [ , ] . notably, cyclosporine was found to delay colchicine-induced diarrhea in an animal model system, likely due to modulation of intestinal abcb . hence, it is suspected that cyclosporine could mask the gastrointestinal side effects of colchicine in humans that might otherwise be a clue to the development of systemic colchicine toxicity. cyp a is the most abundant of the human p enzymes and metabolizes multiple structural classes of drugs (e.g. cyclosporine, quinidine, testosterone, nifedipine, etc) [ , ] . cyp a is a focal point of many drug-drug interactions and dietary and herbal interactions. cyp a may be stimulated by its substrates ("normotropic cooperativity") or its effectors ("heterotropic cooperativity"), which renders prediction of drug-drug interactions difficult [ , ] . these substances may be divided into three groups. the first group contains drugs, such as cimetidine, which have an inhibitory effect on the entire cytochrome system. indeed, in animal studies, it was shown that concomitant administration of cimetidine and colchicine resulted in a significant rise in serum colchicine concentration. the second group contains substances that have a specific inhibitory effect on the isoform cyp a which metabolizes colchicine. these include erythromycin, ketoconazole and other medications. the third group includes drugs that are also metabolized by cyp a , such as cyclosporine and nifedipine, and may compete with colchicine for binding to the enzyme. the interaction in these cases is dictated by the affinity of each medication for the enzyme. thus, coadministration of medications and substances metabolized by the same cytochrome system may lead, in principle, to an increase of one or more of the drugs, exposing the patient to a higher risk of toxicity. certain drugs increase the potential for colchicine toxicity via dual modulation of abcb and cyp a (table . ). these include the macrolide antibiotics erythromycin and clarithromycin, and the statins, e.g. lovastatin, simvastatin and atorvastatin [ ] . clarithromycin is a particularly striking case in point and has been associated with at least fatalities with concomitant colchicine use [ ] . however, azithromycin (a weak cyp a inhibitor) had minimal effects on colchicine concentration and terminal elimination half-life, and decreased total apparent oral clearance by only %. azithromycin should be recommended as a safer alternative to clarithromycin in patients taking colchicine [ ] . mutual potentiation by colchicine and statins of myopathy (sometimes including rhabdomyolysis) is a notable concern [ , ] . significantly, case studies have reported acute myopathy after concurrent use of colchicine with a statin that was either not metabolized or minimally metabolized by the cyp a isoenzyme [ , ] . in this context, fluvastatin can disrupt the integrity of the cytoskeleton and is linked with vacuolization and other pathology in muscle, which is pertinent given the capacity of colchicine to promote vacuolization in muscle by disruption of the microtubule network. a new set of evidence-based guidelines provides an algorithm for reducing colchicine doses to prevent toxicity in patients who are concomitantly taking other drugs [ ] . the researchers conducted a series of studies designed to show the effects of a single-dose of colchicine given with known inhibitors of cyp a or pgy . among the drugs tested were: cyclosporine, ketoconazole, ritonavir, clarithromycin, azithromycin, verapamil extended release (er), and diltiazem er. it was shown that the mean maxi-mum concentration of colchicine was % higher when colchicine was co-administered with ketoconazole compared with colchicine alone. the mean maximum concentration of colchicine was % higher and the mean total colchicine exposure was % higher when colchicine was combined with ritonavir as compared with colchicine alone [ ] . colchicine treatment can alter absorption of other compounds or medications from the intestines. it may induce malabsorption of vitamin b by reducing the number of b intrinsic factor receptors as shown in the intestinal mucosa of guinea pigs [ ] . colchicine-induced lactose intolerance occurs in a significantly higher per- centage of patients with fmf treated with oral colchicine compared with non-treated patients [ ] . reduction in iron absorption was also observed among patients with fmf taking colchicine. • colchicine rarely causes oligo/azoospermia • colchicine does not affect sperm motility • it is safe to take colchicine during pregnancy and nursing provided the liver and kidney functions are normal • colchicine is relatively safe in treating children and toddlers and does not affect their growth colchicine is a drug which may affect the function of microtubules in various cells. in high concentrations, it may inhibit mitosis within the process of cell division. therefore, concern was raised as to the effect of colchicine on sperm proliferation and motility in patients taking colchicine. a case report by merlin described a patient with gout who developed azoospermia following treatment with colchicine [ ] . re-challenge again induced azoospermia. because patients with fmf receiving colchicine are often in their child-bearing years, the concern about fertility is pertinent. indeed, rabbits treated with a relatively high dose of colchicine showed various degenerative changes of the testes, including loss of differentiation from spermatogonia to spermatozoa [ ] . however, cohen et al. performed cytogenetic evaluation in patients with fmf receiving long-term colchicine. mitotic rates, percentage of tetraploidy, and chromosomal breakage rates were determined in lymphocytes [ ] . no significant differences were found between the patients and controls. in a study by levy et al., patients receiving long term colchicine therapy were evaluated. no effect on fertility was noted and levels of spermatocytes, testosterone stimulating hormone, luteinizing hormone and prolactin were all within normal limits [ ] . another study showed that four out of males with fmf receiving colchicine suffered from infertility [ ] . one had azoospermia and the others had a normal spermatogram, but the sperms could not penetrate the ova normally. since sperm motility and hence ovum penetration depends upon microtubule function, we hypothesized that colchicine may affect the movement of sperm. accordingly, we studied the effect of colchicine on sperm motility in an invitro system employing the "swim up" technique for sperm selection [ ] . sperm motility was inhibited significantly only after an incubation period of at least h, with a minimal colchicine concentration of μg/ml. because plasma colchicine concentration under therapeutic dose is about - ŋg/ml, the amount of colchicine needed for affecting sperm motility in vitro is -fold higher. thus, it seems unlikely that standard colchicine treatment would inhibit sperm motility unless the drug has a very high and special affinity to the testes. the frequency of oligo or azoospermia with colchicine depends on the underlying disease. bremner and paulson failed to show any effect on spermatogenesis in six healthy volunteers who received commonly used doses of colchicine for - months [ ] . conversely, in a study of turkish men treated with colchicine for behçet disease, oligospermia was evident in ( %) patients and azoospermia in two patients [ ] . if corroborated, these findings suggest that infertility and disturbed spermatogenesis result not only from colchicine use but also may depend on other factors such as genetic background or underlying disease. the vasculitis associated with behçet disease may further contribute to this complication by adding local ischemia to the potential toxicity of colchicine. based upon the above observations, it is tempting to ascribe the development of azoospermia in patients with fmf to colchicine. however, in three cases of azoospermia we performed a testicular biopsy which demonstrated amyloido-sis of the testes [ ] . thus, amyloidosis of the testes should also be considered in patients with fmf presenting with azoospermia (see chaps. and ). another concern related to male fertility is the question on the outcome of pregnancies induced by male patients with fmf. in a study by zemer et al. of patients with fmf, females conceived while their male partners were treated with colchicine [ ] . there was no mention concerning fertility or delivery problems. due to limited data on this issue, some physicians in the past used to advise to discontinue colchicine months before attempting to conceive. in a prospective study, we followed the outcome of pregnancies and deliveries of female partners of males with fmf, were on colchicine when their partners conceived [ ] . as a control group, we followed the outcome of pregnancies and deliveries in healthy women married to healthy men. our findings revealed no difference regarding the rate of early or late abortions, or of congenital malformations. therefore, it seems that there is no need for males with fmf to discontinue colchicine prior to planning conception. the potential effects of colchicine on microtubules, cell division and growth raise a serious concern as to the female reproduction system. fmf attacks may be triggered in some patients by their menstrual period [ ] . the association of fmf attacks and menstruation raises the possibility of hormonal influences (see chap. ). indeed, it was shown that estrogen significantly decreases intercellular adhesion molecules [ ] . furthermore, it was demonstrated that estrogens inhibit tubulin assembly by interacting directly with tubulin s sites analogous to colchicine sites [ ] . moreover, estrogen is metabolized by the a liver cyp- complex and competes in binding it with colchicine. thus, it is tempting to speculate that estrogens mimic the effect of colchicine on tubules and adhesion molecules, thereby enhancing the effect of colchicine. during menstruation, there is a sharp decrease in estrogens, thus their accumulative suppressive effect on inflammation is suddenly diminished. in addition, the reduction in estrogen, allows for a more effective metabolism of colchicine by the a cytochrome (less inhibitory competition by estrogen) so that the effective level of colchicine may be further reduced. this situation may lead to menstruation associated fmf attack. to control these attacks, it is recommended to increase the dose of colchicine (by . mg) for days prior to the onset of the menstrual period and for two more days after its onset (see chap. ). theoretically, colchicine may affect female fertility by affecting the ovaries via its potential effect on cell division. however, this has not been shown. serious concern was raised regarding a teratogenic effect of colchicine. therefore, in the s physicians advised their patients to discontinue colchicine months before planning conception and during pregnancy. sporadic reports claimed that colchicine was safe during pregnancy. furthermore, in a study which followed pregnant women with fmf treated with colchicine, the outcome of the newborns was the same as in an untreated control group [ ] . in another study, all women with fmf, who had pregnancies and were on colchicine, gave birth to normal children [ ] . however, rabinovitch et al. reported that newborns out of ( : ) deliveries of fmf patients were born with trisomy , twice the expected rate of a comparable normal population [ ] . it was not clear whether colchicine therapy itself plays a role with this increment outcome. recently, diav-citrin et al. examined the safety of fetuses by following mothers exposed to colchicine during pregnancy. in a prospective observational comparative cohort study between and they found that colchicine did not appear to be a major human teratogen, and, probably, has no cytogenetic effect [ ] . we followed the outcome of pregnancy in a group of patients with fmf who took colchicine during pregnancy and compared them with a group of patients with fmf who were not treated with colchicine and with a group of healthy pregnant individuals [ ] . we showed that colchicine was not associated with a higher rate of miscarriage or stillbirth. there was no reduction of the duration of pregnancy or the birth weight of the babies. based upon these results, we do not recommend amniocentesis for patients with fmf solely because of treatment with colchicine during pregnancy. leaflets of pharmaceutical companies and textbooks of pharmacology warn women not to nurse their babies while treated with colchicine. milunsky and milunsky found that colchicine was present in the breast milk of patients taking the drug [ ] . we also measured the levels of colchicine in sera and milk of women with fmf at various time points after drug ingestion [ ] . colchicine was detected in all samples of sera and milk, with similar concentrations. however, the estimated daily amount of colchicine ingestion by the nursing babies was less than one tenth the therapeutic dose (per kilogram) given to adult patients with fmf. this rough estimation was concordant with our favorable clinical experience in following more than children of mothers who continued to breastfeed while taking colchicine. therefore, we suggest that breast feeding is safe while taking colchicine. since growth depends upon cell division, the potential effect of colchicine on child growth and development may raise concerns. the diagnosis of fmf can be made as early as several months of age. initially, we were reluctant to start treatment with colchicine before the age of years. during this period, children continued to suffer from recurrent attacks of fmf and were also at risk of developing amyloidosis. furthermore, they exhibited growth delay when compared with healthy children of the same age. following the start of colchicine treatment and control of fmf attacks, their appetites improved and a marked growth spurt was evident. we followed seven children since the age of - years and measured their height and weight every months for a period of years [ ] . their growth while treated with colchicine was within the normal expected percentile range. similar results were observed in larger and more recent studies [ ] . savgan-gurol et al. evaluated the growth process and insulin like growth factor- (igf- ) levels in children with fmf [ ] . they found that igf- levels of children with fmf did not differ from their healthy peers. however, there was a positive correlation between the rate of growth and the cumulative colchicine dose. therefore, they suggested that colchicine enhances the growth of children with fmf by suppressing disease activity and inflammation. the literature suggests that the minimal daily dose for preventing the development of amyloidosis in adult fmf patients is mg/day, even if attacks can be suppressed with a lower dose [ ] . nevertheless, several series from japan, reported that their adult patients with fmf were controlled by low-dose colchicine ( . mg daily). it is hypothesized that the reason for that is their carriage of mutations (in exon and ) which are known to cause a milder disease [ ] . the dose of colchicine should not exceed the maximal tolerated dose and should not be more than mg per day in adults without comorbidity and mg per day in prepubertal children [ ] . it is of paramount importance to avoid toxicity due to concomitant administration of cyp a or pgy inhibitors (table . ). drug-drug interactions need to be considered and the dose of colchicine should be appropriately adjusted. it is necessary to closely monitor the kidney and liver function of these patients (see sect. . for details). we recommend taking the entire colchicine dose at once in order to improve compliance. if the patient develops diarrhea due to ingestion of a relatively high single dose of colchicine the dose should be divided to twice per day without affecting its effectiveness [ ] . although colchicine has been used to treat gout for centuries, relatively few controlled trials have assessed its efficacy. the most recent major trial, acute gout flare receiving colchicine evaluation (agree), randomized patients with acute gout to receive a lower-dose colchicine regimen ( . mg dose followed by one . mg dose h later), a traditional higher dose regimen ( . mg dose followed by . mg every hour for a maximum of . mg) or placebo within h of the onset of attack [ ] . the lower-and higher-dose regimens demonstrated similar efficacy ( . % vs. . % achieving ≥ % improvement within h), but adverse events in the lower-dose regimen were similar to placebo. accordingly, the lower-dose regimen was approved by the fda. american college of rheumatology guidelines recommend the lowerdose colchicine regimen as a first-line therapy option for acute gouty attacks [ ] . in addition to its role in acute gout, colchicine is used prophylactically to reduce gout flare frequency, particularly when patients are initiating urate-lowering therapy. an analysis of three randomized controlled trials found that colchicine use for up to months during initial urate lowering provided greater prophylaxis of flares than its use for only weeks [ ] . there is only limited evidence for the use of colchicine in prophylaxis of pseudogout, although this is recommended practice [ ] . the rationale is the shared nlrp -inflammasome and il- driven inflammatory pathogenesis of urate and calcium pyrophosphate crystal deposition. recent european league against rheumatism (eular) guidelines recommend giving colchicine for acute attacks at a dose of . mg three times daily with or without a mg loading dose and for prophylaxis . mg per day. these recommendations are based largely on expert opinion and a single uncontrolled trial [ , ] . colchicine is recommended in the management of behçet disease, particularly for the mucocutaneous manifestations (oral and genital ulcers) and joint symptoms, based on controlled trials performed to date [ , ] . over the years colchicine has been studied for disorders of hepatic fibrosis. primary biliary cirrhosis is a rational potential indication, given the intense concentration of colchicine in bile and the evolving understanding of the capacity of colchicine to modulate bile composition [ ] . however, a meta-analysis combining the results of randomized controlled trials encompassing subjects with alcoholic or non-alcoholic liver cirrhosis, demonstrated no significant effects of colchicine on mortality, liver related mortality, complications and other outcomes [ ] . in a recent double blind, randomized controlled trial in subjects with chronic liver cirrhosis who could not be treated with interferon-α, muntoni et al. demonstrated that colchicine at a dose of mg per day significantly increased survival ( . % vs. . % p = . ), and decreased serum procollagen iii (a biomarker of liver fibrosis) over a follow up period of . years [ ] .this suggests that there may be a beneficial effect of colchicine for selected subjects with liver cirrhosis. additional research has assessed whether colchicine can delay the development of hepatic cell carcinoma (hcc) in patients diagnosed with hepatitis virus-related liver cirrhosis [ ] . while the effect of colchicine on the progression of cirrhosis was disappointing, colchicine suppressed the development of hcc. nine percent of patients treated with colchicine developed hcc versus % of untreated patients (p − . ), and the time to development of hcc was longer in the colchicine-treated group. colchicine is beneficial in neutrophilic skin conditions. sweet syndrome typically occurs in women aged - years and is characterized by fever, neutrophilia, arthralgia, tender erythematous skin lesions, and neutrophilic infiltrates in the upper dermis. maillard et al. gave colchicine to patients with sweet syndrome ( . mg three times daily for - days). they reported that patients experienced resolution of fever, skin lesions, arthralgia and neutrophilia within days [ ] . interestingly, in pyrin associated autoinflammatory disease with neutrophilic dermatosis (paand) colchicine is not effective and anti-il agents are required for disease control [ ] . the accumulating understanding of the role of inflammation in cardiovascular diseases has been accompanied by recognition of the potential anti-inflammatory benefit of colchicine in these settings. practice guidelines from the european society of cardiology advocate that colchicine appears to be effective when added to a nonsteroidal antiinflammatory drug regimen or as monotherapy to treat an initial attack or to prevent recurrence of pericarditis [ ] . these recommendations have subsequently been supported by randomized trials [ , ] . colchicine was as effective at reducing multiple recurrences of pericarditis as it was for first recurrences. [ , ] similar benefits were observed for treatment of an initial attack of acute pericarditis [ , ] . c-reactive protein (crp) is a biomarker of inflammation and infection. elevated highsensitivity (hs) crp is both a predictor and a pathogenic factor in vascular events such as coronary artery disease [ ] . a recent pilot study evaluated whether low-dose colchicine as an anti-inflammatory treatment could lower hs-crp levels in patients with stable coronary artery disease whose crp remained elevated despite aspirin and high-dose atorvastatin therapy [ ] . low-dose oral colchicine ( . mg twice daily) was associated with decreased hs-crp levels by %. however, in a separate, randomized study of patients with acute coronary syndrome or stroke, raju et al. found no significant association between colchicine use ( mg daily) and crp reduction, suggesting either that the dose studied was insufficient in the acute setting, or that colchicine may be more effective when administered as a prophylactic rather than as a treatment agent [ ] . more recently, nidorf et al. evaluated the effect of colchicine ( . mg daily) on secondary cardiovascular events in patients with stable coronary artery disease already on aspirin and/or clopidogrel and statin therapy [ ] . patients were followed for a median of years. the primary outcome,-the composite incidence of acute syndromes, out-ofhospital cardiac arrest, and non-cardio-embolic ischemic stroke-occurred in . % of patients treated with colchicine versus % of patients receiving placebo. these results warrant further study of the potential benefit of colchicine in the prevention of acute coronary syndrome. there is growing evidence that inflammation plays a role in the re-stenosis process. therefore, one study randomized patients with diabetes mellitus and coronary artery disease who underwent percutaneous coronary intervention with bare-metal stent placement to receive either colchicine . mg or placebo twice daily for months [ ] . subsequent angiography indicated that the rate of angiographic in-stent restenosis was % in the colchicine group versus % in the placebo group (p = . ). post-operative atrial fibrillation is a common occurrence after cardiac surgery and is presumed to be driven by surgery-related inflammation. the colchicine for the prevention of the postpericardiotomy syndrome (copps) atrial fibrillation substudy demonstrated that post-operative administration of colchicine was associated with a % reduction in the incidence of post-operative atrial fibrillation [ ] . however, a second multicenter, randomized, double-blind, placebocontrolled trial (copps- ) found no reduction in post-operative atrial fibrillation among patients receiving colchicine [ ] . colchicine may also reduce the risk of recurrence of atrial fibrillation after ablation therapy. in a randomized, double-blind, placebocontrolled study by deftereos et al., months of colchicine monotherapy was associated with a % reduction in the incidence of atrial fibrillation after pulmonary vein isolation with radiofrequency ablation [ ] . the colchicine group also experienced a significant decrease in il- and crp levels. colchicine update- indianapolis: hackett colchicine: new uses of an old, old drug ueber einige neue giftige organische alkalien (on some new poisonous organic alkalis) medea and the microtubule: research has been translational ever since colchis sulla cariocinesi delle cellule epiteliale e dell endotelio dei vasi della mucosa dello stomato e dell' intestino, nello studio gastroenterite sperimentale (nell avvelenamento per colcico) binding of colchicine- h to cellular protein amino-acid composition of "tubulin" constituting microtubules of sperm flagella the role of microtubule movement in bidirectional organelle transport colchicine in dermatology colchicine in the prevention and treatment of the amyloidosis of familial mediterranean fever colchicine for familial mediterranean fever pharmacokinetics of colchicine in pediatric and adult patients with familial mediterranean fever pharmacokinetic bioavailability of colchicine in healthy male volunteers improvement of colchicine oral bioavailability by incorporating eugenol in the nanoemulsion as an oil excipient and enhancer genuine functions of p-glycoprotein (abcb) concise review: clinical relevance of drugdrug and herb-drug interactions mediated by the abc transporter abcb substrateinduced conformational changes in the transmembrane segment of human p-glycoprotein p-glycoprotein: so many ways to turn it on colchicine today pharmacokinetics and absolute bioavailability of colchicine after i.v. and oral administration in healthy human volunteers and elderly subjects association of drug transporter gene abcb (mdr ) c to t polymorphism with colchicine response in familial mediterranean fever colchicine nonresponsiveness in familial mediterranean fever: genetic, pharmacokinetic, and socioeconomic characterization plasma levels of colchicine after administration of a single dose colchicine plasma levels: implications as to pharmacology and mechanism of action colchicine disposition in patients with familial mediterranean fever with renal impairment measurement of colchicine in urine and peripheral leukocytes (abstract) binding of drugs by albumin and plasma protein colchicine disposition in human leukocytes after single and multiple oral administration colchicine: a state-ofthe-art review bioavailability and pharmacokinetics of two formulations in volunteers colchicine clearance is impaired in alcoholic cirrhosis interaction of tubulin with single ring analogue of colchicine effect of colchicine on lymphocytes and monocytes function and its relation to fibroblast proliferation in primary biliary cirrhosis appearance of chemotactic activity following intracellular injection of monosodium urate crystals: effect of colchicine colchicine inhibition of chemotaxis a new mode of action for an old drug: colchicine decreases surface expression of adhesion molecules on both neutrophils (pmns) and endothelium (abstract) p-glycoprotein expression and function in circulating blood cells from normal volunteers does the lack of the p-glycoprotein efflux pump in neutrophils explain the efficacy of colchicine in fmf and other inflammatory diseases rapports entre noyau et centrioles dans les granulocytes étalalés. rúle des microtubules molecular mechanism of colchicine action: induced local unfolding of b-tubulin response of microtubules to the addition of colchicine and tubulin-colchicine: evaluation of models for the interaction of drugs with microtubules inhibition of lpsinduced tumor necrosis factor-α production by colchicine and other microtubules disrupting drugs downregulation of tumor necrosis factor receptors on macrophages and endothelial cells by mictotubule depolarizing agents colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils involvement of secretory phospholipase a activity in the zymosan air pouch model of inflammation the effects of colchicine and hydroxychloroquine on the cyclo-oxygenases cox- and cox- the french consortium. a candidate gene for familial mediterranean fever ancient missense mutations in a new member of the roret gene family are likely to cause familial mediterranean fever effect of colchicine and cytokines on mefv expression and c a inhibitor activity in human primary fibroblast culture the familial mediterranean fever protein, pyrin, associates with microtubules and colocalizes with actin filaments the pharmacologic basis of treatment with colchicine in children with familial mediterranean fever mechanism of the anti-inflammatory effect of colchicine in rheumatic diseases: a possible new outlook through microarray analysis gout-associated uric acid crystals activate the nalp inflammasome colchicine suppresses neutrophil superoxide production in a murine model of gouty arthritis: a rationale for use of low-dose colchicine innate immune sensing of bacterial modifications of rho gtpases by the pyrin inflammasome interaction of pyrin with . . in an isoform-specific and phosphorylation-dependent manner regulates its translocation to the nucleus nucleotide exchange factor gef-h mediates cross-talk between microtubules and the actin cytoskeleton pyrin inflammasome activation and rhoa signaling in the autoinflammatory diseases fmf and hids technical advance: inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments colchicine attenuates renal injury in a model of hypertensive chronic kidney disease curcumin prevents liver fibrosis by inducing apoptosis and suppressing activation of hepatic stellate cells the effects of colchicine on the progression and regression of encapsulating peritoneal sclerosis colchicine use in children and adolescents with familial mediterranean fever: literature review and consensus statement colchicine is a safe drug in children with familial mediterranean fever current trends in colchicine treatment in familial mediterranean fever colchicine intoxication clinical pharmacology, risk factors, features and management colchicine: old and new estimation of colchicine in a poisoned patient by using high performance liquid chromatography treatment of severe colchicine overdose with colchicine specific fab fragments vos fr high flux dialysis membranes improve lipid profile in chronic hemodialysis patients colchicine clearance by high-flux polysulfone dialyzers abc drug transporters as molecular targets for the prevention of multidrug resistance and drug-drug interactions colchicine myotoxicity: case reports and literature review colchicine use in cyclosporine treated transplant recipients: how little is too much? mdr-and cyp a -mediated drug-drug interactions influence of cytochrome p polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects multiple sequential steps involved in the binding of inhibitors to cytochrome p a heterotropic cooperativity of cytochrome p a and potential drug-drug interactions acute myopathy in a patient with concomitant use of pravastatin and colchicine fatal interaction between clarithromycin and colchicine in patients with renal insufficiency: a retrospective study cytoskeletal myotoxicity from simvastatin and colchicine rhabdomyolysis in a patient treated with colchicine and atorvastin possible colchicine rhabdomyolysis in a fluvastatin-treated patient novel evidence-based colchicine dosereduction algorithm to predict and prevent colchicine toxicity in the presence of cytochrome p effect of colchicine on guinea pig intrinsic factor-vitamin b receptor colchicineinduced lactose malabsorption in patients with familial mediterranean fever azoospermia caused by colchicine-a case report the effect of colchicine on the spermatogenesis of rabbits a cytogenetic evaluation of long term colchicines therapy in the treatment of familial mediterranean fever (fmf) testicular function in patients with familial mediterranean fever, long-term colchicine treatment fertility and obstetric history in patients with familial mediterranean fever on long term colchicine therapy the effect of colchicine on human spermatozoal motility in vitro colchicine and testicular function in man urological evaluation of behçet patients and the effect of colchicine on fertility azoospermia in fmf patients-the role of colchicine and amyloidosis daily prophylactic colchicines in familial mediterranean fever the effect of fmf and colchicine on pregnancies outcome of wives of patients with fmf familial mediterranean fever and menstruation effects of hormone therapy on inflammatory cell adhesion molecules in postmenopausal healthy women qualitative study of the interaction mechanism of estrogenic drugs with tubulin safety of colchicine therapy during pregnancy familial mediterranean fever and its implications for fertility and pregnancy colchicine treatment in conception and pregnancy: two hundred and thirty one pregnancies in patients with familial mediterranean fever pregnancy outcome after in utero exposure to colchicine outcome of pregnancies in fmf women with colchicine breast feeding during colchicine therapy for familial mediterranean fever colchicine in breast-milk of patients with fmf colchicine treatment in familial mediterranean fever (fmf)-reappraisal after years colchicine therapy of children with fmf (abstract) growth and igf- levels of children with familial mediterranean fever on colchicine treatment eular recommendations for the management of familial mediterranean fever familial mediterranean fever is no longer a rare disease in japan evidencebased recommendations for the practical management of familial mediterranean fever comparison of the efficacy of once-and twice-daily colchicine dosage in pediatric patients with familial mediterranean fever-a randomized controlled non-inferiority trial high versus low dosing of oral colchicine for early acute gout flare: twenty-four-hour outcome of the first multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-comparison colchicine study american college of rheumatology guidelines for management of gout. part : therapy and antiinflammatory prophylaxis of acute gouty arthritis effect of prophylaxis on gout flares after the initiation of urate-lowering therapy: analysis of data from three phase iii trials eular recommendations for calcium pyrophosphate deposition. part ii: management intravenous colchicine in the treatment of acute pseudogout a double-blind trial of colchicine in behçet's syndrome eular recommendations for the management of behcet disease mechanisms by which camp increases bile acid secretion in rat liver and canalicular membrane vesicles colchicine for alcoholic and non-alcoholic liver fibrosis and cirrhosis colchicine reduces procollagen iii and increases pseudocholinesterase in chronic liver disease colchicine delays the development of hepatocellular carcinoma in patients with hepatitis virusrelated liver cirrhosis colchicine for sweet's syndrome. a study of cases familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation guidelines on the diagnosis and management of pericardial diseases executive summary; the task force on the diagnosis and management of pericardial diseases of the european society of cardiology colchicine as first-choice therapy for recurrent pericarditis: results of the core (colchicine for recurrent pericarditis) trial colchicine for recurrent pericarditis (corp): a randomized trial efficacy and safety of colchicine for treatment of multiple recurrences of pericarditis (corp- ): a multicentre, double-blind, placebo-controlled, randomised trial colchicine for treatment of acute or recurrent pericarditis colchicine in addition to conventional therapy for acute pericarditis: results of the colchicine for acute pericarditis (cope) trial a randomized trial of colchicine for acute pericarditis c-reactive protein and atherogenesis: from fatty streak to clinical event effect of colchicine ( . mg twice daily) on high-sensitivity c-reactive protein independent of aspirin and atorvastatin in patients with stable coronary artery disease effect of colchicine compared with placebo on high sensitivity c-reactive protein in patients with acute coronary syndrome or acute stroke: a pilot randomized controlled trial low-dose colchicine for secondary prevention of cardiovascular disease colchicine treatment for the prevention of baremetal stent restenosis in diabetic patients colchicine reduces postoperative atrial fibrillation: results of the colchicine for the prevention of the postpericardiotomy syndrome (copps) atrial fibrillation substudy colchicine for prevention of postpericardiotomy syndrome and postoperative atrial fibrillation: the copps- randomized clinical trial colchicine for prevention of early atrial fibrillation recurrence after pulmonary vein isolation: a randomized controlled study key: cord- -o qg qta authors: mocchegiani, eugenio; malavolta, marco title: role of zinc and selenium in oxidative stress and immunosenescence: implications for healthy aging and longevity date: - - journal: handbook of immunosenescence doi: . / - - - - _ sha: doc_id: cord_uid: o qg qta aging is a complex process that includes gradual and spontaneous biochemical and physiological changes which contributes to a decline in performance and increased susceptibility to diseases. zn and se are essential trace elements that play a pivotal role in immune functions and antioxidant defense and, consequently, are claimed to play also a role in successful aging trajectories. consistently with their nature of essential trace elements, a plethora of data obtained “in vitro” and “in vivo” (in humans and animal models) support the relevance of zn and se for both the innate and adoptive immune response. moreover, zn and se are strictly involved in the synthesis and regulation of activity of proteins and enzymes, e.g., metallothioneins (mt) and glutathione peroxidase (gpx), that are necessary for our endogenous antioxidant response. this is clearly important to protect our cells from oxidative damage and to slow the decline of our immune system with aging. age-related changes affecting tissue levels of zn and se may indicate that the risk of zn and se deficiency increases with aging. however, it is still unclear which of these changes can be the consequence of a “real deficiency” and which can be part of our physiological compensatory response to the accumulating damage occurring in aging. furthermore, the upregulation of antioxidant proteins (zn and se dependent) may be a manifestation of self-induced oxidative stress. by the way, zn and se dependent proteins are modulated not only by nutritional status, but also by well-known hallmarks of aging that play antagonistic functions, such as the deregulated nutrient sensing pathways and cellular senescence. thus, it is not an easy task to conduct zn or se supplementation in elderly and it is emerging consistent that these kind of supplementation requires an individualized approach. anyway, there is consistent support that supplementation with zn using doses around mg/day is generally safe in elderly and may even improve part of immune performances in those subjects with a baseline deficiency. regarding se supplementation, it may induce both beneficial and detrimental effects on cellular immunity depending on the form of se, supplemental dose, and delivery matrix. the nutritional association of supplements based on “zn plus se” is hypothesized to provide additional benefits, but this will likely need a more complex individualized approach. the improvement of our knowledge around screening and detection of zn and se deficiency in aging could lead to substantial benefits in terms of efficacy of nutritional supplements aimed at ameliorate performance and health in aging. biological aging is a process driven by molecular damage, molecular heterogeneity, and metabolic imbalance which determinates increased susceptibility to diseases and impaired adaptation to changes in environmental conditions (rattan ) . alterations in the immune functions play a fundamental role in aging. the term "immunosenescence" has been coined to describe the changes that occur with age in the immune system and that lead to a higher incidence of infection, cancer, and autoimmune disease (fulop et al. ; pawelec et al. ) . immunosenescence involves a shift in function of both adaptive and innate immune cells, which can be resumed in a reduced capacity to recognize new antigens and in the occurrence of systemic low-grade chronic inflammation. this phenomenon, termed "inflamaging" (franceschi et al. ) is likely the result of immunosurveillance of chronic infections (chronic antigen stress) (pawelec et al. ) combined with accumulating senescent cells which, in turn, are a rich source of pro-inflammatory factors collectively termed the "senescence-associated secretory phenotype" (sasp) (campisi ) . in analogy with aging, immunosenescence in humans is very heterogeneous and can be described as a complex mosaic resulting from the interaction of a variety of environmental, stochastic, and genetic-epigenetic variables. it is likely that, through a favorable combination of these factors, centenarians avoid or delay the major inflammation-driven age-related diseases, such as cardiovascular disease (cvd), diabetes mellitus (dm), alzheimer disease (ad), and cancer (bürkle et al. ) despite showing some markers of inflammation. diet and nutritional factors are known to play a role in the preservation of our immune system in aging. deficiencies of both macro-and micronutrients in aging is strictly related to global impairments of the immune functions and appearance of age-related diseases (lesourd ) . several causes contribute to micronutrient deficiencies in elderly. first of all, the poor socioecon omic condition present in a large part of old people may lead to a consumption of inexpensive foods deficient in micronutrients (kant ) . the gap is worsened by loss of appetite, lack of teeth, intestinal malabsorption, and subclinical diseases that lead to the final result of frailty, disability, and mortality (semba et al. ) . by contrast, changes in diet and exercise patterns appears to be effective in the prevention of late-onset nutritionrelated conditions, especially when these are instituted early in life (chernoff ) . available data do indicate that essential vitamins and trace elements are necessary for normal immune function (maggini et al. ), but conclusive studies demonstrating that vitamin or mineral supplements can boost immune function are currently lacking. hence, there is evidence that further supplementation of micronutrients beyond the recommended daily intake can improve immune function (dharmarajan ) . however, clear roles exist for vitamin and trace element supplementation in states of deficiency and in subgroups of older adults at high risk for deficiency. by the other way, the prevalence of certain nutritional deficiencies in the geriatric population (e.g., zinc and selenium) is sufficiently high that certain supplements may be indicated (ames ) . we herein review the role of zinc and selenium for our immune system (buttriss ) and describe the mechanisms through which they affect immunosenescence. epidemiological and clinical evidence have shown that in most developing countries deficiencies of these micronutrients are partly responsible for the severity of infectious disease, morbidity, and mortality in malnourished children (bhaskaram ; bailey et al. ) as well as in elderly (meydani ) and critically ill patients (mertens et al. ) . these two trace elements display a common pivotal role in establishing the cellular antioxidant response as well as in mounting a proper immune response, which in turn may be useful to prevent excessive accumulation of senescent cells in aging and to reduce the senescence-associated increase of chronic inflammatory mediators. biological role of zinc zinc is one of the most important trace elements in the body, although its presence in nature does not exceed . % (mills ) . the major characteristics of zinc include a highly concentrated charge, a small radius ( . a), no variable valence (low risk of free radical production), ready passage from one symmetry in its surroundings to another without exchange, rapid exchange of ligands (on and off reactions), and binding mostly to s-and n-donors in biological systems. these properties enable zinc to play a major biological role as a catalyst. zinc is essential for the activity of more than enzymes influencing the activity of zinc dependent antioxidant enzymes, such as superoxide dismutase (sod) and various organ functions having a secondary effect on the immune system (rink and gabriel ) . zinc is present in "zinc finger domains" of transcription factors and in a multitude of proteins, peptides, enzymes, hormones, and cytokines, which act in maintaining body homeostasis (coleman ; berg and shi ) . zinc also regulates mrna stability (taylor and blackshear ) and extracellular matrix (vallee and falchuk ) . zinc binds enzymes, proteins, and peptides with different binding affinity (kd) ranging from À to À mol/l (mocchegiani et al. ) . some of these enzymes may be activated by zinc, as for instance the thymic hormone named thymulin, which loses its activity in absence of zinc (fabris et al. ) , while others may be inactivated. zinc also regulates cell cycle and apoptosis (fraker ) with an optimal range of concentration that has been well defined by studies on genomic stability "in vitro." these studies, performed on various cellular models, suggest that genomic instability is minimized when zn concentration in culture medium is comprised between and μm (ho and ames ; ho et al. ; sharif et al. ) . although, it is currently unknown whether these results can be translated "in vivo," where additional factors including blood composition, genetic background, aging, and diseases can affect the response, these studies "in vitro" confirm that an excess of zn is dangerous for cellular health in equal measure to zn deficiency. a very recent study altered the zinc balance within caenorhabditis elegans to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. the authors found that increasing dietary zinc levels decreased the mean and maximum life span of the worms, whereas treatment with a zinc-selective chelator increased their mean and maximum life span. the life span modulating effects were mediated by daf- , hsf- , and skn- proteins, which suggest the involvement of the insulin/igf- pathway. these results are somewhat supporting the idea that zinc itself has a role as an antagonistic pleiotropy player (kumar et al. ) . by the way, it has been just recently hypothesized that reduced zinc levels in serum during human aging may reflect the homeostatic shade from a general systemic "growth and reproduction" status typical of juvenile age to a "repair and maintenance" status that evolved to preserve health status during old age . the cellular basis for the impact of low and high zn status has been studied for many years but it is only recently that the complexity of zn homeostasis has been appreciated. the advance in the knowledge around the major intracellular zn binding proteins, namely the metallothioneins (mt) and the discovery, cloning, and characterization of zn transporter proteins, has highlighted the role of free zn ion and the fluxes of zn ions between extracellular, cellular, and intracellular compartments. indeed, these zn signals can initiate pathways which have tight analogies with those discovered for calcium signaling, albeit in the sub-and nanomolar range (haase and rink ) . metallothioneins (mt) are a group of low-molecular-weight metal-binding proteins who have high affinity for zinc (kd = . Â À m) (kagi and schaffer ) . mt exist in different isoforms characterized by the length of amino acid chain: isoform i, ii, iii, e iv mapped on chromosome in man and on chromosome in mice with complex polymorphisms (west et al. ). the more common isoforms are i and ii; the isoform iii, also called growth inhibitory factor (gif), is mostly expressed in brain tissues while the isoform iv is restricted in squamous epithelia. mt contain cysteines which bind seven zinc atoms through mercaptide bonds that have the spectroscopy characteristics of metal thiolate clusters (maret and vallee ) . the zinc/cysteine clusters are of two different types. in the beta-domain cluster, three bridging and six terminal cysteine thiolates provide a coordination environment that is identical for each of the three zinc atoms. in the alpha-domain clusters, there are two different zinc sites; two of them have one terminal ligand and three bridging ligands, respectively, while the other two have two terminal and two bridging ligands (maret and vallee ) . mt can act as an antioxidant since zinc-sulfur cluster is sensitive to changes of cellular redox state and oxidizing sites in mt (reduced thiol groups) induce the transfer of zinc from its mt-binding sites to those of lower affinity in other proteins (kagi and schaffer ) . in response to oxidative or nitrosative stress, zn-mt release free zinc ions. the released free zinc ions are used to confer the biological activity to some zincdependent antioxidant enzymes and to other proteins involved in dna repair, as well as to upregulate the gene expression of various factors involved in the antioxidant response (mocchegiani et al. ). hence, mt are able to transduce stress signals into free zinc ion signals which are immediately compensated by the activity of zn transporters and subsequently downregulated by a feedback mechanism that involves the gene expression of mt themselves. this mechanism is also involved in regulation of nitric oxide (no) pathways (bogdan et al. ) as no induces the release of zinc from mt, via s-nitrosylation (zangger et al. ) . the release of zinc by mt, via s-nitrosylation, contributing to raise the intracellular free zinc ions concentration, plays a crucial role in modulating the production of pro-inflammatory cytokines and in the activation of immune cells (rink and haase ) . free zinc ion levels are also regulated by special proteins named zinc transporters, which in turn appear to be also specifically involved through regulation of cellular zinc homeostasis via influx, efflux, or vesicular sequestration (cousins and mcmahon ; eide ) . two families of zinc transporters have been identified. the znt family decreases cytoplasmic zinc concentrations by secretion, sequestration, or efflux, whereas the zip family increases cytoplasmic zinc influx or release of stored zinc (eide ) . the huge network based on zn transporters and mt suggest how much critical is a tight regulation of zinc ion availability. moreover, the timely release, efflux, and influx of zn ions is known to virtually affect all aspects of innate and adaptive immunity. several molecular targets, including phosphatases, phosphodiesterases, caspases, and kinases, suggest that zinc ions are like a second messenger that regulate signal transduction in various kinds of immune cells (mocchegiani et al. ; haase and rink ) . this phenomenon clearly explains why in vivo zinc deficiency alters the number and function of neutrophil granulocytes, monocytes, natural killer (nk), t, and b cells. the redox properties of mt contribute to make this protein a crucial defense against ionizing and uv radiations (cai et al. ) , heavy metals (mercury, cadmium), lipid peroxidation, reactive oxygen species, oxidative stress caused by anticancer drugs, and conditions of hyperoxia (sato and kondoh ) . this protective role of mt has been studied especially in young-adult mt knockout mice (null mice) for short periods of exposure to toxic metals, such as cadmium for weeks (habeebu et al. ) or mercury (single injection and the effect of mercury analyzed days after the injection) (satoh et al. ) , or to anticancer agents for - h (kondo et al. ) , or in presence of an excess of zinc or zinc deficiency for weeks (kelly et al. ) . expression of mt is mediated by zinc (directly via mre responsive elements in the promoter), glucocorticoids (via glucocorticoid responsive element in the promoter), and other stressor agents as well as from inflammatory cytokines, such as il- , il- , ifn-a, tnf-a (davis and cousins ) . these findings clearly suggest the existence of a strong interplay between mt and the immune system. an important link between cytokines and mt seems to be stat (signal transducers and activators of transcription) responsive element observed in the promoter of mt as well as the nitric oxide pathway. it is remarkable that cytokines reported to increase inducible no synthase (inos) expression (il- and tnf-a) have also induced the expression of mt (zangger et al. ). this induction is likely the indirect consequence of the release of free zinc from preexisting mt via s-nitrosylation caused by no. this process is particularly important for the production of thymic hormone (thymulin) from thymic epithelial cells. indeed, il- induces thymic uptake of zinc (coto et al. ) , which is subsequently made available for the activity of thymulin by a process that involve the redox status of mt (savino et al. ) . mt have been also found to be released to the extracellular environment in a number of different compartments, including cell culture media, serum, urine, bronchoalveolar spaces, liver sinusoids, and inflammatory lesions. albeit a very small amount of mt can be found in the extracellular environment (e.g., around μm in serum), these proteins may support the beneficial movement of leukocytes to the site of inflammation representing a "danger signal" for the immune cells and modifying the character of the immune response when cells sense cellular stress (yin et al. ) . hence, mt are involved in several mechanisms that support immune function. what is still uncertain is the role of these proteins in aging and immunosenescence. in particular, there have been concerns around the possibility that mt may retain their protective role during chronic stress and inflammatory conditions associated with the major disease of aging (mocchegiani et al. a ). in the case of cancer, for example, mt have been proven as an important barrier against carcinogenesis (dziegiel et al. ; malavolta et al. ) but, once malignant cells are formed these proteins may help cancer cells to survive and proliferate. several studies have disclosed mt expression as a prognostic factor for tumor progression and drug resistance in a variety of cancers, including breast, prostatic, ovarian, head and neck, nonsmall cell lung cancer, melanoma, and soft tissue sarcoma (eckschlager et al. ; dziegiel et al. ). this action is consistent with the original biological role of mt, as these proteins are part of the defensive intracellular stress response. additionally, elevated levels of extracellular mt, as detected at sites of inflammation and in certain types of neoplastic lesions, have been shown to display an immunosuppressive function (youn and lynes ) . by contrast, there are cases where mt have been found to provide additional benefit in chronic inflammation, especially in cancer therapy. indeed, while cancer cells overexpressing mt appear in general less sensitive to chemotherapeutic drugs, cancers where mt are progressively silenced are more sensitive to drugs following expression of specific mt isoforms in presence of zinc (pedersen et al. ; arriaga et al. ) . these studies suggest that there is not a common role and that different mt isoforms might be differently associated with either chemoresistance or sensitivity to cancer drugs. in the case of other chronic age-related diseases, it was hypothesized that the expression of mt, continuously stimulated by the persistence of inflammatory mediators (e.g., il- ), could lead to a detrimental sequestration of zinc ions with potential functional consequences on the immunity of frail aged individuals (mocchegiani et al. a, b) . observational studies performed (a) in pbmcs from centenarians, which display lower mt expression compared to elderly aged - years, (mocchegiani et al. a ); (b) in pbmc from down's syndrome (syndrome which presents some features of accelerated aging and immunosenescence) subjects, which display levels of mt comparable to elderly subjects (mocchegiani et al. a) , and (c) studies in mice models of athropic thymus, which display overexpression of mt (mocchegiani et al. b) , have contributed to argue the hypothesis against a preserved functional role of mt in aging. however, it has been more recently documented that mt expression declines with "in vitro" aging of pbmcs, thus suggesting that the low levels of mt observed in pbmcs of centenarians could simply be the consequence of the increasing accumulation of senescent cells in blood or other phenomena related to epigenetic changes. conversely, the increased levels of mt observed in pbmcs from elderly subjects could be the physiological consequence of a stress response induced by inflammatory agents. a general pro-longevity role of mt emerges also from recent studies in mice. a longevity phenotype has been shown in mt transgenic mice that overexpresses the mt- isoform (mt -tg) in the c bl/ j background , as well as in cardiac-specific mt transgenic mice that overexpress the human mt isoform in the fvb background (yang et al. ) . more recently, the findings that mt ko mice display a shortened life span can be considered a definitive proof that constitutive mt expression from birth to death may play a beneficial role in longevity, at least in mice, independently by the strain studied (kadota et al. ) . however, it is still uncertain if this situation may be comparable in humans and, most importantly, there are no examples of mt-inducible mice models, which might unravel the consequence of reactivation of mt expression in late life. anyway, association studies of snps of mt genes point out a role of these proteins in human longevity or in the susceptibility to agerelated diseases but the functional role is far to be clarified (giacconi et al. ; cipriano et al. ) . taking inspiration from the role of mt in cancer, it should deserve appropriate consideration for future investigation the role of mt in cellular senescence. currently, there is common agreement on the negative role of excessive accumulation of apoptosis-resistant senescent cells in aging and in several age-related diseases. this evidence is supported by the rejuvenating effects observed after inducible removal of senescent cells in transgenic animals (baker et al. (baker et al. , as well as after treatment of mice with senolytic drugs (chang et al. ; roos et al. ) . while late passage senescent lymphocytes clones may display reduced expression of mt protein , some models of oncogene-induced senescence (collado et al. ) and senescent endothelial cells (malavolta et al. unpublished observation) appear to overexpress mt genes. in analogy with the role shown by mt in resistance to apoptosis of cancer cells, it might be possible to build the hypothesis that these proteins can play a role in the resistance to apoptosis of senescent cells. investigation around this hypothesis might be important to optimize the development of senolytic drugs before human studies. increasing evidence suggest that zinc transporters play a major role in intracellular zinc homeostasis, which is critically involved in the signaling and activation of immune cells. while few gerontological studies have specifically addressed the role of zinc transporters, there is evidence that they may play a role in the process of progressive dysregulation of immune responses associated with aging. a recent flow cytometry assay showed that uptake of extracellular zinc is reduced in pbmcs taken from elderly subjects compared to the younger counterpart (giacconi et al. ). this observation argues for a possible downregulation of zinc importers' or upregulation of zinc exporters' function in aged immune cells. in agreement with this observation, a study performed in mice showed reduced zip expression and dysregulation in splenocytes from aged mice (wong et al. ). most importantly, the age-specific zip dysregulation correlated with an increase in zip promoter methylation while reduced zip expression was shown to enhance pro-inflammatory response. similarly, a correlation between the level of methylation at slc a (znt ) promoter region and age has been reported in humans (coneyworth et al. ). moreover, methylation of the znt promoter region resulted in reduction of znt expression and was hypothesized to contribute to the decline in zn status observed with aging. in addition to epigenetic changes occurring with aging, another potential link between immunosenescence and zinc transporters regards the regulation of zip in the liver. zip expression is upregulated through il- , and this mechanism was shown to be responsible of the hypozincemia that accompanies the acute-phase response to inflammation and infection (liuzzi et al. ) . psychological stress, a putative recipe for accelerated aging, has been shown to induce zinc accumulation and upregulation of zip and metallothionein in rat liver (tian et al. ). since chronic inflammation, particularly driven by increased il- , is a usual event in old age (mocchegiani et al. ) , it cannot be excluded that this process may contribute to hypozincemia, depressed immune response, and risk of infection in the elderly. however, it should be noted that a gene expression study on two major zn transporters (znt and zip ) in leukocytes obtained from two different age groups of korean women indicated major changes following supplementation then baseline changes between the two groups (andree et al. ) . hence, it is still uncertain which zn transporters are more likely involved in age-related changes and, most importantly, which is the real impact of these changes on immune function and health of elderly subjects. "in vitro" studies since the discovery that the crude zinc balance is negative in old mice (mocchegiani et al. ) and in old human (turnlund et al. ), a number of studies involving zinc supplementation have been performed. most of these studies have been focused on the effect of zinc on immune function, as "in vitro" experiments have provided considerable support on the immune-modulatory effects of zinc. when pbmcs are stimulated with zinc, il- , il- , and tnf-α, soluble (s)il- receptor and ifn-γ are released (ibs and rink ) . the secretion of il- , il- , and tnf-α is induced directly by zinc in monocytes and is independent by the presence of lymphocytes (driessen et al. ) . however, the effect of zinc on monocytes may depend upon external stimulation. in fact, zinc inhibits lps-induced tnf-α and il- β release from primary human monocytes and monocytic cell lines through the inhibition of cyclic nucleotide phosphodiesterase activity (von bulow et al. ) , suggesting that zinc may display also some anti-inflammatory properties. the dose of zinc used is also a critical variable. in serum-free culture medium, pharmacological concentrations (> μm of zinc) stimulate monocytes but prevent t cells from activating, perhaps due to the lower intracellular content in t cells than in monocytes (ibs and rink ) . treatment with zinc "in vitro" generally displays also beneficial effects on cell survival but the effect largely depends upon the cell type and the dose of zinc used. the optimal concentration of zinc "in vitro" from genomic stability studies performed in different cellular models point out at concentrations in the range - μm (sharif et al. ) . it seems that both apoptosis prevention and induction are mediated by pathways involving zinc and/or zinc-dependent enzymes (clegg et al. ; wiseman et al. ) . therefore, the modulation of the zinc homeostasis plays a key role not only in preventing apoptosis, when oxidative stress is low, but also in inducing apoptosis, when oxidative stress and cellular damage is high. this mechanism could play a role, for example, in the elimination of virally infected or malignant cells (fraker and lill-elghanian ) . the pro-apoptotic function of zinc has been documented under condition of persistent oxidative damage in pbmc from young-adult humans as well as in very old age (ostan et al. ) . the key player that mediates the activity of zinc in this case appears to be the tumor suppressor p , which needs zinc for site-specific dna binding and proper transcriptional activation (hainaut and mann ; loh ) . the response to zinc during stress condition appears be cell-type dependent. experiments in thymocytes have shown that zinc from up to μm prevents old thymocyte apoptosis induced by dexamethasone or serum deprivation (provinciali et al. ) , whereas the direct introduction of free zinc as zincpyrithione inside thymocytes induces apoptosis (mann and fraker ) . it is likely that the continuous presence of intracellular free zinc ions induced by zn-pyrithione can be interpreted by the cell as a presence of irreversible damage with subsequent activation of pro-apoptotic pathways. old literature documented that zinc supplementation performed throughout the whole life span of rodents is able to delay some age-related cell-mediated immune modifications, such as the decreased circulating thymic hormone levels (iwata et al. ). however, an immunomodulatory effect of zn supplementation has been shown with a relatively short time of treatment in old mice. for example, μg/ml zn ++ in the drinking water induced thymus regrowth and functionality (dardenne et al. ; mocchegiani et al. ) as well restoration of nk cell cytotoxicity (mocchegiani et al. ) in old mice in just month. that the benefit of zinc supplementation upon the immune functions in old mice is not to consider an epiphenomenon comes by the analysis of the rate of survival in old zinc-treated mice. old mice (inbreed balb/c mice) treated with daily zinc at the dose reported above in drinking water from the pre-senescent age ( - months of age) display a significant increment of the median and maximal life span (up to months vs. - of controls) (mocchegiani et al. b ). the increased longevity was largely due to significant decrements of deaths due to cancer and infection in the middle age. part of the effects shown in aged mice can be the consequence of an improved thymopoiesis (wong et al. ). however, increased longevity after zinc supplementation was also reported in the short lived thymectomized and nude mice, which display a negative crude balance of zinc (mocchegiani et al. ). taking into account that the liver extrathymic t cell pathway is prominent in nude, thymectomized and old mice (abo et al. ) , it is likely that these effects of zinc may be in part the effects of zinc on this extrathymic pathway (mocchegiani et al. ) . conversely, in aged c bl/ mice, zinc supplementation ( mg/kg for days) was shown to increase thymopoiesis, as assessed by increased total thymocyte numbers (wong et al. ). the improved thymic output was mediated in part by reducing the age-related accumulation of immature cd (À)cd (À)cd (+)cd (À) thymocytes, as well as by decreasing the expression of the thymosuppressive cytokine named stem cell factor. a slightly increased survival has been also recently documented in old (age ! months) c bl/ j mice supplemented for the whole life span with mg/l of zinc in drinking water. however, survival curves of supplemented mice displayed an increased variability and were markedly lower compared to those of transgenic mice overexpressing mt independently of zinc supplementation . the results of mice overexpressing mt are similar to those obtained in worms with zinc chelation (kumar et al. ) . these data may reinforce the idea that zinc has a potential antagonistic pleiotropic. in particular, zinc may act on nutrient-sensing pathways (e.g., insulin/igf- -like receptor pathway and pi k/akt/mtor pathway) with the final result of promoting growth and proliferation. inhibition of this pathway has been proposed as a pro-longevity treatment while its enhancement may help elderly, which in turn display excessive downregulation of nutrient-sensing pathways as a consequence of compensatory phenomena normally occurring in aging. this point of view could explain the beneficial effects observed by supplementation in elderly and old mice. with regard to elderly, there is consistent support that zinc supplementation may slightly impact immune system, but real benefits appears to be evident only in the case of well-documented nutritional zinc deficiency. an exhaustive picture of the zinc supplementation in elderly by different studies has been reported by haase and rink ( ) . many attempts have been performed in the past using different doses of zn for various treatment duration (duchateau et al. ; sandstead et al. ; bogden et al. ; prasad et al. ; boukaïba et al. ; cakman et al. ; fortes et al. ) . immunostimulative effects of zinc have been documented using pharmacological doses largely exceeding the rda. for example, duchateau et al. ( ) and sandstaed et al. ( ) reported an improvement in response to skin-test antigens and taste acuity with doses of zinc around mg/day for month. however, in the majority of the studies, zinc supplements were used in the range proposed by the rda while attempting to not exceed the tolerable upper intake level (usually a range from to mg/day). prasad et al. ( ) and boukaniba et al. ( ) have found an increment of thymulin activity and improvements in response to skin-test antigens and taste acuity (zinc dose = mg/day for months); bodgen et al. ( ) have reported no benefit exclusively for increased lymphocyte mitogen proliferative response (zinc dose = mg/day for year); cakman et al. ( ) have found enhanced ifn-γ production by leukocytes (zinc dose = mg/day for days); fortes et al. ( ) report an increased number of cytotoxic t lymphocytes (zinc dose = mg/day for days). a long-term ( months) supplementation trial with two doses of zinc ( and mg/day) in elderly was recently conducted by the zenith study (hodkinson et al. ). zn supplementation of mg/day significantly lowered b-lymphocyte at month but not later. zn supplementation of mg/day significantly increased the ratio of cd to cd t lymphocytes at month . overall, these findings suggest zn supplementation has minimal longterm effects on immune status of healthy elderly persons. in the framework of a recent european study focused in zinc and immunosenescence, elderly subjects to be supplemented with zinc were chosen according to the presence of a pro-inflammatory genotype for il- and low plasma zinc (mocchegiani et al. ) . supplementation (with mg/day of zn-aspartate for +/À days) was carried out in old subjects (age > years) who presented stable low plasma zinc levels ( . μm at baseline and at year follow-up) and in c-carriers for il- - g/c with unstable plasma zinc ( . μm at baseline and > . μm at year follow-up). most evident effects of zinc supplementation on immune system comprised both corrective, but also noncorrective effects on known immunosenescence markers, including: ( ) an improved susceptibility of t cells to activation-induced cell death (aicd) (varin et al. ); ( ) an increased circulating levels of il- , mcp- associated with increased nk activity that were partly dependent on il- - g/c and + mt a snps (mariani et al. ) ; ( ) an increase of th and th cells in thawed samples and a slight reduction in th /th ratio in fresh whole blood lymphocytes ); ( ) a marked increase in both basal as well as stress-induced hsp levels in lymphocytes from healthy elderly donors with a higher impact on cd + cells (putics et al. ) ; and ( ) reduced spontaneous cytokine release in pbmcs and defects in termination of inflammatory activity ). in the same population, giacconi et al. ( ) studied the influence of zip gln/arg/leu (rs ) polymorphism on zinc homeostasis and inflammatory response following zinc supplementation. leu-(arg arg genotype) elderly showed higher inflammatory markers at baseline than carriers of other alleles that were reduced after zinc supplementation. later, the influence of + a/g mt a snp on plasma ages and ros production by pbmcs at baseline and after zinc supplementation was also studied ). + g+ carriers showed increased plasma ages and ros production in pbmcs at baseline with a significant interaction of genotype on zinc supplementation only in limited markers of intracellular zinc status. more recently, a randomized, double-blind, placebo-controlled study (with mg zn/day) has been performed on n = nursing home elderly (aged ! year;) with low serum zinc concentration (serum zinc < μg/dl) versus n = placebo ( mg zn/day) group. in this case, the treatment group showed an increase in serum zinc concentration that was associated with an increase in the number of t cells (barnett et al. ) . it is also noteworthy to mention the effects of a zinc fortified milk (designed to integrate mg zinc/day for months) on the cytokine production of pbmcs obtained from very old subjects (age > years). in unstimulated pbmcs from these subjects a significant percent change ( p < . or p < . ) of cytokine release is evident and is related to a good functioning of the cell-mediated immunity ( . % il- p and . % ifn-γ) coupled with increase of anti-inflammatory cytokines ( . % il- ) and decrease of pro-inflammatory cytokines ( . % il- α) ). thus, it seems evident from these studies that physiological dose of zinc for a long period or high doses of zinc for short periods might induce limited effects on immune response. although the body of proof of an impact of zinc on immunosenescence is consistent, there is limited knowledge on the long-term effects and outcome (e.g., protection from infection) of zinc supplementation in elderly. current evidence suggests that benefits on immune response and health outcome are likely to be obtained with doses around the rda in elderly with documented zinc deficiency. unfortunately, we do not have in this moment a reliable assessment of zinc status that can undoubtedly identify subjects with zinc deficiency. taking into account this lack of knowledge, it is recommended to educate elderly to consume an appropriate diet with foods that contain the necessary zinc requirements. the effect of zinc supplementation may be related to the levels of other cations such as cadmium, lead, calcium, iron, manganese, and copper. the beneficial effects of zinc on ameliorating toxicity of cadmium and lead, the accentuation of zinc deficiency by administration of calcium and phytate, and production of hypocupremia by excessive zinc intake in humans and animals are some examples of competition phenomena between these cations (hill ) . such a competition occurs because these ions have similar valence shell electronic structure and therefore could be antagonist to each other. for instance, the competition between zinc and iron (fe++) occurs at the level of cysteine-histidine ligands for the formation of iron or zinc "fingers" proteins (prasad ). if iron is in excess, a preferential binding of iron than zinc to the metal free-protein occurs. excess of zinc or zinc deficiency impairs dna-protein interactions of zinc fingers domains with their cognate dna target sites. in these conditions the production of some transcriptional factors like sp or tfiiia is altered (thiesen and bach ) . similar transcriptional alterations occur in excess or deficiency of copper (prasad ) . this reinforces the notion of the relevance of interactions between zinc and copper as well as with other metals in the immune efficiency (sandstead ) . in order to avoid interference with copper homeostasis, it has been proposed to not exceed - times the rda with zinc supplementation and to perform alternate cycles of supplementation not longer than - months (licastro et al. ; feillet-coudray et al. ). combination of zinc in complex micronutrient supplements appears to produce minimal and contrasting effects. health status was recently investigated in older french adults years after a period of daily nutritional-dose supplementation with antioxidant nutrients (assmann et al. ) . during - , participants received a daily combination of vitamin c ( mg), β-carotene ( mg), vitamin e ( mg), selenium ( μg), and zinc ( mg) or placebo. healthy aging was assessed in - with multiple criteria (e.g., absence of major chronic disease and good physical and cognitive functioning). supplementation was associated with a slightly greater healthy aging probability among men, but not among women or all. similarly, another study assessing a micronutrient mix ( mg vitamin e, mg vitamin c, mg beta-carotene, and mg zinc/day for weeks) on immune function parameters of a population of subject aged - years showed only an increase of delayed-type hypersensitivity (dth) responses (in particularly in the oldest subjects) without any change in responses to systemic tetanus and oral typhoid vaccination, phagocytosis, oxidative burst, lymphocyte proliferation, and lymphocyte subset distribution (wolvers et al. ) . importantly, vitamin and mineral supplements (including zinc) in older women were found to be associated with increased total mortality risk compared with nonusers in other studies (bjelakovic and gluud ; mursu et al. ) . these results reinforce the idea that nutritional guidelines instead of supplementation might be the preferred choice, while keeping interventions with micronutrients only for short periods in case of documented deficiencies. selenium is considered since long time ago an essential dietary element for the prevention of some diseases, including cancer and infections (schwarz ). in food, selenium derives from vegetables and animal products and in particular from the consumption of seafood, liver, and cereals. however, in vegetables and cereals the amount of selenium varies in soil in different countries and geographical regions (wasowicz et al. ) . indeed, selenium deficiency and related diseases have been well documented in geographic regions where the soil content is low, such as the chinese province of keshan (li et al. ) . from this region of china, in fact, was taken the name of the keshan disease, a pathology characterized by selenium deficiency and the presence of mutated strains of coxsackievirus . mammals can use both inorganic and organic selenium as a nutrient. most of the biological functions of selenium are attributed to selenoproteins, which contain selenocysteine residues responsible for their specific activity. selenoproteins are present in every cell type. the human selenoproteome consists of selenoproteins, mostly involved in antioxidant defense systems (kryukov et al. ) . glutathione peroxidases (gpxs), a family of the selenoproteins, protect cells against oxidative damage by catalyzing the reduction of hydrogen peroxide and other hydroperoxides (hall et al. ; brigelius-flohé ) . five seleniumdependent gpx isoforms exist in humans and four isoforms in mice. gpx is found in the cytosol of almost all cells and catalyzes the reduction of free hydroperoxides. gpx is expressed in the gastrointestinal tract and has a substrate specificity similar to gpx ; gpx is an extracellular enzyme found in plasma and reduces membrane-bound phospholipid hydroperoxides (brigelius-flohé ) . gpx is expressed in various tissues, and reduces phospholipid hydroperoxide and hydrogen peroxide using also thiols, such as -mercaptoethanol, cysteine, and homocysteine, other than gsh as reductant agents (roveri et al. ). the isoform gpx seems to be specifically expressed in embryonic tissues and olfactory epithelium (kryukov et al. ) . it also exists as a selenium-independent isoform, gpx , which is an epididymis isoenzyme present in mice and humans (hall et al. ), but its mrna was found to be not translated into functional protein in human epididymis (ghyselinck et al. ) . selenium is also involved in the thioredoxin system, a major enzymatic system that plays an important role in maintaining the redox state of the cell (holmgren ) . this system is highly complementary to the gsh system in protecting against oxidative stress (watson et al. ) . it comprises basically of thioredoxin (trx) and the selenoprotein thioredoxin reductase (tr) and uses the reducing power of nadph to act as a potent antioxidant system as well as a general disulfide redox system (rundlof and arner ) . mammalian tr maintains trx in a reduced state (holmgren ) and reduces a variety of other substrates including nondisulfides. the thioredoxin system protects the cell against oxidative stress through a variety of mechanisms. trx can directly quench single oxygen and scavenge hydroxyl radicals (das and das ) , or reduced trx can indirectly serve as an electron donor for trx peroxidase. in addition, human tr is directly capable to efficiently reduce lipid hydroperoxides, hydrogen peroxide, and organic hydroperoxides using nadph, especially in the presence of catalytic amount of selenocysteine, thus serving as an important alternative to the gpx pathway for the elimination of harmful hydroperoxides (björnstedt et al. ) . trx system is also critical for signal transduction (arner and holmgren ) and in the restoration of the reduced form of several antioxidant compounds, including ascorbic acid, lipoic acid, and ubiquinone (nordberg and arner ) . in this context, selenomethionine, a potent catalytic antioxidant in biological system and an aminoacid occurring in proteins in place of methionine (walter and roy ) , reacts more efficiently than methionine (padmaja et al. ) with oxidants forming methionine selenoxide which, in turn, is effectively and rapidly reduced to seleniomethionine by glutathione (assmann et al. ) . in contrast, methionine sulfoxide, which is produced by the oxidation of methionine in presence of oxidants, is not simply reduced by gsh, but it requires a specific enzymatic reaction catalyzed by methionine sulfoxide reductase (levine et al. ) . since selenomethionine can occur in proteins such as hemoglobin (beilstein and whanger ) , these residues may play a defensive role against peroxinitite. another selenoprotein, which reduces phospholipid hydroperoxides in the presence of thiols, is the selenoprotein p (sep) (burk et al. ) . sep is expressed in many tissues and represents the major plasma selenoprotein, which contains % of the total plasma selenium in the form of selenocysteine. sep protects endothelial cells against damage from peroxynitrite and transports selenium from the liver to peripheral tissues. last, but not the least in order of importance, is a class of selenoproteins (iodothyronine deiodinase enzymes), which catalyze the peripheral deiodination of thyroxin (t ) to , -triiodothyronine (t ). these enzymes play crucial roles in determining the circulating and intracellular levels of t and, consequently, the control of growth, development, differentiation, metabolism, and finally also the immune response (kohrle ; beckett and arthur ) . immunologically, the ability of selenoproteins to protect the host from oxidative stress is vitally important, as many host defense systems rely on the microbiocidal effects of macrophage-or neutrophil-generated free-radical species. oxidative species are generated through general metabolism, during the metabolism of xenobiotics and during exposure to ultraviolet radiation (uv) in sunlight. inflammation as a process to clear infection and damaged tissue also generates great oxidative stress. if antioxidant systems are not functioning correctly, host cells will be damaged (mckenzie et al. ) . taking into account that chronic inflammation and oxidative stress are features of aging (franceschi et al. ) , it is likely that se mediates protective effects through upregulating the antioxidant defenses and host immune responses. the influence of selenium on the immune function can be, in part, attributed to the same selenoproteins involved in the protection against oxidative damage and, in part, to still undefined biochemical pathways. the antioxidant gpxs have probably a role in protecting neutrophils from ros that are produced during inflammation arthur ) . selenium supplementation, in mice, increases the expression of subunits alpha (p ) and/or beta (p / ) of il- receptor (il- r) from activated lymphocytes and nk cells, thereby enhancing proliferation and clone expansion of cytotoxic precursor cells. in vitro, selenium enhances the release of tumor necrosis factor (tnf), il- , and il- from lps-stimulated macrophages (beckett et al. ). however, one of the most widely investigated associations between selenium and the immune system is the effect of the micronutrient on neutrophil function. neutrophils produce superoxide-derived radicals to take part in killing of microbes. this type of process is a balance between the production of sufficient radicals to kill invading organisms and the systems that protect the neutrophils themselves from the radicals. thus, although selenium deficiency does not seem to affect neutrophil number, certain aspects of their function are defective (turner and finch ) . neutrophils from selenium-deficient mice, rats, and cattle are able to ingest pathogens in vitro but are less able to kill them than are neutrophils from selenium-sufficient animals. this defective function has been associated with decreased cytosolic gpx (gpx ) activity in the neutrophils, which allows the free radicals that are produced in the respiratory burst to kill the neutrophils themselves . this is the reason why selenium deficiency may involve oxidative damage of biomolecules, such as lipids, lipoproteins, and dna, that is well known to play a role in many age-related diseases. anyway, selenium deficiency is a condition mainly attributed to geographical factors that include a low selenium content in the soil or to long-term parenteral nutrition rather than aging itself. selenium levels in blood have been studied in different age-related diseases such as cancer, cardiovascular disease, and immune dysfunctions. additionally, as for many other micronutrients, se inadequacy likely due to malnutrition or intestinal malabsorption may occur in older people (seiler ) . however, few data reported a marked selenium deficiency in old subjects. in the baseline results of the su.vi.m.ax study (involving women aged - and men aged - ), it was reported that fewer than % of the volunteers had a serum se status under . μm, which has been proposed as the threshold for se sub-deficiency and that age was associated with increased serum se concentrations in women (arnaud et al. ) . however, in the same study, large part of the population displayed a value of blood selenium below the range of levels ( . - . μm) proposed as optimal for gpx and selenoprotein p (combs ; thomson ). in the eva (etude du vieillissement artériel) study, a longitudinal study that explored the relationships between plasma selenium and mortality in an elderly population for over years, mortality rates were significantly higher in individuals with low selenium [increments = . μm; relative risk (rr) = . ; % ci = . - . ] (akbaraly et al. ) . when the underlying causes of death were considered, an association with low selenium and cancer-related mortality was found. the same authors suggest that plasma selenium could be an indicator of longevity in the pre-aging period of life. survival curves illustrate that the relationship between plasma selenium and mortality remained pertinent during the entire -year period (akbaraly et al. ) . however, the mechanism of this potential relationship is still unclear. other authors observed selenium deficiency in elderly people in relation to hypothyroidism (olivieri et al. ) . interestingly, most of human healthy centenarians seem to display selenium values equal or greater than the lowest values reported in normal elderly (savarino et al. ) . the relevance of selenium in the etiology of cardiovascular diseases has been also extensively studied. selenium metabolism is potentially involved in several protective biochemical pathways related to cardiovascular disease, such as reduction of ldl levels and lipoprotein oxidation, inhibition of foam-cell formation, and shift in prostaglandin production from prostacyclin to tromboxane (alissa et al. ) . however, wei et al. ( ) found no association between death for cardiovascular diseases and baseline selenium status in a cohort of adult individuals (mean age, years) with a mean serum concentration of . μm. finally, an intriguing point is the association between selenium deficiency, immune response, and increased incidence of infections in adults and elderly. in a prospective study performed in patients with systemic inflammatory response syndrome, a % decrease in plasma selenium concentrations was observed compared to reference range (forceville et al. ). moreover, low levels of selenium in these patients were associated to morbidity and mortality while survivors at follow-up displayed slightly increased selenium levels. the interrelationships between selenium deficiency, impaired immune response, and infections have been clearly shown in experimental animals. an inoculated avirulent virus in selenium-deficient animals turns into a virulent one due to genomic changes within the virus, provoking an impaired humoral immune defense (beck ). an enhanced oxidative stress, caused by selenium deficiency, is the likely reason of possible viral genetic changes (beck et al. ) and increased progression of viral infections with subsequent impaired immune defense (daniels ) . regarding the role played by selenium deficiency in viral infections it is also noteworthy to mention the: (i) the emergence of newly recognized human disease agent (coronavirus) that causes sars from guangdong province of china (lashley ) as well as from northern vietnam (reynolds et al. ) , where significant areas of overt selenium deficiency exist (xia et al. ) and (ii) the increased risk of enhanced virulence of influenza virus in elderly (ellis et al. ) associated with a possible selenium deficiency (seiler ) . therefore, selenium deficiency may be considered a risk factor for age-related diseases (cancer, cardiovascular diseases, and infections). such a risk is of relevance in elderly because accumulating data suggest that persistent infection with varicella-zoster virus (vzv) (arvin ) , epstein-barr virus (ebv) (stowe et al. ) , and particularly cmv (mcvoy and adler ) impacts upon the immune system in aging and may contribute to the immune risk phenotype (irp), which predicts remaining longevity in the very elderly (pawelec et al. ) . specific study on these aspects should be encouraged taking into account the possible relevant implications for public health. in vitro studies cellular studies with selenium provide evidence that, in analogy with zinc, it may have anticarcinogenic and antioxidant effects at low concentrations, whereas at concentrations higher than those necessary for nutrition, it can be genotoxic and carcinogenic (valdiglesias et al. ) . se toxicity "in vitro" depends primarily on se compound and dose but also on exposure time and cellular model. conversely to the reputation of an antioxidant and protective trace element, various "in vitro" studies found that se compounds can induce dna damage (biswas et al. ; machado et al. ) and produce oxidative stress (wycherly et al. ). an investigation on chromosome breaking activity of various se compounds demonstrated that all may induce this kind of damage with an efficacy that follows this order: selenious acid > sodium selenite > se dioxide > selenic acid > sodium selenite (nakamuro et al. ) . as a consequence, it is not surprising that selenocompounds (mainly sodium selenite but also semet, se dioxide, and methylseleninic acid) induce cell death in various mammalian cell lines (valdiglesias et al. ). an intensive investigation on apoptosis-inducing effects of organic selenium derivatives showed that breast carcinoma cells were highly sensitive to the organic selenium compounds, manifesting apoptosis at . μm of selenium while nontumorigenic mammary epithelial cells required concentration above μm. cell lines derived from hepatoma and neuroblastoma showed intermediate sensitivity and colon carcinoma cells were more resistant to selenium-induced apoptosis (jariwalla et al. ) . a role for se in dna repair has been also documented, likely as a consequence of its modulatory activity on nrf pathway both in tumor and normal tissues (zhang et al. ) . however, the concentrations of selenium compounds, in combination with the medium components and the biological physiology of the cell, affect the redox potential of the compounds that may switch from pro-oxidant to antioxidant (cemeli et al. ) . a recent paper investigated the ability of selenium supplementation in vitro (in the form of na seo nm or nm) to modify monocyte cell viability and ros production under condition of oxidative stress (induced by paraquat mm and s-nitroso-n-acetyl-dl-penicillamine mm) (leighton et al. ) . the experiments indicated that selenium supplementation, especially with the lower dose tested, was effective in improving cell viability and ros quenching ability of monocytes. a previous study addressing the influence of selenium supplementation "in vitro" on macrophages reported enhanced phagocytosis, degranulation, and production of superoxide anion after phorbol myristate (safir et al. ) . in general, it is not easy to give an interpretation of the results "in vitro," especially when addressed at generation of ros and immune function. indeed, ros are not only used to kill pathogens, but they have been recently recognized as important mediators of cell signaling and cell to cell communication in phagocytic and nonphagocytic immune cells . so, even if we know that se compounds in vitro may affect ros production and phagocyte function (huang et al. ) , the overall rationale for supplementation strategies can only derive from "in vivo" supplementation studies. furthermore, there is evidence that as in the case of zinc the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence (yona et al. ; malavolta et al. ; provinciali et al. ) . taking into account that senescent cells accumulate with aging and that cellular senescence is a phenomenon that displays antagonistic pleiotropic effects (campisi ) on physiological and pathological processes, it is likely that many changes observed with aging and attributed to changes in selenium status involve the process of cellular senescence and that their impact on overall health are rather difficult to be correctly predicted. multiple experimental studies in animal models have revealed that se-deficiency impairs immune response to infection, cancer, and other stimuli. for example, se deficiency was reported to reduce cd + t cell response in mice challenged with a peptide/adjuvant (hoffmann et al. ) , to increase tumor growth and burden in a breast cancer mouse model (chen et al. ) , to increase type i allergic response in a mouse model of cutaneous anaphylaxis (arakawa et al. ) , and to increase immunotoxicity of arsenic (rodríguez-sosa et al. ) . the il- and interferon-γ pathways appear to be among the pathway more responsive to dietary selenium intake in mice (tsuji et al. ) . although excessive releases of il- and interferon-γ have been associated with inflamaging, moderate increases and adequate response of these cytokines are known play a pivotal role in host defense through the ability to activate macrophage cell functions thus contributing to keep under control infectious diseases. decreased dietary selenium can change a normally avirulent b coxsackievirus (cbv / ) into a virulent virus (cbv / ) by inducing changes in viral genoma, especially in viral rna polymerase (duarte et al. ). the mutated virus can infect heart muscle and cause myocarditis with the likely development of dilated cardiomyopathy and death (beck and levander ; jun et al. ) . similar to the data reported for coxsackie virus, selenium intake and selenium status affected the host immune response, the mutation rate of the viral genome, and the pathology in mice infected with the human influenza a/bangkok/ / (h n ) virus (nelson et al. ) . however, the impact of selenium status on the outcome of the immune response appears to depend on the virulence of the applied influenza a virus strain. indeed, opposite results with an increased mortality of the selenium adequate group were observed using a the influenza a/puerto rico/ / virus, which is highly pathogenic in mice (li and beck ) . with regard to cancer, many studies have investigated the effects of selenium in carcinogen-exposed animals showing a reduction in tumor incidence and/or pre-neoplastic endpoints (reid et al. ) . various animal experiments have been conducted by combining experimental selenium deficiency with treatment with carcinogens, such as , -dimethylhydrazine (dmh) or dimethylbenz(a)anthracene (dmba), and comparing the results with animals fed with higher content of selenium in the diet. in general, se deficiency appears to affect dmh toxicity with, however, no inhibition of tumor development by nutritional se ( . ppm se) (pence and buddingh ) . three relevant papers report a greater development of carcinoma by dhm or dmba in various organs (colon and mammary gland) of rats under selenium deficiency in comparison with rats treated with ppm of se (jacobs ; liu and milner ; mcgarrity and peiffer ) . these findings further suggest the ability of dietary selenium to inhibit the in vivo metabolism of carcinogens dmba or dmh and, consequently, to inhibit the development of the tumor. selenium supplementation in old mice was also shown to counteract immunosenescence (roy et al. ) . in c bl/ jnia mice aged months, supplementation with selenium ( . ppm as sodium selenite for weeks) corrected age-related defect in lymphocyte proliferation likely as a consequence of an increased number of high-affinity il- receptors. unfortunately, few trials have been carried out up in elderly with a clear focus on immunosenescence. although selenium supplementation improved lymphocyte mitogen responsiveness of institutionalized elderly individuals (peretz et al. ) , it is difficult to conclude on a specific beneficial effect of selenium in immunosenescence. a finnish study adding selenium to fertilizer has shown only an increased selenium status in the general population (young, adult, old) (aro et al. ) without any clear beneficial effect on general health. a recent randomized, doubleblinded, placebo-controlled clinical trial was undertaken to test the effects of se supplementation on a variety of parameters of anti-flu immunity in healthy subjects aged - years (ivory et al. ) . the authors used six doses of se (in different form from to μg/day) for weeks in six groups of individuals with plasma se levels < ng/ml who received flu vaccine (at week ). in this trial, se supplementation resulted in both beneficial and detrimental effects on cellular immunity to flu that was affected by the form of se, supplemental dose and delivery matrix. in general, the dose-dependent increase in t cell proliferation, il- and il- secretion after in vivo flu challenge were contrasted by lower granzyme b content of cd cells or by inhibition of tnf-α synthesis. another recent study was conducted to evaluate the effects of selenium supplementation ( μg selenium supplements for weeks) on metabolic profiles, biomarkers of inflammation, and oxidative stress of patients with type diabetes and coronary artery disease aged - years (farrokhian et al. ) . supplemented patients displayed a significant decrease in insulin, homeostasis model of assessment-insulin resistance, homeostatic model assessment-beta cell function, serum hs-crp, and a significant increase in quantitative insulin sensitivity check index score and plasma total antioxidant capacity concentrations. the positive impact on these biomarkers is, unfortunately, not complemented by a long-term follow-up. however, reduced cardiovascular mortality at -years follow-up was observed in another supplementation trial performed on elderly with selenium and coenzyme q (alehagen et al. ) . in agreement with the protective role of selenium a sweden study performed in elderly subjects showed that persons with serum se in the lowest quartile had % and % increased risk for all-cause and cardiovascular mortality, respectively (alehagen et al. ). this confirms the reduced incidence of cardiovascular diseases observed in previous studies after supplementation with selenium combined in multivitamins supplements (czernichow et al. ; shenoy et al. ) . there is also evidence that a number of hiv-positive patients suffer from deficiencies in vitamins including selenium (coodley ) . enhanced gpx and gsh activity (delmas-beauvieux et al. ) were observed in hiv patients receiving oral selenium supplementation. this finding suggests that gpx and gsh activity may represent natural inhibitors of (aids) viruses and that selenium supplementation may play a role in prevention antiviral strategies (rayman ) . of interest, supplementation with multivitamins and trace elements, including se, during treatment of pulmonary tuberculosis reduced mortality in subjects co-infected with hiv (range et al. ) . large part of the other studies involving selenium supplementation in humans have been conducted in adult subjects with the aim to prevent or counteract various type of cancer. past studies showed that supplementation with μg/day of organic selenium in randomized subjects showed preventive effects in the incidence and the mortality from various types of cancer (prostate, colorectal, and lung cancer) (clark et al. ) . another large supplementation trial with lower amount of selenium ( μg) performed in lixian (north china) showed a small but significant reduction in total and cancer mortality was observed in subjects receiving selenium supplement (blot et al. ) . the impact of se supplementation was better in women than men and, interestingly, the effects were more pronounced in persons under the age of years compared to individuals older than years. moreover, supplementation with mg/day of sodium selenite during therapy of patients with squamous cell carcinoma of the head and neck resulted in a significantly enhanced cell-mediated immune responsiveness (better response of lymphocytes to stimulation with mitogen, generation of cytotoxic lymphocytes, and destruction of tumor cells) (kiremidjian-schumacher et al. ) . considering these results, it might be assumed that younger persons might be more amenable to a protective effect of selenium supplementation. however, a recent large selenium and vitamin e cancer prevention trial (select) carried out in north america has reported unexpected and very concerning findings, especially the increased the risk of high-grade prostate cancer among men with high selenium status (kristal et al. ) . perhaps, the most conclusive data regarding the story of selenium and cancer can be retrieved from an excellent systematic review that included prospective observational studies as well as randomized clinical trials (vinceti et al. ) . the authors of this systematic review concluded that the inverse association between selenium exposure and the risk of some types of cancer cannot be taken as evidence of a causal relation, and that all these studies have many limitations, including issues with the form and the assessment of selenium, heterogeneity, confounding and other biases. moreover, randomized clinical trials reported inverse, null, and direct associations have been reported for some cancer types which overall yielded inconsistent results with even harmful effects of selenium exposure. hence, to date, no convincing evidence suggests that selenium supplements can prevent cancer in humans. dietary zinc and selenium are important nutritional factors for the immune response in protecting against the appearance of age-related diseases. there is a number of biochemical processes in which selenium and zinc interact. selenium compounds that have the capacity to form selenol(ate)s catalytically couple with the glutathione/ glutathione disulfide and metallothionein/thionein redox pairs to either release or bind zinc. (maret ) . when mt are oxidized by glutathion disulfide (gssg) or other disulfides free zinc is released in the cytosol. however, the efficiency of this chemical reaction seems very low even at high concentrations of gssg in the absence of selenium. in contrast, the release of zinc from mt may occur very rapidly in presence of selenium compounds have the capacity to form selenol(ate)s (maret ) . the mechanism of the reaction was suggested to proceed through an activated selenenyl sulfide r-se-s-g intermediate which, in turn, oxidizes the zinc-thiolate cluster of mt to form r-se-s-mt with the concomitant release of zinc during the oxidation (chen and maret ) . the selenol group is subsequently released by the attack of a nearby thiol group of mt that convert r-se-s-mt into thionein generating a catalytic cycle of oxidative zinc release from mt. other oxidized selenium compounds, such as selenoxide and selenic acid, may be directly reduced by mt through the formation of a r-se-s-mt intermediate and the concomitant release of zinc, followed by the formation of an inter-or intramolecular disulfide bond (jacob et al. ; chen and maret ; klotz et al. ) . selenium compounds also catalyze the release of zinc from mt in peroxidation and thiol/disulfide-interchange reactions. in presence of t-butylhydroperoxide, gpx catalyzes the oxidation of mt with subsequent zinc release, suggesting that mt may serve as reducing agents for gpx (or at least some gpx isoforms) in alternative to gsh (jacob et al. ) . these results suggest that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in anti-inflammatory and anticarcinogenic agents. hence, the assessment of intracellular labile zinc and selenoproteome during a challenge with oxidative compounds might be useful to understand the physiology of successful aging. while there are no studies specifically addressing these aspects in humans, it is of interest to consider the results of a recent study that examined elements in the brain, heart, kidney, and liver of mammalian species, and studied their correlation with body mass and longevity (ma et al. ) . zn levels in liver and kidney showed strong, positive correlations with species maximum lifespan and maximum lifespan residual whereas liver se was the only element correlating negatively with all measures of longevity, although the correlations were relatively weak. it is interesting to relate this study with the finding that the naked mole rat (a rodent model of delayed aging with a life span > years) is characterized by a reduced utilization of selenium due to a specific defect in gpx expression (kasaikina et al. ) . a possible interpretation of these results is that there is a delicate balance between the beneficial aspects of se, its toxicity, and other systems that support maintenance functions. supplementation studies with zinc and selenium in humans are also poor, especially with a focus on immunosenescence. girodon et al. ( ) determined the effects of a long-term (for years) daily supplementation with zinc ( mg) plus selenium ( μg) on immunity and the incidence of infections in a large number (n. ) of institutionalized elderly people (> years). the main results of the study were: ( ) selenium-deficient patients decreased from about % to - % in the selenium supplemented group after months of supplementation with respect to placebo group; ( ) antibody titres after influenza vaccine were higher in groups that receive trace elements; and ( ) trace element-supplemented patients were those who remained most free of respiratory tract infections than placebo group. these findings suggest that low dose supplementation of zinc and selenium provides significant improvement in elderly patients by increasing the humoral response after vaccination and decreased influenza compliances (respiratory tract infections) with thus a possible impact on the maintenance of health conditions during aging. however, conversely to the conventional belief and claims that supplementation with zn and se may improve health conditions there are important studies that observed lack of clear effects. the large su.vi.max trial conducted with a supplement of antioxidant including zinc and selenium reported no effects on health-related quality of life after months of supplementation in french adults (briançon et al. ). in addition, supplementation with low or medium doses of zinc and selenium provided no benefits, neither were able to correct low zinc or selenium status in hemodialysis patients (tonelli et al. ) . in conclusion, conversely to the past belief that zinc and selenium supplements could be used as elixir of long life, recent epidemiologic and laboratory studies are changing our perception of the biological effects of these essential trace elements. while several studies suggest that correcting zinc and selenium deficiency in aging provide a beneficial impact on immunosenescence, it is evident that controversial finding exists on the "real" necessity of micronutrient supplementation (dangour et al. ) . the critical step of supplementation strategies with zinc and selenium still remains the diagnosis of zinc and selenium deficiency, as there are no validated methods to assess their status in aging. however, in the case of documented deficiency, it seems to make sense to provide selenium supplements associated with zinc and even with additional supplements. indeed, micronutrient deficiencies are usually not isolated and some biochemical mechanisms involved in the action of selenium are under the control of zinc ion bioavailability. in other words, the attempt to correct selenium deficiency alone may result in lack of beneficial effects due to the presence of zinc or other micro-and macronutrient deficiency. many points still require further investigations, first of all, the possible involvement of mt, zinc, and selenium in antagonistic pleiotropic mechanisms that regulate mammalian life span. one of these mechanisms is cellular senescence, which has gained considerable attention in these last years as a useful anti-aging target. useful tools are available, such as no donors and zinc fluorescent probes (zinpyr- and fluozin- ) to help researcher in the quantitative assessment of labile zinc and on the functional role of mt haase et al. ) in senescent cells. these tools applied in supplementation trials performed in animal models that allow to visualize, sort, and selectively eliminate senescent cells (demaria et al. ) could help to clarify these mechanisms. additional insight could be provided by the investigation of serum trace elements associated with mt induction in pbmc obtained from centenarian's offspring in the framework of markage project (bürkle et al. ) . the results may help to understand the physiological mechanisms involved in healthy aging and may help to understand new strategies for nutritional or pharmacological intervention aimed at increased longevity in healthy conditions. physiological responses of extrathymic t cells in the liver selenium and mortality in the elderly: results from the eva study reduced cardiovascular mortality years after supplementation with selenium and coenzyme q for four years: follow-up results of a prospective randomized double-blind placebo-controlled trial in elderly citizens relatively high mortality risk in elderly swedish subjects with low selenium status the controversy surrounding selenium and cardiovascular disease: a review of the evidence low micronutrient intake may accelerate the degenerative diseases of aging through allocation of scarce micronutrients by triage investigation of lymphocyte gene expression for use as biomarkers for zinc status in humans supplementary seleno-l-methionine suppresses active cutaneous anaphylaxis reaction serum selenium determinants in french adults: the su.vi.m.ax study physiological functions of thioredoxin and thioredoxin reductase effect of supplementation of fertilizers on human selenium status in finland metallothionein g and zinc sensitize human colorectal cancer cells to chemotherapy selenium supplementation: does soil supplementation help and why? selenium in the immune system varicella-zoster virus reduction of methionine selenoxide to selenomethionine by glutathione healthy aging years after a period of daily supplementation with antioxidant nutrients: a post hoc analysis of the french randomized trial su.vi.max the epidemiology of global micronutrient deficiencies clearance of p ink a-positive senescent cells delays ageing-associated disorders naturally occurring p (ink a)-positive cells shorten healthy lifespan effect of zinc supplementation on serum zinc concentration and t cell proliferation in nursing home elderly: a randomized, double-blind, placebo-controlled trial selenium and host defence towards viruses host nutritional status and its effect on a viral pathogen selenium deficiency and viral infection selenium and endocrine systems diet and human immune function deposition of dietary organic and inorganic selenium in rat erythrocyte proteins the galvanization of biology: a growing appreciation for the roles of zinc micronutrient malnutrition, infection, and immunity: an overview chromosome damage induced by selenium salts in human peripheral lymphocytes vitamin and mineral supplement use in relation to all-cause mortality in the iowa women's health study human thioredoxin reductase directly reduces lipid hydroperoxides by nadph and selenocystine strongly stimulates the reaction via catalytically generated selenols the linxian trials: mortality rates by vitamin-mineral intervention group the role of nitric oxide in innate immunity effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly a physiological amount of zinc supplementation: effects on nutritional, lipid, and thymic status in an elderly population long-term antioxidant supplementation has no effect on health-related quality of life: the randomized, double-blind, placebo-controlled, primary prevention su.vi.max trial tissue-specific functions of individual glutathione peroxidases selenoprotein metabolism and function: evidence for more than one function for selenoprotein p pathophysiology of ageing, longevity and age related diseases mark-age biomarkers of ageing nutrient requirements and optimisation of intakes metallothionein in radiation exposure: its induction and protective role zinc supplementation reconstitutes the production of interferon-alpha by leukocytes from elderly persons cell biology: the beginning of the end genotoxic and antigenotoxic properties of selenium compounds in the in vitro micronucleus assay with human whole blood lymphocytes and tk lymphoblastoid cells clearance of senescent cells by abt rejuvenates aged hematopoietic stem cells in mice catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compounds dietary selenium supplementation modifies breast tumor growth and metastasis nutrition and health promotion in older adults polymorphisms in mt a gene coding region are associated with longevity in italian central female population decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial zinc deficiency-induced cell death zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins tumour biology: senescence in premalignant tumours selenium in global food systems does promoter methylation of the slc a (znt ) zinc transporter gene contribute to the ageing-related decline in zinc status? update on vitamins, minerals, and the carotenoids effects of zinc-fortified drinking skim milk (as functional food) on cytokine release and thymic hormone activity in very old persons: a pilot study interleukin regulates secretion of zinc-thymulin by human thymic epithelial cells and its action on t-lymphocyte proliferation and nuclear protein kinase c integrative aspects of zinc transporters effect of supplementation with antioxidants upon long-term risk of hypertension in the su.vi.max study: association with plasma antioxidant levels micronutrient supplementation in later life: limited evidence for benefit selenium: does selenium status have health outcomes beyond overt deficiency? restoration of the thymus in aging mice by in vivo zinc supplementation thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions metallothionein expression in animals: a physiological perspective on function the enzymatic antioxidant system in blood and glutathione status in human immunodeficiency virus (hiv)-infected patients: effects of supplementation with selenium or beta-carotene an essential role for senescent cells in optimal wound healing through secretion of pdgf-aa is vitamin supplementation appropriate in the healthy old? induction of cytokines by zinc ions in human peripheral blood mononuclear cells and separated monocytes rna virus quasispecies: significance for viral disease and epidemiology beneficial effects of oral zinc supplementation on the immune response of old people the role of metallothioneins in carcinogenesis metallothioneins and cancer zinc transporters and the cellular trafficking of zinc influenza-and respiratory syncytial virus-associated morbidity and mortality in the nursing home population thymic hormone deficiency in normal ageing and down's syndrome: is there a primary failure of the thymus? selenium supplementation affects insulin resistance and serum hs-crp in patients with type diabetes and coronary heart disease effect of zinc supplementation on in vitro copper-induced oxidation of low-density lipoproteins in healthy french subjects aged - years: the zenith study selenium, systemic immune response syndrome, sepsis, and outcome in critically ill patients zinc supplementation and plasma lipid peroxides in an elderly population the effect of zinc and vitamin a supplementation on immune response in an older population roles for cell death in zinc deficiency the many roles of apoptosis in immunity as modified by aging and nutritional status inflamm-aging. an evolutionary perspective on immunosenescence aging, frailty and age-related diseases structural organization and regulation of the gene for the androgen-dependent glutathione peroxidase-like protein specific to the mouse epididymis novel- a/g mt a polymorphism in old patients with type diabetes and atherosclerosis: relationship with inflammation (il- ) and zinc comparison of intracellular zinc signals in nonadherent lymphocytes from young-adult and elderly donors: role of zinc transporters (zip family) and proinflammatory cytokines influence of + a/g mt a polymorphism on advanced glycation end-products (ages) in elderly: effect of zinc supplementation effect of zip gln/arg/leu (rs ) polymorphism on zinc homeostasis and inflammatory response following zinc supplementation impact of trace elements and vitamin supplementation on immunity and infections in institutionalized elderly patients: a randomized controlled trial. min. vit. aox. geriatric network the immune system and the impact of zinc during aging zinc signals and immune function flow cytometric measurement of labile zinc in peripheral blood mononuclear cells metallothionein-null mice are more susceptible than wild-type mice to chronic cdcl( )-induced bone injury zinc binding and redox control of p structure and function the majority of human glutathione peroxidase type (gpx ) transcripts are incorrectly spliced: implications for the role of gpx in the male reproductive tract mineral interrelationships. in: prasad as (ed) trace elements, human health and disease low intracellular zinc induces oxidative dna damage, disrupts p nfkappa b, and ap dna binding, and affects dna repair in a rat glioma cell line zinc deficiency induces oxidative dna damage and increases p expression in human lung fibroblasts effect of zinc supplementation on the immune status of healthy older individuals aged - years: the zenith study dietary selenium modulates activation and differentiation of cd + t cells in mice through a mechanism involving cellular free thiols the role of selenium in inflammation and immunity: from molecular mechanisms to therapeutic opportunities zinc-altered immune function selenium supplementation has beneficial and detrimental effects on immunity to influenza vaccine in older adults circulating thymic hormone levels in zinc deficiency selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes selenium inhibition of , -dimethylhydrazine-induced colon carcinogenesis differential sensitivity of various human tumourderived cell types to apoptosis by organic derivatives of selenium selenium deficiency contributes to the chronic myocarditis in coxsackievirus-infected mice deficiency of metallothionein- and - genes shortens the lifespan of the /sv mouse strain biochemistry of metallothionein zinc supplementation in the elderly reduces spontaneous inflammatory cytokine release and restores t cell functions consumption of energy-dense, nutrient-poor foods by adult americans: nutritional and health implications. the third national health and nutrition examination survey, - reduced utilization of selenium by naked mole rats due to a specific defect in gpx expression metallothionein i and ii protect against zinc deficiency and zinc toxicity in mice selenium and immunocompetence in patients with head and neck cancer role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress the deiodinase family: selenoenzymes regulating thyroid hormone availability and action enhanced apoptosis in metallothionein null cells baseline selenium status and effects of selenium and vitamin e supplementation on prostate cancer risk characterization of mammalian selenoproteomes zinc levels modulate lifespan through multiple longevity pathways in caenorhabditis elegans emerging infectious diseases at the beginning of the st century can selenium supplementation modify oxidative stress in-vitro? a role for selenium supplementation in the prevention of cardiovascular disease nutritional factors and immunological ageing methionine residues as endogenous antioxidants in proteins selenium deficiency induced an altered immune response and increased survival following influenza a/puerto rico/ / infection keshan disease: an endemic cardiomyopathy in china detection of enteroviral rna in paraffin-embedded myocardial tissue from patients with keshan by nested pcr oral zinc supplementation in down's syndrome subjects decreased infections and normalized some humoral and cellular immune parameters age, dietary selenium and quantity of , -dimethylbenz(a)anthracene influence the in vivo occurrence of rat mammary dna adducts interleukin- regulates the zinc transporter zip in liver and contributes to the hypozincemia of the acute-phase response the missing zinc: p misfolding and cancer organization of the mammalian ionome according to organ origin, lineage specialization, and longevity -ditrifluoromethyldiphenyl diselenide: a new organoselenium compound with interesting antigenotoxic and antimutagenic activities selected vitamins and trace elements support immune function by strengthening epithelial barriers and cellular and humoral immune responses single and three-color flow cytometry assay for intracellular zinc ion availability in human lymphocytes with zinpyr- and double immunofluorescence: relationship with metallothioneins metallothionein downregulation in very old age: a phenomenon associated with cellular senescence? survival study of metallothionein- transgenic mice and respective controls (c bl/ j): influence of a zinc-enriched environment modulators of cellular senescence: mechanisms, promises, and challenges from in vitro studies with dietary bioactive compounds serum copper to zinc ratio: relationship with aging and health status metallothioneins, longevity and cancer: comment on "deficiency of metallothionein- and - genes shortens the lifespan of the /sv mouse strain zinc pyrithione induces apoptosis and increases expression of bim cellular zinc and redox states converge in the metallothionein/thionein pair thiolate ligands in metallothionein confer redox activity on zinc clusters effect of zinc supplementation on plasma il- and mcp- production and nk cell function in healthy elderly: interactive influence of + mt a and À il- polymorphic alleles selenium and difluoromethylornithine additively inhibit dmh-induced distal colon tumor formation in rats fed a fiber-free diet selenium: an essential element for immune function immunologic evidence for frequent age-related cytomegalovirus reactivation in seropositive immunocompetent individuals low zinc and selenium concentrations in sepsis are associated with oxidative damage and inflammation nutrition interventions in aging and age-associated disease zinc in human biology reversibility of the thymic involution and of age-related peripheral immune dysfunctions by zinc supplementation in old mice zinc, t-cell pathways, aging: role of metallothioneins zinc and immunoresistance to infection in aging: new biological tools zinc, metallothioneins, immune responses, survival and ageing mtmrna gene expression, via il- and glucocorticoids, as potential genetic marker of immunosenescence: lessons from very old mice and humans metallothioneins (i+ii) and thyroid-thymus axis efficiency in old mice: role of corticosterone and zinc supply metallothioneins/parp- /il- interplay on natural killer cell activity in elderly: parallelism with nonagenarians and old infected humans. effect of zinc supply the variations during the circadian cycle of liver cd d-unrestricted nk . +tcr gamma/delta+ cells lead to successful ageing. role of metallothionein/il- /gp /parp- interplay in very old mice zinc, metallothioneins, and longevity-effect of zinc supplementation: zincage study zinc deficiency and il- - g/c polymorphism in old people from different european countries: effect of zinc supplementation. zincage study metallothioneins, ageing and cellular senescence: a future therapeutic target dietary supplements and mortality rate in older women: the iowa women's health study studies on selenium-related compounds. v. cytogenetic effect and reactivity with dna host nutritional selenium status as a driving force for influenza virus mutations reactive oxygen species, antioxidants, and the mammalian thioredoxin system selenium, zinc, and thyroid hormones in healthy subjects: low t /t ratio in the elderly is related to impaired selenium status effect of zinc ions on apoptosis in pbmcs from healthy aged subjects rapid oxidation of dl-selenomethionine by peroxynitrite human immunosenescence: is it infectious? inflammation, ageing and chronic disease the role of metallothionein in oncogenesis and cancer prognosis effect of dietary selenium deficiency on incidence and size of , -dimethylhydrazine-induced colon tumours in rats lymphocyte response is enhanced by supplementation of elderly subjects with selenium-enriched yeast zinc deficiency in elderly patients flow cytometric analysis of cd /tcr complex, zinc, and glucocorticoid-mediated regulation of apoptosis and cell cycle distribution in thymocytes from old mice chapter -nutritional modulators of cellular senescence in vitro zinc supplementation boosts the stress response in the elderly: hsp status is linked to zinc availability in peripheral lymphocytes the effect of multi-vitamin/mineral supplementation on mortality during treatment of pulmonary tuberculosis: a randomised two-by-two factorial trial in mwanza, tanzania molecular and cellular basis of aging the importance of selenium to human health selenium supplementation and lung cancer incidence: an update of the nutritional prevention of cancer trial factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi zinc and the immune system zinc homeostasis and immunity effect of selenomethionine supplementation in food on the excretion and toxicity of arsenic exposure in female mice chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice purification and characterization of phospholipid hydroperoxide glutathione peroxidase from rat testis mitochondrial membranes supplementation with selenium restores age-related decline in immune cell function regulation of the mammalian selenoprotein thioredoxin reductase in relation to cellular phenotype, growth, and signaling events the effect of selenium on immune functions of j . cells requirements and toxicity of essential trace elements, illustrated by zinc and copper zinc nutriture in the elderly in relation to taste acuity, immune response, and wound healing recent studies on metallothionein: protection against toxicity of heavy metals and oxygen free radicals enhanced renal toxicity by inorganic mercury in metallothionein-null mice serum concentrations of zinc and selenium in elderly people: results in healthy nonagenarians/centenarians thymic hormone-containing cells. v. immunohistological detection of metallothionein within the cells bearing thymulin (a zinc-containing hormone) in human and mouse thymuses essentiality and metabolic functions of selenium clinical pictures of malnutrition in ill elderly subjects low serum micronutrient concentrations predict frailty among older women living in the community the effect of zinc sulphate and zinc carnosine on genome stability and cytotoxicity in the wil -ns human lymphoblastoid cell line rationale and design for the cardiovit study (cardiovit, atherosclerotic vascular disease and hyperhomocysteinemia: an epidemiological study in indians, additionally evaluating the effect of oral vitamin supplementation) chronic herpesvirus reactivation occurs in aging zinc inhibits turnover of labile mrnas in intact cells transition metals modulate dna-protein interactions of sp zinc finger domains with its cognate target site assessment of requirements for selenium and adequacy of selenium status: a review psychological stress induced zinc accumulation and up-regulation of zip and metallothionein in rat liver trace element supplementation in hemodialysis patients: a randomized controlled trial dietary selenium levels affect selenoprotein expression and support the interferon-γ and il- immune response pathways in mice selenium and the immune response stable isotope studies of zinc absorption and retention in young and elderly men th and th cell polarization increases with aging and is modulated by zinc supplementation in vitro evaluation of selenium genotoxic, cytotoxic, and protective effects: a review the biochemical basis of zinc physiology in vitro and in vivo effects of zinc on cytokine signalling in human t cells selenium for preventing cancer zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses tnf-alpha and il- beta production in monocytes by elevation of guanosine , -cyclic monophosphate selenomethionine, a potential catalytic antioxidant in biological systems selenium status of low-selenium area residents: polish experience thioredoxin and its role in toxicology prospective study of serum selenium concentrations and esophageal and gastric cardia cancer, heart disease, stroke, and total death human metallothionein genes: structure of the functional locus at q endothelial response to stress from exogenous zn + resembles that of no-mediated nitrosative stress, and is protected by mt- overexpression effect of a mixture of micronutrients, but not of bovine colostrum concentrate, on immune function parameters in healthy volunteers: a randomized placebo-controlled study zinc supplementation increases zinc status and thymopoiesis in aged mice increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation high dietary intake of sodium selenite induces oxidative dna damage in rat liver effectiveness of selenium supplements in a low-selenium area of china metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress metallothionein mediates leukocyte chemotaxis interplay between selenium levels, selenoprotein expression, and replicative senescence in wi- human fibroblasts metallothionein-induced suppression of cytotoxic t lymphocyte function: an important immunoregulatory control nitric oxide selectively releases metals from the aminoterminal domain of metallothioneins: potential role at inflammatory sites attenuating the toxicity of cisplatin by using selenosulfate with reduced risk of selenium toxicity as compared with selenite ros and ros-mediated cellular signaling acknowledgments the study has been partially funded through the revenues of the " per mille" donations received by inrca through the italian ministry of health. the authors declare that there are no conflicts of interest. key: cord- -opojt e authors: dimarco, ross f. title: postoperative care of the cardiac surgical patient date: journal: surgical intensive care medicine doi: . / - - - - _ sha: doc_id: cord_uid: opojt e the subspecialty of interventional cardiology began in . since then, the discipline of interventional cardiology has matured rapidly, particularly with regards to ischemic heart disease. as a result, more patients are undergoing percutaneous catheter interventional therapy for ischemic heart disease and fewer patients are undergoing surgical myocardial revascularization. those patients referred for surgical revascularization are generally older and have more complex problems. furthermore, as the population ages more patients are referred to surgery for valvular heart disease. the result of these changes is a population of surgical patients older and sicker than previously treated. the open-heart patient requires specialized care because physiologic systems are disrupted by cardiopulmonary bypass (cpb). cpb results in a generalized inflammatory response caused by blood contact with the synthetic surfaces of the bypass circuit. the interface between blood elements and the surfaces of the circuit causes a generalized inflammatory response. this inflammatory response results in a series of complex reactions that activate the complement, clotting, and fibrinolytic cascades causing bleeding, microemboli, fluid retention, and an altered hormonal response. [ ] [ ] [ ] [ ] cpb is a nonspecific activator of the inflammatory system. after the discontinuation of cpb, generalized complement activation occurs with elevations of c a and c a anaphylatoxins. the activation of these anaphylatoxins can result in pulmonary sequestration of leukocytes , and the production of superoxides. there then occurs further leukocyte activation and the generation of leukotactic factors that further increase the local inflammatory response. , also, vasoactive amines from platelets are liberated in response to cpb or possibly from protamine administration, which can result in pulmonary hypertension and systemic hypotension. yet another result of the complement activation is an increase in vascular permeability that may predispose the patient to a capillary-leak syndrome with fluid sequestration in the third space, particularly the lung. from a clinical perspective, the generalized inflammatory response results in postoperative pulmonary dysfunction, renal dysfunction, and a resetting of the hypothalamic thermoregulatory center. , the inflammatory response caused by cpb also has direct negative cardiac effects. the inflammatory state caused by cpb involves platelet-endothelial cell interactions and vasospastic responses resulting in low-flow states in the coronary circulation. the anaphylatoxin c a is a potent molecule that is spasmogenic and has leukocyte-activating properties that cause degranulation and release of toxic oxygen free radicals. the complement-exposed leukocytes are attracted to adhere to the vascular endothelium and to aggregate, resulting in margination in blood vessels and leukoembolization. these inflammatory cells mediate injury by increasing their production and releasing oxygen free radicals or proteolytic enzymes. at its worse at - h after cpb. , recovery of ventricular function begins in - h and full recovery usually occurs by - h. the systemic vascular resistance rises as ventricular function worsens. this is a compensatory mechanism in an effort to maintain systemic blood pressure and perfusion in the face of depressed ventricular contractility. the oxygen free radicals and the proteolytic enzymes released by the neutrophils also damage endothelial cells increasing capillary permeability resulting in capillary leak during this period. the capillary leak may last from to days and is related to the duration of cpb. hypothermia has multiple adverse effects on the postoperative open-heart patient. regarding the circulatory status, it predisposes cardiac dysrhythmias, increases svr, precipitates shivering and impairs coagulation. it also indirectly decreases cardiac output by increasing vasoconstriction and causing bradycardia. as a consequence of the inflammatory state after cpb, the postoperative open-heart patient is in a unique physiologic state where rules applicable to other physiologic situations may not apply, and a failure to recognize this concept results in management errors. concerns about the short-and long-term effects of cpb has generated the recent concept of off-pump coronary artery bypass surgery. while there seems to be growing evidence that this off-pump approach to the surgical management of ischemic heart disease is advantageous, there does remain some debate. despite the movement toward avoidance of cpb in selected patients with ischemic heart disease, the majority of these patients as well as virtually all patients with valvular heart disease are operated on using cpb. the cpb circuit is not the only factor responsible for this altered physiologic state. the time of ischemia and reperfusion, hypothermia, hypotension with nonpulsatile flow, altered coagulation, and the administration of blood and products are other factors contributing to the altered postoperative physiologic state. management of the postoperative open-heart patient initial management the patient after open-heart surgery presents with multiple, rapidly changing clinical problems. initially, these patients are unstable and their clinical status is extremely fluid and dynamic. caring for the postoperative open-heart patient requires bedside presence and the knowledge of general fundamental concepts of patient care as well as concepts specific to this set of patients. the initial management of these patients as they return from the operating room is critical, for it may well set the tone for the rest of the recovery period. clinical errors at this time can have farreaching implications. the initial management should begin even before the patient arrives in the intensive care unit (icu). it is vital to review the chart noting indications for surgery, preoperative hemodynamic data, comorbid conditions, medications, and allergies. upon the patient's arrival in the icu, perform a careful systematic assessment of the patient. begin the assessment by speaking directly to the surgical and anesthesia team. ascertain what procedure was done in the operating room and inquire as to any intraoperative events that might impact the patient's postoperative course. then, physically examine the patient as part of this initial evaluation. during the initial assessment, avoid focusing on any one issue and attempt to get a global picture of the patient's clinical status. at this time, the patient is completely dependent on support systems, and dysfunction of any one of these can lead to disaster. the following points must be observed: heart rate and rhythm, blood pressure, temperature, right and left heart filling pressures, hemodynamic profile, pharmacologic support, ventilator status, chest drainage, neurologic status, laboratory results, ekg, and chest x-ray. a thorough knowledge of the specific monitoring and drug delivery lines is imperative as is knowledge of where the drains are placed. once the initial assessment is complete, specific issues can be identified, prioritized, and addressed. the primary objective in managing the postoperative openheart patient is achieving adequate hemodynamic performance by optimizing myocardial oxygen supply and demand. optimal tissue oxygenation is essential to avoid organ dysfunction and can be determined by calculating oxygen delivery and oxygen demand. oxygen delivery is a function of oxygencarrying capacity and cardiac output. oxygen demand is a function of oxygen consumption. the most important concept in the optimization of myocardial oxygen supply and demand, and tissue oxygenation is an adequate cardiac output. cardiac output is expressed as liters per minute and cardiac index as liters per minute per square meter. normal cardiac index is between . and . l/min/m . an uncomplicated recovery from cardiac surgery can be anticipated when the cardiac index is maintained greater than . and . l/min/m . , cardiac output is a function of stroke volume and heart rate, where cardiac output (co) is the product of heart rate (hr) and stroke volume (sv). an optimal heart rate is usually between and beats per minute. this rate allows for optimal filling of the heart at an economic level of myocardial oxygen consumption. stroke volume is determined by preload, afterload, and contractility, and can be influenced by cardiac rhythm. stroke volume is the end-diastolic volume minus the end-systolic volume and in normal states is ml preload refers to left ventricular end-diastolic sarcomere fiber length and is a function of end-diastolic ventricular volume (lvedv). it can be directly measured by echocardiography and is indirectly measured by left heart filling pressures; i.e., pulmonary artery diastolic pressure (padp), pulmonary capillary wedge pressure (pcwp), and left atrial pressure (lap). the former are all reflections of the left ventricular end diastolic pressure (lvedp). the compliance of the left ventricle is determined by the relationship between filling volumes and pressures, or lvedv/lvedp. stiff ventricles have low compliances and require higher filling pressures to achieve adequate volumes. this scenario is almost universal after cardiac surgery. afterload is a reference to left ventricular wall tension during systole. it is determined by intraventricular systolic pressure and ventricular wall thickness. since there is minimal change in left ventricular wall thickness during cardiac surgery, intraventricular systolic pressure has the most impact on afterload. systolic blood pressure (sbp) as a function of systemic vascular resistance (svr) is the major determinant of afterload. an elevated sbp resulting from peripheral vasoconstriction and an elevation of the svr negatively influences both stroke volume and myocardial oxygen demand. myocardial oxygen demand is elevated because a major determinant of myocardial oxygen consumption is ventricular wall tension. contractility is the intrinsic strength of myocardial contraction at a constant preload and afterload. it is best assessed by echocardiography, and can be inferred from an analysis of cardiac output and filling pressures. while cardiac output is an important component of oxygen delivery, it is not the only factor. oxygen delivery is a function of cardiac output, hemoglobin, and arterial oxygen saturation (sao ). most of the oxygen delivered to tissues is bound to hemoglobin. low hemoglobin is a major factor adversely affecting oxygen delivery; therefore, maintenance of optimal hemoglobin is essential. conversely, efforts should be made to limit transfusions, if possible, to avoid transfusion-related illnesses, immunologic compromise, and cost. a strategy should be in place to guide transfusions and should be based on criteria providing adequate oxygen delivery. the optimal postoperative hemoglobin is probably - %. , red blood cell (rbc) transfusions should be considered in patients with hematocrits lower than - % and those patients with poor lv function, marginal sao , ischemic findings on electrocardiogram (ecg), hypotension, tachycardia, and effort-related symptoms. similarly, the optimal oxygen saturation is - %, and maintaining an sao greater than % does not enhance oxygen delivery. mixed venous oxygen saturation (svo ) is a measure of the adequacy of oxygen delivery to the tissues. it can be measured from blood drawn from the distal port of a swan-ganz catheter or continuously using a fiber-optic oximetric pulmonary artery catheter. a diminished svo generally indicates decreased tissue perfusion and/or increased oxygen extraction by tissues. svo is an indirect correlate of the cardiac output. in the absence of factors that increase oxygen utilization, a % decrease in svo is an indication of a low cardiac output and can be seen before any change in other hemodynamic parameters. other causes of a diminished svo are shivering, elevated temperature, anemia, alteration in inspired oxygen, and altered alveolar gas exchange. these conditions cause a diminished svo in the presence of a normal cardiac output by causing increased oxygen utilization. svo measurement can be of particular help in assessing adequate oxygen delivery when thermodilution cardiac output is unreliable (e.g., tricuspid regurgitation, improperly placed swan-ganz catheter, malfunctioning swan-ganz catheter), when thermodilution cardiac output is unavailable because swan-ganz cannot be placed (e.g., mechanical prosthesis in the tricuspid position), or when the clinical situation is unstable requiring online, minute-to-minute cardiac evaluation. another important aspect in the appropriate management of the postoperative patient is minimizing the myocardial oxygen demand (mvo ). the mvo is influenced by afterload, preload, heart rate, and contractility. reducing afterload will reduce oxygen demand. increasing preload, heart rate, and contractility will improve cardiac output but will also increase mvo . providing adequate myocardial oxygen supply is equally important to the postoperative patient. myocardial oxygen supply is determined by coronary blood flow, duration of diastole, coronary perfusion pressure (systemic diastolic pressure minus lvedp), hemoglobin level, and arterial oxygen saturation. postoperatively, myocardial oxygen supply is optimized by avoidance of tachycardia, maintenance of adequate perfusion pressure (avoid hypotension and hypertension), avoiding ventricular distention and inappropriately elevated lvedp, and by managing preload judiciously. the goal of postoperative management is the maintenance of a satisfactory cardiac output. hemodynamically, the cardiac index (ci) should be greater than . l/min/m at a pulmonary capillary wedge pressure (pcwp) of less than mmhg or pulmonary artery diastolic pressure (padp) of less than - mmhg with a heart rate less than bpm. clinically, the patient should be warm, well perfused, and with an appropriate urine output. by definition, a ci greater than . l/ min/m is satisfactory, a ci of . - . l/min/m is marginal, and a ci below . l/min/m is unacceptable and calls for intervention. ninety percent of all postoperative open-heart patients demonstrate a transient low cardiac output (lco) related to the release of oxygen free radicals in response to the induced inflammatory state of cardiopulmonary bypass, or from ischemic/reperfusion injury as a result of cardioplegic arrest. , , , the ventricular function becomes depressed in h and is at its worst at - h. generally, there is significant recovery in about - h and full recovery by - h. lco is more common in patients with preoperative lv systolic dysfunction, diastolic dysfunction, prolonged cardiopulmonary bypass, and in women. , clinical manifestations of low cardiac output as cardiac output deteriorates, compensatory mechanisms develop and are the result of sympathetic autonomic stimulation and endogenous catecholamine production. these compensatory mechanisms result in an increased heart rate, increased contractility, and increased arterial and venous tone (resulting in elevation of preload and afterload). these compensatory mechanisms may increase the cardiac output but at the expense of myocardium oxygen utilization, and consequently the myocardium may become more depressed. as the myocardium becomes depressed, the left ventricular function worsens and the systemic vascular resistance (svr) increases in an attempt to maintain systemic blood pressure. this elevation in the svr is compounded by the vasoconstriction seen with hypothermia. the early clinical manifestations of low cardiac output may be subtle. the only findings may be cool extremities accompanied by progressive tachycardia. as the compensatory mechanisms fail, more advanced clinical manifestations occur. overt findings of poor peripheral perfusion such as pale, cool extremities and diaphoresis, pulmonary congestion and poor oxygenation, oliguria secondary to poor renal perfusion, and metabolic acidosis will be manifest. early intervention is indicated at the onset of the early manifestations to avoid the complications of prolonged hypoperfusion and progression to the advanced manifestations. the etiology of lco can be abnormal preload, afterload, contractility, or heart rate and rhythm or a combination of these. the most common causes of lco after surgery are related to decreased left ventricular preload caused by hypovolemia and bleeding, vasodilatation, rewarming, drugs, cardiac tamponade, right ventricular dysfunction, positive pressure ventilation, and a tension pneumothorax. increased afterload is usually the result of acute vasoconstriction most often related to vasoactive drug therapy. it can also be from preexisting hypertension, pain or awareness, fluid overload, or hypothermia. decreased contractility is causative of lco in patients with preexisting lv dysfunction in association with perioperative ischemia. perioperative ischemia is usually a consequence of poor intraoperative myocardial protection, incomplete revascularization, coronary artery or conduit spasm, coronary artery "trash" syndrome, graft closure, acute anemia, hypoxia, or acidosis of any etiology. tachyarrhythmias adversely affect cardiac output by decreasing cardiac filling time and subsequently decreasing stroke volume coronary perfusion time. tachyarrhythmias also increase myocardial oxygen demand. bradyarrhythmias depresses cardiac output, especially when left ventricular dysfunction limits the compensatory mechanism of an increasing stroke volume. bradyarrhythmias are particularly deleterious in association with aortic insufficiency of any degree. when atrial fibrillation occurs, there is a loss of atrial contribution to cardiac output and subsequent fall in the cardiac output. finally, any ventricular arrhythmia adversely affects the cardiac output. diastolic dysfunction causes lco in a specific set of patients. it is often seen in small women with hypertension, patients with long-standing aortic stenosis, or patients with hyperdynamic left ventricles. all of these situations are associated with left ventricular hypertrophy, poor ventricular relaxation, and near-obliteration of the left ventricular cavity during systole. , diastolic dysfunction presents with nor-mal lv function and normal or elevated pcwp, but a lco syndrome. these patients deteriorate quickly if sinus rhythm or atrial-ventricular synchrony is lost. miscellaneous noncardiac causes of lco include anaphylaxis or anaphylactoid reaction, marked alterations in temperature, sepsis, adrenal insufficiency, and the various protamine reactions. when no obvious diastolic or systolic dysfunction is present, then consider tamponade from blood or clot within the confines of the mediastinum and pericardium. the diagnosis of low cardiac output begins with a bedside physical examination. the early clinical manifestations of lco are apparent to the clinician with a heightened suspicion for their presence. the importance of a careful bedside assessment cannot be overstated. the examination should include the condition and appearance of skin and mucous membranes, breath sounds, murmurs, temperature of extremities, and a level of consciousness. the ekg monitor is a minimum level of monitoring after open-heart surgery. it is a screening device for ischemia and arrhythmias, both causes of lco. all ischemic changes on monitors must be further assessed with a -lead ekg and it is prudent to confirm all but the most obvious arrhythmias with a -lead ekg. hemodynamic monitoring, at a minimum, includes a central venous pressure (cvp) line and can be used to assess preload as well as right ventricular function. clinical lco and low cvp suggests inadequate preload as the cause of lco. clinical lco and an elevated cvp are more complicated. this situation may be the result of right heart failure, volume overload, left heart failure, tamponade, or some preexisting problem such as severe chronic obstructive pulmonary disease (copd). in this circumstance, the information from a swan-ganz catheter or transesophageal echocardiogram (tee) can clarify the situation. swan-ganz catheters (pulmonary artery catheters) are used in all patients in some institutions and selectively in others. oximetric swan-ganz catheters are optional and are used in highly selected situations when minute-to-minute cardiac assessments are necessary. swan-ganz catheters provide an assessment of right and left heart filling pressures, determine cardiac output, stroke volume, svr, and svo . the information acquired from these catheters confirm the diagnosis of clinical lco and provide information as to the etiology. for example, low filling pressures suggest preload as the causative factor, whereas high filling pressures indicate a problem with contractility or afterload. a chest x-ray is a valuable and essential tool in the postoperative period for multiple reasons, but it can also assess the lungs as a cause for low cardiac output. in particular, a chest x-ray can identify a pneumothorax, hemothorax, pleural effusion, adult respiratory distress syndrome, and the endotracheal tube position as potential causes of a low cardiac output. it also assesses the mediastinum for an enlarged mediastinal silhouette suggesting tamponade or incorrect position of intrathoracic monitoring lines. echocardiography has become a first-line tool in evaluating the postoperative patient suspected of having lco. it can either be a transthoracic examination or a transesophageal examination. the surface echocardiography has limited value in the immediate postoperative period because of the presence of dressings and chest tubes, but can provide some information about lv function and recognize obvious tamponade. transesophageal echocardiography is an extremely valuable tool in the postoperative period and can be carried out at the bedside. it provides excellent visualization of cardiac dynamics, the pericardial space, and the mediastinum. it is the best diagnostic modality for lv function, presence of tamponade, and the development of new valvular abnormalities. it is also good for right ventricular assessment. each of the previous diagnostic modalities has an important role in the assessment of the postoperative cardiac surgical patient with suspected lco. once the diagnosis of lco is established and the etiology determined, appropriate treatment actions can be instituted. the management of low cardiac output begins by excluding tamponade as the cause. if there is no indication of tamponade, treat the correctable noncardiac abnormalities such as respiratory abnormalities, acid-base and electrolyte imbalances, and anemia. if lco persists, direct therapy at treatable cardiac abnormalities such as ischemia with a nitroglycerine infusion and consider diagnostic catheterization with catheter or operative intervention if ischemia persists. consider coronary spasm, but this is a difficult diagnosis. suspect coronary spasm when the patient presents with hemodynamic instability and ekg changes, especially st segment elevation. coronary spasm usually responds to calcium channel blockers and is a particular threat in patients with arterial conduits as grafts. arrhythmias can also cause lco. ideally, the patient should be in sinus rhythm at - bpm. in the presence of lco, arrhythmia management should be aggressive and pacing support may be needed to maintain atrial-ventricular synchrony. after the initial steps of correcting obvious noncardiac and cardiac abnormalities, the volume status should be assessed and preload optimized. it is helpful to know what filling pressures resulted in the best cardiac performance in the operating room or catheterization laboratory (cath lab) and adjust the volume accordingly. the cardiac performance should be followed closely as volume is administered, and if filling pressures increase without concomitant improvement of cardiac output, an inotrope will be needed. be mindful that the injudicious use of volume administration will result in distention of the ventricle (right, left, or both) with a shift in the frank-starling curve. as the ventricular wall tension increases, the myocardial oxygen demand increases and contractility becomes impaired. if volume administration fails to improve filling pressures, there may be ongoing volume loss from hemorrhage, diuresis, capillary leak syndrome, or vasodilatation from drugs, warming, or previous comorbid conditions. volume should be given in doses of % of estimated blood volume (blood volume is estimated as . × body weight in kg for adults). orders for volume expansion should be written with a prescribed stop order when the optimal filling pressure is exceeded to prevent ventricular distension. the choice of the appropriate volume expander is important. if the hemoglobin is less than . g, give packed red blood cells (prbcs); if the hemoglobin is . - . g, give prbcs and a colloid of choice; and if the hemoglobin is . g or greater, give a colloid of choice or equivalent dose of crystalloid. pharmacologic support is considered when the cardiac output fails to improve after optimizing preload, afterload, rate and rhythm, and metabolic abnormalities. the threshold for using vasoactive agents should be low in patients with a preoperative history of compromised ventricular function. the choice of the agent depends on multiple factors: the hemodynamic profile of the patient; associated medical conditions; treating physician's understanding of the agent; and, to a lesser extent, cost. of these factors, the most important is the hemodynamic profile of the patient. inotropic agents must be chosen based on the specific hemodynamic abnormality most responsible for the current lco state. often, the causative factors are multifaceted and dynamic, making flexibility and vigilance key. it is not unusual to need a combination of agents to successfully treat lco. at the initiation of therapy for lco, a bedside presence is mandatory to respond minute-to-minute to hemodynamic changes. an understanding of the basic mechanism of action and of the inotropic agents comprises the basis for agent selection. in general, each category of agents exerts their effects differently. catecholamines affect -adrenergic and -adrenergic receptors. they elevate the levels of intracellular cyclic amp (camp) by -adrenergic stimulation of adenylate cyclase. the phosphodiesterase (pde) inhibitors, inamrinone and milrinone, elevate camp by inhibiting camp degradation. elevation of camp augments calcium influx into myocardial cells and increases contractility. the stimulation of -and -adrenergic receptors results in elevation of svr and pvr. cardiac receptors increase contractility and decrease heart rate. stimulation of receptors results in increased contractility, heart rate, and conduction. in contrast, stimulation results in peripheral vasodilatation and bronchodilatation. the overall hemodynamic effect of these agents is dose-related. the need to use these agents in combination is often beneficial and necessary to achieve the desired hemodynamic effect and lessen undesired sequelae. when infusing vasoactive agents, several caveats are noteworthy. first, these agents have a lessened effect in an acid medium; therefore, it is important to maintain the patient in proper acid-base balance to achieve the full effect of the therapy. an increasing dose of the agent may be indicating a falling ph. secondly, the route of administration should always be through a central line and not peripherally. thirdly, these agents should always be administered with a rate-controllable infusion pump. finally, higher blood levels can be attained by infusing them directly into the left atrium to avoid partial deactivation or removal by the lungs. this method can also be employed to lessen the pulmonary vasoconstrictive effects and subsequent rv dysfunction of catecholamines such as epinephrine or norepinephrine. this method of infusion has its own inherent risks and should be reserved for extreme circumstances. epinephrine is the catecholamine of choice for low cardiac output in many institutions. it has potent inotropic effects and increases cardiac output by increasing contractility and heart rate. some of its effects are dose-related. at doses lower than mcg/min (< . mcg/kg/min), its effects result in mild vasodilatation and a decrease in the svr while maintaining an adequate blood pressure. doses greater than mcg/ min (> . mcg/kg/min) produce effects that cause vasoconstriction with an increased svr potentially decreasing cardiac output further as well as increasing myocardial oxygen demand. epinephrine may cause tachycardia, but often less than that with dopamine or dobutamine at comparable doses. it can be arrhythmogenic, usually causing ventricular ectopy. hyperglycemia and metabolic acidosis are not infrequently associated with its use. while epinephrine can be used as a first-line agent in patients with ventricular arrhythmias or brittle diabetes mellitus, it must be done so with care. in some institutions, it is used as a second-line agent if dopamine and/ or dobutamine are not tolerated or ineffective. secondary uses for epinephrine include stimulation of heart rate in patients with bradycardia, bronchospasm, anaphylaxis, and general resuscitation for cardiac arrest. epinephrine is the least expensive of the commonly used inotropes. epinephrine is begun at mcg/min and titrated to effect or to - mcg/min. dopamine is also a first-line agent for low cardiac output in some institutions. it is indicated for lco with a low svr and diminished systemic blood pressure. it also may be beneficial in the face of decreased urine output. aside from its inotropic and chronotropic effects, an added effect is the selective "dopaminergic" effect that increases renal perfusion, glomerular filtration rate, and urine production by directly reducing renal afferent arteriolar tone and indirectly increasing efferent arteriolar tone. the hemodynamic effects of dopamine are largely dose-dependent. despite its ability to increase urine production in some instances, it has never been shown to prevent acute renal failure. , at doses of - mcg/kg/min, the main effects are renal as described, although there can be a mild effect with a decrease in svr and systemic blood pressure. at doses of - mcg/kg/min, effects are predominant increasing contractility. at this dose, there is also a chronotropic effect that increases heart rate and has the potential for arrhythmogenesis. doses of dopamine of greater than mcg/kg/min results in increasing inotropy, but also this dose causes a predominant effect. this effect occurs directly but also indirectly from the release of norepinephrine. the svr increases as do the filling pressures and myocardial oxygen consumption leading to ventricular dysfunction. these adverse effects can be somewhat mitigated by the concomitant use of vasodilator therapy. its use may be limited by profound tachycardia even at low doses and excessive urine production. dopamine is begun as an infusion at . mcg/kg/min and titrated to - mcg/kg/ min if needed. if a favorable response is not achieved at mcg/kg/min, it is unlikely that higher doses will result in hemodynamic improvement. dobutamine has similar effectiveness as dopamine, but does not have its renal dopaminergic effect. dobutamine may augment myocardial perfusion better than dopamine. it is a positive inotrope with strong effect that increases contractility and also heart rate. dobutamine has mild effect and decreases svr; this effect is mild and may be offset by its mild vasoconstricting effect present in some specific circumstances. also, unlike dopamine, dobutamine reduces ventricular wall tension by reducing afterload and preload particularly in the presence of volume overload. , there appears to be augmentation of myocardial blood flow and an improvement of the myocardial oxygen supply and demand curve, but this positive effect may be lessened by tachycardia. the usefulness of dobutamine may be limited by tachycardia that may be profound and may trigger atrial fibrillation. because of its hypotension from the vasodilating effect, dobutamine should be used with caution in the hypotensive or hypovolemic patient and is contraindicated if tamponade is suspected. it is most commonly used for low cardiac output associated with a mildly elevated svr and may have a synergistic effect when used with pde inhibitors. it does have a moderate pulmonary vasodilatory effect and can improve rv dysfunction. it is more expensive than dopamine, yet only minimally more effective. dobutamine is begun as an infusion at mcg/kg/min and can be increased for effect up to mcg/kg/min. inamrinone and milrinone are phosphodiesterase inhibitors known as "inodilators." these agents produce positive inotropic effects and vasodilation independent of adrenergic stimulation. they improve biventricular output by increasing stroke volume index, left ventricular contractility, and producing pulmonary vasodilation. these agents also produce vasodilation in arteriolar and venous smooth muscle, thus reducing preload and afterload, and their use is associated with decreased myocardial oxygen consumption, despite a modest positive chronotropic effect. inamrinone and milrinone decrease coronary vascular resistance, improve coronary perfusion, and improve the myocardial oxygen supply/ demand ratio. pde inhibitors have an additive effect when used with catecholamines because of their differing sites of action. [ ] [ ] [ ] catecholamines stimulate the production of camp whereas pde inhibitors slow the hydrolysis of camp. inamrinone and milrinone are generally considered second-line agents in the treatment of lco. they are usually employed when first-line agents like dopamine or epinephrine are not providing adequate hemodynamic improvement or if side effects are limiting their effectiveness. however, there is evidence that administering these agents preemptively, prior to separation from cardiopulmonary bypass in patients with preoperative lv dysfunction, may eliminate the need for inotropic therapy subsequently. , inamrinone and milrinone are particularly useful in patients with rv dysfunction secondary to pulmonary artery hypertension and elevated pvr. these agents are also useful in treating diastolic dysfunction as they have been shown to have relaxant or lusitropic properties. they also appear to have direct vasorelaxant effects on arterial graft conduits and may be useful in patients with evidence of internal mammary spasm or in the presence of radial artery grafts. , these drugs have a relatively long half-life of - h; consequently, the loading dose will be effective for several hours after administration but the patient should be reassessed at that time for any ongoing need for therapy. since the pde-inhibitors are effective vasodilators, the systemic blood pressure may require support, usually with agonists. vasopressin may be an alternative drug to support the systemic blood pressure while reducing the need for catecholamine pressors. inamrinone is associated with thrombocytopenia, but this is rare with milrinone. there does not appear to be any significant hemodynamic difference between inamrinone and milrinone, but milrinone has largely replaced inamrinone in clinical use because of the latter's thrombocytopenic effects. both are relatively expensive compared to other inotropic agents. inamrinone is given as a loading dose of . mg/ kg over min (may need . mg/kg if bolus given while on cardiopulmonary bypass) followed by an infusion of - mcg/ kg/min. milrinone is given as a loading dose of mcg/kg over min, then an infusion dose of . - . mcg/kg/min. norepinephrine is another naturally occurring catecholamine. it has a pronounced effect on peripheral receptors resulting in peripheral vasoconstriction, elevated svr, and elevated systemic blood pressure. norepinephrine also is a agonist increasing myocardial contractility and heart rate. the increased afterload, contractility, and heart rate result in an increase in myocardial oxygen consumption. the overall increase in myocardial oxygen consumption may have a deleterious effect on ischemic myocardium. the primary effect of norepinephrine is elevation of blood pressure and mildto-moderate elevation of the cardiac output. it also has been shown to cause regional redistribution of blood flow with reduced renal, mesenteric, and peripheral perfusion. the primary indication for norepinephrine is a low cardiac output associated with a low svr. it is a reasonable choice of pharmacologic support if the svr is low and the cardiac output is . - . l/min/m . if the svr is low and the cardiac output greater than . l/min/m , a pure agonist may be used. if the svr is low and the cardiac output is less than . l/min/ m , another inotrope should be used in addition to or in place of norepinephrine. norepinephrine can be used in combination with afterload reduction to titrate the systemic blood pressures to acceptable levels and to maintain a satisfactory systemic blood pressure. it can also be used in combination with epinephrine to augment the effect. the starting dose is mcg/min ( . mcg/ kg/min) and titrated to the desired systemic blood pressure. at doses greater than mcg/min ( . mcg/kg/min), visceral and peripheral perfusion is reduced to such an extent the patient may become acidotic. isoproterenol is a -adrenergic agonist. it has strong effect, some effect, and little action. the effects increase cardiac output by its moderate increase in contractility and marked increase in heart rate. the effect reduces svr. it has been shown to reduce pulmonary vascular resistance and may be effective in treating reactive pulmonary hypertension when right heart failure is contributing to low cardiac output. it can afterload reduce the right ventricle. isoproterenol also has strong bronchodilator effect. the indications include right ventricular failure associated with elevated pvr and bronchospasm, and can be used to stimulate heart rate in patients with bradycardia and no functioning pacemaker wires. its use is limited because it increases heart rate and myocardial oxygen demand. since it is a nonselective -adrenergic agonist, it will predispose to tachyarrhythmias, ventricular irritability, and ventricular dysrhythmias. as a result of the tachyarrhythmias, isoproterenol has been largely replaced by pde inhibitors. phenylephrine has no direct cardiac effects. it is a pure -agonist that increases svr. it does have some usefulness in the treatment of lco resulting from myocardial ischemia secondary to global hypoperfusion. if systemic blood pressure is reduced as a consequence of vasodilatation, coronary perfusion may be compromised leading to myocardial ischemia and ventricular dysfunction. phenylephrine directly stimulates -adrenergic receptors leading to an elevation of the coronary perfusion pressure and resolution of global myocardial ischemia. systemic vasodilatation is most often seen immediately following cpb or in the early hours of recovery as the patient rewarms. in these circumstances, phenylephrine may be helpful. since it provides no direct cardiac benefits, its role is limited. phenylephrine can cause vasoconstriction of an arterial conduit and should be used with caution in patients with arterial conduit grafts. its main indication is to increase svr in patients with low svr and normal or elevated cardiac output. it can also be used as a temporizing measure in a hypotensive, hypovolemic patient until the volume status is corrected. the usual starting dose is mcg/min and the usual dosing range is . - . mcg/kg/min. nesiritide is a recombinant b-type natriuretic peptide. it is identical to the endogenous b-type natriuretic peptide secreted by the ventricles in response to increased cardiac volume and pressure overload. nesiritide decreases sympathetic stimulation and inhibits the neurohumoral responses seen in heart failure. it exerts its effects by inhibiting the renin-angiotensinaldosterone system to decrease aldosterone, norepinephrine, and endothelin levels resulting in natriuresis and diuresis. the net effect is a balanced reduction in preload and afterload, and relaxation of smooth muscle. it indirectly improves cardiac output with no increase in heart rate and no increase in myocardial oxygen demand. nesiritide is lusitropic and dilates native coronary arteries and arterial conduits. it is not proarrhythmic. it has been shown to dilate afferent and efferent renal arterioles increasing glomerular filtration resulting in natriuresis and diuresis. like pde inhibitors, it can be used synergistically with catecholamines to reduce dosages and side effects. while nesiritide has demonstrated favorable clinical results in nonsurgical patients with decompensated heart failure and it has pharmacologic effects possibly beneficial to the postoperative cardiac surgical patient, experience with nesiritide in surgical patients is limited. early results indicated that it may not be any better than milrinone. one clinical trial did demonstrate a trend toward reduced length of stay without adverse effects. its main indication in the surgical patient is in conditions of diastolic dysfunction or lco states associated with elevated pulmonary artery pressures. it is also useful in conditions of fluid overload and postoperative renal failure. nesiritide is given, a dose of a mcg/kg over min followed by an infusion of . - . mcg/kg/min. vasopressin is a peptide hormone synthesized in the hypothalamus and is released from the posterior pituitary upon stimulation by hyperosmolality, hypotension, and hypovolemia. it has two sites of action: kidney and blood vessels. the primary function of arginine vasopressin (avp) is to regulate extracellular fluid volume by affecting renal tubular absorption of water. it acts on the renal collecting tubules by increasing water permeability and results in decreased urine formation. this is its antidiuretic function and is why it is commonly known as antidiuretic hormone (adh). the antidiuretic effect increases blood volume and indirectly increases cardiac output and arterial blood pressure. a secondary function of avp is vasoconstriction. it binds to vascular smooth muscle to cause vasoconstriction. avp is a potent vasopressor even in patients with catecholamine-resistant hypotension. loss of catecholamine pressor effect is a well-established phenomenon. in acute shock states, vasopressin levels increase rapidly and then decrease in prolonged shock states leading to a relative deficiency of vasopressin. , the deficiency of vasopressin is thought to contribute to hypotension refractory to catecholamines, especially in sepsis. , because vasopressin is a potent vasopressor, infusions of vasopressin leads to improved organ perfusion, increased mean arterial pressure, and improved neurological function. , , vasopressin is indicated for the management of severe vasodilatory shock. in patients with "vasoplegia," profound peripheral vasodilatation with preserved cardiac output, vasopressin may have a role. this condition is usually associated with patients on preoperative angiotensin-converting enzyme inhibitors or amiodarone. it may also be the consequence of leukocyte activation and release of proinflammatory mediators caused by the systemic inflammatory response to cpb. , vasopressin is usually successful in reversing the low svr when phenylephrine and norepinephrine are not. , vasodilatory shock is not uncommon in patients with a ventricular assist devices (vad) and may benefit from the vasoconstrictive actions of vasopressin. despite vasopressin's effect in vasodilatory shock, it remains a second-line agent because there is no current evidence to support the use of vasopressin as a first-line agent instead of catecholamines. there is growing evidence that vasopressin may provide comparable or superior efficacy to epinephrine as a resuscitative agent for cardiac arrest and hemodynamic collapse when administered as a single bolus of units intravenously. the recommended infusion rate for vasopressin in the treatment of vasodilatory shock is . - . units/min. doses greater that . units/min may lead to cardiac arrest. rapid rebound hypotension commonly occurs after vasopressin infusion is discontinued. potential adverse sequelae of vasopressin therapy include ischemic cutaneous necrosis, intestinal ischemia, and decreased hepatosplanchnic flow and cardiac output. ionized calcium is critical for excitation-contraction coupling in cardiac muscle. hypocalcemia depresses ventricular contractility and peripheral vascular resistance; the net effect is lco and low systemic blood pressure. the hemodynamic effects of calcium chloride are more profound if the patient is hypocalcemic. serum ionized calcium levels are low postoperatively, particularly just prior to weaning from cpb, and a bolus of calcium is frequently given just prior to weaning from cpb. the effect of a bolus of calcium is increased contractility and increased svr. it has little effect on the heart rate. it is more effective when the patient is hypocalcemic, but is also efficacious even if the patient is normocalcemic. calcium chloride provides ionized calcium, which acts as a strong but very evanescent inotrope. a continuous infusion of calcium does not sustain its hemodynamic effect. ionized calcium is necessary for the effective action of catecholamines. the main indication for calcium chloride is at the termination of cardiopulmonary bypass to augment systemic blood pressure during separation from bypass. it is also used as an emergency resuscitation agent to support hemodynamics until a more complete evaluation can be performed and more specific measures utilized. the dose is in increments of . - . g slow iv bolus. cardiopulmonary bypass and hypothermic arrest results in low levels of circulating thyroid hormone. , , triiodothyronine (t ) has hemodynamic effects based on this reduction in the plasma-free level of t following cardiopulmonary bypass. t remains low for h, but not low enough to cause symptoms of hypothyroidism. augmenting the levels of t can increase myocardial function and has been shown to increase cardiac output and lower svr in patients with ventricular dysfunction. - t exerts its positive inotropic effect by increasing aerobic metabolism and synthesis of highenergy phosphates. it directly stimulates calcium adenosine triphosphatase (atpase) in the sarcolemma and sarcoplastic reticulum. the enhancement of calcium transport decreases intracellular calcium aiding myocardial relaxation, myocardial compliance, and diastolic function. , , t also decreases svr. currently, there are conflicting results on the use of t in the treatment of lco. the current role for t is salvage when cardiopulmonary bypass cannot be terminated despite maximum support including inotropic agents and intra-aortic balloon counterpulsation. there are no studies, to date, that show that t favorably improves outcome in patients failing to separate from cardiopulmonary bypass even though hemodynamics have improved in patients with ventricular dysfunction. the dosage is . - . mcg/kg as an iv bolus. pharmacologic support is the first-line therapy for lco. mechanical support should be considered for the management of lco when there is need for more than two inotropic agents used at the upper range of their therapeutic efficacy, when there are complications from these agents, or when lco progresses to cardiogenic shock. other uses of mechanical support postoperatively include myocardial ischemia or the development of mitral regurgitation that cannot be managed medically. finally, mechanical support is indicated for the patient experiencing acute deterioration and in need of a transplant. available mechanical support devices are the intraaortic balloon and circulatory assist devices such as left and/or right ventricular assist devices. the intra-aortic balloon pump has been an effective tool for the management of lco states, ongoing ischemia, valvular disease, and the complications of myocardial infarction since its development in . intra-aortic balloon pump (iabp) counterpulsation provides hemodynamic support and control of ischemia before and after surgery. it has been shown to be effective in improving the diastolic function of the left ventricle. iabp counterpulsation is very effective in the management of low cardiac output states. unlike most inotropic agents, it provides hemodynamic support to the failing heart by decreasing myocardial oxygen demand and improving coronary artery perfusion. iabp counterpulsation acts to improve the myocardial oxygen supply:demand ratio. it reduces the impedance of left ventricular ejection by rapidly deflating just before systole, thus unloading the lv, and in this way decreases myocardial oxygen demand. as it rapidly inflates just after aortic valve closure, it increases the diastolic coronary perfusion and improves myocardial oxygen supply. the survival rate of patients requiring postoperative iabp support is - %. , the indications for iabp counterpulsation are perioperative ischemia, mechanical complications of myocardial infarction (such as acute mitral regurgitation, ventricular septal defect, and cardiogenic shock), postoperative low cardiac output states not responsive to moderate doses of inotropic agents, and for the acute deterioration of myocardial function to provide temporary support or a bridge to transplantation. iapb counterpulsation is contraindicated in the presence of aortic insufficiency, aortic dissection, and severe aortic and peripheral vascular disease. the iabp can be inserted percutaneously or surgically. the percutaneous approach is favored despite its somewhat higher prevalence of vascular complications. percutaneous insertion is preferred because of ease of insertion and removal. the iabp is inserted percutaneously using the seldinger technique and is positioned fluoroscopically. the balloon tip marker should be positioned just distal to the origin of the left subclavian artery. the surgical insertion requires the exposure of the femoral artery and creation of a sidearm to the femoral artery with a vascular graft, followed by the insertion of the balloon through the graft. an alternative open surgical approach is exposure of the femoral artery and then direct cannulation with a vascular sheath using a guide wire. a hemostatic suture is placed in the femoral artery around the stem of the iabp. the iabp can be inserted by an open supra-inguinal approach in cases of severe femoral arterial disease, or the transthoracic approach via the ascending aorta in cases of severe aortoiliac peripheral disease. triggering of the device is timed using ekg or arterial waveform. if ekg is used, the inflation is set at the peak of t wave, the end of systole. deflation is set just before or on the p wave. arterial waveform triggering is more reliable and a better timing technique when outside electrical impulses (i.e., pacemaker, electrocautery) may interfere with interpretation of the ekg signal. with arterial triggering, the inflation should occur at the dicrotic notch and deflation just before the onset of the aortic upstroke. proper timing will show an arterial waveform with augmentation of the diastolic portion of the curve. support with the iabp is instituted at a : ratio with ventricular systole based either on ekg monitoring or the arterial pressure pulse tracing. there is often immediate hemodynamic improvement and the patient requires less inotropic support. when the required inotropic support reaches moderate levels (generally half the doses required prior to iabp support) consideration for weaning is possible. the iabp is weaned by reducing the assist ratio from : to : or less depending on the system. the weaning process can usually begin after - h of support and completed by - h. if the device was placed percutaneously, it can be removed similarly with firm pressure to the groin for min. since the arterial puncture site is several centimeters proximal to the skin insertion site, a common mistake is to direct the pressure at the skin insertion site instead of the arterial puncture site. when this error occurs, a large hematoma develops in the groin proximally. if a hematoma occurs or if the perfusion to the distal limb is compromised, immediate exploration is required. at times, there is a failure to achieve augmentation from the counterpulsation with the iabp. this can be the result of tachycardia and arrhythmias, inadequate balloon volume, and/or balloon rupture. arrhythmias effect augmentation by disrupting the normal inflation and deflation patterns of the device. rapid heart rates, usually atrial fibrillation with ventricular responses greater than bpm, interfere with the balloon's ability to inflate and deflate. in this circumstance, augmentation can be achieved by changing the triggering ratio to : (one iabp cycle for every second cardiac cycle). inadequate gas volume in the balloon can also result in an inability to augment. volume loss from the balloon can result from a gas leak or from failure of the balloon to unwrap. either circumstance necessitates the removal of the balloon. of more immediate concern is a balloon rupture. this is heralded by blood in the balloon tubing. the balloon must be removed immediately as helium and blood can create a rock-hard thrombus making surgical removal necessary. vascular complications are the most commonly encountered complications of iabp counterpulsation. the most catastrophic complication is an aortic or iliac artery dissection or rupture. fortunately, this is an uncommon occurrence. equally catastrophic is paraplegia from a periadventitial aortic hematoma or as the consequence of embolization of atherosclerotic debris to the spinal cord. embolization or altered perfusion to visceral vessels can also occur with iabp counterpulsation. the most common vessels involved are the renal arteries. this usually occurs in the presence of significant atherosclerotic disease in the aorta. altered perfusion of the kidneys and renal failure can happen if the balloon is situated below diaphragm. the iabp can also restrict perfusion to the lima if it is advanced too far proximally into the subclavian artery. distal limb ischemia is the most common complication of the iabp. the occurrence rate is - % and occurs more commonly with percutaneous placement, in women, and in patients with small femoral arteries. heparin therapy is advisable if the iabp is in place more than - days after surgery. the management of compromised distal perfusion begins by knowing the preoperative vascular status of the patient as well as obtaining a baseline status of the distal extremities with physical examination and doppler assessment as soon as possible after implantation of the iabp. thereafter, the distal pulses or doppler signals should be assessed hourly and recorded along with the vital signs of the patient. if the pulses or doppler signals deteriorate, initially rule out peripheral vasoconstriction from hypothermia, low cardiac output, or as a result of vasopressor agents. if limb ischemia persists, remove the sheath from the femoral artery if the iabp was inserted percutaneously. if distal perfusion remains compromised, then remove the balloon and place it on the contralateral side if counterpulsation remains necessary. femoral artery exploration is necessary if iabp removal does not improve the vascular integrity of the threatened limb. if the patient remains dependent on the iabp and the femoral artery approach is not feasible any longer, consider the transthoracic approach. thrombocytopenia can occur from the mechanical destruction of the platelets by the iabp. thrombocytopenia may also be related to drug interactions (heparin, amrinone, etc.) when the iabp is implanted, a platelet count should be checked daily and if a downward trend develops, then every - h. circulatory assist devices were introduced by cooley and his associates in . these devices, commonly referred to as ventricular assist devices (vads), are used as a bridge to transplantation, a bridge to recovery, and for support after cardiac surgery. they are the ultimate therapy for low cardiac output. they are usually employed intraoperatively for failure to wean from cardiopulmonary, but can also be an option postoperatively if the patient fails to respond to vasoactive agents and the iabp. vads should be considered if the patient does not respond to maximum medical therapy including the iabp. , the therapeutic strategy of vads is to provide sufficient flow to support the systemic and/or pulmonary circulation while the myocardium recovers. short-term devices are used if there is a reasonable chance for recovery, whereas long-term devices are considered if the chances of recovery are remote and the patient is a suitable candidate for transplantation. prior to committing to circulatory assist, a thorough investigation for correctable causes of lco must be made. transesophageal echocardiography is helpful in evaluating ventricular wall motion and excluding other structural conditions related to the cardiac procedure. preload and afterload should be optimized, appropriate inotropic therapy instituted, and placement of the iabp accomplished before considering circulatory assist. circulatory assist can be left or right heart bypass or combined biventricular bypass. the general indications for vad implantation include a complete and adequate cardiac surgical procedure, the correction of all metabolic problems, the inability to wean from cardiopulmonary bypass, the inability to reverse deteriorating hemodynamic embarrassment despite maximum drug therapy and iabp, and a cardiac index less than . - l/min/m . left ventricular assist devices (lvads) provide systemic perfusion while the left ventricle recovers. the indications for lvad support include those general indications for vads as well as a systolic bp less than mmhg, left atrial pressure greater than mmhg, svr greater than dyne s/cm , and urine output less than ml/h. lvads require a left atrial cannula connected to an aortic cannula via a centrifugal pump. the lvad flow is dependent on the intravascular volume and right ventricular function. the goal of management is a lvad flow of . l/min/m . these devices reduce left ventricular wall stress by % and left ventricular myocardial oxygen demand by %. monitoring mixed venous oxygen saturation can assess adequacy of tissue perfusion. after lvad implantation, inotropic support should be discontinued to decrease myocardial oxygen demand. in some circumstances, an inotrope may be needed to support the right ventricle and vasoconstricting agents may be needed to maintain the svr and a mean arterial pressure greater than mmhg. heparin therapy is necessary after postoperative bleeding stops and, particularly, when flow is decreased to less than . l/min. after implant a long-term device as a bridge to transplantation if the patient is an appropriate candidate. right ventricular assist devices (rvads) provide support to the right ventricle (rv) and allow recovery much the same as do lvads. the main contributing factor to right ventricular failure is an elevated pulmonary vascular resistance; however, it can also be the result of an rv infarction, or inadequate intraoperative protection. indications for an rvad include the general indications for vads as well as a right atrial pressure greater than mmhg, left atrial pressure less than mmhg, and no tricuspid regurgitation. right heart bypass is established by connecting the right atrial cannula to a pulmonary artery cannula via a centrifugal pump. despite the presence of an rvad, adequate systemic flows depend on intact left ventricular function. management goals are an rvad flow of . l/min/m and an increase in left atrial pressure to mmhg while maintaining a right atrial pressure of - mmhg. impaired rvad support may be the result of hypovolemia or inadequate cannula drainage. during rvad support, if the patient becomes hypotensive it may be the result of hypovolemia, left ventricular dysfunction, or a decreased systemic vascular resistance. a tee to assess the left ventricular function may be appropriate at this time as well as the use of an inotrope or vasopressor. interval tee examinations may be used to assess the recovery of the right ventricle, and weaning criteria are the same as those for an lvad. from the standpoint of prognosis, generally patients requiring rvad have a poor prognosis. weaning is accomplished in only about % and survival to discharge in about %. biventricular failure occurs in - % of patients requiring postoperative circulatory assist. biventricular assist devices (bivads) support both pulmonary and systemic circulation and can even be used in periods of ventricular fibrillation. the indications for bivad implantation are a right atrial pressure greater than - mmhg, left atrial pressure greater than mmhg, no tricuspid regurgitation, and inability to maintain lvad flow greater than . l/min/m with a right atrial pressure greater than mmhg. it is not an unusual circumstance for lvad implantation to unmask right ventricular dysfunction and the need for an rvad. bivads are managed to create a sequential adjustment of rvad and lvad flow achieving a systemic flow rate of . l/min/m . the heparin requirements, the assessment of recovery, and device weaning are the same as for the lvad and rvad. weaning is accomplished in % of patients and survival to discharge in only %. this poor prognosis is a reflection of the adverse impact biventricular failure has on survival. to be optimally effective, circulatory assist devices as support for lco require adequate pulmonary function and gas exchange. in circumstances of compromised cardiac and pulmonary function, cardiopulmonary function support is also required. cardiopulmonary support (cps) is accomplished with a portable centrifugal pump, membrane oxygenator, heat exchanger, and heparin-coated tubing. this system is generally referred to as extracorporeal membrane oxygenation (ecmo). indications for ecmo or cps are those of vads in association with impaired oxygenation. ecmo can also be used for cath lab catastrophes or in support of high-risk angioplasty. , only two cannulae are required for ecmo/cps support, a venous drainage cannula and arterial perfusion cannula. if the sternum is open, the cannulation technique is the right atrium and aorta. the percutaneous cannulation can also be used using the common femoral artery and vein or the jugular vein. since this system does not completely divert all the blood from the lv (pulmonary venous return to the lv persists), the lv is not completely decompressed, and a beating heart and competent aortic valve is necessary. an iabp is frequently concomitantly used to provide augmented pulsatile coronary perfusion. the management of the patient on ecmo/cps is complicated and labor intensive. it requires an experienced, committed, and well-trained staff. preload must be optimized and the svr may need support with -agonist agents or vasopressin. pulmonary artery hypertension must be controlled and may require using inhaled nitrous oxide. if renal failure occurs, consider early continuous venovenous hemofiltration. ventilation with low tidal volumes is helpful. heparin-coated tubing may eliminate the need for full anticoagulation, but heparin anticoagulation is required to prevent excess fibrin formation in the oxygenator membrane. the activated clotting time (act) is maintained s by continuous heparin infusion. the results of ecmo/cps depend on the degree of organ dysfunction at the time of initiation and the indication for its use. if it was instituted for cardiac arrest, the survival is %. of those patients placed on ecmo/cps for postcardiotomy cardiogenic shock, - % will die on support and only half of those who do not will survive the hospitalization. patients who survived days had a % -year survival. , currently, there are a variety of mechanical assist drive devices available for ventricular assist. selection of the particular device depends on the length of support required. there are short-term devices and long-term devices. the short-term devices are non-implantable and employed if recovery of ventricular function is expected. the long-term devices function as bridges to transplant and may be a long-term alternative to transplant. these devices are pulsatile, implantable, and provide total support of circulation. the selection of a long-term support device is rarely a consideration in the acute care management of the postoperative open-heart patient. however, a working understanding of the short-term devices may be required in the management of the postoperative patient with low cardiac output. the complications of these devices include mediastinal bleeding, mediastinal sepsis, thromboembolic events, renal failure, malignant ventricular arrhythmias, respiratory failure, refractory systemic vasodilatation, and immunocompromise. most patients return to the intensive care unit following openheart surgery with an arterial line, foley catheter, and usually a thermodilutional swan-ganz catheter. the hemodynamic status of the patient can be determined by careful assessment of data provided by these monitoring devices. with information collected by these monitoring devices, an accurate and realtime profile of the patient's hemodynamic status can be calculated and appropriate therapeutic interventions prescribed. the following is a discussion of commonly encountered hemodynamic situations in the postoperative open-heart patient. this is a very common postoperative occurrence. it usually occurs with rewarming and responds well to volume expansion. if hypotension persists despite volume expansion, or if presenting hypotension is severe, consider temporizing with a vasopressor such as phenylephrine or norepinephrine. the systemic vascular resistance (svr) and cardiac output/index must be followed closely when using either drug. the hemodynamic effects of phenylephrine are purely -adrenergic and act to increase the systemic vascular resistance. it has no cardiac effects. the indirect cardiac effects include a decrease in cardiac output caused by an increasing afterload as well as a potential increase in the cardiac output by raising perfusion pressure in coronary arteries. patients may become refractory to the therapeutic effects of phenylephrine after several hours and may require a change to norepinephrine. the starting dose of phenylephrine is mcg/min and increase to effect up to mcg/min, with the usual dosage range of . - . mcg/ kg/min. if there is inadequate therapeutic response to phenylephrine, switching to norepinephrine may prove effective. norepinephrine has powerful -adrenergic properties and some weaker -adrenergic effects. the -adrenergic stimulation will increase the systemic blood pressure by increasing the svr. the -adrenergic effects will increase contractility and heart rate. clinically, the -adrenergic effects predominate and will increase myocardial oxygen demand and may cause a fall in cardiac output despite its -adrenergic effect on contractility. the vasoconstrictive effects of norepinephrine may increase organ perfusion pressure but decrease absolute blood flow and result in visceral ischemia; this is an important potential adverse effect of this agent. the initial dose of norepinephrine is mcg/min ( . mcg/kg/min) and titrate to effect. recall that at doses greater than mcg/min ( . mcg/ kg/min), visceral and peripheral perfusion is reduced to such an extent the patient may become acidotic. this is another common occurrence and is seen in patients with normal left ventricular function. it is related to an increased arterial resistance secondary to hypothermia and increased levels of circulating catecholamines, plasma reninangiotensin, and vasopressin. , , postoperatively, systemic hypertension is more commonly seen in patients with normal left ventricular function, preoperative hypertension, preoperative use of -blockers, and patients having aortic valve replacement. the adverse sequelae of systemic hypertension include exacerbation of any latent myocardial ischemia by increasing afterload, stresses on suture lines, a predisposition to bleeding, and an increased potential for stroke and aortic dissection. , hypertension may be the result of hyperdynamic cardiac function or peripheral vasoconstriction, or both; and a hemodynamic profile must be ascertained before initiating therapy so as to direct therapy at the appropriate cause. the usual criterion for pharmacologic treatment is a mean arterial pressure % above the upper level of the normal patientspecific mean arterial pressure (map), usually greater than mmhg, or arbitrarily, a systolic blood pressure greater than mmhg (map greater than mmhg). in managing the postoperative hypertensive patient, a few caveats are important to keep in mind. first, a patient with a history of longstanding hypertension or critical carotid stenosis may require a higher perfusion pressure to maintain adequate cerebral and renal perfusion. secondly, a patient with a tenuous aorta or thin-walled vein grafts may require a lower pressure to avoid suture line dehiscence and catastrophic hemorrhage. the treatment goal in this scenario is to lower the svr and reduce myocardial oxygen demand without adversely affecting coronary artery perfusion. the treatment of systemic hypertension in the early postoperative period is vasodilator therapy. this can be augmented with -blocker therapy, calcium channel blocker therapy, angiotensin converting enzymes (ace) inhibitor therapy, and sedation, depending on the clinical circumstances. the vasodilator of choice for systemic hypertension postoperatively is sodium nitroprusside (snp). snp has a rapid onset of action and can produce rapid and excessive hypotension, but it has a short half-life. it is imperative that filling pressures are optimized before beginning snp, or a hypotensive collapse will occur. snp relaxes smooth muscle and as such decreases arterial resistance in the systemic and pulmonary circuit. it also relaxes venous capacitance vessels. it should be used with caution in the setting of myocardial ischemia as it can produce a coronary steal phenomenon. it has the potential for either short-term cyanide toxicity or thiocyanate toxicity with prolonged use. snp can also cause hypoxemia by opening intra-pulmonary shunts. the dosage is initiated at . - . mcg/kg/min and titrated to a maximum dose of mcg/kg/min. nitroglycerine (ntg) is primarily a venous dilator that lowers blood pressure by reducing preload, filling pressures, stroke volume, and cardiac output. since its primary action is on venous vessels, it usually maintains arterial diastolic pressure, but at high doses can produce arterial dilatation of varying degree and lower coronary artery perfusion pressure. ntg must be used with care if the patient is hypovolemic or the cardiac output is marginal, as reducing preload further will reduce cardiac output further and produce a reflex tachycardia. ntg works best in the hypertensive patient with active ischemia and high filling pressures. the major adverse effect of ntg is methemoglobinemia and impaired oxygen transport. the dosage begins at . mcg/kg/min and can be titrated up to mcg/kg/min. hydralazine is a direct arterial vasodilator that can be used to unload the left ventricle and treat systemic hypertension. it produces arterial vasodilatation and usually a compensatory tachycardia. in the immediate postoperative period, it is used as a supplement to other agents and not as the primary drug for the management of hypertension. hydralazine most commonly is used in the hemodynamically stable patient that remains hypertensive several days postoperatively but is unable to take oral medications. the dosage is - mg iv bolus every h as needed. calcium channel blockers primarily produce antihypertensive effects by relaxing vascular smooth muscle. they are very effective for managing postoperative hypertension, but do have a variety of cardiovascular hemodynamic effects and conduction alterations specific to each particular agent. calcium channel blockers are also used for the treatment of coronary spasm and rapid atrial tachycardias as well as for hypertension. nicardipine is a strong systemic and coronary vasodilator that does not cause coronary steal or tachycardia. it has little or no effect on the venous system and can be used without great concern for altering preload. the onset of action is rapid and has a relatively long half-life of min. nicardipine is not a negative inotrope and has no effect on av conduction. the dosage is an initial iv bolus of . mg over min and repeat every min to a total dose of . mg, then begin an infusion of - mg/h. diltiazem also acts as a peripheral vasodilator that reduces svr; however, it decreases cardiac output as a result of its negative inotropic and chronotropic (slows av conduction) effect. diltiazem is a good choice when hypertension is associated with coronary spasm because it is a potent coronary artery vasodilator. it is also a good option if hypertension is associated with atrial fibrillation and a rapid ventricular response. the dosage is . mg/kg iv bolus over min and a repeat dose in min of . mg/kg, then an infusion of - mg/h. verapamil is a peripheral vasodilator with moderate negative inotropic and chronotropic effects. its indications for usage are similar to diltiazem. the dosage is . mg/kg iv bolus initially, then - mcg/kg/min infusion. nifedipine, like all calcium channel blockers, lowers blood pressure by reducing the svr. it has potent vasodilatory actions. it causes a slight increase in heart rate and inotropy. when compared to snp, an infusion of nifedipine has a more positive effect on cardiac output and a greater decrease in svr. it has no effect on venous capacitance and preload. nifedipine is a potent coronary vasodilator and is an effective agent for managing suspected coronary spasm or arterial conduit spasm. while an intravenous form is available, it is primarily given sublingually or orally at a dose of - mg every h. amlodipine acts on the svr as do all other calcium channel blockers and may result in an increased cardiac output as a result of decreasing afterload. it has no negative inotropic or chronotropic properties by virtue of its lack of effect on the sa and av nodes. amlodipine exerts its antihypertensive effect gradually over a -h span and is used mainly for the long-term management of hypertension. the dose of amlodipine is . - mg daily. -blockers reduce pressure by negative inotropic and chronotropic actions. they reduce contractility, lower stroke volume and cardiac output, and lower heart rate. these agents are used to control hypertension associated with normal or hyperdynamic cardiac output, especially if the patient is tachycardic. esmolol is an ultrafast, short acting, cardioselective agent. because it is so short acting, it is the -blocker of choice for transient hypertension in a hemodynamically unstable patient. it should be used with caution in a patient with marginal cardiac output. the reduction in blood pressure is generally greater than the reduction in heart rate. it is cardioselective and can be used in a patient with bronchospasm. the dosage is an initial dose of . - . mg/kg over min, followed by mcg/kg/min over min followed by a continuous infusion titrated to effect. if an adequate response is not obtained after the initial dose, another loading can be given followed by mcg/kg/min over min. there is little to be gained by cumulative doses of more than mcg/kg/min. labetalol has -adrenergic and -adrenergic blocking effects as well as a direct vasodilatory effect. the -adrenergic blocking effect prevents reflex vasoconstriction. this agent is used when a longer-acting antihypertensive effect is needed because its duration of action is h. labetalol has a rapid onset of action resulting in a blood pressure response within min. the dosage is . mg/kg iv bolus over min, with subsequent dosing at . mg/kg every min until desired effect is reached or a total dose of mg is administered. metoprolol is a cardioselective -blocker used mainly to control ischemia or to slow ventricular response in atrial fibrillation, but rarely can it be used to treat postoperative hypertension. the onset of action is min and duration of action is about h. the dosage is mg iv bolus every min until the desired effect is reached or a total dose of mg. propranolol is a non-cardioselective agent with a long duration of action and has negative inotropic effect and as such is rarely used to treat postoperative hypertension. the dosage is in . mg increments given every - min until desired effect is reached or a total dose of . mg/kg. enalaprilat is an ace inhibitor that reduces blood pressure by inhibiting the activation of the renin-angiotensin system. it causes a balanced arterial and venous dilatation and acts to reduce myocardial oxygen consumption by its action on preload and afterload. it generally does not cause a reflex tachycardia. enalaprilat can be used alone or as a supplement in situations requiring high doses of nitroprusside or nicardipine. the onset of action is min and usually has a -h duration of action. the dosage is . - . mg iv over min every h. it can be used as a continuous infusion of mg/h with a doubling of the dose every min until the desired effect is reached or a total dose of mg. fenoldopam mesylate is a dopamine receptor agonist that is a rapid-acting peripheral and renal vasodilator. it is indicated for the short-term management of severe hypertension. fenoldopam mesylate causes a rapid fall in blood pressure and a reflex tachycardia. other hemodynamic effects include increase in stroke volume index and cardiac index attributed to the fall in svr. there is also an associated fall in pulmonary vascular resistance that may make its use beneficial in patients with pulmonary artery hypertension and rv failure. these properties make it an option for the management of postoperative hypertension in the cardiac surgical patient. it also has a beneficial effect on the kidneys. it dilates renal afferent arterioles and increases renal blood flow. the dosage of fenoldopam mesylate is an initial infusion of . - . mcg/ kg/min and increases at increments of . mcg/kg/min to the desired effect or a maximum of . mcg/kg/min. the renoprotective dose is . mcg/kg/min and is usually not associated with hypotension. while it has been shown to be effective in the management of postoperative hypertension in the cardiac surgical patient, it is not cost-effective and should be reserved for instances when other agents are ineffectual. the two most common causes of this scenario are right ventricular failure and diastolic dysfunction. right ventricular failure is rarely an isolated clinical situation. when it is, it is the result of poor intraoperative protection or a right ventricular infarct. more commonly, it is associated with pulmonary artery hypertension, either preexisting or the result of infused vasoconstricting adrenergic agents, administration of blood products, a type iii protamine reaction, hypoxemia, acidosis, or a tension pneumothorax. the hemodynamic hallmark of rv failure is a central venous pressure (cvp) higher than the pulmonary artery diastolic pressure (pad) or pulmonary capillary wedge pressure (pcwp). tee is an excellent mode of rv assessment and diagnosis of rv failure. the treatment of rv dysfunction begins by optimizing preload to a cvp of - mmhg. pushing the cvp higher may result in rv dilatation and exacerbation of rv dysfunction. also, a distended rv can have an adverse effect on the lv by shifting the intraventricular septum into the lv and impairing lv filling and stoke volume. hypoxemia, hypercarbia, and acidosis must be corrected as these adversely affect rv function. there must be active transport of volume from the right atrium to the rv, so it is imperative that atrioventricular (av) conduction be maintained or established using sequential av pacing if necessary. the addition of inotropic support is often necessary. inotropes that support biventricular function and are pulmonary vasodilators should be selected. the phosphodiesterase inhibitors are reasonable agents, but their action on the svr may necessitate the use of -adrenergic agents and lead to further vasoconstriction of the pulmonary vasculature. isoproterenol may improve rv contractility, but its proarrhythmic effects may not be well tolerated. when rv failure is associated with an elevated pulmonary vascular resistance (pvr), it is mandatory to decrease rv afterload by using a pulmonary vasodilator. the pulmonary vasodilators have no direct effect on rv or lv inotropy. their effect is indirect by afterload reduction of the rv. nesiritide (see prior description) is a synthetic -type natriuretic peptide that reduces pulmonary artery pressure and unloads the rv. it also has vasodilatory effects on the svr and renal arterioles resulting in improved cardiac output and a synergistic effect with loop diuretics. , inhaled nitric oxide (ino) is a selective pulmonary vasodilator and decreases rv afterload. this results in enhanced rv performance. it has little, if any, effect on the svr. inhaled nitric oxide is administered through a ventilator circuit designed to mix o and no. this generates a low level of no , which must be monitored as it is toxic to lung parenchymal tissue. inhaled nitric oxide is quite effective, but it is cumbersome and expensive. the usual dose is - ppm administered through a ventilator circuit. prostaglandin e and its analogs, epoprostenol and iloprost, are potent pulmonary vasodilators effective in the treatment of pulmonary hypertension. these agents are most frequently used in cardiac transplantation, but have been used effectively after mitral valve surgery. , , diastolic dysfunction is a function of impaired myocardial relaxation. in the postoperative period, it results in lco with normal or elevated filling pressures in patients with normal or hyperdynamic lv function. it is commonly seen in small women with left ventricular hypertrophy from hypertensive cardiovascular disease or aortic stenosis. severe diastolic dysfunction is associated with reduced left ventricular compliance exacerbated by edema often associated with ischemic cross-clamping, reperfusion, and cpb. inotropic agents used to treat the lco in the postoperative period will worsen diastolic dysfunction. diastolic dysfunction is frequently associated with tachycardia. the filling pressures are high and stroke volume reduced because the impaired left ventricular relaxation leads to impaired filling of the lv and a deceased lv end-diastolic volume (lvedv). swan-ganz monitoring confirms high left-sided filling pressures and lco. the svr is elevated as a compensatory mechanism. tee is diagnostic. it confirms a hypertrophic lv with decreased compliance and filling. the lv may be so hyperdynamic as to obliterate the lv cavity at end-systole. diastolic dysfunction is difficult to manage. if not managed successfully, end-organ dysfunction is inevitable. the initial steps in management are to assure av synchrony and adequate preload. volume should be infused until the pcwp is - mmhg to increase lvedv. intuitively, it may seem inappropriate to give volume in the setting of elevated filling pressures, but the elevated filling pressures are the consequence of impaired lv compliance and not volume overload. inotropic agents should be replaced with lusitropic agents. ace inhibitors may improve diastolic compliance. calcium channel blockers also have some lusitropy and may be of benefit. finally, inamrinone and milrinone have lusitropic properties as does nesiritide. there is no one agent shown to be better than the others and often management requires courses of therapy and observation. if the patient can be guided through the first few days, the cardiac output gradually improves. arrhythmias cardiac arrhythmias carry a source of morbidity and mortality in the postoperative surgical patient. these arrhythmia are usually an indicator of some underlying abnormality and should alert the clinician to closely evaluate the patient. in addition to standard electrocardiograms (ekg), the temporary atrial and ventricular pacing wires are useful in the diagnosing and treatment of postoperative arrhythmias. the ideal postoperative rhythm is sinus rhythm at - bpm. sinus tachycardia is frequently seen in the early postoperative period and is most commonly caused by vasodilatation secondary to rewarming, reperfusion injury to the left ventricle secondary to cardiopulmonary bypass, sympathomimetic drugs, pain and anxiety as the patient awakens from anesthesia, normovolemic anemia, withdrawal from -blocker therapy, occasionally fever, and idiopathic. isolated ventricular ectopy may be an indication of ongoing myocardial ischemia, particularly within the first h postoperatively. other causes of ventricular ectopy are hypokalemia, hypomagnesemia, hypoxia, preexisting ectopy, sympathomimetic drugs, and mechanical irritation from the swan-ganz catheter. there remains controversy as to the significance of isolated ventricular ectopy. it is not clear what the incidence of isolated premature ventricular contractions (pvcs) degenerating to malignant ventricular arrhythmias actually is. however, most agree that in the presence of active myocardial ischemia, pharmacologic suppression is indicated and this concept includes those patients in the first h after surgery when the myocardium may be irritable. unlike chronic pharmacologic treatment of isolated ventricular ectopy, treatment in the acute postoperative period is not usually associated with the risk of proarrhythmia. treatment is particularly beneficial in patients with lv dysfunction and ejection fractions less than %. in the first h after surgery, ventricular ectopy is treated if the ectopic beats occur at a rate greater than beats/min or ventricular tachycardia of less than min. the treatment of pvcs begins with the correction of any underlying correctable cause such as hypokalemia or hypomagnesemia. if atrial wires are present, overdrive atrial pacing at a rate greater than the current sinus rate can be tried. lidocaine is the initial drug treatment for ventricular ectopy. the dosage is an initial loading dose of mg/kg as an initial bolus followed by one or two additional doses of . mg/kg mg every min. after the initial bolus, an infusion of - mg/min can be started. an alternative option is an initial bolus of mg followed by a loading infusion of mg over min. the loading dose is followed by a maintenance dose of . - . mg/min. if the ectopy is uncontrolled, an additional bolus of - mg can be given and the infusion rate increased. lidocaine toxicity is a significant risk at infusion rates greater than mg/min, especially in the elderly. if lidocaine does not suppress ectopy, it can be elected not to treat unless ventricular tachycardia occurs or with intravenous amiodarone. sustained ventricular tachycardia (vt) or ventricular fibrillation (vf) are usually associated with acute myocardial ischemia or infarction or an electrolyte imbalance, but can occur without the obvious presence of either. these arrhythmias are most often seen in patients with previous infarcts and subsequent revascularization to the infarcted area, and occur with a frequency of - % after cardiac surgery. reperfusion of areas of ischemia or infarction can precipitate vt of vf as the areas of ischemic myocardium are reperfused. the reperfusion arrhythmias occur in patients with unstable angina, recent infarction, and ejection fractions of less than %. in these circumstances, nonviable myofibrils embedded in the scar are triggered and this leads to an altered dispersion of repolarization and the development of reentry arrhythmias. the resultant ventricular arrhythmia is usually a sustained polymorphic vt with a normal qt interval as compared to the monomorphic vt noted in patients with a previous myocardial infarction and depressed lv function. this reentry arrhythmia rarely responds to lidocaine and usually requires amiodarone and possible -blockade. the treatment of nonsustained vt in patients with preserved lv function is similar to the treatment of pvcs. in patients with ejection fractions less than % and nonsustained vt, the prognosis is poor without treatment, and an electrophysiologic evaluation is necessary as an implantable cardioverter-defibrillator may be indicated. sustained vt without hemodynamic instability can be managed with ventricular overdrive pacing. cardioversion may be necessary if overdrive pacing is not successful or if the patient becomes unstable. an amiodarone bolus of mg infused over min followed by an infusion of mg/min for h, then . mg/ min for h should be prescribed. these patients will ultimately need an electrophysiologic evaluation. all patients with vt or af with hemodynamic instability require immediate defibrillation as per acls protocol. if the patient is unresponsive to defibrillation or persistence of hemodynamic instability, the sternotomy must be reopened emergently at the bedside. torsades de pointes is an uncommon but malignant arrhythmia not often related to the postoperative cardiac surgical patient. on the ekg monitor, the qrs complex appears to "twist" around the isoelectric baseline. its onset is usually pause-dependent, initiated by a pvc occurring at the end of a t wave. it is usually associated with a prolonged qt interval. treatment of torsades de pointes is immediate cardioversion. if the patient is not hyperkalemic, potassium chloride should be administered to shorten the qt interval. magnesium and -blockers may eliminate the trigger and prevent recurrence. finally, ventricular pacing at - bpm or an isoproterenol infusion of - mcg/min will shorten the action potential and prevent early afterdepolarization. , be aware that a wide complex tachyarrhythmia does not necessarily indicate ventricular tachycardia because atrial fibrillation with a rapid ventricular response can result in rbbb with aberrant conduction (so-called ashman phenomenon) mimicking ventricular tachycardia. atrial fibrillation (af) is the most common arrhythmia after cardiac surgery. despite the recent institution of prophylactic regimens for af, the overall incidence remains - %. it has an occurrence of - % after coronary artery bypass graft (cabg) surgery and up to % of patients undergoing combined cabg valve procedures. [ ] [ ] [ ] after on-pump coronary artery bypass surgery, the incidence is - %, , after minimally invasive cabg it is %, and following valve surgery it is - %. , there is controversy as to whether off-pump cabg has a lower incidence of af. patient's age appears to be the most powerful predictor of the occurrence of af. the incidence is . % in patients less than years of age and % in those older than . , other predictors are a history of congestive heart failure, preoperative atrial fibrillation, and chronic obstructive pulmonary disease. [ ] [ ] [ ] atrial fibrillation is most likely to occur - days after surgery. the episodes of these arrhythmias may recur or persist for up to weeks before resolving spontaneously. ten to % of patients are discharged in atrial fibrillation whereas % will return to sinus rhythm within - days with only digoxin or -blockade therapy. , [ ] [ ] [ ] it is a leading cause for readmission after early discharge. the management of postoperative af begins with an assessment of the patient. if the patient is unstable, immediate cardioversion is indicated. a synchronized shock of - j is applied. rarely is this the only treatment necessary, as the patient often reverts back to af, especially if this occurs in the early postoperative period. if the patient is hemodynamically stable, the initial treatment of postoperative af is rate control and is indicated if it lasts longer than - min or is associated with severe symptoms. the most important aspect of the treatment of postoperative af is the control of the ventricular rate. in many protocols, the first-line agent for rate control is the calcium channel blocker diltiazem. therapy is initiated with a bolus of . mg/kg over min and followed by an infusion of - mg/h to titrate the heart rate to less than bpm. slowing of the ventricular rate is usually noted within min and is more effective for atrial fibrillation than atrial flutter. the use of diltiazem is limited by hypotension, which occurs with an incidence of - %. , pretreatment with mg of calcium may lessen the hypotensive effect. diltiazem has a mild negative inotropic effect and must be used with caution in patients with compromised left ventricular function. while diltiazem is extremely effective in slowing the ventricular rate, it converts fewer than % to sinus rhythm. verapamil can be used in lieu of diltiazem for rate control in rapid atrial fibrillation. begin with a bolus of - mg, then an infusion of - mg/h. if the blood pressure is tenuous, pretreat with - , mg of calcium chloride. while calcium channel blockers are effective rate control agents, they are not as effective as -blockers in converting patients back to normal sinus rhythm (nsr). beta-blockers are equally or more effective for rate control and also can effect conversion to nsr % of the time. , they are not used as frequently for postoperative af by some clinicians because of their negative inotropic properties. esmolol is a short acting, selective -blocker. it must be used in an icu setting with appropriate monitoring because of its propensity to cause hypotension, particularly in patients with poor lv function. the loading dose is . - . mg/kg over min followed by an infusion of - mcg/kg/min. metoprolol has less of a tendency to cause hypotension and is more suited for use in a non-icu area. it is a long-acting, selective -blocker. it is dosed at mg iv every min to a total dose of mg. digoxin has only a modest response in the acute setting. there is only a - % decrease in ventricular rate with digoxin alone. at least half of the patients remain in af after the rate has been slowed. an effort should be made to cardiovert the patient back to sinus rhythm. if the patient is hemodynamically unstable, electrical cardioversion is an option. there is a high incidence of recurrent atrial arrhythmia unless an antiarrhythmic regimen is instituted. currently in many institutions the antiarrhythmic of choice is amiodarone. amiodarone has properties of class iii antiarrhythmics and -blockade. it is becoming the drug of choice for postoperative af because it is safe and effective. it is associated with only modest hypotension and has no proarrhythmic effects. it does slow the ventricular rate as effectively as -blockers or calcium channel blockers, which are often used as adjuncts to amiodarone. it does have a higher rate of cardioversion than either calcium channel blockers or -blockers. amiodarone has the same frequency of cardioversion as type c antiarrhythmics, but takes longer. amiodarone has fewer adverse side effects than those antiarrhythmics. it can be given intravenously, but is just as effective orally for non-life-threatening arrhythmias. the half-life of the drug is long, up to days, and its long-term use is associated with visual disturbances, tremors and other neurologic sequelae, hepatitis, pulmonary fibrosis, photosensitivity, skin discoloration, thyroid abnormalities, and cardiac conduction disturbances. these side effects, however, are rarely a factor when used to treat postoperative atrial fibrillation because amiodarone is administered only for weeks. if given intravenously, the initial loading dose is mg over min, followed by an infusion of mg/min for h, then . mg/min for h. an oral taper dose is then prescribed of mg bid for week, mg daily for week, then mg daily for weeks. if the patient has no further episodes of af, it can be discontinued at that time. procainamide is a type a antiarrhythmic that once was a first-line antiarrhythmic for the postoperative cardioversion of af in most centers. it restores nsr in % of patients within min. procainamide is proarrhythmic and has a mild negative inotropic effect. it is associated with more short-term side effects than amiodarone. it has vasolytic properties and as such should not be used until the ventricular rate has been slowed to less than bpm. the loading dose is an intravenous bolus of mg/kg (dose not to exceed g total) at a rate not exceeding mg/min. this can be followed by an infusion of mg/min or converted to an oral procainamide derivative in h. up to one-third of patients cannot tolerate procainamide because of gastrointestinal, hematological, or immunologic side effects. this drug is cleared by the kidneys and blood levels of procainamide and its active metabolite, n-acetyl procainamide (napa), should be monitored, particularly, in patients with renal and hepatic dysfunction. ibutilide is a rather new agent for the treatment of postoperative atrial fibrillation. the incidence of torsades de pointes is about - %, which is considerably higher than with either procainamide or amiodarone. ibutilide is useful in patients with poor left ventricular function or chronic lung disease, but its use is limited by its proarrhythmic effect. conversion to sinus rhythm occurs at a rate of - % for atrial fibrillation and - % for atrial flutter. the dose begins with a bolus of mg over min with a second infusion min later. no further dosing is indicated. the drug must be stopped if qt prolongation occurs as it may contribute to torsades, but sustained polymorphic ventricular tachycardia may occur even in the absence of qt interval prolongation. there are several strictly oral agents that can be used for pharmacologic conversion back to sinus rhythm. sotalol is useful as a single-agent therapy for atrial fibrillation cardioversion. it is a class iii antiarrhythmic with beta-blocking activity. it can cause prolongation of the qt interval and initiation of therapy must be done while monitoring the patient. the drug is limited mainly by its beta-blocking effects such as reactive airway disease, depression, and negative inotropy. the dose is - mg twice daily. quinidine is still used by some clinicians for the conversion of atrial fibrillation to sinus rhythm. it may be slightly more effective than amiodarone, but it is being used with decreasing frequency. , though quinidine is cost-effective and has very little negative inotropy, it is associated with a high incidence of side effects, particularly gastrointestinal, neurological, and hematological. also, the proarrhythmic and frequent dosing make other agents a better choice. flecainide can also be used for the management of atrial fibrillation. flecainide was found to be associated with an increased mortality when given after a myocardial infarction, and created much concern when given with ischemic heart disease. it is not recommended for patients with structural heart disease. postoperative atrial fibrillation is associated with increased morbidity and cost; therefore, there is great interest in the prophylaxis of postoperative atrial fibrillation. multiple trials and multiple protocols have been investigated searching for an effective prophylactic regimen. the most effective and practical regimens all include preoperative -blockade therapy started - h preoperatively. [ ] [ ] [ ] [ ] beta-blockade therapy given preoperatively and through the postoperative period is superior to their use only postoperatively. when given preoperatively and postoperatively, the incidence of af is %. [ ] [ ] [ ] magnesium sulfate has been used as prevention for postoperative af. hypomagnesemia is common after cardiac surgery and is associated with atrial arrhythmias. there is a debate as to whether routine magnesium administration lowers the incidence of postoperative af. it may be effective when used with -blockers and when the serum magnesium is low. , since it is relatively benign and may be potentially effective, some recommend its routine administration through the first postoperative day. sotalol is a -blocker with class iii antiarrhythmic properties. it reduces the incidence of postoperative af by as much as % when given preoperatively and postoperatively. because it has -blocker action, it must be used with caution in patients with lv dysfunction and those with marginal systemic blood pressure. it is excreted by the kidneys and is not recommended in patients with renal insufficiency. sotalol can also cause qt interval prolongation and has been associated with torsades de pointes. it is not well tolerated in % of patients and must be withdrawn. the dose of sotalol is mg twice daily. amiodarone is a class iii antiarrhythmic with some properties of class i, ii, and iv drugs. it is as effective as sotalol in preventing postoperative af and can be used alone or in conjunction with -blockers. [ ] [ ] [ ] [ ] amiodarone is particularly useful in patients with intolerance to -blockers. it is rarely associated with pulmonary toxicity when used as a short-term therapy, but the rare incidence of amiodarone toxicity can cause hypoxemia. as prophylaxis, amiodarone is started in the operating room as a mg bolus over min followed by an infusion of mg/min for h then . mg/min for h. the oral dose of mg twice daily is continued for week. if the patient should develop af, a -week regimen is recommended. in the event the patient should develop af with either the sotalol or amiodarone prophylactic regimen, the ventricular response rate is usually slow and easier to manage. the efficacy of both sotalol and amiodarone as prophylaxis is better if started several days preoperatively. postoperative stroke as a consequence of atrial fibrillation is well documented. the incidence of stroke is between and % in patients with postoperative atrial fibrillation as compared to - . % in patients without atrial fibrillation. , the risk of embolic stroke is substantial after h or more of atrial fibrillation. all patients with postoperative atrial fibrillation should be anticoagulated unless there is a contraindication. anticoagulation should be started within - h of the onset af. bradycardia requiring pacing occurs in approximately % of postoperative patients. the most common defect is right bundle branch block (rbbb). about % of the patients will have permanent conduction abnormalities. the associated bradycardia is treated with temporary epicardial pacing. the most commonly used mode is ventricular pacing. in all the open-heart patients, temporary epicardial ventricular pacing wires are fixed to the right ventricle and, in many, right atrial wires are also placed. bradycardia from any etiology is an indication for ventricular pacing. if the patient is hemodynamically unstable with simple ventricular pacing, physiologic pacing may be required if atrial electrodes are available. if an atrial electrode was not fixed to the heart, a temporary transvenous atrial pacing electrode can be inserted. simple ventricular pacing is accomplished by connecting the temporary electrodes to an external pacemaker. these pacemaker units are bipolar and require the ventricular lead electrode be connected to the negative pole and an indifferent electrode, often a skin wire, connected to the positive pole of the pacemaker. the output is set initially at ma and the threshold adjusted to assure a safe margin of capture. a decision is then made as to the mode of pacing; i.e., synchronous (demand) or asynchronous (fixed). the synchronous mode is chosen to avoid pacer stimulation on the t wave and the resulting ventricular fibrillation. the asynchronous mode is used only in unusual situations, such as the use of electrocautery, when other electrical activity interfere with the sensing in the synchronous mode. the rate must be set depending on the needs of the patient. physiologic pacing requires choosing the desired mode, atrial thresholds, atrioventricular intervals, as well as the ventricular settings. failure to pace may be the result of faulty electrical connections, dislodgment of the epicardial electrodes from the heart, a faulty pacemaker, the development of electrically silent areas of the myocardium in the region of the electrodes, or the development of a rhythm incompatible with pacing such as atrial or ventricular fibrillation. postoperative bleeding is always present to some extent. it is related to mechanical factors and coagulopathy. mechanical factors are considered surgically correctable. less than % of postoperative bleeding is from surgically correctable causes. it is usually indicated by bleeding greater than ml/h with normal or near-normal coagulation studies. mechanical bleeding is characterized by clots in the drainage tubes. coagulopathy is present to some extent in all patients after cardiopulmonary bypass. with the current aggressive use of percutaneous catheter intervention for the treatment of various acute coronary syndromes (acs), drug-induced coagulopathy is frequently seen. following deployment of stents for acs, patients are placed on platelet inhibitors such as glycoprotein iib/iiia inhibitors (eptifibatide, tirofiban, or abciximab) or the adp binding inhibitor clopidogrel. in some instances, acute myocardial infarctions are treated with thrombolytic therapy and this results in a profound coagulopathy. , fibrinolysis results from the activation of the fibrinolytic system either intrinsically from cardiopulmonary bypass or therapeutically from preoperative thrombolytic therapy. , this appears to be the primary cause in coagulopathy following cardiopulmonary bypass (cpb). a progressive fibrinolytic state occurs and its intensity is directly related to the duration of cardiopulmonary bypass. it is associated with the degradation of clotting factors as well as platelet dysfunction. platelet defects are also an important cause of postoperative bleeding. the platelet-related bleeding diathesis is a result of a decrease in the absolute platelet number, and more importantly, secondary to impaired platelet function. , the decrease in the platelet number, or quantitative defect, results from hemodilution, preoperative thrombocytopenia from medications, and the consumption of platelets by the cardiopulmonary bypass circuit. the cpb circuit itself can reduce the platelet count by - % and worsens as the duration of bypass lengthens. the diminished platelet function, or qualitative defect, may be directly related to the duration of cpb. passage of platelets through the cardiopulmonary bypass circuit results in decreased platelet membrane receptors for fibrinogen and glycoprotein ib and glycoprotein iib/iiia complex. thrombocytopenia may also be caused by heparin-induced thrombocytopenia. this usually occurs in patients with a previous exposure to heparin within months. it is the result of heparin antibodies causing platelet aggregation. there is often a history of heparin resistance during cpb. the qualitative defect in platelets may also be related to preoperative medications such as aspirin, heparin, and the glycoprotein iib/iiia inhibitors. , residual heparin effect can account for a postoperative bleeding diathesis. heparin effect is usually reversed by the time the patient gets to the intensive care unit. it should always be considered as a possibility in the bleeding patient. heparin rebound is the recurrence of measurable heparin activity after complete protamine neutralization. it is associated with larger heparin doses given intraoperatively, after long cpb runs, and obese patients. it is thought to be the result of elution of heparin from plasma proteins hypothermia is a significant cause for postoperative coagulopathy. the coagulation cascade is mediated by enzymatic reactions. these reactions are temperature-sensitive and occur most efficiently at normothermia. hypothermia retards the normal coagulation cascade as a result of this altered enzymatic activity. hemodilution of cpb is another source of coagulopathy and affects all blood elements including coagulation factors. most factors are reduced by % and factor v by %. this phenomenon affects patients with small blood volumes more profoundly. also, coagulation factors are lost with cell saving. an attempt should be made to specifically diagnose the coagulopathy. the specific abnormalities can usually be diagnosed if appropriate studies are ordered. platelet defects are both quantitative and qualitative. the diagnosis of quantitative defects, thrombocytopenia, can be made early in the postoperative period with a simple platelet count. if thrombocytopenia occurs later in the course, consider hit and obtain a heparinplatelet aggregation test to confirm the presence of heparin antibodies. qualitative platelet defects, thrombasthenia, can be present with a normal platelet count but platelet function will be abnormal and the clot formation inadequate. the bleeding time is prolonged and indicates abnormal platelet aggregation and adhesiveness. residual heparin effect is diagnosed by a prolonged partial thromboplastin time (ptt) and/or activated clotting time (act). either a ptt or an act should be measured on admission to the intensive care unit because inadequate heparin reversal with protamine is usually seen early in the postoperative period. generally, other laboratory values will be normal. a heparin-protamine titration test can be performed if the hepcon system (medtronic inc., minneapolis, minnesota) is available. this test directly quantifies the amount of heparin circulating. it will detect any residual heparin and also allow for a calculation of the appropriate dose of protamine needed to neutralize the residual heparin. if the ptt or act are elevated h after the last heparin dose, it is unlikely secondary to heparin as the half-life of heparin is h; if heparin effect is suspected at this time, obtain heparin levels to confirm the diagnosis. fibrinolysis is associated with an elevated pt and ptt; decreased levels of factors i, v, and viii; rapid euglobulin clot lysis; and the presence of d-dimers. d-dimers indicate the presence of fibrin monomers, and their presence is diagnostic for fibrinolysis if accompanied by decreased fibrinogen levels. an elevated d-dimer alone is not uncommon, particularly if shed blood is being reinfused and in itself is not diagnostic of fibrinolysis. disseminated intravascular coagulation (dic) is the severest form of coagulopathy. from a laboratory standpoint, it is manifested by an elevated pt and ptt, decreased fibrinogen levels, thrombocytopenia, and an elevated fibrinsplit products (greater than mcg/ml) and d-dimer. dic is rarely seen in the early postoperative period and usually is associated with other complications. thromboelastography and sonoclot analysis are two studies available in some institutions that have been shown to specifically identify the source of the bleeding diathesis. these studies are not commonly available. coagulation factor deficiencies either from hemodilution or true deficiencies can be diagnosed by measuring the specific factors, but in the acute setting this may not be practical as obtaining these results is time-consuming. increased ptt and pt (prothrombin time) usually manifest factor deficiencies. specific studies can be ordered, but it is usually reasonable to proceed with the empiric treatment before results are available. there must be a high degree of suspicion for factor deficiencies in the patient with a previous or family history of abnormal bleeding, liver disease, prior warfarin therapy, hemodilution, or clinical evidence of disseminated intravascular coagulation. the treatment of a postoperative coagulopathy must be prompt and aggressive. the bleeding cycle must be interrupted as "bleeding begets bleeding." the specific treatment consists of blood component therapy based on an accurate diagnosis. initial therapy begins by sending coagulation studies to include a pt, ptt, platelet count, and fibrinogen level. then, notify the blood bank that component therapy will be needed and an adequate supply of cross-matched packed red blood cells, fresh frozen plasma (contains all coagulation factors except platelets), cryoprecipitate (factor viii and fibrinogen), and platelet concentrates should be readily available. next, hypothermia should be corrected. within the first h and even before the coagulation studies are available, consider the empiric use of protamine sulfate in the event residual heparin or heparin rebound is the cause. if the bleeding continues after the hypothermia is corrected and the empiric protamine is given, an algorithmic approach can be used. this algorithm begins by sending coagulation studies. then, transfuse platelets, unit/ kg body weight, and draw post-transfusion platelet count. if the bleeding continues and the posttransfusion platelet count is less than , , repeat the platelet transfusion of unit/ kg body weight. if the posttransfusion platelet count is greater than , , but the fibrinogen is less than mg/ ml, give unit of cryoprecipitate/ kg body weight. if the posttransfusion platelet count is greater than , , but fibrinogen is greater than mg/ ml, and the pt or ptt is less than . times control value, recheck for surgical bleeding and do a bleeding time; and if it is greater than min, give desmopressin . mcg/kg iv. if the posttransfusion platelet count is greater than , , but the fibrinogen is greater than mg/ ml, and the pt or ptt is greater than . times control value, give fresh frozen plasma ml/kg. if bleeding persists at the completion of the algorithm, consult a hematologist. in addition to blood component therapy, there are drugs available for the treatment of postoperative coagulopathy. protamine is the specific drug for the reversal of heparin. the dosage is - mg increments given iv over min. be aware there are three types of adverse reactions to protamine administration. type i reaction is systemic hypotension from rapid administration that usually occurs if the entire neutralizing dose is given in less than min. it is a histamine release reaction that causes a reduction in the svr and pvr. it can be avoided by giving the dose over - min. type ii reaction is an anaphylactic or anaphylactoid reaction resulting in hypotension, bronchospasm, flushing, and edema. it is further divided into type iia that is an idiosyncratic reaction mediated by ige or igg and is caused by the release of histamine or leukotrienes producing a capillary leak syndrome with hypotension and edema. it usually occurs within the first min of administration. type iib is an immediate reaction and is not related to immunoglobulins. type iic is a delayed reaction occurring after min or longer, and seems to be related to complement activation and leukotriene release producing bronchospasm and a capillary leak syndrome that leads to hypovolemia and noncardiac pulmonary edema. type iii reaction is catastrophic pulmonary vasoconstriction with acute pulmonary hypertension, right ventricular failure, and severe peripheral vasodilatation with hypotension and myocardial depression. it occurs - min after the protamine is given and is thought to be secondary to the heparin-protamine complex. this complex incites leukocyte aggregation and the release of liposomal enzymes that damage pulmonary tissue. type iii reactions are highly lethal unless cardiopulmonary bypass can be reinstituted to support the patient. treatment is initially calcium chloride and -agonists to support the svr. it may also be beneficial to add -agonists to reduce the pvr. specific drugs to lower the pvr (such as prostaglandin e) may be helpful, but usually it is necessary to readminister heparin and reinstitute cardiopulmonary bypass. desmopressin (ddapv) has not been shown to be of benefit in the uncomplicated patient, but is of value in patients with platelet dysfunction secondary to uremia, liver dysfunction, and antiplatelet medications. [ ] [ ] [ ] it is specific therapy for patients with an acquired defect in platelet plug formation as a result of a deficiency in von willebrand's factor. the dosage is . - . mcg/kg iv over min. epsilon-aminocaproic acid (eaca) is an antifibrinolytic agent that inhibits conversion of plasminogen to plasmin. it may act to preserve platelet function. eaca is best used when given before cardiopulmonary bypass prophylactically, but it can also be used as a rescue agent for severe bleeding, especially if fibrinolysis is present. it should be used with caution or not at all with aprotinin as the combination appears to cause a prothrombotic state with associated graft closure, renal dysfunction, and stroke. the rescue dose for postoperative bleeding is usually - g iv bolus. aprotinin is a serine protease inhibitor that preserves adhesive platelet receptors (gpib) during the early phase of cardiopulmonary bypass. it also has antifibrinolytic properties by inhibiting plasmin. aprotinin has been demonstrated to reduce blood loss when given before and during cardiopulmonary bypass in patients at high risk for postoperative bleeding, such as thrombocytopenia, uremia, hepatic dysfunction, and long complex procedures, particularly reoperations. it does have a role as a rescue agent for postoperative bleeding, but must be used with caution as it may be prothrombotic in the nonheparinized patient. the rescue dose is two million kiu. aprotinin therapy has been associated with an increased morbidity and mortality in some studies and its use is controversial. blood component therapy includes packed red blood cells (rbcs), fresh frozen plasma (ffp), cryoprecipitate (factor viii and von willebrand's factor), and platelets. rbc transfusion should be managed by protocol and determined by the clinical status of the patient. rbcs are indicated in the anemic patient with normal lv function when the hematocrit is - %. , if the patient is actively bleeding, the hematocrit should be maintained at % to afford a margin of safety. if the patient is elderly or has lv dysfunction and cannot increase the cardiac output in response to anemia, the hematocrit should be maintained at a higher level. platelet transfusions are indicated for a platelet count under , if the patient is bleeding excessively. ffp is recommended in the excessively bleeding patient for an inr (international normalized ratio) of greater than . - . . specific treatment with cryoprecipitate and other components is indicated in the presence of a consumptive coagulopathy as reflected by a diminished fibrinogen level, positive d-dimer assay, or the presence of fibrin degradation products. blood conservation is an important part of managing the postoperative patient both with and without significant bleeding. there are preoperative measures, intraoperative measures, and postoperative measures. the preoperative measures include autologous blood donation for elective cardiac procedures. this must be done with care, particularly in the patient with ischemic heart disease or congestive heart failure secondary to valvular heart disease. therefore, it is not a measure widely practiced. another preoperative measure is the modification of the preoperative antiplatelet regimen within limits of therapeutic prudence. and, finally, preoperative erythropoietin can be used in the anemic patient to improve hemoglobin levels sufficiently to avoid perioperative transfusions. intraoperatively, the crystalloid prime of cardiopulmonary bypass circuit with resultant hemodilution to hematocrit of - % minimizes the loss of red cells. also, blood salvage with reinfusion of washed, centrifuged red cells, both from the field and from the circuit after separation from cardiopulmonary bypass, conserves blood. careful operative hemostasis is a must for blood conservation. postoperative autotransfusion and cell saving also conserve blood and reduce the complications of transfusions. the "cell saver" in most institutions has supplanted traditional autotransfusion techniques. the cell saver is a system that combines washing and centrifuging shed blood before reinfusing, as opposed to directly reinfusing shed blood after passing it though a filter. shed blood does not require an anticoagulant because it has undergone fibrinolysis, unless the hemorrhage was extremely rapid. shed, traditional autotransfused blood has low levels of factors viii and fibrinogen as well as platelets, but the platelets present are dysfunctional. autotransfused blood does contain fibrin-split products. conversely, cell saver blood is devoid of clotting factors and platelets as well as fibrin-split products. transfusion of less than one liter of either autotransfusion blood or cell saver blood is without significant risk of exacerbating a coagulopathy. transfusion of greater amounts can potentially worsen the coagulopathy by infusing fibrin monomers, in the case of autotransfusion, and from platelet and factor depletion with both. , autotransfusion of greater than , ml of shed blood should be avoided and blood component therapy should be used to augment reinfusion of cell saver blood to avoid depletion of platelets and clotting factors. multiple factors contribute to postoperative bleeding. , despite deficiencies in the coagulation cascade and multiple potential sites of surgical bleeding, mediastinal drainage slows over the first few hours in the majority of patients. aggressive management of the bleeding patient is generally successful, such that only about - % of patients require reoperation for persistent bleeding. normally, when the patient returns from the operating room, mediastinal drainage is in the order of - ml/h for the first - h and ml/h thereafter. the initial steps in managing the bleeding patient after openheart surgery are aggressive treatment of hypothermia and hypertension, order coagulation studies, notify the blood bank to have blood products available, and consider an empiric dose of protamine. if coagulation studies indicate a coagulopathy, proceed with the algorithm for management. in any patient with excessive mediastinal drainage, cardiac tamponade must be considered. be alert for the followings signs of tamponade: equalization of filling pressures, low cardiac output, hypotension, wide respiration variation of systolic blood pressure with positive pressure ventilation, and a narrowed pulse pressure. at times, the classic findings of tamponade may be absent, but the following points may signal tamponade: the sudden cessation of chest tube drainage, progressive low cardiac output in a patient with a previously normal cardiac output, an unexplained left or right heart failure, severe peripheral vasoconstriction with cyanosis of the ears and digits, progressive fall in the urine output, an unexplained tachycardia, mediastinal widening on chest x-ray, pleural effusion, and diminished ecg voltage. there are caveats regarding cardiac tamponade in the immediate postoperative setting. first, a pulsus paradoxus is not an applicable sign of tamponade in the patient on positive pressure ventilation. positive pressure ventilation reverses blood pressure response to respiration. on the ventilator, during early inspiration, the positive airway pressure causes a compression of the pulmonary veins augmenting left heart filling and thus blood pressure, whereas, later in the inspiratory cycle, left heart filling is diminished and the blood pressure falls. this early rise in the blood pressure is opposite of the fall in blood pressure seen during spontaneous inspiration and makes pulsus paradoxus an unreliable sign of tamponade during positive pressure ventilation. also, it is not unusual for a clot to accumulate next to the right or left atrium and cause unequal elevations of the ra or la pressures. most important, the diagnosis will be made only if a high degree of suspicion is maintained. the diagnostic modality of choice for cardiac tamponade in the postoperative period is transesophageal echocardiography. the definition of excessive mediastinal bleeding is ml/h for h, ml/h for h, and ml/h for h. if mediastinal bleeding persists despite correction of the coagulopathy or if the patient demonstrates evidence of hemodynamic compromise, mediastinal reexploration in the operating room is indicated. an aggressive approach to mediastinal reexploration is in the best interest of the patient. reexploration is associated with increased mortality and morbidity usually because of a delay in proceeding. early reexploration reduces these complications. an emergency reexploration in the intensive care unit is indicated for exsanguinating hemorrhage or impending arrest from any cause. the technique for emergency reexploration begins with a call for the necessary assistance. intubate the patient if necessary and hand ventilate the patient with inspired oxygen of %. remove the dressing and pour antiseptic over the sternotomy incision and block drape the site with sterile towels. reopen the incision with a scalpel and cut or untwist the wires. the sternum is opened with a sternal spreader. then, evacuate the hematoma and attempt to identify the source of bleeding. if a bleeding site is identified, tamponade it with digital pressure. proceed to complete the resuscitation of the patient. ideally, the site of hemorrhage should be repaired in the operating room, but if this is not practical or feasible, repair it in the icu. if internal cardiac massage is needed, do so with two hands by placing the left hand beneath the heart and compressing the anterior aspect of the heart with the right hand using the palm and flattened fingers and take care not to injure the grafts. if the patient has a prosthetic mitral valve in place, take care not to injure the posterior left ventricle with the struts during internal massage. once some semblance of hemodynamic stability has returned, return the patient to the operating room for repair of the bleeding site, irrigation of the mediastinum, and closure. if the reason for emergency re-sternotomy was hemodynamic collapse not related to bleeding or tamponade, placement of an iabp is highly recommended. after the heart, the lungs are the organs most likely to be dysfunctional after cpb. during cpb, neutrophils are sequestered in the pulmonary vasculature and oxygen free radicals cause peroxidation of membrane lipids. these changes produce pulmonary vasoconstriction and are thought to increase the permeability of the alveolar-capillary barrier and consequently produce interstitial edema within the lungs. leukocytes are also activated and cause an inflammatory response of the pulmonary vasculature. during cpb and diminished pulmonary arterial flow, plasma thromboxane b increases, further contributing to the pulmonary vascular inflammation. the cumulative effect of these responses is a more permeable alveolar-capillary membrane and a predisposition to interstitial pulmonary edema. atelectasis also contributes to pulmonary dysfunction. this appears in some way to be linked to a decrease in pulmonary surfactant, and may partially explain the left lower atelectasis seen almost universally after cardiac surgery. thermal injury to the phrenic nerve and/or diaphragmatic dysfunction as well as effusions, pain, and chest tubes are other contributing factors to altered pulmonary function postoperatively. lung and chest wall compliance decrease significantly following cardiac surgery, with the maximum decrease occurring at days and lasting as long as days. the respiratory management of the postoperative cardiac surgical patient is not unlike any other postoperative patient, but there are several factors that are unique to these patients. the unique factors include: incision pain, the interference of chest tubes with the respiratory function, an element of diaphragmatic dysfunction, elevated left heart filling pressures with alveolar edema and diminished compliance, and capillary permeability. , , atelectasis is the most common pulmonary complication occurring in % of these patients. after cardiac surgery, atelectasis occurs most commonly in the left lower lobe. the exact etiology of this phenomenon remains unclear. it is associated with left phrenic nerve paralysis only in % of patients. alterations of the chest wall result in a decrease in the fev and frc and persist for weeks. these alterations lead to an increased respiratory rate, decrease tidal volume, decreased respiratory efficiency, and increased oxygen utilization. pulmonary infiltrates are the result of pneumonia, pulmonary embolism, and adult respiratory distress syndrome (ards) -although with ards, there is typically more of a diffuse process and is associated with more severe hypoxemia. the basic treatment of pneumonia and ards includes blood and sputum cultures, hemodynamic maintenance, euvolemic fluid management with a consideration of fluid restriction and the use of colloid for ards, and the maintenance of an arterial saturation greater than mmhg with minimum inspired oxygen content. [ ] [ ] [ ] bronchospasm can occur immediately after cpb and may interfere with hemodynamic stability. the probable cause is activation of c a anaphylatoxin by cpb. other causes include pulmonary edema, exacerbation of preexisting reactive airway disease, the use of -blockers, and a reaction to protamine. the treatment for bronchospasm includes the exclusion of heart failure, inhaled -agonists, the addition of cholinergic agents, a short course of systemic steroids for refractory bronchospasm, and intravenous aminophylline. aminophylline is reserved for refractory situations because of its arrhythmogenicity in the postoperative period. during cpb, renal blood flow and glomerular filtration rate are reduced - %, with partial but not complete recovery in the first day after cpb. , this is thought to be secondary to renal artery vasoconstriction, hypothermia, and loss of pulsatile flow. the nonpulsatile blood flow of cpb promotes renal artery vasoconstriction and diminishes renal blood flow to the cortex. in addition, angiotensin ii levels are elevated by nonpulsatile flow. , there appears to be a relationship between length of cpb and renal insufficiency, but not pressure or flow rates while on pump. other factors associated with renal failure include preexisting renal dysfunction (creatinine greater than . mg/dl), older age, poor left ventricular function and congestive heart failure, emergency surgery, the use of deep hypothermic circulatory arrest, moderate hypothermia, a preoperative history of hypertension, diabetes, and peripheral vascular disease, isolated valve operations, and the use of radiocontrast dye agents immediately preoperatively. postoperative factors contributing to renal insufficiency include: low cardiac output; hypotension; vasoconstriction; atheroembolism from the iabp; sepsis; rv failure with systemic venous hypertension; respiratory insufficiency with hypoxemia; and medications such as cephalosporins, aminoglycosides, and ace-inhibitors. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] the incidence of renal complications following open-heart surgery has been reported as high as %. the frequency of oliguric renal failure requiring dialysis is - % with a mortality of %. [ ] [ ] [ ] [ ] the most common form of renal failure after cpb, is nonoliguric renal failure. nonoliguric renal failure has a better prognosis with a mortality rate of - %. , the management goal of nonoliguric renal failure is the maintenance of an appropriate glomerular filtration rate by maintaining an adequate cardiac output and an adequate systemic blood pressure. the use of loop diuretics is controversial. they are unlikely to prevent the progression of nonoliguric to oliguric renal failure. dopamine at a "renal dose" of - . mcg/ kg/min is commonly used to preserve renal function. there are no studies demonstrating a renoprotective effect. dopamine may increase urine output, but it has been shown to be associated with renal tubular necrosis equal to or worse than controls. , in patients with a serum creatinine of > . mg/ dl, infusion of fenoldopam of . - . mcg/kg/min has been shown to preserve renal function. the best management of oliguric renal failure is prevention by early identification and treatment of deteriorating renal function. this prevention begins by avoiding hypotension and low cardiac output states, optimizing volume status, considering the early use of inotropic agents and pressors, and the early use of iabp. once oliguric renal failure occurs, a nephrology consultation is in order. strict euvolemia must be maintained, as well as careful monitoring of metabolic status and electrolyte balance and the daily review of medications looking for drugs excreted by kidneys. if renal failure occurs several days following surgery, it is most likely not related to cpb but more likely as a result of sepsis, nephrotoxic drugs, low cardiac output, and obstruction of the urinary tract. the perfusion of intra-abdominal viscera is also adversely effected by cpb. the blood flow to the liver is reduced by % during cpb and there is concomitant relative hypoperfusion of splanchnic and gastric flow. the decrease in gastric flow results in gradual decreasing of gastric ph and is associated with the appearance of endotoxin in the circulation, suggesting that the intestinal barrier is compromised and translocation is a possibility. , gastrointestinal complications are generally not a common source of significant morbidity after open-heart surgery. they occur at a rate of approximately - %. these complications are the result of a low cardiac output state with its associated sympathetic vasoconstriction and hypoperfusion of the abdominal organs. the most common serious complication after cpb is gastrointestinal hemorrhage from gastritis or gastroduodenal ulcer disease. the pathology is usually hemorrhagic gastritis or duodenitis. , occasionally, the hemorrhage is from previous duodenal ulcer disease and rarely from the colon. gastrointestinal hemorrhage occurs in only about % of cases and the risks are higher in patients with copd, hypotension, excessive postoperative bleeding, reoperation, and a prior history of peptic ulcer disease. it is recommended that these high-risk patients have prophylactic ulcer therapy. an appropriate prophylactic regimen would include sucralfate g q h orally or down a nasogastric tube. another option is omeprazalone mg daily. ranitidine appears to be the best option with a lower rate of gastrointestinal hemorrhage and an equivalent incidence of pneumonia. hepatic dysfunction is marked by transient elevation of liver function tests in % of patients. less than % of the patients will develop significant hepatocellular damage resulting in either chronic hepatitis or liver failure. , the risk factors for these complications are prolonged cpb, multiple transfusions, and multiple valve replacements. elevated lfts in association with hyperbilirubinemia occurring within the first - days is a result of low cardiac output and "shock liver." shock liver may cause hemodynamic instability with low systemic vascular resistance. hyperbilirubinemia without elevated lfts, if it occurs early, may be the result of cholestasis from red blood cell trauma and destruction, as well as from right heart failure with passive congestion of the liver, although the alkaline phosphatase may be elevated in this instance. bilirubin usually normalizes in - days with observation only. if isolated hyperbilirubinemia occurs late, it is caused by infection from transfused blood products. the risk of infection after transfusion depends on the number of units transfused and types of products transfused. the most common infections are non-a, non-b hepatitis (seen more often after clotting factor transfusions), cytomegalovirus, epstein-barr virus, and acute cholecystitis. acute cholecystitis is seen more often in the elderly after prolonged cpb, suggesting hypoperfusion may be a factor. transient hyperamylasemia can be found in as many as % of patients after cpb, yet is associated with pancreatitis in only - % of the patients. the risk factors include long cpb time and multiple transfusions. it is a must to exclude postoperative pancreatitis as this is a serious problem with a high mortality rate. ischemic bowel syndrome as a result of mesenteric ischemia is a catastrophic complication. it is often associated with the hypoperfusion of low cardiac output, particularly the elderly patient requiring inotropic or iabp support. electrolyte imbalances are common after cardiopulmonary bypass. potassium alterations are the result of rapid shifts that occur during cardiac surgery and cpb. the factors related to potassium fluxes are hyperkalemic cardioplegia, renal dysfunction while on cpb, low cardiac output and associated oliguria and acidosis, hemolysis of red cells, diuresis, and diminished potassium uptake in the face of diabetes mellitus. certain medications also impair potassium excretion and cause hyperkalemia. this list of medications include ace inhibitors, potassium-sparing diuretics, non-steroidal anti-inflammatory drugs, angiotensin receptor blockers, and -blockers. the principal adverse effect of potassium alterations is on the electrical activity of the heart and can be lifethreatening. hyperkalemia manifests itself predominantly electrocardiographically. asystolic arrest can occur when potassium rises rapidly to a level exceeding . meq/l. the ekg findings are more related to the rate of rise of potassium level than to an absolute level. they are peaked t waves, st depression, prolonged pr interval, loss of p wave, qrs widening, bradycardia, and asystole. hyperkalemia may result in failure of the heart to respond to the pacemaker stimulus and this may be a factor during resuscitation. treatment includes optimizing cardiac function and shifting potassium into the cells and increasing its excretion. the cardiac function is optimized with calcium gluconate. if there is evidence of cardiac toxicity, . - g of calcium gluconate is given intravenously over min. potassium is shifted into the cells by giving meq of nahco to correct acidosis and giving units of regular insulin and g of % dextrose. potassium excretion is enhanced with furosemide - mg iv, kayexalate enema g in water enema or g po with sorbitol or dialysis. hypokalemia is usually a result of diuresis without adequate replacement of potassium. diuresis is usually profound after cpb owing to hemodilution. diuretics, insulin administration, or alkalosis may exacerbate this diuresis. hypokalemia promotes atrial, junctional, and ventricular ectopy. it can cause life-threatening ventricular tachycardia, but usually does not become clinically evident until serum concentration is less than . meq/l. hypokalemia can also be the cause of metabolic alkalosis as hydrogen ions replace potassium within the cells. the treatment is potassium chloride (kcl) administration through a central line at - meq/h. serum potassium raises approximately . meq/l for each meq of kcl given. a slower rate is recommended in the presence of renal insufficiency. calcium plays a complex role in myocardial reperfusion damage and energetics. ionized calcium should be measured during and after cpb because hemodilution, hypothermia, ph shifts, and use of citrated blood will affect protein binding of calcium. hypocalcium is the most frequently seen calcium abnormality in the perioperative period. the treatment of hypocalcemia is a calcium chloride bolus of . - g. calcium gluconate ml of % solution will have fewer cardiovascular effects than calcium chloride. hypomagnesemia is not uncommon after cpb. the incidence is %. the most common etiology for hypomagnesemia is the diuresis and hemodilution associated with cpb. the effects of hypomagnesemia are mainly cardiac effects and similar to those of potassium on the electrical activity of the heart. manifestations of hypomagnesemia include atrial and ventricular dysrhythmias, potentiation of digoxin-related dysrhythmias, and a predilection to coronary spasm. since magnesium is also related to energy metabolism, prolonged ventilator support has also been related to low serum magnesium levels. treatment is an infusion of g magnesium sulfate in ml of solution to raise the serum level to meq/l. note that magnesium has been shown to inhibit the vasoconstrictive effect of epinephrine but not its cardiotonic effect. hyperglycemia routinely occurs during cpb. modest elevations are present during hypothermia, but more marked elevations of blood glucose happen during rewarming. hyperglycemia is caused by increased glucose mobilization related to increases in cortisol, catecholamines, and growth hormone levels during cpb. there also appears to be a blunted insulin response and impaired insulin production as well as a peripheral insulin resistance during cpb. the impaired insulin secretory response may last h. these changes are exaggerated in the diabetic patient, and insulin requirement may be seven times greater than preoperative requirements in the first h after surgery. hyperglycemia postoperatively is associated with osmotic diuresis, impaired wound healing, increased risk of infection, and impaired blood pressure regulation. hyperosmolar, hyperglycemic, non-ketotic coma is unusual following open-heart surgery. it usually occurs in type ii diabetics - days after surgery. diabetic ketoacidosis is rarely encountered in the postoperative period. the most efficient method of managing the postoperative patient is with an insulin infusion. the usual dose is . unit/kg/h of regular insulin in a saline mix. blood glucose levels must be monitored every h to maintain serum glucose of - mg/dl. type ii diabetics should be restarted on their oral regimen as soon as they are taking po. the most common and most frequent hematologic complication of open-heart surgery is thrombocytopenia and platelet dysfunction. , platelet counts decrease rapidly by % soon after the institution of cpb but usually remain above k. platelet counts less than , /mm occur in approximately % of patients on postoperative day one. platelet counts begin to increase by the third postoperative day. bleeding from thrombocytopenia is usually not a problem until the platelet count falls below , /mm . of greater clinical significance is the progressive deterioration of platelet function during cpb. within minutes of cpb, platelet aggregation is impaired and continues to worsen throughout cpb. this platelet dysfunction is precipitated by contact of the platelets with synthetic surfaces of the cpb circuit as well as by hypothermia. also, the mechanical stresses of cpb cause fragmentation of the platelets and a temporary depletion in the membrane antigen for glycoproteins ib, iib, and iiia. hypothermia impairs platelet thromboxane a synthesis resulting in reversible platelet dysfunction. bleeding time returns to normal in about - h and the platelet count is restored in several days. , , platelet dysfunction occurs less commonly with the use of antifibrinolytic drugs, such as -aminocaproic acid, because these agents act in part by reducing platelet activation during cpb. indications for platelet transfusion are as follows: a platelet count less than - , /mm , ongoing bleeding with a platelet count less than , /mm , and a platelet count less than , /mm if a surgical procedure is planned. cpb also effects the plasma concentration of coagulation factors ii, v, vii, ix, x, and xiii. the plasma concentration of these factors decline during cpb secondary to hemodilution but remain at levels adequate for hemostasis, and, with the exception of fibrinogen, return to normal by h. , fibrinogen and plasminogen decrease during cpb from dilution and not consumption, and usually return to normal by h. heparin-induced thrombocytopenia (hit) is an infrequent but serious complication with a high mortality rate if the fulminant course progresses to heparin-induced thrombotic thrombocytopenia (hitt). hit is caused by the formation of igg platelet membrane antibodies, which, in the presence of heparin, produce platelet aggregates and heparin resistance. the range of intensity of hit and hitt spans from only moderate thrombocytopenia to a syndrome of arterial or venous thrombosis caused by platelet aggregation and bleeding from profound thrombocytopenia. , if the diagnosis of hit is suspected, all heparin must be discontinued including therapeutic infusions, line flushes, heparin-coated monitoring lines, and low-molecular-weight heparins. the laboratory confirmation by platelet aggregation testing is important, but may take at least h to confirm; therapy should be instituted as soon as the diagnosis is suspected. platelet counts must be monitored on a daily basis. in-hospital, postoperative infections after open-heart surgery occur at a rate of - %. the most common infections are the respiratory, urinary, and wound or surgical site infections. while all postoperative infections adversely affect outcomes, it is the sternal wound infection and mediastinitis that have the greatest adverse effects. the overall incidence of sternal wound infections is . - . %. , when sternal wound infections are associated with mediastinitis, the mortality varies from to %. when recognized early and effectively treated, the mortality is - % the rate of mediastinitis is higher in valvular procedures and in combined procedures. the use of bilateral internal mammary arteries increases the risk of sternal wound complications to %. staphylococcus aureus and staphylococcus epidermidis are the most common pathogens encountered accounting for % of infections. preoperative predisposing factors include type and timing of skin preparation, cardiopulmonary failure, need for an iabp, diabetes mellitus, steroid use, a history of mediastinal radiation, osteoporosis, age, and copd. , intraoperative factors are a cpb run greater than h, excessive bleeding, the use of bilateral internal mammary arteries, valve procedures, combined procedures, and inadequate sternal fixation. postoperative bleeding will increase the risk for sternal wound complications, as will low-flow states, concurrent infections, tracheotomies, and prolonged ventilatory support. the most obvious sign of a wound infection is purulent drainage from the incision. there should be a heightened level of suspicion in a patient whose pain begins to increase toward the end of the first postoperative week rather than decrease. also the wound is reddened and swollen and there is a localized area of skin necrosis associated with the drainage. the drainage is serous if the complication is minor, involving only the superficial soft tissue. however, if the complication is a major one with mediastinitis there is extensive purulent drainage with infection extending down to the sternum and mediastinum. these findings may not always be an indication of infection, but could be aseptic necrosis from internal mammary artery mobilization. fever, leukocytosis, or gram-positive bacteremia should raise the suspicion of a sternal wound infection. any fever of undetermined etiology should raise the question of wound sepsis, particularly in diabetics where few other local or systemic signs may be present as a result of a poor inflammatory response. the evaluation begins with a culture of the purulent drainage. if there is no drainage, a likely area of the wound should be opened and careful cultures obtained. radiographic workup is of limited value. routine chest x-rays are of little help. a chest computed tomography (ct) scan may identify indolent, retrosternal infections, particularly if gas-forming organisms are present. [ ] [ ] [ ] minor infections usually respond to treatment with antibiotics and local care, including wound packing. major infections require mediastinal exploration and debridement of infected tissue, including the sternum. if the sternum is necrotic or grossly infected, removal of the sternum is necessary and requires closure with a muscle flap, either a pectoralis major or rectus abdominis flap. omentum can also be used to provide a vascular bed for healing, but omental mobilization is associated with a higher morbidity than the creation of a muscle flap. appropriate parenteral antibiotics are required for a -week period. the incidence of leg wound infections is - %. these complications may result from poor surgical technique with a creation of flaps, failure to eliminate dead space, or hematoma formation. the risk factors are obese women, use of thigh veins, diabetes, and severe peripheral vascular disease. the prevention of leg infections involves careful surgical technique and the use of suction drains to eliminate dead space in the leg. the treatment is appropriate antibiotic coverage, debridement, and a consideration for early plastic surgery involvement. prophylactic antibiotics should be administered for h starting in the operating room just prior to the incision. firstor second-generation cephalosporins are used because of their effectiveness against gram-positive cocci. vancomycin is used in patients with true anaphylactic allergy to penicillin or cephalosporins. if the patient does not have a documented history of a severe anaphylactic reaction to penicillin or a cephalosporin, a cephalosporin should be used. attempts must be made to limit the use of vancomycin for prophylaxis to lessen the likelihood of vancomycin-resistant enterobacter infections. neurologic complications following open-heart surgery are dreaded sequelae. the overall incidence of focal deficits is - %. , these usually occur intraoperatively and are noted in the first - h. some % of the deficits may develop postoperatively as a result of hemodynamic instability or arrhythmia. risk factors of stroke for the open-heart patient include increasing age (a risk up to % in patients older than years), diabetes mellitus, preexisting cerebrovascular disease especially with a history of recent stroke, perioperative hypotension, atherosclerotic plaques and calcifications in the ascending aorta, left ventricular mural thrombus, opening a cardiac chamber, postoperative atrial fibrillation, long duration of cpb, and warm blood cpb. [ ] [ ] [ ] [ ] the presentation of neurologic complications depends on the site and extent of the insult. transient ischemic attacks present with focal deficits of hemiparesis or hemiplegia, aphasia, dysarthria, hand incoordination, visual deficits (either retinal or central), and coma. if an interventional neurologist is available, an immediate consultation should be obtained. an evaluation begins with a careful neurologic examination, then a ct scan of the brain with contrast infusion, an echocardiogram (surface or transesophageal) to exclude a cardiac source, and noninvasive carotid studies. if there is no evidence of an intracranial hemorrhage on ct scan, heparin is started, and then warfarin if the stroke is thought to be embolic. if the deficit occurs during surgery, there is some debate as to the need for anticoagulation versus just antiplatelet therapy. other therapy includes the standard measures to reduce intracranial pressure and even a carotid endarterectomy in patients with severe carotid stenosis and transient neurologic deficits. physical therapy is started soon after the event is diagnosed. as regards prognosis, patients with focal deficits have an excellent prognosis. in patients with coma, the prognosis is poor with a mortality rate of % and a high percentage of survivors staying in the vegetative state. postoperative encephalopathy and delirium occur in approximately % after open-heart surgery. the risk factors include older age, recent alcoholism, preoperative organic brain syndrome, severe cardiac disease, multiple associated medical illnesses, and complex and prolonged surgical procedures on cpb. common causes of delirium are medication toxicity, metabolic disturbances, alcohol withdrawal, low cardiac output syndromes, periods of marginal cerebral blood flow during cpb, hypoxia, sepsis, and a recent stroke. the evaluation of delirium begins with a review of the patient's current medications and drug levels, an identification of a possible history of recent alcoholism or substance abuse, neurologic examination, and abgs, electrolytes, bun, creatinine, cbc, magnesium, and calcium determinations. the management of delirium begins by correcting any metabolic abnormalities, discontinuing inappropriate medications, and psychotropic medications for agitation such as haloperidol . - . mg po/im/iv q h. the treatment of suspected alcohol withdrawal include benzodiazepines, thiamine, and folate. initiation of white cell activation during cardiopulmonary bypass: cytokines and receptors thyroid hormone changes after cardiovascular surgery and clinical implications hypermetabolism after cardiopulmonary bypass hypermetabolic response after hypothermic cardiopulmonary bypass hormonal and metabolic changes during hypothermic coronary artery bypass surgery in diabetic and non-diabetic subjects organ dysfunction after cardiopulmonary bypass. a systemic inflammatory reaction initiated by the extracorporeal circuit complement activation during cardiopulmonary bypass: evidence for generation of c a and c a anaphylatoxins the effects of complement activation during cardiopulmonary bypass. attenuation by hypothermia, heparin, and hem dilution interleukin- and the pathogenesis of the acute phase response superoxide and inflammation: a mechanism for the anti-inflammatory activity of superoxide dismutase cardiorespiratory effects of protamine after cardiopulmonary bypass in man the rapid induction by interleukin- of pulmonary microvascular permeability effects of prolonged aortic cross clamping with potassium cardioplegia on myocardial contractility in man cardiopulmonary bypass and the lung oxygen derived free radical and postischemic myocardial dysfunction reperfusion injury" by oxygen derived free radicals? ischemia-a coagulation problem? acute myocardial dysfunction and recovery: a common occurrence after coronary bypass surgery beating heart surgery: does it make a difference? management of patients after cardiac surgery critical care for the adult cardiac patient. sabiston & spencer surgery of the chest manual of perioperative care in cardiac and thoracic surgery cardiac catheterization. in: grossman w, editor. heart disease. philadelphia: wb saunders early risks of open heart surgery for mitral valve disease management of the cardiovascular subsystem after cardiac surgery myocardial ischemia during cardiovascular surgery as detected by an st segment trend monitoring system ventricular wall stress comparison of two transfusion strategies after elective operations for myocardial revascularization does hemoglobin affect perioperative myocardial lactate flux in patients undergoing coronary artery bypass surgery? myocardial oxygen supply and demand manual of perioperative care in cardiac and thoracic surgery reperfusion injury" by oxygen free radicals? the hemostatic mechanism after open-heart surgery. i. studies on plasma coagulation factors and fibrinolysis in patients after extracorporeal circulation preoperative and intraoperative predictors of inotropic support and long-term outcomes in patients having coronary artery bypass grafting diastolic dysfunction is predictive of difficult weaning of from cardiopulmonary bypass abnormal left ventricular intracavitary flow acceleration in patients undergoing aortic valve replacement for aortic stenosis. a marker for high postoperative morbidity and mortality abnormal systolic intracavity velocities after valve replacement for aortic stenosis. mechanisms, predictive factors, and prognostic significance cardiac surgery hemodynamic advantage to left atrial epinephrine administration in open heart surgery lack of renoprotective effects of dopamine and furosemide during cardiac surgery dopamine therapy for patients at risk of renal dysfunction following cardiac surgery: fact or fiction? dobutamine and dopamine after cardiac surgery: greater augmentation of myocardial blood flow with dobutamine effects of dobutamine on hemodynamic and left ventricular performance after cardiopulmonary bypass in cardiac surgical patients the comparative effects of dopamine and dobutamine on ventricular mechanics after coronary artery grafting: a pressure-dimension analysis hemodynamic comparison of dopamine and dobutamine in the postoperative volume-loaded, pressure-loaded, and normal ventricle use of amrinone in cardiac surgery patients the effects of amrinone versus dobutamine on myocardial mechanics after hypothermic global ischemia a randomized, blinded trial of amrinone, epinephrine, and amrinone/epinephrine after cardiopulmonary bypass (cpb) combination high dose amrinone and dopamine in the management of moribund cardiogenic shock after open heart surgery combined inotropic effects of amrinone and epinephrine after cardiopulmonary bypass in humans pharmacology of bipyridines: amrinone and milrinone the effect of milrinone on hemodynamics and left ventricular function after emergence from cardiopulmonary bypass a single dose of milrinone facilitates separation from cardiopulmonary bypass in patients with pre-existing left ventricular dysfunction direct vasodilator effect of milrinone, an inotropic drug, on arterial coronary bypass grafts vasorelaxant effect of the phosphodiesteraseinhibitor milrinone in the human radial artery used as coronary bypass graft vasopressin as an alternative to norepinephrine in the treatment of milrinone-induced hypotension the effects of milrinone on platelets in patients undergoing cardiac surgery hemodynamic effects of prostaglandin e and isoproterenol early after cardiac operations for mitral stenosis product inserts. natrecor (nesiritide) nesiritide compared with milrinone for cardiac surgery perioperative nesiritide versus milrinone in high-risk coronary artery bypass patients effect of perioperative nesiritide administration on postoperative renal function and clinical outcomes in patients undergoing cardiothoracic surgery (poster , abstract ) arginine vasopressin in advanced vasodilatory shock: a prospective, randomized, controlled study vasopressin: physiology and clinical use in patients with vasodilatory shock: a review role of vasopressin in the management of septic shock vasopressin deficiency contributes to the vasodilation of septic shock vasopressin in the icu risk factors for post-cardiopulmonary bypass vasoplegia in patients with preserved left ventricular function vasoplegic syndrome after open heart surgery refractory vasodilatation after cardiopulmonary bypass for heart transplantation in recipients on combined amiodarone and angiotensin-converting enzyme inhibitor therapy: a role for vasopressin administration arginine vasopressin in the treatment of patients with postcardiotomy vasodilatory shock fixed-dose vasopressin compared with titrated dopamine and norepinephrine as initial vasopressor therapy for septic shock randomized comparison of epinephrine and vasopressin in patients with out-ofhospital ventricular fibrillation terlipressin for norepinephrine-resistance septic shock surviving sepsis campaign for management of severe sepsis and septic shock iononized calcium, the heart, and hemodynamic function cardiopulmonary bypass and thyroid function: a "euthyroid sick syndrome thyroid hormone treatment after coronary-artery bypass surgery inotropic effect of triiodothyronine in low cardiac output following cardioplegic arrest and cardiopulmonary bypass: an initial experience in patients undergoing open-heart surgery triiodothyronine as an inotropic agent after open-heart surgery thyroid hormone treatment after coronary artery bypass surgery acute cellular actions of thyroid hormone and myocardial function triiodothyronine administration in coronary artery bypass surgery: effect on hemodynamics a double blind placebo controlled study of the effect of triiodothyronine upon cardiac performance and outcome following coronary bypass surgery initial clinical experience with intraaortic pumping for cardiogenic shock the intraaortic balloon pump: a review intra-aortic balloon pumping: effects of left ventricular diastolic function intraaortic balloon counterpulsation: patterns of usage and outcome in cardiac surgery patients intraaortic balloon pumping in patients requiring cardiac operations. risk analysis and long-term follow-up intraaortic balloon pump insertion: a randomized study comparing percutaneous and surgical techniques cardiac surgery effects of intraaortic balloon position on renal artery blood flow occlusion of left internal mammary artery with intraaortic balloon: clinical implications orthotopic cardiac prosthesis for two-staged cardiac replacement mechanical circulatory support the continuing evolution of mechanical ventricular support selection criteria for placement of left ventricular assist devices outcome of patients surviving to heart transplantation after being mechanically bridged for more than days experience with right ventricular assist devices for perioperative right-sided circulatory failure analysis of right ventricular function during bypass of the left side of the heart by afterload alterations in both normal and failing hearts emergency cardiopulmonary bypass support in patients with cardiac arrest percutaneous cardiopulmonary bypass: application and indication for use biopump with and without intraaortic balloon pump on the coronary and carotid flow an analysis and results of prolonged resuscitation in cardiac arrest patients by extracorporeal membrane oxygenation clinical experience with adults receiving extracorporeal membrane oxygenation for cardiac failure: survival at years postcardiotomy mechanical support: risk factors and outcomes left ventricular assist pathogenesis of paroxysmal hypertension developing during and after coronary bypass surgery: s study of hemodynamic and humoral factors systemic hypertension associated with coronary artery bypass surgery perioperative management of hypertension and tachycardia effects of postoperative hypertension and its treatment drug therapy: sodium nitroprusside comparison of intravenous nitroglycerine and sodium nitroprusside for treatment of acute hypertension developing after coronary bypass surgery comparison of intravenous nifedipine with sodium nitroprusside for treatment of acute hypertension after cardiac surgery reversal of preexisting vasospasm in coronary artery conduits labetalol for the control of elevated blood pressure following coronary artery bypass grafting cardiorespiratory response to intravenous angiotensin-converting enzyme inhibitor enalaprilat in hypertensive cardiac patients da receptor stimulation by fenoldopam in the treatment of postcardiac surgical hypertension right ventricular dysfunction in low output syndrome after cardiac operations: assessment by transesophageal echocardiography postoperative care of the cardiac surgical patient perioperative use of nesiritide in adult cardiac surgery nesiritide (bnp) in the management of postoperative cardiac patients inhaled nitric oxide. a selective pulmonary vasodilator. current uses and therapeutic potential inhaled epoprostenol (prostacyclin) and pulmonary hypertension before surgery iloprost improves hemodynamics in patients with severe chronic cardiac failure and secondary pulmonary hypertension diastolic dysfunction in post-cardiac surgical management the epicardial electrogram in the diagnosis of cardiac arrhythmias following cardiac surgery postoperative care following cardiac surgery de novo refractory ventricular tachyarrhythmias after coronary artery bypass revascularization new-onset sustained ventricular tachycardia after cardiac surgery current status of the implantable cardioverter-defibrillator guidelines for cardiopulmonary resuscitation and emergency cardiovascular care a practical approach to torsades de pointes refractory postoperative torsades de pointes syndrome successfully treated with isoproterenol atrial fibrillation following coronary artery bypass surgery predictors of atrial fibrillation after coronary artery surgery: current trends and impact on hospital resources atrial fibrillation after cardiac surgery is the incidence of postoperative atrial fibrillation following cardiac valve surgery reduced by minimally invasive surgery (abstract)? atrial fibrillation after coronary artery bypass graft surgery: predictors, outcomes, and resource utilization do off-pump techniques reduce the incidence of post-operatic atrial fibrillation in elderly patients undergoing coronary artery bypass grafting? postoperative atrial fibrillation hazards of postoperative atrial arrhythmias atrial fibrillation and flutter after coronary artery bypass grafting: epidemiology, risk factors and preventive trials risk factors for atrial fibrillation after coronary artery bypass grafting the importance of age as a predictor of atrial fibrillation and flutter after coronary artery bypass grafting influence of clinical and hemodynamic variables on risk of supraventricular tachycardia after coronary artery bypass prevention of supraventricular arrhythmias after coronary artery bypass surgery: a meta-analysis of randomized controlled trials a placebo-controlled trial of continuous intravenous diltiazem infusion for hour heart rate control during atrial fibrillation and atrial flutter: a multicenter study esmolol versus diltiazem in the treatment of postoperative atrial fibrillation/ flutter after open heart surgery esmolol versus diltiazem in atrial fibrillation following coronary artery bypass graft surgery safety and efficacy of intravenous diltiazem in atrial fibrillation or atrial flutter amiodarone versus diltiazem for rate control in critically ill patients with atrial tachyarrhythmias acute circulatory actions of intravenous amiodarone loading in cardiac surgical patients practical approaches to treating atrial fibrillation proacainamide conversion of acute atrial fibrillation after open-heart surgery compared with digoxin treatment manual of cardiovascular medicine. philadelphia: lippincott williams and wilkins invited letter to the editor: amiodarone and quinidine for postoperative atrial arrhythmias intravenous amiodarone bolus versus oral quinidine for atrial flutter and fibrillation after cardiac surgery efficacy of ibutilide for termination of atrial fibrillation and flutter cardiac surgery: postoperative arrhythmias management of atrial fibrillation after cardiac surgery, part ii: prevention and treatment pharmacological approach for the prevention of atrial fibrillation after cardiovascular surgery meta-analysis of the effectiveness of prophylactic drug therapy in preventing supraventricular arrhythmia early after coronary artery bypass grafting atrial fibrillation after coronary artery bypass grafting: is it a disorder of the elderly? arrhythmia prophylaxis after aorto-coronary bypass: the effect of minidose propranolol supraventricular tachyarrhythmias after coronary bypass surgery: a double blind randomized trial of prophylactic low dose propranolol intravenous magnesium sulfate prophylaxis for atrial fibrillation after coronary artery bypass surgery postoperative atrial tachyarrhythmias in patients undergoing coronary artery bypass graft surgery without cardiopulmonary bypass: a role for intraoperative magnesium supplementation antiarrhythmic effect of magnesium sulfate after open heart surgery: effect of blood levels preoperative use of sotalol versus metoprolol in the prevention of atrial fibrillation after cardiac surgery amiodarone vs. sotalol as prophylaxis against atrial fibrillation/flutter after heart surgery. a meta-analysis role of amiodarone in reducing atrial fibrillation after cardiac surgery in adults postoperative oral amiodarone as prophylaxis against atrial fibrillation after coronary artery surgery amiodarone reduces the incidence of atrial fibrillation after coronary artery bypass grafting acute pulmonary toxicity after low-dose amiodarone therapy stroke following coronary artery bypass surgery role of prophylactic anticoagulation for direct current cardioversion in patients with atrial fibrillation and atrial flutter postoperative care in cardiac surgery high-dose aprotinin in emergency coronary artery bypass after thrombolysis immediate coronary bypass following failed streptokinase infusion in evolving myocardial infarction enhanced fibrinolytic activity during cardiopulmonary bypass in open-heart surgery in man caused by extrinsic (tissue-type) plasminogen activator kinetics of anticoagulation effect of heparin hematologic changed during and after cardiopulmonary bypass and their relationship to the bleeding time and nonsurgical blood loss platelet function in cardiac surgery: influence of temperature and aprotinin bleeding after cardiopulmonary bypass the platelet function defect of cardiopulmonary bypass heparin-induced thrombocytopenia and cardiac surgery a prospective study of aspirin's effect on red cell losses in cardiac surgery inhibition of platelet function by heparin: an etiologic factor in postbypass hemorrhage heparin management protocol for cardiopulmonary bypass influences heparin rebound but not bleeding heparin binding proteins: contribution to heparin rebound after cardiopulmonary bypass alterations in hemostasis associated with cardiopulmonary bypass; pathophysiology, prevention, diagnosis, and management adverse effects of postoperative infusion of shed mediastinal blood diagnosis and therapy of disseminated intravascular coagulation and activated coagulation guidelines for transfusion support in patients undergoing coronary artery bypass grafting. transfusion practices committee of the american association of blood banks desmopressin usage in elective cardiac surgery the effect of desmopressin acetate on postoperative hemorrhage in patients receiving aspirin therapy before coronary artery bypass operations treatment of severe platelet dysfunction and hemorrhage after cardiopulmonary . postoperative care of the cardiac surgical patient bypass: reduction in blood product usage with desmopressin reduction of bleeding after heart operations through the prophylactic use of epsilonaminocaproic acid aprotinin for primary coronary artery bypass grafting: a multicenter trial of three dose regimens postoperative aprotinin: effect of blood loss and transfusion requirements in cardiac operations comparison of two transfusion strategies after elective operations for myocardial revascularization preoperative use of erythropoietin for cardiovascular operations in anemia apparent coagulopathy caused by infusion of shed mediastinal blood and its prevention by washing of the infusate safety and therapeutic effectiveness of reinfused shed blood after open-heart surgery adverse effects of postoperative infusion of shed mediastinal blood mediastinal bleeding after cardiac surgery: etiologies, diagnostic consideration, and blood conservation methods bleeding complications associated with cardiopulmonary bypass re-exploration for bleeding is a risk factor for increased morbidity and mortality re-exploration after coronary bypass surgery: risk factors, outcomes, and the effect of time delay complement activation during cardiopulmonary bypass: evidence for generation of c a and c a anaphylatoxins cytokine responses to cardiopulmonary bypass with merman and bubble oxygenation solute permeability of the alveolar capillary barrier cardiopulmonary bypass significantly reduces surfactant activity in children the influence of pleural and mediastinal chest tubes on respiration following coronary artery bypass grafting (cabg) cardiopulmonary bypass and the lung postoperative phrenic nerve palsy in patients with open-heart surgery the acute respiratory distress syndrome new concepts and therapies of adult respiratory distress syndrome relationship between alteration in renal hemodynamics during cardiopulmonary bypass and postoperative renal function renal arteriography during extracorporeal circulation in dogs with a preliminary report upon the effects of low molecular weight dextran changes in renal blood flow and pah extraction during extracorporeal circulation of short and long duration prevention of supraventricular arrhythmias after coronary artery bypass surgery: a meta-analysis of randomized control trials pulsatile and nonpulsatile cardiopulmonary bypass: review of a counterproductive controversy is kidney function altered by the duration of cardiopulmonary bypass? renal dysfunction after myocardial revascularization intravenous diltiazem and acute renal failure after cardiac operations report of a substudy of warm versus cold cardiopulmonary bypass: changes in creatinine clearance valvular heart surgery is an independent risk for acute renal failure acute renal failure in the patient undergoing cardiac operation. prevalence, mortality rate, and main risk factors acute renal failure associated with cardiac operations. a case study survival of patients with acute renal failure requiring dialysis after open-heart surgery: early prognostic indicators noncardiac complications of open-heart surgery acute renal failure after open-heart surgery the effect of "renal dose" dopamine on renal tubular function following cardiac surgery; assessed by measuring retinol binding protein (rbp) fenoldopam for renal protection in patients undergoing cardiopulmonary bypass association between gastric intramucosal ph and splanchnic endotoxins, antibody to endotoxin, and tumor necrosis factor concentrations in patients undergoing cardiopulmonary bypass the reasons for gastrointestinal consultation after cardiac surgery abdominal complications following cardiac surgery gastrointestinal complications after cardiac surgery the acute surgical abdomen after cardiac surgery involving extracorporeal circulation gastrointestinal complications after cardiac surgery gastrointestinal complications after coronary artery bypass grafting a comparison of sucralfate and ranitidine for the prevention of upper gastrointestinal bleeding in patients requiring mechanical ventilation hyperbilirubinemia after cardiac operation. incidence, risk factors and clinical significance severe ischemic early liver injury after cardiac surgery blood transmitted and clotting factor transmitted non-a, non-b hepatitis hyperamylasemia after cardiac surgery. incidence, significance, and management acute pancreatitis after cardiopulmonary bypass hypomagnesemia is common following cardiac surgery magnesium inhibits the hypertensive but not the cardiotonic actions of low-dose epinephrine diabetes and coronary artery surgery clinical features of hyperosmolar nonketotic diabetic coma associated with cardiac operations the effect of cardiopulmonary bypass on platelet function and platelet kinetics platelet surface glycoproteins: studies on resting and activated platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective a-granule release hypothermia-induced reversible platelet dysfunction heparininduced thrombocytopenia heparin-induced thrombocytopenia the impact of nosocomial infections on patient outcome following cardiac surgery a prospective study of sternal wound complications sternal and costochondral infections following open-heart surgery. a review of , cases mediastinitis after cardiac valve operations: impact upon survival recent experience with major sternal wound complications major sternal wound infections after open-heart surgery: a multivariate analysis of risk factors in , consecutive operative procedures rigid internal fixation of the sternum in postoperative mediastinitis does bilateral internal mammary artery grafting increase surgical risk? approaches to sternal wound infections occurrence of and microbiological findings in postoperative infections following open-heart surgery. effect on mortality and hospital stay use of computed tomography to assess mediastinal complications after median sternotomy clinical-radiological evaluation of poststernotomy wound infections infectious mediastinitis after cardiac operations: computed tomographic findings leg wound complications associated with coronary revascularization an year evolution of coronary arterial bypass grafting ( - ) stroke following coronary artery bypass grafting: a ten year study usefulness of atrial fibrillation as a predictor of stroke after isolated coronary artery bypass grafting differential effects of advanced age on neurologic and cardiac risks of coronary artery operations risk factors for stoke after coronary artery bypass central nervous system complications of open-heart surgery postcardiotomy delirium: conclusions after years? key: cord- -d gxew authors: grimble, robert f. title: the interaction between nutrition and inflammatory stress throughout the life cycle date: journal: nutrients, stress, and medical disorders doi: . / - - - : sha: doc_id: cord_uid: d gxew the human race inhabits a world in which it is surrounded by a myriad of different microorganisms—yeasts, bacteria, protozoa, and viruses. most of these are benign, and some, such as the normal gut flora, play an important part in promoting health via the synthesis of vitamins and stimulation of normal function of gut epithelia. approximately . % of microbes in our environment have catastrophic effects if they penetrate the epithelial surfaces of the body (bryson, ). history reveals many instances in which armies have been defeated and civilizations have collapsed because of encounters between humans and such microorganisms (diamond, ). the human race inhabits a world in which it is surrounded by a myriad of different microorganisms--yeasts, bacteria, protozoa, and viruses. most of these are benign, and some, such as the normal gut flora, play an important part in promoting health via the synthesis of vitamins and stimulation of normal function of gut epithelia. approximately . % of microbes in our environment have catastrophic effects if they penetrate the epithelial surfaces of the body (bryson, ) . history reveals many instances in which armies have been defeated and civilizations have collapsed because of encounters between humans and such microorganisms (diamond, ) . humans, like all mammals, have evolved with a complex immune system, which is present as specialized organs (spleen, thymus) or cell types (lymphocytes, macrophages, and mast cells) throughout the body. the system can detect and destroy any cell or particle that is not"self," i.e., a normal component of the body. a complex series of events follows from contact between components of the immune system and microbes invading the body ( fig. ) . the response can be divided into two main categories. the first is the acquired immune response, in which the immune system recognizes specific chemical motifs on the invader and "remembers" the encounter so that a more rapid, specific, and intense response can be produced at any future meeting. the second category is the nonspeciflc response in which the response to each encounter is similar for all invaders of the body. the process of inflammation is a central part of the second category of response. the immune response is also activated by a wide range of adverse events, such as surgery, bums, and trauma. the primary purposes of the response are to kill pathogens and initiate the curative processes that will restore body function to normal. the first purpose is achieved by creating a hostile tissue environment through production of oxidant molecules and activation of t and b lymphocytes. part of the response ensures a supply of substrate, from endogenous sources, for supporting the activity of t and b lymphocytes and enhancement of antioxidant defenses. the latter event is important for protecting healthy tissue from the oxidants produced as part of the inflammatory response (grimble, a) . the response exerts considerable biological demands and stress on the body. a central part of substrate provision is the release of amino acids into the blood from the breakdown of proteins in skeletal muscle, skin, and bone matrix, and fatty acids released from triglycerides stored in adipose tissue. enhanced gluconeogenesis, catabolic hormone production, and decreased insulin sensitivity occurs to facilitate this redistribution of tissue infective or cellular fig. . diseases and conditions in which inappropriate or excessive amounts of pro-inflammatory cytokines exert adverse or lethal effects on the host. components (fig. ) . the animal loses the desire to carry out many day-to-day activities. physical weakness ensues, exploratory activity declines, appetite is decreased, and apathy and sleep may occur. the response thus exerts physiological and mental stress upon the body. inflammation comes under the control of signaling proteins (cytokines) that possess hormone-like actions. the pro-inflammatory cytokines interleukin (il)- , il- , and tumor necrosis factor (tnf)-t~, are major activators and modulators of the events described above. to modulate the degree of stress imposed on the body, in achieving the essential functions of inflammation, the response comes under the control of powerful anti-inflammatory mechanisms. these will impose their biological effects with increasing vigor as the original stimulus for the inflammatory response (infection, injury) declines in intensity. heat-shock proteins, endorphins, glucocorticoid hormones, and cytokine receptor antagonists are important components of this anti-inflammatory system.this system is essential for closing down the inflammatory response once it has achieved its primary purposes because of the high biological cost it imposes on the body (grimble, a) . although cytokines play an important role in the response to infection and injury, they can exert damaging and lethal effects on the host. many studies have shown that excessive or prolonged production of cytokines is associated with increased morbidity and mortality in a wide range of acute and chronic inflammatory conditions (fig. ) . these include sepsis, adult respiratory distress syndrome, malaria, meningitis, cancer, cystic fibrosis, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, and asthma. events similar to those seen in the inflammatory response to injury and infection can be observed during the course of overt inflammatory diseases such as rheumatoid arthritis and crohn's disease and in diseases that have a covert inflammatory basis, for example, atherosclerosis and diabetes mellitus (fig. ) . clearly the inflammatory response in these situations does not have a purposeful nature and contribute to the disease process. recent studies indicate that low-intensity inflammation occurs in elderly and obese individuals (grimble (grimble , . thus, the inflammatory response, which has evolved to allow humankind to survive infection and injury, is indiscriminate in both its triggers and targets. as a result, the process is a two-edged sword capable of both defending and damaging its bearer. during the remainder of this chapter we will be exploring the biological and nutritional factors that determine the intensity of, and outcome from, the inflammatory process. various components of the inflammatory response interact to modulate its intensity. predominant among these interactions are the relative amounts of pro-and anti-inflammatory cytokines produced during the response to microbes and injury and the effect of oxidant molecules on cytokine production. early work on cytokines and the response to infection linked excessive pro-inflammatory cytokine production with increased morbidity and mortality in a wide range of conditions, such as malaria, meningitis, and sepsis. however, research in the last yr has shown that the balance in production between pro-and anti-inflammatory cytokines has a more direct bearing on the outcome of infection and injury. for example, in sepsis, plasma il- concentrations were higher and il- concentrations were lower in patients who died than in those who survived (arnalich et al., ; taniguchi et al., ) . a survey of over patients admitted to hospital in the netherlands with fever showed that, independently of how the patients were clinically classified (positive blood cultures, presence of endotoxin), those who subsequently died had a higher plasma il- :tnf-o~ ratio than patients who survived (van dissell, van langervelde, westendorp, kwappenberg, & frolich, ) . powerful oxidant molecules (e.g., superoxide, hydrogen peroxide, hypochlorous acid) are produced as part of the inflammatory response. their biological purpose is to destroy invading microbes. however, these molecules also have the capacity to damage host tissues and to increase the intensity of the inflammatory response. clearly both of these biological events can have adverse effects upon the host. the oxidant molecules activate at least two important families of proteins in the host that are sensitive to changes in cellular redox state. the families are nuclear transcription factor k b (nf-rd ) and activator protein (ap ). these transcription factors act as "control switches" for biological processes, not all of which are of advantage to the individual. nf-rd is present in the cytosol in an inactive form, by virtue of being bound to an inhibitory unit i-r,b. phosphorylation and dissociation of i-r,b renders the remaining nf-~cb dimer active. the dissociated i-rd is degraded, and the active nf-r,b is translocated to the nucleus, where it binds to response elements in the promoter regions of genes. a similar translocation of ap , a transcription factor composed of the protooncogenes c-fos and c-jun, from cytosol to nucleus, also occurs in the presence of oxidant stress. binding of the transcription factors is implicated in activation of a wide range of genes associated with inflammation and the immune response, including those encoding cytokines, cytokine receptors, cell adhesion molecules, acute-phase proteins, and growth factors (schreck, rieber, & baeurerle, ) (fig. ) . activation of nf-rd can be brought about by a wide range of stimuli including pro-inflammatory cytokines, hydrogen peroxide, mitogens, bacteria and viruses and their related products, and ultraviolet (uv) and ionizing radiations. the extent of activation of nf-rd will depend in part upon the strength and efficiency of the antioxidant defenses of the body. these comprise endogenous components such as glutathione (gsh) and enzymatic components of antioxidant defenses, such as catalase, superoxide dismutase (sod), and gsh peroxidase, and dietary components that have antioxidant properties (e.g., vitamins c and e and polyphenolic compounds). the influence of modulation of inflammation by these dietary factors are dealt with later. an unfortunate side effect of activation of nf-r,b arises from the ability of the transcription factor to activate transcription of the genes of some viruses, such as human immunodeficiency virus (hiv) (fig. ) . this sequence of events, in the case of hiv, accounts for the ability of minor infections to speed the progression of individuals who are infected with hiv towards acquired immunodeficiency syndrome (aids). thus, if antioxidant defenses are poor, each encounter with general infections results in cytokine and oxidant production, nf-~zb activation, and an increase in hiv replication. it is thus unfortunate that reduced cellular concentrations of gsh are a common feature of infections, including that from the hiv (staal, ela, & roederer, ) . oxidant damage to cells will indirectly create a pro-inflammatory effect by the production of lipid peroxides. this situation may also lead to upregulation of nf-tcb activity. as will be seen in later sections, genetic and dietary factors change the intensity of the inflammatory response. thus, although the inflammatory response has evolved to ensure the survival of the human species, individuals may die as a result primarily of the response to invasion rather than from the invasive agent itself. it has recently become apparent that single base changes (single-nucleotide polymorphisms [snps]), usually in the promoter region of genes responsible for producing the molecules involved in the inflammatory process, exert a modulatory effect on the intensity of inflammation. in vitro production of tnf-o~ by peripheral blood mononuclear cells (pbmcs) from healthy and diseased subjects stimulated with inflammatory agents shows remarkable individual constancy in males and postmenopausal females (jacob et al., ) . this constancy suggests that genetic factors exert a strong influence. a number of studies have shown that snps in the promoter regions for the tnf-o~ and lymphotoxin table single nucleotide polymorphisms (snps) in cytokine genes associated with altered levels of cytokine production # gene and location of polymorphism in promoter region genotype associated with raised cytokine production and~or altered clinical outcome to inflammation b pro-inflammatory cytokines tnf-t~ - lt-t~ + il-l~- il- - anti-inflammatory cytokines il- - c tgf- [~ + (arg- -pro) c tnf-ct - a allele (tnf ) lt-t~ + aa (tnfb : ) ct or tt g allelle gg gg tnf, tumor necrosis factor; lt, lymphotoxin; il, interleukin; tgf, transforming growth factor; c, cytosine; g, guanosine; t, thymidine, a, adenine. athe location of the polymorphism is indicated by the nucleotide position in the promotor region. bpoor clinical outcome for pro-inflammatory cytokines. clmproved clinical outcome for anti-inflammatory cytokines. (lt)-o~ genes are associated with differential tnf-t~ production (allen, ; messer et al., ; wilson et al., ) . the tnf (a) and tnfb (a) alleles (at - and + for the tnf-o~ and lt-t~ genes, respectively) are linked to high tnf production, particularly in homozygous individuals. the snp in the lt-ot gene (+ ) is found in linkage disequilibrium with major histocompatibility molecules hla-a , b , dr (messer et al., ; wilson et al., ) . this genotype has also been reported to define a tnf "high expresser" haplotype (warzocha et al., ) , in addition to modifying expression of lt-o~ itself (messer et al., ) . a large body of research has indicated that snps occur in the upstream regulatory (promoter) regions of many cytokine genes (bidwell et al., ) . many of these genetic variations influence the level of expression of genes and the outcome from the inflammatory response. both pro-and anti-inflammatory cytokines are influenced by the differences in genotype (allen ; turner, williams, & sankeran, ) . a number of snps that have been implicated in the outcome of inflammatory stress are shown in table . nf-lcb is activated by oxidants and switches on many of the genes involved in the inflammatory response (cytokines, adhesion molecules, and acute-phase proteins). enhancement of antioxidant defenses is important in protecting healthy tissues and in preventing excessive activation of nf-~zb by the oxidative cellular environment during inflammation (schreck et al., ) . nf-rd upregulates cytokine and adhesion molecule expression, increasing the risk of host damage (jersmann, hii, ferrante, & ferrante, ) . genetic factors also influence the propensity of individuals to produce oxidant molecules and thereby influence nf-rd activation. natural resistance-associated macrophage protein (nramp ) has effects on macrophage functions, including tnf-ct production and activation of inducible nitric oxide synthase (inos), which occurs by cooperation between the nramp , tnf-o~, and lt-o~ genes (ables et al., ) . there are four variations in the nramp gene, resulting in different basal levels of activity and differential sensitivity to stimulation by inflammatory agents. alleles , , and are poor promoters, whereas allele causes high gene expression. hyperactivity of macrophages, associated with allele , is linked to autoimmune disease susceptibility and high resistance to infection, whereas allele increases susceptibility to infection and protects against autoimmune disease (searle & blackwell, ) . as indicated earlier, a number of molecules suppress production of pro-inflammatory cytokines and exert an anti-inflammatory influence. these include antioxidant defenses and il- (chernoff et al., ; espevik et al., ) . production is modulated by genetic factors. there are at least three polymorphic sites (- , - , - ) in the il-l promoter that influence production (perrey, pravice, sinnott, & hutchinson, ) . snps also occur in genes encoding enzymatic components of antioxidant defenses, such as catalase, sod, and gsh peroxidase, which influence levels of activity (chorazy, schumacher, & edlind, ; forsberg, lyrenas, de faire, & morgenstern, ; mitrunen et al., ) . there is circumstantial evidence, that at an individual level, an inflammatory genotype exists that can adversely effect the host. in a study of inflammatory lung disease caused by exposure to coal dust, the tnf (lt-a+ a) allele was almost twice as common in miners with the disease than in those who were healthy (zhai, jetten, schins, franssen, & borm, ) . development of farmer's lung from exposure to hay dust was % greater in individuals with the tnf allele than in those without the allele (schaaf, seitzer, pravica, aries, & zabel, ) . the tnf allele was also twice as common in smokers who developed chronic obstructive pulmonary disease than in those who remained disease-free (sakao et al., ) . in addition to disease progression, genetic factors have important effects on mortality and morbidity in infectious and inflammatory disease. during malaria, children who were homozygous for tnf had a sevenfold greater risk of death or serious pathology than children who were homozygous for the tnf allele (mcguire, hill, allsopp, greenwood, & kwiatkowski, ) . in intensive-care patients the occurrence of "g high-producing allele for il- was present in those who developed multiorgan failure with a frequency of one-fifth of that of the normal population (reid, hutchinson, campbell, & little, ) . in sepsis, patients possessing the tnf allele had a . -fold greater risk of death than those without the allele, and patients who were homozygous for the lt-~ + a allele had twice the mortality rate and higher peak plasma tnf-ot concentrations than heterozygotic individuals (mira et al., ; stuber, peterson, bokelmann, & schade, ) . the tnf allele also been found in increased frequencies in systemic lupus erythromatosus, dermatitis hepetiformis, and insulin-dependent diabetes mellitus and noninsulin-dependent diabetes mellitus (niddm) (jacob et al., , wilson, clay, & crane, wilson, gordon, & di giovine, ) . thus, it now appears that each individual possesses combinations of snps in their genes associated with inflammation corresponding to inflammatory drives of differing intensities when microbes or tissue injury are encountered. at an individual level this may express itself as differing degrees of morbidity and mortality (fig. ) . the strength of the genomic influence on the inflammatory process may affect the chances of an individual developing inflammatory disease, particularly if their antioxidant defenses are poor. in addition to disease progression, genetic factors have important effects on mortality and morbidity in infectious and inflammatory disease and following injury (paolini-giacobino, grimble, & pichard, ) . there are sex-linked differences in the influence of genotype on the inflammatory processes. in general, males are more sensitive to the genomic influences on the strength of the inflammatory process than females. in a study on lt-a genotype and mortality from sepsis, it was found that men possessing a tnfb (lt-o~+ aa) genotype had a mortality of % compared with men who were tnfb (lt-o~+ gg), who had a % mortality rate. in female patients the mortalities for the two genotypes were % and %, respectively (schroder, kahlke, book, & stuber, ) . in a study on patients undergoing surgery for gastrointestinal cancer, it was found that postoperative c-reactive protein (crp) and il- concentrations were higher in men than in women. multivariate analysis showed that males possessing the tnf (tnf-o~- a) allele had greater responses than men without it. the genomic influence was not seen in females (table ) (grimble, thorell, et al., ) . furthermore, possession of the il- - t allele was associated with a % greater length of stay in hospital in old men admitted for geriatric care (table ) (grimble, anderson, et al., ) . women were unaffected by these genetic influences. paradoxically, with improvements in hygiene and vaccination programs against infectious diseases, two major changes in public health and population characteristics have led to a general increase in inflammatory stress in populations of industrialized countries in the last half century. these are, respectively, an increase in the number of overweight and obese subjects and an increase in longevity. we will now examine the mechanisms underlying this phenomenon. it has been recognized for many years that there is a strong link between the "diseases of affluence"--obesity, insulin sensitivity, and atherosclerosis. however, it is only quite recently that the realization came that inflammation provided a link between the three ( ) tnf, tumor necrosis factor; il, interleukin; crp, c-reactive protein. a d postoperatively. bl d postoperatively. *significantly different from females with same genotype by multivariate analysis allowing for longer operation time and greater blood loss; p = . andp = . for crp and il- , respectively. means + sd, values in parentheses are the number of patients. ( ) + ( ) + ( ) + ( )* + ( ) tnf, tumor necrosis factor; il, interleukin; c, cytosine; t, thymidine; a, adenine; g, guanine. athe location of the polymorphism is indicated by the nucleotide position in the il- ~ and lt-t~ genes, tnfb (gg), tnfb (ag), tnfb (aa). *significantly different from value for same sex possessing the other genotype; p < . using mann-whitney test. means + sd, values in parentheses are the number of patients. biological phenomena (fig. ) . many studies have shown a clear link between obesity, oxidant stress, and inflammation (grimble ) . the link may lie in the ability of adipose tissue to produce pro-inflammatory cytokines, particularly tnf-t~. there is a positive relationship between adiposity and tnf production. a positive correlation ,i, plasma triglyceride concentrations fig. . interaction between leptin and tumor necrosis factor (tnf) with adipose tissue mass, lipid metabolism, and inflammation. tnf and leptin stimulate the immune system and adipose tissue, respectively. both also act on lipid metabolism and plasma triglyceride concentrations. between serum tnf-~ production and body mass index (bmi) has been noted in niddm patients and healthy women (nilsson, jovinge, niemann, reneland, & lithell, ; yaqoob, newsholme, & calder, ) . leptin has been shown to influence proinflammatory cytokine production ( fig. ). thus, plasma triglycerides, body fat mass, and inflammation may be loosely associated because of these endocrine relationships. a number of population studies have been conducted to explore the extent and nature of the relationship of inflammation to these diseases of affluence. the studies have examined populations in which there is a high incidence of insulin insensitivity, such as pima indians and individuals with a south asian background. tnf-o~ is overexpressed in adipose and muscle tissues of obese subjects compared with tissues from lean individuals (hotamisligl & spiegelman. ) . in a study of a group of nondiabetic pima indians, employing the hyperinsulinemic euglycemic clamp to assess insulin action, strong evidence of the links between inflammation, insulin insensitivity, and obesity emerged. plasma il- was found to be related positively to adiposity and negatively to insulin sensitivity. the investigators concluded that the relationship between il- and insulin action appeared to be mediated through adiposity (vozarova, weyer, & hanson, ) . a number of studies have looked at the extent of the interaction between insulin insensitivity and inflammation by studying the extreme form of diabetes, type diabetes mellitus. a study assessed endothelial cell perturbation by measurement of von willebrand factor and tissue-plasminogen activator (t-pa), in type diabetics who had had the disease for < or > yr. compared with normal subjects, children with diabetes for <lyear had the highest concentrations of von willebrand factor, indicating that endothelial perturbation represents an early event in type diabetes (romano, pomilio, & vigneri, ) . studies that have attempted either to remove the cause of inflammation, to lower plasma lipids, or improve insulin sensitivity have supported the hypothesis that inflammation, insulin sensitivity, and atherosclerosis are intimately interlinked. when a study of the potential anti-inflammatory effect of weight loss was conducted in obese women, it was found that a yr weight-reduction program resulted in lowering of plasma il- , tnf-cx, and adhesion molecule concentrations (ziccardi, nappo, & giugliano, ) . since the s large population studies have indicated that inflammation plays a key role in cardiovascular disease (cvd) (grimble, ; ross, ) . periodontal disease and other low-grade infections, such as chlamydia pneumoniae infection, have been linked closely with atherosclerosis. development of atherosclerotic plaques to which macrophages have already been recruited occurs by cytokines inducing hypertriglyceridemia and hypercholesterolemia (kol, sukhova, lichtman, & libby, ; saldeen & rand ) . chlamydial infection induces production of tnf-~. the cytokine inhibits the action of lipoprotein lipase, leading to changed lipid metabolism, elevation of serum triglycerides, and a decrease in serum high-density lipoprotein (hdl) cholesterol, thereby exerting an atherogenic influence (arrnitage, ) . in the bruneck study, raised plasma bacterial lipopolysaccharide (lps) was associated with an increased rate of thickening of the coronary artery intima. smoking, a further inflammatory stress, exacerbated this effect (williet & kiechl, ) . lps binds in human serum to both low-density lipoprotein (ldl) and hdl cholesterol and makes ldl cholesterol immunogenic or toxic to endothelial cells. thus, the link between specific infections and atherosclerosis may be a nonspecific effect of chronic inflammation on the atherosclerotic process. it is well recognized that alterations in plasma protein concentrations invariably occur among the many metabolic changes that occur during inflammation. proteins that increase during inflammation (positive acute-phase proteins, e.g., crp and fibrinogen) and those that decrease (negative acute-phase proteins, e.g., serum albumin and retinolbinding protein) are used to diagnose inflammation in population nutritional surveys. the early indications that inflammation was involved in atherosclerosis came from the findings that there were links between concentrations of positive and negative acute-phase proteins in blood and cvd (grimble, ). in the bruneck study of a group of to -yr-old italians, it was found that impaired glucose tolerance and, to a greater extent, type diabetes were strong independent predictors of advanced atherosclerosis (measured by high-resolution ultrasound of the carotid artery) (bonora, kiechl, & oberhollenzer, ) . a cross-sectional study of obesity, plasma crp, fibrinogen, and carotid artery intima media thickness (an index of atherosclerosis) in more than multiethnic subjects showed a positive relationship between plasma crp and body fat. intima media thickness was related to crp and fibrinogen in men. the relationship was attenuated by adjustment for bmi (festa, d'agostino, & williams, ) . a cross-sectional study of more than men and women examined the link between elevated plasma crp concentrations and prevalent cvd, ankle/brachial blood pressure, and carotid artery intima media thickness. after adjustment for age and family type, there was a weak association between crp and intima media thickness in both sexes and with prevalent heart disease in women (folsom, pankow, & tracy, ) . a study on a turkish population of individuals with low cholesterol concentration but a high prevalence of other risk factors for coronary heart disease investigated whether crp acted as a predictor of coronary heart disease. among the risk factors, only crp and systolic blood pressure were independent risk factors for cvd (onat, sansoy, & yildirim, ) . in a study in which crp concentrations and conventional risk factors for cvd in healthy indian asians were compared with values in a similar number of healthy european white subjects, crp and cvd risk factors were higher in the former group. however, differences were eliminated when adjustment was made for central obesity and insulin resistance score (chambers, eda, & bassett, ) . a study on a brazilian population found that markers of inflammation correlated with components of the metabolic syndrome---cardiovascular and diabetes risk factors, insulin resistance, and central obesity (duncan & schmidt, ) . in a study of healthy elderly subjects in the netherlands, acute-phase proteins, soluble adhesion molecules, il- , and insulin were measured and associated with cholesterol and obesity. the association between insulin, obesity, and cholesterol was as strong as between insulin, acute-phase proteins, and adhesion molecules (hak, pols, & stehouwer, ) . insulin insensitivity occurs as part of the normal inflammatory response to pathogens. during inflammation the secretion of catabolic hormones, which enhances muscle protein breakdown and glutamine release, will oppose insulin action. paradoxically, however, although insulin insensitivity may initially exert a beneficial effect during the response to infection and injury, it has an adverse influence in chronic disease processes. glucose and glutamine act as major fuels for immune cells during the normal response to infection. large increases in glucose and glutamine utilization by immune cells occur during the response to infection and injury (spitzer, bagby, meszaros, & lang, , . studies in rats given lps and observations in patients with sepsis show that the flow of amino acids into the circulation increases and gluconeogenesis is enhanced under the influence of pro-inflammatory cytokines. an insulin-insensitive state will reduce glucose uptake by tissues in which the process is insulin dependent (muscle), thereby increasing availability to tissues in which the process is not insulin dependent (immune tissue). a study in the united states investigated whether elevated plasma il- and crp was associated with the development of type diabetes mellitus in more than , healthy women. in the -yr follow-up period, women developed type diabetes. for these women, baseline il- and crp were higher than in controls. the relative risk of future type diabetes in women between the highest and lowest quartiles of these inflammatory markers was . for il- and . for crp (pradhan, manson, & rifai, ) these data suggest a possible role for inflammation in diabetogenesis. furthermore, data collected from the third national health and nutrition examination survey (nhanes iii) in the united states provide further evidence for a possible role of inflammation in insulin resistance and glucose intolerance. more than men and women were studied for associations between plasma crp, fasting insulin, glucose, and glycosylated hemoglobin (hba c). elevated crp was associated with higher insulin and hba c in both sexes and with raised glucose in women (wu, dorn, & donahue, ) . a study on the link between crp, central adiposity, and fasting glucose and insulin in more than healthy italian women showed an independent relationship of adiposity to insulin resistance and crp concentrations (pannacciulli, cantatore, & minenna, ) . in a review, nishimura and murayama ( ) discussed the possibility that treatment of periodontal infection may improve insulin sensitivity. their conclusions about the efficacious effect of an anti-infective approach are supported by a study in which type diabetic patients with periodontal disease were given antimicrobial treatment with minocycline. blood tnf-t~ concentrations and glycosylated hemoglobin decreased (iwamoto, nishimura, & nakagawa, ) . conversely, improvement in insulin sensitivity exerted an anti-inflammatory effect. a group of hyperlipidemic patients and normolipidemic controls who were insulin resistant and hypertriglyceridemic with low hdl cholesterol concentrations and raised tnf-o~ production and plasma il- and fibrinogen concentrations were studied. all subjects were treated with the lipid-lowering drug bezafibrate. the drug normalized all parameters.the drug thus exerts an anti-inflammatory effect associated with its ability to normalize lipid metabolism and insulin sensitivity (jonkers, mohrschladt, & westendorp, ) . in an in vitro study on a lung epithelial cell line, the oral hypoglycemic agent thiazolidinedione exerted an anti-inflammatory influence by suppressing production of monocyte chemoattractant protein (mcp- ) (momoi, murao, & imachi, ). it can be concluded from the studies reported in the above sections that inflammation is closely linked to obesity, cvd, insulin insensitivity, and diabetes mellitus. although this interaction is a relatively novel concept, it has been known for many years that these diseases are interlinked and that insulin insensitivity is a common factor in their pathology. a number of recent studies have examined the mechanisms underlying the link between inflammation and the conditions and diseases in which insulin insensitivity plays a part (fig. ) . a difficult question to address is whether chronic inflammation leads to a condition of insulin insensitivity and associated diseases, or whether insulin insensitivity, which is associated with obesity, diabetes, and atherosclerosis, brings about a condition of chronic inflammatory stress. many studies suggest that the former is more likely to be the case. reviews have highlighted the risk of inflammation and infection of atherosclerotic plaques associated with poor diabetic control and the importance of elevated nonesterified fatty acid concentrations, glucocorticoids, and low-grade inflammation as causative agents in atherosclerosis and insulin insensitivity (bell, ; corry & tuck, ) . a study on type diabetes mellitus patients determined the extent to which pbmcs contributed to oxidative stress and inflammation. a linear correlation between hbalc and superoxide release was found. the authors concluded that type diabetes mellitus is accompanied by priming of pbmcs and increased self-necrosis. the necrosis may start a chain of events that results ultimately in oxidant stress and endothelial dysfunction (shurtz-swirski, sela, & herskovitis, ) . in a study on pima indians, who are characterized by high incidence of obesity and insulin resistance but not atherosclerotic disease, crp, icam- , and secretory phospholipase a are correlated with body fat but not e-selectin and von willebrand factor. in addition to showing that markers of inflammation increase with adiposity, the study showed that markers of endothelial dysfunction increase in proportion to insulin resistance and inflammation (weyer, yudkin, & stehouwer, ) . a further study on pima indians examined whether a raised white blood cell count predicted a worsening of insulin action, insulin secretory function, and the development of type diabetes. a high white blood cell count predicted the development of diabetes with a relative risk of . when adjusted for age and sex (vozarova, weyer, & lindsay, ) . tnf-o~ has been shown to play a key role in mediating insulin resistance as a result of obesity in patients and in numerous rodent models of obesity---diabetes syndromes (hotamiligil & spiegelman, ) . multiple mechanisms have been suggested to account for these metabolic effects of tnf-ot. these include the downregulation of genes that are required for normal insulin action, direct effects on insulin signaling, induction of elevated plasma free fatty acids via stimulation of lipolysis, and negative regulation of ppar- , an important insulin-sensitizing nuclear receptor (moiler, ) . the induction of insulin resistance is mediated through its ability to produce serine phosphorylation of insulin receptor substrate (irs)- , decreasing the tyrosine kinase activity of the insulin receptor (hotamisligil, budavari, murray, & spiegelman, ) . neutralization of tnf-t~ in obese fa/fa rats by intravenous administration of a soluble tnf receptor immunoglobulin g chimeric protein substantially improved insulin sensitivity and restored the tyrosine kinase activity in fat and muscle (hotamisligil et al., ) . in an in vitro study using t -l adipocytes, tnf-t~ was shown to induce sustained suppressor of cytokine signaling protein (socs- ) production. socs- has been shown to decrease insulin-induced irs tyrosine phosphorylation and its association with the p regulatory subunit of phosphatidylinositol- kinase (emanuelli, peraldi, & filloux, ) . these observations therefore suggest that socs- may be a key mediator in the development of insulin sensitivity during inflammation. obesity is associated with insulin resistance, particularly when body fat has a central distribution. while elevated plasma leptin concentrations are associated with obesity, some studies have suggested that insulin sensitivity is an additional determinant of circulating leptin concentrations. leptin is produced by adipose tissue in proportion to adipose tissue mass and is a pleiotropic molecule. in addition to playing a role in appetite and adipose tissue regulation, leptin influences immune functions. leptin concentrations increase acutely during inflammation and regulate t-cell responses, polarizing t-helper (th) cells toward a th phenotype. thus, increased leptin production during obesity, may exert a pro-inflammatory influence (faggioni, feingold, & grunfeld, ) . further complexity is added to the concept of a link between insulin sensitivity, inflammation, and obesity by the results of a study of individuals selected from the health professionals follow-up study in the united states (chu, spiegelman, & hotamisligil, ) . in the study plasma insulin, leptin, and soluble tnf receptor (stnf-r, an index of tnf-o~ production) concentrations were measured and correlated with bmi and the cvd risk factors insulin, triglyceride, t-pa antigen levels, and apolipoprotein (apo)-a . in a multivariate regression model controlling for age, smoking, alcohol intake, physical activity, and diet, bmi was inversely associated with hdl cholesterol and apo-a and positively associated with trigyceride, apo-b, and t-pa antigen levels. the associations between bmi and these cvd risk factors were only slightly changed after adjusting for leptin and/or stnf-r, but were substantially attenuated after controlling for insulin levels. these data suggest that the association between obesity and biological predictors of cvd may be mediated through changes in plasma insulin, rather than leptin or stnf-r levels, and that insulin may be exerting an anti-inflammatory effect. (cnop, landchild, & vidal, ) . genetic factors may play a part in the interaction between inflammation, insulin insensitivity, and obesity. nf-~:b is an important mediator of inflammation by increasing transcription of a range of genes central to the inflammatory process (cytokines, adhesion molecules, acute-phase proteins). snps in the nf-r,~b gene were investigated in a group of type diabetic patients and compared with gene frequencies in normal controls. it was found that there was a higher frequency of allele bp (a ) (high bioactivity) and a lower frequency of allele bp (a ) (less bioactive) in diabetics than in controls. genotype may thus contribute to inflammatory stress in diabetes mellitus (hegazy, o'reilly, & yang, ) . nf-~:b may also provide an important focus for ppar- action, because it has been shown in a number of studies that ppar-~/in combination with retinoid x receptor is able to inhibit nf-~:b activation (wada, nakajima, & blumberg, ; fruchart, staels, & durlez, ; debril, renaud, fajas, & auwerx, ) . it is interesting to note that the n- polyunsaturated fatty acids (pufas), found in abundance in fish oil, are ppar-y agonists, raising the possibility that the oil may exert its anti-inflammatory effects partly via this mechanism. in an investigation of cytokine production in healthy males, the author found that in the study population as a whole there were no statistically significant relationships between bmi, plasma fasting triglycerides, and the ability of pbmcs to produce tnf-a. however, individuals with the lt-c~+ aa genotype (associated with raised tnf production) showed significant relationships between tnf production and bmi and fasting triglycerldes (fig. ) . thus, despite the study population being comprised of healthy subjects, within that population were individuals with a genotype that resulted in an "aged" phenotype as far as plasma lipids, bmi, and inflammation were concerned (paolini-giacobino et al., ) . it is well known that the incidence of diseases of affluence and recognizable inflammatory diseases, such as rheumatoid arthritis, increase with aging. are these phenomena an unfortunate side effect of maturity, or is there a common mechanism that determines the appearance of these diseases patterns in the elderly.'? paradoxically, aging is associated with a decline in t-lymphocyte function and an increase in inflammatory stress. a number of elements of the chronic inflammatory response are apparent in otherwise healthy elderly subjects. the elements of the response include muscle protein loss, a rise in plasma acute-phase protein concentrations, and a decrease in plasma zinc. an age-related increase in il- concentration has been found in serum, plasma, and supernatants of mononuclear blood cell cultures from apparently healthy elderly people and centenarians (fagiolo et al., ; baggio, donnazzan, monti, & marl, ; ershler & kerller, ) . because il- is a pleiotropic cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types (ershler & kerller, ) and plays a pivotal role in neuroendocrlne and immune system homeostasis, it is not surprising that the rise in production of pro-inflammatory cytokines might have long-term pathological effects (bethin, vogt, & muglia, fasting triglycerides fig. . relationships between tnf production, bmi and fasting triglycerides, and the ability of pbmcs to produce tnf-o~ in the study populations: (a) all subjects irrespective of genotype, (b) individuals with the lt-ct+ aa genotype, associated with raised tnf production. ns, nonsignificant relationship; bmi, body mass index; pbmcs, peripheral blood mononuclear cells. the number of subjects is shown in parentheses; the correlations were examined by spearman' s rank correlation. levels of this cytokine have also been found as early as at - yr of age (mysliwska, bryl, foerster, & mysliwski, ) , particularly in men (young, skibinski, mason, & james, ) . population studies have shown that the magnitude of increase in the concentration of il- is a reliable marker for functional disability and a predictor of mortality in the elderly (ferrucci, harris, guralnik, & tracy, ; harris, ferrucci, tracy, & corti, ) . antioxidant status may decline with age (nuttall, dunne, kendall, & martin, ) and may thus be linked to increased tnf-~ production (kudoh, katagai, takazawa, & matsuki, ; rink, cakman, & kirchner, ). an enhanced capacity for the release of pro-inflammatory cytokines by white blood cells may contribute to the pathogenesis of ischemic stroke. grau et al. ( ) investigated the lps-induced release of il- [ , il- , il- , and tnf- ~ in whole blood from patients with a history of ischemic stroke under the age of and age-and sex-matched healthy control subjects. release ofll- was significantly higher in young stroke patients than in control subjects (grau et al., ) . the question of whether aging is associated with chronic elevation of cytokine production or whether an increased capacity for cytokine production following the normal inflammatory challenges of life develops during aging is an interesting one to consider. insight into this issue can be gained from the response to surgery where an inflammatory stimulus is applied at a defined moment in time, making it easy to follow the subsequent response. ono, aosasa, tsujimoto, ueno, and mochizuki ( ) investigated the agerelated changes in the inflammatory response in patients with gastric cancer undergoing distal gastrectomy. patients were divided into two groups: > yr of age (elderly group) and -< years of age (young group). serum il- concentrations, tnf-ct production and cd b/cd expression by monocytes, and the postoperative clinical course were compared between the two groups to assess the inflammatory response to surgery. tnf-~ production by lps-stimulated monocytes and cd l b/cd expression on monocytes were significantly higher in the elderly than in the young group. moreover, serum il- concentrations on the first postoperative day in the elderly group were significantly higher than those in the young group. paradoxically, both loss of body weight and lean tissue and obesity are found in elderly populations. is there, therefore, a link between this phenomenon and increased levels of inflammation? the loss of muscle mass and strength that occurs with aging is described clinically as sarcopenia (rosenberg, ; roubenoff, ) . it is an important contributor to the development of frailty and functional impairment during aging. it is well established that aging is associated with a significant decline in muscle strength that becomes functionally important by the seventh decade of life. the relationship between chronic inflammation owing to disease during aging and the prevalence of low body mass are well illustrated in rheumatoid arthritis. in a study on patients with rheumatoid arthritis, the loss of body mass was greater for lean tissue than fat, with over % of the rheumatoid group falling into the lowest th percentile of a reference population for skeletal muscle mass assessed from the upper arm muscle area. in female patients there was a significant correlation between reduced fat-free mass and two indicators of inflammatory stress---erythrocyte sedimentation rate and plasma crp concentration (munro & capell, ) . clinical and animal studies show a relationship between raised plasma cytokine concentrations and low muscle mass. visser et al. ( ) investigated whether markers of inflammation are associated with muscle mass and strength over a time course of several years in over healthy well-functioning black and white elderly persons ( - yr). mid-thigh muscle cross-sectional area, appendicular muscle mass, and muscle strength were assessed. plasma concentrations of il- and tnf-~ were also measured. higher cytokine concentrations were associated with lower muscle mass and lower muscle strength. the most consistent relationship across the gender and race groups was observed for il- and grip strength. when an overall indicator of elevated cytokine production was created by combining the concentrations of il- and tnf-o~, with the exception of white men, elderly persons having high concentrations of il- (> . pg/ml) as well as high levels of tnf-~ (> . pg/ml) had a smaller muscle area, less appendicular muscle mass, and lower muscle strength compared to those with low levels of both cytokines. thus, raised plasma concentrations of il- and tnf-~ are associated with lower muscle mass and lower muscle strength in well-functioning older men and women as well as those suffering frank inflammatory disease. nutrient intake is clearly another important determinant of lean body weight and fat mass and may play a part in the decline in lean tissue with age as well as an increase in inflammatory stress. a recent survey of , subjects in communities in nhanes iii in the united states also included a survey of about elderly people ranging in age from to yr, to yr, and + yr (marwick, ) . the report indicated that the median intake of total energy was in general lower than the recommended kcal for men and kcal for women (marwick, ) . chronic inflammation is either a causative agent or a closely associated process in the pathology of obesity, insulin insensitivity, and atherosclerosis.the incidence of these conditions increases with aging. a fundamental question is which precedes the other-the general increase in inflammation or the development of diseases with overt and covert inflammatory bases? this "chicken-and-egg" question is difficult to answer. however, examination of data from studies conducted in elderly populations may throw some light on the answer to this conumdrum. there are at least two potential mechanisms for the higher level of chronic inflammation observed in elderly than in younger subjects. the first of these is that the elderly are experiencing a higher level of asymptomatic urinary infection. this possibility was studied in consecutive patients ( - yr) admitted to the hospital for functional disability. patients were examined for the presence or absence of bacteria in the urine. twenty subjects had a positive urine culture, and sex-and age-matched subjects had a negative urine culture. inclusion criteria were temperature < . °c, no clinical signs of infection, and no current antibiotic treatment. patients with asymptomatic bacteriuria had significantly increased levels of tnf receptors and a higher number of neutrophils in the blood compared to the group without bacteriuria. thus, the study provides some support for the hypothesis that asymptomatic urinary infections are associated with low-grade inflammatory activity in frail, elderly subjects (prio, bruunsgaard, roge, & pedersen, ) . a second potential mechanism resides in endocrine changes during aging. in aging, dysregulation of secretion of hormones that come under the regulation of the hypothalamic-pituitary-adrenal axis may occur. this may have an effect on the regulation of cortisol secretion, as mentioned earlier. cortisol is important as an anti-inflammatory agent. the effect of aging on glucocorticoid sensitivity of pro-inflammatory cytokine production was examined in elderly men, testosterone-treated elderly men, and young controls. stress-induced increases in cortisol did not differ significantly between experimental groups, but glucocorticoid sensitivity increased significantly in young controls and testosterone-treated elderly men, whereas a decrease was found in untreated elderly men. as the increase in glucocorticoid sensitivity after stress serves to protect the individual from detrimental increases of pro-inflammatory cytokines, the disturbed mechanism in elderly men may result in an increase in inflammatory stress (rohleder, kudielka, hellhammer, wolf, & kirschbaum, ) . there is now a large body of evidence suggesting that the decline in ovarian function with menopause is associated with spontaneous increases in pro-inflammatory cytokine production. as mentioned earlier, studies in men and postmenopausal women indicate a remarkable individual constancy in the ability of pbmcs to produce tnf-cz ex vivo, and genetic determinants underlie this constancy. however in premenopausal women production is highly variable at an individual level, indicating how ovarian hormones are able to override the influence of genotype (jacob et al., ) . the exact mechanisms by which estrogen interferes with cytokine activity are still incompletely known but may include interactions of the estrogen receptor with other transcription factors, modulation of nitric oxide activity, antioxidative effects, plasma membrane actions, and changes in immune cell function. experimental and clinical studies also strongly support a link between the increased state of pro-inflammatory cytokine activity and postmenopausal bone loss (pfeilschifter et al., ) . recent evidence indicates the presence of snps, associated with the strength of the inflammatory response, affects longevity. human longevity may be directly correlated with optimal functioning of the immune system. therefore, it is likely that one of the genetic determinants of longevity resides in polymorphisms for genes influencing the activity of the immune system. it has been estimated that up to variations in the genome contribute to life span (martin, ) . those contributing to loss of muscle and bone mass during aging are related to the inflammatory process and include pro-and anti-inflammatory cytokines and their receptors. studies in mice have shown that the genes controlling the major histocompatibility complex (mhc), known to control a variety of immune functions, are associated with differences in the life span of different strains of mice, but a major difference between observations in mice and humans is that the latter have a lifetime experience of exposure to pathogens, whereas for laboratory animals this exposure is kept to a minimum. thus, although hla studies in mice of different genotypes may be interpreted to support studies of mhc effects on longevity in humans, in mice the association may be by way of altered susceptibility to lymphomas, whereas in human beings the effect on longevity is likely to be via an altered response to pathogens and susceptibility to infectious disease. a number of cross-sectional studies have examined the role of hla genes on human longevity by comparing hla antigen frequencies between groups of young and elderly persons. conflicting findings have been obtained. when this topic was reviewed (caruso et al., ) , it was concluded that in humans there may be an association between longevity and some hla-dr alleles or the hla-bs,dr haplotype. these genotypes are involved in the antigen nonspecific control of immune response, in other words, the component of immune function associated with inflammation and cytokine biology. recent evidence indicates that presence of snps in certain pro-and anti-inflammatory cytokine genes influences life span. when individuals between and yr of age were studied, it was noted that not only was plasma il- concentration positively related to age but individuals with a snp in the promoter region of the il- gene, which predisposes to high levels of production of the cytokine (- gg), decreased in frequency with age. the effect was seen in men but not in women (bonafe, olivieri, & cavallone, ) . although men with snps made up % of the -to -yr-old age group, the percentage fell to % in subjects < years of age. conversely, one of three snps in the il- gene (- gg), which is closely linked to higher production of the anti-inflammatory cytokine il- (hutchinson, pravica, hajeer, & sinnott, ; turner et al., ) , was found in higher proportions in male centenarians than in younger controls ( vs %). in females this genotype exerted no effect upon longevity (lio et al., ) . thus, it would appear that genetic characteristics that might influence the balance between proand anti-inflammatory cytokines influence mortality in men but not in women (franceschi et al., ) . a study on snps that influence interferon (ifn)-~t production further reinforces the concept that possession of a genotype that predisposes to a raised pro-inflammatory status is not compatible with a long life span (lio et al., ) . in women, possession of the a allele, which is associated with low production of ifn-'~, significantly increased the possibility of reaching old age. it might be concluded that possession of high-producing alleles of the il- is universally protective against morbidity as well as mortality. possession of a genotype that results in low levels of il- production (- aa) increases the risk of developing inflammatory diseases (hajeer, lazanes, & turner, ; huizinga, keijsers, & yanni, ; tagore, gonsalkorale, & pravica, ) . however, as already mentioned, in a large survey of hospitai admission in the netherlands, patients with raised il- :il- ratios had higher mortality rates (van dissel et al., ) . not all studies implicate cytokine gene snp in longevity. cytokine gene polymorphisms at il- o~, il- ~, il- ra, il- , il- , and tnf-o~ were measured in finnish nonagenarians ( men and women) and in healthy blood donors ( - yr) used as controls. no statistically significant differences were found in the distribution of genotype, allelic frequencies, and a + carrier status between nonagenarians and younger controls (wang, hurme, jylha, & hervonen, ) . in a review on the different impact of genetic factors on the probability of reaching old age, franceschi et al. ( ) concluded from studies conducted in italy that emerging evidence (regarding mtdna haplogroups, thyrosine hydroxylase, and il- genes) suggests that female longevity is less dependent on genetics than male longevity and that female centenarians are more likely to have had a healthier lifestyle and more favorable environmental conditions than males. however, a recent study conducted by our group suggests that although a pro-inflammatory genotype may be disadvantageous to elderly males, it may confer a survival benefit in females. subsequent survival was studied in elderly geriatric patients ( + yr) after a period of hospitalization for a range of conditions necessitating geriatric care. although women possessing a pro-inflammatory genotype (tnf-o~- a allele or il- - gg) had improved yr survival rates, men possessing pro-inflammatory genotypes (il-l~- t allele or lt-ct + aa) had shortened survival rates (grimble, thorell, et al., ) (table ) . as outlined in the preceding sections, the inflammatory response, although essential for survival in the presence of pathogens, can exert deleterious effects on the host.the clear need to find ways of modulating cytokine production and other aspects of inflammation has fostered the research area of immunonutrition. in a clinical context the purpose of immunonutrition is to find nutritional means of altering the patient' s inflammatory response to infection and injury, from the detrimental to the beneficial side of the pivot on which an individual undergoing a response is positioned. while inflammation may be exerting deleterious effects most obviously in patients, people on the borderline of health and disease living in the general population table nutrients commonly used in immunonutrient supplements and their potential mode of action • n- polyunsaturated fatty acids: act as anti-inflammatory agents and reverse immunosuppression • sulfur amino acids and their precursors: enhance antioxidant status via gsh synthesis • glutamine: nutrient for immune cells, improves gut barrier function, precursor for gsh • arginine: stimulates nitric oxide and growth hormone production, improves helper t-cell numbers • nucleotides: rna and dna precursors, improve t-cell function may also require nutritional modulation of ongoing inflammatory processes. during the last years the pace of evolution ofimmunomodulatory feeds and intravenous solutions has accelerated. these products contain combinations of a number of components to which various functional attributes are ascribed them (table ) (grimble, a) . many studies have indicated that n- polyunsaturated fatty acids (pufas), glutamine, arginine, sulfur amino acids, and nucleotides are all potentially capable of shifting the balance from a disadvantageous to an advantageous response to infection and injury. the examples used here are illustrative rather than comprehensive. a number of studies indicate that improvement of antioxidant status is associated with an increase in cellular aspects of immune function. meta-analyses have been conducted on the efficacy of immunonutrients that influence antioxidant status. in clinical trials, indices such as infection rates, mortality rates, and length of stay are often measured in the absence of functional and biochemical aspects of the response, such as t-cell function, cytokine production, and antioxidant status, and vice versa, giving a rather incomplete picture of the mechanisms of any observed effects of immunonutrition. however, beale, bryg, & bihari ( ) , in a meta-analysis of studies containing more than patients receiving enteral immunonutrition, observed that although there was no effect upon mortality, there were significant reductions in infection rates, time spent on a ventilator, and length of hospital stay. while this finding indicates that immunonutrition may be useful in modulating the inflammatory process in patients experiencing severe inflammation, the consistency of the effects observed was disappointing. there are at least three major reasons why it is difficult to demonstrate a consistent effect. first, patients used as the subjects of clinical trials of immunonutrients will constitute a diverse population--different ages, at different stages of a disease process, and undergoing complex clinical treatment in addition to nutrient therapy. second, patients will have differing genetic backgrounds that will influence the intensity of the inflammatory and immune responses they are undergoing. this issue is dealt with below. third, nutrients may exert paradoxical effects, as illustrated by the findings of the first observations of the effects of fish oil on cytokine production in healthy subjects. the findings of endres et al. ( ) that a daily supplement of g/d of fish oil given to nine young men for wk was able to reduce ex vivo production ofll- and tnf-~ by lps-stimulated pbmcs aroused great interest in fish oil as an anti-inflammatory nutrient. this perception was supported by a large amount of animal data. however, endres' data showed a wide variability in the effect of the fish oil supplements. the standard deviations of the mean for il- and tnf-cx production were and %, respectively. this indicates that cytokine production could have risen or fallen as a consequence of taking the supplement. the effects of supplementing healthy young men with g/d offish oil for wk, on tnf-ct production by pbmcs stimulated with endotoxin have been studied in the author' s laboratory. it was found that % of subjects experienced a fall in production and % a rise. although the ability of fish oil to increase tnf-t~ production is at first sight paradoxical, earlier work of dinarello, bishai, rosenwasser, and coceani ( ) and kunkel, remick, spengler, and chensue ( ) indicated that fish oil could potentially change cytokine production in either direction. what mechanisms could result in this divergent effect? inflammation will result in activation of phospholipase a , which releases arachidonic acid (aa) (c : n- ) from the cell membrane for prostaglandin e (pge ) or leukotriene b (lt b ) synthesis. the in vitro studies (kunkel et al., ) showed that pge suppressed tnf-t~ production, whereas ltb had the opposite effect (dinarello et al., ) .. fish oil is rich in eicosapentaenoic acid (c : n- ), which will replace aa in the cell membrane and results in the production of pge and lt b . pge and lt b are considerably less potent than the corresponding compounds produced from aa, and thus dietary fish oil may lessen the inhibitory influence of pge or the stimulatory influence of ltb on tnf-o~ production, resulting in a potential increase or decrease, respectively, in production of the cytokine. fish oil could thus result in an inflammatory cytokine response, which could fall on either side of the pivot. the response to bacterial invasion of the body, or injury, contains a paradox. although the inflammatory response and the t-cell response both play a part in defeating the invader, the inflammatory response may in some clinical circumstances exert an inhibitory influence on t-cell function. in severely infected or traumatized patients, an enhanced inflammatory state occurs, which is associated with immunosuppression. in vitro studies support this inverse relationship. pbmcs taken from healthy young subjects and incubated with gsh show decreased pge and ltb production (reduced inflammation) and an increase in mitogenic index and il- production (enhanced immune function) (wu, meydani, sastre, hayek, & meydani, ) . thus, enhancement of antioxidant defenses reduces the likelihood of the inflammatory response suppressing t-cell function (grimble, (grimble, , b . although all antioxidants are important owing to the linked nature of antioxidant defense (fig. ) , gsh plays a pivotal role as it acts directly as an antioxidant and maintains other components of defense in a reduced state through enzymic conversion between the oxidized and reduced states. various compounds can be used to increase gsh synthesis (fig. ). n-acetyl cysteine (nac) and the gsh prodrug oxothiazalidine- -carboxylate (procysteine) have been used in a number of clinical studies. tissue gsh content is also influenced by protein and sulfur amino acid intake. unfortunately, surgery, a wide range of diseases that have an inflammatory component, and aging and protein energy malnutrition decrease gsh concentration in blood and other tissues (boya et al., ; loguercio et al., ; luo, hammarqvist, anderson, & wernerman, ; micke, beeh, schlaak, & buhl, ; nuttal et al., ; reid et al., ) (table ). within h of elective abdominal surgery, muscle gsh content falls by > %. values return to normal h postoperatively. a smaller perturbation in blood gsh occurs over a shorter time course. modification of the gsh content of liver, lung, spleen, and thymus in young rats by feeding diets containing a range of casein (a protein with a low sulfur amino acid content) concentrations changed immune cell numbers in lung (hunter & grimble, found that in unstressed animals the number of lung neutrophils decreased as dietary protein intake and tissue gsh content fell. however, in animals given an inflammatory challenge (endotoxin), liver and lung gsh concentrations increased directly in relation to dietary protein intake. lung neutrophils, however, became related inversely with tissue gsh content. addition of methionine to the protein-deficient diets normalized tissue gsh content and restored lung neutrophil numbers to those seen in unstressed animals fed a diet with adequate protein content (fig. ) . why does tissue gsh content have differing effects on immune cell populations depending on whether or not an inflammatory response is occurring? a partial explanation may come from an in vitro study using hela cells and cells from human embryonic kidney. in the study, both tnf-o~ and hydrogen peroxide resulted in activation of nf-~cb and ap (wesselborg, bauer, vogt, schmitz, & schulze-osthoff, ) . addition of the antioxidant sorbitol to the medium suppressed nf-~fl activation as expected, but unexpectedly activated ap . thus, the antioxidant environment of the cell might exert opposite effects upon transcription factors closely associated with inflammation (e.g., nf-~fl ) and cellular proliferation (e.g., ap ). evidence for this biphasic effect was seen when gsh was incubated with immune cells from young adults (wu et al., ) . a rise in cellular gsh content was accompanied by an increase in il- production and lymphocyte proliferation (enhancement of t-cell function) and a decrease in production of the inflammatory mediators pge and ltb (anti-inflammatory influence). without doubt, a decline in antioxidant status in the presence of oxidant stress will increase inflammatory stress. the interaction between oxidant stress and an impaired ability to synthesize gsh, a situation that stimulates inflammation, is clearly seen in cirrhosis, a disease that results in high levels of oxidative stress and an impaired ability to synthesize gsh . in pena an inverse relationship between gsh concentration and the ability of monocytes to produce il- , il- , and tnf-t~ was observed. treatment of cirrhotic patients with the procysteine increased monocyte gsh content and reduced il- , il- , and tnf-t~ production. septic patients given an infusion of nac ( mg/kg bolus followed by infusion of mg/kg over -h) showed a decrease in plasma il- and soluble tnf receptor p , had a reduced requirement for ventilator support, and spent fewer days in intensive care than patients not receiving nac (spapen et al., ). de rosa et al. ( showed that nac was able to restore tissue gsh concentrations in individuals with hiv infection. in a study on hiv-positive patients, brietkreutz et al. ( ) showed that a dose of mg/d of nac for mo resulted in a decrease in plasma il- (decreased inflammation), an increase in natural killer cell activity, and increased responsiveness of t lymphocytes to tetanus toxin stimulation (improved lymphocyte function). antioxidants might act to prevent nf-r,b activation by quenching oxidants. however, nf-rd and ap may not respond to changes in cell redox state in the same way. when rats were subjected to depletion of effective tissue gsh pools by administration of diethyl maleate, there was a significant reduction in lymphocyte proliferation in spleen and mesenteric lymph nodes (robinson et al., ) . an increase in inflammatory stress would be expected in this study. thus, it can be hypothesized that antioxidants exert an immunoenhancing effect by activating transcription factors that are strongly associated with cell proliferation (e.g., ap ) and an anti-inflammatory effect by preventing activation of nf-r,b by oxidants produced during the inflammatory response (dr&ouml; ge et al., ) .thus, inclusion of antioxidants or substances that increase gsh synthesis in immunonutrient mixes would seem to be beneficial. improvement of antioxidant defenses is also possible by feeding other components of antioxidant defenses. supplementation of the diet of healthy subjects and smokers with iu/d t~-tocopherol for wk suppressed the ability of pbmcs to produce tnf-t~ (mol, de rijke, demacher, & stalenhoef, ) . the same dose given to healthy elderly subjects for d increased delayed-type hypersensitivity and raised antibody titers to hepatitis b (meydani et al., ). an enteral feed enriched with vitamin e, vitamin c, and taurine given to intensive-care patients decreased total lymphocyte and neutrophil content in bronchioalveolar lavage fluid (decreased inflammation) and resulted in a reduction in organ failure rate, a reduced requirement for artificial ventilation, and a reduction of d in intensive-care stay (gadek et al., ) . a number of roles have been ascribed to glutamine as an immunonutrient: (a) as an essential nutrient for immune cells, (b) as an important modulator of gut barrier function, and (c) as a substrate for gsh synthesis. a number of reviews have been written about the first two of these roles (newsholme, crabtree, salleh, & ardawi, ; elia, ) ; we will consider the last one here. could glutamine be exerting an anti-inflammatory influence via an effect on gsh that enhances immune function? in a study in rats, glutamine supplementation resulted in an increased production of gsh by the gut (cao, feng, hoos, & klimberg, ) , and total parenteral nutrition (tpn) with glutamine raised plasma gsh concentrations in these animals (denno, rounds, faris, halejko, & wilmore, ) . in randomized controlled trials the administration of glutamine, either as a dipeptide during tpn to surgical patients or as a glutamine-enriched enteral feed to trauma patients, resulted, respectively, in improved nitrogen retention (less tissue protein depletion) and a . -d reduction in hospital stay, a concomitant suppression of the rise in plasma-soluble tnf receptors (reduced inflammation), and a lower incidence ofbacteremia, pneumonia, and sepsis (improved immune function) (houdijk et al., ; morlion et al., ) in the previous section the influence of antioxidants on severe inflammation was considered. do the general findings from this type of study also apply to modulation of low-grade chronic inflammation, such as has been observed in the elderly and obese? because aging is so closely associated with increased oxidative stress, which might both result from and contribute to a stimulation in the level of inflammation in the elderly, antioxidant therapy could produce beneficial effects. the effects would be seen in a decrease in oxidant damage, downregulation of inflammation, and, because of the inverse link between inflammation and immune function, an improvement in t-lymphocyte function. meydani, meydani, & verdon ( ) reported that supplementation of aged mice ( mo old) with dietary vitamin e ( ppm) improved several indices of the immune system to levels comparable to those seen in young animals. supplementation of aged mice with this vitamin also increased clearance of influenza virus from the lung to that observed in animals supplemented with other antioxidants such as melathonine, gsh, or strawberry extract, which contains a high level of flavonoids with antioxidant activity . in a double-blind, placebo-controlled study, meydani and colleagues (meydani, barklund, & lui, ; meydani et al., ) also reported that supplementation of elderly subjects with vitamin e for a short ( mo) or long ( . too) period of time also improved several in vitro and in vivo indices of immune response. the optimal immune response was observed with iu of vitamin e per day in the long-term study. it is worth noting that this level of vitamin e has also been reported to be the optimal level for reducing plasma f -isoprostane, a reliable index of lipid peroxidation (dillon, vita, & leeuwenburgh, ) . improving the immune response in the elderly may result in a lower incidence of infections, which are prevalent among the elderly, and thus may contribute to a longer and healthier life. many observational and clinical trials have also indicated that a high intake or high plasma level of this vitamin is associated with a low risk of cardiovascular disease. the vitamin may be operating at two levels; first, by protecting ldl from peroxidation, thereby reducing its atherogenicity, and second, by lowering the level of chronic inflammation by downregulation of nf-r,b. a reduction in platelet aggregability may also arise out of this action (huang et al., ; tanus-santos et al., ) . indeed, several lines of evidence indicated that supplements of vitamin e may prevent cardiovascular disease by reducing the susceptibility of ldls to oxidation (jailal, fuller, & huet, ) , reducing the expression of chemokines, adhesion molecule expression, and monocyte adhesion (wu, koga, martin, & meydani, ) , decreasing smooth muscle proliferation (azzi, boscoboinik, & marilley, ) , and decreasing platelet aggregation (steiner ) . another anti-inflammatory approach using nutrients would be to supplement diets of the elderly with n- pufas. supplementation with n- pufas from fish oil, however, has been reported to suppress the immune response (meydani, endres, & woods, ; meydani, ) , which hampers enthusiasm for the use of n- pufas for their benefits in cvd. however, the latter concern could be addressed by including a vitamin e supplement along with fish oil supplements. in a recent study it was found that supplementing elderly persons with (n- ) fatty acids of fish oil in combination with vitamin e while maintaining the anti-inflammatory properties of (n- ) pufas did not reduce immune indices in the elderly . fish oil supplementation is not universally efficacious in the treatment of inflammatory disease (grimble, ) . rheumatoid arthritis and inflammatory bowel disease have been the most successfully treated of all inflammatory diseases (calder, ) . the antiinflammatory mechanism may be through suppression of tnf-tx production. endres et al. ( ) reported that large doses ( g/d for wk) of oil in nine healthy volunteers resulted in a small but statistically significant reduction in tnf-o~ and il- [ production from pbmcs. subsequently, fewer than half of similar small intervention studies were able to demonstrate a statistically significant reduction in cytokine production. to understand the differences in response more closely, the author's laboratory conducted a study on young men fed g fish oil daily for wk and measured tnf-tx production by pbmcs before and after supplementation in relation to the snp at - in the tnf-o~ and at + in the lt-tx genes. no significant effect of fish oil on cytokine production was noted in the group as a whole. however, when data were examined according to tertile of tnf-ct production prior to supplementation, homozygosity for tnfb (lt-o~+ a) was . times more frequent in the highest than in the lowest tertile of production. the percentage of individuals in whom fish oil suppressed production was lowest ( %) in the lowest tertile and doubled with each ascending tertile. in the highest tertile, mean values were decreased by % (p < . ). in the lowest tertile, mean values were increased by % (p < . ). tnfb (lt-cz+ aa) homozygotes were strongly represented among unresponsive individuals in the lowest tertile of tnf-cz production prior to supplementation. in this lowest tertile, only tnfb l/b (lt-o~+ ga) heterozygous subjects were responsive to the suppressive effects of fish oil. in the medium tertile, this genotype was six times more frequent than other lt-t~ genotypes among responsive individuals. no relationship between possession of tnf or (tnf-ot- g or a) alleles and responsiveness to fish oil was found. clearly, although the level of inflammation determines whether fish oil will exert an anti-inflammatory influence or not and is influenced by the tnfb (lt-ct+ a) allele, the precise genomic mechanism for an anti-inflammatory effect is unclear at present . antioxidant intake also modifies cytokine production. in a study on healthy men and women and smokers, dietary supplementation with iu/d ct-tocopherol for mo suppressed the ability of pbmcs to produce tnf-t~. production was reduced by and % in nonsmokers and smokers, respectively (mol et al., ) . in a similar dietary intervention study on normolipaemic and hypertriglyceridaemic subjects given iu/ d of o~-tocopherol for wk, reduced tnf-o~, il- , and il- production by lps-stimulated blood mononuclear cells occurred (mol et al., ; van tits, demacker, de graaf, hak-lemmers, & stalenhoef, ) . a similar effect of cx-tocopherol was noted in a study on normal subjects and type diabetics (devaraj & jialal, ) . however, there were large standard deviations in the data from these studies, indicating major intraindividual variability in the ability of vitamin e to suppress production of the cytokine. although a number of studies have shown that o~-tocopherol suppresses superoxide production, the situation with regard to nitric oxide is less clear (mol et al., ; van tits et al., ) n the a-tocopherol derivative pentamethyl-hydroxychromane inhibited lps-stimulated nf-gb and inos activation in cultured j macrophages (hattori, ) . at present it is not known whether antioxidants interact differently with snps in the genes associated with oxidant stress and inflammation than they do with the other anti-inflammatory nutrient, n- pufa. this topic is currently an area of active research at the author's laboratory. proteomic studies have shown that inos and sod are both influenced by the nramp gene (kovorova, necasova, porkertova, radzoich, & macela, ) . the production of oxidant molecules enhancing pro-inflammatory cytokine production via high levels of nf-kb activation may thus be under a genomic influence owing to the aforementioned variations in the nramp gene (formica, roach, & blackwell, ) . a better understanding of this interaction and of the interaction of n- pufas and antioxidants with genotype may allow the better design of nutrient products for the treatment of inflammatory disease. it is clear from the current understanding about the purpose and functioning of the immune system throughout the life cycle that it is a powerful biological entity that profoundly alters body function while it is carrying out its prime purpose of defending the body against invasion by pathogens. however, within the response lie the seeds of disaster at an individual level, for the inflammatory component of the response can turn against the body, particularly as the body ages or becomes obese. the response, which can be devastating when directed against microbes entering the body, also sows the seeds of atherosclerosis, degradation of brain function, and insulin insensitivity and hastens the passage of hiv-infected individuals towards full-blown aids. along with the insights arising from the unraveling of the human genome has come evidence that the inflammatory response is able to protect the human species from invasion by pathogens but not all individuals within the species from ill health. the differing ability of humans, particularly the male of the species, at an individual level to mount an inflammatory response of different levels of intensity owing to genotype can result in widely contrasting outcomes of invasion of the body by pathogens. on the one hand, individuals may effectively fight off invasion provided the immune response follows a normal pathway, whereas other individuals within the same community encoutering the same pathogens will die from the strength and nature of the response rather than from the direct effects of the invader. insights gained from the genomic influences on cytokine production and the response to malaria suggest that the retention of alleles in pro-inflammatory cytokine genes that resulted in enhanced cytokine production within the human gene pool over generations could be a result of the heterozygotes' better capacity for fighting pathogens, whereas homozygotes of the high-producing genotype run an increased risk of a strong adverse inflammatory response. in the case of sickle cell anemia, where heterozygotic individuals reap an advantage in resistance to malaria by possession of only one copy of the anemia allele, homozygous individuals for the sickle cell trait pay the price for possession of two copies of the allele and die young. because of the overall advantage of this situation to the species, the potentially disadvantageous allele will be retained within the human gene pool over generations. with the twin discoveries that nutrients can modulate the inflammatory response and that cytokine genotype can modulate the effectiveness of nutrients in controlling inflammation, nutritional science sits at an exciting moment in its development. the mapping of how pro-and anti-inflammatory cytokine genotypes interact with responsiveness to immunonutrients at an indivividual level will allow tailor-made nutritional treatments of all diseases that have an underlying inflammatory basis. furthermore, a better understanding of how nutritonal therapies and genetics interact will allow the twin adverse biological factors increasing the level of inflammatory stress in populations---obesity and aging--to be tackled by targeted nutritional therapy. the roles of nramp and tnfo~ genes in nitric oxide production and their effect on the growth of salmonella typhimurium in macrophages from nrampl congenic and tumor necrosis factor-alpha-/-mice polymorphism of the human tnf-cz promoter--random variation or functional diversity? molecular immunolology periodontal infections and cardiovascular disease--how strong is the association? oral disease predictive value of nuclear factor kappab activity and plasma cytokine levels in patients with sepsis vitamin e: a sensor and an information transducer of the cell oxidation state lipoprotein(a) and lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors immunonutrition in the critically ill: a systematic review of clinical outcome inflammation, insulin resistance, infection, diabetes, and atherosclerosis. endocrinological practice interleukin- is an essential, corticotropin-releasing hormone-independent stimula-torof the adrenal axis during immune system activation cytokine gene polymorphism in human disease: online databases, supplement a gender-dependent genetic predisposition to produce high levels of il- is detrimental to longevity impaired glucose tolerance, type diabetes mellitus and carotid atherosclerosis: prospective results of the bruneck study antioxidant status and glutathione metabolism in peripheral blood mononuclear cells from patients with chronic hepatitis c improvement of immune functions in hiv infection by sulfur supplementation: two randomized trials a short history of nearly everything n- polyunsaturated fatty acids and cytokine production in health and disease glutamine enhances gut glutathione production immunogenetics of longevity. is major histocompatibility complex polymorphism relevant to the control of human longevity? a review of literature data c-reactive protein, insulin resistance, central obesity, and coronary heart disease risk in indian asians from the united kingdom compared with european whites a randomized, controlled trial of il- in humans. inhibition of inflammatory cytokine production and immune responses glutathione peroxidase in rheumatoid arthritis: analysis of enzyme activity and dna polymorphism prostaglandin-e regulation of tumor necrosis factor receptor release in human monocytic thp- cells plasma insulin, leptin, and soluble tnf receptors levels in relation to obesity-related atherogenic and thrombogenic cardiovascular disease risk factors among men the concurrent accumulation of intra-abdominal and subcutaneous fat explains the association between insulin resistance and plasma leptin concentrations : distinct metabolic effects of two fat compartments selective aspects of the insulin resistance syndrome the pleiotropic functions of peroxisome proliferator-activated receptor gamma glutamine enriched tpn enhances plasma glutathione in resting state n-acetylcysteine replenishes glutathione in hiv infection low-density lipoprotein postsecretory modification, monocyte function, and circulating adhesion molecules in type diabetic patients with and without macrovascular complications: the effect of alpha-tocopherol supplementation guns, germs and steel: the fate of human societies ~-tocopherol supplementation reduces systemic markers of oxidative damage in healthy adults the influence of lipoxygenase inhibitors on the in vitro production of human leukocytic pyrogen and lymphocyte activating factor (interleukin- ) functions of glutathione and glutathione disulphide in immunology and immunopathology chronic activation of the innate immune system may underlie the metabolic syndrome glutamine in parenteral nutrition socs- inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice the effect of dietary supplementation with n- polyunsaturated fatty acids on the synthesis ofinterleukin- and tumor necrosis factor by mononuclear cells age-associated increased interleukin- gene expression, late diseases, and frailty inhibition of cytokine production by cyclosporin a and transforming growth factor beta leptin regulation of the immune response and the immunodeficiency of malnutrition serum il- level and the development of disability in older persons the relation of body fat mass and distribution to markers of chronic inflammation the investigators of the nhbli family heart study. association of c-reactive protein with markers of prevalent atherosclerotic disease interaction with extracellular matrix proteins influences lsh/ity/bcg (candidate nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release a common functional c-t substitution polymorphism in the promoter region of the human catalase gene influences transcription factor binding, reporter gene transcription and is correlated to blood catalase levels. free radicalbiology andmedicine do men and women follow different trajectories to reach extreme longevity? ppars, metabolic disease and atherosclerosis effect of enteral feeding with eicosapentaenoic acid, - inolenic acid, and antioxidants in patients with acute respiratory distress syndrome increased cytokine release by leucocytes in survivors of stroke at young age serum albumin and mortality theory and efficacy of antioxidant therapy effect of antioxidative vitamins on immune function with clinical applications dietary lipids and the inflammatory response nutritional modulation of immune function stress proteins in disease inflammatory status and insulin resistance. current opinion in clinicainutrition and metabolic care inflammatory response in the elderly. current opinion in clinical nutrition and metabolic care gene:gene interactions influence the outcome in elderly patients the ability of fish oil to suppress tumor necrosis factor-alpha production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes which influence tnf-alpha production cytokine genotype and gender influence the inflammatory response to surgery il- gene promoter polymorphisms in rheumatoid arthritis markers of inflammation and cellular adhesion molecules in relation to insulin resistance in nondiabetic elderly: the rotterdam study effect of long-term dietary antioxidant supplementation on influenza virous infection associations between elevated interleukin- and c-reactive protein levels with mortality in the elderly pentamethyl-hydroxychromane, vitamin e derivative, inhibits induction of nitric oxide synthase by bacterial lipopolysaccharide nfkappab polymorphisms and susceptibility to type diabetes reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes irs- -mediated inhibition of insulin receptor tyrosine kinase activity in tnf-alpha-and obesity-induced insulin resistance tumor necrosis factor a: a key component of the obesitydiabetes link randomised trial of glutamine-enriched enteral nutrition on infectious morbidity in patients with multiple trauma inhibition of the expression of inducible nitric oxide synthase and cyclooxygenase- in macrophages in macrophages by & hq derivatives: involvement of ikappab-alpha stabilisation are differences in interleukin production associated with joint damage? cysteine and methionine supplementation modulate the effect of tumor necrosis factor c~ on protein synthesis, glutathione and zinc content of tissues in rats fed a lowprotein diet identification of high and low responders to allographs the effect of antimicrobial periodontal treatment on circulating tumor necrosis factor-alpha and glycated hemoglobin level in patients with type diabetes heritable major histocompatibility complex class ii-associated differences in production of tumor necrosis factor-co relevance to genetic predisposition to systemic lupus erythematosus immunocompetence and oxidant defense during ascorbate depletion of healthy men bacterial lipopolysaccharide and tumor necrosis factor alpha synergistically increase expression of human endothelial adhesion molecules through activation of nf-kappab and p mitogen-activated protein kinase signaling pathways. infection and immunology the effect of alpha-tocopherol supplementation on ldl oxidation severe hypertriglyceridemia with insulin resistance is associated with systemic inflammation: reversal with bezafibrate therapy in a randomized controlled trial chlamydial heat shock protein localizes in human atheroma and regulates macrophage tumor necrosis factor-a andmatrix metalloproteinase expression a natural resistance to intracellular pathogens: modulation of macrophage signal transduction related to the expression of the bcg locus plasma proinflammatory cytokine response to surgical stress in elderly patients modulation of macrophage-derived interleukin- and tumour necrosis factor by prostaglandin e allele frequencies of + tga single nucleotide polymorphism at the first intron of interferon-gamma gene in a group of italian centenarians ethanol consumption, amino acid and glutathione blood levels in patients with and without chronic liver disease skeletal muscle glutathione after surgical trauma genetics and the pathobiology of ageing nhanes iii health data relevant for aging nation variation in the tnfalpha promoter region associated with susceptibility to cerebral malaria polymorphic structure of the tumour necrosis factor (tnf) locus: an nco i polymorphism in the first intron of the human tnf-~ gene correlates with a variant amino acid in position and a reduced level of tnfc~ production vitamin e supplementation enhances cell-mediated immunity in healthy elderly subjects oral (n- ) fatty acid supplementation supresses cytokine production and lymphocyte proliferation: comparison between young and older women immunological effects of national cholesterol education panel (ncep) step- diets with and without fish-derived (n- ) fatty acid enrichment vitamin e supplementation and in vivo immune response in healthy subjects. a randomized controlled trial vitamin e supplementation suppresses prostaglandin e synthesis and enhances the immune response of aged mice oral supplementation with whey proteins increases plasma glutathione levels in hiv-infected patients association of tnf , a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study association between manganese superoxide dismutase (mnsod) gene polymorphism and breast cancer risk plasma levels of lipid and cholesterol oxidation products and cytokines in diabetes mellitus and smokers: effect of vitamin e treatment potential role of tnf-alpha in the pathogenesis of insulin resistance and type diabetes inhibition of monocyte chemoattractant protein- expression in cytokine-treated human lung epithelial cells by thiazolidinedione total parenteral nutrition with glutamine dipeptide after major surgery. a double blind controlled study prevalence of low body mass in rheumatoid arthritis: association with the acute phase response increase in il- and decrease of interleukin production during the ageing process are influenced by health status glutamine metabolism in lymphocytes. its biochemistry, physiology and clinical importance relation between plasma tumor necrosis factor-alpha and insulin sensitivity in elderly men with non-insulin-dependent diabetes mellitus periodontal inflammation and insulin resistance--lessons from obesity age-dependent oxidative stress in elderly patients with non-insulin-dependent diabetes mellitus c-reactive protein and coronary heart disease in western turkey increased monocyte activation in elderly patients after surgical stress c-reactive protein is independently associated with total body fat, central fat, and insulin resistance in adult women genomic interactions with disease and nutrition treatment with glutathione precursor decreases cytokine activity genotyping for polymorphisms in interferon-gamma, interleukin- , transforming growth factor-beta and tumour necrosis factor-alpha genes: a technical report changes in proinflammatory cytokine activity after menopause c-reactive protein, interleukin , and risk of developing type diabetes mellitus asymptomatic bacteriuria in elderly humans is associated with increased levels of circulating tnf receptors and elevated numbers of neutrophils genetic variation in cytokine production may be protective of icu admission and may influence mortality in vivo rates of erythrocyte glutathione synthesis in children with severe protein-energy malnutrition altered cytokine production in the elderly glutathione depletion in rats impairs t-cell and macrophage immune function age and sex steroid-related changes in glucocorticoid sensitivity of proinflammatory cytokine production after psychosocial stress endothelial perturbation in children and adolescents with type diabetes: association with markers of the inflammatory reaction epidemiologic and methodologic problems in determining nutritional status of older persons the pathogenesis of atherosclerosis: a perspective for the s origins and clinical relevance of sarcopenia association of tumor necrosis factor alpha gene promoter polymorphism with the presence of chronic obstructive pulmonary disease interactive role of infection, inflammation and traditional risk factors in atherosclerosis and coronary artery disease reactive oxygen intermediates as apparently widely used messengers in the activation of nuclear transcription factor-~j and hiv- tumor necrosis factor-alpha - promoter gene polymorphism and increased tumor necrosis factor serum bioactivity in farmer's lung patients gender differences in sepsis: genetically determined? shock evidence for a functional repeat polymorphism in the promoter of the human nramp gene that correlates with autoimmune versus infectious disease susceptibility involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type diabetic patients does n-acetyl cysteine inflkuence cytokine response during early human septic shock? alterations in lipid and carbohydrate metabolism in sepsis altered control of carbohydrate metabolism in endotoxemia glutathione deficiency in human immunodeficiency virus infection vitamin e, a modifier of platelet function: rationale and use in cardiovascular and cerebrovascular disease a genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-alpha concentrations and outcome of patients with severe sepsis interleukin (il- ) genotypes in inflammatory bowel disease change in the ratio of interleukin- to interleukin- predicts a poor outcome in patients with systemic inflammatory response syndrome effects of endothelial nitric oxide synthase gene polymorphisms on platelet function, nitric oxide release, and interactions with estradiol an investigation ofpolymorphisms in the interleukin- gene promoter antiinflammatory cytokine profile and mortality in febrile patients alphatocopherol supplementation decreases production of superoxide and cytokines by leukocytes ex vivo in both normolipidemic and hypertriglyceridemic individuals relationship of interleukin- and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and women: the health abc study circulating interleukin- in relation to adiposity, insulin action, and insulin secretion high white blood cell count is associated with a worsening of insulin sensitivity and predicts the development of type diabetes ppar-gamma and inflammatory bowel disease: a new therapeutic target for ulcerative colitis and crohn's disease lack of association between human longevity and polymorphisms of il- cluster, il- , il- and tnf-alpha genes in finnish nonagenarians genetic polymorphisms in the tumor necrosis factor locus influence non-hodgkin's lymphoma outcome activation of transcription factor nf-kappa b and p mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways humoral markers of inflammation and endothelial dysfunction in relation to adiposity and in vivo insulin action in pima indians biology of arterial atheroma comparative genetic association of human leukocyte antigen class ii and tumor necrosis factor-alpha with dermatitis herpetiformis an allelic polymorphism within the human tumor necrosis factor alpha promoter region is strongly associated with hla-a , b , and dr alleles a genetic association between systemic lupus erythematosus and tumor necrosis factor alpha effect of vitamin e on human aortic endothelial cell production of chemokines and adhesion to monocytes effect of dietary supplementation with fish oil in combination with different levels of vitamin e on immune response in healthy elderly human subjects in vitro glutathione supplementation enhances interleukin- production and mitogenic responses in peripheral blood mononuclear cells from young and old subjects associations of serum c-reactive protein with fasting insulin, glucose, and glycosylated hemoglobin: the third national health and nutrition examination survey production of tumour necrosis factor--alpha increases with age and bmi in healthy women the influence of age and gender on serum dehydroepiaandrosterone sulphate (dhea-s), il- , il- soluble receptor (il- sr) and transforming growth factor beta (tgf-beta ) levels in normal healthy blood donors polymorphisms in the promoter of the tumor necrosis factor-alpha gene in coal miners reduction of inflammatory cytokine concentrations and improvement of endothelial functions in obese women after weight loss over one year the author is grateful to the bbsrc for funding much of the work reported in this chapter. the author is also grateful to collegues in the united kingdom, sweden, and switzerland for scientific collaboration and advice. key: cord- -y jgjera authors: bauer, maria title: cardiovascular anatomy and pharmacology date: - - journal: basic sciences in anesthesia doi: . / - - - - _ sha: doc_id: cord_uid: y jgjera this chapter reviews the cardiovascular anatomy, the effects of medications on the cardiovascular system, and current guidelines. patients in the perioperative period often receive agents that affect hemodynamic variables such as heart rhythm and rate, blood pressure, or cardiac output. are coronary blood flow and arterial o content. coronary blood flow is determined by the patency of the coronaries, coronary perfusion pressure, and coronary vascular resistance. . the major determinants of myocardial o demand are heart rate, inotropic state, and wall tension (which is the function of intracavitary pressure, radius and wall thickness, preload and afterload). . critical o delivery is the point at which the extraction ratio is maximized, and any further incongruence between demand and supply will lead to tissue hypoxia. . in the perioperative setting, continuation of longstanding beta blocker therapy is recommended. . beta blockers should not be started on the day of surgery in beta blocker naïve patients. . patients with coronary artery disease undergoing surgical coronary revascularization should be administered a beta blocker before surgery. . although recent perioperative guidelines suggested continuing angiotensin converting enzyme (ace) inhibitors/angiotensin receptor blockers (arbs) before non-cardiac surgery, adverse circulatory effects during anesthesia in patients chronically treated with ace inhibitors/arbs has led to the recommendation that these drugs be discontinued h before surgery. . epinephrine inhibits uterine contractions especially during the second stage of labor. the heart is a conical hollow muscular organ located between the lungs in the mid-mediastinal portion of the thorax, suspended in the pericardial sac. as a dual pump, it maintains unidirectional blood flow to the body and the lungs by its rhythmical torsion and untwisting throughout the series of cardiac cycles. its receiving chambers (the left and right atrium, composed of myocardial layers), and its pumping chambers (the left and right ventricle, composed of layers of myocardium building up the separate vortices each ventricle) are structurally separated by their respective interchamber septa corresponding with the long axis of the heart, and are electrically isolated by the left and right fibrous rings comprising the fibrous cardiac skeleton in the short axis of the heart, perpendicular to the long axis. the development of the cardiovascular system begins on day of gestation, when mesoderm-derived blood islands, con-sisting of endothelial cells and hemoblasts, begin to form. these blood islands coalesce to form a pair of endothelial heart tubes, which on day fuse into a single primitive heart tube with a cranial inflow (arterial) and a caudal outflow (venous) end. this primitive heart tube is divided into regions: truncus arteriosus, bulbus cordis, primitive ventricle, primitive atrium, and sinus venosus. initial contractions occur on day - , and unidirectional circulation is established by week . from weeks - , folding and septation of the heart and the great vessels takes place, critical to the development of the chambers and the normal embryonic vascular circuit. in the developed heart, the atrial chambers lie cephalad and to the right of their corresponding ventricles, and the right-sided chambers lie anterior to their corresponding left-sided chambers. the atrial and ventricular septation is summarized later. the fibrous skeleton of the heart is a framework of dense collagen rings around the atrioventricular (av) and semilunar valves, as well as the left and right fibrous trigones (also known as the central fibrous body), and the membranous portion of the interatrial and interventricular septum. its main component is the central fibrous body, where the leaflets of all cardiac valves converge. the fibrous skeleton reinforces the ostia of the valves and prevents the annuli from overdistension by resisting forces of pressure developing through the cardiac cycle. it provides attachment for the valvular leaflets and cusps, as well as for some of the musculature of the atrial and ventricular wall. it is electrically impermeable, only allowing electrical propagation from the atrioventricular node across the right fibrous trigone to the bundle of his. chronic degenerative changes and calcium deposition into the collagen skeleton results in delayed conduction and depolarization, arrhythmias, rigidity of the valvular ostia, restricted leaflet opening and/or leaflet malcoaptation. the interatrial septum is a blade-shaped wall between the left and right atrium. its development begins during the fifth gestational week with the septum primum growing from the posterior wall of the primary atrium toward the endocardial cushion, formed within the atrioventricular canal. with the incomplete fusion of the septum primum and the endocardial cushion, a progressively diminishing space above the endocardial cushion, the ostium (also known as foramen) primum remains. during this process, small coalescing perforations in the upper portion of the septum primum create the ostium (or foramen) secundum. concurrently, a second septum, the septum secundum begins to form to the right of the septum primum, growing from the anterior wall, partially covering the foramen primum. its growth stops at the seventh week of gestation, leaving a gap known as the foramen ovale. this is essential in reducing pulmonary blood flow through the inactive fetal lungs. the foramen ovale is continuous with the ostium secundum, and is covered by the septum secundum on its left side. as a result of normal adaptive changes in the neonatal circulation, the decreasing pulmonary vascular resistance (pvr) and the resultant reversal of interatrial pressure gradient results in the permanent functional closure of the foramen ovale. in onethird of the population incomplete fusion between the septum primum and the septum secundum results in a residual flap valve and a probe patent foramen ovale (pfo). excessive apoptosis within the septum primum, or incomplete development of the septum secundum results in an atrial septal defect (asd), the most common congenital heart defect to manifest in adulthood: there are types of asd, the most common being the secundum type asd, accounting for - % of all asds. the secundum type asd is located centrally, it is larger than a pfo, and it is commonly associated with mitral valve prolapse. the primum type asd is less common, it represents - % of asds. it is located in the lower portion of the interatrial septum, and it is associated with endocardial cushion defects, av-valve abnormalities, or ventricular septal defects such as in down syndrome. the sinus venosus asd represents - % of asds. it is associated with abnormal pulmonary venous return. the fourth type, the coronary sinus asd or unroofed coronary sinus is the least common. it results from incomplete septation between the inferior portion of the left atrium and the coronary sinus, and is commonly associated with a persistent left superior vena cava. asds often remain clinically silent with preserved normal left atrial size, however, longstanding left-to-right shunting and resultant dilatation of the right-sided chambers along with persistent pulmonary hypertension may reverse the direction of the shunt, resulting in hypoxemia and eisenmenger physiology. the interventricular septum (ivs) separates the left and right ventricles from one another. under physiological pressure and filling conditions its convexity is bowing into the right ventricle. its margins are marked externally by the anterior and posterior interventricular grooves. the upper, posterior part of the interventricular septum is thin, and constitutes the membranous interventricular septum. the greater, anterior portion is the muscular interventricular septum. during cardiogenesis, along with the atrial septation a concurrent ventricular septation is also taking place. with the development of the endocardial cushions and the atrioventricular separation, there is tissue growth between the left and the right sides of the developing atrioventricular canal. this is the muscular portion of the interventricular septum, growing from the inferior portion of the ventricle toward the endocardial cushion. as the muscular ivs reaches the endocardial cushion, a small interventricular foramen remains. this is closed by tissue growth from the endocardial cushion, connective tissue from the muscular ivs, as well as tissue from the septation of the truncus pulmonalis and the conus arteriosus. inadequate contribution from either component results in different types of ventricular septal defects. the right atrium is the cardiac chamber receiving the systemic and cardiac venous return via the superior and inferior venae cavae (svc and ivc) and the coronary sinus. it is divided into components. the venous component receives deoxygenated blood from the svc and the ivc. at the inferior cavoatrial junction lies the eustachian valve, important before birth in directing blood from the ivc to the left atrium across the foramen ovale. a network of fine filamentous strands, the chiari network, may be seen in this region. the variably sized eustachian valve and chiari network are normal variants within the right atrium. the second component is the vestibule, which converges into the leaflets of the tricuspid valve, and the third is the appendage (also known as auricle). the right atrial appendage has a wide junction with both the vestibule and the venous component. the vestibuloauricular junction is identified by the terminal groove, the external marking of the prominent terminal crest on the internal surface. a consistent feature of the right atrial anatomy is the extension of the auricular pectinate muscles beyond the appendage to the atrioventricular junction. the sinus node, a cluster of specialized myocardial cells capable of spontaneous electrical impulse generation, lies at the superior cavoatrial junction at the terminal groove. it is supplied by the nodal artery, arising from the right coronary artery in % of cases, or the proximal left circumflex artery in the remaining %. the atrioventricular node is located in the floor of the right atrium, near the opening of the coronary sinus. preserving blood flow to the sinus node and other conductive tissues is key to avoiding arrhythmias and other conduction abnormalities. the tricuspid valve is located nearly vertically behind the aortic valve. with a valve area of - cm , it is the largest of the valves in the heart. its annular plane is saddle-shaped and is apically displaced relative to that of the mitral valve. a displacement index greater than mm/m (body surface area) suggests the presence of ebstein's anomaly. its leaflets are the septal, anterior, and posterior tricuspid valve leaflets, attached by their chordae tendineae to the right ventricular papillary muscles (true chords), or, not uncommonly, directly to the septum (false chords). the subvalvular apparatus of the tricuspid valve is variable. most commonly, the chordae tendineae originate from or papillary muscles, the anterior being the most prominent; the posterior, which is usually less prominent and may have several subdivisions; and the septal, which is the least prominent or may be entirely absent. the right ventricle is the most anterior of the chambers of the heart, located immediately behind the sternum. its geometry is complex: the right ventricle is crescent shaped when viewed in the short axis, and triangular when viewed longitudinally. current guidelines identify its walls as anterior, inferior, and lateral free wall. medially, the right ventricle shares the septum with its left-sided counterpart. for purposes of functional quantification, these walls are further divided into basal, mid, and apical segments. anatomically and functionally, the right ventricle is composed of portions: the smooth muscular right ventricular inflow tract, the apical trabecular, and the right ventricular outflow tract. the inlet portion encircles the tricuspid valve. the trabecular portion of the right ventricle extends into the apical region. the wall of the ventricle is thin here, making it vulnerable to perforation by catheters and electrodes. the most prominent of the trabeculae is the septomarginal trabecula. it carries part of the right bundle branch of the conduction system. it was once wrongly thought to prevent the right ventricle from overdistending, hence the name "moderator band. " the septomarginal trabecula originates from the base of the septum, it divides into an anterior limb that supports the pulmonic valve cusps, and a posterior limb, from which the medial papillary muscle arises. its main mass extends from the base of the septum to the apex and divides into smaller muscle ridges. two of these are particularly prominent, one giving rise to the anterior papillary muscle, and the other crossing the right ventricular cavity as the moderator band. the outlet portion consists of the circumferential muscular infundibulum that supports the pulmonic valve, which is the only one of the cardiac valves with no single ringlike annulus. it is supplied by the conus branch of the right coronary artery. the pulmonic valve separates the right ventricular outflow tract from the pulmonary artery. it is located anteriorly and to the left of the aortic valve. it is a semilunar valve formed by cusps, labeled as anterior (nonseptal), left, and right, each with a fibrous nodule at the midpoint of their free edges. the leaflets coapt via their crescent-shaped surfaces. the cusps of the pulmonic valve are formed by endocardial folds that are supported by internal dense collagenous plates as well as elastic connective tissue, continuous with the fibrous skeleton of the heart. they are thinner than the cusps of the aortic valve, and, lacking fibrous continuity with the septal leaflet of the tricuspid valve, are supported only by a prominent ridge of the posterior subpulmonary infundibular musculature. this is the supraventricular crest that separates the pulmonic and tricuspid valves from one another, and supports the semilunar attachments of the pulmonic valve. surgical incisions made through this crest may run toward the interventricular septum, and jeopardize the right coronary artery. oxygen-rich blood returning from the lungs via the pulmonary veins is received first by the left atrium. the left atrium has basic components: first, the largest, smoothwalled pulmonary venous component; second, the vestibule; and third, the left atrial appendage. the wall of the vestibule is continuous with both the venous component and the posterior (mural) leaflet of the mitral valve, and may affect normal valvular function when left atrial dilation is present. pectinate muscles are confined almost exclusively to the appendage in the left atrium. the flap valve of the fossa ovalis is found on the left atrial side of the interatrial septum. recurrent, symptomatic, drug-refractory atrial fibrillation most often originates from the pulmonary veins or their pulmonary venous ostia. circumferential or segmental ablation of potential triggers is considered effective means to control refractory atrial fibrillation, as scar forming is thought to prevent propagation of abnormal signals to the rest of the atrial musculature. the ablation and mapping catheters are inserted via the femoral or jugular veins, and are placed through separate transseptal punctures. most often, radiofrequency energy is used to make ablation lesions around the pulmonary vein ostia. in the presence of a left atrial appendage thrombus, or inability to anticoagulate in the -day peri-and post-procedural period, pulmonary vein isolation for atrial fibrillation ablation is contraindicated. a rare, frequently disabling, sometimes fatal complication of the procedure is an atrio-esophageal fistula resulting from thermal damage. other potential complications are cardiac tamponade, arrhythmias or atrioventricular block, embolic events, valvular complications, or peripheral vascular damage. the bileaflet mitral valve is located between the left atrium and left ventricle, embedded into the saddle-shaped mitral valve annulus. it serves as a unidirectional valve directing blood from the atrium toward the ventricle by passively opening during diastole and closing in systole, as determined by the pressure gradient between the chambers. the mitral valve apparatus consists of the left atrial wall, the annulus, the anterior leaflet attached to the aortic root, the posterior leaflet attached to the left atrial myocardium, the chordae tendineae attached to the ventricular side of the mitral valve leaflets, and the anteromedial and posterolateral papillary muscles. the anterior leaflet of the mitral valve is wide. it occupies about one-third of the annular circumference, and forms the anterior portion of the left ventricular outflow tract. in contrast with the posterior leaflet, its free edge has no indentations. the posterior (mural) leaflet is narrower, it occupies about two-thirds of the annular circumference, and it is further divided into scallops by clefts. because the posterior aspect of the mitral valve annulus contains little fibrous tissue, the posterior leaflet, especially the middle scallop, is more prone to prolapse than the anterior. the circumflex artery courses near the left half of the posterior leaflet, whereas the right half is in close proximity to the coronary sinus and the arterial branch supplying the av node. the mitral valve leaflets are supported by a dense collagen annulus. the most vulnerable portion of the mitral valve annulus is its aspect continuous with the right fibrous trigone, due to the proximity of the atrioventricular node and the penetrating bundle of his. the muscular components of the mitral valve apparatus are the anteromedial and posterolateral papillary muscles. the anteromedial papillary muscle is more prominent and is supplied by the left coronary system. the posteromedial papillary muscle is smaller, and is supplied by the right coronary artery. the mitral valve closes with the contraction of the papillary muscles, and open when they relax. papillary rupture, as a result of ischemia, will result in acute mitral regurgitation. because the anterolateral papillary muscle is supplied by both the left anterior descending (lad) artery and the left circumflex artery, its ischemic dysfunction is relatively uncommon. in about - % of individuals, the posteromedial papillary muscle was found to be perfused by a single vessel, most commonly by the right coronary artery. the left ventricle (lv) is the largest and thickest chamber of the heart. it receives oxygenated blood from the left atrium during diastole, to transfer it to the body during across the aortic valve during systole. relative to the right ventricle, it is located laterally and posteriorly. its superior, widest aspect is termed the base, leaning upward and toward the right shoulder, opposing its apex, pointing down and to the left. by consensus, its walls are termed septal, and the free anterior, inferior, and lateral walls. for purposes of functional quantification, these are further divided into basal, mid, and apical segments. morphologically, the left ventricle also has an inlet, a fine trabecular and an outlet component. the inlet portion extends from the mitral valve annulus to the origins of the papillary muscles, and contains the mitral valve apparatus. in contrast to the coarsely trabeculated right ventricle, the left ventricular free wall is finely trabeculated. the trabeculation extends to the apex, where, due to the progressively decreasing number of myocardial fibers, the wall thickness is thin. its outflow tract differs from that of the right ventricle in that it is anatomically and functionally contiguous with the inlet via the aortic-mitral continuity. the left ventricular outflow tract supports the aortic valve, and its septal portion, in contrast with the primarily muscular right ventricular infundibulum, includes the membranous interventricular septum. the left bundle branch of the conduction system courses posteriorly to the membranous septum between the right and noncoronary cusps of the aortic valve. the interventricular septum is formed by subendocardial myocardial fibers of both the left and the right ventricle, intermingled with circumferentially arranged fibers derived from the left ventricle. for this reason, in a normally func-tioning heart, the septum thickens toward the left ventricle during systole. during contraction, the high left ventricular pressure along with the septum provides a "splint" against which the right ventricle is able to shorten, hereby contributing to the emptying of the right ventricle (systolic ventricular interdependence). under pathologic conditions, for example, during acute right ventricular volume or pressure overload, or due to conduction abnormalities or pacemaker activation, the septum may appear flat, or move paradoxically toward the right ventricle during systole. blood flow to the anterior / of the septum is supplied by the septal perforators of the left anterior descending artery. the posterior / is supplied by the distal branches of the right coronary artery. the papillary muscles of the left ventricle arise from the anterolateral and posteromedial portions of the left ventricular free wall. their arrangement ensures parallel alignment of the chordae tendineae with the lv axis during systole, allowing for complete leaflet coaptation and closure of the mitral valve in the normally functioning heart. all coronary branches contribute to the blood supply of the left ventricle, acute ischemic events therefore predictably manifest as regional wall motion abnormalities based on the coronary distribution. the left anterior descending artery and its branches, the septal perforators and the diagonals perfuse the anterior wall, the anterior two-thirds of the septum, and the apex. the obtuse marginal branches of the left circumflex artery supply the lateral wall. the right coronary artery perfuses the medial portions of the inferior wall, as well as the posterior one-third of the interventricular septum. anastomotic connections between the left and the right coronary system allows for preserved myocardial perfusion distal to a significant obstruction or total coronary occlusion. the aortic valve is part of the aortic root consisting of the annulus, the aortic valve cusps, the sinuses of valsalva, the junction between the sinuses and the proximal ascending aorta (the sinotubular junction), and the proximal ascending aorta. it is located at the left ventricular outlet, separating the aorta and the ventricle. owing to its central location, it is the only valve in the heart that is related to all chambers and each of the valves. as a trileaflet semilunar valve, it consists of cusps: the left and right coronary cusps giving rise to their respective coronary arteries, as well as the non-coronary cusp, which faces the interatrial septum. the right coronary cusp is the most anterior and is adjacent to the right ventricular outflow tract. the left coronary cusp is located posteriorly relative to the other . histologically, its cusps are composed of dense collagenous plates, which are in continuity with the fibrous skeleton of the heart, covered by endocardium. each cusp has its attachment to the aorta and the left ventricle. immediately behind the cusps, the wall of the proximal ascending aorta bulges outward to form the aortic sinuses of valsalva. the function of these sinuses is to prevent coronary ostial occlusion by creating eddy currents of blood flow during rapid ejection. as with the pulmonic valve, each cusp has a fibrous nodule at the midpoint of their free edges, forming the nodule of arantius. the leaflets coapt via their crescentshaped surfaces. incomplete coaptation of the valve leaflets during diastole results in aortic regurgitation, whereas limited systolic leaflet excursion due to excessive leaflet thickening resulting from degenerative or rheumatic disease process suggests the presence of aortic stenosis. the severity of aortic stenosis can be assessed by various methods. a peak transaortic velocity of greater than m/second, a peak transaortic gradient of at least mm hg and a mean gradient of at least - mm hg, as well as an aortic valve area of less than cm is consistent with severe aortic stenosis. the first pair of arteries branching off the proximal ascending aorta are the left and right coronaries, originating from their respective sinus of valsalva in the aortic root. while there is some variability in their point of origin and their distribution, the right coronary artery (rca) almost always supplies the right ventricle (rv), whereas the left coronary artery (lca) supplies the anterior and lateral wall of the left ventricle (lv), as well as the anterior portion of the interventricular septum. blood supply to the remainder of the left ventricle is determined by the coronary dominance. this refers to the artery that supplies the posterior descending artery (pda) and the posterior lateral branch (plb). right dominance (when the right coronary artery gives off the pda and the plb) occurs in - % of normal individuals. left dominance (the pda and the plb originating from the left circumflex artery) is slightly more common in men than in women. if the pda is supplied by the rca and the plb branches off the left circumflex artery, the system is codominant. the left coronary artery arises from the left coronary sinus as the left main stem, which then bifurcates into segments: the left anterior descending (lad) and the circumflex (lcx) artery. the relatively short left main stem courses between the left atrial appendage and the pulmonary trunk before giving off the lad and the lcx, and, in - % of cases the variant coronary ramus intermedius, the branch supplying the anterior aspect of the heart. the left anterior descending artery traverses the anterior interventricular groove and perfuses the anterior wall via the diagonal branches, as well as the anterior two-third of the interventricular septum via its septal perforators coursing along the anterolateral wall, and a small portion of the anterior wall of the right ventricle, adjacent to the interventricular groove, via the right ventricular branches. the circumflex artery courses in the left atrioventricular groove. its primary branches are the obtuse marginals, supplying the lateral aspect of the left ventricle, including the posteromedial papillary muscle. in % to % of individuals, it gives off the posterior descending artery (left dominance). further branches of the left circumflex artery supply the left atrium, and, in % of the cases, the sinus node. a left dominant coronary system also supplies the atrioventricular node. the right coronary artery emerges from the right sinus of valsalva and is carried by the right atrioventricular groove. in about half of the cases it gives off the conus branch that supplies the right ventricular outflow tract. the rca perfuses the anterior free wall of the right ventricle via its acute marginal branches, the sinus node via the nodal artery in % of individuals, and the atrioventricular node in patients with right-dominant coronary circulation. it bifurcates into the posterior descending artery and the posterolateral branch at the posterior interventricular groove. the posterior descending artery gives off the posterior septal perforators that supply the posterior one-third of the interventricular septum. the posterolateral branch supplies the inferoposterior wall of the left ventricle along with the acute marginal branches of the rca and/or the circumflex artery. the kugel's artery is an anastomotic branch between the rca and the lcx that may give off a branch that supplies collateral circulation to the atrioventricular node. of the cardiac veins, the great cardiac vein courses in the anterior interventricular groove upward from the apex, alongside the lad. it drains into the coronary sinus. the middle cardiac vein courses in the inferior interventricular groove, upward from the apex, accompanying the pda. the coronary sinus courses in the posterior atrioventricular groove, alongside the left circumflex artery. it receives the great cardiac vein proximally and the middle cardiac vein distally, and drains into the right atrium. its orifice is guarded by the thebesian valve. the anterior right ventricular veins course on the anterior surface of the right ventricle, where they may drain directly into the right atrium, or coalesce to form the small cardiac vein. the small coronary vein runs posteriorly through the right av groove. the thebesian veins are small veins draining directly into the cardiac chambers, primarily into the right atrium and the right ventricle. patients in the perioperative period often receive agents that affect hemodynamic variables such as heart rhythm and rate, blood pressure, or cardiac output. the effect of these agents is governed predominantly by transmembrane ion fluxes. drugs used for rhythm and rate control act by modulating na + , k + , and ca + currents. intracellular calcium is a key mediator in coupling electrical excitation to mechanical contraction, and is an important determinant of the contractile state of the myocardium. the vascular tone of resistance vessels is regulated by ion fluxes via different types of k + , ca + and cl − channels. much of human physiology (for example, responses to stimuli by hormones, neurotransmitters, ions, or photons) is regulated by gtp binding (g) protein-linked signal transduction. g-proteins serve as points of communication between the extracellular and intracellular environment. adrenergic receptors are membrane-spanning molecules coupled to adenylate cyclase via a g-protein located on the inner membrane of the cell. their activation ultimately leads to the modulation of downstream effectors. g-proteins consist of (α, β and γ) subunits. the binding of an agonist to the adrenergic receptor replaces guanosine diphosphate (gdp) by guanosine triphosphate (gtp), and causes the α-subunit of the g-protein to break free from the β-γ complex, and act as a primary messenger: in beta receptors, it stimulates adenylate cyclase and triggers cyclic adenosine monophosphate (camp) production, which, as a second messenger in the process of signal transduction, activates its target kinases that phosphorylate regulator proteins and ultimately increases intracellular calcium levels. pure alpha-adrenergic agonists also increase intracellular calcium levels by stimulating phospholipase c, an enzyme that catalyzes hydrolysis of phosphatidyl inositol to diacyl glycerol and inositol triphosphate. inositol triphosphate stimulates the release of calcium from the sarcoplasmic reticulum, and both molecules act as myofilamental calcium sensitizers. under physiologic conditions, electrical impulse generation, conduction, and propagation occurs in specialized excitatory and conductive tissues within the myocardium, and is driven by a sequence of ion fluxes through sarcolemmal ion channels. the pacemaker of the normal cardiac muscle is the sinoatrial (sa) node, located in the right atrium, below and lateral to the ostium of superior vena cava, supplied by the nodal branch of the right coronary artery (rca). its intrinsic rate ( - beats per minute) is determined by the resting transmembrane potential, the threshold potential, and the rate of phase spontaneous diastolic depolarization. the sa node has the steepest slope of phase depolarization. for this reason, its intrinsic rate is the highest, therefore this is the dominant, primary pacemaker of the heart. rhythmical, automatic impulses generated by the sinus node spread directly to the right atrium via its own noncontractile sinus fibers that fuse with excitable and contractile atrial fibers, as well as anteriorly to the left atrium via the bachmann's bundle. electrical impulse from the right atrium propagates via other internodal tracts, including the posterior (thorel's) and median (wenkebach's) pathways to the atrioventricular (av) node. the av node, supplied by the av nodal branch of the distal rca and the septal perforators of the left anterior descending (lad) coronary artery, and located in the right atrial side of the interatrial septum behind the tricuspid valve and superior to the coronary sinus, delays impulses from the atria by approximately - ms before allowing them to pass to the bundle of his. these slow conduction velocities allow the atria to empty before ventricular contraction begins. the av node is the primary regulator of ventricular rate in atrial fibrillation and flutter. its intrinsic rate is - beats per minute, and it is considered a latent intrinsic pacemaker. the bundle of his is located just above the interventricular septum (ivs) and is supplied by the septal perforators of the lad, as well as the posterior descending artery. as a latent pacemaker, its intrinsic rate is - beats per minute. from the bundle of his, impulses rapidly travel down the bundle branches on either side of the membranous portion of the interventricular septum, to depolarize the left (left bundle branch, lbb, which also depolarizes the ivs) and the right (right bundle branch, rbb) ventricles. the left bundle branch further divides into the anterior and posterior fascicles. both bundles terminate in the purkinje fibers that penetrate the ventricular myocardium, initiating its contraction from the endocardium toward the epicardium. an organized sequence of impulse generation and conduction is required for the cardiomyocytes to synchronously contract, and, by coordinated chamber contraction, to maintain cardiac output. understanding normal cardiac electrophysiology is the foundation for understanding the basic principles of disease mechanisms and antiarrhythmic pharmacotherapy. the following is a brief review. in the normal resting myocardium, transmembrane potential is determined primarily by potassium conductance, and is maintained by the na + /k + atpase. the resting transmembrane potential is stable around − mv (the intracellular compartment being more negative relative to the outside of the cell), approaching the potassium equilibrium potential. an action potential is triggered by either the cardiac pacemaker cells, or by the surrounding cardiomyocytes. this results in a transient increase in na + conductance, which in turn initiates an increase in na + -influx through fast sodium channels. when the threshold potential is reached at around − mv, a large enough number of sodium channels have been opened to generate a self-sustaining sodium influx. above − mv, long-opening (l-type) ca + -channels open with resultant ca + -influx down its concentration gradient. at around mv, fast na + -channels start to close. this is phase (early rapid depolarization), peaking at + mv (na + -equilibrium potential). the increase in na + -conductance is inactivated. this, with the outward movement of k + via transient k + -channels results in a brief period of initial repolarization. the transmembrane potential returns from slightly positive to approximately mv. this is phase . following the initial repolarization, a constant ca +influx via slow (l-type, "long-opening") ca + -channels, and an increase in k + conductance via delayed rectifiers main-tains the membrane potential just below mv. the electrical countercurrents are balanced. this is phase , the plateau. the phase between the initiation of the upstroke and the end of the plateau is the absolute refractory period, during which the cell is unable to be depolarized regardless of the strength of the stimulus, and corresponds with phase between the beginning of the qrs complex and the beginning of the t wave on the electrocardiogram (ecg). the next phase of the cardiac action potential is the rapid repolarization. ca + -conductance via the l-channels is inactivated, but the k + -channels remain open. potassium is rapidly shifted out of the cell, and the transmembrane potential rapidly approaches the k + -equilibrium. this is phase . the phase between the initiation of the upstroke and the transition between phase and is the relative refractory period, during which the cell may be able to be depolarized to allow for a non-propagated stimulus. this corresponds with the early upstroke of the t wave. during phase , the cell is able to be depolarized by supranormal stimuli (relative refractory period), resulting in an action potential. this corresponds with the dome of the t wave on the ecg. membrane hyperpolarization at the end of this phase results in a hyperexcitable period, during which even weak stimuli can trigger an action potential. this brief phase corresponds with the downward slope of the t wave. during phase , na + and ca + channels are closed. there is a constant outflow of potassium ions through inward rectifier channels. this is the resting phase in the contractile cells, or spontaneous diastolic depolarization in the cardiac pacemaker cells. pacemaker cells are found primarily in the dominant and subsidiary rhythm generators (sa node, av node, his-purkinje system) of the heart. however, impulses may originate from other sites; for example, cells at the coronary sinus, atrioventricular valves, or cells at the crista terminalis. abnormally, impulses may originate from around the pulmonary veins. these cells are different from contractile cardiomyocytes in that they share the characteristic to generate spontaneous cardiac action potentials. they exhibit automaticity, have an unstable membrane potential, and have no rapid depolarization phase. automaticity means that pacemaker cells are capable of initiating their own action potential without external stimulus. these cells undergo a spontaneous diastolic depolarization and action potential is triggered when threshold potential is reached. pacemaker cells undergo spontaneous diastolic depolarization during phase (as opposed to the resting phase during phase in contractile cells), and have no rapid depolarization phase. these cells have fewer inward k rectifier channels than contractile myocytes do, and their transmembrane potential is never lower than − mv. therefore, the fast na + -channels that need − mv to get activated are permanently inactivated in these cells. multiple ion currents are responsible for the generation of spontaneous action potentials. the synergy of different currents-( ) the decay of the k + -efflux; ( ) an inward depolarizing "funny" mixed na + -k + inward current, activated by membrane hyperpolarization, and playing a major role in the spontaneous depolarization of the sinoatrial node; and ( ) an inward ca + -current in the late phase of the spontaneous diastolic depolarization-determines the rate and shape of action potentials in cardiac pacemaker cells. physiologically, the dominant pacemaker is the sinoatrial node. when its rate drops below the intrinsic rate of a latent, non-dominant pacemaker-for example, as a result of parasympathetic stimulation or sa nodal disease-the removal of the sinus overdrive leads to "escape-activation" of these non-dominant centers. junctional rhythm may also occur when the av junctional pacemaker rate exceeds and suppresses the sinoatrial rate. cardiac arrhythmias are commonly defined as abnormalities of the normal sequence of impulse generation and propagation within the myocardium. while benign arrhythmiasfor example, premature atrial contractions, isolated premature ventricular contractions, or atrial fibrillation in the absence of structural heart disease-are very common, malignant ventricular arrhythmias (monomorphic or polymorphic ventricular tachycardia [vt], ventricular fibrillation) accounted for an estimated , - , sudden cardiac deaths in the united states. underlying provoking factors are, for example, arterial hypoxemia, acidosis or alkalosis, electrolyte abnormalities, ischemia, increased sympathetic activity, atrial or ventricular dilation, or proarrhythmogenic drugs. in some cases, correction of these factors is sufficient to suppress cardiac ectopies. however, when this alone does not control cardiac arrhythmias, and/or hemodynamic compromise ensues, or the presence of a disturbance increases the risk of a life-threatening arrhythmia, administration of antiarrhythmic agents may be warranted. the main mechanisms of cardiac dysrhythmias have been identified as: focal activity due to abnormal impulse generation this results from: enhancement or reduction of normal automaticity (such as in sinus tachycardia and sinus bradycardia, accelerated junctional rhythm). enhanced abnormal automaticity of the purkinje-fibers, atrial or ventricular myocytes; ie, cells that do not normally display automaticity (for example, atrial tachycardia or accelerated idioventricular rhythm. purkinje fibers are catecholamine-sensitive, and their activation can be augmented by increased sympathetic tone, for example during myocardial ischemia). triggered activity. this occurs when early afterdepolarizations and delayed afterdepolarizations initiate spontaneous multiple depolarizations, such as in torsades de pointes, or ventricular arrhythmias in the setting of digitalis toxicity. abnormal conduction delayed conduction (such as in atrioventricular blocks), or re-entrant mechanisms (for example, sa nodal re-entry, atrial flutter, av nodal re-entry tachycardia, or ventricular tachycardia). re-entrant tachycardias can be generated by different mechanisms: reflection, circus movement re-entry, and phase re-entry. circus movement re-entry due to an anatomical or functional block is a major mechanism and the most commonly described. for a re-entrant circuit to occur, roughly parallel conductive pathways must be connected with conductive tissue, capable of forming an electrical circuit. one of these pathways must have a refractory period substantially longer than that of the other pathway (pathway a). finally, the pathway with the shorter refractory period (alternate pathway, pathway b) must conduct impulses more slowly than pathway a. once an extra impulse (for example, premature contraction) encounters these separate pathways of conduction when pathway a is unable to be depolarized due to being in the refractory period but pathway b is capable of depolarization, the impulse will be conducted via pathway b. the signal then travels to the distal end of pathway a to reconnect with it if it is no longer refractory, then it is conducted retrograde to the site where it reconnects with pathway b. pathway b has a shorter refractory period and therefore it recovers faster: the impulse will travel into pathway b where it will reenter that portion of the circuit, completing the loop. micro-re-entry occurs with ventricular tachycardia from conduction around scar tissue such as in myocardial infarction (mi). macro-reentry occurs via conduction through concealed accessory pathways, such as in wolff-parkinson-white syndrome. pharmacological management of arrhythmias is warranted when treatment of the underlying causes does not break the arrhythmia, the arrhythmia results in hemodynamic compromise, or it increases the risk of development of a lifethreatening arrhythmia. the most widely used classification system of antiarrhythmic drugs was proposed by vaughan williams. this system classifies the antiarrhythmic agents based on their ability of abolishing an arrhythmia by blocking specific ion currents during the action potential. ion-specific channels exist in different stages. during the upstroke phase (phase ) of the action potential the channels are in the activated state. during the plateau phase of repolarization (phase ), the inactivated state occurs: during the effective refractory period channels are unresponsive to new or continued stimulus. ion channels are closed during the resting phase (phase ). the effects on the action potential and the effective refractory period of the cardiac action potential determine the clinical effect of antiarrhythmic drugs. drugs blocking inward sodium ion flow will slow conduction and result in suppression of the maximum upstroke velocity of the cardiac action potential. potassium channel blockers prolong repolarization by prolonging the duration qtc prolongation. l and t type calcium channels are present in the myocardium, and are targets of calcium channel blockers. class i class i antiarrhythmic drugs are fast na-channel inhibitors. fast sodium channels are blocked during phase (upstroke) and phase depolarization of the action potential with resultant decreases in the amplitude and rate of the action potential and conduction velocity. class ia for example, quinidine, procainamide, disopyramide. these drugs lengthen both the action potential and the effective refractory period reflecting sodium channel inhibition and lengthening of repolarization reflecting potassium channel blockade. these drugs are considered membrane stabilizers in the treatment of atrial, av nodal, and ventricular arrhythmias. they may prolong the qrs and qt intervals. quinidine -quinidine has mild parasympatholytic and alpha-blocking effects and decreases systemic vascular resistance (svr). procainamide -procainamide is used to suppress premature atrial and ventricular contractions and to prevent precipitation of atrial fibrillation or flutter. it increases refractoriness and can prevent accessory pathway re-entry tachycardias. high serum levels may cause direct myocardial depression and bradycardia requiring temporary pacing or administration of beta-agonists. procainamide and its active metabolite n-acetylprocainamide may induce qt-prolongation and torsades de pointes. long-term therapy may cause lupus-like symptoms. disopyramide -disopyramide has a vagolytic effect that is dose-dependent and reversible by pyridostigmine. it depresses myocardial contractility and increases svr, and may thus precipitate or exacerbate congestive heart failure. class ib for example, lidocaine, tocainide, mexiletine. these are less powerful na-channel blockers. they shorten action potential duration and refractory period in normal ventricular myocytes. class ii drugs have minimal effect on inotropy or svr. in the ischemic tissue, lidocaine may block adenosine triphosphate-dependent channels, preventing ischemicmediated shortening of ventricular depolarization. lidocaine depresses the slope of phase depolarization in purkinje fibers and increases the fibrillation threshold in ventricular myocytes. signs of toxicity are frequent with concentrations above mcg/ml. class ic for example, flecainide, propafenone. class ic drugs are potent sodium channel blockers, indicated for the treatment of ventricular arrhythmias. they markedly decrease the rate of phase depolarization and speed of conduction. they have little effect on the duration of the action potential and the effective refractory period in ventricular muscular cells, but do shorten the duration of the action potentials in the purkinje fibers. this inhomogeneity on the rate of cardiac repolarization plus the slowing of cardiac conduction may contribute to the prodysrhythmic effects of these drugs particularly in patients with history of myocardial infarction, left ventricular dysfunction, or previous sustained ventricular tachycardia. class ic drugs, especially flecainide, significantly depress inotropy and prolong the pr and qt intervals. class ii drugs are beta-adrenergic receptor blockers. betaadrenergic receptor antagonists decrease the rate of spontaneous phase depolarization, important in suppression of ventricular arrhythmias during ischemia and reperfusion. they are effective in the treatment of adrenergically mediated disease states, in which increased phase depolarization, enhanced conduction velocity, and a shorter refractory period all contribute to increased automaticity. beta-blockers decrease the rate of v max of the action potential, prolongs its duration as well as the effective refractory period. druginduced slowing of the heart rate with resulting decreases in myocardial oxygen requirements is desirable in patients with coronary artery disease. beta blockers slow the sinus rate, prolong av-nodal conduction and enhance refractoriness. they prolong the pr interval on the ecg. esmolol is used to convert atrial fibrillation with rapid ventricular response to normal sinus rhythm, or to maintain a slow ventricular rate. it also predictably reverses the fibrillation threshold lowering effects of catecholamines. class iii drugs are potassium channel blockers; for example, amiodarone, bretylium, sotalol. these drugs prolong cardiac repolarization, action potential duration, and the effective refractory period possibly by interference with sodium and calcium exchange. these effects are beneficial in preventing cardiac dysrhythmias by decreasing the proportion of the cardiac cycle during which myocardial cells are excitable and susceptible to a triggering event. reentrant tachycardias may be suppressed if the action potential duration becomes longer than the cycle length of the tachycardia circuit. amiodarone -in addition to class iii effects, amiodarone exhibits class i na + -channel blocking, class ii beta blocking, and class iv ca + -channel blocking properties. it prolongs repolarization and cardiac action potential, it produces negative chronotropy in nodal tissues and as a ca + and k + -channel blocker, it slows sa-nodal conduction speed and prolongs refractory period. it is an αand β-receptor antagonist with potent vasodilating and myocardial depressant potential. it may produce sinus bradycardia or heart block, necessitating administration of positive chronotropes or initiation of temporary pacing. long-term side effects include pulmonary fibrosis, corneal microdeposits, liver cirrhosis, hyperthyroidism, or hypothyroidism. sotalol -sotalol is a mixture of isomers that possess similar class iii effects, used for the treatment of atrial fibrillation or atrial flutter, as well as to treat ventricular arrhythmias. the l isomer of sotalol acts as a beta antagonist, whereas the d isomer may increase mortality in patients with ventricular dysfunction and recent myocardial infarction. sotalol lacks intrinsic sympathomimetic activity or membrane-stabilizing properties. the lower incidence of dysrhythmia effects seen with amiodarone and racemic sotalol may be related to the beneficial class ii effects. bretylium is no longer available in the united states for clinical use. calcium channel blockers: these agents act by inhibiting inward slow inward calcium currents that may contribute to the development of ventricular arrhythmias. these drugs are useful in the treatment of idiopathic ventricular tachycardias. calcium channel blockers prolong neuromuscular blockade. prodysrhythmia effects of antiarrhythmic agents describe bradydysrhythmias or tachydysrhythmias that represent new dysrhythmias associated with chronic antidysrhythmic drug treatment. types of dysrhythmias include torsades de pointes, incessant vt, and wide complex ventricular rhythm. torsades de pointes (polymorphic vt) is the most common dysrhythmia and is triggered by early after-depolarizations in a setting of a delayed depolarization and increased duration of refractoriness manifesting as prolonged qt c interval on the ecg. class ia (especially quinidine) and class iii drugs prolong qt c by potassium channel blockade and provide the setting for torsades de pointes. drug-induced torsades is often associated with bradycardia, because the qt c interval is longer at slower heart rates. exacerbating factors such as hypokalemia, hypomagnesemia, poor lv function, and concomitant administration of other qt-prolonging drugs are important predisposing factors in the development of torsades de pointes. incessant ventricular tachycardia may be precipitated by cardiac antidysrhythmic drugs that slow conduction of cardiac impulses (class ia and class ic) sufficiently to create a continuous ventricular reentry circuit. incessant ventricular tachycardia is more likely to occur with high doses of class ic drugs and in patients with prior history of sustained ventricular tachycardia and poor lv function. ventricular tachycardia due to this mechanism is generally slower because of the drug effect, but may be resistant to drugs or electrical therapy. wide complex ventricular rhythm is usually associated with class ic drugs in the setting of structural heart disease with excessive plasma concentrations or abrupt changes in the dose. wide complex rhythm is thought to reflect a reentrant tachycardia and easily degenerates to ventricular fibrillation. under normal physiological conditions oxygen delivery (do , the amount of oxygen delivered to the cells) is adequate to meet cellular oxygen demand (vo , the amount of oxygen extracted from arterial blood) and maintain aerobic metabolism. the ratio of oxygen consumption to the oxygen available to tissues is the oxygen extraction ratio, which varies for different organs. during periods of increased workload and increased cellular demand, and/or decreased supply, aerobic metabolism can still be maintained independently of blood flow by increasing o extraction. critical o delivery is the point at which the extraction ratio is maximized, and any further incongruence between demand and supply will lead to tissue hypoxia and subsequent activation of anaerobic metabolic pathways. when myocardial oxygen consumption exceeds the reserve in coronary blood supply to meet a given o demand, ischemia is precipitated. the heart's high, - % o extraction ratio (compared to - % for the rest of the body) can make it susceptible to even short periods of ischemia. the major determinants of myocardial o supply are coronary blood flow and arterial o content. coronary blood flow is further determined by the patency of the coronaries, coronary perfusion pressure, and coronary vascular resistance. the major determinants of myocardial o demand are heart rate, inotropic state, and wall tension (which is the function of intracavitary pressure, radius, and wall thickness, as well as preload and afterload). antianginal pharmacotherapy is indicated when the supply/demand imbalance results in ischemia. in accordance with the american college of cardiology/american heart association (acc/aha) guideline for the management of patients with non-st-elevation acute coronary syndromes, standard medical therapy includes the use of beta blockers, nitroglycerin, calcium channel blockers, analgesics, and cholesterol management. this section will focus on role of nitroglycerin, beta-blockers and calcium channel blockers in the management of angina pectoris. nitroglycerin nitroglycerin (ntg) is an endothelium-independent smooth muscle relaxant that acts predominantly on venous capacitance vessels and large epicardial coronary arteries, maximizing blood flow to the subendocardial areas. peripheral venodilatory effects are prominent even at low doses and are not dose-dependent, whereas dilation of peripheral conductance and resistance vessels occur at higher doses, and further increases in dose result in more pronounced vasodilation. nitroglycerin reduces myocardial o demand and increases oxygen supply by reducing preload and decreasing ventricular dimension and wall tension, dilating normal and atherosclerotic coronary arteries, and enhancing collateral circulation. the degree to which coronaries are able to dilate is dependent on their baseline vascular tone. nitroglycerin produces a dose-dependent systemic and pulmonary arterial vasodilatory effect, and predictably decreases cardiac filling pressures. its use is indicated for initial treatment of nearly all types of myocardial ischemia, as well as for managing hypertension and heart failure. elimination half-life of nitroglycerin is approximately . min. ntg enters the smooth muscle cells and generates nitric oxide through a glutathione-dependent pathway, which stimulates cyclic gmp production and causes peripheral vasodilation via a sequence of protein phosphorylation and dephosphorylation within the smooth muscle. nitric oxide production via guanylate cyclase stimulation and cgmp production is a sulfhydryl (sh)-group dependent process. depletion of sh-groups by prolonged exposure leads to doseand duration-dependent tolerance, which may manifest within the first hours of treatment. restoring sh-supplies with reducing agents (for example, n-acetylcysteine) does not reliably reverse nitrate tolerance, therefore a drug-free interval of - hours is recommended to maintain drug efficacy. rebound myocardial ischemia may occur during drug-free intervals. nitroglycerin is available in intravenous (iv), sublingual, or topical formulations. a typical iv dose to treat acute ischemia is - mcg. for a continuous infusion, the dose range is . - mcg/kg/min. nitroglycerin at low doses causes predominantly splanchnic venodilation that results in venous pooling, decreased cardiac filling, and decreased biventricular end-diastolic volume and pressure. excessive decreases in diastolic blood pressure may decrease coronary blood flow and trigger reflex tachycardia and increased contractility mediated by the baroreceptor reflex. the combination of decreased coronary blood flow and increased myocardial o demand may provoke angina pectoris. with administration of intravenous phenylephrine, adequate coronary perfusion pressure can be maintained. in the lungs, nitroglycerin inhibits hypoxic pulmonary vasoconstriction and worsens hypoxia and gas exchange. it has been shown to have platelet-inhibitory effects, the clinical significance of which has not been elucidated. although an uncommon complication of nitroglycerin therapy, the nitrite metabolite of ntg is capable of oxidizing the ferrous (fe + ) ion in the hemoglobin into the ferric (fe + ) state, producing methemoglobin. ferric (oxidated) iron is unable to carry o effectively, and in sufficiently high concentrations (> %) it can impair tissue oxygenation. treatment of methemoglobinemia is methylene blue, to facilitate the conversion of methemoglobin to hemoglobin. high doses of ntg is more likely to produce methemoglobinemia in patients with hepatic dysfunction. the acc/aha guidelines recommend that nitrates should not be administered to patients with non-st-elevationacute coronary syndrome (acs) who recently received a phosphodiesterase inhibitor (class iii, level of evidence: b). nitrates should not be administered to patients with signs of hypovolemia or hypotension, and it should be used with caution in patients with right ventricular infarction. as discussed in the previous section, beta-blockers are class ii antiarrhythmics with a multitude of favorable properties utilized in the treatment of cardiac ischemia. their negative inotropic, negative chronotropic, and antihypertensive properties allow for reduction in o consumption and an increase in blood supply during diastole. beta blockers slow spontaneous diastolic depolarization and shorten the duration of cardiac action potentials. ventricular fibrillation threshold is increased. beta blockers reduce myocardial infarct size. in the absence of contraindications (cardiogenic shock, decompensated heart failure, severe sinus bradycardia, second-or thirddegree atrioventricular block, or active bronchospasm), beta blockers should be administered early in the treatment of myocardial ischemia. while early administration has not been shown to improve short-term survival, beta blockers decrease reinfarction and the frequency of ventricular dysrhythmias, and their use has been associated with mortality benefit after myocardial infarction. perioperative beta blockers should be continued in patients already receiving beta blockers, according to class indications for perioperative beta blockade from the acc/aha recommendations; patients at high perioperative risk for adverse cardiac events should be started on beta blockers preoperatively, and continued up to days postoperatively. in these patients, it is reasonable to titrate beta blockers to heart rate and blood pressure. beta blockers are competitive, reversible inhibitors of beta adrenergic receptors. beta adrenergic receptors are g-protein coupled receptors. stimulation by their agonists activates adenylate cyclase to produce cyclic amp. cyclic amp in turn activates protein kinases pathways with subsequent phosphorylation of l-type calcium channels and troponin c, the net effect being enhanced inotropy, positive chronotropy and dromotropy. these responses are all blunted by receptor occupancy by beta antagonists. propranolol -propranolol is a non-selective β(beta)- and β(beta)- receptor blocker with no alpha-receptor activity. as the most soluble beta blocker, its use is associated with the highest frequency of central nervous system side effects. it is well absorbed when taken by mouth. for a comparable effect, higher oral than intravenous doses are required due to its very high ( %) hepatic first-pass metabolism. its active metabolite does not add to the primary clinical effect due to its short half-life. propranolol decreases cardiac output by decreasing the heart rate and reducing myocardial contractility, effects that are especially prominent in sympathetically driven disease states. owing to its β(beta)- receptor antagonism, propranolol may increase systemic vascular and coronary vascular resistance. increased airway resistance may be evoked by propranolol in asthmatic patients. renal and hepatic blood flow is reduced with the administration of propranolol. metoprolol -metoprolol was the first β(beta)- selective receptor antagonist used in clinical practice to prevent increased chronotropy and inotropy in response to sympathetic stimulation. its receptor selectivity is dose related. metoprolol is lipid-soluble, it diffuses more readily into ischemic regions than hydrophilic drugs. fifty percent of the drug administered is metabolized during first-pass hepatic metabolism. at the intravenous dose of . mg/kg, maximum beta receptor blockade is achieved. esmolol -esmolol is a short-acting cardioselective agent metabolized rapidly by red blood cell esterases. it is primarily a β- blocking agent, producing significant decreases in heart rate and contractility. it lacks the ability to block peripheral vascular β- receptors, therefore decreases in blood pressure and cardiac index is more pronounced due to unopposed peripheral vasodilation. esmolol has been safely used in patients with reactive airway disease. sotalol -sotalol is a class iii antiarrhythmic agent with both β-receptor and k + -channel antagonistic effects. a relatively recent cochrane database review found significant reductions in incidence of postoperative atrial fibrillation in the cardiac surgical population. despite the conclusions of this review, the use of sotalol in the post-cardiac surgical population remains limited, owing to sotalol's undesired side effects, such as hypotension, bradycardia, qt-prolongation, and inducing torsadetype ventricular arrhythmias. calcium channel blockers (ccbs) comprise a diverse group of agents that selectively inhibit calcium influx into myocardial and vascular smooth muscle cells. dihydropyridines (such as nifedipine, nicardipine, nimodipine, amlodipine, felodipine, isradipine) exert their effect on the peripheral arteriolar beds (nimodipine favors cerebral vessels), and produce marked peripheral vasodilation with little direct effect on heart rate, av conduction, and inotropy. they may elicit reflex sympathetic activation via the baroreceptor reflex. non-dihydropyridines (phenylalkylamines, for example, verapamil; and benzothiazepines, for example, diltiazem), on the other hand, block av nodal calcium channels, and have significant negative inotropic, chronotropic, and dromotropic effects. their main anti-ischemic effects are due to their ability to reduce myocardial o consumption by depressing contractility, decreasing heart rate and systemic afterload, and increasing o supply by coronary and collateral vasodilation. calcium channel blockers are preferred in vasospastic (variant) angina, as beta-blockers may provoke or aggravate ischemia in some patients. all calcium channel blockers are effective in the treatment and prevention of coronary vasospasm. they may reduce reinfarctions and long-term events in hypertensive patients with acute myocardial infarction. calcium channels display selectivity to calcium ions, and are present among others in myocardial, smooth muscle, skeletal muscle, and neural tissues, as well as in membranes of subcellular organelles, such as the mitochondria or the sarcoplasmic reticulum. calcium channel blockers bind to voltage-gated transient (t), long (l) type channels in the myocardium and smooth muscle, or to neural (n) type channels, rendering them inactive. t-type calcium channels are activated at low voltages, play a major role in the phase depolarization, and are not affected by calcium channel blockers. l-type channels are activated at higher voltages, playing a role in the phase plateau of the cardiac action potential. these are the ca + -channels that are able to be blocked by calcium antagonists. dihydropyridines prevent calcium entry into the vascular smooth muscle cell by extracellular modulation of the l-type channels. the primary target of dihydropyridines is the peripheral arteriolar bed except for nimodipine, which favors cerebral vessels. reflex tachycardia may be elicited with their use. examples include nifedipine, nicardipine, nimodipine, amlodipine, and felodipine. the predominant action of these agents is decreasing systemic, coronary, and cerebrovascular vasomotor tone. dihydropyridine calcium channel blockers, with the exception of the primarily antianginal nifedipine, will be discussed later. nifedipine -nifedipine was the first dihydropyridine calcium channel blocker in clinical use for its coronary and peripheral vasodilator properties. it has greater coronary and peripheral vasodilatory properties than verapamil with negligible effects on venous capacitance vessels. it has little or no effect on cardiac impulse generation and on sa and av nodal conduction. peripheral vasodilation and the resultant decrease of systemic blood pressure activate baroreceptors, leading to reflex sympathetic nervous system activity manifesting as tachycardia. in the absence of concomitant beta-receptor blockade, it may increase the risk of myocardial infarct or recurrent angina. excessive peripheral vasodilation can be antagonized with phenylephrine. it may be combined with beta blockers without increasing the risk of av-block. nifedipine is used in angina pectoris due to coronary artery vasospasm. no iv preparation is available due to its extreme instability when exposed to light. its abrupt discontinuation has been associated with coronary artery vasospasm. phenylalkilamines bind to the intracellular portion of the l-type calcium channel when it is in the open state, and occlude the channel. verapamil -verapamil is a synthetic derivative of papaverine. it is supplied as a racemic mixture, in which the d-isomer lacks ca + -channel blocking properties and acts as a fast na + -channel blocker, accounting for local anesthetic effects, and the l-isomer is specific for slow ca + -channels. the predominance of this action accounts for the classification of this drug as a calcium channel blocker. verapamil decreases the heart rate by depressing sinoatrial and av-nodal activity (hence its utility in the treatment of supraventricular arrhythmias), lowers systemic blood pressure due to myocardial depression and peripheral vasodilation, and produces moderate coronary artery dilation (preferred in essential hypertension and vasospastic angina). its negative inotropy is more pronounced in patients who already have a depressed left ventricular function, therefore verapamil should be avoided in symptomatic heart failure, severe bradycardia, sinus node dysfunction, and av nodal block. these effects of verapamil may be enhanced with concomitant β(beta)-blockade. in the presence of drug-induced heart block, isoproterenol may be useful to increase heart rate. verapamil may also precipitate dysrhythmias in patients with wolff-parkinson-white (wpw) syndrome, and has proven effective in the treatment of hypertrophic cardiomyopathy with or without left ventricular outflow tract (lvot) obstruction. verapamil may be useful in the treatment of premature labor, as well as fetal and maternal tachydysrhythmias. it may decrease uterine blood flow, and should be administered with caution to parturients with impaired uteroplacental perfusion. benzothiazepines block l-type channel via a mechanism that is not well understood. diltiazem may act on the na + /k + pump, decreasing the amount of intracellular na + available for exchange with extracellular calcium, and it may inhibit the calcium-calmodulin binding. diltiazem -diltiazem, like verapamil, blocks the calcium channels at the av node. it is considered first-line treatment for supraventricular tachydysrhythmias. it may also be used for the control of chronic essential hypertension. diltiazem has minimal cardiodepressant effects and is unlikely to interact with β-blockers to decrease contractility. alpha and beta adrenergic receptors are g-protein-coupled receptors present on various types of cells: pre-and postsynaptic sympathetic nerve terminals; as well as cardiac, skeletal, or smooth muscle cells; hepatocytes; pancreatic islets (beta cells); adipose tissue; platelets; and the renal juxtaglomerular apparatus. they bind endogenous and synthetic catecholamines. there are discrete types of adrenergic receptors, each with several subtypes: alpha and beta. alpha receptors are predominant on the peripheral and pulmonary vasculature. they have subtypes, α- and α- . the α- subtype is g q protein-coupled, expressed by smooth muscle, adipose tissue, the liver, sweat glands, and the kidneys. g q protein-coupled receptors activate the phospholipase c (plc) pathway: plc cleaves phosphatidylinositol , -bisphosphate (pip ) into diacyl-glycerol (dag) and inositol triphosphate (ip ). ip in turn interacts with intracellular calcium stores and increases the intracellular calcium content. dag activates protein kinase c, which further modulates ion channels. stimulation of α- receptor results primarily in vascular smooth muscle contraction. other effects include contraction of the pregnant uterus, contraction of urogenital smooth muscle (bladder neck, prostate), bronchoconstriction, mydriasis, glycogenolysis and gluconeogenesis, secretion from sweat glands, and renal sodium reabsorption. alpha- receptors are g i -coupled receptors expressed by central presynaptic nerve terminals. g i -proteins inhibit the production of camp from atp, reduce ca + permeability, and increase k + permeability. presynaptic neuronal α agonists cause inhibition of acetylcholine as well as norepinephrine release by negative feedback from norepinephrine present in the synaptic cleft. stimulation of central presynaptic α receptors inhibits sympathetic nervous system output and causes sedation. peripheral α receptor stimulation causes decreased insulin secretion from pancreatic beta cells, inhibition of lipolysis, platelet aggregation, and vascular smooth muscle contraction. beta- adrenergic receptors are found on the sinus node, av-node, and the myocardium. these are g s -protein coupled receptors and are expressed predominantly in the cardiac pacemaker cells, myocardium, salivary glands, as well as eccrine and apocrine sweat glands. their stimulation activates adenylate cyclase, increasing intracellular campsynthesis. camp as a second messenger activates intracellular signaling pathways that result in increased ca + levels during systole. calcium binds to troponin c, facilitates actin-myosin cross-bridge formation, and increases sarcomere contraction. this translates into faster heart rate, increased myocardial excitability, increased conductivity, and more forceful contractions. beta- receptors are located on the smooth muscle of the bronchial tree, some coronary vessels, skeletal muscle arteries, gastrointestinal tract, and the urinary bladder. stimulation of a β receptor may activate g-proteins that regulate adenylate cyclase differently: the excitatory g s and the inhibitory g i proteins. stimulation of the gi protein results in decreased smooth muscle tone. epinephrine is an endogenous catecholamine produced by the adrenal medulla. it is a potent direct α -, α -, β -, and β adrenergic agonist. in all dose ranges it has strong positive inotropic effects. peripheral vascular resistance varies according to the dominant effect of the receptors activated: at low doses (β > α) svr is usually decreased. at moderate doses (β comparable to α), svr may remain unchanged. at high doses (α > β), svr is increased. it increases myocardial oxygen consumption. . hemodynamic effects . preload: increased due to α -effect on capacitance vessels . contractility: augmented due to β -effect on the myocardium; its lusitropic effects account for an enhanced rate of relaxation. . lusitropy: as a β -agonist, epinephrine enhances early diastolic filling by accelerating relaxation, augmenting filling, and reducing end-systolic ventricular size. . afterload: dose-dependent effect. pulmonary hypertension may occur. . heart rate: tachycardia due to β -effect, increased excitability and conduction, increased risk of arrhythmias. . cardiac output: augmented. . dopamine is an endogenous catecholamine, a precursor to epinephrine and norepinephrine, found in the adrenal medulla and peripheral nerve terminals. it has effects on alpha, beta, and dopaminergic (da) receptors. at the lowest doses ( - mcg/kg/min) its dopaminergic effects are dominant, resulting in the dilation of the cerebrovascular, renal, and mesenteric vascular beds. in low to moderate dose ranges its β -adrenergic effects are predominant, resulting in increased heart rate, increased contractility, and increased cardiac output. at high doses (greater than - mcg/kg/ min) its α -agonist effects predominate: high-dose dopamine increases svr, pvr, and decreases peripheral perfusion. it is an effective mixed inotrope and vasoconstrictor; however, it is not recommended as first-line therapy in the treatment of septic shock due to the side effects associated with its use. dopamine has positive chronotropic effects in all dose ranges, and in higher dose ranges it may induce arrhythmias due to increased excitability and conduction. dobutamine is a synthetic derivative of dopamine that does not affect endogenous norepinephrine release. it is a racemic mixture of enantiomers of (−) dobutamine with vasoconstrictor (α -agonist), and (+) dopamine with vasodilator (α antagonist) properties. it is a myocardial β -agonist with a predominant positive inotropic effect. its β and α effects are limited, and it completely lacks α effects. in the heart, it augments myocardial contractility and o consumption, and increases cardiac output by increasing heart rate and stroke volume. increased myocardial work is compensated by increased coronary blood flow. on all systemic vascular beds and pulmonary blood vessels it acts as a vasodilator and may cause hypotension. this is primarily mediated by its mild β effects, which are partially unopposed by dobutamine's α effects. dopexamine is a synthetic dobutamine-analogue with positive inotropy, positive chronotropy and peripheral vasodilator action, as well as minimal alpha-receptor activity. it is a potent agonist of da -receptors (and thus decreases renovascular resistance). it is indicated for treatment of low cardiac output states. it is widely used in europe, but it is not approved by the food and drug administration (fda) in the united states. ephedrine is an alkaloid-derivative with sympathomimetic effects. it has mild direct agonist on α -, β -, and β -receptors, and leads to indirect norepinephrine-release from the neurons. it is an easily titratable pressor and inotrope with a short duration of action that does not reduce placental blood flow, and is therefore safe to administer in pregnancy. phenylephrine is a synthetic noncathecolamine with nearly selective postsynaptic α and minimal β effects. due to its vasoconstrictor effects on resistance vessels, it is widely used for the treatment of intraoperative drug-induced hypotension. its vasoconstrictor potency is less than that of epinephrine and norepinephrine. it is a direct α-agonist with short duration of action. it raises both systemic and pulmonary vascular resistance. phenylephrine constricts coronary blood vessels and augments coronary blood flow. it constricts cerebral and renal vessels, but does not compromise blood flow to these organs. phenylephrine may reduce renal, skeletal muscle, mesenteric, and skin blood supply. myocardial o requirement is usually unchanged unless hypertension is present. vagally mediated reflex bradycardia commonly occurs as a response to increased vascular resistance and usually responds well to atropine; this response can be blocked by atropine. its effects on cardiac output appears to be determined by preload-dependency. . isoproterenol is a pure direct β -and β -receptor agonist. it increases cardiac output by increasing heart rate, augmenting myocardial contractility as well as by afterload reduc- vasopressin is a naturally occurring antidiuretic nonapeptide synthetized as a pro-hormone primarily in the supraoptic, and secondarily in the paraventricular nuclei of the posterior hypothalamus. after binding to carrier protein neurophysin, it is transported via the supraoptic hypophyseal tract to the posterior hypophysis, where it is stored and released into the circulation when plasma osmolality is higher than physiologic, or when profound hypovolemia is present. normal plasma concentrations are less than pg/ml. the half-life of endogenous vasopressin is - min, and it is metabolized via renal and hepatic pathways. due to trypsin activity in the gastrointestinal tract it must be administered parenterally or intranasally. in concentrations higher than required for its antidiuretic effect, it acts as a non-adrenergic vasoconstrictor on peripheral vascular beds. it completely lacks beta-adrenergic effects; therefore, it produces less tachycardia compared to adrenergic agonists. in severe shock states, or in conditions where refractory vasoplegia is present, a deficiency in vasopressin levels may necessitate restoration of its physiological concentration by administration of low-dose exogenous vasopressin. the effects of vasopressin are mediated by a family of g-protein coupled vasopressin receptors. v a and v b are g q protein-linked, whereas v is g s protein-linked receptors. v a is located primarily on the vascular smooth muscle of the systemic, coronary, splanchnic, and renal vessel bed, mediating pressor response by increasing peripheral vascular resistance. intra-arterial infusion of vasopressin constricts all major celiac artery branches except the hepatic artery. a less well appreciated aspect is vasopressin's pulmonary vasodilatory effect by inducing constitutive endothelial nitric oxide synthase and increasing nitric oxide production. vasopressin's vasodilatory effects are mediated by endothelial oxytocin receptors. v a is also expressed in diverse tissues such as hepatic, central neuronal, and myometrial tissues, as well as platelets. v a receptors are g q -protein linked, activate phospholipase c, and ultimately increase intracellular calcium, leading to vasoconstriction. v b receptors activate adrenocorticotropic hormone (acth) release from the anterior pituitary gland. v receptors are located primarily in the distal tubules and collecting ducts of the kidneys, and play a role in the homeostatic regulation of plasma volume and preservation of serum osmolality. these receptors are gs-protein-linked, and activate adenylate cyclase to ultimately increase intracellular camp levels. this in turn leads to mobilization of aquaporin channels and their insertion into the luminal surface of the collection tubules, and the subsequent increase of water reabsorption. v receptors are also expressed in endothelial cells, where they are involved in the release of von willebrand factor (vwf) and factor viii (f viii). vwf protects f viii from breakdown, and plays an important role in binding platelets to the site of bleeding. . hemodynamic effects: adrenergic-independent direct peripheral vasoconstriction via v a receptors without affecting chronotropy or inotropy. redistributes blood to the coronaries and the cerebral vasculature without β -induced increase in myocardial o consumption. milrinone is a noncatecholamine and nonglycoside inodilator with potent positive inotropic and vasodilator properties. it acts as a competitive inhibitor of phosphodiesterase- (pde- ) in the cardiac and vascular smooth muscle cells, independently of β-adrenergic mechanisms. pde- is an enzyme that hydrolyses intracellular camp into its inactive metabolite. inhibition of the camp-breakdown allows for the camp levels to remain elevated. this in turn increases the ca + influx into the myocardium, and increases ca + efflux from the vascular smooth muscle, an effect translating into increased inotropy in the heart, and vasodilation of the peripheral arteries and veins. in the myocardium, milrinone augments contractility, facilitates diastolic relaxation, enhances automaticity, and shortens av-nodal conduction time. afterload reduction in the peripheral vessels usually has little effect on chronotropy. overall, milrinone improves myocardial contractility, cardiac output, and ejection fraction. myocardial o consumption is unchanged or slightly increased. . levosimendan is a pde- inhibitor that acts as an intracellular calcium-sensitizer to troponin c. it improves contractility in a calcium-dependent manner by binding to cardiac troponin c and stabilizing its calcium-induced conforma-tional changes, and has vasodilatory effects by opening atpsensitive potassium (k atp ) channels of the vascular smooth muscle. k atp channels close as intracellular atp concentration increases, their selective blockade results in arteriolar vasoconstriction. activation of k atp channels may also account for its anti-stunning effects without increasing intracellular calcium concentrations or energy consumption in the myocardium. the effects of levosimendan are greatest during systole when intracellular calcium levels are the highest, and diminish along with the decreasing intracellular calcium availability during diastole. levosimendan acts independently of camp and has been used in beta-blocked patients. it does not increase myocardial oxygen consumption. it does not impair the baseline diastolic function and is tolerated without arrhythmogenicity. it is a systemic and coronary vasodilator used for treatment of acute decompensated congestive heart failure. the levosimendan infusion versus dobutamine (lido) study demonstrated a significant survival benefit of levosimendan over dobutamine, and smaller studies described its benefits for pulmonary hypertension complicated by right ventricular failure. it is not fda-approved for clinical use in the united states. papaverine is a nonspecific phosphodiesterase inhibitor, derived from opium; however, as a benzyl-isoquinolon, it is structurally and functionally unrelated to opioid alkaloids. it is used by surgeons to treat and prevent spasm of the internal mammary artery during cardiac surgery. hypertension is a major risk factor for cardiovascular morbidity and mortality; essential hypertension accounting for the vast majority of adult cases. based on recommendations of the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc- ), the classification of blood pressure (bp) for adults has been as follows: diuretics, beta-blockers, angiotensin converting enzyme (ace) inhibitors/angiotensin receptor blockers (arbs), calcium channel blockers (ccbs), and aldosterone antagonists are mainstays of pharmacologic management of essential hypertension. specific antihypertensive classes target specific end-organ damage, hence the difference in drug combinations when coexisting risk factors are taken into consideration: heart failure: diuretics, beta-blockers, ace inhibitors/ arbs, aldosterone antagonists post-myocardial infarction: beta-blockers, ace inhibitors, aldosterone antagonists high coronary disease risk: diuretics, beta-blockers, ace inhibitors, ccbs diabetes: diuretics, beta-blockers, ace inhibitors/ arbs, ccbs chronic kidney disease: ace inhibitors/arbs recurrent stroke prevention: diuretics, ace inhibitors this section will focus on drugs used in the management of systemic hypertension, pulmonary hypertension, and heart failure. the primary determinants of blood pressure are cardiac output and peripheral vascular resistance. cardiac output is maintained by heart rate and stroke volume. stroke volume is determined by preload, contractility, and afterload, as well as the left ventricular and vascular compartmental size. modulation of these factors by controlling input from vasoactive hormones, neurotransmitters, and local endotheliumderived factors will bring about a change in blood pressure: peripheral resistance is decreased by peripheral α- blockers, central α- agonists, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, diuretics, or direct vasodilators. heart rate is modulated by beta-blockers, calcium channel blockers, or the novel agent ivabradine. stroke volume is reduced by preload-reducer diuretics and venodilators, negative inotropy of calcium channel blockers, or afterload-reducing agents. labetalol -labetalol is a nonselective beta-blocker with selective α receptor blocking properties. the potency of β-blockade is approximately -fold greater than α blockade. its partial β agonism promotes peripheral vasodilation. labetalol can be considered a peripheral vasodilator not triggering reflex tachycardia. in a dose-related fashion it decreases heart rate and peripheral vascular resistance, leaving the stroke volume and cardiac output unchanged. after oral administration labetalol is completely absorbed. its significant first-pass metabolism produces inactive metabolites. phentolamine -phentolamine is a reversible nonselective competitive antagonist at the adrenergic α and α receptors, as well as at serotonin receptors. its main action is arterial vasodilation with minimal venodilation. svr and pvr is therefore decreased. heart rate and contractility drastically increases due to both the baroreceptor reflex and phentolamine's direct effect on central presynaptic α receptors, allowing for increased norepinephrine release and decreased reuptake. this results in unopposed β effects that may provoke arrhythmias and myocardial ischemia, and respond well to β-blockers. in patients pre-treated with phentolamine, administration of epinephrine may result in hypotension due to unopposed β effects. phentolamine is a gastrointestinal stimulant, may lower blood glucose, and causes histamine release. it is used for the diagnosis of pheochromocytoma (a drop in sbp > mm hg and dbp > mm hg is diagnostic for pheochromocytoma), management of hypertensive episodes associated with the resection of pheochromocytoma, as well as local treatment of skin necrosis associated with norepinephrine extravasation. phenoxybenzamine -phenoxybenzamine is a nonselective, noncompetitive adrenergic α -and α antagonist at postganglionic synapses in smooth muscle and exocrine glands. its main action is arterial vasodilation with minimal venodilation. it decreases svr and pvr. heart rate and contractility are increased due to both the baroreceptor reflex and phentolamine's direct effect on central presynaptic α receptors, allowing for increased norepinephrine release and decreased reuptake. this results in unopposed β effects that may provoke arrhythmias and myocardial ischemia, and respond well to β-blockers. phenoxybenzamine is available in po form only. it is used for treatment of pheochromocytoma; it is started - days preoperatively to allow for expansion of blood volume. to avoid unopposed alpha-stimulation and life-threatening hypertensive episodes, β-blockers are not introduced until after adequate alpha-blockade. due to its side effects-for example, orthostatic hypotension and tachycardia-especially after administering the initial dose, its dose should be increased slowly. alpha-and beta-blockers should be continued until the morning of surgery. prazosin, doxazosin, terazosin, tamsulosin -prazosin, doxazosin, terazosin and tamsulosin are selective α -antagonists. their main cardiovascular action is decreasing svr and pvr with only minimal increases in heart rate. prazosin acts on arteries and veins. prazosin and doxazosin are indicated for treatment of chronic hypertension. terazosin blocks vascular α b , as well as α a receptors in the prostate and bladder neck. it is used for treatment of benign prostate hypertrophy (bph) and hypertension. tamsulosin is a selective α a receptor blocker used for symptomatic bph and to help with passage of kidney stones. orthostatic hypotension with syncope may occur with the use of peripheral α-blockers, especially after the first dose. tolazoline -tolazoline is a nonselective competitive α-adrenoceptor antagonist, structurally similar to phentolamine. it acts as a sympathomimetic and also stimulates muscarinergic ach-receptors and causes histamine release. it decreases systemic and pulmonary vascular resistance. it markedly increases heart rate and may provoke arrhythmias due to its sympathomimetic effects and the reflex sympathetic activation associated with its use. although it is used to treat persistent pulmonary hypertension in neonates, it is not a selective pulmonary vasodilator, and may worsen the svr/pvr ratio when fixed pulmonary hypertension is present. central α receptor agonists reduce sympathetic outflow by stimulating bulbar α activity and reducing peripheral norepinephrine release without eliciting reflex tachycardia and increased contractility. these agents potentiate the effects of anesthetics and may substantially reduce anesthetic and narcotic requirements. central α receptor agonists may exacerbate depression and are contraindicated for management of hypertension in patients treated with monoamine oxidase inhibitors. clonidine -clonidine is a centrally acting α-agonist with near selectivity on α -receptors ( : α to α effect). as a partial agonist-antagonist, it elicits submaximal response on central pre-and post-junctional α -receptors while blocking the effects of other agonists. it is a coronary vasodilator and acts as a sympatholytic-a property utilized to manage withdrawal symptoms in opioid and alcohol addicts. clonidine effectively reduces svr, pvr, and renal vascular resistance. its half-life is hours. clonidine blocks pain signal transmission in the brain. it may potentiate opioid effects on the central nervous system and has local anesthetic properties. when administered epidurally, it produces dose-dependent analgesia not reversed by opioid antagonists-an effect utilized in the treatment of cancer pain. it doubles the duration of intermediate-duration local anesthetics when used as adjunct to peripheral nerve blocks. due to risk of hypotension and bradycardia, epidural clonidine is not recommended in the perioperative, obstetric, and postpartum setting. it causes sedation in a dosedependent manner. due to lack of adequate data, it should not be administered above the c dermatome. clonidine should not be discontinued abruptly, as rebound hypertension is frequently triggered. its immediate-release oral formulations should be discontinued within hours before surgery and resumed as soon as possible in the early postoperative period. methyldopa -methyldopa is a centrally acting antihypertensive. its mechanism of action is not fully elucidated. its antihypertensive effects are most likely due to its metabolism to α-methyl-norepinephrine, which stimulates central inhibitory α-receptors and lowers blood pressure by false neurotransmission and possibly by reducing plasma renin activity. it has no direct effect on cardiac and renal function. reversible thrombocytopenia and leukopenia, hemolytic anemia, a positive coombs test, and liver dysfunction including fatal liver necrosis has been reported with the use of methyldopa. despite these adverse effect, methyldopa is still commonly used for the treatment of pregnancy-induced hypertension due to the lack of adverse effects of long-term treatment on the fetus. guanabenz -guanabenz is an oral antihypertensive active on central α -receptors. its action is mediated by bulbar α activation, leading to attenuated sympathetic outflow at the level of the brain stem. it effectively controls blood pressure without significant effects on renal function. it frequently causes sedation; therefore, additive sedative effects should be considered when used with other centrally acting depressants. its abrupt discontinuation may rarely result in an increased production of endogenous catecholamines and subsequent rebound hypertension. in patients with coexisting liver and kidney disease, careful monitoring of blood pressure is recommended. guanfacine -guanfacine is a selective oral central postsynaptic α a -receptor agonist with a duration of action longer than that of clonidine. it reduces sympathetic outflow and with resultant decrease in heart rate and vasomotor tone. guanfacine preferentially binds postsynaptic α a -receptors in the prefrontal cortex, and modulates behavioral responses. its immediate release form is used for management of hypertension, whereas its extended release form is used for treatment of attention deficit/hyperactivity disorder as monotherapy or as adjunct to central nervous system stimulants. its dose should be reduced with concomitant use with cyp a inhibitors, and slowly increased with the use of cyp a inducers. dexmedetomidine -dexmedetomidine is a selective central α a and peripheral α b -agonist with sedative and anesthetic properties. centrally, it reduces sympathetic outflow and inhibits norepinephrine release at regular doses. at high doses, it activates peripheral α b receptors with resultant vasoconstriction, increased svr, pvr, pulmonary artery occlusion pressure, and central venous pressure. its use is indicated for procedural sedation for awake fiberoptic intubation, as well as sedation of intubated and mechanically ventilated patients in the intensive care unit. its initial loading dose is mcg/kg over min, followed by a maintenance rate of . - . mcg/kg/hour, administered via a controlled infusion device, and titrated to effect. no dose adjustments are required in patients with severe renal impairment. in patients with severe hepatic impairment, dose reduction is recommended. it may cause episodes of hypotension, bradycardia, or sinus arrest. these adverse effects are enhanced in the presence of beta-blockers and antihypertensive agents, as well as in patients with advanced age, diabetes mellitus, pre-existing heart block, bradycardia, hypovolemia, or depressed ventricular function. limited information is available regarding its use in pregnancy. dexmedetomidine is expected to cross the placenta. perioperative continuation of α agonists are not recommended for prevention of major adverse cardiac events. when the circulating blood volume is low and renal perfusion is reduced (the afferent glomerular arteriolar pressure is decreased), the renal juxtaglomerular apparatus converts proenzyme prorenin into renin, which is then released into the circulation. upon entering the circulation, renin cleaves a decapeptide from plasma protein angiotensinogen to produce angiotensin i, precursor to the potent vasoconstrictor angiotensin ii. angiotensin ii (at-ii) is the primary vasoactive hormone of the renin-angiotensin system. it is generated from angiotensin i by the proteolytic action of angiotensin converting enzyme (ace). it also stimulates aldosterone secretion by the adrenal cortex. at-ii is therefore a potent vasoconstrictor of arteries and veins. it is responsible for increased aldosterone secretion and sympathetic nervous system stimulation. angiotensin ii normally binds to the at receptor that ultimately leads to the increased release of calcium from sarcoplasmic reticulum to produce vasoconstriction. besides its effects on vasomotor tone, at ii also mediates alveolar permeability and lung injury: excessive ace inhibition is related to worse lung injury, an important consideration in the management of critically ill patients. decreased generation of angiotensin ii (for example, by inhibition of ace) results in decreased vasoconstrictive effects, usually without eliciting reflex tachycardia or an increase in cardiac output. in addition, decreased concentrations of plasma aldosterone results in decreased sodium and water retention. ace inhibitors block the at-i to at-ii conversion, as well as the breakdown of bradykinin, an endogenous vasodilator substance, which contributes to the antihypertensive effects of these drugs. ace inhibitors reduce activation of low density lipoprotein (ldl) receptors and decrease the concentrations of ldl cholesterol. if the concentration of ldl is already sufficiently low, ace inhibitors may no longer be effective in reducing the rate of cardiovascular events. ace inhhibitors can be classified according to the structural element that interacts with the zinc ion of the enzyme, as well as the form in which the drug is administered (prodrug or active). administration of ace inhibitors (for example, benazepril, fosinopril, ramipril, or quinapril) as prodrugs increases the bioavailability prior to their hepatic metabolism to the active drug. enalapril is the prodrug of the active ace inhibitor enalaprilat, and its conversion may be altered in patients with hepatic dysfunction. captopril, enalaprilat, and lisinopril are not prodrugs. the major difference among the clinically used ace inhibitors is in duration of action. in the absence of contraindications, ace inhibitors and angiotensin receptor blockers are recommended in patients with systolic heart failure to reduce morbidity and mortality. they may be preferred in hypertensive patients with chronic kidney disease and/or diabetes mellitus. unless contraindicated, ace inhibitors are prescribed together with a beta blocker. contraindications to the use of ace inhibitors are prior life-threatening reactions (angioedema) and known or planned pregnancy. caution should be used in the presence of low baseline blood pressure, elevated levels of serum potassium, increased serum creatinine, or bilateral renal artery stenosis. common side effects are cough, upper respiratory congestion, rhinorrhea, and allergic-like symptoms. it is speculated that these airway responses reflect potentiation of the effects of kinins due to drug-induced inhibition of peptidyldipeptidase activity and subsequent breakdown of bradykinin. if respiratory depression develops, prompt injection of epinephrine is advised. angioedema is a potentially lifethreatening complication of treatment with ace inhibitors. decreases in glomerular filtration rate may occur. for this reason, ace inhibitors are used with caution in patients with preexisting renal dysfunction and are not recommended for patients with renal artery stenosis. hyperkalemia is possible due to decreased production of aldosterone. the risk of hyperkalemia is greatest in patients with recognized risk factors (congestive heart failure with renal insufficiency). measurement of plasma concentrations of potassium may be indicated in treated patients. ace inhibitors are to be avoided in pregnancy due to their potential to induce oligohydramnios, other fetal developmental abnormalities, or fetal demise. although recent perioperative guidelines have suggested continuing ace inhibitors/arbs before non-cardiac surgery, adverse circulatory effects during anesthesia have been recognized in patients chronically treated with ace inhibitors/arbs, leading to the recommendation that these drugs be discontinued hours before anesthesia and major, low-risk, urgent, or emergent surgery. prolonged hypotension has been observed in patients undergoing general anesthesia for minor surgery in whom ace inhibitor therapy was maintained until the morning of surgery. surgical procedures with major fluid shifts have also been associated with hypotension in patients treated with ace inhibitors. treatment with ace inhibitors does not increase the incidence of hypotension after the induction of anesthesia in patients with infarction-induced myocardial dysfunction. exaggerated hypotension attributed to continued ace inhibitor therapy has been responsive to crystalloid fluid infusion and/or administration of sympathomimetics such as ephedrine or phenylephrine. if hypotension is refractory to treatment, treatment with vasopressin is usually effective. ace inhibitors may increase insulin sensitivity and hypoglycemia, which is a concern when these drugs are administered to patients with diabetes mellitus. captopril -captopril is an oral vasodilator that inhibits the formation of angiotensin ii in the lung. the subsequent decrease in plasma at-ii levels leads to decreased arterial and venous vasomotor tone, generally without eliciting tachyphylaxis or reflex hemodynamic changes. in common with all ace inhibitors, captopril reduces preload and afterload on the heart. it promotes renal excretion of sodium and water, reducing the circulating blood volume. treatment with captopril is associated with survival benefit after myocardial infarction, congestive heart failure, or hypertension. captopril may delay the development of renal disease in patients with diabetes mellitus, and counteracts ventricular remodeling after myocardial infarction. also in common with all ace inhibitors, captopril may reversibly reduce kidney perfusion and decrease kidney function. patients with bilateral renal artery stenosis are at risk for kidney failure. due to suppressed aldosterone activity, hyperkalemia may occur. a common side effect is chronic nonproductive cough, secondary to the inhibition of the breakdown of bradykinin. angioedema, a life-threatening condition potentially causing airway obstruction, is a rare adverse effect. enalapril and enalaprilat -enalapril is an oral ace inhibitor used for the treatment of hypertension and congestive heart failure. the therapeutic and side effects of enalapril are very similar to those of captopril. as an inactive prodrug, it first must undergo hepatic metabolism into its active form, enalaprilat. enalaprilat is an intravenous ace inhibitor, used primarily to treat severe hypertension. its therapeutic and side effects are similar to those of captopril. it has a duration of action longer than nitrates, eliminating the need for continuous infusion. enalaprilat decreases peripheral vascular resistance without eliciting reflex tachycardia, an increase in cardiac output and myocardial oxygen consumption. lisinopril and ramipril -lisinopril and ramipril are oral ace inhibitors-prodrugs used for the treatment of hypertension. they must first undergo hepatic metabolism into their active form. their therapeutic and side effect profile is very similar to that of captopril. in renal insufficiency, dose adjustment is required. fosinopril -fosinopril is an oral ace inhibitor used for the treatment of hypertension. it must be converted into an active metabolite in the liver and the gastrointestinal tract. it is eliminated by biliary excretion, therefore, unlike other ace inhibitors, it does not need dose adjustment in patients with kidney insufficiency. perindopril, trandolapril, moexipril -perindopril, trandolapril, and moexipril are oral ace inhibitors used for management of left ventricular dysfunction after myocardial infarct, as well as treatment of hypertension. these prodrugs must be hydrolyzed in the liver into their active metabolites. their action and side effect profile is similar to that of captopril. angiotensin ii levels may increase and maintain elevated blood pressure despite adequate treatment with ace inhibitors, due to at-ii production via non-ace-dependent pathways. angiotensin ii receptor blockers produce antihypertensive effects by blocking the vasoconstrictive actions of at ii without affecting ace activity. because these drugs do not inhibit ace, they do not cause an increase in bradykinin, which contributes to some of the side effects, for example, dry cough and angioedema, of ace inhibitors. their effects and indications (hypertension, post-myocardial infarction and heart failure, prevention of renal insufficiency in diabetics) and are similar to angiotensin converting enzyme inhibitors. arbs are preload-and afterload-reducers, they downregulate sympathetic activity by blocking at-ii effect on peripheral norepinephrine release and reuptake, they act as diuretics and natriuretics by blocking aldosterone secretion, and counteract cardiac remodeling associated with hypertension, heart failure and myocardial infarction. arbs may be preferred to ace inhibitors in hypertensive patients with heart failure, ischemic heart disease, and/or post-myocardial infarction. angiotensin receptor blockers are generally well tolerated with a low incidence of side effects and drug interactions. in common with ace inhibitors, arbs are contraindicated in pregnancy. in the presence of bilateral renal artery stenosis, at-ii constricts the efferent glomerular arteriole more than the afferent arteriole, allowing for adequate glomerular capillary pressure and filtration. ace inhibitors and arbs eliminate this effect of at-ii. in the presence of at least unaffected kidney, sufficient filtration can still be maintained even after at- receptors are blocked; however, when a solitary kidney or bilateral renal artery stenosis is present, kidney function may worsen. losartan -losartan is an oral arb indicated for the treatment of hypertension and lowering fatal and nonfatal cardiac and cerebrovascular events, especially when left ventricular hypertrophy is present. both losartan and its first metabolite are active and effectively antagonize at-ii action on the at- receptor. volume and salt deficit should be corrected before treatment with losartan. in the presence of liver and kidney disease, or in patients whose kidney function depends on the activity of the reninangiotensin-aldosterone system, dosage adjustment, withholding, or discontinuation may be required, and periodic monitoring of kidney function and potassium is necessary. it is contraindicated in pregnancy. valsartan -valsartan is an arb used as monotherapy or in combination with amlodipine and/or hydrochlorothiazide for the initial management of hypertension and heart failure with reduced ejection fraction. it conveys morbidity and mortality benefit in clinically stable patients with symptomatic heart failure or left ventricular dysfunction after mi. valsartan may be given as part of the standard post-mi regimen, along with aspirin, beta-blockers, statins, and thrombolytics. it is contraindicated in pregnancy. irbesartan -irbesartan is an oral arb. its indications, therapeutic and side effect profile is similar to that of losartan and valsartan. olmesartan, candesartan, telmisartan, eprosartan -olmesartan and candesartan are taken as prodrugs, and are metabolized into their active form during their absorption from the gastrointestinal tract. candesartan is the only arb depending on its metabolism for clinical effect. dose adjustment is required in the presence of kidney disease. olmesartan, telmisartan, and eprosartan do not require dose adjustment in the presence of mild to moderate kidney disease. beta blockers have been shown to reduce mortality after myocardial infarction, congestive heart failure, hypertension, and chronic angina. the properties of these antiarrhythmic agents have been discussed previously. this section will briefly review the role of beta blockers in the treatment of hypertension and congestive heart failure. beta adrenoceptor antagonists blunt the effect of sympathetic stimulation on the cardiovascular system. the magnitude of this effect depends on the density of receptors at the effector sites, and the availability and relative concentration of both the agonist catecholamines, and the antagonist receptor blockers. beta blockers inhibit myocardial and peripheral vascular β -receptors to reduce heart rate, contractility, and myocardial o consumption. by decreasing heart rate, beta blockers increase diastolic filling time, and improve o and substrate delivery to the left ventricle. by decreasing contractility, beta blockers reduce left ventricular ejection velocity and decrease shear forces on the aorta in the presence of dissection, and reduce dynamic ventricular outflow tract obstruction; for example, in hypertrophic obstructive cardiomyopathy or the tetralogy of fallot. svr may increase due to inhibition of β vasodilation; beta blockers should therefore be used with caution in patients with peripheral vascular disease. beta blockers are generally not preferred as a first-line agent for initial management of hypertension, but may be considered as add-on therapy in patients who do not respond adequately to treatment with other drug classes. they can precipitate congestive heart failure, especially when used with other myocardial depressants; for example, calcium channel blockers. abrupt perioperative discontinuation of beta blocker therapy may produce rebound tachycardia and hypertension. traditionally, beta-receptor antagonists are classified on the basis of their relative selectivity for β -and β -receptors, the presence or absence of intrinsic agonistic (sympathomimetic) activity and their pharmacokinetic features. the first generation of beta blockers (propranolol) nonselectively blocked both β -and β -receptors. second-generation cardioselective beta blockers have greater affinity for β -than for β -adrenoceptors, and are less likely to produce undesired side effects (bronchoconstriction, increased svr). β selectivity is dose-dependent; therefore, caution should be exercised when administering a beta-blocker to a patient with reactive airway disease. examples of cardioselective beta blockers: metoprolol, atenolol, bisoprolol, esmolol, betaxolol, and acebutolol. examples of nonselective beta blockers: labetalol, carvedilol, nadolol, timolol, sotalol, and propranolol. timolol eye drops can produce systemic beta blockade. labetalol -labetalol is a long-acting nonselective beta-receptor blocker with selective alpha -receptor blocking properties. its alpha to beta receptor blocking ratio is : when administered by mouth, and : when administered intravenously. it produces dose-related vasodilation without eliciting reflex tachycardia. labetalol decreases blood pressure and systemic vascular resistance. heart rate may slightly increase; stroke volume and cardiac output remain unchanged. due to its long duration of action, it is usually not preferred for intraoperative minute-to-minute control of hemodynamic variables. carvedilol -carvedilol is a nonselective beta-adrenergic blocking agent with selective activity on peripheral alpha receptors. it is used for management of hypertension, either as monotherapy or in combination with other agents. carvedilol has demonstrated survival benefit and is now also part of the standard treatment regimen for clinically stable patients who have survived the acute phase of mi and have a left ventricular ejection fraction of less than %. in hypertensive patients with left ventricular dysfunction (those who depend on beta-adrenergic stimulation in order to maintain cardiovascular compensation), the usual (lower) heart failure dosage applies (instead of that for hypertension). contraindications to the use of carvedilol include high-degree atrioventricular block, bronchial asthma or other reactive airway disease, cardiogenic shock, decompensated congestive heart failure (chf) requiring inotropes, severe liver dysfunction, and history of severe hypersensitivity reactions. abrupt discontinuation of carvedilol may precipitate cardiac ischemia or malignant ventricular arrhythmias, as well as thyroid storm in patients with thyrotoxicosis. certain beta blockers may act as a competitive partial agonist-antagonist on peripheral β-receptors, and elicit a submaximal response at maximal occupancy. this phenomenon is referred to as intrinsic sympathomimetic activity, or isa. these agents will prevent a beta-agonist from binding to its receptor, and will decrease blood pressure and systemic vascular resistance while resulting in less resting bradycardia and maintaining resting cardiac output than is observed with beta-blockers without isa. long-term treatment with agents with isa result in a decrease of blood pressure due to decreased vascular resistance, rather than decreased cardiac output. these agents may be useful in patients who are unable to tolerate excessive bradycardia resulting from treatment with beta blockers. agents with isa have not been shown to be beneficial after myocardial infarction and are not included in standard post-mi treatment regimens. they are less effective than other beta blockers in the treatment of angina and tachycardia. examples of beta blockers with isa: pindolol and acebutolol. perioperative cardiac complications are not uncommon, with % of patients suffering major cardiac complications, and % showing evidence of significant myocardial injury. according to the acc/aha perioperative clinical practice guideline, continuation of long-standing beta blocker therapy is recommended. beta blockers should not be started on the day of surgery in beta-blocker-naïve patients. in patients with intermediate or high perioperative risk, or in patients with at least revised cardiac risk index (rcri) risk factors, it may be reasonable to initiate beta blocker therapy at least h prior to surgery. it may also be reasonable to initiate perioperative beta blocker therapy long enough in advance to assess safety and tolerability; however, it is uncertain whether starting beta blockers benefits those with long-term indications, but no other rcri risk factors. in the perioperative setting, beta blockers have been confirmed to reduce the incidence of postoperative atrial fibrillation when started before or immediately after surgery. initiation of beta blocker therapy prior to surgery is currently reserved for patients with significant coronary artery disease undergoing surgical coronary revascularization. calcium channel blockers have a multifaceted profile of therapeutic effects. calcium channel blockers are used primarily as anti-ischemic agents for treatment and prevention of stable angina pectoris. common to all calcium channel blockers, but to a different extent in each class, they act as peripheral vasodilators without eliciting reflex tachycardia, they induce coronary vasodilation, they are negative inotropes, and have electrophysiologic depressant properties. the non-dihydropyridine diltiazem and verapamil are used for rate control in acute cardiac ischemia, when beta blockers are contraindicated. most importantly, calcium channel blockers are first-line agents as potent coronary vasodilators in the treatment of prinzmetal (variant, vasospastic) angina. as discussed previously, dihydropyridines act on the peripheral arteriolar beds, and produce marked peripheral vasodilation with direct and indirect effect on heart rate, av conduction, and inotropy. examples are the rapid-acting antianginal nifedipine, the long-acting vasodilator nicardipine, the highly lipid-soluble nimodipine (favoring cerebral vessels), amlodipine, felodipine, or isradipine. nifedipine -nifedipine is primarily indicated in the management of prinzmetal angina. only its extendedrelease formulations are recommended for the treatment of hypertension, as well as the management of raynaud's disease. conventional (immediate-release) formulations are contraindicated in the management of acute myocardial infarction due to its negative inotropy and reflex sympathetic activation. nicardipine -nicardipine is a highly potent peripheral vasodilator that inhibits calcium influx into the myocardium and vascular smooth muscle. nicardipine has no effects on the sa node and av node. it produces clinically insignificant negative inotropy, and may be combined with a beta-blocker for the treatment of angina. this drug has the greatest vasodilating effects of all the ccbs, with vasodilation being particularly prominent in the coronary arteries. because of all the antianginal drugs dihydropyridines produce the greatest peripheral vasodilation, either nifedipine or nicardipine may be useful in patients who have residual hypertension despite adequate beta-adrenergic blockade. nicardipine produces dose-related decrease in both systolic and diastolic blood pressure. nicardipine is frequently used as a tocolytic. when administered, it binds to the inside of the myometrial l-channels causing them to remain closed, and inhibiting uterine contractions. pulmonary edema has been reported when nicardipine was used as tocolytic. its use is contraindicated in patients with severe aortic stenosis: a decrease in diastolic pressure may worsen myocardial oxygen balance. nimodipine -nimodipine is a highly lipid-soluble analogue of nifedipine. this high degree of lipid solubility facilitates its penetration into the central nervous system, where it blocks the influx of extracellular calcium necessary for contraction of large cerebral arteries. this is especially valuable during the treatment and prevention of cerebral vasospasm after subarachnoid hemorrhage. nimodipine has minimal negative inotropic effect on the myocardium. amlodipine -amlodipine is a dihydropyridine calcium antagonist only available in oral form, with minimal cardiodepressant effects. its anti-ischemic effects are comparable to beta-blockers in patients with acute coronary syndrome. the combination of amlodipine and a beta blocker is more effective in the treatment of myocardial ischemia than either drug alone. its actions are resembling those of nifedipine. it is used primarily for oral treatment of hypertension. felodipine -felodipine is primarily a peripheral vasodilator with no clinically significant negative inotropy. its actions are resembling those of nifedipine. it is used for oral treatment of hypertension. isradipine -isradipine is a peripheral vasodilator with no clinically significant negative inotropy. its actions are resembling those of nifedipine. it is used for oral treatment of hypertension. direct vasodilators: hydralazine, nitroglycerine, nitroprusside hydralazine -hydralazine is a direct systemic arteriolar vasodilator, with minimal venodilator (preload and postural) effects. its mechanism of action is not fully understood. it appears to interfere with calcium movements within the cell that initiate and maintain the contractile state. hydralazine decreases systemic blood pressure (systolic less than diastolic). hydralazine may produce reflex sympathetic nervous system stimulation: it increases renin and at-ii activity, which leads to aldosterone stimulation and sodium reabsorption; tachycardia and increased myocardial contractility results in an increase in cardiac output, and may provoke angina. patients with coronary artery disease should be monitored for myocardial ischemia. the use of hydralazine to treat pulmonary hypertension is not recommended since the associated systemic vasodilation may result in systemic hypotension. hydralazine has a relatively slow onset of action with peak effect occurring by min. with chronic po use, a lupus-like reaction may occur. nitroglycerine -nitroglycerine is a direct coronary vasodilator with greater effects on the venous than on the arterial system. its mechanism of action and effects are described in a previous section. nitroprusside -sodium nitroprusside (snp) is a direct-acting, nonselective vasodilator. it is indicated for rapid correction of hypertensive emergencies. it produces vasodilation by directly increasing intracellular nitric oxide levels; its effects on arteries and veins are balanced. nitroprusside lacks effect on nonvascular smooth muscle and the myocardium; however, reflex tachycardia and increased inotropy may occur, making it an undesirable drug of choice for the treatment of aortic dissection, where shear forces should be minimized. its immediate onset of action and short duration allows for iv infusion and precise titration of dosage. as nitroprusside interacts with oxyhemoglobin (hbfe + ), it dissociates immediately and forms methemoglobin (hbfe + ) while releasing nitric oxide (no) and the highly toxic free cyanide ions. in contrast to the organic nitrates (for example, nitroglycerin), nitroprusside does not require the presence of sulfhydryl compounds to generate no, instead it spontaneously produces them and therefore acts as a prodrug. nitric oxide is the active mediator in the vasodilating effects of snp: it activates the guanylate cyclase present in vascular smooth muscle and increases cgmp, which in turn decreases ca + entry into the cell and intracellular ca + concentration. nitroprusside's degradation products are rapidly cleared via non-enzymatic pathways. each snp molecule releases cyanide ions. cyanide ions undergo sulfurilation by the hepatic and kidney enzyme rhodanase (also known as thiosulfate sulfurtransferase, indicating that thiosulfate must be available for the trans-sulfuration to take place) to form thiocyanate, and are excreted in the urine. availability of thiosulfate is the rate-limiting step in cyanide detoxification. excess cyanide ions immediately react with methemoglobin to form cyanmethemoglobin. when sulfur donors and methemoglobin are exhausted, unscavenged free cyanide radicals may accumulate and bind to tissue cytochrome oxidase, to inhibit mitochondrial oxidative phosphorylation. this leads to tissue hypoxia despite adequate levels of available oxygen. the most common adverse effect of snp is hypotension and dysrhythmias. tachyphylaxis may occur, requiring adjustments in dosage for the necessary effect. thiocyanate toxicity is infrequent, and presents with nausea, abdominal pain, hyperreflexia, tinnitus, seizures, and changes in mental status. thiocyanate clearance can be facilitated by dialysis. rarely, cyanide toxicity ensues. signs and symptoms of cyanide toxicity are hypertension (secondary to tachyphylaxis), arrhythmias, st segment changes, altered mental status, seizures, coma, elevated mixed venous po (due to inhibition of cellular o utilization), increasing base deficit, and metabolic acidosis. no cyanosis is seen; spo remains high. treatment includes immediate discontinuation of nitroprusside, mechanical ventilation with % oxygen, and correction of acidosis with bicarbonate. mild toxicity (stable hemodynamics with discontinuation of snp, base deficit less than ) can be treated with thiosulfate ( mg/kg iv bolus over min). severe toxicity (worsening hemodynamics even after discontinuation of snp, base deficit greater than ) is treated with % sodium nitrite ( - mg/kg over min). sodium nitrite converts oxyhemoglobin to methemoglobin, which competes with cytochrome oxidase for the cyanide ions. hydroxocobalamin is an alternative to thiosulfate or sodium nitrite. it binds cyanide to form cyanocobalamin, which acts as a nontoxic reservoir and is excreted by the kidneys. trimethaphan dilates peripheral arteries by blocking cholinergic transmission on nicotinergic autonomic ganglia by binding to the postsynaptic ach-receptor. it is a short-acting competitive acetylcholine-antagonist with both sympatholytic and parasympatholytic effects. it causes vasodilation and tachycardia. common side effects are mydriasis, urinary retention, and ileus. its use is very limited; it has been used for inducing controlled hypotension during surgery, reduction of blood pressure during hypertensive emergencies (for example, in patients with a dissecting aortic aneurysm), or for the emergency treatment of pulmonary edema. pulmonary hypertension-sustained elevated pressure within the pulmonary artery-is a heterogenous and frequently progressive disorder of the pulmonary vasculature that ultimately leads to increased pulmonary vascular resistance, right heart failure, and death, due to constrained pulmonary blood flow and vascular remodeling of the resistance arteries. endothelin- (et- ) is a proinflammatory mediator-a direct vasoconstrictor that stimulates pulmonary vascular smooth muscle cell proliferation and induces fibrosis. its effects are mediated through the et a receptors that mediate vasoconstriction and smooth muscle proliferation, and the et b receptors that induce production of nitric oxide and prostacyclin, and mediate et- clearance. clearance of et- is diminished in pulmonary hypertension. bosentan -bosentan is an orally active dual et a and et b antagonist. it is approved for treatment of who group pulmonary hypertension to slow clinical deterioration and enhance functional capacity in patients who are not candidates for treatment with calcium channel blockers. the use of bosentan increases the risk of severe hepatic injury, liver cirrhosis, and liver failure. liver function should be monitored monthly while taking bosentan. bosentan may cause serious birth defects. pregnancy should be ruled out prior to initiation of treatment and monthly thereafter. it should be avoided in patients with elevated transaminases and is contraindicated in pregnancy. angioedema, fluid retention, and possible dose-related decreases in hemoglobin and hematocrit has been reported. prostacyclin (prostaglandin i , pgi , pgx) and thromboxane a are the main metabolites of arachinoid acid with opposing effects on the vascular smooth muscle. thromboxane a induces vasoconstriction and promotes platelet activation, whereas pgi is a vasodilator with platelet inhibitor effects. in pulmonary arterial hypertension, their physiologic balance is shifted toward thromboxane a . this promotes smooth muscle cell proliferation, vasoconstriction, and thrombogenesis. the activity of prostacyclin synthase is decreased in pulmonary hypertension, leading to low levels of the vasodilator and antiproliferative pgi . restoring the balance between vasodilating and vasoconstricting factors, including the administration of prostacyclin analogues, is a mainstay of the medical management of pulmonary hypertension. epoprostenol -pgi -analogue epoprostenol is used for the long-term treatment of idiopathic pulmonary hypertension, and pulmonary hypertension associated with scleroderma spectrum diseases. it improves functional class, exercise tolerance, and hemodynamics. its main pharmacological actions are: ( ) direct pulmonary and systemic vasodilation, and ( ) platelet inhibition. it is an afterload reducer for both the left and the right ventricle. it produces dose-related decreases in pulmonary vascular resistance; it increases stroke volume and cardiac output. epoprostenol is usually administered as a continuous infusion into a central vein. dosing must be individualized; however, optimal dose range for long-term therapy is believed to be - ng/kg/minute, when used as monotherapy. alternatively, it may be delivered as an inhaled aerosol, for example for the treatment of acute right ventricular failure in the intraoperative or early postoperative setting. common side effects are headache, flushing, nausea, diarrhea, and musculoskeletal pain. contraindications to its chronic use include congestive heart failure due to severe left ventricular systolic dysfunction, pulmonary edema during initial treatment, or known hypersensitivity to epoprostenol or structurally related agents. abrupt withdrawal or sudden dose adjustments should be avoided. its use should be limited to centers experienced with its administration and with systematic follow-up with patients. iloprost -iloprost is a stable prostacyclin-analogue delivered as an inhaled aerosol to patients with idiopathic pulmonary hypertension, pulmonary hypertension associated with scleroderma spectrum diseases or appetite suppressants, or pulmonary hypertension related to chronic thromboembolic disease. in the acute setting, iloprost decreases pulmonary vascular resistance; with long-term use, in addition to its pulmonary vasodilator effects it suppresses pulmonary vascular smooth vessel proliferation. iloprost is delivered as an inhaled aerosol. it is approved for functional class iii and iv to relieve symptoms, enhance exercise tolerance, and diminish disease progression. its adverse effects are similar to those of epoprostenol. its use should be avoided in pre-existing hypotension. it may induce bronchospasm in patients with reactive airway disease. caution should be used with its administration in the presence of increased risk of bleeding, anticoagulation, or other bleeding disorders. abrupt withdrawal or sudden dose adjustments should be avoided. the use of iloprost should be limited to centers experienced with its administration and with systematic follow-up with patients. treprostinil -treprostinil is a stable prostacyclinanalogue that can be taken by mouth, administered into a central vein, or subcutaneously (preferred). its main actions are pulmonary arterial vasodilation and inhibition of platelet aggregation. it is approved to decrease exercise-related symptoms and diminish clinical deterioration. treprostinil may induce symptomatic hypotension. it may increase risk of bleeding. there appears to be an increased risk of gram-negative bloodstream infections, especially in patients receiving intravenous treatment via a chronic indwelling catheter. its side effects are similar to those of epoprostenol. abrupt withdrawal or sudden dose adjustments should be avoided. its use should be limited to centers experienced with its administration and with systematic follow-up with patients. phosphodiesterase- (pde- ) is the intracellular enzyme responsible for degrading cyclic nucleotide monophosphates. it limits signal transduction by camp and cgmp-second messengers implicated in pathogenetic pathways leading to pulmonary hypertension. cgmp is involved in mechanisms of pulmonary vasodilation; its rapid hydrolysis by pde- significantly limits its effect on vasomotor tone. sildenafil -sildenafil is a pde- inhibitor initially indicated for treatment of erectile dysfunction. it enhances the effect of nitric oxide in the corpus cavernosum and the pulmonary arterial smooth muscle. it is approved for treatment of who group pulmonary hypertension to improve functional capacity and delay disease progression; its unlabeled indication is treatment of pulmonary hypertension after recent left ventricular assist device placement. common side effects associated with the use of sildenafil are headache, flushing, dyspepsia, epistaxis, and disturbances of color discrimination. it may potentiate the effects of antihypertensive agents. the use of sildenafil for pulmonary hypertension should be avoided with strong cyp a inhibitors. the use of sildenafil is contraindicated with concurrent use of organic nitrates and guanylate cyclase stimulant riociguat. tadalafil -tadalafil is a longer-acting pde- inhibitor approved for treatment of erectile dysfunction, benign prostate hypertrophy, and who group pulmonary hypertension in patients who are not candidates for treatment with calcium channel blockers. its mechanism of action is similar to that of sildenafil. it is taken by mouth as a once-daily dose. it may be considered for combination therapy with a prostacyclin analogue or et- receptor antagonist. its side effects are similar to those of sildenafil. it may potentiate the effects of antihypertensive agents. its use is not recommended in patients with recent myocardial infarction or stroke within months; uncontrolled arrhythmias, hypotension, uncontrolled hypertension, heart failure, or unstable angina. it should be avoided when severe aortic stenosis or dynamic left ventricular outflow tract obstruction is present. concomitant use of organic nitrates is contraindicated. nitric oxide (no) is a potent, endothelium-derived, cgmpdependent direct vasodilator generated from the terminal guanidine nitrogen of l-arginine. it is produced by isoforms of nitric oxide synthase (nos) in response to hypoxia, pulsatile flow, and flow-induced sheer stress on the arterial wall. it exists in the gaseous form and is administered as an inhalational agent. once inhaled, it diffuses to target cells and activates guanylate cyclase to increases cgmp production: an increased cgmp concentration subsequently leads to vasorelaxation. no diffuses across the pulmonary capillary endothelium into the circulation. once in the circulation, it avidly binds to the iron of heme-based proteins; it is rapidly inactivated by hemoglobin. in pulmonary arterial hypertension decreased nos activity is present, resulting in decreased no-induced pulmonary vasodilation. the use of nitric oxide is favored in the treatment of pulmonary hypertension for its selective pulmonary vasodilator and antiproliferative effects. it vasodilates the well-ventilated areas, and improves v/q matching. it inhibits platelet activation, aggregation, and adhesion. it is synergistic with pgi , allowing the endothelium to maintain its antithrombotic properties. nitric oxide is used to treat persistent pulmonary hypertension of the newborn. as with inhaled prostaglandin analogues, it does not appear to improve clinical outcomes in acute respiratory distress syndrome (ards). heart failure is a constellation of clinical symptoms (primarily fatigue and dyspnea) secondary to impaired left ventricular systolic (reduced ejection fraction) or diastolic (preserved ejection fraction) function, and/or elevated intracardiac pressures. the aha/acc guidelines classify the syndrome by its evolution from asymptomatic preclinical stages to progression to advanced structural heart disease and symptomatology at rest, refractory to maximal medical management. these guidelines also complement the widely accepted functional classification of symptomatic heart failure by the new york heart association (nyha i-iv). chronic heart failure is a condition characterized by vasoconstriction, volume overload, and neurohormonal activation. the goal of its pharmacological management is to reduce vascular tone, reduce sympathetic activation, and improve cardiac function. conventional treatment options include angiotensin-converting enzyme inhibitors, angiotensin ii receptor blockers, aldosterone antagonists, beta blockers, and the combination of isosorbide dinitrate and hydralazine and diuretics. novel approaches are now the use of ivabradine and angiotensin receptor-neprilysin inhibitor/ arb combinations. nesiritide is a recombinant human b-type natriuretic peptide with vasodilatory properties. it binds to vascular endothelial and smooth muscle a-and b-type natriuretic peptide receptors. it increases cgmp levels. cgmp serves as a second messenger to produce arterial and venous dilation. much like endogenous natriuretic peptides, it suppresses the sympathetic nervous system, the renin-angiotensin-aldosterone system, and endothelin. based on the results of initial trials and the observed reduction of pulmonary capillary wedge pressure and symptomatic relief, it was first approved for treatment of patients with acute decompensated heart failure; however, the acute study of clinical effectiveness of nesiritide in decompensated heart failure (ascend-hf) demonstrated that nesiritide did not convey advantage over standard treatment of acute decompensated heart failure. ivabradine is a novel sinoatrial modulator used for the treatment of chronic stable angina pectoris and heart failure with an ejection fraction lower than % inadequately controlled by beta blockers, in patients with native sinus rhythm. it reduces the heart rate by inhibiting cardiac pacemaker inward "funny" current in the sinoatrial node, which, unlike beta-blockers or calcium channel blockers, produces selective heart rate control without affecting ventricular repolarization or myocardial contractility. it may prove beneficial for the treatment of heart failure with reduced ejection fraction. it is contraindicated in sick sinus syndrome, as well as concomitant use of cyp a inhibitors; for example, azole antifungals, macrolides, and protease inhibitor antiretrovirals. it is contraindicated with the concomitant use of verapamil or diltiazem. arni combinations have been shown to convey mortality benefit in patients with chronic heart failure, a condition in which neurohormonal activation, volume overload, and vasoconstriction play important roles. natriuretic peptides are potent vasodilators with natriuretic properties. they reduce sympathetic tone and inhibit the renin-angiotensinaldosterone (raas) axis. neprilysin is an endopeptidase that degrades vasoactive and natriuretic peptides. sacubitril is a neprilysin inhibitor, therefore it increases the concentration of natriuretic peptides. its combination with a raasinhibitor angiotensin receptor blocker conveys cardiovascular and renal protection. the paradigm-hf (prospective comparison of arni with acei to determine impact on global mortality and morbidity in heart failure) trial demonstrated significant reduction in cardiovascular and allcause mortality, as well as heart failure-related hospital readmissions in patients with class ii-iv heart failure and reduced ejection fraction when treated with sacubitril/valsartan compared to enalapril alone. it should be avoided in pregnancy and it should not be administered concomitantly with ace inhibitors. the updated acc/aha/heart failure society of america (hfsa) guidelines for management of heart failure include the addition of ivabradine and sacubitril/valsartan to the list of treatment options for patients with heart failure and reduced ejection fraction. digitalis is a cardiac glycoside that selectively and reversibly inhibits the integral membrane protein na + /k + atpase ion transport system: the na + /k + atpase maintains gradients for na + and k + that determine myocardial excitability and action potential. digoxin is the only commercially available digitalis preparation available in the united states. it binds on the external alpha subunit, increases intracellular na + concentration, decreases ca + efflux, and augments inotropy by releasing calcium from the sarcoplasmic reticulum into the cytoplasm, making more calcium available to generate contraction. digoxin makes resting potential less negative with a resultant increase in myocardial excitability. m vo is decreased due to its effects on wall tension, preload, and afterload. common clinical uses treatment of supraventricular tachyarrhythmias, ventricular rate control for atrial fibrillation with rapid ventricular response. it has been used for treatment of paroxysmal supraventricular tachycardia due to av-nodal reentry or av-reentry. historically, it is used for the treatment of symptomatic heart failure associated with left ventricular dysfunction. treatment may be initiated in patients who have not yet responded to the conventional treatment regimen with an ace inhibitor or a beta-blocker. digoxin does not provide overall mortality benefit, but alleviates symptoms and decreases heart failure related hospital admissions. digoxin does not appear to have significant effect on disease progression in asymptomatic individuals. administration po or iv. assuming normal renal function, the loading dose for an adult is typically . - . mg increments iv or im for a total of - . mg. maintenance dose is . - . mg/day, based on serum levels and clinical effect. adverse effects dizziness, mental disturbances, diarrhea, nausea, vomiting, cardiac dysrhythmias, heart block, and visual disturbances. warning/contraindication administration of digoxin is not recommended for asymptomatic (nyha class i) patients. digoxin is contraindicated when dynamic lvot obstruction is present and in pre-excitation arrhythmias. caution should be used with concomitant use of beta-blockers, calcium channel blockers, or calcium. digoxin has a very low therapeutic index with a nonlinear dose response. dose should be reduced in hypokalemia, hypothyroidism, extensive myocardial or renal damage, and in geriatric patients. due to its very narrow therapeutic range, increased latency of onset and long duration of action, careful dosage determination is essential to avoid digitalis toxicity. in addition to drug pharmacokinetics and pharmacodynamics, patient characteristics-for example, age, cardiac and renal functional status, other medical comorbidities and their pharmacotherapyshould be considered. doses should be individualized. digoxin toxicity has a relatively high mortality rate. therapeutic digoxin levels fall between . - . ng/ml. levels of lower than . ng/ml are non-toxic. levels higher than ng/ml are definitely toxic. infants and children appear to be more tolerant of higher digoxin levels without manifesting signs and symptoms of toxicity. signs of toxicity include extracardiac (primarily central nervous system and gastrointestinal) and cardiac effects. extracardiac manifestations are similar in both acute and chronic intoxication. in pediatric patients, drowsiness, nausea, and vomiting are common signs of early toxicity, and may present before or after the manifestation of cardiotoxicity. in neonates and infants, sinus bradycardia is a premonitory sign of digitalis cardiotoxicity. in adults, early gastrointestinal effects include nausea, vomiting, and anorexia. these may present before or after the manifestation of cardiotoxicity. central nervous system effects include headache, drowsiness, generalized weakness, visual disturbances (color vision is commonly affected), disorientation, confusion, delusions, hallucinations, delirium, or amnesia. cardiovascular toxicity may develop in the absence or presence of other signs and symptoms of toxicity, and includes new arrhythmias, especially those that exhibit features of increased automaticity and av-block, premature atrial and ventricular beats, and malignant ventricular arrhythmias. acute toxicity is usually associated with hyperkalemia, whereas chronic toxicity is associated with hypokalemia or normokalemia. factors that predispose to toxicity are electrolyte derangements such as hypokalemia, hypomagnesemia, or hypercalcemia, diuretic use, alkalosis or acidosis, impaired kidney function, hyperventilation, hypocapnia, arterial hypoxemia, treatment with quinidine, and decreased muscle mass. hypokalemia increases myocardial binding of glycosides; binding of cardiac glycosides to the na + /k + atpase enzyme complex is inhibited by elevated potassium levels. treatment of digitalis toxicity immediate discontinuation of digoxin, correction of predisposing factors, treatment of cardiac dysrhythmias (phenytoin, amiodarone, beta-blockers), and temporary pacing. supplemental k decreases the binding of digitalis to myocardial tissue. digoxin fab is a special digoxin-binding antidote that prevents and reverses toxic effects and enhances elimination; as there is no comparable alternative treatment, it should be promptly administered to patients with digitalis toxicity. b. decrease afterload to promote forward flow. c. allow the heart rate to increase above beats per minute to raise aortic diastolic pressure. d. minimize iv hydration to avoid volume overload. . the preferred agent to treat drug-induced hypotension in the patient from question is a. ephedrine b. phenylephrine c. epinephrine d. norepinephrine . in patients with an intact circulation, rapid infusion of intravenous amiodarone leads to a. rapid restoration of native sinus rhythm and hemodynamic stability b. shortened refractory period c. hypotension and bradycardia d. increased speed of conduction in the sinoatrial node . which of the following statements about the cardiovascular effects of norepinephrine is correct? a. the primary effect of norepinephrine is direct β(beta) -agonism. b. norepinephrine increases cardiac output to a greater extent than phenylephrine. c. norepinephrine augments renal and mesenteric blood flow. d. the arrhythmogenicity of norepinephrine is significantly greater than that of epinephrine, dobutamine, dopamine or isoproterenol. flattened t-wave, or the osborne-waves, typically seen in hypothermia. treatment is hydration with normal saline to decrease plasma calcium levels by dilution, administration of non-thiazide diuretics, and avoidance of thiazides. non-depolarizer muscle relaxant doses should be decreased in patients with hypercalcemia and muscle weakness. the cardiac effects of hyperkalemia are peaked t-waves at mildly elevated potassium levels ( - meq/l), and wide or flat p wave, prolonged pr interval, wide qrs, prolonged qt, ventricular fibrillation, and asystole at higher serum potassium levels ( - meq/l). treatment is: ( ) temporary membrane stabilization with iv calcium (commonly administered dose: mg cacl or g ca-gluconate), ( ) inducing intracellular k + −shift by administering intravenous insulin and glucose (commonly administered dose: to g intravenous dextrose along with to units of insulin), and ( ) promoting excretion (diuretics, hemodialysis, resins). slightly prolonged pr interval, shallow or biphasic t-waves, prominent u-waves, and st-segment depression reflects hypokalemia; whereas prolonged qt c , third degree av-block, torsades de pointes or ventricular tachycardia may reflect hypocalcemia. . b. according to the most recent acc/aha guidelines, there is class i recommendation for continuing beta blocker therapy in patients using beta blockers chronically. it may be reasonable to start beta blockers in patients with or more rcri risk factors; however, beta blockers should not be started on the day of surgery. alpha- agonists are not recommended for risk reduction of major adverse cardiac events. it is a class i recommendation to wait at least days after percutaneous myocardial revascularization with balloon coronary angioplasty, days after bare metal stent placement, and days after drug eluting stent placement. . a. propofol is a gabaergic agent. it causes central nervous system inhibition by decreasing the rate of dissociation of gaba from its receptor, and prolonging the gaba-mediated chloride influx into the cell, causing membrane hyperpolarization. its induction dose for a normovolemic adult patient with normal cardiac function is - . mg/kg. propofol decreases mean arterial pressure by producing direct myocardial depression and decreasing svr. it alters the baroreceptor reflex and causes a disproportionally small compensatory increase in heart rate relative to the decrease in blood pressure. it decreases cerebral metabolic rate of oxygen consumption, cerebral blood flow, and icp; however, at high doses, it may significantly decrease cerebral perfusion pressure. propofol may trigger histamine release, and allergic reactions are therefore possible. it is not associated with increased incidence of postoperative nausea and vomiting. a. ketamine is a phencyclidine derivative interacting with numerous receptors: primarily, it acts as a glutamate-antagonist at both nmda and non-nmda receptors; additionally, it is an opioid receptor agonist (opioid-sparing effects) and an m muscarinergic acetylcholine receptor antagonist (bronchodilation). it interacts with nicotinergic and monoaminergic receptors, as well as with voltage-dependent na +and l-type ca + channels. its actions on the nmdareceptors account for its psychomimetic, analgesic, and amnestic effects. the incidence of emergence delirium may be reduced by concomitant administration of a benzodiazepine and by minimizing external stimulation during emergence. ketamine produces centrally mediated sympathetic activation: it increases plasma epinephrine levels, heart rate, and mean arterial pressure. it is the only anesthetic that increases peripheral arteriolar tone. in catecholamine-depleted patients, or in those with limited inotropic reserve, its intrinsic cardiodepressant properties predominate, and may cause hypotension. absolute contraindications to the use of ketamine are hypersensitivity to ketamine or chemically related agents, significantly elevated blood pressure, stroke, intracranial hemorrhage, active delirium or psychosis, porphyria, pregnancy, and preeclampsia. . c. etomidate is a lipid-soluble nonbarbiturate hypnotic and anesthetic without analgesic effects. it is a carboxylated imidazole, structurally unrelated to other intravenous anesthetics. it is frequently used for rapid intravenous induction of anesthesia at standard doses of . - . mg/kg. its short duration of action is subsequent to its rapid redistribution to peripheral compartments. etomidate causes minimal cardiorespiratory depression even in the presence of cardiac and pulmonary disease. it may increase heart rate and cardiac output by % to %, and may produce a - % decrease in systemic vascular resistance and mean arterial pressure. it decreases cerebral blood flow, cmro , and icp. even a single induction dose of etomidate suppresses cortisol production by inhibiting the activity of -β(beta)-hydroxylase. etomidate causes pain on injection and it is associated with increased incidence of postoperative nausea and vomiting. there are no absolute contraindications to the use of etomidate as an induction agent. . a. aortic stenosis, congenital or acquired, is the most common valvular disease in the united states. most common etiologies are senile degeneration and congenital bicuspid aortic valve. asymptomatic patients even with severe aortic stenosis carry a small risk of sudden cardiac death, whereas the occurrence of symptoms are ominous signs of poor outcome. the classic triad of symptomatic aortic stenosis are angina pectoris (life expectancy is about years, unless the aortic valve is replaced), syncope (average life expectancy: - years), and congestive heart failure (average life expectancy: - years). treatment is surgical. as the severity of the stenosis progresses, a pressure gradient develops between the left ventricle and the aorta, causing the left ventricular wall tension to increase (fixed increase in lv afterload). this leads to a compensatory increase in wall thickness. according to the law of laplace, the wall tension within a sphere filled to any given pressure depends on the thickness of the sphere: pressure = ( * wall thickness * wall tension)/radius. consequently, at a constant pressure, wall tension can be decreased by increasing the thickness of the sphere's wall: left ventricular wall tension = (lv pressure * radius)/ * lv wall thickness. wall stress is a major determinant of myocardial o demand. concentric left ventricular hypertrophy with an increasing wall thickness but unchanged chamber size therefore reduces wall stress and o demand. chamber size, contractility, and stroke volume are usually preserved until late in the disease process. the increasing lv wall thickness will eventually lead to impaired left ventricular relaxation and decreased compliance, and eventually a fixed stroke volume. to maintain an adequate stroke volume, preload augmentation is necessary. a thick, noncompliant heart has an increased basal myocardial o consumption. in the hypertrophied left ventricle, capillary density does not adapt to the increased wall thickness, and any decrease in coronary perfusion pressure will compromise myocardial o supply. maintenance of adequate afterload to ensure adequate systemic diastolic and coronary perfusion pressure is essential in preventing hypotension and the resultant risk of subendocardial ischemia. extremes of heart rate are poorly tolerated in patients with hemodynamically significant aortic stenosis. maintenance of normal rate and sinus rhythm is essential, as a noncompliant left ventricle is unable to augment stroke volume to maintain cardiac output during bradycardic episodes to restore cardiac output, and excessive tachycardia will reduce coronary perfusion during diastole. atrial contraction contributes up to % to % of the left ventricular filling, maintenance of sinus rhythm is essential, and any supraventricular arrhythmias should be aggressively treated. . b. patients with concentric lvh and a hemodynamically significant aortic stenosis are unable to augment cardiac output in response to any sudden decrease in systemic vascular resistance. administration of an α -adrenergic agent-for example, phenylephrine-improves coronary perfusion pressure without adding to the already existing fixed afterload, and increasing myocardial work. concomitant venoconstriction increases venous return and augments preload. reflex bradycardia may improve myocardial o consumption. it is essential not to delay treatment of hypotension, as the thick myocardium is at risk for ischemia, further worsening cardiac output and coronary perfusion, leading to sudden death. agents with the potential to increase heart rate or myocardial o consumption are not first choices for treatment of hypotension in this patient with severe aortic stenosis. . c. amiodarone is a class iii antiarrhythmic agent used for the treatment of atrial and ventricular arrhythmias. it is used during cardiac arrest to aid defibrillation of recurrent or refractory ventricular fibrillation. amiodarone has na + , k + , ca + − channel, as well as alpha-and beta-adrenergic receptor blocker properties. its rate-dependent (rapid) administration may cause profound hypotension and bradycardia. treatment is volume expansion, administration of vasoconstrictors and positive chronotropes, or temporary pacing. . b. norepinephrine is an endogenous catecholamine with peripheral post-synaptic α -and β effects. its peripheral α effects predominate over its myocardial β effects. the effects of norepinephrine mediated by β -receptors are not clinically significant. norepinephrine is a potent vasoconstrictor (map and svr are increased) with mild positive inotropy. it does increase myocardial o requirements. its β-induced positive chronotropy is counteracted by the reflex bradycardia resulting from the α-mediated increase in afterload. norepinephrine does not significantly increase cardiac output; an effect different from that of phenylephrine (phenylephrine may decrease cardiac output). norepinephrine is less likely to induce tachyarrhythmias than epinephrine, dobutamine, dopamine, or isoproterenol. inhibits na efflux during phase shortens duration of phase . this results in decreased duration of the action potential decreases the slope of phase depolarization. this results in digoxin's characteristic effect on the stsegment of the ekg increases the slope of phase repolarization. this leads to increased automaticity and ectopic beats shortens av conduction time. shortens the refractory period in the atria and the ventricles increases the pr interval due to delayed av-conduction characteristic scaphoid st segment depression due to decreased slope of phase depolarization flat or inverse t-waves. the diminished amplitude or negative deflection does not correlate with serum levels this leads to a more rapid ventricular repolarization. this is independent of parasympathetic activity afterload: decreased . heart rate: digoxin reduces ventricular response rate in atrial fibrillation or flutter. it sensitizes arterial baroreceptors in the carotid sinus, increases parasympathetic activity by activating the vagal nuclei and the ganglion nodosum with resultant decreases in sa node activity, prolonged effective refractory period and av conduction time, resulting in slowed heart rate cardiac output: increased chapter . surgical anatomy of the heart acc/aha guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery. a report of the american college of cardiology/ american heart association task force on practice guidelines esc guidelines for the diagnosis and treatment of acute and chronic heart failure philadelphia: lippincott williams &wilkins perioperative beta blockade in noncardiac surgery: a systematic review for the acc/aha guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: a report of the american college of cardiology/american heart association task force on practice guidelines accf/aha expert consensus document on pulmonary hypertension a report of the american college of cardiology foundation task force on expert consensus documents and the american heart association and the pulmonary hypertension association withholding versus continuing angiotensinconverting enzyme inhibitors or angiotensin ii receptor blockers before noncardiac surgery: an analysis of the vascular events in noncardiac surgery patients cohort evaluation prospective cohort clinical anesthesia key: cord- - yy y n authors: freye, enno; levy, joseph victor title: mechanism of action of opioids and clinical effects date: journal: opioids in medicine doi: . / - - - - _ sha: doc_id: cord_uid: yy y n nan the opium poppy probably reached china about the seventh century a.d. through the efforts of arab traders who advocated its use for medicinal purposes. in chinese literature, however, there are earlier references to its use. the noted chinese surgeon hua to of the three kingdoms ( - a.d.) used opium preparations and cannabis indica for his patients to swallow before undergoing major surgery. the opium poppy, papaver somniferum, is an annual plant, i.e., the plant matures one time, and does not regenerate itself. new seed must be planted each season. from a small seed, it grows, flowers, and bears fruit (a pod) only once. the entire growth cycle for most varieties of this plant takes about days. the tiny seeds (like the seeds on a poppy seed roll) germinate quickly in warm air and sufficient soil moisture. in less than weeks, the young plant emerges from the soil, grows a set of four leaves, and resembles a small cabbage in appearance. the lobed, dentate (jagged-edged) leaves are bluish-green with a dull gray or blue tint. the major legal opium production areas in the world today are in governmentregulated opium farms in india, turkey, and tasmania (australia). the major illegal growing areas are in southwest asia (afghanistan, pakistan, and iran) and in the highlands of mainland southeast asia (burma, laos, vietnam, and thailand)popularly known as the golden triangle ( figure ii- ) . opium poppy is also grown in colombia, mexico, and lebanon. opium poppies containing small amounts of opium alkaloids were, at one time, widely grown as an ornamental plant and for seeds in the united states. the opium poppy control act of declared the possession of this plant illegal. from the cut capsule latex is exuded, which is collected and further processed in order to gain the different ingredients ( figure ii- ) . within the secreted latex collectors will find the major constituents of opium poppy, which are as follows: . morphine ( %- %), the most important alkaloid, which was discovered by the pharmacist sertürner in a small town of einbeck, located in lower saxonia in germany in . he decided to name the extract from the opium poppy morphine ( figure . thebaine ( . %- %) a precursor of many of semi-synthetic opioid agonists (i.e. etorphine, oxymorphone) and antagonists (naloxone, naltrexone, diprenorphine, cyprenorphine), mixed agonist/antagonists (nalbuphine) as well as the partial agonist buprenorphine ( figure c ). . benzylisoquinolines are a group of agents, which do not interact with the opioid receptor. the most important one is papaverine ( . %- %) a phosphodiestrase inhibitor, which relaxes the smooth muscle, and noscapine ( %- %), which is used as a cough suppressant ( figure d) . raw or cooked opium contains more than different alkaloids, including morphine, codeine, and thebaine ( figure ii- ) . in mainland southeast asia, the morphine alkaloid alone accounts for approximately % of the total weight of opium. heroin manufacturers must first extract the morphine from the opium, before converting the morphine to heroin. the extraction is a simple process, requiring only a few chemicals and a supply of water. morphine sometimes is extracted from opium in small clandestine laboratories, which are typically set up near the opium poppy fields. since the morphine base is about one-tenth the weight and volume of raw opium, it is desirable to reduce the opium to morphine before transporting the product from the field to a heroin laboratory. the following is a step-by-step description of morphine extraction in a typical mainland southeast asian laboratory. an empty -gallon oil drum is placed on bricks about a foot above the ground and a fire is built under the drum. thirty gallons of water are added to the drum and brought to a boil. ten to fifteen kilograms of raw opium are added to the boiling water. with stirring, the raw opium eventually dissolves in the boiling water, while soil, leaves, twigs, and other non-soluble materials float in the solution. most of these materials are scooped out of the clear, dark brown liquid opium solution. slaked lime (calcium hydroxide) or, more often, a readily available chemical fertilizer with a high content of lime, is added to the solution. lime will convert the water-insoluble morphine alkaloid into water-soluble calcium morphenate. (other opium alkaloids do not react with lime to form water-soluble calcium salts, as does morphine.) codeine is an opium alkaloid that is slightly water-soluble and some codeine will be carried over with the calcium morphenate in the liquid. otherwise, for the most part, the other alkaloids will become a part of the sludge. as the solution cools, the morphine solution is scooped from the drum and poured through a filter. cloth rice sacks are often used as filters and can then be squeezed in a press to remove most of the solution from the wet sacks. liquid saponated cresol (lysol) is commonly added to the solution to facilitate filtering. the morphine-rich solution is then poured into large cooking pots and reheated but, this time, not boiled. ammonium chloride (a powder) is added to the heated calcium morphenate solution to adjust the alkalinity to a ph of to , and the solution is then allowed to cool. within or h, morphine base precipitates (crashes) out of the solution and settles to the bottom of the cooking pot. the solution is then poured off through cloth filters. any solid morphine base chunks in the solution will remain on the cloth. the morphine base is removed from both the cooking pot and from the filter cloths, wrapped and squeezed in cloth, and then dried in the sun. when dry, the crude morphine base is a coffee-colored coarse powder. this form of morphine is commonly known by the chinese term pi-tzu in mainland southeast asia. if morphine base is to be stored or transported to another location, it may be pressed into blocks. crude morphine base is generally %- % morphine, and is an intermediate product in the heroin process. addicts do generally not use this morphine base. this crude morphine base may be further purified (and changed to morphine hydrochloride) by dissolution in hot water and hydrochloric acid, then adding activated charcoal, reheating, and filtering. the solution is filtered several times before being allowed to cool. as the solution cools, morphine hydrochloride precipitates out of the solution and settles to the bottom. the precipitate is trapped (or captured) by filtration. if the morphine hydrochloride is to be stored or transported to another location, it may be pressed into bricks. morphine hydrochloride (often tainted with codeine hydrochloride) is usually pressed into brick-sized blocks in a press and wrapped in paper or cloth. the most common block size is in. by in. by in., and weighs about lb ( . kg). it takes a full day to extract morphine from opium. as described in the preceding paragraphs, the chemicals used to isolate morphine from opium (known as extraction) include calcium hydroxide (slaked lime) and ammonium chloride. the precursor chemical normally used in the conversion of morphine to heroin (known as acetylation) is acetic anhydride. chemical reagents used in the conversion process include sodium carbonate and activated charcoal. chemical solvents needed are chloroform, ethyl alcohol (ethanol), and ethyl ether. other chemicals may be substituted for these preferred chemicals, but most or all of these preferred chemicals are readily available from smugglers and suppliers. laboratory equipment includes large chinese cooking woks, measuring cups, funnels, filter paper, litmus paper, and enamel (or stainless steel) pots. only the most sophisticated heroin laboratories use glass flasks, propane gas ovens, vacuum pumps, autoclaves, electric blenders, venting hoods, centrifuges, reflux condensers, electric drying ovens, and elaborate exhaust systems. it is common to find portable, gasoline-powered generators at clandestine heroin conversion laboratories. generators are used to power various electrical devices. heroin synthesis from morphine (either morphine base or morphine hydrochloride) is a two-step process that requires between and h to complete . heroin base is the intermediate product. typically, morphine hydrochloride bricks are pulverized and the dried powder is then placed in an enamel pot. acetic anhydride is added, which then reacts with the morphine to form heroin acetate. (this acetylation process will work either with morphine hydrochloride or morphine base.) the pot lid is tied or clamped on, using a damp towel for a gasket. the pot is carefully heated for about h, below boiling, at a constant temperature of c ( f). it is never allowed to boil or to become so hot as to vent fumes into the room. tilting and rotation agitate the mixture until all of the morphine has dissolved. when cooking is completed, the pot is cooled and opened. during this step, morphine and the anhydride become chemically bonded, creating an impure form of diacetylmorphine (heroin). water is added to the thick, soupy mixture and the mixture is stirred as the heroin dissolves in the solution. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates heroin base, which is then filtered and dried by heating in a steam bath. for each kilogram of morphine, g- g of crude heroin base is formed, depending on the quality of morphine. the tan-colored heroin base (about % pure heroin) may be dried, packed, and transported to a heroin-refining laboratory, or it may be purified further before conversion to heroin hydrochloride (a water-soluble salt form of heroin) at the same site. mainland southeast asian heroin base is an intermediate product that can be further converted to either smoking heroin (heroin no. ) or injectable heroin (heroin no. ). to make heroin no. , the crude base is mixed with hydrochloric acid, resulting in heroin hydrochloride (hcl). adulterants, including caffeine, are added after this conversion. for each kilogram of crude heroin base, about kg of caffeine is used. various flavorings such as quinine hydrochloride or strychnine hydrochloride are sometimes added to heroin no. . next, the wet paste mix is stirred to dryness over a steam bath. the resulting dry heroin no. will be in the form of coarse lumps. the lumps are crushed and passed through a mesh sieve, and the grains (pieces) are then packaged for sale. the entire process takes about h and requires only minimal skill. while extra attention to stirring is required to assure dryness, one person can prepare kg of heroin no. during this time. the reaction of morphine with acetic anhydride produces heroin acetate. to the heroin acetate mixture in the pot, water is added and mixed by stirring. a small amount of chloroform is added. the mixture is stirred and then allowed to stand for min. doing so dissolves highly colored impurities and a red, greasy liquid is formed at the bottom of the container. the water layer is carefully poured off and saved in a clean pot, leaving the red grease in the pot. in a clean pot, activated charcoal is stirred into the aqueous solution and is filtered to remove solid impurities. the decolorizing effects of the charcoal, combined with the chloroform treatment, will leave a light yellow solution. the use of charcoal is repeated one or more times, until the solution is colorless. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates the heroin base, which is then filtered and dried by heating on a steam bath. the heroin base is heated until dried. the powder should be very white at this stage. if not white, the base is redissolved in diluted acid, treated repeatedly with activated charcoal, re-precipitated, and dried. the ultimate purity and color of the resulting heroin hcl will depend largely on the quality of the heroin base. the heroin base is then dissolved in ethyl ether. conversion to the hydrochloride salt is achieved by adding hydrochloric acid in ethanol to the heroin mixture. the heroin then precipitates. the process of extracting morphine from opium involves dissolving opium in boiling water, adding lime (calcium oxide), or slaked lime (calcium hydroxide), or limestone (calcium carbonate) to precipitate non-morphine alkaloids, and then pouring off the morphine in solution. ammonium chloride is then added to the solution to precipitate morphine from the solution. the chemicals used to process opium to morphine have a number of legitimate purposes and are widely available on the open market. an empty oil drum, some cooking pots, and filter cloths or filter paper are needed. in the united states, opium preparations became widely available in the nineteenth century and morphine was used extensively as a painkiller for wounded soldiers during the civil war. the inevitable result was opium addiction, contemporarily called the army disease or soldier's disease. these opium and morphine abuse problems prompted a scientific search for potent, but nonaddictive, painkillers. in the s, chemists developed an opium-based and supposedly nonaddictive substitute for morphine. the bayer pharmaceutical company of germany was the first to produce the new drug in large quantities under the brand name heroin. this product was obtained by acetylation of morphine. soon thereafter studies showed heroin to have narcotic and addictive properties far exceeding those of morphine. although heroin has been used in the united kingdom in the treatment of the terminally ill, its medical value is a subject of intense controversy. among the commonly known classes of opioids/opiates being used in practice are morphine, codeine, heroin, and the antagonist naloxone ( figure ii- ). morphine by itself is still made from opium and although there is a major first-pass effect (i.e. degradation by liver enzymes), oral administration is still possible, but requires substantial dosage increase. codeine, which is also taken orally, has a strong ability to inhibit coughing, but it induces less analgesia. among the phenylpiperidines a number of synthetic compounds have entered the market. the most known is meperidine/pethidine (demerol®), which is very similar to morphine, but is more efficacious when given orally for the control of pain. another derivative is loperamide (imodium®), an agent being used as a common antidiarrheal, which does not enter the brain, as it is incapable of crossing the blood-brain barrier. hence it is not abused and therefore is sold as a doc (drug over the counter). contrary, fentanyl (sublimaze®) is an opioid, which is at least times as potent as morphine. this agent is used with nitrous oxide or droperidol (a neuroleptic) in intravenous anesthesia (neuroleptanesthesia), but it is also a used in a transdermal patch for the control of chronic pain. another known opioid is methadone, which has a good oral efficacy, a much longer half-life than morphine ( - h) , and in regard to its effect much like morphine. it is used for treatment of heroin addiction and for the control of chronic pain. a methadone congener, which is being used solely in the methadone substitution programs is laam ( -levoacetylmethadol), only needs to be taken once every h. the opioid propoxyphene (darvon®) has figure ii- . molecular structure of different opioid ligands with agonistic or antagonistic properties the lowest analgesic potency ( . times morphine). it is almost always given together with aspirin for the control of mild to moderate pain. it is very popular clinically due to misplaced concerns about the abuse potential of codeine. it is interesting to note that all commonly used opioids have a similar structure in regard to their terminal morphine ring and the distance between the ring and the n-substitution. such common traits suggests that opioids must have a common structure in order to interact with a specific receptor site ( figure ii- ) . thus, central analgesics mediate their action by means of an interaction with specific opioid receptor sites located within specific areas of the central nervous system, which are engaged in the transmission of nociceptive afferences and the identification of pain. there, opioids act as agonists at highly definite receptor sites, and there is general agreement on the existence of at least three types of opioid receptor sites (table ii- ). . the morphine mu receptor ( ) at which the prototype morphine binds, . the kappa receptor ( ) at which the prototype agonist is ketocyclazocine, and . the delta receptor ( ) at which the prototype endogenous opioid ligand enkephalin binds. opioid receptors are distributed widely in brain and found in spinal cord and peripheral sensory and autonomic nerves. there are the three well-characterized members of the opioid receptor family, designated by the greek symbols , and . the more recently discovered orl receptor is placed with this family due to its high degree of structural homology. these receptors were renamed op , op , op and op , respectively, by an international union of pharmacology (iuphar) nomenclature committee in [ ] . this nomenclature has proved unpopular. the nomenclature (x-opioid peptide receptor) has been proposed giving , mu or mop; , delta or dop; , kappa or kop and orl or nop receptors. in order to keep matters straightforward the original nomenclature is used in the following chapters. the products of endogenous opioid peptide genes activate opioid receptors physiologically: proenkephalin (giving methionine-and leucine-enkephalin; metenk and leu-enk, respectively; figure ii- ), prodynorphin (dynorphins a and b and -neo-endorphin) pro-opiomelanocortin ( -endorphin) and pronociceptin (nociceptin, also known as orphanin fq). met-enk and leu-enk have highest affinity for -receptors, less affinity for , and very low affinity for -receptors; the dynorphins have preferential affinity for -receptors, but bind to the and types with high affinity; -endorphin binds with high affinity to and receptors, but has little affinity for receptors. all the peptides are full agonists at their cognate receptors. endomorphin- and - , derived from an unknown precursor, are endogenous peptides with high selectivity for -receptors. these peptides are unusual in that they are partial agonists. none of the proenkephalin, prodynorphin or pro-opiomelanocortin peptide products or the endomorphins displays affinity for the orl receptor. similarly, the orl receptor agonist nociceptin has no appreciable affinity for , or receptors. the four receptor types have been cloned and shown to be -transmembrane receptors activating g proteins of the pertussis-toxin insensitive g i/o family, but including g z. evidence for subtypes of , and opioid receptors exists, but the molecular basis for the observed functional and pharmacological differences is unclear. putative and receptors are differentiated by several agonist and antagonist ligands. however, there is only one receptor gene, the protein product of which has properties of the putative receptor. the distinction between the proposed and receptors is based largely on the apparent preferential blockade of the type by the antagonist, naloxonazine [ ] . there is only one cloned receptor gene, corresponding to the putative receptor, but several forms of the -receptor mrna arising from alternative splicing have been reported. the receptors these encode differ at the end of the c-terminal tail and show subtle differences in the binding profile of opioid ligands; a role for the variants is not known. the cloned -receptor, with high affinity for u is the subtype. the proposed and subtypes are poorly defined in both molecular and pharmacological terms (table ii- ) . a recent explanation for subtypes has evolved with the identification of opioid receptor heterodimers or hetero-oligomers that appear to have properties different from the monomeric receptors. an interesting addition to ligands that bind to the receptor is the hallucinogen salvinorin-a. this is a highly efficacious and potent agonist, but is most unusual in that it has no nitrogen atom. endogenous opioid systems have a functional role in modulating pain perception; opioid agonists are therefore potent analgesics. opioid receptors are also present in hypothalamus (figure ii- ), where they influence temperature regulation and control of hormonal secretion. in the forebrain, endogenous opioids are involved in behavioral reinforcement and appear to play a role in anxiety and in the expression of emotions. in addition, opioids influence gastrointestinal and autonomic nervous system function. originally, a fifth binding site, the sigma receptor, was included in this group. however, actions mediated through this receptor are not reversed by naloxone so it is not a true opioid receptor. the -receptors have been further sub-classified into two distinct subtypes ( and ), as have the -receptors. kappa receptors have been divided into , , and sub-types. recently, several of these receptors have been cloned successfully. in animal models, some laboratories have cloned up to -receptor subtypes [ ] . however, the functional significance of these "spliced variants" remains unclear at present. originally suggested by martin and coworkers [ ] , all three opioid receptor types mediate different opiate effects as they figure ii- . difference in topographic density of the three opioid receptor sites within the central nervous system. adapted from [ ] normally serve endogenous opiates (the endorphins, dynorphins, and enkephalins; table ii- ): . activation of the mu receptors ( ) results in analgesia, euphoria, respiratory depression, nausea, gi slowdown, and miosis. receptors of this type are mostly located in periaquaductal gray (pag), spinal trigeminal nucleus, caudate and geniculate nuclei, thalamus, and spinal cord. . binding at the kappa receptors ( ) induces modest analgesia, dysphoria, feelings of depersonalization and disorientation, miosis, and mild respiratory depression. these receptors are mainly found in basal ganglia, nucleus accumbens, ventral tegmentum, cortex, hypothalamus, periaqueductal grey, the spinal cord, and in the periphery. . occupation of the delta receptors ( ) results in anxiolysis and central pain relief, although its overall significance is not all that well understood. they are mainly found in the nucleus accumbens and the limbic system (table ii- ) . molecular biology techniques have enabled the primary amino acid sequence of the human -, -, and -opioid receptors to be determined. the pharmacological and functional properties of the cloned receptors, the development of "knockout" animals, which are deficient in a receptor or part of a receptor, and the manipulation and substitution of various amino acids in critical domains of the various opioid receptors have provided new insights in opioid action. in this regard, the three opioid receptor genes, encoding mu (mor), delta (dor), and kappa (kor) have been cloned. the binding affinities of a range of opioids to the -, -, and -opioid receptors and also to the cloned orphinan receptor have been examined in animals. the animal data indicate that while the commonly prescribed opioids (agonists and antagonists) bind preferentially to the -receptor, they also interact with all three receptor types. morphine and normorphine (a minor metabolite of morphine) show the greatest relative preference for the -receptor. methadone (which also has some nmda-receptor blocking activity) shows significant binding to -receptors, while buprenorphine, and to a lesser extent naloxone, avidly binds to all three receptor types. there is evidence (albeit inconsistent) that the d-enantiomer of methadone blocks the nmda receptor [ ] . the binding affinity of buprenorphine to the receptor is much greater than that of naloxone, which explains why the latter only partially reverses buprenorphine overdose. animal data also indicate that codeine and diamorphine have very poor binding to opioid receptors, which reinforces the possibility that both are prodrugs where the pharmacologically active species are morphine [ ] and -monoacetyl morphine, respectively [ ] . oxycodone may also act through an active metabolite, though there are some data, which suggest that this is not the case [ ] . pethidine is considered to be a potent -receptor agonist, but it does bind weakly to all three opioid receptors (table ii- ) . ketobemidone has a lower affinity for the -receptor than does morphine, but it shows greater discrimination for this receptor compared to -receptors. the binding of both of these opioids to the -receptor is similar [ ] . this difference in opioid action is also mirrored in the difference in affinity of various narcotic ligands interacting with the three relevant opioid receptor sites (table ii- ) . it should be noted that some of those ligands, either pure antagonists, mixed agonist/antagonists or partial agonists, are characterized by displacement potency at a specific receptor site. from the above binding and displacement values it can be seen, that opioid practically bind to all three receptor sites, however with different affinity. the preference in binding to one receptor site manifests itself in the visible clinical effect, which may either be of agonistic or of antagonistic nature. the binding of morphine, methadone, buprenorphine, and naloxone to the cloned human -receptor shows excellent congruence with the animal data [ ] . fentanyl shows a similar binding affinity, while codeine demonstrates greater binding affinity to the cloned human receptor (table ii- ; figure ii - ). thus, for these commonly administered opioids, there is no great variability in their affinity for the humanreceptor. the clinical relevance of these data is that different opioids act in different ways. from anecdotal clinical experience there is considerable interindividual adapted from [ , , , , ] variability in response to each opioid and this reinforces the need to assess an individual's response to opioid analgesia carefully. it also is premature to extrapolate from laboratory data, which in many instances have not yet been replicated, to the clinic. however, data increasingly inform the clinical use of these drugs and will be particularly relevant to new approaches to their use such as "opioid switching". figure ii- shows the putative analgesic effect mediated by the main -opioid receptor depicting that higher affinity also correlates closely with analgesic potency. but aside from -receptor interaction, analgesia can also be mediated through a -receptor and a -receptor site. the classification of different opioid receptor types is based on the original description by martin and coworkers from [ ] . the effects presumed to be mediated at -receptors have been defined as a result of both human and animal studies, while the effects mediated at -receptors derive predominantly from animal models. receptors mediate analgesia that persists in animals made tolerant to -agonists. the -agonists produce less respiratory depression and miosis than -agonists. it is assumed that opioid receptors mediate opioid receptors are found in several areas of the brain, particularly in the periaqueductal grey matter, and throughout the spinal cord ( figure ii- ). supraspinal systems have been described for -, -, and -receptors, whereas -and -receptors modulate pain at the spinal level [ , , ] . the different distribution of the various opioid subsites suggests different mechanisms of action in the mediation of analgesia. thus, -selective opioids like morphine, fentanyl and sufentanil, due to the high density of binding sites, mediate their main action within the brain stem and the midbrain. due to their close vicinity to respiratory and cardiovascular regulating centers in the brain stem, selective -opioids accordingly induce a marked depression of respiration and blood pressure. on the other hand, due to the main distribution of the -receptors within the cortex (lamina v, vi) [ ] it is conceivable that these ligands induce a lesser respiratory and cardiovascular depressive effect. as a consequence and contrary to -ligands, -ligands induce a marked sedative appearance. in addition, there is a lesser addiction liability with -ligands, which is easily derived from the fact that the relevant areas in the limbic system show a low concentration of -binding sites. also, the lesser analgesic potency of -ligands is enlightened by the fact that most of the -selective receptors can be found in the deep lamina vi of the cortex. since their dendrites retrograde descend to the thalamus, all ascending nociceptive input is modified, resulting in a depression of nociceptive afferences and a reduction in arousal. certain dendrites of petrosal cells of the cortex also descend down to the brain stem, whereby the activating, ascending reticular system (ars) is affected resulting in a reduction of vigilance [ ] . in summary, due to the dissimilarity of distribution of the three opioid receptor subtypes with the spinal cord and the supraspinal areas of the cns, a functional differentiation can be expected. this effect is reflected in difference of binding affinities with the brain where % of all receptor sites are referred to the -, % to the -and % to the -opioid receptor [ , ] . the present understanding of the effect profiles of opioid receptors, however, remains incomplete, as new advances make it clear that their disposition and structure are extremely complex. opioids inhibit pain signals by different mode of actions: • inhibition of ca ++ -influx into the buttons of the presynaptic cell (e.g. the one releasing substance p; figure ii - ) . this is because ca ++ -influx is necessary for neurotransmitter release, whereby opioids reduce or prevent substance p from being released. • acting as an inhibitory neurotransmitter, since the opioid hyperpolarizes the postsynaptic cell by enhancing k + -flow out of the neuron, which makes it more difficult for all incoming nociceptive afferences to stimulate the next neuron, and thus more difficult to send painful information. • moderation of central perception of painful information in the limbic system so as to make it less aversive when it is perceived. several facts have led to the assumption that opioids interact with specific binding sites in the cns. a slight molecular substitution at the side chain of the morphine molecule structure results in considerable changes of potency (table ii- ) . whereas pethidine (meperidine, usp), a piperidine derivative, may be considered a weak analgesic, fentanyl, a piperidine derivative, is about - times more potent than morphine. the opioid antagonists levallorphan and naloxone are noted for a low and a analgesic effect, respectively. furthermore, only the levorotator (levo-) isomers of opioids, which appear in their natural form (i.e. compounds which, when in solution, rotate plane-polarised light to the left) are pharmacologically active (e.g. levorphanol). their dextrorotatory (dextro-) isomers, which can be synthesized in the laboratory (e.g. dextrophane), shows a negligible pharmacological effect. both substances are structurally the mirror image of each other ( figure ii- ) . in this context it is important to note that only the levo-stereoisomer of the racemic mixture is the pharmacologically active ingredient. this observation supports the part ii figure ii- . mechanism of action of opioids at the central nervous system. by binding at the same receptor site as the endogenous opioids (i.e. enkephalins, endorphins), the release of excitatory neurotransmitters such as acetylcholine and glutamate is decreased thereby reducing the receiving cells excitatory input. the degree of opiate receptor binding is proportionally to the net release of excitatory transmitters and the reduction of depolarization produced by the arriving nociceptive nerve impulse. this enkephalin inhibitory system normally modulates the activity of the ascending pain pathways within the spinal cord and the brain. opioid agents act by binding to unoccupied enkephalin receptors, thereby potentiating the analgesic effects of the system notion that stereroselectivity of an opioid analgesic is a prerequisite in order to bind to the opiate receptor site, thus inducing analgesia. based on their interactions with the various receptor subtypes, opioid compounds can be divided into agonist, agonist/antagonist, and antagonist classes (table ii- ) . by definition an agonist is a drug that has affinity for and binds to cell receptors to induce changes in the cell that stimulate physiological activity. the agonist opioid drugs have no clinically relevant ceiling effect to analgesia. as the dose is raised, analgesic effects increase in a log linear function, until either analgesia is achieved or dose-limiting adverse effects supervene. efficacy is defined by the generally opioids exist in optical isomers, which are a mirror image in the molecular form. only the levorotatory (levo)-isomer, which in solution rotates plane-polarized light to the left, produces the characteristic analgesic effect of an agent. the dextrorotatory isomer is totally inactive. this sterospecificity of opiate action supports the concept of selective receptor binding to a site, which is able to distinguish in "handedness or goodness of fit" of an opioid molecule maximal response induced by administration of the active agent. in practice, this is determined by the degree of analgesia produced following dose escalation through a range limited by the development of adverse effects. potency, in contrast, reflects the dose-response relationship. potency is influenced by pharmacokinetic factors (i.e. how much of the drug enters the body systemic circulation and then reaches the receptors) and by affinity to drug receptors. the concepts of efficacy and potency are illustrated in the following figure, which shows the dose-response curves for two drugs with a full agonistic and a partial agonistic action. if the logarithm of dose is plotted against response an agonist will produce an s-shaped or sigmoid curve. the efficacy of the two drugs, defined by maximum response is the same. the full agonist produces the same response as a partial agonist but at a lower dose, and therefore is described as more potent ( figure ii- ). an antagonist by definition is an agent that has no intrinsic pharmacological action but can interfere with the action of an agonist. competitive antagonists bind to the same receptor and compete for receptor sites, whereas non-competitive antagonists block the effects of the agonist in some other way. contrary the mixed agonist/antagonists analgesics can, in turn, be subdivided into the mixed agonist/antagonists and the partial agonists, a distinction also based on specific patterns of drug-receptor interaction. both the partial agonist and the agonist/antagonist drugs have a ceiling effect for analgesia, and although they produce analgesia in the opioid-naive patient, in theory they can precipitate withdrawal in patients who are physically dependent on morphine-like drugs. for these reasons, they have been considered generally to have a limited role in the management of patients with cancer pain. the mixed agonist/antagonist drugs produce agonist effects at one receptor and antagonist effect at another. pentazocine is the prototype agonist/antagonist: it has agonist effects at the -receptors and weak to medium antagonistic action at the figure ii- . typical dose-response curves of a full agonist, a partial agonist and an antagonist on opioid-related effects -receptor thus in addition to analgesia, pentazocine may produce -mediated psychotomimetic effects not seen with full or partial agonists. when a mixed agonist/antagonist is administered together with an agonist, the antagonist effect at the -receptor can generate an acute withdrawal syndrome. a partial agonist has low intrinsic activity (efficacy) so that its dose-response curve exhibits a ceiling effect at less than the maximum effect produced by a full agonist ( figure ii- ) . buprenorphine is the main example of a partial agonist opioid. increasing the dose of such a drug above its ceiling does not result in any further increase in response. this phenomenon is illustrated in the figure in which buprenorphine is the partial agonist. the full agonist is more potent than the partial agonist (in the lower part of the curve it will produce the same response at a lower dose), but is less effective than both coadministered ligands because of its ceiling effect. when a partial agonist is administered together with an agonist, displacement of the agonist can cause a net reduction in pharmacological action, which may be sufficient to generate an acute withdrawal syndrome. while this is a theoretical possibility with morphine and buprenorphine, no such interaction has been reported clinically. similarly, it has been suggested that the effects of morphine may be blocked in a patient switched from buprenorphine, because of the prolonged action of buprenorphine and the assumption that it will "antagonize" the effect of morphine. this has been one of the reasons, why buprenorphine has not been in cancer pain management. however, the recent development of a transdermal formulation of buprenorphine may encourage its use in chronic cancer pain (and chronic noncancer pain). an analgesic ceiling with buprenorphine is only reached at doses of - mg or more in h [ , ] . when used in usual recommended doses (e.g., two patches of g/h of transdermal buprenorphine, equivalent to - mg per h) buprenorphine can be considered a full -agonist since at these doses its effect will lie on the linear part of the dose-response curve [ ] . relative potency is the ratio of the doses of two analgesics required to produce the same analgesic effect. by convention the relative potency of each of the commonly used opioids is based upon a comparison with mg of parenteral morphine. data from single-and repeated-dose studies in patients with acute or chronic pain have been used to develop an equianalgesic dose table (table ii- ) that provides guidelines for dose selection when the drug or route of administration is changed. the information contained in the equianalgesic dose table does not represent standard doses, nor is it intended as an absolute guideline for dose selection. many variables may influence the appropriate dose for an individual patient, including intensity of pain, prior opioid exposure in terms of drug, duration, and dose (and the degree of cross-tolerance that this confers), age, route of administration, level of consciousness, metabolic abnormalities (see below), and genetic polymorphism in the expression of relevant enzymes or receptors. in addition, a substitution at the side chain, for example the substitution of a methyl-group by an allyl-group or the substitution by a cyclopropyl-group results in the new opioid antagonist naloxone, diprenorphine and naltrexone respectably, or mixed agonists/antagonists (nalorphine, levallorphane), which have the capability of antagonizing the effect of the parent compound ( figure ii- ) . similarly, when the n-methyl group of the highly potent opioid oxymorphone or the pure agonist etorphine ( times of morphine) is replaced by a cyclopropylmethyl group, the highly potent antagonists naltrexone and diprenorphine are derived. these compounds are . times as potent as naloxone and while the former is used as an oral preparation in the rehabilitation of the earlier opiate addict, the latter is used in veterinary medicine for the reversal of immobilization of wild animals. in addition, diprenorphine is also the original substance of buprenorphine where additional three methyl groups are incorporated in the molecule ( figure ii- ). such minor changes in the molecular structure and their resultant major pharmacological effect suggest, that similar to hormones and catecholamines, opioids bind with specific receptor sites which results in the characteristic effects such as analgesia. various research groups corroborated this hypothesis almost simultaneously. pert and snyder [ ] , terenius [ ] and kosterlitz [ ] were the first research group to identify selective binding sites in the cns using radioactive labeled opioids. these so-called opiate binding sites were found mainly in neuronal structures and nervous pathways involved in the transmission of nociceptive signals such as the first relay station of pain transmission, the substantia gelatinosa of the spinal column. in the posterior horn the impulse is passed over to the second neuron while, simultaneously, descending nerve fibers from the reticular system (the cortico-and reticulo-spinal tract) induce either a facilitation or an attenuation of pain transmission, which results in a modulation of pain impulses at the spinal level ( figure ii- ). the course of pain transmission is to the contralateral side of the spinal cord where impulses have already undergone a distinct separation. it is the paleospinothalamic pathway, consisting of nonmyelinated c-fibers, which mediate the excruciating, dull pain component, which is difficult to localize as it ends in the nonspecific nuclei of the medial thalamus [ ] . en route, the paleospinothalamic tract sends off afferent fibers to the midbrain area, such as the periaqueductal grey matter and the reticular formation [ ] . the pathway ends in intralaminar nuclei of the thalamus and the nucleus limitans, a patch of pigmented nerve cells border the mesencephalon ( figure ii- ) . from there, subcortical pain pathways link with the pallidum, the alleged psychomotoric center that sends fibers to all areas of the brain hemisphere. the neospinothalamic pathway, in contrast, consists of myelinated a -fibres, which transfer impulses to the nucleus ventrocaudalis-parvocellularis (n.v-c parvocellularis). from there pain sensations are projected to the postcentral gyrus, which enables the patient to localize the source of pain ( figure ii- ) . both, the central grey matter and the pallidum are characterized by a dense accumulation of opiate binding sites [ , ] . it is worth noting that nervous pathways transmitting the dull, chronic and less pinpointed pain components are more affected by opioids, while , is an important relay station in the mediation of nociceptive afferents to higher pain modulating and discriminating centers of the cns, which is necessary for the nonspecific feeling of pain and is closely coupled with emotions the neospinothalamic pathway conveys the sharp and well localized pain components which accompany any injury and are always the first to be perceived. the indefinable, dull, emotional component is perceived later, giving pain its negative character. this separation in pain pathways is of special importance. impulses from the fast pathway usually antagonize the mediation of slow afferent impulses on all levels in the cns: substantia gelatinosa and reticular formation, as well as the specific and the nonspecific projecting nuclei of the thalamus [ ] . opioid binding sites, as they are visualized with receptor-binding techniques, strikingly map the paleospinothalamic pain pathway ( figure ii- ) . furthermore, there is a high density of opioid binding sites in various other parts of the brain [ , , , ]: . the corpus striatum, being part of the limbic and the extrapyramidal motor system, is responsible for triggering opioid-induced muscular rigidity. it is not only the regulatory center for locomotion but it is also the center for the regulation of attention and perception. . the nucleus solitary tract in the brain stern, which is the origin of the noradrenergic dorsal pathway bundle, which is in command of vigilance and the cough reflex. . the nuleus amygdala, being part of the limbic system, is in charge of the mediation of euphoria (or "kick") when opioids are used for other purposes than pain. . the locus coeruleus being the origin of the neurosympathetic system in the brain stem, regulates peripheral vasodilatation. . lastly, a dense accumulation of opioid binding sites is found in the substantia gelatinosa at the dorsal horn of the spinal cord. -current thinking is that effective opioid analgesics work through stimulating mu ( ) receptors, which also produce euphoria. -euphoria is mediated by the actions of opiates at a cluster of brain areas that include the nucleus accumbens and ventral segmental area. dopamine influx seems to cause subjective pleasure, or euphoria. -opioids may have a "disinhibiting" (inhibition of inhibitory neurons) effect that allows greater dopamine influx. because the main property of opioids is the blockade of nociceptive transmission in the mesencephalon (i.e. the nucleus limitans and the limbic system) the following effects can be observed: . no pain (analgesia), since any sensation is not identified as painful. . a lack of the negative emotional component of pain. on the contrary, euphoria may result and pain is no longer experienced as an emotional distress, even though pain impulses are transmitted via the ventrocaudal-parvocellular nucleus to the postcentral cortex. . during the opioid-induced pain-free state, the site of pain, however, still can be localized. as a consequence pain has lost its negative character and is no longer experienced or perceived as uncomfortable and distressing. in contrast to the analgesics that have a peripheral site of action (e.g. acetylsalicylic acid; asa), opioids act at the relay station of nociceptive-propagating pathways at the synapse of nerve conduction. within the nerve, pain impulses are transmitted as a change in electric conduction. and in order to guarantee maintenance of the nociceptive impulse, the excitatory impulse releases a neurotransmitter at the terminal nerve. due to its chemical configuration, the transmitter fits exactly into a binding site at the opposite nerve ending resulting in an increase of excitability and a change in the electrical nerve conduction. opioids have the property of binding to specific receptor sites at pre-and post terminal nerve endings resulting in an inhibition of a release of the excitatory neurotransmitter. the continuity of the impulse is interrupted, the nociceptive signal is no longer transmitted and thus can no longer be perceived as such ( figure ii- ). due to the difference in stereoconfiguration, opioids differ in their affinity (i.e. goodness of fit) at these binding sites ( figure ii- ). this explains why different opioids are characterized by a large variety in potency. in addition, opioids also differ in their intrinsic activity (i.e. the degree of conformational change of the receptor site) resulting in different intracellular effects. taken together affinity and intrinsic activity results in the efficacy of a drug within the system ( figure ii- ) . thus, binding properties are reflected in varying analgesic potencies. contrary, the intensity of binding with the receptor site (i.e. the intensity with which the opioid adheres to the binding site) is reflected in the duration of effects (table ii- and figure ii- ) [ , , ] . for instance opioid analgesics such as sufentanil or lofentanil have an exceptional goodness of fit to the opioid receptor site, which results in high potency. on the other hand, the low dissociation coefficient from the receptor of buprenorphine or lofentanil is characterized by a long duration of action, while the high association coefficient demonstrates increase of affinity to the binding site. contrary to agonists, antagonists are able to displace an opioid from its receptor binding site and take up his position. displacement is only possible because the antagonist has a greater affinity to the binding site. therefore, affinity of an opioid antagonist is expressed in its antagonistic potency. naloxone or naltrexone have a very high affinity to the receptor and easily displace an opioid whereas levallorphan is five times weaker ( figure ii- ) . in order to induce a similar antagonistic effect, a higher dose of levallorphan is necessary. in order to induce increasing effects with opioids, the goodness of fit not only is a prerequisite. of additional importance is the conformational change the receptor undergoes after binding, which is expressed in the "the intrinsic activity". an opioid must, therefore, not only fit to the receptor; it must also induce a chain reaction in the transmembrane receptor domain resulting in a net effect ( figure ii- ) . the reaction after opioid binding seems to depend on the side chain of the molecule. thus it appears that one portion of the opioid molecule provides the binding to the receptor whereas another portion is responsible for the induction of a conformational change (i.e. intrinsic activity), which will either be of agonistic or antagonistic nature. in a sensitive and specific opiate-receptor assay, the guinea pig ileum with its dense accumulation of receptor binding sites, it was possible to demonstrate receptor affinity and pharmacological efficacy ( figure ii- ) . this assumption is underlined by the effects induced by "pure" opioid antagonists such as naloxone or naltrexone, which also have a good fit with the receptor site, however when given on their own do not induce an analgesic effect. for instance, if naloxone is given by itself, the compound does not induce effects similar to its parent compound oxymorphone ( figure ii- ) . also, in contrast to a potent opioid like fentanyl, the antagonist naloxone has a lower dissociation coefficient resulting in a shorter duration of action, which may result in a reoccurrence of an opioid-like effects such as respiratory depression. however, due to its high source: adapted from [ , , ] part ii figure ii- . difference in affinity and intrinsic activity of various opioids. note, that codeine has a similar intrinsic activity as sufentanil. however, due to the higher affinity of the latter the net analgesic potency is much larger association coefficient (i.e. affinity), it induces a rapid displacement of the agonist and a reversal of all opioid effects. on the other hand mixed agonist/antagonists, such as pentazocine, nalorphine, levallorphan, nalbuphine and butorphanol, demonstrate characteristics, which enable them to displace a pure agonist at the receptor site (antagonistic effect), but at the same time when administered by themselves, they induce opioid related effects such as analgesia and respiratory depression (agonistic effects; table ii- ) . such dual activity is only possible by means of their intrinsic activity at two distinct and different receptor sites: one the antagonistic activity at the -and its agonistic action art the -receptor site. and lastly, partial agonists like meptazinol and buprenorphine induce their analgesic potency via the -opioid receptor. although having a high affinity, their analgesic ceiling effect at the higher dose range is due to a lesser intrinsic activity, resulting in a lesser net analgesic appearance than pure agonists. such difference in the characteristic traits of opioids can be summarized as follows: . the affinity to the receptor (displacement properties or extrinsic activity) . the intensity of binding to the receptor (duration of effect) . the ability to change the conformation of the receptor (intrinsic activity) . the competitive potency (antagonism) . the degree of metabolism (duration of effect) note the relatively high antagonistic potency of buprenorphine, however, is due to its high affinity to the receptor site resulting in the displacement of a ligand at the preoccupied receptor site. similarly like a hormone or other extracellular "first messengers" that bind to its receptor on a cell surface, a signal is transmitted or "transduced" to the cells interior, thus setting a series of events that produce a biological response. such "events" include both chemical reactions and physical reactions like a conformational change in the protein molecules. the biological responses include cell differentiation, altered metabolism and cell growth and division. there are three signaling pathways that share many of the same intracellular events. each pathway is characterized by its receptor and by the cascade of intracellular events that lead to a biological response. each receptor has an extracellular, transmembrane, and intracellular component and the binding of a ligand to the receptor represents the "primary message". the term "secondary messenger" is used for those mediators that diffuse from one part of the intracellular space to its spatially removed target. among these secondary messengers are adenosine- ,- -cyclic phosphate (c-amp). many integral membrane glycoprotein membranes share a seven transmembrane alpha-helix motif ( figure ii- ) . the ß-adrenergic receptor, whose natural ligands are epinephrine and norepinephrine, is an example of such receptors. similarly in the opiate receptor, binding of a ligand presumably initiates a conformational change in the membrane protein that is transmitted to the cell interior. this physical reaction can then facilitate other physical or chemical reactions, which are conveyed to ion channels, resulting in a change of transmembrane ion flow. the transduction of the signals from external messengers, including opiate ligands involves intracellular heterotrimeric g-proteins, which are bound to the inner cell (plasma) membrane, a secondary messenger system, involving cyclic amp, and a target response. as the name implies, these proteins are trimers, consisting of an , , and subunit. they are bound to the inner membrane and the subunit can bind the guanine nucleotides, gtp and gdp. g-proteins are involved in vision, smell, cognition, hormone secretion and muscle contraction in humans, and in mating in yeast. there are more than receptors (not including odor receptors) that utilize g-proteins, and there are at least members of the g-protein family, with each member having its characteristic , , and subunits. while the subunit is different for each g-protein, the ß/ pair can be the same. however, all of the g-proteins share a similar structure. in regard to the opioid receptor, specifically the g-proteins transmit the signal from the intracellular part of the receptor to the effector. adenylyl cyclase (ac), which is an inner membrane-bound enzyme, regulates the production of the secondary messenger, adenylyl cyclase. other effectors that are g-proteindependent include additional enzymes, like cyclic gmp phosphodiesterase, and transmembrane ion channels ( figure ii in its resting conformation, the g-protein consists of a complex of the three subunit chains and a gdp molecule bound to the alpha subunit. the alpha subunit is in close proximity to the intracellular part of the transmembrane receptor and, when a ligand binds to the receptor, the change in its conformation causes it to bind to the g-protein at the alpha subunit. this results in an exchange of bound gdp for gtp, which is more abundant in the cell than gdp. gtp causes a conformational change in the alpha subunit, thus "activating" it so that the alpha subunit dissociates from the -pair. the alpha subunit diffuses along the membrane until it binds to an effector, thereby activating it. the alpha subunit is also a gtpase, so the signal transduction is regulated at this level by hydrolysis of gtp to gdp and inorganic phosphate. such hydrolysis can occur spontaneously or upon interaction with a gtpase activating protein, "gap". the gdp-alpha subunit complex then binds to the ß/ complex to reform the original trimeric protein. since the stimulation of the external receptor can activate a number of g-proteins, signal amplification can occur. while this is a desired response in many instances, control at this level is needed to modulate it. g-proteins, then, are nano-switches when they turn on the effector by binding of the alpha subunit and turning it off when the gtp is hydrolyzed. the duration of production of secondary messenger, like cyclic amp, is determined by the rate of hydrolysis. in this sense, the g-protein acts as a nano-timer. although there is controversy over the role of the ß/ subunits in modulation of signals, it is likely that there are both inhibitory and stimulatory effects. if different receptors act on the same g-protein, or if different g-proteins act on the same effector, the potential exists for a "graded" response to an extracellular signal. if the same receptor acts on many g-proteins, or if one g-protein acts on many effectors, then there may be many simultaneous responses to the primary messenger. following binding the g-proteins activates the membrane-bound effector, adenylyl cyclase (ac). this enzyme catalyzes the synthesis of cyclic amp resulting in the formation of atp, camp and pyrophosphate. because this molecule is freely diffusing through the cytoplasm, it is a "secondary messenger" (figure ii- ). the reverse reaction, the formation of atp from camp and pyrophosphate, is catalyzed by a specific phosphodiesterase. camp is involved in a number of physiologic processes. for the breakdown of glycogen, stimulation of the ß-adrenergic receptor involves activation of adenylyl cyclase and synthesis of cyclic amp. the activity of camp-dependent protein kinase (capk) requires camp in order to phosphorylate ser and thr residues on other cellular proteins. glycogen phosphorylase is activated by capk, making glucose- -phosphate available for glycolysis. adenylyl cyclase activity is regulated at a number of levels, including modulation of gtpase activity of ga, phosphodiesterase activity, and protein phosphatases. inhibitory g proteins, gi, are analogous to the stimulatory g proteins, gs, except for the exchange of gtp by gdp by the -subunit and the subsequent inhibitory action of gia on adenylyl cyclase. rather, it has been determined that ligand induced dimerization is the mechanism through which the receptor ptks are activated. this dimerization brings the tyrosine kinase catalytic domain on each receptor into close enough arrangement so that each kinase can phosphorylate tyr residues in the other's tyrosine kinase domain. such activated catalytic domains can then phosphorylate tyrosines outside of the catalytic domains, which can then modify other intracytoplasmic proteins, either by phosphorylation or by other means. all these changes are reversed with an overexpression of activation when an opioid is antagonized by a specific antagonist such as naloxone with activation of the excitatory nmda-(n-methyl-d-aspartate) receptor, resulting in a rebound with an increase in transmission of stimuli ( figure ii- ) . the next step in the signaling pathway involves activation of an inner membranebound monomeric g protein known as ras, which initiates a series of kinase reactions that ultimately carry the signal to the transcriptional apparatus of the nucleus. ras, being a g protein, is activated when its bound gdp in the resting state is replaced by gtp. it, too, has gtpase activity, but the half-life is too slow to allow for effective regulation of a signal. another gtpase activating protein, gap, increases the rate of gtp hydrolysis by ras. a "kinase cascade" ensues, involving raf (a ser/thr kinase), map kinase (also known as mek, which is both a tyr kinase and ser/thr kinase, and a family of proteins known as mapks or erks. opioids induce a variety of clinical relevant effects, which can be subdivided in being advantageous and/or even detrimental. one of the major consequences following opioid administration is that of analgesia, or antinociception. and while nsaids induce their antiniociceptive effect via cyclooxygenase (cox) inhibition, local anesthetics selectively block ion-channels, thus inhibiting the transmission of nociceptive efferent to the higher pain modulating centers in the cns. contrary, opioids bind to those areas, which not only are involved in transduction but also in the modulation and identification of painful afferences. although the majority of opioids are able to induce a maximal analgesic effect, the dosages necessary to induce such a result differ significantly. for instance, an opioid like sufentanil part ii needs a much lower dosage than the less potent opioid morphine. this is due to the higher affinity and intrinsic activity of sufentanil, suggesting that only a lesser portion of receptors needs to be occupied in order to induce the desired effect. however, a high analgesic potency necessarily does not reflect a better efficacy. this is because in certain painful conditions, some opioids are more efficacious than others. on the other hand, not all painful conditions, as the patient expresses them, can be treated successfully with an opioid. therefore, before starting an opioid therapy it is mandatory to evaluate the kind of painful condition the patient has, use the specific opioids as indicated, and avoid those painful states where opioids are contraindicated or result in a lesser therapeutic effect. however, there is the general position: in intense to severe, excruciating pain, opioids are the sole agents, which are able to induce sufficient analgesia -pain from muscular dysfunction. in patients who present pain of myofacial nature, opioids are contraindicated since they will not result in an alleviation of nociception. due to muscle spasm or an increase in tension physical therapy presents the first defense line in the therapeutic approach. this is accompanied by the administration of a benzodiazepine, which induces a muscle relaxant effect and/or the injection of a corticoid together with a local anesthetic ( . % bupivacaine) in so-called trigger points ( figure ii- ) . trigger points are typical points which are sore and from which the pain radiates to referred areas. such points can be felt as knots or bumps under the palpating finger, which can be moved over the underlying musculature. following the in injection of the local anesthetic the circulus vituosus of increased muscle tension and myofacial pain is interrupted. local ischemia is alleviated and local accumulation of pain mediating substances is flushed out. a typical example is tension-type headache, which is the moist common type of headache. originating from increased stress, it is accompanied by emotional factors and fear. thus the painful condition can be considered of psychosomatic nature. -pain of neurogenic or deafferentiation origin, also termed as complex regional pain syndrome (crps), this type of pain is mostly seen after injury of peripheral nerves leading to spontaneous and paroxysmal discharges. such pain typically is seen as post-herpetic pain, central pain after stroke, diabetic peripheral neuropathy, phantom limb pain, traumatic nerve avulsion, trigeminal neuralgia, lumbosacral plexopathy, all being circumscribed as neuropathic pain. it originates proximal of the peripheral nociceptor ( figure ii- ) , and characteristically is due to a dysfunction or lesion of the peripheral nerve fibers and/or centrally located nervous structures. typically this type of pain is accompanied by a sensory deficit: it is of a burning, shooting, stabbing, piercing, tearing or electricshock-like, paroxysmal and vice-like nature. this pain is of paresthetic, hypoor hyperesthetic quality often is refractory to any opioid therapy. the causes for such painful conditions may be quite different: compared to a normal situation (top), there is spontaneous ectopic firing from increased sodium-channel activity after peripheral nerve injury (middle), which eventually results in central sensitization with stimulus independent pain (bottom). modified from [ ] ectopic spontaneous discharges originating from a lesioned or a cut nerve, this results in an upregulation of sodium-channels being the source of spontaneous discharge. continuous nociceptive barrage later results in central sensitisation and "wind-up" (figure ii- ). partial denervation with spontaneous discharge of nerve activity is followed by an induced release of a nerve growth factor (ngf). such release induces sprouting of fibers into adjacent afferent somatic nerve fibers resulting in an enlargement of the receptive field and an increased conduction of nociceptive impulses to higher pain centers ( figure ii- other topical formulations with capscaicin or emla cream present additional therapeutic options for treatment of neuropathic pain. in addition, transcutaneous electrical stimulation (tens) or even spinal cord stimulation (scs) may present an alternative and effective strategy, resulting in the attenuation of pain. in the latter technique, analgesia is induced by the electrically induced release of endogenous opioids (enkephalins, endorphins, dynorphin) in the spinal cord and within the hypothalamus activating the descending serotoninergic and noradrengergic pathways. -opioids in visceral painful condition. another type of pain, which cannot be treated sufficiently with an opioid, is visceral pain. such a painful condition may arise from the intestine (e.g. the irritable bowel syndrome or ibs) or pain emerging from other internal organs such as the gall bladder, the urinary tract or pain following an appendectomy, cholecystectomy or hysterectomy ( figure ii- ). due to the participation of smooth muscles in such a condition a peripheral analgesic with a muscle relaxant effect can be of advantage. because the sympathetic nervous system to a major part is involved in such a condition, therapeutic implications incorporate a ganglionic blocker, surgical sympathectomy, or intravenous conduction anesthesia with guanethidine. -psychosomatic painful conditions, if treated with an opioid, in the long run are bound to end in opioid resistance. such a painful condition is mainly seen in the depressive patient or it may even be a premonitoring sign of schizophrenia. however, pain can also be part of a conversion-neurotic syndrome [ , ] , where aside from pain fear, phobia, and obsessive-compulsive symptoms are the dominant elements. painful conditions being sensitive to opioids all agonizing painful states, which are due to • posttraumatic • postoperative • ischemic, or of • tumor origin, can be treated sufficiently with an opioid. the rational for the therapy with a central analgesic is related to the fact, that the nociceptive impulses are transmitted via specific pain afferents reaching supraspinal areas. by blockade of specific receptor sites within the brain, opioids induce a reduction and/or result in a total blockade of nociceptive impulses. opioids therefore present the main line of defense in all medium to severe painful conditions. when using opioids one has to realize that this group of ligands, besides their beneficial analgesic effect at the same time also induces a detrimental respiratory depression. this is a mayor drawback when using opioids in acute pain and is directly proportional to their analgesic potency. for instance a potent opioid such as fentanyl already is able to induce respiratory depression in the lower dose range. however, a less potent opioid like codeine or tramadol, even when given in dosages higher than their therapeutic margin, will not induce a clinically relevant respiratory depressive effect ( figure ii- ) . because opioids given for alleviation of chronic pain are given orally and in a controlled release formulation, there is no acute rise in opioid plasma level, which otherwise would induce respiratory impairment. in addition, chronic pain patients cannot be considered as being opioid naïve. their respiratory center already has developed some degree of habituation, being less sensitive to the opioid agent. those opioid ligands, which inherit a lesser respiratory depressive effect, however, are characterized by a comparable reduced analgesic potency. also, a pure -type ligand such as morphine, fentanyl or sufentanil is characterized by a doserelated decrease in respiration until total apnea becomes apparent. contrary, the potent partial agonist buprenorphine with increasing doses demonstrates a ceiling effect, which is seen at a dose of g/kg ( figure ii- ) . typically, when administering high dosages of potent -type ligands such as fentanyl or alfentanil, a time related sequence of effects on respiration can be observed. the progression of respiratory depression is a characteristic trait, which develops within seconds to minutes: . a reduction in respiratory rate (bradypnea) with a partial compensation of tidal volume. . a respiration, which is only kicked off by external stimuli, such as noise or pain. changes in tidal volume (liter/min) in subjects being exposed to increasing dosages of the pure -type ligand fentanyl and the partial agonist buprenorphine. note, the ceiling effect in respiratory depression at higher doses of buprenorphine. adapted from [ ] . a short period where respiration is forgotten, originally termed in europe as "oublie respiratoire", as it was observed in the early times of neuroleptanesthesia when using fentanyl together with a neuroleptic agent for the induction and maintenance of anesthesia. at this stage, however, the patient can be ordered to take deep breaths. . the total apnea, where in spite of any external stimuli or the command to breathe the patient spontaneously will not be able to take a deep breath. he needs immediate respiratory assistance. this centrally-induced opioid-related respiratory depression is due to a blockade of the respiratory regulating centers within the brain stem (pons and medulla), resulting in a lesser sensitivity to an increase in arterial pco and/or a reduction of arterial po [ , ] . in addition the activating reticular system (ars), which descends down into the brain stem, acts as a regulatory pacemaker for the inspiratory center, by which respiratory depressive effect of opioids are affected. this is reflected in the clinics when in addition to an opioid a benzodiazepine is added, which by depressing vigilance, results in an immediate cessation of respiration [ ] . any opioid-induced respiratory depression instantaneously and effectively can be reversed by the administration of a specific opioid antagonist such as naloxone. because of the higher affinity of the antagonist, naloxone displaces the agonist from the receptor site (competitive antagonism), and after binding respiratory depression is reversed and normal ventilation is instigated ( figure ii- ) . clinically, an opioid-related respiratory depression is reversed by titrating the dose of naloxone necessary to • initiate a sufficient spontaneous respiration, however • avoiding an acute abstinence syndrome with tachycardia and hypertension, and at the same time • remaining a sufficient level of analgesia during reversal one should consider the half-life of naloxone, which is between and min [ , ] . therefore "remorphinisation" with a reoccurrence of respiratory depression may appear if the half-life of the agonist is longer than the antagonist, or if high concentrations of the agonist are still circulating in the blood plasma [ ] . following successful reversal it therefore is mandatory to administer an additional dose of naloxone intramuscularly, which acts like a depot, or hook the patient up to a continuous intravenous drip of a naloxone solution, sufficient for long-term receptor occupation by the antagonist. all these procedures, however, do not replace the need for a continuous surveillance, which is necessary in order to detect any possible gradual development of respiratory impairment. respiratory depression can also be reversed by a mixed agonist/antagonist such as nalbuphine. although being less potent than naloxone, it however is one of the mixed ligands having a sufficient antagonistic potency (table ii- ) . at the same time the ligand has a -fold longer duration of action than [ , ] , while the lesser antagonistic potency results in a more gradual and not in abrupt displacement, resulting in lesser sympathetic overdrive [ ] ( figure ii- ) . another pure antagonist, nalmefene shows the longest duration of antagonism with up to h of action [ ] . because of its high antagonistic potency ( . -fold of naloxone) an acute abstinence syndrome can be induced if the necessary dose is not titrated to patients need [ ] . it had been proposed that different receptor sites in the cns mediate opioid-related analgesia and respiratory impairment [ ] . such difference has also been demonstrated for fentanyl-analogues in the animal [ ] , suggesting a clinical relevance for reversal of respiratory impairment, however at the same time maintaining antinociception. such connotation was further corroborated by experimental data where the selective antagonist naloxonazine was able to reverse opioid induced analgesia, but not respiratory impairment. this led to the assumption that -opioid subreceptors are involved in the mediation of opioid-induced respiratory depression (i.e. and ) [ , ] . clinical data seem to underline this assumption, as low doses of sufentanil demonstrated a lesser respiratory depressive effect and a higher analgesic potency when compared to fentanyl. such difference in action reportedly is due to a disparity in receptor affinity to -subsites, with a higher affinity to the -and a lesser affinity to the -receptor [ ] . other experiments, however, suggest that the co-binding of -selective ligands to the -receptor results in respiratory impairment. subanalgesic doses of the -selective ligand d-ala -d-leu-enkephalin, when co-administered with morphine, induced a potentiation of analgesia, while another -ligand d-ala -met-enkephalinamid produced a reduction in analgesia [ ] . such -related differentiation in efficacy was also seen with the potent ligand sufentanil. there respiratory impairment was reversed while at the same time maintaining antinociception using the highly selective -antagonists naltrindol and naltribene respectively [ ] ( figure ii- ) . the implication of / -receptor interaction is further supported by receptor binding studies, where sufentanil demonstrates higher -selectivity than fentanyl (table ii- ) . such putative interaction between -and -receptors is further corroborated when co-administering intrathecally a -and a -selective ligand resulted in a potentiation of effects [ ] . from such data it can be concluded that a coupling mechanism between the -and -opioid receptor not only seems to result in an increase in analgesia, but at the same time also seems to cause respiratory depression. such coupling mechanisms may result in a modulation to potentiation of effects whereby it is still uncertain whether both sites independently operate from each other or whether the -receptor only accentuates the effects induced by -binding. besides a direct action of opioids on the sensitivity of the respiratory center to changes of arterial po and pco , also centrally-induced sedative effects very likely influence respiration. such sedative effects can be derived with the aid of the electroencephalogram where clinically different potent opioids qualitatively induce a different in the eeg pattern. since such eeg changes are dose-related, one is able to derive a dose-relationship. at the same time such eeg-changes reflect the bioavailability of centrally active agents acting on nervous structures of the cns, depicting the effect-concentration site [ , ] . thus, following intravenous administration of an agent, it is not the plasma concentration, which is responsible for a centrally induced effect. more importantly, it is the actual concentration of the opioid at the receptor site, which is affected significantly by issues such as distribution of an agent, its lipophilicity, or the present brain perfusion. therefore vigilance changes can be considered as important aspects in an opioidrelated respiratory impairment being derived in two relevant experiments: . wakefulness by itself already is a fact resulting in sufficient respiration. this could be demonstrated nicely in volunteers where hyperventilation and the resultant hypocapnia resulted in a rhythmic respiratory pattern. if however, the same volunteers were asleep or in anesthesia, hypocapnia was followed by apnea [ ] . . in the animal laryngeal stimulation during anesthesia resulted in apnae, without, however, initiating a cough reflex. being awake, a cough reflex without apnae was induced following laryngeal stimulation [ ] . . there is a close exponential correlation of the physiologic regulatory mechanism affecting respiration. this had been demonstrated after sufentanil application in the canine, whereby increasing dosages of a selective antagonist not only dose-related reversal of sufentanil-induced hypercarbia and hypoxia with the two selective -antagonists naltrindol (nti) and naltribene (ntb) respectively, in the canine. due to the higher lipophilicity of naltribene being able to pass through the blood-brain barrier, there is a superior reversal effect. in spite of increasing doses of the antagonist there is a blockade of response to the electrically induced evoked potential, which is only reversed by the highly specific -antagonist cyprodime. adapted from [ ] reversed the depressed respiratory drive but at the same time induced an increase of power in the high frequency beta band ( - hz) of the eeg ( figure ii- ) , reflecting increase in vigilance [ ] . . clinically such sedative related respiratory depression can also be derived in patients, when cumulative dosages of an opioid reach a point where the respiratory center "forgets" to respond adequately by initiating deep breaths (oublie respiratoire). this is seen in classical neuroleptanalgsia where the patient's vigilance can be increased to a point by external stimuli (e.g. pain, auditory stimuli) resulting in the initiation of an inspiratory effort [ ] . from all these data it can be derived that the simultaneous binding of opioid within the activating reticular system (ars) in the brain stem, vigilance is depressed, which secondarily affects the response of the respiratory center following hypercapnia. at such instances the overall mesencephalic reticular control mechanism is no longer able to adequately respond to a stimulus and only with an increase in vigilance there is an accelerated reactivity, being able to sufficiently respond to an increase in arterial pco . since the reticular mechanism is coupled with reticulo-cortical afferences, such changes can be derived from cortical changes in the eeg. such a "forgotten" reaction to sufficiently respond to a given stimulus [ ] is also seen in the clinical environment when a benzodiazepine is given on-top of an opioid resulting in a further deterioration of respiratory drive. this is because a benzodiazepine depresses the reaction of the ars, and the concomittant reduction in vigilance results in a lessened reaction to external stimuli, producing a clinically relevant suppression of respiration. in general prolongation of respiratory depression after opioid administration has to be expected when the following agents are coadministered: . all agents that inhibit the biotransformation of opioids such as contraceptives, anti-tumor agents, anti-arrhythmics, antidepressants, systemically administered antimycotics, neuroleptic drugs, and volatile anesthetics [ , , , , ] . by inhibition of conjugation of glucoronide and oxidative dealkylation, the necessary metabolic pathways for degradation and termination of activity of most agents, a prolongation of action has to be expected. . all agents, which are able to displace the opioid from protein binding within the plasma, resulting in a higher portion of the pharmacologically active agent. preparations such as cumarine derivatives, and phenylbutazone, which when coadminstered are prone to result in a prolongation of effects [ , , , ] . . in addition, hypoproteinemia and acidosis of the blood, both of which result in lesser protein binding, cause a higher concentration of non-bound opioid in the blood plasma. such increase in plasma concentration now is able to bind to the receptor site with an increase of efficacy and a longer duration of action [ ] . following opioid-based anesthesia, several factors cause an overhang of opioid action, which may even result in a "re-morphinisation" and the re-occurrence of respiratory impairment: . the excessive intramuscular premedication with an opioid, which may act like a depot. . the premedication with a long-acting benzodiazepine, which is able to induce a reduction in vigilance lasting into the postoperative period. . the uncritical intraoperative use of high concentrations of a volatile anesthetic, which results in a lesser biodegradation of the opioid. . the intraoperative administration of fractional doses of an opioid, which results in an accumulation. due to the fact that a portion of each dose of an intravenously administered opioid is also taken up by peripheral sites (e.g. fatty tissue, musculature, skin, internal organs) there is an accumulation of the agent, which act like a depot. from there the drug later diffuses into the blood-stream, resulting in a prolongation of effects ( figure ii- ). . an insufficient loading dose of the opioid, which may result in the necessity of re-administration of small amounts of the drug intraoperatively with consequent peripheral accumulation. . long-term intravenous administration of an opioid by drip, resulting in the increase of the agent in the peripheral compartment with later recirculation into the blood stream. . the combination of opioids with different half-lifes, which may result in an unforeseen potentiation of effects. . uncritical administration of bicarbonate resulting in alkalosis of the blood, which induces a faster release of the opioid from the peripheral compartment. . a non-corrected hypovolemia, which coincides with lesser protein binding of the agent and a higher portion of the free active compound. . uncritical use of a selective antagonist such as naloxone, not considering that its half-life is shorter than the agonist, resulting in a later reoccurrence of respiratory impairment. the sedative effect of opioids goes in hand with their capability to induce sleep (lat. hypnos). such an effect is mostly seen with the mixed agonist/antagonists, while morphine takes a medium position ( figure ii- ) . the hypno-sedative effect of opioids is useful in premedication and during postoperative analgesia, where a sedated status of the patient is advantageous. in contrast to a potent sedative nature of mixed agonist/antagonists, the pure -type ligand fentanyl is characterized by a very low sedative potency. when in the beginning of use of neuroleptic analgesia for anesthesia, fentanyl was used together with the neuroleptic agent droperidol, often patients reported of intraoperative "awareness". although being an obligatory part of anesthesia, sleep, was not sufficiently maintained throughout the whole procedure. therefore in order to guarantee a sufficient level of sleep in patients receiving a fentanyl-based anesthetic technique, an additional hypnotic (propofol), a benzodiazepine (midazolam), a neuroleptic agent (i.e. dehydrobenzperidol), or a volatile anesthetic (sevoflurane, desflurane, or enflurane) has to be given on-top the opioid. nowadays the problem of awareness again has gained much attention [ ] , since the technique of total intravenous anesthesia (tiva) with remifentanil and [ ] propofol, completely omitting nitrous oxide (n o), often results in an insufficient level of sleep with awareness. typically pure -ligands such as bremacozine and tifluadom (table ii- ) , which in comparison to morphine have a -fold analgesic potency [ , ] , do not induce a respiratory depressive effect [ ] . their lack in respiratory impairment is due to the selective binding in deep layers of the cortex [ , ] , where in comparison to the brain-stem, a more than % higher concentration of -binding sites is found [ , ] . their predominant sedative effect is due to centripetal fibers descending down from deep layers of the cortex to the thalamus, thus decreasing the nociceptive input [ ] . although having the advantage of an increased sedative effect combined with the lack in respiratory impairment, clinically, the use of -ligands had to be abandoned. this is because of their intense dysphoric side effects, which lasts for several hours. in addition, their analgesic potency, in comparison to pure -ligands is much lower. therefore such agents cannot be regarded as suitable for intraoperative use, where an intense nociceptive barrage can only be blocked by a potent -opioid. only the mixed agonist/antagonists (e.g. nalbuphine, butorphanol), which exert their analgesic action through binding at the -site, currently are in clinical use mainly for postoperative analgesia [ , ] . this is because cumulative dosages, contrary to a typical -ligand like morphine, result in a ceiling effect for respiratory depression ( figure ii- ). in addition, because of their wide margin of safety, high dosages part ii figure ii- . ceiling effect of respiratory depression following cumulative doses of the mixed agonist/antagonist nalbuphine. in contrast to morphine, at a certain dose there is no further increase in the degree of respiratory impairment. adapted from [ ] have been advocated in balanced anesthesia where the opioid resulted in an up to % reduction in mac (minimal alveolar concentration) of the volatile agent [ , ] . opioids in general induce a dose-related hyposedative component, which is mirrored in the electroencephalogram by an increase of activity in the slow -with concomitant decrease of power in the fast -domain. however, when giving a large bolus dose of fentanyl ( - g/kg body weight) alfentanil ( g/kg body weight) morphine ( - mg/kg body weight) or sufentanil ( - g/kg body weight) an immediate dominance of delta-waves in the eeg becomes evident, being accompanied by sleep. for instance, such effects clinically are seen when high-dose opioid anesthesia is used in cardiac patients for the induction of anesthesia. such a sleepinducing effect is due to a short-term blockade of all afferences being switched in the activating reticular system (ars) of the mesencephalon. aside from a blockade within the nucleus limitans a deep level of analgesia is initiated [ ] . such a "narcotic component", with dominance of delta-activity in the eeg, and contrary to equi-analgesic doses of fentanyl, it is more apparent after sufentanil [ ] , which makes this agent more suitable for the induction of cardiac patients ( figure ii- ) . following induction with a potent -ligand such as fentanyl or sufentanil the initial "narcotic component" later transforms into a "pure analgesic component". this is because the opioid is redistributed, which results in a lesser concentration within the cns and a lesser binding in areas within the ars. at this stage there is a note, a pronounced delta activation after injection and a lesser arousal reaction induced by laryngoscopy and intubation, which in the sufentanil group reflected in a lesser decline of power in the slow delta-domain of the eeg. adapted from [ ] dominance in the -band ( - hz) of the eeg, which is stable, not being affected by any nociceptive stimuli [ , ] . clinically, such an effect has been described for the precursor of fentanyl, the opioid phenoperidine [ ] and for fentanyl [ ] . after a period of - min the deep narcotic component changes into a sedative state, which is stable and cannot be reversed to desynchronization by any nociceptive stimulus. during such "analgesic state" the patient again is able to respond to verbal commands, while at the same time having a deep analgesic level ( figure ii- ) . without the addition of nitrous oxide, such patients are awake, however, nociceptive afferents are not able to modulate the ars, the endotracheal tube is tolerated while at the same time nociception is only sensed as a touch. such phenomena are due to afferents ascending along the spinothalamic tract, which directly ascend to the postcentral cortical area by which the impulse can be localized. collaterals, which ascend through the nucleus limitans within the limbic system and convey nociception, are sufficiently blocked by the opioid and the patient does not perceive pain ( figure ii- ) . the opioid receptor system bordering the fourth cerebral ventricle and the underlying activating reticular system is the relevant anatomical structure in mediating sedation. selective perfusion of increasing concentrations of the opioid fentanyl in the awake canine induced a dose-related enlargement of slow-wave high amplitude delta-activity within the eeg, characterized by a sleep-like behavior ( figure ii- ) . this effect was reversed by the levo-isomer of naloxone inducing an arousal reaction. it, however, was not reversible by the dextro-isomer of the antagonist [ ] . the physiological significance of opioid receptors in the control of vigilance is also reflected in the high density of opioid binding sites in the mesencephalon [ ] . physiologically this is mirrored by an arousal reaction following intense acoustic or a nociceptive stimulus, both of which induce a reversal from the low frequency delta-to high frequency beta-activity in the eeg ( figure ii- ) . in summary, it is concluded that opioids primarily affect the limbic system, the specific site for inducing the negative component of nociception. lastly such assumption is underlined by the result from mc kenzie and coworkers in the animal where the opioids morphine and pethidine were not able to sufficiently block any pain related nervous transmission from the mesencephalon to the higher cortical areas [ ] . in contrast, both ligands were able to block nociceptive transmission from the mesencephalon to hippocampal areas of the limbic system, the part of the cns, which is responsible for the identification of pain, causing the negative, grief, stinging and an intense emotional feeling associated with pain ( figure ii- ) . such differences in pain modulation were corroborated in patients undergoing stereotactic, painful stimulation within specific areas of the cns [ ] . nociceptive afferents of the spinothalamic tract end in the nucleus ventrocaudalis parvo-cellularis part ii figure ii- . selective perfusion of increasing doses of fentanyl through the fourth cerebral ventricle of the awake canine. note, the direct sedative (delta-synchronisation in the eeg) effect via the underlying ars. this effect is mediated through opioid receptors located on the floor of the ventricle, since it was reversible with naloxone (desynchronisation with beta activation in the eeg). adapted from [ ] thalami, from where they further ascend to different cortical areas. since these nuclei reflect a specific somatotopic differentiation, electrical stimulation within this area induced painful sensations in different parts of the body. decoding of painful afferents was only possible when the stimulating electrode was placed within the nucleus limitans, where collaterals of the spinothalamic tract switch to the limbic system. there stimulation induced a less well-localized, however, intense unspecific displeasure [ ] . following the administration of high dosages of opioids with different potency, epileptogenic activities in the eeg with tonic-clonic seizures can be induced in the animal. pethidine (meperidine), morphine, alfentanil, fentanyl and sufentanil when administered in doses above , , , and mg/kg body weight respectively, induced epileptogenic discharges [ ] . because such massive dosages are never used in anesthesia or for analgesia in acute or chronic pain, epileptogenic effects, although being cited in the literature after fentanyl [ , ] and sufentanil [ ] are of insignificant nature. this is because the clinical picture resembles tonicclonic seizures, however, in the eeg no such discharges could be derived [ ] . therefore, those high doses of opioids, which induced epileptogenic activity in the rat [ ] or the canine [ ] are far off from therapeutic range. thus, in general, an epileptogenic activity of opioids can be canceled out. one exception is the use of high dosages of pethidine (meperidine), where the metabolic product norpethidine is a potent epileptogenic compound, which especially in the newborn is able to induce epileptogenic activity [ ] . the cause for the few observations of a pseudoepileptogenic activity of opioids when being administered within the therapeutic range very likely is due to a desinhibition of the cortical motor center within the cns, as this phenomenon was observed during the induction of anesthesia or following a decline in plasma concentration. such assumption is underlined by part ii figure ii- . two main components of painful afferents: localization of nociception (cortical area) and the initiation of the negative component (i.e. the limbic system within the mesencephalon). opioids mainly modify the latter area "epileptogenic activity" during the induction of anesthesia with the pure hypnotic etomidate, where desinhibition of the motor cortex activity was the cause of cortical discharges [ ] . each cough involves a complex reflex arc beginning with the stimulation of sensory nerves that function as cough receptors. there is evidence, primarily clinical, that the sensory limb of the reflex exists in and outside of the lower respiratory tract. although myelinated, rapidly adapting pulmonary stretch receptors (rars), also known as irritant receptors, are the most likely type of sensory nerve that stimulates the cough center in the brain, afferent c-fibers and slowly adapting pulmonary stretch receptors (sars) also may modulate cough. rars, c-fibers, and sars have been identified in the distal esophageal mucosa; however, studies have not been performed to determine whether they can participate in the cough reflex. although gastroesophageal reflux disease can potentially stimulate the afferent limb of the cough reflex by irritating the upper respiratory tract without aspiration and by irritating the lower respiratory tract by micro-or macroaspiration, there is evidence that strongly suggests that reflux commonly provokes cough by stimulating an esophageal-bronchial reflex. each involuntary cough involves a complex reflex arch beginning with the stimulation of sensory nerves in the airway epithelium that function as "cough receptors." efferent impulses from these receptors are conducted by means of the vagus nerve to the "cough center" in the brain stem. because cough can be voluntarily suppressed, controlled, or initiated, there also can be afferent input from the cerebral cortex. the function of this "cough center" is to receive these impulses and produce a cough by activating efferent nervous pathways to the diaphragm and laryngeal, thoracic, and abdominal musculature. the possibility that there might be afferent input other than the vagus nerve and cerebral cortex was based on clinical observations described in case reports and a few animal studies [ ] . histologic studies of the respiratory tract in both animals and humans have revealed sensory nerve endings within the basal layer of the epithelium and between epithelial cells of the larynx, trachea, and bronchi [ ] . these nerve endings are thought to be cough receptors. they contain neuropeptides, such as substance p and calcitonin-gene related peptide (cgrp), which mediate neurogenic inflammatory events in the airways. these sensory nerve endings have been found to be most numerous in the posterior wall of the trachea, at branching points of large airways, and less numerous in the more distal, smaller airways. none have been found beyond terminal bronchioles. it is not known for certain which type of afferent nerve mediates cough. a model that summarizes the current understanding of cough is schematically depicted in the figure. it shows the myelinated, rapidly adapting pulmonary stretch receptors (rars), also referred to as irritant receptors, as the most likely type of sensory nerve that stimulates the cough center in the brain. both mechanical and chemical stimulation of rars have been shown experimentally to cause cough. another type of sensory nerve in the airways, c-fibers, may also participate in regulating cough. they are unmyelinated, vagal afferent fibers that may be activated by the same triggers as rars. their activation releases neuropeptides locally that may secondarily stimulate cough by activating rar nerves. however; impulses transmitted by c-fibers alone probably do not stimulate cough, because experimental evidence has shown that they inhibit cough centrally in the brain. a third type of sensory nerve, the slowly adapting pulmonary stretch receptors (sars), may modulate cough. although these nerves do not directly respond to chemical and mechanical triggers, they do appear to be activated by the deep breath of a cough and may enhance cough by making the expiratory effort more forceful. in addition to mechanical and chemical stimuli, cough has been caused in animals by thermal and electrical provocation. the sites most sensitive to all stimuli are the larynx, trachea, and cannulae of the larger airways. outside of the lower respiratory tract, cough receptors have been demonstrated histologically only in the hypopharynx [ ] . however, it has been inferred from clinical studies that sensory nerve endings subserving the cough reflex via the vagus nerve probably exist in the extemal auditory canals and eardrums, hypopharynx, pericardium, stomach, and esophagus, because stimulation of these sites has been reported to cause cough [ ] . based on the fact that cough can be voluntarily initiated, postponed, and/or suppressed, this provides evidence that there also can be afferent input from the cerebral cortex. in addition to directly stimulating cough by carrying impulses from cough receptors to the cough center, vagal afferents may indirectly provoke cough by another mechanism. they may stimulate neurotransmitter release or mucus secretion from airway submucosal glands that, in turn, stimulate the cough reflex [ ] . the existence of a discrete central cough center is controversial. what is known is that afferent pathways first relay impulses to an area in or near the nuc eus tractus solitarius. these impulses then are integrated into a coordinated cough response in the medulla oblongata of the brain stem, probably separate from the medullary centers, which control breathing. although electrical stimulation studies of different areas in the medulla have evoked cough in animals, suggesting that the cough center is diffusely located [ ] a discrete cough center still may exist, because these electrical stimulations may have activated afferent pathways of the cough reflex. the motor outputs trom the cough center are in the ventral respiratory group, with the nucleus retro-ambigualis sending impulses via motoneurons to the respiratory skeletal muscles and the nucleus ambiguus sending impulses to the larynx and bronchial tree. more specifically, the efferent impulses of the cough reflex are transmitted from the medulla to the expiratory musculature, through the phrenic nerve and other spinal motor nerves, and to the larynx through the recurrent laryngeal branches of the vagus nerves ( figure ii- ) . vagal efferents also innervate the tracheobronchial tree and mediate bronchoconstriction [ ] . although stimulation of cough and bronchoconstriction can be experimentally separated using nonpermeant anions to stimulate cough without bronchoconstriction, these two phenomena normally are activated simultaneously to facilitate the most effective cough. bronchoconstriction may improve clearance of secretions by narrowing the cross-sectional area of the airways, thereby increasing the velocity of air leaving the patients lower respiratory tract during the expiratory phase of coughing [ ] . experimentally, it has been shown in animals that the efferent pathways of the cough reflex are anatomically distinct and separate from the efferent pathways of normal spontaneous ventilation. blockade of the cough reflex arch by means of opioids, known as the antitussive action, refers to the fact that they suppress this protective reflex. this is of benefit during anesthesia and/or in patients being artificially ventilated in the intensive care unit (icu) because it results in the tolerance of an endotracheal tube. however, the antitussive potency differs significantly among the various opioids ( figure ii- ) . the action is not related to a specific receptor site, because a stereoselective action of opioids in regard to their antitussive effect could not be demonstrated. in addition, reversal with the selective antagonist naloxone is less selective [ ] . the mode of action is a blockade of the cough center within the brainstem. three of the most commonly used suppressors of the cough reflex are hydrocodone, codeine and hydromorphone. all of them are characterized by low analgesic potency, they model for afferent limb of cough reflex in airways. myelinated. rapidly adapting pulmonary stretch receptors (rars) and unmyelinated c-fibers are sensory nerves that participate in the afferent limb of the cough reflex. mechanical and chemical stimuli activate sensory nerve enelings in the epithelial layer. rars appear to be the main type of sensory nerve stimulating cough centrally. although c-fibers may inhibit cough centrally, neuropeptides released in the periphery upon stimulation of c-fibers may indirectly stimulate cough by activating rars. slowly adapting pulmonary stretch receptors (sars) do not respond to irritant stimuli that initiate cough but may enhance cough centrally by making expiratory muscular effort more forceful demonstrate a negligible dependence liability, and they are common components in doc (drugs over the counter) cough medicine. a similar antitussive potency, however, is also seen with the more potent opioids such as diamorphine, fentanyl or sufentanil. the latter are used in an opioid-based anesthetic regimen or in icu patients who are in need of ventilatory support. when a potent opioid such as sufentanil is used, the patient is adapted much easier to the respiratory cycle of the ventilator resulting in lesser doses of additional sedative agents. morphine in this regard has a much weaker antitussive activity while pethidine (meperidine) and all mixed agonist/antagonists are characterized by a negligible antitussive action ( figure ii- ) . it can be summarized that potent opioids also inherit a marked antitussive effect, while weak opioids and especially the mixed opioid analgesics are unable to sufficiently suppress the cough reflex. during the induction of anesthesia, while injecting an intravenous bolus dose of a potent opioid such as fentanyl or sufentanil, often a cough reflex is initiated. such part ii dependence or addictive liability (how addictive a drug is likely to be) depends upon how quickly the drug enters/leaves the brain. also, it is directly proportional to the analgesic potency and it depends on the opioid receptor sites with which the ligand interacts. for instance, members of mixed agonist/antagonists (i.e. nalbuphine, pentazocine, butorphanbol) demonstrate a predominant interaction with the -opioid receptor, which is characterized by a low dependence liability ( figure ii- ). in addition, development of dependence also is related to the speed for instance, an opioid like buprenorphine is characterized by a low dissociation constant reflecting long binding to the receptor, a long duration of action and a slow separation from the receptor. the definition of addiction is based in two different terms: . physiological dependence produced by repeated drug-taking that is characterized by a withdrawal syndrome, when drug is removed (e.g. alcohol, opiates). . psychological dependence produced by repeated drug taking that is characterized by obsessions and compulsive drug-seeking behaviors; results in a detrimental impairment in physical, mental or social functioning. there are five classes of abused psychoactive drugs . opioids produce a dream-like state; effects include: analgesic (reduction in pain), hypnotic (sleep inducing), euphoria (sense of happiness or ecstasy) using morphine, heroin, or the cough suppressant codeine. . depressants produce feelings of relaxation/sedation and a dream-like state, anxiolytic (anxiety-reducing) and hypnotic effects; reduce central nervous system activity. members of the class are alcohol, barbiturates, and benzodiazepines. . stimulants increase alertness, arousal, and elevated mood; activate central nervous system (sympathomimetic = mimic the activation of the sympathetic nervous system). members of this class are cocaine, amphetamines, nicotine, caffeine. . psychedelics produce distortions of perception and an altered sense of reality. typical representatives are lsd, psilocybine, mescaline, mdma (ecstasy), and pcp (phencyclidine). . marijuana with one of its major active ingredient thc produces feelings of well-being and sense of acuity (sharpness). it also can produce feelings of relaxation. also, it is recognized, that opioids, which demonstrate a fast onset of action or due to their galenical preparation (injection, smoking) result in an immediate high plasma concentration, followed by an instantaneous receptor binding, results in a "kick" with an euphoric feeling. such preparations therefore are more prone to induce a behavior pattern of abuse. therefore addictive liability or how addictive a drug is likely to be, depends upon how quickly the drug enters/leaves the brain (table ii- ) . most importantly, the tendency of opioids to result in an abuse is very much linked to the fact of why and when the opioid is ingested. for instance, an opioid taken only for the mere pleasure will rapidly result in the development of dependency. contrarily, if an opioid is taken for the attenuation of pain, the likelihood to develop an abuse behavior is very low. aberrant drug related behavior has to be suggested when the following signs of abuse are obvious: in physical examination. a routine physical examination can elucidate common complications of heroin use or assist in diagnosing opioid dependence. chronic intravenous use can be confirmed by the presence of "track" marks, which are callouses that follow the course of a subcutaneous vein. these are caused by repeated injections into adjacent sites over an accessible vein. tracks are often found in easily accessible body areas, such as the backs of the hands, antecubital fossae, on the legs, or in the neck. signs of recent injection may be found in unusual places in patients attempting to hide their sites of injection. a thorough examination for tracks or recent injection sites should include looking between the table ii- . factors that influence how quickly a drug will enter the brain . chemical structure: how fatty is the drug (i.e. its lipophilicity)? does the drug have nutrients that our brain uses? . how fast does the drug cross the blood brain barrier (bbb) lipophilic drugs (i.e. heroin, fentanyl) cross the bbb much faster than hydrophilic agents (i.e., morphine); they mimic nutrients our brain needs and can "slip" through transporters in the bbb. . route of administration: is the drug entering directly into the blood stream or is it entering first into the stomach? the routes of administration increase the likelihood that a drug enters the blood stream whereby it increases the addictive liability of the drug. intravenous injection > smoking/snorting > sublingual application > inhalation via nostrils and lungs because they have abundant blood capillaries and more blood supply under the tongue and the lung respectively. fingers and toes, under the fingernails and toenails, in the axillae, breast veins, and the dorsal vein of the penis. one complication of drug use that can be found on examination is nasal septal perforation from repeated intranasal insufflation (especially when cocaine is mixed with heroin and snorted). a heart murmur may indicate subacute bacterial endocarditis, a complication of intravenous injection without using good sterile technique. posterior cervical lymphadenopathy may suggest early viral infection, especially with hiv. hepatic enlargement may indicate acute hepatitis; a small, hard liver is consistent with chronic viral hepatitis due to hepatitis b or c virus, which are common among injection drug users who share needles. signs of opioid intoxication may include pinpoint pupils, drowsiness, slurred speech, and impaired cognition. signs of acute opioid withdrawal syndrome include watering eyes, runny nose, yawning, muscle twitching, hyperactive bowel sounds, and piloerection. on the other hand the following behaviors are more suggestive of an addiction disorder: • selling prescription drugs • prescription forgery • stealing or "borrowing" drugs from others • injecting oral formulations • obtaining prescription drugs from nonmedical sources • concurrent abuse of alcohol or illicit drugs • multiple dose escalations or other non-compliance with therapy despite warnings • multiple episodes of prescription "loss" • repeatedly seeking prescriptions from other clinicians or from emergency rooms without informing the prescriber or after warnings to desist • evidence of deterioration in the ability to function at work, in the family, or socially that appear to be related to drug use • repeated resistance to changes in therapy despite clear evidence of adverse physical or psychological effects from the drug the following behavior pattern is less suggestive of an addiction disorder. • aggressive complaining about the need for more drugs. • drug hoarding during periods of reduced symptoms. • requesting specific drugs. • openly acquiring similar drugs from other medical sources. • unsanctioned dose escalation or other noncompliance with therapy on one or two occasions. • unapproved use of the drug to treat another symptom. • reporting psychic effects not intended by the clinician. • resistance to a change in therapy associated with "tolerable" adverse effects with expressions of anxiety related to the return of severe symptoms. and while addiction is characterized by a compulsive drug-using and drug-seeking behavior that interferes with normal functioning and causes use of the drug despite increasingly damaging consequences, there are different mechanisms from dependence as addicts can experience intense cravings for drugs even years after sobriety (after body set-points, etc. should have adjusted back to normal). the nervous pathways involved in the development of dependence is the mesolimbic-dopaminergic reward system, where direct activation (i.e. cocaine, nicotine, alcohol) or inhibition of the inhibitory gabaergic neurons in the ventral tegmental area (vta) directly project to dopaminergic neurons in the nucleus accumbens (i.e. opioids such as heroin) results in an increased release of dopamine in the nucleus accumbens and stimulation of the prefrontal area resulting in euphoria. on the other hand, insufficient release of dopamine in this area results in dysphoria, which is seen in abstinence ( figure ii- ). in the acute phase, opioids inhibit adenylyl cyclase and camp. over time, the downstream transcription factor (creb) is activated which increases adenlyly cyclase production ( figure ii- ) . chronic opioid use leads to upregulation of camp and creb, directing to tolerance, dependence and withdrawal symptoms. the significance of intracellular changes in creb is supported by data in mice without creb, which are less likely to develop addiction. also, recent research has also implicated an opioid peptide, dynorphin, in this pathway. besides initial changes in creb, chronic administration of an opioid results in the induced increased formation of peptides syntheses fosb within the nucleus accumbens. such overexpression of fosb increases the sensitivity to cocaine and opioids resulting in an increased likelihood of relapse. in general an opioid-related dependency has to be distinguished from a dependency of the barbiturate-, alcohol-or cocaine-type. this is because they all result in different psychopathological and withdrawal symptoms. the latter is a set of physiological reactions that occur in response to removal of a drug following repeated treatment; often (although not always), the reactions are opposite those produced by the drug itself. in the beginning it is the pleasure seeking behavior that results in repetitive drug administration until finally, the drugs of abuse that produce physical dependence (e.g., opiates or alcohol), results in the avoidance of the unpleasant withdrawal syndrome can contribute to repeated drug-taking ( figure ii- ). the main physiological consequence of nausea and emesis is the removal of toxins, which is an important protective reflex mechanism being induced during food intoxication. it however, is also seen after radiation or chemotherapy, after the administration of an opioid-based anesthetic regimen or during long-term therapy for alleviation of chronic pain. about % of all patients experience nausea and/or emesis after opioid anesthesia. the cause of such reaction is a stimulation of the chemoreceptor biochemical changes induced during addiction and the development of tolerance, which is followed by withdrawal when an antagonist is administered or by lack in maintenance dosages trigger zone (ctz), which lies in close vicinity to the emetic center, bordering the fourth cerebral ventricle, above the area postrema ( figure ii- ). this area is richly supplied with dopaminergic, histaminergic, serotonergic ( -ht ), and cholinergic receptor sites, being the origin of metabolic or drug induced vomiting [ ] . contrary to the other areas within the cns, the ctz is characterized by leaking capillaries (windowed capillaries), through which opioids as well as toxins can disseminate. such anatomical difference indicates that this area does not contain the usual blood-brain barrier (bbb). being located within the dorsal part of the activating reticular formation (ars), all visual, cortical and limbic efferences, as well as efferences of nearby nuclei of the vasomotor center and the center for salivation and respiratory control are switched, resulting in a controlled succession during vomiting. once the vomiting center is stimulated by any of the efferent stimuli, a coordinated sequence of events is commenced: • stop of rhythmical contractions of the stomach, followed by an accumulation of food in the abdomen, resulting in. • retroperistaltic action. • contraction of the cardia with increase of pressure in the stomach. • due to the coordinated contractions of diaphragm, intercostal muscles and the rectus abdominis muscle, food is being expelled forcefully via the opened orifice of the stomach, the dilated esophagus and the opened glottis. because the ctz shows a dense accumulation of serotonin receptors, the serotoninantagonist ondansetron (zofran®) is able to induce an antiemetic effect [ , ] . other agents, which are given for reversal of emesis, are metoclopramide, and/or the neuroleptic agents haloperidol, triflupromazine, or alizapride-hcl, all of which interact through direct binding with the dopaminergic d -receptor. another antiemetic is diphenhydramine, which exerts its action via binding at the cholinergic and histaminergic receptors ( figure ii- ) . postoperative nausea and emesis (ponv), however, still present a problem specifically related to anesthesia. in a large survey with over patients and using multivariance analysis, the following main risk factors for ponv were identified: • female sex • young age • history of ponv/motion sickness • nonsmoking status • long duration of anesthesia each min increase in duration increases ponv risk by %, and a baseline risk of % is increased to % after min [ ] . the type of operation, the addition of nitrous oxide (n o), high age and/or the addition of an opioid to the anesthetic regimen, in comparison to the above risk factors, had a lesser impact on the incidence on ponv [ , ] . commonly the key strategic antiemetic agents for reducing patients ponv are as follows (figure ii- ): the -ht -receptor antagonists are used for both the prevention and treatment of ponv and have a low side-effect profile. they are given toward the end of surgery for greatest efficacy, and are more effective in preventing vomiting than in preventing nausea. dolasetron, granisetron, and ondansetron all have favorable side-effect profiles. no evidence has revealed differences in efficacy and safety among the -ht -receptor antagonists used for the prophylaxis of ponv. a recent study demonstrated the equivalent efficacy and safety of granisetron and ondansetron when these agents were used in combination antiemetic therapy. in this study, low-dose granisetron ( . mg) plus dexamethasone mg was found to be not inferior to ondansetron mg plus dexamethasone mg in patients undergoing abdominal hysterectomy with general anesthesia. the combinations prevented vomiting in % and % of patients, respectively, in the first h after tracheal extubation, and in % and % of patients, respectively, in the h after extubation. dexamethasone has been found to be effective for the management of ponv and their proposed mechanism of action is that of membrane stabilization and inhibition of inflammation. use of this agent is controversial because of its alleged association with delayed wound healing. it has a slow onset but a prolonged duration of action, and therefore it is advised that dexamethasone be administered upon induction of anesthesia. the most commonly used dose for adults is - mg i.v. smaller doses of . - mg have also been used and found to be as effective. based on a quantitative, systematic review of the data, no adverse side effects, especially delayed wound healing, have been noted following a single antiemetic dose of dexamethasone [ ] . the neuroleptic drug droperidol, a butyrephenone derivative, is widely used for ponv prophylaxis and is comparable with ondansetron as a prophylactic antiemetic. similar to haloperidol it acts as a dopamine antgonist at the ctz and the area postrema. for greatest efficacy, droperidol is administered at the end of surgery or concomitantly with morphine via patient-controlled analgesia systems. the use of low doses ( . - . mg) of droperidol has not been associated with the typical side effects of higher doses of this drug (hypotension, extrapyramidal symptoms, sedation, akathisia, dysphoria). in , the food and drug administration began requiring that droperidol labeling include a "black box" warning stating that the drug may cause death or life-threatening events resulting from qtc prolongation and the possibility of life-threatening torsades de pointes. the labeling requirement was based on reported cases associated with droperidol use (at doses of . mg) during its approximately years on the market [ ] . however, no case reports in peer-reviewed journals have linked droperidol with qtc prolongation, cardiac arrhythmias, or death at the doses used for the management of ponv. also, in a randomized, doubleblind, placebo controlled trial, droperidol was not associated with a significant increase in the qtc interval in comparison with saline solution [ ] . in another recent study, droperidol did not increase the qtc interval any more than did ondansetron [ ] . transdermal scopolamine (transderm scop® . mg), an antimuscarinic ganet, works by blocking the cholinergic receptor. it has an antiemetic effect when applied the evening before surgery or h before the end of anesthesia preventing the patient from post-discharge nausea, vomiting and retching. the phenothiazines, promethazine, and prochlorperazine act both as d -and the h -receptor antagonist. they also inhibit histamine receptors and possibly cholinergic receptors in the gut. both have been shown to be effective antiemetics when administered intravenously at the end of surgery. all three drugs may cause sedation, dry mouth, and dizziness. metoclopramide is benzamide that blocks d -receptors both centrally and peripherally in the gastrointestinal tract increasing gastric emptying. the antihistamines, especially diphenhydramine act on both the ctz and the vestibular pathways of the inner ear. at higher doses however, they can prolong general anesthesia and recovery times. consensus guidelines agree that patients at high or moderate risk for ponv are most likely to benefit from prophylaxis. patients at low risk for ponv are usually not candidates for prophylaxis unless their condition may be compromised by the medical sequelae or vomiting. those at moderate risk for ponv should receive antiemetic monotherapy or combination therapy. those at high risk should receive combination therapy with two or three antiemetics from different classes. drugs with different mechanisms of action can be combined for optimal efficacy. for example, the -ht -receptor antagonists (more effective against vomiting) can be combined with droperidol (more effective against nausea). a multimodal approach that incorporates both baseline risk reduction and antiemetic therapy should be adopted for ponv prophylaxis. a recent prospective, double blind, randomized, controlled trial compared three strategies for the prevention of ponv in patients undergoing laparoscopic cholecystectomy: ( ) a multimodal approach using ondansetron, droperidol, and total intravenous anesthesia (tiva) with propofol; ( ) a combination of ondansetron and droperidol, with isoflurane and nitrous oxide-based anesthesia; and ( ) tiva with propofol alone. the complete response rate was higher in the multimodal group ( %) than in the combination group ( %) or tiva-only group ( %), as was the degree of patient satisfaction. nausea and vomiting may persist in some patients after they leave the postanesthesia care unit (pacu). after medication and mechanical causes of ponv have been excluded, rescue therapy with antiemetics can be initiated. for patients who received no prophylaxis, low-dose therapy with -ht -receptor antagonists may be initiated. consensus guidelines also recommend low-dose therapy with a -ht -receptor antagonist for patients in whom dexamethasone prophylaxis has failed. for patients in whom initial -ht -receptor antagonist prophylaxis has failed, a -ht -receptor antagonist rescue therapy should not be given within the first h after surgery. similarly, patients in whom prophylactic combination therapy with a -ht -receptor antagonist plus dexamethasone has failed should be treated with an antiemetic from a different class. as a general guideline, patients who experience ponv within h after surgery should be treated with an antiemetic other than the one used for prophylaxis. for the treatment of patients who experience ponv > h after surgery, drugs from the prophylactic antiemetic regimen may be repeated, except for dexamethasone and transdermal scopolamine, which have a longer duration of action. also, propofol may be used in small doses ( mg as needed) for the treatment of ponv in a supervised environment. the preliminary results of a recent analysis support the recommendation that a rescue antiemetic should be from a class other than that of the original antiemetic agent [ ] . this analysis of a previous trial reported that in patients who failed prophylaxis with ondansetron or droperidol, promethazine was significantly more effective in controlling ponv than the original agent. dimenhydrinate was also more effective than droperidol in patients who failed prophylaxis with droperidol. in summary, the first step in the management of ponv is to identify surgical patients at high or moderate risk for ponv, then reduce baseline risk factors in these patients. combination antiemetic therapy is recommended for patients at high risk for ponv for patients at moderate risk, monotherapy or combination therapy may be considered. a multi modal approach for the prevention of ponv including the use of antimetics with different mode of action, hydration and tiva with propofol has been shown to be most effective. patients who have not received prophylaxis and experience ponv can be treated with a low dose of a -ht -receptor antagonist. in patients who fail prophylaxis treatment with an antiemetic, another agent than the one used for prophylaxis is recommended. opioids can induce muscular rigidity, which is due to an increased tone of the striatal muscle. especially, the muscles of the thoracic cage and of the abdomen show this rigidity, a phenomenon, which is observed after the bolus injection of a potent opioid, such as the fentanyl series (i.e. fentanyl, sufentanil, alfentanil and remifentanil; figure ii- ) . increase in muscle tone is directly correlated to the -receptor interaction, because mixed agonist/antagonists and highly selective -opioid antagonists (i.e. ctap), but not -(e.g. nor-binaltorphine) nor -antagonists (e.g. naltrindole) were able to reverse such muscular rigidity [ , ] . in addition, administration of the selective antagonist methylnaltrexone in the nucleus raphe pontis was able to reverse increased muscle tone after alfentanil in the animal [ ] suggesting this nucleus is an additional important site of action of opioids to induce rigidity. clinically this rigidity is a disadvantage because it results in an insufficient ventilation of the patients and is characterized by the following features [ , ] : • it appears shortly after intravenous injection of a potent opioid. • it can be induced especially in the elderly patient population. • it is potentiated by nitrous oxide (n o). • it is more likely to develop in patients with parkinson's disease. the anatomical correlate by which opioids induce muscular rigidity is the striatum, and, being part of the basal ganglion system, it has the task to control locomotion ( figure ii- ) . within the striatum there is a dense accumulation of opioid binding sites, which interact with dopaminergic d -receptors. similar as in parkinson's disease, there is a reduction in the dopamine level with an ensuing imbalance of the cholinergic transmitter system, both of which are in balance with each other and a necessary prerequisite for the control of muscle tone [ ] . while in parkinson's disease, increased muscle tone is induced by decrease of dopaminergic neurons in the striatum, opioid-induced rigidity is due to an enhanced degradation of the transmitter dopamine resulting in a functional deficit of a sufficient level in the nigro-striatal pathway [ ] . the exact mode of action of opioids to reduce dopamine level within the nigrostriatal system and induce muscular rigidity, very likely is induced by inhibition of tyrosine hydroxylase, the necessary enzyme for the synthesis of dopamine [ ] . due to the interconnection with the inhibitory gabaminergic system, output of gaba in the pallidum declines ( figure ii- ) . this, in turn, causes an overactivity of cholinergic neurons projecting to thalamic neurons. from here the area a of the cortical premotor center is activated and the corticospinal tract leads efferents to the anterior horn of the spinal cord [ ] . although opioids do not directly affect muscle tone, rigidity rapidly can be reversed by the injection of a competitive or non-competitive muscle relaxant [ ] . although the increased efferent output at the neuromuscular junction is not reduced, muscle relaxants induce their action by inhibiting the binding of acetylcholine at the motor endplate ( figure ii- ) . because the gabaminergic system in the putamen is involved in the mediation of opioid-induced muscular rigidity, any increase in gabaminergic transmission can also ease this side effect. such a notion has been supported by results, where a benzodiazepine reduced the increased muscle tone, an effect that could be reversed by the specific benzodiazepine antagonist flumazenil [ ] . in addition, significance of the neurotransmitter dopamine in basal ganglia of the cns, which are involved in the regulation of muscle tone = pallidum externum; = putamen; = nucleus caudatus; = thalamus; = hypothalamus; = lobus parietalis; = central grey; = corticospinal tract; = inhibitory dopaminergic pathway; = thalamo-cortical neurons; = substantia nigra since neighboring -receptors interact with the substantia nigra, opioid-related rigidity could be attenuated by the additional administration of the -agonist dexmedetomidine [ ] . the miotic action of opioids on the pupil is an easily recognizable and quantifiable effect in man. the neural pathways responsible for regulating pupil size are reasonably well defined. yet, the mechanisms behind this and related effects of opioids on the eye in humans and laboratory animals have just begun to be explored. opioid-induced miosis in the human, dog and rabbit is thought to be mediated through the central nervous system. this action is a specific opioid effect as demonstrated by its antagonism by naloxone. theories have been advanced suggesting comparison between groups p < . p < . figure ii- . alfentanil-induced truncal rigidity in patients following induction of anesthesia. in comparison to the rapid bolus injection of the drug, slow injection over a period of min resulted in a significant lesser reduction of thoracic compliance. the increase in truncal rigidity was instantly reversed by a low dose ( mg/ kg body weight) of the fast acting muscle relaxant succinylcholine. adapted from [ ] that morphine produces its effects by direct stimulation of the edinger-westphal (preganglionic parasympathetic) nucleus [ ] . an alternative view has been postulated that morphine depresses cortical centers, which normally inhibit the edinger-westphal nucleus. others have suggested that miosis is caused by stimulation of opioid receptors located on the iris sphincter, although this opinion seems to be in the minority. the exact site, or sites, of action within the cns, which are responsible for opioid-induced miosis remain obscure. it is generally accepted, however, that sympathetic innervation is not essential, the miotic effect being entirely dependent on the integrity of the parasympathetic system. for example, lee and wang [ ] have shown that dogs with a sectioned oculomotor nerve fail to show miosis even with a fold increase in the dose of morphine. in contrast, dogs show normal responses following sympathectomy. while local application of a muscarinic antagonist (scopolamine) that blocks the pupil sphincter completely abolishes the pupillary effects of morphine in the rabbit, application of a sympathetic neuronal blocker (guanethidine) or of an alpha-adrenergic antagonist (phentolamine) that block the pupil dilator had no effect in those experiments. thus, pharmacologic dissection of the autonomic innervation of the pupil suggests that opioid-induced miosis is mediated solely through the parasympathetic system. other cns structures may also be involved in opioid-induced miosis. lee and wang [ ] have shown that removal of the cerebral hemispheres potentiates the miotic action of morphine in the dog. they interpreted this effect as being a reflection of the loss of tonic inhibition originating in the occipital lobes. the latter are known to play a regulatory role in pupillary function, particularly with respect to the near-response (accommodation, convergence and miosis) and, hence, their removal might be expected to alter the pupillary response to drugs. the same authors also observed that acute or chronic optic nerve section did not alter the miotic response to morphine in dogs. in humans, it was shown that morphine produced a dose-related miosis under conditions of low ambient light. taken together, these findings suggest that morphine may cause miosis through more than one mechanism. the main neural structures, which are thought to regulate pupillary size are found in the midbrain, mainly the pretectal area and the edinger-westphal nucleus of the oculomotor complex. because neuronal unit activity in the edinger-westphal nucleus has been shown to correlate with light-induced pupillary constriction. opioids therefore depress or abolish spontaneous and light-induced firing of pupilloconstrictor neurons in the pretectal area, while the opposite effect is observed in the edinger-westphal nucleus where a marked increase in spontaneous firing rate resulting in madriasis. it is because of this increase in activity that certain animal species (rat, cat, monkey) demonstrate an opioid-induced mydriasis. in addition, the brain stem region regulating pupil size is known to have multiple inputs, including the cortex and midbrain, and several others can be assumed to exist. depression by morphine of tonic inhibitory input trom the cortex may partially account for the miosis observed by lee and wang [ ] . these findings suggest that opioids may act directly on the neurons subserving the parasympathetic light reflex. also, in contrast to other workers, morphine has no local action on the iris. for example, lee and wang [ ] could not produce miosis by injecting % of an effective systemic dose of morphine ( mg) directly into the anterior chamber of the eye in dogs. although opioid binding sites have been found in the retina of the rat, cow, toad and skate, opioids injected into the anterior chamber may stimulate retinal receptors in some species, causing miosis via reflex parasympathetic output. following oral ingestion, but also after the systemic administration, opioids also bind to selective receptors located within the intestinal tract. the physiological significance of peripherally located opioid binding sites within the intestine is that of regulation of the propulsive transit. the intestine with a total surface of nearly m is an underestimated important anatomical site as it has a high accumulation of neuronal tissue, which has been termed the enteral nervous system (ens), which acts like a second brain. since there is a close interconnection of the ens with the cns via the vagus nerve, regular impulses to and from the ens are being exchanged. anatomically the intestine is surrounded by two separate syncytial, netlike nervous structures. one is the myentericus plexus (auerbach) located between the longitudinal and the circulatory muscle fibers ( figure ii- ) . the second is the submucosal meissner plexus, located between circulatory and the submucosal muscle fibers. within the auerbach plexus of the intestinal tract, there is a balance between the cholinergic and enkephalinergic neurons: binding of systemically applied opioids to enkaphalinergic receptor sites results in an inhibition of transit followed by constipation. contrarily, cholinesterase inhibitors induce an accumulation of acetylcholine at ach-receptors with an increase in motility and an enhancement of transit. presently, however, not very much is known of the long-term effect of central analgesics on opioid-receptors within the myenteric plexus, and if opioid ligands induce only a constipating effect, whether they also depress the immune system in the intestine or result in a distress the neuroregulatory and endocrine function. while analgesia, respiratory depression, bradycardia, antitussive action and miosis all are centrally induced opioid effects, the most relevant peripheral opiod action is that of constipation [ ] [ ] . this is most relevant in patients with chronic pain taking an opioid for its attenuation. being one of the major side effects, it often results in the necessity to take a laxative on a routine basis. the cause for such constipation is the constriction of the pylorus resulting in a delay in emptying of the stomach [ ] . however, most important, opioids induce a constriction of the small intestine resulting in a delay of the propulsive transit. because selective opioid binding sites are mainly located in the small intestine, opioids inhibit the release of local acetylcholine [ , , ] , followed by a concomitant loss of coordinated propulsive movements of the gut. a constipating effect of opioids on the large intestine is of significantly lesser degree, because this part of the intestinal tract contributes to a much lesser extent to the overall constipating effect. this is because continuous propulsive movements are not seen on this area, and contrary to the small intestine, the percentage of enkephalinergic neurons is significantly lower [ , ] . in addition, enkephalin derivatives are able to inhibit transit in the small while at the same time increasing contractions in the large intestine [ ] . systemicallyselective applied opioids therefore primarily interact with enkephalinergic neurons in the antrum, the duodenum, and the small intestine, all of which results in a delay of transit [ , , , ] . the constipating effect of an opioid can be reversed by a selective peripheral acting antagonists such as methylnaltrexone [ , ] or alvimopane [ ] . opioids, which interact primarily with the -opioid receptor induce a lesser constipating effect [ , ] , while -selective ligands induce no effect on gastrointestinal transit [ ] . comparable to a ketamine-or a volatile anesthetic based regimen, an opioid-based anesthesia results in a longer delay of gastrointestinal emptying in the postoperative period [ , ] (figure ii- ) . this, however, is clinically of little significance, as the potential constipating effect does not last longer than h after anesthesia. contrarily to many other anesthetics, opioids in general do not depress the cardiovascular system. this is also reflected in the higher therapeutic range (ld /ed ) being derived in the animal (table ii- ) . such data can also be conveyed to the human, since a wide therapeutic margin of safety is directly correlated with a lack in cardiovascular impairment. while carfentanil, with a potency twice that of sufentanil, is solely used in veterinary medicine for the immobilization of wild animals [ ] , lofentanil ( fold potency of fentanyl), due to its intensive receptor binding, is characterized by a duration of action of h [ ] . both fentanyl derivates are not in clinical use, because the high potency and the intense receptor binding would be difficult to handle in patients. from the table, however, it is obvious that the higher the selectivity to the receptor site, and the higher the potency, the lesser the amount of cardiovascular depression [ , , , , , ] . following the injection of potent opioids, bradycardia is the most prominent cardiovascular effect seen in patients. this is due to a direct central stimulation of the nucleus nervi vagi and a typical effect of -ligands. thereafter, a reduction of the sympathetic drive is initiated resulting in an overexpression of parasympathetic activity. also, a direct peripheral negative inotropic activity with a potentiation of acetylcholine release at the sinus node of the heart is discussed [ ] . the increase in vagal tone and the reduction of sympathetic drive on the peripheral vasculature results in a decline of mean arterial pressure. a reduction of sympathetic tone on vessel tone and a reduction of resistance is also termed as "pooling" of circulating blood volume. such a reduction of peripheral resistance in certain cases may be of benefit for the patient, as it is accompanied by a reduction in afterload of the heart [ , ] . bradycardia, the reduced peripheral resistance (i.e. afterload of heart) as well as the pooling effect with a reduction of preload of the heart, can be of benefit for a patient with myocardial infarction. this is because those three variables are major determinants in myocardial oxygen consumption (mvo ) [ , ] . it, however, should be noted that the sympatholytic action with pooling of blood volume induced by potent opioids might demask a previously compensated hypovolemic condition in a patient resulting in significant hypotension. for instance, in patients with multiple trauma a reduced dose of the opioid should be given, either diluted or slowly injected while measuring blood pressure continuously. in general, however, especially in polytraumatized patients, opioids are of benefit, as they reduce the stress-related release of hormones and particularly of angiotensin ii, maintaining the effect of circulating catecholamines on the vasculature. opioid-related bradycardia with an accompanying hypotension can rapidly be reversed with increasing doses of the vagolytic agent atropine ( . - - . mg/kg body weight). the incidence and the severity of such a drop in blood pressure cannot be foreseen. it is related to the autonomic basal tone of the patient and the dose of the injected potent -ligand ( figure ii- ) . depending on the product, the autonomic basal tone of, and the applied dosages to the patient, either parasympathetic (inhibitory) and/or a sympathetic (excitatory) symptoms are induced (table ii- ) . such clinical effects can be diminished by atropine, an -blocker (e.g. phenoxybenzamine), a ß-blocker (e.g. propranolol), and a ganglionic blocker (e.g. hexamethonium) respectively [ ] . the stimulatory effects of opioids can also be explained in the laboratory where stimulation of cyclic amp formation, phosphinoside hydrolysis, and the elevation of intracellular calcium, resulting from mobilization of calcium stores and by stimulating influx, which leads to an increased neurotransmitter release and neurotransmission [ ] . thus, at the cellular level these changes may underlie the opioid stimulatory effect. in addition, such stimulation is also discussed as playing a part in the development of tolerance to opioid drugs [ ] . the effect of increasing doses (mg/kg body weight) of potent opioids on the cardiovascular system of the canine, where low amounts result in parasympathetic activation, and high to massive doses induce an increase of sympathetic drive. adapted from [ ] in opioid-based anesthesia, vagal-or sympathetic-induced side effects can be reduced or eliminated by the following techniques: . the preliminary administration of atropine (up to mg/kg body weight). . the simultaneous administration of a volatile anesthetic (n o, enflurane, desflurane, sevoflurane). . the simultaneous use of a neuroleptic agent (e.g. droperidol, haloperidol). . the simultaneous use of a benzodiazepine (e.g. diazepam, midazolam, lorazepam). . the simultaneous use of a hypnotic (e.g. barbiturate, etomidate, propofol). adapted from [ , ] all these agents induce a depression of cns activity in different areas of the central nervous system, which results in equilibrium of the autonomic nervous system discharge, thus, reducing the overshoot of sympathetic and/or parasympathetic tone ( figure ii- ) . mixed agonist/antagonists, when given in dosages above the therapeutic range, induce a cardiostimulatory sympathomimetic effect, which purportedly is induced via stimulation of -receptor sites [ ] . as a result, tachycardia, an increase in peripheral vascular resistance, and an increase in pulmonary artery pressure are induced (table ii- ) , all of which increase myocardial oxygen consumption (mvo ). therefore agonist/antagonists should not be given above their therapeutic range in patients with mi or with a preexistent cardiovascular disease [ ] . a malfunction at the atrio-ventricular node in the myocardium, followed by prolongation of the p-q interval is a phenomenon, which can be induced in patients demonstrating a preexisting abnormal conduction system in the heart. such prolongation manifests itself especially when potent opioids are being administered (fentanyl, sufentanil), whereby the opioid-induced acetylcholine release induces a stimulation of vagal activity. thus, patients already having a prolongation of p-q time or who present a sick-sinus syndrome, extreme bradycardia has to be anticipated, which could result in concomitant cardiac arrest. in order to prevent such a scenario, the opioid should not be given as a bolus, but rather as a diluted solution. in addition, the solution should be injected slowly over a long period of time neuroleptics block afferents from entering the ascending reticular formation, which increase vigilance; tranquillizers protect the hippocampus from an excitatory activation, while barbiturates, hypnotics and volatile anesthetics primarily block the cerebral cortex from arousal table ii- . different cardiovascular effects of -ligands, mixed agonist/antagonists, and partial agonists resulting in a decrease (⇓) or an increase (⇑) blood pressure heart rate pulmonary artery pressure adapted from [ , , ] of at least min. if, however, extreme bradycardia is recognized on the monitor, atropine is the agent of choice ( . - . mg/kg body weight) for rapid reversal. in very extreme cases, the antiarrythmic agent metaproterenol may become necessary, as it is able to increase atrio-ventricular conduction. high doses of methadone or its derivative -levoacetylmethadol (laam) may result in life threatening torsades de points with the potential of ensuing ventricular fibrillation. predisposing factors for the development of such a situation are a prolongation of atrio-ventricular conduction time, hypopotassemia, and/or the simultaneous intake of agents, which inhibit metabolism of the opioid (e.g. tricyclic antidepressants, imidazol derivatives, antimalaria agents, or antihistaminics). a direct negative inotropic effect on the myocardium has been demonstrated in the isolated papillary muscle and in the langendorff preparation of the heart for a variety of opioids [ , ] . such direct effects, however, are not of clinical significance, because such a depression is only evident in concentrations above the therapeutic range. in addition, compensatory cardiovascular and the autonomic regulatory mechanisms come into play when an opioid is given to a subject. following the intravenous injection of pethidine (meperidine, usp), hypotonia and syncope may result. because of the atropine-like molecular structure of this agent, tachycardia, as well as reflex bradycardia can be observed [ ] . for the reason of these potential side effects pethidine should not be given to patients with myocardial infarction [ ] . in addition, it is observed that in a shock-like situation, due to the release of endogenous opioids (enkephalins, endorphins), the additional administration of an exogenous opioid results in an additional occupation of opioid binding sites within the myocardium. this aspect is followed by a negative inotropic effect with an unfavorable consequence on hemodynamics [ ] . some experimental work has postulated a putative direct negative inotropic effect of n o in an opioid-based anesthetic regimen [ ] . since this is mainly seen when n o is given in concentrations above % with a resultant drop in fio , this very both agents dose-dependently reduce adrenaline-induced ventricular extrasystoles. adapted from [ ] pvc-premature ventricular countraction likely is due to an insufficient myocardial oxygen supply. in addition, high concentrations of n o have a direct vasodilatory effect, resulting in a reduction of venous return to the heart and a drop in blood pressure [ ] . it therefore is advocated that in patients receiving opioid anesthesia with a preexisting cardiovascular disease, the optimal concentration in fio should be around . . opioids also have been demonstrated to induce an anti-arrhythmic effect. this has been shown in the animal for meptazinol [ ] and in experimental coronary artery occlusion, using fentanyl, sufentanil and carfentanil respectively [ , , ] (figure ii- ). the reason for such an antifibrillatory effect seems to be due to the increase in vagal tone [ ] . international union of pharmacology. xii. classification of opioid receptors separation of opioid analgesia from respiratory depression: evidence of different receptor mechanism opiate receptors: different ligand affinity in various brain regions identification and characterization of three new alternative spliced mu-opioid receptors the effects of morphine and nalorphine-like drugs in the nondependant and morphine-dependant chronic spinal dog the d-and the l-isomers of methadone bind to the non-competitive site on the nmda receptor in the rat forebrain and spinal cord plasma concentrations of codeine and its metabolite morphine, after single and repeated oral administration novel receptor mechanisms for heroin and morphine- ß-glucoronide analgesia the intrinsic antinociceptive effects of oxycodone appear to be k-opioid receptor mediated ketobemidone, methadone and pethidine are non-comptitve n-methyl-d-aspartate (nmda) antagonists in the rat cortex and spinal cord the binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties selectivity of ligands for opioid receptors, in opioids i. handbook of experimental pharmacology comparison between epidural fentanyl, sufentanil, carfentanil, lofentanil and alfentanil in rats: analgesia and other in vivo effects interaction of -opioid receptor agonists and antagonists with the analgesic effect of buprenorphine in mice european federation of the international association for the study of pain chapters opiate receptor: demonstration in nervous tissue multiple opiate receptors: support for unique mu, delta and kappa sites autoradiographic localization of opiate k-receptors in the guinea pig brain autoradiographic localization of kappa opiate receptors to deep layers of the cerebral cortex may explain unique sedative and analgesic effects characterization of opioid receptors in nervous tissue buprenorphine causes ceiling effect in respiratory depression but not in analgesic effect the clinical pharmacology of buprenorphine: extrapolating from the laboratory to the clinic clinical pharmacology of buprenorphine: ceiling effects at high doses specific uptake of narcotic analgesics by subcellular fractions of the guinea pig ileum in vitro models in the study of structure-activity relationships of narcotic analgesics Über die antagonistischen systeme der schmerzempfindung und des schmerzgefühls im peripheren und zentralen nervensystem a morphine-like factor "enkephalin" in rat brain: subcellular localization determination of methionine enkephalin in discrete regions of rat brain pain mechanisms: a new theory neurotransmitter receptor binding in the brain interaction of naloxone with mu-and delta-opioid agonists on respiration of rats h-sufentanil, a superior ligand for the mu-opiate receptor: binding properties and regional distribution in rat brain and spinal cord alfentanil (r ) -a particularly shortacting narcotic analgesic in rats n- -substituted -( arylethyl)- -piperidinyl-nphenylpropanamides, a novel series of extremely potenet analgesics with unusually high safety margin neuropathic pain: aetiology, symptoms, mechanisms, and management current views on the role of psychiatrists in the management of the chronic pain psychodynamics and psychotherapy in the treatment of patients with chronic pain the effects of morphine and meperidine on the central respiratory mechanisms in the cat: the action of levallorphan in antagonizing these effects respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem fentanyl in the fourth cerebral ventricle causes respiratory depression in the anesthetized but not in the awake dog pharmacokinetics of naloxone in rats and man. basis for its potency and short duration of action prevention of late fentanyl-induced respiratory depression after the injection of the opiate antagonists naltrexone and s- as compared to naloxone pharmacokinetics of fentanyl as a possible explanation for recurrence of respiratory depression analgesic effectiveness of the narcotic agonist-antagonists opioid agonists, antagonists and mixed narcotic analgesics: their use in postoperative and chronic pain management reversal of narcotic-induced respiratory depression with nalbuphine hydrochloride reversal of fentanyl-related respiratory depression with nalbuphine; effects on the co -response curve of man antagonism of postoperative opioid-induced respiratory depression: nalbuphine versus naloxone nalbuphine reverses fentanyl-related eeg changes in man prolonged antagonism of opioid action with intravenous nalmefene in man antagonism of fentanyl-induced respiratory depression with nalmefene enkephalins: isolation, distribution and function multiple opioid receptors mediate the respiratory depressant effect of fentanyl-like drugs in the rat minireview: multiple mu opiate receptors multiple morphine and enkephalin receptors and the relief of pain differences in magnitude and duration of opioid induced respiratory depression and analgesia with fentanyl and sufentanil mu and delta receptors: their role in analgesia and in the differential effects of opioid peptides on analgesia opioid-induced respiratory depression and analgesia may be mediated by different subreceptors the delta receptor is envolved in sufentanilinduced respiratory depression delta opid receptor enhancement of mu opioid receptor-induced antinociception in spinal cord the pharmacokinetics and pharmacodynamics of gi b. anesthesiology context-sensitive half-time in multicompartment pharmacokinetic models for intravenous anesthetic drugs influence of cerebral activity in wakefulness on regulation of breathing waking and ventilatory responses to laryngeal stimulation in sleeping dogs respiratory safety association des analgésiques centraux et des neuroleptiques en cours d'intervention part ii drug interactions of clinical significance with opioid analgesics drug interaction and side effects index™ biotransformation von fentanyl. ii. akute arzneimittelinteraktion -untersuchungen bei ratte und mensch the magnitude and the duration of respiratory depression produced by fentanyl and fentanyl plus droperidol in man a quick guide to common drug interaction, in patient care plasma protein binding of phenytoin after cholecystectomy and neurosurgical operations intravenous fentanyl kinetics morphine and phenytoin binding to human plasma protein in renal and hepatic failure plasma concentrations of fentanyl in normal surgical patients and those with severe renal and hepatic disease reduction in the incidence of awareness using bis monitoring bremazocine: a potent, long-acting opiate kappa-agonist tifluadom (kc- ) induces suppression and latency changes of somatosensory-evoked potentials which are reversed by opioid antagonists bremazocine: an opiate which induces sedation and analgesia but no respiratory depression cellular distribution of the mrna for the k-opiod receptor in the human neocortex: a non-isotopic in situ hybridization study opiate receptor binding sites in human brain utilisation pratique des analgesiques centraux en anesthesie et reanimation patient-controlled analgesia using butorphanol for postoperative pain control: an open label study nalbuphine versus pentazocine in postoperative pain after orthopedic surgery the enflurane sparing effect of morphine, butorphanol, and nalbuphine mac reduction of enflurane and isoflurane and postoperative findings with nalbuphine hcl and fentanyl: a retrospective study ceiling effect for respiratory depression by nalbuphine the "narcotic" component of fentanyl. l'anesthese vigile et subvigile die hypnotische wirkung von fentanyl und sufentanil kardiovaskuläre und zentralnervöse effekte unter fentanyl versus sufentanil bei der intubation herzchirurgischer patienten eeg findings in neuroleptanalgesia central nervous effects of neuroleptanalgesia as induced by haloperidol and phenoperidine exzitatorische und inhibitorische phänomene am zentralnervensystem, verursacht durch fentanyl perfusion of the fourth cerebral ventricle with fentanyl induces naloxone reversible hypotension, bradycardia, baroreflex depression and sleep in unanaesthetized dogs regional distribution of opiate receptor binding in monkey and human brain the effects of morphine and pethidine on somatic evoked responses in the midbrain of the cat, and their relevance to analgesia wechselwirkungen zwischen dem system der schnellen schmerzempfindung und dem des langsamen Über die zweiteilung der schmerzempfindung und des schmerzgefühl, in schmerz comparative study of cardiovascular, neurological, and metabolic side effects of eight narcotics in dogs genralized grand mal seizure after recovery from uncomplicated fentanyl-etomidate anesthesia another case of grand mal seizure after fentanyl aministration tonic-clonic activity after sufentanil seizure-like movements during fentanyl infusion with absence of seizure activity in a simultaneous eeg recording the effects of high-dose fentanyl on cerebral circulation and metabolism in rats opioid analgesics and antagonists, in the pharmacological basis of therapeutics diagnose and treatment of symptoms of the respiratory tract h-codeine binding in the guinea pig lower brain stem physiology and pharmacology of vomiting treatment of postoperative nausea and vomiting after outpatient surgery with the -ht antagonist ondansetron comparison of ondansetron versus placebo to prevent postoperative nausea and vomiting in women undergoing ambulatory gynecologic surgery coinsensus guidelines for managing postoperative nausea and vomiting postoperatives erbrechen -ein score zur voraussage des a meta-analysis of nausea and vomiting following maintenance of anaesthesia with propofol or inhational agents dexamethasone for the prevention of postoperative nausea and emesis-a quantitative systemic review food and drug admistration black box warning on the perioperative use of deoperidol: a review of the cases effect of low-dose droperidol on the qt interval during and after general anesthesia: a placebo-controlled study prolongation of qtc interval after postoperative nausea and vomiting treatment by droperidol or ondansetron the effectiveness of rescue antiemetics after failure of prophylaxis with ondansetron or droperidol: a preliminary report role of opioid receptors in the substantia nigra in morphine-induced muscular rigidity antagonism of dermorphin-induced catalepsy with naloxone, trh-analog cg and the benzodiazepine antagonist, ro - catatonia" produced by alfentanil is reversed by methylnaloxonium microinjections into the brain abdominal muscular rigidity induced by morphine and nitrous oxide studies in muscular rigidity, nitrous oxide and narcotic analgesic agents the effect of fentanyl and droperidol on the dopamine metabolism of the rat striatum morphine katalepsy in the rat: relation to striatal dopamine metabolism tyrosine hydroxylation in the rat striatum after fentanyl and droperidol in vivo decrease of neocortical choline acetyltransferase after lesion of the globus pallidum in the rat attenuation of fentanyl-induced truncal rigidity die lungencompliance wird durch die rasche injektion von alfentanil beeinträchtigt dexemedetomidine, acting through central alpha -adrenoceptors, prevents opiate-induced muscle rigidity in the rat gastrointestinal effects of opioids naloxone and morphine inhibit gastric emptying of solids effect of synaptic transmission blockade on morphine action in the guinea pig myenteric plexus enkephalin-like immunoreactivity in the human gastrointestinal tract autoradiographic localisation of opiate receptors in rat small intestine influence of opiates on colonic motility relative involvement of mu, kappa, and delta receptor mechanisms of opiate-mediated antinociception in mice inhibition of gastrointestinal transit by morphine in rats results primarely from direct drug action on gut opioid sites effect of morphine on gastric emptying a comparison of the effect of oral controlled release morphine and intramuscular morphine on gastric emptying methylnaltrexone prevents morphine-induced delay on oral-cecal transit time without affecting analgesia: a double-blind randomized placebo-controlled trial oral methylnaltrexone for opioid-induced constipation effects of adl - , a peripherally restricted mu opioid anntagost, on gut motility in methadone and laam-dependent patients with opioidinduced constipation. a dose-ranging study the effects of morphine and nalbuphine on intestinal transit in mice peptide opioid antagonist seperates peripheral and central opioid antitransit effects die gastro-coekale transitzeit nach fentanyl/midazolam-im vergleich zur enfluran-und ketamin/midazolam-narkose keine hemmung der intestinalen motilität nach ketamin-/midazolamnarkose immobilization of free-ranging wild animals using a new drug carfentanil and lofentanil sufentanil, a very potent and extremely safe intravenous morphine-like compound in mice, rats and dogs the development of new synthetic narcotics pharmacotherapy of opioids: present and future developments hämodynamische wirkungen hoher dosen von fenanyl, meperidine und naloxon beim hund cardiovascular effects of high doses of fentanyl, meperidine and naloxone in dogs filling pressures of the heart and pulmonary circulation of the patient with coronary artery disease after large doses of morphine control of myocardial oxygen consumption utilisation pratique des analgésiques centraux en anesthésie et réanimation determinants of the stimulatory opioid effect on transmitter release and possible cellular mechanisms: overview and original results stimulatory effects of opioids sigma receptor ligands: function and activity. neurotransmissions comparative effects and analgesic efficacy of the agonist-antagonist opioids a review of its pharmacological properties and therapeutical uses, in new drug series contractile responses to morphine, piritramid and fentanyl: a comparative study of effects on the isolated myocardium myocardial opiate receptor activity is stereospecific, independent of muscarinic receptor antagonism, and may play a role in depressing myocardial function utilisation de la pentazocine comme analgesique pour le traitement des douleurs post-operatoires. etude comparative entre le pethidine, la piritramide et la pentazocine, in utilisation de la pentazocine en anesthesie et reanimation hemodynamic changes following corticosteroid and naloxone infusion in dogs subjected to hypovolemic shock without resuscitation nitrous oxide as an adjunct to narcotic anesthesia does nitrous oxide or a reduced fi alter the hemodanymic function during high dose sufentanil anesthesia? the antiarrhythmic effect of meptazinol effects of high doses of fentanyl on myocardial infarction and cardiogenic shock in the dog the antifibrillatory effect of fentanyl, sufentanil, and carfentanbil in the acute phase of local myocardial ischemia in the dog antifibrillatory action of the narcotic agent fentanyl protective effect of the vagotonic action of morphine sulfate on ventricular vulnerability les effets anti-arrythmiques des opiaces. comparison avec un beta-bloqueur chez le chien. cah d'anesthesiol opposite pupillary effects in the cat and the dog after microinjection of morphine, normorphine and clonioline in the edinger-westphal nucleus mechanism of morphine -induced miosis in the dog key: cord- -x qozizw authors: kato, masamichi title: radiofrequency biology: in vivo date: journal: electromagnetics in biology doi: . / - - - - _ sha: doc_id: cord_uid: x qozizw nan carcinogenesis is a complex, multistage process. normal cells develop into tumor cells as a result of genetic changes (initiation stage). once a cell has been initiated, typically by the accumulation of genetic changes as a result of damage and faulty dna repair, several kinds of chemicals and physical forces, such as ionizing radiation, can act as "promoter" (or co-promoter) to increase the rate of growth of tumor cells (promotion stage). la regina et al. ( ) carried out a study to determine whether chronic exposure to microwave fields from cellular phones increased incidence of spontaneous tumors in f rats. eighty male and female rats were randomly placed into one of three groups. the sham-exposed group received no irradiation; the frequency division multiple access (fdma) group was exposed to . mhz, fdma-modulated microwaves; and the code division multiple access (cdma) group was exposed to . mhz, cdma-modulated microwaves. rats were exposed for h/d, d/w for over yrs. the nominal, time-averaged brain sar was . w/kg. there were no differences in final body weights or survival for either males or females in any group. no differences were found between treated and sham-exposed animals for any tumor in any organ. the authors conclude that chronic exposure for up to years to . mhz (fdma) or . mhz (cdma) microwaves had no effect on the incidence of spontaneous tumors or the initiation stage of carcinogenesis. rat liver is the most commonly used experimental model for investigating multistage carcinogenesis in tissues. imaida et al. ( a) reported on a medium-term liver bioassay in which near-field exposure of f male rats to mhz or to . ghz electromagnetic fields resulted in slightly decreased numbers and areas of given its critical importance for humans, the effect of exposure to rf energy on the central nervous system (cns) has been studied extensively. the following sections review experiments assessing brain morphology, the blood-brain barrier (bbb), electroencephalogram (eeg), evoked potentials, behavior, the hippocampus, microwave field detection, and neurotransmitters. very few investigators have assessed the cellular morphology of brains of animals that had been exposed to microwaves. perhaps this is because it is a daunting challenge, and perhaps because there is little reason to expect any change when the microwave doses used do not produce excessive heating. albert et al. ( b) studied the effects of either . ghz (sar of . w/kg) or mhz (sar w/kg) applied to young rats. then the histological appearance of cerebellar purkinje cells was assessed. environmental stress, hypoxia, alcohol or fatigue can easily damage these cells, and glia cells subsequently replace the damaged neurons. exposure to both frequencies had similar effects, producing irreversible decreases of purkinje cells in rats irradiated either during fetal or fetal and early postnatal periods. decreases in the relative number of purkinje cells were apparent in animals exposed postnatally. tsurita et al. ( ) investigated the effects of exposure to . ghz tdma (time division multiple access) signals on purkinje cells in cerebellum of rats. mature male sd rats were divided into three groups of eight. the rats in the fieldexposed group, which had their heads arrayed in a circle near the central antenna of an exposure system, were exposed to microwaves for h/day for either or wks. the rats in the sham-exposed group also were placed in the exposure system, but no microwave exposure was given. the cage-control group was neither placed in the system nor exposed. the sar for the brain was w/kg; the whole body sar was . w/kg. no morphological changes were observed in any group. the different outcomes from albert et al. ( b) and from tsurita et al. ( ) might be due to the very different ages of the rats used. albert et al. ( a) exposed pregnant squirrel monkeys to . ghz microwaves (sar . w/kg) for h/d, d/w; post-natal exposure continued until the offspring were . months of age. they then studied the effects of microwave exposure on the density of purkinje cells in the cerebellar uvula. in contrast to the rat data (albert et al. b) , there was no effect on the monkey's purkinje cells. albert et al. ( a) point out that differences in the anatomical structures of the head and in the exposure conditions (free field vs. multipath) could have contributed to the difference in results. also, . ghz might be closer to resonant frequency of the rat than the squirrel monkey, and the depth of penetration of rf energy can be important. further the structure of the brain in each species presents a different exposure configuration. in the rat, the cerebellum is exposed in the dorsal and posterior aspect and is covered by a thin calvaria. in the squirrel monkey, the cerebellum is overlapped by the occipital lobes, a thicker calvaria, and thick muscles in the cervical region (albert et al. a ). based on these three studies, it is impossible to know whether or not microwave exposure can affect the microscopic anatomy of regions of the brain. however, based on the relatively low energy levels and the presumed lack of tissue heating associated with them, it is unlikely that environmental exposures produce frank brain pathology. presumably experiments associated with ongoing research programs intended to address the safety of cell phones will produce a few more studies relevant to this specific question. the blood-brain-barrier (bbb) maintains the homeostatic environment of the brain by regulating the entry of vital substances and nutrients into brain and the expelling of carbon dioxide and metabolic waste products out of the brain. this barrier protects the brain from foreign toxic substances but allows passage of the molecules that are necessary for metabolism. brain capillaries are composed of endothelial cells, pericytes, that have smooth muscle-like properties and reside adjacent to capillaries, and of astroglial processes that ensheath more than % of the capillary surface (fig . ). among these structures, the endothelial cells are the principal anatomic site of the bbb. the endothelial anatomical features of general capillaries and of brain capillaries. general capillaries have inter-endothelial clefts, fenestrae, and prominent pinocytotic vesicles. these features allow relatively non-selective diffusion across the capillary wall. endothelial cells of the brain capillary contain an increased number of mitochondria to support energy-dependent transport systems and are inter-connected by complex, inter-endothelial "tight" junctions. these anatomical features, in conjunction with specific transport systems, result in highly selective transport of water-soluble compounds across the barrier endothelium. astrocyte processes surround more than % of the brain capillaries. functionally, this results in what is called the blood-brain barrier. cells of the bbb are interconnected by complex arrays of "tight" junctions. these junctions block diffusion across the capillary wall. in capillaries of other organs, and in the relatively few brain capillaries that do not form a barrier, blood-borne, polar molecules diffuse passively across vessels through "spaces" between endothelial cells, i.e., through specialized cytoplasmic fenestrations. normal brain function requires a large number of compounds that must be able to cross brain capillaries. entry into the cerebrospinal fluid is achieved primarily in three ways: ( ) by diffusion of lipid-soluble substances, ( ) by energy-dependent, receptor-mediated transport of specific, water-soluble substances, and ( ) by ion channels. most substances that must cross the bbb are not lipid soluble and therefore cross by specific carrier-mediated transport system. glucose is transported through the glut system, which is not energy dependent. the net flux of glucose is driven by the relatively higher concentration of glucose in plasma. amino acids are transported across barrier endothelial cells primarily by three distinct carrier systems: the l, and asc systems. for further study, the readers are advised to consult with textbook of neuroscience (e.g., kandel et al ) in many diseases the bbb does not function effectively and substances that are normally excluded enter the brain. a number of causes -such as inflammation of brain membrane (i.e., meningitis), edema, anoxia, hypertension, and ionizing radiation -have been shown to induce bbb changes, often increasing permeability of substances to the brain. radiotracer, immunohistochemistry, or the classical staining method by evans blue often are used to reveal permeability changes. with a functional bbb, evans blue does not enter the brain. thus, when areas of brain are stained blue, it indicates a breakdown of the bbb in those areas. the existence of a microwave-induced bbb permeability increase was controversial for many years. now most researchers believe that the permeability change is associated with an increase in temperature, and no permeability change is observed with athermal microwave exposure. ikeda et al. ( ) used mhz rf exposure to investigate the distributions of temperature changes produced by interstitial hyperthermia in agar phantoms and in brains of dogs. the heating limits of normal dog brains were • c for min or • c for min; breakdown of the bbb, assessed using evans blue, was observed with heating at • c for min. ohmoto et al. ( ) used rats to investigate the temperature distribution, early histological changes, bbb disruption, and sequential changes in cerebral blood flow (cbf) following hyperthermia, ranging from to • c, produced by rf-induced, localized cerebral hyperthermia. histological changes and bbb disruption were observed in brain regions heated to • c and above. fritze et al. ( ) investigated the effects of gsm microwave exposure on the permeability of the bbb. rats were restrained in a carousel of circularly arranged plastic tubes and sham-exposed or microwave irradiated at mhz for h at sars of . , . or . w/kg. the extravasation of protein was assessed, either at the end of exposure or days later, by immunohistochemistry staining of serum albumin. an increase in serum albumin extravasation after microwave exposure was observed only in the group exposed to the highest sar of . w/kg and the extravasation was present only immediately after exposure. histological injury was not observed in any of the examined brains. the observed albumin extravasations were very modest and, moreover, were totally reversible. finnie et al. ( ) studied the effect of short-term exposure to gsm microwaves on vascular permeability in the brain. mice (n = ) were given a single, far-field, whole-body exposure at . mhz for min at a sar of w/kg. control mice were either sham-exposed (n = ) or permitted free movement in a cage (n = ) to exclude any stress-related effects. vascular permeability changes were detected using albumin immunohistochemistry. no differences among groups were detected. finnie et al. ( ) further studied the effect of long-term exposure to gsm fields at mhz. mice were given a min, far-field, whole-body exposure dy/wk for wks at sars of . , . , . , or . w/kg. albumin staining was used to detect increased permeability. the results suggest that prolonged exposure to mobile telephone-type radiation produced negligible disruption to bbb integrity under these experimental conditions. tsurita et al. ( ) investigated the effects of athermal exposure to . ghz modulated by the tdma protocol on the permeability of the bbb. adult male sd rats were assigned to one of three groups of eight rats. the rats in the microwave-exposed group were exposed hr/dy. the rats in the sham-exposed group were placed in the exposure system without microwave delivery. the animals in the cage control group were not placed in the exposure system. the exposure period was or wks. the sar was w/kg in the brain, and the whole body sar was . w/kg. core body temperature was measured; no increase in body temperature was detected. changes in the bbb were sought using evans blue injection and immunostaining of serum albumin. microwave exposure had no effect on the bbb. contrary to the negative findings of these five papers, two other groups have reported that non-thermal microwave irradiation did cause bbb permeability changes. salford et al. ( ) reported that weak, non-thermal level, pulsed microwaves give rise to a significant leakage of albumin through the bbb. salford et al. ( ) further investigated whether a pathologic leakage across the bbb might be accompanied by neuronal damage. three groups each of eight rats were exposed for hr to a gsm microwave field at whole-body sars of , , and mw/kg, all well below the level expected to produce temperature elevation. the authors found a positive relationship between sar and number of "dark" neurons, evidence for neuronal damage, in the cortex, hippocampus, and basal ganglia of exposed rats. the authors claim that they presented, for the first time, evidence for neuronal damage caused by a nonthermal microwave exposure. leszczynski et al. ( ) examined whether athermal exposures of cultures of a human endothelial cell line to mhz gsm radiation could activate a cellular thermal stress response. non-thermal ( ± . • c), hr microwave exposure caused a transient increase in phosphorylation of heat shock protein- . the authors hypothesized that mhz gsm radiation induced activation of heat shock protein- to cause an increase in bbb permeability through stabilization of endothelial cell stress fibers. efforts to reproduce the results of salford et al. were reported by four groups at the annual meeting of the bioelectromagnetics society. mcquade et al. ( ) and haro et al. ( ) examined albumin leakage and degenerative neurons, which were originally reported by salford's group. both parties found no differences between the microwave-exposed and sham-control groups. two other groups also assessed other measures in addition to albumin leakage. shirai et al. ( ) examined alteration of bbb-related genes, such as p-glycoprotein, aquaporin- , and claudin- , and masuda et al. ( ) studied microcirculation. neither group found any differences between the irradiated and the control animals. combined effects of microwave irradiation and other agent(s) have been reported by two groups. neilly and lin ( ) studied the combined effects of ethanol and microwaves on the bbb in male wister rats. anesthetized rats, each with an implanted venous cannula, were infused with . , . , . or . gm/kg of absolute ethanol. a control group was given . g/kg of isotonic saline. the left hemisphere of each brain was irradiated by . ghz microwave energy at . w/cm (rms) for min. the rectal temperatures remained at . • c. immediately after irradiation, evans blue dye was injected through the cannula. the results showed that as the quantity of alcohol was increased, the degree of staining was decreased or eliminated. the temperature of the irradiated area of the brain increased for the first to min of irradiation and then stabilized for the remainder of the irradiation period. the steady-state temperature was highest in animals receiving saline or the smallest dose of alcohol. as the quantity of alcohol was increased, the steady-state temperature was reduced. these results indicate that ethanol inhibits microwave-induced permeation of the bbb through reduced heating of the brain. there is another report which investigated combined effect of microwave exposure and virus infection. the expression of japanese encephalitis virus (jev) lethality in mice requires entry of the virus into the cns. this entry is presumably through the capillary endothelial cells, because entry between these cells is inhibited by bands of circumferential tight-junctions. a viremic stage occurs during the first to days after virus administration in mice. lange and sedmak ( ) assessed how both microwave radiation ( . ghz, continuous wave, min exposure) and hypercarbia (co exposure) affected capillary endothelial cells permeability to jev in adult swiss-cox mice. exposure to microwaves at very high sars of approximately - w/kg resulted in a dose-dependent increase in jev-induced lethality. similarly, hypercarbia produced by exposure to , , and % co was observed to produce a dose-dependent increase in virus-induced lethality. both microwave radiation and hypercarbia are thought to promote pinocytosis within capillary endothelial cells of the cns. this may be one mechanism by which they enhance jev-induced lethality in adult swiss-cox mice. historically, a reasonably sized set of papers has addressed the possibility that microwave exposure can diminish the effectiveness of the bbb. the pattern of results is clear. if microwave exposure produces hyperthermia, bbb integrity is diminished. if the microwave exposures used do not produce hyperthermia in the brain, i.e., if they are athermal, there is no effect. the salford saga appears to be a recapitulation of a process that has occurred repeatedly over the past four decades. some group claims an "athermal" effect. because this would be important, if true, other investigators rapidly attempt to replicate the claim. invariably, the alleged athermal effect is not substantiated. the human eeg changes according to a h circadian rhythm of behavior in response to the h astronomical cycle. sleep states lasting approximately h during night form the unconscious part of that cycle. a basic principle of sleep cycle control in human has been articulated by borbely ( ) as a "two process model," in which sleep-wake state transitions result from the combined effects of circadian factors and homeostatic factors. during sleep, a third regulator, the ultradian, rem-nrem oscillator comes into play. in terms of eeg, sleep comes in two forms, rapid eye movement (rem) -when dreaming occurs -and non-rem (nrem). johnson and guy ( ) demonstrated thermographically that metal electrodes in a cat brain increased the local sar by times. therefore the use of metallic elec-trodes for eeg recordings made most early results questionable. glass electrodes filled with ringers solution (johnson and guy ) or carbon-loaded teflon electrodes with conductivity close to that of tissue have been used to minimize field perturbation (chou and guy ) . even with minimally field perturbing electrodes, eeg electrodes can pick up rf fields and induce current into the head, making it difficult to differentiate between the direct effect of the rf field and an effect of the induced currents in acute experiments. takashima et al. ( ) reported on the effects of modulated rf fields ( - mhz, or hz modulation) on the eegs of male rabbits following acute ( - h) and chronic ( h/d for - weeks) exposures. although acute exposure up to v/m did not cause effects, chronic exposure above v/m enhanced the low frequency components of the eeg and decreased high frequency activities. the acute study showed that metal electrodes caused artifacts during recording. however, the effects of chronic exposure were not due to the presence of electrodes, because electrodes were not present during rf exposure. the effect of mw/cm , . ghz exposure for min on eeg of rabbits was described by chizhenkova ( ) . exposure of the head increased the number of slow waves and spindle-shaped firings in the eeg. it also changed the discharge frequency of neurons in the visual cortex, producing an enhancement of the evoked response of visual cortex neurons to a light stimulus. thuroczy et al. ( ) reported that the total power of eeg spectra increased in rats after whole-body . ghz, continuous wave microwave exposure ( mw/cm ) for min; changes occurred at mw/cm . the cerebral blood flow (cbf) increased after mw/cm . the power of eeg δ waves ( . - hz) was increased by thermal level of brain localized ghz (continuous wave) exposure at mw/g with simultaneously increase of the cbf. vorobyov et al. ( ) analyzed average eeg frequency spectra in eight unanesthetized adult rats with chronically implanted carbon electrodes in symmetrical somesthetic areas. microwaves of mhz, at . - . mw/cm , amplitude modulated at hz were applied for min on and min off during min sessions. there were no differences, other than an elevation of eeg asymmetry in the - hz range observed during the first sec after onset of the exposure. in summary, it appears that elf-modulated microwave fields produced changes in eeg patterns by enhancing the low-frequency components and decreasing highfrequency activities. hietanen et al. ( ) examined the possible influence of microwave radiation on human brain function by recording eeg activity of volunteers. the sources of exposure were five different cellular phones (analogue and digital models) operating at a frequency of mhz or . ghz. the eeg was recorded in an awake, closedeyes situation. six min sessions, including sham exposure, were completed for each subject. the duration of the real exposure phase was minutes. exposure to the microwave fields emitted by cellular phones had no abnormal effects on human eeg. kramarenko and tan ( ) recorded eeg changes during exposure of human head mobile phone emissions. the spatial distribution of the electromagnetic field was concentrated around the ipsilateral eye adjacent to the basal surface of the brain. slow-wave activity ( . - . hz) appeared in the contralateral frontal and temporal areas; the activity lasted for about sec and reappeared every - sec. after turning off the mobile phone, the slow-wave activity disappeared - min later. the authors interpreted these results as indicating that cellular phones might reversibly influence human brain function by inducing abnormal slow waves in the eeg of awake persons. krause et al. ( a krause et al. ( : b studied the effects of the mhz microwave field emitted by one model of a cell phone on the event-related desynchronization and synchronization of the - hz, - hz, - hz, and - hz eeg frequency bands of human subjects performing either ( ) an auditory memory task or ( ) a visual sequential letter task with three different working memory load conditions . all subjects performed the memory task both with and without exposure to emf in counter-balanced order. the microwave exposure increased eeg power in the - hz frequency, only when examined as a function of memory load. however, in their own replication studies, which were conducted under a double-blind procedure, krause et al. ( ) were not able to replicate the positive finding from their own earlier studies. they mentioned that microwave-exposure effects on the eeg and on the performance on memory tasks might be variable and not easily replicable, for reasons yet to be clarified. studies of sleep eeg by mann et al. ( a) and wagner et al. ( ) showed no effects were produced by irradiation with a mhz microwave field, pulsed at hz and with an average power density of . mw/cm . during an entire night-time sleep episode, borbely et al. ( ) exposed subjects to mhz, at a maximum sar of w/kg, using an intermittent schedule consisting of alternating min on and min off intervals. spectral power of the eeg increased in the - hz and . - hz bands during the initial part of nrem sleep and then subsided. huber et al. ( ) reported that pulse-modulated, mhz electromagnetic field exposure increased relative cbf (rcbf) in the dorsolateral prefrontal cortex ipsilateral to exposure. this microwave exposure also enhanced eeg power in the alpha frequency range prior to sleep onset and in the spindle frequency range during stage- sleep, although the effects were subtle. huber et al. ( ) extended the analysis. unilateral exposure during waking induced a similar effect in both hemispheres. microwave exposure during sleep reduced waking after sleep onset and affected heart rate variability. huber et al. ( ) observed an increase in relative rcbf in the dorsolateral prefrontal cortex on the side of exposure (ipsilateral) after min of unilateral head exposure to pulse-modulated, mhz microwaves emf. two types of emf exposure were applied: a 'base-station-like' and a 'handset-like' signal (sar of w/kg for both conditions). the effect depended on the special power in the amplitude modulation of the rf carrier such that only 'handset-like' exposure, with its stronger low-frequency components, but not the 'base-station-like' exposure affected rcbf. the authors stated that pulse modulation of cell phone signals is necessary to induce changes in the eeg, both waking and sleeping. overall, outcomes of the various human studies have been inconsistent, and comparison between individual studies is difficult. enhanced power in the alpha band was observed in both human and some animal studies. effects reported on sleep eeg are more likely to involve nrem alpha waves, compared to other bands, as they do in these eeg experiments on young human subjects. motor actions often are self-initiated without an outside cue. almost second before a self-initiated volitional movement begins, a characteristic negative shift in cortical potentials is seen in the eeg record of medial premotor areas, where the supplementary motor area is located. this slow negative potential, referred to as the preparatory potential, signals the planning that occurs before movement is executed. the region responsible for this negative potential was localized more precisely in a study comparing increase in rcbf during simple, complex, and imagined sequences of finger movements. complex movement sequences require more planning than do simple repetitive movements. imagining complex movements might require the same amount of planning as real movements (krakauer and ghez ) . freude et al. ( ) searched for effects on preparatory potentials during exposure to cell phone emissions. in the first experiment, healthy male human subjects performed simple, self-paced finger movements to elicit a preparatory potential. in the second experiment, they performed a complex and cognitively demanding visual monitoring task. both tasks were performed with and without microwave exposure, in counter-balanced order. exposure produced a significant decrease of preparatory potentials at central and temporo-parieto-occipital brain regions while performing the visual monitoring task. no effect on the slow potentials was seen in the simple finger movement task. freude et al. ( ) studied healthy male subjects who were exposed to modulated . mhz microwaves ( hz pulse frequency, pulse width µsec; . w/kg sar) emitted near the left ear. two experiments were completed, about months apart. in the first experiment, microwave exposure produced a decrease of preparatory potential while performing a complex visual monitoring task. this effect was replicated in the second experiment. exposure effects on preparatory potentials were analyzed further with two, less demanding tasks: a simple finger-movement task and a two-stimulus task eliciting a contingent negative variation (cnv) response. cnv was explained in chapter . in comparison to the complex visual monitoring task, no effects were found with the less demanding tasks. the results suggested a selective microwave-exposure effect on particular aspects of human information processing but did not indicate any influence on human performance. hinrichs and heinze ( ) investigated potential effects of gsm on verbal memory encoding by recording event-related magnetic fields from the brain during subsequent memory retrieval. after encoding words from a study list presented in the first phase, the subjects had to discriminate old from new words mixed together in a test list. subjects completed two experimental sessions, one with microwave exposure during the study phase, and one without. field exposure produced changes an early ( - msec), task-specific component of the event-related magnetic field. the authors suggest field exposure interfered with item encoding, although behavioral measures were not affected. the literature on possible microwave exposure effects on the eeg is not small, and it suggests effects can be measured. unfortunately, eeg has proven to be of limited utility as a general neurotoxicology screening measure, and the problem of suitable electrodes for recording during rf exposure is an added special difficulty. the literature does suggest that a scattering of eeg changes can be measured as a consequence of microwave exposure. it would take a considerable amount of additional research to build this hint into useful knowledge. exposure can produce subtle effects on human eeg. however, there is little agreement on either what mental processes are affected or the meaning of the changes reported. most studies test of cell phone signals and human cognitive performance used only one dose, had poor dosimetry, inadequate control groups, and poor traceability. possible interference and reflection of microwaves should be assessed in exposure setups. boredom and heat can induce sleep, and sleep-related changes in the eeg could account for positive findings. as a result no conclusions can be drawn from the presently available research on possible effects of microwave exposure and eeg. in both humans and animals, microwave fields are suspected of being able to affect cognitive functions. more specifically, several studies performed in rodents have suggested that spatial learning can be impaired by electromagnetic field exposure. a major effect of exposure to rf frequencies above khz is heating. microwave heating is known to affect memory and learning (saunders et al. ) . at ghz, because of the shorter wavelength, exposure is mostly superficial; the rf energy is absorbed by skin rather than the deeper tissues, as it is with the lower frequencies (longer wave lengths) of and mhz (adair et al. (adair et al. : a . these frequencies are used by current mobile communication systems. threshold sars were estimated to be . w/kg at mhz (near body resonance, for the human) and - w/kg at the higher frequencies. however, colonic temperature rose in all cases by about • c. the lowest sar threshold for learning effects was found during exposure to more deeply penetrating fields at lower frequencies, such as mhz (saunders et al. ) . resonant frequencies for humans and animals differ, because of their different body sizes. referring to body temperature changes, yamaguchi et al. ( ) studied two behavioral tasks, using a t-maze for the assessment of memory: subjects first performed a spatial discrimination task, being rewarded on one side in the training session; they then performed on a reversal task, being rewarded on the other side in the test session. reversal discrimination is described by a -stage model. in the first stage, after the reward location has been reversed, the undoing of old habits is required. the second stage is the period when the animal responds by chance. in the third stage, the new habit is acquired. working memory is required in the first stage, where the rats have to remember which of the two baited arms contains the food reward, whereas reference memory is involved in the following stage. sd rats were exposed daily for h for either days or weeks. exposure consisted of a pulsed (tdma) mhz microwave field in a carousel-type exposure system; two different sars were used. in one group, the sar at the brain was . w/kg, and the whole-body sar was . w/kg, which did not cause an increase in core body (intraperitoneal) temperature. other subjects were exposed at the brain average sar of w/kg and the whole body average sar of . w/kg for min daily for days. in this group intraperitoneal temperature began to rise soon after the beginning of exposure and rose by about • c within minutes. the rats with a brain sar of w/kg for days showed decreases in the transition in number of correct choices in the reversal task, compared to cage-control or sham-exposed subjects. however, rats exposed with a brain sar of . w/kg, for either days or for weeks, showed no impairment of t-maze performance. these results suggest that the exposure to a tdma microwave field at levels about -fold stronger than emitted by cellular phones does not affect the learning and memory processes when there are no thermal effects. wang and lai ( ) reported that the exposure to pulsed . ghz with a whole body sar of . w/kg for h daily for days caused deficits in spatial reference memory in rats using a morris water-maze. microwave-exposed rats were slower than sham-exposed and cage-control rats in initial learning to locate the platform. however, there was no difference in swim speed among the three groups of animals, indicating that the difference in learning was not due to a change in motor functions or motivation. during the probe trial, microwave-exposed animals spent less time in the quadrant that had contained the platform. also, their swim patterns were different from those of the sham-exposed and cage-control animals. these observations indicated that microwave-exposed rats used a different strategy in learning the new location of the platform. the authors indicate that acute exposure to pulsed microwaves caused a deficit in spatial "reference" memory in the rat. lai ( ) investigated the effect of a temporally incoherent magnetic field ('noise') on microwave-induced spatial learning deficit in the rat. rats were trained in six sessions to locate a submerged platform in a circular water-maze. four treatment groups of rats were studied: ( ) microwave-exposure ( . ghz, continuous wave; whole-body sar . w/kg), ( ) magnetic field 'noise' exposure ( mg), ( ) microwave + magnetic field exposure, and ( ) sham exposure. animals were exposed to these conditions for h immediately before each training session. one hour after the last training session, animals were tested in a min probe trial in the maze, during which the platform was removed. the time spent the quadrant of the maze in which the platform had been located was scored. results show that microwave-exposed rats ( ) had a deficit in learning to locate the submerged platform, when compared with performance of the sham-exposed animals. exposure to magnetic field noise alone ( ) did not affect the performance of the animals. however, simultaneous exposure to magnetic field noise ( ) attenuated the microwave-induced spatial learning deficit. thus, simultaneous exposure to a temporally incoherent magnetic field attenuated microwave-induced spatial learning and memory deficits in the rat. however, there are several reports which found no effects of microwave exposure on learning and memory tasks. sienkiewicz et al. ( ) studied the effect of repeated, acute exposures to a low-intensity mhz microwave field pulsed at hz on a spatial learning and working memory task. adult male c bl/ j mice were exposed under far-field conditions in a gtem cell for min each day for days at a sar of . w/kg. their performance in an -arm radial maze was compared to that of sham-exposed control animals. animals were tested in the maze immediately following exposure or after delays of or min. no field-dependent effects on performance were observed in choice accuracy or in total times to complete the task. dubreuil et al. ( ) used a head-only exposure system emitting -mhz gsm microwaves, pulsed at hz, to expose rats for min with sars of either . or . w/kg. two behavioral tasks were employed to demonstrate performance deficits in spatial learning after microwave exposure: an -arm radial maze and a spatial navigation task in an open-field arena, i.e., another version of the morris water-maze. there were no differences in performance on the two spatial learning tasks among microwave-exposed, sham-exposed, and cage-control rats. dubreuil et al. ( ) extended their study by using a more complex spatial learning task and a non-spatial object recognition task. altogether, this set of experiments provides no evidence indicating that spatial or non-spatial memory can be affected by a min, head-only exposure to a mhz (gsm) microwave field. cobb et al. ( ) examined the possibility of changes in 'working memory' (baddeley ) of rats following whole body exposure to microwave radiation. during each of days, rats were exposed within circularly polarized waveguides for min to pulsed ( µsec pulses, pps) . ghz fields at whole body sar of . w/kg, followed by testing in a -arm radial maze. rats received a pre-exposure injection of one of three psychoactive compounds or saline, to determine whether the drugs would interact with microwave exposure to affect maze performance. there was no evidence that exposure to pulsed . ghz microwaves caused decrements in the ability of rats to learn the spatial memory task. cosquer et al. ( ) studied whether whole-body exposure to . ghz electromagnetic fields affected anxiety responses of rats in a plus maze rats were exposed for min to pulsed . ghz microwaves ( µsec pulse width, pps) with a whole-body sar of . w/kg and brain sar of . w/kg. exposure failed to induce any effects on anxiety responses. d'andrea et al. ( ) studied the effect of very high peak-power microwave pulses in the absence of whole-body heating on a time-related behavioral task. five rhesus monkeys, macaca mulatta, were exposed to peak-power densities of . w/cm (rms) while performing a time-related behavioral task. the task was composed of a multiple schedule of reinforcement consisting of three distinct behavioral components: inter-response time, time discrimination, and fixed interval. trained monkeys performed the task during exposure to . ghz pulses at low pulserepetition rates ( - hz). no changes in performance were observed during fieldexposure compared to sham-exposure sessions. these might be species difference between rodents and monkeys. it is difficult to make broad generalizations about the presence or absence of microwave-exposure effects on learning and memory in animals. there are many studies reporting positive effects, but there are just as many reporting absence of effects. when attempting to compare and contrast experiments, the many differences in task, subject size, microwave exposure conditions, etc. prevent closure and produce frustration. science would be better served if investigators stopped independently shot gunning experiments, (looking for an effect, any effect) and focused on key questions, comparing hypotheses with single experiments and conducting replicative experiments across laboratories. koivisto et al. ( a koivisto et al. ( : b examined possible influences of a mhz microwaves on cognitive function of healthy human subjects. microwave exposure shortened response times in simple reaction time and vigilance tasks, and time to complete a mental arithmetic task was decreased . also, microwave exposure speeded up response times when the memory load was three items or more but had no effects with lower loads ). these results indicate that microwave exposure with a signal like that of a cell phone actually can improve human cognitive performance. however, in their own replication study, which was conducted with improved methodology including multi-center testing and a double-blind design, researchers from the same laboratory could not replicate their previous results. the authors argue that the inability to replicate previous findings could have been caused either ( ) by the lack of actual effects, or ( ) the magnitude of effects being at the sensitivity threshold the tests used. maier et al. ( ) examined effects of exposure to pulsed electromagnetic fields (gsm standard) on cognitive performance of humans by using a psychophysiological test paradigm. eleven subjects performed an auditory discrimination task. following a first test cycle, the volunteers relaxed for min while being either field-exposed or sham-exposed. subsequently, the test was repeated. data acquired before and after the resting phase were compared for both experimental conditions. nine of the test participants ( . %) showed worse auditory discrimination performance after microwave exposure, as compared with the sham-control condition. two recent papers reported facilitating effects on human attention from exposure to the microwave fields used by mobile phones. edelstyn and oldershaw ( ) investigated the effects of acute mobile phone exposure on a range of tasks assessing capacity and processing speed of the attentional system. cognitive performance was assessed at three points (prior to mobile phone exposure, and at and min post-exposure) using six cognitive neuropsychological tests (digit span and spatial span forwards and backwards, serial subtraction and verbal fluency). thirty-eight subjects were assigned randomly to either an experimental group, which was exposed for min to the microwaves emitted by a mhz mobile phone, or to a control group in which the mobile phone was switched off. subjects remained blind to mobile phone status throughout duration of study. differences between the two groups were evident after min on two tests of capacity (digit span forwards and spatial span backwards) and one of processing speed (serial subtraction). in all three instances, performance was facilitated following mobile phone exposure. no deficits were evident. lee et al. ( ) studied the relationship between the facilitating effect and the duration of exposure. seventy-eight university students were assigned randomly to either an experimental or a control group, and then performance on the administered attention tasks was compared. participants in the experimental group performed better on one of the two measures of attention only after they had been exposed to the electromagnetic field emitted. in summary, the results of recent studies of cell phone effect on human cognitive performance suggest that attentional functions can be enhanced by exposure to the low-intensity microwaves emitted by mobile phones. the noting of any bioeffect is of interest, given the small amount of energy involved. the discovery of improved performance is an ironic development in a research program that is focused on hazard assessment. note also that other research suggests cell phone use while driving is adverse, apparently because it distracts drivers from the driving task. these laboratory tasks do not duplicate all aspects of the real world situation. in order to obtain some clues to help elucidate the mechanisms of microwave irradiation on the cns, two groups have carried out experiments on hippocampal slices. the hippocampal slice preparation, in which a portion of the hippocampus is placed in vitro and an input pathway is stimulated and activity in an output pathway is recorded has been used extensively to study the electrophysiology and neuropharmacology of processes related to learning and memory. one classic approach is to study long-term potentiation (ltp). the amplitude of the response gets bigger as after some initial input stimulation is applied, and the change persists over time. tattersall et al. ( ) reported the possibility that low-intensity microwave fields can have effects on hippocampal tissues in vitro. slices of rat hippocampus were exposed to mhz, continuous wave ( . - . v/m, - min exposure) microwaves in a stripline waveguide. at low field intensities, the predominant effect on the electrically evoked field potential in ca was a potentiation of the amplitude of the population spike by up to %. however, higher intensity fields could produce either increases (up to %) or decrease (up to %). to eliminate the possibility of microwave-induced artifacts associated with the use of a metal stimulating electrode, the effect of microwave exposure on spontaneous epileptiform activity induced in ca by -aminopyridine ( - µm) also was investigated (tattersall et al. ). exposure to a microwave field ( v/m) reduced or abolished epileptiform bursting in % of the slices tested. the maximum field intensity used in these experiments, . v/m, was calculated to produce a sar of between . and . w/kg in the slices. measurements with a luxtron fiberoptic probe confirmed that there was no detectable temperature change (+/− . • c) during a min exposure to this field intensity. furthermore, imposed temperature changes of up to • c failed to mimic the effects of rf exposure. the authors claim that low-intensity microwaves can modulate the excitability of hippocampal tissues in vitro in the absence of gross thermal effects. pakhomov et al. ( ) studied effects of short, extremely high-power microwave pulses on neuronal network function using electro-physiological techniques in the rat hippocampal slice model. population spikes in the ca area were evoked by repeated stimulation ( per sec) of the schaffer collateral pathway. brief tetanic stimulation ( sec at hz) was used to induce ltp of synaptic transmission. in three different series of experiments with a total of brain slices, the extremely highpower microwave pulse irradiation was performed before, during or after the tetanus. the carrier frequency was . ghz, the pulse width was from . to µsec, and the repetition rate was from . to hz. the peak sar in brain slices reached up to mw/kg. (the instantaneous power is incredible.) microwave heating of the preparation ranged from . • c (at . kw/kg) to • c (at . kw/kg). the experiments demonstrated that the only effect caused by extremely high peak power microwave exposure, within the studied range of parameters, was a transient and fully reversible decrease in the population spike amplitude. recovery took no more than a few minutes after the cessation of exposure and return to the initial temperature. the effect's features were characteristic of an ordinary thermal response. also, the effect was proportional to the temperature rise but not to any specific parameter of microwave exposure. the decrease in the amplitude of the population spike also could be induced by a continuous wave irradiation or by conventional heating. irradiation did not affect the ability of neurons to develop ltp in response to tetanus or to retain the potentiated state that was induced before irradiation. no lasting and delayed effects were observed. the results are consistent with a thermal mechanism action for extremely high peak power microwaves, and no indication of other mechanisms on neuronal function is suggested. in comparing pakhomov et al. ( ) with tatersall et al. ( ) , pakhomov et al. pointed out that of the over brain slices studied to date, no positive effect could be found without a temperature increase of the preparation of at least . • c, whereas tattersall et al. elicited variable effects, comparatively, with no measurable increase in preparation temperature. pakhomov et al. also indicated that "if the reported nonthermal effects are unambiguously replicated under artifact-free conditions, this finding could have a major impact on modern microwave biology and rf exposure safety guidelines". further study is warranted in this area. . . . perception koivisto et al. ( ) studied whether healthy humans sense the presence of microwave fields from digital gsm mobile phones ( mhz, hz pulse modulation). the subjects were assigned in two single-blind experiments. the duration of the microwave exposure was about min in experiment and min in experiment . each subject rated symptoms (headache, dizziness, fatigue, itching or tingling of the skin, redness on the skin, and sensations of warmth on the skin) or sensations in the beginning of the experimental session and at the end of both the exposure and the non-exposure conditions. the results did not reveal any differences between exposure and non-exposure conditions, suggesting that a - min exposure to the present microwave field for a cell phone does not produce subjective symptoms in healthy humans. many terms are used to name hypersensitivity to electromagnetic fields; such as electromagnetic hypersensitivity, electro-hypersensitivity, hypersensitivity to electricity, hypersensitivity to electric and magnetic fields, and electrical hypersensitivity (ehs). these terms are used to describe people who claim to be sensitive to electric and magnetic fields from a variety of sources, such as power lines, mobile telephones, mobile phone base stations, household appliances, visual display monitors, and light sources (comar ) . there is no independent measure for verifying this condition; currently it is totally dependent on verbal report. the comar report mentioned skin problems associated with visual display units in norway (linden and rolfsen ) ; later reports have come from other nordic countries, elsewhere in europe, and north america. individuals claiming ehs generally report a prevalence of symptoms that are related to the nervous system, such as fatigue, stress, and sleep disturbances. the second most prevalent are skin symptoms, which include facial pricking, burning sensations, and rashes. these are followed in importance by various body symptoms (body ache and pain), eye symptoms (burning sensations), and less commonly ear, nose, throat, and digestive symptoms (comar, ) . generally, the field that elicit ehs are reported to be very weak, well below what is known to affect normal individuals, and far below currently accepted safety standards. nevertheless ehs is a real phenomenon which is an annoying problem for the affected persons. the prevalence rate of self-reported ehs among the general population was studied recently in sweden and california. hillert et al. ( ) analyzed a cross-sectional questionnaire completed in by , men and women between and years in stockholm county. the response rate was %, and . % of the respondents reported hypersensitivity to electric or magnetic fields. prevalence was highest among women in the -to year age group. levallois et al. ( ) completed a study based on questions regarding electromagnetic fields and chemical sensitivities that were added to the california adult tobacco survey. self-reported electric and magnetic field sensitivity was defined as "allergic or very sensitive to getting near electrical appliances, computers or power lines". self-reported chemical sensitivity was defined as considering oneself "allergic or unusually sensitive to everyday chemicals". among a sample of , californians, subjects ( . %) reported to be "allergic or very sensitive" to being near electrical devices as well as to chemicals. among the subjects, subjects ( . %) reported sensitivity to electrical devices but not to chemicals. three sets of researchers have conducted provocation studies in which individuals claiming ehs were exposed to electric or magnetic fields in an attempt to probe any possible links between reported symptoms and exposure conditions. researchers have tried to find objective changes, because a major problem in studying ehs is the lack of established pathophysiological markers. several studies were performed to evaluate ehs under controlled laboratory conditions, and one provocation study was carried out under everyday conditions. andersson et al. ( ) tested if psychological treatment of ehs patients was effective. seventeen patients were assigned randomly to a treatment group or to a waiting-list control group in a retest-posttest control group design. the patients also participated in double-blind provocation tests before and after the treatment. the provocation test showed that this group of ehs patients did not react to the electromagnetic fields. a 'treatment package' of cognitive-behavioral methods were used. first, information about a model for understanding of how somatic symptoms could interact with the person's interpretation of the symptoms was provided, using a 'vicious circle' model. second, with the help of a homework assignment, each patient registered symptoms, the situations in which they occurred, and the interpretations made. with this psychological perspective, the patients could more realistically "test" the evidence. the patients in the experimental group reduced their evaluations of the disability more than the control group did, indicating that psychological approaches can be of value for these patients. also, blood samples were analyzed for prolactin, cortisol, dehydroepiandrosterone, and cholesterol: no differences were found. using double-blind provocation experiments, hietanen et al. ( ) tested the hypothesis that ehs persons can reliably report subjective symptoms from exposure to the microwaves emitted by handheld mobile phones (cellular phones). they also tested whether sensitive subjects were able to determine whether a phone was on or off by sensing microwaves. the study group consisted of volunteers ( women and men) who reported themselves as being sensitive to cellular phones. the exposure sources were one analog nmt phone ( mhz) and two digital gsm phones ( mhz and . ghz). the duration of a test session was min, and three or four sessions were performed in random order for each subject during a single day. the subjects were asked to report symptoms or sensations as soon as they perceived any abnormal feelings. various symptoms were reported; most of them appeared in the head region. however, the number of reported symptoms was higher during sham-exposure than during real-exposure conditions. furthermore, none of the subject could distinguish real exposure from sham exposure. also, blood pressure, heart rate, and respiration rate were monitored every min, without finding any changes. from these results, the authors concluded that adverse subjective symptoms, though unquestionably perceived by the ehs subjects were not produced by cellular phones. flodin et al. ( ) carried out a provocation study in the homes or workplaces of the ehs patients; hours after the test sessions, the subjects reported their symptoms and judgments as to on-off status. fifteen subjects selected as having 'fast and distinct reactions' from electric equipment were provoked on four occasions: usually two true and two sham challenges were completed. the intervals between exposures were a few or more days, in order to provide the subjects with an opportunity to recover before the next provocation. a control group of subjects verified that the provocations were performed in a blind manner, i.e., they could not discriminate real and sham exposures. the ehs patients were no better than the control group in deciding whether or not they were exposed to electric and magnetic fields. from these data the authors concluded that exposure to electric and magnetic fields per se does not seem to be the cause of the symptoms experienced by this patient group. several groups have tried to find out indicators that might explain the complaints of ehs persons. sandstrom et al. ( ) hypothesized that there are other factors in the office environment that can affect the autonomic nervous system and/or cns, resulting in the symptoms reported. flickering light is one such factor, and it was therefore chosen as the exposure parameter in this study. ten patients complaining of ehs and the same number of control subjects were exposed to amplitude-modulated light. the sensitivity of the brain to this type of visual stimulation was tested by means of two objective electrophysiological methods, electroretinography and visual evoked potential. a higher amplitude of brain cortical responses at all frequencies of stimulation was found when comparing ehs patients with controls. no differences in retinal responses were revealed. lonne-rahm et al. ( ) assigned ehs patients to one of two groups and tested them in a double-blind provocation study. these patients, who reported increased skin symptoms when exposed to electromagnetic fields, were compared with controls. both groups were exposed to min periods of high-or low-stress situations, with and without simultaneous exposure to the electric and magnetic fields from a visual display unit (vdu). the matched controls were tested twice as were the patients, but the fields were turned off both times for the controls. stress was induced by requiring the participants to act in accordance with a random sequence of flash-ing lights while simultaneously solving complicated mathematical problems. blood samples were analyzed for concentrations of melatonin, prolactin, acth, neuropeptide y, and growth hormones, and the expression of different peptides, cellular markers, and cytokines (somatostatin, cd , factor xiiia, and tnf-α). skin biopsies also were analyzed for the occurrence of mast cells. stress provocation resulted in feelings of more intense mental stress and elevated heart rate. the ehs patients reported increased skin symptoms when they knew or believed that the vdu's "electromagnetic field" was turned on. with the blind conditions there were no differences between 'on' or 'off'. inflammatory mediators and mast cells in the skin were not affected by the stress exposure or by exposure to electromagnetic fields. hormones and cytokines, etc. all showed no differences among groups or conditions. the authors concluded that the patients did not react to the electric and magnetic fields associated with a vdu device. hillert et al. ( ) investigated the nature and possible etiology of fatigue in people claiming ehs. the aim was to test the hypotheses that perceived fatigue was due to alterations in cholinesterase activity. the rationale of the study is that fatigue has reportedly been associated with cholinesterase inhibition due to exposure to organophosphates (markowitz ) . symptoms have been suggested to appear even when cholinesterase activity is near or within the range of normal or very slightly depressed (< %). the study group consisted of people who reported a hypersensitivity to electricity, including disabling fatigue. cholinesterase activity was assessed three times: twice based on current symptoms reported by the subjects. and once at a randomly selected time. no significant reduction in acetylcholinesterase was identified in any subject. the results do not support the hypothesis that a change in cholinesterase activity mediates fatigue in people reporting hypersensitivity to electricity. although not in microwave frequencies, but at hz, lyskov et al. ( a) performed a neurophysiological study of ehs patients by comparing visual functions, blood pressure, heart rate, electrodermal activity, respiration, eeg and visual evoked potentials. the authors found that the patients had a trend to hyper-sympathetic tone, hyper-responsiveness to sensory stimulation, and heightened arousal. lyskov et al. ( b) further investigated possible neurophysiological effects of intermittent ( sec on/off), hz, µt magnetic field exposure on patients with ehs and on control subjects during rest and performance of a mental arithmetic task. eeg, visual evoked potentials, electrodermal activity, ecg, and blood pressure were recorded from ehs patients and control volunteers. the total duration of the test was min, divided into two min rest periods and two min periods of mathematical performance. magnetic field and sham exposures were presented randomly during these periods, resulting in four different conditions: field-rest, sham-rest, field-math, and sham-math. the data showed main effects of the group factor (ehs vs. control subjects) on heart rate (p < . ), heart rate spectrum ratio, i.e., heart rate variability (p = . ); these measures were higher in the ehs group. also, electrodermal activity was shorter in latency and higher in amplitude in the ehs group. on the other hand, eeg characteristics did not differ between groups. the condition factor (math task vs. relaxed) showed main effects for heart rate (p < . ), heart rate spectrum ratio (p = . ), electrodermal activity (p < . ), and alpha and theta spectrum bands of eeg. however, the field factor (real or sham magnetic field exposure) did not affect either the autonomous functions mentioned above or the eeg variables of either group. these data do not indicate that ehs patients or controls were affected by low-level hz magnetic field exposure. however, persons reporting ehs differed from the control subjects in baseline values of several physiological characteristics. in summary, these studies all suggest that ( ) the ehs individual cannot detect electric and magnetic fields, and ( ) that the symptoms reported by ehs individuals are not related to electric or magnetic field exposures. given this, perhaps it is no surprise that, so far, researchers have not found any hormonal, immunological, or neurochemical 'markers' which are associated with ehs. however, as suggested by studies of autonomic nervous functions, such as heart rate and electrodermal activity (lyskov et al. b) , or study of brain cortical responses (sandstrom et al. ) , it might be possible to demonstrate that ehs patients might have a physiological predisposition to hyper-sensitive responding to physical and psychological environmental stressors. ehs patients might falsely attribute their real symptoms. because neurotransmitters are critical messenger of information within the nervous system, many researchers have focused their attention on the issue of whether microwave radiation can affect transmitter actions. (an outline of neurotransmitters is provided in section . . .) gandhi and ross ( ) exposed rats to mhz at mw/cm , that raised the core temperature by . • c. of six brain regions investigated, only the hypothalamus showed significant changes in receptor states, confirming its pivotal role in thermoregulation. adrenergic receptors showed a % decrease in binding following radiation after a . • c increase in body temperature, suggesting a mechanism to facilitate noradrenaline release, which maintains thermal homeostasis by activating heat dissipation. muscarinic cholinergic receptors showed a % increase in binding at the onset of radiation, which might be attributed to the release of acetylcholine (ach) in the hypothalamus in response to heat accumulation. inaba et al. ( ) exposed rats to . ghz at an ambient temperature of - • c. microwave exposure at power densities of and mw/cm increased the rectal temperature by . • c and . • c, respectively. the noradrenaline content in the hypothalamus was significantly reduced after microwave exposure at a power density of mw/cm . there were no differences in the dopamine contents of any region of the brain between microwave-exposed rats and control rats. the dihydroxyphenyl acetic acid content, which is the main metabolite of dopamine was increased in the pons and medulla oblongata at a power density of mw/cm . the dopamine turnover rates and the ratio of metabolite to dopamine in the striatum and cerebral cortex were increased at a power density of mw/cm . the serotonin content in all regions of the brain did not change. the -hydroxyindoleacetic acid content in the cerebral cortex was increased at power densities of and mw/cm . clearly microwave exposures producing a modest rise in core body temperature produce a variety of changes in cns neurotransmitters. the mechanisms for, and the significance of, the observed changes remains to be determined. mausset et al. ( ) developed a protocol of neurotransmitter detection based on immunohistochemistry and image analysis. gamma-vinyl-gaba, an inhibitor of gaba-transaminase, was injected in rats to increase gaba concentration in the cns. the cellular gaba contents then were revealed by immunohistochemistry and semi-quantified by image analysis using three parameters: optical density, staining area, and number of positive cells. the increase in gaba content induced by gamma-vinyl-gaba in the molecular and the granular layers of cerebellum was reflected by these three parameters. this protocol was used to investigate the effects of exposure to mhz. both pulsed microwave exposure with a sar of w/kg and continuous wave exposure with a high sar of w/kg were tested. a selective diminution of the stained processes in the purkinje cell layer after exposure to pulsed microwaves, in addition, a decrease in optical density in the three cell layers were apparent after continuous wave exposure. whether this effect is, at least partly, due to a local heating of the tissues is not known. overall, it appears that microwave exposure with a relatively high sar of w/kg, one that might produce hyperthermia, induces a diminution in cellular gaba content in the cerebellum. testylier et al. ( ) studied ach release in the brain of freely moving rats exposed to a . ghz continuous wave microwave field ( or mw/cm ) or to a mhz field amplitude-modulated at hz. the rats were exposed for hr during the day or exposed for either h or h during the night. serial neurotransmitter measurements were performed by microdialysis using a membrane implanted through the upper ca region of the hippocampus. after irradiation with the . ghz microwaves, at mw/cm , rats did not show a significant modification of ach release, whereas those exposed at mw/cm showed a % decrease in ach release from hippocampus. this decrease was maximal at h post exposure. exposure to the mhz microwaves for hr do not cause any effects, but exposure for hrs induced a % decrease in ach release during the period p.m. - a.m. compared to control rats. this work indicates that neurochemical modification of the hippocampal cholinergic system can be observed during and after an exposure to a low-intensity microwave field. pakhomov et al. ( ) investigated the effects of high-power microwave ( . ghz) pulses (pulse widths from . to µsec at a rate of . or . hz) on synaptic transmission and ltp in rat hippocampal slices. (see pakhomov et al. ( ) in section . . . .) microwave heating of the preparation ranged from . • c (at a sar of . kw/kg) to • c (at . kw/kg). the only effect produced by the high-power pulsed microwave exposure was a transient and fully reversible decrease in the population spike amplitude. irradiation did not affect the ability of neurons to develop ltp. these results are consistent with the view that the only mechanism of action for extremely high power microwave pulses is thermal. the available literature clearly indicates that, if the sar is high enough, microwave exposure can alter neurotransmitter function in the cns. psychoactive agents affect neuronal function by modifying neurotransmitter activities, resulting in some sort of behavioral changes or altered physiological parameters, depending on the experimental conditions. if microwave irradiation caused interaction with the known effects of psychoactive agents, one could investigate, as the next step, the affected transmitter substances with established physiological and pharmacological experimental techniques. lai et al. ( ) studied the effects of various psychoactive drugs in rats exposed for min to a circularly polarized, pulsed ( µsec pulses, pps) microwave field ( . ghz) at a sar . w/kg. apomorphine-induced hypothermia and stereotypy were enhanced by irradiation. amphetamine-induced hyperthermia was attenuated, but stereotypy was unaffected. morphine-induced catalepsy and lethality were enhanced by irradiation, at certain dosages of the drug. these results suggest that microwave exposure interact with certain types of psychoactive agents. lai et al. ( ) measured sodium-dependent, high-affinity choline uptake in various regions of the brain, including frontal cortex, hippocampus, hypothalamus, inferior colliculus and striatum, of rats irradiated for min with either pulsed or continuous wave, low-level microwaves ( . ghz; power density, mw/cm ; average whole-body sar . w/kg). pulsed microwave irradiation ( µsec pulses, pps) decreased choline uptake in the hippocampus and frontal cortex. pretreatment with a narcotic antagonist (naloxone or naltrexone; mg/kg) blocked the effect of pulsed microwaves on hippocampal choline uptake but did not alter the effect on the frontal cortex. continuous wave exposure decreased the uptake in the frontal cortex. this is the first report that microwave irradiation reduced sodium-dependent, high-affinity choline uptake, an index of cholinergic activity. lai et al. ( a) studied the effects of single ( min) and repeated (ten daily min sessions) pulsed ( pps, pulse width µsec) . ghz exposures (sar of . w/kg) on the concentration and affinity of benzodiazepine receptors in the cerebral cortex, hippocampus, and cerebellum of the rat. a receptor-binding assay, with h-flunitrazepam as ligand, was used. benzodiazepine receptors in the brain are responsive to anxiety and stress. immediately after a single exposure, an increase in the concentration of receptor was observed in the cerebral cortex. however, in rats subjected to repeated exposures, no change in receptor concentration was found in the cerebral cortex immediately after the last exposure, which might indicate an adaptation to repeated exposures. lai et al. ( b) performed experiments to investigate subtypes of opioid receptors in the brain involved in the effect of acute ( min) pulsed ( µsec pulses, pps) microwave exposure ( . ghz; sar . w/kg) on cholinergic activity in the rat brain. rats were pretreated by microinjection of specific antagonists of µ, δ, and κ opioid-receptors into the lateral cerebroventricle before microwave exposure. the data showed that all three subtypes of opioid receptors are the intermediate step in the microwave-induced decrease in cholinergic activity in the hippocampus. lai et al. ( ) studied effects of microwave irradiation on radial-arm-maze performance of rats. after min of exposure to pulsed . ghz microwaves ( µsec pulses, pps; sar . w/kg), rats showed retarded learning, indicating a deficit in spatial 'working memory' function. this behavioral deficit was reversed by pretreatment before exposure with the cholinergic agonist physostigmine or the opiate antagonist naltrexone. however, pretreatment with the peripheral opiate antagonist naloxone methiodide showed no reversal of the effect. these data indicate that both cholinergic and endogenous opioid neurotransmitter systems in the brain are involved in the microwave-induced spatial memory deficit. lai et al. ( ) reported that intra-septal microinjection of β-funaltrexamine blocked a microwave-induced decrease of hippocampal cholinergic activity in the rat after min exposure to pulsed . ghz microwaves ( µsec pulses, pps; sar . w/kg). these data indicate that µ-opioid receptors in the septum mediate a microwave-induced decrease in cholinergic activity in the hippocampus and support the hypothesis that microwaves at a whole body sar of . w/kg can activate endogenous opioids in the brain. work from several investigators shows that microwave exposure can modulate brain neurotransmitter activity, especially that involve with thermoregulation. additonally, an interesting series of papers from one laboratory clearly suggests that psychoactive agents can modulate the effects of microwave exposure on neurotransmitters (and behavior). these results indicate that ( ) endogenous opioids play an important role in some of the neurological effects of microwaves, and ( ) parameters of microwave exposure are important determinants of the outcome of the microwave effects. further research in this area should be a fruitful activity, contributing to an understanding of the mechanisms connecting microwave exposure to behavior. matsumoto ( : ) studied the effect of . ghz microwave irradiation on crayfish peripheral nerve. response characteristics of discharging impulses of the slowly adapting stretch receptor were obtained by calculating the cross-correlation function between input and output of the receptor neurons. the m-sequence signal of the . ghz microwave was applied as the input. temperature change of the receptor was measured by a thermistor. results showed a strong correlation between the impulse frequency and temperature change. the authors concluded that the impulse frequency change was caused by the thermal change produced by the microwave irradiation. courtney et al. ( ) and chou and guy ( ) used a temperature-controlled waveguide to expose frog sciatic nerves, cat saphenous nerves, rabbit vagus nerves, and rabbit superior cervical ganglia, and rat diaphragm muscles to . ghz in either continuous wave or pulsed modes. no specific microwave effects were observed. these results show that under a constant ambient temperature, microwave effects on the selected electrically excitable tissues could not be detected. however, it was shown that a temperature rise in the solution as small as . • c could induce observable effects on action potential (ap) latency or muscle contraction (chou and guy, ) . mcree and wachtel ( ) reported that exposure to . ghz, continuous wave microwave fields (at a sar of w/kg) would consistently lower the survival time of isolated frog sciatic nerves stimulated at high repetition rates ( pulse-pairs per sec). to assess the role that these microwaves might have on active transport of k and na ions, mcree and wachtel also performed a series of experiments in which the active na-k pump was substantially blocked by ouabain prior to microwave exposure. paired nerves were soaked for min in a high concentration ( − g/liter) of ouabain to rapidly produce blockage of the na-k pump. with stimulation at pulses/sec, the 'rundown time course' was, as expected, accelerated in all ouabain-treated nerves. however, the microwave-exposed nerves showed no additional shortening of survival time. the experiments were repeated at a slower stimulation rate ( pulses/sec) so that the survival time of the nerves more closely approximated that of nerves not treated with ouabain ( to h vs. min or less for ouabain-treated nerves). results at the lower stimulation rate also showed that there was no difference in the survival times of ouabain-treated exposed and ouabain-treated, unexposed-control nerves. these results lend support to the view that the relative loss of excitability in microwave-exposed nerves is related to an interference with or counteraction of the na-k pump. pakhomov et al. ( ) studied effects of an acute, continuous wave exposure to millimeter waves ( - ghz) on compound action potential (cap) conduction in an isolated frog sciatic nerve preparation. caps were evoked by either low-rate ( paired pulses/sec) or high-rate ( paired pulses/sec) electrical stimulation of the nerve. the low-rate stimulation did not alter the functional state of the nerve, and the amplitude, latency, and peak latency of caps could stay stable for hours. microwave irradiation for - min at . - . mw/cm , either at various constant frequencies or with a stepwise frequency change ( . or . ghz/min), did not cause any detectable changes in cap conduction or nerve refractoriness. at a higher field intensity of - mw/cm , a subtle and transient reduction of cap latency and peak latency along with a rise of the test cap amplitude were observed. rogers et al. ( ) used a classical electrophysiology method and preparation to assess the strength-duration (s-d) curve for the frog sciatic nerve and gastrocnemius muscle preparation. with a pulse of nsec, the voltage threshold for elicitation of a muscular twitch was . kv and a. the authors expressed the s-d curve in terms of all relevant units, including tissue current density, tissue electric field, calculated temperature increase in tissue, tissue sar, external power density, and external sar. the computations suggest that a single nsec stimulus of a in tissue is athermal; the estimated temperature rise was . × − • c. from the experimental results presented in these five papers, it certainly appears that the effects of microwave exposure on the nerves are caused primarily by tissue heating. kolosova et al. ( ) studied the effects of exposure to . ghz at power density mw/cm on the recovery of function in damaged rat sciatic nerve. lesions were produced by nerve section followed by microsuturing. irradiation was applied to the skin of the thigh in the area of suturing. total number of aps recorded from the nerve was used to study the functional properties of regenerated fibers mon after damage. microwave exposure had a stimulatory effect on regeneration processes: conduction velocity increased by - % with increase in total ap as compared with the shamoperated control group. it is not known whether this effect is due to local temperature changes or some other factors, such as increased local blood flow, because there is no description of temperature measurement. the limited data available suggests rf or microwave exposure can modulate functional properties of nerves. it seems most likely that the effects are related solely to heating. classic electrophysiology techniques applied to nerves would appear to be an excellent preparation to be used for studying microwave and rf effects and mechanisms. in many ways, the endocrine system is a key regulatory system, using diffuse chemical messages (hormones) placed into the general circulation for dispersal throughout the body. (see section . for background.) the pituitary gland is the primary interface between the brain and the endocrine glands. the pituitary has been recognized as the 'master gland' for many decades. the primary targets of the hormones produce by the pituitary gland are other endocrine organs, such as adrenal or thyroid glands. a linked system, such as brainpituitary-adrenal is called an axis. endocrine responses can be an adaptive response to environmental stimuli; such responses are not necessarily adverse, analogous to a febrile response when there is a need for host immune defense. the adrenal is a "dual gland". acth acts on the cortex, i.e., the outer layer, of the adrenal gland and stimulates the secretion of steroid hormones. catecholamines are synthesized in the medulla, i.e., core, of the adrenal which release adrenalin and noradrenalin. noradrenalin is a transmitter substance of sympathetic fibers, as well. to characterize the response of the pituitary-adrenal axis to microwave exposure, lotz and michaelson ( ) measured plasma corticosterone and colonic temperature in unanesthetized male rats exposed to continuous wave . ghz. the rats were exposed in the far field of a horn antenna for or min at power densities of , , , , , or mw/cm . other exposures were conducted for min at , , , or mw/cm . the average energy absorption rate of the rats was . w/kg absorbed per mw/cm . thus, for example, at mw per cm , the sar was about . w/kg. colonic temperature was significantly elevated after exposures to power densities of mw/cm or greater, with progressively larger increase after high intensity exposures. plasma corticosterone concentration was elevated above that of sham-exposed controls only after exposures at or mw/cm for exposures of either or min. with min exposures, plasma corticosterone concentration was increased at , , and mw/cm . the relationship between the increased levels of circulating corticosterone and colonic temperature suggested that the increases in corticosterone levels reflected the level of physiological response to the body temperature elevations caused by microwave exposure. lotz and michaelson ( ) further studied this effect by examining the effects of acute microwave exposure of normal, hypophysectomized, or sham-hypophysectomized rats. (hypophysectomized means the pituitary has been removed.) plasma corticosterone levels in acutely hypophysectomized rats exposed to mw/cm for min were below control levels, indicating that the microwave-induced corticosterone response observed in normal, intact rats is dependent on acth secretion by the pituitary. in other groups of rats pretreated with dexamethasone before being exposed microwaves for min, the corticosterone response to a mw/cm exposure was completely suppressed by doses equal to greater than . µg dexamethasone/ g body weight. however, the corticosterone response to a mw/cm exposure was only partially suppressed by prior administration of . or . micrograms dexamethasone/ g body weight. the evidence obtained in these experiments, combined with the results of previous report (lotz and michaelson, ) , indicate that the stimulation of the adrenal axis in the microwave-exposed rat is a systemic, integrative process resulting from to general hyperthermia. lu et al. ( ) studied changes in thyroid stimulating hormone (tsh) concentration in unanesthetized rats exposed to . ghz, amplitude-modulated ( hz) microwaves in the far field for or h, using power densities between and mw/cm . individual rats were exposed from to times to seek any possible accumulation, acclimation, or sensitization. colonic temperature was measured using a thermistor. tsh concentration decreased in rats after microwave exposure. the influence of microwave exposure on serum tsh concentration was independent of the number of exposures, indicating absence of accumulation, acclimation, or sensitization. decreased tsh concentration usually was accompanied by increased colonic temperature. for h exposure, the lowest irradiance was mw/cm , which produced a . • c increase in colonic temperature that was independent of the number of exposures. for h exposure, the lowest irradiance was mw/cm ,which produced or a . • c increase in colonic temperature, regardless of the number of exposures. the rats exposed at mw/cm for hr had a lower tsh concentration than did sham-exposed rats. these results suggest that the microwave effect of serum tsh could be dependent on duration of exposure. none of the rats exposed at an irradiance lower than mw/cm had any change in tsh concentration. the effect of microwave exposure on tsh concentration was not persistent after exposure. the relation between tsh concentration and colonic temperature was curvilinear (exponential). imaida et al. ( a) measured acth, corticosterone, and melatonin after weeks of near-field exposure of f rats to a . mhz microwave field like that used by cellular phones. increases occurred for all three hormones in the exposed group compared with the sham-exposed control group. experimental conditions are described in section . . . these examples make it clear that microwave exposure can alter hormones, when body temperature is elevated. these hormonal changes are part of the normal physiological response to hyperthermia. de seze et al. ( ) conducted an experiment to evaluate the effect of an mhz signal emitted by a gsm cell phone ( hz impulses, one-eight duty cycle, w peak power) on endocrine function of male volunteers, aged from to . end points were serum adrenocorticotropin, thyrotropin, growth hormone, prolactin, lh, and fsh concentrations. each subject was exposed to microwaves through the use of a cellular phone h/d, d/w, for mon. the hormone levels were determined in weekly blood samples, obtained starting weeks before the commencement of the exposure and ending weeks after exposure ended. (all but one blood sample was drawn h after each weekly session. the seventh drawing was performed in the morning after the last weekly exposure.) within each individual, the pre-exposure hormone concentration was used as the control value. there was a change only in thyrotropin concentration, and it occurred only on one occasion; on the seventh sampling, a % decrease was found. this change recovered fully during the postexposure period. therefore, cell phone exposure did not induce a long-lasting effect on the hormone-secretion rate of the anterior pituitary gland in humans. mann et al. ( b) investigated the influence of mhz microwaves (pulsed at hz, average power density . mw/cm ) from a circularly polarized antenna on the endocrine system in healthy humans. nocturnal hormone profiles of growth hormone, cortisol, lh, and melatonin were determined under polysomnographic control. only one alteration in activity of the hypothalamo-pituitary-adrenal axis was found: a slight, transient elevation in the cortisol serum level immediately after onset of field exposure, which persisted for hour. for growth hormone, lh and melatonin, no effects were found on either total hormone production during the entire night or the dynamic characteristics of the secretion pattern. radon et al. ( ) tested healthy male students to see whether or not the microwave fields used by the gsm standard have any noticeable effects on salivary melatonin, cortisol, neopterin, and immunoglobulin a levels during and several hours after exposure. in a shielded experimental chamber, the circularly polarized electromagnetic field was transmitted by an antenna position cm behind the head of sitting subjects. the carrier frequency of mhz was pulsed with hz, and the average power flux density was w/m . in double blind trials, each subject received a total of , randomly allotted, hour periods of exposure and sham exposure, equally distributed among day and night. the salivary concentrations of melatonin, cortisol, neopterin and immunoglobulin a did not differ between exposure and sham exposure. based on the experiments conducted with animals and humans, it appears that microwave exposure at the athermal levels produced by cell phones causes no important changes in hormones. experiments with animals in which higher doses can be applied, resulting in increased body temperature. under this circumstance, microwave exposure can alter hormones. these hormonal changes are part of the normal physiological response to hyperthermia. there is ample experimental evidence that changes of earth-strength static magnetic fields and elf magnetic fields can depress the nocturnally enhanced melatonin synthesis in the pineal gland of mammals, including humans. thus, it is reasonable to ask if the electromagnetic fields associated with cell phones might also influence secretion of melatonin. vollrath et al. ( ) reported that exposure to mhz microwaves, both continuous or pulsed at hz, for min to hr, at day or night, had no notable, short-term effect on pineal melatonin synthesis in male or female sd rats and djungarian hamsters. the sars were approximately . to . w/kg in rats and . w/kg in hamsters. pineal synaptic ribbon profile numbers, which were studied in rats only, likewise were not affected. with such low whole-body sars, thermal effects are very unlikely. however, brain sars presumably were somewhat higher, and the possibility of local hot spots can not be excluded without additional study. it should be noted that much of the brain is highly vascularized, meaning high blood flow rates will dissipate heat into the large heat sink provided by the rest of the body; thus the brain is protected against local heating. hata et al. ( ) investigated the effect of hour exposure during the dark to a . ghz tdma signal on serotonin and melatonin synthesis in a total of male and female sd rats. the brain sar was . w/kg; the whole body sars were . w/kg for the somewhat heavier males and . w/kg for the somewhat lighter females. no differences in melatonin or serotonin levels were observed among the microwave-exposed, sham-exposed, and cage-control groups. bakos et al. ( ) found no changes in the -sulfatoxymelatonin excretion of exposed wistar rats (n = ) compared to a sham-exposed or control group (n = ) after exposure to mhz ( µw/cm ) and . ghz ( µw/cm ), gsmlike radiation. the animals were exposed daily for h, between : am and noon, during a day exposure period. the exposure occurred during the morning, when melatonin production is low. one wonders what would have been found if exposure had been scheduled during the night, particularly in the earlier portion of the night when nocturnal melatonin production is rising. imaida et al. ( a) mentioned increased daytime melatonin concentration after weeks of near-field exposure to . mhz microwaves. (exposure conditions were described in section . . .) it should be noted the elevation of melatonin was observed during daytime; blood samples were acquired between : a.m. and noon). a similar daytime increase after weeks of exposure to a hz circularly polarized rotating magnetic fields was observed by kato et al. ( ) . (see section . for a complete review of the elf literature.) stark et al. ( ) investigated the influence of - mhz rf fields on salivary melatonin concentration in dairy cattle. two commercial dairy herds at two farms were compared. the exposed group was located at a distance of m from the transmitter: the average nightly field strength was . ma/m. the control group was located at a distance of , m from the antenna: the average nightly field strength was . ma/m. at each farm, salivary melatonin concentration in five cows was monitored over a period of consecutive days. saliva samples were collected at hour intervals during the dark phase of the night. as an additional intervention, the short-wave transmitter was switched off during three of the ten days (off phase). the salivary melatonin values of the two initial nights did not show a difference between exposed and unexposed cows. however, on the first night of re-exposure after the transmitter had been off for days, the difference between groups ( . pg/ml) in salivary melatonin concentration of the two groups differed. there are three reports which observed no effect following short-term microwave exposure ( min, hr, and day) on rodents, but there is one positive result from a week study. in addition, one environmental study of rf suggests a subtle effect occurred in dairy cows. in summary, the picture is suggestive, but not convincing, that microwave signals also can reduce nocturnal melatonin production. clearly, additional research is required, particularly with longer exposure intervals. de seze et al. ( ) examined whether microwave fields from cell phones would alter melatonin levels in the human. volunteers were two groups totaling men, - yr old. exposures were to commercially available cellular telephones of the gsm type ( mhz) or to the dcs type (digital communication system, . ghz), for hr/day, days/week, for wks. the phones were at their maximum power. blood samples were collected hourly during the night and every hr in the daytime. four sampling sessions were performed at day intervals: before the beginning of the exposure period, at the middle and the end of the exposure period, and days later to evaluate the persistence or late appearance of potential effects. the melatonin circadian profile was not affected. bortkiewicz et al. ( ) evaluated the effect of cell phone on -hydroxymelatonin sulfate ( -ohms) excretion of nine healthy males, aged - years. the experiment was performed under controlled lighting conditions: light intensity was lx until midnight and lx during the remainder of the night. each person was sampled twice: once on a day without exposure, and once on a day with cellular phone exposure for min, from to p.m. the phone used operated at mhz, pulsed with hz (pulse width µsec), and the sar was . w/kg. the subjects did not know sham-exposed or field-exposure days. from p.m. until midnight, the subjects listened to music, and then they slept till a.m. urine samples were collected at p.m., at midnight, and at a.m. the -ohms concentration in both phone-type experiments did not differ between exposed and control sessions at any of the three time points. circadian variation of -ohms level was detected in all subjects. the results demonstrated that emf emitted by cellular phones has no distinct influence on the melatonin level. jarupat et al. ( ) studied the effects of . ghz microwave fields emitted from a cellular phone on nocturnal melatonin secretion in saliva under carefully designed experimental protocol. the subjects were eight females (mean age; , range - years), all in the follicular phase of the menstrual cycle. the subjects had not used a cellular phone for at least one week before the experiment. a cell phone was attached to the left ear for min every hour, from : h to : h: on one day it was on, and on the other day was off. in the within-subjects comparison, melatonin concentration on the field-exposed day was significantly (p < . by paired test) lower than it was on the sham-exposure day. santini et al. ( ) studied the effects of more than one month of exposure to vdus (khz frequency range) on nocturnal urinary excretion of -ohms by women. six women, who worked at least h/d, d/w, in front of a vdu, constituted the exposed group. seven women served as the unexposed control group. the -ohms concentration in urine was % less (p < . ) in the field-exposed group as compared with the control group. burch et al. ( ) evaluated the relationship between cellular telephone use and excretion of the melatonin metabolite -ohms in two populations of male electric utility workers (study , n = ; study , n = ). participants collected urine samples and recorded cellular telephone use over three consecutive workdays. personal -hz magnetic field and ambient light exposures were characterized on the same days using emdex ii meters. no change in -ohms excretion was observed among those with daily cellular telephone use > min in study ( worker-days). however, in study , workers with > min cellular telephone use per day ( worker-days) had lower creatinine-adjusted mean nocturnal urinary ohms concentrations and lower overnight total -ohms excretion, compared to subjects not using cellular telephones. there also were linear trends of decreasing nocturnal -ohms/creatinine concentrations (p = . ) and total overnight -ohms excretion (p = . ) across categories of increasing cellular telephone use. a combined effect of cellular telephone use and occupational hz magnetic field exposure also was observed in study . from these results, the authors concluded that prolonged use of cellular telephones can lead to reduced melatonin production. furthermore, elevated -hz magnetic field exposure might potentiate the effect. three of the four studies of microwave exposure and melatonin in animals are negative. one possibility is that the exposure durations examined were too short to detect an effect. in studies with humans, two short-term ( hour and hour) laboratory experiments with cell phone exposure during the light phase revealed no influence on melatonin rhythm in young men. two experiments using women as subjects reported melatonin reduction. in a laboratory experiment, acute ( hr) cell phone use was examined. in an occupation study, subacute ( month) vdu use was examined. an additional occupational exposure study suggests melatonin reduction in men with greater cell phone use. in addition, an additive effect of hz magnetic field exposure and cell phone use was noted. although the initial database is small, it does suggest that microwaves, like elf electric and/or magnetic fields, can depress melatonin production. certainly additional studies are required. the physiological importance of melatonin and the economic importance of mobile telephony make mandatory acquisition of additional scientific knowledge. jauchem et al. ( ) investigated the thermal distribution and cardiovascular effects produced by sustained exposure of rats to ghz electromagnetic radiation. sixteen anesthetized sd rats were exposed individually at a power density of mw/cm under far-field conditions in the "e" orientation. irradiation began when colonic temperature was • c and continued until death. large, immediate increases in subcutaneous temperature on the irradiated side were accompanied by more moderate, delayed increases in colonic temperature. during irradiation, arterial blood pressure initially increased and then precipitously decreased until death. the heart rate increased throughout the exposure period. the patterns of body temperature, heart-rate and blood pressure changes that occurred before death were similar at ghz and ghz. because of the very short wavelength, exposure to ghz produces extreme peripheral heating without similar levels of core heating, and this pattern of heat deposition can be sufficient to produce circulatory failure and subsequent death. jauchem et al. ( ) exposed male sd rats (n = ) individually to one of three conditions: ( ) ghz, ( ) ghz, or ( ) combined ghz and ghz, all at an equivalent whole-body sar of w/kg. microwave exposure was started when colonic temperature was . • c, and it was continued until lethal temperatures were attained. during exposure, arterial blood pressure initially increased, but it then decreased until death. heart rate increased throughout the exposure period in all groups, indicating no unusual physiological responses occur during multi-frequency microwave exposure, compared with single-frequency microwave exposure. in general, the cardiovascular system is influenced by body temperature changes in the same manner, no matter how the heating is produced. mann et al. ( a) investigated the influence of pulsed, high-frequency electromagnetic fields emitted by digital mobile radio telephones on heart rate during slow-wave as well as rem sleep in healthy humans. no significant effects were observed on heart rate and heart rate variability. the authors conclude that autonomic control of heart rate was not affected by weak, pulsed, high-frequency electromagnetic fields. braune et al. ( ) found no non-thermal influence of the fields emitted by mobile phones on the vascular autonomic nervous system of healthy humans. the exposure was implemented using a gsm-like signal ( mhz, pulsed at hz; w) using a mobile phone mounted on the right-hand side of the heart in a typical telephoning position. each period of sham-exposure and of field-exposure consisted of min of supine rest, min of degrees upright tilt on a tilt table, and another min of supine rest. (assessment cardiovascular performance with a tilt table is a standard technique in cardiovascular physiology.) blood pressure, heart rate and cutaneous capillary perfusion were measured continuously. in addition, serum levels of norepinephrine, epinephrine, cortisol and endothelin were analyzed in venous blood samples taken every min. all parameters measured showed no changes under a nonthermal electromagnetic exposure. tahvanainen et al. ( ) evaluated cardiovascular responses in terms of blood pressure and heart rate during controlled breathing, spontaneous breathing, head-up tilt table test, valsalva maneuver, and deep breathing test in a randomized, doubleblind, placebo-controlled cross-over trial in which healthy subjects were submitted to mhz ( w), to . ghz ( w) cellular phone exposure and to sham exposure, in separate sessions. arterial blood pressure (arm cuff method) and heart rate were measured during and after the min microwave and sham-exposure sessions. compared to sham exposure, arterial blood pressure and heart rate did not change significantly during or after the min microwave exposure at mhz or . ghz szmigielski et al. ( ) studied the autonomic nervous system's regulation of cardiovascular function by assessing the time course of diurnal rhythms of blood pressure and heart rate in a group of workers ( healthy workers; - years) exposed to various intensities of rf fields ( . - . mhz). they found a reduction in the amplitudes of blood pressure and heart rate rhythms and a shift of the acrophase to an earlier time. these changes were more pronounced among workers exposed to higher intensities of rf electromagnetic fields. summarizing these results from animal and humans, two primary conclusions about the data seem apparent. first, when there is no heating, there are no cardiovascular effects. second, in animal experiments where exposure sufficient to produce lethality from hyperthermia can be conducted, numerous, expected cardiovascular effects become manifest. these few studies are preceded by decades of work establishing the same points. thus, one can conclude that there are no important effects on the cardiovascular system in the usual environments. as a secondary point, it must be noted that the "engineering" aspects of these human studies often are not very good. well-trained medical personnel who are experts in the biology of the dependent variables need to understand they must obtain engineering support to deal with independent variable issues relating to exposure, dosimetry, etc. studies on ocular effects from microwave exposure started with daily et al. ( ) , and many papers have been published since then, particularly in the s and s. some researchers have radiated the whole body of the experimental subjects, and others have limited exposure to the eyes. short-term (e.g., minutes), repeated or chronic (e.g., several weeks or months) exposures have been used. it has been reported that microwaves can cause a variety of ocular effects, most often cataract in the lens. however, effects on the retina, cornea, and other ocular systems also have been reported. cataract formation has been observed in some experimental animals when one eye was exposed to a localized, very-intense microwave field and the other eye was the sham-exposed control. daily et al. ( ) used the dog as the experimental animal. however, the most frequently used animal has been the albino rabbit; in recent years, non-human primates also have been used. microwaves at . ghz have been used in many recent studies, although other frequencies, such as . ghz or ghz, also are used. experimental results obtained using rabbits will be presented first; monkey and human data follow. it is known that, under conditions of partial-body exposure to intense microwaves, significant thermal damage can occur in vulnerable tissues. kramar et al. ( ) investigated microwave-induced ( . ghz) cataract formation in the rabbit eye. there was a direct relationship between maximal energy absorption and maximal temperature in the vitreous body at a point midway between the posterior surface of the lens and the retinal surface. the locus of peak energy absorption and peak temperature correlated well with cataract formation in the posterior cortical lens, after a latent period of a few days, when the microwave exposure was at or above threshold levels, e.g., w/kg for min and retrolental temperature above • c. kramar et al. ( ) tried to explain cataractogenesis by introducing hot water into the ocular cavity of rabbit. cataract was consistently produced by retrolental temperatures between • c and • c. the authors claim that these findings support the assumption that microwave cataractogenesis is due to the local production of elevated temperatures. hirsch et al. ( ) studied effects of repeated microwave irradiation to the albino rabbit eye. they used . ghz microwaves irradiated to the eye of albino rabbits for min/day for a month. clinical examination was carried out for a period up to year. no change occurred below a power density of mw/cm . however, at and above this value, posterior subcapsular iridescence and posterior cortical cataracts were produced. chou et al. ( ) investigated the effects of exposure of rabbits to . and mw/cm , . ghz continuous wave radiation for days. sixteen male new zealand rabbits were divided into two groups: eight of them were exposed hr/dy, dy/wk for wks; the other eight rabbits were sham exposed. eyes were examined for cataract formation. the only difference identified was decrease of food consumption during the mw/cm exposure. there were no ocular differences between microwave-exposed and sham-exposed groups. foster et al. ( ) reported that a single, min exposure to . ghz radiation, with . w being absorbed, produced a cataract in half of the exposed eyes of new zealand white rabbits. saito et al. ( ) investigated the effects of acute microwave exposure at . ghz on the eye of unanesthetized rabbits, with the contralateral eye serving as the control. nine restrained, adult japanese white rabbits were irradiated unilaterally by . ghz continuous wave microwaves for min to min. using phantom material, the estimated sar was . w/kg. the average increment in corneal surface temperature increment was . • c for min. miosis occurred in all rabbits within min. post-exposure ophthalmologic signs included ) miosis and papillary congestion; ) keratoleucoma and corneal edema; ) endothelial cell detachment and floating in aqua oculi, ) fibrogenesis in the anterior chamber, and ) conjunctiva edema. these signs disappeared within week after exposure. there was no cataract formation. more sophisticated experimental equipment and methods have been employed in recent investigations. kojima et al. ( ) investigated the effect of systemic anesthesia on ocular effects and temperature in rabbit eyes exposed to microwaves. one eye of male pigmented rabbits was exposed at . ghz for - min ( mw/cm ; w/kg), either under anesthesia (ketamine hydrochloride + xylazine) or without anesthesia. temperatures within the eye were measured during microwave exposure by a fluoroptic thermometer. changes in the anterior segment were evaluated by image analysis utilizing a scheimpflug camera, specular microscopy, and a laser flare cell meter. the exposed eyes showed miosis, conjunctival congestion, corneal edema, and an increase in the light scattering of the anterior shallow cortex in the papillary area of the lens. the group under systemic anesthesia showed much stronger symptoms than those without anesthesia. the highest temperature during exposure was in the vitreous, followed by the anterior chamber, and the retrobulbar cavity of the orbit. the ocular temperatures of the rabbits under systemic anesthesia were - • c higher than those without anesthesia. body temperature showed an increase of • c during the exposure. the more pronounced ocular effects in the anesthetized rab-bits were associated with the significantly higher ocular temperatures. the authors stressed that the influence of systemic anesthesia on ocular changes should be considered when examining microwave-induced cataract formation. considerable research has been done on cataractogensis in rabbit eyes exposed to microwaves. cataracts result from excessive heating. safe and dangerous exposure levels can be predicted. kramar et al. ( ) investigated possible species differences relating to cataract formation following microwave exposure. the authors irradiated both rabbits and monkeys (macaca mulatta) in the near field of continuous wave . ghz to determine the cataractogenic threshold. rabbits developed cataracts and transient changes such as miosis, dilated vessels etc. at "apparent" incident power densities of mw/cm or greater. monkeys sustained facial burns, but no lens damage, even at "apparent" incident power densities of mw/cm . these rabbit and monkey experiments showed clearly that the same incident power density microwave exposure did not produce similar effects on the face and eye of these two experimental animals (kramar et al. ) . the dissimilar effects reflect the different patterns of . ghz energy absorption in the monkey and rabbit heads due to their different facial structures. rabbit eyes protrude in comparison to monkey eyes, which are more embedded within the eye sockets. the sar threshold for cataracts in the monkey eye might be the same as the sar threshold for cataracts in rabbit eyes. these results showed that cataractogenic power density levels in rabbit and dogs should not be directly extrapolated to primates, including human beings. kues et al. ( ) examined ocular effects associated with exposure to ghz waves on rabbits and monkeys (macaca mulatta). an antenna that produced a uniform energy distribution was used at an incident power density of mw/cm . acute exposure of both rabbits and monkeys consisted of a single, h exposure; the repeated exposure protocol consisted of five separate h exposures on consecutive days. one eye was exposed, and the contralateral eye served as the sham-exposed control. after post-exposure diagnostic examinations, ocular tissue was examined by both light and transmission electron microscopy. no ocular changes were found in either rabbits or monkeys that could be attributed to millimeter-wave exposure at mw/cm . as mentioned above, kramar et al. ( ) also saw no cataracts in microwave-exposed monkeys. mcafee et al. ( ) trained unrestrained monkeys (macaca mulatta) to expose their own face and eyes to pulsed microwave radiation at a frequency of . ghz and an average power density of mw/cm . twelve monkeys were individually irradiated during for periods of to days, for to minutes each day. the subjects then were observed for a period of one year. no deleterious effects, such as cataracts, were been observed. mcafee et al. ( ) further reported the outcome of microwave irradiation of rhesus monkeys' eyes at . ghz over months and . ghz for months at average power density of mw/cm . irradiation of monkeys (macaca mulatta) was accomplished without restraint or anesthesia by training the monkeys to irradiate themselves. no cataracts, no effects on cornea, aqueous and vitreous humors or retina, and no loss of visual capability were found. lu et al. ( ) studied the effects of . ghz, high-peak power microwaves on retina in rhesus monkeys. pre-exposure fundus photographs, retinal angiograms, and electroretinograms (erg) were obtained to screen for normal ocular structure and function. seventeen monkeys were randomly assigned to receive either sham or pulsed microwave exposure. the pulse characteristics were . mw (approximately . mw/kg, temporal peak retinal sar), . µsec pulse length at pulses repetition rates of , . , . , and . hz. (these are extremely high power values, but the pulses are very short.) microwaves were delivered to the face with retinal sars of , . , . , or . w/kg. nine exposures were given at hr/dy, dy/wk for weeks. pre-exposure and post-exposure fundus pictures and angiograms all were within normal limits. the response of cone photoreceptors to light flash was enhanced in monkeys exposed at . or . w/kg, but not in monkeys exposed at . w/kg. enhanced cone photoreceptor b-wave responses were observed in a sardependent manner; these could be an early indicator of mild injury. however, no evidence of degenerative change or erg depression was seen. from these results, retinal injury is very unlikely at w/kg delivered to the retina. functional changes probably were reversible, because no evidence of histopathologic correlation with erg changes was observed. chalfin et al. ( ) evaluated anterior segment bioeffects of pulsed, ghz and ghz microwave exposure in the nonhuman primate eye. five juvenile rhesus monkeys (macaca mulatta) underwent baseline anterior segment ocular assessment consisting of slit lamp examination, corneal topography, specular microscopy, and pachymetry. these studies were repeated after exposure of one eye to pulsed ghz or ghz microwaves at varied fluences, with the other eye serving as a control. the fluence required to produce a threshold corneal lesion, i.e., faint epithelial edema and fluorescein staining, was . j/cm at ghz and . j/cm at ghz. transient changes in corneal topography and pachymetry were noted at these fluences. endothelial cell counts remained unchanged. threshold corneal injury from ghz and ghz microwave exposure is produced at fluences below those previously reported for co laser radiation. these data might help elucidate the mechanism of thermal injury to the cornea. magnetic resonance imaging (mri) has been widely used worldwide since . high-field-strength/high-frequency mri systems can cause tissue heating. the eye is particularly susceptible to temperature elevations because of its relatively poor blood supply. shellock and crues ( a) and shellock and schatz ( ) measured corneal temperatures in patients immediately before and after mri performed with a . t, mhz imager and a transmit/receive head coil; estimated peak sars ranged from . to . w/kg. there was an increase in the average corneal temperature: . ± . • c before imaging and . ± . • c after. the changes in corneal temperature ranged from . • c to . • c (mean . • c), and the highest corneal temperature measured after imaging was . • c. in animal models, the eye temperature threshold for microwave-induced cataractogenesis is between • c and • c. from these results, it was concluded that clinical mri with use of a head coil delivering microwaves at the sars studied causes relatively minor increases in corneal temperature that do not appear to pose any thermal hazard to ocular tissue. from the results described above, one can conclude that microwave-induced cataracts are caused by an elevation in corneal temperature. whole body (or farfield) exposure studies show that cataracts do not form in rabbit eyes unless intense microwave fields, at or near lethal levels, are applied. because microwave-induced cataracts and other ocular changes have been reported in experimental animals, especially rabbits, several researchers have been concerned with human eyes. dozens of papers, referring to communication facilities and equipment, were published during s and s. however, cataracts caused by microwave exposure have not been reported in humans. accidental exposures have produced either sub-clinical changes to the lens or no ocular effects (elder ) . in summary, experimentally induced cataracts have been reported in rabbits, but not monkeys following microwave exposure. the presumed mechanism for cataract initiation is hyperthermia. at frequencies of about khz or greater, the dominant interaction mechanism in biological tissues is heating. at lower frequencies, the dominant interaction mechanism in biological tissues is induced current. the photon energy of microwave radiation is far too low to affect chemical bonding directly. the electric fields induced in tissues by rf radiation result in energy absorption due to the polarization of electrically charged structures and the flow of ions. it is assumed that the increase in linear and rotational energy is rapidly dissipated by molecular collision, resulting in generalized heating. investigations on auditory responses to microwaves can be classified into two categories. one is perception, i.e., hearing of microwaves, and the other is any possible effects on the auditory pathway. audible frequency ranges for sound waves are hz to , hz in humans; hz to , hz in dogs; and khz to khz in bats. the receptor cells for hearing are the inner and outer hair cells in the organ of corti of the cochlea within the inner ear. both hair cells and the auditory nerve fibers are tonotopically organized; at any position, they are most responsive to a particular frequency. the tectorial membrane arises from the organ of corti. the longest stereocilia of the outer hair cells are tightly attached to the lower surface of the tectorial membrane. when the basilar membrane vibrates in response to a sound, the organ of corti and the overlying tectorial membrane are carried mechanically with it. because the basilar and tectorial membranes pivot about different lines of insertion, their oscillating displacements are accompanied by back-and-forth shearing motions between the upper surface of the organ of corti and the lower surface of the tectorial membrane. the mechanical deflection of the hair cell bundle is the proximate stimulus that excites each hair cells of the cochlea. this deflection is transduced into a receptor potential. the receptor potentials trigger aps that eventually are transmitted to the cortical auditory areas, which are responsible for perception of sound. the localization of sound sources sets stringent limits on the speed of direct mechano-electrical transduction of hair cells. if a sound source lies directly to one side of an animal, an emitted sound will reach the nearer ear somewhat sooner than the farther ear. for a human, this delay is at most µsec. both humans and owls can locate sound sources on the basis of much smaller temporal delays, about µsec. for this to occur, hair cells must be capable of detecting acoustical waveforms with microsecond-level resolution (hudspeth ) . at mhz, the wave duration is µsec. any frequencies higher than mhz are therefore "out of limit" for activation of hair cells in the organ of corti. indeed, there are no known reports of continuous wave signals causing microwave-induced sound in humans or microwave-induced auditory responses in experimental animals (elder and chou ) . all the reports on 'hearing' of radiofrequency deal with auditory response to pulsed ( to around , hz) microwaves, which commonly is called radiofrequency hearing. the 'sound was something like that of a bee buzzing on a window, but with, perhaps, more high frequencies' (ingalls ) . it also has been reported to be similar to other common sounds, such as a click, hiss, knock ,or chirp. a quiet environment is required for the mf hearing, because the normal noise levels in outdoor or laboratory settings mask the hearing of microwave sounds. the necessary condition for hearing the radiofrequency-induced sound is the ability to hear audio frequencies above approximately khz and bone conduction hearing at lower acoustic frequencies. cochlear microphonics are the alternating potential changes which follow the stimulus frequency. microphonics are recorded from near the cochlea and are mainly composed of receptor potentials from outer hair cells. chou et al. ( ) recorded cochlear microphonics that were similar to those evoked by acoustic stimuli, from the round window of guinea pigs during irradiation by pulsed mhz (repletion rate hz) microwaves. recording of cochlear microphonics demonstrated that the microwave auditory effect was due to mechanical distortion of the cilia of cochlear hair cells. these experiments indicate that the primary site of transduction of microwave energy is outside or at the cochlea. chou and galambos ( ) recorded the brain-stem evoked response from guinea pigs stimulated at various intensities by acoustic pulses. either blocking of the external ear or destruction of the middle-ear produced little change in the brain-stem evoked responses elicited by microwave pulses. from these results, it was suggested that conduction of pressure waves through the bones appear to be the mechanism responsible in perception of pulsed microwaves. using conventional glass microelectrodes, seaman and lebovitz ( ) recorded extracellular aps of neurons in cat dorsal and postventral cochlear nuclei while the head of the cat was exposed to microwave pulses at mhz. response thresholds to acoustic tones, acoustic clicks, and microwave pulses were determined for auditory units with characteristic frequencies from hz to . khz. the midline brainstem sar threshold was as small as . mw/g/pulse, and specific absorption threshold was as small as . µj/g/pulse. microwave thresholds were generally lower for characteristic frequency less than khz, as were most acoustic thresholds. these results show that microwave pulses directly stimulated cochlea, then the evoked aps propagated along the normal auditory system, i.e., eighth cranial nerve, medial geniculate nucleus, and primary auditory cortex. foster and finch ( ) proposed thermoelastic expansion as a mechanism for rf hearing. they conducted experiments in water and in kcl solution exposed to microwave pulses similar to those that produce sounds in humans. they showed that pressure changes would result from the absorption of microwave pulses that could produce significant acoustic energy in the solution. thus, they concluded that audible sounds were produced, via bone conduction, by rapid thermal expansion caused by absorption of the energy in the microwave pulses. arai et al. ( ) investigated whether the pulsed microwaves emitted by a mobile phone have short-term, adverse effects on the human central auditory system. using volunteers with normal hearing, the auditory brainstem response (abr), the abr recovery function, and the middle latency response were recorded before and after using a mobile phone for min. none of the three measures were affected by exposure to the field for min. bak et al. ( ) reported no effects of microwave exposure at mhz, mhz, or . ghz on abr during and after exposure for min. the subjects were young, healthy volunteers of both sexes. the abr evaluation was performed before, during, and immediately after the exposure, and the latencies of waves i, iii, and v, and inter-waves i-v were analyzed. the authors concluded that brief mobile phone use does not affect propagation of electrical stimuli along the auditory nerve to auditory brainstem centers. ozturan et al. ( ) studied the effects of mobile phone use for minutes on human hearing. using volunteers with normal hearing, evoked otoacoustic emissions (oaes) were measured before and after cell phone exposure. no measurable changes in evoked oaes were detected, and none of the subjects reported a deterioration in hearing level. the authors concluded that a min exposure to the microwaves emitted by a mobile telephone had no effect on hearing, a least at outer ear, middle ear, and cochlea. then the same group (kizilay et al. ) studied the effects of chronic exposure to the microwaves emitted by a mobile phone on the inner ear of adult and developing rats using distortion-product oaes. seven of adult rats and four newborn rats were exposed hr/day for days; the other seven adult rats were assigned to control group. no measurable difference in distortion-product oaes were found between exposed and control groups, and no changes were found in developing rats. it was concluded that days of exposure at hr/dy did not cause any hearing deterioration in either adult or developing rats. the rf hearing effect occurs because the elf-modulated pulses stimulate the cochlea, which then responds in the normal manner. apparently the cochlea is stimulated via bone conduction caused by rapid thermal expansion resulting from the absorption of the energy in the microwave pulses. these available data from animals and humans suggest that the microwave fields associated with cell phones have no effect on the auditory pathway. however, the data base is small, and the durations of experimental exposure are short, especially in humans. because so many people already make extensive use of their cell phones, finding unexposed (or not recently exposed) subject will become increasingly difficult. perhaps laboratory tests show no differences, because the auditory system has been affected before the subject arrives at the laboratory. it is essentially important for the existence of animals or humans to maintain body temperature within a certain limited range, while living environments of diverse and varying temperatures. said the most basic level, the maintenance of body temperature is achieved by balancing heat production within the body and heat loss to the surroundings. change of body temperature is detected, especially externally at the skin and internally by a specialized region of the brain. the information is integrated in the cns, and regulation is achieved by autonomic and behavioral thermoregulatory reactions (fig . ) . these reactions act to overcome the thermal load for maintaining the normal body temperature. the balance of body temperature is expressed by the following equation; where m is the rate at which thermal energy is produced through metabolic processes; w is the rate at which work is produced; e is the rate of heat exchange with the surroundings via evaporation; r is the rate of heat exchange with the surroundings via radiation; c is the rate of heat exchange with the environment via convection; d is the rate of heat exchange with the surroundings via conduction; and s is the rate of body heat storage. heat production is achieved by non-shivering thermogenesis (nst) and shivering heat production. the basal metabolic rate, a part of nst, is the heat production of a resting human being in a thermo-neutral condition ( - • c) at a time exceeding hours from the last meal. nst consists of both basic nst, which is related to energy demand of the body, and thermoregulatory nst, which takes place during cold exposure. body tissues that produce thermogenesis are skeletal muscles, thoracic and abdominal organs (such as heart, liver, and alimentary canal), brain, and brown adipose tissues. nst is humorally controlled by β-adrenergic activity. shivering is non-voluntary contraction of skeletal muscles that occurs simultaneously on both flexor and extensor muscles at - hz. the produced heat is conducted by blood flow throughout the body. heat exchange between body and the surroundings is achieved by heat loss responses and evaporation. thermosensors are located under the skin and deep inside the body. the deep sensors are mainly located in medial preoptic/anterior hypothalamic region. however, some additional thermosensors are found in medulla oblongata, spinal cord, and deep abdominal organs. among these receptors, the skin sensors are the most impor-tant, but medial preoptic/anterior hypothalamic receptors also contribute greatly to production of thermoregulatory responses. integration of temperature information from these sensors takes place in hypothalamus. the thermoregulatory system can be described as a "closed loop, with negative feedback", as are other biological regulatory systems, like endocrine system. exposure to rf at frequencies above khz, i.e., exposure to microwaves, generates heat in body tissues. the sensation of tissue warming is necessary to initiate appropriate behavioral action, although physiological responses such as sweating and peripheral circulation changes also can be initiated automatically and autonomously by thermal stimuli. therefore, any microwave experiments should be planned and interpreted vis-à-vis thermoregulatory mechanisms. spiers et al. ( ) reported neonatal rats ( - days of age) showed a . • c increase in the colonic temperature at the end of a min continuous wave exposure to . ghz microwaves ( mw/cm , sar = w/kg) at cold ambient temperature of • c, without any change in metabolic heat production. colonic temperature was increased by . • c after exposure to mw/cm for min. the results indicate that the hypothermic rat pup can be effectively warmed by low-level microwave irradiation. furthermore, the pup is capable of altering metabolism in response to such heating. adair et al. ( ) studied changes in thermoregulatory physiological responses and behaviors in squirrel monkeys chronically exposed to continuous wave, . ghz microwaves, for hr/wk for wk at power densities of or mw/cm . the whole body sar was . w/kg per mw/cm . three different, controlled environmental temperatures, , or • c, were used. most previous studies paid no attention to the factor of environmental temperature. physiological responses were measured three times: ( ) during a pre-exposure phase of - weeks, ( ) during a -week exposure period, and ( ) during a post-exposure period of - weeks. variables measured were body mass, blood properties, metabolic heat production, sweating, skin temperature, deep body temperature and behavioral responses by which the monkeys selected a preferred environmental temperature. results showed no reliable alteration of metabolic rate, internal body temperature, blood indices, or thermoregulatory behavior. an increase in sweating rate occurred in the • c environment. skin temperature, reflecting vasomotor state, was reliably influenced by both ambient temperature and microwaves. the most robust consequence of microwave exposure was a reduction in body mass. adair et al. ( ) studied whether exposure to microwave fields at the resonant frequency generated heat deep within the body. adult male squirrel monkeys, held in the far field of an antenna within an anechoic chamber, were exposed ( min or min) to either resonant mhz or supra-resonant . mhz continuous wave fields (e polarization) in cool environments. whole-body sars ranged from - w/kg ( mhz) and - w/kg ( , mhz). colonic and several skin temperatures, metabolic heat production, and evaporative heat loss were monitored continuously. during brief microwave exposures in the cold, the reduction of metabolic heat production was directly proportional to the sar, but , mhz energy was a more efficient stimulus than was the resonant frequency ( mhz). detailed analyses of the data indicate that temperature changes in the skin were the primary source of the neural signal for a change in physiological interaction processes during microwave exposure in the cold. the essential message from these experiment is that microwave irradiation is just another source of heat, which the body can deal with by the normal processes of the thermoregulatory system. microwaves are not an extraordinary, highly-dangerous stimulus that is beyond the body's coping mechanisms. microwaves are just a heat source, like a heat lamp or hot air. however, too much heat, like a very high sar or a forest fire, is a serious threat to existence. adair et al. ( ) measured thermoregulatory responses of heat production and heat loss in seven adult volunteers ( women and men, aged - years) during min dorsal exposures of the whole body to continuous wave, mhz microwaves. two exposure levels, sars of . or . w/kg, were tested in each of three ambient temperatures ( , and • c). no changes in metabolic heat production occurred under any of the exposure conditions. vigorous increases in sweating rate on back and chest, directly related to both ambient temperature and power densities, cooled the skin and ensured efficient regulation of the deep body (esophageal) temperature to within . • c of the normal level. these results indicate that dorsal exposures of humans to microwaves at the supra-resonant frequency of mhz, at local peak sar up to . w/kg, are mildly thermogenic and are counteracted efficiently by normal thermophysiologic heat loss mechanisms, principally sweating. adair et al. ( ) further measured thermoregulatory responses of heat production and heat loss in two different groups of seven adult volunteers (males and females) during min dorsal exposures of the whole body to continuous wave mhz or . ghz microwave fields. at each frequency, two power densities were tested at each of three ambient temperatures [t(a)] = , and • c) plus t(a) controls (no rf). the normalized peak sar was the same for comparable power densities at both frequencies, i.e. peak surface sar = . and . w/kg. no change in metabolic heat production occurred under any exposure conditions at either frequency. the magnitude of increase in those skin temperatures under direct irradiation was directly related to frequency, but local sweating rates on back and chest were related more to t(a) and sar. both efficient sweating and increased local blood flow contributed to the regulation of the deep body (esophageal) temperature to within . • c of the baseline level, which agreed with the previous study. many reports present data showing that continuous wave and pulsed microwave fields, at the same frequency and average power density, produce similar responses in the exposed organism. during whole-body exposure of squirrel monkeys at . ghz using either continuous wave or pulsed fields, heat production and heat loss responses were nearly identical. to explore this question in humans, adair et al. ( a) exposed two different groups of volunteers to . ghz using either continuous wave (two females, five males) and pulsed ( µsec pulse width; three females, three males) microwave fields. thermophysiological responses of heat production and heat loss were measured under a standardized protocol ( min pre-exposure baseline, min field or sham exposure, min post-exposure baseline), conducted in three t(a) levels: , , and • c. at each t(a), the sars were , . and . w/kg. data for each group showed minimal changes in core temperature and metabolic heat production for all test conditions. local skin temperatures showed similar patterns for continuous wave vs. pulsed exposure; skin temperature depended only on sar. however, there was one reliable difference between continuous wave and pulsed exposure. only the skin temperature of the upper back (i.e., the area facing the antenna) showed a greater increase during pulsed exposure than during continuous exposure. for all other measurements, no clear evidence for a differential response to continuous vs. pulsed microwave fields was found. adair et al. ( b) studied thermoregulatory responses of human beings following partial body exposure to . ghz continuous wave microwaves at higher peak power densities of and mw/cm . seven volunteers (four males and three females) were tested at each power density at three t(a) values: , and • c. the lab's standard protocol of min baseline, min exposure, and min baseline was used. esophageal and six skin temperatures, metabolic heat production, local sweating rate, and local skin blood flow were measured. no change in esophageal temperature or metabolic heat production was recorded at any power density at any t(a). at peak density of mw/cm , skin temperature on the upper back (irradiated directly) increased . • c with t(a) at • c; . • c with t(a) at • c, and . • c with t(a) at • c. these differences were due primarily to increases in local sweating rate, which was greatest in ambient temperature at • c. also at peak power density at mw/cm , local skin blood flow on the back increased % over baseline level with t(a) at • c; the increase was only % at a t(a) of • c. vigorous heat loss responses of blood flow and sweating maintain thermal homeostasis efficiently. adair et al. ( ) measured thermophysiological responses of heat production and heat loss in seven adult volunteers (six males and one female, aged - years) during min dorsal exposures of the whole body to a mhz (continuous wave) microwave field. sars of . , . and . w/kg were tested in each of three t(a) ( , , and • c), along with sham exposure. a standardized protocol consisting of min pre-exposure baseline, min microwave or sham exposure, and min post-exposure baseline was used. measured responses included esophageal and skin temperatures at seven locations, metabolic heat production, local sweating rate, and local skin blood flow. no changes in metabolic heat production occurred under any test condition. unlike published results of similar exposure at mhz and . ghz, local skin temperatures, even those on the back that were irradiated directly, changed little or not at all during mhz exposures. during the min microwave exposure, esophageal temperature showed modest changes (range = − . to . • c) and never exceeded . • c. thermoregulation principally was controlled by appropriate increases in evaporative heat loss (sweating) and, to a lesser extent, by changes in skin blood flow. because of the relatively deep penetration of microwave energy at this frequency, effectively bypassing the skin, these changes must have been stimulated by thermal receptors deep in the body, rather than by those located in the skin. from these results, the authors argued that continuous microwave radiation with an intensity less than mw/cm is unlikely to affect physiology significantly through athermal mechanisms. walters et al. ( ) studied the role of baseline skin blood flow on the rate of cutaneous heating induced by ghz microwave energy in humans ( female, male). exposure intensities were high power, w/cm for sec, and low power, mw/cm for sec. at the time of exposure, skin blood flow was (a) normal, (b) eliminated using a blood pressure cuff to occlude forearm blood flow, or (c) elevated by heating the skin prior to irradiation. results showed that only a two-fold elevation in baseline skin blood flow had a profound impact on the subsequent rate of heating, resulting in a substantially lower rate of heating. occlusion to block increased blood flow to the skin reversed this lower rate of heating. these results demonstrate that relatively small changes in skin blood flow can produce substantial alterations in the rate of skin heating during prolonged ghz exposure. in summary, results from humans tell the same story as experiments with animals. microwaves produce tissue heating, and the body uses its thermoregulatory system in the usual manner to deal with the added heat load. the strong implication is that microwaves and rf are not hazardous, so long as they do not exceed the thermoregulatory capacity of the body. for clinical investigation of patients, mri devices are used widely. the primary concern about this technology is the possibility of tissue heating caused by microwave exposure. shellock and crues ( b) measured body and skin temperatures in patients immediately before and after clinical mri. mri was performed with a . t system using a cm, open-bore microwave transmit/receive head coil specifically designed for examination of the brain. body temperature did not change. however, forehead skin temperature and outer canthus skin temperature increased by . • c and . • c, respectively. shellock et al. ( ) further studied physiological responses to an mri procedure performed at a sar of . w/kg at . t, mhz. assessment was made before, during a min mri procedure, and immediately after mri. statistically significant (p < . ) increases in temperature of the tympanic membrane, the skin of the chest, abdomen, upper arm, hand and thigh, plus increases in heart rate and cutaneous blood flow, were associated with exposure to high sar. however, these small changes all are well within the limits that can be tolerated by persons with normal thermoregulatory function. mammals have evolved elaborate systems for regulating optimal body temperature in hot or cold environments. experiments with animals show that microwaves are just another heat source, like hot air or an infrared heat lamp. if heat load produced by microwave (or rf) exposure is small, the thermoregulatory system adjusts and there are no adverse effects. however, if the heat load applied is too great, lethality can result. experiments with humans, including those with mri, are directly in agreement with the viewpoint based on animal experiments. however, with humans, only low sars can be used. many details enter the story, such as differences in penetration depth of different wavelengths (i.e., frequencies), but the big picture is clear. biological systems are fundamentally noisy -on the molecular scale as a consequence of thermal agitation -and macroscopically -as a consequence of physiological functions and animal behavior. if electromagnetic fields are to significantly affect physiology, their direct physical effect must be greater than that from the ubiquitous, endogenous noise. thermal noise is an essential attribute of living systems, and the lack of thermal noise means death. one of the classic problems in bioelectromagnetics has been the 'needle in the haystack' problem. how can an imposed stimulus producing tiny energy effects have any influence under conditions where the noise is many orders of magnitude bigger than the signal? scientists have offered theoretical formulations by which the signal/noise ratio could be overcome, but it has been very difficult to illustrate the workings of such theoretical mechanisms in data from real biological systems. physiological interaction processes and radiofrequency energy absorption partial-body exposure of human volunteers to mhz pulsed or cw fields provokes similar thermoregulatory responses human exposure to mhz cw energy at levels outside the ieee c . standard does not increase core temperature thermophysiological consequences of whole body resonant rf exposure ( mhz) in human volunteers human exposure at two radio frequencies ( and mhz): similarities and differences in physiological response thermophysiological responses of human volunteers during controlled whole-body radio frequency exposure at mhz thermoregulatory consequences of long-term microwave exposure at controlled ambient temperatures effect of nonionizing radiation on the purkinje cells of the uvula in a squirrel monkey cerebellum effect of nonionizing radiation on the purkinje cells of the rat cerebellum a cognitive-behavioral treatment of patients suffering from 'electric hypersensitivity thirty minutes mobile phone use has no short-term adverse effects on central auditory pathways working memory no effects of acute exposure to the electromagnetic field emitted by mobile phones on brainstem auditory potentials in young volunteers gsm modulated radiofrequency radiation does not affect -sulfatoxy-melatonin excretion of rats chronic exposure to a gsm-like signal (mobile phone) does not stimulate the development of dmba-induced mammary tumors in rats: results of three consecutive studies from slow waves to sleep homeostasis: new perspectives pulsed highfrequency electromagnetic field affects human sleep and sleep electroencephalogram the excretion of -hydroxymelatonin sulfate in healthy young men exposed to electromagnetic fields emitted by cellular phone -an experimental study influence of a radiofrequency electromagnetic field on cardiovascular and hormonal parameters of the autonomic nervous system in healthy individuals melatonin metabolite excretion among cellular telephone users millimeter wave absorption in the nonhuman primate eye at ghz and ghz slow potentials and spike unit activity of the cerebral cortex of rabbits exposed to microwaves middle-ear structures contribute little to auditory perception of microwaves effects of electromagnetic fields on isolated nerve and muscle preparation carbon-loaded teflon electrodes for chronic eeg recordings in microwave research cochlear microphonics generated by microwave pulses chronic exposure of rabbits to . and mw/cm mhz cw microwave radiation radial arm maze performance of rats following repeated low level microwave radiation exposure electromagnetic hypersensitivity whole-body exposure to . ghz electromagnetic fields does not alter anxiety responses in rats: a plus-maze study including test validation microwave effect on rabbit superior cervical ganglion the effects of microwave diathermy on the eye lack of behavioral effects in the rhesus monkey: high peak microwave pulses at . ghz gsm radiocellular telephones do not disturb the secretion of anterior pituitary hormones in humans evaluation in humans of the effects of radiocellular telephones on the circadian patterns of melatonin secretion, a chronobiological rhythm marker does head-only exposure to gsm- electromagnetic fields affect the performance of rats in spatial learning tasks? head-only exposure to gsm -mhz electromagnetic fields does not alter rat's memory in spatial and non-spatial tasks the acute effects of exposure to the electromagnetic field emitted by mobile phones on human attention ocular effects of radiofrequency energy auditory response to pulsed radiofrequency energy effects of global system for mobile communication (gsm)-like radiofrequency fields on vascular permeability in mouse brain effect of long-term mobile communication microwave exposure on vascular permeability in mouse brain provocation of electric hypersensitivity under everyday conditions microwave hearing: evidence for thermoacoustic auditory stimulation by pulsed microwaves dosimetric study of microwave cataractogenesis effects of microwaves emitted by cellular phones on human slow brain potentials microwaves emitted by cellular telephones affect human slow brain potentials effect of global system for mobile communication (gsm) microwave exposure on bloodbrain permeability in rat alterations in alpha-adrenergic and muscarinic cholinergic receptor binding in rat brain following nonionizing radiation mhz mobile phone does not affect short term memory in humans effects on brains of fisher rats of exposure to gsm- signals: preliminary data of a confirmation study of the salford experiments short-term exposure to mhz tdma field does not alter melatonin synthesis in rats hypersensitivity symptoms associated with exposure to cellular telephones: no causal link human brain activity during exposure to radiofrequency fields emitted by cellular phones prevalence of self-reported hypersensitivity to electric or magnetic fields in a population-based questionnaire survey environmental illness: fatigue and cholinesterase activity in patients reporting hypersensitivity to electricity effects of gsm electromagnetic field on the meg during an encoding-retrieval task effects of repeated microwave irradiations to the albino rabbit eye electromagnetic fields, such as those from mobile phones, alter regional cerebral blood flow and sleep and waking eeg radio frequency electromagnetic field exposure in humans: estimation of sar distribution in the brain, effects on sleep and heart rate exposure to pulse-modulated radio frequency electromagnetic fields affects regional cerebral blood flow hearing experimental study on thermal damage to dog normal brain lack of promoting effects of the electromagnetic near-field used for cellular phones ( . mhz) on rat liver carcinogenesis in a medium-term liver bioassay the . ghz electromagnetic near-field used for cellular phones does not promote rat liver carcinogenesis in a medium-term liver bioassay inhibitory effects of low doses of melatonin on induction or preneoplastic liver lesions in a medium-term liver bioassay in f rats: relation to the influence of electromagnetic near field exposure effects of whole body microwave exposure on the rat brain contents of biogenic amines sensation of hearing in electromagnetic fields effects of the mhz electromagnetic field emitted from cellular phone on nocturnal melatonin secretion cardiovascular and thermal responses in rats during ghz irradiation cardiovascular and thermal effects of microwave irradiation at and/or ghz in anesthetized rats non-ionizing electromagnetic wave effects in biological materials and systems effects of chronic exposure of electromagnetic fields from mobile phones on hearing in rats effects of mhz electromagnetic field emitted by cellular telephones on response times in humans the effects of electromagnetic field emitted by gsm phones on working memory gsm phone signal does not produce subjective symptoms influence of anesthesia on ocular effects and temperature of rabbit eyes exposed to microwaves effect of low-intensity millimeterrange electromagnetic irradiation on the recovery of function in lesioned sciatic nerves in rats voluntary movement thermal cataract formation in rabbits acute microwave irradiation and cataract formation in rabbits and monkeys the ocular effects of microwaves on hypothermic rabbits: a study of microwave cataractogenic mechanisms effects of high-frequency electromagnetic fields on human eeg: a brain mapping study effects of electromagnetic field emitted by cellular phones on the eeg during an auditory memory tasks: a double blind replication study effects of electromagnetic field emitted by cellular phones on the eeg during a memory task effects of electromagnetic fields emitted by cellular phones on the electroencephalogram during a visual working memory task absence of ocular effects after either single or repeated exposure to mw/cm from a ghz cw source interaction of microwaves and a temporally incoherent magnetic field on spatial learning in the rat microwave irradiation affects radial-arm maze performance in the rat single vs. repeated microwave exposure: effects on benzodiazepine receptors in the brain of the rat opioid receptor subtypes that mediate a microwave-induced decrease in central cholinergic activity in the rat intraseptal microinjection of beta-funaltrexamine blocked a microwave-induced decrease of hippocampal cholinergic activity in the rat psychoactive-drug response is affected by acute low-level microwave irradiation low-level microwave irradiations affect central cholinergic activity in the rat japanese encephalitis virus (jev): potentiation of lethality in mice by microwave radiation the effect of chronic exposure to . mhz fdma or . mhz cdma radiofrequency radiation on the incidence of spontaneous tumors in rats the effect of the duration of exposure to the electromagnetic field emitted by mobile phones on human attention non-thermal activation of the hsp /p mark stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer-and blood-brain barrier-related effects study of self-reported hypersensitivity to electromagnetic fields in california video computer terminals and occupational dermatitis provocation with stress and electricity of patients with 'sensitivity to electricity temperature and corticosterone relationships in microwaveexposed rats effects of hypophysectomy and dexamethasone on rat adrenal response to microwaves the relationship of decreased serum thyrotropin and increased colonic temperature in rats exposed to microwaves effects of high peak power microwaves on the retina of the rhesus monkey neurophysiological study of patients with perceived 'electrical hypersensitivity' provocation study of persons with perceived electrical hypersensitivity and controls using magnetic field exposure and recording of electrophysiological characteristics absence of deleterious effects of chronic microwave radiation on the eyes of rhesus monkeys absence of ocular pathology after repeated exposure of unanesthetized monkeys to . ghz microwaves effects of mhz exposure on the integrity of the blood-brain barrier elimination of microwave effects on the vitality of nerves after blockage of active transport effects of pulsed electromagnetic fields on cognitive processes -a pilot study on pulsed field interference with cognitive regeneration no effects of pulsed high-frequency electromagnetic fields on heart rate variability during human sleep effects of pulsed high-frequency electromagnetic fields on the neuroendocrine system poisoning of an urban family due to misapplication of household organophosphate and carbamate pesticides real-time measurement of brain microcirculation during rf-emf exposure using an " "-shaped loop antenna effects of radiofrequency exposure on the gabaergic system in the rat cerebellum: clues from semi-quantitative immunochemistry interaction of ethanol and microwaves on blood-brain barrier of rats sequential changes in cerebral blood flow, early neuropathological consequences and blood-brain barrier disruption following radiofrequency-induced localized hyperthermia in the rat effects of the electromagnetic field of mobile telephones on hearing search for frequencyspecific effects of millimeter-wave radiation on isolated nerve function effects of high power microwave pulses on synaptic transmission and long term potentiation in hippocampus no effects of pulsed radio frequency electromagnetic fields on melatonin, cortisol, and selected markers of the immune system in man strength-duration curve for an electrically excitable tissue extended down to near nanosecond reversible irritative effect of acute . ghz microwave exposure on rabbit eyes -a preliminary evaluation permeability of the blood-brain barrier induced by mhz electromagnetic radiation, continuous wave and modulated at , , and hz nerve cell damage in mammalian brain after exposure to microwaves from gsm mobile phones neurophysiological effects of flickering light in patients with perceived electrical hypersensitivity video screen exposure and -sulfatoxymelatonin urinary excretion in women biological effects of exposure to nonionizing electromagnetic fields and radiation: iii. radiofrequency and microwave radiation thresholds of cat cochlear nucleus neurons to microwave pulses corneal temperature changes induced by high-field-strength mr imaging with a head coil temperature changes caused by mr imaging of the brain with a head coil increased corneal temperature caused by mr imaging of the eye with a dedicated local coil physiologic responses to an mr imaging procedure performed at a specific absorption rate of . w/kg lack of effects of mhz electromagnetic near field exposure on the blood-brain barrier in immature and young rats low-level exposure to pulsed mhz microwave radiation does not cause deficits in the performance of a spatial learning task in mice acute thermoregulatory responses of the immature rat to warming by low-level , mhz microwave radiation absence of chronic effect of exposure to short-wave radio broadcast signals on salivary melatonin concentrations in dairy cattle alteration of diurnal rhythms of blood pressure and heart rate to workers exposed to radiofrequency electromagnetic fields cellular phone use does not actually affect blood pressure or heart rate of humans effects of modulated rf energy on the eeg of mammalian brains effects of low intensity radiofrequency electromagnetic fields on electrical activity in rat hippocampal slices effects of exposure to low level radiofrequency fields on acetylcholine release in hippocampus of freely moving rats simultaneous response of brain electrical activity (eeg) and cerebral circulation (reg) to microwave exposure in rats biological and morphological effects on the brain after exposure of rats to a mhz tdma no short-term effects of high-frequency electromagnetic fields on the mammalian pineal gland effects of weak microwave fields amplitude modulated at elf on eeg of symmetric brain areas in rats human sleep eeg under the influence of pulsed radio frequency electromagnetic fields. results from polysomnographies using submaximal high power flux density effects of blood flow on skin heating induced by millimeter wave irradiation in humans acute exposure to pulsed -mhz microwaves affects water-maze performance of rats mhz pulsed tdma fields affect performance of rats in a t-maze task only when body temperature is elevated dynamic characteristics of crayfish stretch receptor for microwave radiation (japanese text) formulation and simulation of the neuron response to temperature stimulation (japanese text) key: cord- - zxoq m authors: volochnyuk, dmitriy m.; grygorenko, oleksandr o.; gorlova, alina o. title: fluorine-containing diazines in medicinal chemistry and agrochemistry date: - - journal: fluorine in heterocyclic chemistry volume doi: . / - - - - _ sha: doc_id: cord_uid: zxoq m the combination of a fluorine atom and a diazine ring, which both possess unique structural and chemical features, can generate new relevant building blocks for the discovery of efficient fluorinated biologically active agents. herein we give a comprehensive review on the biological activity and synthesis of fluorine containing, pyrimidine, pyrazine and pyridazine derivatives with relevance to medicinal and agrochemistry. abstract the combination of a fl uorine atom and a diazine ring, which both possess unique structural and chemical features, can generate new relevant building blocks for the discovery of effi cient fl uorinated biologically active agents. herein we give a comprehensive review on the biological activity and synthesis of fl uorine containing, pyrimidine, pyrazine and pyridazine derivatives with relevance to medicinal and agrochemistry. although being present in very small amounts, they are highly odiferous and can be detected at extremely low concentrations. unlike other heterocycles found in many important natural products, pyridazines were discovered only after , and relatively few pyridazines have thus far been isolated from natural sources. as synthetic compounds, all diazines constitute an important pharmacophoric moiety present in many drugs acting on various pharmacological targets as well as agrochemicals. inspite of organofl uorine compounds are almost absent as natural products, ~ % of drugs in the pharmaceutical pipeline and ~ % of agrochemicals contain at least one fl uorine atom. one of the earliest synthetic fl uorinated drugs is the antineoplastic agent -fl uorouracil, derivative of pyrimidine, an antimetabolite fi rst synthesised in . since the advent of -fl uorouracil, fl uorine substitution is commonly used in contemporary medicinal and agrochemistry to improve metabolic stability, bioavailability and protein-ligand interactions. in this review only compound bearing fl uoro or fl uoroalkyl substituent in diazine ring are discussed. among fl uorine containing diazines now drugs and agrochemicals are presented on the market. this review provides an information about fl uorinated diazines as drugs or agrochemicals and their mode of action as well as synthesis. the review is divided in two parts. first part dedicated to the medicinal and synthetic chemistry of fl uorinated diazines that have reached at least clinical development phase. the second one dedicated to the biological role and the chemistry of the marketed agrochemicals based on fl uorinated diazines. it is widely accepted that compounds containing fl uorine atoms have a remarkable record in medicinal chemistry and play a continuing role in providing lead compounds for potential therapeutic applications. the reasons for that have been discussed extensively in a number of books and reviews [ , ] . in this view, fl uorine-containing diazines are not the exception; they have attracted attention of medicinal chemists since s when fluorouracil ( ) was introduces as anti-cancer drug. analysis of mddr (mdl drug data report) data retrieved , hits derived from fl uorine-containing diazines [ ] . nearly a third part of them is represented by anti-cancer agents (fig. ) ; other important classes (more than examples) include compounds with antiviral (mainly anti-hiv) and antiarthritic activity. according to mddr, compounds containing a fl uorinated diazine moiety have entered pre-clinical studies, of them have reached clinical phase, and of these have become drug substances (fig. ) . in the following sections, fl uorinecontaining diazine derivatives that have reached at least clinical development phase will be discussed, focusing on their aspects related to medicinal and synthetic organic chemistry. the use of fl uorinated diazines as anti-cancer agents is the major fi eld of their application in medicinal chemistry. the fi rst representative of this class, fluorouracil ( ) was developed by charles heidelberger and co-workers in [ ] . it was approved by u.s. fda [ ] in as antineoplastic agent in the treatment of advanced colorectal cancer. fluorouracil represents a class of rationally designed anticancer agents which act as antimetabolites. the observation that rat hepatomas utilized radiolabeled uracil more avidly than normal tissues [ ] implied that the enzymatic pathways for utilization of uracil or its close analogs differed between malignant and normal cells -a feature which might provide a target for antimetabolite chemotherapy. a minimal modifi cation of uracil by introducing a single fl uorine atom allowed for implementation of cellular uptake and metabolic activation of via the same transport processes and enzymes involved in the case of uracil. however, in the case of essential biological targets, remarkable differences are observed due to unique properties of the fl uorine atoms, which result in inhibition of the metabolic and signal pathways involved. although all the details of the mechanism by which fluorouracil gives its biological effect are not elucidated, a remarkable progress has been made over the past half a century in elucidating its cellular and clinical pharmacology [ , ] . the key steps in fluorouracil metabolism are shown in scheme . up to % of administered as injection is transformed to dihydrofl uorouracil (dhfu, ) by dihydropyrimidine dihydrogenase (mostly in liver tissues). however, this metabolite is not involved into antineoplastic activity; instead, itself and its further metabolites are responsible for most of the toxic effects of . the main mechanism of activation of fluorouracil is conversion to fl uorouridine monophosphate (fump, ), either directly by orotate phosphoribosyltransferase, or via fl uorouridine (fur, ) through the sequential action of uridine phosphorylase and uridine kinase. is then phosphorylated to give fl uorouridine diphosphate (fudp, ), which can be either phosphorylated again to the active metabolite fl uorouridine triphosphate (futp, ) , or reduced to fl uorodeoxyuridine diphosphate (fdudp, ) by ribonucleotide reductase. in turn, can either be dephosphorylated or phosphorylated to generate an alternative activation pathway involves the thymidine phosphorylase catalysed conversion of to floxuridine (fudr, ), which is then phosphorylated by thymidine kinase to give . the metabolite of -floxuridine -is itself used as an anti-cancer agent [ ] . it was launched in by hospira inc [ ] . upon rapid injection, most of floxuridine is catabolized to fluorouracil; hence similar effects on the organism are obtained in this case. on the contrary, when is slowly administered into the arterial blood, it is mostly transformed to ; thus toxic effects are diminished comparing to [ ] . it has long been recognized that one of the main mechanisms underlying fluorouracil action is inhibition of thymidylate synthase by fl uorodeoxyuridine monophosphate ( ) [ ] . thymidylate synthase belongs to a class of enzymes required for dna replication, and its activity is higher in rapidly proliferating cells. in particular, thymidylate synthase is responsible for methylation of deoxyuridine monophosphate (dump, ) to deoxythymidine monophosphate (dtmp, ) with the use of , -methylenetetrahydrofolate ( ) as a cofactor (scheme ) [ ] . with fl uorodeoxyuridine monophosphate, a slowly-reversible ternary complex is formed instead. inhibition of thymidylate synthase leads to deoxyribonucleotide imbalance, and hence to interference with dna synthesis and repair. alternative mechanism of dna-directed fluorouracil effect is misincorporation of fl uorodeoxyuridine triphosphate ( ) into dna. analogously, fl uorouridine triphosphate ( ) is extensively incorporated into different rna species, disrupting their normal processing and function [ , , ] . two principal approaches were used for the preparation of fluorouracil (scheme ). one of the fi rst methods [ , ] commenced from ethyl fl uoroacetate which was subjected to claisen condensation with ethyl formate to give . the salt was introduced into reaction with s -alkylisothiourea to give fl uoropyrimidines , which were hydrolysed to give . several variations of this method were also described; their common drawback was the use of highly toxic fl uoroacetic acid derivatives. in an alternative approach, fluorouracil was prepared by direct fl uorination of different pyrimidine derivatives, including uracil [ ] , cytosine [ ] , and orotic acid [ ] . in the latter method, the initially obtained fl uoroorotic acid was subjected to decarboxylation. the use of two-step reaction sequence was claimed to be advantageous due to simplifi ed product isolation and purifi cation. early synthesis of floxuridine commenced from fluorouracil ( ) which was transformed into its mercury salt and then allowed to react with -deoxy-dribofuranosyl chloride derivative (scheme ) [ ] . the product was subjected to alkaline hydrolysis to give floxuridine ( ). as in the case of fluorouracil, newer syntheses of floxuridine relied on direct fl uorination of uracil derivatives. fluorination of uridine was done using fl uorine [ ] , acetyl fl uoride [ ] , and cf of [ ] . the latter reagent gave good but still moderate yield of the product ( %). the use of a two-step reaction sequence, i.e. fl uorination of diacetoxy derivative and hydrolysis, improved the yield of to % over two steps [ , ] . despite fluorouracil remains the main agent for the treatment of certain cancer types ( i.e. colorectal) [ ] , it displays various side effects due to its nonspecifi c cytotoxicity, poor distribution to tumor sites, and serious limitations in effectiveness due to drug resistance. apart from modulation of fluorouracil biological action through combination therapies [ , ] , a number of drugs and clinical candidates acting as prodrugs of and/or were developed (table ) . the fi rst example of fluorouracil prodrug is tegafur ( ) developed in s in latvia [ , ] . tegafur is an oral slow-release prodrug formulation of fluorouracil which is readily absorbed through the gastrointestinal tract. the major pathway of metabolic activation of includes hydroxylation by hepatic cytochrome p enzymes, mostly cyp a (scheme ) [ ] . apart from fluorouracil, -hydrohybutyraldehyde and succinic dialdehyde are also formed, which are further transformed into γ-butyrolactone and -hydrohybutyric acid [ ] . tegafur was shown to be - times more potent and less toxic than ; hence lower doses of can be utilized, resulting in decreased neurotoxicity without compromising the antitumor effects. another prodrug of fluorouracil -doxifl uridine ( ), which also implies the idea of attachment of sugar-like moiety to the molecule of , was launched in japan in [ ] . the mechanism of metabolic activation of is rather simple and includes hydrolysis to fluorouracil by thymidine phosphorylase [ ] . since the level of thymidine phosphorylase is signifi cantly higher in several types of solid tumours (in particular, colorectal, breast, and kidney cancers) as compared with normal tissues, doxifl uridine possesses a higher therapeutic index for these types of cancers. the use of is somewhat limited by gastrointestinal toxicity after oral administration due to release of by intestinal pyrimidine nucleoside phosphorylase [ ] . yet another sugar-modifi ed fluorouracil derivative -ogt ( ), in which galactose is incorporated onto the fl uoropyrimidine moiety, was developed by oxford glycosciences and had reached phase i clinical study [ ] . in , the company decided to discontinue development of as the results of phase i/ii clinical study were not suffi ciently strong to justify large scale phase ii studies. ogt was rationally designed to reduce the systemic toxicity normally associated with fluorouracil while retaining activity against tumors localized in the liver, in which it may be preferentially localized through the asialoglycoprotein receptors [ ] . these receptors are present on the surface of hepatocytes and recognise various sugar-containing biomolecules through terminal galactose and n -acetylgalactosamine residues. the metabolic activation of ogt occurs once the compound enters hepatocytes, where the galactose molecule is cleaved from the fluorouracil residue. two derivatives of floxuridine -tt- ( ) and t- ( ) have reached phase ii clinical trials in japan [ ] . the compounds showed signifi cant antitumor activity by oral administration; moreover, they slowly released floxuridine, and the effective level of was prolonged [ , ] . the gastro-intestinal disturbances and loss of body weight were serious side effects of and . several prodrugs of flourouracil were obtained by acylation or carbamoylation of n- and/or n- atoms of the pyrimidine ring of . in particular, an oral drug carmofur ( ) which is -hexylcarbamoyl derivative of was launched in japan in and later -in other countries [ ] . the carbamate moiety in decomposes gradually in neutral water or in basic conditions, but it is strongly resistant to acidic hydrolysis and hence can survive acid in the stomach. the -hexylcarbamoyl moiety also facilitates the rapid uptake of through the cell membrane [ ] . the metabolic activation of carmofur involves oxidation and scission of the side-chain with slow release of [ ] . two main routes of the side chain transformation are ω-oxidation and (ω- )-oxidation: metabolites - were detected after administration of carmofur (fig. ) [ ] . non-enzymatic hydrolytic decomposition of and its metabolites also contributes to release of . another oral prodrug of fluorouracil, atofl uding ( ) is a diacyl derivative of . atofl uding has reached phase iii clinical trials in china [ ] . the activation of includes its fast non-enzymatic hydrolysis to o -toluyl- -fluorouracil ( ) following oral administration; is then slowly metabolized to (scheme ) [ ] . since the acetyl group of atofl uding is not stable and prone to decompose, impairing quality control for the preparation, a possibility of direct application of was also considered [ ] . an interesting idea was behind design of emitefur ( ), a prodrug of fluorouracil which was developed by otsuka pharmaceutical and has reached phase iii clinical trials in japan [ , , ] . the structure of contains the fragments of two biologically active components: fluorouracil ( ) and -cyano- , -dihydroxypyridine ( ), which is a potent inhibitor of dihydropyrimidine dehydrogenase. therefore, is a double prodrug which not only delivers fluorouracil but also prevents its enzymatic biotransformation to the dihydropyrimidine derivative . metabolic activation of occurs via rapid cleavage of the ester bonds by esterase to give and -ethoxymethyl- -fl uorouracil ( ) (scheme ). the intermediate is further metabolized to by microsomal enzymes in the liver [ ] . all the prodrugs of fluorouracil discussed above contained the fragment of in their structure; their transformation to included hydrolysis reaction as the key step. on the contrary, -fl uoro- -pyrimidinone ( -fp, ) which has been studied in phase i clinical trials [ ] is activated through oxidative process. in particular, pyrimidine is transformed to by aldehyde oxidase, which is present in high concentrations in the human livers but not in the gastrointestinal tract [ ] . two prodrugs of , capecitabine ( ) and galocitabine ( ), are -fl uorocytidine derivatives. both the compounds were developed by hoffman la roche; whereas capecitabine was launched in , galocitabine was terminated at phase ii clinical trials [ ] . both the compounds are close analogues as well as prodrugs of doxifl uridine ( ), which was used as the lead compound in their design. the main goals of such design were to minimize the mielotoxicity and to increase the tumor selectivity of . in fact, capecitabine ( ) indeed demonstrated minimal mielotoxicity in clinical studies. although the therapeutic indices of were much higher in mice tumor models than in the case of , it was not effi ciently metabolised to the active species in humans. the metabolic activation of and includes their hydrolysis by carboxylesterase or acylamidase in liver to give ′-deoxy- -fl uorocytidine ( ), which is then transformed to by cytidine deaminase (scheme ) [ ] . syntheses of fluorouracil prodrugs relied on either chemical modifi cation of or direct fl uorination of the corresponding pyrimidine derivatives. in particular, tegafur ( ) was obtained from by reaction with , -dihydrofuran [ - ] , -chloro- [ , ] , -alkoxy- [ ] , -acetoxytetrahydrofuran [ , , ] , and -trimethylsilyloxybutyraldehyde dimethyl acetal ( ) (scheme ) [ ] . alternatively, was prepared via fl uorination of compound [ ] or ester [ ] . one of the early syntheses of doxifl uridine ( ) [ , ] commenced from floxuridine ( ) which reacted with thionyl chloride to give cyclic sulphite (scheme ). methanolysis of upon treatment with sodium methylate gave , which was reduced with tributyltin to give . in an analogous approach, the compound was prepared via iodide , in turn obtained from in two steps (scheme ) [ ] . it should be noted that direct transformation of into the corresponding iodide was done with low yield of the product, hence the protection strategy was necessary to use. bromide was a key intermediate in one more analogous scheme [ ] . several syntheses of doxifl uridine relied on glycosylation of fluorouracil derivative . in particular, ′-deoxyrybose derivatives , , and were used for that purpose (scheme ) [ , ] . finally, direct fl uorination of ′-deoxyuridine derivatives with f /n [ ] or acof [ ] was also described. syntheses of ogt ( ) relied on glycosylation of the compound (scheme ). reaction of with bromide [ , ] or acetate [ ] gave tetraacetyl derivative , which was transformed to upon deprotection. with as the glycosylating reagent, in situ generation of from fluorouracil was also described [ ] . synthesis of carmofur ( ) and atofl uding ( ) was performed in obvious and straightforward manner. carmofur ( ) was prepared by reaction of fluorouracil ( ) and n -hexylisocyanate (scheme ) [ , ] . alternative approach included reaction of with phosgene and then -with n -hexylamine. early syntheses of -fl uoro- -pyrimidinone ( ) relied on desulfurization of fluorouracil thio-derivatives. in particular, reaction of pyrimidine derivatives with p s followed by treatment with raney nickel and gave alkoxy derivative , which was transformed to upon acidic hydrolysis (scheme ) [ ] . a more straightforward transformation sequence was also described; including reaction of fluorouracil ( ) with p s and reduction of thione with raney nickel [ , ] . alternatively, the thione was alkylated to give derivative , which was either oxidated and then hydrolyzed [ ] or subjected to reaction with hydrazine and then -silver oxide [ ] ; in both cases, was obtained. a completely different synthetic scheme commenced from fl uoroacetic acid which was subjected to vilsmeiertype formylation to give -fl uoro- -dimethylamino-acrolein ( ) [ ] . reaction of with triethyloxonium tetrafl uoroborate and dimethylamine gave the salt , which led to upon reaction with urea. finally, was also obtained by direct fl uorination of -pyrimidinone [ , ] . syntheses of capecitabine ( ) started from -fl uorocytosine ( ) (see further sections for the preparation of , which is used as antifungal drug). in particular, compound reacted with , , -tri-o -acetyl- -deoxy-β-d -ribofuranose ( ) to give diacetyl derivative , which was acylated with n -pentylchloroformate and then hydrolyzed, resulting in the formation of (scheme ) [ - ] . variations of this method using a silyl derivative of instead of itself [ , ] , as well as -o -acetyl- , -o-isopropylidene- -deoxy-d -ribofuranose ( ) (scheme ) [ ] or , , -tri-o -methoxycarbonyl- -deoxy-d -ribofuranose [ ] as the sugar sources were also reported. syntheses of galocitabine ( ) were performed analogously to that of capecitabine, , , -trimethoxybenzoyl chloride being used instead of npentylchloroformate at the corresponding steps [ , , , ] . [ ] . the active principle of both tas- and ftc- with anti-cancer effect is trifl uridine ( ). as in the case of fluorouracil, one of the mechanisms by which compound exhibits its antitumor activity is inhibition of thymidylate synthase [ ] . more precisely, trifl uridine is transformed into α,α,α-trifl uorothymidine monophosphate ( ) by thymidine kinase (scheme ); similarly to the fluorouracil derivatives discussed in the previous sections, compound is true inhibitor of thymidylate synthase. however, compound exhibits an anticancer effect on colorectal cancer cells that have acquired fluorouracil resistance as a result of the overexpression of thymidylate synthase. therefore, an alternative mechanism of action is also in operation, namely, incorporation of α,α,α-trifl uorothymidine triphosphate ( ) into dna, which results in single-strand breaks, followed by double-strand breaks when the cells progress to a subsequent dna replication phase [ ] the major drawback of trifl uridine ( ) is its high susceptibility to biodegradation, which is catalysed by thymidine phosphorylase and gives α,α,α-trifl uorothymine ( ) and -deoxy-α-dribose -phosphate ( ) [ ] . in the case of tas- , this issue is overcome by co-administration of thymidine phosphorylase inhibitor tipiracil ( ) [ ] , whereas improved biological effect of ftc- upon oral administration is achieved by its gradual biotransformation, mainly through the action of liver microsomes, releasing over a long period [ ] . the fi rst synthesis of trifl uridine commenced from trifl uoromethylacrylonitrile ( ) which reacted with hbr and then with urea to give amide in moderate yield. hydrolysis of was accompanied by cyclization and led to dihydropyrimidine (scheme ). two-step aromatization of gave α,α,α-trifl uorothymine ( ). compound was transformed to in low yield ( %) by enzymatic glycosylation [ ] . the yield of the last step in this sequence was signifi cantly improved when was preliminarily transformed to bis-silyl derivative , and chloride was used for glycosylation [ , ] an alternative approach to was based on direct trifl uoromethylation of the corresponding deoxyuridine derivatives or , using cf cooh-xef [ ] and cf i-cu-hmpa [ ] as the reagents, respectively (scheme ). ftc- ( ) was prepared by regioselective benzylation of trifl uridine ( ) (scheme ) [ ] . as in the case of , direct trifl uoromethylation was also used for synthesis of . the following sequence was established as the most practical: tritylation of ′-deoxy- -iodouridine ( ), ′-o -benzylation, n -benzoylation, crosscoupling reaction with cf cu reagent, and acidic deprotection (scheme ) [ ] . alternatively, was prepared in low yield by glycosylation of α,α,α-trifl uorothymine using the bis-silyl derivative (scheme ) [ ] . an approach to cancer treatment which relies on using fl uorinated uracil analogues as antimetabolites is the most recognised in the fi eld of fl uorinated diazines relevant to medicinal chemistry. however, other strategies are also gaining momentum; in particular, several compounds which act as kinase inhibitors ( i.e. - ) have reached clinical development phase (table ) . compound ly- ( ) is currently being developed by eli lilly and co.; monomesylate salt of has entered phase i clinical trials in patients with advanced cancer in [ ] . it acts as a potent oral inhibitor of the cyclin-dependent kinases and (cdk / ), playing a key role in regulating cellular proliferation [ ] . in particular, these cyclin d-dependent kinases facilitate progression of gap cell cycle phase (g ) by phosphorylating retinoblastoma susceptibility protein (rb), which prevents association of rb with e f transcription factor, and thus relieves transcriptional repression by the rb-e f complex. in addition, these fluorine-containing diazines in medicinal chemistry and agrochemistry kinases also sequester cdk interacting and kinase inhibitory proteins (cip/kip) from their complexes with cyclin-dependent kinase (cdk ), facilitating activation of cdk with cyclin e [ ] monomesylate salt of inhibits cdk and cdk with ic values of and nm, respectively; moreover, it is able to cross blood-brain barrier and therefore has the potential for the treatment of brain tumors and metastases [ ] . fostamatinib disodium (tamatinib fosdium, ), which is prodrug of tamatinib ( ) (scheme ), was discovered by rigel; it is currently studied in phase ii clinical trials by rigel and astra zeneca plc. for treatment of b-cell lymphoma [ ] . apart from that, compound is also investigated as agent for treatment of autoimmune thrombocytopenia and rheumatoid arthritis. because of its poor pharmaceutical properties, tamatinib ( ) is orally administered as the methylene phosphate ( ) is quickly cleaved to by alkaline phosphatases that are present on the apical brush-border membranes of the intestinal enterocytes, after which the more hydrophobic can be readily absorbed [ ] . tamatinib ( ) acts as an atp-competitive inhibitor of spleen tyrosine kinase (syk) -a non-receptor tyrosine kinase which is a key component of the b-cell receptor (bcr) signaling pathway [ ] . it is shown that bcr-mediated signaling through syk occurs to a greater degree and for a longer duration in neoplastic cells than in nonmalignant b-cells. inhibition of the syk pathway prevents chronic lymphocytic leukemia (cll) cells from interacting with the microenvironment, and promotes proapoptotic signals. r- ( ), also known as as- , is another kinase inhibitor discovered by rigel. it was investigated in phase i clinical trials for several types of tumors by rigel and merck serono; the latest study was terminated in , concerning a review of the available clinical data and low probability of completing the trial based on the observed recruitment rate [ ] . compound inhibits aurora kinases -serine/threonine kinases which are essential for cell proliferation, mainly due to regulation of gap and mitotic cell cycle phases (g /m). over-expression of aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. although the biological functions of two types of aurora kinases (a and b) are different, in both cases their inhibition induces apoptosis of the cell, leading to similar phenotypes. some other kinases are also inhibited by , in particular fms-like tyrosine kinase (flt ) [ ] . one more aurora kinase inhibitor -pf- ( ) -was developed by pfi zer; it has been investigated in phase i clinical trials for treatment of solid tumors (the study completed in ) [ ] . pf- was generally well tolerated with manageable toxicities, and a recommended phase ii dose could be established; however, clinical or metabolic antitumour activity was limited [ ] . similarly to r- ( ), compound inhibits both aurora a and b kinases; other kinases are affected to a lesser extent [ ] . therefore, pf- ( ) produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. azd- ( ) was developed by astrazeneca and studied in phase i clinical trials for treatment of advanced solid malignancies (the study terminated in ) [ ] . azd- is an atp-competitive inhibitor of janus kinase (jak ) -an intracellular non-receptor tyrosine kinase that transduce cytokine-mediated signals via the janus kinase -signal transducer and activator of transcription (jak-stat) signaling pathway. in particular, inhibition of jak blocks stat signaling, associated with chronic cytokine stimulation in some tumors [ ] . x-ray diffraction study of complex formed by and jak shows that the donor-acceptor-donor hydrogen-bonding motif provided by aminopyrazole fragment forms three hydrogen bonds with an adenine binding pocket, whereas the fl uoropyrimidine ring occupies a nearby hydrophobic pocket [ ] . synthesis of ly- ( ) relied on selective functionalization of , -dichloro- -fl uoropyrimidine ( ), which can be easily obtained from fluorouracil ( ) (scheme ) [ ] . first, boronic ester was prepared from aniline in three steps, including benzimidazole ring construction and palladiumcatalyzed coupling with pinacol diborane. suzuki-type reaction of and resulted in selective functionalization at c- of the pyrimidine ring and gave chloride . buchwald-hartwig coupling of with amine (prepared in two steps from -ethylpiperazine ( ) and ( )) gave the fi nal product . analogously, selective functionalization of was used for the preparation of fostamatinib disodium ( ) (scheme ). in particular, reaction of with equimolar amount of amine and then -with , , -trimethoxyaniline ( ) gave tamatinib ( ) [ ] . it should be noted that no detailed procedures of performing these transformations were given in the initial patent; moreover, synthesis of the starting compound (amine ) is not documented to date. to obtain fostamatinib disodium ( ), compound was treated with chloride and cs co ; further deprotection subsequent and salt formation gave the target product [ ] . similar approach was used for the synthesis of r- ( ) (scheme ) [ ] . in this case, lactam , which was obtained from norbornadiene ( ) and graf isocyanate (clso nco), was protected with boc o and then subjected to ringopening with aqueous ammonia to give amide . deprotection of followed by arylation with gave an intermediate , which was then treated with n -arylpiperazine derivative (prepared in two steps from -fl uoro- -methylnitrobenzene ( )) to give racemic . optically pure was obtained either by chiral stationary phase hplc applied at different steps of the synthesis, or via enzymatic resolution of boc-protected lactam . it is not surprising that synthesis of pf- ( ) also followed analogous strategy, , -dichloro- -trifl uoromethylpyrimidine ( ) being used as a key fl uorinated diazine building block instead of (scheme ) [ ] . the synthetic scheme commenced from amine which was n -trifl uoroacetylated, then nitrated, and subjected to a change of the protecting group to give boc derivative . two alternative pathways were developed for further transformations. in the fi rst one, compound was reduced into fused aniline derivative which reacted with to give compound . deprotection of followed by coupling with n -acetylglycine led to the formation of chloride . alternatively, compound was deprotected, coupled with n -acetylglycine, reduced catalytically and then arylated with to give . finally, compound reacted with cyclobutyl amine to give the fi nal product as racemate. both enantiomers of were also obtained using this scheme if boc derivative was subjected to chiral stationary phase hplc prior further transformations. although a similar strategy was used for the preparation azd- ( ), in this case the fl uorinated diazine moiety is not in a central part of the molecule; hence a different approach was used for the construction of the fl uorinated the fi ght against hiv infection is another important fi eld where fl uorinated diazines have remarkable record, including approved drug emricitabine ( ) and compounds that have reached clinical development phase (compounds - ) ( table ) . all these compounds act as hiv reverse transcriptase inhibitors and fall into two categories: fl uorocytidine analogues ( and - ) and trifl uoromethyl-substituted quinazolone derivatives ( - ). emtricitabine ( ) was discovered in emory university (atlanta, usa); development of the drug was completed by gilead sciences, and the compound was approved by fda under trade name emtriva ® in . it is also marketed in combinations with other anti-hiv agents, i.e. tenofovir ( , used as a prodrug) (truvada ® ), efavirenz ( ) and tenofovir (atripla ® ), rilpivirine ( ) and tenofovir (complera ® ), and elvitegravir ( ), cobicistat ( ), and tenofovir (stribild ® ) [ ] emricitabine is a close analogue of lamivudine ( ), which is an example of nucleoside analogs -an important class of reverse transcriptase inhibitors, which has gained much attention since the initial success of the fi rst representative, zidovudine ( ) [ ] (fig. ) . emtricitabine ( ) was discovered in emory university (atlanta, usa); development of the drug was completed by gilead sciences, and the compound was approved by fda under trade name emtriva ® in . it is also marketed in combinations with other anti-hiv agents, i.e. tenofovir ( , used as a prodrug) (truvada ® ), efavirenz ( ) and tenofovir (atripla ® ), rilpivirine ( ) and tenofovir (complera ® ), and elvitegravir ( ), cobicistat ( ), and tenofovir (stribild ® ) [ ] emricitabine is a close analogue of lamivudine ( ), which is an example of nucleoside analogs -an important class of reverse transcriptase inhibitors, which has gained much attention since the initial success of the fi rst representative, zidovudine ( ) [ ] . emtricitabine ( ) is very similar to lamivudine ( ) with respect to its activity, convenience, safety and resistance profi le; the only remarkable difference is longer intracellular half-life of . analogously to , the biologically active form of is triphosphate , which is formed by a stepwise phosphorylation of (scheme ). compound can be considered as , -dideoxycytidine trifosphate analogue and acts as a competitive inhibitor and alternate substrate of the normal deoxycytidine triphosphate ( ). as a competitive inhibitor of the normal substrate, inhibits incorporation of into the growing dna chain by viral reverse transcriptase; as an alternate substrate, it is incorporated into this chain (as ) and acts as a chain terminator (since is missing the ′-hydroxyl group required for further chain elongation) [ , ] . although emtricitabine might have the potential for toxicity caused by interaction with human mitochondrial dna enzymes, both in vitro and in vivo testing results show that this is not a serious issue. low toxicity of as compared to other nucleoside reverse transcriptase inhibitors is a remarkable advantage of this drug. as with all representatives of this class, the major drawback of is rapid development of drug resistance by a single point mutation of viral reverse transcriptase [ ] . the main route of elimination of is renal excretion, mostly unchanged ( % of the dose). the metabolic transformations of emtricitabine include oxidation of the sulphur atom to form the ′-sulfoxide diastereomers ( %) and conjugation with glucuronic acid to give ′-o -glucuronide ( %) [ ] . a racemic form of emtricitabine, racivir, was also studied in clinics by pharmasset and has reached phase ii trials [ ], designed to measure its effi cacy in patients harbouring virus resistant to lamivudine. it was shown that d (+)enantiomer is less potent and more toxic than emtricitabine itself. one of the reasons behind lower potency of is that is phosphorylated by deoxycitidine kinase to a greater extent; therefore, the active form ( ) is formed more readily for (-)-enantiomer [ , ] . elvucitabine ( ) and its enantiomer dexelvucitabine ( ) were discovered in yale university (new haven, usa) and emory university (atlanta, usa), respectively. both compounds were further developed by commercial companies (achillion pharmaceuticals and incyte co., respectively), and have reached phase ii clinical trials [ ] . development of was terminated due to inability to pair with other cytidine analogues and higher risk of hyperlipasemia. phase ii studies of were suspended because of bone marrow suppression in several patients [ ] . the mode of action of elvucitabine is quite similar to that of emtricitabine; the major advantages of include long plasma half-life (up to ten times greater than that of ) and superior potency against common resistance mutations [ ] . four compounds dpc- ( ), dpc- ( ), dpc- ( ), and dpc- ( ) were developed by dupont pharmaceuticals as non-nucleoside reverse transcriptase inhibitors. al the compounds have reached phase i clinical trials; dpc- ( ) was further progressed into phase ii trials by bristol-myers squibb after the company had acquired dupont pharmaceuticals; however, the development was stopped in due to poor pharmacokinetics [ ] . the compounds are close analogues of efavirenz ( ) -a non-nucleoside reverse transcriptase inhibitor approved by fda in [ ] . all the compounds - showed similar to efavirenz activity towards wild-type virus in vitro ; however, they were more effective towards singlemutation variants and showed lower plasma serum protein binding [ , ] . it might be assumed that mechanism of action of - is similar to that of efavirenz, which is known to bind within the non-nucleoside inhibitor binding pocket of reverse transcriptase [ ] , both spatially and also functionally associated with the substrate-binding site. metabolism of dpc- ( ) was studied in rats. analogously to efavirenz, the main metabolite is glucuronide conjugate (more than % of excreted dose in the bile) (scheme ). however, a glutatione conjugate was also isolated, which is presumably formed via oxirene intermediate ; in this view, metabolism of was different from that of [ ] . approach starting from d-mannose or d-galactose was used for the preparation of d-enantiomer [ ] . most of the methods describing the preparation of emtricitabine (and racivir) rely on the construction of , -oxathiolane ring by reaction of glycolaldehyde or glyoxalic acid derivatives with mercaptoacetic acid or mercaptoacetic aldehyde (which exists as , -ditiane ). for example, one of the fi rst of syntheses of this type commenced from allyl alcohol which was silylated and then subjected to ozonolysis to give glycolaldehyde derivative (scheme ) [ ] . reaction of with mercaptoacetic acid afforded , -oxathiolane , which was reduced with lialh(o t bu) or dibal and then acetylated to form . finally, reaction of with silylated fl uorocytosine derivative followed by deprotection led to the formation of racemic (racivir). more than preparations described in patents are variations of the above synthetic scheme. in particular, to obtain optically pure emtricitabine, lipase-catalyzed enzymatic resolution, as well as chiral stationary phase hplc was used [ ] . however, the most effective procedure included separation of menthyl derivatives. this method evolved signifi cantly since the fi rst publication (which in fact relied on separation of all the possible diastereomers) [ ] ; one of the recent multigram preparations is shown in the scheme [ ] . the fi rst step of the synthesis included formation of methyl ester from glyoxalic acid and l -menthol. reaction of with , -ditiane gave , -oxathiolane as a mixture of cis diastereomers. compound was transformed to chloride by treatment with thionyl chloride and methanesulfonic acid. reaction of and led to the formation of , which was separated as a single diastereomer by transformation to oxalate and subsequent crystallization. finally, reduction of with nabh gave emtricitabine ( ) which was isolated as hydrochloride. an interesting variation of the method was patented by glaxo wellcome inc [ ] . their synthesis was started from , -dichloro- -fl uoropyrimidine ( ) (scheme ). reaction of with naoet and then -with anion of , -dimethoxyethanol gave pyrimidine derivative , which upon detection formed aldehyde . reaction of and led to the formation of , -oxathiolane , which was acetylated to give . treatment of with tmsotf resulted in rearrangement leading to , which was transformed to racemic (racivir) by reaction with ammonia. a number of methods for the preparation of elvucitabine ( ) were reported in the literature. in the fi rst synthetic scheme developed in yale university [ ] , ′-deoxy- -fl uoro-β-l-uridine ( ), which is enantiomer of floxuridine ( synthesis of elvucitabine ( ) developed by chemists from vion pharmaceuticals commenced from lactone (scheme ), which can be obtained in steps from d-glutamic acid [ ] . phenylselenation of enolate generated from proceeded highly diastereoselectively and led to . phenylselenide was reduced with dibal and then acetylated to give acetate as a mixture of anomers. reaction of with was also diastereoselective due to the steric effect of bulky phenylselenyl substituent and gave β anomer in almost quantitative yield. oxidative elimination of the selenide substituent from and subsequent deprotection gave elvucitabine ( ) as a single enantiomer. an analogous synthesis was described by chemists from emory university [ ] . syntheses of dexelvucitabine ( ) [ ] and later -elvucitabine ( ) [ ] were described, starting from d-and l-xylose, respectively, both using almost the same methodology. in particular, d-xylose was transformed into the dibenzoyl derivative [ ] . compound was subjected to bromoacylation with excess of -acetoxy- methylpropionyl bromide ( ) to give a mixture of esters and . this mixture was subjected to reductive elimination to give , which was transformed to upon alcoholysis. another synthesis of relied on palladium mediated ferrier rearrangementtype glycosidation of a furanoid glycal (scheme ) [ ] . the initial steps of fluorine-containing diazines in medicinal chemistry and agrochemistry the synthesis were quite similar to those shown in scheme . the major difference was the use of polymer-supported pph at the glycal generation step, which allowed for isolation of unstable glycal with more than % purity. palladium-catalyzed reaction of with -fl uorocytosine ( ) was accompanied by ferrier-type rearrangement and led to derivative , which was transformed to upon deprotection. all the reported syntheses of dpc- ( ) and dpc- ( ) commenced from the corresponding o -amino-α,α,α-trifl uoroacetophenones (scheme ). in the fi rst preparations of and , reacted with tmsnco to give adducts , which were transformed to cyclic imines upon dehydratation. reaction of with lithium cyclopropylacetylenide gave racemic and , which were subjected to chiral stationary phase hplc to isolate and as pure enantiomers [ , ] . several improvements were reported for this synthetic scheme. in particular, diastereoselective additions of lithium cyclopropyl acetylenide to the derivatives of containing residues of α-phenylethyl amine or campheic acid were developed [ , ] . moreover, an enantioselective modifi cation of this method employing amino alcohol as an asymmetric catalyst was discovered [ , ] . another enantioselective method involved reaction of the derivatives of and cyclopropyl acetylene itself, catalysed by amino alcohol derivatives ( e.g. ) and zn(otf) [ ] . dpc- ( ) and dpc- ( ) were obtained by reduction of and , respectively, with lialh [ , ] . recently, an alternative approach to the synthesis of was reported, which relied on enantioselective organocatalytic mannich-type reaction of imine derivative and cyclopropyl methyl ketone (scheme ) [ ] . although enantioselectivity of the key step was moderate ( ee %), it could be easily enhanced to > % by a single recrystallization of intermediate . apart from anti-hiv drugs discussed in the previous section, two additional antiviral agents can be mentioned: trifl uridine ( ) and favipiravir ( ) . trifl uridine ( ) was mentioned above as a component of phase iii investigational drug tas- . it is however more known as an ophthalmic anti-herpes agent launched by glaxo wellcome (now merged into glaxosmithkline) in [ ] . it is effective against herpetic keratitis, and seems to be especially useful in 'diffi cult' cases [ ] . high susceptibility to biodegradation of trifl uridine is advantageous for its use as ophthalmic drug, as its action in other tissues is thus prevented. as in the case of anti-tumor activity, the mechanism of antiviral action of involves the inhibition of viral replication. trifl uridine does this by incorporating into viral dna during replication, which leads to the formation of defective proteins and an increased mutation rate [ ] . inhibition of thymidylate synthetase also seems to contribute into antiviral effect of . the details of these processes, as well as synthesis of were discussed in the above sections. favipiravir ( ) has been discovered by toyama chemicals; it is currently in phase iii (japan) and phase ii (usa) clinical trials [ , ] . favipiravir is under development as an agent against infl uenza virus, however, it was also tested against other rna viruses, including arenaviruses, bunyaviruses, west nile virus (wnv), yellow fever virus (yfv), and foot-and-mouth disease virus (fmdv) [ ] . a proposed mechanism of action of includes its biotransformation into ribofuranosyltriphosphate derivative (scheme ), which inhibits infl uenza virus rna polymerase in the host cells [ ] . [ , ] . acidic hydrolysis of gave amide , which upon mild alkaline hydrolysis led to . alternatively, compound was obtained by mild alkaline hydrolysis of followed by reaction with h o -naoh, or by reaction of with allyl or benzyl alcohol, removal of the protection, and hydrolysis. recently, an improved version of this method was patented, which allowed authors to claim its industrial applicability [ ] . one more method for the preparation of commenced from pyrazine derivative , which was transformed to dichloride using sandmeyer reaction (scheme ) [ ] . hydrolysis of the ester moiety in followed by one-pot chloroanhydride formation, introduction of fl uorine atom and amination gave derivative , which was transformed into by diazotization and subsequent hydrolysis. several other approaches to the synthesis of favipiravir were also described, most of them relying on direct fl uorination of pyrazine derivatives with molecular fl uorine [ ] all they were low-yielding and allowed for the preparation of milligram quantities of the fi nal product. a single compound is discussed in this category, namely gsk- ( ), which was developed by glaxosmithkline and has reached phase ii clinical trials in bacterial skin infections [ ] and phase iii -in community-acquired bacterial pneumonia [ ] . compound acts as an inhibitor of peptide deformylase -an enzyme that removes the formyl group during eubacterial peptide elongation. bacterial protein synthesis initiates with formyl-methionyl-trna and, consequently, all polypeptides newly synthesized in bacteria contain an n -formylmethionine terminus. this residue is further removed in two steps catalyzed by peptide deformylase and methionine aminopeptidase, respectively. inhibition of peptide deformylase increase production of bacterial n -formylated polypeptide, which prevents bacteria growth and possibly triggers an enhanced immune response [ ] . peptide deformylase is a metalloprotease, which mostly utilizes fe + in its active site. it was shown for analogs of that n -formyl-n -hydroxylamine function coordinated to metal ion when the inhibitor was bound to the enzyme [ ] . synthesis of was started from preparation of chiral diamine (scheme ) [ ] . in particular, d -serine methyl ester was converted to n -benzyl derivative , which was transformed into carboxylic acid using reaction with chloroacetyl chloride and subsequent hydrolysis. carboxylic acid was subjected to coupling with benzyl amine, reduction, reaction with ethyl oxalyl chloride and reductive cyclization to give bicyclic compound . finally, two-step reduction of led to the formation of diamine , which was isolated as dihydrochloride. reaction of with dichloro derivative and then -hydrazine hydrate gave the product , which was coupled with carboxylic acid and subjected to catalytic hydrogenation to give . two drugs were launched as anti-fungal agents to date: flucytosine ( ) (valeant, ) and voriconazole ( ) (pfi zer, ) (fig. ) [ ] . flucytosine itself has no antifungal activity; its activity results from the rapid conversion into fluorouracil ( ) within susceptible fungal cells [ ] . the mechanism of cytotoxic effect of fluorouracil has been discussed in the previous sections. flucytosine is taken up by fungal cells by cytosine permease, which is the transport system for cytosine and adenine. inside the fungal cells, is deaminated to by cytosine deaminase. the specifi city of this step is crucial for the narrow antifungal spectrum of : mammalian cells as well as fungi lacking cytosine deaminase are not sensitive to . on the other hand, fluorouracil itself cannot be used as an antifungal drug, since it is only poorly taken up by fungal cells and is too toxic to human cells. the major drawback of flucytosine is rapid development of resistance in fungi, either by mutations or by increased synthesis of pyrimidines; this limits the use of as a single antifungal agent. monotherapy with flucytosine is currently only used in some cases of chromoblastomycosis and in uncomplicated candidosis; in all other cases, is used together with other agents, usually amphotericin b [ ] . the effect of voriconazole ( ) is exerted within the fungal cell membrane. in particular, cytochrome p -dependent -α-lanosterol demethylase is inhibited, which prevents the conversion of lanosterol ( ) to ergosterol ( ) -an important component of yeast and fungal cell membranes which does not occur in mammalians (scheme ). this mechanism results in the accumulation of toxic methylsterols and inhibition of fungal cell growth and replication [ ] . voriconazole is active against many fungal infections, including invasive aspergillosis, pseudallescheria , scedosporium , fusarium infections [ ] . it is also proposed for empirical antifungal therapy [ ] . an important advantage of voriconazole is high oral bioavailability ( %). the most common side effect, which is unique for voriconazole among other azole antifungals, is a reversible disturbance of vision (photopsia): it occurs in nearly a third of patients but rarely leads to discontinuation of the drug [ ] . resistance to voriconazole still remains uncommon, although an increase of resistance and continued surveillance with greater use of the drug has been reported [ ] . the fi rst synthesis of flucytosine ( ) has been reported in [ , ] . the synthetic scheme is quite similar to that for fluorouracil ( ); in the case of , compound was subjected to reaction with pcl and then -liquid ammonia to give , which was transformed to upon hydrolysis (scheme ). in an alternative method, compound (prepared from fluorouracil) reacted with socl to give , which was transformed to upon reaction with ammonia in methanol [ ] . another synthesis commenced from , -difl uoro- -chloropyrimidine, which, however, is not readily accessible [ ] . flucytosine was also obtained by direct fl uorination of cytosine using cf of ( % yield) [ , ] , fl uorine [ , ] , and acof [ ] . despite numerous syntheses of voriconazole ( ) were documented, they all followed the same synthetic strategy, namely, addition of anion to ketone , followed by isolation of necessary diastereomeric pair and its resolution with -camphorsulphonic acid (scheme ). three different approaches were used for the generation of anion or the corresponding organometallic species. first of them relied on deprotonation of the pyrimidine derivative (prepared from the fl uorinated keto ester or dichloro derivative ) by strong bases such as lda (scheme ) [ - ] . the main drawback of this method was low diastereoselectivity of the key step; therefore tedious separation of diastereomers was necessary. another approach to generation of relied on zncl -catalyzed decarboxylation of salts , prepared from (scheme ) [ ] . in this case, the desired diastereomeric pair was obtained with much better selectivity ( . : ). the last approach relied on reformatsky-type reaction involving and bromides (prepared from [ , ] or its thio analogues [ - ] ) or sulfonates (prepared from ) (scheme ) [ , ] . in this case, good diastereoselectivities were obtained. seven compounds designed as agents acting at central and/or peripheral nervous system have reached at least phase ii clinical trials, and only one of them was launched (table ) [ , ] . these compounds address different biological targets and act as skeletal muscle relaxants (afl oqualone ( )), antipsychotics a representative of fl uorinated diazines, afl oqualone ( ), was launched in in japan as a central acting muscle relaxant [ ] . it is an analogue of methaqualone ( ) (fig. ) -a drug widely used as a hypnotic, for the treatment of insomnia, and as a sedative and muscle relaxant in s, but reclassifi ed as a schedule i controlled substance in usa in [ ] . the mechanism of action of afl oqualone is not well studied. it was shown that its site of action is different from that of other central acting muscle relaxants, i.e. mephenesin, chlormesazone or diazepam [ ] . gaba-enhancing effect was also demonstrated [ ] . the main routes of metabolism of in human include n -acetylation, followed by hydroxylation at the ′-methyl and acetyl methyl carbons, as well as glucuronidation of the aromatic amino group. this pattern of metabolism is similar to that observed in monkeys and rats, but drastically different from that in dogs [ ] . synthesis of afl oqualone commenced from -nitroanthranilic acid ( ) which was transformed to amide via the corresponding chloroanhydride (scheme ) [ ] . catalytic reduction of followed by acetylation gave , which reacted with chloroacetyl chloride to form quinazoline . nucleophilic substitution of chlorine atom in with fl uorine led to the formation of , which upon deprotection gave afl oqualone ( ). alternatively, compound was subjected to acylation with fl uoroacetyl chloride or anhydride to give amide [ ] . refl uxing of with acetic anhydride gave quinazoline , which was reduced to afl oqualone either by catalytic hydrogenation or using sncl . all three compounds discussed in this section ( i.e. - ) have reached phase ii clinical trials as agents for treatment schizophrenia. development of bmy- ( ) was discontinued more than years ago. fluorine-containing diazines in medicinal chemistry and agrochemistry noted that relative role of these two targets in biological effect of was debated in the literature. whereas in pigeons, the effect was serotonergically mediated primarily through -ht a receptors [ ] , in other model systems, these interactions did not seem to contribute signifi cantly to the potential antipsychotic action of the compound [ ] . although studies in animal models supported for the suggestion that bmy- ( ) may possess antipsychotic properties [ ] , clinical trials showed lack of effi cacy in schizophrenia treatment [ ] . recently, bmy- was proposed as a promising candidate for clinical trials of l -dopa-induced dyskinesia -a common side effect observed during prolonged use of l -dopa in parkinson disease patients [ ] . it was shown that the compound suppresses abnormal involuntary movements related to l -dopa-induced dyskinesia via its -ht a agonistic effect. abt- ( ) developed by abbott is a selective d receptor antagonist [ ] . it was suggested that selective antagonists of d receptor might be promising antipsychotic agents lacking the presumed d receptor-mediated side effects, although d antagonists may express their effect via mechanisms that cannot be refl ected by the commonly used animal models [ ] . it was shown that abt- produced cognitive signals but did not achieve suffi cient d receptor occupancy at the doses used in clinical studies [ ] . nevertheless, these studies allowed for the assumption that the development and clinical testing of newer d receptor antagonists with higher potency at d receptors, enabling suffi cient receptor occupancy, is highly warranted [ ] . on the contrary, jnj- ( ) is a d highly selective receptor antagonist and hence acts in a mode analogous to that of most marketed antipsychotics [ ] . jnj- is characterized by a rapid dissociation rate from the dopamine d receptor, which was hypothesized to confer antipsychotic effi cacy and improved tolerability [ ] . clinical studies in patients with an acute exacerbation of schizophrenia showed that jnj- had similar biological activity but lesser tendency to induce weight gain compared to a known antipsychotic drug, olanzapine ( ) [ ] (fig. ) . synthesis of bmy- ( ) commenced from pyrimidine derivative which reacted with piperazine to give derivative (scheme ) [ , ] . reduction of the compound followed by deprotection gave amine , which was alkylated with chloride and then subjected to acidic hydrolysis to form ketone . reduction of allowed bmy- ( ) to be obtained. pure enantiomers of were also obtained. to achieve this, the following methods were used: resolution of with using reaction with α-phenylethyl isocyanate [ ] or lipase-catalyzed acetylation or hydrolysis [ ] , alkylation of with enantiopure alcohols [ ] ; and microbial reduction [ ] or ru-catalyzed enantioselective hydrogenation [ ] of . abt- ( ) was obtained starting from amidine and ethyl trifl uoroacetoacetate to give pyrimidine (scheme ) [ ] . reaction of with socl and then -piperazine led to the formation of amine . selective alkylation of with -bromo- -chloropropane gave chloride , which reacted with thiouracil anion to form abt- ( ). bmy- ( ) was developed by bristol-myers squibb as nootropic agent ( i.e. for cognition disorders) and has reached phase ii clinical studies. the compound was effective in vitro [ ] as well as in animal models [ - , ] that may predict cognitive enhancement. the mode of action of bmy- is poorly understood. it was shown that the compound has an anti-anoxic action, and activation of the cns cholinergic system is involved as one of the causative mechanisms for this effect [ ] . clinical trials showed that bmy- was not significantly superior to placebo in alzheimer's disease; moreover, although generally well tolerated, also had a higher rate of discontinuations [ , ] . synthesis of bmy- ( ) optimized for large scale preparations commenced from malonodiamide and ethyl trifl uoroacetate, which reacted to give pyrimidine (scheme ) [ ] . compound was transformed into dichloro derivative upon treatment with pocl . reaction of with piperidine (prepared from -pyridinylmethyl chloride in two steps) gave , which was reduced catalytically to form bmy- ( ). alternatively, bmy- was obtained by arylation of with -chloro- -trifl uoromethylpyrimidine ( ) [ ] . synthesis of jnj- ( ) was quite trivial and relied on selective functionalization of -aminopiperidine core, fi rst with -chloro- -trifl uoromethylpyridazine ( ) and then -with , -difl uorobenzaldehyde (scheme ) [ ] . . bw- w ( ) was developed as a cns-acting antihyperalgesic agent ( i.e. for treatment of increased sensitivity to pain). it is an analogue of anticonvulsant drug lamotrigine ( ) (fig. ) , used n the treatment of epilepsy and bipolar disorder [ ] . like lamotrigine, bw- w binds to the transmembrane segment s in domain iv of α subunit of voltage-gated sodium channels (na v ), thus acting as a pore blocker [ ] . it is assumed that neuropathic pain is partially mediated by an increase in the density of na v channels in injured axons and their dorsal root ganglions. clinical studies in patients with chronic neuropathic pain showed that although bw- w signifi cantly lowered allodynia severity at the fi rst day, the effect did not maintain in further treatment [ ] . gw- x ( ) is a selective cb receptor full antagonist which has potent analgesic, anti-infl ammatory and anti-hyperalgesic actions. it was selected as a clinical candidate after lead optimization of a pyrimidine ester (gk , fig. ) , identifi ed in a focused screen as a partial agonist at the cb receptor with micromolar potency [ ] . the compound was evaluated as an analgesic for treatment of infl ammatory pain (phase i trials) and dental pain (phase ii trials) [ ] . in the latter study, single doses of gw failed to demonstrate clinically meaningful analgesia in the setting of acute dental pain [ ] . fluorine-containing diazines in medicinal chemistry and agrochemistry trifl uoride (dast) to give racemic . alternatively, nitrile reacted with ethyl fl uoroacetatet -buok and then -ethyl iodide to give enol ether , which was transformed to racemic by reaction with guanidine. resolution of enantiomers of was achieved by crystallization of dibenzoyl-l -tartaric acid salt; the more active r -enantiomer was isolated. in the synthesis of gw- x ( ), commercially available pyrimidine reacted with , -dichloroaniline to give ester , which was subjected to hydrolysis followed by amide coupling with -aminomethyltetrahydropyran ( ) to afford (scheme ) [ , , ] . in the previous sections, compounds targeting cancer cells or nervous system, as well as those fi ghting foreign organisms were discussed. three compounds do not fall into any of these categories. fostamatinib disodium ( ) was mentioned above as an anti-cancer investigational drug, but it was also studied as agent for autoimmune diseases, i.e. rheumatoid arthritis (currently in phase iii) and autoimmune thrombocytopenia (in phase ii). gemigliptin ( ) was approved as an anti-diabetic drug in south korea in . pf- ( ) was discontinued after phase i studies as an agent for liver fi brosis; nevertheless, it is currently investigated in diabetic nephropathy (fig. ) (phase ii, october ) [ ] . as it was mentioned in the previous sections, the active principle of fostamatinib disodium ( ) is tamatinib ( ), which is formed by enzymatic hydrolysis of in the intestine. as in the case of lymphoma, the effect of in autoimmune diseases is related to inhibition of spleen tyrosine kinase (syk) by [ , ] . as syk has the central role in transmission of activating signals within b cells, inhibition of this enzyme lowers expression of a number of proinfl ammatory cytokines and hence leads to immunosuppression [ ] . fostamatinib has shown signifi cant effi cacy in the treatment of patients with rheumatoid arthritis not responding to methotrexate ( ) (a drug which is used conventionally in therapy), although a number of adverse events were observed [ ] . if these results are confi rmed once phase iii studies are completed, it may fi nd a place in the treatment of patients with rheumatoid arthritis with poor response to conventional therapy (fig. ) . gemigliptin ( ) was developed by lg life sciences as an inhibitor of dipeptidyl peptidase (dpp- ) -a target of oral drugs used to treat used to treat type diabetes (characterized by high blood glucose in the context of insulin resistance and relative insulin defi ciency) [ ] . the fi rst representative of this class, sitagliptin ( ) was launched in . in human body, gemigliptin is metabolized to lc - , which is a major active metabolite, by cytochrome p a isozyme [ ] . inhibition of dpp- results in increase of incretin levels (which is normally inactivated by dpp- ), in particular glucagon-like peptide- (glp- ) and gastric inhibitory peptide (gip) [ ] . incretins inhibit glucagons release and stimulate insulin secretion, which leads to decrease in glucose blood levels. clinical trials showed effi cacy and safety of gemigliptin administered once daily as a monotherapy, [ ] as well as in addition to metformin ( ) [ ] for type diabetes patients. pf- ( ) is a phizer's investigational drug, initially developed as agent for liver fi brosis -formation of excess fi brous connective tissue in liver [ ] . the development of the compound was discontinued since february after phase i trials. recently, a phase ii study of pf- in diabetic nephropathya progressive kidney disease caused by angiopathy of capillaries in the kidney glomeruli [ ] -was registered [ ] . pf- is an antagonist of chemokine receptors ( i.e. ccr and ccr ) [ ] . these chemokine receptors are important players in the traffi cking of monocytes/macrophages and in the functions of other cell types relevant to pathogenesis of many diseases [ ] , including liver fi brosis [ ] and diabetic nephropathy [ ] . gemigliptin ( synthesis of optically active pf- ( ) based on commercially available (-)-vince lactam as chirality source. starting from (-)-vince lactam the chiral key -amino- -cyclopentene- -carboxylic acid derivative was synthesized. the compound is dimethyl pyrrole protected form of corresponding aminoacid, which was subjected to amide coupling with boc-protected diamine to give amine (scheme ) [ ] . removing of the pyrrole function followed by catalytic hydrogenation gave amine , which was subjected to reductive amination of ketone , separation of diastereomers, deprotection and then -arylation with pyrimidine derivative to afford the fi nal product, . agrochemistry is one of more important fi eld of application of the fl uorinated compounds which is widely recognized [ , ] (fig. ). uracil derivatives butafenacil ( , inspire®, rebin®) and benzfendizone ( ) were introduced as herbicides in , whereas their pyridazine-derived analogue flufenpyr-ethyl ( ) -in [ ] . butafenacil (developed by syngenta ag) is used for weed control in grapes, nut crops, pome and stone fruits and also as a cotton defoliant [ ] . it was registered in australia and approved by u. s. environmental protection agency. benzfendizone (developed by fmc corporation) is a post-emergence herbicide that provides good control of grass and broadleaf weeds in tree fruits and vines, as a cotton defoliant, and in total vegetation control [ ] . flufenpyr-ethyl (developed by sumitomo chemical company) was registered in usa for use on corn, soybeans and sugarcane [ ] . the most recent example is safl ufenacil ( , kixor®), introduced by basf in for preplant burndown and selective pre dicot weed control in multiple crops, including corn. [ ] . compounds - act as inhibitors of protoporphyrinogen oxidase (protox) -an enzyme in the chloroplasts of the plant cells that oxidizes protoporphyrinogen ix ( ) to produce protoporphyrin ix ( ) (scheme ) [ ] . in turn, is a precursor molecule for both chlorophyll and heme. when protoporphyrinogen oxidase is inhibited, protoporphyrinogen ix is accumulated and transferred from chloroplasts into the cytoplasm, where non-enzymatic conversion of to occurs. when present in cytoplasm, is cytotoxic due to interaction with oxygen upon action of light, which results in formation of singlet o molecules. o causes lipid peroxidation, membrane disruption and plant cell death. butafenacil is known to be eye, skin and respiratory tract irritant in humans [ ] . it also demonstrated very high toxic effect to algae, and moderate toxicity to fi sh, aquatic invertebrates and honeybees. for benzfendizone and flufenpyr-ethyl, no reports on toxic effects are available. acute mammalian toxicology studies of safl ufenacil indicate that herbicide has low toxicity for mammals after ingestion, dermal exposure or inhalation. it is not an irritant for eyes and skin and does not act as a sensitizer. studies of the structure-activity relationship (sar) of uracile derivatives as protox inhibitor showed that presence of a polyfl uorinated alkyl group at position of the uracil ring critical. alkyl groups such as methyl at position of the uracil ring resulted in compounds with low or no biological activity [ ] . limited data are available on the synthesis of butafenacil ( ). in particular, it was prepared by esterifi cation of carboxylic acid , [ ] as well as by reaction of isocyanate with ester (scheme ) [ ] . preparation of neither nor was disclosed in the corresponding patents, although synthesis of carboxylic acid was partially described elsewhere [ ] . benzfendizone ( ) was obtained from ethyl trifl uoromethylaminocrotonate ( ) which reacted with isocyanate in the presence of nah and then directly methylated to give (scheme ) [ ] . demethylation of phenol moiety in followed by alkylation with benzyl chloride gave benzfendizone. in the preparation of flufenpyr-ethyl ( ), hydrazones or were the key synthetic intermediates (scheme ) [ - ] . both compounds and were prepared by reaction of dibromoketone and the corresponding hydrazines and , in turn obtained by reduction of diazonium salts and . alternatively, hydrazone was prepared by reaction of and ethyl trifl uoroacetoacetate, followed by hydrolysis and decarboxylation. further transformations of included reaction with (carbethoxylidene)triphenylphosphorane resulting in the formation of pyridazine derivative . acidic hydrolysis of led to , which was alkylated with ethyl bromoacetate to give (scheme ). alternatively, either or reacted with methylmalonic acid to give adducts or , which underwent cyclization upon heating with carboxylic acid and a base to give and , respectively. both and were transformed to flufenpyr-ethyl ( ) as described above (scheme ). compounds discussed in this section are derivatives or analogues of sulfonylurea herbicides -agrochemicals which began the present low-dose era of herbicide chemistry in s [ ] . primisulfuron-methyl ( ) (from ciba-geigy corporation and syngenta ag) is a sulfonylurea derivative introduced in [ ]. it is used for post-emergence control of actively growing weeds in corn and in non-cropland areas [ ] . cloransulam-methyl ( ), florasulam ( ), and diclosulam ( ), all developed by dow agrosciences, are examples of the triazolopyrimidine sulfonanilide herbicides; they were introduced in , , and , respectively. cloransulam-methyl is used for soil-applied and post-emergence control of broadleaf weeds in soybeans [ ] . florasulam is a highly-selective broadleaf herbicide which is registered for use in cereals in many countries around the world. diclosulam-based products are registered for use to control annual and certain perennial broadleaf weeds; they can be can be applied as soil, foliar, or burndown treatments in crops such as sugar cane, peanuts and soybeans and in forestry applications. compounds - inhibit acetohydroxy acid synthase (ahas), formerly known as acetolactate synthase. its activity is not present in animals, but it has been found in all plants where measurements have been attempted. acetohydroxy acid synthase catalyses the fi rst step in production of branched amino acids (leucine, valine and isoleucine) (scheme ), which are obviously needed for the protein synthesis and cell growth. the compounds - seem to bind within the substrate-access channel of the enzyme, thus blocking α-ketocarboxylate access to the active site. while these herbicides are undoubtedly highly successful, resistance developed due to mutations within ahas is becoming a serious problem [ , ] . primisulfuron-methyl is a slightly toxic for skin, inhalation and eye exposure, with little metabolic activity in mammalian. it is slightly toxic to freshwater fi sh, aquatic organisms and to marine shrimp and has no toxic effect on birds and honeybees [ ] . cloransulam-methyl can be highly toxic to certain aquatic plants and algae on an acute basis; it is practically nontoxic to other non-plant organisms. florasulam is highly toxic to aquatic organisms and slightly toxic to birds, and diclosulam is very highly toxic to aquatic organisms [ ] . in contrast to uracile herbecides in which cf -group is critical for activity in fl uorinated triazolopyrimidine series fl uorine atom responsible for the methabolitic transformation of the herbecides. the different metabolic pathway of the triazolopyrimidine herbicide diclosulam and cloransulam-methyl are guided by the fl uorine atom at the -position on the triazolopyrimidine ring system (scheme ). the predominance of one pathway is very crop specifi c. in cotton, and are metabolized by the displacement of the -fl ouro substituent on the triazolopyrimidine ring by a hydroxy group, forming . its soybean selectivity is attributed to facile conjugation with homo-glutathion (homogsh), which displaces the -fl uoro substituent ( ). this mechanism was found to only occur in soybeans for these herbecides. in maize and wheat, and are detoxifi ed by hydroxylation at the -th position on the aniline moiety ( ) followed by subsequent glycosidation [ ] . cloransulam-methyl ( ) and diclosulam ( ) were obtained by reaction of sulfonyl chloride with the corresponding aniline derivatives (scheme ). synthesis of commenced from dichloropyrimidine [ ] , which reacted with kf and then -hydrazine hydrate to give . reaction of with cs /et n and then -benzyl chloride was accompanied by dimroth rearrangement and gave the synthesis of primisulfuron-methyl ( ) started from reaction of diethyl malonate and thiourea (scheme ) [ ] . the resulting pyrimidine derivative was methylated, difl uoromethylated and then oxidized to give sulfone . reaction of with aqueous ammonia gave heteroaromatic amine , which was transformed to primisulfuron-methyl ( ) upon treatment with isocyanate . fluoxastrobin ( ) is a pesticide from bayer cropscience for the control of fungal diseases, which was registered by u. s. environmental protection agency (epa) in [ ] . fluoxastrobin is used on peanuts, tuberous and corm vegetables, leaf petiole vegetables, fruiting vegetables and turf. fluacrypyrim ( ) was discovered by basf ag and introduced in by nippon soda co., shows acaricidal effect against all stages of tetranychid [ ] . both and are representative of strobilurin family with parent compound strobilurin a ( ) (fig. ) , discovered in late s [ ] . interestingly, fluacrypyrim ( ) is the fi rst representative of strobilurin family which is not used as a fungicide. strobilurins are the part of the larger group of the so-called quinone outside inhibitors (qoi) -compounds which act at the quinol outer binding site of the cytochrome bc complex. this enzyme, also referred to as ubiquinol: ferricytochrome c reductase, or complex iii, is the third complex in the electron transport chain -a cascade of enzymes which couples electron transfer between nadh and o with the transfer of h + ions across a membrane to generate chemical energy in the form of adenosine triphosphate (atp) [ ] . the overall result of the reaction catalyzed by cytochrome bc complex is reduction of ferricytochrome c by oxidation of ubiquinol ( ) and the concomitant pumping of protons from the mitochondrial matrix to the intermembrane space. the mechanism of this process is too sophisticated to be discussed herein. it is important that the enzyme has two binding sites for the substrate or its oxidized form (fig. ) , i.e. outer (q ) and inner (q ), and the quinone outside inhibitors bind to the outer site. this leads to inhibition of mitochondrial respiration -a process which is essential to all living organisms. the selective biological effect of quinone outside inhibitors on certain organisms ( i.e. fungi or mites) is achieved by differential penetration and degradation between various species, leading to a combination of high fungicidal (or acaricidal, in the case of ) potency and good crop safety [ ] . unfortunately, resistance has already evolved to this class of pesticides in some plant pathogens in certain geographical areas [ ] . although the in vitro fungicidal activity of the natural strobilurin a was discovered soon, its agrobiological testing in vivo was diffi cult because of its volatility and the inherent lability of the ( e,z,e )-triene system, which resulted in rapid photolytic or metabolic degradation. the unusual structural simplicity of this natural product soon made it a target for chemical derivatization. below a set of isosterical replacement in a course of lead optimization of natural strobirulin a leading to commercial synthetic products shown on the fig. . the fi rst sequence leads to fi rst commercialized strobilurin azoxystrobin ( , amistar®, syngenta) and than to fl uoxastrobin ( ), which structure combines a methoximino , -dihydro- , , -dioxazin- -yl toxophore (bayer toxofore) with an optimally adjusted side-chain bearing a -( -chlorophenoxy)- -fl uoro-pyrimidin- yl-oxy moiety as an essential element. fluoxastrobin ( ) has an advantage as no reorientation of the toxophore is necessary for binding to the target. the sars studies indicate that the fl uorine atom has a benefi cial effect on the phytotoxicity and leaf systemicity. another sequence leads to picoxystrobin ( , acanto®, syngenta), which has a -cf -pyridin- -yl moiety in its arylalkyl ether side-chain. an indication switch from the fungicidally to acaricidally active strobilurin type with β-methoxyacrylate pharmacophore is achieved by exchange of the -cf pyridin- -yl moiety in the arylalkyl ether side-chain of picoxystrobin with a -i pro- -cf -pyrimidin- -yl moiety to give fl uacrypyrim ( ). fluoxastrobin ( ) was obtained by reaction of compounds and in the presence of k co (scheme ) [ ] . compound was prepared by reaction of , , -trifl uoropyrimidine ( ) with potassium o -chlorophenolate. in turn, was obtained from -chloro- , -difl uoropyrimidine ( ) by reaction with kf. preparation of fluacrypyrim ( ) started with reaction of o -isopropylisourea hydrochloride and ethyl trifl uoroacetoacetate to give pyrimidine (scheme ) [ ] . alkylation of with bromide (or the corresponding chloride [ , ] ) in the presence of alkali or k co gave fluacrypyrim. cu o-catalyzed alkylation of was also developed for the synthesis of [ ] . compounds and were obtained using several closely related methods. in particular, ticl -mediated reaction of chloride and methyl orthoformate was used to obtain (scheme ) [ , ] . alternatively, reacted with methyl formate in the presence of ticl -et n to give , which was treated with p -toluenesulfonic acid in methanol to give . yet another method included reaction of with methyl orthoformate to give , which was transformed to upon treatment with methanesulfonic acid. another approach to fluacrypyrim ( ) commenced from pyrimidine derivative , which reacted with methyl formate in the presence of ticl -et [ , ] . methylation of using methyl orthoformate or dimethyl sulphate and alkali led to the formation of . the last pesticide from this section is flufenerim (flumfen® ), which is under development by ube industries as an insecticide. it is reported to control aphids, whitefl ies, and cotton leafworm, but has no activity against thrips [ ] . since flufenerim is chemically related to pyrimidifen (miteclean® ) (fig. ) , it was initially believed to have similar mechanism of action, i.e. inhibition of the mitochondrial electron transport of nadh dehydrogenase (nadh: ubiquinone oxidoreductase, complex i) -an enzyme which transfers electrons from nadh to ubiquinone and hence opens the electron transport chain cascade. nevertheless, it was shown that reduced activity of acetylcholinesterase -an effect which possibly can be addressed to interaction with other systems [ ] . flufenerim ( ) was prepared from , -dichloro- -ethylpyrimidine ( ) (scheme ) [ ] . compound was chlorinated with chlorine gas; the product thus obtained was subjected to nucleophilic substitution with acok to give acetate , which upon hydrolysis and subsequent reaction with diethylaminosulphur trifl uoride (dast) gave fl uoride . finally, reaction of with amine led to the formation of flufenerim ( ). since discovery of the fi rst fl uorinated diazine -antineoplastic agent -fl uorouracil more than compounds from the class were introduced into the market. undoubtedly the success was achieved due to joint progress of medicinal chemistry, agrochemistry as well as synthetic methods of heterocyclic and fl uoroorganic chemistry. the continued progresses in these fi elds of science allow us to predict that the number of fl uorine containing diazines as drugs or agrochemicals on the market will be increased. recent trends in using of perfl uorinated diazines as core scaffold for the synthesis of a diverse array of polysubstituted fl uorinated diazines for hts increases probability of these compounds as potential hits and leads. also the new methodologies of direct introduction of fl uorinated substituent, like baran approach, continue to appear facilitating further investigation. moreover in the chemical space covered by fl uorinated diazines remains "white spots". thus diazine scaffold decorated by important in med and agrochem fl uorinated fragments such as -chf , -ch cf , -ocf , -scf , -sf not investigated because the synthetic chemistry of these compounds on development phase or not developed at all. therefore the comprehensive investigations in the fi eld of fl uorinated diazines still are interesting both for academic and industrial scientists. fluorine in medicinal chemistry fluorine in medicinal chemistry: a century of progress and a -year retrospective of selected highlights elsevier mdl, version . fluorinated pyrimidines, a new class of tumourinhibitory compounds united states food and drug administration www.fda.gov studies in -acetylaminofl uorene carcinogenesis. . the utilization of uracil- -c- by preneoplastic rat liver and rat hepatoma -fl uorouracil: mechanisms of action and clinical strategies -fl uorouracil: forty-plus and still ticking. a review of its preclinical and clinical development medicinal chemistry of anticancer drugs cancer drug discovery and development: combination cancer therapy: modulators and potentiators fluorouracil: biochemistry and pharmacology the catalytic mechanism and structure of thymidylate synthase the synthesis of -fl uoropyrimidines process for fl uorinating uracil and derivatives thereof process for production of -fl uorouracil and its derivatives process for producing -fl uorouracil preparation of thymidine and deoxyfl uorouridine, and intermediates therefor Über die reaktion von uracil und seinen nucleosiden mit elementarem fluor reaction of acetyl hypofl uorite with pyrimidines. part . synthesis, stereochemistry, and properties of -fl uoro- , -dihydropyrimidine nucleosides nucleic acid related compounds. . direct fl uorination of uracil and cytosine bases and nucleosides using trifl uoromethyl hypofl uorite. mechanism, stereochemistry, and synthetic applications nucleic acid related compounds. iii. facile synthesis of -fl uorouracil bases and nucleosides by direct fl uorination clinical colorectal cancer: ode to -fl uorouracil -fl uorouracil derivatives: a patent review verfahren zur herstellung von n -( ′tetrahydrofuryl)-und n -( ′tetrahydropyranyl)-derivaten -substituierter urazile und deren alkalimetallsalzen ussr n -( ′-furanidyl)-derivatives of -substituted uracils involvement of microsomal cytochrome p and cytosolic thymidine phosphorylase in -fl uorouracil formation from tegafur in human liver formation pathways of γ-butyrolactone from the furan ring of tegafur during its conversion to -fl uorouracil comparison of gastrointestinal toxicity of -fu derivatives a phase i safety and pharmacokinetic study of ogt in patients with liver cancerю a novel, orally administered nucleoside analogue, ogt , inhibits the liver invasive growth of a human colorectal tumor, c hm antitumor effect and tumor level of -fl uoro- ′-deoxyuridylate following oral administration of tetradecyl ′-deoxy- -fl uoro- ′-uridylate antitumor activity of t- , a novel synthetic fudr derivative, on murine colon cancer and its hepatic metastasis synthesis and antitumor activity of -fl uorouracil derivatives sensitivity to six antitumor drugs differs between primary and metastatic liver cancers metabolism of -hexylcarbamoyl- -fl uorouracil (hcfu), a new antitumour agent, in rats, rabbits and dogs low dose -hyxylcarbamoyl- -fl uorouracil (hcfu) recommended for cirrhotic patients with hepatocellular carcinoma phase iii clinical study of a new anticancer drug atofl uding atofl uding n( )-o-toluyl-fl uorouracil inhibits human hepatocellular carcinoma cell growth via sustained release of -fu a phase ii trial of a new -fl uorouracil derivative, bof-a (emitefur), for patients with advanced gastric cancer effi cacy of a new -fl uorouracil derivative, bof-a , in advanced non-small cell lung cancer. a multi-center phase ii study phase i assessment of the pharmacokinetics, metabolism, and safety of emitefur in patients with refractory solid tumors phase i clinical trial of -fl uoro-pyrimidinone ( fp), an oral prodrug of -fl uorouracil ( fu) -fl uoro- -pyrimidinone, a liver aldehyde oxidase-activated prodrug of -fl uorouracil capecitabine preclinical studies: from discovery to translational research metabolism of capecitabine, an oral fl uorouracil prodrug: f nmr studies in animal models and human urine process for producing -fl uorouracil derivative with a calcium chloride catalyst process for the preparation of -( -tetrahydrofuryl)- -fl uorouracil method for the preparation of derivatives of uracil process for the preparation of n.sup. -( ′-furanidyl)- -fl uoro-uracil synthesis of tetrahydro- -furyl derivatives of -substituted uracils liepin'sh e ( ) nitrogen-containing organosilicon compounds c. reaction of -fl uoro- , -bis-o-(trimethylsilyl)uracil with , -dihydrofuran analogs of pyrimidine nucleosides i. n,(atetrahydrofuryl) derivatives of natural pyrimidine bases and their antimetabolites the synthesis of (tetrahydro- -furanyl)- -fl uorouracil (ftorafur) via direct fl uorination synthetic studies on chemotherapeutics. ii. synthesis of phenyl-substituted , -dihydro- -oxonicotinic acid derivatives. [studies on the syntheses of heterocyclic compounds. part analogs of pyrimidine nucleosides facile synthesis of tetrahydro- -furylated pyrimidines and purines using a new catalyst of cesium chloride a novel synthesis of -fl uorouracil derivatives having oxacycloalkane moieties synthesis of -(tetrahydro- -furanyl)- -fl uorouracil (ftorafur) via direct fl uorination studies on fl uorinated pyrimidines. i. a new method of synthesizing -fl uorouracil and its derivatives ′-halogeno- ′, ′-cyclic sulphite isomers in the preparation of ′-halogeno nucleosides. synthesis of ′-deoxyuridine and ′-deoxy- -fl uorouridine ′-halogeno- ′, ′-sulphites in the synthesis of ′, ′-dideoxy- -fl uorouridine and related analogues fluorinated pyrimidine nucleosides. . synthesis and antitumor activity of a series of ′-deoxy- -fl uoropyrimidine nucleosides . stereospezifi sche synthese des cancerostatikums '-desoxy- -fl uor-uridin ( -dfur) und seiner ′-deuterierten derivate rapid continuous synthesis of -deoxyribonucleosides in fl ow via brønsted acid catalyzed glycosylation preparation, antibacterial effects and enzymatic degradation of -fl uorouracil nucleosides reaction of acetylhypofl uorite with pyrimidines. part . synthesis, stereochemistry and properties of -fl uor- , -dihydropyrimidine-nucleosides gb chem abstr synthesis and antitumor activity of phagocytic conjugates of -fl uorouracil with albumin synthesis of f- labeled nucleoside analogues therapeutic compounds with pyrimidine base -fl uoro- ′-deoxyuridine derivatives and a process for the preparation thereof novel -fl uoro- -deoxyuridine derivatives and salts thereof, process for producing the same, and antitumor agents containing the same -fl uorouracil derivatives. i. the synthesis of -carbamoyl- -fl uorouracils -carbamoyl- -fl uorouracil derivatives studies on the syntheses of heterocyclic compounds. . studies on the synthesis of chemotherapeutics. . synthesis and antitumor activity of n -acyl-and n -(alkoxycarbonyl)- -fl uorouracil derivatives synthesis of -fl uorouracil derivatives containing an inhibitor of -fl uorouracil degradation -fl uorouracil derivatives -fl uorouracil derivatives derivatives of pyrimidine some derivatives of -fl uoropyrimidine antitumor properties of ( h )-pyrimidinone riboside (zebularine) and its fl uorinated analogs synthesen mit substituierten malondialdehyden, xix. darstellung fl uorsubstituierter carbo-und heterocyclen verfahren zur herstellung von -fl uorpyrimidin- -on und seinen n - -substituierten derivaten a simple synthesis of -fl uoro- -pyrimidinone and its n -substituted derivatives n -(substituted-oxycarbonyl)- ′-deoxy- -fl uorocytidine compounds, compositions and methods of using same the design and synthesis of a new tumor-selective fl uoropyrimidine carbamate, capecitabine processes related to making capecitabine process for the preparation of capecitabine combination therapy for the treatment of cancer using cox- inhibitors and dual inhibitors of egfr synthesis and biological activity evaluation of cytidine- ′-deoxy- -fl uoro-n-[(alkoxy/aryloxy)] carbonyl-cyclic ′, ′-carbonates process for preparing capecitabine methods for preparing capecitabine and beta-anomer-rich trialkyl carbonate compound used therein a novel combination antimetabolite, tas- , exhibits antitumor activity in fu-resistant human cancer cells through a mechanism involving ftd incorporation in dna trifl uorothymidine exhibits potent antitumor activity via the induction of dna double-strand breaks thymidine phosphorylase. substrate specifi city for -substituted ′-deoxyuridines potentiation of the antitumor activity of α, α, α-trifl uorothymidine by the co-administration of an inhibitor of thymidine phosphorylase at a suitable molar ratio in vivo antitumor activity of ftc- , a masked -trifl uoromethyl- ′-deoxyuridine derivative syntheses of -trifl uoromethyluracil and -trifl uoromethyl- ′-deoxyuridine alternative synthesis of ′-deoxy- -(trifl uoromethyl)-uridine and the α-anomer thereof the synthesis of ′-deoxy- -trifl uoromethyluridine utilizing a coupling reaction direct perfl uoroalkylation including trifl uoromethylation of aromatics with perfl uoro carboxylic acids mediated by xenon difl uoride studies on organic fl uorine compounds. part . trifl uoromethylation of pyrimidine and purine nucleosides with trifl uoromethyl-copper complex studies on antitumor agents. . antitumor activities of o -alkyl derivatives of ′-deoxy- -(trifl uoromethyl)uridine and ′-deoxy- -fl uorouridine studies on antitumor agents. ix. synthesis of ′-o -benzyl- ′-deoxy- -trifl uoromethyluridine us . clinicaltrials.gov: a service of the u.s. national institutes of health abstract b : ly , a potent oral inhibitor of the cyclindependent kinases and (cdk / ) that crosses the blood-brain barrier and demonstrates in vivo activity against intracranial human brain tumor xenografts cyclin-dependent kinase pathways as targets for cancer treatment metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleen tyrosine kinase inhibitor r in humans: contribution of hepatic and gut bacterial processes to the overall biotransformation tyrosine kinase inhibitors as potential drugs for b-cell lymphoid malignancies and autoimmune disorders preclinical characterization of aurora kinase inhibitor r /as identifi ed through an imagebased phenotypic screen phase i, open-label, multicentre, dose-escalation, pharmacokinetic and pharmacodynamic trial of the oral aurora kinase inhibitor pf- in advanced solid tumours pf- , an orally bioavailable small molecule aurora kinase inhibitor for cancer therapy the jak inhibitor azd potently blocks stat signaling and oncogenesis in solid tumors discovery of -chloro-n -[( s )- -( -fluoropyrimidin- -yl)ethyl]-n -( -methyl- h -pyrazol- -yl)pyrimidine- , -diamine (azd ) as a novel inhibitor of the jak/stat pathway pyrimidinediamine compounds for use in the treatment or prevention of autoimmune diseases -aminopyrazole)pyrimidine derivatives for use as tyrosine kinase inhibitors in the treatment of cancer anti-hiv drugs: compounds approved within years after the discovery of hiv emtricitabine, a new antiretroviral agent with activity against hiv and hepatitis b virus pharmacokinetics of antiretrovirals in pregnant women the ′-triphosphates of the (-) and (+) enantiomers of cis- -fl uoro- -[ -(hydroxymethyl)- , -oxathiolane- -yl]cytosine equally inhibit human immunodefi ciency virus type reverse transcriptase the antihepatitis b virus activities, cytotoxicities, and anabolic profi les of the (-) and (+) enantiomers of cis- -fl uoro- -[ -(hydroxymethyl)- , -oxathiolan- -yl]cytosine new nucleoside reverse transcriptase inhibitors for the treatment of hiv infections overview of antiretroviral agents expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodefi ciency virus type inhibition of clinically relevant mutant variants of hiv- by quinazolinone nonnucleoside reverse transcriptase inhibitors structural basis for the resilience of efavirenz (dmp- ) to drug resistance mutations in hiv- reverse transcriptase characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (s)- -chloro- -(cyclopropylethynyl)- -(trifl uoromethyl)- , -dihydro- ( h)-quinazolinone (dpc ), in rats. possible formation of an oxirene metabolic intermediate from a disubstituted alkyne asymmetric synthesis and biological evaluation of β-l-( r , s )-and α-l-( r , r )- , -oxathiolane-pyrimidine and -purine nucleosides as potential anti-hiv agents structure-activity relationships of β-d-( s, r)-and α-d-( s, s)- , -oxathiolanyl nucleosides as potential anti-hiv agents method for the synthesis, compositions and use of ′-deoxy- -fl uoro- ′-thiacytidine and related compounds intermediates in the synthesis of , -oxathiolane nucleoside enantiomers processes for the diastereoselective synthesis of nucleoside analogues novel process for the preparation of cis -nucleoside derivative design and synthesis of ′, ′-dideoxy- ′, ′-didehydro-beta-l-cytidine (beta-l-d c) and ′, ′-dideoxy ′, ′-didehydro-beta-l- -fl uorocytidine (beta-l-fd c), two exceptionally potent inhibitors of human hepatitis b virus (hbv) and potent inhibitors of human immunodefi ciency virus (hiv) in vitro stereoselective syntheses of β-l-fd c and β-l-fddc synthesis and biological evaluation of ′, ′-didehydro- ′, ′-dideoxy- -fl uorocytidine (d fc) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against hiv- reverse transcriptase synthesis and comparative evaluation of two antiviral agents: β-l-fd c and β-d-fd c method for synthesizing beta-l-fl uoro- ′, ′didehydcytidine (β-l-fd c) method for the synthesis of ′, ′-dideoxy- ′, ′-didehydronucleosides synthesis of d-d fc, a biologically active nucleoside via an unprecedented palladium mediated ferrier rearrangement-type glycosidation with an aromatization prone xylo-furanoid glycal a new asymmetric , -addition method: application to the synthesis of the hiv non-nucleoside reverse transcriptase inhibitor dpc general scope of , -diastereoselective additions to a ( h)-quinazolinone: practical preparation of hiv therapeutics an effi cient chiral moderator prepared from inexpensive (+)- -carene: synthesis of the hiv- non-nucleoside reverse transcriptase inhibitor dpc nmr spectroscopic investigations of mixed aggregates underlying highly enantioselective , -additions of lithium cyclopropylacetylide to quinazolinones highly enantioselective construction of a chiral tertiary carbon center by alkynylation of a cyclic n-acyl ketimine: an effi cient preparation of hiv therapeutics highly enantioselective construction of a quaternary carbon center of dihydroquinazoline by asymmetric mannich reaction and chiral recognition trifl uridine: a review of its antiviral activity and therapeutic use in the topical treatment of viral eye infections physical and biological consequences of incorporation of antiviral agents into virus dna fluorine-containing diazines in medicinal chemistry and agrochemistry t- (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections mechanism of action of t- against infl uenza virus nitrogen-containing heterocyclic carboxamide derivatives or salts thereof and antiviral agents comprising the same novel pyrazine derivatives or salts thereof, pharmaceutical composition containing the same, and production intermediates thereof organic amine salt of -fl uoro- -hydroxy- -pyrazinecarbonitrile and method for producing the same method for producing dichloropyrazine derivative antibiotic r&d gets a dose of funding method for producing dichloropyrazine derivative antibiotic activity and characterization of bb- , a novel peptide deformylase inhibitor peptide deformylase inhibitors flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions ii pharmacology and clinical use of voriconazole voriconazole: a new triazole antifungal agent voriconazole compared with liposomal amphotericin b for empirical antifungal therapy in patients with neutropenia and persistent fever multiple-triazole-resistant aspergillosis process for the preparation of -fl uorocytosine nucleic acid related compounds. . direct fl uorination of uracil and cytosine bases and nucleosides using trifl uoromethyl hypofl uorite. mechanism, stereochemistry, and synthetic applications a direct synthesis of -fl uorocytosine and its nucleosides using trifl uoromethyl hypofl uorite Über synthesen von difl uoraminopyrimidinen process for preparing -fl uorocytosine salt triazole antifungal agents novel antifungal -aryl- -( h - , , -triazol- -yl)butan- -ol derivatives with high activity against process for the preparation of voriconazole process for preparing voriconazole improved process for the preparation of ( r , s )- -( , -difl uorophenyl)- -( -fl uoropyrimidin- -yl)- -( h - , , -triazol- -yl)butan- -ol (voriconazole) improved process for the preparation of ( r , s )- -( , -difl uorophenyl)- -( -fl uoropyrimidin- -yl)- -( h - , , -triazol- -yl)butan- -ol process for the preparation of voriconazole a process for making voriconazole preparation of triazoles by organometallic addition to ketones and intermediates therefor an improved process for the preparation of voriconazole and intermediates thereof process for the production of voriconazole intermediates of voriconazole and preparation method of voriconazole using the same process for preparing voriconazole by using new intermediates a novel process to manufacture ( r, s)- -( , -difl uorophenyl)- -( -fl uoropyrimidin- -yl)- -( h- , , -triazol- -yl)butan- -ol section vii. worldwide market introductions the encyclopedia of addictive drugs pharmacological studies on -amino- -fl uoromethyl- -( o -tolyl)- ( h )-quinazolinone (afl oqualone), a new centrally acting muscle relaxant (i) studies on biologically active halogenated compounds. . synthesis and central nervous system depressant activity of -(fl uoromethyl)- -aryl- ( h )-quinazolinone derivatives discriminative stimulus characteristics of bmy in the pigeon a role for sigma binding in the antipsychotic profi le of bmy the sigma ligand bmy- as a potential antipsychotic: evidence from the latent inhibition model in rats bmy , a sigma receptor ligand for the treatment of schizophrenia the sigma- antagonist bmy- inhibits l -dopa-induced abnormal involuntary movements by a way- -sensitive mechanism in vitro characterization of the selective dopamine d receptor antagonist a- . th annual meeting, society of neuroscience effect of dopamine d antagonists on ppi in dba/ j mice or ppi defi cit induced by neonatal ventral hippocampal lesions in rats a double-blind, randomized, placebocontrolled study of the dopamine d receptor antagonist abt- in patients with acute schizophrenia dopamine d receptor antagonism -still a therapeutic option for the treatment of schizophrenia third generation antipsychotic drugs: partial agonism or receptor functional selectivity pharmacology of jnj- , a specifi c and fast-dissociating d antagonist for the treatment of schizophrenia a double-blind, randomized, placebo-controlled study with jnj- , a novel, highly selective, fast dissociating d receptor antagonist in the treatment of acute exacerbation of schizophrenia agents for treatment of brain ischemia antipsychotic -fl uorophenylbutyl- -( -pyrimidinyl)piperazine derivatives synthesis and biological characterization of α-( -fl uorophenyl)- -( -fl uoro- -pyrimidinyl)- -piperazinebutanol and analogues as potential atypical antipsychotic agents resolution of α-( -fl uorophenyl)- -( -fl uoro- -pyrimidinyl)- -piperazinebutanol (bms ) and α-( -chloropropyl)- -fl uorobenzenemethanol using lipase-catalyzed acetylation or hydrolysis evaluation of the effects of the enantiomers of reduced haloperidol, azaperol, and related -amino- -arylbutanols on dopamine and σ receptors asymmetric hydrogenation of amino ketones using chiral rucl (diphosphine)( , -diamine) complexes piperidin- -yl-pyridazin- -ylamine derivatives as fast dissociating dopamine receptor antagonists a -heteroaryl- -piperidinyl-methyl pyrrolidinone, bmy , delays the decay of hippocampal synaptic potentiation in vitro effect of bmy on acquisition of shape discrimination and memory retention in monkey effect of bmy on classical conditioning of the eyeblink response in young and older rabbits bmy and piracetam facilitate performance of two-choice win-stay water-escape in normal rats effects of oral bmy on morris water task performance in - month old f- rats effects of bmy- on anoxia in mice effi cacy and safety of bmy in alzheimer disease the use of the computerized neuropsychological test battery (cntb) in an effi cacy and safety trial of bmy , in alzheimer's disease process for large-scale production of bmy cerebral function enhancing diazinylpiperidine derivatives small molecule blockers of voltage-gates sodium channels molecular determinants of voltage-dependent gating and binding of pore-blocking drugs in transmembrane segment iiis of the na + channel α subunit a multicenter, double-blind, randomized, placebo-controlled crossover evaluation of a short course of bw- w in patients with chronic neuropathic pain discovery of -[( , -dichlorophenyl)amino]-n -[(tetrahydro- h -pyran- -yl)methyl]- -(trifl uoromethyl)- -pyrimidinecarboxamide, a selective cb receptor agonist for the treatment of infl ammatory pain a randomized, controlled study to investigate the analgesic effi cacy of single doses of the cannabinoid receptor- agonist gw , ibuprofen or placebo in patients with acute pain following third molar tooth extraction optically active phenyl pyrimidine derivatives as analgesic agents pyrimidine derivatives and their use as cb modulators combination of cb modulators and pde inhibitors for use in medicine role of spleen tyrosine kinase inhibitors in the management of rheumatoid arthritis of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of syk fostamatinib, a syk inhibitor prodrug for the treatment of infl ammatory diseases the status of fostamatinib in the treatment of rheumatoid arthritis recent advances in non-peptidomimetic dipeptidyl peptidase inhibitors: medicinal chemistry and preclinical aspects effects of ketoconazole and rifampicin on the pharmacokinetics of gemigliptin, a dipeptidyl peptidase-fluorine-containing diazines in medicinal chemistry and agrochemistry iv inhibitor: a crossover drug-drug interaction study in healthy male korean volunteers an update in incretin-based therapy: a focus on dipeptidyl peptidase inhibitors a multicentre, multinational, randomized, placebo-controlled, double-blind, phase trial to evaluate the effi cacy and safety of gemigliptin (lc - ) in patients with type diabetes effi cacy and safety of the dipeptidyl peptidase- inhibitor gemigliptin compared with sitagliptin added to ongoing metformin therapy in patients with type diabetes inadequately controlled with metformin alone liver fi brosis infl ammation and the pathogenesis of diabetic nephropathy -aminocyclopentanecarboxamides as chemokine receptor modulators dual targeting of ccr and ccr : therapeutic potential for immunologic and cardiovascular diseases chemokine receptor genotype is associated with diabetic nephropathy in japanese with type diabetes dipeptidyl peptidase-iv inhibiting compounds, methods of preparing the same, and pharmaceutical compositions containing the same as an active agent fluorine-containing agrochemicals: an overview of recent developments. in: tressaud a (ed) fluorine and the environment: agrochemicals, archaeology, green chemistry & water agricultural products based on fl uorinated heterocyclic compounds. in: petrov va (ed) fluorinated heterocyclic compounds: synthesis, chemistry, and applications a history of weed control in the united states and canada -a sequel the pesticide book, th edn. meisterpro information resources advanced technologies for parasitic weed control protoporphyrinogen oxidase inhibitor: an ideal target for herbicide discovery synthesis and chemistry of agrochemicals v process for the production of -aryl-uracils synthesis and chemistry of agrochemicals vi method for producing sulfonic acid diamides pyridazin- -one derivatives, their use, and intermediates for their production production of pyridazine herbicides herbicidal composition. us structure and mechanism of inhibition of plant acetohydroxyacid synthase acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids environmental protection agency offi cial site www.epa.gov weed management in peanut ( arachis hypogaea ) with diclosulam preemergence n -arylsulfi limine compounds and their use as catalysts in the preparation of n -arylarylsulfonamide compounds preparation of n -arylarylsulfonamide compounds process for heterocyclic sulfonyl chloride compounds preparation of n-arylarylsulfonamide compounds pyrimidinyl)amino)carbonyl)amino)sulfonyl)benzoic acid methyl ester (primisulfuron) acaricides -biological profi les, effects and uses in modern crop protection review of strobilurin fungicide chemicals lehninger principles of biochemistry recent developments in the mode of action of fungicides mechanisms of resistance to qoi fungicides in phytopathogenic fungi halogen pyrimidines and its use thereof as parasite abatement means -alkoxy- -trifl uoromethylpyrimidin- -yl)oxymethylene]phenylacetic acid derivatives, their preparation and intermediate therefor, and use thereof processes for producing acrylic acid derivative processes for producing acrylic acid derivative methods for highly selectively o -alkylating amide compounds with the use of copper salts flufenerim, a novel insecticide acting on diverse insect pests: biological mode of action and biochemical aspects inhibitors of mitochondrial electron transport: acaricides and insecticides -phenethylaminopyrimidine derivative, and agricultural and horticultural chemical for controlling noxious organisms containing the same kinetics and metabolism of a new fl uoropyrimidine, ′-deoxy- -fl uorouridine studies on tetrahydrofuryl- -fl uorouracils. iv. mode of reaction of -fl uorouracil with -acetoxytetrahydrofuran preparation of -pyrimidinone and derivatives pyrimidine derivatives for the treatment of abnormal cell growth effects of afl oqualone on vestibular nystagmus and the lateral vestibular nucleus identifi cation and measurement of urinary metabolites of afl oqualone in man biocatalytic synthesis of some chiral drug intermediates by oxidoreductases the nootropic compound bmy- improves spatial learning ability in brain injured rats modifi cation of chemokine pathways and immune cell infi ltration as a novel therapeutic approach in liver infl ammation and fi brosis key: cord- - j m authors: campbell, duncan j. john title: ace inhibition in heart failure and ischaemic heart disease date: journal: frontiers in research of the renin-angiotensin system on human disease doi: . / - - - - _ sha: doc_id: cord_uid: j m nan angiotensin converting enzyme (dipeptidyl carboxypeptidase i, kininase ii, ec . . . , ace) plays a major role in the metabolism of many different peptides, including angiotensin (ang) i, bradykinin, kallidin, and n-acetyl-seryl-aspartyllysyl-proline (acsdkp). ace inhibitors are established therapy for heart failure and ischaemic heart disease, and alterations of ang ii, bradykinin, kallidin, and acsdkp peptide levels are implicated in the mechanisms of this therapy. this chapter briefly describes the renin angiotensin, kallikrein kinin, and acsdkp systems, and their role in cardiovascular physiology and disease. the role of ace inhibition in treatment and prevention of heart failure and ischaemic heart disease is summarised, and the possible mechanisms of the therapeutic benefits of ace inhibitors are described. this is not an exhaustive review, but focuses on those aspects most relevant to the clinical application of ace inhibitors. figure shows an outline of the pathways of ang peptide formation and metabolism. in addition to the classical pathway involving renin and ace, alternative pathways have been proposed (campbell ) . there remain many questions concerning the mechanisms of ang peptide formation in discrete tissue compartments such as the heart. serine proteases, for example, may form ang ii by processes independent of renin at sites of inflammation or coagulation, where kallikrein and/or cathepsin g may be active. figure . pathways of ang peptide formation and metabolism. adapted from (campbell ) studies of nephrectomised animals show the main mechanism of ang peptide formation in the heart involves kidney-derived renin (campbell et al ; danser et al ) . renin messenger rna (mrna) levels in the heart are very low or undetectable (de mello et al ) . cardiac renin expression may, however, be induced by myocardial infarction and macrophages and myofibroblasts may express renin at the site of repair (sun et al ) . all ang peptides are derived from angiotensinogen. although angiotensinogen may be produced in low levels in the heart (dostal et al ; paul et al ) , plasma is the main source of angiotensinogen for ang peptide formation in the heart. ace is a membrane-bound zinc-containing metallopeptidase, some of which is cleaved from membranes and released as soluble ace found in plasma and other fluids (erdos ). ace has two catalytic domains with differential substrate specificities and susceptibility to ace inhibitors (wei et al ; wei et al ; jaspard et al ) . table lists the many substrates of ace. those ace substrates most related to cardiac function are ang i, the bradykinin and kallidin peptides, and acsdkp. both catalytic domains of ace posses dipeptidyl carboxypeptidase and endopeptidase activities and can cleave ang i, bradykinin-( - ), bradykinin-( - ), and substance p. however, the n-terminal catalytic domain cleaves of lutein- bradykinin-( - ), bradykinin-( - ), and bradykinin-( - ) lys -bradykinin-( - ) (kallidin), lys -bradykinin-( - ), and lys -bradykinin-( - ) substance p n-acetyl-seryl-aspartyl-lysyl-proline (acsdkp) chemotactic peptide neurotensin luteinising hormone-releasing hormone (lh-rh) enkephalins cholecystokinin gastrin adapted from (ehlers et al ; erdos ; hooper ; rieger et al ) ising hormone-releasing hormone (lh-rh) and acsdkp more efficiently than the c-terminal domain (jaspard et al ; rousseau et al ) . the two catalytic domains of ace interact differently with ace inhibitors. captopril, enalapril, lisinopril, and trandolapril are all highly potent inhibitors of both domains. whereas trandolapril, lisinopril and enalapril show preference for the c-terminal catalytic domain, captopril shows preference for the n-terminal catalytic domain (wei et al ) . ace has a widespread tissue distribution, including vascular endothelium and smooth muscle cells, the brush border of proximal tubule cells of the kidney, and the brain (erdos ) . ace is expressed by the endothelium of the coronary vasculature, and by the endocardium and epicardium, but not by the valves in the human heart (dostal et al ) . ace is also expressed by cardiac fibroblasts, and fibroblast expression of ace is increased in the border zone of myocardial infarction (dostal et al ; burrell et al ) . cardiac ace expression is up-regulated in heart failure (hirsch et al ; studer et al ) . many different cell types express ang receptors in the heart. the type ang (at ) receptor is expressed by coronary smooth muscle and endothelial cells, cardiomyocytes, fibroblasts, nerves, and conduction tissue (regitz-zagrosek et al ) . at receptors are expressed by fibroblasts and endothelial cells (regitz-zagrosek et al ) . in heart failure, cardiomyocyte at receptor expression may be down-regulated, whereas fibroblast expression of both at and at receptors is increased (ohkubo et al ) . the at receptor mediates most of the known actions of ang ii. there is continuing uncertainty about the role of the at receptor, which may mediate actions of ang ii in the vasculature and heart that differ from those of the at receptor (carey et al ; voros et al ) . the at receptor is described further by danser in chapter of this volume. human heart chymase was initially discovered in homogenates of human heart and proposed to be the major pathway of conversion of ang i to ang ii in the heart (urata et al ) . given that chymase is not inhibited by ace inhibitors, it represented a potential pathway of continued ang ii formation in patients taking ace inhibitor therapy (dell'italia et al ) , and thereby provided a rationale for a possible superiority of at receptor blocker (arb) therapy over ace inhibitor therapy. however, studies of the effects of ace inhibition in rats, mice, and humans, and of ace gene knockout in mice, show ace is the dominant pathway of ang ii formation in the heart (campbell et al ; campbell et al ; zeitz et al ; campbell et al a) . ace-related carboxypeptidase (ace ), like ace, is a membrane-associated and secreted metalloprotease expressed predominantly on endothelium (donoghue et al ; tipnis et al ; hamming et al ) . ace is expressed in all human tissues, with relatively high levels in renal and cardiovascular tissues, and also in the gut (harmer et al ) . in contrast to the dipeptidyl carboxypeptidase activity of ace, ace cleaves ang i to ang-( - ) and also cleaves ang ii to ang-( - ). ace is not inhibited by ace inhibitors. kinetic considerations make it unlikely that ace contributes to ang i metabolism in vivo (jaspard et al ; vickers et al ) . ace and ace have similar k m for ang i ( and . μmol/l, respectively) but the k cat for ace ( s − ) is approximately -fold higher than that for ace ( . s − ), such that the k cat /k m ratio is approximately -fold higher for ace ( . x l/mol per s) than for ace ( . x l/mol per s). by contrast, the k m ( μmol/l), k cat ( . s − ), and k cat /k m ratio ( . x l/mol per s) of ace for ang ii (vickers et al ) make it more likely to participate in ang ii metabolism. initial genetic studies suggested an important role for ace in ang peptide metabolism in the heart. the ace gene knockout mouse was reported to have a cardiomyopathic phenotype associated with increased ang ii levels in plasma, heart, and kidney. additionally, the cardiomyopathic phenotype was ameliorated by concomitant ace gene knockout, suggesting that altered ang peptide metabolism contributed to the phenotype (crackower et al ) . in subsequent studies the ace gene knockout mouse had a normal cardiac phenotype, although it had an enhanced pressor response to ang ii administration (gurley et al ) . ace activity is reported to be increased in the hearts of patients with heart failure (zisman et al ) . however, measurement of ang peptides in coronary venous blood of patients with heart failure or ischaemic heart disease does not support an important role for ace in either ang i or ang ii metabolism in the human heart (campbell et al b) . elucidation of the role of ace in ang ii metabolism must await the development of specific ace inhibitors. effects of the ras on the heart and vasculature both systemic and local actions of ang ii impact on the heart. systemic actions of ang ii include its vasoconstrictor action to increase blood pressure and the stimulation of aldosterone secretion. increased aldosterone levels may produce hypokalaemia and contribute to cardiac fibrosis (brilla et al ) . local cardiac actions of ang ii include inotropic and hypertrophic effects, and cardiac remodelling (paul et al ) . at receptor stimulation induces both myocyte hypertrophy and collagen synthesis (regitz-zagrosek et al ) . moreover, ang ii may contribute to oxidative stress, inflammation, and thrombosis (dzau ; duprez ) . at -mediated nadph oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis (li et al ; mehta et al ) . ang ii also activates gene transcription factors involved in vascular inflammation and remodelling (oettgen ) . ang ii and its metabolite ang iv may promote thrombosis by stimulating plasminogen activator inhibitor type (pai- ) and pai- production by the vasculature (van leeuwen et al ; feener et al ; kerins et al ) . additionally, ang ii may promote thrombosis by activation of nuclear factor b-dependent proinflammatory genes and accelerating vascular expression of tissue factor (dielis et al ) . ang ii stimulates endothelin release (kohno et al ; moreau et al ) and endothelin blockade prevents some of the cardiovascular actions of ang ii (webb et al ; rajagopalan et al ; herizi et al ) . ang-( - ) is a biologically active peptide (ferrario et al ) . the main pathway of ang-( - ) formation is by cleavage of ang i by neutral endopeptidase (nep, endopeptidase . ) (yamamoto et al ; duncan et al ) (fig. ). ang-( - ) may also be formed by ace cleavage of ang ii, but the significance of this pathway remains to be established. many actions of ang-( - ) are contrary to those of ang ii, and ang-( - ) is proposed to function as a counter-regulatory hormone in blood pressure control, and in other cardiovascular actions of ang ii. ang-( - ) reduces blood pressure and produces endothelium-dependent vasodilatation (benter et al ; pörsti et al ; benter et al ; nakamoto et al ; brosnihan et al ; le tran et al ) , actions that may be due in part to potentiation by ang-( - ) of the hypotensive effects of kinins (paula et al ; lima et al ) and/or to stimulation of vascular prostaglandin production (benter et al ; paula et al ) . in support of a role for kinin-mediated nitric oxide production in its vasodilator effects, ang-( - ) induced vasodilatation and hypotension were attenuated by nitric oxide synthase (nos) inhibition (pörsti et al ; gorelik et al ) , by the type bradykinin (b ) receptor antagonist icatibant (pörsti et al ; abbas et al ; lima et al ; gorelik et al ) , and also by at receptor antagonism (lima et al ) . moreover, ang-( - ) stimulation of nitric oxide release from coronary vessels was blocked by icatibant (brosnihan et al ) . high concentrations of ang-( - ) inhibit ace, leading to the suggestion that ang-( - ) potentiates the effects of bradykinin through ace inhibition (li et al ) . however, the ic for ang-( - ) inhibition of ace was nmol/l and it is unlikely endogenous ang-( - ) levels would be sufficient to produce this effect. ang-( - ), like other ace inhibitors, may potentiate the actions of a b receptor agonist by an indirect mechanism that is independent of bradykinin hydrolysis (deddish et al ) , possibly by sensitisation of the b receptor (marcic et al ) . this mechanism of potentiation of kinin-induced hypotension by ang-( - ) is unlikely to operate in vivo, however, because micromolar concentrations of ang-( - ) were required to produce this effect (deddish et al ) . plasma ang-( - ) levels are less than ang ii levels, except during ace inhibition when ang-( - ) levels increase several-fold, in parallel with the increase in ang i levels (lawrence et al ; menard et al ) . tissue levels of ang-( - ) are very low or undetectable, even with ace inhibition (campbell et al ; ) . there is, therefore, uncertainty whether ang-( - ) levels are sufficient to play a role in cardiovascular physiology and disease states in humans. . . figure shows an outline of the pathways of kinin peptide formation. a proportion of kininogens is hydroxylated on pro of the bradykinin sequence, leading to the formation of hydroxylated kinin peptides. the kininogens are the sole precursors of the kinin peptides and are coded by a single gene. differential splicing of the initial mrna transcript produces two different mrna coding for either high or low molecular weight kininogen. each is a glycoprotein that contains the kinin sequence in its mid portion. tissue kallikrein and plasma kallikrein are both serine proteases. whereas a single gene codes for plasma kallikrein there is a large family of tissue kallikrein genes, although klk is the only tissue kallikrein known to generate kinin peptides (yousef et al ) . kininogens and tissue kallikrein are expressed in many different tissues. plasma kallikrein is predominantly expressed in liver, although recent studies suggest expression of plasma kallikrein in the brain (takano et al ) . in humans, plasma kallikrein forms bradykinin from high molecular weight kininogen, whereas tissue kallikrein forms kallidin from high or low molecular weight kininogens (fig. ) . by contrast, both plasma and tissue kallikrein generate . an outline of the formation of kallidin and bradykinin peptides in humans. a proportion of high molecular weight kininogen is hydroxylated on pro of the bradykinin sequence, giving rise to both hydroxylated and non-hydroxylated peptides. adapted from (campbell ) bradykinin in rodents (bhoola et al ) . bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin. alternative pathways of kinin formation involving enzymes other than kallikreins may operate in disease states. although low molecular weight kininogen is a poor substrate for plasma kallikrein, it will form bradykinin in the presence of neutrophil elastase which, by cleaving a fragment from low molecular weight kininogen, renders it much more susceptible to cleavage by plasma kallikrein (sato et al ) . moreover, the combination of mast cell tryptase and neutrophil elastase releases bradykinin from oxidized kininogens that are resistant to cleavage by kallikreins (kozik et al ) . kinin production in vivo is controlled in part by endogenous inhibitors of the kallikrein enzymes. the main inhibitors of plasma kallikrein are c inhibitor, -macroglobulin and antithrombin iii (bhoola et al ) . an important inhibitor of tissue kallikrein is kallistatin, although the function of kallistatin in vivo is uncertain (chao et al ) . all components of a functional kks are expressed in the heart . the heart and vasculature express tissue kallikrein (oza et al ; xiong et al ; nolly et al ; nolly et al ) . in addition, plasma kallikrein, a member of the contact system, generates bradykinin at the endothelial surface of blood vessels (campbell ) . . dose related effects of the ace inhibitor perindopril on ang ii, ang i, and bradykinin levels in the cardiac ventricles of control rats and rats with myocardial infarction. *, p < . compared to mg/kg per day perindopril. data adapted from (campbell et al ; duncan et al ) kinins act via two types of kinin receptor, the b and the b receptors. the b receptor normally predominates, whereas the b receptor is induced by tissue injury. the kks generates bioactive kinin peptides: bradykinin, hyp bradykinin, kallidin, and hyp -kallidin act on the b receptor, whereas their carboxypeptidase metabolites des-arg -bradykinin, des-arg -hyp -bradykinin, des-arg -kallidin, and des-arg -hyp -kallidin act on the b receptor. hydroxylated kinins have similar biological activity to non-hydroxylated kinins. of particular interest is the recent report that the human b receptor is activated by both plasma and tissue kallikrein (hecquet et al ) . cathepsin g and trypsin similarly activate the b receptor and activation is blocked by icatibant. thus, the b receptor may belong to a new group of serine-protease-activated receptors (hecquet et al ) . ace is one of many enzymes that metabolise kinin peptides (campbell ) and the efficiency of metabolism is an important determinant of their levels in blood and tissues. consequently, inhibition of any single enzyme that contributes to kinin metabolism causes only a modest increase in kinin levels. kinin peptides have a broad spectrum of activities and both systemic and local cardiac actions impact on the heart (bhoola et al ). kinin peptides act through many different second messenger systems, in particular nitric oxide and prostaglandins (bhoola et al ) . the b receptor participates in an inhibitory interaction with endothelial nos (enos) that is reversed by bradykinin (ju et al ) . this interaction may recruit enos to the b receptor and allow for effective coupling of bradykinin signalling to the nitric oxide pathway. kinins are potent vasodilators and promote diuresis and natriuresis. kinins in high concentration also participate in the cardinal features of inflammation, producing vascular permeability, neutrophil chemotaxis and pain (bhoola et al ) . cardiac bradykinin levels are increased during the acute phase of myocardial infarction in rats (duncan et al ) . by contrast, we found decreased kallidin levels in coronary sinus blood of subjects with heart failure, suggesting downregulation of the cardiac kks in heart failure (duncan et al ) . there is a large body of evidence demonstrating anti-hypertrophic and cardioprotective actions of the kks (griol-charhbili et al ; koch et al ; park et al ; spillmann et al ) . the cardioprotective effects of bradykinin included the reduction of arrhythmias, reduction of lactate, lactate dehydrogenase, and creatine kinase release, and increase in myocardial contractility and myocardial levels of glycogen, adenosine triphosphate and creatine phosphate during postischaemic reperfusion of the isolated working rat heart (linz et al ) . moreover, bradykinin suppressed endothelin release from the post-ischaemic rat heart (brunner et al ) . kinins protect against ischaemia-reperfusion injury by decreasing endothelial adherence of leukocytes, leading to attenuation of post-ischaemic leukocyte adherence, attenuation of disruption of the microvascular barrier and reduced tissue injury (shigematsu et al ) . many of the actions of kinins counteract those of ang ii, by causing endothelium-dependent vasodilatation through endothelial release of nitric oxide and prostacyclin (pelc et al ; lamontagne et al ; gallagher et al ) . kinins also counteract the hypertrophic actions of ang ii and reduce collagen formation (gallagher et al ; ritchie et al ) . administration of kinin receptor antagonists indicates a role for endogenous kinins in the regulation of the coronary vasculature and in the myocardial response to myocardial infarction. icatibant reduced flow-dependent vasodilatation of human coronary arteries, indicating a role for kinins in the regulation of coronary vasculature (groves et al ) . icatibant enhanced myocardial interstitial deposition of collagen following myocardial infarction in the rat, indicating a role for endogenous kinins in the modulation of collagen deposition; however, icatibant did not modify morphological and molecular markers of cardiomyocyte hypertrophy (wollert et al ) . kinins participate in the process of ischaemic preconditioning, and have also been shown to limit reperfusion injury (baxter et al ) . kinins may also protect against thrombosis by stimulating endothelial release of nitric oxide, prostacyclin, and tissue plasminogen activator (dielis et al ) . new properties of kinin peptides are being discovered. for example, b receptors may have an important role in angiogenesis (emanueli et al ) . acsdkp is an inhibitor of pluripotent haemopoietic stem cell proliferation (lenfant et al ; bonnet et al ) , and is normally present in human plasma and mononuclear cells (pradelles et al ) . acsdkp is released from its precursor thymosin-ß by prolyl oligopeptidase (cavasin et al ) and it is cleaved to an inactive form by the dipeptidyl carboxypeptidase activity of the n-terminal catalytic domain of ace (rousseau et al ) . acsdkp has a . min half-life in the circulation and is probably released continuously . the importance of ace in acsdkp metabolism is shown by the -fold increase in acsdkp plasma levels that accompany ace inhibition . acsdkp inhibits dna and collagen synthesis by cardiac fibroblasts (rhaleb et al ) , and both prevents and reverses myocardial inflammation and fibrosis in rats with heart failure after myocardial infarction . acsdkp and thymosin-ß stimulate coronary vasculogenesis and angiogenesis (wang et al ; smart et al ) , and acsdkp increases myocardial capillary density in rats with myocardial infarction (wang et al ) . many clinical trials demonstrate the therapeutic benefit of ace inhibition in heart failure and ischaemic heart disease. it is of note, however, that the effects of ace inhibitors are dose related. large clinical trials, by necessity, use only one dose of any drug. the results of such trials are just as much a measure of the effect of the dose as they are a measure of the effect of the drug. use of a less than optimal dose may fail to reveal a drug's true therapeutic potential. this is of particular concern in a head-to-head comparison of two active drugs, where the result may be more due to choice of dose than to choice of drug. clinicians should strive to achieve drug doses that have proven to be of benefit in clinical trials. at present, a large proportion of patients receiving ace inhibitor therapy are receiving less than optimal doses (lenzen et al ) . measurement of plasma ang peptide levels is not feasible for the monitoring of ace inhibitor therapy, but measurement of plasma acsdkp levels may assist in this regard (struthers et al ) . heart failure is associated with neurohormonal activation that includes increased renin, ang ii, and aldosterone levels, and activation of the sympathetic nervous system (francis et al ) . increased ang ii, aldosterone, noradrenaline, and adrenaline levels predict increased mortality in heart failure patients (swedberg et al ) . therapies that counteract the effects of ras and sympathetic nervous system activation are the cornerstone of heart failure therapy (hunt et al ; swedberg et al ) . acute ace inhibition in heart failure patients promotes arterio-and venodilatation, with reduction in both afterload and preload, and an associated increase in cardiac output, stroke volume, and stroke work index, along with a decrease in pulmonary capillary wedge pressure, indicating improved left ventricular (lv) function (gavras et al ; ader et al ) . the cooperative north scandinavian enalapril survival study (consensus) demonstrated reduced mortality and improved symptoms with enalapril therapy in patients with severe heart failure (the consensus trial study group ). moreover, mortality was lower with enalapril therapy than with hydralazine-isosorbide dinitrate therapy in the second veterans administration cooperative vasodilator-heart failure trial (v-heft ii) (cohn et al ) . the studies of left ventricular dysfunction (solvd) confirmed the survival benefits of enalapril therapy in patients with reduced lv ejection fraction and heat failure (the solvd investigators ) and also demonstrated the prevention of heart failure in asymptomatic subjects with reduced lv ejection fraction (the solvd investigators ). ace inhibition improves survival, symptoms, and functional capacity, and reduces hospitalisation in patients with moderate and severe heart failure and lv systolic dysfunction (flather et al ; abdulla et al ) . ace inhibition is recommended as first-line therapy in patients with a reduced lv ejection fraction with or without symptoms, and should be up-titrated to the doses shown to be effective in clinical trials (hunt et al ; swedberg et al ) . although the patients recruited to the consensus, v-heft ii, and sovd studies had reduced lv ejection fraction due most often to ischaemic heart disease, they were enrolled several months or more after a myocardial infarction. studies in rats demonstrated survival advantage of ace inhibitor therapy commenced days after myocardial infarction (pfeffer et al b) . additionally, ace inhibition reduced arterial pressure and total peripheral resistance, attenuated lv remodelling, prevented deterioration in cardiac output and stroke volume index, and prevented the increase in lv volume, lv chamber stiffness and lv end diastolic pressure in rats with myocardial infarction (pfeffer et al a) . these benefits of ace inhibition in rats with myocardial infarction were confirmed in patients. the survival and ventricular enlargement (save) trial showed reduced mortality with ace inhibitor therapy when commenced - days after myocardial infarction in patients with asymptomatic lv dysfunction (pfeffer et al ) . in addition, ace inhibitor therapy reduced the incidence of both fatal and nonfatal major cardiovascular events, including the development of severe heart failure and recurrent myocardial infarction. the benefits of ace inhibitor therapy after myocardial infarction were confirmed in the acute infarction ramipril efficacy (aire) and the trandolapril cardiac evaluation (trace) studies (the acute infarction ramipril efficacy (aire) study investigators ; kober et al ) . the aire study recruited patients - days after myocardial infarction who had shown clinical evidence of heart failure at any time. the trace study recruited patients - days after myocardial infarction who had a lv ejection fraction ≤ %. both the aire and trace studies showed survival advantage with ace inhibitor therapy and the trace study showed less development of severe heart failure. other large clinical trails confirmed the benefits of ace inhibition after myocardial infarction (gissi- gruppo ; isis- collaborative group ) . in addition to mortality benefit and reduction of severe heart failure, ace inhibition after myocardial infarction attenuates lv remodelling, lv enlargement and increase in lv mass, and improves lv ejection fraction after myocardial infarction (pfeffer et al ; sharpe et al ; sogaard et al ; johnson et al ) . by contrast, the consensus ii trial found the commencement of ace inhibitor therapy within hours of myocardial infarction did not improve survival (swedberg et al ) . the failure of ace inhibition to improve outcomes in the consensus ii trial may have been due to its protocol. ace inhibitor treatment was started with intravenous infusion of mg enalaprilat within hours after the onset of chest pain, followed by administration of oral enalapril. intravascular administration of ace inhibitor had a negative inotropic effect in several human studies (foult et al ; haber et al ; zeitz et al ) , although not in another (friedrich et al ) . thus, the failure of ace inhibitor therapy to produce benefit in the consensus ii trial may have been due to the negative inotropic effect of intravenously administered enalaprilat, in addition to its administration within hours of chest pain. current european society of cardiology guidelines recommend the initiation of ace inhibitors after the acute phase of myocardial infarction in patients with signs or symptoms of heart failure, even if transient, to improve survival and to reduce re-infarctions and hospitalisations for heart failure . the hope study was based on emerging evidence that ace inhibition reduced the risk of myocardial infarction in patients with low ejection fraction (pfeffer et al ; yusuf et al ; lonn et al ) . it examined the effects of addition of mg ramipril to standard therapy in patients aged at least years with a history of coronary artery disease, stroke, peripheral vascular disease, or diabetes, plus at least one other cardiovascular risk factor (hypertension, elevated total cholesterol level, low high-density lipoprotein cholesterol level, cigarette smoking, or microalbuminuria). patients were excluded if they had heart failure, were known to have a low ejection fraction, were taking an ace inhibitor or vitamin e, had uncontrolled hypertension or overt nephropathy, or had had a myocardial infarction or stroke within weeks before the study began. during a mean follow-up of years ramipril reduced the primary outcome (composite of myocardial infarction, stroke, or death from cardiovascular causes) from . % to . % (relative risk . , % confidence interval . to . ; p < . ). treatment with ramipril reduced the rates of death from cardiovascular causes and all-cause mortality, myocardial infarction, revascularisation procedures, cardiac arrest, heart failure, and complications related to diabetes. the europa study examined the effects of addition of mg perindopril to standard therapy in patients with previous myocardial infarction, angiographic evidence of coronary heart disease, coronary revascularization, or a positive stress test. past history of heart failure was recorded in . % of subjects, but none had clinical signs of heart failure, with % in new york heart association class i and none in class ii or higher. during a mean follow-up of . years, perindopril reduced the primary outcome (composite of cardiovascular death, non-fatal myocardial infarction, cardiac arrest with successful resuscitation) from . % to . % (relative risk . , % confidence interval . to . ; p< . ). the main contributor to this reduction in the primary outcome was the reduction in non-fatal myocardial infarction. perindopril also reduced the incidence of heart failure requiring hospitalisation. by contrast, the prevention of events with angiotensin converting enzyme inhibition (peace) study failed to show an effect of ace inhibition on its primary endpoint (the peace trial investigators ). the peace study examined the effects of addition of mg trandolapril to standard therapy on cardiovascular events in patients with stable coronary heart disease and preserved lv function. during a median follow-up of . years, trandolapril produced non-statistically significant reductions in the primary endpoint (composite of cardiovascular death, myocardial infarction, and coronary revascularization) from . % to . %, and in cardiovascular death and non-fatal myocardial infarction from . % to . %, although trandolapril reduced hospitalisation or death due to heart failure from . % to . %. participants in the peace study were at lower risk of cardiovascular events than those in the hope and europa studies. the baseline blood pressure of peace participants was less than that of patients in the hope and europa studies, and was similar to the level achieved with active therapy in the hope and europa studies. in addition, peace participants received more intensive management of risk factors than did those in the hope and europa studies, with % of peace participants receiving lipid lowering therapy ( % in hope, % in europa), and % had undergone coronary revascularization before enrollment ( % in hope, % in europa). thus, peace participants had an event rate similar to that of the general population ( . % annualised rate of death), and the more aggressive management of their risk factors may have negated any potential benefit from ace inhibitor therapy. there has been debate about the reasons for the failure of the peace study to show an effect of trandolapril on the primary endpoint (pitt ; fox et al a) . although the dose and type of ace inhibitor may be implicated, the most likely explanation is the low event rate in its relatively low risk population (necessitating the inclusion of revascularisation as part of the primary endpoint), such that the study did not have sufficient statistical power to achieve its aim. the ischemia management with accupril post bypass graft via inhibition of angiotensin converting enzyme (imagine) study similarly showed a lack of benefit from mg quinapril in optimally treated low-risk patients after coronary artery bypass grafting (keuper et al ) . pooled analysis of the hope, europa, and peace trials showed ace inhibition reduced all cause and cardiovascular mortality, non-fatal myocardial infarction, stroke, heart failure, and coronary artery bypass surgery, leading to the recommendation that ace inhibitors be considered in all patients with atherosclerosis (dagenais et al ) . a meta-analysis of the hope, europa, peace, and other studies came to a similar conclusion (al-mallah et al ) . however, the number needed to treat for . years to prevent either one death, one nonfatal myocardial infarction, or one coronary revascularisation procedure was (al-mallah et al ) . current european society of cardiology guidelines state: "ace inhibition is well established in the treatment of heart failure or lv dysfunction and in the treatment of diabetic patients. thus, it is appropriate to consider ace inhibitors for the treatment of patients with stable angina pectoris and co-existing hypertension, diabetes, heart failure, asymptomatic lv dysfunction and post-myocardial infarction. in angina patients without co-existing indications for ace inhibitor treatment the anticipated benefit of treatment (possible absolute risk reduction) should be weighed against costs and risks for side-effects, and the dose and agent used of proven efficacy for this indication" (fox et al b) . et al ) . the systemic effects include the reduction of circulating ang ii and aldosterone levels and the increase in kinin and acsdkp levels. decreased ang ii and increased kinin levels contribute to the reduction of blood pressure by ace inhibition. there is ongoing debate about the extent to which the benefits of ace inhibition are related to blood reduction, as opposed to intrinsic benefits of ace inhibition (sever et al ) . a major contributor to the benefits of ace inhibition in heart failure and ischaemic heart disease may be the reduction in systemic blood pressure, and consequent reduction in heart work. ace inhibition may improve cardiac function by reducing coronary vascular resistance in patients with heart failure, thereby augmenting cardiac blood flow (dietz et al ) . ace inhibition reduces circulating and tissue levels of ang ii in both animals and humans (campbell et al ; duncan et al ; campbell et al ; zeitz et al ) . ace inhibition produced a modest reduction in ang ii levels in europa participants (ceconi et al ) . however, the effects of ace inhibition on ang ii levels can be variable, and depend on the responsiveness of renin secretion . in situations where renin shows little increase in response to ace inhibition, the levels of ang ii and its metabolites show a marked fall, with little change in the levels of ang i and its metabolites. by contrast, a large increase in renin levels in response to ace inhibition also increases the levels of ang i and its metabolites. the increased ang i levels promote ang ii formation by residual uninhibited ace and by serine protease pathways of ang i conversion, thereby buffering any fall in ang ii levels during ace inhibition . improved survival of heart failure patients with ace inhibitor therapy is associated with reduction in ang ii and aldosterone levels (swedberg et al ) . the role of renin in determining the response of ang ii levels to ace inhibition is most evident in heart failure, where many patients continue to have elevated ang ii levels despite ace inhibitor therapy (roig et al ; campbell et al ) . it is of note that maximally recommended doses of ace inhibitor do not completely prevent ace mediated formation of ang ii in heart failure (jorde et al ) . the beneficial therapeutic effects of concomitant ß-blocker therapy in heart failure may be due in part to the associated reduction in renin and ang ii levels (campbell et al ) . the effects of ace inhibitors on ang ii levels are dose dependent (fig. ) . studies in rats showed tissue-specific differences in the dose-related effects of ace inhibition on ang ii levels (campbell ) . renal ang ii levels were reduced by lower doses of ace inhibitor than were required to reduce ang ii levels in other tissues such as the heart (fig. ) . ang-( - ) . this is due in part to the increase in ang i levels, with subsequent conversion to ang-( - ). another mechanism for the increase in ang-( - ) levels during ace inhibition is the inhibition of ang-( - ) metabolism, given that ace is an important pathway of ang-( - ) metabolism yamada et al ) . studies in rats led to the proposal that increased ang-( - ) levels mediate in part the hypotensive effects of ace inhibition (iyer et al a; iyer et al b) . however, there is as yet no evidence that these mechanisms operate in patients receiving ace inhibitor therapy. there is ample evidence that kinin peptides contribute to the therapeutic effects of ace inhibitors (linz et al ; bönner ) . ace inhibitors increase circulating and tissue levels of bradykinin in animals (fig. ) and humans (campbell et al ; duncan et al ; zeitz et al ) . the effect of ace inhibition on kinin peptide levels in any tissue compartment depends on the contribution of ace, relative to other kininases, to kinin peptide metabolism in that compartment. ace inhibitor therapy did not increase either bradykinin or kallidin peptide levels in cardiac atria of patients with ischaemic heart disease, despite the reduction in ang ii levels . the maintenance of low levels of kinin peptides by their efficient metabolism is relevant to the success of ace inhibitor therapy. ace inhibition has only a modest effect on kinin peptide levels because of the many other kininases that contribute to kinin metabolism. it is for this reason that ace inhibitors are generally free of the side effects, such as angioneurotic oedema, that one might expect from increased kinin peptide levels (nussberger et al ; nussberger et al ) . studies with kinin receptor antagonists indicate a role for kinins in the cardiovascular actions of ace inhibitors in animals and humans (linz et al ) . studies in humans indicate a role for the b receptor in flow-dependent vasodilatation in normal volunteers (hornig et al ) and in the hypotensive effects in patients with hypertension (gainer et al ; squire et al ) . a role for the b receptor is indicated in the systemic haemodynamic effects of ace inhibition in patients with heart failure (witherow et al ; cruden et al ) . cardioprotective effects of ace inhibition that were attenuated by icatibant included the reduction of arrhythmias, reduction of lactate, lactate dehydrogenase, and creatine kinase release, and increase in myocardial contractility and myocardial levels of glycogen, adenosine triphosphate and creatine phosphate during reperfusion of the ischaemic isolated working rat heart (linz et al ) . icatibant attenuated the ace inhibitor-induced increase in coronary flow and nitric oxide levels in dogs with myocardial ischaemia (kitakaze et al ) . icatibant also prevented the potentiation of ischaemic preconditioning by ace inhibition in human atria (morris et al ) . the post-ischaemic anti-arrhythmic effect of ace inhibition may be mediated by kinin-induced suppression of endothelin release (brunner et al ) . icatibant prevented the reduction in myocardial infarct size and the reduction in post-infarct remodelling by ace inhibition in animal models (linz et al ; hartman et al ; stauss et al ; mcdonald et al ; hu et al ) . however, a subsequent study in an in vivo canine model of myocardial ischaemic injury did not show an effect of ace inhibition on infarct size (black et al ) . moreover, icatibant did not modify the antihypertrophic effect of ace inhibition in rats with myocardial infarction, although it partially reversed the reduction in myocardial collagen deposition by ace inhibitor therapy in one study (wollert et al ) . possible mechanisms by which kinin peptides mediate the therapeutic benefits of ace inhibition include the promotion of endothelial production of nitric oxide and prostacylin, thereby contributing to the correction of endothelial dysfunction and reduced oxidative stress (linz et al ; bönner ; münzel et al ) . ace inhibition induced endothelial nos (enos) in vasculature of control rats, and attenuated the induction of inducible nos (inos) in rats administered bacterial lipopolysaccharide (bachetti et al ) . icatibant prevented the increase in nitric oxide formation in the heart and reduction in myocardial oxygen consumption that accompany ace inhibition in dogs (zhang et al ) . icatibant also prevented the antiproliferative effect of ace inhibition in neointima formation following endothelial injury to the rat carotid artery (linz et al ) , and the increase in capillary density induced by chronic ace inhibitor treatment in stroke-prone spontaneously hypertensive rats (gohlke et al ) . part of the benefits of ace inhibition may be due to the enhancement of insulin-mediated muscle glucose uptake, that is also attenuated by icatibant (henriksen et al ; henriksen et al ) . ace inhibition also affects the kks by mechanisms separate from prevention of kinin degradation. for example, chronic ace inhibition in mice and rats induced both renal and vascular b receptor expression without modification of b receptor expression (marin-castano et al ) . moreover, enalaprilat and other ace inhibitors in nanomolar concentrations were shown to directly activate the human b receptor, in the absence of ace and b receptor ligands (ignjatovic et al ) . several studies show ace inhibitors may potentiate the effects of bradykinin by a mechanism independent of prevention of kinin metabolism, that involves direct interaction between ace and the b receptor (fleming ) and attenuation of the sequestration of the b receptor (benzing et al ; chen et al ) . additionally, membrane ace appears to have its own signalling cascade that is activated by binding of ace inhibitors (fleming ) . one approach to differentiation of the respective roles of the ras and kks in mediating the therapeutic benefits of ace inhibition is the comparison of ace and arb therapy. comparison of ace inhibitor and arb therapy after myocardial infarction, or in patients with heart failure, did not show any difference in outcomes (pitt et al ; dickstein et al ; pfeffer et al ; mcmurray et al ) . these studies suggest ace inhibitor and arb therapy act through blockade of the ras, but a role for bradykinin cannot be excluded because losartan was shown to increase bradykinin levels in hypertensive humans (campbell et al ) . maximally recommended doses of ace inhibitors do not completely prevent ace mediated formation of ang ii in heart failure (jorde et al ) . combination of ace inhibitor and arb therapy produces more complete blockade of the ras that is dependent on the dose regimens of the individual therapies (menard et al ; azizi et al ) . this combination therapy improves outcomes in heart failure patients (cohn et al ; mcmurray et al ) , but not following myocardial infarction mcmurray et al ) . ace inhibition causes a several-fold increase in acsdkp levels that may contribute to decreased cardiac inflammation and fibrosis, and to increased myocardial capillary density after myocardial infarction (wang et al ; yang et al ) . elevated acsdkp levels during ace inhibitor therapy may also contribute to the anaemia experienced by heart failure patients receiving ace inhibitor therapy (van der meer et al ). heart failure patients have increased plasma aldosterone levels consequent to stimulation of aldosterone secretion by increased ang ii levels (weber ) . evidence that reduced aldosterone levels may contribute to the therapeutic benefits of ace inhibition is the reduced hypokalaemia in patients receiving ramipril therapy in the hope study (mann et al ) . in addition to promotion of sodium retention and oedema formation, aldosterone may promote cardiac fibrosis and deterioration in cardiac function (brilla et al ) . the possible clinical importance of this mechanism is shown by the benefits of aldosterone receptor antagonists in patients with heart failure, and in patients with lv dysfunction after myocardial infarction (pitt et al ; pitt et al ) . many authors have suggested the reduction in sympathetic activity that may accompany ace inhibition is due to a reduction in the stimulation of sympathetic activity by ang ii. however, although ace inhibitor therapy leads to reduction in sympathetic nervous system activity in heart failure, this is thought to be mainly secondary to the improvement of cardiovascular haemodynamics, rather than the specific consequence of reduced stimulation of the sympathetic nervous system by ang ii (esler et al ) . cardiac hypertrophy is well recognised as a risk factor for death and cardiovascular events (levy et al ) . ace inhibitors reduce cardiac hypertrophy in hypertensive patients (dahlof et al ) and also reduce progressive lv remodelling after myocardial infarction (ferrari ) . ventricular remodelling has a dominant role in the pathogenesis of heart failure, and the prevention of remodelling is considered to be an important mechanism of the benefit of ace inhibitor therapy in heart failure and after myocardial infarction (cohn ; abdulla et al ) . reduction of myocardial infarction and other ischaemic events by ace inhibition raises the possibility that these drugs inhibit atherosclerosis. ace inhibitors correct endothelial dysfunction in patients with heart failure and ischaemic heart disease mancini et al ; ceconi et al ) . these effects of ace inhibition may be due to the reduction of oxidative stress, vascular remodelling and inflammation by reduced ang ii levels and increased kinin levels. however, current evidence does not allow these data to be extrapolated to a reduction in atherogenesis by ace inhibition in humans. despite the prevention of atherosclerosis in animal models, ace inhibitor therapy was not able to reduce atherogenesis in patients. ace inhibition with cilazapril did not prevent restenosis after angioplasty (mercator), (mercator study group ; faxon ) . similarly, quinapril did not reduce restenosis after coronary stenting; in fact, late loss in minimum lumen diameter was significantly higher in the quinapril group than in controls (meurice et al ) . additionally, ace inhibition with enalapril failed to reduce progression of coronary atherosclerosis, as assessed by intravascular ultrasound, in patients with coronary artery disease (nissen et al ) . a meta-analysis of randomised controlled studies of the effect of antihypertensive therapies in progression of carotid intima-media thickness showed only a weak, non-significant reduction in progression of carotid intima-media thickness by ace inhibitor therapy, with significant heterogeneity between studies (wang et al ) . some studies showed a reduction in progression of intima-media thickness by ace inhibition and some did not. of note, calcium channel blockers were significantly more effective than ace inhibitors in their reduction of progression of intima-media thickness (wang et al ) . reduced rates of myocardial infarction with ace inhibitor therapy may also be due to an effect of this therapy on the mechanisms of thrombosis and fibrinolysis. ace inhibition reduced plasma levels of pai- antigen and activity in normal subjects on low salt diet and in subjects following myocardial infarction (wright et al ; moriyama et al ; oshima et al ; vaughan et al ; brown et al ; brown et al ) , although this effect of ace inhibition was not confirmed in other studies of patients with previous myocardial infarction (zehetgruber et al ; pedersen et al ) . ace inhibition also reduced pai- antigen, but not pai- activity, in subjects with congestive cardiac failure (goodfield et al ) . diabetes is well recognised to accelerate the processes of cardiovascular disease, and reduction of diabetes incidence may contribute to the therapeutic benefits of ace inhibition. many large clinical trials, including the hope, peace, and solvd studies, showed a reduced incidence of type diabetes with ace inhibitor therapy (abuissa et al ) . however, the diabetes reduction assessment with ramipril and rosiglitazone medication (dream) study found ramipril did not reduce diabetes incidence among persons with impaired fasting glucose levels or impaired glucose tolerance, although it significantly increased regression to normoglycaemia (the dream trial investigators ) . this improvement in insulin resistance may be due in part to the enhancement of insulin-mediated muscle glucose uptake by ace inhibition (henriksen et al ; henriksen et al ) . aortic compliance is an important determinant of coronary blood flow (o'rourke et al ) . a recent meta-analysis showed ace inhibitors decrease arterial stiffness (mallareddy et al ) . ace inhibitors, by increasing aortic compliance, may reduce central systolic blood pressure and maintain diastolic blood pressure, thereby reducing heart work without compromising myocardial perfusion. decrease in arterial stiffness by ace inhibition may be due to reduced collagen deposition, as suggested by studies in spontaneously hypertensive rats (benetos et al ) . reduction of aortic collagen deposition by ace inhibition was not affected by icatibant, suggesting that this effect of ace inhibition was not mediated by kinins (benetos et al ) . atrial fibrillation is an important contributor to poor prognosis in heart failure (wang et al ) , and prevention of atrial fibrillation by ace inhibition may contribute to the therapeutic benefits of this therapy (vermes et al ). given that kinin peptides mediate in part the therapeutic benefits of ace inhibition, and that some of the actions of kinins are mediated by prostaglandins, the question arises whether a drug that inhibits prostaglandin synthesis may attenuate the effects of ace inhibition. this question was addressed in a systematic review of the interaction between aspirin and ace inhibitor therapy (teo et al ) . the solvd study found aspirin prevented the reduction of death by ace inhibition, but this interaction between aspirin and ace inhibitor therapy was not significant in the other trials examined. however, both solvd and the other trials showed aspirin attenuated the prevention of myocardial infarction or reinfarction by ace inhibition. by contrast, there was no evidence that aspirin attenuated the prevention of stroke, hospital admission for heart failure, or revascularisation by ace inhibitor therapy. when the composite of major vascular events including death, myocardial infarction or reinfarction, hospital admission for heart failure, stroke, and revascularisation was examined, aspirin did not significantly attenuate the benefits of ace inhibitor therapy. this analysis shows, therefore, that aspirin does interact with ace inhibitor therapy, at least in the case of myocardial infarction. however, in the absence of clear contraindications, concomitant use of aspirin and ace inhibitors should be considered in all patients at high risk of major vascular events (teo et al ) . ace inhibitors have a major role in the treatment and prevention of heart failure and ischaemic heart disease. reduction in ang ii levels, and increase in kinin and acsdkp levels, are implicated in the mechanisms of the therapeutic effects of ace inhibitors. much of the detail of these mechanisms, however, remains to be discovered. azizi, m., and menard, j., , combined blockade of the renin-angiotensin system with angiotensinconverting enzyme inhibitors and angiotensin ii type receptor antagonists. circulation, : - . bachetti, t., comini, l., pasini, e., cargnoni, a., curello, s., and ferrari, r., , ace- esler, m., and kaye, d., , ß-blockers, angiotensin ii, and ace inhibitors in patients with heart failure. lancet, : - . swedberg, k., held, p., kjekshus, j., rasmussen, k., rydén, l., and wedel, h., , effects of the early administration of enalapril on mortality in patients with acute myocardial infarction-results of the cooperative new scandinavian enalapril survival study ii (consensus ii). n engl j med, : - . takano, m., horie, m., narahara, m., miyake, m., and okamoto, h., , expression of kininogen mrnas and plasma kallikrein mrna by cultured neurons, astrocytes and meningeal cells in the rat brain. immunopharmacology, : - . teo, k.k., yusuf, s., pfeffer, m., torp-pedersen, c., kober, l., hall, a., pogue, j., latini, r., and collins, r., , effects of long-term treatment with angiotensin-converting-enzyme inhibitors in the presence or absence of aspirin: a systematic review. lancet, : - . the acute infarction ramipril efficacy (aire) study investigators, , effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure. angiotensin-( - ) induces bradykininmediated hypotensive responses in anesthetized rats a meta-analysis of the effect of angiotensin-converting enzyme inhibitors on functional capacity in patients with symptomatic left ventricular systolic dysfunction a systematic review: effect of angiotensin converting enzyme inhibition on left ventricular volumes and ejection fraction in patients with a myocardial infarction and in patients with left ventricular dysfunction angiotensin-converting enzyme inhibitors or angiotensin receptor blockers for prevention of type diabetes: a meta-analysis of randomized clinical trials immediate and sustained hemodynamic and clinical improvement in chronic heart failure by an oral angiotensinconverting enzyme inhibitor angiotensin-converting enzyme inhibitors in coronary artery disease and preserved left ventricular systolic function: a systematic review and meta-analysis of randomized controlled trials high plasma level of n-acetylseryl-aspartyl-lysyl-proline -a new marker of chronic angiotensin-converting enzyme inhibition effect of reduced angiotensin-converting enzyme gene expression and angiotensin-converting enzyme inhibition on angiotensin and bradykinin peptide levels in mice angiotensin converting enzyme inhibition modifies angiotensin, but not kinin peptide levels in human atrial tissue nephrectomy, converting enzyme inhibition and angiotensin peptides effects of converting enzyme inhibitors on angiotensin and bradykinin peptides losartan increases bradykinin levels in hypertensive humans evidence against a major role for angiotensin converting enzyme-related carboxypeptidase (ace ) in angiotensin peptide metabolism in the human coronary circulation role of the angiotensin at receptor in blood pressure regulation and therapeutic implications prolyl oligopeptidase is involved in release of the antifibrotic peptide ac-sdkp. hypertension ace inhibition with perindopril and endothelial function. results of a substudy of the europa study: pertinent kallistatin, a novel human tissue kallikrein inhibitor: levels in body fluids, blood cells, and tissues in health and disease metabolism of angiotension-( - ) by angiotensin-converting enzyme human ace and bradykinin b receptors form a complex at the plasma membrane structural basis for heart failure. ventricular remodeling and its pharmacological inhibition a comparison of enalapril with hydralazineisosorbide dinitrate in the treatment of chronic congestive heart failure a randomized trial of the angiotensin-receptor blocker valsartan in chronic heart failure angiotensin-converting enzyme is an essential regulator of heart function bradykinin contributes to the systemic hemodynamic effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure angiotensin-converting-enzyme inhibitors in stable vascular disease without left ventricular systolic dysfunction or heart failure: a combined analysis of three trials reversal of left ventricular hypertrophy in hypertensive patients. a metaanalysis of treatment studies cardiac renin and angiotensins: uptake from plasma versus in situ synthesis angiotensin ii and the heart : on the intracrine renin-angiotensin system n-domainspecific substrate and c-domain inhibitors of angiotensin-converting enzyme angiotensin-( - ) and keto-ace dissecting the role of chymase in angiotensin ii formation and heart and blood vessel diseases effects of losartan and captopril on mortality and morbidity in high-risk patients after acute myocardial infarction: the optimaal randomised trial the prothrombotic paradox of hypertension: role of the renin-angiotensin and kallikrein-kinin systems improvement of cardiac function by angiotensin converting enzyme inhibition: sites of action a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - the cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function? effect of chronic angiotensinconverting enzyme inhibition on endothelial function in patients with chronic heart failure effects of angiotensin converting enzyme inhibition on angiotensin and bradykinin peptides in rats with myocardial infarction kinins in humans angiotensin and bradykinin peptides in rats with myocardial infarction interaction between neutral endopeptidase and angiotensin converting enzyme inhibition in rats with myocardial infarction: effects on cardiac hypertrophy and angiotensin and bradykinin peptide levels role of the renin-angiotensin-aldosterone system in vascular remodeling and inflammation: a clinical review tissue angiotensin and pathobiology of vascular disease: a unifying hypothesis angiotensin-converting enzyme: biochemistry and molecular biology targeting kinin b receptor for therapeutic neovascularization angiotensin i converting enzyme and the changes in our concepts through the years does the renin-angiotensin system exert an important stimulatory influence on the sympathetic nervous system? effect of high dose angiotensin-converting enzyme inhibition on restenosis: final results of the marcator study, a multicenter, double-blind, placebo-controlled trial of cilazapril angiotensin ii induces plasminogen activator inhibitor- and - expression in vascular endothelial and smooth muscle cells effects of angiotensin-converting enzyme inhibition with perindopril on left ventricular remodeling and clinical outcome: results of the randomized perindopril and remodeling in elderly with acute myocardial infarction (preami) study a new hormone of the angiotensin system long-term ace-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients signaling by the angiotensin-converting enzyme direct myocardial and coronary effects of enalaprilat in patients with dilated cardiomyopathy: assessment by a bilateral intracoronary infusion technique should angiotensin-converting enzyme-inhibitors be used to improve outcome in patients with coronary artery disease and 'preserved' left ventricular function? guidelines on the management of stable angina pectoris: executive summary: the task force on the management of stable angina pectoris of the european society of cardiology plasma norepinephrine, plasma renin activity, and congestive heart failure: relations to survival and the effects of therapy in v-heft ii intracardiac angiotensin-converting enzyme inhibition improves diastolic function in patients with left ventricular hypertrophy due to aortic stenosis effect of bradykininreceptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive and hypertensive subjects bradykinin-induced reductions in collagen gene expression involve prostacyclin angiotensin converting enzyme inhibition in patients with congestive heart failure gissi- : effects of lisinopril and transdermal glyceryl trinitrate singly and together on -week mortality and ventricular function after acute myocardial infarction blockade of bradykinin b receptors prevents the increase in capillary density induced by chronic angiotensin-converting enzyme inhibitor treatment in stroke-prone spontaneously hypertensive rats effects of acute angiotensin ii type receptor antagonism and angiotensin converting enzyme inhibition on plasma fibrinolytic parameters in patients with heart failure angiotensin - induces bradykinin-mediated relaxation in porcine coronary artery role of tissue kallikrein in the cardioprotective effects of ischemic and pharmacological preconditioning in myocardial ischemia role of endogenous bradykinin in human coronary vasomotor control altered blood pressure responses and normal cardiac phenotype in ace -null mice intracoronary angiotensin-converting enzyme inhibition improves diastolic function in patients with hypertensive left ventricular hypertrophy tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme reduction of myocardial infarct size in rabbits by ramiprilat: reversal by the bradykinin antagonist hoe human bradykinin b( ) receptor is activated by kallikrein and other serine proteases glucose transport activity in insulinresistant rat muscle: effects of angiotensin-converting enzyme inhibitors and bradykinin antagonism ace inhibition and glucose transport in insulinresistant muscle: roles of bradykinin and nitric oxide prevention of the cardiovascular and renal effects of angiotensin ii by endothelin blockade tissue-specific activation of cardiac angiotensin converting enzyme in experimental heart failure angiotensin converting enzyme: implications from molecular biology for its physiological functions role of bradykinin in mediating vascular effects of angiotensin-converting enzyme inhibitors in humans chronic effects of early started angiotensin converting enzyme inhibition and angiotensin at -receptor subtype blockade in rats with myocardial infarction: role of bradykinin acc/aha guidelines for the evaluation and management of chronic heart failure in the adult: executive summary. a report of the american college of cardiology/american heart association task force on practice guidelines (committee to revise the guidelines for the evaluation and management of heart failure) novel mode of action of angiotensin i converting enzyme inhibitors: direct activation of bradykinin b receptor isis- : a randomised factorial trial assessing early oral captopril, oral mononitrate, and intravenous magnesium sulphate in patients with suspected acute myocardial infarction vasodepressor actions of angiotensin-( - ) unmasked during combined treatment with lisinopril and losartan angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system differences in the properties and enzymatic specificities of the two active sites of angiotensin i-converting enzyme (kininase ii). studies with bradykinin and other natural peptides angiotensin-converting enzyme inhibitor therapy affects left ventricular mass in patients with ejection fraction > % after acute myocardial infarction maximally recommended doses of angiotensin-converting enzyme (ace) inhibitors do not completely prevent ace-mediated formation of angiotensin ii in chronic heart failure inhibitory interactions of the bradykinin b receptor with endothelial nitric-oxide synthase determinants of angiotensin ii generation during converting enzyme inhibition angiotensin induction of pai- expression in endothelial cells is mediated by the hexapeptide angiotensin iv hotline sessions of the th european congress of cardiology angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers synergistically increase coronary blood flow in canine ischemic myocardium: role of bradykinin a clinical trial of the angiotensin-converting-enzyme inhibitor trandolapril in patients with left ventricular dysfunction after myocardial infarction cardiac function and remodeling is attenuated in transgenic rats expressing the human kallikrein- gene after myocardial infarction angiotensin ii stimulates endothelin- secretion in cultured rat mesangial cells a novel mechanism for bradykinin production at inflammatory sites. diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens prostacyclin and nitric oxide contribute to the vasodilator action of acetylcholine and bradykinin in the intact rabbit coronary bed an alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal-directed antisera: measurement of eight angiotensin peptides in human plasma angiotensin-( - ) and the rat aorta: modulation by the endothelium inhibitor of hematopoietic pluripotent stem cell proliferation: purification and determination of its structure under-utilization of evidence-based drug treatment in patients with heart failure is only partially explained by dissimilarity to patients enrolled in landmark trials: a report from the euro heart survey on heart failure prognostic implications of echocardiographically determined left ventricular mass in the framingham study endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology angiotensin-( - ) augments bradykinin-induced vasodilation by competing with ace and releasing nitric oxide potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-( - ) in normotensive and hypertensive rats role of bradykinin in the cardiac effects of angiotensin-converting enzyme inhibitors contribution of kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors emerging role of angiotensin-converting enzyme inhibitors in cardiac and vascular protection effect of angiotensin-converting enzyme inhibitors on arterial stiffness in hypertension: systematic review and meta-analysis angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease -the trend serum potassium, cardiovascular risk, and effects of an ace inhibitor: results of the hope study enhancement of bradykinin and resensitization of its b receptor induction of functional bradykinin b( )-receptors in normotensive rats and mice under chronic angiotensin-converting enzyme inhibitor treatment bradykinin antagonism inhibits the antigrowth effect of converting enzyme inhibition in the dog myocardium after discrete transmural myocardial necrosis the effect of valsartan, captopril, or both on atherosclerotic events after acute myocardial infarction: an analysis of the valsartan in acute myocardial infarction trial (valiant) effects of candesartan in patients with chronic heart failure and reduced left-ventricular systolic function taking angiotensin-converting-enzyme inhibitors: the charm-added trial angiotensin ii cell signaling: physiological and pathological effects in the cardiovascular system synergistic effects of ace inhibition and ang ii antagonism on blood pressure, cardiac weight, and renin in spontaneously hypertensive rats does the new angiotensin converting enzyme inhibitor cilazapril prevent restenosis after percutaneous transluminal coronary angioplasty? results of the mercator study: a multicenter, randomized, double-blind placebo-controlled trial effect of ace inhibitors on angiographic restenosis after coronary stenting (paris): a randomised, double-blind reactive hyperreninemia is a major determinant of plasma angiotensin ii during ace inhibition angiotensin ii increases tissue endothelin and induces vascular hypertrophy -reversal by et a -receptor antagonist captopril reduced plasminogen activator inhibitor activity in patients with acute myocardial infarction angiotensin-converting enzyme inhibitors potentiate preconditioning through bradykinin b receptor activation in human heart are ace inhibitors a "magic bullet" against oxidative stress? circulation angiotensin-( - ) and nitric oxide interaction in renovascular hypertension effect of antihypertensive agents on cardiovascular events in patients with coronary disease and normal blood pressure: the camelot study: a randomized controlled trial a local kallikrein-kinin system is present in rat hearts the kallikrein-kinin system in cardiac tissue plasma bradykinin in angio-oedema bradykinin-mediated angioedema arterial stiffness regulation of vascular inflammation and remodeling by ets factors angiotensin type receptors are reexpressed by cardiac fibroblasts from failing myopathic hamster hearts and inhibit cell growth and fibrillar collagen metabolism the effects of the angiotensin-converting enzyme inhibitor imidapril on plasma plasminogen activator inhibitor activity in patients with acute myocardial infarction rat aortic smooth muscle cells in culture express kallikrein, kininogen, and bradykininase activity bradykinin prevents reperfusion injury by targeting mitochondrial permeability transition pore through glycogen synthase kinase beta physiology of local renin-angiotensin systems angiotensin-( - ) potentiates the hypotensive effect of bradykinin in conscious rats does long-term angiotensin converting enzyme inhibition affect the concentration of tissue-type plasminogen activator plasminogen activator inhibitor- in the blood of patients with a previous myocardial infarction mechanism of coronary vasodilation produced by bradykinin influence of chronic captopril therapy on the infarcted left ventricle of the rat effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. results of the survival and ventricular enlargement trial effect of captopril on progressive ventricular dilatation after anterior myocardial infarction valsartan, captopril, or both in myocardial infarction complicated by heart failure, left ventricular dysfunction, or both survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril ace inhibitors for patients with vascular disease without left ventricular dysfunction-may they rest in peace? effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: randomised trialthe losartan heart failure survival study elite ii eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction the effect of spironolactone on morbidity and mortality in patients with severe heart failure. randomized aldactone evaluation study investigators release of nitric oxide by angiotensin-( - ) from porcine coronary endothelium: implications for a novel angiotensin receptor negative regulator of pluripotent hematopoietic stem cell proliferation in human white blood cells and plasma as analysed by enzyme immunoassay role for endothelin- in angiotensin ii-mediated hypertension myocardial angiotensin receptors in human hearts effect of n-acetyl-seryl-aspartyl-lysyl-proline on dna and collagen synthesis in rat cardiac fibroblasts involvement of human plasma angiotensin i-converting enzyme in the degradation of the haemoregulatory peptide n-acetyl-seryl-aspartyl-lysyl-proline bradykinin blocks angiotensin ii-induced hypertrophy in the presence of endothelial cells clinical implications of increased plasma angiotensin ii despite ace inhibitor therapy in patients with congestive heart failure the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme mechanism of kinin release from human low-molecular-masskininogen by the synergistic action of human plasma kallikrein and leukocyte elastase blood pressure reduction is not the only determinant of outcome early prevention of left ventricular dysfunction after myocardial infarction with angiotensin-converting-enzyme inhibition bradykinin prevents postischemic leukocyte adhesion and emigration and attenuates microvascular barrier disruption thymosin beta induces adult epicardial progenitor mobilization and neovascularization effects of captopril on ischemia and dysfunction of the left ventricle after myocardial infarction cardioprotective mechanisms of the kallikrein-kinin system in diabetic cardiopathy bradykinin b( ) receptor antagonism attenuates blood pressure response to acute angiotensin-converting enzyme inhibition in normal men angiotensin-converting enzyme inhibition in infarct-induced heart failure in rats: bradykinin versus angiotensin ii nonadherence with angiotensin-converting enzyme inhibitor therapy: a comparison of different ways of measuring it in patients with chronic heart failure increased angiotensin-i converting enzyme gene expression in the failing human heart. quantification by competitive rna polymerase chain reaction renin expression at sites of repair in the infarcted rat heart guidelines for the diagnosis and treatment of chronic heart failure: executive summary (update ): the task force for the diagnosis and treatment of chronic heart failure of the european society of cardiology hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality ventricular dysfunction: insight from the studies of left ventricular dysfunction (solvd) trials. circulation hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase interaction between at and at receptors during postinfarction left ventricular remodeling n-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo carotid intima-media thickness and antihypertensive treatment: a meta-analysis of randomized controlled trials temporal relations of atrial fibrillation and congestive heart failure and their joint influence on mortality: the framingham heart study the endothelin receptor antagonist, bq- , inhibits angiotensin ii-induced contractions in rabbit aorta aldosterone in congestive heart failure the two homologous domains of human angiotensin i-converting enzyme are both catalytically active the two homologous domains of human angiotensin i-converting enzyme interact differently with competitive inhibitors bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure differential effects of kinins on cardiomyocyte hypertrophy and interstitial collagen matrix in the surviving myocardium after myocardial infarction in the rat effects of captopril therapy on endogenous fibrinolysis in men with recent, uncomplicated myocardial infarction identification, purification, and localization of tissue kallikrein in rat heart converting enzyme determines plasma clearance of angiotensin-( - ) in vivo metabolism of angiotensin i by neutral endopeptidase (ec . . . ) in spontaneously hypertensive rats ac-sdkp reverses inflammation and fibrosis in rats with heart failure after myocardial infarction the new human tissue kallikrein gene family: structure, function, and association to disease effect of enalapril on myocardial infarction and unstable angina in patients with low ejection fractions the aceinhibitor lisinopril affects plasma insulin levels but not fibrinolytic parameters myocardial uptake and biochemical and hemodynamic effects of ace inhibitors in humans ace inhibitors promote nitric oxide accumulation to modulate myocardial oxygen consumption - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace this work was supported by a senior research fellowship from the national health and medical research council of australia (id ), and by the national heart foundation of australia (id g m ). key: cord- - yo ni authors: gralinski, michael; neves, liomar a. a.; tiniakova, olga title: methods to induce cardiac hypertrophy and insufficiency date: - - journal: drug discovery and evaluation: pharmacological assays doi: . / - - - - _ sha: doc_id: cord_uid: yo ni animal models of cardiac hypertrophy and insufficiency have been reviewed by hasenfuss ( ), muders and elsner ( ), vanoli et al. ( ), patten and hall-porter ( ), dubi and arbel ( ), gomes et al. ( ), and szymanski et al. ( ). male sprague dawley rats weighing - g are fasted h before surgery. anesthesia is induced by i.p. injection of mg/kg hexobarbital. the abdomen is shaved, moistened with a disinfectant, and opened by a cut parallel to the linea alba. the intestine is moistened with saline and placed in a plastic cover to prevent desiccation. the aorta is prepared free from connective tissue above the left renal artery and underlaid with a silk thread. then, a cannula no. ( .  mm) is placed longitudinally to the aorta and both aorta and cannula are tied. the cannula is removed, leaving the aortic lumen determined by the diameter of the cannula. the intestine is placed back into the abdominal cavity with the application of . mg rolitetracycline (reverin). in shamoperated controls, no banding is performed. the skin is closed by clipping. the animals are treated once daily over a period of weeks with doses of the ace inhibitor or other antihypertensive drugs found previously effective to lower blood pressure in rats. at the end of the experiment, blood pressure is measured under hexobarbital anesthesia ( mg/kg i.p.) via indwelling catheters in the left carotid artery. blood pressure measurement in conscious rats with the conventional tail-cuff method is not possible due to the large pressure difference across the ligature. therefore, only one measurement at the end of the study is possible. the hearts are removed, rinsed in saline until free of blood, and gently blotted to dryness. total cardiac mass is determined by weighing on an electronic balance to the nearest . mg. the atria and all adjacent tissues are trimmed off, and the weight of the left ventricle including the septum as well as the remaining cardiac tissue representing the right ventricle is determined separately. weights are calculated per g body weight. the total cardiac mass and weight of left and right ventricle of treated rats are compared with operated controls and sham-operated controls. uetmasu et al. ( ) described a simple method for producing graded aortic insufficiencies in rats and subsequent development of cardiac hypertrophy. selective perforation of the right cup of the aortic valve or in combination with that of the left valve cup was performed using a plastic rod inserted from the right common carotid artery. hypertrophy of the heart, but no hypertension or cardiac insufficiency, was observed. similar methods were used by yamazaki et al. ( ) to study the alterations of cardiac adrenoceptors and calcium channels subsequent to aortic insufficiency, by umemura et al. ( ) to study baroreflex and β-adrenoceptor function, and by ishiye et al. ( ) to study the effects of an angiotensin ii antagonist on the development of cardiac hypertrophy due to volume overload. hyperplastic growth response of vascular smooth muscle cells in the thoracic aorta was found following induction of acute hypertension in rats by aortic coarctation by owens and reidy ( ) . changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with aortic banding were studied by feldman et al. ( ) . muders et al. ( ) produced aortic stenosis in rats by placing a silver clip (inner diameter . mm) on the ascending aorta. schunkert et al. ( ) studied alteration of growth responses in established cardiac pressureoverload hypertrophy in rats with aortic banding. prevention of cardiac hypertrophy after aortic banding by ace inhibitors probably mediated by bradykinin could be shown (linz et al. (linz et al. , a (linz et al. , b, (linz et al. , linz and schölkens ; schölkens et al. ; weinberg et al. ) . weinberg et al. ( ) studied the effect of angiotensin at receptor inhibition on hypertrophic remodeling and ace expression in rats with pressure-overload hypertrophy due to ascending aortic stenosis. molina et al. ( ) described a novel experimental model of pressure-overload hypertrophy in young sprague dawley rats created by placing a small titanium clip (internal diameter - . mm) in the aorta proximal to the right brachiocephalic artery. a decrease of % in fs was observed - weeks after aortic constriction. increased expression of β-myosin heavy chain, atrial natriuretic peptide, interleukin- , interleukin- , and tnf-α was also reported. bruckschlegel g, holmer sr, jandeleit k, grimm d, muders f, kromer ep, riegger ga, schunkert h ( ) blockade of the renin-angiotensin system in cardiac pressureoverload hypertrophy in rats. hypertension : - feldman am, weinberg eo, ray pe, lorell bh ( ) selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with aortic banding. circ res : - gohlke p, stoll m, lamberty v, mattfeld t, mall g, van even p, martorana p, unger t ( ) cardiac and vascular effects of chronic angiotensin converting enzyme inhibition at subantihypertensive doses. j hypertens (suppl ):s -s ishiye m, umemura k, uematsu t, nakashima m ( ) effects of losartan, an angiotensin ii antagonist, on the development of cardiac hypertrophy due to volume overload. biol pharm bull : - linz w, schölkens ba ( ) a specific b -bradykinin receptor antagonist hoe abolishes the antihypertrophic effect of ramipril. br j pharmacol : - linz w, schölkens bw, ganten d ( ) converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats. clin exp hypertens a ( ): - linz w, henning r, schölkens ba ( ) role of angiotensin ii receptor antagonism and converting enzyme inhibition in the progression and regression of cardiac hypertrophy in rats. j hypertens (suppl ):s -s linz w, schaper j, wiemer g, albus u, schölkens bw ( a) ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. br j pharmacol : - linz w, wiemer g, schölkens ba ( b) contribution of bradykinin to the cardiovascular effects of ramipril. j cardiovasc pharmacol (suppl ):s -s linz w, wiemer g, schölkens ba ( ) bradykinin prevents left ventricular hypertrophy in rats. j hypertens (suppl ):s -s linz w, wiemer g, gohlke p, unger t, schölkens ba ( ) the contribution of bradykinin to the cardiovascular actions of ace inhibitors. in: lindpaintner k, ganten d (eds) the cardiac renin angiotensin system. futura publishing company, armonk, pp - linz w, wiemer g, schmidts hl, ulmer w, ruppert d, schölkens ba ( ) scholkens ba, linz w, martorana pa ( ) experimental cardiovascular benefits of angiotensin-converting enzyme inhibitors: beyond blood pressure reduction. j cardiovasc pharmacol (suppl ):s -s schunkert t, weinberg eo, bruckschlegel g, riegger aj, lorell bh ( ) alteration of growth responses in established cardiac pressure overload hypertrophy in rats with aortic banding. j clin invest : - uetmatsu t, yamazaki t, matsuno h, hayashi y, nakashima m ( ) a simple method for producing graded aortic insufficiencies in rats and subsequent development of cardiac hypertrophy. j pharmacol methods : - umemura k, zierhut w, quast u, hof rp ( ) baroreflex and β-adrenoceptor function are diminished in rat cardiac hypertrophy due to volume overload. basic res cardiol : - weinberg eo, schoen fj, george d, kagaya y, douglas ps, litwin se, schunkert h, benedict cr, lorell bh ( ) angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis. circulation : - weinberg eo, lee ma, weigner m, lindpaintner k, bishop sp, benedict cr, ho kkl, douglas ps, chafizadeh e, lorell bh ( ) angiotensin at receptor inhibition: effects on hypertrophic remodelling and ace expression in rats with pressure-overload hypertrophy due to ascending aortic stenosis. circulation : - yamazaki t, uematsu t, mizuno a, takiguchi y, nakashima m ( ) alterations of cardiac adrenoceptors and calcium channels subsequent to production of aortic insufficiency in rats. arch int pharmacodyn ther : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] chronic heart failure in rats purpose and rationale rat models of heart failure were reviewed by . chronic heart failure can be induced in rats by occlusion of coronary arteries. one of the first reports was by . more recent reports are by pfeffer et al. ( ) , hodsman et al. ( ) , van veldhuisen et al. ( , kajstura et al. ( ) , gómez et al. ( ) , liu et al. ( a, b) , and jadavo et al. ( ) . itter et al. ( ) described a model of chronic heart failure (chf) in spontaneously hypertensive rats. adult male -month-old shr/nhsd and wky/nhsd rats (harlan sprague dawley, winkelmann, germany) weighing - g were used. cardiovascular failure was induced by permanent ( weeks) occlusion of the left coronary artery mm distal to the origin from the aorta resulting in a large infarction of the free left ventricular wall. eight weeks after surgery, parameters indicating chf were measured. cardiac hypertrophy, function, and geometric properties were determined by the "working heart" mode and in vivo determinations by mri and heart weight. hydroxyproline/proline ratio was measured as an indicator of heart fibrosis. the rats were anesthetized with a mixture of ketamine/xylazine ( / mg/kg) i.p. the left ventrolateral thorax was shaved and prepared to create a disinfected surgical access area. when a stable anesthesia was achieved, the animals were placed on a small animal operation table, intubated, and ventilated with room air using a small animal ventilator hugo sachs elektronik, germany) . the level of anesthesia was deemed as adequate following loss of the pedal withdrawal reflex and absence of the palpebral reflex. the tidal volume was adjusted at - ml and the ventilation rate was breaths/ min. left thoracotomy was performed via the third intercostal space. the heart was exposed and the pericardium opened. the left main coronary artery was ligated with perma-hand silk - usp (ethicon, norderstedt, germany) near its origin at the aorta ( mm distal to the edge of the left atrium). ligation resulted in infarction of the free left ventricular wall. ligation was deemed successful when the anterior wall of the left ventricle turned pale. at this point, the lungs were hyperinflated by increasing the positive end-expiratory pressure, and the chest was closed. the rats were placed on a heating pad. they were continuously monitored until they start moving in their cages. to avoid ventricular arrhythmias, lidocaine ( mg/kg i.m.) was given before surgery. the sham procedure consisted of opening the pericardium and placing a superficial suture in the epicardium of the lv. to prevent acute lung edema, the rats received furosemide mg/kg twice daily for days via the drinking water. measurements at the end of the study before killing the animals weeks after mi, noninvasive sequential nuclear magnetic resonance (nmr) measurements of heart geometric properties were done. thereafter the animals were anesthetized with pentobarbitone ( mg/kg i.p.) and subsequently heparinized (heparin sodium iu/ g body weight i.p.). once stable anesthesia was achieved (stage iii , reflexes absent), the animals were connected to an artificial respirator via a pe tube inserted into the trachea and ventilated with room air. the right carotid artery was cannulated with a polyethylene catheter to monitor mean blood pressure, systolic blood pressure, diastolic blood pressure, and heart rate over a stable time course of min. a transverse laparotomy and a right anterolateral thoracotomy were performed, and the heart was rapidly removed for the evaluation of its function in the working heart mode. thereafter the heart weight, and the left and right ventricular weights were determined. for infarct size determination, the left ventricle was sectioned transversely into four slices from the apex to the base. the infarct size was determined by planimetry and expressed as a percentage of lv mass. lung weight and further lung histology sections were evaluated. hydroxyproline/proline ratio was determined in paraffin-embedded slices of the left ventricle. the animals were monitored by mri at day and day post-mi. the rats were anesthetized with a mixture of % halothane and / n o/oxygen with a specially manufactured rat mask. the fully anesthetized rats (phase iii) were placed on a cradle made of plexiglas in a supine position. respiration and ecg were monitored continuously. mri experiments were performed according to rudin et al. ( ) . the images were acquired by a spin-echo sequence se ( / ), the field of view was mm, and the image resolution was  pixels with a dimension of .  . mm. four adjacent transverse slices were recorded; slice thickness was . mm. before the acquisition of data, a coronary pilot scan was measured for adequate positioning of the transverse slices. mri data acquisition was gated to the cardiac cycle by a physiograd sm mr monitoring system (bruker, karlsruhe, germany). two sets of transverse images were acquired, one at end-systole and another at end-diastole. end-diastole was defined as the image obtained ms after the onset of the r wave of the ecg, corresponding to the largest cavity area. end-systole was defined as the image with the smallest lv cavity area. the image analysis was done using bruker software (karlsruhe, germany). the parameters of left ventricular end-diastolic volume (lvedv), left ventricular end-systolic volume (lvesv), septum size, infarct size, ejection fraction (ef), left ventricular chamber diameter (r), and circumference were measured. ef was estimated in percentage terms by the subtraction of lvesv from lvedv. after the procedure, the rats were ventilated with oxygen, the mask was replaced, and they were brought back into their cages. they were monitored until they started moving in the cage. blood pressure/heart rate the animals were anesthetized with pentobarbitone ( mg/kg i.p.) and subsequently heparinized (heparin sodium iu/ g body weight i.p.). once stable anesthesia was achieved, the animals were connected to an artificial respirator via a pe tube inserted into the trachea and ventilated with room air. the right carotid artery was cannulated with a polyethylene catheter. the catheter was connected to a plugsys measuring system (hugo sachs elektronik, march-hugstetten, germany) to monitor mean blood pressure, systolic blood pressure, diastolic blood pressure, and heart rate over a stable time course of min. for the final investigations, the heart of the anesthetized rat was rapidly removed and immersed in physiological buffer chilled to c. the aorta was dissected free and mounted onto a cannula (internal diameter: . mm) attached to a perfusion apparatus. the hearts were perfused according to the method of langendorff with an oxygenated ( % o / % co ) noncirculating krebs-henseleit solution of the following compositions (mm): nacl, ; kcl, . ; cacl , . ; mgso , . ; nahco , . ; kh po , . ; glucose, . ; . at a perfusion pressure of mmhg. any connective tissue, thymus, or lung was carefully removed. a catheter placed into the pulmonary artery drained the coronary effluent perfusate that was collected for the determination of coronary flow and venous po measurements. the left atrium was cannulated via an incision of the left auricle. all pulmonary veins were ligated close to the surface of the atria. when a tight seal with no leaks had been established and after a -min equilibration period, the hearts were switched into the working mode, using a filling pressure (preload) of mmhg in wky/nhsd and mmhg in sh rats. the afterload pressure was mmhg in wky/nhsd and mmhg in sh rats. after validation of the basis parameters, the afterload pressure was enhanced in a cumulative manner from an additional - mmhg. thereafter, the isovolumetric maxima were determined by enhancing the preload pressure in steps of - mmhg. flow and pressure signals for computation were obtained from the plugsys measuring system. computation of data was performed with a sampling rate of hz, averaged every s, using the software aquire plus v . f (po-ne-mah, hugo sachs elektronik, march-hugstetten, germany). after the evaluation of the external heart work, the total heart weight and the left and right ventricular weights were determined. the left ventricle was then sectioned transversely into four slices from the apex to the base. eight pictures were taken of each rat heart, two from each slice. total infarct size was determined by planimetry of the projected and magnified slices. the area of infarcted tissue as well as the intact myocardium of each slice were added together and averaged. the infarcted fraction of the left ventricle was calculated from these measurements and expressed as a percentage of the lv mass. the left ventricular perimeter, diameter, infarct scar length, as well as wall thickness and infarct wall thinning were determined as well. according to pfeffer et al. ( ) and , rats with infarct sizes < % and > % were excluded from the study. after lung weight determination, the organ was immersed in % formalin (ph . - . ; . m). the lung was cut into small pieces, dehydrated, and embedded in paraffin. hematoxylin and eosin (he) sections were evaluated by light microscopy. hydroxyproline/proline ratio after embedding, the rest of the fixed left ventricular tissue was freeze-dried. proline and hydroxyproline was then analyzed according to the method of lópez de león and rojkind ( ) and the ratio of both were calculated. the data are given as mean ae sem. statistics were performed using the sas system statistics package (sas institute, cary, n.c., usa) with a sequential rejection t-test. jain et al. ( ) studied the effects of angiotensin ii receptor blockade after coronary ligation and exercise training on treadmill in rats. medvedev and gorodetskaya ( ) induced heart failure in rats by microembolization of coronary vessels with -μm plastic microspheres. katona et al. ( ) found that selective sensory denervation by capsaicin aggravates adriamycin-induced cardiomyopathy in rats. a simple and rapid method of developing high output heart failure and cardiac hypertrophy in rats by producing aortocaval shunts was described by garcia and diebold ( ) . rats weighing - g were anesthetized with mg/kg i.p. pentobarbitone. the vena cava and the abdominal aorta were exposed by opening the abdominal cavity via a midline incision. the aorta was punctured at the union of the segment two-thirds caudal to the renal artery and one-third cephalic to the aortic bifurcation with an -gauge disposable needle. the needle was advanced into the aorta, perforating its adjacent wall and penetrating in the vena cava. a bulldog vascular clamp was placed across the aorta caudal to the left renal artery. once the aorta was clamped, the needle was fully withdrawn, and a drop of cyanoacrylate glue was used to seal the aorta-punctured point. the clamp was removed s later. the patency of the shunt was verified visually by swelling vena cava and the mixing of arterial and venous blood. the peritoneal cavity was closed with silk thread stitches and the skin with metallic clips. rats with aortocaval shunts developed cardiac hypertrophy with significantly higher absolute and relative heart weights. other studies with aortocaval shunts in rats were published by flaim et al. ( ) and liu et al. ( ) . isoyama et al. ( ) studied myocardial hypertrophy after creating aortic insufficiency in rats. terlink et al. ( ) studied ventricular dysfunction in rats with diffuse isoproterenol-induced myocardial necrosis. studies (inoko et al. ; klotz et al. ) have shown that dahl-salt-sensitive rats when placed on a high-salt diet from the th week of age will develop concentric lv hypertrophy without chamber dilation around the th week and decompensate heart failure between the th and the th week. introduction of the high-salt diet at or weeks of age will result in diastolic heart failure or systolic heart failure phenotypes, respectively (doi et al. ) . another rat model of systemic hypertension inducing heart failure is created by clipping one renal artery while leaving the contralateral kidney untouched. this induces systemic hypertension and lv concentric remodeling within weeks (junhong et al. ; rizzi et al. ) . extensive lv fibrosis and diastolic dysfunction was also reported (junhong et al. ). purpose and rationale rockman et al. ( rockman et al. ( , developed a model of ventricular hypertrophy in the intact mouse by use of microsurgical techniques. eight-week-old adult mice weighing - g are anesthetized by intraperitoneal injection of a mixture of mg/kg ketamine, mg/kg xylazine, and . mg/kg morphine. animals are placed under a dissecting microscope in the supine position, and a midline cervical incision is made to expose the trachea and carotid arteries. after endotracheal intubation, the cannula is connected to a volume-cycled rodent ventilator on supplemental oxygen with a tidal volume of . ml and a respiratory rate of per min. both left and right carotid arteries are cannulated with flamestretched pe tubing. catheters are connected to modified p statham transducers. the chest cavity is entered in the second intercostal space at the left upper sternal border through a small incision, and the thymus is gently deflected out of the field of view to expose the aortic arch. after the transverse aorta is isolated between the carotid arteries, it is constricted by a . nylon suture ligature against a -gauge needle, the latter being promptly removed to yield a constriction of . -mm diameter and provide a reproducible transverse aortic constriction of - %. the hemodynamic effects of acute and chronic constriction are followed by monitoring the pressure gradient between the two carotid arteries in anesthetized animals. systolic and mean arterial pressure at baseline, during total occlusion when the ligature is tied, and early ( min) and late ( days) after transverse aortic constriction are recorded. the increase in systolic pressure provides an adequate mechanical stimulus for the development of cardial hypertrophy. to confirm myocardial hypertrophy, both shamoperated and aortic-constricted hearts are examined days after operation. hearts examined for cell size are perfused with % paraformaldehyde/ % glutaraldehyde through the apex, immersed in osmium tetroxide, dehydrated in graded alcohols, and embedded in araldite. tissue blocks are sectioned at a thickness of μm, mounted on slides, and stained with toluidine blue. cell areas are measured by manually tracing the cell outline on an imaging system connected to a computer. at the end of the experiment, mice were sacrificed in anesthesia, heart excised, and weighed, the atria and ventricles separately frozen in liquid nitrogen for northern blot analysis. total rna is extracted by a single-step extraction with guanidinium thiocyanate. the rna is size fractionated by agarose gel electrophoresis, transferred to nylon membranes by vacuum blotting, and hybridized with the appropriate complementary dna probes labeled with p by random priming to a specific activity of . - .  cpm/ng. variables measured are expressed as mean ae sd. statistical significance of differences between sham-operated and thoracic aortic-constricted animals is assessed by student's t-test. dom et al. ( ) studied myosin heavy chain regulation and myocytes' contractile depression after lv hypertrophy in aortic-banded mice. okada et al. ( ) subjected mice to transverse aortic constriction. echocardiographic analysis demonstrated cardiac hypertrophy and failure and weeks after surgery. cardiac expression of endoplasmatic reticulum chaperones was significantly increased, indicating that pressure overload by transverse aortic constriction induced prolonged endoplasmatic reticulum stress. stansfield et al. ( ) described a minimally invasive murine model of transverse aortic constriction debanding, in which the band is removed up to weeks after constriction through the same suprasternal incision. this reversible model of pressure overload was shown as an interesting model to study the molecular mechanisms involved in lv reverse remodeling. dom gw, robins j, ball n, walsh ra ( ) myosin heavy chain regulation and myocytes contractile depression after lv hypertrophy in aortic-banded mice. am j physiol ( pt chronic heart failure in mice purpose and rationale several authors reported the development of murine models of cardiac failure (kaplan et al. ; rockman et al. ; balasubramaniam et al. ; suzuki et al. ; walther et al. ; wang et al. ; liao et al. ) . xu et al. ( ) studied cardioprotection in mice with heart failure by dual inhibition of angiotensin-converting enzyme (ace) and neutral endopeptidase (nep). mice with a targeted deletion of the b kinin receptor gene or c bl/ j mice at an age of - weeks were anesthetized with mg/kg sodium pentobarbital i.p., intubated and ventilated with room air using a positive-pressure respirator. a left thoracotomy was performed via the fourth intercostal space; the lungs were retracted to expose the heart, and the pericardium was opened. the left anterior descending coronary artery was ligated with an - nylon suture near its origin between the pulmonary outflow tract and the edge of the left atrium. acute myocardial ischemia was considered successful when the anterior wall of the left ventricle turned pale and an obvious st segment elevation was observed. the lungs were inflated by increasing positive end-expiratory pressure and the thoracotomy site was closed. sham-operated mice were subjected to the same procedure except that the suture around the left anterior coronary artery was not tied. systolic blood pressure was measured in conscious mice using a noninvasive computerized tail-cuff system. cardiac geometry and function were evaluated with a doppler echocardiographic system. where lvad is the lv diastolic area and lvas is the lv systolic area. four weeks after surgery, each strain was separated into one group treated with an ace inhibitor, one group treated with a nep inhibitor, one group treated with both inhibitors, and one control group. all drugs were administered in drinking water for weeks. at the end of the study, all mice were anesthetized with pentobarbital and the heart stopped at diastole by intraventricular injection of % kcl. the heart, lungs, and liver were weighed to assess hypertrophy and congestion. infarct size was determined by gomori trichrome staining and expressed as the ration of the infarcted portion to total lv circumference. sections ( μm) from each slice were double stained with fluorescein-labeled peanut agglutinin to delineate the myocyte cross-sectional area and interstitial space and rhodamine-labeled griffonia simplicifolia lectin i to show the capillaries. to calculate interstitial collagen fraction, the total surface area (microscopic field), interstitial space (collagen plus capillaries), and area occupied by capillaries alone were measured by computerassisted videodensometry. after weeks of treatment, plasma renin was measured. data were expressed as mean ae se. mortality rates were compared using χ tests. for the echo, blood pressure, heart weight, lung weight, infarct size, plasma renin concentration, and histology data, paired or two-sample tests using nonparametric methods were used to perform all comparisons of interest. by an aortocaval shunt. congestive heart failure was induced, which resulted in the development of myocardial hypertrophy, impaired cardiac function, and increased expression of the natriuretic peptides in the left ventricle. transgenic mice and heart failure purpose and rationale several hundreds of papers on transgenic mice and heart failure are published. only a few can be mentioned here. chien ( ) described cardiac muscle diseases in genetically engineered mice. edwards et al. ( ) described severe cardiomyopathy in transgenic mice overexpressing the skeletal muscle myogenic regulator myf . arber et al. ( ) found that mlp-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. graham et al. ( ) described a mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoforms of the adenine nucleotide translocator. iwase et al. ( ) studied cardiomyopathy in transgenic mice induced by overexpression of the cardiac stimulatory g protein α-subunit. knollmannn et al. ( ) reported remodeling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin. beggah et al. ( ) described reversible cardiac fibrosis and heart failure induced by conditional expression of an antisense mrna of the mineralocorticoid receptor in cardiomyocytes. verheule et al. ( ) found increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of tgf-β . duncan et al. ( ) found that chronic xanthine oxidase inhibition prevents myofibrillar protein oxidation and preserves cardiac function in a transgenic mouse model of cardiomyopathy. hartil and charron ( ) reviewed mouse models where transgenic technology has been utilized to alter expression of genes involved in cardiac uptake and metabolism of either lipid or carbohydrate. hilfiker-kleiner et al. ( ) reported that stat knockout mice harboring a cardiomyocyterestricted deletion of stat showed enhanced susceptibility to cardiac injury caused by myocardial ischemia, systemic inflammation, or drug toxicity. studied reversal of amyloidinduced heart disease in desmin-related cardiomyopathy. cardiac insufficiency in guinea pigs congestive heart failure in man is characterized by cardiac hypertrophy, peripheral edema, lung and liver congestion, dyspnea, hydrothorax, and ascites. effective treatment is achieved by cardiac glycosides. based on techniques reported by , a method was developed to induce congestive heart failure in guinea pigs with symptoms very close to human pathology (vogel and marx ; vogel et al. ). male guinea pigs weighing - g are used. the fur at the ventral thorax is shaved and the skin disinfected. the animal is anesthetized with ether. the skin is cut with scissors on the left side at a length of cm. the left musculus pectoralis is cut at the costal insertion and elevated. the forth intercostal space is opened with two blunted forceps. the heart is pressed against the opening with the left hand. the pericardium is opened with a fine forceps and pulled back to the basis of the heart. the beating heart is extruded from the thorax wound by pressure with the left hand on the right thorax wall. a ring-shaped clamp covered with a thin rubber tube is placed around the basis of the heart, keeping the heart outside of the thorax without closing off the blood circulation. a thread soaked with diluted disinfectant solution is placed as a loop around the apex of the heart and tightened so that the apical third of both ventricles is tied off. the degree of tightening of the loop is essential. complete interruption of blood supply to the apical third resulting in necrosis has to be avoided as well as the loop's slipping off. technical skill is necessary to place the loop around the beating heart into the correct position. after removal of the clamp, the heart is placed back, the incision between the fourth and fifth costal rib closed, and the musculus pectoralis placed over the wound. intrathoracal air forming a pneumothorax is removed by pressure on both sides of the thorax. after application of an antibiotic emulsion, the skin wound is closed. the surgical procedure has to be finished within a short period of time. the animals develop symptoms of severe congestive heart failure with a death rate of % within days. lung weight and relative heart weight are significantly increased. exudate in the thorax cavity and ascites amount between . and . ml with extreme values of . ml. lung edema and liver congestion are found histologically. peripheral edema and preterminal dyspnea and tachypnea are observed. when treated with various doses ( . - μg/kg) of cardiac glycosides s.c. or i.m. over a period of days, the symptoms of cardiac insufficiency, e.g., volumes of transudate as well as death rate, are dosedependent diminished. from survival rate, ed values of cardiac glycosides can be calculated which are in the same dosage range as therapeutic doses in man. the experimental model in guinea pigs reflects very closely the symptoms of cardiac insufficiency in man, e.g., lung congestion, hydrothorax, liver congestion, ascites, peripheral edema, and cardiac hypertrophy. the therapeutic potency of cardiac glycosides can be evaluated with this method. additional factors being known to enhance the symptoms of congestive heart failure in man, like salt load and diphtheria toxin, further increase mortality and hydropic symptoms. the method can be used for special purposes; however, it needs considerable training and technical skill. siri et al. ( siri et al. ( , produced left ventricular hypertrophy in the guinea pig by gradually increasing ventricular afterload. a mildly constricting band was placed around the ascending aorta of very young guinea pigs ( - g). with growth to - , g, left ventricular systolic pressure increased and ventricular hypertrophy developed. only some of the animals developed dyspnea and severe ventricular dysfunction. kiss et al. ( ) studied the effects on ca + transport and mechanics in compensated pressureoverload hypertrophy and congestive heart failure in guinea pigs. the descending aorta was banded for and weeks in adult guinea pigs. tweedle et al. ( ) assessed subrenal banding of the abdominal aorta as a method of inducing cardiac hypertrophy in the guinea pig. induced myocardial infarction in rats by ligation of the left coronary artery and found hemodynamic benefits and prolonged survival with long-term captopril therapy. acute ischemic left ventricular failure can be induced in anesthetized dogs by repeated injections of plastic microspheres into the left coronary artery (see chapter "▶ coronary drugs", section "acute ischemia by injection of microspheres in dogs"). huang et al. ( ) created congestive heart failure in sheep by selective sequential intracoronary injection of μm microspheres under . % isoflurane injection. pfeffer ma, braunwald e ( ) hemodynamic benefits and prolonged survival with long-term captopril therapy in rats with myocardial infarction and heart failure. circulation :i- -i- selye h, simple techniques for the surgical occlusion of coronary vessels in the rat. cardiomyopathic syrian hamster cardiomyopathy in syrian hamsters has been described by bajusz et al. ( ) , bajusz and lossnitzer ( ) , bajusz ( ) , bajusz et al. ( a, b) , homburger and bajusz ( ) , and gertz ( ) . the disease originates from an autosomal, recessively transmissible disorder, which leads to degenerative lesions in all striated muscles and in particular in the myocardium. histopathological changes consist of myocytolytic necrosis followed by fibrosis and calcification. the evolution of the cardiomyopathic disease can be characterized by five distinct phases: a prenecrotic stage, in which no pathology is evident, a time of active myocytolysis and cellular necrosis, a phase of fibrosis and calcium deposition, an overlapping period of reactive hypertrophy of the remaining viable myocytes, and a final stage of depressed myocardial performance and failure. the model of cardiomyopathy in syrian hamsters has been used by several authors. one has to note that several strains of cardiomyopathic hamsters have been used: strain bio : by capasso et al. ( capasso et al. ( , ) and by chemla et al. ( chemla et al. ( , , strain bio . by tapp et al. ( ) and by sen et al. ( ) , strain chf cm by van meel et al. ( ) and by haleen et al. ( ) , strain bio . by ver donck et al. ( ) , strain j- -n by kato et al. ( ) , and strain chf by desjardins et al. ( ) and hanton et al. ( ) . various experimental protocols have been described. most authors use survival rate and heart weight as end point (e.g., van meel et al. ; ver donck et al. ; hanton et al. ) . generally, the experiments are started with animals at an age of - days. capasso et al. ( capasso et al. ( , ) studied the mechanical and electrical properties of cardiomyopathic hearts of syrian hamsters using isolated left ventricular posterior papillary muscles. tapp et al. ( ) tested stress-induced mortality in cardiomyopathic hamsters by five consecutive daily -h periods of supine immobilizations at c. sen et al. ( ) tested the inotropic and calcium kinetic effects of calcium channel agonists and antagonists in primary cultures of isolated cardiac myocytes. haleen et al. ( ) tested the effects of an angiotensin-converting-enzyme inhibitor not only on survival but also on left ventricular failure in the isolated langendorff heart by measurement of left ventricular end-diastolic pressure, dp/ dt max , and mean coronary flow. dixon et al. ( ) tested the effect of an at receptor antagonist on cardiac collagen remodeling in the cardiomyopathic syrian hamster. in addition to the effects on left ventricular papillary muscles strips, chemla et al. ( ) tested the effects on diaphragm contractility in the cardiomyopathic syrian hamster. whitmer et al. ( ) and kuo et al. ( ) tested sarcolemmal and sarcoplasmatic reticulum calcium transport in the cardiomyopathic syrian hamster. nigro et al. ( ) identified the syrian hamster cardiomyopathy gene. tanguay et al. ( ) tested the coronary and cardiac sensitivity to a vasoselective benzothiazepine-like calcium antagonist in isolated, perfused failing hearts of syrian hamsters. bilate et al. ( ) recommended the syrian hamster as a model for the dilated cardiomyopathy of chagas disease. female hamsters were infected via the intraperitoneal route with trypanosoma cruzi y strain blood trypomastigotes. survival was monitored, echocardiography was performed after and months, and histopathological examinations were carried out at the end of the study period. positive effects of various drugs have been found in the cardiomyopathic hamster, such as cardiac glycosides, inotropic compounds, beta-blockers, calcium antagonists, and ace inhibitors. the specificity of the effects has to be challenged. the tight skin (tsk) mouse is a genetic model of pulmonary emphysema connected with right ventricular hypertrophy (martorana et al. ; gardi et al. ). bajusz e ( ) purpose and rationale rabbit models of heart failure were reviewed by . rapid pacing was used by masaki et al. ( ) , porsa et al. ( ) , eble et al. ( ), li et al. ( , and rose et al. ( ) ; coronary artery ligation by pennock et al. ( ) , currie and smith ( ) , romanic et al. ( ), and miller et al. ( ) ; combined pressure and volume overload by ezzaher et al. ( ) , mohammadi et al. ( ) , dekker et al. ( ), and baartscheer et al. ( a, b) ; aortic insufficiency and aortic constriction by bouanani et al. ( ) and pogwizd et al. ( ) ; regurgitation after damage of the mitral valve by gunawardena et al. ( ) ; and regurgitation after aortic valve destruction by magid et al. ( magid et al. ( , , yoshikawa et al. ( ) , king et al. ( ) , liu et al. ( ), and luchner et al. ( ) used a rabbit model of progressive left ventricular dysfunction to investigate differential expression of cardiac atrial natriuretic peptide and brain natriuretic peptide. ventricular pacing-induced heart failure could be induced with a transvenously implanted pacemaker system. male rabbits (chinchilla bastard) underwent implantation of a programmable cardiac pacemaker (medtronic minix , minneapolis, mn., usa). under anesthesia (ketamine mg/kg xylazine mg/kg i.m.), the right internal jugular vein was dissected and cannulated with a single-lumen central venous catheter (braun, germany). the catheter was then advanced into the right ventricle under pressure guidance. a transvenous screw-in pacemaker lead (medtronic) was advanced through the catheter into the ventricular apex and implanted endocardially. the pacemaker was implanted subcutaneously into the right abdominal wall, and the pacemaker lead was connected subcutaneously with the pacemaker. rapid ventricular pacing-induced heart failure could be induced with a transvenously implanted pacemaker system. all rabbits were allowed to recover for at least days after surgery before the pacemaker was started for the induction of heart failure. proper pacemaker function was checked intraoperatively, at the time of programming, and subsequently all days. rabbits (chf group) underwent pacing with a stepwise increase of stimulation frequencies over days. during the first days, animals were paced at beats/min (bpm). this protocol results in elvd, as defined by significant lv systolic dysfunction with cardiac enlargement and decreased perfusion pressure, but no clinical signs of heart failure. the pacing rate was then increased to bpm for days and bpm for another days, and elvd evolved to chf with further cardiac enlargement and further decreased perfusion pressure together with clinical signs of fluid retention (ascites). at baseline (control), after being paced at bpm for days (elvd) and at the end of the protocol (chf), conscious arterial pressure was measured invasively via the medial ear artery and a -dguided m-mode echocardiogram was obtained. at the end of the pacing protocol, rabbits were killed by i.v. euthanasia and tissue was rapidly harvested. hearts were trimmed on ice, snap frozen in liquid nitrogen, and stored at À c until further processing. a long-and short-axis echocardiogram (hp sonos , mhz probe) was performed under light sedation ( mg midazolam i.m.) in a supine position from the left parasternal window. lv end-diastolic (lvedd) and end-systolic (lvesd) dimensions and diastolic and systolic thickness of the left ventricular anterior wall (aedth and aesth) and posterior wall (pedth and pesth) as well as left atrial diameter (lad) were determined from three repeated -d-guided m-mode tracings using the ase convention. from those measurements, fractional shortening (fs) was calculated as analytical methods for analysis of cardiac natriuretic peptide expression, mrna was extracted from all atrial and left ventricular samples utilizing a commercial kit (fasttrack, invitrogen). as a probe for brain natriuretic peptide (bnp), a -bp ecor /hindiii dna restriction fragment containing the gene for rabbit bnp was used. results of the quantitative studies were expressed as mean ae sem. comparisons between the control, elvd, and chf groups were performed by analysis of variance (anova) followed by fisher's least significant difference test. comparison between the atrial and lv tissues as well as between atrial natriuretic peptide (anp) and bnp was performed by paired student's t-test. statistical significance was defined as p < . . arnolda et al. ( ) studied adriamycin cardiomyopathy in the rabbit. klimtova et al. ( ) performed a comparative study of chronic toxic effects of daunorubicin and doxorubicin in rabbits. alexander et al. ( ) studied electrographic changes following coronavirus-induced myocarditis and dilated cardiomyopathy in rabbits. purpose and rationale several methods are described, to induce congestive heart failure in dogs, such as rapid ventricular pacing (armstrong et al. ; freeman et al. ; wilson et al. ; komamura et al. komamura et al. , perreault et al. ; travill et al. ; cheng et al. ; redfield et al. ; cory et al. ; kiuchi et al. ; ohno et al. ; vatner et al. ; williams et al. ; eaton et al. ; spinale et al. ; wolff et al. ; zile et al. ; ravens et al. ; shinbane et al. ; o'rourke et al. ; winslow et al. ) . luchner et al. ( ) assessed circulating, renal, cardiac, and vascular angiotensin ii in a canine model of rapid ventricular pacinginduced heart failure that evolves from early left ventricular dysfunction to overt congestive heart failure. male mongrel dogs underwent implantation of a programmable cardiac pacemaker (medtronic). under pentobarbital sodium anesthesia and artificial respiration, the heart was exposed via a small left lateral thoracotomy and pericardiotomy, and a screw-in epicardial pacemaker lead was implanted into the right ventricle. the pacemaker was implanted subcutaneously into the left chest wall and connected to the pacemaker lead. the dogs were allowed to recover for at least days after surgery before the pacemaker was started. during the first days, dogs were paced at beats/min (bpm), resulting in early left ventricular dysfunction as defined by significant systolic dysfunction with decreased cardiac output, cardiac enlargement, and increased filling pressures but maintained systemic perfusion pressure and renal sodium excretion and no clinical signs of heart failure. the pacing rate was then increased weekly to , , , and bpm, and early left ventricular dysfunction evolved to overt congestive heart failure with avid sodium retention and clinical signs of congestion. at baseline (control), after dogs had been paced at bpm for days and at the end of the protocol (overt chf), urine was collected for measurement of sodium excretion; conscious mean arterial pressure was measured via a port catheter; a -dguided m-mode echogram was obtained; and arterial blood was drawn. cardiac filling pressures and cardiac output were measured by the thermodilution method at baseline and at the end of the protocol. arterial blood was collected in edta tubes for measurement of anp, bnp, cgmp, pra, aldosterone, and ang ii. after euthanasia, hearts were rapidly trimmed and left ventricles weighted for calculation of the index lv weight to body weight. results were expressed as mean ae se. comparison between the control, early lv dysfunction, and overt chf were performed by anova followed by fisher's least significant difference test. kleaveland et al. ( ) and nagatsu et al. ( ) used the technique of experimental mitral regurgitation in dogs to induce left ventricular dysfunction. a -cm, -f sheath was introduced across the aortic valve through the carotid artery. a urologic calculus retrieval forceps was advanced through the sheath to the mitral valve apparatus and was used to sever chordae tendineae. when pulmonary capillary wedge pressure rose to mmhg and forward stroke volume was reduced to % of its baseline, a ventriculogram was performed to confirm angiographically that severe mitral regurgitation had been created. dell 'italia et al. ( ) and su et al. ( ) induced mitral regurgitation by percutaneous chordal rupture in dogs. kinney et al. ( ) published a method to induce acute, reversible tricuspid insufficiency in anesthetized dogs. a wire spiral is advanced through the atrioventricular canal from the right atrium. the spiral causes regurgitation by preventing complete apposition of the valve leaflets while permitting retrograde flow to occur through the spiral lumen. the degree of regurgitation can be controlled by the use of spirals of different size. creation of tricuspid insufficiency was demonstrated by onset of right atrial pressure v waves, a ballooning of the right atrium during ventricular systole, palpation of an atrial thrill, or color doppler echocardiography. the model is reversible and allows repeated trials of various grades of regurgitation. carlyle and cohn ( ) described a non-chirurgical model of chronic left ventricular dysfunction. the method is accomplished by repetitive dc shock with a guidewire introduced percutaneously and positioned in the left ventricle along the intraventricular septum and an external paddle at the left ventricular apex. produced localized left ventricular necrosis without obstruction of the coronary blood flow in dogs by transmyocardial direct-current shock. sabbah et al. ( , sabbah et al. ( , and gengo et al. ( ) produced chronic heart failure in dogs by multiple sequential intracoronary embolizations with microspheres. the dogs underwent three to nine intracoronary embolizations with polystyrene latex microspheres ( - μm in diameter) performed - weeks apart. embolizations were discontinued when left ventricular ejection fraction was less than %. used multiple coronary microembolizations in dogs, whereby three to nine embolizations were performed week apart. the first three embolizations consisted of ml of microsphere suspension injected subselectively into either the left anterior descending or left circumflex coronary artery in an alternating fashion. subsequent embolizations consisted of - ml of microspheres divided equally between the left anterior descending or left circumflex coronary artery until lv ejection fraction was < %. magovern et al. ( ) described a canine model of left ventricular dysfunction caused by five weekly intracoronary infusions of adriamycin. koide et al. ( ) described premorbid determinants of left ventricular dysfunction in a model of gradually induced pressure overload in dogs. mongrel dogs were studied through weeks of gradually imposed ascending aortic constriction with the use of a novel banding technique. during banding, an initial gradient of mmhg was created. before banding, at , , and weeks after banding, hemodynamics and left ventricular mechanics were examined at cardiac catheterization; then the pressure overload was increased by tightening the band. valentine et al. ( ) and devaux et al. ( ) described x-linked muscular dystrophy in dogs with cardiac insufficiency similar to duchenne muscular dystrophy in men and recommended this as an animal model for cardiac insufficiency. bilateral renal wrapping model in adult male dogs ( - kg) has been described previously (page ; hart et al. ; maniu et al. ) ; in this model, the kidneys were wrapped with silk without constriction of renal vessels. increased in systolic blood pressure and lv mass index was observed at weeks post renal wrapping. at weeks post renal wrap, an increase in lvedp was observed. lv end-diastolic volume, ejection fraction, stroke volume, and cardiac output were not changed in this model. no changes in circulating angiotensin ii, endothelin, catecholamines, and plasma renin activity were also noted. a modification of this method has been published by hayashida et al. ( hayashida et al. ( , ; in these studies only the left kidney was wrapped. the dogs developed hypertension and diastolic dysfunction with increased lv weight/body weight ratio and lvedp and without significant changes in fractional shortening or lv diameters an increased ang ii levels. ( pt cardiac failure was studied in pigs using several experimental procedures. chow et al. ( ) recommended rapid ventricular pacing in pigs as an experimental model of congestive heart failure. farrar et al. ( ) studied pacing-induced dilated cardiomyopathy in pigs. congestive heart failure was produced by rapid ventricular pacing at bpm for week. spinale et al. ( a spinale et al. ( , b, spinale et al. ( , ) examined the consequences of chronic supraventricular tachycardia on various parameters of ventricular dysfunction and subendocardial changes in pigs. carroll et al. ( ) investigated gene expression in a swine model of ventricular hypertrophy during pressure overload. multani et al. ( ) studied long-term angiotensin-converting enzyme and angiotensin i receptor inhibition in pacing-induced heart failure in pigs. heart failure was induced by rapid atrial pacing ( bpm for weeks). kassab et al. ( kassab et al. ( , investigated remodeling of right ventricular branches after hypertrophy in pigs. krombach et al. ( ) studied the effects of amlodipine in congestive heart failure in pigs at rest and after treadmill exercise. left thoracotomy was performed in yorkshire pigs under anesthesia. catheters connected to a vascular access port were placed in the thoracic aorta, the pulmonary artery, and the left atrium. the access ports were then placed in a subcutaneous pocket. a -mm flow probe was placed around the pulmonary artery immediately distal to the pulmonary catheter and the electrical connection exteriorized through the thoracolumbar fascia. a shielded stimulating electrode was sutured onto the left atrium, connected to a programmable pacemaker, and buried in a subcutaneous pocket. the thoracotomy was closed in layers and the pleural space evacuated of air. after a - -day recovery, measurements were performed under normal resting conditions and after exercise. the pacemakers were activated to bpm for a period of days. during the last days, one group was treated with drug, the other served as control. at the day of the study, electrocardiograms were performed, and the pacemakers deactivated. after a -min stabilization period, -d and m-mode echocardiographic studies were used to image the left ventricle from the parasternal approach. left ventricular fractional shortening was calculated as (end-diastolic dimensionend-systolic dimension)/diastolic dimension and was expressed as a percentage. the access ports were entered and pressures obtained using externally calibrated transducers. the flow probe was connected to a digital flowmeter. from the digitized flow signal, stroke volume was computed on a beat-to-beat basis and averaged for a minimum of ejections. pulmonary and systemic vascular resistances were computed as the mean pressure divided by cardiac output multiplied by the constant to convert to resistance units of dyne Á s Á cm À . samples were drawn from the pulmonary artery and atrial catheters for measurement of oxygen saturation and hemoglobin content. the plasma samples were assayed for renin activity, endothelin concentration, and catecholamine levels. results were presented as mean ae sem. pairwise tests of individual group means were compared using bonferroni probabilities. zhang et al. ( ) studied functional and bioenergetic consequences of postinfarction left remodeling in a porcine model. proximal left coronary artery occlusion was used to generate a myocardial infarction in young pigs. the animals were then followed over several months while remodeling of the left ventricle developed. left ventricular wall thickness, ejection fraction, and wall stress were measured by mri. myocardial atp, creatine phosphate, and inorganic phosphate levels were measured by spatially localized p-nmr spectroscopy, and regional myocardial blood flow was measured with radioactive microspheres. all mri studies were performed on the standard siemens medical system vision operating at . t. the animals were anesthetized with sodium pentobarbital. a catheter was placed into the femoral artery and advanced into the lv chamber for lv pressure recording. animals then were placed on their left side in a helmholtz coil with a diameter of cm, which was used to improve signal to noise. to compute lv wall stress, the image acquisition was triggered by the lv pressure through the fluid-filled lv catheter. all of the imaging sequences were synchronized to the lv pressure trace. the electronic lv pressure signal was recorded and fed to a comparator set to a threshold level of % of the upslope of the lv pressure curve at the beginning of systole. the signal from the comparator was sent to a pulse former and then fed to the ecg port of the magnetic resonance system, where it was treated like the standard electrographic input to run the pulse sequences. scout images were taken in the axial plane with a single-shot, ultrafast gradient echo sequence wilke et al. ; geiger et al. ) . from the axial image, both horizontal and vertical long-axis images were obtained. by alternating back and forth several times, a true vertical long axis of the left ventricle was obtained. from the longaxis scout image, short-axis segmented cine turboflash slices were prescribed to cover the myocardium from apex to base. the double oblique, short-axis turboflash images cover the heart from apex to base with a slice thickness of mm, with no interslice gap. the parameters of the segmented cine sequence were tr/te/flip angle = ms/ . ms/ with an fov = . cm and a matrix of  (pixel size,  . mm) and slice thickness of - mm (atkinson and edelman ) . the sequence used segmented k-space acquisition such that three phase-encoded lines were gathered per cardiac phase per heartbeat. total image acquisition required approximately heartbeats for each slice location. the temporal image resolution (data acquisition window) of this sequence was ms per cardiac image. each myocardial level took < . min to acquire, since two acquisitions were used and the average heart rate of the animals was bpm. the average number of short-axis slices needed to image the entire myocardium from apex to base was - . this -min protocol provided high signal-to-noise cine sequences covering the entire heart. to obtain high-resolution anatomic heart images, multislice, single-phase spin-echo images trigged in the systolic phase were acquired to cover the entire heart. these images permitted the precise delineation of the extent of the scar region of the heart. images were taken with a slice thickness of mm and a fov of . cm, resulting in a true spatial resolution of  . mm pixel size. the tr for this sequence equals the rr interval ( ms) and the echo time te was set to ms. total measurement time for an average of - slices was min. the imaging data were archived to optical disk and copied to a sun sparc workstation for evaluation with the use of an automatic segmentation program (imageview, siemens cooperate research). the program is based on robust deformable models of endocardial and epicardial border segmentation of ventricular boundaries in cardiac magnetic resonance images. this segmentation technique has been combined with a user interface that allows one to load, sort, visualize, and analyze a cardiac study in < min. the segmentation algorithm is based on the steepest descent as well as dynamic programming strategies integrated via multiscale analysis for minimizing the energy function of the resulting contour. the ventricular boundaries are used to construct a three-dimensional model for visualization and to compute hemodynamic parameters. automatic segmentation of endocardial and epicardial boundaries was performed for calculation of ventricular volumes, ef, lv diastolic and systolic volumes, and absolute myocardial mass from multislice, multiphase magnetic resonance cine images. starting with a user-specified approximate boundary or an interior point of the ventricle for one starting image in one slice, the algorithm generated automatic contours corresponding to the epicardium and the endocardium and automatically propagated them to other slices in the cardiac phase (spatial propagation) and to other phases for a given slice location (temporal propagation) of the cardiac study. the observer then could make some manual corrections to the six or seven pairs of contours in the first column of the temporal-spatial matrix. manual modifications generally were made on the apex and base levels. mean lv wall thickness for each short-axis ring was averaged from three measurements of the remote zone (anterior wall and septum wall). the thickness of the scar was averaged from three measurements of the scar area. lvsa measurement in each slice was computed by subtracting the total area enclosed by the endocardium from that enclosed by the subepicardium; the resultant area was multiplied by the slice thickness to obtain the volume of each slice; the total lv mass volume was calculated by adding up the volumes of all the short-axis slices. the total lvsa was obtained by dividing the total lv wall mass volume by the mean of lv wall thickness of each slice. similarly, the lvssa was obtained by dividing the total scar volume, which was the sum of the scar volume of each short axis, by the mean of the scar thickness of each short axis. lv mass was computed by the total lv wall mass volume multiplied by . (specific gravity of myocardium) to calculate the lv mass. the lv end-diastolic volume (v d ) and end-systolic volume (v s ) of each slice were represented by the area enclosed by the endocardium. the total lv volume was computed by adding the volumes of all slices. lvef was calculated by  (v-v s )/v d %. interobserver and intraobserver errors for the calculations of lv mass and lv volumes have been shown to be < mg and ml, respectively (mcdonald et al. ) . meridional wall stress was computed from the lv pressure and simultaneously obtained short-axis view of lv mri (lv cavity diameter and average thickness the remote lv wall) as described by grossman et al. ( ) . measurements were performed in a -cm-bore, . -t magnet interfaced with a sisco (spectroscopy imaging systems corporation) console. the lv pressure signal was used to gate nmr data acquisition to the cardiac cycle, while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions (robitaille et al. ). p and h-nmr frequencies were and . mhz, respectively. spectra were recorded in late diastole with a pulse repetition time of - s. this repetition time allowed full relaxation for atp and p i resonances and % % relaxation for the cp resonance (zhang and mcdonald ) . cp resonance intensities were corrected for this minor saturation; the correction factor was determined for each heart from two spectra recorded consecutively without transmural differentiation, one with -s repetition time to allow full relaxation and the other with the - -s repetition time used in all the other measurements. radiofrequency transmission and signal detection were performed with a -mm-diameter surface coil. the coil was cemented to a sheet of silicone rubber . mm in thickness and % % larger in diameter than the coil itself. a capillary containing μl of m phosphonoacetic acid was placed at the coil center to serve as a reference. the proton signal from water detected with the surface coil was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenters. this was accomplished with a spin-echo experiment and a readout gradient. the information gathered in this step also was used to determine the spatial coordinates for spectroscopic localization. chemical shifts were measured relative to cp, which was assigned a chemical shift of À . ppm relative to % phosphoric acid at ppm. spatial localization across the lv wall was performed with the rapp-isis/fsw method (hendrich et al. ) . signal origin was restricted with the use of b gradients and adiabatic inversion pulses to a column coaxial with the surface coil perpendicular to the lv wall. the column dimensions were  mm. within this column, the signal was further localized using the b gradient to five voxels centered about , , , , and spin rotation increments. fsw localization used a nine-term fourier series expansion. the fourier coefficients, the number of free induction decays acquired for each term in the fourier expansion, and the multiplication factors used to construct the voxels have been reported previously. the position of the voxels relative to the coil was set using the b magnitude at the coil center, which was experimentally determined in each case by measurement of the pulse length for the phosphonoacetic acid reference located in the coil center. each set of spatially localized transmural spectra was acquired in min. a total of scans was accumulated within each -min block. resonance intensities were quantified with the use of integration routines provided by the sisco software. atpγ resonance was used for atp determination. since data were acquired with the transmitter frequency being positioned between the atpγ and cp resonance, the off-resonance effects on these peaks were negligible. the numeric values for cp and atp in each voxel were expressed as ratios of cp/atp. p i levels were measured as changes from baseline values (Δp i ) with the use of integrals obtained in the region covering the p i resonance. , volume loading after myocardial infarction , rapid ventricular pacing (rademaker et al. (rademaker et al. , (rademaker et al. , moreno et al. ) , coronary microembolization , and thrombus-induced heart failure ). rademaker et al. ( ) studied combined angiotensin-converting enzyme inhibition and adrenomedullin in an ovine model of heart failure induced by rapid ventricular pacing. coopworth ewes ( ae kg) were instrumented via a left lateral thoracotomy. under general anesthesia (induced by mg/kg thiopentone; maintained with halothane/nitrous oxide), two polyvinyl chloride catheters were inserted in the left atrium for blood sampling and left atrial pressure (lap) determination; a konigsberg pressuretip transducer was inserted in the aorta to record mean arterial pressure (map); an electromagnetic flow probe was placed around the ascending aorta to measure cardiac output (co); a -french swan-ganz catheter was inserted in the pulmonary artery for infusions; and a -french his bundle electrode was stitched subepicardially to the wall of the left ventricle for left ventricular pacing. all leads were externalized through incisions in the back. a bladder catheter was inserted per urethra for urine collections. the animals were allowed to recover for days before commencing the study protocol. during the experiments, the animals were held in metabolic cages, had free access to water, and ate a diet of chaff and sheep pellets (containing mmol/day sodium and mmol/day potassium). a further mmol of sodium was administered orally daily as nacl tablets using an applicator. heart failure was induced by days of rapid left ventricular pacing ( bpm) (rademaker et al. ) and maintained by continuous pacing for the duration of the study. on four separate days with a rest day between each, the sheep received, in random order, a vehicle control (haemaccel), human adrenomedullin alone ( ng/min per kg infusion for h), an ace inhibitor alone (captopril: mg bolus + mg/h infusion for h), and both agents combined. infusions were administered in a total volume of ml via the pulmonary artery catheter, commencing at : h. mean arterial pressure, left atrial pressure, cardiac output, and calculated total peripheral resistance (ctpr = mean arterial pressure/cardiac output) were recorded at -min intervals in the h prior to infusion (baseline) and at , , , , , , and min during both the -h infusion and post-infusion periods. hemodynamic measurements were determined by online computer-assisted analysis. blood samples were drawn from the left atrium at min and immediately pre-infusion (baseline) and at , , , and min during the -h infusion and post-infusion periods. samples were taken into tubes on ice, centrifuged at , g for min at c, and stored at either À or À c before assay for immunoreactive (ir-) adrenomedullin, camp, plasma renin activity, angiotensin ii, aldosterone, atrial natriuretic peptide, brain natriuretic peptide, endothelin- , catecholamines, and cortisol. all samples from individual animals were measured in the same assay to avoid inter-assay variability. plasma electrolytes and hematocrit were measured in every sample taken. urine volume and samples for the measurement of urine camp, sodium, potassium, and creatinine excretion were collected every h. creatinine clearance was calculated as urine creatinine/plasma creatinine. results are expressed as mean ae sem. baseline hemodynamic and hormone values represent the means of the four and two measurements, respectively, made in the h immediately pre-infusion. statistical analysis was performed by repeatedmeasures anova. baseline data from all treatments were compared. treatment-and timerelated differences between all four study limbs were determined using a two-way anova (treatment-time interactions are quoted in the text). statistical significance was assumed when p < . . investigated the role of hypertension in ischemic heart disease in the cynomolgus monkey with coarctation of the aorta. studied cardiotoxic effects of adriamycin in macaques. various studies were performed by the group of hoit and walsh in baboons (hoit et al. , b, a . studied the effects of thyroid hormone on cardiac β-adrenergic responsiveness in conscious baboons. adult male baboons (papio anubis) weighing - kg were pre-instrumented for physiological monitoring in a lightly anesthetized, sedated state. animals were pre-instrumented with a konigsburg micromanometer and a polyvinyl catheter in the lv apex, miniaturized sonomicrometer pairs ( mhz, mm) across the lv anteroposterior minor axis, a polyvinyl catheter in the right atrium for central venous access, and pacing wires on the right atrial appendage. wires and tubes were tunneled subcutaneously into the interscapular area for later use. postoperative pain was reduced by the use of buprenex ( . mg/kg i.m., q h), and postoperative antibiotic (monocid mg/kg) was administered for days to reduce the risk of infection. baseline hemodynamic studies were performed after a minimum of week for postoperative recovery. hemodynamic data acquisition and analysis the micromanometers and fluid-filled catheters were calibrated with a mercury manometer. zero drift of the micromanometer was corrected by matching the lv end-diastolic pressure measured simultaneously through the lv catheter. the fluid-filled lv catheter was connected to a pre-calibrated statham -db transducer with zero pressure at the level of the mid-right atrium. the transit time of ultrasound between the ultrasonic dimension crystals was measured with a multichannel sonomicrometer (triton technology) and converted to distance assuming a constant velocity of sound in blood of . mm/ms. the analog lv dp/di signal was obtained online by electronic differentiation of the highfidelity lv pressure signal. τ was derived from the high-fidelity lv pressure tracing by the method of weiss et al. ( ) , which assumes a monoexponential decay of lv pressure to a zero asymptote and has been shown to be directionally equivalent to other mathematical approaches for quantification of isovolumic pressure decay. τ is equal to the time in milliseconds for lv pressure to decay to /e; thus, decreases in τ reflect improved isovolumic ventricular relaxation. fractional shortening of the lv minor axis was calculated as (edd-esd)/edd, where edd is the lvend-diastolic dimension and esd is the lv end-systolic dimension. lv end-diastole was defined as the time in which lv dp/dtmax increased by ! mmhg/s for ms, and lv end-systole was defined as the time of the maximum ratio of lv pressure to lv minor-axis dimension. lv volumes were derived from minoraxis diameter (d) measurements: v cf was calculated as lv fractional shortening divided by lv ejection time; lv ejection time was defined as the time from peak-positive to peaknegative dp/dt. analog signals for high-fidelity and fluid-filled lv pressures, lv short-axis dimension, lv dp/dt, and the ecg were recorded online on a gould multichannel recorder at and mm/s paper speed and digitized through an analog-to-digital board (dual control systems) interfaced to an ibm at computer at hz and stored on a floppy disk. data were analyzed using an algorithm and software developed in our laboratory. steady-state data were acquired over - s during spontaneous respiration and averaged. hemodynamic studies were performed a minimum of week after instrumentation and were repeated after - ( . ae . ) days of thyroid t administration. animals were tranquilized with valium ( - mg) and ketamine ( mg), and cholinergic blockade was achieved with atropine ( . - . mg i.v.); additional ketamine was administered as necessary, to a maximum cumulative dose of mg/kg. animals were atrially paced at a rate - % greater than the control heart rate in order to obtain data at matched heart rates after thyrotoxicosis was produced. after hemodynamic stability was ensured and baseline data were recorded, intravenous dobutamine was infused at -min intervals at upwardly titrated rates of . , . , . , and . μg Á kg À Á min À to examine the effects of β -adrenergic stimulation. the dose range of catecholamine for these studies was chosen to alter inotropic and lusitropic states without causing an untoward increase in heart rate. steady-state hemodynamic measurements were made during minutes and of each infusion period. at each level, the pacemaker was briefly turned off to determine the effect of dobutamine on the heart rate. four of the animals in this group were studied with incremental pacing both before and after β-adrenergic blockade with esmolol ( . mg Á kg À Á min À i.v.). the pacing protocol and the results from a larger group of animals studied before β-adrenergic blockade were detailed in a previous report. briefly, atrial pacing was instituted at a rate above the intrinsic heart rate to avoid competing rhythms and was increased at . -hz increments until the critical heart rate was achieved. the critical heart rate was defined as the rate at which dp/dt max and τ reached a maximum and minimum, respectively, during progressive increases in heart rate. we showed previously that hyperthyroidism significantly increases the critical heart rates for both dp/dt max and τ. the ec of dobutamine for lv dp/dt max was determined by fitting log(dose)-transformed data to a sigmoidal relation with software from graphpad. additional animals were chronically instrumented so that we could examine the effects of β -adrenergic stimulation. one animal died suddenly after receiving thyroid hormone for days. in the remaining three animals, the β -adrenergic agonist terbutaline was infused both before and after production of the hyperthyroid state. incremental doses of terbutaline ( min/dose) were infused over a dosing range of - ng Á kg À Á min À . thyroid function tests were performed before the baseline experiment in the euthyroid state and before the terminal experiment (within h of the last dose of t ) in the hyperthyroid state. t radioimmunoassay, t , and free t levels were measured at each state. paired mean data were compared by student's t-test. the effects of thyroid status, catecholamine dose, and β-blockade on hemodynamic and dimension variables were examined with repeated-measures anova (superanova, abacus concepts). when significant differences were found, group means were compared with contrasts. a value of p < . was considered significant. unless specified, data are expressed as mean ae sd. weiss jl, frederickson jw, weisfeldt ml ( ) hemodynamic determinants of the time course of fall in canine left ventricular pressure. j clin invest : - cardiac failure in other species various species have been used to study experimental cardiac failure. breisch et al. ( ) studied the effects of pressure-overload hypertrophy in the left myocardium of young adult cats. hypertrophy was induced by a % constriction of the ascending aorta. recommended dilated cardiomyopathy in turkeys as an animal model for the study of human heart failure. studied energy metabolism in normal and hypertrophied right ventricle of the ferret heart. studied ca + handling and myofibrillar ca + sensitivity in ferret cardiac myocytes with pressure-overload hypertrophy. bovine hereditary cardiomyopathy was recommended as an animal model of human dilated cardiomyopathy by . primary cultures from cardiomyocytes are prepared from ventricles of -day-old neonatal wistar kyoto rats. according to the method of , the cultures are treated for days with . mm bromodeoxyuridine to suppress proliferation of nonmyocardial cells. elastic culture dishes (   cm) are made by vulcanizing liquid silicone rubber consisting of methylvinyl polysiloxane and dimethyl hydrogen silicone resin using platinum as a catalyst. the bottom of the disk is mm thick, and it is highly transparent because of no inorganic filler in either component. cells are plated in a field density of  cells/cm in culture medium consisting of dulbecco's modified eagle's medium with % fetal bovine serum. mechanical stress on cardiac cells is applied by gently pulling and hanging the dish on pegs. a % change in length of the dish results in an almost identical change in the length of the cell along a single axis ). cardiocytes are stretched by %, %, or %. drugs, e.g., an angiotensin ii receptor antagonist, are added min before stretch. for protein analysis, the silicone dishes are stretched for h after days of serum starvation and [ h]phenylalanine ( μci/ml) is added for min. at the end of each stress, the cells are rapidly rinsed four times with ice-cold phosphate-buffered saline and incubated for min on ice with ml of %trichloroacetic acid. the total trichloroacetic acid-insoluble radioactivity in each dish is determined by liquid scintillation counting. for the determination of mitogen-activated protein kinase, cardiomyocytes are lysed on ice and centrifuged. aliquots of the supernatants of myocyte extracts are incubated in kinase buffer ( mm/l tris-hcl, ph . , mm/l mgcl , mm/l dithiothreitol, μm/l apt, μci [γ - p]atp, μm/l protein kinase inhibitor peptide, and . mm/l egta) and substrates ( -μg myelin basic protein). the reaction is stopped by adding stopping solution containing . %hcl, mm/l atp, and % bovine serum albumin. aliquots of the supernatant are spotted on p paper (whatman), washed in . % phosphoric acid, dried, and counted. for determination ofc-fos mrna, northern blot analysis is performed. values are expressed as mean ae sem. comparisons between groups are made by one-way anova followed by dunnett's modified t-test. the interesting approach to induce hypertrophy of cardiac cells in vitro has been used predominantly by one research group. confirmation by other research groups including modifications of the mechanical procedures seems to be necessary. transition from compensated hypertrophy to intrinsic myocardial dysfunction during development of left ventricular pressure-overload hypertrophy in conscious sheep. systolic dysfunction precedes diastolic dysfunction an ovine model of tachycardia-induced degenerative dilated cardiomyopathy and heart failure with prolonged onset induction of early biomarkers in a thrombusinduced sheep model of ischemic heart failure natriuretic peptides in sheep with pressure overload left ventricular hypertrophy natriuretic peptides maintain sodium homoeostasis during chronic volume loading post-myocardial infarction in sheep remodeling of chronic severely failing ischemic sheep heart after coronary microembolization: functional, energetic, structural, and cellular responses selective microembolization of the circumflex coronary artery in an ovine model: dilated, ischemic cardiomyopathy and left ventricular dysfunction disparate effects of early pressure overload hypertrophy and force-dependent indices of ventricular performance in the conscious baboon effects of angiotensin ii generated by an angiotensin converting enzyme-independent pathway on left ventricular performance in the conscious baboon effects of thyroid hormone on cardiac β-adrenergic responsiveness in conscious baboons the effects of a thyroid hormone analog on left ventricular performance and contractile and calcium cycling proteins in the baboon role of hypertension in ischemic heart disease and cerebral vascular disease in the cynomolgus monkey with coarctation of the aorta effects of thyroid hormone on left ventricular performance and regulation of contractile and ca + -cycling proteins in the baboon cardiotoxic and possible leukemogenic effects of adriamycin in nonhuman primates myocardial characteristics of pressure overload hypertrophy. a structural and functional study energy metabolism in normal and hypertrophied right ventricle of the ferret heart bovine hereditary cardiomyopathy: an animal model of human dilated cardiomyopathy dilated cardiomyopathy in turkeys: an animal model for the study of human heart failure ca + handling and myofibrillar ca + sensitivity in ferret cardiac myocytes with pressure-overload hypertrophy angiotensin ii receptor antagonist tcv- induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intercellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro control of cardiac gene expression by mechanical stress stretching cardiac myocytes stimulates protooncogene expression mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells mechanical loading activates mitogen-activated protein kinase and s peptide kinase in cultured cardiac myocytes in vitro methods to study hypertrophy of cardiac cells molecular aspects of mechanical stress-induced hypertrophy references and further reading large animal models for diastolic dysfunction and diastolic heart failure-a review of the literature rodent models of heart failure: an updated review animal models of human cardiovascular disease, heart failure and hypertrophy animal models of chronic heart failure small animal models of heart failure: development of novel therapies, past and present animal models of cardiorenal syndrome: a review experimental models of heart failure aortic banding in rats blockade of the renin-angiotensin system in cardiac pressure-overload hypertrophy in rats selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with aortic banding cardiac and vascular effects of chronic angiotensin converting enzyme inhibition at subantihypertensive doses effects of losartan, an angiotensin ii antagonist, on the development of cardiac hypertrophy due to volume overload a specific b -bradykinin receptor antagonist hoe abolishes the antihypertrophic effect of ramipril converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats role of angiotensin ii receptor antagonism and converting enzyme inhibition in the progression and regression of cardiac hypertrophy in rats ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats contribution of bradykinin to the cardiovascular effects of ramipril bradykinin prevents left ventricular hypertrophy in rats the contribution of bradykinin to the cardiovascular actions of ace inhibitors ace inhibition decreases postoperative mortality in rats with left ventricular hypertrophy and myocardial infarction novel experimental model of pressure overload hypertrophy in rats central vasopressin in experimental aortic stenosis in the rat regulation of aortic atrial natriuretic factor and angiotensinogen in experimental hypertension hyperplastic growth response of vascular smooth muscle cells following induction of acute hypertension in rats by aortic coarctation experimental cardiovascular benefits of angiotensin-converting enzyme inhibitors: beyond blood pressure reduction alteration of growth responses in established cardiac pressure overload hypertrophy in rats with aortic banding a simple method for producing graded aortic insufficiencies in rats and subsequent development of cardiac hypertrophy baroreflex and β-adrenoceptor function are diminished in rat cardiac hypertrophy due to volume overload angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis angiotensin at receptor inhibition: effects on hypertrophic remodelling and ace expression in rats with pressure-overload hypertrophy due to ascending aortic stenosis alterations of cardiac adrenoceptors and calcium channels subsequent to production of aortic insufficiency in rats development of different phenotypes of hypertensive heart failure: systolic versus diastolic failure in dahl salt-sensitive rats chronic arteriovenous shunt: evaluation of a model for heart failure in rat simple, rapid, and effective method of producing aortacaval shunts in the rat defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure neurohumoral responses to chronic myocardial infarction in rats transition from compensatory hypertrophy to dilated, failing left ventricles in dahl salt-sensitive rats the effect of myocardial adaptation to volume overload in the rat a model of chronic heart failure in spontaneous hypertensive rats (shr) nhe- inhibition improves impaired mitochondrial permeability transition and respiratory function during postinfarction remodelling in the rat angiotensin ii receptor blockade attenuates the deleterious effects of exercise training on post-mi ventricular remodelling in rats proteomic analysis of left ventricular diastolic dysfunction hearts in renovascular hypertensive rats myocyte cellular hyperplasia and myocytes cellular hypertrophy contribute to chronic ventricular remodelling in coronary artery narrowing-induced cardiomyopathy in rats selective sensory denervation by capsaicin aggravates adriamycin-induced cardiomyopathy in rats development of heart failure in chronic hypertensive dahl rats: focus on heart failure with preserved ejection fraction regional changes in hemodynamics and cardiomyocyte size in rats with aortacaval fistulas developing and established hypertrophy effects of angiotensin-converting enzyme inhibitors and angiotensin ii type receptor antagonists in rats with heart failure chronic heart failure induced by coronary artery ligation in lewis inbred rats a simple method for collagen and total protein determination in formalinfixed paraffin-embedded sections systemic and regional hemodynamic effects of perindopril in experimental heart failure animal models of chronic heart failure ventricular enlargement and reduced survival after myocardial infarction myocardial infarct size and ventricular function in rats survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril matrix metalloproteinase inhibition improves cardiac dysfunction and remodeling in -kidney, -clip hypertension determination of rat heart morphology and function in vivo in two models of cardiac hypertrophy by means of magnetic resonance imaging a canine model of chronic heart failure produced by multiple sequential coronary microembolizations simple techniques for the surgical occlusion of coronary vessels in the rat effect of left ventricular sphericity on the evolution of ventricular dysfunction in rats with diffuse isoproterenol-induced myocardial necrosis neurohumoral and hemodynamic effects of ibopamine in a rat model of chronic myocardial infarction and heart failure relation between myocardial β-adrenoceptor density and hemodynamic and neurohumoral changes in a rat model of chronic myocardial infarction: effects of ibopamine and captopril myosin heavy chain regulation and myocytes contractile depression after lv hypertrophy in aortic-banded mice prolonged endoplasmatic reticulum stress in hypertrophic and failing heart after aortic constriction. possible contribution of endoplasmatic reticulum stress to cardiac myocyte apoptosis segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy in vivo murine cardiac hypertrophy. a novel model to identify genetic signaling mechanisms that activate an adaptive physiological response characterization of a model to independently study regression of ventricular hypertrophy nifedipine and diltiazem suppress ventricular arrhythmogenesis and calcium release in mouse hearts cardiac adaptations to chronic exercise in mice benidipine, a long-acting calcium channel blocker, inhibits cardiac remodeling in pressure-overloaded mice molecular and physiological alterations in murine ventricular dysfunction a new model of congestive heart failure in the mouse due to chronic volume overload ) βark inhibition improves survival in a mouse model of heart failure induced by myocardial infarction angiotensin deficiency in mice leads to dilated cardiomyopathy role of a selective aldosterone blocker in mice with chronic heart failure dual inhibition of ace and nep provides greater cardioprotection in mice with heart failure chronic xanthine oxidase inhibition prevents myofibrillar protein oxidation and preserves cardiac function in a transgenic mouse model of cardiomyopathy cardiomyopathy in transgenic myf mice a mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoforms of the adenine nucleotide translocator genetic modification of the heart: transgenic modification of cardiac lipid and carbohydrate utilization many good reasons to have stat in the heart cardiomyopathy induced by cardiac gs α overexpression remodelling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin reversal of amyloidinduced heart disease in desmin-related cardiomyopathy increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of tgf-β cardiac insufficiency in guinea pigs a stable ovine congestive heart failure model. a suitable substrate for left ventricular assist device assessment differential changes in cardiac phospholamban and sarcoplasmic reticular ca + -atpase protein levels. effects on ca + transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure hemodynamic benefits and prolonged survival with long-term captopril therapy in rats with myocardial infarction and heart failure simple techniques for the surgical occlusion of coronary vessels in the rat compensatory hypertrophy and failure in gradual pressure-overloaded guinea pig heart depressed intracellular calcium transients and contraction in myocytes from hypertrophied and failing guinea pig hearts assessment of subrenal banding of the abdominal aorta as a method of inducing cardiac hypertrophy in the guinea pig untersuchungen an der experimentellen hydropischen herzinsuffizienz des meerschweinchens Über die experimentelle hydropische herzinsuffizienz am meerschweinchen. operationstechnik, einfluß zusätzlicher faktoren und effekt von herzglykosiden hereditary cardiomyopathy: a new disease model a new disease model of chronic congestive heart failure: studies on its pathogenesis the heart muscle in muscular dystrophy with special reference to involvement of the cardiovascular system in the hereditary myopathy of the hamster spontaneous hereditary myocardial degeneration and congestive heart failure in a strain of syrian hamsters dissociation of factors influencing myocardial degeneration and generalized cardiocirculatory failure the syrian hamster as a model for the dilated cardiomyopathy of chagas' disease: a quantitative echocardiographical and histopathological analysis mechanical and electrical properties of cardiomyopathic hearts of syrian hamsters chronic calcium channel blockade prevents the progression of myocardial contractile and electrical dysfunction in the cardiomyopathic syrian hamster effects of perindopril on myocardial inotropy, lusitropy and economy, and on diaphragm contractility in the cardiomyopathic syrian hamster myocardial effects of early therapy with perindopril during experimental cardiomyopathy effects of milrinone treatment in cardiomyopathic hamsters (chf ) with severe congestive heart failure non-invasive evaluation of the cardiac function in golden retriever dogs by radionuclide angiography cardiac collagen remodelling in the cardiomyopathic syrian hamster and the effect of losartan the pathogenesis of clinical and experimental congestive cardiomyopathy: recent concepts microvascular spasm in the cardiomyopathic syrian hamster: a preventable cause of focal myocardial necrosis myocardial contractility in relation to hypertrophy and failure in myopathic syrian hamsters cardiac collagen changes during the development of right ventricular hypertrophy in tight-skin mice with emphysema cardiomyopathic syrian hamster: a possible model of human disease effects of quinapril, a new angiotensin converting enzyme inhibitor, on left ventricular failure and survival in the cardiomyopathic hamster effects of hydrochlorothiazide and captopril on the survival and heart weight of cardiomyopathic hamsters myopathy of hamster dystrophy: history and morphological aspects new models of human disease in syrian hamsters hereditary polymyopathy and cardiomyopathy in the syrian hamster: i. progression of heart and skeleton muscle lesions in the um-x . line calcium and myocardial cell injury. an appraisal in the cardiomyopathic hamster effects of angiotensin converting enzyme inhibitors and the role of the renin-angiotensin-aldosterone system in j- -n cardiomyopathic hamsters simultaneous reduction of the sarcolemmal and sr calcium aptase activities and gene expression in cardiomyopathic hamster multiple cardiac contractile protein abnormalities in myopathic syrian hamsters (bio : ) tsk mice with genetic emphysema. right ventricular hypertrophy occurs without hypertrophy of muscular pulmonary arteries or muscularization of arterioles identification of the syrian hamster cardiomyopathy gene inotropic and calcium kinetic effects of calcium channel agonist and antagonist in isolated cardiac myocytes from cardiomyopathic hamsters hereditary and acquired cardiomyopathies in experimental animals: mechanical, biochemical and structural features coronary and cardiac sensitivity to the vasoselective benzothiazepine-like calcium antagonist, clentiazem, in experimental heart failure alprazolam reduces stressinduced mortality in cardiomyopathic hamsters pimobendan increases survival of cardiomyopathic hamsters nebivolol increases survival in cardiomyopathic hamsters with congestive heart failure calcium transport properties of cardiac sarcoplasmic reticulum from cardiomyopathic syrian hamsters (bio . and . ): evidence for a quantitative defect in dilated myopathic hearts not evident in hypertrophic hearts increased sensitivity of ventricular myocardium to intracellular calciumoverload in syrian cardiomyopathic hamster cardiac failure in rabbits electrographic changes following rabbit coronavirus-induced myocarditis and dilated cardiomyopathy adriamycin cardiomyopathy in the rabbit: an animal model of low output cardiac failure with activation of vasoconstrictor mechanisms sr calcium handling and calcium after-transients in a rabbit model of heart failure increased na + /h + -exchange activity is the cause of increased [na + ] i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model betaadrenoceptors and adenylate cyclase activity in hypertrophied and failing rabbit left ventricle enhanced phosphorylation of phospholamban and downregulation of sarco/ endoplasmatic reticulum ca + atpase type (serca ) in cardiac sarcoplasmatic reticulum from rabbits with heart failure cellular uncoupling during ischemia in hypertrophied and failing rabbit ventricular myocardium: effects of preconditioning cellular uncoupling during ischemia in hypertrophied and failing rabbit ventricular myocardium: effects of preconditioning myosin heavy chain synthesis during progression of chronic tachycardia induced heart failure in rabbits increased negative inotropic effect of calcium-channel blockers in hypertrophied and failing rabbit heart rapidly adapting receptors in a rabbit model of mitral regurgitation protein turnover in compensated chronic aortic regurgitation comparative study of chronic toxic effects of daunorubicin and doxorubicin in rabbits attenuated outward potassium currents in carotid body glomus cells of heart failure rabbit: involvement of nitric oxide fibrosis, myocyte degeneration and heart failure in chronic aortic regurgitation differential expression of cardiac anp and bnp in a rabbit model of progressive left ventricular dysfunction hypertrophic and functional response to experimental chronic aortic regurgitation heart failure due to chronic aortic regurgitation dynamic arterial baroreflex in rabbits with heart failure induced by rapid pacing altered oscillatory work by ventricular myofilaments from a rabbit coronary artery ligation model of heart failure protein kinase c activity and expression in rabbit left ventricular hypertrophy animal models of chronic heart failure echocardiographic changes after myocardial infarction in a model of left ventricular dysfunction upregulation of na + /ca + exchanger expression and function in an arrhythmogenic rabbit model of heart failure tachycardia heart failure alters rabbit aortic smooth muscle responsiveness to angiotensin ii matrix metalloproteinase expression in cardiac myocytes following myocardial infarction in the rabbit molecular correlates to altered expression of potassium currents in failing rabbit myocardium transgenic model for human troponin i-based hypertrophic cardiomyopathy sequential changes in sympatho-neuronal regulation and contractile function following aortic regurgitation in rabbit heart cardiac failure in dogs rapid ventricular pacing in the dog: pathophysiological studies of heart failure a nonchirurgical model of chronic left ventricular dysfunction effect of heart failure on the mechanism of exercise-induced augmentation of mitral valve flow compensatory asymmetry in down-regulation and inhibition of the myocardial ca + cycle in congestive heart failure in dogs by idiopathic dilated cardiomyopathy and rapid ventricular pacing increased ace and chymase-like activity in cardiac tissue of dogs with chronic mitral regurgitation early left ventricular dysfunction elicits activation of sympathetic drive and attenuation of parasympathetic tone in the paced canine model of congestive heart failure influence of heart rate on left ventricular performance in conscious dogs myocardial beta receptor and voltage sensitive calcium channel changes in a canine model of chronic heart failure load versus humoral activation in the genesis of early hypertensive heart disease diastolic properties in canine hypertensive left ventricular hypertrophy: effects of angiotensin converting enzyme inhibition and angiotensin ii type- receptor blockade load-sensitive diastolic relaxation in hypertrophied left ventricles acute, reversible tricuspid insufficiency: creation of a canine model factors involved in delaying the rise in peripheral resistance in developing heart failure volume overload hypertrophy in a closed chest model of mitral regurgitation premorbid determinants of left ventricular dysfunction in a novel model of gradually induced pressure overload in the adult canine alterations in left ventricular diastolic function in conscious dogs with pacing-induced heart failure exhaustion of frank-starling mechanism in conscious dogs with heart failure angiotensin ii in the evolution of experimental heart failure a model of left ventricular dysfunction caused by intracoronary adriamycin hemodynamic and humoral effects of vasopeptidase inhibition in canine hypertension hemodynamic, left ventricular structural and hormonal changes after discrete myocardial damage in the dog native β-adrenergic support for left ventricular dysfunction in experimental mitral regurgitation normalizes indexes of pump and contractile function mechanisms of altered excitationcontraction coupling in canine tachycardia-induced heart failure. i: experimental studies mechanism of altered patterns of left ventricular filling during the development of congestive heart failure a method for producing persistent hypertension by cellophane abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure tachycardia induced failure alters contractile properties of canine ventricular myocytes cardiorenal and neurohumoral function in a canine model of left ventricular dysfunction a canine model of chronic heart failure produced by multiple sequential coronary microembolizations decreases proportion of type i myofibers in skeletal muscle of dogs with chronic heart failure effects of long term monotherapy with enalapril, metoprolol, and digoxin on the progression of left ventricular dysfunction and dilation in dogs with reduced ejection fraction tachycardiainduced cardiomyopathy: a review of animal models and clinical studies angiotensin-converting enzyme inhibition and the progression of congestive cardiomyopathy differential expression of angiotensin-converting enzyme and chymase in dogs with chronic mitral regurgitation hemodynamic and neurohumoral response in heart failure produced by rapid ventricular pacing canine x-linked muscular dystrophy. an animal model of duchenne muscular dystrophy: clinical studies experimental models of heart failure decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure defective endothelium-mediated control of coronary circulation in conscious dogs after heart failure endomyocardial gene expression during development of pacing tachycardia-induced heart failure in the dog experimental congestive heart failure produced by rapid ventricular pacing in the dog: cardiac effects mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, ii: model studies calcium sensitivity of isometric tension is increased in canine experimental heart failure effects of chronic supraventricular pacing tachycardia on relaxation rate in isolated cardiac muscle cells cineangiography of the heart in a single breath hold with segmented turboflash sequence gene expression in a swine model of ventricular hypertrophy: intercellular adhesion molecule, vascular endothelial growth factor and plasminogen activators are upregulated during pressure overload rapid ventricular pacing in pigs: an experimental model of congestive heart failure pacing-induced dilated cardiomyopathy increases left-to-right ventricular systolic interaction dynamic programming for detecting, tracking and matching deformable contours wall stress and patterns of hypertrophy in human left ventricle phase modulated rotating frame spectroscopic localization using an adiabatic plane rotation pulse and a single surface coil coronary arterial tree remodelling in right ventricular hypertrophy remodeling of the bifurcation asymmetry of right ventricular braches in hypertrophy amlodipine therapy in congestive heart failure: hemodynamic and neurohormonal effects at rest and after treadmill exercise rapid, accurate and simultaneous noninvasive assessment of right and left ventricular mass with nuclear magnetic resonance imaging using the snapshot gradient method abnormal myocardial bioenergetics in canine asymptomatic left ventricular dysfunction long-term angiotensin-converting enzyme and angiotensin i receptor inhibition in pacing-induced heart failure. effects on myocardial interstitial bradykinin levels transmural high energy phosphate distribution and response to alterations in workload in the normal canine myocardium as studied with spatially localized p nmr spectroscopy chronic supraventricular tachycardia causes ventricular dysfunction and subendocardial injury in swine relationship between bioimpedance, thermodilution, and ventriculographic measurements in experimental congestive heart failure collagen remodelling and changes in lv function during development and recovery from supraventricular tachycardia relation between ventricular and myocytes function with tachycardiainduced cardiomyopathy contrast-enhanced first pass myocardial perfusion imaging: correlation between myocardial blood flow in dogs at rest and during hyperemia bioenergetic consequence of left ventricular remodeling secondary to discrete myocardial infarction functional and bioenergetic consequences of postinfarction left remodelling in a new porcine model cardiac failure in sheep transition from compensated hypertrophy to intrinsic myocardial dysfunction during development of left ventricular pressureoverload hypertrophy in conscious sheep. systolic dysfunction precedes diastolic dysfunction an ovine model of tachycardiainduced degenerative dilated cardiomyopathy and heart failure with prolonged onset induction of early biomarkers in a thrombus-induced sheep model of ischemic heart failure natriuretic peptides in sheep with pressure overload left ventricular hypertrophy natriuretic peptides maintain sodium homoeostasis during chronic volume loading post-myocardial infarction in sheep remodeling of chronic severely failing ischemic sheep heart after coronary microembolization: functional, energetic, structural, and cellular responses selective microembolization of the circumflex coronary artery in an ovine model: dilated, ischemic cardiomyopathy and left ventricular dysfunction effect of remodelling, stretch and ischemia on ventricular fibrillation frequency and dynamics in a heart failure model beneficial hemodynamic and renal effects of adrenomedullin in an ovine model of heart failure combined angiotensin-converting enzyme inhibition and adrenomedullin in an ovine model of heart failure four-day urocortin-i administration has sustained beneficial hemodynamic, hormonal, and renal effects in experimental heart failure cardiac failure in monkeys disparate effects of early pressure overload hypertrophy and force-dependent indices of ventricular performance in the conscious baboon effects of angiotensin ii generated by an angiotensin converting enzyme-independent pathway on left ventricular performance in the conscious baboon effects of thyroid hormone on cardiac βadrenergic responsiveness in conscious baboons the effects of a thyroid hormone analog on left ventricular performance and contractile and calcium cycling proteins in the baboon role of hypertension in ischemic heart disease and cerebral vascular disease in the cynomolgus monkey with coarctation of the aorta effects of thyroid hormone on left ventricular performance and regulation of contractile and ca + -cycling proteins in the baboon cardiotoxic and possible leukemogenic effects of adriamycin in nonhuman primates hemodynamic determinants of the time course of fall in canine left ventricular pressure cardiac failure in other species breisch ea, white fc, bloor cm ( ) myocardial characteristics of pressure overload hypertrophy. a structural and functional study energy metabolism in normal and hypertrophied right ventricle of the ferret heart bovine hereditary cardiomyopathy: an animal model of human dilated cardiomyopathy dilated cardiomyopathy in turkeys: an animal model for the study of human heart failure ca + handling and myofibrillar ca + sensitivity in ferret cardiac myocytes with pressureoverload hypertrophy angiotensin ii receptor antagonist tcv- induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intercellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro control of cardiac gene expression by mechanical stress stretching cardiac myocytes stimulates protooncogene expression mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells mechanical loading activates mitogenactivated protein kinase and s peptide kinase in cultured cardiac myocytes in vitro methods to study hypertrophy of cardiac cells molecular aspects of mechanical stress-induced hypertrophy key: cord- -for qa s authors: chandrappa, ramesha; chandra kulshrestha, umesh title: major issues of air pollution date: - - journal: sustainable air pollution management doi: . / - - - - _ sha: doc_id: cord_uid: for qa s environmental issues change from place to place and time to time. the issues include local as well as global issues. the understanding of issues is necessary to find solution. air pollution issues have changed over a period of time. issues like atmospheric brown cloud, climate change, hazardous air pollutants, black/muddy snow which are hardly discussed few decades back have now gaining importance. this chapter elaborates major issues due to air pollution. atmosphere and its layers when the solar system condensed out of "primordial solar nebula" which is nothing but interstellar cloud of gas and dust, the situation was not as complex as today and air pollution was not an issue. the early atmosphere of this planet was believed to be blend of carbon dioxide, nitrogen, water vapour and hydrogen. the early atmosphere of this planet was slightly reducing chemical mixture as compared to present atmosphere which is strongly oxidizing. with lapse of time, distinct layers of the atmosphere were formed with distinct characteristics. these are described below. troposphere: it is the lower most layer of atmosphere extending from the earth's surface to - km altitude depending on time and latitude ( fig. . ). this layer is characterised by declining temperature with height and rapid vertical mixing. temperature at the ground is about °c and it decreases gradually till the tropopause is reached. the tropopause is the boundary having constant temperature which separates the troposphere and stratosphere. the decrease of atmospheric temperature (t) with height (z) is called as lapse rate (α) which is expressed as follows the change in temperature of a mass of air as it moves upwards is called as adiabatic lapse rate. the dry adiabatic lapse rate (dalr) and the moist adiabatic lapse rate (malr) are . and . °c/km respectively. actual change of temperature with altitude for the stationary atmosphere or temperature gradient is known as environmental lapse rate (elr). generally, decrease of . °c/km is considered as environmental lapse rate. in the troposphere, atmospheric pressure also decreases rapidly with the altitude. one can understand that climbing to an altitude of . km, would put you above % of the atmosphere's molecules where atmospheric pressure is mb only. actually, the heating of the surface creates warm air at surface. the warm air rises, but air expands as it rises and cools as it expands (adiabatic cooling). typically, the troposphere is characterized by warm air at surface and cooler air above. if the cooler air exists at surface and the warmer air above, this is called as buoyancy. if a rising air parcel becomes saturated condensation occurs. the condensation warms the air parcel due to the release of latent heat. so, a rising parcel cools less if it is saturated the atmosphere near earth's surface is divided into different layers based on wind behaviour: the laminar sublayer also called the viscous sublayer, is the region in which the flow is laminar. with respect atmosphere it is usually less a centimetre. the surface layer extends from ≤ - m. the heat and vertical turbulent fluxes in this layer are constant. the ekman layer is the layer in a fluid where force balance between pressure gradient force, turbulent drag and coriolis force are balanced. it was named after scientist vagn walfrid ekman who explained the phenomena for the first time. ekman layer extends to height of - m. in this layer wind direction is affected by earth's rotation. wind speed his layer generally increases with height. free atmosphere is the layer above ekman layer in which the effect of the surface friction on the air motion is negligible. stratosphere: it is positioned just above the troposphere extending from - km. in the stratosphere, temperature increases with altitude, from − °c at base to °c at the top of the stratosphere. the increase in temperature is basically due to the absorption of solar energy by the ozone layer. ozone (o ) is effective absorbing species for solar uv radiation ( - nm with a maximal absorption at about nm) in the stratosphere. generally, uv-c ( - nm) which is highly harmful is entirely screened out by dioxygen (< nm) and ozone (>about nm) before km height. uv-b ( - nm) radiation is mostly screened by ozone layer. uv-a ( - nm) reaches earth surface having small damage e.g. premature ageing of skin etc. mesosphere: just above the stratosphere, the mesosphere exists extending from - km altitude, the space shuttles orbit in this layer of the atmosphere. due to decrease in solar heating, temperature decreases with altitude in the mesosphere, °c at base, − °c at the top of the mesosphere. the top of the mesosphere is the coldest region of atmosphere. polar mesospheric clouds of water ice are seen in this layer which are known as noctilucent clouds. these are highest clouds in earth atmosphere and are seen mostly during summer months between and degree n and s. the d layer of ionosphere also exists in mesosphere which is seen during the day time. meteors burn up in the mesosphere while entering the earth atmosphere. thermosphere: thermosphere is the last layer of the atmosphere which exists at km and above up to exosphere. in the thermosphere, the temperature increases with altitude as atoms of this layer are accelerated by solar radiation. temperature at the base of the thermosphere is − °c but it is °c at km and °c at upper part. though the temperature of this layer is very high but the heat content negligible. auroras exist in thermosphere. the auroras are seen due to the effect of energetic particles (electrons and photons) coming in the solar wind. charged particle entering the atmosphere ionize atmospheric constituents. the aurora can be seen best in the dark sky or 'magnetic midnight' time. in northern latitudes, the effect is known as the aurora borealis (or the northern lights), named after the roman goddess of dawn, aurora, and the greek name for the north wind, boreas, by galileo in . international space station orbits in upper part of thermosphere ( - km). in the thermosphere, ionization occurs due to uv rays. e and f regions of ionosphere exist in this layer. at the exosphere (beginning - km), the atmosphere turns into space. ionosphere: in fact, the ionosphere extends from - km covering partly mesosphere and thermosphere. it has diurnal and seasonal variation as the ionization depends upon sun and its activity. as mentioned earlier, the d region of the ionosphere exists in the mesosphere while e and f regions of the ionosphere exist in the thermosphere. ionosphere is a shell of electrons and electrically charged atoms and molecules. e and f regions are present at nights. d region is formed during day time when the e and f regions become much stronger. often during the day, the f region is further differentiated into f and f regions. in the d region, uv rays ionize no and x-rays ionize o and n . in the e region, x-rays and far uv ionize o À . e region can reflect radio waves lower than mhz. extreme uv rays ionize o in the f-region during nights. f layer is responsible for short wave (hf) radio communication for long distances. ever since the discovery of fire, air pollution has been a problem. "heavy air of rome" in a.d., has been recorded by roman philosopher seneca. in , king edward i prohibited burning of coal in london (william and lou ) . by the s, people were using coal as fuel in processes like limekilns and metalworking leading to air pollution which had black smoke as well as oxides of sulphur. late th and early th centuries saw dramatic changes in manufacturing, agriculture, mining, production as well as transportation. invention of electric power in the nineteenth century resulted in coal fired electric generation in s. very famous example of air pollution is the smog formation around los angeles during the s which led to the passing of first state environmental legislation in usa. in , the air pollution control act was enacted in usa which was the first federal environmental legislation in the country. later on in s, oil overtook coal as the source of primary energy. extensive use of oil led to the emissions wherever vehicles moved. with the industrial revolution in the post eighteenth century economy changed to machine-based manufacturing in many of the present developed countries. mechanization of the textile industries and iron-making techniques increased demand for fuel and their by air pollution in those areas of such activities. the developments in th century led to second industrial revolution. the construction activity also saw shift in construction material as well as technology. the invention of cement replaced mud walls ( fig. . ) and increase in cement demand lead to emissions from this sector. as the european and american markets were saturated, asian markets opened up for vehicles which are currently unbalanced where in poor people ride on bus top or trucks while rich people ride in individual cars ( fig. . ) . while the economic crisis in greece resulted in reduction of air pollution (vrekoussis et al. ) china witnessed dramatic air quality detonation last decade. analysis of data from monitoring network created by who and unep in cities in developed as well as developing countries shows that over the past - year indicate that the lessons of earlier experiences in the now developed countries have yet to be learned. air pollution in of the megacities shows that ambient air pollution at levels where serious health effects (david et al. ) . the rise of population in the developing countries in future with a lack for air pollution control will worsen in many more cities. in the beginning of s when the rapid growth in europe lead to environmental pollution and air pollution of london which resulted in death of more than people was fresh in memory the united national conference of the human environment in stockholm in lead to foundation of international cooperation in this regard. this is followed by series of development that aimed to bring down air pollution. convention on long-range transboundary air pollution in was signed unece countries. governments of unece member states signed the convention on long-range transboundary air pollution on november . the lphur protocol or % protocol aimed to bring down % so at national level by countries of unece region. all business decisions affect the air and atmosphere. hence, like water which is purified, packaged and priced, soon pure air will also be priced. there are oxygen bars opened up in many parts of the world to supply oxygen to customers. however, inspite of urgent need of stringent air pollution policies and regulations in several parts of the world, air pollution control is still not a political priority as compared to the business and economy in many parts of the world. as a result the pollution is continued in one form of other, many forms are not even monitored and controlled. over the years only few conventional air pollutants such so , no , fig. . traffic from a developing asian country particulate matter, o etc. are monitored by the researchers and the pollution control authorities. pollutants such as persistent organic pollutants (pops) were neglected in the past but have been considered recently for continuous monitoring due to their severe health effects. both organic as well as inorganic air pollutants cause deadly deceases and hence, their monitoring is very important for humans and environment. while many developing countries took the matter seriously others were only keen to satisfy international community. even though environmental legislations were enacted all over the world, the capacity of enforcing agencies was limited mainly due to insufficient knowledge and research capacity with enforcing agency. many organisations had very few staff to start with limited budget to monitor and travel. the absence of expertise had either lead to improper monitoring by selecting improper sampling site/methodology and poor analysis. many organisations till date are dogged with insufficient manpower to the extent of one to ten technical/scientific staff for a million of citizens. what makes air pollution most challenging compared to other pollution is its complexity. as mentioned above, unlike water which can be contained in a container for easy study it is difficult to simulate the atmospheric setup in a laboratory. further, aerodynamics at earth's surface cannot be easily explained by mathematics as it occurs in nature. a variety of factors like radiation, friction, flow pattern, chemical reaction, influence by biological setup, changing climate, changing weather, changing living style, new inventions, social changes, law of the land, attitude of the people, physiology of people, economic changes of the region together is responsible for the scenario at a given time at a given reason. due to complexity of problem, air pollution has not been thoroughly understood by many developing countries and is not a priority. issues like the poor governance, low research capability, illiteracy, corruption, national/international conflicts and political instability has often cause of low attention to air pollution in spite that millions die due to air pollution all over the world. in spite of magnitude of the problem, the loss of life and wealth due to air pollution is invisible to many government servants. this could be attributed to low emotional intelligence of people responsible to serve the people who act as trustees of the country to protect interest of people and property of the country. illiteracy among the citizens was also cause for not complaining about the pollution. the use of staff for other duties like election/census/sports has also one of the many reason for poor implementation of environmental laws. many of the enforcing agency are worried about financial expenditure rather pollution control as misappropriation of financial resource could end up officer responsible for appreciation in jail. on the other hand, the unaccounted pollution is not at all fault as serious as financial misappropriation. the environmental laws can also be misused to raise funds or cause inconvenience to rivals by people in power. as shown in fig. . , issues, causes, influencing factors and impacts of air pollution can be attributed to many aspects which are not quantifiable. the corruption among governance, low ethics among industries, non availability of technology, incapability to adopt new technology and low research capability plague many countries. in spite of enthusiasm shown by many international agencies to support the cause it is often denied or poorly adopted by beneficiary countries. the major sources of pollution are combustion process, industry, transportation, waste disposal, use of agro chemicals, and respiration of living organisms (famhy et al. ). none of these sources can be avoided as they are meant for survival of the humans. apart from these sources other sources like accidental fire; wind storms; natural disasters; education/research; decomposition of dead and decaying matter; wars; bursting of crackers; use of explosives; sports/events; testing/practicing of use of war weapons; launching satellites; volcanic eruption; construction; methane generation in rice fields due to biodegradation; demolition of buildings; methane generated by ruminant animals during digestion of food; painting; processing of grains; soil erosion and weathering of rock/minerals add to pollution. service sectors like healthcare, software, business process out sourcing (bpo) also contribute air pollutants while using equipments/air conditioners/ transportation. release of pathogens from health care establishment, animal rearing, slaughtering, and research can be far more detrimental compared to conventional pollutants. unlike war and crime effect of air pollution usually happen in slow manner taking years before actual impact is visible. some of the acute impact in recent past like episode in chernobyl and bhopal has been faded away from memory of people due to other burning issues at individual regions. common air pollution issues are discussed below. the term "acid rain" is commonly used to refer the wet (fog, rain, cloud water, snow, sleet, and dew) and dry (acidifying gases and particles) deposition of acidic components. in this regard, it is important to know that "clean" or natural rain is also slightly acidic (but usually will not be lower than . ) due to carbonic acid carbonic acid formation in atmosphere according to the following reaction (charlson and rodhe ) : due to this fact, ph value of . is considered as the ph of natural precipitation, below this value, the precipitation is called as acid rain. sometimes, natural rain can also contain nitric acid formed due to electric discharge such as lightning. it can also have acidity due to organic acids contributed by vegetation (galloway et al.) . basically, acid-base reactions in the atmosphere determine the ph of precipitation. as we know that the atmosphere is highly oxidizing medium, gases such as so and no x are oxidised to sulphuric and nitric acids respectively. such oxidation occur through homogeneous or heterogeneous pathways as described below oxidation of so gas phase homogeneous oxidation reaction so þ oh ! hso ð : Þ aqueous phase oxidation reaction homogeneous aqueous-phase oxidation of so takes place by its dissolution and dissociation in water heterogeneous oxidation however, so is also oxidized through heterogeneous reactions involving calcareous soil dust and carbon soot infact, pathway of the reaction ( . ) is the major route of so oxidation which controls occurrence of acid rain in dusty regions. the soil-dust which is generally dominated by caco effectively scavenge atmospheric so forming calcium sulphate in the atmosphere (kulshrestha et al. ) . due to this reason, in the regions where ambient levels of suspended particulate matter are very high violating the limits of national ambient air quality standards (naaqs), the ph of rain water has also been reported very high (kulshrestha ) . in these regions, the levels of ambient so are recorded very low due to same reason. figure . a shows that even at higher sulphate levels, the ph of rain water is higher in indian region but in the acidified regions such as united states, the ph of rain water is highly acidic decreasing with the increase in sulphate concentrations ( fig. . b ). this suggests that in the acidified regions, sulphate is present as sulphuric acid whereas in dusty regions, sulphate is present as calcium sulphate in rain water. rain water dominated by crustal components or mineral dust such as calcite or dolomite or dolomite has lower acidity. most of indian soils have very high ph. the ph of rain water in india (table . ) has been termed as the mirror image of the ph of soil of that area (kulshrestha ) . long-term measurement in lhasa, of tibetan revealed that ph values, such as . were observed during - period due to alkaline as well as soil-borne continental dusts (zhang et al. ) . the ph of precipitation during - , in israel is mostly alkaline with ph . ± . (mamane et al. ). oxidation of no no is oxidized by oh no þ oh ! hno ð : Þ such no oxidation process is faster than so oxidation by oh. further, through ozone and no radical reactions, nitric acid is formed. during daytime, no radical is formed during nighttime, no radical so formed reacts with no resulting in the formation of hno hno further reacts with dust and sea salt particles acid rain can damage monuments and buildings. figure . shows the corrosion impact of acid rain on a statue and the roofing of a building. the damaged walls of the buildings and monuments leave a rough surface along with the moisture, which is a favorable place for the growth of microorganisms. acid rain can corrode sculpture and architecture, railway tracks, paints of cars, and joints of bridges and flyovers. over the past few decades, huge amount of coal and petroleum are consumed for meeting energy demand of mankind. burning of such fuels has ended with the accumulation of excess concentrations of gaseous and particulate pollutants in the atmosphere disproportionately. as seen in figs. . and . , the sulphur and (berresheim et al. berresheim et al. ) . around tg of no x are emitted by the lightening, stratosphere and the soils representing the natural input of no x . fossil fule and biomass burning are the major sources of no x which contribute around tg no x per year (ipcc ). in addition, anthropogenic activities such as industries and automobiles also inject an array of metals and other chemicals in the atmosphere which ultimately have adverse effects on human health and the environment. since industrial emissions have contributed gigatonnes of green hosue gases (ghgs). according to latest report published in climate change journal, / of these emissions ( gigatonnes) are contributed by companies across the globe (ref) out which belong to oil and natural gas sector, coal producers and cement producers. former ussr entities, chinese government run the change in land use has also changed the generation and migration of natural air pollutants like volcanic ash, dust, pollen grains, spores, virus and bacteria. the changing climate has changed wind patterns leading to migration of pollutants in a pattern which was totally different as compared to past. global competition for creation of wealth and infrastructure has engulfed hills, river sand, minerals and rocks beneath the earth. the waste this activity created has contributed to methane, chemical pollutants yet to be identified and named. the new multistoried building has changed path air traversed before it was setup. the population explosion followed by urban migration has left agricultural fields barren and increased urban foot print within a span of few decades. the "greenhouse effect" is the rise in temperature of the earth's atmosphere due to the existence of greenhouse gases (ghgs) in atmosphere. generally, the solar radiation received by earth drives the climate system. about % of the incoming 'short wave' solar radiation is absorbed by the earth while around % of it is reflected back to the space. when earth cools down, it releases the heat as long wave radiation. ghgs present in the atmosphere absorb this outgoing 'long wave radiation' from earth which enhances atmospheric temperature. greenhouse is a phenomenon used in greenhouses to increase temperature capturing long wave radiation to facilitate optimum growth condition for plants. the term "greenhouse" is borrowed to explain increase in temperature due to trapping of long wave radiation in the atmosphere. swedish scientist svante arrhenius first time demonstrated global warming. arrhenius ( ) who was awarded nobel prize in chemistry in showed that a doubling of the atmospheric co concentration would lead to - k warming of the earth surface due to greenhouse effect. ghg increase has resulted in an increase of . °c in global mean surface temperature since the th century. it is expected that this will further increase up to . - . °c by . according to ipcc, ghgs have altered the climate in past few decades resulting in impact on food, water, economy, raw materials, plant/animal health, energy, and biodiversity. it is worth mentioning that the existence of most of biosphere is due to greenhouse effect. fig. shows that without greenhouse effect earth's temperature would have been − °c (fig. . ) . a rise of > °c in the atmospheric temperature made human life possible on the earth. what worries is the sharp rise as seen during past few decades which has adverse direct and indirect effects such as global warming, sea level rise, monsoon disturbance, floods, cyclones etc. net radiative forcing is affected by the presence of aerosols in the atmosphere. aerosols can absorb or reflect radiation depending on the properties of aerosols. dark aerosols such as soot absorb radiation and light colored aerosols such as showing its importance for sustainable temperature for biosphere sulphate reflect radiation. it is to be noted that the combined effect of present increase in co , n o and ch would lead to an increase of atmospheric heating by . w m- which is % lesser that of expected from observed increase in concentrations of major ghgs due to aerosol effect. however, aerosol radiative forcing calculations have huge uncertainties which need to be corrected. the higher uncertainties are primarily due to lack of measurements from the sites of different characteristics such as rural, background, urban, desert, forest, high altitude especially from tropical regions. considerable uncertainties also exist in quantifying the role of dust aerosols in climate variability due to the difficulty in assessing direct and indirect effects of aerosols on clouds. aerosols alter radiative balance as well as the cloud density of the atmosphere resulting in changes in atmospheric stability and cloud microphysics, which can either foster or suppress the development of clouds and precipitation (li et al. ) . increase in particulate matter in the atmosphere can affect cloud development resulting in reduce precipitation in dry regions/seasons and increasing precipitation in wet regions/seasons. figure . shows impact of climate change. global warming is expected bring changes in weather patterns which may lead to disasters like storms, flood and droughts. ghg sources can be tracked to-energy generation, industry, transport, residential and commercial building, land use change, agriculture, waste disposal. these activities have resulted in rise in ghgs over past decade. combustion of fuel, degradation of organic matter in rice cultivation and waste disposal has contributed to ghg in addition to escape of ghgs manufacture in industries. the naturally occurring ghgs are water vapor, carbon dioxide, methane, nitrous oxide and ozone. table . gives major ghgs and their abundance and characteristics. anthropogenic activity has increased the concentration of these ghgs and contributed gases like perfluorocarbons and hydroflurocabons (hcfs) (ramesha et al. ) . some selected ghgs were phased out during execution of montreal protocol. per capita co emissions of different countries are shown in fig. . . australia, usa canada are the three top nations which have highest ghg emissions while china, egypt and india contribute the least ghgs. overall, the consequences of global warming (especially indirect) would be region specific and can not be generalized. this will mainly depend upon overall tolerance of that geographical area against the change in atmospheric composition and circulations due to increase of ghgs and other pollutants. the increase in atmospheric temperature due to presence of ghgs in higher concentrations will not be uniform throughout the earth. the uneven temperature which is also cause for wind patterns across the globe will change both in direction and speed bringing shift in normal pattern of rain which means rains could come during harvest time and destroy crop and rain may not come at sowing season. further the shortage of rain could cause drought in one region while excess rain could cause flood in other reason. the excess temperature could favor reproduction of pests where as it could alter reproduction patter in plants and animals. flowering date can be shifted and so as rise in sea level due to excess water inflow due to melting ice caps in mountains as well as polar ice can submerge coastal area and bring in loss of economy. due to climate change global land precipitation has increased by nearly % since the beginning of the twentieth century (jones and hulme ; hulme et al. ) and change is not uniform neither spatially nor temporally (karl and knight ; doherty et al. ) . as a result of climate change annual snow-cover extent (sce) has decreased by nearly % in northern hemisphere since mainly due to decline in spring and summer from the mid- s over eurasian as well as american continents (robinson ) . the flood, storm, tornadoes, extreme temperate, cyclone formed due to climate change can trigger mass movement (land slide and avalanche) in hills and mountains leading to further disaster. the climate change has resulted in drought in north china and summer floods in south china. change in precipitation pattern since a.d. is mainly due to human-made absorbing aerosols. long range transport (lrt) and trans-boundary driven air pollution long range transport (lrt) and trans-boundary air pollution affect distance sites. convention on long-range transboundary air pollution signed on is considered as pioneering international instrument which has paved the way for fruitful cooperation among parties in europe (harald et al. ). since, air has no boundaries, pollutants are transported globally from one geographic area. several times remote and background sites are affected by lrt. there are examples where states having no air pollution sources are affected by lrt of pollution from distance or nearby countries or states. rodhe ( ) demonstrated air pollution transport through air mass trajectory calculations and established that acid rain occurrence in northern europe was mainly due to industrial sources located in the south and west. dust from the thar desert has been the primary potential source of particulate pollution in the indian subcontinent. also, the suspended particulate matter from the dust storms originated in oman and other middle eastern regions has been reported to have influence on air quality in south asia (begum et al. ). long-range transport of dust aerosols over the arabian sea and indian region has been reported by badarinath et al. ( ) . kulshrestha and kumar ( ) have reported lrt of pollution and its influence on rain water chemistry at himalayan sites. according to the study, most of the acidic components such as sulphate and nitrate are transported from western airmasses and are deposited through precipitation having long term adverse impact on himalayan ecosystem. similarly, air pollution is the issue of transboundary transport of air pollution is an important issue between and within the countries. for example recent air pollution problem in new delhi can be termed as transboundary pollution problem (kulshrestha ) . in s, exhaust of diesel driven buses was mainly responsible for air pollution in delhi city but after the supreme court decision, all the busses were replaced by cng driven buses. this gave a big relief to the citizens of delhi. but surprisingly, so and no levels were recorded extremely high during winter season of crossing micrograms per cubic meter violating the naaqs limit of micrograms per cubic meter. such observations were never reported in recent past winters. such sudden increase can be explained on the basis of transboundary air pollution contributed by a number of new established brick kilns being operated in nearby states such as uttar pradesh, haryana and punjab (kulshrestha ) . in addition, diesel driven heavy duty trucks entering from outside of delhi also contribute significant amount of carbon and sulphur oxides making delhi air polluted (gupta et al. ) . global competition for creation of wealth and infrastructure has engulfed hills, river sand, minerals and rocks beneath the earth. the waste this activity created has contributed to methane, chemical pollutants yet to be identified and named. the new multistoried building has changed path air traversed before it was setup. the population explosion followed by urban migration has left agricultural fields barren and increased urban foot print within a span of few decades. the abundance of ozone at a point in the stratosphere is controlled by production, destruction, and transport of ozone as well as other substance. the key mechanism for the generation of stratosphere ozone is the breaking of molecular oxygen by solar uv with wavelengths of less than nm by photolysis to make nascent oxygen atoms which in turn combines with molecular oxygen to make ozone. ozone destruction occurs by reactions of oxygen atoms with ozone and reactions involving certain naturally occurring species like nitrogen oxide radicals (no x : mostly no and no ), odd-hydrogen radicals (ho x : oh and ho ), and/or halogen radicals. the natural ozone concentration vary daily (due to changing weather); seasonally; and multiannually and interannually. the concentration of stratospheric ozone can be changed by anthropogenic activity. chemicals in emissions from anthropogenic activities responsible for deletion of stratospheric ozone are called the ozone-depleting substances (odss). since the s it has been recognized that a numerous chemicals emitted by anthropogenic activities deplete stratospheric ozone. in order to understand the impact on layer ozone, national oceanic and atmospheric administration (noaa) has developed the ozone depleting gas index (odgi) derived from noaa's measurements of chemicals containing chlorine and bromine at various remote surface sites around earth's surface. index is defined as at the crest in ozone depleting halogen profusion and zero for the level. two different indices are derived one relevant for the ozone hole over antarctica (the odgi-a), and other relevant to ozone layer at mid-latitudes (the odgi-ml). odgi-a in was . and odgi-ml in the beginning of was . . benchmark halogen level over antarctica will be reached sometime around considering and that over mid latitude will be reached sometime in (daniel et al. ) . ods restricted by the montreal protocol, were declining in the atmosphere by with notable exceptions being halon- (a bromine-containing chemical used mostly in fire extinguishers) and hcfcs, that are used as substitution for cfcs in various applications. decline in reactive halogen concentrations are mainly due to the rapid phase-out as well as atmospheric decline of short-lived odgs such as methyl bromide and methyl chloroform (montzka et al. (montzka et al. , . the reduction related to cfc- as well as cfc- , has been less because of continuing emissions as well as their lifetimes are long ( - years). though concentrations of hcfc's continue to rise in the background atmosphere production is not scheduled for a total phase-out until . main pathways of ozone destruction are hydroxy radical (oh) chlorine and bromine radical (cl and br) the montreal protocol adopted in montreal in , [and subsequently amended in london ( ), copenhagen ( , vienna ( ) , montreal ( ) and beijing ( ) ] control of production and consumption of ods. nitrogen oxides from natural and anthropogenic activity catalytically destroy ozone through following reaction (crutzen ; jonston ) . the relative contributions of odss to depletion of ozone layer are quantified by ozone depletion potential (odp). in spite of many similarities between n o and odss it is not considered to be an ods in montreal protocol (ravishankara et al. ). air pollution transported across the continents as well as ocean basins has resulted in trans-continental and trans-oceanic plumes of atmospheric brown clouds (abcs) made up of sub micron size aerosols. abcs interrupt sunlight by reflecting and absorbing reflecting resulting in large surface dimming. on the other hand, black carbon as well as some organics augments atmospheric heating increasing global warming. black carbon is mainly emitted due to wildfires; and combustion of biofuel, coal, diesel and gasoline. as per ramanathan and feng ( ) abc warms atmosphere at elevated levels from to km, resulting in retreating of glaciers as well as snow packs in the hindu kush-himalaya-tibetan glaciers. aerosols may nucleate additional cloud droplets enhancing dimming effect ( fig. . ) . the dimming decreases evaporation of water from the earth's surface, slowing down the hydrological cycle. according to some studies, in south asia due to abc dimming north-south gradients in sea surface temperatures as well as landocean difference in surface temperatures, have slowed down the monsoon circulation and reduced rainfall over the continents. the following figure shows global estimates of dimming. impact of air pollution on flora can be observed in the form of discolourations of the leaf due to internal cellular damage thereby reducing the market value of agricultural crops such as tobacco and spinach where visibility is important. the air pollution can also result in reduction of the leaf surface and can provide points of entry for pathogens. air pollutant can affect plant physiological or biochemical processes resulting in significant loss of growth/yield as well as changes in nutritional quality (ashmore and marshall ) . so entering leaves through stomata are absorbed by mesophyll (cells between the lower and upper epidermis layers of a leaf) of leaves causing toxicity due to reducing property of gas. when the limiting concentration exceeds the cells are inactivated by plasmolysis and then killed. many industries that emit fluorides like aluminium reduction; smelting of iron and non-ferrous ores; ceramics and phosphate reduction as well as phosphate fertilizers. the fluorides are emitted due to volatilization of molten cryolite in aluminium industry and volatilization fluoride which may present as impurity in raw material in other industry. fluorides are of great concerns with respect to air pollution as all fluorides tend to accumulare in forage and build up concentration which causes fluorosis when consumed by cattle or sheep. further hydrogen fluoride and silicon tetrafluoride are toxic to some plants even at the concentration as low as . ppb (thomas ) . air pollution has been recognized as reason for injury to vegetation in europe and north america in the past few centuries. entire forest communities were lost up to km downwind of the smelter complex located at sudbury, canada in s due to so and metal emissions with other ecological effects observed at a greater distance (winterhalder ) . the areas where cells are killed collapse and dry up. if only few cells in an area are injured the area will become chlorotic (yellowing c) dimming due to abcs (w/m ) fig. . global dimming estimates (ramanathan and carmichael ) or whitening of usually green plant tissue due to decreased amount of chlorophyll) or brownish red in color. the lesions (abnormality in the tissue) due to ozone are usually confined to upper surface and are uniformly distributed as brown or white flecks or stipples or blotches. ozone flecking was observed on grape, avocado, citrus, and other broad leaf plants in outlying regions of los angels valley as well as surrounding hills (thomas ) . among the many mechanisms by which foot and mouth disease can be spread by transport of virus through the wind. even though this is uncommon infection can be carried across borders and seaways (donaldson and alexandersen ) . air pollutants affect photosynthesis and respiration of plants. the effect differs from species to species and each species will have threshold limits after which it is vulnerable for adverse effect. the pock mark type injury on the upper side of leaf has been attributed to acid aerosols associated with fog as it contains acid and other toxicants. the no x concentration in atmosphere is too low to cause plant damage. chlorine is around three times phytotoxic as so . hydrogen chloride at a concentration of ppm for few hours can cause pant damage. ammonia has around same phytotoxicity as hcl. h s is slightly phytotoxic (thomas ) . the change in land use has also affected the production and transport of natural air pollutants like dust, pollen grains, spores, virus and bacteria. vegetation cover is drastically reduced in the urban areas due to sudden increase in built area. developing countries are facing significant air pollution impact of land use change due to rapid urbanization. more significant effect is seen due to expansion of the cities for housing purpose which emits a huge amount of dust during the construction of buildings. the emissions of air pollutants from these residential colonies become a continuous source of polluted air. according to the data, about % of agricultural land in delhi has been transformed into nonagricultural area resulting in decrease in net sown area from , to , ha (mohan et al. ) . same is the case for most of the cities. the urban dust has been reported highly rich in carbon and sulphate (kulshrestha et al. ; kumar et al. ; gupta ) . dust sulphate significantly affect various biochemical constituents and the morphology of the plants (gupta ) . figure . shows an increase in ascorbic acid and proline content of foliar with the increase in sulphate levels correlating with the increase in stress level. more severe effect is reported for the industrial area as compared to the residential areas due to more stress of pollutants at industrial site which affects plant physiology and morphology. soil dust coming out from the digging of land and the construction activities could be the sources of microbes. the relationship between different types of house dust and the concentration of fungi has reported by kaarakainen et al. ( ) . investigation of animals following the air pollution at donora, pennsylvania in disclosed that many animals were ill and died during the week of smog. dogs were most susceptible species during the episode with . % reported to be sick and canine deaths. investigators attributed canine sickness to three syndromes-signs of respiratory syndrome, digestive syndrome, and anorexia (poor apatite). % of the birds affected by episode died. during the london fog in a number of prise cattle were severely affected. five of the cattle died where as were subjected to emergency slaughter. post mortem examination revealed emphysema, bronchiolitis and right heart failure. hydrogen sulphide released during pollution disaster in at poza rica, mexico was responsible for death of % canneries and about % of the other exposed animals in the area. the important long-term effect due to air pollution of radioactive substance are cancer; shortening of life span, and genetic or mutation effect (catcott ) . health forests across the world is being affected by air pollutants due to increasing tropospheric ozone concentrations, raise atmospheric carbon dioxide concentrations, as well as acidic precipitation. emissions of sulphur dioxide, oxides of nitrogen, and other pollutants like ammonia affect forests in rapidly industrializing areas. increasing levels of uv-b radiation from stratospheric ozone depletion are possibly a threat to health of forest (percy and ferretti ) . bioaccumulation and biomagnification of chemicals in food web would further worsen the ecological balance. bioaccumulation is an increase in the pollutants concentration over time in a biological organism. compounds accumulate in living organisms as they are stored faster than they are disintegration or excretion. biomagnification is raising accumulation of a pollutant in living beings as it moves from one level to other in a food chain. lead which is now drastically reduced in ambient air in many parts of the world is toxic to flora, fauna and micro-organisms and bioaccumulates in most organisms. generally, . ppm of lead in the blood of water birds is considered toxic although toxic symptoms may appear at . ppm lead. lead in at . ppm or more in liver of water birds is considered to be lethal. reduced survival has been reported for some sensitive species of birds, at of - ppm body weight. further, dietary levels of ppm affect reproduction. mortality during experimental studies in water birds normally occurs at dose concentrations of - ppm lead in with lethal levels varying from doses of - ppm (unep ) . lead concentrations of - mg/kg soil cause visible effects on, growth, photosynthesis or other parameters (ipcs ) . crop failure can occur due to-(a) impact of air pollutants on crop's health, ( ) climate change, ( ) uv ray penetration due to damage to ozone layer. figure . shows dust deposition on the three. such deposition is likely to cause impact on health of tree depending on the constituents of dust. ozone is the most destructive air pollutant to crops as well as ecosystems (heagle ) as it is a strong oxidant. surface level ozone is a secondary air pollutant created in the atmosphere from the oxidation of no x under bright sunlight as well as volatile organic. ozone as well as its secondary byproducts damage plants by reducing photosynthesis as well as other physiological functions, resulting in weaker, undersized plants with inferior crop quality and decreased yields (fiscus et al. ; booker et al. ; avnery et al. ) . crops next to thermal power plants in india suffered most in past decades due to inefficient air pollution control equipments which emitted fly ash that deposited on the crop. the pollution during mining, construction roads and operation of hot mixing plats in developing countries have resulted damage to crop to great extent. air pollution damages materials and involved three components-expenditures to repair, preventive measures, and loss of amenity. impact of materials fall into four categories: soiling, material loss, discoloration, and structural failure. the atmospheric corrosion of materials depends on acidification of the air, due to gases such as so and hno . as per the reviews damage to siliceous stones is not significant, and therefore attention is restricted to calcareous stones (harter ; lipfert ; lipfert ; napap ; ) . impact on material sulphuric acid aerosols attack building material mainly those containing carbonates like marble ( fig. . , . and . ) , limestone, slate and mortar which can be explained by following equation. the calcium sulphate formed is washed away. the deterioration of calcareous stone can be explained in three stages: simple dissolution of calcium carbonate; dissolution of calcium carbonate followed by the fall-out of less soluble particles; and steady build-up of salts if the calcium carbonate is not washed away. statues and other ornamental material may get discolored due to deposition of air pollutants. figure . shows statues covered with plastic covers in bangalore, india to avoid dust deposition and soiling of statues. mortar contains sand, calcium hydroxide as well as other carbonate phases. cement in concrete is susceptible to acid attack leading to discoloration/soiling (fig. . ) , surface erosion, spalling and corrosion of embedded steel. damage to reinforced cement concrete structure will be insignificant if the steel is provided with sufficient cover of good quality concrete. paint and polymeric materials can be affected due to acidic deposition as well as photochemical oxidants, particularly ozone. impacts on this material include fig. . marble within siliceous stones damaged due to air pollutants erosion of polymer surfaces, loss of gloss and soiling, interaction with sensitive pigments and fillers loss of paint adhesion from and substrates. contamination of substrate before to painting can lead to premature failure due to embrittlement and cracking. atmospheric corrosion of metals ( fig. . ) is an electrochemical process occurring when the surface is wet. corrosion rate mainly depends on humidity, precipitation, temperature as well as levels of atmospheric pollutants. so causes most damage, on metals even though chloride plays play a significant role in coastal area. as per the studies conducted by tzanis et al. ( ) in athens, greece metal and alloy specimens exposed the highest weight change is observed for unalloyed carbon steel at the level of m, and lowest weight change was observed at m. values of weight change for zinc and copper specimens were about four times lower than steel and almost constant with the height. the weight loss of unalloyed carbon steel was found to be more than times higher than other materials. copper and bronze suffered less by atmospheric corrosion with almost equal mass loss. zinc mass loss was nearly % greater than that of copper and bronze. sulfuric acid mist can damage, nylon, cotton, linen and rayon. excess exposure to so accelerates corrosion of metals. hydrogen fluoride will corrode many substances except polyethylene, lead, wax, and platinum. human health can be adversely affected by air pollution depending on the pollutant, quantity of pollutant and path of entry into body. human receptors include the eyes, nose, skin, or respiratory system. eye is more susceptible to damage as well as irritation compared to the skin. figure . shows common forms of exposure response relationship. relationship between the level of exposure (or dose) as well as the degree of effect (or response) usually occur in four ways: ( ) effect/response increases with exposure without threshold, ( ) effect/response increases with exposure with threshold, ( ) effect/ response versus exposure takes curvilinear shape, or ( ) effect/response versus exposure takes s-shape (david ) the clinical course of a disease is shown in fig. . . the living organism after exposing to disease casing factor like air pollutant can either recover or recover with disability or die. before end results human beings usually starts therapy as soon as symptoms of disease appear. the disease need not have to occur only to organs exposed to air pollutants. the toxins easily get absorbed by blood and varied to all parts of the body. for example chronic inhalation of cyanide compounds affects central nervous system, cardiovascular system, thyroid gland, respiratory system, eyes as well as skin. some gases like so cause irritation and some gases like acetylene and carbon monoxide cause asphyxiation (cause death without significant physiological effect). carbon monoxide causes a chemical asphyxiation since it impairs the oxygen transport in blood vessels as affinity of co for haemoglobin is three hundred-fold more than oxygen. lead in air can cause severe health damage as it is toxic at very low exposure levels. organo-lead compounds, like tetra-alkyl-lead and tri-alkyl-lead compounds, are more toxic compared to inorganic forms of lead. lead has acute as well as chronic effects on health of humans as it is a multi-organ system toxicant. it can the evidence indicates humans who are responsible for air pollution are adversely affected. children's health is adversely affected due to air pollution due to incomplete metabolic systems, ongoing process of lung development and growth, high rates of infection by pathogens immature host defenses, and activity specific to children can result in higher exposure to air pollution (who ) . figure . shows impact of air pollution on human health. figures . , . , . , . and . shows lungs affected by air pollution. figure . shows food items exposed to atmosphere are often contaminated with air pollutants leading to food spoiling. the lung of developing fetal and the infant is more vulnerable to injury by lung toxicants. air pollutants interact with environmental exposures, like four million deaths between and all over world were due to respiratory infections (who ) . particulate and gaseous pollutants can act on upper and lower airways to initiate as well aggravate cellular inflammation. nasal allergy to pollen is termed as pollinosis, and allergy particularly to grass pollen is termed hay fever. plants whose pollens are dispersed by air currents are air pollutants can get into the body through food contamination through food or water exposed to polluted air. pollutant entering into body can remain on skin or in the respiratory/digestive system or move into the blood. pollutants entering the bloodstream can be transported to all body parts. as pollutants moves through the body it can undergo many chemical changes, becoming more or less toxic (figs. . and . ) . the contamination of meuse river valley of belgium resulted in death of people in the first week of december . the findings of investigation revealed that weather patterns had a major impact on sulfur dioxide concentrations, especially during temperature inversion (shy ) . air pollution along the monongahela river near pittsburgh, usa in october , , resulted in death of people due to cardiac as well as respiratory diseases (goldsmith and friberg ) . as per ostro ( ) outdoor air pollution was accounted for approximately . % of total mortality and it is the cause for . % of all disability-adjusted life years (dalys) as well as % of all cardiopulmonary disease. in estimates released by who ( a), around million people died in as a result of air pollution exposure. this confirms that air pollution is now the world's largest single environmental health risk. as per the who ( b), . million deaths related to household air pollution in . . million deaths occurred in the south east asia and . million deaths occurred in western pacific regions. about , deaths occurred in africa, , in europe, , in the eastern mediterranean region, , in the americas and , deaths occurred in high income countries. in a total . million deaths linked to ambient air pollution (who c). . million death occurred in western pacific region and , deaths occurred in south east asian. about , deaths occured in the eastern mediterranean region, , in europe, , in the americas, , in africa, , in high-income countries of europe , in americas, , in western pacific, , in eastern mediterranean ( , ) . breakdown by disease due to ambient air pollution are: ( ) %-ischaemic heart disease; ( ) %-stroke; ( ) %-chronic obstructive pulmonary disease (copd); ( ) %-lung cancer; and ( ) %-acute lower respiratory infections in children (who a, c) . breakdown by disease due to household air pollution are: ( ) %-stroke; ( ) %-ischaemic heart disease; ( ) %-copd; ( ) %-acute lower respiratory infections in children; and ( ) %-lung cancer (who a, b) ( fig. . ) . lung cancer is one the leading reason of cancer death in both men as well as women. apart from smoking air pollution is one of the main reason. cancer can originate from lung or other parts of the body. cancer originating from lung cells is known as primary lung cancer. it can start in the bronchi) or in the alveoli. as per studies by siegel et al. ( ) lung and bronchus cancer would cause death of % of male cancer patients and % of female cancer patients in usa. smoking key reason of lung cancer (u.s. department of health and human services ), other causes include radon (national academy of sciences ) secondhand smoke (u.s. department of health and human services ) and air pollutants like benzene, formaldehyde, as well as asbestos. the size of lung may increase (due to increase in mucus) or collapse partially/wholly due to lung cancer. many people affected due to sars (severe acute respiratory syndrome), avian flu and swine flu due to transmission of virus through air in last decade. diesel and gasoline exhaust are probably and possibly carcinogenic to humans (iarc ) and children living near places with high traffic density have more risk of cancer (savitz and feingold ) . more than , people lost their lives in the year due to deadly acute air pollution that occurred due to temperature inversion over london. more than people lost their lives instantly due to methyl isocyanate poisoning in and hundreds of thousands were disabled permanently. dioxins and furans created during manufacture of certain chemicals like herbicides; pulp and paper industry during bleaching the wood pulp; burning waste (like municipal solid waste and medical waste) or substance which has chlorine; secondary copper smelting; forest fires; cement kilns; coal fired power plants; residential wood burning; burring bodies in crematorium or open fire. dioxins and furans are known carcinogens and can change hormone levels. high doses of dioxin can lead to skin disease called chloracne. dioxins and furans can bring changes in development of the fetus, decrease ability to reproduce and suppress immune system. mercury which is emitted from combustion of coal and waste/substance with mercury can lead to neurological and behavioral disorders, kidney disorder ranging from augmented protein in the urine as well as kidney failure. aerobiology plays an important role in the spreading of infectious diseases. aerobiology is the study of the course of action involved in the microorganisms' movement in the atmosphere from one location to another. airborne particles can remain in air for many days (wells ; wells and stones ; duguid ) . large quantities of infectious pathogens expelled in hospitals can spread via airborne. blockage of sunlight due to air pollution can promote spread of harmful microbe in atmosphere resulting in spread of infection. epidemiological studies have revealed that exposure to air pollutants during fetal development as well as early postnatal life is connected with numerous health problems including very low birth weight (vlbw); low birth weight (lbw); congenital defects; intrauterine growth restriction (iugr); preterm birth (ptb); behavioural problems, intrauterine and infant mortality; decreased lung growth, childhood asthma, increase in rates of respiratory tract infections, and neurocognitive decrements (wang and pinkerton ) . silicosis due to inhaling silica particles and asbestosis due to inhaling asbestoses is long known to scientific community. studies by kathleen and boguang ( ) revealed that occurrence rates of silicosis increased by years of dust exposure. pm air pollution increases risk for cardiovascular diseases (miller et al. ; pope et al. ) and people with diabetes are more susceptible to cardiovascular health effects connected with pm air pollution (o'neill et al. ; whitsel et al. ; ursula et al. ) . certain microorganisms, transforming a less toxic species into toxic derivative like produce methyl iodide, which reacts with substance like mercury, arsenic, selenium, and tellurium to form their methyl derivatives by process is known as methylation. black snow and outbreak of red-tide in lacustrine systems was observed during early in the kashmir himalayan valley, india. the occurrence of muddy snowfall in may at more than m above sea level in afarwat glaciers of kashmir, india has been attributed to changing environmental scenario of the region (lone et al. ) . the occurrence of "black snow" on the mountain peaks of gund, sonamarg in kashmir, india observed in march, was due to the burning of oil-fields for the period of gulf war (kawosa ) . occasional muddy rains were also observed in some regions of the kashmir valley in april (lone ) . the occurrence of muddy snowfall in the alpine zone of kashmir himalaya evoked environmental concern as it has bearing on drinking water supplies as well as ecology of many natural snow-fed streams. the chemical composition shows that the particulate deposition approximately comprises % of lime stone, . % of clay, % of gypsum and . % of iron ore. such a chemical composition suggests that these particulates might have origin from stone quarrying activities or cement manufacturing units. air pollution contributes substantial quantities of contaminants to snow. the asian part of the russian federation as well as europe contributes all but a minute percentage of the lead reaching the arctic through air out of which - % is anthropogenic (unep ). most of the air pollutants in atmosphere either settles down itself or get scrubbed by rain/snow/hail/dew unless otherwise it reacts with other pollutants to from secondary pollutants or absorbed/adsorbed by other living/nonliving things. the pollutants entrapped in precipitation would either stay in snow for year to come or reach surface/ground water bodies over the course of time. the material settled on the surface of solid bodies also gets washed over course of time. the acids/alkali formed in the atmosphere can dissolve chemical on the surface of the earth before reaching the water bodies. the water not only gets contaminated due to conventional pollutants, it also scrubs radioactive material released into atmosphere. rainwater was collected in the nijmegen area of netherlands after chernobyl accident, in first three weeks of may revealed presence of radioactive material (dennis ) . summarises the findings of studies of rainwater from scandinavia and great britain after chernobyl accident shows that the issue is far from easily understood (lucas and john ) . the organic compounds with diverse chemical structures, sources, as well as uses such as industrial compounds, pesticides, persistent degradation products, byproducts of fossil fuel combustion, and impurities during manufacture of chemicals are transported atmospherically and deposited into remote environments. such deposition can affect plants and animals including human health. nitrogen controls productivity and eutrophication of estuaries. elevated nitrogen inputs to water bodies leads to harmful algal blooms; loss of sea grasses; hypoxic and anoxic bottom waters; and reduced fish stocks (valiela and costa ; hallengraeff ) . these eutrophication problems are consequence of increased population growth and air pollution (lee and olsen ; nixon ; vitousek et al. ) . a significant nitrogen inputs to estuaries in new york may be due to atmospheric deposition (jaworski et al. ) . lead is important for scientific community in environmental science as may enter surface waters due to erosion of lead-containing soil though lead is not very mobile in soil (unep ) . estimated residence times of biological particles with lead in surface waters, up to two years(unep ). many of the air pollutants do not remain in atmosphere for ever. most of the air pollutants will settle on soil or water causing soil and water pollution. airborne pollutants from anthropogenic as well as natural sources deposit on land and water bodies. air pollutants can travel to great distances from the source. pollutants in soil and water bodies include nitrogen compounds, heavy metal, sulfur compounds, pesticides, and other toxics. airborne pollutants can fall to the ground simply due to gravity. such deposition is termed as "air deposition" or "atmospheric deposition". mercury extracted for centuries from sulfide ore or cinnabar has become a global pollutant. mercury can be released into the atmosphere from many anthropogenic activities, such as municipal trash incineration, combustion of high sulfur coal which contains cinnabar, use of mercury based fungicides. mercury has low reactivity in its elemental state and has long atmospheric residence time while the oxidized forms are removed by wet/dry deposition (fogg and fizgerald ) . oxidized reactive gaseous mercury is very soluble in water and hence is effectively deposited on water and land by rain/snow/hail/due. particulate forms of mercury settle as dry deposition (keeler et al. ) . the total quantity of mercury in the atmosphere is around - metric tons, and nearly % of that was produced by human activities (fitzgerals and watras ) . concentrations mercury in atmosphere peaked between s and s (engstrom and swain ) . anthropogenic activities contribute - % of the gross annual mercury emissions and % of mercury vapor exists as elemental mercury (fitzgerald ) . rest of the mercury exists as reactive gaseous mercury as complexes of divalent mercury, and/or in the organic form (stratton and lindberg ) . while deposition of h þ , so À and ca þ in numerous central european forests has declined in the last decade, deposition of no À and nh þ remained high or increased resulting in depletion of soil al-pools, release of so À formerly stored soil, accumulation of nitrogen in soil increasing nitrogen availability to trees and reducing ca þ concentration in the soil. soil acidification as well as increased nitrogen availability will decrease the fine root and change the rooting zone to upper layers of soil. such shift in rooting zone will decrease the root/shoot ratio causing increased drought susceptibility of trees (matzner and murach ) . thirty-three years study of atmospheric heavy metal deposition in denmark along with european emission inventories revealed concentrations of lead, cadmium, copper, zinc, vanadium, nickel and arsenic in soil at remote forest plantation on the island of laesoe, denmark. the accumulation of heavy metals was more in the top soil (hovmand et al. ) . deposition of particles from mineral processing and stone crushing can settle on soil leading to change in quality of top soil. the escape of materials which include by-product and products from chemical industry can contaminate soil to great extend. as per data published by unep ( ) concentration in soil next to roadways and in towns was up to several thousand mg pb/kg, and soils adjacent to smelters as well as battery factories were up to , mg pb/kg. study of production, distribution as well as consumption of goods and services is called economics. an economic system of a region or nation is referred as economy. the economy differs widely between, and within, countries due to differing environments, cultures, and government systems. the direct consequences of air pollution can cause millions of dollars if measured in terms of cost of treatment to heal sickness, lost productivity, missed educational/development opportunities. the economic loss due to climate change and disasters triggered by air pollution can take away good share of gross domestic product (gdp) of any country. even though air pollution is spread across the world, it is most severe in cities of developing countries (david et al. ) . but considering the spatial distribution, it is difficult to generalize the issue. every country will have pockets of polluted areas as well as clean area. but developing world has disadvantage of high population growth rate in cities and poor infrastructure. it means poorly paved roads which rises dust increasing suspended particulate matter. the adulteration of fuel and old vehicles would add smoke to atmosphere. air pollution in cities of developing countries can cost the nation after a decade due to unhealthy senior citizens. the immediate air pollution threat can come due to loss of tourism and crop. the soiled buildings, corroded structures and degraded construction material due to increase in acidic air pollutants can cost the owners a hefty amount even though it contributes to gdp of a country. fires to clear forest to accommodate large scale rubber and oil palm plantations in indonesia resulted in major air pollution in asia during april to november of due to wide spread of forest fires. the disaster was cause of thick smoky haze which covered indonesia, malaysia, singapore, brunei, southern thailand and philippines. the fires destroyed a large area of rainforest that included endangered species. while the fire destroyed commercial timber, plantations and farmland, air pollution resulted in tourism. the smoke was also cause of temporary shutdown of industry and commerce as well as increase in health care costs. much mortality occurred malaysia in the period due to air pollution from indonesia. as per, teresa and leonardo ( ) reduced cognitive potentials due to preventable childhood lead exposure translate into economic losses of $ . , $ . , and $ . billion international dollars at africa, asia, and latin america/caribbean, respectively amounting to . % of the global gdp. lifetime economic productivity (lep) associated due to childhood lead exposure in low and middle income countries (lmic) currently amounts to $ billion annually (teresa and leonardo ) . visibility is the maximum distance in a given direction at which an object can be visually recognized with unaided eyesight (wark et al. ). the destruction of visibility is mainly due to the absorption and scattering of visible light by suspended particles (chan et al. ) . pm . , are believed to be mainly responsible for the scattering of visible light (sloane et al. ) . fine particulate matter is the main pollutant in the majority fig. . reduced visibility with time . am, . am and . am on clear day in another location in bangalore urban areas in china (zhang et al. (zhang et al. , and other countries. hence it has acquired worldwide attention for its bad impacts on visibility (ghim et al. ) as well as public health (hong et al. ) . the visibility is affected by meteorology and concentration of pollutants. as could be seen from figs. . and . the visibility is not affected much at . am clock due to less traffic and low concentration of pollution. at . am as the number of vehicles on road increased visibility decreased as concretion of suspended particles in atmosphere increased and low temperature hindered dispersion of pollutants. at . am the pollutants dispersed mainly due to increase in temperature and therefore dispersion of pollutants. on the inûuence of carbonic acid in the air upon the temperature of the ground. the london, edinburgh and dublin philosophical magazine ozone impacts on agriculture: an issue of global concern global crop yield reductions due to surface ozone exposure: . year potential crop production losses and economic damage under two scenarios of o pollution long-range transport of dust aerosols over the arabian sea and indian region-a case study using satellite data and ground-based measurements long range transport of soil dust and smoke pollution in the south asian region chemical composition and climate of the atmosphere the ozone component of global change: potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species effect of air pollution on animals source apportionment of visibility degradation problems in brisbane (australia) using the multiple linear regression techniques factors controlling the acidity of natural rainwater the influence of nitrogen oxides on the atmospheric ozone content a focus on information and options for policymakers environmental pollution and the global burden of disease urban air pollution in megacities of the world radioactivity in rainwater following the chernobyl accident a gridded reconstruction of land and ocean precipitation for the extended tropics from - predicting the spread of foot and mouth disease by airborne virus the size and the duration of air carriage of respiratory droplets and droplet nuclei recent declines in atmospheric mercury deposition in the upper midwest air pollution control technologies compendium. international centre for sceince and high technology and united nations industrial development organization crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning is mercury increasing in the atmosphere? the need for an atmospheric mercury network (amnet). water, air, and soil pollution mercury in surficial waters of rural wisconsin lakes mercury in southern new england rains visibility trends in korea during the past two decades effects of air pollution on human health industrial dust sulphate and its effects on biochemical and morphological characteristics of morus (morus alba) plant in ncr delhi a review of harmful algal blooms and their apparent global increase clearing the air - years of the convention on long-range transboundary air pollution acidic deposition-materials and health effects ozone and crop yield effects of air pollutants on acute stroke mortality atmospheric heavy metal deposition accumulated in rural forest soils of southern scandinavia precipitation sensitivity to global warming: comparison of observations with hadcm simulations diesel and gasoline engine exhausts and some nitroarenes. lyon: international agency for research on cancer lead-environmental aspects. environmental health criteria atmospheric deposition of nitrogen oxides onto the landscape contributes to coastal eutrophication in the northeast united states reduction of stratospheric ozone by nitrogen oxide catalysts from supersonic transport exhaust calculating regional climatic time series for temperature and precipitation: methods and illustrations microbial content of house dust samples determined with qpcr secular trends of precipitation amount, frequency, and intensity in the usa risk of silicosis in a colorado mining community environmental chemistry of a rare muddy snowfall occurrence on alpine zone glaciers of gulmarg particulate mercury in the atmosphere: its significance, transport, transformation and sources proceedings of first composition of atmospheric deposition (cad) workshop held at tsukuba. japan acid rain some facts about recent air pollution problem in delhi estimation of so contribution by dry deposition of so onto the dust particles in india airmass trajectories and long range transport of pollutants: review of wet deposition scenario in south asia carbon as major constituent of atmospheric aerosols at urban sites in india eutrophication and management initiatives for the control of nutrient inputs to rhode island coastal lagoons long-term impacts of aerosols on the vertical development of clouds and precipitation effects of acidic deposition on the atmospheric deterioration of materials atmospheric damage to calcareous stones: comparison and reconciliation of recent findings environmental chemistry of a rare muddy snowfall occurrence on alpine zone glaciers of gulmarg radioactive contamination in nijmegen rainwater after the chernobyl accident contribution of alkaline and acidic sources to precipitation in israel. the science of the total environment soil changes induced by air pollutant deposition and their implication for forests in central long-term exposure to air pollution and incidence of cardiovascular events in women dynamics of urbanization and its impact on land-use/land-cover: a case study of megacity delhi present and future trends in the atmospheric burden of ozone biological effects of ionizing radiation (beir) vi report: the health effects of exposure to indoor radon coastal marine eutrophication: a definition, social causes and future concerns quantitative assessment of worldwide contamination of air, water and soils by trace metals diabetes enhances vulnerability to particulate air pollution-associated impairment in vascular reactivity and endothelial function outdoor air pollution: assessing the environmental burden of disease at national abd local levels. geneva: world health organisation an assessment of global and regional emissions of trace metals to the atmosphere from anthropogenic sources worldwide air pollution and forest health: toward new monitoring concepts cardiovascular mortality and long-term exposure to particulate air pollution: epidemiological evidence of general pathophysiological pathways of disease global and regional climate changes due to black carbon air pollution, greenhouse gases and climate change: global and regional perspectives coping with climate change: principles and asian context nitrous oxide (n o): the dominant ozone-depleting substance emitted in the st century critical review on natural global and regional emissions of six trace metals to the atmosphere. (prepared for the international lead zinc research organisation, the international copper association hemispheric snow cover and surface albedo for model validation a study of the sulfur budget for the atmosphere over northern europe association of childhood cancer with residential traffic density atmospheric chemistry and physics of air pollution health effects of air pollution cancer statistics size-segregated fine particle measurements by chemical species and their impact on visibility impairment in denver measurement of gas phase ionic mercury (ii) species in ambient air. abstracts of papers of the economic costs of childhood lead exposure in low-and middle-income countries effects of air pollution on plants on the corrosion and soiling effects on materials by air pollution in athens final review of scientific information on lead-version of traffic-related air pollution and incident type diabetes: results from the salia cohort study the health consequences of smoking. a report of the u.s. surgeon general the health consequences of involuntary exposure to tobacco smoke: a report of the surgeon general-executive summary eutrophication of buttermilk bay, a cape cod coastal embayrnent: concentrations of nutrients and watershed nutrient budgets human alterations of the global nitrogen cycle: causes and consequence economic crisis detected from space: air quality observations over athens/greece air pollutant effects on fetal and early postnatal development air pollution-its origin and control on air-borne infection: study ii. droplets and droplet nuclei on air-borne infection heart rate variability, ambient particulate matter air pollution, and glucose homeostasis: the environmental epidemiology of arrhythmogenesis in the women's health initiative the world health report -health systems: improving performance. geneva: world health organization effects of air pollution on children's health and development a review of the evidence million premature deaths annually linked to air pollution burden of disease from household air pollution for burden of disease from ambient air pollution for principles and practices of air pollution control student manual environmental degradation and rehabilitation of the landscape around sudbury, a major mining and smelting area chemical characterization and source apportionment of pm . in beijing: seasonal perspective alkaline rains on the tibetan plateau and their implication for the original ph of natural rainfall asian emissions in for the nasa intex-b mission key: cord- -rtg fs authors: lim, t. k. title: punica granatum date: - - journal: edible medicinal and non-medicinal plants doi: . / - - - - _ sha: doc_id: cord_uid: rtg fs nan the pomegranate tree is native from the middle east to the himalayas in northern india. it has been cultivated and naturalised since ancient times throughout the mediterranean region of asia, caucasus, northern africa and europe. the fruit has manifold uses as it is today and was featured in egyptian mythology and art, in the old testament of the bible and in the babylonian talmud. from its native range, it was introduced to central and southern india and southeast asia. it was reported growing in indonesia in . it was introduced into latin america and california by the spanish in , it is now grown in california and arizona. it has been widely cultivated throughout india and drier parts of southeast asia and tropical africa. the most important growing regions are egypt, china, afghanistan, turkey, syria, pakistan, bangladesh, iran, iraq, india, myanmar and saudi arabia. there are some commercial orchards in israel on the coastal plain and in the jordan valley. pomegranate is primarily mild-temperate to subtropical and naturally adapted to regions with cool winters and hot summers, but can also be grown in warm tropical areas, such as in southern india, southeast asia and various islands in the caribbean. areas with mean annual temperature of - °c is ideal. it suffers severe, irrecoverable injuries at temperatures below - . °c. the plant thrives in a semi-arid condition with mean annual rainfall of to , mm and is extremely drought-tolerant. it does not fl ower and fruit well in very humid and wet climates. it is cultivated up to altitudes of , m as occur throughout the western range (baluchistan, n. & s. waziristan, nwfp, kurram, dir, chitral) in pakistan. the species is adaptable to a wide range of soil types including soils on which other fruit species will not grow. it thrives on calcareous soil, alkaline soil, gravelly soil and on deep, acidic loams. for commercial cultivation well-drained, heavy, light and medium soils are preferred although it can withstand seasonal waterlogging. irrigation is required to sustain high yields in drier areas. the fruit is relished fresh, out of hand by quartering the fruit and lifting out the rind to exposed the juice-laden arils around the seeds, both of which are eaten. the fruit is also consumed as juice which is the basis for lemonades or a beverage similar to wine. in the middle east, caucasus and india, pomegranate juice is a very popular beverage. for beverage purposes, the juice is usually sweetened. pomegranate juice is widely made into grenadine syrup for use in mixed drinks, cocktails and often processed into wine. in saudi arabia, the juice sacs may be frozen intact or the extracted juice may be concentrated and frozen, for future use. the juice can be processed into jellies by the addition of pectin and sugar. pomegranate is also used in food and as a spice condiment. fresh pomegranate arils are used in preparation of curd rice dadhojanam (telugu) in andhra pradesh in india. in northern india, the reduced juice is used for desserts and for marinating and tenderising meat due to its proteolytic enzymes. dried pomegranate arils are used in various cuisines such as trail mix, granola bars, or as toppings for ice-cream, yogurt and salads. dried whole arils are commonly sold in ethnic indian subcontinent markets. they impart a subtle, sweet-sour and tart fl avour popular in punjab and gujarat. dried pomegranate seeds, ' anardana ', has culinary importance as spice for vegetable and legume dishes in northern india and in pakistani cuisine. these dried seeds are used as an acidic condiment for chutney and curry preparation. the dried seeds can also be ground and used, which results in a deeper fl avoring in dishes and prevents the seeds from getting stuck in teeth. seeds of the wild pomegranate variety known as daru from the himalayas are renown as quality sources for this spice. in turkey, pomegranate seeds are also used in salads and sometimes as garnish for desserts such as güllaç . in greece and cyprus, pomegranate is used to make kolliva , a mixture of pomegranate seeds and sugar. pomegranate juice can also be processed into a concentrate, syrup and sauces for juice in food dishes and desserts. in iran, a traditional recipe fesenjan is made from a thick pomegranate sauce and ground walnuts used for duck and poultry or in a popular pomegranate soup ash-e nar . in azerbaijan, pomegranate sauce narsharab , made from pomegranate juice, is served with fi sh or tika kabab (grilled, roasted or stewed meat). in turkey, pomegranate sauce called nar ekşisi is used as a salad dressing, to marinate meat, or simply to drink straight. pomegranate syrup used in muhammara , a roasted red pepper, walnut, and garlic spread popular in syria and turkey. pomegranate is also popular in greek cuisine such as kollivozoum i, a creamy broth made from boiled wheat, pomegranates and raisins, legume salad with wheat and pomegranate, traditional middle eastern lamb kebabs with pomegranate glaze, pomegranate eggplant relish, and avocadopomegranate dip. pomegranate is also processed into a liqueur and fruit confectionery used as icecream toppings or mixed with yogurt and jam on toast. a deciduous, much-branched, small tree or shrub . - m high with a smooth, dark grey bark (plate ). branches are terete, opposite and branchlets usually ending in spines. leaves are opposite, glabrous, coriaceous, glossy green, entire, simple, oblong-lanceolate (plates , and ) to obovate or elliptic, - (− ) × - (− ) mm, subpetiolate, apex sub-actue to obtuse. flowers are large, showy, scarlet red or white, bisexual, to cm across, solitary or clustered at the shoot apex (plates and ). calyx campanulate, reddish or purplish with six triangular, persistent lobes, petals , broadly obovate, wrinkled, alternating with the sepal lobes, stamens numerous, multiseriate, persistent, inserted on fl ower tube, ovary subglobose, inferior with three cells in two-series, style one thick, reddish, stigma simple slightly bilobed. fruit globose to subglobose, - cm in diameter, pale red to scarlet to purple or brownish,; the rind thick and coriaceous (plates , , and ). internally, the fruit is partitioned by thin leathery yellow septa into compartments fi lled with transparent sacs (arils) fi lled with tart, fl avourful, fl eshy, juicy, red, pink or whitish pulp (plates and ). in each sac, there is one white, pink or red, angular, soft or hard seed - mm long. food value of raw, pomegranate fruit (refuse % skin and membrane) per g edible portion based on the california wonderful variety is as follows (usda ) : water . g, energy kcal ( kj), protein . g, total lipid (fat) . g, ash . g, carbohydrate . g; fi bre (total dietary) g, total sugars . g, minerals -calcium mg, iron . mg, magnesium mg, phosphorus those of seeds, except potassium which was . ppm in the juice. the seed lipids had a refractive index of . , melting point . °c, iodine value . , acid number . , unsaponi fi able matter . %, saponi fi cation value . , ester value . and glycerol content . %. the lipids contained fatty acids, with caprylic ( . %), the predominant acid, followed by stearic acid ( . %); linoleic acid ( . %) and oleic acid ( . %). the saturated fatty acids of the seed lipids constituted . % of the total fatty acids content. vitamin c content in different turkish cultivars of pomegranate had a range of - . mg/ g, oil content a range of . - . %, sterol content a range of . - . %, anthocyanin content a range of , - , mg/l, potassium a range of - , ppm, calcium a range of - ppm, magnesium a range of - ppm, iron a range of - ppm, sodium a range of - ppm, and phosphorus a range of - ppm (dumlu and gürkan ) . elfalleh et al. ( ) found total sugars of pomegranate juice comprised about g/ ml fructose and about g/ ml glucose, soluble proteins about g/l, . mg/ ml of phosphorus, and . mg/ ml of potassium. the peel contained . and . mg/ g of phosphorus and potassium respectively. the sodium contents were nearly mg/ ml in both peel and juice. nutrient composition of the juice for most components was comparable to the whole fruit. protein and fat values were higher in the whole fruit compared to the juice due to the seeds, which are % of the aril (juice sac) weight (thomas and gebhardt ) . pomegranate aril juice was reported to provide about % of an adult's daily vitamin c requirement per ml serving, and to be a good source of vitamin b (pantothenic acid), potassium and antioxidant polyphenols. the tocopherol ( a -tocopherol, g -tocopherol, and d -tocopherol) contents were, respectively, . , . , and . mg/ g from dry pomegranate seed (elfalleh et al. a, b ) . a total of compounds were found in pomegranate aroma pro fi les, including monoterpenes, aldehydes, alcohols, monoterpenoids and linear hydrocarbons . the most abundant compounds were trans - -hexenal, -carene, a -terpinene and a -terpineol. the total concentration of volatiles ranged from . to . g/kg. overall consumer preference of pomegranate juices was associated with the presence of monoterpenes such as a -pinene, b -pinene, b -myrcene, limonene and g -terpinene. the presence of aldehydes such as hexanol, hexanal and cis - -hexenol was correlated with poor overall consumer liking. high overall consumer liking was associated with intense and acceptable fresh pomegranate odour and fl avour (high scores of satisfaction degree), medium intensity of red colour and low sourness. pomegranate is a fruit rich in polyphenols that include fl avonoids, tannins and hydrolyzable tannins (gil et al. ; seeram et al. a, b ) . pomegranate contain a complex mixture of gallotannins, ellagitannins, ellagic acid and anthocyanins (madrigal-carballo et al. ) . pomegranate juice was found to be rich in tannins, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins (gil et al. ) . the predominant organic acid in pomegranate was citric acid followed by malic acid (pande and akoh ) . the peel fraction had the highest total hydrolyzable tannins content ( , . - , . mg/ g of fw). a total of dimers of fl avanol-anthocyanin adducts were detected, consisting of mono-and disubstituted hexoside derivatives of the adducts between the fl avan- -ols (epi)gallocatechin, (epi) catechin and (epi)afzelechin and the anthocyanidins delphinidin, cyanidin and pelargonidin in pomegranate juice (sentandreu et al. ) . anthocyanidins found in pomegranate fruit included: delphinidin, cyanidin, and pelargonidin (noda et al. ) . pomegranate fruit was reported to contain ellagic acid, gallagic acid, punicalins and punicalagins (reddy et al. ) ; ellagic acid, caffeic acid, luteolin and punicic acid (lansky et al. a, b ) ; pelargonidin- -galactose, cyanidin- -glucose, gallic acid, quercetin, and myricetin (naz et al. ) ; gallic acid, methyl gallate, ellagic acid, (+) catechin, isoquerecitrin, d-mannitol, ursolic acid, oleanolic acid, b -sitosterol and daucosterol (rena et al. ) . pomegranate fruit was found to have a low level of indolamines ( - m g/g serotonin, - m g/g tryptamine, and - ng/ g melatonin) (badria ) . gözlekçi et al. ( ) found that in all the turkish pomegranate cultivars the highest levels of total phenolic content were obtained from the peel extracts. the total phenolic content ranged from , . to , . mg gallic acid equivalent (gae)/l among the cultivars. however, the total phenolic content of pomegranate juice and seed extract ranged from . to , . mg gae/l and . - . mg gae/l, respectively. four phenolic compounds were identi fi ed and quanti fi ed in pomegranate peel and pulp: hydroxybenzoic acids (gallic and ellagic acids) and hydroxycinnamic acids (caffeic and p -coumaric acids) (elfalleh et al. a ) . ellagitannins isolated from pomegranate pericarp included: inhibitors punicalin, punicalagin, granatin b, gallagyldilactone, casuarinin, pedunculagin, tellimagrandin i, gallic acid, granatin a, corilagin and ellagic acid (satomi et al. ) . pomegranate fruit peel had been reported to be a rich source of hydrolyzable tannins called ellagitannins (ets); pomegranate ets were found to show potent antioxidant, antiatherosclerotic and anticancer activities (seeram et al. b ) . the major fruit peel ets were punicalagin ( - % w/w) and ellagic acid (ea; . % w/w) and unquanti fi ed amounts of punicalin and ellagitannin-glycosides (hexoside, rhamnoside and pentoside). pomegrante fruit peel is currently an underutilized food by-product with potential to develop phytoceuticals with potential health bene fi ts or to develop products for use in the cosmetic and food biopreservative industries (seeram et al. b ) . prodelphinidins and gallocatechins including gallocatechin, gallocatechin-( - )-catechin, gallocatechin-( - )-gallocatechin and catechin-( - )-gallocatechin were identi fi ed from pomegranate peels (plumb et al. ) . luteolin, luteolin -o -glucoside, kaempferol, kaempferol- -o -glucoside, kaempferol- -orhamnoglycoside and quercetin were found in the fruit peel (van elswijk et al. ) . of the ellagi-tannins isolated from pomegranate pericarp, seven namely punicalin, punicalagin, granatin b, gallagyldilactone, casuarinin, pedunculagin and tellimagrandin i were found to be active carbonic anhydrase inhibitors and four namely gallic acid, granatin a, corilagin and ellagic acid to be weakly active inhibitors. the type of inhibition by three and seven punicalagin and gallagyldilactone was found to be noncompetitive. a new antifungal peptide designated as pomegranin with a molecular mass of kda, was isolated from fresh pomegranate peels (guo et al. ) . epicatechin, epigallocatechin -gallate, fl avan- -ol, catechin were found in the fruit peel and juice (de pascual-teresa et al. ) . pomegranate fruit and juice were found to contain the following lignans: isolariciresinol, medioresinol, matairesinol, pinoresinol, secoisolariciresinol and syringaresinol (bonzanini et al. ). total lignin contents in the seeds was determined as . m g/g, in wood knots . m g/g, in fruit pulp . m g/g and in the endocarp. . m g/g. syringaresinol was most abundant in the seed ( . m g/g), pinoresinol in knots ( . m g/g), pulp ( . m g/g) endocarp ( . m g/g) and juice ( . m g/g). lignans were also found in two concentrated juices and three pomegranate beverages at levels of . - . m g/g. in addition to the peel, mesocarp, and twigs, lignans were detected in two juices obtained from entire pomegranate fruits, four commercial juices, and three encapsulated pomegranate extracts (fischer et al. ) . isolariciresinol was the predominant lignan with contents of . , . , and . mg/kg dry matter in processed pomegranate mesocarp, peel, and twigs, respectively. six anthocyanin pigments identi fi ed as delphinidin -glucoside, delphinidin , -diglucoside, cyanidin -glucoside, cyanidin , -diglucoside, pelargonidin -glucoside and pelargonidin , -diglucoside were found to be responsible for the red colour of pomegranate juice (cv 'mollar') (gil et al. ) . the fruit skin contained only the cyanidin and pelargonidin derivatives. generally, there was an increase in juice pigmentation with fruit ripening. the concentration of pigments in juice obtained from mature pomegranates ranged between and m g of anthocyanin per gram fresh weight of arils. six anthocyanin pigments delphinidin -glucoside and , -diglucoside, cyanidin -glucoside and , -diglucoside and pelargonidin -glucoside and , -diglucoside were found to be responsible for the red color of pomegranate juice (hernández et al. ) . generally, juice pigmentation increased as the fruit ripened. in the early fruit-ripening stages, delphinidin , -diglucoside was the major pigment, followed by cyanidin , -diglucoside, while in the later stages, the monoglucoside derivatives cyanidin -glucoside and delphinidin -glucoside increased considerably. the pelargonidin derivatives were always present in small amounts. rp-hplc analysis of pomegranate arils' anthocyanins revealed mono-and diglucosylated delphinidins and cyanidins as the major anthocyanins and pelargonidins as minor components (borochov-neori et al. ) . anthocyanin accumulation changed inversely to the season's temperatures. cyanidins were generally more abundant but delphinidin accumulation was enhanced in cooler season. monoglucosylated anthocyanins prevailed at cooler temperatures and subsided during seasonal warming with a concomitant rise in diglucoside proportion. the major anthocyanins detected in the iranian pomegranate varieties were as follows: delphinidin -glucoside ( . - . mg/l), delphinidin , -diglucoside ( . - . mg/l), pelargonidin -glucoside ( . - . mg/l), pelargonidin , -diglucoside ( . - . mg/l), cyanidin -glucoside ( . - . mg/l), and cyanidin , -diglucoside ( . - . mg/l) (alighourchi et al. ) . the major anthocyanins in the juice of iranian pomegranate cultivars were delphinidin , -diglucoside ( - , mg/l), cyanidin , -diglucoside ( - , mg/l), delphinidin -glucoside ( - , mg/l) and pelargonidin , -diglucoside ( - mg/l) (mousavinejad et al. ) . the cultivar, saveh black leather had the highest level of ellagic acid ( mg/l). pomegranate juices obtained from six iranian pomegranate cultivars were found to have . - . total soluble solids content (brix), ph values of . - . , titrable acidity concentration from . to . g/ g, total sugars content from . to . g/ g (faroogh), total antho-cyanins . - . mg/ g, ascorbic acid . - . mg/ , total phenolics content . - . mg tannic acid/ g, the total tannins level . - . mg tannic acid/ g, condensed tannins from . mg to . catechin/ g, antioxidant activity from . to . % (zarei et al. ) . phenolics, fl avonoids, anthocyanins, and tannins of pomegranate juices, obtained from nine tunisian ecotypes were quanti fi ed by el kar et al. ( ) . phenolics ranged from , to , mg gallic acid equivalents/l and fl avonoids from to mg quercetin equivalent/l of juice. highest anthocyanin content was mg cyanidin- -glucoside equivalent/l and highest tannin content was , mg catechin equivalent/l of juice. tartaric and quinic acids were con fi rmed in pomegranate juice at concentrations of - and ~ mg/l, respectively (ehling and cole ) . twenty-one volatile compounds were found in fresh pomegranate juices from nine spanish cultivars, including aldehydes, monoterpenes, and alcohols . the most abundant compounds were hexanal, limonene, trans - -hexenal, and cis - -hexenol. the presence of monoterpenes ( a -terpineol) was correlated with overall consumer preference of pomegranate juice while high aldehydes ( trans - -hexenal) concentrations were correlated with poor overall consumer liking. -hydroxymethyl furfural was determined to be at a signi fi cant level in traditional sour concentrate of pomegranate juice (orak ) . pomegranate was known to contain estrogens (estradiol, estrone, and estriol) (mori-okamoto et al. ) . polysaccharide (psp ) was isolated from pomegranate rind (joseph et al. ) . pomegranate seed oil was found to have % saturated fatty acids, % monounsaturated, % diunsaturated and approximately % conjugated acid, most probably punicic acid (el-shaarawy and nahpetian ) . pomegranate seed was found to have high contents of a -tocopherol ( . - . mg/ g) and g -tocopherol ( . - . mg/ g). the seeds of punica granatum also contained ursolic acid and b -sitosterol along with a long straightchain hydrocarbon -nonacosene (ahmed et al. ) . presence of estrogens and glycosides were also detected. estrone, an estrogen, was identi fi ed in pomegranate seeds (heftmann et al. ) . cold pressed pomegranate seed oil was found to contain punicic acid ( . %), palmitic acid ( . %), stearic acid ( . %), oleic acid ( . %), linoleic acid ( . %) and three unidenti fi ed peaks from which two ( . %) were probably isomers of punicic acid (schubert et al. ) . pomegranate seed had an average lipid content of . % with punicic acid as the predominant fatty acid (pande and akoh ) . pomegranate seed oil was found to be rich in -o -trans , cis , trans - , , -octadecatrienoyl glycerol and also to have small amounts of -o -isopentyl- -o -octadec- -enoyl glycerol and the known cis - -octadecenoic, octadecanoic and eicosanoic acids (fatope et al. ) . pomegranate seed oil (pgo) was reported to be rich in % cis (c) , trans (t) ,c - : as conjugated linolenic acids (cla) (kohno et al. ) . a triglyceride, di-o -punicyl-o -octadeca- z , z , e -trienylglycerol, was isolated and characterized from the seeds of punica granatum from india and iran (yusuph and mann ) . four compound were isolated from pomegranate seeds namely . pomegranate seed oil from pomegranate cultivars was found to have mainly unsaturated fatty acids (about %) (el kar et al. ) . the predominant fatty acid was linolenic acid ( . - . %), followed by linoleic acid ( . - . %), oleic acid ( . - . %), palmitic acid ( . - . %), stearic acid ( . - . %), gadoleic acid ( . - . %), lignoceric acid (< . %), arachidic acid (< . %) and myristic acid (< . %). pomegranate seed linolenic acid isomers, punicic acid and α-eleostearic acid were found in pomegranate seeds (tran et al. ) . a high yield ( . - . %) of unsaponi fi able matter containing tocopherol, aliphatic alcohol (including policosanol), squalene, phytosterols and triterpene was obtained from pomegranate seed oil (caligiani et al. ) . the levels of squalene (up to mg/kg), policosanol ( - mg/kg), b -sitosterol (up to , mg/kg) and cycloartenol ( , - , mg/kg) were found while b -and d -tocopherol were the most abundant vitamin e forms. the seed oil of p. granatum may be an interesting alimentary source of substances of nutraceutical value involved in the modulation of cholesterol metabolism. linolenic acid isomers like punicic acid and αeleostearic acid were reported from pomegranate seeds (tran et al. ) . qualitatively, the pomegranate fatty acid composition of pomegranate cultivars ( tunisian and chinese) seed oil was identical comprising mainly unsaturated about % (elfalleh et al. b ). the predominant fatty acid was linolenic acid ( . - . %), followed by linoleic acid ( . - . %), oleic acid ( . - . %), palmitic acid ( . - . %), stearic acid ( . - . %), gadoleic acid ( . - . %), lignoceric acid ( < . %), arachidic acid ( < . %) and myristic acid ( < . %). ) isolated the following bioactive compounds from pomegranate seeds: coniferyl methy lellagic acid; , ¢ , ¢ -tri-o -methylellagic acid; phenethyl rutinoside; icariside d and daucosterol. a new class iii chitinase (pomegranate seed chitinase) with a molecular weight of approximately kda was isolated and puri fi ed from pomegranate seeds (yang et al. ) . this chitinase was found to naturally bind calcium ions with high capacity and low af fi nity, suggesting it to be a calcium storage protein. this enzyme was found to be widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. two new b -sitosterol esters elucidated as stigmast- en- b -ol- b -dodecanoate ( b -sitosterol laurate) and stigmast- -en- b -ol- b -tetradecanoate ( b -sitosterol myristate) along with the known com pounds n-tricosane, n-heptacosanyl n-hexanoate olean- , -dien- b -ol- -oic acid and olean- -en- bol- -oic acid were isolated from pomegranate fl owers (bagri et al. b ) . a new polyphenol compound named pomegranatate, together with, ellagic acid, , ¢ , ¢ -tri-o -methylellagic acid, ethyl brevifolincarboxylate, urolic and maslinic acids, and daucosterol were isolated from the ethanolic extract of the fl owers of punica granatum . maslinic acid exhibited antioxidant activity as evaluated by measurement of ldl susceptibility to oxidation. a taraxastane-type triterpene, punicanolic acid; two galloyl glucoses, , , -tri-o -galloyl b -d-glucopyranoside, , -di-o -galloyl- , -o -(s)-hexahydroxydiphenoyl b -d-glucopyranoside; fl avones, luteolin; triterpnenes oleanolic acid, maslinic acid; and b -sitosterol were isolated from pomegranate fl owers (xie et al. ) . an alkaloid -( -propenyl)-d -piperideine was isolated from pomegranate leaves (roberts et al. ) . pomegranate leaves were found to contain tannins granatin a, granatin b, corilagin, strictinin, , , , -tetra-o -galloyl-b -d-glucose and , , , , -penta-o -galloyl-b -d-glucose and an ellagitannin, punicafolin elucidated as , , -tri-o -galloyl- , -(r)-hexahydroxydiphenoyl-b -dglucose (tanaka et al. (tanaka et al. , . pomegranate leaves were found to be rich in polyphenols: brevifolin carboxylic acid, brevifolin, corilagin, ellagic acid, , , , , -pentahydroxydibenzo[ b,d ] pyran- -one, granatin-b and punicafolin (nawwar et al. b ) ; n-( ¢ , ¢ -dihydroxyphenyl) pyridinium chloride, as well as the known fl avone glycosides, apigenin (nawwar et al. a ) ; ellagitannin, punicafolin, tannins, granatins a and b, corilagin, strictinin, , , , -tetra-o -galloyl-b -d-glucose and , , , , -penta-o -galloyl-b -d-glucose (tanaka et al. ) ; gallotannins, , , -tri-o -galloylb -glucopyranose and , , -tri-o -galloyl-b -glucopyranose together with the hitherto unknown ellagitannins, , -di-o -galloyl- , -( r )-hexahydroxydiphenyl-b -glucopyranose and brevifolin carboxylic acid -monopotassium sulphate (hussein et al. ) . a hydroquinone pyridinium alkaloid in the form a mixture of a con jugated and a cross-conjugated heterocyclic mesomeric betaine was isolated from the leaves of punica granatum (schmidt et al. ) . balwani et al. ( ) isolated a novel compound, -methyl-pyran- -one- -o -b -d-glucopyranoside from pomegranate leaves. these alkaloid isopelletierine, methylisopelletierine, pelletierine, pseudopelletierine were isolated from pomegranate bark (chilton and partridge ; wibaut et al. ) and roots (chilton and partridge ) ; isopelletierine, methylisopelletierine and y pelleterine from bark (wibaut and hollstein ) ; and n-acetyl-sedridine from bark and root (neuhöfer ) . the bark is rich in punicotannic acid (about %) and also contains gallic acid, mannite and four alkaloids isopelletierine, methylisopelletierine, pelletierine, pseudopelletierine (grieve ) . the following alkaloids were isolated from pomegranate bark and roots: pelletierine, methylisopelletierine, pseudopelletierine and from roots norpseudopelletierine, sedridine, -( ¢ -hydroxypropyl) d -piperidine; -( ¢ -propenyl) d -piperidine, hygrine and norhygrine (neuhöfer et al. ) . tannins and related compound were isolated from pomegranate bark and included punicalin and punicalagin elucidated as to , -(s, s)-gallagyl-d-glucose ( ) and , -(s)hexahydroxy-diphenoyl- , -(s, s)-gallagyl-dglucose ( ), respectively and a hydrolyzable tannin, -o -galloyl- , -(s, s)-gallagyl-d-glucose (tanaka et al. a ) ; ellagitannins, punicacorteins a, b, c and d, punigluconin, casuariin and casuarinin (tanaka et al. b ) . punicacor teins a, b, c and d were established as novel c-glycosideic ellagitannins, the former two possessing a unique tetraphenyl (gallagyl) ester group, and the latter two containing a galloyl group in place of the gallagyl group, while punigluconin was elucidated as , -di-o -galloyl- , -(s)-hexahydroxydiphenoyl gluconic acid. a fl avonoid diglycoside, quercetin- , ¢ -dimethyl ether- -o -a -l-arabinofuranosyl ( → )-b -dglucopyranoside, quercetin, pelargonidine- , diglucoside and ellagic acid were isolated from pomegranate bark (chauhan and chauhan ) . the heartwood of punica grantum was found to contain ellagitannins: diellagic acid rhamnosyl toumy and rauwald ) ; ¢ -o -methyl- , methylenedioxyellagic acid, as well as eight known ellagitannins and gallotannins (el-toumy et al. ) . a new dimeric gallic acid glycoside named humarain was isolated from stem bark of punica granatum (tantray et al. ) . punica granatum is a unique medicinal plant with a long and extensive ethnomedicinal uses since ancient times. various parts of the plant viz. seed, aril, fruit juice, peel, leaf, fl ower, bark, and roots have been reported to contain bioactive phytochemicals with interesting medicinal values and pharmacological activities. the phytochemistry and pharmacological properties of pomegranate plant parts suggest a wide range of clinical applications for the treatment and prevention of ailments such as cancer as well as other diseases where chronic in fl ammation is believed to play an essential etiologic role . the synergistic action of the pomegranate constituents appears to be superior to that of single constituents. in the past two decade, numerous invitro, in-vivo and preclinical studies on the antioxidant, anticarcinogenic, and anti-in fl ammatory properties of pomegranate constituents have been published, focusing on treatment and prevention of cancer, cardiovascular disease, diabetes, dental conditions, erectile dysfunction, bacterial infections and antibiotic resistance, and ultraviolet radiation-induced skin damage (jurenka ) . other potential applications include infant brain ischemia, male infertility, alzheimer's disease, arthritis, and obesity. aqueous and ethyl acetate extracts of pomegranate arils, juice and peels exhibited good antioxidant activity (ricci et al. ) . pomegranate juice, peel, and seed oil antioxidants were con fi rmed by ferric reducing antioxidant power (frap) and oxygen radical absorbance capacity (orac) methods (elfalleh et al. ) . the highest values were recorded in peels with . mmol trolox equivalent/ g and . mmol te/ g for frap and orac assay, respectively. the tocopherol ( a -tocopherol, g -tocopherol, and d -tocopherol) contents were, respectively, . , . , and . mg/ g from dry pomegranate seed. four phenolic compounds were identi fi ed and quanti fi ed in pomegranate peel and pulp: hydroxybenzoic acids (gallic and ellagic acids) and hydroxycinnamic acids (caffeic and p -coumaric acids). results showed that the antioxidant potency of pomegranate extracts was correlated with their phenolic compound content. in particular, the highest correlation was reported in peels. high correlations were also found between peel hydroxybenzoic acids and frap orac antioxidant capacities. identi fi ed tocopherols appeared to contribute in major part to the antioxidant activity of pomegranate seed oil. gil et al. ( ) found that the antioxidant activity of commercial pomegranate juices ( − teac) was three times higher than those of red wine and green tea ( − teac). commercial juices extracted from whole pomegranates showed higher antioxidant activity than in experimental juices obtained from the arils only ( − teac). further, they found that commercial juices contained the pomegranate abundant tannin punicalagin ( , − , mg/l) while only traces were detected in the experimental juice obtained from arils showing that pomegranate industrial processing extracts some of the hydrolyzable tannins present in the fruit rind. also, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins were found in the pomegranate juices. the results of studies by tzulker et al. ( ) showed that the antioxidant activity in pomegranate aril juice correlated signi fi cantly to the total polyphenol and anthocyanin contents. however, the homogenates prepared from the whole fruit exhibited an approximately -fold higher antioxidant activity than the level found in the aril juice. unlike the arils, the antioxidant level in the homogenates correlated signi fi cantly to the content of the four hydrolyzable tannins in which punicalagin was predominant, while no correlation was found to the level of anthocyanins. pomegranate juice was found to be a potent inhibitor of superoxide anion-mediated disappearance of nitric oxide . it was much more potent than concord grape juice, blueberry juice, red wine, ascorbic acid, and dl-α-tocopherol. as little as three μl of a six-fold dilution of pomegranate juice, in a reaction volume of , μ l, produced a marked antioxidant effect, whereas μ l of undiluted blueberry juice or nearly , μ l of undiluted concord grape juice were required to produce similar effects. pomegranate juice and other antioxidant-containing products were found to augment the anti-proliferative action of nitric oxide (deta/no) on vascular smooth muscle cell (rat aorta) proliferation. and other antioxidant-containing products were found to augment the antiproliferative action of no on vascular smooth muscle cell (rat aorta) proliferation. pomegranate juice was much more effective than the other products tested and elicited no effects when tested alone in the absence of added no. pomegranate juice elicited no effects on enos protein expression or catalytic activity and did not enhance promoter activity in the enos gene. the observations indicated that pomegranate juice possessed potent antioxidant activity that resulted in marked protection of nitric oxide against oxidative destruction pande and akoh ( ) in their study found the highest antioxidant capacity to be in pomegranate leaves followed by peel, pulp, and seed. the tannin rich mixtures from pomegranate by-product exhibited ic values against reactive oxygen species (ros) generation at . - m g/ ml. the antioxidant capacity (orac) of pomegranate juice was , m mol te/ g pomegranate juice which was comparable to blueberry and grape juice (thomas and gebhardt ) . oral administration of fl avonoid rich fractions from pomegranate fruits to rats at a dose of mg/kg/day exhibited potential antiperoxidative activity (sudeesh and vijayalakshmi ) . malondialdehyde, hydroperoxides and conjugated dienes levels in the liver were signi fi cantly decreased antioxidative enzymes catalase, superoxide dismutase (sod), glutathione peroxidase and glutathione reductase were signi fi cantly elevated. glutathione content in the tissues were also increased. pomegranate fermented juice and cold pressed seed oil exhibited potent antioxidant activity almost equivalent to butylated hydroxyanisole (bha) and green tea ( thea sinensis ), but signi fi cantly higher than that of red wine ( vitis vitifera ) (schubert et al. ) . flavonoids extracted from cold pressed pomegranate seed oil exhibited - % inhibition of sheep cyclooxygenase and - % inhibition of soybean lipoxygenase. flavonoids extracted from pomegranate fermented juice displayed - % inhibition of soybean lipoxygenase but showed no signi fi cant inhibition of sheep cyclooxygenase. total polyphenols in cold pressed pomegranate seed oil showed a concentration by weight of approximately . %. fatty acid composition in cold pressed pomegranate seed oil showed punicic acid ( . %) along with palmitic acid ( . %), stearic acid ( . %), oleic acid ( . %), linoleic acid ( . %) and three unidenti fi ed peaks from which two ( . %) are probably isomers of punicic acid. acetone extract ( %) of pomegranate fruit displayed scavenging activity against hydroxyl (·oh) and superoxide (o ·-) radicals (noda et al. ) . its three major anthocyanindins, delphinidin, cyanidin, and pelargonidin, scavenged o ·in a dose-dependent fashion with id values of . , , and m m, respectively but did not effectively scavenge nitric oxide. the anthocyanidins inhibited a fenton reagent ·oh generating system. further, the anthocyanidins inhibited hydrogen peroxide-induced lipid peroxidation in the rat brain homogenates with id values . , . , and m m, respectively for delphinidin, cyanidin, and pelargonidin (noda et al. ) . in another study, pomegranate elagitanninsellagic acid, gallagic acid, punicalins and punicalagins from pomegranate fruit showed ic values of . , . , . and . m m, respectively, against reactive oxygen species (ros) generation and no toxicity up to . m g/ml against hl- cells (reddy et al. ) . the good antioxidant action of punicalagin a high molecular weight polyphenol isolated from pomegranate fruit pith and carpellary membrane was expressed not only through its scavenging reactions but also by its ability to form metal chelates (kulkarni et al. ) . binding of punicalagin with bovine serum albumin and metal ions such as iron and copper revealed different binding af fi nities, whereas its binding with dna was very weak and non-speci fi c. in-vitro cytotoxic studies against three cell lines, namely, vero (normal african green monkey kidney cell line), hep- (human larynx epithelial cancer cell line), and a- (human small cell lung carcinoma cell line) showed that punicalagin, was toxic only at higher concentration. studies found that pomegranate peel had the highest antioxidant activity among the peel, pulp and seed fractions of kinds of fruits commonly consumed in china as determined by frap (ferric reducing antioxidant power) assay (guo et al. ) . in a subsequent study pomegranate peel extract was shown to have markedly higher antioxidant capacity than the pulp extract in scavenging or preventive capacity against superoxide anion, hydroxyl and peroxyl radicals as well as inhibiting cuso -induced ldl oxidation. the contents of total phenolics, fl avonoids and proathocyanidins were also higher in peel extract than in pulp extract. the large amount of phenolics contained in peel extract may cause its strong antioxidant ability. the authors concluded that pomegranate peel extract appeared to have more potential as a health supplement rich in natural antioxidants than the pulp extract. separate studies showed pomegranate peel extracts to have both antioxidant and antimutagenic properties and may be exploited as biopreservatives in food applications and neutraceuticals (negi et al. ) . all the pomegranate peel extracts (ethyl acetate, acetone, methanol and water) exhibited marked antioxidant capacity, but the water extract was the lowest. the order of antioxidant capacity varied because of differential responses at four concentrations ( , , and m g/ml) in each solvent (negi et al. ) . studies in male rats showed that pomegranate fruit peel extract decreased lipid peroxidation in hepatic, cardiac, and renal tissues and serum glucose concentration (parmar and kar ) . pomegranate peels were found to contain potent antioxidant contents, as evidenced by free radical dpph scavenging value of . m g/ml and abts scavenging value of . mm trolox equivalent antioxidant capacity/ g dry weight (elfalleh et al. ) . aqueous and alcoholic extracts of pomegranate rind showed good antioxidant effect with ic ranging from . to . /ml for aqueous and . - . m g/ml for alcoholic extracts (rajan et al. ) . phenolic compounds, tannins and fl avonoids were the major phytochemicals present in both the extracts. the aqueous and alcoholic extract yielded . and mg/g gallic acid equivalent phenolic content, . snf . mg/g quercetin equivalent fl avonoid and . and . mg/g tannic acid equivalent tannins respectively. plumb et al. ( ) found that the prodelphinidin dimers from pomegranate peels were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer in scavenging of the radical cation of , -azinobis ( -ethyl-benzothiazoline- -sulphonate, abts) relative to the water-soluble vitamin e analogue trolox c (expressed as trolox c equivalent antioxidant capacity, teac). in the lipid phase, only one of the dimers (gallocatechin-( - )-catechin) was signi fi cantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. the water, methanol, acetone and ethyl acetate (etoac) extracts of pomegranate peel phenolics showed enhanced inhibitory effect on lard peroxidation as the phenolic concentrations increased . acetone extract exhibited the highest antiliperoxidant activity followed by water, methanol and etoac extracts. acetone extract at . % (w/w) and water extract at . % (w/w) exhibited an antiliperoxidant effect close to that of tea polyphenols ( . %, w/w) and higher than that of bht (butylated hydroxytoluene) ( . %, w/w). at . % (w/w), acetone extract exerted a higher inhibitory activity on lard oxidation than that of tea polyphenols and bht. studies by guo et al. ( ) showed that showed that red pomegranate peel extract had the best effect on the scavenging ability of superoxide anion with lowest ic value ( . m g/ml) among all pomegranate extracts (peel, juice, and seed of three varieties). the peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide with the lowest ic value ( . m g/ml) of the nine extracts. the seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the ic value . m g/ ml). the seed extract of white pomegranate (the ic value was . m g/ml) was the most powerful on the dna damage-preventing effect of the extracts. the results of studies by xu et al. ( ) indicated that pomegranate peel extracts exerted protective effects on oxidative stress in mice loaded with restraint stress which may be attributed to its free radical scavenging activity and lipid peroxidation inhibitory effect. the extract decreased alanine aminotransferase and malondialdehyde levels and increased antioxidant capacity in the liver and glutathione levels in plasma as compared with restraint stress control mice. the methanol fraction of pomegranate peel showed highest antioxidant activity by all the four in vitro assays viz. dpph free radical scavenging, phosphomolybdenum, frap (fe( +) reducing power) and cuprac (cupric ions (cu( +)) reducing ability) comparable to ascorbic acid and butylated hydroxy toluene (bht) followed by activity in ethanol, acetone, and ethyl acetate fractions (zahin et al. a ) . in cell free-systems, preparations from various parts of pomegranate displayed displayed good antioxidant capacity as assayed by , -diphenyl- -picrylhydrazyl (dpph), chemiluminescence luminol/xanthine/xanthine oxidase and lipoxygenase assays, with relative potency sequence of rind extract > pomegranate juice > aril juice (sestili et al. ) . however, only the rind extract was capable of preventing the deleterious effects -cytotoxicity, dna damage and depletion of non-protein sulphydrils (npsh) pool, caused by treatment of cells with hydroxide peroxide, tert-butylhydroperoxide or oxidized lipoproteins (ox-ldl) via a mechanism which was postulated to involve both direct scavenging of radical species and iron chelation. the results suggested that the aril juice the major and tasty part of pomegranate fruit, did not contain ellagic acid and punicalagin (i.e. the polyphenols highly represented in the rind which appeared to be responsible for the antioxidant capacity) in amounts suf fi cient to exert cytoprotection in oxidatively injured, living cells. based on these results, the authors advocated that development and evaluation of rinds-only based derivatives of pomegranate for antiatherogenic preventive purposes in humans should be encouraged. the antioxidant activity (percentage of inhibition of on peroxidation in linoleic acid system) of cpj (traditional sour concentrate of pomegranate juice) was determined to be higher ( . %) than that of pj (pomegranate juice) ( . %) (orak ). during the concentration process, the reducing sugars, glucose and fructose level of cpj showed an increase to . , . , and . %, respectively. in cpj the amounts of sodium, iron, zinc, copper and lead were found lower than those of pj. in contrast, potassium and magnesium mineral contents increased during concentration. the total phenolics were also found to be , and , m g/ml in pj and cpj, respectively. the total anthocyanin content of pj was found to be . mg/l but it was not determined in cpj. -hydroxymethyl furfural was determined to be at a signi fi cant level in cpj as a result of the heat process. sezer et al. ( ) found that pomegranate and red wines decreased low-density lipoprotein (ldl) diene levels following a -min incubation period compared with controls. however, pure pomegranate wine demonstrated a greater antioxidant effect on diene level ( m mol/mg of ldl protein) than pure red wine ( m mol/mg of ldl protein). the phenol levels of pomegranate and red wines ( , mg/l gallic acid equivalents and mg/l gallic acid equivalents, respectively) were in accordance with their total antioxidant activity ( . and . %, respectively). four compound from pomegranate seeds namely coniferyl ( ) displayed antioxidant activity, which was evaluated by measurement of low-density lipoprotein (ldl) susceptibility to oxidation and by in-vitro determination of malondialdehyde (mda) levels in the rat's brain . ethanolic extract of pomegranate fl owers was found to contain a large amount of polyphenols and to exhibit potent reducing ability, both indicative of potent antioxidant ability (kaur et al. ) . the extract showed . % antioxidant activity in dpph model system. the fl ower extract was found to signi fi cantly scavenge superoxide (o − ) (by up to . %), hydrogen peroxide (h o ) (by up to %), hydroxyl radicals ( − oh) (by up to %) and nitric oxide (no) (by up to . %). the extract also inhibited ( − oh) induced oxidation of lipids and proteins in vitro. these results indicated pomegranate fl ower extract to exert a signi fi cant antioxidant activity in-vitro. daily consumption of pomegranate juices was found to be potentially better than apple juice in improving antioxidant function in the elderly (guo et al. ) . as the plasma ascorbic acid, vitamin e, and reduced glutathione contents did not differ signi fi cantly between the apple and pomegranate groups in the study, the phenolics may be the functional components contained in pomegranate juice that accounted for the observations. recent in-vitro studies and preclinical animal studies have shown that pomegranate extracts selectively inhibit the growth of breast, prostate, colon and lung cancer cells (adhami et al. ) . an initial phase ii clinical trial of pomegranate juice in patients with prostate cancer reported signi fi cant prolongation of prostate speci fi c antigen doubling time. some of these researches are further elaborated herein. various parts of the pomegranate fruit e.g. seed oil, juice, fermented juice and peel extract, had been shown to exert suppressive effects on human breast cancer cells in-vitro and in this context, three estrogenic compounds, i.e. luteolin, quercetin and kaempferol, were detected in the fruit peel extract (van elswijk et al. ) . studies showed pomegranate fruit possessed chemopreventive and adjuvant therapeutic potential for human breast cancer (kim et al. ) . polyphenols from fermented pomegranate juice, pericarp, and oil inhibited aromatase activity by - % indicating its ability to effect a blockade of endogenous active estrogen biosynthesis. fermented juice and pericarp polyphenols, and whole seed oil, inhibited -b -hydroxysteroid dehydrogenase type from to %, at concentrations ranging from to , m g/ml in the sequence seed oil > > fermented juice polyphenols > pericarp polyphenols. lyophilized fresh pomegranate juice elicited a % inhibition of the estrogenic activity of -b -estradiol; whereas the lyophilized juice by itself displayed only minimal estrogenic action. inhibition of cell lines by fermented juice and pericarp polyphenols was according to estrogen-dependent (mcf- ) > > estrogen-independent (mb-mda- ) > normal human breast epithelial cells (mcf- a). in both mcf- and mb-mda- cells, fermented pomegranate juice polyphenols consistently displayed about twice the anti-proliferative effect as fresh pomegranate juice polyphenols. pomegranate seed oil elicited % inhibition of proliferation of mcf- at m g/ml medium, % inhibition of invasion of mcf- across a matrigel membrane at m g/ml, and % apoptosis in mda-mb- estrogen receptor negative metastatic human breast cancer cells at m g/ml. in a murine mammary gland organ culture, fermented juice polyphenols effected % inhibition of cancerous lesion formation induced by the carcinogen , -dimethylbenz[a] anthracene (dmba). pomegranate seed oil and fermented pomegranate juice polyphenols were found to have anti-angiogenic activity (toi et al. ) . in-vitro studies showed that these pomegranate fractions strongly suppressed vascular endothelial growth factor in normal human breast epithelial cells (mcf- a) and in estrogen sensitive (mcf- ) human breast cancer cells, but upregulated migration inhibitory factor in estrogen resistant (mda-mb- ) human breast cancer cells. an anti-proliferative effect on angiogenic cells was shown in human umbilical vein endothelial cell (huvec) and in myometrial and amniotic fl uid fi broblasts, and inhibition of huvec tubule formation was also demonstrated in an invitro model employing glass carrier beads. additionally, they showed a signi fi cant reduction in new blood vessel formation using the chicken chorioallantoic membrane (cam) model in-vivo. in another study, pretreatment of mouse mammary organ culture with pomegranate fermented juice polyphenols (w), a high-performance liquid chromatographic (hplc) peak separated from w (peak b), or pomegranate seed oil prior to exposure to the to the carcinogen , -dimethylbenz[a] anthracene (dmba) resulted in a % reduction in the number of lesions for w compared with control, peak b and pomegranate seed oil each effected an % reduction (mehta and lansky ) . both pomegranate extracts and genistein inhibit the growth of mcf- breast cancer cells through induction of apoptosis, with combination treatment being more ef fi cacious than single treatments (jeune et al. ) . more recent studies demonstrated that pomegranate fruit extract dose-dependently inhibited nf-kb-dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while suppressing rhoc and rhoa protein expression ) . the bioactive components of the fruit extract comprised mainly ellagitannins and phenolic acids in the aqueous fruit extract and conjugated octadecatrienoic acids in the lipid fruit extract derived from seeds. the results suggested a role of pomegranate fruit extract in lowering the metastatic potential of aggressive breast cancer species. pomegranate extract inhibited the proliferation and viability of mmtv-wnt- mouse mammary cancer stem cells in-vitro in a timeand concentration-dependent manner (dai et al. ) . its constituents ellagic ursolic acid and luteolin also caused a time-and concentrationdependent reduction of cell proliferation and viability, suggesting that they contribute to the inhibitory effect of the extract, while caffeic acid had no effect. the methanolic pomegranate fruit peel extract was found to reduce cell proliferation and induce apoptosis on mcf- breast cancer cells (dikmen et al. ) . in addition, expression of the pro-apoptotic gene bax was increased, and that of the anti-apoptotic gene bcl- was decreased after pomegranate extract treatment. the extract exhibited high antioxidant activity and yielded total phenolic content of . mg of gallic acid equivalents/g of extract with ellagic acid as the most abundant constituent. among the ten pomegranate ellagitanninderived compounds (namely ellagic acid, gallagic acid, urolithins a and b and their acetylated, methylated, and sulfated analogues), urolithin b (ub) was shown to most effectively inhibit aromatase activity in a live breast cancer cell assay (adams et al. ) . ub signi fi cantly inhibited testosterone-induced mcf- aro cell proliferation. the remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than ub. the results suggested pomegranate et-derived compounds to have potential for the prevention of estrogen-responsive breast cancers. pomegranate seed linolenic acid isomers, punicic acid and α-eleostearic acid were found to be selective estrogen receptor modulators (serms) in-vitro (tran et al. ) . punicic acid inhibited (ic ) estrogen receptor (er) α at . m m, estrogen receptor β at . m m. α-eleostearic acid (aea) inhibited erα/erβ at . / . m m. punicic acid agonized erα/erβ (ec ) at . / m m, antagonizing at / m m. α-eleostearic acid antagonized erα/erβ at / m m. both isomers induced erα and erβ mrna expression in mcf- breast cancer cells, but not in mda-mb- breast cancer cells. punicic acid, an omega- fatty acid in pomegranate seed oil, was found capable of inhibiting breast cancer proliferation (grossmann et al. ) . proliferation was inhibited and % for mda-mb- and mda-erα cells, respectively. further punicic acid induced apoptosis in the mda-mb- and mda-erα cells by and %, respectively compared to untreated control cells and disrupted cellular mitochondrial membrane potential. the results suggested the breast cancer inhibitor properties of punicic acid were dependent on lipid peroxidation and the protein kinase c signalling pathway. treatment of human lung carcinoma a cells with pomegranate fruit extract resulted in a decrease in the viability of a cells and dosedependent arrest of cells in g -g phase of the cell cycle (khan et al. a, b ) . treatment of cells with pomegranate fruit extract inhibited (i) phosphorylation of mapk proteins, (ii) pi k, (iii) phosphorylation of akt at thr , (iv) nf-kappab and ikkα, (v) degradation and phosphorylation of ikappabα, and (vi) ki- and pcna. oral administration of pomegranate fruit extract ( . and . %, wt/vol) to athymic nude mice implanted with a cells resulted in a signi fi cant inhibition in tumour growth. treatment of mice with pomegranate juice prior to exposure to carcinogens benzo(a)pyrene (b(a) p) and n-nitroso-tris-chloroethylurea (ntcu), resulted in statistically signi fi cant lower lung tumour multiplicities than mice treated with carcinogens only (khan et al. a ) . treatment of cells with pomegranate fruit extract caused inhibition of (a) activation of nuclear factor-kappab and ikappabα kinase, (b) degradation and phosphorylation of ikappabα, (c) phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinase / , c-jun nh( )-terminal kinase / , and p ), (d) phosphatidylinositol -kinase (p and p ), (e) phosphorylation of akt at thr( ), (f) activation of mammalian target of rapamycin signaling, (g) phosphorylation of c-met, and (h) markers of cell proliferation (ki- and proliferating cell nuclear antigen) and angiogenesis (inducible nitric oxide synthase, cd , and vascular endothelial growth factor) in lungs of b(a)p-and ntcu-treated mice. overall, the results suggested that pomegranate fruit extract could be a useful chemopreventive/chemotherapeutic agent against human lung cancer. flavonoid-rich polyphenol fractions from the pomegranate fruit had been reported to exert antiproliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in-vitro and in-vivo (kawaii and lansky ) . they found that various fruit extracts had proportional inhibitory effects on human hl- promyelocytic leukemia cell proliferation. fermented pomegranate juice and aqueous extract of pomegranate pericarps were found to be strong promoters of differentiation in all settings, while fresh juice extract showed only a relatively mild differentiation-promoting effect. li et al. ( ) found that pomegranate ellagitannins bound with gelatin to form self-assembled nanoparticles. ellagitannins encapsulated in nanoparticles were less effective in inducing the early stage of apoptosis on human promyelocytic leukemia cells hl- . but they had similar effects in inducing late stage of apoptosis and necrosis. differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies (kawaii and lansky ) . pomegranate emulsion treatment ( or g/ kg) to rats, started weeks prior to the dietary carcinogen diethylnitrosamine (dena) challenge and continued for weeks thereafter, showed striking chemopreventive activity demonstrated by reduced incidence, number, multiplicity, size and volume of hepatic nodules, precursors of hepatocellular carcinoma (bishayee et al. ) . both doses of the emulsion signi fi cantly attenuated the number and area of g -glutamyl transpeptidase-positive hepatic foci compared with the dena control. the emulsion also attenuated dena-induced hepatic lipid peroxidation and protein oxidation and elevated protein and messenger rna expression of the hepatic nuclear factor e -related factor (nrf ). the methanolic extract of punica granatum fl owers was exhibited inhibitory effect on tumour necrosis factor-α (tnf-α, ng/ml)induced cytotoxicity in l (murine fi broblast) cells (xie et al. ) . a new taraxastane-type triterpene, punicanolic acid ( ), was isolated from the active fraction (ethyl acetate-soluble fraction) together with four triterpenes ( - ), two galloyl glucoses ( , ), two fl avones ( , ) , and b -sitosterol. among the constituents, , oleanolic acid ( ), maslinic acid ( ), , , -tri ( ), and luteolin ( ) signi fi cantly inhibited tnf-α-induced cytotoxicity in l cells at m m. four pure chemicals, ellagic acid (e), caffeic acid (c), luteolin (l) and punicic acid (p), all important components of the aqueous compartments or oily compartment of pomegranate fruit exhibited anticancerous activities by inhibiting human pc- prostate cancer cell invasion of matrigel arti fi cial membranes (lansky et al. a ) . all compounds signi fi cantly inhibited invasion when employed individually. when c, p, and l were equally combined at the same gross dosage ( m g/ml) as when the compounds were tested individually, a supra-additive inhibition of invasion was observed. pomegranate cold-pressed seed oil, fermented juice polyphenols (w), and pericarp polyphenols (p) each acutely inhibited in-vitro proliferation of human prostate cancer, lncap, pc- , and du human cancer cell lines (albrecht et al. ) . these effects were mediated by changes in both cell cycle distribution and induction of apoptosis. for example, the androgen-independent cell line du showed a signi fi cant increase from to % in g( )/m cells by treatment with pomegranate oil ( m g/ ml) with a modest induction of apoptosis. in other cell lines/treatments, the apoptotic response predominated, for example, in pc- cells treated with pomegranate pericarp polyphenols, at least partially through a caspase -mediated pathway. all agents potently suppressed pc- invasion through matrigel, and furthermore pomegranate pericarp polyphenols and seed oil demonstrated potent inhibition of pc- xenograft growth in athymic mice. overall, the study demonstrated signi fi cant antitumour activity of pomegranatederived materials against human prostate cancer. in another study, combinations of the anatomically discrete pomegranate fractions: fermented pomegranate juice polyphenols (w), pomegranate pericarp (peel) polyphenols (p) or pomegranate seed oil (oil) exhibited synergistic prostate cancer suppression (lansky et al. b ) . supraadditive, complementary and synergistic effects were proven in all models. proliferation effects were additionally evaluated with compusyn software median effect analysis and showed a concentration index ci < , con fi rming synergy. pomegranate fruit extract (pfe) exhibited antiproliferative and proapoptotic activities against human prostate cancer cells (malik et al. ; malik and mukhtar ) . pfe ( - m g/ ml; h) treatment of highly aggressive human prostate cancer pc cells resulted in a dosedependent inhibition of cell growth/cell viability and induction of apoptosis. immunoblot analysis revealed that pfe treatment of pc cells resulted in (i) induction of bax and bak (proapoptotic); (ii) down-regulation of bcl-x(l) and bcl- (antiapoptotic); (iii) induction of waf /p and kip /p ; (iv) a decrease in cyclins d , d , and e; and (v) a decrease in cyclin-dependent kinase (cdk) , cdk , and cdk expression. findings established the involvement of the cyclin kinase inhibitor-cyclin-cdk network during the antiproliferative effects of pfe. oral administration of pfe ( . and . %, wt/vol) to athymic nude mice implanted with androgen-sensitive cwr rnu cells resulted in a signi fi cant inhibition in tumour growth concomitant with a signi fi cant decrease in serum prostate-speci fi c antigen levels. the results suggested that pomegranate juice may have cancer-chemopreventive as well as cancerchemotherapeutic effects against prostate cancer in humans. in a phase ii, simon two-stage clinical trial for men with a rising prostate-speci fi c antigen (psa), daily consumption of pomegranate juice was found to have a positive effect following surgery or radiation for prostate cancer (pantuck et al. ) . there were no serious adverse events reported and the treatment was well tolerated. mean psa doubling time signi fi cantly increased with treatment from a mean of months at baseline to months posttreatment. in-vitro assays comparing pretreatment and posttreatment patient serum on the growth of human prostate cancer lncap showed a % decrease in cell proliferation and a % increase in apoptosis, a % increase in serum nitric oxide, and signi fi cant reductions in oxidative state and sensitivity to oxidation of serum lipids after versus before pomegranate juice consumption. in further studies, a standardized ellagitannins (ets)-enriched pomegranate extract (pe), signi fi cantly inhibited lapc- xenograft growth in severe combined immunode fi cient (scid) mice as compared to vehicle control seeram et al. ) . ellagic acid and several synthesized urolithins were shown to inhibit the growth of human prostate cancer cap cells in-vitro. the chemopreventive potential of pomegranate ets and localization of their bioactive metabolites in mouse prostate tissue suggested that pomegranate may play a role in cap treatment and chemoprevention. the results of studies demonstrated that an ellagitannin-rich pomegranate extract could inhibit tumour-associated angiogenesis as one of several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications (sartippour et al. ) . a pomegranate extract standardized to ellagitannin content (pomx) inhibited the proliferation of lncap and huvec cells signi fi cantly under both normoxic and hypoxic conditions. hif- α (hypoxia-inducible factor- α) and vegf (vascular endothelial growth factor) protein levels were also reduced by pomx under hypoxic conditions. pomx decreased prostate cancer xenograft size, tumour vessel density, vascular endothelial growth factor (vegf) peptide levels and hif- α expression after weeks of treatment in severe combined immunode fi cient (scid) mice. studies showed that pomegranate extract inhibited androgen-independent prostate cancer growth through a nuclear factor-kappabdependent mechanism . pomegranate extract (pe) inhibited nf-kappab and cell viability of prostate cancer cell lines in a dose-dependent fashion in vitro. maximal pe-induced apoptosis was dependent on pe-mediated nf-kappab blockade. in the lapc xenograft model, pe delayed the emergence of lapc androgen-independent xenografts in castrated mice through an inhibition of proliferation and induction of apoptosis. the scientist also showed that pomegranate polyphenols inhibited gene expression and androgen receptor (ar) most consistently in the human prostate cancer lncap-ar cell line (hong et al. ) . therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the ar may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where ar is up-regulated. koyama et al. ( ) demonstrated that pomegranate extract derived from rind and arils (minus seeds) inhibited cell proliferation and induced apoptosis in human lapc prostate cancer cells by modulation of the igf-igfbp (insulin growth factor -insulin growth factor binding proteins) axis. pomegranate extract treatment also decreased igf- mrna expression in a dose-dependent manner indicating that its actions also involved tumour-speci fi c suppression of igf- . pomegranate peel extracts increased the levels of oxygen radical absorbance capacity (orac) in plasma and the density of lecithin and the levels of zn in prostatitic rats (kuang et al. ) . it decreased the levels of malondialdehyde of prostate and the activity of acid phosphatase and the number of white blood cell and adjusted the levels of no in plasma compared with the prostatitis model group. the results indicated that pomegranate peel extracts could markedly improve the protective function of oxidation resistance. pomegranate ellagitannins/microbial metabolites were found to have cyp b (a target in prostate cancer chemoprevention) inhibitory activity in prostate cancer cells (kasimsetty et al. ) . urolithin a, a microbial metabolite, was the most potent uncompetitive inhibitor of cyp b -mediated ethoxyresoru fi n-o -deethylase (erod) activity, exhibiting two-fold selectivity over cyp a , while urolithin b was a noncompetitive inhibitor with three-fold selectivity. the punicalins and punicalagins exhibited potent cyp a inhibition with - -fold selectivity over cyp b . cellular uptake experiments demonstrated a fi ve-fold increase in urolithin uptake by rv cells. western blots of the cyp b protein indicated that the urolithins interfered with the expression of cyp b protein. thus, urolithins were found to display a dual mode mechanism by decreasing cyp b activity and expression. wang et al. ( ) showed that in addition to causing cell death of hormonerefractory prostate cancer cells, pomegranate juice also increased cell adhesion and decreased cell migration of the unkilled cells. pomegranate juice was found to upregulate genes involved in cell adhesion such as e-cadherin, intercellular adhesion molecule (icam- ) and down-regulated genes involved in cell migration such as hyaluranan-mediated motility receptor (hmmr) and type i collagen. in addition, pomegranate juice signi fi cantly decreased the level of secreted pro-in fl ammatory cytokines/chemokines such as il- , il- p , il- b and rantes, thereby having the potential to decrease in fl ammation and its impact. pomegrante juice also inhibited the ability of the chemokine sdf a to chemoattract these cancer cells. faria et al. ( ) found that pomegranate juice consumption decreased total hepatic cytochrome p (cyp) content as well as the expression of cyp a and cyp a in male mice. prevention of procarcinogen activation through cyp activity/expression inhibition may be involved in pomegranate juice's effect on tumour initiation, promotion, and progression pomegranate juice showed greatest antiproliferative activity against all cell lines namely human oral (kb, cal ), colon (ht- , hct , sw , sw ) and prostate (rwpe- , rv ) tumour cells by inhibiting proliferation from to % (seeram et al. a ) . at m g/ml, pomegranate juice, ellagic acid, punicalagin and a standardized total pomegranate tannin (tpt) extract induced apoptosis in ht- colon cells. however, in the hct colon cells, ellagic acid, punicalagin and tpt but not pomegranate juice induced apoptosis. the trend in antioxidant activity was pomegranate juice > tpt > punicalagin > ellagic acid. their data indicated the superior bioactivity of pomegranate juice compared to its puri fi ed individual polyphenolic active ingredients illustrating the multifactorial effects and chemical synergy of the action of multiple compounds. in further studies, they (adams et al. ) found that pomegranate juice signi fi cantly suppressed tnf-α-induced cox- protein expression by %, total pomegranate tannin extract (tpt) %, and punicalagin % in ht- colcon cells. in addition, pomegranate juice reduced phosphorylation of the p subunit and binding to the nfkappab response element . -fold, tpt suppressed nfkappab binding ten-fold, punicalagin . fold, whereas ellagic acid was ineffective. pomegranate juice also abolished tnfαinduced akt activation, needed for nfkappab activity. pomegranate fruit rich in ellagitannins may have bene fi cial effects against colon cancer. in the stomach and gut, ellagitannins were reported to be hydrolyzed to release ellagic acid (ea) and were converted by gut microbiota to urolithin a ( , -dihydroxy- h-dibenzopyran- one) type metabolites (sharma et al. ) . they reported that pomegranate ellagitannin extract, ellagic acid, and their colonic metabolite, urolithin a inhibited wnt signaling, which plays a pivotal role in human colon carcinogenesis, suggesting that et-rich foods may have potential against colon carcinogenesis and that urolithins were relevant bioactive constituents in the colon. studies by gonzález-sarrías et al. ( ) showed that elagic acid and its colonic metabolites, urolithin-a ( , -dihydroxy- h-dibenzo [b,d] pyran- -one) and urolithin-b ( -hydroxy- hdibenzo[b,d]pyran- -one), modulated phase i and phase ii detoxifying enzymes in colon cancer caco- cells. ellagic acid and urolithins may exert some blocking chemopreventive effects in the colon but this effect may be critically affected by interfering factors, such as the food matrix nature. saruwatari et al. ( ) found that pomegranate juice potently inhibited the sulfoconjugation of -naphthol in caco- human colon carcinoma cells. the inhibition was both doseand culture time-dependent, with a % inhibitory concentration (ic ) value of . % (vol/vol). punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in caco- cells with an ic of m m. additionally pomegranate juice and punicalagin both inhibited phenol sulfotransferase activity in caco- cells. the data also suggested that constituents of pomegranate juice, most probably punicalagin, impaired the enteric functions of sulfoconjugation and that this may have effects upon the bioavailability of drugs and other compounds and may be related to the anticarcinogenic properties of pomegranate juice. pomegranate seed oil (pgo) rich in % cis (c) , trans (t) ,c - : as conjugated linolenic acids (cla) could suppress by azoxymethane -induced colon carcinogenesis, and the inhibition was associated in part with the increased content of cla in the colon and liver and/or increased expression of peroxisome proliferator-activated receptor (ppar) γ protein in the colon mucosa (kohno et al. ) . pomegranate extract was found to induce cell cycle arrest and alter cellular phenotype of human pancreatic cancer cells panc- and aspc- (nair et al. ) studies by weisburg et al. ( ) showed that pomegranate extract exerted greater antiproliferative effects towards cancer (such as hsc- carcinoma), than to normal, cells, isolated from the human oral cavity. the antiproliferative mechanism of pomegranate extract was, in part, by induction of oxidative stress. the mode of cell death was by apoptosis, as activation of caspase- , and cleavage of parp. reduction of caspase- activation and of parp cleavage in cells co-treated with pomegranate extract and either cobalt or pyruvate, respectively, as compared to pomegranate extract alone, indicated that apoptosis was through the prooxidant nature of pomegranate extract. pomegranate seed oil ( %) signi fi cantly decreased mice skin tumour incidence, multiplicity, and -o -tetradecanoylphorbol -acetate (tpa)-induced ornithine decarboxylase activity, an important event in skin cancer promotion (hora et al. ) . the results suggested the potential of pomegranate seed oil as a safe and effective chemopreventive agent against skin cancer. afaq et al. ( a, b ) demonstrated that topical application of pomegranate fruit extract (pfe) prior to -o -tetradecanoylphorbol- -acetate (tpa) application on mouse skin afforded signi fi cant time-dependent inhibition, against tpa-mediated increase in skin edema and hyperplasia, epidermal ornithine decarboxylase (odc) activity and protein expression of odc and cyclooxygenase- . also, application of pfe resulted in inhibition of tpa-induced phosphorylation of erk / , p and jnk / , as well as activation of nf-kappab and ikkα and phosphorylation and degradation of ikappabα. pretreatment of pfe on tpa-induced skin tumour promotion in , -dimethylbenz(a)anthracene-initiated cd- mouse substantially reduced tumour incidence and lower tumour body burden when assessed as total number of tumours per group, percent of mice with tumours and number of tumours per animal as compared to animals that did not receive pfe. skin application of pfe prior to tpa application also resulted in a signi fi cant delay in latency period from to weeks and afforded protection when tumour data were considered in terms of tumour incidence and tumour multiplicity. studies by george et al. ( ) suggested that pomegranate fruit extract (pfe) and diallyl sul fi de (das) in combination afforded better suppressive activity of mouse skin tumours than either of these agents alone. pfe and das alone delayed onset and tumour incidence by ~ and ~ %, respectively, while their combination at low doses synergistically decreased tumour incidence more potentially (~ %,). further, regression in tumour volume was seen with continuous combinatorial treatment. the inhibition was associated with decreased expression of phosphorylated erk / , jnk and activated nf-k b/p , ikk a , i k b a phosphorylation and degradation in skin tissue/ tumour. polysaccharide (psp ) isolated from pomegranate rind was found to have antioxidant, antitumour and immunomodulatory properties (joseph et al. ) . psp exhibited a dosedependent enhancement in antioxidant activity using concentrations from to , m g/ml when evaluated using various assays such as, ferric reducing antioxidant power assay, linoleic acid emulsion thiocyanate assay, and superoxide, hydroxyl and nitric oxide radical scavenging assays except for the dpph assay for which the highest activity was obtained at m g/ml. psp exhibited anticancer activity with ic values of . and . m g/-ml following h incubation for mcf- (breast cancer), and k (leukemia) cells, respectively. all the pomegranate peel extracts (ethyl acetate (etoac), acetone, methanol and water) decreased sodium azide mutagènicity in salmonella typhimurium strains (ta and ta ), either weakly or strongly (negi et al. ) . at , m g/ plate all the extracts showed strong antimutagenicity. the antimutagenicity of the water extract was followed by acetone, etoac and methanol extracts. the methanol pomegranate peel fraction with promising antioxidant activity showed antimutagenic activity against sodium azide and methyl methane sulphonate with percent inhibition of mutagenicity ranging from . to . % in a concentration-dependent manner using the ames salmonella/microsome assay (zahin et al. a ) . similar trend of inhibition of mutagenicity ( . - . %) against indirect mutagens ( -amino fl uorene and benzo(a)pyrene) was also recorded. phytochemical analysis by hplc, lc-ms of total phenolic content revealed high content of ellagitannins which might be responsible for promising antioxidant and antimutagenic activities of p. granatum peel extract. methanol extract of punica granatum fl owers ( mg/plate) showed the highest antimutagenic activity in salmonella typhimurium ta and ta , respectively (wongwattanasathien et al. ) . the protective effects of these fl ower extracts might be due to the presence of antimutagenic components that were supposed to be fl avonoids. studies demonstrated that tannin from the pericarp of punica granatum was an effective agent against genital herpes simplex virus (hsv- ) (zhang et al. ) . the tannin not only inhibited hsv- replication, but also showed stronger effects of killing virus and blocking its absorption to cells. punica granatum extract showed anti-human herpes simplex virus type (hsv- ) activity, which was possibly contributed by the polyphenolic compounds in the herbal extract (li et al. ) . studies by neurath et al. ( ) indicated that hiv- entry inhibitors from pomegranate juice adsorbed onto corn starch and the resulting complex blocked virus binding to cd and cxcr /ccr and inhibited infection by primary virus clades a to g and group o. their results suggested the possibility of producing an anti-hiv- microbicide from inexpensive, widely available sources. pomegranate juice containing polyphenols, β-sitosterol, sugars and ellagic acid) was reported to inactivate hiv and further shown to inactivate in fl uenza, herpes viruses and poxviruses (kotwal ) . a formulation consisting of fulvic acid, a complex mixture of compounds was previously reported to render vaccinia virus, hiv and sars virus non-infectious. recently, both fulvic acid and pomegranate juice were shown to inactivate genetically diverse strains of in fl uenza including h n , further con fi rming the broad spectrum nature of these agents. sundararajan et al. ( ) found that the acidity of pomegranate juice and concentrated liquid extract contributed to rapid anti-in fl uenza activity, but this was not a factor with pomegranate polyphenols powder ( %) extract. studies using pomegranate powder extract showed that min treatment at room temperature with m g/ml pomegranate polyphenols resulted in at least a log reduction in the titers of in fl uenza viruses pr (h n ), x (h n ), and a reassortant h n virus derived from a human isolate. however, the antiviral activity was less against a coronavirus and reassortant h n in fl uenza viruses derived from avian isolates. electron microscopic analysis indicated that viral inactivation by pomegranate polyphenols was primarily a consequence of virion structural damage. pomegranate polyphenol extract was shown to have anti-in fl uenza virus properties (haidari et al. ) . of four major polyphenols in pomegranate polyphenol extract (ppe) (ellagic acid, caffeic acid, luteolin, and punicalagin) punicalagin was the effective, anti-in fl uenza component. punicalagin blocked replication of the virus rna, inhibited agglutination of chicken rbc's by the virus and had virucidal effects. further, the combination of ppe and oseltamivir synergistically increased the anti-in fl uenza effect of oseltamivir. the data showed ppe inhibited the replication of human in fl uenza a/ hong kong (h n ) virus in-vitro. exposure of foodborne virus surrogates feline calicivirus (fcv-f ), murine norovirus (mnv- ), and ms (ssrna) bacteriophage to pomegranate juice and pomegranate polyphenols resulted in titer reductions after one hour at room temperature, suggesting promise for use in hurdle technologies and/or for therapeutic or preventive use (su et al. ) . ethanolic extracts of garcinia mangostana , punica granatum and quercus infectoria were found to have good antimicrobial activity of nine thai medicinal plants with mics for methicillin-resistant staphylococcus aureus (mrsa) isolates of . - . , . - . and . - . mg/ml, respectively, and for s. aureus atcc of . , . and . mg/ml, respectively (voravuthikunchai and d kitpipit ) . mbcs for mrsa isolates were . - . , . - . and . - . mg/ml, and for s. aureus atcc were . , . and . mg/ml, respectively. punica granatum was found to have anti-quorum-sensing activity and may be useful in combating pathogenic bacteria and reduce the development of antibiotic resistance (koh and tham ; zahin et al. b ) . in another study the ethanolic extract of p. granatum exhibited bacteriostatic and bactericidal activities against two enterohemorrhagic escherichia coli strains (voravuthikunchai and limsuwan (pai et al. ) . ethanol extract of p. granatum exhibited strong antibacterial activity against escherichia coli (sharma et al. ) . studies showed that punica granatum (pomegranate) methanolic extract (pgme) dramatically enhanced the activity of all antibiotics tested (braga et al. a ) . synergic activity was detected between pgme and the fi ve antibiotics tested, chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, ranging from to %. using pgme ( . × mic) in combination with ampicillin ( . × mic), cell viability was reduced by . and . % in methicillin-sensitive staphylococcus aureus (mssa) and methicillin-resistant staphylococcus aureus (mrsa) populations, respectively. pgme increased the post-antibiotic effect (pae) of ampicillin from to h. pomegranate extract inhibited staphylococcus aureus growth and subsequent enterotoxin production (braga et al. b ) . of several thai medicinal plants, the ethanol extract of p. granatum fruit rind displayed the most outstanding in-vitro antibacterial activity with mic of . and . mg/ml and mbc of . and . mg/ml against staphylococcus aureus and escherichia coli respectively (chansakaow et al. ) . the extract was found to contain both hydrolysable and condensed tannins. the methanol pomegranate pericarp extract exhibited maximum antibacterial activity against salmonella typhimurium , salmonella typhi and shigella dysenteriae serotype (pradeep et al. ) . studies showed that the antibacterial activity of pomegranate rind can be enhanced by the addition of metal salts and vitamin c (mccarrell et al. ) . pomegranate rind extracts (pre) exhibited activity against the gram positive organisms at h were inactive against gram negative bacteria. addition of cu + salts to pre solutions extended the activities resulting in no detectable growth being observed for the pre/cu + combination against escherichia coli , pseudomonas aeruginosa and proteus mirabilis . minimal antimicrobial activity was observed following incubation with fe + , mn + or zn + salts alone or in combination with pre against any of the organisms in the test panel. the addition of vitamin c markedly enhanced the activities of both pre/fe + and pre/cu + combinations against staphylococcus aureus . pelargonidin- -galactose, cyanidin- -glucose, gallic acid, quercetin, and myricetin isolated from the methanolic extract of pomegranate fruit exhibited appreciable activity against species of corynebacteria , staphylococcus , streptococcus , shigella , salmonella , bacillus subtilis , vibrio cholera , and escherichia coli (naz et al. ) . however, all these compounds were more inhibitory against gram-positive species. gallic acid exerted the highest inhibitory activity against all the tested bacteria. various tannin-rich fractions from pomegranate byproduct and the ellagitannins, ellagic acid ( ), gallagic acid ( ), punicalins ( ), and punicalagins ( ) displayed antimicrobial activity when assayed against escherichia coli , pseudomonas aeruginosa , candida albicans , cryptococcus neoformans , methicillin-resistant staphylococcus aureus (mrsa), aspergillus fumigatus and mycobacterium intracellulare (reddy et al. ) . compounds and showed activity against p. aeruginosa , c. neoformans , and mrsa. a new antifungal peptide designated as pomegranin, isolated from fresh pomegranate peels, was found to inhibit mycelial growth of the fungi botrytis cinerea and fusarium oxysporum with an ic of and . m m, respectively (guo et al. ) . lyophilized pomegranate juice (lpj) exhibited antilisterial activity in-vitro and in ground beef (lucas and were ) . against fi ve listeria monocytogenes strains, lpj had a mic of . - . % (wt/vol). the lpj ( , , , and min of heating) signi fi cantly inhibited growth of all fi ve l. monocytogenes strains in refrigerated ground cooked beef by . - . log cfu/g at day . heating did not negatively impact lpj antilisterial activity. ethanol peel extract of pomegranate exhibited in-vitro and in-vivo antimicrobial activity against salmonella typhimurium (choi et al. ) . the minimal inhibitory concentrations of their extract were in the range of . - , m g/ml. in a s. typhimurium infection mouse model. the extract was found to have signi fi cant effects on mortality and the numbers of viable s. typhimurium recovered from faeces. although clinical signs and histological damage were rarely observed in the treated mice, the untreated controls showed signs of lethargy and histological damage in the liver and spleen. the results of this study indicated that the peel extract had the potential to provide an effective treatment for salmonellosis. studies on patients with denture stomatitis showed that gel extract of p. granatum may be used as a topical antifungal agent for the treatment of candidosis associated with denture stomatitis (vasconcelos et al. ) . in subsequent studies, punica granatum phytotherapeutic gel and miconazole (daktarin oral gel) exhibited antimicrobial effect against three standard streptococci strains ( streptococcus mutans , streptococcus sanguis and streptococcus mitis ), s. mutans clinically isolated and candida albicans either alone or in association (vasconcelos et al. ) . the minimum inhibitory concentrations of adherence of punica granatum gel against the test organisms were: : for s. mutans (atcc), s. mutans (ci) and s. sanguis ; : for s. mitis and : for c. albicans . the minimum inhibitory concentrations of adherence of miconazole against the same organisms were: : , : , : , : and : , respectively. in experiments with three and four associated microorganisms, the punica granatum gel had greater ef fi ciency in inhibiting microbial adherence than the miconazole. the results of this study suggest that this phytotherapeutic agent might be used in the control of adherence of different microorganisms in the oral cavity. studies showed that the hydroalcoholic extract from punica granatu m fruits was very effective against dental plaque microorganisms, decreasing the colony forming units per milliliter (cfu/ml) by % (menezes et al. ) . while similar values were observed with chlorhexidine, used as standard and positive control ( % inhibition). however, another study found that the gel containing % punica granatum extract was not ef fi cient in preventing supragingival dental plaque formation and gingivitis (salgado et al. ) . methanolic extract of pomegranate peel exhibited antibacterial activity against oral pathogens: staphylococcus aureus and s. epidermidis (abdollahzadeh et al. ) . only at concentration of mg/ml and mg/ml the extract was effective against lactobacillus acidophilus , streptococcus mutans and streptococcus salivarius . the extract did not inhibit actinomyces viscosus and candida albicans . pomegranate rind extract (pre) singularly showed limited ef fi cacy against methicillin-sensitive and -resistant staphylococcus aureus ( mssa, mrsa) respectively but in combination with cu(ii) ions (cupric sulphate), it exhibited moderate antimicrobial effects against clinical isolates of mssa, mrsa and panton-valentine leukocidin positive community acquired mssa (pvl positive ca-mssa) isolates. (gould et al. ) . sastravaha et al. ( ) showed that adjunctive local delivery of extracts from centella asiatica in combination with p. granatum signi fi cantly improved clinical signs of chronic periodontitis such probing pocket depth, attachment level, gingival index at and months and of bleeding index at months in the test group as compared to control. no signi fi cant differences in plaque index were found between the two treatment modalities. the test group also showed statistically greater reduction of interleukin il- β at both and months and lower il- concentration. a study of young adults showed that weeks of thrice daily mouth rinsing with the extract improved salivary measures relevant to oral health including gingivitis (disilvestro et al. ) . salivary changes observed included a reduction in total protein (associated with plaque forming bacteria readings), activities of aspartate aminotransferase (an indicator of cell injury) and α-glucosidase activity (a sucrose degrading enzyme). the changes also included increased activities of the antioxidant enzyme ceruloplasmin and radical scavenging capacity. pomegranate mouth-rinse was found to have an antiplaque effect (bhadbhade et al. ) . aggregatibacter actinomycetemcomitans , porphyromonas gingivalis , and prevotella intermedia strains in-vitro. pomegranate mouth-rinse could be explored as a long-term antiplaque rinse with prophylactic bene fi ts. probiotication improved the antioxidant activity of sweet pomegranate aril juice from . to . %, and sour pomegranate juice from . to . % (fazeli et al. ) . based on the ferric reducing antioxidant power (frap) value, the reducing power of the probioticated pomegranate juices was also much stronger than the nonprobioticated juices. the frap values for sweet and sour probioticated pomegranate juices were . and . mmol/l, respectively, which were notably higher than . and . mmol/l for sweet and sour nonprobioticated juices. total counts of lactobacillus casei gg increased by about three log in sweet and two log in sour juices after h incubation. both fermentated and nonfermentated juices exhibited a potent and widespectrum antibacterial effect, with the highest activity against pseudomonas aeruginosa with the sweet juice showing wider zones of growth inhibition. the results showed that probiotication of sweet and sour pomegranate juices could add to their bene fi cial antioxidant activities. pomegranate byproducts and punicalagins inhibited the growth of pathogenic clostridia and staphylococcus aureus (bialonska et al. b ) . the growth of probiotic lactobacilli and bi fi dobacteria were generally not affected by ellagitannins. the effect of pomegranate ellagitannins on bi fi dobacteria was species-and tannin-dependent. the growth of bi fi dobacterium animalis ssp. lactis was slightly inhibited by punicalagins, punicalins, and ellagic acid. pomegranate ellagitannin-enriched polyphenol extract (pomx) supplementation signi fi cantly enhanced the growth of bi fi dobacterium breve and bi fi dobacterium infantis. bialonska et al. ( a ) found that products of the intestinal microbial transformation of pomegranate ellagitannins may account for systemic antioxidant effects. while moving through the intestines, pomegranate ellagitannins namely ellagic acid and punicalagins are metabolized by gut bacteria into urolithins that readily enter systemic circulation. their study found that the antioxidant activity of urolithins was correlated with the number of hydroxy groups as well as the lipophilicity of the molecule. the most potent antioxidants were urolithins c and d with ic values of . and . m m, respectively, when compared to ic values of . and . m m of the parent ellagic acid and punicalagins, respectively. the dihydroxylated urolithin a showed weaker antioxidant activity, with an ic value . m m, however, the potency was within the range of urolithin a plasma concentrations. numerous laboratory research, animal and human pilot studies had reported on the effectiveness of pomegranate fruit, pomegranate juice and pomegranate fruit polypehnols in reducing heart disease risk factors ldl oxidation, blood pressure, serum angiotensin converting enzyme (ace) activity, cholesterol esteri fi cation, macrophage oxidative status, and macrophage foam cell formation, all of which are steps in atherosclerosis and cardiovascular disease (aviram et al. (aviram et al. , (aviram et al. , aviram and dornfeld ; kaplan et al. ; esmaillzadeh et al. ; fuhrman et al. ; de nigris et al. ; rosenblat et al. a, b ; fuhrman and aviram ; bagri et al. ) . in healthy humans, pomegranate juice consumption decreased ldl susceptibility to aggregation and retention and increased the activity of serum paraoxonase by % (aviram et al. ) . paraoxanase an hdlassociated esterase, could protect against lipid peroxidation. in apolipoprotein e-de fi cient e o mice, oxidation of ldl by peritoneal macrophages was reduced by up to % after pomegranate juice consumption and this effect was associated with reduced cellular lipid peroxidation and superoxide release. the uptake of oxidized ldl and native ldl by mouse peritoneal macrophages obtained after pomegranate juice administration was reduced by %. further, pomegranate juice supplementation of e o mice reduced the size of their atherosclerotic lesions by % and also the number of foam cells compared with control e o mice supplemented with water. the potent antiatherogenic effects in healthy humans and in atherosclerotic mice may be attributable to its antioxidative properties. anti-atherosclerotic properties was attributed to pomegranate potent anti-oxidative characteristics. after consumption of pomegranate juice, a % reduction in serum angiotensin converting enzyme (ace) activity and a % reduction in systolic blood pressure were noted in hypertensive patients . similar dose-dependent inhibitory effect ( %) of pomegranate juice on serum ace activity was observed also in-vitro. additional studies showed that pomegranate juice supplementation to atherosclerotic mice reduced macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis (kaplan et al. ) . pomegranate juice supplementation reduced each of the proatherogenic variables. it signi fi cantly induced serum paraoxonase activity and reduced mouse peritoneal macrophage (mpm) lipid peroxide content compared with placebo-treated mice and control mice. pomegranate juice administration to apolipoprotein e-de fi cient e o mice signi fi cantly reduced the oxidized (ox)-ldl mpm uptake by % and mpm cholesterol esteri fi cation and increased macrophage cholesterol ef fl ux by % compared with age-matched, placebo-treated mice. pomegranate juice consumption reduced macrophage ox-ldl uptake and cholesterol esteri fi cation to levels lower than those in -month-old, unsupplemented controls. pomegranate juice supplementation to e o mice with advanced atherosclerosis reduced the lesion size by % compared with place botreated mice. in a separate study, supple mentation of young ( -month-old) e o mice for months with a tannin fraction isolated from pomegranate juice reduced their atherosclerotic lesion size, paralleled by reduced plasma lipid peroxidation and decreased ox-ldl mpm uptake. studies indicated that the proatherogenic effects induced by perturbed shear stress in cultured human coronary artery endothelial cells could be reversed by chronic administration of pomegranate juice (de nigris et al. (de nigris et al. , . pomegranate juice concentrate and pomegranate fruit extract rich in punicalagin reduced the activation of redox-sensitive genes elk- , p-jun, p-creb, and increased enos expression (which was decreased by perturbed shear stress) in cultured endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice. furthermore, oral administration of pomegranate juice to hypercholesterolemic mice at various stages of disease reduced signi fi cantly the progression of atherosclerosis and isoprostane levels and increased nitrates. de found that pomegranate juice reverted the potent downregulation of the expression of endothelial nitricoxide synthase (nosiii) induced by oxidized low-density lipoprotein (oxldl) in human coronary endothelial cells. their data suggested that pomegranate juice could exert bene fi cial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the nitric-oxide synthase bioactivity. aviram et al. ( ) reported that pomegranate polyphenols protected low-density lipoprotein (ldl) against cell-mediated oxidation via two pathways, including either direct interaction of the polyphenols with the lipoprotein and/or an indirect effect through accumulation of polyphenols in arterial macrophages (aviram et al. ) . pomegranate polyphenols were shown to reduce the capacity of macrophages to oxidatively modify ldl, due to their interaction with ldl to inhibit its oxidation by scavenging reactive oxygen species and reactive nitrogen species and also due to accumulation of polyphenols in arterial macrophages; hence, the inhibition of macrophage lipid peroxidation and the formation of lipid peroxide-rich macrophages. additionally, pomegranate polyphenols increased serum paraoxonase activity, resulting in the hydrolysis of lipid peroxides in oxidized lipoproteins and in atherosclerotic lesions. these antioxidative and antiatherogenic effects of pomegranate polyphenols were demonstrated in-vitro, as well as in-vivo in humans and in atherosclerotic apolipoprotein e de fi cient mice. dietary supplementation of polyphenol-rich pomegranate juice to atherosclerotic mice signi fi cantly inhibited the development of atherosclerotic lesions and this may be attributed to the protection of ldl against oxidation. subsequent studies indicated that that pomegranate juice consumption by patients with carotid artery stenosis cas decreased carotid carotid intima-media thickness (imt) and systolic blood pressure and these effects could be related to the potent antioxidant characteristics of pomegranate juice polyphenols (aviram et al. ) . for all studied parameters, the maximal effects were observed after year of pomegranate juice consumption. further consumption of pomegranate juice, for up to years, had no additional bene fi cial effects on imt and serum paraoxonase (pon ) activity, whereas serum lipid peroxidation was further reduced by up to % after years of pomegranate juice consumption. the antiatherogenic properties of pomegranate juice (pj) were attributed to its antioxidant potency and to its capacity to decrease macrophage oxidative stress, the hallmark of early atherogeneis (rosenberg et al. ). pomegranate juice polyphenols and sugar-containing polyphenolic anthocyanins were shown to confer pj its antioxidant capacity. their study showed that pj sugar consumption by diabetic mice for days resulted in a small but signi fi cant decrement in their peritoneal macrophage total peroxide levels and an increment in cellular glutathione content, compared to mouse peritoneal macrophages harvested from control diabetic mice administrated with water. these antioxidant/antiatherogenic effects could be due to the presence of unique complex sugars and/or phenolic sugars in pj. they further showed the anti-oxidative characteristics of pj unique phenolics punicalagin and gallic acid could be related, at least in part, to their stimulatory effect on macrophage paraoxonase (pon ) expression, a phenomenon which was shown to be associated with activation of the transcription factors papr γ and ap- (shiner et al. ) . similar results were obtained by pomegranate byproduct (which includes the whole pomegranate fruit left after juice preparation) ( or . m g of gallic acid equiv/kg/day) administration to apolipoprotein e-de fi cient mice that resulted in attenuation of atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized low-density lipoprotein (rosenblat et al. ) . in-vitro studies showed that preincubation of j .a macrophages with pomegranate juice resulted in a signi fi cant reduction in ox-ldl degradation by % (fuhrman et al. ) . macrophage cholesterol biosynthesis was inhibited by % after cell incubation with pomegranate juice. this inhibition, however, was not mediated at the -hydroxy- methylglutaryl coenzyme a reductase level along the biosynthetic pathway. it was concluded that pomegranate juice-mediated suppression of ox-ldl degradation and of cholesterol biosynthesis in macrophages could lead to reduced cellular cholesterol accumulation and foam cell formation. studies in iran reported that consumption of concentrated pomegranate juice may modify heart disease risk factors in hyperlipidemic ( cholesterol ³ . mmol/l or triacylglycerol ³ . mmol/l) patients (esmaillzadeh et al. (esmaillzadeh et al. , . after consumption of concentrated pomegranate juice, signi fi cant reductions were seen in total cholesterol, low-density lipoprotein (ldl)-cholesterol, ldl-cholesterol/high-density lipoprotein (hdl)-cholesterol, and total cholesterol/hdl-cholesterol. but, there were no signi fi cant changes in serum triacylglycerol and hdl-cholesterol concentrations. anthropometric indices, physical activity, kind and doses of oral hypoglycemic agents, and the intakes of nutrients and fl avonoid-rich foods showed no change during the concentrated pomegranate juice consumption period. rosenblat et al. ( ) reported that pomegranate juice consumption by diabetic patients did not affect serum glucose, cholesterol and triglyceride levels, but it resulted in a signi fi cant reduction in serum lipid peroxides and tbars (thiobarbituric acid reactive substance) levels by and %, whereas serum sh (sulfhydryl) groups and paraoxonase (pon ) activity signi fi cantly increased by and %, respectively. in the patients versus controls monocytes-derived macrophages (hmdm), they observed increased level of cellular peroxides (by %), and decreased glutathione content (by %). pomegranate juice consumption signi fi cantly reduced cellular peroxides (by %), and increased glutathione levels (by %) in the patients' hmdm. the patients' versus control hmdm took up oxidized ldl (ox-ldl) at enhanced rate (by %) and pomegranate juice consumption signi fi cantly decreased the extent of ox-ldl cellular uptake (by %). they thus concluded that pomegranate juice consumption by diabetic patients did not worsen the diabetic parameters, but rather resulted in anti-oxidative effects on serum and macrophages, which could contribute to attenuation of atherosclerosis development in these patients. pomegranate juice was found to have potent antiatherogenic activity . in-vitro studies demonstrated a pomegranate juice dose-dependent antioxidant capability against lipid peroxidation in plasma (by %), in ldl (by %), and in hdl (by %). pomegranate juice consumption by hypertensive patients reduced their systolic blood pressure (by %), along with inhibition (by %) of angiotensin converting enzyme (ace). pomegranate juice supplementation to atherosclerotic apolipoprotein e-de fi cient (e°) mice reduced their atherosclerotic lesion size by % and the number of foam cells in their lesion. consumption of pomegranate juice by ten patients with carotid artery stenosis (cas) for year reduced the patients' carotid intima-media thickness (imt) by %. these effects were associated with exvivo reduced lipid peroxidation in plasma and in isolated lipoproteins in humans and mice. furthermore, pomegranate juice consumption by humans increased the activity of their serum paraoxonase (pon ), an hdl-associated esterase that protects against lipid peroxidation. macrophage atherogenicity was studied in mouse peritoneal macrophages (mpm) harvested from e° mice. following pomegranate juice consumption, uptake of oxidized ldl and cell-mediated oxidation of ldl by macrophages was reduced by and by %, respectively, in association with reduced cellular lipid peroxidation, reduced superoxide anion release due to decreased nadph-oxidase activation, and elevated glutathione content. in-vitro studies demonstrated that pomegranate juice reduced macrophage ox-ldl degradation by %, and macrophage cholesterol biosynthesis by %. overall, the results of the above studies demonstrated that pomegranate juice consumption had very potent antiatherogenic properties, which could be associated mainly with pomegranate juice hydrolysable tannin antioxidative properties. in a recent study ) pomegranate juice (pj), fruit peels (pomxl, pomxp), arils (poma), seeds (pomo), and fl owers (pomf), extracts all were found to possess antioxidative properties in-vitro. after consumption of pomegranate juice, fruit peel, aril and fl ower extracts the atherosclerotic lesion area in atherosclerotic apolipoprotein e-de fi cient (e ) mice was signi fi cantly decreased by , , , , or %, respectively, as compared to placebo-treated group, while pomegranate seed oil had no effect. pomegrante fl ower consumption reduced serum lipids, and glucose levels by - %. consumption of the extracts except for the seed oil resulted in a signi fi cant decrement, by , , , , or %, respectively, in mpm (mouse peritoneal macrophage) total peroxides content, and increased cellular paraoxonase (pon ) activity, as compared to placebo-treated mice. the uptake rates of oxidized-ldl by e ( )-mpm were signi fi cantly reduced by approximately % after consumption of juice and the two fruit peel extracts. similar results were obtained on using j a. macrophage cell line. finally, pomegranate phenolics (punicalagin, punicalin, gallic acid, and ellagic acid), as well as pomegranate unique complexed sugars, could mimic the antiatherogenic effects of the pomegranate extracts. rock et al. ( ) reported that after weeks of pomegranate juice consumption by male patients, basal serum oxidative stress was signi fi cantly decreased by %, whereas serum concentrations of thiol groups signi fi cantly increased by %. moreover, hdlassociated paraoxonase (pon ), arylesterase, paraoxonase, and lactonase activities increased signi fi cantly after pomegranate juice consumption by - %, as compared to the baseline levels. pon protein binding to hdl was signi fi cantly increased by % following pomegranate juice consumption, and the enzyme became more stable. in male patients that consumed pomegranate polyphenol extract and in female patients that consumed pomegranate juice, a similar pattern was observed, although to a lesser extent. these bene fi cial effects of pomegranate consumption on serum pon stability and activity could lead to retardation of atherosclerosis development in diabetic patients. results of a randomized, double-blind, parallel trial involving men ( - years old) and women ( - years old) with moderate coronary heart disease risk suggested that in subjects at moderate coronary heart disease risk, pomegranate juice consumption had no signi fi cant effect on overall carotid intima-media thickness progression rate but may have slowed carotid intima-media thickness progression in subjects with increased oxidative stress and disturbances in the triglycerides-rich lipoprotein/high-density lipoprotein axis (davidson et al. ) . lei et al. ( ) reported that the pomegranate leaf extract could inhibit the development of obesity and hyperlipidemia in high-fat diet induced obese mice. mice treated with the extract presented a signi fi cant decrease in body weight, energy intake and various adipose pad weight percents and serum, serum total cholesterol (tc), triglyceride (tg), glucose levels and tc/highdensity lipoprotein cholesterol (hdl-c) ratio after weeks treatment. further, the extract signi fi cantly attenuated the raising of the serum tg level and inhibited the intestinal fat absorption in mice given a fat emulsion orally. the effects were postulated to be partly mediated by inhibiting the pancreatic lipase activity and suppressing energy intake. yamasaki et al. ( ) found that mice fed dietary pomegranate seed oil (pso) high in levels of punicic acid showed signi fi cant increases in serum triacylglycerol and phospholipid levels but not in total cholesterol. punicic acid could be detected in serum, liver, and adipose tissues in mice fed the . or . % pso diet. oral administration of streptozotocin-induced diabetic wistar rats with of and mg/kg of aqueous pomegranate fl ower extract for days resulted in a signi fi cant fall in fasting blood glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol , very low density lipoprotein, lipid peroxidation level (bagri et al. ) . pomegrante extract elevated levels of high density lipoprotein cholesterol (hdl-c), reduced glutathione (gsh) and the antioxidative enzymes, glutathione peroxidase (gpx), glutathione reductase (gr), glutathione-s-transferase (gst), superoxide dismutase (sod) and catalase (cat). mcfarlin et al. ( ) found that weight gain in high fat diet mice was associated with an increase in biomarkers of cholesterol pro fi le, glucose sensitivity, adipose tissue accumulation and systemic low-grade in fl ammation. despite a similar level of energy intake, high-fate diet mice had a greater concentration of leptin and a lower concentration of adiponectin compared to high fat + pomegranate seed oil diet mice. pomegranate seed oil, a rich source of -cis , -trans conjugate linolenic acid, only altered body weight accumulation, fi nal body weight, leptin, adiponectin and insulin. pomegranate seed oil intake was associated with an improvement in insulin sensitivity, suggesting that risk of developing type two diabetes may have been reduced; however, cvd risk did not change. lan et al. ( ) demonstrated that ellagic acid in pomegranate leaf tannins could be transported into hepg cells and this correlated with total cholesterol alteration in the cells. vroegrijk et al. ( ) found that pomegranate seed oil, a rich source of punicic acid, ameliorated high-fat diet induced obesity and insulin resistance in mice, independent of changes in food intake or energy expenditure. compared to high fat diet mice, its intake resulted in a lower body weight and improved peripheral insulin sensitivity but did not affect liver insulin sensitivity. in a randomized, double-blind, placebo-controlled clinical trial of obese adult volunteer, pomegranate juice administration for month did not modify insulin secretion and sensitivity in the obese patients, however, the natural evolution to increased weight and adiposity was halted (gonzález-ortiz et al. ) . rosenblat and aviram ( ) found that the inhibitory effect of pomegranate juice on triglyceride biosynthesis could be attributed to a direct effect of pomegranate juice on the activity of diacylglycerol acyltransferase (dgat ) the rate-limiting enzyme in triglyceride biosynthesis. pomegranate juice and its constituent punicalagin signi fi cantly and dose-dependently decreased the triglyceride content and triglyceride biosynthesis rate in j a. macrophages or in c bl/ mouse peritoneal macrophages. both pomegranate juice and punicalagin increased ( . -fold) mouse peritoneal macrophages paraoxonase (pon ) mrna expression, and pon was previously shown to inhibit dgat activity. however, the addition of pj or punicalagin ( m m) to microsomes from pon -de fi cient mouse peritoneal macrophages still resulted in a signi fi cant reduction ( - %) in dgat activity. in a randomised block design study of student volunteers, supplementation of pomegranate juice caused a fall in diastolic blood pressure and this could be related to ros scavenging activity rather than to angiotensin-converting enzyme inhibitors (wright and pipkin ) oral administration of pomegranate juice extract ( and mg/kg) to angiotensin-ii treated rats for weeks signi fi cantly reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines (waghulde et al. ) . pomegranate juice administration signi fi cantly decreased the serum levels of ace (angiotensin converting enzyme) and the levels of thiobarbituric acid reactive substances (tbars); while enzyme activity of superoxide dismutase (sod), catalase (cat), glutathione reductase (gsh) in kidney tissue showed a signi fi cant elevation in pomegranate juice treated angiotensin-ii induced hypertensive rats. the results suggested that pomegranate juice extract could prevent the development of high blood pressure induced by angiotensin-ii probably by combating the oxidative stress and antagonizing the physiological actions of angiotensin-ii. chronic administration of pomegranate fruit juice (pj) extract ( and mg/kg; p.o. for weeks) reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines and also reversed the biochemical changes induced by diabetes and angiotensin ii (ang ii) (mohan et al. b ) . acute subcutaneous administration of angiotensin ii causes a rise in blood pressure in streptozotocin-induced diabetic wistar rats. pj treatment also caused a signi fi cant decrease in levels of thiobarbituric acid reactive substances (tbars) in the kidney and pancreas while activities of enzymes superoxide dismutase (sod), catalase (cat), and glutathione reductase (gsh) showed signi fi cant elevation. pj treatment prevented the tubular degenerative changes induced by diabetes. the results suggested that the pj extract could prevent the development of high blood pressure induced by ang ii in diabetic rats probably by combating the oxidative stress induced by diabetes and ang ii and by inhibiting ace activity. pomegranate in particular its fl owers, seeds, and juice have been employed for the treatment of various diseases in traditional unani and ayurvedic systems of medicine in india but only the fl ower has been prescribed for the treatment of diabetic disorders katz et al. ) . the mechanisms for it hypoglycaemic effects are largely unknown, though recent research suggested pomegranate fl owers and juice may prevent diabetic sequelae via peroxisome proliferator-activated receptor (ppar) α/γ binding and nitric oxide production (katz et al. ; huang et al. a, b ; li et al. ; xu et al. ) . pomegranate compounds associated with such effects include oleanolic, ursolic, and gallic acids (katz et al. ) . another study suggested that punica granatum fl ower (pgf) extract inhibited increased cardiac fatty acid uptake and oxidation in the diabetic condition (huang et al. b ) . pgf extract and its component oleanolic acid enhanced peroxisome proliferator-activated receptor (ppar)-α luciferase reporter gene activity in human embryonic kidney cells. this effect was completely suppressed by a selective ppar-α antagonist mk- , consistent with the presence of ppar-α activator activity in the extract and this component. the fi ndings suggested that pgf extract improved abnormal cardiac lipid metabolism in zucker diabetic fatty rats by activating ppar-α and thereby lowering circulating lipid and inhibiting its cardiac uptake. excess triglyceride (tg) accumulation and increased fatty acid (fa) oxidation in the diabetic heart contribute to cardiac dysfunction. in subsequent in-vitro studies, the scientists (huang et al. a ) demonstrated that -week oral administration of methanol extract from pgf ( mg/kg, daily) inhibited glucose loading-induced increase of plasma glucose levels in zucker diabetic fatty rats (zdf), a genetic animal model for type two diabetes, whereas it did not inhibit the increase in zucker lean rats (zl). the treatment did not lower the plasma glucose levels in fasted zdf and zl rats. further, rt-pcr results demonstrated that the pgf extract treatment in zdf rats enhanced cardiac ppar-γ mrna expression and restored the down-regulated cardiac glucose transporter (glut)- (the insulin-dependent isoform of gluts) mrna. these results suggest that the anti-diabetic activity of pgf extract may result from improved sensitivity of the insulin receptor. from the in-vitro studies, it was demonstrated that the pgf extract enhanced ppar-γ mrna and protein expression and increased ppar-γ-dependent mrna expression and activity of lipoprotein lipase in human thp- -differentiated macrophage cells. phytochemical investigation demonstrated that gallic acid in pgf extract was mostly responsible for this activity. further in-vitro studies showed that punica granatum fl ower extract and its components oleanolic acid, ursolic acid, and gallic acid inhibited lipopolysaccharide-induced nf-kappab activation in macrophages. the fi ndings indicated that punica granatum fl ower extract reduced cardiac fi brosis in zucker diabetic fatty rats, at least in part, by modulating cardiac et- and nf-kappab signalling. recent studies suggested that pomegranate fl ower (pgf) medicine ameliorated diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation ) . pgf-treated zdf rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. these effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (ppar)-α, carnitine palmitoyltransferase- and acyl-coa oxidase (aco), and reduced stearoyl-coa desaturase- . in contrast, pgf showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. pgf treatment also increased ppar-α and aco mrna levels in hepg cells. li et al. ( ) reviewed the dual ppar-α/-γ activator properties of pomegranate fl ower and its potential treatment of diabetes and its associated complications. ppars are nuclear transcription factors and are the major regulators of lipid and glucose metabolism. ppar-α is involved in the regulation of fatty acid (fa) uptake and oxidation, in fl ammation and vascular function, while ppar-γ participates in fa uptake and storage, glucose homeostasis and in fl am mation. synthetic ppar-α or ppar-γ agonists have been widely used in the treatment of dyslipidaemia, hyperglycaemia and their complications. however, they are associated with an incidence of adverse events. given the favourable metabolic effects of both ppar-α and ppar-γ activators, as well as their potential to modulate vascular disease, com bined ppar-α/-γ activation has recently emerged as a promising concept, leading to the development of mixed ppar-α/-γ activators. hontecillas et al. ( ) demonstrated that punicic acid (pua), a conjugated linolenic acid isomer found in pomegranate, caused a dosedependent increase ppar α and γ reporter activity in t -l pre-adipocyte cells and bound although weakly to the ligand-binding domain of human ppar γ. dietary pua decreased fasting plasma glucose concentrations, improved the glucose-normalizing ability, suppressed nf-kappab activation, tnf-α expression and upregulated ppar αand γ-responsive genes in skeletal muscle and adipose tissue. pua improved glucose homeostasis and suppress obesity-related in fl ammation studies in india showed that pomegranate seed extract ( , and mg/kg) administered orally to streptozotocin (stz)-induced diabetic rats caused a signi fi cant reduction of blood glucose levels by and %, respectively, at the end of h (das et al. ) . kim et al. ( ) found that administration of pomegranate extract to streptozotocin (stz)-induced diabetic mice for weeks improved postprandial glucose regulation. further elevated na(+)-dependent glucose uptake by brush border membrane vesicles isolated from stz mice was normalized by pomegranate treatment. the results suggested that pomegranate extract could play a role in controlling the dietary glucose absorption at the intestinal tract by decreasing sodium-coupled glucose transporter sglt expression, and may contribute to blood glucose homeostasis in the diabetic condition. oral administration of pomegranate fl ower (pgf) extract markedly lowered plasma glucose levels in non-fasted zucker diabetic fatty rats (a genetic model of obesity and type two diabetes), whereas it had little effect in the fasted animals, suggesting it affected postprandial hyperglycemia in type two diabetes . the extract was found to markedly inhibit the increase of plasma glucose levels after sucrose loading, but not after glucose loading in mice, and it had no effect on glucose levels in normal mice. in-vitro, pgf extract demonstrated a potent inhibitory effect on α-glucosidase activity (ic : . m g/ml). these fi ndings strongly suggested that pgf extract improved postprandial hyperglycemia in type two diabetes and obesity, at least in part, by inhibiting intestinal α-glucosidase activity. postprandial hyperglycemia plays an important role in the development of type two diabetes and has been proposed as an independent risk factor for cardiovascular diseases. in a recent paper, bagri et al. ( a ) reported that oral administration of pomegranate aqueous extract at doses of and mg/kg for days to stzinduced diabetic rats resulted in a signi fi cant reduction in fasting blood glucose, total cholesterol (tc), triglycerides (tg), low-density lipoprotein cholesterol (ldl-c), very low density lipoprotein (vldl), and tissue lipid peroxidation levels coupled with elevation of high density lipoprotein cholesterol (hdl-c), glutathione (gsh) content and antioxidant enzymes in comparison with diabetic control group. the results suggested that pg could be used, as a dietary supplement, in the treatment of chronic diseases characterized by atherogenous lipoprotein pro fi le, aggravated antioxidant status and impaired glucose metabolism and also in their prevention. in-vitro studies showed that pomegranate juice polyphenols increased recombinant paraoxonase- binding to high-density lipoprotein (hdl) beyond their antioxidative effect (fuhrman et al. ) . further recombinant paraoxonase- was found to be associated more ef fi ciently with hdls isolated from diabetic patients after pomegranate juice consumption versus hdls isolated before pomegranate juice consumption. studies by ahmed et al. ( ) showed that pomegranate fruit extract or compounds derived from it may inhibit cartilage degradation in osteoarthritis and may also be a useful nutritive supplement for maintaining joint integrity and function. the extract inhibited interleukin (il)- β induced expression of matrix metalloproteinases by suppressing the activation of mitogen-activated protein kinases and nuclear factor-kappab in human chondrocytes in-vitro. pomegranate methanol extract was found to dose-dependently inhibit tumour necrosis factor α (tnf-α) production in lipopolysaccharide (lps) stimulated cells (jung et al. ) . the data suggested that the extract may suppress lps-stimulated tnf production through inhibition of nfkappab in bv microglia cells. shukla et al. ( b ) reported that consumption of hydrolyzable tannins-rich pomegranate extract potently delayed the onset and reduced the incidence and severity of collagen-induced arthritis in mice. pomegranate extract -fed mice had reduced joint in fi ltration by the in fl ammatory cells, and the destruction of bone and cartilage were alleviated. levels of interleukin il- were signi fi cantly decreased in the joints of pomegranate-fed mice with collagen-induced arthritis. in mouse macrophages, pomegranate extract abolished multiple signal transduction pathways and downstream mediators implicated in the pathogenesis of rheumatoid arthritis. in another study, rabbit plasma samples collected after oral ingestion of polyphenol rich pomegranate fruit extract were found to inhibit the il- β-induced pge and no production in chondrocytes (shukla et al. a ) . these same plasma samples also inhibited both cox- and cox- enzyme activity ex-vivo but the effect was more pronounced on the enzyme activity of cox- enzyme. the studies suggested that pomegranate fruit extract-derived bioavailable compounds may exert an anti -in fl ammatory effect by inhibiting the in fl ammatory cytokineinduced production of pge and no in-vivo. pomegranate extract rich in polyphenols was found to inhibit the interleukin- b -induced activation of mkk- , p a -mapk and transcription factor runx- in human osteoarthritis chondrocytes (rasheed et al. ) . this pharmacological actions of pomegranate extract suggest that the extract or its derived compounds may be developed as mkk and p -mapk inhibitors for the treatment of osteoarthritis and other degenerative/ in fl ammatory diseases. in a pilot week openlabelled study, pomegranate consumption reduced the composite disease activity index (das ) and tender joint count in rheumatoid arthritis patients, and this effect could be related to the antioxidative property of pomegranates (balbir-gurman et al. ) . the results suggested dietary supplementation with pomegranates may be a useful complementary strategy to attenuate clinical symptoms in rheumatoid arthritis patients. supplementation of obese zucker rats with pomegranate fruit extract (pfe) or pomegranate juice (pj) signi fi cantly decreased the expression of vascular in fl ammation markers, thrombospondin (tsp), and cytokine tgfβ , whereas seed oil supplementation had a signi fi cant effect only on tsp- expression (de nigris et al. a ) . plasma nitrate and nitrite (no(x)) levels were signi fi cantly increased by pfe and pj. in addition, the effect of pfe in increasing endothelial no synthase (enos) expression was comparable to that of pj. the data suggested possible clinical applications of pfe in metabolic syndrome (clinical conditions such as obesity, hypertension, dislipidemia, and diabetes). in-vivo studies revealed that aqueous pomegranate peel extract inhibited neutrophil myeloperoxidase activity and attenuated lipopolysaccharide-induced lung in fl ammation in mice (bachoual et al. ) . inhibition of myeloperoxidase activity by pomegranate extract could explain its antiin fl ammatory action. balwani et al. ( ) demonstrated that -methyl-pyran- -one- -o -b -d-glucopyranoside (mpg) isolated from pomegranate leaves, inhibited tnf a -induced cell adhesion molecules expression by blocking nuclear transcription factor-k b (nf-k b) translocation and activation. the results suggested that mpg could be useful as a novel lead molecule for developing future antiin fl ammatory agents. oral pomegranate extract decreased reactive oxygen species concentration and acute in fl ammation in the tympanic membrane in rats after myringotomy (kahya et al. ) . the density of in fl ammatory cells was signi fi cantly less in rats treated with the extract and the lamina propria thickness and vessel density were also signi fi cantly reduced. pretreatment of wistar rats with a methanolic extract of pomegranate peel followed by carbon tetrachloride treatment retained catalase, peroxidase, and superoxide dismutase to values comparable with control values, whereas lipid peroxidation was reduced by % as compared to control (chidambara murthy et al. ) . histopathological studies of the liver supported the hepatoprotective effects exhibited by the extract by restoring the normal hepatic architecture. kaur et al. ( ) demonstrated that pre-treatment of mice with pomegranate fl ower extract at a dose regimen of - mg/ kg body weight for a week signi fi cantly and dose dependently protected against ferric nitrilotriacetate (fe-nta)-induced oxidative stress as well as hepatic injury. the extract conferred up to % protection against hepatic lipid peroxidation and preserved glutathione (gsh) levels and activities of antioxidant enzymes viz., catalase (cat), glutathione peroxidase (gpx) glutathione reductase (gr) and glutathione-s-transferase (gst) by up to , . , . , . and . % respectively. a protection against fe-nta induced liver injury was apparent as inhibition in the modulation of liver markers viz., aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), bilirubin and albumin in serum. the histopathological changes produced by fe-nta, such as ballooning degeneration, fatty changes, necrosis were also alleviated by the extract. the fl ower extract was found to signi fi cantly scavenge superoxide radicals by up to . %, hydrogen peroxide by up to %, hydroxyl radicals by up to % and nitric oxide by up to . %. the extract also inhibited (.)oh induced oxidation of lipids and proteins in vitro. the potent antioxidant property of the fl ower extract was postulated to be responsible for its hepatoprotective effects. in another study, pomegranate fl ower infusion was found to exhibit hepatoprotective and antioxidant effect against trichloroacetic acid (tca)-exposed rats (celik et al. ) . the infusion signi fi cantly decreased levels of aspartate aminotransferase and alanine aminotransferase; increased signi fi cantly glutathione s-transferase activity in the liver, brain and spleen and maintained superoxide dismustase level in the liver. intake of pomegranate juice by mice for weeks was found to confer hepatic protection against protein and dna oxidation (faria et al. b ) . there was also a signi fi cant decrease in gsh (reduced glutathione) and gssg (oxidized glutathione), without change in the gsh/gssg ratio. all studied enzymatic activities (superoxide dismutase (sod), glutathione peroxidase (gpx), glutathione s-transferase (gst) and glutathione reductase (gr) and catalase) were found to be decreased by pomegranate juice treatment. also, glutathione s-transferase and glutathione synthetase transcription were also decreased in this group. chronic pomegranate peel extract administration to rats alleviated the bile duct ligation (bdl)-induced oxidative injury of the liver and improved the hepatic structure and function (toklu et al. ) . plasma antioxidant capacity and hepatic glutathione levels were signi fi cantly depressed by bdl but were increased back to control levels in the pomegranate extract-treated bdl group. increases in tissue malondialdehyde levels and myeloperoxidase activity due to bdl were reduced back to control levels by pomegranate extract treatment. similarly, increased hepatic collagen content in the bdl rats was reduced to the level of the control group with extract treatment. sumner et al. ( ) showed that daily consumption of pomegranate juice may improve stressinduced myocardial ischemia in patients who have coronary heart disease in a randomized, placebo-controlled, double-blind study. after months, the extent of stress-induced ischemia decreased in the pomegranate group (sds − . ± . ) but increased in the control group (sds . ± . ). this bene fi t was observed without changes in cardiac medications, blood sugar, hemoglobin a c, weight, or blood pressure in either group. mohan et al. ( a ) demonstrated that pre-treatment of male wistar rats with pomegranate juice ( and mg/kg, p.o.) and its butanolic extract( mg/kg., p.o.) for a period of days signi fi cantly inhibited the effects of isoproterenol-induced myocardial infarction such as heart rate, pressure rate index, ecg patterns, levels of lactate dehydrogenase, creatine kinase, superoxide dismutase and catalase in the serum and vascular reactivity changes. treatment with pj and b-pj ( mg/kg., p.o.) alone did not alter any of the parameters as compared to vehicletreated wistar rats. hassanpour et al. ( ) found that pomegranate fruit extract displayed cardioprotective doxorubicin (dox)-induced cardiotoxicity in rats. rats administered the extract showed decreased qt and increase in heart rate compared to the dox group signi fi cant decrease in creatine kinase-mb isoenzyme, lactate dehydrogenase and no such signi fi cant decrease in aspartate aminotransferase were observed as compared to the dox group. there was signi fi cant increase in the level of reduced glutathione, whereas inhibition of lipid peroxidation and increase in superoxide dismutase concentration was not signi fi cant in the extract treated group compared to the dox group. histopathological study of the extract -treated group showed slight protection against myocardial toxicity induced by dox. p. granatum fruit peel extract elicited % precipitation of ovine haemoglobin in-vitro and when orally administered to ethanol-induced gastric-damaged rats produced a signi fi cant decrease in gastric lesions (gharzouli et al. ) . the acid content of the stomach was signi fi cantly increased by pomegranate ( %) suggesting that monomeric and polymeric polyphenols could strengthen the gastric mucosal barrier. administration of % methanolic pomegranate rind extract inhibited aspirin-and ethanol-induced gastric ulceration (ajaikumar et al. ) . treated animals showed increased antioxidant levels of superoxide dismutase (sod), catalase, glutathione (gsh) and glutathione peroxidase (gpx) and decreased level of tissue lipid peroxidation. no erosion of gastric mucosa, sub-mucosal edema and neutrophil in fi ltration was observed in treated animals. pomegranate tannins ( , , mg/ kg) signi fi cantly inhibited ulcerative formation induced by both water immersion stress and pylorus ligation, and decreased the gastric mucosa damages induced by intragastric absolute ethanol, in dose-dependent manner in rats (lai et al. ) . its antiulcer effect was found to be due to increasing secretion of adherent mucus and free mucus from the stomach wall, which may inhibit generation of oxygen-derived free radicals, and decrease the consumption glutathione peroxidase (gsh-px) and superoxide dismutase (sod), decrease absolute alcohol-induced elevation of malondialdehyde and maintain content of no at normal level. punica granatum peel extract (ppe) supplementation of irradiated rats reduced oxidative damage in the ileal tissues and protected against ionizing radiation-induced enteritis and leukocyte apoptosis in rats, probably by a mechanism associated with the decreased production of reactive oxygen metabolites and enhancement of antioxidant mechanisms (toklu et al. ) . ppe treatment reversed all these biochemical indices induced by irradiations such as the decrease in glutathione and total antioxidant capacity associated with increases in malondialdehyde levels, myeloperoxidase activity, collagen content of the tissue with a concomitant increase -hydroxy- ¢deoxyguanosine (an index of oxidative dna damage) and increases in pro-in fl ammatory cytokines (tnf-α, il- β and il- ) and lactate dehydrogenase. histopathological alterations and the increase in leukocyte apoptosis and cell death induced by irradiation was also reversed by ppe. oral administration of aqueous methanolic extract of pomegranate ( and mg/kg bw) signi fi cantly reduced the ulcer lesion index produced by alcohol, indomethacin, and aspirin, at both doses in rats (alam et al. ) . in pylorusligated rats the extract signi fi cantly reduced the ulcer lesions, gastric volume, and total acidity and prevented the ulceration by increasing the ph and mucus secretion. oral administration of pomegranate extract and its ellagic acid rich fraction ( and mg/ kg) signi fi cantly attenuated dextran sulfate sodium -induced colonic in fl ammation in mice along with attenuation of histamine, myeloperoxidase and oxidative stress (singh et al. ) . the antiulcerative effect was comparable to sulphasalazine ( mg/kg, p.o.) and sodium cromoglycate ( mg/kg i.p). the authors stated that the antiulcerative effects may be attributed to mast cell stabilizing, antiin fl ammatory and antioxidant actions. pomegrante peel extracts exhibited remarkable in-vitro anti-helicobacter pylori activity against the clinical isolates of h. pylori (mean of inhibition zone diameter ranging from to mm/ m g disc). helicobacter pylori infection causes lifelong chronic gastritis, which can lead to peptic ulcer, mucosa-associated lymphoid tissue (malt) lymphoma and gastric cancer. pretreatment of rats with hydroalcoholic extract of pomegranate fl owers ( and mg/kg p.o. twice daily for days) signi fi cantly attenuated hypertonic glycerol-induced myoglobinuric renal dysfunction in a dose-dependent manner . the mechanism of renoprotective effects of punica granatum was found to involve activation of ppar-g and nitric oxide-dependent signalling pathway. pomegranate fruit rind powder at the dose of mg/kg orally as aqueous suspension was found to stimulate the cell-mediated and humoral components of the immune system in rabbits (gracious ross et al. ) . the pomegranate powder elicited an increase in antibody titer to typhoid-h antigen. it also enhanced the inhibition of leucocyte migration in leucocyte migration inhibition test and induration of skin in delayed hypersensitivity test with puri fi ed protein derivative (ppd) con fi rming its stimulatory effect on cell-mediated immune response. punicalagin isolated from pomegranate fruit was found to be a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated t cells (nfat). punicalagin downregulated the mrna and soluble protein expression of interleukin- from anti-cd /anti-cd -stimulated murine splenic cd + t cells and suppressed mixed leukocytes reaction (mlr) without exhibiting cytotoxicity to the cells. in vivo, the punicalagin treatment inhibited phorbol -myristate -acetate (pma)induced chronic ear edema in mice and decreased cd + t cell in fi ltration of the in fl amed tissue. the results suggested that punicalagin could be a potential candidate for the therapeutics of various immune pathologies. yamasaki et al. ( ) found that dietary pomegranate seed oil (pso) high in levels of punicic acid ( c, t, c-octadecatrienoic acid), may enhance b-cell function in mice. splenocytes isolated from mice fed . or . % pso produced larger amounts of immunoglobulins g and m but not immunoglobulin a irrespective of stimulation with or without phorbol -myristate -acetate and the calcium ionophore a . dietary pso did not affect the percentages of b cells or cd -positive or cd -positive t cells in splenocytes. a polysaccharide (psp ) isolated from pomegranate rind was found to have immunomodulatory activity (joseph et al. ) . psp showed in-vitro growth stimulatory effect on isolated normal lymphocytes, and a proliferative index of . at a concentration of , m g/-ml was obtained, indicating immunomodulatory activity. wistar rats with excision wounds treated with % water-soluble gel formulated from the methanolic extract of dried pomegranate rind, showed good complete wound healing after days (chidambara murthy et al. ) . in comparison in rats treated with . % gel, healing was observed on day , and in the positive control animals receiving the blank gel took - days for complete healing. collagen content in terms of hydroxyproline level increased by two-fold in the group treated with . % gel. the gel extract was found to contain gallic acid and catechin as major components. aslam et al. ( ) found that pomegranate seed oil, but not aqueous extracts of fermented juice, peel or seed cake, stimulated human keratinocyte proliferation in monolayer culture. contrariwise, pomegranate peel aqueous extract (and to a lesser extent, both the fermented juice and seed cake extracts) stimulated type i procollagen synthesis and inhibited matrix metalloproteinase- (mmp- ; interstitial collagenase) production by dermal fi broblasts, but had no growth-supporting effect on keratinocytes. the results suggested that pomegranate peel aqueous extract could promote regeneration of dermis and pomegranate seed oil could promote regeneration of epidermis. pomegranate peel methanol extract-based ointment signi fi cantly enhanced wound contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile strength), the biochemical (increasing of collagen, dna and proteins synthesis) and the histopathological characteristics in guinea pigs (hayouni et al. ) . the extract displayed antioxidant activity as potent as natural and synthetic compounds (trolox, butylated hydroxyanisole, quercetin). in addition, the extract exhibited signi fi cant antibacterial and antifungal activity against pseudomonas aeruginosa , staphylococcus aureus , escherichia coli , klebsiella pneumoniae , salmonella anatum , salmonella typhimurium , streptococcus pneumoniae , and fungi candida albicans , candida glabrata , trichopyton rubrum and aspergillus niger. the results suggested that the pomegranate formulated ointment may be used as skin repair agent without hazard to human health. the ethanol extract of p. granatum fl owers showed signi fi cant wound healing activity when topically administered in rats (pirbalouti et al. ) . the extract signi fi cantly increased the rate of wound contraction and collagen turnover. in-vitro studies using normal human epidermal keratinocytes, showed that pre-treatment with pomegranate fruit extract rich in anthocyannins and hydrolyzable tannins protected against the adverse effects of uv-b radiation by inhibiting uv-b-induced modulations of nuclear factor kappa b (nf-kappab) and mitogen-activated protein kinases (mapk) pathways (afaq et al. a, b ) . similarly, they reported pomegranate fruit extract to be an effective agent for ameliorating uva-mediated skin damages by modulating cellular pathways in-vitro (syed et al. ) . uva-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. pretreatment of normal human epidermal keratinocytes with the extract ( - m g/ ml) for h before exposure to uva resulted in a dose-dependent inhibition of uva-mediated phosphorylation of signal transducers and activators of transcription (stat ), protein kinase b/ akt and mitogen activated protein kinases (mapks) viz. extracellular signal-regulated kinase (erk / ). the extract pretreatment also inhibited uva exposure-mediated increases in ki- antigen and pcna (proliferating cell nuclear antigen) and increased the cell-cycle arrest induced by uva in the g phase and the expression of bax and bad (proapoptotic proteins), while suppressing bcl-x(l) antiapoptotic protein expression. studies by zaid et al. ( ) showed that pretreatment of human immortalized hacat keratinocytes with polyphenol-rich pomegranate fruit extract inhibited uvb-mediated decrease in cell viability, decrease in intracellular glutathione content and increase in lipid peroxidation. immunoblot analysis showed that pretreatment of hacat cells with pomegranate fruit extract inhibited uvb-induced ( ) upregulation of mmp- , - , - and - , ( ) decrease in timp- , ( ) phosphorylation of mapks and (iv) phosphorylation of c-jun, whereas no effect was observed on uvb-induced c-fos protein levels. the results suggested that pomegranate fruit protected hacat cells against uvb-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products. pomegranate fruit extract ( - mg/l) was effective at protecting human skin fi broblasts from cell death following uv irradiation (pacheco-palencia et al. ) . this photoprotective effect was postulated to be related to a reduced activation of the pro-in fl ammatory transcription factor nf-kappab, suppression of proapoptotic caspase- , and an increased g /g phase, associated with dna repair. higher polyphenolic concentrations ( - , mg/l) were required to achieve a signi fi cant reduction in uv-induced reactive oxygen species levels and increased intracellular antioxidant capacity. pretreatment of reconstituted human skin "epiderm" with pomegranate-derived products pomx juice, pomx extract and pomegranate oil inhibited uvb-induced cyclobutane pyrimidine dimers (cpd), -dihydro- ¢deoxyguanosine ( -ohdg), protein oxidation and proliferating cell nuclear antigen (pcna) protein expression (afaq et al. ) . further, pretreatment of epiderm with pomegranate-derived products resulted in inhibition of uvb-induced collagenase (mmp- ), gelatinase (mmp- , mmp- ), stromelysin (mmp- ), marilysin (mmp- ), elastase (mmp- ), and tropoelastin. mmp- and mmp- activities were also inhibited. overall, the results suggested that all three pomegranate-derived products may be useful against uvb-induced damage to human skin. park et al. ( ) using cultured human skin fi broblasts, demonstrated that pomegranate fruit rind extract rich in polyphenols catechin, quercetin, kaempferol, and equol signi fi cantly protected against uvb-induced skin damage. the synthesis of collagen was increased and the expression of mmp- was decreased. oral feeding of pomegranate fruit extract to mice provided substantial protection from the adverse effects of uvb radiation via modulation in early biomarkers of photocarcinogenesis (afaq et al. ) . the extract inhibited uvb-induced: skin edema; hyperplasia; in fi ltration of leukocytes; lipid peroxidation; hydrogen peroxide generation; ornithine decarboxylase (odc) activity; and odc, cyclooxygenase- and proliferating cell nuclear antigen protein expression. the extract enhanced repair of uvbmediated formation of cyclobutane pyrimidine dimers (cpds) and -oxo- , -dihydro- ¢deoxyguanosine ( -oxodg). the extract inhibited uvb-mediated nuclear translocation of nf-k b; activation of ikk a ; and phosphorylation and degradation of i k b a . additionally, the extract further enhanced uvb-mediated increase in tumour suppressor p and cyclin kinase inhibitor p . in further studies, khan et al. ( ) reported that oral feeding of pomegranate fruit extract to skh- hairless mice inhibited uvbinduced epidermal hyperplasia, in fi ltration of leukocytes, protein oxidation and lipid peroxidation. immunoblot analysis demonstrated that oral feeding of pomegranate fruit extract to mice inhibited uvb-induced ( ) nuclear translocation and phosphorylation of nuclear factor kappa b/p , ( ) phosphorylation and degradation of i k b a , ( ) activation of ikk a / i k k b and ( ) phosphorylation of mitogen-activated protein kinase proteins and c-jun. pomegranate fruit extract consumption also inhibited uvb-induced protein expression of ( ) cox- and inos, ( ) pcna and cyclin d and ( ) matrix metalloproteinases- ,- and - in mouse skin. overall, the data showed that pomegranate fruit extract consumption afforded protection to mouse skin against the adverse effects of uvb radiation by modulating uvbinduced signalling pathways. in a double-blind, placebo-controlled trial involving female subjects age - s, kasai et al. ( ) found that oral administration of an ellagic acid-rich pomegranate extract had an inhibitory effect on a slight pigmentation (stains and freckles, brightness of face) in the human skin caused by uv irradiation. methanolic pomegranate extract showed . % invitro mushroom tyrosinase inhibitory activity (adhikari et al. ) . a pomegranate rind extract was found to have skin whitening activity (yoshimura et al. ) . the extract exhibited inhibitory activity against mushroom tyrosinase invitro comparable to that of the skin whitening agent, arbutin. when taken orally the extract inhibited uv-induced skin pigmentation on the back of brownish guinea pig. the results suggested the skinwhitening effect of the extract was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. a pomegranate polysaccharide fraction inhibited the formation of advanced glycation end-products (ages) by % and also inhibited the formation of fructosamine in the bsa/glucose system (rout and banerjee ) . the fraction inhibit , -diphenyl- -picrylhydrazyl (dpph) and , ¢ -azinobis[ ethylbenzothiazoline- -sulfonate] abts(+) radical activities by and %, respectively with m g/ ml concentration it also inhibited mushroom tyrosinase by % at m g/ml concentration suggesting its ef fi cacy as a potential skin whitener. pomegrante juice was shown to have a protective effect against ethylene glycol-induced nephrolithiasis in rats (tugcu et al. ) . ethylene glycol caused hyperoxaluria characterised by severe crystalization in renal tubules and granulovacuolar epithelial cell degeneration, marked elevation in malondialdehyde and nitric oxide levels and decrease of reduced glutathione (gsh) in rats there was no crystal formation in the rats treated with ethylene glycol and pomegranate juice. administration of pomegranate juice at medium and high dosage to rats was found to have inhibitory effects on renal tubular cell injury and oxidative stress caused by oxalate crystal deposition by reducing ros, inos, p -mapk, and nf-kb expression (ilbey et al. ) . rats treated with methanol pomegranate seed extract exhibited signi fi cant inhibitory activity against castor-oil induced diarrhoea and pge induced enteropooling (das et al. ) . the extract also displayed a signi fi cant reduction in gastrointestinal motility in charcoal meal test in rats. pomegranate juice and polyphenol-rich pomegranate fruit extract reduced platelet aggregation, calcium mobilization, thromboxane a( ) production, and hydrogen peroxide formation, induced by collagen and arachidonic acid (mattiello et al. ) . the polyphenol -rich fruit extract was more potent in reducing platelet activation. studies showed that pomegranate fruit components (mainly ellagic acid) modulated human thrombin amidolytic activity (cuccioloni et al. ) . pomegranate fruit ethanol extract was found to signi fi cantly increase the growth of osteoblastic mc t -e cells and caused a signi fi cant elevation of alkaline phosphatase (alp) activity and collagen content in the cells (kim and choi ) . treatment the extract decreased the tnfα-induced production of interleukin il- and nitric oxide in osteoblasts. promprom et al. ( ) found pomegranate seed extract to be a potent stimulator of phasic activity in rat uterus. pomegranate seed extract and b -sitosterol, the main constituent of the extract ( %), increased spontaneous contractions of the rat uterus in a concentration-dependent manner. their data suggested that the uterotonic effect was due to nonestrogenic effects of b -sitosterol acting to inhibit k channels and sarcoplasmic reticulum calcium atpase and thereby increasing contraction via calcium entry on l-type calcium channels and myosin light chain kinase. two b -secretase (bace ) inhibitors (anti-dementia agents) were isolated from pomegranate rind and identi fi ed as ellagic acid and punicalagin with ic values of . × − and . × − m (kwak et al. ) . ellagic acid and punicalagin were less inhibitory to α-secretase (tace) and other serine proteases such as chymotrypsin, trypsin, and elastase, thus indicating that they were relatively speci fi c inhibitors of bace . b -secretase is an aspartic-acid protease involved in the pathogenesis of alzheimer's disease studies showed that ethanolic extract of p. granatum seeds signi fi cantly exhibited the anxiolytic activity animal models of elevated plus maze test, barbiturate-induced sleeping time, tail suspension test, hot-plate and tail-fl ick tests (kumar et al. ) . the extract ( and mg/kg) signi fi cantly increased the sleeping latency and reduced the sleeping time. tail suspension test showed that the extract ( and mg/kg) was able to induce a signi fi cant decrease in the immobility time, similar to imipramine, a recognized antidepressant drug. tail-fl ick and hot-plate tests exhibited antinociceptive property of pomegranate extract, similar to morphine, a recognized antinociceptive agent. phytochemical screening and measurement of reducing power revealed the central nervous system (cns) activity of ethanol extract of pomegranate seeds may be due to its antioxidative pro fi le. supplementation of pomegranate fl owers led to improvements in learning and memory performances of streptozotocin-induced diabetic rats (cambay et al. ) . supplementation of pomegranate fl owers restored the elevated levels of lipid peroxidation and decreased level of glutathione towards their control values. daily pomegranate fl ower supplementation to diabetic rats reduced the increase in glial-fi brilar acidic protein (gfap) contents induced by diabetes in the hippocampus. the observations suggested that pomegranate fl ower supplementation decreased oxidative stress and amelioratedimpairment in learning and memory performances in diabetic rats and may be clinically useful in treating neuronal de fi cit in diabetic patients. loren et al. ( ) found that maternal dietary supplementation with pomegranate juice was neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury. dietary supplementation with pomegranate juice resulted in markedly decreased brain tissue loss (> %) in all three brain regions assessed, with the highest pomegranate juice dose having greatest signi fi cance pomegranate juice also diminished caspase- activation by % in the hippocampus and % in the cortex. in further studies, the scientists showed that pomegranate polyphenols and resveratrol reduced caspase- activation following neonatal hypoxic-ischemic injury (west et al. ) . in separate study, transgenic mice (app(sw)/tg ) treated with pomegranate juice had signi fi cantly less (approximately %) accumulation of soluble aβ and amyloid deposition in the hippocampus as compared to control mice (hartman et al. ) . mice administered pomegranate juice learned water maze tasks more quickly and swam faster than controls. the results suggest that pomegranate juice may be useful in alzheimer's disease and warrant further studies. choi et al. ( b ) found that the ethanol pomegranate extract mitigated h o induced oxidative stress in pc cells. additionally, the extract inhibited neuronal cell death caused by a b -induced oxidative stress and a b -induced learning and memory de fi ciency in mice. studies showed that pomegranate fruit extract exhibited embryo protective effect against adriamycin-induced oxidative stress in chick embryos (kishore et al. ) . pre-administration of pomegranate fruit extract signi fi cantly ameliorated to normal, embryo gross morphological deformities and signi fi cant changes in the levels of biochemical parameters in amniotic fl uid observed in the adriamycin-treated group. pomegranate juice consumption by healthy male rats provided an increase in epididymal sperm concentration, sperm motility, spermatogenic cell density and diameter of seminiferous tubules and germinal cell layer thickness and antioxidant activity, and it decreased abnormal sperm rate when compared to the control group (türk et al. ) . a signi fi cant decrease in malondialdehyde level and marked increases in glutathione, glutathione peroxidase and catalase activities, and vitamin c level were observed in rats treated with different doses of pomegranate juice. studies showed that ethanolic extract of pomegranate could be useful for the treatment of the deleterious effect of lead acetate administration on sperm production in rats (leiva et al. ) . the extract exhibited antioxidant activity similar to that of ascorbic acid and prevented lead acetate -induced spermatogenic disruption in rats. its antioxidant activity could explain its capacity to reverse the damage produced by lead acetate on spermatogenesis. in a randomized, placebo-controlled, doubleblind, crossover study involving male patients with mild to moderate erectile dysfunction, of the subjects who demonstrated improvement in global assessment questionnaires (gaq) scores after beverage consumption, reported improvement in erectile function after drinking pomegranate juice (forest et al. ) . subjects were more likely to have improved scores when pomegranate juice was consumed. although overall statistical signi fi cance was not achieved, this pilot study suggested the possibility that larger cohorts and longer treatment periods may achieve statistical signi fi cance. studies in rabbits with atherosclerosis-induced erectile dysfunction showed that pomegranate extract signi fi cantly improved intracavernosal blood fl ow, erectile activity, smooth muscle relaxation and fi brosis of the atherosclerotic group in comparison with the atherosclerotic group receiving placebo, but did not normalize them to the agematched control levels . pomegranate extract appeared more effective in diminishing oxidative products, preventing superoxide dismutase and aldose reductase gene upregulation, and protecting mitochondrial, endothelial and caveolae structural integrity of the atherosclerotic group. the study showed that dietary antioxidants could improve arteriogenic erectile dysfunction. pomegranate known to contain estrogens (estradiol, estrone, and estriol) exhibited estrogenic activities in mice (mori-okamoto et al. ) . administration of pomegranate extract (juice and seed extract) for weeks to ovariectomized mice prevented the loss of uterus weight and shortened the immobility time compared with % glucose-dosed mice (control). further, ovariectomy-induced decrease of bone mineral density was normalized by administration of the pomegranate extract. the bone volume and the trabecular number were signi fi cantly increased and the trabecular separation was decreased in the pomegranate-dosed group compared with the control group. some histological bone formation/ resorption parameters were signi fi cantly increased by ovariectomy but were normalized by administration of the pomegranate extract. these changes suggested that the pomegranate extract inhibited ovariectomy-stimulated bone turnover. the authors concluded that pomegranate may be clinically effective on a depressive state and bone loss in menopausal syndrome in women. studies in human volunteers, found that in human liver microsomes, the mean % inhibitory concentrations (ic ) for pomegranate juice (pj) and grapefruit juice (gfj) versus cyp a (triazolam α-hydroxylation) were . and . %, (v/v) respectively without preincubation of inhibitor with microsomes (farkas et al. ) . after preincubation, the ic for pj increased to . % whereas the ic for gfj decreased to . % suggesting mechanism-based inhibition by gfj but not pj. administration of pj also did not affect c(max), total area under the curve (auc), or clearance of oral midazolam. however, gfj increased midazolam c(max) and auc by a factor of . and . , respectively, and reduced oral clearance to % of control values. the results suggested pj did not alter clearance of intravenous or oral midazolam, whereas gfj impaired clearance and elevated plasma levels of oral midazolam. jarvis et al. ( ) reported a potential interaction between pomegranate juice and warfarin as laboratory studies hade shown that pomegranate juice inhibited cytochrome p enzymes involved in warfarin metabolism. in a an open-label, randomized, single-center, two-period crossover study in healthy japanese volunteers, a single subtherapeutic doses of midazolam following weeks consumption of pomegranate juice did not signi fi cantly alter the pharmacokinetic pro fi le of midazolam compared with that of the control (misaka et al. ) . results of a -week randomized, double-blind, placebo-controlled study involving patients suggested that polyphenol-rich pomegranate juice (pj) supplementation added no bene fi t to the current standard therapy in patients with stable chronic obstructive pulmonary disease (cerdá et al. ) . the high teac (trolox equivalent antioxidant capacity) of pj could not be extrapolated in-vivo probably due to the metabolism of its polyphenols by colonic micro fl ora. the understanding of the different bioavailability of dietary polyphenols was thus critical before claiming any antioxidant-related health bene fi t. elbow fl exion strength was signi fi cantly higher during the -to -h period post-exercise with pomegranate juice compared with that of placebo (trombold et al. ) . elbow fl exor muscle soreness was also signi fi cantly reduced with pomegranate juice compared with that of placebo and at and h post-exercise. isometric strength and muscle soreness in the knee extensors were not signi fi cantly different with pomegranate juice compared with those using placebo. the results indicated a mild, acute ergogenic effect of pomegranate juice in the elbow fl exor muscles of resistance trained individuals after eccentric exercise. seven highly active ellagitannin inhibitors against carbonic anhydrase, punicalin ( ), punicalagin ( ), granatin b ( ), gallagyldilactone ( ), casuarinin ( ), pedunculagin ( ) and tellimagrandin i ( ), and four weakly active ellagitannin inhibitors, gallic acid ( ), granatin a ( ), corilagin ( ) and ellagic acid ( ), were isolated from pomegranate pericarps (satomi et al. ) . the type of inhibition by compounds ( ) and ( ) using p -nitrophenyl acetate as a substrate, was noncompetitive. carbonic anhydrase inhibitors are used as antiglaucoma drugs, and many potent carbonic anhydrase inhibitors have also been shown to inhibit the growth of several tumour cell lines in-vitro and in-vivo providing interesting leads for developing novel antitumour therapies (supuran et al. ) using the hot plate method in mice, pomegranate fl ower extracts showed signi fi cant analgesic activity at a dose of mg/kg body weight (chakraborthy ) . maximum analgesic activity was observed at min after drug administration, which was equivalent to the standard drug used morphine sulphate. two milliliters of aqueous extract of pomegranate roots exhibited higher activity on cultures from entamoeba histolytica than from entamoeba invadens strains, producing growth inhibitions of about and % respectively (segura et al. ) . alkaloid concentrations of mg/ml had no amoebicide activity, however tannins at concentrations of m g/ml for e. histolytica , and m g/ml for e. invadens were suf fi cient to produce an growth inhibition about %. tannic acid was also tested on the cultures of e. histolytica producing a high inhibitory activity on growth, this effect was produced at . mg/ ml similar to that observed with the tannin mixture. the methanolic extract of pomegranate was reported to in-vitro inhibit the growth of the malarial parasite, plasmodium berghei (dell'agli et al. ) . in another study, gallagic acid and punicalagin from pomegranate by-product exhibited antiplasmodial activity against plasmodium falciparum d and w clones with ic values of . , . , . and . m m, respectively (reddy et al. ) . pomegranate extract exhibited strong antimalarial activity against plasmodium falciparum (valdés et al. ) . p. grantum plant extract also exhibited in-vitro activity against the vaginal parasite, trichomonas vaginalis (el-sherbini et al. ) . hydroalcoholic pomegranate extract inhibited the growth of intracellular amastigotes of leishmania amazonensis with ic value of . m g/ml (garcía et al. ) . additionally, a low toxicity on macrophage from balb/c mice was observed. wibaut and hollstein ( ) found that the anthelminthic activity of pomegranate bark extract was mainly due to isopelletierine, methylisopelletierine while y pelleterine was less active the molluscicidal activity of p. granatum bark and canna indica root against the snail, lymnaea acuminata was found to be both time and dose dependent (tripathi and singh ) . the toxicity of p. granatum bark was more pronounced than that of c. indica . the h lc of the c. indica was . mg/l whereas that of the bark of p. granatum was . mg/l. the ethanol extract of p. granatum ( h lc : . mg/l) was more effective than the ethanol extract of c. indica ( h lc : . mg/l) in killing the test animals. p. granatum and c. indica may be used as potent molluscicides since the concentrations used to kill the snails were not toxic to the fi sh colisa fasciatus , sharing the same habitat with the snail. in a subsequent study, tripathi et al. ( ) reported that sub-lethal h exposure to active fraction of pomegranate bark separately or in combination with canna roots signi fi cantly inhibited the activity of acetylcholinesterase, acid/alkaline phosphatase, na(+)k(+)atpase and lactic dehydrogenase in the nervous tissue of lymnaea acuminata. lei et al. ( ) found that ellagic acid, the principal bioactive component of pomegranate leaf extract, had poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach. studies in rats showed that only - % of the ingested punicalagin was detected as such or as metabolites in urine and faeces (cerdá et al. b ) . only traces of punicalagin metabolites being detected in liver or kidney. the transformation of ellagic acid derivatives to h-dibenzo[b,d] pyran- -one derivatives in the rat was con fi rmed. studies of cerdá et al. ( ) found that the potential systemic biological effects of pomegranate juice ingestion should be attributed to the colonic micro fl ora metabolites rather than to the polyphenols present in the juice. neither the potent antioxidant punicalagin nor ellagic acid present in pomegranate juice were detected in both plasma and urine on ingestion of pomegranate juice. three microbial ellagitannin-derived metabolites were detected: , -dihydroxy- h-dibenzo[b,d]pyran- -one glucuronide, an unidenti fi ed aglycone (tentatively, trihydroxy- h-dibenzo[b,d]pyran- -one) and hydroxy- -hdibenzo [b,d] pyran- -one glucuronide in the plasma and urine. the metabolites did not show signi fi cant antioxidant activity compared to punicalagin from pomegranate juice. in separate studies, ellagic acid was detected in human plasma at a maximum concentration ( . ng/ml) after h post-ingestion of pomegranate juice but was rapidly eliminated by h (seeram et al. ) . six hours post-ingestion of pomegranate juice, ellagic acid (ea) was detected in plasma of all healthy human volunteers with a maximum concentration of . m mol/l, area under concentration time curve of . ( m mol × h) × l(- ), time of maximum concentration of . h, and elimination half-life of . h . ellagic acid metabolites, including dimethylellagic acid glucuronide (dmeag) and hydroxy- h-benzopyran- -one derivatives (urolithins), were also detected in plasma and urine in conjugated and free forms. dmeag was found in the urine obtained from of subjects on day , but was not detected on d − (day before) or + (day after), demonstrating its potential as a biomarker of intake. urolithin a-glucuronide was found in urine samples from subjects on d and in the urine from subjects on d + . urolithin b-glucuronide was found in the urine of three subjects on d and in the urine of fi ve subjects on d+ . the scientists asserted that urolithins, formed by intestinal bacteria, may contribute to the biological effects of pomegranate juice as they may persist in plasma and tissues and account for some of the health bene fi ts noted after chronic juice consumption. studies by seeram et al. ( ) found that pomegranate juice, pomegranate polyphenol liquid extract and pomegranate polyphenol powder extract provide similar levels of plasma and urinary ellagitannin metabolites such as urolithin a, in human subjects. there was a delay in time of maximum concentration of pomegranate powder extract compared to pomegranate juice and pomegranate polyphenol liquid. mertens-talcott et al. ( ) found ellagic acid from pomegranate extract to be bioavailable at h after consumption by healthy volunteers. its metabolites urolithin a, urolithin b, hydroxyl-urolithin a, urolithin a-glucuronide, and dimethyl ellagic acidglucuronide were found in the plasma. the antioxidant capacity measured with the oxygen radical absorbance capacity (orac) assay was increased with a maximum effect of % after . h, whereas the generation of reactive oxygen species (ros) was not affected. vidal et al. ( ) found that in chick embryo model doses of hydroalcoholic pomegranate fruit extract of less than . mg per embryo were not toxic. the ld of the extract, determined in of- mice of both sexes after intraperitoneal administration, was mg/kg. con fi dence limits were - mg/kg. at the doses of . and . mg/kg of extract, the repeated intranasal administration to wistar rats produced no toxic effects in terms of food intake, weight gain, behavioural or biochemical parameters, or results of histopathological studies. cerdá et al. ( a ) found that repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for days was not toxic. punicalagin and related metabolites were identi fi ed in plasma, liver, and kidney. five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic acid, , -dihydroxy- h-dibenzo [b,d] pyran- -one glucuronide, and , , -trihydroxy- h-dibenzo [b,d] pyran- -one. feedstuff intake, food utility index, and growth rate were lower in punicalagin treated rats during the fi rst days without signi fi cant adverse effects, which could be due to the lower nutritional value of the punicalagin-enriched diet together with a decrease in its palatability (lower food intake). no signi fi cant differences were found in punicalagin treated rats in any blood parameter analyzed (including the antioxidant enzymes glutathione peroxidase and superoxide dismutase) with the exception of urea and triglycerides, which remained at low values throughout the study. clinical studies by heber et al. ( ) demonstrated the safety of a pomegranate ellagitannin-enriched polyphenol dietary supplement in overweight individuals with increased waist size and provided evidence of antioxidant activity in humans re fl ected by a signi fi cant reduction in thiobarbituric acid reactive substances (tbars) linked with cardiovascular disease risk. patel et al. ( ) found that the no observed-adverse-effect level (noael) for a standardized pomegranate fruit extract was determined as mg/kg body weight/day, the highest dose tested in rats. compared to the control group, administration of the extract did not result in any toxicologically signi fi cant treatmentrelated changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. toxicological evaluation of pomegranate seed oil (pso) showed that the no observable adverse effect level (noael) was , ppm pso (= . g pso/kg body weight/day) (meerts et al. ) . no mutagenicity of pso was observed in the absence and presence of metabolic activation up to precipitating concentrations of , m g/ plate (ames test) or m g/ml (chromosome aberration test). the acute oral toxicity study revealed no signi fi cant fi ndings at , mg pso/ kg body weight. results from reversion and gene-conversion test in microorganisms, sister chromatid exchanges, micronuclei and sperm-shape abnormality assays in mice, clearly showed that the hydroalcoholic extract of pomegranate whole fruit was genotoxic when tested both in-vitro and in-vivo (sánchez-lamar et al. ) . the bark of the roots, the fl owers, the rind of pomegranate fruit and the seeds, are of fi cial in many pharmacopoeias. various parts of the pomegranate plant have been extensively used for thousands of years in traditional medicine in the middle east, ancient greece and asia (burkill ; grieve ; stuart ) ; and in india such as in the ayurveda and unani systems of medicine (nadkarni and nadkarni. ; sharma et al. ; kapoor ; pradeep et al. ) . the fruit rind and stem bark have been used as a traditional remedy for diarrhoea, dysentery and intestinal parasites. pomegranate pericarp has been commonly employed as a crude drug in indian traditional medicine for the treatment of diarrhoea as well as for use as an astringent, antihelminthic, asphrodisacs, laxative, diuretic, stomachic, cardiotonic and refrigerant. the seeds and juice are considered as bitter and astringent and employed as a tonic for hear and throat ailments. the astringent qualities of the fl ower sap, fruit rind and tree bark are considered useful remedies for nose bleeds and gum bleeds, toning skin, (after mixing with mustard oil) fi rming-up sagging breasts and treating haemorrhoids. a syrup prepared from the fruit is useful in all bilious complaints. the juice of the fresh fruit is much esteemed in dyspepsia and as a cooling, thirst-quenching beverage in fever and sickness. the fruit juice is also found bene fi cial in leprosy. pomegranate fruit juice has been used as eyedrops to treat cataracts. dried, pulverized fl ower buds are employed as a remedy for bronchitis. pomegranate has been reported as a remedy for diabetes in the unani system of medicine practiced in the middle east and india. the ancient greeks and egyptians used the fruit rind, fl owers and root bark as astringents and the last as vermicide for treating tapeworms. in malaysia, the root bark is used as vermifuge and powdered root bark is administered to children for stomach pains. the root is also used for diarrhoea and tits sap used for treating sore-eyes. leaves are used in jamu preparations with a raft of other herbal ingredients for many medicinal complaints. pounded leaves are used in a complex bolus for stomach ache and the fruit juice is recommended for coughs. in singapore, the root bark has been used in as a component in a compound infusion or decoction taken by women for days after childbirth. other traditional uses of the fruit rind and root include as a treatment for snakebite (jain and puri ) , diabetes (singh ) , burns (siang ) , leprosy and assorted gynecological problems (singh et al. ) . in sri lanka, the fresh fruit has been used as a refrigerant to lower fever (arseculeratne et al. ) . in the philippines, a decoction of the tender leaves is used as a gargle for affections of the buccal cavity. the rind of the fruit is used internally in decoction as anthelmintic and taenifuge. in mexico, a decoction of the fl owers is gargled to relieve oral and throat in fl ammation. in korea, traditional uses of the fruit and rind include as an anthelminthic and for phlegm, cholethiasis, tineapedis and laryngitis. punica granatum is a drought tolerant tree suitable for arid and semi-arid zone afforestation. pomegranate has deep rooting system and is used for erosion control, planted along rivers to stabilize banks. an ideal suitable ornamental plant for gardens and amenity parks. pomegranate grows along well as intercrop with grapes in mediterranean countries. the tree is sometimes used for fencing and planted as boundary plants. pomegranate leaf litter decomposes slowly and is suitable for mulching. the leaves are foraged by domesticated stock. ink can be made by steeping the leaves in vinegar. both the fruit rind and the fl owers yield dyes for textiles. the light-coloured wood is hard and durable, mostly used in making farm implements, walking-sticks and in woodcrafts as it is only available in small dimension. tree branches are used as fi rewood. the bark is used in tanning and dyeing providing the yellow hue for moroccan leather. root bark yields a black ink rich in tannins. in japan, an insecticide is derived from the bark. studies revealed that pomegranate peel can prepared as an adsorbent in treating industrial ef fl uents containing phenols and safely disposed of by stabilizing into cement (bhatnagar and minocha ) . studies showed that that pomegranate peel waste can be used as adsorbent bene fi cially for nickel removal from aqueous solution (bhatnagar and minocha ) . pomegranate husk when converted into activated carbon exhibited its ability to remove hexavalent chromium from wastewater (nemr ) . studies showed that the nutritive value and the antioxidant capacity of pomegranate peel could be enhanced by ensiling into a favorable healthpromoting constituent of feedlot beef cattle diet (shabtay et al. ) . dietary supplementation with fresh peels promoted signi fi cant increases in feed intake and a-tocopherol concentration in the plasma, with positive tendency toward increased weight gain of bull calves. the pomegranate fruit is steeped in religious and cultural signi fi cance in judaism, christianity, islam, hinduism religions, persian, armenian, azerbaijani and chinese cultures (wikipedia ) . pomegrante germinates readily from seeds and are established from seedlings, rooted hardwood cuttings, from air layering and suckers. screening of nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition pomegranate fruit extract modulates uv-bmediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa b in normal human epidermal keratinocytes paragraph sign anthocyanin-and hydrolyzable tannin-rich pomegranate fruit extract modulates mapk and nf-kappab pathways and inhibits skin tumorigenesis in cd- mice protective effect of pomegranate-derived products on uvb-mediated damage in human reconstituted skin oral feeding of pomegranate fruit extract inhibits early biomarkers of uvb radiation-induced carcinogenesis in skh- hairless mouse epidermis short communication: studies on punica granatum -i isolation and identi fi cation of some constituents from the seeds of punica granatum punica granatum l. extract inhibits il- beta-induced expression of matrix metalloproteinases by inhibiting the activation of map kinases and nf-kappab in human chondrocytes in vitro the inhibition of gastric mucosal injury by punica granatum l. (pomegranate) methanolic extract protective effects of punica granatum in experimentally-induced gastric ulcers pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells anthocyanins characterization of iranian pomegranate ( punica granatum l.) varieties and their variation after cold storage and pasteurization antimicrobial activity of pomegranate ( punica granatum l.) fruit peels studies on medicinal plants of sri lanka. part pomegranate as a cosmeceutical source: pomegranate fractions promote proliferation and procollagen synthesis and inhibit matrix metalloproteinase- production in human skin cells pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduces systolic blood pressure pomegranate juice consumption reduces oxidative stress, atherogenic modi fi cations to ldl, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein e-de fi cient mice pomegranate juice fl avonoids inhibit low-density lipoprotein oxidation and cardiovascular diseases: studies in atherosclerotic mice and in humans pomegranate juice consumption for years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and ldl oxidation pomegranate phenolics from the peels, arils, and fl owers are antiatherogenic: studies in vivo in atherosclerotic apolipoprotein e-de fi cient (e ) mice and in vitro in cultured macrophages and lipoproteins an aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung in fl ammation in mice flora of java (spermatophytes only), . noordhoff melatonin, serotonin, and tryptamine in some egyptian food and medicinal plants antidiabetic effect of punica granatum fl owers: effect on hyperlipidemia, pancreatic cells lipid peroxidation and antioxidant enzymes in experimental diabetes new sterol esters from the fl owers of punica granatum linn consumption of pomegranate decreases serum oxidative stress and reduces disease activity in patients with active rheumatoid arthritis: a pilot study -methyl-pyran- -one- -o -b -d-glucopyranoside isolated from leaves of punica granatum inhibits the tnf a -induced cell adhesion molecules expression by blocking nuclear transcription factor-k b (nf-k b) the antiplaque ef fi cacy of pomegranate mouthrinse adsorptive removal of , -dichlorophenol from water utilizing punica granatum peel waste and stabilization with cement biosorption optimization of nickel removal from water using punica granatum peel waste urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay the effect of pomegranate ( punica granatum l.) byproducts and ellagitannins on the growth of human gut bacteria pomegranate-mediated chemoprevention of experimental hepatocarcinogenesis involves nrf -regulated antioxidant mechanisms climate effects on anthocyanin accumulation and composition in the pomegranate ( punica granatum l.) fruit arils synergic interaction between pomegranate extract and antibiotics against staphylococcus aureus pomegranate extract inhibits staphylococcus aureus growth and subsequent enterotoxin production a dictionary of the economic products of the malay peninsula, revised reprint, vols. ministry of agriculture and co-operatives characterization of a potential nutraceutical ingredient: pomegranate ( punica granatum l.) seed oil unsaponi fi able fraction volatile composition and sensory quality of spanish pomegranates ( punica granatum l.) pomegranate ( punica granatum l.) fl ower improves learning and memory performances impaired by diabetes mellitus in rats hepatoprotective role and antioxidant capacity of pomegranate ( punica granatum ) fl owers infusion against trichloroacetic acidexposed in rats repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for days is not toxic evaluation of the bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from pomegranate juice the potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxy- h-dibenzopyran- -one derivatives by the colonic micro fl ora of healthy humans pomegranate juice supplementation in chronic obstructive pulmonary disease: a -week randomized, doubleblind, placebo-controlled trial analgesic activity of various extracts of punica granatum (linn) fl owers antibacterial activity of thai medicinal plant extracts on the skin infectious microorganisms flavonoid diglycoside from punica granatum studies on antioxidant activity of pomegranate ( punica granatum ) peel extract using in vivo models study on wound healing activity of punica granatum peel the partition chromatography of alkaloids: part iii.-the alkaloids of punica granatum in vitro and in vivo antibacterial activity of punica granatum peel ethanol extract against salmonella punica granatum protects against oxidative stress in pc cells and oxidative stress-induced alzheimer's symptoms in mice the wealth of india: a dictionary of indian raw materials and industrial products pomegranate extract inhibits the proliferation and viability of mmtv-wnt- mouse mammary cancer stem cells in vitro studies on antidiarrhoeal activity of punica granatum seed extract in rats studies on the hypoglycaemic activity of punica granatum seed in streptozotocin induced diabetic rats effects of consumption of pomegranate juice on carotid intima-media thickness in men and women at moderate risk for coronary heart disease bene fi cial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells the in fl uence of pomegranate fruit extract in comparison to regular pomegranate juice and seed oil on nitric oxide and arterial function in obese zucker rats effects of a pomegranate fruit extract rich in punicalagin on oxidation-sensitive genes and enos activity at sites of perturbed shear stress and atherogenesis quantitative analysis of fl avan- -ols in spanish foodstuffs and beverages antiplasmodial activity of punica granatum l. fruit rind the antioxidant potency of punica granatum l. fruit peel reduces cell proliferation and induces apoptosis on breast cancer pomegranate extract mouth rinsing effects on saliva measures relevant to gingivitis risk antimicrobial activity of six pomegranate ( punica granatum l.) varieties and their relation to some of their pomological and phytonutrient characteristics elemental and nutritional analysis of punica granatum from turkey analysis of organic acids in fruit juices by liquid chromatography-mass spectrometry: an enhanced tool for authenticity testing pomegranate ( punica granatum ) juices: chemical composition, micronutrient cations, and antioxidant capacity physico-chemical properties and dpph-abts scavenging activity of some local pomegranate ( punica granatum ) ecotypes antioxidant capacities of phenolic compounds and tocopherols from tunisian pomegranate ( punica granatum ) fruits fatty acids from tunisian and chinese pomegranate ( punica granatum l.) seeds chemical composition of juice and seeds of pomegranate fruit studies on pomegranate seed oil ef fi cacy of two plant extracts against vaginal trichomoniasis two ellagitannins from punica granatum heartwood two new ellagic acid rhamnosides from punica granatum heartwood ellagiand gallotannins from punica granatum heartwood concentrated pomegranate juice improves lipid pro fi les in diabetic patients with hyperlipidemia cholesterol-lowering effect of concentrated pomegranate juice consumption in type ii diabetic patients with hyperlipidemia pomegranate juice effects on cytochrome p s expression: in vivo studies effect of pomegranate ( punica granatum ) juice intake on hepatic oxidative stress pomegranate juice does not impair clearance of oral or intravenous midazolam, a probe for cytochrome p - a activity: comparison with grapefruit juice monoacylglycerol from punica granatum seed oil effect of probiotication on antioxidant and antibacterial activities of pomegranate juices from sour and sweet cultivars determination of lignans in edible and nonedible parts of pomegranate ( punica granatum l.) and products derived there from, particularly focusing on the quantitation of isolariciresinol using hplc-dad-esi/ms(n) ef fi cacy and safety of pomegranate juice on improvement of erectile dysfunction in male patients with mild to moderate erectile dysfunction: a randomized, placebo-controlled, double-blind, crossover study foundation for revitalisation of local health traditions pomegranate and cardiovascular diseases: pomegranate juice polyphenolic antioxidants protect against oxidative stress and atherosclerosis development pomegranate juice inhibits oxidized ldl uptake and cholesterol biosynthesis in macrophages pomegranate juice polyphenols increase recombinant paraoxonase- binding to high-density lipoprotein: studies in vitro and in diabetic patients screening of medicinal plants against leishmania amazonensis synergistic growth inhibition of mouse skin tumors by pomegranate fruit extract and diallyl sul fi de: evidence for inhibition of activated mapks/nf-k b and reduced cell proliferation effects of aqueous extracts from quercus ilex l. root bark, punica granatum l. fruit peel and artemisia herba-alba asso leaves on ethanolinduced gastric damage in rats arte's f, toma's-barbera'n f ( ) changes in pomegranate juice pigmentation during ripening antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing effect of pomegranate juice on insulin secretion and sensitivity in patients with obesity dissimilar in vitro and in vivo effects of ellagic acid and its microbiota-derived metabolites, urolithins, on the cytochrome p a anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of s. aureus , mrsa and pvl positive ca-mssa total phenolic distribution of juice, peel, and seed extracts of four pomegranate cultivars immunomodulatory activity of punica granatum in rabbits -a preliminary study punicic acid is an omega- fatty acid capable of inhibiting breast cancer proliferation antioxidant activities of peel, pulp and seed fractions of common fruits as determined by frap assay evaluation of antioxidant activity and preventing dna damage effect of pomegranate extracts by chemiluminescence method pomegranate juice is potentially better than apple juice in improving antioxidant function in elderly subjects pomegranin, an antifungal peptide from pomegranate peels pomegranate ( punica granatum ) puri fi ed polyphenol extract inhibits in fl uenza virus and has a synergistic effect with oseltamivir in vitro antibacterial activity of some iranian medicinal plant extracts against helicobacter pylori pomegranate juice decreases amyloid load and improves behavior in a mouse model of alzheimer's disease cardioprotective effect of whole fruit extract of pomegranate on doxorubicin-induced toxicity in rat hydroalcoholic extract based-ointment from punica granatum l. peels with enhanced in vivo healing potential on dermal wounds safety and antioxidant activity of a pomegranate ellagitanninenriched polyphenol dietary supplement in overweight individuals with increased waist size identi fi cation of estrone in pomegranate seeds evolution of juice anthocyanins during ripening of new selected pomegranate ( punica granatum ) clones pomegranate polyphenols down-regulate expression of androgensynthesizing genes in human prostate cancer cells overexpressing the androgen receptor activation of ppar gamma and alpha by punicic acid ameliorates glucose tolerance and suppresses obesity-related in fl ammation chemopreventive effects of pomegranate seed oil on skin tumor development in cd mice anti-diabetic action of punica granatum fl ower extract: activation of ppargamma and identi fi cation of an active component pomegranate fl ower improves cardiac lipid metabolism in a diabetic rat model: role of lowering circulating lipids pomegranate fl ower extract diminishes cardiac fi brosis in zucker diabetic fatty rats: modulation of cardiac endothelin- and nuclear factor-kappab pathways tannins from the leaves of punica granatum pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide effects of pomegranate juice on hyperoxaluria-induced oxidative stress in the rat kidneys possible interaction between pomegranate juice and warfarin anticancer activities of pomegranate extracts and genistein in human breast cancer cells evaluation of antioxidant, antitumor and immunomodulatory properties of polysaccharide isolated from fruit rind of punica granatum suppressive effect of punica granatum on the production of tumor necrosis factor (tnf) in bv microglial cells therapeutic applications of pomegranate ( punica granatum l.): a review antioxidant effect of pomegranate extract in reducing acute in fl ammation due to myringotomy pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis crc handbook of ayurvedic medicinal plants effects of oral administration of ellagic acidrich pomegranate extract on ultraviolet-induced pigmentation in the human skin effects of pomegranate chemical constituents/intestinal microbial metabolites on cyp b in rv prostate cancer cells punica granatum : heuristic treatment for diabetes mellitus punica granatum (pomegranate) fl ower extract possesses potent antioxidant activity and abrogates fe-nta induced hepatotoxicity in mice differentiation-promoting activity of pomegranate ( punica granatum ) fruit extracts in hl- human promyelocytic leukemia cells oral consumption of pomegranate fruit extract inhibits growth and progression of primary lung tumors in mice pomegranate fruit extract inhibits prosurvival pathways in human a lung carcinoma cells and tumor growth in athymic nude mice pomegranate fruit extract impairs invasion and motility in human breast cancer pomegranate fruit extract inhibits uvb-induced in fl ammation and proliferation by modulating nf-k b and mapk signaling pathways in mouse skin stimulation of osteoblastic differentiation and inhibition of interleukin- and nitric oxide in mc t -e cells by pomegranate ethanol extract chemopreventive and adjuvant therapeutic potential of pomegranate ( punica granatum ) for human breast cancer inhibitory effect of pomegranate on intestinal sodium dependent glucose uptake embryo protective effect of pomegranate ( punica granatum l.) fruit extract in adriamycin-induced oxidative stress screening of traditional chinese medicinal plants for quorum-sensing inhibitors activity pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats genetic diversity-independent neutralization of pandemic viruses (e.g. hiv), potentially pandemic (e.g. h n strain of in fl uenza) and carcinogenic (e.g. hbv and hcv) viruses and possible agents of bioterrorism (variola) by enveloped virus neutralizing compounds (evncs) pomegranate extract induces apoptosis in human prostate cancer cells by modulation of the igf-igfbp axis effect of pomegranate peel extracts on experimental prostatitis rats in vitro studies on the binding, antioxidant, and cytotoxic actions of punicalagin central nervous system activity of acute administration of ethanol extract of punica granatum l. seeds in mice beta-secretase (bace ) inhibitors from pomegranate ( punica granatum ) husk effects of pomegranate tannins on experimental gastric damages transport behavior of ellagic acid of pomegranate leaf tannins and its correlation with total cholesterol alteration in hepg cells punica granatum (pomegranate) and its potential for prevention and treatment of in fl ammation and cancer pomegranate ( punica granatum ) pure chemicals show possible synergistic inhibition of human pc- prostate cancer cell invasion across matrigel possible synergistic prostate cancer suppression by anatomically discrete pomegranate fractions immune-suppressive activity of punicalagin via inhibition of nfat activation pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet induced obese mice effect of punica granatum (pomegranate) on sperm production in male rats treated with lead acetate antiviral activities of medicinal herbs traditionally used in southern mainland china punica granatum fl ower extract, a potent alpha-glucosidase inhibitor, improves postprandial hyperglycemia in zucker diabetic fatty rats evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract pomegranate fl ower: a unique traditional antidiabetic medicine with dual ppar-alpha/-gamma activator properties fabrication of nanoparticles using partially puri fi ed pomegranate ellagitannins and gelatin and their apoptotic effects maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury anti-listeria monocytogenes activity of heat-treated lyophilized pomegranate juice in media and in ground top round beef pomegranate ( punica granatum ) supplements: authenticity, antioxidant and polyphenol composition prostate cancer prevention through pomegranate fruit pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer effects of pomegranate juice and extract polyphenols on platelet function antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin c pomegranate seed oil consumption during a period of high-fat feeding reduces weight gain and reduces type diabetes risk in cd- mice toxicological evaluation of pomegranate seed oil breast cancer chemopreventive properties of pomegranate ( punica granatum ) fruit extracts in a mouse mammary organ culture volatile composition of pomegranates from spanish cultivars using headspace solid phase microextraction punica granatum (pomegranate) extract is active against dental plaque absorption, metabolism, and antioxidant effects of pomegranate ( punica granatum l.) polyphenols after ingestion of a standardized extract in healthy human volunteers effect of weeks' consumption of pomegranate juice on the pharmacokinetics of a single dose of midazolam: an open-label, randomized, single-center, -period crossover study in healthy japanese volunteers cardioprotective potential of punica granatum extract in isoproterenol-induced myocardial infarction in wistar rats effect of pomegranate juice on angiotensin ii-induced hypertension in diabetic wistar rats pomegranate extract improves a depressive state and bone properties in menopausal syndrome model ovariectomized mice fruits of warm climates. julia f identi fi cation and quanti fi cation of phenolic compounds and their effects on antioxidant activity in pomegranate juices of eight iranian cultivars indian materia medica with ayurvedic pomegranate extract induces cell cycle arrest and alters cellular phenotype of human pancreatic cancer cells medicinal plants in the republic of korea nmr spectral analysis of polyphenols from punica granatum antibacterial activity directed isolation of compounds from punica granatum antioxidant and antimutagenic activities of pomegranate peel extracts potential of pomegranate husk carbon for cr(vi) removal from wastewater: kinetic and isotherm studies the existence of pelletierine derivatives in punica granatum alkaloids in the bark of punica granatum l. (pomegranate) from yugoslavia punica granatum (pomegranate) juice provides an hiv- entry inhibitor and candidate topical microbicide antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin evaluation of antioxidant activity, colour and some nutritional characteristics of pomegranate ( punica granatum l.) juice and its sour concentrate processed by conventional evaporation agroforestree database: a tree reference and selection guide version protective effects of standardized pomegranate ( punica granatum l.) polyphenolic extract in ultraviolet-irradiated human skin fi broblasts antifungal ef fi cacy of punica granatum , acacia nilotica , cuminum cyminum and foeniculum vulgare on candida albicans : an in vitro study antioxidant capacity and lipid characterization of six georgia-grown pomegranate cultivars phase ii study of pomegranate juice for men with rising prostate-speci fi c antigen following surgery or radiation for prostate cancer antimicrobial ellagitannin from pomegranate ( punica granatum ) fruits extract of punica granatum inhibits skin photoaging induced by uvb irradiation medicinal values of fruit peels from citrus sinensis , punica granatum , and musa paradisiaca with respect to alterations in tissue lipid peroxidation and serum concentration of glucose, insulin, and thyroid hormones safety assessment of pomegranate fruit extract: acute and subchronic toxicity studies the wound healing activity of fl ower extracts of punica granatum and achillea kellalensis in wistar rats antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel searchable world wide web multilingual multiscript plant name database antibacterial activity of punica granatum l. against gastro intestinal tract infection causing organisms the effects of pomegranate seed extract and {beta}-sitosterol on rat uterine contractions antioxidant potentials of punica granatum fruit rind extracts pomegranate extract inhibits the interleukin- b -induced activation of mkk- , p a -mapk and transcription factor runx- in human osteoarthritis chondrocytes antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from punica granatum l studies on the chemical constituents from xinjiang punica granatum pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappab-dependent mechanism antioxidant activity of punica granatum fruits the occurrence of -( -propenyl)-d -piperideine in the leaves of pomegranate consumption of wonderful variety pomegranate juice and extract by diabetic patients increases paraoxonase association with high-density lipoprotein and stimulates its catalytic activities pomegranate juice protects macrophages from triglyceride accumulation: inhibitory effect on dgat activity and on triglyceride biosynthesis anti-oxidative effects of pomegranate juice (pj) consumption by diabetic patients on serum and on macrophages pomegranate byproduct administration to apolipoprotein e-de fi cient mice attenuates atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized lowdensity lipoprotein free radical scavenging, antiglycation and tyrosinase inhibition properties of a polysaccharide fraction isolated from the rind from punica granatum pomegranate juice sugar fraction reduces macrophage oxidative state, whereas white grape juice sugar fraction increases it antiplaque and antigingivitis effects of a gel containing punica granatum linn extract: a double-blind clinical study in humans assessment of the genotoxic risk of punica granatum l. (punicaceae) whole fruit extracts ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo pomegranate juice inhibits sulfoconjugation in caco- human colon carcinoma cells carbonic anhydrase inhibitors from the pericarps of punica granatum l investigation of a betainic alkaloid from punica granatum antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice fl avonoids bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate ( punica granatum l.) juice in vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice rapid large scale puri fi cation of ellagitannins from pomegranate husk, a by-product of the commercial juice industry pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to hours pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to the mouse prostate gland pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects growth inhibition of entamoeba histolytica and e . invadens produced by pomegranate root ( punica granatum l.) lc-dad-esi/ms(n) determination of direct condensation fl avanol-anthocyanin adducts in pressure extracted pomegranate ( punica granatum l.) juice pomegranate wine has greater protection capacity than red wine on low-density lipoprotein oxidation nutritive and antioxidative potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed central council for research in ayurveda & siddha ( ) database on medicinal plants used in ayurveda antibacterial activity of medicinal plants against pathogens causing complicated urinary tract infections effects of fruit ellagitannin extracts, ellagic acid, and their colonic metabolite, urolithin a, on wnt signaling macrophage paraoxonase (pon ) expression is up-regulated by pomegranate juice phenolic anti-oxidants via ppar gamma and ap- pathway activation bioavailable constituents/metabolites of pomegranate ( punica granatum l.) preferentially inhibit cox activity ex vivo and il- beta-induced pge production in human chondrocytes in vitro consumption of hydrolyzable tannins-rich pomegranate extract suppresses in fl ammation and joint damage in rheumatoid arthritis use of combined traditional chinese and western medicine in the management of burns traditional medicine in fiji: some herbal folk cures used by fiji indians medicinal plants from ujjain district madhya pradesh. part ii exploring the ameliorative potential of punica granatum in dextran sulfate sodium induced ulcerative colitis in mice pharmacological investigations of punica granatum in glycerol-induced acute renal failure in rats philippine alternative medicine. manual of some philippine medicinal plants in vitro effects of pomegranate juice and pomegranate polyphenols on foodborne viral surrogates flavonoids from punica granatum : potential antiperoxidative agents punica granaum l effects of pomegranate juice consumption on myocardial perfusion in patients with coronary heart disease in fl uenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins designing of novel carbonic anhydrase inhibitors and activators photochemopreventive effect of pomegranate fruit extract on uva-mediated activation of cellular pathways in normal human epidermal keratinocytes punicafolin, an ellagitannin from the leaves of punica granatum xl: revision of the structures of punicalin and punicalagin, and isolation and characterization of -o -galloylpunicalin from the bark of punica granatum l tannins and related compounds. xli: isolation and characterization of novel ellagitannins, punicacorteins a, b, c, and d, and punigluconin from the bark of punica granatum l tannins and related compounds. c. reaction of dehydrohexahydroxydiphenic acid esters with bases, and its application to the structure determination of pomegranate tannins, granatins a and b humarain: a new dimeric gallic acid glycoside from punica granatum l. bark nutritive value of pomegranate fruit and juice preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo pomegranate peel extract prevents liver fi brosis in biliary-obstructed rats punica granatum peel extract protects against ionizing radiationinduced enteritis and leukocyte apoptosis in rats pomegranate ( punica granatum ) seed linolenic acid isomers: concentrationdependent modulation of estrogen receptor activity molluscicidal activity of punica granatum bark and canna indica root enzyme inhibition by the molluscicidal agent punica granatum linn. bark and canna indica linn. root the effect of pomegranate juice supplementation on strength and soreness after eccentric exercise protective effect of a potent antioxidant, pomegranate juice, in the kidney of rats with nephrolithiasis induced by ethylene glycol effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats antioxidant activity, polyphenol content, and related compounds in different fruit juices and homogenates prepared from different pomegranate accessions usda) ( ) usda national nutrient database for standard reference, release . nutrient data laboratory home page in vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants rapid dereplication of estrogenic compounds in pomegranate ( punica granatum ) using on-line biochemical detection coupled to mass spectrometry use of punica granatum as an antifungal agent against candidosis associated with denture stomatitis minimum inhibitory concentration of adherence of punica granatum linn (pomegranate) gel against s. mutans , s. mitis and c. albicans studies on the toxicity of punica granatum l. (punicaceae) whole fruit extracts activity of medicinal plant extracts against hospital isolates of methicillin-resistant staphylococcus aureus medicinal plant extracts as anti-escherichia coli o :h agents and their effects on bacterial cell aggregation pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice punica granatum attenuates angiotensin-ii induced hypertension in wistar rats bioactive compounds from the seeds of punica granatum (pomegranate) constituents of the fl owers of punica granatum pomegranate: constituents, bioactivities and pharmacokinetics cellular and molecular mechanisms of pomegranate juice-induced anti-metastatic effect on prostate cancer cells pomegranate extract, a prooxidant with antiproliferative and proapoptotic activities preferentially towards carcinoma cells pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury investigation of the alkaloids of punica granatum investigation of the alkaloids of punica granatum l. by partition chromatography antimutagenicity of some fl owers grown in thailand pomegranates ( punica granatum ), kiwifruit ( actinidia deliciosa ) and blood pressure: a pilot study medicinal fl owers. xxiii. new taraxastane-type triterpene, punicanolic acid, with tumor necrosis factor-alpha inhibitory activity from the fl owers of punica granatum effect of pomegranate peel extracts on oxidative stress in restrained mice pomegranate fl ower ameliorates fatty liver in an animal model of type diabetes and obesity dietary effect of pomegranate seed oil on immune function and lipid metabolism in mice chitinase iii in pomegranate seeds ( punica granatum linn.): a high-capacity calcium-binding protein in amyloplasts inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultravioletinduced pigmentation a triglyceride from punica granatum broad spectrum antimutagenic activity of antioxidant active fraction of punica granatum l. peel extracts screening of certain medicinal plants from india for their anti-quorum sensing activity inhibition of uvb-mediated oxidative stress and markers of photoaging in immortalized hacat keratinocytes by pomegranate polyphenol extract pomx studies on physicochemical properties and bioactive compounds of six pomegranate cultivars antiviral activity of tannin from the pericarp of punica granatum l. against genital herpes virus in vitro antiliperoxidant activity of pomegranate peel extracts on lard dietary antioxidants improve arteriogenic erectile dysfunction key: cord- -h o yqv authors: nan title: oral communications and posters date: - - journal: inflamm res doi: . /bf sha: doc_id: cord_uid: h o yqv nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : ( ) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? ( ),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; ( ) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd + tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp( - ) peptide in complex with hla-dr b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr b gene and a human t-cell receptor (tcr) that recognises the mbp - peptide in the context of hla-dr b either spontaneously or after immunization with mbp - develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far genes in this concert have been identified; the mhc class ii ab gene in the mouse ( ) and the ncf gene in the rat ( ) and in the mouse ( ) . the ncf protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf deficiency linking the ncf pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats ( ). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to % of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry- (azd ) is an inhibitor of mek / currently in development for cancer. phase determined the msd ( mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (- %) and proliferative index (- %). the trough plasma concentration ( ng/ml) corresponded to~ % inhibition of perk. about % of pts had stable disease after months. these results demonstrate that arry- (azd ) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase studies, in melanoma, colorectal, lung and pancreatic cancer. arry- is another potent, selective mek / inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry- inhibited tnfa, il- b and il- (ic s of , and nm, respectively). % inhibition of perk required nm. arry- was highly efficacious in cia and aia rat models, with ed s of and~ mg/ kg, respectively. in normal volunteers arry- was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry- monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry- in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock- isoform.these rock- inhibitors, typified by slx- and slx- , are potent (ic < nm), selective for rock- (> fold selectivity for rock- over rock- ) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx- inhibits cell proliferation and migration in several tumor cell lines including ht- , panc- and mda-mb- . moreover in xenograft studies using nude mice, slx- significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx- prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx- is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock- selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx- and slx- will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox- is constitutively expressed.the cox- gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox- are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox- can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox- plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox- and tumorigenesis come from genetic studies.overexpression of cox- has been observed to drive tumor formation whereas cox- deficiency protects against several tumor types.selective cox- inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox- inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox- -derived pge are found in both inflamed and neoplastic tissues.the fact that pge can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox- contributes to both wound healing and tumor growth.taken together, it seems likely that cox- induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. ( ), k hagihara( ), t nishikawa( ), j song( ), a matsumura ( ) ( ) health care center, osaka university, japan ( ) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il- in the inflammatory status. to know the pathogenic role of il- and the efficacy of il- blockade in inflammation, a humanized anti-il- receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il- blocking therapy improved the clinical symptoms and the laboratory findings, the il- function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il- , il- and tnf-alpha. in result, il- was an essential cytokine in induction of saamrna through the activation of stat which constructed the complex with nf-kappab p and a cofactor p . although there was no stat consensus region on saa promoter, stat bound at the site of nf-kappab re. the above research proved that il- signal is essential on the synergistic induction of saa via newly discovered stat transcriptional mechanism, suggesting the presence of this stat mechanism in inflammation, and confirming the normalization of serum saa level by il- blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada( ), k matsushima( ), s kaneko ( ) ( ) kanazawa university, japan ( ) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)- / monocyte chemotactic and activating factor (mcaf)/ ccl , a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. ( ), p lacamera ( ), b shea ( ), g campanella ( ), b karimi-shah ( ) , n kim ( ), z zhao( ), v polosukhin ( ), y xu( ), t blackwell ( ) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa , are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa -deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r receptor. we have shown that tnf-r is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor- (par- ) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par- is implicated in innate defense responses associated with lung inflammation. we showed that par- is expressed by human alveolar (a ) and bronchial ( hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh . in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par- , but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh . preincubation of a par- transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par- , generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par- in the respiratory tract, thereby altering the host innate defense mechanisms. caspase- -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor (par ) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars , and , suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (> %), which is not seen in dizygotic twins (< %) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before . incidence has increased since now leading to a lifetime prevalence of up to . % in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome being most consistent between different populations. in three coding variations in the card gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card gene. variations in the card gene do not fully explain the linkage finding in the pericentromeric region of chromosome . after stratification for card variants, the broad linkage peak is reduced to two more defined peaks on p and q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes and , respectively). dlg- is the example of a low-risk susceptibility gene with a modest associated odds ratio ( . - . ) . interestingly, the associa-tion signal appears to be confined to young males. slc a / which encode the kation-transporters octn and have been suggested to represent the disease gene in the + kb haplotype block on chromosome q . mdr has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a k illumina scan has been published identifying il- r as a further disease gene. we used a genome wide candidate gene approach (with appr. . csnps) to identify atg l as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr , teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr +/ cd + t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx -b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr .the compound proved to be highly efficacious in the tnf-aare and mdr a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx -b was well-tolerated, and no drug-related saes were reported.a -day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx -b was shown to be both safe and to have encouraging clinical results: % of patients on ccx -b (cdai ! , crp > . mg/l) exhibited a -point drop in cdai compared to % on placebo. furthermore, a crp decrease of mg/l was seen in the ccx -b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il- p , ifn-gamma, and the chemokine rantes was shown in the ccx -b group while levels remained stable in the placebo group. ccx -b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg d in a model of inflammatory bowel disease, where cd +cd -t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg d antibody therapy. the expression of nkg d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg d on the cell surface as well as increased levels of nkg d mrna in cd + t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg d antibody (cx ) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx treatment decreased the cellsurface expression of nkg d and prophylactic administration of cx attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx administration to mildly colitic animals, whereas cx did not attenuate severe colitis. thus, nkg d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg d in cd + t cells increased markedly during the development of disease and since administration of cx early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than have gained approval. the pipeline contains many more potential biopharmaceuticals and at present in new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled % human biologicals are potentially immunogenic ( ) ( ) . i shall discuss two examples: ) recombinant human cytokines (ifn-beta- a and - b), and ) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used ( - ). it took more than years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized ( , ). ) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, - % of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnß antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnß in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnß antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit , was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, : - , ) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par and par also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par activation in joint inflammation and pain. male c bl/ mice received an intra-articular injection of either the par activating peptide aypgkf-nh or the inactive peptide yapgkf-nh ( mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par activating peptide caused knee joint blood flow to gradually increase by up to % over the succeeding hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par antagonist pepducin p pal ( mg i.p.). the control peptide yapgkf-nh had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par receptors in mice knees causes joint inflammation and pain. vincent lagente ( ), e boichot ( ) ( ) air liquide, centre de recherche claude-delorme, jouy en josas, france ( ) inserm u , universitØ de rennes matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp- ) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp- /timp- ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that members of the par family (par , par and par ) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par and par in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par and par -dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par and par dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par -dependent mechanism.in contrast, activation of par on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled consecutive dogs (group b). results: dogs were excluded from group a where / dogs ( %) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in , a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including consecutive dogs with a final diagnosis known to predispose for dic ( % were finally diagnosed with dic). outcome was -day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim : ; - ) . relative risk according to bpw (rr [ % confidence interval]) for (a) a dic diagnosis and (b) -day mortality, were assessed. -day mortality in the study population was %. % were bpw positive. bpw was not a significant diagnostic factor for dic (rr= . [ . ; . ] ), but strongly so for outcome (rr= . [ . ; . ] ) with a % ( / ) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. ( ), b hideo ( ) ( ) department of molecular pathology, kumamoto university, japan ( ) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin- expression (graÅa-souza et al., ) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., ) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. ( ) ( ) university of melbourne, victoria, australia ( ) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx ), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx -/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx -/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx -/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx -/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase- (mmp ) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx -/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp . chris bolton( ), c paul( ), s barker ( ), r mongru ( ) ( ) william harvey research institute, london, uk ( ) university west of england, bristol ( ) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a subunit (glyr a ) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a , the reduction of glyr a was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a , while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a may be a target molecule for pkg to induce allodynia. ( ), r leite( ), ys bakhle ( ) ( ) federal university of minas gerais, belo horizonte, brazil ( ) medical college of georgia, augusta, usa ( ) imperial college, london, uk selective cyclooxygenase inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats ( - g; - animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and min later given cx ( mg/kg, s.c.). after a further min, rats were injected (ipl) with the inflammatory stimulus, carrageenan ( mg/paw). mechanical pain threshold was hourly measured over the next h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin % reversal = . nanomoles), higher doses of microtubule inhibitors (taxol % reversal = . nanomoles) with no effect of acrylamide ( up to nanomoles).we conclude that ) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and ) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox- in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox- inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for hrs daily for a period of seven days. nimesulide ( . mg/kg, i.p.) was administered daily for days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in . ul. ngf ( . , and ug) injected in the tmj challenged h prior by cg ( ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k a ( and ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production and h after the tmj cg ( ug) injection. the tmj injection of ngf ( ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf ( ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd +/f / -ve/cd c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam- , although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl /mcp- , and its receptor ccr , and in response to anti-cxcr .this was supported by in vitro experiments showing that mif induced ccl /mcp- release from cultured murine endothelial cells.finally, mice lacking cd , the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd .this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf- ) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap- activities. gm-bmm preferentially produced tnfa, il- , il- and il- while, conversely, bmm generated more il- and ccl ; strikingly the latter population could not produce detectable il- and il- . following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap- activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il- following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type diabetes mellitus (t dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad , ia- and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd and cd t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th , tc ) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b: - cause diabetes . knockouts of the insulin gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin gene (islet expression) prevents % of diabetes . dual insulin knockout with transgenic insulin with altered peptide (b :a) prevents all diabetes . islets with native b: - sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells .anti-b - t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain . alpha chain as transgene sufficient to engender anti-insulin autoantibodies . kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as % of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a -step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam- and lfa- , have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa- antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, düsseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il- and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin- , tnf-µ, p mapk, msk / , mk , nf-kappab and ap- . histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h receptor (h r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h r deficient mice and mice treated with h r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th responses. ex vivo restimulation of primed t cells showed decreases in th cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h r was responsible for the the t cell effects. the influence of h r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h r deficient mice. the h r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h r was minor. surprisingly the h r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h r antagonists. ( ), bsp reddy ( ) ( ) nizams institute of medical sciences, hyderabad; india ( ) genix pharma, india rupatidine, carries the majority of the histamine h receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either mg rupatidineformulation after overnight fast. washout was days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc - mmsq/hr and auc - %/hr) both for untransformed and log transformed data were found to be within - % of % ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet ( ), h yoshimura( ), k ohara ( ), y mastui ( ), h hara ( ), h inoue ( ), h kitasato ( ), c yokoyama( ), s narumiya ( ), m majima ( ) ( ) kitasato university school of medicine, kanagawa, japan ( ) tokyo dental medical colledge, tokyo, japan ( ) kyoto university school of medicine, kyoto, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitorsconstitute revascularization. we hypothesized that txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggrega- inflamm. res., supplement ( ) tion.to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out(ipko) and thromboxane (tx) a receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf- and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. purpose: the s calcium-binding proteins, a , a and a are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a /a is anti-microbial; a has oxidant-scavenging functions. a is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys to ala a mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s s in asthmatic lung. the s s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a . a , a or the a /a complex had relatively low ability to induce il , tnf, il , and chemokines mrna from pbmc compared to a . only a induced significant levels of il ; none induced il or gm-csf mrna compared to lps. in contrast to a which is activating, a significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys . conclusions: the cytokine profile generated by a in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a in oxidant defence, particularly to hypobromite generated by activated eosinophils. ( ), d mankuta( ), g gleich ( ), f levi-schaffer ( ) ( ) hebrew university of jerusalem, israel ( ) hadassah university hospital, israel ( ) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson ( ), d kashanin ( ), f odowd( ), v williams( ), g walsh ( ) ( ) cellix ltd, institute of molecular medicine, dublin, ireland ( ) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam- and vcam- , is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. - dynes/cm ). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam- , icam- , and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam- are high ( - %, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam- , with pma-stimulated cells showing a higher affinity for icam- than nonstimulated cells (approx. % and %, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph . ). male c bl/ were instilled with ml/kg of gj. after or h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. . ae . , sg h: . ae . ; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il- ratio in balf at the nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il- ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c bl/ mice were subjected to min of intestinal ischemia and challenged with . mg/kg of intranasal lps at the th hour of reperfusion (two-hit). balf and culture of lung explants were performed h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r ( . ae . vs . ae . , mg/mg). lung mpo was increased ( . ae . vs . ae . ; od nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group ( . ae . vs . ae . ; x e cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf ( . ae . vs . ae . ; mm) and in lung explants ( . ae . vs . ae . ; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. ( ) ( ) novartis institutes for biomedical research, horsham, uk ( ) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over % (p< x - ) for exercise capacity, measured by running on a treadmill.after months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p< . ); however there was a - -fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p< . ).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p< . ). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. ( ), ap ligeiro-oliveira( ), jm ferreira-jr( ), sr almeida( ), w tavares de lima( ), shp farsky ( ) ( ) university of s¼o paulo, brazil ( ) regional integrated university of alto uruguai and missðes, brazil methods: male wistar rats were exposed to vehicle or hq ( mg/kg; ip.;daily, days, two-day interval every five days). on day , animals were ip sensitized with ovalbumin (oa). assays were performed on day . results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound / . in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd and cd on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missðes / university of s¼o p, department of clinical and toxicological analyses, s¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br ( ) ( ) radboud university nijmegen, medical centre, nijmegen, the netherlands ( ) university hospital, zürich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr and tlr was associated with the expression of ifna, tnfa, il- b, il- , il- , and il- and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr / tlr expression in synovial tissue was associated with the presence of ifna, il- b and il- , but not tnfa, il- and il- . to investigate whether ifna, il- b and il- could induce tlr / tlr upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr / mrna expression. ifna incubation resulted in significant tlr /tlr upregulation, whereas il- b and il- did not. pre-incubation with ifna and subsequent stimulation of tlr /tlr significantly enhanced il- , tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il- b were produced upon stimulation with atp, but not upon tlr / tlr stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il- b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il- b / il- in synovial tissue. first, involvement in tlr /tlr regulation and second, involvement in atp-induced production of biologically active il- b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod (nucleotide/ binding oligomerization domain) and nod in model of acute joint inflammation induced by intraarticular injection of tlr (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod and nod occurs also in humans, we exposed pbmcs carrying either nod frameshift or nod frameshift mutation with scw fragments in vitro. both tnfa and il- b production was clearly impaired in pbmcs carrying the nod fs compared to pbmcs isolated from healthy controls. in line with the nod gene deficient mice, human pbmcs bearing the nod mutation produced enhanced levels of proinflammatory cytokines after h stimulation with scw fragments. these data indicated that the nlr family members nod and nod have a different function in controlling tlr -mediated pathways. we hypothesize that intracellular nod -nod interactions determine the cellular response to tlr triggers. whether lack of controlling tlr -driven pathways by nod signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp- cells with vitamin-d . lilra expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra expression (p= . ). treatment of macrophages with lps, tnf-a, il- b and ifn-g but not il- caused significant down-regulation of lilra (p< . ). function of lilra was assessed by cross-linking with plate-immobilised lilra -specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at h. correlation between lilra expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra could potentially provide therapeutic benefit. ( ) ( ) university of valencia, spain ( ) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm- ) on an animal model of human rheumatoid arthritis. dba- /j mice were treated with corm- ( , or mg/kg/day, i.p.) from day to after collagen-induced arthritis (cia) and sacrificed on day . administration of corm- resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm- treatment with the most pronounced protective effect observed at mg/kg. the levels of pro-inflammatory mediators (pge , il- beta, tnfalpha, il- and il- ) in the hind paw homogenates were significantly inhibited by corm- . in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm- alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm- appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without ng/ml il- a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a - were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < . ). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il- a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr and tlr activation in the expression of arthritis was studied using interleukin- receptor antagonist deficient (il- ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il- ra-/-tlr -/-mice developed more severe arthritis compared to il-ra-/-tlr +/+ littermates; whereas, tlr -/-il- ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr and tlr differentially regulated the disease expression, we studied the role of these tlrs in il- production and th development, both important in il- ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il- upon stimulation with tlr and tlr ligands; however, il- ra-/-bmdcs produced less il- than wild type dcs upon tlr stimulation and more il- than wild type dcs upon tlr stimulation. furthermore, il- ra-/-t cells produced lower amounts of il- when cultured with tlr -activated apcs and higher amounts of il- when cultured with tlr -stimulated apcs, both in combination with cd stimulation. facs analysis of th (cd +/il- +) cells from both spleen and draining lymph nodes revealed % reduction in il- ra-/-tlr -/-mice compared to il- ra-/-tlr +/+ littermates. specific cd /cd stimulation of non-adherent splenocytes confirmed lower il- production in il- ra-/-tlr -/-. these findings suggest important roles for tlr and tlr in regulation of th development and expression of arthritis. prostaglandine (pge ) stimulates the transactivational activity of p through p map kinase-dependent ser phosphorylation (jbc ) .p controls cell-cycle progression, in part, by differential regulation of ap- proto-oncogenes (jun/fos).currently we studied pge control of cyclin d promoter activity with particular attention to the role of ap- oncogenes.pge induced a . fold increase in junb mrna expression (northern blot), a . -fold increase in junb promoter activity (luciferase assay), and increased ser junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra / , and fosb expression were upregulated by pge .in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g v) resulted in a . fold increase in cyclin-d promoter activity, cyclin-d synthesis, thr /tyr phosphorylation of erk / ( . fold) and ap- (c-jun)-dependent transactivity ( . fold); cyclin d /cdk - inhibitor p ink a synthesis was suppressed. addition of excess rass n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d promoter activity/protein synthesis, blocked p ink a synthesis; the latter effects were reversed by the addition of excess junb.pge exerted temporal and bi-phasic dose-dependent control of the cyclin d promoter activity, largely through differential ap- activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla / cox/pges biosynthetic axis and highlight the complexity of pge action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., , inflammation res. (s ) s ) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider ( , pnas. : - ) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha ( mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c bl/ mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day . hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic < . mm). diga ( ml, mm) i.a. prevented hour aia (- . +/- . mm).diga at mg/kg reduced aia when administered i.v. (- . +/- . mm, p< . ) and i.p. (- . +/- . mm, p< . ), but is hydrolysed p.o. ( . +/- . mm ns). polysulphated diga (diga s) was unstable, but stabilised by n-acetylation (nacdi-ga s).tnf synthesis was potently inhibited by both nacdiga and nacdiga s (ic < . mm).nacdiga s ( mg/kg p.o.) inhibited aia (- . +/- . mm), and nacdiga s with lower degrees of sulphation (mw and kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on persons, divided into groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: ) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. ) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs expression following exposure of murine macrophages to lps implicated socs in the control of lps-mediated signaling. socs regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs in regulating the production tnf by lps and pam csk -activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs (adv-socs ), control vector (adv-gfp) or left untreated. adv-socs monocytes were exposed to tlr and tlr ligands, lps ( ng/ml) or pam csk ( ng/ml). facs analysis demonstrated infection efficiencies of ae % and ae % (n= , mean ae sem) of monocytes expressing adv-gfp or adv-socs at moi . adv-socs blocked lps and pam csk induced tnf mrna and protein production in a dose-dependent manner. in contrast, il- and il- production by adv-socs -infected monocytes was not blocked. adv-socs also blocked lps and pam csk induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of ae % or ae % were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p activation. furthermore, the kinetics of lps and pam csk induced ikba phosphorylation and degradation in adv-socs monocytes remained unaffected (n= and donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p or erk mapk pathways. these data suggest that socs regulation of lps and pam csk -induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels ( - mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: ) measuring cytosolic free calcium ([ca +] i) in fura- loaded hdsmc using spectrofluorometry and ) force measurements in - mg detrusor strips. hdsmc responded to beta-nad+ ( - mm) with an immediate and transient increase in [ca +] i. the ca + transient was followed by one or two much slower and transient increases in [ca +] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca + responses persisted in the absence of extracellular calcium. the ca + responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p x purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp -induced neurotoxicity. we recently showed that tat induces the overexpression of il- via cellular kinase pkr and activation of transcription factor ets- . in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat tyrosine phosphorylation, but had no effects on the serine residue of stat and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat was abrogated by tat. the inhibition of phospho-stat led to the deformation of stat homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and , oligoadenylate synthetase ( , oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of , oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase- belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin- (il- ), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase- in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase- and il- . interestingly, increased caspase- activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p mapk dependent increased secretion of procaspase- and active caspase- . furthermore, anisomycin increased the mrna expression of il- through a p mapk dependent but caspase- independent mechanism, reaching a maximum level after hours of stimulation. finally, anisomycin caused a rapid ( hours) increase in the secretion of proil- and active il- . secretion of active il- was mediated through a p mapk/caspase- dependent mechanism, whereas secretion of proil- was mediated by a p mapk dependent but capsase- independent mechanism. these data demonstrate that the activity of caspase- is increased in psoriatic skin and that il- secretion is regulated by a p mapk/caspase- dependent mechanism, making caspase- a potential target in the treatment of psoriasis. prostaglandin e (pge ) regulates the stability of cyclooxygenase- (cox- ) mrna through adenylate/uridylate-rich elements (ares) in the untranslated region ( utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge dependent stabilization of cox- in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox- utr] reporter fusion mrna ( ae . % decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il- b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~ kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox- gene transcription, thus enabling ttp to regulate cox- gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox- mrna stability and transcription and whose localization and interaction with other factors is regulated by pge . these data can provide important insight into deciphering the role of pge in fine-tuning physiological and pathophysiological gene regulation. ( ) ( ) chinese academy of sciences, shanghai, pr china ( ) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)- is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp- has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp- is regulated is still not fully understood. here, we show that inhibition of p map kinase by specific inhibitor sb or rna interference (rnai) markedly reduced the expression of mkp- in lps or pgn-treated macrophages, which is correlated with prolonged activation of p and jnk. depletion of mapkap kinase (mk ), a downstream substrate of p , by rnai also inhibited the expression of mkp- . the mrna level of mkp- is not affected by inhibition of p , but the expression of mkp- is inhibited by treatment of cycloheximide. thus, p mapk plays a critical role in mediating expression of mkp- at a posttranscriptional level. furthermore, inhibition of p by sb prevented the expression of mkp- in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p -mk -dependent induction of mkp- in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il- and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd /cd mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th cytokine il- is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar -/-and ifnar -/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p x receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin- beta (il- b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin- , a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin- is required for processing of caspase- and release of mature il- b induced by p x receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il- b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin- -independent phase induced by p x receptor activation, and this was unaltered by pannexin- inhibition.nigericin did not induce dye uptake.inhibition of pannexin- blocked caspase- and il- b processing and release in response to this two stimuli.thus, while pannexin- is required for il- b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin- hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il- . here we verify the effect of saa on the mrna expression and release of mip- alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip- alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa ( ug/ml) induced the expression of mip- alpha mrna at , and hours. mip- alpha protein was found in the suppernatant of and hours cultures (p< , ) and the addition of sb (p inhibitor) and pd (erk / inhibitor) completely abolished the release of mip- alpha. conclusions: saa is an inducer of mip- alpha expression in pbmc and p and erk / are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p decrease mediator production and therefore can produce profound anti-inflammatory effects. arry- is a potent inhibitor of p enzyme (ic < nm) with a novel pharmacophore and physiochemical properties distinct from those of other p inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic < nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed ~ mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of , , , or mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry- was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il , pge and tnffz with > % inhibition observed at the mg dose level. the plasma concentrations of drug peaked at~ ng/ml at the mg dose and cytokine inhibition was sustained for > hours, showing that low doses of arry- produced profound effects on clinical biomarkers. further evaluation of arry- in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, mg/kg; oral route; days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam- on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il- b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name ( mg/kg; oral route; days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained hours after i.p. injection of oyster glycogen ( %; ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of x cultured cells before and hours after lps stimulation ( m;g/ml). results: levels of no, tnf and il- b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il- b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin- (il- )-induced mmp- expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il- beta alone or in combination. mmp- mrna was analyzed by rt-pcr. mmp- protein, phospho-stat and p / mapk levels were measured by western blotting. mmp- promoter-luciferase, cmv-cbp/p plasmids and stat sirna were transfected by calcium phosphate method. ap- activity was monitored by elisa. stat -cbp/p interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il- -induced expression of mmp- and promoter activity. blockade with neutralizing ifn-gr antibody revealed that mmp- inhibition by ifn-¼ was mediated by the ifn-¼ receptor. ifn-beta-stimulated activation of stat was directly correlated with mmp- suppression. knockdown of stat gene by specific sirna or its inhibition with fludarabine partially restored the il- induction of mmp- expression and promoter activity. ifn-g did not alter activator protein (ap- ) binding ability but promoted physical interaction of stat and cbp/p co-activator. p overexpression reversed ifn-g inhibition of endogenous mmp- mrna expression and exogenous mmp- promoter activity. conclusions: ifn-g through its receptor activates stat , which binds with cbp/p co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp- gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for ( ), p asmawidjaja ( ), r hendriks( ), erik lubberts ( ) ( ) erasmus medical center, department of rheumatology, rotterdam, the netherlands ( ) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il- in th polarization in the prone autoimmune dba- mice with and without collagen-induced arthritis and to evaluate th specific cytokine and transcription factor expression. il- induced th cells in vitro from spleen cells of naïve and collagen-type ii (cii) immunized dba- mice. the percentage of th cells is markedly higher in cii-immunized versus naïve dba- mice. adding il- to tgf-beta/il- stimulated cd + t cells did not significantly increase the percentage of th cells. tgfbeta/il- in contrast to il- induced a relatively high percentage of il- +/ifn-gamma-cells and low il- -ifn-gamma+ cells. tgf-beta/il- did not increase il- receptor expression, which may explain why adding il- directly or two days after tgf-beta/il- did not result in an increase in the percentage of th cells. elevated expression of il- a and il- f as well as the th specific transcription factor rorgammat was found under il- as well as tgfbeta/il- conditions. interestingly, il- but not tgf-beta/il- is critical in the th cytokine il- expression in t cells from ciiimmunized dba- mice. these data show that il- was more pronounced in inducing il- +/ifn-gamma-(th ) cells under cii-immunized conditions. furthermore, il- did not markedly increase the percentage of th cells induced by tgf-beta/il- . however, il- is critical for the induction of il- expression, suggesting a unique role for il- in the induction of specific th cytokines ebi was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p of il- . ebi protein has been shown to form heterodimers with p . p /ebi termed il- , can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi and p is very limited. these data lead to the hypothesis that ebi may play a role independently from its association with p . thus, to define the function of ebi , we generated ebi transgenic (tg) mice expressing in multiple tissues. ebi tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd +, cd + t cells, b cells, nk cells and nkt cells. cd +t cells isolated from spleens of ebi tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd and anti-cd antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi tg mice were recovered with anti-ebi polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi antibody. in contrast, anti-p antibody suppressed chs responses in wt mice. these data suggest that ebi acts in different from il- , and reduces th responses. ( ), o thaunat( ), x houard ( ), o meilhac ( ), g caligiuri( ), a nicoletti ( ) ( ) inserm u and university denis diderot-paris , chu xavier bichat, paris, france ( ) inserm umr s , universitØ pierre et marie curie-paris , centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep -i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk / m and vascular inflammation leads to formation of leukotrienes through the -lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b (ltb ) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb -induced atherogenesis. targeting ltb -induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox- ). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox- ) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox- and the inducible enzyme responsible for pge synthesis (mpges- ) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi is the major biological active prostanoid produced in the normal physiological conditions. however, when cox- is induced, pgi and pge are equally produced. the role of cox- , cox- and mpges- activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep , ep , ep or ep ) preferentially stimulated by pge in the vascular wall. for these reasons, we have characterized the receptors activated by pge in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin- â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox- inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn (a gene that regulates treshold of lumphocyte activation) the allele c t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in dutch ra cases and unrelated dutch controls. carrier analysis of the snp (rs ) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p = . and or = . ). in a meta-analysis of all studies comparing individuals, the or for the cc genotype to develop ra was . and the p-value < . . in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd / q , card /nod and il r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card /nod is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card appear to show opposite effects to other models and human systems. in humans, card mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp /cryopyrin/ cias gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp protein associates with asc and caspase- (thereby forming a molecular machine termed inflammasome that displays high proil- beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il- beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp -dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. ( ) ( ) erasmus mc, rotterdam, the netherlands ( ) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd c-diphtheria toxin (dt) receptor (cd cdtr) transgenic mice by airway administration of dt h before ova aerosol ( x) challenge or during an ongoing inflammation (depletion after x ova aerosols continued with additional ova aerosols). numbers of balf eosinophils, th cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th -type cytokines (e.g. il- and il- ), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th -type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi -kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd r, cd r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a (spla ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla , hdl and ldl and of their hydrolysis products on mitogenesis and pge and ltb release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla . spla hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge from vsmc was enhanced by group x spla s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla .native ldl and its spla hydrolysis products enhanced the release of pge in order x>v>iia.the release of ltb from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla .migration of vsmc was significantly enhanced by spla iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca ( ) ( ) ipmc-cnrs umr , valbonne, france ( ) university of washington, seattle, usa ( ) inserm umrs , paris, france ( ) university of naples, italy the superfamily of phospholipase a comprises at least intracellular enzymes and up to secreted pla s (spla s). elucidating the biological roles of each pla member is currently the most challenging issue in the pla field. the different spla s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla can trigger secretion of tnf alpha, il and il in a non-enzymatic manner. on colorectal cancer cells, hgx spla stimulates cell proliferation, produces potent eicosanoids including pge , and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla s, we have examined fatty acid release by spla siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla was observed when the residual sm/ pdcho molar ratio had reached . compared to a starting ratio of . .group v spla showed preferential release of linoleate at residual sm/ptdcho molar ratio . , while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla , possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla . the fatty acid specificity of pla is significantly affected by sm. thus spla x exhibited enhanced specificity for the release of arachidonic acid ( : ) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of : by spla v. ceramide selectively stimulated the release of : by both enzymes. only the long chain ceramides (> carbons) were effective, while ceramide phosphate did not stimulate spla activity. sm-deficient cells released more : in response to spla -treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a (spla ) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a- , an inhibitor of spla enzymes, on atherosclerosis and cholesterol levels over weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study ( % fat; . % cholesterol, . % casein) and were treated with vehicle or a- bid at mg/kg or mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a- significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for weeks, oral dosing of a- ( mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a- is a potential novel therapeutic agent for the treatment of atherosclerosis. ( ), s doty( ), c antonescu ( ), c staniloae ( ) ( ) saint vincents hospital manhattan, new york, usa ( ) hospital for special surgery, new york, usa ( ) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene (tsg- ) is induced by tnf-a during inflammation and its secreted product tsg- glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox- and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg- is expressed in atherosclerotic plaques and investigated tsg- protein expression and cellular distribution on superficial femoral artery endarterectomy specimens from diabetic and non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg- antibody. tsg- expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg- expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg- stained the foamy macrophages. tsg- expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg- gene (more that fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg- in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg- is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. ( ), r krohn ( ), h lue ( ), jl gregory( ), a zernecke ( ), rr koenen ( ), t kooistra ( ), p ghezzi( ), r kleemann ( ), r bucala( ), mj hickey ( ), c weber ( ) ( ) university hospital of the rwth aachen, germany ( ) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr , inducing rapid integrin activation. mif directly bound to cxcr with high affinity (kd of . nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr as well as cd , a recently identified membrane receptor moiety for mif. accordingly, cxcr and cd were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr , serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. ( ), r toes ( ), h van bockel( ), paul quax ( , ) ( ) tno bioscienses, leiden, the netherlands ( ) department of vascular surgery, leiden university medical center, the netherlands ( ) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c bl/ mice depleted for natural killer (nk)-cells by anti-nk . antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jµ knockout mice that lack nk . + natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c bl/ mice depleted for cd + tlymphocytes by anti-cd antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd + t-lymphocytes. this impairment was even more profound in anti-nk . treated mhc-class-ii-deficient mice that lack both nkand cd + t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c bl/ mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd -positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd + t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf: u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by % (p= . ), when compared to saline treated controls (n= ).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c convertases). crry-ig ( mg every other day) led to % decrease in vein graft thickening (p= . ) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. ( ), m-c koutsing tine( ), p borgeat( ), h ong ( ), sylvie marleau ( ) ( ) universite de montreal, quebec, canada ( ) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep , a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd , elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from weeks of age. at weeks old, mice were treated daily with a s.c. injection of ep ( mg/kg) for days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for minutes, followed by minutes reperfusion. our results show that ep significantly reduced leukocyte accumulation by % in the lungs, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice (n = - per group), as assessed by myeloperoxidase assay. this was associated with a % reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd -/-deficient mice, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice. we conclude that ep protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd -mediated pathway. glycogen synthase kinase beta (gsk- beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk- beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk- beta, tdzd- and sb , on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to mmhg for min) and subsequently resuscitated with shed blood for h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd- ( mg/kg, i.v.) or sb ( . mg/kg, i.v.) min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd- , but not sb , attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin . neither of the gsk- beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk- beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. ( ), y ito ( ), h yoshimura ( ), h inoue ( ), n kurouzu ( ), h hara ( ), y mastui ( ), h kitasato ( ), s narumiya( ), c yokoyama ( ), m majima ( ) ( ) kitasato university school of medicine, japan ( ) kyoto university school of medicine, japan ( ) tokyo medical and dental university, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out mice (ipko) and thromboxane (tx) a receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf- and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from patients with synovitis of "t months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from patients undergoing vitrectomy for vitreoretinal disorders. one dimensional h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il- , ccl ), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. ( ), am artoli( ), a sequeira( ), c saldanha ( ) ( ) instituto de medicina molecular,faculdade de medicina de lisboa, portugal ( ) cemat, instituto superior tØcnico, universidade de tØcnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn- , slfn- , and slfn- were maximally induced after hours. the maximal induction of slfn- and slfn- was observed after hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn- , slfn- , slfn- and slfn- were elevated in joints affected by cia. to investigate the role of slfn- , we have generated a transgenic mouse line, which over expresses slfn- specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal . further characterisation of the slfn- over expressing mouse line will be used to assess the function of slfn- in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin , lcn- , is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn- determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn- is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn- . ( ), y cao ( ), s adhikari ( ), m wallig ( ) ( ) national university of singapore, department of pharmacology, singapore ( ) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein ( mg/kg) for , and hours respectively. neutralizing monoclonal anti-il- antibody ( . mg/kg) was administered either with or without crambene ( mg/kg) hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp- , tnf-a and il- â and up-regulating anti-inflammatory mediators like il- . phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd .the anti-inflammatory mediator il- plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il- is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. ( , ) ( ) northern ontario school of medicine, thunder bay, ontario, canada ( ) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, , : l -l ). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn delivery system.using several fluorescence-based detection systems, we established that the natural beta integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a a receptor (a ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b formation while potentiating that of prostaglandin e through the up-regulation of the cyclooxygenase (cox)- pathway. moreover, our laboratory determined that a ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a ar, migrated neutrophils expressed less cox- than their wild type counterpart while displaying higher mrna levels of tnf-and mip- . mononuclear cells from a ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p and pi- k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a amino acid protein that contains a s domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr (tandem repeat unit b), rhrnr (tandem repeat unit a) and rhrnr (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. ( ), n lu( ), r jonsson( ), d gullberg ( ) ( ) department of biomedicine, university of bergen, norway ( ) the gade institute, university of bergen, norway a ß is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a ß expression. specifically, we have analyzed the influence of cell passage, growth factors and the -d microenvironment. using sv immortalized as well as primary fibroblasts, we show that a ß integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a ß is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a ß that need to be to taken into consideration when evaluating the role of a ß in wound healing and fibrotic disorders. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par in the process of inflammation in the gut.colonic inflammation in c bl wildtype and par -/-mice was induced by treatment with . % dss (in drinking water) or tnbs ( mg or mg in ul of % ethanol, single intracolonic injection) or pre-sensitizing mice with % oxazolone (in olive oil) applied to the skin of the abdomen, and days later, a single intracolonic injection of % oxazolone (dissolved in % ethanol).intravital microscopy was performed, days (tnbs/dss) or days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par -/-.our evidences indicate that deficiency in par attenuates inflammatory responses in the experimental models of colitis associated with either th (tnbs/dss) or th (oxazolone) cytokine profile.therefore, par deficiency in the gut exerts antiinflammatory properties that are independent of th or th cytokine profile.the present study further highlights par as a potential target for inflammatory bowel diseases. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par (par -/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par deficiency. aim of this study was to assess the effects of par deficiency (via par -/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp ( mm) or paf ( nm) or by intraperitoneal injection of tnf-a; ( . mg -given hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par -/-versus par +/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par -/-mice compared to wild types as well. no difference was observed between par -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par activation in mouse colon leukocyte trafficking highlights par as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. ( ), kk hansen( ), k chapman( ), n vergnolle ( ), ep diamandis ( ), md hollenberg ( ) ( ) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada ( ) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks and to activate pars , and in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk is able to activate both of pars and and to prevent thrombin from activating par . on the other hand, klk was a specific activator of par . kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars and and by inactivating par , kallikreins, like klks and , may play a role in regulating the inflammatory response and perception of pain. ( ), d park ( ), b short( ), n brouard( ), p simmons( ), s graves ( ), j hamilton ( ) ( ) melbourne university, melbourne, victoria, ( ) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p subunit and nrf into hepg or other cells, as well as sirna technique to knockdown endogenous p in cells, we found that nf-kb p subunit repressed the anti-inflammatory and anticarcinogenetic nrf -are pathway at transcriptional level. in p -overexpressed cells, the are-dependent expression of heme oxygenase- was strongly repressed. in the cells where nf-kb and nrf were simultaneously activated, p unidirectionally antagonized nrf transcriptional activity. the p -mediated are inhibition was independent of the transcriptional and dna-binding activities of p . co-transfection and rna interference experiments revealed two mechanisms which coordinate the p -mediated repression of are: ( ) p selectively deprives creb binding protein (cbp) from nrf , but not mafk, by competitive interaction with the ch -kix domain of cbp, resulting in inactivation of nrf transactivation domain and concomitant abrogation of the nrf -stimulated coactivator activity of cbp; ( ) p promotes recruitment of histone deacetylases (hdac ) to are by enhancing the interaction of hdac with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p subunit. since various inflammatory and tumor tissues constitutively overexpresses p in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd l, only lps-matured dc expressed par and par on their surface. stimulation of dc with thrombin, par -or par -activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk / and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain (mlc ). the erk / -and rock-i-mediated phosphorylation of mlc was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine- ( f) by either nh -reactive n-succinimidyl- -[ f]fluorobenzoate or sh-reactive n-[ -( -[ f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, - %; specific radioactivity, - gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of f-nldl and f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of f-nldl and f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-Þb (nf-Þb), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p mapk and the down stream kinases msk and in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il- b before kinase activation was determined by western blotting. a significant inhibition of both msk and activations was seen after pre-incubation with dmf and stimulation with il- b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p (ser ), which is known to be transactivated by msk . furthermore, incubation with dmf before stimulation with il- b resulted in a significant decrease in nf-kb binding to the il- kb and the il- kb binding sites as well as a subsequent decrease in il- and il- mrna expression. our results suggest that dmf specifically inhibits msk and activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki- and ck- . epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol ; , - ) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer ( ), e hasselager( ), j clausen( ), l iversen ( ), k kragballe ( ) ( ) aarhus university hospital, denmark ( ) novo nordisk a/s, denmark interleukin- (il- ) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il- are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il- is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il- expression in non-lesional psoriatic skin ex vivo. mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il- beta ( ng/ml) for , , , , , and hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il- beta rapidly induced il- mrna expression in the biopsies. the highest level of il- mrna was detected after hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il- mrna. increased levels of il- protein were detected in the supernatant of the il- beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il- protein in the keratinocytes, whereas the il- protein was present in epidermal cd a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il- expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il- protein in epidermal dendritic cells whereas no il- was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il- . one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp- was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp- activity and mrna levels induced by tnf was inhibited by~ % following h treatment with calcitriol. using specific inhibitors we established that the induction of mmp- was dependent upon the erk pathway, while p -mapk and pkc inhibited, and jun-kinase, pi- -kinase and src did not affect it. levels of c-fos, a component of ap- transcription complex known to mediate mmp- induction, were elevated by tnf and further increased by calcitriol. the induction of mmp- by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp- by egf. we conclude that calcitriol inhibits the induction of mmp- gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. ( ), p verzaal ( ), t lagerweij ( ), c persoon-deen ( ), l havekes ( ), a oranje ( ) ( ) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands ( ) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd + t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th -mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. ( ), y sawanobori ( ), u bang-olsen ( ), c vestergaard( ), c grønhøj-larsen ( ) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il- mrna has been shown. also, transgenic mice over-expressing il- exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il- antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il- ratanti-mouse mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il- as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il- antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il- antibody treatment in the time interval day - . our results suggest an anti pruritic role for il- antibody in an atopic dermatitis-like animal model. anti il- antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. ( ), p ferro ( ), hm asnagli ( ), v ardissone ( ), t ruckle ( ), f altruda ( ), ch ladel ( ) ( ) rbm merck serono/university of torino, italy ( ) merck serono pharmaceutical research institute, geneva, switzerland ( ) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide -kinases (pi ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi k-g inhibitor as on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose , mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi k-g are demonstrable after oral administration of selective inhibitors like as in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box (hmgb ) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb absorber. the purpose of this study was to test the hypothesis that hmgb removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb absorber group (group i),hmgb nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at hours after lps injection. the rats were sacrificed hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb with anova. results: survival rate was % in the group i at hours after lps injection, as compared with % in the group ii and % in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb therapeutically reverses lethality of established sepsis indicating that hmgb inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp ( ) ( ) department of pharmacology, national university of singapore, singapore ( ) dso national laboratories, singapore hydrogen sulfide (h s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, mg/kg i.p.), an inhibitor of h s formation or sodium hydrogen sulfide (nahs) ( mg/kg, i.p.), a h s donor. pag was administered either hour before or hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam- , p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs ( mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-x×b inhibitor, bay - . h s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay - . moreover, expression of cxcr in neutrophils obtained from h s treated mice was significantly upregulated leading to an obvious elevation in mip- directed migration of neutrophils. therefore, h s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid (trpv ) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv antagonists are not available for use in the mouse in vivo, thus established trpv knockout (-/-) mice were used. c bl wt and trpv -/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv -/-mice, with trpv -/-mice showing an enhanced drop at h. in a separate group temperature, a proposed pre-mortality marker was also reduced by h, again with a significantly increased drop in trpv ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv -/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv -/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv plays a protective role in sepsis. trpv is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. ( ), da souza-junior( ), l de paula ( ), mc jamur ( ), c oliver ( ), sg ramos ( ), cl silva ( ), lucia helena faccioli ( ) ( h rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il- , kc, mcp- and mip- were detected in the lungs by day . in vivo degranulation of mast cells by c / led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c / . histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c / . of interest, the number of mycobacterial bacilli recovered from the lungs was log higher after treatment of infected mice with c / . these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. ( ), ms chadfield ( ), db sørensen ( ), h offenberg ( ), m bisgaard ( ), he jensen ( ) ( ) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark ( ) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: female balb/ c-j mice (app. g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp ) and porcine (p ) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp- and metalloproteinase inhibitor timp- . results: all mice infected with the avian strain were euthanized after hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after , and h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp- and timp- between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp- and timp- . ( ), r molinaro ( ), a franÅa ( ), m bozza ( ), f cunha( ), s kunkel ( ) ( ) universidade do rio de janeiro, brazil ( ) universidade de s¼o paulo, brazil ( ) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr knockout mice (ccr -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr -/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c /bl mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since h after and until days. ccr -/-mice subjected to clp presented an increase in survival rate ( %) compared with wildtype mice ( %), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr -/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr -/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr -/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -µ and il- â) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa ( ), commiphora mukul ( ) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il- , cxcl and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl , which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr , the receptor for cxcl . while plasmablasts migrate in response to cxcl , plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr , the receptor for the interferon-gamma-induced chemokines cxcl , and , which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, ( ), h lee ( ) c a is a potent inflammatory mediator produced during complement activation. unregulated c a signalling through its receptor (c ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c ar antagonists for human therapy. we took neutrophils from genetically modified human c ar knock-in mice in which the mouse c ar coding region was replaced with human c ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c ar. these mabs inhibit c a-induced neutrophil migration and calcium-flux, and bind to a region of the nd extracellular loop of c ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c ar knock-in mice were given - mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single mg/kg dose of anti-c ar mab given days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g arrest in macrophage cell line raw . , their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba- mice by immunisation with bovine type ii collagen/adjuvant on day and . cor , synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day . etanercept ( mg/kg ip q d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry- is a potent (ic = nm), selective, atp-uncompetitive mek / inhibitor.arry- is highly efficacious in cia and aia rat models, with ed s of and mg/kg, respectively, equal to or better than standard agents. addition of arry- to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il- and il- production (ic s of , and nm, respectively). in contrast, inhibition of perk required nm to achieve a % reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of , , , or mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry- was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il and tnffz with > % inhibition observed at plasma concentrations of and ng/ml, respectively. similar inhibition of perk required ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry- and helped define tolerability. clinical evaluation of arry- in combination with methotrexate in patients with rheumatoid arthritis is on-going. p (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il- a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p activity is expected to regulate the levels of tnf-a and il- b thereby alleviating the effects of inflammation in ra. a new class of p inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. ( ), h aaes ( ), w-h boehncke( ), j pfeffer( ), t skak-nielsen( ), i teige ( ), k abell ( ), ph kvist ( ), e ottosen ( ), tk petersen ( ), lars svensson ( ) ( ) discovery, leo pharma, industriparken, ballerup, denmark ( ) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il- b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately % of psoriasis patients develop psoriatic arthritis. leo is a member of newly developed class of selective p map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice ( % reduction in mice and % in rats at mg/kg p.o.). in the humanised psoriasis model, leo at a dose of mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by %) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx- is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock- . its ic for rock- and rock- inhibition is nm and > mm respectively. the ability of slx- to inhibit septic liver injury was investigated in c bl/ j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps ( mg/mouse) and d-gal ( mg/ mouse) i.p and . hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a > fold increase in serum alt and ast levels. this increase was reduced by > % in mice pretreated with slx- either orally ( mg/kg, and hrs prechallenge) or i.p. ( - mg/kg, min pre-challenge). slx- inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by > %. to assess the kinetics of slx- s benefit, slx- ( mg/kg i.p.) was given min prior to, or or min after the lps/ d-gal. slx- was effective at inhibiting the rise in alt and ast levels at all time points suggesting that inhibiting rock- even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, out of mice given lps/d-gal were dead by hrs whereas in mice given slx- ( mg/kg orally) animal died at hours and the remaining mice were alive hrs later. these results show that specific inhibitors of rock- may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il- secretion in human purified t-cells stimulated with anti-cd and anti-cd or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. ( ), j zhang ( ), k henley ( ), m white ( ), d hilton ( ), b kile ( ) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c bl/ control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments . f(ab) or iv. f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il- beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt ( ), u hüffmeier ( ), i kçnig ( ), j lacorz ( ), a reis ( ), k reich ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*- a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*- t was detected (or= . , % ci . - . ; pcorr= . ) also in patients negative for the psors risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*- a and psoriasis seems to primarily reflect ld with psors , tnf*- t may represent a risk factor for psa independent of psors . ( ), n modi ( ), m stanford ( ), e kondeatis ( ), r vaughan ( ), f fortune ( ), w madanat ( ), c kanawati( ), p murray ( ) methods: dna was obtained from patients with bd, from the uk and from the middle east (me), and controls individuals, from the uk and from me. dna was prepared by proteinase k digestion, and salt extraction and - and snp were detected by a pcr-ssp. results: there was no significant difference in expression of - c/t or a/g when all bd patients were compared to all control individuals, (p= . and . , respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p= . , p= . ) or me bd patients (p= . , p= . ) when compared to the appropriate controls. ( ), da brown ( ), h johnen ( ), mr qiu ( ), t kuffner ( ), pgm curmi ( ), l brown ( ), m mazzanti ( ), sn breit ( ) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il- gene is single copy gene located on chromosome q . - in humans. interleukin- is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin- concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il - snp in the children with the cp. methods: dnas obtained from peripheral blood of cp patients and unrelated healthy volunteers were genotyped for the il - snp pcr-rflp method. results and conclusions: il - genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il fngene polymorphisms and cerebral palsy has to be investigated in the future studies. ( ), d delbro ( ), e hansson ( ) ( ) kalmar university, sweden ( ) gçteborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: . the expression of alpha nachrs and alpha nachrs by immunofluorescence and western blot. . intracellular ca +-transients spread within the astroglial networks after stimulation with nicotine. . pro-inflammatory cytokines, il- b, il- , and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca + transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca + responses were blocked by the alpha nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database ). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at mg/kg dose, and above. in the th test weak % effect was seen at side effect free dose ( mg/kg). in the arthritis model b.i.d. mg/kg dose of tramadol given on days - after cfa caused a maximum % effect on weight bearing incapacitance (day ). however, its effect seemed to diminish ( % on day ) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at but not at mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge and pgd in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk- .biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge and pgd levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk- was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo( ), lm yshii ( ) ( ), sk costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college, london, uk ( ) butantan institute, s¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr , a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin ( %, h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il- b and il- , but not tnfa) and anti-inflammatory (il- ) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr ( nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. ( ) ( ) university of s¼o paulo, s¼o paulo, brazil ( ) butantan institute, s¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or , -naphthoquinone ( , -nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and , -nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep ( mg/kg) and , -nq ( nmol/kg). after min, dep and , -nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following h treatment with pollutants. this treatment contributes to augment the ach ( - - - m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: , -nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and , -nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv ( , , mm) dose-dependently inhibited ng/ml lps induced tnfa, il- b, il- , mcp- and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk / and jnk but not p . nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk / , jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. ( ), cs patil( ), sv padi ( ), vp singh ( ) ( ) university institute of pharmaceutical sciences, panjab university, chandigarh, india ( ) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia days after the nerve ligation. sildenafil ( and fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) and glyt are expressed in glia and neurons, respectively. glyt make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt (sarcosine and org ) and glyt (alx and org ) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt and glyt inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp ff mouse to assess the effect of the gp ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il- family cytokine receptor gp preventing shp /ras/erk signaling and leading to constitutive stat transcriptional activation. tumour development is inflammation dependent, there is a requirement for il- , and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s inflamm. res., supplement ( ) posters dispensable for tumorigenesis. in gastric antrum the gp ff mutation results, decreased erk / activation and constitutive phosphorylation of stat , abnormal signaling is replicated in macrophages. antral stat activation is unaffected by depletion of il- . however in macrophages an absence of il- results in higher stat activation demonstrating the anti-stimulatory role of il- on macrophages. the gp ff mutation results in macrophages with decreased il- and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp ff mice display aberrant gp signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. ( ), y riffo-vasquez( ), s brain( ), s costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and , -naphthoquinone ( , -nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv knockout (ko) mice exposed to dep and , -nq. dep ( mg/kg) and , -nq ( nmol/kg)-induced airway inflammation was assessed via i-labelled albumin h post pollutants injection. mpo assay and histopathology were performed h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and , -nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv ko, suggesting these pollutants act via a trpv -mediated mechanism. in contrast, mpo activity in the airways of trpv ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv ko, indicating that the lack of trpv receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv receptors. acknowledgements: capes, cnpq, fapesp. ( ), n sato( , ), y endo ( ), s sugawara ( ) ( ) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan ( ) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j . . female balb/c mice ( weeks old) received a basal diet or a biotin-deficient diet for weeks.ten days after sensitization with intraperitoneal injection of lps and nicl , the mice were challenged with intradermal injection of nicl into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j . cells were cultured in biotin-sufficient or deficient medium for weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il- beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j . cells produced il- beta significantly higher than biotinsufficient j . cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il- beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il- , icam- and e-selectin were studied in patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in patients and severe -in patients. the highest levels of il- were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il- and adhesion molecules and severity of pancreatitis. during first week the levels of il- gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam- levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il- and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il- and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for â-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd and dnam- on mc, and b and nectin- on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells ( - weeks with scf, interleukin- and prostaglandin e , cbmc). cbmc were activated, after their culture for days with myeloma ige ( mg/ml), by rabbit anti-human ige antibodies ( mg/ml), for , and h at c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl- , bcl- , bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl- that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl- and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl- family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. ( ), c armishaw( ), z yang ( ), h cai ( ), p alewood( ), c geczy ( ) ( ) university of new south wales, faculty of medicne, sydney, australia ( ) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s a , s a and s a are closely related proteins associated with inflammation. s a is expressed in the human genome, but not in the mouse. s a is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s a and human (h) s a share % structural identity, but the hinge domains are more divergent. the ms a hinge (ms a - ) is a potent chemoattractant for leukocytes whereas this sequence in hs a (hs a - ) is inactive. methods: s a hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp cells or murine mc in vitro and mouse footpad injection. results: s a hinge domain (s a - ) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s a , the hinge domain only weakly induced cytokine production (il , il ) by macrophages. residues essential for oedema were hydrophobic in nature (leu , isoleu , isoleu and isoleu ). n and i were essential for responses provoked by - m s a - whereas mutation of k , l , n , i , d , k and i significantly reduced migration with - m s a - . conclusions: s a and ms a may be functional chemattractant equivalents; s a may have arisen by duplication of human s a . isoleucine residues in s a hinge domain are essential for its proinflammatory properties. ( ), g kiriakopoulou ( ), e tsimara( ), a voultsou( ), k zarkadis ( ) ( ) general hospital of zakynthos, greece ( ) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes . eo . %, hb . g/dl, ht . %, mcv , fl, mch . pg, thrombocytes . , blood sedimentation mm, fe mg/ml, ferritin . ng/ml. normal biochemical examinations. u/s: livers size lightly increased . mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules ( - mm) with a thin wall and quills on the side, as well as three imago worms (> mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites . , eo . %, hb , g/dl, ht . %. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement ( ) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average . mg/ l; p < . ) and with attacks (average . mg/l; p < . ) were significantly higher than those of normal controls (average . mg/l). serum hs-crp levels were inversely correlated with fev . % in asthmatic patients (r = - . , p < . ). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was % hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. ( ), sk bk( ), v sharma( ), rp bhandari ( ) ( ) nepal medical college teaching hospital, nepal ( ) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s inflamm. res., supplement ( ) posters from january to december . demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was , , giving an incidence of . per deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over % were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were . and . per deliveries (p <. );the corresponding cases of deepvenous-thrombosis diagnosed were . and . per deliveries, respectively (p <. ). the majority ( %) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of < weeks, a bmi of , or more and maternal-age of or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. ( ), ch ladel ( ), t ruckle( ), c rommel( ), r cirillo ( ) ( ) rbm-merck serono, colleretto giacosa, italy ( ) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi k signalling inhibition resulted in clinical amelioration of disease symptoms. as , a novel isoform non-selective, yet specific class-i-pi k inhibitor, administered at mg/kg twice-a-day for days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as was able to decrease its levels down to values comparable with naïve animals. controls and as treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi kg) and immunosuppressive (via pi kd/a/b) activity. ( ) ( ) institute of biomedical science, university of sao paulo, brazil ( ) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol ( ìg/kg) or progesterone ( ìg/kg) h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement ( ) s the release of il- and reduced that of il- and tnf. the release of il- by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il- and increased il- production in bal and increased that of il- and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam- and mac- expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after (ovx- ) or days (ovx- ) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, %; aerosol) and sacrificed h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam- , mac- and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day , e-selectin expression increased(ovx- = , ae , ) and at day , decreased (ovx- = , ae , ) as compared to respective controls (sh- = , ae , ; sh- = , ae , ). estrogen treatment reverted this profile in both groups (ovx- +e= , ae , ; ovx- +e= , ae , ). mean fluorescence intensity of bal cells showed increase of mac- expression (ovx- = ae , vs sh- = , ae , ), icam- (ovx- = , ae , vs sh- = , ae , ) and l-selectin (ovx- = , ae , vs sh- = , ae , ) at day i.e., ovx- group. on the other hand, we observed a decrease in icam- (ovx- = , ae , vs sh- = ae , ) and mac- expression (ovx- = , ae , vs sh- = , ae , ) was seen in ovx- group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx- ) and decreased (ovx- ) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell ( ), s lim ( ), y lin ( ), bp leung ( ), c thiemermann ( ), wsf wong ( ) ( ) national university of singapore ( ) the william harvey research institute, london, uk glycogen synthase kinase b (gsk- b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk- b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd- , a non-atp competitive gsk- b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd- significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il- , il- and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd- also suppressed the mrna levels of icam- , vcam- and chitinase proteins in the lung. histological studies revealed that tdzd- substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd- also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd- . our findings suggest that inhibition of gsk- b may have therapeutic potential for the treatment of allergic airway inflammation. ( ), a yildirim ( ), f ercan ( ), n gedik ( ), m yuksel( ), inci alican ( ) ( materials and methods: sprague-dawley rats ( - g) were exposed to oc (burn group) or oc water bath (control group) for s under ether anesthesia. adm ( ng/kg; s.c) was administered min before burn and all rats were decapitated h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p< . - . ) and mpo activity showed a marked increase in all tested tissues (p< . ) of the burn group. adm reversed these parameters effectively (p< . - . ). luminol chemiluminescence levels showed increases in both ileum and lung (p< . - . ) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p< . ) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p< . ). adm reduced plasma tnf-alpha level (p< . ) which showed a significant increase in burned animals compared to controls (p< . ). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday ( ), sd joshi ( ), kr reddy ( ) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg +, and an inhibitor, hql- , with anti-inflammatory activities in vivo, at . resolution. hql- was found to reside within the catalytic cleft between trp and gsh in the quaternary complex. hql- inhibited h-pgds competitively against the substrate pgh as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql- bound to h-pgds with an affinity that was -fold higher in the presence of gsh and mg + than in their absence. hql- inhibited selectively pgd production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql- inhibited antigen-induced production of pgd , and airway inflammation in mice without affecting the production of pge and pgf f¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats ( - g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for h ( breath/min). one lung was subjected to hyper-ventilation ( ml/kg/lung) and the other was ventilated with a normal volume ( ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip- was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip- expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip- between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. ( ), j-y gillon( ), v lagente ( ), e boichot ( ) ( ) university of rennes , rennes, france ( ) serono international s.a., geneva, switzerland macrophage elastase (mmp- ) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp- in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp- (rhmmp ) on il- /cxcl release from alveolar epithelial cell line a and we explored the underlying mechanisms. a cells were stimulated with rhmmp- ( . - - . - u/ml) during hours and il- /cxcl level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p kit. we observed that mmp- elicited il- /cxcl release in a dose-dependent manner. this production could be prevented by a pretreatment for hour with a selective mmp- inhibitor (as - mm) or with the nonselective inhibitor batimastat ( - mm) . the il- /cxcl production was also inhibited by actinomycin d ( mg/ml), erk / inhibitors (u mm and pd mm) and nfkb inhibitors including bay - ( mm) and nfkb activation inhibitor ( nm), whereas p kinase inhibitor (sb ) had no effect. stimulation with mmp- was rapidly followed by a phosphorylation of erk / ( min) and an nfkb nuclear translocation and activation ( h). the nfkb activation was not inhibited by a treatment with u . these data suggest that alveolar epithelium is a target of mmp- since it upregulates gene expres-s inflamm. res., supplement ( ) posters sion and release of il- /cxcl , via erk / and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)- , in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h cells were exposed to serial dilutions of test agents in serum-free medium for hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il- , matrix-metalloprotease- (mmp- ), the gel-forming mucin muc ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p s cyp a and cyp b were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il- alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th ) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase- (cox- ) and cox- dependent phases of prostaglandin d (pgd ) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic values of . mm and . mm, respectively. this compound inhibited cox- and -dependent conversion of the exogenous arachidonic acid to pgd in a dose-dependent manner with an ic values of . mm and . mm, respectively. however, dpt did not inhibit cox- protein expression up to a concentration of mm in the bmmc, indicating that dpt directly inhibits cox- activity. furthermore, this compound consistently inhibited the production of leukotriene c (ltc ) in a dose dependent manner, with an ic value of . mm. these posters inflamm. res., supplement ( ) s results clearly demonstrate that dpt has a dual cox- / -lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf- , could block the inflammation induced by an acute exposure to cigarette smoke in strains of mice, balb/c and c /bl .animals were administered the compound ( - mg/kg) either per os (p.o.) or intranasally (i.n.) hour before and hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf- dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~ % at mg/kg; p < . )or i.n. (~ % at mg/kg; p < . ).however, the compound had no clear effect on bal neutrophil infiltration in c /bl mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < . ). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il- beta, kc, and mip- , pkf- had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table ) cytokine analysis showed significant increases in tnfalpha, il- , kc, gm-csf, mcp- , il- beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in ( ) tnfa + gm-csf ( ) gm-csf ( ) il- + gm-csf ( ) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst- assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd a low cd b + cd c + cd + cd + cd low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd + t cell proliferation, il- production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after weeks. the hmscs were cultured and in passage ,the cell surface antigens of both groups were analyzed by flow cytometory.in passage , in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after weeks, alp staining and activity were measured in each group.after weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for weeks and weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal høgh ( ), hm lindegaard ( ), gl sorensen ( ), a høj ( ), c bendixen ( ), p junker ( ), u holmskov ( ) ( cytosolic phospholipase a (cpla ) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla -deficient mice are resistant to cia. thus we aimed to study whether cpla is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla antibodies revealed low positive cpla protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla were detected. in addition, robust cpla protein expression in the skeletal muscles surrounding the joints and strong cpla positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla protein, due to an inflammatory infiltrate and increased cpla expression, suggests an important role of cpla in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b .q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome a disease protective locus (cia ) and another on chromosome a disease-promoting locus (cia ). nod.q allele at cia promotes arthritis whereas cia , has a protective effect in contrast to the b .q allele on chromosome . a promising candidate gene in cia locus is complement factor (c ) as the nod.q allele produced defective c protein. cia locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf , fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf mutated and wildtype mice. we could show that ncf mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf wildtype dc. in addition, ncf mutated dc induced higher levels of il- production by hybridoma t cells. to analyze the role of ncf in dc on arthritis, mice were developed expressing functional ncf restricted to dc (b .qdcn). these mice are characterized for burst, ncf expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf deficient mice. this suggests that ncf plays a role in the uptake and processing of posters inflamm. res., supplement ( ) s antigens, probably by dc. this will be further investigated with the b .qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days and in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day and % incidence was observed on day . mif -/-mice exhibited delayed arthritis, with onset on day and % incidence on day . mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo ( ), a denadai-souza ( ), lm yshii ( ), a schenka ( ), ma barreto ( ), d boletini-santos ( ), c lima ( ), v rioli ( ), mn muscar ( ), e ferro ( ), sk costa ( ) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin ( or ìg/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day . results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day , there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag , we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo ( ), r saldanha-gama ( ), ja moraes ( ), r zingali ( ), c marcienkewicz ( ) ( ) universidade do estado do rio de janeiro, rio de janeiro, brazil ( ) universidade federal do rio de janeiro, rio de janeiro, brazil ( ) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb , a b and/or avb ) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a /a b integrins, vlo , induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo interaction with a b integrin, activating the focal adhesion cascade. vlo effects on the delay of neutrophil is modulated by pi k, erk- mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl- family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a b integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) ( ), h serezani ( ), m peters-golden( ), sonia jancar ( ) '( ) university of s¼o paulo, brazil ( ) university of michigan, usa it is been shown that leukotriene (lt) b and cysteinyl lt (ltc , ltd and lte ) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin - um), pkc-a (ro- - - nm), pi k (ly - um and wortmannin- nm), erk / (pd - um), cpla (aacocf - um), p mapk (sb - um) and ca++ (bapta/am- um), before stimulation with ltb or ltd and addition of igg-opsonized red blood cells for min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb -enhanced phagocytosis is dependent on pkc-a, while ltd effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin- mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin- is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin- as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin- at gene and protein level in monocytic thp- cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin- on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin- and - expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin- and- expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc was incubated with pap ( ng/ml) and/or tnfa ( ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p . results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u macrophages. methods: ha was added to u macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule- (icam- ) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)- b, and il- were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il- b, and il- . in contrast to kda ha, kda ha at mg/ ml inhibited lps-induced cytokine production. anti-icam- antibody blocked the effects of ha on the lps actions on u cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam- antibody reversed the inhibitory effects of ha on phosphorylation of p nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam- through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox- is prostacyclin (pgi ), which is a potent vascular patency factor. induction of endothelial cox- under hypoxic conditions is well documented. this response, along with associated pgi release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox- expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox- promoter region in huvec. cox- induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox- expression than those for nfkb. furthermore, expression of cox- was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi but not vaso-occlusive thromboxane a was seen. thus the putative protective induction of cox- in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th -predominant immune responses remains to be investigated. therefore, the effects of a th selective inhibitor tak- were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak- was administered orally, starting at the time of induction of glomerulonephritis. in group , the drug was administered daily for the initial days. tak- was administered on day only in group , and from day to in group . in each group, nephritic rats were killed on days and . results: in group , glomerular damage, including crescent formation, was improved on day , with reductions in the numbers of cd , cd and ed- positive cells, as well as in urinary protein excretion. protein and transcript levels of th cytokines in the diseased kidneys were markedly decreased by tak- treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group , in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group , partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s inflamm. res., supplement ( ) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd- mice were injected intraperitoneally with mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline ( mg/kg), lisofylline ( mg/kg) or prednisolone ( mg/kg). the tissues were harvested , , , , and h following lps administration and stored at - c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin- beta (il- beta), interleukin- (il- ), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at or h after lps administration, whereas the expression of tnf-alpha and il- beta in lungs and il- in liver reached the peak values at h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately %), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz( ), m losada ( ), c company ( ), c lope-gines( ), l piqueras( ), j cortijo ( ) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx cl (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii microm or with tnfalpha ( ng/ml) for , and h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after h and h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for and h. these effects were caused by the interaction of aii with its at receptor since they were abolished by losartan (at receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for and h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th -type cytokine profiles, i.e. m. globosa induced il- , il- , and il- secretion from the keratinocytes, whereas m. restricta induced il- secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il- and il- , respectively. the m. globosainduced increase in il- secretion was inhibited by mabs against cd and cd . in case of m. restricta, mabs against toll-like receptor (tlr ) and cd suppressed significantly il- secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl ) and tarc (thymus and activation-regulated chemokine/ccl ) are th type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e sub-fraction showed inhibitory activity on the stat protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il- family now consists of members, most of which have assigned functions.there are members of the il- receptor family (including the decoy receptor type ii il- r).many of the il- family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il- family members il- f , f and f signal through a complex of the il- r family member rp in association with il- r acp to activate common inflammatory pathways.the specific activity is is low, on the order of - ug/ml ec .we have found that removal of a few n-terminal amino acids from il- f , f , f and f can increase their bioactivity approximately -fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il- f is capable of antagonizing signaling via il- rrp , but full-length f is inactive. ( ) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr (hvem/tnfrsf ), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf ), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr pathway. c bl/ tr knockout mice and their wild-type control were fed a high-fat diet for weeks and the obesity phenotypes were determined in the obese tr knockout mice and the control. the obese tr mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, mg/kg, i.v.) and controls were sensitized with ova ( ìg) and al(oh) ( mg, s.c.) days after alloxan or saline injection. the animals were challenged days later by the intratracheal instillation of ova ( mg/ . ml). the following analyses were performed h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il- beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils ( %) and mononuclear cells ( %); reduced levels of tnf-alpha ( %) and il- beta ( %), and reduced p-selectin expression ( %) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin ( iu, s.c.), h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il- beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il- and the adhesion molecule icam- by real-time pcr. icam and il- were induced rapidly peaking after h, their mrna levels increased again from h to reach a plateau between h to h after exposure to tnf, whereas tnf mrna levels increased steadily between h and h. h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il- but did not affect that of icam- or tnf h following exposure while calcitriol markedly inhibited the induction of all pro-inflammatory genes h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p by tnf. this action was mimicked by the posters inflamm. res., supplement ( ) s jnk inhibitor sp and the p inhibitor sb .the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p cascades. ( ), cm lotufo ( ), p borelli ( ), zs ferreira ( ), rp markus ( ), shp farsky ( ) ( ) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of s¼o paulo, brazil ( ) department of physiology, bioscience institute,university of s¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru -treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru -treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam- , vcam- and pecam- . participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru -treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. ( ), y naito( ), t okuda ( ), k mizushima ( ), t okayama ( ), i hirata ( ), h tsuboi ( ), t suzuki ( ), o handa ( ) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co( ppm- ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc- mrna/protein and p-p mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc- mrna/protein and p-p mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero ( ), m maróstica ( ), m saad( ), j pedrazzoli jr ( ) ( ) s¼o francisco university medical school, brazil ( ) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass ( . +- . and . +- . % of body weight for colitis and control, respectively; p< . ) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter ( . +- . and . +- . um for colitis and control, respectively; p< . ), higher basal lipolysis ( . +- . and . +- . ug.mg- for colitis and control, respectively; p< . ) and tnf-alpha production ( . +- . and . +- . ng.mg- for colitis and control, respectively; p< . ). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis ( . +- . and . . +- . ug.mg- for colitis and control, respectively; p< . ), increased tnfalpha ( . +- . and . +- . ng.ml- for colitis and control, respectively; p< . ), leptin ( . +- . and . +- . pg.ml- for colitis and control, respectively; p< . ) and adiponectin production ( +- and +- ng.ml- for colitis and control, respectively; p< . ). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il- â, il- , il- , kc, cox- , igg heavy chain, and phospho-stat in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p mapk, the activation of the p mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p mapk and of mk and hsp , two down-stream targets, were not increased in dss treated animals compared to controls. leo , a potent inhibitor of p activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr + cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/- ng/ml lps. for mlr, balb/c tcells were added for days. cells were incubated with sb ( -( -fluorophenyl)- -( -ethylsulfinylphenyl)- -( -pyridyl) h-imidazole, sb) or ml ((rs)-{ -[ -( -fluorophenyl)- -methylsulfanyl- h-imidazol- -yl]pyridine- -yl}-( -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il- production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic spdc . mm, bmdc . mm) and ml (ic spdc . mm, bmdc . mm). preincubation with dcs had no effect, despite reduced lps stimulated il- and tnfa synthesis by sb (ic il- . mm, tnf . mm) and ml (ic il- . mm, tnf . mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p mapk in the mlr is under investigation. p inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. ( ), s luik( ), s laufer( ), m seed ( ), v holan( ), s fiorucci ( ) ( ) synovo gmbh, tübingen, germany ( ) university of tübingen, germany in vivo anti-inflammatory activity of certain p kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically ( d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at mmolkg- d- . we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il- , il- ) and adhesion molecules (e-selectin and icam- ) were measured in patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the , , and day. all patients were divided on two groups: first group compiled patients, in which dexamethasone ( mg/day during - days) was applied in the complex management of acute pancreatitis, and control group - patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il- , il- , il- , icam- , and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th transcription factor gata- can inhibit th /th cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day . at day , t cell specific gata- tg mice did not show any difference in arthritis score compared to wild type mice. however, at day , wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata- tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata- protects against development of severe joint inflammation. facs analysis revealed low levels of il- +/ifn-gammacells in wild type as well as in gata- tg mice at day . as expected, il- positive cells were higher in gata- tg mice compared with wild type mice. interestingly, at day , the percentage of il- +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata- tg mice, suggesting prevention of th expansion under gata- overexpression in vivo. these data revealed that t cell specific overexpression of the th transcription factor gata- protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il- +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata- overexpression in t cells prevents expansion of the il- +/ifn-gamma-t cell subset. pingping jiang ( ), pt sangild( ), t thymann ( ), hh-y ngai ( ), w-h sit ( ), k-l chan ( ), jm-f wan ( ) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis ( d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to days of parenteral feeding followed by day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = ) were compared with corresponding pigs remained healthy (n = ). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase and chain a, medium-chain acyl-coa dehydrogenase with -thiaoctanoyl-coa etc.), inflammation (peptide-binding protein (pbp ) and snail homolog ), signal transduction proteins (thyroid hormone binding protein precursor, park protein and chain b, structure of the rho family gtp-binding protein cdc in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli :b ) by inhalation. the lungs were lavaged hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of mg ( ml/animal) of lps (p.aeruginosa) to c bl/ j mice. control animals received a single dose of sterile ml . % saline/animal. the mice were terminated , , , and h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by %). treatment with daivonex / dovobet also showed an anti psoriatic effect with a % reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res ( ): ) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or % protein diet (c-group) during the first days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o -, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin (k ) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k /vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with -o-tetradecanoylphorbol- -acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone- -valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd -positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone- -valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type diabetes (t d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t d. we aim to detect how gimap affects the lifespan of t cells. results: overexpression in c cells of both wt gimap and gimap with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap causes apoptosis in jurkat cells and primary naïve t cells but not in activated t cells. finally, gimap -mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap causes rapid death of the cells. this suggests that gimap is pro-apoptotic. the results with human wt gimap support this conclusion: recently, much focus has been on the cellular cd / cadpr signaling system during inflammatory processes. the cd /cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il- . to our knowledge, the expression and function of the cd /cadpr signaling system in the human detrusor muscle have not been described. cd protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca + concentration ([ca +] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd which could be upregulated by il- ( ng/ml). in hdsmc briefly activated with il- ( ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca +]i. cyclic adpr-mediated ca + increase was greatly reduced in ca + free medium suggesting ca + entry as well as ca + release. cyclic adpr -elicited ca + increase was mimicked by -deaza-cadpr, and blocked by -bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il- , cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that ) hdsmc express cell surface cd , ) the expression and function of cd are augmented by il- , ) externally added cadpr elicited a rapid, il- -dependent, and -bromo-cadpr-inhibitable ca + mobilization, ) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd /cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for days before induction of arthritis and days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin ( mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration ( mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t ), and compared with the original formula (t ) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t cream (n= ) or no cream (n= ) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment ( . ml ; stirred continuously at c). min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ,) or not in the presence of % t , % t , or no treatment. hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < . ). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t with zinc only was more effective than t (treat:f , = . , p < . ; lps:f , = . , p < . ; treat by lps:f , = . , p < . ). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. ( ) ( ) osprey pharmaceuticals limited, canada ( ) probetex, inc., texas, usa the ccl /ccr chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl chemokine fused to a truncated form of the enzymatically active a domain of shiga toxin has been developed. the ccl portion binds specifically to ccr -bearing leukocytes and enters the cells, where the sa portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day and treated intravenously with vehicle, or mg/kg of the recombinant protein q d from day until day . urine and blood collections were made prior to ats injection and on days and . animals were sacrificed on day . no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of , , , and % for glomerular lesions, m/m count, fibronectin and µ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement ( ) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning ( h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd b expression on resting monocytes harvested from h blister was lower in patients as compared to controls (p= . ). lower expression of cd b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p= . and p= . , respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip- µ was higher among patients (p= . ) and similar levels were seen in blister fluids. concentration of mcp- was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru inhibited no production, and inos protein and mrna expression in murine j macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac selective mc . hdac inhibitors reversed the effects of dexamethasone and ru on inos expression or no production. stably transfected a / cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv -dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd mice ( - g) were given intraplantar injections (i.pl.) of tnfa ( pmol/ microl) and tyrode (as vehicle, contralateral paw; microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and h after injection. indomethacin ( mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle ( % nahco ). another group of mice were injected with tnfa i.pl. plus % nahco with the contralateral paw injected with tyrode plus indomethacin ( mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa ( pmol) leads to significantly reduced (p< . compared to baseline values) paw withdrawal latency in both paws h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin ( mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox- inhibitor, nimesulide. in conclusion, cox- derived prostaglandins are important in the development of hyperalgesia. local cox- inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats ( - g) were administered dexamathasone ( mg/kg, i.p.) either h before or h after lps ( mg/kg, i.p.) injection. animals were killed h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il- b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h s concentration and liver and kidney h s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h s and tissue h s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps ( ng/ml, h, oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone ( nm) significantly (p< . ) reduced expression of both cse and inos. these data provide further evidence that h s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h s production. ( ), u jalonen ( ), h kankaanranta ( ), r tuominen( ), e moilanen ( ) ( ) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland ( ) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup , tis , g s and zfp , is a factor that binds to utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s inflamm. res., supplement ( ) posters concentration of pma for h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro (inhibits pkcb, & and e), gÖ (inhibits pkca, b and &) and cgp (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp did not affect ttp mrna half-life and therefore we measured the effects of cgp on the activation of transcription factors involved in ttp expression. cgp had no effect on the activation of nf-kb, egr or sp . in contrast, cgp reduced the activation of transcription factor ap- , which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap- . the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il- , il- and no) in raw . cells. among them, -oxooctadec- -enoic acid and -oxooctadec- -enoic acid inhibited the production of tnf-a, il- , il- and no at the concentration of mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il- , il- and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen( ), p hjarnaa ( ), j fensholdt ( ), p-p elena( ), k abell ( ), tk petersen ( ) ( ) discovery, leo pharma, ballerup, denmark ( ) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr- . additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd induced il- response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to mg/kg i.p. or mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi kg gene depletion and pi kd kinase-inactive mutation, pik cgd koi, exhibited thymus atrophy, similar to previously reported pi kg and d double knockout (p g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg / igg a ratio and ige level were elevated in pik cgd koi mice corresponding to a skewed th profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi k d or together with pi k g selective inhibitors, while th cytokines were suppressed th cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th reaction but attenuate th medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, h before i.d. injection of . ml pbs containing des-arg -bradykinin (dapk, nmol right hind paw) and . ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) - minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk / . dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp and erk / pd inhibitors. western blot analysis shows that phosphorylation of jnk and erk / is important in the up-regulation of b receptors. our results clearly show that the phosphorylation of both erk / and jnk mapkinases is an important step for the in vivo up-regulation of b receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b receptor require further investigation. continued interest in development of small molecule inhibitors of p mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p leads to increase production of pro-inflammatory cytokines such as tnf-a and il- b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified -( -chlorophenyl)- -(( -methoxyphenoxy)methyl)- [ , , ] triazolo [ , -b] [ , , ] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the -substituted- -amino- -mercapto- h- , , -triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position and . synthesis and sar will be presented. cytokines like il- b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il- b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il- b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p mapk inhibitor sb and a highly active compound developed in our lab. a data set was generated by determining whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il- and tnf a. inhibition of p map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p map kinase. scope of this work was to investigate -thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised -alkylsulfanyl, -( -fluorophenyl), -( -aminopyridin- -yl) substituted imidazoles as p map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the -aminopyridin- -yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the -thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met , for inhibitory activity. less clear is the importance of the hydrogen bond between n of the imidazole ring and lys of p map kinase.to investigate the role of lys in interacting with the scaffold we prepared two sets of , diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys , contrary to the observation on the compound vk- and p map kinase, that a nitrogen atom bearing a lone pair in position of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys rather than to form an attractive interaction with p map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of -substituted and unsubstituted , -diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p . these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw . cells. treatment of c bl/ mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il- b in the serum. the serum level of the anti-inflammatory cytokine il- was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk / and p mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw . cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide mg for days followed by mg daily for weeks. at week all patients started infliximab mg/kg, and received a further four infusions at weeks , , and . results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a , the active metabolite of leflunomide, and adverse events. the mean disease activity score (das ) fell from . at week to . (p< . ) at week and remained between . and . up to week . in those patients remaining on treatment, more than % achieved an acr response from week to week , and up to % achieved an acr response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il- those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of anticollagentype ii mabs on day , followed by i.p. injection of lps on day to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide ( mg/kg/day) starting on the day of first immunization (day ) in prophylactic treatment group and after the onset of arthritis (day ) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il- b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group ( . ae . ) and therapeutic treatment group ( . ae . ) compared to untreated group ( . ae . , p = . and p = . respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p< . ). steady state mrna levels of il- b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p< . ). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine ( ìm) to detect oxygen free radical production. the cells ( , x cell/ml) were then incubated with a range of concentrations of fibrinogen ( - mg/dl) for minutes. our results show that posters inflamm. res., supplement ( ) s fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations ( - mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox- and cox- ) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox- is mainly involved in homeostatic processes, while the inducible isoform (cox- ) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox- and cox- of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox- and anti-cox- effects by prostaglandin e (pge ) measurement after a (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing reference nsaids, selective or not for cox- or cox- . fifty % inhibitory concentration (ic ) values against cox- and cox- and cox- :cox- ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= . ). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox- and cox- . for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day . the inflammatory response was measured after , and hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at / hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp- were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd b, were cd lo, ssclo and rp- + (granulocyte marker). in humans, transmigrated granulocytes expressed cd and cd b. these cells had a significantly higher cd b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated consecutive patients with mean age ae . years who were admitted within h after ischemic stroke. a control group of patients with mean age ae . years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: . ae . and ae . nmol/l. correlation analysis revealed p< . . discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p< . ) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from ffae in the control to ffae at the concentration of ffmg/ml (p< . ). in the ischemic phase, cynodon dactylon ( ffmg/ml) also decreased the incidence of vt from % (control) to %. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p< . for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during min followed by h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox- (hypothalamus) associated to a marked reduction of ca +-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca +-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson ( ), h qui ( ), m wang ( ), i vedin ( ), jz haeggstrçm ( ), j palmblad ( ) ( ( ), r carnuccio( ), p romagnoli ( ), f rossi ( ) ( ) second university of naples, italy ( ) university of naples, italy ( ) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury ( ) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox- , nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone ( mg/kg/ die, by gavage) for days before carotid injury and days after injury. control rats received physiological solution. days after injury cox- expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p< . ). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by % in treated versus control carotids (p< . ). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h o production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h o production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( ( ), b sehnert ( ), h lanig( ), s päßler( ), r holmdahl ( ), h burkhardt ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander university of erlangen-nuremberg, germany ( ) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic that is highly somatically mutated and its epitope on type ii collagen (cii, aa - ). methods: the establishment of a dynamic simulation modelling of a ciic single-chain fragment (scfv) in complex with the triple helical ciic epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c scfvs and the respective ciic epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr and cdr regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c scfv sequence into the respective germline does not affect cii binding affinity (kd x - ) could be confirmed experimentally by the mutagenesis of (!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd + t-cell proliferation and active brain lesions, on the one hand, and il- , il- and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il- production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il- , but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il- responses, and disease activity is associated with mbp-induced proliferation of cd + t cells. ( ), k goula ( ), p georgakopoulos ( ) ( ) renal unit, st. anrdew hospital, patras, greece ( ) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men ( %). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of maintenance haemodialysis patients at our center over the past years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture. patients ( . %) from the study group were diabetic. results: cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli ( cases), enterobacter aerogenes ( case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone ( mg/kg/day). thereafter the amount of steroid was reduced to . mg/kg/day and a low dose of paquinimod ( . mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd +, cd + and on cd -cd -t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at : . mp were separated on -d gels and tested in wb ( : ) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around , , , kda ( total positive/ tested patients). the dominant antigen was at kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m muscarinic receptor was overcome. proteins were separated on d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for h, exposed to uvb and further cultured for another h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase (msk ) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin- â (il- ß) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase- (mmp- ) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp- activity and mmp- expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp- with the ic s of . - . ìm, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp- induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin ( . - . ìm) strongly inhibited mmp- induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap- , whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p mapk activation, and kaempferol inhibited the p mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp- inhibition and mmp- down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. ( ) ( ) university of valencia, spain ( ) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol- -thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-Þb activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta ( mg/ml) produced a % inhibition of oedema and a strong reduction of pge ( %), ltb ( %) and mpo activity ( %) in skin homogenates. the inhibitory effect of avarol at the same dose was % for oedema, % for pge , and % for ltb and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-Þb nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-Þb activation and can be mediated by the downregulation of intracellular signal-transduction ( ), ams silva( ), cmm santos( ), dcga pinto( ), jas cavaleiro( ), jlfc lima ( ) ( ) requimte, departamento de química-física, faculdade de farmµcia da universidade do porto, porto, portugal ( ) departamento de química, universidade de aveiro, aveiro, portugal -styrylchromones are a novel class of chromones, vinylogues of flavones ( -phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of -styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic -styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several -styrylchromones on the proinflammatory enzymes cyclooxygenase (cox- ), cyclooxygenase (cox- ) and -lipoxygenase ( -lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied -styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested -styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of -styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers f-fluorodeoxyglucose (fdg), -o-methyl- - f-fluoro-l-dopa (omfd), and f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp- was compared with that by squamous cell carcinoma (fadu), mamma (mcf- ) and colorectal adenocarcinoma (ht ) cell lines (without or in the presence of specific inhibitiors). several thp- phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp- cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp- cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp- phenotypes and between thp- phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a -end sage ( sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by sage in colon cancer cell lines treated with epigenetically affecting agents, -aza- deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin- (il- ) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il- is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il- contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il- . serum levels of il- are elevated in patients with colorectal cancer, however serum levels of il- were found to be independent of il- mrna expression in tumor tissue. in this study we analyzed il- mrna expression by real-time pcr in sporadic colon cancer tissue as well as corresponding normal mucous tissue. il- mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p= , ). there was no correlation betweenil- mrna expression and tumor grade or stage. thus we can conclude that il- produced at the tumor site is not involved in sporadic colon cancer progression. ( ), t aiamsa-ard ( ), v chinswangwatanakul ( ), ki techatraisak ( ), s chotewuttakorn ( ), a thaworn ( ) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with % of serum and incubated for hours. cells were analyzed by using [ h] thymidine and immunoblotting assay for cell proliferation and cox- /nos- protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox- but not nos- in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox- . conclusions: this result suggested that some factors in serum of cancer patients could induce cox- protein expression in huvec. the increasing of cox- might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok- @on neutrophil-derived superoxide production and tumor growth. methods: ah a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok- , a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok- on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok- . ( ), m jokic ( ), v zjacic-rotkvic( ), s kapitanovic ( ) ( ) university hospital sestre milosrdnice, bucharest, romania ( ) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il- is a pleiotropic cytokine mapped to chromosome p - . its promoter snp - g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from patients diagnosed with gep-net and age and sex-matched volunteers were analyzed for - g/c snp of the il- gene. to analyze il - c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis Ä test and fishers exact test were used. the level of significance was . . results: there were no differences observed in the frequencies of the - high expression (gc and gg) genotypes between the patients and healthy volunteers (p= . ), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p= . ). - g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p= . ). conclusions: high expression genotypes of il- - snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type receptor (nts ). here we show that hmc- human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc- cell rna yielded a band with % sequence identity to pront and a band corresponding to the pront processing enzyme, pc a.immunocytochemistry on hmc- cells showed specific staining for pront. stimulation of hmc- cells with a + pma, pge , c / or mastoparan released immunoreactive nt.rt-pcr on hmc- cell rna yielded a band with % sequence identity to human nts . western blotting gave bands corresponding to unglycosylated ( kda) and glycosylated ( kda) nts .immunocytotochemistry on hmc- cells showed specific staining for nts . these finding have significance for the role of mast cells in tumor growth. ( ), j buddenkotte ( ), mp schçn( ), m steinhoff ( ) ( ) university hospital münster, germany ( ) university hospital würzburg, germany the proteinase-activated receptor par- has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par- in cutaneous cancerogenesis is still unknown. here we could show a protective role of par- in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par- -deficient and wild-type mice were painted once with dmba ( , -dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate ( -o-tetradecanoylphorbol- -acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after weeks, par- -deficient mice showed a significantly increased number of skin tumors ( per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par- stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase / (erk / ) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta (tgf-â ). thus, our results provide the first experimental evidence for a tumor-protective role of par- . ( ), ma arbós ( ), a fraga ( ), i de torres( ), j reventós ( ), j morote ( ) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf /cxcr and mcp /ccr chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf /cxcr and mcp /ccr expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b f (sc). paf-r antagonist, web ( mg/kg, ip) was given daily for days. we found that eat growth was significantly reduced by pretreatment with web , and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web pretreatment. eat growth was accompanied by increased production of prostaglandin e , vegf and no which was reduced significantly by web treatment. in b f melanoma, web , alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web- reducedactive caspase- expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. ( ), mt quiles ( ), a figueras( ), r mangues( ), f vinals( ), jr germa( ), g capella ( ) ( ) institut de recerca vall de hebron, barcelona, spain ( ) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl (sdf- )/ cxcr chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys and asp nih t transfectants. cys tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with µ-smooth muscle actin positives cells surrounded by f / macrophages were only observed in asp tumors associated with a shorter growth period. asp tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp- protein levels were similarly diminished in both transfectants. higher mmp and mmp activities and expression were observed in asp tumors probably produced by macrophages or stromal cells. total and active mmp levels were higher in cys tumors. the expression of sdf- and cxcr remained unchanged while sdf- g isoform was selectively induced in cys tumors, suggesting sdf- a or b are induced in asp tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf- expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase- and substrates; tnf-alpha; il- ). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher ( -fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ and gencat_agaur_ xt_ ). ( ), m spinola( ), c pignatiello( ), w cabrera ( ), og ribeiro ( ), n starobinas( ), t dragani ( ) ( ) butantan institute, sao paulo, brazil ( ) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c bl/ and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing , mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin (vnn ) and major histocompatibility antigen e alpha (h -ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome ( . cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr- and - , pdgfr, kit, csf- r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability ( %) has translated into superior in vivo anti- inflamm. res., supplement ( ) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk . thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a and du- ) or low passage patient derived tumors (cxf ; colon cancer, rxf ; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. ( ), p bobrowski( ), m shukla ( ), t haqqi ( ) ( ) albany medical college, usa ( ) rainforest nutritionals, inc, usa ( ) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol # . proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il- b ( ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado ( - mg/ml) was tested for its ability to maintain optimal igf- transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity ( mg/kg po) leading to gsh safety rating of /unclassifiable. oligomeric proanthocyanidin content was high ( mg/kg) with the majority of oligomers > mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il- b induced glycosaminoglycan release from human cartilage explants. progrado prevented il- b induced suppression of igf- production from human cartilage explants as well as stimulating basal igf- production (p< . ). comparable changes in igf- gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf- . this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h o -induced cytotoxicity and oxidative dna damage in the nih/ t fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of , -diphenyl- picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h o -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h o , without cytotoxicity except for the hexane extract at high concentrations (> mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk project from korea research foundation). sirt is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt activity, comparing heart (h) and adipose (a) tissue of sedentary young (n ), sedentary old (n ) and trained old (n ) rats. the trained old rats performed a -weeks moderate training on treadmill. on h and a tissue of all rats sirt activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts -hydroxynonenal ( -hne), mnsod, catalase and foxo a by western blot, and gadd a, cyclin d and foxo a mrna by rt-pcr. aging reduced sirt activity in h (p< . ) without effects in a, producing an increase of mda (h, p< . ; a, p< . ) and -hne (h, p< . ; a, p< . ), and a decrease of mn-sod (p< . ) and catalase (p< . ) expression in both h and a. aging did not affect foxo a protein expression in h, and foxo a mrna in a. exercise produced an increase in h foxo a protein expression (p< . ) and in a foxo a mrna, associated to higher mn-sod (h, p< . ; a, p< . ) and catalase (h, p< . ; a, p= . ) levels in both h and a of aged rats. in heart exercise-induced higher sirt activity bring on decrease in cyclin d and increase in gadd a mrna expression. in a we found a similar decrease in cyclin d , without changes in gadd a mrna expression. these findings suggest that exercise is able to increase sirt activity in aged rats. ( ), t horiguchi( ), k abe( ), h inoue( ), t noma ( ) ( ) institute of health biosciences, the university of tokushima graduate school, tokushima, japan ( ) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl -induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with f×l/ kg bw of ccl w/wo mg/ kg bw of gl. after hours and hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between hour-treated-livers (ccl or ccl and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at hours compared to that in ccl treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a , hsp , and procollagen were still very high in gl-treated liver. after hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl -treated liver. then, we screened , genes by microarray and found that genes were up-regulated and genes were down regulated in ccl +gl compared to ccl treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn ). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp- monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp- cells. we also demonstrate that such modulation involves both erk / and p mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. ( ), b arnold( ), g opdenakker ( ) ( ) jagiellonian university, department of evolutionary immunobiology, krakow, poland ( ) german cancer research center, department of molecular immunology, heidelberg, germany ( ) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase (mmp- -/-) at least one compensatory mechanism operates as there are elevated levels of pge of cox- origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp- -/-mice as at hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp- -/-mice. for this numbers of apoptotic (annexin v) and necrotic ( -aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase were significantly lowered in mmp- -/-mice while levels of caspase , and were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp- -/mice during zymosan peritonitis and it is due to the decreased levels of active caspase . the increased activity of other examined caspases is most probably independent of apoptosis. ( ), h james ( ) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), and mg/kg produced a dose-dependent inhibition of paw edema ( % and % at h; % and % at h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), mg/kg( % at h; % at h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of mg/ kg( % at h; % at h) but not with a dose of mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin mg and mg/kg ( % and % at h; % and % at h respectively) and selective cox- inhibitor rofecoxib, mg/kg ( % and % respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. ( ), p hart( ), j edwards ( ), c quirk ( ) ( ) molecular pharmacology limited (usa), australian division, perth, western australia ( ) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il- â production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen ( %) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at min, or at hrs after injury. a third injury site was not treated. photographs were taken before, hrs, and weeks after injury. cytokines: human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of % or % active ingredient. hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at hrs after oxsoralen-uv, the min treatment site showed slight erythema, the hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. weeks after injury, the min site was normal, the hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il â secretion by . % and . %, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. ( ), p hart( ), j snowden ( ), maud eijkenboom ( ) ( ) molecular pharmacology limited (usa), australian division,perth, western australia ( ) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of %, . %, %, . %, %, % or % bov-zn (two pooled experiments). hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for %, %, %, % fcs against %, . %, %, % bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter ) in treated and untreated cell cultures over - hrs at intervals. the ic for tnf-a suppression was reached at . % bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by % bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-£\ and il- fÒ. these cytokines are known to be regulated by the stress-activated p fnfnmap kinase pathway. because of this, inhibition of p map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last years, numerous groups have reported on the development of p map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p vs other kinases, enabling the development of p selective molecules. inflamm. res., supplement ( ) posters anti-inflammatory activities. a series of labdane-type diterpenoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, and were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of and % were noted in the -o-tetradecanoyl-phorbol- -acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e , and tumour necrosis factor-alpha in bacterial endotoxin-activated raw . macrophage cells with ic in the range - mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase- , as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbß, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of mg/kg and substance ffw- Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect ( %) at a dosage of mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first min and hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw- Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th -response with increased levels of proinflammatory cytokines like tnfa and il , and mouse models of crohns disease show that the balance of il / is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il , il and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d , which were able to suppress the cocktail induced il secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p - inflammation and human incisional hernia pathophysiology maria antonia arbos via( ) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney ( ae vs. ae ae ; p< . ), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of s¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge mimetic combination therapy (dp- ) that inhibits basal and lps/tlr induced tnf-?, il- ß, and mmp- , , , in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp dramatically inhibited il- ß, tnf-?, mip , mcp- and il- expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il- ß, mmp- and to a lesser extent mmp- expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data ( , anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb from the -lipoxygenase pathway, pge through the inducible cyclooxygenase (cox- ) pathway and cytokines/chemokines as tnf-alpha, il- beta, il- , mip- alpha, mip- beta, mip- alpha and mip- alpha. engagement of the adenosine a a receptor (a ar) blocks the in vitro synthesis of ltb while it potentiates the cox- pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip- alpha, mip- beta, mip- alpha and mip- alpha in toll-like receptor- -stimulated human neutrophils. little effect was observed on il- beta and il- . using the murine air pouch model of inflammation with a ar-knockout mice, we observed that the activation of a ar positively impacts the expression of cox- in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a a receptor, neutrophils that migrated into the air pouch h following lps injection expressed higher mrna levels of tnf-alpha, mip- alpha and mip- beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a ar-knockout mice and in view of the potent inhibitory actions of pge on inflammatory cells, an increased cox- expression and a prevented release of tnf-alpha, mip- alpha and mip- beta caused by a ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis ( d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw . after challenged with lps (escherichia coli :b ) for , and hours. we identified proteins from approximately detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein , capg protein, poly(rc) binding protein , isocitrate dehydrogenase (nad+) alpha, lactate dehydrogenase , a chain, guanine nucleotide binding protein (g protein), beta polypeptide like , triosephosphate isomerase and proteasome alpha subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p , malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified -amino- -benzyl- -( -bromophenyl)- -methyl- , -dihydropyrano[ , c] pyrazole- -c arbonitrile as a potential novel lead structure as p map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol- ( h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha -acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from . to . ìg/ml in male, and from . to . ìg/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at . - . ìg/ml or days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from to ìg/ml in male, and from to ìg/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at - , ìg/ml. despite a high value of , - , ìg/ml being observed for serum aag levels in pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid ( ìg/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi- , a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi- .the ability of pbi to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi- treated animals, n= ) were used to assess leukocyte infiltration and to measure by elisa tnf-µ, mcp- and pge production.in vitro, pbi- is able to significantly decrease by . % ae . % (p< . ), tnf-a production by human lpsactivated pmn.in vivo, pbi- significantly decreased the production of tnf-a ( . % ae . %; p< . ), mcp- ( . % ae . %; p< . ) and pge ( . % ae . %; p< . ) induced by lps injection.however, pbi- did not significantly inhibit leukocyte infiltration.these results show that pbi- is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. ( ), lj lowenstine,( ), aj norris( ), t spangler( ), lm woods ( ) ( ) zoological society of san diego, usa ( ) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n= each) were selected for parasitology and em (group ), and gross and histopathology (group ). all outbreak cases and group controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in % ( / ) of outbreak cases and in % ( / ) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in archived crows submitted to the vmth from - , suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk / by mek / is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as , a selective mek inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for days (twice daily), by oral route at the doses of , and mg/kg. as at and mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o -.), hydrogen peroxide (h o ), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. ( ), h spalteholz ( ), u reibetanz ( ), p salavei ( ), m fischlechner ( ), h-j glander( ), j arnhold ( ) ( ) university of leipzig, medical faculty, institute for medical physics and biophysics, germany ( ) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects - % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h o ) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor- (par ) in inflammation and innate immunity. par is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par . par is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation ( hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. ( ), k bendtzen ( ), f sellebjerg ( ), ch nielsen ( ) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from ms patients and healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd + monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il- , tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione( ), g tarantino( ), a spanò( ), p ladogana ( ), fl perrone( ), s padula( ), a riccio ( ) ( ) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy ( ) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: patients with arthritis were selected in a population of subjects affected by hcv infection. each patients was evaluated by clinical examination ( denoted poliarticular and mono-oligoarticualr involvement), by x-graphic aspects of joint involvement ( patients presented join erosions), by ana, rf and anti-ccp positiveness.results: , % of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in out of the patients with joint erosions, and only in out of the patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg d on cd + t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type diabetes. the monoclonal antibody cx recognizes murine nkg d and has been shown to block ligandbinding and mediate internalization of nkg d. furthermore, cx can inhibit and/or ameliorate disease in animal models of type diabetes and inflammatory bowel disease. thus, it is very likely that nkg d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb .scid mice. different doses of cx antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx elisa in serum) and flow cytometry (down-regulation of nkg d on cd b+ nk cells) for up to two weeks after administration. we found that cx very efficiently down-regulate nkg d on cd b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx antibody in vivo. ( ), k mehta( ), n deo( ), j chaudhary( ), p bobrowski ( ) ( ) albany medical college, usa ( ) vedic lifesciences, usa ( ) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n= ) were screened and randomized to receive glucosamine sulfate (n= , mg/day) or reparagen (n= , mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a % improvement in womac pain, with assessments at , , , and weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p< . ), with a similar, progressive improvement over the course of the week treatment protocol ( - % reduction in total womac or vas scores). the response rate was substantial for both glucosamine ( %) and reparagen ( %), which exceeded placebo responses ( %, p < . ) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p < . ), and serum igf- levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin- (il- ) and monocyte chemoattracting protein- , two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by % and %, respectively. corresponding reduction in ibuprofen group was % and %. protective mechanisms involved were assessed in human cultured u macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha ( % and % reduction by supplementation with nm and ibuprofen, respectively) and il- ( % and % in corresponding reduction). nm supplementation dramatically reduced prostaglandin e secretion by stimulated macrophages along with cyclooxigenase- (cox ) cellular protein expression. mrna levels forcox and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. ( ), hp kim ( ), kh son ( ) ( ) college of pharmacy, kangwon national university, south korea ( ) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw . cells. among the tested compounds, -methoxy- , -dichlorochalcone (ch ), -hydroxy- -methoxychalcone (ch ), -hydroxy- -bromo- -methoxychalcone (ch ) and -hydroxy- , -dimethoxychalcone (ch ) potently inhibited no production (ic s, . - . mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position ( or ) to carbonyl moiety with/without -(or -)hydroxyl group and -halogen substitution in b-ring. when the cellular action mechanisms of ch , ch and ch were further examined, it was revealed that ch and ch clearly down-regulated inos expression while ch did not. moreover, ch and ch were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)- activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox- expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox- activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, cyclohexylimino- -methyl- , -dihydro- h-benzo [ , ] oxathiol- -one (lyr- ) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw . . the lyr- reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr- could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr- inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys- to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser- and to glu). therefore, lyr- could inhibit lps-induced nf-kb activating pathway by targeting ser- and/or residues on the activation domain of ikkbeta. as pharmacological actions, lyr- prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox- , or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw . . furthermore, lyr- protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline ( mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, nm, , and mm, increased guinea pig plasma dao activity by , , and %, respectively, while sertraline increased it at mm (by %). at higher concentrations ( and mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at and nm amitriptyline the activity of hnmt increased by and %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider( , ), c macarthur ( ), d trune ( ), s mccoy ( ) ( ) oregon health and science university, portland, oregon, usa ( ) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p , for treatment of inflammation induced by either lps or bacteria.peptide p was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p inhibited lps-induced production of serum inflammatory mediators.moreover, p treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p .simultaneous injection of bacteria and peptide p resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p treatment.-subcutaneous and oral administrations of p , but not intravenous administration, were also efficacious in reducing inflammation. administration of p after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. ( ), c zhou( ), y zhang( ), m sun( ), x wan ( ), h yu( ), x yang( ), rd ye ( ), j-k shen ( ) formyl peptide receptor-like (fprl ) is a structural homologue of fpr, which binds chemotactic peptides of as small as amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl ligands include peptides of - amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl -transfected cell line. although substitution of d-met resulted in loss of activity, removal of val together with d-met produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl ligand. further substitution of lys with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl through aromatic interactions involving the side chains of trp and tyr , hydrophobic interaction of nle , and the thio-based hydrogen bonding of met , with the respective residues in fprl which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl -related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il- , tnf-a and il- levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects ( , and mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses ( . and mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin ( and mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin . mg/ mouse/day in combination with glycine or mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. ( ), p sambrook( ), k fukudome( ), m xue ( ) ( ) university of sydney, st leonards, nsw, australia ( ) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr . cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein- -induced chemotaxis of monocytes by approximately %. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than % in ra monocytes. the inhibitory effects of apc were reversed by rcr , indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord- -wh hes authors: mocchegiani, eugenio; malavolta, marco title: role of zinc and selenium in oxidative stress and immunosenescence: implications for healthy ageing and longevity date: - - journal: handbook on immunosenescence doi: . / - - - - _ sha: doc_id: cord_uid: wh hes ageing is an inevitable biological process with gradual and spontaneous biochemical and physiological changes and increased susceptibility to diseases. some nutritional factors (zinc and selenium) may remodel these changes leading to a possible escaping of diseases with subsequent healthy ageing, because they are especially involved in improving immune functions as well as antioxidant defense. experiments performed “in vitro” (human lymphocytes exposed to endotoxins) and “in vivo” (old mice or young mice fed with low zinc dietary intake) show that zinc is important for immune response both innate and adoptive. selenium provokes zinc release by metallothioneins (mt), via reduction of glutathione peroxidase. this fact is crucial in ageing because high mt may be unable to release zinc with subsequent low intracellular free zinc ion availability for immune response. taking into account the existence of zinc transporters (znt and zip family) for cellular zinc efflux and influx, respectively, the association between znt and mt is important in maintaining satisfactory intracellular zinc homeostasis in ageing. improved immune performance occur in elderly after physiological zinc supplementation, which also induces prolonged survival in old, nude and neonatal thymectomized mice. the association “zinc plus selenium” improves humoral immunity in old subjects after influenza vaccination. therefore, zinc and selenium are relevant for immunosenescence in order to achieve healthy ageing and longevity. ageing is an inevitable biological process that is accompanied with gradual and spontaneous biochemical and physiological changes including increased susceptibility to diseases, adverse environmental conditions and loss of mobility and agility. alterations in the immune functions play a fundamental role in ageing. the inability of an organism in remodeling these immune changes may lead to the appearance of some degenerative age-related diseases. as a result, the "remodeling theory of ageing" has been proposed (paolisso et al . ) . various nutritional factors are directly linked with these phenomena as for instance in restoring the immune functions as well as in the capacity to respond to oxidative stress (meydani ) , which is in turn the main cause of the immune derangement in elderly (pawelec ) . approximately, micronutrients (vitamins, essential minerals and other compounds required in small amount for normal metabolism) have been reported as essential components in the diet (shenkin ) . the dietary intake of essential macro and micronutrients is usually inadequate in the elderly (ames ) . several causes contribute to this gap. first of all, the poor socio-economic condition present in a large part of old people may lead to a consumption of inexpensive foods deficient in micronutrients, such as carbohydrates (kant ) . the gap is worsened by loss of appetite, lack of teeth, intestinal malabsorption and decreased requirement of energy that lead to the final result of frailty, disability and mortality (semba et al. ) . some authors have reported that the deficiency of macro and micronutrients in ageing is strictly related to global impairments of the immune functions with subsequent limited defense against external noxae and appearance of age-related diseases (lesourd ) . by contrast, recent longitudinal studies in dietary daily intake in human nonagenarian/centenarians (successful ageing) have shown that an adequate consumption of micro and macronutrients as well as a satisfactory content of some trace elements in the cells lead to good performances in several immune functions, especially in innate immune performances (chernoff ; mocchegiani et al. ) . therefore, nutritional factors may play a pivotal role for immunosenescence in order to reach healthy ageing and longevity. we herein review the role of zinc and selenium, taking into account the pivotal role played by these two micronutrients in the efficiency of the immune functions (buttriss ) . recent epidemiological and clinical evidence have shown that in most developing countries deficiencies of these micronutrients are partly responsible for the severity of infectious disease, morbidity and mortality in malnourished children (bhaskaram ) as well as in ageing (meydani ) . indeed, these two trace elements form an important pillar in the nutrition of young and elderly persons because also involved in tissue integrity (enwonwu and sanders ) . thus, it is evident that a deficiency of these elements could lead to a crucial impairment of the organ and tissue function with subsequent influence on many body homeostatic mechanisms, including the immune functions. zinc is one of the most important trace elements in the body, although its presence in nature does not exceed . % (mills ) . the major characteristics of zinc include a highly concentrated charge, a small radius ( . a), no variable valence [low risk of free radical production], ready passage from one symmetry in its surroundings to another without exchange, rapid exchange of ligands (on and off reactions), and binding mostly to s-and n-donors in biological systems. these properties enable zinc to play a major biological role as a catalyst. removal of the catalytic zinc results in an active apoenzyme that usually retains the native tertiary structure (vallee and falchuk ) . thus, it is not surprising that zinc is essential for the activity of more than enzymes influencing the activity of zinc dependent antioxidant enzymes, such as superoxide dismutase (sod) and various organ functions having a secondary effect on the immune system (rink and gabriel ) . zinc also regulates the balance between the gene expression of metalloproteinases (mmps) and the tissue inhibitors of matrix metalloproteinases (timps; nagase and woessner ) . the main function of mmps is the removal of extracellular matrix (ecm) during tissue resorption and progression of many diseases. however, it is notable that mmps also alter biological function of ecm macromolecules by specific proteolysis (shapiro ) . therefore, since mmps are induced especially by proinflammatory cytokines (il- and tnf-alpha), an overexpression of mmps may lead to excessive proteolysis of ecm, as it occurs in chronic inflammation (gueders et al. ) . as a consequence, degradation of ecm and limited cell-cell adhesion may occur, so "trapping" bioactive mediators (moot and werb ) . thus, the expression of mmps genes are under the control of some inhibitors of mmps, such as timps gene products, α- macroglobulin (α- m) and -amyloid precursor protein (nagase and woessner ) . as a result, a balance in the expression of the metalloproteinases [either as activators (mmps) or as inhibitors (timps)] is necessary for an optimal function of many biological systems. examples of altering the balance between mmps and timps or α- m have been recorded in certain types of cancer, infections and ageing (nagase and woessner ) that are conditions characterized by zinc deficiency (fabris and mocchegiani ) . zinc also regulates g /g phase of cell cycle through cyclins/cdk complexes in a dose dependent manner. specifically, high doses of zinc ( μm) result in cell cycle arrest (paramanantham et al. ) , whereas low doses of zinc ( μm) inhibit apoptosis (fraker ) . zinc is present in "zinc finger domains" of many proteins, peptides, enzymes, hormones, transcriptional factors and cytokines, which act in maintaining body homeostasis (coleman ; berg and shi ) . zinc also regulates mrna stability (taylor and blackshear ) and extracellular matrix (vallee and falchuk ) . moreover, zinc binds enzymes, proteins and peptides with different binding affinity (kd) ranging from - to - mol/l ( see review mocchegiani et al. ). these compounds display low biological activity when the zinc-binding doesn't occur, as for instance for thymic hormone named thymulin, which loses its activity in absence of zinc (fabris et al. ) . finally, zinc plays a critical role in structure, function, stabilization and fluidity of biomembrane due to its binding to sulphydryl groups forming mercaptides (vallee and falchuk ) . zinc also maintains the enzymatic activity of inducible nitric-oxide synthase (inos; bodgan et al. ) , with a binding between zinc and two cysteine residues, which are part of the structures of the heme domain of inos (li et al. ) . as: (i) nitric oxide (no), via no synthases, affects the gene expression of metallothioneins (mt) in order to protect the host from oxidative stress (arizono et al. ) and (ii) no is involved in zinc release from mt, via s-nitrosylation (zangger et al. ) , the structural task of zinc in no production is crucial. in this context, the release of zinc by mt, via s-nytrosilation, contributing to raise the intracellular free zinc ions concentration, plays a crucial role in modulating the production of proinflammatory cytokines and in the activation of immune cells (rink and haase ) . therefore, the interrelationships between zinc and mt is crucial in maintaining the immune response especially in ageing where the production of proinflammatory cytokines is chronic leading to a constant presence of inflammatory status coupled with low intracellular zinc ion bioavailability (mocchegiani et al. ). the interrelationship between zinc and mt is also regulated by the special proteins named zinc transporters (znt), which in turn appear to be also specifically involved through regulation of cellular zinc homeostasis via influx, efflux, or vesicular sequestration (cousins and mcmahon ; eide ). the znt, some of which are tissue specific, maintain intracellular zinc concentration in a narrow physiological range in order to avoid cellular zinc toxicity or deficiency when dietary zinc intakes fluctuate. two families of znt have been identified. the znt family decreases cytoplasmic zinc concentrations by secretion, sequestration, or efflux, whereas the zip family increases cytoplasmic zinc influx or release of stored zinc (eide ) . therefore, the balance of znt is fundamental to maintain an optimal intracellular zinc homeostasis in ageing, because reduced zinc intake by the diet or intestinal zinc malabsorption or loss of zinc through urine by high levels of proinflammatory cytokines are usual events in elderly (prasad et al. b ). mt, are a group of low-molecular-weight metal-binding proteins who have high affinity for zinc (kd = . × - m; kagi and schaffer ) . mt exist in different isoforms characterized by the length of aminoacid chain: isoform i, ii, iii e iv mapped on chro-mosome in man and on chromosome in mice with complex polymorphisms (west et al. ). the more common isoforms are i and ii; the isoform iii, also called growth inhibitory factor (gif), is a brain-specific member of the mt family and the isoform iv is restricted in squamous epithelia. mt contain cysteines, all in reduced form, and bind seven zinc atoms through mercaptide bonds that have the spectroscopy characteristics of metal thiolate clusters (maret and vallee ) . the zinc/cysteine clusters are of two different types. in the beta-domain cluster, three bridging and six terminal cysteine thiolates provide a coordination environment that is identical for each of the three zinc atoms. in the alpha-domain clusters, there are two different zinc sites; two of them have one terminal ligand and three bridging ligands respectively, while the other two have two terminal and two bridging ligands (maret and vallee ) . following these biochemical characteristics, mt distribute intracellular zinc as zinc undergoes rapid inter-and intracluster exchange (kagi and schaffer ) . moreover, mt act as antioxidant since zinc-sulfur cluster is sensitive to changes of cellular redox state and oxidizing sites in mt (reduced thiol groups) induce the transfer of zinc from its mt binding sites to those of lower affinity in other proteins (kagi and schaffer ) . this transfer confers biological activity to antioxidant metalloenzymes. therefore, the redox properties of mt and their effect on zinc in the clusters are crucial for the protective role of mt in presence of ionizing and uv radiations (cai et al. ) , heavy metals (mercury, cadmium), lipid peroxidation, reactive oxygen species, oxidative stress caused by anticancer drugs, and conditions of hyperoxia (sato and kondoh ) . this protective role of mt has been studied especially in young-adult mt knockout mice (null mice) for short periods of exposure to toxic metals, such as cadmium for weeks (habeebu et al. ) or mercury (one single injection and the effect of mercury analyzed days after the injection; satoh et al . ) , or to anticancer agents for - hrs. (kondo et al. ) or in presence of an excess of zinc or zinc deficiency for weeks (kelly et al. ) . therefore, the protective role of mt is evident in transient stress condition, as it may occur in young adult-age, in which the chronic status (by stress or inflammation) is a rare event (mocchegiani et al. ) . in contrast, this role may be questionable in ageing because the stress-like condition and inflammation by high levels of il- are chronic (ashok and ali ) , with also a different response to stress with respect to young (degroot et al. ). since il- affects the gene expression of mt (hernandez et al. ) , these proteins may turn off from protective to harmful agents in ageing following the "antagonistic pleiotropy theory of ageing" (williams and day ) . in fact, despite mt increase in ageing, a limited release of zinc by mt leading to an impaired immune and antioxidant response has been proposed (mocchegiani et al. a, b) . in contrast, in presence of lower stress and inflammation, as it occurs in centenarians, mt production is low coupled with satisfactory zinc ion bioavailability (mocchegiani et al. a) . indeed, since il- acts on the cells through its subunit receptor gp (bravo and heath ) , the relative lower gene expression of gp with respect to elderly found in centenarians (moroni et al. ) may imply that a quota of il- is inactive in centenarians leading to low gene expression of mt, satisfactory free zinc ion availability and low degree of inflammation (mocchegiani et al. a) . as a result, the satisfactory immune performances and antioxidant activi-ties lead to a good healthy status in these exceptional individuals (mecocci et al. ; mocchegiani et al. a) . therefore, the interrelationships among inflammatory status, mt and zinc are pivotal in order to achieve successful ageing, furtherly suggesting a different role of mt in ageing that is crucial for immune response (mocchegiani et al. a ). whether mt might play an antagonistic pleiotropic role remains however to be clearly demonstrated also taking into account that they may play different role in different organs. on this aspect, recent findings in cardiac-specific metallothionein transgenic mice suggest that the expression of these proteins in cardiocytes may alleviate aging-induced cardiac contractile defects and oxidative stress prolonging life span (yang et al. ). in addition, daf- mutant nematodes other than a longevity phenotype, display an altered expression of mt which, in turn, seems to interact with the insulin signaling pathway (barsyte et al. ) . therefore, even if the specific function of mt in ageing is still a matter of discussion, all these reports associated to recent findings on the possible role played by mt in modulating cellular respiration and energy metabolism (feng et al. ; ye et al. ) strongly suggest that these proteins are involved in the maintenance of health status and in successful aging. on the other hand, recent findings show a novel polymorphisms of mt a (a/c at position + leading to an asparagine/threonine aminoacid substitution) involved in successful ageing, lower inflammation and satisfactory intracellular zinc ion bioavailability (cipriano et al. ). with regard to the role played by the znt in ageing and immunosenescence, a paucity of data exists in literature. after an increase from the birth up to adult age in some tissues, pancreas (clifford and macdonald ) or brain (nitzan et al . ) , significant decrements of both znt and zip families in peripheral leukocytes from elderly women occur, in particular the subtypes znt and zip (andree et al. ) . taking into account that zip family increases cytoplasmic zinc influx (eide ) , an intriguing point is that zip expression is up-regulated through il- , and that this zinc transporter most likely plays a major role in the mechanism responsible for an excess of intracellular zinc and, at the same time, for hypozincemia that accompanies the acute-phase response to inflammation and infection (liuzzi et al. ). since chronic inflammation by high il- , hypozincemia and risk of infections are usual events in old age (mocchegiani et al. ) , the possible alterations of the znt in ageing coupled with the inability of high zinc-bound mt in zinc release, may thus allow still more synergistic deleterious effects on immune response that it may be due or to low or excess of zinc within the cells. this last assumption is supported by the discovery that both low and high levels of intracellular zinc lead to cell death (fraker ) . therefore, the intracellular zinc ion availability should be maintained within a strict range in order to exert beneficial effect, otherwise it may trigger pathological pathway cascades possibly contributing to the onset and progression of degenerative diseases (mocchegiani et al. ). for a prompt immune response against stressor agents and inflammation, macrophages produce some cytokines, such as il- , il- , ifn-α, tnf-α, which, in turn, provoke a new synthesis of mt in the liver but, at the same time, an alteration in the zinc status (bui et al. ). these findings clearly suggest the existence of interplay between mt and the immune system. il- affects mt mrna in thymic epithelial cells (tecs) by means of pkc, which is, in turn, zinc-dependent (coto et al. ) and participates in metal-induced mtmrna (yu et al. ) . moreover, mt are donors of zinc for thymulin reactivation in tecs (coto et al. ). mt act both as a reservoir of zinc during zinc deficiency and as a zinc buffering protein in presence of excessive amount of zinc in order to prevent zinc toxicity (kelly et al. ) . following these findings, mt are, out of doubt, protective agents with also the task in preventing zinc deficiency during an inflammatory status. it has been recently reported that, under inflammatory conditions, mt in the extracellular environment may support the beneficial movement of leukocytes to the site of inflammation representing a "danger signal" for the immune cells and modifying the character of the immune response when cells sense cellular stress. however, high mt produced in chronic inflammation, may alter the normal chemotactic responses that regulate leukocyte trafficking (yin et al . ) . taking into account that zinc ions attract leukocytes by inducing and promoting the chemotactic response (hujanen et al. ) , high mt production might be dangerous for immune response in presence of chronic inflammation. moreover, (i) the existence of high mt and low zinc ion bioavailability in the atrophic thymus from old mice (mocchegiani et al. ); (ii) the presence of high mt in lymphocytes from old people and down's syndrome subjects (syndrome of accelerated ageing) coupled with impaired innate immunity (mocchegiani et al. a ) and (iii) the occurrence of atrophic thymus in young stressed mice overexpressing mt (mocchegiani et al. b) , furtherly suggest this dangerous role played by mt in immune function during ageing. additionally, elevated levels of extracellular mt, as it can be found especially in chronic inflammatory sites, can cause a dramatic decreases in cytotoxic t lymphocyte (ctl) activity against allogeneic target cells, reduces the proliferative response of ctll- cells to cytokines, and decreases the level of major histocompatibility complex (mhc) class i and cd molecules detectable on the surface of lymphocytes (youn and lynes ) . therefore, high mt may also have an immunosuppressive effect worsened by the fact they are not donors of zinc in ageing but rather sequester zinc. on the other hand, high mt induce down-regulation of many other biological functions related to zinc, such as metabolism, gene expression and signal transduction (kagi and schaffer ). an unbalance between mt isoforms leads to impairments of zinc-dependent body homeostatic mechanisms within the brain, as reported in samp mice (model of accelerated ageing; wen et al. ) . moreover, high mt are an index of unfavorable prognosis in cancer (ebadi and swanson ) . however, the limited capability of mt in zinc release is still unresolved problem in ageing, especially regarding to the precise mechanism involved. the zinc release from mt under oxidative stress conditions is accompanied by more mt disulfide bond formation (feng et al. ) . but, an intriguing point is that also no provokes the zinc release by mt, via s-nitrosylation (zangger et al. ) . despite inos increases in ageing, the release of zinc by mt is very limited. one hypothesis might be an unbalance between no synthases (inos and cnos; mocchegiani et al. a ). however, no donors and zinc fluorescent probes are useful tools in order to study the zinc release from mt and to evaluate the intracellular labile zinc in ageing. using a methodology for testing intracellular free zinc ion availability in pbmc recently developed in our laboratory , it has been shown that the no-induced release of zinc can be preserved at least in nonagenarians carrying mt a polymorphism favorable for successful ageing (cipriano et al. ). moreover, a flow cytometric assay for the measurement of intracellular labile zinc was recently developed by haase et al. ( ) the zinc-sensitive fluorescent probe named fluozin- was used to quantify the amount of labile zinc in peripheral blood mononuclear cells isolated from human blood. with this method, the intracellular concentrations of labile zinc in resting cells were estimated to be . nm in monocytes and . nm in lymphocytes (cd +; haase et al. ) . therefore, the combination of these two novel methodological procedures will permit to study in depth the cause of limited zinc release from mt in ageing and, at the same time, to evaluate the intracellular labile zinc. anyway, a limited zinc release from mt exists in ageing provoking a low free zinc ion availability for immune response and antioxidant activity. the recent discovery of another novel polymorphism of mt (- a/g mt a) may indirectly support this assumption. indeed, old subjects carrying aa genotype display high mt, low zinc ion availability, enhanced il- and impaired innate immune response with subsequent possible risk for atherosclerosis and diabetes type ii (giacconi et al. ) . therefore, mt may have a different role in immunosenescence, following the concept that several genes/proteins that increase fitness early in life may also have negative effects later in life: named "antagonistic pleiotropy theory of ageing" (williams and day ) . since the crude zinc balance is negative in old mice and in old human (turnlund et al. ), zinc supplementations in old mice and in elderly have been carried out in order to improve the immune response. the scientific rationale for the immune supporting role of zinc supplementation "in vivo" finds consistent support by data obtained "in vitro" in immune cells. at this regard, many effects of zinc on immune cells have been shown by assessing the cytokine concentration in the samples after zinc stimulation. when pbmcs are stimulated with zinc, il- , il- , tnf-α, soluble (s)il- receptor and ifn-γ are released (ibs and rink ) . the secretion of il- , il- and tnf-α is induced directly by zinc in monocytes and is independent by the presence of lymphocytes (driessen et al. ) . however, the effect of zinc on monocytes may depend upon external stimulation. in fact, zinc inhibits lps-induced tnfα and il- β release from primary human monocytes and monocytic cell lines through the inhibition of cyclic nucleotide phosphodiesterase activity (von bulow et al. ) , suggesting that zinc may display also some anti-inflammatory properties. the dose of zinc used is also a critical variable. in serum-free culture medium, concentrations > μm of zinc/l stimulate monocytes but prevent t-cells from activating, perhaps due to the lower intracellular content in t-cells than in monocytes (ibs and rink ) . treatment with zinc "in vitro" generally displays also beneficial effects on cell survival but, the effect largely depends upon the cell type and the dose of zinc used. it seems that both apoptosis prevention and induction are mediated by pathways involving zinc and/or zinc-dependent enzymes (clegg et al . ; wiseman et al. ) . therefore, the modulation of the zinc homeostasis plays a key role not only in preventing apoptosis, when oxidative stress is low, but also in inducing apoptosis, when oxidative stress and cellular damage is high, in order to down regulate immune responses and to eliminate virally infected or malignant cells (fraker and lill-elghanian ) . taking into account the strict correlation existing between oxidative stress and immune function especially in response to specific stimuli through the production of proinflammatory cytokines for a prompt immune response (franceschi et al. ) , this role of zinc in inducing apoptosis of only damaged cells in presence of high oxidative stress is evident in young-adult age and with a great surprising in very old age (ostan et al. ) , perhaps due to the presence of satisfactory zinc ion availability (mocchegiani et al. a ) that regulates p activity for health lifespan (bauer and helfand ) , being p a zinc binding protein (hainaut and mann ) . experiments in thymocytes also support this point of view, since media supplemented with zinc from up to μm prevents old thymocyte apoptosis induced by dexamethasone or serum deprivation (provinciali etal. ) , whereas the direct introduction of free zinc as zinc-pyrithone inside thymocytes induces apoptosis (mann and fraker ) . in this last case, the continuous presence of intracellular free zinc ions can advice the cell that permanent oxidative stress and irreversible damage are present, thus activating proapoptotic pathways. old literature reports that a physiological zinc supplementation in the diet throughout the life span in adult rodents prevents some age-related cell-mediated immune modifications, such as the decreased circulating thymic hormone levels (iwata et al. ) . more recently, a physiological zinc supplementation ( μg/ml zn ++ in the drinking water for -month) in old mice induces thymus re-growth and functionality (dardenne et al. ; mocchegiani et al. ) and restoration of nk cell cytotoxicity . that the benefit of zinc supplementation upon the immune functions in old mice is not to consider an epiphenomenon comes by the analysis of the rate of survival in old zinc treated mice. old mice (inbreed balb/c mice) treated with daily zinc at the dose reported above in drinking water from the pre-senescent age ( - months of age) display a significant increment of the rate of survival up to th month of age when this strain of mice usually lives up to - th month of age. the increment of old survivor zinc treated mice is particularly significant in the middle age ( - th month of age; mocchegiani et al. b ). the increased rate of survival is largely due to significant decrements of deaths due to cancer and infection in the middle age (mocchegiani et al. b) . of interest, the crude zinc balance is negative, other than in old mice, also in nude and neonatal thymectomized mice (mocchegiani et al. b (mocchegiani et al. . a zinc supplementation increases the rate of survival also in nude and neonatal thymectomized mice (mocchegiani et al. ), which display a very short survival due to thymus absence (piantanelli and fabris ) . taking into account that the liver extrathymic t-cell pathway is prominent in nude, thymectomized and old mice in order to compensate the thymic failure (abo ) , it is evident the zinc also affects the liver extrathymic t-cell pathway with good performances of the immune functions against external noxae (mocchegiani et al. ) coupled with increased rate of survival. with regard to elderly, undefined data exist on the beneficial effect of zinc supplementation upon the immune efficiency due to different doses of zinc used and to the length of the treatment (bodgen et al. ; boukaiba et al. ; cakman et al. ; duchateau et al. ; fortes et al. ; prasad et al. b; sandstead et al. ) . although zinc was used at the dose recommended by rda (from to mg/day) in the majority of the studies, prasad et al. ( b) and boukaniba et al. ( ) have found an increment of thymulin activity and improvements in response to skin-test antigens and taste acuity (zinc dose = mg /day for months); bodgen et al . ( ) have reported no benefit exclusively for increased lymphocyte mitogen proliferative response (zinc dose = mg/day for -year); cakman et al. ( ) have found enhanced ifn-γ production by leukocytes (zinc dose = mg/day for days); fortes et al. ( ) report an increased number of ctls (zinc dose = mg/ day for days); duchateau et al. ( ) and sandstaed et al. ( ) have observed an improvement in response to skin-test antigens and taste acuity (zinc dose = mg/day for -month). thus, it seems evident from these studies that physiological dose of zinc for a long period or high doses of zinc for short periods might induce limited effects on immune response perhaps due to a zinc accumulation in various organs and tissues with subsequent toxic effect of zinc upon the immune functions (fosmire ; sandstead ) . in this context, it is useful to remind that high doses of zinc trigger apoptosis of the immune cells in presence of high oxidative stress, as reported above. therefore, zinc supplementation has to be used with caution for short periods and on alternate cycles. following that, in our experience, zinc treatment at the dose of mg zn ++ /day for -month in down's syndrome subjects, in elderly and in old infected patients restores thymic endocrine activity, lymphocyte mitogen proliferative response, cd + cell number, peripheral immune efficiency (nk cell cytotoxicity), th /th paradigm (franceschi et al. ; kahmann et al. ; mocchegiani et al. ) and dna-repair (chiricolo et al. ) . at clinical level, significant reductions of infection relapses occur in down's syndrome (licastro et al. ) in elderly and in old infected patients with a faster outcome from the pathology (mocchegiani et al. ) . physiological zinc supplementation was reported to lead to a decrement in plasma lipid peroxide concentrations in elderly people living in a public home (fortes et al. ) . the positive effect of zinc on lipid peroxide could derive from its protective effects on sulphydryl groups against oxidation and the fact that zinc is a component of superoxide dismutase (sod; mills ) . zinc supplementation is also useful in reducing the oxidative stress in old patients with diabetes type ii (roussel et al . ) because it inhibits nf-kb activation and decreases inducible no synthase. as such, the generation of ros decreases, thus zinc provides a protective effect on β cells against death (ho et al. ). an intriguing point of the zinc supplementation is the increment of znt. elderly women treated for days with mg of zinc gluconate /day display significant increments of znt gene expression in peripheral leukocytes (andree, et al. ) , even if the gene expression of the znt is sensitive in relation to the immune cells considered (whitney et al. ) . such increments of znt have been also observed in human lymphoblastoid cells adding in vitro or μmol/l of zinc (andree et al. ) , furtherly suggesting the relevance of zinc supplementation also in affecting the gene expression of znt and, consequently, the correct maintenance of intracellular zinc homeostasis. that the beneficial effects of zinc supplementation are not to be considered as epiphenomena, it comes by the increased survival also in nude and neonatal thymectomized (ntx) mice treated with physiological zinc ( μg zn++/day for -month) in the drinking water, taking into account that they display a very short survival due to thymic absence and negative crude zinc balance (mocchegiani et al. , b (mocchegiani et al. , . the prolonged survival is largely due to mortality reduction (about %) by infections because zinc also affects the extrathymic t-cell pathway that is prominent in old, nude and ntx mice for t-cell maturation and host defense (abo et al . ) . indeed, in vivo and in vitro studies have shown that zinc is a key trace element for liver t-cell maturation and function, particularly for liver nkt cells bearing tcr γδ with high production of ifn-γ (mocchegiani et al. ) . of interest, the increment and function of nkt cells (miyaji et al. ) and tγδ cells (colonna-romano et al. ) also occur in human centenarians, who in turn display satisfactory zinc ion bioavailability and good immune response (mocchegiani et al. a ). all these "in vitro" and "in vivo" studies in ageing, some age-related diseases, and syndrome of accelerated ageing (nude mice, ntx mice, down's syndrome) demonstrate the pivotal role played by zinc supplementation in maintaining or improving global immune response and in fighting the oxidative stress, strengthen by findings observed in human centenarians. however, since zinc also affects mt gene expression (maret ) , the question arises whether zinc supplementation in old age may furtherly increase mt causing possible major harmful effects. old zinc treated mice exhibit no further significant increments of liver mt mrna, suggesting that mt in ageing may be already over-expressed before supplementation (mocchegiani et al. b ). moreover, the effects observed during zinc supplementation on the immune system, such as reduced inflammation and restored th /th paradigm (prasad ) , suggest that intracellular zinc may return available despite over-expressed mt (mocchegiani et al. b) with a maintenance of their original protective role. therefore, the possible harmful effect of mt in ageing seems to not constitute a problem during physiological zinc supplementation. the beneficial effect of physiological zinc supplementation must be, however, related to the levels of other cations such as cadmium, lead, calcium, iron, manganese and copper. the beneficial effects of zinc on ameliorating toxicity of cadmium and lead, accentuation of zinc deficiency by administration of calcium and phytate, and production of hypocupremia by excessive zinc intake in humans and animals, are some examples of competition phenomena between these cations (hill ) . such a competition occurs because these ions have similar valence shell electronic structure and, therefore could be antagonist to each other. for instance, the competition between zinc and iron (fe++) occurs at the level of cysteine-histidine ligands for the formation of iron or zinc "fingers" proteins (prasad a ). if iron is excess, a preferential binding of iron than zinc to the metal free-protein occurs. excess of zinc or zinc deficiency impairs dna-protein interactions of zinc-fingers domains with their cognate dna target sites. in these conditions the production of some transcriptional factors like sp or tfiiia is impaired (thiesen and bach ) . the same impairment of zinc fingers dna domains occurs in excess or deficiency of copper (prasad a ). this reinforces the notion of the relevance of interactions between zinc and copper as well as with other metals in the immune efficiency (sandstead ) . thus a limited range of bioavailability exists for each metal. as such, immune responses are optimum. indeed, the beneficial effect of zinc is strictly dependent by the dose and the length of treatment. zinc accumulation or imbalance zinc-to-copper ratio may occur despite low doses of zinc (fosmire ). as such, harmful side effects in the cardiovascular system and in the brain may appear with increased low-density lipoprotein and cholesterol (fosmire ) and neural cell-death (kim et al. ) , respectively. therefore, caution in zinc supplementation is necessary for avoiding undesirable and harmful unexpected side effects. zinc supplementation must not exceed - times the rda/day, for short periods ( - months) and on alternate cycles. this treatment doesn't interfere in copper absorption (faillet-coudray et al. ; licastro et al. ) . zinc picolinate form may be the best supplement (wapnir et al. ). selenium (se) is an essential dietary element for the prevention of some diseases, including cancer and infections (schwarz ) . such an assumption has been subsequently confirmed in animals with a selenium deficiency in the diet and concomitant treatment with various carcinogens, such as , -dimethylhydrazine (dmh) or dimethylbenz(a)anthracene (dmba), compared with animals fed with higher content of selenium in the diet. in this context, although se deficiency appears to affect dmh toxicity with however no inhibition of tumor development by nutritional se ( . ppm se; pence and buddingh ) , three relevant papers report a greater development of carcinoma by dhm or dmba in various organs (colon and mammary gland) in rats fed with selenium deficiency in the diet in comparison with rats treated with ppm of se (jacobs ; liu and milner ; mcgarrity and peiffer ) . these findings further suggest the ability of dietary selenium to inhibit the in vivo metabolism of carcinogens dmba or dmh with subsequent less development of the tumor. with regard to infection, decreased dietary selenium can change a normally avirulent b coxsackievirus (cbv / ) into a virulent virus (cbv / ) by inducing changes in viral genoma, especially in viral rna polymerase mutations (duarte et al. ) that infect heart muscle and cause myocarditis with subsequent possible development of dilated cardiomyopathy and death (beck and levander ) . in food, selenium derives from vegetables and animal products and in particular from the consumption of seafood, liver, and cereals. however, in vegetables and cereals the amount of selenium varies in soil in different countries and geographical regions (wasowicz et al. ) . indeed, selenium deficiency and related diseases have been well documented in geographic regions where the soil content is low, such as the chinese province of keshan (li et al. ) . from this region of china, in fact, keshan disease is named the pathology characterized by selenium deficiency and presence of substantial number of virulent viruses, including coxsackieviruses . mammals can use both inorganic and organic selenium as a nutrient. most of the biological functions of selenium are attributed to selenoproteins, which contain selenocysteine residues responsible for their specific activity. selenoproteins are present in every cell type. the human selenoproteome consists of selenoproteins, mostly involved in antioxidant defence systems (kryukov et al. ) . glutathione peroxidases (gpxs), a family of the selenoproteins, protect cells against oxidative damage by catalysing the reduction of hydrogen peroxide and other hydroperoxides (brigelius-flohe ; hall et al. ) . five selenium dependent gpx isoforms exist in humans and four isoforms in mice. gpx is found in the cytosol of almost all cells and catalyses the reduction of free hydroperoxides. gpx is expressed in the gastro-intestinal tract and has a substrate specificity similar to gpx ; gpx is an extracellular enzyme found in plasma and reduces membranebound phospholipid hydroperoxides (brigelius-flohe ) . gpx is expressed in various tissues, and reduces phospholipid hydroperoxide and hydrogen peroxide using also thiols, such as -mercaptoethanol, cysteine and homocysteine, other than gsh as reductant agents (roveri et al. ). the isoform gpx seems to be specifically expressed in embryonic tissues and olfactory epithelium (kryukov et al. ) . it also exist a selenium independent isoform, gpx , which is an epididymis isoenzyme present in mice and humans (hall et al. ), but its mrna was found to be not translated into functional protein in human epididymis (ghyselinck et al. ) . selenium is also involved in the thioredoxin system, a major enzymatic system that plays an important role in maintaining the redox state of the cell (holmgren ) . this system is highly complementary to the gsh system in protecting against oxidative stress (watson et al. ) . it comprises basically of thioredoxin (trx) and the selenoprotein thioredoxin reductase (tr) and uses the reducing power of nadph to act as a potent antioxidant system as well as a general disulfide redox system (rundolf et al. ) . mammalian tr maintains trx in a reduced state (holmgren ) and reduces a variety of other substrates including nondisulphides. the thioredoxin system protects the cell against oxidative stress through a variety of mechanisms. trx can directly quench singlet oxygen and scavange hydroxyl radicals (das and das ) , or reduced trx can indirectly serves as an electron donor for trx peroxidase. in addition, human tr is directly capable to efficiently reduce lipid hydroperoxides, hydrogen peroxide and organic hydroperoxides using nadph, especially in the presence of catalytic amount of selenocysteine, thus serving as an important alternative to the gpx pathway for the elimination of harmful hydroperoxides (bjornstedt et al. ) . trx system is also critical for signal transduction (arner and holmgren ) and in the restoration of the reduced form of several antioxidant compounds, including ascorbic acid, lipoic acid, and ubiquinone (nordberg and arner ) . in this context, selenomethionine, a potent catalytic antioxidant in biological system and an aminoacid occurring in proteins in place of methionine (walter and roy ) , reacts more efficiently than methionine (padmaja et al. ) with oxidants forming methionine selenoxide which, in turn, is effectively and rapidly reduced to seleniomethionine by glutathione (assmann et al. ) . in contrast, methionine sulphoxide that it is produced by the oxidation of methionine in presence of oxidants, is not simply reduced by gsh, but it requires a specific enzymatic reaction catalyzed by methionine sulphoxide reductase (levine et al. ) . since selenomethionine can occur in proteins such as haemoglobin (beilstein and whanger ) , these residues may play a defensive role against peroxinitite. another selenoprotein, which reduces phospholipid hydroperoxides in the presence of thiols, is the selenoprotein p (sep; burk et al. ) . sep is expressed in many tissues and represents the major plasma selenoprotein, which contains % of the total plasma selenium in the form of selenocysteine. sep protects endothelial cells against damage from peroxynitrite and transports selenium from the liver to peripheral tissues (burk et al. ) . last, but not the least in order of importance, is a class of selenoproteins (iodothyronine deiodinase enzymes), which catalyse the peripheral deiodination of thyroxin (t ) to , ' -triiodothyronine (t ). these enzymes play crucial roles in determining the circulating and intracellular levels of t and, consequently, the control of growth, development, differentiation, metabolism and finally also the immune response (kohrle ; beckett and arthur ) . immunologically, the ability of selenoproteins to protect the host from oxidative stress is vitally important, since many host defence systems rely on the microbiocidal effects of macrophage-or neutrophil-generated free-radical species. oxidative species are generated through general metabolism, during the metabolism of xenobiotics and during exposure to ultraviolet radiation (uv) in sunlight. inflammation as a process to clear infection and damaged tissue also generates great oxidative stress. if antioxidant systems are not functioning correctly, host cells will be damaged (mckenzie et al. ) . taking into account that the inflammation is chronic in ageing as well as oxidative stress (franceschi et al. ) , the role played by selenium through the selenopreoteins in immune response is therefore vital in elderly. the influence of selenium on the immune function can be, in part, attributed to the same selenoproteins involved in the protection against oxidative damage and, in part, to still undefined biochemical pathways. the antioxidant gpxs have probably a role in protecting neutrophils from ros that are produced during inflammation (arthur et al. a, b) . selenium supplementation, in mice, increases the expression of subunits alpha (p ) and/or beta (p / ) of il- receptor (il- r) from activated lymphocytes and nk cells, thereby enhancing proliferation and clone expansion of cytotoxic precursor cells. in vitro, selenium enhances the release of tumor necrosis factor (tnf), il- and il- from lps stimulated macrophages ( see review beckett et al. ) . however one of the most widely investigated associations between selenium and the immune system is the effect of the micronutrient on neutrophil function. neutrophils produce superoxide-derived radicals to take part in killing of microbes. this type of process is a balance between the production of sufficient radicals to kill invading organisms and the systems that protect the neutrophils themselves from the radicals. thus, although selenium deficiency does not affect neutrophil numbers in a range of species, certain aspects of their function are defective (turner and finch ) . neutrophils from selenium-deficient mice, rats and cattle are able to ingest pathogens in vitro but are less able to kill them than are neutrophils from selenium-sufficient animals. this defective function has been associated with decreased cytosolic gpx (gpx ) activity in the neutrophils, which allows the free radicals that are produced in the respiratory burst to kill the neutrophils themselves (arthur et al. b ). therefore, taking into account all these mechanisms, selenium deficiency has been mainly studied in relation to ageing/mortality and in some age-related diseases, whose pathogenesis is related to preservation of membrane integrity and to oxidative damage of biomolecules, such as lipids, lipoproteins and dna. selenium deficiency is a condition, mainly attributed to low selenium content in the soil or to long-term parenteral nutrition. selenium is essential for several biochemical mechanisms and selenium blood decline concentrations relate to chronic age-related disease such as cancer, cardiovascular disease and immune dysfunctions (seiler ) . during ageing, selenium deficiency may occur in relation to intestinal malabsorption. however, few data report a marked selenium deficiency in old subjects (seiler ) . more recently, a paper has explored the relationships between plasma selenium and mortality in an elderly population for a long period of observation ( years): the eva (etude du vieillissement artériel) study (akbaraly et al. ) . the authors have observed during this long period that the mortality rates were significantly higher in individuals with low selenium [ . μmol/l: a value below the cutoff considered as optimal ( . - . μmol/l; thomson ; combs ) ]. when the underlying causes of death were considered, an association with low selenium and cancer-related mortality was found. the same authors suggest that plasma selenium could be an indicator of longevity in a preaging, independently living population not specifically at risk for cancer and cardiovascular diseases. survival curves illustrate that the relationship between plasma selenium and mortality remained pertinent during the entire -year period (akbaraly et al. ) . however, the mechanism of this potential relationship is still under debate and further research needed especially on the role played by selenoproteins on this phenomenon. other authors demonstrate selenium deficiency in elderly people in relation to hypothyroidism (oliveri et al. ) . interestingly, human healthy centenarians display selenium values quite similar to normal elderly (savarino et al. ). as few trials have been carried out up to date in elderly, it is difficult to report a specific beneficial effect of selenium in immunosenescence, even if beneficial effects of selenium supplementation on lymphocyte mitogen responsiveness have been reported in institutionalized elderly individuals (peretz et al . ) and in old animals (roy et al. ) . moreover, a finnish study adding selenium to fertilizer has shown only an increased selenium status in the general population (young, adult, old; aro et al. ), but not on its possible beneficial effects. the major evidence of the beneficial effects of selenium relate to age-associated diseases. many studies have investigated the effects of selenium in carcinogen-exposed animals showing a reduction in tumor incidence and/or preneoplastic endpoints (reid et al. ) . a supplementation with μg/day of organic selenium in randomized subjects showed preventive effects in the incidence and the mortality from various types of cancer (prostate, colorectal and lung cancer; clark et al. ; reid et al . ) . another large supplementation trial in which a physiological amount of selenium ( μg) was recently performed in lixian (north china) in order to test its possible beneficial effect in preventing cancer. a small but significant reduction in total and cancer mortality was observed in subjects receiving selenium supplement. the reduction were shown to be greater in women than men and interestingly more pronounced in persons under the age of years compared to individuals older than years (blot et al. ) . considering these results, it can be assumed that younger persons might be more amenable to a protective effect of selenium supplementation with thus a role of selenium in preventing age-related diseases or in enhancing the innate immune defenses in the course of the pathology, as observed in selenium supplemented cancer patients ( mg/d of sodium selenite; kiremidjian-schumacher et al. ) . the relevance of selenium in the etiology of cardiovascular diseases has been also studied. selenium metabolism is potentially involved in several protective biochemical pathways related to cardiovascular disease, such as reduction of ldl levels and lipoprotein oxidation, inhibition of foam-cell formation and shift in prostaglandin production from prostacyclin to tromboxane (alissa et al. ) . however, wei et al. ( ) found no association between death for cardiovascular diseases and baseline selenium status in a cohort with a mean serum concentration of . μmol/l in younger individuals (mean age, years). the major studies on the incidence of cardiovascular diseases in these last years have been performed in adult people using a combinations of multivitamins and some trace elements, including selenium, as possible prevention of cardiovascular diseases (atherosclerosis, myocardial infarction, thrombosis). all these studies have shown a less incidence of cardiovascular diseases after supplementation with these combinations in comparison to placebo groups (czernichow et al. ; shenoy et al. ) . therefore, the existence of a clear link between selenium deficiency "in se" and cardiovascular disease remains to be clearly defined. finally, an intriguing point is the association between selenium deficiency, immune response and increased incidence of infections in adults and elderly. patients with systemic inflammatory response syndrome display a strong impairment in immune efficiency, a decrease of % in plasma selenium concentrations coupled with increased morbidity and mortality rates (forceville et al. ) . the interrelationships between selenium deficiency, impaired immune response and infections have been clearly shown in experimental animals. an inoculated avirulent virus in selenium deficient animals turns into a virulent one due to genomic changes within the virus, provoking an impaired humoral immune defence (beck ) . in humans, a relevant clinical trial with multivitamins and selenium has shown an increment of cd + counts over the baseline levels (coodley ) and enhanced gpx and gsh activity (delmas-beauvieux et al. ) in hiv infected patients this finding suggests that cysteine/gsh are effective natural inhibitors/combaters of (aids) viruses and thereby capable in preventing the development of chronic virus diseases that can lead to aids (rayman ) . moreover, supplementation with multivitamins and trace elements, including se, during treatment of pulmonary tuberculosis may reduce mortality in subjects co-infected with hiv (range et al. ). therefore, an enhanced oxidative stress, caused by selenium deficiency, is the reason of possible viral genetic changes (beck et al. ) and increased progression of viral infections with subsequent impaired immune defense (daniels ) . additionally, it is also of interest for the role played by selenium deficiency in viral infections the following points: (i) the emergence during these last years (from ) of a newly recognized human disease agent (coronavirus) that causes sars from guangdong province of china (lashley ) as well as from northern vietnam (reynolds et al. ) , where significant areas of overt selenium deficiency exist (xia et al. ) ; ii) the increased risk of enhanced virulence of influenza virus in elderly (ellis et al. ) associated with a possible selenium deficiency (seiler ) . therefore, the selenium deficiency may be considered as a relevant risk factor for the appearance of age-related diseases (cancer, cardiovascular diseases and infections by viruses, which may become more virulent or mutated). such a risk is of relevance in elderly because accumulating data suggest that persistent infection with varicella-zoster virus (vzv; arvin ) , epstein-barr virus (ebv; stowe et al. ) and particularly cmv (mcvoy and adler ) impacts upon the immune system in aging and may contribute to the immune risk phenotype (irp), which predicts remaining longevity in the very elderly (pawelec et al. ) . specific study on these aspects should be encouraged taking into account the possible relevant implications for public health. dietary zinc and selenium are important nutritional factors for the immune response in protecting against the appearance of age-related diseases. the regulation of zinc ion bioavailability by selenium and selenoproteins has been recently investigated (maret ) . zinc/thiolate coordination occurs in mt affecting the binding and release of zinc from mt. zinc/thiolate cluster of mt can be oxidized by glutathion disulfide (gssg) or other disulphides in order to release zinc. however, the efficiency of this chemical reaction seems very low even at high concentrations of gssg in the absence of selenium. in contrast, the release of zinc from mt occur very rapidly following the addition of selenium compounds that has the capacity to form a catalytic selenol(ate), releases zinc (maret ) . the mechanism of the reaction was suggested to proceed through an activated selenenyl sulphide r-se-s-g intermediate which, in turn, oxidizes the zinc-thiolate cluster of mt to form r-se-s-mt with the concomitant release of zinc during the oxidation (chen and maret ) . the selenol group is subsequently released by the attack of a nearby thiol group of mt that convert r-se-s-mt into thionein generating a catalytic cycle of oxidative zinc release from mt. other oxidized selenium compound, such as selenoxide and selenic acid may be directly reduced by mt through the formation of a r-se-s-mt intermediates and the concomitant release of zinc, followed by the formation of an inter-or intramolecular disulfide bond (chen and maret ; jacob et al. ; klotz et al. ) . selenium compounds also catalyze the release of zinc from mt in peroxidation and thiol/disulfide-interchange reactions. in presence of t-butylhydroperoxide, gpx catalyses the mt oxidation with subsequent zinc release, suggesting that mt may serve as reducing agents for gpx (or at least some gpx isoforms) in alternative to gsh (jacob et al. ) .therefore, the assessment of zinc ion bioavailability, mt and selenium concentrations could represent useful tools for studying the physiology of successful ageing. indeed, a recent study shows that . % of the 'healthy' nonagenarian/centenarians display both zinc and selenium levels equal or greater than the lowest values in the elderly (savarino et al. ) . moreover, healthy nonagenarians display low mt, good zinc ion bioavailability (mocchegiani et al. a) and satisfactory gpx activity (mecocci et al. ) . these findings suggest that an adequate zinc and selenium content in cells and tissues are crucial to achieve health ageing and longevity. in this context, girodon et al. ( ) determined the effects of a long-term (for years) daily supplementation with zinc ( mg) plus selenium ( μg) on immunity and the incidence of infections in a large number (n. ) institutionalized elderly people (> years). the main results of the study were: ( ) selenium deficient patients decreased from about % to - % in the selenium supplemented group after months of supplementation with respect to placebo group; ( ) antibody titres after influenza vaccine were higher in groups that receive trace elements; ( ) trace element supplemented patients were those who remained most free of respiratory tract infections than placebo group. these findings suggest that low dose supplementation of zinc and selenium provides significant improvement in elderly patients by increasing the humoral response after vaccination and decreased influenza compliances (respiratory tract infections) with thus possible achievement of health longevity. beneficial effects obtained by zinc and selenium supplementation alone or associated on immune response and at clinical level are summarized in table . therefore, even if some controversial finding exists on the "real" necessity of micronutrient supplementation (dangouret al. ) , the huge amount of data reported associated to observational data, clearly suggests that zinc and selenium play a pivotal role for immunosenescence in order to achieve healthy ageing and longevity. however, zinc seems to plays the major role because some biochemical mechanisms involved in the action of selenium are under the control of zinc ion bioavailability, which in turn is affected by mt and znt expression. one of the most relevant biochemical pathways is the release of zinc by mt through interactions with gpx and intracellular disulphides. however, some points require further investigations. first of all, the reason of a possible limited zinc release in ageing and the biochemical mechanism involved, in particular addressing no-related intracellular pathways. such an investigation is relevant taking into account the double face of no action: or as antioxidant or as inducer of cell death (colasanti and suzuki ) . although useful tools are now available, such as no donors and zinc fluorescent probes (zinpyr- and fluozin- ), in order to test the capacity of the cells in the zinc release by mt, the quantity of labile intracellular zinc in old age remains to be furtherly explored. this last point is also crucial because a fine modulation of intracellular labile zinc is fundamental in order to avoid an excessive zinc release by mt that can result toxic for the cell with subsequent cell-death. moreover, the association of these studies with the role played by znt in ageing may give a more exhaustive picture of the role played by zinc in ageing. the results may form a rationale to select old individuals who effectively need zinc supplementation because zinc, in a some extent, may be also toxic for the immune system leading to a further worsening of the already dysfunctional immune functions in ageing. in fact, many clinical trials of zinc supplementation in elderly report contradictory data on the benefit of zinc supplementation upon the immune functions. thus, it is necessary to have many useful tools to screen real zinc-deficient old subjects. among these tools, the genetic screening for some polymorphisms of mt, such as mt a, might constitute a useful additional value in screening old subjects healthy ageing and longevity. indeed, old subjects noncarriers of the c allele for mt a + polymorphism display a better preservation of intracellular zinc homeostasis at advanced age, less inflammation, and are predisposed to the longevity with respect to old subjects carrying the c allele for the same mt polymorphism. this finding further suggests that only a certain number of old subjects are prone to zinc supplementation, and not all old population. in the case herein reported, a simple genotype screening might be useful to check the old subjects who should more frequently assess their zinc status for a possible zinc supplementation. in this context, genetic studies and the effect of zinc supplementation exclusively in old subjects with determinate polymorphisms for mt and il- are studied in zincage project (www.zincage.org) funded by european commission (ec) in fp . another project funded by ec in fp (zenith) confirms the presence of defects in zinc status and immune response in elderly. however, the biology of zinc is very complex and further studies are necessary in ageing especially addressed to the zinc-binding proteins strictly related to the inflammation and oxidative stress because both these conditions are the basis for the onset of a possible zinc dyshomeostasis in elderly (mocchegiani et al. ) . with regard to selenium, the mechanisms of action of se through selenoproteins against oxidative damage have been clear established, even if some aspects at genetic level especially regarding to the glutatione peroxidases require further studies. indeed, while on one hand the genomic sequence of all gpxs isoforms has been established, the evolutionary reasons of an incorrect splicing of the selenium-independent gpx in humans is still to investigate. anyway, selenium through selenoproteins has a wide range of action affecting the antioxidant system, the thyroid hormones turnover and the immune functions with a special focus on innate immune response. on the other hand, a correct thyroid hormone turnover affects the immune performances (mocchegiani et al. ). as such, selenium treatment has been performed in various pathologies characterized by selenium deficiency, high oxidative stress and impaired immune function, such as cancer, infections, cardiovascular diseases as well as ageing. in this context, the more intriguing finding is the discovery that selenium deficiency in the diet or in soil is implicated in the mutation of a normally avirulent b coxsackievirus (cbv / ) into a virulent virus (cbv / ) by inducing changes in viral genoma. moreover, a marginal selenium deficiency ( . μmol/l) causes a higher rate of mortality (by cancer) in old people with respect to old individuals with baseline selenium values ( . μmol/l). therefore from the review data herein reported, zinc and selenium in the daily diet during ageing may be relevant in order to preserve immune and antioxidant functions, which can lead to healthy ageing and longevity. alternatively, a combined oral supplementation of these micronutrients can be recommended taking into account the beneficial effects of zinc and selenium in improving the humoral immune response in old vaccinated individuals (duchateau et al. ; girodon et al. ) . however, the gap between the estimated average requirement of zinc and the upper limit of safe intake is relatively narrow, because excessive zinc may be toxic (fosmire ). concerning to selenium, even if few reported cases have been associated with an excessive intake of selenium, it has to be taken into account that the institute of medicine of the national academy of sciences has set a tolerable upper intake level for selenium at micrograms per day for adults to prevent the risk of developing selenosis (johnson et al. ) . therefore, supplementation with zinc and selenium can be recommended in old people who effectively need zinc and/or selenium supplementation after a careful evaluation of the "zinc/selenium status" through plasma measurement, clinical features and possibly evaluating the intracellular content of zinc and selenium. the usefulness of mt polymorphisms in identifying subjects at risk for zinc deficiency might be an additional tool. as such, the impaired immune functions in elderly, through these two trace elements, may be restored with subsequent healthy ageing and longevity. physiological responses of extrathymic t cells in the liver selenium and mortality in the elderly: results from the eva study the controversy surrounding selenium and cardiovascular disease: a review of the evidence low micronutrient intake may accelerate the degenerative diseases of aging through allocation of scarce micronutrients by triage kirschke cp et al investigation of lymphocyte gene expression for use as biomarkers for zinc status in humans nitric oxide mediated metallothionein induction by lipopolysaccharide physiological functions of thioredoxin and thioredoxin reductase effect of supplementation of fertilizers on human selenium status in finland selenium supplementation: does soil supplementation help and why? selenium in the immune system varicella-zoster virus the aging paradox: free radical theory of aging reduction of methionine selenoxide to selenomethionine by glutathione longevity and heavy metal resistance in daf- and age- long-lived mutants of caenorhabditis elegans new tricks of an old molecule: lifespan regulation by p selenium and host defence towards viruses host nutritional status and its effect on a viral pathogen selenium deficiency and viral infection selenium and endocrine systems selenium immunity and disease deposition of dietary organic and inorganic selenium in rat erythrocyte proteins the galvanization of biology: a growing appreciation for the roles of zinc micronutrient malnutrition, infection, and immunity: an overview human thioredoxin reductase directly reduces lipid hydroperoxides by nadph and selenocystine strongly stimulates the reaction via catalytically generated selenols the linxian trials: mortality rates by vitamin-mineral intervention group the role of nitric oxide in innate immunity effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly a physiological amount of zinc supplementation: effects on nutritional, lipid, and thymic status in an elderly population receptor recognition by gp cytokines tissue-specific functions of individual glutathione peroxidases zinc status and interleukin- beta-induced alterations in mineral metabolism in rats selenoprotein metabolism and function: evidence for more than one function for selenoprotein p nutrient requirements and optimisation of intakes metallothionein in radiation exposure: its induction and protective role zinc supplementation reconstitutes the production of interferon-alpha by leukocytes from elderly persons catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compounds nutrition and health promotion in older adults enhanced dna repair in lymphocytes of down syndrome patients: the influence of zinc nutritional supplementation polymorphisms in mt a gene coding region are associated with longevity in italian central female population zinc deficiency-induced cell death decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial survey of mrnas encoding zinc transporters and other metal complexing proteins in pancreatic islets of rats from birth to adulthood: similar patterns in the sprague-dawley and wistar bb strains update on vitamins, minerals, and the carotenoids the dual personality of no zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins gamma/delta t lymphocytes are affected in the elderly impact of selenium and cancer-prevention findings on the nutrition-health paradigm interleukin regulates secretion of zinc-thymulin by human thymic epithelial cells and its action on t-lymphocyte proliferation and nuclear protein kinase c integrative aspects of zinc transporters effect of supplementation with antioxidants upon long-term risk of hypertension in the su.vi.max study: association with plasma antioxidant levels micronutrient supplementation in later life: limited evidence for benefit selenium: does selenium status have health outcomes beyond overt deficiency? restoration of the thymus in aging mice by in vivo zinc supplementation thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions responses to mild cold stress are predicted by different individual characteristics in young and older subjects the enzymatic antioxidant system in blood and glutathione status in human immunodeficiency virus (hiv)-infected patients: effects of supplementation with selenium or beta-carotene induction of cytokines by zinc ions in human peripheral blood mononuclear cells and separated monocytes rna virus quasispecies: significance for viral disease and epidemiology immunosenescence and the lung beneficial effects of oral zinc supplementation on the immune response of old people the status of zinc, copper, and metallothionein in cancer patients zinc transporters and the cellular trafficking of zinc influenza-and respiratory syncytial virus-associated morbidity and mortality in the nursing home population nutrition: impact on oral and systemic health zinc, human diseases and aging thymic hormone deficiency in normal ageing and down's syndrome: is there a primary failure of the thymus? effect of zinc supplementation on in vitro copper-induced oxidation of low-density lipoproteins in healthy french subjects aged - years: the zenith study metallothionein disulfides are present in metallothioneinoverexpressing transgenic mouse heart and increase under conditions of oxidative stress metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts selenium, systemic immune response syndrome, sepsis, and outcome in critically ill patients zinc supplementation and plasma lipid peroxides in an elderly population the effect of zinc and vitamin a supplementation on immune response in an older population zinc toxicity roles for cell death in zinc deficiency the many roles of apoptosis in immunity as modified by aging and nutritional status inflamm-aging. an evolutionary perspective on immunosenescence oral zinc supplementation in down's syndrome: restoration of thymic endocrine activity and of some immune defects genes involved in immune response/inflammation, igf /insulin pathway and response to oxidative stress play a major role in the genetics of human longevity: the lesson of centenarians structural organization and regulation of the gene for the androgen-dependent glutathione peroxidase-like protein specific to the mouse epididymis novel - a/g mt a polymorphism in old patients with type diabetes and atherosclerosis: relationship with inflammation (il- ) and zinc impact of trace elements and vitamin supplementation on immunity and infections in institutionalized elderly patients: a randomized controlled trial. min. vit. aox. geriatric network matrix metalloproteinases (mmps) and tissue inhibitors of mmps in the respiratory tract: potential implications in asthma and other lung diseases flow cytometric measurement of labile zinc in peripheral blood mononuclear cells metallothionein-null mice are more susceptible than wildtype mice to chronic cdcl( )-induced bone injury zinc binding and redox control of p structure and function the majority of human glutathione peroxidase type (gpx ) transcripts are incorrectly spliced: implications for the role of gpx in the male reproductive tract metallothionein induction by restraint stress: role of glucocorticoids and il- mineral interrelationships. in: prasad as (ed) trace elements, human health and disease dietary zinc supplementation inhibits nf-kappab activation and protects against chemically induced diabetes in cd mice polymorphonuclear leukocyte chemotaxis induced by zinc, copper and nickel in vitro zinc-altered immune function circulating thymic hormone levels in zinc deficiency selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes selenium inhibition of , -dimethylhydrazine-induced colon carcinogenesis the antioxidants-vitamin c, vitamin e, selenium, and carotenoids biochemistry of metallothionein effect of improved zinc status on t helper cell activation and th /th ratio in healthy elderly individuals consumption of energy-dense, nutrient-poor foods by adult americans: nutritional and health implications. the third national health and nutrition examination survey, - metallothionein i and ii protect against zinc deficiency and zinc toxicity in mice zinc-induced cortical neuronal death with features of apoptosis and necrosis: mediation by free radicals selenium and immunocompetence in patients with head and neck cancer role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress the deiodinase family: selenoenzymes regulating thyroid hormone availability and action enhanced apoptosis in metallothionein null cells characterization of mammalian selenoproteomes emerging infectious diseases at the beginning of the st century nutritional factors and immunological ageing methionine residues as endogenous antioxidants in proteins keshan disease: an endemic cardiomyopathy in china crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. implications for dimer stability and comparison with endothelial nitric-oxide synthase detection of enteroviral rna in paraffin-embedded myocardial tissue from patients with keshan by nested pcr oral zinc supplementation in down's syndrome subjects decreased infections and normalized some humoral and cellular immune parameters age, dietary selenium and quantity of , -dimethylbenz(a)anthracene influence the in vivo occurrence of rat mammary dna adducts interleukin- regulates the zinc transporter zip in liver and contributes to the hypozincemia of the acute-phase response single and three-color flow cytometry assay for intracellular zinc ion availability in human lymphocytes with zinpyr- and double immunofluorescence: relationship with metallothioneins zinc pyrithione induces apoptosis and increases expression of bim thiolate ligands in metallothionein confer redox activity on zinc clusters cellular zinc and redox states converge in the metallothionein/thionein pair selenium: an essential element for immune function selenium and difluoromethylornithine additively inhibit dmh-induced distal colon tumor formation in rats fed a fiber-free diet immunologic evidence for frequent age-related cytomegalovirus reactivation in seropositive immunocompetent individuals plasma antioxidants and longevity: a study on healthy centenarians nutrition interventions in aging and age-associated disease zinc in human biology functional alteration of granulocytes, nk cells, and natural killer t cells in centenarians nutrient-gene interaction in ageing and successful ageing. a single nutrient (zinc) and some target genes related to inflammatory/immune response mtmrna gene expression, via il- and glucocorticoids, as potential genetic marker of immunosenescence: lessons from very old mice and humans metallothioneins (i+ii) and thyroid-thymus axis efficiency in old mice: role of corticosterone and zinc supply the variation during the circadian cycle of liver cd d-unrestricted nk . +tcrγδ +cells lead to successful ageing. role of metallothionein/il- /gp /parp- interplay in very old mice zinc, metallothioneins and longevity. effect of zinc supplementation: zincage study zinc, t-cell pathways, aging: role of metallothioneins metallothioneins/parp- /il- interplay on natural killer cell activity in elderly: parallelism with nonagenarians and old infected humans. effect of zinc supply zinc and immunoresistance to infection in aging: new biological tools zinc, metallothioneins, immune responses, survival and ageing reversibility of the thymic involution and of age-related peripheral immune dysfunctions by zinc supplementation in old mice interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool in very old age regulation of matrix biology by matrix metalloproteinases matrix metalloproteinases postnatal regulation of znt- expression in the mouse brain reactive oxygen species, antioxidants, and the mammalian thioredoxin system selenium, zinc, and thyroid hormones in healthy subjects: low t /t ratio in the elderly is related to impaired selenium status effect of zinc ions on apoptosis in pbmcs from healthy aged subjects rapid oxidation of dl-selenomethionine by peroxynitrite metabolic age modelling: the lesson from centenarians flow cytometric evaluation of the dna profile and cell cycle of zinc supplemented human chang liver cells human immunosenescence: is it infectious? effect of dietary selenium deficiency on incidence and size of , -dimethylhydrazine-induced colon tumours in rats lymphocyte response is enhanced by supplementation of elderly subjects with selenium-enriched yeast hypopituitary dwarf and athymic nude mice and the study of the relationships among thymus, hormones, and aging effects of zinc deficiency on th and th cytokine shifts zinc deficiency in elderly patients flow cytometric analysis of cd /tcr complex, zinc, and glucocorticoid-mediated regulation of apoptosis and cell cycle distribution in thymocytes from old mice the importance of selenium to human health the effect of multi-vitamin/mineral supplementation on mortality during treatment of pulmonary tuberculosis: a randomised two-by-two factorial trial in mwanza, tanzania selenium supplementation and lung cancer incidence: an update of the nutritional prevention of cancer trial factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi zinc and the immune system zinc homeostasis and immunity antioxidant effects of zinc supplementation in tunisians with type diabetes mellitus supplementation with selenium restores age-related decline in immune cell function regulation of the mammalian selenoprotein thioredoxin reductase in relation to cellular phenotype, growth, and signaling events requirements and toxicity of essential trace elements, illustrated by zinc and copper zinc nutriture in the elderly in relation to taste acuity, immune response, and wound healing recent studies on metallothionein: protection against toxicity of heavy metals and oxygen free radicals enhanced renal toxicity by inorganic mercury in metallothionein-null mice serum concentrations of zinc and selenium in elderly people: results in healthy nonagenarians/centenarians essentiality and metabolic functions of selenium clinical pictures of malnutrition in ill elderly subjects low serum micronutrient concentrations predict frailty among older women living in the community matrix metalloproteinase degradation of extracellular matrix: biological consequences micronutrients in health and disease rationale and design for the cardiovit study (cardiovit, atherosclerotic vascular disease and hyperhomocysteinemia: an epidemiological study in indians, additionally evaluating the effect of oral vitamin supplementation) chronic herpesvirus reactivation occurs in aging zinc inhibits turnover of labile mrnas in intact cells transition metals modulate dna-protein interactions of sp zinc finger domains with its cognate target site assessment of requirements for selenium and adequacy of selenium status: a review selenium and the immune response stable isotope studies of zinc absorption and retention in young and elderly men the biochemical basis of zinc physiology zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses tnf-alpha and il- beta production in monocytes by elevation of guanosine ', '-cyclic monophosphate selenomethionine, a potential catalytic antioxidant in biological systems absorption of zinc by the rat ileum: effects of histidine and other low-molecular-weight ligands selenium status of low-selenium area residents: polish experience thioredoxin and its role in toxicology prospective study of serum selenium concentrations and esophageal and gastric cardia cancer, heart disease, stroke, and total death changes of metallothionein and mrna levels with age in brain of senescence-accelerated mice and the effects of acupuncture human metallothionein genes: structure of the functional locus at q individuality and variation in gene expression patterns in human blood antagonistic pleiotropy, mortality source interactions, and the evolutionary theory of senescence endothelial response to stress from exogenous zinc (zn +) resembles that of no-mediated nitrosative stress, and is protected by mt- overexpression effectiveness of selenium supplements in a low-selenium area of china metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress zinc metallothionein imported into liver mitochondria modulates respiration metallothionein mediates leukocyte chemotaxis metallothionein-induced suppression of cytotoxic t lymphocyte function: an important immunoregulatory control metal-induced metallothionein gene expression can be inactivated by protein kinase c inhibitor nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites zincage project key: cord- -ralxw ad authors: oishi, peter; fineman, jeffrey r. title: diseases of the pulmonary vascular system date: - - journal: the respiratory tract in pediatric critical illness and injury doi: . / - - - - _ sha: doc_id: cord_uid: ralxw ad nan although historically considered the lesser circulation, pathology of the pulmonary circulation is a great source of pediatric morbidity and mortality. this is most commonly displayed in neonates with persistent pulmonary hypertension; neonates, infants, and children with congenital heart disease; and adolescents and young adults with primary pulmonary hypertension. recent evidence indicates that normal pulmonary vascular tone is regulated by a complex interaction of vasoactive substances that are locally produced by the vascular endothelium [ ] [ ] [ ] [ ] [ ] [ ] . these substances, such as nitric oxide (no) and endothelin- (et- ), are capable of producing vascular relaxation and/or constriction, modulating the propensity of blood to clot, and inducing and/or inhibiting smooth muscle cell migration and replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in fact, mounting data implicate endothelial injury and the subsequent aberration in the endogenous production of these substances in the pathophysiology of pulmonary hypertensive disorders [ ] [ ] [ ] [ ] [ ] . this chapter discusses the normal regulation of the fetal, transitional, and postnatal pulmonary circulations, the pathophysiology of pediatric pulmonary hypertensive disorders, and new therapeutic and preventative strategies for pulmonary hypertension. particular emphasis is placed on the role of the pulmonary vascular endothelium in these processes and treatment modalities. fl ow and decreases pulmonary vascular resistance but not to newborn values [ ] . a proportion of this decrease relates to alterations in the physical architecture of the alveoli and small pulmonary vessels that occur with mechanical distention [ ] . in addition, physical expansion of the lung results in the release of vasoactive substances, such as pgi , which increases pulmonary blood fl ow and decreases pulmonary vascular resistance in the fetal goat and lamb independent of the changes in oxygen tension [ ] [ ] [ ] [ ] [ ] [ ] . when ventilation is accompanied by changes in oxygen tension (i.e., ventilation with ambient air or supplemental oxygen), fetal pulmonary blood fl ow increases and pulmonary vascular resistance falls to newborn values. the exact mechanisms of this oxygen-induced pulmonary vasodilation remain unclear. alveolar and/or arterial oxygen may directly dilate pulmonary resistance vessels or may trigger the release of vasoactive substances, such as pgi or no. in fact, data indicate that no, in particular, participates in the decrease in pulmonary vascular resistance that accompanies increases in alveolar and arterial oxygen tension [ , ] however, despite its important role, inhibition of no does not impair the immediate fall in pulmonary vascular resistance seen after birth, further suggesting that multiple mechanisms are involved in this transitional physiology. in fact, recent data implicate fl uid shear forces across endothelial cells, which result in the production of both no and pgi , as an additional mechanism by which vasodilation occurs after birth [ ] . it is possible that this particular mechanism acts to maintain pulmonary vasodilation once it has been established by the mechanisms described earlier. in general, the dramatic increase in pulmonary blood fl ow with the initiation of ventilation and oxygenation at birth refl ects a shift from active pulmonary vasoconstriction in the fetus to active pulmonary vasodilatation in the newborn. failure to undergo this normal transition contributes substantially to the pathophysiology of many neonatal pulmonary hypertensive disorders, including bronchopulmonary dysplasia, persistent pulmonary hypertension of the newborn, chronic lung disease, and congenital heart disease [ , . the successful transition from the fetal to the postnatal pulmonary circulation is marked by the maintenance of the pulmonary vasculature in a dilated, low-resistance state [ ] . recent evidence suggests that basal no release, and the subsequent increase in smooth muscle cell cyclic guanosine monophosphate (cgmp) concentrations, in part mediate the low resting pulmonary vascular resistance of the newborn [ ] . other vasoactive substances, including histamine, -hydroxytryptamine, bradykinin, and metabolites of arachidonic acid by the cyclooxygenase and lipoxygenase pathways, have also been implicated in mediating postnatal pulmonary vascular tone; however, their roles are not well elucidated. two of the most important factors affecting pulmonary vascular resistance in the postnatal period are oxygen concentration and ph. decreasing oxygen tension and decreases in ph elicit pulmonary vasoconstriction [ ] . alveolar hypoxia constricts pulmonary arterioles, diverting blood fl ow away from hypoxic lung segments, toward well-oxygenated segments, thus enhancing ventilationperfusion matching [ ] . this response to hypoxia, unique to the pulmonary vasculature, is greater in the younger animal than in the adult [ ] . indeed, in most vascular beds (e.g., cerebral vasculature), hypoxia is a potent vasodilator. the exact mechanism of hypoxic pulmonary vasoconstriction remains incompletely under-stood but likely involves changes in the local concentration of reactive oxygen species that in turn regulate voltage-gated potassium channels and calcium channels [ , ] . acidosis potentiates hypoxic pulmonary vasoconstriction, whereas alkalosis reduces it [ ] . the exact mechanism of ph-mediated pulmonary vascular reactivity also remains incompletely understood but appears to be independent of paco [ ] . recent data suggest that potassium channels play an important role in mediating these responses as well [ ] . manipulating alveolar oxygen tension and systemic arterial ph are fundamental approaches to changing pulmonary vascular tone in the critical care setting. alveolar hyperoxia and alkalosis are often used to decrease pulmonary vascular tone because they generally relieve pulmonary vasoconstriction with little effect on the systemic circulation as a whole. however, severe alkalosis is generally avoided because of the detrimental effects of severe hypocarbia or alkalosis on cerebral and myocardial blood fl ow (see general treatment approach, later) [ , ] . despite extensive innervation of the lung, neural input is not a major determinant of basal pulmonary vascular tone. however, pulmonary neurohumoral receptors are sensitive to α-adrenergic, β-adrenergic, and dopaminergic agonists [ , ] . therefore, vasoactive agents that stimulate these receptors will affect the vascular tone of both the pulmonary and systemic circulations. alterations in vascular tone, in response to a given agent, are dependent on the relative tone of the vascular bed at a given time. therefore, the response of these agents is diffi cult to predict in an individual critically ill patient. pulmonary vascular resistance changes throughout gestation and after birth. the resistance of the pulmonary circulation at any one time is related to several factors and can be estimated by applying the resistance equation and the poiseuille-hagen relationship [ ] . the resistance equation (the hydraulic equivalent of ohm's law) states that the resistance to fl ow between two points along a tube equals the decrease in pressure between the two points divided by the fl ow [ , ] . for the pulmonary vascular bed, where rp is pulmonary vascular resistance and qp is pulmonary blood fl ow, the decrease in mean pressure is from the pulmonary artery (ppa) to the pulmonary vein (ppv) or left atrium, where la is mean left atrial pressure: rp = [ppa − ppv or la (mean)]/qp therefore, the calculated pulmonary vascular resistance increases when pulmonary arterial pressure increases or when pulmonary blood fl ow decreases. changes in pulmonary venous pressure or mean left atrial pressure are somewhat more complicated. in isolation, increases in pulmonary venous pressure and left atrial pressure would decrease the calculated pulmonary vascular resistance. however, increases in pulmonary venous pressure are generally accompanied by a greater increase in pulmonary arterial pressure (which maintains driving pressure), resulting in an increase in the calculated resistance across the pulmonary vascular bed. furthermore, changes in left atrial pressure, which occur independent of alterations in pulmonary vascular resistance, must be considered. for example, large intracardiac shunts (e.g., ventricular septal defect) may result in congestive heart failure with an elevation in left atrial pressure. closure of the ventricular septal defect may acutely decrease left atrial pressure, resulting in an elevation in the calculated pulmonary vascular resistance (provided that pulmonary arterial pressure does not decrease to the same extent), when in fact no change in pulmonary vascular tone has occurred [ ] . other factors that affect pulmonary vascular resistance can be defi ned by applying a modifi cation of the poiseuille-hagen relationship, which describes the resistance (r) to fl ow of a newtonian fl uid through a system of round, straight glass tubes of constant cross sectional area: where l is length of the system of vessels, n is vessel number, r is the internal radius of the system of vessels, and η is the viscosity of the fl uid. according to this relationship, increasing the viscosity of blood perfusing the lungs or decreasing the radius or crosssectional area (πr ) of the pulmonary vascular bed increases pulmonary vascular resistance. because the above equations describe steady, laminar fl ow of a newtonian fl uid in rigid, glass tubes, differences between physical and biologic systems should be considered. first, blood is not a newtonian fl uid. however, this is probably of little importance at normal hematocrit levels [ ] . the viscosity of blood is related to red cell number, fi brinogen concentration, and red cell deformability. an increased hematocrit (secondary to fetal hypoxemia, twin-to-twin transfusion, maternal-to-fetal transfusion, or delayed clamping of the umbilical cord) will increase viscosity [ , ] as pulmonary vascular resistance increases logarithmically when the hematocrit increases. second, pulmonary vessels are not rigid tubes. their walls are deformable, and their size and shape are infl uenced by transmural pressure. for example, as pulmonary blood fl ow or left atrial pressure increases, vessel diameter may change, and/or the recruitment of additional pulmonary vessels may occur. therefore, the fall in calculated pulmonary vascular resistance with increases in pulmonary blood fl ow is nonlinear [ , , ] . third, blood fl ow through the pulmonary circulation is pulsatile, not laminar, and the small pulmonary arteries are branched, curved, and tapered, not smooth [ ] . in addition, the small pulmonary arteries are in parallel, and the radii of these arteries may differ in different lung zones. despite these differences from physical models, the general effects of changes in physical factors, such as viscosity and radius, do apply [ ] [ ] [ ] . in fact, a change in luminal radius is the major factor responsible for maintaining a high pulmonary vascular resistance in the fetus. consideration of these factors, particularly viscosity and cross-sectional area of the vascular bed, is important in evaluating the pathophysiology of pulmonary hypertensive disorders. finally, it is important to note the overall relationship between lung volume and pulmonary vascular resistance, which has been described by several investigators [ , ] . these studies have shown that this relationship to be u-shaped (figure . ) with minimal pulmonary vascular resistance noted at functional residual capacity. using an open-chest model, pulmonary vascular resistance decreased as lungs were infl ated from a collapsed state and then progressively increased at higher lung volumes, which was thought to be related to infl ation pressure on the alveolar vessels. these observations support the concept that lung infl ation may have a variable effect on the distribution of pulmonary blood fl ow. when pressure is expressed in mm of hg and fl ow in l/min, units of resistance are derived as mm of hg/l/min (wood unit, u). however, comparisons among patients of differing weight and age are problematic. therefore, resistance is more commonly expressed in relation to body surface area, as u·m . multiplying u by converts to dynes/sec/cm − , a common form utilized to express resistance in other settings. pulmonary vascular resistance may be as high as - u·m immediately after birth and then, under normal conditions, falls as previously described to adult levels of - u·m by to weeks of life [ , ] . pulmonary hypertensive disorders are a signifi cant source of morbidity and mortality in the pediatric population. pulmonary hypertension is defi ned as a mean pulmonary artery pressure of greater than mm of hg at rest or greater than mm of hg during exercise. in addition, a calculated pulmonary vascular resistance of greater than u is generally considered abnormal. in neonates, the most common etiology results from a failure to undergo the normal fall in pulmonary vascular resistance at birth termed persistent pulmonary hypertension of the newborn (pphn) that has an incidence of ∼ per , live births. however, other pulmonary abnormalities, such as congenital diaphragmatic hernia, respiratory distress syndrome, and bronchopulmonary dysplasia, may also result in neonatal pulmonary hypertension. beyond the neonatal period, the majority of pediatric pulmonary hypertensive disorders are associated with congenital heart defects. other, less common causes of pediatric pulmonary vascular disease include primary (idiopathic) pulmonary hypertension, hypoxiainduced pulmonary vascular disease, rheumatologic disorders, sickle cell disease, portal hypertension, chronic thromboembolic disease, human immunodefi ciency virus disease, and drug-toxin induced disease. a number of clinical classifi cation systems for vascular endothelial cells are capable of producing a variety of vasoactive substances that participate in the regulation of normal vascular tone. a schematic of some of these endothelial factors is shown in figure . . these substances, such as no, et- , and prostacyclin are capable of producing vascular relaxation and/or constriction, modulating the propensity of the blood to clot, and inducing and/or inhibiting smooth muscle cell migration and replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nitric oxide is a labile humoral factor produced by nitric oxide synthase (nos) from l-arginine in the vascular endothelial cell [ ] [ ] [ ] . nitric oxide diffuses into the smooth muscle cell and produces vascular relaxation by increasing concentrations of guanosine ′ ′-monophosphate (cgmp) via the activation of soluble guanylate cyclase [ , ] . nitric oxide is released in response to a variety of factors, including shear stress (fl ow) and the binding of certain endothelium-dependent vasodilators (such as acetylcholine, adenosine triphosphate [atp], and bradykinin) to receptors on the endothelial cell [ , ] . basal no release is an important mediator of both resting pulmonary and systemic vascular tone in the fetus, newborn, and adult, as well as a mediator of the normal fall in pulmonary vascular resistance that occurs immediately after birth , , ] . in addition, aberrant no-cgmp signaling is integral to the pathophysiology of pulmonary hypertension, as well as a number of other biologic vascular disorders [ , , , , , , , ] . endothelin- is a amino acid polypeptide also produced by vascular endothelial cells [ ] . the vasoactive properties of et- are complex, and studies have shown varying hemodynamic effects on different vascular beds [ ] [ ] [ ] [ ] [ ] . however, its most striking property is its sustained hypertensive action. in fact, et- is the most potent vasoconstricting agent discovered, with a potency times that of angiotensin ii. the hemodynamic effects of et- are mediated by at least two distinct receptor populations, et a and et b [ , ] . the et a receptors are located on vascular smooth muscle cells and mediate vasoconstriction, whereas the et b receptors are located on endothelial cells and smooth muscle cells and thus may mediate both vasodilation and vasoconstriction, respectively. individual endothelins occur in low levels in the plasma, generally below their vasoactive thresholds. this suggests that they are primarily effective at the local site of release. even at these levels, they may potentiate the effects of other vasoconstrictors, such as norepinephrine and serotonin [ ] . the role of endogenous et- in the regulation of normal vascular tone is unclear at present [ ] . nevertheless, alterations in et- have been implicated in the pathophysiology of a number of disease states, including pulmonary hypertensive disorders, and has been implicated in the so-called rebound effect of inhaled no [ , , , , ] . endothelial-derived hyperpolarizing factor (edhf), a diffusible substance that causes vascular relaxation by hyperpolarizing the smooth muscle cell, is another important endothelial factor. endothelial-derived hyperpolarizing factor has not yet been identifi ed, but current evidence suggests that its action is dependent on k + channels [ ] . activation of potassium channels in the vascular smooth muscle results in cell membrane hyperpolarization, closure of voltage-dependent calcium channels, and ultimately vasodilation. potassium channels are also present in endothelial cells. activation within the endothelium results in changes in calcium fl ux and may be important in the release of no, prostacyclin, and edhf. potassium channel subtypes include atp-sensitive k + channels, ca + -dependent k + channels, voltage-dependent k + channels, and inward-rectifi er k + channels [ ] . the breakdown of phospholipids within vascular endothelial cells results in the production of the important byproducts of arachidonic acid, including prostacyclin (pgi ) and thromboxane (txa ). prostacyclin activates adenylate cyclase, resulting in increased camp production and subsequent vasodilation, whereas txa results in vasoconstriction via phospholipase c signaling. other prostaglandins and leukotrienes also have potent vasoactive properties. increasing evidence suggests that endothelial injury and the resulting alteration in the balance of these and other vasoactive substances has a signifi cant role in the development of pulmonary hypertension and increased vascular reactivity [ , , ] . support for this hypothesis is strengthened by observations that endothelial injury precedes pulmonary hypertension and its associated vascular remodeling in several animal models of pulmonary hypertension [ , ] . in humans, endothelial dysfunction, including histologic abnormalities of the endothelium, impairment of endothelium-dependent pulmonary vasodilation, and increased plasma et- concentrations have been described in children with congenital heart defects and pulmonary hypertension before the development of signifi cant vascular remodeling [ , , ] . in addition, neonates with pphn and adults with advanced pulmonary vascular disease have evidence of endothelial dysfunction, impairment of endothelium-dependent pulmonary vasodilation, increased plasma et- concentrations, and decreased prostacyclin production [ , , , ] . the mechanism of injury to the vascular endothelium is unclear but is likely multifactorial and in part dependent on the etiology of the pulmonary hypertension. for example, in children with congenital heart disease and increased pulmonary blood fl ow, the initiating endothelial injury is likely mediated by increased shear stress. however, once pulmonary arterial pressure is elevated, shear stress-mediated endothelial injury appears to promote the progression of the disease, independent of the underlying etiology. finally, a genetic disposition appears to be important in some subtypes of pulmonary vascular disease and remains an area of active research. for example, up to % of patients with familial idiopathic pulmonary hypertension have mutations resulting in the loss of function of bone morphogenetic protein receptor ii [ ] [ ] [ ] [ ] . following an initial endothelial injury, smooth muscle proliferation and progressive structural remodeling occurs. the progression of anatomic changes is best characterized in congenital heart disease (see later discussion) [ ] [ ] [ ] [ ] . however, regardless of the etiology, advanced disease is characterized by medial hypertrophy, intimal hyperplasia, angiomatoid formation, in situ thrombi, and eventual vascular obliteration. if the underlying stress remains untreated (e.g., delayed repair of cardiac shunt), these structural changes can progress to the point of becoming functionally "fi xed" or irreversible. an important goal of therapy is to halt this progression and reverse the early vascular remodeling if possible. regardless of the underlying etiology, the general treatment approach is similar and can be subdivided into four major goals: ( ) prevent and acutely treat active pulmonary vasoconstriction, ( ) support the failing right ventricle, ( ) treat the underlying etiology, and ( ) chronically promote, if possible, the regression of pulmonary vascular remodeling. in the intensive care setting, the prevention and treatment of active pulmonary vasoconstriction is a primary focus for the care of patients with underlying pulmonary vascular disease. it is well appreciated that these patients have augmented pulmonary vasoconstriction in response to such stimuli as hypoxia, acidosis, the catecholamine-mediated α -adrenergic stimulation associated with pain and agitation, and increases in intrathoracic pressure [ ] [ ] [ ] . in fact, acute increases in pulmonary vascular resistance can lead to signifi cant cardiopulmonary compromise (i.e., a pulmonary hypertensive crisis). the pathophysiology of such a crisis in outlined in figure . . following an acute increase in pulmonary arterial pressure, there is an acute increase in right ventricular afterload, producing right ventricular ischemia and, ultimately, failure [ , ] . the resulting increase in right ventricular end diastolic volume shifts the intraventricular septum to the left, decreasing left ventricular volume and cardiac output. decreased cardiac output results in decreased systemic perfusion and metabolic acidosis. increased pulmonary vascular resistance and right ventricular failure also decrease pulmonary blood fl ow, increasing dead space ventilation. distention of the pulmonary arteries and perivascular edema produce large and small airways obstruction, respectively, which impairs ventilation-perfusion matching and decreases lung compliance. in fact, the decrease in lung compliance can be so dramatic that chest wall movement is impaired, even with manual ventilation. the ensuing hypoxemia, hypercapnia, and acidosis (metabolic and/or respiratory) further increase pulmonary vascular resistance and perpetuate this cascade. prevention of pulmonary hypertensive crises may be accomplished by avoiding stimuli known to increase pulmonary vascular resistance, including hypoxia, acidosis, agitation, pulmonary overdistention, and polycythemia [ ] . various regimens have been utilized for this purpose, including the judicious use of supplemental oxygen, analgesics, sedatives, and muscle relaxants (especially before noxious stimuli, such as suctioning); the maintenance of an alkalotic ph with the use of controlled ventilation and buffer; aggressive evacuation of pneumothoraces and pleural effusions; the utilization of low lung volume ventilator strategies; the minimization of positive end-expiratory pressures; and the maintenance of the hematocrit below % [ , ] . in addition, data suggest that the use of pulmonary vasodilator therapy may decrease the incidence of pulmonary hypertensive crises [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . treatment of active pulmonary vasoconstriction is accomplished with the use of pulmonary vasodilator therapy. the mainstay of acute pulmonary vasodilator therapy remains supplemental oxygen and moderate alkalosis, as these therapies have minimal effects on the systemic vasculature. interestingly, the dose-dependent response of the pulmonary vasculature to these agents has not been well established. studies in newborn lambs demonstrate dosedependent pulmonary vasodilation in response to increasing ph from . to . , and a dose-dependent response to increasing inspired oxygen concentrations from . to . , with minimal effects at higher concentrations [ ] . several intravenous agents have been utilized to promote pulmonary vasodilation, including tolazoline, sodium nitroprusside, nitroglycerin, prostacyclin, prostaglandin e , nifedipine, and α-adrenergic antagonists, such as phenoxybenzamine [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the effi cacies of these agents are variable, at least in part because of their effects on the systemic vasculature. systemic afterload reduction can be advantageous in the setting of left ventricular dysfunction; however, signifi cant reductions in pulmonary arterial pressure without unacceptable systemic hypotension are often not possible [ ] [ ] [ ] . in addition, intravenous vasodilators can override intrinsic hypoxic pulmonary vasoconstriction, resulting in an increase in dead space ventilation, which may not be tolerated in some critically ill patients [ ] [ ] [ ] [ ] [ ] . more recent treatment modalities, most notably inhaled no, deliver short-acting vasodilators to the pulmonary vasculature via an inhalational route [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . when administered to the lung in its natural gaseous form, no diffuses through the alveolar wall to reach small pulmonary arteries. it then enters vascular smooth muscle cells, initiating a cascade that results in pulmonary vasodilation via increases in cgmp. after entering the blood vessel lumen, no is rapidly inactivated by hemoglobin, which confi nes its effects to the pulmonary vasculature. because of these properties, inhaled no has several advantages over other vasodilators, including ( ) selective pulmonary vasodilation caused by rapid inactivation by hemoglobin, ( ) rapid onset and elimination, and ( ) an improvement in ventilation-perfusion matching because of the limitation of delivery to well-ventilated lung regions. accordingly, inhaled no has become a mainstay of treatment for acute pulmonary hypertensive disorders and the assessment of pulmonary vascular reactivity. inhaled prostacyclin has similar pulmonary selectivity, secondary to rapid inactivation by hemoglobin. its vasodilating effects are secondary to increasing camp concentrations. currently, studies on the use of inhaled prostacyclin for pediatric pulmonary hypertension are sparse, and comparison studies between inhaled no and inhaled prostacyclin are lacking [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . inhibitors of phosphodiesterases (pdes), a family of enzymes that hydrolyze the cyclic nucleotides camp and cgmp, are a relatively new class of agents that have potent vasodilating and inotropic effects [ ] . milrinone is a pde inhibitor that increases camp concentrations. animal and human data demonstrate pulmonary vasodilation in response to milrinone that can be in excess of its systemic effects if the pulmonary vasculature is constricted [ ] [ ] [ ] [ ] . in addition, a large, randomized study demonstrates that its use decreases the incidence of low cardiac output syndrome following surgery for congenital heart disease [ ] . given these properties, milrinone is increasingly utilized in the postoperative management of patients with congenital heart disease and pulmonary hypertension. sildenafi l, a pde inhibitor, which increases cgmp concentrations, also has potent pulmonary vasodilating effects [ ] . the oral formulation is currently being investigated for chronic pulmonary hypertensive therapy, and recent short-term studies demonstrate benefi cial effects in children with advanced pulmonary vascular disease [ ] . the intravenous formulation is currently being investigated for acute pediatric pulmonary hypertensive disorders (pphn and perioperative pulmonary hypertension) [ , ] . increasing data implicate alterations in et- in the pathophysiology of pulmonary hypertension (see earlier) and suggest that et-receptor antagonism may be a useful therapeutic strategy [ , , , , ] . in fact, bosentan, an oral combined et a and et b receptor antagonist, has demonstrated effi cacy as a chronic therapy for advanced pulmonary vascular disease [ , ] . to date, there have been no large studies on the use of et receptor antagonists for acute pulmonary hypertensive disorders. in addition, the use of selective et receptor antagonism is under investigation but has not yet reached clinical trials. a signifi cant component of the pathophysiology of both acute and chronic pulmonary hypertension is the development of right ventricular dysfunction, which often requires pharmacologic support. maintenance of adequate preload is necessary to optimize cardiac output in patients with pulmonary hypertension. continuous central venous pressure monitoring may be helpful to guide volume therapy, keeping in mind that patients with a poorly compliant right ventricle or increased right ventricular afterload may require elevated central venous pressures to maintain an adequate cardiac output. frequent clinical assessment of liver size can be helpful, particularly in infants. despite adequate preload, cardiac output may be compromised secondary to elevated right ventricular afterload and/or biventricular myocardial dysfunction after cardiac surgery and cardiopulmonary bypass, necessitating the use of inotropic agents [ , ] . these agents increase stroke volume at a given preload and afterload by stimulating ß -adrenergic receptors [ , ] . however, some of these agents also stimulate ß -or α -adrenergic receptors, which are found on the smooth muscle cells of both the pulmonary and systemic arteries. agents that stimulate ß -adrenergic receptors decrease both pulmonary and systemic vascular resistance and improve right and left ventricular function [ , ] . agents that stimulate α -adrenergic receptors may increase both systemic and pulmonary vascular resistance. therefore, a rational approach to using inotropic agents in the setting of pulmonary hypertension is to utilize agents with ß -receptor selectivity and minimal α -adrenergic stimulation (i.e., dobutamine). although animal studies have shown that high doses of dopamine increase pulmonary vascular resistance, human studies have shown increased cardiac output with minimal effects on pulmonary vascular resistance [ , ] . milrinone is also a useful therapy for patients with pulmonary hypertension and myocardial dysfunction, given its vasodilatory and inotropic properties [ ] . in the setting of pulmonary hypertension secondary to congenital heart defects, an atrial communication can be benefi cial in that it allows the failing right ventricle to decompress when right atrial pressure rises [ ] . accordingly, atrial septal defects can be left unclosed (i.e., patent foramen ovale) or created at the time of surgery. the existence of an atrial level communication decreases the risk of right ventricular failure and maintains left-sided cardiac output. the resulting right-to-left shunt is generally well tolerated, particularly if high hemoglobin concentrations are maintained. as right ventricular function improves, right-to-left shunting decreases and oxygenation improves. atrial septostomy as a part of management for chronic pulmonary hypertension (e.g., primary pulmonary hypertension) has been advocated but must be considered carefully on an individual basis [ ] [ ] [ ] [ ] [ ] . in patients with refractory pulmonary hypertension, short-term postoperative extracorporeal support has been useful during the postoperative period of extreme vasoreactivity. however, its use should be limited to support those patients in which the underlying pulmonary vascular disease is deemed reversible. whenever possible, treatment of the underlying disorder must coincide with symptomatic treatment for pulmonary hypertension if attenuation and/ reversal of the disease are to be successful. for example, in neonates, this may involve correction of underlying metabolic disturbances, antibiotics for infectious etiologies, and exchange transfusions for polycythemia. for patients with congenital heart disease, repair of the underlying defect, after determining that the pulmonary vascular disease is reversible (see later), is mandatory. for hypoxia-induced disease, tonsillectomy and adenoidectomy may be required for sleep apnea, and a descent to sea level may be needed for high-altitude-related disease. finally, for rheumatologic disease, immunosuppression may be required. the mainstay of chronic therapy has been aimed at decreasing pulmonary vascular resistance, thereby assisting right ventricular function and perhaps attenuating the progression of vascular remodeling by decreasing the pressure to which the vasculature is exposed. the continuous infusion of prostacyclin (epoprostin) has been the most successful therapy to date in this regard [ ] [ ] [ ] [ ] [ ] . in fact, several studies in humans with advanced pulmonary vascular disease demonstrate improved -year survival and improved exercise tolerance. interestingly, even those patients without an initial vasodilating response to the infusion show signifi cant longterm benefi t, suggesting that effects beyond vasodilation, such as antiplatelet effects, camp-mediated inhibition of smooth muscle cell growth, or other unknown mechanisms may be responsible for the treatment effect [ ] . despite the impressive results, several factors limit its utilization, including the need for chronic intravascular assess with the associated infectious and thrombotic risks, and many other untoward effects, including headache, fl ushing, and acute cardiopulmonary compromise with disruption of the infusion [ ] . with the increasing appreciation for the role of et- in the pathophysiology of pulmonary vascular disease, et receptor antagonists have been developed as a potential treatment modality. to date, bosentan, a combined et a and et b receptor antagonist, is the most widely studied agent and is the only receptor antagonist approved for the treatment of pulmonary hypertension [ , ] . recent studies in adults with primary pulmonary hypertension demonstrate similar improvements in survival and exercise tolerance as those demonstrated with epoprostenol [ ] . the use of et receptor antagonists for pediatric pulmonary hypertensive disorders is currently under investigation. defi ciencies in the no-cgmp cascade in pulmonary vascular disease are well documented. in addition, the vasodilating effects, antiplatelet effects, and antiproliferative effects of augmenting this cascade are well appreciated. therefore, new chronic therapies that augment no-cgmp signaling, which include chronic inhaled no delivered by nasal cannula and sildenafi l, are currently under investigation [ ] . in fact, the short-term benefi t of sildenafi l in children with advanced pulmonary hypertension has recently been reported [ ] . data indicate that several of these new oral therapies, such as bosentan and sildenafi l, may offer additional benefi t by virtue of their ability to inhibit vascular smooth muscle growth and fi brosis [ ] . a number of other treatment strategies, including combination drug therapies, are currently under investigation. to date they have been used predominantly in advanced pulmonary vascular disease, but, due to these favorable characteristics, several potential applications warrant investigation. this includes their use in lung hypoplastic syndromes, in hypoxia-associated disease, and in congenital heart disease in order to improve the operability of patients with modest vascular changes [ ] . in a number of clinical conditions, pulmonary vascular resistance does not decrease normally at birth. as a result, pulmonary blood fl ow remains reduced and pulmonary arterial pressure remains high. the pathophysiologic effects are hypoxemia, myocardial dysfunction, and a resulting reduction in systemic oxygen delivery. the hypoxemia is most often secondary to extrapulmonary rightto-left shunting of blood at the atrial and/or ductal levels but may also be secondary to intrapulmonary right-to-left shunting of blood when associated with parenchymal lung disease. the pathophysiologic mechanisms preventing the normal pulmonary vasodilatation at birth remain unclear and are most likely multifactorial in etiology. within this defi nition of pphn, three major subgroups are often characterized: those with underdevelopment of the lung, those with maladaptation of the lung, and those with maldevelopment of the lung [ ] . these subgroups represent a spectrum of etiologies and pathophysiology. for example, underdevelopment of the lung represents disorders of vascular hypoplasia, which are usually associated with varying degrees of lung hypoplasia. within this subgroup, patients with congenital diaphragmatic hernia have been most thoroughly investigated. although the structural abnormalities are greatest on the side of the hernia, both of the lungs of these patients are smaller and have fewer alveoli than do lungs from a normal control population [ ] [ ] [ ] . their lungs also have fewer vessels per unit of lung [ ] . thus, the total cross-sectional area of the vascular bed is markedly decreased. furthermore, the existing pulmonary arteries have increased muscle mass with medial hypertrophy in normally muscularized arteries and an abnormal extension of muscle into the intra-acinar arteries. the increased muscularization may explain the labile, right-to-left extrapulmonary shunting of blood seen in such patients [ , ] . the response to therapy and long-term outcome is dictated by the degree of hypoplasia of the underlying vasculature. following acute therapies, which often include surgical repair, mechanical ventilation with inhaled no, and extracorporeal support, subacute and chronic pulmonary hypertension has been increasingly recognized as a major outcome variable in these patients. because ultimately lung and vascular growth are necessary to reverse the disease process, aggressive long-term support with agents that inhibit vascular remodeling (i.e., et receptor antagonists and pde inhibitors) is an emerging treatment approach to support these infants as they grow. maladaptation of the lung represents a stress event at the time of delivery that does not allow the normal dilating stimuli, such as increases in systemic arterial ph and oxygen tension, to occur. this may occur in the setting of apnea, pneumonia, sepsis, and aspiration of meconium or amniotic fl uid [ ] [ ] [ ] . the underlying pulmonary vasculature is often normal, and, thus, these neonates are likely to respond to vasodilator therapy and the correction of contributory metabolic abnormalities. maldevelopment of the lung represents a group of conditions in which the vasculature is thickened and abnormally distributed. for example, some newborns who die from persistent pulmonary hypertension have abnormally muscular pulmonary vascular beds, even when they die on the fi rst day of life. in particular, they have thickened muscular coats in the normally muscular preacinar arteries, and extension of muscle into the normally nonmuscular intra-acinar arteries [ , ] . because vascular remodeling takes time to develop, it has been hypothesized that this increased muscularization is caused by a chronic intrauterine stress. in animal models, this pathophysiology can be mimicked by chronic placental insuffi ciency, fetal hypoxemia, chronic constriction of the ductus arteriosus, and chronic no inhibition [ , , [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, pphn has been associated with maternal indomethacin use, which causes constriction of the ductus arteriosus [ , ] . the response to therapy in neonates with maldevelopment of the lung is variable and may be related to the extent and type of underlying structural vascular pathology. the primary therapeutic approach is to decrease pulmonary vascular resistance and support myocardial function. the specifi c treatment modality depends on the underlying etiology. if the cause is perinatal asphyxia, correcting alveolar hypoxia, hypercarbia, and metabolic acidosis by ventilation with % oxygen, and by administration of buffer, should decrease pulmonary vascular tone toward normal levels. if parenchymal disease (i.e., respiratory distress syndrome, meconium aspiration, or pneumonia) is causing pulmonary vasospasm due to alveolar hypoxia and hypercarbia, then infl ation of the alveoli with positive end-expiratory pressure, surfactant administration, and mechanical ventilation may reverse the pulmonary hypertension [ ] [ ] [ ] . the near-term child can exert substantial intrathoracic pressure opposing mechanical ventilation; thus, sedation and occasionally muscle paralysis may be necessary to obtain stable mechanical ventilation [ ] . when treatment of the underlying pulmonary parenchymal, infectious, or infl ammatory disease is ineffective, or if there is no such underlying disease, therapy is directed at reversing abnormal pulmonary vasoconstriction. this is generally accomplished with sedation, mechanical ventilation with % oxygen, and alkalinization. when further pulmonary vasodilation is needed, inhaled no is utilized with or without high-frequency ventilation. in fact, several multicentered, randomized trials have demonstrated that inhaled no improves oxygenation and decreases the need for extracorporeal life support in newborns with persistent pulmonary hypertension [ , , ) , although no differences in mortality were noted. the use of extracorporeal membrane oxygenation has substantially decreased overall mortality for most subsets of pphn. however, overall mortality rates remain substantial at %- % [ ] [ ] [ ] [ ] . the development of pulmonary hypertension and increased pulmonary vascular reactivity is associated with two major types of congenital heart disease: ( ) those with increased pulmonary blood fl ow and pulmonary arterial pressure and ( ) those with increased pulmonary venous pressure [ ] [ ] [ ] ] . after birth, large communications at the level of the ventricles or great vessels result in increased pulmonary blood fl ow and pulmonary arterial pressure, which produces progressive structural and functional abnormalities of the pulmonary vasculature [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . similarly, elevated pulmonary venous pressure results in progressive increases in pulmonary venous and arterial pressure, which produces structural abnormalities of the pulmonary vasculature. heath and edwards fi rst described the progression of these pulmonary vascular changes in [ ] . in their classifi cation, changes progress from medial hypertrophy (grade i) to intimal hyperplasia (grade ii), lumen occlusion (grade iii), arterial dilatation (grade iv), angiomatoid formation (grade v) and fi brinoid necrosis (grade vi). in addition, morphometric analysis shows progression of disturbed arterial growth and remodeling of the pulmonary vascular bed, which correlates with the aberrant hemodynamic state of the pulmonary circulation [ , , [ ] [ ] [ ] . these changes are characterized by ( ) abnormal extension of vascular smooth muscle into small peripheral pulmonary arteries and mild medial hypertrophy of normally muscular arteries (grade a), ( ) severe medial hypertrophy of normally muscular arteries (grade b), and ( ) decreased pulmonary arterial number (grade c). uncorrected, these vascular changes result in decreased cross-sectional area and obliteration of the pulmonary vascular bed and death secondary to severe cyanosis or myocardial failure. different congenital heart defects vary considerably in the frequency and severity of pulmonary hypertension. the risks and frequencies of developing advanced pulmonary vascular disease (pvd) for particular heart defects are summarized in table . . importantly, children with trisomy and congenital heart defects often have an accelerated development of advanced pulmonary vascular disease [ ] . this may be secondary to confounding factors such as airway obstruction or another unidentifi ed predisposition. after surgical correction, early vascular changes (grades i-iii, grades a, b) are reversible; however, more severe changes are irreversible and progressive. therefore, the pathophysiologic state of the pulmonary circulation is the main determinant of the clinical course and the success of surgical treatment, and it explains the trend toward early repair of congenital heart defects [ ] . although early surgical repair of congenital heart defects has decreased the incidence of irreversible pulmonary vascular disease, even those children with reversible vascular changes suffer morbidity and mortality in the perioperative period secondary to chronic and/or acute elevations in pulmonary vascular resistance ] . chronic elevations are related to the structural changes that decrease the cross-sectional area of the pulmonary vascular bed. these alterations may take several months to normalize following surgical repair. acute elevations in pulmonary vascular resistance are often seen immediately following surgery with cardiopulmonary bypass, when there is often a period of enhanced pulmonary vascular reactivity [ , , ] . this period may last up to - days and is most likely a manifestation of preexisting aberrant endothelial cell-smooth muscle cell interactions that are exacerbated at the time of surgery. during cardiopulmonary bypass, several factors including the disruption of pulmonary blood fl ow, complement activation, and neutrophil activation induce pulmonary vascular endothelial dysfunction. this results in an increase in the production and/or release of endothelial factors that promote vasoconstriction, such et- and txa , and a decrease in endothelial relaxing factors, most importantly no [ ] . this period of extreme reactivity may produce severe hypoxemia, acidosis, low cardiac output, and death if not treated immediately. classically, a preoperative determination of pulmonary vascular reactivity is made in the cardiac catheterization laboratory in order to assess the operability of a given patient, that is, the degree to which the pulmonary vascular disease is reversible, as well as the postoperative risk. this testing involves measuring pulmonary arterial pressure and calculating pulmonary vascular resistance under varying conditions. the vascular resistance following acute maximal vasodilator therapy (e.g., oxygen and no) represents the degree of structural pulmonary vascular disease that is present. despite the frequent utilization of such testing, there is no absolute pulmonary vascular resistance that is universally considered inoperable. in general, a larger reduction in resistance in response to vasodilator therapy correlates with an increased chance of reversibility and a lower risk of perioperative morbidity from pulmonary hypertension. recent studies suggest that the combination of % oxygen and inhaled no produces maximal pulmonary vasodilation and has some perioperative predictive value [ , ] . in fact, a % decrease in the ratio of the pulmonary-to-systemic vascular resistance with vasodilator therapy, and a nadir in this ratio of less than %, was % sensitive and % accurate in predicting a good surgical outcome. therefore, the combination of oxygen and inhaled no is now most commonly used for pulmonary vascular reactivity testing. reactivity testing may also be helpful in the intensive care unit in the setting of a persistent postoperative elevation of pulmonary arterial pressure in order to differentiate between residual anatomic defects and prolonged periods of increased tone [ ] . the optimal treatment for perioperative pulmonary hypertensive morbidity is prevention with early surgical repair. it is increasingly clear that the longer the pulmonary vasculature is exposed to the abnormal forces associated with increased blood fl ow and/or pressure, the greater the risk of perioperative pulmonary vascular reactivity. following surgery, the goal of perioperative management is to minimize active pulmonary vasoconstriction during the period of exaggerated reactivity and support the right ventricle. to this end, avoidance of those stimuli that increase pulmonary vascular resistance (hypoxia, acidosis, pain, agitation, increased intrathoracic pressure) is critical. continuous pulmonary arterial and right atrial pressure monitoring is often helpful by allowing prompt recognition of pulmonary hypertensive crises and evaluation of the response to therapeutic maneuvers. monitoring systemic arterial pressure and systemic arterial oxygen saturation is essential in that it allows changes in pulmonary arterial pressure to be interpreted in the context of the total cardiopulmonary response. for example, if systemic and pulmonary arterial pressures increase in response to pain and agitation, but right atrial pressure does not increase, and systemic perfusion and oxygen saturation remain adequate, then specifi c treatment directed at the pulmonary vasculature is not necessary. conversely, increases in pulmonary arterial pressure that are associated with increased right atrial pressure, decreased systemic pressure, and/or decreased systemic saturation might herald imminent collapse. the objective of vasodilator therapy is to decrease right ventricular afterload and prevent acute increases in pulmonary arterial pressure. inhaled no, in combination with oxygen, has been increasingly utilized because of its potent vasodilating effects, pulmonary selectivity, and rapid onset and elimination (see earlier). several studies demonstrate its potent vasodilating effects in this population [ ] [ ] [ ] [ ] ; however, large, randomized trails are lacking. one randomized trial did demonstrate that inhaled no decreased postoperative pulmonary vascular resistance, the incidence of pulmonary hypertensive crises, and the days of mechanical ventilation compared with placebo [ ] . in patients with a history of pulmonary venous hypertension (total anomalous pulmonary venous return, mitral valve disease), aggressive diuresis may be helpful because interstitial pulmonary edema may contribute signifi cantly to elevations in pulmonary vascular resistance. therapies that maintain an adequate cardiac output in this patient population are not dissimilar to therapies utilized in other patient populations, with the exception of the particular emphasis placed on right ventricular afterload reduction and support. it is noteworthy that patients with a poorly compliant right ventricle or with increased right ventricular afterload may require elevated central venous pressures to maintain an adequate preload. in addition, the use of inotropic agents with signifi cant α -adrenergic effect should be minimized to avoid the associated pulmonary vasoconstriction. agents such as dobutamine, milrinone, and dopamine are routinely utilized. the use of high levels of positive end-expiratory pressure (peep) is somewhat controversial. mechanical hyperventilation with high peep increases intrathoracic pressure and pulmonary vascular resistance [ , ] . this therapy should be avoided if adequate systemic arterial saturation can be achieved by other means. however, at low lung volumes, the use of peep may increase lung volume until very recently, pulmonary arterial hypertension of unknown etiology was termed primary pulmonary hypertension. however, recent evidence indicates a genetic disposition in a subset of patients with primary pulmonary hypertension, and a number of diseases that lead to pulmonary arterial hypertension with similar histological and pathophysiologic features have been uncovered [ ] [ ] [ ] [ ] . thus, at the third world symposium on pulmonary arterial hypertension, a new classifi cation was proposed to further classify primary pulmonary hypertension into the following subgroups: ( ) idiopathic pulmonary arterial hypertension (ipah), ( ) familial pulmonary arterial hypertension (fpah), and ( ) pulmonary arterial hypertension related to risk factors or associated conditions (apah) [ ] . unfortunately, mortality from primary pulmonary hypertension remains high and may be higher for children than adults. in fact, the primary pulmonary hypertension national institutes of health registry reports a median survival of only months for pediatric patients [ ] . however, recent data suggest that pediatric patients may respond differently than adults to new therapies and that these differences may portend a better outcome in younger patients [ , ] . the frequency of primary pulmonary hypertension in pediatric patients is not known, but it appears that the number of confi rmed cases is increasing. the incidence is slightly increased in females [ ] . the most common causes of death in children with primary pulmonary hypertension are right ventricular failure and sudden death, which may be related to malignant cardiac arrhythmias, pulmonary emboli, or acute right ventricular ischemia [ ] . physicians caring for children in an intensive care unit setting must be cognizant of this disorder, albeit rare, because relatively benign disease processes, such as pneumonia, can be life threatening for children with primary pulmonary hypertension, which may not have been previously identifi ed. as opposed to adults with primary pulmonary hypertension, who often have severe plexiform lesions resulting in relatively fi xed vascular changes, children display greater medial hypertrophy with less intimal fi brosis and fewer plexiform lesions [ , ] . in addition, pediatric patients have a decreased pulmonary arterial number and increased pulmonary vascular reactivity compared with adult patients. the molecular mechanisms underlying primary pulmonary hypertension remain speculative; however, studies suggest an integral role for endothelial dysfunction, resulting in an increase in factors that favor both vasoconstriction and mitogenesis, such as et- and txa , and a decrease in factors that promote vasodilation and smooth muscle antiproliferation, such as no and prostacyclin [ , , , [ ] [ ] [ ] [ ] . other mechanisms have been investigated including, altered gene expression, coagulation abnormalities (resulting in intravascular thrombosis), and defects of pulmonary vascular smooth muscle cell potassium channels [ ] [ ] [ ] . recent advances in the understanding of pulmonary hypertension have established an association with a number of disease processes and toxins. thus, it is now known that pulmonary hypertension can be related to collagen vascular disease, portal hypertension, human immunodefi ciency virus infection, chronic obstructive pulmonary disease, interstitial lung disease, sleepdisordered breathing, alveolar hypoventilation disorders, chronic exposure to high altitudes, thromboembolic disease, sickle cell disease, schistosomiasis, sarcoidosis, thyroid disorders, glycogen storage disease, gaucher disease, hereditary hemorrhagic telangiectasia, myeloproliferative disorders, and pulmonary capillary hemangiomatosis. in addition, drugs or toxins, most notably anorexigens, have been associated with the development of pulmonary hypertension [ , ] . in general the diagnostic work-up includes history and physical examination, electrocardiography, chest radiography, echocardiography, and cardiac catheterization. serologic evaluation in order to exclude secondary causes is required, and v/q scanning to evaluate for pulmonary emboli may be necessary. treatment strategies for pediatric pulmonary arterial hypertension are evolving. when the disease is associated with a known disorder, treatment must include specifi c therapy aimed at the underlying condition. however, general treatments include the approach reviewed above, with oxygen, calcium channel blockers, anticoagulation, et receptor antagonists, prostacyclin analogues, acute and chronic inhaled no, pde type inhibitors, atrial septostomy, and lung or heart-lung transplant considerations as indicated [ ] . patients with pulmonary arterial hypertension have histologic evidence of in situ pulmonary vascular thrombosis, which is the rationale for anticoagulation therapy. although several adult studies have demonstrated its effi cacy, pediatric studies are lacking [ , ] . currently, warfarin is the treatment of choice for adult patients and in large pediatric centers with signifi cant experience with pediatric pulmonary arterial hypertension. low-molecularweight heparin is another alternative [ ] ; aspirin does not have any demonstrated effi cacy. chronic calcium channel blockade is effi cacious for a subset of adults and children with pulmonary arterial hypertension. in fact, whereas less than % of adults respond to calcium channel blockers, up to % of children are positive responders [ , ] . it is worth noting that calcium channel blockers are not utilized in the management of other common causes of pediatric pulmonary hypertension, such as pphn and congenital heart disease. indeed, calcium channel blockade should be avoided in neonates and after congenital heart surgery. however, studies indicate that long-acting calcium channel blockers, such as nifedipine and amlodipine, are well tolerated in children with primary pulmonary hypertension. an important caveat is that a positive response to calcium channel blockers (i.e., an acute reduction in pulmonary arterial pressure) must be demonstrated as a part of acute vasodilator reactivity testing. children without a positive acute response do not benefi t from chronic treatment. prostaglandins are a mainstay of therapy for patients with pulmonary arterial hypertension. in general, prostacyclin (epoprostenol) is administered as a continuous infusion, necessitating a permanent indwelling central catheter, with its associated risks [ ] [ ] [ ] [ ] [ ] . however, various other formulations including oral, inhaled, and subcutaneous prostacyclin analogues have been developed and are in various stages of clinical investigation [ ] [ ] [ ] [ ] . supplemental oxygen, cardiac glycosides, antiarrhythmic therapy, and inotropic agents are also variably utilized in certain patients [ , ] . diuretic therapy is also often benefi cial, keeping in mind that these patients may require elevated right ventricular preload. based on an expanding understanding of the disease process, future therapies might include elastase inhibitors and gene therapy [ , ] . as noted previously, atrial septostomy may have a role in the management of a select group of patients [ ] . however, atrial septostomy in the setting of an acute exacerbation of chronic pulmonary hypertension may lead to unacceptable hypoxemia because of excess right-to-left atrial shunting. finally, heart-lung, single-lung, or bilateral lung transplantation has been successful in pediatric patients with terminal pulmonary hypertension [ , ] . the international society for heart and lung transplantation reports survival of approximately % at years in pediatric patients [ ] . consensus is lacking as to the best type of transplant. increases in pulmonary arterial pressure in response to hypoxia are well described. clinical and experimental evidence suggests that prolonged exposure or chronic intermittent exposure to hypoxia can result in functional and structural derangements of the pulmonary vasculature, leading to pulmonary hypertension [ ] [ ] [ ] . fortunately, elevations in pulmonary arterial pressure that occur in response to acute hypoxia (such as an acute ascent in altitude) are rapidly reversible. interestingly, there is great clinical variability in the response to hypoxia. for example, increased susceptibility to high-altitude pulmonary edema, which is associated with increased pulmonary arterial pressure, has been linked to certain major histocompatibility complexes [ , ] . the mechanisms of hypoxia-induced pulmonary hypertension continue to be an area of intense investigation. to date the precise mechanisms remain unclear, but it is known that a number of endothelial derived factors, such as no, et- , leukotrienes, and potassium channels, participate [ , [ ] [ ] [ ] [ ] . furthermore, additional genetic polymorphisms are also under investigation. pediatricians must consider this physiology in patients with conditions such as upper airway obstruction, central hypoventilation, and neuromuscular disorders that affect ventilation. in fact, many of these patients do develop evidence of pulmonary hypertension, with right ventricular enlargement. in most cases, addressing the underlying pathology is curative, but it can take some time to fully reverse the structural changes that have occurred. the pathophysiology of acute lung injury involves damage to both the alveolar epithelium and pulmonary vascular endothelium. vascular endothelial injury accounts for key features of acute lung injury, including intravascular thrombosis and capillary permeability that increases alveolar fl uid [ ] . in fact, pulmonary vascular injury, in the setting of acute lung injury, can lead to pulmonary arterial hypertension, resulting in increased intrapulmonary shunting, hypoxia, pulmonary edema, and right ventricular dysfunction [ ] [ ] [ ] [ ] . in children with acute lung injury, persistently elevated pulmonary arterial pressures have been associated with worse outcomes [ ] ; therefore, vasodilators have been utilized in the management of these patients. however, intravenous vasodila-tors that dilate both the systemic and pulmonary vasculature have signifi cant problems, including systemic hypotension, right ventricular ischemia, increased intrapulmonary shunting (i.e., increased v/q mismatch), and increased hypoxemia [ ] [ ] [ ] [ ] [ ] . consequently, selective pulmonary vasodilation with inhaled no has been utilized, as it improves v/q matching and oxygenation without untoward systemic effects [ , ] . unfortunately, improvements in oxygenation associated with inhaled no are transient, and large randomized trials have failed to demonstrate an improvement in mortality with its use [ , , ] . the routine use of inhaled no in patients with acute lung injury, therefore, cannot be justifi ed; however, it may be indicated in individual patients, particularly those with an acute hemodynamic compromise and refractory hypoxemia caused by elevated pulmonary arterial pressures. clearly, physicians caring for pediatric patients with acute lung injury must include an awareness of the pulmonary vascular aberrations associated with the disease in their management considerations. historically, diseases of the pulmonary vasculature, although not uncommon, have been underrecognized. this was caused, in part, by the paucity of effective treatments as well as an incomplete understanding of the vascular biologic mechanisms. in fact, over the past decade, the therapeutic gold standard has been the continuous infusion of prostacyclin. although certainly extending and improving the lives of many patients, intravenous prostacyclin administration has been predominantly limited to patients with irreversible disease, given the inconvenience and morbidity associated with its delivery. fortunately, an expanded understanding of the vascular endothelium, vascular smooth muscle cells, and the role of their interactions in the pathophysiology of pulmonary vascular disease have resulted in new effective treatments, with additional potential therapies evolving rapidly. oral agents such as bosentan and sildenafi l are two examples with great promise. in addition, accumulated experience and focused research have uncovered a multitude of disease processes that contribute directly or indirectly to the development of pulmonary hypertension. physicians caring for critically ill children must be aware of these illnesses, the pathophysiology of pulmonary hypertension, and the available treatment options in order to translate these advances into improved outcomes for patients. cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate a novel potent vasoconstrictor peptide produced by vascular endothelial cells vascular endothelial cells synthesize nitric oxide from l-arginine flow-induced release of endothelium-derived relaxing factor molecular mechanisms of endothelium-mediated vasodilation the l-arginine-nitric oxide pathway n omega-nitro-l-arginine attenuates endothelium-dependent pulmonary vasodilation in lambs endothelial modulation of pulmonary vascular tone nitric oxide-generating vasodilators and -bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells edrf inhibition augments pulmonary hypertension in intact newborn lambs in vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue m&b , a cgmp phosphodiesterase inhibitor, is a pulmonary vasodilator in lambs nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia endothelial dysfunction in pulmonary hypertension vascular endothelial growth factor increases endothelinconverting enzyme expression in vascular endothelial cells tone-dependent responses to endothelin in the isolated perfused fetal sheep pulmonary circulation in situ endothelin- produces pulmonary vasodilation in the intact newborn lamb developmental effects of endothelin- on the pulmonary circulation in sheep circulatory effects of endothelin in newborn piglets maturational changes in endothelium-derived relaxations in newborn piglet pulmonary circulation circulatory changes during growth in the fetal lamb impairment of endotheliumdependent pulmonary artery relaxation in children with congenital heart disease and abnormal pulmonary hemodynamics reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension expression of endothelin- in the lungs of patients with pulmonary hypertension altered endothelium-dependent responses in lambs with pulmonary hypertension and increased pulmonary blood fl ow morphological development of the pulmonary vascular bed in fetal lambs effects of birth-related events on blood fl ow distribution fetal and neonatal pulmonary circulation changes in the lungs of the new-born lamb control of the fetal and neonatal pulmonary circulation distribution and regulation of blood fl ow in the fetal and neonatal lamb regulation of pulmonary vascular tone in the perinatal period effect of lung expansion on the fetal lamb circulation the onset of breathing at birth stimulates pulmonary vascular prostacyclin synthesis increasing oxygen tension dilates fetal pulmonary circulation via endothelium-derived relaxing factor oxygen modulates endotheliumderived relaxing factor production in fetal pulmonary arteries pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn effects of birth-related stimuli on l-arginine-dependent pulmonary vasodilation in ovine fetus ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs endothelin- in congenital heart disease increased pulmonary blood fl ow alters the molecular regulation of vascular reactivity in the lamb altered regulation of the et- cascade in lambs with increased pulmonary blood fl ow and pulmonary hypertension alterations in nitric oxide production in -week-old lambs with increased pulmonary blood fl ow increased endothelial nos in lambs with increased pulmonary blood fl ow and pulmonary hypertension emergence of smooth muscle cell endothelin bmediated vasoconstriction in lambs with experimental congenital heart disease and increased pulmonary blood fl ow sgc and pde are elevated in lambs with increased pulmonary blood fl ow and pulmonary hypertension altered endothelial function in lambs with pulmonary hypertension and acute lung injury alterations in tgf-beta expression in lambs with increased pulmonary blood fl ow and pulmonary hypertension alterations in et- , not nitric oxide, in -week-old lambs with increased pulmonary blood fl ow in utero placement of aortopulmonary shunts. a model of postnatal pulmonary hypertension with increased pulmonary blood fl ow in lambs the pathophysiology of pulmonary hypertension in congenital heart disease altered endothelium-dependent relaxations in lambs with high pulmonary blood fl ow and pulmonary hypertension endothelin- vasoactive responses in lambs with pulmonary hypertension and increased pulmonary blood fl ow failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs persistent fetal circulation: newly recognized structural features the structural basis of persistent pulmonary hypertension of the newborn infant pharmacologic therapy for persistent pulmonary hypertension of the newborn: as "poly" as the disease itself structure and function in pulmonary hypertension. new perceptions elevated immunoreactive endothelin- levels in newborn infants with persistent pulmonary hypertension loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia an imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension pulmonary pkg- is upregulated following chronic hypoxia models of persistent pulmonary hypertension of the newborn (pphn) and the role of cyclic guanosine monophosphate (gmp) in pulmonary vasorelaxation physical factors affecting normal and serotonin-constricted pulmonary vessels response of the pulmonary vasculature to hypoxia and h+ ion concentration changes site and sensitivity for stimulation of hypoxic pulmonary vasoconstriction infl uence of alveolar oxygen on pulmonary vasoconstriction in newborn lambs versus sheep hypoxic pulmonary vasoconstriction. physiologic signifi cance, mechanism, and clinical relevance hypoxic pulmonary vasoconstriction increased arterial ph, not decreased paco , attenuates hypoxia-induced pulmonary vasoconstriction in newborn lambs the effect of hypocarbia on the cardiovascular system of puppies pulmonary vascular k+ channel expression and vasoreactivity in a model of congenital heart disease the nervous control of the circulation in the foetal and newly expanded lungs of the lamb physiology and pharmacology of the pulmonary circulation in the fetus and newborn poiseuille's law and its limitations in vascular systems mechanics of the pulmonary circulation applied hydro-and aeromechanics congenital diseases of the heart: clinical-physiological considerations relative viscosity of blood at varying hematocrits in pulmonary circulation effect of hematocrit and inertial losses on pressure-fl ow relations in the isolated hindpaw o the dog mechanical infl uences on the pulmonary microcirculation recruitment vs. distensibility in the pulmonary vascular bed relationship between regional lung volume and regional pulmonary vascular resistance infl uence of state of infl ation of the lung on pulmonary vascular resistance biosynthesis of nitric oxide and citrulline from l-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical pharmacology of endotheliumderived nitric oxide and nitrovasodilators activation of purifi ed soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid cyclic guanosine monophosphate as a mediator of vasodilation nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation cloning and expression of a cdna encoding an endothelin receptor cloning of a cdna encoding a non-isopeptide-selective subtype of the endothelin receptor regulatory functions of the vascular endothelium regulation of the cerebral circulation: role of endothelium and potassium channels pulmonary artery endothelial abnormalities in patients with congenital heart defects and pulmonary hypertension. a correlation of light with scanning electron microscopy and transmission electron microscopy impairment of pulmonary endothelium-dependent relaxation in patients with eisenmenger's syndrome development of crotalaria pulmonary hypertension: hemodynamic and structural study plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. evidence for increased production of endothelin in pulmonary circulation heterozygous germline mutations in bmpr , encoding a tgf-beta receptor, cause familial primary pulmonary hypertension. the international pph consortium sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding bmpr-ii, a receptor member of the tgf-beta family mutation in the gene for bone morphogenetic protein receptor ii as a cause of primary pulmonary hypertension in a large kindred bmpr germline mutations in pulmonary hypertension associated with fenfl uramine derivatives lung biopsy in congenital heart disease: a morphometric approach to pulmonary vascular disease ultrastructural fi ndings in lung biopsy material from children with congenital heart defects quantitative structural analysis of pulmonary vessels in isolated ventricular septal defect in infancy pulmonary vascular disease in different types of congenital heart disease. implications for interpretation of lung biopsy fi ndings in early childhood pulmonary vascular disease with congenital heart lesions: pathologic features and causes pulmonary hypertension in children: perioperative management diagnosis and management of postoperative pulmonary hypertensive crisis transient myocardial ischemia of the newborn infant: a form of severe cardiorespiratory distress in full-term infants persistence of atrioventricular valve regurgitation and electrocardiographic abnormalities following transient myocardial ischemia of the newborn lung mechanics in congenital heart disease with increased and decreased pulmonary blood fl ow a randomized, controlled study of the -hour and -hour effects of inhaled nitric oxide therapy in children with acute hypoxemic respiratory failure inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. the neonatal inhaled nitric oxide study group inhaled nitric oxide in the neonate with cardiac disease low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. clinical inhaled nitric oxide research group effects of inhaled nitric oxide in patients with acute respiratory distress syndrome: results of a randomized phase ii trial. inhaled nitric oxide in ards study group multicenter randomized controlled trial of the effects of inhaled nitric oxide therapy on gas exchange in children with acute hypoxemic respiratory failure inhaled nitric oxide therapy for persistent pulmonary hypertension of the newborn inhaled nitric oxide in congenital hypoplasia of the lungs due to diaphragmatic hernia or oligohydramnios randomized, multicenter trial of inhaled nitric oxide and high-frequency oscillatory ventilation in severe, persistent pulmonary hypertension of the newborn inhaled nitric oxide therapy inhaled nitric oxide and persistent pulmonary hypertension of the newborn. the inhaled nitric oxide study group the effects of inhaled nitric oxide on postoperative pulmonary hypertension in infants and children undergoing surgical repair of congenital heart disease inhaled nitric oxide in premature infants with the respiratory distress syndrome inhaled nitric oxide, oxygen, and alkalosis: dose-response interactions in a lamb model of pulmonary hypertension effects of nitroprusside and dopamine on pulmonary arterial vasculature in children after cardiac surgery comparison of effects of prostaglandin e and nitroprusside on pulmonary vascular resistance in children after open-heart surgery hemodynamic effects of nifedipine and oxygen in children with pulmonary hypertension. cardiovasc does prostacyclin enhance the selective pulmonary vasodilator effect of oxygen in children with congenital heart disease? hemodynamic effects of prostaglandin e in patients with congenital heart disease and pulmonary hypertension comparison between prostaglandin e and epoprostenol (prostacyclin) in infants after heart surgery use of atp-mgcl in the evaluation and treatment of children with pulmonary hypertension secondary to congenital heart defects refractory hypoxemia associated with neonatal pulmonary disease: the use and limitations of tolazoline hemodynamic effects of pulmonary arterial infusion of vasodilators in newborn lambs comparative studies on the hemodynamic effects of prostaglandin e prostacyclin, and tolazoline upon elevated pulmonary vascular resistance in neonatal swine comparison of ketanserin and sodium nitroprusside in patients with severe ards prostaglandin e and nitroglycerin reduce pulmonary capillary pressure but worsen ventilation-perfusion distributions in patients with adult respiratory distress syndrome the pathophysiology of failure in acute right ventricular hypertension: hemodynamic and biochemical correlations prostaglandin e in the adult respiratory distress syndrome. benefi t for pulmonary hypertension and cost for pulmonary gas exchange prostacyclin for the treatment of pulmonary hypertension in the adult respiratory distress syndrome: effects on pulmonary capillary pressure and ventilation-perfusion distributions inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right heart dysfunction, and refractory hypoxemia after cardiothoracic surgery inhaled epoprostenol (prostacyclin) and pulmonary hypertension before cardiac surgery inhaled prostacyclin for term infants with persistent pulmonary hypertension refractory to inhaled nitric oxide the use of inhaled prostacyclin in nitroprusside-resistant pulmonary artery hypertension inhaled prostacyclin (pgi ) is an effective addition to the treatment of pulmonary hypertension and hypoxia in the operating room and intensive care unit aerosolized prostacyclin (epoprostenol) as an alternative to inhaled nitric oxide for patients with reperfusion injury after lung transplantation inhaled aerosolized prostacyclin and pulmonary hypertension during anesthesia for lung transplantation effects of inhaled prostacyclin analogue on chronic hypoxic pulmonary hypertension dose-response to inhaled aerosolized prostacyclin for hypoxemia due to ards inhaled prostacyclin in the treatment of pulmonary hypertension inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fi brosis cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms milrinone decreases both pulmonary arterial and venous resistances in the hypoxic dog van trigt p rd. milrinone improves pulmonary hemodynamics and right ventricular function in chronic pulmonary hypertension hemodynamic and inotropic effects of milrinone after heart transplantation in the setting of recipient pulmonary hypertension milrinone: systemic and pulmonary hemodynamic effects in neonates after cardiac surgery effi cacy and safety of milrinone in preventing low cardiac output syndrome in infants and children after corrective surgery for congenital heart disease sildenafi l for primary and secondary pulmonary hypertension benefi cial effect of oral sildenafi l therapy on childhood pulmonary arterial hypertension: twelve-month clinical trial of a single-drug, open-label, pilot study interaction between inhaled nitric oxide and intravenous sildenafi l in a porcine model of meconium aspiration syndrome intravenous sildenafi l and inhaled nitric oxide: a randomised trial in infants after cardiac surgery effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebocontrolled study bosentan therapy for pulmonary arterial hypertension surgical implications of pulmonary hypertension in congenital heart disease the effects of dopamine and isoproterenol on the pulmonary circulation acute circulatory effects of dopamine in patients with pulmonary hypertension dobutamine pharmacokinetics and cardiovascular responses in critically ill neonates dobutamine: a hemodynamic evaluation in children with shock effects of vasoactive drugs on thromboxane a mimetic-induced pulmonary hypertension in newborn lambs factors that infl uence the outcome of primary pulmonary hypertension atrial septostomy as palliative therapy for refractory primary pulmonary hypertension double balloon technique for dilation of valvular or vessel stenosis in congenital and acquired heart disease successful palliation of primary pulmonary hypertension by atrial septostomy blade balloon atrial septostomy in patients with severe primary pulmonary hypertension graded balloon dilation atrial septostomy in severe primary pulmonary hypertension. a therapeutic alternative for patients nonresponsive to vasodilator treatment long-term treatment of primary pulmonary hypertension with continuous intravenous epoprostenol (prostacyclin) survival in primary pulmonary hypertension with long-term continuous intravenous prostacyclin a comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. the primary pulmonary hypertension study group primary pulmonary hypertension: improved long-term effects and survival with continuous intravenous epoprostenol infusion reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension vasodilator therapy for primary pulmonary hypertension in children pulmonary arterial hypertension in children survival with fi rst-line bosentan in patients with primary pulmonary hypertension treatment of rebound and chronic pulmonary hypertension with oral sildenafi l in an infant with congenital diaphragmatic hernia ventilatory predictors of pulmonary hypoplasia in congenital diaphragmatic hernia, confi rmed by morphologic assessment unsuspected pulmonary vascular abnormalities associated with diaphragmatic hernia morphologic analysis of the pulmonary vascular bed in congenital left-sided diaphragmatic hernia mortality from neonatal diaphragmatic hernia vasodilator response and prediction of survival in congenital diaphragmatic hernia pulmonary hypertension in the perinatal aspiration syndromes beta-hemolytic streptococcal infection appearing as persistent fetal circulation persistent pulmonary hypertension: assessment of perinatal risk factors umbilical cord compression produces pulmonary hypertension in newborn lambs: a model to study the pathophysiology of persistent pulmonary hypertension in the newborn hemodynamic, pulmonary vascular, and myocardial abnormalities secondary to pharmacologic constriction of the fetal ductus arteriosus. a possible mechanism for persistent pulmonary hypertension and transient tricuspid insufficiency in the newborn infant ligating the ductus arteriosus before birth causes persistent pulmonary hypertension in the newborn lamb ligating the ductus arteriosus before birth remodels the pulmonary vasculature of the lamb chronic pulmonary hypertension in utero impairs endothelium-dependent vasodilation pulmonary endothelial no synthase gene expression is decreased in fetal lambs with pulmonary hypertension possible association between maternal indomethacin therapy and primary pulmonary hypertension of the newborn morphologic analysis of the pulmonary vascular bed in infants exposed in utero to prostaglandin synthetase inhibitors the therapeutic application of end-expiratory pressure in the meconium aspiration syndrome effects of peep and tolazoline infusion on respiratory and inert gas exchange in experimental meconium aspiration surfactant treatment of full-term newborns with respiratory failure respiratory paralysis to improve oxygenation and mortality in large newborn infants with respiratory distress national experience with extracorporeal membrane oxygenation for newborn respiratory failure. data from cases extracorporeal membrane oxygenation and conventional medical therapy in neonates with persistent pulmonary hypertension of the newborn: a prospective randomized study extracorporeal membrane oxygenation trials ecmo for neonatal respiratory failure the pulmonary circulation: remodeling in growth and disease. the j. burns amberson lecture vascular structure in lung tissue obtained at biopsy correlated with pulmonary hemodynamic fi ndings after repair of congenital heart defects quantitative structural study of pulmonary circulation in the newborn with aortic atresia, stenosis, or coarctation the pathology of hypertensive pulmonary vascular disease; a description of six grades of structural changes in the pulmonary arteries with special reference to congenital cardiac septal defects down's syndrome, complete atrioventricular canal, and pulmonary vascular obstructive disease effects of surgical closure of ventricular septal defects upon pulmonary vascular disease use of inhaled nitric oxide and acetylcholine in the evaluation of pulmonary hypertension and endothelial function after cardiopulmonary bypass combined effects of nitric oxide and oxygen during acute pulmonary vasodilator testing inhaled nitric oxide as a preoperative test (inop test i): the inop test study group inhaled nitric oxide and congenital cardiac disease inhaled nitric oxide therapy after fontan-type operations inhaled nitric oxide in pediatric cardiac surgery inhaled nitric oxide and prevention of pulmonary hypertension after congenital heart surgery: a randomised double-blind study clinical classifi cation of pulmonary hypertension survival in patients with primary pulmonary hypertension. results from a national prospective registry comparison of primary plexogenic arteriopathy in adults and children. a morphometric study in patients primary pulmonary hypertension in children: clinical characterization and survival primary pulmonary hypertension. a national prospective study relation of arachidonate metabolites to abnormal control of the pulmonary circulation in a child practical method for quantifying hearing aid benefi t in older adults increased plasma endothelin- in pulmonary hypertension: marker or mediator of disease? nitric oxide and endothelin- in pulmonary hypertension dysfunctional voltage-gated k+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension abnormal endothelial factor viii associated with pulmonary hypertension and congenital heart defects von willebrand factor abnormalities in primary pulmonary hypertension primary pulmonary hypertension: natural history and the importance of thrombosis the effect of anticoagulant therapy in primary and anorectic drug-induced pulmonary hypertension inhibition of hypoxic pulmonary hypertension by heparins of differing in vitro antiproliferative potency the effect of high doses of calciumchannel blockers on survival in primary pulmonary hypertension short-term hemodynamic effect of a new oral pgi analogue, beraprost, in primary and secondary pulmonary hypertension effect of orally active prostacyclin analogue on survival of outpatients with primary pulmonary hypertension long-term treatment of primary pulmonary hypertension with aerosolized iloprost, a prostacyclin analogue treatment of pulmonary hypertension with the continuous infusion of a prostacyclin analogue, iloprost the short-term effects of digoxin in patients with right ventricular dysfunction from pulmonary hypertension elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-c antisense prevents progression, of vascular disease complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor role of atrial septostomy in the treatment of pulmonary vascular disease paediatric lung transplantation lung transplantation for pulmonary vascular disease registry for the international society for heart and lung transplantation: seventh offi cial pediatric report- reversal of high altitude pulmonary hypertension lung vascular smooth muscle as a determinant of pulmonary hypertension at high altitude endothelial and subintimal changes in rat hilar pulmonary artery during recovery from hypoxia. a quantitative ultrastructural study abnormal circulatory responses to high altitude in subjects with a previous history of high-altitude pulmonary edema association of high-altitude pulmonary edema with the major histocompatibility complex oxygen causes fetal pulmonary vasodilation through activation of a calciumdependent potassium channel isolated pulmonary resistance vessels from fetal lambs. contractile behavior and responses to indomethacin and endothelin- endothelin a receptor blockade decreases pulmonary vascular resistance in premature lambs with hyaline membrane disease k+ channel pulmonary vasodilation in fetal lambs: role of endothelium-derived nitric oxide the acute respiratory distress syndrome pulmonary hypertension in severe acute respiratory failure the pulmonary vascular lesions of the adult respiratory distress syndrome effect of increased vascular pressure on lung fl uid balance in unanesthetized sheep biventricular function in the adult respiratory distress syndrome cardiopulmonary abnormalities in severe acute respiratory failure inhaled nitric oxide for the adult respiratory distress syndrome acute and sustained effects of early administration of inhaled nitric oxide to children with acute respiratory distress syndrome low-dose inhaled nitric oxide in patients with acute lung injury: a randomized controlled trial inhalation of nitric oxide in acute lung injury: results of a european multicentre study. the european study group of inhaled nitric oxide key: cord- -i reanan authors: nan title: ecb : th european congess on biotechnology date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: i reanan nan in the last years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b , biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like , -propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of mio m . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only % of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼ k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., ) to generate high-resolution immunohistochemistry images representing normal tissues and different cancer types (uhlen and ponten, ) . each antibody is used to generate more than individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, ) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag , palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse , heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk (cdc p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t , and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., ) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., ) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a ( ). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine ( -hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the -hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete -hydroxymandelic acid synthase ( -hmas, encoded by hpd) from s. coelicolor mt and , using the delivery plasmid pzmh . there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt hpd and hpd. exogenous supply of -hydroxyl phenylglyoxylate and -hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer , c. flores , l. pérez-méndez , r. sangüesa , e. espinosa , j. villar : research institute, hospital universitario n.s. de candelaria, s/c tenerife , spain; department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife , spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl chemokine (or mip- ) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl gene variants and susceptibility to severe sepsis in hospitalised patients and healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs and a newly described polymorphic short tandem repeat d s ) were associated with the syndrome. we found a significant association of common variants at d s with the development of severe sepsis (heterozygote carriers or . ; % ci . - . , and homozygote carriers or . ; % ci . - . ; mantel-haenszel χ test for linear trend p = . ). the risks remained significant even after a genomic control adjustment, based on additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl gene variants may contribute to the development of severe sepsis. kasper møller , ana paula oliveira , jens nielsen , mark johnston : center for microbial biotechnology, biocentrum, technical university of denmark, denmark; department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt /snf -rgt ) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben , dk- vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y by means of dna microarrays david bartolomé-martín , david juck , m a teresa del peso-santos , charles w. greer , julián perera : departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, madrid, spain; environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h p r . e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa and paa gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y , in p. sp. y t (a paa deletion mutant) and in p. sp. y c (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi gene of saccharomyces cerevisiae anssi törmä , , juha-pekka pitkänen , , laura huopaniemi , risto renkonen : medicel ltd., haartmaninkatu , helsinki, finland; rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box , helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi . the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected samples from continuous cultivations over h and measured genome-wide gene expression levels, enzyme activities, and concentrations of intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi led to two major types of activation profiles, one peaking at the time when pmi p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste p, tec p, dig p, and mcm p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn p and msn p. skn p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan , france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that % of the variability is due to the genes. two hundred fifty one regulated genes (from the expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced and ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. non-redundant unigenes for each microalga, only % of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like- , flavohemoglobin and adp ribosylation factor- . in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca + -atpase (serca, "the calcium pump"), is responsible for pumping the ca + released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda , anette henriksen , per hägglund , christine finnie , birte svensson : biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk- kgs. lyngby, denmark; biostructure group, carlsberg laboratory, dk- valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh and hvtrxh that share % sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the d-structures of hvtrxh and hvtrxh are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh , hvtrxh , e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human kda (p ) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg . the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of % by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into aminopropenoic acid and o-glycosylated threonine into -amino- butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of -aminopropenoic acid and -amino- -butenoic acid, resulting in a complete map of o-linked glycans precisely located at different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt- in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt- and wnt- , and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt- mrna levels. probes also for wnt- and wnt- have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a c substituted form, or a c n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the proteins quantified, we identified signaling proteins, including virtually all known egfr substrates and novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider , dave g smith , steven skaper , alastair d. reith : discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, ) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase (gsk ) signalling (coghlan et al., ) . novel findings will be presented on a study of gsk inhibition in a primary neuronal cell line by differential d gel electrophoresis, which resulted in the identification of more than proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., ) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., . anal. chem. , - . plant cytochromes p (p s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about plant p s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p reductase was replaced with the gene of a p reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p expression and activity (pompon et al., ) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no ) . reference pompon, d., louerat, b., bronine, a., urban, p., . methods enzymol , - . folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej , dk - aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the -helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, ) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, ) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., . j. proteome res. , - . adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, ) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., ) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. n and n labeled proteins were separated using d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., ) . we quantified proteins, using on average peptide peak pairs per protein. this analysis revealed that proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a / , vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, - milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim , hye jin hwang , kwang bon koo , jang won choi , jong won yun * : department of biotechnology; department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to . %. pancreatic proteome were analyzed by -de system, which separated more than individual spots. the -de analysis demonstrated that thirty-four proteins from a total of about matched spots were differentially expressed, of which spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < . ) between the healthy and diabetic rats, and the altered proteins were restored (p < . ) upon eps treatment. it was first found that carbonyl reductase ( . -fold, p < . ) and mawdbp ( . -fold, p < . ) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t i) amidase from pseudomonas aeruginosa s. martins , j. andrade , a. karmali , a.i. custódio , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly % of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f g ; e a ). the dissociation of ag-ab complex required % of propylene glycol and either . m (nh ) so or . m nacl. the binding of purified mab of igm class (e a ) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e a showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e a on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of % as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel , a. ristič , , v. gaberc-porekar , v. menart , : national institute of chemistry, ljubljana, si- ; lek pharmaceuticals d.d., ljubljana, si- . e-mail: spela.peternel@ki.si (Š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at • c classical insoluble ibs are formed while at • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure ( . - . kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the -year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang , sang-woo kim , kwang-bon koo , jang-won choi , jong-won yun * : department of biotechnology, daegu university, kyungsan, kyungbuk - , korea; department of bioindustry, daegu university, kyungsan, kyungbuk - , korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to . %. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of proteins of the -de gel were expressed differentially between diabetic and healthy rats. among them, proteins were upregulated and proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤ , -xylose and ␣ , -fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤ , -galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤ , -galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤ , -galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤ , galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal , gerald r. smith , pakize dogan : department of biochemistry, hacettepe university medical school, , ankara, turkey; divisions of basic sciences, fred hutchinson cancer research center, seattle, wa - , usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from to ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch ) (och ch ) cooh and hs(ch ) (och ch ) oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta , , naser amirizadeh , alireza zomorodipour * , fariba ataei : department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: - , tehran, iran; islamic azad university of jahrom, jahrom, iran; department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t promoter (novagen). two dna fragments from light-and heavy-chains of hfviii ( bp-c c and bp-a a , respectively) were subcloned in the expression vector. the use of his -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c c -expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c c -related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of mg/l (of culture) of the purified c c -related peptide was obtained. the recombinant hfviii c c -derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc- juan carlos cruz-jiménez , lorenzo saliceti-piazza , rafael montalvo : chemical engineering, university of puerto rico-mayagüez campus, mayagüez , puerto rico; biology, university of puerto rico-mayagüez campus, mayagüez , puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require . m nacl to sustain growth and structural integrity. among the . genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is , located between the and bases (halobacterium sp. nrc- genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc- genome project. the forward ( -ccgcatgcgcc cacagtccc- ) and reverse ( -ccggcctctagaacggcctgg- ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej , dk- copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to . fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decyder tm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, - oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive - exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s are involved in the binding of the enzyme to the rna substrate, being the s domain the most important. in rna-binding proteins it has been shown that the s domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s -dependent. in order to characterize the s domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s domain was substituted by the s of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg , anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco- spectropolarimeter. protein concentrations of - g/ml and path length of cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ± . at • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej c, dk- aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein (rpl ) followed by expression in an rpl deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl and gfp. we therefore purified rpl -gfp-his, rpl -his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of , and g per gram cellular wet weight, respectively. however, rpl -gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl -gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl -his and gfp to select for monoclonal antibodies specific for each protein. this resulted in antibodies directed against rpl and antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin- , a phagedisplay antibody and yeast elongation factor a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decydertm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s and s protein of sars-cov for modeling d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays recombinant origins for this virus have been found. s and s subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s and s subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv- showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv- shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than % of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej , dk- valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to , ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, - month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above % in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose ff, spanning molecular weights from . to kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins ( conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > . ) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency ( - %) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of . - . × expansion using flow rates at - cm h − . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent ( - l/cycle) was applied to a column ( cm × m) containing l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a -channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk- aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au( ) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., . nature , . otero, r., et al., . angewandte chemie int. ed. , . otero, r., et al., . nat. mater. , . otero, r., et al., . angewandte chemie int. ed. , . rosei, f., et al., . science , . schunack, m., et al., . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md , usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po and pco and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs , frank kensy , markus jeude , tibor anderlei , barbara dittrich , doris klee : biochemical engineering, rwth aachen university, aachen, germany; ac biotec, jülich, germany; textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between -and -folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk- lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from to l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples , italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco ( g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley , : process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a- , austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential -d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o consumption, co evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen , daniel pioch , le thi hoi , jörg albers , rainer hintsche , stefan evers , karl-heinz-maurer , michael hecker , thomas schweder : institut für pharmazie, ernst-moritz-arndt-universität, greifswald, germany; ebiochipsystems, itzehoe, germany; vtb-enzymtechnologie, henkel kgaa, düsseldorf, germany; institut für mikrobiologie, ernst-moritz-arndt-universität, greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, ) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, ) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: ( ) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and ( ) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to % less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene , j. liesiene , b. niemeyer , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for h at room temperature using three solvent mixtures (chloroform/ethanol/water : : . , v/v/v); (chloroform/ethanol : , v/v), and (chloroform/ethanol : , v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica m. the elution was performed using mixture of chloroform and ethanol / (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = . , and n-palmitoyl-sphingosine cer(np), r f = . , were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was . and . min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow, russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin-␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin-␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in % methanol at a temperature of − • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv- nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at mg of high-purity nef. this was supposed to be produced campaign-wise in - batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk- lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in -l bioreactors. a p. pastoris gs mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step ( l of basal salts medium, g/l glycerol, . ml biotin . %, . ml ptm , l antifoam, ph adjusted to with % nh oh) was carried out until glycerol was completely exhausted ( h). the glycerol fed-batch step was carried out feeding with % glycerol ( ml/l biotin . % and ptm ) at . ml/min by h and min of posterior starvation. the methanol fed-batch step was carried out feeding with % methanol ( ml/l biotin . % and ptm ) by h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to g/l. in all the fermentations the air flow rate and the agitation were set at l/min and - rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of g/l wet weight were obtained (od ). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was . ml/min, after h decreased . times for h, increased to . ml/min for h and afterwards it was decreased manually to a constant value of . ml/min for that the dissolved oxygen will not decrease to values less than % (last h). the total protein amount in the culture medium supernatant increased during the production step until values of . g/l (assay at ph ), . times more than the worst assay (ph ) recombinant shrimp trypsinogen production was confirmed by sds-page (about mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya - , japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., . febs lett. , - . yoshikawa, y., et al., . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in . l of aqueous solutions at the temperature of • c and ph and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using , , , , and % (w/v) substrate concentrations; and . , . , . , . and . , % (v/v) enzyme concentrations; and , , and % (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc in the first and second stages were in order of urea > niso > sucrose >kh po > k hpo > cacl > yeast extract > mgso and yeast extract > sucrose > k hpo > kh po > urea > cacl > niso , respectively. the optimal concentrations of nutritional components for improved urease production were determined as g/l sucrose, . g/l urea, . g/l yeast extract, . g/l niso · h o, . g/l mgso · h o, . g/l cacl , . g/l kh po , and . g/l k hpo . these results showed that urea, niso , yeast extract and sucrose had significant effect on urease production by a. niger ptcc . tween and mgso had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about . and . times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engineering, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, ) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta e) (Ç alık et al., ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant e. coli on a defined medium with . kg/m glucose were investigated in order to finetune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q /v r = . vvm and n = , , and min − and; q /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by d and d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, old westport road, north dartmouth, ma - , usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j - t fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j - t cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j - t cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina , , longina paegle , liga zala , , maija ruklisha : faculty of biology, university of latvia, kronvalda blvd. , riga lv- , latvia; institute of microbiology and biotechnology, university of latvia, kronvalda blvd. , riga lv- , latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine -diphosphate diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa and of hcmv gb (termed antigenic domain , ad- ) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad- of hcmv glycoprotein gp , which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad- with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad- as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow , russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about mg/l of cell culture, corresponding to , , and units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin ( -(␤-d-ribofuranosyl)- , , -triazole- -carboxamide), cladribine ( -chloroadenine- -␤-d- -deoxyribofuranoside) and fludarabine ( -fluoroadenine- -␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of , and %, referred to , , -triazole- -carboxamide, -chloroadenosine and -fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon - , korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about and kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were nm, ± mv, respectively. iso-electric point(ph iep )of fibroin was ph . . the amount of fibroin adsorbed on a gold surface was less than . g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazarİleri , pınar Ç alık , ali Şengül : ib lab, department of chemical engineering, metu, ankara, turkey; gülhane sch med, dept immunol, ankara, turkey. e-mail: e @metu.edu.tr (n.İleri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec . . . ), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph . with u/cm enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan , megumi kitagawa , yoichi kumada , naoya hashimoto , masami shiiba , shigeo katoh , masaaki terashima : graduate school of science and technology, kobe university, nada, kobe - , japan; department of human science, kobe college, okadayama, nishinomiya - , japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at • c in a modified gastric juice, . mg/ml nacl containing g/ml pepsin (ph ). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil c -ms-ii, . cm × cm). amino acid sequences and ic values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time - min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, - , campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodÖ. aydogan , e. bayraktar ,Ü. mehmetoglu , m. parlaktuna , t. mehmetoglu : department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara , turkey; department of petroleum and natural gas engineering, middle east technical university, ankara , turkey. e-mail: mehmet@eng.ankara.edu.tr (Ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using ml of . m ammonium fumarate (ph . ) as substrate at circc in an orbital shaker at rpm for h. e. coli (atcc ) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co is used to form gas hydrates crystalline structure. back extraction experiments were carried out between - bar g pressure and at circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of . %. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no − are required as electron acceptor (denitrification). this process consists of no − reduction to nh + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about % of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin- -o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin- ␤ (il- ␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin- -o-␤-d-glucopyranoside by lc-ms, ir, h nmr, and c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c- /i treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il- ␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin- -o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin- -o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a ml shake-flask containing ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin , karim senni , jacqueline ratiskol , farida guéniche , jean guézennec , gaston godeau , sylvia colliec-jouault : ifremer, nantes cedex , france; ea université rené descartes, montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, ; colliec-jouault et al., ) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., ) . the structure of the native eps has been described (rougeaux et al., ) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini , christina saarimaa , arne matzen , peter neubauer : bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi- , finland; sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., ) proteomic analysis was carried out by d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka , paweł parniewski : department of microbiology and immunology, university of Łódź, - Łódź, banacha / , poland; centre for medical biology, polish academy of sciences, - Łódź, lodowa , poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant p a . performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi - , taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., ) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., . contr. eng. pract. , - . modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building , dtu, lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes -phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, - bydgoszcz, ul. seminaryjna , poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod , alvin w. nienow , graham poulter , reg wilson , henri tapp , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh , yoichi kumada , nanae maeshima , daisuke nohara : graduate school of science and technologykobe university, kobe - , japan; department of biomolecular sciencegifu university, gifu - , japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - , são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and autoclaved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues % (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch , andreas luebbert , rimvydas simutis : institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl /de /gfp) for which more than full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a- graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale ( l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of . m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = ) rather than in the u-loop section (bo = ). it was also observed that the time for mixing to % homogeneity after tracer pulse addition was a function of the number of cycles through the reactor ( - . ) within the range of flow velocities (u) studied (u = . m/s to u = . m/s). the mixing time to % homogeneity was between . s (at u = . m/s) and s (at u = . m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: ( ) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. ( ) it has been tested for production of more than recombinant proteins. ( ) it is easily scalable from lab scale to kg of processed protein. ( ) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. ( ) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). ( ) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. ( ) a number of therapeutics is currently being developed using tagzyme tm . ( ) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol- -phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors ( . , and l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega / / alkaline phosphatase (ap, e.c. . . . ) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a . m potassium phosphate buffer with ph . . the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from to • c. ap in cow's milk was completely inactivated at • c during s but approximately % of activity remained at • c after min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from to min as the temperature increased by • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from to • c reduced the inactivation time from min to s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of th framework program of eu, project foodpro, no. sme- - - . during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph - . the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph . the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph . another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb % of total amount of igg was about . - h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by % in less than min in all cases of dynamic adsorption measurements. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, , ankara, turkey. email: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about , g/mol) or hydrodynamic radius ( . nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range - × g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than nm), whereas some adsorbents contained a significant fraction of pores larger than nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of nm (corresponding to mabs) was about . - . at ion-exchange and hydrophobic adsorbents and . - . at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a dm stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was , u g − . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in and dm mechanically stirred bioreactors and in air-lift bioreactors, and dm whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about u g − . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was u g − in the dm air-lift reactor and u g − in the dm air-lift bioreactor. the lower yield of ftase in dm bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega / / and / / and by science and technology assistance agency, grant number apvt- - . the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august and november . in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to - g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. g/h (≈ % purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt- was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte , j. liesiene , b. niemeyer : department of chemical technology, kaunas university of technology (ktu), radvilenu pl. , kaunas, lithuania; institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg , hamburg, germany d- nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − m, and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at % of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at psi ( %, w/v, w/w, pass) was found to be u/ml of adsorbent while dbc of samples disrupted at psi ( % w/v, w/w, pass) was u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei , niels mathiasen , timothy hobley , owen rt thomas , : center for microbial biotechnology, building , biocentrum-dtu, technical university of denmark, , lyngby, denmark; department of chemical engineering, university of birmingham, edgbaston, b tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch and ) were examined, which had ionic capacities measured to be and mmol cl − /ml support respectively. in all adsorption experiments a mm tris-hcl ph buffer was used. maximum static binding capacities of adsorbent batches and were determined to be . and . mg dna/ml particle, respectively. dynamic binding capacity at % breakthrough (dbc % ) was measured in a -cm diameter eba column containing . ± . cm settled bed with a feed of g/ml dna. dbc % of the adsorbents were . and . mg dna/ml support for batches and , respectively in buffer containing no salt. however, the maximum dbc % for batch ( . mg dna/ml support) and ( . mg dna/ml support) were obtained in buffers containing . and . m nacl, respectively. further increases in salt concentration led to a decrease in dbc % for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity ( ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than . m. the effect of different concentrations of dna during loading in the presence of . m nacl was studied. it was found that increasing dna concentrations in the feed from to g/ml, to g/ml resulted in a decrease of dbc % by , and %, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost % of the pdna precipitated when . vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré , , u. jørgensen , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a cm diameter column with - cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu + was used and a feedstock of denatured hat-tagged human beta- microglobulin (hat-h␤ m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to % of dbc working lead to a recovery of . % at a purity of % and . -fold concentration. the eba-based process performed unimpeded and productivity was calculated to be % higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. . mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period ( - min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. % of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in m urea) inclusion bodies of recombinant histidine affinity tagged human beta microglobulin (hat-h-beta m), were refolded by dilution in a pipe reactor ( s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor ( s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran , j. joseph , a. sinclair , y. zhou , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices ( - %), at various aspect ratios, fluid flow rates and varying viscosities ( . - . mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg- ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process ( % enzyme release with a specific activity of . u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by -times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of % ( -times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of > % (after h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system % of the dimer is isolated with purity (hplc) of %. the application of a dialysis membrane technique allows the separation of % of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building , lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from to m, whereas the series was reversed for monovalent cations at concentrations up to . m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately . ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra , knut olav straetkvern , bjørg egelandsdal , gerd vegarud : department of natural science & technology, hedmark university college, n- hamar, norway; norwegian university of life sciences, as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about % of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in -d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella , m. leinonen , t. nieminen , a. breitenstein , p. neubauer : bioprocess engineering laboratory, university of oulu, finland; scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., ; leskelä et al., ) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a h pre-enrichment period gave reliable results, had a detection limits of about - cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, ) . rhodococcus erythropolis igts is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way ( s route (oldfield et al., )), obtaining hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., a (del olmo et al., , b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: , , and rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, )). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl (de ) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a . % of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a . and . % were obtained respectively. the freezing and thawing disruption treatment released less than % of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of kda, and a pi around . the optimum temperature ranged between and • c, while optimum ph was around . pox appeared remarkably thermostable since it retained % of its activity at • c for at least h. in addition, the enzyme was stable at a ph range from , to . kinetic constants were calculated, the apparent k m values were and m for gaïacol and h o , respectively. the high thermostability of pox may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h o and other peroxides. immobilization of pox was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained % of its activity at • c for mn while the immobilized pox retained full activity for mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at • c, and lost m of its activity after months. the immobilized form of pox retained complete activity for months at the same temperature. the immobilized enzyme was used to detect h o in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h o to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e , was purified through q-sepharose, gel filtration chromatography and deae-sephadex a- . the mw of the pure enzyme was kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph . and in range of . - . , respectively. the optimum temperature and thermostability were at • c and up to • c by h, respectively. the k m and v max were . mg/ml and . mol/min mg and . mg/ml . mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was . and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was . : . : . and : : with maltodextrin and soluble starch at . %. application of magnetic separation technology for recovery of immobilised lipases nadja schultz , anke neumann , george metreveli , matthias franzreb , christoph syldatk chair of technical biology, university of karlsruhe, engler bunte ring , d- karlsruhe; forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; inst. für wasserchemie, engler bunte ring , ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a -phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a -phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a -phase-system with the help of the new high gradient magnetic separator (hgms) is examined in ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), - m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., ) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a -phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures ( , , , − • c) and at different ph values (ph , and ). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a -phase-system. challenging approaches and first results on the recovery of immobilized lipase from a -phase-system in ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − , and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie , forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least %. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box , se- lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka , prague cz- , czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the . kda transcriptional regulator merr of mercury-inducible mer operon of tn exhibiting high affinity and selectivity towards hg + , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg + , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg + in a range of - nm by simply monitoring the growth in the hg + /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing m hg + when modified cells accumulated . -fold more hg + than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon - , korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader , boris neumann , karima schwab , milan popovic , rakesh bajpai : studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., berlin, germany proteome factory ag, dorotheenstr. , berlin, germany; department of chemical engineering, university of missouri-columbia, w ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., was extended to two component feeding. the key idea, discussed in this presentation, was using the po and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., . probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. , - . antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, rue pierre et marie curie, paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between . and % of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk- copenhagen, denmark glp- (glucagon-like peptide- ), a peptide of amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp- administered to patients with type diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp- is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin- , was found to be a full agonist of the glp- receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now ( / ) been approved for diabetes treatment by the fda. other approaches include acylation of glp- whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp- and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp- from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to % of postprandial insulin secretion. the incretin hormones are glucagon-like peptide- (glp- ) and glucosedependent insulinotropic polypeptide (gip). in patients with type diabetes ( dm) the incretin effect is severely reduced or absent. in dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp- secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp- might therefore be possible. glp- is a product of the glucagon gene and its actions include: ( ) potentiation of glucose-induced insulin secretion; ( ) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; ( ) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; ( ) inhibition of glucagon secretion; ( ) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and ( ) inhibition of appetite and food intake. these actions make glp- particularly attractive as a therapeutic agent for dm. thus, continuous subcutaneous administration of glp- for weeks resulted in a mmol/l reduction in mean plasma glucose and a reduction in hgba c of . %; a weight loss of kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp- is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: ( ) the development of metabolically stable analogues of glp- viz. activators of the glp- receptor; and ( ) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin- or analogues thereof, as well as glp- derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type diabetes, and have been employed in clinical studies of up to weeks' duration. on the basis of these studies it can be concluded that a therapy of type diabetes mellitus based on stimulation of glp- receptors is likely to be effective and to become a clinical reality within the not too distant future( - ). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of - % in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum . . , blegdamsvej , dk- copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym , is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k) . in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k and present a loosely arranged keratin filament network and low levels of k protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k . three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k . the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) % glycerol and % hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in % volume of sugar either and m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob . human fetal osteoblast cell line with vigorous shaking overnight and incubating at • c and % co . observation of the seeded scaffolds was made after days and days of incubation. the seeded cells were stained with bcip/nbp at • c overnight. the cell proliferation in the and m scaffolds appeared approximately the same with a possible advantage of m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d- bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in . if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a , s- lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e was found to be ± (pa), the equilibrium young"s modulus e ∞ ± (pa), and the apparent viscosity ± (pas). after exposed to m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to % and the apparent viscosity increased by %. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are million persons aged years or over and this number is predicted to triple by the middle of the st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to %. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d- , germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt- (wnt family) possesses conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, ) recently despite various attempts for many years. the other target protein is human collagen prolyl- -hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv- replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv- escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position − upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the − mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv- with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed . % cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl ( g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl ( g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching u/g dry cell mass. high cell density cultures carried out in the l bioreactor, in which it was attained g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of , u/l and productivities of u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu , biocity th floor, turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh ) with the liberation of excess free energy in the form of a blue-green light at nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, ) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., ; bechor et al., ) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, ; galluzzi et al., ) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after min (black downward arrow) of incubation at • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every min by means of the wallac victor multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at nm with the same device. multi- white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at + • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with , % d-glucose and g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration ( g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from - % to more than %, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl cells) and at much higher levels than in induced rn cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. . xl /pqaclux growing cells were incubated with the following concentration of trimethoprim: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn and xl cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, ) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, ) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's ( and kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a -l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to % of hghs of total proteins in crude fermentation medium and - mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores , * , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; patients had invasive amoebiasis and patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was %, specificity was %, positive predictive value was %, negative predictive value was % and accuracy was %. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar , sema tan , aysun ergene , perihan güler , semra mirici , ergin hamzaoglu , ahmet duran , sinem yildirim : mersin university, faculty of science and literature, department of biology, mersin, turkey; kırıkkale university, faculty of science and literature, department of biology, yahsihan-kırıkkale, turkey; akdeniz university, faculty of education, department of biology education, antalya, turkey; selcuk university, faculty of education, department of biology education, konya, turkey turkey is covered yearly with a huge number of plant species. about species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than species on the world. this variety is represent with species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide ( c ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than % of dna was encapsulated in this nanovesicles under the condition of m nacl and % (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at • c was smaller than that prepared at room temperature. the resultant c ab/ctab nanovesicle was ca. nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule- (vcam- ) as a target for inhibition, and intercellular adhesion molecule- (icam- ) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam- in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an % down-regulation of vcam- mrna while the expression of icam- remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: ( ) does vcam- -specific sirna affect the amount of vcam- on the surface of huvec? ( ) is adhesion between leukocytes and endothelial cells affected by vcam- -specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs ( %) are related to fermentation components. economic analysis indicates a unit production cost of $ /g pdna. for a selling price of $ /g, the payback time was . years and the roi was . %. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials ( . %), (ii) reduce the environmental impact associated with isopropanol ( %) and (iii) reduce costs associated with the treatment/disposal of liquid waste ( . %). preparation of chitosan microspheres for controlled release of somatotropin s. simsek , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution ( %) containing polysorbate . sodium sulphate solution ( % w/v) containing somatotropin was added into the chitosan solution and mixed rpm for a hour. resulting suspension was centrifuged , rpm min at • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph . phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between and %. average particle size of microspheres was between and m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi - , japan. e-mail: h d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b melanoma. in an in vitro study, when immature dcs were pulsed with b cells heated at • c for min, mhc class i/ii, costimulatory molecules cd /cd , and chemokine receptor ccr in the dcs were up-regulated, thus resulting in dc maturation. c bl/ mice bearing a b melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in % of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb , els maas , eiichi akaho , hiroto kambara , debbie hulston , anna kilimnik : marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; faculty of pharmaceutical sciences, kobe gakuin university, kobe - , japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc- . isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co- gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was . % and . % respectively, the linearity is ranging from to %, and the recovery for three different sera is ranging from to %. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni , a. tavares , g.a. monteiro , d.m.f. prazeres : centro de engenharia biológica e química (cebq), instituto superior técnico, - lisboa, portugal; instituto gulbenkian de ciência, rua da quinta grande , - oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty , , promod kumar verma : department of cancer biology, lerner research institute, euclid avenue cleveland, oh , usa, department of biotechnology, guru nanak dev university, amritsar, punjab , india. e-mail: riar @rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. . mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. . mg was adequate as effective dosage for kleb-siella pneumoniae. mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be %, while that of garlic was between . and %. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and . % garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the randomly selected subjects serologically examined for hbv, and were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in conserved sequence of viral genome. results: of the hbsag positive carriers, hbv dna was detected in . and . % using kit and kit respectively. only . % were positive in both kits. in hbcab positive subjects (n = ), . % were positive by kit and all samples were negative when tested by kit . there was a significant difference between two kits. sensitivity of kit and kit were . and . %, respectively. overall, kit increased the detection rate of hbv dna by . %. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard , x. liu , d. oupicky , f. besenbacher , j. kjems : inano, university of aarhus, denmark, department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range - nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣ , -galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣ , -galactosyltransferase gene, human cd , cd and cd genes controlling complement activation and human genes encoding ␣ , -fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease a( a-pro) of coxsackievirus b (cvb ) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif g (eukaryotic initiation factor g-formerly called p ) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet b(+) and since a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de ) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p was prepared by synthesizing small peptides, corresponding to antigenic site of p coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p ) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p cleavage by apro was assessed by sds-page and western blot analysis. cleavage of p by r a-pro was prominent after h. so recombinat a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya , suruga-ku, shizuoka - , japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤ , -n-acetylglucosaminyltransferase were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, % of total expressed ␤ , -n-acetylglucosaminyltransferase was secreted into hemolymph of silkworm. using bacmid system mu/ml of ␤ , -n-acetylglucosaminyltransferase was expressed in hemolymph of silkworm, which was -three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for min. the pollen germinated for h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the kc gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul - , korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the kc gene, which was originally obtained from a silkworm. the expression of kc inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a -fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing kc showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of kc . the positive effects of kc expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the kc gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer , f. hatipoglu , z. ogurtan , a.l. baş , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at + • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, . ± . kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps ( . cm ) at • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at , and st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after days treatment except controls. after days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after days treatment. the wound epithel elongation and thickness values were measured; a ( and m), b ( and, m), c ( and, m), d ( and, m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e region of hcv with the amino acid sequence e antibody [ghrmawdmm). materials and methods: hyper-immune hcv e antibodies were incubated over night at • c with serum samples from patients positive for hcv rna, with different viral load, ranged - million copes/ml, then incubated min to hepg cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e antibody. direct immunostaining of e antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e antibody compared with samples without e . of positive serum samples, ( %) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp ) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp , this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (< years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, c > t and a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < . ) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, c > t and a > c in mothers of ds children (case mothers) (n = ) was compared with controls (n = ). frequencies of mthfr genotypes (cc, ct, and tt) at position demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr a > c genotypes in egypt. our results showed that mthfr a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg cells infected with native hcv rna of genotype . the culture system presented herein was shown to support hcv replication on the following bases ( ) consistent detection of both plus and minus rna strands for weeks in cells and in fresh culture supernatent, ( ) ability of supernatent to infect naive hepg cells ( ) consistent expression of core and e envelope proteins in infected cells throughout the week culture. s-odns against aug translation start site (s-odn- , nt - ) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn , nt - ) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype was completely arrested after h in culture using either s-odn molecule (with more efficacy of s-odn than s-odn ) at concentrations as low as m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde -phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype , the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to -fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was . - cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about h. isolation, cloning, expression and purification of snap- as a substrate of botulinum neurotoxin type a and e m.l. mossavi , f. ebrahimi , j. amani * , h. basiry , z. ahmadi : department of biology, faculty of science, imam hussein university, tehran, iran; department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of kda (snap- ); synaptobrevin/vamp- and syntaxin. snap- is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap- in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap- was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha- antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya - , japan. e-mail: h d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about % higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from clade f hiv- isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) base pairs, approximately aligning with the standard sequence of hiv- (accession nc in genbank) over the interval - bp. this segment, which is currently used for the standard identification and assessment of hiv- strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length bp and is located in the second interval ( - bp) of the analyzed dna segment, respectively along the - bp region of nc . taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari , , a. zomorodipour : islamic azad university of jahrom, tehran, iran; national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari @yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked . and . kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from ml culture approximately mg protein for feldi chain and mg of feldi chain were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk- , denmark in we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of - mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited % to % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül Şentürk , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engng, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc , and then sub-cloned to pmk e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin ␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin ␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral ( - ) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl -hydroxylase production in e. coli a. neubauer , m. bollok , j. myllyharju , p. collagen prolyl -hydroxylase (p h) involved in the biosynthesis of collagens is an ␣ ␤ tetramer. recombinant expression of p h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., ) for optimization of the fermentation procedure. a high p h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited - % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo / , mäkinen et al., ) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e and e (e rns ) and e . glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e and e we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei , taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer department of biotechnology, altana pharma ag, konstanz, germany; imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu- : ( ) the ability of calu- cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). ( ) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than % or even % are possible, while a high over all yield can be achieved (∼ %). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in , a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤- , -n-acetyl glucosamine and ␤- , -glucuronic acid repeating disaccharide units with a molecular weight (mw) up to mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in the amount of threonine produced worldwide was , t and raised to , t in and , t in . concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. ( ); fermentation process for the preparation of l-threonine; wo/ . ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. ( ) . using a recently described protein sequence motif fraaije et al. ( ) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼ • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. ( ) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. ( ) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (> %) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar , j. friedrich , v. alphand : laboratory of biotechnology, national institute of chemistry, ljubljana si- , slovenia; groupe biocatalyse et chimie fine cnrs fre , université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., . j. mol. catal. b , - . carnell, a., willetts, a., . biotechnol. lett. , - . ten brink, g.j., et al., . pyranose oxidase (p ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c- oxidation of several aldopyranoses to the respective -keto derivatives, transferring electrons to molecular oxygen to yield h o . p ox is of interest for carbohydrate conversions, as its reaction products ( -keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e k and e r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p monooxygenases. cytochrome p monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp a from bacillus megaterium, cyp a and cyp a from bacillus subtilis are soluble fusion proteins comprising p monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp a also known as p bm- is well characterized and shows high activity compared to other p monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa - , japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose -epimerase (dte), that epimerize all ketohexoses at c- position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all aldohexoses with ketohexoses using the enzyme. finally, all hexoses, ketohexoses, hexitols and aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d- , germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c -compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of % could be achieved, without optimizing the system. the system remained active for more than h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by % through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in . the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$ . billion per year) could be paid by re-assigning cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to - % of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce - gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, ) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by %. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within - years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and -hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs (a commonly used laboratory strain) and tmb (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. ( ) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck ( , ) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. ( ) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck ( ) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck ( ). he, j., hertweck, c., chem. biol. , , - chem. bio. chem. , , glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the -kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., ) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., ) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., ) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., ) , and the aromatic side chains are interconnected by p monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., ) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., (weist et al., , . bischoff et al., . angew. chem. int. ed. , - . pfeifer et al., . j. biol. chem. , - . puk et al., . j. bacteriol. , - . puk et al., . chem. biol. , - . recktenwald et al., . microbiology , - . weist et al., . angew. chem. int. ed. , - . weist et al., spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc e je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., ) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., ( ) . genome-scale analysis of streptomyces coelicolor a ( ) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s . the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde- -phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be . - . -fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about and % compared to the native tef promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b ts - ). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed - -fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b ts - produced . g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as • c and ph . , respectively. one of the isolates that could grow at - % salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag - t . in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii -like endogenous plasmid psb and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a . -fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory ( ), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among transformants tested ldha and ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only . times of the wild type while this was times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of ␣h-epoxypregnanolone ( ␣h) and -epoxy-pregnenolone ( ) into their ␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing g l − of the steroid substrate and . g l − cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: - -keto-; ␣-hydroxy-and ␣, ␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating ␣h without any significant accumulation of the - -keto-derivative. the most effective accumulation of hydroxylated derivatives of was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after day ( nkat). the optimum biobleaching conditions are • c and ph: . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brigthness from to . in day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m- ayşegül ersayin , aytaç kocabaş , b. zümrüt ogel , ufuk bakir : biotechnology department, middle east technical university, ankara, turkey; food engineering department, middle east technical university, ankara, turkey; chemical engineering department, middle east technical university, ankara, turkey, . e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to % of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d- , -linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤- , -xylanases (ec . . . ). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m- , has been shown. bacillus m- produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of kda upon sds-page and a pi of . upon ief was partially purified by liquid chromatographic techniques -fold with % recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h o released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h o from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h o concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h o concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose . m and vinyl ester . m has been mixed in dimethylformamide at rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations ( , , and mg/ml) as catalyst. afterwards, the influence of the temperature ( and • c). after that, the influence of the molar ratios ( : ; : ; : m/m) between vinyl laurate (ch (ch ) cooch ch ) and sucrose. subsequently, we investigated the effects of water amount, using , , and % of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using mg/ml of protease from bacillus subtilis at • c, molar ratio of : (vinyl laurate:sucrose) and adding % of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after hours of reaction by c nmr. the results confirmed the c substituted sugar mono-ester ( -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engng, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld ( ) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta e) Ç alik et al. ( ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer ( ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., . methods in enzymol. : - . Ç alık, p., Ç alık, g.,Özdamar, t.h., . enzyme microb. technol. , - . rainer, b.w., . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was -fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last h experiment hydroxyectoine accounted for - % of the total content, accumulating up to - % of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between • c and • c using a ratio : /biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration ( mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q (coq ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from to %, the intracellular coq content increased about -fold, yielding mg per g-dry cell weight at % dissolved oxygen level. azide significantly increased the intracellular coq content, with the highest value of . mg per g dry cell weight in the presence of . mm of sodium azide. however, dnp (up to m) and h o (up to m) did not affect the intracellular coq content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building , dk- lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. ( ) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami ( ) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. ( ) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi , , a. zomorodipour : islamic azad university of jahrom, iran; department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg (cgg) and arg (cga) and codons for leu (tta) and pro (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe (ttc) → leu (ctg) and ala (gcg) → met (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf ) was subjected for further expression analysis in a t -based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf ) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip ) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. - w that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. - w ) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of amino acids that is synthesized as preprotein with a signal sequence of amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in mm diameter were inoculated into the center of malt extract agar and incubated at , , , and • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at • c. at • c the e group mycelium and at • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as and • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of and nm, were produced by q-switched nd + :yag laser in its third and forth harmonic, respectively. the energy of each pulse for nm was . mj/cm and for nm was . mj/cm . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of : before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, . tcid /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the . - j/cm of puvls with the wavelength of nm and also, was exposed into the . - . j/cm of puvls with the wavelength of nm. furthermore, bvdv suspended in fbs was exposed into the , , and j/cm of puvls with the wavelength of nm and also, was exposed into the . , . , . , and . j/cm of puvls with the wavelength of nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the and nm wavelengths of puvls, were and . j/cm , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the and nm wavelengths of puvls, were and j/cm , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of . j/cm of nm puvls was used to grow vero cell line in successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki , y. ishii , k. kino , k. kirimura : dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, , -dihydroxy-benzoic acid) from resorcinol (re, , dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu- ) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be kda by gel-filtration, and that of the subunit was determined to be kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of and in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, . mm ␥-ra was produced from mm re at • c for h, with a yield of %. ishii, y. et al., . biochem. biophys. res. commun., , - . laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl - , poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec. . . . . p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume dm ; fas- of working volume . dm ). experiments in both fermenters were made at impeller revolutions and min − , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed min − . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm ): white sugar, . ; nh no , . ; mgso · h o, . ; kh po , . ; feso · h o, . . fermentations were carried out in the lab bioreactor biomer . the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of dm . high citric acid concentration in culture medium (p = . g/dm ), high yield of citric acid (y p/s = . %) and high efficiency coefficient (k ef = . ) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık, iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant escherichia coli on a defined medium with . kg/m glucose were investigated in order to fine-tune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = . vvm and n = , , , min − and; q o /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of -androstene- , -dione (ad) and , -androstadien- , -dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr p c of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were ( ) unbleached pine pulp from portucel tejo; ( ) unbleached eucalyptus pulp from portucel setúbal; ( ) bleached eucalyptus pulp from portucel setúbal; and ( ) pulp made from recycled paper from renova s.a. several types of handsheets were produced with different grammage namely, and g/m . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of nm), temperature ( • c) and moisture ( %); and thick saline fog at a concentration of % and temperature of • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied ( , and %), while the temperature was held constant ( • c); in the second the temperature varied ( , and • c) and the moisture was held constant ( %). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/ / . aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol- -p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul , nur koçberber , gönül dönmez , serpil takaç department of biology faculty of science ankara university beşevler ankara turkey; department of chemical engineering faculty of engineering ankara university tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at • c and ph . lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween , glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of % whey + % glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = and • c for h was resulted in the extracellular and intracelluar lipase activities of and u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: - and - ) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from to mosm kg − . in case of mscs cells, the maximal specific growth rate [] of . [h − ] associated with the highest specific glucose consumption rate [-q gluc ] of . × − [mol cells − h − ] was obtained in medium of mosm kg − . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of mosm kg − . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca , , andreas k. gombert , elmar heinzle , christoph wittmann : biochemical engineering, saarland university, saarbrücken, germany; chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of . , . and . h − . the specific rate of o consumption (qo ) increased with dilution rate from . to . mmol (g dw) − h − . the respiratory coefficient remained almost stable around . for all metabolic states investigated. even at the dilution rate of . h − , which is close to the maximum growth rate of the strain of . h − , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below % of the carbon consumed as glucose. all carbon balances closed near % underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm ). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about %, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = . l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < ). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm , was shown to produce a novel bacteriocin named bacthuricin f . the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f , the specific activity was increased -fold. this bacteriocin was heat-stable up to • c and resisted up to ph . . its molecular mass, determined by mass spectrometry was . da. direct n-terminal sequencing of bacthuricin f revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm , and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi showed an open reading frame of bp, encoding a amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc - kwon-hye ko , gee-sun yoon , gi-sub choi , joo-won suh , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an . kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon - , korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., ) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., ) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., ) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., and aljanabi et al., ) or spores (ferreira et al., and xu et al., ) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc ). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of operational conditions. the region of maximum production is outlined by the following intervals: temperature around • c; lactose concentration between and g/l; concentration of yeast extract between and g/l; ph between and . . fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview . , which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with • gl. the fermentation operated with concentrations of ethanol at • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) g glucose, g whey powder and g corn steep powder. for a homofermentative process, ph . was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were g and . g/lh, but in fed batch system, after feeds of glucose, production and productivity increased up to g and g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s- . the molecular weight of t. harzianum pgii was , da by gel filtration and sds-page. pgii had isoelectric point of . and optimum ph of . . pgii was very stable at the ph . . the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were . mg/ml and s − , respectively. pgii was found to have temperature optimum at • c and was approximately stable up to • c for min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. , phenanthroline, tween , tween , triton x- and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of mg of enzyme from kg of horseradish roots. the one mg of enzyme contains units of peroxidase. this value is similar to that produced by sigma ( - unit mg − powder). the final preparation is salt free reddish brown powder with free ammonia content less than . g − units. the rz value (a /a ) of the enzyme, which is a good criterion of purity and heme content, was . . the lyophilized enzyme was stable at − • c for at least one year. the liquid form of the enzyme in presence of . % sodium azide was stable up to days at • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to l scale mikkel nordkvist , per munk nielsen , peter budtz , john villadsen : center for microbial biotechnology, technical university of denmark, dk- lyngby, denmark; novozymes a/s, dk- bagsvaerd, denmark; chr. hansen a/s, dk- hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in l scale, experiments were performed in a l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose -epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep / , and gel filtration chromatography on sephacryl s- hr with a final specific activity of . u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of , da and a monomeric structure. the purified enzyme exhibited maximal activity at • c and ph . without the addition of metal ions and at • c and ph . with . mm mn + . among various metal ions, mn + was the most effective divalent cation for d-fructose epimerization activity. the addition of mn + significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose -epimerase but d-psicose -epimerase. the equilibrium ratio between d-psicose and d-fructose was : at • c with . mm mn + . when the enzyme was used at u/ml, dpsicose was produced at g/l from g/l d-fructose containing mm mn + after min, corresponding to a conversion yield of . %. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni , f. wayman , m. mattey : department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki , greece; department of bioscience, university of strathclyde, glasgow, g xw, uk. e-mail: mp @vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in -l stirred tank reactor, indicates that nh + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh ) so addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as g/l. about microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb was much faster than a reference strain of erwinia rhapontici. fmb is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and s rdna analyses, fmb was determined to be enterobacter sp. the maximum conversion of % sucrose to palatinose and trehalulose by enterobacter sp. fmb was achieved within h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb revealed that it showed % similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth ( • c) and optimal proteolytic activity ( • c) yielding biomass specific growth rate of . and . h − , respectively. the achieved proteolytic activities were . and . u/ml of protease, respectively. optimised medium composition ( g/l beef extract, g/l yeast extract, g/l peptone and . g/l cacl ) yielded a specific growth rate of . h − and . ku/l of protease, in shake flask experiments. bioreactor experiments (from to l) with the selected medium were performed at • c in order to test aeration rate ( and vvm), stirring ( - rpm) and ph (uncontrolled, controlled at and ). best protease activity was u/ml in l bioreactor without ph control at rpm and vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., ) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri levansucrase (van hijum et al., ) was secreted in significant amounts (∼ mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. . mg his -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui , naoya shinzato , hisashi saeki , hitoshi matsuda : center of molecular biosciences, university of the ryukyus, okinawa - , japan; japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. %ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around %ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, - lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size ( - kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: bp and mature section: bp) and the restriction site on mcs of pqe- vector were digested. in this study, pqe- expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a- . isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to -methyl- -butanol, -methyl- -butanol, and -methyl- -butanol but not to -methyl- -butanol. -methylpentane was oxidized to -methyl- -pentanol, -methyl- pentanol, and -methyl- -pentanol but not to -methyl- -pentanol or -methyl- -pentanol. -methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent . -diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher , : division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse , a- vienna, austria; research centre applied biocatalysis, petersgasse , a- graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about %. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via . -diketo-d-gluconic acid ( . -kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent . -diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate -keto-l-gulonic acid ( -klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic -klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp promoter designated as hela c cells, respond sensitively to luciferase grp induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp , and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c- and/or c- oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj , usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., . - weeks for fed-batch operation and - months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes - days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw - , poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of dm . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from . to . dm /(dm h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = . %), high volumetric rate of its production (r pc = . g/(dm h)) and the highest biosynthesis efficiency coefficient (k ef = . ) were obtained with dilution rate d = . dm /(dm h). there was also high citric acid concentration (p = . g/dm ) and low residual sugar concentration (s k = . g/dm ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc . the medium composition for culture was as follows (in g/l): lactose pa , yeast extract , (nh ) so and kh po . the fermentations was carried out at • c, ph . , at rpm starting with an initial cellular concentration of × cels/ml, with different aeration rates. the cells were disruped with chloroform % (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with . vvm was % higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of . - . vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only % at • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for , and %, in the presence of commercial gelatin at the concentration of % at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature • c, ph . with a gl − lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained % of glutaraldehyde. after daily determinations of enzymatic activities, a fall of , and % was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at , and %, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia - , brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec . . . ), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t / ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = . . − mol/ml) and the glucose effect was noncompetitive inhibition (k g = . . − mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution ( . × − cm /s at • c). the half-time of enzyme inactivation (t / ) was h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr p c of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec. . . . . p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the th day of culture, and the amount of laccase produced was higher by, ca. % as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for - weeks at the temperature • c in a certomat bs shaker at rpm. the best results in the c. unicolor strain culture were achieved using a -day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiÜnlü department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with , , and % concentrations of methanol (m), ethanol (e), -propanol ( p) and -butanol ( b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at + • c: (i) stirring crl with phosphate buffer for h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. the activity of crls was followed for h at • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. b-crls and e-crls showed higher activities than m-crls and p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and p-crls. the highest and the lowest activities were obtained with % b-crl and % p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: ) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the -propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiÜnlü department of chemical engineering, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with -propanol ( p) at the ph values of . , , , . , and to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at + • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for h; (ii) treating the solutions with % p; (iii) stirring treated-crls for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at rpm and • c for h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of . u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph . as , , and %, respectively. these values were followed with p treated crl at ph as , , and %. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between . and . the effects of fatty acids, nitrogen (as nh no ), phosphorus (as kh po ), ph value, manganese (mn + ), iron (fe + ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from to . with a medium containing g fatty acids/l, the production reached a maximum of g oxalic acid/l after days. the addition of . % (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity ( - g/l days) was maintained for - days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara , turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. . . . , p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr and abr , were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in probable laccase genes, one of which was the previously cloned abr gene. in this study, one of these genes (aflac ) was further characterized. after sequence alignment and characterization studies, aflac was predicted to have bp having six introns, which makes the protein amino acid long, and the predicted protein sequence showed % homology with the dihydrogeodin oxidase of aspergillus terreus and % homology to the laccase of botryotinia fuckeliana. aflac gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac gene, followed by cloning onto pan - and pan - vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than , the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after - h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers ( . %dw), in every samples the presence of s-adenosyl-methionine in traces ( . % dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached . % dw after h incubation. moreover, gsh levels can be related to sam production (up to . % dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from to g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed g/l. based on the results of these experiments, smooth strain awg- was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes ( h). in the steady state, the highest citrate productivity ( . g/lh) was obtained with this strain, when the feed medium contained g/l of glucose and dilution rate (d) was d = . /h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by -fold by using pet a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented % of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a % recovery and with and specific activity of . u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from . to mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as -butyl- -methylimidazolium tetrafluoroborate (bmim-bf ) and -butyl- -methylimidazolium hexafluorophosphate (bmim-pf ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu + -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar , john dahlbacka , noora sirén , niklas von weymarn , kaj fagervik : faculty of chemical engineering,Åbo akademi university, finland; laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from to • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture ( • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces -keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of , -diketo-d-gluconic acid, was selected as a suitable host for a , -diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two -keto-reductases in the host organism that diminish the yield of -keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of % of synthesis yields of the sporulating cells, in contrast, salt caused increase of % of spores counts, corresponding to % toxins production improvement. combined effects of both stressors lead to toxins production improvement of %, yield improvement of %. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. % increase of icps production was reached into l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than % improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg , javier menendez , christopher koth , matthew freeman : mrc laboratory of molecular biology, cambridge, uk; ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and -keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin , abd-elaziem farouk , hamzah mohd salleh , ralf greiner : biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, kuala lumpur, malaysia; centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße , d- karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of % referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about u mg − . gel filtration of the native enzyme on a calibrated sephacryl s- column gave a molecular mass of the phytase of , ± da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph . and . and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of , ± da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph . and was virtually inactive above ph . . at ph . , % and at ph . , % of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range . - . at • c. within days the phytase did not lose any activity in the ph range from . to . , but at ph values below . a rapid decline in activity was observed. at ph . , % and at ph . , % of the initial activity was lost during h. in the range of temperatures studied, - • c, the optimum temperature for the enzyme was found to be • c. the apparent activation energy was estimated at ph . from the slope of log v max versus /t. the data showed excellent linearity from to • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be . kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to • c and assayed using the standard phytase assay. the enzyme lost no activity in min at temperatures up to • c. when exposed for min at • c, it retained % and at • c % of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = . mmol l − and k cat = s − at ph . and • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations > mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between − and − m. mg + , ca + , mn + , co + , ag + , hg + , and cu + had little or no effect on enzyme activity, while zn + , fe + , and fe + showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe + and fe + is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from − to − m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of mol l − . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin subunit; hal ) in escherichia coli. the recombinant hal amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh , which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as . h − . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below . h − . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinÖzbek department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in . l of aqueous solutions containing % (w/v) corn gluten and . % (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures , , , , and • c; and at the ph values . , , . , and . to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of . - . cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method ( ) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures ( . % of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay , ufuk bakir , zumrut b. ogel : chemical engineering department, middle east technical university, inonu bulvari, ankara, turkey; food engineering department, middle east technical university, inonu bulvari, ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. . . . , p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. . . . , o-diphenol oxidoreductase) and tyrosinases (e.c. . . . , monophenol monooxygenase). another group of enzymes, peroxidases (e.c. . . . ), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, % glucose and . % yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;Äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr- núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal , microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, ) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, were active on one or more of the substrates evaluated, and of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., ) . among them, strain cr- , one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr- was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., ) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, ) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta . maximum activity was achieved on muf-butyrate at • c and ph . , suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., ) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p- , aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as . mmol o /l h and . g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to . g-xylitol/g-xylose from . g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol -phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol- phosphate (l-g p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol- -phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux , louise johansson , jo maertens , peter vanrolleghem , gunnar lidén : biomath, ghent university, coupure links , gent, belgium; department of chemical engineering, lund university, p.o. box , lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from . to . h − ) and different limitations (phosphate and glucose). the e. coli strain used was w with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu- l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., ) and did not produce citric acid when cultivated with sham (kirimure et al., ) . in this study, the transcript levels of alternative oxidase gene (aox ) (kirimure et al., ) and activities of alternative oxidase under the conditions of citric acid production were examined during days-cultivation. the amount of aox mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at days at log-phase, decreased during and days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., . curr. genet. , - . kirimura, k., et al., . biosci. biotechnol. biochem. , - . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect . a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect tss promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose- -phosphate dehydrogenase based on c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, c-metabolic flux analysis ( c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose- -phosphate dehydrogenase (g pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., ) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g pdh activity ; . ; . and times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., , metab. eng., , . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box , lund university, se- lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than % of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached . g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of . g/g. in contrast, only about % of the fermentable sugars were consumed within h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building , dk- lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at • c with wheat straw hydrolysate as a substrate in concentrations from to wt.% equivalent to total sugar mixture of - g/l. wheat straw hydrolysate (woh) [ g/l wheat straw, . % dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast . , and novozym (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than %, and high ethanol yields were achieved. reactor shows good long-term performance ( days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt . mtt gave lager strain a the ability to grow on yp/ % maltotriose in the presence of mg/l of the respiratory inhibitor antimycin a. this transporter gene shares % similarity with mph and mph , % similarity with agt and % similarity with mal and mal . moreover, mtt shares even higher similarity ( %) with the s. pastorianus mty gene (m. salema-oom, unpublished, ncbi accession number aj ). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a and ws / , and of a containing mtt or mtt alt, a more efficient, altered version of this gene lacking the basepairs from the end and containing base-pairs of vector sequences. these transport studies show that mtt and, especially, mtt alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk -ct- - ) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre laboratory, department of chemical engineering, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (Özçelik et al., ) , aroh for the reaction r at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r ) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to the portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc , and then sub-cloned to pmk e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk ::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,İ., Ç alık, p., Ç alık, g.,Özdamar,t.h., . metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. ( - ), - . the % respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. % in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez , puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a -year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak , istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was . u/mg and after % saturation this value increased . u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of , , , and % by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under h operation condition. Øyvind m. jakobsen , , michael c. flickinger , svein valla , trond e. ellingsen , trygve brautaset : department of biotechnology, norwegian university of science and technology, norway; sintef applied chemistry, sintef, norway; biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to g/l of glutamate upon methanol growth at • c (thermotolerant and methylotroph) and mutants producing - g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. the penicillium chrysogenum oat gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into -aminoadipic semialdehyde is important in providing -aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine- -aminotransferases and to lysine- -aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an -bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat promoter region. deletion of the oat gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat gene after induction by the basic amino acids may provide additional -aminoadipic acid for the formation of the -aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. -methylsalicylic acid synthase ( -msas) was chosen as a model pks. replicative plasmids carrying the genes encoding -msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding -msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing -msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box , aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt- useful in textile fabric treatment rita pyc , jadwiga sojka-ledakowicz , tadeusz antczak , joanna lichawska : institute of technical biochemistry, the technical university of lodz, lodz - , poland; textile research institute, lodz - , poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt- in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: pm for total pectinolytic activity, j/ml for endoglucanase and j/ml for endoxylanase and they were . - . times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph . - . . woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt- . this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [ - c] glucose as carbon source. the c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza , a. noohi , j. hamedi * : department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were rpm, days at • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed putative orfs, out of which are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a -bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with -bp insert as a probe, a -bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production h earlier and showed a . -fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and -fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño , gonzalo de gonzalo , gianluca ottolina , giacomo carrea , dick b. janssen , marco w. fraaije , biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijen- baeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring , stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, ) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm . a p -like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p -like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., ) . the full size gene encoding a cytochrome p enzyme was amplified by pcr from genomic dna and cloned into the vector pet a(+) for heterologous expression in escherichia coli bl (de ) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p enzyme shows hydroxylation activity towards -ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., . directional genome walking using pcr. biotechniques , - . werck-reichhart, d., feyereisen, r., . cytochromes p : a success story. genome biol. ( ), . - . . selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe , , yoshie yamauchi-sato , toshihiro nagao , satoshi negishi , tadamasa terai , takashi kobayashi , rolf d. schmid , yuji shimada : osaka municipal technical research institute, osaka, japan; stuttgart university, stuttgart, germany; the nisshin oillio group, ltd, yokosuka, japan; osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of cis, trans-and trans, cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; ( ) sequencial esterification-glycerolysis, ( ) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce % monoglyceride at % esterification. improving the thermal stability of cellobiohydrolases i (cel a) from t. reesei by site directed evolution frits goedegebuur , lydia dankmeyer , peter gualfetti , brad kelemen , edmundo larenas , paulien neefe , pauline teunissen , colin mitchinson : genencor international bv, archimedesweg , cn leiden, the netherlands; genencor international inc., page mill road, palo alto, ca , usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh (hypocrea jecorina cel a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., ) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: . ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were , -dimethyl- -octen- , diol and , -dimethyl- , -epoxy- -octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at • c and rpm. when the medium was cassava, the production of rose oxide, , -dimethyl- octen- , -diol and , -dimethyl- , -epoxy- -octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching mg/l for the cis-isomer and mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp - ) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler , m. retzlaff , k.-h. van pée , r. ulber : technische universität kaiserslautern, germany; technische universität münchen, germany; technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., ; keller et al., ) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., ) , e.g. halogenation of tryptophan in other positions than position . our current research is focussed on tryptophan- -halogenases, because the -br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan- -halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph . , . m) for h at + • c and centrifuging. the hydrolyses were carried out in shaking flasks for h at rpm and • c with the initial substrate concentration of . m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of h were , , , and %, respectively. this study was supported by ankara university biotechnology institute (project no: ). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi . to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. . this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before ]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya , pınar Ç alık , tunçer h. ozdamar : iblab, department of chemical engineering, metu, ankara, turkey; bre laboratory, department of chemical engng, ankara university, ankara, turkey. e-mail: e @metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec . . . ) that catalyses the synthesis the enantiopure -hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax ) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm ) chromosomal dna was used as sap gene (accession number x ) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc ::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc plasmid, thereafter sub-cloned into pbr , pmk and phv shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t promoter and expressed in e. coli bl (de ) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec . . . ) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a . kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose -phosphate/d-fructose -phosphate phosphoketolase gene, xpkl, was located from nucleotide to . the xpkl orf encoded a amino acids-residue polypeptide (xpkl) with a calculated molecular mass of , da, a value coincident with that estimated by comparative sds-page (about , da). a putative ribosome binding site (gggagc) is present - nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was % identical to dxylulose -phosphate/d-fructose -phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il in the broad host range pls rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a -fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl (de ) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was . -fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., ) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation ( . g g − ) our immobilisate exhibits an activity of , u g − . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : . ± . m versus k m,free : . ± . m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity ( % as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by %. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade , po box , dk copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, ) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., ) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., ) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, ) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., ; lebouder et al., ; panchaud et al., ) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., ) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., ) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., ) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., ; smid et al., ; de vos et al., ) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of , sage (serial analysis of gene expression) tags of nt representing , different tags were analyzed. the tags seen or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., ) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf , ∆snf and ∆snf ∆snf was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about h in ∆snf or ∆snf mutants, and for more then h in ∆snf ∆snf mutant, whereas it only lasted h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of . h − . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf and ∆snf mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, ) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan , ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with , -dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg , wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: ( ) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. ( ) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. ( ) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. ( ) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. ( ) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. ( ) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately . million genomic shotgun sequences and . expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent -end sequences from a total of non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. ( ) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is . % of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d . billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping ( . of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a -week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb- and raftilose synergy (prebiotic) was performed in colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t , week ) midway through (t , weeks) and following intervention (t , weeks). rectal biopsies were obtained at t and t . the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces : in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < . ) and lactobacilli increased although not significantly (p = . ) while coliforms decreased (p < . ). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < . ), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t versus t (p = . ) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < . ), ifn-␥ production by pbmcs but il- , il- , il- , and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaÅrsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box , sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature ( - • c), ph ( . - . ) and sucrose concentration ( - g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with fermentations revealed that the highest eps formation was found at • c, ph . and g/l of sucrose. sucrose did not further affect the eps formation above a concentration of g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th , th and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il- , il- and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- , il- and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il- production remained unaffected. those lactobacilli with greatest capacity to induce il- were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il- -inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk- , denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde -phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum v were able to bind to caco- intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from to mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of and . , to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), , -dihexylocarbocyanine iodide (dioc ( )) and to-pro- . results show that an enteric coating material, eudragit l - , is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and -hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok , usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp a , cyp e , and ugt b from sorghum bicolor accumulated % dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp a accumulated % dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp a plus cyp e resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp a and cyp e , was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp a and cyp e were complemented by retransformation with s. bicolor ugt b . accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. , loci annotated with full-length cdnas were analysed and loci ( . %) containing utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. of the isolated loci ( . %) contained only one utr intron and these were used for further analysis, where it was investigated if the utr introns had a characteristic size distribution. genes containing transcripts with utr introns were more subjected to alternative splicing ( . % versus . %) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate , and bp compared to only one for orf introns. this suggests widespread multiple splicing events in utr introns. the results presented here suggest that utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp f constitutively expressing cyp a cdna of the bovine cytochrome p scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc . growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was . giu/ml, after h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, . times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about . % of total intracellular enzyme. the best length of time for sonication was found to be min. experiments showed that optimum ph and temperature of the enzyme were . - and • c, respectively. the highest activity of the enzyme was observed at ph . for up min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of , and • c, by the enzyme, caused %, % and % of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan , a. alirezasadat barikani : department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of , , and s, and the effect of the uv mutation was assessed. as a result, the period of s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer . m with the ph of . , then ml nitrous acid . m was added and was mixed for - min. finally the sampling was done in the periods of , , , and min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol %, acetozyme gz . g/l, acetozyme d g/l, acetic acid . % and l double distilled water. the acetic acid was produced in the rate of g/ ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek ). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya , turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka - , japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., ) . in the present work, we have clarified that there are two types of sul genes (scsul and non-scsul ) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and % identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul and non-scsul has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu . the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul or non-scsul failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul and non-scssu resulted in two-fold higher sulfite production compared with overexpression of only non-scssu and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., . functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s, respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to and brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e . the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about , , da. sugar content of brm was . % (w/w) and glucosamine ( . mol%) was the high level. protein content of brm was . % (w/w) and serine ( . mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e . potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to / and clarifying by centrifugation. total-n: . g n/l; ␣-amino-n: . % (w/v) (as glycine); soluble protein (bradford): . mg/l (as bsa); total carbohydrates: . g/l; reducing sugars: . g/l; dry weight: . % (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about h. cultivations of a. oryzae atcc were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. . ) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, ) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against -ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of -ketogluconic acid is optimal at ph . - . with significantly lower values at ph . and . , respectively. data on the kinetics of the conversion of both glucuronate and -ketogluconate will be shown. nakase, t., suzuki, m., . j. gen. appl. microbiol. , - . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, ; hugenholtz et al., ) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc and ln. mesenteroides fsc . after digestion by gi enzymes, the juice was supplied to fully differentiated caco- cells to study iron uptake and transepithelial transport by caco- cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc fermented juice. surprisingly, the l. pentosus fsc fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc fermented juice than from l. pentosus fsc fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately billion people worldwide. in sub-saharan africa, more than million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. in the case of demre sivrisi anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at • c for week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv- was tested in this work in batch experiments in synthetic media at constant initial substrate concentration ( g l − ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = . g l − ) and yield (y p/s = . g g − ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk- , denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en and en . considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: ( ) testing against the specific spoiling species of the food product, ( ) testing on surface, not only in suspension, ( ) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to • c and ph values between and . were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to % (w/w) of total sugars were achieved when the initial lactose concentration was g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until to about million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot ( strains) was tested on cheese curd. by a limited number of cultures, successively, out of , out of and out of strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar , casilla -d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between . and . h − . composition of the culture media was that of the mrs broth. biomass concentration presents higher values ( . - . g l − ) at dilution rates between . and . h − . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt . high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as − • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at - mpa, cells were plated under optimal conditions to assess cell viability. treatments at - mpa and − • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at − • c compared to the same treatment at − • c because both the extra-and intracellular surrounding remained fully frozen. at − • c a solid-solid phase transition was observed when pressure was higher than mpa. a metastable state of ice i was observed at mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w ), middle (s. cerevisiae w ) and final fermentation (s. cerevisiae k ). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium ( % glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and -phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about %) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. . g/dm ), methanol ( . mg/dm ) and isoamyl alcohols ( . mg/dm ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. . g/dm ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa , india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh were treated with different biofertilizers using nitrogen level of , and kg ha − and one level of p, i.e. kg ha − in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e at ( ± . kg ha − ) as well as kg ha − ( ± . kg ha − ) followed by a. chroococcum ht ( ± . kg ha − ) and avk ( ± . kg ha − ). whereas, with kg ha − highest yield was observed with mac ( ± . kg ha − ) followed by e ( ± . kg ha − ) and avk ( ± . kg ha − ). maximum height at kg ha − was observed with mac inoculation ( . ± . cm) followed by avk ( . ± . cm) and ht ( . ± . cm). various chosen strains were tested with desi (hd ) and american cotton (h ) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after , , and days. highest survival rate was observed in mac (( . ± . ) × ), which decreased after and days, respectively. ( . ± . ) × , ( . ± . ) × with mac and ( . ± . ) × and ( . ± . ) × with ht , respectively. maximum boll weight was with avk ( . ± . g boll wt. plant − ) followed by pseudomonas ( . ± . g), ac ( . ± . g) and ala ( . ± . g) boll no. plant − was maximum with ala and avk ( ± . plant − ) followed by pseudomonas ( ± . plant − ). maximum height and dry matter was obtained with pseudomonas ( . ± . cm) and avk ( . ± . cm) with variety hd under field conditions. net saving of % nitrogen was observed using a. chroococcum (e and avk ) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of , -diphenyl- -picrylhydrazyl (dpph) and , -azinobis-( ethylbenzothiazoline- -sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts ( . % and . %) than raw seeds ( . % and . %) and bsa ( . % and . %), respectively. it has been also found that tempeh fermentation process increased . times sulfhydryl groups content ( . m/mg of albumins) as compared to raw seeds ( . m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds ( . fi and . fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas , j.m. domínguez , p. torre , j.c. parajó : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a % of this waste. soluble sugars ( . %), cellulose ( . %) and pectin ( . %) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at • c and liquid:solid ratio of g/g, containing . g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect (atcc , nrrl ) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect cultures with naringin as inducer (pérez-mateos et al., ) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, ) . immobilized enzymes reduced % the naringin content at • c and ph . . furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles ( h) remaining % and % catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl - /ali). gray and olson, . j agric. food chem. , - . pérez-mateos, et al., ( ), . otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and auto-claved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues percent (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, Árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box , astaxanthin ( , -dihydroxy-␤,␤-carotene- , -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations ( . - , v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym ) and lipase from mucor miehei (lipozyme rm m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of % in h. for hexane, the same conversion was reached after h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec . . . ). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to %. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of , -d ( , . and mg l − ) and n medium supplemented with five level of , -d ( . , , . , and . mg l − ). for deilamani cultivar the best medium was n with . mg l − , -d and for tarom mahalli the same medium with mg l − were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l − ) [( : ), ( : ), ( : . ), ( : ), ( : ), respectively] and . mg l − , -d and mg l − bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for min [prior to transfer to the regeneration medium], and third factor was factor of with substitution of sucrose with maltose [after weeks; sucrose: maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to %. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova , brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh ␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg with inserted crt genes was used for transformation of chemically competent e. coli dh ␣ cells, while in s. cerevisiae shuttle vector paur was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: . - . g/g of d.w., beta-carotene: . - . mg/g of d.w.). the highest yield obtained in transformed strain was . g/g of lutein and . g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains ( . mg of total carotenoids + mg ergosterol/l; g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures- , , and • c. after heating, grape seeds were extracted with % ethanol ( . g grape seed/ ml of % ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at • c for min, while that of powdered grape seed extract (pgse) were at • c for min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at • c for min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, , canon frome court, ledbury hr td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene ( g/g) in medium with % nacl and mm h o than in control sample. similar result was observed in r. glutinis cultivated in presence of % nacl in inoculation medium only ( g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from to , mycelial growth ( . g/l) and eps production ( . g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from to at day , which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec . . . ) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with -methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. , kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for , ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , -d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde- -phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il . we constructed a number of l. lactis strains in which the tpi activity was modulated from % to % of the wild-type level. the enzyme was found to be present in high excess with % tpi activity supporting % of the wildtype glycolytic flux, and with % of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose- -phosphate (g p), fructose- , bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from % to %, and only in the strain with % tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level ( % tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at • c in the presence of . % isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of - kb bands. more than bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, - . e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads , technical university of denmark, dk- kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten , s. borges , p. cravo , l.a. cardoso : instituto de investigação científica tropical, cvz, lisboa, portugal; instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of % of ad libitum feeding in the wild animals (decrease of % lw) and % of that ingestion in the halop breed (decrease of % lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t and igf- in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in , the area under their cultivation has globally increased from . million hectares in to . in . the number of countries adopting gm crops also rose from one country, the usa, in to in . despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of shoppers in seven food outlets between november and december of . it revealed knowledge at %, with positive attitudes and good perceptions towards gm foods (χ = . , d.f. , p < . ). seventy nine percent of shoppers were willing to buy and consume gm foods (χ = . , d.f. , p < . ). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = . ). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version . . the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting . million kenyans today, mostly children. consumers and gm foods: the case of turkeyÖzlenÖzgen , mustafa yildiz : department of family and consumer sciences, school of home economics, university of ankara, ankara , turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsÖzlenÖzgen , haluk emiroglu , mustafa yildiz , ayşe sezen taş : department of family and consumer sciences, school of home economics, university of ankara, ankara, turkey; faculty of law, university of bilkent, ankara, turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of university educated consumers, consisting of males and females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years - , being analyzed a total of valid surveys. the designed questionnaire included questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that % of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group ( %) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec . . . ) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as • c, ph = . , h and mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = − . ). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = . − . ) and acetate production (c jacetate pk = . − . ), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of amino acid residues, with a signal peptide and a proenzyme region of and amino acids, respectively. the caprine preprochymosin has % and % identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm - c, k. lactis sel , characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g / s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from . to . h − , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of . h − , nitrogen limitation induced lipid synthesis ( % tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being %. finally, the batch and continuous culture methods are compared for docosahexaenoic acid ( : , n − ) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn t c . bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain bvl and microbacterium sp. strain a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain bvl required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain a only used nadph. several studies were made on strain bvl intracellular soluble extracts. a k m of . m cr(vi) and a v max of . ± . nmol cr(vi) min − mg − protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were . • c and . , respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., ) . h. butylicus grows at up to • c (optimally between and • c) at ph . it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains , , bp on a single circular chromosome. protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., . j. bact. , - . hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann , , anke henne , gerhard gottschalk : göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb and hb , provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb and hb share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from Ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between . and . and two isolates at . - . . they grow at temperature between and • c and salt concentration between % and %. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag - t . the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that . % of the transportation fuel has to come from biofuels in year . a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to % of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about % of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around . - . euro/l ethanol assuming a yearly capacity of tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita - , japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about % of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when kg of wet spent grain was treated under the kg/cm pressure for min using a l steam explosion reactor, g of total sugar was obtained from the liquefied spent grain. furthermore, . % (w/v) of glucose, . % (w/v) of xylose, and . % (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. . % (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted % of initial sugar concentration after days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid ( -hydroxy- octadecenoic) that represents % of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn c, rmn h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym , liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with . n hcl and confirmed by tlc analysis and the results showed conversion rates about %. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , -pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about % of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about . euro/kg n. gas lift reactors could sustain the highest anammox capacity at . kg n removed/m reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was . kg n/m reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a -week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n l g bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj , usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: ( ) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, ( ) estimation and control of volatile organic compound (voc) emissions from open tanks, and ( ) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design k (k = ) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in ml sealed bottles with different concentrations (cr(iii)- ppm, cu(ii)- ppm, mn(ii)- ppm, zn(ii)- ppm) of metal sulfate and g l − of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn ( - %), however with cu ( - %), mn ( - %) and cr ( - %). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten Øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk- odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. Øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df and the plasmid putk that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df /putk and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df /putk over incubation period of min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df /putk were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df /putk was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and -butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area inİzmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of isolates required salt for growth. the salinity ranges at which growth was detected were: - % for two isolates, - % for one isolate, - % for two isolates and - % for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within - % salt concentration range at ph . . extracellular protease activity was detected in the medium of all isolates grown at , , , and % salinity at both ph . (optimum growth ph) and ph . . out of isolates, , and were found to produce cellulase enzyme when the salt concentrations were , and %, respectively. at % salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at - % salt concentration range. this project was supported by tubitak through project tbag - t . chemical engineering department, middle east technical university, ankara , turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio ), tin oxide (sno ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio and sno coated glasses with respect to the control glass reduced by - % and - %, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f- narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m g vs − ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the -plus variants of foot protein type (fp- ), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type variant a (mgfp- a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼ % purification with affinity chromatography. recombinant mgfp- a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type (mgfp- ). the adsorption abilities and adhesion forces of purified recombinant mgfp- a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp- using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp- a was comparable to that of cell-tak but lower than that of recombinant mgfp- . collectively, these results indicate that recombinant mgfp- a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using -aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to mg/l of p-cresol within h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to % (measured as decrease in cod) were obtained after days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector a, chandigarh , india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj . role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in days, whereas free cells were able to deplete % pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of . log units in colony forming units of free cells at the end of the study ( days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of . % and - -fold purification. the activity of pxb was stable at the ph ranging from . to . . andreja kresal, vanja kokol, vera golob textile department, university of maribor, , slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani , l. kredics , a. szekeres , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungary; hungarian academy of sciences, university of szeged, microbiological research group, hungary; pilze-nagy ltd. kecskemét, p.o. box , hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of , , , , , and m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, - lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod , cod, toc, vfa, tkn, n-nh + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h o . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod ) and n-nh + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to %). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at , , , , , , , , and • c, at ph , in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was h. initial cod of the stillage amounted to . g o /l, the main carbon sources being reducing substances ( . g/l), organic acids (determined as their sum) ( . g/l) and glycerol ( g/l). at • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from . % ( • c) to . % ( • c). without ss separation, cod reduction ranged between . % ( • c) and . % ( • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was • c and that there was a local optimum at about • c. according to the temperature applied, the content of reducing substances decreased by . - %, that the organic acids by . - . %, and that of glycerol by . - %. the experiments also produced the following two findings: ( ) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and ( ) temperature was a factor affecting the demand for ammonia nitrogen (n-nh ), which was the highest at and • c. the high n-nh demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc- was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately % homology with the general pce dehalogenase). according to the substrate utility of this strain kbc- and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene , -dioxygenase and cis- , -dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt and we developed three microbial systems carrying: ( ) benzene dioxygenase, ( ) dihydrodiol dehydrogenase and ( ) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was % and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt or gtt genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt ∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt ∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt p and gtt p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt ∆ and gsh ∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd + conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione ( -methyl- , -napthoquinone), we have also observed that gtt p and gtt p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt ∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt ∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to - % degradation in days depending on the culture conditions, whereas the latter showed a degradation percentage above % in days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik , , marianna turkiewicz , jaroslaw dziadek , pawel parniewski : centre for medical biology pas, lodowa street, - lodz, poland; faculty of biotechnology and food sciences technical university of lodz, stefanowskiego / street, - lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from to • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly %. the main object of our further research is to clone hsp family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i / / . out of plant species found in the flora of turkey, about are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over bulblets/explants) from single immature embryos on nutrient media in most species tested after months of culture initiation. regenerated bulblets were kept at • c for weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of -fluorophenol ( -fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use -fp up to concentrations of . mg l − and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during -fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after days ( nkat). the optimum biobleaching conditions are • c and ph . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brightness from to . in -day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about - weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of . l is used. in this method, butachlor with concentration of mg/l are treated. removal percent of butachlor for concentration of . and mg/l are calculated to . % and . %, respectively. removal percent of cod is also calculated to %. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas , p. torre , j.m. domínguez , j.c. parajó , a. converti : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was . % of xylitol ( g/l), . % of other sugars and . % of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of . in these conditions this treatment allows to remove a . % of the impurities. the resulting solution was evaporated and crystallized containing % of ethanol and a xylitol concentration of g/l. crystallization was performed at t = • c with slightly agitation. after h were separated xylitol crystals with a recovery yield of % and a purity degree of %. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was > mg of amplified genomic dna corresponding to about a billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn + , cu + and pb + at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez , , carla crespo , bo mattiasson : department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box , s- lund, sweden; instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately mg/l of zn + , mg/l of cu + and mg/l of pb + . the maximum concentration of hydrogen sulphide obtained was approximately mm. removal efficiencies of %, % and % for zinc, cooper, and lead, respectively, were achieved in the present work. at • c during days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) ppm, ni(ii) . ppm, zn(ii) ppm. the maximum precipitation percentages were approximately: % ( ppm cr(iii)), % ( . ppm ni(ii)) and % (for d. vulgaris- ppm zn(ii) and desulfovibrio sp.- ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co evolution and o consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of . l. complete suspension of the coal particles of - m mean diameter was achieved at an agitation rate of rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co production rate and o consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal s rdna-pcr products, bp and bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy and cy , respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes oligonucleotide probes representing different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b (fb ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as mm, whereas for arsenate, this bacterium could resist to concentrations upper than mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level . (f-test). it took place the cod degradation at . % with the average yield coefficient y chsk/x . g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (> . %)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the . % cod decrease after the steady state stabilization. this work was supported by project msm . fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of -cp, -cp, , -dicp, and , , -tricp as sole source of carbon and energy at • c (ra) and • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over months. total chlorophenol removal capacities of and mg l − day − were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of mg l − day − . the population of ␤-proteobacteria was the major bacterial community of the biofilm ( - %) followed by the ␥-proteobacteria ( - . %). two bacteria with the ability to mineralize mg chlorophenols l − were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by • c (in two steps of • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from % of the ␥-proteobacteria at • c to % at • c. the pollutant removal capacity decreased to and mg l − day − in ra ( • c) and rb ( • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of urban consumers and professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: . immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l , hsp , hsp . the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. . diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp c-end, l n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. . water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. . development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. . human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. . nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent ). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez , la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in , and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo - a planta, edificio urbis, las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between and % of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl -hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. . van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus , -anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣- , -glucan lyase ␣- , -glucan lyase, a new class of starch and glycogen degrading enzyme ␣- , -glucan lyase, a new starch processing enzyme for production of , -anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣- , -glucan lyases from fungi ␣- , -glucan lyases producing , -anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣- , -glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe* -leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd soluble forms of toll-like receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq - -c - ) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk program. this study was supported by ankara university biotechnology institute (project no: ). this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c- of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c- and c- of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of -keto- this work was partially financed by the scientific and technological research council (cicyt, spain), grant ren - , by the iii pla de recerca de catalunya (generalitat de catalunya), grant sgr- , and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: / / and / / . this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). financed by a marie curie re-integration grant merg-ct- - and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f , d ) and gvop- . . .- - - . the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt pxib pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet Ç etin , sedat dönmez , gönül dönmez : department of biology, faculty of science, ankara university, beşevler, ankara, turkey; department of food engineering, faculty of engineering, ankara university, dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at mg l − initial chromium(vi) concentrations was determined as . chromium(vi) reduction by srb was investigated at . - . mg l − initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than % of the initial chromium(vi) levels which ranged from . to . mg l − within - days period. the effects of initial - . g l − concentrations of sulfate and - % (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including mg l − chromium(vi). when the % whey was used as carbon source in the pc medium, . % of the . mg l − initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt pxib pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa , rh. aurantiaca , rhodotorula sp. , williopsis californica , candida krusei t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was and × − mol (g sorbent) − for rh. aurantiaca and s. cerevisiae , respectively. in the case of living biomass, the high- this work has been financed by the spanish ministry of science and technology and european feder (project ctm - ). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo - , japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in cm culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl · h o, feso · h o and cacl · h o (ph . ). light was supplied by panels of blue or red led lamps. the temperature was kept at • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres , l. kredics , l. hatvani , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungry; hungarian academy of sciences, university of szeged, microbiological research group, hungry; pilze-nagy ltd. , kecskemét, p.o. box , species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of - amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about % of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., . integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit , - . glucansucrases from family of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family and the family of glycoside hydrolases. among the natural diversity existing in family in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b- f (dsr-s), which catalyses almost essentially the synthesis of ␣- , -linkages, alternansucrase from l. mesenteroides nrrl b- (asr), which produces alternan polymer formed of ␣- , and ␣- , -alternated linkages and finally dextransucrase from l. mesenteroides nrrl b- (dsr-e), which is responsible for the synthesis of a branched dextran composed of about % of ␣- , -linkages in the main chain and % of ␣- , -branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter Østergaard novozymes a/s, dk- bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in , it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from to ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo , johan patring , jelena jastrebova , thomas andlid : department of chemical and biological engineering, chalmers university of technology, po box , gothenburg, sweden; department of food science, swedish university of agricultural sciences, po box , uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about . times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr ) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr genes (fu et al., ) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., ) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of primers out of the issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction ( . ), and the percentage of informative polymorphisms ( %). the three combinations of ecori/msei primers revealed reliable aflp markers, ( %) of witch were polymorphic. the range of fragment sizes varied from to bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > . ) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = . ), fortuna and clementine (s = . ), kara and king of siam (s = . ). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in - at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several -year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ . ) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = . ) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = . ) and clementine (s = . ). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about mg l − initial reactive dye concentrations was determined as . lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x- ) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to -fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x- had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with . n edta. isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core ( . m, . m diameter). cores were collected from depths of to m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at • c on board the vessel for - days. the core samples were plated on several different agar types and incubated aerobically for weeks at • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromatocity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at ppm was . % equal to . mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo was studied. cells of ifo did not flocculate even in the stationary phase without mg + ("mg + -deficient cells") although they began to flocculate strongly h after inoculation in the presence of mg + ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg + -deficient cells". co-flocculation between "complete cells" and "mg + -deficient cells" was investigated by chemical modification. treatment of "mg + -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg + -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg + -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg + -deficient cells". these findings suggest that "mg + -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo . the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a - kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules ( days old) in vitro. for further identification of the portal serum factors in the range of - kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing % fetal calf serum, % portal human serum or % peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight . kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa - , japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c ) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism. srdna phylogenetic analysis of the isolates, totally ca , showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost % of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent , entl ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black , cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of . for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at • c for rb , cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada- , spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in % and % (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant ( % versus %, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, ) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., . appl. microbiol. biotechnol. , - . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromaticity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of mm s − ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches %.both reactors have been operated for days, until a volumetric olr of g cod l − j − , with more than % of carbon removal. the total amount of cod removed and methane produced were higher in reactor b ( . and . %, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to % cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about % dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h and co . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h production in the acidogenic fermentation reactor (a l cstr). preliminary results show that the effluent of the acidogenic reactor fed with g/l total sugars and operated at ph and d = . h − (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h production yield has been estimated at . mol h /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s ) and biomass concentration (x ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm /pbb a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about . g l − leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume . m ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than % of total nitrogen at hydraulic retention time (hrt) of h. at hrt of h, the bioreactor accomplished a maximum rate (the transformation of nh + to n ) of . g n/day m of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with % sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = . day − . yield of sulphur conversion to sulfate after days was η = %. in the presence of bottom ash from the incineration of refinery sludges µ = . day − and the yield of sulphur conversion was η = . %. a η fe = % removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in ml sealed bottles with different concentrations of studied metals ( - mg/l), with % of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than - %) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni ( days old) were incubated in rpmi medium containing % fetal calf serum, % hamster portal venous or % hamster peripheral venous serum (highly susceptible host) or % rat portal venous or % rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus % fetal calf serum, blis were increased by % in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules ( days old) in the lungs. cell proliferation was detected in schistosomules of days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at ppm was . % equal to . mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology ( ). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik ( ). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au ( ): topics were "gene diagnosis" ( ) and "genome research-what is in it for me?" ( ) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school ( ), high school students spend - weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age - ) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. ( ) european commission. eurobarometer . , december ; ( ) www.dialog-gentechnik.at; ( ) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord- - yoilsho authors: nan title: abstracts of the (nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the (th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - -y sha: doc_id: cord_uid: yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise % of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only . % of the volume, but % of the total cell number (kmiec ) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. ) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr breast cancer cells were exposed to the anti-cancer drug herceptin ( μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm- cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around % of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm- cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu th health program grant agreement no . deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases and (erk / ) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk / activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine as a prerequisite for nuclear erk / signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk / signaling: since activation of erk / triggers erk dimerization, a prerequisite for erk t autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t -phosphorylation. first, we investigated the impact of monomeric erk on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk ∆ - and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk ∆ - overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆ - in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk and prevents its dimerization. moreover, the peptide effectively inhibited erk t -phosphorylation and nuclear translocation of yfp-tagged wild-type erk after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype (aav )-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i knockout on cardiac function and survival in a murine heart-failure model of cardiac β adrenoceptor overexpression. methods and results: β -adrenoceptor overexpressing mice lacking gα i (β -tg/gα i -/-) were compared to wild-type (c bl/ ) mice and littermates either overexpressing cardiac β -adrenoceptors (β -tg) or lacking gα i (gα i -/-). at days of age mortality of mice only lacking gα i was higher compared to wild-type or β -tg, but similar to β tg/gα i -/mice. beyond days mortality of β -tg/gα i -/mice was enhanced compared to all other genotypes (mean survival time: ± days). echocardiography revealed similar cardiac function of wild-type, β -tg and gα i -/mice, but significant impairment for β -tg/gα i -/mice (e.g. ejection fraction ± % versus ± % in wild-type mice). significantly increased ventricle-to body-weight ratio ( . ± . % versus . ± . % in wild types), left-ventricular size (length . ± . cm versus . ± . cm in wild types) and anp and bnp expression (mrna: % and % of wild type, respectively) clearly indicated hypertrophy. gα i was significantly upregulated in gα i -knockouts (protein compared to wild-type mice: ± % in gα i -/and ± % in β -tg/gα i -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β adrenoceptors in β -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than -fold higher (b max : ± fmol/mg) than on the otherwise used c bl/ background. accordingly, fvb/n-based β -tg mice showed a significantly impaired cardiac function at an age of d while c bl/ -based β -tg mice did not. conclusions: gα i -deficiency combined with cardiac β -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c bl/ background β adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day while there was overt cardiomyopathy in mice additionally lacking gα i . we propose an enhanced effect of increased β -adrenergic drive by lack of protection via gα i . the observed gα i upregulation was not sufficient to compensate for gα i deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i is currently addressed in a subsequent study using β -tg and gα i deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; mg/kg per d) for and hours and days. overall days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; d iso ± ) vs. wt cardiomyocytes ( d iso ± ). at this time point a % decrease of cardiac output and a % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after h ( -fold), and declined after h ( -fold) to . fold increase after days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: regulated genes (up-regulated: %) in untreated; altered genes (up %) after h; changed genes (up %) after h and altered genes (up %) after days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m receptors, while m receptors appear to exert a heart-specific function only at later stages. continuous m blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m inhibition. taken together, we postulate that muscarinic m receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir- has proved effective against fibrosis of the myocardium and in other organs. mir- has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir- in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir- fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir- in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav -icre vector allowed for specific and almost complete deletion of mir- in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir- in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir- gene in mice. aav -mediated deletion of mir- , albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir- demonstrated that mir- exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir- therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c acts as a multikinase inhibitor. results of a thermal shift assay revealed that c shows by far the highest binding affinity to the bmp- -inducible kinase (bmp k/bike). bmp k represents an as yet largely uncharacterized protein, which is not regulated by bmp- in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c and (ii) the role of bmp k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c affected the viability of human umbilical vein endothelial cells (huvecs). both c and the knock-down of bm k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec- ). also the proliferation of hmec- was reduced by c treatment (ic : µm). a tube formation assay on matrigel demonstrated that c significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c treatment nor by bmp k gene silencing. in regard to inflammatory processes, c treatment or bmp k silencing of huvecs decreased the adhesion of thp- cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c or bmp k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c treatment, the knock-down of bmp k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c in vitro. the precise role of bmp k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp k silencing and c treatment will be further elucidated. moreover, since the inhibition of bmp k seems not to be responsible for all actions of c , we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine (cxcl ). cxcr seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr agonists or antagonists could result in reduced tlr signaling. however, the role of cxcr and the influence of different cxcr ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose ( mg/body weight) in mice. the plasma stable cxcl analog ctce- d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr , cxcl and tlr expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce- d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately %, % or %, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce- d diminished the lps induced expression of cxcr , cxcl , tlr , nf-κb, cleaved caspase- and gp phox, whereas heme oxygenase expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce- d in all organs. the cxcr is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce- d attenuated tlr signaling, induced heme oxygenase activity and mitigated apoptosis. thus, the administration of cxcl analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c bl/ j mouse strains with a loxp flanked exon of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef - (fxn -/-) and fx-mef - (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over days, with medium and substance changes at day and , which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day , , and and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor which leads to disruption of protein synthesis and finally causes cell death [ ] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [ , ] . in detail, we confirmed the involvement of the host cell chaperone hsp and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [ , , ] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp and fk -binding protein (fkbp) as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp , cypa, cyp , fkbp and fkbp . the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [ ] murphy ( ) toxins , - . [ ] barth ( ) naunyn-schmied arch pharmacol , - . [ ] kaiser et al. ( ) cell. microbiol. , - . [ ] dmochewitz et al. ( ) cell. microbiol. , - . [ ] ratts et al. ( ) j. cell biol. , - . [ ] schnell et al. ( ) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins ( ). prompted by these result, we found that only human α-defensin- (hnp- ) but not human β-defensin- (hbd- ), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin- prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp- causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy -asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a . % knockdown (p< . student's t-test, n= ) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n= ). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s mix), as was the synthesized ba-epoxide (-s mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr -positive colon stem cells, which coincided with atr-chk -p signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h ax, suggesting the usefulness of γ-h ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [ ] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines ( ). in particular, the chemokines monocyte chemoattractant protein (mcp- , ccl ), interleukin- (il- /cxcl ) and stromal cell-derived factor (sdf- a/cxcl ) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl was shown to attenuate obesity in mice ( ) . the cc chemokine ccl is known to stimulate ccr receptors, and the cxc chemokines cxcl and cxcl to activate cxcr and cxcr or cxcr and cxcr , respectively. the receptor(s) activated by cxcl is currently unknown. a role of cxcl in modulating cxcr signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr and the related receptor fprl , and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr receptors were absent ( ) . yet, the analysis revealed appreciable levels of mrna encoding ccl , cxcl , and cxcl , and ccr , cxcr , cxcr , fpr , and fprl , and cxcr . of interest, cxcr -and cxcr -mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl , cxcl , cxcl . while sgbspreadipocytes and adipocytes did not accumulate ccl -mcherry upon stimulation, they showed a small accumulation of cxcl -mcherry, and a strong accumulation of cxcl -mcherry in the endosomal compartment. similar results were obtained in murine t l- preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl -binding putative receptor protein(s) in murine t l- preadipocytes. ( ) makki, k. et al., ( ) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic ', and to some extent of the corresponding cytidylyl analogue ccmp , . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac -camps in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell , - ( ) . koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. ( ): p. - ( ) . nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem. , - ( ) cardiovascular pharmacology hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel ( %: %: . mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology ( % strain, hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen . ; collagen + cms . ; collagen + cms + cfbs . , collagen + cms + ffbs . . we next generated ehm with cms and ffbs or cfbs. after weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p< . ) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm : . ± . , n= vs. . ± . , n= ). cross sectional area (csa) of tissues was greatly increased (p< . ) in cfb-ehms (csa in mm : . ± . , n= vs. . ± . , n= ) and nonmyocyte content was higher in cfb-ehms ( . ± . , n= vs. . ± . , n= ; x cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase (has ) enzyme was significantly (p< . ) upregulated. based on these findings, we pharmacologically intervened with has mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p< . ) reduced csa (csa in mm : control . ± . , n= ; hyaluronidase of concentrations from . u to u . ± . , n= ) with a concurrent, statistically significant (p< . ), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm : control . ± . , n= ; hyaluronidase of concentrations from . u to u . ± . , n= ). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type receptor (vegfr ), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with µm of the camp analog -pcpt- '-o-camp ( ) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac in huvec decreased the basal and vegf-induced sprouting. surprisingly, µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than , indicating an additional role of pka. in accordance, µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh cre ) were created using the cre/loxp system. mr cdh cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received % nacl with drinking water for weeks. the mineralocorticoid deoxycorticosterone acetate (doca, . mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype ( . ± . vs. ctrl . ± . mmhg, p< . ) and mr cdh cre ( . ± . vs. ctrl . ± . mmhg, p< . ) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh cre mice (ventricle weight . ± . mg vs. mr wildtype . ± . mg, p< . ). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam ) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: . ± . vs. ctrl . ± . , p< . ) and mr cdh cre ( . ± . vs. ctrl . ± . , p< . ) mice. mrna expression of the fibrosis marker gene col a (mr wildtype . ± . -fold; mr cdh cre . ± . -fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype . ± . fold; mr cdh cre . ± . -fold vs. ctrl). in the kidney vcam mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca + concentration due to ca + entry from the extracellular space is critical for platelet activation and aggregation. ca + entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a (txa ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca + entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca + entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc and trpc in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca + signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc /c proteins for procoagulant activity. in addition, we detected the expression of s trpc transcripts in mouse platelets. therefore, we tested trpc /c /c -deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc /c /c inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc /c /c -deficient mice and we observed that the aggregation was reduced after adp ( µm) or txa -analogue ( µm) stimulation, but not after collagen stimulation ( µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca + response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/ mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/ mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/ mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/ mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/ mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp b ( -fold in females, -fold in males) in -day old offspring of adult female mice exposed one week prior mating to tcpobop ( mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop ( mg/kg) prior to mating. the analysis of hepatic cyp b expression in -day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a -fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e . followed by crossfostering proved transmission to be mediated predominantly via lactation (f hepatic cyp b induction -fold) and only to a minor part via intra-uterine exposure ( fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f and f generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp b catalytic activity. in both tcpobop-pretreated f and in their f descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by > %. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by % in -weeks-old offspring. in summary, the car-mediated cyp b induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with alzheimer´s disease patients indicated that the combined treatment with donepezil and egb ® had less side effects than donepezil alone (yancheva et al., ) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment ( days of pretreatment) with donepezil ( , mg/kg p. o.), egb ® ( mg/kg p. o.), or the combination of the two drugs, or vehicle in - month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. ( ) . ginkgo biloba extract egb (r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, ( ) , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations ( - µm; just et al. expert opin emerging drugs : - , ) observed in patients treated with dantrolene. although not yet proven directly, oat may be involved in renal secretion of dantrolene and -oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp . since oat was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. : - , ) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr , by influx through oat , where it inhibits calcium efflux by ryr thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat . in addition, we identified, besides dantrolene and -oh dantrolene, several further fdaapproved drugs such as tyrphostin ag , ceefourin , glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat . controls with other defects and controls with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥ mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with % confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls and additionally adjusted for maternal age in controls . results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were / ( . %) for cases, / ( . %) for controls and / ( . %) for controls and adjusted ors ( %cis) were . ( . - . ) and . ( . - . ), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-( . vs. . for controls , and . vs. . for controls ). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being and . times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac protects non-transformed cells from topoisomerase ii-(top )-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac -mediated regulation of topoisomerase ii activity. however, the link between rac and top -poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top -poison-induced cytotoxicity. here, we investigated the relevance of rac -regulated actin cytoskeleton integrity as well as mitochondrial integrity in top -poison-induced dna damage responses as well as cytotoxicity under situation of rac inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h v) as well as cardiomyocytes (h c ) were chosen as in vitro model systems for top -poisoning. the cells were pre-treated with rac inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top -poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc -assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac -inhibitor. top -poison-induced raise in mitochondrial mass was slightly enhanced by the rac -inhibitor and statins. interestingly, inhibition of rac counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top -poisoninduced cytotoxicity in h c and h v cells. the data point to a role of rac -regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top -poisons. poly(adp-ribose) polymerase (parp ) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [ ] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp as well as by non-covalent poly(adp-ribose)-wrn interaction [ ] [ ] [ ] . furthermore, pharmacological parp inhibition as well as a genetic parp ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [ ] . here we report a novel role for parp and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h o -induced oxidative stress, parp is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp -dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [ , ] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [ ] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [ ] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [ - ] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases methylcytosine and -hydroxymethylcytosine as well as their derivatives ( formylcytosine and -carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene , , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb , xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle , mangerich and bürkle ) . here we generated a genetic knock out of parp in one of the most widely used human cell systems, i.e. hela parp ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp ko cells with a series of artificial and natural parp variants, we analyzed structure-function relationships of parp in a cellular environment without interfering with endogenously expressed wt-parp . we confirmed that the parp e k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp l f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp -par interaction via at least one specific par binding motif located in zinc finger of parp . expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp l f itself triggered apoptosis, parp e k expression led to a strong g -arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt mitigated effects of the e k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp ko cells with a natural cancer-associated parp snp variant (v a), as well as a newly identified parp mutant present in a patient of pediatric colorectal carcinoma (f l-v a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp -dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle ( ) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer ( ): - . mangerich, a. and a. bürkle ( ) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: - . leukemic cells frequently overexpress the transcription factor wilms tumor (wt ) and the persistence of wt expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt and cell death. in addition, we show that an rna interference-mediated elimination of wt sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt is a novel biological target of hydroxyurea and they suggest that wt has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res : , ) and stably transfected to hek cells (hekß -fret). to monitor the camp response the epac -based fret sensor of camp, was stably transfected alone (hekwt-e ) and together with a moderate level of native ß ar to hek cells (hekß -e ; nikolaev et al. jacc : , ) . fret-activity was measured with recently developed fluorescence detectors ( channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using nm for excitation and / nm for the emmission ratio. cells were cultivated in -format -well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß -fret cells exhibited ec -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: . ± . , adrenaline . ± . , noradrenaline . ± . ). ßar-expression levels were controlled by radioligand binding with [ h]-(-)-cgp , resulting in different densities of ~ x and ~ . x receptors/cell in hekß -fret and hekß -e , respectively, whereas in hekwt-e cells only ~ ß ar were found. surprisingly, the low level of ßar in hekwt-e cells allowed the measurement of the action of ß -sympathomimetics (ß sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~ . ) to an effective regulation of fret activity in the presence of . mm ibmx (pec ~ . ± . ), nearly matching the ~ -fold amplification of iso (pk d~ . ; pec ~ . ). in order to determine ß ar-mediated side effects of ß sym, hekß -e cells characterized by a -fold higher level of ß ar over ß ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec ~ . whereas the high affinity ß sym salmeterol acted a partial agonist (~ % of iso maximum, pec ~ . ), both compounds being rather insensitive against the highly effective ß ar-blockade with µm cgp , a. for that reason hekß -fret cells were used characterizing fenoterol as partial agonist (~ %) whereas salmeterol activated less than % of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß ar present in hekß -e cells precludes the exact determination of ß ar-mediated side effects of ß sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin- affinity and trafficking. the affinity of arrestin- to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin- and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin- dissociation from the β ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin- into the bleached region was prolonged between two-and fourfold for the β ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek t cells transfected with the appropriate cdnas. here we focused on muscarinic m -, m -, and m -receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/ -proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m -and m -achrs was found to be -fold faster in comparison to gq. similarly, we observed a -fold right shift of the concentration-response curves of go proteins binding to m -achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m -achr by using a fret-based assay. our results showed that gq activation required -fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m -achr for gi family members by comparison of dissociation kinetics of gi -, gi , gi -, and go-proteins from m -achr under nucleotide depleted conditions. k off of gi and gi were found to be two-fold higher in comparison to gi and go proteins, indicating the higher affinity of the latter ones to m -achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine that is predominantly catalyzed by g protein-coupled receptor kinase . as a consequence, the selective morphine-induced s phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s but also drive higher order phosphorylation on the flanking residues threonine , threonine , and threonine in a hierarchical phosphorylation cascade that specifically requires grk and grk isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels hcn in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn and hcn isoforms for brain function and epilepsy has been demonstrated, however the role of hcn , the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn in controlling oscillations in the thalamocortical network. hcn is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn -deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc and channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc / / channels are directly activated by diacylglycerol (dag), trpc and channels are commonly regarded as daginsensitive. in contrast to trpc / / channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) and . interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc confers dag-sensitivity on trpc channels. trpc channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf and proteins and overexpression of a nherf mutant incapable of trpc binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip depletion itself caused slight trpc current increases while during pip depletion, the membrane permeable dag analogue oag evoked even higher trpc currents suggesting that pip depletion induces an active and dag-sensitive channel conformation. receptor mediated pip depletion also resulted in dissociation of nherf and from the c-terminus of trpc thereby eliciting a dag-sensitive trpc channel conformation. thus, our findings suggest that dag-sensitivity of trpc is the result of an activation cascade starting with pip depletion and subsequent dynamic dissociation of nherf and from the c-terminus of trpc . altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm -deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm may exert antinociceptive properties. fluorometric ca + assays and a compound library containing approved drugs were used to identify trpm inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura- -loaded single cells was applied to monitor [ca + ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm inhibitors. the compounds exhibited half-maximal inhibitory concentrations of . - µm. the selectivity profiles of maprotiline and primidone for trpm were promising with no inhibitory effects on trpm , trpm , trpa , trpv , trpc , trpc and p x receptor channels. primidone inhibited pregs-induced [ca + ] i signals in rat drg neurones, indicating a block of native trpm channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm -related effects by acutely challenging the channel´s function. pharmacological interference with trpm applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il- ) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e /k can be crosslinked efficiently in both states, the closed-and open state of the p x r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e /r , e /k , e /r and e /k and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p x r. the comparison of the closed-state and open-state model of the rat p x r showed that atp promotes a marked rearrangement of the side chains of the residues r and k to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r and k with atp competitively releases e , e and e from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem , tmem and tmem . expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem proteins exhibit to plasma-membrane spanning domains, but fluorescently labeled tmem fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem -eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem -deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura- or indo- indicate higher resting ca ++ concentrations in tmem -/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem -yfp-knock-add-on mice we identified tmem in organelles of the apical acinar cell pole and a partial colocalisation with lamp proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh cl application. the role of tmem for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck- stimulation indicating that tmem inactivation leads to an enhancement of the globalization of cck- evoked ca + release from intracellular organelles. taken together, our study identifies tmem as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [ ] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [ ] gundert-remy, u. et al. ( ) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol : - . during the past decades, considerable progress has been made in implementing r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes ( x gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed h after the last irradiation by γh ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht , a small molecule inhibitor of the small rho-gtpase rac , was applied in vivo, pointing to an involvement of rac in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh -interacting domain death agonist) knockout cell line in neuronal ht- cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas -knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht- cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin- and the bid inhibitor bi- c on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas -bid-knockout cells. bi- c inhibited glutamate-induced morphological changes of ht- cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin- in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi- c abolishes lipid peroxide formation in the erastin model and ferrostatin- in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi- c and ferrostatin- in the respective model systems. investigating the crispr/cas -bid-knockout ht- cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type (aav ) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange at the azo-and other weak bonds in our experimental set-up (fig. a) . amongst other substances, the carcinogens aniline (max. conc. . ± . µg/ml) and , -dichlorobenzidine (max. conc. . ± . µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. b) . here, , -benzenedicarbonitrile (max. conc. . ± . µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. . ± . µg/ml) and the human carcinogen benzene (max. conc. . ± . µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥ µg/ml evolving upon ruby laser irradiation of > . mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep , ( ) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco- model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). ; ( ) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [ , ] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > °c [ ] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [ ] . we here present results of a nine-day human intervention study with healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [ ] . the study design included washout periods with an aa-minimized diet ( - ng /kg bw), a low aa intake day ( . - . µg /kg bw) as well as a high aa intake day ( . - . µg /kg bw). after a three-day washout period an aama baseline level of ± nmol/d was determined. low aa intake led to an aama excretion within h of ± nmol/d, high intake to ± nmol/d corresponding to an aama excretion rate of about % of the ingested aa dose within h, whereas aama output within h corresponded to % of the respective aa intake, the aama baseline after days washout corresponds to a net exposure level of . - . μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [ ] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste , doi: . iarc, iarc monographs on the evaluations of carcinogenic risks to humans , . [ ] tareke et al., j. agric. food chem. , , - . [ ] efsa panel on contaminants in the food chain (contam), efsa journal ; ( ): [ pp.] . [ ] ruenz et al., arch. toxicol. doi: . /s - - heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf /skn- , daf ), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + %). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn- (homolog to mammalian nrf ), but not daf- (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct cells, baicalein activates nrf ; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf . the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf- transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn- but not daf- . experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf /skn- activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [ ] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in healthy postmenopausal women ( - yrs).in a randomized doubleblind protocol each subject received four treatments, doses of intranasal progesterone ( . mg mpp ; . mg mpp ), mg of zolpidem and placebo. the conditions consisted of experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from : to : . simultaneously blood was collected every min between : and : by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after . and . mg mpp . univariate f-tests in the manovas pointed to the following results (significant p-values at α= . ). sei was higher after zolpidem than after the other three treatments. after . mg mpp sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after . mg mpp and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after . mg mpp and after zolpidem. after all active treatments with mpp and zolpidem the time spent in sleep stage was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after . mg mpp . no other changes of hormone secretion were found. our study show sleep promoting effects of . mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after . mg mpp . no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january and june from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of or and possible anticholinergics with a score of ). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of . a patient's individual score of or higher is considered to be clinically relevant. in total, , geriatric patients (median age years, . % female, median no. of drugs ) were evaluated. of these, , ( . %) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in , ( . % of the patients taking anticholinergic drugs) patients. , ( . % of the patients taking anticholinergic drugs) patients had a score of or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine ( ), amitriptyline/quetiapine ( ) and amitriptyline/carbamazepine ( ). , ( . %) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct and mate expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. , although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct (encoded by slc a ), oct (slc a ), oct (slc a ) and mate (slc a ). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek stably expressing the organic cation transporters oct , oct , oct or mate . intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct and mate in a time-and concentration-dependent manner, but not by oct or oct . timolol is only transported by mate , but not by the octs. as shown by immunolocalization studies, the oct and mate transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct and mate may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: . zhang et al., nat rev drug discov. jun ; ( ) : - . . lavik et al., eye (lond). may; ( ): - . . gaudana et al., pharm res. may; ( ): - . . kraft et al., invest ophthalmol vis sci. ( ): - . . nies et al., plos one. ( ) :e . supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel mg, prasugrel mg or prasugrel mg (n= ). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s = . ; p= . ), hipf (r s = . ; p= . ), ipc r s = . ; p< . ). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p< . ). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p c polymorphisms (* and * ), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s was the strongest predictor of on-treatment platelet reactivity (partial η = . ; p< . ) followed by intrinsic platelet reactivity (partial η = . ; p < . ). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp c * polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [ ] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [ ] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [ ] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki- index -fold in the cscc model (p£ . ). while the presence of claudin- and occludin were distinctly reduced, zonula occludens protein- was more wide-spread, and claudin- was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [ ] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for . -fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [ ] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb r with integrated graduate education was launched in . the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the rs and to find alternatives to animal experimentation within the next years. a panel of r experts has been set up to provide advice and assistance and to raise awareness in society for r-related issues. research in bb r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on r including modules on ethics and legislation. currently, phd students are qualified for management positions in professional areas related to the rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd c and d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz- -derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer , -dinitrochlorobenzene (dncb) induced il- and il- secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd and pd-l in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine ( rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than , compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. %) fail in the late drug development due to toxicity and adverse effects [ ] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about , compounds and cancer cell lines from the national cancer institute's (nci) nci- growth inhibition data [ ] with focus on five immune cell lines (rpmi- , ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [ ] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [ ] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [ ] , and encourage the reduction of in vivo testing according to the principles of the rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype e activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult e ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for d pharmacophore development. eight specific d pharmacophores were created that allow identification of sult e substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over , experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult e inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult e for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. ( ) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. ( ) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p= . ). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p= . ). % of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in . by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the -organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the -organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused -organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd -values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the -organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [ , ] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck , which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il- alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [ ] vávrová, k., henkes, d., strüver, k., sochorová, m., Školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a d skin construct. the journal of investigative dermatology. , - ( bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive / has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the -rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the -r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c /bl j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored and minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb r is funded by bmbf. in the r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the r-concept is the overall goal of scientific animal welfare. article of the 'directive / /eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [ ] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately % [ ] [ ] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [ ] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from . to . mg/kg per bodyweight [ ] [ ] [ ] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [ ] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [ ] rl / /eu. [ ] bmel ( ) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [ ] eu commission ( ) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [ ] carbone l ( ) pain in laboratory animals: the ethical and regulatory imperatives. plos one , e . [ ] gv-solas, a.f. a.d. ( ) . empfehlung zur schmerztherapie bei versuchstieren. [ ] carpenter, j.w., t.y. mashima, and d.j. rupiper ( ) . exotic animal formulary, nd edition, w.b. saunders co., phila. [ ] flecknell, p. ( ) . laboratory animal anaesthesia, nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a -slice scanner were collected from female minipigs, consisting of animals aged months (group , n= ) and animals (group ; n= ) examined at the age of and months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to % where the largest volume was , ml and the lowest , ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between and months of age. comparing and months old minipigs, no significant difference in distance could be observed. from the age of months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between and months of age indicates ongoing anatomical changes of this parameter until the age of months. therefore animals should be older than months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [ ]. worldwide it is estimated that over million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [ ] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [ ] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals ( r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin ( -ht) system, in particular the -ht a receptor, playing a major role. activating -ht a receptors by the -ht a receptor agonist -hydroxy- -(dipropylamino)tetralin ( -oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic -ht a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the -ht a receptor in serotonergic projection areas, bert et al. ( , pmid: ) revealed exaggerated -oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic -ht a receptors to thermoregulation, more precisely to the hypothermic effect of -oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of -oh-dpat ( . mg/kg - mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the -ht synthesis inhibitor parachlorophenylalanine (pcpa; mg/kg, i. p. on four consecutive days) would alter the effects of -oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the -ht a receptor. results: -ht a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: . °c; nmri wild-type mice: . °c). in both genotypes, systemic administration of -oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (- . °c compared to - . °c in nmri wild types). dose response curves of -oh-dpat revealed an ed = . mg/kg in transgenic and an ed = . mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to -oh-dpat in mice. the dose-response curves indicate a higher potency of -oh-dpat in transgenic mice. the exaggerated hypothermic response to -oh-dpat in mutant mice implies that postsynaptic -ht a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that -oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing -ht a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg gly in the human beta -adrenoceptor (adrb ). the adrb carrying arginine at position in helix in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta -adrenoceptor variants arg -adrb and gly -adrb is poorly understood. despite its hyperfunctionality, we found the arg -variant of the adrb to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly -variant. using adrb sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg -adrb exerted faster activation speed and arrestin recruitment than the gly -variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved -residue sequence, trehpstant , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine (s ) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase (grk ). by contrast, high-efficacy opioids not only induce phosphorylation of s but also drive higher-order phosphorylation on the flanking residues threonine (t ), threonine (t ), and threonine (t ) in a hierarchical phosphorylation cascade that specifically requires grk / isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk and grk to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk / . in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk / isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk or grk . conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk or grk . mutation of s to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/ , and g / , which interact with distinct cellular effectors to shape the final cellular response . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/ / inhibitor fr on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m acetylcholine receptor (cho-m ). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor , . we show that fr can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m membranes. in contrast to this, bim- , a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation . interestingly, inhibition of high affinity agonist binding by fr was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr , combined uncoupling of both gi and gs proteins from m was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [ ] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [ ] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [ ] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m machr which preferentially activates g proteins of the g q/ type but can also promiscuously stimulate g s proteins. g q/ -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m machr in ip and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper- -naph completely lost intrinsic activity for both g q/ and g s activation at the m machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper- -phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper- -phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/ -mediated signaling, activation of the g s protein in m machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades -( h-imidazol- -yl)propylguanidine (sk&f- , ( ) [ ] ) is known as the prototypic pharmacophore of highly potent histamine h -receptor (h r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [ ] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f- , , we characterized newly synthesized derivatives including several bivalent compounds (e.g., ) by using different pharmacological in-vitro methods. [ ] the potential h r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf cells [ ] (hh , , , r). compounds were also functionally characterized in the [ s]gtpgs assay (hh r, sf cell membranes). [ ] selectivity vs. hh , , r was determined for selected derivatives also using this technique. organ bath studies (gph r (ileum), gph r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f- , analogues displayed partial or full agonism via hh r and gph r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine , was a partial agonist (e max = %) and -times as potent as histamine vis-à-vis the gph r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa values for the antagonist ( . - . ) which are in accordance with literature data. however, in the functional in-vitro assay on gph r, the positive chronotropic response evoked by sk&f- , was surprisingly resistant to antagonism by cimetidine and other typical h r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h r agonist. this behaviour is unique within the large series of sk&f- , analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [ ] parsons, m.e. et al.; agents actions , , . [ ] buschauer, a.; j. med. chem. , , - [ ] pockes, s.; dissertation, univ. regensburg . [ ] seifert, r. ; j. pharmacol. exp. ther. , ( ) , - . the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine (s ) and threonine /serine (t /s ), which enabled us to selectively detect either the s -or the t /s -phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch and ro - induce a stably phosphorylation at s and t /s followed by a profound internalization of the receptor. the nociceptin-induced s and t /s phosphorylation can be blocked by selective nop receptor antagonists such as j or sb , . nnc - , buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s phosphorylation occurred at a faster rate than phosphorylation at t /s indicating that s is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol -myristate -acetate facilitated receptor phosphorylation only at s but not at t /s , indicating that s can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t /s in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling ( ). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation ( ) . in man, two cc chemokine receptors ccr a and ccr b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin ( ) . interested in differential signaling of ccr a and ccr b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk / ) in the presence of ccl , ccl , ccl , and ccl . all these chemokines have been shown to interact with human ccr receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr b receptors by ccl induced both rho-dependent srf activation and receptor internalization, while ccl -stimulation resulted in srf but little if any receptor internalization. in contrast, ccr a-expressing cells showed ccl dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr receptors for coupling to g proteins revealed that arginine within the putative 'eighth helix' of the carboxyl-terminal portions of ccr a and ccr b is not involved in ga i -mediated induction of erk / and plays a minor role in ccr b receptor internalization, but is specifically required for the ccr a/ and ccr b/ga q -mediated activation of srf. serotonin -ht c receptors ( -ht c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). -ht c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express -ht c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa - cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where -ht c r in vivo expression has been detected. thus, we tested mhypoa - cells for putative -ht c r expression by performing single cell calcium imaging. we observed that serotonin and the specific -ht c r agonist, way , induced robust calcium transients, which were strongly inhibited by two unrelated -ht c r-selective antagonist (sb , , rs , ). serotonin and way , also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases- / in a sb , and rs , sensitive manner, providing further evidence for functional -ht c r expression in mhypoa- / cells. intrinsic activity of way , ranged between . and . compared to serotonin in all assays, defining way , as a partial -ht c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa- / cells endogenously express -ht c r and thus represent the first cell line to analyse -ht c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with h-adenine and calculation of the conversion rate to h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α a or µ opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc or heteromeric kir . /kir . or kir . /kir . channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp a mice leading to double transgenic mice (h xpp a = dt). pp a mice (j biol chem ; : ) overexpress the catalytic subunit of protein phosphatase a (pp a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about days we noted reduced ef in pp a ( . ± . %, n= ) compared to wt ( . ± . %, n= ) and tg ( . ± . %, n= ). interestingly, in dt the ef ( . ± . %, n= ) was higher than in pp a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp a is involved in h -receptor signaling and we tentatively conclude that the h -receptor is able to ameliorate systolic but not diastolic cardiac function of pp a mice. serotonin ( -ht) can exert positive inotropic and chronotropic effects in humans via -ht -receptors. we have generated transgenic mice (tg) which overexpress the human -ht -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and , and hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to . ± . % seven hours after lps in wt and to . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] p< . vs pre-lps value). however, hours after lps, diastolic function, measured as e/a, amounted to . ± . in p< . tg vs. wt). moreover, after hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after and hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from . ± . to . ± . in wt and from . ± . to . ± . in tg (tg vs. wt, p> . , n= - ; after hours). we tentatively conclude: -ht -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h -receptors. we have generated transgenic mice (tg) which overexpress the human h -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec = nm) or amthamine (ec = nm), a more selective and potent h -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to mm potassium chloride. thereafter, histamine ( µm) induced a pie ( . ± . % of control, n= ) in tg but not in wt preparations whereas isoprenaline ( µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound / , a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine ( µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk , a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek t cells transfected with yfp-tagged µor and mturquoise-tagged grk as well as non-fluorescent gα i , gβ and gγ subunits showed a robust increase in fret upon superfusion with µm [d-ala , n-mephe , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine ( µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately - % of that of the corresponding damgo signal. grk binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α a -adrenergic receptor or the m muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk interaction with that of the µor-gβγ interaction in the presence of grk revealed similar time constants both for the on-and off-kinetics (grk : k on . s ). this suggests that, in the absence of grk , the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk , however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk -gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α a -adrenergic receptor or the m muscarinic acetylcholine receptor in the absence and presence of grk and compared the kinetics with the kinetics of grk binding and unbinding to these receptors. in both cases, we found that grk and gβγ in the presence of grk associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c bl/ mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin- (et- ) for sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et- treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et- had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin- was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn and ccn to be moderately up-regulated after h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et- treatment. finally, we could show that the perinuclear localization of cav and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production ( ). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs ( ) ( ) ( ) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac -camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine -phosphate (s p) is generated by sphingosine kinase (sk)- and - and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s p - . after activation, s p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s p mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru- . in addition, in vivo studies showed that dexamethasone downregulated s p expression in glomeruli isolated from c bl/ mice treated with dexamethasone ( mg/kg body weight). functionally, we identified s p as a key player mediating s p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s p-induced migration of mesangial cells. this effect was again reversed in the presence of ru- . conclusion: we suggest that dexamethasone inhibits s p-induced mesangial cell migration via downregulation of s p . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. ; : - ) . the g protein-coupled receptor mrgd is a receptor for angiotensin-( - ) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-( - ) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-( - ) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-( - ) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-( - ). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek cells, we provided final pharmacological proof for mas to be a receptor for ang-( - ). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-( - ). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type receptor at as a receptor for the heptapeptide, but discovered that the at blocker pd can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-( - ) but excluding the at receptor, and by enforcing the revisit of such publications which concluded at function by using pd as a specific at blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within ms . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between - ms . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor (mglur ) sensors by fret after uncaging of an inert glutamate derivative. -methoxy- -nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α aadrenergic receptor, among others receptors, has been extensively studied in single cell assays ( ) ( ) . the activation kinetics of the labeled α a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology ( ) . additionally, we used halotag in combination with the nanoluc ( ) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec value in accordance with the previously published data. this suggests the functionality of the fret-based α a -halo/cfp sensor. similar results were obtained with the α a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin biosensors. upon stimulation, β -adrenergic receptors bound β-arrestin with a time constant τ = . ± . s, indicating that β-arrestin binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin with a τ = . ± . s. stimulation of β -adrenergic vs. m muscarinic or ffa receptors resulted in different patterns of conformational changes in the various β-arrestin sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin activation. upon agonist removal, first the interaction (delay = . ± . s) and only then the active state of β-arrestin (delay = . ± . s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β -adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about , fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi (historical strain), strain (serotype f, variant tcdbf), strain r (hypervirulent strain, ribotype o ), and strain r (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain ) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi . in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in d and d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p rhogef-knockout mice were adenovirally transduced for to h with recombinant adenoviruses or directly used. for d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p rhogef implies that p rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il- and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/ , g / and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr (r h), a bmp (bone morphogenic protein) type receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c c cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp -induced osteoblast differentiation in c c cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr r h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for h in culture. for activation of the g q/ -signaling the cells were treated with endothelin- (et- ), angiotensin ii (ang ii) or phenylephrine (pe) for s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p rhogef is involved in the regulation of the et- -induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor (kdelr ) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin- the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p rhogef in the transport of membrane proteins, we overexpressed p rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr and its co-localization with p rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr was found in control-transduced cells. further, p rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p dn and detected similar changes with respect to kdelr localization and golgi structure suggesting that this regulatory function of p rhogef is not dependent on its activity. conclusion: p rhogef mediates the activation of rhoa from gpcrs coupled to g q/ proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/ , g / ) exist in mammals and these must suffice to convey signals from about g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr , a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/ / over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek cells that lack functional gq/ alleles by crispr-cas genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/ using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept , , and accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc , borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [ ] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [ ] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [ ] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at hours post fertilization, larvae at days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [ ] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac ). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [ ] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [ ] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences , - ( ) . ccmp, ', '-cump and ', '-cimp were ineffective. to further characterize the action of ', '-cgmp on hut- cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). ', '-cgmp significantly increased apoptosis (ec = µm) and inhibited proliferation (ec = µm) of okt -activated hut- cells. interestingly, also ', '-cgmp exhibited comparable effects on apoptosis and proliferation with ec values of µm and µm, respectively, while ', '-camp, ', '-ccmp and ', '-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested ', '-cgmp degradation products under the same experimental conditions and found that both '-gmp and guanosine increased apoptosis and inhibited proliferation with ec -values between and µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of ', '-cgmp, ', '-cgmp, '-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [ ]. ', '-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut- cells (e.g. enpp ), yielding '-gmp, which could be further degraded to guanosine by the '-ekto-nukleotidase cd . a similar pathway may lead from ', '-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms a and b as well as the cgmp-selective pde a also degrade the emerging second messenger cump [ , ] . we analyzed the enzyme kinetics of pde b-mediated cump-hydrolysis using recombinant gst-tagged pde b and a highly sensitive and specific hplc-ms/ms method. our data show that pde b is a low-affinity enzyme for cump with a cump k m -value of > µm. the pde -selective competitive inhibitor milrinone inhibited pde b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde b is highly expressed in adipose tissue [ , ] . thus, we differentiated murine t -l -mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde b expression and basal cnmp-concentrations. in both differentiated and undifferentiated t -l cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by % and %, respectively, while ccmp was reduced by % and cump even by %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde b was upregulated ~ -fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde a-mediated cump hydrolysis. pde a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi . they comprise a "green" membrane-targeted version (mcgi ) and a "red" variant (red cgi ) that contains the fluorophores tsapphire and dimer . methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi sensor showed a good signal-to-noise ratio, an ec value of ≈ . µm for cgmp, and a high selectivity for cgmp over camp (> -fold). flincg , a non-fret-based cgmp sensor, showed similar properties as cgi . the new membrane-targeted mcgi and the new red cgi displayed ec values of ≈ . µm cgmp and a high selectivity for cgmp over camp (> -fold). in vsmcs, the red cgi showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de . the "green" fret-based camp sensor, epac -camps, showed a signal-to-noise ratio comparable to that of cgi , an ec value of ≈ µm for camp and a selectivity for camp over cgmp of ≈ -fold. finally, imaging of cells expressing both the epac -camps and the red cgi demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi would be useful to investigate membrane-associated cgmp compartments and red cgi to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at -receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [ ] . whether this causes an activation of cgks is not known. these enzymes exist in different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [ ] , [ ] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet ( . % na + ) and enalapril ( mg/kg/d) for weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay - , a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw /lrmp (lymphoid-restricted membrane protein) is a type membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [ ] , [ ] . jaw and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw and irag share the highest homology [ ] , [ ] . the coiled coil domains of irag and jaw are important for the interaction with ip rs. as already known, irag forms a trimeric complex with cgkiβ and ip r and gets phosphorylated by cgkiβ [ ] . hence we examined if jaw is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw possible phosphorylation sites can be found. our in vitro studies with jaw and cgki showed that jaw gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw and cgkiβ was detected. to examine the importance of jaw in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw . mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw . hence, the function of jaw in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine ', '-cyclic monophosphate (camp) and guanosine ', ' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp and , hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine ', '-cyclic monophosphate (ccmp) and uridine ', '-cyclic monophosphate (cump) also fulfill the criteria of second messengers [ , ] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [ ] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [ ] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap ) were examined as potential ccmp and cump target proteins. hek cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor (npr , also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase a (pde a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor (npr ) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e . embryos. because npr binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr could lower the activity of the cnp-npr -cgmp signaling cascade in drg neurons. in the absence of npr , the majority of sensory axons showed normal bifurcation, but » % of the axons turned only in rostral or caudal direction. this study shows ( ) that pde a is important for the degradation of cgmp in embryonic drg neurons, ( ) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde a, and ( ) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi ). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d- -amino- -phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac -camps fused to a phosphodiesterase (pde a ). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families , and ) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde . however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde a (v max = µmol/min/mg) to a camp fret sensor (epac -camps) we monitor camp concentrations in direct vicinity of pde a . in comparison to pde , we show that pde a , albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j macrophages, arglabin strongly induced accumulation of lc type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc -ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp and pro-interleukin (il)- β, we further analyzed whether arglabin inhibits the nlrp inflammasome. arglabin reduced expression of nlrp and pro-il- β, inhibited activation of caspase- , and release of mature il- β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp inflammasome. intraperitoneal injection of arglabin into female apoe .ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il- β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par activating peptide sligrl ( µg/kg), hydralazine ( mg/kg), isoproterenol ( mg/kg), losartan ( mg/kg) and furosemide ( mg/kg) in conscious wild-type (wt) and par -deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet ( . % nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par -deficient mice than in wild-type mice ( ± versus ± ). renin mrna expression was ± % of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par -deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par (-/-) and wt mice. administration of sligrl increased renin secretion about -fold (p< . ). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par (-/-) and wt mice. the absolute changes (delta prc) were similar ( ± , ± , ± , ± in wt, and ± , ± , ± , ± in par (-/-)). in conclusion, chronic absence of par reduces basal renin expression and renin release. however, par -deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca + sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin- -gallate (egcg), which has been shown to decrease myofilament ca + sensitivity, on cardiac myocyte contractility and force-ca + relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc -targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca + transients at °c under hz pacing using the ionoptix system in the absence or presence of egcg ( . µm). furthermore, force-ca + relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg ( µm). results: in baseline settings and absence of fura- , ki cardiomyocytes displayed higher sarcomere shortening ( type- serine/threonine protein phosphatase (pp ) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca + handling and contractility. all pp heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor (i- ). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i- resulted in an improved basal ca + handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i- , regulated by gsk -dependent phosphorylation at thr , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with . % nacl (n= ). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca + transients were measured in isolated, indo- -loaded myocytes. the peak amplitude of [ca] i was reduced by % in tg nacl compared to wt nacl (p< . ), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by % in tg iso compared to wt iso (p< . ). consistently, sr ca + load was diminished by % in tg nacl compared to wt nacl (p< . ). chronic iso stimulation led to an unchanged sr ca + content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than -fold higher phospholamban phosphorylation at ser in tg (p< . ). thus, these findings suggest that overexpression of i- is able to reduce the progression of heart failure by an improvement of myocyte ca + handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp- macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a (abca ) expression in human thp- macrophages by this leoligin derivative. the transporters abcg and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock and rock are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock and rock are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock and rock by % was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with μm fasudil or µm h p for general rock inhibition and µm slx- for inhibition of rock . protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock and rock influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock and rock in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock , but not of rock led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h p and qpcr analysis of cf with a downregulation of rock and rock showed that both rocks are involved in the regulation of ecm proteins, such as collagens a , a, and a , biglycan, decorin, elastin and its respective degrading enzyme mmp . conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, ] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, ] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm- as a nucleus-specific marker was used to enrich cm nuclei to > %. proteins were extracted from nuclear lysate with % sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from murine cell lines served as silac (lys /arg )-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified proteins, of which are annotated to the nucleus. %/ % (adult / p ) of nuclear proteins are annotated as dna-binding. . %/ . % belong to transcription factor complexes; . %/ . % are able to bind transcription factors. . %/ . % have chromatin modification functions; . %/ . % modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. % of proteins are shared between p and adult nuclei. proteins are exclusive to p , are only found in adult. proteins are enriched ( . -fold increase in abundance) in p hearts, proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p cip /waf , senescence-associated ß-galactosidase (sa-ß-gal) and p ink a were increased -, -and -fold (n= - ; p < . ), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β -adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin ( ± . %), platelet derived growth factor receptor α ( ± . %) and α-smooth muscle actin ( ± . %), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor (cb ) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, , mg/kg/day) with osmotic minipumps for days. treatment with cb antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna- a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb antagonist treatment and foxb (a direct target of mir- a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir- a/b family seems to be sensitive to cb antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type diabetes mellitus (t dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: -week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing mg/g dabigatran or respective placebo for weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in t -l cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il- from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using t l cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca + -atpase (serca a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca + cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca + kinetics were measured in isolated, fura -loaded and electrically paced ( . hz) cardiomyocytes as the time to % decay of the ca + signal (t % ). the time to % baseline of sarcomere length (t % baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed . -fold stronger phosphorylation signals if pentamers were absent (p< . ). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t % [ms]: ± and ± , respectively, p< . ). notably, t % of pln-ko myocytes was ± ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca + clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t % baseline [ms]: ± in tgpln versus ± in tgafa-pln and ± in pln-ko, p< . ). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca + kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the ' end of mrnas ( ' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous ' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih- t fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor- (par ) regulates inflammatory responses including monocyte migration and cytokine release. par is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine- -phosphate (s p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s p on the expression of par , tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp and u cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par , tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with ng/ml pma (phorbol -myristate -acetate) over h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp and u cells s p induced a time-( to h) and concentration-dependent ( . to µm) significant upregulation of par mrna and total protein expression. par total protein was upregulated maximally (about . -fold, n= ) with µm s p after h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp monocytes and tm was not regulated by s p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s p resulted in a time-( to h) and concentration-dependent ( . to µm) significant upregulation of tf expression within to h of incubation. conversely, par total protein expression was reduced by about % after h s p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp cells as tf source. timedependent incubation with s p ( µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from . ± . to . ± . min and elevated total the thrombin generation capacity from ± to ± nm. peak thrombin formation was elevated from ± to ± nm/min (control versus s p for h, mean±sd, n= , respectively). these data suggest that s p induces an enhanced expression of par in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s p does upregulate tf expression but attenuates par levels. since par is involved in regulation cell migration, s p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin- (il- ) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il- expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il- mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp- cells was determined by western blot. immunostainings for il- and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa ( nm) induced a time-dependent ( - h) increase in il- mrna expression. maximum expression was observed within h to a . ± . fold increase (mean±sd, n= , p< . ). incubation with an inhibitor of p map kinase (sb , µm) or pi k (ly , µm) significantly attenuated fxa-induced il- mrna expression (n= ). inhibition of p / mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for h resulted in a markedly increased il- secretion into the smc culture media from . ± . to . ± . ng/ml (p< . , n= ). stimulation of thp- cells with il- ( ng/ml) induced a time dependent ( - h) increase of up to . ± . fold (p< . , n= ) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il- expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il- regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il- expression and secretion in venous smc which may be regulated via p and pi k signaling. il- enhanced tf expression in thp- monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il- release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p ). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week - with vpa ( . % in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week and ). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week : tg veh : ± %, wt veh : ± %; week : tg veh : ± %, wt veh : ± %, p< . ). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week : ± %, tg vpa at week : ± %, p< . vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p< . ) proteins were significantly changed while proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p< . ). proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p< . ), whereas vpa affected proteins in tg atria (tg vpa vs. tg veh , p< . ), out of which proteins were common. prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine ( mm), shifted the concentration response curves for the pie of histamine from ec = nm to nm (p< . ). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine ( µm), shifted the pie of histamine from ec = nm to nm and increased the efficacy of histamine for the pie (p< . ). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp , pp a, pp b (calcineurin) and pp in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase cβ (pp cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased ( . ± . mg/g) compared to wild type (wt) littermates ( . ± . mg/g; p< . ) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg ( . ± . mg/g) vs. wt ( . ± . mg/g; n= - ; - months of age; p< . ). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: . ± . % (tg) versus . ± . % (wt); n= - ; - months; p< . and fractional shortening: . ± . % (tg) versus . ± . % (wt); n= - ; - months; p< . ). the left ventricle was dilated (systolic diameter: . ± . mm (tg) versus . ± . mm (wt); n= - ; - months; p< . ; diastolic diameter: . ± . mm (tg) versus . ± . mm (wt); n= - ; - months; p< . ). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein (igfbp ) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein (igfbp ) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp in murine cardiac tissue at different developmental stages by qpcr normalized to tpt (tumor protein, translationally-controlled ). this analysis showed temporal changes of cardiac igfbp expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp expression in different heart cells showed a very low igfbp in adult cardiomyocytes in contrast to a high expression in undifferentiated sca- positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp was found up-regulated (p< . ). further we found an increased igfbp expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p< . ). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p< . ). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp in angiotensin or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin , showed the opposite results, namely a reduction in its expression (p< . ; p< . , respectively). summary and conclusion: our results show active igfbp transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp in a conserved manner. we hypothesize that igfbp may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c bl/ j mice fed a high-fat diet (hfd) for weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last weeks with crataegus extract ws® ( mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® , neither in pvat nor in aorta. phosphorylation at serine is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine in pvat, but not in aorta. ws® treatment significantly improved enos serine phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® treatment. in addition, ws® treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin- (sirt ), known also as a longevity gene. the activity of sirt depends, among others, on the intracellular content of its cofactor nad. ws® treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt is enos. deacetylation of enos at lysine residues and by sirt enhances the activity of the enos enzyme. currently, we are studying the effect of ws® on nad synthesis, sirt activity, and enos (de)acetylation. in conclusion, crataegus extract ws® reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine and upregulation of sirt . the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk ) and the raf-erk / pathway. these two functions of rkip could counteract each other. while grk inhibition is cardio-protective, inhibition of the pro-survival erk / axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. ). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp ) under control of the alpha-mhc promoter. two different rkip-transgenic lines with . -fold and . -fold increased cardiac rkip protein level were generated (ref. , and jax id number ). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of . ± . % (n= ; ±s.d.) were documented at an age of months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk and raf-erk / axis by the human rkip gene (pebp ) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp ), which does not seem to inhibit the raf-erk / axis in vivo but instead confers grk inhibition- heterodimerization between the at receptor (at r) for the vasopressor peptide, angiotensin ii, and the b receptor (b r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at r--b r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at r--b r heterodimer in vivo, we generated a transgenic model of at r--b r heterodimerization (tg-b r+) by transgenic expression of the b r gene (bdkrb ) in the b r-deficient tg-b r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at r and b r at a distance of less than nm in tg-b r+ mice whereas the at r--b r heterodimer was absent in tg-b r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b r in the aorta of tg-b r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b r. the efficient at r--b r heterodimerization in tg-b r+ mice was accompanied by an enhanced angiotensin ii at r-stimulated vasopressor response relative to that of tg-b -/-mice, which lack the at r--b r heterodimer. as a control, the endothelin- -stimulated vasopressor response mediated by the etar, which did not dimerize with b r, was not significantly different between tg-b r+ and tg-b r-/-mice. together these findings provide strong evidence that at r--b r heterodimerization occurs in vivo and enhances the angiotensin ii at r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase (grk ) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk , grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk / axis and involved the erk / -dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine- . as a consequence of erk-dependent phosphorylation of pparg on serine- , the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine- phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine- a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as weeks of age in homozygous mice and - weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for weeks with vpa ( . mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of . ± v/s (tg: n= cells) vs. . ± . v/s (wt: n= , p= . ). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at % (tg: . ± . ms, n= vs. wt: . ± . ms, n= , p< . ), at % (in ms: . ± . vs. . ± , p< . ) and at % (in ms: . ± . vs. ± . , p< . ) repolarization. vpa treatment reduced ap amplitude in wt mice by mv (n= , p< . ) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced ( %: . ± . ms, %: . ± . ms, %: . ± . ms, n= , p< . vs. untreated tg), the amplitude was increased by mv (n.s.) and the slope of depolarization was increased by % (p= . , n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: ± . pf, n= vs. wt: ± . pf, n= , p< . ), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: . ± pf, n= n.s. vs. wt; tg-vpa: ± pf, n= , p< . vs. wt-vpa; p< . vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca + exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca + imaging revealed that the transduction rate of spontaneous sub-threshold ca + -waves into supra-threshold transient-like ca + -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca + transport rate (r serca ) was enhanced in tg vcms potentially limiting ca + extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during - m isoproterenol challenge, tg: . *, ctl: . ; n= /condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx , serca a and decrease of kchip protein levels. however, the respective mrna levels (slc a , atp a and kcnip ) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir- (fold change in tg: . *) and mir- (fold change in tg: . *) (n= /condition). atp a is a predicted target of mir- and mir- has recently been shown to regulate ncx and i to related potassium channel subunits. (*p< . vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from . (mg/g) in untreated control animals to . (mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of . (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr ( . mg/g) compared to saline treated animals ( . mg/g) and pka mutants ( . mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: . vs . mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh / ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged ( . mg/g) or a strongly elevated hbr ( . mg/g). however, additional hypertrophic factors including anp, myh / ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of µm after and h (ctb assay) nor increase in the apoptosis rate after , , or h up to µm. measuring adenosine triphosphate (atp) levels revealed that µm soraphen a does not alter the atp levels in huvecs after h treatment. in contrast, a h treatment significantly lowered the atp levels by %. also gene silencing of acc in huvecs attenuated the atp levels by %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels ( %) in soraphen a-treated cells after h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic value of µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g /g phase by % and increases the number of cells in the g /m phase by %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, µm soraphen a lowered the migration of endothelial cells by %. gene silencing of acc in huvecs strongly decreased endothelial migration, whereas a knockdown of acc had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for , fu / - ) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type (pcsk ) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october . we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor (klf ) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w domain containing protein (bzw ), a phylogenetically conserved protein, as a β-catenin and klf interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p< . , cm n= , cfb n= , cpc n= ), and skeletal muscle. bzw was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw , containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf knockout (ko) mice **p< . n= , and in a cardiac specific β-catenin stabilized mouse model, **p< . n= ). a mouse model with constitutively bzw loss of function (bzw ko) showed cardiac specific upregulation of β-catenin on rna level (***p< . , p< . , ctrl n= , bzw ko n= ) and on protein level (**p< . , ctrl n= , bzw ko n= ). echocardiography analysis in eight-weeks-old bzw ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p< . ctrl n= , angii n= ) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p< . ctrl n= , dcm n= , icm n= ). conclusion: these data demonstrated that bzw is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine- -phosphate (s p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s p lyase [ ] . mice that lack s p lyase are characterized by the accumulation of s p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [ ] [ ] [ ] . on the other hand, embryonic fibroblasts from s p lyase-deficient mice (sgpl -/--mefs) are resistant to chemotherapy-induced apoptosis [ ] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [ ] . interestingly, s p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [ ] . the aim of the present study was to analyze the link between s p lyase deficiency and altered cholesterol homeostasis using sgpl background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine- (p-ser ) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser -pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> . fpkm, gene expression rank < , ) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser -pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type (tnni ) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin (acta ) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt fl/fl mice with heterozygous dnmt fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl -cre) resulted in embryonic deletion of dnmt (ko). embryos without myl -cre served as controls (ctl). embryos were dissected and genotyped at e . , e . , e . and e . . rna-seq and pyrosequencing of genomic dna was performed on e . hearts, histology on e . hearts and electron microscopic imaging on e . hearts. for deletion of dnmt in adult mice, homozygous dnmt fl/fl mice expressing an inducible cre recombinase (myh -mcm) were given tamoxifen i.p. over days. homozygous dnmt fl/fl mice not carrying myh -mcm as well as myh -mcm carrying mice without dnmt fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl -cre mediated loss of dnmt resulted in progressive embryonic lethality with absence of living ko embryos after e . . ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet . methods: female mice homozygous for tet flox and male mice heterozygous for tet flox and heterozygous for myl -cre were mated and offspring were genotyped after weaning. mice homozygous for tet flox and heterozygous for myl -cre (ko) or homozygous for tet flox without myl -cre (controls) were sacrificed at weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet and tet . cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp a gene and at the binding site of the transcription factor gata but not near the promotor and the binding site of the transcription factor tbx . higher methylation levels were not accompanied by changes in atp a expression. however, both myh and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet is involved in dna demethylation in cardiomyocytes. loss of tet expression resulted in embryonic lethality. compensatory upregulation of tet and tet isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor (pi ). a recent gwas analysis showed an association of a genetic variant in the pi genomic locus (rs ) with chemerin plasma levels. here we tested the hypothesis that pi determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi , which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi -deficient animals compared to littermate controls. to investigate whether pi and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi . when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi to potently inhibit cathepsin k activity. taken together, we propose pi to act as a regulator of chemerin processing. the transient receptor potential canonical (trpc ) is a second messenger-gated cation channel, which mediates depolarization and ca + entry. it is known to be activated by diacylglycerol derivatives (dag, -oleoyl- -acetyl-sn-glycerol oag) [ ] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [ ] thus, the discovery of potent inhibitors of trpc may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc . [ ] larixyl acetate, another component of the resin showed an even higher potency in trpc inhibition (ic = . µm) and a -fold selectivity compared to trpc . these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc . hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc with similarly low ic values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c position, however, seems to play no important role for the inhibition of trpc currents. larixyl methylcarbamate lead to trpc inhibition with an ic value of . ± . µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc , this compound bears the benefit of high subtype selectivity towards trpc . even with concentrations up to µm of larixyl methylcarbamate, no complete inhibition of the ca + influx via trpc channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α nachr using whole-cell patch clamping under voltage-clamping conditions (- mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, -tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes ( ). among the mammalian trp channels, the canonical channel (trpc ) is a ca + -permeable ion channel, which is predominantly expressed in the brain ( ) . many aspects of trpc function are still elusive although behavioral experiments with trpc -knock-out mice suggest a role in innate fear-response ( ) and some studies indicate a trpc -mediated down regulation of neurite outgrowth in nerve cells ( , ) . to elucidate trpc function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo ( , ) . thus, there is still a need for identifying novel and efficient trpc modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc activator. hek cells heterologously expressing trpc upon tetracycline induction (hek trpc ) show a btd-induced concentration-dependent activation in both ca + assays (ec = . µm) and in electrophysiological whole cell patch clamp recordings (ec = . µm). btd elicits currents with an n-shaped i/v curve, typical for trpc . the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc inhibitor ( ) . mtt assays revealed that incubating hek trpc cells for h with btd concentrations above µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca + -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to µm. ca + assays showed no influence of btd on closely related trpc channels, as well as trpc / / at concentrations up to µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc subunits can also assemble to heteromeric channel complexes with their closest relatives trpc and trpc ( ) . trpc / and trpc / heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc subunit. transient receptor potential canonical channels / and are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc , based on the recently available structural information on trpv , we performed structure-guided mutagenesis and identified a single residue in transmembrane domain (g ), which is conserved within the canonical family of trp channels. single point mutations at position in trpc largely eliminated lipid sensitivity. trpc g a expressed in hek cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc / / channels (gsk a) activated wild-type trpc and trpc channels as well as the respective lipid insensitive mutants (trpc g a, trpc g a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g to gsk recognition by trpc . our results demonstrate the existence of two different mechanisms of trpc / activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc / involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s provided by g . lipids and synthetic activators of trpc / may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb ( , '-(propane- , -diyl) bis ( -tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c -c ) of mb were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of µm carbamoylcholine in the functional assay. mb and several bispyridinium structure analogues (mainly c -c linker) exhibited no regular displacement curves at [ h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the -tert-butyl-substituted bispyridinium compound mb showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb or mb , could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations ( - µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field ( , , hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb ( , ′-(propan- , -diyl) bis ( -tertbutylpyridinium) di(iodide)) and mb ( , ′-(propan- , -diyl) bis ( -ethylpyridinium) di(iodide)) in the tested concentration range ( - µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low ( hz) and high ( and hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p x receptor-induced current by the opening of asic channels g. stephan , p. illes universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p x receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel (asic ) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p x and asic subunits has to be taken into consideration. we transfected rat (r)p x r and rasic constructs individually or together in a ratio of : into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p x r-and asic -constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from . to . caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to . . the opening of asic channels failed to facilitate the p x r current when -guanidine- -methylquinazoline was used to stimulate a non-proton ligand-sensor of asic . then, we substituted ca + in the extracellular medium by ba + or decreased the intrapipette concentration of egta, to modify the free intracellular ca + concentration. in cells individually transfected with the receptor-channels, external ba + increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p x r-and asic -specific currents in a similar manner as external ba + . in cells co-transfected with p x r/asic , the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic activation. taken together, our results suggest that p x r and asic interact with each other, since the activation of asic had a marked impact on the p x r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca + and the proton sensor of asic appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned- . in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned- support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv has been identified in endothelial cells ( ), epithelial cells ( ) and arterial smooth muscle cells ( ) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in ). it is a matter of debate however if trpv activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc ( ) which is also expressed in both cell types. to analyze the potential role of trpv on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after minutes of ischemia in trpv -deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv /trpc double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc deficiency protected lungs from iri-induced edema ( ) . moreover, we identified reduced expression levels of aquaporin in trpv -deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily (trpm ) has been shown to be expressed in human kidney , . using newly developed anti-trpm antibodies, we are able to visualize trpm protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm (trpm the atp-gated p x receptor (p x r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p x -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p x r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p x r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p x r are overexpressed under the control of the endogenous p x promoter. the egfp-tagged p x rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p x -egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map -and neun-positive cells indicating that under non-pathological conditions, the p x receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd protein was observed in the p x r-overexpressing mice. these results suggests that overexpression of p x rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd primarily localizes to lysosomes and endosomes, this further supports a role of the p x r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc and tpc -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca + channels or such as trp channels. due to their biophysical properties, tpc and tpc are assumed to be involved in the efflux of ca + from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc for vesicle fusion & fission processes we investigated a potential impact of tpc on the uptake of bacterial toxins. first we determined the precise localization of tpc in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc -deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland , . the trpm gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists , . variations include the presence or absence of five stretches of to amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini . furthermore, three different aminotermini of human and mouse proteins have been described , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified variants that differ in exons , , , and . however, only variants bearing the short, ca + permeable pore loop were detected. ´ rapid amplification of cdna ends ( ´race) revealed that trpm proteins of the pituitary gland carry truncated c-termini exclusively. ´race identified five independent regions of transcription initiation within the trpm gene implying the presence of five independent promotors. the different transcripts encode four trpm amino termini α, β, γ and δ of - amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~ %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to different bilayers at a time. here we utilize the "orbit" multichannel system with a meca chip by ionera to measure tpc channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav ) and potassium channels (like kv , kv ). the voltage-gated na + channel nav . initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv . is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav . and kv . have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) is known to interact with the kv . channel complex, modulating kinetics and voltage dependence. the overexpression of dpp in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv channels. to study the potential influence of dpp on nav . channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k cells, expressing scn a alone or with dpp . surprisingly, we observed significant effects of dpp on nav . channel voltage dependence and kinetics. thus, the co-expression of dpp significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav . channels alone. in addition we analysed the effects of dpp on the kinetics nav . currents. while time to current peak was not affected in cells co-expressing nav . and dpp compared to nav . alone, dpp slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav . and dpp compared to nav . alone. in summary, we provide first evidence that dpp not only interacts with kv channels, but also influences nav . channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv . and nav . behave very differently depending on the expression of its associated proteins like dpp . cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc /c -deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca + -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc and trpc (camacho londoño et al., ) . nevertheless, the role of trpc /c for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal days. our results show that there is no difference in nm at-ii induced ca + -release or ca + -entry in trpc /c -deficient cf compared to wt. to evaluate whether the lack of trpc /c can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca + -release and ca + -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca + ] i like isoproterenol, -ht, thrombin and endothelin- . cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv . channels, which are known to be expressed in vascular smooth muscle cells. k v . voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l (k v . opener), hmr , chromanol- b (k v . inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq -/-mice to determine whether k v . plays a role in the regulation of arterial tone. we found that r-l produces similar concentration-dependent relaxations (ec ~ , µm) of wild-type (kcnq +/+) and kcnq -/-arteries pre-contracted with either phenylephrine or mm kcl. this relaxation was not affected by µm chromanol- b, µm hmr or µm xe (pan-k v blocker). the anti-contractile effects of pvat were normal in kcnq -/-arteries. chromanol- b and hmr did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq -/-mice exhibited normal peak k v currents. the k v . - opener retigabine caused similar relaxations in kcnq -/-and wild-type vessels. we conclude that k v . channels are apparently not involved in the control of arterial tone by alpha adrenergic vasoconstrictors and pvat. in addition, r-l is an inappropriate pharmacological tool for studying the function of native vascular k v . channels in mice. introduction: according to international guidelines [ ] [ ] [ ] [ ] [ ] [ ] [ ] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [ ] [ ] . material and methods: tissue the skin samples are descended from female pigs of german landrace ( kg weight, approx. month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from different skin area (fig. ) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of . µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of mm were punched out from the frozen skin stripes and stored at - °c. hplc and skin absorption waters corporation hplc containing sample manager, binary gradient pump, pda detector (optional: electron spray mass spectrometer); column: nucleodur® - c ec mm x . mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, -cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of h at °c. the dosage compartments of each cell were filled with approximately µl of the caffeine solution ( mg/ml). samples of ml are taken after , , , and hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. ) . thickness was in the order of magnitude between to . µm and intra variations were less than %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [ ], demonstrating intact barrier properties of the membranes after month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in , the mak commission derived a new threshold value of . mg/m for gbs with a density of , recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day , and ) and the lung toxicity after intratracheal instillation in rats (day and ): tio (rutile, micro), tio (anatase, nano), eu o (micro-nano mixed), baso (micro), zro (micro) and amorphous sio (nano). two doses of . and . µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio (rutile) and baso (pmn < % after days in the low dose group; < % in the high dose group; full recovery after days). in contrast, the tio (anatase) showed a stronger response (pmn > % after and days). the rare earth eu o (micro-nano) dust showed the strongest effect (approx. % pmn after and days) including a red-coloured lung lavage fluid. µ-zro and amorphous sio showed a strong acute response after days, however, mostly complete recovery after days. the low solubility criterion was met by the following dusts: tio (both) and zro . -similar volumetric lung burdens were deposited in a parallel validation experiment ( -day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, µm of pyr and µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp a gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp b in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from ) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and ) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of µm (bpa), µm (fen) and . µm (keto), for concentrations in target organs resulted in estimated oral loels of , and . mg/kg. when calculations were made for plasma levels oral loels were estimated to be , and mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of mg/kg bw day for bpa ( ), mg/kg day for fen ( ) and mg/kg day for keto ( ) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal /uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp a promotor activity was investigated using a pxr-dependent cyp a reporter gene assay. cyp a induction was analyzed at the mrna and protein levels in hepg cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp a promotor activity could only be detected for echimidine. cyp a induction by echimidine was verified at the mrna and protein level in hepg wildtype and hepg pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases ( isoforms are known, nox - , duox & ) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox -, nox -and nox -deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of ng/kg during days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox and nox could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox -knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox -knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox -knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox . conclusion: separate eliminations of nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last years about drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin , podocin, synaptopodin, retinol-binding protein , clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of , -intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c exoenyzme (c ) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c . knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c . moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c . proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c -induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c compared to wild type cells. as vimentin knock out did not completely inhibit c uptake into cells, an additional uptake mechanism or additional receptor for c is likely. nevertheless, our data reveals that c employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for h before toxicants were added to the cells. migration and viability of the cells were then measured after another h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein (rip ) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht- cells erastin promotes activation of rip and subsequent rip -rip necrosome formation which has been investigated as a hallmark of regulated necrosis . in fact, silencing of rip by sirna or by the rip inhibitor necrostatin- prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin- fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein- (rip ) and rip kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) , . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip /rip -dependent necroptosis and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip -rip complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi- , was from medchem express. murine embryonic stem cells (es-d ) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of . % in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between . and mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in , and wells out of wells in total at concentrations of , and mg/l. at mg/l no differentiation was observed. effects on cell viability were observed at mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of mg/l this nsa -inibitor showed a clear impact on differentiation with out of wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi- at concentrations up to mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg ) and developmental toxicity studies (oecd tg ) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the -generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some -days old pups. for all effects clear noaels were determined. in the -generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies ( -generation toxicity study and a -week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in , dimoxystrobin was classified with r (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r (which has been translated into the ghs classification repr. , h d) was based on offspring body weight and heart effects seen in the -generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in and a restriction program for pfoa was initiated in . therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and , , , -tetrafluoro- -(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek- t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a -fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a -fold repeated estrogen response element (ere). both constructs were stably transfected into t d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone- and -mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of betaestradiol (e ) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) . furthermore, rats fed idd until pnd were fed ird for days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e and estrone (e ) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e and diet (idd vs ird and idd vs idd+ird). e levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e and ratios e /e in mg and ratio of e levels in pl/mg were not affected by diet. in contrast, diet tended to affect e concentrations in pl (p= . ) due to an increase in the ird group (p= . ) whereas e levels in the idd+ird group only tended to be elevated (p= . ). in mg, ird and idd+ird increased e levels only weakly (p= . each). likewise, besides significant changes in transcript levels of cyp a and a , putatively decreasing oxidation of e to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp , mt a) was observed in the idd+ird group. e mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e levels but not diet were significantly associated with gata transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt ) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e (increase in gdf , hgf, igf r, wnt a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e and even decreased not significantly by idd+ird. taken together, despite an increase in e levels in pl, less er activation was observed after dietary exposure to if. whereas e and transcript levels of enzymes involved in e (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le / - . august copenhagen irish (aci) rats with β-estradiol (e )-releasing implants are an accepted model to study the etiology of breast cancer, but neither e (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) and , placebo (-e ) or silastic implants containing mg e were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd . palpable mg tumors (pt) and - mg per animal without pt were characterized histologically and categorized into normal (-e group, n= ), hyperplasia and non-pt and pt with and without solid tumors (+e group, n= ). e , estrone (e ), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e and e in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of transcripts involved in (biotrans)formation of e and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e , plasma e as well as e and (borderline) e levels in mg were higher in ird. plasma e as well as e and e levels in mg were lower in the -e group than that in the +e group. e levels as well as e /e and e mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki ). ird increased e /e ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata ) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd ) tissues. levels of e and -meo-e were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd b (conversion e to e ) and cyp a . transcript levels of gstm and gstm were decreased in the whole +e group and of gstt and gstt in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp and mt a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of -meo-e nor cellular stress (gclc, mt a, tp ). of note, neither -meo-e , nor e catechols, nor e catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e or e were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e or e . taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e and cellular (oxidative) stress, pt exhibited highest e levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e /e ratio in pt. supported by dfg le- / - . level of beta-estradiol (e ) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from mammoplasty specimen obtained from women (age - years old) not taking estrogen active drugs. of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n= ) and bacterial metabolites (n= ) as well as the estrogens estrone (e )-sulfate, e , e and -methoxy-e were determined by uhplcand gc-ms/ms, respectively and transcript levels of enzymes involved in e (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e /e ratio with agl(+con) was observed in glandular tissues (r= . , p= . ), accompanied by a significant negative correlation of e levels with agl (r=- . /p= . ), possibly due to reduction of beta-hydroxysteroid dehydrogenase (conversion of e to e ) expression as indicated by a weak negative correlation of transcript levels of beta-hydroxysteroid dehydrogenase with agl+con (r=- . , p= . ). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e /e ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le- / - . allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp- cells were used as a model system and treated with the sensitizer , dinitrochlorobenzene (dncb; , and µm) and the irritant sodium dodecyl sulfate (sds; µm). samples were taken after , and hours. thp- activation was analyzed by measuring the established activation markers cd and cd after hours. a targeted lc-ms/ms approach was used to analyze metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., ) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp- were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp- -macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp- cells. evaluation of hepatic cell stress showed the strongest effect on thp- -macrophages when phh were incubated with apap or dic for h.treatment of kc and thp- -macrophages with supernatants of phh challenged for h with hepatotoxic compounds indicates that thp- -derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp- derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to , . , . , and mg/cm³ ceo as well as mg/m³ baso nanoparticles ( h/day, days/week, weeks). animal dissections were conducted at five time points (exposure day and ; recovery day , and ) aiming for endpoints mandatory according to oecd guideline . additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo and baso retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to days was detected by balf and histopathology examination. based on our current results effects of ceo nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt- project is funded by the german federal ministry of education and research (bmbf) - x a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco- model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco- cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana . expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana . project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana . provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana . is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around % of total pm emissions are emitted by wood burners (uba ) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a ) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted : and directly delivered to the exposure chamber. after h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. part fibre toxicol : ), or amosite asbestos at µg/cm for h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed significantly differentially regulated genes for mwcnt a, for mwcnt d, for amosite, and for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht ). for this purpose ht cells were exposed for hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of - µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of . pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about %; control about %), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about %; control about %) for concentrations above µg/ml. induction of apoptosis was determined for silver particles (up to % early apoptotic and % late apoptotic cells) as well as for titanium dioxide and zinc oxide ( % each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to %). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [ ] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of nm and µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in -well plates and exposed to doses of , , and µg/cm² growth area of the respective cnt type for h. the wst- assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h so /hno mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [ ] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union th framework program (grant agreement no. ), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of µg/cm ( h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il- liberation (elisa) as primary endpoints, and quartz dq and al o as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) nanoplatelets (mg ; mean lateral size: , - µm; mean thickness: - nm) represented the least, and pristine few layers graphene nanoplatelets (gr ; mean lateral size: µm; mean thickness: nm; graphene layers: ± . ) the most biologically active nm. clear concentration dependencies were detected for gr in follow-up experiments. mg and gr were further tested in other lung-relevant cell types, i.e. mrc- primary human lung fibroblasts and a lung adenocarcinoma epithelial cells. interestingly, mrc- cells were less sensitive towards biological effects of gr , compared to am, whereas a cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for h to . or µg/cm of gr on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr , i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc- cells and limited uptake in a cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc- cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. with several protocol extensions. female rats ( /group) were exposed to cerium dioxide (nm- , . ; . ; ; mg/m³) for two years; a control group was exposed to clean air. after one year exposure, µg/lung was found in animals exposed to . mg/m³ and . mg/lung in animals exposed to mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of . and . mg/m³ ceo . neither pre-neoplastic nor neoplastic changes were observed after -months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in . this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. with several protocol extensions. female rats ( /group) were exposed to cerium dioxide (nm- , . ; . ; ; mg/m³) and barium sulfate (nm- ; mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo lung burdens were maximally . mg/g lung tissue at the highest exposure concentration of mg/m³. in comparison, highest ceo burdens in organs remote to exposure were liver and spleen with maximally roughly x - g/g tissue. in brain, maximum ceo levels were x - mg/g lung tissue. baso lung burdens were comparatively low ( mg/g) within the first weeks of exposure and steeply increased to mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in . the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df nano) that consists of tiers to assign nanomaterials to main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass ( ) soluble nanomaterials, ( ) biopersistent high aspect ratio nanomaterials, ( ) passive nanomaterials, and ( ) active nanomaterials. the df nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df nano for nanomaterials were validated against grouping by results of existing in vivo data and demonstrated concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo np with an average diameter of - nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations ( . , . , and %) and were used to expose human lung adenocarcinoma cells (a ) for up to hours. at µg/ml ceo np showed little to no toxic effecton growth arrested a cells at fcs concentrations of % or below, but cell viability was decreased to about % in proliferating cells in cultures with % fcs. the proinflammatory effect of ceo np was investigated through measurement of il- mrna expression after hours and cellular il- secretion after hours. the qrt-pcr showed that the expression of il- mrna in cells treated with µg/ml ceo np in % fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il- secretion, which showed a stronger increase by cells treated at % fcs. differences in cellular uptake of ceo np was determined by fluorescence-activated cell sorting (facs) after and hours of exposition. after hours, the cells treated with ceo np in % fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about nm ( % fcs) to nm ( . % fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase- (ho- ) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)- β, il- , il- and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il- β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio np by wst- assay overall revealed only minimal toxicity in comparison to sio np which were used as reference material. ho- and g-gcs mrna analyses indicated that specific tio np triggered a moderate induction of oxidative stress. il- was only induced after sio treatment, whereas il- was not affected by any of the tested np. in contrast, various tio np caused a significant induction of il- β mrna expression. however, no significant induction of il- β and tnf-α protein secretion was observed for any of the tio np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp programme. specific types of tio particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase (nat ) activity j. lichter , b. blömeke trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase (nat ), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat has not been analyzed yet. to address whether human nat is a target of silver nanoparticles and released irons on protein, purified nat was exposed to silver ions (agno ) and silver nanoparticles (ag -cooh, average size nm, carboxyl functionalized), and nat enzyme activity was analyzing the n-acetylation of the nat substrate para-aminobenzoic acid (paba). therefore, purified nat ( ng/µl) was co-exposed for min to paba ( mm) and agno or ag -cooh ( . , . , , and µg/ml each), resulting in a nat :silver relation of : . - (w/w). both, agno and ag -cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat (w/w, µg/ml) enzyme activity was reduced about ± . % (agno ) and ± . % (ag -cooh). the lowest concentration analyzed ( . µg/ml) reduced nat activity about ± % (agno ) and ± % (ag -cooh). fifty percent activity reduction was caused by . ± . µg/ml of agno and . ± . µg/ml of ag -cooh, which is -fold lower compared to the published ic values for other metal oxide nanoparticles ( - µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat -days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of and mg/kg body weight for nanosilver and corresponding amounts of silver acetate ( . and . mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by -d gel electrophoresis and differentially expressed spots were identified by maldi-ms. unique proteins, showing a log ratio of ≤ - . for downregulation and ≥ . for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf -mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated - - protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below % for mg/ml after h). in general the effects of both nps were almost similar. additionally the effects of the baso -nps were compared more in detail. uptake of the baso was quantified by icp-ms after h and h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso of plc and nhbec to . mg/ml over h and h leads to ~ µg baso / mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il- and il- could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after h. summing up baso -nps showed toxic effects in primary human lung cell cultures after h for concentrations under mg/ml. c exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht revealed a c -mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., ) . former results of studies on map kinase signalling indicated c -induced modulations of downstream signalling modules. therefore, ht cells treated with nm c for h were applied for screening of the activity of various transcription factors followed by luciferase reporter assays. five transcription factors namely sp , atf , e f- , cbf, stat were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp target genes p and cox- as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd , a target gene of atf , was decreased. our results suggest that c is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of . g/cm , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from to xg resulted in higher yields. in general, large variations in yield ( . % ± . %) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl- -naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or -tert-butyl- -methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system ( qtrap) and a hybrid quadrupole time-of-flight instrument (tt + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. ( ) chem res toxicol , resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of fmol per injection ( pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and -monochloropropane- , -diol ( -mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and -mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and -mcpd. the metabolite , -dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is -mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary -mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound c -dhpma. the limit of quantification is µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = ) of one male volunteer collected over ten days was ± µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing . mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b c f mice and nasal neoplasms in male f /n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic -sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n -[(furan- -yl)methyl]- ′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase (nat ) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n= ), dortmund (n= ) and neuss (n= ). nat was genotyped using seven characteristic polymorphisms (rs , rs , rs , rs , rs , rs , rs ). haplotypes were reconstructed using phase v. . . . we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat * b/* b and * b/other slow haplotypes as well as between the ultra-slow * a/* a and * a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of ( %) patients showed a relapse within the first yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators ( % vs. %, p= . ). this frequency is even higher in ultra-slow acetylators ( %, or= . , p= . ) but not in slow * b/* b genotypes ( %, p= . ). ultra-slow acetylators had a significantly shorter relapse free time within yr after bc diagnosis than rapid acetylators (median . vs. . yr, hr= . , p= . ). this trend was not that pronounced in all slow acetylators combined ( . yr, hr= . , p= . ) nor in the subgroup of nat * b/* b genotypes ( . yr, hr= . , p= . ). the effect of ultraslow nat is even more pronounced in smokers (hr= . , p= . ) but absent nonsmokers (hr= . , p= . ). ultra-slow nat seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand β-estradiol (e ). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf- cells treated with e or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [ ]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; / ). urine specimens from children aged from to months from different day-care centers were analyzed. in total, preschool children were recruited with mean age of months. our study results show that nearly all children (> %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, oh-mehp, oxo-mehp, oxo-minp), for bisphenol a and methylparaben. triclosan was detected in % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about - µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about µg/l. median urinary concentration for methylparaben and bisphenol a were about µg/l and µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were µg/l, . µg/l and , µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study ( / ) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of µg/kg benzo[a]pyrene and µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [ ]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl ). first, the time-dependent internalization of hemin and fecl into hct cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco- and hct -cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion -oxoguanine ( -oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue (mth ), which protects the nucleotide pool by hydrolysis of -oxogtp, -oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct and caco- cells using western blot analysis, which was followed by a reduction in cell viability at high doses after h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between . % and %. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [ , ] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [ ] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [ ] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [ ] göggmann, schimmer ( ) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. , [ ] [ ] [ ] [ ] wiseman et al. fatty acid esters of -chloro- , and of -chloro- , are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free -mcpd and -mcpd. -mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which mg/kg b.w./day of either -mcpd or -mcpd were orally administered to rats over a period of days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis ( de). among others, the redox sensor protein dj- was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with -mcpd or -mcpd. up to six different isoforms of dj- were identified by de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either -mcpd or -mcpd predominantly led to a shift of the abundance between two dj- isoforms in the rat tissues. this effect was more pronounced with -mcpd compared to -mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys ) of dj- to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either -mcpd or -mcpd. dj- is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys was shown to be crucial for the activity of dj- , and the irreversible oxidation of cys to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of -mcpd and -mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. , -unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure ( h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure ( d) comparatively to short-term exposure ( h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp a (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp , which results in cell stress and caspase / dependent cell death. recently, we discovered that higher concentrations of suba ( mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [ ] . in contrast to hela and caco- cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of -mcpd and its esters. high amounts of -mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related -mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of -mcpd, pointing to kidney and testes as main target organs. -mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for -mcpd, showing similar organ toxicity compared to -mcpd and identifying heart as additional target organ. no such data exist for -mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for -mcpd, -mcpd and their esters. hence, an oral -days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by -mcpd and -mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of -mcpd + -mcpd ( mg/kg body weight), and -mcpd-dipalmitate ( + . mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by -d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of unique proteins deregulated at a log ratio of ≤ - . for downregulation and ≥ . for upregulation at p ≤ . were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by -mcpd versus -mcpd. dipalmitate treatment even caused more deregulation than -mcpd. upregulated cytochrome b-c complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after -mcpd and -mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after -mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after -mcpd treatment. protein dj- (park ), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with -mcpd and the higher dose of the -mcpd diester. tropomyosin beta chain (tpm ) was commonly upregulated after -mcpd and -mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of -mcpd-and -mcpd dipalmitate-induced proteomic changes exist compared to -mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as α-hydroxy-chol ( α-ho-chol), β-hydroxy-chol ( β-ho-chol), -keto-chol ( -o-chol), , α-epoxy-chol (α-epoxy-chol) and , β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where β-ho-chol (milk) and -o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: α-cholestan- β-ol), respectively in breast adipose tissues of healthy women undergoing mammoplasty. furthermore, tissue levels of fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: . nmol/g tissue, range: . - . nmol/g), followed by α-epoxy-chol > -o-chol > α-ho-chol> β-ho-chol ( . nmol/g, range: . - . nmol/g). tissue levels of chol ( . micro mol/g, range: . - . micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of α-ho-, β-ho-, and -o-chol (r = - . , p= . - . ) median oxychol/chol ratios ranged from . ( , to . ( β-ho-chol). -o-chol and -ho-chols correlated strongly with each other (r= . - . , p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r= . , . , and . , respectively, p= . , . , . , respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products ( ) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin ( ) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them ( ) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation ( ) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument ( ). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently ( , ). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points ( ). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas - - ), icaridine (cas - - ) and ebaap (cas - - ) ( ). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than % as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than , international publications have been transferred to a database, with more than , entries. according to the database, pharmaceuticals have been found in the environment of countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, chemicals (ca. %) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a and days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet ( - mg/l) and els study ( . - . mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat- (slc a ) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat- for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat- expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat- in human ea.hy endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat- mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb ( , -dichloro- -β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat- gene responsible for this regulation. to our surprise and in contrast to data published by others the chromosomal region up to kb upstream of the first hcat- exon exhibited no promoter activity in either endothelial or dld- colon carcinoma cells that exhibit a very strong endogenous hcat- expression. we could however identify a promoter element within the first intron of the hcat- gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat- expression. our results indicate starvation-induced transcriptional regulation of hcat- through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc -family consisting of the two multidrug and toxin extrusion transporters mate- (slc a ) and mate -k (slc a ). while mate- is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate -k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with different anti-neoplastic agents comparative transport experiments with the model substrate -methyl- -phenylpyridinium (mpp) have been carried out. therefore stably transfected hek -cells expressing mate- or mate -k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek -cells were used. the interaction analyses were carried out by determining the uptake of [ c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) b that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp b substrate estrone sulfate (e s), and observed a significantly lower k m for mdck-hoatp b compared to . ± . µm, *p< . student´s t-test) whereas there was no difference in v max (mdck-hoatp b . ± . fmol/min/µg protein; mdck-roatp b . ± . fmol/min/µg protein). the human oatp b exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [ ] . to identify whether rat oatp b owns the same characteristics, the cellular accumulation of µm e s (low affinity site) or . µm e s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp b . as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e s in mdck-hoatp b cells ( . ± . fmol/min/µg protein vs. . ± . fmol/min/µg protein, *p< . student´s t-test), there was no inhibition of e s accumulation in mdck-roatp b cells ( . ± . fmol/min/µg protein vs. . ± . fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp b while human oatp b is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per ng of total rna) in different tissues of rat revealed a high expression of oatp b in liver ( . ± . ), a moderate expression in small intestine ( . ± . ) and colon ( . ± . ), and a low expression level in kidney ( . ± . ) . the latter is in accordance with previous findings showing that oatp b is abundant in rat kidney as quantified by absolute proteomics technics [ ] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v. , targetscan . ) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of mirnas, were detected in all tissue types. out of ten transporters five showed significant inverse correlations with putatively targeting mirnas (e.g. pept and hsa-mir- a, r= - . , p= . ). reporter gene assays indicated interactions of mir- a/b with pept '-utr (p = . and p = . ) as well as of mir- a with abcb '-utr (p = . ). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept and abcb . background: the human uptake transporter nact [gene symbol slc a ; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc a gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc a gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g r, -p.t m and -p.l p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of tested) inhibited nact-mediated citrate transport at the tested drug concentration of µm. from these, benzbromarone shows the strongest inhibition with an ic value of . µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g r, -p.g e,p.t m, -p.l p and -p.l p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc a gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member (abcb ) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol- , -bisphosphate (pip ) is a known regulator of abcb . the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht- served as a model. we employed various in-vitro methods for the measurement of abcb activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb activity and re-sensitize our cell model against chemotherapeutic agents like -fluorouracil which are known to be substrates of abcb . taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb might be a new approach to target resistant tumors without interfering with the natural function of abcb in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ h]-labeled homoarginine as substrate and human embryonic kidney (hek ) cells stably overexpressing human cat [gene: slc a (solute carrier family )], cat a (slc a a) or cat b (slc a b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat ( -fold), cat a ( . -fold) or cat b ( . -fold) after . min using µm substrate (each p< . ). apparent k m values for cellular net uptake of homoarginine were . µm for cat , µm for cat a and . µm for cat b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat , cat a and cat b. compared to other cat substrates, the cat -and cat b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter (oct , alternative name slcc a ) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b ), known oct substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct has been shown to have strong inter-species differences. in rodents oct is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct orthologs. methods: overexpression of oct orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ . the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct . human oct showed a fold higher v max for the uptake of asp + and -fold increase of v max for sumatriptan in comparison to the rodent oct orthologs. conversely, human oct showed a -fold lower v max for the uptake of fenoterol compared to rodent oct s. there was no difference between rodent and human oct in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct constructs and by comparing the effects of key point mutations in mouse and human oct orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct orthologs. therefore oct pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct . ranitidine is a histamine h -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately % of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct [ ]. oct is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct alleles are known [ ] . thereof alleles confer partial or complete loss of oct activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct on the uptake of ranitidine. we used hek cells stably transfected to overexpress wild type or variant oct isoforms. the variant oct alleles oct * a (met val), oct * c (phe leu), oct * d (pro leu/met val), oct * (met del), oct * (arg cys), oct * (gly ser), oct * (gly arg/met del), oct * (cys arg/met del), oct * (ser phe), oct * (arg met), oct * (pro leu), oct * (ser leu), oct * (ile thr), oct * (ser leu) and oct * (thr met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct isoforms. wild type oct showed a time and concentration dependent uptake of ranitidine with a k m of . ± . µm and a v max of . ± . pmol/min/mg protein. variants oct * , oct * , oct * and oct * completely lacked ranitidine transport activity. variants oct * , oct * , oct * and oct * showed v max values decreased by , , and %, respectively. oct * variant showed an increase of v max by %. there was no significant changes in ranitidine uptake by variants oct * a, oct * c, oct * d, oct * , oct * and oct * compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct substrate and demonstrated that genetic polymorphisms in oct can strongly affect ranitidine uptake. the effects of oct polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of -ht a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate -ht a receptors, we here compare lsd and dmt with regard to processes of -ht a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the -ht selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective -ht a antagonists ketanserin and mdl . after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ s]gtpуs binding) is paralleled by a desensitisation of frontocortical -ht receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha- -ht a transfected) hek and (endogenously -ht a expressing) c glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise -ht a receptors, fails to activate the phospholipase d (which is needed for -ht a internalisation), and fails to inhibit the synthesis of -ht a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of -ht a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of -ht a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., ) and sunset (schmidt et al., ) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div . in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor- kinase inhibitor a and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a and spermidine had no effect on de novo protein synthesis in juvenile neurons (div ) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div ). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin a receptors ( -ht a r) apparently play a pivotal role in the development of depression. the involvement of an altered, -ht a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located -ht a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic -ht a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic -ht a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment ( days) with the selective -ht a r agonist -oh-dpat ( , mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; mg/kg/day). mice are sacrificed either one day (proliferation) or days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic -ht a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia ( days), but not after spontaneous remission (age days). as indicated by unaltered homeoboxprotein nkx . (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc and cavii mrna, used as markers for the gaba-switch, were unchanged in and day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha subunit). by stereological determination, we found a % decrease in alpha subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in ( %) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem ( . and . mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam ( . and . mg/kg i.p.) in the dt sz hamster. examinations of αlpha gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha gaba a r subunits. changes in αlpha gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. ) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh- ( x - m) and jwh- ( x - m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh- was not affected by pregnenolone ( - m), the inhibition by jwh- was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone ( - m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ -tetrahydrocannabinol ( x - m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ tetrahydrocannabinol were not changed by pregnenolone ( - m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, - m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol : - , ) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β -antagonist sr a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor (trh-r ) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par- , par- and par- . in human adipose tissue, par- is found in vascular stromal cells and par- in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in t l cells. results: differentiation of t l preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par- is time-dependently upregulated in maturing cells while par- expression is detectable but not altered. in preadipocytes, thrombin ( u/ml) activates the mitogenic kinase erk / , promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki and the inflammatory cytokine interleukin (il)- are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par -activating peptide (gypgkf, µm) and cathepsin g, a proteolytic par- activator released from neutrophils and mast cells. repeated exposure of maturing t l cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il- expression. pretreatment of mccm with a par- inhibitor further drives lipid accumulation, the induction of il- by contrast is suppressed. conclusion: par- activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par- expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr antagonist amd exerts deleterious effects in endotoxemia in vivo s. seemann , a. lupp the chemokine receptor cxcr is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine (cxcl ). although a blockade of the cxcr /cxcl axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr seems to be part of the lipopolysaccharide sensing complex, cxcr agonists or antagonists may have a positive impact on tlr signaling. additionally, cxcr is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr blockade on endotoxemia by applying a sublethal lps dose ( mg/body weight) in mice. the selective cxcr inhibitor amd was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp phox as well as of heme oxygenase in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than % or % when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about % in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp phox to occur above average, whereas heme oxygenase expression was distinctly decreased. our results indicate that a blockade of the cxcr in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr antagonist cannot be recommended, hence indicating the activation of cxcr to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr , tlr and tlr mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr expression in a time-dependent and concentration-dependent manner and reduced tlr / expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il- β further increased tlr mrna levels. gene expression of mapk phosphatase- (mkp- ) was also upregulated by dexamethasone. glucocorticoid-induced tlr expression was negatively regulated by p mapk signalling under inflammatory conditions through mkp- induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr signalling as demonstrated by reduced il- , il- β, mmp- and mcp- levels. however, the expression of traf , a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il- cytokines and t helper (th ) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il- β, propranolol induced the th priming cytokines il- and il- in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il- γ and lps failed to increase il- release in molc and modc in the presence of propranolol but further induced secretion of il- β. autophagy has been linked with the secretion of il- family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p and lc -i to lc -ii conversion, induced accumulation of lc positive vesicles, as well as expression of il- signalling downstream adapter molecule traf , indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz- (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr expression and responded robustly to tlr engagement, confirmed by increased cd and cd expression, upregulated il- , il- p and il- p mrna levels and il- release. tlr activation reduced ccr and elevated ccr mrna expression and induced migration of mutz-lcs towards ccl . similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il β whereas ligands of tlr and tlr failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr -activated mutz-lcs, co culture with naive cd + t cells led to significantly increased ifn-γ and il- levels indicating th differentiation independent of il- . tlr -mediated effects were blocked by the putative tlr / antagonist cu-cpt , however, no selectivity for either tlr / or tlr / was observed. computer-aided docking studies confirmed non-selective binding of the tlr antagonist. taken together, our results indicate a critical role for tlr signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep - . , a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep - . and the structurally related compound pep - lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr (fsl- ) but not tlr (lps) activation by increased il- production, as determined by elisa. western blot analysis showed that both salps inhibited fsl- -induced phosphorylation of nf-κb p and p mapk. furthermore, the peptides significantly reduced il- release and gene expression of il- β, ccl (mcp- ) and hbd- as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il- secretion, downregulated expression of the maturation markers cd and cd , as analysed by flow cytometry, and inhibited ccr -dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il- levels and cd /cd expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep - . affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid -related factor (nrf ), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf -deficient (nrf -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for min (v t = ml/kg, f= min - , peep= cmh o, fio = . ), before µl hydrochloric acid (hcl) with ph= . or ph= . were instilled intratracheally, controls received nacl. mice were then ventilated for further h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph= . induced mild lung injury, indicated by hypoxemia (po /fio ~ mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf -/mice. in contrast, hcl ph= . caused more severe lung injury (po /fio ~ mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf -/mice. conclusion: we conclude that the presence of nrf augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [ ] [ ] [ ] [ ] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [ ] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [ ] . adma is degraded by dimethylarginine dimethylaminohydrolase and (ddah) enzymes [ ] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide- kinase (pi k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh ] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah and ddah enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah and enzymes. the pi k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-( - ) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-( - ) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-( - ) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-( - ) infusion provided best protection from experimental ali when administered by continuous infusion starting min after oa infusion till the end of the experiment ( - min). both pretreatment (- - min before oa) and short-term therapy ( - min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-( - ) min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-( - ) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y- and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of µm y- under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens- (zo- ). under conventional culture, y- induced higher growth rate of primary ebec and increased the passage number up to passages with retained epithelial cell behavior. in the presence of y- , ebecs under ali showed higher teer values. expression of zo- correlated with the increase in teer, but in y- -treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y- facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic values for the viability in the low micromolar range [ ] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe + . the radicals were trapped by -methyl- -nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = . g, and a h = . g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic , -dimethyl- pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe + a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = . and a h = . g. according to previously published data [ ] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [ ] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine : - , . nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca + -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the ' untranslated region ( 'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld . using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c a-enos). normal enos (enos-tg) or a novel dimer-destabilized c a-enos described previously (antioxid redox signal. sep ; ( ) : - ) were introduced in c bl/ in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for weeks or treatment with sod mimetic tempol. results: c a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos / phosphorylation and amp kinase (ampkα) phosphorylation at thr in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd . exercise training increased phosphorylation of enos at ser / and of ampkα at thr in wt but not in c a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp- cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka ( . mg/ml) and mya ( mg/ml) treatment down to % and % resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka ( mg/ml) treatment by % and % resp.; after mya ( . - mg/ml) treatment by % and % resp. lps-induced tnfα release from thp- cells was inhibited concentrationdependently by my (ic = . μg/ml; % inhibition), ka (ic = μg/ml; % inhibition) and cc (ic = μg/ml; % inhibition). furthermore, ka ( µg/ml) and cc ( µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to -fold (for cxcl ). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a a receptors (a ar) and adenosine a b receptors (a br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a ar agonist (cgs ) and an a br antagonist (psb- ) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw with a ar as a possible mechanism of the protective effect of stw in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of , , -trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a , a b, and a receptor with specific agonists reduced the acetylcholine (ach, µm)-induced contractions, while activation of a br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs ( µm) or with psb ( µm). the same effects with smaller intensity were found for stw ( µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw interact with the a ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw as a therapeutic approach in ibs. (allescher ) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw (ottillinger et al. ) . stw (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw ) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than pharmacological tests, is needed. all data from studies including stw alone, or stw and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of d histograms (lorkowski et al. ) . results: more than pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw contribute differently to the overall effect of stw . altogether, the evaluation of the data shows that stw is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw consists of a mixture of standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw ii lacks the last components. stw was shown to be effective clinically to treat functional dyspepsia ( ) and irritable bowel syndrome ( ) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis ( ) and in the management of ulcerative colitis ( ) . the present study was initiated to show whether stw ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with % dextran sodium sulfate in drinking water for week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw in doses of and ml/kg or stw ii in a dose of ml/kg for week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of mg/kg. conclusions: the modified preparation, stw ii thus proved to be as effective as stw , thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. ( ) schmulson mj ( ) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor (maropitant) and serotonin receptor (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk ) and channel opening (activation of ht ) on the intracellular calcium homeostasis (using fluorometric ca + analysis) and cell proliferation rates in endogenously nk and ht receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk or ht cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk and ht receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every - days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh promoter, further corroborated that rasal expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa- ht], urb ) on a lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, -ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases- (timp- ). using sirna approaches, a causal link between the timp- -upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp- expression. inhibitor experiments point toward a decisive role of cb and transient receptor potential vanilloid in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa- ht and urb . as underlying mechanism of its anti-invasive properties an upregulation of timp- was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, -arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb and arachidonoyl serotonin (aa- ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p / mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p / mapk activation, a blockade of pparα left p / mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p / mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes and leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k . an inverse correlation of mir- expression and protein levels of the efflux transporter atp-binding cassette transporter g (abcg ) was observed in cells resistant to different im-concentrations, pointing to a relation of mir- to im-resistance. hence, we investigate in current studies, how the influence of mir- on im-sensitivity could be explained. methods: we transfected k cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir- or inhibitory anti-mir- , challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst- -, caspase glo -assay and cell counting. in addition, we analyzed changes in abcg expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k showed an effect of mir- -inhibition using anti-mir- . this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p< . ) and cell membrane integrity and reduced capase- activity (p< . ). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg expression, we found that µm im-resistant k do not express higher amounts of abcg , but showed higher transport rates of im or the abcg -substrate hoechst . conclusions: overall, these experiments indicate that mir- does not only affect abcg -expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg . in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe( %)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of - gy using a kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [ ] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [ ] to % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to % of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h s production in the nociceptive system are only poorly understood. here we investigated the expression of the h s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h s. this work was supported by the deutsche forschungsgemeinschaft (sfb -a ) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt- ) and mesenchymal-like (j- ) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt- r , j- r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh ax and bp foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j- and j- r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt- cells showed an about - -fold higher resistance to cispt than j- cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt- r and j- r revealed a - -fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt- r cells disclosed a reduced apoptosis and enhanced g /m arrest following cispt exposure as compared to rt- wild-type cells. by contrast, induction of cell death was similar in j- and j- r cells. notably, j- r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j- r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt- r and rt- cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j- r and rt- r . unexpectedly, j- r and rt- r shared the upregulation of xaf- . treatment of j- r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk- and, moreover, re-sensitization to cispt by chk- inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt- cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j- cells. furthermore, our findings indicate that chk- might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein (satb ) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb in the upregulation of her after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb expression in gastric cancer cells, and the downregulation of satb by sirnas completely prevented the induction of her upon c-met inhibition. in contrast, her or her expression levels were not affected by satb -specific sirnas. the function of satb as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her expression after c-met inhibition. interestingly, the upregulation of her in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb and pkc are critically involved in the regulation of her expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb- breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki- proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb- breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for , pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb- ) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam- , vcam- , e-selectin, n-cadherin, and galectin- . moreover, the chemokine system cxcl /cxcr was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl could be excluded, since blocking the cxcl receptor cxcr on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β -integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β -integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for , fu / - ). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse ( ). based on an evolutionary cancer model ( ) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc and hcc cell lines; both erlotinib sensitive) ( ). plasma concentrations from two single-dose groups, mg/kg and mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens ( mg/kg daily, mg/kg daily, mg/kg every days, mg/kg every days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem . . results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the mg/kg dose. a -compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: and days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec = . μm for both cell lines), and on the partially sensitive transit phase (ec = . μm and . μm, for hcc and pc cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and -lipoxygenase ( -lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of min whereas the increase in vocalisation threshold persisted over a period of min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox- and -lo but not of cox- . we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox- and -lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. , million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., and buchner et al., documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > , . in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over purified compounds and over extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of , %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d receptor partial agonist bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d receptor partial agonist -bromoterguride ( -bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [ ]. to determine its atypical character in vivo, the ability of -bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of -bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: week old male sprague-dawley rats were injected with -bt ( . or . mg/kg; i.p.) followed by pcp ( . mg/kg; s.c.) or apomorphine ( . mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of -bt ( . or . mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp ( . mg/kg; i.p. twice daily for days) was assessed in the nor task consisting of a min acquisition trial and a min retention trial separated by a h inter-trial interval. clozapine ( . mg/kg; i.p) or haloperidol ( . mg/kg; i.p) were used as positive controls. results: the dopamine d receptor partial agonist -bt ( . mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly -bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that -btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that -bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of -bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of -bromoterguride [ ], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of -bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid- receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in . - . year old cb -/and control mice, fed with a standard diet and assessed effects of the cb agonist, hu during high fat diet in - week old mice. results: the cb -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb agonist hu prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m -like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb agonists may fortify cb -mediated anti-obesity signaling without the risk of anti-cb mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic : xan µm, res µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic : xan µm, res µm) while had less influence on the viability of pmm (ic : xan µm, res > µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin ( -ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph ), or the raphe nuclei of the brainstem (tph ) and the respective -ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral -ht is mainly produced by tph -expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, -ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral -ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg as a hcc tumor cell line and the fibroblast cell line fi as stroma simulation. additionally caco cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of . - µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: . µg/ml -cr: µg/ml -es: µg/ml. performance of the caco -transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with -fluorouracil ( fu), which is used as a chemotherapeutic agent, caused a reduction of its ic values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin- , - , - , - . claudin- is known to be the key tj protein tightening the bbb. therefore, claudin- has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin- and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin- containing barriers. the claudin- targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin- and immortalized brain endothelial cells (bend. ). the peptides decreased the amount of claudin- and zo- at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein ( da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin- (cldn ) which tightens the bbb for molecules < da. consequently, cldn is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn assay, the barrier opener (bo ) was identified as a cldn modulator. initially, a significant removal of cldn from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw da) demonstrated the effect of bo . concentration dependent treatment ( - µm) of cell monolayers with bo reduced tightness of the tjs between some hours and h. applying -hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo became detectable in mice. due to short stability (< h) of bo in the bloodplasma repeated administration ( . mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw da). the small molecule bo is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about h of incubation with concentrations varying from to µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow ( da) and fitc-dextran ( kda); the localization of tric elongated from ttjs towards btjs and cldn was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c bl/ mice. c bl/ mice without transplantation were used as normal controls. donor c bl/ mice to recipient c bl/ mice were served as the isograft group. days after transplantation, mice were scanned using an in vivo small animal µct (skyscan ). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf- pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf- (igf r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf- pathway is the transcription factor daf- (hfoxo a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf- pathway. recently, a cohort study showed that a decreased mortality rate in humans between age - correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about %) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf- overactivity. consequently we found an increased nuclear localization of daf- in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf- -ko mutant background. rt-qpcr brought jnk- , a known activator of daf- , into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf- dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf- pathway and particularly the transcription factor daf- . the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. ( ) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance ( ). in hfd/stz model, the rats are fed with high-fat diet for - weeks or for a relatively long time (≥ months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz ( - mg/kg), some of the diabetic rats receive treatment ( ) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats ( . ± . mg/dl vs. . ± . mg/dl, p < . ). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats ( ± . gvs. . ± . g, p < . ). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for weeks. further details can be found in table . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency ( ) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type diabetes ( ). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: . srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. ( ) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type diabetes and pharmacological screening. pharmacol res ( ): - . . oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. ( ) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem ( ): - . . franz, m. j. ( ) . the dilemma of weight loss in diabetes. diabetes spectr ( ) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt dystonia, which is caused by a gag deletion in tor a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d receptor mediated acetylcholine release has not been examined yet. the aim of present study was to ( ) generate more powerful tests to detect behavioural alterations in the dyt knock-in mouse and to ( ) examine the behavioral effects of the d receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole ( . - mg/kg i.p.) were tested in dyt knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt knock-in mice showed increased thigmotaxis at a dose of . mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay - after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay - was ( mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay - administration influenced the activity of the ecm degrading matrix metalloproteases (mmp and mmp ) and their inhibitor timp- , the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor- (pai- )) and the secretion of cytokines, e.g. il- . thereby, bay - increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay - might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly (sh -domain protein expressed inlymphocytes ) is essential for the generation of a fully functional t-cell response. the lack of sly leads to reduced survival rates of infected mice. the increased susceptibility of sly ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly ko t cells displayed a dysregulation of forkhead box protein o (foxo ) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo shuttles to the cytoplasm after phosphorylation in a protein complex including - - proteins. interestingly, we observed a similar regulation for the adapter protein sly , where tcr stimulation results in sly phosphorylation and sly export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly to - - proteins. altogether, this study describes sly as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly regulates t-cell proliferation (schäll et al., eur j immunol. ) . the catalytical isoforms p γ and p δ of phosphatidylinositol- , -bisphosphate kinase γ (pi kγ) and pi kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p γ and p δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi kγ and pi kδ in p γ ko δ d a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p γ and p δ might exacerbate asthma. here we analysed p γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il- and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p γ and p δ causes eosinophilia and ige hyperproduction, p γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type receptors (b ) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med : ; - ) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually - min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a , kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately µm below the surface. after stabilization of the green fluorescent signal, a , kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b changes physiologic endothelial permeability. this newly developed transgenic mouse line (b tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie- -promotor, the human b cdna, the sv poly-a-signal and a tie- intron fragment. we observed that b tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the , kda dextran at each time point (fig. ) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type (b ). using a plasmid consisting of pbluescript ii sk+ -vector, the tie- -promotor, the human b cdna, the sv poly-a-signal and a tie- intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b was generated and backcrossed to c bl/ for more than times (b tg ). lung mrna using primers specific for the human or the mouse b cdna revealed a . -fold stronger expression of human b in b tg (n= ), while the expression of murine b mrna was unchanged and similar to transgene negative littermates (b n ). we have evaluated the specificity of several antibodies directed against b and found that a rabbit monoclonal anti b antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b -/mice. however, this antibody primarily stains rodent b and has only little cross-reactivity to human b . hence, we were not able to detect a significant increase of b protein in tissues of b tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b n , we found a significant icatibant sensitive aortic dilation in b tg following preincubation with diclofenac which indicates functional overexpression of b in conductance vessels of b tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching . ± . fold at . nmol bradykinin in c bl/ (n= each, p< . vs. vehicle). a similar increase was found in b n ( . ± . fold, n= , p< . vs. vehicle), while there was a stronger response in b tg ( . ± . fold, n= , p< . vs. vehicle) which was significantly different to b n (p< . ) and c bl/ (p< . ). in another set of experiments the specific and ace-resistant b agonist labradimil ( . nmol) was used instead of bradykinin. labradimil increased extravasation by . ± . fold in c bl/ (n= each, p< . vs. vehicle), by . ± . fold in br n (n= each, p< . vs. vehicle) and . ± , fold in b tg (n= , p< . vs. vehicle) which was significantly different to c bl/ (p< . ) but not to b n (p> . ). the effects of bradykinin and labradimil were largely blocked by nmol icatibant (i.v.) in c bl/ , b n and b tg mice (p< . ) and hence mediated by activation of b . these data suggest that overexpression of b in b tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller universitätsklinikum, innere , nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = . …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd + and cd + b-cells completely persists for month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = mcg/ml and the infusion time t = hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t / = hours (not shorter than the time t of infusion). with the terminal half-life of hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t / < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: ) are the observed metabolites ruminant specific and formed directly in the rumen? ) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro > days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph . ) and redox potential (~- mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within h to . . .-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides ( c-polydatin, octyl- c-β-d-glucopyranosid) were cleaved within hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd and contribute significantly to animal welfare ( r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. ( ) and including an empiric correction factor, mrd for the training set was . whereas bias was . µmol/l. by applying the developed pbpk model to the test set the respective values were mrd= . and bias= . µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco- cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco- cell system under different experimental conditions. the permeation characteristics of hypericin ( µm) in absence or presence of hypericum extract , . µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from to . %). the majority of hypericin was found after cell extraction ( % in absence and % in presence of the extract). the recovery was in the range of %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in healthy foals ( ♂ and ♀, age: - days, body weight: - kg) which were treated once with rif alone ( mg/kg s.i.d., p.o., a) followed by the administration of gam ( mg/kg once weekly, i.v., b) for weeks. study period ("rif-gam acute", c) includes the administration of gam and rif for days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for weeks. all periods were completed with blood sampling for pharmacokinetic analysis for ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c h : . ± . ; balc/c h : . ± . ). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c h -ratio was unaffected (peters et al. ) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp d . direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp d . however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about % of caucasians are compound heterozygous carriers of loss-of-function oct polymorphisms. therefore, oct polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct alleles. methods: mg desvenlafaxine (pristiq®) was orally administered to healthy subjects preselected according to their oct genotypes. oct * allele was regarded full active, oct * to * alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the subjects carried two active, one active, and zero active oct alleles. age, height and weight were . ± . years (mean ± standard deviation), . ± . m and . ± . kg with no significant differences among the oct genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc -infinity of desvenlafaxine varied between . and . min*mg/l and auc -infinity of didesmethylvenlafaxine between . and . mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average % greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct genotype groups. conclusions: our results suggest that oct genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly ( ). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression ( ) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september and december using the medical records of elderly patients (≥ years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss . statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n= ). median age was (min: , max: ), patients were female ( . %). according to medical records of patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug ( . % vs. % . ) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics ( . % vs. . %) and angiotensin receptor blockers ( . % vs. . %) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women ( . % vs. %, . % vs. . %, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over % of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e guideline [ ], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [ ] the ability to detect qtc-prolongations of about ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [ , ] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like- (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin- , - and - . apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in patients with nstemi, stemi patients and controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap ) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for sec to min reduced platelet aggregation in lta studies by up to % for apelin- . this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name ( µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group ( , % ± , % in mi versus % ± , %in controls; p = . ). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin- in patients with mi ( . ± . pg/ml versus . ± . pg/ml; p = . ). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = - . ; p = . ). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin- . upon platelet activation, apj is internalized and surface expression is reduced by about %. in mi patients, plasma levels of apelin- and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin- levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from to . inclusion criteria comprised the initial absence of dementia and age of years or older in . anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn , , s. schmiedl , , k. janhsen , , p. thuermann , , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of ( ) drugs with a known association to a certain adverse event (=positive controls) and ( ) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for % of the positive control drugs, and for none of the negative controls. ryan et al. developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. ; suppl :s - ) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in to were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of % (relative risk . ) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with % statistical power. results: from a total of index drugs in the reference sets of ryan et al., ( . %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of , , index drug initiators were included in the analysis. for a total of index drugs, a relative risk of . could be detected with % power. the numbers of index drugs for each of the outcomes of interest were: ami ( positive controls; negative controls); aki ( positive controls; negative control); ugib ( positive controls; negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with % power to detect a relative risk of . was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of -nor- -carboxy- administration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study -nor- -carboxy-∆ -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > . ). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term / ) at technische universität münchen (tum). the course consisted of a -day teaching period, followed by a -day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of out of ( %) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. ). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n= rd -year medical students, n= th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the items were presented as statements for restudy, items were tested using single-choice questions, and items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of single-choice questions, questions of which had not been used before (as a standard of comparison). the remaining questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of of these reworded questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [ ] . in order to address the persisting prescribing deficiencies [ ] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [ ] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least . % of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [ ] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising indications, drugs representing mechanisms, and up to adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. / chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, % to % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., ) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category . this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of - t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to ml/m³ and in mice in response to ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category with a mak-value of ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl- cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm ( mhz) and umts ( mhz). additionally, lte ( . mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from to w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ % (m) in % (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in ( ) a simple non-probabilistic way, ( ) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or ( ) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector ( ) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between . and . µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox , which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s assays in addition to the h r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than control groups with each males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., ) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = . , the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately to mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca + -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of and mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca +/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: , mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to mg per day over the period of up to weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of mg of hca for weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include and , respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg and especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt cells were either continuously incubated for h (approximately . cell cycles duration) with methyl methanesulfonate (mms; - µm), benzo[a]pyrene (b[a]p; . - µm), vincristine ( - nm) , and colcemid ( - nm) or cells were irradiated with xrays ( . - sv) and then cultured for h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation ( w, min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for out of the treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤ -fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk- e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s phosphorylated h ax (γh ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk- e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p , p-chk , p-chk and p-kap . furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk- e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann , g. fritz institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a ´- ´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic ) and moderate toxic (ic ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic and ic were determined after h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, h after permanent tbhq treatment. pictures were taken at the time points h, h and h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht- cell line on the ea.hy monolayer was investigated. wrn-deficient or non-deficient cells were treated with µm and µm tbhq or with tnfα. results: for ea.hy cells, µm and µm tbhq were determined as ic and ic , respectively. performing the migration assay, ea.hy cells showed % gap closure, whereas wrn-deficient cells showed a closure of only % after h. the gap was closed of % and % after µm and µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after µm tbhq treatment, whereas the treatment with µm tbhq showed a slight decrease in migration of about % compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after µm tbhq treatment a slight increase of adhesion was detected in ea.hy cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht- adhesion to ea.hy , which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl , jurkat and tk ) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk cells showed similar increasing trends, whereas hl cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration ( nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk cells. the results for the highest investigated concentrations ( . nm and nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk cells. in contrast the hl cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p status of hl (null), jurkat (multiple mutations), tk (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc- were applied. accumulation of p and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p and pakt analysis, and quantification of the oxidized dna base -oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than bacterial species and population densities of up to cfu per cm . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., ) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of - hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and - photoproducts ( - pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [ ] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct . similar results were obtained in sw and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p as observed by flow cytometry and western blot analysis in hct , sw and hcec. cdt-and irinduced autophagy rates in hct increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein (lamp- ) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser . pharmacological inhibition of akt in hct cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc b and lamp after h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after h and significantly lower apoptosis/necrosis rates after h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n -guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n -ethylthioethyl- ´desoxyguanosine (n -ete-dg) and n -ethylthioethyl- ´desoxyadenosine (n -ete-da) and their thermal hydrolysis products n ethylthioethyl-guanine (n -ete-gua) and n -ethylthioethyl-adenine (n -ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone ax (gh ax), which can be visualised as gh ax foci by immunocytochemistry. in the present study, we tried to assess if gh ax is a reliable biomarker for detecting the cellular response to dna damage. we selected well-characterised genotoxic compounds and compared them with non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh ax by manual and automatic scoring of γh ax foci, and by flow cytometry counting of γh ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh ax positive cells showed a significant correlation. further we compared dna damage induced by selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh ax assay detects a specific response to dna damage which can persist longer. the γh ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh ax), nuclear division (phospho-h , dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for and hrs in triplicates on a microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide -chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h events at and hrs and polyploidy at hrs time point. eto induced a clear increase of yh ax with a simultaneous reduction of phospho-h at and hrs. finally, cccp caused a reduction of phospho-h events, increased cleaved parp events and did not influence γh ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh ax and phsopho-h pilot study results as well as cleaved parp with caspase / . in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage , additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih t , mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g /g = ~ %) and semi-confluent cultures, similarly arrested in the g /g phase by serum-starvation or the mek inhibitor u , ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk . however, inhibition of atm, the kinase responsible for chk activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip , since the chemical rip kinase inhibitor necrostatin- (nec- ) blocked cell death up to averagely %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp- and p are probably not involved. deeper analyses could give evidence that nec- did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec- , possibly rip , acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca + signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc and mtorc ) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c t cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for days with the tumour initiator mca ( -methylcholanthrene) followed by days with the promotor tpa ( -o-tetradecanoylphorbol- -acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi- . surprisingly, an incubation with µm osi- led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s . our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi- , which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi- by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c t cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator -methylcholanthrene (mca) and the tumour promotor -otetradecanoylphorbol- -acetate (tpa). in the first experiment several metformin concentrations ( . - mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using mm metformin. this effect was not caused by growth inhibition of metformin itself since mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to , , and ppm for years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [ ]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: . ppm) after whole body exposure to vapor [ ] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of , , , ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, -pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [ ] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day (e ) at . °c and % humidity. on e , the eggs were cracked open and transferred into petri dishes. on e , cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with -methylcholanthrene as a tumour initiator and -o-tetradecanoylphorbol- -acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in -well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march ). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between - %. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's ( r), described by russel and burch in . in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches , resulting in a refinement of in vitro testing. in this study, human tk cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of . (± . ) h was observed while fbs supplemented cells showed a doubling time of . (± . ) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by ) determining reactivity class cut offs based on published data on substances for the dpra performed according to oecd c to predict un ghs sensitizer classes, ) a variant of the dpra assessing reaction kinetics (time and concentration) for substances or ) an extended protocol testing several test substance concentrations for reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of . % (ec . %). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes a and b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between min and hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. , , and mm) indicated that potency classes could be assigned using the ec . % value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec . % value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase (nat ) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u and thp- ) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p -dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or -amino- -methylphenol, respectively. in contrast, covalent peptide adducts were identified for putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a ' out of ' prediction model provided a high sensitivity of % for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the ' out of ' prediction model in a set of direct haptens was comparable (sensitivity = % when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and two-test and three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. ) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [ ] [ ] [ ] [ ] . in recent supplement, -methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [ , ] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [ ] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of mg/ml ( %), finite dosing and min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [ ] and electrostatic potentials using gamess [ ] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. ). toluene- , -diamine shows a higher accumulated mass due to increased lipophilicity (fig. ) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [ ] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [ ] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba ] contains data for about substances on dermal absorption which has been found through the echemportal [ ] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. chemicals have been investigated in a comparable test system; from these were shown to have a low dermal absorption of less than % and compounds showed a high absorption rate of more than %. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than % to more than %. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in [ ] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [ ]who ( ) ehc dermal absorption [ ] scholz et al ( ) naunyn-schmiedeberg´s arch pharmacol (suppl ):s [ ] www.echemportal.org [ ] http://edetox.ncl.ac.uk [ ] jung et al ( ) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett : - , ) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology : - , ) . we modelled the concentration time profile exploring the dose which would lead to a concentration at hour and at hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between and µm /sec (truisi et al., ) and an absorption of % and an absorption rate of /h (cristofoletti and dressman, j pharm sci : - , ). results: the data of the in vitro study with µm ibuprofen could well be modelled. when assuming a clearance of µm /sec the dose of mg resulted in an hour concentration of . µm in the hepatic vein of pbhkm equal to . nmol/well (volume of the well = ml) in the in vitro study in which the measured concentration was . nmol/well. the concentration at hours of . µm (equal to . nmol/well) corresponded with the in vitro concentration ( . nmol/well). the modelling approach was less successful with in vitro dosing of µm. the fold higher dose of mg lead to nearly double the concentration at hour than measured in vitro. with a dose of mg/kg an approximation was feasible resulting in . µm in the hepatic vein at hour which is equal to . nmol/well whereas the measured concentration in vitro was . nmol/well. even with a clearance value as low as the . percentile ( µm /sec), the concentration at hours was modelled to be lower than the in vitro measured value (in vivo model: . µm which corresponds to . nmol/well; measured in vitro concentration: . nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro ( µm) corresponds to a dose of mg (note: the highest approved daily dose is , mg). for the high in vitro dose modelling was successful only for the concentration hour after dosing and a dose of , mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and . fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score ( positive out of possible genotoxicity categories) was assigned to of the tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [ ], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin (trpa ) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk / , are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk / and increased protein biosynthesis of sre-regulated genes in hek cells overexpressing htrpa . cees increased perk / levels already after . min which could be prevented by the trpa blocker ap . activation of target genes through perk / signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa channel activation. thus, trpa might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst- ) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort (" rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a ), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg is often used as a model for liver toxicity studies. however, under two-dimensional ( d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for days in a three-dimensional ( d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg cells. in our present study we extended previous studies and compared hepg cell cultivation in three different d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after , , , and days of cultivation. our results show that the previously reported increase of metabolic competency of hepg cells is not primarily the result of d culture but a consequence of the duration of cultivation. hepg cells grown for days in d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg is not due to d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [ ]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [ ] , whereas asfas were less toxic [ ] . a substantial transfer across an intestinal barrier model (caco- ) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [ ] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [ ] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic ~ - . µm) in a concentration range comparable to that of arsenite (ic ~ . µm), whereas asfas were less cytotoxic (ic > µm). after incubation with ashcs the cellular arsenic concentrations increased - fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [ , ] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic values of . ± . µm for hache and . ± . µm for hbche. ic values with chloroquine were . ± . µm for hache and . ± . µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after minutes, only paraoxon-inhibited hache ( %) and cyclosarin-inhibited hbche ( %) were reactivated by µm chloroquine. on the contrary, µm amodiaquine reactivated all tested ops after minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [ ] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., , , - . [ ] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., , , - bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional ( d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a d model was used consisting of the permanent breast epithelial cell lines mcf a (er -, estrogen receptor negative) and mcf a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the d culture of mcf a and mcf a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf a) and er-independent (mcf a and mcf a) effects. the d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg ) cells and (b) co-culture systems of human monocytic or macrophage-like (thp- ) and hepg cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg and thp- monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about % cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg . we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator ( -methylcholanthrene) and promotor ( -o-tetradecanoylphorbol- -acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day , morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that ( ) several substances have a different impact on the transformation process, ( ) it is possible to identify for each substance the phase with the greatest effectiveness and ( ) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c t assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg ) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg ) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg ) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et (exposure time reducing viability of treated tissue to %) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg , one of the five severe ocular irritant formulations (un ghs cat ) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat formulations was just above the un ghs cat classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins- e) and exocrine (ar j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, . ( ): p. . briefly, pancreas was infused and embedded with agarose. tissue sections of app. µm were prepared using a vibratome and maintained in cell culture medium for up to days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins- e cells) or amylase/lipase (ar j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins- e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers ( , -dinitrochlorobenzene, -mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology ; : - ) . dendritic cells generated in fcs supplemented medium were cd a+/cd c+, whereas dendritic cells from serum free culture conditions were cd a−/cd c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd a+/cd c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp- cells, as model for dc, enhanced cyp enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd on thp- cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp- cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp- cells by microarray technology. we identified differentially expressed mirnas in cocultured thp- cells compared to monocultured thp- cells irrespective of the treatment (medium, . % dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after h) additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp- cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp- cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp- and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm and . trpa has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in ). although recently two reports identified trpa in pulmonary epithelial and endothelial cells ( , ) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa -deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa on a cellular level we cultured a hek cell line stably expressing trpa ( ) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca + -influx in this cell line, which was blocked by the specific inhibitor a . in the future, we will utilize the isolated perfused lung model ( ) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa -deficient mice after exposure to potential toxic inhalation hazards (tih see ) to challenge the hypothesis of trpa as an important toxin sensor in the lung. by this strategy we hope to understand trpa function in lung cells and to evaluate trpa proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for days to male rats including blood sampling for metabolic profiling after , and days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole- -acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole- -propionic acid for aminoglycosides, taurine for fluoroquinolones, indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., ; luo, schomaker, houle, aubrecht, & colangelo, ; yamazaki et al., ) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low ( mg/kg) and a high ( mg/kg) dose. rats were sacrificed following , , or consecutive daily doses, or after recovery days following consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) and days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide (oatp ) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/ proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann background: in a -day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp r , gc, cyp a, ugt a) in liver was also affected, while initial indications for modulation of renal cyp a and renal and hepatic cyp b could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n= for single substances, n= for combinations) were treated for days at dose levels tested based on noaels from -day subchronic feeding studies and ranged from noael/ to noaelx . quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total ( -oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n= ) were . ± . ng/ml (min/max: / ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c ( ppm in food, n= ), there was a statistically nonsignificant reduction of vitd levels to . ± . % of the concurrent control (n= ). however, for of animals of this group, measured vitd level were below the range observed in pooled controls (n= ). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p for c, e and pz; cdk and gadd a for e; cdkn c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp a / and ugt a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with ppm cyproconazole, there were indications for a moderate reduction of -oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a -year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the th week, and she has been taking a multidrug therapy (dapsone mg/day, rifampicin mg/month, clofazimine mg /day and clofazimine mg/month) for the past months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about weeks. in the th, nd, th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the th week of gestation without perinatal complications. the baby was also healthy (apgar - , g, cm), and its growth and development were normal during a -month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in in berlin, germany, to evaluate kidney function in people ≥ years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january until april ). the probability of dabigatran use rose with increasing age (+ %), and the probability of phenprocoumon use rose in case of egfr < ml/min/ . m (+ %) or male sex (+ %). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ years in nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > mmhg and/or dbp decrease of > mmhg within min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > mmhg and/or mean dbp > mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with nhr (mean age = . ± . years). in total, subjects ( . %) had oh. the mean change in sbp from sitting to standing was . ± mmhg (range + . to - . mmhg) in patients with oh and . ± . mmhg (range + . to - mmhg) in patients without oh. mean sbp was significantly higher ( . ± . mmhg) in people with oh than in those without ( . ± . mmhg). all of the nhr with oh were hypertensive compared to % of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p> . ). medication data was available for patients. all individuals with oh and nhr without oh ( %) had antihypertensive medication. more than different antihypertensive drugs were present in patients with oh ( . %) and in patients without oh ( . %). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than % of patients in both groups reached points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around % of patients with oh as well as patients without oh needed more than sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer ( : ) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from mg/d clom citrate to up to mg/d in case of non-ovulation. however, therapy outcome is variable and approximately - % of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via -hydroxylation of trans-clom by the highly polymorphic cytochrome p (cyp) d leading to the major active metabolite trans- hydroxyclomiphene (trans- -oh-clom) [ ] . recently, we identified a less active trans- -oh-clom which is also formed by cyp d . besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of mg clom citrate. results: incubations with human liver microsomes revealed an almost -fold higher glucuronidation rate for trans- -oh-clom, which is exclusively catalyzed by ugt b , compared to the more potent trans- -oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans- -oh-clom to its sulfate is -fold higher compared to -oh-clom. for both metabolites a participation of sult a and sult e was identified. these results were in line with previous studies, which identified the same sults [ ] and ugts [ ] responsible for the conjugation of the structurally related trans- -hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans- -oh-clom and trans- -oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom- -o-glucuronide. conclusion: our results suggest a new metabolic route via trans- -oh-clom which appears to be a potential inactivation pathway of clom. institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of . µm sm (ic ). today the cells are able to tolerate a concentration of . µm sm (ic ), which reflects to a concentration of which % of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h o , glutathione, ca + -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening significant up-regulated and significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp a expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm -mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in . the program was accredited in by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates places per year to an average of applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about % of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler , m. hermanns-clausen uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from to in total cases of human exposures to chlorine developed from mixtures of hypochlorite and acids ( . of cases) were registered. in cases the exposure was due to mixtures of products intended for domestic use. % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough ( %), dyspnea ( %), irritated upper airway ( %), abdominal discomfort (pain, nausea, vomiting) ( %), thoracic pain ( %), irritated eyes ( %), dizziness ( %), and bronchospasm ( %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in patients ( %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( % ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were patients in the same period exposed to hypochlorite evaporation alone. of them ( %) had symptoms of which in cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway ( %), nausea or vomiting ( %), cough ( %), irritated eyes ( %). dyspneoa was less fequent than in the mixture group ( %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh ax assay for genotoxic and nongenotoxic agents: comparison of h ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant - is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [ ] mürdter t, et al. hum mol genet, , : - [ ] nishiyama t. et al., biochemical pharmacology, , : - [ ] sun d. et al., drug metabolism and disposition, , : background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: bd patients ( / m/f, . ± . y, tbsa . ± . %) and patients ( . ± . y) from the icu were included in this observational study. blood samples were taken within the rd interval of the h-lasting dosing period of piperacillin/tazobactam ( / . g within . h) at , and . h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least xmic but preferably xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at , , and . h, were as follows: c max . ± . vs. . ± . mg/l, p< . , half-life . ± . vs. . ± . h, p> . , clearance . ± . vs. . ± . l/h, p< . , volume of distribution . ± . vs. . ± . l, p< . . free concentrations (which were included in tdmx calculations) were ± vs. ± % (p< . ) of total concentrations. duration per day while concentrations exceeded xmic ( . ± . vs. . ± . h, p< . ) or xmic ( . ± . vs. . ± . h, p< . ) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for xmic could be enhanced to . ± . h if the piperacillin amount will be increased to x g/d and the infusion duration to h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as > mg mtx/m bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years - were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. patients aged - years (male = , female = ) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, mtx treatment cycles corresponding to mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph < . , trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [ ] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [ ] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [ ] , we speculated that repression of gene expression as the predominant transcriptional response to ota [ , ] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [ ] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk- ) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h a, h b, h and h . we further selected acetylation at histone h lysine (h k ), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [ ] and which was reduced from ~ % in controls to < . % in response to ota, to establish a link between loss of h k acetylation and expression of genes consistently shown to be down-regulated in response to ota [ , ] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h k acetylation at promoter regions of the selected genes (% of controls: amigo : %, clasp : %, ctnnd : %). overall, these data provide first evidence for a mechanistic link between h k hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays ( most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to - % (average prediction accuracy was %). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [ ] [ ] started in and is a public-private partnership project within the european innovative medicines initiative (imi) [ ] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to week) repeated dose studies, mostly in rodent. overall more than study reports for approximately investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [ ] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [ ] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than active substances approved for being used in pesticides and over chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg , heparg, rat: h iie) and adrenal gland (human: h r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a -day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg , heparg, hek ). cytoxicity has been analysed by wst- and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp- as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd on thp- cells after incubation with these prohaptens (hennen et al., ) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp- with b[a]p and eugenol in coculture with hacat cells augmented cyp a and/or cyp b mrna levels, while this was not found for thp- monoculture. augmentation of cyp a mrna needed continuous presence of hacat cells. in coculture, levels of -oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of -oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp- cells by , -dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp- cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci : - .toxicology -toxic pathway analysis/aop background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel mg or prasugrel mg (n= ). adp-induced platelet reactivity was assessed by impedance aggregometry to minutes and day after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at min (r= . ; p< . ). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations min/day : p= . for clopidogrel, p= . for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so ) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc- and miapaca- cells in comparison to the non-target cell line nih t with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc- cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so by subcutaneous injection at the neck. after the tumor reached a diameter of to mm in size treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k ). complete blood count analysis was done by labor gmbh, berlin. results: the adenine release mediated by dianthin-egf was . pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic value of approximately nm for egf receptor expressing miapaca- and bxpc- cells as compared to nm for non-target nih t cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from pm to µm. toxicity studies in balb/c mice revealed . µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be % less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet % less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc- cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp a and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp a at different concentrations with and without nadph to determine the ic shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version . and admetsar. results: fractions (f -f ) indicated ic shift reduction ≥ post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = . ) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic = . µg/ml) across mdr -mdckii cells. niaziminin indicated . % bioavailablity via the human intestinal membrane with % chance of inhibiting cyp a . βsitostenone showed strong p-gp inhibition ( . %) with % absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp a or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than departments of all berlin hospitals from october until december . through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases ( . %). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient , pelargonium sidoides in patient ), and eight times as possible (valeriana in patients , , , , , mentha piperita in patient , hypericum perforatum in patient , eucalyptus globulus in patient ). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient ). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. ). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. , fig. ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil for one of our patients ( fig. & ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for % of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, ) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n= per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at °c throughout the experiment and electrical resistance barrier integrity testing was performed at the start ( h) and end of the experiment ( h). four test preparations at final bpa concentrations of . , , , and mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca %, v/v), uridine '-diphosphoglucuronic acid (udpga, mm) and '-phosphoadenosine- '-phosphosulfate (paps, µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration ( mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between . and . % of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between and % of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z and instead of the molecule ions (m/z and ) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were . ng/ml and . ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter (mct ), encoded by the slc a gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct and its epigenetic regulation by slc a promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct /slc a might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct protein expression was analysed in paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v. . (definiens ag). dna methylation in the slc a promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in paraffin embedded metastasis samples by maldi tof-ms. mct protein expression data and dna methylation at the specific cpg site in the slc a promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately % and % in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc a promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the '-regulatory region of mct may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) -hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in postmenopausal breast cancer patients of european origin who were treated with mg/day of tamoxifen for at least months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of premenopausal breast cancer patients mainly of european descent , . twelve snps close to or above genome-wide significance (p < e- ) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes , and for (e) metabolite e and to chromosomes and for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < % were confirmed as putative candidates: rs was associated with a - % variant allele-dependent increase of (e) and (z) metabolite e isomers (p< . ), and rs , mapping to a gene encoding zinc finger protein znf , was associated with increased risk of reccurrence or death (hr carriers . , % ci: . - . ; p < . ). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water ( : ) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from . nm to nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the % tolerance range for all analytes in all matrices. matrix effects were between - % (plasma) and - % (urine), apparent recoveries in plasma were above %. basal plasma ba level (mean ± sd) in fasting healthy subjects were ± nm (unconjugated bas), ± nm (glycine-conjugated bas) and ± nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were ± nm (unconjugated bas), ± nm (glycine-conjugated bas) and ± nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord- -mq h t authors: nan title: executive summary date: - - journal: allergy doi: . /j. - . .tb .x sha: doc_id: cord_uid: mq h t allergic rhinitis is now recognized as a major cause of morbidity that significantly impairs function and quality of life. moreover, it is now widely held that the pathophysiologic mechanisms causing nasal allergy contribute, or predispose many individuals, to the development of other airway diseases, including asthma. allergic rhinitis may well be a factor in % of children with otitis media with effusion (ome), and perhaps % of cases of chronic sinusitis. as many as % of persons with asthma aged to years have elevated serum ige antibodies to five common aeroallergens. in many instances, nasal allergy signals the presence of more severe disease. considerable evidence now suggests that early and appropriate intervention can improve the quality of life and productivity of patients with allergic rhinitis, enhance the academic performance of children, and reduce the prevalence of airway complications. the goal of treatment has shifted from mere symptom alleviation to blocking the pathophysiologic mechanisms that cause chronic allergic inflammation and leave patients vulnerable to airway infections. the earlier in a patient's life that this can be accomplished, the better the anticipated consequences. a panel of experts was convened in amsterdam, the netherlands, on september , to explore these issues and their impact on allergy prevention and treatment in primary care. their undertaking was supported by an unrestricted educational grant from schering‐plough pharmaceuticals. allergic inflammation appears to be the first and primary occurrence in the chain of events leading to asthma and other airway disorders. chemical mediators released during hypersensitivity reactions give rise to the symptoms of allergic rhinitis, and induce the cell infiltration and activation that results in chronic inflammation. a key event in this process is the upregulation of specific intercellular adhesion molecules, including members of the immunoglobulin superfamily intercellular adhesion molecule- (icam-l), that permits inflammatory cells to migrate into nasal, sinus, and lung tissue. in certain conditions, inflammatory mediators stimulate the expression of icam- , which is also a receptor for the subtype of human rhinoviruses that accounts for % of human rhinovirus infections. it is currently under investigation that allergen exposure and viral infection in the first years of life may alter pulmonary and immune function irreversibly in genetically predisposed children. * moreover, these same mechanisms may contribute to other respiratory problems associated with eosinophilic inflammation, lymphoid hyperplasia, mucosal edema, and viral infection. therapies that downregulate icam- are being investigated as a means of preventing or minimizing allergic inflammation. treatments presently known to downregulate icam- include antihistamines (loratadine, terfenadine, cetirizine, and azelastine). allergic rhinitis is associated with impairments in how patients function physically, emotionally, socially, and at work or school. adults may complain of role and activity limitations, frustration, sleep disturbance, irritability, embarrassment over symptoms, cognitive impairment, decreased alertness, and performance deficits. in children, chronic nasal allergies are associated with learning deficits. unfortunately, sedating antihistamines can exacerbate these effects by hampering performance and cognition even when subjects have no sense of being impaired. at least one of the newer nonsedating agents, by contrast, can partially reverse allergic rhinitis' effect on cognition and performance. nasal allergy may usually be differentiated from other forms of rhinitis by an allergy diagnosis. this includes personal history of symptoms, the timing of their expression, triggers, and physical examination. skin prick tests and eventually in vitro diagnosis of specific ige are important. imaging studies are usually indicated only if the diagnosis is in doubt, if related airway disorders complicate the presentation (e.g., nasal polyps), or when occupational rhinitis is suspected. the the impact of allergic rhinitis temporal association of nasal or ocular symptoms with the workplace strongly suggests occupational rhinitis. treatment of chronic rhinitis and nasal allergies should be individualized, with therapeutic measures aimed at the underlying etiology, likely pathophysiology, and dominant symptoms. medication is best taken prophylactically for seasonal symptoms before the anticipated onset of symptoms in seasonal rhinitis or for episodic exposures to specific ailergensand regularly, rather than as needed. such usage of antihistamines and intranasal steroids has been found to alleviate exacerbations in patients with both nasal allergy and seasonally induced asthma. avoidance of inciting factors (e.g., allergens, irritants) can reduce the expression of nasal symptoms and minimize the need for medications. the avoidance measures that are easiest to implement are the ones most likely to be used and therefore to be successful. antihistamines. although traditional agents have some benefits (e.g., low cost), they can no longer be recommended due to their potential for causing cns impairment. nonsedating anthstamines are the preferred option. oral decongestants. although topical steroids are the first-line treatments for nasal blockage, a-adrenergic agonists such as pseudoephedrine, phenylpropanolamine, or phenylephrine also ameliorate nasal congestion. this symptom is a major factor in perennial rhinitis. careful dosing of agonists is required to avoid aggravating hypertension. moreover, these agents generally should be avoided in patients with cardiovascular disease, thyrotoxicosis, glaucoma, or diabetes. oral corticosteroids. a brief course is indicated only to relieve severe nasal blockage so that topical therapy can proceed. in such cases, patients should be referred to an allergist. intranasal corticosteroids are highly effective in reducing inflammation, rhinorrhea, itch, and nasal blockage. they are the first choice for treating nasal blockage and largely reduce, but may not eliminate, the need for other medications to treat ocular symptoms. they may be used safely in children, though youngsters often have difficuity with sprays. in addition to treating nasal allergies, these medications can be used to treat nonallergic rhinitis with eosinophilia syndrome (nares) and to shrink small nasal polyps or prevent their recurrence after surgery. the onset of action of topical corticosteroids is slow compared to antihistamines. topical nasal decongestants may be used to open nasal passages in preparation for the use of other agents, such as intranasal steroids. to prevent rebound congestion, they should be used for to days and no more than to days. mast-cell stabilizers include intranasal cromolyn and nedocromil sodium. cromolyn may be used for the prophylaxis of allergic symptoms, and is considered especially safe for elderly patients, children, and pregnant women. the need for frequent administration can raise compliance issues. nedocromd sodium, unlike cromolyn, can both prevent an allergic reaction and control a reaction in progress. topical antihistamines allay allergen-induced itching, sneezing, rhinorrhea, and ocular symptoms but are less useful for nasal blockage. intranasal anticholinergics such as ipratropium bromide are effective in controlling the excessive watery rhinorrhea associated with neurogenic stimuli (e.g., cold air, spicy foods) or the common cold. however, they have no effect on nasal blockage, sneezing or itching, or ocular symptoms. patients with rhinitis or asthma caused by allergens for which the clinical efficacy and safety of sit have been documented by placebo-controlled, doubleblind studies, and those requiring daily pharmacotherapy for longer periods (e.g., preventive treatment during a pollen season or perennially) are candidates for sit. sit is most effective if a single allergen is identified, rather than multiple allergens. injections should be prescribed by specialists and given by physicians only if a specific allergen has been identified. sit should never be initiated in pregnant women, though the continuation of maintenance therapy is safe. considerable care must be taken if allergic rhinitis coexists with moderate or severe asthma. local immunotherapy offers improved safety and equivalent effectiveness. when patients respond poorly to standard medical care, clinicians should a) ascertain whether compliance has been poor, b) adjust drug doses, c) consider combination therapy, d) reconsider the diagnosis, e) reassess the patient for a nasal structural defect or a complication of allergic rhinitis, or f) refer the patient to a specialist. referral can be helpful under the following circumstances: in most cases when significant airway comorbidity is present (asthma, chronic sinusitis, nasal polyps, or otitis media with effusion) when the diagnosis is in question or special diagnostic testing is required when occupational rhinitis is suspected, to distinguish between clear-cut allergic reactions and toxic or nonallergic reactions when poor symptom control necessitates a consultation for environmental control measures, pharmacotherapy, or specific immunotherapy when medication side-effects are intolerable when rhinitis is only part of a complex series of mucosal allergies. allergic rhinitis is a seasonal or perennial disorder characterized by mild to severe upper respiratory symptoms such as nasal congestion, rhinorrhea, sneezing, and itching. these symptoms arise from an underlying inflammatory process initiated by a reaction between the allergen and immunoglobulin e (ige), neurogenic stimuli, and other complex cellular processes. a panel of experts was convened in amsterdam, the netherlands, on september , to explore these issues and their impact on allergy prevention and treatment in primary care. their undertaking was supported by an unrestricted educational grant from schering-plough pharmaceuticals. conclusions and recommendations from that meeting are summarized subsequently and presented in expanded form later in this monograph. allergens are primarily responsible for provoking chronic inflammatory processes; viral infections underlie acute exacerbations of asthma, chronic sinusitis, and otitis media with effusion. there is a growing awareness of how allergic rhinitisand some of the medications used to treat itcan affect patients' quality of life, work or school performance, and emotional well-being. besides physical symptoms, patients may exhibit fatigue, psychomotor sluggishness, irritability, and mood and cognitive disturbances ( - ). learning is impaired in children ( ), while some adults may report reduced productivity and concentration ( ). this combination of physical, emotional, and functional problems may diminish quality of life in both adults ( ) and adolescents ( ). moderate to severe perennial allergic rhinitis has been found to affect quality of life significantly compared to healthy subjects in eight of the nine dimensions of the medical outcomes study short-form health survey (sf- ) ( ). the prevalence of allergic respiratory disorders is high and is a burden on the health-care system: one study of patients in a london general practice found that nearly one in seven adults had allergic rhinitis ( ). by the age of years, % of children followed from birth in the tucson children's respiratory study had physician-diagnosed allergic rhinitis ( ). those who developed rhinitis before the age of were more likely to have asthma by age ( ). as many as % of persons with asthma aged to years have been shown to exhibit high levels of ige antibodies to five common aeroallergens ( ). of patients with chronic sinusitis, . % also had allergy in one study ( ). about - % of children with ome had allergic rhinitis ( ) or nasal allergy ( ). for comparison, about one-third of the general population has chronic sinusitis or ome. in general, the prognosis for patients with asthma or allergic rhinitis is mixed: only % of patients are cured, about % improve, % show no change, and % deteriorate over time ( ). medicine has begun to revise its traditional therapeutic approach to allergic rhinitis. it is now acknowledged that impeding the natural course of airway allergies by interfering with the pathophysiologic mechanisms that cause mediator release and chronic inflammation may help prevent related airway disorders. this would reduce overall morbidity and improve patients' quality of life. it also should help make patients less vulnerable to the consequences of viral infection, as well as to environmental factors such as pollution. recent advances in allergy treatmentsmost notably the introduction of nonsedating selective histamine hiantagonists and intranasal corticosteroid sprayshold the promise of achieving these goals. of particular interest are the studies showing that tgeatment of allergic rhinitis reduces the incidence and severity of asthma ( ) ( ) ( ) . the purpose of this monograph is to introduce new ways of thinking about and managing allergic rhinitis in the hope that physicians will look for related airway disorders and individualize therapeutic programs, considering tbe special needs of populations such as pregnant women, children, and the elderly. persons with allergic rhinitis have ige antibodies bound to high-affinity receptors on mast cells and basophils, and to low-affinity receptors on other cells, such as eosinophils, monocytes, and platelets. allergic inflammation is initiated when allergens deposited in the airway bind to ige molecules, causing cellular degranulation and releasing a number of inflammation mediators ( ) . this process entails both an early-and a late-phase rqsponse (fig. ) . the interaction of antigen with specific ige antibodies leads to the degranulation of mast cells and basophils and the prompt release of preformed and newly generated mediators, such as histamine, neutral proteases, leukotriene c,, prostaglandin d,, cytokines, and kinins ( ). the interaction of these chemical messengers with blood vessels, mucous glands, and nerves produces the symptoms of allergic rhinitis, such as sneezing, rhinorrhea, and itching. histamine is one of the most important mediators of the early-phase allergic response in the nasal mucosa. its release stimulates sensory nerves, this reaction, manifested by some patients, entails a process of cell infiltration and activation occurring over - h. late-phase allergic reactions begin when autocoid mediators and cytokines released from mast cells during the early-phase response upregulate the expression of leukocyte endothelial adhesion molecules in the postcapillary venules of the nose. especially sensitive to histamine, postcapillary venules are also the site of blood cell extravasation. once adherent to the endothelium, these cells pass between adjacent cells to the perivascular space. various chemoattractants, essentially il- and the chemokines, draw primed leukocytes (eosinophils, basophils, neutrophils, and mononuclear cells) into the submucosal tissue. once there, by interacting with additional stimuli by matrix proteins, they release their own mediators. this perpetuates the inflammatory response and augments aspects of the immediate hypersensitivity reaction, such as mucosal congestion and mucus secretion. although pruritus the impact af allergic rhinitis and sneezing occur, the major symptoms in latephase reactions are hypersecretion and congestion. the chronic inflammation caused by repeated allergen exposure lowers the threshold for other provocative stimuli reactions ( ) . as a result, allergic individuals react more strongly to a) low levels of the primary allergen, b) other allergens to which they are only mildly sensitive, or c) nonspecific "triggers", such as cold air, cigarette smoke, spicy foods, and strong chemical odors. a key event in chronic respiratory allergy is the upregulation of specific adhesion molecules that permit inflammatory cells (e.g., eosinophils) to migrate into nasal, sinus, and lung tissue. the first step is the experience of such molecules on endothelial cells and on activated circulating inflammatory cells. the accumulation of eosinophils in these tissues is injurious, and contributes to the pathogenesis of allergic rhinitis, sinusitis, and asthma ( - ) . in certain conditions, histamine is known to stimulate the expression of adhesion molecules on nasal epithelial cells of normal subjects ( ) . an important adhesion molecule required for effective cell recruitment in allergic disease is intercellular adhesion molecule (icam- ) ( ) . vascular inflammation induced in a primate madel of allergic sensitization and exposure ( ) ; b) on bronchial epithelial and on vascular endothelial cells in symptom-free asthmatics ( ) ; c) on epithelial cells of the nose and conjunctiva in patients with perennial and seasonal ( ) allergic r~n i t i s (fig. ) ; and d) on the nasal and conjunctival epithelium of patients with asymptomatic perennigl rhinitis when exposed to allergen, though not in cohabiting relatives or heqltby volunteers ( ) . besides its role in allergic inflammation, icam- serves as a receptor for the major subtype of humqn rhinoviruses; this subtype accounts for % of all human rhinoviruses ( , ) . rhinoviruses are a major cause of the common cold. evidence of minimal, persistent inflammation in asymptomatic allergic patients suggests that by inducing icam- , subclinical allergen exposure may incregse the susceptibility of allergic patients to rhinovirus infection, and thereby explain the greater frequency of colds in asthmatic children ( ) . in short, it now appears that both icam- and viral infection play a role in the patbophysiology of asthma (fin. ) . the epidemiologic link between viral respiratpry infection and asthma is. strong. researchers have shown that such viral infections are associated with: % of wheezing episodes that last longer than h in noqhospitalized children ( ) % to % of asthqa exacerbations in to -year-old children ( ) . among the viruses associated with childhood asthma exacerbations are rhinoviruses, respiratory syncytial virus (rsv), adenovirus, and coronaviruses ( ) . rhinovirus infection has been implicated in % of acute asthmatic episodes in schoolchildren, followed by coronaviruses in '% of cases ( ) . levels of eosinophil, a major basic protein in children with asthma, have been shown to be higher during exacerbations associated with rhinovirus infection than during asymptomatic periods adults with allergic rhinitis exhibit bronchial hyperresponsiveness to histamine and antigen following rhinovirus infection. rhinovirus infection also predisposes the allergic patient to allergenprovoked late asthmatic reactions ( ) . in asthmatics, but not in nonasthmatics, eosinophilia in the bronchial mucosa is still evident at - weeks after rhinovirus infection, probably due to ongoing secretions initiated by the virus in the allergenprimed mucosa ( ) . ( ) . rhinitis can be divided into three categories: allergici.e., acute or chronic conditions characterized by seasonal or perennial symptoms, or both infectiousi.e., nasal disorders caused by viral, bacterial, or fungal agents nonallergichoninfectiousi.e., a heterogeneous group of disorders comprising eosinophilic conditions, such as aspirin idiosyncracy; or noneosinophihc conditions, such as gustatory rhinitis, rhinitis of pregnancy, and vasomotor rhinitis. table catalogues the characteristic features of the various forms of chronic rhinitis ( , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite the considerable overlap, it is usually possible to diagnose the disorder by skin prick test and the patient's symptoms, history (including relevant behavioral practices, such as smoking), and physical examination. the elements of a diagnosis include elicitation of a detailed patient history and physical examination of the nose, eyes, ears, and lungs. among the many factors that must be explored are patterns of symptom expression, environmental triggers, medication use, family history, and exposure to workplace allergens or irritants. it is important to determine the severity of symptoms and to uncover possible complications such as asthma, sinusitis, otitis media, or nasal polyps. questions related to psychosocial or quality-of-life problemssuch as fatigue or cognitive impairmentalso should not be overlooked. the differential diagnosis of rhinitis primarily depends on use of an allergy skin prick test. pharmacologic agents that may affect results should be withdrawn for a sufficient time before testing ( table ) . because of its long duration of action (approximately - days), the antihistamine astemizole can suppress skin test reactivity for - weeks after its discontinuation ( ) . by contrast, this effect lasts only - days with most other antihistamines. most patients who present with rhinitis exhibit clusters of symptoms that define them as either "sneezers and runners" or "blockers". those with seasonal allergic rhinitis are usually sneezers and runners. some patients with rhinitis have only a single symptom or many nonnasal complaints, such as headache, sore throat, postnasal drip, a "full" or a "stuffy" head, recurrent head colds, chronic "sinusitis", chronic cough, plugged ears, hyposmia, loss of the sense of taste, fatigue, or poor concentration. often, patients use the term "sinuses" or "sinusitis" to describe symptoms caused by nasal pathology. a thorough patient history yields information needed to classify nasal symptoms, determine their cause, and make appropriate therapeutic decisions. family studies indicate that environment generally influences the expression of allergic disease (ll), but genetics determines the severity and specificity of the symptoms ( ) . genetics also is an important component of atopy as a general predisposition, involving multiple as yet unidentified genes. children not only inherit a tendency to atopy from their parents, but are likely to develop the same allergic disorders ( , ) . consequently, the following information should be sought: symptom type, occurrence, and frequency symptom duration and severity aggravating factors current medications illicit drugs. the age at which symptoms began (episodic, seasonal, or perennial) allergen exposure in home or work environment although the physical examination of patients with chronic rhinitis centers on the nose, eyes, and throat, clinicians also should evaluate the ears, sinuses, and lungs to identlfy any related airway disorders, which often are present. general signs of an atopic predisposition, such as eczema, also are helpful. imaging studies are usually indicated only in specific circumstancesmost commonly, when the diagnosis is in doubt, when symptoms persis despite appropriate therapy, or when related airway disorders complicate the diagnosis. persistent symptoms or airway complications may be factors in perennial allergic rhinitis. plese.g., limiting outdoor activities during the height of the pollen season, shielding young children from passive tobacco smoke and gas heating or wood-burning stoves. they also may entail fairly complex undertakings aimed at specific allergens; e.g., cat dander. as people spend increasing amounts of time indoors, environmental control is focused largely on containing exposure to house dust, animal dander, mold, and cockroaches. as a rule, measures to avoid allergens are effective when they are likely to be usede.g., simple to follow and inexpensive to implement. patients should be informed that even partial compliance can help control symptoms and lessen the need for medicalion. they should be encouraged to persevere, as it may lake weeks or months of avolddnce before an improvement in sympfoms is noticeable. (see appendix for a patient information sheet about allergen-control measures.) the selection of an appropriate and effective medication for the treatment of chronic rhinitis entails consideration of several factors: although impaired olfaction is frequently overlooked in cases of nasal allergy, it is relatively easy to assess by olfactory threshold tests. patients who require more sophisticated testingsuch as assessment of mucociliary function or nasal airway patencyshould be referred to a specialist. underlying eriology. especially in patients for whom immunotherapy is being considered, this requires careful diagnosis of the allergen (s), primarily through skin prick testing. a likely parhophysiology. symptoms due to inflammatory processes (as in nasal allergy) require differed medications than those caused treatment options consist of allergen avoidance, pharmacologic therapy, or allergen-specific immunotherapy. efforts aimed at environmental control may involve relatively simple activities based on general princi-by-noninflammatory neurogenic mechanisms (as in gustatory, idiopathic, or atrophic rhiniiis). dominant symptoms. medications should address the patient's most prominent complaints. combination therapy may be warranted for patients with a mix of moderately severe lo severe symptoms. safety. it is important determine whelher medication side-effects may impede a patient's perfarmance at work or scho , diminish quality of life, or enhance risk of sustairiing personal the impact of allergic rhinitis table withdrawal of pharmacologic agents that affect skin test reactions ( ) individuals. regular antihistamine use over weeks or months has been found to reduce asthma symptoms significantly in allergic rhinitis patients with seasonal and chronic rhinitis ( ) ( ) ( ) . injury or significant morbidity. the potential for drug interactions also must be considered when patients are taking multiple medications. patient age and other special considerations. the needs of special populationspediatric patients, the elderly, pregnant women, and competitive athletes must be evaluated. coexistence of related airway disorders. in patients with coexisting airway disease, the treatment of nasal allergy is important to prevent exacerbation of sinusitis or asthma. patient preferences and compliance historv. in some countries, patients prefer oral medications to sprays ( ) . additionally, individuals differ greatly in their capacity to adhere to therapeutic regimens. consequently, an attempt should be made to determine each patient's understanding of abd willingness to comply with a specific treatment program. factors that may adversely affect compliance include poor symptom relief, the nature or severity of the side-effects, and the inconvenience or complexity of the therapeutic rkgimen ( , ). as a rule, it is best for patients to start therapy before the anticipated onset of symptoms, to suppress those immunologic and mediator mechanisms that cause inflammatory reactions and to minimize the priming effect. prophylactic therapy is usually possible in seasonal allergic rhinitis, when the onset of symptoms is relatively easy to predict, or for episodic exposures to specific allergens (e.g., before visiting the home of someone with pets). as a rule, patients with seasonal or perennial rhinitis should take their medication regularly, rather thhn as needed, because consistent use best controls mucosal inflammation. in turn, this helps lessen the risk of related airway complications in susceptible in its consensus statement, the international rhinitis management working group advocated a stepwise approach to therapy that took into account specific diagnoses and patient characteristics ( ) . this approach is summarized in table . stepwise management certainly represents a reasonable starting point for therapeutic decisions in cases of chronic rhinitis. nonetheless, the complexity and interrelationship of these disorders suggest that, while taking account of the guidelines, a case-by-case approach provides added value. what follows is a review of the benefits and limitations of various medications used to treat chronic rhinitis. among the agents considered are topical and oral antihistamines, topical and oral corticosteroids, topical and oral decongestants, crornolyn sodium, nedocromil, sodium intranasal anticholinergic agents, and saline sprays. the discussion concludes with a review of combination drug therapy and the ihdications for immunotherapy. there are at least three classes of histamine receptors, designated hi, h,, and h,. histamine can exert local or widespread effects on smooth muscles and glands. bronchoconstriction and contrktion of the intestine are mediated by h,-receptors, while gastric secretion results from h,-receptor activation ( ) . h,-receptors appear to exist predominantly in the central nervous system (cns) and at presynaptic nerve endings ( ) . histamine causes capillary dilation; hi-receptor stimulation leads to a rapid, brief dilator response, while h,-receptors mediate a response that is slowkr to develop and more sustained ( ) . it also plays a role in extravascular smooth-muscle contraction and (more rarely) relaxation, with h,receptor activation responsible for contraction and h,-receptor stimulation usually resulting in relaxation ( ) . the class of agents known as hi-receptor antagonists comprises drugs that act rapidly to antagonize the histamine activity of hi-receptors, thereby relieving the maiq symptoms of allergic rhinitis (sneezing, watery rdnorrhea, and itching of the ndse, eyes, and palate). in addition, some of the hewer agents have been shown to possess antiallergy properties that may contribute to diagnosis and treatment. these properties include the ability to: inhibit histamine release from human basophils block histamine, and partly block prostaglandin d, release, in the nasal secretions of highly allergic subjects ( ) directly inhibit eosinophil activation ( , ) block histamine activation of airway epithelial cells by suppressing the expression of surface markers, such as icam- , and the major histocompatibility complex class i antigen hla-dr, involved in antigen presentation ( ) . the ability both to inhibit histamine release and downregulate icam- makes certain anthistamines (e.g., terfenadine [ , ] , loratadine [ ] , cetirizine [ ] , azelastine [ ] , oxatomide [ ] , and levocabastine [ ] particularly suitable for treating allergic inflammation, because icam- is a marker of allergen-induced inflammation as well as a receptor for human rhinoviruses ( , ) . ( , reduce vascular permeability ( ) sedating vs nonsedating antihistamines. "sedating" agents are distinguished from "nonsedating" ones by the higher incidence of drowsiness associated with the former at the doses used to control allergic ,symptoms. in part, this difference is due to the speed with which antihistamines cross the bloodbrain barrieri.e., rapidly in the case of the sedating agents and slowly in the case of the nonsedating agents. the sedative potential of the newer agents is limited further by their specificity for binding to peripheral hi-receptors. sedating antihistamines, in contrast, affect central hi-receptors and receptors of other types (e.g., muscarinic cholinergic, a-adrenergic, and tryptaminergic) ( ) . this may contribute to their sedative and other side-effects ( ) . sedating agents' effect on central hi-receptors may be especially relevant, as hstaminergic transmission originating from the posterior hypothalamus sustains waking arousal ( ) . in short, by having a reduced capacity to cross the blood-brain barrier and specifically blocking hireceptors, the nonsedating agents are less able to cause cns effects and associated adverse reactions. (see subsequent discussion of cns effects.) efficacy. table presents selection criteria for of the antihistamines most commonly used in europe. studies have shown that the newer nonsedating antihistamines produce moderate to the impact of allergic rhinitis excellent symptom response in up to % of patients with seasonal allergic rhinitis ( , making them as effective as, if not more effective than, the older (i.e., sedating) agents ( , ) . as a group, the newer agents demonstrate comparable efficacy ( , ) . a comparison of loratadine and astemizole in patients with seasonal rhinitis found equivalent efficacy in the two ( ) . cetirizine has been shown to lessen nasal blockages ( ) . although the use of antihistamines in asthma is still controversial, the new nonsedating agents seem to offer some benefit for allergic patients with concomitant asthma. data indicate that terfenadine ( , ) and loratadine alone ( ) , and combined with pseudoephedrine ( ) , astemizole ( ), certirizine ( ) , and azelastine ( ) exhibit modest bronchodilating effects ( ), reduce bronchial sensitivity to histamine ( ) , and guard against exercise-( ) and antigen-induced bronchospasm. such effects may require higher than currently recommended doses, however. loratadine ( . , cetirizine ( ), and terfenadine ( ) have been shown to reduce asthma symptoms in rhinitic patients with seasonally induced asthma when taken over weeks or months, though this evidence is disputed. together, such findings suggest that timely and appropriate use of antiallergy therapy may help prevent lower airway symptoms in susceptible patients if used in an adjunctive manner. moreover, they dispel the fallacy that antihistamines are contraindicated in asthmatic patients. cns effects. sedating antihistamines can exacerbate the cognitive and functional problems caused by untreated allergic rhinitis ( ) . among their most troubling cns effects are fatigue, sedation, and performance impairment ( ) . patient reports of unusual daytime drowsiness have been noted in - % of users of sedating antihistamines ( ) -e.g., diphenhydramine, pyrilamine, brompheniramine, chlorpheniramine, triprolidine, hydroxyzine, promethazine, and cyproheptadine. by contrast, when given at the recommended doses, the "nonsedating" antihistaminesastemizole, ebastine, fexofenadine, loratadine, and terfenadinecause no more drowsiness than placebo. confusion exists as to the sedating properties of two new agents, acrivastine and cetirizine. both medications have been associated with significantly more reports of daytime drowsiness than placebo at recommended doses: acrivastine, mg, % versus % ( ); cetirizine, mg, % to % versus % ( ) . although the lower dose ( mg) of cetirizine is no more sedating than placebo ( ) , it has been shown that, if allowed, more than % of cetirizine users will titrate the dose upward, to achieve symptom relief ( ). after repeated dosing, some patients may become tolerant of the sedating effects of antihistamines ( ) . however, it is unwise to depend upon tolerance to abolish the antihistamine- induced sedation and performance impairment that commonly occurs for at least week after the medication is started ( ) . it is generally agreed that tolerance is a highly idiosyncratic phenomenon that is not universal, complete, or predictable. virtually all antihistamines are equally effective in relieving allergy symptoms (see "efficacy" section). consequently, before recommending a particular antihistamine, physicians must weigh the benefits of that agent against its drawbacks. the lower cost of the older "sedating" antihistamines, and their general availability without prescription, may argue in their favor, but their use can no longer be recommended. the nonsedating agents offer the potential long-term benefits of safety and improved performance at work or in school compared to sedating antihistamines, and greater therapeutic compliance owing to their longer half-life and the need for less frequent dosing ( , ) . - ' consequences of cns effects. unfortunately, one's subjective sense of sleepiness or impairment is an unreliable gauge of whether it is safe to operate heavy machinery while taking sedating antihistamines. this is because impairment can occur without the patient's awareness ( , ) . these factors have important safety implications in areas such as building consltuct ion, manufacturing, pub ic transpottation, and automobile driving. simulated and real-life driving studies verify that these agents carl impair performance significantly ( , , ) . in one study, lane weaving by drivers given triprolidine mg (emtamed release), was comparable to that ib pers hs with a blood alcohol level of . %; moreover. at - h after drug use, the drivers stopped feporting any sense of being impaired ( ) (fig. ) . another study found that the effects of a single -mg dose of cetirizine resembled those of alcohol ( g/kg lean body mass), and that the impairment appeared to be greater when alcohol and cetirizine were taken together ( ) (fig. ) . by contrast, no psychomotor deficits have been observed in drivers given loratadine ( , ) , or in individuals tested while taking astemizole ( ); nor does the use of alcohol with these agents produce an additive effect. studies have also shown that terfenadine did not impait psychomotor performance or reaction time ( ) , and that ebastine at , , or mg given for days did not impair driving performance compared to triprolidine mg ( ). besides causing psychomotor problems, sedating antihistamines also adversely affect cognition and functional performance ( , , , ), compromising sustained attention and cerebral processing speed in adults ( ) and children ( ) . the effects of nonsedating agents on somnolence, cognition, and performance, by contrast, are comparable to those of placebo ( , , , ) . specific findings to date include the following: performance on a digit symbol substitution test impaired by diphenhydramine ( mg t.i.d.), but not by loratadine ( mg q.d. and mg visuavmotor coordinationimpaired by promethazine, chlorpheniramine, and clemastine, but not by terfenadine ( , ) clerical skills - skills impaired by diphenhydramine; none by loratadine ( ) learning in children with seasonal rhinitisimpaired by diphenhydramine ( mg) compared to nonallergic, unmedicated children, with the impairment in allergic children offset partly by loratadine ( mg) ( ). nonallergic, unmedicated children performed significantly better on tests of learning ability than atopic children given placebo, diphenhydramine, or loratadine; performance among atopic children was q.d.) ( ) the impact of allergic rhinitis best among subjects receiving loratadine and worst among those receiving diphenhydramine. this suggests that the learning problems associated with allergy are augmented by sedating antihistamines and offset somewhat by nonsedating agents. cardiovascular effects. two nonsedating agentsterfenadine and astemizolehave been found to cause malignant ventricular arrhythmias, such as torsades de pointes ( , ), when patient factors or drug interactions result in greatly elevated serum drug levels ( , ). although rare, these phenomena can delay cardiac repolarization and prolong the qt interval through blockage of a k rectifier channel, resulting in possible sudden death. at least case reports involving patients have been published in which patients taking astemizole experienced torsades de pointes, qt prolongation, and sometimes other cardiac effects ( ) . eight such reports affecting patients have appeared for terfenadine ( ) . acrivastine, cetirizine, fexofenadine and loratadine appear to be the least likely of the newer antihistamines to be arrhythmogenic ( ) . the risk of cardiac arrhythmias can be minimized by reviewing the medical and pharmacologic risk factors for torsades de pointes before prescribing terfenadine or astemizole for any individual. drug interactions associated with the potential for cardiotoxicity are a major concern of clinicians, as patients frequently fail to report all of the medications they are tdking. concomitant administration of certain antifungal agents (especially ketoconazole and itraconazole) or macrolide antibiotics (patticularly erythromycin), along with terfenadine or astemizole, inhibit metabolism of these two antihistamines through competition for hepatic p enzymes. this results in increased plasma cohcentrations of these antihistamines. coadministratiori of terfenadine or adtemiztde in users of such medications is thus sttictly contraindicated. hepatic dysfunction and preexisting cardlovascular disease also ate risk factors for cardiovascblar complication$ with these ztrltihfstamines. because the causes of qt prolongation are additive ( ), patients with more risk factors have a potentially greater chance of developing ventricular arrhythmias while taking either terfenadine or astemizole. the use of these drugs at recommended doses without the aforemefitioned concomitant medications by patiedts lacking known rtsk factors carries a low risk of ah adverse catdiac event. still, such low-risk patients taking terfenadine or &temizble should be warned not to inmease antihistamine doses without !dnsulting their physician. cytochrome p enzymes are responsible for the metabolism of many drugs. all of the currently available nonsedating antihistamines are metabolized by the cytochrome p hepatic enzyme system. cytochrome p a is involved in the metabolism of terfenadine, and erthromycin attenuates the metabolic effect of cyp a . loratadine is metabolized primarily by cyp a and secondarily by cyp d . other considerations. side-effects involving the digestive tract (e.g., loss of appetite, nausea, vomiting, epigastric distress, and constipation or diarrhea) are among the most common for all antihistamines. many of the older agents are associated with significant anticholinergic or antimuscarinic sideeffects, such as dryness of the mouth and respiratory passages, urinary retention or frequency, palpitation, blurred vision, hypotension, headache, and tingling or weakness of the hands ( ) . use of astemizole can cause weight gain. in a comparative trial with loratadine, weight gain was significantly greater with astemizole (pco. ) ( ) . this sideeffect may argue against astemizole's use in selected individuals. nasal blockage is a major factor in perennial rhinitis, and a possible incitement to related airway disorders such as ome and chrohic sinusitis. topical steroids are the agents of choice for nasal blockdge (see subsequent section, "intranasal steroids"); however, leukotriene antagonists or oral decongestants also control this symptom. examples of the last class of agents include pseudoephedrine, phenylpt-opanolamine, and phenylephrine. these a-adrenergic agonists constrict blood vessels supplying the nasal mucosa, thereby decreasing congestion; they do not control the itchihg, sneezing, or rhinorrhea associated with upper-respiratory allergies. when such syifiptoni coexist with significant blockage, optimal rhariagement requires the use of intranasal steroids. another option is a combination decongestant/antihistamine medication (see next section). a-adrenoreceptor agonists are stimulatory agents that constrict vascular beds throughout the body, not just in the nose. this raises the potential for significant systemic side -effectse.g., hypertensibn, palpitations, tachycardia, and extrasystole, as well as restlessness, insomnia, and headache. as side-effects are dose-related, clirliciahs must choose the ptescribed dosage carefully to prevent aggravating or "unmasking" hypertension. patiefits shobld the impact of allergic rhinitis be cautioned against increasing dosages on their own. as a rule, oral decongestants should be avoided in patients who have cardiovascular disease, thyrotoxicosis, glaucoma, or diabetes. medications that combine an antihistamine with a long-acting oral decongestant offer another option for managing nasal blockage. the value of this practice has been proven in trials using formulations containing pseudoephedrine and either loratadine ( ), acrivastine ( ), or chlorpheniramine ( ) . such combinations may be especially beneficial for patients at risk of chronic, allergen-induced eustachian tube obstruction, a condition that may favor the development of ome ( ) . combined antihistamine/ decongestants can provide greater overall symptom relief than possible with either agent alone ( , ), though the tradeoff may be a higher incidence of adverse events ( ) . nevertheless, combination agents decrease the number of drugs patients must take, a fact which may facilitate therapeutic compliance in selected individuals. oral corticosteroids and systemic injected depot preparations carry a high risk of systemic sideeffects. consequently, the only justification for their use in chronic rhinitis is to improve nasal patency in patients with severe or urgent blockage, so that topical therapy can proceed. even then, systemic steroids should be given only by a specialist, and only to patients who lack contraindications to their use. these agents generally should not be used in children or pregnant women. a course of systemic steroids may be needed to gain control of nasal polyps, after which the patient can be managed with topical steroids ( ) . in addition, oral corticosteroids can rapidly relieve anosmia associated with polyps and, by opening the nasal passages, facilitate the access of locally acting nasal corticosteroids. alternate-day dosing may improve the riskbenefit ratio of oral corticosteroids, but studies proving this point have yet to be undertaken. various topical agents are now available for relieving the symptoms and, in some cases, addressing the underlying pathology, of chronic rhinitis. the benefits, appropriate usage, and drawbacks of specific agents are discussed in this section. general limitations of topical treatments are reviewed in table . the value of any topical agent in the treatment of allergic or nonallergic rhinitis syndrome may be limited by several factors. physical anomalies. severe septal deviation or enlarged turbinates may impede direct delivery of topical agents to the nasal mucosa. poor therapeutic compliance. compliance studies have shown that patients generally prefer taking pills to using medicinal sprays ) slow therapeuric onset. in the case of intranasal steroids and crornolyn sodium. a slower onset of action may leave some patients frustrated and noncompliant. par!ent handicaps in particular. children and elderly persons may find it difficult to aim sprays or manipulate inhalers. intranasal steroids can relieve most of the symptoms of seasonal and perennial allergic rhinitis ( ) ( ) ( ) ( ) ( ) ( ) . they are more effective than cromolyn sodium ( ). intranasal steroids are probably the most effective drug for all symptoms but they are particularly effective on nasal blockage topical steroids are the agents of choice for relieving nasal blockage. they permit direct delivery to the nasal mucosa, thus minimizing systemic sideeffects. the following specific actions are noteworthy: inhibition of both the early and late hypersensitivity response to allergens ( ) reduction of the number of eosinophils in the nasal mucosa ( , ) substantial inhibition of the nasal secretory response ( ) reduction of mast cells and t cells desensitization of nasal irritant receptors ( ). ( - ) . appropriate use. intranasal steroids may be used to: relieve seasonal or perennial allergic rhinitis, when the main complaint is moderate to severe nasal blockage treat nares (only effective therapy at present) shrink small nasal polyps, or manage patients with polyps once control is gained through a course of oral steroids ( ) prevent polyp recurrence after surgery reduce the risk of chronic sinusitis, ome, or bronchial asthma in allergic patients with a history of these complications. drawbacks. side-effects of intranasal steroids are generally minor and local, e.g., irritation, burning, and reactive sneezing, which usually disappear after several days; and nasal dryness followed by blood-stained crusts ( ). in rare cases, treatment has to be stopped due to epistaxis. a few reports of septal perforation following intranasal steroid use have been published ( , ). some adverse effects (e.g., mucosal drying) can be avoided by using sprays with an aqueous base. the risk of systemic side-effects is very small, with one published report of a posterior cataract after intranasal steroid use and one report of children's growth being inhibited while taking an adult dose of budesonide ( ). it is prudent to use the lowest efficacious dose in children and pregnant women ( ). once-daily usage probably is sufficient for all glucocorticoids if the nose is patent and dry when the spray is used ( ). some patients or agents may require multiple daily dosing, however, or multiple sprays per nostril. patients should be informed that onset of action is delayed compared with that of antihistamines and decongestants, and requires a few days for patients with seasonal allergic rhinitis ( ). many patients are reluctant to use steroids in any form, and parents may be especially hesitant to sanction their use in children. individuals should be reassured that intranasal steroids are much safer than oral corticosteroids and that the former should not be equated with the latter. this anti-inflammatory agent has been proved safe and effective for the treatment of allergic rhinitis ( , ), although in one comparative study ( ) it proved less effective than intranasal corticosteroids. owing to its control of both early-and late-phase hypersensitivity responses, cromolyn can relieve nasal itching, sneezing, hypersecretion, and nasal blockage. its need for frequent administration (four to six times daily [ ], with possible reduction to two to three times daily in periods of low antigen load [ ]), may impede therapeutic compliance. appropriate use. cromolyn sodium is best used for the prevention of allergic symptoms. given its wide safety margin, it often is prescribed for elderly patients, children, and pregnant women with seasonal or perennial allergies. it has virtually no utility in nonallergic rhinitis syndromes. the ocular formulation of cromoglycate is very effective in controlling eye symptoms. like cromolyn, nedocromil sodium controls both the early-and late-phase allergic reaction. unlike cromolyn, nedocromil can both prevent an allergic reaction and control a reaction in progress ( ). nedocromil has significantly improved symptoms of rhinorrhea, congestion, itching, and sneezing ( ). it is about times more potent than cromolyn sodium. recommended dosing is two to four times daily ( ). appropriate use. like cromolyn, nedocromil sodium is very effective at blocking symptoms when used immediately prior to an anticipated allergen exposure ( ). both nasal challenge studies ( , ) and those involving seasonal exposures ( ) indicate that topical antihistamines can inhibit allergen-induced nasal symptoms, including sneezing and rhinorrhea. side-effects are locale.g., stinging and itching in the nose and, occasionally, the throatwith the incidence ranging from % to % of patients ( - ). azelastine has been shown to lessen nasal blockage, although reported side-effects include burning in the nose and altered taste ( ) . the lack of major improvement in nasal blockage ( , ) in some other studies, however, suggests that congestion entails more than the direct effect of histamine on receptors on the capacitance vessels of the nasal mucosa. levocabastine is a potent antihistamine available as nasal spray and eye-drops. minute amounts applied topically are sufficient to result in an antiallergic effect. appropriate use. on the basis of their safety profile, topical antihistamines may be used to relieve ocular or nasal symptoms in patients with mild to moderate seasonal allergies. when patients have severe ocular symptoms, specific topical preparations for ocular application should be used. azelastine ( ) and levocabastine ( , ) , for example, have been found to be nonsedating. topical nasal decongestants constrict the smaller arterioles of the nasal passages, thus reducing blood flow and relieving mucosal edema. these agents can relieve nasal congestion rapidly and effectively, and generally are available without a prescription. this probably explains their popularity among patients with perennial forms of allergic or nonallergic rhinitis. unfortunately, the overuse of decongestant sprays can cause rebound nasal congestion and rhinitis medicamentosa. moreover, like oral sympathomimetics, topical sprays are associated with systemic side-effects such as cardiac arrhythmias and exacerbation of hypertension ( ) . thus, the impact of allergic rhinitis patients who overuse decongesting nose-drops should be assessed for hidden allergic nasal disease. appropriate use. the primary use for topical decongestants in chronic rhinitis is to open severely blocked nasal passages, so that slower acting drugs (e.g., cromolyn or intranasal steroids) can reach the necessary nasal passages to provide ongoing symptom control. their potential for causing rebound congestion is such, however, that these agents should never he used for inore than consccurive days. ipratropium bromide, an anticholinergic drug, acts on the glandular cholinergic receptors in the nose to control excessive watery rhinorrhea ( ) . rhinorrhea generally improves within min, and remains in check for s- h. "here is no improvement, however, in nasal blockage, sneezing, or itching. nasal dryness, the main local side-effect of topical anticholinergics, may be alleviated by adjusting the dose. although rarely required, high doses of these agents may cause systemic side-effects. appropriate use. intranasal anticholinergics may be used to relieve the excessive rhinorrhea associated with allergic or nonallergic rhinitis syndromes e.g., gustatory or idiopathic rhinitis ("skier's nose"). the latter, perennial form of rhinitis entqils little if any nasal blockage, and tends not to respond to other treatments. intranasal anticholinergics also may help relieve the rhinorrhea associated with common colds ( ) . various symptoms of perennial rhinitise g , nasal stuffiness, sneezing, rhinorrhea, and nose-blowing may improve with the use of saline or propyleneand-polyethylene glycol sprays. in one trial, patients also showed decreased eosinophil counts with propylene-and-polyethylene glycol spray ( ) . symptom amelioration also may be accompanied by an improvement in nasal airway resistance and nasal biopsy findings ( ) . although wetting agents act slowly, their lack of side-effects makes them attractive therapeutic options for some patients. appropriate use. wetting agents are indicated for the relief of mucosal irritation or dryness, the prevention of mucosal atrophy, and the removal of encrusted or thickened mucus. they also may be used immediately before intranasal steroid dosing, to prevent or mitigate drug-induced local side-effects. when the use of any of these medications results in poor control of chronic rhinitis, physicians have several options: determine whether there has been therapeutic noncompliance. if so, try to ascertain the reason e.g., poor symptom control, inconvenient dosing schedule, high medication cost. once the cause is known, engage the patient in therapeutic decision-making, as this may enhance treatment satisfaction and compliance. two or more antiallergy medications may be prescribed if a single agent fails to relieve adequately a patient's symptoms. combination therapy generally is indicated in the following situations. w h e n syrnptonis are moderately severe, severe, or diverse patients with severe nasal symptoms often benefit from taking both an antihistamine/decongestant and a topical nasal steroid. when patients have severe ocular symptoms, specific topical preparations for ocular application should be used. in a study comparing solo and combined drug use in patients with seasonal rhinoconjunctivitis, juniper et al. found that a ) nasal symptoms responded better to beclomethasone alone than to astemizole monotherapy, b) ocular symptoms were relieved more effectively by asteniiznle alone than by beclomethasone monotherapy, and c) the combination provided the best relief of ocular symptoms ( ). when symptoms are intense or long-lasting patients who suffer from perennial rhinitis with seasonal exacerbations may benefit from using intranasal steroids on a continual basis and employing "rescue" antihistamines as needed. persons with allergic rhinitis and asthma might require a nasal corticosteroid and a bronchodilator to control their upper and lower airway problems. reliance on specific immunotherapy (sit) varies from country to country. physicians in the south of europe are more inclined to offer sit, while a decreasing number of patients receive this therapy in scandinavia and the uk ( ) . immunotherapy used to be viewed as indicated only for patients responding inadequately to, or developing side-effects from, drug therapy ( ) . current recommendations are to consider sit for a broader range of patients (see table ). sit, unlike pharmacotherapy, modifies the immune system and plays a preventive role in asthma development. it is used in conjunction with drug therapy but should reduce the need for symptomatic pharmacotherapy. the efficacy of sit for table specific immunotherapy for allergic rhinitis ) grass pollen, ragweed pollen, and house-dust mites is very well established ( , ) . results are best when the patient has a single allergic sensitivity rather than allergies to multiple substances. it must be stressed, however, that sit should be undertaken only by physicians working in facilities equipped to handle possible adverse reactions (urticaria, laryngeal edema, bronchospasm, and anaphylaxis). although its safety under these conditions is good, a careful riskhenefit analysis is required for each patient before injections commence. some experts believe that children may be especially responsive to sit ( ) . although childhood allergies show a strong tendency toward spontaneous improvement, sit may hasten this effect in a greater percentage of case% disagreement exists, however, about the age at which sit may be started ( , ) . noninjective immunotherapy is another option to consider. nasal, sublingual, and oral immunotherapy have been demonstrated to be safe and effective ( ) . perhaps the most enmuraging thing about sit is that it holds out the possibility at present, more than conventional pharmawlogic therapyof "curing" seasonal allergies and even preventing the progression to asthma ( , ). children a variety of genetic and environmental factors favor the development of respiratory allergies in children (table ) . allergy should be considered in the differential diagnosis when children have asthma, ige levels exceeding iu/ml at age , or a maternal history of allergy ( ). these were found on logistic regression to be risk factors for development of allergic rhinitis by age in the tucson children's respiratory study, which followed healthy children from birth to age ( ). presence of dogs as household pets and serum ige exceeding iu/ml were significant risk factors for development of atopic (vs nonatopic) allergic rhinitis ( ). wheezing in infancy usually implies no increased risk of asthma or allergies, but for the minority ( %) of children in whom wheezing persists at age , this symptom may signal a predisposition to asthma ( ) . a prospective study following children from birth to age years found that risk factors for persistent wheezing were elevated serum ige at months old and a maternal history of asthma ( ) . preventive therapy may be especially relevant for these children. special concern is also indicated for those with severe or persistent nasal polyps. such children should be referred to a specialist, as polyps in the very young may be associated with cystic fibrosis or primary ciliary dyskinesia. environmental control assumes particular importance in children with allergic rhinitis, as considerable evidence shows that chronic respiratory symptoms are associated with significant early exposure to allergens ( , ). the tucson children's respiratory study of -year-olds found that % had allergic rhinitis, and half of them had been diagnosed with this condition in their first year of life ( ). accordingly, it is important to instruct parents about environmental control measures and the need to.implement them early in the course of their child's disease. early avoidance not only improves symptoms, but also may affect the natural history of nasal allergy and related disorders such as ome. moreover, if implemented aggressively at home, it has the added benefit of possibly preventing similar allergies in any siblings that the patient may have. when drug therapy is needed, a conservative approach to selection is warranted. nonsedating oral antihistamines are a good choice for children, owing to their lack of cns side-effects and ease of use. moreover, once-or twice-daily administration elunnates the need for dosing during school hours. pediatric formulations of the nonsedating antihistamines generally are available throughout europe. cromolyn sodium, nedocromil, or intranasal steroid sprays, although safe and effective, may be difficult for small children to use. this can lead to compliance problems. moreover, cromolyn must be dosed at least four times a day, whereas nedocromil is used twice a day. intranasal steroids usually are given at half the adult dose, once daily in the morning. as a rule, systemic steroids and topical vasoconstrictors should be avoided in very young children. perennial rhinitis in the elderly is generally idiopathic in nature ( ) , with the underlying cause being autonomic imbalance, alteration in muscarinic receptors, or the sequelae of previously treated nasal disorders. almost invariably, the only symptom of nasal hyperresponsiveness in older persons is profuse rhinorrhea. treatment with ipratropium bromide, an anticholinergic drug, has been shown to be effective in some cases ( ) . for elderly persons with true allergic rhinitis, treatment plans must take into account age, concomitant illnesses, and the use of other pharmacologic agents. nonsedating topical or oral antihistamines are a reasonable choice for this population. older antihistamines are best avoided, not only because of their sedative properties, but also because of the potential for urinary retention, problems with visual accommodation, and cns impairment. because of their sympathomimetic effects, oral vasoconstrictors (e.g., phenylephrine, phenylpropanolamine, or pseudoephednne) are generally contraindicated or need to be used with considerable caution in patients with diseases that frequently occur in the elderly such as hypertension, the impact of allergic rhinitis coronary artery disease, diabetes, glaucoma, or prostate hypertrophy. finally, elderly patients may require lower doses of certain antihistamines owing to slowed hepatic metabolism. moreover, because they often take multiple prescription and nonprescription medications, the elderly also run a greater risk of drug interactions than most other patients. pregnancy-associated hormones affect the nasal mucosa, and may indirectly affect the nose through increasing circulating blood volume ( ) . as a result, some women with allergic rhinitis experience an exacerbation of symptoms, especially congestion, during pregnancy. this phenomenon generally starts toward the end of the first trimester and resolves rapidly after parturition. other women experience the apparent onset of allergic rhinitis during pregnancy ( ) . although pharmacologic agents may help control nasal symptoms in pregnant women, chicians should prescribe them cautiously, bearing the following facts in mind: despite its probable safety in pregnant women, clinical experience with loratadine is still relatively limited. the safety of diphenhydramine remains uncertain ( ) . all oral antihistamines are excreted in breast milk. in the usa, pseudoephedrine is approved for use in pregnancy at recommended dosages, and is widely prescribed in this population. owing to their overall safety, saline wetting agents and cromolyn sodium may be used to relieve congestion during pregnancy. in some european countries, topical antihistamines are recommended for the treatment of allergic rhinitis during pregnancy ( ) . if cromolyn proves ineffective, intranasal steroids may be tried. their use at recommended doses has not been associated with teratogenicity or other adverse effects in pregnant women ( ) . unfortunately, nasal congestion in pregnant women is often refractory to medical therapy. in such cases, patients may obtain some relief by using an external nasal dilator and saline douches. under no circumstances should immunotherapy be started during pregnancy, though continuation of maintenance doses is safe ( ) . physical exercise causes vasoconstriction that lasts for about h after exertion stops. however, endurance athletes (e.g., long-distance runners or cyclists) may experience long-lasting rebound congestion after a brief period of improved nasal patency. this rebound effect may impair athletic performance. when managing allergic rhinitis in competitive athletes, physicians must be careful to avoid prescribing medications with doping properties or therapy that may adversely affect performance. nonsedating antihistamines and topical steroids are generally the best choices for serious athletes. the international olympic committee and other international regulatory authorities prohibit sympathomimetic decongestants (e.g., phenylpropanolamine and pseudoephedrine). in large enough quantities, these agents can act as psychostimulants as strong as d-amphetamine. immunotherapy is recommended for athletes who wish to avoid being dependent on anti-allergy medications, though discomfort at the injection site may impede physical performance for several days at a time. the successful management of chronic rhinitis depends greatly on how well patients cooperate with the therapeutic program. educating patients about their disease and its management can promote that cooperation. it has been shown, for example, that allergic symptoms can be well controlled and a reasonable quality of life maintained when patients are given the appropriate medications, education, and written instructions before the pollen season commences ( ) . specifically, patients benefit from knowing or understanding the following: h o w and why rhinitis develops. simple avoidance measures that can minimize exposure to allergens or rhinitis triggers. the appendix provides a handout about allergen control measures for patients in primary care. complications and related airway diseases that can develop f r o m poor allergy control. patients who understand the scope of their disease are apt to take it seriously and thus comply better with therapy. this, in turn, may help minimize their risk of developing related airway diseases. how specific medications work, and how to use nasal sprays correctly. patients must understand that antihistamines and intranasal steroids are most effective when taken on a regular basis and not as needed. for those who require two or more medications, an explanation of how each drug works provides a rationale for their use and may aid compliance. the necessity for prophylactic medication. patients should be informed that early use of medication can prevent the onset of severe symptoms. individuals with seasonal symptoms should start treatment at the outset of the spring or autumn seasons or in late summer. those with perennial allergies need to take medication in advance of specific exposuresfor example, before visiting a relative who keeps indoor pets. the side-effects associated with prescribed medications. patients should be told about common side-eff ects of particular pharmacologic agents, and what to do in the event of their occurrence. possible drug interactions. patients should understand the importance of informing their physician of all the medications they are taking, both prescription and nonprescription. because of the risk of torsades de pointes, this is especially vital for users of terfenadine or astemizole. for the same reason, patients taking either of the latter two agents should also be instructed not to increase the dosage without consulting their physician. the appropriate use of topical decongestants. the risks and consequences of overuse should be discussed. simply written instructions help to reinforce key messages. still, clinicians should ascertain whether patients truly understand what they have been told. consider having patients repeat instructions rather than merely asking whether everything is clear. reviewing key points with patients during subsequent office visits is advisable, especially when therapeutic goals are not being met. building a trusting alliance with patients is important in gaining their cooperation with therapy. in part, trust is established by attending to patients' concerns and preferences. specifically, patients should be asked the following types of questions: how does rhinitis affect you daily? what medications or measures have you tried to reduce your symptoms? what do you expect from anti-allergy therapy? do you anticipate any problems from treatment? do you have any preferences in medications? asking patients about their opinions and experiences in these matters fosters empathy and trust, and so encourages compliance. other pathways to improved compliance include the following: prescribing drugs with a longer half-life, so that less frequent dosing is possible. choosing combination agents when more than one medication is needed to control symptoms. selecting pharmacologic agents with few sideeffects. educating patients about their disease and its management (see prior section about patient education). for example, allergic patients who appreciate that poor compliance may increase the risk of asthma may be more motivated to follow pharmacologic and avoidance protocols. selecting medications that target what the patient considers to be his or her most troubling symptoms. impressing upon patients that compliance improves disease control and quality of life. allergists and immunologists find that compliance can be good, even when patients are not symptomatic, because failure to take medications or practice avoidance guarantees the return of symptoms. in conclusion, iherapeutic intervention in allergic rhinitis aims to relieve symptoms, improve longterm outcome and quality of life, and encourage patient satisfaction with medical management. patients who are compliant with therapy generally obtain better symptom relief and feel more satisfied with their care. allergy and depression: a neurochemical threshold model of the relation between the finesses effects of allergy season on mood and cognitive function allergic diseases from infancy to adulthood the allergic irritability syndrome: four case reports and a position statement from the neuroallergy committee of the american college of allergy assessment of quality of life in adolescents with allergic rhinoconjunctivitis: development and testing of a questionnaire for clinical trials assessment of quality of life in patients with perennial allergic rhinitis with the french version of the sf- health status questionnaire epidemiology of acute asthma: ige antibodies to common inhalant allergens as a risk factor for emergency room visits middle meatus antrostomy: patency rates and risk factors the role of ige-mediated hypersensitivity in the development of otitis media with effusion the role of nasal allergy in otitis media with effusion: a clinical study increase in hospital admissions for childhood asthma: trends in referral, severity, and readmissions from to in a health region of the united nasal beclomethasone prevents the seasonal increase in bronchial responsiveness in patients with allergic rhinitis and asthma treatment of allergic rhinitis with intranasal corticosteroids in patients with mild asthma: effect on lower airway responsiveness once daily intranasal fluticasone propionate ( mg) reduces nasal symptoms and inflammation but also attenuates the increase in bronchial responsiveness during the pollen season in allergic rhinitis the role of inflammatory mediators in allergic rhinitis quantitative intranasal pollen challenges. . the priming effect in allergic rhinitis weller pe the immunobiology of eosinophils the effects of activated eosinophils and neutrophils on guinea pig epithelium in vitro eosinophil-and neutrophil-mediated injury of human lung fibroblast cells inhibitory activity of loratadine and descarboethoxyloratadine on expression of icam- and hla-dr by nasal epithelial cells the expression of leukocyte-endothelial adhesion cells is increased in perennial allergic rhinitis intercellular adhesion molecule- (icam- ) in the pathogenesis of asthma mucosal inflammation in asthma evidence of intercellular adhesion molecule- expression on nasal epithelial cells in acute rhinoconjunctivitis caused by pollen exposure minimal persistent inflammation is present at mucosal level in patients with asymptomatic rhinitis and mite allergy the major human rhinovirus receptor i s icam- a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses a longitudinal study of the role of viral infections in exacerbations of asthma in school children in the community community study of role of viral infections in exacerbations of asthma in - year old children increased levels of eosinophil, a major basic protein in nasal secretions in rhinovirus infection rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects international consensus report on the diagnosis and management of rhinitis nonallergic rhinitis with eosinophilia syndrome a precursor of the triad: nasal polyposis, intrinsic asthma, and intolerance to aspirin the diagnosis and treatment of recurrent and chronic sinusitis in children gustatory rhinitis: a syndrome of food-induced rhinorrhea nonallergic chronic rhinitis syndromes seasonal variation in dust mite and grass-pollen allergens in dust from the houses of patients with asthma rhinitis of pregnancy intranasal cocaine abuse in an allergist's office nasal, pharyngeal, and laryngeal manifestations of hypothyroidism allergy in twin pairs parental asthma, parental eczema, and asthma and eczema in early childhood the familial incidence of allergic disease comparison of patients' compliance with prescribed oral and inhaled asthma medications determinants of patient compliance with allergen immunotherapy is your asthmatic patient really complying? treatment of allergic rhinitis with loratadine plus pseudoephedrine in patients with mild asthma: effects on lower airway symptoms and function prophylactic treatment of grass pollen-induced asthma with cetirizine terfenadine, a potent histamine hi-receptor antagonist in the treatment of grass pollen sensitive asthma goodman gilman's pharmacological basis of therapeutics inhibitory effect of the h, antagonist loratadine on histamine release from human basophils in vitro inhibition, by loratadine and descarboxyethoxyloratadine, of histamine release from human basophils, and of histamine release and intracellular calcium fluxes in rat basophilic leukemia cells (rbl- h ) antiallergic activity of hi-receptor antagonists assessed by nasal challenge effect of loratadine on human eosinophil function in vitro effect of terfenadine on human eosinophil and neutrophil chemotactic response and generation of superoxide the effect of antihistamines on the immediate allergic response: a comparative review topical azelastine reduces eosinophil activation and intercellular adhesion molecule expression on nasal epithelial cells: an antiallergic activity terfenadine exerts antiallergic activity reducing icam- expression on nasal epithelial cells in patients with pollen allergy position paper. the clinical safety of hi-receptor antagonists. an eaaci position paper histaminergic transmission in the mammalian brain a multicentric study of loratadine, terfenadine, and placebo in patients with seasonal allergic rhinitis non-sedating antihistamines a comparison of loratadine, a new nonsedating antihistamine, with clemastine and placebo in patients with fall seasonal allergic rhinitis once daily loratadine versus astemizole once daily the role of antihistamines in the treatment of chronic urticaria perennial allergic rhinitis: clinical efficacy of a new antihistamine treatment of bronchial asthma with terfenadine: a randomised controlled trial effect of terfenadine in asthmatic patients effect of a non-sedative antihistamine (loratadine) in moderate asthma: a double-blind controlled clinical crossover trial the effect of astemizole on exercise-induced asthma comparison of the bronchodilatory effects of cetirizine, albuterol, and both together versus placebo in patients with mild-tomoderate asthma corticosteroidsparing effect of azelastine in the management of bronchial asthma comparative safety of hi-antihstamines physicians' desk reference double-blind comparison of cetirizine and placebo in the treatment of seasonal rhinitis dose-ranging comparative evaluation of cetirizine in patients with seasonal allergic rhinitis sleepiness and performance during three-day administration of cetirizine or diphenhydramine hi-receptor antagonists. comparative tolerability and safety antihistamines and driving performance: the netherlands cetirizine effects on objective measures of daytime sleepiness and performance does cetirizine belong to the new generation of antihistamines? an investigation into its acute and subchronic effects on highway driving, psychometric test performance and daytime sleepiness effects of terfenadine on actual driving performance hanlon je effects of loratadine and cetirizine on actual driving and psychometric test performance, and eeg during driving psychomotor effects of astemizole and chlorpheniramine, alone and in combination with alcohol acute and subchronic effects of the hi-histamine receptor antagonist ebastine in , , and mg dose, and triprolidine mg on car driving performance double-blind, placebo-controlled trial of terfenadine in seasonal allergic rhinitis and conjunctivitis benefit/ risk ratio of the antihistamines (h,-receptor antagonists) the effects of loratadine, diphenhydramine and placebo on worker productivity: results of a double blind trial [abstract differential cognitive effects of terfenadine and chlorpheniramine antihistamines, drowsiness, and psychomotor impairment: central nervous system effect of cetirizine antihistamines and visual function: studies on dynamic acuity and the pupillary response to light a double-blind study of the effects of loratadine versus placebo on the performance of pilots comparison of the effect of the macrolide antibiotics erythromycin, clarithromycin, and azithromycin on terfenadine steady-state pharmacokinetics and electrocardiographic parameters torsades de pointes associated with drugs and toxins: recognition and management loratadine and pseudoephedrine sulfate: a double-bhd, placebo-controlled comparison of a combination tablet (sch ) and its individual components in seasonal allergic rhinitis a cross-over comparison of acrivastine, pseudoephedrine and their combination in seasonal allergic rhinitis fireman l? effect of an antihistamineldecongestant on nasal and eustachian tube function following intranasal pollen challenge effects of terfenadine and pseudoephedrine, alone and in combination in a nasal provocation test and in perennial rhinitis intranasal allergen challenge during corticosteroid treatment the impact of allergic rhinitis efficacy of oncea-day intranasal administration of triamcinolone aceto once daily triamcinolone acetonide nasal spray is effective for the treatment of perennial allergic rhinitis topical corticosteroids and nasal reactivity fluticasone propionate nasal spray for allergic rhinitis intranasal fluocortin butyl in patients with perennial rhinitis: a -month efficacy and safety study including nasal biopsy a comparative study of beclomethasone dipropionate aqueous nasal spray with terfenadine tablets in seasonal allergic rhinitis pegelow k- . efficacy of an oral antihistamine, astemizole, as compared to a nasal steroid spray in hay fever a comparative study of dexchlorpheniramine maleate sustained release tablets and budesonide nasal spray in seasonal allergic rhinitis comparison of beclomethasone dipropionate aqueous nasal spray, astemizole, and the combination in the prophylactic treatment of ragweed pollen-induced rhinoconjunctivitis oral antihistamine or nasal steroid in hay fever: a double-blind double-dummy comparative study of once daily astemizole vs twice daily nasal beclomethasone dipropionate local effect of intranasal beclomethasone dipropionate aerosol in hay fever glucocorticosteroids and rhinitis nasal septa perforation associated with topical corticosteroid therapy the effect of cromolyn on nasal disease blychert l- . effects of topical treatment with h, and h, antagonists on clinical symptoms and nasal vascular reactions in patients with allergic rhinitis comparison of levocabastine, a new selective hi-receptor antagonist, and disodium cromoglycate, in a nasal provocation test with allergen topical levocabastine, a selective hi-antagonist, in seasonal allergic rhinoconjunctivitis azelastin in der allergologischeu praxis doubleblind assessment of azelastine in the treatment of perennial allergic rhinitis a review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential absence of central effects with levocabastine eye drops effects of topical administration of levocabastine on psychomotor and cognitive function topical nasal sprays: treatment of allergic rhinitis anticholinergic treatment of watery rhinorrhea the common cold: current therapy and national history beneficial effects of propylene and polyethylene glycol and saline in the treatment of perennial rhinitis a novel therapy concept for the treatment of allergic rhinitis eaaci position papers report of bsaci working party alternative routes of immunotherapy: a review asthma and wheezing in the first six years of life the effect of genetic and environmental factors on the prevalence of allergic disorders at the age of two years ipratropium (atrovent) in the treatment of vasomotor rhinitis of elderly patients the course and management of asthma and allergic diseases during pregnancy drugs in pregnancy and lactation pharmacotherapy of allergic rhinitis aqueous beclomethasone dipropionate nasal spray in treatment of seasonal (ragweed) rhinitis key: cord- - yrftdp authors: zhang, runzhi; gao, xi; bai, hong; ning, kang title: traditional chinese medicine and gut microbiome: their respective and concert effects on healthcare date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: yrftdp advances in systems biology, particularly based on the omics approaches, have resulted in a paradigm shift in both traditional chinese medicine (tcm) and the gut microbiome research. in line with this paradigm shift, the importance of tcm and gut microbiome in healthcare, as well as their interplay, has become clearer. firstly, we briefly summarize the current status of three topics in this review: microbiome, tcm, and relationship of tcm and microbiome. second, we focused on tcm's therapeutic effects and gut microbiome's mediation roles, including the relationships among diet, gut microbiome, and health care. third, we have summarized some databases and tools to help understand the impact of tcm and gut microbiome on diagnosis and treatment at the molecular level. finally, we introduce the effects of gut microbiome on tcm and host health, with two case studies, one on the metabolic effect of gut microbiome on tcm, and another on cancer treatment. in summary, we have reviewed the current status of the two components of healthcare: tcm and gut microbiome, as well as their concert effects. it is quite clear that as the holobiont, the maintenance of the health status of human would depend heavily on tcm, gut microbiome, and their combined effects. with the rapid advancement of research in traditional chinese medicine (tcm) and gut microbiome, studies involving both fields have significantly accelerated around the world. tcm and gut microbiome are closely related to human health. therefore, exploring the relationship between tcm and gut microbiome is essential for healthcare. the microbiome is a novel research field related to human health, bioenergy, agriculture, and the environment. it has recently emerged as a crucial factor in human physiology and disease. taking the microbiome into consideration, human beings could be considered "super organisms," with around % human cells and % microbial cells, most of which can be found in the gut (lederberg, ) . the human gut is a biological niche, home to a variety of microbes that affect nearly all aspects of human biology through their interactions with their hosts (blaser, ) . the human intestinal flora is a complex micro-ecosystem, containing approximately microbes from more than , microbial species, of which the phyla firmicutes, bacteroidetes, and actinobacteria (gill et al., ) account for more than % of the species above. in addition, the genetic factors, dietary habits, and various environmental factors of the host lead to the diversity and the specificity of the human intestinal flora, while human intestinal flora in turn would play important roles in exerting host's physiological functions, such as metabolism, energy intake, and immune regulation. under normal circumstances, the intestinal flora is in a state of homeostasis. environment, drugs, and the other factors that destroy the structure of intestinal flora will cause dysbiosis, which may influence human health. recent studies have already accumulated ample evidences that the gut microbiome plays pivotal roles in many forms of chronic diseases. for example, inflammatory bowel disease (ibd) is a chronic bowel disease with clinical symptoms of abdominal pain, diarrhea, and unknown etiology, it includes ulcerative colitis and crohn's disease (cd) . the fecal microbiome of cd patients contains a significant reduction of firmicutes diversity, and it's worth mentioning that the clostridium leptum phylogenetic group has less abundance in cd patients than healthy (manichanh et al., ) . constipation is a common health problem with high mortality, as well as a predisposing factor for many conditions. bifidobacterium adolescentis exhibited strain-specific effects in alleviation of constipation . obesity is a global problem in that nearly % of the people worldwide are obese. obese mice had % fewer bacteroidetes and more correspondingly firmicutes than their lean littermates (ley et al., ) . besides, they reached similar conclusions for obese people (ley et al., ) . dietary interventions might lead to the modulation of gut microbiome, which will contribute to weight loss, enhance the integrity of the intestinal barrier, and reduce the antigenic load in the circulation, ultimately improving the inflammatory and metabolic phenotypes (xiao et al., ) . type diabetes (t d) is a complex disorder affected by genetic and environmental components (wellen and hotamisligil, ; risé rus et al., ) , and become a major public health problem on a global scale. a protocol has been developed for the metagenome-wide association study (mgwas) and a two-phase approach has been taken based on high-throughput sequencing of the intestinal microbiome of chinese individuals. mgwas analysis revealed that the intestinal microbiome of patients with t d was mal-regulated, which was manifested by the decrease in the abundance of certain universal butyrate-producing bacteria and the increase of pathogenic bacteria in various conditions (qin et al., ) . metformin is a widely used drug in the treatment of t d. a recent study discovered the mechanism of metformin by regulating the composition of gut microbiome (wu et al., ) , indicating a close relation between the gut microbiome and t d. human gut microbiome are structurally dynamic over time and plastic under different conditions, as the bacterial composition as well as the collection of genes varied with xenobiotics and the environment (zhao et al., ) . due to the close relevance between the microbiome and the human genome, the taxonomic and functional composition changes of the intestinal microbiome inevitably regulate gene expression and host immunity, which may lead to the occurrence of diseases, in particular, chronic diseases. in recent years, with the launching of various international projects on human microecology [e.g., hmp: http://www. hmpdacc.org/, metahit: http://www.metahit.eu/ and ihmp (proctor et al., ) ], gut microbiome have become the hotspot research between chronic diseases and the gut microbiome has steadily increased, promoting the most significant paradigm shifts in modern medicine. tcm is in constant development and inheritance along the long chinese traditional culture. it has been developed in clinics over thousands of years and has accumulated abundant clinical experience, forming a field with unique experiences and specific theories. the tcm system is complex, as it contains components of natural plants, animals, and mineral materials. tcm uses therapeutic herbs to treat the disease and restore the balance of physical function according to the patient's syndrome, based on the combination principle of "king, vassal, assistant and delivery servant" (yi and chang, ) . each prescription combination of these herbs is called a tcm preparation or prescription, such as liuweidihuangwan (lwdhw) pills. many chinese herbal medicines are known for their therapeutic effects compared to chemical and biological agents (chan, ; corson and crews, ; qiu, ) , which contain not only bioactive components but also various nutrients such as proteins and polysaccharides. for example, glycyrrhiza uralensis fisch., also called "gan-cao" in china, as a health-preserving and therapeutic agent, it has been widely utilized for more than , years. it is one of the most broadly used ingredients in tcm preparations in china, exerting a wide range of pharmacological efficacies. glycyrrhiza uralensis fisch. contains many bioactive ingredients, including glycyrrhizin, glycyrrhetinic acid, glycyrol, coumarin, and alkaloids. in addition, previous studies have shown that glycyrrhiza uralensis fisch. has a variety of pharmacological effects, including antitumor (shibata et al., ) , antiviral (nakashima et al., ) , and anti-inflammatory effects (kimura et al., ) . the traditional discovery pathway for most new drugs is to identify or design pharmacologically effective agents that specifically stimulate or inhibit a specific set of target receptors. nevertheless, the influence of the gut microbiome on humans has been largely ignored in this process. therefore, the pharmacological effects of these single target drugs on chronic diseases are limited. in contrast, since one of the characteristics of tcm is oral administration, the drug will interact with the intestinal flora inevitably. previous studies have proven that tcm is conductive to maintain the homeostasis of the intestinal flora (chang et al., ; zhou et al., ) , and the gut microbiome could also exert pharmacological effects of the tcm on host (park et al., ) , which render tcm a potential new drug in the western markets. thus, research about relationship between tcm and gut microbiome is significant, which helps researchers to further study the pharmacological effects of tcm on the human body and the causal links among intestinal microbiome with disease. in this review, we summarize the current status of microbiome, tcm, relationship of tcm and microbiome, as well as the therapeutic effects of tcm and gut microbiome's mediation roles. among them, databases and tools that contribute to the molecular understanding of tcm and the role of the gut microbiome in diagnosis and treatment are also presented. finally, we introduce the impacts of gut microbiome on tcm and host health, with two case studies, one on the metabolic effect of gut microbiome on tcm, and another on cancer treatment (figure ). an increasing number of studies have been conducted on tcm, gut microbiome, and their interplay. statistics on the published papers in "public medicine (pubmed)" (https://pubmed.ncbi. nlm.nih.gov) and "china national knowledge infrastructure (cnki)" (https://www.cnki.net) showed that the number of literature on tcm and gut microbiome continued to rise in recent years ( figure ). most importantly, due to the standardization of tcm, research on tcm and gut microbiome has received more and more attention around the world, and the tcm-related research outputs have shown explosive growth in . to maintain the health of humans under various physiological conditions and environments, tcm and the gut microbiome must coordinate with each other to cope with the challenges. the interactions between tcm and the gut microbiome can mainly be categorized into four types ( figure ): the regulatory effects of tcm on gut microbial communities are mainly reflected in treatment of dysbiosis to restore homeostasis of the intestinal flora. ganoderma lucidum (g. lucidum), called "ling-zhi" in china, has been widely used as a health-preserving and therapeutic agent. a water extract of g. lucidum has antiobesogenic effects, as it could modulate the taxonomical composition of the gut microbiome (chang et al., ) . the water extract of g. lucidum mycelium can reverse high-fat diet (hfd)-induced gut dysbiosis, indicated by the decreased firmicutes-bacteroidetes ratios and endotoxin-bearing proteobacteria levels. it can also maintain intestinal barrier integrity and reduce metabolic endotoxemia. in addition, it can reduce the expression and secretion of the proinflammatory cytokines tumour necrosis factor (tnf), interleukin- -beta (il- b), and interleukin- (il- ). the glucose tolerance and insulin sensitivity are improved by the extract as well. these effects are especially mediated by the gut microbiome, as it has already been observed from experiments on mice that the weight loss effect induced by g. lucidum is transmissible via transfer of feces from g. lucidum-treated mice to hfd-fed mice. the study has also demonstrated that the antiobesogenic effect of the g. lucidum water extract is mainly owing to its high molecular weight polysaccharide fraction (> kda). g. lucidum polysaccharide can inhibit colon expansion of colorectal cancer (crc) mice and reduce mortality by %, by alteration of reduced the relative abundance (ra) of cecal oscillospirawhich was first observed in crc mice, and down-regulation of four cancer-related genes (acaa b, fabp , mgll, and scd ) expression of colonic epithelial cells (luo et al., ) . gegen qinlian decoction (gqd), a traditional prescription, could effectively alleviate t d by modulating the gut microbiome (xu et al., ) . in their study, compared to the patients with a low dose gqd and the placebo, patients who received high dose and moderate dose gqd showed remarkable reductions in adjusted mean changes from baseline fasting blood glucose (fbg) and glycated hemoglobin (hba c). faecalibacterium prausnitzii was a species enriched significantly by gqd; the species was negatively correlated with hba c and fbg, while -h postprandial blood-glucose levels were positively correlated with homeostasis model assessment of b-cell function, which suggests that the structure of gut microbiome was changed by gqd. furthermore, the amounts of beneficial bacterium, such as faecalibacterium spp., could be enriched by gqd treatment. it has been reported that another tcm, polygonatum kingianum, is effective in the treatment of diabetes and related diseases (deng et al., ) . a phytochemistry investigation demonstrated that the major types of active chemical constituents of p. kingianum were total saponins (tspk) and total polysaccharides (pspk). tspk and pspk could prevent the increase of fasting blood glucose after oral administration, and tspk could increase the content of fasting insulin. since tspk and pspk improved the intestinal microecology by increasing the abundance of firmicutes and decreasing that of bacteroidetes and proteobacteria, tspk and pspk could prevent t d by changing the regulation of the gut microbiome (yan et al., ) . huang-qin decoction (hqd) is widely used to alleviate gastrointestinal disorders such as inflammatory bowel disease. hqd significantly inhibited the weight loss, tissue damage, colon shortening, and inflammatory cytokine changes caused by dextran sulfate sodium (dss). the relative abundance of lactococcus in the dss + hqp group was higher than that in the dss group, while desulfovibrio and helicobacter were decreased, indicating that hqd can improve dss-induced inflammation through its regulation in the gut microbiome. in addition to tcm directly acting on gut microbiome to ameliorate diseases, some tcms exert indirect effects on the intestinal flora . ginsenosides and polysaccharides in du-shen-tang made from panax ginseng are used to investigate their pharmacological effects on acute cold stress and overfatigue. it was demonstrated that the intestinal metabolism and absorption of certain ginsenosides were improved by polysaccharides. in addition, in recovery of the disordered gut microbiome, polysaccharides especially boost the growth of two important probiotics, lactobacillus spp. and bacteroides spp. (zhou et al., ) . glycyrrhiza uralensis fisch., also named licorice, has been commonly used for sore throat and gastrointestinal diseases. after administering different doses of licorice aqueous extracts to mice, they found that the proportion of bacteroides gates decreased significantly, and the proportion of phylum firmicutes increased and became dominant. bacteroides was reported to be associated with iga in humans and may cause colitis, and more phylum firmicutes than bacteroides lead to more efficient absorption of food calories. therefore, it is speculated that licorice aqueous extract can promote intestinal absorption, anti-inflammatory effect, and treatment of abdominal pain . diammonium glycyrrhizinate (dg), the main component of licorice root extracts, is a compound of the natural bioactive pentacyclic triterpenoid glycoside, can protect against nonalcoholic fatty liver disease (nafld) in mice through the decreasing of firmicutes-to-bacteroidetes ratio and endotoxin-producing bacteria such as desulfovibrio, and elevate the abundance of probiotics such as proteobacteria and lactobacillus. dg can also augment the levels of short-chain fatty acid (scfa)-producing bacteria such as ruminococcaceae to promote scfa production, and restoration of intestinal barrier . the regulation of tcm on human health can also be reflected in other microbial communities, including saliva and tongue coating (jiang et al., ; liang et al., ; li, ) . for figure | number of published papers related to tcm-gut microbiota in pubmed and cnki. papers in pubmed were searched using keywords "gut/intestinal microbiota," "chinese," and "medicine"; papers in cnki were searched by chinese name of "tcm" and "gut microbiota/microbiome." the red bar chart represents the papers in chinese; the blue bar chart represents the papers in english. instance, through a network pharmacology approach (li, ) , the tongue coating microbiome have been found to be associated with various diseases including colorectal cancer (liang et al., ) and "cold-disease" (jiang et al., ) . therefore, the regulation of tcms can be reflected in microbial communities of various human body habitats, including gut and oral habitats, indicating quite diverse microbial-based approaches for noninvasive probing of human health status. in recent years, with the widespread use of synthetic and semisynthetic antibiotics, the problem of bacterial resistance has become increasingly serious in clinical fields. pathogenic bacterial infections are critical factors that may affect the development and severity of the disease. tcm was reported to possess a broad-spectrum antibacterial effect. therefore, systematic study on the antibacterial activity of tcm and the further development of new drugs have become the focus of more and more researchers. currently, more and more research indicate that tcm has antibacterial effect ( table ) . in the long history of tcm treatment, diet has always been considered as an important category of resources. development of the research of gut microbiome involves not only the drug and tcm but also the daily diet of humans. recently, a rapidly increasing number of studies have indicated the crucial role of the diet for the treatment of disease (leboeuf et al., ; li et al., b; moayyedi et al., ; naudin et al., ; yu et al., ) . in addition, studies about the close relationship between the diet and the intestinal flora have gradually aroused attention, which may offer us some new perspective for disease prevention. carbohydrates are important components of the daily diet and include monosaccharides, disaccharides, and polysaccharides. both fructooligosaccharide (fos) and polyphenols can be transferred directly into the large intestine of mammals alone, reshaping the composition of gut microbiome, which is beneficial to human health. the combination of different phenolic compounds and fos had a distinct impact upon the host, and gut microbiome improved by using prebiotics can delay the onset of senescencerelated health problems (zheng et al., ; tanabe et al., ) . adding catechin to an fos diet could inhibit firmicutes and enhance bacteroidetes. in addition, it turns out that antibiotic treatment influences the diversity and composition of the gut microbial community. furthermore, the use of probiotics or prebiotics to modulate antibiotic-induced gut microbiome destruction has been considered a desired treatment or prophylaxis (bron et al., ) . cefixime can reduce the diversity of the microbial community and lead to a significant decrease in the relative abundance of firmicutes. the composition of gut microbiome of lactobacillus and fos probiotic mixture treatment group was much more diverse than that of the natural recovery group, indicating a better recovery effect of the probiotic cocktails on the gut microbiome (shi et al., ) . moreover, the composition of the gut microbiome significantly changed in the hfd + fructose (hff)-fed and the hfd + sucrose (hfs)-fed rats compared with the control diet (c)-fed rats; body-fat mass, metabolic inflexibility, glucose intolerance, lipopolysaccharide (lps), insulin, renal reactive oxygen species (ros), malondialdehyde (mda), nadphox, and srebp- were significantly higher, and antioxidant enzymes and lean body masses were significantly lower in the hfs group with respect for the hff group (rosas-villegas et al., ) , indicating the harmful effect on the hfs group and the hff group on gut microbiome as well as the health of humans. carrageenan, agarose, and alginate are algae-derived undigested polysaccharides that have been used as food additives for hundreds of years. bacteroides uniforms l could degrade agarose completely, and the major enzyme secreted was b-agarase. the enzyme produced by isolate f (bacteroides xylanisolvens and escherichia coli), which degrades k-carrageenan oligosaccharides, is b-carrageenase, and alginate, guluronic acid oligosaccharides, and mannuronic acid oligosaccharides could be degraded by two enzymes from bacteroides ovatus g . vitamin d is an important pro-hormone for optimal intestinal calcium absorption for mineralization of bone (khazai et al., ) , which is common in high-fat food such as cream and salmon. vitamin d deficiency is a disease caused by air pollution, insufficient sunlight exposure, and altered dietary composition, which have also been associated with other diseases, including autoimmune diseases, hepatitis, and cancer (holick et al., ) . the functionality of the vitamin d-vitamin d receptor (vdr) axis in up-regulating and the activation/process of ileal a-defensins was identified. conversely, dietary vitamin d deviancy resulted in the loss of paneth cellspecific a-defensins, which may consequently lead to intestinal dysbiosis and endotoxemia, indicating that vitamin d is essential for the homeostasis of intestinal flora (su et al., ) . acetate has been widely distributed in nature, such as in fruits or vegetable oils. a recent study showed that acetate produced by protective bifidobacteria could improve intestinal defense that is mediated by epithelial cells, and this could protect the host against negative infection (fukuda et al., ) . in addition, moderate consumption of wine has shown the potential to delay the onset of neurodegenerative diseases: after drinking, microbial metabolites may effectively protect neuroblastoma cells from nitrosative stress injury (esteban-fernandez et al., ) . with the application of network pharmacology and related databases and tools, the research on tcm and gut microbiome has been further developed (figure ) . commonly used databases of tcm gut microbiome research include the tcm database [tcm-mesh , tcmsp (ru et al., ) , tcmid , and hit (ye et al., ) ], as well as pathogen-host databases [phi-base (winnenburg et al., ) and ehfpi ]. tcm-mesh has integrated information about tcm ingredients, compound-protein interactions, as well as protein-disease relationships, to establish the largest resource for tcm target genes and diseases. based on the biological databases and clinical trial results, a researcher can analyze the "tcm-host-gut microbiome" interaction network from a holistic perspective. virtual metabolic human database (noronha et al., ) contains information on human and gut microbial metabolism, which is linked to hundreds of diseases and nutritional data by mathematical models. many tools are usually used for analysis and visualization as well. for example, qiime (caporaso et al., ) is a software that provides an open-source bioinformatics pipeline for performing microbiome analysis from raw dna sequencing data. it includes demultiplexing, quality filtering, operational taxonomic units (otu) picking, taxonomic assignment, phylogenetic reconstruction, diversity of analyses and visualizations. picrust (langille et al., ) is designed to predict microbiome functional content from marker gene (e.g., s rrna) surveys and full genomes. metaphlan (segata et al., ) is a computational tool for microbial communities analysis based on metagenomic sequencing data, in which the data were compared with the markers of each species obtained from the known database to determine the species category. muscle (edgar, ) and clustalw (thompson et al., ) are two algorithms used for multiple sequence alignments; muscle is a computer program for creating multiple alignments of protein sequences. mega (kumar et al., ) is a computer program package for estimating evolutionary distances, reconstructing phylogenetic trees, and computing basic statistical quantities from molecular data. additionally, itol (letunic and bork, ) is an online tool for visualization, annotation, and management of phylogenetic trees. furthermore, with the development of deep learning, many methods have been used for studying the gut microbiome and diseases. for example, a model was constructed to predict responses to anti-integrin biologic therapy for inflammatory bowel diseases through gut microbiome function (ananthakrishnan et al., ) . although many approaches are not currently used in tcm study on gut microbiome, related methods will become a future trend. therefore, additional investigations are warranted. furthermore, interactive visualization tools are of equal importance. cytoscape is an open-source platform that could be used for complex network analysis and visualization (shannon et al., ) , which is useful for analyzing the network in network pharmacology studies. similar functions are also offered by the web services of many databases. the involvement of the gut microbiome in maintaining the health status of every person will undoubtedly affect the effectiveness of drug treatments, including tcm treatment. here, we provide two case studies to illustrate this, one on the metabolic effect of gut microbiome on tcm, and the other on cancer treatment, to illustrate the mediation role of the gut microbiome in medicinal treatment in general. previous studies have found that many of the chemical constituents of tcm can be transformed and metabolized by the intestinal flora, making it easier for the body to absorb. in other words, the gut is like a processing factory of tcm. the effects of the intestinal flora on different kinds of chemical constituents vary: flavonoids represent a major class of secondary metabolites of plants and can be classified into subclasses: anthocyanins, chalcones, dihydrochalcones, flavanols, flavanones, flavones, flavonols, isoflavones, flavan- -ols, flavan- , -diols, aurones, and proanthocyanidins (crozier et al., ) . the effects of these polyphenols in the prevention of cancer, cardiovascular disease, t d, and cognitive dysfunction are supported by an increasing number of studies (del et al., ; rodriguez-mateos et al., ) . to exert their effects, flavonoids are metabolized by the gut microbiome, and the resulting compounds may have bioactivity. such bacterial conversion and potential health consequences for the human host (chiou et al., ) are hardly unique: baicalin, puerarin, and daidzin, which are widely found in tcm prescriptions, can also be metabolized by gut microbiome (kim et al., ) . as baicalin is difficult to be absorbed in the gut, it has to be hydrolyzed by gut microbiome to baicalein to exert its effect. metabolism of baicalin was hardly detected in germ-free rats compared to conventional rats, indicating that gut microbiome play a key role in the metabolism of baicalin (akao et al., ) . (mochizuki et al., ; sun et al., ; shi et al., ) . ginsenoside compounds k (ck) is one of the metabolites of ginsengoside, which exerts various pharmacological effects, including antiallergic (shin and kim, ) , antidiabetic (kim et al., ) , anticarcinogenic (zhou et al., ; zhang et al., ) , anti-inflammatory (joh et al., ; chen et al., ) , and antiaging effects (kim et al., ) . the occurrence of ck in rat plasma is required for intestinal bacterial hydrolysis after oral administration of ginsengosides (akao et al., ) . when human intestinal flora was cultured anaerobically with ginsengosides rb , rb , and rc, these ginsengosides were metabolized into ck, where the transformation of rc is conducted by the concert efforts of bacteroides spp., eubacterium spp., and bifidobacterium spp. (bae et al., ; bae et al., ) . in addition, intestinal bacterial metabolism of ginsengosides was mainly dependent on the composition of intestinal flora, such as bacteroides spp., ruminococcus spp., and bifidobactetium spp. (kyung-ah kim et al., ) . additionally, the metabolism of panax notoginseng saponins (pnss) was influenced by gut microbiome, of which proteobacteria may have an effect on the deglycosylated metabolism of pnss by regulating the activities of glycosidases (xiao et al., ) . up-regulation of bacteroidetes may increase the activity of redox metabolic enzymes in intestinal flora and accelerate the redox metabolism of pnss (xiao et al., ) . oxymatrine and matrine are two main alkaloids in the radix sophorae flavescentis, and oxymatrine could be transformed into matrine by intestinal bacteria when taken orally, and both can be absorbed by the blood . scopolamine was incubated with rat intestinal flora in vitro under limited oxygen conditions, and scopolamine was transformed by gut microbiome into scopine (chen et al., ) . anthraquinones have widespread applications throughout industry and medicine, and are naturally distributed in many plants, including rheum officinale baill., fallopia multiflora (thunb.) harald., catsia tora linn., folium sennae, and aloe vera (linn.) n. l. burman var. chinensis (haw.) berg. through a chemical constituent study on the metabolism of rhubarb extract by rat intestinal bacteria, a total of components, including emodin-o-glucosides, aloe-emodin-o-glucosides, physcion-oglucosides, chrysophanol-o-glucosides, and their aglycones were found to be biotransformed by rat intestinal bacteria. twelve major metabolites were identified in the incubation sample, suggesting the importance of the intestinal flora during the metabolism of anthraquinones (song et al., ) . carcinogenesis may be mediated by microbiome influencing cellular metabolism, inflammation, and proliferation (zitvogel et al., ) . the microbiome regulate cancer at the level of predisposing conditions, initiation, genetic instability, susceptibility to host immune response, progression, comorbidity, and response to therapy. several examples are as follows: there is an increasing number of studies involving mice and humans on the role of intestinal flora in cancer treatments. in mice, the procarcinogenic phenotype expressed by genetically mutated mice has been shown to be transferred to wild-type mice by microbiome transfer (garrett et al., ; couturier-maillard et al., ; hu et al., ) , and transfer of fecal microbiome from patients who are sensitive to cancer treatment into germfree mice has been found to endow these animals with an ability to respond efficiently to therapy (vetizou et al., ) , indicating that changes in the composition of intestinal flora indirectly affect carcinogenesis and response to cancer treatment through lifestyle and host genetic. furthermore, the intestinal flora affects anticancer activity, toxicity, as well as pharmacokinetics of drugs at various levels (dzutsev et al., ; spanogiannopoulos et al., ) . several anticancer drugs have experimentally been shown to be affected by the intestinal flora , including the hydrolysis of methotrexate, the nitroreduction of misonidazole, and the deconjugation of irinotecan. in germfree mice or mice in which gut commensals were exhausted by broad-spectrum antibiotics, the antitumor effect of cisplatin or oxaliplatin treatment on subcutaneous transplantable tumors was decreased evidently (lida et al., ) . probiotics lactobacillus acidophilus in antibiotic-treated mice restored cisplatin-induced inflammatory gene expression (gui et al., ) , indicating the mediation role of gut microbiome on drug efficacy. additionally, oral administration with bifidobacterium bifidum and l. acidophilus, a probiotic combination, was found to prevent intestinal toxicity in cancer patients who were treated with both cisplatin and radiotherapy (chitapanarux et al., ) . in addition to the direct effects on the enzymes and microbiome on the absorption and metabolism of drugs (maurice et al., ; feng et al., ; montassier et al., ) , the indirect effects of gut microbiome on the metabolism of drugs were reported to occur through regulation of gene expression and the physiological function of the local mucosal barrier and of distant organs, such as the liver (bjorkholm et al., ; selwyn et al., ; selwyn et al., ) . radiotherapy is one of the traditional methods for cancer treatment. because radiotherapy alters the microbiome composition and destroys the intestinal barrier (barker et al., ) , pathobionts can easily access the intestinal immune system (belkaid and hand, ) . with the development of research on cancer and microbiome, probiotics have been reported to be beneficial in prevention of radiation-induced enteropathy in some clinical studies. for example, patients | recent studies on the close relationship between colorectal cancer (crc) and the gut microbiome. year reference fecal metabolomic signatures are associated with gut microbiome and colorectal cancer pathogenesis: ( ) the levels of polyunsaturated fatty acids and sphingolipids are further increased in patients with crc, which may represent early changes in crc. ( ) regardless of disease status, age, or gender, there is a strong overall correlation between microbiome and metabolomics data, and the correlation is higher in women than in men. ( ) the abundance of multiple bacteria in firmicutes, actinobacteria, and bacteroides is strongly correlated with elevated metabolite pathways in patients. (kim et al., ) non-nucleatum fusobacteria common in southern chinese populations are associated with colorectal cancer: ( ) irrespective of crc disease status, the prevalence, relative abundance, and diversity of fusobacterium in southern chinese populations are higher than those in other regions. ( ) in addition to non-nucleatum fusobacteria, fusobacterium varium and fusobacterium ulcerans and other fusobacterium groups are also enriched in crc patients. ( ) several non-nucleatum taxa possess fada homologues and were enriched in crc cohorts. (yeoh et al., ) a pathological imbalance of the gut microbiome (dysbiosis) is present in colorectal cancer patients: ( ) bacteria affect crc directly or indirectly, by secreting metabolites, by invading tissues, and by modulating the host immune response. ( ) fusobacterium, peptostreptococcus, porphyromonas, prevotella, parvimonas, bacteroides, and gemella are among the most prominent crc-associated bacteria. (ternes et al., ) changes in gut microbiome can reduce the carcinogenic effect of colitis-related colorectal cancer: ( ) enterobacter strains are associated with molybdenum-dependent metabolic pathways in the colitis model. oral sodium tungstate can inhibit the activity of e. coli molybdenum and reduce the colonization of e. coli in the intestine. ( ) limiting the growth of enterobacteriaceae can control intestinal inflammation and reduce the incidence of colorectal cancer. ( ) oral sodium tungstate treatment can reduce intestinal inflammation and inhibit tumorigenesis caused by colicin. (zhu et al., ) colorectal cancer can be diagnosed by lachnoclostridium as a marker: ( ) metagenomic analysis identified "m " from a lachnoclostridium sp., fusobacterium nucleatum (fn), and clostridium hathewayi (ch) to be significantly enriched in crc. ( ) the combination of m with fn, ch, bacteroides, and fit can diagnose crc most accurately. (liang et al., ) intestinal fungal disorders in colorectal cancer, expected to be used for diagnosis: ( ) crc intestinal fungal disorders, increased ratio of basidiomycetes/ascomycetes, increased malassezia, decreased yeast and pneumocystis, genus enrichment, species abundance changes. ( ) the co-occurrence association between fungi and the mutual exclusion of bacteria-fungi in crc increased, and the positive interaction between proteobacteria and ascomycota became mutually exclusive in crc. (coker et al., ) different intestinal flora species increased or decreased the effectiveness of drugs: ( ) bacterial nucleotide metabolism genes in caenorhabditis elegans affect drug efficacy. ( ) -fluorouracil and -fluoro- '-deoxyuridine act through bacterial ribonucleotide metabolism to exert their cytotoxic effects in c. elegans rather than by thymineless death or dna damage. two-way bacterial mediation effects of fluoropyrimidine on host metabolism may contribute to drug efficacy: ( ) microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin b , b , and ribonucleotide metabolism. ( ) disturbances in bacterial deoxynucleotide pools amplify -fu-induced autophagy and cell death in host cells. (scott et al., ) levels of p. anaerobius are higher in human colon tumor tissues and adenomas compared with nontumor tissues; this bacterium increases colon dysplasia in a colorectal cancer mouse model. p. anaerobius interacts with tlr and tlr in colon cells to increase the abundance of reactive oxidative species, which promotes cholesterol synthesis and cell proliferation. (tsoi et al., ) f. nucleatum plays a key role in colorectal carcinogenesis through suppression of the hosts' immune response to tumor. a diet rich in whole grains and dietary fiber is associated with a lower risk of f. nucleatum-positive colorectal cancer. (mehta et al., ) key microbial markers are critical in the classification of crc cases and are commonly used in the diagnosis of disease. (shah et al., ) ( ) aim -deficient mice are hypersusceptible to colonic tumor development. ( ) susceptibility of aim -deficient mice to colorectal tumorigenesis is enhanced by dysbiotic gut microbiome compared with healthy wildtype mice. (man et al., ) zhang et al. with head and neck cancer received lactobacillus brevis cd lozenges during chemotherapy and radiation, and the incidence of treatment-induced mucositis decreased while the treatment completion rate increased (sharma et al., ) . immunotherapy is another approach that has shown potential in treating solid cancers and hematopoietic (couzin-frankel, ) . the proliferation and antitumor cytotoxic activity of transferred t cells in the tumor were increased by the total-body irradiationinduced translocation of intestinal flora into the mesenteric lymph nodes (paulos et al., ) . moreover, many studies have revealed the crucial role of intestinal flora in colorectal cancer treatment recently ( table ) , indicating the close relationship between gut microbiome and cancer treatment. in recent years, with the modernization of tcm and the development of systems biology, our understanding of tcm has been significantly advanced. tcm has gradually transformed from an experience-based approach to an evidence-based medicinal system, of which chinese scientists have made great contributions. we modify this sentence to "for example, it was discovered that arsenic induces the degradation of a key leukemogenic protein and exerts a pharmacological effect on the treatment of acute promyelocytic leukemia (zhang et al., ) . youyou tu, the nobel prize winner, has also focused on tcm research and research on the combination of western medicine and tcm for many years (tu et al., ; tu et al., ) . the trend has become clear that more and more tcm researches use molecular biology approach. however, with the systems biology approach enabled, the potential of tcm studies at the molecular level remains to be better exploited. on the other hand, research about the microbiome, in particular the gut microbiome, has made enormous progress during recent years. with the launch of various international projects on the human microbiome, research on the gut microbiome has become a hot research area. studies have proven that tcm could be used as a perfect agent to treat many kinds of diseases, many of which were mediated by gut microbiome. therefore, combination of the research of tcm and gut microbiome is important to maintain the healthy status of the host-microbiome holobiont. in addition, with the advancement of biological big data research, databases can now provide a more holistic perspective for tcm research and intestinal flora research. however, with the regulation or mediation principles of gut microbiome on human health still lacking except for a few diseases, more microbiome experiments, sequencing data, and analytical methods have yet to be conducted, collected, or developed to better understand these principles. it has become urgent that several problems remain to be solved for both tcm and gut microbiome research areas, especially in the era of biological big-data. with the constant increase in research in related fields, various experimental data have been generated. these all require better ways to curate, analyze, and interpret the concerted effects of tcm and the microbiome on human health. first, a more comprehensive database on tcm and gut microbiome is needed, which should not only include the interaction between the tcm and the gut microbiome but should also represent the advantages of tcm databases and microbiome databases in accelerating the application of tcm on a global scale. second, analytical methods must keep pace with the rapid development of modern systems biology. a more powerful data mining tool is required to investigate the complex and multi-scale "tcm-hostmicrobiome" network. third, investigations on the mechanisms of mutual regulation between tcm and gut microbiome are limited. how do various tcms regulate dysbiotic microbiome in concert? what kinds of enzymes produced by gut microbiome are responsible for metabolizing tcm and exerting the effect of tcm? these only represent a few questions that are to be answered. fourth, the microbiome has been indicated as a precision medicine frontier (kuntz and gilbert, ) , as interindividual differences in microbiome patterns have been reported, even between co-raised twins. finally, it is only when enough time-series samples are collected can we answer questions about dynamic patterns for the "tcm-host-microbiome" network. such dynamic pattern is the basis for the "shelf life" and "personalized medicine" for the microbiome-mediated tcm treatment, and could push for the modernization of tcm. in summary, researchers might take advantage of tcm and microbiome for better health care and treatment, as both possess the great potential in health care. this might lead to another paradigm shift: from genome-centric precision medicine to systems biology approach enabled holobiont-centric precision medicine. kn and hb conceived and proposed the idea. kn, hb, and rz jointly designed the main idea of this work. kn, rz, and xg contributed to the interpretation of data for the work. kn, hb, xg and rz drafted the manuscript, kn, hb, xg, and rz revised it critically for important intellectual content. all authors read and approved the final manuscript to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. drug metabolism: intestinal bacterial hydrolysis is required for the appearance of compound k in rat plasma after oral administration of ginsenoside rb from panax ginseng balicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form gut microbiome function predicts response to anti-integrin biologic therapy in inflammatory bowel diseases constitutive beta-glucosidases hydrolyzing ginsenoside rb and rb from human intestinal bacteria metabolism of ginsenoside r(c) by human intestinal bacteria and its related antiallergic activity the in vitro growth-inhibitory effect of euphorbia humifusa willd. (ehw) on bacteria the tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence role of the microbiota in immunity and inflammation intestinal microbiota regulate xenobiotic metabolism in the liver the microbiome revolution emerging molecular insights into the interaction between probiotics and the host intestinal mucosa qiime allows analysis of high-throughput community sequencing data progress in traditional chinese medicine ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota study on the metabolism of scopolamine in rat intestinal flora ginsenoside metabolite compound k alleviates adjuvant-induced arthritis by suppressing t cell activation metabolic and colonic microbiota transformation may enhance the bioactivities of dietary polyphenols randomized controlled trial of live lactobacillus acidophilus plus bifidobacterium bifidum in prophylaxis of diarrhea during radiotherapy in cervical cancer patients enteric fungal microbiota dysbiosis and ecological alterations in colorectal cancer molecular understanding and modern application of traditional medicines: triumphs and trials nod -mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer breakthrough of the year dietary phenolics: chemistry, bioavailability and effects on health dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases saponin rich fractions from polygonatum odoratum (mill.) druce with more potential hypoglycemic effects the role of the microbiota in inflammation, carcinogenesis, and cancer therapy muscle: multiple sequence alignment with high accuracy and high throughput neuroprotective effects of selected microbial-derived phenolic metabolites and aroma compounds from wine in human sh-sy y neuroblastoma cells and their putative mechanisms of transforming berberine into its intestine-absorbable form by the gut microbiota bifidobacteria can protect from enteropathogenic infection through production of acetate synergists from portulaca oleracea with macrolides against methicillinresistant staphylococcus aureus and related mechanism bacterial metabolism affects the c. elegans response to cancer chemotherapeutics colitis-associated colorectal cancer driven by t-bet deficiency in dendritic cells metagenomic analysis of the human distal gut microbiome study of the anti-bacteria activity of the extracts from the leaves of five herbs well-balanced commensal microbiota contributes to anti-cancer response in a lung cancer mouse model developing a metagenomic view of xenobiotic metabolism evaluation, treatment, and prevention of vitamin d deficiency: an endocrine society clinical practice guideline microbiota-induced activation of epithelial il- signaling links inflammasome-driven inflammation with transmissible cancer curcuma longa (turmeric): an auspicious spice for antibacterial, phytochemical and antioxidant activities integrating nextgeneration sequencing and traditional tongue diagnosis to determine tongue coating microbiome ginsenoside rb and its metabolite compound k inhibit irak- activation-the key step of inflammation calcium and vitamin d: skeletal and extraskeletal health metabolism of puerarin and daidzin by human intestinal bacteria and their relation to in vitro cytotoxicity compound k induces expression of hyaluronan synthase gene in transformed human keratinocytes and increases hyaluronan in hairless mouse skin ginsenoside metabolite compound k stimulates glucagon-like peptide- secretion in nci-h cells via bile acid receptor activation fecal metabolomic signatures in colorectal adenoma patients are associated with gut microbiota and early events of colorectal cancer pathogenesis effects of chalcones isolated from licorice roots on leukotriene biosynthesis in human polymorphonuclear neutrophls mega : molecular evolutionary genetics analysis version . for bigger datasets introducing the microbiome into precision medicine comparative analysis of the gut microbiota in people with different levels of ginsenoside rb degradation to compound k predictive functional profiling of microbial communities using s rrna marker gene sequences activation of oxidative stress response in cancer generates a druggable dependency on exogenous non-essential amino acids infectious history interactive tree of life (itol) v : an online tool for the display and annotation of phylogenetic and other trees obesity alters gut microbial ecology microbial ecology: human gut microbes associated with obesity degradation of marine algae-derived carbohydrates by bacteroidetes isolated from human gut microbiota diammonium glycyrrhizinate protects against nonalcoholic fatty liver disease in mice through modulation of gut microbiota and restoration of intestinal barrier lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov- ) prebiotic-induced anti-tumor immunity attenuates tumor growth mapping ancient remedies: applying a network approach to traditional chinese medicine dynamic microbe and molecule networks in a mouse model of colitis-associated colorectal cancer a novel faecal lachnoclostridium marker for the non-invasive diagnosis of colorectal adenoma and cancer commensal bacteria control cancer response to therapy by modulating the tumor microenvironment chemical composition and antibacterial activity of the essential oil of phellodendron lavallei ehfpi: a database and analysis resource of essential host factors for pathogenic infection ganoderma lucidum polysaccharide alleviating colorectal cancer by alteration of special gut bacteria and regulation of gene expression of colonic epithelial cells critical role for the dna sensor aim in stem cell proliferation and cancer reduced diversity of faecal microbiota in crohn's disease revealed by a metagenomic approach xenobiotics shape the physiology and gene expression of the active human gut microbiome association of dietary patterns with risk of colorectal cancer subtypes classified by fusobacterium nucleatum in tumor tissue evidence-based and mechanistic insights into exclusion diets for ibs inhibitory effect of tumor metastasis in mice by saponins, ginsenoside-rb , (r)-and (s)-ginsenoside-rg , of red ginseng chemotherapy-driven dysbiosis in the intestinal microbiome a new anti-human immunodeficiency virus substance, glycyrrhizin sulfate; endowment of glycyrrhizin with reverse transcriptase-inhibitory activity by chemical modification lactococcus lactis subsp. cremoris elicits protection against metabolic changes induced by a western-style diet the virtual metabolic human database: integrating human and gut microbiome metabolism with nutrition and disease intestinal bacteria activate estrogenic effect of main constituents puerarin and daidzin of pueraria thunbergiana microbial translocation augments the function of adoptively transferred self/tumor-specific cd + t cells via tlr signaling the integrative human microbiome project: dynamic analysis of microbiome-host omics profiles during periods of human health and disease a metagenome-wide association study of gut microbiota in type diabetes traditional medicine: a culture in the balance dietary fats and prevention of type diabetes bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update differential effect of sucrose and fructose in combination with a high fat diet on intestinal microbiota and kidney oxidative stress tcmsp: a database of systems pharmacology for drug discovery from herbal medicines host-microbe co-metabolism dictates cancer drug efficacy in c. elegans computational meta'omics for microbial community studies rna-seq quantification of hepatic drug processing genes in germ-free mice regulation of hepatic drug-metabolizing enzymes in germ-free mice by conventionalization and probiotics leveraging sequence-based faecal microbial community survey data to identify a composite biomarker for colorectal cancer cytoscape: a software environment for integrated models of biomolecular interaction networks ethnobotany of the genus taraxacum-phytochemicals and antimicrobial activity lactobacillus brevis cd lozenges reduce radiation-and chemotherapy-induced mucositis in patients with head and neck cancer: a randomized double-blind placebo-controlled study pharmacokinetics and dopamine/acetylcholine releasing effects of ginsenoside re in hippocampus and mpfc of freely moving rats restoration of cefixime-induced gut microbiota changes by lactobacillus cocktails and fructooligosaccharides in a mouse model inhibitory effects of licochalcone a isolated from glycyrrhiza inflata root on inflammatory ear edema and tumour promotion in mice antipruritic effect of ginsenoside rb and compound k in scratching behavior mouse models metabolic analysis of rhubarb extract by rat intestinal bacteria using liquid chromatography-tandem mass spectrometry the microbial pharmacists within us: a metagenomic view of xenobiotic metabolism vitamin d signaling through induction of paneth cell defensins maintains gut microbiota and improves metabolic disorders and hepatic steatosis in animal models ginsenoside rg and aluminum hydroxide synergistically promote immune responses to ovalbumin in balb/c mice dietary fructooligosaccharide and glucomannan alter gut microbiota and improve bone metabolism in senescence-accelerated mouse microbiome in colorectal cancer: how to get from meta-omics to mechanism? clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice peptostreptococcus anaerobius induces intracellular cholesterol biosynthesis in colon cells to induce proliferation and causes dysplasia in mice studies on the constituents of artemisia annua l studies on the constituents of artemisia annua part ii chemical composition and allelopathic, antibacterial, antifungal, and antiacetylcholinesterase activity of fish-mint (houttuynia cordatathunb.) from india anticancer immunotherapy by ctla- blockade relies on the gut microbiota interconverting flavonostilbenes with antibacterial activity from sophora alopecuroides studies on metabolism of oxymatrine by human intestinal bacteria the in vitro growth-inhibitory effect of magnolia officinalis rehd.et wils.(mor) on bacteria bifidobacterium adolescentis exerts strain-specific effects on constipation induced by loperamide in balb/c mice effects of turnip on intestinal flora of mice coptidis rhizoma: a comprehensive review of its traditional uses, botany, phytochemistry, pharmacology and toxicology inflammation, stress, and diabetes phi-base: a new database for pathogen host interactions metformin alters the gut microbiome of individuals with treatment-naive type diabetes, contributing to the therapeutic effects of the drug a gut microbiota-targeted dietary intervention for amelioration of chronic inflammation underlying metabolic syndrome qualitatively and quantitatively investigating the regulation of intestinal microbiota on the metabolism of panax notoginseng saponins structural modulation of gut microbiota during alleviation of type diabetes with a chinese herbal formula effect of glycyrrhiza uralensis decoction on intestinal flora in mice tcmid: traditional chinese medicine integrative database for herb molecular mechanism analysis antibacterial activity of disodium succinoyl glycyrrhetinate, a derivative of glycyrrhetinic acid against streptococcus mutans intake of total saponins and polysaccharides from polygonatum kingianum affects the gut microbiota in diabetic rats a study on antibacteral effect of six varieties traditional chinese medicine against neisseria gonorhoea gut microbiota drives the attenuation of dextran sulphate sodium-induced colitis by huangqin decoction two thymol derivatives from the flower buds of lonicera japonica and their antibacterial activity hit: linking herbal active ingredients to targets southern chinese populations harbour non-nucleatum fusobacteria possessing homologues of the colorectal cancer-associated fada virulence factor an overview of traditional chinese herbal formulae and a proposal of a new code system for expressing the formula title pth induces bone loss via microbial-dependent expansion of intestinal tnf(+) t cells and th cells arsenic trioxide, a therapeutic agent for apl compound k, a ginsenoside metabolite, inhibits colon cancer growth via multiple pathways including p -p interactions tcm-mesh: the database and analytical system for network pharmacology analysis for tcm preparations five new alkaloids from the roots of sophora flavescens targeting the human genome-microbiome axis for drug discovery: inspirations from global systems biology and traditional chinese medicine microwave-assisted extraction, purification, partial characterization, and bioactivity of polysaccharides from panax ginseng impact and consequences of polyphenols and fructooligosaccharide interplay on gut microbiota in rats studies on the preparation, crystal structure and bioactivity of ginsenoside compound k gut microbiotainvolved mechanisms in enhancing systemic exposure of ginsenosides by coexisting polysaccharides in ginseng decoction editing of the gut microbiota reduces carcinogenesis in mouse models of colitis-associated colorectal cancer anticancer effects of the microbiome and its products the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zhang, gao, bai and ning. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ugoxns authors: jensen, bent borg title: extensive literature search on the ‘effects of copper intake levels in the gut microbiota profile of target animals, in particular piglets’ date: - - journal: nan doi: . /sp.efsa. .en- sha: doc_id: cord_uid: ugoxns nan background and terms of reference as provided by the requestor . . in the context of the development of the scientific opinion linked to the mandate related to the revision of maximum levels of copper in feed, the experts of the ad-hoc working group (wg) of the feedap panel have considered relevant to evaluate the effects of copper intake levels in the gut microbiota profile of target animals. published scientific papers have addressed this issue and it seems there is a relationship between levels of dietary copper in animals and differences in their gastrointestinal microbiota, which in turn may result e.g. in more nutrients becoming available for absorption; the mode of actions have also been recently studied. a possible outcome of the opinion might be a recommendation of different maximum levels compared to those currently authorised in feed (table ) . therefore it is necessary to know the potential effects of a variation in copper intake levels on the gut microbiota profile of the target animals which might be accounted in the feedap panel opinion. piglets should be particularly considered in this literature search, as this is animal category for which the maximum copper in feed is allowed by commission regulation (ec) no / ; at least, two other relevant animal species/categories (chickens for fattening, dairy cows, fish, cat, dog) shall be included. the project should be accomplished in a structured manner, that is following an extensive literature search (els) process. the steps listed below should be followed and duly documented under the present call for tender: -development of an els protocol, which will be subject to efsa´s approval the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. -databases to be consulted -criteria to select the papers -criteria for data extraction -assessment of the quality of the data -synthesis of the data the purpose of the contract was to provide scientific assistance to the feed unit of efsa in the completion of an extensive literature search on the effects of copper intake levels in the gut microbiota profile of target animals, in particular piglets. the procedure includes the provision of a report collecting, collating, analysing and synthesising the scientific data and information on the els topic. the specific objectives of the contract resulting from the present procurement procedure are as follows: the searches in web of science resulted in hits (shown in appendix a ). the search in cab abstracts resulted in hits of which one (p in appendix a and appendix b) was not among the hits from the search in web of science. consulting the reference list of relevant papers from the search another studies/papers were retrieved (p to p in appendix a and appendix b). search terms and boolean operators (strings): ts=(copper or cu or copper sulphate) and ts=(gut or intestinal or intestine or ileal or digestive tract or gastrointestinal tract or git) and ts=(bacteria or microbiota or microflora or microorganism or microbial community) and ts=(chickens or layer or hen or broiler) the searches in web of science resulted in hits (shown in appendix a ). the search in cab abstracts resulted in hits of which one (ch in appendix a and appendix b) was not among the hits from the search in web of science. no further studies/papers were found by consulting the reference lists from relevant papers from the search. search terms and boolean operators (strings): ts=(copper or cu or copper sulphate) and ts=(gut or intestinal or intestine or ileal or digestive tract or gastrointestinal tract or git) and ts=(bacteria or microbiota or microflora or microorganism or microbial community) and ts=(cow or cattle or rumen or ruminant or bovine). the searches in web of science resulted in hits (shown in appendix a ). the search in cab abstracts resulted in hits of which all were included in the hits from the search in web of science. no further studies/papers were found by consulting the reference lists from relevant papers from the search. effect of copper intake on gut microbiota (pigs/piglets, chickens, dairy cows) www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. for the general approach to complete this els the contractor considered the technical manual for performing electronic literature searches in food and feed safety and the efsa guidance: application of systematic review methodology to food and feed safety assessments to support decision making. the inclusion criteria for the scientific papers were no limitation on timespan, no limitation on language and inclusion of primary research and review articles (the later to get a comprehensive view on the subject). the els protocol was primarily based on primary research. papers on foreign languages, where translation was not possible, were excluded.  the study had to contain a control diet and diet supplemented with one or several copper concentrations in the range of to mg/kg.  studies including the effect of any sources of dietary copper on any aspect of the microbiota in the gastrointestinal tract or faeces (profile, activity, metabolites and ph) were selected. the total number of references retrieved, the references rejected at the title and abstract screening and the references from which data was extracted are shown as a flow chart in annexes a to a . the list of included and excluded references are shown in appendix b. data collected was the effect of dietary copper in the range from to mg/kg on any aspect on the microbiota in the gastrointestinal tract or faeces. copper content: background content of feed, supplemented copper, concentration and source (data given in table for piglet/pigs and in table for broilers/layers). animals: species/category and age (weight) of the animals (data given in table for piglet/pigs and in table for broilers/layers). number of animals; animals per treatment; replicates (data given in table for piglet/pigs and in table for broilers/layers). composition and activity of the microbiota in the gastrointestinal tract or faeces (profile, activity, metabolites and ph). an overview of the data obtained is given in table for piglets/pigs and in table for broilers/layers. the data obtained is narratively described in the text and an overview of the effect of copper concentrations and sources on the concentrations on bacterial populations is shown in tables , and for piglets/pigs and in table for broilers/layer. if available, performance parameters (data given in table for piglet/pigs and in table for broilers/layers). the results are narratively described in the text and shown in tables , , , and , for piglets/pigs and in tables , and for broilers/layers. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the study design (incl. e.g. the duration of the study), the conduction of the study and the description of the study was evaluated. it was checked if the number of animals in each group in the studies were adequate. it was checked if the methods used to describe the microbiota profiles were adequate. it was checked if the statistical methods (incl. e.g. randomisation of the assignment of animals in treatment groups) used were adequate. the methodological quality of the selected studies is captured in table for piglets/pigs and in table for broilers/layers. of the references found for pigs and piglets by the literature search, were considered appropriate to be included in the els (annex a ). references were duplicates. of the excluded references, were excluded when screening the title and abstract and after consulting the full paper. the list of included and excluded references is provided in appendix b. of the references found for chickens by the literature search, were considered appropriate to be included in the els (annex a ). references were duplicates. of the excluded references, one full paper could not be retrieved, were excluded when screening the title and abstract and after consulting the full paper. it should be mentioned that out of the included papers were written in foreign languages ( in korean and in chinese) with english abstracts and table and figure legends. the list of included and excluded references is provided in appendix b. of the references found for cows by the literature search, were considered appropriate to be included in the els (annex a ). references were duplicates. of the excluded references, were excluded when screening the title and abstract and after consulting the full paper. a closer examination of the five studies first selected to be included in the els study on cows, revealed the papers not to be appropriate to be included in the els. one of the studies (cow ) was published in polish, two of the studies were dealing with antibiotic resistance (cow and cow ), one was dealing with the effect of copper on parasite control (cow ) and the last one on the effect of copper on abomasa ulcers (cow ). the list of included and excluded references is provided in appendix b. effect of copper intake on gut microbiota (pigs/piglets, chickens, dairy cows) www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. pigs and piglets . . assessment of the methodological quality of the studies table : assessment of the methodological quality of the piglet/pig studies as shown in table the quality of the papers retrieved from the search on piglet/pig was generally judged to be good although some of them were rather old. five out of papers were written in foreign languages ( in german and in chinese) with english abstracts and table and figure legends four types of copper sources were used in the research with pigs (table ) : ) inorganic copper, ) organic bound copper, ) clay bound copper and ) copper-loaded nanoparticles. inorganic copper was used in of the studies. the sources were cuso (concentrations - mg cu/kg feed, in studies p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p and p ), cuo (concentrations - mg cu/kg feed, in studies p and p ) and elemental copper (concentration mg cu/kg feed, in study p ). organic bound copper was used in of the studies. the sources were cu-aa complex (cupric amino acid complex, concentration mg cu/kg feed, in study p ), cu-cit (cupric citrate, concentrations , and mg cu/kg feed, in studies p and p ), cu-met (copper the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj methionate, concentrations and mg cu/kg feed, in study p ) and cu-gly (copper glycinate, concentrations , and mg cu/kg feed, in study p ). clay bound copper was used in of the studies. the sources were cu-mm (copper-bearing montmorillonite from aluminosilicate clay). it was used in studies p , p , p , p and p at concentrations from to mg cu/kg. copper-loaded nanoparticles were used in of the studies. the source was copper loaded chitosan nanoparticles, it was used at a concentration of . mg cu/kg feed in study p . the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. usa ----- ppm cuso . experiment ----- ppm cuso . ----- ppm cucit . ----- ppm cucit . ----- ppm cucit . ----- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the microbial characteristics measured in each of the selected studies with piglets/pigs are shown table . eighteen of the studies were done with piglets (p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p , p and p ) and were done with growing pigs (p , p , p , p , p , p , p , p and p ). the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj pigs (hu et al., ) the population of clostridium was significantly reduced in as well si as caecum content in pigs fed mg/kg cu as cu-montmorillonite compared to the control fed pigs; however, although numerically lower, the effect was not significantly different from pigs fed copper free montmorillonite. in study p (kroger et al., ) addition of mg/kg cu as cuo or cuso to diets of growing pigs were shown significantly to reduce pigs containing costridium perfringens in gut content from the small intestine and colon. no effect was found of elementary copper. the effects of cuo and cuso addition were only seen if cu was supplemented until the pigs were slaughtered at kg live weight. if it was withdrawn from the feed at kg live weight no effect was detected when the pigs were slaughtered at kg. in conclusion low concentrations of copper as clay bound copper seem to reduce the population of clostridium both in piglets and in growing pigs. further high concentrations of inorganic copper as either cuso or cuo may reduce the population of clostridium in growing pigs. coliform bacteria. the effect of copper on the population on coliform bacteria was investigated in studies with piglets (p , p , p , p , p , p , p , p , p , p , p , p , p , p ) and four studies with growing pigs (p , p , p , p ). the results from the studies are summarised in table . it may be concluded from the experiments that supplementing piglet (and growing pigs) diet with low additional copper amounts (below mg/kg cu) seems to inhibit the population of coliform bacteria in the gastrointestinal tract. especially clay bound copper seems to be effective. when supplemented to the feed in amounts above mg/kg copper it does not seem to have any effect on the population of coliform bacteria in the gastrointestinal tract of pigs. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. enterococci. the effect of copper on the population density enterococci was investigated in one study with piglets. dietary doses of mg/kg cu as cuso significantly reduced the counts of enterococci in stomach content of piglets while no effect was seen in content from the si, caecum or colon (study p ). lactic acid bacteria. the effect of copper on the population density of lactic acid bacteria was investigated in one study with piglets. dietary doses of mg/kg cu as cuso significantly reduced the counts of enterococci in stomach content of piglets while no effect was seen in content from the si, caecum or colon (study p ). the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. lactobacilli. the effect of copper on the population density and/or the composition of lactobacilli was investigated in studies with piglets (p , p , p , p , p , p , p , p , p , p , p and p ) and in four studies with growing pigs (p , p , p and p ). the results from the studies are summarised in table . in study p with piglets (højberg et al., ) dietary doses of mg/kg cu as cuso significantly reduce the counts of lactobacilli in stomach content while no effect was seen in content from the si, caecum or colon. in study p (jensen, ) the population of lactobacilli was reduced throughout the gastrointestinal tract in piglets fed mg/kg cu as cuso . in study p (ma et al., ) no effect of addition of mg/kg cu as cumm was detected on the population of lactobacilli in content from the si or colon. in study p (mei et al., ) a numeric reduction in the population of lactobacilli was detected by addition of , and mg/kg cu as cuso to piglet diets when the population density were enumerated using selectively cultivation, however if the population of lactobacilli were quantified by use of qpcr the reduction found for and mg/kg cu as cuso were significant different from the results found in the control fed piglets. in study p (mei et al., ) no significant effect of addition of mg/kg cu as cuso was found on the population of lactobacilli in cecal content. in study p (namkung et al., ) the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj from piglets was found by supplementing the diet with mg/kg in study p (kellog et al., ) . in the study with growing pigs (p , hawbaker et al., ) no significant effect on the population of staphylococci in fecal samples were detected in any of the three experiments carried out with mg/kg cu as cuso . in conclusion the effect of dietary copper on the gastrointestinal populations of staphylococci in pigs seems to be non-existent or very week. streptococci. the effect of copper on the population density and composition of streptococci was investigated in two studies with piglets (p and p ) and four experiments with growing pigs (p , p , p and p ). with piglets no significant effect in the fecal counts of streptococci were found in study p experiment and study p experiment (bunch et al., ) neither by feeding the piglets , or mg/kg cu as cuo or by feeding them mg/kg cu as cuso . there was, however, a numerical reduction in streptococci in both experiments by feeding mg/kg cu as cuso . no significant reduction in the population of streptococci was found by feeding piglets with mg/kg cu as cuso in study p (kellog et al., ) . in study p (varel et al., ) with growing pigs, mg/kg cu as cuso reduced the number of ureoluytic organisms in fecal samples with a marked decrease in the streptococcus spp., which made up % of the ureolytic bacteria in fecal samples from pigs fed the control diet. a marked fall in the number of streptococci in fecal samples was found in study p (fuller et al., ) when growing pigs were fed mg/kg cu as cuso compared to pigs fed a control diet. the extent of the reduction varied but the counts of streptococci in the copper treated pigs were always lower than in the control pigs and the reduction could be as much as from to cfu/g faeces. in study p (experiments , and ), a significant reduction of streptococci in fecal samples of growing pigs was found in experiment and while only a numerical reduction was found in experiment by supplementing the diet of growing pigs with mg/kg cuso . in study p (hendericks et al., ) , it was concluded that cuso (concentration of cu not given) strongly inhibited the growth of streptococci in an ex vivo experiment with si content from growing pigs. in conclusion the results strongly indicate that supplementation with mg cu/kg, or higher, as cuso significantly reduces the population of streptococci in the gastrointestinal tract of growing pigs. the effect of cuso on the population of streptococci in piglets is more questionable. total anaerobe bacteria. the effect of supplementary copper on the population of total anaerobic bacteria is shown in table . no effect on the anaerobic population was observed if the amount of copper added to the diet was below mg/kg copper. however, addition of mg/kg cu as cuso to the diet may reduce the population of total anaerobic bacteria in the colon, while copper as cuo does not seem to have any effect even at cu concentrations of mg/kg. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. table : effect of copper source and copper concentration on the population of anaerobic bacteria in the gastrointestinal tract of piglets and growing pigs. total aerobe bacteria. the effect of copper on the population of total aerobe bacteria was investigated in two studies with piglets (p and p ) and in one study with growing pigs (p ). in study p experimet (bunch et al., ), piglets were fed either a control diet or diets supplemented with , or mg/kg cu as cuo and a diet supplemented with mg/kg cu as cuso . a significant reduction in the population of total aerobe bacteria in faecal samples was found in the piglets fed mg/kg cu as cuso . in contrast cu as cuo resulted in a significant linear increase with increasing cuo supplementation in the population of total aerobic bacteria in faecal samples. in study p experiment , no significant effect of either mg/kg cuo or mg/kg cuso was found on the population of total aerobic bacteria in piglet faecal samples. in agreement with that no effect on the population of aerobic bacteria in fecal samples from piglets was found in study p (kellog et al., ) by supplementing the diet with mg/kg cu as cuso . in the study with growing pigs (p , hawbaker et al., ) a significant reduction in the population of total aerobic bacteria in fecal samples was detected in all three experiments carried out with mg/kg cu as cuso . in conclusion the effect of copper concentration and source on the population on total aerobe bacteria seems to be very variable. ureolytic bacteria. the number of ureolytic bacteria was investigated in study p (varel et al., ) . addition of copper sulfate to the diet at a concentration of mg/kg copper significantly reduced the number of ureolytic bacteria compared to control fed pigs, in fecal samples collected after , and weeks of feeding the experimental diets, with a marked decrease occurring in streptococcus spp., which make up % of the ureolytic isolates. quantitative real-time polymerase chain reaction (qpcr) has been used in one investigation with piglets (p , mei et al., ) to detect the effect of copper supplementation on bacterial populations (lactobacilli, enterobacteria or total anaerobic bacteria) in gastrointestinal content. it was shown that the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj the population of lactobacilli in caecum content from piglets was significantly decreased in piglets fed diets supplemented with or mg/kg cu as cuso while no effect was seen by a diet supplemented with mg/kg cu as cuso . no effect was found of any of the cuso supplemented diets on the population of enterobacteria or total anaerobic bacteria. use of traditional microbiological culturing methods did also show a reduction in the population of lactobacilli with increasing cuso addition; however these results were not significant. the microbial community was investigated in four studies with piglets (p , p , p and p ). in study p (namkung et al., ) , study p ( the results obtained from the microbial community structure studies are summarized in table . . . in all studies the number of bands per sample was measured and in study p and p the similarity indices for bands between samples were calculated. the number of bands is indicative for the number of bacterial species within a sample; the similarity coefficient is indicative of how similar the bacteria within a treatment group are. table : effect of copper sources and copper concentrations on microbial diversity and microbial similarity obtained from microbial community structure studies. no effect of copper concentration or sources was found in study p neither on the number of species nor on the similarity of the microbiota in faecal samples. however in studies p , p and p it was shown that supplementing piglet diets with high amounts of copper ( to mg/kg) as cuso affect the number of bacterial species in ileal (p ), in caecal (p and p ) and in colonic (p ) digesta. furthermore, in study p addition of mg/kg cu as cuso significantly reduced the degree of similarity of the microbiota in ileal samples compared to pigs fed a diet without supplementary copper addition. as also pointed out by the authors in study p , the combination of faecal samples and the microbial methods used in their study may not have been sensitive enough to detect differences among the dietary treatments. in study p , the similarity analysis further shows that the piglets did restore a diverse microbiota weeks after withdrawal from the high cu diet, but the community structure still seems to be different from that in the control fed pigs. in general, the community structure studies strongly suggest that supplementing piglet diets with to mg/kg cu as cuso change the microbial community in the small intestine, the caecum and the colon. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. digesta content. the amount of gastrointestinal content was investigated in study p (højberg et al., ) . the addition of high concentration of cuso ( mg/kg cu as cuso ) to piglet diets had no significant influence on digesta content in any part of the gastrointestinal tract compared to piglets fed a control diet. dry matter. the dry matter content in gastrointestinal digesta was investigated in piglets in study p (højberg et al., ) . in contrast to high concentrations of zno (addition of mg/kg zn as zno to the diet) -which significantly increase the dry matter content in the stomach and decrease the dry matter content in caecum and colon-, high concentration of cuso ( mg/kg cu as cuso ) had no significant influence on the dry matter content in any part of the gastrointestinal tract (stomach, si, caecum and colon). ph. the effect of copper on faecal ph and ph of digesta were investigated in three studies with piglets (p , p and p ) and one study with growing pigs (p ). in study p (højberg et al., ) and p (jensen ) no effect of feeding piglets diets supplemented with either mg/kg (p ) or mg/kg of cu (p ) as cuso was found on ph in any segments of the gastrointestinal tract (stomach, si, caecum and colon) compared to piglets fed control diets. in study p (zhu et al., ) a significant effect on ph in caecum and colon content was detected by feeding copper as cu-chitosan ( and mg/kg cu) or as cuso ( mg/kg cu) to piglets compared to piglets fed a control diet. in study p (huang et al., ) the effect of feeding copper supplemented diets to growing pigs on ph in faecal samples taken at the beginning of the experiment and after three and five weeks on the experimental diets were investigated. four different copper supplemented diets were used: either or mg/kg cu as cuso or or mg/kg cu as cucitrate. apart from a small but significant decrease in ph in faecal samples taken after weeks from the pigs fed the diet supplemented with mg/kg cu as cuso (ph . in faecal samples from the control fed pigs versus . in the copper fed pigs), no effect on ph was detected. in general the effect of supplementing pig diet with copper on faecal or gastrointestinal ph seems to be limited. diarrhoea. the effect of copper on diarrhoea incidence was investigated in three studies with piglets (p , p and p ) and in one study with growing pigs (p one where an antibiotic was included in the all diets tested ( mg/kg mecadox) and one without antibiotic in the diets. in both, experiment faecal samples were taken at day for measurement of the odour characteristics. in the study with antibiotic the pigs were kept on the experimental diets through the growing-finishing phase and samples taken after days for measurements of odour characteristics. in samples taken after days from the piglets fed the diets with antibiotics addition effect of copper intake on gut microbiota (pigs/piglets, chickens, dairy cows) www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. of mg/kg cu as cuso and and mg/kg cu as cu citrate significantly reduced two of the all three odour characteristics measured (odour intensity and irritation intensity) while no significant effect were found on the third characteristic (odour quality). no significant effect of diets with mg/kg cu as cuso or mg/kg cu as cuso was found on any of the odour characteristics measured. in samples taken after days from the piglets fed the diets without antibiotics, odour intensity was significantly reduced in piglets fed , and mg/kg cu as cu citrate while no significant effects were found for piglets fed or mg/kg cu as cuso . none of the experimental diets affected irritation intensity. however, mg/kg cu as cuso and , and mg/kg cu as cu citrate all significantly improved odour quality. no effect of mg/kg cuso was found on odour quality. in samples taken after days from the pig fed the diets with antibiotics odour intensity and irritation intensity were significantly reduced by all experimental diets except the diet supplemented with mg/kg cu as cu citrate which had no effect on odour intensity and irritation intensity. odour quality was significantly improved in faecal samples from all pigs fed copper supplemented diets compared to faecal samples from the control pigs. in contrast to the above mentioned results no effect of adding copper ( or mg/kg as cu as cuso or , , or mg/kg cu as cu citrate) to antibiotic supplemented diets were found in study p (armstrong et al., ) on either odour intensity, irritation intensity or odour quality of faecal samples from piglets (weaned at to days) after days feeding the experimental diets. in conclusion supplementing pig diet with or mg/kg as cu citrate or mg/kg as cuso may improve odour characteristics of swine waste. the mechanism of action was believed to be attributed to an antibiotic-like function of copper on the microbiota in the gastrointestinal tract. short chain fatty acids (scfa) concentration. lactate. microbial production of lactate was investigated in two studies with piglets (p and p ). addition of mg/kg zn as zno to the diets in study p significantly reduced the concentration of lactate in digesta in the stomach and si. however in the caecum and colon, where lactate is usually not detectable, a significant accumulation of lactate was observed in the piglets receiving the high zno diet. in contrast, the addition of mg/kg cu as cuso to the feed had no significance on the concentration of lactate in any segments of the gastrointestinal tract, but counteracted the accumulation of lactate found in the high zno fed piglets in caecum and colon. in study p (song et al., ) , the concentration of d-lactate was measured in plasma samples. d-lactate is produced by the microbiota in the gastrointestinal tract and absorbed to the blood. mammals do not have the enzymatic system to metabolise d-lactate so plasma d-lactate may be a measurement of the microbial activity in the gastrointestinal tract. a significant reduction in plasma d-lactate was found in study p in pigs fed mg/kg cu as either cu-camontmorillonite or cu-namontmorillonite compared to the control fed pigs, indicating a reduced fermentation in the gastrointestinal tract in the piglets fed the copper supplemented diets. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj gastrointestinal tract, but counteracted the accumulation of succinate in caecum and colon found in the high zno fed piglets. absorption of ammonia. effect of copper on ammonia absorption was investigated in growing pigs in study p (yen and nienaber, ) . the addition of mg/kg cu as cuso to the diet significantly reduced the net absorption of ammonia to the portal vein compared to pigs fed a diet without the addition of extra copper. as concluded by the author, that may reflect decreased urease activity and ammonia production by the gastrointestinal microbiota in the pigs receiving the copper supplemented diet. deamination and decarboxylation of amino acids. the effect of copper on microbial formation of ammonia by deamination of amino acids and production of amines (histamine, putrescine, cadaverine, tyramine and phenylethylamine) by decarboxylation of amino acids in the si has been investigated using ex vivo incubations in study p (dierick et al., ) using si content from growing pigs. both processes were severely decreased if mg/kg cu as cuso was added to the incubations, indicating that copper has a sparing action on microbial degradation of amino acids in the small intestine, leaving more amino acids available for absorption. urease activity. effect of copper on urease activity has been investigated in two studies, one study with piglets (study p ) and one study with growing pigs (study p ). in the study with piglets (p , højberg et al., ) urease activity was investigated in content from the stomach, si, caecum and colon in piglets fed diets either added mg/kg cu as cuso or control diets without the addition of extra cuso both diets were fed with or without addition of mg/kg zno. the urease activity was below the detection limit in stomach and si content, highest urease activity was found in the colon. addition of cuso to the diets had no significant effect of the urease activity. in contrast to piglets, a significant effect (p< . ) of mg/kg cu as cuso was found on urease activity in fecal samples from growing pigs in study p (varel et al., ) . atp concentrations. bacterial activity as determined by the concentration of adenosine triphosphate (atp) was measured in study p and study p . in study p (højberg et al., ) no significant effects were found on atp concentration in any of the gastrointestinal segment investigated (stomach, si caecum and colon) by addition of mg/kg cu as cuso to piglet diets compared to a control diet without additional addition of cuso . similar results were found in study p (jensen, ) , where no effect of the addition of mg/kg cuso to a diet containing mg/kg cu was found on the atp concentrations in content from the stomach, three segments from the si, the caecum and three segments from the colon. microbial enzyme activity. the effect of copper on microbial enzyme activity of β-glycosidase and βglucuronidase has been investigated in one study (p , xia et al., ) . no significant effect was found by the addition of mg/kg cu as cuso to the diets on either β-glycosidase or βglucuronidase activity in si or colon content. addition of mg/kg cu as cu-montmorillonite reduced the activity of both enzymes in si as well as colon content compared to the control fed pigs. however apart from the activity of β-glucuronidase in the colon content, the effects were not significantly different from the activities found in pigs fed copper free montmorillonite. feeding high copper diets to germ free pigs has been investigated in one study with growing pigs (p , shurson et al., ) . germ free pigs tended to have higher average daily gain (adg) and average daily feed intake (adfi) than conventionally reared pigs. addition of mg/kg cu as cuso to the feed tended to reduce adg and adfi in germ-free pigs while it increases adg and adfi in conventionally reared pigs. the high copper diets did not appear to make organ weight or intestinal characteristics in conventionally reared pigs different than those of the germ free pigs. high copper diets seem to reduce intestinal cell turnover in germ free pigs while it tended to accelerate intestinal turnover in conventionally reared pigs, perhaps via an interaction with the gut microbiota. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. coliform bacteria. it may be concluded from the experiments that supplementing piglet and growing pigs diet with low additional copper amounts (below mg/kg cu) seems to inhibit the population of coliform bacteria in the gastrointestinal tract. especially clay bound copper seems to be effective. when supplemented to the feed in amounts above mg/kg copper, it doesn't seem to have any effect on the population of coliform bacteria in the gastrointestinal tract of pigs. lactobacilli. from the results it may be concluded that supplementing pig diets with copper streptococci: in conclusion the results with growing pigs strongly indicate that the addition of mg cu/kg, or higher, as cuso significantly reduces the population of streptococci in the gastrointestinal tract of growing pigs. the effect of cuso on the population of streptococci in piglets is more questionable. in general, the studies on community structure strongly suggest that supplementing piglet diets with to mg/kg cu as cuso change the microbial community in the small intestine, as well as in the caecum and the colon. in conclusion, supplementing pig diets with or mg/kg as cu citrate or mg/kg as cuso may improve odour characteristics of swine waste. the mechanism of action was believed to be attributed to an antibiotic-like function of copper on the microbiota in the gastrointestinal tract. in general, the effect of supplementing pig diets with copper on faecal or gastrointestinal ph seems to be limited. the overall conclusion from the studies with piglets and growing pigs is that copper even at low concentrations (< mg/kg feed) may affect the microbiota in the gastrointestinal tract. especially the population of clostridia and coliform bacteria seems to be affected by low concentrations of copper. in particular, copper bound clay minerals seem to have an effect on the populations of coliform bacteria and clostridia. at higher concentrations (> mg/kg feed) cu as cuso reduce the population of lactobacilli in piglet as well as growing pigs. in growing pigs, the addition of cu as cuso reduces the population of streptococci in colonic and fecal samples, the population of ureolytic bacteria in the colon of which streptococci make up %, the urease activity in the colon, and decarboxylation and deamination of amino acids in the small intestine. however, no effect of cu as cuso on the population of streptococci and on urease activity was seen in piglets. supplementing piglet diets with to mg/kg cu as cuso significantly affects the community structure of the microbiota in the small intestine, the caecum and the colon. effect of copper intake on gut microbiota (pigs/piglets, chickens, dairy cows) www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj chickens . . assessment of the methodological quality of the studies table : assessment of the methodological quality of the chicken studies. as shown in table the quality of the papers retrieved from the search on chickens was generally judged to be low. ten out of papers were written in foreign languages ( in korean and in chinese) with english abstracts and table and figure four types of copper sources were used in the research with chickens (table ) : ) inorganic copper, ) organic bound copper, ) clay bound copper and ) copper-loaded nanoparticles. inorganic copper was used in of the studies. the sources were cuso (concentrations - mg cu/kg feed, in studies). organic bound copper was used in of the studies. the sources were cu-soy proteinate (concentrations - mg cu/kg feed, in studies ch , ch , ch , ch , and ch ), cu-met (concentrations - mg cu/kg feed, in studies ch , ch and ch ), cu-fish meal (concentration mg cu/kg feed, in study ch ), starch cu complex (concentration mg cu/kg feed, in study ch ), starch methionine cu complex (concentration mg cu/kg feed, in study ch ), and na-alginate cu complex (concentration mg cu/kg feed, in study ch ). clay bound copper was used in of the studies. the source was copper-bearing montmorillonite from aluminosilicate clay (cu-mmt). it was used in study ch and ch at concentrations from to mg cu/kg feed. copper-loaded nanoparticles were used in of the studies. the source was copper-montmorillonite nanomaterial (concentrations - mg cu/kg feed, in study ch ) and copper silicate nanoparticles (concentration mg cu/kg feed, in study ch ). the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the microbial characteristics measured in each of in the selected studies with chickens are shown in table . fifteen of the studies were done with broilers (ch , ch , ch , ch , ch , ch , ch , ch , ch , ch , ch , ch , ch , ch , ch and ch ) and were done with laying hens (ch and ch ). the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. ), a significant increase in the population of bifidobacteria in content from the cecum was found by addition of or mg/kg copper as cuso to the diet both in broilers slaughtered after and weeks feeding the treatment diets. in contrast to that, addition of mg/kg copper to the diet as cuso resulted in a significant reduction in the population of bifidobacteria in cecal content in broilers slaughtered after weeks while no effect was found in broilers slaughtered after weeks. in all studies the population density of bifidobacteria were determined by counting the bacteria on selective media but several studies has shown that no really good selective media exist for the enumeration of bifidobacteria. campylobacter. the effect of copper on the population density of campylobacter was investigated in study ch (aydin et al., ) . no significant effect of an addition of mg/kg copper as cuso to the diet was found on the population density of campylobacter in ileal content. clostridia. the effect of copper on the population density on clostridia in content from the small intestine was investigated in studies with broilers (ch , ch , ch , ch , ch and ch ) and in two studies with layers (ch and ch ). the effect of copper concentration and copper sources on populations of clostridia in chicken gut content is shown in table . a significant reduction in the population of clostridia in si content from broilers were found by the addition of mg/kg cu as cuproteinate in study ch (kim et al., a) the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. cu-proteinate. similarly, no effect on the population of clostridia was found in study ch (min et al., ) by the addition of mg/kg cu as either cuso , cu-methionine or cu-fish meal to the diet; or in study ch by the addition of mg/kg cu as cuso . in the studies with layers a significant reduction of the population of clostridia were found in both investigations (ch and ch ). in study ch (choi et al., ) the population of clostridia was reduced by the addition of and mg/kg cu whatever it was added to the diet as cuso or cu-proteinate, and in study ch (paik et al., ) , by addition of and mg/kg cu whatever it was added as cu-methionine or cu-proteinate. the effect of copper on the density of clostridia in content from the caecum was investigated in two studies (ch and ch ). in study ch (xia et al., ) a significant reduction in the population of clostridia was found by the addition of mg/kg cu as cu-montmorillonite to the diet while no effect was found by the addition of mg/kg cu as cuso to the diet. in study ch (xu et al., ) no effect on the population of clostridia in caecum content was found by the addition or mg/kg cu-montmorillonite to the diet. supplementing chicken (broilers and layers) diets with additional copper seems to reduce the population of clostridia in the gastrointestinal tract of the birds, even at low copper concentrations. in particular, clay bound copper seems to be effective in reducing the population of clostridia. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. coliform bacteria. the effect of copper on the population density of coliform bacteria in content from the gizzard was investigated in three investigations (ch , kim et al., and ch experiments and , min et al., ) . as shown in the effect of copper on the population density of coliform bacteria in si content was investigated in experiments with broilers and in experiments with layers (table ) . a significant reduction in the population of coliform bacteria was found in study ch (choi et al., ) by the addition of , or mg/kg cu as cuso to the diet, while no effect of an addition of mg/kg cu as cuso was found in studies ch (kim et al., ) , ch (min et al., ) , ch experiments and (min et al., ) ; and of mg/kg cu as cuso in study ch . furthermore, no effect was found in layers (ch , kim et al., b) fed diets containing or mg/kg cu as cuso . in study ch (kim et al., ) a significant reduction in the population of coliform bacteria in broiler si content was found by the addition of mg/kg cu as cu-methionine to the diet while it had no effect at a concentration of mg/kg. no effect was found by the addition of mg/kg cu as cu-methionine in study ch (min et al., ) and no effects were found in layers at concentrations of and mg/kg (ch , paik et al., ) . copper as cu-protionate was shown to reduce the population of coliform bacteria in study ch (kim et al., ) when it was added to the diet at a concentration of mg/kg cu while it had no effect at a concentration of mg/kg. no effect of cu-protionate was found in study ch (aydin et al., ) at a concentration of mg/kg cu or in study ch (kim et al., a) at a concentration of mg/kg cu. cu-propionate at concentrations of and mg/kg cu had no effect on the density of coliform bacteria in si content from layers neither in study ch nor in study ch (paik et al., ) . a significant reduction in the population of coliform bacteria in si content from broilers were found in study ch (ma et al., ) conducted where small intestinal content was incubated for hours with or without addition of copper. in both experiments it was found that mg/kg as cuso reduced the growth of coliform bacteria, while mg/kg cu as cuso or tribasic copper chloride at concentrations of mg/kg and mg/kg had no effect on the growth of coliform bacteria. the effect of copper on the population of coliform bacteria in caecal content has been investigated in seven studies with broilers (ch , ch experiment , ch experiment , ch , ch , ch and ch ). a summary of results from the experiments are shown in table . no effect on the population of coliform bacteria were found by feed concentrations of mg/kg cu as cuso in study ch (kim et al., ) the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj (ch experiment , min et al., ) , or with mg/kg cu as a cu-starch/methionate complex (ch experiment and ch experiment ) or with a na-alginate cu complex (ch experiment ). in general the effect of copper on the gastrointestinal populations of coliform bacteria seems to be rather weak, in most cases where an effect was seen, the concentrations were higher than mg/kg cu; the exception may be copper bound clay particles, especially copper bound nanoparticles that seems to have an effect at concentrations below mg/kg cu (ch and ch ). that high concentrations of copper may reduce the population of coliform bacteria in gut content from chickens is in agreement with the ex vivo experiment studies (ch and ch ) that show that cu as cuso inhibit the growth of coliform bacteria at a concentration of mg/kg cu but not at a concentration of mg/kg cu. enterococci. the effect of copper on the population of enterococci has only been investigated in a single experiment (ch , aydin et al., ) . supplementation of the diet with cu-proteinate corresponding to a cu concentration at mg/kg cu had no significant effect on the population of enterococci in broiler ileal content. lactobacilli. the effect of copper concentration and copper sources on populations of lactobacilli in gut content is shown in table . the population lactic acid bacteria (lab) in content from the gizzard was investigated in three experiments with broilers: ch (kim et al., ) , ch experiment and ch experiment (min et al., ) . no effect of either copper concentration or copper source was found (table ). the effect of copper on the population density of lactobacilli in si content was investigated in experiments with broilers and in experiments with layers. a significant reduction in the population of lactobacilli was found in study ch (choi et al., ) by addition of or mg/kg cu as cuso to the diet, while no effect was found by addition of mg/kg cu as cuso . no effect of the addition of mg/kg cu as cuso was found in studies ch (kim et al ) , ch (min et al., ) , ch experiments and (min et al., ) ; and of mg/kg cu as cuso in study ch (xia et al., ) . in the studies with layers (ch , kim et al., b ) the population of lactobacilli was significantly increased when the diet was supplemented with either or mg/kg cu as cuso . cu-methionat at a cu concentration of mg/kg cu had no effect on the population of lactobacilli (ch , min et al., ) while at cu concentrations of mg/kg, it had a significant stimulating effect on the population of lactobacilli in broiler si content (ch , kim et al., ) . only a tendency to an increase (ch ) was found when it was added to a diet at a concentration of mg/kg cu. in layers fed a diet supplemented with mg/kg cu as cu-methionate a significant increase in the population of lactobacilli was observed (ch , paik et al., ) , while at a concentration of mg/kg it resulted in a tendency to an increase (ch ). addition of cu-propionate corresponding to cu amount of mg/kg to broiler diets resulted in an increase in the population in study ch while no effect was seen in study ch (kim et al., a) . at an addition corresponding to a cu concentration of mg/kg no effect was found of cu-propionate on the population of lactobacilli. in layers addition of cu-propionate corresponding to an addition of mg/kg cu resulted either in a significant reduction in the population of lactobacilli (study ch ) or to a tendency to an increase (study ch ). both in study ch and ch , an increase was detected in the population of lactobacilli when layer diets were supplemented with ppm cu as cu-propionate. no effect on the population of lactobacilli in si content from broilers were found in study ch by addition of or mg/kg cu as cu- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the population lactobacilli in content from the caecum of broilers was investigated in seven experiments (table ) : ch , ch experiment , ch experiment , ch , ch , ch and ch ). in experiment ch , no effects of supplementing feed with mg/kg cu as cuso were found on the population of lactobacilli in cecal content. the same was the case in study experiments and when feed was supplemented with mg/kg cu-starch, cu starch/met and cu-naalginate; in experiment ch , for supplementation with mg/kg cuso ; and in experiments ch and ch for supplementation with and mg/kg cu-montmorillonite. in study ch , addition of mg/kg cu as cu-montmorillonite-nanomaterial to broiler fed stimulated the population of lactobacilli in cecal content. the same was found in study ch when it was added at concentrations of mg/kg and mg/kg cu as cuso to broiler diets, while cu as cuso at a concentration of mg/kg resulted in a significant reduction in the population of lactobacilli. from the investigation of copper concentrations and sources on the population of lactobacilli in gastrointestinal content it may be concluded that copper concentrations at or below mg/kg seems to stimulate the population of lactobacilli in chicken gut content, while it does not seem to have any effect at concentrations from to mg/kg but has an inhibitory effect at concentrations above streptococci. the effect of copper on the population of streptococci was investigated in three experiments with broilers from two studies (ch and ch experiment and ). in study ch (choi et al., ) the population of streptococci were higher after week but lower after weeks in content from the si of broilers fed either , or mg/kg cu as cuso compared to the control fed birds, while no effect were seen of any of the cu concentrations after or weeks on the experimental diets. in study ch experiment (min et al., ) the effect of the addition of mg/kg cu as cuso , cu-starch or cu-starch/methionine to diets to broilers on the population of streptococci in content from the gizzard, upper and lower si and cecum was investigated. no effect of any of the added copper sources was found on the population of streptococci in any of the investigated git segments. in study ch experiment , the effect of the addition of mg/kg cu as cuso , a cu-starch /methionine complex, a cu-starch/methionine complex (added to a diet where the methionine was reduced with the amount of methionine added with the cu-starch/methionine complex) and a na-alginate cu complex to diets of broilers on the population of staphylococci in content from the gizzard, upper and lower si and cecum was investigated. no effect of any of the added copper sources was found on the population of streptococci in any of the investigated git segments. in general supplementing broiler diets with additional copper dose not seems to have any effect on the population of streptococci in the gastrointestinal tract. total aerobe bacteria. the effect of copper on the population of total anaerobic bacteria was investigated in three studies (ch , ch and ch ). no effect was found in study ch (ma et al., ) the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the microbial community was investigated in two studies (ch and ch ). in both studies the microbial community was determined by isolating bacterial dna, amplifying the v region of the s ribosomal dna and performing denaturing gradient gel electrophoreses (dgge). in both studies the number of bands per sample and the similarity indices for bands between samples were calculated. the number of bands is indicative for the number of bacterial species within a sample whereas the similarity coefficients indicate how similar the bacteria within a treatment group are. in study ch (thompsen et al., ) broilers were fed either a control diet, the control diet supplemented with mg/kg bacitracin or the control diet supplemented with mg/kg cu as cuso from day to day . at day , six birds per treatment group were slaughtered and the bacterial community structure analysed in samples of ileal content and samples of ileal mucosa. amplicon profiles from the ileal content revealed that dietary treatment had no effect on band numbers or similarity coefficients. likewise, band numbers of the mucosa-associated bacteria were not affected by dietary treatments. the comparison similarity coefficients, however, revealed that the mucosa-associated bacterial communities of birds fed mg/kg cu as cuso had higher similarity indices than either the control or bacitracin fed broilers. the high similarity coefficient observed in birds fed copper might indicate that addition of high concentrations of copper may select for copper resistant bacteria. in study ch (pang et al., a) broilers were fed either a control diet, a diet supplemented with mg/kg cu as cuso or a diet supplemented with mg/kg cu as tribasic copper chloride (tbcc) from hatch to day . on day eight birds per treatment were slaughtered and the bacterial community structure analysed in samples of ileal content and samples of ileal mucosa. neither cu supplementation at . mg/kg nor cu sources affected the number of predominant bacterial species (dgge band number) in ileal content (mean = . ) or ileal mucosa (mean = . ). furthermore, neither cu supplementation at . mg/kg nor cu sources (cuso or tbcc) affected the similarity coefficient of the microbial community structure in ileal content. however supplementation of the diet with . mg/kg cu as tbcc increased the similarity coefficient of ileal mucosal bacteria, while supplementing the diet with . mg/kg cu as cuso did not affect the similarity coefficient of ileal mucosal bacteria. this shows that the mucosa associated microbiota was more similar between individual birds for the tbcc fed broilers, compared to the control and the cuso fed birds. in conclusion the effect of copper supplementation of broiler diets seems to have little effect on the community structure of the microbiota in the small intestine. no effect of supplementing broiler diets with either or mg/kg cu as cuso or mg/kg cu as tbcc were found on band numbers (number of bacterial species) neither in digesta samples nor in mucosal samples. moreover, no effect of copper www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj supplementation was found on population similarity in digesta samples, while mg/kg cu as cuso affected the population similarity in mucosa samples in study ch but no effect of mg/kg cu as cuso was found on the population similarity in mucosal samples in study ch . in study ch , however, an effect of mg/kg cu as tbcc was found on the population similarity in mucosal samples. fecal moisture content. fecal moisture content was investigated in one study (ch , choi et al., ) . it was measured each week during the six weeks of the experiment. the moisture content was significantly higher at week in the chickens fed and mg/kg cu as cuso and numerically higher in the group fed mg/kg cu as cuso compared to the control group ( . , . , . and . % for the control group and the groups fed diets supplemented with , and mg/kg cu as cuso , respectively). no effect on the moisture content of any of the copper concentrations investigated was found from week to . ph. the effect of copper on ph of gastrointestinal or fecal content was investigated in five experiments with broilers (ch , ch , ch experiment , ch experiment and ch ). in study ch (aydin et al., ), no effect of mg/kg cu as cu-proteinate was found on ph of ileal content in broilers slaughtered at day . in study ch experiment (min et al., ) , the addition of mg/kg cu as either cuso , a cu-starch complex or a cu-starch/methionine complex to broiler diets had no effect on ph of content from gizzard, upper si, lower si or cecum taken from broilers slaughtered at day . in study ch (min et al., ) , supplementation of broiler diets with mg/kg cu as cuso , cu-methionine or cu-fish meal significantly reduced the ph in content from the gizzard ( . , . , . and . for the control group and the three cu supplemented groups, respectively) from broilers slaughtered at day . no effects of any of the copper sources were found on ph in content from the upper or lower si or the caecum. in study ch experiment (pang et al., ), supplementation of broiler diets with mg/kg cu as either cuso , a cu-starch /methionine complex, a cu-starch/methionine complex (added to a diet where the methionine was reduced with the amount of methionine added with the cu-starch/methionine complex) and a na-alginate cu complex significantly reduced the ph in content from the gizzard ( . , . , . , . , . for the control group and the four cu supplemented groups respectively) from broilers slaughtered at day . no effect of any of the copper sources was found on ph in content from the upper or lower si, while the cu starch complex and the na-alginate cu complex significantly reduced the ph in content from the caecum ( . , . and . for the control group and the two cu supplemented groups, respectively). in study ch (shao et al., ) , supplementation of broiler diets with mg/kg cu as cu silicate nanoparticles significantly reduce the ph of content from the caecum from birds slaughtered at day ( . and . for the control fed and the copper fed birds, respectively). no effect of copper supplementation were found on ph of caecal content from birds slaughtered at days or on ph of fecal samples taken throughout the experimental period of days. in conclusion the effect of supplementing broiler diets seems to reduce ph in gizzard content while the effect on the rest of the gastrointestinal tract seems to be marginal. ammonia emission. effect of copper on ammonia emission was investigated in study ch (shi et al., ) . supplementation of broiler diets with mg/kg cu as cu silicate nanoparticles significantly reduce the ammonia emission at day , , , , and to about half of the emission found for the control fed birds. gizzard erosion. the effect of copper on gizzard erosion was investigated in five experiments with broilers (ch , ch , ch , ch experiment , and ch experiment ). in study ch (choi et al., ) , gizzard erosion was investigated in broilers fed four different diets (a control and three diets supplemented with mg/kg, mg/kg and mg/kg cu as cuso ). gizzard erosion was measured in three birds per treatment after , and weeks on the experimental diet. gizzard erosion indices were scored from (normal) to (severe). the incidence of gizzard erosion increased as the level of copper in the diet increased and with time on the experimental diets. after weeks on the experimental diet the gizzard erosion indices were . , . , . and . for the birds fed the www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj control diet and the birds fed , and mg/kg cu as cuso , respectively. in study ch (kim et al., ,) gizzard erosion was investigated in broilers in a x factorial experiment including levels of added fat ( % and %) and levels of added copper (no addition, mg/kg cu as cuso .h o and mg/kg cu as cuso . h o). again increasing gizzard erosion indices were evident in birds fed the copper supplemented diets; both in the birds fed the low and the high fat diet. in experiment ch (min et al., ) , gizzard erosion was investigated in birds fed a control diet and three diets supplemented with three different copper sources ( mg/kg cu as cuso , mg/kg cu as cu-methionine and mg/kg cu as cu-fish meal). gizzard erosion was measured as described for study ch in four birds per treatment after days on the experimental diets. again severe gizzard erosion was observed in the birds fed the copper supplemented diets ( . , . , . and . for the control fed birds and the birds fed mg/kg cu as cuso , cu-methionine and cu-fish meal, respectively). in study ch experiments and (min et al., ) , gizzard erosion was investigated for five sources of copper all added to the diets to increase the cu concentration with mg/kg. the five copper sources were cuso , a cu-starch complex, a cu-starch/methionine complex, a cumethionine complex and a na-alginate cu complex. regardless of copper sources, severe gizzard erosion was observed in all broilers fed copper supplemented diets in all four studies where it was investigated. in conclusion, supplementation of broiler diets with copper at concentrations above mg/kg strongly increases gizzard erosion. ammonia concentration. effect of copper on ammonia concentration in fecal samples was investigated in study ch (shi et al., ) . supplementation of broiler diets with mg/kg cu as cu silicate nanoparticles had no effect on ammonia concentration in fecal samples neither on day nor on day . microbial enzyme activity. the effect of copper on microbial enzyme activity of β-glycosidase and βglucuronidase has been investigated on two studies with broilers (ch and ch ). in study ch , broilers were fed a control diet and two experimental diets supplemented with either or mg/kg cu as cu-montmorillonite-nanomaterial (cu-mmn). the diets were fed to the broilers for days. eight birds from each treatment were slaughtered at day and day and the enzyme activity measured in content from the si and the caecum. the activities of β-glucosidase and β-glucuronidase in the small intestine of birds fed on the diet supplemented with both concentrations cu-mmn were significantly lower than the control, both for the birds slaughtered at day and for the birds slaughtered at day . similarly both enzyme activities were reduced in the cu-mmn fed birds compared to the control fed birds in caecal content at day but not at day . in study ch (xu et al., ) , broilers were fed a control diet and two experimental diets supplemented with either g/kg montomorillonite (mm) or g/kg cu-montomorillonite (cu-mm) (resulting in an addition of mg/kg cu to the feed). the diets were fed to the broilers for days. eight birds from each treatment were slaughtered at day and day and the enzyme activity measured in content from the si and the caecum. the activities of β-glucosidase and β-glucuronidase in the small intestine of birds fed on the diet supplemented with cu-mm were significantly lower than the control both for the birds slaughtered at day and for the birds slaughtered at day . β-glucosidase activities were reduced in the cu-mm fed birds compared to the control fed birds in caecal content at day but not at day . the caecal β-glucuronidase was not significantly affected by cu-mm. the enzyme activities in the mm fed birds were somehow between the control fed birds and the birds fed the copper enriched mm (cu-mm). clostridia: supplementing chicken (broilers and layers) diets with additional copper seems to reduce the population of clostridia in the gastrointestinal tract of the birds, even at low concentrations of copper. especially clay bound copper seems to be effective in reducing the population of clostridia. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. coliform bacteria: in general the effect of copper on the gastrointestinal populations of coliform bacteria seems to be rather weak; in most cases where an effect was seen, the concentrations was higher than mg/kg cu, the explanation may be copper bound clay particles, especially copper bound nanoparticles seem to have an effect at concentrations below mg/kg cu (ch and ch ). that high concentrations of copper may reduce the population of coliform bacteria in gut content from chickens is in agreement with the ex vivo experiment studies (ch and ch ) that show that cu as cuso inhibit the growth of coliform bacteria at a concentration of mg/kg cu but not at a concentration of mg/kg cu. lactobacilli: from the investigation of copper concentrations and sources on the population of lactobacilli in gastrointestinal content it may be concluded that copper concentrations at or below mg/kg seem to stimulate the population of lactobacilli in chicken gut content, while it does not seem to have any effect at concentrations from to mg/kg but has an inhibitory effect at concentrations above mg/kg. in agreement with that, the ex vivo experiments (ch and ) show that mg/kg cu as cuso stimulated the growth of lactobacilli in broiler si content. however no inhibitory effect on the growth was seen by a copper concentration of mg/kg cuso . that no effect of tribasic copper chloride (tbcc) was seen in the ex vivo experiment is properly due to the low solubility of tbcc in water (ch ). streptococci. supplementing broiler diets with additional copper does not seem to have any effect on the population of streptococci in the gastrointestinal tract. aerobe bacteria: only three studies could be found and copper supplementation showed no effect (the effect in study ch may be an effect of the nanoparticles rather than an effect of copper). total anaerobe bacteria: copper supplementation had no effect in most studies (only in one study with high copper concentration). ex vivo studies. mg/kg cu as cuso inhibit the growth of coliform bacteria in chicken si content. no effect of mg/kg cuso on growth of lactobacilli. no effect on growth of either coliform bacteria or lactobacilli by tbcc (properly due to low water solubility). ph. supplementing broiler diets with copper seems to reduce ph in gizzard content while the effect on the rest of the gastrointestinal tract seems to be marginal. community structure. the effect of copper supplementation of broiler diets seems to have little effect on the community structure of the microbiota in the small intestine. neither cu supplementation at . mg/kg or mg/kg nor cu sources (cuso or tbcc) affected the number of predominant bacterial species (dgge band number) in ileal content or ileal mucosa or affected the similarity coefficient of the microbial community structure in ileal content. in study ch , it was found that supplementation of the diet with . mg/kg cu as tbcc increase the similarity coefficient of ileal mucosal bacteria while supplementing the diet with . mg/kg cu as cuso had no effect. in contrast to that, it was found in study ch that supplementing broiler diets with mg/kg cu as cuso increase the similarity coefficient of the mucosa associated microbiota. gizzard erosion. supplementation of broiler diets with copper at concentrations above mg/kg strongly increase gizzard erosion. of the references found for cows by the literature search, were in the first phase considered appropriate to be included in the els. however, a closer examination of the five studies revealed the papers not to be appropriate to be included in the els. one of the studies was published in polish, two of the studies were dealing with antibiotic resistance, one was dealing with the effect of copper on parasite control and the last one on the effect of copper on abomasal ulcers. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. of the references found for pigs and piglet by the literature search, were considered appropriate to be included in the els. in total different characteristics related to the gastrointestinal microbiota were investigated in the selected studies. the number of characteristics investigated in the individual studies varied from to . the number of studies in which each characteristic was studied varied from to . the characteristic studied could be divided in themes: plate count of bacteria ( studies), qpcr of bacterial populations ( study), microscopic counts of bacteria ( study), community structure analyses ( studies), gut content characteristics ( studies), microbial metabolites ( studies), microbial activity ( studies) and studies in germ free pigs ( study). the copper concentrations used in the studies varied from . to mg/kg feed. four types of copper sources were used in the studies: inorganic bound copper ( studies at concentrations from - mg/kg feed), organic bound copper ( studies at concentrations from - mg/kg feed), copper bound to clay minerals ( studies at concentrations from - mg/kg feed) and copper-loaded nanoparticles ( study at a concentration of . mg/kg). it may be concluded from the experiments that supplementing pig diets with low additional copper amounts (below mg/kg cu) seems to inhibit the population of coliform bacteria in the gastrointestinal tract of as well piglets as growing pigs. in particular, clay bound copper seems to be effective. when supplemented to the feed in amounts above mg/kg copper, the effect on the population of coliform bacteria in the gastrointestinal tract of piglets as well as pigs was marginal. supplementing pig diets with copper concentrations above mg/kg as cuso has a reducing effect on the population of lactobacilli in the gastrointestinal tract of as well piglets as growing pigs. the effect of cu as cuo did not affect the population of lactobacilli at high concentrations ( and mg/kg feed). diets with copper concentration below mg/kg feed seem to have marginal effect on the population of lactobacilli in the gastrointestinal tract both in piglets and in growing pigs. the results with growing pigs strongly indicate that the addition of mg cu/kg, or higher, as cuso significantly reduces the population of streptococci in the gastrointestinal tract of growing pigs. the effect of cuso on the population of streptococci in piglets is more questionable. community structure studies strongly suggest that supplementing piglet diets with to mg/kg cu as cuso affects the community structure of the microbiota in the small intestine, the caecum and the colon. the overall conclusion from the studies with piglets and growing pig is that copper, even at low concentrations (< mg/kg feed), may affect the microbiota in the gastrointestinal tract. especially the population of clostridia and coliform bacteria seems to be affected of low concentrations of copper, and especially copper bound clay minerals seem to have an effect. at higher concentrations (> mg/kg feed) cu as cuso reduces the population of lactobacilli in piglets as well as in growing pigs. in growing pigs cuso reduces the population of streptococci in colonic and fecal samples, the population of ureolytic bacteria in the colon of which streptococci make up %, the urease activity in the colon, and decarboxylation and deamination of amino acids in the small intestine. no effect of cu as cuso on the population of streptococci and on urease activity was seen in piglets. supplementing piglet diets with to mg/kg cu as cuso significantly affected the community structure of the microbiota in the small intestine, the caecum and the colon. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. of the references found for chickens by the literature search, were considered appropriate to be included in the els. in total different characteristics related to the gastrointestinal microbiota were investigated in the selected studies. the number of characteristics investigated in the individual studies varied from to . the number of studies in which each characteristic was studied varied from to . the characteristic studied could be divided in themes: plate count of bacteria ( studies), community structure analyses ( studies), gut content characteristics ( studies), microbial metabolites ( study) and microbial activity ( studies). the copper concentrations used in the studies varied from to mg/kg feed. four types of copper sources were used in the studies: inorganic bound copper ( studies at concentrations from - mg/kg feed), organic bound copper ( studies at concentrations from - mg/kg feed), copper bound to clay minerals ( studies at concentrations from - mg/kg feed) and copper-loaded nanoparticles ( study at a concentration from - mg/kg). from the plate count investigations of bacterial population it can be concluded that supplementing chicken (broilers and layers) diets with additional copper seems to reduce the population of clostridia in the gastrointestinal tract of the birds, even at low concentrations of copper. especially clay bound copper seems to be effective in reducing the population of clostridia. in general the effect of copper on the gastrointestinal populations of coliform bacteria seems to be rather week, in most cases where an effect were seen the concentrations was higher than mg/kg cu, the exception may be copper bound clay particles, especially copper bound nanoparticles seems to have an effect at concentrations below mg/kg cu. that high concentrations of copper may reduce the population of coliform bacteria in gut content from chickens is in agreement with the ex vivo experiment studies that show that cu as cuso inhibits the growth of coliform bacteria at a concentration of mg/kg cu but not at a concentration of mg/kg cu. from the investigation of copper concentrations and sources on the population of lactobacilli in gastrointestinal content it may be concluded that copper concentrations at or below mg/kg seems to stimulate the population of lactobacilli in chicken gut content, while it does not seem to have any effect at concentrations from to mg/kg. however, it has an inhibitory effect at concentrations above mg/kg. in agreement with that, the ex vivo experiments show that mg/kg cu as cuso stimulated the growth of lactobacilli in broiler si content. however, no inhibitory effect on the growth was seen by a copper concentration of mg/kg cuso . in conclusion the effect of supplementing broiler diets with copper at concentrations of mg/kg either as inorganic or organic bound copper seems to reduce ph in gizzard content while the effect in the rest of the gastrointestinal tract seems to be marginal. in conclusion the effect of copper supplementation of broiler diets seems to have little effect on the community structure of the microbiota in the small intestine. neither cu supplementation at . mg/kg or mg/kg nor cu sources (cuso or tbcc) affected the number of predominant bacterial species (dgge band number) in ileal content or ileal mucosa or affected the similarity coefficient of the microbial community structure in ileal content. in study ch , it was found that supplementation of the diet with mg/kg cu as tbcc increase the similarity coefficient of ileal mucosal bacteria while supplementing the diet with mg/kg cu as cuso had no effect. in contrast to that, it was found in study ch that supplementing broiler diets with mg/kg cu as cuso increase the similarity coefficient of the mucosa associated microbiota. supplementation of broiler diets with copper at concentrations above mg/kg strongly increases gizzard erosion. the overall conclusion from the studies with broilers is that copper even at low concentrations (< mg/kg feed) may affect the microbiota in the gastrointestinal tract. especially the population of clostridia seems to be affected by low concentrations of copper. in particular, copper bound clay minerals seem to have an effect. at higher concentrations (> mg/kg feed) inorganic or organic effect of copper intake on gut microbiota (pigs/piglets, chickens, dairy cows) www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. bbj bound copper also seems to affect the population of lactobacilli, and coliform bacteria, to reduce the ph in gizzard content and to produce severe gizzard erosion. of the references found for cows by the literature search, were in the first phase considered appropriate to be included in the els. however, a closer examination of the five studies revealed the papers not to be appropriate to be included in the els. one of the studies was published in polish, two of the studies were dealing with antibiotic resistance, one was dealing with the effect of copper on parasite control and the last one on the effect of copper on abomasal ulcers. while implementing this extensive literature search some suggestions and remarks were raised:  the quality of the papers retrieved from the search on chickens was generally judged to be low. out of papers were written in foreign languages ( in korean and in chinese) with english abstracts and table legends .  a closer examination of the five studies first selected to be included in the els study on cows, revealed the papers not to be appropriate to be included in the els. one of the studies (cow ) was published in polish, two of the studies were dealing with antibiotic resistance (cow and cow ), one was dealing with the effect of copper on parasite control (cow ) and the last one on the effect of copper on abomasa ulcers (cow ). www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. schole j, szasz e, peters j, eikemeyer j and sickel e, . the total anabolic activity of the blood as a connecting link between the intestinal-flora theory and the intermediary action of growth the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. new bacterium comprising lactic acid bacterium and bacterium of genus bifidobacterium comprising metal nanoparticle bound to its surface, useful e.g. as contrast agent for mri of digestive tract of subject, and for treating cancer contribution to the study of gut hypersensitivity reactions to soybean proteins in preruminant calves and early-weaned piglets ddesaturation of organ lipids and tissue-lipids and influence on depot triglyceride structure by feeding high levels of different copper-compounds composite preblend material for pig capable of improving immunologic function includes ferrous sulfate, copper sulfate, zinc sulfate, manganese sulfate, composite microbial ecological agent, ethoxyquinoline, choline chloride and bran nutritional alternatives for antimicrobial control in pigs scientific opinion on the safety and efficacy of copper compounds (e ) as feed additives for all animal species: cupric sulphate pentahydrate based on a dossier submitted by manica s swine. proceedings of the rd western nutrition conference, optimizing efficiency of animal production how to avoid making mistakes when choosing alternatives to feed antibiotics. svinovodstvo (moskva) silver nanoparticles as a potential antimicrobial additive for weaned pigs utilization of fermented flocculated poultry sludge as a feed constituent for pigs nutritional compound additive alleviates jejunal mucosal structure disruption of piglets challenged with feed containing corn contaminated with mycotoxins this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document antibacterial effects of the cu(ii)-exchanged montmorillonite on escherichia coli k and salmonella choleraesuis antibacterial effects of the cu(ii)-loaded montmorillonite in vitro and its on mechanisms effect of supplementing tylosin and olaquindox or copper on growth, tissue mineral contents and enteric bacteria counts of weanling pigs bacterial resistance to copper in animal feeds feed without antibiotic by microbial fermentation for later period of growing/fattening pigs comprises batch mixture and compound fermented material e.g. lactobacillus acidophilus, aspergillus niger in specific weight ratio microbially fermented piglet early growth feed without antibiotic comprises e.g. bacillus subtilis, aspergillus based fermentation material and composite material action of antimicrobial agents on intestinal bacteria of pigs in vitro gastrointestinal health and function in weaned pigs: a review of feeding strategies to control post-weaning diarrhoea without using in-feed antimicrobial compounds antimicrobial use in swine production and its effect on the swine gut microbiota and antimicrobial resistance effects of dietary oligosaccharides on the growth performance and faecal characteristics of young growing pigs effects of montmorillonite-zinc oxide hybrid on performance, diarrhea, intestinal permeability and morphology of weanling pigs effects of cu +-exchanged montmorillonite on intestinal microflora, digestibility and digestive enzyme activities of nile tilapia compound feed additive for improving pig intestinal tract includes yeast extract, kelp, glucagon-like peptide- , potassium, wheat bran, fermented straw, fructooligosaccharide, stachyose, cellulase effect of high dietary zinc oxide on the caecal and faecal short-chain fatty acids and tissue zinc and copper concentration in pigs is reversible after withdrawal of the high zinc oxide from the diet analysis of antibiotic and copper resistance of escherichia coli isolated from feces of sows and piglets before and after weaning preparation of microbial feed additive for reducing stress of piglet involves preparing culture medium of specified ph, culturing strain to specified bacterial impact of exocrine pancreas on digestion in pigs protective efficacy of azomite enriched diet in oreochromis mossambicus against aeromonas hydrophila effect of pharmacological intakes of zinc and copper on growth performance, circulating cytokines and gut microflora of newly weaned piglets challenged with coliform lipopolysaccharides lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology in nursery pigs influence of diet on microbial community structure and activity in the intestinal tract of weanling pigs impact of the diet on digestive disorders of pigs, with special emphasis on proliferative enteropathy and swine dysentery gut health in the pig the effect of including additives in feeds for pigs on the appearance of resistance in e-coli prevalence of strains with multiple resistance to antibiotics, and the transferability in vitro of the r factor of strains of effect of organic acid supplementation on the performance and ileal microflora of broiler during finishing period new microelement organic chelate complex compound useful e.g. for inhibiting pathogenic bacteria e.g. salmonella, and treating or preventing disease e.g. poultry enteric diseases, swine enteric diseases and bovine enteric diseases the evolution of codon usage in structural and non-structural viral genes: the case of avian coronavirus and its natural host gallus gallus effect of two microbial phytases on mineral availability and retention and bone mineral density in low-phosphorus diets for broilers compound feed additive for improving pig intestinal tract includes yeast extract, kelp, glucagon-like peptide- , potassium, wheat bran, fermented straw, fructooligosaccharide, stachyose, cellulase viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens probiotics preventive nutrition in chicken intensive production practices. lucrai stiinifice -medicina veterinara, universitatea de stiinte agricole si medicina veterinara "ion ionescu de la brad dietary role of phytase to improve minerals bioavailability for bone conformation characterisation and histopathological observations of a selected brazilian precocious line of eimeria acervulina apparent total tract macronutrient and energy digestibility of -to- -day-old whole chicks, adult ground chicken, and extruded and canned chicken-based diets in african wildcats (felis silvestris lybica) effects of dietary supplementation of copper-sulfate and copper-soy proteinate on the performance and small intestinal microflora in laying hens effects of supplemental coppermethionine chelate and copper-soy proteinate on the performance, blood parameters, liver mineral content, and intestinal microflora of broiler chickens effects of interaction of copper sulfates and dietary fat on the performance of broiler chickens effect of dietary copper source and level on gi copper levels and ileal e. coli survival in broiler chicks free-flowing particulate powder useful in animal composition for preventing or treating intestinal infection in animal, and/or increasing growth of animal effects of freeze-dried mitsuokella jalaludinii culture and natuphos (r) phytase supplementation on the performance and nutrient utilisation of broiler chickens chromium cr(vi) biosorption property of the newly isolated actinobacterial probiont streptomyces werraensis ld . biotech influence of dietary inclusion of bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens bacillus amyloliquefaciens supplementation alleviates immunological stress and intestinal damage in lipopolysaccharide-challenged broilers green compound premix feed useful for laying hens in laying period, prepared by uniformly mixing micronutrient substances of trace elements, vitamins, synthetic amino acids antibiotic growth promoters enhance animal production by targeting intestinal bile salt hydrolase and its producers three stages ecological feed, useful for broilers, comprises fish powder, composite premix, corn and soybean oil and rest of vegetables, where the compound premix comprises microelement premix and feed additive effects of dietary supplementation of quercetin on performance, egg quality, cecal microflora populations, and antioxidant status in laying hens effect of phytate and phytase on the ileal flows of endogenous minerals and amino acids for growing broiler chickens fed purified diets comparative study of the cellular immune response of chickens and turkeys vaccinated with fowl cholera vaccine the effect of administering glycolytic enzymes associated with fatty acids in feed for meat-type fowls. agricultura -revista de stiinta si copper promotes tff -mediated helicobacter pylori colonization performance of single comb white leghorn layers fed corn-soybean meal and barley-corn-soybean meal diets supplemented with a direct-fed microbial evaluation of litter material and ventilation systems in poultry production: iii. litter reuse, darkling beetle populations and intestinal parasites effects of clay on performance, moisture of droppings and health status of poultry: an overview effect of dietary supplementation of cu-methionine chelate and cu-soy proteinate on the performance, small intestinal microflora and immune response in laying hens effect of the physical form of feeds and growth promoting substances added to the diet on performance and some physiological parameters in broiler chickens mintrex (r) zn and mintrex (r) cu organic trace minerals improve intestinal strength and immune response to coccidiosis infection and/or vaccination in broilers pperformance promoters in animal nutrition . . quantitative factorial analysis of effectiveness effects of dietary polysaccharides from the submerged fermentation concentrate of hericium caput-medusae (bull.: fr.) pers. on performance, gut microflora, and cholesterol metabolism in broiler chickens effects of tribasic copper chloride on growth, copper status, antioxidant activities, immune responses and intestinal microflora of blunt snout bream (megalobrama amblycephala) fed practical diets effect of organic acid supplementation on the performance and ileal microflora of broiler during finishing period relationship of messenger rna reverse transcriptasepolymerase chain reaction signal to campylobacter spp. viability effects of zinc-bearing clinoptilolite on growth performance, cecal microflora and intestinal mucosal function of broiler chickens meat hygienic and feed technological investigations on the application of industrial-dried cage layer excreta for fattening beef-cattle . . microbiological, feed-analytical and residual conditions of dried cage layer excreta and of cornsilage, used as basic feedstuff microelements in the circulation of coccidiosis-infected chicks effects of dietary grape seed polyphenols on plasma lipid and mineral contents, and intestinal microflora in broiler chicks effects of copper-loaded chitosan nanoparticles on growth and immunity in broilers protective effects of zinc-bearing clinoptilolite on broilers challenged with salmonella pullorum effects of copper-loaded chitosan nanoparticles on intestinal microflora and morphology in weaned piglets effect of substrate adaptation on the microbial fermentation and microbial composition of faecal microbiota of weaning piglets studied in vitro the effect of defaunation on nitrogen retention in sheep on a diet containing protein of varying degrees of degradability. byulleten' vsesoyuznogo nauchno-issledovatel'skogo instituta fiziologii new microelement organic chelate complex compound useful e.g. for inhibiting pathogenic bacteria e.g. salmonella, and treating or preventing disease e.g. poultry enteric diseases, swine enteric diseases and bovine enteric diseases novel polyphenol oxidase mined from a metagenome expression library of bovine rumen -biochemical properties, structural analysis, and phylogenetic relationships oral immunization of balb-c mice with salmonellatyphimurium aroa mutant carrying the brucella-abortus copper zinc superoxide dismutase gene effect of vermicomposting on the transformation of some trace elements in fly ash metabolism of sulfur in the gastro intestinal tract pseudomonas pickettii: a common soil and aerobic groundwater bacteria with pathogenic and biodegradation properties interaction between copper oxide wire particles (cowp) and duddingtonia fagrans in hair breed lambs evaluation of gastro-intestinal nematode parasite control strategies for first-season grazing cattle in sweden new bacterium comprising lactic acid bacterium and bacterium of genus bifidobacterium comprising metal nanoparticle bound to its surface, useful e.g. as contrast agent for mri of digestive tract of subject, and for treating cancer contribution to the study of gut hypersensitivity reactions to soybean proteins in preruminant calves and early-weaned piglets animal feed composition useful for e.g. reducing intestinal bacillus count of ruminant and monogastric animals, comprises e.g. corn, meal, rice bran, wheat fine bran, syrup, palm oil, coconut oil, limestone, dicalcium monophosphate and salt. cn -a. cow * www this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author bison and copper iron in human milk: a study on the role of lactoferrin and the distribution of iron and some other trace elements in milk. abstracts of uppsala disseratations from the faculty of medicine effect of supplementary magnesium on reproduction and balance of selected mineral elements in sheep. prace i materialy zootechniczne effects of a polymer-coated urea product on nitrogen metabolism in lactating holstein dairy cattle biological attributes of bio-yoghurt versus the conventional one fed in spray dried form development of protein-a gold immunoelectron microscopy for detection of bovine coronavirus in calves -comparison with elisa and direct immunofluorescence of nasal epithelial-cells metal-mediated oxidative dna damage induced by methylene blue importance of colostrum for raising newborn calves pathogenesis of neurological signs associated with bovine enteric coccidiosis -a prospective-study and review enzymatic properties of cellobiose -epimerase from ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme effects of nitrilotriacetic acid on apparent absorption and duodenal flow of manganese, iron, zinc and copper in sheep alternative approaches to control -quo vadit? effects of feeding elevated concentrations of copper and zinc on the antimicrobial susceptibilities of fecal bacteria in feedlot cattle copper and manganese in hay samples from scrapie-free, scrapie-prone and scrapie-afflicted farms in iceland icelandic agricultural sciences microbial decomposition of organic cpds.|comprises a mixt. of magnetic field-treated gastrointestinal microorganism culture and micronutrients this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document copper and manganese in hay samples from scrapie-free, scrapie-prone and scrapie-afflicted farms in iceland icelandic agricultural sciences microbial decomposition of organic cpds.|comprises a mixt. of magnetic field-treated gastrointestinal microorganism culture and micronutrients rumen bacterial metabolism as affected by extracellular redox potential antibiotic growth promoters in animal nutrition assessment of sustainable vermiconversion of water hyacinth by eudrilus eugeniae and eisenia fetida alterations in ruminal utilization of magnesium and zinc in lambs fed different ratios of concentrate -forage chronic botulism in a saxony dairy farm: sources, predisposing factors, development of the disease and treatment possibilities influence of experimentally induced theileriosis (theileria annulata) on the pharmacokinetics of a long-acting formulation of oxytetracycline (otc-la) in calves effects of experimentally induced theileria annulata infection on the pharmacokinetics of oxytetracycline in cross-bred calves effects of freeze-dried mitsuokella jalaludinii culture and natuphos (r) phytase supplementation on the performance and nutrient utilisation of broiler chickens effects of altered dietary iron intake in mycobacterium-paratuberculosis-infected dairy-cattle -sequential observations on growth, iron and copper-metabolism and development of para-tuberculosis intracellular iron storage and the pathogenesis of para-tuberculosis -comparative studies with other mycobacterial, parasitic or infectious conditions of veterinary importance new biologically pure culture of a probiotic bacteria strain for producing bacteriocins useful in animal feed formulation for promoting growth and feed conversion in monogastric animal e.g. avian, dog, rabbit, mink effect of copper deficiency upon erythro kinetics in growing cattle preliminary results praseodymium and copper cation-exchange capacities of neutral-detergent fibers relative to composition and fermentation kinetics this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author purification and characterization of bacterial cm-cellulase from camel rumen fluid ( camelus dromedarius) studies on the mechanism of toxicity of the mycotoxin sporidesmin . . inhibition by copper-chelating agents of the generation of superoxide radical by sporidesmin protective efficacy of azomite enriched diet in oreochromis mossambicus against aeromonas hydrophila the diagnosis of adult bovine diarrhea -trial to put forward a new technique acute sheep poisoning from a copper sulfate footbath combined control of the principal diseases of sheep in the spring khimioprofilaktika, patogenez i epizootologiya parazitozov sel'skokhozyaistvennykh zhivotnykh phosphorus digestion and metabolism in ruminants: applications to production disease and environmental considerations. production diseses in farm animals th international conferences east lansing copper deficiency in an infant with giardiasis fed with cows milk biogenic amines in animal nutrition: origin, metabolism and physiological aspects uses of plants, animal and mineral substances in mediterranean ethno-veterinary practices for the care of small ruminants prevalence of strains with multiple resistance to antibiotics, and the transferability in vitro of the r factor of strains of escherichia coli and salmonella of various origins. lucrari stiintifice, institutul agronomic isolation of clostridiumperfringens from neonatal calves with ruminal and abomasal tympany, abomasitis, and abomasal ulceration annual report of studies in animal nutrition and allied sciences annual report of studies in animal nutrition and allied sciences. volume parasitic coenoses in the intestine of sheep and their control. ussr, akademiya nauk ukrainskoi ssr, institut zoologii the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. the present document has been produced and adopted by the bodies identified above as author. this task has been carried out exclusively by the author in the context of a contract between the european food safety authority and the author, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author. key: cord- -tvqpv fp authors: corrin, bryan; nicholson, andrew g. title: occupational, environmental and iatrogenic lung disease date: - - journal: pathology of the lungs doi: . /b - - - - . - sha: doc_id: cord_uid: tvqpv fp nan in practice the term is confined to the effects of mineral dust on the lungs. diseases caused by organic dusts are not included among the pneumoconioses and, in medicolegal practice at least, the presence of dust alone is insufficient to indicate pneumoconiosis: for compensation to be considered, the mineral dust must alter the structure of the lung and cause disability. the british industrial injuries advisory council defined pneumoconiosis as 'permanent alteration of lung structure due to the inhalation of mineral dust and the tissue reactions of the lung to its presence, excluding bronchitis and emphysema' . parkes recommends that cancer and asthma caused by mineral dust should also be excluded from the definition, an opinion with which we concur. to reach the lung, dust particles have to be very small. particle density and shape also affect the aerodynamic properties of dust. host factors such as airflow characteristics, airway branching patterns and airway disease also affect dust deposition. three deposition mechanisms are recognised ( fig. . . ): . inertial impaction: when air streams change direction or velocity, the inertia of the entrained particles causes them to maintain their original direction for a distance that depends upon their density and the square of their diameter. the same rules govern a car approaching a bend too fast: the car crashes into the outside of the bend. . sedimentation (gravitational settlement): under the influence of gravity, particles settle with a speed that is proportional to their density and the square of their diameter. . diffusion: very small airborne particles acquire a random motion as a result of bombardment by the surrounding gas molecules. inhaled dust particles are liable to sediment out in the alveoli if they have a diameter in the range of - µm, are roughly spherical in shape, and in density approximate to that of water. larger or denser particles impact or precipitate on the walls of the conductive airways and are rapidly removed by ciliary action. smaller particles may reach the alveoli but do not sediment so readily and many are therefore exhaled. very small particles are deposited on the walls of alveoli by diffusion but because they are so small the total amount of dust deposited in this way is insignificant compared with that deposited by sedimentation ( fig. . . ). direct measurement shows that most lung dust ( %) has a particle diameter less than . µm. fibrous dust particles behave differently. fibres over µm in length may reach the alveoli if they are very thin and remain aligned with the air stream. fibre penetration is inversely related to path length and the number of bifurcations. tall people have longer conductive airways and experience more deposition in these sites than short people who have greater alveolar deposition for the same level of exposure. slightly more dust is deposited in the right lung than the left, probably because the right main bronchus is more in line with the trachea, and is broader and shorter than the left, and carries % of the inhaled air. , dust clearance from the lung inhaled dust that settles in the conductive airways is removed within a day or two by ciliary action. only dust that reaches the alveoli is liable to cause pneumoconiosis and much of this is also removed, but the clearance rate here is much slower: many coalminers continue to expectorate mine dust years after retirement. alveolar clearance is gravity largely effected by macrophages, principally via the airways to the pharynx but also via lymphatics to the regional lymph nodes. the airway and interstitial routes interconnect at the bronchiolar level where some dust-laden macrophages leave the interstitium for the air space. this interconnection is probably the route utilised by circulating macrophages clearing other parts of the body of endogenous or exogenous particulate matter via the lung. long asbestos fibres present a particular problem to macrophage clearance. some minerals, notably chrysotile asbestos, undergo slow physicochemical dissolution in the lungs. only a small fraction of the inhaled dust gains access to the interstitium, a necessary step if it is to cause pneumoconiosis. some free dust enters through the bronchus-associated lymphoid tissue , and some is taken up by, or pierces, the alveolar epithelium ( fig. . , p. ). [ ] [ ] [ ] some of this is transported within hours to the hilar lymph nodes. so rapid is this translocation that it is thought not to involve most, the lesions are more numerous and better developed in the upper lobes than the bases but the reverse is true of asbestosis. the reasons for this are complex but undoubtedly involve the dust deposition:clearance ratio for the effect of the dust will depend upon both its amount and the duration of its stay in the lungs. there are well-recognised regional differences in the distribution and clearance of inhaled material, which in turn are dependent upon man's upright posture, the consequent gravitational forces being maximal at the apices. when standing at rest, the apices of the lungs are hardly perfused, so that lymph formation and clearance are much better at the bases. [ ] [ ] [ ] similarly, the apices are relatively less well aerated; alveoli in the lower lobes receive more air than those in the upper lobes. , the greater respiratory excursions at the bases are thought to promote macrophage mobility there. it is to be expected therefore that the bases would both receive and clear more dust than the apices, rendering it difficult to predict on theoretical grounds which parts of the lungs carry the heaviest dust burden. in fact, more dust of all types is found in the upper lobes, the part most severely affected by every type of pneumoconiosis except asbestosis. , the predilection of asbestos to affect the periphery of the lower lobes is attributed to the dangerous long asbestos fibres preponderating there. , pulmonary reactions to mineral dust the main tissue reaction to mineral dust is fibrosis. silica is highly fibrogenic and is therefore very likely to cause pneumoconiosis. carbon is non-fibrogenic and therefore, unless there are complications, coal pneumoconiosis causes little disability. tin too is harmless, and stannosis therefore unimportant, although the chest radiograph is highly abnormal because tin is very radiopaque. stannosis is one of several terms that specify pneumoconiosis due to a particular mineral, the best known being silicosis, asbestosis and anthracosis. the blackness of carbon and red-brown colour of iron give ample evidence, both naked-eye and microscopically, of the type and amount of these dusts when they are present in the lung ( fig. . . ), but other inorganic dusts may be more difficult to identify. however, a flick-out substage condenser and polaroid filters to test for refractility and birefringence respectively are useful adjuncts that are too often neglected by the histopathologist. crystalline silica is traditionally regarded as being only weakly birefringent, in contrast to silicates which generally show up brightly with simple crossed polaroid filters. however, with modern microscope lamps, if the light source is set at high intensity when using polaroid filters, both silica and silicates are birefringent. mineralogists use polarising microscopy for analysis, but only by studying large polished crystals with controlled orientation of the light. the small dust particles found in tissue sections are too small to permit analysis by this technique but it is nevertheless very useful for detecting their presence ( fig. . . ) . particle shape gives a useful indication of mineral type but appearances are sometimes deceptive: the plate-like crystals of talc are seldom observed as such, usually being viewed edge-on, when they appear to be needle-shaped. occasionally, stains can be used to identify minerals, e.g. a modified perls' reaction for inhaled iron, and irwin's aluminon stain for aluminium, but these too have largely been replaced by modern analytical techniques. ultrafine dust particles are particularly liable to be transported across the alveolar epithelium. the integrity of the alveolar epithelium is very important to dust translocation from the air spaces to the interstitium. much more dust reaches the interstitium if the epithelium is damaged. , it is widely thought that macrophages that have left the interstitium for the alveolar space never return, , but this is probably untrue. heavily laden macrophages accumulate in alveoli bordering the terminal and respiratory bronchioles, eventually filling them completely. erosion of the alveolar epithelium permits re-entry of these macrophages into the interstitium, very close to foci of bronchial mucosaassociated lymphoid tissue (malt), which are found near the terminal bronchioles. these aggregates guard the mouths of lymphatics, which commence at this point; alveoli are devoid of lymphatics. dustladen interstitial macrophages accumulate in and around the bronchial malt, which macklin therefore referred to as dust sumps. most pneumoconiotic lesions are found in the region of the dust sumps and are therefore focal. asbestosis is diffuse rather than focal because the long asbestos fibres are not readily mobilised and cannot be concentrated in the centriacinar dust sumps. this is also seen on occasion with platy non-fibrous dusts such as talc, mica, kaolinite and feldspar. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] within the dust sumps the dust particles are not static. they are constantly being freed and reingested by interstitial macrophages and, because these cells are mobile, successively inhaled dusts soon become intimately mixed. macrophages play an important role in pneumoconiosis and if the dust is fibrogenic the repeated phagocytosis of indestructible mineral particles results in constant fibroblast stimulation. the zonal distribution of pneumoconiosis pneumoconiosis affects both lungs but seldom evenly and some pneumoconioses show characteristic patterns of lung involvement. in microincineration combined with dark-field microscopy can also be used to demonstrate small particles. incombustible mineral particles that cannot be seen with bright-field or polarising microscopy are rendered visible by this technique and their position on the slide can be compared with tissue reactions evident in a serial section that has not been incinerated. microincineration has, however, also been largely replaced by modern analytical techniques that will now be considered. analytical electron microscopy is very helpful in identifying minerals, whether applied to lung digests or tissue sections. [ ] [ ] [ ] [ ] scanning electron microscopy permits the examination of thicker sections than transmission electron microscopy but does not detect very small particles. however, scanning electron microscopy allows more tissue to be examined and avoids the difficulty of cutting mineral particles with an ultramicrotome. mineral particles in a -µm thick deparaffinised section can be recognised in a scanning electron microscope set to collect the back-scattered electrons. the instrument can then be focused on points of potential interest and switched to x-ray diffraction, which provides information on crystal structure (fig. . . ) . alternatively, elemental analysis may be undertaken with either energy-dispersive or wavelength-dispersive x-ray spectroscopy. with energy-dispersive x-ray spectroscopy, all elements of atomic number above are identified, whilst with wavelength-dispersive x-ray spectroscopy the section can be scanned for one particular element. with the former technique different elements are shown graphically as individual peaks, the heights of which are proportional to the amounts of the different elements within the particle studied, thereby giving information on probable molecular formula ( fig. . . ) . thus, different silicates can be distinguished from each other and also from silica, which registers as pure silicon, oxygen (atomic number ) not being detected. the fact that the elements of low atomic number that constitute organic chemicals are not detected means that any minerals present (except beryllium, atomic number ) can be recognised easily in tissue sections. only particles can be analysed however: elements present in only molecular amounts cannot be detected by x-ray analysis. the detection of trace amounts of substances such as beryllium requires bulk chemical analysis or techniques that are not widely available such as atomic absorption spectrometry, neutron activation analysis and microprobe mass spectrometry. , the last of these techniques can also provide molecular (as opposed to elemental) analysis of organic as well as inorganic particles. another analytical technique of interest is microscopic infrared spectroscopy which provides data on the compound nature of microscopic particles in tissue sections ( fig. . . ) . micro-raman spectroscopy is also useful in this respect. some metals cause hypersensitivity, which can be identified by exposing the patient's lymphocytes to metals and measuring their reaction in vitro. radiological grading of pneumoconiosis a scheme for grading pneumoconiosis radiologically by comparison with standard radiographs has been adopted by the international labour organisation (ilo) and is widely used. small opacities (up to cm diameter) are graded by their profusion, , and indicating increasing numbers, and by their size, increasing through p, q and r if rounded and s, t and u if irregular. type p opacities are described as punctiform and measure up to . mm in diameter; larger lesions up to mm in diameter (type q) are described as micronodular or miliary; and those over mm and up to cm in diameter (type r) are described as nodular. irregular opacities cannot be sized so accurately, s, t and u indicating fine, medium and coarse respectively. large opacities (over cm diameter) are graded by their combined size, increasing through a, an opacity measuring between and cm in diameter; b, one or more opacities whose combined area does not exceed the equivalent of one-third of the area of the right lung field (when they are regrouped in the mind's eye or measured with a transparent ruler); and c, one or more opacities whose combined area exceeds one-third of the area of the right lung field (when similarly regrouped). in coalworkers, small opacities (up to cm diameter) correspond to simple coalworker's pneumoconiosis and large opacities (over cm diameter) to complicated coalworker's pneumoconiosis, which is also known as progressive massive fibrosis. silicotic lesions have been identified in the lungs of egyptian mummies, and the injurious effects on the lungs of inhaling mine dust have been recognised for more than years. as long ago as the sixteenth century in joachimsthal, bohemia (now jachymov, czech republic), diseases of miners' lungs were attributed to the dust the miners breathed. silicosis, tuberculosis and lung cancer are all now known to have been prevalent among the miners in this region, the cancer being largely attributable to the high level of radioactivity in the mines. silicosis was recognised in the uk soon after the discovery in that the addition of calcined flint to the clay from which china is made produced a finer, whiter and tougher ware. the preparation and use of this flint powder were highly dangerous, causing the condition known as potter's rot, one of the first of the many trade names by which silicosis has since been known. aluminium oxide (alumina) now provides a safe, effective substitute for flint in this industry. in it was noted that sheffield fork grinders who used a dry grindstone died early, and amongst other preventive measures it was recommended that the occupation should be confined to criminals: fortunately for them, the substitution of carborundum (silicon carbide) for sandstone was effective enough. however, silicosis still occurs in some miners, tunnellers, quarrymen, stone dressers and metal workers. silica in one form or another is used in many trades -in the manufacture of glass and pottery, in the moulds used in iron foundries, as an abrasive in grinding and sandblasting, and as a furnace lining that is refractory to high temperatures. rocks such as granite and sandstone are siliceous and their dusts are encountered in many mining and quarrying operations. in coal mining in the uk the highest incidence of the disease was in pits where the thinness of the coal seams required the removal of a large amount of siliceous rock, a process known as 'hard heading' . in south africa, silicosis causes a high mortality among the gold miners on the witwatersrand, where the metallic ore is embedded in quartz. slate is a metamorphic rock that contains both silica and silicates, and slateworkers develop both silicosis and mixed-dust pneumoconiosis. , nor are rural industries immune from the disease, particularly if ventilation is inadequate, as it is in certain african huts where stone implements are used to pound meal and the occupants develop mixed-dust pneumoconiosis. silicosis and mixed-dust pneumoconiosis have also been reported in dental technicians. desert sand is practically pure silica but the particles are generally too large to reach the lungs. however, silicosis has been reported in inhabitants of the sahara, libyan and negev deserts and those living in windy valleys high in the himalayan mountains, [ ] [ ] [ ] [ ] [ ] [ ] [ ] whilst in california the inhalation of dust raised from earth has led to silicate pneumoconiosis in farm workers, horses and a variety of zoo animals. the silica in rocks such as granite, slate and sandstone is largely in the form of quartz and this is therefore the type of silica encountered in most of the industries considered above. cristobalite and tridymite, which are possibly even more fibrogenic than quartz, are more likely to be encountered in the ceramic, refractory and diatomaceous earth industries where processing involves high temperatures. many workers with silicosis are asymptomatic. as a general rule, exposure to silica dust extends over many years, often or more, before the symptoms of silicosis first appear: by the time the disease becomes overt clinically, much irreparable damage has been inflicted on the lungs. the initial symptoms are cough and breathlessness. from then onwards, respiratory disability progresses, even if the patient is no longer exposed to silica dust. ultimately, there may be distressing dyspnoea with even the slightest exercise. silicosis sometimes develops more rapidly, perhaps within a year or so of first exposure. such 'acute silicosis' was observed in the scouring powder industry in the s when these cleansing agents consisted of ground sandstone mixed with a little soap and washing soda. , the additives were considered to have rendered the silica in the sandstone more dangerous but it is possible that the rapidity of onset of the disease merely reflected the intensity of the dust cloud to which the packers were exposed. confusingly, the term 'acute silicosis' has since been applied to a further effect of heavy dust exposure in tunnellers, sand blasters and silica flour workers, namely pulmonary alveolar lipoproteinosis (see below), , whilst the terms 'accelerated silicosis' or 'cellular phase silicosis' have been substituted for 'acute silicosis' in referring to the rapid development of early cellular lesions. , the time from first exposure to the development of symptoms (the latency period) is inversely proportional to the exposure level. however, it is evident that a certain amount of silica can be tolerated in the lungs without fibrosis developing, indicating either a time factor in the pathogenetic process or a threshold dust load that has to be reached before fibrosis develops. silica particles that are roughly spherical in shape and of a diameter in the range of - µm sediment out in the alveoli and are concentrated within macrophages at macklin's dust sumps, as explained previously (see p. ). early lesions, as seen in so-called accelerated or cellular phase silicosis, consist of collections of macrophages separated by only an occasional wisp of collagen. the early lesions have been likened to granulomas and on occasion have been mistaken for langerhans cell histiocytosis or a storage disorder, but langerhans cells are scanty and the histiocytes contain dust particles rather than accumulated lipid or polysaccharide. the macrophages of the early lesion are gradually replaced by fibroblasts and collagen is laid down in a characteristic pattern. the mature silicotic nodule is largely acellular and consists of hyaline collagen arranged in a whorled pattern, the whole lesion being well demarcated ( fig. . . ) and sometimes calcified. small numbers of birefringent crystals are generally evident within the nodules when polarising filters are used, but these mainly represent silicates such as mica and talc, inhaled with the silica. silica particles are generally considered to be only weakly birefringent, but fairly strong birefringence is evident in strong light (see above). silicotic nodules develop first in the hilar lymph nodes and are generally better developed there than in the lungs. [ ] [ ] [ ] indeed, silicotic nodules are occasionally found in the hilar lymph nodes of persons who have no occupational history of exposure to silica and whose lungs are free of such lesions, the silica in the nodes being presumed to represent inhaled particles derived from quartz-rich soil. severely affected lymph nodes often calcify peripherally, giving a characteristic eggshell-like radiographic pattern. this is sometimes the only radiological abnormality. such enlarged lymph nodes may occasionally press upon and obstruct adjacent large bronchi or result in a left recurrent laryngeal nerve palsy, so simulating malignancy. sometimes the nodules develop within the walls of major bronchi, occasionally causing a middle-lobe syndrome (see p. ). silicotic nodules are also found along the lines of the pleural lymphatics , where they have been likened to drops of candle wax on the visceral pleura. very rarely, silica-induced fibrosis is more pronounced in the pleura than in the lungs. lung tissue between the nodules is often quite normal and not until the process is very advanced is there any disability ( fig. . . b). in severe cases large masses of fibrous tissue are formed, which may undergo central necrosis and cavitation ( fig. . . ). on close inspection it is evident that these consist of conglomerations of many silicotic nodules closely packed together. in such severe cases cor pulmonale develops. occasionally, silicotic nodules develop in the abdominal as well as the thoracic lymph nodes, and in the liver, spleen, peritoneum and bone marrow. [ ] [ ] [ ] [ ] [ ] in about % of cases, the typical pulmonary nodules that predominantly affect the upper lobes are accompanied by diffuse fibrosis that is maximal in the lower lobes. , , [ ] [ ] [ ] the latter may show 'honeycombing' and closely resemble idiopathic pulmonary fibrosis. the association is too common to be explained by chance and the diffuse fibrosis is therefore regarded as a further manifestation of the pneumoconiosis, possibly due to an interaction between the dust and the immunological factors discussed below. the pathogenesis of silicosis has excited much interest and many different theories have been advanced over the years. an early theory held that the hardness of the silica was responsible, but this was discounted by the observation that silicon carbide (carborundum) is harder than silica but is non-fibrogenic. theories based on the piezoelectric property and on the solubility of silica were successively abandoned although the latter had a long period of popularity. it gained support from kettle's experiments which showed that fibrosis developed about chambers placed in an animal's peritoneal cavity if the chambers contained silica powder sealed in by a collodion membrane through which solutes such as silicic acid could pass. however, it was later shown that the pores in a collodion membrane are quite irregular in size and when the experiments were repeated using chambers guarded by millipore membranes, no fibrosis developed, despite solutes being able to diffuse out. the solubility theory also fails to take account of the differing fibrogenicity of the various forms of silica despite them being of similar solubility. furthermore, if the outer, more soluble layer of the particles is removed by etching, fibrogenicity is increased although solubility is decreased. in line with this, freshly fractured crystalline silica is more pathogenic in every respect than its aged equivalent, which may partly explain the severity of silicosis in trades such as sandblasting. these observations suggest that the fibrogenicity of silica is connected with its surface configuration. it is now known that uptake of the silica by macrophages is necessary for silicosis to develop. if silica and macrophages are enclosed together in peritoneal millipore chambers, a soluble product of the macrophages diffuses out and causes fibrosis. this observation led to the realisation that the fibrogenicity of the various crystalline forms of silica correlated well with their toxicity to macrophages and for a time macrophage death was thought to be necessary. it is now considered that before the macrophages are killed by the ingested silica, they are stimulated to secrete factors that both damage other con stituents of the lung and promote fibrosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] transforming growth factor-β is one fibrogenic factor that has been implicated in the pathogenesis of silicosis. [ ] [ ] [ ] toxic damage to macrophages is due to silica particles injuring the phagolysosomal membranes, so releasing acid hydrolases into the cytoplasm. it is important in the pathogenesis of the disease indirectly because when the macrophage crumbles, the silica particles are taken up by fresh macrophages and the fibrogenic process continues. it has been suggested that early involvement of the hilar lymph nodes in the fibrogenic process promotes the development of the disease in the lung by delaying dust clearance. immunological factors have been implicated in the pathogenesis of silicosis because many patients with silicosis have polyclonal hypergammaglobulinaemia, rheumatoid factor or antinuclear antibodies, and because there is a well-recognised association between autoimmune diseases such as systemic sclerosis and rheumatoid disease and exposure to silica. , [ ] [ ] [ ] [ ] the relation of immunity to dust exposure appears to be a reciprocal one: on the one hand, the presence of dust results in rheumatoid lesions in the lungs being more florid (see caplan's syndrome, p. ), whilst on the other, non-specific immunisation of rabbits with horse serum results in experimental silicotic lesions being larger and more collagenous. it is doubtful whether pneumoconiosis and autoimmune disease play a causative role in each other but one seems to aggravate the other and may lead to its earlier development. one of the commonest and most feared complications of silicosis is chronic respiratory tuberculosis. once this infection has been added to the silicosis, the prognosis rapidly worsens. it is thought that in the presence of silica, the tubercle bacilli proliferate more rapidly because the ingested silica particles damage phagolysosomal membranes and thereby interfere with the defensive activity of the macrophages. the synergistic action of silica dust has long been held responsible for the inordinately high incidence of respiratory tuberculosis in mining communities. many former south african gold miners now have acquired immunodeficiency syndrome (aids) as well as silicosis and tuberculosis has consequently reached almost epidemic proportions amongst these men. phagocyte damage by ingested dust particles may also cause some cases of chronic necrotising aspergillosis complicating pneumoconiosis. a series of studies suggesting that there might be a link between silica inhalation and lung cancer was reviewed by the international agency for research on cancer in , leading to the conclusion that the evidence for carcinogenicity of crystalline silica in experimental animals was sufficient, while in humans it was limited. subsequent epidemiological publications were reviewed in , when it was concluded that the epidemiological evidence linking exposure to silica to the risk of lung cancer had become somewhat stronger but that in the absence of lung fibrosis remained scanty. the pathological evidence in humans is also weak in that premalignant changes around silicotic nodules are seldom evident. nevertheless, on this rather insubstantial evidence, lung cancer in the presence of silicosis (but not coal or mixed-dust pneumoconiosis) has been accepted as a prescribed industrial disease in the uk since . some subsequent studies have provided support for this decision. in contrast to the sparse data on classic silicosis, the evidence linking carcinoma of the lung to the rare diffuse pattern of fibrosis attributed to silica and mixed dusts is much stronger and appears incontrovertible. , alveolar lipoproteinosis in response to heavy dust exposure a further complication of exposure to silica is the development of alveolar lipoproteinosis (see p. ). , , , very heavy experimental exposure to silica, and indeed other dusts, stimulates hypersecretion of alveolar surfactant to such an extent that the normal clearance mechanism is overwhelmed. [ ] [ ] [ ] [ ] [ ] [ ] [ ] alveolar macrophages are enlarged by numerous phagolysosomes distended by lamellar bodies that represent ingested surfactant. the alveoli are filled by such cells and, having a foamy cytoplasm, they produce the appearances of endogenous lipid pneumonia, similar to that more usually encountered as part of an obstructive pneumonitis distal to a bronchial tumour. the macrophages gradually disintegrate and the free denatured surfactant slowly becomes compacted, during which time its staining with both eosin and the periodic acid-schiff reagents intensifies until the appearances are finally those of alveolar lipoproteinosis. this process prevents the aggregation and concentration of the dust in the usual foci and thereby hinders the development of silicosis. lipoproteinosis and silicosis may be seen in conjunction but, more often, different areas of the lung show one or the other. the lipoproteinosis has its own severe impact on lung function, but, unlike silicosis, is potentially reversible (by massive alveolar lavage). occasional patients exposed to silica develop renal disease. [ ] [ ] [ ] [ ] two mechanisms appear to operate. first, translocation of silica particles from the lungs leads to their deposition in the renal interstitium with resultant nephrotoxity. second, silica stimulates an autoimmune response characterised by the formation of various antibodies, notably rheumatoid factor and antinuclear antibodies, which leads to the development of immune complex-mediated glomerulonephritis. , amorphous silica manmade submicron forms of silica, variously known as amorphous, vitreous, colloidal, synthetic or precipitated silica, are widely used in industry. they consist of pure non-crystalline silicon dioxide. particle size ranges from to nm but aggregates of the particles measure from to µm. industrial surveys suggest that inhalation of such dust is harmless, observations that are in accord with the results of animal experiments. an amorphous silica is the principal component of the fossilised remains of diatoms that constitute the sedimentary rock, diatomite ( fig. . . ). this is generally obtained by open-cast mining, following which the rock is crushed and calcined. the calcined product is used in filters, insulation material and as a filler. being amorphous, the silica in diatomite is harmless, but calcining (> °c) results in its conversion to crystalline forms of silica. diatomaceous earth pneumoconiosis is unusual and its risk appears to be related to the amount of cristobalite and tridymite (two forms of crystalline silica) produced in the calcining process. the silicates are complex compounds in which silicon and oxygen form an anion combined with cations such as aluminium and magnesium: talc, for example, is a hydrated magnesium silicate with the formula mg si o (oh) . silicates include fibrous forms (asbestos and the zeolites), plate-like forms (talc and mica) and clays (kaolinite and fuller's earth). in histological sections, the platy talc and mica particles are generally cut tangentially and therefore appear needleshaped (see fig. . . ). they are strongly birefringent whereas the clays are only weakly so. talc particles in the lung exceeding µm in length should arouse suspicion of intravenous drug abuse. of the fibrous silicates, zeolite is used as a building material in certain communities, notably in central turkey. pneumoconiosis is not a problem but zeolites are of medical interest because, like asbestos, they present a mesothelioma risk. asbestos is dealt with separately (see below). pneumoconiosis has been described with various non-fibrous silicates, notably in the rubber industry, which uses talc and, less commonly, mica as lubricants. other occupations posing a risk include the extraction of kaolinite from china clay (kaolin), , , and in the open-cast and underground mining of fuller's earth (montmorillonite, bentonite and attapulgite clays, which were originally used in 'fulling' (degreasing) wool). , however, all these substances are commonly contaminated with silica, asbestos or both, and it has been questioned whether in pure state they are at all fibrogenic. the modifying effect of inert substances such as iron on that of silica is well known (see mixed-dust pneumoconiosis, below) and it has been suggested that talc, mica and fuller's earth act in a similar way in regard to their more fibrogenic contaminants, the pneumoconioses attributed to them in reality representing mixed-dust pneumoconiosis or asbestosis. contrary evidence comes from reports of pulmonary fibrosis in persons heavily exposed to pure talc, mica or kaolin. all these silicates are evident in the tissues as plate-like birefringent crystals which often provoke a foreign-body giant cell reaction (see fig. . . ) and may result in fibrotic nodules. large focal lesions resembling the progressive massive fibrosis of coalworkers may be produced, and also a diffuse 'asbestosis-like' form of pneumoconiosis, the latter attributed to poor macrophage mobilisation of the plate-like particles. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it would appear therefore that silicates are indeed fibrogenic if enough is inhaled; they appear to vary in fibrogenicity but in all cases they are less fibrogenic than silica. inert dusts are non-fibrogenic and therefore of little clinical consequence, although elements of high atomic number can give rise to a striking chest radiograph. it should be noted however that inert or lowly fibrogenic materials may be associated with substances of medical importance, for example, kaolin, bentonite and barytes (barite) may all be contaminated with silica , , and talc may be contaminated with asbestos. the best known of the inhaled inert mineral dusts is carbon while, of the remainder, iron is the most widespread. others include tin and barium. with all these dusts, particles retained in the lung are gathered at macklin's dust sumps by heavily laden macrophages which are lightly bound together there by a few reticulin fibres. collagen is not formed and the worker suffers no ill-effects. the lungs take on the colour of the dust and in siderosis assume a deep brick-red hue. carbon deposition is commonly found in the lungs, particularly those of city dwellers and tobacco smokers. it is also the principal constituent of coal, which is dealt with separately below, and large amounts of pure carbon may be inhaled by workers involved in the manufacture of carbon black, carbon electrodes and charcoal. [ ] [ ] [ ] [ ] although carbon is regarded as being non-fibrogenic, the very heavy lung burdens encountered in industries such as these may lead to the complicated form of pneumonconiosis known as progressive massive fibrosis that is more commonly encountered in coal workers (see p. ). heavy pure carbon deposition may also be acquired domestically when wood is burnt in buildings devoid of a chimney, so-called 'hut lung', a term that is also applied to the domestic acquisition of carbon mixed with silica or silicates, resulting in forms of mixeddust pneumoconiosis. , , anthracofibrosis is a term introduced by chinese bronchoscopists for bronchial stenosis or obliteration associated with carbon pigmentation of the mucosa. although the original description incriminated tuberculosis, mixtures of various mineral dusts acquired at work or domestically are a more likely cause. [ ] [ ] [ ] [ ] iron dust in the lungs was first described by zenker in , when he also introduced the terms siderosis and pneumonokoniosis. zenker was describing a woman who coloured paper with iron oxide powder ('rouge'), a substance which is still encountered by some workers engaged in polishing silver, glass, stone and cutlery. siderosis is also found in welders, iron foundry fettlers, steel workers, boiler scalers and haematite miners and crushers. iron dust particles are reddish-brown but in the lung may be masked by carbon : when evident, or revealed by microincineration, they resemble haemosiderin and generally give a positive perls' reaction, but particularly with haematite, heat ( - o c) and concentrated ( n) hydrochloric acid may be necessary. haematite miners in both the uk (cumbria) and france (lorraine) have an increased risk of bronchial carcinoma, but radon gas rather than haematite is the suspected carcinogen. radon is a decay product of uranium. minute amounts are present in all rocks but local concentrations occur and these are liable to build up in mines if ventilation is limited. silver, as well as iron, is found in the lungs of silver polishers, where it stains elastin in alveolar walls and pulmonary vessels grey. such argyrosiderosis is as harmless as siderosis. tin miners are subject to silicosis but not stannosis because the ore, which is found in association with siliceous rocks, contains only low concentrations of the metal. tin smelters, on the other hand, and factory workers exposed to high concentrations of tin dust or fume, are liable to inhale large amounts of this inert metal and develop the striking chest radiograph of stannosis. they remain in good health however for tin is completely non-fibrogenic. tin particles in the lung resemble carbon but are strongly birefringent and remain after microincineration: microprobe analysis provides positive identification. other inert dusts include barium, which also has a high atomic number and is therefore radiopaque, and minerals of low radiodensity such as limestone, marble and cement (all chiefly composed of calcium carbonate) and gypsum (hydrated calcium sulphate). however, the extraction of barium ore (almost entirely in the form of barium sulphate, which is known as barytes in europe and barite in the usa) may entail exposure to silica and silicates. pure baritosis resembles stannosis and siderosis. the term 'mixed-dust pneumoconiosis' refers to the changes brought about by inhaling a mixture of silica and some other less fibrogenic substance such as iron, carbon, kaolin or mica. , , [ ] [ ] [ ] the proportion of silica is usually less than %. typical occupations include foundry work and welding and the mining of coal, haematite, slate, shale and china clay. the action of the silica is modified and, although fibrotic nodules are formed, they lack the well-demarcated outline and concentric pattern of classic silicosis. the lesions are found in a centriacinar position and are stellate in outline with adjacent scar emphysema. they are firm and generally measure no more than mm in diameter. they closely resemble the fibrotic nodules of simple coal pneumoconiosis (see below). confluent lesions also occur on occasions. these resemble the progressive massive fibrosis of coalworkers and appear to represent a single large lesion rather than a conglomeration of individual nodules, as in advanced silicosis. abundant dust is generally evident in lesions of all sizes; this consists of black carbon or brown iron mixed with crystals of varying degrees of birefringence, silicates generally being strongly birefringent and silica weakly so. calcification is unusual. mixed-dust pneumoconiosis carries an increased risk of pulmonary tuberculosis, but not to the same degree as silicosis. in some cases the stellate nodules are accompanied by diffuse fibrosis, as in silicosis and again possibly involving interactions between the dust and immunological factors. involvement of the bronchi with consequent stenosis (so-called anthracofibrosis) is described above. the term 'anthracosis' was initially applied to changes observed in a coalminer's lung but is now often extended to include the common carbon pigmentation of city dwellers' lungs, and the term 'coal pneumoconiosis' is more appropriate to a special form of pneumoconiosis to which coalworkers are subject, particularly those who work underground. the principal constituent of coal, carbon, is non-fibrogenic, so suspicion has naturally fallen on the ash content of mine dust, some of which derives from the coal, some from adjacent rock strata and some from stone dust laid in the roadways to minimise the risk of coal dust explosions. coal itself appears to be the responsible agent because coal-trimmers, working in the docks and not exposed to rock dust, also develop the disease. coalminers encountering siliceous rock are, of course, also liable to develop silicosis like other underground workers. coal consists largely of elemental carbon, oxygen and hydrogen with traces of iron ore and clays such kaolinite, muscovite and illite, but no silica. the mineral content varies with the type and rank (calorific value) of the coal. all coal derives from peat, the youngest type being lignite and the oldest anthracite, with bituminous (house) coal in between. as it ages, the oxygen and mineral constituents diminish and the coal hardens. lignite is soft and said to be of low rank, anthracite hard and of high rank, with bituminous coal intermediate. although high-rank coal is of low mineral content, its dust is more toxic to macrophages in vitro and is cleared more slowly in vivo. this observation may explain why, in the uk, high-rank coal is associated with a higher prevalence of coal pneumoconiosis. the low mineral content of high-rank coal is reflected in the mineral content of the lungs of those who hew such coal in the uk, but in the ruhr, in germany, and in pennsylvania, in the usa, anthracite miners' lungs contain more silica than those who hew bituminous coal, the silica presumably deriving from other sources. not surprisingly, the presence of silica is reflected in the tissue reaction to the inhaled dust, resulting in a more fibrotic reaction very analogous to mixed-dust pneumoconiosis. a spectrum of changes is therefore encountered in coalminers' lungs, ranging from coal pneumoconiosis through mixeddust pneumoconiosis to silicosis; the findings in any individual depend upon the nature of the coal being mined and the type of work undertaken. in high-rank british collieries the development of coal pneumoconiosis appears to depend on the total mass of dust inhaled, whereas in low-rank british collieries the mineral content of the lung dust appears to be more important. this may explain apparently contrary data drawn from different coalfields -data based on coals of different composition that are not strictly comparable. some workers have stressed the importance of silica in the dust whereas others, particularly in the high-rank coalfields of south wales, have been unable to detect any association between silica and the level of pneumoconiosis. both findings may be correct, but only for the particular group of miners examined in each case. the lesions of coal pneumoconiosis are generally focal and fall into one or other of two major types, simple and complicated, depending upon whether the lesions measure up to or over cm; simple corresponds to categories - of the ilo grading system (see p. ) and complicated, which is also known as progressive massive fibrosis, to ilo categories a-c. more diffuse interstitial fibrosis has been reported in about % of welsh and west virginian coalminers, usually involving those carrying a particularly heavy dust burden; it runs a more benign course than non-occupational interstitial fibrosis (idiopathic pulmonary fibrosis). similar findings have been reported from france. simple coal pneumoconiosis consists of focal dust pigmentation of the lungs, which may be associated with a little fibrosis and varying degrees of emphysema. its clinical effects are relatively minor. some degree of black pigmentation (anthracosis) of the lungs is common in the general urban population, especially in industrial areas, but much denser pigmentation is seen in coalminers, whose lungs at necropsy are black or slate-grey. black pigment is evident in the visceral pleura along the lines of the lymphatics and on the cut surface where it outlines the interlobular septa and is concentrated in macklin's centriacinar dust sumps ( fig. . . ). the dust is generally more plentiful in the upper parts of the lungs and in the hilar lymph nodes, possibly due to poorer perfusion and consequently poorer lymphatic drainage there (see p. ). two forms of coal dust foci are recognised, macules and nodules, the former being soft and impalpable and the latter hard due to substantial amounts of collagen. both lesions are typically stellate but the more fibrotic the nodules, the more rounded they become, until it is difficult to distinguish them macroscopically from those of silicosis. in these circumstances reliance has to be placed on the whorled pattern of the collagen that is evident microscopically in silicosis. the stellate nodules are analogous to those seen in mixed-dust pneumoconiosis caused by mixtures of silica and inert dusts other than carbon (see above). with polarising filters, small numbers of birefringent crystals may be seen in both macules and nodules, usually representing mica or kaolinite derived from rock that bordered the coal. macules consist of closely packed dust particles, free or within heavily laden macrophages, so that the lesion appears black throughout ( fig. . . ). appropriate stains show that the dust-laden macrophages and free dust are lightly bound by reticulin. very little collagen is evident. although striking in their appearance, dust macules are thought to have little effect on lung function. nodules contain substantial amounts of collagen and are thought to have an adverse, but limited, effect on respiration. they vary from a heavily pigmented, stellate lesion, which apart from its collagen content resembles the dust macule ( fig. . . ), to one that is less pigmented and more circumscribed. the stellate, heavily pigmented type of nodule is seen in lungs that have a relatively low ash content whilst the more rounded and less pigmented nodule is seen in lungs with relatively high ash loads. radiologically (see p. ), p-type opacities correspond to macules, q-type opacities to the stellate nodules that resemble those of mixeddust pneumoconiosis and r-type opacities to the rounded nodules that resemble those of silicosis. , thus, the radiological changes of simple coalworker's pneumoconiosis are due to the dust and the small amount of collagen present and do not reflect any emphysema that may also be present. however, pulmonary dust foci are often associated with emphysema ( fig. . . ) and the severity of the emphysema appears to correlate with the dust load. the prevalence of chronic bronchitis and emphysema is high in the coal industry and it has long been debated whether occupation or cigarette smoking is the major factor contributing to emphysema in coalminers. [ ] [ ] [ ] [ ] as well as mineral dust, nitrous fumes from shot-firing form another occupational hazard of coal mining. heppleston made a special study of the emphysema found in coalminers, claiming that it differs from centriacinar emphysema, as seen in smokers in the general population, and attributing it to the dust. he introduced the term 'focal emphysema of coalworkers' to describe this special process. others find it very difficult to identify any convincing difference between the emphysema of coalworkers and that encountered outside the industry but heppleston based his claims on the study of serial sections. by this means he showed that, although both forms affect respiratory bronchioles, the focal emphysema of coalworkers affects more proximal orders of these airways and is not associated with the bronchiolitis seen with centriacinar emphysema. furthermore, focal emphysema is a dilatation lesion whereas coniosis, also known as progressive massive fibrosis, can have very serious consequences. particularly when the lesions are large, it is associated with productive cough, breathlessness, significant impairment of lung function and premature death. the major factor accounting for the development of progressive massive fibrosis appears to be the sheer bulk of coal dust in the lung, rather than coal rank or the silica content of the mine dust. progressive massive fibrosis has occasionally been recorded in dockers loading silica-free coal into the holds of ships and in workers exposed to pure carbon in the manufacture of carbon black and carbon electrodes. [ ] [ ] [ ] progressive massive fibrosis is characterised by large (over cm) black masses, situated anywhere in the lungs but most common in the upper lobes. the lesions may be solitary or multiple and very large, occupying most of the lobe and even crossing an interlobar fissure to involve an adjacent lobe (figs . . b, . . ). they cut fairly easily, often with the release from a central cavity of black fluid flecked by cholesterol crystals. for many years it was believed that the condition was the result of synergism between mycobacterial infection and dust but the failure of the attack rate to decrease as tuberculosis declined negated this view. today, more emphasis is placed on total dust load for the lesions tend to affect lungs that carry an unduly heavy dust burden. if the remainder of the lung shows little evidence of dust accumulation, the possibility of the masses representing caplantype lesions (see below) should be considered. centriacinar emphysema involves destruction of adjacent alveolar walls. by definition, therefore, focal emphysema is not a true emphysema at all (see p. ). however, it has been shown that mineral dusts cause elastin and collagen breakdown in the rat lung. focal emphysema may progress to the destructive centriacinar form and this has strengthened claims that mine dust plays a causal role in centriacinar emphysema. , [ ] [ ] [ ] [ ] [ ] [ ] in the uk, these claims have been accepted and chronic bronchitis and emphysema in coalminers and metal production workers have been accepted as prescribed industrial diseases since . in germany too, chronic obstructive pulmonary disease is now compensatable as an occupational disease. the conditions for compensation in the uk were initially: • underground coal mining for a minimum of years in aggregate • forced expiratory volume in second at least litre below that expected or less than litre in total • radiological category of at least / . however the last of these criteria has now been dropped. the inclusion of a time element and the omission of some estimate of dust load (such as radiological category) have been criticised, with some justification. as with lung cancer caused by chromates benefit is paid irrespective of smoking habits. whereas simple coal pneumoconiosis, particularly the macular variety, has little effect on lung function, complicated coal pneumo- microscopically, the lesions consist of dust and connective tissue intermixed in a random fashion. central necrosis and cavitation commonly occur. the necrosis is thought to be ischaemic. it is amorphous or finely granular, and eosinophilic apart from abundant dust particles and cholesterol crystal clefts. the fibrotic component in a complicated pneumoconiotic lesion is rich in fibronectin, with collagen only more abundant at the periphery. two types of progressive massive fibrosis are recognisable, corresponding to the two types of nodule described in simple coal pneumoconiosis. the first appears to have arisen by enlargement of a single nodule, whereas the second is a conglomeration of individual lesions, each of which corresponds to the more circumscribed type of nodule seen in simple coal pneumoconiosis. the ash content of the lungs bearing these two types of progressive massive fibrosis varies in the same way as with the two types of simple pneumoconiotic nodules, the enlarged single lesion being found in lungs with a relatively low ash content, and the conglomerate lesion in lungs with a relatively high ash content. the second type resembles the conglomerate nodules of large silicotic lesions but lacks the characteristic whorled pattern of the latter. the diffuse interstitial fibrosis found in a minority of coalworkers is associated with heavy dust deposition. it may progress to honeycombing but, as with the focal forms and unlike idiopathic interstitial fibrosis, it is better developed in the upper zones, the reasons for which are discussed above (see the zonal distribution of pneumoconiosis, p. ). the pathogenesis of coal pneumoconiosis has much in common with that of silicosis, and indeed many other pneumoconioses. it involves the promotion of fibrogenic factor synthesis and release by cells phagocytosing the inhaled dust. several such factors have now been identified, the degree of fibrosis produced varying with the amount of dust inhaled and the ability of its constituents to promote the production of the responsible cytokines. these include plateletderived growth factor, insulin-like growth factors and , transforming growth factor-β and tumour necrosis factor-α. , , as with other minerals, the indestructability of the dust perpetuates the process. as in silicosis, immunological factors appear to be involved, for there is an increased prevalence of rheumatoid arthritis and of circulating autoantibodies [ ] [ ] [ ] in miners with coal pneumoconiosis. rheumatoid factor has also been demonstrated within the lung lesions. these abnormalities are generally more pronounced in miners with complicated pneumoconiosis but are also found in those with the simple variety. it is also possibly pertinent to the immunological basis of coal pneumoconiosis that some of the pulmonary manifestations of rheumatoid disease are more pronounced in coalminers. this was first pointed out by caplan and will be considered next. caplan described distinctive radiographic opacities in the lungs of coalminers with rheumatoid disease, and it is now recognised that similar lesions may develop in rheumatoid patients exposed to siliceous dusts. the development of such rheumatoid pneumoconiosis does not correlate with the extrapulmonary or serological activity of the rheumatoid process. nor is there a strong relation to dust burden: caplan lesions are characteristically seen in chest radiographs that show little evidence of simple coal pneumoconiosis. pathologists recognise the lesions as particularly large necrobiotic nodules similar to those seen in rheumatoid patients who are not exposed to dust (fig. . . ) . however, because of their large size (up to cm diameter) they may be confused with progressive massive fibrosis undergoing central ischaemic necrosis (see above) or silicosis complicated by caseating tuberculosis. such errors will be less likely if the radiological evolution of the lesions is considered for they tend to cavitate and undergo rapid remission, only to be succeeded by others. they are also well demarcated radiologically. pathologically, they resemble rheumatoid nodules in showing peripheral palisading but differ in their large size and the presence of dust. the dust accumulates in circumferential bands or arcs within the necrotic centres of the lesion (fig. . . ), an arrangement that suggests periodic episodes of inflammatory activity. caplan lesions differ from tuberculosis in lacking satellite lesions and tubercle bacilli, and from progressive massive fibrosis in showing characteristic bands of dust pigmentation (table . asbestosis is defined as diffuse interstitial fibrosis of the lung caused by exposure to asbestos dust. , it does not cover asbestos-induced carcinoma of the lung or asbestos-induced pleural disease. the development of asbestosis depends on the presence of fairly large dust burdens: this is in contrast to mesothelioma and other forms of asbestos-induced pleural disease, which, although also dose-related, occur following the inhalation of far smaller amounts of asbestos dust. asbestos is a generic term for more than naturally occurring fibrous silicates, fibre being defined as an elongated particle with a length-tobreadth (aspect) ratio of at least . asbestos fibres have a high aspect ratio, generally over . based on their physical configuration they can be divided into two major groups, serpentine and amphibole. the physical dimensions and configuration of asbestos fibres are strongly linked to their pathogenicity. chrysotile (white asbestos) is the only important serpentine form. it accounts for most of the world production of asbestos of all types ( being a serpentine mineral, chrysotile consists of long, curly fibres that can be carded, spun and woven like cotton ( fig. . . ). the curly chrysotile fibres are carried into the lungs less readily than the straight amphibole asbestos fibres, and once there undergo physicochemical dissolution and are cleared more readily. they readily fragment into short particles that are easily ingested by macrophages and in the acidic environment of the macrophage phagolysosome they are particularly unstable. the half-life of chrysotile in the lungs is estimated to be in the order of only a few months. , not surprisingly therefore chrysotile is the least harmful type of asbestos in respect of all forms of asbestos-induced pleuropulmonary disease. [ ] [ ] [ ] it may nevertheless cause pulmonary fibrosis if sufficient is inhaled. , in contrast to chrysotile, amphibole forms of asbestos consist of straight rigid fibres that are stable within the lung. they do not fragment, they are insensitive to chemical attack and their clearance halflives are in the order of decades rather than months. the main amphibole forms of asbestos of commercial importance are crocidolite (blue asbestos) and amosite (brown asbestos). crocidolite, reputedly the most dangerous in regard to all forms of asbestos-related disease, was formerly mined in western australia (wittenoom) and south africa (cape province and the transvaal); it was the principal amphibole used in the uk. amosite, the name of which derives from the acronym for the former asbestos mines of south africa company in the transvaal, was the principal amphibole used in north america. amphiboles are no longer imported by the developed countries but much remains in old lagging and presents a considerable dust hazard when this is removed. tremolite, a further amphibole asbestos, contaminates quebec chrysotile deposits, montana vermiculite and many forms of commercial (non-cosmetic) talc and is responsible for much of the asbestos-related disease in chrysotile miners and millers. another amphibole asbestos, anthophyllite, was formerly mined in finland. it causes pleural plaques (see p. ) but not lung disease, possibly because its fibres are relatively thick ( fig. . . ) . erionite is a zeolite rather than a type of asbestos but is comparable in form to amphibole asbestos and is also biopersistent. it is found . these coated structures are termed 'asbestos bodies' . because other fibres may gain a similar coat, the non-specific term 'ferruginous body' has been advocated. however, coated carbon fibres (so-called coal bodies) are easily recognised as such by their black core. in practice, ferruginous bodies with the appearance of asbestos bodies almost always prove to have an asbestos core. , long fibres are more likely to be coated than short ones, which are cleared more quickly: in one study few fibres less than µm in length were coated and few fibres over µm in length were uncoated. amphiboles form bodies more readily than chrysotile. a comparison of light and electron microscopic fibre counts found that . % of chrysotile, % of crocidolite and . % of amosite formed bodies. nevertheless, sufficient chrysotile fibres are coated to permit recognition of asbestosis by standard histological criteria (diffuse fibrosis and asbestos bodies), even if chrysotile is the only asbestos present. despite the biodegradability of chrysotile, asbestos body numbers do not materially diminish with time. very occasionally however a patient with diffuse pulmonary fibrosis and a history of asbestos exposure has no evident asbestos bodies but analysis shows a fibre burden within the range found in asbestosis, justifying fibre analysis in such cases. a there is evidence that alveolar macrophages are involved in the coating of asbestos fibres to form asbestos bodies and that the bodies are less harmful to the macrophages than uncoated fibres. asbestos bodies give a prussian blue reaction for iron when stained by perls' method and their yellow-brown colour makes them easily recognisable in unstained films of sputum or in unstained histological sections. sections may be cut µm thick to increase the yield and help identify bodies that lie at an angle to the microtome blade. there is a good correlation between the numbers of asbestos bodies seen in lung sections and those in tissue digests. , the bodies may be found singly or in irregular clumps or stellate clusters. they are unevenly distributed but in well-established asbestosis they are easily found. if they are not evident, asbestos burden may be assessed quantitatively in tissue digests (see below). their presence in lung tissue, sputum or bronchoalveolar lavage fluid merely confirms exposure, not the presence of disease. however, the number of asbestos bodies in lavage fluid correlates well with lung asbestos burden , and the number in sputum correlates with the duration and intensity of exposure. [ ] [ ] [ ] fibre counts , , [ ] [ ] [ ] [ ] [ ] quantitation is desirable in certain circumstances (box . . ), in which case it is best effected on -cm blocks of fixed or fresh lung tissue obtained from three different sites, avoiding tumour and thickened pleura. the tissue blocks are digested with caustic soda or bleach, following which the fibres may be collected on a millipore membrane or viewed in suspension in a red blood cell-counting chamber. if phase contrast optics are used both coated and uncoated fibres can be assessed. alternatively, dark ground illumination can be used to demonstrate uncoated fibres. however, electron microscopy is to be preferred as it detects far more fibres than are visible by light microscopy and can also provide information on fibre type. it is important that the laboratory is well practised in fibre analysis and has established its own control range for the general population as well as asbestosis as most lungs contain some asbestos. ambient fibres are generally shorter than µm and some workers therefore confine their counts to fibres that are at least as long as this. justification for this comes from animal experiments demonstrating that long fibres cause more inflammation, chromosomal damage, fibrosis, lung tumours and mesotheliomas than short fibres, [ ] [ ] [ ] [ ] and from studies in humans suggesting that long fibres in parts of central turkey where it causes both mesothelioma and a pattern of interstitial pulmonary fibrosis that is comparable to asbestosis. , asbestos use and exposure exposure to asbestos occurs in countries where it is extracted ( asbestos is used particularly for fireproofing, in heat and sound insulation and for strengthening plastics and cement. thus, unless adequate precautions are taken, exposure is experienced by dockers unloading asbestos in the close confines of a ship's hold, by thermal insulation workers (laggers and strippers) in shipyards, power stations, train maintenance depots, factories and other large buildings, by construction workers such as carpenters cutting asbestos building panels, and by workers making asbestos products such as fireproof textiles, brake and clutch linings, and specialised cement. as well as such direct exposure, exposure may also be: • indirect, as experienced by the families of asbestos workers • paraoccupational, as experienced by those working alongside an asbestos worker • neighbourhood, as experienced by those living downwind of an asbestos works or mine • ambient, as experienced by those living or working in a building containing asbestos. exposure to asbestos incorporated in the structure of a building carries a negligible health risk if the asbestos material is well maintained to prevent shedding of dust. stripping asbestos out is more dangerous than maintaining it in situ, but maintenance is sometimes neglected. the near indestructibility of asbestos accentuates the health problems that its ubiquity poses. because of their aerodynamic properties, fibres of µm or more in length may reach the finer bronchioles and alveoli. once impacted, the sharp asbestos fibres become coated with a film of protein that is rich in iron. the coating is thickest at the ends of the fibres, giving a other human studies have shown that, although asbestos load is maximal in the upper lobes, more long fibres are found at the bases, where fibrosis is most marked. , a further reason for limiting attention to the longer fibres is that the shorter ones are cleared more easily and their number therefore varies with the time lapsed since last exposure. for these reasons asbestos regulations in many countries now limit attention to fibres that are over µm in length and have a length-to-diameter (aspect) ratio greater than : such fibres have become known as regulatory or world health organization (who) fibres. values are best expressed as fibres/g dry lung. by light microscopy, normal values range up to : over is seen with mesotheliomas, and over in asbestosis (table . . ). , , , however, compared with electron microscopy, light microscopy is relatively insensitive, showing only . % of the amosite, % of the crocidolite and . % of the chrysotile. light microscopic counts correlate poorly with severity of asbestosis and electron microscopy non-asbestos fibres commonly found in the lung include mullite, which derives from fly ash. this may constitute up to % of the total fibre burden (see table . . ) and is thought to be harmless. there is no firm evidence that manmade fibres present a health hazard but in certain localities natural non-asbestos mineral fibres, zeolites for example, are important causes of mesothelioma (see p. ) and also cause interstitial pulmonary fibrosis. in contrast to the first half of the twentieth century, much of the asbestosis encountered today is asymptomatic, identified radiologically or histologically in lungs resected for carcinoma or removed at autopsy. symptomatic cases are characterised by an insidious onset of breathlessness, a dry cough and crackles over the lower lung fields. finger clubbing is a variable feature. lung function tests show a restrictive respiratory defect. radiology initially shows small irregular basal opacities that gradually coalesce to become linear, coarsen and eventually progress to a honeycomb pattern of small cysts. the principal differential diagnosis, both clinically and pathologically, is from idiopathic pulmonary fibrosis. this is aided by the slow progression of asbestosis, which often extends over years, as opposed to an average course of - years from presentation to death for the idiopathic condition. most cases of asbestosis are diagnosed solely on the occupational history and these clinicoradiological features. recourse to histology is unusual but biopsy (preferably as a wedge of lung) may be undertaken if the clinical features are atypical. histology also arises when the pathologist samples lung parenchyma remote from a resected carcinoma (the universal importance of which cannot be overemphasised). asbestosis (established) over over the light microscopic counts include total fibres (coated and uncoated). the electron microscopic counts include only amphibole asbestos. results from different laboratories vary and these figures, derived from several sources, , , provide only a general guide. reliable results depend upon counts being made regularly and the normal range from that laboratory being ascertained. ratios of counts obtained by electron and light microscopy vary greatly but approximate to . is better in this respect. [ ] [ ] [ ] by transmission electron microscopy, values may range up to in controls, with asbestosis generally above and mesotheliomas found at any level down to , all these figures representing amphibole fibres/g dried lung (see table . . ). , , it should be noted that counts from different parts of the same lung may vary widely; , - caution should therefore be exercised in interpreting a count obtained on a single sample. there is also wide discrepancy between laboratories, even when analysing the same sample. results obtained in an individual case therefore have to be evaluated against a standard set of values unique to that laboratory. electron microscopy also provides valuable information on the type of fibre. chrysotile differs physically from the amphiboles in two respects: its fibres are both curved and hollow (figs . . and . . ). with an electron microscope equipped for microprobe analysis, the various forms of asbestos may also be distinguished from other fibres and from each other (box . . ), , an important point as the amphibole forms of asbestos are far more dangerous than chrysotile (table . . ). [ ] [ ] [ ] coroners require autopsy verification of the diagnosis in all suspected cases and this also necessitates hystology. when the lungs from a patient with asbestosis are seen at autopsy, pleural fibrosis is often found, and although this may also be attributable to asbestos exposure it is to be regarded as an independent process and not part of the asbestosis: it is dealt with separately on page . slicing the lung affected by asbestosis shows a fine subpleural fibrosis, especially of the lower lobes ( fig. . . ). in severe cases the fibrosis often extends upwards to involve the middle lobe and lingula, and sometimes the upper lobes also. microcystic change associated with the fibrosis develops in advanced cases and in severe disease there may be cysts over cm in diameter. however, these classic changes are seldom seen in developed countries today. following decades of dust suppression in asbestos factories, current patients have mild to moderate asbestosis and are dying of related cancer or of non-pulmonary disease. in some of these cases the asbestosis is only detectable microscopically. fixation of the lungs through the bronchi and the use of heard's barium sulphate impregnation technique facilitate demonstration of the fibrosis (see p. and fig. . . ). the mild degree of asbestosis currently encountered is of little functional significance but is often critical in determining whether an associated carcinoma of the lung should be attributed to asbestos exposure (see below). the histological diagnosis of asbestosis requires an appropriate pattern of interstitial fibrosis associated with the presence of asbestos bodies. both components must be present. the fibrosis is paucicellular, lacking any significant degree of inflammation and being collagenous rather than fibroblastic. it is generally considered that asbestosis begins about the respiratory bronchioles and alveolar ducts where most of the asbestos fibres impact. alveolar walls attached to these bronchioles show fine interstitial fibrosis. however, this early lesion has to be interpreted with caution because it is not specific to asbestos, being found with other inhaled mineral dusts , and even in many cigarette smokers who have not been so exposed. it more likely represents a non-specific reaction to a variety of inhaled particles. it may cause mild airflow obstruction but is not associated with the radiographic, clinical or restrictive changes of classic asbestosis. as the disease progresses, the focal changes join up so that the basal subpleural regions show widespread interstitial fibrosis and eventually complete destruction of the alveolar architecture. in severe cases there may be honeycombing and metaplastic changes in the alveolar and bronchiolar epithelium. apart from the presence of asbestos bodies the changes resemble those of non-specific interstitial pneumonia, or more rarely usual interstitial pneumonia. fibroblastic foci may be found but they are uncommon. there is often an increase in alveolar macrophages but the desquamative interstitial pneumonia that has been reported in association with asbestos , is not to be regarded as a variant of asbestosis ; concomitant smoking is a more likely cause. a variety of other non-specific inflammatory processes such as organising pneumonia have been reported in asbestos workers and if localised some have been suspected of representing malignancy until biopsied. several schemes have been proposed for grading the extent and severity of asbestosis. these are of value in epidemiological studies but should only be applied to cases meeting the histopathological criteria for a diagnosis of asbestosis. one such scheme is shown in box . . . , , in well-established asbestosis asbestos bodies are numerous and easy to find, aggregates of them sometimes forming clumps ( fig. . . ) . in earlier lesions a detailed search may be necessary, in which fibrosis confined to the walls of respiratory bronchioles and the first tier of adjacent alveoli b extension of fibrosis to involve alveolar ducts and/or two or more tiers of alveoli adjacent to the respiratory bronchiole, with sparing of at least some alveoli between adjacent bronchioles fibrotic thickening of the walls of all alveoli between at least two adjacent respiratory bronchioles honeycomb change a an average score is obtained for an individual case by adding the scores for each slide ( - ), then dividing by the number of slides examined b grade and, to a lesser extent, grade need to be distinguished from smoking-induced peribronchiolar fibrosis and mixed-dust pneumoconiosis. case the examination of unstained or perls-stained sections facilitates their identification. minimum criteria for the diagnosis of asbestosis require the identification of diffuse interstitial fibrosis in well-inflated lung tissue remote from a lung cancer or other mass lesion and the presence of either two or more asbestos bodies in tissue with a section area of cm or a count of uncoated asbestos fibres that falls in the range recorded for asbestosis by the same laboratory. , there are marked variations in the concentration of asbestos fibres between samples from the same lung , and it is therefore recommended that at least three areas be sampled, the apices of the upper and lower lobes and the base of the lower lobe. the equivalent of mallory's alcoholic hyalin of the liver has been described in the lungs in asbestosis, , and subsequently in other [ ] [ ] [ ] [ ] it is seen as small eosinophilic cytoplasmic inclusions within hyperplastic type ii alveolar epithelial cells (fig. . . a ). electron microscopy shows that the inclusions consist of a tangle of tonofilaments ( fig. . . b ) and by immunocytochemistry a positive reaction is obtained with antibodies to cytokeratin, both these features being typical of mallory's hyalin in the liver. the inclusions also react for ubiquitin, the accumulation of which is indicative of cellular damage, in particular faulty proteinolysis. the differential diagnosis of asbestosis includes pulmonary fibrosis due to many other causes, any of which may of course affect an asbes-tos worker as much as members of the general population. the proportion of diffuse pulmonary fibrosis in asbestos workers that is not attributable to asbestos has been estimated to be as high as % and likely to rise as the risk of asbestosis diminishes with better industrial hygiene. the principal differential diagnosis of asbestosis is from idiopathic pulmonary fibrosis. both diseases affect the bases and periphery of the lungs predominantly. in the late stages, cystic change is more evident in idiopathic pulmonary fibrosis but this criterion is not totally reliable. nor is the presence of pleural fibrosis, although it is usually present in asbestosis and is seldom found in idiopathic pulmonary fibrosis. asbestosis seldom progresses or does so very slowly after exposure ceases , whereas idiopathic pulmonary fibrosis typically proves fatal within - years from onset. the fibrosis of asbestosis is generally paucicellular: inflammation is not a feature and the fibroblastic foci that characterise the usual interstitial pneumonia pattern of fibrosing alveolitis are seldom observed in asbestosis. very often the distinction from idiopathic pulmonary fibrosis has to be based on the amount of asbestos in the lung and, if asbestos bodies are not readily identifiable, this has to depend on fibre counts. errors are made both by overlooking substantial numbers of asbestos bodies completely and by ascribing undue importance to scanty bodies. if considering the possibility of minimal asbestosis (to justify attributing carcinoma of the lung to asbestos, for example) it should be remembered that a little peribronchiolar fibrosis is also characteristic of smokers' lungs, centriacinar emphysema and early mixeddust pneumoconiosis. [ ] [ ] [ ] as described above, at least two asbestos bodies/cm in the presence of interstitial fibrosis distant from any lung cancer or other mass lesion is required for a diagnosis of asbestosis. although the causes of asbestosis and idiopathic pulmonary fibrosis are very different, they resemble each other in several ways, suggesting that similar pathogenetic mechanisms may operate. , [ ] [ ] [ ] in both these diseases there is degeneration of the alveolar epithelium and capillary endothelium, with patchy loss of the former, and bronchoalveolar lavage shows an increase in macrophages that might perpetuate the damage by releasing lysosomal enzymes, nitric oxide and hydroxyl radicals. , [ ] [ ] [ ] both diseases are also characterised by an increased prevalence of circulating non-organ-specific autoantibodies. experimentally, asbestos exposure leads to the activation of a variety of fibrogenic cytokines at sites of lung injury. , [ ] [ ] [ ] [ ] [ ] [ ] inhaled asbestos activates a complement-dependent chemoattractant for macrophages and macrophage stimulation involves the secretion of fibroblast stimulating factors, [ ] [ ] [ ] asbestos being intermediate between haematite and silica in regard to macrophage-mediated fibrogenicity. the epithelial damage could be mediated directly by the needle-like asbestos fibres or indirectly through enhanced phagocyte generation of free radicals (which is much greater with amphibole asbestos than with either chrysotile or silica). , fibrogenic cytokines released by activated pulmonary phagocytes and regenerating alveolar epithelial cells in asbestosis include tumour necrosis factor-α and transforming growth factor-β, as in idiopathic pulmonary fibrosis. as a result of better industrial hygiene, asbestosis is less severe today than in earlier years when it followed much heavier exposure, with the consequence that death from respiratory failure and cor pulmonale is less common and sufferers are surviving longer. there is therefore now a greater risk of asbestos-related cancer eventually developing. asbestos exposure predisposes to two varieties of malignant neoplasm, carcinoma of the lung and mesothelioma of the pleura and peritoneum. the risk of malignancy increases with dose but the relative risk of carcinoma is much smaller than that of mesothelioma. for example, with heavy exposure, as in lagging, the risk of mesothelioma is increased -fold whereas it is increased only fivefold for lung cancer. hence, with light exposure there is a substantial risk of mesothelioma but only a small risk of lung cancer. asbestosis requires heavy exposure and in one group of patients with asbestosis, % died of pulmonary carcinoma, % of mesothelioma and % of other respiratory diseases. although there were many earlier reports, the link with carcinoma of the lung may be considered to have been firmly established by , that between crocidolite asbestos and mesothelioma by , and that between amosite asbestos and mesothelioma by . mesothelioma is considered on page . in regard to carcinoma of the lung, asbestos is not such a potent pulmonary carcinogen as cigarette smoke but together their effects are multiplicative rather than additive (table . . ). , however, the risk attributable to asbestos is the same regardless of smoking history, being increased fivefold in both smokers and non-smokers. there is usually a latent period in excess of years between first exposure to asbestos and the development of lung cancer and the risk increases the greater the cumulative exposure. the increased risk involves carcinomas of all the histological types encountered in the lung, although adenocarcinoma has been disproportionately overrepresented. , [ ] [ ] [ ] [ ] [ ] [ ] it is uncertain whether the increased risk of carcinoma is caused by the asbestos , [ ] [ ] [ ] [ ] [ ] [ ] or the asbestosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the latter view envisages the carcinoma arising in the foci of alveolar epithelial hyperplasia and dysplasia that commonly accompany any interstitial fibrosis (see carcinoma complicating idiopathic pulmonary fibrosis, p. ). however, most carcinomas complicating asbestosis arise in the bronchi rather than the alveolar tissue. on the other hand, more arise in the sites worst affected by asbestosis, the lower lobes and the periphery of the lung, than in the general population ( fig. . . ) . , , [ ] [ ] [ ] [ ] [ ] the majority view has been that asbestosis is a necessary precursor of the carcinoma but evidence to the contrary is finding increasing support (table . . ). in the uk, industrial compensation was formerly only awarded to an asbestos worker for carcinoma of the lung if there was also asbestosis or diffuse pleural fibrosis but new rules were introduced in . asbestosis remains a sufficient criterion but diffuse pleural thickening is not and asbestosis is no longer a necessary criterion: asbestos is deemed to have been responsible if the patient worked in asbestos textile manufacture, spraying, lagging or gas mask manufacture for at least years before or years after . the basis for these changes is the premise that heavy asbestos exposure is sufficient in itself to account for carcinoma of the lung. together, these factors have a multiplicative rather than additive effect mortality ratio non-smoking controls non-smoking asbestos workers cigarette-smoking controls cigarette-smoking asbestos workers asbestosis associated with carcinoma of the lung. the asbestosis has been highlighted by barium sulphate impregnation and is seen as a grey subpleural band to the right of the picture. although the carcinoma has arisen in the same lobe as the asbestosis it has not obviously arisen in an area affected by asbestosis. . asbestosis diagnosed clinically, radiologically or histologically or a minimum count of asbestos bodies per gram dry lung tissue (/g dry), or an uncoated asbestos fibre burden of million amphibole fibres more than µm in length/g dry, or million amphibole fibres more than µm in length/g dry or estimated cumulative exposure to asbestos of at least fibres/ ml-years or an occupational history of year of heavy exposure to asbestos (e.g. manufacture of asbestos products, asbestos spraying) or - years of moderate exposure (e.g. construction or ship-building) and . a minimum lag time of years lung fibre counts in the asbestosis range (see table . . ) provide valuable evidence of such exposure. compensation standards for asbestos-associated lung cancer in different countries are shown in box . . . , asbestos-induced airway disease although asbestosis causes a restrictive respiratory defect, airflow limitation is also seen in this disease. much of the airflow limitation is attributable to cigarette smoking but it is also seen in non-smoking asbestos workers and is worse in those with asbestosis. the pathological basis of this appears to be small-airways disease (see p. ). it is possibly a non-specific reaction to inhaled dust or cigarette smoke. because it is not established that this lesion progresses to interstitial alveolar fibrosis (asbestosis) the term 'asbestos airways disease' is suggested. fibrosis limited to the bronchioles is specifically excluded from the definition of asbestosis in the latest guidelines (although these retain grade for fibrosis limited to the bronchiolar walls). it should also be noted that, although emphysema is considered to be a destructive rather than fibrotic condition, a little focal the presence of asbestos-related bilateral pleural plaques or asbestos-related bilateral pleural thickening and occupational exposure and a lag time of at least years the presence of asbestosis or pleural plaques or diffuse pleural thickening or fibre-years of exposure only fibre-years of exposure are taken into account exposure, at least years' latency and asbestos-related pleural or parenchymal changes asbestosis is not required but smoking is taken into consideration attempts are made to quantify separately the attributability to asbestos, smoking and other factors (e.g. radon) fibrosis is generally evident in this common condition and does not necessarily indicate early asbestosis. aluminium has been implicated in the development of respiratory disease during the refining of its principal ore, bauxite, to yield various aluminium oxides (aluminas), in the preparation of the metal by smelting alumina, in the production of corundum abrasive and in the production of special aluminium powders used in explosives. bauxite is a mixture of various aluminium oxides, hydroxides and silicates, iron oxide and titanium dioxide. the oxides of aluminium are obtained by differential heating of the ore and the respiratory effects of this work appear to be no more than mild airway irritation. it is generally accepted that aluminium oxide is inert. aluminium is prepared by the electrolytic reduction of its oxide dissolved in sodium aluminium fluoride (cryolite), a process releasing a considerable amount of fluoride-rich effluent. exposed workers have complained of what is termed pot-room asthma. the pathology of this condition is not well described but the pathogenesis is thought to involve irritation rather than allergy. the abrasive corundum is formed from bauxite mixed with coke and iron heated in an electric arc furnace, a process in which workers may be exposed to the fumes of alumina and free silica. in the past some of these workers developed diffuse pulmonary fibrosis (shaver's disease) and, although this was initially attributed to the aluminium, it is now agreed that the free silica was the responsible agent. the exposure to free silica has been reduced and the disease is now regarded as historic. aluminium powder holds a paradoxical position in regard to lung disease. in certain industries it has caused very severe pulmonary fibrosis, yet in others it has proved harmless. indeed, at one time canadian miners breathed aluminium dust before work, in the belief that this would reduce the danger of silica in the mine dust and more recently silicosis has been treated by such means in france. it is questionable whether this practice is effective but it at least appears to cause no harm. the explanation for these contradictory observations probably lies in differing methods of manufacture of aluminium powder. aluminium metal appears to be an inert substance but this is only because it has a high affinity for oxygen and the surface layer of aluminium oxide so formed is firmly bound to the underlying metal, unlike ferric oxide which permits further rusting of iron. granular aluminium powders, produced in a ball mill or from a jet of molten aluminium, therefore acquire a protective coat of surface oxide and are inert. with stamped aluminium powders, however, surface oxidation is prevented by lubricants added to aid the separation of these flake-like particles. the usual lubricant (stearin) contains stearic acid and this polar compound combines with the underlying metal, which is thereby protected from both atmospheric oxidation and the action of body fluids when such dust is inhaled. in certain circumstances, however, non-polar lubricants in the form of mineral oils have been substituted for stearin. this happened in germany during the second world war when munition production was stepped up but stearin was difficult to obtain, , and in the uk in the s to make the powder darker for purely commercial reasons. in vitro, oil-coated stamped aluminium powder reacts with water to produce aluminium hydroxide, which affords the underlying metal no protection against further attack, so that aluminium hydroxide continues to be formed. this substance is a protein denaturant, once used in the tanning industry, and it is believed that this property underlies the very ex ceptional cases of severe pulmonary fibrosis that have occurred in connection with stamped aluminium powder produced with mineral oil rather than stearin. , the fibrosis has a very characteristic pattern, affecting the upper lobes and progressing rapidly, the interval from onset of symptoms to death being as short as years. there is marked shrinkage of the lungs with gross elevation of the diaphragm and buckling of the trachea (fig. . . ). the lungs are grey ( fig. . . ) and microscopically, numerous small black jagged particles are seen. these can be shown to contain aluminium with irwin's aluminon stain or by microprobe analysis. what appears to be a different pathological effect of aluminium dust on the lungs is the rare development of granulomatous disease resembling sarcoidosis and berylliosis. , this represents hypersensitivity to the metal, amenable to confirmation with a lymphocyte transformation test similar to that used to diagnosis berylliosis (see below). rare cases of desquamative interstitial pneumonia and pulmonary fibrosis have been reported in aluminium welders. , elements with atomic numbers from (lanthanum) to (lutetium) are known as the lanthanides or rare earth metals. they are used in many manufacturing processes, including the production of hightemperature ceramics and the grinding of optical lenses. carbon arc lamps used in reproduction photography emit appreciable quantities of oxidised lanthanides, particularly cerium oxide, and there are reports of pneumoconiosis in exposed individuals. the pathological changes reported have varied from granulomatous nodules to diffuse interstitial fibrosis indistinguishable from the idiopathic variety except for the presence of rare earth elements (usually cerium) detected by polarising light microscopy and electron microprobe analysis. hard metal is a tungsten alloy containing small amounts of cobalt, titanium, molybdenum and nickel. it is exceptionally tough and once formed can only be worked with diamond. it is used in the tips of drill bits, on abrasive wheels and discs, and in armaments. interstitial lung disease is liable to arise in its manufacture or in those using hard metal as an abrasive. experimental work suggests that cobalt is the dangerous constituent but this element is soluble and, unless industrial contact has been recent, analysis of lung tissue usually shows tungsten and titanium but no cobalt. the role of cobalt is also indicated by the development of similar interstitial lung disease in diamond polishers using high-speed polishing discs made with a diamond-cobalt surface that lacked tungsten carbide and the other constituents of hard metal. , hard-metal lung disease and cobalt lung take two forms, an industrial asthma and interstitial fibrosis. the latter has a diffuse lower zonal distribution and the appearances mimic idiopathic pulmonary fibrosis. however, an unusual feature is the presence of moderate, or perhaps only small numbers, of giant cells (fig. . . a, b) . , not only are there multinucleate alveolar macrophages but syncytial cell forms develop in the alveolar epithelium. electron microscopy confirms that these are multinucleate type ii pneumocytes (fig. . . c ). such epithelial changes are well known in measles pneumonia but the viral inclusion bodies that characterise this infection are not found in hard-metal pneumoconiosis. the changes are those initially described as a particular pattern of idiopathic interstitial pneumonia termed giant cell interstitial pneumonia or gip (see p. ). elemental analysis shows that many, but not all, cases of gip represent hardmetal disease. the exceptions seldom give a history of cobalt exposure and must be presumed to represent true idiopathic cases. conversely, epithelial giant cells are not always found in hard-metal pneumoconiosis and so their presence, although highly characteristic, is neither totally specific nor totally sensitive. beryllium is the lightest of metals. it has an atomic weight of and special properties that make it especially useful in many applications. it is more rigid than steel, has a high melting point and is an excellent conductor of heat and elecricity. unfortunately, the inhalation of beryllium dust or fume is exceedingly dangerous. , those who worked with beryllium compounds before precautionary measures were taken suffered a high morbidity and mortality. sometimes, the escape of dangerous fumes from the factories was on such a scale that people living in nearby houses, downwind from the places in which these materials were being worked, contracted and occasionally died from berylliosis ('neighbourhood cases'). alternatively, contamination of a beryllium worker's clothes might lead to berylliosis in a temperatures. the alloys of beryllium are also now widely used, especially those with copper, on which it confers elasticity and resistance to fatigue. alloy manufacture and the machining of beryllium alloys are therefore further activities that entail a risk of berylliosis, as is the recovery of the metal in the recycling of scrapped electronic and computer parts. seemingly innocuous occupations such as dental laboratory technician are not without risk of chronic berylliosis. there are good grounds for regarding chronic berylliosis as being an allergic condition. many of those affected react strongly to skin tests with dilute solutions of beryllium salts, although these must be undertaken with care: occasionally in a highly sensitised person even so small an exposure may evoke a systemic reaction. the skin reaction is of the delayed type, occurs in only % of exposed individuals, is not associated with a clear-cut dose-response curve and represents a granulomatous response. further evidence for the disease having an allergic basis derives from bronchoalveolar lavage, which demonstrates an excess of t-helper lymphocytes that proliferate in vitro on exposure to beryllium salts. a positive transformation test given by these lymphocytes is a more reliable indicator of disease than in vitro blood lymphocyte transformation testing, which is safe but not wholly reliable and indicates only sensitization, rather than berylliosis. susceptibility to berylliosis varies widely from person to person and it is notable that chronic pulmonary disease is strongly associated with the hla antigen dpβ and the glu gene. , the importance of genetic factors is supported by a report of the disease in identical twins. chronic berylliosis is thought to be initiated by the metal binding to tissue proteins and acting as a hapten to initiate a delayed hypersensitivity response characterised by a proliferation of t-helper lymphocytes. these sensitised cells in turn secrete a variety of cytokines (e.g. interleukin- , tumour necrosis factor-α and interferon-γ) that recruit and activate macrophages, which mature into epithelioid cells. the resultant epithelioid cell granulomas destroy the lung tissue and lead to pulmonary fibrosis. if beryllium enters the subcutaneous tissues through a cut or abrasion, as often happened in the earlier days of fluorescent lamp manufacture, a sarcoid-like granuloma soon appears at the site; in time, the overlying epidermis may break down to form an ulcer. even more serious are the lesions produced by the inhalation of beryllium. chronic pulmonary berylliosis takes the form of a widespread granulomatous pneumonia with a histological picture identical to that of sarcoidosis (fig. . . a ). both berylliosis and sarcoidosis affect the upper lobes more than the lower (fig. . . b ) and in both diseases the granulomas are preferentially distributed along lymphatics and may involve adjacent blood vessels. in neither condition is there widespread necrosis but in both diseases the granulomas occasionally display a little central necrosis or hyalinisation. as in sarcoidosis, the hilar lymph nodes may be involved but, unlike sarcoidosis, not in isolation. over a period of many years, the sarcoid-like granulomas gradually undergo progressive fibrosis, with consequent impairment of pulmonary function. in the later stages, when the disease has become chronic, dispersal of beryllium from its site of initial absorption may lead to generalisation of the disease and to the appearance of similar granulomas elsewhere, particularly in the liver, kidneys, spleen and skin, but this is unusual. relative. beryllium compounds may also cause contact dermatitis and conjunctivitis. beryllium is also classified as a probable pulmonary carcinogen, but this is controversial. two forms of berylliosis are recognised, acute and chronic. acute berylliosis was first reported in germany in and is now largely of historical interest, being only encountered as a result of rare accidental or unexpected exposure. it follows the inhalation of a soluble beryllium salt and represents chemical injury, the pathology being that of diffuse alveolar damage (see p. ). further consideration will be confined to chronic berylliosis, which is allergic in nature. chronic berylliosis was first reported in in the fluorescent lamp industry. beryllium has now been replaced in this application but it has since proved to be of great value in the nuclear, electronic, computer and aerospace industries and the production of refractory materials and crucibles that are to be subjected to particularly high and there is a lifelong risk of disease. progression often entails alternating exacerbations and remissions, long after exposure has ceased. in keeping with the view that berylliosis is a hypersensitivity reaction, very little beryllium is necessary to cause the disease. particulate beryllium is so scanty in the affected tissues and the atomic number of beryllium so low that electron microprobe analysis is generally unsuitable for its detection. furthermore conventional detectors are protected by a beryllium window. however, the substitution of a polymeric window has enabled beryllium to be detected by electron microprobe analysis, presumably in a patient with fairly heavy exposure. ion or laser microprobe mass spectroscopy can also detect very small amounts of beryllium in tissue sections but these techniques are not widely available. the differential diagnosis of chronic berylliosis is from sarcoidosis, to which it is identical morphologically. [ ] [ ] [ ] however, as noted above, it is unusual for berylliosis to cause significant hilar lymphadenopathy in the absence of pulmonary disease, which is a common feature of sarcoidosis. extrathoracic granulomas, erythema nodosum and uveitis, which are all common in sarcoidosis, are unusual in berylliosis. however, one group found that % of patients initially diagnosed as having sarcoidosis actually had chronic berylliosis. similar findings have been reported by others. , any patient thought to have sarcoidosis who has worked with or near metals should be offered a beryllium lymphocyte transformation test. a list of laboratories performing this test can be found at www.dimensional. com/~mhj/medical_testing.html. although polyvinyl is not a mineral and the reaction of the lungs to its presence is therefore not a true pneumoconiosis, it is generally so termed and is dealt with here for convenience. workers are exposed to polyvinyl chloride dust in the milling and bagging of this plastic and micronodular opacities may be detected in their lungs radiologically. however, the material is non-fibrogenic and histology merely shows a foreign-body reaction to the dust particles. the radiological opacities may abate when exposure ceases. nevertheless, one polyvinyl chloride worker developed systemic sclerosis, which is a recognised complication of silicosis (see p. ). polyvinyl chloride is produced from vinyl chloride monomer, which has a causal association with angiosarcoma of the liver and probably other forms of cancer, including carcinoma of the lung (see p. ). in the late s a characteristic lung disease was identified in workers at several factories producing plush material by spraying nylon flock on to an adhesive backing material. [ ] [ ] [ ] [ ] the flock fibres are too large to be inspired but may be mixed with smaller nylon shards of respirable size. the workers complained of cough and breathlessness and were found to have a restrictive ventilatory defect with interstitial markings on radiography. their symptoms improved on removal from the workplace but relapsed on return to work. pathologically, there was lymphocytic bronchiolitis and peribronchiolitis with widespread lymphoid hyperplasia represented by lymphoid aggregates. chronic berylliosis is characterised by the gradual onset of cough, shortness of breath, chest pain, night sweats and fatigue. these symptoms may develop within a few weeks of exposure or many years later. once the worker is exposed, the beryllium is retained in the tissues granulomas were not identified. the histological appearances suggest a severe immunological reaction and raise possibilities such as rheumatoid disease and sjögren's syndrome but consideration of the clinical and serological setting and the occupation should permit recognition of the cause. the industrial production of popcorn and other foodstuffs appears to carry a risk of obstructive airway disease. [ ] [ ] [ ] [ ] biopsy of affected workers has shown peribronchiolar fibrosis and granulomas and air sampling has identified many volatile organic compounds, of which the flavouring agent diacetyl ( , -butanedione) is suspected of being responsible for the bronchiolitis. it is difficult to continue paint spraying (air brushing, aerographics) without adequate respiratory protection but in the early s several small aerographic factories operated in the neighbourhood of alicante, southeastern spain without any concern for the workers' health. the workers were required to paint patterns on textiles using a hand-held spray gun. the atmospheric pollution was intense but complaints of respiratory difficulties were met with reassurances and the workers urged to continue. this they did because of the otherwise poor economy, often returning to work when disabling breathlessness had settled down. a change of paint (to acramin f) may have contributed because the worst-affected workers were employed at two plants that had made this switch. their illness has been described as the 'ardystil syndrome' after the name of one of these factories. some workers were left with permanent respiratory disability. one required a lung transplant and others died. [ ] [ ] [ ] [ ] transbronchial biopsy showed organising pneumonia, which in the fatal cases had progressed to irreversible interstitial fibrosis. a similar outbreak of respiratory disease was subsequently reported in algerian textile factories where acromin f was applied by the same technique. , acromin f is marketed as a paste and used as such without ill-effect. its use in heavy spray form appears to be responsible for the 'ardystil syndrome' . workers in engineering workshops may be exposed to the prolonged inhalation of fine sprays or mists of the longer-chain hydrocarbons that constitute many mineral oils. this may result in exogenous lipid pneumonia, which is described on page , or extrinsic allergic alveolitis. [ ] [ ] [ ] the vapour of shorter-chain hydrocarbons such as paraffin oil (kerosene: c - ) and petrol (gasoline: c - ) and gaseous hydrocarbons such as propane may act as acute asphyxiants or central nervous system depressants but have negligible pulmonary toxicity. however, if they are ingested or aspirated in their liquid form they are acutely toxic to the lungs, producing a chemical pneumonitis with the features of diffuse alveolar damage. ingestion may be accidental or deliberate (see fig. . , p. ) whereas aspiration is generally inadvertent, occurring in siphoning accidents, such as those experienced by fairground operatives who 'breath or eat fire' ('fire-eater's lung'). , animal experiments involving the intratracheal injection of kerosene resulted in acute pulmonary exudates, which cleared except for residual bronchiolitis. welder's pneumoconiosis, first recognised in , essentially represents the fairly harmless deposition of iron in the lungs (siderosis -see p. ). however, welders may suffer various ill-effects from the inhalation of substances other than iron (table . . ). some of these are para-occupational risks, that is, encountered by welders because they work near another process and are inadvertently exposed: thus, shipyard welders may be exposed to asbestos, and those in foundries to silica. welders may therefore develop a mixed-dust pneumoconiosis (see p. ), rather than just siderosis. however, one analytical investigation identified excess amounts of iron alone in association with pulmonary fibrosis; the silicon content did not differ from that in controls. more directly, welders may be exposed to asbestos insulation that they themselves use, while welders of special steel alloys run the risk of metal-induced asthma, metal fume fever, polymer fume fever and the consequences of toxic metal fume inhalation, all of which are described separately in this chapter, as is lung disease in aluminium welders. chronic bronchitis has been attributed to the inhalation of low concentrations of irritants such as ozone and nitrogen dioxide by welders but this risk is unproven and the subject of much controversy. welders may also inhale carcinogenic hexavalent chromium compounds in the course of their work and therefore develop lung cancer. the term 'welder's lung' is often applied indiscriminately to any of these diseases and, as it has no specific meaning, is best avoided. dust, fume and gas are some of the terms used to describe different physical forms of respirable agents. they are defined in table . . on the finely divided fume of several metals is highly toxic to the lungs and capable of producing severe acute and chronic damage to both the conductive airways and the alveoli, resulting in acute tracheobronchitis and bronchiolitis, diffuse alveolar damage, obliterative bronchiolitis and pulmonary fibrosis. important metal fumes in this respect include aluminium, which is released together with silica fume in bauxite smelting (see shaver's disease, above), cadmium from welding or cutting special steels, chromium from cutting its alloys or in the manufacture of chromates, cobalt released in the production and use of its alloys (see hard-metal disease, above), mercury released in various industries and in the home, nickel carbonyl released during the purification of metallic nickel or the manufacture of nickel alloys and beryllium (see above). many irritant gases cause severe acute and chronic damage to both the conductive airways and alveoli. the changes are non-specific and similar to those wrought by toxic metal fumes (see above) and viruses amongst other agents. they consist of acute tracheobronchitis and bronchiolitis, obliterative bronchiolitis, diffuse alveolar damage and pulmonary fibrosis. the gases liable to produce such damage include oxides of nitrogen, sulphur dioxide, ozone, phosgene, chlorine, ammonia and various constituents of smoke, notably acrolein. some of these are also touched upon in chapter . because they are of general as well as occupational importance, although there is no rigid difference between general and occupational pollution. ozone, sulphur dioxide and nitrogen dioxide are oxidising gases that may be found together as industrial atmospheric pollutants. each is capable of producing diffuse alveolar damage by means of its oxidising properties and the release of free active radicals. in addition, they cause damage to distal airways, particularly terminal and respiratory bronchioles, with resulting bronchiolitis. oxides of nitrogen may be encountered with fatal consequences by farmhands seeking to free a blockage in a silo when they encounter pockets of this gas that have accumulated on top of the fermenting silage: the term 'silo-filler's disease' is generally applied to the initial haemorrhagic oedema or the obliterative bronchiolitis that develops in those who survive the initial chemical injury. [ ] [ ] [ ] [ ] asphyxia due to the farmhand encountering pockets of carbon dioxide is a further hazard within agricultural silos. other farmhands have suffered from the inhalation of toxic gases or bacteria when handling liquid manure. [ ] [ ] [ ] [ ] welding, which is considered below, may also involve exposure to toxic gases such as oxides of nitrogen. ozone, the principal oxidant gas of photochemical smog, produces pulmonary changes at relatively low levels and may be encountered at higher concentrations in various industries. potentially dangerous levels of ozone are produced from atmospheric oxygen by ultraviolet radiation given off in welding while ozone is used in industry to sterilise water, bleach paper, flour and oils, and mask the odour of organic effluents. the damage wrought by ozone is predominantly centriacinar in distribution, affecting terminal and respiratory bronchiolar epithelium and proximal alveolar epithelium. [ ] [ ] [ ] there is loss of cilia and necrosis of centriacinar alveolar type i epithelial cells. the changes are dose-dependent and, in one study, the youngest animals were most sensitive. in long-term experiments, hyperplastic bronchiolar clara and ciliated cells extended peripherally to line alveolar ducts. the role of granulocytes is stressed in some experimental studies and it is notable that neutrophil migration is prominent when the human lungs are damaged by ozone. aldehydes such as acetaldehyde, formaldehyde and acrylic aldehyde (acrolein) are widely used in the plastics and chemical industries. the first is a liquid and the others are water-soluble gases. pathologists are of course familiar with formaldehyde solution from its use as a disinfectant and histological fixative. all these aldehydes are intensely irritant and their acute effects generally prevent prolonged exposure to high concentrations. chronic effects include skin sensitivity and asthma, and in rats nasal carcinoma. however, the doses to which these experimental animals were exposed far exceed any that are likely to be encountered by humans, in whom there is no convincing evidence of aldehyde-induced cancer. ammonia gas is extensively used in industry as a raw material, notably in the manufacture of nitrogenous products such as fertilisers and plastics. it is highly soluble and its acute irritative effects are mainly felt in the eyes, nose and throat, but high levels affect the major airways, possibly leading to them being blocked by exudates. survival usually brings full recovery but bronchiectasis and obliterative bronchiolitis have been described. chlorine gas is widely used in the chemical industry. it is transported and stored under pressure in liquid form. heavy exposure through its accidental release or use as a war gas has proved fatal through its acute toxicity causing exudative airway occlusion and pulmonary oedema. survivors usually recover completely but, as with nitrogen dioxide and ammonia, there is a risk of obliterative bronchiolitis. phosgene (carbonyl chloride, cocl ) is a poisonous, colourless gas that was responsible for thousands of deaths during world war i, when it was used in chemical warfare. it is used industrially in the preparation of some organic chemical compounds and is formed, perhaps inadvertently, by the combustion of methylene chloride in products such as paint strippers. phosgene causes injury to terminal bronchioles and alveoli, with resulting oedema and hyaline membrane formation. the mechanism of cell damage is uncertain but it may depend on inactivation of intracellular enzymes by the gas. longterm problems are rare but chronic bronchitis and emphysema have been described in survivors. mustard gas (bichloroethyl sulphide, c h cl s) is a further agent that has been used in chemical warfare. it is primarily a skin vesicant but when inhaled it results in widespread epithelial destruction and pulmonary oedema. survivors may be left with irritant-induced asthma (reactive airways dysfunction), chronic bronchitis, tracheobronchomalacia, bronchiectasis and bronchiolitis obliterans. [ ] [ ] [ ] thionyl chloride is used in the manufacture of lithium batteries where it is liable to result in the release of sulphur dioxide and hydrochloric acid fumes. workers in such factories have developed lung injury varying from mild, reversible interstitial disease to severe obliterative bronchiolitis. hydrogen sulphide is the principal chemical hazard of natural gas production. high levels of the gas also buid up in sheds housing large numbers of pigs, the source here being the pig manure. once inhaled the gas is rapidly absorbed into the blood stream. the effects are therefore widespread but include the usual respiratory effects of irritant gases, varying from sneezing to pulmonary oedema and acute respiratory distress, depending upon the exposure. in alberta cases were identified over a -year period. the overall mortality was %; % of victims were dead on arrival at hospital. most required admission to hospital but the survivors experienced no long-term adverse effects. a the danger of asphyxia from the inhalation of gases devoid of oxygen is fairly widespread in industry. it generally arises from the use of inert gases, which, being non-toxic, give a false sense of security. pockets of these gases tend to form in confined spaces. anoxic death from the accumulation of methane is well known in mines and has also occurred in slurry pits and sewers. anoxic asphyxia in diving (and anaesthesia) has resulted from the incorrect connection of gas cylinders or failure to notice that a mixed gas contains insufficient oxygen. deaths have occurred in welding when argon or carbon dioxide has been used to shield the weld and prevent oxidation of the metals at the high temperatures employed. deaths have also resulted from inadvertent entry to discharged oil tanks filled with nitrogen to reduce the risk of explosions, or from the formation of pockets of nitrogen gas applied in liquid form to freeze the contents of damaged pipes so that they can be repaired without the necessity to drain down. the respiration of a gas devoid of oxygen causes loss of consciousness within seconds because it not only fails to provide oxygen but removes that present in the pulmonary arterial blood. the changes at autopsy are those common to cellular hypoxia. they include cerebral and serosal petechiae and pulmonary congestion and haemorrhage but these features are not specific and are not always present. the cause of death can generally only be surmised from the circumstances surrounding the death. occupational asthma is the commonest cause of work-related respiratory disease in many western countries (table . . ). [ ] [ ] [ ] the reported incidence ranges from per million workers in south africa to per million workers in finland. , it occurs in many industries (table . . ) and occupational factors can be identified as contributing to asthma in about % of adult cases. over aetio- in the uk a third are organic, a third chemical, % metallic and the rest miscellaneous. the commonest, in descending order, are isocyanates, flour and grain, laboratory animals, glutaraldehyde, solder or colophony and hardening agents. atopy appears to predispose to occupational asthma when the allergen is of high molecular weight but not when it is of low molecular weight. for example, atopic individuals are particularly prone to develop asthma if employed in the manufacture of biological detergents, whereas atopy does not increase the risk of asthma from sensitisation to toluene di-isocyanate, which is a serious health problem in the manufacture of polyurethane. similarly, platinum salts are such potent sensitising agents that nearly all those exposed to them develop asthma. asthma-provoking metals other than platinum include chromium, cobalt, nickel and vanadium, all of which are used in steel alloys, and possibly aluminium (see pot-room asthma, p. ). other asthma-inducing factors encountered in industry include grain and flour dust, certain wood dusts, soldering fluxes containing colophony (pine resin), epoxy resin hardeners such as phthallic anhydride, isocyanate-containing foams and paints, formaldehyde and the excreta of laboratory animals. contaminated humidifiers may cause occupational asthma as well as humidifier fever and extrinsic allergic alveolitis. pathologically, occupational asthma is identical to nonoccupational asthma (see p. ). byssinosis is a further form of occupational asthma, one encountered in the cotton industry. the sensitising agent is a component of the cotton bract, which is the part of the cotton harvest other than the cotton fibre. bract consists of dried leaf, other plant debris and soil particles and contains a variety of fungal and bacterial residues, including lipopolysaccharide endotoxin, but the exact nature of the sensitising agent remains unknown. the endotoxin is unlikely to be responsible for byssinosis but may be the cause of so-called mill fever, a self-limiting illness characterised by malaise, fever and leukocytosis that is experienced by many people on first visiting a cotton mill. dust levels and the risk of byssinosis are particularly high in the carding rooms where the raw cotton is teased out before it is spun. affected workers are worse when they return to work after the weekend break, a feature attributed to antibody levels having built up during this brief respite from the cotton dust. there is no link with atopy and the fluctuating antibodies are precipitins of the immunoglobulin g class. complement activation by both arms of the complement cascade has been reported. , when the lancashire economy was largely cotton-based, necropsies on workers suffering from byssinosis generally showed gross emphysema, and this came to be accepted as evidence of byssinosis. however, it is now realised that in this heavily industrialised part of the uk, emphysema is as common in the general population as in cotton workers and it can no longer be considered a component of byssinosis. other findings in byssinosis are more commensurate with asthma, namely an increase in bronchial muscle and mucous cells. no granulomas or other evidence of extrinsic allergic alveolitis are found. fever may be the predominant feature in a variety of occupational illnesses and the unifying term 'inhalation fever' has been proposed. however, the individual occupations are of interest and these conditions will therefore be considered separately. mill fever has been mentioned above under byssinosis. humidifier fever is an acute illness characterised by malaise, fever, myalgia, cough, tightness in the chest and breathlessness, all of which are worse on monday mornings if the humidifier responsible is at work rather than home. the chest complaints, and their aggravation on return to work after the weekend, are features shared with byssinosis (see above) but the general complaints fit better with extrinsic allergic alveolitis (see p. ). humidifier fever develops in circum-stances that also lead to the development of a form of extrinsic allergic alveolitis, and not surprisingly the same name has been extended to this latter condition, with inevitable confusion. both diseases are caused by microbiological contamination of humidifiers or air conditioners so that a fine spray of microorganisms is emitted into the office, factory or home. investigations have generally shown the baffle plates of the air conditioner to be covered with a slime of bacteria, fungi or protozoa (mainly amoeba and ciliates), and extracts of this have been used to identify precipitins in the patient's sera, as in extrinsic allergic alveolitis. however, unlike extrinsic allergic alveolitis, humidifier fever resolves within a day and leaves no permanent injury. for this reason there is seldom the opportunity to study the tissue changes, and partly for this reason it remains unclear whether the disease is mediated by immune complexes, as in extrinsic allergic alveolitis, or by endotoxins derived from the contaminants. a febrile illness occurring in precipitin-negative farm-workers after heavy exposure to fungi in their silos was attributed to inhaled fungal toxins and named pulmonary mycotoxicosis. it is also known as precipitin test-negative farmer's lung and organic dust toxic syndrome. the condition is generally self-limiting and is seldom biopsied but desquamative interstitial pneumonia and diffuse alveolar damage have been reported. , metal fume fever this is a self-limiting acute illness characterised by fever, sweating, myalgia, chest pain, headache and nausea, that comes on monday mornings when occupational exposure is experienced after a weekend's respite, as with bysinnosis and humidifier fever; during the week tolerance develops. , the disease involves the release of cytokines such as tumour necrosis factor and is presumed to have an allergic basis. the metals involved are chiefly zinc, copper and magnesium, and, to a lesser extent, aluminium, antimony, iron, manganese and nickel. occupations at risk include any that generate such metal fumes, but particularly welding. it is most commonly associated with welding zinc-coated surfaces. if the symptoms persist, alternative diagnoses, such as acute cadmium poisoning and other specific toxic metal fume diseases, should be suspected: these are not self-limiting and may cause severe bronchiolitis or diffuse alveolar damage (see above). this illness resembles metal fume fever except that it occurs without regard to previous exposure: no tolerance develops and there is therefore no particular susceptibility on mondays. the polymers concerned are quite inert, except when heated to produce fume: polytetrafluorethylene (ptfe, teflon, fluon, halon) is a notable example. as with other self-limiting diseases, little is known of the tissue changes. environmental irradiation chiefly affects the skin but in some parts of the world rocks near the surface release significant amounts of radon gas. this carcinogen is liable to accumulate in buildings and be inhaled, so subjecting the occupants to an increased risk of lung cancer. the installation of underfloor ventilation is therefore advocated in such areas. this subject is explored more fully on page . the body is vulnerable to both increases and decreases in pressure and it is the lungs that often bear the brunt of the damage. increased pressure may result in blast injury or crushing of the chest while decreased pressure may result in the lungs literally bursting or dissolved gases being released within the blood (caisson disease), or the vascular alterations that underlie mountain sickness developing. some of these pressure changes entail a risk of pneumothorax and it is essential that this is properly investigated postmortem by the chest being opened under a water seal. loud music has been incriminated as a specific form of air pressure change causing pneumothorax and metereologists have shown that 'spontaneous' pneumothoraces tend to occur in clusters associated with natural drops in atmospheric pressure. , explosions may cause injury by the body being violently thrown against a less moveable object, by objects being thrown against the body or by the blast wave hitting the body. these mechanisms often act together but sometimes there is only blast injury, to which the lungs are particularly vulnerable. for a time it was considered that the damage was direct, the blast wave travelling down the airways to injure the lungs. however, at the start of the second world war, experiments conducted in the uk showed that the lungs were injured indirectly, the blast wave being transmitted to them through the chest wall: pulmonary blast injury is worst on the side of the body towards the explosion, and can be reduced by protective clothing. underwater explosions are particularly dangerous because water is incompressible. there may be severe internal injury but no external evidence of damage other than a trickle of blood from the mouth or nose. this is because the injury is rate-dependent. quite small thoracic deform-ation may produce severe pulmonary damage if peak compression is attained very quickly, typically in less than ms. conversely, severe chest wall distortion may produce only minor pulmonary contusion if this time is extended beyond ms. at necropsy, the lungs are contused, with blood evident in the airways and parenchyma. depending on the force of the blast, the haemorrhage may be pinpoint, patchy or confluent. it tends to follow the lines of the ribs and may be accompanied by pleuropulmonary lacerations having the same distribution. in this case there will also be haemothorax, pneumothorax and possibly air embolism. patchy pulmonary haemorrhages cuff the blood vessels. , in patients who survive for a few days, the lungs resemble the liver macroscopically and histologically show chronic interstitial inflammation and fibrosis as well as haemorrhage. other injuries are often present and fat embolism, aspiration pneumonia, fluid overload and infection may all be added to the effects of the blast wave. 'chest squeeze' is another form of barotrauma caused by high pressure but here the body is compressed rather than subject to a sudden wave of pressure as in blast injury. it is experienced by divers who descend very deeply, thereby subjecting their bodies to such high pressure that their chest walls are literally crushed, so that their ribs break and their lungs are severely compressed. more common mishaps experienced by divers include drowning and decompression sickness, both of which are dealt with below, and neurological syndromes such as nitrogen narcosis, which will not be considered further. 'burst lung' is the most acute form of decompression sickness. it is experienced by divers and submariners making rapid ascents from depth and by aviators who ascend too rapidly in unpressurised aeroplanes, experience failure of a plane's pressure system or have to eject at high altitudes. injury to the lung is caused by trapped alveolar gas expanding so rapidly that it exceeds total lung capacity before it can escape through the trachea. the lungs literally burst: the alveolar walls rupture and blood mixes directly with alveolar air. the victim experiences chest pain and there may be blood-stained froth at the mouth or frank haemoptysis. air may enter the alveolar walls to cause interstitial emphysema or air embolism. asthmatics may be at particular risk because of regional air-trapping. . diving mammals such as porpoises and whales are protected from such dangers of peripheral air-trapping by cartilage extending far out into the finest conductive airways so that these passages never close, even at the end of full expiration (fig. . . ) . , patients requiring positive-pressure artificial respiration are also at risk of burst lung, but the complications of the resultant interstitial emphysema differ from those experienced by divers. in divers, the chest wall is buttressed by the surrounding water and air in the interstitium is liable to track towards the hilum of the lungs and enter pulmonary veins, with resultant cerebral and coronary air embolism, either of which may prove fatal. iatrogenic burst lung, on the other hand, takes place in patients whose chest wall is not so buttressed, and then outward rupture of the interstitial air is more likely, resulting in pneumothorax. extension of the interstitial emphysema to the mediastinum, neck and chest wall is also more likely in such patients, resulting in surgical emphysema at these sites. however, there are exceptional cases marked by both cerebral embolism and extensive air tracking. the same circumstances that lead to burst lung may also cause decompression sickness, which is also known as caisson disease. in this condition there is a sudden release of nitrogen gas that has gone into solution in the lipids of adipose tissue and of myelinated nervous tissue at the higher pressure: the released nitrogen gains access to the blood stream in which it forms bubbles. doppler ultrasound techniques show that this is quite customary when divers ascend from depth, but the lungs generally provide an effective filter so that there are no untoward systemic effects, although there may be sudden chest pain on deep inspiration ('the chokes'). gradual decompression permits the nitrogen to diffuse across the alveolar membranes and be exhaled. if, however, substantial amounts of nitrogen are released from solution, sufficient pulmonary arteries may be blocked to cause pulmonary hypertension, with resultant opening of arteriovenous communications or a patent foraman ovale, so permitting the gas to enter the systemic circulation. this is often followed by limb pains ('the bends') and perhaps cerebral symptoms ('the staggers'). fatal cases are characterised by gas bubbles within blood vessels throughout the body and froth in the heart chambers. delayed effects include ischaemic necrosis of bones and other tissues. deep-diving mammals are protected by the same mechanism that prevents them suffering from burst lung. they exhale before diving and during the dive the chest is compressed to the extent that virtually all the gas in the lungs passes into the cartilage-buttressed nonrespiratory airways (see fig. . . ) , resulting in very little to be absorbed by the blood. the pulmonary collapse also serves to reduce buoyancy. the distribution of the little gas that is absorbed is minimised by bradycardia. many viscera experience anaerobic respiration but hypoxia is minimised in the heart and musculature by high levels of haemoglobin and myoglobin. the brain is further protected by the supplying arteries drawing on oxygen stored in an unusual spongelike cervical organ known as the rete mirabilis. mountain sickness is due to reduced atmospheric pressure brought about more slowly than that responsible for decompression sickness . , it may be acute or chronic. acute mountain sickness is likely to be experienced by anyone who ascends above - m without a period of acclimatisation at intermediate levels. symptoms are as liable to occur in people born at high altitude who return after a few weeks spent at sea level as in those who go to the mountains for the first time: acclimatisation is obviously short-lived and is therefore necessary whenever an ascent is to be made. the ill-effects are commonly precipitated by exercise. in the susceptible, acute mountain sickness commonly appears within days of ascent. the basis of acute mountain sickness is tissue hypoxia. it results in deteriorating intellectual and psychological function, headache, nausea, vomiting, and more rarely pulmonary and cerebral oedema. high-altitude pulmonary oedema is characterised by increasing dyspnoea, cyanosis and a dry cough, and later the production of copious, frothy sputum, which sometimes becomes blood-stained. the pulmonary artery pressure is markedly raised but wedge pressures are normal, indicating that the left side of the heart is unaffected and that pulmonary venous constriction is unlikely to be an important contributory factor. the pulmonary oedema fluid has a high protein content and the condition has been characterised as a non-cardiogenic high-permeability oedema associated with excessive pulmonary hypertension. , hypoxia is a well-known cause of pulmonary arteriolar constriction but in acute mountain sickness the vascular response appears to be exaggerated for the pulmonary artery pressure is considerably higher than is usual for the altitude. an association with certain hla complexes (hla-dr and hla-dq ) suggests that this has a genetic basis. although arteriolar constriction only tends to protect the pulmonary capillaries, it could explain the oedema if the process was patchy -as is the resultant oedema -for patchy arteriolar constriction would subject the rest of the lung to abnormally high pressures and lead to capillary stress failure in these areas (see pp. and ). , measurements of capillary pressure suggest that this is indeed the case. furthermore, vasodilators such as calcium channel blocking agents and inhaled nitric oxide gas , , have been used with success to counter acute mountain sickness, supporting the idea that hypoxic vasoconstriction plays a central role. autopsy shows the lungs to be heavy and firm. the cut surface weeps oedema fluid, which is often blood-stained, but a striking feature is the patchy distribution of the changes. areas of haemorrhagic oedema alternate with others that contain clear oedema fluid and others that are normal apart from overinflation. pulmonary arterial thrombi are commonly found. microscopy confirms the presence of haemorrhagic oedema and may show neutrophils and hyaline membranes in the alveoli. the alveolar capillaries are congested and may contain thrombi. there may also be an increase in mast cells and rarely pulmonary infarction. the right ventricle is commonly dilated whereas the left ventricle is normal. highlanders generally show right ventricular hypertrophy and increased muscle in their pulmonary arteriesm, changes that are not apparent in lowlanders. , chronic mountain sickness prolonged residence at high altitude leads to hypoxic pulmonary hypertension (see p. ), an increase in red cell mass and cor pulmonale. livestock taken from lowland plains to high-altitude pastures suffer similarly but the natural stock of the himalayas and ethiopian highlands are apparently immune. so too are other species long established at high altitude such as the llama and yak. these species are said to have adapted to their climate, that is, the forces of natural selection have bred out the pulmonary vasoconstrictive response to hypoxia. cattle of european origin and humans acclimatise to high altitude by processes such as increasing their red cell mass but generally they are not adapted like native species and suffer hypoxic pulmonary hypertension at altitudes in excess of m. certain himalayan highlanders may be an exception to this in that their small pulmonary arteries are reported not to show the muscularisation that characterises hypoxic pulmonary hypertension. in cattle of european origin, the dependent oedema of right-sided cardiac failure caused by hypoxic pulmonary hypertension affects the breast (brisket) particularly and in the rocky mountains of north america such cattle are said to have 'brisket disease' . a human counterpart of this has been described in children of chinese ancestry who have been taken to reside in tibet and who have developed a fatal form of subacute infantile mountain sickness. a small minority of permanent residents in the andes develop the changes of chronic mountain sickness to a marked degree and are said to suffer from monge's disease. the basis of this is alveolar hypoventilation, which leads to a progressive fall in systemic arterial oxygen saturation and elevation of haemoglobin concentration to an unusually severe degree. the latter averages about g/dl, which exceeds even the g/dl found in healthy high-altitude residents. patients with monge's disease are so deeply cyanosed that their lips are virtually black. their pulmonary artery resistance is also markedly raised. the cause of the alveolar hypoventilation is uncertain but the only cases of monge's disease that have come to necropsy had conditions such as kyphoscoliosis that predispose to alveolar hypoxia. drowning is defined as suffocation by submersion, and usually occurs in water. it is the commonest cause of accidental death among divers but % of drowning accidents do not involve deep descents. falling into quite shallow water is a particularly common cause of drowning in young children. in adults, men outnumber women by to . more die in fresh water than the sea, not because it is more hazardous to the lungs than sea water, but because unguarded inland waters and swimming pools are visited more frequently. alcohol consumption contributes to many deaths by drowning. drowning is not simply a matter of being unable to keep one's head above water. this may be merely a secondary event. for example, the entry dive may result in underwater head injury, or the exertion of swimming may precipitate a heart attack. furthermore, the struggling swimmer going down for the third time ('drowning not waving') is the exception: most drowning is characterised by the swimmer failing to surface or quietly dropping beneath the surface without anyone noticing. swimming underwater can be extremely hazardous if it is preceded by hyperventilation, a danger that needs to be more widely appreciated. hyperventilation results in undue loss of carbon dioxide so that instead of hypercapnia forcing the swimmer to surface to breathe, progress under water may be continued until hypoxia causes sudden loss of consciousness. panic contributes to many swimming accidents and is often precipitated by the inadvertent aspiration of just a little water. most people are naturally buoyant, but only slightly so. with the lungs fully expanded the average adult has a positive buoyancy of about . kg, which is sufficient to keep the head out of the water if the rest of the body is submerged. if an arm (weight about kg) is raised to wave for help, the head will go down. if the swimmer shouts, exhalation reduces buoyancy to neutral at normal end-expiration and to negative at residual volume. buoyancy cannot be regained when the head is submerged and unless able to swim to the surface, the person will continue to sink. autopsy generally shows that the lungs are full of water, but some victims die of 'dry drowning' due to laryngospasm. events may also be modified by the temperature of the water. sudden immersion in cold water may result in tachycardia, hypertension and hyperventilation, making it difficult for the victim to keep the airways free of water. it may also result in sudden death due to ventricular fibrillation. even a good swimmer loses consciousness within an hour of immersion in very cold water. drowning is then inevitable unless a correctly fitted life jacket is worn, in which case there is a danger of death from hypothermia. however, as in open heart surgery, cold prolongs the interval before there is irreversible brain damage. if the person is rescued, water in the lungs is quickly absorbed, even if it is saline, and therefore hyperosmolar:aspirated sea water is quickly equilibrated by pure water joining it from the blood but the alveolar epithelial barrier remains impermeable to protein and once osmotic equilibrium is reached, all is quickly reabsorbed. [ ] [ ] [ ] fresh water is absorbed even more quickly. it is unnecessary to tip the patient to hasten this process. any water recovered in this way comes from the stomach and time that should be devoted to mouth-to-mouth breathing and cardiac massage is lost. these resuscitative efforts may need to be prolonged as fresh water in particular inactivates alveolar surfactant, leading to alveolar collapse which persists until the surfactant is replenished. very few victims who are resuscitated on site fail to survive, and very few who cannot be resuscitated on site recover later. interchange of fluid between the blood and air spaces may cause major fluctuations in plasma volume with consequent changes in ionic concentrations and haemolysis. hypervolaemia may cause circulatory problems but hyperkalaemia consequent upon the haemolysis is not thought to be as important as was formerly believed: ventricular fibrillation following submersion is more likely to be a complication of hypothermia than of electrolyte imbalance. circulatory collapse may ensue shortly after rescue. this is due to loss of the circulatory support provided by the pressure the water exerts on the body, which results in a considerable increase in cardiac output while the body is immersed. on leaving the water the loss of this support results in a tendency to venous pooling. although this is countered by baroreceptor responses, these are reduced by prolonged immersion in cold water. circulatory collapse is believed to be the cause of death in many persons who perish within minutes of rescue. to counter this effect, patients should be lifted out of the water in the prone position. it can be seen that, in fatal cases, the pathologist is faced with several possibilities. thus, death may have been due to: • natural causes before the body entered the water • unnatural causes before entry, the body merely being disposed of in the water • natural causes in the water • injuries received in the water from impact with rocks, a boat or a ship's propeller, or in tropical waters from predators such as a crocodile or a shark (any of which may also be incurred after death, as may disfigurement by fish and rats) • 'dry drowning' • true drowning • hypothermia • circulatory failure after rescue. true drowning is indicated by froth in the airways and heavy waterfilled lungs. both fresh and salt water contain numerous microscopic algae known as diatoms and those representative of the water in which the drowning occurred are found in the lungs. unless death occurred before submersion, diatoms are also found in other viscera because these tiny life forms easily enter the circulation. thus, the presence of diatoms in digests of organs such as the kidneys, liver, brain and bone marrow suggests that death was due to drowning. because they have a siliceous capsule, diatoms are resistant to putrefaction as well as digestion and can be identified in the body long after death. however, a positive test is not always accepted as proof of drowning and a negative test does not exclude drowning. the various physical forms in which respirable environmental agents may be encountered are defined in table . . . some effects of inhalant lung injury are recognised as distinct disease entities and are dealt with elsewhere: for example, the pneumoconioses on page , extrinsic allergic alveolitis on page , chronic bronchitis on page and lung cancer on page . other respirable agents, such as lead fume and carbon monoxide gas, exert their harmful effects elsewhere in the body and will not be considered further. this section is concerned with toxic substances that may be inhaled by the general public. those that are more likely to be encountered in the workplace or in war zones are considered on page . the lungs have a rather stereotyped pattern of response to inhaled toxins, displaying degenerative changes and inflammation of varying degree, the former sometimes amounting to necrosis. in general, the site of maximal absorption or injury is related to solubility (for gases and vapours) and particle size (for aerosols such as dusts, fog, fumes, mists, smog and smoke): the less water-soluble and the smaller the particle size, the further down the respiratory tract the agent will penetrate ( fig. [ ] [ ] [ ] thus, ammonia produces intense congestion of the upper respiratory passages and laryngeal oedema whereas phosgene has little effect on these sites but causes pulmonary oedema. air pollution [ ] [ ] [ ] [ ] [ ] the toxic (as opposed to allergenic) air pollutants thought to pose the greatest threat to the lungs comprise smoke particles, sulphur dioxide, oxides of nitrogen, various aldehydes and ozone. smoke and sulphur dioxide derive particularly from the combustion of fossil fuels in domestic fires and power stations, nitrogen dioxide is an important car exhaust and domestic gas appliance pollutant and ozone is the principal photochemical product of smog. aldehydes such as formaldehyde and acrylic aldehyde (acrolein) also contribute to general air pollution because they are released in the combustion of diesel oil and petrol. collectively, these pollutants have been incriminated in the exacerbation (rather than causation) of asthma. they also predispose to respiratory infection and result in airway inflammation and hypersecretion. , their effect on children is of particular concern because development of the lungs is known to continue well into childhood and damage to the lungs before their growth is complete is likely to be irreparable. at the other extreme of life episodes of severe air pollution are known to hasten the deaths of many patients with chronic airway disease. particularly high concentrations of the agents responsible for air pollution may be encountered in industry and their effects are therefore also considered in chapter . , on occupational diseases of the lung. many of the polycyclic hydrocarbons found in polluted air are carcinogenic (see p. ) and it is therefore not surprising that urban air pollution has been found to be associated with excess mortality from lung cancer. domestic air pollution is rife in many of the poorer parts of the world due to the burning of biomass (wood, dried cow dung, bagasse, straw) in unventilated living rooms for heating and cooking. the women are particularly at risk of developing chronic bronchitis while their children have an increased incidence of acute respiratory infections. , , a volcanic ash (tephra) irritates the eyes, skin and respiratory tract and in some eruptions may contain much free silica (e.g. montserrat in and mount st helens, washington state, usa in ) or be associated with the release of radon gas (e.g. the azores in ). the destruction of the world trade center in caused massive air pollution of new york city that had lasting respiratory effects on survivors, rescue workers and local residents. [ ] [ ] [ ] at the time of the disaster there was much smoke from combustion of aeroplane fuel and flammable materials in the building while the collapse of the twin towers released dust from cement and dry-wall partitions that was highly alkaline. [ ] [ ] [ ] this caused considerable irritation of the eyes and the conductive airways. a year later many victims were still suffering from bronchial hyperreactivity and poor ventilatory function, in a so-called reactive airways dysfunction syndrome , and there was continuing spirometric decline years later. the respirable portion of the dust formed only a small fraction of the whole but given the level of exposure its future effects cannot be discounted, particularly as it contained substances such as asbestos. unusual effects attributed to the disaster include acute eosinophilic pneumonia and granulomatous pneumonitis. , allergenic air pollutants are dealt with in detail in the sections on asthma (see p. ) and extrinsic allergic alveolitis (see p. ). allergenic air pollution is generally occupational or domestic but periodic widespread air pollution was responsible for the epidemics of asthma seen in barcelona in the s, which were eventually traced to ships discharging cargoes of soya flour (see p. ). smoking-related diseases figure large throughout this book and in this section they are merely summarised collectively. of the greatest importance, both in the number of patients they affect and in their clinical effects on the individual, are the various forms of chronic obstructive lung disease and lung cancer, but there are many other respiratory diseases associated with smoking, and a few that are less common in smokers (box . . ). not surprisingly, these diseases are often encountered in combination and sometimes one may obscure another. for example, a cigarette smoker may have emphysema in the upper lobes and idiopathic pulmonary fibrosis in the lower lobes. , alternatively, langerhans cell histiocytosis and desquamative interstitial pneumonia may affect the same parts of the lungs, in which case the focal lesions of the former may be masked by the latter condition. the term 'smoking-related interstitial lung disease' has been introduced to cover a spectrum of interstitial diseases related to smoking , , as well as being used in a more restricted sense to describe a combination of air space enlargement and interstitial fibrosis predominantly affecting the lower lobes. , , a quite advanced interstitial fibrosis has been reported in smokers with no clinical evidence of interstitial lung disease. b early changes detectable in smokers include chronic bronchiolitis, fibrosis of the bronchiolar wall and mild peribronchiolar interstitial fibrosis. , even earlier changes are detectable at the molecular level: as many as smoking-responsive genes that are significantly up-regulated or down-regulated have been identified in normal cigarette smokers. there is marked individual variation, which may explain why many lifelong heavy smokers experience no respiratory problems. histological evidence that a patient smokes is provided by an increase in the number of alveolar macrophages and a characteristic brown discoloration of cytoplasm due to the phagocytosis of tar and other particulate matter derived from tobacco smoke (fig. . . ) . cigarette smokers are at greater risk of lung disease than cigar and pipe smokers, probably because they inhale more deeply. they do this because cigarette smoke is more acid than cigar and pipe smoke and its nicotine content is therefore absorbed more easily through the lungs than the buccal mucosa. smokers obviously put their own health at greatest risk but the lesser hazards of passive smoking are now well recognized (see p. ). passive smoking involves both the smoke exhaled by others and that coming from smouldering tobacco between puffs, the latter being known as sidestream smoke. the harmful effects of maternal smoking on the unborn child also come in this category. they include increased airway responsiveness and reduced lung function during the neonatal period and an increased risk of sudden infant death syndrome. reduced numbers of alveolar attachments to the bronchioles have been demonstrated in such infants. smoking is also associated with disease of other organs (e.g. carcinoma of the oesophagus and bladder) but these are outwith the remit of this text. tobacco smoking by waterpipe (shisha, hubble-bubble) is enjoying a rise in popularity, both in its heartland, the middle east, and western countries, and wherever it is practised it is widely perceived as being less dangerous than smoking cigarettes. this is probably a mis conception. what evidence there is suggests that waterpipe tobacco smoking is just as harmful as cigarette smoking, if not more so. the lungs may be injured in burned patients in many ways (box . . ) , but an important consideration when a body is recovered from a fire is whether death was due to the fire or took place beforehand, the latter raising the possibility of foul play. a vital reaction to the skin burns and the presence of soot in the lower airways provide evidence that death occurred in the fire but an absence of soot from the airways may be due to death occurring rapidly, from asphyxia or poisoning by gases released in the conflagration. soot is cleared rapidly and if the patient survives a few days an absence of soot from the airways is to be expected. lung injury may result directly from heat and smoke inhalation or indirectly from the release of mediators associated with blast injury or shock. although air temperature in a fire may reach very high temperatures thermal injury seldom extends beyond the carina but more extensive injury from heat alone was seen in men exposed to steam escaping from a fractured boiler pipe. those dying immediately showed coagulative necrosis of the respiratory mucosa down to the level of the alveolar ducts and alveolar congestion and oedema, while those surviving a little longer exhibited diffuse alveolar damage. the diffuse alveolar damage probably represented a manifestation of shock from their extensive cutaneous scalding whereas the mucosal necrosis is directly attributable to heat. diffuse alveolar damage is extrinsic allergic alveolitis sarcoidosis blast injury asphyxia poisoning by combustion products (e.g. carbon monoxide, cyanide) direct thermal injury (largely limited to the trachea) irritant smoke, fume and gas (e.g. oxides of nitrogen, ammonia, acrolein, sulphur dioxide) hypovolaemic shock secondary to skin loss septicaemic shock from: infected skin burns infected central lines secondary viral and bacterial pneumonia fluid overload tracheostomy complications, including tracheobronchitis, pneumonia and barotrauma oxygen toxicity absorption of toxic topical disinfectants thromboembolism uraemia usually part of systemic multiorgan failure in these patients, and is the leading cause of death in burns. the ubiquity of plastics today means that smoke contains numerous irritants, including isocyanates, aldehydes and fluorinated organic chemicals. irritant smoke products have two principal effects. firstly, they cause an immediate painful stimulation of the eyes and respiratory tract which at low concentrations may prevent escape and at high concentrations may cause laryngeal spasm and death. secondly, they cause bronchopulmonary injury some hours after exposure. burned patients dying within - days often show tracheobronchial necrosis and diffuse alveolar damage with prominent hyaline membranes. , , secondary herpesvirus infection is often present. , the respiratory changes caused by heat and smoke are non-specific and careful consideration of the many causes of lung injury in burned patients listed in box . . and of the clinical circumstances and management is generally required. often it will be concluded that the cause of the lung injury is multifactorial. long-term consequences of smoke inhalation include bronchiectasis and obliterative bronchiolitis. methyl isocyanate, the chemical released at bhopal the bhopal catastrophe of was caused by the accidental release of tons of methyl isocyanate gas (ch -n=c=o) from a pesticide plant. over people were exposed, of whom died, mostly within hours of exposure, and were seriously injured. the victims complained of intense ocular and respiratory irritation. some survivors were left with persistent respiratory impairment, which was thought to be due to obliterative bronchiolitis. , methyl isocyanate is an extremely potent respiratory irritant, destroying the epithelium throughout the conducting airways, with comparatively less parenchymal injury. in survivors, epithelial regeneration, often involving squamous metaplasia, quickly commences, but not before endobronchial granulation tissue projections have developed, resulting in obliterative bronchiolitis. tear gases are chemical irritants delivered as an aerosol for the purpose of riot control. they react with mucocutaneous sensory nerve receptors causing intense irritation of the eyes, mucous membranes and skin. the respiratory effects are mainly concentrated on the upper tract so that there is violent sneezing, severe rhinorrhoea and cough but there may also be tracheobronchitis and rarely pulmonary oedema. patients with pre-existent asthma or chronic obstructive pulmonary disease are most severely affected while others may be left with reactive airways dysfunction. toxins reaching the lungs via the blood stream may be drugs, food contaminants, metabolites produced elsewhere in the body, or chemicals ingested intentionally or accidentally, either in the home or the workplace. the lungs are selectively damaged by certain blood-borne toxins for a variety of reasons. for example, the herbicide paraquat is preferentially taken up by the lungs because of its molecular homology with certain endogenous substances. as detailed below, the type i alveolar epithelial cells are the cells that bear the brunt of the damage in paraquat poisoning. on the other hand, the alveolar capillary endothelium has its own selective uptake mechanisms (see metabolic functions of the pulmonary endothelium, p. ) which may be responsible for it being selectively damaged by other chemicals. the bronchiolar clara cells are selectively injured by some ingested chemicals because they are equipped to deal with inhaled xenobiotics, but occasionally this activity results in metabolites that are extremely toxic. an example of this from veterinary medicine is provided by the furan-derivative -ipomeanol, which is found in mouldy sweet potatoes and results in acute pulmonary oedema in cattle fed such a diet. when this chemical is injected into mice, the bronchioles are denuded of clara cells whereas the intervening ciliated cells are completely unaffected. the selective damage to the bronchiolar clara cells appears to stem from the oxidative efficiency of their p- cytochromes, which is much higher than those of the liver. chemicals having a similarly selective effect on bronchiolar clara cells include -methylfuran, carbon tetrachloride, naphthalene and , -dichloroethylene, the last of which is a volatile compound that is widely used in the plastics industry. procarcinogens may be activated in the airways by similar mechanisms. paraquat is a dipyridylium compound that is widely used in agriculture as a herbicide. it kills all green plants but is inactivated on contact with the soil. it is applied as a spray and if the manufacturer's instructions are followed there is no danger to health. most fatal cases of paraquat poisoning, both accidental and suicidal, have been due to ingestion of the % aqueous solution gramoxone. the less concentrated granular form weedol is unlikely to be ingested accidentally but may be taken suicidally. paraquat is not absorbed by the intact skin but repeated or prolonged application damages the epidermis so that absorption into the blood stream with consequent systemic effects is possible, but rare. although paraquat has toxic effects on the liver, kidneys and myocardium, these are transient and attention has centred on the pulmonary changes, which are usually fatal. following suicidal ingestion of large amounts of paraquat, death from multiorgan failure and pulmonary haemorrhage occurs within a few days, whereas most victims of accidental paraquat poisoning die from progressive pulmonary fibrosis between and days after ingestion. in those who survive longer, a honeycomb pattern of pulmonary fibrosis may be apparent. paraquat is a powerful oxidant and owes its toxicity to the production of active oxygen radicals. the lungs are particularly susceptible because paraquat is concentrated there by an active uptake mechanism in the alveolar epithelium. the inadvertent uptake of paraquat probably stems from a similarity between the molecular arrangement of its quaternary nitrogen atoms and the amine groups of endogenous oligoamines such as putrescine, spermidine and spermine, which are concerned in alveolar epithelial cell division and differentiation (fig. . . ) . this results in paraquat levels being - times higher in the lung than in the plasma. once taken up by the lung, paraquat is not metabolised but participates in redox cycling so that superoxide radicals are constantly produced. epithelial injury is proportional to the concentration of paraquat, while it is lessened by hypoxia and antioxidants such as superoxide dismutase, and potentiated by increased concentrations of oxygen. [ ] [ ] [ ] [ ] the high concentration of oxygen in the alveoli is a further reason why the lungs are particularly vulnerable to paraquat. knowledge of the toxic effects of paraquat comes from observations on autopsy series , , and from experimental studies that have enabled the sequence of pulmonary changes to be observed. [ ] [ ] [ ] [ ] in accordance with paraquat being taken up by the alveolar epithelium, electron microscopy shows that these cells suffer more profound damage than the endothelium. type i epithelial cells swell and undergo necrosis (fig. . . ), whilst type ii cells, although remaining capable of proliferation, show ultrastructural evidence of damage with derangement of cell organelles. , histological changes in the lungs follow the pattern of diffuse alveolar damage, with a characteristic feature of the early exudative phase being intense vascular congestion and alveolar haemorrhage. , , hyaline membranes are most clearly seen by about days (fig. . . ) and epithelial proliferation and fibrosis are conspicuous by about days. the pattern of pulmonary fibrosis in paraquat poisoning has been disputed. some authors have stressed its interstitial position, whereas others have clearly demonstrated that it is intra-alveolar. , , [ ] [ ] [ ] [ ] however, as described on page , it generally assumes an obliterative pattern of intra-alveolar fibrosis in which the lumina of several adjacent alveoli are totally effaced, rendering them completely airless (see fig. . , p. ). a new multisystem disease appeared abruptly in the environs of madrid in . [ ] [ ] [ ] over people were affected and about in died. the disease was initially thought to be mycoplasma pneumonia but was soon found to be associated with the use of adulterated oil sold illicitly by door-to-door salesmen. although it was sold for culinary purposes the oil had been produced for industrial use in steel manufacture. it consisted of rapeseed and olive oil mixed with liquified animal fat, aniline and other organic chemicals. it has not been possible to identify the exact chemical responsible for the disease or to reproduce the changes in other species but the later induction of similar pathological changes by another substance contaminated with an aniline derivative is possibly relevant (see l-tryptophan-induced eosinophilia-myalgia syndrome, p. ). some clinical and pathological features of the disease suggest that immune mechanisms may also be involved. the initial clinical features included fever, respiratory distress, cough, haemoptysis, skin eruptions and marked eosinophilia. radiographs suggested pulmonary oedema and sometimes showed pleural effusion. about % of patients died at this stage but most recovered quickly. however, within a few weeks many were readmitted to hospital with nausea, vomiting, diarrhoea and abdominal pain. about a quarter then proceeded to develop weakness, myalgia, weight loss, scleroderma-like skin signs and pulmonary hypertension. , many of these patients died after a long, wasting illness or are permanently disabled with neurological and hepatic disorders. in the early phase the lungs showed the most severe changes, which consisted of a combination of diffuse alveolar damage, eosinophilic infiltrates and arterial luminal narrowing by endothelial swelling and vacuolation, intimal foam cell infiltration and a non-necrotising vasculitis. , , there was also capillary thrombosis, which later extended into arteries and veins, culminating in fibrosing obliteration of these blood vessels. in some patients dying of haemoptysis, dilated thin-walled blood vessels were identified in the mucosa of major blood-filled airways. late features in the lungs included plexogenic arteriopathy (see p. ), possibly secondary to changes in the liver. similar inflammatory and vascular changes were seen in many other tissues. notable extrapulmonary features included fasciitis, vasculitis, neuronal degeneration, perineuritis, hepatic injury and tissue eosinophilia. sauropus androgynus is a vegetable that is widely cultivated for the table in many south-eastern asian countries. it is apparently harmless when cooked but recently there has been a vogue in taiwan for consuming large amounts of its unprocessed juice, blended with that of guavas or pineapple, because of its supposed efficacy as a slimming aid and in blood pressure control. coincident with this fad there has been an upsurge in patients with symptoms of obstructive lung disease. within a -month period more than such patients were seen at one hospital. [ ] [ ] [ ] they had four features in common: recent consumption of uncooked s. androgynus juice, fixed ventilatory obstruction, radiological evidence of bilateral bronchiectasis and an absence of any previous chronic respiratory disease. four patients agreed to undergo open-lung biopsy. this showed chronic bronchiolitis or obliterative bronchiolitis of constrictive pattern. the lymphocytes were mainly t cells but immunofluorescent and electron microscopy showed no evidence of an immune process. four patients underwent single-lung transplantation. the excised lungs showed sclerotic obliteration of bronchial arteries in the walls of bronchi - mm in diameter with segmental necrosis of bronchi - mm in diameter. the changes were considered to fit best with segmental ischaemic necrosis of bronchi at the watershed zone of the bronchial and pulmonary vasculature. further patients have required lung transplantation but public education of the dangers of this herbal medicine now appears to have been successful. alcohol and nicotine outstrip all other recreational drugs in popularity and their effects are of course well known. those of tobacco smoking are summarised above and dealt with in detail in the chapters on obstructive lung disease (chapter ) and carcinoma of the lung (chapter . ). less well known is the lung disease that results from smoking blackfat tobacco, a practice popular with guyanese indians. blackfat is the trade name of a type of tobacco that is flavoured with mineral oil, some of which vaporises and is inhaled when the tobacco is smoked, to cause exogenous lipid pneumonia (see p. [ch ] ). in recent years the smoking of two other substances, marijuana and cocaine, has gained in popularity. it would not be surprising if the long-term effects of smoking these substances were similar to those of cigarette smoking but as yet it is too early to judge. however, the short-term effects are similar to those of tobacco smoking and this bodes badly for their ultimate effects. marijuana consists of the dried leaves of the cannabis plant, also known as hemp, as opposed to hashish, which is the plant's resin, and a further extract known as 'weed oil' . all these substances are smoked because they contain cannabis alkaloids which have psychoactive effects. however, this habit also exposes the lungs to many of the same respiratory irritants that are found in tobacco smoke. initial exposure to marijuana smoke often results in coughing while habitual smokers produce black sputum. bronchial biopsy shows inflammation and squamous metaplasia and bronchoalveolar lavage demonstrates increased numbers of cells, which are predominantly macrophages but also include neutrophils. [ ] [ ] [ ] [ ] [ ] these changes are virtually identical to the short-term effects of tobacco smoke and are therefore likely to be similarly followed by the development of chronic obstructive lung disease and lung cancer. indeed, the dangers of smoking marijuana are probably greater than those of smoking tobacco as compared with tobacco smoking it is associated with a fivefold greater increase in blood carboxyhaemoglobin and a threefold increase in the amount of tar inhaled. it is estimated that three cannabis cigarettes result in the same degree of bronchial damage as tobacco cigarettes. there is also evidence that the effects of smoking marijuana and tobacco are additive. not surprisingly therefore, epidemiological studies report a doserelated impairment of large-airway function in marijuana smokers. there are also several reports attributing pneumothorax to marijuana smoking ( fig. . . ) . , the pneumothorax may be spontaneous or develop during the deep, sustained inspiratory effort involved in smoking marijuana (or cocaine), which may be enhanced by a partner applying positive ventilatory pressure by mouth-to-mouth contact. thoracoscopy in such cases has shown predominantly apical, irregular bullous emphysema, while lung biopsy has demonstrated widespread alveolar filling by heavily pigmented macrophages. , evidence is also beginning to accumulate that long-term cannabis use increases the risk of lung cancer. smoking cannabis in the form of weed oil is also reported to result in exogenous lipid pneumonia. cocaine cocaine hydrochloride is a fine white powder derived from the leaves of the plant erythroxolon coca by a complex chemical process. it is heat-labile and therefore cannot be smoked. users inject it intravenously or inhale it unheated through the nose, the latter practice being known as 'snorting' . however, a heat-stable free-base form that can be smoked is easily prepared from the hydrochloride with baking powder and a solvent such as ether. this process results in a crystalline deposit that is known as 'rock' because of its appearance or 'crack' because of the crackling sound it emits when heated. when smoked, the cocaine is readily absorbed and an intense surge of euphoria is experienced within seconds. the intravenous route takes twice as long and 'snorting' several minutes. the hard addict therefore prefers to smoke 'crack' . a variety of pulmonary complications of smoking free-base cocaine has been reported. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] acute effects include cough, shortness of breath, chest pain and haemoptysis. asthma may be aggravated, black sputum is produced, and pneumothorax and interstitial emphysema have resulted from valsalva manoeuvres undertaken in the belief that they promote even more rapid absorption. biopsy has shown pulmonary congestion and oedema, organising pneumonia, haemorrhage, haemosiderosis, diffuse alveolar damage and interstitial pneumonia or fibrosis. less common effects include eosinophilic pneumonia, extrapulmonary eosinophilic angiitis, medial thickening of pulmonary arteries and the barotrauma described above (see fig. . . ) . severe burning of the airways has also been seen due to 'crack' being smoked before all the ether used in its preparation has evaporated. 'snorting' unheated cocaine has its own complications: substances such as cellulose or talc with which the drug is 'cut' (mixed as a diluent) are liable to provoke a foreign-body giant cell reaction in the lungs (fig. . . ). however, particles of foreign material larger than those in the usual respirable range (allowing for the fibrous shape of substances such as cellulose) should suggest intravenous use (see 'filler embolism', below). heroin is usually injected, but it may be smoked, when, as with marijuana, it is liable to lead to a very pronounced macrophage response. intravenous heroin abuse sometimes causes the sudden onset of a potentially fatal high-permeability pulmonary oedema (fig. . . ). intravenous abuse of heroin and other drugs is also liable to cause 'filler embolism', which will now be considered. 'filler embolism' is the result of illicit drug usage in which compounds designed for oral use are injected intravenously to heighten their effects. oral preparations consist largely of fillers such as talc or starch and this insoluble particulate matter accumulates in the pulmonary capillaries. it provokes a foreign-body giant cell reaction, thrombosis and fibrosis and may cause pulmonary hypertension ( fig. this 'designer' drug, taken for its central stimulant activity (street names 'ice' or 'u- -e-uh', pronounced euphoria), is related to the appetite suppressor aminorex, discussed on page , and has similarly been associated with pulmonary hypertension. assessing whether a particular clinical manifestation represents an adverse drug reaction considers previous experience with the drug, alternative aetiological agents, the timing of events, drug levels, and the effect of withdrawing the drug and rechallenge with the drug. it is worth bearing in mind that: • one drug may cause several patterns of disease. • one pattern of disease may be produced by a variety of drugs. • a drug reaction may develop long after the drug has been withdrawn. • a drug reaction may develop suddenly even though the dose of the drug has not been altered. • drug effects may be augmented by factors such as age, previous radiotherapy and elevated oxygen levels. • drug reactions may be localised. • many drugs cross the placenta to affect the fetus. an alternative classification of adverse drug reactions, which is more appropriate to pathology practice and which will be followed here, is one based on the pattern of disease. some pathological patterns of drug-induced lung disease are shown in table central depression of respiration occurs as a side-effect of barbiturates, morphine and its derivatives, and even mild sedatives, and may be particularly troublesome in patients suffering from chronic obstructive lung disease. ventilation in such patients may be largely dependent on hypoxic respiratory drive and treatment with oxygen may therefore also have an adverse effect on respiration by lowering the degree of hypoxia and so diminishing the stimulation of the respiratory centre. peripheral impairment of the respiratory drive may be brought about by aminosides and other antibiotics, while corticosteroids may result in a myopathy affecting the respiratory muscles. other iatrogenic hazards affecting the peripheral nerves controlling respiration include nerve root disease complicating immunisation and surgical damage to the spinal and phrenic nerves. asthmatic patients are particularly susceptible to exacerbations of their disease by drugs (box . . ). this effect may occur either as a predictable pharmacological side-effect of the drug or as an idiosyncratic response. examples of the former include β-adrenergicic antagonists and cholinergic agents while examples of the latter include sensitivity to the colouring agent tartrazine, for which reason many manufacturers have eliminated tartrazine from their red, orange and yellow tablets. allergic bronchoconstriction also forms part of generalised anaphylactic reactions induced by vaccines and antisera and occurs as a localised response to penicillin, iodine-containing contrast media, iron dextran and other medicaments. bronchospasm may also be initiated by the non-specific irritant effect of inhaling nebulised drugs if they are prepared as a hypotonic solution, a side-effect that is prevented by using isotonic solutions. aspirin and other non-steroidal anti-inflammatory agents aspirin-induced asthma has been recognised for many years and more recently several of the newer anti-inflammatory drugs have been found to exacerbate asthma in certain sensitive individuals. the basis for this is uncertain but the likelihood of an individual antiinflammatory drug provoking an asthmatic response is related to its potency as an inhibitor of prostaglandin cyclooxygenase pathway, resulting in the production of leukotrienes. [ ] [ ] [ ] as well as asthma being exacerbated by drugs, the disease has been caused by occupational exposure in the pharmaceutical industry to certain drugs which can be inhaled during manufacture, notably penicillin, cephalosporin, methyldopa, cimetidine and piperazine. obliterative bronchiolitis of the constrictive type has been reported with penicillamine , and gold , but in many cases it is possibly the underlying condition rather than the drug that is res ponsible (see p. ). this is often rheumatoid disease, which is sometimes complicated by bronchiolitis obliterans whether the patient is under treatment or not. organising pneumonia extending into peripheral bronchioles (see p. ) may be seen with a variety of drugs but results in a restrictive rather than obstructive lung defect and is to be regarded as a cytotoxic effect of the drug acting primarily at the alveolar level (see below). raw sancropus androgyns taken as a slimming aid causes severe obliterative bronchiolitis (see p. ). the cytotoxic effects of drugs may be acute or chronic, leading to changes as varied as pulmonary oedema, diffuse alveolar damage, pulmonary haemorrhage and haemosiderosis, organising pneumonia, interstitial pneumonitis and interstitial fibrosis. , some of the most severe acute effects are seen with the chemotherapeutic agents used in malignant disease but they are also recorded with drugs that are not traditionally thought to be cytotoxic, e.g. desferrioxamine administered as a prolonged intravenous infusion in acute iron poisoning. pulmonary toxicity due to busulphan was first described in , and has been the subject of several subsequent studies. [ ] [ ] [ ] [ ] it remains the mainstay of treatment for chronic myeloid leukaemia. like other alkylating agents, it acts by cross-linking dna strands. clinical estimates of the incidence of pulmonary toxicity vary around % but subclinical damage is thought to be much more common. although not strictly dose-dependent, toxicity is rarely seen with a total cumulative dose of less than mg. synergy with radiation and other cytotoxic drugs occurs. similar effects have been reported for most cytotoxic agents, particularly bleomycin. pulmonary toxicity is seen less commonly with other alkylating agents, such as cyclophosphamide and melphalan. [ ] [ ] [ ] [ ] bleomycin is a cytotoxic antibiotic derived from streptomyces species. it is widely used in the treatment of neoplasms such as lymphomas and germ cell tumours, and is thought to produce its therapeutic and toxic effects by altering the normal balance between oxidants (active oxygen radicals) and antioxidant systems. bleomycin produces superoxide radicals when incubated with oxygen and iron in vitro. oxygen enhances its effects, a fact well known to anaesthetists who accordingly take care to limit concentrations of inspired oxygen to % in patients on bleomycin who are undergoing surgery. [ ] [ ] [ ] radiotherapy and cytotoxic agents such as bleomycin are also synergistic. bleomycin is preferentially concentrated in the lungs and pulmonary fibrosis can be produced in animals when it is administered intravenously, intraperitoneally or by intratracheal instillation. electron microscopy shows that the early changes consist of swelling and vesiculation of endothelial cells, interstitial oedema and type i epithelial cell necrosis. , the reported incidence of bleomycin toxicity varies from to % depending on the type of patient being treated and on dosage. in general, toxic effects increase with age and cumulative dose: above a total dose of about units they rise significantly. the acute morphological changes attributable to drugs include pulmonary oedema and diffuse alveolar damage. acute pulmonary oedema is seen in heroin addicts who die while injecting themselves intravenously but it is also seen in patients administered a variety of drugs therapeutically, for example hydrochlorothiazide, salicylate, opiates, vinorelbine,and desferrioxamine. the oedema is of the high permeability type (see p. ), rich in protein, and is occasionally haemorrhagic or accompanied by the hyaline membranes of diffuse alveolar damage. diffuse alveolar damage has alveolar epithelial necrosis as its basis (figs . . and . . ). however, the continuing action of many cytotoxic drugs affects the regeneration process so that atypical type ii epithelial cells develop, a characteristic feature that was first described with busulphan and subsequently with bleomycin. , these two drugs differ chemically but both act (by different mechanisms) on dna. the atypical cells have abundant deeply eosinophilic or amphophilic cytoplasm and large nuclei, which may be multiple but are usually single. the nuclei measure up to µm and are densely stained throughout or contain either large homogeneous deeply eosinophilic inclusions or clear vacuoles (fig. . . ) . electron microscopy distinguishes the inclusions from nucleoli and shows them to consist of tubular aggregates derived from the internal nuclear membrane. airway epithelium shows similar nuclear changes and often undergoes squamous metaplasia. the presence of such cells in sputum specimens submitted for cytology can lead to a misdiagnosis of malignancy. fibrosis may follow diffuse alveolar damage or develop insidiously, perhaps many years after drug therapy ceased (fig. . . ) . it may be both interstitial and intra-alveolar. the interstitial component is often accompanied by a non-specific chronic inflammatory infiltrate. the proportions of inflammation, which is potentially reversible, and fibrosis, which when collagenous is irreversible, obviously bear on the prognosis. however, most case reports antedate the recent classification of interstitial pneumonia described in chapter and it is uncertain how their pathological appearances would now be classified. the majority lack the classic features of usual interstitial pneumonia and fibrotic non-specific interstitial pneumonia. many show overlapping patterns of intersitital pneumonia and this alone should arouse suspicion that a drug may have been responsible. however, some cytotoxic drugs result in pulmonary changes by more than one mechanism: for example, methotrexate may produce hypersensitivity reactions with granuloma formation [ ] [ ] [ ] [ ] or pulmonary eosinophilia as well as diffuse alveolar damage. pulmonary toxicity is also occasionally seen in patients undergoing treatment with gold salts for rheumatoid disease: in addition to diffuse alveolar damage, there may be eosinophilia and dermatitis in these cases, again indicating possible hypersensitivity. nitrofurantoin is another example of a drug resulting in a variety of patterns of alveolar injury: diffuse alveolar damage, desquamative interstitial pneumonia, giant cell interstitial pneumonia, organising pneumonia and eosinophilic pneumonia have all been recorded in association with this drug. [ ] [ ] [ ] it should also be noted that in patients with neoplastic disease, clinical features suggestive of a pulmonary drug reaction may be due to factors other than drugs. in leukaemic patients, for example, these include direct infiltration of the lungs by leukaemic cells, opportunist infection and, if bone marrow transplantation has been undertaken, the effects of irradiation and possibly graft-versus-host disease. phospholipidosis is encountered with drugs such as the antidysrhythmic agent amiodarone, which block lysosomal enzymes involved in the breakdown of complex lipids. this leads to their accumulation throughout the body but the effect is most marked in tissues that take up the drug and contain cells rich in lysosomes. the lung fulfils both these requirements through its rich complement of alveolar macrophages. these cells accumulate the enzyme substrate (phospholipid) in their cytoplasm with the result that large foam cells fill the alveoli (fig. . . ). the appearances are those of endogenous lipid pneumonia, similar to that seen in obstructive pneumonitis. however, with amiodarone cytoplasmic vacuolation is also seen in epithelial and interstitial cells. the phospholipid inclusions contained within the vacuoles are particularly well seen in unstained frozen sections viewed by polarised light. identical changes to those induced by amiodarone were seen in the lungs of rats exposed to very high levels of the antidepressant drug iprindole and the anorectic drug chlorphentermine. these three compounds, iprindole, chlorphentermine and amiodarone, all belong to the amphiphilic group of drugs which block lysosomal phospholipase and sphingomyelinase. although their pharmacological actions are very different, a molecular homology is apparent (fig. . . ) . it is likely that all patients receiving substantial amounts of amiodarone develop phospholipidosis throughout the body, but this is generally well tolerated. only a minority experience respiratory impairment and in these there is also evidence of pulmonary inflammation and fibrosis, which is possibly mediated immunologically. these patients generally have a restrictive lung deficit, the onset of which may be acute or chronic. bronchoalveolar lavage shows foamy macrophages but these cells indicate exposure to the drug rather than drug toxicity; nor are they specific to amiodarone, being observed on occasion with other drugs. lymphocytes of suppressor type may also be detected on lavage. histologically, amiodarone toxicity is diagnosed on a combination of phospholipidosis and interstitial pneumonia and fibrosis. occasionally the hyaline membranes of diffuse alveolar damage are superimposed on the interstitial changes (see fig. . . ) . [ ] [ ] [ ] in some patients the fibrosis is intraalveolar rather than interstitial and the appearances are those of organising pneumonia. the process may be localised and mimic a neoplasm radiologically. , amiodarone toxicity is probably dose-dependent but there is considerable individual variation in the amount required, , which appears to be under genetic control. amiodarone toxicity is uncommon in patients taking daily doses of mg or less whereas the there are drugs that undoubtedly cause a usual interstitial pneumonia pattern, for example the chemotherapeutic agents and nitrofurantoin (fig. . . ), while others, for example the statins, are recorded as having induced a non-specific interstitial pneumonia pattern. a drug history is therefore imperative when assessing any patient with diffuse parenchymal lung disease. organising pneumonia similar to the cryptogenic condition described on page , and probably similarly reversible with steroids, has been encountered with a variety of drugs, including amiodarone, sulphasalazine and pencillamine. penicillamine has also been incriminated in the development of both diffuse alveolitis and bronchiolitis obliterans, but both these changes could well be due to the underlying rheumatoid disease for which the pencillamine is administered. in busulphan lung there may be an organising intraalveolar fibrinous exudate, which at its most extreme results in irreversible effacement of the alveolar architecture by sheets of loose connective tissue (see p. ). with continued experimental administration of the drug iprindole mentioned above, the phospholipidosis it produced gradually evolved into alveolar proteinosis (more properly called lipoproteinosis; see p. ), but this has not been reported as a drug effect in humans. alveolar proteinosis has however been recognised in a number of patients receiving chemotherapy for conditions such as leukaemia. the mechanism here is probably based on the cytotoxic action of the drug and the material filling the alveoli may represent the detritus of degenerate alveolar cells rather than excess pulmonary surfactant, as in the primary auto-immune form of alveolar proteinosis. eosinophilic pneumonia, the pathology of which is described on page , may be caused by several drugs, including nitrofurantoin, para-aminosalicylic acid, sulphasalazine, phenylbutazone, gold compounds, aspirin and penicillin (see box . , p. ). , it may also follow radiation to the chest. the tissue eosinophilia is generally accompanied by a rise in the number of eosinophils in the blood. the clinical picture varies from transient asymptomatic opacities on a chest radiograph to a life-threatening illness with severe respiratory distress and hypoxaemia, so-called acute eosinophilic pneumonia (see p. ). the reaction is often associated with a florid rash. withdrawal of the drug may be all that is required to effect resolution but corticosteroids are usually given as they produce a marked improvement. this syndrome of necrotising granulomatosis, vasculitis and eosinophilia in asthmatic patients, which is described more fully on page , has been reported when leukotriene receptor antagonists have been used to treat asthma. however, it is likely that the syndrome has been merely unmasked by the antileukotriene permitting a reduction in corticosteroid dose rather than representing a direct effect of the antileukotriene. , mesalazine has also been implicated in inducing a vasculitis during treatment for inflammatory bowel disease. the eosinophilia-myalgia syndrome was identified in the usa in and quickly identified as being due to the ingestion of ltryptophan from one particular japanese supplier. withdrawal of this substance led to the virtual elimination of the disease, but not before patients had been affected, in fatally. [ ] [ ] [ ] [ ] [ ] cases were subsequently described in europe where there were further fatalities. l-tryptophan is an essential amino acid that is freely available to the public: its purchase does not require a medical prescription. it has been promoted as a dietary supplement and as an agent against insomnia and premenstrual tension. women in the reproductive years preponderated in the patients affected by the resultant eosinophiliamyalgia. the clinicopathological features of the syndrome are similar to those of the spanish toxic oil syndrome (see p. ) and differ more in degree than type. the discovery of an aniline-derived contaminant in the tryptophan-induced condition is a further link connecting these two syndromes. an immune basis is suggested by the identification of t lymphocytes activated against fibroblasts in the eosinophilia-myalgia syndrome. the illness is a multisystem disorder and besides blood eosinophilia and myalgia there may be arthralgia, fever, rash and involvement of the lungs, liver and central nervous system. as in the toxic oil syndrome, there is fasciitis, wasting and muscle pain associated with blood and tissue eosinophilia. the lungs are affected in % of cases. pulmonary symptoms have included cough, dyspnoea and chest pain. radiographs have shown diffuse bilateral infiltrates and pulmonary hypertension has been documented in a few cases. histology of the lungs shows an oedematous myxoid intimal thickening affecting small pulmonary blood vessels and a diffuse interstitial lymphocytic and eosinophilic infiltrate. , , , , these cells may also be seen within the walls of the thickened blood vessels (fig. . . ) . , massive ingestion of l-tryptophan has resulted in the appearances of an organising pneumonia. as an adverse drug reaction, granulomatous alveolitis is best exemplified by the extrinsic allergic alveolitis of pituitary snuff-takers, but it is also encountered on rare occasions with cytotoxic and other drugs, including methotrexate, bacille calmette-guérin (bcg) immunisation, interferons, ciprofloxacin, antiviral therapy and tumour necrosis factor antagonists. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the histological appearances may suggest extrinsic allergic alveolitis or sarcoidosis but the centri-acinar or lymphangitic concentration of these conditions is usually lacking. however, unless an infective agent can be demonstrated the diagnosis generally requires consideration of the clinical and environmental details, including any drug regimen. exogenous lipid pneumonia may result from the unintentional aspiration of various fat-based medicaments such as liquid paraffin, oily nose drops and petroleum jelly or of fat-rich dietary supplements in the form of ghee. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the consumption of liquid paraffin as an aperient is common in some countries and may be taking place without the knowledge of the patient's medical practitioner. regurgitation and aspiration of ingested oil are especially likely to happen during sleep in the presence of a hiatus hernia or when the oesophagus fails to empty completely into the stomach because of achalasia of the cardia. the aspiration of vegetable oil occurred in the past from the use of menthol in olive oil for the treatment of tuberculous laryngitis, and occasionally from the use of iodinated vegetable oils for bronchography. [ ] [ ] [ ] [ ] more recently exogenous lipid pneumonia has developed from the constant sucking of lollipops formulated for the administration of the analgesic fentanyl but also containing a stearate component. the treatment of epistaxis by nasal packing with paraffin gauze has also led to exogenous lipid pneumonia. the pathology of exogenous lipid pneumonia is described on page . other medicines may also be aspirated unwittingly, for example a ferrous sulphate tablet may cause brown iron staining and necrosis of the bronchus at the point of impact, progressing to bronchial stenosis. [ ] [ ] [ ] distal infection is then likely, as with any foreign body. barium sulphate aspiration may complicate gastrointestinal radiography. large amounts may impair ventilation but being inert there is no permanent injury to the lungs, although the striking changes are evident on the chest radiograph. an outbreak of pulmonary hypertension affecting many swiss, austrian and german patients in the period - was probably due to the anorectic drug aminorex, which was accordingly withdrawn with regression in the number of new cases. the pathology in these patients was identical to that of primary pulmonary hypertension (see p. ) and it proved impossible to reproduce the condition in laboratory animals but the epidemiological evidence that aminorex was to blame is very strong. fenfluramine and phentermine, further anorectic drugs that are chemically similar to aminorex, have also been associated with such plexogenic pulmonary hypertension, [ ] [ ] [ ] [ ] [ ] and with fibroproliferative plaque on the tricuspid valve and pulmonary arteries. pulmonary hypertension due to pulmonary veno-occlusive disease has sometimes complicated the use of cytotoxic chemotherapeutic agents or followed bone marrow transplantation. non-steroidal anti-inflammatory agents such as indomethacin and diclofenac cross the placenta and, if given in late pregnancy, may cause premature closure of the ductus arteriosus, resulting in severe neonatal pulmonary hypertension. , pulmonary hypertension is a well-recognised association of human immunodeficiency virus (hiv) infection but until recently has been unexplained. now, however, evidence is emerging that the highly active antiretroviral therapy administered to hiv-positive patients might be responsible for the pulmonary hypertension. the older high-oestrogen contraceptive drugs carried a slight risk of thromboembolism but this is not seen with the newer preparations. pulmonary thromboembolism has also occurred with a drug-induced lupus syndrome associated with anticardiolipin antibodies. chemotherapeutic drugs such as mitomycin may cause widespread smallvessel thrombosis resulting in the haemolytic-uraemic (thrombotic microangiopathic) syndrome. there is prominent involvement of pulmonary vessels and patients often suffer from respiratory as well as renal insufficiency, and pulmonary hypertension. the syndrome can develop during treatment or up to several months after the drug has been withdrawn. pulmonary thromboembolism is also recorded as a complication of immunoglobulin infusion. non-traumatic fat embolism has resulted from the agglutination or 'creaming' of fat emulsions administered intravenously as a source of calories to debilitated patients. [ ] [ ] [ ] [ ] [ ] [ ] the agglutinated liposomes occlude fine blood vessels throughout the body, causing effects such as priapism, osteonecrosis and pancreatitis. they may be demonstrated in the pulmonary capillaries but the lungs have considerable vascular reserve and it is uncertain what effect the vascular occlusion has on pulmonary function. agglutination of these fat emulsions is particularly common in severely ill patients and this has been attributed to the elevated blood levels of acute-phase proteins, especially c-reactive protein, that are found in the very ill. the agglutination is also induced by calcium and may be brought about by administering calcium and other mineral supplements through the same venous line as the fat. once agglutinated, the fat is less soluble and may be demonstrated in paraffin sections. sudan black is especially useful for this purpose (fig. . . ). microvascular crystal embolism is a further risk of parenteral nutrition, the crystals representing various calcium salts that may precipitate in the circulation. transient diffusion abnormalities attributed to oil embolism are very common in patients undergoing lymphangiography but serious respiratory impairment is limited to those patients with pre-existing lung disease or in whom substantial amounts of contrast medium are injected rapidly. [ ] [ ] [ ] [ ] other emboli of an iatrogenic nature described in pulmonary arteries include the broken-off ends of intravenous catheters and cannulas, particles from dialysis tubing, prosthetic implants of substances such as teflon and silicone , [ ] [ ] [ ] [ ] and various materials injected to occlude abnormal blood vessels. , diffuse pulmonary haemorrhage diffuse pulmonary haemorrhage may result from interference with the clotting mechanism by anticoagulants or from widespread pulmonary capillaritis, the latter reported in leukaemic patients treated with retinoic acid. pulmonary haemorrhage has also been reported as an idiosyncratic reaction to lymphangiography media and as a complication of immunoglobulin infusion, while the development of anti-basement membrane antibodies resulting in goodpasture's syndrome has been attributed to penicillamine. a infection is a common pulmonary hazard in any patient receiving corticosteroids, chemotherapy or any other immunosuppressant drug. viral, bacterial, fungal and protozoal infections, often in combination, may all develop in the lungs of such patients and tissue reactions may be atypical. pneumocystis jiroveci, for example, may elicit a granulomatous reaction or cause diffuse alveolar damage rather than the usual foamy alveolar exudate (see p. ). metastatic calcification, described on page , may result from any drug causing hypercalcaemia, e.g. high doses of vitamin d, calcium and inorganic phosphate or excessive alkali intake in the treatment of peptic ulceration. carcinoma of the lung may be promoted by drugs. arsenicals cause squamous metaplasia of the bronchi and occasionally squamous carcinoma, while peripheral scar cancers, usually adenocarcinomas, have developed in lungs showing fibrosis due to drugs such as busulphan. drugs may result in a variety of pleural diseases. common examples include effusions, chronic inflammation and fibrosis. these are usually encountered in isolation but may be associated with chronic interstitial pneumonia or fibrosis. sometimes there is also serological evidence of systemic lupus erythematosus: many drugs, including hydantoin, practolol, procainamide, hydralazine and sulphonamides, are associated with the development of a syndrome resembling systemic lupus erythematosus that includes pleural disease. whether the drugs are directly responsible for the syndrome or merely promote the development of latent natural disease is uncertain. ergotamine derivatives such as methysergide and bromocriptine are notable for the production of pleural fibrosis, which is sometimes associated with mediastinal and retroperitoneal fibrosis large amounts or prolonged treatment are generally required to produce this effect. [ ] [ ] [ ] in patients given practolol, pleural thickening has become evident several years after the drug was discontinued. this shows the need for a careful drug history in any patient with unexplained pleural fibrosis. reports of radiation-induced lung damage began to appear soon after ionising radiation became widely used in the treatment of malignant disease. [ ] [ ] [ ] despite refinements in radiotherapy techniques it is often impossible to avoid irradiating small areas of lung when treating cancer of the lung, breast, spine, thymus and oesophagus. parts of the lungs are also included in 'mantle' irradiation of mediastinal lymph nodes affected by lymphoma. occasionally, the whole of both lungs is irradiated, as in the treatment of widespread pulmonary metastases or as part of whole-body irradiation prior to marrow transplantation for the treatment of leukaemia. radiation pneumonitis, usually localised, is estimated to affect about % of patients. therapeutic irradiation is given as divided doses over several weeks in order to minimise damage to adjacent tissue. the effects of such fractionated treatment are cumulative. in the lungs an early exudative phase soon passes and progressive damage becomes apparent only after months or even years. , the changes are generally confined to the area of lung that is irradiated but are widespread when the whole body is irradiated prior to bone marrow transplantation or there is accidental whole-body irradiation. however, localised irradiation of the lung has been followed by abnormalities in non-irradiated areas. these include bilateral alveolar exudates, migratory organising pneumonia affecting both lungs , and fulminant bilateral interstitial pneumonia. the likelihood of lung injury is increased by the simultaneous use of cytotoxic drugs and oxygen therapy. furthermore, chemotherapy following irradiation may result in exacerbation of the injury in areas previously irradiated, a phenomenon termed 'recall pneumonitis' . , in the long term, irradiation also results in an increased incidence of lung carcinoma. this was seen in patients given therapeutic irradiation to the spine for ankylosing spondylitis and is still encountered on occasion following irra diation for breast cancer. the pathogenesis of radiation injury is described on page . radiation damage to the lung is traditionally separated into fulminant acute injury coming on within days, subacute pneumonitis developing within several weeks (typically - months) and interstitial fibrosis slowly evolving from the subacute stage or making itself apparent years later. the migratory organising pneumonia referred to above is an unusual further effect, as is chronic eosinophilic pneumonia. in the pleura, radiation causes fibrinous effusions and adhesions. pleural effusion and pulmonary oedema may be augmented by the long-term effects of radiation on the heart. fulminant acute injury is an unusual and unexpected effect of therapeutic radiation but one that is likely to come to the attention of the pathologist as an autopsy is often requested. the clinical features are those of acute lung injury and the pathological changes are those of diffuse alveolar damage. the cause is likely to be accidental overdosage, augmentation of the radiation damage by accompanying oxygen therapy or treatment with cytotoxic drugs. occasionally however these factors can be excluded, in which case the damage has to be ascribed to 'hypersensitivity' . subacute radiation pneumonitis is encountered more commonly. after an interval of about - months the patient complains of shortness of breath and a non-productive cough. the chest radiograph shows hazy opacification proceeding to more dense consolidation. lung biopsy shows alveolar and interstitial oedema, possibly with residual hyaline membranes, proliferation of atypical alveolar epithelial cells and interstitial fibroblasts and organising thrombosis. later, as the process advances, there is widespread fibrosis comparable to that illustrated in figure . on page and ultimately dense scarring (fig. . . ) . tracheal and aortic injury may complicate radiation treatment of tracheal lesions, sometimes resulting in an aortotracheal fistula. patients requiring mechanical ventilation are liable to suffer lung injury in a number of ways. in addition to effects of barotrauma such as pneumothorax and surgical emphysema, they often develop diffuse alveolar damage. the high oxygen tension that is often combined with mechanical ventilation is a major factor - but mechanical forces other than the high pressures responsible for barotrauma can also contribute to this form of lung injury, notably by resulting in excessive end-expiratory stretch and repeated collapse/recruitment of the alveolar walls. , low tidal volume ventilation is therefore a fundamental part of the management of diffuse alveolar damage. although oxygen is necessary to life, it is cytotoxic in high concentrations. severe hyperoxia damages dna, inhibits cellular proliferation and ultimately kills cells. its toxicity is thought to be due to the intracellular production of active oxygen radicals, some of which derive from activated neutrophils attracted to the site of injury. [ ] [ ] [ ] [ ] under normal conditions most of the oxygen is reduced to water by cytochrome oxidase, and any active radicals produced are eliminated by superoxide dismutase, catalase and other antioxidants. however, these defence mechanisms may prove inadequate when active radicals are produced in excess. problems are likely to arise in clinical practice when lung disease necessitates the concentration of oxygen in the inspired air being raised in order to maintain normal blood levels of oxygen and prevent cerebral hypoxia. [ ] [ ] [ ] a 'safe' level for oxygen administration is not firmly established and, because of species differences in susceptibility to oxygen, caution is needed in extrapolating from animal studies. however, animal experiments have shown that previous damage to the lungs renders them unduly sensitive to oxygen , and conversely that prior exposure to high levels of oxygen confers some resistance to subsequent oxygen exposure. clinical studies suggest that less than % oxygen (at atmospheric pressure) can be tolerated for long periods without ill effect. little, if any, serious lung damage results from administration of % oxygen for up to hours but concentrations between % and % carry a risk of damage if this period is exceeded. , extracorporeal oxygenation of the blood circumvents the problem but if it is to be prolonged it becomes a major undertaking that poses its own hazards; it is therefore generally reserved for patients who remain hypoxaemic despite other measures. intravenous blood oxygenators are employed to minimise the supplementation of inspired oxygen and partial liquid ventilation utilising perfluorocarbon has also been used. experimentally, disruption of cd binding to reduce the release of proinflammatory cytokines has shown promising results in blunting oxygen-induced lung injury. none of the morphological changes attributable to oxygen toxicity is specific. the earliest ultrastructural change in experimental oxygen poisoning is swelling of endothelial cells, the cytoplasm of which becomes grossly oedematous and vacuolated. swelling and fragmentation of type i epithelial cells follow and these cells become separated from their basement membrane, which is then coated by thin strands of protein. this coating is replaced by proliferating type ii cells by the th day. with recovery in room air the lungs practically return to normal. the full clinical picture of oxygen poisoning is the acute respiratory distress syndrome and the corresponding pathological changes are those of diffuse alveolar damage, as described on page . patients with hypovolaemic shock or undergoing major surgery often require massive blood transfusions and this provides another possible cause of pulmonary damage. although hypervolaemia is the commonest cause of pulmonary oedema after blood transfusion, transfusion-related acute lung injury is more often fatal. platelet and white cell aggregates are known to develop in stored blood, but a relationship between the number of microaggregates transfused and the degree of respiratory impairment has not been convincingly demonstrated. leukocyte antibodies are a more likely cause of lung injury in these patients. such antibodies are often found in multiparous female donors as a result of sensitisation by fetal white cells during pregnancy. alternatively, the recipient may have developed them during pregnancy or as a result of previous blood transfusions. the implicated antibodies are thought to initiate alveolar capillary damage within hours of transfusion by stimulating granulocyte aggregation. , electron microscopy has shown capillary endothelial damage with activated granulocytes in contact with alveolar basement membranes. cardiopulmonary bypass entails oxygenation and circulation of the blood by extracorporeal devices, so permitting major heart surgery. in the early days of such surgery it was not unusual for patients to develop fatal respiratory insufficiency in the postoperative period. this led to the term 'postperfusion lung' . electron microscopic studies showed alveolar damage with degranulation of neutrophils in pulmonary capillaries. , the syndrome is now less common but infants remain susceptible. the most likely explanation is that the synthetic materials with which blood comes into contact during the bypass procedure are able to activate complement. this is mediated by hageman factor (factor xii) and the alternative pathway. aggregation of neutrophils leads to their sequestration in the lungs and damage results from their release of lysosomal enzymes and active radicals. [ ] [ ] [ ] the process is delayed by hypothermia. a postcardiac injury syndrome develops after a variety of myocardial or pericardial injuries: it has been described after cardiac surgery (postpericardiotomy syndrome), myocardial infarction (dressler's syndrome), blunt trauma to the chest, percutaneous puncture of the heart and implantation of a pacemaker. there is a delay of anything between a few days and a few months between the cardiac injury and the onset of symptoms, which comprise chest pain, breathlessness, dyspnoea and fever. examination usually reveals haemorrhagic pleural or pericardial effusions and pulmonary infiltrates. the syndrome usually resolves spontaneously and few pathological studies have therefore been conducted. however, the changes of diffuse alveolar damage have been reported, principally hyaline membrane formation and type ii pneumocyte hyperplasia. the pathogenesis is obscure. antibodies reacting with myocardial antigens often develop after cardiac surgery but there is no relationship between these and the development of the syndrome. [ ] [ ] [ ] this minimally invasive technique is used to destroy lesions as varied as pulmonary metastases and the connection between the left atrium and ectopic foci in the muscular sleeves that surround the terminations of the pulmonary veins (see p. ). the former may be complicated by pneumothorax and the latter by pulmonary vein stenosis. , central venous cannulation (synonym: catheterisation) is widely used in treating seriously ill patients and may give rise to serious complications. the commonest early complications related to the respiratory tract are caused by local trauma: they include pneumothorax, subcutaneous emphysema, haemothorax and air embolism. infection occurs later, causing endocarditis, septic emboli and lung abscesses. thrombosis is another common late complication: one autopsy study of patients with central venous lines showed that % had major pulmonary emboli and % had microscopic emboli in their pulmonary arteries. pulmonary artery cannulation, for example with a swan-ganz catheter, may result in pulmonary infarction or any of the traumatic complications of central venous catheterisation mentioned above. , tracheotomy entails a small immediate risk of haemorrhage from damaged subthyroidal arteries, while an endotracheal tube predisposes to infection, as with all foreign bodies. infection is also promoted by the filtering action of the upper respiratory air passages being bypassed. the latter factor also necessitates humidification of the inspired air and on occasion the humidifier or ventilator has become contaminated so that an aerosol of bacteria is introduced directly into the lower respiratory tract. high-pressure ventilation may also lead to interstitial emphysema, pneumothorax and surgical emphysema. asphyxia may follow an endotracheal tube becoming blocked by secretions or through it being badly positioned. secretions need to be constantly removed yet repeated suctioning to achieve this has led to cardiac dysrrythmia and even cardiac arrest. if the balloon on the endotracheal tube is too near the tracheostomy it may act as a fulcrum, causing the tip of the tube to press into the tracheal wall. pressure necrosis and perforation may follow, leading to mediastinitis, tracheo-oesophageal fistula or erosion of a large blood vessel. these are also complications of tracheobronchial laser therapy. pressure from the balloon may lead to a tracheal diverticulum and after the tube is withdrawn the trachea may become narrowed at either the site of the incision or further down where the balloon on the tracheal tube causes pressure. small, shallow ulcers generally heal quickly but deeper ulcers cause necrosis of the tracheal cartilage, and healing is then often accompanied by fibrous stenosis (fig. . . ) or web formation. this results in wheezing and dyspnoea but not before the trachea has narrowed to % of its original size, which may take months. earlier narrowing may be caused by oedema or a fibrinous pseudomembrane. , sometimes the stenosis takes the form of a large mass of granulation tissue at the tracheostomy site, a so-called granuloma ball. in children especially, intubation may lead to tracheomalacia so that after the tube is removed the airway collapses. necrotising sialometaplasia is a further complication of prolonged intubation. the incidence of such posttracheostomy complications can be minimised by careful placement of the stoma and tube, avoidance of large apertures and high cuff pressures, elimination of heavy connecting equipment and meticulous care of the tracheostomy. nasogastric feeding tubes may of course lead to aspiration lesions in the lungs and even fatal asphyxia if they are inadvertently allowed to enter the trachea rather than the oesophagus. bronchoscopy is generally a safe, almost routine procedure. a review of patients who underwent bronchoscopy identified severe complications in ( . %), of whom three died. the fatal cases comprised a -year-old with coronary heart disease who developed cardiac arrest and two patients who had had tracheal transplantation for oesophageal cancer and required bronchoscopic laser treatment but died of airway obstruction. the pleural cavity is intubated in the treatment of pneumothorax and pleural effusions the tube being placed anteriorly to drain air and posteriorly to drain fluid. complications include laceration of an intercostal artery or vein, the lung, the diaphragm and the heart. pneumonectomy has been practised since the s, since when the mortality associated with this operation has dropped from over % to near zero in the best hospitals. risk factors include underlying lung disease, other medical conditions and more extensive procedures such as pleuropneumonectomy and pneumonectomy combined with chest wall resection. the anatomical changes that take place soon after pneumonectomy have been extensively studied by radiologists who describe the air-filled postpneumonectomy space gradually filling with fluid and contracting as the mediastinum shifts and the ipsilateral dome of the diaphragm rises. much of the space is filled by fluid within weeks but complete opacification may take up to months. rapid filling in the immediate postoperative period suggests haemorrhage or chylothorax. however, fluid accumulation is normally rapid after pleuro-pneumonectomy and may compromise the function of the other lung. pathologists conducting autopsies long after the operation may find complete fibrous obliteration of the postpneumonectomy space, coupled with mediastinal shift and elevation of the hemidiaphragm, but often there is persistent brown fluid, which may be clear, cloudy or occasionally purulent. the remaining lung is generally enlarged, with its volume greater than predicted. animal studies have shown that if one lung is excised early in life the enlargement is partly due to enhanced growth but later it represents only dilatation of existing air spaces. hepatocyte growth factor is thought to be involved in the proliferation of residual lung cells following pneumonectomy. pulmonary complications include those typically seen after other thoracic procedures, such as haemorrhage and infection, and those unique to the postpneumonectomy state, namely anastomotic dehiscence and postpneumonectomy pulmonary oedema. the latter presents as the acute respiratory distress syndrome and represents the early stages of diffuse alveolar damage. it follows severe shift of the heart and mediastinum, which is commoner in children and young adults, in whom the tissues are more compliant. [ ] [ ] [ ] [ ] the condition complicates up to % of lung resections , and is commoner following excision of the right lung when severe herniation of the left lung into the postpneumonectomy space stretches the trachea and left main bronchus and the latter is compressed between the left pulmonary artery in front and the arch of the aorta behind. in the long term the compression can result in bronchomalacia and postobstructive bronchiectasis. if postpneumonectomy oedema develops the immediate postoperative mortality is high - % following pneumonectomy, % following lobectomy and % following sublobar resections. , the pathogenesis is probably multifactorial but apart from factors such as fluid overload and high inspired oxygen concentrations there is probably an element of alveolar wall injury, induced by oxidant generation secondary to lung stretching and general surgical trauma. , occupational dust exposure and chronic obstructive pulmonary disease -a systematic overview of the evidence occupational exposures and the risk of copd: dusty trades revisited the origin of the term 'pneumonokoniosis' industrial injuries advisory council. pneumoconiosis and byssinosis. london: hmso; aerosols: their deposition and clearance human lung parenchyma retains pm . airway branching patterns influence asbestos fiber location and the extent of tissue injury in the pulmonary parenchyma and lymph nodes in infants' lungs pulmonary sumps, dust accumulations, alveolar fluid and lymph vessels candida lung abscesses complicating parenteral nutrition a diffuse form of pulmonary silicosis in foundry workers inhalation of china stone and china clay dusts: relationship between mineralogy of dust retained in the lungs and pathological changes talc pneumoconiosis. a report of six patients with postmortem findings diffuse 'asbestosis-like' interstitial fibrosis of the lung pulmonary pathology in workers exposed to nonasbestiform talc talc pneumoconiosis -a pathologic and mineralogic study diffuse interstitial fibrosis in nonasbestos pneumoconiosis -a pathological study the disposal of coal and haematite dusts inhaled successively vertical gradient of alveolar size in lungs of dogs frozen intact apical localization of phthisis distribution of blood flow and ventilation perfusion ratio in the lung, measured with radioactive co apical localization of pulmonary tuberculosis, chronic pulmonary histoplasmosis, and progressive massive fibrosis of the lung effect of inspiratory flow rate on the regional distribution of inspired gas apical scars and their relationship to siliceous dust accumulation in non-silicotic lungs topographic distribution of asbestos fibres lung structure as a risk factor in adverse pulmonary responses to asbestos exposure. a case-referent study in quebec chrysotile miners and millers particle deposition in casts of the human upper tracheobronchial tree differences in particle deposition between the two lungs pulmonary retention of ultrafine and fine particles in rats experimental study of the dust-clearance mechanism of the lung the lung: an excretory route for macromolecules and particles phagocytic potential of pulmonary alveolar epithelium with particular reference to surfactant metabolism chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages the uptake of mineral particles by pulmonary epithelial cells translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships patterns of particle deposition and retention after instillation to mouse lung during acute injury and fibrotic repair silica deposition in the lung during epithelial injury potentiates fibrosis and increases particle translocation to lymph nodes the permeability of lung parenchyma to particulate matter the migration of bronchoalveolar macrophages into hilar lymph nodes etude au microscope electronique du granulome pulmonaire silicotique experimental the postnatal development of lymphoreticular aggregates in human lung in relation to occupational and non-occupational exposure the distribution of amosite asbestos in the periphery of the normal human lung diseases associated with exposure to silica and nonfibrous silicate minerals detection of silica particles in lung tissue by polarizing light microscopy a practical method for the identification of particulate and crystalline material in paraffin-embedded tissue specimens particles in paraffin sections demonstrated in the backscattered electron image quantitative analysis of inorganic particulate burden in situ in tissue sections microanalysis in histopathology ion microprobe mass analysis of beryllium in situ in human lung: preliminary results laser probe mass spectrometry (lamms) analysis of beryllium, sarcoidosis and other granulomatous diseases molecular identification of foreign inclusions in inflammatory tissue surrounding metal implants by fourier transform laser microprobe mass spectrometry value of beryllium lymphocyte transformation test in chronic beryllium disease and in potentially exposed workers radiographic and pathologic correlation of coal workers' pneumoconiosis slateworker's pneumoconiosis transkei silicosis three cases of dental technician's pneumoconiosis related to cobaltchromium-molybdenum dust exposure: diagnosis and follow-up deposition of silicosis dust in the lungs of the inhabitants of the sahara regions sulla possibilita e sulla frequenza della silicosi pulmonare tra gli abitanti del deserto libico simple siliceous pneumoconiosis of bedouin females in the negev desert simple siliceous pneumoconiosis in negev bedouins silicosis in a pakistani farmer silicosis in a himalayan village population -role of environmental dust non-occupational pneumoconiosis at high altitude villages in central ladakh silicate pneumoconiosis of farm workers silicate pneumoconiosis and pulmonary fibrosis in horses from the monterey-carmel peninsula comparative pathology of silicate pneumoconiosis two cases of acute silicosis -with a suggested theory of causation acute silicosis silicose aigue fatale par inhalation volontaire de poudre à recurer acute silicosis in tombstone sandblasters accelerated silicosis in scottish stonemasons the relation between fibrosis of hilar lymph glands and the development of parenchymal silicosis distribution of silicotic collagenization in relation to smoking habits silicosis presenting as bilateral hilar lymphadenopathy silicotic lymph node lesions in nonoccupationally exposed lung carcinoma patients obliterative central bronchitis due to mineral dust in patients with pneumoconiosis left recurrent laryngeal nerve palsy associated with silicosis anthracotic and anthracosilicotic spindle cell pseudotumors of mediastinal lymph nodes: report of five cases of a reactive lesion that simulates malignancy right middle lobe atelectasis associated with endobronchial silicotic lesions pleural pearls following silicosis: a histological and electronmicroscopic study silicainduced pleural disease: an unusual case mimicking malignant mesothelioma factors associated with massive fibrosis in silicosis silicosis of systemic distribution hepatosplenic silicosis hepatic silicosis silicotic lesions of the bone marrow: histopathology and microanalysis peritoneal silicosis pulmonary fibrosis in non-ferrous foundry workers irregular opacities in coalworkers' pneumoconiosis -correlation with pulmonary function and pathology pneumoconiosis-related lung cancers -preferential occurrence from diffuse interstitial fibrosis-type pneumoconiosis the effects of dusts on peritoneal cells within diffusion chambers freshly fractured quartz inhalation leads to enhanced lung injury and inflammation: potential role of free radicals an examination of the cytotoxic effects of silica on macrophages activity of macrophage factor in collagen formation by silica fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages effect of sio -liberated macrophage factor on protein synthesis in connective tissue in vitro the regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis the ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro cytokines and cytokine network in silicosis and coal workers' pneumoconiosis activation of murine macrophages by silica particles in vitro is a process independent of silica-induced cell death localization of type i procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis transforming growth factor-beta (tgfbeta) in silicosis mechanisms in the pathogenesis of asbestosis and silicosis changes in the composition of lung lipids and the 'turnover' of dipalmitoyl lecithin in experimental alveolar lipo-proteinosis induced by inhaled quartz case report: pulmonary alveolar proteinosis and aluminum dust exposure induction of surfactant protein (sp-a) biosynthesis and sp-a mrna in activated type ii cells during acute silicosis in rats silicon nephropathy: a possible occupational hazard rapidly progressive silicon nephropathy silica and glomerulonephritis: case report and review of the literature rapidly progressive crescenteric glomerulonephritis in a sandblaster with silicosis diatomaceous earth pneumoconiosis particle size for differentiation between inhalation and injection pulmonary talcosis kaolin pneumoconiosis. a case report kaolin pneumoconiosis. radiologic, pathologic and mineralogic findings silicosis in wyoming bentonite workers pneumoconiosis due to fuller's earth pulmonary disease caused by the inhalation of cosmetic talcum powder talc dust pneumoconiosis mica-associated pulmonary interstitial fibrosis baritosis: a benign pneumoconiosis silicosis in barium miners scleroderma in gold-miners on the witwatersrand with particular reference to pulmonary manifestations immunological factors in the pathogenesis of the hyaline tissue of silicosis mini-review -immunologic aspects of pneumoconiosis adverse effects of crystalline silica exposure chronic necrotizing pulmonary aspergillosis in pneumoconiosis -clinical and radiologic findings in patients silica and some silicates. monographs on the evaluation of the carcinogenic risk of chemicals to humans occupational lung disease: . exposure to crystalline silica and risk of lung cancer: the epidemiological evidence do silica and asbestos cause lung cancer? report by the industrial injuries advisory council in accordance with section of the social security administration act on the question whether lung cancer in relation to occupational exposure to silica should be prescribed. london: hmso; crystalline silica exposure, radiological silicosis, and lung cancer mortality in diatomaceous earth industry workers acute silicoproteinosis acute silicolipoproteinosis atypical reaction to inhaled silica alveolar proteinosis. its experimental production in rodents experimental endogenous lipid pneumonia and silicosis pathogenesis of experimental pulmonary alveolar proteinosis pneumoconiosis in carbon electrode makers graphite pneumoconiosis of electrotypers case report: a case of wood-smoke-related pulmonary disease hut lung. a domestically acquired particulate lung disease bronchial stenosis due to anthracofibrosis anthracofibrosis attributed to mixed mineral dust exposure: report of three cases anthracofibrosis, bronchial stenosis with overlying anthracotic mucosa: possibly a new occupational lung disorder occupational anthracofibrosis pneumoconiosis and other causes of death in iron and steel foundry workers histochemical study of certain iron ore dusts mixed-dust pneumoconiosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation chronic interstitial pneumonia in silicosis and mix-dust pneumoconiosis: its prevalence and comparison of ct findings with idiopathic pulmonary fibrosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation coal pneumoconiosis coal workers' pneumoconiosis pneumoconiosis in coal trimmers dust exposure, dust recovered from the lung, and associated pathology in a group of british coalminers the relationship between the mass and composition of coal mine dust and the development of pneumoconiosis interstitial fibrosis in coal miners -experience in wales and west virginia coal worker's pneumoconiosis: ct assessment in exposed workers and correlation with radiographic findings variations in the histological patterns of the lesions of coal workers' pneumoconiosis in britain and their relationship to lung dust content comparison of radiographic appearances with associated pathology and lung dust content in a group of coalworkers small airways disease and mineral dust exposure. prevalence, structure and function coal mining, emphysema, and compensation revisited coal mining, emphysema, and compensation revisited -reply coal mining and chronic obstructive pulmonary disease: a review of the evidence the pathogenesis of simple pneumokoniosis in coal workers mineral dusts cause elastin and collagen breakdown in the rat lung: a potential mechanism of dust-induced emphysema emphysema in coal workers' pneumoconiosis pulmonary disability in coal workers' pneumoconiosis quantified pathology of emphysema, pneumoconiosis, and chronic bronchitis in coal workers emphysema and dust exposure in a group of coal workers coal worker's lung: not only black, but also full of holes contributions of dust exposure and cigarette smoking to emphysema severity in coal miners in the united states report by the industrial injuries advisory council in accordance with section of the social security administration act on the question whether bronchitis and emphysema in coal miners and metal production workers should be prescribed. london: hmso; the new prescription: industrial injuries benefits for smokers? exposure to respirable coalmine dust and incidence of progressive massive fibrosis the attack rate of progressive massive fibrosis ischemic necrosis in anthracosilicosis the composition of massive lesions in coal miners mechanisms of fibrosis in coal workers' pneumoconiosis: increased production of platelet-derived growth factor, insulin-like growth factor type i, and transforming growth factor beta and relationship to disease severity secretion and mrna expression of tnf alpha and il- in the lungs of pneumoconiosis patients a broader concept of caplan's syndrome related to rheumatoid factors differential agglutination test in rheumatoid arthritis complicated by pneumoconiosis circulating antinuclear antibody and rheumatoid factor in coal pneumoconiosis serologic changes in pneumoconiosis and progressive massive fibrosis of coal workers immunological investigations of coalworkers' disease certain unusual radiological appearances in the chest of coal-miners suffering from rheumatoid arthritis pathological studies of modified pneumoconiosis in coal-miners with rheumatoid arthritis (caplan's syndrome) asbestos, asbestosis, and cancer: the helsinki criteria for diagnosis and attribution report of the asbestosis committee of the college of american pathologists and pulmonary pathology society the asbestos cancer epidemic rapid short-term clearance of chrysotile compared with amosite asbestos in the guinea pig persistence of natural mineral fibers in human lungs: an overview a pathological and mineralogical study of asbestos-related deaths in the united kingdom in correlation between fibre content of the lungs and disease in naval dockyard workers correlation between fibre content of the lung and disease in east london asbestos factory workers the effects of the inhalation of asbestos in rats mass and number of fibres in the pathogenesis of asbestos related lung disease in rats fiber burden and patterns of asbestos-related disease in chrysotile miners and millers carcinogenesis and mineral fibres lung diseases due to environmental exposures to erionite and asbestos in turkey prospective study of mesothelioma mortality in turkish villages with exposure to fibrous zeolite analysis of ferruginous bodies in bronchoalveolar lavage from foundry workers analysis of the cores of ferruginous (asbestos) bodies from the general population fiber counting and analysis in the diagnosis of asbestos-related disease concentrations and dimensions of coated and uncoated asbestos fibres in the human lung comparison of the results of asbestos fibre dust counts in lung tissue obtained by analytical electron microscopy and light microscopy asbestos bodies and the diagnosis of asbestosis in chrysotile workers asbestos fiber content of lungs with diffuse interstitial fibrosis: an analytical scanning electron microscopic analysis of cases comparison of phagocytosis of uncoated versus coated asbestos fibers by cultured human pulmonary alveolar macrophages numbers of asbestos bodies on iron-stained tissue sections in relation to asbestos body counts in lung tissue digests asbestos content of lung tissue in asbestos associated diseases: a study of cases analysis and interpretation of inorganic mineral particles in 'lung' tissues asbestos bodies in bronchoalveolar lavage in relation to asbestos bodies and asbestos fibres in lung parenchyma asbestos bodies in the sputum of asbestos workers: correlation with occupational exposure the optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to human pathology reaction assessment of mineral fibres from human lung tissue detection of asbestos fibres by dark ground microscopy analysis of lung asbestos content guidelines for mineral fibre analyses in biological samples: report of the ers working group relationship between occupations and asbestos-fibre content of the lungs in patients with pleural mesothelioma, lung cancer, and other diseases inflammation-generating potential of long and short-fibre amosite asbestos samples asbestos-stimulated tumour necrosis factor release from alveolar macrophages depends on fibre length and opsonization the pathogenicity of long versus short fibre amosite asbestos administered to rats by inhalation and intra-peritoneal injection cytogenetic and pathogenic effects of long and short amosite asbestos exposure and mineralogical correlates of pulmonary fibrosis in chrysotile asbestos workers quantitative assessment of inorganic fibrous particulates in dust samples with an analytical transmission electron microscope fibre distribution in the lungs and pleura of subjects with asbestos related diffuse pleural fibrosis malignant mesothelioma in women observations on the distribution of asbestos fibres in human lungs analysis of asbestos fibres and asbestos bodies in tissue samples from human lung: an international interlaboratory trial distribution and characteristics of amphibole asbestos fibres in the left lung of an insulation worker measured with the light microscope the identification of asbestos dust with an electron microprobe analyser symposium on man-made mineral fibres mortality in cases of asbestosis diagnosed by a pneumoconiosis medical panel small-airway lesions in patients exposed to nonasbestos mineral dusts small airways disease and mineral dust exposure relation of smoking and age to findings in lung parenchyma: a microscopic study electron microscopic studies in desquamative interstitial pneumonia associated with asbestos desquamative interstitial pneumonia associated with chrysotile asbestos fibres localized inflammatory pulmonary disease in subjects occupationally exposed to asbestos criteria for the diagnosis and grading of asbestosis report of the pneumoconiosis committee of the college of american pathologists and the national institute for occupational safety and health cytoplasmic hyalin in asbestosis further observations on cytoplasmic hyaline in the lung lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore lung pathology of fatal severe acute respiratory syndrome immunohistochemical detection of ubiquitin-positive intracytoplasmic eosinophilic inclusion bodies in diffuse alveolar damage idiopathic pulmonary fibrosis in asbestosexposed workers mortality of workers certified by pneumoconiosis medical panels as having asbestosis radiographic evidence of asbestos effects in american marine engineers chronic airflow obstruction in lung disease. philadelphia: saunders fine structural changes in cryptogenic fibrosing alveolitis and asbestosis pulmonary asbestosis and idiopathic pulmonary fibrosis: pathogenetic parallels crocidoliteinduced pulmonary fibrosis in mice hydroxyl radicals are formed in the rat lung after asbestos instillation invivo asbestos fibers and interferon-gamma up-regulate nitric oxide production in rat alveolar macrophages oxidant sand antioxidant mechanisms of lung disease caused by asbestos fibres antibodies in some chronic fibrosing lung diseases asbestos causes translocation of p protein and increases nf-kappa b dna binding activity in rat lung epithelial and pleural mesothelial cells rapid activation of pdgf-a and -b expression at sites of lung injury in asbestos-exposed rats upregulation of the pdgf-alpha receptor precedes asbestos-induced lung fibrosis in rats tnf-alpha receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers alveolar cell stretching in the presence of fibrous particles induces interleukin- responses the molecular basis of asbestos induced lung injury inhaled asbestos activates a complementdependent chemoattractant for macrophages alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis cellular and molecular basis of the asbestos-related diseases up-regulated expression of transforming growth factor-α in the bronchiolar-alveolar duct regions of asbestos-exposed rats a study of macrophage-mediated initiation of fibrosis by asbestos and silica using a diffusion chamber technique enhanced generation of free radicals from phagocytes induced by mineral dusts mortality from lung cancer in asbestos workers diffuse pleural mesotheliomas and asbestos exposure in northwestern cape province carcinogenicity of amosite asbestos asbestos exposure, cigarette smoking and death rates smoking, exposure to crocidolite, and the incidence of lung cancer and asbestosis pathology of carcinoma of the lung associated with asbestos exposure a study of the histological cell types of lung cancer in workers suffering from asbestosis in the united kingdom lung cancer cell type and asbestos exposure histological type of lung carcinoma in asbestos cement workers and matched controls incidence of lung cancer by histological type among asbestos cement workers in denmark lung cancer in asbestos cement workers in denmark asbestos and lung cancer in glasgow and the west of scotland exposure to crocidolite and the incidence of different histological types of lung cancer asbestos and lung cancer asbestos and lung cancer: is it attributable to asbestosis or to asbestos fibre burden? in: corrin b, editor. pathology of lung tumours asbestos fibreyears and lung cancer: a two phase case-control study with expert exposure assessment the quantitative risks of mesothelioma and lung cancer in relation to asbestos exposure is asbestos or asbestosis the cause of the increased risk of lung cancer in asbestos workers? relation between asbestosis and bronchial cancer in amphibole asbestos miners asbestosis as a precursor of asbestos related lung cancer -results of a prospective mortality study asbestos exposure, asbestosis, and asbestosattributable lung cancer asbestos and lung cancer -reply criteria for attributing lung cancer to asbestos exposure asbestosis: a marker for the increased risk of lung cancer among workers exposed to asbestos asbestos, asbestosis, and lung cancer: a critical assessment of the epidemiological evidence airway function in lifetime-nonsmoking older asbestos workers severe diffuse small airways abnormalities in long term chrysotile asbestos miners morphology of small-airway lesions in patients with asbestos exposure does aluminum smelting cause lung disease? the treatment of silicosis by aluminum powder silicose aigue. caractéristiques cliniques, radiologiques, fonctionnelles et cytologiques du liquide bronchoalveolaire. a propos de observations pathologie and pathogenesis der aluminiumlunge die aluminiumlunge -eine neue gewerbeerkrankung. z f d ges pulmonary fibrosis in workers exposed to finely powdered aluminium aluminium pneumoconiosis i. in vitro comparison of stamped aluminium powders containing different lubricating agents and a granular aluminium powder effect on the rat lung of intratracheal injections of stamped aluminium powders containing different lubricating agents and of a granular aluminium powder desquamative interstitial pneumonia in an aluminum welder sarcoidlike lung granulomatosis induced by aluminum dusts aluminum induced pulmonary granulomatosis aluminum welding fume-induced pneumoconiosis rare earth (cerium) pneumoconiosis rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review hard metal disease interstitial lung disease and asthma in hard-metal workers: bronchoalveolar lavage, ultrastructural, and analytical findings and results of bronchial provocation tests the biological action of tungsten carbide and cobalt: studies on experimental pulmonary histopathology cobalt lung in diamond polishers rapidly fatal progression of cobalt lung in a diamond polisher hard metal pneumoconiosis and the association of tumor necrosis factor-alpha the pulmonary toxicity of beryllium beryllium and lung disease recent chronic beryllium disease in residents surrounding a beryllium facility nonoccupational beryllium disease masquerading as sarcoidosis -identification by blood lymphocyte proliferative response to beryllium beryllium disease international agency for research on cancer. an evaluation of carcinogenic risk to humans. overall evaluation of carcinogenicity: an updating of iarc monographs vols - uber eine apparatur zur erzeugung niedriger staubkonzentrationen von grosser konstanz und eine methode zur mikrogravinctrischen staubbestemmung. anwendung bei der untersuchung von stauben aus der berylliumsgewinnung delayed chenical pneumonitis occurring in workers exposed to beryllium compounds chronic beryllium disease in a dental laboratory technician maintenance of alveolitis in patients with chronic beryllium disease by berylliumspecific helper t cells hla-dpb glutamate : a genetic marker of beryllium disease immunogenetic basis of environmental lung disease: lessons from the berylliosis model beryllium disease in identical twins beryllium detection in human lung tissue using electron probe x-ray microanalysis diagnostic criteria for chronic beryllium disease (cbd) based on the uk registry - united kingdom beryllium registry: mortality and autopsy study lung disease and the lightest of metals misdiagnosis of sarcoidosis in patients with chronic beryllium disease diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients the pathology of interstitial lung disease in nylon flock workers flock worker's lung: broadening the spectrum of clinicopathology, narrowing the spectrum of suspected etiologies polyethylene flock-associated interstitial lung disease in a spanish female popcorn workers' lung clinical bronchiolitis obliterans in workers at a microwave-popcorn plant clinical bronchiolitis obliterans in workers at a microwave-popcorn plant bronchiolitis obliterans syndrome in popcorn production plant workers popcorn worker's lung' in britain in a man making potato crisp flavouring toxicity in textile airbrushing in spain outbreak of organising pneumonia in textile printing sprayers epidemic outbreak of interstitial lung disease in aerographics textile workersthe 'ardystil syndrome': a first year follow up organizing pneumonia in textile printing workers: a clinical description outbreak of pulmonary disease in textile dye sprayers in algeria five-year follow-up of algerian victims of the ''ardystil syndrome mineral oils and petroleum . skorodin ms, chandrasekhar aj. an occupational cause of exogenous lipoid pneumonia machine operator's lung. a hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols metalworking fluid-associated hypersensitivity pneumonitis: a workshop summary an outbreak of hypersensitivity pneumonitis at a metalworking plant: a longitudinal assessment of intervention effectiveness stenius-aarniala b. fire-eater's lung fire-eater's lung: seventeen cases and a review of the literature pulmonary mechanics in dogs given different doses of kerosene intratracheally x-ray appearance of the lungs of electric arc welders the health of welders in naval dockyards: the risk of asbestosrelated diseases occurring in welders welders' pneumoconiosis: tissue elemental microanalysis by energy dispersive x ray analysis the respiratory health of welders toxic fumes and gases . nemery b. metal toxicity and the respiratory tract lung changes associated with the manufacture of alumina abrasives acute inorganic mercury vapor inhalation poisoning nickel poisoning ii. studies on patients suffering from acute exposure to vapors of nickel carbonyl silo-filler's disease -a syndrome caused by nitrogen dioxide silo-filler's disease: nitrogen dioxide-induced lung injury silo filler's disease silo-filler's disease in new york state dung lung: a report of toxic exposure to liquid manure death caused by fermenting manure acute toxic exposure to gases from liquid manure slurry lung': a report of three cases pulmonary lesions in rats exposed to ozone pathology of adult respiratory distress syndrome response of macaque bronchiolar epithelium to ambient concentrations of ozone ultrastructural alterations of alveolar tissue of mice differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for months ozone-induced acute tracheobronchial epithelial injury -relationship to granulocyte emigration in the lung ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy a cohort study of workers exposed to formaldehyde in the british chemical industry -an update pulmonary toxicity following exposure to methylene chloride and its combustion product, phosgene the diversity of the effects of sulfur mustard gas inhalation on respiratory system years after a single, heavy exposure: analysis of cases tracheobronchomalacia and air trapping after mustard gas exposure an international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients thionyl-chloride-induced lung injury and bronchiolitis obliterans hydrogen sulfide poisoning: review of years' experience anoxic asphyxia anoxic asphyxia -a cause of industrial fatalities: a review sword ' : surveillance of work-related and occupational respiratory disease in the uk occupational asthma as identified by the surveillance of work-related and occupational respiratory diseases programme in south africa incidence of occupational asthma by occupation and industry in finland sword ' : surveillance of work-related and occupational respiratory disease in the uk american thoracic society statement: occupational contribution to the burden of airway disease aetiological agents in occupational asthma reported incidence of occupational asthma in the united kingdom, - occupational asthma in a factory with a contaminated humidifier the pathology of byssinosis byssinosis: a review in vitro alternative and classical activation of complement by extracts of cotton mill dust: a possible mechanism in the pathogenesis of byssinosis antibody independent complement activation by card-room cotton dust occupational fevers . raskandersen a, pratt ds. inhalation fever -a proposed unifying term for febrile reactions to inhalation of noxious substances humidifier fever pulmonary mycotoxicosis organic dust toxicity (pulmonary mycotoxicosis) associated with silo unloading desquamative interstitial pneumonitis and diffuse alveolar damage in textile workers: potential role of mycotoxins pulmonary mycotoxicosis: a clinicopathologic study of three cases pulmonary involvement in zinc fume fever cytokines in metal fume fever music: a new cause of primary spontaneous pneumothorax atmospheric pressure influences the risk of pneumothorax: beware of the storm experimental study of blast injuries to the lungs biophysics of impact injury to the chest and abdomen histologic, immunohistochemical, and ultrastructural findings in human blast lung injury histologic, immunohistochemical, and ultrastructural findings in human blast lung injury the ultrastructure of rat lung following acute primary blast injury disorders associated with diving decompression disorders in divers intrapulmonary air trapping in submarine escape training casualties airway structure and alveolar emptying in the lungs of sea lions and dogs the structure and function of the small airways in pinniped and sea otter lungs mechanisms of death in shallow-water scuba diving pulmonary barotrauma in submarine escape training the pathophysiology of decompression sickness hemodynamic changes induced by recreational scuba diving dysbaric osteonecrosis: aseptic necrosis of bone deep diving mammals: dive behavior and circulatory adjustments contribute to bends avoidance current concepts: high-altitude illness illnesses at high altitude high altitude pulmonary edema high altitude pulmonary oedema; characteristics of lung lavage fluid prevention of high-altitude pulmonary edema by nifedipine pulmonary circulation at high altitude association of high-altitude pulmonary edema with the major histocompatibility complex pathogenesis of high-altitude pulmonary oedema: direct evidence of stress failure of pulmonary capillaries high-altitude pulmonary edema: current concepts high-altitude pulmonary edema is initially caused by an increase in capillary pressure when lungs on mountains leak -studying pulmonary edema at high altitudes inhaled nitric oxide for high-altitude pulmonary edema lung pathology in high-altitude pulmonary edema pathological features of the lung in fatal high altitude pulmonary edema occurring at moderate altitude in japan lack of smooth muscle in the small pulmonary arteries of the native ladakhi -is the himalayan highlander adapted? aspects of hypoxia pulmonary blood vessels and endocrine cells in subacute infantile mountain sickness the human pulmonary circulation: its form and function in health and disease pulmonary edema associated with salt water near-drowning -new insights the effect of salt water on alveolar epithelial barrier function near-drowning: clinical course of lung injury in adults haggard hw. action of irritant gases upon the respiratory tract regional respiratory tract absorption of inhaled reactive gases respiratory irritants encountered at work health effects of outdoor air pollution committee of the environmental and occupational health assembly of the committee of the environmental and occupational health assembly of the fine particulate air pollution and mortality in us cities, - health effects of diesel exhaust emissions respiratory changes due to long-term exposure to urban levels of air pollution: a histopathologic study in humans chronic glandular bronchitis in young individuals residing in a metropolitan area an association between air pollution and mortality in united states cities chronic bronchitis in women using solid biomass fuel in rural peshawar indoor air pollution: a poverty-related cause of mortality among the children of the world risk of copd from exposure to biomass smoke human health impacts of volcanic activity persistent hyperreactivity and reactive airway dysfunction in firefighters at the world trade center the world trade center residents' respiratory health study: new-onset respiratory symptoms and pulmonary function pulmonary function after exposure to the world trade center collapse in the new york city fire department characterization of the dust/smoke aerosol that settled east of the world trade center (wtc) in lower manhattan after the collapse of the wtc chemical analysis of world trade center fine particulate matter for use in toxicologic assessment longitudinal assessment of spirometry in the world trade center medical monitoring program acute eosinophilic pneumonia in a new york city firefighter exposed to world trade center dust granulomatous pneumonitis following exposure to the world trade center collapse smoking-related interstitial lung diseases: a concise review concomitant upper-lobe bullous emphysema, lower-lobe interstitial fibrosis and pulmonary hypertension in heavy smokers: report of eight cases and review of the literature heterogeneity in combined pulmonary fibrosis and emphysema smoking-related interstitial lung diseases challenges in pulmonary fibrosis : smoking-induced diffuse interstitial lung diseases combined pulmonary fibrosis and emphysema: a distinct underrecognised entity smoking-related changes in the background lung of specimens resected for lung cancer: a semiquantitative study with correlation to postoperative course clinical assembly contribution to the celebration of years of the ers. smoking-related lung diseases: a clinical perspective clinically occult interstitial fibrosis in smokers: classification and significance of a surprisingly common finding in lobectomy specimens bronchiolar inflammation and fibrosis associated with smoking -a morphologic cross-sectional population analysis relation of smoking and age to findings in lung parenchyma: a microscopic study variability in small airway epithelial gene expression among normal smokers airway alveolar attachment points and exposure to cigarette smoke in utero patterns of global tobacco use in young people and implications for future chronic disease burden in adults tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic pathology of the lung in fatally burned patients respiratory tract pathology in patients with severe burns heat injuries to the respiratory system death in the burn unit: sterile multiple organ failure pulmonary interstitial edema and hyaline membranes in adult burn patients herpesvirus infections in burn patients pulmonary herpes simplex in burns patients long-term course of bronchiectasis and bronchiolitis obliterans as late complication of smoke inhalation disaster at bhopal: the accident, early findings and respiratory health outlook in those injured chronic lung inflammation in victims of toxic gas leak at bhopal respiratory morbidity years after the union carbide gas leak at bhopal: a cross sectional survey management of the effects of exposure to tear gas ingested toxic agents . boyd mr. evidence for the clara cell as a site of cytochrome p -dependent mixed-function oxidase activity in lung pulmonary histological appearances in fatal paraquat poisoning pulmonary damage due to paraquat poisoning through skin absorption paraquat induced pulmonary fibrosis in three survivors kinetics and cellular localisation of putrescine uptake in human lung tissue enhancement of oxygen toxicity by the herbicide paraquat hypoxic protection in paraquat poisoning the effects of variable o tension and of exogenous superoxide dismutase on type ii pneumocytes exposed to paraquat paraquat-induced injury of type ii alveolar cells the changing pattern of paraquat poisoning in man remodeling of the alveolar structure in the paraquat lung of humans: a morphometric study experimental paraquat poisoning the pathogenesis and structure of paraquatinduced pulmonary fibrosis in rats pulmonary ultrastructure after oral and intravenous dosage of paraquat to rats patterns of pulmonary structural remodeling after experimental paraquat toxicity experimental models of interstitial pneumonia: paraquat, iprindole fine structure of the lung lesion in a case of paraquat poisoning fatal pulmonary intra-alveolar fibrosis after paraquat ingestion paraquat lung: a reappraisal an immunohistochemical study of the fibrosing process in paraquat lung injury toxic-allergic syndrome caused by ingestion of rapeseed oil denatured with aniline pathology of a new toxic syndrome caused by ingestion of adulterated oil in spain clinical epidemiology of toxic oil syndrome -(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome pulmonary hypertension due to toxic oil syndrome. a clinicopathologic study pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome pulmonary vascular lesions in the toxic oil syndrome in spain outbreak of bronchiolitis obliterans associated with consumption of sauropus androgynus in taiwan histopathological study of sauropus androgynus-associated constrictive bronchiolitis obliterans: a new cause of constrictive bronchiolitis obliterans sauropus androgynus-constrictive obliterative bronchitis/bronchiolitishistopathological study of pneumonectomy and biopsy specimens with emphasis on the inflammatory process and disease progression segmental necrosis of small bronchi after prolonged intakes of sauropus androgynus in taiwan sauropus bronchiolitis -reply the lipoid pneumonia of blackfat tobacco smokers in guyana a morphometric analysis of the male and female tracheal epithelium after experimental exposure to marijuana smoke differential examination of bronchoalveolar lavage cells in tobacco cigarette and marijuana smokers tracheobronchial changes in habitual, heavy smokers of marijuana with and without tobacco airway inflammation in young marijuana and tobacco smokers an unusual cause of patchy ground-glass opacity pulmonary hazards of smoking marijuana as compared with tobacco british lung foundation. a smoking gun? tracheobronchial histopathology in habitual smokers of cocaine, marijuana, and/or tobacco effects of cannabis on pulmonary structure, function and symptoms large lung bullae in marijuana smokers the pulmonary pathology of illicit drug and substance abuse bong lung: regular smokers of cannabis show relatively distinctive histologic changes that predispose to pneumothorax cannabis use and risk of lung cancer: a case-control study exogenous lipid pneumonia related to smoking weed oil following cadaveric renal transplantation a review of the respiratory effects of smoking cocaine medical complications of cocaine abuse blackened bronchoalveolar lavage fluid in crack smokers -a preliminary study pulmonary histopathology in cocaine abusers pulmonary complications of crack cocaine: a comprehensive review cocaine-induced churg-strauss vasculitis pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization crack lung: an acute pulmonary syndrome with a spectrum of clinical and histopathologic findings effects of 'crack' cocaine on pulmonary alveolar permeability pulmonary hemorrhage and antiglomerular basement membrane antibody-mediated glomerulonephritis after exposure to smoked cocaine (crack): a case report and review of the literature cellulose granulomas in the lungs of a cocaine sniffer intravenous injection of talc-containing drugs intended for oral use. a cause of pulmonary granulomatosis and pulmonary hypertension autopsy findings in drug addicts pulmonary 'mainline' granulomatosis: talcosis of intravenous methadone abuse self induced pulmonary granulomatosis. a consequence of intravenous injection of drugs intended for oral use the pulmonary vascular lesions of intravenous drug abuse microcrystalline cellulose pulmonary embolism and granulomatosis talc granulomatosis: laboratory findings similar to sarcoidosis postmortem findings of pulmonary lesions of older datum in intravenous drug addicts. a forensic-pathologic study recreational use of aminorex and pulmonary hypertension fatal drug reactions among medical inpatients drug-related hospital admissions: a review of australian studies published - incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies (see comments) the quality in australian health care study adverse reactions to drugs drug-induced pulmonary disease -an update an algorithm for the operational assessment of adverse drug reactions. ii. demonstration of reproducibility and validity aspirin idiosyncrasy and tolerance aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks; diagnostic and physiopathological implications mechanism of aspirin sensitivity bronchiolitis and bronchitis in connective tissue disease. a possible relationship to the use of penicillamine bronchiolitis obliterans in a patient with localized scleroderma treated with d-penicillamine constrictive bronchiolitis obliterans following gold therapy for psoriatic arthritis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease progressive airway obliteration in adults and its association with rheumatoid disease drug-induced infiltrative lung disease drug-induced and iatrogenic infiltrative lung disease interstitial lung disease associated with drug therapy pulmonary toxic effects of continuous desferrioxamine administration in acute iron poisoning interstitial pulmonary fibrosis following busulfan therapy busulphan lung report of a case and review of the literature busulfan lung: report of two cases and review of the literature a -year-old man with pulmonary infiltrates after a bone marrow transplantation -busulfan pneumonitis drug-induced pulmonary disease lung parenchymal injury induced by bleomycin pathology of high dose intermittent cyclophosphamide therapy cyclophosphamide pneumonitis pulmonary histopathologic changes associated with melphalan therapy melphalan-induced pulmonary interstitial fibrosis potentiation of bleomycin-induced lung injury by exposure to % oxygen factors influencing postoperative morbidity and mortality in patients treated with bleomycin bleomycin therapy and anaesthesia risk factors of anesthesia and surgery in bleomycin-treated patients the pathogenesis of bleomycin-induced pulmonary damage in mice experimentally induced bleomycin sulfate toxicity origin and significance of intranuclear tubular inclusions in type ii pulmonary alveolar epithelial cells of patients with bleomycin and busulfan toxicity late bcnu lung -a light and ultrastructural study on the delayed effect of bcnu on the lung parenchyma statin-induced fibrotic nonspecific interstitial pneumonia bronchiolitis obliterans organizing pneumonia during treatment with acebutolol and amiodarone methotrexate-induced pneumonitis (baltimore) bronchoalveolar lavage findings suggest an immunologic disorder pneumonitis complicating low-dose methotrexate therapy for rheumatoid arthritis -discrepancies between lung biopsy and bronchoalveolar lavage findings methotrexate pneumonitis: review of the literature and histopathological findings in nine patients the pulmonary toxicity of antineoplastic agents relationship of gold and penicillamine therapy to diffuse interstitial lung disease desquamative interstitial pneumonia following long-term nitrofurantoin therapy two unusual pathological reactions to nitrofurantoin: case reports bronchiolitis obliterans organising pneumonia associated with the use of nitrofurantoin amiodarone lung toxicity: a human and experimental study effect of amiodarone on the lung shown by polarized light microscopy fine structural alterations in the lungs of iprindole-treated rats effects of chlorphentermine on the rat lung amiodarone-induced hypersensitivity pneumonitis. evidence of an immunological cell-mediated mechanism amiodarone pneumonitis: three further cases with a review of published reports amiodarone-associated pulmonary toxicity. a clinical and pathologic study of eleven cases amiodarone lung: pathologic findings in clinically toxic patients amiodarone-induced bronchiolitis obliterans organizing pneumonia (boop) amiodarone-related pulmonary mass and unique membranous glomerulonephritis in a patient with valvular heart disease: diagnostic pitfall and new findings nodular amiodarone lung disease amiodarone pneumonitis: no safe dose amiodarone pneumonitis: no safe dose susceptibility to amiodarone-induced pulmonary toxicity: relationship to the uptake of amiodarone by isolated lung cells acute amiodarone-induced pulmonary toxicity following lung resection pulmonary alveolar proteinosis developing from desquamative interstitial pneumonia in long term toxicity studies of iprindole in the rat eosinophilic lung diseases mesalazine-induced eosinophilic pneumonia chronic eosinophilic pneumonia after radiation therapy for breast cancer churg-strauss syndrome in patients receiving montelukast as treatment for asthma inhaled corticosteroids and churg-strauss syndrome: a report of five cases pulmonary migratory infiltrates and pachypleuritis in a patient with crohn's disease immunodeficiency virus-infected patients receiving antiretroviral therapy ciprofloxacin-induced acute interstitial pneumonitis pulmonary granulomas after tumour necrosis factor alpha antagonist therapy pulmonary sarcoidosis developing during infliximab therapy sarcoid-like granulomatous disease following etanercept treatment for ra pneumonia due to liquid paraffin: with chemical analysis a case of chronic paraffin pneumonitis liquid paraffin pneumonia -with chemical analysis and electron microscopy paraffinoma confirmed by infrared spectrophotometry foreign body granulomata of the lungs due to liquid paraffin exogenous lipoid pneumonia due to nasal application of petroleum jelly reaction of human lungs to aspirated animal fat (ghee): a clinicopathological study clinical reactions following bronchography the reaction of pulmonary tissue to lipiodol experimental study of bronchographic media on lung a method for the identification of lipiodol in tissue sections of lungs, lipids, and lollipops bronchial necrosis and granuloma induced by the aspiration of a tablet of ferrous sulphate syndrome of iron pill aspiration pulmonary disease associated with l-tryptophan-induced eosinophilic myalgia syndrome. clinical and pathologic features a case of the eosinophilia-myalgia syndrome associated with use of an l-tryptophan product an investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use l-tryptophan and the eosinophiliamyalgia syndrome: pathologic findings in eight patients histopathologic features of the l-tryptophan-related eosinophilia-myalgia (fasciitis) syndrome -(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome immunemediated mechanisms and immune activation of fibroblasts in the pathogenesis of eosinophilia-myalgia syndrome induced by l-tryptophan pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome tryptophan-induced lung disease -an immunophenotypic, immunofluorescent, and electron microscopic study bronchiolitis obliterans organizing pneumonia associated with massive l-tryptophan ingestion pulmonary disease complicating intermittent therapy with methotrexate case records of the massachusetts general hospital. a -year-old man with increasing dyspnea, dry cough, and fever after chemotherapy for lymphoma interferon-alpha therapy associated with the development of sarcoidosis proliferation and differentiation in mammalian airway epithelium sarcoid-like pulmonary disorder in human review of the literature severe barium sulfate aspiration into the lung: clinical presentation, prognosis and therapy aminorex and the pulmonary circulation pulmonary hypertension and fenfluramine irreversible pulmonary hypertension after treatment with fenfluramine dietary pulmonary hypertension appetite-suppressant drugs and the risk of primary pulmonary hypertension fatal pulmonary hypertension associated with short-term use of fenfluramine and phentermine autopsy findings of heart and lungs in a patient with primary pulmonary hypertension associated with use of fenfluramine and phentermine pulmonary veno-occlusive disease following therapy for malignant neoplasms pulmonary veno-occlusive disease in an adult following bone marrow transplantation: case report and review of the literature indomethacin in the treatment of premature labor. effects on the fetal ductus arteriosus prenatal diagnosis of intrauterine premature closure of the ductus arteriosus following maternal diclofenac application roles and mechanisms of human immunodeficiency virus protease inhibitor ritonavir and other anti-human immunodeficiency virus drugs in endothelial dysfunction of porcine pulmonary arteries and human pulmonary artery endothelial cells pulmonary embolism after intravenous immunoglobulin fat embolism in infancy after intravenous fat infusions pulmonary fat accumulation after intralipid infusion in the preterm infant intralipid microemboli the pathogenesis of fat embolism pulmonary lipid emboli in association with long-term hyperalimentation the impact of intravenous fat emulsion administration in acute lung injury microvascular pulmonary emboli secondary to precipitated crystals in a patient receiving total parenteral nutrition -a case report and description of the high-resolution ct findings pulmonary complications following lymphangiography with a note on technique changes in pulmonary function due to lymphangiography pulmonary complications of lymphangiography respiratory distress syndrome from lymphangiography contrast medium spallation and migration of silicone from blood-pump tubing in patients on hemodialysis acute pneumonitis after subcutaneous injections of silicone in transsexual men pulmonary granulomas secondary to embolic prosthetic valve material pulmonary teflon granulomas following periurethral teflon injection for urinary incontinence acute pneumonitis after subcutaneous injections of silicone for augmentation mammaplasty a pathological study following bronchial artery embolization for haemoptysis in cystic fibrosis isobutyl- -cyanoacrylate pulmonary emboli associated with occlusive embolotherapy of cerebral arteriovenous malformations hemorragie alveolaire diffuse secondaire a l'utilisation d'anticoagulants oraux diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome intrapulmonary hemorrhage with anemia after lymphangiography alveolar hemorrhage as a complication of treatment with abciximab d-penicillamine induced goodpasture's syndrome in wilson's disease drugs and the pleura pleuropulmonary changes induced by ergoline drugs pleuropulmonary disease as a side-effect of treatment with bromcriptine pleuropulmonary disease due to pergolide use for restless legs syndrome fibrosis of the lung following roentgen-ray treatments for tumor radiation reaction in the lung radiation pneumonitis: experimental and pathologic observations radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors the pathogenesis of radiationinduced lung damage radiation pneumonitis: a review adult respiratory distress syndrome after limited thoracic radiotherapy migratory bronchiolitis obliterans organizing pneumonia after unilateral radiation therapy for breast carcinoma migratory organizing pneumonitis 'primed' by radiation therapy hamman-rich syndrome 'primed' by radiation? recall' pneumonitis: adriamycin potentiation of radiation pneumonitis in two children recall lung pneumonitis due to carmustine after radiotherapy pulmonary radiation injury mortality from cancer and other causes after radiotherapy for ankylosing spondylitis increased risk of lung cancer after breast cancer radiation therapy in cigarette smokers aortotracheal fistula secondary to bacterial aortitis respirator lung -a misnomer pathology of adult respiratory distress syndrome pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project lung injury caused by mechanical ventilation ventilator-induced lung injury oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes in vitro damage of rat lungs by oxygen metabolites intercellular adhesion molecule- contributes to pulmonary oxygen toxicity in mice -role of leukocytes revised intercellular adhesion molecule- expression on the alveolar epithelium and its modification by hyperoxia normobaric oxygen toxicity of the lung oxygen pneumonitis in man pathology of pulmonary oxygen toxicity diffuse alveolar damage -the role of oxygen, shock and related factors diffuse interstitial pulmonary fibrosis. pulmonary fibrosis in mice induced by treatment with butylated hydroxytoluene and oxygen potentiation of diffuse lung damage by oxygen: determining values resistance and susceptibility to oxygen toxicity by cell types of the gas-blood barrier of the rat lung ultrastructural observations on the development of the alveolar lesions extracorporeal membrane oxygenation for adult respiratory failure pulmonary pathology of patients treated with partial liquid ventilation disruption of the cd -cd ligand system prevents an oxygen-induced respiratory distress syndrome pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. ii ultrastructural and morphometric studies diffuse alveolar damage, respiratory failure and blood transfusion pulmonary injury -secondary to extracorporeal circulation fine structural changes in the lungs following cardiopulmonary bypass complement and the damaging effects of cardiopulmonary bypass acute lung injury during cardiopulmonary bypass: are the neutrophils responsible? inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies the pleuropulmonary manifestations of the postcardiac injury syndrome the postmyocardial infarction syndrome. the nonspecificity of the pulmonary manifestations the postcardiac injury syndromes antiheart antibodies following open heart surgery: incidence and correlation with postpericardiotomy syndrome analysis of the factors associated with radiofrequency ablation-induced pneumothorax irreversible intrapulmonary vascular changes after pulmonary vein stenosis complicating catheter ablation for atrial fibrillation pulmonary venous stenosis after treatment for atrial fibrillation right sided infective endocarditis as a consequence of flow directed pulmonary artery catheterisation complications and consequences of endotracheal intubation and tracheotomy. a prospective study of critically ill adult patients pathologic changes of the trachea after percutaneous dilatational tracheotomy pseudomonas aeruginosa respiratory tract infections in patients receiving mechanical ventilation cardiac arrhythmias resulting from tracheal suctioning obstructive fibrinous tracheal pseudomembrane -a potentially fatal complication of tracheal intubation -h pretreatment with methylprednisolone versus placebo for prevention of postextubation laryngeal oedema: a randomised double-blind trial tracheobronchomalacia in children necrotizing sialometaplasia (adenometaplasia) of the trachea severe complications of bronchoscopy the postpneumonectomy state evaluation of post-pneumonectomy space by computed tomography the postpneumonectomy space: factors influencing its obliteration hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice severe airway obstruction caused by mediastinal displacement after right pneumonectomy in a child. a case report postpneumonectomy syndrome: diagnosis, management, and results treatment of left pneumonectomy syndrome with an expandable endobronchial prosthesis postpneumonectomy syndrome: another twist acute lung injury and acute respiratory distress syndrome after pulmonary resection the mortality from acute respiratory distress syndrome after pulmonary resection is reducing: a -year single institutional experience prevalence and mortality of acute lung injury and ards after lung resection lung injury following pulmonary resection in the isolated, blood-perfused rat lung the pathogenesis of lung injury following pulmonary resection it is estimated that % of all hospital admissions are due to effects of therapeutic drugs, that - % of inpatients experience a drug reaction and that % of deaths in hospital may be related to drug therapy. [ ] [ ] [ ] [ ] the lungs are often involved in these adverse reactions. the mechanism of an adverse drug reaction may be based on:• overdosage: toxicity linked to excess dose, or impaired excretion, one classification of adverse drug reactions is that based upon the type of drug (box . . ). this is not adopted here but in passing it is worth noting that pharmacists are generally very helpful in supplying details of adverse reactions to specific drugs. alternatively, information on the long list of potentially pneumotoxic drugs may be obtained at http://www.pneumotox.com. a useful scheme for chapter key: cord- -dtbm ue authors: malbrain, manu l. n. g.; langer, thomas; annane, djillali; gattinoni, luciano; elbers, paul; hahn, robert g.; de laet, inneke; minini, andrea; wong, adrian; ince, can; muckart, david; mythen, monty; caironi, pietro; van regenmortel, niels title: intravenous fluid therapy in the perioperative and critical care setting: executive summary of the international fluid academy (ifa) date: - - journal: ann intensive care doi: . /s - - - sha: doc_id: cord_uid: dtbm ue intravenous fluid administration should be considered as any other pharmacological prescription. there are three main indications: resuscitation, replacement, and maintenance. moreover, the impact of fluid administration as drug diluent or to preserve catheter patency, i.e., fluid creep, should also be considered. as for antibiotics, intravenous fluid administration should follow the four ds: drug, dosing, duration, de-escalation. among crystalloids, balanced solutions limit acid–base alterations and chloride load and should be preferred, as this likely prevents renal dysfunction. among colloids, albumin, the only available natural colloid, may have beneficial effects. the last decade has seen growing interest in the potential harms related to fluid overloading. in the perioperative setting, appropriate fluid management that maintains adequate organ perfusion while limiting fluid administration should represent the standard of care. protocols including a restrictive continuous fluid administration alongside bolus administration to achieve hemodynamic targets have been proposed. a similar approach should be considered also for critically ill patients, in whom increased endothelial permeability makes this strategy more relevant. active de-escalation protocols may be necessary in a later phase. the r.o.s.e. conceptual model (resuscitation, optimization, stabilization, evacuation) summarizes accurately a dynamic approach to fluid therapy, maximizing benefits and minimizing harms. even in specific categories of critically ill patients, i.e., with trauma or burns, fluid therapy should be carefully applied, considering the importance of their specific aims; maintaining peripheral oxygen delivery, while avoiding the consequences of fluid overload. intravenous fluids have been in clinical use for over a century, yet the medical and scientific community have only recently begun to appreciate the importance of judicious fluid administration, the necessity to handle them as any other drug we prescribe [ ] [ ] [ ] [ ] , and the considerable side effects with which they may be associated [ , ] . three major indications exist for intravenous fluid administration [ , , [ ] [ ] [ ] : resuscitation, replacement, and maintenance. resuscitation fluids are used to correct an intravascular volume deficit or acute hypovolemia; replacement solutions are prescribed to correct existing or developing deficits that cannot be compensated by oral intake alone [ ] ; maintenance solutions are indicated in hemodynamically stable patients that are not able/allowed to drink water in order to cover their daily requirements of water and electrolytes [ , ] . in addition to these classical indications, the quantitative relevance of fluids administered as drug diluents and to guarantee catheter patency, the so-called fluid creep, has been recently underlined [ , ] . although the use of intravenous fluids is one of the most common interventions in medicine, the ideal fluid does not exist. in light of recent evidence, a reappraisal of how intravenous fluids should be used in the perioperative and critical care setting is warranted. here, we present the executive summary on this area of the international fluid academy (https ://www.fluid acade my.org). similarly to antibiotics, the ds of fluid therapy need to be considered (table ) [ ] . fluids are drugs with indications, contraindications, and side effects. different indications need different types of fluids, e.g., resuscitation fluids should focus on rapid restoration of circulating volume; replacement fluids must mimic the fluid that has been lost; maintenance fluids must deliver basic electrolytes and glucose for metabolic needs. the dose makes the poison, as stated by paracelsus. however, timing and administration rate are equally important for fluids [ , ] . of note, in contrast to most drugs, there is no standard therapeutic dose for fluids. the duration of fluid therapy is crucial and volume must be tapered when shock is resolved. however, while "starting triggers" for fluid resuscitation are quite clear, clinicians are less aware of "stopping triggers" of fluid resuscitation. the final step in fluid therapy is to withhold/withdraw fluids when they are no longer required, thus reducing the risk of fluid overload and related deleterious effects [ ] . intravenous "balanced" solutions include crystalloids and colloids with minimal effect on the homeostasis of the extracellular compartment, and in particular on acid-base equilibrium and electrolyte concentrations [ ] . in addition, the term "balanced" has been recently applied also to fluids with a low chloride content (cl − ). therefore, there are two main categories of balanced solutions ( table ): ( ) fluids causing a minimal effect on acid-base equilibrium, having an electrolyte content with an in vivo strong ion difference (sid), i.e., the sid after metabolism of the organic anion, close to - meq/l; ( ) fluids having a normal or sub-normal cl − content (cl − ≤ meq/l). according to the quantitative approach to acid-base equilibrium [ , ] , the three variables regulating the ph of biologic fluids independently are ( ) partial pressure of carbon dioxide (pco ); ( ) the concentration of non-volatile weak acids (a tot ); ( ) the strong ion difference (sid), defined as the difference between the sum of all strong cations and the sum of all strong anions [ ] . these principles clearly suggest that intravenous fluids may affect ph due to (i) the specific electrolyte content characterizing the solution, therefore altering the sid of the extracellular compartment and (ii) the dilution effect due to the volume infused, thus reducing the concentration of a tot [ ] [ ] [ ] . ideally, the fluid able to leave plasma ph unchanged after its administration, at constant pco , should balance these variations. recent studies clearly showed that, in this regard, the ideal balanced solution should have an in vivo sid equal to the baseline concentration of hco − [ ] . if the sid of the infused fluid is greater than plasma hco − , plasma ph will tend toward alkalosis; if the sid of the infused fluid is lower than plasma hco − , plasma ph will tend toward acidosis, as it is always the case for nacl . %, the so-called "normal" saline [ ] . as stated above, the definition of "balanced" solution includes also a category of iso-and near-isotonic fluids with a low cl − content (equal to or lower than meq/l), as compared to nacl . %. nonetheless, the final composition of such a fluid, especially when considering crystalloids, will depend on ( ) tonicity; ( ) electrical neutrality and ( ) sid. indeed, an isotonic balanced solution leaving unaltered acid-base equilibrium (i.e., with an sid close to meq/l) will necessarily have a cl − content > meq/l (as in sterofundin-iso). in contrast, a fluid with an sid of meq/l and a lower cl − content will necessarily be slightly hypotonic (as with lactated ringer's). finally, an isotonic fluid with a low cl − content will necessarily have a higher sid (as with plasmalyte), with a consequent alkalizing effect. [ , ] . on the other hand, in an experimental model of near-fatal hemorrhagic shock, a lower dose of balanced solution was needed, as compared to nacl . % to restore a target blood pressure [ ] . these conflicting results underline the fact that findings about fluid therapy are condition-specific, and that results obtained from septic patients or experimental models should not be extrapolated to all situations. despite these controversies, which need further clarification, several definitive differences exist between these two categories of drugs. first, chloride-rich nacl . % causes a higher dose-dependent degree of acidosis and hyperchloremia, which possibly favors the contraction of vascular smooth muscles [ , ] , potentially leading to a reduced renal perfusion. when healthy volunteers received l of either saline or plasma-lyte over h, saline significantly decreased renal artery blood velocity, decreased renal cortical tissue perfusion, decreased urine output, and increased extravascular fluid accumulation compared with plasma-lyte [ ] . these findings may support the idea that hyperchloremia may cause increased tubule-glomerular feedback and decreased renal cortical perfusion [ ] . indeed, a large-scale propensity-matched observational analysis of u.s. insurance data showed that the use of plasmalyte ® versus nacl . % on the first day of major abdominal surgery led to significantly less renal failure requiring dialysis [ ] . in addition to the effect on renal perfusion, nacl . %, being slightly hypertonic, likely causes an increased incretion of arginine vasopressin. these two effects can conceivably contribute to the slower renal excretion of nacl . % as compared to balanced solutions [ , ] . indeed, more fluid will be retained in the interstitial space, with the consequent propensity to cause more edema [ , ] . however, it is not merely the renal function that could be deranged by high chloride concentrations; infusion of nacl . % can cause abdominal discomfort in healthy volunteers [ ] and a reduced gastric perfusion in elderly surgical patients [ ] . two important and large randomized controlled trials comparing the use of balanced solutions and normal saline have been published in the last years. the split in-vivo sid-all organic molecules contained in balanced solutions are strong anions. the resulting calculated sid (in vitro-sid) is equal to meq/l, due to electrical neutrality. once infused, the organic molecules are metabolized to co and water; the resulting in vivo-sid corresponds to the amount of organic anions metabolized a sterofundin-iso or ringerfundin b in vivo-sid of tetraspan reported in the table results from the sum of organic anions; of note, there is a discrepancy as compared to the sid calculated as the difference between inorganic cations and inorganic anions ( meq/l vs. meq/l). no study was the first multi-center double-blind randomized controlled trial performed on patients, comparing balanced and unbalanced fluids in intensive care units. it showed no significant difference in the main outcome, i.e., incidence of acute kidney injury [ ] . while providing a high level of evidence, this trial did not give a definitive answer. indeed, the median volume of study fluid was only l over days. moreover, both study groups had received a median volume of . - . l of plasmalyte within h prior to enrolment, therefore making it plausible that prior administration of plasmalyte counterbalanced the effects of low-dose nacl . %. the smart-trial was a large study performed in five intensive care units of a single academic center [ ] . a total of , patients were randomized to receive either nacl . % or a balanced solution (plasma-lyte a or lactated ringer's). in both groups, patients received an extremely small amount of fluids: a median of l from admission to day or discharge, whichever came first. despite the unexpectedly low volume of crystalloids, the authors found a small difference in the primary outcome, i.e., the incidence of major adverse kidney events within days (composite of death, new renal replacement therapy or persistent renal dysfunction) in favor of balance solutions. looking at the overall outcome, it is important to emphasize that there was no reduction of in-hospital mortality and that neither the incidence of renal replacement therapy ( . % vs. . %, p = . ) nor the incidence of persistent renal dysfunction ( . % vs. . %, p = . ) was statistically significant. a similar study performed by the same authors and published in the same issue of the new england journal of medicine, the salt-ed trial, found a similar difference in the incidence of major adverse kidney events in noncritically ill adults [ ] . in summary, we can avoid fluid-induced metabolic acidosis and excessive chloride loading simply using balanced solutions. there is increasing evidence that an excessive chloride administration may have a detrimental effect on renal function, even at low doses. therefore, the use of balanced solutions, particularly in patients that potentially need a significant amount of intravenous fluids, seems to be a reasonable pragmatic choice [ ] . on the contrary, saline may be an intuitive choice for patients with hypovolemic hyponatremia or hypochloremic metabolic alkalosis. in any other settings, the most important reason to choose nacl . % over balanced solutions is likely economic in nature. therefore, the patient's serum chloremia is an important factor to determine the appropriate type of fluids. albumin accounts for approximately % of the plasma protein content [ ] and is the main determinant of plasma oncotic pressure, playing a crucial role in the regulation of microvascular fluid dynamics [ , ] . normal plasma concentration of albumin ranges between and g/l, corresponding to approximately . - . mmol/l, and to an in vitro pressure of approximately . mmhg. in contrast, in vivo colloid-oncotic pressure is lower, since the permeability of the endothelial barrier to albumin is variable, even in healthy subjects. nonetheless, according to starling's law, oncotic pressure is the force counteracting intravascular hydrostatic pressure, therefore acting to reabsorb water and small solutes from the interstitium to the intravascular space. the crucial role of albumin's oncotic property in the regulation of microcirculatory fluid dynamics also seems to apply to the endothelial glycocalyx layer [ , ] . this gel-like layer, lining the luminal side of the endothelium, is thought to comprise % of the intravascular volume. the current view of the glycocalyx is that it holds many compounds that are mandatory for the functioning of the endothelium and mediates several key physiological processes, such as maintaining the vascular barrier, hemostasis, prevention of cell adhesion to the endothelium and transmission of shear stress [ ] . the role of the glycocalyx is however under continuous investigation and its role and function might need to be revised in the future [ ] . of note, shedding of the glycocalyx occurs in the presence of reactive oxygen species, hyperglycemia, cytokines, and endotoxin, and is therefore common in critically ill patients [ ] . in the context of fluid homeostasis, loss of barrier function induced by glycocalyx shedding is associated with the formation of edema [ ] . furthermore, fluid therapy itself is known to be potentially deleterious for endothelial function [ ] , likely because of the resulting oxidative stress. however, the risks probably relate to the specific clinical context. indeed, while volume loading did not cause glycocalyx shedding in surgical patients and healthy volunteers [ , ] , the amount of glycocalyx shedding was proportional to the volume of fluid given in septic shock patients [ ] . the albios study, a large italian randomized controlled trial, gave some suggestions on whether or not albumin administration improves outcomes in severe sepsis and septic shock [ ] . patients with severe sepsis were randomized to receive either % albumin and crystalloids or crystalloids alone after initial early goal-directed resuscitation. in patients randomized to albumin treatment, albumin was supplemented for days, to maintain an albumin concentration ≥ g/l. despite some beneficial physiologic effects (lower heart rates, higher mean arterial pressure, and lower daily net positive fluid balance over the first days), no difference was observed either in mortality at days ( . % vs. . %) or in overall organ failure scores. however, when analyzing the results according to disease severity, patients with septic shock randomized to albumin supplementation showed a lower risk of death (relative risk . ; % confidence interval-ci . - . ) as compared to those just receiving only crystalloids. it is worth mentioning that this trial did not utilize albumin as a resuscitation fluid, but as a drug to correct hypoalbuminemia. colloids should remain in the intravascular space longer than crystalloids, provided that the endothelial barrier is intact, which is often not the case in critically ill patients [ ] . given the recent discussion on the potential adverse effects of artificial colloids, especially of hydroxyethyl starches (hes), a renewed interest in the use of albumin has emerged. however, despite the strong physiologic rationale and significant scientific effort [ , ] , to date, no randomized controlled trial has shown any significant benefit of fluid resuscitation using albumin over other types of fluids, including crystalloids [ ] . some reports have even suggested that albumin administration in the setting of cardiac surgery may be associated with the development of acute kidney injury [ ] . as stated previously, one of the largest albumin trials to date, the albios study, reported a reduction in -day mortality in a subgroup of patients with septic shock. however, this result was based on a post-hoc rather than predefined analysis and should, therefore, be interpreted with caution. the results of two ongoing randomized trials, the albumin italian outcome septic shock-balanced trial (albioss-balanced) and the albumin replacement therapy in septic shock (ariss), may provide some answers to the above-mentioned issues. the significant cost and the availability of equally effective low-cost alternatives do not play in favor of albumin, although a subgroup analysis of the albios dataset may suggest that albumin infusion is likely cost-effective in patients' septic shock [ ] . up to date, the theoretical benefits of albumin are not supported by sound clinical evidence, and the case for albumin remains controversial. the aim of perioperative fluid therapy, in parallel with the maintenance of the effective circulating blood volume, is to avoid both fluid overload and under-hydration, while maintaining patients' fluid balance as close as possible to zero. despite this rationale, it is not unusual for surgical patients to receive - l of fluid and - mmol of sodium, leading to edema and adverse outcomes [ ] , which is also favored by the marked and mean arterial pressure-dependent reduction of the elimination capacity of crystalloids [ , ] . on the other hand, overnight fasting and bowel preparation, when traditionally applied, lead to fluid deficits. apparently, patients develop postoperative complications when fluid retention exceeds . l [ , ] . of course, fluid gain depends not only on the amount of fluid administered, but also on the capacity of the kidney to excrete the excessive fluid and salt [ ] . fluid therapy is not only meant to compensate intraoperative losses but should also take into account those occurring prior to surgery, induced by poor water intake, bowel preparation, major inflammation associated with a stress response, and possibly, hemorrhage. dehydration, however, is difficult to detect through clinical methods. many studies examined whether a fluid load is capable of reducing hypotension caused by the induction of general/regional anesthesia. however, results regarding a preload strategy have been discouraging [ , ] . in response to the ongoing administration of large volumes of crystalloid to patients undergoing major surgery, a 'fluid restrictive' strategy has been proposed. for example, brandstrup et al. demonstrated in a multicenter randomized controlled trial that a more restrictive regimen was associated with better outcomes following colorectal surgery [ ] . however, the regimen was restrictive compared with the standard of care that was excessive (e.g., l positive balance due to high crystalloid volumes) [ ] . it is therefore conceivable that the group with a better outcome rather benefitted from the avoidance of fluid excess than from fluid restriction. the interpretation of the literature on the topic is hampered by the use of very heterogeneous definitions [ ] . what is however clear from observational studies is that both too much and too little fluid are associated with poor outcomes [ ] [ ] [ ] [ ] . recently, a large cohort study from u.s. hospitals including adult patients having colon, rectal or primary hip or knee surgery was concluded [ ] . a significant association was found between liberal fluid administration on the day of surgery and worse outcomes (increased total costs and length of stay in all patients), as well as increased presence of postoperative ileus, in patients undergoing colorectal surgery. interestingly, the authors also observed that restrictive fluid utilization (the lowest % by volume) was also associated with worse outcomes. it is common in enhanced recovery after surgery (eras) protocols to find the term "intraoperative fluid restriction" [ ] . however, alternative terms, such as "zero balance" or the avoidance of salt and water excess, are also available. protocols advocate the infusion of balanced crystalloid of - ml/kg/h and to give additional boluses of fluid only to match needs judged by either measured volumes lost during surgery, or the assessment of peripheral perfusion (such as according to the so-called 'goal-directed fluid restriction') [ ] . overall, the literature suggests that algorithm-based perioperative fluid regimens result in improved patient outcomes. fluid management in postoperative patients is a key determinant of their outcomes. while restoring effective volume is critical for these patients, fluid management should not compromise healing processes. optimal fluid management should thus target efficient central hemodynamics and tissue perfusion while avoiding positive net fluid balance. in theory, colloids offer the advantages over crystalloids of higher plasma expansion capacity and longer plasma half-life. they have the theoretical disadvantage of delaying clotting time and increasing the risk of kidney injury. in randomized trials, the ratio of the cumulative dose of colloids over the cumulative dose of crystalloids ranged roughly from . to [ ] . in patients with overt clinical hypovolemia, colloids were superior to crystalloids in improving cardiac filling pressures and performance [ ] . likewise, in a large multinational randomized trial performed in critically ill patients with acute hypovolemia, colloids reduced vasopressor and ventilator dependency when compared to crystalloids [ ] . a recent systematic review of resuscitation with hes in surgical critically ill patients identified randomized trials [ ] . however, this review found no statistically significant difference between hes and crystalloids, in terms of mortality (risk ratio . ; % ci . to . ; i = %), need for renal replacement therapy (risk ratio . ; % ci . to . ; i = %), and major infectious complications (risk ratio . ; % ci . to . ; i = %). it is worth mentioning that eligible trials were too small to draw firm conclusions on this issue. it should also be stated that there are opposing views regarding the use of starches [ ] . for example, several criticisms regarding the chest trial have been put forward which still require to be addressed [ , ] . furthermore, it can be stated that in the chest trial starches were administered to patients that were not hypovolemic. on the other hand, the cristal trial (where % of the colloid group received hes) concluded that significantly less volume was required to achieve hemodynamic stability for hes vs. nacl in the initial phase of fluid resuscitation in severe sepsis patients without any difference for adverse events in both groups [ ] . taking these opposing views into consideration, the ongoing debate about the use of starches in hypovolemic critically ill patients still requires more data. among patients undergoing major abdominal surgery, the recent results of the flash trial, showed no significant difference in a composite outcome of death or major postoperative complications within days after surgery [ ] . pending the results of ongoing trials, there are currently insufficient data to ban the use of colloids in the surgical intensive care unit. many patients undergoing surgery are not able to ingest food or fluids for some time following surgery and will require maintenance fluids. recently, a debate emerged on the tonicity of these solutions: although guidelines traditionally recommended the use of hypotonic maintenance fluids, in pediatric literature, these were shown to be associated with an increased incidence of symptomatic hyponatremia [ , ] . the recent randomized controlled topmast trial in adults undergoing major thoracic surgery found this problem to be mild in these patients. isotonic maintenance fluids, on the other hand, were associated with a considerably larger positive cumulative fluid balance (estimated at . l more positive under fluids containing compared to mmol/l of sodium) [ ] . the problem with fluid overload in the perioperative setting a certain degree of hypervolemia is necessary to maintain organ perfusion during anesthesia and surgery. however, fluid given after the induction of anesthesia mainly increases "unstressed" blood volume, because vasodilatation occurs as a consequence of anesthesia. at this point, additional fluid administration is needed to optimize stroke volume, i.e., to add to the "stressed" intravascular volume [ ] . many clinicians still consider this "wet" approach as the gold standard for intraoperative fluid therapy [ ] , although intravascular volume expansion certainly bears some dangers. myocardial work and cardiac pressures increase when infused fluids have exceeded the degree of anesthesia-induced vasodilatation. moreover, fluid overload reduces the colloid osmotic pressure that, together with raised cardiac pressures, might promote pulmonary edema [ ] . these issues are of particular relevance in patients with poor cardiovascular status. finally, hypervolemia may be responsible for another important effect: the release of atrial natriuretic peptides (anps) to the circulation caused by the stretching of atrial myocardial fibers [ , ] . indeed, in response to a rapid infusion of crystalloids, anp levels increase -to -fold [ ] [ ] [ ] , therefore reducing strain on the circulation by promoting natriuresis and capillary leakage of albumin. fluid administration is one of the cornerstones of hemodynamic resuscitation in critically ill patients. how much fluid to give has been the subject of lively debate over the years. too much fluid can have harmful consequences on multiple organ systems, e.g., worsening gas exchange, renal function and wound healing. fluid overload is particularly likely to arise in conditions when capillary permeability is altered due to an inflammatory response, such as during sepsis. a positive fluid balance has been associated with worse outcomes in several studies in various groups of intensive care unit (icu) patients [ , [ ] [ ] [ ] . in patients with septic shock, fluid administration and positive fluid balance were independently associated with increased mortality rates [ , ] . similarly, in patients admitted to the icu after major surgery, fluid balance was an independent risk factor for death [ ] . indeed, a multimodal restrictive fluid strategy aiming for negative fluid balance (pal-treatment) in patients with acute lung injury (ali) was associated with improved outcomes in a retrospective study [ ] . it has to be acknowledged that a positive fluid balance could be a marker of disease rather than a pure iatrogenic or preventable problem and it would be erroneous to assume the default position of underresuscitation. indeed, inadequate resuscitation due to insufficient fluid administration may result in poorer tissue perfusion and hence organ dysfunction and failure, particularly in the early phase of treatment. a balance needs to be achieved, such that each patient receives sufficient, but not excessive, fluid for her/his needs. crucially, different patients will have different needs and baseline fluid status depending on multiple factors including age, co-morbid disease and current diagnosis. in addition, it is mandatory to consider indices of fluid tolerance, such as cvp, lung water, oxygenation and hemoglobin levels. fluid requirements vary during the course of illness. as such, fluids must be prescribed on an individual patient basis; the prescription should be regularly reviewed and tailored to the evolving clinical stage. the answer to the question of whether fluid overload is a problem in the icu will thus depend on when it is asked. in the acute resuscitation/ salvage phase, fluid administration is generous. while fluid overload should always be a concern, a positive fluid balance is a specific target of this phase. the term deresuscitation/de-escalation was first suggested in [ ] and finally coined in [ ] . it specifically refers to 'late goal-directed fluid removal' , which involves "aggressive and active fluid removal through diuretics and renal replacement therapy with net ultrafiltration". deresuscitation/de-escalation is sometimes also used to more loosely refer to the phase of critical illness and/or the care of a critically ill patient, after initial resuscitation, stabilization, and optimization. it is characterized by the discontinuation of invasive therapies and a reduction of a spurious fluid balance. late conservative fluid management is defined as consecutive days of negative fluid balance within the first week of icu stay, and is an independent predictor of survival in icu patients [ ] . fluid overload and a positive cumulative fluid balance are associated with increased morbidity and worse outcomes, as previously discussed. the natural course of events after a first insult (such as infection, trauma, etc.) is a systemic inflammatory response with increased capillary permeability and organ dysfunction [ ] . the presence of fluid overload and interstitial edema may thus trigger a vicious cycle. this is what has been referred to as the ebb phase of shock [ ] . in the majority of patients, shock reversal occurs (with correct antibiotics and proper source control) and excess fluids can be mobilized: this is called the flow phase [ ] . however, some patients will not transfer spontaneously from the ebb to flow phase and will remain in a state of unresolved shock with positive cumulative fluid balance, and this is where active deresuscitation/de-escalation might have an important role. it is unclear which is the best therapeutic option for deresuscitation/de-escalation. the administration of albumin in combination with diuretics ( % albumin to achieve a serum albumin levels of g/l and furosemide bolus of mg followed by continuous infusion of mg/h) and the association of this strategy with the sequential application of peep set to counteract intraabdominal pressure (iap) have been proposed [ ] . in addition, renal replacement therapy and aggressive ultrafiltration can be used to achieve a negative fluid balance in selected patients [ ] . when it comes to deresuscitation/de-escalation, it is important to decide on when, how and for how long. for this purpose, we need to use the right targets to reach our goals. "over-deresuscitation" has its drawbacks and may cause neurologic dysfunction in the long run [ ] . in conclusion, it is crucial to ensure that the indication for fluid resuscitation no longer exists (e.g., absence of vasopressor, no lactate, adequate venous oxygen saturation of hemoglobin) before starting with deresuscitation. furthermore, the steps of deresuscitation/de-escalation need to be kept in mind: ( ) define a clinical endpoint (e.g., improvement in oxygenation); ( ) set a fluid balance goal (e.g., l negative balance in h); ( ) set perfusion and renal safety precautions (e.g., vasopressor need, % serum creatinine increase); ( ) re-evaluate after h unless safety limits reached; ( ) adjust the plan accordingly. two articles were published recently, almost simultaneously, referring to the dynamics of fluid therapy [ , ] . these conceptual models identified four dynamic phases. the acute dialysis quality initiative (adqi) group proposed s.o.s.d. (salvage, optimization, stabilization, de-escalation) as acronym [ ] . however, during the international fluid academy day (ifad) meetings there was a clear preference for the r.o.s.e. acronym (resuscitation, optimization, stabilization, evacuation) as summarized below, in fig. and table . we tried to suggest endpoints and targets for the different phases; however, it was decided not to include them because there cannot be a specific target of cardiac index and ppv must be considered only if cardiac output is low. a high ppv is often a physiological state and defining a "normal" state when a low ppv value is reached might lead to unnecessary fluid infusion [ ] . also, defining a given preload level as a target of resuscitation is senseless as it may shift from patient to patient and from time to time. in the first, salvage/resuscitation phase, when a patient presents with hemodynamic shock, the aim of the treatment is resuscitation and correction of shock with the achievement of an adequate perfusion pressure. a rapid fluid bolus should be given (although the exact amount can vary, usually - ml/kg given over to min and repeated when necessary), normally in association with vasopressor administration. in parallel, emergency procedures to resolve any obvious underlying cause should be performed, with hemodynamic monitoring initiated. in this phase, the goal is early adequate goal-directed fluid management: fluid balance must be positive. we do not support blind adherence to the surviving sepsis campaign guidelines adagio to administer ml/kg of fluids within the first hour for all patients, as explained previously [ ] . this may lead to either over-or under resuscitation in some patients. every patient needs an individual and personalized approach. the optimization phase starts when the patient is no longer in overt absolute/relative hypovolemia, but remains hemodynamically unstable. some form of monitoring will by now be in place. fluids should be administered according to individual needs, reassessed on a regular basis, e.g., using fluid challenge techniques [ , ] . fluid challenges must be conducted carefully, bearing in mind the four essential components (trol): type of fluid (e.g., a balanced crystalloid-like plasmalyte); rate (e - ml over min); objective (e.g., normal arterial pressure or heart rate); and limits (e.g., high central venous pressure level) (fig. ) [ ] . the aim of this phase is to optimize and maintain adequate tissue perfusion and oxygenation in order to prevent and limit organ damage. the patient must be carefully monitored during the optimization phase: often several types of monitoring (e.g., arterial catheter, echocardiography, central venous pressure, arteriovenous blood gas) are required to obtain the most complete picture of a patient's hemodynamic status. although a resuscitation based on microcirculatory endpoints is expected to result in analogous amelioration in the microcirculation, a lack of coherence may exist between macro-and microcirculation. thus, markers of hypoperfusion should include also lactate, prolonged capillary refill time and mottling score [ ] . once the patient is stable, the stabilization phase begins and evolves over days. in this phase, the aim of fluid management is to ensure water and electrolytes to replace ongoing losses and provide organ support. the target should be a zero or slightly negative fluid balance. it might be of interest, in this context, to underline the weeks fact that in the major trials suggesting a harmful effect of starches [ , ] , these colloids were given abundantly also in the stabilization phase, i.e., in a phase that possibly did not require these drugs. the final phase is evacuation or de-escalation, with the purpose of removing excessive fluid. this will be frequently achieved by spontaneous diuresis as the patient recovers, although ultrafiltration or diuretics might be necessary. of note, it was recently shown that diuretics might favor the recruitment of microcirculation, thus decreasing diffusion distances and improving oxygen extraction [ ] . fluid management in acute hemorrhagic shock following trauma although traumatic brain injury remains the commonest cause of death following severe blunt injury, concomitant major hemorrhage will result in cerebral hypoperfusion, which undoubtedly contributes to secondary brain injury and death. as such, hemorrhage remains the most preventable cause of trauma mortality. an adequate intravascular volume, hemoglobin concentration and oxygen saturation are essential to maintain aerobic metabolism. humans do not tolerate anaerobic metabolism and % of oxygen consumption is used in the formation of adenosine triphosphate (atp), the major energy source for cell function. rapid reversal of anaerobic metabolism is imperative to restore atp and prevent irreversible cellular apoptosis and death [ ] . recognizing that hypovolemia is the consequence of hemorrhagic shock, past strategies utilized crystalloids to restore intravascular volume, followed by blood transfusion. crystalloids, however, do not carry oxygen, and oxygen delivery may only be enhanced by an adequate hemoglobin concentration. furthermore, major hemorrhage is accompanied by a unique coagulation disorder, the acute coagulopathy of trauma and shock (acots) [ ] , leading to poor clot formation, as a result of increased binding of thrombin to thrombomodulin and enhanced fibrinolysis. dilution of coagulation factors, acidosis, and hypothermia play a secondary role in this scenario. the approach to resuscitation must therefore be proactive and not reactive with the combined administration of packed red blood cells, plasma, platelets, and cryoprecipitate. the use of clear resuscitation fluids should be minimized. based on military experience, the recommended ratio of packed red blood cells to plasma and platelets should be : : . the endpoints for hemoglobin concentration of g/dl, a platelet count of > , , an inr < . and a fibrinogen concentration of > g/l cannot be generally recommended. in addition, the ionized calcium level should be > . mmol/l. while the above is a general recommendation, not all patients will require such an aggressive approach [ ] . indeed, over-zealous transfusion is associated with unwanted complications. the standard approach has been to use conventional laboratory coagulation testing to determine the need for component therapy. these, however, are performed at room temperature and do not reflect individual steps in coagulation. thromboelastometry has now been recognized as an essential tool to monitor coagulopathy in trauma [ ] . this device reflects the entire process of coagulation and can graphically determine the need for specific coagulation factors. unlike laboratory coagulation studies, modern thromboelastometry machines may be set to the patient's core temperature and accurately reflect the in vivo coagulation status. these instruments should be the standard of care in centers handling major trauma. following the crash- trial indicating the benefit of tranexamic acid given within h from injury, such treatment has been included in many protocols for major hemorrhage [ ] . in the presence of a sophisticated trauma system, the benefits are doubtful and further data are warranted [ ] . the understanding of burn shock pathophysiology and subsequent development of fluid resuscitation strategies resulted in dramatic outcome improvements in burn care during the last decades [ ] . however, while under-resuscitation has become rare in clinical practice, there is growing concern that over-resuscitation, leading to increased morbidity and mortality, has become more of an issue in burn care. in the late sixties of the previous century, baxter and shires developed their landmark formula at the parkland memorial hospital, which has lasted decades as the gold standard for fluid resuscitation in acute burn care across the world [ ] . the formula advocates ml crystalloids per kg per % of total body surface area for h, of which half is given during the first h. diuresis (target ml/kg/h) is used to guide the amount of intravenous fluids. during the second h of resuscitation, colloids are allowed, and resuscitation volume is adapted according to diuresis (with a gradual decrease if diuresis is adequate). however, over the last years, multiple centers have reported excess fluid administration [ , ] . this fluid excess often leads to "resuscitation morbidity", a group of complications linked to fluid overload, such as delayed wound healing, delayed recovery of gastrointestinal function (with ileus), pulmonary edema (due to capillary leak and increased extravascular lung water), limb compartment syndrome, orbital compartment syndrome, intra-abdominal hypertension and abdominal compartment syndrome leading to multiple organ failure [ ] [ ] [ ] . this discrepancy between the predicted and the administered fluid is known as "fluid creep", a term brought to life by basil pruitt [ ] . recommendations for fluid resuscitation in burns are listed in table . the most well-known adverse effect of nacl . % is hyperchloremic metabolic acidosis. given the large infusion volumes administered to burn patients, balanced solutions are preferred. indeed, since the beginning of burn resuscitation, most formulae advocate the use of balanced crystalloid solutions. of note, an observational study reported lower sequential organ failure assessment (sofa) scores in severely burned patients resuscitated with acetated ringer's [ ] . the use of colloids in the first h has been controversial since it was thought that the existing capillary leak would allow large molecules to leak out into the extravascular space and exert an osmotic pull increasing the formation of edema [ ] . in the last years, renewed interest in colloids has arisen during burn resuscitation, instigated by the awareness of morbidity related to resuscitation and fluid creep. until recently, the low molecular weight hes solutions were widely used as a resuscitation fluid in critically ill icu, surgery and burn patients. however, after large fluid trials, including the chest and s trials, showing increased mortality and a higher rate of renal replacement therapy have raised alarming conclusions regarding the safety of hes solutions, starches can no longer be used in burn injuries as recommended by the pharmacovigilance risk assessment committee (prac) [ , , ] . albumin is a natural plasma protein that contributes most to intravascular oncotic pressure in humans (see above). the most common solutions are %, % or % albumin. it is a relatively expensive solution and its availability may be limited in some countries. although albumin resuscitation has been used with some reservations, especially in the acute phase of burn resuscitation, trials provide promising data regarding the use of albumin as an adjunctive therapy in burn resuscitation [ , ] . similarly, hypertonic saline has been used for decades in burn resuscitation; theoretically, it expands the circulating volume by an intravascular water shift. proponents claim that this process will decrease tissue edema and will lower the rate of complications. this hypothesis, however, needs to be confirmed by further studies. consider the ps of fluid prescription as shown in fig. and tailor the iv fluids to the patient's need via individualized and personalized care (table ) [ ] . prescription safety can be summarized by the ' ds' principle as explained above [ ] : the bottom line is "give the right fluid in the right dose to the right patient at the right time" the prescription of fluid therapy is one of the most common medical acts in hospitalized patients but many of the aspects of this practice are surprisingly complex. it is time to introduce fluid stewardship in your icu. the physician should engage in writing a prescription that accounts for drug, dose, duration and whenever possible de-escalation. c pharmacy: the prescription is sent to the pharmacy and is checked for inconsistencies by the pharmacist to get a more holistic view. d preparation: the process by which the prescription is prepared and additions (e.g., electrolytes) made. e patient: the filled prescription goes back to the patient and fluid stewards should observe administration, response, and debrief to avoid fluid-induced harm, we recommend a careful evaluation of the chosen solution and a phase-wise approach to its administration, taking into account the clinical course of the disease or surgical procedure. fluids should be prescribed with the same care as any other drug and every effort should be made to avoid their unnecessary administration. c. replacement and redistribution if patients have ongoing abnormal losses or a complex redistribution problem, the fluid therapy is adjusted for all other sources of fluid and electrolyte losses (e.g., normal saline may be indicated in patients with metabolic alkalosis due to gastro-intestinal losses) d. fluid creep all sources of fluids administered need to be detailed: crystalloids, colloids, blood products, enteral and parenteral nutritional products, and oral intake (water, tea, soup, etc.) precise data on the concentrated electrolytes added to these fluids or administered separately need to be collected fluid creep is defined as the sum of the volumes of these electrolytes, the small volumes to keep venous lines open (saline or glucose %), and the total volume used as a vehicle for medication the following information is included in the iv fluid prescription: the type of fluid the rate of fluid infusion the volume or dose of fluid the iv fluid prescription is adapted to current electrolyte disorders and other sources of fluid intake patients have an iv fluid management plan, including a fluid and electrolyte prescription over the next h the prescription for a maintenance iv fluid only changes after a clinical exam, a change in dietary intake or evaluation of laboratory results fluid management before, during and after elective surgery hydroxyethyl starch / . versus ringer's acetate in severe sepsis intravenous balanced solutions: from physiology to clinical evidence it is time to consider the four d's of fluid management association between a chloride-liberal vs chloride-restrictive intravenous fluid administration strategy and kidney injury in critically ill adults resuscitation fluids national institute for h, care excellence guideline development g. intravenous fluid therapy for adults in hospital: summary of nice guidance intravenous fluid therapy for hospitalized and critically ill children: rationale, available drugs and possible side effects principles of fluid management and stewardship in septic shock: it is time to consider the four d's and the four phases of fluid therapy effect of isotonic versus hypotonic maintenance fluid therapy on urine output, fluid balance, and electrolyte homeostasis: a crossover study in fasting adult volunteers maintenance intravenous fluids in acutely ill patients maintenance fluid therapy and fluid creep impose more significant fluid, sodium, and chloride burdens than resuscitation fluids in critically ill patients: a retrospective study in a tertiary mixed icu population normal saline to dilute parenteral drugs and to keep catheters open is a major and preventable source of hypernatremia acquired in the intensive care unit mortality after fluid bolus in african children with severe infection unintended consequences: fluid resuscitation worsens shock in an ovine model of endotoxemia fluid overload, de-resuscitation, and outcomes in critically ill or injured patients: a systematic review with suggestions for clinical practice independent and dependent variables of acid-base control stewartìs textbook of acid-base lulucom electrolyte shifts across the artificial lung in patients on extracorporeal membrane oxygenation: interdependence between partial pressure of carbon dioxide and strong ion difference crystalloid strong ion difference determines metabolic acid-base change during in vitro hemodilution effects of intravenous solutions on acid-base equilibrium: from crystalloids to colloids and blood components in vivo conditioning of acid-base equilibrium by crystalloid solutions: an experimental study on pigs the rule regulating ph changes during crystalloid infusion rapid saline infusion produces hyperchloremic acidosis in patients undergoing gynecologic surgery kinetics of isotonic and hypertonic plasma volume expanders influences of red blood cell and platelet counts on the distribution and elimination of crystalloid fluid balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic shock and the effects on renal oxygenation, oxidative stress, and inflammation chloride regulates afferent arteriolar contraction in response to depolarization regulation of renal blood flow by plasma chloride acetate-buffered crystalloid fluids: current knowledge, a systematic review an acetate-buffered balanced crystalloid versus . % saline in patients with end-stage renal disease undergoing cadaveric renal transplantation: a prospective randomized controlled trial major complications, mortality, and resource utilization after open abdominal surgery: . % saline compared to plasma-lyte effect of volume loading with liter intravenous infusions of . % saline, % succinylated gelatine (gelofusine) and % hydroxyethyl starch (voluven) on blood volume and endocrine responses: a randomized, three-way crossover study in healthy volunteers ab)normal saline and physiological hartmann's solution: a randomized double-blind crossover study should chloride-rich crystalloids remain the mainstay of fluid resuscitation to prevent 'pre-renal' acute kidney injury?: con iatrogenic salt water drowning and the hazards of a high central venous pressure the effect of intravenous lactated ringer's solution versus . % sodium chloride solution on serum osmolality in human volunteers the effects of balanced versus saline-based hetastarch and crystalloid solutions on acid-base and electrolyte status and gastric mucosal perfusion in elderly surgical patients effect of a buffered crystalloid solution vs saline on acute kidney injury among patients in the intensive care unit: the split randomized clinical trial balanced crystalloids versus saline in critically ill adults balanced crystalloids versus saline in non-critically ill adults balanced crystalloid solutions human serum albumin: from bench to bedside the clinical use of albumin: the point of view of a specialist in intensive care albumin in critically ill patients: the ideal colloid? the endothelial glycocalyx: composition, functions, and visualization the role of endothelial surface glycocalyx in mechanosensing and transduction the structure and function of the endothelial glycocalyx layer glycocalyx degradation is independent of vascular barrier permeability increase in non-traumatic hemorrhagic shock in rats the endothelium in sepsis reactive oxygen species mediate modification of glycocalyx during ischemia-reperfusion injury long intravascular persistence of % albumin in postoperative patients minimal shedding of the glycocalyx layer during abdominal hysterectomy intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation albumin replacement in patients with severe sepsis or septic shock role of albumin, starches and gelatins versus crystalloids in volume resuscitation of critically ill patients impact of albumin compared to saline on organ function and mortality of patients with severe sepsis colloids versus crystalloids for fluid resuscitation in critically ill people albumin administration is associated with acute kidney injury in cardiac surgery: a propensity score analysis economic model of albumin infusion in septic shock: the emaiss study effects of intravenous fluid restriction on postoperative complications: comparison of two perioperative fluid regimens: a randomized assessor-blinded multicenter trial arterial pressure and the rate of elimination of crystalloid fluid volume kinetics of ringer's lactate solution in acute inflammatory disease fluids and gastrointestinal function is crystalloid preloading useful in spinal anaesthesia in the elderly? volume preloading is not essential to prevent spinal-induced hypotension at caesarean section variability in practice and factors predictive of total crystalloid administration during abdominal surgery: retrospective two-centre analysis a rational approach to perioperative fluid management perioperative fluid management: turning art to science perioperative fluid management: science, art or random chaos? importance of intraoperative oliguria during major abdominal surgery: findings of the restrictive versus liberal fluid therapy in major abdominal surgery trial perioperative fluid utilization variability and association with outcomes: considerations for enhanced recovery efforts in sample us surgical populations guidelines for perioperative care in elective colorectal surgery: enhanced recovery after surgery (eras((r))) society recommendations effect of a perioperative, cardiac output-guided hemodynamic therapy algorithm on outcomes following major gastrointestinal surgery: a randomized clinical trial and systematic review fluid resuscitation in sepsis: a systematic review and network meta-analysis greater cardiac response of colloid than saline fluid loading in septic and non-septic critically ill patients with clinical hypovolaemia effects of fluid resuscitation with colloids vs crystalloids on mortality in critically ill patients presenting with hypovolemic shock: the cristal randomized trial comparison of hydroxyethyl starch colloids with crystalloids for surgical patients: a systematic review and meta-analysis ema recommendation to suspend hes is hazardous data too important to share: do those who control the data control the message? the great fluid debate: methodology, physiology and appendicitis effect of hydroxyethyl starch vs saline for volume replacement therapy on death or postoperative complications among high-risk patients undergoing major abdominal surgery: the flash randomized clinical trial mmol/l of sodium versus mmol/l of sodium in maintenance intravenous fluid therapy for children in hospital (pims): a randomised controlled double-blind trial nice cg : intravenous fluid therapy in adults in hospital compared to mmol per liter of sodium in intravenous maintenance fluid therapy for adult patients undergoing major thoracic surgery (topmast): a single-center randomized controlled double-blind trial volume and its relationship to cardiac output and venous return fluid management and goal-directed therapy as an adjunct to enhanced recovery after surgery (eras) effect of elevated left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema the endothelial glycocalyx: the great luminal barrier plasma atrial natriuretic peptide concentration and renin activity during overhydration with . % glycine solution in conscious sheep changes in serum electrolyte and atrial natriuretic peptide concentrations, acid-base and haemodynamic status after rapid infusion of isotonic saline and ringer lactate solution in healthy volunteers population volume kinetics predicts retention of . % saline infused in awake and isoflurane-anesthetized volunteers a positive fluid balance is an independent prognostic factor in patients with sepsis positive fluid balance as a prognostic factor for mortality and acute kidney injury in severe sepsis and septic shock the effect of excess fluid balance on the mortality rate of surgical patients: a multicenter prospective study fluid administration in severe sepsis and septic shock, patterns and outcomes: an analysis of a large national database aiming for a negative fluid balance in patients with acute lung injury and increased intra-abdominal pressure: a pilot study looking at the effects of pal-treatment fluid management in critically ill patients: the role of extravascular lung water, abdominal hypertension, capillary leak, and fluid balance continuous veno-venous hemofiltration to adjust fluid volume excess in septic shock patients reduces intra-abdominal pressure the adult respiratory distress syndrome cognitive outcomes study: long-term neuropsychological function in survivors of acute lung injury four phases of intravenous fluid therapy: a conceptual model liberal versus restrictive fluid therapy in critically ill patients fluid challenges in intensive care: the fenice study: a global inception cohort study fluid challenge revisited let's give some fluid and see what happens" versus the "mini-fluid challenge effects of fluids on the macro-and microcirculations hydroxyethyl starch or saline for fluid resuscitation in intensive care recruitment of sublingual microcirculation using handheld incident dark field imaging as a routine measurement tool during the postoperative de-escalation phase-a pilot study in post icu cardiac surgery patients recovery of hepatocellular atp and "pericentral apoptosis" after hemorrhage and resuscitation acute coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and hyperfibrinolysis early and individualized goal-directed therapy for trauma-induced coagulopathy effects of tranexamic acid on death, vascular occlusive events, and blood transfusion in trauma patients with significant haemorrhage (crash- ): a randomised, placebo-controlled trial do all trauma patients benefit from tranexamic acid? an overview on fluid resuscitation and resuscitation endpoints in burns: past, present and future. part -historical background, resuscitation fluid and adjunctive treatment physiological response to crystalloid resuscitation of severe burns fluid creep: the pendulum hasn't swung back yet! a systematic review on intra-abdominal pressure in severely burned patients intra-abdominal hypertension and the abdominal compartment syndrome: updated consensus definitions and clinical practice guidelines from the world society of the abdominal compartment syndrome protection from excessive resuscitation: "pushing the pendulum back elevated orbital pressure: another untoward effect of massive resuscitation after burn injury safety of resuscitation with ringer's acetate solution in severe burn (voltrab)-an observational trial fluid volume and electrolyte changes of the early postburn period assessment of hemodynamic efficacy and safety of % hydroxyethyl starch / . vs. . % nacl fluid replacement in patients with severe sepsis: the crystmas study burn patient characteristics and outcomes following resuscitation with albumin colloid administration normalizes resuscitation ratio and ameliorates "fluid creep it is time for improved fluid stewardship publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations mlngm and nvr are co-founders of the international fluid academy (ifa). this open access article is endorsed by the ifa. the mission statement of the ifa is to foster education, promote research on fluid management and hemo- ds: drug-dose-duration-de-escalation; anp: atrial natriuretic peptide; atp: adenosine triphosphate; ci: confidence interval; eras: enhanced recovery after surgery; hes: hydroxyethyl starch; iap: intra-abdominal pressure; icu: intensive care unit; peep: positive end-expiratory pressure; rose: resuscitation-optimization-stabilization-evacuation; sid: strong ion difference; sofa: sequential organ failure assessment. mlngm wrote the concept. mlngm wrote first draft. all other authors reviewed and edited the manuscript. all authors read and approved the final manuscript. dr. manu malbrain is professor at the faculty of medicine and pharmacy at the vrije universiteit brussels (vub) and member of the executive committee of the abdominal compartment society, formerly known as the world society of abdominal compartment syndrome (https ://www.wsacs .org/). he is a cofounder, past-president and current treasurer of wsacs. he is a co-founder of the international fluid academy (ifa). not applicable. not applicable. not applicable. not applicable. key: cord- - q abusm authors: luna, beatriz; tervo-clemmens, brenden; calabro, finnegan j. title: considerations when characterizing adolescent neurocognitive development date: - - journal: biol psychiatry doi: . /j.biopsych. . . sha: doc_id: cord_uid: q abusm nan disentangle pandemic-related effects (e.g., due to stress, time out of school, etc) from agerelated developmental effects. cohort-sequential/accelerated longitudinal designs, which follow multiple cohorts with various starting ages, may be better able to isolate developmental and visit effects through the inclusion of cross-sectional effects. however, they may be more limited in the ability to leverage within-subject developmental effects, given the potential moderating effects of initial age. importantly, both designs have limitations in attrition and scan failures, which can introduce bias (e.g., over representing willing participants who may be more mature). we suggest investigators optimize data collection for their planned analyses and known sources of error. when the aim is to model subject-specific developmental trajectories, as in the neurodevelopment of individual differences, a single cohort design is optimal, but with consideration to confounding non-developmental visit effects. alternatively, group-level normative developmental changes and their physiological underpinnings may be better suited by an accelerated longitudinal design that can control for non-developmental visit effects. simulation studies can be particularly useful in modeling these effects prior to data collection, informing sample size and requisite power that can be tailored to study-specific factors (e.g., reliability of included measurements, predicted attrition rate). a growing number of large-scale big data collection efforts, in both the us (e.g., philadelphia neurodevelopmental cohort (pnc); pediatric imaging, neurocognition, and genetics (ping) study; national consortium on alcohol and neurodevelopment in adolescence (ncanda); adolescent brain cognitive development (abcd) study; lifespan human connectome project development (hcp-d)) and europe (e.g., neuroscience in psychiatry network (nspn), braintime; center for lifespan changes in brain and cognition (lcbc)), as well as multi-site aggregation of existing data (e.g., enigma consortium)) have the potential, for the first time, to provide a rigorous understanding of the replicability and effect sizes of various developmental neuroimaging outcomes. this is critically needed especially with the multiplecomparison thresholding techniques used in neuroimaging and the relatively low reliability of many functional measures. initial investigations in the abcd dataset have already delineated important implications in this regard, including effects of data collection site and scanner manufacturer ( ) . importantly, large datasets can also be leveraged to replicate findings regarding normative development and deviations associated with psychopathology ( ). modeling frameworks that allow for curvilinear longitudinal trajectories and age as a continuous variable, such as multi-level and structural equation models, have the ability to capture the uniqueness of the adolescent period. inverse forms of age (e.g., /age) are particularly important for modelling adolescence as they can capture fast growth during childhood, deceleration in adolescence, and stabilization in adulthood. in comparison, quadratic models provide the opportunity to identify peaks during adolescence. exploratory analyses that test several functional forms should balance model fit with model complexity and use information criteria (e.g., akaike information criterion (aic) / bayesian information criterion (bic)) for model selection. more recently, algorithmic approaches have been adopted for fitting non-linear developmental trajectories (e.g., general additive models). these approaches have many key advantages, including flexible and quantitatively-defined functional forms and permit the examination of age-periods of significant change ( ), which can delineate plasticity and growth that can inform predictive models for risk for psychopathology ( ) and opportunities for effective interventions, though they often require very large sample sizes. in addition, wellpowered models of normative development can be used as a template from which to assess impairment given the age of subjects ( ) . potential confounds in developmental neuroimaging studies are well-recognized including head motion, non-developmental visit effects, and missing data. thus, limiting artifacts during imaging acquisition and use of state-of-the-science approaches to identify and mitigate these effects in the acquired data (e.g., global signal regression, despiking, and template-based artifact removal) are critical. given that artifact removal techniques can also remove meaningful signal, it becomes important to characterize information loss that varies systematically with age (e.g., global signal regression ( )). after addressing artifacts in "pre-processing" stages, it is also important to test, report, and potentially control for any remaining associations with age/pubertal status. additionally, missing data needs to be integrated in analyses to ensure representative sampling. the interpretation of findings on adolescent brain development should always be informed by conceptual models of neurodevelopment, broader theories from the neuroscience literature, and in consideration of limitations of data. this is particularly important given our stillemerging field, which may lead to multiple interpretations that are critical to move towards increased transparency and reproducibility. for example, task-based fmri group differences showing lower bold activation in adolescents or clinical groups compared to adults or healthy populations could be due to limited regional engagement or alternatively, varied strategies, or compensatory processes ( ) . consideration of alternative interpretations, including behavioral performance (e.g., task difficulty, distinguishing between correct and error trials, response speed), and systems-level changes are thus critical. within this context, both exploratory and hypothesis driven designs are needed, but with clear rationale and predictions based on the current status of the literature and informed by open science approaches. critically, wellvalidated negative findings are as important as "positive results" in moving the field forward. a next step in developmental cognitive neuroscience is to characterize the physiological neural mechanisms underlying development. multimodal imaging approaches that concurrently assess multiple aspects of brain maturation, including those that animal and postmortem studies have shown undergo unique changes through puberty (myelination, neurotransmitters), can inform underlying developmental mechanisms ( ) . several acquisitions that go beyond structural, task-, and resting state-fmri, such as characterizing white matter microstructure (e.g, diffusion tensor imaging and magnetization transfer ratio), tissue iron as a marker for dopamine (e.g., quantitative susceptibility mapping, r ', and neurotransmitter systems (positron emission tomography, mr spectroscopy), can move our understanding of normative developmental mechanisms forward and inform the etiology of psychopathology and potential interventions. overall, methodological and analytical approaches that characterize developmental change are critical in characterizing adolescent development and informing normative neurocognitive growth and risk for psychopathology. notably, development through adolescence must be conceptualized in a nonlinear fashion characterizing the transition to adult-level trajectories and integrating the multiple and independent brain maturational mechanisms that underlie behavior and determine adult trajectories ( figure ). depiction of curvilinear trajectories from childhood through adolescence and into adulthood, including processes that increase (green) (e.g., myelination, cognitive control, and prefrontal gaba) or decrease (blue) (e.g., synaptic pruning, cortico-subcortical functional connectivity and prefrontal glutamate) and stabilize into adulthood or show unique peaks in adolescence (red) (e.g., cortico-subcortical connectivity, dopamine function, affective processes) ( ) . the age of adolescence identifying reproducible individual differences in childhood functional brain networks: an abcd study development of white matter microstructure and intrinsic functional connectivity between the amygdala and ventromedial prefrontal cortex: associations with anxiety and depression development of hippocampal-prefrontal cortex interactions through adolescence early cannabis use and neurocognitive risk: a prospective functional neuroimaging study. biological psychiatry: cognitive neuroscience and neuroimaging age-associated deviations of amygdala functional connectivity in youths with psychosis spectrum disorders: relevance to psychotic symptoms topography and behavioral relevance of the global signal in the human brain what has fmri told us about the development of cognitive control through adolescence? maturation of the human striatal dopamine system revealed by pet and quantitative mri adolescence as a neurobiological critical period for the development of higher-order cognition the authors are supported by the national institute of mental health: r mh , r mh , r mh , and the staunton farm foundation. the authors report no biomedical financial interests or potential conflicts of interest. key: cord- -xta e j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - - sha: doc_id: cord_uid: xta e j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, . mg/kg/d days) via minipumps. chronic iso increased mrna levels of ndpk c by . ± . -fold and its protein levels by . ± . -fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a . -fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~ %. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. , , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin- ', ', '-trimethylether in the human colon carcinoma cell line hct and the human hepatoma cell line hepg as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin- ', ', '-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h dcf-da assay in hct cells) decreased with increasing methyl groups in the b-ring. myricetin- ', ', '-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly arg variant of the β -adrenergic receptor (β ar) on receptor conformation we used β ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg -β ar did not show altered activation kinetics after prestimulation, the gly -β ar became slower compared to the initial stimulation suggesting that β ars possess a memory of previous activation. we then permeabilized β ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β ar variants did not display receptor memory, suggesting that the β ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna- or mirna- a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir- , which shows decreased levels in colon carcinoma. in contrast, while mir- a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir- a and identify the protooncogenic kinase pim- as a target of mir- a. pim- harbours a highly conserved mir- a binding site within its '-utr, and seed mutagenesis of this target sequence abolishes the mir- a-mediated downregulation of pim- . the knockdown of pim- by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir- a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna- through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk . likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir- a. this is due to the mir- a-mediated downregulation of pim- expression and resembles the pim- knockdown through rnai / pim- sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir- and mir- a may be promising mirnas in colon carcinoma therapy. md , a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md was also investigated. the potential of md to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v lung fibroblasts (v cells). v cells were treated with . µm, . µm and . µm md or the positive control, the direct mutagen -nitroquinoline-n-oxide (nqo, µm) for h. on day , mutants exhibiting loss of hprt function were selected with -thioguanine . whereas at µm chloroquine, a % decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, µm md were needed to observe a similar decrease ( %) in fluorescence intensity of eb. therefore, md is fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous -tg resistant mutants per colony-forming cells was ± . as expected, µm nqo caused a significant increase in the mutant frequency (mf, ± ) . in contrast, mf was not significantly affected by treatment with md at both noncytotoxic ( . µm: ± ) and cytotoxic ( . µm: ± ) concentrations. in conclusion, md is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md does not cause gene mutations in the hprt test. since current studies show that various metabolites of md are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca + entry or depolarisation. recently, we showed that trpm acts as a ca + -activated non-selective cation channel and critically determines the driving force for ca + influx in mast cells following fcεri-stimulation ( ) . in addition to trpm we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc , trpc , trpc , trpc and trpm . in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc , trpc , trpc , trpc , trpm , trpm and trpm . to identify the functional role of those trp channel proteins for mast cell activation we analysed ca + signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp- and flufenamic acid (ffa), but could not evoke a rise in the [ca + ]i in both pmc and bmmc. sphingosine phosphate and lysophosphatidylcholine, which were reported to activate trpc channels, induced only minor rise in [ca + ]i in bmmcs, respectively. here, we will present our analysis of ca + signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca + -dependent mast cell activation such as adenosine, endothelin- , substance p and compound / in bmmcs and pmcs derived from mouse lines with inactivation of trpc , trpc , trpc , trpc or trpc since specific antagonists are still lacking for these trp channels. the α a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α a-ar activation with very high temporal resolution, we took advantage of the previously described α a-ar flash/cfp sensor [ ] . the results indicate that in our system the efficacy of ne in eliciting α a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ ms. the ec values decrease in an exponential manner and arrive at ~ µm with a half-life of ~ ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [ ] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ ms and ~ , ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine- -phosphate (s p) is an immune modulator produced by sphingosine kinase (sphk ) and sphingosine kinase (sphk ) and de-phosphorylated or degraded irreversibly by s p phosphatases and a lyase, respectively. we recently showed that tlr -induced il- p is selectively counter regulated by sphk , s pr and its extracellular ligand s p. on the other hand, spiegel et al. have demonstrated that specific, sphk -dependent, binding of s p to traf enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s p. in a first approach we focused our investigations on sphk effects on both traf and traf stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr or cpg-tlr signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il- p secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf activated via tnf-alpha interacted with the traf pathway to reduce il- p . further series with dcs derived from sphk -deficient mice confirmed our former results that il- p in contrast to other cytokines is specifically sensitive to sphk -s p feedback, but did not change the effects of tnf-alpha on wt dcs il- p release. in comparison, cpg-tlr -induced il- p release reached only % of lps-induced il- p levels and was less sensitive to tnf-alpha costimulation. however, sphk -deficiency strongly augmented cpg-dependent il- p production. in ongoing experiments we started to analyze the details of traf /rip and traf /tak activation by ubiquitination blots in wt, sphk -and s plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. , besche v. , bros m. , li h. , handler n. , bauer f. , erker t. , behnke f. , mönch b. , förstermann u. , dirsch v. m. , werz o. , kleinert h. , pautz a. university of vienna department of pharmacognosy, althanstr. , wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin (sirt ) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld- cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna- function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p mapk activation or activity. in addition we have evidence that sirt is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety ( ) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude , homburg, germany tmem proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit to membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek cells. tmem -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura- revealed that the elevation of the cytosolic ca + concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem -deficient acinar cells. tmem is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek cells the tmem -eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem -eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem -eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) , ± , , n= using lysotracker ® dye). analysis of subcellular localization with independent tmem fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem -/mice. the small molecule bcl- /mcl- inhibitor tw- shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl- family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl- or bcl- , with high expression correlating to high risk phenotype. co-expression can be detected in approximately % and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl- and other proteins, but not or to a lesser extend mcl- , elucidated the importance of mcl- inhibition for cytotoxicity in nb. tw- is a small molecule inhibitor that showed almost equal affinity to bcl- and mcl- . to explore the effect of combined bcl- /mcl- inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr- , sy y and kelly) were treated with tw- and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw- . using sirna, we investigated the functional relevance of mcl- and bcl- in kelly cells. for in vitro cell viability we observed ic values of . ± . µmol/l. on treatment with µmol/l dose of tw- , all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl- as well as mcl- knockdown induced apoptosis in kelly cells. interestingly, tw- was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl- and mcl- using tw- exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl- /mcl- may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein (oscp ) and the organic anion transporting polypeptides oatp a and oatp c are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ the hepato-intestinal induction of the detoxifying enzymes cyp a and cyp a by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp a , cyp a is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp a promoter we demonstrate that the cyp a expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang (yy )-binding site from the cyp a promoter which occurred in haplorrhine primates. this yy site is conserved in cyp a , but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy binding site from promoters of the cyp a gene lineage during primate evolution may have enabled the utilization of cyp a both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp a and cyp a . serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap- ) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant d) . barlow s. harrington house, harrington road, brighton, bn re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of in a million of developing cancer during a lifetime. in , from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of . micrograms/kg of diet ( . ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of . ppb is equivalent to . micrograms/person per day, assuming that g of food and g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in , kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of . micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova ( mg/ml) was dissolved in sodium borate buffer ( . m, ph . ) . for chemical treatment various amounts ( - mg) of -fluoro- , dinitrobenzene (dnfb; sensitizer) or , -dichloro- -nitrobenzene(dcnb; non-sensitizer) was added and stirred for hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with µg/ml ova and blocked with % fcs in pbs. ova samples (native or conjugated; . - µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace % of ab to plate-bound ova (ic ) was calculated (minimum of n= independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic value, whereas mock treatment resulted in comparable ic values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. ´-deoxy-camp in human cell lines: another second messenger? beckert c. , hinz c. we have shown that ´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~ was observed. as a remarkable exception, in hl- promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than -fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek cells after hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek cells ac assays (mn + /forskolin-or mn + /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde and pde hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h r -h r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h r and h r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h r and h r. in order to analyze these signaling pathways, we established a cellular model using transfected hek cells which stably express recombinant mh r or mh r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca + concentration ([ca + ]i) in cells expressing either of both, mh r (pec = . ) or mh r (pec = . ). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h r-expressing cells were blocked by the h r antagonist mepyramine ([ca + ]i: pkb = . ) and those in the h r-expressing cells by the h r antagonist jnj ([ca + ]i: pkb = . ) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj , which behaves as a partial mh r agonist in the steady-state gtpase assay using membranes of infected sf cells, was without effect on [ca + ]i and forskolin-induced camp-accumulation in the mh r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h r and the h r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk / , p , jnk, creb, pkb (akt), and mkk / were detected in cells expressing either of both, mh r and mh r. in summary, the hek cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β regulatory subunit of voltage-activated calcium channels belkacemi a. cavβ subunits of voltage-activated ca + channels are required for trafficking the poreforming cavα subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih t fibroblasts do express cavβ and cavβ subunits, but we could not detect any voltage-activated ca + influx. whereas in mouse cardiomyocytes or hek cells coexpressing cavβ and cavα subunits a dihydropyridin-sensitive voltage-activated ca + influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca + influx. among the proteins potentially interacting with cavβ are the inositol , , -trisphosphate receptors (ip rs) [ , ] . we therefore coexpressed mouse cavβ and mouse ip r type , or in cos- cells and found coimmunoprecipitation of ip rs using an antibody for cavβ and vice versa. to study the release of ca + from ip -sensitive stores we performed fura- measurements on fibroblasts isolated from wild type and cavβ -deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par- , lpa or bradykinin. receptor-activated ca + release was more pronounced in β -deficient mefs and cfs, whereas thapsigargin-induced ca + release was the same in cells from both genotypes. in addition, ip production measured by a radioreceptor assay was already increased in β -deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β knockout may result, in part, from the increased amount of ip -releasable ca + . [ ] berggren, yang, murakami, et al., removal of ca channel β subunit enhances caoscillation frequency and insulin exocytosis. cell , ( ) - . [ ] müller, haupt, bildl, et al., quantitative proteomics of the cav channel nanoenvironments in the mammalian brain. pnas , ( ) - . bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat- -mediated arginine transport by pma (vina-vilaseca et al, j biol chem : ) . this mechanism requires nedd e ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u glioblastoma cells overexpressing hcat- a-egfp. in addition, sirna-mediated knock down of cdc prevented the decrease of hcat- amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase . by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp -dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap . whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap is a specific target of the kinase pink (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase . we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table ) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac , mac , mac , soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca + homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca + -channels and the expression of ca + -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation minipigs underwent ventricular pacing at beats/min (ddd mode) for one year. minipigs were paced from the rvfw and minipigs from the lva, respectively. minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca + currents. western-blots were carried out to investigate the expression of the ca +handling proteins l-type ca + -channel, serca and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca + -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca + -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca -expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca + -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd ) and irritation (oecd ) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp a, b and a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo / activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [ ] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [ ] or propanal (aldh) oxidation [ ] . nat activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [ ] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project ( d). signalling pathway indicates that in b cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about . µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to % of the tolerable weekly intake (twi) of . µg/kg bw for cd defined by the european food safety authority (efsa) in . beverages and vegetables are the food groups most relevant for exposure to pb. about . µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects ( . µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is . µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with %. for pb a weekly intake of µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα / and gαq via specific rhogefs. the p rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p rhogef and characterize the dynamics of p rhogef-gαq-interaction in single living cells. the fusion of p rhogef with venus resulted in a functional gαq-regulated p rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h and cholinergic m -recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p rhogef was verified by introducing point mutations rendering p rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p rhogef and gαq upon cotransfection of rgs , suggesting a very short lifetime of the p rhogef-gαq-complex or the ability of rgs to bind to p rhogef-associated gαq. taken together, fret-based imaging of the interactions between p rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs . toxicity of silver nanoparticles in intestinal cells boehmert l. the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco- is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco- cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of . nm stabilised with tween- und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco- cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine- -phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. , halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase a (pp a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n= ) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein (hsp ) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp protein was increased (p< . , n= - ) but not camkii (p> . ). protein phosphatase type (pp ) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p< . , . in contrast, pp protein was upregulated (p< . , in tg compared to wt. for comparison the regulatory a-subunit of pp a and hsp were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. , hassan m. there is increasing evidence that hydrogen sulfide (h s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e -related factor (nrf ) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy- -induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf may constitute a protective mechanism during glomerular inflammatory disease. rac knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. , düsseldorf, germany rac belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap . anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac may be involved in the ddr. to study the in vivo function of rac we used an inducible cre-based knockout mouse model (rac flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp mrna expression was reduced in the liver of rac -/animals as compared with rac proficient animals. in addition we found more apoptotic cells in rac -/mice. hours after treatment with the anthracycline derivative doxorubicin the number of gh ax foci in rac -/animals was reduced in comparison to rac +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac -/mice. none of these protective effects resulting from rac deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of days. there were no significant differences in the number of gh ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac -/animals. furthermore the number of mitotic events was almost two times higher in the rac -/mice compared to the rac +/+ mice. summarizing, our findings show that impaired hepatic expression of rac protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse , tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine t cells with tbhq in -well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf pathway. s angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type receptor brand s. , amann k. , schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacherstr. , würzburg, germany universität erlangen-nürnberg institut für pathologie, krankenhausstraße , erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c bl/ mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c bl/ mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c- t cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm , hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c- t cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c- t cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c- t cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c- t cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s . in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c- t cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac and ac being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac and ac share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac , as well as overexpression of ac both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from - week old homozygote ac knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine '-triphosphate (gtp) and '-o-( thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac - , gs-, gi-a and β -, β -ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac knockout, mrna expressions analysis of ac - , gs-, gi-a and β -, β -ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac . whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac and ac , or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk- / and lkb- in hypothalamic gt - cells breit a., ellen d., gudermann t. goethestrasse , münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin- receptor (mc r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt - cells) and monitored ampk phosphorylation at thr which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases- / (erk- / ), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk- / was blunted by pka inhibitors, we propose that erk- / serves as a link between pka and ampk in gt - cells. furthermore, down-regulation of liver kinase b- (lkb- ), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase- decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk- / and lkb- in gt - cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav . gene (cacna c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell ; : - ) . functionally similar biophysical effects can be induced by structural variation β -and β -subunits of the voltagedependent calcium channels (herzig et al., faseb j. ; : - ; jangsangthong et al., pflugers arch. ; : - ) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. ; : - ) , we are investigating a function-based candidate gene hypothesis linking the β subunit gene (cacnb ) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb in patients with asd. we found three rare missense mutations in asd patients, but not in unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav . subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav . . β subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. ( ) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta ( ) subunits. pflugers arch , - . herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. ( ) . mechanism of ca(v) . channel modulation by the amino terminus of cardiac beta -subunits. faseb j , - . splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. ( ) . ca(v) . calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell , - . trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. ( ) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] background: brain serotonin ( -ht) has been implicated in the regulation of food-intake. the ingestive effects of -ht are mediated by a number of different receptor subtypes under which the -ht a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by -ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic -ht a autoreceptors in the raphe nuclei or by postsynaptic -ht a heteroreceptors in serotonergic terminal structures. methods: the effect of the -ht a receptor agonist -oh-dpat ( . , . or . mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l mice. l mice are characterized by an overexpression of postsynaptic -ht a receptors. results: the administration of the -ht a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the -ht a receptor in food intake. further, we make the assumption that the hyperphagic effect of -oh-dpat is mediated by presynaptic -ht a autoreceptors in the raphe nuclei which decreases -ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic -ht a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc (mrp ) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) β on abcc mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc expression while inflammation. three different cell lines a) hepg cells (liver tissue) and b) caco (colon tissue) both without naïve il β expression and c) skhep cells representing physiological liver tissue with naive il β expression, were stimulated with different concentrations of il β (range pg/ml to ng/ml). over a period of h samples were taken at defined time points. abcc mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p mapk, akt, erk and jnk) we analysed the signal transduction pathways associated with il β-mediated transcriptional abcc regulation. on abcc mrna level an up-regulation in caco cells ( , fold) and hepg cells ( , fold) within the first hour after stimulation with ng/ml il β was shown. in contrast skhep cells demonstrated a decreased abcc mrna expression ( , - , fold) in comparison to unstimulated controls. the abcc protein expression exhibited a time and il β dependent regulation as well. the analysis for the signal transduction showed for p mapk a moderat time dependet down regulated phosphorylation ( %) in hepg cells whereas it showed no effect in caco cells. concluding, the expression of abcc is regulated moderately by il b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of micrornas. statistical analysis using the -∆ct method was performed. micrornas showing a p-value< . and a fold change> were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan , mirdb , pictar , microcosm , diana microt ), selected putative target genes were further functionally investigated by the david bioinformatics database . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target genes, especially transcription regulators ( %). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad , nras, rb , raf ). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators ( %), but also cellular transporters ( %, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta -adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. , buschauer a. activation of the β -adrenergic receptor (β ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [ ] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β ar ligands on cyclic adenosine ', '-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [ , ] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β ar antagonists in the camp assay and the o •assay are different. such differences were previously reported for β ar antagonists in other test systems [ ] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human -ht a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße , magdeburg, germany the serotonin a ( -ht a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of -ht a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of -ht a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged -ht a receptor by internalization. using immunocytochemical techniques in stably transfected hek cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the -ht a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized -ht a receptors colocalize with a -labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of -ht a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces -ht a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the -ht a internalization to occur. overall, we conclude that the internalization of the human -ht a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of -ht (a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of -ht a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung , kurt-georg-kiesinger-allee , bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma ). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma ) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/ ). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method . . ) and for atropine (ph. eur. method . . or . . ) based on lhrd leads in each case to a suggested fsd of d related to g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method . . or . . ) based on food legislation emerged a proposed fsd of d related to g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin- `, `, `-trimethylether in c. elegans büchter c. , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin- `, `, `-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin ( µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives ( µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf- , the nad + -dependent protein deacetylase sir- . and the heat-shock transcription factor hsf- , respectively. lifespan extension by myricetin disappeared in daf- and sir- . loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf- loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf- . in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf- and sir- . , probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee , ulm, germany activation of phospholipase c-γ (plcγ ) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology (sh ) domains and one src homology (sh ) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ is also regulated by autoinhibitory elements within its specific array (walliser et al., ; everett et al., ) . our recent data demonstrated that plcγ is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh (sh c) and the sh domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ . plcγ has been shown to be phosphorylated at tyrosine residues and upon bcr stimulation (kim et al., ) . both tyrosines are located in the linker between the sh c and the sh domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ . interestingly, a novel phosphorylation site in plcγ was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue (rikova et al., ) . in this work, we demonstrate, for the first time, the activation of plcγ by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ at tyr . molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. - , berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between µm and µm. at concentrations higher than µm the substance was cytotoxic to the cells (ic µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor α (hnf α) by incubating the cells already with moderate concentrations of pfoa at a level of about µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. , bumke scheer m. (kao et al., ) . two daughter cell lines were developed from m sv after x-ray irradiation (m sv r ) and an additional transfection with a mutated erbb oncogene (m sv r -n ), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., ) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. ( ) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h -receptor (h r) shows no ameliorating effects on asthmatic symptoms, h r antagonists may be new drugs for asthma therapy. in addition to the h r-and h rselective antagonist thioperamide, the selective h r antagonist -[( -chloro- h-indol- yl)carbonyl]- -methylperazine (jnj ) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj as well as the selective h r antagonist -({ - [ -(piperidin- -yl) propoxy]phenyl}methyl)piperidine (jnj ) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and . m znso using µl of plasma. analyte extraction from lung tissue was achieved by treating - mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp a mercury column ( x mm; µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph ) and methanol at a flow rate of . ml/min. the analytical run-time was minutes. for plasma samples the assay was linear over a concentration range of . - µg/ml for jnj and jnj , and . - µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of . - µg/sample, jnj and jnj in a range of . - µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk , one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk -dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk is already expressed during embryogenesis and we can detect it in the developing heart. however, grk has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk on cardiac signalling and development. tools for grk specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk may potentially serve as a candidate gene for congenital heart disease. identification of a hcn interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn - ) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn , hcn and hcn channels have been studied in quite some detail there is only little information on the particular role of the hcn channel. as an important step towards achieving a better understanding of hcn function we set out in this study to identify proteins that are assembled with hcn in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn using heterologous coexpression in hek cells. here, we provide an in-depth analysis of the functional interaction between hcn and one of the identified interacting proteins (hip . ). we show that hip . physically binds to hcn channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn and generated a series of tpc antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc knock-out model by our self-generated tpc antibodies. tpc knockout mice are viable and do not show any obvious deficits. to isolate tpc containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc . foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf- breast cancer cells and mcf- cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo overexpressing cells was a a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo -dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a is a direct target of foxo , a luciferase reporter containing a . kb of the a promoter was co-transfected with different amounts of foxo wild-type expression construct. foxo induced a dosedependent increase in a promoter activity, supporting the assumption that a is a direct transcriptional target of foxo . overexpression of foxo led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf- cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a , an ubiquitin modifying enzyme, as a novel foxo target gene. our data implicate that sustained foxo expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm is essential for motility, proliferation and cell growth. up-regulation of trpm function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm channel is inhibited by the known modulators of sk - channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns , ska , ucl . the most potent of these compounds, ns (ic . µm), interferes with the regulation of trpm by cytosolic mg + . here we show that ns ( µm) fully and reversibly inhibits native trpm -like currents in hek cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm currents by ns would impact cellular processes known to be affected by a genetic inactivation of trpm . we found that ns ( - µm) suppressed motility of hek cells without a detectable effect on cell viability. taken together, our findings indicate that ns is a potent and reversible inhibitor of endogenous trpm currents and may be a good candidate drug for pharmacological targeting of trpm . sulfur mustard ( , '-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance -chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed minutes after treatment with mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp- becomes activated upon treatment with mustards. parp- is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp- is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape and ercc , i.e. endonucleases involved in ber and ner, respectively. the reduction of ape expression had no effect on parp activity. surprisingly, the knockdown of ercc almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc -parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt- . expression and activity of g protein coupled receptor kinase (grk ) are elevated in several conditions of compromised heart function. although grk inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf but it also acts as a physiological inhibitor of grk upon phosphorylation by pkc at serine . a detailed understanding of the rkip/grk interaction may help to identify inhibitory compounds for grk . since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf -into a grk -inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk , we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk association. this implicates, that dimerization of rkip is essential to bind grk . to determine whether rkip dimers consequently inhibit grk activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk interaction and will help to develop specific grk inhibitors. expression and function of trpm ion channels in epithelial mdck cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. , homburg, germany trpm proteins build ca + permeable cation channels [ ] activated by steroids [ ] and sensitive to increased temperatures [ ] . trpm channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [ ] or as nociceptors of noxious heat, respectively [ ] . however, northern blots and in situ hybridization experiments revealed that trpm is also expressed in epithelial cells of the choroid plexus and the ciliary body [ ] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm is also expressed in madin-darby canine kidney (mdck) cells. quantitative analysis of trpm expression by qrt-pcr revealed a ~ fold upregulation in mdck cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm , a related ion channel described as regulator of proliferation in other cell types, remained constant. hek cells overexpressing trpm channels did not proliferate in the presence of the trpm agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca + imaging experiments using fura revealed that pregnenolone sulphate induces ca + entry in well separated mdck cells but not in cells growing in confluency. we hypothesize that trpm might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck cells. inhibition of grk by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase (grk ). grk initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk are upregulated in human heart failure, it has been proposed that grk inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r c ). pln r c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi and gαi with gαi as the predominant isoform. gene-targeted mice lacking either gαi or gαi were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi -deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi -deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi -deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi deficiency. to assess the pathophysiological consequences of platelet gαi function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi -deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi -deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi deficient mice were strongly protected from injury. thus we suggest that gαi and gαi play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog -br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase . anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk / and p phosphorylation, growth and migration of b melanoma cells. similar results were obtained with wm human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav . (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) , which is associated with absence epilepsy. cav . is one of the two t-type calcium channels known to be expressed in the rt . it has been demonstrated that ablation of either cav . or cav . reduces susceptibility to experimentally induced epilepsy ; and in addition that both channels share several pharmacological properties [ ] [ ] [ ] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav . (-|-), cav . (-|-) and cav . (-|-)xcav . (+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav . and cav . calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip /rip -dependent necroptosis in neuronal cells diemert s. , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip / -dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring , bldg. n, trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat enzyme activities after incubation of thp- cells with cyanamide for h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat protein inhibition was observed for and µm cyanamide using over-expressed human recombinant nat (insect cell cytosol containing recombinant human nat * ). however, no inhibition was found in the presence of recombinant nat * . as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat or nat cytosol, by thp- cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat . further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat , leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat , which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [ ] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca + influx and the subsequent contraction of pasmc [ ] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [ ] . trpc-expression in freshly isolated and passaged pasmc cultured in low ( %) and high fetal bovine serum ( %) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc and trpc the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc and inducing acute hpv in pasmc will be presented. references [ ] staub, n. c. ( ) . site of hypoxic pulmonary vasoconstriction, chest , s- s. [ ] weissmann, n. et al. ( ) . classical transient receptor potential channel (trpc ) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. , - trpv is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv is assumed to contribute to the maternal-fetal calcium transport. most probably trpv is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv protein in the placenta that might contribute to the regulation of the trpv protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv (aa - and - ) as trpv -gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv protein is calpain . in contrast to the classical calpains (calcium activated cystein proteases), calpain is unique in that it lacks the cysteine residue in the active site. calpain is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv and calpain was confirmed in reciprocal pulldown experiments and the trpv binding region for calpain was narrowed down by using overlapping n-terminal trpv -gst fusion proteins. after injection of trpv crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain crna. in further experiments we want to study potential regulatory effects of the trpv protein on the calpain function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. , münster, germany it has been demonstrated that chronic ingestion of - µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. , j toxicol. we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g /m-and s-phase arrest as well as subg peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg , caspase activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h o induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h o induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp- gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse , basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr /app/ps ) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of months, resulting in a depolarized mitochondrial membrane potential. at months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c /bj -thy -appsl (ad-mice) mice that were fed with mg olesoxime/kg feed for months. drug plasma levels reached approx. ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca + -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca + -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca + -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the th framework program for rtd -project mitotarget -grant agreement health-f - - . several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide ( µm) for min. thereafter, the cell lysates were subjected to diagonal d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf -keap -antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq . the lusens assay was in house validated with a panel of chemicals and cosmetic ingredients including the performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of % compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il- and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße , hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il- concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of . x eosinophils. we stimulated eosinophils with varying concentrations of il- , eotaxin- (ccl ), eotaxin- (ccl ), and eotaxin- (ccl ). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of - pm/ . x eosinophils. neither il ( - pg/ml), nor any of the eotaxins ( ng/ml either alone or in varying combinations) did stimulate anandamide production after min of incubation. our results suggest that chemotactic molecules like eotaxin and il- are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse , ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg or . however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. µl of test substance preparation are applied to the skin preparation. after h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above %. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p is an attractive target in cancer therapy and several approaches were pursued to restore p function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a and saos osteosarcoma cells. the internalized biotin-p also partially co-localized with early endosomes. importantly, the delivery of biotin-p into p -deficient saos cells resulted in impaired cell viability and upregulation of caspase / activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to , , , tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb . activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb . thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a ( , ) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb . already h after treatment, genes were found to be upregulated and genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch- and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq as a novel ahr target protein in rat liver oval cells. although the function of foxq is poorly understood, it has been shown very recently that foxq is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h and h receptors in the brain can be easily deduced since h receptor antagonists of the first generation have a sedative effect and an inverse h agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h and h receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h agonist impromidine µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was %, the pec was . and the pa of the h antagonist ranitidine against impromidine was . . with respect to h receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h agonist -methylhistamine - µm but inhibited by histamine µm via h receptors by and %, respectively. in mouse cortex membranes, -methylhistamine µm also failed to affect s-gtpγs binding although r-α-methylhistamine µm, acting via h receptors, increased it by %. in conclusion, h receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of s-gtpγs binding (mice), could not be shown. murine cx promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße , münster, germany connexin (cx ) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx down-regulation, suggesting that creb related transcription factors are involved in cx gene regulation. in order to study the functional role of creb in the regulation of the cx promoter we have generated a luciferase based murine cx promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx promoter activity (cacreb % ± % vs control % ± %; p< . vs control , n= ), dncreb % ± % vs control % ± %; p< . vs control, n= ). the activity of the murine connexin promoter is modulated by creb. both cacreb and dncreb led to cx down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf transcription factor family, which in turn could suppress cx promoter activity. (supported by the dfg) results: fxa increased par- mrna, protein and cell-surface expression and augmented par- -mediated mitogenesis. par- expression was not influenced. the regulatory action of fxa on par- was concentration-dependent and mimicked by a par- selective activating peptide. the thrombin inhibitor argatroban or par- gene silencing did not influence fxa-stimulated par- expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox- in smc. nox- gene silencing prevented fxa-stimulated par- regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par- expression and mitogenic activity. fxa induced nuclear translocation and par- dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par- expression. in separate studies, fxa promoted par- mrna stabilisation through increased human antigen r (hur)/par- mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par- expression. conclusion: expression and mitogenic activity of vascular par- , but not par- , is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox- -containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par- may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor aim: we recently reported that functional expression of par- thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par- . methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par- mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose ( mm vs . mm) slowed par- mrna degradation in the presence of actinomycin d. par- mrna decay was not affected. hur binding to par- mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par- mrna. hydrogen peroxide (h o ) also induced cytosolic hur shuttling and increased par- mrna and total protein expression. the role of endogenously generated h o in the regulatory effect of high glucose on par- expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h o generation) and cell-permeant catalase (to degrade cellular h o ). both approaches prevented the stimulatory effect of high glucose on par- expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par- transcript stability. cicaprost also attenuated high glucose-induced hur binding to par- mrna and as a consequence normalised par- expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par- thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h o , increase par- expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par- expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day ) and on postoperative days , , , , , and . a significant reduction of recovery was observed in the ndpk b depleted mice at days and . thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than % in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for hours to constant atmospheric et concentrations of , , or ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was . ± . nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was . ± . nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw on human dendritic cells fink c. , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw on the expression of maturation related genes (cd a, cd ), cytokines (tnfα, il- , il- ), chemokines (ccr ), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr , tlr ). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in well plates. non-adherent cells were eliminated. to induce idc development, ng/ml il- and ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day , maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of µg/ml and cultured for additional days. after incubation with different concentrations ( . in idc, compared to control, we found a significantly increased expression of cd ( . - . fold) and tnfα ( . - . -fold) genes after treatment with . - % stw , respectively, but no effect was found on the expression of cd a, il- , il , adam , cd c, cd ,tlr , tlr , mhc-ii and ccr . in summary, these data demonstrate a stimulatory effect of stw in idc and especially in mdc, concerning an increase of various genes related to maturation (cd ), immunomodulation (tnfα, cd ), adhesion (ccr ) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring a, karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of -beta-estradiol (e ) and estrone (e ). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p -catalyzed metabolic activation to catechols (wang et al., chem res toxicol , . like e and e , zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res , . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which -oxo- , -dihydro- 'deoxyguanosine ( -oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of -oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp a , and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of -oxo-dg could be demonstrated with each extract. the levels of -oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. -hydroxylated zen/zel, which is the major catechol, was more reactive than -hydroxylated zen/zel to form -oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e and e . the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me / - ) and "food and health" of kit. thrombin regulates expression of sphingosine kinase- (sphk- ) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk- expression and atherosclerotic burden in vivo flößer a. results: thrombin induced a time-and concentration-dependent ( - nmol/l) increase in sphk- mrna and protein expression in human saphenous vein smc, n= - . this was mimicked by a synthetic par- ligand. inhibition of sphk- attenuated thrombin-induced smc proliferation but not smc migration (n= ). the regulatory action of thrombin on sphk- expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk- mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk- mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk- expression by % (n= ) and plaque size by % compared to control animals (n= ). conclusions: thrombin induces sphk- expression and s p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk- expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to . µg/g don and . µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t- toxin, ht- toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to µg/g don, µg/g ennb, µg/g ergotamine, ng/g ht- toxin) exert pronounced cytotoxicity in v cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp a . recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp a and proinflammatory cox- , respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct -formylindolo [ , b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc ) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 'methoxy- '-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase (chk ), an important cell cycle regulator that arrests the cell in g /m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: . ± . min. vs. mpa: . ± . min., n = - , p < . ). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: . ± . min. vs. drospirenone: . ± . min., n = - ) and levonorgestrel (placebo levonorgestrel: . ± . min. vs. levonorgestrel: . ± . min., n = ) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase (mmp- ) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp- , a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg , wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a -well plate format using glutamate/malate ( mm/ . mm) and succinate ( mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. ( ) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., ) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), -octyn- -ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd expressed on dcrc (ec = half maximal effective concentration). a pronounced induction of cd at low concentrations was seen with la, lna and oa (ec : , and µmol/l, respectively). ua exhibited an intermediate response (ec : µmol/l). with oc and ma, we observed effects at higher concentrations only (ec : and µmol/l). no significant increase of cd was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter (oct ) and oct , respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph . and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least million ddd each) for their inhibitory interaction with oct and oct . human embryonic kidney (hek) cells stably overexpressing oct or oct and the prototypical oct substrate -methyl- phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at µm, % and %, respectively, of the tested compounds significantly decreased oct -and oct -mediated uptake of mpp+. the most potent inhibitors (inhibition > %) of oct were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct . in contrast, neither at µm nor at µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct or hek-oct cells. there was a significant correlation between the degree of oct and oct inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between and µm in humans, inhibitory interactions of these compounds with hepatic oct have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. ex b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: ± %, n= ) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: ± %, n= ) and failing patients (ef: ± %, n= ). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation beadchip (illumina). this microarray allows analysis of more than , methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified cpg sites in hypertrophic samples and cpg sites in failing samples which were differentially methylated compared to control specimens (delta > %; p< . ). cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and `utr regions. specifically probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase (srpk ) showed diminished cpg-methylation in hypertrophic (- . ± . %) and failing (- ± . %) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh or myh . these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln , u -mg and gl . the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora , a a, a b and a . they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora or a a, respectively . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. abundance of adora , a a, a b and a mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora b being the most abundant. adora , adora a and adora are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora is upregulated . fold (+/- . fold) and fold (+/- . fold) at day and at day , respectively, as compared to preadipocytes (n= ). adora a is fold (+/- . fold) upregulated at day and fold upregulated (+/- . fold) at day , respectively (n= ). adora was found upregulated . fold (+/- . fold) at day (n= ). in contrast to this, ador b was downregulated to . fold (+/- . fold) at day and to . fold (+/- . ) day compared to preadipocytes (n= ). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora a activation by cgs significantly increased lipolysis by % (+/- . %) compared to untreated control. moreover, adora antagonist psb increased lipolysis by % (+/- . %) (n= ). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora a agonist or adora antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin- (myoz ) and g protein signaling modulator (gpsm , also termed ags ) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa ( -methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of - µg/ml or prolonged maa elimination half-life of up to hours. in one case with manifest hepatic insufficiency an maa concentration of more than µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b + amino acid transporters recognize fet as substrate (lat and , y + lat , and b + at, respectively). in contrast, y + lat and atb ,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat is the dominant amino acid transporter in all glioblastoma cells investigated (ln /u /u mg/u /a /t g). a strong lat expression was also shown on the protein level. to find out whether lat is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln glioblastoma cells. [ h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat in ln cells led to a concomitant decrease of lat mrna and tyr transport (down to % and %, respectively). these results indicate that tyr is exclusively transported by lat in ln cells. we are currently performing transport studies using [ f]fet to investigate whether fet transport is also exclusively mediated by lat in glioblastoma cells. a further question is if lat , a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße , düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan ( mg/kg), valsartan ( mg/kg) or valsartan ( mg/kg) from weeks of age for weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac -positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht- cells, and their doxorubicin ic was four times higher. comparative protein expression levels showed that the primary cells had less rad and as well as less topoiiα, while ku and levels were similar. another very interesting protein is the mrn (mre -rad -nbs ) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre . the toxicity of mirin in ht- cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of µm and incubation times of hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [ ] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [ ] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e and hdac expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin- -gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e protein level in the human colon carcinoma cell line ht after h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ µm. hdac expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac- and sumo e . these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin- -gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. , marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into -oh-pregnenolone, the placenta tissue highly depends on the supply of c- steroids for their conversion into c- estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e ) and estradiol (e ), while αoh-dheas supplied by the fetus contributes to over % of placental estriol (e ) synthesis. soat, a member of the slc family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e s), and pregnenolone sulfate (pregs) [ ] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg- as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg- cell line that transformed dhea into e and αoh-dhea into e . by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg- cells. currently we investigate the transformation of dheas of these soat-jeg- cells by lc-ms-ms. we could demonstrate transport of αoh-dheas for stably transfected soat-hek cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [ , ] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [ ] for polymer toxicity testing. fertilized chicken eggs were incubated and after h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after - h (fig. ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative ( . % nacl) and positive controls ( % sodium dodecyl sulphate (sds) and . n naoh) were investigated. additionally ld values for different cationic polymers have been determined. within the selected incubation times ( - h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps ( ) hazard identification and characterisation, based on substance-specific toxicological hazard data, ( ) estimates of the level of exposure toward the substance and ( ) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires ( ) information on human exposure, for which it is essential that exposure is fully captured and ( ) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally , based on toxicological testing in animals (munroe et al., ) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., ) by classifying the chemicals into three toxicity classes using a decision tree based on a series of questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., ) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the th percentiles and dividing them by the default uncertainty factor of . it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn ) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning bp of the 'flanking region of kibra into the pgl -vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 'cdna ends ( 'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p was separated by ~ bp into two distinct regions, promoter p a and p b. deletion constructs harbouring promoter p b were transcriptionally active only in ihke cells. 'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_ ). exclusively in ihke cells, two additional tss were detected in intron , resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p and p ) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf l (transcription factor -like [t-cell specific, hmgbox]) resulted in a ~ -fold increase of promoter p a and intron promoter p ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p and three alternative promoters p b, p and p . the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon could result in truncated kibra protein isoforms. tcf l is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg and a corresponding technical guidance document (gd ) give technical guidance how to perform valid experiments , . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: - . - . ). finite dose experiments using rat and human skin were performed with c-labeled testosterone, caffeine, mcpa ( -chloro- -methylphenoxyacetic acid) and its -ethylhexyl-ester mcpa- ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the c-penetrants. teer, tewl and ³h o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status '-upstream of dna regions encoding for selected candidate mirnas. results: out of mirnas, were detected in both tissue types. the expression of one mirna was . fold higher (q= . ) and another was . fold lower (q= . ) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of '-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox , mecp , bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm . recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il- -and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c bl/ b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/ , cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt mice hamann m. early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. ( , j. neurosci. [ ] , - ) initially described a transgenic mouse model (dyt mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine ( , and mg/kg) as well as a long-term treatment over days ( mg/kg/d) did not induce pronounced effects in dyt mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt mice compared to controls after repeated local striatal applications of pilocarpine ( and µg/ . µl/hemisphere) let presume an altered synaptic plasticity in dyt mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd which inhibits cdk and cdk is currently tested in patients with solid tumors such as glioma. we found that pd suppressed il- -induced expression of il- suggesting that cdk or cdk may have unknown anti-inflammatory properties. to study the effects of cdk on the il- -signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk , cdk s p, in asynchronized hela cells. cdk -expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk s p enhanced il- -induced il- and il- mrna expression. moreover, shrna-mediated suppression of endogenous cdk confirmed a role of this kinase in regulation of maximal il- -induced gene expression of il- . these data also revealed that the contribution of cdk to inflammatory gene expression is highest in g , when activity of endogenous cdk is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g , g /s, g or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk in hela fucci or inhibition by pd suppressed inducible il- expression. microarray experiments identified many additional genes that required active cdk for maximal il- -or tnf-inducible gene expression. we also found that cdk co-immunoprecipitated with p nf-κb, colocalized with p in the nucleus and was recruited together with the p subunit to the proximal il- promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc mutations. since the first descriptions at least different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc mutations, we have analysed all mutated trpc channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring a, karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. , hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h -receptor (h r) is involved in acute inflammation and th cytokine production. consequently, we intended to analyze the role of h r in a murine th lymphocyte transfer-based model of asthma. specifically the ability of h r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd + t cells were polarized in vitro under th -favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il- production in t cells polarized in the presence of h r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about % eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h r -/-dcs yielded only about - % eosinophils in bal fluids. in summery, the h r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h r on dcs reduced their ability to stimulate proper th polarization of cd + t cells, we conclude that ha via the h r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf (skn- ) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box , düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn- is the nrf homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf ::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf in cells, it has no effect on skn- localisation. further the effect on the nrf protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are and µg/(kg d), resp. for children, food accounts for % of the total exposure, followed by mouthing ( %) and house dust ( %). the adult exposure is almost entirely ( %) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter ( %) and dressings (mayonnaise) ( %) as well as highly consumed foods e.g. bread and bakery ( %) and vegetables ( , %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed , µg/(kg d). high exposures were estimated ( th percentile) up to , µg/(kg d). on average people in germany are exposed to dehp below the current tdi of µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) ). on the basis of the available measurements of dehp, the exposure assessment has been focused on food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier ) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier ) was then used to rank the cumulative probability distributions of the exposure assessments of food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the food groups to groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm ; mean±sem; creb-ko ± vs. wt ± ; n= / cells, n= mice, p< . vs. ctr.). the nuclear factor of activated t-cells c , nfatc , is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an . ± . fold increase of the nfat model promoter activity (n= ; n= transfections), but had no impact on kv . promoter activity which is known to be regulated by nfatc ( . ± . fold; n= ; n= ; p< . vs. ctr.). nfatc overexpression led to a . ± . fold increase of the nfat-dependent model promoter activity (n= ; n= ) and to an inhibition of kv . promoter activity ( . ± . fold; n= ; n= ; p< . vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p x -rezeptors -perazine-type neuroleptic drugs are potent modulators of p x receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany p x receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p x is a promising target for pharmacological intervention. accordingly, p x blockers are currently tested in phase ii clinical trials. in an attempt to identify p x -modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek hp x cell line. with ic values of - µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp ( mm)-triggered increases in the intracellular ca + concentration ([ca + ]i) that was mediated by human p x (hp x ). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp x mediated ca + influx. whole-cell inward currents, measured at - mv, were blocked by more than % by - µm prochlorperazine. the inhibitory effects of perazines developed within about ms, hinting to a direct mode of action by binding to the p x protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro- uptake when preincubated before p x stimulation with mm atp or when applied subsequent to the agonist. interestingly, when added to a hek cell line expressing the rat p x , perazines potentiated the atp-induced increase in [ca + ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca + ]i, changes in yo-pro- permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p x activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p x . similarly, pre-treatment with lov also lowered dox-induced stabilisation of p and phosphorylation of chek and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h c ), it decreased dox-and eto-induced phosphorylation of histone h ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il , ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p , wee , cjun/fos and hmox- following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore genes from the significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb and abcg transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change . and . ). moreover, cyp a mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change . ). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc a , abcg , npt , and urat as predicitive for the serum levels of urate . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate , , , . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc a have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc a . therefore, the aim of our study was to investigate which sequences in the slc a gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc a gene. dna from human kidney samples was then genotyped for rs being part of this region. next total slc a mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs showed lower slc a mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc a , urat , npt , and oat , respectively was observed. conclusion. our data suggest that the slc a snps rs and rs might influence slc a mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc a and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p enzymes yielding ´-hydroxymethyleugenol ( ´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)- ´-ohme (separated into its enantiomers) served as test compound. we could show that hsult a , standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult a -proficient and sult-deficient bacteria was examined after incubation with µm of (+)-or (-)- ´-ohme. for selective detection and quantification of me-derived ´-deoxyadenosine (da) and ´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult a . the concentration dependence of adduct formation in hsult a -proficient bacteria was examined for (+)- ´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered mg/kg bw (±)- ´-ohme (i.p.) to mice carrying the hsult a / a gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy ) and in human kidney cell lines (hek t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha and beta and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx ) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse , erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca + -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx ) to pacemaking. ncx is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca + -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx knockout (cpncx ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx ko animals as compared to controls. in conclusion, these initial results establish ncx as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg ). the ultimate carcinogenic phase i bp metabolite anti-bp- , dihydrodiol- , -epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco- cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco- monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc , abcc and abcc knockdown cell lines were generated allowing to demonstrate that abcc mediates the basolateral, abcc the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco- cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. , regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura -am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of -br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without -br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after hours of stimulation with forskolin (fsk) in immortalized rat vsmcs ( . ± . ; n= from isolations). in a r rat smooth muscle cells the icer promoter showed a maximum stimulation of . ± . fold after two hours of fsk stimulation (n= from isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. - , leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of members (tmc -tmc ) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc cause deafness in human and mice whereas tmc and tmc (also referred to as ever and ) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc and tmc in hek cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc or tmc , stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc -or tmc -expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc or tmc . recently, it has been reported that tmc and tmc might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc and tmc mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc , hacat cells revealed the same phenotype as described above for the hek cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between . thz and thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, . thz, . thz and . thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at . thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around . thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [ ] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t- by resazurin fluorescence and compared it with the placebo supplement. additionally, the t- cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il- -beta. after the cytokine stimulation the mrna expression of ip- , il- and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t- cells increased the expression of ip- (gamma-interferon inducible protein ), tnf-alpha and il- . by preincubation with fn at µm the mrna expression of ip- was strongly reduced (log -ratio - ). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [ ] h. hoensch, b. groh, l. edler, w. kirch ( ) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, , - . the cxcr c-terminal domain mediates efficient cxcl uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. , jena, germany cxcl -signaling mediated by the g protein-coupled cxcr receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl -receptor cxcr modulates cxcl /cxcr -signaling by acting as a cxcl -scavenger. given the distinct functions of cxcr and cxcr , we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek ) with respect to trafficking, stability, i-cxcl radioligand degradation, and g protein-coupling. we found that the c-terminal residues of cxcr were sufficient for cxcr to undergo rapid spontaneous internalization. replacement of the cxcr c-terminal domain with that of cxcr (cxcr - tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr c-terminal domain by that of cxcr caused ligand-independent internalization of cxcr . receptor-mediated i-cxcl -uptake, release of i-cxcl -degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr c-terminus. while the cxcr c-terminus was sufficient to abolish g protein coupling in the cxcr - tail mutant, replacement of the cxcr c-terminus, cxcr second intracellular loop or both domains with the corresponding cxcr domain did not generate a g protein-coupled cxcr chimera. taken together, we provide evidence that the cxcr c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr -c-terminal domain may effectively control cxcr functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap and bat marker genes ucp , pgc α, cidea. the α and β isoforms of sgc were highly expressed in bat whereas α sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of -pcpt-cgmp. in contrast, staining was lower in sgcβ -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ -/brown adipocytes contain approximately % less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in - % decrease in c/ebpα, pparγ and ap expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately % in sgcβ -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. , muller et al. , ma-hock , pauluhn . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to mg/m . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße , münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding -hydroxy- -( -pyridyl)- -butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of -pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the -pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, -methyl- , -bispyridin- -ylmethyl- h-pyrimidin- , -dion and -( ''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of -pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( h, c, cosy, hmqc, hmbc). for synthesis of -( ''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with -([ , ,-dichlorotriazin- -yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty / - ). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon , pon and pon . while pon is found predominantly in the circulation, pon and pon are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon and pon are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon and pon were shown to interact with coenzyme q which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon and pon reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon -knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon and pon as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. , darmstadt, germany stw (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco- cells after stimulation with lps ( ng/ml) for hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps ( ng/ml) stimulation of differentiated thp- cells was measured using a commercially available elisa. stw ( . - . µg/ml) reduced lps ( ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of . %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw to different extents. the maximum inhibition differed in a wide range between the components. stw ( . µg/ml) reduced significantly the release of tnf-α by % in lps ( ng/ml)-stimulated differentiated thp- cells while having no effect in untreated cells. in concentrations equivalent to stw caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw , indicating a possible synergistic effect. our results indicate a multi-target effect of stw . the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw . immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis ( ) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp a , encoding p ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized h- during the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of mirnas in placentas of different gestation times revealed a significant expression of mirnas by comparing placentas of early gestation (< .week), preterm (< .week) and term (> .week). when comparing the analyzed groups, of these mirnas expose a continuous up-(including mir- a, mir- ) or downregulation (including mir- , mir- a). while comparing placentas of early gestation to term placentas, % (e.g. mir- , mir- ) were up-and % (e.g. mir- , mir- ) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, % of mirnas showed higher (e.g. mir- ) and % lower expression (e.g. mir- , mir- ). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir- and mir- . specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir- ( %) and mir- ( %) in term placentas, while crh is upregulated fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the ´-utr sequence complementary to crh was exhibit for the predicted mir- binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~ %, whereas the decreased luciferase activity for the predicted mir- binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides - %), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a ) and experimental (a ,a ) of rats weighing - g local application of dichloromethane extract of s.i in vehicle olive oil preparations of and mg were used. animals were anaesthetized ( ether pro narcosi ), trauma cm long and mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for days with λ of each preparation. group a showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a . the macroscopic ulcus dimensions were better in group a while the microscopic view were similar in a and a and better in comparison to control a . a tendency to trauma remodelling was observed, without statistical significance (t = . ) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~ mg particles/m ) or clean air (controls) for or weeks ( days/week and hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf ( - ), the dfg graduate school grk and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi k/akt, as activation of pi k/akt using a pi k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase- activation in tcsl-treated fibroblasts. conclusions: . rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. . the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a (anxa ) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca + binding anxa undergoes conformational changes, which lead to oligomerization of anxa to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa , there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic value of . µm [ . to . ] which is much higher than ic values of sirolimus described in literature [ . nm to nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf and ifitm was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße , jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine (s ) threonine (t ) and serine (s ) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s a/t a/s a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s a/t a/s a/t a/t a/t a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t and t which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s was the primary phosphorylation site, whereas t , t and t were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s but not of t , t of t . our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. methylene dianiline (mda; cas no. - - ) is on the usepa list for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of . mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at . mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at . mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral -week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: . mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein (arfrp ) is a member of the arf-family and controls the arf-like (arl )-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp expression was suppressed by sirna in caco -cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco -cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp vil-/was . ± % lower than that of control mice at the age of days) arfrp vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp . in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp vil-/mice. arfrp vil-/enterocytes as well as arfrp mrna depleted caco- cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp vil-/epithelium and was predominantly colocalized with rab . our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about % resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by . -fold and in perimeter by . fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about % of the rhoa knockdown cells reattach, but only % of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by %. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung , albertstraße , freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the -kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. ; : ) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor (pi ). here we report the generation of a mouse line where pi can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi allele. global pi deficiency (pi lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n = ), p > . ; fibrosis (%): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n = ), p > . ; fractional shortening (%): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n= ), p > . ). in addition we carried out an immunohistochemical analysis of pi expression using pi -deficient mice as negative controls. pi localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [ , , ] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [ ] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec . . . ) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [ ] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [ ] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [ ] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [ ] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β -adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase (grk ) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on -week-old c /bl mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c eps , c sh , phenyl , cn , and c were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a , an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [ ] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp /cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk -binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [ ] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. ( ) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h ) on top and microvascular endothelial cells (iso-has- ) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin- and - labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il- increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp- (prestimulated with nm pma for d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h /iso-has- with thp- ) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il- release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [ ] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [ ] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [ , ] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, pro-and retrospective studies with herbal medicinal products containing ethanol at doses of to mg per single dose, depending on the age group, have been analyzed, covering patients of - years of age. in these studies, altogether adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other herbal medicinal products it was shown that during the past few years, more than mio daily doses have been sold, corresponding to more than mio of patients (data obtained from manufacturers; figures available partly from onwards, partly from / onwards). from the packages sold in germany in the years between and , . mio were attributable to self-medication, . mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on may . she has initiated this work. prucalopride was introduced as a new selective -ht receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that -ht receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other -ht receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human -ht a receptor. isolated electrically driven ( hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven ( hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr , a -ht receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec of - . and - . , respectively (p< . vs. wt, n= ). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by µm gr . we could demonstrate that prucalopride acts as an agonist at the -ht receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases ( , ) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw (a component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome ( , ) ). in one model, maternal separation ( ) was performed on weanling rats starting from postnatal day for h each day for weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) ( , ) . adult animals were restrained for min/ day for week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw in restoring the deranged parameters. a group of animals received stw orally once daily starting treatment week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the -oxoguanine dna glycosylase (ogg ), which generates harmful repair intermediates [ , ] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single -oxo- , -dihydro-deoxyguanosine ( -oxodg) varies between the opposing dna strands of the gene [ ] . we now have addressed the question, to which extent the transcription inhibitory potential of -oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single -oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single -oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for -oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of -oxodg present in the ′-versus ′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of -oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single -oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg protein. the results thus support the initiatory role of ogg in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg , mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [ ] . an efficient method for incorporation of a single -oxo- , -dihydroguanine ( -oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing -oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg [ ] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing -oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a ′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites ( ′-gcaatgnn). adapters containing the restriction sites for directional cloning into the ′-or the ′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single -oxog in the same sequence context into opposing dna strands in the 'utr and in the 'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv , for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above °c. less is known about the influence of cholesterol on trpv signalling. we modified the cholesterol content of hek cells stably transfected with trpv and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv , another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv -/mice, our results imply that a cholesterol-regulated trpv signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution ( ml min - ). after equilibration in a stable perfusion and ventilation mode for min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po was adjusted to mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po of mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc blocker larixol acetate ( µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during h of reperfusion from . ± . g to . ± . g. this weight gain was inhibited by supplementation of the trpc blocker (from . ± . g to . ± . g h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from . ± . g to . ± . g h after reperfusion), and the trpc blocker had no additional effect (initial . ± . g, h reperfusion . ± . g). we conclude that a trpc -blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. , mainz, germany the transcriptome analyses of human cells showed that additionally to the . protein coding sequences the human dna codes for much more ( . ?) non-coding rnas . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il- β-induced inos expression by an antisense rna (as- -utr-rna) in rat hepatocytes . a promoter located on the antisense strand ' to the last exon (exon ) of the rat inos gene drives the expression of an as-rna complementary to the '-utr of the rat inos mrna. using different sense primers with homology to the '-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as- -utr-rna in human cells. in addition, transient transfection analyses using constructs containing a . kb fragment of the '-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld- cells revealed no promoter activity of these sequences. korneev et al. described the expression of a kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld- cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld- cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna , rna , - rna , ( . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse , zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som ) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s , s , t , t , t and t , which in turn leads to a robust endocytosis of the sst receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s and s . somatoprim and ke are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke on sst somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke were tested for functional selectivity at sst receptor mutants, which possess a carboxyl-terminal sst tail. this approach allows detection of receptor activation by phospho-specific sst antibodies. compared to octreotide, somatoprim activates sst but has a higher activity on sst . ke and pasireotide are partial agonists at the sst receptor. pasireotide, ke and somatoprim show comparable effects on sst receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. , , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where beta-estradiol (e ) and cdcl modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi . /s - - -x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium ( mg/kg b.wt cdcl (cd )) or steroid estrogen ( . mg/kg b.wt. alpha-ethinylestradiol (ee )) on ahrassociated gene expression (i.e., ahr, cyp a , gsta , nqo ) in the small intestine of rats after oral exposure ( days gavage). the animals were also co-treated with cd and pure anti-estrogen ( . mg/kg b.wt zk (zk)) or ee , to asseess whether ermediated processes are involved. we also measured cyp a mrna expression in two estrogen receptor negative colon cancer cell lines (ht- and caco ) treated for days with cdcl ( µm) and e ( . µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for days ( . - mg/kg b.wt cdcl equivalent to , , and ppm) and ee ( . mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o -methylguanine-dna methyltransferase (mgmt), o meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u- mg and ln- glioma cell lines. the maximum amount of autophagy was observed several days ( h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o meg, are repaired preferably by homologous recombination (hr) ln- cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor -methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf ( nuk ). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb a) have been found in patients suffering from rp. we used cngb -deficient (cngb -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb a cdna (approx. kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype ) into the subretinal space of -week-old cngb -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb -/mice expressed full-length cngb a and cnga , which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category ) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg ) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [ ] . for many nanomaterials published genotoxicity studies did not give a consistent picture [ ] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [ ] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [ ] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco- human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco- cells by activated pmn and this effect increased with increasing pmn to caco- cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco- cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h o was increased in buthionine sulphoximine (bso) pre-treated caco- cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco- cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg cells incubated with oa in the absence and presence of s mix. pure oa, as well as oa pre-activated with liver homogenisates (s mix) were used to treat human hepg cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s fraction plus cofactors of phase i enzymes. the cell viability was measured after h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by ´, ´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s mix was toxic at nm oa. strong effects could be observed when oa was pre-activated with human s -mix at a concentration of nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg cells treated with oa in the presence of all investigated s -mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse , berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october . here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. , magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p mapk and phospholipase d . in line with this, we observed marked phosphorylation of p mapk and activation of phospholipase d induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to fold induction of il- , which was dependent on p . in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il- . by inducing il- opioids significantly modulate the t helper cell balance into the type direction, which influences various immune responses, e. g. the antiviral, t helper cell type -mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse , bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde , the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde , the ca +/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca + concentrations. and lastly pde , the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde . cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has - ). the has gene is alternatively spliced. has transcript variant (has v ) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has transcript variant . the aim of the present study was to investigate whether has v is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has v mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has v fusion protein and a ddk-tagged has v construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has v mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has v protein expression in vsmc and platelets. in vsmc has v mrna expression was strongly up-regulated in response to interleukin- β (il- β, ng/ml) whereas stimulation with interleukin- ( ng/ml), platelet-derived growth factor-bb ( ng/ml), transforming growth factor β ( ng/ml), tumor necrosis factor α ( ng/ml) and interferon-γ ( ng/ml) had no effect. transfection of hek cells with eyfp-has v fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has v proteins were precipitated together suggesting formation of multimeric has v complexes. transfection of has v did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has v is present in vascular cells and responds to inflammatory cytokines such as il- ß. because of the intracellular localization and the lack of ha secretion in has v transfected cells, has v may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p , in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig) -vpgkg-(vpgig) , were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after h adhesion ( %) and an enhanced proliferation ( %, h adhesion, h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. ( ) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure ( - -fold, p< . , n= ). notably, pde a protein abundance is -fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p< . , n= ). to this end we tested whether pde a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde a overexpression induced α-sma expression ( . -fold p< . , n≥ ) and to a lower extent ctgf synthesis ( . -fold, p< . , n≥ ). mechanistically, pde a showed preferential subsarcolemmal localisation with diminished total cgmp levels (- %, p< . , n≥ ). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (- %, p< . , n≥ ). these data implicate pde a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. ) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: , or nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at mg/m , all three tested polymers -including the np ( and nm) -did not cause any changes in lavage fluid and in histopathology at mg/m . the organic pigment was a poorly soluble pyrrol with an intense orange color. the np ( to nm width and to nm length) and the coarse pigments ( to nm width and . to µm length) are both needle-like. they were tested at , , and mg/m for the np and , and mg/m for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso , zno, ceo , al-doped ceo , zro , amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after days and weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the substances ranged between < . and > mg/m . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. , wächter t. the eu reach regulation no / requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go r, accessible free of charge at www.go r.org, has been created to allow quickly finding relevant hazard information and data. go r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid chapters. hereby, the user can browse directly through the entire million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h -receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. , hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin has been approved as orphan drug for the consolidation treatment of aml ( ) . it is assumed that histamine exerts its effects by activating the histamine h -receptor (h r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation ( ) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml ( ), we initiated a study to explore the possibility that h r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl- cells. in hl- cells, histamine and various h -receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h r conformations. h r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h r agonists showed no signs of cytotoxicity. intriguingly, following h r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) , indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure ( minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/ protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca + release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca + imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/ proteincoupled metabotropic glutamate receptor (mglur ) was activated by superfusion of (rs)- , -dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca + concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca + stores by thapsigargin, cyclopiazonic acid and ip . the results indicate that after activation of the gαq/ protein-coupled metabotropic glutamate receptor (mglur ) of the postsynaptic neuron ca + is released from the endoplasmic reticulum. this ca + release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb receptors. the guanine nucleotide exchange factor dock controls reelin dependent cdc effects on radial migration pichler m. the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi -deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi -deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low ( mm) and high ( mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi -deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi -deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi -targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. , ) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r (molecular probes, . mg/ml, excitation nm, emission nm). the affinity of compounds in radioligand binding was slightly higher in sur b than in sur a-type channels, but the enantiomeric ratio in sur a channels matched that one determined for the sur b-type indicating some conformity of the binding pockets of sur a and sur b-proteins. surprisingly, however, the membrane potential tests revealed that the ( r, s)-enantiomer acted as agonist (a) whereas the ( s, r)-enantiomer acted as antagonist (b): ( r, s)-bms- induced membrane hyperpolarisation whereas ( s, r)-bms- repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms- is not selective for sur a as compared to sur b-type k atp channels. its enantiomers activate and block sur -type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur b-type katp channels act as partial agonists at cardiac sur a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. , düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur -type katp channels (nielsen et al., j med chem : , ) were characterized in sur b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal , r , ) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek (kir . /sur a)-cells and compared to hek (kir . /sur b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation nm, emission nm) using . mg/ml of the dye r (molecular probes). standard-kco induced hyperpolarisation with ~ -fold smaller potency (pec ) in sur a. ttd-compounds with ch -cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur a and sur b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca ) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca channels in nonneuronal cells. the aim of this study was to investigate the expression of kca channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca . and kca . channel activator cyppa ( µm) and specific inhibitory peptides ( µm) were applied to distinguish effects mediated by the kca channel subtypes. all kca channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps ( ng/ml) induced microglial proliferation. the kca . /kca . channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca . channels, but not of kca . channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il- , but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il- production while tnf-a production was not affected. moreover, using inhibitory sk /kca . channel peptides, we demonstrated that sk /kca . channels modulate lps-induced cytokine il- production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca . channel stimulation reversed microglial activation. thus, kca . channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. ( r, s)-( s, r)- intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. , hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r. to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase (cdk ) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk in cancer. cdk was shown to regulate tumor growth, and our group discovered that cdk regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk in hcc. histology of tissue micro arrays indicates an increased expression of cdk in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk in hcc, we analyzed the impact of both pharmacological inhibition of cdk and specific downregulation of cdk with rna interference on hcc cells. pharmacological inhibition of cdk with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g /m cell cycle arrest and cell death, and reduced motility of huh and hepg cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk also reduced proliferation, clonogenic survival, migration and invasion of huh cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat at ser that is directly phosphorylated by cdk , and tyr . in line with this, the expression of the antiapoptotic protein mcl- is reduced by inhibition of cdk . our results point to an important function of cdk in hcc and suggest cdk as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c toxin lillich m. , chen x. clostridium botulinum produces the adp-ribosyltransferase c , which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [ ] . thus, c toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c bot mutant (c mut) and streptavidin. the c portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j a. macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j a. macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j a. macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h iie rat hepatoma cells. in this context cw (a diphosphane ligand with azoyl substituents r p(ch ) pr , r= thiazol- -yl) turned out to be the compound with the highest cytotoxic potential with an ic value of , mm ( h incubation). further investigations revealed that cw induced an apoptotic cell death in h iie demonstrated by the activation of caspase / ( h incubation with mm cw ). in addition the induction of apoptosis was confirmed by the dna ladder formation ( h incubation with mm cw ). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi k/akt but after h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw shows strong toxic effects in h iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: - hrs vs. hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for , biedersteiner str , münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc -, and trpc -, and trpc /c -double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc -, and trpc /c -double knock-out mice as compared to aorta from ctr and trpc -knock-out mice indicating a functional link between cgki and trpc channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by -br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc -, trpc -, and trpc /c -double knock-out mice. no effect of -br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc -, and trpc -knockout mice. trpc could be detected in both smooth muscle and endothelial cells whereas trpc was only detected in endothelial cells. the results suggest that absence of cgki or trpc impairs the vasodilator tone induced by endothelial no production but that cgki and trpc channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc -/-, and trpc -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb in human hepatic cell models lohr c. , neser s. after the treatment with tcdd, however, a total of genes were more than -fold up regulated in hepg e.g. cytochrome p a (cyp a ) ( -fold) a sensitive marker for ahr activation. additional up regulated genes in hepg were; arylhydrocarbon receptor repressor (ahrr) -fold, aldehyde dehydrogenase a (aldh a ) -fold, and cytochrome p b (cyp b ) -fold. genes were more than -fold down regulated in hepg cells e.g. -proprotein convertase subtilisin/kexin type . markedly different findings were obtained in hheps, i.e., genes were up regulated, the highest up regulated gene was cyp b with a -fold increase in gene expression, followed by cyp a ( -fold) and aldh a ( -fold). only a small group of genes were significantly down regulated ( in total), e.g., solute carrier family (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only in common genes were up regulated, including cyp a , cyp a , cyp b and aldh a . only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb /a ). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. - , - and - ) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac , expressed in sf insect cell membranes, with cam, cam- , - , - and - by measuring the catalytic activity of ac . cam-mutants show a - -fold lower potency than cam, but they are more efficacious than cam. most prominently, cam- was % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam- , it is more hydrophobic than cam and this leads to a better ppi with ac . in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac . because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee - , potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the th week of age and total pancreatic insulin content indicated diabetes prevalence of % in males and % in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = . ) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about - % non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after , , , or weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p e and a isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep receptors for prostaglandin e convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was ( ) the pharmacological characterization of ep receptors in human pulmonary arteries and ( ) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l- served as the ep antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep /ep agonist sulprostone ( nm - mm) concentrationdependently contracted human pulmonary artery rings (pec and emax; . ± . and . ± . %, relative to the contraction induced by kcl mm). the concentrationresponse curve of sulprostone was not affected by the ep antagonist sc ( µm) but shifted to the right by l- ( µm) (apparent pa . ). extending the exposure time to l- from . to h increased its antagonistic potency to . (schild plot-based pa ; concentrations . , and µm). in pithed rats electrical stimulation ( . hz, ms, v for s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline ( . nmol/kg) increased heart rate (hr) by beats/min. sulprostone ( - nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by %. l- ( µmol/kg) diminished the inhibitory effect of sulprostone nmol/kg on the neurogenic tachycardia by %. in conclusion, ep receptors ( ) located postsynaptically strongly contract human pulmonary arteries and ( ) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. - -bromo-n- [ -( - voltage-gated ca + channels of the central nervous system control a multitude of ca + dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav . ca + channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca + -channels. ca + influx via cav . ca + channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav . ca + channels play a crucial role in synaptic transmission. the global cav . knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav . ca + channels for learning and memory. therefore, we crossed a floxed cav . mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav . in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav . channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav . knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav . for learning and memory. helicobacter hepaticus-infected rag -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., , pnas : - . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f / -positive macrophages, by wks postinfection (pi), progressing into colon carcinoma by wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as -oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [ ] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c rf , which shares , % amino acid sequence identity with the drosophila protein. it covers amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c rf and the ca v . channel in xenopus oocytes reduced the amount of the α b and cavβ subunits of the ca + channel in the plasma membrane but did not affect the gating properties of the cav . channel. expression of c rf alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c orf -fusion in e.coli (yield mg at mg/ml). we are currently preparing the c orf part of the his-sumo-c orf fusion protein by ulp -protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c orf sequence. we could not identify any homologues of c orf in the mouse genome and to analyze its function we are currently generating c orf deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c orf by expression of the bgalactosidase gene under the control of the endogenous c orf promoter. by southern blot analysis we´ve already identified homologous recombinant embryonic stem cell clones out of analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of β-estradiol in cultured v cells expressing human cytochrome p a martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany oxidative metabolism of the female sex hormone β-estradiol (e ) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e undergoes metabolic activation by cytochrome p -dependent monooxygenase (cyp) isozyme a to -hydroxy-( -ho) and to a lesser extent to -ho-e . if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e was determined in male chinese hamster lung fibroblasts (v cells) expressing human (h) cyp a and (ii) the metabolite profile of e in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp a and dimethylsulfoxide as solvent control. v hcyp a were treated with nm e for weeks and the resulting -thioguanine ( -tg) resistant mutants selected at weeks (w) and . the frequency of spontaneous -tg resistant mutants per colony-forming cells ranged from ± (w ) to ± (w ). as expected, . µm bap induced a significant increase in mutant frequency (mf, ± , w and ± , w ) . treatment with nm e resulted in a -fold ( ± , w ) and a -fold ( ± , w ) increase in mf, suggesting slight mutagenic activity. in culture medium of v hcyp a treated with nm e , -ho-e , -methoxy-(meo)-e , -o-methyl- ho-e and -meo-e (suggesting intracellular formation of -ho-e ) were detected. while -meo-e concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w for methylcatechols and at w for -ho-e , correlating with the maximum increase in mf, observed after weeks as well. in conclusion, e possessed a slight mutagenic potential after hcyp a -mediated activation to -, -ho-e and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis ( -β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg- and transactivation via estrogen receptors α and β in stably-transfected hek cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of β-estradiol and progesterone in cell culture supernatants of jeg- cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of > . µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [ ng - µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm and trpm have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm -deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm regulates catecholamine release. besides trpm we recently identified transcripts encoding additional trp channels including trpc and trpc in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation ( µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc /c /c triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp (ec . vs. . µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at a and at b receptors as mechanosensors in myogenic vasoconstriction blodow s. , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/ -proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/ -coupled receptors such as angiotensin ii at b, vasopressin v a, endothelin eta and etb and α a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v a receptor and α a and α ab adrenoceptors showed no differences of myogenic tone, blocking of at a and at b receptors by losartan and candesartan, eta receptor by bq and α a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at a -/mice with and without additional blocking of at b receptors by candesartan suggested that especially at b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at b -/mice. these findings suggest that mechanosensitive gq/ -protein coupled receptors, especially at b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [ ] . they encode a substantial variety of isoformes and so far we have identified more than distinct trpm proteins in mouse and rat each varying in exons , , , , , and . these variants differ enormously in their biophysical properties. for example splicing within exon affects the channel pore and causes significant changes of the ionic selectivity of trpm channels [ ] , whereas splicing of nucleotides encoded by exon leads to dormant trpm proteins. however, the frequency of these different isoformes in trpm expressing tissues is completely unknown. to get insight into the significance of the different trpm isoformes we investigated the abundance of alternative trpm transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon ranges from up to % in different cell types and tissues. furthermore we analyzed the trpm transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm transcripts are most abundant. for that purpose we sequenced more than clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon . in cells of the choroid plexus nearly all ( / clones) carried the short ca + permeable pore. furthermore, we identified seven variants spliced in exon encoding truncated trpm proteins. however, the composition of the trpm transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of , or µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of . µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of µg/person/day, which corresponds to a dose of . µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has , - , - ), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at weeks coincided with ha deposition and induction of has in aortic root plaques. in human symptomatic carotid artery plaques has was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has was specifically induced via activation of nfkb by interleukin- β (il- β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay - . has was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il- β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f / positive macrophages as shown by immunohistochemistry. to study the effects of has mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has were employed. overexpression of has resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has were mediated by both pi k signaling and mapk signaling via hyaluronan receptors cd and rhamm. conclusion: the present results suggest that has -dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has -mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for weeks and were pretreated by inhalation of the arginase inhibitor ( )s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il- levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn -deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. - , münchen, germany hcn encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn -/and hcn +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, ) silva et al., ) . we simulated . the alcohol concentration in a breastfed neonate and a -month-old suckling infant after the nursing mother had consumed alcohol, . the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman . the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was . ‰ after consuming . l of wine, peak concentration was . ‰ in the newborn, . ‰ in the -month-old infant and . ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was . ‰ in the neonate and . ‰ in the -month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation " to glasses of wine on occasion" (agence nationale d'accréditation et d'Évaluation en santé, assante ) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. ( ) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. ( ) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type (gαi ) and adenylyl cyclase type v (ac ). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai -cfp and yfp-ac . after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. ) . to further analyze the properties of the dissociation between gai and ac we measured in parallel the offset kinetics of the interaction between gai -yfp and gβg-cfp (gi -fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs accelerated the deactivation of gi proteins and the dissociation of gai -cfp from yfp-ac . these experiments revealed that in the absence of rgs the dissociation of gai from ac after agonist withdrawal takes about times longer than the deactivation of gi proteins. in the presence of rgs this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai from ac was only marginally accelerated by rgs . these observations lead us to hypothesize, that ac might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai -ac interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi / ) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by % ± % in pkgi / as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha ( % ± . %), ppargamma ( % ± . %) and ap ( % ± . %) expression in pkgi / cells as compared to pkgifl/fl. treatment of t -l cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in t -l cells. concomitant activation of pkgi in t -l preadipocytes and treatment with the demethylating agent -aza-deoxycytidine significantly increased expression of uncoupling protein- (ucp- ) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser- in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp- promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex and , mtorc and . under amino-acidrich conditions, activated mtorc promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc . bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the , -bz flunitrazepam (flu), clonazepam (clo) and chlorodiazepam ( -cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas -cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc . cells with -cd, flu and clo for , and hours up to genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at hours revealed that the expression of genes was regulated by both flu and clo but only genes were regulated by both -cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of -cd. the difference between the gene regulation by flu and clo on the one hand and that of -cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the , -bz could be mediated by the recognition sites targeted by the , -bz, i.e. the gria -encoded ionotropic glutamate receptor ampa , we investigated by quantitative pcr whether hmc . cells express gria , tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra , gabrb , gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra , gabra , gabrb , gabrg and gabrr . expression of gria was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee - , nuthetal, germany -hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v cells genetically engineered for expression of human sult a suggesting that hmf is converted into the reactive sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n -(( -formylfuran- -yl)methyl)- 'deoxyadenosine (n -ffmda) and n -(( -formylfuran- -yl)methyl)- '-deoxyguanosie (n -ffmdg). these adducts were also detected in dna from v -sult a cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta engineered for expression of human sult a . the putative mutagen -sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta -sult a and in dna of liver, lung and kidney of fvb/n mice that had received about mg ffa/kg body weight per day via the drinking water for days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp (methyl cpg binding protein ) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp is known to be a target gene for several micrornas including the cluster mir- / in the brain. recently, our group could show that mecp expression is downregulated in human heart failure suggesting that mecp might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp by the cluster mir- / in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir- / expression, we treated cultured nrcms with µm norepinephrine for hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir- ( . ± . -fold of untreated cells, p< . ) and of mir- - p ( . ± . -fold of untreated cells, p< . ) and downregulated mecp mrna and protein levels ( . ± . -fold of untreated cells, p< . ). to check whether mecp downregulation also occurs by direct mir- / activation we increased levels of mir- and mir- in cardiac myocytes by transfecting precursor mir- and mir- - p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp mrna and protein downregulation ( . ± . -fold of control, p< . ) after mir- overexpression. similar results were obtained by overexpression of mir- - p. to test the effects of adrenoceptor activation on the mir- / -mecp axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps ( mg/kg/day each). after days, cardiac ventricles were analyzed. nppa gene expression ( . ± . -fold of control animals), mir- and mir- - p levels ( . ± . and . ± . -fold of control animals, p< . and p< . , respectively) were increased while mecp protein levels decreased to % (p< . ) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir- / expression and to downregulation of mecp in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at- receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at- receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a % increase of control (pec %) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec %= . ± . (mean±sem, n= ); maximum increase= ± %]. neither rosiglitazone nor pioglitazone ( . - µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec %= . ± . , . ± . and . ± . ; maximum increase= ± %, ± % and ± %, in the presence of . , and µm of rosiglitazone, respectively, n= - , each). in contrast, pioglitazone in concentrations up to µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer (dlc ) is a tumor suppressor whose allele is lost in % of liver, breast, lung and % of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia and (mkl / ) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc . moreover, dlc loss and mkl nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl in dlc -deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl phosphorylation. dlc loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr , myl and myh . furthermore, we identified a novel target gene, integrin a , with a key role in cell migration and metastasis, that exhibited a dlc -and mkldependent regulation. depletion of mkl / suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc loss. our data provide insight into the mechanism by which dlc loss initiates tumorigenesis. as mkl and have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p y receptors (p y , , , , , , , ) , and homo-or heterotrimeric ligand-gated ion channel or p x receptors (subunits: p x - ). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p y and p x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p y and p x receptors [ ] [ ] [ ] [ ] [ ] [ ] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm . has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm . concentration in a telephone booth ( , m volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with sec time resolution; laser scatter allowed a size resolution from , µm to µm. for the pm . concentration the following values were calculated: cumulative pm . concentration as auc-pm . (µg/m /sec), peak pm . concentration as cmax-pm . (µg/m ) and average pm . concentration cmean-pm (µg/m ). in closed door condition both cigarettes produced particulate auc-pm . values of ± µg/m /sec (rc) to ± µg/m /sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin- (il- ) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has - ) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il- participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il- . therefore, the aim of the present study was to elucidate whether il- regulates the expression and function of ha-matrix in cfs. cells were isolated from c bl/ j mice and used during passage - for experiments. cfs were stimulated with il- or hyper-il- which is a fusion protein of il- and soluble il- receptor (sil- r). after and min signal transducer and activator of transcription (stat ) was phosphorylated in response to hyper-il- but not in response to il- . rt-pcr revealed rapid upregulation of has ( . ± . fold of unstimulated control, h) in response to hyper-il- . has was induced to a lesser degree ( . ± . fold of unstimulated control, h) whereas has was not responsive ( . ± . fold of unstimulated control, h). in contrast, il- had no effect on transcript levels of has isoenzymes. in turn, expression of has and has in response to hyper-il- was inhibited by ag , which indicates the involvement of stat signaling. interestingly, despite induction of has and has the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il- . this may indicate that il- regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il- trans-signaling (hyper-il- ) via the soluble il- r and subsequent stat signaling with increased ha-synthesis. the fact that il- had no significant effect suggests that the expression of the non-signaling membrane-bound il- α-receptor (il- r) in cultured murine cardiac fibroblasts is not sufficient to induce has and - gene expression. therefore, il- trans-signaling mediated by il- and the circulating sil- r might be necessary to mediate the il- -induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by -aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße , bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura- fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified -dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec µm). fura- fluorimetry showed an increase in intracellular ca + levels by -dl-aminoisobutyric acid ( µm). moreover, -dlaminoisobutyric acid reduced the forskolin-induced camp production by up to % (ec µm). in addition to -dl-aminoisobutyric acid, we identified -dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of . (nfat assay), thioridazine with an apparent pkb of . (nfat assay) and . (camp assay) and rimcazole with an apparent pkb of . (nfat assay) and . (camp assay). in conclusion we show for the first time that -dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf- -chemokine receptor cxcr plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr is involved in disease states including inflammation and cancer. it is well established that sdf- -stimulated cxcr receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr c-terminal domain contains serine and threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr . here, we analyzed the phosphorylation pattern of cxcr at c-terminal sites after stimulation of the receptor with sdf- and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s / , s / and s / in immunoblot analyses, we showed that the sites were phosphorylated after stimulation with sdf- or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf- -induced phosphorylation at s / , s / and s / was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s / and then at s / and s / . taken together, these results indicate that the c-terminus of cxcr is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. in [ ] ), but information on transfer from maternal blood to milk is scarce: published data [ ] indicate that levels of ota in milk are roughly one tenth ( . ) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [ ] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was ± ng/l, and no major variations were observed over time (p = . ). on the other hand, ota levels found in colostrum ( ± ng/l) were higher than in mature milk (p < . ). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p . ± . ) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [ ] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [ ] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [ ] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= ) and turkey (n= , only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [ ] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [ ] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of ng/kg-bw/day set for adults [ ] . in both cohorts, most of the urine samples tested positive for ota (chile %, turkey %). ota levels observed in urine samples from the turkish infants (range: - , ng/l) were fold higher than levels found in chilean samples (range positive samples: - ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to % of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p system. using the estrogen receptor response assay (e)- -hydroxyclomiphene and (e)- -hydroxy-n-desethylclomiphene (ec : . and . nm, respectively) turned out to be times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp d revealed to be the major isoenzyme involved in the formation of -hydroxlated metabolites. n-deethylation was catalysed by cyps a / , d , c and c . rates of -hydroxylation in microsomes from human liver donors correlated with the number of functional cyp d genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp d genes (poor metabolizers) cmax of (e)- -hydroxyclomiphene and -hydroxy-ndesethylclomiphene was and times lower, respectively, when compared with subjects with at least one fully functional cyp d allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was and -fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound proved to be the most active derivative, showing a significant toxicity at a concentration of , µm. compounds and showed significant toxic effects at a concentration of µm. the compound showed no toxicity up to a concentration of µm. all derivatives , and have a ec between and µm. we further proved the induction of apoptosis by apo-one assay (caspase / activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb using whole genome microarray analysis neser s. , lohr c. , van ede k. i. , andresen k. interaction with the aryl hydrocarbon receptor (ahr), with , , , -tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p isoenzymes (cyps), i.e., cyp a , a , and b . one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c bl/ mice were treated with single doses of six dl-congeners (tcdd, pcb , pcb , and pcb ) , and the 'non-dioxin-like' (ndl) pcb . quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array ( x k) system. the quantity of genes affected (≥ fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated genes, and down-regulated , -pncdf-treatment had impact on (up), and (down). treatment with pcb led to marginal numbers of up and down-regulated genes. with (up), and (down) genes shared, the most extensive overlap occurred between tcdd-and -pncdd-treatment. no overlap was found due to treatments with the ndl pcb ( up, down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of up, and down-regulated remain, most of them being related to drug metabolism. while pcb regulated only genes involved in drug metabolism, omission of pcb -regulated genes resulted in consistently (up), and (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years and were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, enquiries were registered in total. in . % of those enquiries systemic antibiotics were named with a total number of drugs. . % of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions ( . %), diagnosis/treatment ( . %), drug application ( . %) and drug-druginteractions ( . %). the majority of the requesting patients ( . %) was born before . a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter , oat (slc a ), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like -fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat localization in human liver. because interindividual variability of oat expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat expression. methods: an expression profile of oat for human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat mrna expression was analyzed in well-characterized human liver samples from caucasians that were accompanied by detailed demographic and clinical data. oat was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc a gene region. results: oat mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat was identified in human liver. hepatic expression of full-length oat mrna and oat protein varied -fold and fold, respectively. oat mrna and protein levels did not correlate with each other. oat was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the exons, the '-flanking region, or the '-untranslated region of the slc a gene were identified. univariate analysis showed that oat mrna is reduced in patients diagnosed for cholestasis (p= . ) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat expression. this indicates consequences for hepatic drug elimination of and response to oat drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [ ] . we investigated the interaction of mb and several structure analogous at the orthosteric binding site of human α nachr (hα nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα nachrs were investigated with radioligand binding experiments performed as high-throughput method [ ] . membrane preparations of gh c cells stably expressed hα nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb , mb and mb exhibited ki values at micromolar concentrations while three other compounds, mb , mb and the pharmacological inactive mb (negative control) did not show any interaction with the orthosteric binding site of hα nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase- and - , and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh ax and bp foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku formed at early times after acnu treatment less γh ax and bp foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku mutant cells show foci levels similar to the wt indicating restitution of h ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh ax foci increased significantly in the s-phase and remained at a high level during g where a fraction of cells remained arrested. rad , atm, mdc- and rpa- foci were also formed and shown to co-localize with γh ax. these foci were ameliorated significantly in s-and g -phase, which was similar to the time course of γh ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk and less phosphorylation of chk in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb globally knock-out animal as background, we induce the expression of cb specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c a, fmlp, and leukotriene b are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi and gαi isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice days after i.p. injection of % thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi -deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi was upregulated in gαi -deficient macrophages. vice versa gαi was upregulated in gαi -deficient macrophages. concerning gβ isoforms, both gβ and gβ were strongly reduced in the gαi -deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi -deficient macrophages showed reduced gβ protein levels only which caused a change in the gβ / gβ quotient in favour of gβ . we are currently establishing an in vivo infection model with l. monocytogenes in gαi and gαi -deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin biosensors reveal conformational changes upon binding to the β -adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr. , würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor ( tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin , in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin . upon β -adrenergic receptor (β ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin flash ), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin /β ar interaction on a timescale of seconds. a β ar mutant that was previously shown not to interact with β-arrestin was utilized as control and did not induce a conformational change in the β-arrestin molecule. our data provide evidence that β-arrestin changes it`s conformation upon binding to the activated β ar in living cells. the β-arrestin flash sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a in the heart nunes f. the calcium binding protein annexin a has been examined in the context of heart failure in the past. annexin a expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res ). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a gene trap model (gt) in which the annexin a protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of . hz, hz and hz as well as an increased sarcomere shortening at hz and hz in anxa gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt ( , ± . vs. . ± . , *=p< . vs. wt; n= - / ). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca + -atpase (serca a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n= ). however, co-immunoprecipitation experiments suggest, that anxa is able to interact with hax , which acts as a repressor of serca a (n= ). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations ( - m- - m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at - m iso [%]: wt: ± ; gt: ± *= p< . vs wt; n= - ). in conclusion, annexin a contributes to the regulation of cardiomyocyte contractility. the anxa up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. mar; ( ) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr i subfamily of nuclear receptors. based on a -dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of µl serum with oasis hlb cartridges allowed a reliable quantification down to ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp column ( mm x mm; µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai , frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl ( - ) has antagonistic function for cxcr , cxcl (p g ) is able to inhibit cxcr and the herpesvirus encoded protein vmip-ii interferes with ccr , - and - as well as with cxcr , - and cx cr . their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl ( - ), cxcl (p g ) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th- cell clone if with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl ( - ), cxcl (p g ) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type- -diabetes and contact dermatitis. small heterodimer partner (shp- ) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp- is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp- expression, suggesting that shp- is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp- , including hnf α, lrh , lxr, fxr, srebp c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp- and to determine their impact on the transactivation of shp- . dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely - t>c (rs ), - g>c, - c>t (rs ) and del- ctga (rs ). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh , lxr, fxr, srebp c and pparγ) of shp- expression. only the transactivation by hnf α was decreased in the presence of the - c>t polymorphism to % and the - g>c polymorphism to %. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about in . individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [ ] . mutations in the pkhd gene cause arpkd. more than different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [ ] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [ ] [ ] [ ] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [ , ] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [ ] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda - ) and tcdb (tcdb - ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda - and tcdb - induced time and concentration dependent cell rounding and rac -glucosylation. however, depending on the cell line, truncated toxins exhibit up to -fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda - predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh -only protein bid, a member of the bcl- family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi c in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi- c were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht- cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified novel molecules that significantly prevented glutamate-induced toxicity in ht- cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. , selinski s. in the s, an uncommonly high percentage of glutathione s-transferase m (gstm ) negative bladder cancer cases ( %) was reported in the greater dortmund area (golka et al., ) . the question arose whether this uncommonly high percentage of gstm negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the s. in total bladder cancer patients from the st.-josefs-hospital dortmund-hörde and controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm , gstt and the n-acetyltransferase (nat ) tag snp rs . the frequency of the gstm negative genotype was % in bladder cancer cases and thus much lower, compared to a previous study performed from - in the same area ( %). nat genotypes were distributed equally among cases and controls ( % slow acetylators). less gstt negative genotypes were present in cases ( %; controls %). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht- neurons. silencing of aif expression by sirna ( nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone ( nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht- cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca + influx through cav . channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav . cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type a (csnb ), aland island eye disease (aied) and cone-rod dystrophy type (cordx ). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav . function we analyzed the visual phenotype of cav . -deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav . -deficient mice. heterozygous cav . mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav . knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav . channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. , freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase b enzymes (ugt b ) revealed that ugt b . and ugt b . had markedly lower intrinsic clearance as compared to ugt b . (hanioka et al., ) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, , mielke et al., ) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt b . variant (v ) was fold and those of the ugt b . variant (v ) was fold higher than that of the ugt . variant. with dermal exposure at a relevant exposure level, the cmax values were . (v ) and . fold (v ) of ugt . variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in . fold (v ) and . fold (v ) higher cmax values as compared to ugt . variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose . fold (v ) and . fold (v ), for relevant dermal exposure . fold (v ) and . fold (v ), and for combined exposure . fold (v ) and . fold (v ) of ugt . variant. from the results we conclude: ( ) polymorphism of udpglucuronyltransferase ( b . and b . ) has an impact on the blood concentrations which, however, is less than fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. ( ) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase b polymorphism on bisphenol a glucuronidation. arch toxicol. , ( ) : - . mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. ; ( ) : - . mielke h, partosch f, gundert- the heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway ( ) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation ( ) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. ), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion ( ) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde ) is expressed in cm ( ). sil may act on other pdes, such as pde c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase ( days of angii infusion at mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst and sst in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines (t ) and (t ), which enabled us to selectively detect either the t -or the t -phosphorylated form of sst . we show that agonist-mediated phosphorylation occurs at t , whereas t is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst -selective ligand l- , but not octreotide or ke were able to promote a clearly detectable t phosphorylation. however, none of these compounds was able to stimulate t phosphorylation and sst internalization to the same extent as the natural somatostatin. agonist-induced t phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase (grk ). like that observed for the sst receptor, phosphorylation of sst occurred within seconds. however, unlike that seen for the sst receptor, dephosphorylation and recycling of sst were complete within minutes. we also identify protein phosphatase g (pp g) as sst receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t . in addition, we identify grk -mediated phosphorylation and pp g-mediated dephosphorylation of t as key regulators of rapid internalization and recycling of the human sst receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß adrenergic receptor, d dopamine receptor, parathyroid hormone receptor , thromboxane a receptor and the vasopressin receptor . however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase beta (pp ß) as the phosphatase for the cluster of phosphorylated threonines ( ttetqrt ) within the sst a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase gamma (pp γ) as mor phosphatase that catalyzed t and s dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst a chimera with the rsst a c-terminal tail and a rsst a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c -carbonyl function of pat. the exocyclic amino group of the dna base is linked to c of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of top prescribed psychiatric drugs. for drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in spcs individual data points ( . ± . per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of . (range: - ) months. the respective spcs covered on average . ± . % (range: . - %) of all mentioned indications and . ± . % (range . - %) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average . ± . % (range: . - . %) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the spcs of the drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. ex b. protein kinase ck (former name 'casein kinase ') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [ , ] . elevated ck activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [ ] . ck is a heterotetramer consisting of two catalytic subunits (ck α) attached to a dimer of regulatory subunits (ck β) [ ] . ck β stabilizes ck α against denaturation and modulates the substrate specificity of the catalytic subunit [ ] . due to the relatively small and hydrophobic ck α-ck β interface ( Ų) [ ] , low molecular weight inhibitors are able to interfere with the ck subunit interaction and thus affect the kinase activity [ ] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [ ] . we have developed an elisa-based ck α-ck β binding assay using recombinant human ck subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck subunit interaction. primary hits were further characterized by determination of the parameters ic and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < . µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy were exposed for days to ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for hrs on top of matrigel-coated inserts. differential expressions of phosphoproteins were found in cb-treated cells while an altered expression of phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [ ] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße - , berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in . since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: . applicability domain. the chemical space of the ttc dataset (> compounds) has to be compared with that of cosmetic ingredients (> compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz , bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene - , -diol- , -epoxide (anti-bpde)-dna adducts as well as cytochrome p (cyp) a / b enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after h and h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp a / b activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of . ± . and . ± . anti-bpde/ nucleotides after incubation with µm ( h) and µm b[a]p ( h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after h than after h. increased cyp a / b activities were observed at > . - µm ( h) and > . - µm ( h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than % cytotoxicity in relation to the negative control were found after h incubation with ≥ µm b[a]p and after h with ≥ µm b[a]p. overall, incubation of a cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav . channel complex has been shown to play a key role in cav . channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb gene coding for cavβ , exon , contains several potential phosphorylation sites, e.g. for protein kinase a or ca + /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav . . potential phosphorylation sites are ser and ser (in the β subunit in rat, perez-reyes et al. a ) and ser in the c-terminus of the poreforming α c subunit (lemke et al. b ) . in cardiomyocytes camkii regulates ca + release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav . channel complex and camkii are thr in the β subunit (in rat, grueter et al. c ) and ser and ser in the cterminus of the α c subunit (blaich et al. d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon after aa pro . this mutation prevented translation of the cavβ c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ stop mice with s a mice (lemke et al. b ) lacking the pka phosphorylation site s in the α c subunit (s aβ stop mouse) or with sf mice (blaich et al. d ) lacking the camkii phosphorylation sites s and s in the α c c-terminus (sfβ stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s aβ stop mice. electrophysiological investigations on cardiomyocytes of s aβ stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav . channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing % fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: . ± . pf, n= ; caf: . ± . pf, n = ) and membrane potential (sr: - . ± . mv, n = ; caf: - . ± . mv, n = ) . in both groups, we observed fast activating outward currents with a mean threshold at - mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: . ± . pa/pf, n = ; caf: . ± . , n = ; p < . ). when maintained in culture for - weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: %; sr: %). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: . ± . pa/pf, n = ; caf: . ± . pa/pf, n = ; p < . ). na + currents were not altered by nm tetrodotoxin (ttx), but µm ttx reduced current amplitude to % of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav . . conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha a-adrenergic receptor. hek t cells were transfected with yfp-tagged alpha a-adrenergic receptor and the three subunits of gi . the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha aadrenergic receptor recruits gi with a half-life of around milliseconds. when arrestin and grk were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around seconds. there seemed to be no effect of grk +arrestin cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin -cfp to yfp-tagged alpha a-adrenergic receptors in the presence of grk . upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin , confirming previous observations that the alpha a-adrenergic receptor recruits arrestin . co-transfection of the three subunits of gi had no effect on the kinetics of arrestin binding to the alpha a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b , b , b ) in detrusor and urothelium. we have shown previously that only b -ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b -ar in the absence of urothelium. but in intact detrusor strips b -ars seem to have an additional inhibitory effect. affinity of the selective b -ar antagonist l , is unchanged, therefore the intact urothelium does not interact with the function of b -ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. , würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator (crtc ) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc is involved in this process, we investigated the expression of crtc in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc mutant mice the expression of crtc was significantly higher than in the controls ( . -and . -fold, respectively; n= ). in both forms of human hypertrophy, we found a significantly -fold upregulation of crtc protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n= - for each group). conclusions: crtc is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse , zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (< %). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥ -fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at -receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for months with telmisartan (tel mg/kg/d) or amlodipine (aml, mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after months by more than g. tel normalized sbp whereas it remained > mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first weeks, but raised beyond control levels during the last weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β -adrenoceptor -camp mediated, immediate stimulation of β -adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., bonn, germany based on their bronchodilatory effects, β -adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β -adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β -adrenoceptor expression was addressed here. mrc- human lung fibroblasts were cultured for up to h in absence or presence of test substances, followed by β -adrenoceptor mrna determination by qpcr. β -adrenoceptor mrna decreased with a half-life of min after inhibition of mrna synthesis with actinomycin d ( µm), but increased by ± %, ± % and ± % (means±sem) within . , and . h, resp. after inhibition of protein synthesis by cycloheximide ( µm). the β -adrenoceptor agonists formoterol and olodaterol ( - nm) induced a rapid increase in β -adrenoceptor mrna (maximally within h by ± % and ± % at nm, resp.). however, after h exposure to nm formoterol or olodaterol a reduction in β -adrenoceptor mrna by ± % and ± %, resp., was observed. both, the stimulatory and inhibitory effects of β adrenoceptor agonists were mimicked by forskolin ( µm, increase by ± % and inhibition by ± %) and cholera toxin ( ng/ml, increase by ± % and inhibition by ± %). the formoterol-induced up-regulation of β -adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β -adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β -adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β adrenoceptor gene expression by β -adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β -adrenoceptor gene. et- appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et- and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et- expression in hlf was explored. mrc- hlf were cultured for up to h in absence or presence of test substances, followed by prepro-et- (ppet- ) mrna determination by qpcr. the muscarinic agonist oxotremorine ( µm) induced an increase in ppet- mrna by %, an effect prevented by nm tiotropium. the β -adrenoceptor agonist olodaterol (up to nm) caused a reduction of ppet- mrna expression by %. the effect of nm olodaterol was prevented by ici , ( µm), but not affect by cgp ( µm) . the pka agonist -bnz-camp ( µm) caused a reduction in ppet- mrna expression by %, whereas the epac agonist -cpt- '-o-me-camp ( µm) caused only a marginal inhibition by %. olodaterol ( nm) strongly opposed the stimulatory effect of µm oxotremorine. an increase in ppet- mrna expression by % caused by . ng/ml tgf-β was effectively opposed by and nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet- mrna caused by a maximally effective concentration of tgf-β ( ng/ml, increase by %) was not significantly affected by or nm olodaterol. likewise, the pkaagonist -bnz-camp ( µm) opposed the increase in ppet- mrna expression caused by . ng/ml tgf-β, but not that caused by ng/ml tgf-β. tgf-β caused, with an ic of . ng/ml, a marked down-regulation of β -adrenoceptor mrna expression, maximally by % within h. et- expression in hlf is stimulated by muscarinic receptors and inhibited by β adrenoceptors. the effect of β -adrenoceptors may be mediated via pka. et- expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β -adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β -adrenoceptors. since et- can promote pro-fibrotic features in hlf, inhibition of et- expression could contribute to long-term beneficial effects of long-acting β -adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h c embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv . and kv . , the cytosolic β-subunit kchip and the transmembrane subunits dpp and dpp using rt-pcr. treatment with doxorubicin ( µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h c cardiac myoblasts. while kv . was detected in h c cells and hearts from human and rat, kv . mrna was only expressed in adult rats. during hypertrophy kv . was downregulated in human tissue as well as in doxorubicin-treated h c cells compared to the controls. in rat hearts kv . expression was increased after doxorubicin treatment. interestingly, kv . was also found to be up-regulated in rat heart tissues and h c cells after treatment with doxorubicin. as previously shown, kchip mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip mrna was upregulated in hypertrophic rat hearts and h c cells. the expression of dpp and dpp was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp and dpp were not expressed in the adult rat heart or h c cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h c cells, the mrna of dpp and dpp was up-regulated. h c cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h c cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp and dpp in doxorubicin-induced hypertrophic h c cells. in preliminary experiments specific short-hairpin rna, targeting dpp and dpp , has been designed and tested in heterologous expression systems. the nonsynonymous c. t>c germline genetic variation in the liver-specific organic anion transporter slco b is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc , slco a , and slco b , have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc , slco a , and slco b contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c h, mtx auc - h, and mtx clearance cl) in an unselected population of children with all from the all-bfm trial (clinicaltrials.gov: nct ) who received courses of mtx at g/m as h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. ( ) . abcc c.- c>t (rs ), slco a c. g>a (p.his arg, rs ), slco b c. t>c (p.val ala, rs ) and slco b a>g (p.asn asp, rs ) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c h ( . ± . µmol/l), auc - h ( . ± . h*mg/l), and cl ( . ± . ml/min/m²). the rgc values of c h, auc - h, and cl ranged from . - . suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco b c. t>c snp was significantly associated with c h (p< . ), auc - h (p< . ), and cl (p= . ) of mtx. compared with the wildtype genotype, in patients with the tc genotype c h and auc - h increased by % (p= . ) and % (p= . ), respectively, whereas cl significantly decreased by % (p= . ). pharmacokinetic variables significantly changed with increasing number of variant c. t>c alleles (p< . , jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc c.- c>t, slco a c. g>a, and slco b a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c. t>c polymorphism in slco b contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase- (cox- ) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a , h , h ), the present study investigates the contribution of cox- and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner ( - µm), whereas structurally-related cox- inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox- inhibitor ns- , the pparγ antagonist gw- and by sirna targeting cox- or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox- and pparγ at both mrna and protein level. using an established cox- activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns- was shown to suppress celecoxib-induced cox- activity when added prior to arachidonic acid. among the cox- -dependent pgs analyzed, pgd and its dehydration product -deoxy-∆ , -pgj were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns- which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox- -and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox- and pparγ and a subsequent nuclear translocation of pparγ by cox- -dependent pgs. uncoagulated ppp contained - nm thrombin throughout. fxa was initially measured in clots at nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for h only marginally influenced smc apoptosis but increased mitogenesis over -fold. this was reduced by all inhibitors. clot-stimulated induction of tnfα and interleukin- mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. , stephan-schnatz k. the guanine nucleotide exchange factor rhogef is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first hours after seeding. cell counting revealed that to hours after seeding the percentage of adherent cells was significantly lower in the rhogef knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef was paralleled by a loss in rhoa and cadherine expression. as rhogef mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon ( -pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first hours. in contrast, the adhesion of rasmc after rhogef knockdown was no longer stimulated by cgmp. as these data indicate that rhogef mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef , we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h receptors (h r). activation of fpr leads to a release of reactive oxygen species (ros). h r activation results in an increase of intracellular '- '-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and -methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites ( mg) were incubated in gc vials with ml dest. water or ml methanol, each at °c for hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n= )( fig. ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram ( ± µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr in tumor metastasis is discussed. subsequently, detection of cxcr expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr antibody umb- . methods: in comparison to negative and positive control samples (cxcr -knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr -expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb- may prove to be of great value in the assessment of cxcr expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β -adrenoceptor-mediated inhibition of formyl-peptide-induced o β -adrenoceptor (adrb )-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb gene contains a total of polymorphisms. this variability could cause part of the ~ % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb agonists for each individual. (ortega et al. ; chung et al. ) our present study connects sequence data with pharmacological data of prototypical adrb -ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o .production and its inhibition by the agonists are examined in a -well cytochrome-c assay. characteristic pharmacological values (pic , emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb -sequence variant is determined by sanger-sequencing. complete determination of a , bp sequence, including the entire adrb exon and part of the flanking '-and '-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine ', '-monophosphate (camp) and cyclic guanosine ', '-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine ', '-monophosphate (cump). these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using '-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine ', '-monophosphate (cimp). using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde a. this finding is consistent with data published by hardman and sutherland who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde a. as cump has furthermore been proven to be present in mammalian cells , to differentially activate camp-and cgmp-dependent protein kinases and to be synthesized from utp by mammalian soluble guanylyl cyclase , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat and identify signal proteins involved in this pathway. rhoa-dependent stat stimulation requires rock and junkinase activation as well as ap -induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl /i- as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat -dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. christian-albrechts universität institut für immunologie, michealisstrasse , kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about %. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt= ml/kg and f= min - with fio = . and peep= cmh o while lung mechanics were followed. half of the mice received µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at °c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht- cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high µmol; medium µmol; low µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of . ± . % (high), . ± . % (medium) and . ± . % (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: . ± . nm*h - (high); . ± . nm*h - (medium) and . ± . nm*h - (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p x ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany the homomeric p x receptor (p x r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p x receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p x receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p x wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek cells, heterologously expressing the human p x receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p x r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p x agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany purinergic p x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p x receptor family, the p x receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p x receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp x model that we developed from the known zebra fish p x crystal structure in the closed state. voltage-dependent modulation of alpha a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. , marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations ( nm: % inhibition) but almost absent at saturating agonist concentrations ( µm: % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine ( µm, vm=- mv) resulted in a fret response that was inhibited by % at + mv. in contrast to ne, strong receptor inhibition at + mv was present even at super-saturating concentrations of clonidine ( µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = + mv. therefore we conclude that negative membrane potentials promote active conformations of the a a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has , in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal , ) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e ) mediated effects on ha-rich ecm during actinic aging. weeks of uvb irradiation ( x med ( mj/cm ), weekly) of hairless skh- mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e ) by means of controlled release pellets abolished these effects confirming the stimulatory role of e . the increase of dermal ha correlated with induction of ha synthase has by e . in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e . however in cultured skin fibroblasts e reduced the expression of versican and had no effect on has . therefore, direct upregulation of has and versican in fiborblasts by e was excluded. however, e increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between and at least hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno solutions with . and . mg/cm , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. , berlin, germany c exoenzyme (c bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c bot to the hippocampus-derived ht cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c bot. the binding assays established that c bot bound in a concentration-dependent manner to ht cells. in the overlay assay we detected one clear band of kda. to elucidate whether glycosylation is important for the c bot-protein interaction, ht cells were incubated with glycosidase f resulting in a decreased binding of c to the kda band. to explore the involvement of phosphorylation in the binding of c to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c bot. pretreatment greatly reduced the c bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c botprotein interaction in the following overlay. thus, interactions between c bot and ht cell proteins may require phosphorylation. to further characterize the kda band as binding target of c bot, the kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this kda single gel band proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c bot-protein interaction. s solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. , köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide ( % to %) on the assessment of k m, vmax and clint with regard to the -hydroxylation of midazolam via cyp a and the cyp a catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for -hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er rommel c. , rösner s. introduction: the transcription factor er (ets related ) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er expression. thus, the aim of the present study was to investigate the cardiac function of er in genetically modified mouse models. we previously generated transgenic mice overexpressing er under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day after birth (p ) and in adult mice ( months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine of phospholamban (pln) was reduced in er αmhc mice. in addition, protein phosphatase (pp ) expression was significantly increased in er αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca a protein in er αmhc atria. electron microscopy revealed the significant structural remodeling of er αmhc atria at months of age. conclusion: increased cardiac expression of the ets-transcription factor er leads to structural and electrical remodeling of the atria. thus, er may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. , halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p -purinoceptors which are further divided in p x - and p y - -receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. µm atp as well as utp transiently increased phosphorylation of erk / and p mapk with a maximum effect at to minutes after application of atp and utp in neonatal cardiac myocytes (n= preparations each). the maximum phosphorylation of p increased with atp to % ± % (p< . ) at minutes and with utp up to % ± % (p< . ) at minutes. the phosphorylation with erk / mapk increased with atp to % ± . % (p< . ) at minutes and to % ± % (p< . ) with utp at minutes of basal values, respectively. after minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk / and p mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students ( th or th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving - previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of points could be obtained as a sum of both tests. results: in the means % of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk / ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany background and aims: the extracellular regulated kinases and (erk / ) play an important role in cardiac hypertrophy and cell survival. erk / are phosphorylated at the so-called tey motif, which in turn activates erk / . hypertrophic stimuli lead to an additional autophosphorylation threonine (thr ). this autophosphorylation of erk / stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk / activity and prevention of erk thr phosphorylation, we either inhibited erk activity with pd or overexpressed a mutant of erk , which cannot be phosphorylated at thr phosphorylation. while inhibition of overall erk activity with pd attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr phosphorylation by overexpression of the phosphorylation deficient mutant (erk t a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk t a significantly reduced tac-induced hypertrophy compared to wild-type erk overexpressing mice. in line with the in vitro experiments, erk thr inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk / . therefore, specific targeting of erk thr phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde , a pde in principle capable of hydrolysing camp as well as cgmp. pde contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde was mostly based on the analysis of mrna distribution, we generated antisera against pde to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde inhibitor papaverine as well as specific pde inhibitors suggest pde as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde to camp degradation in striatum, to identify the physiological pathways pde is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse , regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp r-ii and the insp r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca + -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp r interactions. the insp r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp r-i and insp r-iii but not with insp r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type enhancer binding protein (hivep ) is regulated by proinflammatory stimuli and statins salomon a. , , schmitz b. objective: hivep binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned kb upstream (rs ) and another in exon (rs ) of the hivep gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, ; plos one, ) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy ) with proinflammatory cytokines or statins ( h). serial hivep promoter deletion constructs were cloned into the pgl -basic vector, a potential enhancer fragment, harbouring rs c/t, into the pgl -promoter vector. in ea.hy cells and thp monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy cells, endogenous hivep expression was increased by proinflammatory cytokines tnfα and il- β. simvastatin ( . and . µm) and atorvastatin ( µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep expression. the construct harbouring rs t exerted significantly higher transcriptional activity (ta) compared to rs c (p< . ). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep expression in ea.hy and thp cells. cotransfection of sp and egr led to an increase in ta, while wt exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp to the '-flanking region and the intronic modulator of hivep . conclusion: increased hivep expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep expression is regulated by sp combined in a transcription factor module with egr and wt under basal and/or inflammatory conditions. the rs site harbours potential activational capacity for hivep gene transcription and may communicate with the sp /egr /wt module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv . / channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv . - . channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv . / channels, we examined the acute effects of the preferred kv . / channel opener ica on lid in this animal model. four weeks post -ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with mg/kg l-dopa and mg/kg benserazide for days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from to over min. for drug testing, ica ( , and mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined min after drug administration using the block and the stepping test. ica reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first min after the application of mg, it lasted up to min in rats treated with mg ica . a higher dose of mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v channel opener retigabine was based on its action on striatal kv . / channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n= - ). myotoxic cerivastatin ( . , . , µm for days) caused a concentration-dependent decrease of contractile force (p< , , n= - ) paralleled by an increase in inos transcript (mean±sem: . µm ± . -fold, n= p< . ; . µm . ± . -fold, n= p< . ) and protein ( . µm . ± -fold, n= p< . ; . µm . ± . -fold, n= p< . ). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with w, a specific inos inhibitor. we applied µm of w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n= - ). also, l-name ( mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n= - , p< . ). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp ( µm) caused a reduction of contractile force. combined treatment with cerivastatin and . µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with m syndrome and mutations in the cul gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. , münchen, germany m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< - sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul ( %) or obsl ( %) gene, respectively. cul constitutes an e ubiquitin ligase that is involved in the regulation of the insulin-like growth factor (igf- ) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate (irs- ). to investigate the role of cul mediated irs- degradation in the pathogenesis of m syndrome. primary skin fibroblasts of seven m syndrome patients (six with cul mutations, one with a obsl mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs- protein concentrations and activation of the igf- signaling pathway (western blot). the proliferation rate of m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs- protein levels and activation of the pi k/akt and erk mapk pathway were only increased in a subset of m cells that carried cul mutations, but not in cells from a patient with the obsl mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs- levels to the observed phenotype, human imr fibroblasts were stably transfected with retroviral vectors encoding irs- . despite -fold overexpression of irs- compared to empty vector controls, no significant effect of igf- stimulation on proliferation rate or pi k/akt and erk mapk signaling was observed. skin fibroblasts of m patients with cul mutations displayed an increased proliferation rate and enhanced activation of the igf- signaling pathways. despite accumulation of irs- in fibroblasts from a subset of m patients with cul mutations, no pathomechanistic role for irs- could be demonstrated. collectively, our data indicate that a dysregulated igf- signaling may contribute to the pathogenesis of m syndrome, yet in an irs- independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. , münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present ( / ) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was days. to date, the website consists of pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has visitors per month with the following evaluation results: "excellent": %, "good": % and "satisfactory": % (n= ). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. , bad nauheim, germany tgf-β activated kinase (tak ) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin- or tnfa to ikk, p and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t with animals carrying floxed alleles of the tak gene. adipoqcreer ; tak fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin- . stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak deficient adipocytes show reduced activation of jnk and p which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/ and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/ -regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk and subsequently the erk / cascade. the role of pyk for the growth of sclc cells was further supported by the fact that inhibition of pyk using a sirna approach or a novel specific inhibitor, pf , exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g / signaling by sirna-mediated g(alpha) or g(alpha) knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk versus the role of calcium-independent signaling via g / , we tested the effect of pyk inhibition in cells with impaired g / signaling. notably, pyk and g / double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk activation or g / -dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp- were used for days and stimulated with various cytokines (il- , gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd c, cd , cd ) and by immunfluorescence compared to monocytes. the bronchial tract contains up to dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between to hz and the transepithelial resistance values were stable between to Ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git and cavb schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, homburg, germany high-voltage activated ca channels are assembled from pore-forming α subunits and two distinct types of auxiliary subunits, cavβ -β and, maybe, α δ -δ . by a cavβ -specific antibody based affinity chromatography the cavβ protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ were identified by mass spectrometry (lc-esi-ms/ms) and include α -subunits, α δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ protein, including the g protein-coupled receptor kinase-interactor (git ). the aa git is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git and cavβ proteins were co-immunoprecipitated by the antibodies for cavβ and git, respectively. we cloned the git cdna from mouse brain and co-expressed it with the cavβ subunit in hek cells. like in brain lysates the git protein was retained by cavβ precipitated by the antibody for cavβ and cavβ was retained by the git protein precipitated by the antibody for git . both proteins, cavβ and git are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ -git interaction in the absence of functional cav channels. in addition, using mefs from cavβ -deficient mice enables us to control the impact of cavβ on git function. vice versa down-regulation of git by specific sirnas might allow to control the impact of git on cavβ function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine- -phosphate and fty on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine -phosphate (s p) is a mediator that modulates various physiological functions of skin cells. s p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s p, but not fty reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s p via the s p receptor, which is not activated by fty . there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h ) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il- ). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp- ) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd ) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo- ) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above µm are accompanied with inhibition of nat activity in human keratinocytes [ ] . in the following we investigated the impact of ppd on nat activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp- cells. measured nat activity of thp- was comparable to those found in primary keratinocytes. a h treatment of thp- cells with physiologically relevant concentrations of ppd ( - µm) led to a % reduction of nat activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already h after application of ppd or mappd while nat mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat protein after h. the greatest inhibition was found for oxidised ppd (up to %). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec . . . ). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened bp of the '-flanking region of gla in patients with fabry's disease and controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy ) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs ; rs ; rs ; rs ; rs ; all minor alleles p< . ). we identified two regions, a proximal one between - and - and a distal region between and - with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to . -fold (p< . ). in ihke cells, insertion of the minor t allele (rs ) significantly enhanced basal ta of the proximal promoter region (p= . ), while insertion decreased basal ta (p< . ) of the distal promoter portion. the combined insertion of the minor c alleles (rs ; rs ), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p= . ). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c : -cer levels were . fold increased in ms patients. this translates into the finding that c : -cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c : -cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers and its product c : -cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c : -cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers and therefore to limit the inflammatory effects of c : -cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a lung epithelial cells, hepg liver epithelial cells and hk- proximal tubule epithelial cells. the used nanomaterials incorporated five tio samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from . to µg/cm for cytotoxicity and from to µg/cm for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst- assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp project enpra (fp -nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v cells expressing human cytochrome p b schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v lung fibroblasts (v cells) represents a widely-used mammalian test system to detect gene mutations. since v cells do not express any cytochrome-p dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v cells expressing human (h) cyp isozymes have been commercialized. to test these v cells for their use in the hprt test, v h a and h b cells were characterized regarding (i) spontaneous frequency of -thioguanineresistant clones per clonable cells (smf), (ii) the stability of which over weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v cells (w : ± ; w : ± ) and v h a (w : ± ; w : ± ) only varied within the range of historical controls, smf of v h b increased continuously over time (w : ± ; w : ± ). (iii) although the smf of v and v h a were similar, the mutational spectrum of v cells was characterized by as many transversions as transitions and deletions of exon or exon + , whereas the mutational spectrum of v h a was characterized exclusively by transversions and deletion of exon + . surprisingly, with out of cdnas derived from v h b mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v h b , cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between . ± . and . ± . . yet its smf was unstable reaching up to ± . in conclusion, the mutational spectrum differed between the v cell lines. furthermore, h b expression seemed to enhance smf in v cells. even though a temporary reduction of the smf by cloning was possible, smf of v h b cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and d/ d h/ c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into fractions by gradient elution open cc on silica gel. the most active fractions - were separated again yielding subfractions. this afforded , -di-o-α-linolenoyl- -o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol- -o-β-dglucopyranoside, the structures of which were determined by esi-ms and d/ d h/ c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid , -di-o-α-linolenoyl- -o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats ( - weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca + selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai and trpc in plasma membrane microdomains of rbl- h mast cells. orai -mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc . this negative impact of trpc on icrac was independent of channel function as the trpc pore dead mutant (e k) inhibited icrac to a similar extent as wild type trpc . importantly, despite a reduction in icrac, nfat translocation in trpc overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc e k but substantially reduced by trpc mutants with either i) eliminated fkbp /calcineurin binding (p q) or ii) deficiency in pkc phosphorylation (s a). moreover, inhibition of pkc phosphorylation by (gfx x; µm) strongly suppressed nfat signaling. we suggest trpc as a scaffold that links orai-mediated ca + -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to ± % of the initial airway area (%-iaa) and ± %-iaa, respectively. in frequency response curves half maximal response was found at . ± . hz for guinea pig pcls and . ± . hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to ± %-iaa. % of human pcls were responsive to capsaicin and airways contracted to ± %-iaa, respectively. in gp ruthenium red and skf blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk / -pathway is involved in pkc-induced nox up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy endothelial cells with phorbol myristate -acetate (pma) for h leads to an up-regulation of nox expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox up-regulation can be attenuated by pd , (an erk / inhibitor), but not by sp (a jnk inhibitor), indicating in the involvement of erk / . consistently, pma treatment leads to a sustained activation of erk / , and sirnamediated knockdown of erk / markedly reduces the pma-induced nox up-regulation. h , an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox expression, indicating that msks are not the target molecules of erk / in this scenario. on the contrary, knockdown of the transcription factor elk- by sirna significantly reduces the pma-induced nox up-regulation. in conclusion, erk / and elk- are involved in the pkcα-induced nox up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, wuerzburg, germany annually, over , women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p , seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron of p gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with g of normal mammary gland tissue a smf of . ± . x - per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin- (il- ) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β -agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il- mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac expression, but did not affect epac and pka expression. importantly, epac expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il- release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac . polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt ) exposed to . µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt ) were exposed to . µm bap for h (n= ), wk (n= ) and wk (n= ). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. ) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. % of the differentially expressed proteins after h of treatment are involved in the processing of pre-mrna ( %) and protein metabolism ( %). this pattern changed after wk of treatment. then, % of the proteins affected the cytoskeleton whereas still % were categorized to protein metabolism and only % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins and , are currently being validate by pcr. fused master gel : representative -de gel of rt cells exposed to . µm bap for wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd ( . - µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am- (cb receptor antagonist) and by o- (g protein-coupled receptor [gpr] agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) / . blockade of erk activation by pd prevented cbd-stimulated msc migration, whereas inhibition of p mapk by sb was inactive in this respect. furthermore, am- and o- were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c bl/ wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c bl/ wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße , erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr i ) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco a and slco b and a significantly decreased expression of transporters such as slco b , slco a and abcc in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco b mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ = . ; p= . ; n= ). bisulfite sequencing revealed that promoter cpg islands of abcc and slco a were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc- and scc- cell lines, transcript expression of transporters (e.g., abcc , slco a ; p< . ) and pxr (nr i ; p< . ) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc and slco a expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco b may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc- and scc- cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn in neuropathic and inflammatory pain schnorr s. , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn - ) and contributes to cellular excitability in the heart and the nervous system. hcn and hcn are the two most abundant hcn subunits in peripheral sensory neurons with hcn being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn by using a nociceptor specific cre-transgene driven by the nav . promoter. the nociceptor-specific knockout of hcn in drg neurons (nosphcn ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn was mainly restricted to small sized drg neurons. the conditional loss of hcn resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. - , leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv , trpv , trpv , and trpv and the cold receptor trpm were insensitive towards apomorphine treatment, trpa could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa channels in a stably transfected cell line (hek -trpa ), as well as natively expressed trpa in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [ ] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [ ] , agonists of the muscarinic m receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp a and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p (cyp) a and a . the most prominent regulator of cyp a is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp a induction in murine hepatoma cells (hepa- c c ). indeed, aoh and ame enhanced the levels of cyp a in hepa- c c cells but not in cells with inactivated ahr (hepa- c c ) or arnt (hepa- c c ). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp a expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than , , scientific abstracts, and , are newly added every year. the protein sequence database uniprotkb stores over , , sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße , münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β -adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca + -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age - weeks) and subsequently used for experiments within hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca + cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. , bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca + concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca + concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc , with angiotensinii ( . mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study hours after a single instillation of µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size ( -, -and -nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to -nm and -nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. , bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc ), b f melanoma cells or human neuroblastoma cells (sh-sy y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc - deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc and b cells was indistinguishable between wild-type mice and animals lacking munc - , the number of metastases in the lung was strongly reduced in munc - -deficient animals. the strong decrease in formation of metastases in munc - deficient mice was also observed after i.v. injection of llc and b f cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient methoxy- -indolylmethyl glucosinolate and its break-down product -methoxy- indolylmethyl alcohol schumacher f. , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that -methoxy- -indolylmethyl ( -mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [ ] . we have identified and synthesized the major dna adducts n -( -mim)-dg and n -( -mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n -( -mim)-dg and n -( -mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated -mim glucosinolate. the peak levels of n -( -mim)-dg and n -( -mim)-da in the murine large intestine were reached h after a single administration of µmol -mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite -mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered -mim alcohol generated significant levels of n -( -mim)-dg and n -( -mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [ ] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., ( ) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. , , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb ) and multidrug resistance protein (abcc ), and the nuclear pregnane x receptor (pxr/nr i ), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n= ) dgf occurred in . %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs tt genotype was significantly associated with dgf (recessive model: or, . ; %ci, p= . unadjusted) , even after correction for multiple testing (p= . holm-adjusted). when we performed multivariate analysis including genetic and clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il- receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs tt genotype of the donor (recessive model: or, . ; % ci, . - . ; p= . unadjusted) which held true after correction for multiple testing (p= . ). for abcc variants only the donor rs t>a genotype correlated with dgf by univariate (or, . ; %ci, p= . unadjusted) as well as by multivariate analysis (or, . ; %ci, p= . ; table ) but not after correction for multiple testing (p= . ). for variants in the abcb gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant is c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs /fe- ). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. , freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas ) . in the cytosol, cdt adp-ribosylates actin at arg , thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (> µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp and acf . besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim that usually functions as a calcium sensor in the er and activates the store operated orai calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd- mice, and dialysates were obtained while a monofilament was inserted for min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma analyzer -rose extensively during ischemia to more than % of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after min. when bilobalide ( µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only % of baseline level (p< . ). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to - % of sham-operated mice. in the ischemic hemisphere, several values were lower at % of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of % of baseline. direct addition of bilobalide ( µm) to post-ischemic mitochondria caused a -fold increase of complex i activity in vitro. pretreatment of mice with bilobalide ( mg/kg i.p.) one hour before mcao caused a significant, -fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as -hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod- ), catalase (cat) or glutathione peroxidase (gpx / ). raw . mouse macrophages were exposed to hema ( - mm) for h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine ( µm bso), or enhanced by -oxothiazolidine- -carboxylate ( mm otc) and n-acetyl cysteine ( mm nac). expression of sod- found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod- expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx / was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h o decomposition. the inhibitory effect of hema on gpx / expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h o is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h o production leads to the activation of cat expression and a feed-back inhibition of sod- expression. the hema-induced reduction of gpx / expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e - ) , fixed to coverslips and incubated in roller tubes for about - weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx ( . µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime ( µm) was added spontaneous muscle activity in the co culture recovered from . ± . hz to . ± . hz (control . + . hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to . ± . % by obidoxime compared to control. muscle force production after indirect stimulation was stable for hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a . fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n= from isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling (rgs ) and peptidylprolyl isomerase a (ppia) were . - . fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n= ). transcripts of rgs ( . fold, ) and ppia ( . fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n= - ). we conclude that crem deficiency is associated with transcriptional changes of rgs , pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p and p subunits in regulating phosphoinositide kinase γ by gβγ and h-ras shymanets a. phosphoinositide -kinase γ (pi kγ) controls a plethora of cellular responses. pi kγ, a heterodimer formed by non-catalytic p or p and catalytic p γ subunits, is regulated by gβγ and ras. earlier we speculated that p binds to gβγ to translocate cytosolic pi kγ to the plasma membrane, enabling direct activation of p γ (brock et al., j. cell biol. ) . however, the p subunit does not function as gβγ adapter (kurig et al., pnas ) . since the impact of each non-catalytic subunit in regulating p γ by gβγ and ras still remains elusive, we studied their role in detail. gβ γ variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf cells and tested for their ability to activate pi kγ. we observed that p , but not p , was able to rescue the stimulatory activity of gβ γ mutants incapable to activate p γ (shymanets et al., biochem. j. doi: . /bj ). to further study the functional impact of the non-catalytic subunits on pi kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi kγ variants. although p /p γ exhibited stronger sensitivity to gβ γ than p γ, the activity of vesicles-associated p /p γ was significantly higher as compared to vesicles-associated p /p γ or p γ in the absence and in the presence of gβ γ . to study an effect of ras proteins on pi kγ variants, recombinantly expressed h-ras was purified from sf cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p /p γ and p /p γ differed, which may explain integration of pi kγ variants in different signalling pathways. taken together, p and p subunits implement discrete functions in respect to (i) membrane recruitment of pi kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. , leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive / /ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, ) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive / /ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, ) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. % of all saxonian citizens are inscribed to this service) for the years to was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/- % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a - % above average prescription figure during spring time (february to may) and a to % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. , freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα / and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα / family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st- cells and in primary osteoblasts from rat calvaria. st- cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st- cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c bl/ and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c bl/ or balb/c were subjected to high ( cmh o) or low pressure ( cmh o) ventilation for minutes. after minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c bl/ mice showed higher tnf, il- β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl- , cxcl- ) and il- . kinase activities (jnk, akt, erk / , p map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by % and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c bl/ and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh ) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh nanoparticles for up to days. analysis of cell viability revealed that ps-nh but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh trigger apoptosis in macrophages. we further polarized macrophages to either m or m using ifn-γ and lps or il- , respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m and m macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp . loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. , ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh ) polystyrene nanoparticles of ~ nm in diameter are internalized by human macrophages, thp- monocytic leukemia cells, and by pmadifferentiated thp- cells via different mechanisms. in buffer, macrophages and thp- rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp- cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp- cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp . specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh ) of ~ nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp inflammasome activation and the subsequent release of il- β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp protein resulting in a conformational change of the pyrin domain of the nlrp protein as predicted by molecular modeling. txnip interaction with nlrp led to assembly of the nlrp inflammasome complex, to caspase- activation, and release of il- β. using an in vitro knockdown approach, we showed that ps-nh induced activation exclusively of nlrp , whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase- activation and the subsequent release of il- β caused by ps-nh nanoparticles. these data reveal a novel mechanism of the nlrp activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp . the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a and s a . addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p /rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph . , helenalin interacts with rela (k d = . µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic of . µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg , lys , gly and ile of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph . , helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of µm, though no significant interaction was observed at ph . . thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp . prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [ ] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [ , ] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [ ] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [ ] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v . induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with ', -diamidino- -phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a (pga ), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the dpgj and pga -induced significant decrease in the tgsh level in v may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd . determination of the concentration required to cause a half-maximal increase in cd -expression (ec ) allowed a quantitative evaluation. the irritative potential of the substances was assessed by -aad ( -amino-actinomycin d)-staining. the concentration required to devitalize % of the examined cells compared to a zero control was termed ec %. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec -values ranging from . µmol/l (pentylparaben) to . µmol/l (methylparaben). due to a pronounced cytotoxicity (ec % = . µmol/l), we could not estimate an ec -value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin by protein kinase dyrk a at serine affects protein stability soppa u. septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin (sept ) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase a (dyrk a). dyrk a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept and overexpression in hela cells we identified serine as the major phosphorylation site of dyrk a and generated a phosphospecific antibody. transient coexpression of sept and dyrk a in hela cells increased phosphorylation of serine by % in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk a mutants k r and d n did not increase serine phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk a inhibitor harmine reduced phosphorylation of exogenous sept at serine about % in hela cells. furthermore, down regulation of dyrk a by rna interference lead to decreased phosphorylated serine . these results indicate that endogenous dyrk a contributes to sept phosphorylation in hela cells. finally we analyzed protein stability of wild type sept compared to the phosphorylation resistant s a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept s a mutant is more stable than wild type sept . in summary, our results suggest phosphorylation at serine by dyrk a as a novel mechanism to regulate sept stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee , bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. ) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. ) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca + influx. in the present study we analyzed whether kca channels of sk and bk type play a role in sustaining ca + oscillations at g -to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca + ]i oscillations and peak amplitude were determined using ca + indicator fura- am. results: sk , but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk inhibition by tram- dose-dependently ( , to µm) inhibits the growth of primary mammary tumor cells probably by a g cell cycle arrest. ca + oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk channels. -dependent cell cycle progression is dependent on sk activity. blocking sk disrupts a feed-forward loop that coordinates ca + influx via trp or crac channels in tumor cells. the consequences of sk inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of -hydroxymethyl- -methylpyrroline n-oxide stolze k. esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap , -dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a -carboxybutyltriphenylphosphonium-substituent to the spin trap -hydroxymethyl- -methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. , würzburg, germany type diabetes mellitus (dm ) is a growing health problem affecting more than million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o -) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn + quenching and qpcr analysis were applied. furthermore, the effect of trpc knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc was not able to function as a homomeric channel. instead, trpc subunits formed functional receptor-operated heteromeric channel complexes with trpc , , , , and . heteromers containing trpc subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc further reduced calcium permeability. in gnrh neurons endogenously expressing trpc , , , and , downregulation of trpc by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc was not involved in store-operated cation influx in gnrh neurons. moreover, trpc suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin- (trpm ) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm -like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm blockers. furthermore, we could show that these blockers are effective on endogenous trpm in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm activity in vivo. in sensory neurons, trpm may exert similar functions as trpv . thus, trpm blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec ) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): / × ec , / × ec , / × ec , and × ec . each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of hours. induced dna double-strand breaks (dsbs) were tested by the γh ax focus assay, which is a direct marker for dsbs using anti γh ax antibodies. for quantitative γh ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec values of substances were found (mmol/l;mean +/-sem): tmp(eo) ta a, b . ± . ; , -hddma b, c . ± . ; etma a, c ; . ± . ; a significantly (p < . ) differently to , -hddma, b significantly (p < . ) differently to etma, c significantly (p < . ) differently to tmp(eo) ta. after six hours of exposure with tmp(eo) ta at . mm there were induced . γ-h ax foci-formations in hgfs, at . mm . foci, at . mm . foci and at . mm . foci. after exposure with , -hddma at . mm there were induced . γ-h ax foci, at . mm . foci, at . mm . foci and at . mm . foci. after exposure with etma at . mm there were induced . γ-h ax foci, at . mm . foci, at . mm . foci and at mm . foci. the negative controls dmso and medium cultures displayed . - . γ-h ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: , -hddma > tmp(eo) ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and -hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of hours. induced dna double-strand breaks (dsbs) were tested by the γh ax focus assay, which is a direct marker for dsbs using anti γh ax antibodies. for quantitative analysis of the γ-h ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec (mmol/l) of triethylenglykol dimethacrylat (tegdma) and -hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < . ) higher dsbs compared to hema ( . ± . vs . ± . ). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed ( . ± . ), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed ( . ± . ), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed ( . ± . ), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed ( . ± . ), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys was replaced by ala (c a) resulting in destabilization of enos has been generated (enos-c a). transgenic mice carrying c a were generated on a c bl/ background using the endotheliumspecific tie- promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c a (enos-ko/enos-c a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c bl/ , enos-ko, and enos-ko/ enos-c a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c a-tg in aorta ( . ± . %, n= ), skeletal muscle ( . ± . %, n= ) and myocardium ( . ± . %, n= ) and revealed an increased phosphorylation of enos on ser / ( ± %) as compared to c bl/ (p< . , n= ). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c a-tg (p< . , n= - ), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c a as a source of elevated radical generation. endothelium-specific introduction of enos-c a at ~ % of c bl/ level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc) indicated that endothelium-dependent relaxation in enos-ko/enos-c a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p< . , n= - ). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n= - , p< . ). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c a-tg was substantially lower then in enos-ko and tended to be similar to c bl/ . conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle- tn and hbe o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk / were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn o or mno with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g / , or gq/ g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/ signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga selectively in nociceptors to investigate the contribution of the entire gq/ signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/ in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/ results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/ deficient mice, suggesting a novel function for gq/ in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/ , whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/ branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße , münster, germany ap duration and ca + cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for second ( hz; hz; hz + s -stimulus after - ms) followed by a s rest period. the resting-membrane-potential (rmp) was observed over cycles. we observed rmp-fluctuations of different length (amplitude < mv; % of observed cardiomyocytes, mean events/cell; < s: %, . ; < s: %, . ; > s: %, . ), spontaneous depolarizations (> mv; %, . ) and spontaneous aps ( %, . ). intracellular ca + transients and sarcomere shortening were measured after loading cardiomyocytes with indo- /am. after preconditioning ( min/ hz) cells were measured under basal and continuous isoprenaline ( - m) stimulation (iso). a min pacing period was followed by a min interval of no pacing. pacing frequency was reduced after each cycle ( hz, . hz, . arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca + overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord , bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi and cgi with apparent cgmp affinities of nm and nm, respectively. one mouse line expresses cgi driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm alpha promoter directs cgi expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase , and , activated caspase , and aif). exposure of hepatocytes to α-ama at concentrations of , µm, , µm and µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of µm and mm, silibinin at µm and µm and a combination of both ( µm benzylpenicillin and µm silibinin, mm benzylpenicillin and µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept µm silibinin at , µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations ( , µm and µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy ) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh ), we analyzed the amount of bh in ea.hy by hplc. a concentration-dependent reduction of bh and also bh (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch , acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh involving dihydrofolate reductase, dhfr). treatment of ea.hy cells with dex decreased mrna and protein expression of both gch and dhfr. because bh is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap- and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh tert foreskin fibroblasts (behaving like primary cells) and sv -immortalized gm fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap- binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression ( - min after exposure) was not translated into the protein, the second wave of transcription ( - h after uvc exposure) resulted in c-fos protein expression, - h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p k and erks) were immediately induced upon uvc exposure and stayed active for at least h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed - h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch / - ). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp c alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel ( mg qd), albeit carriage of cyp c loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp c genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type (octn /slc a ) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc a gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn expression in monocytes and thp- cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp- and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn expression was characterized on monocytes and thp- cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn specific signals as well as a localization in the plasma membrane. following thp- cells were activated using lps ( ng/ml) for up to h, thereby indicating the expected cytokine response as demonstrated by increased tnfα ( fold induction) and il- β ( fold induction) mrna levels. in addition, octn expression was analyzed identifying an initial reduction of around % compared to untreated cells. in parallel activated thp- cells were coincubated with increasing concentrations of the octn substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by % in the presence of mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp- cells represent a useful model to study octn expression and function. in addition, we demonstrate immunosuppressive effects of octn substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg- cells to ricinoleic acid caused an increase in [ca + ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca + ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e receptors ep and ep as mediators of ricinoleic acid-induced effects. to test if ep and ep receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep -deficient (ep -/-) or ep -deficient mice (ep -/-). while ep -/mice responded similarly to the wt mice, ep -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep , consistent with a preferential expression of the ep receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in . in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an -times (cipralex ® : → ) and -times (onbrez ® : → ), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : of ; onbrez ® : of ). an additional amount of conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports ( of ). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße , düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type and type patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc ). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in h as measured by an ha-binding protein linked assay (starved, g/l glucose: ± . %; g/l: . ± . %; g/l: . ± . %; full medium ± . %; . ± . %; . ± . %). surprisingly, total ha concentrations were about . - . fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with mg/l insulin for h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, g/l glucose: ± . % vs . ± . %; g/l: ± . % vs . ± . %). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut , glut ) were analyzed by qrt-pcr. the relative abundance was . ± . in favor of glut indicating the presence of insulin-independent glucose transport. conclusion: in osc the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type , where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße , tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb -mutated tumours. larch-derived diterpenes are potent and selective trpc blockers urban n. , kübler w. the transient receptor potential channel trpc is a poorly ca + -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc and trpc . indeed, turpentines and resins, but not coniphere oils blocked trpc and trpc in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc -prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc -selective block. larixol acetates displayed an ic towards the dag-or receptor-stimulated trpc activity of about . - µm, but did not strongly inhibit a number of other trp channels, including trpv , trpm , trpm , trpm , or trpa . selectivity for trpc compared to its closest relative, trpc , was about -fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc . electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc -/mice. we conclude that trpc blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt cells were cultured in mccoy's a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) mg of protein sample was mixed with % of coomassie blue g- (cbb g- ) and loaded in each lane of - % polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a % sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. , , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina n, a serine protease inhibitor, which is homologous to human a -antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany introduction. activated β -adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk / at threonine (erk thr phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β -receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk showed a significant increase in [ h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr -phosphorylation deficient mutants (erk t a and erk t s ) or pretreatment with the erk inhibitor, pd , significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for days to wild-type mice and transgenic mice overexpressing either wild-type erk t t or erk t s . echocardiography and histological analyses revealed that erk t s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk ) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk t a or erk t s are retained in the cytosol and prevented elk -phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr -phosphorylation. conclusion. taken together, gβγ-subunits participate in β -adrenergic receptor mediated hypertrophy by enhancing erk thr -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. , burke m. introduction: alpha-toxin, a kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation ( ). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets ( ) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp , pp or su (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr- -phospho-src from calbiochem and cell signaling, respectively ( ). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner ( . - . µg/ml of toxin). pre-incubation with src inhibitors (pp , pp or su ) reduced α-toxin-induced platelet aggregation by about %. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr- followed by a fast and complete dephosphorylation within min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr- phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin ( µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr- typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m and m prefer coupling to gi proteins, whereas the odd-numbered receptors m , m and m prefer coupling to gq proteins. with respect to ligand binding and m receptor activation, the conserved epitope trp . at the beginning of tm displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m receptor [ ] . in the inactive m receptor, it provides subtype-independent baseline affinity for allosteric antagonists [ ] . in the active receptor, m trp . affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [ ] . to study the role of trp . for m receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm wild-type-cells and hm trp . →ala-cells, amounting to . and . x receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m trp . →ala relative to m wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m and the corresponding mutant. in the case of pilocarpine, replacement of trp . by alanine in m did not reduce intrinsic efficacy. this finding is in contrast to m , where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m trp . →ala mutant relative to m wild-type. this finding is also in contrast to m , at which pilocarpine's potency was not sensitive to the trp . →ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp . →ala mutation between the m and the m receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in t and a cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca + handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. background: in atrial myocytes ca + entry through l-type ca + channels (ica,l) triggers a larger ca + release (ca + transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo- ) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, µm) and the nonselective phosphodiasterase (pde) inhibitor -isobutyl- -methylxanthine (ibmx, µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l ( . ± . pa/pf, n= / [myocytes/patients] vs . ± . pa/pf, n= / , p< . ) and cat ( . ± . nm vs . ± . nm, p< . ) were lower than in ctl myocytes, whereas diastolic [ca + ]i levels were unchanged (caf, . ± . nm; ctl, . ± . nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to . ± . pa/pf (n= / ) in caf and to . ± . pa/pf (n= / ) in ctl. the corresponding cats increased to . ± . nm in caf and to . ± . nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca + ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, + . ± . % vs ctl, + . ± . %, p< . ) and cat (caf, + . ± . % vs ctl, + . ± . %, p= . ) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, . ± . pmol/mg, n= vs ctl, . ± . pmol/mg, n= , p< . ), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ . mm /s, g-proteins ~ . mm /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g . molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee , ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s and n ) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz /bcl or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac -mediated phospholipase c-β (plcβ ) and plcγ activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ , plcβ , and plcδ , but that it had little or no effect on the activity of plcγ and plcε. the amino acid residues s and n , likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac caused inhibitory effects on rac -mediated plcβ and plcγ stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ distinct from those which are necessary for rac interaction, as the split pleckstrin homology domain of plcγ , which is essential for its interaction with activated rac , is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ and plcγ , both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ activation. effect of rac inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse , düsseldorf, germany background: the small gtpase rac is a well characterized member of the rashomologous (rho) family. rac is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap ). furthermore, rac can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac is still unclear. here, we address the question how rac influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac , human hepatoma cells were pretreated with the rac -inhibitor eht before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac inhibitor showed a higher viability, less phosphorylation of h ax (s ) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac resulted in a reduced phosphorylation of topo iiα (s ) and an increased interaction of topo iiα with hsp in doxorubicin treated cells. the data indicate that inhibition of rac protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg ) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg cdna-clone sequences were submitted to the ncbi genebank (eu , gq and gq ). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst was measured and the selectivity was determined by the bcrp inhibitor ko . inhibition studies using hoechst identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. , kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [ ] . as an α,b-unsaturated aldehyde, ac forms , -michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-( -hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-( -carbamoylethyl)-cysteine (aama), and (n-acetyl-s-( -hydroxy- -carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [ ] . nevertheless, in a pilot study in humans urinary -hpma excretion of non-smokers was reported to be about three fold higher, as compared to aama [ ] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips ( g), equivalent to an uptake of µg aa (absolute amount), together with an as yet unknown amount of acrolein [ ] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to h after test meal uptake. the results demonstrated kinetics of -hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of -hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [ ] stevens, j.f. and maier, c.s. ( ) molecular nutrition & food research ; [ ] osorio, v. m. and de lourdes cardeal, z., ( ) journal of chromatography a ; [ ] schettgen, t., musiol, a., and kraus, t., ( ) rapid communications in mass spectrometry ; [ ] ewert, a., granvogl, m., and schieberle, p., ( ) lebensmittelchemikertag protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str. , konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase (parp- ) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to fold in unstimulated [ ] and . fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine ': ' monophosphate (camp) and guanosine ': ' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic ': ' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [ ] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [ ] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr . we chose this gene because it is regulated by numerous stimuli including camp [ ] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr . esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am ( , , , µm) over to min h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb , sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr expression in hela cells. methyl-cpg-binding protein (mecp ) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp is diminished in murine and human heart failure. prevention of mecp downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp . in order to investigate the cardiac function of mecp , two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp in cardiac myocytes under control of the tetracycline-system (mecp -tg) and mice with targeted ablation of mecp in myocytes (mecp mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp expression could be completely prevented by the mecp transgene. cardiac contractility and relaxation were significantly decreased in mecp -tg animals. upon electron microscopical investigation, transgenic mecp expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp caused a rightward shift in the size distribution of mitochondria as compared with mecp -tg hearts. epigenetic processes, including the recognition of dna methylation by mecp , may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass , würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of and weeks. untreated ren rats exhibit increased blood pressure from the age of weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren animals, a significant higher superoxide production could be observed in kidneys already in week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren rats compared to control rats. as another organ affected by hypertension the heart of the ren animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d a pore mutation leads to complete inactivation of trpv channels in epididymis weißgerber p. , kriebs u. replacement of aspartate residue by alanine (d a) in the pore of trpv channels in mice disrupts ca + absorption by the epididymal epithelium resulting in abnormally high ca + concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv d a proteins (sci signal , ra , ) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv d a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv -/and trpv d a/d a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv -/and trpv d a/d a males. the profound reduction in ca + uptake by the epididymal epithelium was identical in trpv -/and trpv d a/d a males. this direct comparison of these parameters indicate that deleting trpv does not further aggravate the phenotype observed in trpv d a/d a mice, and -in our opinion -allows the conclusion that the d a pore mutant of the trpv protein leads to complete inactivation of the trpv channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n i) on herg channel function in hek cells sellmaier v. , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome (lqt ) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh or kv . ). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as isoforms ( a and b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg gene (n i) in a patient with lqt. both herg a and herg b subunits were cloned from a human heart cdna library and the specific n i mutation introduced by site directed mutagenesis. hek cells were transiently transfected with equal amounts of mutated herg a and wild type (wt) herg b cdnas and the resulting potassium current compared to herg a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at - mv for wt and - mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = ms and tslow = ms versus tfast = ms and tfast = ms in wt channels, determined from the tail current at - mv after a -second pulse to + mv. the half-maximal steady-state inactivation of the tail current was shifted by mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc haplotypes by the influence of micrornas. methods: abcc cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 'utr sequence of abcc whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc were identified form an mirna array after rifampicin stimulation of hepg cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 'utr vs. mod. 'utr) concerning the - c/ g/ c (cgc) abcc wt in hepg cells. using the fl vector construct, the expression level of cac haplotype protein was increased ( , % ± %). transfection assays with the mir- , which was identified as a candidate mirna targeting abcc mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir- was able to down regulate the abcc protein expression. there was no significance in downregulation for one abcc haplotype, respectively (modified 'utr: cgc - %; cac - %, fl 'utr: cgc - %; cac - % down regulation compared to mir-negative control). discussion: differences of abcc protein expression level are due to the epigenetic regulation of abcc haplotypes. to further characterize the effect of mir- on tcg, tgt and cgt abcc haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg , jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk effectively reduced ltb pleural levels in female but not in male rats treated with carrageenan, and mk increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk . in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, : , ): male wistar rats were exposed to the test items for h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of %, the instilled dose corresponded to the aerosol concentration of mg/m used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of - nm. in the last years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd ) with zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v cells, both showing negative results whereas the mouse lymphoma mutation test / l y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in -days, -days and -days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of . mg/m in the -day study. in a prenatal developmental toxicity study according to oecd tg , with repeated inhalation exposure to female wistar rats from gestation day through , maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. , leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. ( ) . the initial report used a database of organic substances compiled from publicly available sources. in total, noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per november , containing data for more than registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. , schumann b. b-and l-moc can be cultivated up to days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h to tpm ( mg/l) reduced viability to % or % after or h, respectively. in a viability was % after h with tpm ( mg/l). the same dose of tpm lead to a decrease in viability to % ( h) or % ( h) in nhbec and to % ( h) or % ( h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm ( mg/l). in h the induction was . and . fold after or h, respectively. while in a it was . ( h) and . fold ( h). in nhec and plc tpm ( mg/l) did not have a significant effect on gsh-levels after h. in n-moc tpm ( mg/l) also did not modulate gsh after h, but it diminished gsh-level by . fold upon prolonged contact ( h). in b-moc gsh concentrations also decreased to . or . fold the level of controls after or h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by % (wt , ± , mg/g vs. tg , ± , mg/g, p< . , n= ). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, µl mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet ( %) was performed. after days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration ( ± . ms vs. ± . ms, p< . ) and qtc interval ( ± ms vs. ± ms, p< . ) in tg (n= ) compared to wt (n= ) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße , würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca + -atpase serca a, thereby decelerating cytosolic ca + clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca + transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l a, i a and c f) or favor pentamer formation (i a, v a). transfected hek cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with . µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with . µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature ( °c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. , , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a -fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r ) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n -mb-camp and into '-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s wt und s kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s wt nor in s kincells. interestingly, we observed apoptosis in s wt and s kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. ( ) wolter s, golombek m, seifert r ( ) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s cells : induction of apoptosis in s wt and in s cells lacking the catalytic subunit of pka (s kin-) with a) db-cnmps and b) cnmp-ams for h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. , mainz, germany nebivolol is a third generation β receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment ( mg/kg/day for days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam- , vcam- ) and pro-inflammatory cytokines (e.g. il- ). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde inhibitors using two novel pde reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg a, wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde reporter cell lines. plasmid constructs encoding human pde b or pde d were stably co-transfected with the beta -adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde and non-pde inhibitors, we could show that our novel pde b and pde d reporter cell lines specifically monitor pde inhibition with high sensitivity. pde -selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta -adrenoceptor reporter cell line with no recombinant pde expression, pde inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde d from cell lysates. two different groups of pde inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d , showed high cellular activity and much better inhibition of full-length versus truncated pde d . the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde d . the results imply that these novel pde reporter cell lines are well-suited for the characterization of the cellular activity of pde inhibitors and may also support a better understanding of the complex pde pharmacology. plexin-b is required for kidney regeneration after acute renal failure xia j. , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b during kidney regeneration we generated mice lacking plexin-b specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within weeks after injury, plexin-b knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p phox) and several cytosolic regulatory components including p phox, p phox, p phox and the small gtpase rac . we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox and nox in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin (sirt ). we then wanted to find out whether the effect of resveratrol on rac was mediated by sirt . sirt is a histone/protein deacetylase. in vitro incubation of rac with sirt led to a reduction of lysine acetylation. deacetylation of rac on lysine could be identified by mass spectrometry analyses. the lysine lies within the p phox-binding region of rac . consistently, in vitro incubation of rac with sirt markedly reduced its binding activity to p phox. in conclusion, we provide evidence that rac is a direct target molecule of sirt . sirt deacetylates rac on lysine and thereby inhibits its interaction with p phox. this is a novel mechanism of nadph oxidase inhibition by sirt /resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitÄtsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany dexrazoxane (drz, icrf- ) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top ). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top b, the dominant cardiac top isoform. this was investigated using top b mutants resistant to drz, which were expressed in cells depleted of wild-type top isoforms. top b-mediated double-strand dna breaks were assessed as γ-h ax. the levesl of dsb generated by the top b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top b. preincubation with drz depleted wild-type top b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top b depletion nor the reduction of dsb by drz was seen in drz-resistant top b mutants. furthermore, the cardially ineffective drz analog icrf- , capable of iron chelation but not of top binding, affected neither the stability of top b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top b rather than by iron chelation. they also suggest a cardioprotective function of top b, which is currently under investigation using cardiomyocyte-specific top b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw were cultivated for , , and days in dmem supplemented with three different concentrations of amikacin, streptomycin ( mg/l, mg/l, mg/l), gentamicin or tobramycin ( mg/l, mg/l, mg/l). we determined the expression of two junctional proteins (connexin , ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested ( mg/l) connexin and n-cadherin were reduced to ± % and ± % of the controls (n= ). no change was recognized for vimentin ( ± %). effects obtained with streptomycin were less pronounced for these these proteins ( ± %, ± %, and ± %, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of mg/l (connexin : ± % and ± %; n-cadherin: ± % and ± %; vimentin: ± % and ± %) and mg/l (connexin- : ± % and ± %; n-cadherin: ± % and ± %; vimentin: ± % and ± %). after incubation for and days the effects occurred in the same range. the substances showed no influence on the viability of serw sertoli-cells up to mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ % inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s e) in bk-strex, resulted in a ~ % reduction of basal channel activity, whereas the s a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c : a) or by pharmacological inhibition of palmitoyl transferases with -bromopalmitate ( -bp). both, mutation and pretreatment with -bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ % inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s a channel mutant pretreated with -bp. in a clonal rat somatomammotroph pituitary cell line (gh b ), in which pcr products without (zero) and with the bp strex exon could be identified, the pkc activator pma blocked channel activity by ~ %. this inhibition was increased to over % when gh b cells were pretreated with -bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser , probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met- a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. x a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv transformed, non-malignant pleural mesothelial cell line met- a was chosen as the main in vitro model in this project. in the present study part, met- a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos ( , , and µg/cm ) was demonstrated in met- a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg modified comet assay. thus, met- a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met- a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than % cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met- a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm channels zierler s. transient receptor potential melastatin (trpm ) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm . waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg + ) concentration. mutating a mg + -sensitive site on the trpm kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg + . waixenicin a failed to inhibit the closely homologous trpm channel and did not significantly affect trpm , trpm , and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm ion channels. consistent with trpm inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg + -dependent block of trpm , waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg + . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße - , hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total water pipe strands of domestic installation systems were examined. they comprised single water samples and single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. , homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical (mtrpc ) were identified. the trpc protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc gene exist, trpc a ( aa) and trpc b ( aa), trpc b lacks aa to of the trpc a variant. both trpc variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc -interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc a (aa to ; aa to ). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc in hek cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc . in addition, changes of cytosolic calcium were monitored and trpc currents were recorded in hek cells expressing the candidate cdnas and stably expressing the trpc a or trpc b and the muscarinic receptor type cdnas. the tarbp protein, one of the candidates shown to interact with trpc , changed calcium influx when coexpressed with trpc . in order to identify the domains of trpc responsible for its interaction with the tarbp protein, six trpc -gst-fusion proteins covering the carboxyl terminal aa of trpc a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc could be identified to interact with tarbp . one of these domains is well conserved within the trpc protein, corresponding to the result, that trpc and tarbp effectively co-immunoprecipitate, too. the tarbp protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer , eif c /ago and tarbp (chendrimada et al. ) . by its interaction it may link trpc to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. , würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c bl/ -mice were equipped with osmotic minipumps, delivering ang ii in a concentration of ng/kg · min during days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type (at ) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine ´, ´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek cells that stably express hcn , hcn , hcn or hcn , respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn and hcn channels, ccmp shifted the membrane potential for half maximal activation (v . ) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec for ccmp was µm which is about times higher than the ec of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v . of ih by about + mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [ ] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [ , ] , outperformed the current standard for the therapy of actinic keratosis, -fluoruracil, when tested in the tumour cell line scc , in normal human keratinocytes and fibroblasts [ ] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the d construct of scc developed by höller and co-workers [ ] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc seeding to grow an aklike construct with scc cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki- , marker for scc: cytokeratin- , axl, marker for invasion: mmp , marker for apoptosis: caspase- , nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin- and its caspase-induced cleavage product into the culture medium following drug exposure for up to days. efficacy of a . % oxbu solution proved close to or even better when compared to both a . % fluorouracil and . % aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [ ] . -therefore, these d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other d models of skin diseases currently gaining increased interest as test platforms. ima , a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd + and cd + t-cell responses associated with improved survival walter s. , kuttruff s. ima is a novel peptide-based vaccine consisting of hla-a* and hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima vaccinations in combination with low-dose gm-csf (cohort ; n= ) or ima / gm-csf plus topically applied imiquimod (cohort ; n= ). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd + t-cell responses and by ics assay for cd + tcell responses. as immune status biomarkers, phenotypically defined myeloid derived suppressor cell populations (mdsc - ) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima overall was immunogenic in / ( %) evaluable patients, with % and % of patients mounting multiple cd + and cd + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd + cell responses as detected by ics (p= . ). multiple cd + and multiple cd + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd + and multiple cd + responses. these patients had significantly higher dcr at months (p= . ), improved ttp (p= . ) and improved pfs (p= . ) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p= . , hazard ratio . ). in the study population, levels of different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc and mdsc high frequencies were associated with shorter os (p= . and p= . , respectively). to summarize, both hla-a* and hla-dr restricted peptides in ima were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima . acrylamide (aa), a genotoxic carcinogen (iarc class a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp e into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n of guanine (n -ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-( -carbamoylethyl)-cysteine (aama), and n-acetyl-s-( -hydroxy- carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n -ga-gua dna adducts in liver, kidney and lung was measured h after application, a time point where cmax of n -ga-gua was reached. the untreated control group was found to excrete about . nmol (aama plus gama) in the urine ( h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of . µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n -ga-gua adduct levels in any organ tested (limit of detection, lod, . adducts/ nucleotides). at the tenfold higher dose ( µg/kg bw), adducts were found in kidney (about adduct/ nucleotides) and lung (< adduct/ nucleotides), but not in liver. at and µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at µg aa/kg bw (about - adducts/ nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure ( . - µg/kg bw ) leads to n -ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. , dresden health insurance costs in germany have grown by % p.a. over the last ten years and amount to approx. bn eur in . while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from to . the data of a private health insurance company with more than . customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. , , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for ) replacement of tests on juveniles and (sub-) adult rodents, ) stages with impossibility of sensation of pain in the individuals, ) highly sensitive prospects of modes of action of chemicals, which ) might show consequences in the next generations. channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav . e/r-type voltage-gated calcium channel transcriptional upregulation of cav . mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav . t-type calcium channels histidine residues in the is -is loop are critical for nickel-sensitive inhibition of the cav . calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct (slc a ) functional characterization of the human organic cation transporter variant p. ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp- tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc- (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago for microrna processing and gene silencing index a , , , , objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr c ), rs modulating blood pressure, renin, and aldosterone levels, and rs , which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels > mmol/l were defined as 'high'. after application of µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤ %), or 'strong' (> %). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n= ), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p= . ). both nr c variants were associated with a change > % in endothelial stiffness after amiloride treatment. the rs c allele was significantly associated with a strong amiloride response (p= . ), while the rs a allele only showed a trend towards stronger amiloride effects (p= . ). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr c variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. , essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p (cyp) enzymes, including cyp e . cytotoxicity of acetaminophen (aap) is based on its cyp e -catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp e -overexpressing ctnnb mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb -mutated tumors. administration of a single dose of aap ( mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about % necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb -mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp e -overexpressing hepatoma. release of , -epoxyeicosatrienoic acid ( , -eet) upon neuronal activity induces trpa -dependent mechanical pain hypersensitivity sisignano m. , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp ) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of , -eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception , -eet-levels increased in the drg and it is released from activated sensory neurons in vitro. , -eet potently induced a calcium flux [ nm] in cultured drg-neurons which was completely abolished when trpa was deleted or inhibited. in spinal cord slices , -eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, , -eet-induced enhancement of sepsc frequency was abolished in trpa null mice, suggesting that , -eet pre-synaptically facilitates spinal cord synaptic transmission via trpa . finally, intrathecal injection of , -eet caused mechanical hyperagesia in wild type but not trpa null mice. we conclude that , -eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa at central afferent terminals in the spinal cord. sisnaiske j. , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep / ). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca +, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox by histone deacetylases siuda d. , , zechner u. , prawitt d. nox is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox -mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox transcription by histone deacetylase (hdac). in cultured human ea.hy endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox mrna expression. a similar down-regulation of nox mrna expression can be achieved with sirna-mediated knockdown of hdac . hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox promoter is significantly inhibited as well, which may explain the reduction in nox transcription. in conclusion, hdac inhibition decreases nox transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v cells solecki g. m. key: cord- - pjajl authors: moriconi, eleonora; feraco, alessandra; marzolla, vincenzo; infante, marco; lombardo, mauro; fabbri, andrea; caprio, massimiliano title: neuroendocrine and metabolic effects of low-calorie and non-calorie sweeteners date: - - journal: front endocrinol (lausanne) doi: . /fendo. . sha: doc_id: cord_uid: pjajl since excessive sugar consumption has been related to the development of chronic metabolic diseases prevalent in the western world, the use of sweeteners has gradually increased worldwide over the last few years. although low- and non-calorie sweeteners may represent a valuable tool to reduce calorie intake and prevent weight gain, studies investigating the safety and efficacy of these compounds in the short- and long-term period are scarce and controversial. therefore, future studies will need to elucidate the potential beneficial and/or detrimental effects of different types of sweeteners on metabolic health (energy balance, appetite, body weight, cardiometabolic risk factors) in healthy subjects and patients with diabetes, obesity and metabolic syndrome. in this regard, the impact of different sweeteners on central nervous system, gut hormones and gut microbiota is important, given the strong implications that changes in such systems may have for human health. the aim of this narrative review is to summarize the current evidence for the neuroendocrine and metabolic effects of sweeteners, as well as their impact on gut microbiota. finally, we briefly discuss the advantages of the use of sweeteners in the context of very-low calorie ketogenic diets. on the basis of their energy content, sweeteners can be classified into calorie, low-calorie and non-calorie compounds. calorie-sweeteners include natural sugars ( ), such as sucrose, glucose, fructose, maltose, lactose, and trehalose. they are mainly present in fruits, honey, milk, dairy products, and mushrooms ( ) and their caloric values is on average kcal/g. their sweetening power is measured in relation to sucrose, which is considered as a reference sugar ( ) . low calorie and non-calorie sweeteners provide no or few calories and are characterized by a high sweetness taste. low-calorie sweeteners include polyols or sugar alcohols, which are low-digestible compounds obtained from the replacement of an aldehyde group with a hydroxyl one ( ) . the most common polyols are sorbitol, xylitol, maltitol, mannitol, erythritol, isomalt, and lactitol; they are naturally found in fruits, vegetables, and mushrooms ( ) . non-calorie sweeteners are mostly obtained by chemical synthesis (except stevia rebaudiana), and are characterized by minimal or absent nutritional content ( ) . they include saccharin, aspartame, acesulfame-k, and sucralose ( ) . in the last few decades, intake of sugar (free and added sugars) is dramatically increased, especially in western world ( ) . high intake of sugars has been related to the development of several diseases, including obesity, type diabetes, cardiovascular disease, non-alcoholic fatty liver disease ( ) ( ) ( ) , as well as tooth decay ( ) , neurocognitive diseases ( ) , and chronic inflammatory disorders ( ) . in , the world health organization (who) recommended the consumption of free sugars below % of total daily energy intake. however, a further reduction in free sugars intake below % of total energy intake has been strongly suggested ( ) . for a healthy adult, % of total energy intake is equivalent to ∼ g of sugar per day. free sugars include all sugars added to foods by the manufacturer, as well as sugars naturally present in non-intact fruit and vegetables (i.e., juiced or pureed). free sugars do not include sugars naturally present in intact fruit, vegetables, and dairy products ( ) . with regard to children and adolescents, a scientific statement published by the american heart association (aha) in recommends < g of added sugars per day, although added sugar should not be included in the diet for children < years of age ( ) . according to a recent study, in uk added or free sugar intake has been estimated between % and % of total energy intake, respectively, and it is higher in children than in adults ( ) . the prevalence of obesity and its comorbidities (such as type diabetes, cardiovascular diseases and cancer) has dramatically increased ( ) and several governments started to promote policies aimed to encourage a healthy diet and lifestyle ( ) . in denmark introduced a tax on saturated fat, which was repealed in , since it demonstrated a positive but not consistent effect on health ( ) . in the same year, hungary added levy on foods with high fat, sugar, salt and caffeine content; soft drinks and alcohols were also taxed. in , france introduced a tax on sweetened beverages ( , ) . in united states, where sweetened beverages consumption is still high, taxes on sugarsweetened beverages have been approved since ( , ) . in california, the first state which approved the tax, a % reduction of sweetened beverage consumption was reported ( ) and a positive impact on health care cost savings was observed ( ) . since , uk approved several policies in order to reduce childhood obesity, including the "sugar tax" on sweetened beverages, called the soft drinks industry levy, which became effective in april ( ) . in hungary, sugar tax resulted in a qualitative improvement and reformulation of food products ( , ) . in addition, nutrition labeling has been encouraged in order to help consumers to choose healthy foods. in , the food standards agency in the uk promoted a color coding in labeling system. in this color coding system, red, yellow, and green labels correspond to high, medium, and low percentages of fat, salt, sugar, and total energy present in the product, respectively ( ) . in this regard, some studies found that front-of-package nutrition labels can readily convey to consumers key information on the nutritional profile of different food products, showing that green labels are associated with the highest healthfulness perception of these products ( ) . however, vasiljevic et al. found that nutritional labels of snack foods had limited impact on perceptions of healthiness and no effects on the snack choice, whereas emoticon labels had stronger effects on perceptions of taste and healthfulness of snacks compared to color labels ( ) . a recent cochrane review evaluated the effects exerted in the general population by the taxation of unprocessed sugar or sugar added foods in terms of consumption of these foods and changes in prevalence and incidence of overweight, obesity, and other diet-related diseases ( ) . the authors concluded that there is still limited (and low-quality) evidence to support that taxing unprocessed sugar or sugar added foods has a significant impact on reducing their consumption and preventing overweight, obesity or other adverse health outcomes ( ) . therefore, future studies are needed to draw concrete conclusions in this direction. notably, an article by fernandez and raine ( ) recently reviewed the impact of sugar-sweetened beverage taxation on obesity, concluding that current evidence is still limited. importantly, authors suggest that sugar-sweetened beverage taxation will likely fail to have a significant impact on the prevalence of obesity and associated non-communicable diseases until this policy will not be associated with interventions aimed to increase access to non-sweetened beverages, educate consumers about healthy beverage alternatives and explore taxation of other beverages and non-nutritive foods ( ) . to address the growing health issue of obesity, sweetener consumption has gradually increased over the last years ( ) ( ) ( ) ( ) . indeed, when used judiciously, non-calorie sweeteners may facilitate reductions in the intake of added sugars, leading to decreased total energy, weight loss, prevention of weight gain and/or subsequent beneficial effects on related metabolic parameters ( ) . nonetheless, these potential benefits may not be fully achieved without reductions in total food intake and/or in presence of a compensatory increase in energy intake from other sources ( ) . in addition, animal and human studies have reported controversial results on the safety of non-calorie sweeteners ( ) . besides their potential to reduce daily calorie content, non-calorie sweeteners were reported to potentially display detrimental metabolic (weight gain) ( ) and neuroendocrine (addiction) effects ( ) . conversely, some intervention studies reported that consumption of non-calorie sweeteners is associated with weight loss and improved metabolic parameters ( , ) . despite a growing use of non-calorie sweeteners, which is gradually increasing in both healthy and obese/overweight individuals, there is indeed a knowledge gap regarding their safety and efficacy in the long term-period. therefore, there is an urgent need to update the current positions from international agencies on the use of these compounds. we will review here the metabolic and neuroendocrine properties of the most commonly used low-calorie (polyols) and non-calorie sweeteners, along with their safety profile and main use in food industry. sugar alcohols (also referred to as polyols) are characterized by a lower calorie content ( to kcal/g) ( ) than sucrose ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , or high gi (≥ ) ( ) . c source ( , ) . d source ( ) . e source ( ) . ( table ) ( ) . low amounts of polyols are naturally present in vegetables, mushrooms and fruits (melon, peach, apple, pear, apricot), but also in oat ( , ) . they include hydrogenated mono-, di-, oligo-, and polysaccharides ( ) and are mainly used in "sugar-free" products, sweets, and chewing gums ( ) . polyols are stable compounds at high temperatures and do not interfere in the maillard reaction ( ) . the late stages of maillard reaction lead to the generation of the so-called ages (also referred to as "advanced glycation end-products") in foods and biological systems ( , ) . of note, ages contribute to the development of micro-and macrovascular complications of diabetes ( ) ( ) ( ) , by inducing oxidative stress and activating inflammatory pathways ( , ) polyols do not affect glucose homeostasis ( , ) . also, polyols have long been suggested as valid sugar substitutes able to exert a beneficial role on insulin resistance and glucose control in patients with type diabetes and/or metabolic syndrome. nonetheless, robust evidence on the longterm effects of polyols in terms of glucose control and chronic complications in diabetic patients is still scarce and inconclusive ( ) . interestingly, most of these compounds do not undergo fermentation by oral bacteria flora ( ); therefore, polyols can reduce the risk of tooth decay because they represent a poor source of energy to resident bacteria of the oral cavity and do not create an acidic environment ( , ) . polyols increase saccharolytic anaerobic and aciduric bacteria in the colon and give rise to the production of short-chain fatty acids which play a key role in the maintenance of the intestinal epithelial barrier ( ) . although acceptable daily intake (adi) dose has not been established for polyol increased polyol consumption may cause gastrointestinal discomfort and laxative effects in healthy individuals ( , ) . the european union legislation approved the use of seven different polyols, including erythritol, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol ( ) . herein, we summarize the main properties of the polyols that are most commonly used in food and beverage industry, also discussing their potential impact on human health. sorbitol provides . kcal/g. sorbitol is naturally present in grapes, prunes, cherries, peaches, apples, pears, and fruit juices ( ) . sorbitol is poorly absorbed in the small intestine, while in the colon it is converted by gut microbiota into gases and shortchain fatty acids, providing energy ( ) . sorbitol has osmotic effects and it acts as a laxative when ingested in high doses ( - g) ( ). in addition, chronic ingestion of sorbitol through chewing gums can cause increased intestinal motility regardless of its osmotic effect ( ) . therefore, sorbitol use should be avoided by individuals with irritable bowel syndrome ( ) . in fda approved sorbitol use as generally recognized as safe (gras) ( ) . mannitol is naturally present in mushrooms, marine algae, strawberries, onions, and pumpkins ( ) . only % of ingested mannitol is absorbed in the gut, whereas the remaining part is excreted in the urine. in the gut, mannitol is slowly fermented ( ) . mannitol is virtually inert and does not interfere with pharmacological compounds. due to this reason, it is used also in hygiene products, drug filler and intravenous fluid solutions ( ) . moreover, the osmotic diuretic properties of mannitol account for its use as intravenous solution in the management of elevated intracranial pressure and cerebral edema ( ) . xylitol is a natural sweetener found in fruits, vegetables and oats, and it is extracted from birch trees ( ) . due to its low caloric content ( . kcal/g) and low glycemic index, xylitol has long been suggested as a valid alternative to glucose and sucrose in patients with diabetes ( ) . in a pre-clinical study, week administration of xylitol at high doses has been shown to improve glucose tolerance in rats ( ) . conversely, a randomized, placebo-controlled, crossover trial conducted in lean and obese volunteers showed that acute xylitol and erythritol ingestion did not significantly affect circulating levels of glucose and insulin, despite being able to stimulate the secretion of the gut hormones cholecystokinin (cck) and glucagon-like peptide- (glp- ). of note, the marked increase in cck and glp- levels was accompanied by a significant slowing in gastric emptying ( ) . these findings are interesting and do not exclude that chronic ingestion of these sweeteners may play a role in the regulation of glucose homeostasis. also, the increase in glp- levels may have relevant clinical implications beyond the insulinotropic action of glp- , considering the well-known role exerted by glp- receptor agonists in the reduction of cardiovascular risk among diabetic patients, along with the anorexigenic properties of glp- and its analogs ( ) ( ) ( ) . remarkably, cck has also been shown to induce short-term satiety and to play a role in the regulation of insulin secretion and overall β-cell function and survival, displaying complementary biological actions with those exerted by glp- ( ). erythritol is a polyol contained in fruits (e.g., melon, peach), wine and beer ( ) . it is chemically derived from the fermentation of natural sugars (e.g., glucose and sucrose) by trichosporonoides megachiliensis ( ) . its sweetening power corresponds to - % of that of sucrose ( ) . erythritol is poorly absorbed in the jejunum and is excreted unmodified in the urine ( ) . only a small fraction of erythritol undergoes gut fermentation. therefore, an excessive consumption of erythritol can be associated with laxative effects ( ) . gastrointestinal discomfort is generally observed when erythritol intake is > , mg/kg of body weight ( ) . erythritol intake does not appear to have detrimental effects on glucose control and its use is generally deemed as safe in patients with diabetes ( ) . similarly to other polyols, erythritol does not participate in maillard-type reactions and, therefore, does not cause the production of ages. in addition, by acting as a scavenger for hydroxyl radicals, erythritol exerts anti-oxidant and endothelium-protective properties ( ) . erythritol provides a negligible amount of energy ( . kcal/g) ( ) . thus, it is commonly used as part of the dietary patterns recommended for people with obesity ( ) . due to its sweet taste and high digestive tolerance, and the fact that it is virtually calorie-free and non-cariogenic, erythritol is widely used in the food and beverage industry. non-calorie sweeteners (also known as artificial sweeteners or non-nutritive sweeteners) are defined as compounds with high sweetening power. although most of them do not provide calories upon ingestion, some of these compounds (such as aspartame and stevia rebaudiana) have a measurable caloric value that is considered negligible at the doses commonly used by humans. non-calorie sweeteners can be of synthetic or natural origin ( table ) . stevia rebaudiana has a natural origin. it is commonly called stevia and derives from a plant that grows in south america ( ) . stevia contains steviol glycosides, stevioside, and rebaudioside a, that account for its sweet taste, and other minor glycosides, such as rebaudioside b, rebaudioside c, rebaudioside d, rebaudioside e, rebaudioside f, dulcoside a, rubusoside, and steviolbioside. stevia also contains a complex of terpenes, tannins, sterols, vitamins, carotenes, flavonoids, and other microelements ( ) . after ingestion, the steviol glycosides contained in stevia are ( , ) . b source ( , ) . c source ( , ) . d source ( ) . not digested in the upper gastrointestinal tract ( ), but they are metabolized by bacteria of the bacteroidaceae family in the colon, resulting in the production of steviol ( ) , which is subsequently processed in the liver and converted into steviol glucuronide ( ) . energy from fermentation of steviol glycosides (usually assessed as kcal/g) is low ( ) . stevia has a strong sweetening power, -to -fold higher than that of sucrose ( ) . its maximal recommended daily intake is mg/kg, and it is considered unsafe at higher doses (eu regulation / ) ( ) . this quantity corresponds to approximately nine tablets per day. considering that stevia is to times sweeter than sugar, it is extremely unlikely for an individual to ingest the maximum dose of mg/kg over a h period. stevia offers several advantages over other non-calorie sucrose substitutes. in vitro, stevia displayed antiviral effects ( ) , immunomodulatory activity ( ) and antiinflammatory properties, through inhibition of nf-κb and pro-inflammatory cytokines expression ( ) . in rats, stevioside showed antihyperglycemic effects through the enhancement of the first-phase of insulin secretion with a concomitant suppression of glucagon levels; stevia also caused a pronounced reduction of both systolic and diastolic blood pressure in rats ( ) . intriguingly, pre-clinical evidence suggests that steviol glycoside derivatives can exert antiproliferative properties in several cancer cell lines, including pancreatic ( ), breast ( ) , and gastric cancer ( ) cell lines. aspartame was discovered in ( ) . it provides kcal/g, but it is included in the group of non-calorie sweeteners, due to its strong sweetening power ( ) . it is composed by phenylalanine, aspartic acid and methanol ( ) . given the high content in phenylalanine, aspartame use is contraindicated in individuals with phenylketonuria, a rare autosomal recessive inborn error of metabolism characterized by a decreased metabolism of the amino acid phenylalanine ( ) . although the use of aspartame has been approved in united states since ( ) and in europe since ( ), its safety is still debated. after several studies performed during the s and the s ( - ), a long term study was carried out in rats to assess its carcinogenic potential ( ) . rodents treated with different dosages of aspartame until their natural death showed an increase in the frequencies of lymphomas and leukemias, carcinomas of the renal pelvis and ureter, and schwannomas ( ) . these results were confirmed even at doses of mg/kg body weight, which are lower than the recommended maximum daily intake in europe and in united states ( ) . the potential carcinogenicity of aspartame was first attributed to methanol, that is converted into formaldehyde and then into formic acid both in rats and humans ( ) . based on data obtained from different studies, the european food safety authority (efsa) was called in to re-evaluate the safety of aspartame on human health. efsa concluded that aspartame is safe at a dose of mg/kg body weight/day ( ) . however, aspartame safety on human health is still under debate; in fact, a recent study highlighted several important shortcomings in the efsa document ( ) . acesulfame-k is a potassium salt of -methyl- -axanthiazine- ( h)-one , -dioxide. its sweetening power is -fold higher than sucrose ( ). acesulfame-k does not provide calories. since it is not catabolized in humans, acesulfame-k does not affect serum potassium levels despite its potassium content ( ) . the acceptable daily intake (adi) of acesulfame-k is mg/kg body weight. it is used in various sweet foods and beverages ( ) . hydrolysis of acesulfame-k gives rise to acetoacetamide, a degradation product that can be toxic if produced in large amounts ( ) . acesulfame-k carcinogenicity has been investigated in rats, where no carcinogenic effects were observed ( ) . the majority of studies noted that it displays neutral effects on body weight or glucose tolerance ( , ) . sucralose is derived from sucrose after replacement of three chloride atoms with three hydroxyl groups ( ) . it was discovered in and has a sweetening power -to fold higher than sucrose. the adi of sucralose is mg/kg body weight in united states ( ) and mg/kg body weight in europe ( ) . only up to - % of sucralose is absorbed in the gastrointestinal tract, while the remaining undergoes intestinal excretion unmodified ( ) . sucralose is stable during baking and it is considered safe in beverages and foods that require cooking ( ) . sucralose consumption does not affect glycemic control or insulin sensitivity in healthy individuals when administered alone, whereas its use in combination with carbohydrates showed a negative impact on glucose metabolism ( ) . several studies in vitro ( , ) and in vivo ( , ) demonstrated that sucralose is not a carcinogenic compound. only two studies noted a positive relationship between sucralose and mutagenic activity. in one, two human colon cancer cell lines (caco- and ht- ) and one human embryonic kidney cell line (hek- ) were exposed to very high doses of sweetener solutions for up to , , and h, leading to cell alterations and dna fragmentation ( ) . in the other, mouse lymphoma cells showed doubtful results when exposed to mg/ml sucralose concentrations ( ) . however, in both studies very high sucralose concentrations were used. recently, soffritti et al. raised questions regarding sucralose safety ( ) , although efsa revaluation judged that these results were not supported by the available data ( ) . saccharin is a non-calorie sweetener derived from , benzoisothiazol -( h). its sweetening potency is almost -fold higher than that of sucrose. saccharin has an unpleasant bitter or metallic taste ( ) . experiments conducted in the s have showed a link between saccharin and an increased incidence of bladder cancer in a rat strain genetically susceptible to bladder tumors, when exposed to % saccharin in the diet for weeks ( ) . nonetheless, saccharin generates a urinary precipitate mainly composed of calcium phosphate, which can exert cytotoxic effects on urothelial cells of rats and induce mild hyperplasia ( ) . however, very high saccharin concentrations were tested in animal models, if compared to the doses commonly ingested by humans ( ) . with regard to metabolic parameters, a recent study evaluated the administration of saccharin (at different doses, namely: . , , and mg/kg) in male wistar rats ( ) . an increased body weight was noted in rats after and days of mg/kg saccharin treatment. authors also observed an increase in glucose, uric acid and creatinine levels, as well as in oxidative status in the liver of saccharin-treated rats, suggesting that saccharin may impair glucose homeostasis, induce obesity and lead to impairments in kidney and liver function ( ) . the world health organization and the eu scientific committee for food declared saccharine as safe up to the approved daily intake doses ( mg/kg body weight) ( ) . nowdays, saccharine is commonly used in soft drinks, baked foods, jams, canned fruit, candy, dessert toppings, and chewing gum ( ) . the consequences of low-calorie and non-calorie sweeteners on daily food consumption and eating behavior are still controversial. eating causes an amplification of dopamine release in the nucleus accumbens, similar to what occurs upon substance abuse ( ) . however, the ingestion of palatable foods causes an increase in dopamine production greater than standard food ( ) . given that palatable foods stimulate the same neural pathways involved in drug addiction, it has been suggested that an excessive sugar intake can lead to addiction. moreover, after long-term consumption of sugar, withdrawal symptoms have been described in rats, similarly to what has been observed in morphine and nicotine dependence ( ) . food addiction leads to changes in the expression of dopamine receptors ( ) . neuroimaging studies revealed that obese individuals exhibit lower dopamine sensitivity in nucleus accumbens accompanied by a decrease in dopamine d receptor expression, similar to what has been observed in drug-addicted subjects ( ) ( ) ( ) ( ) . interestingly, dopamine d receptor expression is inversely related to body mass index in obese patients ( ) . these findings suggest that chronic exposure to sugar decreases dopamine-d receptor expression. furthermore, it has been hypothesized that obese subjects respond to dopamine deficiency by overeating palatable foods ( ) . several brain regions are involved in food-reward, namely: lateral hypothalamic area (lha), ventral tegmental area (vta), nucleus accumbens (nac) and prefrontal cortex (pfc). several neurotransmitters (gaba, glutamate and opioids) are involved in different aspects of reward in the above-mentioned brain regions ( , ) . in particular, the dopaminergic circuitry from lha to vta and from vta to the nac is involved in hedonic processes ("liking"), reinforcement ("learning"), and motivation ("wanting") ( ), while acetylcholine is involved in the aversive aspects of withdrawal ( ) . during withdrawal state, extracellular dopamine decreases in the accumbens, while acetylcholine is released from accumbens neurons. intermittent or excessive sugar consumption induces neurochemical modifications, mimicking the effects of opioids ( ) . food choice and food intake are physiologically regulated by metabolic and neural signals. in particular, metabolic signals act as nutritional status sensors and mediate the ingestion of a sufficient amount of energy. on the other hand, sensory signals regulate food choice and are linked to subsequent metabolic adaptation, resulting in conditioned responses to these foods ( ). the combination of learned responses with metabolic and sensory signals results in a specific pattern of food intake. the responses to sensory inputs, such as taste, texture, and sight of food, include consecutive preabsorptive physiological responses, which are collectively referred to as cephalic phase responses. such digestive preparation confers to the body the ability to anticipate the particular challenge a food poses for maintaining energy homeostasis ( ) . among these responses, the cephalic-phase insulin response, elicited by sugars, enhances glucose tolerance in humans ( ) ( ) ( ) . although replacing calorie with non-calorie sweeteners definitely reduces the energy density of foods and beverages, this does not necessarily translate into metabolic advantages and improved health status. it has been hypothesized that daily intake of non-calorie sweeteners can "trick" the brain by encouraging sugar craving and addiction ( ) . indeed, lack of calories generally abolishes the post-ingestive food reward mediated by the hypothalamus ( ) . in keeping with this, it has been suggested that uncoupling sweet taste from energy causes progressive weakening of conditioned responses to sweet taste ( ) . as previously mentioned, sweet taste is able to evoke physiological adaptations which play an important role in the finely-tuned regulation of energy homeostasis, by sensing the presence of caloric nutrients in the gut and facilitating the absorption and subsequent utilization of energy. when sweeteners are not associated with caloric intake, their ability to sense energy is altered, with a subsequent reduced ability to use energy and a mitigated activation of the peripheral and central pathways that promote the feeling of satiety ( ) . it is also known that non-calorie sweeteners evoke different brain responses compared to calorie sugars. in particular, sucralose is known to display reduced ability to activate midbrain areas related to reward, including lha, vta, and nac ( ) . indeed, given the critical role of melanin-concentrating hormone (mch) neurons in the lha in establishing nutrient preference, a preclinical study showed that sucrose activated mch neurons, resulting in dopamine release (da). by contrast, sucralose was able to induce da release in mice only in the presence of light stimulation, which led to the activation of mch neurons. these findings suggest that non-calorie sweeteners require additional stimuli to obtain the same rewarding effect of sucrose (figure ) ( ) . significant controversy exists over the effects of low-calorie sweeteners on metabolic health. studies conducted on rodents ( ) and humans reported a positive association of low-calorie sweetener consumption with weight gain and/or diabetes ( ) ( ) ( ) , other studies a positive association with lower bmi and weight loss ( , ) , in other cases their use was not related to metabolic parameters ( , ) . such heterogeneity is probably due to methodological limitations of some of these studies ( ) . a recently published study shed some light on the controversy regarding the effects of low-and non-calorie sweeteners on metabolic health ( ) . authors reported that consumption of sucralose was able to rapidly impair glucose metabolism and brain response to sweet taste in healthy subjects only when administered in the presence of carbohydrates. in fact, insulin sensitivity as well as neural responses to sugar were not altered by sucralose or carbohydrate alone. notably, the combined effect of sucralose and carbohydrates was even more pronounced in adolescents, who showed a dramatic increase in insulin resistance measured by the homeostatic model assessment for insulin resistance index (homa-ir). these findings refute the "sweet uncoupling hypothesis, " which is based on the concept that uncoupling sweet taste from caloric content could determine metabolic dysfunctions and reduce the potency of sweet taste ( ) . these data may help explaining the obesogenic potential of low-calorie sweeteners in the context of western diets, especially considering the frequent use of "diet drinks, " often containing non-calorie sweeteners, associated with carbohydrates-rich meals. the sweet taste perception begins with the activation of taste receptors of the tongue, which are located within the taste buds of lingual papillae ( ) . taste receptors include g protein-coupled figure | brain reward circuitry involved in central effects of sweeteners. the dopaminergic pathway is strictly involved in hedonic processes ("liking"), reinforcement ("learning"), and motivation ("wanting"). midbrain dopaminergic circuits include lateral hypothalamus (lha), ventral tegmental area (vta), and nucleus accumbens (nac). dopamine release is driven by orexin (orx) peptides and melanin-concentrating hormone (mch) secreted by lha. in particular, orx and mch neurons from lha project to vta, where orx peptides and mch mediate the activation of dopamine (da) neurons and increase the release of da in projection areas such as the nac. it has been established that dopamine reward pathway response induced by caloric sweeteners consumption, such as sucrose, is greater compared to non-calorie sweetener sucralose. interestingly, a preclinical study provided evidence that mch neurons account for the natural preference for sucrose over sucralose and that such effect can be reversed by stimulating mch neurons with light. this suggests that non calorie-sweeteners require additional stimuli to obtain the same rewarding effect of sucrose. receptors and ion channels. type taste receptors (t rs; sweettaste and umami receptors) and type taste receptors (t rs; bitter-taste receptors) are both g protein-coupled receptors ( , ) . the activation of these receptors generates second messengers, such as inositol trisphosphate and diacylglycerol, leading to the activation of taste-transduction channels ( ) . these metabolic pathways project to brain circuits, allowing the appreciation of taste. sweet-taste receptors are also expressed outside of the oral cavity. they have been found throughout the gastrointestinal tract, particularly in the enteroendocrine l and k cells ( , ) . besides the gastrointestinal tract, sweet-taste receptors have also been found in pancreatic β-cells ( ) , bile ducts ( ) , and lungs ( ) . in the gut, glucose is absorbed through sodium-dependent glucose cotransporter- (sglt- ) localized on the luminal membrane and the passive glucose transporter (glut ) on the basolateral membrane of the enterocyte ( ) . glucose binding to sweet-taste receptors present on enteroendocrine cells leads to glp- and peptide yy (pyy) secretion from l-cells, promoting satiety ( ) . since both non-calorie and low-calorie sweeteners bind to sweet-taste receptors present in the oral cavity and subsequently lead to the sweet taste perception, it has been hypothesized that noncalorie sweeteners and low-calorie sweeteners may activate the same sweet-taste receptors expressed on enteroendocrine cells, promoting gut hormone secretion. in vitro, sucralose has been shown to stimulate glp- secretion from a human l-cell line (nci-h cells) ( ) and to increase glp- and glucosedependent insulinotropic peptide (gip) release in a murine enteroendocrine cell line ( ) (figure ) . however, these results were not confirmed in vivo. several studies showed no effect of oral sucralose ( ) , aspartame, and acesulfame-k on glp- , pyy, ghrelin, or gip secretion ( ) . these results have been confirmed also for sucralose administration before a solid meal, which did not elicit any effects on gip or glp- release ( ) . nevertheless, other studies showed controversial results. in fact, non-calorie sweeteners ingested through diet soda have been shown to synergize with glucose to enhance glp- release in healthy subjects, even if it is unclear whether this effect was due to the activation of sweet-taste receptors present on taste buds of lingual papillae and/or enteroendocrine cells, or if it was due to other mechanisms ( ). moreover, non-calorie sweeteners (such as sucralose, acesulfame-k and saccharin) have been shown to increase glucose absorption in the small intestine by up-regulating sglt expression in mice through the activity of enteric neurons ( ) . indeed, glp- stimulates the release of neuropeptides, which in turn bind to g protein-coupled receptors (expressed on the basolateral membrane of enterocytes) and induce sglt expression ( ) . in vivo, these sweeteners displayed the same effects by increasing glut expression at the level of the apical membrane ( ) . giving the ability of non-calorie sweeteners to increase sugar absorption during a meal, it is worth considering their potential implications in terms of metabolic effects. the recent report that simultaneous consumption of sucralose and maltodextrin-derived glucose acutely disrupts glucose tolerance and insulin sensitivity ( ) may be a likely consequence of the increased intestinal glucose absorption following the upregulated expression of sglt and/or glut . in vitro studies demonstrated that sucralose, saccharin, and acesulfame-k stimulate pancreatic β-cell insulin secretion in the frontiers in endocrinology | www.frontiersin.org presence of glucose ( ) . the natural non-calorie sweetener stevia is also able to enhance glucose-induced insulin release, through inhibition of atp-sensitive k-channels ( ) and stevioside has been shown to reduce plasma glucose levels in mice ( ) , postprandial glucose and glucagon levels in subjects with type diabetes ( ) . finally, mechanistics studies are needed to better elucidate the effects of different sweeteners on gut hormone-mediated glucose homeostasis in humans. figure depicts some of the effects of non-calorie sweeteners on enteroendocrine cells and gut hormones secretion, which have mainly been inferred from in vitro and animal studies. it has been hypothesized that the relationship between sugar, metabolic syndrome and its related disorders may be mediated, at least in part, by changes in the gut microbiota ( , ) . in particular, increased added sugar and novel sweeteners consumption may alter the carbohydrate pools in the gut, thus creating distinct environments that can favor adaptation and enhance colonization and virulence of some endogenous and/or exogenous pathogenic microbes ( ) . since most sugars and sweeteners are absorbed at the level of the small intestine by sugar transporters, only up to % of these compounds reach the large intestine ( ) . therefore, the small intestine environment is more enriched in sweeteners compared to large intestine. moreover, microbes in the small intestine have a greater number of carbohydrate uptake and utilization genes and transcripts compared to those in the large intestine ( ) . gut microbiota composition displays a remarkable spatial heterogeneity across the gastrointestinal tract, with different microbial communities creating distinct microenvironments at different gut locations (a phenomenon also known as "gut biogeography") ( ) . di rienzi and britton recently proposed that the variation in microbial communities found along the gastrointestinal tract can also depend on the variation in sugars and sweeteners present at different gut locations ( ) . furthermore, changes in sugar and sweetener pool can lead to adaptation of the gut microbiota involving transcriptional, metabolic and compositional changes in gut microbes. these changes allow for increase in abundance of microbes whose niche and microenvironment are best filled. in addition, genetic changes can also allow existing microbes to alter their niche in order to better utilize the new nutrient pool ( ) . suez et al. demonstrated that saccharin-fed mice developed glucose intolerance as a consequence of compositional and functional alterations to the gut microbiota ( ) . similarly, germ-free mice receiving fecal transplantation from saccharin-fed animals displayed glucose intolerance, suggesting that derangements in glucose metabolism are mediated by saccharin-induced alterations to the gut microbiota ( ) . similar findings were reproduced in healthy human subjects consuming saccharin for week ( ) . importantly, saccharin-fed mice showed a reduction in akkermansia muciniphila, a mucin-degrading bacteria with probiotic properties associated with favorable metabolic effects ( ) . saccharin consumption has also been shown to affect microbiota composition in rats; the growth of six bacterial strains (three lactobacillus species and three escherichia coli strains) was inhibited and the fermentation of glucose was decreased ( , ) . mice treated with saccharin ( . mg/ml) for months showed gut dysbiosis, which is broadly defined as any change in the composition of resident commensal microbial communities relative to the community found in healthy subjects ( ) , along with an increased expression of pro-inflammatory inducible no synthase (inos) and tnf-α in liver ( ) . sousa et al. clearly demonstrated that a specific diet can alter gut microbiota by changing the abundance of specific strains. of note, authors inoculated mice with an escherichia coli strain carrying a mutation disrupting the ability to consume galactitol (a galactose-derived sugar alcohol) ( ) . surprisingly, part of the e. coli population regained galactitol metabolism, resulting in the coexistence of two distinct strains within the gut microbiota that could or not consume galactitol (galactitol-positive and galactitol-negative strains, respectively) ( ) . apart from the role of sweeteners as nutrients for gut microbiota, it is worth considering that some of these compounds may exert direct toxict effects on specific microbes. for instance, xylitol cannot be metabolized by oral bacteria ( ) and is therefore added to oral products or chewing-gums. also, stevia glycosides (stevioside and rebaudioside a) have been shown to inhibit the growth of strains of lactobacillus reuteri, a symbiotic lactobacillus species which inhabits the gastrointestinal tract of mammals and is often administered as a probiotic additive in healthy foods ( ) . however, inulin and fructans-contained in the roots of stevia-favored the proliferation of bifidobacteria and lactobacilli in a pre-clinical study ( ) . stevia also showed a bactericidal effect on enterohemorrhagic escherichia coli ( ) . also, some sugar alcohols have been shown to promote an increase in the number of beneficial gut microbes both in rats and in healthy human volunteers ( , ) . mice fed a high-fat diet plus xylitol showed a reduced fecal content of bacteroidetes and barnesiella, along with an increased abundance of firmicutes and prevotella ( ) . moreover, xylitol promoted a substantial change in rodent fecal microbiota, decreasing gram-negative and increasing gram-positive bacteria ( ) . of note, the ability of sweeteners to affect gut microbiota composition may also have important immunological implications. in this regard, a recent study conducted on streptozotocin-induced diabetic mice and non-obese diabetic (nod) mice-which are both widely used as animal models of human type diabetes-has shown that trehalose, a natural caloric disaccharide derived from a rodent intestinal nematode and characterized by antioxidant properties ( ) , is able to affect gut microbiota by increasing the abundance of ruminococcus spp. ( ) . such change in intestinal microbiota composition appears to account for the induction of cd + regulatory t cells, which play a role in inhibiting the onset of diabetes and reducing blood glucose levels in diabetic animals ( ) . in addition, authors found that patients with type diabetes, when compared to healthy volunteers, had fewer cd + regulatory t cells, as well as lower serum trehalose concentrations and fecal content of ruminococcus ( ) . these results suggest that trehalose may have a potential prophylactic and/or therapeutic role in humans (e.g., use of trehalose and ruminococcus strains as a prebiotic and probiotic, respectively), as a tool to induce cd + regulatory t cells in order to prevent the development of type diabetes and/or counteract the immune-mediated β-cell destruction shortly after the onset of the disease. since sweeteners cross the placenta ( ) and are found in the maternal milk ( ) , there is the potential for a relationship between prenatal exposure to different sweeteners and gut microbiota composition later in life. animal studies indicate that acesulfame-k crosses the placenta during pregnancy and can potentially lead to an increased sweet preference during adulthood ( ) . on the other hand, prenatal sucralose exposure does not affect fetal organogenesis ( ) , but increases the risk for hematopoietic neoplasia in male mice ( ) and favors adipocyte differentation in cultured pre-adipocytes ( ) . these results suggested that non-calorie sweeteners consumption during pregnancy could impact on offspring adipose tissue differentiation, promoting childhood obesity ( ) . moreover, exposure of pregnant mice to a mixture of acesulfame-k and sucralose at different concentrations demonstrated that non-calorie sweeteners were able to affect gut microbiota composition in the offspring in a dose-dependent manner, increasing firmicutes content and reducing the amount of beneficial species, including akkermansia muciniphila ( ) . in conclusion, early prenatal exposure to specific non-calorie sweeteners could favor the occurrence of metabolic diseases later in life, by inducing detrimental changes in the gut microbiota composition. the aforementioned findings support the notion that sweetener consumption modifies the nutrient environment in the gut and induces a series of functional changes in the gut microbiota, which potentially result in transcriptional, metabolic, compositional, and/or genetic adaptation by gut microbes. in turn, microbial adaptation to sweeteners may affect host-microbe interaction and influence the subsequent immune responses (e.g., pro-inflammatory, anti-inflammatory, immune responses that promote microbe survival or clearance) ( ) . however, the mechanisms underlying the microbial adaptation to a given sweetener are not fully understood yet. in particular, it is still not clear if dietary sugars and sweeteners can also induce changes in the host environment. the exact impact on the host exerted by microbial metabolites derived from added sugar and sweetener metabolism also needs to be clarified and addressed in future studies. to date, the european union (eu) and efsa approved the use of non-calorie sweeteners, namely: acesulfame-k (e- ), advantame (e- ), aspartame (e- ), aspartame-acesulfame salt (e- ), cyclamic acid and its sodium and calcium salts (e- ), neohesperidin dihydrochalcone (e- ), neotame (e- ), saccharin (e- ), stevia (e- ), sucralose (e- ), and thaumatin (e- ). eu and efsa confirmed that non-nutritive and low-calorie sweeteners are safe for human health if used within the adi ( ). amongst low-calorie sweeteners, eu approved the following compounds: sorbitol and sorbitol syrup (e ), mannitol (e- ), isomaltose (e- ), polyglycitol syrup (e- ), maltitol and maltitol syrup (e- ), lactitol (e- ), xylitol (e- ), and erythritol (e- ). unlike non-caloric sweeteners, polyols and low-calorie sweeteners are classified as gras and adi is not reported for them ( ) . an international consensus statement on the use of low-and non-calorie sweeteners has been signed in lisbon in july ( ) . the consensus concluded that low-and noncalorie sweeteners consumption is safe, as also supported by who, fda ( ) and efsa ( ) , and the dietary consumption of low-and non-calorie sweeteners promotes dental health when these compounds replace free sugars ( , ) . therefore, the consensus encourages the education of consumers on the use of products containing low-and non-calorie sweeteners, in order to increase awareness of general population on their correct use ( ) . finally, consumption of low-and non-calorie sweeteners during pregnancy showed neutral effects on offspring health and data obtained from animal studies were not confirmed in humans ( ) . although more research is needed to fully assess the effects of in utero exposure to sweeteners, current evidence does not suggest adverse effects in pregnancy. nevertheless, it is recommended that sweeteners are consumed in moderate amounts, adhering to the acceptable daily intake standards established by regulatory agencies ( ) . fda has approved several types of sugar substitutes, considering them as safe. nonetheless, the american heart association and the american diabetes association suggest to limit the use of sweeteners due to the lack of strong evidence for their effects on body weight and cardiometabolic risk factors in the long-term period ( ) . with regard to effects on gut microbiota, most of the sweeteners affect bacterial gut composition, potentially inducing dysbiosis. among sweeteners, polyols seem to show a good safety profile. moreover, they are non-cariogenic, do not negatively affect gut microbiota and are characterized by a very low-energy value ( ) . moreover, the potential favorable effects of polyols on glucose homeostasis may suggest their use as a valid option in subjects with type diabetes and metabolic syndrome, although further research is needed in this area. in this context, a careful nutritional advice is essential for a conscious use and for a correct transition, through the use of sweeteners, from sweetened foods to sugar-free foods. the role of nutrition specialists appears therefore crucial to recommend a diet with a proper use of sweeteners, avoiding the risk of an excessive use of these compounds. given the scarcity of data on sweetener safety in the longterm period, it is important to carefully evaluate the use of these compounds particularly in selected patients, such as those affected by metabolic derangements. indeed, different studies and meta-analyses found an association between the consumption of sweeteners and artificially sweetened beverages with increased risk of overweight, obesity, metabolic syndrome and type diabetes ( , , ( ) ( ) ( ) , thus highlighting the need for future prospective studies aimed at evaluating the exact impact of different types of sweeteners on human health from a metabolic perspective. however, when consumed in moderate amounts, sweeteners may be used as part of a nutritional rehabilitation program aimed to limit daily consumption of refined sugars ( , , ( ) ( ) ( ) . over the last decades several healthy dietary patterns have been proposed to tackle the growing obesity epidemic. dietary approaches based on marked reduction of carbohydrate and refined sugar consumption are emerging in clinical practice and are highly debated. during the last few years, interest in very low-calorie ketogenic diets (vlckd) has gradually grown due to their safety and their marked potential in inducing weight loss ( ) . in this context, non-calorie sweeteners represent a valuable tool for improvement of patient adherence to a strict nutritional regimen and rehabilitation program. the addition of non-calorie sweeteners to food replacements allowed for a marked reduction in carbohydrate and sugar content (< g/day, ≃ % of total energy intake), with preservation of food palatability and diet satisfaction ( ), thereby avoiding craving and increase in appetite, which may reduce the efficacy of a vlckd. on the basis of the recent findings on the effects of sucralose ( ) , the association of non-calorie sweeteners to a very low-carbohydrate nutritional regimen represents a valid approach to prevent the detrimental metabolic effects on insulin sensitivity and the altered neural response to sugars induced by an excessive carbohydrate consumption (figure ). dietary consumption of sweeteners has progressively increased over the last decades in order to reduce the burden of cardiovascular and metabolic diseases caused by modern western diets, which are characterized by a high content in refined and added sugars. also, the introduction of sugar taxes in several countries is likely to cause an even greater use of these compounds. nonetheless, at present there is still scarce evidence to establish conclusively whether the consumption of different types of sweeteners (e.g., low-calorie sweeteners vs. non-calorie sweeteners) can result in significant beneficial or detrimental effects on energy balance, appetite, body weight, and/or cardiometabolic risk factors in healthy subjects and patients with metabolic diseases (particularly obesity and type diabetes). indeed, the health impact of sweetener consumption, as well as the potential health consequences resulting from switching from one sweetener to another, still remain poorly understood. therefore, future prospective studies aimed to address short-and long-term safety and efficacy of different types of sweeteners in various clinical settings (e.g., obesity, type diabetes, metabolic syndrome) and in different age groups are needed. another area that warrants further investigation is the impact of different types of sweeteners on gut microbiota. emerging evidence supports how different food components (including sweeteners) can drive changes in the gut microbiota, resulting in relevant implications for human health and disease ( , , , , ) . mechanistic studies using gut organoids or animal models will certainly help to better elucidate: (i) how different types of sweeteners can reshape the gut microbiota, (ii) the interactions between sweeteners/sweetener metabolites, gut microbiota and host, and (iii) the consequences of these interactions on host physiology and biological processes in the short-and long-term period. future studies will also be helpful to evaluate which sweeteners are able to promote the growth of beneficial or detrimental gut microbes, resulting in potential human health benefits or harms. additionally, clinical studies evaluating the impact of different sweeteners on gut microbiota composition will further help to fully address all these unanswered questions regarding the sweetener-gut microbiotahost triad. finally, the extent to which all the aforementioned sweetenerinduced changes at different levels (central nervous system circuits, gut hormone secretion and gut microbiota) are clinically relevant in terms of human health is still not clear. genetic, anthropometric and dietary differences may, at least in part, account for the high interindividual variability in the response to different types of sweeteners ( ) . future studies based on epidemiological approaches combined with tools used in precision medicine may help to better establish the subset of individuals who are more likely to receive benefit or harm from sweetener consumption ( ) . all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. mc and em conceived, wrote, and revised the manuscript. afe and vm prepared the figures and wrote part of the manuscript. mi wrote part of the manuscript and carefully revised it. ml and afa revised the manuscript. we wish to thank dr. elisa fabbrini for critical reading of the manuscript. figures generation has been performed by using biorender software. this work is dedicated to the shining memory of dr. italo nosari, and to all other healthcare workers who died of covid- . natural sweeteners in a human diet the health implications of sucrose, high-fructose corn syrup, and fructose: what do we really know? academy of nutrition and dietetics. position of the academy of nutrition and dietetics: use of nutritive and nonnutritive sweeteners sugar alcohols sugar alcohols-their role in the modern world of sweeteners: a review nonnutritive sweeteners: where are we today? a review of sugar consumption from nationally representative dietary surveys across the world sugar-sweetened beverages and weight gain in children and adults: a systematic review from to and a comparison with previous studies fructose-containing caloric sweeteners as a cause of obesity and metabolic disorders fructose and sugar: a major mediator of non-alcoholic fatty liver disease is sugar consumption detrimental to health? a review of the evidence - nutritional patterns associated with the maintenance of neurocognitive functions and the risk of dementia and alzheimer's disease: a focus on human studies guideline: sugars intake for adults and children (who guidelines approved by the guidelines review committee). geneva: world health organization perspective: total, added, or free? what kind of sugars should we be talking about? added sugars and cardiovascular disease risk in children: a scientific statement from the american heart association free and added sugar consumption and adherence to guidelines: the uk national diet and nutrition survey comorbidity associated with obesity in a large population: the apna study global, regional, and national prevalence of overweight and obesity in children and adults during - : a systematic analysis for the global burden of disease study the danish tax on saturated fat: why it did not survive european nations launch tax attack on unhealthy foods world cancer research fund international. nourishing _use economic tools. world cancer research fund international viewpoint: can u.s. local soda taxes continue to spread? food policy cost-effectiveness of a us national sugar-sweetened beverage tax with a multistakeholder approach: who pays and who benefits public health product tax in hungary: an example of successful intersectoral action using a fiscal tool to promote healthier food choices and raise revenues for public health the economics of taxes on sugar-sweetened beverages: a review of the effects on prices, sales, cross-border shopping, and consumption food policy approaches to obesity prevention: an international perspective does green mean healthy? nutrition label color affects perceptions of healthfulness making food labels social: the impact of colour of nutritional labels and injunctive norms on perceptions and choice of snack foods taxation of unprocessed sugar or sugar-added foods for reducing their consumption and preventing obesity or other adverse health outcomes insights on the influence of sugar taxes on obesity prevention efforts low-calorie sweetener consumption is increasing in the united states trends in the consumption of low-calorie sweeteners consumption of low-calorie sweeteners among children and adults in the united states nonnutritive sweeteners: current use and health perspectives: a scientific statement from the american heart association and the american diabetes association sugar substitutes: health controversy over perceived benefits fueling the obesity epidemic? artificially sweetened beverage use and long-term weight gain intense sweetness surpasses cocaine reward increased postprandial glycaemia, insulinemia, and lipidemia after weeks' sucrose-rich diet compared to an artificially sweetened diet: a randomised controlled trial sucrose compared with artificial sweeteners: different effects on ad libitum. food intake and body weight after wk of supplementation in overweight subjects available online at: www health potential of polyols as sugar replacers, with emphasis on low glycaemic properties the expanding world of nutritive and non-nutritive sweeteners glycemic index and glycemic load: measurement issues and their effect on diet-disease relationships food and food products on the italian market for ketogenic dietary treatment of neurological diseases / of the european parliament and of the council of december on food additives american dietetic association. position of the american dietetic association: use of nutritive and nonnutritive sweeteners sugar substitutes: mechanism, availability, current use and safety concerns-an update. open access maced dietary intake of individual (free and intrinsic) sugars and food sources in the spanish population: findings from the anibes study sweeteners as food additives in the xxi century: a review of what is known, and what is to come toxicity of the ages generated from the maillard reaction: on the relationship of food-ages and biological-ages food protein-polysaccharide conjugates obtained via the maillard reaction: a review accumulation of advanced glycation end products is associated with macrovascular events and glycaemic control with microvascular complications in type diabetes mellitus glycation and carboxymethyllysine levels in skin collagen predict the risk of future -year progression of diabetic retinopathy and nephropathy in the diabetes control and complications trial and epidemiology of diabetes interventions and complications participants with type diabetes clinical review: the role of advanced glycation end products in progression and complications of diabetes isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface food processing and maillard reaction products: effect on human health and nutrition low-digestible carbohydrates in practice bioavailability of cellobiose and other non-digestible and/or non-absorbable sugar substitutes and related topics impact of polyols on oral microbiome of estonian schoolchildren scientific opinion on the substantiation of health claims related to the sugar replacers xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, d-tagatose, isomaltulose, sucralose and polydextrose and maintenance of tooth mineralisation by: sugar replacers xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, d-tagatose, isomaltulose, sucralose and polydextrose related hea sorbitol as a sweetener in the diet of insulin-dependent diabetes short-chain fatty acid production from mono-and disaccharides in a fecal incubation system: implications for colonic fermentation of dietary fiber in humans carbohydrate malabsorption and the effect of dietary restriction on symptoms of irritable bowel syndrome and functional bowel complaints safety assessment of hydrogenated starch hydrolysates quantification of fructans, galacto-oligosacharides and other short-chain carbohydrates in processed grains and cereals mannitol: a review of its clinical uses use of fructose, xylitol, or sorbitol as a sweetener in diabetes mellitus xylitol improves pancreatic islets morphology to ameliorate type diabetes in rats: a dose response study gut hormone secretion, gastric emptying, and glycemic responses to erythritol and xylitol in lean and obese subjects from glucose lowering to treatment of cardiovascular disease: the repositioning of glucose-lowering agents an overview of glp- agonists and recent cardiovascular outcomes trials anorexigenic effects of glp- and its analogues cholecystokinin (cck) and related adjunct peptide therapies for the treatment of obesity and type diabetes erythritol: a review of biological and toxicological studies high-level production of erythritol by mutants of osmophilic moniliella sp advances in sweeteners erythritol: an interpretive summary of biochemical, metabolic, toxicological and clinical data erythritol is a sweet antioxidant serum glucose and insulin levels and erythritol balance after oral administration of erythritol in healthy subjects carbohydrate issues: type and amount non-nutritive sweeteners and their implications on the development of metabolic syndrome the truth about artificial sweeteners -are they good for diabetics? sweeteners permitted in the european union: safety aspects stevia leaf to stevia sweetener: exploring its science, benefits, and future potential stevia rebaudiana bertoni: a natural alternative for treating diseases associated with metabolic syndrome metabolism of stevioside by healthy subjects metabolism of stevioside and rebaudioside a from stevia rebaudiana extracts by human microflora pharmacokinetics of rebaudioside a and stevioside after single oral doses in healthy men phylogeny and biogeography of the genus stevia (asteraceae: eupatorieae): an example of diversification in the asteraceae in the new world european food safety authority scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (e ) as a food additive analysis of anti-rotavirus activity of extract from stevia rebaudiana immune up regulatory response of a non-caloric natural sweetener, stevioside stevioside suppressed inflammatory cytokine secretion by downregulation of nf-κb and mapk signaling pathways in lps-stimulated raw . cells antihyperglycemic and blood pressure-reducing effects of stevioside in the diabetic goto-kakizaki rat no effect of dietary aspartame or stevia on pancreatic acinar carcinoma development, growth, or induced mortality in a murine model anticancer potential of steviol in mcf- human breast cancer cells steviol, a natural product inhibits proliferation of the gastrointestinal cancer cells intensively structure-taste relationships of aspartic acid amides soft drinks | chemical composition acute effect of aspartame-induced oxidative stress in wistar albino rat brain artificial sweeteners -a review fda (food and drug administration). food additives permitted in food for human consumption directive / /ec of june on sweeteners for use in foodstuffs aspartame: commissioner's final decision preclinical studies of aspartame in non primate animals incidence of brain tumors in rats fed aspartame toxicity of aspartame and its diketopiperazine for wistar rats by dietary administration for weeks results of long-term carcinogenicity bioassay on sprague-dawley rats exposed to aspartame administered in feed mega-experiments to identify and assess diffuse carcinogenic risks first experimental demonstration of the multipotential carcinogenic effects of aspartame administered in the feed to sprague-dawley rats comparative metabolism of aspartame in experimental animals and humans scientific opinion on the re-evaluation of aspartame (e ) as a food additive efsa's toxicological assessment of aspartame: was it even-handedly trying to identify possible unreliable positives and unreliable negatives? bitter taste of saccharin and acesulfame-k analysis of multiple sweeteners and their degradation products in lassi by hplc and hptlc plates - - ) in genetically modified (fvb tg.ac hemizygous) mice and carcinogenicity studies of acesulfame potassium in genetically modified [b . -trp (tm brd) (n ) haploinsufficient] mice (feed studies)mice the impact of low and no-caloric sweeteners on glucose absorption, incretin secretion, and glucose tolerance non-nutritive sweeteners: no class effect on the glycaemic or appetite responses to ingested glucose sucralose metabolism and pharmacokinetics in man us food and drug administration (us fda). food additives permitted for direct addition to food for human consumption / /ec of the european parliament and of the council of december amending directive / /ec on sweeteners for use in foodstuffs the development and applications of sucralose, a new high-intensity sweetener an overview of the safety of sucralose short-term consumption of sucralose with, but not without, carbohydrate impairs neural and metabolic sensitivity to sugar in humans lack of dna-damaging activity of five nonnutritive sweeteners in the rat hepatocyte/dna repair assay the absence of genotoxicity of sucralose a combined chronic toxicity/carcinogenicity study of sucralose in sprague-dawley rats a carcinogenicity study of sucralose in the cd- mouse the effect of five artificial sweeteners on caco- , ht- and hek- cells sucralose administered in feed, beginning prenatally through lifespan, induces hematopoietic neoplasias in male swiss mice statement on the validity of the conclusions of a mouse carcinogenicity study on sucralose (e ) performed by the ramazzini institute differences in susceptibility to sodium saccharin among various strains of rats and other animal species comparative analysis of the proliferative response of the rat urinary bladder to sodium saccharin by light and scanning electron microscopy and autoradiography saccharin deemed "not hazardous" in united states and abroad long-term saccharin consumption and increased risk of obesity, diabetes, hepatic dysfunction, and renal impairment in rats sugar content in artificial sweetener drug addiction as a disorder of associative learning. role of nucleus accumbens shell/extended amygdala dopamine mesolimbic dopaminergic system activity as a function of food reward: a microdialysis study evidence that intermittent, excessive sugar intake causes endogenous opioid dependence excessive sugar intake alters binding to dopamine and mu-opioid receptors in the brain sugar addiction: from evolution to revolution relation between obesity and blunted striatal response to food is moderated by taqia a allele changes in dopamine release and dopamine d / receptor levels with the development of mild obesity overlapping neuronal circuits in addiction and obesity: evidence of systems pathology similarity between obesity and drug addiction as assessed by neurofunctional imaging: a concept review separate magnitude and phase regularization via compressed sensing the neuroscience of natural rewards: relevance to addictive drugs modulation of feeding-induced activation of mesolimbic dopamine transmission by appetitive stimuli and its relation to motivational state dopamine microdialysis in the nucleus accumbens during acute and chronic morphine, naloxoneprecipitated withdrawal and clonidine treatment cephalic phase responses and appetite predictive models of glucose control: roles for glucose-sensing neurones how neural mediation of anticipatory and compensatory insulin release helps us tolerate food sugar-induced cephalic-phase insulin release is mediated by a t r +t r -independent taste transduction pathway in mice physiological mechanisms by which non-nutritive sweeteners may impact body weight and metabolism food reward in the absence of taste receptor signaling artificial sweeteners produce the counterintuitive effect of inducing metabolic derangements sucrose activates human taste pathways differently from artificial sweetener hypothalamic melanin concentrating hormone neurons communicate the nutrient value of sugar do low-calorie sweeteners promote weight gain in rodents? sugar-sweetened beverages, weight gain, and incidence of type diabetes in young and middle-aged women sugar-sweetened and artificially-sweetened beverages in relation to obesity risk beverage consumption patterns in elementary school aged children across a two-year period does low-energy sweetener consumption affect energy intake and body weight? a systematic review, including meta-analyses, of the evidence from human and animal studies low-calorie sweeteners and body weight and composition: a meta-analysis of randomized controlled trials and prospective cohort studies understanding the metabolic and health effects of low-calorie sweeteners: methodological considerations and implications for future research mammalian sweet taste receptors the cell biology of taste taste buds: cells, signals and synapses the receptors and cells for mammalian taste the functional involvement of gut-expressed sweet taste receptors in glucosestimulated secretion of glucagon-like peptide- (glp- ) and peptide yy (pyy) incretin release from gut is acutely enhanced by sugar but not by sweeteners in vivo sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic amp signaling systems and stimulates insulin secretion ccaat/enhancer-binding protein beta regulates expression of human t r taste receptor gene in the bile duct carcinoma cell line, hucct taste isn't just for taste buds anymore t r and gustducin in gut sense sugars to regulate expression of na+-glucose cotransporter molecular forms of human enteroglucagon in tissue and plasma: plasma responses to nutrient stimuli in health and in disorders of the upper gastrointestinal tract gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide- effects of oral ingestion of sucralose on gut hormone response and appetite in healthy normal-weight subjects effects of carbohydrate sugars and artificial sweeteners on appetite and the secretion of gastrointestinal satiety peptides effects of different sweet preloads on incretin hormone secretion, gastric emptying, and postprandial glycemia in healthy humans ingestion of diet soda before a glucose load augments glucagon-like peptide- secretion sweet taste receptors in rat small intestine stimulate glucose absorption through apical glut rebaudioside a directly stimulates insulin secretion from pancreatic beta cells: a glucose-dependent action via inhibition of atp-sensitive k-channels rebaudioside a potently stimulates insulin secretion from isolated mouse islets: studies on the dose-, glucose-, and calcium-dependency antihyperglycemic effects of stevioside in type diabetic subjects diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome adaptation of the gut microbiota to modern dietary sugars and sweeteners the human small intestinal microbiota is driven by rapid uptake and conversion of simple carbohydrates gut biogeography of the bacterial microbiota artificial sweeteners induce glucose intolerance by altering the gut microbiota the effect of sodium saccharin in the diet on caecal microflora acesulfame k, cyclamate and saccharin inhibit the anaerobic fermentation of glucose by intestinal bacteria defining dysbiosis and its influence on host immunity and disease saccharin induced liver inflammation in mice by altering the gut microbiota and its metabolic functions recurrent reverse evolution maintains polymorphism after strong bottlenecks in commensal gut bacteria biochemical principles of the use of xylitol in medicine and nutrition with special consideration of dental aspects the influence of stevia glycosides on the growth of lactobacillus reuteri strains chemical characterization and prebiotic activity of fructo-oligosaccharides from stevia rebaudiana (bertoni) roots and in vitro adventitious root cultures bactericidal activity of a fermented hot-water extract from stevia rebaudiana bertoni towards enterohemorrhagic escherichia coli o :h and other food-borne pathogenic bacteria combination of polydextrose and lactitol affects microbial ecosystem and immune responses in rat gastrointestinal tract a human volunteer study to assess the impact of confectionery sweeteners on the gut microbiota composition effects of consuming xylitol on gut microbiota and lipid metabolism in mice gut microflora interactions with xylitol in the mouse, rat and man trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies cd + regulatory t cells are critical in prevention of autoimmune-mediated diabetes sugar substitutes during pregnancy nonnutritive sweeteners in breast milk effects of mother's dietary exposure to acesulfame-k in pregnancy or lactation on the adult offspring's sweet preference sucralose: assessment of teratogenic potential in the rat and the rabbit nonnutritive sweetener consumption during pregnancy, adiposity, and adipocyte differentiation in offspring: evidence from humans, mice, and cells maternal exposure to nonnutritive sweeteners impacts progeny's metabolism and microbiome ibero-american consensus on low-and no-calorie sweeteners: safety, nutritional aspects and benefits in food and beverages the safety and regulatory process for low calorie sweeteners in the united states european food safety authority (efsa) efsa panel on food additives and nutrient sources added to food consensus statement on benefits of low-calorie sweeteners: benefits of lowcalorie sweeteners role of sugar and sugar substitutes in dental caries: a review low calorie beverage consumption is associated with energy and nutrient intakes and diet quality in british adults intake of artificially sweetened soft drinks and risk of preterm delivery low-calorie sweetened beverages and cardiometabolic health: a science advisory from the american heart association sugar and artificially sweetened beverages linked to obesity: a systematic review and meta-analysis prospective associations and population impact of sweet beverage intake and type diabetes, and effects of substitutions with alternative beverages the association between artificial sweeteners and obesity very-low-calorie ketogenic diet (vlckd) in the management of metabolic diseases: systematic review and consensus statement from the italian society of endocrinology (sie) interplay between food and gut microbiota in health and disease how non-nutritive sweeteners influence hormones and health key: cord- -lv fy e authors: dávalos, alberto; henriques, rossana; latasa, maría jesús; laparra, moisés; coca, maría title: literature review of baseline information on non‐coding rna (ncrna) to support the risk assessment of ncrna‐based genetically modified plants for food and feed date: - - journal: nan doi: . /sp.efsa. .en- sha: doc_id: cord_uid: lv fy e this report is the outcome of an efsa procurement (np/efsa/gmo/ / ) reviewing relevant scientific information on ncrna and on rna interference(rnai) that could support the food and feed risk assessment of ncrna‐based genetically modified (gm) plants. information was retrieved through key words and key questions covering the stability and degradation of ncrnas after oral ingestion, the passage of ncrnas from food and feed to human and animal organs and tissues via the gastrointestinal tract and other barriers, as well as the potential effects on the gastrointestinal tract, the immune system or the entire organism.full description of the strategy used for the literature search and for studies selectionis provided and the number of retrieved publications is reported. this report is divided into four partsdiscussing the kinetics of exogenous ncrnas in humans and animals, with focus on ingested ncrnas (part ); the possible effects of ncrnas on the gastrointestinal tract (part ), systemically(part )and on the immune system (part ). this report suggests that some plant ncrnas (e.g mirnas and sirnas) show higher stability as compared to other ncrnas due to peculiar chemical characteristics ( ’‐o‐methylation at ’ end).however, ingested or administered ncrna must overcome many extracellular and cellular barriers to reach the intended target tissue or functional location in sufficient amount to exert any biological effect. literature data indicate that chemically unmodified and unformulated ncrnas exhibit very low stability in the gastrointestinal tract and in biological fluids and, in general, do not elicit major biological effects.this report also provides an overview of the rna content in plant‐derived foods and diets and discusses the controversies on the presence of dietary exogenous rnas in the biological fluids of humans and animals and their effects. finally, gaps in the scientific literature are highlighted and recommendations provided . introduction this scientific report is the result of a contract awarded by the european food safety authority (efsa) to the madrid institute of advanced studies (imdea)-food, under the contract title: "literature review of baseline information on non-coding rna (ncrna) that could support the food/feed risk assessment of ncrna-based gm plants" (contract number np/efsa/gmo/ / ). for the literature review of the available scientific information on foreign (exogenous) ncrnas that could support the risk assessment of ncrna-based gm plants, the following tasks were defined by efsa. based on available scientific literature (narrative review) to review the kinetics profile of foreign (exogenous) ncrna in humans and animals (experimental animals, livestock and pets). this to be based on information from research and development of ncrnas intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area) and focused primarily on the oral route of administration of ncrna, with detailed information on the absorption, distribution, metabolism and excretion of ncrna molecules. other routes of administration, however, not to be excluded. based on available scientific literature (narrative review), to review the effects (physiological, paraphysiological, and pathological) of foreign (exogenous) ncrnas on the gi tract and annex glands (e.g. liver, pancreas, salivary glands) in humans and animals (experimental animals, livestock and pets). this to be based on information from research and development of ncrna molecules intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area). based on available scientific literature (narrative review), to provide information on the possibility of systemic effects of foreign (exogenous) ncrnas in humans and animals (experimental animals, livestock and pets) with focus on gastrointestinal barrier to absorption, and other barriers as relevant (e.g. placenta). based on available scientific literature (narrative review), to assess the plausibility of effects on the immune system of humans and animals (experimental animals, livestock and pets) of foreign (exogenous) ncrnas. gm plants based on silencing approaches by rnai (through ncrna expression) are developed for food and feed purposes and assessed by efsa within the eu gmo regulatory framework. the team considered that pursuing the four tasks defined by efsa to support the risk assessment of gm plants in this regulatory context required gathering preparatory baseline information on ncrna and refinement of the scope of the tasks. therefore, further elaboration on the terms of reference provided by efsa has been conducted and it is described below. it should be emphasized that the available information on ncrnas which could be possibly relevant to food and feed safety, overlaps with and is barely distinguishable from the broader information on rnas. information on rna in general has therefore been included in the search as warranted; when possible, the relevance of distinguishing peculiar ncrnas features is described. the term "foreign ncrna" or "exogenous ncrna" used in this report refers to ncrna molecules to which humans/animals can be exposed through the diet or via therapeutic treatment. similarly, "foreign rna" or "exogenous rna" refers to rna molecules to which humans/animals can be exposed through the diet or via therapeutic treatment. part : kinetics of exogenous ncrna in humans and animals (efsa task ) efsa task : based on available scientific literature (narrative review) to review the kinetics profile of foreign (exogenous) ncrna in humans and animals (experimental animals, livestock and pets) . this to be based on information from research and development of ncrnas intended to be used as therapeutics and/or from research on biomarkers in humans (pharmaceuticals/medicine area) and to be focused primarily on the oral route of administration of ncrna, with detailed information on the absorption, distribution, metabolism and excretion of ncrna molecules. other routes of administration, however, not to be excluded. to address efsa task , preparatory baseline information is provided i) on general features of plant ncrnas including their function, movement in the plant, biogenesis and degradation (section . . ), ii) on their stability (i.e. for how long ncrna molecules retain their original structure, and how they resist degradation over time and in various conditions, both inside and outside the plant), and turnover, also in comparison with turnover of endogenous ncrna in animals (section . . ). if necessary, comparisons between the ncrnas class and other rna classes are made. general information on rnas used/intended for use as therapeutics (including ncrnas) is provided, expanding on the chemical modifications necessary to avoid degradation and to achieve a target effect after administration (section . . ). lessons from rna-based therapeutics as regards the pharmacokinetics of exogenous rna are discussed, with a focus on non chemically modified (naked)ncrnas. in addition, aspects of the pharmacodynamics of rnas as learnt by therapeutics are addressed (section . . ). a description of cellular uptake of rna (in general and with focus on ncrnas), and of the many barriers represented by human/animal cells after oral ingestion is addressed insection . . . further relevant information on dietary exposure to plant rnas (i.e. exposure to plant rnas following dietary consumption) by different diet types, and presentation of the gaps in the literature on dietary plant rna exposure is provided in section . . . efsa task : based on available scientific literature (narrative review), to review the effects (physiological, paraphysiological, pathological) of foreign (exogenous) ncrna on the gastrointestinal tract and annex glands (e.g. liver, pancreas, salivary gland) to address efsa task preparatory baseline information is provided on i) the gi tract barriers to ingested exogenous rnas, including ncrnas (section . . ); ii) experience in rna-based therapeutics for oral (gi) administration, with focus on delivery of naked (non chemically modified) ncrnas (section . . ). biological effects of dietary exogenous ncrnas (plant and nonplant origin) on the gi tract and its annex glands is presented in section . . . www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to address efsa task , preparatory baseline information is provided on the molecular pathways relevant for the uptake of exogenous ncrnas by human and animal cells and the intracellular trafficking that follows, with description of the effects by exogenous ncrnas once they reach the specific subcellular location (section . . ). to exert systemic biological effects, in most cases the ingested exogenous rna must enter the systemic circulation to reach the target tissues. information is therefore provided on the landscape of exogenous rnas in biological fluids and tissues from humans and animals, with focus on ncrna when possible (section . . ). a description of possible systemic biological effects of dietary exogenous ncrnas is provided in section . . , with details on tissue barriers (i.e. placenta, brain) encountered. a detailed review is made of the contradictory information on the systemic effects exerted by plant-derived exogenous ncrnas. finally, studies on the safety and in general on risk assessment on ncrnas from gmos are reviewed (section . . ). efsa task : based on available scientific literature (narrative review), to assess the plausibility of effects on the immune system of humans and animals (experimental animals, livestock and pets) of foreign (exogenous) ncrna molecules. to address efsa task , preparatory baseline information is provided on ncrna mediated regulation of the immune system in humans and animals. the many pathways of sensing exogenous rnas, both at the endosomal compartment and in the cellular cytosol, and the mechanism of triggering the immune system are reviewed (section . . ). an overview is done of the plausibility of biological effects of exogenous plant ncrnas on the regulation and function of the immune system upon uptake by mammalian cells (section . . ). in addition, since the gut microbiota influences the immune system, a review on the possible effects of exogenous ncrnas as gut microbiota modulators was considered relevant and it is provided (section . . ). in the final section of this report, concluding remarks inform on the gaps existing in the scientific literature, and highlight areas needing further investigations to better understand and support the food and feed risk assessment of ncrna-based gm plants. an extensive search was done to identify as many studies as possible relevant to the literature review questions. to include all possibly relevant information multidisciplinary databases and information resources were explored. unpublished research reports ("grey literature") including dissertations, thesis or other scientific reports were also included, which were retrieved using general search engines (i.e. google). the following information sources, databases or search engines ( table ) were used for the literature review: pubmed (biomedical), world wide science (multidisciplinary), web of science (multidisciplinary), springerlink (multidisciplinary), scopus (multidisciplinary), scielo (multidisciplinary) and biorxiv (preprint biology). finally, the publications reporting the outcome of two efsa procurements aiming respectively at investigating and summarising the state of knowledge on the mode-of-action of dsrna and mirna pathways, the potential for non-target gene regulation by dsrna-derived sirnas or mirnas, the determination of sirna pools in plant tissues and the importance of individual sirnas for silencing ; and reviewing relevant scientific information on rna interference that could serve as baseline information for the environmental risk assessment of rnai-based gm plants ) were also used. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the methodology used for this literature review process and report preparation followed three basic principles: i) methodological rigour and coherence in study retrieval and selection; ii) transparency; and iii) reproducibility. to ensure that these principles were implemented in this literature review process, the search methodology design, study selection and data and results presentation follow, whenever possible, most of the methods and techniques described in the efsa guidelines on application of systematic review methodology for food and feed safety assessments to support decision-making (efsa, ) . the following section describes the general key steps followed in the literature search methodology; detailed descriptions of the literature search methodology for each topic are also provided in this section. to find a comprehensive set of scientific literature related to exogenous ncrnas and their biological effects on a target organism, either local or systemically, key questions were identified and key words were used as search queries for each task section according to a pre-defined review process. to provide a fit-for-purpose literature search, the team defined key questions based on the efsa tender specifications and their interpretation as above described. to support this phase, the team identified key elements of the questions considering the pico approach of efsa ( ). the key elements identified include the population, in this case humans or animals (experimental animals, livestock and pets) as defined by the terms of reference of efsa. the intervention was identified as exogenous rnas (focusing on ncrnas). the comparator was identified as the control or reference group (not exposed to exogenous rnas) in most studies, whenever possible. the comparator could also be identified as the modified (either chemically or biologically) version of the exogenous rnas when referring to the stability or pharmacokinetic of the exogenous rna. the outcome is the biological effect (if any) as a response to the intervention. other specific key questions are determined for every part of the specific tasks defined by efsa. evidence is generally scarce for most of the topics covered by this scientific review, and in some cases the information was available from not directly related studies (i.e. pharmaceutical industry studies). the literature review was helpful in identifying both knowledge gaps and unexpectedly relevant studies or evidence not previously known to exist. these knowledge gaps are used to make informed proposals for future research designs (section . ). the key methodological steps in the literature search, selection and review process are shown in figure . three phases were defined in this process. the preparatory phase started with team discussions of topics to be included in the literature review. during this phase key elements addressing the efsa tender specifications and their interpretation as above described were identified and key questions were defined. the key questions aimed to to assist the literature search in order to produce a comprehensive overview of the topic that best fits tender specifications. the bibliography search and selection phase included several steps. the initial search in bibliographic databases and search engines was done using task-specific key words. the key words used for the search were derived from the efsa tender specifications and their interpretation (tasks - ) and were complemented with other key words identified in the preparatory phase. identified key words are detailed for each task in section . the final phase of review and final selection involved two additional steps, i.e. a detailed review of all relevant previously selected documents and a systematic evaluation of the final selected studies. a detailed review of the selected documents was done for studies with full text availability (either freely available or available for purchase) and, based on this, a final selection was made of full-text studies most relevant to answer the key questions for tasks ( - ) and apt for review in this report. a systematic evaluation of these final documents was done to develop a comprehensive overview of the topic. conflicting or contradictory results were reported in a neutral way and supported by references and the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. citations of all available evidence, both from the published and grey literature. retracted papers were carefully identified and noted. finally, each section of the narrative review was assessed by one reviewer. if more specific data was needed because of missing or novel information, additional bibliographic research was performed. additional key words that could directly address the specific data needed were identified and used to run a new specific search. the search among full-text studies was focused on obtaining only specific missing or required data (e.g. quantitative data on rnas levels in plants, earlier studies on exogenous rna exposure to mammalian cells). this new specific search generally represented less than % of the literature included in this report and it was performed only once for certain topics. finally, to reduce the risk of introducing bias into the literature review process and to assure reproducibility in the review methodology the following general approach was applied: when the search of the identified "key words" in different databases resulted in a myriad of duplicate documents, manual removal of these duplicates was performed in each section by each team expert; when relevant studies that might answer the identified key questions were identified after the first search run, these were used to further refine the literature search, for example refining the keywords; each selected full-text study to be included in the result section was manually examined by the team. to identify literature relevant to the topic, the methodology described in section . . . ( figure ) was applied, with adaptations. a multiple-step approach was followed based on the expertise of the team members who carried out research in the area. following a first search based on identified keywords, a careful selection of retrieved review studies was done, followed by a search of specific publications referred to in these reviews. additional specific key words were identified, and an additional search was run. this search retrieved a relatively small number of studies; therefore, team members reviewed all these to determine which were relevant to the topic. duplicates, irrelevant or erroneously assigned publications were removed. team members also cross-checked each others' findings, sharing discussing the results of their searches. the following areas were investigated. the purpose of this search was to provide general information on rnas function, movement in the plant and aspects of biogenesis and degradation, with a focus on ncrna (either small or long). by comparing animal and plant ncrna pathways, the aim was to identify similarities between animal and plant ncrna processing machineries that would indicate the possibility of ingested plant-derived ncrnas being processed in animals. in addition, this section was organized to avoid details that were outside the scope of this literature review in the topic of plant ncrna biology. this serves as an introductory chapter allowing easier integration of the knowledge presented in later chapters. the following combination of key words or phrases was used in some of the databases indicated above: plant small rnas; plant and sirna; plant and mirnas; plant and lncrnas; plant and circrnas; plant rna and movement. www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the initial search using the above-mentioned key words or phrases yielded a large number (≈ ) of documents ( table ) , most of which were not relevant to the topic. particularly, the databse web of science retrieved documents not directly related to the topic. to assist and refine the search, studies were further filtered by their relevance to the key questions identified: -what type of ncrnas are described in plants? -what are the main differences between mammalian and plant ncrnas? -what is the function of plant ncrnas? -what is known about the movement of plant ncrnas? title and abstracts were evaluated, and studies further refined using the key questions. detailed study of the most relevant full-text documents answering the key questions, retrieved documents which were finally selected as relevant to give an overview of plant ncrnas. search was then refined by using other key words including "half-life", "turnover", "fate" or "degradation". the identified studies were then manually evaluated by title and abstract and their number further limited due to their low relevance to the below identified key questions for this specific subchapter: -are plant ncrnas more stable than animal (mammalian) ncrnas? -if so, what makes a plant ncrna more stable than its animal (mammalian) counterpart? -why is it relevant to consider ncrna stability outside the organism of origin (plant)? -are certain types of plant ncrnas more or less stable than others? -what is known about the half-life of ncrnas? documents on the stability of rnas (not only ncrnas) were further screened for relevance to the question. most of the studies in the literature describe the stability of a specific class of intracellular rnas (i.e. mrnas). the stability of rnas other than ncrnas (e.g. mrna, trna), unless are used as model of general ncrna stability, is outside the scope of this literature review, since it is very unlikely they would be used as baseline information for food/feed risk assessment of ncrna-based gm plants. these keywords were specifically selected since they describe the kinetic profile of exogenous ncrnas in human and animals. the word "pharmacokinetics" was included in the search because it is a general term that retrieves data on absorption, distribution, metabolism or excretion of any molecule, particularly those intended for use as therapeutics (pharmaceutical/medicine areas). the keywords "increased intestinal permeability" or "increased plasma clearance" are intended to retrieve any study indicating changes in these parameters. although a large number of publications unrelated to ncrnas were expected, these keywords were chosen to initially explore the general aspects of physiological or pathological conditions associated with modification in these parameters. the search was further refined after this initial exploration. the search of the above-mentioned key words on different databases yielded ≈ . entries ( table ) . a quick exploration of titles and abstracts suggested that (as expected) a number were unrelated to or were not useful to appropriately address the topic. it is worth noting that the search in the previous section (section . . . .) indicated that the chemical modifications generally introduced for rna-based therapeutics are normally absent in nature. scientific data that could serve as a baseline to support risk assessment of ncrna-based gm plants should contain primarily information on ncrnas resembling those present in the nature or in gm plants. therefore, studies on naked exogenous rnas were analysed to prepare this narrative review. to promote comparisons between natural and synthetic rnas, a very few examples of slightly chemically modified rnas were included. heavily chemically modified rnas were not considered due to their unlikely existence in nature. only in vivo studies were considered, both in animals (mammals) or humans. studies were further evaluated for their relevance to answer the key questions identified for this section: -what are the pharmacokinetic properties of rna-based therapeutics? -what routes of administration have been used for rna-based therapeutics? -what is it known about the pharmacodynamic properties of rna-based therapeutics? -do disease conditions modify the pharmacokinetic properties of rnas when administered to mammals? the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after detailed evaluation of the full-text reports, documents meeting the described criteria were finally selected for review. understanding whether and how exogenous rnas are taken up by cells and what barriers are encountered is relevant to study the possible absorption and systemic exposure to ncrnas introduced by food and feed. this topic is reviewed in this dedicated section and excluded from the section on the pharmacokinetics of exogenous rnas (section . . . . above). to search and select the relevant studies concerning the uptake of exogenous rnas in mammals, the following combination of key words was used in some of the databases listed above: rna and cellular uptake; ncrna and cellular uptake; mirna and cellular uptake; lncrna and cellular uptake. these key words were used to screen for studies where "cellular uptake" was mentioned in the title or abstract for any type of rnas. it was also decided to include specific types of ncrnas including "mirna" or "lncrna" to expand the search to literatures where the more general term "rna" was not necessarily used. the initial search of scientific documents using the above keywords yielded ≈ documents ( table ) . these were explored by their relevance (title and abstract), and only studies addressing naked ncrnas were considered for full text analysis, since highly chemically modified rnas are not common in nature. to address the reference terms for task- of this procurement, and after expert discussion for this section (preparatory phase) key questions were identified and the studies then further filtered for their relevance to the these: -are exogenous rnas taken up by mammalian cells? -how does exogenous rnas uptake in mammalian cells occur? -what are the biological barriers to consumed rnas? -what are the biological barriers for the exogenous rnas after absorption and entering into the mammalian circulatory system? only studies where exogenous rnas were evaluated were included in the literature review. after detailed evaluation of full-text reports and expert judgement on their relevance to efsa task , documents were finally selected for review. to select relevant studies for plant rna exposure, the following combination of keywords were used: plant and rna content; plant and mirna content; plant and sirna content; plant and lncrna content; plant and dsrna content; rna intake and vegetarians; vegetables/fruit intake and vegetarians. these specific keywords were used because the wording "rna (ncrna, mirna, sirna, dsrna, lncrna) content" would yield most of the documents related to quantities of rnas in a specific substrate. these were combined with "plant" to retrieve only those studies where plants were used to evaluate rna content. it was also decided to use "rna intake", "vegetable intake" or "fruit intake" to specifically refer to consumption, and to combine this with "vegetarian" to search only for studies where comparisons are made between vegetarian and other types of diets. alternative wordings including "rna amount" or "vegan" were used to search for alternative information. using the above keywords, ≈ studies were retrieved (table ) during the initial search. to more appropriately cover this section and to refine the search, the following key questions were identified during the preparatory phase: -what is the general amount of rnas naturally occurring in edible plants? -what percentage of total plant rnas are ncrnas, mirnas, sirnas, lncrnas or dsrnas? -does exposure to rnas change in different diet types? -do changes in rnase activity, due to pathologies or human polymorphisms, influence dietary exposure? titles and abstracts were evaluated, and studies further filtered using the key questions above. the search was also refined to focus on those studies where quantitative rnas data were mentioned. only in vivo exposure studies were used (either epidemiological or experimental). common types of diets were only considered (i.e. vegetarian, vegan or omnivorous). for rna exposure by different diet types, studies where comparative analysis of at least two different diets (one of which was the general population diet type) were mainly considered. in all cases, full-text studies were evaluated to screen for relevance to answer the identified key questions. a detailed study of the full-text documents resulted in the identification of studies most relevant to address this specific topic of task- , and which were reviewed for this section of the report. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to identify literature relevant to the topic, the methodology described in section . . . (figure ) was applied. during the preparatory phase key questions were identified to assist in the literature search. the document search was first done using specific key words and then refined according to the key questions. detailed analysis of the full-text documents was done by team members with expertise in the area. search and analysis results were discussed collectively among team members. the following areas were investigated: in this subsection, there is a general description of the biological barriers within the gi tract that exogenous rnas would need to overcome to exert a biological effect, either locally or systemically. to select relevant studies, the following combination of key words or phrases were used: "gastrointestinal tract anatomy"; "gastrointestinal tract physiology"; intestinal transport pathway; physicochemical properties of rnas. an exploration of titles and abstract of the yielded entries (table ) suggested that a large number of documents were unrelated to the topic. only one key question was identified in the preparatory phase: -what are the biological barriers within the gastrointestinal tract to ingested exogenous rnas? the search for relevant studies addressing this key question was based mainly on team member judgement. detailed study of the most relevant full-text documents answering the key question retrieved documents which were finally selected as relevant to this section of the report. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this search was intended to collect data from rna-based therapeutics employing oral administration (and other gi routes) which could support the search on the possible biological effects of plant dietary exogenous rna and ncrnas on the gi tract and its annex glands. the initial search was done using the following key words on the different databases: ncrna oral delivery; rna oral delivery; sirna oral delivery; aptamer and oral; sirna gastrointestinal delivery; mirna gastrointestinal delivery; oral exosome rna; extracellular vesicle and gastrointestinal. these key words were selected to search for any documents related to rnas and oral administration. any information on oral administration was considered relevant. to expand the search, different terms referring to different types of ncrnas (mirna, aptamer, sirna or others), and a synonym for "administration", i.e. "delivery", were included. the initial search using the above key words or phrases yielded a small number (≈ ) of documents ( table ). the different databases generated similar number amounts of publications, most of which were duplicates. using the key word "lncrnas" in combination with the other key words did not yield any information relevant to the topic. indeed, just the search using "lncrna exosome" identified fewer than documents, but none was relevant to oral delivery or gi administration. the wording "dsrna and oral" produced ≈ documents, but none of them was relevant to mammalian organisms. the documents were selected by relevance, evaluating title and abstract. in this section, only studies in animals (mammals) or humans were considered. when replacing the word "delivery" with "administration", ≈ additional documents were identified, but almost % were unrelated to the review topic. since most of the documents in the literature describe minimally modified rnas for oral use, all chemically modified rnas were considered in this section. these can provide baseline information to support food/feed risk assessment of ncrna-based gm plants. to assist and refine the search, studies were further filtered for their relevance to the key question identified: is the oral route of administration relevant for rna-based therapeutic development? -what types of formulation (delivery vehicles) are tested for rna-based therapeutics for the oral route of administration or other gastrointestinal tract routes of administration? the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. -do exogenous ncrnas (naked rnas) resist the conditions of the gastrointestinal tract environment? -could ncrnas-based therapeutics -delivered by oral administration or other gi tract routes of administration-exert a biological effect in the gi tract or its annex glands? -are there any natural vehicles for oral administration of exogenous ncrnas? from the initial ≈ entries identified using the above key words and after filtering for relevance to the key question with evaluation of titles and abstracts and detailed study of the full-text reports, documents were selected for review in this section. twenty ( ) of these documents reports selected examples of local effects of nucleic acids-based drugs (mainly exogenous rnas) within the gi tract, using oral administration. since efsa required a literature review also including livestock species, an additional specific search was run for birds and fish. this search was intended to compile information on administration of exogenous ncrnas in either fish or birds, regardless of administration method, exerting any biological effects in vivo. the initial search was done using the following key words on the different databases: exogenous rna and fish; exogenous rna and bird; rna oral delivery and fish; rna oral delivery and bird; ncrna administration and fish; ncrna administration and bird. to expand the search, different terms referring to different types of ncrnas were used (sirna, mirna, circrna, or dsrna). the initial search using the key words or phrases produced a small number (≈ ) of documents (table ) in most of the databases. to assist and refine the search, only one key question was identified during the preparatory phase for this subsection: -are exogenous ncrnas also used to exert rnai effects in fish or birds? from the initial ≈ entries identified using the above key words, and after filtering for relevance to the key question, and evaluating titles and abstracts and detailed study of the full-text reports, documents were selected for the review in this subsection. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this chapter provides baseline information supporting food/feed risk assessment of ncrnas-based gm plants. in the effort to cover the widest amount of literature containing relevant scientific data on exogenous ncrnas and their possible biological effects, the following key words or phrases were used in the search: dietary plant non coding rnas; food plant non coding rnas; exogenous plant non coding rnas; diet plant micrornas; food plant mirnas; breast milk exosomes; breast milk mirna; breast milk ncrnas; breast milk lncrnas; breast milk rna; dietary exogenous rna. these key words were selected to cover most, if not all, available studies on the topic ( table ) . the search of other common examples of exogenous ncrnas (non-plant origin) consumed by oral intake was focused on breast milk ncrnas, due to their relevance to human nutrition (table ) . almost all documents yielded by the three databases (pubmed, scopus or web of science) were duplicates. in this section, studies reporting local effects (in the gi tract or its annex glands when consuming exogenous ncrnas) were preferentially (but not exclusively) evaluated. other databases including the preprint server for biology (biorxiv), scielo or search engines (i.e. google) were also used. the initial search in some databases using the above key words yielded a number of entries (table and ) not exceeding ≈ documents. because this specific topic is especially salient to future understanding of the possible food/feed risk assessment of ncrnas-based gm plants, all the identified studies were further evaluated by title and abstract and their relevance to answering the identified key questions: does the literature describe the effect of exogenous plant-origin ncrnas when consumed orally? -does the literature describe negative results or contradictory results regarding the possible biological effect of ncrnas when consumed orally? -are ncrna stability properties under gi tract conditions normally evaluated? -are exogenous ncrnas evaluated quantitatively? -are exogenous ncrnas consumed in an amount sufficient to exert a local biological effect?are there other common examples of exogenous ncrnas (other than plant-origin) that could exert a biological effect on the gi tract and its annex glands when ingested orally? local effects (in the gi tract or its annex glands) are mainly described in this section. the possible mechanisms of plant exogenous ncrnas cellular uptake, their intracellular trafficking and systemic biological effects (including plasma levels) are reviewed in another section (section . .). of the initial ≈ documents retrieved using the above key words, and after filtering for relevance to the key questions by evaluating titles and abstracts, and detailed study of the full-text reports, documents were selected for the review in this subsection. from these, documents were selected for plant-origin ncrnas and for non-plant origin (breast milk) ncrnas. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in line with the methodology described in section . . . . (figure ), a multiple-step approach based on the expertise of the team members responsible for this section was applied to identify literature relevant to the possible systemic effects of exogenous ncrnas. the document search was first done using specific key words in the databases and refined based on the appropriate key questions. detailed analysis of the full-text documents was done by team members and the results discussed collectively. the below areas were investigated. the search objective was to obtain information on basic molecular cellular uptake pathways of exogenous ncrna, including proteins involved in this process (if any). a review was also done on several other aspects of intracellular trafficking, once an exogenous ncrna is internalized, and how it exerts a biological effect. tissue barriers to exogenous ncrna function were also investigated. the content of this section deepens on molecular pathways previously described in section . . . to assist the search the following key words were used in the different databases: exogenous ncrna and mechanism and absorption; ncrna and mechanism and absorption; sidt (sid transmembrane family member ) and ncrna; sidt (sid transmembrane family member ) and ncrna; ncrna and uptake; intracellular trafficking and ncrna. these key words and terms were selected to identify any document related to the mechanisms of uptake and intracellular trafficking of ncrnas exerting a biological effect. the search was expanded by adding and combining different terms for exogenous ncrnas (see table ), including the general term "rna", www.efsa.europa.eu/publications efsa supporting publication : en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. or specific ncrnas such as "mirna". the initial search using the above key words or combinations thereof produced a moderate number of documents (table ). the different databases generated similar amounts of documents, most of which were duplicates. the largest proportion of the references was obtained using the key wording "ncrna and uptake" (≈ ). however, most of these had no relation to this specific section. the search was refined by filtering for relevance to answering the identified key questions: is there any clear transport of exogenous ncrnas into mammalian cells? -are orthologous of environmental rnas transporters in lower organisms (i.e. sid family of proteins) involved in the mammalian exogenous ncrna mechanism of uptake? -what molecular mechanisms are involved in exogenous ncrnas trafficking inside the cell? -what is the cellular fate of exogenous ncrnas once inside the cell? -what are the specific tissue barriers to exogenous ncrna function? very few documents evaluate the molecular mechanism of exogenous ncrnas uptake, intracellular trafficking and function. from the initial ≈ documents retrieved using the above key words, and after evaluation of their titles and abstracts and filtering for relevance to the key question, less than documents initially seemed relevant to the topic and were finally selected for review after detailed study of the full-text reports. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this subsection is intended to retrieve available information on the presence of exogenous rnas in human and animal biological fluids (i.e. blood, plasma, serum or any other biological fluids) and tissues. the search focused on the presence of dietary plant-derived exogenous ncrnas, due to their relevance to the overall literature review. to cover the largest possible portion of the relevant literature, the following key words were used in the search: exogenous rna and human plasma; exogenous rna and serum; plant ncrna and human tissue; plant lncrna and human plasma; plant mirna and blood; plant mirna and tissue; plant rna and tissue. to expand the search, the above key words were used in different combinations for the same search (i.e. blood, serum or plasma for biological fluids) ( table ). the general wording "rna" was also incorporated into some searches. specific types of ncrnas were also added, including "mirna" or "lncrna", to avoid missing documents possibly related to these specific molecule types. the search using these key words or phrases in different databases yielded more than ≈ . publications (table ) . a quick exploration of the titles and abstracts suggested that only few studies were related to the review topic. only one key question was identified as relevant which was related to the presence or absence of evidence of exogenous ncrnas in biological fluids and tissues. since these findings are important for the risk assessment of ncrna-based gm plants, the key question was presented in its positive and negative forms as follow: are there studies describing the presence of exogenous rnas in the biological fluids and tissues of humans and/or animals? -are there studies contradicting the presence of exogenous rnas in the biological fluids and tissues of humans and/or animals? after a detailed assessment of the full-text, documents were finally selected for review. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. this search primarily aimed to gather information relevant to understand the possibility of systemic effects of dietary exogenous ncrnas after intake (oral administration) by humans and/or animals (mammals). information on the possible passage through specific biological barriers (i.e. placenta or brain) was also searched. the search was guided by the following key words to identify studies providing any data on the possibility of systemic effects of dietary exogenous ncrnas: plant ncrna and diet; plant exogenous rna and diet; plant rna and diet, plant environmental rnai and diet; plant rna and placenta; plant rna and brain; dietary rna and cross kingdom. these key words were used to guarantee that every single document potentially providinginformation on the possibility of systemic effects of exogenous ncrnas was found. to widen the search, other wording such as "environmental rnai", "cross kingdom" or "mobile rnas" were used. although the wording "systemic effects" was not used in the key words during the search, the above key words were used to retrieve any documents in which a biological effect (local or systemic) might be reported. different wording for the term ncrnas was also used (including lncrna, circular rna, mirna, or dsrna). the initial search in some databases using the above key words produced a small number of entries (table ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. approximately ≈ documents were initially identified using the above key words (table ) . after filtering for their relevance to the key question by detailed assessment of the full-text reports, documents were finally selected for review. within this specific subsection, the selected documents are mainly grouped as studies that "support the evidence" or studies that "contradict the evidence" of systemic biological effects of dietary exogenous ncrnas in humans and animals (mammals). although not specifically included in the scope of this literature review, search for toxicological effects of dietary exposure to exogenous ncrnas in humans or animals is relevant to risk assessment. this search was aimed at identifying information on possible toxicological effects of plant-derived exogenous ncrnas following food/feed consumption. the initial search was conducted using the following key words in the different databases: the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. few documents were initially retrieved and after a quick evaluation of titles and abstracts, it became evident that very little information is available in this area. therefore, different terms were used to refer to exogenous ncrnas, including circular rna, lncrna and mirnas. the search using the above key words yielded a small number of documents (table ) . only in vivo studies were considered, both in animals (mammals) and humans (if available), with focus on dietary plant-origin exogenous ncrnas. the key questions identified to refine the literature search for this specific topic were: -is there evidence of any toxicological effects of plant-derived dietary exogenous ncrnas? -are the toxicological effects related to rnai-mediated interaction with the human or animal genome? -are there possible interactions related to unintended rnai-mediated gene regulation? approximately documents were identified using the above key words. after filtering for relevance to the key questions, and assessing the full-text reports, only documents were finally selected for review. a systematic and comprehensive review and collection of the literature relevant to the topic was performed with the aid of the team members' expertise. an extensive search using key words was initially done on multidisciplinary databases to avoid publication bias, followed by a grey literature search using general search engines. the resulting documents were screened by title and abstract to determine relevance to the topic and refined using the identified key questions. after full-text analysis of the documents, irrelevant documents were eliminated. all stages of the screening process were independently assessed and, in case of uncertainty, discussed with other experts to avoid personal biases. the methodology followed the key steps described in section . . . . (figure ) and a total of papers were identified, investigating the areas below described. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the final goal of this search was to identify relevant information on ncrnas-mediated processes associated with innate and adaptive immune responses. this section serves as an introduction to the studies presented later, and it is based on the results of the seach described in the next paragraph. sixty-four ( ) documents were reviewed for this section. the search was aimed primarily at gathering state-of-the-art knowledge on the possible biological effects of exogenous (plant) ncrnas on the regulation and function of the immune system. to cover this specific section, a combination of the following key words was used in the different databases: exogenous rna and immune and human/mice/rat; exogenous plant rna/mirna/sirna/lncrna/dsrna/transgenic rna and immune function/inmmunity and human/mice/rat; genetically modified plants and immune function/immunity and human/mice/rat; exogenous plant rna/mirna/sirna/lncrna/dsrna and primary cells and lymphocyte/monocyte/macrophage; exogenous rna/genetically modified plants and immune function/immunity and zebrafish. the key words were designed to identify all publications that use in vivo, including non-mammalian animal models, and in vitro ncrnas testing to evaluate the influence on tolerance processes as well as immune cell function. the search also included publications on in silico methods. the different key words were entered one by one into the search field 'topic' of the databases ( table ). the search was designed to identify documents including methods that could use non-mammalian animal models (i.e. zebrafish embryos). all search terms within one key word were combined by the operator 'or' and 'and' copied in a second or third search field. 'review' and 'meeting' documents were excluded. a search for grey literature was done in general search engines (i.e. google) using the different key words in the 'search' function of the respective websites. all the information on in vivo studies, using mammalian and/or non-mammalian organisms, and in silico studies was collected. these key words were chosen because this section aimed to describe the possible effects that exogenous ncrnas, specifically of plant dietary origin, might exert on the immune system of humans and animals. the search yielded ≈ entries (table ) . publications were excluded if one of the following criteria was met: the biological effect could not be unambiguosly attributed to ncrnas; the publication addressed immunodeficiency or autoimmunity; the publication reported no immunity (innate or adaptive)-associated endpoint or it is not specific for immune function and/or homeostasis evaluation; evaluation of exposure of and/or effect on cells other than immune cells (e.g. epithelial cells); the study was unable to measure processes related to immune function and/or homeostasis; the study reported data on potentially immunocompetent cells not fully differentiated as mature immune cell; no ncrnabased plant was tested in the publication; and the publication did not address plant-derived exogenous ncrna. the unexpectedly low number of publications retrieved using the key words proved a challenge in the selection process. for example, selection for in vivo studies produced only articles after title/abstract screening. to expand the number of documents, additional key word searches were included and less conservative criteria were set for the title/abstract screening. retrieval of grey literature was also based on information containing "preliminary results" or on-going projects that could possibly answer the key questions. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. studies were further evaluated for their relevance to answering the key questions. the key questions identified using the above key words were: -which test methods or approaches are available to investigate the possible role of plant-derived exogenous ncrnas on the immune system of humans or animals? -what human or animal immune response(s), innate or adaptive, are possibly associated with administration of plant-derived exogenous ncrnas? -which mediators in humans and animals have been identified and/or ascribed to immune response(s) possibly mediated by administration of plant-derived ncrnas? -which innate immunity mediators (i.e. chemokines, cytokines) have been identified in humans and animals as associated to effects of plant-derived ncrnas? -which human or animal cells (i.e. lymphocytes, macrophages, myeloid) are possibly involved in immune responses mediated by plant-derived ncrnas? after the selection of the full-text documents, publications were excluded if not providing sufficient information (i.e. endpoint, exposure time), if investigating a mechanism other than immune-related processes, or if describing an operational procedure or guideline rather than a research study. from the initial ≈ documents identified using the above key words, and after filtering for the relevance to the key questions and detailed study of the full-text reports, documents were finally selected for review in the three sections of part (efsa task ). of these, documents were selected for review in this section. gut microbiota is important for the function of the immune system, therefore the purpose of this additional search was to provide general information on the effect of dietary exogenous ncrnas on the gut microbiota and the possible consequent secondary modulation of human or animal immune systems. this would serve as an introduction to identifying information gaps and experimental needs for future studies relevant to food/feed risk assessment of ncrna-based plants. selected full-text studies were analysed for their relevance to answering the following key question: -is there evidence for influence of exogenous plant ncrnas on the gut microbiota related to the immune system? five documents were finally selected for review in this section. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. results are presented narratively. when appropriate, tables and figures are included to allow better understanding of the topic covered by the literature review. some sections of this review provide only limited amount of quantitative analysis due to the nature of the available evidence and the key questions identified for each subchapter. at the end of each subchapter, a short summary presents the general points identified in the reviewed literature. gaps in the literature and open questions identified in the field are presented in a subchapter of certain sections. these gaps in the literature may serve as suggestions for further research in each topic. part : kinetics of exogenous ncrnas in humans and animals (efsa task ) ncrnas are transcripts that are not translated into any functional peptides or proteins. these transcripts include structural (transfer, ribosomal, small nuclear, and small nucleolar rnas) and regulatory ncrnas. the latter comprise both small and long ncrnas that can regulate gene expression by acting on chromatin, transcription, rna processing, rna stability and translation. this part of the report introduces general features of regulatory ncrnas including their function and aspects of their biogenesis and degradation. it also establishes possible comparisons between animal and plant ncrna pathways. this can be considered background information to clarify the possibility of plant ncrna biological effect in humans and animals that ingest food/feed from ncrna-expressing gm plants. in addition, ncrna movement inside the plant is also described. specific details on the structural ncrnas and the broad topic of plant ncrna biology are outside the scope of this literature review. following an extensive literature search as described above and based on the team expert decisions on the topic, this section is a review of scientific documents. small ncrnas (srnas) srnas are common to both plants and animals. two major classes of srnas are found in eukaryotes: micrornas (mirnas) and small interfering rnas (sirnas). they function as regulators of endogenous genes or as defenders from invasive nucleic acids. they are characterized by the double-stranded nature of their precursors, and differ from the less abundant piwi-interacting rnas (pirnas) which derive from fragmentation of single-stranded rnas (juliano et al., ) , and are primarily found in animals; they have not been described in plants. pirnas exert their functions in germ and stem cells through interaction with piwi-proteins (juliano et al., ) . by contrast, both mirnas and sirnas are bound to argonaute (ago) proteins, where they identify 'target' rnas by base-pairing interactions, and act in both somatic and germ cells. all srnas in plants are modified at the ' -terminus by '-o-methylation, including mirnas and sirnas, which lack this modification in animals. '-o-methylation is essential to conferring stability and protection from ' uridylation and degradation (borges and martienssen, , ) . mirnas and sirnas are distinguished by their origin and biogenesis (figure ) . mirnas are derived from processing of single-stranded precursors with a hairpin structure, whereas sirnas are generated from long, fully complementary double-stranded rna (dsrna) precursors (carthew and sontheimer, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endonucleases (dcls) that excise rna precursors into short double-stranded fragments, - nucleotide (nt) long, with -nt ' overhangs; and argonaute proteins (agos) that engage these duplexes and support their silencing activies based on target complementarity (carthew and sontheimer, ). further details on sirnas and mirnas are provided below. sirnas several different classes of sirnas with specialized biological roles have been identified in plants. the most abundant class consists of heterochromatic sirnas (hetsirnas), which are usually - nt long and originate from the repetitive and intergenic regions in the chromosome and transposable elements. hetsirna are crucial in dna methylation and chromatin modification through a process known as rnadirected dna methylation (daxinger et al., ) . hetsirnas are processed by dcl nuclease and preferentially loaded into ago . mature ago -sirna complex can interact with complementary noncoding nascent rna polymerase triggering the recruitment of dna methyltransferase (domains rearranged methyltransferse ) to silence target loci at the transcriptional level via dna methylation and repressive chromatin modifications (vaucheret, ; lee and carroll, ) . these hetsirnas may account for approximately half of a plant's total mass of srnas. another class of sirnas are naturalantisense transcript sirnas (natsirnas) which can be generated from dsrna precursors through hybridization of independently-transcribed complementary rna strands (vaucheret, ) . in addition, secondary sirnas generated as a "secondary effect" of mirna-mediated target cleavage are found in plants (axtell, ; vaucheret, ) . the mirna-mediated cleaved target is occasionally used by rna-dependent rna polymerase (rdr) to produce secondary sirnas, which can either give rise to a phased set of sirnas (phasirnas) or trans-acting sirnas (tasirnas) with the ability to target genes different from their loci of origin. this secondary pool of sirnas can greatly amplify and sustain a systemic silencing throughout the organism. recognizable rdr-encoding genes are present in the genome of many rnai-competent eukaryotes, with the notable exceptions of insect and vertebrate species in which these secondary sirnas are absent (carthew and sontheimer, ). the lack of these secondary sirnas might have a positive effect on specificity (as sirna amplification can lead to the silencing of multiple transcripts, specifically if they share a highly conserved sequence or a common exon) and a negative effect on the amplification of rna silencing in these species. plants have more diversified and specialized sirna-based pathways than other organisms, which are thought to contribute to plant plasticity . it is generally accepted that these pathways evolved as a cellular defence mechanism against rna viruses and transposable elements, which were later adapted to regulate the expression of endogenous genes. this is consistent with the fact that most small rna classes have a recognized role in defence responses, as well as in epigenetic regulation, and that plants have larger and repetitive genomes (borges and martienssen, ) . the diversification of srna-directed silencing pathways in plants occurred through the expansion of the rdr-polymerases, dcl and ago proteins. rdr genes are found in rna viruses, plants, fungi, protists and some animals, but are absent in flies, mice and humans; this is consistent with the fact that the vast majority of srnas in humans are mirnas (kurzynska-kokorniak et al., ) . absence of rdr activity may also justify the lack of intercellular spreading of rna silencing in vertebrates, whereas systemic silencing is a phenomenon widely reported in plants and nematodes. dicer or dicer-like proteins in plants constitute a four-member gene family, whereas vertebrates have only one member (mukherjee et al., ) . the diversification of agos has resulted in development of distinct gene-silencing processes based on differential ago affinities to small-rna duplexes (borges and martienssen, ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mirnas mirnas are well-studied srnas deeply conserved over long evolutionary distances, even between the plant and animal kingdoms. however, there are substantial differences between these two kingdoms with regard to mirna biogenesis, and mechanism and scope of mirna-mediated gene regulation (jones-rhoades et al., ) . detailed pathway comparisons and species information are described elsewhere . transcription of mirnas is typically performed by rna polymerase ii, and transcripts are capped and polyadenylated. in animals, most mirnas are derived from longer hairpin transcripts (pri-mirna) by the consecutive processing by the rnase iii-like enzymes drosha and dicer, whereas in plants only dicer (particularly dcl ) is responsible for mirna processing. thus, the biogenesis of most mirnas in plants occurs in the nucleus, whereas in animals it requires the sequential cleavage of pri-mirna in the nucleus and then in the cytoplasm by distinct rnaseiii enzymes (rogers and chen, ) . once processed, one strand of the hairpin duplex is loaded into an ago protein to form the core of the mirna-induced silencing complexes (miriscs). miriscs silence the expression of target genes predominantly at the post-transcriptional level (ptgs). the targets to be silenced are recognised through base-pairing interactions between the loaded mirna and mrna target, which contains a partially or fully complementary sequence (huntzinger and izaurralde, ) . plant mirnas recognize fully or nearly complementary binding sites, which are generally located within the open reading frames (orfs) of the mrna target. of importance is that mirna nt - are usually engaged in base pairing, which allows target cleavage by ago proteins (between nt and ). by contrast, animal mirnas recognize partially complementary binding sites, which are generally located in ' utrs. in both plants and animals, complementarity to the ' end of the mirna (the 'seed' sequence, containing nt - ) is a major determinant in target recognition and is sufficient to trigger silencing. for most mirna-binding sites complementarity is limited to the seed sequence (seed-matched sites) or to the seed sequence plus mirna nucleotide . however, in some rare cases complementarity to the ' region of the mirna might contribute to target recognition, particularly when the mrna has a weak seed match. even for these sites, however, mirna nucleotides - generally bulge out, preventing endonucleolytic cleavage by agos. in both animals and plants the mirna ' terminal nucleotide is buried in the mid domain of agos and is not available for pairing with the target (huntzinger and izaurralde, ) . therefore, there is an important difference in complementarity between plant and animal mirnas and their targets; this is less extensive in animals than in plants. in both animals and plants mirnas can move from cell-tocell. while in mammalian cells mirnas and other types of rnas can be transferred through secretory vesicles (ruvkun, ; valadi et al., ) , in plants they mostly use a different mechanism (kobayashi and zambryski, ) (see section . . . . for details). however, recent evidences also suggest that plants can send small rnas in extracellular vesicles to pathogens to silence virulence genes (cai et al., ) . regarding the silencing mechanism, mirnas were initially thought to inhibit translation in animals and to predominantly promote target endonucleolytic cleavage in plants. however, recent evidence has changed this view by showing that mirnas can trigger translational repression and mrna destabilization in both kingdoms. in both plants and animals, the current evidence suggests that target mrna degradation provides a major contribution to silencing by mirnas (huntzinger and izaurralde, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. next generation sequencing approaches have revealed a new transcriptional landscape in which many novel lncrnas have been identified. these are transcripts longer than bp without any clear protein coding potential. lncrnas are transcribed either from intergenic regions (lincrnas), introns (incrnas) or the opposite strand to protein-coding genes or other lncrnas; they are thus natural antisense transcripts (nats) (ariel et al., ; chekanova, ; shafiq et al., ; yamada, ) . as a confirmation of their widespread nature and possible biological relevance, lncrnas have been identified in plants, fungi and animals. in fact, lncrnas are involved in diverse biological processes across the eukaryotes, ranging from regulation of mating type in yeast to the pluripotency of embryonic stem cells in mammals (zofall et al., ; flynn and chang, ) . in plants, lncrnas play key roles in flowering time regulation, gene silencing, root organogenesis, seedling photomorphogenesis, and reproduction (ariel et al., ; chekanova, ; shafiq et al., ; yamada, ) . although plant lncrna biological characterization is still limited, detailed analysis of over arabidopsis thaliana transcriptome data sets identified , putative lncrnas, including approximately , nats and over lincrnas. both in plants an mammalians (jin et al., ; liu, j et al., ) these lncrnas do not show association with small rnas and are also expressed at lower levels ( -fold to fold less) than mrnas. plant nat-lncrnas can overlap completely ( %) or have complementary sequences in the ' or ' regions of mrnas. they accumulate in a tissue-specific manner and many are modulated in respond to biotic or abiotic stresses, suggesting fine-tuning regulatory roles wang et al., ; ben amor et al., ; xin et al., ) . nat-lncrnas also accumulate under specific environmental conditions such as light exposure, and their expression overlaps with accumulation of histone acetylation marks, suggesting some transcription regulation effect . in plants there are other types of lncrnas. such is the case of the lncrnas involved in: ) the rna-dependent dna methylation silencing pathway (rddm); and ) the lncrnas generated from phasloci which serve as precursors of -nt and -nt secondary phased phasirnas in many plant genomes (zhai et al., ; fei et al., ; zheng et al., ) . however, the function of most plant lncrnas is still unknown (yamada, ; shafiq et al., ; ariel et al., ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. many lncrnas in mammals and yeast originate from specific genomic locations such as the regions around transcription start sites (tsss), enhancer regions, intron splicing sites, and transcription termination sites. most of the studied lncrnas are expressed around the tss, including exosomesensitive yeast cryptic unstable transcripts (cuts) and stable un-annotated transcripts (suts) (xu et al., ) , and upstream antisense rnas (uarnas) (flynn et al., ) , among others. many mammalian non-polyadenylated lncrnas also correspond to divergently transcribed, exosome-sensitive ernas mapped to enhancer regions (andersson et al., ) ; so far plant ernas have not been reported. although most plant lncrnas are transcribed by rna pol ii, there are two plant-specific rna polymerases, pol iv and pol v, that also produce lncrnas (wierzbicki et al., ; li et al., ; yamada, ) . like many yeast and mammalian lncrnas, most plant lncrnas are polyadenylated, although non-polyadenylated lncrnas do exist (heo and sung, ; shin and chekanova, ; kim and sung, ; andersson et al., ) . in fact, hundreds of non-polyadenylated lncrnas induced by specific abiotic stresses were identified in arabidopsis . the expression of lncrna is regulated at transcriptional level and in combination with the pathways involved in their biogenesis, ' end processing and degradation. the main '- ' exoribonuclease complex may regulate lncrna levels since various groups of polyadenylated ncrnas were originally identified in arabidopsis exosome mutants (chekanova et al., ) . rna exosome is an evolutionary conserved cellular rna processing/degradation complex (lange and gagliardi, ). some of these lncrnas originate from the tsss of protein-coding genes, resembling cuts, or they overlap with the ' ends of protein-coding transcripts and extend into the first intron (chekanova et al., ) . gene expression can be regulated by lncrnas (either in cis or in trans) by sequence complementarity or homology with other rnas or dna, and/or by their structure; lncrnas can thus form specific scaffolds or platforms for assembly of specific complexes (chekanova, ) . most lncrnas regulate transcription. in animals this can be achieved by: ) modulation of transcription factor dna-binding activity; ) control of rna pol ii pausing; or ) recruitment of chromatin modellers, which will ultimately affect chromatin topology and nuclear organization (bonasio and shiekhattar, ) . in plants, there are two lncrnas (hid , apolo) that associate with chromatin, promote loop formation, and modulate transcription ), (ariel et al., (figure ). some lncrnas act at the post-transcriptional and translational level (chekanova, ; jabnoune et al., ) . specifically, lncrnas can act as "decoys" or mimics by blocking certain rnas and/or dnas to access their protein regulators (e.g. ips ) (franco-zorrilla et al., ; huang et al., ) . bioinformatics analyses have also identified other putative mirna target mimics in animals that act similarly to plant mirna sponges (chekanova, ) . another plant lncrna (asco) also acts as a decoy of nuclear speckle rna binding proteins leading to different alternative splicing events and consequent altered root development (bardou et al., ) . other lncrnas with biological function are the enhancerrnas (ernas), described in yeast and mammals, but still unknown in plants. these ernas can act in cis as scaffolds to recruit co-activators and thus promote chromosome looping between enhancer and promoter regions; they can interact with other lncrnas to form specific chromosome structures to control gene expression. ernas are regulated by the exosome which can affect either rna synthesis or degradation in these regions (pefanis et al. ) . the exosome can also protect ernas from genomic instability by resolving r loops, which are stable rna-dna triplexes naturally formed during transcription, which can persist in regions that have divergent transcription, eventually becoming deleterious (skourti-stathaki and proudfoot, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. action of ernas and exosome can control gene expression and nuclear organization in these enhancer regions. interactions between lncrna and chromatin modifiers can be either dependent or independent of srnas. in animals they are srna-independent and occur via the trithorax complex (h k trimethylation) ; or prc (polycomb repressive complex , that regulates h k methylation) (tsai et al., ) . in plants they are -srna-dependent, with the rna-dependent dna methylation pathway (rddm) (matzke and mosher, ) (see below).further details on specific aspects are provided below. the rddm pathway seems to be a plant-specific pathway that relies on lncrnas transcribed from pol iv that will produce nt sirnas. in parallel, lncrnas produced by pol v act as a scaffold that can then recruit the sirna-ago complex by sequence complementarity (matzke and mosher, ) . however, pol v-dependent lncrnas are difficult to identify probably due to their low accumulation rates. nevertheless, the few identified lncrna are non-polyadenylated and can be either tri-phosphorylated or capped at their ' end (wierzbicki et al., ) . in addition, pol v also seems to collaborate with pol ii to promote rddm (zheng et al., ) . another group of regulatory ncrnas that are pol iv -rdr dependent were identified at intergenic regions overlapping mostly with transposons or sequence repeats. these ncrnas are also non-polyadenylated and at their ' end have a monophosphate instead of ' tri-phosphate or cap (zheng et al., ) . the arabidopsis exosome is involved in metabolism or processing of these lncrnas generated by pol iv, v and also some from pol ii (chekanova, ) . also worth mentioning is that the exosome is constituted by different subunits that are functionally diverse and can affect metabolism of smrnas and dna methylation (chekanova, ) . lncrnas and regulation of flowering some of the best characterized plant lncrnas are those involved in regulation of flowering upon "vernalisation" (the need of plants to experience a period of cold -winter-to enable flowering in spring). therefore, transgenic plants expressing any of these lncrnas may have biotechnological value. to assess the future relevance of these lncrnas it is important to understand the molecular mechanisms behind their function, since they are quite different from those of srnas (sirnas and mirnas).the best studied lncrnas in this process are: ) coldair (heo and sung, ) ; ) coldwrap (kim and sung, ); ) coolair (swiezewski et al., ) and asl (shin and chekanova, ) . all these lncrnas modulate the flclocus, which encodes a transcription factor able to repress flowering (berry and dean, ) . interestingly, these four lncrnas originate from different regions within the flclocus (promoter, first intron, and ' end antisense) suggesting a function in cis. they all contribute to prevent flc transcription by recruiting the prc silencing complex and the accumulation of repressive chromatin marks (crevillén and dean, ; csorba et al., ; rosa et al., ; sun et al., sza; swiezewski et al., ; liu, f et al., ) . of interest is that certain aspects of this regulation are similar to that of the mammalian lncrnas hotair and xist. it has been proposed that some of these lncrnas could directly bind to prc components, but findings showing that mammalian prc can bind very strongly to unrelated rnas (davidovich et al., ) question the need for specific lncrnas in this recruitment step. the study of components of the arabidopsis exosome (rrp l and rrp l ) revealed their role in coolair and asl expression or processing, although this seemed to occur independently of the exosome core complex. both rrp ls could process the ' end of coolair and promote asl accumulation, similarly to xist in humans (shin and chekanova, ; ciaudo et al., the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. regulate the levels of different lncrnas and probably act in different mechanisms to silence flc (chekanova, ) . lncrnas and r-loop formation coolair transcript levels are decreased due to r-loop formation at its promoter (sun et al., b) , which could promote exosome recruitment through the ' end processing pathway (chekanova, ) . this function is also seen in mammals where the exosome also resolves/degrades deleterious r loops (pefanis et al.) . surprisingly, in mutants affected in r loop formation, coolair may accumulate together with flc, suggesting that this regulation is still not fully understood (chekanova, ) . lncrnas and nuclear architecture animal lncrnas are involved in tethering rna, dna and proteins, and thus affect nuclear d structure (engreitz et al., ; quinodoz and guttman, ; hacisuleyman et al., ) . in plants, extensive evidence supports a similar role, either within the rddm pathway (moissiard et al., ) , in the regulation of flowering time (hepworth and dean, ) or in auxin signalling and root development (ariel et al., ; ariel et al., ) . plant and animal lncrnas can be intergenic, intronic or natural antisense transcripts, depending on their location in the genome. although only a few lncrnas have been characterized in detail, they are known to have different modes of action, ranging from chromatin modifications including prc recruitment, to promotion of translation, mirna target mimicry, hijacking splicing factors and formation of chromatin loops. adapted from (ariel et al., ) . not all processes occur simultaneously. different types of lncrnas are shown schematically. circrnas earlier studies of rnas structure proved that viroids (existing as uncoated rna molecules and are known to infect plants) have circular rna (circrna) molecules. the nature of covalently closed circular rna molecules was determined due to: i) the inability to phosphorylate at the '-terminus; ii) resistance to metaperiodate oxidation or borohydride reduction of the '-terminal ribose; or iii) resistance to venom phosphodiesterase degradation (sanger et al., ) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. al., ), plants (daros and flores, ) , and mammals (nigro et al., ) including humans memczak et al., ) , and across the eukaryotic spectrum. circular rnas' lack of coding potential was identified early on by the inability of circular mrna (kozak, ) or rna (konarska et al., ) to assemble/adhere to eukaryotic ribosomes. circular rnas are also unable to be translated in plants extracts . as with other ncrnas, circrnas in plants exhibit tissue-specific expression, have a considerable number of isoforms, alternative backsplicing (canonical and noncanonical) and alternative circularization patterns (lu et al., ; sun et al., ; wang et al., ; darbani et al., ) . the biogenesis of circrnas is a conserved feature in animal and plant cells. transcribed by rna polymerase ii and backsplicing reactions of pre-messenger rnas, they occur in the spliceosome and require a repeated sequence and rna-binding proteins. spliceosome formation is initiated by sequential assembly of small nuclear ribonucleoproteins onto a specific region of the pre-mrna downstream of the ' donor splice site (dinucleotide gt) and upstream of the ' acceptor site (dnucleotide ag). thus, exonic or intronic circrnas are generated depending on the initial small nuclear ribonucleoprotein binding site (lee et al., ; sun et al., ) . although their mechanisms of action in plants are still unclear, some studies in rice suggest that they do not imply any significant enrichment (as occurring in humans) for mirna target sites, and that circular rna and its linear form may act as a negative regulator of its parental gene (lu et al., ) . other studies have shown that fluctuations in circrnas do not correlate with the levels of their parentalloci encoded linear transcripts (darbani et al., ) . some circrnas have also been shown to contain putative mirna binding sites lu et al., ) and have been identified as mirna sponges (zuo et al., ) . by binding to mirnas, and consequently repress their function, circrnas act as mirna sponge to regulate the response to stress . recent data suggest that circrnas in plants may affect plant response to abiotic and biotic stress. for example, circrnas may be involved in chilling injury in tomato (zuo et al., ) , or may respond to imbalances in iron and zinc (darbani et al., ) . in another study, tan and colleagues (tan et al., ) showed that overexpression of a tomato circrna derived from phytoene synthase (psy ), reduced psy mrna abundance, and lycopene and β-carotene accumulation. this was likely due to the continuous highly expressed circrna and/or the low abundance of linear rna from the overexpression vector. similar results were reported for another circrna derived from the phytoene desaturase gene (tan et al., ) . their role in developmental processes has also been established (cheng et al., ) , including their possible role in different aspects of development and senescence in arabidopsis cheng et al., ) . other biological processes such as photosynthesis (dou et al., ) or mitochondrion function (darbani et al., ) have also been proposed as involving circrnas. circrnas are a popular topic in animal research because of their potential as post-transcriptional regulators (memczak et al., ; piwecka et al., ; guarnerio et al., ; hansen et al., ; ashwal-fluss et al., ) , their recognized function as mirna sponges, as sponges for rna-binding proteins or their competition with linear splicing; and their role as diagnostic markers (zhao et al., a; zhao et al., b) . indeed, circrnas have also been found in biological fluids including saliva (bahn et al., ) , seminal fluid (dong et al., ) and plasma . research in plants is just emerging and functional studies are still lacking. it is unknown if this type of novel ncrnas could resist gastrointestinal tract conditions, due to their expected high stability, and have a biological effect on an organism. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ncrnas can regulate many biological processes through their impact on gene expression by acting on chromatin structure and affecting transcription, rna processing, rna stability and translation. among these, srnas are produced by cleavage of dsrnas intermediates, either from hairpin precursors (mirnas) or from the synthesis of dsrnas by rdrs (sirnas); most lncrnas regulate gene expression without being processed. despite size and biogenesis differences, most ncrnas share sequence-specific inhibitory functions. important differences are present between plants and other organisms in srnadirected silencing pathways, which are highly diversified and specialized in plants through the expansion of rdr, dcl and ago proteins that mediate srnas biogenesis and function. the vast majority of small regulatory rnas in humans and animals are mirnas. in contrast to plants, no evidence for systemic spreading of rna silencing in humans and vertebrates have been found. although very few plant lncrnas have been studied in detail, they seem to share with their animals' counterparts the ability to recruit chromatin modifiers and thus regulate gene expression. in the same context, very few studies are available on plant circrnas. their functional characterization will bring novel insights into their possible role as a novel class of noncoding regulators. when considering the potential presence of plant ncrnas in food or feed it is important to determine the mobility potential of these molecules inside the source plant (figure ). there are several comprehensive reviews (chitwood and timmermans, ; dunoyer et al., ) addressing sirna/mirna movement in detail. it has been hypothesiezed that srnas may move throughout the plant. although the initial model would suggest non-cell autonomous (one whose action extends beyong the cell producing the signal) rna silencing by srnas, other intermediates could also account for this signal: dsrnas produced by rdr activity (plant specific), or fold back rna acting as silencing trigger (dunoyer et al., ; dunoyer et al., ; himber et al., and smith et al., ) . these intermediates could also diffuse from one cell to another and be processed again by dcl , which has been associated with nt sirna movement (dunoyer et al., ) . in detail, secondary sirnas processed from dsrna by dcl could move cell-tocell to propagate silencing by signal amplification. it has been shown that dcl expression from phloem companion cells is critical to the short-term spread of the silencing effect of a sirna duplex. this was reported in a study in which the viral suppressor p was found to be expressed in phloem companion cells and able to bind to nt small dsrna (vargason et al., ) . dcl -dependent sirna movement has also been shown to occur within certain organs, such as leaves, to create gradients (chitwood et al., ; levine et al., ) . other dcls-dependent sirnas are involved in sirna movement and these can also be sorted into different risc complexes (montgomery et al., ; mi et al., ) . other examples of mobile sirnas could be the ta-sirnas and dcl -dependent nt sirnas, also acting through ago . dcl processing of long dsrna substrates generated by pol iv (a plant-specific rna polymerase) and rdr (another plant-specific protein) leads to production of nt sirnas that are incorporated into ago and will transcriptionally silence transposons (chapman and carrington, ; mi et al., ) . in fact, sirnas derived from transposons or methylated regions in the genome have been shown to be associated with chromatin silencing. these -nt mobile sirnas could then promote epigenetic changes transmissible to following generations. when transgenes are expressed in plants, the generated dsrnas can be processed by dcl and dcl . another dcl, dcl , processes dsrnas into nt sirnas which can induce gene silencing of viral origin or from transgene expression (deleris et al., ) . sirnas may also function as mobile signals able to promote epigenetic modifications. grafting experiments with roots from dcl dcl dcl mutants and shoots expressing gfp sirnas could the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. indicate that sirnas to nt long move throughout the plant, but still unclear if in free or proteinbound form (i.e. bound to ago or other proteins) (molnar et al., ) . within the plant, sirnas are known to move in a source-to-sink direction and into growing meristems (molnar et al., ; palauqui et al., ; schwab et al., ) . their accumulation in reproductive cells could promote epigenetic changes in subsequent generations. in fact, slotkin et al. (slotkin et al., ) showed that dcl dependent sirnas generated by transposon activation in the pollen vegetative nucleus can silence transposons in pollen sperm cells. these cells are then able to transmit genetic material to the following generation. a similar effect may occur if maternally-derived sirnas in the endosperm moved to the embryo and influence genome integrity or even lead to the creation of new epialleles (molnar et al., ) . similar findings of sirnas silencing transposable elements in the animal germ line have also been reported in an animal cell line and tetrahymena (malone and hannon, ). considering that plant development relies more on positional effects than cell lineage, the role of mirnas in the control of several developmental processes has to be strictly regulated at the mobility level (chitwood et al., ; levine et al., ) . for example, as small rnas diffuse into regions of low mrna (targets) expression, it eliminates target molecules therein, but cannot affect regions of high mrna levels (levine et al., ) . as mentioned previously, dcl processes imperfect hairpins into nt mirnas, with a ' u nucleotide incorporated into ago , and cleavage the mrna targets. ago can also incorporate these mirnas (brodersen et al., ) . while sirna movement is better understood, this is not the case for mirna movement. mirnas can be isolated from phloem sap (mir , mir , mir ) but it is unclear if they can leave the phloem (buhtz et al., ; pant et al., ; yoo et al., ) . some reports support both endogenous mirna and artificial mirna movement (chitwood et al., ; schwab et al., ) . it has been shown that mirnas can move from below the shoot apical meristem into the meristematic layers (chitwood et al., ) , and, similarly to sirna movement, from the phloem towards the root meristems (molnar et al., ) . although initial evidence suggested sirna mobility, with only dcl responsible for this, it is now accepted that almost every known rnai pathway in plants has non-cell autonomous activity. movement has been shown for srnas ) of viral origin; ) induced by transgene expression; ) ta-sirnas; ) mirnas and ) repeat-associated sirnas (dunoyer et al., ; carlsbecker et al., ; chitwood et al., ; molnar et al., ; slotkin et al., ) . however, it is still unclear if srnas can move in a free state or associated with specific proteins, and in a single or double strand form. whereas some reports suggest that the srna molecule movement happens in a duplex form, the strand bias found in the srnas present in dcl dcl dcl grafted roots suggests that single strand sirnas can also diffuse (molnar et al., ) . unlike in animals, in which sirnas can move from cell-to-cell through secretory vesicles (ruvkun, ; valadi et al., ) , in plants the size of small rna would allow cellto-cell movement through plasmodesmata channels connecting different plant cells (kobayashi and zambryski, ) . this movement seems to occur in a source-to-sink direction, and could be regulated in some tissues through the formation of sirnas-ago complexes (molnar et al., ; schwach et al., ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. plant small rnas (srnas) can move either cell-to-cell or long distance. cell-to-cell movement occurs through plasmodesmata (green arrows) that allow spreading of srnas from the cell where they are generated to - neighbouring cells (a). this cellto-cell movement can be extended over the surrounding cells by signal amplification (b). in this case, srna targets will be converted into new dsrnas by the combined action of dcls and rdrs. several grafting experiments have demonstrated that - nt sirnas can move over long distances in the plant (c). whether they move as ssrna, dsrna or associated with proteins is still unclear. adapted from (dunoyer, p. et al., ) , (melnyk et al., ) and (molnar et al., ) . in terms of mirna mobility, it is still unclear if all mirnas, or only a subset, can diffuse. different mobility could be associated with different tissues/structures, and/or the size and stability of the passenger strand mirna (a.k.a. mirna*, star strand). in addition, mirna movement regulation could involve subcellular compartmentalization (by nuclear export proteins such as hasty (park et al., ) ) and sequestration via ago proteins (e.g. mir / can be sequestered by ago and affect shoot apical meristem differentiation (liu et al., ; tucker et al., ) ). most of the information is available for srnas since they represent the most studied class of ncrna to date. in addition to the role of srna movement when considering ingestion of transgenic plants expressing altered levels of srnas, it is also important to address the issue of their biogenesis and turnover to assess their temporal availability when ingested. this is especially important since transgenic plants expressing rna interference constructs will accumulate the different srna intermediary forms (pri-mirnas, pre-mirnas, mature mirnas, drnas, sirnas), which may also be processed outside the plant when ingested. several reviews published in high-impact journals (rogers and rogers and chen, ; or scientific reports the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. view of the processes of mirna maturation and degradation in plants. it is important to consider the homology between the mirna biogenesis pathways in plants and animals as well as mirna stability. this knowledge allows understanding ) whether any aspect of animal metabolism or other biological processes could be modulated by plant mirnas; and ) if mirnas can survive for long periods after ingestion. as typical pol ii transcripts, pri-mirnas are stabilized by addition of a ' -methylguanosine cap (xie et al., ) and a ' polyadenylated tail (jones-rhoades and bartel, ; zhang et al., ) . mirna genes are also subjected to transcription factor regulation and can be alternatively spliced. the size of these transcripts varies and their processing normally occurs in a base-to-loop direction (rogers and . in plants, there are multiple dcl endonucleases that possess dexd/h-box rna helicase, duf , paz, tandem rnaseiii, and dsrna-binding domains. dcls cleave the dsrna precursor generating a -nucleotide ' overhang (margis et al., ) . processing of the pri-mirna requires at least two catalytic cycles to free the mirna:mirna* duplex. in animals, this requires sequential cleavage of the pri-mirna in the nucleus first and then in the cytoplasm by different rnase iii enzymes. the biogenesis of most mirnas in arabidopsis requires dcl (park et al., ; reinhart et al., ) , although other dcls may be involved, but are not essential (xie et al., ; gasciolli et al., ) . in contrast to animals, all plant mirna processing steps occur in the nucleus (papp et al., ) . dcl activity will result in mature mirnas of - nucleotides (nt), although variations in size can occur (rogers and chen, ) . confirming its fundamental biological relevance, dcl mutants are lethal (golden et al., ) . other dcl proteins that function in different srna biogenesis pathways might also have minor roles in mirna biogenesis. for instance, several mirna genes are partially processed into - -nucleotide mature mirnas species whose accumulation depends on dcl (vazquez et al., ) . in the absence of dcl , mature -nucleotide mirnas can accumulate due to dcl activity. in contrast, processing of mir , mir , and mir depends primarily on dcl , probably as a consequence of highly complementary fold-backs in these pri-mirnas (ben amor et al., ; rajagopalan et al., ) . dicer cleavage of animal pri-mirnas is facilitated by the action of dsrnabinding domain (dsrbd) proteins (drbs) (jiang et al., ; parker et al., ) . in arabidopsis, there are five drbs that have been shown to bind dsrna in vitro (hiraguri et al., ) . the best studied member of this family is hyl (hyponastic leaves ) which is involved in pri-mirna processing, but also in pri-mirna intron splicing (laubinger et al., ; szarzynska et al., ) . hyl seems to increase the accuracy of dcl processing of most, but not all, pri-mirnas, suggesting that other mechanisms may also be involved (liu, c et al., ) . from the same family, drb and drb are also involved in pri-mirna processing, by associating with dcl (rogers and . another protein involved in mirna biogenesis is serrate (se), which can interact with hyl and dcl (lobbes et al., ; machida et al., ) . like hyl , se also seems to increase dcl pri-mirna cleavage efficiency (dong et al., ) . of interest is that these two proteins (hyl and se) can be regulated by phosphorylation. although hyl phosphorylation can decrease its activity (manavella et al., ) , the biological relevance of this modification in se is not known (rogers and . another regulator, dawdle (ddl), a phosphothreonine-binding forkhead associated (fha) domain protein, seems to stabilize pri-mirnas, possibly by direct binding (yu et al., ) . like se and hyl , ddl can also interact with dcl , but does not seem to be involved in pri-mirna processing. ddl most likely facilitates dcl access or recognition of pri-mirnas. in addition, ddl can recruit phosphorylated se or hyl to the pri-mirna. the human homologue of ddl, snip , seems, among other functions, to be involved in mirna biogenesis and interacts with drosha (yu et al., ) . ddl could therefore be an evolutionarily www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. conserved factor in mirna biogenesis. other examples of conserved srna biogenesis machinery include ago, dicer, hen and exportin (yu et al., ) . two components form the cap structure of rna pol ii transcripts in plants: cap-binding proteins (cbps) and . there is substantial overlap among cbp , abh /cbp , and se mutants in terms of: ) the specific pri-mirnas affected; ) the specific mrna splicing defects observed, and ) a bias for accumulation of first introns (laubinger et al., ) . this connection between pri-mirna processing and pre-mrna splicing seems to converge at se and cbps. in fact, splicing defects are also observed in cbp and abh /cbp in plants. in animals, the cap and its associated cap-binding complex are essential for the correct splicing of the first intron (lewis et al., ) . the connection between pri-mirna processing and splicing also converges in tough (tgh), a g-patch domain rna-binding protein. the tgh mutant accumulates pri-mirnas in vivo and has been shown to affect processing of pri-mirnas into -nucleotide mature mirnas (ren et al., ) . tgh may have either a structural or a regulatory role in pri-mirna processing. tgh binds both pri-and pre-mirnas in vivo, but this probably occurs via association with the loop structure since tgh binds ssrna but not dsrna in vitro (ren et al., ) . tgh paralogs have been described in metazoans (calderon-villalobos et al., ) , but their roles in mirna biogenesis are still unknown. a human tgh paralog is reported in spliceosomal preparations (jurica et al., ) . a conserved connection may exist between tgh and splicing and possibly pri-mirna processing (rogers and chen, ). mirna processing seems to occur in specific subnuclear regions. hyl , se and dcl can make pairwise interactions that occur in certain subnuclear particles (fang and spector, ) . tgh can also interact with dcl , hyl , and se in subnuclear foci (ren et al., ) . these foci seem to be zones of functional pri-mirna processing (fang and spector, ; fujioka et al., ; manavella et al., ) . some of these components (dcl , hyl , se, tgh) also co-localize with components of the splicing machinery, further strengthening the connection between the two processes (calderon-villalobos et al., ; fang and spector, ; fujioka et al., ) . processed mirna:mirna* duplex will leave the nucleus to carry out its function in the cytoplasm. several components have been implicated in this transport and function and are described below. nucleo-cytoplasmic transport hasty (hst) is a member of the importin-ß family of arabidopsis that accumulates in the nuclear periphery (bollman et al., ) . hst is a paralog of human exportin- involved in pre-mirna transport. hst can participate in dcl -dependent mirna:mirna* duplex export (zeng and cullen, ) . but this is probably not the sole mechanism for mirna nucleocytoplasmic transport, since passive diffusion via the nuclear pore may also occur (jacob et al., ; park et al., ) . ema /sad is another importin-ß family protein involved in mirna function. ema is an orthologue of human importin- which facilitates nuclear import of ago and reduces its association with target mrnas (weinmann et al., ) . it is still unclear if ema ) sequesters mirnas; ) is involved in transport of other risc loading factors or; ) affects loading of certain mirnas into risc (rogers and . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after leaving the nucleus the mirna:mirna* duplex is loaded into ago, a risc component. the arabidopsis ago family consists of members that associate with small rnas possessing specific ' nucleotides (mi et al., ; takeda et al., ) . in plants, the slicer activity of agos has been shown for ago , ago , ago , ago , and ago (rogers and . ago seems to have a predominant role since it preferably associates with 'u small rnas (most common ' nucleotide in mirnas) (mi et al., ; takeda et al., ; vaucheret et al., ) . however, other agos can also associate with specific mirnas depending on mirna length or even their accumulation in different tissues (ebhardt et al., ; rogers, kestrel and chen, xuemei, ) , suggesting a certain flexibility in this association. in plants, mirna guide strands generally accumulate in higher levels than mirna* strands, although the exact mechanism for strand selection is still not fully understood (rogers and chen, ). hyl (drb) seems to be involved in this process (eamens et al., ; manavella et al., ) . in animals, loading of sirnas into risc requires the cytosolic drb protein loq, but loading of mirnas into risc requires dicer rather than loqs (liu et al., ; liu, x et al., ) , suggesting different mechanisms in plants and animals. hyl is predominantly nuclear, therefore its association with the mirna:mirna* duplex suggests that ) hyl may be transported to the cytoplasm together with the processed mirna duplex; or ) loading may also occur in the nucleus. in addition, other risc loading mechanisms can exist (eamens et al., ). in animals, sirna passenger strand unwinding requires ago slicer activity; mirnas duplexes are unwound by a slicing-independent mechanism (matranga et al., ) . in plants, ago slicer activity removes sirna passenger strand but this function is not required for mirna passenger strand removal (carbonell et al., ; iki et al., ) . as occurs in animals, plant mirnas use an alternative slicerindependent unwinding mechanism. ago loading seems to require heat shock protein (hsp ) too (iki et al., ) . in addition, via hsp , ago is able to interact with squint (sqn) and the phosphatase pp (iki et al., ) . these interactions seem to regulate ago loading and consequently risc activity. ago phosphorylation has been detected in arabidopsis (de la fuente van bentem et al., ) in manner similar to human ago . apparently, phosphorylated ago exhibits reduced srna binding (rüdel et al., ) . phosphoregulation by pp or other proteins may modulate arabidopsis ago loading capacity. unlike in animals, in which mirna biogenesis occurs in the nucleus and cytoplasm, the mirnas in plants are generated in the nucleus in a mostly dcl- -dependent manner. studies confirm that several of the major components are evolutionary conserved, such as the dicer, ago, hen and exportin paralogs involved in nucleocytoplasmic transport of the mirna:mirna* duplex. however, shared pathway components among the different species do not reflect totally identical processes, such as, loading of mirnas into risc which occurs differently in plants and animals. one interesting common aspect remains the pri-mirna processing and pre-mrna splicing which seems to occur both in plants and animals. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section . . . and following the methodology described in section . . . ., a total of publications were selected as relevant to the stability and turn-over of non-coding rnas. of these, only four analyse ncrnas stability at a genome-wide scale, and one (enuka et al., ) simultaneously addresses the stability of several circrnas. using these studies, the stability of small ncrnas, lncrnas and circrnas can be compared. while earlier studies report the degradation of plant nucleic acids when exposed to hard conditions (i.e. cooking), six recent studies suggest that some ncrnas (i.e. mirnas) from plants can resist these conditions. turnover of ncrna molecules in plants and mammals depends both on their stability and degradation rate and it is described that chemical modifications can increase stability of different types of srnas (e.g. sirnas, mirnas). in mammalian cells, artificially introduced -o-methyl groups can stabilize sirnas without affecting their rna interference activity (czauderna et al., ) . considerable work has been done to address the issue of mirna modifications and how these affect their stability. among these, '-o-methylation has been identified as relevant. plant mirnas have a naturally occurring methyl group on the ' nucleotide ribose ( figure ). in this case, methylation does not require guide rnas, since hen (hua enhancer ) can methylate mirna:mirna* duplexes. this hen -dependent '-omethylation on the ' terminal ribose is a mg + dependent methylation mechanism that will ultimately stabilize mirnas (abe et al., ; molnar et al., ; yu et al., ; yang et al., ) .the hen mg +-dependent methylation mechanism relies on its two dsrbds binding the substrate duplex, and the la motif-containing domain interacting with the ' end of the substrate strand (huang et al., ). this methylation step occurs after dcl processing creates the mirna:mirna* duplex but before duplex www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. unwinding and selection of the mirna guide strand. however, it is still not clear whether hen can methylate free mirnas or can bind hyl , dcl and ago , although it has been shown to interact with hyl and dcl . in animals, the hen homolog has a different structure since it does not possess the dsrbd and la motif-containing domains. therefore, the animal hen homolog methylates singlestranded srnas associated with ago or piwi proteins (saito et al., ; horwich et al., ) . the degradation of ncrna molecules in plants and mammals is mediated by ' nucleotidyl transferases and exoribonucleases. the arabidopsis hen suppressor (heso ) belongs to the dna polymerase β gene family (zhao et al., ; ren et al., ) . in other organisms, heso putative homologs are ribonucleotidyl transferases that are able to add specific nucleotides to the ' end of different rnas (martin and keller, ) . arabidopsis heso can also add nontemplated nucleotides to the ' end of unmethylated mirnas in vitro and exhibits a preference for uridine (ren et al., ; zhao et al., ) . analysis of hen and heso mutants indicates that this ' oligo uridylation triggers degradation of mirnas (ren et al., ; zhao et al., ) . the arabidopsis family of small rna degrading nucleases (sdns) of '- ' exonucleases has similarity to the yeast rex exonucleases. sdn has '- ' exonuclease activity versus short ssrnas but is not active against longer or double-stranded substrates (ramachandran and chen, ) . in plants, ' truncated mirnas can be modified by the addition of ' oligonucleotide tails (see above) (ibrahim, fadia et al., ; li, j et al., ; lu et al., ). in addition, these ' truncated mirnas also seem to associate with ago . this interaction delays their degradation and allow addition of the ' oligonucleotide tail leading to their degradation at a later stage (zhao et al., ) . whereas '-o-methylation may protect against ' oligouridylation by heso (see above) and consequent targeting to degradation, this modification would not protect against sdn . it is possible that sdn can promote mirna ' truncation and then facilitate subsequent ' polyuridylation by heso . however, after ' polyuridylation sdn would be unable to degrade these tailed mirnas, suggesting the existence of other nucleases (ramachandran and chen, ) .for instance, the addition of ' polyuridylated tails in mirnas seems to attract exosome-mediated degradation in algae (ibrahim, f. et al., ) , but in arabidopsis this regulation has not been addressed in detail (rogers and rogers, kestrel and chen, xuemei, ) . another possible mechanism would include loss of ago association and protection of mirnas with long ' tails due to heso activity. in addition, ' polyuridylation may act as a nucleases recruitment platform, thus promoting mirna degradation. nevertheless, ' polyuridylation and nuclease processing seem to be interconnected events that will ultimately result in degradation of the tagged mirnas. the overall turn-over of rnas is mainly described using half-life studies in both plants and animals. most of the available reports assessing ncrna half-life focus on the role of ' truncation, ' uridylation and consequent degradation (see above) as the main processes regulating mirna turnover in plants. however, a recent report identifies the enzymes (sdn and sdn exonucleases) responsible for ' truncation in vivo and its relationship with ' tailing (uridylation). also of importance, this report shows the opposite role of ago and ago in mir / degradation by sdns, specifically sdn . ago promotes mir degradation by sdn , an unexpected role for a plant ago which has been associated with a protecting role in mirna stability (yu et al., ) . this function of ago (enhancing mir degradation) keeps mir out of the stem cell niche in the shoot apical meristem (where ago is not expressed). accumulation of ectopic mir in the shoot apical meristem fails to maintain the stem cell population. this kind of mechanism is thus needed to clear mir since this particular mirna can move between cell layers (see mirna movement section, above). another report evaluates the activity of nucleotidyl transferases in ' uridylation of mirnas and in vitro assays indicated a fast enzymatic process (tu et al., ) . also, xrn - nucleases that affect mir homeostasis in c. elegans do not www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. seem to process mature plant mirs but rather the by-products of pri-mirna processing (ji, lijuan and chen, xuemei, ) . in summary, there are no specific studies on mirna half-life in plants. the reports mentioned here evaluate mirnas levels at steady state in different mutants, for example, when either the exonucleases and/or nucleotidyl transferases involved in mirna turnover are deleted. the half-life of mirnas in mammals has been addressed in studies of dicer ablation in mouse embryonic fibroblasts showing that mirnas are highly stable inside the cell. indeed, turnover assays revealed that the average mirna half-life is h (i.e. ≈ days) (figure ). although some mirnas have a shorter half-life, these data generally indicate that mirnas are much more stable (≈ x more) than messenger rnas. (gantier et al., ) . several features have been described that might explain this high half-life for mirnas. for instance, partial pairing of mirna and the mrna target site generally produces translational repression, while extensive pairing of mirna and mrna produce a mrna cleavage (pasquinelli, ) . in the former case the mirna remains stable, but in the latter it degrades. further studies in mammalian cells have also shown that argonaute proteins stabilize mature mirnas in a slicing-independent manner, increasing mature mirna stability (winter and diederichs, ). for specific mirnas, it has been found that certain rna-binding proteins (i.e. roquin) can enhance mature mirna- stability by reducing its ' end uridylation (srivastava et al., ) . while intracellular levels of mirnas are mostly affected by cell division (ghosh et al., ; gantier et al., ) , mirna activity and turnover can also be controlled by subcellular distribution of microribonucleoproteins; that is, polysome sequestration contributes to an increase of cellular mirna levels, but without an increase in mirna activity (ghosh et al., ) . plant small rna duplexes can be '-o-methylated at their ' terminal ribose by hen , loaded into ago and protected from uridylation and degradation by '- ' exonucleases. ago -bound unprotected small rnas, however, are uridylated by heso and degraded by sdn . adapted from (borges and martienssen, ) and (ji and chen, ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. widespread variation of mirna decay in slow-dividing primary macrophages. bone marrow derived macrophages from when analysing lncrnas stability in mammals at a genome-wide scale, lncrnas half-life generally varies over a wide time range, comparable to, although on average less than, that of mrnas (clark et al., ) . in a study of all transcripts, including mammals, it was found that the range of half-lifes for ncrnas (all types) was similar to that of the mrna half-life (tani et al., ) . indeed, a large number of ncrnas transcripts have a half-life of less than h (tani et al., ) , which agrees with the < h half-life for hundreds of unstable lncrnas (clark et al., ) . however, although there are lncrnas with extreme stability (half-life > h) they are still much less stable than mirnas (gantier et al., ) (see above), but more stable than protein-coding rnas (wang, l et al., ) . circrnas have a covalently closed loop structure with neither '- ' end nor a polyadenylated tail, which confers high resistance to rna exonuclease or rnase r treatment when compared to that of their linear sequence counterparts (memczak et al., ) . indeed, earlier studies of the structure of rnas from viroids, which were found to be circular, suggested that the nature of covalently closed circular rna molecules confers certain properties including resistance to metaperiodate oxidation or borohydride reduction of the '-terminal ribose; the inability to phosphorylate at the '-terminus; or resistance to venom phosphodiesterase degradation (sanger et al., ) . in mammals, some studies have evaluated the stability of circrnas compared to that of their linear isoform derived from the same host gene. for example, circrnas were found to be less abundant and dynamic than their counterparts (enuka et al., ) . calculating the half-life of circrnas and their linear counterparts, enuka et al., showed that the media half-life of circrnas of mammary cells ( . - . h) was at least . times longer than the median half-life of their linear counterparts ( . - . h). this suggests that cirrnas are generally more stable and static compared to linear species (enuka et al., ) (figure frame a). in a stability study on some circrnas it was found that while the associated linear transcripts exhibited a half-life of < h, the circular rna isoforms were highly stable, with transcript half-life exceeding h (jeck et al., ) . in plants, covalently circularized exogenous rna incubated with wheat embryo cell extract in vitro have shown that the circular rnas exhibited considerable stability compared to the linear version (makino et the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. al. , ) . while linear rna degraded within the first few minutes, its circular counterpart was stable for more than min in vitro (figure frame b). by contrast, circularization dramatically reduced the translation capability of the rna, which was restored to some extent after linearization. plant srnas are naturally methylated at their ' nucleotide ribose. this modification depends on hen and protects srna from degradation. unmethylated or truncated srnas can be uridylated by heso and thus be targeted for degradation by '- ' exonucleases of the sdn family. although ago binding to mirnas was believed to protect them from degradation, recent reports suggest that ago in particular may promote mirna degradation by sdns. differently from the plant situation, animal srnas are not methylated. a) mcf a cells were metabolically labelled using su, for or h. the rna was then extracted, biotinylated and purified on streptavidin magnetic beads. flow-through rna was also collected. next, rna was reverse transcribed and quantified using highthroughput real time pcr (fluidigm). the half-lives of circrnas and their corresponding linear counterparts are shown. halflife values were calculated from two samples, labelled with su for or h and then averaged. all data were corrected for any bias introduced due to low uridine (short length) of rna species (see 'materials and methods' section). the circrnas (red dots) and their linear counterparts (blue dots) were sorted from high to low according to the difference between their half-lifes. error bars represent standard errors. source: from (enuka et al., ) . b) stability of circular rnas in the wheat germ cell-free translation system. a denaturing polyacrylamide gel separating the synthesized circular and linear rnas with a (gaaa) sequence after incubation with the wheat embryo extract for the indicated time periods. untreated rna is in lane n. positions of circular, linear, and ribosomal rnas are indicated as c, l, and rrna, respectively, on the right. source: from . compelling evidence supports the significant contribution of srnas to communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. (baulcombe, ; (ghag et al., ; koch et al., ; vega-arreguín et al., ; helber et al., ) . srnas or dsrnas, efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. for example, can be transferred from plant to pests such as insects that eat leaves or nematodes that infect roots. in fact, transgenic plants can be created that express dsrna homologous to essential genes of insect pests or nematodes and thus control them (baum et al., ; fairbairn et al., ; mao et al., ) . thus, this silencing transfer mechanism is very relevant for the food and feed risk assessment of ncrna gmo plants, highlighting the need to evaluate the stability of ncrnas outside the plant. earlier studies suggested that food nucleic acid content was hydrolysed when cooked (colling and wolfram, ) . most of ncrnas, including srnas and lncrnas have been discovered in the last years, and thus new data has been added to the body of knowledge. in a study of srna stability under cooking conditions, plant mirnas were detected after cooking although at significantly lower levels when compared to fresh plant tissues (see table ) (zhang, lin et al., ) . differences were observed for mirnas from different food/feed sources (rice, cabbage, wheat or potato), ranging from nearly undetectable values to almost % persistance of mirnas after cooking. levels of mirnas were monitored with a stem-loop quantitative reverse transcription polymerase chain reaction (qrt-pcr) assay using the u snrna for normalization, a commonly used housekeeping gene in mirnas analyses. the same study found that most of the plant mirnas and mammalian mirnas could survive under acidic conditions that mimic the gastric acidic environment. of note is that the degradation rate of mammalian mirnas under acidic conditions was similar to that of their synthetic form (without '-omethylated ' ends), whereas plant mirnas had a much slower degradation rate compared with their synthetic form (without '-o-methylated ' ends), suggesting that methylation had a protective effect on the stability of plant mirnas (table ) . another study reported that endogenous mirnas can be detected by qpcr analysis in rice and soybean plant materials after storage, processing and cooking (philip et al., ) . for processing and cooking, soybeans were soaked in rnase-free water with . % w/v nahco overnight at ºc, then separated from the soaking liquid, rinsed in fresh rnase-free water, and then boiled in rnase-free water for www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. min until they became soft in texture. by constrast, a synthetic mirna showed a significantly higher susceptibility to simulated food processing conditions as compared to plant mirnas, as well as high molecular weight rnas (total rna). the synthetic mirna used for comparisons was the cel-lin- , which is a caenorhabditis elegans mirna. the synthetic mirna was synthesized without '-o-methylated ' ends (philip et al., ) . these results suggest that the methylation and the small size of plant mirnas make them more resistant to degradation than synthetic (not chemically modified) mirnas. in addition, the study showed significant plant mirna stability in a simulated digestion system for min prior to absorption or transport into the blood stream. however, this study provided most of the quantitative data from q-pcr analysis without normalization, and data should be considered as relative compared to the treated control in the experiments. studying the stability of plant srna in vitro, liang and colleagues exposed total rna ( µg), isolated from brassica oleracea, to freshly drawn serum from mice. in serum, a large amount of srnas survived after h of incubation at ºc, but after h only about % of the rna could be detected. the authors also exposed the plant rna molecules to fecal suspensions at ºc. after h incubation significant amounts of srnas were still detectable, but after h rna molecules were undetectable . suggesting that srnas were more resistant to degradation in the presence of serum suspension than in fecal suspensions. these results were produced by gel electrophoresis of srnas, which is known to be less sensitive than qrt-pcr or rna sequencing methods. this study also reports that, when orally ingested, plant mir (chosen because it is the most highly enriched plant mirna from b. oleracea) can pass intact through the gastrointestinal tract in mice (a maximum of . % recovered from the stomach of some individuals), and can be detected in the blood, liver, spleen and kidney. levels of mir were monitored by qrt-pcr using a taqman probe, which can distinguish differences of one nucleotide. since only one mirna was evaluated, the above findings cannot be generalized to other mirnas. other studies have reported mirnas to be stable in watermelon and mixed fruit juices produced by simple extraction without additives . plant mirnas were detected by stem-loop qrt-pcr using taqman probes and some were validated by northern-blot analysis. to reduce the nonspecificity signal of qrt-pcr, no-template controls were used. yang and colleagues studied artificial in vitro digestion systems that simulate mammalian gastric and intestinal conditions, and found that mir was between - fold more stable than other mirnas . this effect was observed for cabbage extracts when compared to either the 'o-methylated or the non-modified form. detection was also done by qrt-pcr. the same research group reported recently that several plant mirnas, including mir , were degraded during storage at ºc in cabbage extracts obtained by mechanical maceration . degradation of these mirnas correlated with that of s rrna, whereas mir accumulation was increased over -fold after hours of storage, and then gradually decreased over a span of days. mirna (mir ) increasing in macerated tissues (rather than degrading) is uncommon for most mirnas. indeed, the authors indicated that, in terms of genesis and amplification, mir is an atypical mirna, which could explain the disparity in serum detection with respect to canonical plant mirnas. thus, mir was shown to be derived from s rrna and processed in a dcl -independent manner, meaning that when s rrna is degraded mir is produced. this study presents the possibility that certain ncrnas can have non canonical features for synthesis or stability, and should be evaluated individually. in another recent study, the in vitro stability of curcuma longa mirna (clo-mir ) was assessed. when exposed to foetal bovine serum (fbs) for up to h, the mir exhibited high resistance . in summary, food nucleic acid content are generally hydrolysed during processing, with some studies suggesting that mirnas from plants may be more resistant to degradation than synthetic or animal www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mirnas due to endogenous modifications ( '-o-methylated ' ends). however, these findings are not consistent with mirna stability studies in the animal gastrointestinal tract, and further research is required. although there are many studies on plant ncrnas stability (see . . . .), these mainly focus on ncrna stability within the plant itself. mammalian ncrna stability differs from that of plants because lack the natural '-o-methylation at the ' ends occurring in plant ncrnas, mostly in mirnas (yu et al., ) . very few reports were identified in which plant ncrna stability is investigated in a mammalian organism ) (see sections . . and . .) . understanding the molecular basis that confers plant ncrna stability is of vital importance and needs to be further evaluated to better inform on the relevance of plant ncrna for food and feed risk assessment. for instance, modifications plant mirnas not occurring in mammals, such as the methyl group located on the ' nucleotide ribose, could hypothetically have an impact on plant mirnas stability in mammalian systems due to the lack of the appropriate enzymes for their recognition and degradation, or due to increased stability to mammalian rnases . should this be the case, plant mirnas could manifest a much longer than expected half-life in mammalian organisms when compared to mammalian mirna, which could increase the probability of encountering target molecules. the literature contains descriptions of possible in vitro interaction of plant mirnas with mammalian mirna silencing complexes which may trigger target repression chin et al., ) . however, this hypothesis needs to be experimentally validated since there are gaps in the literature. moreover, it needs to be determined if sufficient levels of ingested plant mirnas reach a target cell to determine an effect in mammalian cells. for instance, in mammalian studies it has been suggested that the threshold for target gene regulation is between and copies of mammalian mirna per cell . accumulation of plant exogenous ncrnas has not yet been reported, but recent next generation sequencing studies have reported a plethora of plant mirnas and other rnas in circulating in the human organism (see section . ). the biological significance of their presence is still unknown. it remains unknown if under certain pathological conditions (i.e. compromised intestinal permeability or renal function) exposure to these plant ncrnas could change. all these gaps in the literature would need to be experimentally validated. to determine the half-life of plant ncrnas in mammalian cells would require experiments utilizing labelled plant ncrnas, first transfected into mammalian cells (in vitro studies) and then orally administered to experimental animals (in vivo studies). the markers used for these experiments could be radioactive probes or labelling molecules of very low molecular weight, such as biotin or fluorescein, to prevent distorting the actual size of the plant ncrna under study. due to their small size, this is important when studying mirnas stability. there is evidence that certain plant ncrnas can induce silencing in some eukaryotic pathogens, pests, parasites or symbiotic microorganisms as a defence strategy. in return, various pathogens develop mechanisms to evade this defence system, proving the existence of a two-way sirnas traffic between pathogens and their plant hosts. plant ncrnas stability outside the plant and in animal systems may play a role in cross-kingdom effects but has received limited attention. the resistance of plant ncrna to the gastrointestinal environment or processing has been partly investigated but more research would be needed. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following the methodology described in the section . . . and based on a literature search as described in section . . . . , a total of documents were selected as relevant to describing the state of the art in the field of ncrnas used/intended for use as therapeutics in humans (pharmaceuticals/medicine area). this constitutes valuable preparatory material on the pharmacokinetics (and pharmacodynamics) of ncrna, as required by task . of these publications at least indicate that to ensure the biological activity of exogenous rnas for therapeutic requires numerous chemical modifications and delivery methods. although composed mainly of only four basic building blocks, rnas form simple to very complex structures. its intrinsic base-pair property, enhanced by the ability of the extra hydroxyl groups in the ribose sugar to form hydrogen bonds, are the foundations of its diverse structure and functions (lu et al., ) . rna can fold into various secondary structures including stem, loop, bugle, pseudoknots, gquadruplex and kissing hairpin. it can perform a wide range of biological functions, ranging from regulation of gene expression at various levels to catalysing chemical reactions which closely depend on the ribonucleotide chain's spatial structure. other rnas, such as ribozymes (doudna and cech, ) , can form tertiary structures with catalytic activity. certain rnas can also undergo transformation between alternative structures, depending for instance on binding to specific ligands or environmental changes, as is the case of riboswitches (mironov et al., ) . riboswitches are noncoding rna structures located within mrnas that bind endogenous ligands to regulate gene expression or rna splicing events (cheah et al., ) . the sirna mechanism of action involves dicing of endogenous dsrnas from longer rnas transcripts by dicer and loading into the protein complex of the rna-induced silencing complex (risc). argonaute (ago) proteins are the catalytic core of the risc complex in plants and animals (tolia and joshua-tor, ) . one strand (the passenger strand) is discarded and the guide strand is paired to a complementary mrna sequence via the risc complex. gene silencing can be achieved mainly through post-transcriptional gene silencing (ptgs) or transcriptional gene silencing (tgs). in ptgs mechanisms, the mrna undergoing translation can be sequence-specific cleaved by the risc complex and degraded when the target mrna is perfectly complementary to the sirna. when the interaction is partial or only limited complementarity exists, translational repression and rna degradation occurs, which is a mechanism exerted by mirnas. the mechanism of action for mirna differs somewhat. the mirnas are endogenous encoded singlestranded rnas of about nt long that are important post-transcriptional regulators of gene expression by pairing through a sequence-specific complementary binding to the ' utr of the target mrna. this is usually mediated by a sequence of - nucleotides, known as the seed region, at the ' end of the mature small rna (bartel, ). however, mirnas can also bind to other mrna sites including ' utr or protein coding exons (forman and coller, ) . depending on the degree of sequence complementarity, the interaction will result in mrna degradation and/or translational repression (ameres et al., ; bartel, ) , with destabilization of target mrna being the predominant efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mechanism to reduce protein output exerted by mammalian micrornas (guo et al., ) . most animal mirnas are transcribed by rna polymerase ii into a long primary transcript (pri-mirna) containing a stem-loop structure. the pri-mirna is then processed by a multiprotein complex containing the nuclear rnase iii drosha into a ≈ nt long hairpin-shape precursor (pre-mirna) and exported to the cytoplasm via an exportin- and ran-gtp-dependent mechanism. the cytoplasmic rnase iii dicer further processes the pre-mirna by removing the terminal loop to produce a small rna duplex, containing the functional microrna and the passenger (*) strand (mirna/mirna*). the duplex mirna is then separated and the functional mirna incorporated into ago proteins within the rna-induced silencing complex (risc), guiding it to the target mrna to induce its translational repression or mrna destabilization (krol et al., ; bartel, ). in , zamecnik first reported the use of an exogenous synthetic oligodeoxynucleotide complementary to the rous sarcoma virus s rna as a potent inhibitor of protein translation (stephenson and zamecnik, ) and virus replication (zamecnik and stephenson, ) . since then, the ability to use exogenous (synthetic) agents to control gene expression has revolutionized many aspects of biological research and catalysed the development of a new promising class of exogenous molecules to treat human diseases. however, despite the obvious promise of this approach, progress has been slow because of the need to overcome myriad technical hurdles, particularly those related to the pharmacokinetics, pharmacodynamics and delivery of antisense molecules. indeed, as most antisense-based potential therapies have not yet produced significant clinical results, very few rnabased or dna-based antisense drugs have been approved for clinical use. in , the u.s. food and drug administration (fda) approved the first aso-based therapeutics for the treatment of a human disease. in , fomivirsen (marketed as vitravene) was approved for clinical use by the european medicines agency (ema) for the european market for treatment of cytomegalovirus (cmv) retinitis in immunocompromised patients. fomivirsen is a synthetic -mer aso (dna) that binds complementary to the sequence of the mrna encoding the ie protein from cmv. the first rna-based therapeutic approved for clinical use was pegaptanib (marketed as macugen), which was approved by both the fda (in ) and ema (in ), for treatment of neovascular (wet) age-related macular degeneration. pegaptanib is also the only approved single strand rna aptamer drug, and it targets the vascular endothelial growth factor as an antagonist. in both cases, fomivirsen and pegaptanib, the route of administration is intravitreal injection, and the unique properties of this route provide benefits in terms of pharmacokinetic and immunological response. in , the fda (but not the ema) approved the second rna-based therapeutic, mipomersen (marketed as kynamro) for treatment of homozygous familial hypercholesterolemia. mipomersen is a dna-rna aso that binds to the mrna of apolipoprotein b (apob), thus reducing apob protein and concomitantly ldl cholesterol levels. antisense oligos like fomivirsen (dna) and mipomersen (dna-rna) act by binding to a complementary sequence of a mrna, thus preventing production of its encoded protein. nusinersen (marketed as spinraza) approved by the fda (in ) and ema (in ) as an orphan-drug for treatment of spinal muscular atrophy is a rnabased aso that increases the production of the full-length survival motor neuron (smn ) protein. eteplirsen ( the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (bobbin and rossi, ) and many others, which suggests that exogenous rnas can be delivered to humans and used to modify gene function, protein accumulation and treat human diseases. the ability of nucleic acids, both dna and rna, to form duplexes by base complementarity, has been used to develop oligonucleotide-based drugs for gene silencing. in principle, an oligonucleotide binds a target rna through watson-crick base pairing and exerts gene silencing through different mechanisms (kole et al., ) . a single strand antisense oligonucleotide (aso) binds to the mrna, which is also single-stranded, and is translated into proteins. different molecular mechanisms to either block gene expression or degrade the rna duplex formed by watson-crick base pairing have been described. cleavage of the rna strand of dna•rna hybrids is predominantly mediated by the enzyme rnase h (walder and walder, ) , which is an abundant enzyme in both the nucleus and cytoplasm of eukaryotic cells (wu et al., ; ten asbroek et al., ) . asos can also bind rna and block ("steric blockers") gene expression rather than facilitating rna cleavage (dias et al., ) . also considered to be within the aso family are peptide nucleic acids (pnas) or phosphorodiamidate morpholino oligomers (pmos or "morpholinos"). their modification usually acts through this mechanism (michel et al., ) , which in some cases exerts high efficacy in vivo (iversen et al., ; kole et al., ) . exogenous rna molecules or asos can alternatively target and bind a splicing site on the target pre-mrna and thus modify its exon content (mayeda et al., ; skordis et al., ) , which results in the production of alternative splicing products with potential applications in exon skipping therapy (goemans et al., ) . because many of these asos contain dna with multiple modifications, they are not discussed here in detail. the present literature review focuses mainly on ncrna and particularly on exogenous ncrnas. the discovery of rna interference (rnai) (fire et al., ) increased the interest in using exogenous chemically-synthesised rnas for silencing genes in mammals, and promoted their potential usage in human therapeutics (song et al., ) . to achieve therapeutic results, rnai can be administered via delivery to the cell of small exogenous rna duplexes -including short interfering rnas (sirnas) (zuckerman and davis, ), mirnas mimics (bouchie, ), short hairpin rnas and dicer substrate rnas (dsirnas) (foster et al., ; kim et al., ) -to the cell for further processing into an rna interfering silencing complex. although double-stranded rnas have been widely used for sirna therapy, single-stranded sirna (ss-sirna) is capable of activating the rnai pathway and exerting rnai effects (holen et al., ; lima et al., ; prakash et al., ) . moreover, singlestranded mirna mimics have also been shown to activate the mirna pathway (chorn et al., ) . in principle, then, both dsrnas and ss-sirnas can trigger gene silencing of complementary messenger rna sequences (holen et al., ) . other small rnas with imperfect matches have also been described to repress mrna translation (saxena et al., ) . for mirna therapeutics, different pharmacological tools have been developed which involved either inhibiting or enhancing mirna function (van rooij and olson, ; janssen et al., ) . approaches to inhibiting mirna function include small-molecule inhibitors; mirna masking; mirna sponges; and aso, such as anti-mirs, locked-nucleic acids (lna), or cholesterol-modified antagomirs (krutzfeldt et al., ) . the latter's complementarity allows them to bind to mirnas, inducing duplex formation or mirna degradation. the lna therapeutic approach successfully led to the first mirna-based clinical trials for the treatment of hepatitis c (miravirsen; santaris pharma, denmark) with promising results (janssen et al., ) . there are also approaches that can be utilized to enhance mirnas function called "mimic". these include small-molecule activators of mirna expression and mirna mimics, which are www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. exogenous mirnas delivered by several methods aiming to repress the function of their endogenous targets. they are also referred to in the literature as "mirna replacement therapy". as proof of concept, a synthetic version of mir- a (mrx , mirna therapeutics), delivered using a liposomal delivery formulation, was the first mirna to enter a clinical trial for cancer (beg et al., ) . the literature also describes other types of rnas that can exert physiological functions similar to that of mirnas and sirnas. for example, a -nt long guide hairpin rna (ghrna), that might not require ago or dicer, has been shown to exert mirna or sirna activity in vivo (ohno et al., ) . this novel rna interference technology may act as a novel platform for rna-based therapy. indeed, systemic or local injection of the ghr-form mirna- a (ghr- a) suppressed tumour growth in a mouse model of ras-induced lung cancer. understanding the manifold mechanisms of rna function requires detailed knowledge of the rna tertiary structure (magnus et al., ) , which can lead to therapeutic development (digiusto et al., ; strobel et al., ) . indeed, different experimental (lu et al., ; weinreb et al., ) and computational methods (magnus et al., ) have been developed to predict rnas structure and interactions. the capacity of rna to fold in various ways can generate unique three dimensional secondary structures capable of specific molecular recognition of their target cognate (zhou and rossi, ; tuerk and gold, ) . aptamers are short single-strand rna molecules ( - nt) with a defined structure that can specifically bind to a molecular target via tertiary structures. aptamers can be rationally designed using selex technology (tuerk et al., ) , and can therefore be incorporated into chemically modified rnas with high nuclease resistance properties suitable for animal and clinical studies (sullenger and nair, ). unlike other ncrnas therapeutics, aptamers can target soluble extracellular proteins or extracellular domains of cell-surface receptors. the latter characteristic is unique and can be used to deliver sirnas, mirnas or other compounds to target tissues, binding them to a cell-specific aptamer (zhou and rossi, ) . aptamer therapeutics have rapidly progressed to clinical studies, and some are already in phase clinical trials (sullenger and nair, ; zhou and rossi, ) . the unique properties of aptamers led to development of the first therapeutic aptamer and the first rna-based drug approved for clinical use, pegaptanib. however, aptamer and other ncrnas therapeutics are not free of adverse effects (lincoff et al., ) . when compared to sirna, dsirna was found to be comparable in potency and duration of effect. however, dsirna was found to be more immunostimulatory when compared to the shorter sirnas (foster et al., ) . preclinical studies suggest that when administered in lipid nanoparticles (lnp) dsirnas can exert biological effects in different mouse models with tumours of diverse origin (ganesh et al., ) . although preliminary data in nonhuman primates suggest acceptable tolerance for this specific lnp formulation (ganesh et al., ) , the dsirna therapeutic arena still needs to demonstrate its efficacy and safety in humans. catalytic rnas, the so-called ribozymes, are another family of rnas for which therapeutics have been actively developed (kobayashi et al., ) , reaching advanced clinical trials (burnett and rossi, ; mitsuyasu et al., ), but this review will not focus on this type of rna molecules. unmodified nucleic acids have been shown to possess limited stability in biological media and are subject to rapid enzymatic-mediated degradation (braasch et al., ; . there are three major classes of intracellular rna-degrading enzymes (houseley and tollervey, ) (a.k.a. ribonucleases or rnases): (i) endonucleases acting within the rna chain, ii) ' exonucleases and iii) ' exonucleases degrading rna from the ' or ' end respectively. very large amounts of nuclear rnas efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. are rapidly degraded (brandhorst and mcconkey, ) , this being considered either as translation noise (struhl, ) or part of the biological function of ncrnas. instead, most cellular rnas are modified to resist to exonuclease degradation (ren et al., ) . most genomes encode a plethora of rnases, often with overlapping activities, making redundancy a general feature of rna degradation systems, presumably with the goal of enhancing the overall efficiency and robustness of these degradation pathways (houseley and tollervey, ). normal physiological degradation of endogenous rna molecules or degradation of endogenous rna molecules with defects in processing, folding or assembly are not addressed in this literature review. abundant rnase activity has been described in different tissues, including human body fluids (blank et al., ) (weickmann and glitz, ) and with differential catalytic properties (leimoni et al., ) . different tissues contribute to body fluid rnases (neuwelt et al., ) , supporting the cellular defence system against, for instance, viruses (barrangou et al, )(gupta et al., . the mammalian ribonuclease a family seems to contribute to this host defence by exerting antimicrobial activity (harder and schroder, ; dyer and rosenberg, ) . rnase , for instance, is a rnase a superfamily member with potent ribonuclease activity that is secreted by epithelial tissues including skin, respiratory tract, genitourinary tract, and the gut (harder and schroder, ) . the abundant rnase a produced in humans very probably functions to reduce rna contamination, whether endogenous or exogenous, preventing entry into unwanted rna-processing pathways (houseley and tollervey, ). as above described, unmodified rnas (naked) are generally unstable in biological systems, due to the large amount of ubiquitously expressed nucleases. thus, several chemical modifications have been tested for exogenous rna developed for in vivo therapies to increase resistance to nucleases, enhance binding affinity, facilitate cellular uptake, improve the pharmacokinetic and pharmacodynamics profiles, and reduce immunological response or toxicity (wan and seth, ; bennett et al., ) . there are several sites of an exogenous ncrna molecule susceptible to chemical modification ( figure ) without interfering with its ability for base-paring or enhancing its drug-like properties. the several sites on rnas that can be modified include the base, the sugars, and the backbone. this allows them to conjugate with a wide variety of molecules. different chemical modifications have been tested in the context of developing rna-based therapeutics aimed at increasing the pharmacological properties of exogenous rnas and allowing their successful use in different pathologies/targeted therapies. the following section briefly reviews the types of rna modifications included in different studies in the available literature. nucleobase modifications can affect base-paring properties, binding affinity or specificity for the target mrna, as well as exert adverse effects due to competition with natural nucleotide pools or interfere with polymerases (wan and seth, ). replacement of adenosine in the sirna guide strand with adenosine analogues has been found to modify immunomodulatory activity. watson-crick face-localized n-ethylpiperidine triazole modification has been found to be less reactive than the hoogsteen edge modification, and the ' end was more effective at blocking cytokine production than those placed at the ' end (valenzuela et al., ) . c- substitution in pyrimidines in the rna guide strand increase thermal stability of sirna duplexes. indeed, smaller -methyl group substitutions resulted in better sirna activity (terrazas and kool, ). major groove modifications have also been found to increase the serum stability of sirnas (terrazas and kool, ). -position purine modifications, where the major groove edge (i.e. the hoogsteen face) is modified, have also been tested. thiazole modifications of guide strand sirna at position is well tolerated in sirnas, but -ethynyl at this location reduces efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. potency (ibarra-soza et al., ) . several other nucleobase modifications have been evaluated to modify thermal stability of the sirna duplex, hydrogen bonding and sterics, or off-target effects (peacock et al., ) . although not exactly a base modification, movement of the nucleobase from c- to another position on the ribose (i.e. isonucleosides) in sirnas has been evaluated (zhang, j et al., ) . while maintaining binding capacity to rna and stability toward nucleases, passenger strand modifications with isonucleoside at the ' or ' terminals can retain the silencing activity and minimize the passenger strand specific off-target effect (zhang, j et al., ) . chiral inversion of the natural d-forms (spiegelmers) of rna has also been tested in aptamers, leading to their clinical evaluation (ludwig et al., ) . increase nuclease resistance modify plasma protein binding prevent rapid renal excretion improve pharmacokinetics enhance rna-binding affinity enhance thermal stability increase nuclease resistance enhance rna-binding affinity enhance cellular uptake reduce renal filtration enhace delivery to certain tissues modulate protein binding enhance specific cellular uptake increase possibility of formulations for specific tissues/mucosal mebranes ability to traverse biological barriers using extracellular vesicles ´to d irection sites susceptible for modification on an rna molecule. exogenous rnas for therapeutic use can be subjected to different chemical modifications without interference on their base-pairing ability and enhancing their drug-like properties. conjugates at the ´-end or the ´-end may have the same objectives. earlier studies showed that the '-oh of the rna ribose sugar is not required for sirna activity (chiu and rana, ) and '-modifications influence the sugar to adopt a '-endo sugar pucker due to the gauche effects between the o '-and '-substitutes, improving affinity properties (egli et al., ) . therefore, the furanose ring structure at the ' position of rnas has been extensively modified to efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. achieve pharmacological properties including increasing nuclease resistance, stability and efficacy (egli et al., ) . the '-o-methyl ( '-ome) or '-o-methoxyethyl ( '-moe) modifications increase resistance to nucleases and duplex melting temperature (lamond and sproat, ; prakash and bhat, ) , and have been widely used to develop mirnas (meister et al., ) , sirnas (judge et al., ; choung et al., ) or aptamer-based (burmeister et al., ) therapies. for example, '-ome in sirna has been shown to selectively protect the particularly vulnerable '-end of the guide strand against exonucleolytic degradation in human blood serum (hoerter and walter, ) . moreover, '-ome is a naturally occurring modification of rna found in trna and other small rnas. '-ome has also been used to modulate rna splicing ( toxicity related to lna modifications has also been described (swayze et al., ) . although sugar-modified oligonucleotides or exogenous rnas, including sirnas or antimirs, are more effective rna inhibitors than their unmodified counterparts, they are still susceptible to serum in addition, the phosphorothioate linkage promotes plasma protein binding (srinivasan et al., ; wan and seth, ; sands et al., ) . this increases pharmacokinetic benefits by reducing rapid renal clearance (sands et al., ) and facilitating tissue delivery, binding to cell surface proteins and cellular uptake (miller et al., ; overhoff and sczakiel, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. binding affinity (tm) for its target complementary nucleic acids (≈ . ºc/linkage) (freier and altmann, ) including mrnas and mirnas (krutzfeldt et al., ) . thus, the use of a mixture of phosphodiester and phosphorothioate bonds may be preferred over all phosphorothioate bonds for in vivo applications (ghosh et al., ; krutzfeldt et al., ) . also, the phosphorothioathe linkage confers chirality at phosphorus and might be relevant for sirna activity (jahns et al., ) . although initial studies have shown exogenous homologous rna uptake by cell suspension, there was very rapid degradation after uptake (shanmugam and bhargava, ) . duplex rnas may be more stable than single-stranded rnas, even in the absence of phosphorothioate modifications (braasch et al., ) . indeed, '-ome hairpin-designed antimir oligonucleotides do not seem to require phosphorothioate modification to exhibit in vitro activity (lennox and behlke, ). minimally-modified phosphodiester aso has been reported to have less (brigui et al., ) , similar or superior activity than its complete phosphorothioate counterparts (uhlmann et al., ) , but sugar modifications in phosphodiester backbones can compensate the loss of activity (monia et al., ) . some adverse effects related to phosphorothioate modifications, compared to their phosphodiester backbone counterparts, have been described in aso and sirnas, including nonspecific protein binding (brown et al., ) , the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (dsrna). alpha-tocopherol conjugated sirnas can be delivered to liver tissue (nishina et al., ) or the brain when administered locally (uno et al., ) . other lipid conjugates of rnas have been evaluated including bile acids, long-chain fatty acids, and medium-chain fatty acids (murakami et al., ; lorenz et al., ; wolfrum et al., ) . conjugation with specific peptides has also targeted sirnas to specific tissues (hsu and mitragotri, ; yamayoshi et al., ) . another approach includes conjugation with high-molecular-weight polyethylene glycol (peg) to overcome renal filtration. this strategy was used in rna aptamers either approved (macugen) or in advanced clinical trials for ocular disorders (zhou and rossi, ; drolet et al., ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. rna delivery to a target tissue for appropriate and specific cellular uptake is currently a main hurdle in rna therapeutics. several synthetic non-viral methods for rnas delivery have been developed for local and systemic rna therapeutics ( multiple studies have suggested that exosomes, naturally-released extracellular vesicles, can deliver rnas (mrnas, mirnas, lncrnas) to recipient cells (zhang, l et al., ; . due to their ability to traverse biological barriers, the use of exosomes or exosome-mimetic nanovesicles for rna delivery is an open field for future rna therapeutics (lunavat et al., ; zhou et al., ) . indeed, preclinical studies suggest that exosome-mediated delivery of rnas is feasible in vivo (didiot et al., ; el-andaloussi et al., ) . different rnas have been found in milk exosomes from different species, including humans zhou, q et al., ) , but whether these vesicle-protected exogenous rnas are bioavailable is still in debate (zempleni et al., ) . it is important to note that most of the above described chemical modifications are not found in nature and have been developed generally to increase the drug-like properties of rnas and other nucleotides. to the best of our knowledge there is no evidence of clinical trials using unconjugated naked or unmodified exogenous rna for human use with any administration route. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. early studies suggested that rnas were poor drug candidates due to their relatively high instability, relatively short half-life in vivo, immunomodulatory effects and the many hurdles to their cellular absorption caused by their negative charge. however, several improvements in stabilization chemistry have been identified in recent decades. indeed, a number of chemical modifications and conjugation strategies have improved their rna-binding affinity, in vivo nuclease stability and pharmacokinetic and pharmacodynamic properties. an exogenous rna can be amenable for chemical modification without interfering with its base-pairing ability. several parts of the exogenous rna molecule can be chemically modified including the nucleobase, the backbone or the ribose (sugar). rnas can also be conjugated with a variety of molecules which can exert different biological properties. in addition to these, chemical and biological approaches can be applied to deliver exogenous rna to cells or specific tissues. although many aspects are still to be fully understood in the field of chemical modification of exogenous rnas for therapeutic use, several exogenous rnas (sirnas, aptamers, mirnas, etc.) have entered advanced clinical trials for human use. adams the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section . . . and . . . ., a total of papers were reviewed on the pharmacokinetics of foreign exogenous ncrnas used/intended to be used as therapeutics (hereafter referred as exogenous ncrnas). this section focuses on the pharmacokinetics of exogenous ncrnas (naked, i.e. nonchemically modified or minimally chemically modified) other than those consumed orally from plants; these will be covered in chapter . . moreover, only in vivo studies, in mammals and humans, are reviewed here. of the documents included here, were used for review of ncrna pharmacokinetics in disease and other conditions, which may be relevant to risk assessment considerations. although not specifically requested by the mandate, information on pharmacodynamics aspects of ncrnas was considered. pharmacodynamics is the study of a drug's effect on an organism, meaning its biological effects and other aspects of its xenobiotic action, (efficacy, potency, and toxicity) and this is considered relevant for ncrnas (see . ). pharmacokinetics is dedicated to determining the fate of substances administered to a living organism. it describes the trajectory of a xenobiotic (in this case exogenous ncrna) after delivery into an organism, and encompasses from the movement of administration, to its movement through the organism, to its complete elimination; in other words, absorption, distribution, metabolism and excretion. a drug's pharmacokinetics depends on an individual's physiology (patient-related factors, i.e. renal function, sex, age) or pathology (i.e. renal failure, hepatic failure), and drug chemical properties. pharmacokinetic relevant parameters include: dose, the amount of drug administered at a single point in time; dosing interval, the time between drug dose administration; c max , the peak plasma concentration of a drug post administration; t max , the time to reach c max ; elimination half-life, the time required for the drug concentration to reach half its original value; area under the curve (auc), the integral of the concentration-time curve (after a single dose or in steady state); and clearance, the volume of plasma from which a substance is completely removed per unit time. pharmacokinetics modelling is done using noncompartmental or compartmental methods; the former estimate exposure to a drug by estimating the auc of a concentration-time graph, and the latter estimate the concentration-time graph using different kinetic models. considering an organism as a homogeneous compartment (monocompartmental model) implies that a drug is distributed equally throughout the entire body (tissues and fluids). therefore, blood plasma drug concentrations are a true reflection of drug concentration in other fluids or tissues, and drug elimination is directly proportional to its concentration in the organism. not all tissues in an organism receive the same blood supply and different drugs have different characteristics (i.e. variation in drug passage through natural barriers, such as the brain blood barrier, due to a drug's biochemical properties), meaning other models are sometimes needed. the two-compartmental model assumes that there is a compartment where distribution is more rapid (central compartment) and another compartment www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (peripheral compartment) consisting of organs with a lower relative blood flow, which consequently exhibits a slower drug distribution rate. saturation of the enzymes responsible for drug metabolisation, the presence of active transmembrane transport mechanisms independent of drug plasma concentration, and many other factors not taken into account by the two-compartmental model, require the use of multi-compartmental models. very few studies have focused on the pharmacokinetic profile of exogenous rnas (naked or unmodified), with most of the knowledge on the absorption, distribution, metabolism and excretion profile of ncrnas coming from the development of antisense oligonucleotides therapeutics (dirin and winkler, ) . scaling dosing from preclinical animal studies to humans (i.e. based on body weight or surface area) is challenging and apparently depends on the mechanism of clearance. for example, the pharmacokinetics of a naked sirna formulated into polymer-based nanoparticles in mice, rats, monkeys and humans exhibited blood c max shortly after intravenous administration and rapid elimination across all species in correlation with the body weight (zuckerman et al., ) . the pharmacokinetic profile investigated during the development of exogenous rna therapeutics has shown to be influenced by the route of administration, as described below. a comprehensive summary of available information of in vivo studies and related pharmacokinetics of exogenous rnas is provided in table . most studies on exogenous ncrnas have focused on iv administration. studies in mice have shown that naked unconjugated sirna ( µg/kg) injected intravenously disappears from peripheral blood min after administration, restricting the likelihood of accumulation to peripheral organs (gao et al., ). similar results were found following iv administration of naked sirna ( i labelled), which distributed to the kidney and liver within the first minute, peaking in the kidney, liver and bladder within the first minutes (braasch et al., ) . although distribution of sirna ( i labelled) decreased markedly after h, it persisted in the kidney and liver up to h, and lower concentrations were observed in the lung, spleen and heart (braasch et al., ) . in rats, sirnas were distributed to the kidney and excreted into the urine one hour after injection (van de water et al., ) . exogenous mirna iv administration has also been tested in animal models and entered human clinical trials. in this case (mirna mimics), they are either chemically modified dsrnas formulated in different nanoparticles (xue et al., ) to preferentially target a tissue (trang et al., ), or incorporated into naturally circulating extracellular vesicles (bala et al., ) . for example, mirnas mir- and let- b formulated in a neutral lipid emulsion have been iv administered to mice and have shown to exert a biological effect (kasinski et al., ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. administration site for less than one day, while in a chitosan-formulated version it persisted for up to days (ma et al., ) . peptide-modified and chemically modified sirnas applied topically to the skin have been found to exert a local biological effect, as compared to the sirna alone (hsu and mitragotri, ) . mirnas formulated with transfection agents or nanocomplexes have also been injected intradermally (srivastava et al., ) , or subcutaneously (urgard et al., ) , and demonstrated to exert local biological effects. intranasal administration of sirnas in rats for brain targeting has resulted in very low levels of radioactive sirnas in the brain or plasma when naked sirnas were administered compared to a formulated version (perez et al., ) . mirnas administered by intranasal injection using transfection reagents reached the dorsal root ganglia and the olfactory bulb (cheng et al., ) and, using another nanovector delivery system through intranasal administration (as drops), it reached the brain, exerting a local biological effect (zhuang et al., ) . due to ncrnas susceptibility to degradation, very few studies have focused on exogenous ncrna administration in the gi tract. non chemically modified naked sirna administered by oral gavage to female mice ( µg in total), was found intact and at high levels (analyzed by northern blot and by quantitative pcr analysis) and hours after dosing in the stomach, small intestine and colon when formulated with chitosan nanoparticles (ballarin-gonzalez et al., ) . by contrast only low sirnas levels were observed in the stomach, intestine and colon of mice hour after oral administration of the non formulated naked sirna. at this timepoint intact sirna was almost exclusively present in the stomach while partial degradation products were observed in the proximal and distal regions of the small intestines. further reduction of sirna levels was observed hours after administration, with traces only found in the colon (ballarin-gonzalez et al., ) . orally administered sirnas, formulated using specific vehicles including thioketal nanoparticles ( µg kg - d - the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. counterparts were present in all epithelial cells (martirosyan et al., ) . oral administration of exogenous exosomes containing mirnas of bovine origin has been shown to attenuate arthritis in mice models. bovine milk derived extracellular vesicles were administered daily by oral gavage starting at week till week after birth and arthritis was induced by collagen gavage. animals receiving exosomes ameliorated the clinical condition, although no clear mechanisms for this effect have been described . enteral (large intestine) sirna administration for therapeutic gene silencing targeting the liver (via the lymphatic route) has also been described, but using chemically modified and formulated sirnas (murakami et al., ) . enzyme-and ph-responsive microencapsulated nanogels are also being developed for oral sirna delivery, as reported in recent in vitro tests (knipe et al., ) . naked sirna formulated in a water-in-oil microemulsion has been administered via the rectal mucosa to mice along days ( - µg sirna/kg, administrations) and has reached the brain where exerted a biological effect through downregulation of the prion protein expression (lehmann et al., ) . prion infected mice presented an improvement of some neuropathological conditions after receiving the formulated sirna following the rectal mucosa route of administration. being the eye the target for the first rna-based therapy, sirna intraocular administration has been investigated. intravitreally injected naked sirna (slightly modified, dtdt) distributed throughout the eye (vitreous, iris, retina, retinal pigment epithelium and sclera) of rabbits when administered at mg/eye, and the pattern of ocular distribution was similar in male and female rabbits ( . conjugated or formulated sirnas have also been tested in animal models (zhang et al., ; janout et al., ) . intravitreal injection of mirnas has been described in the literature using either naked mirnas in mice , rats (qin et al., ) or using transfection reagent in rats (mcarthur et al., ) or exosomes in rats (mead and tomarev, ) . some studies have evaluated ip administration of naked sirnas (slightly modified, dtdt) in animal models (wilcox et al., ; shimizu et al., ) . the half-life of ip administered naked sirna (dtdt) was found to be ≈ times lower than the formulated version (perepelyuk et al., ) . sirna (dtdt) was also quickly excreted into the urine compared to a formulated version (shimizu et al., ) . in a comparison of a formulated (complexed) and naked sirna after ip administration in mice it was found that after min the formulated version was detectable in muscle, kidney, liver and tumour tissue, while the naked sirna was completely absent (urban-klein et al., ) . while not a common administration option, chemically modified sirnas formulated in ph-responsive nanoparticle complexes have been administered in the submandibular gland of mice (by retroductal injection) and found to exert rnai effects (arany et al., ) . the monitoring of the distribution of the chemically modified sirna h post-administration indicated that the formulated sirna was internalized www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in the cytoplasm of duct cells via the endosomal pathway. at h post-administration, the signal was concentrated in the acinar compartment as well. after h, ≈ % of the submandibular gland cells were positive for the sirna. the delivery of the sirna formulated in nanoparticles was more efficient compared to that of naked sirna (arany et al., ) . in another study, iv administration of chemically modified sirna ( . mg/kg) was shown to accumulate remarkably in the submandibular gland just min after injection, and had a half-life in this tissue of ≈ . h, but did not have a rnai biological effect . in contrast, direct local injection of liposome-encapsulated sirna ( . mg/kg) into the submandibular gland was shown to have strong gene-silencing effects, while naked sirna injection ( mg/kg) did not . few studies have focused on this administration route. senn and colleagues compared central nervous system administration of sirna vs. aso (senn et al., ) . compared to phosphothioate and 'methoxyethyl protected aso (up to µg), naked (slightly-modified, dtdt) sirna ( µg) administered to rats via icv was not found in any brain region, including the area around the injection site. in contrast, h after aso injection it was distributed in the thalamus and hypothalamus. the naked sirna clearly exhibited very poor distribution and uptake in the brain compared to the aso. although aso exhibited a capacity to reach different brain structures, its administration at a total dose of µg over two days produced no rnai effect. in the same context, icv administration of sirna (up to µg/rat/day) on two consecutive days did not exert rnai effects. however, in another study, a sirna chemically modified to allow for delivery in the absence of transfection reagents (accell sirna) was administered via the icv route at µg/rat. the sirna was incorporated into different types of neurons, although not glia, where it exerted a biological effect in regions like the cortex, the caudate subregion of the striatum and in the pyramidal cells of the hippocampal ca subregion (nakajima et al., ) . in a different study, a mirnas mimic (chemically modified double-stranded rnas) administered by icv ( pmol/g body weight, mixed with cationic lipid dotap) was found to increase forty fold the level of this mirna in the brain (stary et al., ) . several chemically modified mirnas (agomirs) were also found to be active when administered via this route in rats (huang et al., ; ge et al., ) . a double-stranded modified pre-mirnas was also reported as active in rats (davis et al., ), but a naked mirna (double-stranded -bp rna) injected by icv ( pmol) in this study was unable to increase its levels or exert a biological effect, suggesting that the naked mirna had a higher susceptibility to rnase degradation (davis et al., ) . lipid-complexed naked sirna is reported to be absorbed by mouse epithelial and lamina propria cells of the vagina and ectocervix (palliser et al., ) . however, uncomplexed and unmodified sirnas were immediately degraded (< min) when exposed to genital wash fluid (wu et al., ) , suggesting high rnase activity in the cervicovaginal secretion. also, slightly modified but still naked sirna (dtdt) was unable to exert a clear biological effect (zhang et al., ) . in summary, different administration routes have been tested during ncrna therapeutics development. the administration route used for exogenous ncrna delivery clearly influences pharmacokinetic profile of chemically synthesized ncrna. no matter the administration route, it seems that naked and unformulated rnas rapidly degrade and exert very limited or no biological activity. very few studies have evaluated the distribution profile of administered naked oligonucleotides in organs and tissues or compared it to the distribution profile of their chemically modified counterparts. using the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. imaging techniques in baboons (positron emission tomography -pet -and [ f]-labelling) it was shown that naked oligonucleotides are poor drug candidates due to limited distribution into organs and tissues and very rapid elimination (tavitian et al., ) . compared to '-o-methyl or phosphorothioatemodified oligonucleotides, naked oligonucleotides were found in the heart, liver and kidney and, to a lower extent, in other tissues min after iv injection. at that timepoint, naked oligonucleotides were already found in the bladder suggesting that naked oligonucleotides are very rapidly eliminated compared to '-o-methyl or phosphorothioate ones. moreover, one hour after injection, almost all naked oligonucleotides were found in the bladder or residually in the kidney, while the phosphorothioate oligonucleotides were found largely in the liver and kidney, and the '-o-methyl ones were in an intermediate distribution (tavitian et al., ) . kinetics studies with radiolabelled oligonucleotides also showed a dramatic reduction of naked oligonucleotides in the plasma within the first min, while '-o-methyl and phosphorothioate-modified ones showed a much slower reduction during the first min. naked sirna have been reported to distribute to the kidneys and bladder of mice min after iv administration (braasch et al., ; naeye et al., ) . intravenous administration of mg kg - partially-modified unconjugated sirna (partial phosphorothioate backbone and '-o-methyl sugar modification) or cholesterol-modified sirna in mice resulted in a broader tissue distribution (liver, heart, kidney, adipose and lung) of the cholesterol-conjugated sirna h after injection (soutschek et al., ) . in another study, naked sirna (free sirna) iv administered (retro-orbitally) was found in the kidney min after injection (naeye et al., ) . ip administration of synthetic sirna formulated with low molecular weight polyethylenimine resulted in its presence in muscle, liver, kidney and tumour tissues min after injection, while the naked counterpart was completely absent (urban-klein et al., ) . in rats, chemically modified naked sirnas were distributed in a similar manner to those of '-o-methyl or '-f modified sirnas (viel et al., ) , even though the circulation time of the '-f was increased. in other studies with rats, iv injection of sirna (chelated to indium- ) resulted mainly in renal distribution hour after exposure (van de water et al., ) . some liver distribution of sirnas have been also reported in mice, when using either the two phosphodiester rna strands duplexes or one phosphodiester strand and one phosphorotioate strand sirna duplex (braasch et al., ) . orallyadministered naked sirnas ( µg sirna) were found to be present in the stomach, proximal and distal small intestine and colon tissues hour after administration but at very low levels, further lowering hours after administration (ballarin-gonzalez et al., ) . the same sirna formulated with chitosan nanoparticles was found to distribute systemically and reach the liver, spleen and kidney (ballarin-gonzalez et al., ) . in an experimental model of arthritis in mice, iv administered naked sirna ( µg) was found to distribute to arthritic joints immediately after injection, but disappeared within hours, compared to that of specific formulations (komano et al., ) . overall, these finding suggest that naked sirna, although distributing at very low levels and with short residence times compared to other formulated counterpart versions, does distribute to the gi tract when administered orally or other tissues when administered systemically. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. formulation type (i.e. cationic lipids, neutral lipids, or others) can drive preferential distribution to specific tissues. as described for certain mirnas, a neutral lipid emulsions (nle) formulation has been shown to distribute to the lung after iv injection ( µg) (trang et al., ) . the lack of cationic lipids in the nle seems to bypass some of the shortcomings that can be attributed to the charge, including formation of aggregates in biofluids, filtration by the liver, adherence to the endothelium or uptake by scavenging macrophages. the result is excellent distribution to lung tissues (trang et al., ) . extracellular vesicles (exosomes)-formulated synthetic mir- was found to distribute to liver, adipose, lung, muscle and kidney tissues min after iv injection (bala et al., ) . administration of exosomeloaded mir- resulted in its detection by isolated hepatocytes and liver mononuclear cells, suggesting cellular uptake (bala et al., ) . in summary, naked oligonucleotides, including ncrnas, rapidly distribute to the liver, lung and kidney after iv injection. within the first minutes they are found in the bladder, suggesting rapid renal elimination. chemical modifications or formulations increase their distribution to other tissues or delay clearance. exosome-loaded rnas have recently been shown to be an alternative tool for distribution to different tissues. ncrnas by oral administration seem to distribute to the gi tract or, if formulated, systemically. there are few studies which have evaluated the metabolism of exogenous rnas administered to animals or humans, most of which only investigate their degradation. naked sirnas seem to be rapidly degraded when administered iv (viel et al., ) , decreasing by ≈ % within the first min, although certain chemical modifications (i.e. 'f, 'ome) can slightly increase this time. stability of naked sirna was reduced (< min) compared to that of other chemically modified or formulated sirna when administered by iv injection in mice (gao et al., ) . indeed, incubation of naked sirna with plasma or foetal bovine serum causes a very rapidly degradation of the genetic material (jiang et al., ; braasch et al., ; urban-klein et al., ) . unmodified aptamers in general are also very rapidly degraded (≈ min) (zhou and rossi, ) . when naked sirna is administered as formulated (i.e. nanoparticles) it was shown that the renal filtration barrier can separate the sirna from its carrier (naeye et al., ; zuckerman et al., ) . in the eye, sirnas are degraded by endonucleases without preferences for one side of the duplex (as observed also for chemically modified sirnas) (beverly et al., ) . early in vitro studies also showed that exogenous rnas taken up by cells are rapidly degraded (shanmugam and bhargava, ) , and exogenous rna injected into the blood is rapidly degraded to nucleosides, ribose phosphate and free bases (sved, ). the renal filtration barrier owns an effective size cut-off of ≈ Å (batsford et al., ; bohrer et al., ) , but is also influenced by net molecular charge discrimination (brenner et al., ) . this is supposed to facilitate rapid renal clearance of small molecules and free sirna (zuckerman et al., ; naeye et al., ) since aso, sirnas and aptamers are commonly smaller than this cut-off (huang et al., ) . by comparing naked sirnas and ribose-modified sirnas ( '-o-methyl or 'f) using pet scans in rats and mice, the kidney was identified as the main organ for sirna elimination (viel et al., ) . however, it is worth noting that also the liver participates in sirna elimination. in other studies, sirna appeared to accumulate in the bladder shortly after administration (hatanaka et al., ) . this was also the case for modified sirnas (lna) compared to their versions conjugated with peg administered in mice, in which their levels peaked in the bladder min post injection and they were found in the urine as soon as min after injection (iversen et al., ) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. some studies have described rapid elimination of naked sirna and renal excretion after iv injection at doses including µg (jiang et al., ) and µg/kg (gao et al., ) in mice or rats (van de water et al., ) . other studies using dynamic imaging techniques or radiolabelled sirnas have confirmed that most of the naked sirna (free sirna) administered to mice was found in the bladder after just min up to min, faster than the formulated counterparts (naeye et al., ; zuckerman et al., ) . the literature mostly addresses chemically modified rnas (dyke et al., ) in clinical trials (zhou and rossi, ) . the small size of rna aptamers renders them susceptible to rapid clearance by renal filtration which has driven efforts to increase their size by conjugation for therapeutic use (healy et al., ; haruta et al., ) . overall this indicates that naked exogenous small rnas are rapidly cleared from systemic circulation and excreted in the urine. iv injection of a cocktail of four different plant-based small rnas (mir , mir a, mir a, and mir ; pmol each) showed them to be rapidly cleared from circulation (< min) . the exception was mir , which peaked min after injection compared to the other mirnas administered at equal dosages, and then dramatically decreased at min. when compared to other small rnas, anomalous or atypical stability and genesis were proposed for this specific mirna. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. although scarcely described, there may be several conditions in which, theoretically, the pharmacokinetic properties of exogenous rnas could be modified. increased intestinal permeability has been described in different conditions including consumption of nonsteroidal anti-inflammatory drugs, celiac disease, crohn's disease or other intestinal inflammatory disease (oman et al., ; sundqvist et al., ; michielan and d'inca, ) . for instance, absorption may be altered in cases where vasculature permeability of the gut is increased, as it has been observed in murine models of ageassociated inflammation (jeong et al., ) . increased intestinal permeability ("leaky gut") has been described in alcoholism, contributing to the extra-intestinal tissue damage caused by alcohol (bjarnason et al., ) , including liver damage (keshavarzian et al., ) . similarly, leaky gut has been linked to liver steatosis in obese patients ( theoretically, leaky gut may increase the possibilities of passively absorbing larger amounts of exogenous rnas with consequent release into the circulatory system. however, very few studies are available in which sirnas, aptamers, mirnas or other exogenous ncrnas have been tested under these conditions. absorption of naked sirna formulated into nanoparticles following intrarectal administration was tested in mice models of dextran sulphate sodium (dss) induced colonic inflammation. uptake of sirna (observed mainly in epithelial cells, lamina propria lymphocytes, and cells from the mesenteric lymph nodes including dendritic cells and t cells) was increased in mice suffering from acute colitis compared to uptake (in the same cell types) under healthy conditions (frede et al., ) , suggesting that inflammatory conditions may alter ncrna absorption. although there are no studies using naked unmodified exogenous ncrnas, this topic deserves further attention. acute kidney injury or chronic kidney disease are important public health issues. the kidneys play a key role in elimination of xenobiotics and metabolism products and thus renal clearance should be considered in drug discovery and drug interaction research (feng et al., ) . very few studies have focused on the clearance of exogenous ncrnas in pathological renal conditions. for example, plasma clearance of chemically modified sirna administered by iv injection ( mg/kg) was slightly reduced in renally impaired rats than in normal animals (thompson et al., ) . although sirna concentration in residual kidney tissue of partially nephrectomised animals was slightly greater than the mean in normal animals, the differences did not result in appreciably greater sirna distribution into other organs (thompson et al., ) . in general, similar results, but using asos, were observed in rats after cisplatin-induced renal injury (masarjian et al., ) . also in a study of an aso against mirnas, it was recently reported that in end stage renal disease patients, only relatively modest increases in aso plasma levels were observed when compared to control subjects (http://regulusrx.com/wpcontent/uploads/ / / -aasld-rg- -pk-and-safety-in-esrd-vs-normal.pdf). also using asos, distribution within the kidney was found to be altered in a mice model with chronic kidney disease (ckd) compared to controls (donner et al., ) . moreover, concentration of `-moe asos in the liver of mice with ckd was elevated as compared to mice without ckd, suggesting that a reduction of renal function and aso excretion can potentially influence the systemic delivery of asos (donner et al., ). www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. liver disease may also lead to alterations in drug pharmacokinetics, and thus the potential effects of hepatic impairment are considered in drug development (allen et al., ) . although exogenous ncrnas have not been analysed quantitatively, one study showed that chemically modified and formulated sirnas in lipid nanoparticles were found to be distributed and similarly accumulated in pathological or normal liver tissue from mice (yu et al., ) . other physiological, pathological or lifestyle factors, such as aging (cohen, ) and exercise activity (van baak, ) , may influence the pharmacokinetics of exogenous molecules. however, no information is yet available regarding exogenous rnas and their behaviour under these conditions. pharmacodynamics refers to the relationship between the concentration of a drug at the site of action and the resulting effect, including the time course and intensity of therapeutic and adverse effects. there is scarce data on the pharmacodynamics of naked unmodified exogenous ncrnas. some information is available on chemically modified rnas (or nuclease-stabilized rnas, the latter being outside the scope of this literature review). for example, in a mouse model of viral hepatitis the administration (hydrodynamic injection) of naked or chemically modified (methyl-modified) sirnas was associated with clear dose-related effects of various rnai constructs (peng et al., ) . in this study the naked sirnas had the highest inhibitory effects, but these rapidly declined (shorter-lasting inhibitory effects); in contrast, the methyl-modified sirna duplexes were more stable inside cells and exerted their effect over a longer time period (peng et al., ) . non-compartmental analysis of rna therapeutics is used in most preclinical studies when analysing pharmacokinetic and pharmacodynamic parameters. for example, a non-compartmental model was used to estimate the properties of intravitreal injection of slightly chemically modified sirna (dtdt) (dejneka et al., ) . also, in a mirna mimic therapy by iv administration of a liposomal formulation, a non-compartmental model was used to evaluate different parameters (beg et al., ) . systemic delivery of naked sirna has generally failed to produce significant biological effects (gene silencing). no biological effects have been reported when naked sirna ( µg/eye) was administered by intravitreous injection in rats . few studies report gene silencing effects following local administration, although some of the tested rnas were slightly chemically modified (i.e. dtdt overhangs). intrathracheal administration of naked (but slightly modified, dtdt) sirna was effective through gene silencing effects in a mouse model of haemorrhagic shock and sepsis (perl et al., ) . in non-human primates, naked (but slightly modified, dtdt) and unformulated sirna administered intranasally was effective in severe acute respiratory syndrome sars coronavirus infection reducing viral load and host symptoms (li et al., ) . it looks that naked ncrnas require proper formulation to attain efficacy. naked sirna administered intranasally to mice was effective in reducing viral replication when complexed with lipofectamine (fulton et al., ) . naked sirna formulated in ph-triggered nanoparticles exerted gene silencing effects when administered by iv injection in mice ( mg/kg) (kolli et al., ) . a formulation of naked sirnas using cyclodextrin-containing polycations and transferrin, as a targeting ligand for delivery to transferrin receptor-expressing tumor cells, also showed gene silencing effects when administered by iv injection ( . mg/kg) in a mouse model (hu-lieskovan et al., ) . when administered orally efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ( µg/kg) to a mice model of thioglycollate-elicited inflammation, a formulated sirna reached macrophages in the peritoneum, spleen, liver and lung, exerting a sirna effect (aouadi et al., ) . a liposome-formulated naked mirna iv administered twice a week was efficacious in clinical trials (beg et al., ) . a cationic liposome formulated dicer-substrate sirna (dsirna) (a solid dsirna core in an envelope of cationic lipids and cholesterol) was found to be efficacious in mice when administered by iv injection ( mg/kg) (ganesh et al., ) . preliminary results within this formulation demonstrated a tolerability profile in non-human primates upon repeated administration (ganesh et al., ) . when comparing the naked sirna and to a highly formulated sirna in cyclodextrin-containing polycations with surface peg and transferrin as the targeting ligand, only the formulated version ( . mg/kg iv) was effective in reducing the expression of the ews-fli gene and tumour engraftment, while the naked version exerted no biological effect (hu-lieskovan et al., ) . similarly, in a mouse model of concanavalin a-induced hepatitis a reduction of hepatic injury by liver-specific induction of rna interference was observed upon iv administration a of galactose-conjugated liposome nanoparticles (gal-liponp) bearing naked sirna, while no effects were noted following administration of the unformulated naked sirna (jiang et al., ) . in a collagen-induced arthritis mouse model and using systemic administration, naked sirna (dtdt) was used against tnf-α, and was completely unable to repress tnf-α mrna expression, whereas a formulated version repressed it (komano et al., ) . in a mouse tumour model, intraperitoneal injection of naked sirna exerted no rnai effects, while its complex-formulated version did (urban-klein et al., ) . while developing a model to study sirna pharmacodynamics, intramuscular administration ( µg) of naked sirna (chemically stable) in mice showed no effects without use of electroporation (stevenson et al., ) . there are several ongoing clinical trials using sirnas, rna aptamers, mirnas and other noncoding rnas intended for use in humans to treat diseases including cancer, cardiovascular disease, inflammation, ocular disease and others (zuckerman and davis, ; li and rana, ; bobbin and rossi, ; zhou and rossi, ; sullenger and nair, ) . most of these ncrnas are chemically modified or formulated. adverse effects were identified in some of these studies. the delivery component of the formulation (i.e. liposomes), rather than the naked sirna component, was primarily responsible for the adverse effects observed in different preclinical toxicity studies (zuckerman et al., ) . for the mir- a mimic, adverse effects in humans have been reported including acute renal injury, back pain and fatigue, all likely attributed to the liposomal carrier rather than the mirna (beg et al., ) . liposomerelated toxicities are considered due to activation of the complement and have been well characterized with the most frequent symptoms observed being flushing, rash, dyspnoea, chest pain, back pain and subjective distress (szebeni et al., ) . in non-human primates, repeated iv administration of escalating doses of a non chemically modified sirna led to increased il- and inf-gamma levels, as well as kidney and liver toxicity at the highest dose tested ( mg/kg) (heidel et al., ) . in several clinical trials on rna therapeutics, dexamethasone premedication was required to reduce infusion-related adverse effects or reactions (i.e. hypersensitivity, flushing, oedema, etc.) (beg et al., ) . for some sirnas used in the clinic, a predosing hydration protocol has been used to protect the kidneys (zuckerman et al., ), thus reducing in humans the toxicities observed in animals. there are also animal studies in which no toxic effects are reported. for instance, when highly formulated naked sirnas were administered by iv route at . mg/kg, no apparent liver or kidney toxicity, immunogenicity-related toxicity or organ damage were detected in a murine model of metastatic ewing's sarcoma (hu-lieskovan et al., ) . no histological toxic effects were seen in liver, kidney, lung, heart and spleen in the same model following repeated treatment with the same formulation the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (twice weekly for weeks) (hu-lieskovan et al., ) . the same results were obtained for the naked sirna administered at the same dose and for the same duration (hu-lieskovan et al., ) . in addition, when administered daily via oral gavage ( µg sirna/kg) for days in mice, sirna nanoparticles in galactose modified trimethyl-cysteine conjugates were reported to have no histological toxicity in major organs (brain, heart, kidney, lung, liver or spleen) (han et al., ) . since there are so few studies in the current literature on the pharmacokinetics of naked/unmodified rna molecules, this section included studies of other, minimally-modified, exogenous rnas to compare their pharmacokinetic and pharmacodynamic properties to that of naked rnas, when possible. the findings suggest that naked or unmodified rnas are rapidly cleared from circulation and have been reported in the kidney and urine immediately after administration (generally intravenously). in general, no major gene silencing effects have been observed for naked rnas when compared to chemically modified or formulated ones. this lack of efficacy has probably discouraged studies investigating the toxicity and safety of naked unmodified rnas. while limited studies have reported minor biological effects of certain naked rnas in animal models (very much dependent on the route of administration), the lack of studies in humans prevents scientists from understanding the kinetic profile of exogenous rnas and developing effective therapeutics. finally, scientific literature on toxicological or safety studies provides no evidence for concern about the systemic toxicity of naked exogenous small rnas under normal physiological conditions. allen the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a systematic literature search as described in section . . . . and following the methodology described in the section . . . , a total of papers were selected as relevant to reviewing the topic of rnas uptake. information on naked (non chemically modified) exogenous rnas is mainly presented in this section. of these publications, support the uptake of exogenous ncrnas. one study reports no intestinal absorption of rna (baintner and toth, ) , and three others suggest that rna is degraded before absorption (loretz et al., ; o'neill et al., ; sonoda and tatibana, ) . uptake of plant exogenous rnas, after oral intake is not included here, and will be reviewed in chapter . ; and uptake of exogenous rnas from the pharmaceutical/medicinal area is described in section . . . herein, uptake refers to passage of the molecule (in this case a ncrna) through the cellular membrane as a part of the absorption process. an important feature for the possible biological effect that exogenous rnas, including ncrnas, may have within an organism is their cellular uptake. because of their size and negative charges, exogenous rnas cannot easily passively cross cell membranes (see below). however, earlier studies suggested that exogenous rnas, either homologous (shanmugam and bhargava, ; galand and ledoux, ; sirdeshmukh and bhargava, ) or heterologous (i.e. from e. coli, yeast or others) (natural academy of sciences, ; herrera et al., ; galand et al., ) , could be taken up by mammalian cells (bensch and king, ) . intact, rna may be present inside the cell but is reported to degrade rapidly in some studies (herrera et al., ; shanmugam and bhargava, ) . extracellular ribonuclease activity seems to inhibit uptake (shanmugam and bhargava, ) or function (niu et al., ) of exogenous rnas. uptake of exogenous rna is apparently not due to altered membrane permeability, impaired cell viability, or ribonuclease degradation of the macromolecule during incubation experiments (herrera et al., ) . radioautographic evidences (radioactive assays) have shown the uptake of macromolecular rnas by normal and neoplastic mouse cells, but degradation was also shown to occur prior to or after rna uptake (schwarz and rieke, ) . although intracellular degradation may occur, early studies also described biological actions in response to uptake of exogenous rnas (niu et al., ; niu et al., ; ; esposito, ) , suggesting that once taken up by a cell, rna could eventually exert a biological effect. studies on other types of cellular uptake of exogenous rnas, such as transfer rnas, either homologous or heterologous (depending on donor rna and recipient cell type) have been conducted (gallagher et al., ; sakai and cohen, ; crooke et al., ) . however, as noted in rna therapeutics development (see section . . . ), the large number of barriers encountered by rnas from intake to cell uptake needs to be considered when evaluating possible exogenous rna bioavailability (figure ). the gi tract offers a large surface area potentially relevant for ncrnas cellular uptake and following systemic release, since it contains a large variety of cell types at different maturational levels (e.g. stem cells in the crypts of lieberkühn) located only a few microns from an extensive capillary network (lozier et al., ) . however several barriers exist that could prevent ncrnas uptake by mammalian cells (o'neill et al., ) . for example, in a study conducted in neonatal pigs, no intestinal absorption of rna was observed (baintner and toth, ), suggesting possible rna degradation or an inability to cross the intestinal barrier. gastrointestinal barriers can be distinguished in extracellular and cellular barriers and are presented below. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. orally ingested exogenous ncrna would have to pass different barriers in the human gi tract. for clarity, only the general barriers are shown. gi barriers to the uptake of orally ingested rnas. the first environment encountered by any orally ingested rna is the oral cavity, where lytic enzymes such as amylase or lipase are secreted in the saliva, and the ph ranges from . to . . the stomach is a more acidic environment (ph . - . ) (dressman et al., ) , and nucleic acids are known to be denatured and depurinated (hydrolysis of nucleic acids to release purine bases) over time in acidic gastric fluid (loretz et al., ) . the gastroenteric fluid flow and peristaltic activity in the gi tract could reduce the contact time between ncrnas and the epithelial layer, therefore diminishing the opportunities for cellular uptake, while nuclease enzymes present in the gi lumen could degrade nucleic acids before any cellular uptake (o'neill et al., ) . indeed, purines and pyrimidines in nucleic acids are believed to be absorbed mainly in the form of nucleosides (sonoda and tatibana, ) . the gut flora, predominant in the distal ileum and in the large intestine, produces a range of enzymes that could degrade ncrnas as well. the gi tract is lined by a viscous sticky layer of mucus entrapping foreign particles before reaching the underlying epithelium (figure ) . this "trapping" effect could be mediated by mucus composition, which confers a net negative charge to the mucous layer. since nucleic acids present a net negative charge as well, this mucus could represent an electrostatic barrier difficult to overcome by rnas, and particularly ncrna, if not properly delivered or chemically modified. furthermore, mucus turnover efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (secretion, shedding and discard) happens in a relatively short time period ( - min) (lehr, ), preventing rnas from traversing the mucous layer and to reach the underlying epithelium. in conditions such as ulcerative colitis this mucus layer can be reduced or missing in areas of acute inflammation (pullan et al., ) , which would allow nucleic acids to reach the epithelial cell layer. another barrier is the glycocalyx, a glycoprotein and polysaccharide layer ( - nm thick) associated with the apical cellular membrane of enterocytes (frey et al., ) . this layer prevents the access of certain viruses, bacteria and particles (such as rnas) to the underlying plasma membrane by acting as a size-selective diffusional barrier. the intestinal mucosa is structured as a three-layer barrier: a single layer of epithelial cells, the lamina propria ( furthermore, the epithelial cells (enterocytes), which represent approximately % of the epithelium, have a short lifetime of - days, and are continuously shed and replaced (quastler and sherman, ; jung et al., ) . the enterocyte apical membranes are characterized by a high number of microvilli, structures approximately mm long and nm wide (johnson, ) . since endocytosis occurs primarily at the base of microvilli (jung et al., ) , particles with a diameter greater than nm could not be efficiently endocytosed (uduehi et al., ) . m-cells (cells dedicated to trans-cellular transport and associated to intestinal lymphoid follicles) are fewer in number than enterocytes, but, due to their role in the transport of foreign material from the lumen to the immune cells of the lamina propria, are characterized by a high endocytic activity. moreover, m-cells have microfolds in their apical membrane, and are covered by a reduced glycocalyx and mucous layer. this determines a low breakdown capacity (e.g. low alkaline phosphatase activity, and a lower number of lysosomes) so intracellular degradation of ncrnas could be spared. conversely the capillary network underlying the m-cells is less dense and less permeable (o'neill et al., ) . although the literature has described these cellular barriers when studying several molecules, there is little specific data (o'neill et al., ) on the relevance of these to exogenous rna molecules. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. assuming that ncrnas could overcome the gi barriers and reach the circulatory system, they would still encounter other biological barriers before reaching the appropriate machinery to exert any effects. significant degradation occurs of sirna duplexes (non chemically modified) in min when they are incubated in foetal calf or human sera (haupenthal et al., ; urban-klein et al., ) . sirna in human plasma is rapidly degraded, with nearly % degraded within min (layzer et al., ) . nuclease activity in plasma and tissues is relevant to degrading rnas, including ncrnas. the major activity in plasma is a ' exonuclease, but cleavage of internucleotide bonds can also take place (juliano et al., ) . encapsulation methods, which protect rnas from degradation, include exosomes, microvesicles and apoptotic bodies, all of which are extracellular vesicles distinguished by size, biogenesis and cargos (ela et al., ; van niel et al., ) . exosomes, for instance, contain various species of rna, conferring them protection against degradation and providing them a vehicle for cellular uptake of rnas by endocytosis (cui et al., ). the reticuloendothelial system (res) is comprised of phagocytic cells, including circulating monocytes and tissue macrophages, whose physiological function is to clear the body of foreign pathogens, remove cellular debris generated by tissue remodelling, and clear cells that have undergone apoptosis. additionally, the res plays an important role in uptake and clearance of individual "free" oligonucleotides. phagocytic cells of the res, particularly the abundant kupffer cells in the liver and splenic macrophages, express a number of cell surface receptors, including integrins and scavenger receptors, that are potentially involved in uptake, although the role of scavenger receptors in uptake of free oligonucleotides is somewhat controversial (juliano, ). following internalization of an oligonucleotide, the phagosome fuses with lysosomal compartments, where the contents are subjected to enzymatic degradation by proteases and hydrolases that operate efficiently in the low-ph lysosomal environment. tissues macrophages are most abundant in the liver and spleen, and both these tissues receive high blood flow (juliano et al., ) . the capillary lumen is surrounded by a layer of endothelial cells interlinked by adherence junctions (vecadherin containing) and by tight junctions (occluding and claudin-containing) and tightly adherent to the underlying extracellular matrix. this structure forms a barrier between blood and the parenchymal space. molecules in the blood can be transported across the endothelial barrier by two routes. paracellular transport occurs through the junctions between cells and is limited to molecules of approximately nm diameter or less; only micrornas would be able to transit this barrier by this method. caveolar-mediated transcytosis carries large proteins across the endothelium within vesicles of about nm. in some tissues, such as liver and spleen, there are gaps or fenestrations of - nm diameter between the endothelial cells, which allow egress of larger molecules (juliano, ). endothelial permeability is also increased in sites of inflammation and in some tumours, in which increased permeability is associated with fenestrations between the endothelial cells that result from rapid and disorganized angiogenesis in the tumour. this is known as the epr effect (enhanced permeability and retention), although some argue that this is mostly due to poor lymph flow in the area the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. and many minutes to several hours for the elimination phase; (ii) oligonucleotides are accumulated in most tissues, particularly in kidney and liver, but not in the central nervous system (probably due to the blood brain barrier); (iii) the major route of elimination is via the kidneys; and (iv) although the most detailed studies have been performed in rodents, the pharmacokinetic behaviour of the modified oligonucleotides in humans is similar to that observed in lower animals (juliano et al., ) . the pharmacokinetics and biodistribution of sirna duplexes is similar to that of single-stranded antisense molecules, with the highest uptake in the kidney followed by the liver (juliano et al., ). sirna and uncharged oligonucleotides do not bind extensively to plasma proteins, and are thus cleared by the kidney much more readily than modified oligonucleotides and tend to accumulate at lower levels in tissues (juliano, ) . typically, molecules with sizes of - nm or less can be ultrafiltered by the kidney; micrornas could therefore be rapidly excreted by the renal route, unlike long ncrnas. in fact, sirna molecules intravenously injected can be visualized in renal filtrate within seconds of injection (molitoris et al., ) . plasma sirna concentrations are reported to decline by more than % within min (relative to levels at min post dose) and decline by more than % within h (thompson et al., ) . only about - % of the iv administered dose is absorbed by tissues and the majority of this uptake occurs in the kidney (thompson et al., ) , most of it being cleared from tissues within h of administration . to reach their target, ncrnas must enter mammalian cells and arrive at the appropriate subcellular location (the nucleus or the cytoplasm, depending where the target molecules are located). like other large, polar and charged biological macromolecules, ncrnas are internalized by endocytosis and then trafficked through multiple membrane-bound intracellular compartments. the ability of ncrnas to reach their targets depends on both cellular internalisation (uptake) and intracellular trafficking. (figure ). there are five major classes of endocytosis: (i) the clathrin-coated pit pathway; (ii) the caveolar pathway; (iii) the noncaveolar, clathrin-independent pathways (clic pathways); (iv) phagocytosis (mainly taking place in "professional phagocytes" such as macrophages and granulocytes); and (v) macropinocytosis (in which internalized macromolecules are simply dissolved in the ambient medium) (juliano et al., ). non-coding rnas (ncrnas) can enter cells via different endocytic pathways, including micropinocytosis. internalized ncrnas can traffic from early endosomes (ee) to late endosomes (le) and to lysosomes. ncrnas must escape endosomal organelles to reach the cytosol and nucleus and act on the target molecules. some proteins may direct ncrna to non-productive pathways. the micropinocytosis pathway may represent a non-productive pathway for naked ncrnas. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the first four classes often involve a cell surface receptor and are collectively termed receptor-mediated endocytosis. these pathways are mainly utilized in cellular delivery of oligonucleotides. in general, receptor-mediated endocytosis includes three major steps. (i) receptor binding and internalization represents the primary barrier for oligonucleotide transport; the ligand-receptor binding determines which target cells and tissues oligonucleotides are delivered to. (ii) sequential intracellular trafficking leads oligonucleotides into a variety of low ph endomembrane compartments, including early/sorting endosomes, late endosomes/multivesicular bodies, and lysosomes. in some cases, receptors/ligands can traffic to the golgi complex. in many instances, receptor and ligand are dissociated in the low ph endosome environment. vesicular trafficking can prevent ncrnas from reaching their targets, for example, by sorting to secretory or lysosomal vesicles which may lead to export of ncrnas out of cells or degradation in the lysosomes. (iii) ncrnas must exit from the endosome to reach the site of action in the cytoplasm or nucleus. endosomal trapping represents an important barrier for ncrnas final functional objective. on the other hand, oligonucleotides are able to continuously shuttle between the nucleus and the cytoplasm mediated by nuclear pore structures, and do not require classical nuclear localization signals (juliano et al., ). the available data clearly describe several barriers to be considered when evaluating exogenous rna absorption, circulation, cellular uptake or intracellular trafficking. exogenous rna must overcome numerous biological barriers to reach its intended target within mammalian cells. considering oral administration as the most relevant for exogenous plant ncrnas, the first major limiting step to exogenous rna bioavailability is the gi tract itself. the gi tract encompasses both extracellular and cellular barriers. the extracellular barriers include the presence of enzymes in the lumen, such as amylase or nucleases, a harsh environment with ph ranging from . to . , and a net negative charged mucous layer with a very rapid turnover ( - min). the cellular barriers include a single layer of epithelial cells, the lamina propria and the muscularis mucosa, constituting the threelayer intestinal mucosa barrier. ncrnas could cross the epithelium between cells but this is limited by the presence of tight junctions. pore size in the human intestine would prevent passage of all ncrnas other than mirnas, the smallest ncrnas. ncrnas could also cross this epithelium through transcytosis by traversing the cells; this mechanism implies exposure to intracellular nucleases, recycling of ncrnas back to the lumen and nuclear uptake. many biological barriers and environmental conditions between the gi epithelium and the target tissue are then encountered by ingested rnas. ncrnas would be exposed to nucleases both in the plasma and in the target tissues. once inside the circulatory system, these rnas would be subjected to distribution and elimination, accumulating mostly in the liver and kidney. due to their small size, mirnas are especially rapidly cleared by the kidney, unlike long ncrnas. therefore, a very small percentage of rnas could actually be absorbed by tissues. furthermore, ncrnas would need to escape the reticuloendothelial system, the function of which is to clear the organism of foreign molecules. then rnas must cross the vascular endothelial barrier to reach the target tissue. for ncrnas to exert their functions they must enter the target cells. rna cellular uptake is achieved by endocytosis after which they would enter the intracellular trafficking system through multiple membrane-bound compartments. ncrnas would need to exit these compartments to reach their functional sublocation within the cell, be it the cytosol or the nucleus, while simultaneously escape degradation in the lysosomes. efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. seeds, corn kernels and rice grains, all of which are common food and feed, and numerous endogenous plant srnas are reported to have perfect complementarity to genes in humans, and other animals . however studies in mammals and humans with sirna indicates that the exposure levels required to produce regulatory effects exceed the levels achieved following ingestion . all animal and plant-related foods and feeds contain naturally occurring coding (i.e. mrnas) and ncrnas. early (srivastava, ; lassek and montag, ) and more recent studies ) have estimated that the total rna content in plants is about mg/g plant tissue. plants contain an array of ncrnas, including highly abundant transfer rnas (trnas) and ribosomal rnas (rrnas), single-stranded antisense rnas, and the mirnas and sirnas that trigger rnai and their precursor dsrnas . long dsrnas from non-plant, exogenous sources are particularly common in plants, including plants used for food and feed, due to infection from rna-containing viruses. animalderived foods are generally richer in rna than plant-derived foods, and are likely to contribute significantly to overall rna consumption (jonas et al., ) . of the mg/g tissue of total rna content in plants, relative percentages (by weight) of the major rna forms are approximately % rrna, - % mrna, and - % trna, with srnas accounting for less than % of total rna in plants . srnas are present at levels of up to . µg/g of conventional soybean grain (average = . µg/g grain) and comparable amounts are present in the grains of conventional corn and rice . in tobacco plants engineered to overexpress a dsrna under the control of a constitutive promoter, sirnas levels in leaves reached about . % of total rna (chau, b. l. and lee, k. a., ) . total exposure to construct-derived srnas from a putative biotech soybean product was estimated to be µg/kg/day in the general population and µg/kg/day for children aged six and under . using these same assumptions for rna expression levels in sweet corn, consumption of corn at the highest possible rate would result in an estimated intake of µg/kg/day in the general population and µg/kg/day for children aged in the absence of encapsulation or chemical stabilization to prevent degradation or without the addition of penetration enhancers, the absorption of rna -including sirnas -across the gi tract is described as negligible (akhtar, ; jain, ) . one study suggests that activity could be possible for certain highly expressed plant mirnas following food intake (zhang et al., a) . this study reports that in mice fed a diet consisting entirely of cooked rice (i.e. human equivalent of about kg/day of cooked rice) , several rice mirnas were detectable in mouse serum and liver. some of the results presented in this study were contested in later studies (chen et al., ) . petrick et al. clearly stated the unlikelihood of achieving such high concentrations of mature plant mirnas in the serum, plasma and organs of humans or animals via food intake since the high levels of rice administered the experimental animals did not reflect anticipated dietary exposure levels. in another study, it was concluded that plant mirnas identified in animal srna sequencing data can originate from artefacts of the sequencing process (zhang et al., b) . even in the case of -mer oligonucleotides modified to increase their stability, oral bioavailability in rats was only . % (nicklin et al., ) and intestinal absorption was less than % in a model that bypasses the gastric acidic environment. most of the labelled oligonucleotide was associated with the luminal epithelial cell membrane and very little efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. was localized intracellularly (khatsenko et al., ) . regarding the possibility of dietary sirnas and other dsrnas effects on gastrointestinal tissues, studies with radiolabelled or immunostained dna oligonucleotides demonstrate that these are primarily located extracellularly within the intestinal tract (lumen and luminal wall) (khatsenko et al., ; nicklin et al., ) , and are thus presumed to be minimally absorbed (negligible bioavailability, < %). it has also been observed that absorption decreased as the oligonucleotide length increased (from to nt) (khatsenko et al., ) . the only published quantitative data on srnas content in plant-derived foods ) (detailed above) indicate that . µg of srna is obtained per gram of soybean, rice seeds or corn kernels. as total rna content in plant-derived foods appears to be around mg/g tissue (lassek and montag, ), srnas could therefore represent . - . % of plant seed materials. the amount of sirnas in plants genetically modified to carry hairpin transgenes is estimated at about ng sirna/ µg total rna/g tobacco leaf tissue (chau, bess l. and lee, kevin aw, ) . the resulting estimated human dietary exposure to plant small rnas from rna-based biotech crops was derived from this (table ). . (b) assuming % of the specific food is consumed from a biotech crop, with total rna levels of ≈ mg/g grain and . % of these small rnas derived from the transgene. for details see . these doses are significantly lower than those required to elicit adverse effects in rodents or monkeys ( or mg/kg of chemically-stabilized sirna, respectively) by iv administration (thompson et al., ) . moreover, high mirna concentration is required to target suppression, and levels below copies/cell mirnas have little regulatory capacity (mullokandov et al., ) . according to snow et al., dietary plant mirnas are present at less than one copy per cell in target organs assuming reliable quantification . in another study the calculated dietary exposure in humans of a specific dsrna (dvsnf ) was estimated to be around . x - µg/kg/day in the us population (petrick et al., b) . the same study reported that mg dvsnf /kg/day in a -day repeated dose oral (gavage) toxicity study in mice did not produce any toxic effects (petrick et al., a) . levels for this specific dsrna in this biotech crop were reported to be ≈ . x - µg/g grain tissue on a dry weight basis for corn food, and ≈ . x - µg/g in the whole plant on a dry weight basis . of note is that there is little information on the impact of intended rnai in gm plants on the coding and non-conding plant rna. knowledge of which other rnas are modified, or how other rnas are compensated or desregulated in the gm plant needs to be experimentally evaluated for each specific case. different studies have evaluated the amount of plant-derived foods consumed by the general population. in a study of cohort of women in the uk, the difference between the mean intake of overall fruit and vegetables consumption was three times greater in high consumers vs. low consumers (pollard et al., ) ; vegans and vegetarians were the highest consumers of fruit and vegetables. in an efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. evaluation of the flemish population it was observed that vegans, while having the lowest total energy intake compared to omnivores, have the highest total fruit and total vegetables consumption. the intake of fruit, nuts and/or legume about twice in vegans, using either the healthy eating index (clarys et al., ) or total mean intake (clarys et al., ) . in the epic cohort study, which includes ten european countries, mean daily consumption was between and g/day vegetables and between and g/day fruit (agudo et al., ) . although not homogeneous among countries and centres, the ratio between the highest and lowest consumers was around . for vegetables and for fruit (agudo et al., ) . countries in southern europe had the highest vegetables and fruit consumption. for example, consumption of vegetables and fruit in healthy adults in spain (epic data), considered higher than most european countries and the usa, was g ( . servings) of vegetables and g ( . servings) of fruit (agudo et al., ) , equal to ≈ g/d vegetables and fruit. in the united states (us) the total vegetable and fruit intake has increased slightly (up to ≈ . servings/day) in all consumer categories from the late s (krebs-smith and kantor, ) and remained stable over the last decade (rehm et al., ) . actual quantities, however, are around . servings/day (rehm et al., ) , that is . for fruit and . for vegetables (eaton et al., ) . in cup-equivalents, this is . cup equivalents for vegetables and . cup equivalent for fruit (moore and thompson, ) . mean total vegetable intake in the us for vegetarians is reported to be around g/day and that for non-vegetarians as g/day. total fruit intake was around g/day for vegetarians and g/day for non-vegetarians (haddad and tanzman, ) . in other words, there is less than two-fold difference in the amount of fruit and vegetables consumed between vegetarians and non-vegetarians in the general us population. in a specific population (i.e. the adventist health study) in the us, daily mean consumption of fruits was higher (≈ g/day) for vegans than for other types of vegetarians or non-vegetarians (≈ g/day). the same trend was observed for daily mean consumption of fruit which was higher in vegans (≈ g/day) than other vegetarians or non-vegetarians (≈ g/day) (orlich et al., ). based on these data, dietary exposure to rnas from plants can in general be assumed to be higher in vegans and vegetarians than omnivores. although total rna content in plant-derived foods varies (lassek and montag, ), a person consuming an estimated daily dose of total fruit and vegetables ≈ g/day would theoretically ingest mg of total rnas assuming about mg/g of plant tissue. this value agrees with the dietary rna intake, which typically ranges from . - g/person/day (jain, ) . considering that srnas represent up to . % of plant seed materials ), estimated daily intake in the general population would be around . mg srnas. since vegans and vegetarians normally consume higher amounts of vegetables and fruits (see above), they could be exposed to an average of three times more plant exogenous rna ( . mg/day). no pharmacokinetics studies were identified in these groups. changes in rnase activity can occur in physiological or pathological conditions, this possibly impacting exposure after dietary intake. for instance, increased serum rnase activity has been observed in pancreatic carcinoma, pancreatitis, and renal failure (peterson, the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. et al., ) . human argonaute (ago- ) is a catalytic core component of rnai machinery. ago- mediated gene silencing has been proposed to be linked to the mitogen activated proteine kinase signalling pathway (zeng et al., ) , which is activated in response to cellular stress. since various kinds of stress on human cells induce formation of stress granules, ago- can be recruited to these granules, modifying its intracellular localization and the efficacy of rnai activity (detzer et al., ) . ago- is thus regulated at both the transcriptional and post-translational levels, suggesting that ago- levels can influence cellular mirna activity (adams et al., ). it has also been proposed that inactivation of mitochondria could lead to a strong decrease in mirna-mediated rnai efficiency, and, to a lesser extent, to sirna-mediated rnai (huang et al., ) . due to the interaction of p-bodies with mitochondria, reduced ago- activity could be due to changes in location of endogenous ago- from pbodies (huang et al., ) . association of ago- with cytoplasmic rna granules is known to regulate the translational repression activity of the protein, but phosphorylation of certain residues may prevent this inhibition, allowing the protein to remain active in the cytoplasm (lopez-orozco et al., ) . overall, it seems clear that ago- activity can be modified by cellular stress conditions. however, whether this can influence the function or the exposure to dietary ncrnas remains unknown and would benefit from further research. most studies have been focused on mirnas since these ncrnas were the first to be described. these studies have consequently extended to transgenic dsrnas as these have increasingly used to induce gene silencing. as mentioned previously, little is known about dietary exposure to other ncrnas. given the role of lncrnas in plant physiology, it is important to better understand human and animal exposure to these ncrnas. further research is needed on how transgenic ncrnas affect expression levels of other ncrnas, and other rnas such as mrnas. alteration of the expression levels of certain ncrnas within the plant may modify the levels of other ncrnas due to putative compensatory circuits and codifying rnas. this in turn could lead to changes in protein and enzymatic content, with putative consequent alteration of the modified plant's nutritional value. controlling for these changes would require complete transcriptome sequencing, and comparison of rna levels in unmodified to those of the altered plant . another point to consider is whether special diets (e.g. vegetarian or vegan) modulate ncrnas uptake throughout the gi tract, possibly leading to increased exposure. humans and animals have been continuously exposed to naturally-occurring plant rnas. estimated amounts of ingested plant ncrnas contrast with the much higher doses administered to experimental animals and in clinical trials to humans. although some reports on the presence of plant mirnas in serum, plasma and organs of humans or animals do not agree, the extremely low reported concentrations prevent them from being functional, even in the case of rnas modified artificially to augment their bioavailability. humans following special diets, such as vegetarians and vegans, have high intake of plant rnas, but their increased estimated exposure to such rnas is barely on average fold greater than in omnivorous diets. there are no specific studies determining the effects of increased dietary exposure to plant ncrnas. adams bd, claffey kp, white ba. . argonaute- expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells. endocrinology , - . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to exert a biological effect, exogenous ncrnas need to overcome the physical and biological barriers present in both the gi tract and the circulatory system, reach the target tissue, and enter the appropriate intracellular pathway at sufficient dose. this section describes the obstacles and challenges encountered by exogenous ncrnas following oral intake. following an extensive literature search, and based on expert judgement in the topic, publications were reviewed for this section. rna molecules are large, hydrophilic and negatively charged. even the smallest rna molecules (mirnas) have a high molecular weight (∼ kd) and their net charge is negative (akhtar and benter, a; akhtar and benter, b) . due to these physicochemical properties, rna molecules are highly water soluble with low membrane permeability, thus falling into class iii of the biopharmaceutical classification system (fda, ; amidon et al., ) . like many other class iii compounds, they are not absorbed at all or only to a very small degree. being highly charged species, rna molecules resist partitioning across lipid bilayers, and higher molecular weights effectively restrict their movement by function of the tight junctions present in the gi tract epithelium (tillman et al., ) . in vitro and in vivo preclinical studies have assessed the use of medium chain fatty acids ( - carbon atoms), as well as bile salts, as absorption enhancers to cross the intestinal mucosa (gonzalez ferreiro et al., ; tillman et al., ; raoof et al., ) . orally introduced rna molecules encounter relevant obstacles in the gi tract, these precluding their absorption and activity. in general oligonucleotides are rapidly degraded in the harsh biological milieu of the acidic stomach and enzyme-rich gi tract, as described previously, and show poor transcytosis across the gut (akhtar, ) . in humans the ph in the gi lumen can vary from in the stomach to in the intestine (gamboa and leong, ) . exposure to these ph values can cause ph-induced oxidation or de-amination of rna, resulting in loss of activity (pridgen et al., ) . in addition, nucleases present in the gi lumen for digestion of biological molecules enzymatically degrade rna molecules too (gamboa and leong, ; kriegel et al., ) . the presence of a rich bacterial population, mostly in the intestines (the human microbiome project consortium, ) may also contribute to degradation of these molecules, as bacterial degradation of ribonucleic acids through the secretion of extracellular nucleases has been early described (nishimura, ; eaves and jeffries, ) . finally, if the above-mentioned obstacles are overcome, rna molecules still face the intestinal extrinsic and intrinsic barriers (see also . . ). the extrinsic barrier consists of the mucus layer covering the epithelial cells. this is a complex hydrogel material composed of proteins, carbohydrates, lipids, salts, antibodies, bacteria, and cellular debris (o'neill et al., ; pridgen et al., ) . it consists of loosely and firmly adherent layers that vary in thickness along the gi tract and can fluctuate based on diet. the mucus is secreted by cells presenting a rapid cell turnover, approximately - days in the small intestine in humans (gamboa and leong, ; o'neill et al., ; atuma et al., ) . among other functions, the mucus layer protects www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. epithelial surfaces by trapping pathogens and foreign particulates, and rapidly clearing them. penetration of this mucus barrier is necessary to reach the absorptive epithelial cells. several mucuspenetrating and mucoadhesive materials can be used to enable penetration of molecule to the mucus layers, increasing residence time and contact of the delivered molecules to the epithelium (pridgen et al., ) . the intrinsic barrier consists of the epithelial cell monolayer, constituting the greatest barrier to material from the intestinal lumen to the lamina propria and bloodstream. cells maintain this barrier by forming tight junctions, whose permeability can be modulated through specific combinations of different proteins (o'neill et al., ) . there are several possible pathways across the intrinsic (epithelial) barrier (figure ).  the transcellular pathway passes through the apical and basolateral cell membranes, as well as the cytoplasm. this pathway is very restrictive to the passive flow of hydrophilic solutes such as rna molecules, because of the lipid bilayer membrane and its impermeability to large molecules. transport mechanisms for this pathway can be passive for hydrophobic molecules, or active with membrane pumps for specific molecules such as ions.  the paracellular pathway is the major passive permeation pathway and allows diffusion of small molecules in the space between epithelial cells. the tight junctions regulate permeability of this pathway based on molecules' size and charge.  finally, transcytosis is an active transport pathway that relies on molecule-specific receptors guiding the molecule through the cell without entering degradation pathways. because of their large hydrodynamic size, macromolecules such as rnas are restricted to this pathway (pridgen et al., ) . the m cell transcytosis pathway is the most extensively studied for oral delivery of rnas. this pathway, which is used to transport antigens across the epithelium for immune surveillance, is attractive because m cells lack mucus secretion and have a sparse glycocalyx, among other features. however m cells are closely associated with immune cells in the lamina propria and peyer's patches, such as dendritic cells and macrophages, this from one side limiting the ability of the delivered molecule to reach the bloodstream and on the other side increasing the risk of triggering an immune response (florence, ; kriegel et al., ; pridgen et al., ) . moreover, m cells only make up a small percentage ( - %) of the non-absorptive epithelium in humans. the surface properties of m cells, as well as the number of peyer's patches vary among species, which could make difficult extrapolating animal model data to humans (pridgen et al., ) . an additional obstacle is represented by the immune system, which is intimately associated with the epithelium. numerous types of immune cells patrol the lamina propria, including t cells, macrophages and dendritic cells. if the ingested rna molecules reach the bloodstream, they must also evade the mononuclear phagocytic system (reischl and zimmer, ; pridgen et al., ) . the presence of endogenous and luminal nucleases, together with gi ph conditions, in general determine rapid degradation and low rna molecules' biological half-life (bhavsar and amiji, ) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the paracellular pathway allows diffusion of molecules in the space between epithelial cells and is regulated by intercellular tight junctions. the transcellular pathway passes through the apical and basolateral cell membranes and cytoplasm. it is restricted to hydrophobic molecules or molecules transported by membrane pumps. the transcytosis pathway is an active transport system that relies on molecule-specific receptors guiding the molecule through the cell escaping degradation pathways. transcytosis pathways are found in both epithelial and m cells. from pridgen et al, . transepithelial transport: paracellular, transcellular and transcytosis pathways. first-pass elimination occurs when a compound is metabolised between its site of administration and the site of sampling for drug concentration measurement. this greatly affects the bioavailability of orally administered compounds since the concentration of the active substance reaching systemic circulation is decreased. the liver is usually assumed to be the major site of first-pass metabolism of an orally administered drug, but other sites are the gi tract, blood, vascular endothelium and lungs (pond and tozer, ) . in fact, the obstacles previously described for rna molecule absorption in the gi tract can be considered a first-pass effect. rna molecules reaching the circulatory system must avoid filtration by the kidneys, accumulation in non-target reticuloendothelial system (res) of the liver, kidney, lungs and spleen, degradation by endogenous nucleases, aggregation with proteins in the serum, and uptake by non-targeted cells such as phagocytes (kriegel et al., ; reischl and zimmer, ). once cellular uptake has taken place in the potential target tissue (surmounting the obstacle of crossing the vascular walls and the lipid bilayer cellular membranes), nucleic acids have to undergo efficient intracellular trafficking to ultimately produce the intended pharmacological effects. this includes endosomal transportation (escaping endolysosomal degradation), cytoplasm release, and efficient incorporation into the relevant intracellular pathways such as risc loading (for sirna, mrna, and mirna) or nuclear uptake (for lncrna) kriegel et al., ; pouton and seymour, ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. rna molecules are large, hydrophilic and negatively-charged molecules. to achieve effect via the oral route, these molecules need to overcome the physical and biological barriers present in both the gi tract, where they encounter variable and harsh conditions along with degrading nucleases, and the circulatory system. rna molecules must reach the target tissue, escaping endo-lysosomal degradation, and enter the appropriate intracellular pathway in sufficient doses to exert their effects. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a literature search as described in section . . . . and based on the methodology described in section . . . , a total of documents were selected as relevant to a general review of rna-based therapeutics administered by gi route. of these documents, at least report the use of specific vehicles for gi administration of exogenous ncrnas (i.e. nanoparticles). six of these documents simultaneously evaluated formulated exogenous rnas and their naked version, and reported either rapid degradation under gi conditions, or reduced or absence of biological effect of the naked version compared to the formulated one (table ) . twenty ( ) of these documents provide specific examples of local effects of rna-based drugs administered orally. in addition, papers were selected as relevant to inform on administration of exogenous ncrnas in either fish or birds and possibly exerting biological effects. orally delivered rna has been used as a dietary supplement or empirical therapeutic agent since s (jain, ). for example, yeast rna supplementation as a source of dietary ribonucleic acids has been used in different animals (choudhury et al., ; jha et al., ) including mammals (sukumar et al., ; heaf and davies, ) and in human studies (gianotti et al., ; tepaske et al., ) . in human studies, up to g per day (in divided doses) of exogenous rnas have been administered (for - days), producing an increase in plasmatic uric acid levels (zollner and grobner, ; zollner, ) . the absence of attempts to study the gi absorption of pure rna in these studies suggests that its effects, if any, could be related to rna molecules as a source of nucleotides. indeed, nucleotide supplementation in rats has been shown to affect the healing of ulcerative conditions, ameliorating indomethacin-induced ileitis and aggravating the severity of dextran sulfate sodium (dss)-induced colitis (sukumar et al., , sukumar et al., . also, a study of body fluid composition of rats orally administered yeast rna, mixtures of its constituent nucleosides or its constituent bases and ribose was performed (heaf and davies, ) . the authors showed that ingestion of rna increased intestinal levels of ribose, inorganic phosphate, uridine, pseudouridine, uracil, inosine or uric acid. the effect of orally administered mixed nucleosides on blood and urine composition was similar to that of rna, while some differences were noted for some equivalent mixture of free bases (heaf and davies, ) . this study showed that the dietary rna-phosphate passed to the urine from the gut, suggesting that most of the rna-ribose was probably metabolized (heaf and davies, ) . it has been shown in a murine model of staphylococcus aureus infection that the oral administration of rna was ineffective while the intraperitoneal nucleoside-nucleotide mixture was more effective in maintaining host resistance against bacterial infection (adjei et al., ) . supplementation with nucleotides has also been shown to exert biological effects, including expression regulation of certain genes (sanchez-pozo and gil, ; gil, ) , although the mechanisms of action are still unknown. it is generally assumed that nucleotides are not essential nutrients, but under certain conditions (i.e. low dietary intake, tissue needs increased or stress) dietary nucleotides may play a role as conditionally essential nutrients (jung and batal, ; carver and walker, ) . in this context, dietary supplementation with yeast rna promoted ulcer healing in a rat model of indomethacin-induced ulcerative ileitis (sukumar et al., ) . similar effects were observed when nucleosides and nucleotides were administered intravenously (veerabagu et al., ) . in both cases, the possible mechanism of action was partly due to increased cell proliferation in the damaged tissue (sukumar et al., ; veerabagu et al., ) . similar effects were observed when rna was administered orally or its constituents (nucleotides) were injected iv, suggesting that the effects are due to the absorbed nucleotides. dietary nucleotide supplementation in formula-fed infants efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. has been shown to improve gut microbiota composition (singhal et al., ) . in studies on certain fish model, supplementation with dietary rna at a range of concentrations ( . - . % of nucleic acids in the diet compared to the non-suplemented control group) enhances immunological response (choudhury et al., ; jha et al., ) . in human studies, postoperative enteral diet supplementation with rna, arginine and omega- fatty acids has been shown to modulate the postoperative immune response after surgery for upper gastrointestinal cancer (senkal et al., ) , overcoming more rapidly the immunologic depression after surgical trauma (kemen et al., ) . also in humans, oral supplementation with rna (from yeast) during days before surgery improved host defence in patients undergoing cardiac surgery (tepaske et al., ) or gi cancer surgery (gianotti et al., ) . in both cases, rna supplementation was also accompanied with arginine and omega- fatty acids administration. in summary, the specific contribution of orally administered rna to these effects cannot be ascertained. the instability of orally administered rna molecules and the presence of a large array of rnaases within the gi tract (see sections . . and . . ) suggest low absorption, if any. exposure to a highly active enzymatic environment in the gi tract, extreme ph conditions, and the existence of a mucosal epithelial barrier are the main challenges for oral delivery of rna therapeutics (martirosyan et al., ) . although a considerable amount of food rna is ingested, which varies widely between individuals but is typically in the range . - g/person/day, it is assumed it is degraded and absorbed in minimal amount (jain, ) . however, further data are required to document the extent to which this conclusion pertains to rna molecules with complex structures. to overcome the multiple barriers encountered by exogenous rnas for oral delivery, several strategies in the laboratory or clinical trials have been developed (see . . . below). the oral administration route is advantageous because it increases patient compliance and comfort over injection, provides for simple, repeatable administration, and offers a large surface area for absorption (forbes and peppas, ) . rna delivery may also benefit from advances in the oral delivery of aso for animal (raoof et al., ; raoof et al., ; . indeed, polymeric nanoparticles containing alginate, dextran, chitosan, polyethylenimine, polyethylene glycol, polylactide, yeast derived β-glucans and/or several other natural or chemically synthesized molecules have been tested by the pharmaceutical/medicine field. the literature describes several strategies for the oral delivery of rna oligonucleotides. the target is to stabilize and protect rnas during gi transit, enable cellular uptake in the intestine, and in some cases, promote endosomal escape and increase biological action. thioketal nanoparticles (tkns) were formulated from a polymer, poly-( , -phenyleneacetone dimethylene thioketal) that degrades selectively in response to reactive oxygen species ( the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. (plga) nanoparticles were also tested in the same study, but with no efficacy in diminishing tnfα expression. to obtain efficient mucus transportation, targeted cellular uptake and endosomal/lysosomal escape, different nanoparticles have been tested ( nanoparticles of galactosylated trimethyl chitosan-cysteine containing sirna against map k were tested and found to target activated macrophages in colonic tissue (zhang et al., b) . compared to nanoparticle-protected sirnas, naked sirnas were completely degraded by the intestinal fluids. when administered orally ( µg sirna/kg) for days, nanoparticles improved the dss-induced model of colitis in mice (zhang et al., b) . other ternary polymeric nanoparticles formed by thiolated trimethyl chitosan with tripolyphosphate have been tested in mice and found to efficiently deliver sirnas to the intestine and other tissues when administered orally (zhang et al., a the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ( %, % and %) were also evaluated in trimethyl chitosan-cysteine conjugate nanoparticles . in a mouse model of ulcerative colitis, the mannose density of % was the most effective for sirna knockdown of tnfα following oral administration ( µg/kg) of '-o-methyl-modified tnfαspecific sirna duplex . the use of supramolecular self-assembly nanoparticles (ssnps) -which contain oleyl trimethyl chitosan, poly(γ-( -(((piperidin- -yl)ethyl)amino)methyl)benzyl-lglutamate), oleyl-peg-mannose, oleyl-peg-cysteamine, and sodium tripolyphosphate -with specific functions of mucoadhesion, transepithelial permeation, membrane penetration and active targeting have also been tested ( however, not all nanoparticle types can efficiently deliver rnas. in a study evaluating the ability of unassisted epithelial entry of nucleic acids as a consequence of nanocarrier contact with mucus, antisense oligonucleotides but not sirna (naked) were shown to be delivered to mouse intestine when formulated with nanocarriers composed by chitosan (martirosyan et al., ). multi-compartmental formulations -consisting of one or more internal compartments surrounded by protective external compartments -have been developed to surmount the many barriers to oral rna delivery (kriegel et al., ; . one example is the "nanoparticles in the microspheres oral system" (nimos), a solid-in-solid multi-compartmental system (kriegel et al., ) . gelatin nanoparticles entrapped in poly(epsilon-caprolactone) microspheres were prepared carrying tnfα sirna (slightly chemically modified dtdt) to treat dss-induced colitis. oral administration of sirna ( . mg/kg) to female balb/c mice decreased colonic tnfα expression and supressed the expression of proinflammatory cytokines (kriegel, c and amiji, m, ) . when combined with sirna against cyclin d (naked sirna, also at . mg/kg/day), the silencing effects were more potent than with tnfα sirna alone (kriegel c and amiji mm, ) . dectin- recognizes beta- , and beta- , linked glucans rich particles and intact yeast and is particularly expressed on the monocyte/macrophage and neutrophil lineage (herre et al., ) . β- , -d-glucan has been used to deliver exogenous rna targeting macrophages (aouadi et al., ). delivery of β- , -d-glucan sirna particles containing µg/kg unmodified sirna by oral gavage to mice for days reduced map k expression in different macrophages from different tissues and protected them from lipopolysaccharide-induced lethality (aouadi et al., the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the cd gene was the target of the antigen-presenting dendritic cells and oral administration of the recombinant yeast. short hairpin rna delivered in lactobacillus casei have also been tested to target the intestine (kuwahara et al., ) . other organisms, including attenuated salmonella typhi, were used as vectors to deliver rnas, even ribozymes (bai et al., ) or sirna (jiang et al., ) , using oral administration in mice to target either local or systemic effects (bai et al., ; jiang et al., ) . the the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. extracellular vesicles are a heterogeneous family of vesicles delimited by membranes. these can be classified by their size as i) exosomes ( - nm in diameter), which originate from the endosomal system, ii) microvesicles ( - nm) formed by budding out of the plasma membrane, and iii) apoptotic bodies (> nm) formed by blebbing of the plasma membrane during apoptosis (reviewed in yanez-mo et al., ; colombo et al., ) . increasing evidence suggests that extracellular vesiclemediated communication can take place in vivo, but the nature of the extracellular vesicles involved in these effects remains to be clarified (reviewed in tkach and thery, ; pitt et al., ) . exosomes, which are formed in multivesicular compartments, are secreted when these compartments fuse with the plasma membrane (kowal et al., ; abels and breakefield, ) . exosomes have been shown to contain or transport a myriad of molecules including proteins, carbohydrates, lipids, and a variety of genetic material including dna, mrna and ncrnas (reviewed in choi et al., ; abels and breakefield, ) . exosomes are also described as participating in intercellular communication (costa-silva et al., ; pironti et al., ; nojima et al., ) , delivering their parental cell-derived molecular cargo to a recipient cell. some of these processes are thought to be mediated by ncrnas, including mirnas thomou et al., ) . detailed information on how all these biological processes occur is outside the scope of this review. given the above characteristics of exosomes, these vesicles hold promise as delivery vehicles for therapeutics (munagala et al., ; van der meel et al., ) . indeed, exosomes have been used to deliver sirna to different tissues when administered systemically, including the brain (alvarez-erviti et al., ; cooper et al., ; didiot et al., ) . mirnas have also been delivered via exosomes to different tissues including xenograft breast cancer tissue (ohno et al., ), brain and other tissues when administered systemically or locally (zhang, d et al., ) . interestingly, oral administration of exosomes or extracellular vesicles has also been described in animal models agrawal et al., ; oliveira et al., ; oliveira et al., ) . however, their biological effects were not related to transport of ncrnas. some extracellular vesicles have been shown to resist digestion under in vitro simulated gi conditions (benmoussa et al., ; , particularly those from bovine milk (vashisht et al., ) . moreover, milk exosomes seem to exhibit cross-species tolerance and are not described to induce adverse immune and inflammatory responses (munagala et al., ) . however, whether exosomes or other extracellular vesicles resist the harsh in vivo conditions of the gi tract and the digestive process remains poorly described and needs to be studied (tomé-carneiro et al., ) . exosome-like nanoparticles from plants have been isolated from different species including ginger, grape, grapefruit and carrot (mu et al., ; ju et al., ; zhang et al., ) . some authors indicated that the approximate amount of exosome-like nanoparticles in these edible plants were reported to be ≈ mg/ g edible plant (mu et al., ) , while other authors reported only ≈ mg/kg ginger . most of these studies reported the presence of rnas, either small or large, including ncrnas (mu et al., ; zhang et al., ; ju et al., ) . the amount of rna present in these exosome-like nanoparticles was reported to be ≈ µg rna/ mg exosomelike nanoparticles for grape and grapefruit, and ≈ µg rna/ mg exosome-like nanoparticles for ginger and carrot (mu et al., ) . the in vivo biological effects of these nanovesicles have been assessed. oral delivery of ginger nanovesicles during days at a dose of . mg/day was found to target the colon, to be taken up by epithelial cells and macrophages, and to reduce dss-induced colitis in mice . treatment for weeks with the same dose also exhibited antiinflammatory activity in a mouse model of chronic colitis . ginger-derived lipid vehicles have also been generated from ginger lipids and loaded with sirna-cd (zhang m et al., www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. ). oral administration of these ginger-derived lipid vehicles targets the colon tissue and they are effective in treatment of induced ulcerative colitis in mice. grapefruit exosomes at a dose of mg/kg per day for days also ameliorated the dss-induced colitis targeting intestinal macrophages . when administered orally at a dose of mg of nanovesicles/day, grape exosomes-like nanoparticles were also found to protect mice from a dssinduced model of colitis by induction of intestinal stem cells (ju et al., ) . local intestinal macrophages and stem cells from the small and large intestine were also found to be the target of these exosome-like nanoparticles when administered by oral gavage ( mg/day) (mu et al., ) . even though mirnas were reported to be present in these exosome-like nanovesicles (ju et al., ; mu et al., ; zhang et al., ) , the biological effects of these ncrnas were not evaluated. whether these mirnas resist the gi tract conditions remains unknown. a main hurdle in rna-based therapeutics is the successful delivery of naked rna (non-chemically modified) molecules to specific (target) tissues in the gi tract. the literature contains few studies in which exogenous rnas are administered in vivo using routes of administration other than oral, and most are in the field of inflammatory bowel disease. an amphiphilic cationic cyclodextrin (cd) vector was developed for a complex sirna against tnfα, where the complex was concentrated to µg/ µl in % glucose (mccarthy et al., ) . cd.tnfα.sirna was found to be efficient against dss-induced colitis in c bl/ mice treated twice (day and post-dss) by intrarectal administration with the test solution ( µl). cd.sirna administration reduced tnfα and il- expression as compared to the non-silencing sirna control or the naked tnfα sirna (not delivered in formulated cyclodextrin). enteral delivery of sirnas for systemic effect was also tested. a nuclease resistance and chemically modified sirna against apolipoprotein b was conjugated with α-tocopherol and was administered ( mg/kg) as lipid nanoparticles in the large intestine of mice. the lipid nanoparticle was composed of mixed micelles comprised of linoleic acid and peg- hydrogenated castor oil. the sirna efficiently reached the liver and other tissues using the chylomicron-mediated pathway via the lymphatic route (murakami et al., ) . delivery of sirna into hepatocytes was markedly reduced when the sirna was not bound to α-tocopherol, indicating that conjugation with α-tocopherol was essential to this delivery system using this route. a calcium phosphate (cap) core coated with sirnas and encapsulated in poly(d,l-lactide-co-glycolide acid) (plga) nanoparticles containing an outer layer of polyethyleneimine (pei) was developed for intrarectal delivery (frede et al., ) . in a dss-induced model of colonic inflammation, intrarectal administration ( µg/day) of nanoparticles containing ng sirna against either tnfα, ip- or kc from day to after % dss treatment, ameliorated the colitis. indeed, while intestinal epithelial cells, dendritic cells, macrophages and t cells could uptake the tnfα sirna nanoparticles, reduction of tnfα was only detected in epithelial cells and t cells (frede et al., ) . the stability of sirna within the cap/plga/pei nanoparticles against enzymatic nucleases under colonic conditions was evaluated. while the sirna-loaded cap/plga/pei nanoparticles were detectable after h incubation at ºc with colonic fluid or homogenate, the same sirnas not formulated in nanoparticles was not detected and was assumed to be degraded. even after exposure to colonic homogenate for h at ºc, the nanoparticles retained biological activity. since the supernatant of the homogenate subsequently incubated with a cell line, still reduced the expression of tnfα by % (frede et al., ) , suggesting that sirna-loaded nanoparticles retain its biological activity compared to that of nacked sirnas (not formulated). www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. other alternatives for rna delivery have been tested. ultrasound-mediated rna delivery has been efficiently achieved in mice (schoellhammer et al., ) . chemically modified sirna against tnfα ( ng sirna in µl water) was delivered in the rectum using ultrasound exposure to khz for . s and after a -day treatment tnfα was efficiently repressed in colonic tissue of a mouse model of induced colitis. in the same study, a mrna (≈ kda) complexed in lipid nanoparticles (lnps) was administered in the colon ( µl lnps solution) with ultrasound and found to be successfully translated locally in the colonic tissue (schoellhammer et al., ) . oral administration of exogenous rnas to exert a biological effect, either locally or systemically, is successful if combined with specific delivery technologies, such as vehicles developed for pharmaceutical/medical purposes. nanoparticles have been designed to release their cargo in specific regions of the gi tract (i.e. where inflammation occurs) or to resist the gi conditions. in contrast to other routes of administration, where normally highly chemically modified rnas are used, oral delivery normally involves use of naked rna or minimally modified rnas formulated with complex delivery vehicles. achieving the local desired effect (i.e. targeting inflammation in the colon) has contributed to the development of delivery technologies and the usage of other routes of administration different than the oral as described above. however, the available literature also suggests that naked or unmodified exogenous rnas are rapidly degraded when exposed to the gi conditions without incorporation into delivery vehicles. extracellular vesicles are natural lipid particles released by many cell types with the potential to mediate cell-to-cell communication. these extracellular vesicles have a tremendous potential as delivery vehicles for therapeutics. however, there are still very few studies evaluating their resistance to the harsh conditions of the gi tract following oral administration. knowledge of their biological effects as transporters of exogenous ncrnas is still very limited. nucleic acid-based therapeutics have the potential to treat numerous diseases by correcting abnormal expression of specific genes. as indicated previously, the oral route of drug administration poses serious delivery challenges due to the gi degrading environment, as well as the need to overcome other barriers preventing nucleic acid delivery. because of these obstacles, efforts in developing oligonucleotide-based therapeutics using the oral route have focused on local gastrointestinal delivery, i.e. directly delivering the drug to the target tissue for localized effects, and, therefore, increasing local bioavailability and maintaining doses low while diminishing possible effects to non-target tissues kriegel et al., ) . crohn's disease and ulcerative colitis are the two principal forms of ibd, characterised by being a chronic relapsing inflammatory condition of the gi tract. the pathogenesis of ibd is dependent on the interaction between local immune and environmental factors in genetically-susceptible individuals (o'neill et al., ) . inflammation in crohn's disease is characterised by high production of the cytokines interferon (ifn)-gamma, il- and tnf-alpha, representing a t helper (th )-th response. in ulcerative colitis, the immune response is characterised by th and an atypical th , with high production of il- , il- and il- (o'neill et al., ) . conventional treatment consists of antiinflammatory and immune-suppressive drugs but, while some medications are effective to combat inflammation in the acute phase, they are ineffective in maintaining remission due to toxicity, dependency and higher relapse rates (kriegel, c and amiji, m, ) . the goal for ibd treatment is local delivery of therapeutics to intestinal immune cells. initial approaches involved il- supplementation www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. due to its huge potential in blocking proinflammatory cytokines and inflammatory tissue damage. but human clinical trials have not proven that systemic il- supplementation can help prevent or improve the ibd symptoms, probably due to il- low mucosal levels . separate studies have used gene therapy to increase il- mucosal availability by engineering il- plasmid dna, and achieving more success in rodent models (barbara et al., ; steidler et al., ; lindsay et al., ; bhavsar and amiji, ) . deregulation of tumour necrosis factor (tnf)-alpha production, a proinflammatory cytokine primarily produced by activated macrophages along with other cell types, is associated with the onset and progression of a number of inflammatory diseases (such as ibd, rheumatoid arthritis), alzheimer's disease and psoriasis . the harmful effects of tnf-alpha are thought to be mainly mediated by activation of nf-kappab, therefore regulating expression of over genes. systemic blockage of tnf-alpha is accompanied by several side effects that can be avoided if local inhibition occurs. rnai-mediated local suppression of tnf-alpha can be beneficial for overcoming many of these side effects. different groups have developed various delivery systems for sirna molecules targeting tnf-alpha expression in the gi tract, assaying them both on cells and several rodent models for ibd (wilson et al., ; kriegel, c and amiji, m, ; kriegel, c and amiji, mm, ; laroui et al., ; yin et al., ; he et al., b, a; he et al., ) . even though therapeutic rnai development is in its infancy, current approaches and tools for delivery of these biomolecules via the oral route are improving. in fact, recent reports describe the effects of an oral smad antisense oligonucleotide in a randomized, placebo-controlled, double-blind, phase clinical trial in patients with crohn's disease (monteleone et al., ; monteleone et al., ) . although in this instance the biomolecule used is an antisense oligonucleotide and not a sirna, the design of the oral formulation could be very similar. iron is an essential metal required for numerous physiological functions, but in excess it is a well-defined risk factor in the pathogenesis of several diseases, including cardiovascular and neurodegenerative diseases. since there is no recognised active pathway of iron excretion, disposal of excess iron from the body is the primary therapeutic goal of treating patients with iron overload. use of iron chelators is limited due to nonspecific distribution in non-target tissues, which results in a number of serious side effects and toxicity . another way to treat iron excess is by limiting absorption of exogenous iron. the divalent metal transporter (dmt ) protein plays a well-established role in iron absorption. the primary site of dmt activity is the intestinal epithelium. dmt expression is regulated in response to body iron levels, with expression enhanced when iron stores are low and, conversely, reduced when iron stores are high. oral delivery of sirna-encapsulated nimos to selectively suppress intestinal dmt could decrease intestinal uptake of dietary iron, thereby mitigating iron overload and preventing iron-mediated toxicity . celiac disease is caused by a t-cell mediated immune response in the small intestine against deamidated cereal gluten peptides modified by the enzyme transglutaminase (tg ). the only way to prevent celiac symptoms is strict adherence to a gluten-free diet. the pathophysiology of celiac disease involves a combination of environmental, genetic and immunological factors. among several immune mediators, enzyme tissue tg and the proinflammatory cytokine interleukin- (il- ) have emerged as key players in promoting inflammatory responses against dietary gluten. given their important role in the pathophysiology of celiac disease, sirna mediated silencing of intestinal tg and il- (i.e. by oral administration using nimos) may result in neutralizing proinflammatory effects and could therefore alleviate disease symptoms . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. colorectal cancer is one of the most common forms of cancer, and rnai therapy has great potential in the treatment of local intestinal cancers. however, rnai therapy faces major challenges since the genotypic footprints of the various cancers differ widely, even from individual to individual. depending on the tumour, several different sets of genes may be misexpressed, making it extremely difficult to determine a common/unique "culprit" gene. moreover, mice and rats do not generally spontaneously develop colon cancer, so induction of tumour growth is required. but these developed animal models do not reflect the genetic changes observed in human tumours (o'neill et al., ) . genetic mouse models that spontaneously develop intestinal cancer have also been developed. mutations in the apc gene are associated with development of colon cancer as well as a condition called familial adenomatous polyposis (fap). fap is an inherited condition characterised by development of numerous polyps in the colon. some of these polyps can subsequently become malignant adenocarcinomas (o'neill et al., ) . regarding fap and human gene therapy, one study explored oral delivery of escherichia coli expressing shrna against beta-catenin, and found significant silencing activity in healthy mice (xiang et al., ) . the gi tract provides the possibility of acting at systemic level by targeting local resident cells, such as m cells. m cells can take up encapsulated biomolecules to be phagocytosed by macrophages. in fact, aouadi et al. reported that glucan encapsulated sirna particles (gerps) loaded with sirna were phagocytosed by the underlying gut-associated lymphatic tissue (galt) macrophages and then translocated to distal organs of the reticuloendothelial system such as the liver, spleen and lung (aouadi et al., ) . another report claims functional cd shrna expression delivered into dendritic cells in mice by oral administration of recombinant yeast (xu et al., ) . in this manner, the targeted cells located in the intestine can undergo sirna-mediated gene silencing and migrate into tissues throughout the body. when successfully administered, rnai is a very useful therapeutic strategy for efficiently modulating gene expression. orally administered local rnai therapeutics have great potential due to the increasing bioavailability of the therapeutic biomolecule at the targeted tissue or cell type, coupled with simultaneous evasion of systemic side effects and immunogenic reactions. most advanced studies are focused on inflammatory bowel disease, although other potential diseases are being explored. the current main challenge is development of suitable encapsulation methods to protect the rna molecules from the harsh and varying conditions of the gi tract. the literature on rna delivery in fish and birds is scarce, with some examples of dietary delivery of exogenous rnas. in birds, most of the studies have been done using central nervous system administration of sirnas. for example, in the zebra finch bird, sirnas (three - nt long sequences against tyrosine kinase b, trkb) administration via direct injection into the brain was effective in reducing the volume of the high vocal centre by diminishing the robust nucleus of the arcopallium and the relative number of cells within it (beach et al., ) . in another study, direct administration of sirnas in the brain of white-crowned sparrows reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations (ubuka et al., ; ubuka et al., ) . in the japanese quail, central administration of the same sirnas against gonadotropin-inhibitory hormone the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. sirnas for rna interference in chicken embryos has also been used to target marek's disease virus in vivo (chen et al., ). in fish models, systemic exosome-mediated sirna delivery to zebrafish resulted in effective crossing of the brain blood barrier and localization in the brain in vivo (yang t et al., ) . exosome-delivered sirnas decreased fluorescence-labelled tumour cells in the brain more than fourfold by inhibiting vascular endothelial growth factor (vegf) in a xenograft zebra fish brain tumour model (yang t et al., ) . these results suggested that brain endothelial exosomes could be used to deliver exogenous sirnas to the target site in the brain for treatment of brain cancer. the proposed higher delivery efficiency, lower immunogenicity, and better compatibility than existing foreign rna carriers have promoted the possible use of exosomes for exogenous rna delivery (mei et al., ) . several other studies have evaluated systemic delivery of sirnas to fish animal models, including delivery to the heart using peg-pla nanoparticles (diao et al., ) or using a neutralized non-charged polyethyleniminebased system . the literature describes other examples of sirna delivery into zebrafish embryos to inhibit specific gene function (gruber et al., ) . in the context of other types of ncrnas, direct injection of mir- a mimic rnas at the one-cell stage of zebrafish embryos resulted in inhibition of formation of the caudal vein plexus by means of targeting the endothelial cell bone morphogenic protein smad signalling (icli et al., ) . also in the zebrafish model, injection of a mir- mimic into embryos at the one-cell stage to deliver mir- ubiquitously resulted in protection against sterile inflammation (hsu et al., ) . dsrna microinjection administration to zebrafish embryos ( - cell stage) at a concentration of - pg rna/embryo resulted in rna interference, an effect that was diminished when ssrna was used (wargelius et al., ) . in cultured european sea bass, intramuscular injection ( weeks, µg of dsrna per dose) followed by in vivo electrically mediated dsrna delivery resulted in a reduction of myostatin gene expression (terova et al., ) . delivery of exogenous rnas has also been tested in lower aquatic organisms. in the crustacean red claw crayfish cherax quadricarinatus, systemic injection of sequence specific dsrnas against viral protein b of macrobrachium rosenbergii nodavirus (mrnv) was conducive to rnai effects that were able to functionally prevent and reduce mortality in infected individuals (hayakijkosol and owens, ) . dsrnas (intramuscular injection) have been used to induce antiviral immunity in invertebrates as they can also recognize dsrna as a virus-associated molecular pattern, resulting in activation of an innate antiviral response (robalino et al., ) . oral administration of encapsulated dsrnas (inactivated bacteria expressing dsrna) in the prawn macrobrachium rosenbergii fed twice daily at a rate of % total biomass for days resulted in their protection against white tail diseases caused by mrnv (naveen kumar and karunasagar, ) . dsrnas designed against the capsid and b genes of mrnv reduced the mortality of prawns by ≈ % due to sequence specific-mediated silencing of the viral genes (naveen kumar and karunasagar, ) . injection of viral protein -targeted dsrna (≥ µg dsrna/g shrimp body weight) into shrimp resulted in a high level of nt sirnas mapping across the entire white spot syndrome virus genome after virus challenge, which resulted in protection against the virus (nilsen et al., ) . in planarians, ingestion of bacterially-expressed dsrnas or direct microinjection into the animals can inhibit gene expression through rna interference (newmark et al., ) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. mongersen, an oral smad antisense oligonucleotide, and crohn's disease. n engl j med , - . mu j, zhuang x, wang q, jiang h, deng zb, wang b, et al. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. - . zollner n. . purine and pyrimidine metabolism. proc nutr soc , - . zollner n, grobner w. . influence of oral ribonucleic acid on orotaciduria due to allopurinol administration. z gesamte exp med , - . following a literature search as described in section . . . and based on the methodology described in section . . . , a total of documents were selected as relevant to reviewing the effect of dietary exogenous ncrnas on the gi tract and annex glands. very few studies have focused on the gi tract and their annex glands. four documents specifically evaluated the resistance of plant-derived srnas to simulated in vitro digestion system, ex vivo digestion system or in vivo digestion samples (philip et al., ; yang, et al., a; and reported that the percentage of unaltered mirnas after digestion might be below . %. due to their relevance in early human nutrition, literature on breast milk ncrnas was chosen to describe the biological effects of dietary exogenous ncrnas of non-plant origin, for which full-text reports were studied and reviewed. zhang et al. were the first to report on the transfer of mirnas from food plants to the mammalian circulatory system causing effects on recipient cells . this study reported the detection by high-throughput sequencing of plant srnas in human serum, as well as in tissues of the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. humans, mice and calves. the plant mirnas were distinguished from human mirnas by their '-omethyl modification, which made them resistant to periodate whereas the human mirnas having free ' hydroxyl were periodate sensitive. it was then assumed that the identified plant mirnas were coming from the food uptake. two rice mirnas named osa-mir a and osa-mir a were particularly abundant in human serum samples, since rice is common in human diets. additional in vitro experiments showed that these plant mirnas can endure conditions that mimic the acidic environment of the gut, suggesting that plant mirnas can survive being eaten and digested, pass through the mammalian gi tract and reach target organs. the authors also showed that diet-derived plant mir a can target the low density lipoprotein receptor adapter protein (ldlrap ) mrna based on sequence complementarity. this mirna was able to reduce the ldlrap protein level in the blood and liver of mice fed rice, and eventually increase their plasma low-density lipoproteins (ldl) content. this publication raised questions about whether food-derived srnas could play an active role in human/animal health. however, it was controversial and its results were questioned due to nutritional imbalances in the test-diet studies or lack of rnai-mediated modulation of ldlrap protein levels in mouse liver (witwer and hirschi, ; chen et al., ; . scientists from the monsanto and miragen companies were unable to reproduce these findings . measuring ldlrap protein levels in the liver of mice fed with mir a-containing rice diets by elisa instead of western-blot analysis, dickinson and colleagues did not detect any protein change modulated by mirna a-diets. although changes in the ldl plasma levels were indeed detected, the authors concluded that ldl increases resulted from nutritional imbalances rather than from a mir consumption mediated effect. additionally, plant mirnas including the highly abundant and stable osa-mir a were not detected in liver and plasma samples of mice fed under different rice-based regimes by srna sequencing with the hiseq illumina system . other independent research groups failed to detect dietary plant mirnas in plasma and tissues when conducting controlled feeding studies in humans and in animal models using different detection methods, such as real time quantitative pcr (rt-qpcr), droplet digital pcr (ddpcr) and next generation sequencing (ngs) analysis . these studies provide contradicting evidence of mirnas dietary absorption. in all the attempts, plant mirnas were detected at substantial levels in the diets but were undetectable in animal fluids and tissues. snow and colleagues studied three plant mirna species (mir a, mir a, and mir a) highly abundant in fruits routinely ingested by a group of ten healthy athletes. examination of the athletes' plasma by rt-qpcr analysis using taqman probes revealed high levels of endogenous mirnas but not the three dietderived plant mirnas . the same results were obtained with mice fed vegetarian diets rich in these mirnas. furthermore, using a mir null mutant mouse they showed that none of the dietary mirna including mirna was detected in blood and body tissues, suggesting that dietderived mirnas were not absorbed or maintained in mouse body fluids or organs. another group reported negative results on dietary mirna absorption in non-human primates . using qrt-pcr analysis and the same timeframe of as in zhang's report a, plant-specific mirnas were undetectable in tissues from macaques given a vegetarian diet. another feeding study in humans showed no significant differences in the levels of the plant mir a and mir in the plasma after consumption of broccoli sprouts highly rich in these two mirnas (baier et al., ) . more recently, a different research group using by ngs analysis failed to detect plant mirnas in the plasma of healthy volunteers that normally consume olive oil . the possibility that the plant rnas detected in human tissues and body fluids in some studies were contaminants remains a serious concern . by searching the composition of srna-seq data generated by the zhang's team from amphioxus animals, www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. it was found that the amphioxus data contained rice mirnas as previously reported for human serum. given that amphibia have an exclusive algae-based diet, this strongly suggests that the rice mirnas in both studies are the result of samples contamination . spurious detection of such sequences could arise due to contamination during sample handling, library preparation or sequencing, or result from errors in data analysis. since next-generation sequencing is very sensitive, just a few molecules could cause a false positive detection. more recently a comprehensive meta-study surveying for the presence of cross-species mirnas in sequencing data sets from various human tissues and body fluids indicated that xenomirs (cross-species mirnas) are technical artefacts rather than the results of dietary intake . a bioinformatics study identified plant mirnas in human and porcine milk exosomes . by searching publicly available ngs datasets, the authors identified several plant mirna species, most of them belonging to evolutionarily conserved and highly abundant mirna families. the target prediction suggests that these mirnas may interact with mrnas coding several transcription factors, protein receptors, transporters and immune-related proteins, thus potentially influencing biological functions in humans. of note is that the plant mirna profiles found in mammalian breast milk were similar to the composition in human blood presented by zhang and colleagues . liang and colleagues reported positive results on the absorption of food-derived mirnas from feeding studies in mice using cabbage (brassica oleracea) . they showed that mir , the most abundant mirna in cabbage, was detected in blood and various organs after cabbage consumption. however, there was no absolute quantification of mir or a description of an endogenous control for normalization of qrt-pcr data. a more recent study by zhang's group described how plant mirna from honeysuckle (lonicera japonica) inhibited influenza a virus replication in mice when entering through the gi tract, and they hypothesized that this mirna might be the active component in the traditional chinese medicine based on honeysuckle herb to treat influenza infection . the authors demonstrated that mirna is highly stable in honeysuckle decoction by solexa sequencing and northern blot analysis. they also showed that mir levels increased in mouse peripheral blood and lung following continuous honeysuckle decoction intake (drinking) or gavage feeding, as measured by qrt-pcr using taqman mirna probes. additionally, luciferase reporter assays and in vivo experiments showed that mir was able to target the influenza a virus, and consequently inhibited its replication and reduced mice mortality. the same authors reported that plant mirnas can be found in human umbilical cord blood and amniotic fluid, transferred from mother to the foetus, and that these influence foetal development and health . they showed that mir content increased in the maternal plasma and foetal liver of mice fed honeysuckle. a fluorescently labelled sirna was used to trace the transplacental transmission through feeding. furthermore, they showed that after feeding mice with different amounts of sirnas targeting the alphafetoprotein mrnas the levels of this protein were down regulated in foetal tissues. these results suggest that dietary sirnas delivered from the mother to the foetus could regulate foetal gene expression. more recently, the anti-proliferative effect of a plant mirnas on breast tumours was demonstrated in feeding experiments in mice (chin et al., ) . the plant mir , particularly abundant in broccoli, was found in sera from women, and its levels were inversely correlated with breast cancer morbidity and progression. in human sera, mir was detected in extracellular vesicles by qrt-pcr using a taqman probe. transfection of breast cancer cells with a synthetic mimic mir was shown to reduce their proliferative growth by targeting the transcription factor mrnas. more importantly, oral administration of synthetic mir significantly inhibited the growth of xerograph breast tumours in mice. these results agree with a previous report in which oral administration of a cocktail of three www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. tumour suppressor mirnas that were '-o-methylated to mimic plant mirnas reduced colon tumour burden . additional research is needed to address discrepancies among different studies and verify whether plant srnas derived from diet are absorbed at functional levels in animal species. most of the available data suggests either that gastrointestinal absorption of dietary plant mirnas does not occur in healthy consumers or mirnas levels in blood and tissues of consumers are too low to have a biological impact. however, the biological activity of food mirnas may be more dependent on their accumulation in exosomes than on their abundance per se, because of their reported low concentration. development of sensitive sensors to help to detect the functionality of low-level absorbed mirnas would facilitate assessment of the effects of dietary mirnas in consumers. future studies are needed to establish how exogenous ncrnas absorption and tissue distribution occurs, as well as to address their bioavailability and their biological function. reported that increased mir was detected in sera and urine after consumption of particular foods (i.e. honeysuckle), and disappeared h after the honeysuckle was removed from the diet; this supports the idea that these small rnas were of dietary origin. the role of kidney damage in mirna retention in the animals was also evaluated. using two models of acute renal failure (baliga et al., ) , i.e. the cisplatin (a chemotherapeutic agent) and the glycerol-induced models, it was observed that only mice receiving cisplatin exhibited measurable levels of dietary mirnas in sera and urine. this suggests that kidney damage alone did not lead to enhanced mirna retention in the mouse circulatory system . of interest is that cisplatin treatment, but not glycerol treatment or honeysuckle feeding, disrupted the organization of small intestine epithelial cells as shown by histological analysis. in this study, yang et al. used droplet pcr to demonstrate that the amplified products were likely to be specific and not due, for example, to nonspecific amplification of endogenous rnas . whether particular foods and/or alterations in intestinal permeability could improve the capacity to absorb small rnas from the diet or influence the biological effect within the gi tract, annex glands and systemically is poorly described, but still relevant for risk assessment of exogenous ncrnas in a subset of population. no studies specifically report the biological effects of dietary exogenous plant ncrna on the gi tract. only a few studies (described above) report the possible biological effects in their annex glands, including the liver . for gastrointestinal tissues or the liver, liang et al. reported that icr mice (a strain of albino mice) fed plant total rna ( - µg) extracted from brassica oleracea showed detectable levels of mir in the stomach (up to ≈ copies), intestinal (up to ≈ copies) and faecal content (up to ≈ copies), with the maximum amount present at to h after feeding . estimation of the proportion of orally administered rna that survived digestion suggested that the stomach contained . - . %, the intestines . - . % and the faeces . - . %, while the blood contained about . - . % and the spleen about . - . % . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. which was similar to that of their synthetic form (without '-o-methylated ' ends) . whereas plant mirnas had a much slower degradation rate compared to their synthetic form without '-o-methylated ' ends. it is important to note that extreme ph conditions are not the only physicochemical or biological condition within the gi tract (see section . . . . for details). using a simulated human digestion system in vitro, philip et al. evaluated the resistance of soybean and rice mirnas to simulated gastric fluid early stage digestion (philip et al., ) . the study found continual survivability of plant mirnas in an in vitro simulated digestion system for over min without any significant decrease in their levels (philip et al., ) . mirna levels have been analysed qualitatively by real-time pcr using taqman mirna assays. stability of the plant-derived small rna mirna was also assessed using an artificial in vitro digestion system that simulates mammalian gastric and intestinal conditions . yang et al. compared three samples which contained a) ml cabbage extract with pmol plant-derived mir (measured by qrt-pcr), and pmol spiked-in synthetic '-o-methylated mir a and artificial mirna termed c ; b) ml phosphate buffered saline (pbs) with pmol each of synthetic '-o-methylated mir , mir a and c ; and c) ml pbs with pmol each of synthetic mir , mir a and c without the '-o-methylation. time course analysis during both gastric and intestinal phase digestion showed that most mirnas displayed modest resistance in the acidic gastric environment (up to min), while in the intestinal phase the levels of all mirnas were drastically reduced after five minutes (figure ) . regardless of synthesis origin and '-o-methylation, the digestive stability of mir was up to -fold higher than that of the other mirnas. the most stable form of mir was the plant-derived form in the cabbage extract, since . % survived, compared to . % for the '-o-methylated form, and . % for the non-modified form. similar results were observed when pmol of synthetic '-o-methylated mir , mir a and c were digested in vitro using the ex vivo intestinal fluids ( figure ) . indeed, mir appeared to be more stable after -hour digestion than the other tested mirnas. finally, by directly measuring mir levels in vivo in the small intestines of mice fed the plant-based diet (daily dietary mir intake pmol) it was observed that this particular mirna reached level more than -fold higher than those observed in the animals consuming a single pmol dose of the synthetic mir . mir a, a mirna also present in the plant-based diets, was also detected in the small intestines at a level times lower than that of mir . using transgenic arabidopsis lines expressing the artificial mirna amir-rice, the murine mirna mmu-mir- a or their controls, yang et al. studied gastrointestinal digestion and bioavailability of transgenic mirnas (yang et al., b) . levels of transgenic mirnas in plants (qrt-pcr quantification) were similar ( . and . fmol/g of fresh weight for amir-rice and mmu-mir- a, respectively) to that of srna mir ( - fmol/g in fresh shoot tissue). however, in diets stored at room temperature, the abundance of amir-rice and mmu-mir- a decreased gradually by ≈ -fold (≈ fmol/g diet) while that of the srna mir increased ≈ fold after h. in vitro gastric and intestinal simulated digestion of the synthetic forms of the plantbased transgenic mirnas showed that amir-rice and mir had similar digestive stability, while mmu-mir- a was significantly less stable. the surviving percentage in the intestinal phase after min was . % for mmu-mir- a compared to ≈ . % for mir or amir-rice (figure , frame a). the surviving percentage in the in vivo assays (gavage-fed pmol of synthetic srnas) further decreased the stability of the mirnas, reducing their level to . % for amir-rice and . % for mir (figure , frame b) . however, the levels of mirnas in the small intestines were higher for mir ( fmol/mouse) than amir-rice ( . fmol/mouse), while mmu-mir- a levels were undistinguishable from the host mouse mirna (figure , frame c). amir-rice was not found in serum (neither are mirnas reported in previous studies and present in transgenic plants) while mir was found only in the serum ( . fm, or . x copies per mouse) of mice fed the plant transgenic diets (yang, j. et al., b) www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. altogether, these results suggest that the plant-derived mir might be more stable than other plantbased small rnas. whether this exceptional stability could be generalized to other ncrnas is unknown. however, recent evidence, including some studies evaluating mir and mir , suggests that there may be some misidentified alternative mirnas . indeed, several other rarer but consistently mapped plant mirnas also have % or near % matches to human transcripts or genomic sequences (i.e. rrna), and some do not map to plant genomes at all, suggesting artefactual results of plant mirnas in mammalian sequencing datasets. this emphasizes the need for rigorous filtering strategies when assessing possible dietary exogenous mirnas . indeed, the small rna mir is derived from s rrna and does not undergo canonical mirna processing, as it does not depend on dcl (yang, j. et al., a) . mir is also inefficiently assembled into the risc complex in human cells and modestly regulates gene expression. this suggests that this exogenous srna is different from the canonical plant-based mirnas (yang, et al., b) . these results highlight the need to evaluate other ncrnas as they could originate from precise processing at the ' or ' end of mature or precursor rnas generating other abundant small rnas (lee et al., ) . also, the rna degradome is a crucial component of the total cellular rna pool of plants (nowacka et al., ) . they can be stable degradation intermediates present in a high copy number, or they can derive from various rna species including trna, rrna, mrna and snrna (addo-quaye et al., ; nowacka et al., ) . among the few examples in the literature of possible biological effects of exogenous non-plant origin ncrnas, milk has been studied because it constitutes a rich source of secreted mirnas. human breast milk is an essential source of nutrition for new-borns. in addition to its nutritional function, breast milk contains myriad biologically-active components that influence the development of the the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. more than % of the unique mirnas identified in porcine milk exosomes (gu et al., ) or % in other studies (chen et al., ) . in human exosomes, the ten most abundant mirnas account for % of the unique mirnas identified . lncrnas previously reported as being important for developmental processes were found in human breast milk extracellular vesicles (karlsson et al., ) . maternal factors such as the mother's diet seem to influence mirna expression in the breast milk fat globule (munch et al., ) or exosomes (sun j, ) . maternal weight (xi et al., ) also seems to influence mirna expression. certain mirnas found in breast milk have also been found in placenta tissue (munch et al., ) , although their physiological role is unknown. other studies have shown that colostrum mirnas (early lactation period) are more abundant than in the later lactation period (gu et al., ; xi et al., ; sun et al., ) . bovine milk mirnas isolated from the colostrum whey fraction were more abundant than in the mature milk whey fraction (izumi et al., ) . immunerelated mirnas expression is reported to be higher in colostrum than mature milk (izumi et al., ; na et al., ) . in vitro studies of human milk mirnas using either rnase digestion, freeze-thaw cycles, low ph or other gi conditions have shown that these mirnas are relatively highly stable (kosaka et al., ; liao et al., ) . similarly high stability of mirnas was found when using one or all of the above mentioned hard conditions for milk from cow (hata et al., ; izumi et al., ), pig (gu et al., or panda . indeed, it has been proposed that rna in milk is present in microvesicles and is protected from rnases and the other gi conditions by surrounding membranes (hata et al., ; gu et al., ; . however, technological conditions such as pasteurization (golan-gerstl et al., ) have been shown to reduce mirnas abundance in cow and goat milk by up to ≈ %, or up to ≈ % for certain mirnas (howard et al., ) , and in human exosome (personal communication, faseb conference ). microwave heating also reduces the amount of certain mirnas (howard et al., ) , while ultrasonication influences exosome membrane integrity and thus increases cargo instability (sun et al., ) . among dairy products, mirnas concentrations varied considerably, but were generally lower than the concentration in pasteurized whole milk (howard et al., ) . the exception was fresh cheese ("queso fresco") dip, which contained higher concentrations of mirnas than observed in pasteurized milk (table ). in vitro studies using mirnas isolated from breast milk exosomes, either human or bovine, were found (liao et al., ) , and crl , k or lim (golan-gerstl et al., ) . when subjected to proteinase k treatment or when the vesicle structure was destroyed, uptake or transport of these exosomal mirnas was compromised (kusuma et al., ; wolf et al., ; sun et al., ) . different studies have attempted to quantify the amount of rnas present in breast milk. rna content in microvesicles isolated from cow milk has been estimated to be ≈ ng for colostrum and ≈ for mature milk when isolated from ml sample (hata et al., ) . in human milk, total rna was estimated to be ≈ ng/ cells of the milk cell fraction and ≈ ng/µl fat from milk fat (alsaweed et al., c) . exogenous plant mirnas have also been found in panda milk exosomes , the whole milk or exosomes of human breast milk the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. et al., ). however, levels of mirnas or total rnas were very low (golan-gerstl et al., ; alsaweed et al., c) compared to that of breast milk. indeed, alsaweed et al. reported that the total amount of rna found in bovine milk-based infant formula was ≈ . ng/µl formula ( human mirnas detected) as compared to . ng/µl for a soy-based formula ( human mirnas detected) (alsaweed et al., c). in terms of possible breast milk mirna absorption, plasma levels of mirnas were found to be higher in colostrum-fed piglets compared to mature milk-fed piglets (gu et al., ) . in humans, plasma mirnas (mir- b and mir- c) increased when different doses of cow milk were consumed (baier et al., ) . in contrast to the studies above, the literature also describes several studies that show either different or contradictory results regarding uptake of exogenous rnas from breast milk. auerbach et al. re-analysed a study in which mir- b- p and mir- c- p were found in plasma after bovine milk ingestion by healthy humans (baier et al., ) and found no significant altered mirna levels after milk ingestion (auerbach et al., ) . these results were confirmed by qpcr and rna-sequencing. several technical issues including low mir- b expression, use of control mirnas (normalization), and variation level may have contributed to these discrepancies (auerbach et al., ) . piglets fed cow milk for weeks showed very low levels of cow-specific mirnas in the bloodstream, which were compared to the levels measured from animals fed maize, which also showed similar counts of cow-specific sequences , suggesting a lack of transfer of exogenous small rnas. using a transgenic mouse model that overexpresses mir- b precursor in the mammary epithelial cells, and thus increases levels of mir- b in milk (≈ -fold), no differences were found in blood, liver, small intestine, kidney or lung tissues of pups from these dams when compared to pups from wild type dams (laubier et al., ) . interestingly, the level of mir- b was ≈ -fold higher in the stomach of mice consuming the transgenic milk. oral ingestion of . µg and . µg of total rna from porcine milk exosomes administered daily during three weeks to mice, significalty increased villus height and crypt depth of the duodenum and jejunum relative to the control group, suggesting an effect improving the development of the gi tract in mice . using genetic knock-out (ko) mouse models for mirnas mir- and mir- c/ , title et al., evaluated the uptake of maternal-milk derived mirnas using the foster mother offspring exchange model to prevent the confounding effects of mirnas derived from tissues of suckling offspring . wild type (wt) offspring consuming mir- ko milk at day exhibited basal levels of mir- in milk from the stomach, which likely comes from stomach epithelial cells. at day of suckling, it was confirmed that most mir- /mir- c came from the milk itself rather than the offspring www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. genotype, because there was no significant difference between wt pups and ko pups receiving wt milk. evaluating different parts of the gi tract (jejunum, ileum and colon), no evidence of uptake was observed because there was no measurable increase in mir- levels in enterocytes of ko pups receiving wt milk compared to ko milk. moreover, no changes in plasma, liver or spleen levels of mir- were observed in ko pups receiving wt or ko milk . similar effects were observed at day of treatment and upon evaluating mir- c on day . when the fate of milkderived mirnas downstream of the stomach was analysed, the levels of mir- in the intestinal content of ko pups receiving wt milk was seen to dramatically decrease compared to that of the stomach contents. these data suggest that milk mirnas are being degraded by the digestive system (since no evidence of uptake was observed). indeed, when the spike in cell-mir- was tested, it was degraded more rapidly than milk mir- , suggesting that milk mirnas exhibited a certain degree of resistance (perhaps because of their exosome content). but less than % of mir- copies remained after h incubation of intestinal content, suggesting a possible digestive enzymatic degradation of the milk-derived (exosomes) mirnas . observational studies also suggest that human breast milk mirnas are not related to preventing atopic dermatitis in infancy months postpartum . in general, rna contamination has been proposed as the main source of controversy in mirnas studies reported in the field of breast milk (bagci and allmer, ) . the literature contains very few studies on the biological effects of dietary exogenous ncrnas in the gi tract and its annex glands. indeed, these few studies only report a biological effect in the liver. the available literature suggests that dietary exogenous plant small ncrnas seem to resist the harsh conditions of the gi tract. however, the stability of plant mirnas under gi conditions in vitro, ex vivo or in vivo is low. moreover, these studies suggest that the percentage of surviving mirnas in the gi tract in the best-case scenario is around ≈ %, although this depends on the specific small ncrnas evaluated. several studies report the presence of dietary exogenous mirnas in breast milk of different mammals, including humans. some protected mirnas (i.e. transported in extracelluar vesicles) have been found to be stable under in vitro degradative conditions. while some evidence suggests a possible absortion through oral feeding, other studies indicates a very limited biovailability or lack of accumulation within the gi tract or its annex glands. recent evidence supports the significant contribution of srnas to communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. this silencing transfer phenomenon is very relevant to food and feed risk assessment of ncrna gm plants. although this section (efsa task ) will focus on studies related to the possible trafficking of ncrnas between plants and humans and animals, studies supporting the movement of rna-silencing signals between plants and pathogens are also reviewed. virus-induced gene silencing represents the model of gene silencing in the host by exogenous sirnas. in this case, the sirna specificity determinant is derived from viral rnas. typically, with a discrete length of nt they form perfect duplexes which are produced in plants by dcl and dcl . virusinduced gene silencing is a very effective defence system, and, consistently with the normal dynamics of host-pathogen interactions, all viruses encode silencing suppressors as a counter defence (baulcombe, ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. cross-kingdom rnai implies that a translocation of gene silencing signals occurs between hosts and these organisms. this implies a two-way traffic of sirnas between pathogens and their plant hosts. several studies report that rnas produced in a host plant can be transferred to a fungus or oomycete, symbiont or pathogen, to induce rna silencing in the interacting microorganism (ghag et al., ; koch et al., ; vega-arreguín et al., ; helber et al., ) . transgenic plants carrying a rnai construct, usually with a sense-intron-antisense palindromic structure that produces dsrnas and sirnas, can induce silencing of the targeted transcripts in the interacting organism. furthermore, treatment of fungal conidia with dsrnas of essential genes led to fungal growth inhibition, demonstrating rna interference from environmental silencing signals (koch et al., ) . conversely, the rna produced in a fungus can affect a host plant's defence system (weiberg et al., ) . remarkably, scientists have developed an effective disease control strategy, called host-induced gene silencing (higs), by generating transgenic plants that express exogenous rnai triggers to successfully silence essential genes in pathogens and pests (weiberg et al., ) . in this context, transgenic plants expressing dsrnas or hairpin rnas targeting vital fungal genes of fusarium sp. developed resistance to these important phytopathogens (ghag et al., ; koch et al., ) . small rnas or dsrnas can also be transferred from plant to pests, such as insects eating leaves or nematodes infecting roots. indeed, transgenic plants that express dsrna homologous to essential genes of insect pests or nematodes became resistant to these specific parasites through the silencing activity produced within the target organism when srnas expressed from the plant transgenes is consumed (baum et al., ; fairbairn et al., ; mao et al., ) . many aspects of these cross-kingdom rna interference phenomena are still poorly understood. one of them is how these rna molecules 'travel,' sometimes over long distances through diverse cellular boundaries between plants and interacting organisms. these silencing signals may utilize conserved cell-to-cell as well as systemic rnai pathways present in plants and animals, and may also use organismspecific pathways. rna-protective factors such as agos, other rna-binding proteins, or encapsulation into extracellular vesicles likely play important roles in protecting mobile rnas against degradation during transport (weiberg et al., ; lefebvre and lecuyer, ) . another important question is how these srnas use the target cell rna interference machinery to convey the silencing effect. based on current knowledge, rna-mediated gene silencing seems to be an ubiquitous phenomenon that exists in almost all eukaryotes, and which always follows the principle of complementary nucleotide base-pairing between regulatory srna and mrna sequences (weiberg et al., ) . despite the tremendous differences present in the structural features of regulatory rnas, and the completely unrelated or highly divergent rna gene-silencing mechanisms and pathways that have evolved in diverse organisms, complementary sequence matches seem to be sufficient enough to trigger cross-kingdom gene silencing, as exemplified by plants-parasites, bacteria-to-worms and other lower organism interactions (weiberg et al., ) . following a literature search as described in section . . . and based on the methodology described in the section . . . , a total of documents were selected for review of the topic molecular mechanisms of exogenous ncrna uptake and function. the uptake of exogenous ncrnas is described from the molecular mechanisms point of view (i.e. the presence of a specific transporter, if any). efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. although plant-derived exogenous ncrnas are the main topic of this subsection, some other examples of general molecular mechanisms of exogenous ncrnas uptake, intracellular trafficking and function are included as relevant to understanding the possible fate of plant-derived exogenous ncrnas. other general aspects of rnas uptake are reviewed in chapter . . , and the uptake of exogenous rnas from the pharmaceutical/medicinal area is covered in section . . . as previously mentioned in section . . , cellular uptake of ncrnas presents great challenges due to the physicochemical properties of these molecules. their size and negative charges prevent their easy entry into mammalian cells. cell membranes pose a major barrier to ncrnas entrance since entry into cells is tightly controlled and regulated (mcerlean et al., ) . the anionic lipophilic bilayer prevents entry of macromolecular anionic nucleic acids in their naked form, both restricting their binding to and passive diffusion across these lipophilic cell membranes . furthermore, even though their internalization may depend on the endocytic pathway, endosomal entrapment and lysosomal degradation can be major issues because of the reduced accessibility of ncrnas to their sites of action (nucleus, cytoplasm or mitochondria) (won et al., ; . many different carrier molecules have been developed to achieve the entrance of ncrnas (mostly mirnas and dsrnas) into target cells (bolhassani, ; lindgren and langel, ; mao et al., ; ragelle et al., ; rudzinski and aminabhavi, ; shum and rossi, ) . exogenous ncrnas must be carried by polymers, synthetic cationic lipids or cell-penetrating peptides neutralizing the negative charges of the nucleic acids. most of these cationic lipids tend to form liposomes when dispersed in an aqueous phase, such as blood. endocytosis has been suggested as the main pathway for this nucleic acids-cationic lipid complex internalization by the cell (el ouahabi et al., ) . most reports suggest that so-called cell-penetrating peptides (cpps) bind initially to negatively-charged proteoglycans at the cell surface and are internalized into endosomes (juliano et al., ) . there are multiple pathways of endocytosis (see also section . . ). i. the clathrin-coated pit pathway is the archetype of endocytosis pathways. cell surface receptors and their associated ligands interact with adapter proteins and accessory factors, clustering the receptors into specialized membrane areas subtended by a network of clathrin triskelions. the clathrin network is invaginated by means of membrane curvature promoting specialized proteins, giving rise to a clathrin-coated endosome. this endosome quickly uncoats, generating an uncoated vesicle that will begin its intracellular journey (juliano et al., ) . ii. the caveolar pathway implies the presence of small cell membrane invaginations rich in cholesterol and sphingolipids containing caveolin . cavins, which coat proteins helping to stabilize caveolar structures, are present in these invaginations. there is controversy as to whether caveolae generate independent intracellular vesicles or whether they remain as tubular structures linked to the plasma membrane; however, some data suggest that caveolae generate vesicles able to participate in intracellular traffic, generally presenting a smaller size than other forms of endocytotic vesicles (juliano et al., ) . iii. a number of clathrin-and caveolin-independent pathways have been described. these pathways are often defined in terms of the morphologies of the vesicles they generate or in terms of the cargo that is preferentially internalized. for instance, the flotillin pathway involves the presence of flotillin-rich membrane microdomains, where flotillins are membrane-inserted proteins that may be involved in ordering lipid domains and subsequent endocytosis, similar efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to caveolin (juliano et al., ) . another such clathrin-and caveolin-independent pathway is the clic/geec pathway, which seems to be particularly important for fluid phase endocytosis. clic/geec stands for clathrin and dynamin independent carriers/gpi-ap enriched early endosomal compartments. this pathway gives rise to high-volume tubular endosomes rich in gpi-proteins and that typically contain fluid phase markers such as dextrans (juliano et al., ) . additional clathrin-and caveolin-independent pathways exist, some involving dynaminmediated disjunction of vesicles from the plasma membrane (juliano et al., ) . iv. macropinocytosis is a process by which cell protrusions pinch off large volumes of extracellular fluid and is therefore an important pathway in fluid phase endocytosis. it is also involved in internalization of clustered, activated receptor tyrosine kinases. v. phagocytosis and entosis are large-volume internalization mechanisms as well. they come into play in specialized cells or unusual circumstances but do not play much of a role in oligonucleotides processing in most cell types (juliano et al., ) . vi. the actin cytoskeleton plays an important role in most of the endocytotic processes described above, although certain arenaviruses enter cells by a pathway independent of clathrin, caveolin, dynamin and actin. it has been reported that phosphorothioate antisense oligonucleotides seem to enter the cells by this pathway as well (juliano et al., ) . there have been many attempts to identify endogenous receptors for antisense or sirna molecules. however, no direct evidence for their involvement in oligonucleotide trafficking has been provided (juliano et al., ) . integrins of the beta- subclass as well as scavenger receptors have been suggested as candidates, but this is controversial. toll-like receptor (tlr) family members seem to be the most convincing examples of cellular receptors for oligonucleotides; tlr binds dna having cpg motifs, tlrs / binds single-stranded rna, while tlr binds double-stranded rna. although these tlrs are usually found within endosomes rather than at the cell surface, in some cases they seem to be able to assist in accumulation of oligonucleotides by cells (juliano et al., ). one interesting candidate as a receptor for oligonucleotides is the mammalian homolog of the doublestranded rna (dsrna) transport protein sid- found in caenorhabditis elegans (feinberg and hunter, ) . the human homolog of this protein, sidt , has been described as facilitating rapid contactdependent intercellular small rna transfer (elhassan et al., ) and as selectively binding long doublestranded rna (li, w et al., ) . another member of the sid transmembrane family, sidt , has been shown to take up extracellular double-stranded rna in drosophila s cells (mcewan et al., ) via endocytosis, although in mammalian cells this protein has been located in lysosomal membranes (jialin et al., ) . in fact, some authors have proposed that sidt could mediate cellular rna degradation inside lysosomes through a novel type of autophagy called rnautophagy (aizawa et al., ) . another research group recently reported that sidt is required to transport internalized dsrna from endocytic compartments into the cytoplasm for immune activation (nguyen et al., ), while others have described that this protein mediates "gymnosis", facilitating uptake of naked single-stranded oligonucleotides into living cells (takahashi et al., ) . a more recent report indicates that both sidt and sidt not only do not transport rna, but are involved in cholesterol transport (mendez-acevedo et al., ) . once an oligonucleotide has entered a cell in an endosome, it encounters a complex maze of intracellular pathways leading to multiple destinations and regulated by an intricate protein machinery. key subcellular membrane bound compartments include early and recycling endosomes, late www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endosomes/multi-vesicular bodies, lysosomes, the golgi apparatus and the endoplasmic reticulum. intracellular trafficking is not a random process, but rather a carefully orchestrated choreography that allows the cells to transport endogenous and exogenous materials to the most appropriate places. for nucleotides to exert their function, they need to leave the membrane bound compartments and access the cytosol and/or nucleus (juliano et al., ) . knowledge as to how endocytotic cargos are delivered to subcellular compartments is still partial. many of the internalization pathways previously described converge at the stage of early endosomes, raising the question of how the differentially internalized components traffic to different destinations. certain evidences suggest that membrane domains originating from different internalization pathways maintain their identity within early endosomes, thus providing the means for specific sorting and trafficking to distinct downstream destinations (juliano et al., ) . all membrane traffic proceeds through the same basic steps: i) a coated vesicle is pinched off from a larger donor membrane compartment; ii) the vesicle uncoats, allowing display of the tethering and fusion proteins; iii) the vesicle is carried to its destination along "tracks" provided by actin-or tubulin-based cytoskeletal structures; iv) the vesicle recognizes its target membrane compartment using tethering proteins and then utilizes snare proteins to complete the fusion process and deliver membrane and contents to the target compartment (juliano et al., ) (figure ). ncrnas must escape from the endomembrane compartments and reach the cytosol to exert their function. as mentioned above, intracellular trafficking involves a highly dynamic flux of membrane vesicles engaged in a multitude of fusion and disjunction events. there are a few key points in these processes to be considered when designing non-viral oligonucleotide delivery strategies: ) fusion involves localized stress on the fusion partners, including formation of non-bilayer lipid domains; ) non-bilayer regions of membranes can be much leakier than bilayer regions; ) many enveloped viruses fuse with cells via specialized membrane interacting proteins that, while differing in sequence, act in a manner similar to cellular snare proteins; in many cases these proteins can also induce increments in membrane permeability. therefore, there is an intrinsic relationship between the fusion events inherent to intracellular trafficking and transient leakage of vesicular contents. thus, the innate activity of oligonucleotides taken up by cells is likely due to a modest amount of continuous leakage from endomembrane compartments spontaneously occurring during intracellular trafficking (juliano et al., ) . some ncrnas function is exerted in the nucleus, although nuclear entry may not be the rate-limiting step for oligonucleotide action. studies have shown that oligonucleotides, particularly those with phosphorothioate backbones, are able to continuously shuttle between the nucleus and cytoplasm. this is an active process mediated by nuclear pore structures but does not require classic nuclear localization signals. for phosphodiester oligonucleotides, both passive diffusion and active transport have been described as nuclear entry mechanisms (juliano et al., ) . in terms of the uptake and trafficking of "free" or "naked" oligonucleotides, the co-existence of both productive and non-productive uptake routes has been suggested. the non-productive pathway seems to involve trafficking to lysosomes, while the pathway that results in rnase h-dependent antisense effects eventually leads to interaction with cellular pre-mrna (koller et al., ) . other studies have involved so-called "gymnotic" uptake of antisense oligonucleotides modified with lna (locked nucleic acid) moieties (stein et al., ; zhang et al., ) . subcellular distribution studies surprisingly suggested that the deoxy lna compounds became associated with p-bodies that are usually thought to be sirna action sites (stein et al., ) . unlike the study performed with phosphorothioate www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. oligonucleotides in which the concentrations used were in the nanomolar range, the antisense effects of "naked" lna required micromolar concentrations (juliano et al., ). as described in section . . , exogenous ncrnas must overcome a series of barriers to be functional at systemic level. in addition to the extracellular barriers specifically related to oral absorption, defensive humoral and cellular barriers efficiently prevent the intrusion of exogenous entities into the organism (jeong et al., ) . systemically-administered nucleic acids are rapidly degraded by nucleases in a few minutes and, even in nucleic acid drug therapy using positively-charged polyplexes, they readily interact with serum components to form larger aggregates, resulting in rapid clearance by res or phagocytes (jeong et al., ) . if the nucleic acids survive in the blood stream, they must still reach the appropriate tissues and enter the target cells. in systemic nucleic acid therapy, continuous endothelial walls in the microvasculatures are one of the main barriers limiting the access of oligonucleotides to target cells. in certain pathological cases, such as cancer, the presence of leaky vasculatures in the vicinity of highly penetrating solid tumours allows nanoparticulates to penetrate and accumulate in tumours due to the enhanced permeation and retention effect (jeong et al., ) . caveolae-mediated transcytosis has been considered one of the important mechanisms of macromolecules transport across the endothelium (schnitzer, ) . another possible limiting barrier is the extracellular matrix consisting of various proteoglycans, which are proteins covalently cross-linked with carboxylic or sulphated glycosaminoglycans (gags) (ruponen et al., ) . since gags, such as heparin sulphate, chondroitin sulphate and hyaluronic acid, are polyanions, they may repel the negatively-charged oligonucleotides, affecting mobility of the nucleic acids in the tissue extracellular matrix and therefore limiting their access to target cells. in the case that oligonucleotides reach the target cell membrane, they must enter the cell. the first barrier is the anionic plasma membrane itself, and the second is cellular uptake via endocytosis (see section . . ). without a specific ligand-receptor interaction mechanism, the entrance of ncrnas results in a lack of cell specificity (jeong et al., ) . once taken up by cells, the nucleic acids are localized within the endosomal compartments, where the ph rapidly drops to about by the action of membrane-bound atp-driven proton pumps. the endosomes mature to lysosomes where the oligonucleotides can be degraded by various enzymes (jeong et al., ) . as previously indicated, any surviving nucleic acids must then escape from the endosome to exert their functional effects. in nucleic acid drug therapy, certain endosomal disruptive agents, such as lysomotropic chloroquine, fusogenic peptides and ph-sensitive neutral lipid, are used to facilitate or promote endosomal escape through membrane disruption (jeong et al., ) . after endosomal escape, ncrnas should move through the cytoplasm to encounter their molecular targets, or to the peri-nuclear space where nuclear translocation takes place if the nucleus is the target compartment. passive diffusion of high-molecular weight macromolecules is limited in the cytoplasm. a complex network of microfilaments and microtubules, highly concentrated proteins and various subcellular organelles provide the cytoplasm with a high fluid phase viscosity and mesh-like structure with to Å pores from, making the diffusion process size-dependent (jeong et al., ) . in fact, microinjection of plasmid dna into the cytoplasm showed that normal dna was practically immobile in the cytoplasmic space (dowty et al., ) . however, dna molecules with less than base pairs seem to undergo free diffusion in the cytoplasm (lukacs et al., ) , suggesting that small oligonucleotides (i.e. mirnas) do not face limitations regarding their intracellular trafficking, contrary to large ncrnas (i.e. lncrnas) which might encounter more restrictions reaching the peri-nuclear space. finally, some ncrnas need to enter the nucleus to be functional. the nuclear envelope consists of a double-layer membrane with nuclear pores. the nuclear pore complex allows free diffusion of ions and small molecules, but restricts passage of macromolecules with molecular efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. weights greater than kda, unless accompanied by a nuclear localization signal peptide (nls) (jeong et al., ) , which is not the case with lncrnas oligonucleotides are initially accumulated in an endomembrane compartment (the donor compartment, e.g. early endosomes) and are then trafficked by shuttle vesicles to various other endomembrane compartments (the recipient compartment, e.g. the trans-golgi). the first step ( ) involves disjunction ('pinching off') of a shuttle vesicle under the influence of a coat protein as well as other accessory proteins. at this stage there are non-bilayer regions at the junction between the membranes of the donor compartment and the shuttle vesicle. this provides an opportunity for some oligonucleotides to escape into the cytosol. step involves uncoating of the vesicle; and rab proteins can contribute to this step. step comprises movement of the shuttle vesicle toward its destination along cytoskeletal tracks. motor proteins such as various myosins (for the actin system) or dyneins or kinesins (for the microtubular system) propel the vesicle. rab proteins are involved in forming the appropriate linkages to the cytoskeleton. step entails recognition of the recipient ('target') compartment by the shuttle vesicle. tether proteins work with rab proteins to provide interaction specificity while v-snare proteins in the vesicle membrane interact with t-snare proteins in the recipient compartment membrane to provide firm bridging, as well as contributing to specificity. in step the snare proteins undergo major conformational changes, and, with the assistance of accessory proteins, trigger fusion of the shuttle vesicle membrane with the membrane of the recipient compartment. at this stage non-bilayer regions exist at the junction between shuttle and recipient membranes potentially allowing oligonucleotide escape. reprint with permission from (r l juliano, x ming, o nakagawa. cellular uptake and intracellular trafficking of antisense and sirna oligonucleotides. bioconjugate chemistry : - ). copyrigth ( ) american chemical society. figure : proposed mechanism of vesicular trafficking of oligonucleotides some research has been done on cellular uptake, trafficking and tissue distribution of oligonucleotides without specific targeting or carrier mechanisms. in terms of tissue distribution, certain cell types exhibit a preferential in vivo uptake, particularly kidney proximal tubule cells and liver kupffer cells for phosphorothioate (ps) antisense compounds and sirna (juliano et al., ) . however, only one report has studied systemic level uptake and function of orally administered mirnas . these authors studied intestinal uptake of maternal milk-derived mirnas by new-born mice employing genetically-modified models to distinguish endogenous mirnas from milk-derived exogenous mirnas. their analysis of the intestinal epithelium, blood, liver and spleen revealed no evidence for mirna www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. uptake, even though milk in lactating mothers has been shown to be a particularly rich source of secreted mirnas. sequencing and microarray analysis of mirnas in the milk of various mammalian species has led to the discovery of hundreds of mirnas in cow, pig, rat and human milk, which are derived from exosomes and cellular components. furthermore, their study revealed rapid degradation of milk mirna in intestinal fluid, indicating that mirnas in the milk play a nutritional rather than a generegulatory role in new-born mice . there are certain tissues containing special physical structures that can function as additional barriers to ncrnas entry. such is the case of the central nervous system (cns), where delivery of nucleic acids is particularly challenging because of its anatomical and physiological complexities. the cns is protected by the blood-brain barrier (bbb), which consists of tightly joined capillary endothelial cells, restricting access of large molecules into the brain. the cns extends to include the spinal cord, which is protected by the blood-cerebrospinal fluid barrier (bcsfb), made up of choroid plexus epithelial cells limiting the free diffusion of molecules into the cerebrospinal fluid (csf). the bbb and the bcsfb express numerous transporters and receptors which contribute to the transfer of essential nutrients such as glucose and amino acids into the cns. within the cns, different cell types exist including neurons, astrocytes and other glial cells. entry of nucleic acids into neurons is notoriously difficult for unclear reasons, although it is likely related to their post-mitotic nature, as well as the complex structures and intricacies of neuronal networking (o'mahony et al., ) . for instance, variances in the uptake of sirna lipoplexes (liposome and nucleic acid complexes) were reported at different parts of the neuron structure, with greater uptake efficiency at the cell soma compared to the neuritis, which may occur because of different membrane compositions in these domains (o'mahony et al., ) . circumventing the bbb is only possible by direct administration of ncrnas to a target region of the brain or by administration to the spinal cord where trans-synaptic retrograde transport to the brain is possible. some delivery systems exploit the receptor-mediated uptake of molecules such as transferrin, lactoferrin and insulin receptors by attaching a ligand for the receptor to the non-viral delivery system, or promoting a transient mechanical disruption of the bbb (o' mahony et al., ) . in certain neurological diseases, and in particular in brain cancers, anatomical and physiological changes occur that can impact bbb integrity and the cns accessibility. delivery to the csf to bypass the bbb presents its own obstacles, including rapid clearance from the csf, the bcsfb and limited diffusion from the csf into the brain parenchyma (o'mahony et al., ) (figure ) . another specialized barrier is the placenta. the placenta serves as the interface between the maternal and foetal circulatory systems, and regulates the transfer of oxygen, nutrients and waste products. when exogenous substances are present in the maternal bloodstream, the extent to which these affect the foetus is determined by transport and biotransformation processes in the placental barrier. this barrier is formed by fusion of the outer blastocyst trophoblast cells facing the uterine epithelium into multinucleated syncytiotrophoblast. proliferation of syncytiotrophoblast is mediated by the fusion of precursor cytotrophoblast cells. maternal blood in the intervillous space and foetal blood in the villous capillaries are separated by a continuous layer of syncytiotrophoblast, a discontinuous layer of cytotrophoblast cells, basal lamina, connective tissue, and foetal endothelial cells (al-enazy et al., ) . the placenta undergoes several changes as pregnancy progresses. overall, the placental barrier becomes thinner over time, the distance separating the maternal circulation from the foetal circulation decreasing from - µm in the second month to only - µm at term. passive diffusion across the placenta is favoured for lipophilic substances, allowing them to cross the phospholipid bilayer. smaller compounds tend to cross the placenta more readily, with compounds having a molecular weight less than da crossing it most easily (al-enazy et al., ) . since nucleic acids are polarized, negatively charged molecules, it is unlikely they would be able to cross the placenta by passive diffusion. nevertheless, the placental syncytiotrophoblast is capable of efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. endocytosis (al-enazy et al., ) . no reports refer to exogenous ncrnas entering the placental barrier. however, the presence of placental mirnas has been described in the blood circulation of pregnant women. these mirnas have been reported to be actively secreted into microvesicles and exosomes. these specific exosomal mirnas may be important for embryo implantation, playing a significant role in the intercellular communication pathways that potentially contribute to placentation and development of maternal-foetal vascular exchange (mitchell et al., ) . since exosomes can cross the blood brain barrier, meaning they can cross several layers of cells, it has been speculated that exosomes could directly move from maternal to foetal tissues, and vice-versa, during pregnancy (record, ). the the molecular mechanisms of exogenous ncrna cellular uptake have been inferred from studies performed when developing strategies for nucleic acid delivery in therapeutics. few studies have been done with "naked" oligonucleotides, and even in these cases, the oligonucleotides were chemically modified. these molecular mechanisms of cellular uptake include the different types of endocytosis mechanisms known to date. the existence of receptor-mediated uptake of oligonucleotides through the sidt and sidt proteins has also been described, although their function has been contradicted since they have been described as cholesterol transporters. among the numerous challenges ncrnas must overcome once taken-up by the cell, escaping the endosomal compartments is the most relevant and significant, although reaching their site of action is also crucial to exerting their function. specialized barriers such as the blood-brain barrier or the placental barrier present further obstacles to overcome. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. zhang y, qu z, kim s, shi v, liao b, kraft p, et al. . down-modulation of cancer targets using locked nucleic acid (lna)-based antisense oligonucleotides without transfection. gene ther , - . zhou g, zhou y, chen x. following a literature search as described in section . . . . and based on the methodology in section . . . ., a total documents were selected for reviewing the landscape of exogenous rnas in biological fluids of human and animals. using public databases of rna sequencing datasets, several documents report the application of in silico bioinformatics analysis (table ) clearly describing the widespread presence of exogenous ncrnas in biological fluids(from multiple sources, including diet) wang et al., ; beatty et al., ; yeri et al., ; . however, four documents suggest that exogenous ncrnas present in the public datasets may represent contamination from the laboratory environment since their abundance is extremely low, or they are of non-dietary origin, or could originate from technical artefacts or contamination bagci and allmer, ) . five documents report the development of bioinformatics resources to either predict transportable mirnas, predict functional analysis, or list and compare the exogenous mirnas found in samples chiang et al., ; zhang et al., ; zheng et al., ) . the generalized use of highly parallelized next generation (nexgen) sequencing technologies has promoted the use of sequencing to study complex biological systems, including human and animal biological fluids and tissues. initial studies evaluating the possible presence of exogenous ncrnas in biological fluids were done in silico using public databases of rna-seq results. several studies report the presence of exogenous rnas (i.e. dietary plant ncrnas) in the biological fluids of humans and animals. initial studies of the srnas spectra in human plasma showed that a small fraction of sequence reads from plasma mapped to human mirnas (≈ . %) or human transcripts and human genome sequences (≈ %) . this fraction increased by up to ≈ % when a higher sequence mismatch tolerance was allowed (under two mismatch allowance). surprisingly, a significant number of the unmapped reads aligned with various bacterial and fungal sequences (i.e. ribosomal rnas and trnas). also, many processed reads mapped to common food items, the most abundant being rna sequences from corn (zea mays) and rice (oryza sativa). compared to data from a serum sample from a chinese individual, the sequence abundance between corn and rice was reversed , suggesting the influence of dietary habits. other sequences from other foods in human plasma samples included soybean (glycine max), tomato (solanum lycopersicum), grape (vitis vinifera) and others. including exogenous mirnas, various common household insects were identified . treating plasma samples with dnase, protease, triton x- , and additional rnase reduced the total number of reads compared to the addition of rnase alone, suggesting that some of the exogenous rna molecules are probably associated to circulating protein and/or lipid complexes . in the same study, a lower percentage of exogenous sequences was observed in mouse plasma samples compared to human samples . another study evaluating a larger number of human plasma samples (n= young male athletes) reported exogenous srnas from different bacteria, but very few srnas were consistently detected in all samples (yeri et al., ) . in a study on public srna datasets from various tissues from mammals, chicken and insects, plant mirnas were found in out of datasets analysed, with mir- being extremely over-represented efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. . in datasets ( humans, mice, pig and others) the plant mirnas reads ranged from ≈ . - . % of the total animal mirna reads. insects fed with plant foods containing mir- , did not show accumulation of mir- when evaluated by northern-blot. however, in a very few samples sequencing data detected plant mirnas not present in the food source, including mir- , in addition to mir- at a moderate read, suggesting that plant mirnas observed in some public srna databases may be artefactual and due to sequencing methodology . plant mir- a from monocots was also found to be the most abundant exogenous plant mirna, in most cases, in other srna sequencing datasets studies with levels typically < % of human mirnas . but when analysis was performed in the absence of any known sources of plant contamination, plant mirnas were not detected. this suggests that precaution should be taken to prevent cross-contamination of the samples due to the high sensitivity of next-generation sequencing methods . other human public srna sequencing datasets have also been questioned for the presence of exogenous plant mirnas . of analysed human plasma samples, plant mirnas were found when compared to the genome of different plant model organisms (arabidopsis thaliana, triticum aestivum, oryza sativa, zea mays, and brachypodium distachyon). at least one read was present in selected samples. peu-mir- , found in all samples, is a singular mirna found in selected samples with more than counts. mir- is found in zea mays and conserved in fruits and vegetables including melon, sorghum, tomato, tea and oil palm. other highly-expressed plant mirnas found in up to hundreds of copies in selected samples include peu-mir- (found in samples), peu-mir- (found in samples), and tae-mir- (found in samples) . plant mir- (found in samples) and mir- a (found in samples) were detected in relatively small amounts (less than copies). using bioinformatics tools, the same study showed that seed sequences of mir- and mir- are similar to hsa-mir- and mir- - p for the former and has-mir- - p for the latter, and thus potentially target several gene targets of their human mirna counterparts . other studies have also reported in mouse plasma samples the presence of exogenous rnas matching exogenous species including bacteria, fungi, viridiplantae and food items . these included mir- d and mir- a (corn or rice as the most likely source), and mirnas from worms and insects . abundant non-human rnas were found in other healthy individuals human plasma samples (n= ) assigned to metazoan, bacteria or fungi (beatty et al., ) . several exogenous srnas were found that could potentially derive from food items including fragaria vesca, medicago truncatula, lotus japonicus, glycine max, arabidopsis thaliana, and solanum lycopersicum (beatty et al., ) . twelve public srna sequencing databases were analysed for the presence of plant exogenous mirnas. breast milk exosomes from humans ( samples, . million reads) and pigs ( samples, . million reads) were analysed. although plant mirna species belonging to mirna families ( samples) were found, they showed low abundance (< read counts for the highest expressed mirna). zma-mir- a, zma-mir- a, ath-mir- a, ath-mir- b and ptc-mir- d showed the highest abundance levels, while in humans only samples contained plant mirnas from mirna families (< read counts, for the highest expressed mirna, average of samples). ath-mir- a, pab-mir- , pc-mir- a, bdi-mir- , aly-mir- d, osa-mir- b. and zma-mir- a showed the highest abundance level in human samples . however, bagci and allmer reanalysed the data from lukasik and zielenkiewicz study and found that exogenous plant-derived mirnas in milk are likely to be contamination (bagci and allmer, ) . in a later observational study, lukasik et al. evaluated plant mirnas mir- a, mir- a, mir- a, mir- a and mir- a and reported the presence of plant mirnas in human (n= ) breast milk, both in whole milk and exosomes . out of mirnas, only were found in exosomes (mir- a and mir- a). while the human has-mir- a was found in all samples in a concentration range of . - pm for whole milk efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. and . - . pm in exosomes fractions, plant-derived mirna mir a was found in all samples at a concentration of - fm (≈ -fold higher than other plant mirnas) . compared to endogenous mirnas, the levels of exogenous plant mirnas were rather low and varied significantly between the samples . kang et al. analysed public sequencing srnas datasets from various human tissues and body fluids (n= ), including from serum and plasma samples, from exosomes, from cerebrospinal fluids, from liver, from blood cells and from brain . of note is that exogenous mirnas (xenomirs) were only found in % of the tissue samples (from total samples), with several studies being completely void of any xenomirs. xenomirs were absent from most human tissues samples, or present at very low abundances (with a median of . read counts). no enrichment of xenomirs was found in tissues relevant to dietary intake (i.e. hepatocytes and blood cells). by contrast, out of human body fluids datasets, xenomirs were found in % of samples, although at very low levels (median of read counts). even though the blood brain barrier separates the cerebrospinal fluid from the bloodstream, xenomirs were found in comparable quantities in both , suggesting no depletion (as expected by the presence of the barrier) of xenomirs in body fluids separated from the main bloodstream. moreover, the lack of co-occurrence of xenomirs in cerebrospinal fluid and serum of the same individuals, do not support that xenomirs detected in cerebrospinal fluid samples have entered through the bloodstream, and thus do not support a dietary origin of xenomirs. the most common xenomirs in body fluid samples belonged to the clades for rodents ( %), dicots ( %), insects ( %) and euphyllophytes ( %), suggesting that xenomirs composition does not reflect human food sources. from billion sequence reads (all samples), xenomirs were in low abundance, comprising only . % of the total reads ( , ). the same study reported a controlled feeding trial in rats and piglets (see section . . below for details). shu et al. ( ) used bioinformatics tools to characterize the structural characteristics of exogenous mirnas that can contribute to cross-species transportation and thus be transferred into human circulation . thirty-four thousand ( . ) mirnas sequences were compiled from species (from five kingdoms including animal, plantae, fungi, protista and viruses), including dietary mirnas ( mirnas) from types of common food species such as cow milk, breast milk, tomato, grape, and apple. using a support vector machine (smv)-based feature elimination strategy, features based on sequence, structure, and physicochemical properties were analysed to distinguish human circulating mirnas from the remaining sequences. predictions were made for features (categorized into groups) to better predict transportable mirnas in human circulation. known human plasma mirnas were ranked among the top of the list (dominated by animalia origin). only dietary mirnas were ranked among the top mirnas, five of which have a sequence identical to that in humans (bta-mir- b, bta-mir- , bta-mir- , gga-mir- a- p, gga-mir- b- p) . while only one mirna from plantae ranked among the top mirnas, plantae mirnas ranked in the top as possibly transportable , suggesting that animal-borne mirnas may be subject to more significant absorption in humans than in plant mirnas . it is important to note that this study of evaluated , animalia mirnas and plantae mirnas, whether this discrepancy in the original number of evaluated mirnas may have biased the smv-based classifiers to mammalian transportable mirnas was not addressed. in the same context, a database called "dietary mirna database" that describes different dietary mirna parameters and functional analysis has been described and is freely available. additional databases for exogenous mirna discovery using rna-seq data are also available in the scientific community . using bioinformatics tools (i.e. rnahybrid database), other studies have also predicted the potential function of dietary mirnas in the human body . in a bioinformatics study of plant mirnas, some similar functions between human and plant target genes were identified, such as ion transport and stress response . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analysing public srna sequencing datasets from humans ( ), mice ( ), pig ( ), chicken ( ), fly ( ), c. elegans ( ) and many other nematodes, protozoa or prokaryota, zheng et al. developed a database (exo-mirexplorer) for exploring and comparatively analysing exogenous mirnas (zheng et al., ) . two hundred and thirty-seven ( ) plant-derived mirnas were detected in sequencing samples. common mirna families included mir- , mir- , mir- , mir- and mir- . mir- a was the most frequently found mirna, and was detected in human samples from different studies (zheng et al., ) . the average abundance of mir- a was , . reads per million. in total, mir- a was found in samples from multiple species. mir- a is another frequently observed exogenous mirna from plants, and was detected in samples with an average abundance of . read per million (zheng et al., ) . regarding other biological fluids, exogenous rnas of bacterial origin were found in (urine) and (saliva) samples from young male athletes analysed for srna-seq (yeri et al., ) . plant small rna mir- was detected in urine of mice fed honeysuckle decoction (yang et al., b) . mir- a has been also detected in serum when honeysuckle was supplemented with pmol synthetic mir- a. mir- and mir- a were detected in the urine of mice that had received cisplatin treatment (cisplatin administration produces acute renal failure) and consumed a honeysuckle decoction alone or supplemented with synthetic mir- a. the increased detection of plant small rnas was attributed to disruption of the organization of small intestine epithelial cells produced by cisplatin treatment or longterm consumption (pre-fed) of honeysuckle (yang et al., b) , by mechanisms as yet unknown. mir- levels in urine reached ≈ fm. in tissues, the overall percentage of exogenous sequences in human lung tissue (normal lung rna samples), was found to be less than % . in spermatozoa samples, using public srna sequences datasets, tosar et al. found high amounts of exogenous sequences in three samples . however, in their own analysis of three spermatozoa samples they found no detectable traces of any plant mirnas, suggesting that exogenous mirnas present in the public datasets may have been caused by laboratory environment contamination . in terms of the possible passage of plant mirnas through the brain-blood barrier, kang et al. analysed samples from cerebrospinal fluid (csf) (from one study), which is separated from the bloodstream by the brain-blood barrier, and brain samples (from studies) . in general, xenomirs were absent from most of the human tissue samples, and, when present, they were found at very low abundance ( % of analysed tissues). although the brain-blood barrier would relatively protect the brain from dietary molecules, xenomirs were present in similar fractions in the exposed liver and in the relatively protected brain samples , suggesting, according to the authors, a nondietary origin . xenomirs were present in more body fluids samples (≈ % of samples) than tissue samples (≈ % of samples) and were identified in comparable proportions in the serum and cfs. this study does not provide support for the dietary origin of xenomirs (including plant exogenous rnas) meaning it may originate from technical artefacts . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. in addition to mirnas reported by the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. very few studies have reported the influence of dietary or physiological factors on levels of exogenous ncrnas in biological fluids. in a small study (n= per group), wang and colleagues compared some abundant exogenous mirnas species identified in plasma samples from healthy subjects, colorectal cancer and ulcerative colitis patients . although ulcerative colitis patients apparently had increased levels of circulating mirnas of certain species (i.e. from a bantam family), a large variability of mirnas levels were observed among subjects of the three groups , suggesting that health/disease status may influence the presence of circulating mirnas in human plasma. tissue injury produced by excessive consumption of common pharmaceutical drugs can also influence exogenous rnas levels in plasma . wang et al. showed that acetaminophen overdose in a mouse model produced liver injury, and an increase of rna concentration in plasma compared to the control group was observed (from ≈ to ≈ ng/ml of total rna h after treatment). plasma ncrnas levels were affected by acetaminophen overdose, both endogenous and exogenous. plantderived exogenous mirnas levels were reduced, and this drop in plant rna was related to the observation that mice lose appetite after acetaminophen injection , suggesting the influence of diet on plasma plant-derived mirna levels. levels of exogenous rnas can be affected in case of drug-induced kidney damage. sera and urine from mice treated with cisplatin, a chemotherapeutic agent also producing small intestines epithelial cells disruption, showed increased plant exogenous ncrnas (i.e. plant mirnas), not observed in untreated mice (yang et al., b) . the effect was not observed in a glycerol-induced model of acute renal failure (which does not affect small intestine organization). detection levels of exogenous plant mirnas, including mir- and mir- a, were below background levels in mice pre-fed with chow diet devoid of honeysuckle. however, when mice were pre-fed with honeysuckle and honeysuckle was administered (alone or mir- a-supplemented) dose-dependent levels of mir- a was detected in serum and elevated serum levels of mir- were observed (yang et al., b) . the study shows that long-term honeysuckle feeding potentiates absorption of dietary mirnas through a mechanism that likely differs from cisplatin treatment, suggesting that particular diets can modify the capacity to absorb small rnas, and that altered or damaged guts resulting from illness and/or therapeutic treatment may enhance dietary rna uptake (yang et al., b) . while comparing the presence of exogenous srnas in three human plasma samples, beatty et al. reported that the largest proportion of reads matching plant sequences were found from one individual who reported following a vegetarian diet (beatty et al., ) , suggesting that the diet can influence levels of exogenous srnas of food origin. in another study comparing human breast milk for mirnas derived from plants, no significant differences were observed for exosomal breast milk plant-derived mirnas (mir- a and mir- a) between vegetarian or non-vegetarian volunteers . in the whole milk an increase in plant mirna mir- a was observed in non-vegetarian versus vegetarian diet volunteers . although some studies (see above) have reported the presence of exogenous rnas in human and animal circulation and tissues, very few have focused on quantitative data relevant to risk assessment. wang et al. estimated the concentration of rna in plasma to be < . pm based on an average of ng/ml of rna in plasma with an average of nt in length and a selected rna (i.e. mirna) sequence representing less than . % of the total rna population. after days feeding of herbals, yang et al. reported an increase of mir- in the sera of mice from fm ( -fold higher than control chow-fed animals) following ginseng intake to fm ( -fold higher than control chow-fed animals) following honeysuckle intake . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the lack of enrichment of plant-derived small ncrnas in samples to be analysed by rna-sequencing (i.e. through periodate oxidation) might also influence the low enrichment of these exogenous sequences in biological fluids. however, although '-o-methyl modification of synthetic rna oligonucleotides '-ends can affect ligation activity of the t ligase in srna library preparation and may potentially result in srna sequencing bias and under-representation of srna with '-o -methyl '-ends in quantification experiments (munafo and robb, ) , others suggest a broad capability to detect plant mirnas during the sequencing procedure of small rna library construction . the available literature clearly suggests the widespread presence of exogenous rnas from multiple species, including plants, microbiota and many others, in human and animal biological fluids. evidence for plant-origin ncrnas, possibly relevant to risk assessment of ncrna-based gm, suggests that plantderived mirnas can be found in the biological fluids of humans and animals. whether these sequences are directly derived from dietary sources has not been clearly demonstrated. their overall low abundance, lack of enrichment in tissues most exposed to dietary changes, and their high variation between samples suggest that some may have originated as technical artefacts or contamination, as indicated by some authors. although few reports were available in this area, recent evidence also suggests that the levels of exogenous plant ncrnas in biological fluids may be influenced by diet, subject physiological or pathological conditions, or certain therapeutic treatments. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. following a literature search as described in section . . . . and based on the methodology described in the section . . . ., a total of papers were selected as being relevant to review of the systemic effects of dietary exogenous ncrnas. very few of these studies ( ) provide supporting evidence (in favour) that dietary exogenous ncrnas can be detected in biological samples and/or exert a biological effect after dietary ingestion. by contrast, studies provide contradicting evidence (against) that dietary exogenous ncrnas are either bioavailable or exert a systemic biological effect. one study provides evidence of the possible transfer of small ncrnas through the mammalian placenta . in addition, following a literature search as described in section . . . . and based on the methodology described in the section . . . , papers were selected as relevant to review toxicological effects of dietary exposure to exogenous ncrnas in humans or animals. the first report of systemic effects of dietary exogenous mirna in animals was published in by . plant mirnas were detected in human chinese healthy donors, among which mir- a and mir- a were highly expressed. these mirnas were also detected in calves and mice. plant mirna levels were relatively lower, but at a similar concentration range as endogenous mammalian mirnas present in serum. periodate oxidation, which oxidize mirnas but not plant mirnas (which are '-o-methyl modified), confirmed that these mirnas were genuine plant mirnas as most mammalian mirnas in human sera were oxidized and failed to be sequenced by solexa . the plant mirnas mir- a and mir- a were also detected in different mice tissues, while plant mir- a was not, although it was present in the sera. both mir- a and mir- a were primarily detected in microvesicles in c bl/ j mouse plasma. foods were found to contain the plant mirnas mir- a, mir- a and mir- a, including a chow mice diet at a concentration of . , . and . fmol/g, respectively. fresh rice contained between to -fold higher amounts than the chow diet. plant mirnas were detected in other foods including chinese cabbage, wheat and potato. plant mirnas were found to be stable in cooked foods. plant mir- a and mir- a were higher in the sera and liver of mice after h feeding with fresh rice. mir- a also increased in the stomach and small intestines, but not in the kidney, of mice fed a chow diet or fresh rice. mice fed with total rna extracted from rice or synthetic mir- a (methylated) showed an increased level of mir- a in serum and liver h after feeding. in vitro stability assays suggested that plant mirnas had a much slower degradation rate than their synthetic form (without '-o-methylated 'ends), suggesting that methylation has a protective effect on the stability of plant mirna . of note efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. is that mir- a targets the liver-enriched gene low-density lipoprotein receptor adapter protein (ldlrap ), which plays a role in facilitating the removal of ldl from the circulatory system (garcia et al., ) . direct binding of mir- a to exon of ldlrap , which is in the orf (open reading frame), was demonstrated by luciferase reported assays. transfection of both pre-mir- a and mature mir- a resulted in decreased ldlrap protein expression (but not that of mrna) in hepg cells. moreover, transfection of the intestinal epithelial caco- cell line with mir- a produced microvesicles with plant mir- a which can be taken up by hepg and reduce ldlrap protein expression. mir- a seemed to be associated to ago protein and other cells like t cells which were also able to package mir- a in microvesicles and then deliver mir- a into hepg . injection (iv) of antisense oligonucleotide against mir- a into mice during fresh-rice feeding reduced mir- a levels in the liver and reversed ldlrap repression. mice were finally fed with fresh rice for days to evaluate the physiological function of food-enriched in plant mir- a. serum and liver mir- a levels were induced already at day of feeding and the liver ldlrap protein was repressed after , and days. consequently, ldl levels in mouse plasma were significantly elevated on days and after fresh rice feeding. administration of an antisense oligonucleotide against mir- a reduced plasma levels of mir- a, repressed reduction of ldlrap in the liver and blocked the rice-induced elevation of plasma ldl levels, suggesting that elevation of fresh-rice derived mir- a in the mouse liver specifically decreased liver ldlrap expression and that this caused elevated ldl levels in mouse plasma (figure ). this study provides the first overall evidence of cross-kingdom regulation of gene expression through dietary plant mirnas. the authors further tested a mature mammalian mir- added to the chow diet for mice, and reported an increase in serum and liver levels of mir- after three days , leading to downregulation of liver c-myc expression . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. by analysing western human sera samples from female stage ii-iii breast cancer patients, chin et al. detected multiple plant mirnas from arabidopsis thaliana and soybean (glycine max), but with sparse counts (chin et al., ) . among the detected plant mirnas, ath-mir- a, gma-mir- a- p and gma-mir- e- p had the highest counts (≤ read counts) and were over times more abundant than other plant mirnas. circulating mir- was more abundant in female healthy donors (n= ) than breast cancer patients (n= ) as analysed by qrt-pcr. moreover, circulating mir- was less abundant in patients who relapsed with metastatic disease, suggesting that human serum mir- levels inversely correlate with breast cancer incidence and progression (chin et al., ) . mir- in human serum was detected in an extracellular vesicle-enriched serum fraction obtained by ultracentrifugation and was resistant to sodium periodate oxidation, suggesting its plant origin due to '-o-methylation of the ' end (which makes plant mirnas more resistant to periodate oxidation). in situ hybridization confirmed the presence of mir- in tumour samples, suggesting that mir- in human serum is capable of entering breast tissue (chin et al., ) . the authors analysed several plant food items for the presence of mir- and found that broccoli is particularly rich in this mirna and most of this mirna was still present after cooking (boiling for min, > % remained stable, compared to fresh). in vitro studies showed that synthetic mir- (both double-stranded and '-o-methylated) reduced cell proliferation in breast cancer cell lines but not the non-cancerous breast cell line. extracellular vesicles from healthy human donors also have a similar effect. luciferase assays determined that mir- targets transcription factor (tcf ) and binds to two different sites within the ' utr. tcf is a transcription factor of the wnt signalling pathway and is upregulated in breast cancer. in vitro assays also showed that mir- inhibits breast cancer cell growth by targeting tcf . oral gavage of synthetic '-o-methylated mir- ( mg/kg mir- or scramble control oligos, daily) reduced tumour growth from day of treatment until the end of xenograph experiments ( weeks) (figure ) , an effect that was lost when tumour cells stably overexpress tcf without the ' utr (chin et al., ) . mir- was detected in xenografts of tumours by in situ hybridization analysis. overall, these experiments suggest that plant mirnas can inhibit cancer growth in mammals. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. liang et al. evaluated and quantified plant mirnas in human plasma after consuming a single dose of watermelon juice or a mixture of fruits (liang et al., b) . using taqman probe-based qrt-pcr assays and appropriate non-template controls, sixteen plant mirnas including ath-mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- , mir- and mir- were evaluated and a standard curve generated for quantification. mirnas concentration ranged from . pm for mir- a to , . pm for mir- in watermelon juice, and from . pmol/kg for mir- a to , . pmol/kg for mir- in the fruit mixture. watermelon juice was orally administered to nine healthy male adult volunteers and plasma samples were taken . , , , and h after drinking . l of juice. different mirnas followed different peak time or appearance kinetics in plasma after administration (liang et al., b) . using six plant mirnas ( table ) that showed typical kinetic absorption curves, the total amount of plant mirnas absorbed by the body was calculated. using the plant mirnas concentration in watermelon juice and the areas under the plasma concentration-vs-time curves (auc) of plant mirnas, the total uptake amount, total absorption amount and absorption rate for each plant mirna was calculated (table ) . absorption rate ranged from . % to . %. mir- a, mir- a and mir- a were found almost exclusively encapsulated in microvesicles. mirnas were validated in watermelon juice or plasma by northern blotting. using the same protocol, a fruit eating study was performed. each subject consumed an equal portion of watermelon, apple, banana, orange, grape, mango and cantaloupe ( . kg in total). while mirnas concentration varied, the kinetics of plant mirnas after fruit administration appeared similar to the kinetics after juice administration (liang et al., b) . the authors also suggest that the real concentrations of plant mirnas may be underestimated and, for instance, the real concentration of mir- a in the watermelon juice might be six times higher and its plasma levels four times the previously calculated (table ) concentration (liang et al., b) . adapted from (liang et al., b) in a comment (correspondence) to the editor, the results of above article were criticized as being either a result of technical artefacts or a contamination problem, due to the presence of mir- in monocots and not in the dicot watermelon (witwer, ) . in a reply letter, the original authors indicated that mir- also exists in dicots and that the mirnas display a dynamic physiological kinetic absorption curve in human plasma, supporting the concept of uptake of food-derived mirnas by humans and animals (liang et al., a) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analysing mir- by qrt-pcr in dried flowers and herbs used in chinese medicines or as tea, yang et al. reported different levels in different products including: sophora (≈ fmol/g), honeysuckle (≈ fmol/g), chamomile (≈ fmol/g), blue mallow (≈ fmol/g), ginseng flowers (≈ fmol/g) and as low as . fmol/g in hibiscus . herbal feeding of male mice for days increased plasma circulating levels of mir- compared to that of chow-fed animals. honeysuckle increased -fold ( fm) in sera levels, while chamomile increased -fold ( fm), sophora -fold ( fm), lavender -fold ( fm), blue mallow increased -fold ( fm), ginseng increased -fold ( fm) and no significant changes were detected in hibiscus ( fm). post-feeding urine levels of mir- ranged from -fold higher ( fm) for honeysuckle, and -fold higher ( fm) for chamomile and to -fold higher ( fm) for lavender . however, when chemically synthesized mir- ( '-o-methylated, plant-specific) was administered after single dose feeding of pmols, mice serum levels were elevated roughly -fold within min after gavage but decreased to background levels ≈ h after gavage . mir- was not associated with ago in sera and remained in the unbound fraction. clearance of mir- and other plant-derived synthetic mirnas (mir a, mir a and mir ) were analysed in mice by tail vein injection of fmols. sera collected from mice ( min, min, h, h and h post injection) showed that most mirnas were rapidly cleared from circulation. compared to the other mirnas administered at equal doses, mir- levels were substantially higher at min after injection but after h the apparent clearance of all srnas tested was complete . in the same study, gut microbiota did not seem to influence mir- absorption despite its -fold increase in their microbiome titre after honeysuckle consumption. honeysuckle feeding of mice was also found not to influence gut permeability, discarding its possible absorption due to changes in gut permeability . rapeseed (brassica campestris) bee pollen was evaluated for the possible absorption of plant mirnas in male mice after pollen oral feeding ( g/kg). mirnas were analysed by srna sequencing after or h in mice serum ( µl). from ≈ m clean reads, only reads were plant mirnas, which belonged to mirnas. of the plant mirnas, bna-mir- a ( reads) and bna-mir- ( reads) showed the highest levels in mouse blood and were mapped to the rapeseed genome. the rest of the plant mirnas had ≤ reads, of which mirnas had ≤ reads. both mirnas, mir- a and mir- , were also detected in the rapeseed bee pollen as evaluated by qrt-pcr, with higher abundance for mir- a. serum levels of mir- a after h of rapeseed bee pollen ingestion versus a control were ≈ pm for treated animals while in chow diet fed animals it was ≈ . pm, suggesting that plant mirnas from rapeseed bee pollen can be absorbed by mice by oral ingestion and detected in systemic circulation . evaluated herbal medicine honeysuckle mirnas before and after decoction (boiling for min, ≈ . g/ml water). from ≈ m reads, ≈ . m reads of mirnas were obtained, from which osa-mir- g, peu-mir- and peu-mir- contained more than . copy numbers. most mirnas were degraded by decoction (i.e. mir- g degraded from . to reads), while mir- remained high ( . reads) reaching % of total mirnas in the final honeysuckle decoction. concentration analysis of mir- showed a reduction from . pmol/g in honeysuckle to . pmol/ml in decoction when analysed by qrt-pcr, or from ≈ . pmol/g to . pmol/ml when assessed by northern-blot . compared to other plant mirnas, the apparent high resistance to boiling of mir- was also supported by its high resistance to rnase treatment, which was dependent on its unique sequence and high gc content. oral administration (n= per group) of a single dose ( µl) of honeysuckle decoction (mir- concentration ≈ . pmol/ml) resulted in a maximum peak level in mice plasma and lung tissues h after ingestion (i.e. plasma levels increased from . to . fm). most plasma mir- was found in cell-derived microvesicles and associated efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. to the ago complex. continuous drinking of decoction during three days ( . pmol/ml, ≈ ml/day) increased plasma levels from . to fm, while no changes were observed in the control mice (from . to . fm). levels of mir- in lung and liver tissues increased from to -fold, while the intestines and kidneys remained unchanged. synthetic mir- administration ( nmol/ml, µl given once per mouse, n= per group) resulted in an increase at peak ( h) from . to . fm in plasma and also an increase at peak ( h) in lung tissue, suggesting overall that atypical dietary mirna mir- is absorbed and delivered to tissues . studying its possible biological function, zhou et al. found influenza virus h n encoded mrnas targets of mir- , i.e. virus-encoded proteins pb and ns , which are relevant for viral replication (zanin et al., ) . luciferase activity assays confirmed that mir- binds to pb and ns , which is lost by point-mutation of mir- binding sites of the individual viral gene sequences. in vitro h n replication was reduced by synthetic mir- or total rna extracted from honeysuckle decoction. the inhibitory effect was abolished by co-transfection with anti-mir- antagomir. moreover, no effects in viral replication were observed when pb and ns mutant virus were used with synthetic mir- or honeysuckle decoction rnas . in vivo administration of honeysuckle decoction or synthetic mir- ( . nmol per day) by gavage one day before and during days after inoculation reduced weight loss and viral titre in lung tissues. these results were abolished when the anti-mir- antagomir was used concomitantly. moreover, weight loss and high viral titre were not affected when pb and ns mutant virus were used, and synthetic mir- or honeysuckle decoction administered . the authors also showed similar effects for synthetic mir- or honeysuckle decoction with other influenza viruses, including h n and h n , tested in vitro and in vivo (figure ) . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. regarding this particular mirna, yang et al. observed a different bioavailability of plant-derived mir- vs. its synthetic '-o-methylated counterpart . oral administration of cabbage extract containing . pmol of mir- per µl gavage volume was compared with a -fold higher dose of synthetic '-o-methylated mir- ( pmoles per mouse). after gavage, mice plasma levels of cabbage mir- reached an approximately -fold increase (≈ fm) versus that of synthetic mir- (≈ fm), suggesting that mir- derived from plants is more bioavailable than synthetic rnas . whether this observation can be generalized to other dietary plant mirnas is unknown. the authors also showed that mir- was not present in exosome vesicles that were proteinase-k resistant in circulation . du et al. evaluated the presence of srnas in hong jing tian (hjt, rhodiola crenulata), which is a chinese herbal medicine. male c bl/ j mice were fed with hjt rnas by gavage for three days ( µg/kg per day) and lung tissue collected at , , or h (n= mice per time). the srna sequences of lung tissues (top eight ranked) that were also present in the plant hjt had ≈ k read counts . one of the small rna (hjt-srna-m ) was effective in reducing in vitro transfected gene expression vectors for α-sma, fibronectin and collagen type i α (col a ) in a tgf-β induced fibrosis model. although the type of srna is not clearly described, hjt-srna-m targets the ' utr of these three genes. using the hjt-srna-m agomir (cholesterol-conjugated srna), by intratracheal administration ( mg/kg in µl of saline) (n= mice per group), the authors showed that fibrosis was reduced in a bleomycin-induced fibrosis mouse model . due to the relevance of liposomes in delivering rna molecules, the authors evaluated the possible formation of liposomes during decoction (boiling for min) with plant lipids. they found that phosphocholine lipid molecules were also present in the decoction and suggested that liposomes may be formed that might facilitate the entry of rnas from plants to human cells and tissues; this was not tested . in another study, mlotshwa et al., described oral administration of a cocktail of tumour suppressor mirnas (normally downregulated in colon cancer) and a reduction of tumour burden in the apc min/+ mouse (c bl/ j) model of colon cancer . mir- a, mir- and mir- were chemically synthesized with the same mice sequence, but with a methyl group on the ' position of the ribose at the ' end, which is a chemical characteristic of plant mirnas (see section . . ). for days (starting at weeks of age), mice (n= per group) received either total plant rna spiked with the mix of the three mirnas, total plant rna alone (from a. thaliana) or water. oral dose was µg of total plant rna alone or plus . to . µg (corresponding to . to . nmole) of each species of tumour suppressor mirnas. tumour burden was dramatically reduced in mice receiving the mirna mixture. moreover, mice receiving the plant rna alone also showed a reduced tumour burden, although it was not statistically significant. after periodate oxidation, intestinal levels of mir- a were -fold higher than that of the group receiving water alone . mir- and mir- intestinal levels failed to be detected due to high background of endogenous mice mirnas after periodate oxidation. although the study does not directly evaluate the mirnas produced by the plant, mirnas designed to mimic small rnas produced in plants seemed to be taken up by the digestive tract of apc min/+ mice upon ingestion and exert a biological effect . oral ingestion of total rna isolated from brassica oleracea ( µg) resulted in the detection of plant mir- (the most highly enriched exogenous plant mirna in b. oleracea) in blood of icr mice between h and h after feeding . mir- was also detected in the spleen ( . - . % recovered of orally administered mir- ), and merely detected in the liver or kidney. no evidence of sex differences was reported . at lower doses of total rna ( µg or µg), no mir- was detected in the blood of mice . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. using pigs as experimental animals, luo et al. evaluated the detection of plant-derived mirnas in the serum and tissues after feeding fresh maize to pigs (sus scrofa). jinhua female pigs consumed a fresh maize diet and water ad libitum for days (n= ), and blood and different tissue samples were obtained for plant-derived mirna analysis. eighteen maize mirnas were evaluated ( were detected) in serum and tissues, and exhibited relatively low abundance in the pancreas and longissimus dorsi muscle tissue . periodate oxidation treatment of the samples confirmed the detection of zma-mir- a- p, zma-mir- e- p, zma-mir- a- p, zma-mir- a- p, and zma-mir- a- p (while the rest were completely degraded), suggesting that these mirnas are bona fide plant mirnas (figure ). in a separate experiment, female pigs were fed one meal with fresh maize ( kg per pig) after overnight fasting. serum was collected at different times (i.e. , , , , , and h). after h, pigs were provided with fresh maize diet ad libitum, and serum collected at , , and days and tissue collected after sacrifice at days. the serum levels of some of these mirnas (mir- a- p, mir- a- p, mir- e- p, mir- a- p and mir- a- p) gradually increased after one feeding of fresh meal, reaching peak values at or hours within the first hours and maintaining stable detectable levels (by qrt-pcr) in the serum following days of access to fresh maize. the changes in mirna expression at all measured time-points in most cases were modest (≈ to -fold increase). the five mirnas were primarily present in serum exosomes (≈ . % of concentration in the serum) ( figure ). using bioinformatics, potential targets were determined within the porcine mrnas for zma-mir- a- p. using in vitro luciferase assays with porcine kidney cells, the authors showed that zma-mir- a- p binds to three of these potential target genes including cspg , otx and plagl , an interaction lost when their mutant constructions were used, suggesting overall that dietetically-absorbed mirnas can target endogenous porcine mrnas . the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. several of the above-mentioned studies have reported the presence of dietary plant mirnas in circulation associated to extracellular vesicles or exosomes (chin et al., ; liang et al., b; . this could be relevant for their possible biological effects, as extracellular vesicles can deliver a cargo to several tissues, including crossing the blood-brain barrier, and other biological barriers such as the placenta (matsumoto et al., ; yang, t et al., ; jayabalan et al., ) . their presence in microvesicles could modify their function, for example, it has been described that in some cases delivered naked mirnas cannot exert the same effects as those exerted by delivered exosomal mirnas (thomou et al., ) . also, exosomes as therapeutic drug carriers and delivery vehicles are gaining relevance in therapeutics (aryani and denecke, ; ha et al., ) . recently, it has been described that edible plant-derived exosome-like nanoparticles can be isolated from these plants (ju et al., ) , can transport ncrnas (i.e. mirnas) (mu et al., ) and can exert a biological effect (raimondo et al., ) , highlighting their role as therapeutic nanoparticles . it is still unknown if these exosome-like nanoparticles contribute to the resistance of ncrnas in the harsh conditions of the gi tract and to the absorption of plant ncrnas or the formation of exosomes circulating in biological fluids. regarding other biological barriers, only one study has reported on the transfer of small ncrnas through the placenta. li et al. found that exogenous plant mirnas were consistently detected in the umbilical cord blood and amniotic fluid of healthy chinese pregnant woman . this belongs to foetal circulation system, suggesting that mirnas may transfer through the placenta to the foetal side. three hours after gavage feeding of synthesized mature mirnas of the influenza virus to pregnant mice (at last -day pregnant and with mature placenta) increased mirnas levels were detected in the maternal plasma. an increase of exogenous mirnas levels in the foetal liver was also observed . to determine if mature plant mirnas can pass through the placenta and efficiently enter foetal organs, the traditional chinese herbal honeysuckle was tested. mice were gavage fed with . ml honeysuckle decoction ( . pmol/mice of mir- ) and maternal plasma and foetal liver collected h after gavage feeding. plant mir- levels were elevated ≈ . -fold in the maternal plasma (reaching fm), ≈ -fold in the placenta and ≈ . -fold in the foetuses, suggesting that plant mirna mir- in honeysuckle can efficiently transfer through the placenta to enter the foetal liver (figure ) . the amount of circulating mir- increased only in microvesicles, suggesting that circulating mir- was primarily present in microvesicles and that transplacental transmission may involve a microvesiclesmediated pathway . the same study also tested gavage fed synthetic sirnas ( nmol), which were found in the plasma and foetal liver. protein targeting of the sirna was dramatically reduced, suggesting that exogenous sirnas delivered from the mother to the foetus by trasnplacental transmission may regulate foetal gene expression. direct injection of microvesicles loaded with sirnas also reduced their mrna target in the foetus liver, suggesting overall that exogenous small ncrnas can be transplacentally transmitted from the mother to the foetus. although outside the scope of this review, other non-plant derived exogenous ncrnas have also been reported to exert systemic effects in animals. bovine milk-derived extracellular vesicles (bmevs) were orally administered to il- ra -/mice on the balb/c background, which spontaneously develop polyarticular arthritis, from week to week . drinking water was also supplemented and administered to a collagen-induced arthritis (cia) dba/ j mice model starting at week before immunization to day . mice receiving the higher dose of x bmevs (≈ micrograms/ml) showed a considerable delay in disease onset in the il- ra -/mice model. moreover, in the cia model the higher dose administered in drinking water . x /ml (≈ microgram/ml) reduced arthritis incidence and severity, accompanied by reduced expression of inflammatory cytokine il- and mcp- . although bmevs contained mirnas mir- a, mir- and mir- a, it is unclear if they are efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. responsible for the observed biological effects. in a similar study, oliveira et al. administered bmevs (particle concentration of . x /ml or . x /ml) in the drinking water to female dba/ j mice for weeks and observed an increase in osteocyte numbers. moreover, the highest concentration of bmevs increased the percentage of woven bone compared to the pbs group, and a reduction of the adipocyte area in the bone marrow (oliveira et al., ) . although the bmevs also contained mir- a (oliveira et al., ) , its implication in the biological effects is unknown. exogenous plant-derived ncrnas can pass the placenta and reach the foetus. by analysing small rnas components in royal jelly, honey, beebread and pollen collected during the cabbage (brassica campestris) flowering stage, zhu et al. found that honeybee srnas were present at a far higher level in royal jelly than in beebread and pollen, while the abundance of cabbage small rnas gradually increased from royal jelly to honey, beebread and pollen . several mirnas were detected in the samples, the plant mirnas occurring at far higher concentrations. moreover, beebread and polled mirnas were at a much higher concentration and their composition of individual mirnas was highly similar. the plant representative mirnas with the highest concentration were mir- a, mir- a, mir- a, mir- a, mir- a, mir- a, mir- g, mir- a, mir- a, mir- a, mir- c, mir- a, mir- a, mir- , mir- and mir- a. the same analysis in samples collected during the camellia (camellia japonica) flowering stage showed similar results and out of the enriched mirnas were similar, suggesting that plant mirnas in beebread and pollen may not be very diverse between different sources . absolute concentration of the selected plant mirnas ranged from < . fmol/µg of total rna (almost undetectable) in royal jelly and honey, to a maximum of ≈ fmol/µg total rna in pollen and beebread. feeding experiments to larvae with either rna purified from cabbage pollen (levels of mirnas similar to natural beebread mirnas levels) or a efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. synthetic mix of the plant mirnas, increased whole body accumulation of the representative plant mirnas and developed worker bee-like characteristics (reduced weight and size, extended pre-adult development time and decreased ovary size). ninety-six honeybee genes were predicted to be targeted by the plant mirnas, some of which are known to influence the developmental fate of honeybees (i.e. apis millifera tor, amtor). luciferase assays showed that mir- a directly targets aptor mrna. dietary supplementation of larvae food with synthetic mir- a decreased amtor mrna levels in honeybees and showed reduced body weight, size and ovary size . similar experiments were performed in drosophila melanogaster (dm) with total pollen rna, a synthetic mirna pool or mir- a alone, and in all cases similar phenotypes were obtained (delayed drosophila development, reduced final size, ovary size and fecundity). dmtor was also a target of mir- a. overall, this study implies that plant rnas can be transmitted between species of different kingdoms. the literature also describes some in vitro studies of plant mirnas and their possible effects on mammalian cells. for example, exposure ( h) of plant mirnas extracted from a glycyrrhiza uralensis decoction to human pbmcs produced apparent cell aggregation and increased cell number and hla-dr+ cell proportion (shao et al., ) . in silico studies have also attempted to predict possible plant mirnas with potential targets in humans . as described before (see section . ), in contrast to mammalian mirnas, plant mirnas possess a '-omethylation at the '-end that stabilizes and provides resistance to rnases and oxidation. this characteristic has been used to improve the quantification method for mature exogenous mirnas from plants in biological mammalian matrices . thus, periodate oxidation and βelimination were introduced to evaluate the origin of exogenous mirnas. male c bl/ mice were fed with corn/soy-based rodent chow diet for weeks. plant mir a, mir a and mir a were evaluated. these mirnas were detected in abundant amounts in a corn/soy-based chow diet, but after periodate oxidation and β-elimination . %, . % and . % of mir a, mir a and mir a were determined to be in methylated forms ( . , . and . fmol/g of diet, respectively). the control diet showed no differences between the diet and non-template control ct values after periodate oxidation/β-elimination. this study also suggests the use of exogenous rnas (spike-in), both methylated and un-methylated forms of mirnas, to ensure appropriate periodate oxidation/β-elimination . no detectable levels of plant mirnas were found in plasma, liver or faeces samples from the mice fed the corn/soy-based chow diet, since no differences occurred between the diet and the non-template control after periodate oxidation/β-elimination . this suggests that false positive results can be resolved using treatment with periodate oxidation/β-elimination. plant mirnas from extra virgin olive oil (evoo) and beer have also been evaluated by small rna sequencing. however, very few sequences (≤ reads) were identified, and most aligned to soybean and orange . a feeding study of a single dose ingestion of ml of evoo in healthy adult volunteers (n= ) was performed. mirnas analysis at baseline and after h ingestion was performed. no plant mirnas were detected in plasma after single ingestion of evoo . only one read count was found for other species mirna vvi-mir - p from grape, while two read counts were found from medicago trunculata, grape or brachypodium distachyon when mismatches were accepted . this suggests that beer or evoo do not contain mirnas that contribute to dietary intake, even though mirnas were reported in the pulp of olive (olea europaea) (donaire et al., ) . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. dickinson et al. evaluated a controlled mouse feeding study with rice-containing chow diets (modified ain -g with . % of rice or rice-based with % of rice) or with a purified synthetic chow devoid of plant grain or forage for , and days (figure ) . the "synthetic chow" had negligible levels of osa-mir- a, the "balance rice chow" ( . % of rice) contained reads, while the rice grain contained reads. the concentration of osa-mir- a in each diet was . , and fmol/g of diet for synthetic chow, balance rice chow and rice chow, respectively. plasma and liver analysis of mice fed the diets did not reveal measurable uptake of any rice grain mirnas, including osa-mir- a. indeed, fewer than ten reads were detected in five out of eight samples from mice fed on rice-containing chow and four out of five samples from mice fed on synthetic chow . since the synthetic chow contained no grain or forage from plants, this low number for rice mirnas-mappable reads can only be explained by sequence errors or cross-contamination. in agreement with , the authors indicated that animals fed a chow containing high levels of uncooked rice (%) had significantly increased plasma low-density lipoproteins (ldl) levels at and days after treatment initiation. however, this increase in ldl was not observed in the balanced rice/chow group ( . % rice), which contained fmol/g osa-mir- a. the increase of ldl was attributed to nutritional imbalances between the test and control groups . ldlrap protein was also evaluated by elisa in mouse liver samples, and no changes in levels were observed in any group at any time, suggesting that dietary intake of osa-mir- a (from to fmol/g) does not produce rnai-mediated modulation of ldlrap protein levels in mouse liver . efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. a commercially available plant-based, plant mirna-rich substance containing soy and fruit materials, but not animal products, was administered by gastric gavage (≈ % of estimated blood volume for each animal) to male macaques (macaca nemestrina). relative abundance of plant mirnas mir- , mir- , mir- , mir- , mir- and mir- were analysed by qrt-pcr in the plant-based dietary substance. for instance, for mir- a consistent and efficient amplification through at least pcr cycles was detected. plant mirnas were analysed in plasma samples of macaques (n= ) before and after , and h oral gavage of plant rich mirnas. qrt-pcr results indicated late amplifications of some plant mirnas, but results were highly variable. mir- did not amplify before cycles, and mirs- , - , and - had a median cq greater than . mir- non-template controls also showed regular amplification within the same cq range. mir- exhibited a tendency to increase after ingestion, but was highly variable, and cq cycles were greater than . this was not the case for endogenous animal mirnas or the spike-in control, supporting the case that the late apparent amplification of plant mirnas in plasma was non-specific . moreover, droplet digital pcr analysis showed that counts from plasma were generally very low and of variable intensity, as occurs in non-specific amplifications. this even occurred in mir- (plant mirna with relatively high apparent count by qrt-pcr), which showed large number of counts but with variable intensity plots ("rain" appearance) (figure ) , and is consistent with non-specific amplification . overall, the study (though only subjects were included) suggests that the detection of plant mirnas in plasma does not support a general and consistent uptake of dietary plant mirnas . : substantial levels of mirna in oral diets consumed by macaque but negligible steady-state presence of diet-derived mirnas in plasma or organ tissues. snow et al. ( ) analysed the presence of exogenous plant mirnas after regular intake of a western diet containing fruits in healthy human subjects, and mir- in plasma or organ tissues of mir- -/mice . the conserved plant mirnas mir- a, mir- a and mir- a were highly the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. expressed in many fruits common in the human diet such as ripened avocado, apple, banana, orange and cantaloupe. mir- a and mir- reached up to , copy numbers/mg food. the conserved animal mirna mir- was expressed at substantial levels (≈ , copies/mg food) in meat but not in fruits. mouse vegetarian and soy-enriched diets also contained high quantities (up to , copy numbers/mg food) of mir- a and mir- a, but not mir- . in contrast, mouse diets enriched with animal products contained elevated levels of mir- , suggesting that conserved plant and animal mirnas are commonly present in oral diets for various animal organisms. although the dietary record of of the healthy adults reported the intake of fruits replete in mir- a, mir- a and mir- a the day prior to plasma harvest, these plant-derived mirnas were undetectable in plasma samples. following -week feeding with a custom animal lard diet containing mir- no detectable levels of mir- were found in plasma samples of mir- -/mice, while high quantities of this mirna in plasma were seen in control wild type mice fed the same diet . in tissues (liver, lung, kidney and stomach) mir- was either not detected or detected at exceedingly low levels corresponding to less than one copy of mir- per cell (assuming that the mammalian cell expresses roughly pg of total rna). wild type-mice showed only a non-significant trend toward increased plasma levels of mir- a after consuming either a vegetarian or soy-enriched diet when compared to mice fed a lard diet for the same period ( -week) ( figure ) ; however, levels were very low. indeed, mir- a was detected in organ tissues (liver, lung, kidney and stomach) but at levels of less than one copy of mirna per cell and regardless of diets. mir- a and mir- a were not detected at all. fresh avocado containing three specific plant mirnas was also fed to mice ( . g per mouse over h). even though the three mirnas were found in the stomach content of mice at high levels, very low levels were found in plasma and organ tissues, reaching levels lower than one copy mirna per cell in tissues. this provides no evidence of substantial steady-state presence of these plant mirnas in organ tissues after dietary intake . assuming that, on average, at least copies per cell (> copies/pg srna per cell) are necessary to achieve canonical target gene repression (brown et al., ) , at least copies of a given plant mirna must be ingested and delivered for widespread biological activity in humans (the average human body contains ≈ cells). based on the concentration in ripe cantaloupe (≈ x copies mir- a/mg fruit), an individual would need to consume , kg of cantaloupe just to release this number of copies in the gi tract . in terms of srnas, the literature describes the threshold for target gene regulation to be between and copies of mammalian mirna per cell . in an analysis of activity for hundreds of mirnas, only the most abundant mirnas in cells were found to mediate targeted repression and a large amount of mirnas had no discernible activity (mullokandov et al., ) . this suggests that only a minimum amount of mirnas need be reached to exert a biological activity, although this will also depend on the amount of target transcripts. a study mentioned above the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. analyses of other common plant mirnas reported by others to be bioavailable were not successful. this implies that although the transgenic mirnas had resistance to degradation comparable to that of mir- and high expression levels in plants, they were not readily bioavailable . huang (huang et al., ) analysed the possible bioavailability of orally-ingested corn mirnas in mice when incorporated into a rodent diet and consumed for days. in an initial experiment, male c bl/ mice (n= per group) received either water (control), random nucleotides ( µg, equivalent to the same amount of small rnas) or purified small rnas isolated from corn kernel ( µg, equivalent to the same amout of random nucleotides). in the second experiment, mice received either a control diet (ain- m), ain- + % autoclaved corn kernel powder or ain- + % fresh corn kernel powder. mirna levels in the isolated rna contained . pg mir- a, . pg mir- a, and . pg mir- a per µg corn srna isolates. the diet supplement with fresh corn powder contained pg mir- a, pg mir- a, and pg mir- a per gram of diet. autoclaving the corn powder at ºc for min reduced the amount of initial mirnas by more than %. in the first study, liver and whole blood sample mirnas (analysed by qrt-pcr), showed no differences in ct values between the three groups. after periodate oxidation followed by β-elimination, no differences were detected between the no-template control group and the experimental groups. similar observations were made in the liver and whole blood in the second study using fresh corn powder. also, after periodate oxidation followed by β-elimination, no differences were detected between the no-template control group and the experimental groups in the liver and blood samples. analysis of corn mirnas in cecal and faecal samples www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. showed similar results after periodate oxidation, although certain minor differences remained between the no-template control group and the experimental groups. in all cases, less than . % of total the mirna tested in both studies was recovered from the faecal samples. moreover, analysis of the mirnas in the content collected from different parts of the mouse gi tract from the animal studies administered via gavage or dietary intake showed that calculated recovery accounted for less than . % of the content originally ingested in the stomach, less than . % of that originally ingested in the intestine and faeces, and less than . % of that originally ingested in the colon and cecum (huang et al., ) . further experiments using in vitro digestion system suggested that after the gastric phase, over % of corn mirna mir- a from the diet or from the extract were degraded, suggesting overall that significant degradation of corn mrnas occurred during digestion, which resulted in minimal uptake of corn mirnas after oral intake (huang et al., ) . witwer re-analysed the plasma data evaluated by liu et al., and found that only one putative plant mirna mapped above a median cut-off ( reads), which was the plant mirna peu-mir- . this implies that all rnas -including previously reported exogenous mirnas (xenomirs) such as mir- a, mir- a and the plant ribosomal degradation fragment mir- -are below the level of background noise. it is not clear if mir- is a real mirna as it is found in the highly conserved and expressed subunit rrna of plants . moreover, the mir- sequence is not only a fragment of plant rrna, but also has a % coverage and % identity match with human s rrna, while others do not map to plant genomes at all . perhaps more analyses are needed when assessing possible xenomirs. after in silico xenomirs analysis using public sequencing datasets (see section . . for details), kang et al. performed controlled feeding studies to detect the transfer of exogenous plant mirnas to rat blood or from bovine milk sequences into piglet blood . adult rats (n= per group) followed a -day controlled feeding study with supplementation in three different diets: monocot plant material (rice); dicot material (potatoes); and husbandry chow containing a defined mixture of grains, cereals, vitamins, minerals and fats. small rnas were isolated from serum and sequenced. most samples showed either a lack of plant xenomirs in rat serum, or three or less read counts in certain samples . these extremely low numbers suggested a probable false positive result. in the same study, piglets were fed either cow milk or soy milk for weeks followed by weeks of feeding with maize. few cow-specific sequences were found ( reads) in the piglets fed cow milk, while piglets fed maize as well did show cow-specific sequences ( reads). this suggests likely false positive results. both studies provide no evidence of dietary transfer of xenomirs . the abovementioned studies are summarized in table . although outside the scope of this literature review, pollen plant mirnas ingested as part of a typical diet of adult honey bees were not found to be robustly transferred across the epithelial barrier under normal conditions (masood et al., ) . for example, despite the high levels of plant mir- a in pollen or its detected levels in the bee digestive tract ( . copies per pg total rna in the midgut), less than . copy per pg total rna was found in abdominal tissue (masood et al., ) . www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. large amount of plant mirnas mir- a, mir- a and mir- a detected in vegetarian, soy-enriched, or avocado diets (i.e. ≈ x copies for vegetarian soy/diets or ≈ . x copies for avocado diet of mir- a after h), but very low levels found in plasma and less than copy per/cell in tissues. after weeks mir- rich diet in mir- ko mice, no plasma levels and less than one copy of mir- per cell detected in tissues. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. after periodate oxidation, no major differences observed in mir- a, mir- a, or mir- a among treatment, control groups or no-template control in liver and blood. in cecal and fecal samples no major differences observed among control and treated groups. mirna analysis in the gi content collected from different parts of mouse showed that the recovery (calculated) account for less than . % of originally ingested in the stomach, less than . % of originally ingested in the intestine and feces, and less than . % of originally ingested in colon and cecum. in vitro digestion experiments suggested that after the gastric phase, over % of corn mirna mir- a from the diet or from the extract is degraded. (huang et al., ) the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. exogenous plant mirnas found in umbilical cord blood and amniotic fluid of healthy chinese pregnant women. plant mir- increased in plasma of the mothers, placenta and foetus liver h after oral gavage ( . nmol/mice). exogenous sirna gavage ( nmol) increased plasma levels of mother and foetus liver. protein target in foetus repressed. direct injection of microvesicles loaded with sirnas also increased its levels in foetal liver and reduced mrna expression of its target. exogenous small ncrnas can be transplacentally transmitted from mother to foetus. n.d., not determined. ko, knock out; wt, wild type; mirna, microrna; srna, small rna; dsrna, double-stranded rna; col a , collagen type i α ; α-sma, alpha smooth muscle actin; hjt, hong jing tian; www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). the available evidence presents several examples of systemic effects of plant-derived exogenous ncrnas when administered orally. however, there are also several reports that do not support these findings and contradict the hypothesis of cross-kingdom regulation. in either case, the essential question concerning the existence of this possibility is still heavily debated. important aspects such as the precise mechanism of transport of plant ncrnas from food to the circulatory system, the amount of exogenous ncrnas reaching tissues or the molecular mechanism of cellular uptake need to be understood. it remains unknown if such a transfer could be modulated in particular context (i.e. specific diet, disease status, medication). the available evidence also suggests that plant ncrnas can directly target mammalian genes through rnai effects when they are exposed in in vitro systems. without considering other physiological parameters, if a synthetic plant mirna is exposed to its mammalian target, it can effectively bind by base complementarity and exert a biological effect (i.e. gene repression). however, whether the exogenous plant ncrna can bypass all the biological barriers to reach and interact with the possible target remains to be clarified. apparently, exogenous plant-derived ncrnas are found in exosomes or macrovesicles. it is not yet known how they reach these types of structure in biological fluids, or if novel plant exosome-like nanoparticles participate in any of these processes. www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). immune cells were mainly from studies in human and mouse isolated lymphocytes. however, these studies were descriptive/not comprehensive, reporting specific effects of ncrnas in proinflammatory cytokine production using human cells, and would not allow identifying specific endpoints that can be used to evaluate changes in the regulation of immune function and homeostasis by ncrnas. retrieval of grey literature using the key words 'thesis plant rna immune' provided numerous results (i.e. google = . ), mostly regarding the function of mirnas and rna silencing in plant immunity. for instance, a document could be chosen based on title selection because it included the terms 'plant, double-stranded rna and systemic immunity' (https://uknowledge.uky.edu/plantpath_etds/ ). however, this study was not further considered since it refers to plant immunity. after analysing the documents and refining by key questions, a large number of screened publications were discarded because they refer to the role of endogenous ncrna (human and animals) as well as viral infections in relation to the immune function, which is outside the scope of this literature review. as an example, within original in vivo publications related to the key words, only studies were considered relevant for the proposed review questions. in summary, from the literature search, papers were finally selected as relevant to review of the effects of ncrnas on immunity. very few studies analyse the direct relationship of exogenous plant ncrna with immune function and homeostasis of humans or animals. albeit controversial, exogenous ncrnas derived from plants and microorganisms have also been described in human blood (detailed in section . . .). overall, the results indicate that there is scarce information on the specific impact of plant ncrnas on activation of specific immunocompetent cells. of these references, documents were used for reviewing the general features of ncrnas in immune function and immunity in humans and animals (section . . .), were used for the effect of exogenous plant ncrnas on immune system (section . . .) and for the additional section on the effects of exogenous ncrnas on gut microbial composition (section . . .) ncrnas have been shown to play important roles in immune cell development and function in normal and disease conditions (ansel, ) . human and mouse studies demonstrate that mirnas play a critical role in the regulation of immune cell development and their function. there is also evidence of the relevance of neonatal mirna-mediated immune activation (kosaka et al., ) . here, mirna molecules of maternal origin appear to be stable in the infant's gut conditions, allowing dietary intake and transfer of mirna (kosaka et al., ) . ncrna have also been identified as regulators of metabolic shifts within microbial communities (paul et al., ) . thereby, ncrnas may significantly influence the critical roles of gut microbiota on health and disease. this part of the literature review (efsa task ) presents the current knowledge on the role of exogenous ncrna molecules on the immune system of humans and animals and aims to provide information on the possible effets of dietary ncrnas on the regulation and function of the immune system. in addition, since the gut microbiota influences the immune system, a review on the possible effects of exogenous ncrnas as gut microbiota modulators is provided this chapter provides background information on the modulatory effects of ncrna on the immune system of humans and animals and serves as a general introduction to explore the possibility of dietary plant ncrnas effect on the immune system after dietary intake. sixty-four ( ) documents were finally used to review this section. when activated, the immune system tightly regulates innate and adaptive response(s), aiming at restoring homeostasis, and preventing autoimmunity. the 'amplitude' (i.e. intensity and duration) of the www.efsa.europa.eu/publications efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). interaction with the t-(tcr) and b-(bcr) cell receptors determines cell fate decisions. in the periphery, regulatory t cells (tregs) play a key role in restraining the activity of mature b and t (th , th and th ) cells, and preserving tolerance mechanism(s). these responses depend on transcriptional and epigenetic regulation including regulation by mirnas. emerging systems for measuring mirna activity during immune activation revealed the complex network of genes that may be simultaneously targeted by mirnas to tune distinct cell fate decisions (wells et al., ) . rnas exhibit an intrinsic property of base pairs that is crucial in defining their structure and, thereby, their physiological role (lu et al., ) . plant mirnas are '-o-methyl modified on their terminal nucleotide, making them more difficult to be ligated to the cloning adapter. the inhibitory effect of '-o-methyl modification at the ' end of the rna substrate for the mammalian nuclease family member nef-sp has been described (silva et al., ) . as described elsewhere, most mammalian mirnas require 'pre-processing steps' to become physiologically active. nucleotide sequence complementarity appears as a critical feature driving the predominant result exerted by mirnas. notably, it has been reported that a modification pattern involving alternating '-o-methyl rna bases improves dsirna in vitro stability and evades activation of the innate immune system (collingwood et al., ) . these data were obtained with isolated peripheral blood mononuclear cells (pbmcs) as a mixed 'lymphocyte' population of immune receptors that are encountered in vivo. however, the authors do not provide any molecular mechanism for the observed effects. sirnas have been shown to be potent stimuli of interferon- production by plasmocytoid dendritic cells (hornung et al., ) . this sirna technology induced systemic immune responses in the same range as the toll-like receptor (tlr)- ligand 'cpg', including activation of t cells and dendritic cells in spleen. notably, immunostimulation by sirna was absent in tlr -deficient mice. there is a lack of data related to the effects of plant ncrnas on pge production, implicated in immunosuppression by tlr via cox . several distinct classes of lncrnas are transcribed from different dna elements or are derived from long primary transcripts with noncanonical rna processing pathways, generating new rna species with unexpected formats. these lncrnas can be processed by several mechanisms, including ribonuclease p (rnase p) cleavage to generate mature ' ends, capped by small nucleolar rna (snorna)-protein (snornp) complexes at their ends, or the formation of circular structures. recently, it was reported that several lncrnas mediate their regulatory effects through binding to specific rna-binding proteins . in addition, expression of lncrnas has been defined as highly cell-type specific (guttman et al., ; washietl et al., ) . this high specificity has been reported to be critical for the development and activation of immune lineages. however, lncrnas-mediated regulation of innate immune activation (i.e. il- production) has not been tested for function in vivo, but only evaluated in vitro (roux et al., ) . the regulation of inflammatory mediators or cytokines by lncrnas is still poorly understood. synthetic nucleic acids, such as dsrnas, have been reported to be recognized by toll-like receptor (tlr)- and can stimulate the innate immune system and trigger a type i interferon response (marques et al., ; mian et al., ) . in plants, it has been described that dsrna with ' overhangs contributes to endogenous and antiviral rna silencing pathways (fukunaga and doudna, ) . earlier reports defined a structural basis (i.e. ' overhangs) to cause nonspecific effects on the ds-rna-activated signalling pathways through the interferon responsive factor (irf)- (marques et al., ) . similarly, immune activation by the subcutaneous route with a viral mimic dsrna (poly i:c) of c bl/ mice during neonatal early or late brain development differentially affects depression-related behaviours developed in adolescent and adult mice (majidi-zolbanin et al., ) . the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). specific molecular interactions of the exogenous rna triggering immune responses have been reported. several studies have demonstrated the role of components of innate immunity, including toll-like receptors (tlrs), the retinoic acid-inducible gene i/melanoma-differentiation factor (rig-i/mda ) and the interferon response effectors protein kinase r (pkr) and rnase l, as well as mirnas in the recognition of viral or synthetic dsrna (malathi et al., ; urcuqui-inchima et al., ) . tlrs are a family of proteins recognizing different pathogen and damage-associated molecular patterns, pamps and dmaps, respectively. rig-i/mda , pkr and rnase l constitute cytosolic rna sensing proteins. the pathways responsible for these defined signalling processes can be largely separated (figure ) , despite the existence of some degree of crosstalk between them where the engagement of interferon regulatory factors is a common feature. tlrs play an essential role in innate immune responses in mammals. in humans, ten different tlrs recognize distinct molecular patterns, where tlr- , - , - and - stimulate innate immune responses upon interaction with nucleic acids weber et al., ) . tlr is located on the plasmatic cell membrane, while tlr- , - and - display endosomal localization. it has been reported that tlr recognizes dsrna, viral or the synthetic poly i:c, and sirna has also been identified (in the presence or absence of delivery systems) as a relevant ligand . notwithstanding, rna interference is considered an unlikely mechanism to be engaged for viral sensing (cullen, ) . gurich rna from viruses or synthetic single-stranded (ss) oligoribonucleotides displays an apparent preference for stimulating human tlr / -mediated immune effects (heil et al., ; forsbach et al., ) . a minimum of four nucleotides, uugu, found in the gu-rich region are reported as necessary to stimulate cytokine responses. endogenous or exogenous rnas are internalized into the endosomal compartment. the rna sensor system is composed of i) several innate immune 'toll-like' receptors (tlrs) located at the endosomal compartment that trigger different signalling pathways engaging adaptor distinct molecules (myd and trif), and ii) a cytosolic system (rig-i/mda- ) that coordinates its signalling with tlrs. antigen processing will lead to rearrangement of mhc molecules to activate the adaptive immune response(s). type i interferons (ifn-α/β) are expressed rapidly after infection and serve as a link between the innate and adaptive immune responses. : schematic representation of the eukaryotic rna sensor (single-stranded rna and short/long double-stranded rna) and its relation to innate immunity. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). two classes of ssrna motifs have been described that either preferentially activate tlr -mediated or tlr / -mediated immune responses. au-rich oligoribonucleotides stimulate tlr -but not tlr mediated immune activation (forsbach et al., ) . rna motifs activating tlr -associated signalling fail to induce ifn- from tlr -expressing plasmacytoid dendritic cells but induce the secretion of th like and proinflammatory cytokines from monocytes or myeloid dendritic cells. in addition, rna motifs activating the tlr / -associated signalling pathway stimulate cytokine secretion from both tlr -and tlr -positive immunocytes (forsbach et al., ) . differences in the protein sequence of tlr- between different mammals (human, monkey, chimpanzee, cattle, porcine, mouse, and rat) can be found, and species-specific recognition of ssrna via tlr / had been described (heil et al., ) . the tlr -specific rna sequences are unable to trigger cytokine responses from mouse, rat, and porcine immune cells (forsbach et al., ) . a dual function has also been suggested for the murine coreceptor cd to enhance tlr- , - and - signalling (baumann et al., ; lee et al., ) . however, further studies showed evidenced that human cd did not appear to function as a co-receptor for tlr- or tlr (weber et al., ) .the innate immune system detects rna lacking nucleoside methylation (m c, m a and m u) or otherwise (i.e. pseudouridine or '-o-methyl-u) modified as a mechanism to selectively trigger immune responses to nucleic acids from necrotic tissues or of exogenous origin. a recent systematic study on transcriptional and, especially, post-transcriptional regulation of stressresponsive lncrnas in oryza sativa showed that hundreds of lncrnas with down-regulated polyadenylation are highly conserved in stresses . in the ascidian ciona intestinalis a novel alternative polyadenylation signal activated by the prototypical tlr agonist (i.e. lps) has been described (vizzini et al., ) . moreover, alternative polyadenylation has been identified as a regulatory mechanism during the innate antiviral immune response in macrophages (jia et al., ) . these authors showed an enrichment of polyadenylated genes and mrna abundance change in tlrs as well as rig-i-like receptor, jak-stat and apoptosis-related signalling pathways. studies conducted on the crystal structure of the sensing tlr- ectodomain allowed definition of dsrna recognition of at least bp (liu et al., ; leonard et al., ) . it was concluded that tlr assembles on dsrna as stable dimers and that the minimal signalling unit is one tlr dimer. recognition by secondary structure 'alone' has been reported, independently of the sequence motif, by tlr . this dsrna length is larger than the typical helix occurring in normal mirnas and sirnas. tlr- and - have been found to recognize single-stranded rna (ssrna). the existence of a similar double horseshoe structure for tlr- , - and - upon activation has been proposed, although their binding modes to their ligands remain unclear . often, in a partial view, tlr is considered as the ssrna-sensing tlr and as complementary to tlr . however, higher structural features have been identified as important aspects in tlr rna sensing (jöckel et al., ) . in addition, small molecules that may be viewed as nucleoside analogues are able to activate tlr and/or tlr (hemmi et al., ; lee et al., ) . it has not been defined if the recognition mode for small molecules, including srnas, resembles that of tlr for rna recognition. besides tlrs, rig-i is a cytosolic multi-domain protein sensing viral rna associated to n-terminal caspase activation and recruitment domains (cards) to transmit signalling but preventing it in the absence of rna. the identification of rig-i/mda as cytoplasmic sensors for dsrna, associated to facilitators such as pkr and '- '-olygoadenylate synthetase, suggests that additional 'aspects' (i.e. dependence on atp) other than dsrna structure or short size influence innate immune responses to srnas (i.e. sirnas and mirnas). these aspects remain unclear. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). current information on mirnas points to clear differences in cellular recognition of endogenous and exogenous dsrna. here, '-triphosphate and dsrna represent molecular patterns enabling rig-i to discriminate exogenous from endogenous rna (myong et al., ) . dsrna products as short as - bp are sensed by rig-i independently from the '-termini (marques et al., ) . similarly, the ifninducible '- '-olygoadenylate synthetase (oas) requires dsrna that must be at least nucleotides long for activity, and no modification of the ′-hydroxyl group is tolerated (sarkar et al., ) . the oas family requires synthesizing di-, tri-, and tetrameric '- '-oligoadenylates ( - a), which in turn bind and activate rnase activation of rnase l together with pkr, irf and c-jun-n-terminal kinases, constituting pro-apoptotic mediators in response to dsrna. moreover, human oas may also enhance rig-i mediated signalling by mimicking polyubiquitin . further structural and mechanistic studies identify a key role of domain duplication in the oas family, thus revealing different functions of oas- and oas- in sensing dsrna (donovan et al., ; . rna interference (rnai) as well as antisense oligonucleotides and aptamers have been postulated to exert immune stimulatory effects in mammals (agrawal and kandimalla, ; cullen, ; mustonen et al., ) . however, at present, there is still an important open debate about agents exerting physiologically relevant functions at systems level biology (cullen, ; kleinman et al., ) . moreover, clinical trials of sirna demonstrated that the only population carrying the ff coding variant for tlr was protected from sirna-induced cytotoxicity (kleinman et al., ) . this study concluded that, because multiple cells express surface tlr , the therapeutic approach with sirna might induce unanticipated vascular or immune effects. here, anti-angiogenic innate immunity triggered by sirna was found to occur without the induction of inf-α/β via trif activation being biased toward nf-ĸb rather than irf- . side effects, for example, the immunosupressive effects of t regulatory cell function are inhibited by ifn-α- β (yu et al., ) , are still only vaguely understood in association with exogenous ncrnas administration. naturally occurring mammalian rnas do not display a stimulatory potential on innate immune responses through tlr- , - , - and - in dendritic cells and tlr-expressing cells (karikó et al., ) . by contrast, it has been shown that major innate immune responses to chemically synthesized sirnas are mediated by tlr and/or tlr . notably, replacement of uridines with their '-modified counterparts in the sirnas, reduced immune activation (sioud, ; barik and lu, ) . studies conducted on synthetic sirnas have shown results directly dependent on the nucleotide sequence (judge et al., (judge et al., , hornung et al., ) , and not to silencing effects of a target gene. putative immunostimulatory motifs allowing definition of ifn-α induction by β-gal control sirna or its constituent sense (gu-rich) and antisense ssrna oligonucleotides (judge et al., ) have been identified. these studies were performed in pbmcs, and therefore lack a clear demonstration of these effects at the organism level. intravenous administration of encapsulated sirna induced substantial dose-dependent ( - g sirna) ifn-α responses in outbred institute for cancer research (icr) inbred mice. in this line, reports also exist of increased hepatotoxicity of asos containing locked nucleic acid modifications (swayze et al., ) . in addition, asos carrying '-o-methoxyethylribose modifications showed no cytotoxic effects but were able to reduce targeted mrna. by analysis of sequence variants of either ss or ds sirna, uridine-rich immunostimulatory motifs within sirna were identified in human peripheral blood cells via tlr (sioud, ; jurk et al., ; judge et al., ) . there is a report of sequence-and targetindependent angiogenesis suppression induced by sirna via tlr (kleinman et al., ) . a minimum length for dsrna of about -nucleotide or -nucleotide luc sirna, but not truncated versions the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). suppressed by choroidal neovascularization have been observed in wild-type mice (kleinman et al., ) . recently, it has been established that nuclease-resistant aptamers can play a 'dual' role in immune signalling pathways (gefen et al., ; rajagopalan et al., ) . these aptamers seem to possess a unique feature acting as both agonist and antagonist depending on their degree of oligomerization (nozari and berezovski, ) . aptamers are ss oligonucleotides ( - nucleotides) capable of recognizing, in a specific manner and with high affinity, several types of target molecules by means of a three-dimensional folding of their chain. preclinical studies in murine models have highlighted distinct regulatory points that may be influenced by oligonucleotide aptamers, including clusters of differentiation (cd) (pastor et al., ) , cell-to-cell communication such as immunoglobulins (gefen et al., ) and interleukin (il) signalling (rajagopalan et al., ) . the cd molecules can act in several ways, often acting as receptors or ligands important to the cell initiating signal cascades or serving as adhesion molecules. here, cd is one of the main co-stimulatory receptors responsible for proper activation of t lymphocytes that could be manipulated to modulate the immune response (pastor et al., ) . further studies reported enhancement of t cell functions by specific t cell immunoglobulin- aptamers through negative regulation of ifn- secretion by cd + and cd + cells (gefen et al., ) . positive effects attributed to oligonucleotide aptamer have also been associated to attenuation of il- signalling in cd + cells (rajagopalan et al., ) . while sirna and dsrna in cultured cells have been shown to trigger an interferon response (alexopoulou et al., ; sledz et al., ) , the administration of naked (dtdt) synthetic sirnas ( . mg/kg) to mice (either intraperitoneally or iv via low pressure or high pressure injection) induced no interferon response (heidel et al., ) . this suggests that sirna administration in vivo may elicit little or no immune response. also, modification of nucleotides within rna molecules was shown to reduce immune response (durbin et al., ) , which is relevant when applying nucleotide modifications to rna therapeutics. for example, n- -methyladenosine (m a) and pseudouridine seems to reduce the retinoic acid inducible gene i triggering of the innate immune signalling (durbin et al., ) . thus, rna modification seems to influence recognition of exogenous rnas, for example, ribose '-o-methylation of mrna at the '-end provides a molecular signature for the discrimination of self and non-self rna (züst et al., ) . prior to immune cells performing their immunological action on target cells, they need to receive a suitable 'maturation' signal to enhance clonal expansion and acquire the effector function. activation signals occur in peripheral lymphoid organs through a concerted interaction with mhc molecules. the major histocompatibility complex (mhc) regulates the cell-mediated immune response(s). there are recent reports on the regulatory role of mirnas on mhc class i and ii molecules (wongfieng et al., ; xie et al., ) . for instance, mhc class i chain related protein b present in natural killer (nk) cells was associated to cell-mediated antitumor immune response through engagement with the nkg d receptor (xie et al., ) . the expression level of this receptor has been shown to be regulated by exogenous mir- c mimics in nk cells . based on the temporal and spatial regulation which determines the expression patterns of mirnas, that suggestion was made for the participation of complex regulatory networks composed of several transcription factors influencing the expression level of 'each' mirna. the mhc class ii complex has been identified as a target in mammalian mirnas regulation (wongfieng et al., ) . in addition, some lncrnas have been identified as candidates to be translated into peptide fragments (guttman et al., ) suggesting, as several other 'indirect' lines of evidence, the the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). participation of ncrnas-derived peptides on mhc complexes (yewdell, ; mellman et al., ) . peptides bound to mhc molecules serve as recognizing fragments of antigen for the tcr. here, it has been shown that mirnas can influence the amplitude of tcr signalling modulating t cell fate decisions towards survival and maturation wells et al., ) . here it is worth noting the recently defined dual role of mirna in maintenance of the naive phenotype of cd t cells or clonal expansion, where modulation of let- mirnas levels is required (wells et al., ) . in a similar way to mhc class ii for t cells, bcr determines the fate of developing b cells allowing their adequate maturation or the development of self-reactive b cells leading to immunodeficiency of b cell malignancy. the mirnas set threshold for lymphocyte development targets several genes in the pi k pathway (simpson and ansel, ) . it is also important to highlight that pi k displays an inhibitory interaction on tlrs-induced production of il- , therefore limiting excessive th polarization and suggesting a potential immunoregulatory role for mirnas. a few relatively recent studies address the potential regulatory role of 'specific' mirnas in treg functions (jeker et al., ; warth et al., ) . these studies have shown that inhibition of mirna- a in c bl/ j mice led to reduced foxp expression dependent on the levels of mediators such as tgf and/or retinoic acid (ra) (jeker et al., ) . another study reported induction of treg cell differentiation regulated by a mirna 'network' influencing the promoting factor mtor (warth et al., ) . these studies revealed that the underlying molecular mediators responsible for the mirna regulation of t cell signalling pathways remain unknown. unlike animals, plant mirnas have not been associated to tgf nor ra. intersecting the rapidly emerging field of treg function, it has been discovered that ra controls both the homing and differentiation of treg. in addition, computational and systems biology approaches have identified mtor as a potential pathway to explain the cross-kingdom mirnas-mediated regulatory potential of camptotheca acuminata ). although many reports can be found regarding the immunomodulatory role of endogenous and viral ncrnas, very few evaluated the impact of plant ncrnas on immune function and homeostasis when ingested/absorbed by animals and/or humans. thus, this section was reviewed using scientific documents. however, no studies were found that directly evaluate the effect of plant exogenous ncrna and their effects on the immune system following dietary intervention trials. recent reports highlight the shortcomings of existing mirna measuring systems (i.e. qpcr) in comparison to sequencing procedures to explore mirnas in biological fluids chen et al., a) . these studies point out the considerable number of unmapped ncrnas as a major factor limiting the use of qpcr techniques. notably, the abundance level of identified exogenous rna sequences in biological fluids (i.e. human plasma and breast milk, and serum from mice) was rather low. in addition, the role of food matrix on ncrna delivery to their cellular or tissue targets has not been defined. although the identification of exogenous ncrnas in biological samples led to considerations on their role as cross-kingdom regulators of immune-related processes, the impact (i.e. influence and regulatory role) of exogenous plant ncrnas on immune function and homeostasis is inferential. exogenous rnas sequences have been found in plasma , serum (chen et al., b) and breast milk ) from humans and/or animals. these studies reported the presence of ncrnas in biological fluids, although their impact on immune function remains to be fully clarified. the recent discovery of blood circulating rnas originating from foods seems to indicate that certain dietary plant mirnas can be absorbed at significant levels . however, the potential contribution of distinct 'plant food matrices' on ncrnas the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). absorption has not been addressed as it has been described for viral rnas (seljelid et al., ; zou et al., ) . the existing studies show that adequate antigen-processing processes at the intestinal level can be avoided by ncrnas bound to coadjuvant molecules (i.e. polycations, cationic lipids, phospholipids) (seljelid et al., ; zou et al., ; vertzoni et al., ) . notably, phospholipid homeostasis is central in the response to toll like receptors (tlrs) activation through a type i interferon 'autocrine-paracrine' loop (song et al., ) . in biological samples, rna sequences have been reported from most common gm food matrices, including corn (zea mays), rice (oryza sativa japonica group), soybeans (glycine max), tomato (solanum lycopersicum) and grape (vitis vinifera) . the identification of rice mir- a at fm concentration level in various mouse tissues may indicate the possibility of a cellular active uptake system for circulating rna . these data are in agreement with the role of defined food matrices as effective delivery systems protecting ncrnas from interaction with macrophages, dendritic cells, b-lymphocytes, and t-lymphocytes found in peyer's patches and other sites of gut-associated lymphoid tissue. however, several other studies contradict these results (see section . . . for details). enrichment of mirnas with targets related to immune response has been observed in colostral milk . colostrum represents a primordial food driving the first immunization of offspring and provides the nutritional needs of their immature organs in the earliest stages of life. the facts that immune-related mirnas from host, and exogenous plant mirnas (from bamboo diet to milk exosomes of giant panda) targeting synapse organization, neuron migration or axon guidance are enriched in giant breast milk of panda (restricted to a diet primarily comprising bamboo) indicate that breast milk may facilitate dietary intake of plant mirnas by infants for possible regulation of postnatal development ). very few studies address feeding plant ncrnas to animals (i.e. weaning, adult and old age) to evaluate induction of specific adaptive immune response(s) (i.e. th , th or th ). similarly, there is a lack of data on the impact of exogenous plant ncrnas on expression of mhc-ii molecules to assess the potential allergenicity of these rnas. additionally, the different immune response(s) induced by the administration of distinct exogenous plant ncrnas and well-known weak or strong immune inducers by different routes (intraperitoneal or intragastric) have not been tested. understanding of the allergenic functions of exogenous plant ncrnas is comparatively meagre in relation to proteins. the homology of distinct ncrnas could elicit different immune response(s) as proteins showing homology values up to % on their amino acid sequences induce different igg and igg responses (adel-patient et al., ) . for example, the literature search identified one study on safety assessment of the possible effects of gm-triticale on the immune system using a murine c bl/ model (krzyżowska et al., ) . immunoblotting analysis in serum showed significantly increased il- levels, but not those of ifn-γ. it was concluded that multigenerational use of feeds for rodents containing the gm-triticale leads to expansion of the b cell compartment in the secondary lymphoid organs not associated to allergic response(s) (krzyżowska et al., ) . however, this study did not report the ncrna content in gmtriticale. efforts over the last decade have focused on elucidating the role of ncrnas in immune function. although no immunogenic potential has been found, ncrnas implications in pathways regulating the production of inflammatory mediators (witwer et al., ) , the development of hematopoietic cells (satpathy and chang, ) , regulation of tlrs signalling (carpenter et al., ) and changes in t cells needed to support clonal expansion and growth have been highlighted. papers on ncrnas-mediated regulation of the amplitude and quality of immune response(s) could not be found. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). at the moment, knowledge on the role of ncrnas in immune responses at systems-level remains incomplete. earlier reports defined significant differences in the effects of dsrna (poly i:c) in innate immune responses as a function of 'length' ( . - . kb and . - . kb), depending on cell type (i.e. splenocytes, pbmcs, raw . and thp- ) (mian et al., ) . notably, it was reported that the 'shorter' dsrna caused higher immunomodulatory effects. poly i:c, a synthetic analogue of viral dsrna, has long been known as a strong inducer of innate immune responses by interacting with tlr in the endosome. following ligand recognition, specific ligand cascades involving interferon regulatory proteins and nuclear factor kappa b (nf-b) are activated to produce inflammatory mediators (démoulins et al., ) . similarly, signal-specific lncrnas have been reported in dendritic cells (dcs) stimulated with tlr agonists, % of which clustered with nf-b signalling components (guttman et al., ) . further approaches also identified upregulated responses in a long intergenic noncoding rna (lincrna) in response to tlr- , - , - and - , but not tlr- in macrophages (carpenter et al., ) . specifically, lincrna-cox was found to mediate both activation and repression of immune response genes dependent on interactions of lincrna-cox with heterogeneous nuclear ribonucleoprotein a/b and a /b . a further study determined that lincrna-cox plays a key role as a coactivator of nf-b for the transcription of late-primary inflammatory response genes in macrophages through modulation of epigenetic chromatin remodelling (hu et al., ) . due to the relevance of gut microbiota in immune system development and homeostasis, the possibility of modulating the gut microbiota through dietary exogenous ncrnas was reviewed. as described in section . , during the preparatory phase this novel topic was identified as relevant to this literature review. this additional section serves as a general introduction to emphasize the relevance of the possible modulation of the immune system by dietary exogenous ncrnas through the gut microbiota, and to identify gaps pointing to needs for future studies relevant to food/feed risk assessment of ncrnabased gm plants. specific details of the possible mechanisms of action, if any, are outside the scope of this review. due to the novelty of this topic and the lack of information on the scientific literature, only documents were reviewed for this section. the intestinal tract harbours a complex community (microbiota), which has an enormous impact not only on the nutritional, but also immune status of the host. a balanced gut microbiota composition confers benefits to the host, while microbial imbalances are associated with metainflammatory disorders. the dynamic processes initiated upon birth define the microbiota composition that co-evolves with the host and its environment. thus, colonization of the intestine in early life seems particularly important as it represents one of the major environmental stimuli for immune system maturation. particularly, breast feeding is crucial to influencing early intestinal colonization. as previously indicated, development of emerging 'next generation sequencing technologies' has allowed identification of a significant fraction of the circulating rna in human plasma that originated from exogenous bacterial and fungi species . this study, based on microarray profiling results, reported that plasma can contain ncrnas from common foods influencing the expression profile of a number of genes in the cells. however, several other studies suggest that the quantitative amount of ncrnas are irrelevant for influencing gene expression (see section . . . for details). though the interaction between humans and their environments, particularly microbes, raises the possibility of some sort of feedback signalling process, it is still not fully understood. the literature search showed how few publications address the role of ncrnas in shaping gut microbiota. emerging the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). evidences point out interactions between environmental factors and inter-species gene regulation facilitating host control of the gut microbiota choi et al., ) . however, the impact of exogenous plant ncrnas on gut microbiota modulation remains elusive. increasing evidence demonstrates that a significant number of ncrnas are found in the extracellular media, and it is apparent that mammalian cells release srna that might carry functions which transcend the confines of single cells (valadi et al., ) . few studies have identified changes in mirna expression in human stool. overall, they provide an inconsistent association between their level and dietary habits and lifestyle (tarallo et al., ; . in this context, the survival of exogenous (orally administered) plant mirnas in faeces from mice has been observed . although the amount of particular mirnas such as mir- that survived the gi tract reached a maximum of . %, this study does not support consistent survival rates for distinct ncrnas. a recent study identified mirnas secreted by intestinal epithelial cells in intestinal contents and supports their role in modulating gut microbiota composition . this study reports the potential of host mirnas to enter bacteria and co-localize with microbial nucleic acids. similarly, exogenous plant mirnas that were present in animal faeces were indicated as being primarily acquired orally . recently, bacterial rnas comparable in size to mirnas have been identified that could serve as signalling molecules mediating bacteria-to-human interaction (choi et al., ) . these data were obtained from outer membrane vesicles produced by periodontopathogens and were able to exert 'slight' immunosuppressive effects on t cells cytokine production (il- , il- and il- ). mirna expression levels and amount in stool appear to be modulated by diet (micro-and macronutrients, phytochemicals) without excluding the possibility that the results were affected by other lifestyle factors; however, the influence of gm plant exogenous ncrnas has not yet been exhaustively studied in animals or humans. distinct innate immune mediators participating in the recognition and discrimination between the distinct nucleic acids have been identified; thus, additional definition of the dynamic interplay between the different 'sensors' (i.e. tlrs, rig-i/mda , the interferon response effectors protein kinase r and rnase l) in response to ncrnas have to be clarified. the chemical modifications observed in plant ncrnas highlight the need to address the detection and measurement of exogenous plant ncrnas in body fluids, as well as the sensitivity and specificity of these rnas to cellular and tissue targets. although many reports focusing on synthetic ss-and dsrna mimics can be found in the literature, scientifically assessed direct hazardous impacts of food and feed on fauna and flora are scarce and conflicting. notably, there is a need to evaluate the effects of exogenous plant ncrnas at the system biology level and in human trials. current knowledge on the immune functions of exogenous ncrnas is comparatively lacking. most studies use cell models based on isolated cells that could not mimick the whole set of players that determine immune response(s), which are necessary for mechanistic studies, but need approach cell-to-cell communication in target tissues. additionally, these studies should focus on the relationship of immune responses to production of ifns since tlrs signalling converge on the production of this immune regulatory factor. to better understand the impact of exogenous ncrnas in host microbiota composition and diversity, further studies are needed to establish the microbial targets and their effect on physiological conditions. understanding the basic biological aspects of plant-derived exogenous ncrna (including their half-life and stability), gathering information on human and animal exposure by diet to these molecules, their subsequent bioavailability and possibility to exert local or systemic effects, and reviewing the experience from the development of rna-based therapeutics are relevant aspects for the risk assessment of ncrnabased gm food and feed. the literature includes very few studies evaluating the half-life of plant-derived ncrnas. while no specific studies on plant mirnas and lncrnas half-life were found in this review, the studies on plant circrnas half-life suggest that they exhibit considerable stability as compared to linear rnas (section . . . ). in mammals, the few reports related to ncrnas half-life suggest that mirnas and circrnas are generally much more stable than mrna, while lncrnas have a half-life like that of mrna. however, when assessing the stability of plant ncrnas outside the plant (section . . . .), compelling evidence exists that plant mirnas are highly stable under different conditions including food storage, processing, cooking, or simulated digestion. moreover, they seem to survive after long incubation in serum, or are detected in the gastric content of mice, suggesting that plant mirnas are more resistant to degradation than synthetic or animal mirnas. this high resistance has been attributed to the unique characteristics of plant mirnas (and sirnas), in contrast to mirnas from mammalian and other organisms; the '-omethylation at their ' end which confers plant mirnas more resistance to degradation (section . . . ). efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). the experience from development of ncrna therapeutics (section . . ) shows that unmodified nucleic acids (i.e. rnas) exhibit very low stability in biological media and are subjected to rapid nuclease mediated degradation. indeed, a plethora of rnases are encoded within most genomes, often with overlapping activities, making redundancy a general feature of rna degradation systems. consequently, different chemical modifications or conjugations have been introduced to potential rna-based therapeutics (section . . . ) to improve their rna-binding affinity, their in vivo nuclease stability and their pharmacokinetic and pharmacodynamic properties. the available literature also suggests that naked or unmodified rnas parenterally administered (e.g. iv) are rapidly cleared from circulation and their presence in the kidney and urine has been reported immediately after administration (section . . ). similarly, following oral administration naked or unmodified exogenous rnas are rapidly degraded when exposed to the gi harsh conditions, and different delivery vehicles for exogenous rnas have been developed. in general, and in contrast to chemically modified or formulated rnas, no major biological effects have been observed for naked rnas, which has probably limited further evaluation in humans (section . . . ). although certain disease conditions may influence the pharmacokinetics of exogenous rnas, there are no studies that specifically evaluate this for naked unmodified exogenous ncrnas. to exert a biological effect, an exogenous ncrna first needs to reach the intended target tissue in sufficient quantity levels, this implying the necessity to overcome many biological barriers (section . . ). considering the oral intake as the main possible entry point of exogenous plant ncrnas into both animals and humans, the first major barrier is the mammalian gi tract, which encompasses a group of extracellular and cellular barriers. extracellular barriers include the presence of different enzymes (i.e. nucleases), the harsh environment and a net negative charged mucous layer. the cellular barriers include a three-layer (epithelial cells or enterocytes, the lamina propria and the muscularis mucosa) barrier known as the intestinal mucosa. possible ncrnas passage between cells is limited by the presence of tight junctions and the pore size in the human intestine would prevent the passage of all but mirnas, which are the smallest ncrnas. although crossing the cells by transcytosis may be possible, this mechanism implies facing new intracellular barriers, such as nucleases, recycling of ncrnas back to the lumen and nuclear uptake. however, m-cells, present in the gut epithelium interspersed between enterocytes, present certain characteristics that would make them more amenable for rna uptake. a second barrier is represented by a set of obstacles during the passage from the intestine to the target tissue, which encompasses plasma and tissue nucleases. once in the circulatory system, exogenous ncrnas are subjected to distribution and elimination. for example, small rnas would be rapidly cleared by the kidney due to their small size. furthermore, ncrnas need to escape the reticuloendothelial system, the function of which is to clear foreign pathogens. finally, they would have to cross the vascular endothelial barrier to reach the target tissue, facing hurdles like those in the enterocytes. packaging or incorporation of ncrnas into extracellular transport systems (i.e. exosomes/microvesicles or inclusion in ribonucleoprotein complexes) would confer certain resistance to nucleases and other barriers. ncrnas must enter the target cells to exert their functions. the molecular mechanisms of exogenous ncrna cellular uptake have been inferred from studies performed when developing strategies for nucleic acids delivery as therapeutics and is also largely derived from invertebrates, with little data reported in mammals. descriptions exist of receptor-mediated uptake of oligonucleotides through the sidt and sidt proteins, although this is now in doubt since they have been described as cholesterol transporters. due to the charged nature of rnas, cellular uptake could be achieved by endocytosis after which they would enter the system of intracellular trafficking through multiple membrane-bound compartments. ncrnas must exit these compartments to reach their functional sublocation within the efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). cell, either the cytosol or the nucleus, while simultaneously escaping degradation in the lysosomes. specialized barriers such as the blood-brain barrier or the placental barrier represent additional obstacles to overcome. while rna content in plant-derived foods varies (≈ mg/g of tissue), it could be estimated that when consuming a daily dose of total fruit and vegetables of ≈ g/day, humans theoretically ingest mg of total rnas (dietary intake of plant rna may typically range from . - g/person/day) (section . . . ). humans following special diets, such as vegetarians and vegans, have higher intake of plant origin rnas, but estimated exposure to such rnas implies only a -fold increase on average (section . . . ). it has been estimated that small rnas make up far less than % of total rna in plants, suggesting a very low exposure to plant ncrnas under normal conditions. whether sufficient levels of exogenous plant ncrnas can be consumed in the diet to exert a biological effect is also another aspect that needs to be evaluated. some examples in the literature suggest that for certain plant mirnas (i.e. mir- a) a person would need to consume kg of cantaloupe to reach the copies of mirna per cell possibly determining an effect. these aspects should be evaluated for each plant ncrna (on a case basis), considering that some plant-derived mirnas have shown unexpectedly high resistance to degradation and greater bioavailability. the available literature indicates a widespread presence of exogenous rnas (including plant derived ncrnas, i.e. small rnas) in the biological fluids of humans and animals (section . . ). whether these rnas are derived from dietary intake is unclear. their generally low abundance and lack of enrichment in tissues mostly exposed to dietary changes (i.e. liver) suggest that some of may have be originated as technical artefacts or through contamination. very few studies address biological effects of dietary exogenous ncrnas in the gi tract and its annex glands (e.g. liver) (section . can directly target mammalian genes through rnai effects, but still clarification is needed if exogenous plant ncrnas can overcome all the biological barriers to reach and interact with their possible targets. exogenous plant-derived ncrnas have been found in exosomes or macrovesicles. how they reach these types of structures in biological fluids is unknown. in summary, supporting and contradicting evidence concerning the existence of systemic effects of dietary plant-derived exogenous ncrnas is heavily debated. important aspects such as the precise mechanism/s of transport of plant ncrnas from food into the systemic circulation, the amount of exogenous ncrnas reaching tissues or the molecular mechanisms of cellular uptake need to be determined. while several in vitro studies cover the participation of exogenous ncrnas in the dynamic network to modulate innate immunity responses, few tackle the immunomodulatory effects of plant ncrnas in adaptive immune response (section . ). none of these studies have considered the potential impact of exogenous dietary plant-derived ncrnas at the systemic level. since the first publication suggesting a possible cross-kingdom effect by dietary ncrna (through rice mir- a) the knowledge on exogenous ncrnas, and particularly plant-derived exogenous ncrnas, on efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). their fate when ingested has increased. however, the knowledge useful to support the assessment of dietary ncrna such as ncrna-based gm food and feed still faces several gaps. an important aspect needing further investigation is the stability of plant ncrnas. the meagre available literature contains a very few studies of the half-life of plant ncrnas (i.e. mirnas, lncrnas and circrnas) both inside the plant cell and outside the plant. specifically, the stability of circrnas should be assessed due to their apparently high stability. moreover, very few reports focus on assessment of plant ncrnas stability in mammalian systems, when ingested. understanding the molecular basis that confers plant ncrnas stability is of great importance and needs to be further evaluated. for instance, plant mirnas and sirnas carry modifications that do not occur in mammals. these modifications, such as the presence of a methyl group located on the ' nucleotide ribose, could hypothetically have an impact on the plant mirnas stability in mammalian systems/cells due to either the lack of the appropriate enzymes for recognition and degradation of plant mirnas by mammalian cells, or the increased stability to degradation by mammalian rnases. the circularized structure of plant circrnas would hypothetically confer them increased stability to mammalian rnases. if this is the case, plant mirnas and circrnas could exhibit a much longer half-life in mammalian systems than expected, especially when compared to mammalian mirna or circrnas. this would increase their chances of reaching the appropriate target tissue and encountering suitable target molecules within the cells. the half-life of plant ncrnas within mammalian cells is also an important aspect to consider. experiments are needed with labelled plant ncrnas, first in transfected mammalian cells (in vitro studies) and then orally administered to experimental animals (in vivo studies). radioactive probes or molecules of very low molecular weight, such as biotin or fluorescein, should be used, in order to prevent distorting the actual size of the studied plant ncrna; this is important when studying mirnas stability due to their small size. another aspect possibly relevant for risk assessment is to understand whether and how ingested gm plant-derived ncrnas can affect the expression levels of other ncrnas and other rnas, due to putative compensatory circuits and codifying rnas. whole transcriptome sequencing, comparing the rna levels of unmodified (wild type) to those of modified (transgenic) plants coul be useful to this aim. although available information suggests that many biological barriers exist in mammalian system to ingested plant exogenous ncrnas preventing these to exert a possible local or systemic effect, several gaps are still present. putative pathways of dietary exogenous ncrnas after ingestion and the many transporters potentially involved (i.e, in cellular uptake, or barrier passage transport pathways) in the transit from the gut to the target tissues should be characterized using standard molecular biology gainof-function and loss-of-function approaches. also, the many routes of mammalian intracellular trafficking to the specific intracellular target should be studied. to this aim, ncrnas isolated from human diets should be used preferentially, since most of the incomplete current knowledge comes from the rna-therapeutics field or is derived from the invertebrate world. which molecular mechanisms support the possible passage through specialized barriers (i.e. blood-brain barrier or the placental barrier), is another critical aspect requiring further studies. while there are in vitro studies describing the interaction between plant mirnas and mammalian mirna silencing complexes possibly leading to target repression, these findings need to be experimentally validated in vivo. therefore, more studies are needed to understand the possibility of processing plant ncrnas in mammalian cells. it is also unknown if ingested plant mirnas reaches or needs to reach a necessary level to exert a biological effect. for instance, studies in mammals suggest that the threshold for target gene regulation is > copies per cell for a certain study; however, another study suggests around to . copies of mammalian mirna per cell. clearly, a minimum amount of small rnas efsa supporting publication :en- the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). would be required to achieve biologically relevant effects on gene expression. on a case by case basis it would be useful to evaluate the amount of consumption of a given ncrna in a normal diet. it is also relevant to study if exposure to these plant ncrnas could change under certain pathological conditions (i.e. compromised intestinal permeability or renal function) or dietary patterns (i.e. vegetarian or vegan diets). although tissue accumulation of dietary plant exogenous ncrnas has not been reported to date, it seems clear that plant mirnas and other exogenous rnas are present in biological fluids from humans and animals. the biological significance of their presence is still unknown and needs to be addressed. methods that do not require amplification for detection should be used. more quantitative approaches to determine the levels of plant-derived exogenous ncrnas should be determined both in biological fluids and tissues to understand the magnitude of presence. for example, this could be partially obtained from studies reporting the presence of exogenous rnas in biological fluids using public small rna-seq databases. in the case of plant-derived mirnas, the use of sodium periodate oxidation of samples could be a good start to verifying the presence of genuine plant-derived mirnas from diet in human and animal biological fluids. the immunomodulatory effects of exogenous rnas have been widely described in the literature. however, there is no in vivo information regarding the immunomodulatory effects of dietary exogenous ncrnas. these types of studies should be performed both in vitro and in vivo to determine if the different types of ncrnas consumed in the diet can affect the immune system. in the case of target gene repression by plant ncrnas investigations in specific animal models could be considered. expression levels of other ncrnas and rnas, which may be modified due to putative compensatory circuits could be evaluated in these models, as well as information on the stability and half-life of the specific ncrna in specific cells and systemically following ingestion. genome-wide high-resolution mapping of exosome substrates reveals hidden features in the arabidopsis transcriptome genome-wide identification of circular rnas in arabidopsis thaliana a lariat-derived circular rna is required for plant development in arabidopsis pattern formation via small rna mobility small rnas are on the move literature review of baseline information on rnai to support the environmental risk assessment of rnai-based gm plants identification of circular rnas from the parental genes involved in multiple aspects of cellular metabolism in barley a group i plant intron accumulates as circular rna forms with extensive ' deletions in vivo toward a consensus on the binding specificity and promiscuity of prc for rna a stepwise pathway for biogenesis of -nt secundary sirnas and spreading of dna methylation site-specific phosphorylation profiling of arabidopsis proteins by mass spectrometry and peptide chip analysis hierarchical action and inhibition of plant dicer-like proteins in antiviral defense characterization of stress-responsive lncrnas in arabidopsis thaliana by integrating expression, epigenetic and structural features identification and characterization of human testis derived circular rnas and their existence in seminal plasma the rna-binding proteins hyl and se promote accurate in vitro processing of pri-mirna by dcl genome-wide discovery of circular rnas in the leaf and seedling tissues of arabidopsis thaliana intra-and intercellular rna interference in arabidopsis thaliana requires components of the microrna and heterochromatic silencing pathways dicer-like is required for rna interference and produces the -nucleotide small interfering rna component of the plant cell-to-cell silencing signal plant mobile small rnas the arabidopsis thaliana doublestranded rna binding protein drb directs guide strand selection from microrna duplexes naturally occurring variations in sequence length creates microrna isoforms that differ in argonaute effector complex specificity the xist lncrna exploits three-dimensional genome architecture to spread across the x chromosome identification of nuclear dicing bodies containing proteins for microrna biogenesis in living arabidopsis plants phased, secondary, small interfering rnas in posttranscriptional regulatory networks antisense rna polymerase ii divergent transcripts are p-tefb dependent and substrates for the rna exosome long noncoding rnas in cell-fate programming and reprogramming target mimicry provides a new mechanism for regulation of microrna activity location of a possible mirna processing site in smd /smb nuclear bodies in arabidopsis partially redundant functions of arabidopsis dicer-like enzymes and a role for dcl in producing trans-acting sirnas short integuments /suspensor /carpel factory, a dicer homolog, is a maternal effect gene required for embryo development in arabidopsis oncogenic role of fusion-circrnas derived from cancer-associated chromosomal translocations topological organization of multichromosomal regions by the long intergenic noncoding rna firre natural rna circles function as efficient microrna sponges vernalization-mediated epigenetic silencing by a long intronic noncoding rna flowering locus c's lessons: conserved chromatin switches underpinning developmental timing and adaptation transitivity-dependent andindependent cell-to-cell movement of rna silencing specific interactions between dicer-like proteins and hyl /drb-family dsrna-binding proteins in arabidopsis thaliana phosphate utilization efficiency correlates with expression of low-affinity phosphate transporters and noncoding rna, ips , in barley gene silencing by micrornas: contributions of translational repression and mrna decay cyclophilin facilitates hsp -mediated risc assembly in plants in vitro assembly of plant rna-induced silencing complexes facilitated by molecular chaperone hsp a rice cis-natural antisense rna acts as a translational enhancer for its cognate mrna and contributes to phosphate homeostasis and plant fitness the nuclear pore protein attpr is required for rna homeostasis, flowering time, and auxin signaling dicer- and r d -l catalyze microrna maturation in drosophila molecular insights into mirna processing by arabidopsis thaliana serrate covalent circularization of exogenous rna during incubation with a wheat embryo cell extract small rnas as guardians of the genome fast-forward genetics identifies plant cpl phosphatases as regulators of mirna processing factor hyl the evolution and diversification of dicers in plants passenger-strand cleavage facilitates assembly of sirna into ago -containing rnai enzyme complexes circular single-stranded rna replicon in saccharomyces cerevisiae rna-directed dna methylation: an epigenetic pathway of increasing complexity intercellular and systemic movement of rna silencing signals circular rnas are a large class of animal rnas with regulatory potency sorting of small rnas into arabidopsis argonaute complexes is directed by the ' terminal nucleotide morc family atpases required for heterochromatin condensation and gene silencing small silencing rnas in plants are mobile and direct epigenetic modification in recipient cells specificity of argonaute -mir interaction and dual functionality in tas trans-acting sirna formation evolution of animal and plant dicers: early parallel duplications and recurrent adaptation of antiviral rna binding in plants literature review of baseline information to support the risk assessment of rnai-based gm plants systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions microrna is a long-distance signal for the regulation of plant phosphate homeostasis evidence for nuclear processing of plant micro rna and short interfering rna precursors nuclear processing and export of micrornas in arabidopsis carpel factory, a dicer homolog, and hen , a novel protein, act in microrna metabolism in arabidopsis thaliana rde- preferentially binds long dsrna and its dimerization is necessary for cleavage of dsrna to sirna rna exosome-regulated long non-coding rna transcription controls super-enhancer activity prediction of trans-antisense transcripts in arabidopsis thaliana genome-wide identification of long noncoding natural antisense transcripts and their responses to light in arabidopsis identification and characterization of circrnas in pyrus betulifolia bunge under drought stress analysis of noncoding transcriptome in rice and maize uncovers roles of conserved lncrnas associated with agriculture traits a long noncoding rna maintains active chromatin to coordinate homeotic gene expression arabidopsis noncoding rna mediates control of photomorphogenesis by red light identification of circular rnas and their targets in leaves of triticum aestivum l. under dehydration stress importin is a gene silencing factor that targets argonaute proteins to distinct mrnas noncoding transcription by rna polymerase pol ivb/pol v mediates transcriptional silencing of overlapping and adjacent genes expression of arabidopsis mirna genes genetic and functional diversification of small rna pathways in plants identification and characterization of wheat long non-protein coding rnas responsive to powdery mildew infection and heat stress by using microarray analysis and sbs sequencing bidirectional promoters generate pervasive transcription in yeast functions of long intergenic non-coding (linc) rnas in plants widespread noncoding circular rnas in plants a systemic small rna signaling system in plants wavy leaf , an ortholog of arabidopsis hen , regulates shoot development by maintaining microrna and trans-acting small interfering rna accumulation in rice vigs, higs and figs: small rna silencing in the interactions of viruses or filamentous organisms with their plant hosts control of coleopteran insect pests through rna interference the expanding world of small rnas in plants genome-wide analysis of long noncoding rna stability structural variations and stabilising modifications of synthetic sirnas in mammalian cells circular rnas are long-lived and display only minimal early alterations in response to a growth factor host-delivered rnai: an effective strategy to silence genes in plant parasitic nematodes analysis of microrna turnover in mammalian cells following dicer ablation polysome arrest restricts mirna turnover by preventing exosomal export of mirna in growth-retarded mammalian cells a versatile monosaccharide transporter that operates in the arbuscular mycorrhizal fungus glomus sp is crucial for the symbiotic relationship with plants the drosophila rna methyltransferase, dmhen , modifies germline pirnas and single-stranded sirnas in risc uridylation of mature mirnas and sirnas by the mut nucleotidyltransferase promotes their degradation in chlamydomonas circular rnas are abundant, conserved, and associated with alu repeats regulation of small rna stability: methylation and beyond methylation protects mirnas and sirnas from a ' end uridylation activity in arabidopsis assessing the survival of exogenous plant microrna in mice effective detection and quantification of dietetically absorbed plant micrornas in human plasma adenylation of plant mirnas covalent circularization of exogenous rna during incubation with a wheat embryo cell extract silencing a cotton bollworm p monooxygenase gene by plant-mediated rnai impairs larval tolerance of gossypol rna-specific ribonucleotidyl transferases oral delivery of sirna and antisense oligonucleotides toxicogenomics of non-viral drug delivery systems for rnai: potential impact on sirna-mediated gene silencing activity and specificity nonviral delivery of synthetic sirnas in vivo a theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability oral nucleic acid therapy using multicompartmental delivery systems the adherent gastrointestinal mucus gel layer: thickness and physical state in vivo polymeric nano-and microparticle technologies for oral gene delivery isolation and properties of an extracellular nucleases of serratia marcescens guidance for industry on waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics classification system issues in oral nanoparticle drug carrier uptake and targeting in vitro and in vivo models for the study of oral delivery of nanoparticles alginate/poly-l-lysine microparticles for the intestinal delivery of antisense oligonucleotides multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract extracellular ribonucleases from bacillus subtilis. i. crystallization and specificity intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models first-pass elimination. basic concepts and clinical consequences key issues in non-viral gene delivery polymeric nanoparticle drug delivery technologies for oral delivery applications effect of sodium caprate on the intestinal absorption of two modified antisense oligonucleotides in pigs drug delivery of sirna therapeutics: potentials and limits of nanosystems oral delivery of antisense oligonucleotides in man the human microbiome project consortium. structure, function and diversity of the healthy human microbiome milk-derived exosomes for oral delivery of paclitaxel delivery of sirna to the mouse brain by systemic injection of targeted exosomes orally delivered sirna targeting macrophage map k suppresses systemic inflammation oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models oral nucleic acid therapy using multicompartmental delivery systems oral delivery of rnase p ribozymes by salmonella inhibits viral infection in mice protection and systemic translocation of sirna following oral administration of chitosan/sirna nanoparticles interleukin gene transfer prevents experimental colitis in rats inhibition of trkb limits development of the zebra finch song system commercial dairy cow milk micrornas resist digestion under simulated gastrointestinal tract conditions oral il- gene delivery in a microsphere-based formulation for local transfection and therapeutic efficacy in inflammatory bowel disease the role of nucleotides in human nutrition retroviral delivery of rna interference against marek's disease virus in vivo biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles systemic exosomal sirna delivery reduced alpha-synuclein aggregates in brains of transgenic mice pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver proteomics of extracellular vesicles: exosomes and ectosomes dietary yeast rna supplementation reduces mortality by aeromonas hydrophila in rohu (labeo rohita l.) juveniles effects of mannose density on in vitro and in vivo cellular uptake and rnai efficiency of polymeric nanoparticles peg-pla nanoparticles facilitate sirna knockdown in adult zebrafish heart exosome-mediated delivery of hydrophobically modified sirna for huntingtin mrna silencing oral delivery of small rna and dna colonic gene silencing using sirna-loaded calcium phosphate/plga nanoparticles ameliorates intestinal inflammation in vivo a randomized controlled trial of preoperative oral supplementation with a specialized diet in patients with gastrointestinal cancer a novel class of small rnas: trna-derived rna fragments (trfs) small non-coding rnas transfer through mammalian placenta and directly regulate fetal gene expression assessing the survival of exogenous plant microrna in mice effective detection and quantification of dietetically absorbed plant micrornas in human plasma human milk exosomes and their micrornas survive digestion in vitro and are taken up by human intestinal cells detection of plant mirnas abundance in human breast milk in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? detection of dietetically absorbed maizederived micrornas in pigs exosomal micrornas in giant panda (ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs unsuccessful detection of plant micrornas in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil a novel chemopreventive strategy based on therapeutic micrornas produced in plants annotation of the goat genome using next generation sequencing of microrna expressed by the lactating mammary gland: comparison of three approaches expressional analysis of immune-related mirnas in breast milk identification of stable, high copy number, medium-sized rna degradation intermediates that accumulate in plants under non-stress conditions determination of the potential bioavailability of plant micrornas using a simulated human digestion process human breast milk mirna, maternal probiotic supplementation and atopic dermatitis in offspring the effect of dietary ginseng polysaccharide supplementation on the immune responses involved in porcine milk-derived esrnas immune modulatory function of abundant immunerelated micrornas in microvesicles from bovine colostrum uptake and function studies of maternal milk-derived micrornas mining of public sequencing databases supports a nondietary origin for putative foreign mirnas: underestimated effects of contamination in ngs literature review of information supporting food/feed risk assessment of rnai-based gm plants www.efsa.europa this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document alternative mirnas: human sequences misidentified as plant mirnas in plant studies and in human plasma transfer and functional consequences of dietary micrornas in vertebrates: concepts in search of corroboration: negative results challenge the hypothesis that dietary xenomirs cross the gut and regulate genes in ingesting vertebrates, but important questions persist real-time quantitative pcr and droplet digital pcr for plant mirnas in mammalian blood provide little evidence for general uptake of dietary mirnas: limited evidence for general uptake of dietary plant xenomirs the intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma caco- cells and rat small intestinal iec- cells the levels of human milk micrornas and their association with maternal weight characteristics detection of dietary plant-based small rnas in animals anomalous uptake and circulatory characteristics of the plant-based small rna mir the atypical genesis and bioavailability of the plant-based small rna mir : bulking up while breaking down bioavailability of transgenic micrornas in genetically modified plants exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna analysis of plant-derived mirnas in animal small rna datasets immune-related micrornas are abundant in breast milk exosomes honeysuckle-encoded atypical microrna directly targets influenza a viruses one step forward, two steps back; xeno-micrornas reported in breast milk are artifacts small rnas from plants, bacteria and fungi within the order hypocreales are ubiquitous in human plasma dietary microrna database (dmd): an archive database and analytic tool for food-borne micrornas lack of detectable oral bioavailability of plant micrornas after feeding in mice survey of + data sets from human tissue and body fluid reveals xenomirs are likely artifacts plant mirnas found in human circulating system provide evidences of cross kingdom rnai detection of plant mirnas abundance in human breast milk in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? optimization of enzymatic reaction conditions for generating representative pools of cdna from small rna computational characterization of exogenous micrornas that can be transferred into human circulation mining of public sequencing databases supports a nondietary origin for putative foreign mirnas: underestimated effects of contamination in ngs the complex exogenous rna spectra in human plasma: an interface with human gut biota? the spectrum of circulating rna: a window into systems toxicology detection of an abundant plant-based small rna in healthy consumers oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models exosomes as a nanodelivery system: a key to the future of neuromedicine? endogenous microrna can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen micrornas in serum of mice lack of detectable oral bioavailability of plant micrornas after feeding in mice high-throughput sequencing of rna silencingassociated small rnas in olive (olea europaea l.) plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic hjt-srna-m autosomal recessive hypercholesterolemia caused by mutations in a putative ldl receptor adaptor protein exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges extensive degradation and low bioavailability of orally consumed corn mirnas in mice an improved method to quantitate mature plant microrna in biological matrices using modified periodate treatment and inclusion of internal controls this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document cross talk between adipose tissue and placenta in obese and gestational diabetes mellitus pregnancies via exosomes grape exosome-like nanoparticles induce intestinal stem cells and protect mice from dss-induced colitis survey of + data sets from human tissue and body fluid reveals xenomirs are likely artifacts cross-kingdom regulation of putative mirnas derived from happy tree in cancer pathway: a systems biology approach small non-coding rnas transfer through mammalian placenta and directly regulate fetal gene expression assessing the survival of exogenous plant microrna in mice reply to dr. witwer's letter to the editor effective detection and quantification of dietetically absorbed plant micrornas in human plasma plant mirnas found in human circulating system provide evidences of cross kingdom rnai detection of dietetically absorbed maizederived micrornas in pigs negligible uptake and transfer of diet-derived pollen micrornas in adult honey bees the transport mechanism of extracellular vesicles at the blood-brain barrier unsuccessful detection of plant micrornas in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil a novel chemopreventive strategy based on therapeutic micrornas produced in plants interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles high-throughput assessment of microrna activity and function using microrna sensor and decoy libraries milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation corn rootwormactive rna dvsnf : repeat dose oral toxicology assessment in support of human and mammalian safety a -day oral toxicity evaluation of small interfering rnas and a long double-stranded rna targeting vacuolar atpase in mice citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress cml xenograft growth by inducing trail-mediated cell death this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document ineffective delivery of diet-derived micrornas to recipient animal organisms adipose-derived circulating mirnas regulate gene expression in other tissues uptake and function studies of maternal milk-derived micrornas contamination or artifacts may explain reports of plant mirnas in humans alternative mirnas: human sequences misidentified as plant mirnas in plant studies and in human plasma real-time quantitative pcr and droplet digital pcr for plant mirnas in mammalian blood provide little evidence for general uptake of dietary mirnas: limited evidence for general uptake of dietary plant xenomirs detection of an abundant plant-based small rna in healthy consumers anomalous uptake and circulatory characteristics of the plant-based small rna mir bioavailability of trasngenic micrornas in genetically modified plants exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in danio rerio molecular basis of mammalian transmissibility of avian h n influenza viruses and their pandemic potential exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna honeysuckle-encoded atypical microrna directly targets influenza a viruses plant micrornas in larval food regulate honeybee caste development to evaluate oral toxicity of exogenous ncrnas in mice, petrick et al. administered for days a repeated oral dose of sirnas and dsrna and evaluated several parameters including body weight, food consumption, clinical observations, clinical chemistry, haematology, gross pathology, or histopathology endpoints it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). signs (mortality, abnormalities, and sign of pain and distress), body weight and food consumption, haematology, organ weight, or pathology results. the minor differences observed in certain parameters were limited to single intervals, were not dose-related, and were attributed to interanimal variability torula yeast rna (rna negative control) at mg/kg/day or a control vehicle were used. ten animals per sex and per group were used. no treatment-related effects were observed on clinical signs (mortality, or sign of pain and distress), body weight and food consumption, haematology, organ weight, or pathology results. minor changes in selected parameters in selected groups were observed, but these were attributed by the authors to normal variability and not treatment-related effects this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document ecological risk assessment for dvsnf rna: a plant-incorporated protectant with targeted activity against western corn rootworm literature review of baseline information on rnai to support the environmental risk assessment of rnai-based gm plants a comparative evaluation of the regulation of gm crops or products containing dsrna and suggested improvements to risk assessments endogenous small rnas in grain: semi-quantification and sequence homology to human and animal genes literature review of baseline information to support the risk assessment of rnai-based gm plants corn rootwormactive rna dvsnf : repeat dose oral toxicology assessment in support of human and mammalian safety a -day oral toxicity evaluation of small interfering rnas and a long double-stranded rna targeting vacuolar atpase in mice rnai technologies in agricultural biotechnology: the toxicology forum th annual summer meeting no impact of dvsnf rna on honey bee (apis mellifera l.) adults and larvae in dietary feeding tests microstructure and ultrastructure of highamylose rice resistant starch granules modified by antisense rna inhibition of starch branching enzyme a -day toxicology study of high-amylose transgenic rice grain in sprague-dawley rats a three generation reproduction study with sprague-dawley rats consuming high-amylose transgenic rice high-amylose rice improves indices of animal health in normal and diabetic rats studies performed with human cells were mainly carried out ex vivo using lymphocytes and, to a significant extent, macrophages. from the studies retrieved during the screening phase on immune cells in relation to exogenous ncrna %) from rat and ( . %) from mouse. and within the studies on immune cells other than lymphocytes (i.e. macrophages), the studies were allocated into ( . %) from human, ( . %) from rat, and ( . %) from mouse. all these data clearly show that studies retrieved in relation to ncrnas and www this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document immunological and metabolomic impacts of administration of cry ab protein and mon maize in mouse role of toll-like receptors in antisense and sirna recognition of double-stranded rna and activation of nf-kappab by toll-like receptor rna regulation of the immune system therapy of respiratory viral infections with intranasal sirnas cd is a coreceptor of toll-like receptors and a long noncoding rna mediates both activation and repression of immune response genes identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen micrornas in serum of mice identification of dietetically absorbed rapeseed (brassica campestris l.) bee pollen mirnas in serum of mice chemical modification patterns compatible with high potency dicer-substrate small interfering rnas is rna interference involved in intrinsic antiviral immunity in mammals? poly (i:c) induced immune response in lymphoid tissues involves three sequential waves of type i ifn expression structural basis for cytosolic double-stranded rna surveillance by human oligoadenylate synthetase structural mechanism of sensing long dsrna via a noncatalytic domain in human oligoadenylate synthetase rnas containing modified nucleotides fail to trigger rig-i conformational changes for innate immune signaling this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document identification of rna sequence motifs stimulating sequence-specific tlr -dependent immune responses dsrna with ′ overhangs contributes to endogenous and antiviral rna silencing pathways in plants a tim- oligonucleotide aptamer enhances t cell functions and potentiates tumor immunity in mice ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincrnas ribosome profiling provides evidence that large noncoding rnas do not encode proteins chromatin signature reveals over a thousand highly conserved large noncoding rnas in mammals lack of interferon response in animals to naked sirnas species-specific recognition of single-stranded rna via toll-like receptor and small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway sequencespecific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr lincrna-cox promotes late inflammatory gene transcription in macrophages through modulating swi/snf-mediated chromatin remodeling microrna a marks regulatory t cells the role of alternative polyadenylation in the antiviral innate immune response the '-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor activation or inhibition design of noninflammatory synthetic sirna mediating potent gene silencing in vivo sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna immunostimulatory potential of silencing rnas can be mediated by a non-uridine-rich toll-like receptor motif suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna sequence-and target-independent angiogenesis suppression by sirna via tlr microrna as a new immune-regulatory agent in breast milk this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document the effect of multigenerational diet containing genetically modified triticale on immune system in mice cross-kingdom regulation of putative mirnas derived from happy tree in cancer pathway: a systems biology approach double-stranded rna-mediated tlr activation is enhanced by cd molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor the tlr signaling complex forms by cooperative receptor dimerization mir- a is an intrinsic modulator of t cell sensitivity and selection assessing the survival of exogenous plant microrna in mice structural basis of toll-like receptor signaling with double-stranded rna the host shapes the gut microbiota via fecal microrna in silico identification of plant mirnas in mammalian breast milk exosomes--a small step forward? exosomal micrornas in giant panda (ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs microrna- c promotes natural killer cell cytotoxicity via up-regulating the expression level of nkg d neonatal immune activation during early and late postnatal brain development differently influences depressionrelated behaviors in adolescent and adult c bl/ mice small self-rna generated by rnase l amplifies antiviral innate immunity a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells cancer immunotherapy comes of age length of dsrna (poly i:c) drives distinct innate immune responses, depending on the cell type oligonucleotide-based pharmaceuticals: non-clinical and clinical safety signals and non-clinical testing strategies cytosolic viral sensor rig-i is a '-triphosphate-dependent translocase on double-stranded rna aptamers for cd antigens: from cell profiling to activity modulation cd aptamers as powerful immune response modulators influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document aptamer-targeted attenuation of il- signaling in cd + t cells enhances antitumor immunity catalog of differentially expressed long non-coding rna following activation of human and mouse innate immune response. front immunol , long noncoding rna in hematopoiesis and immunity the effect of poly-l-lysine on the uptake of reovirus doublestranded rna in macrophages in vitro characterization of the mammalian rna exonuclease /nef-sp as a testis-specific nuclear ' → ' exoribonuclease microrna regulation of lymphocyte tolerance and autoimmunity advances in rna sensing by the immune system: separation of sirna unwanted effects from rna interference activation of the interferon system by short-interfering rnas type i interferons function as autocrine and paracrine factors to induce autotaxin in response to tlr activation antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals microrna expression in relation to different dietary habits: a comparison in stool and plasma samples interplay between dengue virus and toll-like receptors, rig-i/mda and micrornas: implications for pathogenesis exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects lps injection reprograms the expression and the ' utr of a cap gene by alternative polyadenylation and the formation of a gait element in ciona intestinalis the complex exogenous rna spectra in human plasma: an interface with human gut biota? induced mir- a expression represses mtor cooperatively with mir- to promote regulatory t-cell differentiation evolutionary dynamics and tissue specificity of human long noncoding rnas in six mammals toll-like receptor (tlr) immune modulation by unformulated small interfering rna or dna and the role of cd (in tlrmediated effects) homology modeling of human tolllike receptors tlr , , and ligand-binding domains modulation of let- mirnas controls the differentiation of effector cd t cells this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document microrna regulation of ifn-beta protein expression: rapid and sensitive modulation of the innate immune response . '-utr and '-utr regulation of micb expression in human cancer cells by novel micrornas microrna- is downregulated by histone deacetylase inhibitors and confers resistance to natural killer cytotoxicity in hepatocellular carcinoma cells ncrna-and pc methylation-dependent gene relocation between nuclear structures mediates gene activation programs plumbing the sources of endogenous mhc class i peptide ligands infalpha- b inhibitory effects on cd (+)cd (+)foxp (+) regulatory t cells in the tumor microenvironment of c bl/ j mice with melanoma xenografts stress-responsive regulation of long noncoding rnas' polyadenylation in oryza sativa exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna antiviral activity of human oligoadenylate synthetases-like (oasl) is mediated by enhancing retinoic acid-inducible gene i (rig-i) signaling lipid-mediated delivery of rna is more efficient than delivery of dna in non-dividing cells ribose '-omethylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda the present document has been produced and adopted by the bodies identified above as authors. this task has been carried out exclusively by the authors in the context of a contract between the european food safety authority and the authors, awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). (zhou et al., ) . twenty female and ten male rats per group in the first generation (f ) were fed the different diets. their pups, weanling/female/group and weanling/male/group were randomly selected as an f generation. the f generation was acquired by using the same procedure described above. at weaning, f generation rats were chosen, and rats/sex/group provided the corresponding diets and observed for weeks. no major differences in animal survival, health status, behaviour, body weight, food consumption, or reproductive capacity were observed with the different diets. some statistically significant differences were observed in animals given the transgenic rice diets compared to those receiving the standard diet for certain clinical chemistry parameters, in certain generations (alt, alkp, ldh, cholesterol, hdlc, ldlc). these changes were not considered adverse or biologically significant (zhou et al., ) . moreover, no evidence of altered incidence or altered severity of background changes was observed in any organ or tissues of the rats fed the three diets (zhou et al., ) . although minor changes in the mean relative weight of epididimides in male f adults was observed in rats fed the rice diets compared to the control group, no difference was observed between the transgenic rice fed rats and their near-isogenic line controls, suggesting overall that the transgenic line was unlikely to cause any risk to rat health in reproduction or development, even when consumed for up to three generations (zhou et al., ) . however, these studies did not evaluate any aspect related to rnai.heinemann et al. compared the history of risk assessment of gmos producing dsrnas in australia, new zealand and brazil, with a focus on regulatory context (heinemann et al., ) . the authors suggested some processes to properly assess the safety of dsrna-producing gm plants before their release or marketing. these include i) bioinformatics analysis to identify any likely, unintended targets of the dsrna in human and animals; ii) experimental procedures that would identify all new intended and unintended dsrna molecules in the gm product; iii) testing animal and human cells in tissue cultures for a response to intended and unintended effects of dsrnas from the product; iv) long-term testing on animals; and possibly v) clinical trials on human volunteers (heinemann et al., ) . in response to this article, the regulatory agency food standards australia new zealand (fsanz) published a document addressing regulation of gm crops and foods developed using gene silencing (http://www.foodstandards.gov.au/consumer/gmfood/pages/response-to-heinemann-et-al-on-theregulation-of-gm-crops-and-foods-developed-using-gene-silencing.aspx). this document criticised some of the comments of the above risk assessment review. some key points included the lack of weight of scientific evidence (published up to ) to support the view that small dsrnas in foods are likely to have adverse consequences in humans; the lack of a scientific basis for suggesting that small dsrnas present in gm foods have different properties than naturally-occurring ones; or the adequate acknowledge of many barriers (in the uptake, distribution and targeting) during oral development of small dsrna therapies, among others. the overall suggestion was that there was no need to consider additional studies as proposed by heinemann et al. ( ) .commentary documents reviewing scientific meetings in the context of rnai technologies in agricultural biotechnology discuss some of the aspects reviewed in this document .additional relevant documents of baseline information to support the risk assessment and environmental risk assessment of rnai-based gm plants can be obtained from efsa external scientific reports the present document has been produced and adopted by the bodies identified above as author(s). this task has been carried out exclusively by the author(s) in the context of a contract between the european food safety authority and the author(s), awarded following a tender procedure. the present document is published complying with the transparency principle to which the authority is subject. it may not be considered as an output adopted by the authority. the european food safety authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s). key: cord- -j kg qf authors: jones, samuel l.; blikslager, anthony t. title: disorders of the gastrointestinal system date: - - journal: equine internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: j kg qf nan however, the abdominal wall is too rigid to allow effective palpation of intraabdominal structures. abdominal auscultation is particularly useful for assessing the motility of the large intestine. progressive motility of the small intestine, conversely, is difficult to distinguish by auscultation from nonprogressive motility. the distinct character of the borborygmi produced during propulsive contractions of the cecum and ascending colon allow evaluation of the frequency and strength of retropulsion and propulsion. propulsive contractions of the cecum and ventral colon occur every to minutes and give rise to prolonged rushing sounds heard over long segments of intestine. retropulsive sounds presumably are similar to propulsive sounds, but they occur less frequently. the distinction of propulsion from retropulsion is not important clinically because both types of contractions signify normal motility. inter-and intrahaustral mixing contractions produce nonspecific sounds of fluid and ingesta movement that are difficult to distinguish from other borborygmi, such as small intestinal contractions or spasmodic contractions. auscultation over the right flank and proceeding along the caudal edge of the costal margin toward the xiphoid allows evaluation of the cecal borborygmi. auscultation over a similar area on the left side allows evaluation of the pelvic flexure and ascending colon. typical progressive borborygmi heard every to minutes on both sides of the abdomen indicate normal motility of the cecum and ascending colon. less frequent progressive sounds may indicate a pathologic condition of the large intestine or may result from anorexia, nervousness (sympathetic tone), or pharmacologic inhibition of examination of patients with disease of the gastrointestinal tract must include evaluation of the metabolic and cardiovascular status of the patient, because acute conditions of the proximal or distal intestinal tract have the potential to lead to endotoxemia and sepsis. examination of the cardiovascular system (heart, peripheral pulse, and mucous membranes), lungs, and abdomen is essential to detect clinical signs of systemic inflammation from endotoxemia, coagulation disorders, dehydration, ileus, shock, and other abnormalities resulting from injury to the small or large intestine. chapter . covers clinical signs of systemic inflammation from endotoxemia and sepsis. one performs the physical examination of the abdomen primarily by auscultation, transabdominal ballottement, and transrectal palpation. abdominal distention often indicates distention of the large intestine; however, small intestinal distention also can cause visible abdominal distention if a large proportion of the small intestine is involved. one can perform abdominal palpation in neonatal foals; after several weeks of age, motility (i.e., α -adrenergic agonists such as xylazine). [ ] [ ] [ ] absolute absence of any auscultable borborygmi suggests abnormal motility and indicates ileus resulting from a serious pathologic condition but is not specific to any segment of the intestine. , if borborygmi are audible but progressive sounds are not detectable, determining whether a significant abnormality exists is difficult. borborygmi heard more frequently than normal may result from increased motility following feeding; from excessive stimulation from irritation, distention, or inflammation; or after administration of parasympathomimetic drugs such as neostigmine. large intestinal motility increases in the early stages of intestinal distention regardless of the site. mild inflammation or irritation of the large intestinal mucosa also can stimulate motility. parasympathomimetic drugs stimulate contractions and auscultable borborygmi in the large intestine; however, an increase in parasympathetic tone may result in segmental contractions, which actually inhibit progressive motility. one can detect sand or gravel in the large intestinal ingesta by auscultation behind the xiphoid process. one can hear sand or gravel particles grinding together during progressive contractions of the ascending colon. the presence of sand in the ingesta becomes clinically detectable by auscultation or fecal sedimentation before the amount of sand is enough to produce clinical signs of pain or irritation (diarrhea). if progressive contractions are audible without hearing sand sounds, clinically important quantities of sand likely are not present. if the frequency of progressive contractions is low or absent, detecting sand by auscultation is difficult. percussion of the abdomen during auscultation can reveal gas in the large intestine. the characteristic ping produced by simultaneous digital percussion and auscultation over a gas-filled viscus often is associated with abnormal accumulation of gas under pressure. this technique is particularly useful in foals, ponies, and miniature horses because of the limitations of palpation per rectum. one can use transabdominal ballottement to detect large, firm masses or an abnormal volume of peritoneal fluid. the usefulness of this technique is usually limited to animals too small to palpate per rectum. one can detect soft tissue masses or fetuses by bumping the structures with a hand or fist. if excessive peritoneal fluid is present, one can generate a fluid wave by ballottement; however, this technique is not as useful in horses older than weeks because the abdominal wall is rigid. transrectal palpation is the most specific physical examination technique for investigation of intestinal disease and is particularly valuable when evaluating obstructive diseases. , the primary objectives of transrectal palpation are to assess the size, consistency, and position of the segments of the large intestine; to determine the presence of any small intestinal distention; and to detect intraabdominal masses. evaluation of the wall thickness and texture and the mesenteric structures (blood and lymphatic vessels and lymph nodes) also may aid in diagnosis of large intestinal disease. the interpretation of transrectal palpation findings in light of clinical signs and laboratory results is an important diagnostic aid for developing appropriate treatment strategies for intestinal diseases manifested by abdominal pain. enlargement of one or more segments of large intestine detected by transrectal palpation provides evidence of obstruction at or distal to the enlarged segment. by systematically evaluating each segment, one may determine the site of obstruction. obstruction of the pelvic flexure, for instance, results in enlargement of the pelvic flexure and ventral colon, but the dorsal and descending colons are of normal size. enlargement of a segment of the large intestine usually is accompanied by abnormal consistency of the contents. one may distinguish accumulation of gas, fluid, or ingesta and may detect foreign bodies in palpable segments. accumulation of gas and fluid infers complete and acute obstruction, whereas accumulation of ingesta infers chronic and incomplete obstruction. accumulation of fluid usually indicates ileus. one must evaluate the consistency of the contents in light of the size of the segment; ingesta in the ventral colon of a dehydrated patient may be firm, but the size of the ventral colon will be normal. conversely, if the ingesta is firm because of a distal obstruction, the ventral colon will be enlarged. displacement of a segment of the large intestine may create an obstruction detectable by enlargement of the segment and accumulation of gas and fluid, even if the site of obstruction is not palpable. torsion of the ascending colon at the sternal and diaphragmatic flexures results in acute accumulation of gas and fluid proximal to the torsion, causing distention of the left dorsal and ventral colons. depending on the degree of torsion, the position of the ventral and dorsal colons may not be significantly abnormal. displacement of a segment of large intestine often results in incomplete obstruction, and the diagnosis relies solely on detection of the displaced segment in an abnormal position. the position of the displaced segment may not be palpable, and the diagnosis then relies on the inability to find the segment in a normal position. one must take care to ensure that the segment that appears to be displaced is not in a normal position but has become too small to palpate from a decrease in the volume of ingesta. the cecum, right dorsal and ventral colons, pelvic flexure, and descending colon are palpable in most horses. one should palpate the nephrosplenic space to detect the presence of intestine, usually pelvic flexure, entrapped within the ligament. small intestine is not normally palpable in the horse. distention is an indicator of ileus with gas or fluid retention, usually following a strangulating or nonstrangulating obstruction. strangulating obstructions result from conditions such as volvulus or torsion, lipoma, or entrapments. such conditions often are accompanied by severe pain, dehydration, peritoneal fluid changes, and a varying degree of gastric fluid accumulation. the small intestine in these cases is turgid and firm on palpation. one should assess the mesentery and wall thickness as for large intestinal disorders. careful palpation of the inguinal rings in stallions with small intestinal distention is crucial for determining inguinal herniation. evaluation of the wall thickness and mesenteric vessels can reveal venous congestion (mural edema and enlarged blood and lymphatic vessels) or inflammation (mural edema with normal vessels). disruption of arterial blood flow does not cause venous congestion, but the arterial pulse is not detectable. mesenteric tears may not be palpable, but the entrapped ischemic intestinal segment may be thickened with edema. one may detect acute or chronic inflammation with cellular infiltration of the intestinal wall as thickening of the wall without edema and also may note enlargement of mesenteric lymph nodes. one should interpret abnormalities in the wall or vessels in light of the size, consistency, and position of the segment of intestine and the clinical signs. several conditions involving small intestinal strangulating lesions do not necessarily cause abnormal rectal examination findings until the disease has been present for an extended time. these conditions include diaphragmatic herniae and epiploic foramen entrapments. peritoneal fluid analysis may be normal in these cases as well because the fluid is trapped in the thorax or in the cranial abdomen. surgery is usually necessary for diagnosis. nonstrangulating causes of small intestinal distention can be divided further into intraluminal and extraluminal obstructions. ileal impactions are probably the most common cause of intraluminal obstructions, and on rare occasions one can palpate the impaction in the upper right quadrant, near the ileocecal opening. intraluminal masses caused by lymphoma, eosinophilic enteritis, foreign bodies or ascarid impactions often lead to small intestinal distention and are usually indistinguishable from one another based on palpation alone. small intestine in these cases can be moderately to severely distended, depending on the degree of obstruction. extraluminal obstructions include abdominal masses, abscesses or tumors, and large colon displacement. one always should palpate the rest of the abdomen carefully to help rule out these causes. some cases of small intestinal distention result from a physiologic rather than a mechanical obstruction. ileus may result postoperatively or following inflammatory diseases of the bowel (proximal enteritis) or peritoneal cavity (peritonitis). the bowel is usually mildly to moderately distended and almost always is accompanied by significant amounts of accumulated gastric fluid. the small colon is easily distinguishable by the presence of normal fecal balls and an antimesenteric band. in cases of impaction of the small colon, a long, hard, tubelike structure is present in the caudal abdomen, and the band is palpable along the length. fluid stool is often present in the rectum in these cases, as is tenesmus. one can detect and carefully evaluate rectal tears by palpation. one can detect mural masses in palpable segments of intestine or mesentery; however, if a mass causes obstruction, one can detect the result of the obstruction in proximal segments of intestine even if the mass is unreachable. palpation of the mesenteric vessels may reveal thickening and thrombosis, which can lead to ischemia or infarction. one can perform visual inspection of the mucosa of the rectum and descending colon with a speculum or flexible endoscope and also can evaluate rectal tears or perforations, mural masses, strictures, or mucosal inflammation. one also can perform guided biopsy of the mucosa or masses. the obvious limitations are the amount of fecal material interfering with the examination and the distance of the lesion of interest from the anus. these techniques offer little advantage over palpation in many cases unless the patient is too small to palpate. examination of the oral cavity in cases of dysphagia or weight loss is a necessary part of the physical examination. one should adequately sedate the horse and should use a full-mouth speculum to allow palpation and visualization of all parts of the oral cavity. one should examine the area for abnormal dentition, foreign bodies, fractures, abscesses, and ulceration. the presence of fluid accumulation in the stomach indicates a decrease or absence in propulsive motions of the small intestine or obstruction of gastric outflow. decreased small intestinal motility may result from a functional or mechanical blockage. masses, feed impactions, or strictures in the pylorus or in the proximal duodenum may obstruct gastric outflow. one routinely assesses fluid accumulation in the stomach by siphoning off the gastric contents with a nasogastric tube and examining the fluid for amount, color, and any particular odor. normal fluid is green and may contain foamy saliva. the volume obtained by gastric lavage is usually less than l. large volumes of fluid (> to l) accumulate in the stomach of horses with proximal enteritis, and the fluid is foul smelling and often has an orange to yellow discoloration. if one suspects proximal enteritis, one can submit the fluid for culture and gram staining. salmonella sp. and clostridium sp. have been cultured in some cases. patients with postoperative ileus also frequently accumulate large amounts of gastric fluid. horses with section . examination for disorders of the gastrointestinal tract strangulating obstructions or luminal obstructions often accumulate moderate amounts of gastric fluid, but the amount is generally less than in horses with proximal enteritis or postoperative ileus. hemorrhage in the gastric fluid usually indicates devitalized small intestine, stomach wall, or severe gastric ulceration. fluid with large amounts of food material often indicates a gastric impaction, and one should lavage the stomach until obtaining no more ingesta. horses and foals with chronic gastric ulceration in the glandular mucosa of the stomach or in the duodenum may develop strictures and have fluid accumulate in the stomach. endoscopy or contrast radiography aids in diagnosing gastric outflow obstruction. evaluation of the hemogram is essential when one assesses conditions of the gastrointestinal tract. however, hematologic alterations associated with diseases of the gastrointestinal tract are often nonspecific, reflecting systemic response to inflammation, endotoxemia, or sepsis. neutrophilic leukocytosis and normochromic, normocytic anemia with or without hyperfibrinogenemia commonly are associated with chronic inflammatory conditions of the intestine. anemia from chronic blood loss occurs infrequently in adult horses because of the large iron stores and high concentrations of iron in their diet; usually anemia follows chronic inflammation, as do alterations in the leukon and plasma fibrinogen concentrations. plasma protein concentrations vary depending on gastrointestinal losses of albumin and globulin and elevation of globulin concentration from antigenic stimulation. protein-losing enteropathies may manifest predominantly as a hypoalbuminemia or may have a concurrent hypoglobulinemia. immunoglobulin quantification can be useful in selected cases; immunosuppression with low immunoglobulin m concentration has been shown to occur in some cases of lymphosarcoma. parasitic infections, especially strongylosis, may be characterized by elevated serum immunoglobulin g(t) concentration. significant alterations of the hemogram do not accompany acute disease of the intestine unless severe inflammation, dehydration, endotoxemia, or sepsis is present. during the early stages of endotoxemia, elevations in circulating concentrations of inflammatory mediators, epinephrine, and cortisol produce characteristic changes in the hemogram. leukopenia, with neutropenia and a left shift, toxic changes in the neutrophil cytoplasm, and lymphopenia occur commonly. hemoconcentration and hyperfibrinogenemia are also common. thrombocytopenia and other coagulopathies are also features of endotoxemia. indeed, thrombocytopenia may be the earliest indicator of sepsis. endotoxemia and circulating mediators of inflammation activate the coagulation cascade, causing a hypercoagulable state that can lead to consumption of coagulation factors and coagulation defects manifested as elevated prothrombin time, partial thromboplastin time, fibrin degradation products, and bleeding time, and reduced activity of antithrombin iii. [ ] [ ] [ ] neutrophilic leukocytosis occurs during the later stages of endotoxemia. the most common serum biochemical abnormalities with diseases of the large or small intestine are electrolyte imbalances. serum calcium concentrations are often low with strangulating obstructions and acute inflammatory diseases. inflammation of the mucosa can disrupt electrolyte fluxes severely. diarrhea or gastric reflux greatly exacerbates the loss of sodium, potassium, calcium, magnesium and bicarbonate. ischemia of the intestine causing hypoxia and cellular damage may be reflected by an elevated serum phosphate concentration resulting from phosphate leakage from damaged cells. ischemia and cellular hypoxia in any segment of the intestine also causes a shift in energy metabolism to anaerobic glycolysis, resulting in increased production of lactate and elevated serum lactate concentration. reduced perfusion of peripheral tissues from hypotensive shock and intestinal ischemia can cause elevations in serum lactate. however, obstruction of the intestine during ischemia may result in absorption of lactate from the lumen. , anion gap is an indirect measurement of organic acid production during states of tissue hypoxia and is a reasonable estimate of serum lactate concentration. metabolic acidosis may accompany lactic acidemia, but an inconsistent association exists between the two, especially when mixed acid-base imbalances are present. , elevations of hepatic enzymes, specifically γ-glutamyltransferase, may occur with large colon displacements, duodenal strictures, or anterior enteritis. relative polycythemia from hemoconcentration or splenic contraction and changes in red blood cell deformability from hypoxia or hypocalcemia may increase blood viscosity. blood viscosity increases in patients with acute obstructive disease. hyperviscosity reduces perfusion of capillary beds, thereby exacerbating ischemia and tissue hypoxia. hyperviscosity is one manifestation (along with lactic acidemia, coagulopathies, and clinical signs of shock) of the pathophysiologic events that take place during acute inflammatory or vascular injury to the large intestine. laboratory tests designed to reflect the systemic effects of endotoxemia, ischemia, sepsis, and shock are important to design therapeutic strategies, and monitor response to therapy. abdominocentesis and analysis of peritoneal fluid (pf) is a diagnostic technique performed on many patients with disease of the gastrointestinal tract. one can quantitate cytologic examination of pf; white blood cell and red blood cell counts; protein, fibrinogen, lactate, phosphate, and glucose concentrations; lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity; and ph. the results of pf analysis may help establish a specific diagnosis but more importantly may reflect inflammatory, vascular, or ischemic injury to the intestine requiring surgical intervention. pf reflects a sequence of events that takes place during acute vascular injury to the intestine. the pf protein concentration first increases, followed by an increase in the red blood cell count and fibrinogen concentration. a transudative process resulting from vascular congestion and increased endothelial permeability allows small macromolecules (albumin) to escape into the pf, followed by larger macromolecules (globulin and fibrinogen), and finally diapedesis of cells (red blood cells, then white blood cells). , if ischemic inflammation of the intestine and visceral peritonitis occur, an exudative process ensues. severe inflammation of the intestine and visceral peritoneum causes large quantities of protein and white blood cells, primarily neutrophils, to escape into the pf. as damage to the bowel progresses, the protein concentration and red blood cell and white blood cell counts continue to rise. as the degree of irreversible damage to the intestine increases, the pf characteristics become more exudative. , eventually, bacteria begin to translocate across the intestinal wall and appear in the pf as the mucosal barrier breaks down. neutrophils predominate, and their cytoplasm becomes granulated, and döhle bodies often are visible. if perforation occurs, bacteria and particles of ingesta appear in the pf, and the neutrophils become degenerate, that is, pyknotic, with karyorrhexis, karyolysis, and smudge cells. elevated pf protein concentration is a sensitive indicator of early inflammation, whereas elevated red blood cell counts in the presence of normal white blood cell counts suggest vascular damage without significant tissue ischemia. elevation of the white blood cell count usually indicates severe tissue inflammation or intestinal injury. the gross color of the pf can be helpful in detecting injury and necrosis of the intestine. a serosanguinous appearance indicates vascular injury, whereas orange or brown-red indicates necrosis with the release of pigments such as hemosiderin. serial samples of pf are most useful in determining the nature and extent of damage to the intestine, but in many cases of ischemia, irreversible tissue damage has occurred by the time pf abnormalities appear. tissue hypoxia and ischemia cause a rapid elevation of pf lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity and lactate concentration. , , phosphate concentration increases when cellular disruption occurs. pf enzyme activities, phosphate, and lactate concentration increase faster and higher than serum activities. [ ] [ ] [ ] pf ph and glucose concentration tend to decrease during intestinal ischemia, but not as low as in septic peritonitis. although biochemical alterations may provide early indicators of intestinal ischemia and necrosis, they are nonspecific and offer no advantage over conventional methods of pf analysis in many cases. pf alkaline phosphatase has been shown to arise predominantly from degenerating white blood cells, and elevations of other enzyme activities may occur with many inflammatory diseases. thus the specificity of many tests run on pf is questionable. however, in selected cases in which conventional pf analysis and physical examination do not provide sufficient information to develop a treatment plan, biochemical analysis of the pf may be useful. cytologically examined cells of the pf may reflect chronic inflammatory conditions of the large intestine, especially eosinophilic or lymphocytic processes. infectious and inflammatory conditions often cause increases in the neutrophil count and may be indistinguishable unless bacteria are visible. one also may detect neoplastic diseases by pf examination. chronic infection and inflammation may be associated with elevated pf protein and fibrinogen concentrations. culture of pf usually is required to distinguish bacterial infections from noninfectious inflammation unless bacteria are visible on cytologic examination. however, culture of pf is often unrewarding because factors that are found in inflammatory pf inhibit bacterial growth, and leukocytes phagocytose many bacteria in the pf. decreases in pf glucose concentrations (< mg/dl) and ph (< . ) are early indicators of a septic process. the glucose concentration and ph in the pf should approximately equal the blood glucose concentration and ph. a pf fibrinogen concentration greater than mg/dl also indicates bacterial infection. gross examination of the feces can provide information about digestion and transit time in the large intestine. large fiber particles in the feces represent poor mastication or poor digestion in the large intestine. small, mucuscovered, hard fecal balls indicate prolonged transit through the descending colon, whereas increased fluidity implies decreased transit time. feces containing sand or gravel are not necessarily abnormal. however, a significant amount of sand implies that large quantities are present in the colon. frank blood indicates substantial bleeding into the distal colon (right dorsal colon and/or small colon) from mucosal damage. laboratory analysis of the feces is performed frequently in cases of diarrhea. fecal cytologic examination and tests for occult blood detect mucosal inflammation, section . examination for disorders of the gastrointestinal tract erosion, or ulceration. severe inflammatory diseases in human beings, invasive bacterial infections in particular, have been shown to increase the shedding of leukocytes in the feces. a higher percentage of horses with salmonellosis and diarrhea have fecal leukocyte counts greater than cells per high power field than horses with negative fecal cultures for salmonella. these results suggest that high fecal leukocyte counts indicate salmonellosis in horses with diarrhea. however, the specificity of this test is probably low. low fecal leukocyte counts do not rule out salmonellosis. fecal occult blood tests detect blood in the feces, presumably from erosion or ulceration of the mucosa, but do not distinguish the source of the blood. large volumes of blood ( to l) given by nasogastric tube were required to produce a positive test for occult blood in the feces, but the amount of blood originating from the large intestine required to produce a positive test is unknown. a positive test implies significant hemorrhage into the gastrointestinal tract. newer, more sensitive tests detect not only occult blood but also degraded blood and may be useful to determine the site and quantity of blood loss. a positive test implies significant hemorrhage into the gastrointestinal tract. bacteriologic examination of the fecal flora has been used to quantitate specific bacterial species in the feces of horses with diarrhea. quantitation of clostridial species may be beneficial in diagnosing clostridial infection of the large intestine. tests to detect clostridial toxins in intestinal contents or feces are important to determine whether clostridia cultured from the feces are causing disease. the most common bacterial pathogens isolated from the feces of horses are salmonella and clostridium. the number of salmonella organisms isolated from the feces of horses with clinical salmonellosis is usually higher than from horses with asymptomatic infections. however, the volume of feces in many cases of acute diarrhea is high, and the concentration of salmonella organisms may be lower than would be expected, accounting for many false-negative fecal cultures. the sensitivity of fecal cultures for detecting salmonella infection may be as low as %. culture of five consecutive daily fecal samples is recommended to increase the sensitivity of the test. because salmonellae are intracellular organisms, culture of rectal scrapings or a rectal biopsy sample, along with fecal material, may increase the sensitivity of culture for detecting salmonella infection to %. one can perform a polymerase chain reaction assay on fecal samples to detect dna from salmonella sp. the polymerase chain reaction test is more sensitive than culture and is frequently positive in clinically normal horses that continuously shed small amounts of bacteria. polymerase chain reaction or immunologic tests also may detect clostridium perfringens and c. difficile exotoxins in the feces. qualitative fecal examination is a technique to detect nematode and cestode ova, protozoan oocysts, parasitic larvae, and protozoan trophozoites. a direct smear of fecal material is a rapid method to screen feces for ova and oocysts, to detect parasite larvae and trophozoites, and to observe motility of ciliates and parasite larvae. fecal flotation is a more sensitive technique for isolating and detecting ova and oocysts because the eggs are concentrated from the sample. zinc sulfate and sucrose solutions are often used to concentrate less dense ova and oocysts. zinc sulfate produces less distortion of trophozoites and larvae than sucrose solutions. fecal sedimentation is particularly appropriate for ciliates, giardia, and trichomonads. quantitative techniques such as the cornell-mcmaster method allow one to estimate the number of eggs per gram of feces and are most appropriate in monitoring parasite control programs. survey radiography of the normal esophagus is usually unrewarding but may be useful in horses with esophageal obstructions to determine the extent and location of the obstruction. one may detect foreign bodies or soft tissue masses, and in cases of esophageal rupture, one may see free air and ingesta in the tissues surrounding the esophagus and may observe pneumomediastinum. thoracic radiographs may be necessary to detect intrathoracic esophageal obstructions, megaesophagus, or cranial mediastinal masses causing extraluminal obstruction. one may use barium swallows or double-contrast esophagrams after resolution of the obstruction to determine whether a stricture or diverticulum or other underlying disorder is present. barium sulfate is the usual contrast medium and can be administered orally via a dose syringe or by nasogastric tube ( to ml of a % barium sulfate suspension or barium paste). oral administration is preferred for evaluation of swallowing and lesions in the proximal esophagus. administration of contrast using a nasogastric tube (preferably cuffed) allows for delivery of larger volumes of barium (up to ml) but should be performed without sedation if possible. one can follow administration of contrast material with air insufflation to create a double-contrast effect. if one suspects a rupture of the esophagus or if the likelihood of aspiration of the contrast material is high, one should use iodinated organic compounds in an aqueous solution as contrast material. contrast radiography may be the most definitive method for the diagnosis of primary megaesophagus or other functional disorders such as autonomic dysautonomia (grass sickness) affecting the esophagus. one should take care when interpreting esophageal radiographs if the horse is sedated. acepromazine or detomidine administration causes esophageal dilation in normal horses, especially after passage of a nasogastric tube. radiography of the adult equine abdomen is an effective technique in detecting radiodense material in the large intestine, such as enteroliths, sand, and metallic objects. , one survey demonstrated that radiography has . % sensitivity and . % specificity for diagnosing enterolithiasis. radiography also can be a useful tool for detecting sand accumulation in the colon that causes diarrhea or impactions ( figure . - ) and for monitoring resolution in medically treated horses. the large size and density of the adult abdomen precludes evaluation of soft tissue structures because the detail and contrast of the radiographs are usually poor. one is more likely to obtain diagnostically useful abdominal radiographs from small ponies and miniature horses than from full-size adult horses. accumulation of gas is visible on radiographs of adult horses, but distinguishing normal intestinal gas from obstruction is often difficult. horses should be fasted for to hours to reduce the amount of ingesta in the large intestine before radiography. abdominal radiography is more useful in foals than in adult horses. radiographs are more detailed and contrast can be good. radiographic evidence of gas distention in the large intestine may indicate large intestinal obstruction, and radiographic signs of displacement are often diagnostic. one may diagnose impactions, intussusceptions, foreign bodies, and other disorders with the aid of radiography. functional ileus may be difficult to distinguish from mechanical obstruction. , administration of contrast (barium sulfate % at ml/kg) via nasogastric tube increases the diagnostic capabilities of radiography. gastric ulceration also is recognizable with contrast radiography in the foal, although this is not as accurate a method as endoscopy. contrast administered retrograde via a -f foley catheter inserted into the rectum at a dose of up to ml/kg has excellent potential for diagnosing disorders of the small colon, transverse colon, and large colon in foals. ultrasonographic evaluation of the abdomen can add valuable information in cases of acute or chronic gastrointestinal disease. examination of the adult horse requires a . -to . -mhz transducer at minimum. one may use sector, linear, or curved linear transducers. clipping of the hair over the area to be examined, along with the application of isopropyl alcohol and ultrasound coupling gel, enhances evaluation. to evaluate the abdomen adequately, one must know the anatomic location and normal appearance of the individual organs. in the left cranial abdomen, one can assess the greater curvature of the stomach between the eleventh and thirteenth intercostal space, and one can use the spleen and the large splenic vein as landmarks. cases of gastric dilation from gas or impaction appear as an enlargement of the viewing area to cover greater than five rib spaces. one also can evaluate the stomach for intramural or extramural masses such as abscesses or for squamous cell carcinoma. the lesser curvature is not routinely visible. assessment of the small intestine should include evaluation for changes in thickness, motility, location, and visibility. one may find small intestinal loops easily in the left lower quadrant of the abdomen, but these normally are visible in other locations. one can visualize the duodenum consistently on the right side of the abdomen deep to the liver in the tenth to twelfth intercostal space or deep to the right kidney at the fifteenth to sixteenth intercostal space. mural thickening (> mm) may occur in cases of infiltrative or proliferative diseases, postoperative cases, enteritis, and paralytic or mechanical ileus. thickening of the small intestinal wall in foals, with or without the presence of gas shadows within the wall, should raise suspicions of clostridial enteritis. one can assess motility by monitoring a specific area for contractions over time. ultrasonography is an accurate method of distinguishing strangulating versus nonstrangulating disorders of the small intestine. strangulated small intestine has thicker small intestinal walls and larger intestinal diameter than in nonstrangulating disorders. strangulating lesions have decreased motility in the incarcerated segments with normal motility elsewhere. cases of paralytic ileus or nonstrangulating obstruction have a diffusely decreased peristalsis, but not to the degree observed with strangulating lesions. , one may diagnose some specific lesions of the small intestinal tract using ultrasonography. one may see ascarids in foals in cases of ascarid impaction and epiploic foramen entrapments as edematous loops of small intestine found in the right cranial abdomen. one may note small intestinal intussusceptions as targetlike lesions when viewed in cross sections. the presence of bowel loops, stomach, or liver in the thoracic cavity indicates the presence of herniation through the diaphragm and should be confirmed using radiography or surgical exploration. evaluation of the large intestine may be difficult because of the large amounts of gas within the lumen. however, certain disorders are readily identifiable via ultrasonography. one can assess the nephrosplenic ligament area for bowel entrapment in the left paralumbar fossa. in cases of entrapment, the spleen will be pulled away from the body wall and fluid or gas shadows will be observable dorsal to the spleen, obscuring the kidney, which is normally adjacent and abaxial to the spleen. small colon, small intestine, or pneumoperitoneum also may produce a gas shadow and obscure the kidney from view. sand impactions may appear as hyperechoic bands on the ventral abdominal wall. one may see ileocecal and cecocolic intussusceptions in the upper right paralumbar fossa. in cases of colitis, large, fluid-filled colons may be visible with or without intramural edema. one can find the right dorsal colon consistently abaxial to the liver, within the right thirteenth to fifteenth intercostal space and may be thickened (> mm) in cases of right dorsal colitis. evaluation of the abdomen always should include assessment of the peritoneal space for any evidence of an increased amount of pf or increased cellularity of the fluid as indicated by an increase in echogenicity. ultrasonography also can be useful in determining the ideal location for abdominocentesis. one also should evaluate the liver, kidneys, and spleen. one may detect choleliths, nephroliths, masses, abscesses, or enlargement of any of these organs. abscesses or tumors not associated with visceral organs may be difficult to visualize and interpret via ultrasonography. although more commonly used to diagnose lameness and musculoskeletal problems, nuclear scintigraphy has several uses for evaluation of the gastrointestinal tract. scintigraphy is now available at most universities and many private referral hospitals. one must use proper isolation protocols and waste disposal techniques strictly. the procedure requires special gamma cameras and the injection of radioactive materials into the bloodstream. one can use one of two methods: injection of technetium- m methylene diphosphonate ( m tc-mdp) directly into the blood or injection of m tc-labeled leukocytes. labeling of leukocytes involves aseptically collecting heparinized blood samples, isolating the buffy coat, and mixing those leukocytes with a radioactive dye ( m tc hexamethylpropyleneamine oxime, or m tc-hmpao) in vitro. one then reinjects the labeled leukocytes and obtains images. the principle of nuclear scintigraphy then lies in increased uptake of the dye or the white blood cells into areas of inflammation. one of the most common uses of nuclear scintigraphy in evaluating the gastrointestinal tract is diagnosis of dental disease. scintigraphy using m tc-mdp proved to be more sensitive in cases of dental disease than was radiography. scintigraphy was slightly less specific, however, and therefore should be used with radiography or computed tomography for ultimate accuracy. scintigraphy using radiolabeled white blood cells can support a diagnosis of right dorsal colitis in the horse. images taken of the abdomen hours after injection showed an increased linear uptake of leukocytes in the region of the right dorsal colon in horses with right dorsal colitis compared with normal horses. this technique also may prove useful for diagnosing intraabdominal abscesses in the horse. other uses of nuclear scintigraphy include evaluation of metastasis of abdominal tumors to bony areas, assessment of biliary kinetics, and determination of gastric emptying times. [ ] [ ] [ ] endoscopy endoscopic examination of the gastrointestinal tract begins with evaluation of the pharyngeal area by examination for any signs of collapse or dysfunction. one should evaluate the ability of the horse to swallow. the floor of the pharynx should be clean and free of feed material and foreign bodies. one can examine the oral cavity with the horse under heavy sedation or anesthesia and with the help of a full-mouth speculum. one can examine the teeth for any irregularities, obvious cavities, sharp points, or hooks and the hard and soft palpate for completeness and any evidence of ulceration, masses, or foreign bodies. one should use a -m flexible fiberoptic endoscope to examine the esophagus, which is accomplished best by passing the endoscope into the stomach and viewing the esophagus as one withdraws the endoscope while dilating the lumen with air. the esophageal mucosa normally should be a glistening, light pink color. ulceration can occur with cases of choke, reflux esophagitis or in horses that have had an indwelling nasogastric tube. erosions may be punctate, linear, or circumferential. one should evaluate carefully for any ulcers to ensure that no areas of perforation through the entire thickness of the esophageal wall exist. distinguishing normal peristaltic contractions from areas of stricture requires observation of the area and its motility over time. one also may note diverticula as outpouchings of the mucosa, sometimes associated with a stricture distally. megaesophagus, although rare, appears as a generalized dilation of the esophagus. one may detect food or foreign body impactions of the esophagus via endoscopy. one always should reevaluate the esophagus after removing any obstruction to detect the presence of complications (ulceration, rupture) or initiating causes (strictures, diverticula, and masses). a -m flexible endoscope also allows examination of the stomach. the horse should be fasted for at least hours before endoscopy. one can examine the cardia and fundus easily, as well as the margo plicatus. the squamous mucosa should resemble the esophageal mucosa. the glandular mucosa should be glistening red and may have a reticulated pattern. one should carefully examine for evidence of ulceration or masses. one can obtain transendoscopic biopsy material easily from esophageal, pharyngeal, or gastric masses, and because the biopsy size will be small, one should take several samples for histopathologic examination. pharmacologic agents (bethanechol) to empty the stomach and provide complete visualization of the entire fundic region, the pylorus, and the duodenum may be useful. for a complete description of gastroscopy and evaluation of gastric and gastroduodenal ulceration, please refer to chapter . . d-glucose or d-xylose absorption tests are useful in determining malabsorption of carbohydrates from the small intestine in horses. the protocol for absorption tests using either carbohydrate is similar. the horse should be fasted for to hours before testing. increased periods of fasting actually have been shown to decrease absorption of d-xylose and interfere with results. one administers a dosage of . to g/kg of d-glucose or d-xylose via a nasogastric tube. administration of sedatives may increase the blood glucose levels falsely and interfere with gastrointestinal transit times. one then collects blood samples to measure glucose or xylose concentrations at , , , , , , , , and minutes after administration. one may take additional samples up to hours after dosing if the results are questionable. one should measure glucose in blood samples collected with sodium fluoride as an anticoagulant and measure xylose in samples collected in heparinized plasma. a normal d-glucose absorption test, also known as an oral glucose tolerance test, should have a peak between and minutes, and this peak should be greater than % above the resting glucose value. complete malabsorption is defined as a peak less than % above the resting levels, and partial malabsorption is defined as a peak between % and % above the resting level. one must keep in mind that gastric emptying, gastrointestinal transit time, length of fasting, cellular uptake and metabolism, and endocrine function influence glucose absorption curves. malabsorption demonstrated by the oral glucose tolerance test is sensitive but not specific. diseases that may cause a lowered or delayed peak include infiltrative lymphosarcoma, inflammatory bowel disease (lymphocytic-plasmacytic or eosinophilic), cyathostomiasis, chronic colitis (salmonella sp.), multisystemic eosinophilic epitheliotropic disease, food allergies, and small intestinal bacterial overgrowth. d-xylose absorption tests have some advantages over the oral glucose tolerance test because xylose is not metabolized in the small intestinal mucosa and insulin does not influence its absorption. gastric and intestinal motility, intraluminal bacterial overgrowth, and renal function still influence xylose absorption, because the kidneys clear xylose. the other main drawback to d-xylose is that it is generally available only in research settings. however, xylose measurements are available at most major universities. a normal d-xylose absorption curve should peak between and mg/dl at to minutes after dosing. decreased xylose absorption can occur in horses with inflammatory bowel disease, lymphosarcoma, multisystemic eosinophilic epitheliotropic disease, cyathostomiasis, extensive small intestinal resections, and any cause of villous atrophy. maldigestion is a common occurrence in foals with diarrhea. bacteria (especially clostridium sp.) and viruses (especially rotavirus or coronavirus) may invade and destroy the villous epithelial cells that manufacture lactase and other disaccharidases, resulting in an inability to digest lactose. in this case, continued ingestion of the mare's milk may cause an osmotic diarrhea, which may exacerbate the underlying enterocolitis. one can perform lactose tolerance testing to assess the degree of maldigestion. one administers d-lactose at g/kg as a % solution via nasogastric tube and measures glucose concentrations in the blood at , , , , , , , , and minutes. a normal curve shows doubling of glucose levels compared with baseline by minutes after administration. accurate measurement of gastric emptying can be difficult to assess. several methods are currently available. multiple diagnostic imaging techniques have been used to study gastric emptying times. one can use contrast radiography to assess gastric emptying in foals. in the normal foal, barium remains in the stomach for varying amounts of time, but a significant amount should be gone within hours. gastric emptying of solid, nondigestible, radiopaque markers also has been used in adult horses and ponies, but the results were variable and unpredictable even in the normal horse. nuclear scintigraphy is used commonly in human beings to measure gastric emptying and can be used in horses where available. this technique requires oral administration of m tc pentenate ( mci), and serial images taken of the cranial abdomen. the tracer is usually not visible minutes after administration in normal horses. alternatively, if nuclear scintigraphy is not available, one can use acetaminophen absorption testing as an indirect determination of gastric emptying. , one performs this test by administering mg/kg of acetaminophen orally and measuring subsequent blood values and calculating the time to reach maximum serum concentrations and the absorption constant. in human beings, the proximal small intestine absorbs almost all of the acetaminophen. the median time to reach peak plasma levels using acetaminophen absorption in horses was . minutes. one often requires histopathologic examination of tissues from the intestine to diagnose chronic inflammatory, infiltrative, or neoplastic conditions, and such examination can be useful in evaluating the extent of injury after obstruction or ischemia. rectal mucosal biopsies are easy to collect with few complications. however, to collect a full-thickness biopsy of the intestine requires a surgical approach (flank or ventral midline approach). laparoscopy offers a safer technique to observe the large intestine and other abdominal structures. one can obtain biopsies of masses, lymph nodes, mesentery, or intestinal serosa via laparoscopy and mucosal biopsies of the upper gastrointestinal tract via endoscopy. other diagnostics, specifically laparoscopy and computed tomography, are available but require specialized equipment and personnel with specific training. flexible or rigid endoscopes used for laparoscopic evaluation of the abdomen allow for visualization of visceral organs and potentially for collection of biopsy material from masses or organs. full-thickness biopsies of the intestines are not routinely possible through the laparoscope and usually require flank or ventral midline laparotomy. the laparoscopic procedure can be done in the standing or recumbent horse. advantages of this technique over a flank or ventral midline celiotomy include smaller incisions, less healing time, and less procedure time. disadvantages include the large amount of equipment needed, skill involved, and the limitation as a diagnostic modality, rather than a treatment. clinical applications of diagnostic laparoscopy include rectal tears, percutaneous abscess drainage, assessment of adhesions, displacements, and integrity of the serosa of various bowel segments, and biopsy of abdominal masses. computed tomography scans are available at several universities across the country. they have been used frequently to evaluate dental disease and may be useful in evaluating tumors and masses of the head, larynx, pharynx, and proximal esophagus. computed tomography also has promise for evaluating abdominal disorders in foals. most equipment can accommodate up to lb. restrictions to computed tomography as a diagnostic aid include expense, availability, expertise, and weight and size limitation. permeability increases, cells are recruited rapidly into the tissue, plasma protein cascades are activated, and a myriad of soluble products are released that coordinate the response, trigger innate and adaptive immunity, and mobilize reparative elements. although the cellular and vascular response and the secreted mediators of inflammation are important for killing pathogens and limiting invasion of injured tissues by commensal organisms, they can be damaging to host cells and proteins if not tightly regulated. thus if the inciting stimulus is not eliminated quickly, the inflammatory response itself causes significant tissue injury. the mechanism regulating inflammation has been the focus of much research to identify therapeutic targets to modulate the damage to host tissues during many gastrointestinal diseases. recent work has provided some of the molecular and cellular details of this complex physiology and has led to novel therapeutic strategies for treating inflammation. the gastrointestinal epithelium interfaces with a luminal environment inhabited by potentially hostile microbial organisms. the epithelium presents a physical barrier to invasion by the flora of the gastrointestinal tract, consisting of the apical cellular membrane, intercellular tight junctions the permeability of which is highly regulated, and a secreted layer of mucus. breaching of the mucosal barrier by invading pathogens generates potent soluble and neural signals that initiate an inflammatory response. conceptually, the epithelium can be thought of as a sensory organ detecting pathogen invasion to trigger an appropriate host defense and reparative response. noninfectious mucosal injury or invasion of epithelial cells by pathogenic organisms such as salmonella activates synthesis of proinflammatory chemokines (chemoattractants) by epithelial cells and triggers a robust influx of neutrophils into the tissue within hours of the damage. of the chemoattractants produced by epithelium, interleukin- (il- ) has a particularly important role in initiating inflammation by recruiting neutrophils from blood [ ] [ ] [ ] and regulating neutrophil migration through tissue matrix adjacent to epithelium. , bacteriaderived formylated chemotactic peptides also act as potent chemoattractants that are fully capable of stimulating a robust inflammatory response in the intestine if the epithelial barrier permits the diffusion of the peptides across the mucosa. epithelial cells activated during infection produce cytokines such as tumor necrosis factor α (tnf-α), arachidonic acid metabolites, and other proinflammatory mediators that activate recruited leukocytes. bacterial products, particularly lipopolysaccharide and other cell wall components, are potent activators of leukocytes recruited into the tissue. once the inflammatory response has been initiated, tnf-α, il- β, and other proinflammatory products of neutrophils, monocytes, mast cells, and epithelial cells amplify the inflammatory response. the enteric nervous system has a key role in sensing and regulating inflammatory responses in the intestine. for example, clostridium difficile toxin a activates a neural pathway that triggers mast cell degranulation and neutrophil influx into the tissue. , blockade of this neural pathway is sufficient to abolish the profound inflammatory response induced by toxin a and many of the effects of toxin a on enterocyte secretion. other pathogens and immune-mediated hypersensitivity reactions similarly stimulate inflammation by mechanisms that involve the enteric nervous system. thus the epithelium interacts in a highly complex manner with the intestinal milieu, the enteric nervous system, and inflammatory cells to regulate the tissue response to injury and infection. resident macrophages located in the lamina propria, submucosa, and intestinal lymphoid organs are among the first cells beyond the epithelium to respond to infection or injury. macrophages are activated by bacterial products via pattern recognition receptors and begin to produce proinflammatory molecules important for recruiting and activating neutrophils and monocytes. pattern recognition receptors recognize microbial products ranging from lipopolysaccharide to peptidoglycan and even cpg-containing bacterial dna and signal the invasion by pathogens. of the pattern recognition receptors, the lipopolysaccharide receptor complex is perhaps the best defined. lipopolysaccharide activates macrophages via the cd -toll-like receptor complex to initiate transcription of the inflammatory cytokines tnf-α and il- β, which synergize with lipopolysaccharide to amplify the macrophage response. lipopolysaccharide, particularly in concert with inflammatory cytokines, stimulates macrophages to produce copious amounts of nitric oxide, which is microbicidal and vasoactive. nitric oxide and other nitrogen radicals react with reactive oxygen intermediates (rois) to produce some of the most toxic molecules of the host defense system: the peroxynitrites. il- is produced as well to recruit neutrophils. as the response progresses, other inflammatory mediators, particularly the arachidonic acid-derived lipids, are produced. these lipids have potent vasoactive effects and are important stimuli of endothelial cells, neutrophils, and platelets. four important changes occur in the intestinal vasculature during inflammation: . alteration of blood flow . increased vascular permeability . increased adhesiveness of endothelial cells, leukocytes, and platelets . exposure of the basement membrane and activation of the complement, contact, and coagulation cascades a wide range of mediators alters blood flow during intestinal tract inflammation, from gasses such as nitric oxide (a major vasodilator of the intestinal vasculature) to lipids (prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor), cytokines, bradykinin, histamine, and others. the major sources for these mediators include activated leukocytes, endothelial cells, epithelial cells, and fibroblasts. the primary determinant of blood flow early in inflammation is vascular caliber, which initially decreases in arterioles, but then quickly changes to vasodilation coincident with opening of new capillary beds, increasing net blood flow. the increase in blood flow is short lived, for the viscosity of the blood increases because of fluid loss and tissue edema through leaky capillaries. leukocyte margination, platelet adhesion to endothelial cells and exposed matrix, and areas of coagulation protein accumulation further decrease local circulation. inflammatory mediator actions on the endothelial cells initially increase vascular permeability. histamine, leukotrienes, platelet-activating factor, prostaglandins, bradykinin, and other mediators stimulate endothelial cell contraction, and interendothelial gaps form. , this stage of increased vascular permeability is readily reversible. concurrently, mediators such as the cytokines tnf-α and il- β induce a structural reorganization of the interendothelial junctions, resulting in frank discontinuities in the endothelial monolayer. cytokines also stimulate endothelial cells to express adhesion molecules that support adhesion of leukocytes and platelets, leading to the next and perhaps most devastating event. leukocytes (primarily neutrophils) and platelets adhere to exposed basement membranes and activated endothelial cells. adherent neutrophils and platelets then are exposed to the mediators of inflammation present in the surrounding milieu, which activates the cells to release oxidants and proteases (particularly elastase) that injure the endothelium and have the potential to cause irreparable harm to the microvasculature. [ ] [ ] [ ] marginated neutrophils begin to transmigrate between endothelial cells (as described in later sections), and if their numbers section . pathophysiology of gastrointestinal inflammation are large enough, they disrupt the integrity of the interendothelial junctions, worsening the vascular leakage. conceptually, these stages of enhanced vascular permeability can be thought of as a mechanism to allow plasma proteins to enter the tissues and to potentiate the critical influx of leukocytes into tissues. however, if not regulated precisely, alterations in hydrostatic and oncotic forces and irreversible damage to the vascular bed may have devastating consequences. moreover, inappropriate activation of plasma protein cascades and leukocytes by activated endothelium and exposed matrix proteins can contribute to systemic inflammation (systemic inflammatory response syndrome; see chapter . for more information) characterized by hypotension, generalized vascular leak syndrome, and multiorgan dysfunction, which may be fatal. phosphodiesterase inhibitors reduce endothelial permeability in ischemia-reperfusion injury and other models of inflammation-induced vascular leakage , by increasing endothelial tight junction integrity and thus may be a viable therapeutic strategy to prevent or reduce the permeability alterations associated with inflammation. endothelial cells respond to products of activated epithelial cells and macrophages in the intestinal tissue to recruit cells and humoral mediators of inflammation into the tissue. activated endothelial cells display a range of molecules critical for neutrophil and platelet adhesion. intercellular permeability increases to expose basement membrane proteins that trigger humoral defense systems (complement, coagulation, and contact system cascades) and to provide access for these macromolecules to the tissue. endothelial cells are an important source of inflammatory mediators that amplify the response and vasoactive substances (particularly nitric oxide) that alter blood flow. infection or injury to the gastrointestinal mucosa causes an influx of leukocytes from the blood that lay the foundation of the inflammatory response. neutrophils, being the first to arrive during inflammation, have a dominant role in the acute response. within minutes, neutrophils are recruited into the tissue where they are activated to release products that not only are proinflammatory and lethal to pathogens but also may damage host cells and tissues. not surprisingly, much attention has been paid to the role of neutrophils in the pathophysiology of many inflammatory conditions. neutrophil depletion is protective in many models of gastrointestinal inflammatory disease. of interest to clinicians, blockade of neutrophil migration into inflamed tissues prevents many of the pathophysiologic events associated with infectious enteritis, ischemia-reperfusion injury, and other gastrointestinal diseases. , [ ] [ ] [ ] [ ] [ ] neutrophil transendothelial migration is a multistep process that is temporally and spatially regulated and has a degree of cell type specificity ( figure . - ) . the predominant sites of neutrophil transendothelial migration are in the postcapillary venules and, in some tissues, the capillaries. endothelial cells in these vessels respond to cytokines and other soluble signals by expressing molecules that promote neutrophil adhesion and transmigration, including selectins and counterreceptors for integrins. as neutrophils flow through these vessels, they are first tethered to activated endothelium. tethering is mediated by selectin molecules expressed on neutrophils (l-selectin) and on activated endothelial cells (p-and e-selectins) that bind to p-selectin glycoprotein ligand- (psgl- ), e-selectin ligand- (esl- ), and other mucin counterreceptors. , tethering functions to increase the exposure of the neutrophil to activating chemokines presented on the surface of the endothelial cells. stimulation of neutrophils by il- and other chemokines activates the second step of transendothelial migration. chemokine binding to their receptors on the neutrophil generates signals that activate the binding of integrin adhesion receptors to their ligands, called intracellular adhesion molecules or vascular cell adhesion molecules expressed on endothelial cells in inflamed mucosa. integrin ligation to cellular adhesion molecules arrests the tethered neutrophils, resulting in firm adhesion to the endothelium. of the integrins expressed on neutrophils, the β integrins have a particularly important role in transendothelial migration. calves and human beings with a disorder known as leukocyte adhesion deficiency illustrate the requirement for β integrinmediated adhesion in neutrophil function. leukocyte adhesion deficiency results from an autosomal recessive trait causing the lack of the β integrin expression. the neutrophils from these individuals cannot migrate into most tissues and do not function normally, resulting in poor tissue healing and profound susceptibility to infection, especially at epithelial barriers. , other integrins also have a role in transendothelial migration. β integrins mediate transendothelial migration in some cells and seem to be particularly important for mediating emigration of monocytes into many tissues. following this firm adhesion step, neutrophils migrate through the endothelium along a chemotactic gradient of il- and other chemoattractants such as leukotriene b . , , neutrophils migrate across the endothelial monolayer at intercellular junctions via a mechanism involving a series of integrin-ligand interactions mediated by β and β integrins and other adhesion molecules that is generally capable of maintaining the integrity of the endothelial barrier. however, massive flux of neutrophils through the endothelium alters endothelial tight junctions and injures the basement membrane, resulting in increased endothelial permeability to molecules as large as plasma proteins and even endothelial cell detachment from the basement membrane. , nonintegrin molecules such as platelet/endothelial cell adhesion molecules (pecams) also are involved in transendothelial migration of neutrophils. homotypic binding of pecams on adjacent endothelial cells form part of the intercellular junction. neutrophils express an integrin of the β family that can bind pecam, and via sequential binding of β integrins to pecam, the neutrophil can , and other proinflammatory mediators. endothelial cells stimulated by inflammatory mediators produce chemoattractants (such as il- ) and display adhesion molecules that promote neutrophil emigration. the three steps of neutrophil (polymorphonuclear [pmn]) emigrationcapture/rolling (mediated by selectins), adhesion (mediated by β integrins), and transendothelial migration (mediated by integrins and platelet/endothelial cellular adhesion molecule [pecam])-occur on activated endothelium. chemoattractant molecules, such as il- trigger neutrophil emigration. in inflamed tissues, cytokines (il- β and tnf-α) and a variety of other proinflammatory mediators stimulate the neutrophil oxidase complex to produce reactive oxygen intermediates (rois; o and h o and their derivatives). activated neutrophils degranulate to release proteases and other hydrolases, cationic peptides (defensins), myeloperoxidase, and other products into the tissue. activated neutrophils synthesize a variety of inflammatory mediators, including prostaglandins (pge ) that modulate the inflammatory response. the products of activated neutrophils (rois, proteases, and mediators) stimulate epithelial secretion and alter tight junction permeability, promoting diarrhea. neutrophils eventually migrate across the infected epithelium by a mechanism that involves integrins, disrupting tight junction integrity and increasing permeability to bacterial products, thus exacerbating the inflammatory response. "unzip" the intercellular junction and migrate through, closing it behind itself. a key feature of neutrophils and other leukocytes is the requirement for integrin-mediated adhesion to extracellular matrix proteins (ecms) or other cells to achieve an optimal effector phenotype. critical components of the ecms in inflamed tissues include fibronectin, fibrinogen, and vitronectin, deposited in tissues as a result of plasma leakage and by synthesis of new proteins by stromal cells and resident macrophages in response to inflammatory mediator activation. the changing composition of the matrix proteins deposited in tissues during inflammation serves as a clue as to the nature of the tissue environment for recruited inflammatory cells as they become activated. individual gene expression studies have demonstrated that adhesion to matrix proteins induces the expression of cytokines and chemokines and their receptors, arachidonic acid-derived lipid mediator synthases, metalloproteinases, growth factors, transcription factors, and other genes that influence the differentiation and activation of inflammatory cells. roi production, phagocytosis, degranulation, and other effector functions stimulated by inflammatory mediators and bacterial products are optimal only when neutrophils adhere to the ecms. adhesion to distinct ecm proteins selectively activates signaling pathways and gene expression of neutrophils, monocytes, and other leukocytes with differing abilities to promote certain functions such that the composition of ecms in many ways controls the development of the ultimate effector phenotype. thus integrin-mediated adhesion provides a mechanism by which neutrophils and other leukocytes can sense the complex tissue environment and respond appropriately. of the activators of neutrophils at sites of inflammation, complement (c -opsonized particles), cytokines (tnf-α and il- β), platelet-activating factor, immune complexes, and bacterial products are among the most potent stimuli. other mediators produced during inflammation may modify neutrophil activity, particularly formylated bacterial peptides, chemokines, complement fragments (c a), leukotriene b , and prostaglandins. activated neutrophils are highly phagocytic; produce large amounts of rois; degranulate to release myeloperoxidase, cationic antimicrobial peptides (defensins), serine proteases (mainly elastase), and metalloproteinases; and secrete inflammatory mediators (tnf-α, il- β, prostaglandins, leukotrienes, and others) (see figure . - ). mast cells strategically reside in mucosal tissues, including the submucosa and lamina propria of the gastrointestinal tract, and constitute a crucial first line of defense at epithelial barriers. however, they are also important effector cells of the pathophysiology of inflammatory gastrointestinal diseases. experimental depletion of mast cells, genetic deficiency in the development of mast cells, or pharmacologic stabilization of mast cells to prevent degranulation have a protective effect in a variety of models of gastrointestinal inflammatory disease, including dextran-or trinitrobenzenesulfonic acid-induced colitis, , ischemia-reperfusion injury, , and immediate hypersensitivity responses. mast cells are activated by a wide variety of microbial products and host-derived mediators. among the activators of mast cells the so-called anaphylatoxins (complement fragments c a, c a, and c a), are potent stimuli causing release of mediators of inflammation. in addition, mast cells are the primary effector cells of immunoglobulin e-mediated anaphylaxis (type i hypersensitivity reactions) by virtue of their high affinity receptors for immunoglobulin e. cross-linking of receptor-bound immunoglobulin e on mast cell surface by antigens (i.e., food antigens) causes rapid degranulation, resulting in the explosive release of granule contents. neural pathways in the intestine also regulate mast cells. mast cells respond to enteric pathogen invasion via neural reflexes that stimulate the release of inflammatory mediators. activated mast cells release preformed histamine, -hydroxytryptamine, proteases, heparin, and cytokines from granules. activation also stimulates de novo synthesis of a range of inflammatory mediators, including prostaglandins, platelet-activating factor, and leukotrienes. transcription of a number of peptide mediators, such as the cytokines tnf-α and il- β among many others, also increases on stimulation of mast cells. mast cell products have profound effects on the vasculature, increasing endothelial permeability and causing vasodilation. moreover, mast cell-derived mediators greatly enhance epithelial secretion by a mechanism that activates neural pathways of epithelial secretion and directly stimulates epithelial cells. mast cell products significantly alter intestinal motility, generally increasing transit and expulsion of intestinal contents. mast cell-derived leukotrienes and tnf-α also have a crucial role in host defense against bacterial pathogens, acting to recruit and activate neutrophils. , mast cells have a newly identified role in host defense and inflammatory responses to bacterial pathogens, which in part is caused by the release of proinflammatory mediators during bacterial infection, which is critical for recruiting and activating other innate host defense cells such as neutrophils. however, mast cells are also phagocytic, have microbicidal properties, and can act as antigen-presenting cells to the adaptive immune system. although accumulating evidence was establishing the role of mast cells in innate immunity, a seminal study that unconditionally identified the importance of mast cells in host defense demonstrated that mast cell-deficient w/ w v mice have impaired responses to gram-negative bacterial peritonitis, resulting in a significant increase in mortality. the role for mast cells in host protective responses appears to be as a sensor of bacterial invasion. unlike immunoglobulin e-mediated responses, bacterial products seem to elicit a highly regulated and selective response from mast cells. the complement cascade is a fundamental part of the inflammatory response. activation of the complement cascade by immune complexes (classical pathway) or by bacteria or bacterial products, polysaccharides, viruses, fungi, or host cells (alternative pathway) results in the deposition of complement proteins on the activating surface and the release of soluble proteolytic fragments of several complement components. in particular, activation of either pathway results in the deposition of various fragments of the complement protein c , which are potent activators of neutrophils and monocytes. opsonization of particles with c fragments constitutes a major mechanism of target recognition and phagocyte activation. during the activation of the complement cascade culminating in deposition of c , soluble fragments of c (c a), c (c a), and c (c a) are liberated. these fragments, termed anaphylatoxins, have potent effects on tissues and cells during inflammation. perhaps most notably, anaphylatoxins are chemotactic for neutrophils (particularly c a), activate neutrophil and mast cell degranulation, and stimulate roi release from neutrophils. the termination of the complement cascade results in the formation of a membrane attack complex in membranes at the site of complement activation, and if this occurs on host cells such as endothelium, it may cause irreversible cell injury. although the primary source of complement is plasma, epithelial cells of the gastrointestinal tract also produce c , suggesting that local production and activation of the complement cascade during inflammation occurs in intestinal tissues. clearly, if the regulatory mechanisms of the complement cascade fail, then the inflammatory response may be inappropriate and tissue injury can occur. the role of complement in gastrointestinal inflammation has been most studied extensively in models of ischemia-reperfusion injury. activation of the complement cascades has a major role in altered endothelial and epithelial permeability in these models. several lines of evidence support the importance of complement in intestinal injury. mice deficient in c or c are protected against ischemia-reperfusion injury. moreover, administration of monoclonal antibodies against c reduced local and remote injury and inflammation during intestinal reperfusion injury in a rat model. administration of a soluble form of complement receptor , a regulatory protein that halts the complement cascade by dissociating c and c on host cell membranes, reduced mucosal permeability, neutrophil influx, and leukotriene b production during ischemia-reperfusion injury in rats and mice. , although neutrophils and mast cells mediate many of the pathophysiologic effects of the complement cascade, the membrane attack complex may have a primary role in altered vascular permeability during ischemia-reperfusion injury. four components initiate the contact system of coagulation: hageman factor (hf), prekallikrein, factor xi, and high-molecular-weight kininogen. hf is a large plasma glycoprotein that binds avidly to negatively charged surfaces. bacterial cell walls, vascular basement membranes, heparin, glycosaminoglycans, and other negatively charged surfaces in the intestine capture hf and the other three important initiators of the contact system in a large multimolecular complex. of the surfaces that bind hf, the extracellular matrix is a potent activator of the contact system. once bound, hf is converted to hf-α, which cleaves prekallikrein to kallikrein and factor xi to factor xia. the ultimate result is further cleavage of hf by kallikrein and triggering of the contact system cascade, activation of intrinsic coagulation by factor xia, activation of the alternative pathway by hf, and proteolytic cleavage of high-molecular-weight kininogen by kallikrein, releasing biologically active kinins. the products of the contact system, particularly bradykinin, have several important biologic properties that drive many of the vascular and leukocytic responses during inflammation. bradykinin induces endothelial cell contracture and intracellular tight junction alterations that increase vascular permeability to fluid and macromolecules. bradykinin also affects vascular smooth muscle contracture, resulting in vasoconstriction or vasodilation depending on the location. bradykinin also increases intestinal motility, enhances chloride secretion by the intestinal mucosa, and intensifies gastrointestinal pain. in neutrophils, kinins stimulate the release of many inflammatory mediators, including cytokines, prostaglandins, leukotrienes, and rois. kallikrein cleaves c to release c a, a potent chemotactic factor for neutrophils, and thus has a role in recruiting and activating inflammatory leukocytes. the plasma kallikrein-bradykinin system is activated in a variety of acute and chronic inflammatory diseases of the gastrointestinal tract. , recent evidence has demonstrated that blockade of the pathophysiologic effects of bradykinin has clinical applications. oral or intravenous administration of the bradykinin receptor antagonist icatibant reduces the clinical signs, onset of diarrhea, and many of the histopathologic changes in experimental models of colitis in mice. inhibition of kallikrein by oral administration of p attenuated the intestinal inflammation, clinical score, and systemic manifestations in a model of chronic granulomatous enterocolitis. thus the contact system is a viable therapeutic target for inflammatory diseases of the intestine. changes in blood flow to the mucosa and other regions of the intestine that reduce perfusion of the tissues can potentiate the initial damage caused by infection or injury. for example, reperfusion of ischemic tissues is associated with platelet and neutrophil clumping in the small vessels of the mucosa, which can impede blood flow. platelets are activated and adhere to exposed basement membrane and activated endothelial cells and provide a surface for leukocyte adhesion. the accumulation of platelets and leukocytes can reduce vessel diameter and blood flow significantly while potentiating local coagulation and thrombus formation. soluble mediators released by activated leukocytes and endothelial cells also affect blood flow. histamine and the vasoactive lipids derived from arachidonic acid (leukotrienes, prostaglandins, thromboxane, prostacycline, and platelet-activating factor) have a prominent role in regulating local perfusion during inflammation and also may have systemic effects on blood flow. procoagulant mediators released by inflammatory cells in response to the inflammatory process (i.e., tissue factor produced by macrophages or endothelial cells), exposed basement membrane proteins, and bacterial components can trigger the contact system and the coagulation and complement cascades, the products of which affect blood flow. nitric oxide, whether produced by endothelial cells or leukocytes (macrophages), is a potent regulator of blood flow and has a significant role in the control of perfusion during inflammation. many of the mediators that affect perfusion also affect endothelial permeability, altering osmotic and hydrostatic balance and tissue edema. in extreme cases, local and systemic coagulopathies initiated by vascular injury and absorption of microbial products and inflammatory mediators induce a hypercoagulable state, leading to microthrombus formation, which can reduce blood flow, or macrothrombus formation, which causes tissue infarction. the cellular mediators of inflammation have the potential to inflict severe injury to intestinal tissues. neutrophils have an important role in the pathophysiology of many intestinal diseases, including ischemia-reperfusion injury, , infectious enterocolitis, [ ] [ ] [ ] nonsteroidal antiinflammatory drug-induced mucosal ulceration, and others. depletion of neutrophils, blockade of their emigration into tissues, or inhibition of neutrophil activation reduce the severity of these and other inflammatory diseases. thus many antiinflammatory therapies are emerging that specifically target neutrophil adhesion, migration, and activation. migration of neutrophils through endothelium during emigration into inflamed tissues is remarkable in that the permeability of the endothelial monolayer is preserved under most circumstances. however, a limit exists above which neutrophil migration alters the permeability characteristics of the endothelium. the effect is in part physical in that mere movement of large numbers of neutrophils through the endothelium is sufficient to disrupt the tight junctions mechanically and is caused in part by toxic products of neutrophils that damage endothelial cells and basement membranes. , serine proteases (particularly elastase) and metalloproteinases released by degranulating neutrophils destroy tissue matrix proteins and cell-surface proteins that make up endothelial intercellular junctions. these degradative enzymes are particularly damaging to basement membranes and the cellular barriers of the endothelium, thus contributing to vascular permeability (and local tissue edema) and thrombosis. the permeability may be affected to the extent that not only water but macromolecules (albumin, matrix proteins, complement, etc.) leak into the interstitium. blockade of neutrophil adhesion to endothelium with anti-β integrin antibodies has a sparing effect on the microvasculature in experimental intestinal ischemiareperfusion injury, reducing the alterations in vascular permeability and histopathologic evidence of microvascular damage. similar to the endothelium of inflamed tissues, massive neutrophil transmigration occurs across the epithelium in response to infection or injury. neutrophil transepithelial migration increases epithelial permeability by disrupting tight junctions. like the endothelium, neutrophils disrupt the epithelial barrier mechanically as they migrate through (see figure . - ). proteases, particularly elastase, degrade basement membrane components and tight junction proteins. products of activated neutrophils (tnf-α and interferon-γ) increase tight junction permeability by direct effects on the enterocytes. prostaglandins released by activated neutrophils stimulate epithelial secretion, thus contributing to diarrhea. subepithelial accumulation of neutrophils can lead to deadhesion of the epithelial cells from the basement membrane and mild to severe ulceration. the physiologic results of the effects of neutrophils and their products on the epithelial barrier include protein-losing enteropathy and absorption of bacterial cell wall constituents, which potentiates the local and systemic inflammatory responses. neutrophils in inflamed tissues stimulated by potent host-derived activators (such as il- β and tnf-α) and bacterial products (lipopolysaccharide) release copious amounts of rois (see figure . - ). although these oxygen and oxyhalide radicals are important for killing pathogens, they are also potentially toxic to epithelial and endothelial cells and matrix proteins. reactive nitrogen intermediates produced primarily by macrophages during inflammation combine with rois to form peroxynitrites, which are particularly toxic. in addition to injury to mucosal tissues, rois also have an as yet ill-defined role in recruiting and activating neutrophils, thereby potentiating the inflammatory response. in support of the role of rois in inflammatory diseases of the gastrointestinal tract, administration of inhibitors of roi production or pharmacologic roi scavengers can be protective in many models of reperfusion injury or enterocolitis. many therapies are aimed at inhibiting neutrophil activation and effector functions in tissues have been evaluated for use in intestinal diseases. phosphodiesterase inhibitors, by causing cyclic adenosine monophosphate accumulation in neutrophils, are antiinflammatory by virtue of their ability to suppress neutrophil activation and roi production. new phosphodiesterase inhibitors selective for the predominant neutrophil isoform of phosphodiesterase hold promise for use in many inflammatory diseases. subepithelial mast cells also have an important role in altering epithelial permeability in inflamed intestine. during the intestinal hypersensitivity response, subepithelial mast cell release of mast cell protease ii by degranulation increases epithelial permeability via an effect on tight junctions. , , this alteration in tight junction permeability results in enhanced transepithelial flux of macromolecules, including proteins and bacterial products. cytokines released by mast cells and phagocytes also regulate tight junction permeability. interleukin- , a product of mast cells and macrophages, increases epithelial permeability. moreover, tnf-α and interferon-γ, products of many inflammatory cells, synergistically increase tight junction permeability. acute equine colitis causes rapid, severe debilitation and death in horses (more than % of untreated horses die or require euthanasia). since , several reports have described a number of different acute diarrheal conditions in the horse that appear to share a common characteristic clinical presentation. - diarrhea associated with acute equine colitis occurs sporadically, is highly fatal, and is characterized by intraluminal sequestration of fluid, moderate to severe colic (abdominal pain), and profuse watery diarrhea with resultant endotoxemia, leukopenia, and hypovolemia. , , the condition can affect adult horses of all ages but usually occurs in horses between and years of age. disease onset is sudden with a rapid progression and often is preceded by a stressful event. a definitive diagnosis is made in only about % to % of cases. , most ante-and postmortem diagnostic tests remain speculative. treatment of the condition in horses is costly because of the massive fluid therapy required. currently, no curative treatment is available for acute colitis in horses, human beings, or other mammals. treatment regimens provide rehydration, electrolyte and plasma protein replacement, mitigation of the effects of circulating endotoxin, and antimicrobial therapy when indicated. attempts during the past years to develop appropriate treatments (i.e., vaccines or pharmacologic agents) have been hampered by the unavailability of acceptable equine models and have been unsuccessful because of the complex pathophysiology of the intestinal epithelium. although the mechanisms responsible for the fluid losses are not known, inflammatory cells may play an integral role because this condition is characterized by large numbers of granulocytes infiltrating the large intestinal mucosa. [ ] [ ] [ ] [ ] [ ] equine cecal and colonic tissues collected during the acute stages of experimentally induced acute equine colitis (equine ehrlichial colitis, lincomycin with and without clostridium spp. inoculation, nonsteroidal antiinflammatory drug administration) reveal the presence of numerous neutrophils and eosinophils in the lamina propria and submucosa. , , , granulocytederived reactive oxygen intermediates are crucial to antimicrobial defenses in the gut and stimulate chloride and water secretion by interactions with enterocytes. , normal equine intestinal tissue is unique compared with that in most other mammalian species for a preponderance of eosinophils located in the intestinal mucosa and submucosa. , production of reactive oxygen intermediates by stimulated phagocytic granulocytes following mucosal barrier disruption may be responsible for the massive fluid secretory response that occurs during the early stages of acute equine colitis. colitis refers to inflammation and mucosal injury of the colon and cecum (typhlocolitis) that may occur in response to a number of causes. , the cause of the colonic injury may be well-defined such as in naturally occurring infectious or experimentally induced colitis. however, many cases of human and animal diarrhea have a speculative or unknown diagnosis or no diagnosis. , , irrespective of the underlying or initiating cause of colonic injury, the colon apparently has a limited repertoire of responses to damage because most forms of colitis demonstrate similarities in histopathologic appearance and clinical presentation. various degrees of mucosal erosion and ulceration, submucosal/mucosal edema, goblet cell depletion, and presence of an inflammatory cellular infiltrate within the mucosa and submucosa are common to many types of human and animal colitis. , [ ] [ ] [ ] characteristic clinical manifestations include intraluminal fluid sequestration, abdominal discomfort, hypovolemia, and most often profuse, watery diarrhea. large bowel diarrhea results from abnormal fluid and ion transport by cecal and colonic mucosa. loss of fluid by the large intestine can result from malabsorptive or hypersecretory processes and is often a combination of the two. colonic secretory processes are a function of the crypt epithelium, whereas absorptive processes are limited to surface epithelial cells. under normal baseline conditions, an underlying secretion by crypt epithelium is masked by a greater rate of surface epithelial cell absorption. abnormal forces influencing the rates of secretion and absorption can result in massive, uncontrolled secretion and malabsorption by large intestinal mucosal epithelial cells, leading to rapid dehydration and death. , two intracellular processes control colonic secretion: the cyclic nucleotide (cyclic adenosine monophosphate [camp] and cyclic guanosine monophosphate [cgmp]) and the calcium system. , agents may activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e [pge ]) or guanyl cyclase (bacterial enterotoxins) and induce increases in camp or cgmp, respectively. this reaction causes phosphorylation of specific protein kinases that induce the actual apical and basolateral membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotide-dependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. , calcium may act through calmodulin, which then can activate membranephosphorylating protein kinases. at least four central systems control intestinal secretion: ( ) the hormonal system, ( ) the enteric nervous system, ( ) bacterial enterotoxins, and ( ) the immune system. , hormonal control of colonic electrolyte transport is exerted primarily through the renin-angiotensinaldosterone axis. , the enteric nervous system controls transport through three separate components: ( ) extrinsic nerves of the parasympathetic and sympathetic pathways; ( ) intrinsic ganglia and nerves, secreting a variety of neurotransmitters including peptides; and ( ) neuroendocrine cells (intraepithelial lymphocytes) that reside in the epithelium and release messengers onto the epithelial cells in a paracrine manner. , [ ] [ ] [ ] [ ] many bacterial enterotoxins can induce intestinal secretion by camp or cgmp signal transduction. bacterial enterotoxins can stimulate enteric neurons, providing evidence for interaction between two controlling systems. preformed inflammatory mediators such as histamine, serotonin, or adenosine and newly synthesized mediators such as prostaglandins, leukotrienes, platelet-activating factor, various cytokines, the inducible form of nitric oxide, and reactive oxygen metabolites can initiate intestinal secretion by directly stimulating the enterocyte and by acting on enteric nerves indirectly to induce neurotransmitter-mediator intestinal secretion. for instance, when added to the t colonic cell line, the known mast cell mediators histamine, adenosine, and pgd induce chloride secretion. [ ] [ ] [ ] prostaglandins of the e and f series can cause an increase in chloride secretion in intact tissue and isolated colonic cells. [ ] [ ] [ ] leukotrienes, platelet-activating factor, and a number of cytokines have been shown to have no effect on t cell secretion but have a significant effect on electrolyte transport in intact tissue, suggesting that intermediate cell types may be involved in these secretory responses. [ ] [ ] [ ] the epithelial cell chloride secretory response occurs via prostaglandin-and adenosine-mediated increases in cellular camp, whereas histamine acts by h receptor induction of phosphatidylinositol turnover, production of inositol triphosphate, and mobilization of intracellular calcium stores. , lipoxygenase products (leukotrienes) are capable of activating a colonic secretory response and do not appear to involve the cyclic nucleotides or calcium ions. phagocyte-derived reactive oxygen mediators (roms) can induce colonic electrolyte secretion in vitro, suggesting that oxidants may contribute directly to the diarrhea associated with colitis. [ ] [ ] [ ] [ ] [ ] reactive oxygen species initiate the secretory response by increasing cellular camp or stimulating mesenchymal release of pge or prostacyclin, which in turn stimulates the epithelial cell or enteric neuron, respectively. [ ] [ ] [ ] [ ] [ ] [ ] sodium nitroprusside, an exogenous source of nitric oxide, stimulated an increase in chloride secretion in rat colon that was mediated by cyclooxygenase products and enteric neurons. table . - summarizes inflammatory mediator-induced epithelial cell chloride secretion. acute colitis rarely develops by a simple cause or effect phenomenon but is influenced by many extrinsic and intrinsic host and microorganism factors. inflammatory mediators released from mast cells and monocytic or granulocytic phagocytes cause intestinal chloride and water secretion and inhibit neutral sodium and chloride absorption. , , inflammatory cells, particularly the phagocytic granulocytes, play an important role in mucosal pathophysiology in cases of colitis. , large numbers of these cells are observed on histopathologic examination of tissues from human and animal cases of colitis. products of cell activation stimulate direct and indirect secretory responses in intestinal cells and tissues. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] products of phagocyte secretion may amplify the inflammatory signal or have effects on other target cells in intestine such as enterocytes and smooth muscle cells (table . - ). the nadph-oxidase system of phagocytes (neutrophils, eosinophils, monocytes/macrophages) is a potent inducer of superoxide radicals used as a host defense mechanism to kill invading microorganisms. , during inappropriate stimulation such as inflammation, trauma, or ischemia followed by reperfusion, increased levels of toxic oxygen species are produced, causing damage to host tissues. engagement of any of several receptor and nonreceptor types including phagocytosis mediators, chemotactic agents, various cytokines, and microbial products can stimulate phagocytes. resident phagocytes or those recruited to colonic mucosa early in the disease process are considered to augment mechanisms causing fluid and electrolyte secretory processes, a so-called amplification process. , activation of the respiratory burst results in the production and release of large amounts of superoxide anion (o − ) and h o . , in addition to these roms, activated phagocytes secrete peroxidase enzyme (myeloperoxidase from neutrophils and eosinophil peroxidase from eosinophils) into the extracellular space. the peroxidases catalyze the oxidation of clby h o to yield hocl, the active ingredient in household bleach products. the peroxidase-h o -halide system is the most cytotoxic system of the phagocytes; hocl is to times more toxic than o or h o . hocl is a nonspecific oxidizing and chlorinating agent that reacts rapidly with a variety of biologic compounds including dna, sulfhydryls, nucleotides, amino acids, and other nitrogen-containing compounds. hocl reacts rapidly with primary amines (rnh ) to produce the cytotoxic n-chloramines (rnhcl). the mechanisms by which hocl and rnhcl damage cells and tissue remain speculative, but possibilities include direct sulfhydryl oxidation, hemoprotein inactivation, protein and amino acid degradation, and inactivation of metabolic cofactors of dna. peroxidase-derived oxidants have been shown to degrade hyaluronic acid and collagen. in addition, luminal perfusion of specific roms increased mucosal permeability and serosal application caused increases in clsecretion in vitro. tissue myeloperoxidase activity, an index of tissue granulocyte infiltration, is used clinically and experimentally to assess degree of intestinal inflammation. , myeloperoxidase activity is elevated in acute flare-ups of human inflammatory bowel disease and various animal models of acute colitis. [ ] [ ] [ ] [ ] [ ] the acute inflammatory response in these conditions is characterized predominantly by neutrophils, the predominant source of myeloperoxidase activity. however, this assay measures total hemoprotein peroxidase, which includes monocyte and eosinophil peroxidase in addition to neutrophils. moreover, levels of peroxidase activity in equine circulating eosinophils are greater than in circulating neutrophils, and this may apply to resident tissue eosinophils as well. arachidonic acid metabolites are thought to play a role in intestinal inflammation in diarrheal disease. , , elevated levels of these intermediate metabolites have been demonstrated in natural disease and experimental models of colitis and appear to parallel increases in roms in inflamed intestine. addition of h o or hocl to rat colonic tissue in ussing chambers has been shown to induce pge release and active clsecretion. , prostaglandins can stimulate increases in clsecretion in intact intestinal tissue , , and in isolated colonic t cells. , interactions between roms and mesenchymal release of pge /pgi may be relevant to the mechanisms producing the diarrheic condition. fibroblasts co-cultured or juxtaposed to colonic t cells greatly increased the clsecretory response to h o in vitro through the release of pge . in addition, equine colonic mucosa has an increased sensitivity to endogenously released prostaglandin by exhibiting a significant secretory response under in vitro conditions. endotoxin, the lipopolysaccharide component of the outer cell wall of gram-negative bacteria, is present in large quantities in the large intestine of healthy horses. , endotoxins are released into the immediate surroundings when gram-negative bacteria undergo rapid proliferation or die. , the intact bowel forms an effective barrier to the transport of significant amounts of these highly antigenic toxins, but the diseased gut absorbs these macromolecules in large amounts, causing the subsequent adverse systemic effects that are often life threatening. disruption of the intestinal barrier (i.e., ischemia, trauma, inflammatory conditions) overwhelms the capacity of the liver to clear endotoxins, and systemic endotoxemia ensues. endotoxins have been shown to be potent activators of the inflammatory process, stimulating the production and release of numerous cytokines by activated macrophages and other immunocytes. in vitro studies suggest that endotoxin activates phagocytic granulocytes to secrete roms, increase release of lysozymes, and enhance the migratory response to chemotactic stimuli. prostacyclin and thromboxane a mediate hemodynamic dysfunction, and lipoxygenase products may induce tissue ischemia. the cytokine interleukin- causes a febrile response and initiates the acute phase response. tumor necrosis factor contributes to many of the abnormal physiologic responses, particularly hemostatic functions that potentiate coagulopathy. additional mediators include interleukin- , platelet-activating factor, procoagulant mediators, and various other speculated substances. endotoxins trigger mucosal immune cells and subsequent release of inflammatory mediators in cases of colitis. the first report of experimentally induced endotoxemia described clinical signs and hematologic findings that closely paralleled those reported for severe colitis in horses. studies in which endotoxin was administered intravenously in human beings and laboratory animals caused significant dose-related gastrointestinal changes, ranging from mild diarrhea to bloody, watery diarrhea. , in vitro studies on the effects of endotoxin on intestinal water and electrolyte transport in adult male rats showed a significant decrease in net colonic sodium absorption and increased colonic permeability. in animal models of protein energy deficiency, endotoxin-induced mortality increased compared with that of well-nourished control animals. endotoxin depresses lymphocyte responses to specific mitogens. thus the adverse effects of malnutrition and endotoxin are mutually aggravating. the importance of a normal immune system to the defense of the mucosal surface of the gastrointestinal tract is evident in the immunosuppressed state. primary immunodeficiencies affecting the gastrointestinal tract are well documented. [ ] [ ] [ ] common agammaglobulinemia is the most frequently reported gastrointestinal disease and causes b cell deficiency-associated giardiasis. interestingly, selective immunoglobulin a (iga) deficiency rarely results in intestinal disease because of a speculated increase in mucosal igm response. however, combined iga and igm deficiencies with a higher incidence of intestinal disease occur. a selective deficiency of secretory iga has been associated with intestinal candidiasis. certain mucosal pathogens may enhance their pathogenicity by producing iga proteases. defects in cell-mediated immunity are associated most commonly with intractable diarrhea, and organisms frequently involved include salmonella spp., escherichia coli, and shigella spp. acquired immunodeficiency or immunosuppression in adults can result from infectious diseases (particularly viral), nutritional deficiencies, aging phenomenon, and drugs (corticosteroids, azathioprine, cyclophosphamide). nutrition is a critical determinant of immunocompetence and risk of illness. , impaired systemic and mucosal immunity contribute to an increased frequency and severity of intestinal infections observed in cases of undernourishment. abnormalities occur in cell-mediated immunity, complement system, phagocytic function, mucosal secretory antibody response, and antibody affinity. morbidity caused by diarrheal disease is increased particularly among individuals with stunted growth rate because of malnourishment. the critical role of several vitamins and minerals in immunocompetence has been substantiated in animals deprived of one dietary element and findings in human patients with single-nutrient deficiency. tables . - and . - summarize nutritional-related immune system abnormalities. in summary, nutritional deficiency can cause increased colonization of the intestine with microorganisms, alter the symbiotic characteristics of resident intestinal bacterial populations, and impair defenses of the gastrointestinal tract, allowing increased risk of systemic spread of infection and absorption of macromolecules (in particular, endotoxin). indigenous microflora greatly impede colonization of the gastrointestinal mucosa by pathogenic organisms. the ability of a potential pathogen to initiate an infection depends on its ability to breach the mucosal epithelial barrier. one mechanism by which the normal flora inhibit establishment of pathogens is by preventing adherence of the pathogen to mucosal cells by occupying the site or by stearic hindrance. [ ] [ ] [ ] [ ] resident microbes also produce byproducts such as antibacterial factors that allow symbiosis rather than competition between them. another hindrance mechanism is production of volatile fatty acids by normal microbial digestive processes to create an environment that is toxic to many bacterial populations, particularly the enterobacteriaceae. disturbances in motility patterns occur during inflammatory diseases of the colon, but the role of motility alterations in the pathogenesis of diarrhea remains unclear. invasive bacteria cause characteristic motor patterns in the colon consisting of rapid bursts of motor activity that appear to decrease transit time through the large intestine. the result is reduced clearance of bacteria from the large intestine, which may contribute to the virulence of the organism. absorption of endotoxin and the release of inflammatory mediators such as prostaglandins disrupts the motility patterns of the large intestine, resulting in less coordinated contractions, and may contribute to the alterations in motility seen with invasive bacteria. although the effect of endotoxin and prostaglandins on transit time is not profound, the disruption of coordinated activity may play a role in causing diarrhea. thorough mixing and prolonged retention time of ingesta are important not only in microbial digestion of nutrients but also in absorption of microbial byproducts and fluid. , the ingesta is viscous and therefore must be mixed to bring luminal ingesta in contact with the mucosa for absorption. in addition, poor mixing increases the thickness of the unstirred layer, decreasing contact of ingesta with the mucosa and decreasing absorption. , progressive motility must be present, however, if a diarrheal state is to occur. , ileus may be accompanied by increased fluid in the lumen of the large intestine, pyridoxine, folic acid, impairs cell-mediated immunity and vitamin c, vitamin e reduces antibody response. vitamin b decreases lymphocyte stimulation response to specific mitogens. zinc deficiency affects humoral and cell-mediated immunity. iron inhibits bacterial multiplication. copper depresses antibody response. needed by neutrophils and lymphocytes for optimal function, which may be related to myeloperoxidase and ribonucleotidyl reductase deficiencies. but without progressive motility the fluid is not passed. frequently, acute colitis causes a period of ileus characterized by scant stool. diarrhea is apparent only when motility returns and the ingesta is passed. increased progressive motility has been suggested to cause diarrhea by decreasing transit time and is thought to play a role in irritant catharsis and in the mechanism of action of some laxatives. irritation and distention increase motility and may well decrease transit time, but increased secretion also is thought to contribute to diarrhea caused by these substances. one should direct the clinical investigation of malabsorption at ascertaining and trying to localize the source of the abnormality. in medical practice, impairment of one or more phases of the digestion and absorption of dietary constituents may precipitate clinical signs that are associated primarily with carbohydrate, protein, or fat malabsorption. this level of differentiation is not possible in the horse because of the herbivorous diet and the contribution of large intestinal functions. in human and small animal medicine, disturbances in digestive processes especially from exocrine pancreatic insufficiency or reduced intestinal bile salt concentration are principal determinants of many clinical malabsorption syndromes. the rarity of pancreatic problems in horses and the herbivorous diet makes maldigestion less of a concern and difficult to pursue diagnostically. nevertheless, maldigestion undoubtedly contributes to chronic weight loss conditions in horses, which may be significant with severe infiltrative disease of the small intestine with partial to total villous atrophy and flattened mucosa. impairment of digestive processes may exacerbate diarrhea in the suckling foal through reduced intestinal bile salt concentrations from hepatic or ileal dysfunction. malabsorption is not synonymous with diarrhea, although diarrhea may be a feature. adult horses rarely exhibit diarrhea with small intestinal problems unless large intestinal involvement is concomitant. chronic diarrhea is predominantly a large intestinal disorder that reflects an overload of water and electrolytes and thus may be considered a state of impaired absorption. primary small intestinal disease is more likely to occur in neonates and young foals. for example, acquired small intestinal brush border lactase deficiency may result in increased lactose fermentation in the large intestine and induction of osmotic diarrhea. box . - lists conditions that have been or potentially could be associated with malabsorption syndromes and maldigestion in the horse. malabsorption in horses has no pathognomonic clinical syndrome. case recognition derives from the robust investigation of horses with chronic wasting. prevalence is unknown. no strict case definition exists, even for chronic wasting. interest generated by unusual clinical test results and their related pathologic findings has stimulated publication of reports of cases considered as malabsorption in horses. pathologic description of the predominant cellular infiltrate and the pattern of intestinal distribution have resulted in the classification of many conditions as representing examples of chronic inflammatory bowel disease (cibd) (see box . - ), drawing analogies from human medicine. in the affected animal, coexistent enteric protein loss reflecting changes in mucosal integrity from extensive infiltration and inflammation in the intestinal tract is likely to be more debilitating than the malabsorption. the principal concern of the owner is the weight loss and poor condition of the horse. many clinical examination findings, except for body condition, may appear within normal limits. investigation of the weight loss, together with the clinical pathologic findings, helps to eliminate other more commonly encountered causes of wasting. box . - lists clinical signs that may be associated with malabsorption syndromes. no characteristic clinical pathologic profile of malabsorption exists. findings relate to the stage of the underlying disease process and intercurrent problems. the syndrome tends to cause anemia (normocytic, normochromic) and neutrophilia. hemolytic or macrocytic anemia and thrombocytopenia have been observed in alimentary lymphosarcoma. lymphocytosis (leukemia) rarely is encountered. eosinophilia is uncommon even with suspected immune-mediated conditions and widespread tissue eosinophilia. many animals are hypoalbuminemic and hypoproteinemic; horses with alimentary lymphosarcoma may exhibit hyperproteinemia and hypergammaglobulinemia. serum or plasma may be lipemic. the clinician may find elevated hepatic and biliary tract enzymes (γ-glutamyltransferase and alkaline phosphatase) in multisystemic conditions; for example, eosinophilic granulomatosis (multisystemic eosinophilic epitheliotropic disease; eg [meed]). abdominocentesis has been of diagnostic value in several alimentary lymphosarcoma cases, but rarely in the granulomatous conditions. ultrasonographic examination of the abdomen can yield infomation on intestinal distention, wall thickness, and unexplained masses detected on rectal palpation. rectal biopsy is easy to perform and may provide an indication of cellular infiltration that could be present at more proximal locations. however, pathologists examine few equine rectal samples, and the interpretation is frequently equivocal. adoption of standardized grading or classification would improve the diagnostic value. a proposed classification system was based on a retrospective study of rectal biopsies from horses ages to years with clinical signs of intestinal disease. necropsy results were studied from horses. biopsy specimens ( horses) and necropsy rectal tissue ( horses) from horses ages to years served as controls. simple proctitis, the presence of neutrophils in the crypt or surface epithelium, was an abnormal finding compared with mild scattered neutrophil infiltration in controls. simple proctitis was found in association with malignant lymphoma and other inflammatory disorders. inflammatory bowel disease was diagnosed from rectal biopsy specimens in of eg (meed) cases and of granulomatous enteritis cases confirmed at necropsy. rectal biopsy aided diagnosis for of horses in a series of lymphocytic-plasmacytic enterocolitis cases. eosinophils were demonstrated on impression smears of rectal mucosal biopsies from of horses with eosinophilic enterocolitis. skin biopsies or ultrasound-guided biopsies of liver, lymph node, or lung may reveal evidence of multisystemic disease. one can obtain intestinal and lymph node biopsies via a standing laparotomy. exploratory laparotomy facilitates rigorous inspection of the gastrointestinal tract and associated organs to obtain multiple biopsies from intestinal sites and lymph nodes. the procedure may provide a diagnosis, enabling one to make decisions on potential treatment and management options. cost and potential postoperative complications may limit surgical procedures for diagnosis. laparoscopy may provide an alternative means to facilitate biopsy of certain tissues. however, one should consider surgical exploration as an option early in the process rather than as a last resort. the noninvasive breath hydrogen test used to assess carbohydrate malabsorption in human beings has not proved reliable in equine studies. intestinal function tests can provide a practical and inexpensive means to assess the absorptive capability of the small intestine. for clinical practice purposes this is limited to carbohydrate absorption. abnormality of carbohydrate absorption has become an important precept on which to base a diagnosis of malabsorption in the horse. however, results of the oral glucose tolerance test (ogtt) or d-xylose absorption test require cautious interpretation. pathologic changes in the mucosa and submucosa must be extensive and widely distributed to greatly affect the peak plasma concentration and shape of the curve. the tests are easy to perform in practice and require a baseline blood sample predosing and further samples for up to hours after administration of the solution. many commercial laboratories conduct glucose and xylose assays. the immediate dietary history, gastric emptying rate, intestinal transit, age, and hormonal effects of the horse influence glucose peak and curve shape. higher glucose peaks are recorded from healthy animals eating grass or hay than from those eating concentrates. recent appetite or the level of cachexia may affect test results. maximum plasma glucose level (> % baseline) is reached by minutes in healthy animals given g glucose per kilogram body mass as a % solution. , break points below which the probability increases of carbohydrate malabsorption associated with intestinal morphology changes have been proposed. a referral population of mature horses with chronic weight loss was divided into three groups based on ogtt results to determine if any concurrence with the morphologic diagnoses existed. group (n = ) had a normal ogtt (peak glucose concentration at minutes > % baseline) and contained animals that had normal small intestinal morphology, and a few with large intestinal lesions. group (n = ) had partial malabsorption and included horses with small intestinal infiltrative disease that allowed some glucose uptake. diagnoses included lymphosarcoma, villous atrophy, granulomatous enteritis and eg (meed). seven horses had normal small intestinal histologic findings. peak glucose concentrations were less than % and greater than % of baseline at minutes. seventeen horses in the group had large intestinal pathologic conditions. group horses (n = ) had total malabsorption; the peak concentration at minutes was less than % above baseline. these horses had severe infiltration throughout most of the small intestine that was attributed predominantly to lymphosarcoma or granulomatous enteritis. however, the test is far from definitive; one cannot assume a flat curve indicates malabsorption and a poor prognosis. two horses with chronic weight loss initially diagnosed with malabsorption based on flat ogtt curves subsequently showed more normal ogtt responses. full-thickness intestinal biopsies were unremarkable. one horse had an elevated serum immunoglobulin e to oat allergen. oats and oat straw were removed from access. dexamethasone was given on a tapered protocol, and a repeat ogtt was normal at months. the other horse received oral probiotics to counter suspected small intestinal bacterial overgrowth, was clinically normal in months, and had an improved ogtt with a minute peak. therefore malabsorption, as defined by an absorption test and weight loss, may occur in the horse without significant morphologic changes in the intestine, and the condition may be transient. demonstration of carbohydrate malabsorption in of horses with chronic diarrhea showed poor diagnostic sensitivity for small intestinal involvement. impaired glucose absorption was recorded in horses with predominantly large intestinal problems, cyathostomiasis, chronic colitis, alimentary lymphosarcoma, and meed. although prior dietary history influences peak plasma xylose concentration, xylose is not confounded by hormonal effects or mucosal metabolism. gastric emptying rate, intestinal motility, intraluminal bacterial overgrowth, and renal clearance do affect curve shape. healthy mares not fed for up to hours had flatter curves and a slower decrease in plasma xylose than when deprived for to hours. hence recent appetite or the level of cachexia may influence test results. abnormal d-xylose absorption represented by a flat curve or delayed absorption is considered indicative of significant jejunal disease and has been observed with most examples of cibd, parasitism, and idiopathic villous atrophy. , ponies may have lower peak d-xylose concentrations at and minutes than horses, although the range of peak values at the test dosage ( . g d-xylose per kilogram body mass in a % solution) is wide. potentially diagnostic discriminatory cut off points for peak plasma xylose concentrations have not been determined. abnormal absorption curves have been detected in the absence of small intestinal histologic changes, and interpretation is clouded further by findings from small intestinal resection studies in healthy ponies. nine ponies with % distal small intestinal resection and four shamoperated controls were placed on interval feeding for weeks and then turned out to pasture until months after surgery. grazing was supplemented by twice daily (meal feeding) concentrate rations. all ponies gained weight and were clinically normal, and none developed diarrhea. however, the mean peak xylose concentration at minutes declined progressively (at monthly intervals) over the study period in the resection group to % of that of controls. lack of clinical malabsorption was attributed to adaptation of the residual % of healthy small intestine and of large intestinal function. bacterial overgrowth in the small bowel remnant from refluxed cecal contents (resected ponies had ileocecal valve bypass) may have contributed to the abnormal xylose assimilation. by contrast, xylose absorption decreased over months, associated with substantial weight loss, lethargy, and diarrhea, in an earlier study of extensive (≥ %) small intestinal resection in ponies. an important difference was the feeding pattern; those ponies received pelleted feed twice daily for the entire month follow-up period. consequently, horses with suspected malabsorption may adapt to an interval feeding regimen. the critical factor could be the availability of sufficient unaffected or minimally affected small intestine and large intestinal functional capacity. the outcome for animals with small intestinal disease and some unknown degree of large intestinal pathologic dysfunction may be less successful than shown in the experimental study. abnormal xylose absorption reverted to normal following days of corticosteroid therapy in an adult thoroughbred gelding with a -week history of weight loss and diarrhea for weeks; peak xylose concentration at minutes was within normal limits and the horse had gained weight. d-xylose absorption was abnormal in an adult standardbred gelding with a -month history of poor performance, weight loss, intermittent fever, mesenteric lymphadenopathy, elevated fibrinogen, and decreased albumin and globulin levels. multiple fullthickness small intestinal biopsies revealed evidence of granulomatous enteritis. the horse received antibiotics postoperatively and then corticosteroids parenterally for to months. after weeks, peak plasma xylose had increased, although absorption was delayed. five months after cessation of corticosteroid therapy, the horse had regained weight and was bright and alert, and d-xylose absorption was normal. diagnostic predictions were made retrospectively by examining d-xylose absorption in horses with granulomatous enteritis compared with those with eg. peak xylose concentrations were much lower in horses with granulomatous enteritis than those with eg, whereas in eg the absorption curve shifted to the right with the peak occurring at minutes. the small intestine is affected predominantly in granulomatous enteritis with extensive villous atrophy and more diffuse lesions in the large intestine, whereas in eg (meed) the large intestine is more involved. hence, the extent and distribution of pathologic changes in the small and large intestines may influence xylose absorption test results. the chronic wasting horse with suspected malabsorption and probable enteric protein loss has at best a guarded to poor prognosis. prognosis may be improved through early and aggressive investigation to achieve a diagnosis, and perhaps assess the stage in the natural progression of the disorder. the owner may elect euthanasia of the animal or may be willing to determine whether the condition can be improved. in the short term, intravenous infusion of plasma or colloids, with or without fluids and electrolytes, may be necessary to stabilize the condition. prognosis is much worse for the horse that is inappetent. prolonged intensive total parenteral nutrition and/or oral alimentation may not be a realistic course of action. the overall therapeutic and management plan can prove to be expensive. the owner must be cognizant from the start that the outcome may not be altered, even after protracted therapy. one cannot make predictions for outcome of therapy without meaningful data because only a few case reports of successful responses with long-term follow up exist. some level of digestive and absorptive capability remains in the diseased small intestine. interval feeding of small quantities of food may be beneficial if the horse continues to eat, and particularly for animals with ravenous appetites that seem able to maintain their reduced state of body condition without further losses. diet should include feeds with a high fiber content to favor large intestinal fermentation, including grass hay and access to pasture complemented by commercial high-fiber rations based on beet pulp and soybean hulls. energy intake can be increased through feeding high-energy dense fats that provide . times more calories than carbohydrates. most affected horses should tolerate high fat ( % to %) processed feeds containing vegetable oils or rice bran (up to % of the concentrate mix, equivalent to % vegetable oil) to achieve the higher-fat composition. changeover to a higher-fat concentrate should be gradual. even in healthy animals that can eat up to % added fat, appetite may decrease as the percentage increases, and fecal consistency may change. clearly, the objective for the horse with suspected malabsorption is to sustain, and preferably increase, dietary intake, value, and efficiency. the owner of an affected horse must be prepared to experiment with feeds, must be patient, and must keep records. no standard procedure exists. exposure to a feed component may contribute to the problem as an allergen eliciting a hypersensitivity reaction. identifying the potential allergen through immunologic testing or by stepwise removal and outcome assessment over a longer period may be difficult. the clinician should give immunosuppressive drugs early in the process. immunosuppressive agents have produced the most promising responses to ameliorate the effects of conditions associated with malabsorption, particularly cibd. short-duration, and in some cases more prolonged and sustained, improvements in body condition, weight gain, demeanor, energy and activity levels have occurred following corticosteroid administration. one should start treatment as early as possible. one should follow initial parenteral (intramuscular or intravenous) loading doses of dexamethasone (sodium phosphate) with a series of depot injections, or orally administered prednisolone or prednisone, on a tapered dose protocol over a period of months. interval low-dose therapy may be necessary if clinical signs return after treatment ends. one uses the lowest dose to control the clinical signs for alternate-day therapy. clinical benefits far outweigh concerns over potential adverse effects. chemotherapeutic agents such as vincristine, cytosine, cyclophosphamide, and hydroxyurea have been tried in a few cases of cibd or lymphosarcoma with no apparent success, probably related to the advanced stage of the disease when treatment was initiated and the dose selected. resection of a segment of intestine that is edematous, hemorrhagic, or constricted is an option in localized forms of cibd, , particularly if gross changes are not discernible in adjacent or distant parts of the intestinal tract, that is, malabsorption is not a feature. long-term outcome has been favorable. removal of a substantial proportion of the diseased small intestine may be indicated in a horse with malabsorption, considering that resection of % distal small intestine was performed in healthy animals without inducing adverse effects. however, because pathologic changes may exist in normalappearing small or large intestine that is not resected or biopsied, the prognosis remains guarded. two young horses with granulomatous enteritis had the thickened terminal small intestine resected with positive outcomes; one survived months, the other has a follow up extending more than years. anthony t. blikslager to gain an appreciation of the mechanisms whereby the mucosa is injured and subsequently repaired, one must understand how the integrity of the mucosa is regulated physiologically. regulation of mucosal integrity is referred to as mucosal barrier function, which is vital because it prevents bacteria and associated toxins from gaining access to subepithelial tissues and the circulation. however, the mucosa has two conflicting functions: it must serve as a protective barrier and continue to absorb solutes necessary to maintain well-being of the host. this conflict is most notable at the intercellular (paracellular) space, which allows passage of select solutes and water, - but which does not admit large molecules, including bacterial toxins. the paracellular space is regulated almost exclusively by the tight junction, which is the interepithelial junction at the apical-most aspect of the paracellular space. although these tight junctions originally were viewed as inert cellular adhesion sites, what has become clear in recent years is that tight junction permeability depends on tissue-specific molecular structure and is regulated by a complex array of intracellular proteins and the cytoskeleton. tight junctions consist of a group of transmembrane proteins that interdigitate from adjacent cells. although occludin originally was thought to be the predominant tight junction transmembrane protein, a group of proteins termed claudins appear to be more critical. these transmembrane proteins interact with the cytoskeleton via a series of intracellular proteins, including zonula occludens , , and ; cingulin; and others. in addition, local regulatory proteins such as the small guanosine triphosphatase-rho are also critical to tight junction function. in general the relative contractile state of the actin cytoskeleton determines the degree to which tight junctions are open or closed, but the complexities of regulation of this process are understood poorly. , the most sensitive measure of mucosal barrier function is transepithelial electric resistance, which is measured by mounting mucosa in an ex vivo system called an ussing chamber, because this measurement is largely a reflection of the permeability of mucosa to ions. , ions may follow one of two routes when traversing epithelium: transcellular and paracellular. because cell membranes have a resistance to passive flow of ions . to log units greater than that of the epithelium as a whole, measurements of transepithelial resistance largely reflect the resistance of the paracellular space, and in particular the tight junctions that regulate paracellular flow of ions. because tight junctions differ in structure from different portions of the mucosa, measurements of transepithelial resistance reflect the net resistance of epithelium of variable permeability within a given tissue. for example, tight junctions in the intestinal glandular structures called crypts are leakier than those in the surface epithelium because of fewer and less organized tight junction strands. , conversely, surface epithelium has a greater number of well-organized tight junction strands that result in epithelium with a high resistance. this correlates well with the absorptive function of epithelium located on the mucosal surface and the secretory function of crypt epithelium. structure of tight junctions also varies with the segment of intestine. for example, tight junctions have more strands in the ileum than in the jejunum, which is reflected by a higher transepithelial resistance in the ileum. the stomach has four regions based on the type of mucosal lining (in an orad to aborad order): nonglandular stratified squamous epithelium, cardiac epithelium, proper gastric mucosa, and pyloric mucosa. stratified squamous epithelium has distinct differences in terms of barrier function compared with the remainder of the gastrointestinal tract. this epithelium has baseline transepithelial resistance measurements of approximately to Ω/cm , which is an order of magnitude higher than the adjacent cardiac mucosa. , thus the stratified squamous mucosa is exceptionally impermeable. this in effect is the only mechanism this mucosa has to defend itself against injury. the stratified squamous epithelium consists of four layers: the outer stratum corneum, stratum transitionale, stratum spinosum, and the basal stratum germinativum. however, not all layers contribute equally to barrier function, the barrier being composed mostly of interepithelial tight junctions in the stratum corneum and mucosubstances secreted by the stratum spinosum. , the relative impermeability of stratified squamous mucosa can be demonstrated by the effects of hcl on this type of epithelium in vitro, which has little effect until it reaches a ph of . or lower. thus although most of the literature on equine ulceration pertains to the effects of hcl and inhibitors of hcl secretion, [ ] [ ] [ ] [ ] other factors may be critical to the development of gastric ulcer disease. the site of hcl secretion (proper gastric mucosa) also is protected from so-called back-diffusion of h + by a high transepithelial electric resistance (compared with cardiac mucosa), but a number of other critical mechanisms also exist to prevent acid injury. the gastric mucosa secretes mucus and bicarbonate, which together form a hco --containing gel that titrates acid before it reaches the lumen. , the mucus layer is formed principally by glycoproteins (mucins) secreted by goblet cells but also includes other gastric secretions and sloughed epithelial cells. mucins consist of core peptides with a series of densely packed o-linked polysaccharide side chains that, once secreted, become hydrated and form a viscoelastic gel. however, the mucus layer does not form an absolute barrier to back-diffusion of acid. thus for acid that does back-diffuse into the gastric mucosa, epithelial na + /h + exchangers are capable of expelling h + once the cell reaches a critical ph. recent studies have renewed interest in the protective mechanisms of mucus because of the discovery of a group of compounds secreted by goblet cells called trefoil peptides. the name of these peptides is derived from a highly conserved cloverleaf structural motif, which confers substantial resistance to degradation by proteases including pepsin. three members of this group are known, ps , sp, and intestinal trefoil factor, the latter of which is secreted solely by goblet cells in the small and large intestine. ps and sp are secreted by goblet cells within the stomach and are believed to intercalate with mucus glycoproteins, possibly contributing to the barrier properties of mucus. these peptides also play a critical role in repair of injured mucosa. an additional mucosal function that serves to reduce the level of injury is adaptive cytoprotection, wherein application of topical irritants to gastric mucosa results in subsequent protection of mucosa in response to repeated exposure to damaging agents. for example, pretreatment with % ethanol protected against mucosal damage in response to subsequent application of absolute ethanol, and this effect was abolished by treatment with the cyclooxygenase inhibitor indomethacin. the cytoprotective effect of prostaglandins has been demonstrated directly in studies in which preadministration of prostaglandins protected gastric mucosa from damage by agents such as concentrated hcl and hypertonic saline. prostaglandins appear to be cytoprotective in the stomach at doses less than those used to inhibit gastric acid secretion, ruling out a simple antacid mechanism. although not fully characterized, cytoprotection has been attributed in part to prostaglandin-stimulated mucus production. an associated beneficial effect of prostaglandins is the increased production of bicarbonate, which is trapped within mucus on the surface of the mucosa. , interestingly, prostaglandin e (pge ) appears to lose its cytoprotective activity in the presence of the mucolytic agent n-acetylcysteine. attention also has been directed at enhanced mucosal blood flow as a potential mechanism for prostaglandin-mediated cytoprotection. for example, pretreatment with pgi protected against ethanol-induced mucosal damage as a result of increased mucosal blood flow. although pge , which is also cytoprotective, does not increase blood flow, it may prevent vascular stasis associated with irritant-induced vascular damage resulting from inhibition of neutrophil adherence to damaged endothelium. sensory nerves also have been implicated in cytoprotective mechanisms. these nerves are distributed throughout gastrointestinal mucosa. as an example of their importance in mucosal cytoprotection, pretreatment of newborn rats with capsaicin (to which sensory nerves are sensitive) renders the mature rats more susceptible to gastric injury. alternatively, use of a low dose of capsaicin, which stimulates rather than destroys sensory nerves, protects gastric mucosa against injurious agents. , sensory nerves contain neuropeptides such as calcitonin-gene-related peptide (cgrp) and substance p, which may play a protective role via vascular mechanisms. for instance, cgrp stimulates increased gastric blood flow, which is theorized to reduce injury in much the same way as prostaglandins do. in fact, recent studies suggest that the roles of prostaglandins and cgrp in gastric cytoprotection are intertwined intimately. in particular, pgi is believed to sensitize sensory nerves following treatment with a mild irritant, with resultant increases in cgrp release and mucosal flow. similar studies have shown that antagonists of cgrp inhibit the cytoprotective action of pge . another neural mediator, nitric oxide, also has been implicated in adaptive cytoprotection. interestingly, nitric oxide has a number of actions that are similar to those of prostaglandins, including maintenance of mucosal blood flow. regulation of barrier function in the intestine is not as well characterized as that of the stomach, although mechanisms of barrier function, including secretion of mucus and regulation of mucosal blood flow, are presumed to be similar. the proximal duodenum also has to protect itself from acid damage as it receives gastric contents, and this involves secretion of mucus and bicarbonate in much the same way as the stomach. one other mechanism that helps the stomach and the intestine to maintain mucosal barrier function is the speed with which the mucosa repairs. thus for a defect to develop in the mucosal barrier, injurious factors have to outpace mucosal recovery. such recovery initially involves epithelial migration across denuded regions of basement membrane (restitution), a process so rapid that epithelial defects may be resurfaced within minutes. for example, in bile salt-injured colon, denuded surface mucosa was covered completely by restitution. in the small intestine, villi greatly amplify the surface area of the mucosal luminal surface, which in turn takes far longer to resurface with restituting epithelium once it has become denuded. however, intestinal villi are able to reduce the denuded surface area considerably by extensively contracting. these mechanisms are described in detail under mechanisms of gastrointestinal mucosal repair. although the stratified squamous epithelium is relatively impermeable to hcl, a number of factors can enhance the damaging effects of hcl in this epithelium. in particular, bile salts and short-chain fatty acids are capable of breaking down the squamous epithelial barrier at an acid ph, thereby exposing deep layers to hcl, with subsequent development of ulceration. , high concentrations of short-chain fatty acids normally exist within the equine stomach because of microbial fermentation. these weak acids penetrate squamous mucosa and appear to damage na + transport activity principally located in the stratum germinativum. bile salts also may be present in the proximal stomach because of reflux from the duodenum. although such reflux has a high ph, bile salts appear to adhere to stratified squamous epithelium, becoming lipid soluble and triggering damage once the ph falls below . diet and management (e.g., periods of fasting) also play crucial roles in the development of conditions conducive to gastric ulceration. typically, a ph gradation in horses exists from proximal to distal compartments of the stomach, with the lowest ph values in the distal stomach. however, fasting disrupts this stratification such that low ph values may be recorded in the proximal stomach. fasting conditions also increase the concentration of duodenal contents within the proximal stomach, particularly bile. proper gastric mucosa is exposed to injurious agents, including pepsin, bile, and acid. parietal cells in the horse secrete acid constantly as an adaptation to near-continuous intake of roughage, but the enterochromaffin-like cells within the proper gastric mucosa and g and d cells within the pyloric mucosa tightly regulate acid secretion. histamine released by enterochromaffin-like cells amplifies acid secretion and interacts with h receptors on parietal cells and g cells, which release the prosecretory hormone gastrin. a combination of histamine and gastrin can have a synergistic effect on parietal cell gastric secretion, because these mediators have distinct receptors and second messengers. however, d cells are sensitive to an acidic environment and release somatostatin, which inhibits acid secretion. nonetheless, gastric mucosa may be exposed to acid for prolonged periods of time, particularly in horses that are extensively meal fed and that do not have the benefit of roughage, which tends to buffer stomach contents. , aside from peptic ulceration induced by combinations of acid and pepsin, research in human medicine has revealed the tremendous importance of helicobacter pylori in inducing ulceration. infection with this organism has the effect of raising gastric ph because of disruption of gastric glands and also induces an inflammatory reaction that causes damage. however, little evidence to date indicates that this organism is involved in gastric ulcers in horses. in the absence of a known role for infectious agents in gastric ulceration in animals, ulceration likely develops from injurious factors similar to those found in the proximal stomach, including gastric acid and bile. however, some factors that are important to induction of squamous epithelial ulceration may not be important in development of proper gastric mucosal ulceration. for example, feed deprivation and intensive training reproducibly induce squamous epithelial ulceration in horses but have little effect on proper gastric mucosa in horses. gastric acid likely plays a key role, whereas other factors such as nonsteroidal antiinflammatory drugs (nsaids) serve to reduce gastric defense mechanisms. in particular, inhibition of prostaglandin production reduces mucus and bicarbonate secretion while also reducing gastric mucosal blood flow. some of the nsaids also have a topical irritant effect, although this appears to be of minor significance because the route of administration (oral or parenteral) seems to have little influence on development of ulceration. the source of prostaglandins responsible for gastric protection originally was assumed to be cyclooxygenase (cox- ), because this isoform is expressed constitutively in gastric mucosa, whereas cox- is not expressed in the stomach unless induced by inflammatory mediators. however, mice in which the cox- gene has been knocked out fail to develop spontaneous gastric lesions, possibly because of compensatory increases in prostaglandin production by cox- . this concept agrees with recent data indicating that inhibition of both cox isoforms is required to induce gastric ulceration. from a clinical perspective this data indicate that drugs selective for cox- or cox- may be less ulcerogenic in the horse. because cox- elaborates prostaglandins induced by inflammatory stimuli, selective inhibitors of cox- may be particularly useful because of their ability to serve as antiinflammatory agents that are less ulcerogenic. the most notable cause of intestinal mucosal injury in horses, particularly those suffering from colic, is ischemia. initially, that a reduction in gastrointestinal blood supply leads to mucosal injury seems intuitive. however, the anatomy of the gastrointestinal tract and the differing structure of the intestinal mucosa at various anatomic locations have a significant influence on the extent of mucosal injury. furthermore, ischemic injury may be induced by several different mechanisms, including occlusion of arterial supply by a thrombus, strangulation of intestinal vasculature, and generalized reduction in blood flow associated with various shock states. in addition, a number of seemingly distinct mechanisms of intestinal injury, such as intestinal distention, also trigger mucosal injury via an ischemic mechanism. finally, reperfusion injury also may influence the extent of mucosal injury following an ischemic episode and has been proposed as a potential site of therapeutic intervention. , thus understanding the mechanisms of ischemia-reperfusion injury is critical to developing an understanding of the severity of various clinical conditions and beginning to formulate a therapeutic approach to diseases characterized by this devastating form of injury. the intestinal circulation is capable of closely regulating blood flow during periods of low systemic perfusion pressure. , in particular, local regulation of resistance vessels within the microvasculature is particularly prominent, whereby metabolic end products of adenosine triphosphate (atp) result in continued dilation of resistance vessels despite reductions in systemic arterial pressure. dilation results in continued perfusion of gastrointestinal tissues during the early stages of shock, while other organs such as skeletal muscle undergo massive shunting of blood resulting from increased constriction of resistance vessels. the reasons for these differences in regulation are not entirely clear but may relate to the high level of energy required to fuel the intestinal mucosa and the serious systemic effects of breaches in the mucosal barrier. however, as blood flow falls below a critical level, regulatory systems are no longer effective and oxygen uptake by the gastrointestinal tissue decreases, culminating in tissue damage. the tip of the villus is the most susceptible region affected by hypoxia in the equine small intestine, largely because of the countercurrent exchange mechanism of blood flow in the small intestinal villus. this countercurrent exchange mechanism is attributable to the vascular architecture, which consists of a central arteriole that courses up the core of the villus, arborizes at the tip, and is drained by venules coursing down the periphery of the villus. as oxygenated blood flows into the central arteriole, oxygen tends to diffuse across to the adjacent venules, which flow in the opposite direction. this series of events takes place along the length of the villus, resulting in a tip of the villus that is hypoxic even under normal conditions. furthermore, reduced blood flow as occurs in shock exacerbates the countercurrent exchange of oxygen, and the tip becomes absolutely hypoxic. this mechanism might explain why the small intestinal mucosa is more susceptible to ischemic injury, compared with the colon, which has no villi. for example, the duration required to produce severe morphologic damage to the equine colon is approximately % longer than in the small intestine. intestinal mucosal epithelium is susceptible to hypoxia because of the high level of energy required to fuel the na + /k + -atpase that directly or indirectly regulates ion and nutrient flux. the first biochemical event to occur during hypoxia is a loss of oxidative phosphorylation. the resulting diminished atp concentration causes failure of the energy-dependent na + /k + -atpase resulting in accumulation of sodium, and subsequently intracellular water. the ph of the cytosol drops as lactic acid and inorganic phosphates accumulate from anaerobic glycolysis. the falling ph damages cell membranes, including lysosomal membranes, resulting in the release and activation of lysosomal enzymes into the cytosol, further damaging cellular membranes. damage to the cell membrane allows the accumulation of high concentrations of calcium in the cytosol, which activates calciumdependent degradative enzymes. these events result in cytoplasmic blebbing of the basal membrane with subsequent detachment of cells from the underlying basement membrane. recent studies on epithelial injury during ischemia suggest that most epithelial cells undergo programmed cell death (apoptosis) during ischemia and reperfusion rather than necrosis, allowing retention of reusable components of irreversibly injured cells. in one study, % of detached epithelium during small intestinal ischemia and reperfusion underwent apoptosis. although the most obvious result of apoptosis is loss of surface epithelium, a number of cells on the lower portion of the villus (in the small intestine) and cells within the crypts also may undergo apoptosis that only may become evident up to hours following reperfusion of ischemic tissue. morphologic changes observed in ischemic-injured small intestinal mucosa follow a similar sequence regardless of whether ischemia alone or ischemia and reperfusion induce injury (table . - ). initially, epithelium separates from the underlying basement membrane, forming a fluid-filled space termed grüenhagen's space ( figure . - ). the mechanism of fluid accumulation in this space is not understood entirely but may result from continued epithelial absorption of nacl and water before it has detached fully from neighboring epithelial cells. this fluid accumulation likely exacerbates epithelial separation from the basement membrane. subsequently, epithelium progressively sloughs from the tip of the villus toward the crypts, which are the last component of the intestinal mucosa to become injured. [ ] [ ] [ ] injury of crypts likely relates to the vascular architecture, because crypts receive a blood supply separate from the vasculature involved in the villous countercurrent exchange mechanism. the early morphologic changes observed in the equine large colon during ischemia are different from those described in the equine small intestine because of the lack of intestinal villi. however, as might be expected, the more superficially located surface cells are sloughed before those in crypts. , the orderly progression of tissue injury has been used by one group of investigators to predict accurately the survival of horses with large colon volvulus. the researchers took biopsies from the pelvic flexure, which has been shown previously to reflect mucosal changes along the length of the colon accurately, and examined them histologically for the width of the crypts and intercrypt interstitial space. they expressed the latter measurements as a ratio of interstitium to crypt width (i:c) and defined nonviable colon as that which has greater than % loss of crypt and an i:c ratio greater than . using this methodology, researchers correctly predicted survival in % of horses. because of the dramatic decline in strongylus vulgarisinduced colic, which was associated frequently with infarction of intestinal arterial blood supply, most ischemic lesions are associated with strangulating obstruction. therefore considering mechanisms of ischemic injury in horses with naturally occurring strangulating lesions is important. the majority of experimental work has assessed complete ischemia (complete occlusion of the arterial blood supply) or low-flow ischemia (during which arterial blood flow is reduced). , however, during intestinal strangulation, a disparity between the degree of occlusion of the veins and arteries occurs whereby veins are occluded before arteries because of differences in compliance of vascular walls. thus strangulating lesions are typically hemorrhagic (hemorrhagic strangulating obstruction) as the arteries continue to supply blood to tissues that have little or no venous drainage. the result is ischemic injury, as previously outlined, but also a tremendous congestion of the tissues. such hemorrhagic congestion has two opposing effects: it disrupts tissue architecture, including the mucosa and its epithelium, and continues to provide oxygenated blood to the tissues during much of the ischemic episode. in contrast, when strangulation results in sudden cessation of arterial blood flow (ischemic strangulating obstruction), tissues appear pale, and the mucosa rapidly degenerates because of a complete lack of oxygenated blood. because intestine that may look nonviable (dark red) may in fact have less mucosal injury than that of ischemic strangulated intestine. an additional consideration in clinical strangulating obstruction is the degree of ischemia that intestinal distention may induce. for example, experimental distention ( cm of h o for hours) and decompression ( hours) of jejunum resulted in a significant increase in microvascular permeability and a significant decrease in tissue oxygenation similar to that which would be expected with low-flow ischemia. , in particular, microscopic evaluation of vasculature revealed capillary endothelial cell damage and local edema formation. this data suggest that distended intestine proximal to an obstruction may undergo mucosal injury despite its normal appearance. indeed, in one study, intraluminal pressures greater than cm h o in naturally occurring cases of colic correlated with a poor prognosis for survival. although that reperfusion of ischemic tissues results in exacerbation of mucosal injury recently has been taken for granted, one should remember that mechanisms underlying intestinal reperfusion injury have been defined largely in laboratory animals under specific conditions. [ ] [ ] [ ] [ ] [ ] however, studies on reperfusion injury in horses have had some conflicting results. , , the conflict may be attributable to the way in which the studies have been performed. in particular, the type of ischemia used in most laboratory animal studies has been low-flow ischemia (in which the blood flow typically is reduced to % of baseline flow), whereas studies in horses have used a number of different ischemic models, including various types of strangulating obstruction. although strangulating obstruction is of great clinical relevance, this type of ischemic insult is less likely to develop reperfusion injury. , , conversely, low-flow ischemia appears to prime tissues for subsequent injury once the tissue is reperfused, and considerable evidence supports the presence of reperfusion injury in horses following low-flow ischemia. , , , nonetheless, lowflow ischemia may not be a common clinical entity. in addition to the type of ischemia, other factors are involved in priming tissues for reperfusion injury, including species and anatomic-specific variation in oxidant enzyme and neutrophil levels ( table . - ). for example, the foal appears to have low levels of small intestinal xanthine oxidase, an enzyme that has been shown to play a critical role in triggering reperfusion injury in laboratory animals, , , whereas adult levels are much greater, particularly in the proximal small intestine. in addition, horses appear to have low numbers of resident neutrophils in the intestinal mucosa, and this population of neutrophils (rather than those recruited from the circulation) appears to be most critical for induction of reperfusion injury. however, studies demonstrating reperfusion injury in the equine colon following low-flow ischemia have shown significant accumulation of neutrophils within the mucosa. therefore a complete understanding of mechanisms of neutrophilic infiltration and the mechanisms whereby they damage tissue requires further study. reperfusion injury is initiated during ischemia when the enzyme xanthine dehydrogenase is converted to xanthine oxidase and when its substrate, hypoxanthine, accumulates simultaneously because of atp use ( figure . - ). , however, little xanthine oxidase activity occurs during ischemia, because oxygen is required as an electron acceptor. during reperfusion, xanthine oxidase rapidly degrades hypoxanthine in the presence of oxygen, producing the superoxide radical as a by-product. the superoxide radical contributes to oxidative tissue damage and, most importantly, activates neutrophil chemoattractants. , thus inhibition of xanthine oxidase in feline studies of intestinal ischemiareperfusion injury prevents infiltration of neutrophils and subsequent mucosal injury. , however, inhibition of xanthine oxidase has had no effect on ischemiareperfusion injury in equine small intestine and colon, suggesting that reperfusion injury is simply a continuation of injury initiated during ischemia, as suggested in some equine studies, or that the classic reperfusion injury pathway is activated by alternate sources of reactive oxygen metabolites. the latter has been suggested by studies in feline models of ischemia-reperfusion injury in which the source of a significant proportion of reactive oxygen metabolites is unknown and is independent of xanthine oxidase and neutrophils. a veterinary review of the pathogenesis of intestinal reperfusion injury in the horse suggested the concept of a therapeutic window wherein treatment of reperfusion injury would be beneficial. the basis of this concept is that certain conditions exist under which ischemic injury is minimal and that tissues are damaged severely during reperfusion. thus under conditions of low-flow ischemia, little injury is demonstrated during hours of ischemia, but remarkable injury occurs during hour of reperfusion. [ ] [ ] [ ] however, a therapeutic window may not exist under conditions of strangulating obstruction in which severe injury occurs during ischemia and minimal injury occurs during reperfusion. this in turn greatly reduces clinicians' ability to ameliorate ischemiareperfusion injury with treatments such as antioxidants at the time of reperfusion. mechanisms of gastric repair depend greatly on the extent of injury. for instance, superficial erosions can be covered rapidly by migration of epithelium adjacent to the wound; a process termed epithelial restitution. however, ulceration (full-thickness disruption of mucosa and penetration of the muscularis mucosa) requires repair of submucosal vasculature and extracellular matrix. the formation of granulation tissue initiates repair and supplies connective tissue elements and microvasculature necessary for mucosal reconstruction. connective tissue elements include proliferating fibroblasts that accompany newly produced capillaries that form from proliferating endothelium. recent studies indicate that nitric oxide is critical to both processes, , which likely explains the reparative properties of nitric oxide in the stomach. once an adequate granulation bed has formed, newly proliferated epithelium at the edge of the wound begins to migrate across the wound. in addition, gastric glands at the base of the ulcer begin to bud and migrate across the granulation bed in a tubular fashion. repairing epithelium expresses epidermal growth factor, which appears to facilitate these processes. in addition, a mucoid cap facilitates these events and retains reparative factors and serum adjacent to the wound bed. once the ulcer crater has been filled with granulation tissue and the wound has been reepithelialized, the subepithelial tissue remodels by altering the type and amount of collagen. despite the remodeling process, ulcers tend to recur at sites of previous ulceration, and the concern is that this remodeling can result in excessive deposition of collagen and fibrosis. reparative mechanisms are similar in the intestine, except that in the small intestine, mucosal villi contribute to mucosal repair. once intestinal epithelium is disrupted, two events occur almost immediately to reduce the size of the denuded portion of the villus: contraction of the villus and epithelial restitution ( figure . - ). for example, in porcine ileum subjected to hours of ischemia, villi were % of their former height and % of the denuded villous surface area was covered in flattened epithelium within hours. enteric nerves appear to regulate villous contraction, because inhibition of enteric nerve conduction prevents villous shortening following injury. the contractile component of the villus is a network of myofibroblasts distributed throughout the lamina propria of the villus and along the central lacteal. inhibition of villous contraction results in retarded epithelial repair because of the larger denuded surface that remains to be covered by migrating epithelium compared with similarly injured villi that have contracted. pge also has been implicated in regulating villous contraction, because application of pge resulted in villous contraction when perfused through normal rat ileum. as villi contract, assuming the basement membrane is intact, epithelium from the margins of the wound migrates centripetally to resurface toward the tip of the villus. the process of restitution is similar in denuded colonic mucosa, except that it may proceed more rapidly because of the lack of villi. epithelial restitution is solely a migratory event that does not depend on provision of new enterocytes by proliferation. cellular migration is initiated by extension of cellular lamellipodia that receive signals from the basement membrane via integrins. intracellular signaling converges on the actin cytoskeleton, which is responsible for movement of lamellipodia. specific components of the basement membrane appear to be critical to the migratory process. for example, application of antibodies to collagen types iii and iv, which are important components of intestinal mucosal basement membrane, impeded epithelial restitution. , other elements of the basement membrane, including proteoglycans, hyaluronic acid and noncollagenous proteins such as fibronectin and laminin also may provide important signals. these subepithelial matrix components that facilitate restitution may form the basis for clinical treatments designed to speed up the repair process, analogous to administration of matrix components to horses with articular cartilage damage. although epithelial restitution results in gross closure of previously denuded regions of gastrointestinal mucosa, closure of interepithelial spaces ultimately is required to restore normal epithelial barrier resistance. because the tight junction is principally responsible for regulating the permeability of the interepithelial space, repair and closure of this structure likely is critical to restore intestinal barrier function. recent research indicates that prostaglandins play a vital role in recovery of tight junction resistance, indicating that administration of nonselective cox inhibitors to horses with colic, particularly those recovering from strangulating obstruction, may be deleterious. therefore judicious use of nsaids is appropriate until more selective drugs that allow continued production of reparative prostaglandins are available for use in horse. after restoration of the epithelial barrier, the epithelium must reestablish normal mucosal architecture to allow normal gut absorptive and digestive function. in porcine ileum subjected to hours of ischemia, the epithelial barrier was restored within hours, but villi were contracted and covered in epithelium with a squamous appearance. restoration of normal villous architecture required another days. newly proliferated crypt epithelium replaces the flattened villous epithelium that characterizes restitution. under normal circumstances the dividing stem cells, of which the base of each mucosal crypt has approximately four, form new enterocytes. newly divided enterocytes migrate from the crypt onto the villus. during migration, enterocytes differentiate and acquire specific absorptive and digestive functions. fully differentiated enterocytes reside on the upper third of the villus for to days and then are sloughed into the intestinal lumen. this process accelerates during mucosal repair and requires increased proliferative rates. a variety of locally available gut-derived factors, including luminal nutrients, polyamines, and growth factors, may stimulate increased proliferation within to hours. the return of the normal leaflike shape of the villus occurs following the appearance of normal columnar epithelium. although prostaglandins have been implicated in mucosal cytoprotective function, few studies have assessed their importance in mucosal repair. one study implicated prostaglandins in growth factor-stimulated restitution, but a more prominent role of prostaglandins in mucosal repair is their ability to close interepithelial tight junctions. , , for instance, ischemic-injured small intestine rapidly recovers barrier function (as measured in vitro as transepithelial resistance) in the presence of pgi and pge , despite the fact that these prostanoids had little effect on villous contraction and epithelial restitution. however, electron microscopic examination of tissues reveals dilation of tight junctions in tissues treated with nsaids, whereas those additionally treated with prostaglandins have closely apposed tight junctions (figures . - and . - ). prostaglandins stimulate closure of tight junctions via the second messengers cyclic adenosine monophosphate and ca + , which interestingly were among the first mediators found to modulate tight junction permeability. , such tight junction closure is of importance to patients with intestinal injury that are treated with nsaids, because reduced prostaglandin levels may result in increased intestinal permeability. for example, in a study on ischemic-injured porcine ileum, treatment with the nsaid indomethacin resulted in a significant increase in intestinal permeability to inulin and lipopolysaccharide compared with tissues that were treated additionally with pgi and pge . section . pathophysiology of mucosal injury and repair a b figure . - ultrastructural appearance of repairing ischemic-injured mucosa. a, restituting epithelium hours following a -hour ischemic episode in the presence of the nonselective cyclooxygenase inhibitor indomethacin. dilation of the interepithelial space and the apical tight junction (arrows) correlates with a leaky intestinal barrier, b, similar restituting epithelium had been treated additionally with prostaglandins e and i . the close apposition of the tight junction (arrows) and the interepithelial space correlate with normalization of intestinal barrier function. -cm bar = µm. the process of restitution absolutely depends on a group of compounds called polyamines. , the rate-limiting enzyme in the formation of the polyamines spermine, spermidine, and putrescine is ornithine decarboxylase. in rats with stress-induced duodenal ulcers, systemic administration of the ornithine decarboxylase inhibitor α-difluoromethylornithine significantly reduced polyamine levels and greatly reduced epithelial restitution. furthermore, intragastric treatment of these same rats with putrescine, spermidine, and spermine prevented the delayed mucosal repair induced by α-difluoromethylornithine. interestingly, gastric tissue levels of ornithine decarboxylase increased in rats with stress-induced gastric ulcers, suggesting that tissue injury enhances polyamine production, which may contribute to the normal rapid rate of epithelial restitution. the mechanisms whereby polyamines stimulate epithelial restitution are not clear. mccormack, wang, viar, et al. hypothesized that polyamines increase transglutaminase activity, an enzyme that catalyzes the cross-linking of cytoskeletal and basement membrane proteins. further investigation of the role of polyamines in iec- cell migration showed that depletion of polyamines resulted in disruption of the cytoskeleton and reduced the physical extension of lamellipodia. more recent studies have clarified this pathway. in particular, polyamines have been shown to regulate cytoskeletal cellular migration via activation of the small guanosine triphosphatase-rho-a by elevating intracellular ca + levels. these elevations in ca + result from polyamine regulation of expression of voltagegated k + channels and altered membrane electric potential. polyamines also play a role in the normal physiologic regulation of crypt cell proliferation and differentiation. , polyamines are produced by fully differentiated enterocytes at the tip of the villus and may reach the crypt within sloughed luminal epithelium or via local villous circulation. following intestinal injury, polyamines appear to stimulate enhanced proliferation by increasing the expression of protooncogenes, which control the cell cycle. the mechanism whereby polyamines influence gene expression likely relates to the cationic nature of these compounds, which may influence the tertiary structure of negatively charged dna and rna. locally produced growth factors-including epidermal growth factor (egf), transforming growth factor α (tgf-α), tgf-β, and hepatocyte growth factor-have the ability to modulate mucosal recovery. the most important of these growth factors in early mucosal repair events is tgf-β, which is a potent stimulus of epithelial restitution and modulator of the extracellular matrix. neutralization of tgf-β retards epithelial migration in vitro, and tgf-β apparently may serve as a point of convergence for mediators of restitution, because neutralizing tgf-β also inhibits the effects of other peptides. however, tgf-β paradoxically inhibits epithelial proliferation, thereby reducing the supply of new enterocytes for mucosal repair. conversely, egf, produced by the salivary glands and duodenal brunner's glands, and the related tgf-α, produced by small intestinal enterocytes, are potent stimulants of enterocyte proliferation. these growth factors share approximately % of their amino acid structure, bind to the same receptor on the basolateral surface of enterocytes, and are not related to tgf-β. the physiologic role of egf is difficult to discern because it is present in the intestinal lumen, with no apparent access to its basally located receptor. however, egf has been proposed to act as a "surveillance agent" that gains access to its receptor during epithelial injury (when the egf receptor likely would be exposed) to stimulate proliferation. tgf-α presumably has a similar role, but it is present in greater concentrations in the small intestine because it is produced by differentiated villous enterocytes. the mature peptide is cleaved from the extracellular component of the transmembrane tgf-α precursor and released into the lumen. another group of proreparative peptides produced within the gastrointestinal tract are the trefoil peptides. under physiologic conditions, trefoil peptides are secreted by mucus-producing cells at distinct anatomic sites. for example, gastric epithelium produces the trefoil peptide ps , whereas the small and large intestine mucosa produce intestinal trefoil peptide. however, any of the trefoil peptides may be upregulated within repairing epithelium regardless of anatomic site. , in addition, trefoil peptides have the ability to induce their own expression, amplifying the level of these reparative factors at sites of mucosal repair. trefoil peptides are the most potent stimulants of epithelial migration in vitro, and their effects are independent of growth factors, including tgf-β. however, recent evidence suggests that egf receptor activation is required for induction of ps and another of the trefoil peptides, termed spasmolytic peptide, in gastric epithelium in vitro. the importance of trefoil peptides to the mucosal repair response in vivo is illustrated by gene knockout studies, in which mice deficient in intestinal trefoil factor have greatly reduced ability to repair intestinal injury. in fact, detergent-induced mucosal injury was lethal because of a lack of restitution compared with wild-type mice that fully recovered from similar mucosal injury. the fact that administration of intestinal trefoil factor restored restitution has important therapeutic implications. the mechanism whereby trefoil peptides stimulate epithelial migration is yet to be characterized fully but appears to involve translocation of the adherens junction protein e-cadherin, thereby allowing cells to become untethered from neighboring cells. the principal metabolic fuel of enterocytes is glutamine and of colonocytes, butyrate. however, recent studies suggest that glutamine and butyrate have more specific proliferative actions aside from their role as nutrients. for example, in the piglet ipec-j enterocyte cell line, glutamine enhanced gene transcription by increasing mitogen-activated protein kinase activity. , similarly, butyrate stimulated mucosal growth following colonic infusion in the rat. because of such growth-promoting actions, glutamine was shown to prevent intestinal mucosal atrophy and dysfunction that accompanies starvation , and long-term total parental nutrition. , additionally, glutamine improves function of transplanted small intestine , and protects intestinal mucosa from injury if administered before chemotherapy and radiation therapy. , intestinal nutrients also may synergize with other proliferative agents. for example, administration of glutamine and tgf-α to porcine ileum that had been subjected to hours of ischemia resulted in a synergistic increase in mitogen-activated protein kinase activity, enterocyte proliferation, and villous surface area. although concern has arisen that such early return to normal surface area may result in dysfunctional mucosal digestive and absorptive function because of resurfacing denuded mucosa with immature epithelium, nutrients and growth factors also appear to promote early differentiation. in the case of glutamine and tgf-α restoration of postischemic small intestine, rapid recovery of digestive enzymes also was documented. effective gastrointestinal motility involves a complex interaction between the enteric nervous system, muscular wall, and luminal contents. additional factors that influence the net transit of digesta include gravity, the volume and viscosity of the contents, and pressure gradients created by simultaneous contraction and relaxation of adjacent segments of bowel. casual use of the term intestinal motility in veterinary medicine often underestimates the complexity of the processes involved in the transit of intestinal contents. this is particularly true when the term is used to describe the frequency and or intensity of intestinal sounds, or borborygmi. the existence of borborygmi does not always equate with progressive movement of intestinal contents. disruption to normal motility occurs commonly in horses for a variety of reasons. examples of diseases in which altered motility may be present include gastroduodenal ulceration, intraluminal obstruction or impaction, excessive wall distention, strangulating obstructions, peritonitis, and inflammatory bowel diseases such as duodenitis proximal jejunits or colitis. ineffective intestinal motility is also a feature of several neonatal diseases, including prematurity, systemic sepsis, and perinatal asphyxia. certain parasitic infections, electrolyte derangements, and endotoxemia can modify digesta transit in horses of all ages. general anesthesia and specific sedatives, such as xylazine, romifidine, or detomidine, also disturb motility. the inhibition of propulsive bowel activity usually is referred to as ileus. ileus is ascribed most frequently to the condition that occurs after laparotomy and is termed simple or uncomplicated postoperative ileus (poi). the term complicated or paralytic ileus describes intestinal motility disturbed for longer periods after surgery. poi in horses is associated most commonly with surgery of the small intestine, particularly after resection and anastomosis, , is a common complication of small intestinal surgery, and can have a negative effect on short-term postoperative survival. , motility dysfunction likely is present in all horses after laparotomy, but many are affected subclinically and require minimal or no specific intervention. in symptomatic animals, clinical signs are apparent shortly after recovery and include colic, tachycardia, dehydration, decreased borborygmi and fecal output, and sequestration of fluid within the stomach. rectal examination and ultrasound reveal small intestinal distention with rare or absent wall movement. the severity and duration of intestinal stasis varies, lasting from minutes to days. a specific motility disorder involving the cecum or ileocecocolic region occurs sporadically in horses. [ ] [ ] [ ] the condition most commonly occurs after general anesthesia and extraabdominal surgery, particularly orthopedic and upper airway procedures, and therefore often is categorized as a form of poi. anecdotally, horses at greatest risk are young male performance animals. other cases occur spontaneously, often in animals with painful primary conditions such as uveitis or septic tenosynovitis. the syndrome is frustrating in that clinical signs are often subtle unless cecal perforation has occurred. in horses with a cecal emptying defect after anesthesia, overt signs are usually apparent to days after the procedure. the earliest detectable signs include depression and a reduction in feed intake and fecal output. ineffective emptying results in overfilling of the cecum with moist contents, which is manifest by signs of mild to moderate colic. if the condition is recognized late or untreated, the cecum may rupture and result in fatal peritonitis. the inherent rhythmicity of electric activity in the intestine is controlled by the interstitial cells of cajal, specialized cells that are electrically coupled to myocytes via gap junctions. these cells are responsible for generating and propagating slow-wave activity and may be critically involved in a range of motility disorders. the enteric nervous system primarily controls and coordinates intestinal contraction. a combination of central and autonomic innervation influences events, but contraction does not require external neural input. the parasympathetic supply to the gastrointestinal tract is via the vagus and pelvic nerves, and the sympathetic supply is through postganglionic fibers of the cranial and caudal mesenteric plexuses. a complex network of interneurons within each plexus integrates and amplifies neural input; the intensity and frequency of resultant smooth muscle contractions are proportional to the amount of sympathetic and parasympathetic input. additional binding sites for a number of other endogenous chemicals, including dopamine, motilin, and serotonin exist within the enteric nervous system and on smooth muscle cells. acetylcholine is the dominant excitatory neurotransmitter in the gastrointestinal tract and exerts its action through muscurinic type receptors on smooth muscle cells. sympathetic fibers innervating the gastrointestinal tract are adrenergic, postganglionic fibers with cell bodies located in the prevertebral ganglia. activation of α adrenergic receptors on cholinergic neurons within enteric ganglia inhibits the release of acetylcholine and therefore reduces intestinal contraction. β -, β -, and β-atypical receptors are directly inhibitory to the intestinal smooth muscle. inhibitory nonadrenergic, noncholinergic neurotransmitters include adenosine triphosphate, vasoactive intestinal peptide, and nitric oxide. , these neurotransmitters are critical for mediating descending inhibition during peristalsis and receptive relaxation. substance p is a nonadrenergic, noncholinergic neurotransmitter that may be involved in contraction of the large colon. , the rate and force of intestinal contractions along the small intestine and large colon of the horse are important determinants of intestinal motility; of even greater importance to the net propulsion of digesta are the cyclical patterns of contractile activity. these patterns are known as the small intestinal and colonic migrating motility (or myoelectric) complexes (mmcs). , the colonic complex usually originates in the right ventral colon and variably traverses the ascending and descending colons. many of these complexes are related temporally to a specialized motility event of the ileum, the migrating action potential complex. local inflammation within the intestinal muscularis and inhibitory neural events are important initiators of intestinal ileus. , intestinal inflammation not only is important in primary intestinal diseases in horses, such as duodenitis-proximal jejunitis and colitis but also is induced after simple intestinal handling during laparotomy. experimental data from other species suggests that handling of the small or large intestine at the time of surgery activates resident macrophages with resultant increased expression of p-selectin and intercellular adhesion molecule on endothelial cells within the vasculature of the muscularis. the upregulation of associated ligands on leukocytes leads to sequential "sticking and rolling," followed by neutrophil migration into the interstitium. the subsequent release of neutrophil products interferes with cell signaling and results in reduced intensity of smooth muscle contraction. furthermore, the inflamed intestine fails to contract normally in response to putative prokinetic agents. another key factor in the development of intestinal stasis after inflammation is the local overproduction of nitric oxide caused by the upregulation of inducible nitric oxide synthase (inos) by resident macrophages. nitric oxide is a key inhibitory neurotransmitter of the nonadrenergic, noncholinergic system. nitric oxide synthase inhibition has been a pharmacologic target in the treatment of experimental ileus. the inhibitory effects of α -agonists such as xylazine and detomidine on cecal and large colon motility are well described. [ ] [ ] [ ] [ ] [ ] [ ] intravenously administered xylazine inhibits cecal and large colon motility for to minutes without seriously disrupting small intestinal myoelectric activity, and detomidine can reduce large intestinal myoelectric activity for up to hours. the α -antagonist yohimbine has a weak but positive effect on cecal emptying in normal ponies, suggesting that normal motility is under constant α -adrenergic tone. atropine is a postganglionic blocking agent that binds to muscarinic receptors. when administered at . mg/kg, atropine inhibits individual small intestinal, cecal, and colonic contractions for about minutes but supresses small intestinal and colonic migrating complexes for up to hours. neural reflexes also may mediate inhibition of motility associated with peritoneal inflammation. , the afferent segment is composed partly of capsaicin-sensitive visceral afferent c fibers that terminate in the dorsal horn of the spinal cord, where they can activate inhibitory sympathetic fibers or synapse directly on the sympathetic ganglia. consequently, the efferent limb of the reflex expresses increased sympathetic outflow, primarily mediated through stimulation of α -adrenoreceptors, and inhibition of acetylcholine release, which provides the rationale for α -blockade in treating ileus. intraluminal infusion of capsaicin before abdominal surgery ameliorated the severity of poi in experimental rats. this finding highlights the importance of visceral afferent fibers in the development of poi. ileus also can occur in association with intestinal obstruction or displacement. mild to moderate distention of the bowel, such as that occurring in the early stages of an intraluminal obstruction, evokes an increase in local contractile activity. , excessive distention results in inhibition of motility within the distended segment of bowel. intestinal stasis is not always detrimental and under certain conditions may be protective. endotoxemia is a clinical feature of many diseases of the equine gastrointestinal tract, and endotoxins independently can exert a negative effect on intestinal motility and transit. a variety of mediators likely are involved, but activation of α -adrenoreceptors and production of prostanoids appear to be important, for pretreatment with yohimbine or nonsteroidal antiinflammatory drugs (nsaids; phenylbutazone or flunixin), respectively, ameliorates the inhibitory effects of experimental endotoxin infusion. , endotoxin infusion induced an inflammatory response in the intestine of rats that mimicked the response induced by handling during laparotomy. the similarity of the responses were highlighted in a recent study that demonstrated that prior exposure of the muscularis to endotoxin protected the intestine from the effects of manipulation. the pathophysiology of cecal emptying defect is not known. this syndrome may best mimic poi in human beings and generally is considered a large intestinal disorder. an important difference in horses is that laparotomy is a rare predisposing factor, and most cases occur in horses undergoing routine extraabdominal surgical procedures. general anesthesia itself is a potent inhibitor of gastrointestinal motility in horses, but these effects are short-lived and reversible within hours of anesthetic withdrawal. the return of normal motility in horses after experimental ileus was most delayed in the cecum, suggesting that this may be a common site of ileus in horses. a link between routine postoperative medications, such as phenylbutazone and aminoglycoside antibiotics, has been suspected but not established. an inhibitory effect of nsaids on large colon contractility has been demonstrated using in vitro techniques. primary sympathetic overstimulation could be involved, for many of the affected animals are young, male horses or animals with painful diseases. the duration of surgery influences the development of small intestinal poi, but not cecal emptying dysfunction. , technique may have a weak influence on small intestinal poi after jejunojejunostomy. the duration of intestinal ileus was shorter in animals that received a sideto-side stapled anastomosis than those that had a hand sewn end-to-end procedure. the duration of ileus after stapled end-to-end anastomosis was not different from that after either procedure. reported risk factors for the development of poi in horses include age (> years), small intestinal resection and anastomosis, breed (arabians had a greatest risk than other breeds), and duration of surgery. interestingly, performing a pelvic flexure enterotomy and emptying the colon had a protective effect against poi. the diagnosis of ileus is based on history and physical examination findings. important tests include determination of pulse rate and rhythm, auscultation and percussion of the abdomen, rectal palpation, and passage of a nasogastric tube. a complete blood count with fibrinogen estimation and cytologic analysis of peritoneal fluid may improve the accuracy of diagnosis. affected animals may be colicky because of accumulation of fluid in the upper gastrointestinal tract (classical poi) or cecal contents (cecal emptying defect). decompression of the stomach is important diagnostically and therapeutically in horses with poi after small intestinal surgery. failure to relieve pain with gastric decompression could point toward mechanical obstruction, severe inflammation of the intestine, or peritonitis. most animals with ileus are depressed and have reduced fecal output and intestinal borborygmi. one should interpret intestinal sounds with caution, however, because the presence of borborygmi does not always equate to progressive intestinal motility and merely may reflect local, nonpropagated contractions. rectal palpation findings in cases of persistent poi or duodenitis-proximal jejunitis are usually nonspecific but may reveal dilated, fluid-filled loops of small intestine. the clinician occasionally can palpate roughened peritoneal surfaces if peritonitis is present. one can palpate cecal distention with digesta in horses with advanced cecal dysfunction. distinguishing functional ileus from mechanical obstruction is important and can be difficult, but horses with mechanical obstruction typically have sustained high volumes of gastric reflux that vary little over time. the management of intestinal ileus depends on the segment of gastrointestinal tract involved. therapy for ileus of the proximal gastrointestinal tract involves a combination of gastric decompression, fluid and electrolyte therapy, and antiinflammatory drug therapy. electrolyte therapy is critical, particularly for maintaining adequate extracellular concentrations of potassium, calcium, and magnesium. calculation of the volume of fluid to be administered should include maintenance requirements ( to ml/kg/day) plus an estimate of losses, especially those lost through gastric decompression. one should consider parenteral provision of calories when feed has been withheld for more than hours, particularly after the horse has had surgery. a combination of amino acids and dextrose with or without lipids effectively provides these calories. hand walking also may provide some benefit to these animals but is not likely to have a direct effect on intestinal motility. one should avoid drugs that may impair normal intestinal motility, including the anticholinergics such as atropine and opiate receptor agonists such as morphine and meperidine. butorphanol appears to have little or no adverse effect on small or large intestinal motility. , one should use α -agonists sparingly because of their inhibitory effects on large intestinal motility. fluid therapy is the key component in managing cecal emptying defect, usually in combination with lubricants or laxatives, such as mineral oil or magnesium sulfate, and with careful use of antiinflammatory drugs. horses with primary cecal impaction or impaction caused by an emptying defect frequently require surgery to prevent fatal rupture. the surgical management of these cases is controversial and may include typhlotomy alone, typhlotomy with a bypass procedure such as ileocolic or jejunocolic anastomosis, or a bypass without typhlotomy. most horses that undergo simple typhlotomy have an uneventful recovery, although a small number experience impaction again and require a second laparotomy. experimental and anecdotal evidence provides a strong rationale for using antiinflammatory drugs to prevent and treat gastrointestinal ileus, particularly in animals that may have endotoxemia. flunixin meglumine is used widely in equine practice as an analgesic and antiinflammatory agent, and it ameliorates many of the adverse systemic effects of endotoxin, particularly those on the cardiovascular system. a potential negative effect of nsaids on large intestinal contractility has been suggested. broad-spectrum antimicrobials are indicated when one suspects sepsis or for the compromised immune system, as in cases of moderate to severe endotoxemia. theoretical concerns have been raised regarding the use of aminoglycoside antibiotics in animals with ileus. high concentrations of aminoglycoside antimicrobials inhibited intestinal contractions in exposed sections of intestine in vitro, but this inhibitory effect is unlikely to occur at clinically relevant doses. motility-enhancing drugs have been advocated to treat gastrointestinal ileus. unfortunately, information directly pertinent to horses is limited and must be extrapolated cautiously from that of other species because of the differences in intestinal anatomy and physiology. prokinetic drugs potentially can shorten the length of hospitalization, thereby reducing the cost of treatment and the number of potential complications such as weight loss, thrombophlebitis, and laminitis. experimental evidence indicates that prokinetic drugs can minimize the development of postoperative abdominal adhesions. most prokinetic drugs require a healthy gut wall to enhance intestinal contraction. therefore one should not assume that many of these drugs would be effective in the presence of an inflammatory injury such as that which can occur after intestinal manipulation at surgery or that associated with duodenitis-proximal jejunitis. bethanechol is a parasympathomimetic agent that acts at the level of the myenteric plexus and directly on intestinal smooth cells through muscarinic receptors. bethanechol is a synthetic ester of acetylcholine and is not degraded by anticholinesterase. bethanechol has cholinergic side effects, including abdominal discomfort, sweating, and salivation, although these are minimal when the drug is administered at . mg/kg body mass subcutaneously or orally. bethanechol has efficacy in diseases that involve abnormal gastric emptying and delayed small intestinal transit and has been shown to increase gastric contractility and hasten the emptying of liquid and solid phase markers from the stomach of normal horses. [ ] [ ] bethanechol also increases the strength and duration of wall contractions in the cecum and right ventral colon and consequently speeds up cecal emptying. neostigmine increases receptor levels of acetylcholine by inhibiting cholinesterase. the drug ( . to . mg/kg intravenously) promotes cecal and colonic contractile activity and hastens the emptying of radiolabeled markers from the cecum. neostigmine has been used to manage small intestinal ileus, but it significantly delayed the emptying of -mm beads from the stomach of normal adult horses. metoclopramide acts principally as a -hydroxytryptamine -receptor ( ht- ) agonist and ht- -receptor antagonist. in contrast to newer generation benzamides, metoclopramide is also an antagonist at dopamine (da ) and (da ) receptors. antagonism of prejunctional da receptors facilitates acetylcholine release and smooth muscle contraction. metoclopramide crosses the blood-brain barrier, where its antagonist properties on central da receptors can result in extrapyramidal signs, including seizure. these signs were responsible for poor acceptance of the drug in equine practice. most investigators have failed to demonstrate significant effects of metoclopramide in experimental animals, but constant intravenous infusion ( . mg/kg/hr) in a population of postoperative horses significantly decreased the volume and duration of gastric reflux over control and intermittent drug infusion groups. infusion was well tolerated and appeared to be superior to intermittent infusion or no treatment at all. cisapride is a second-generation benzamide that acts as a ht- agonist and ht- receptor antagonist but is without antidopaminergic action. stimulation of ht- receptors within the enteric nervous system enhances release of acetylcholine from the myenteric plexus. several reports suggest the efficacy of cisapride in managing intestinal disease in horses, including the resolution of persistent large colon impaction, treatment of equine grass sickness, and as a preventative for poi in horses after small intestinal surgery ( . mg/kg body mass intramuscularly during the postoperative period). [ ] [ ] [ ] [ ] the horse erratically absorbs tablets administered rectally, but a method for preparing a parenteral form of the drug from tablets has been described. cisapride has the potential to cause adverse cardiac side effects mediated through blockage of the rapid component of the delayed rectifier potassium current that include lengthening of the qt interval and development of torsades de pointes, a potentially fatal arrhythmia. these adverse effects have resulted in withdrawal of the drug in the united states. domperidone acts as a competitive antagonist at peripheral da receptors. the drug is a therapeutic agent ( . mg/kg/day) for mares grazing endophyte-infected tall fescue, principally because of drug-enhanced prolactin release. the potential prokinetic effects of domperidone have not been studied extensively in horses, but a modest efficacy of domperidone ( . mg/kg intravenously) has been demonstrated in experimental ileus in two ponies. erythromycin is a direct motilin receptor agonist on smooth muscle cells and also may act within the enteric nervous system to facilitate the release of acetylcholine and motilin. erythromycin enhances gastric emptying in normal horses but has a more pronounced effect on the hindgut. , erythromycin lactobionate ( . mg/kg intravenously) hastens cecal emptying in normal animals and induces colonic mmc-like activity across the colon. administration often is associated with defecation and abdominal discomfort. the drug may help prevent cecal impaction in horses after anesthesia, although its effectiveness on cecal motility in the immediate postoperative period may be reduced. high doses, constant infusion, or prolonged use of erythromycin induces receptor downregulation and inhibition of activity. erythromycin can induce diarrhea in adults, therefore one should avoid dosing over many days. naloxone ( . mg/kg intravenously) induces contractile activity in the cecum and left colon. defecation commonly follows administration of naloxone within to minutes. α -adrenoreceptor antagonists such as yohimbine or tolazoline counteract increased sympathetic outflow in response to nociceptive stimulation. yohimbine infusion ( µg/kg) also may attenuate the negative effects of endotoxin on motility. intravenous infusion of lidocaine may suppress primary afferent neurons, thereby limiting reflex efferent inhibition of motility. an infusion dose of to mg/min over to hours has been recommended for horses. lidocaine infusion is associated with reversible side effects that include muscle fasciculations, ataxia, and seizure. consequently, the rate of infusion requires close monitoring. katharina l. lohmann, michelle henry barton endotoxemia is defined as the presence of endotoxin in the bloodstream. most often, however, the term is used to refer to the associated clinical manifestations caused by an overshooting inflammatory reaction. in its pathophysiologic consequences the innate immune response to endotoxin (lipopolysaccharide) is similar to the response to other stimuli; for example, overwhelming bacterial infection, viral infection, or severe trauma. the term systemic inflammatory response syndrome therefore was introduced to describe a general systemic inflammatory process independent of cause. sepsis is defined as the "systemic inflammatory response to infection," and septic shock as "sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction." according to these definitions the diagnosis of sepsis requires documentation of infection by culture in addition to two or more of the following findings: hypo-or hyperthermia, tachycardia, tachypnea or hypocapnia, and leukocytosis, leukopenia, or an increased proportion of immature leukocyte forms. organ failure is a common sequela of endotoxic or septic shock, and the term multiple organ dysfunction syndrome describes insufficiency of two or more organ systems, as evident by clinical or clinicopathologic changes. in horses one should include the laminae of the feet in the list of organs susceptible to failure. german scientist richard pfeiffer ( - ), in working with vibrio cholerae, first described endotoxin as a toxin "closely attached to, and probably integral of, the bacterial body." he observed this toxin to be distinct from the actively secreted, heat-labile, and proteinaceous bacterial exotoxins. endotoxin later was found to be a heat-stable lipopolysaccharide structure, and the terms endotoxin and lipopolysaccharide now are used interchangeably. lipopolysaccharide is a major structural cell wall component of all gram-negative bacteria, including noninfectious species (figure . - ). with to × molecules per cell, lipopolysaccharide makes up about % of the outer layer of the outer cell membrane and is a key functional molecule for the bacterial outer membrane, serving as a permeability barrier against external noxious agents. the lipopolysaccharide molecule consists of four domains, which are essential for the virulence of gram-negative bacteria. three of the domains (inner core, outer core and o-specific chain) represent the hydrophilic polysaccharide portion of the molecule, whereas the lipid a portion represents the hydrophobic lipid portion ( figure . - ). combined, these domains confer the overall amphiphilic properties of the molecule that lead to the formation of micellar aggregates in aqueous solutions. o-specific chains (also called o-antigen polysaccharides or o-chains) are characteristic of any given type of lipopolysaccharide and show enormous structural variability between bacterial serotypes. o-chains are synthesized by addition of preformed oligosaccharide blocks to a growing polymer chain and therefore have a repetitive structure. o-specific chains determine part of the immunospecificity of bacterial cells and, on interaction with the host immune system, serve as antigens for the production of species-specific antibodies. o-specific chains are further responsible for the smooth appearance of gram-negative bacterial colonies on culture plates, and lipopolysaccharide molecules containing an o-chain are termed smooth lipopolysaccharide. the inner (lipid a-proximal) and outer (o-chainproximal) core oligosaccharide portion is more conserved between different strains of gram-negative bacteria than the o-specific chain. the core of all lipopolysaccharide molecules contains the unusual sugar kdo ( -deoxy-dmanno-oct- -ulopyranosonic acid), which links the core region to the lipid a molecule. synthesis of a minimal core is essential for the survival of bacteria, and the smallest naturally occurring lipopolysaccharide structure consists of lipid a and kdo. in contrast to the s-form colonies, colonies of gram-negative bacteria with lipopolysaccharide molecules that lack the o-specific chain but contain a core region show a rough appearance on culture plates. rough lipopolysaccharide molecules are denoted further as ra, rb, etc. to indicate the length of the core region. in re-lipopolysaccharide (also called deep rough lipopolysaccharide), the core region is reduced to a kdo residue. remutants often are used to raise antibodies against the core region in an attempt to provide cross-protection against a variety of bacterial species. the lipid a portion serves to anchor the lipopolysaccharide molecule in the bacterial outer membrane and has been identified as the toxic principle of lipopolysaccharide, and its structure is highly conserved among gramnegative bacteria. the common structure shared by lipid a molecules is a , '-bisphosphorylated β , -linked d-glucosamine disaccharide backbone (lipid a backbone), which is acylated by up to six fatty acids. figure . - shows the acylation pattern for escherichia coli lipopolysaccharide. variation in the lipid a structure between gramnegative bacteria affects the number, length, and position of fatty acids and the backbone structure and the substitution of phosphate by other polar groups. according to its nature as a structural cell wall component, the presence of endotoxin implies the presence of gram-negative bacteria as a source. depending on the nature of the underlying disease, these bacteria may circulate in the bloodstream in their intact form (i.e., bacteremia), may be confined to a localized infectious process, or may be part of the endogenous bacterial flora colonizing the gastrointestinal tract. in any of these scenarios, endotoxin molecules are released as a by-product of bacterial growth and in large numbers on bacterial cell death. common infectious conditions associated with endotoxemia in horses include neonatal gram-negative sepsis, bacterial pneumonia and pleuropneumonia, endometritis, peritonitis, and infectious colitis with bacteria such as salmonella spp., that are not part of the normal intestinal flora. in one study, for example, endotoxin was detectable in plasma of % of foals evaluated for presumed sepsis. the term translocation describes entry of endogenous bacteria and bacterial products from the gastrointestinal tract into tissues and the systemic circulation. the natural intestinal flora of horses consists mainly of gramnegative, anaerobic bacteria, and thus large amounts of endotoxin normally exist in the lumen of the equine intestinal tract. even in health, small amounts of endotoxin cross the intact mucosal barrier and reach the portal circulation and the liver. these molecules are cleared, however, by the mononuclear phagocytic system in the liver and only lead to a localized and restricted activation of the host immune system. for endotoxin translocation to become detrimental, excessive amounts have to cross the intestinal barrier and overwhelm the mononuclear phagocytic system or the capacity of the liver to detoxify lipopolysaccharide must be compromised. the latter may be a concern in conditions such as hepatitis, cholangiohepatitis, or portosystemic shunting of blood. permeability of the intestinal mucosal barrier frequently increases in cases of acute gastrointestinal disease. colic patients are prime candidates to development endotoxemia, and plasma endotoxin was detectable in % to % of colic patients on admission. , a higher percentage of horses tested positive for endotoxin when only patients presented for surgical intervention were evaluated. aside from gastrointestinal rupture, increased permeability to intact bacteria or free endotoxin molecules is thought to be associated most commonly with ischemic insults such as strangulating obstruction and bowel infarction, severe inflammation as in proximal enteritis and colitis, bacterial overgrowth, and intraluminal acidosis, which occurs with grain overload. , one study, however, found no difference in plasma endotoxin detection between disease groups, therefore emphasizing the fact that any disease of the abdominal cavity can induce endotoxemia in horses. in the same study, endotoxin was approximately times more likely to be detected in peritoneal fluid as opposed to plasma samples. similarly, higher cytokine concentrations have been measured in peritoneal fluid than in plasma. the likely explanation for these findings is a local inflammatory response in the peritoneal cavity elicited by translocated bacteria and/or lipopolysaccharide molecules before their absorption into the systemic circulation. although certainly the most important factor in horses, conditions other than gastrointestinal disease may result in translocation of endotoxin and bacteria. in experimental studies using laboratory animals, entry of gutassociated bacteria into the lymphatic system was demonstrated after hypovolemic shock, burn injuries, trauma, malnutrition, and starvation. [ ] [ ] [ ] furthermore, endotoxin itself caused bacterial translocation into mesenteric lymph nodes after intraperitoneal administration to mice. these findings have received much attention in the literature concerning human patients because they serve to explain cases of endotoxic shock in the absence of demonstrable bacterial infection. one should keep in mind the possibility of translocation when evaluating cases of presumed systemic inflammatory response syndrome in horses, in which one cannot demonstrate bacterial infection or gastrointestinal disease. endotoxin translocation also may be associated with strenuous exercise, which results in reduced splanchnic blood flow, hypoxemia, and a higher body temperature. in fit racehorses a significantly increased mean plasma lipopolysaccharide concentration was found after racing, whereas antilipopolysaccharide immunoglobulin g levels were decreased. fit horses showed significantly higher antilipopolysaccharide immunoglobulin g concentrations at rest than sedentary controls, suggesting leakage of small amounts of endotoxin from the intestinal lumen during training and racing. the clinical significance of these findings requires further investigation. the initiating event in the pathophysiology of endotoxemia is the activation of lipopolysaccharideresponsive cells by endotoxin, resulting in altered cellular functions and increased expression of inflammatory mediators. immune cells such as macrophages, which are the first to encounter endotoxin, respond to minute amounts of lipopolysaccharide, which usually allows them to eliminate gram-negative bacteria and free lipopolysaccharide molecules efficiently. an important factor in the exquisite sensitivity to lipopolysaccharide is the presence of lipopolysaccharide-binding protein (lbp). lbp is an approximately -kd plasma glycoprotein synthesized by hepatocytes and belongs to the group of acute phase proteins. under the control of inflammatory agents and cytokines, lbp concentration in plasma increases approximately -fold within hours of an inflammatory stimulus. the main function of lbp is to transfer lipopolysaccharide to endotoxin-responsive cells, which include mononuclear phagocytes, neutrophils, lymphocytes, and endothelial cells. the importance of a highly sensitive response to lipopolysaccharide for protection against gram-negative bacterial infection is demonstrated in experiments using lbp "knock-out" mice (mice that lack the lbp gene and are therefore unable to synthesize lbp). although these animals are resistant to the effects of isolated lipopolysaccharide, they are unable to control bacterial infection and rapidly succumb. despite its crucial importance for an effective host defense, lbp is not essential for lipopolysaccharide-receptor interaction per se, because high concentrations of lipopolysaccharide can activate cells in the absence of lbp. aside from its role as a catalyst of cellular activation by lipopolysaccharide, lbp has opsonizing activity and participates in the phagocytosis of lipopolysaccharide by macrophages and neutrophils. , although phagocytosis of lipopolysaccharide is receptor dependent, it appears to be uncoupled from intracellular signaling events and occurs in the absence of cell activation. lbp further catalyzes transfer of lipopolysaccharide to lipoproteins such as high-density lipoprotein, which neutralizes lipopolysaccharide activity. this detoxifying effect may become important when large amounts of lipopolysaccharide are present. a protective effect of lbp against lipopolysaccharide challenge and infection has been demonstrated in a murine model. the most important lipopolysaccharide receptors known to date are cluster differentiation antigen (cd ) and toll-like receptor (tlr ). both are classified as pattern recognition receptors, which means that they recognize lipopolysaccharide as a pattern common to all gram-negative bacteria. cd is a -kd protein that in its membrane-bound form (mcd ) is inserted into the cell membrane via a glycosylphosphatidyl-inositol anchor. cd is expressed primarily on monocytes and tissue macrophages and to a lesser extent on neutrophils. cd also is found in a free, or soluble, form (scd ) that can bind to cell types lacking cd , such as endothelial cells, and make them lipopolysaccharide-responsive. in addition to this proinflammatory effect, high concentrations of scd can sequester and neutralize lipopolysaccharide. the amount of circulating scd greatly increases during inflammation, which makes it a useful marker of acute and chronic inflammation. although cd is known to be crucial for cellular activation, it cannot transmit signals to the inside of the cell because it lacks a transmembrane domain. the missing link between cd and the cytosolic environment is a toll-like receptor in association with a molecule named md- . the name toll-like receptor stems from the homology of the mammalian receptor with a receptor type in drosophila (toll) that is important for dorsoventral orientation and immune responses in the fly. a number of toll-like receptors have been identified in mammalian species so far, but tlr appears to be the receptor subtype most important for lipopolysaccharide signaling. the importance of cd and tlr in the cellular response to lipopolysaccharide has been demonstrated in a number of experiments. mice deficient in cd are incapable of mounting a normal inflammatory response to lipopolysaccharide, whereas mutation or deletion of the gene encoding for tlr causes lipopolysaccharide hyporesponsiveness. [ ] [ ] [ ] after binding of lipopolysaccharide to cellular receptors, a multitude of signaling events takes place within the cell and results in the alterations of cellular metabolism known as cell activation. signaling pathways are characterized by sequential phosphorylation and thereby activation of enzymatic activities. a typical end result of intracellular signaling is the activation of transcription factors; for example, proteins that bind to dna and promote gene transcription. translational mechanisms are activated in a similar manner. among the bestcharacterized pathways in endotoxin-induced cell signaling are the mitogen-activated protein kinase (mapk) pathways and the activation of transcription factor nuclear factor κb (nfκb) (figure . - ) . , in the nfκb pathway the intracellular domain of tlr associates with the adapter protein myeloid differentiation factor and recruits interleukin- receptor-associated kinase to the complex. activation of interleukin- receptor-associated kinase, tumor necrosis factor receptor-associated factor, nfκb-inducing kinase, and iκb-kinase follow, and lastly, iκb is phosphorylated. iκb is an inhibitor protein complex that sequesters and inactivates nfκb in the cytoplasm. on phosphorylation, iκb is ubiquinated and degraded, and nfκb is translocated to the nucleus where it unfolds its activity. nfκb is a dimeric protein complex with several isoforms of which the p /p heterodimer is the most important inducible complex in mammals. proteins of importance for the pathogenesis of septic shock, the genes of which contain promoter elements for nfκb, include cytokines, inducible nitric oxide synthase, and cyclooxygenase (cox- ). three groups of mapks known to be crucially important for lipopolysaccharide-induced signal transduction are extracellular signal-regulated kinase, c-jun-terminal kinase, and p . final effects of signaling through mapk pathways include the activation of several transcription factors, translation initiation factors, and cytosolic enzymes such as phospholipase a , as well as an increase in the expression of adhesion molecules on the cell surface. despite the characterization of seemingly separate pathways, one should recognize that interaction and synergy between pathways is likely to occur. for example, simultaneous activation of p , c-jun-terminal kinase, and extracellular signal-regulated kinase results in much higher levels of tumor necrosis factor (tnf) reporter expression than activation of a single pathway. , aside from the mechanisms described here, pathways involving atypical protein kinase c , and receptor-independent integration of lipopolysaccharide into the cell membrane and ceramide-like second messenger activity of lipopolysaccharide have been proposed. additional pathways are likely to be uncovered in the ongoing investigation of intracellular signaling mechanisms and their in vivo significance. although endotoxin can exert some direct effects, cytokines are a primary mediator of lipopolysaccharide effects. cytokines are glycoprotein molecules that regulate inflammatory and immune responses by acting as a signal between cells. cytokines of major interest in the pathogenesis of endotoxemia include tnf, the interleukins, chemokines, and growth factors such as granulocyte-monocyte colony-stimulating factor. tnf is thought of as the most "proximal" cytokine released in response to lipopolysaccharide. studies corroborate this by showing that administration of recombinant tnf mimics the effects of lipopolysaccharide, and that antibodies directed against tnf protect against the lethal effects of endotoxin. increased plasma activity of tnf is associated with increased mortality in equine patients with acute gastrointestinal disease and in septic neonates. despite being a structurally diverse group of proteins, cytokines share several characteristics that allow them to execute their complex functions in the inflammatory response. any individual cytokine generally is produced by several different cell types, can act on different cell types, and has multiple effects on any given cell. furthermore, cytokine effects are redundant, meaning that different cytokines can share the same effect. in endotoxemia, this is particularly true for the effects of interleukin- (il- ) and tnf. many of the biologic activities of cytokines in vivo result from synergistic or antagonistic actions involving two or more cytokines. within itself the cytokine response is highly regulated: cytokines induce or suppress synthesis of other cytokines including their own (feedback regulation), regulate expression of cytokine receptors, and regulate cytokine activities. additional regulatory mechanisms include the release of specific cytokine inhibitors such as soluble il- and tnf-α receptors, cytokine receptor antagonists such as il- receptor antagonist, and antiinflammatory cytokines including il- , il- , il- , and transforming growth factor β. glucocorticoids also are produced increasingly in response to endotoxin and inhibit the production of cytokines. during a controlled inflammatory response, therefore, cytokine secretion is a selflimited event, whereas excessive stimulation of cytokine release can lead to the perpetuation of the inflammatory response even after the initial stimulus has been removed. conversely, the compensatory antiinflammatory reaction can become severe enough to cause anergy of the immune system and increased susceptibility to infection, which has been termed the compensatory antiinflammatory response syndrome. overall, excessive and unbalanced stimulation of the immune system therefore may result in predominantly proinflammatory (systemic inflammatory response syndrome), antiinflammatory (compensatory antiinflammatory response syndrome), or combined (mixed antagonist response syndrome) responses. interestingly, tolerance to endotoxin develops after repeated exposure to lipopolysaccharide. tolerance can be demonstrated in vitro and in vivo and encompasses decreased production of cytokines and a diminished clinical response. , mechanisms that likely are responsible for the development of endotoxin tolerance include receptor downregulation and inhibition of intracellular signaling pathways. , cytokines such as tnf are important mediators in the development of endotoxin tolerance. the development of endotoxin tolerance in horses has been reported. , aside from cytokines, a number of other molecules function as inflammatory mediators in the pathogenesis of endotoxemia, the synthesis and release of which are stimulated by endotoxin and by cytokines. these mediators include the arachidonic acid metabolites or prostanoids, platelet-activating factor (paf), oxygenderived free radicals, nitric oxide (no), histamine, kinins, and complement components. table . - summarizes the origins, targets, and effects of the most important inflammatory mediators involved in the pathogenesis of endotoxemia. figure . - shows the pathways of arachidonic acid metabolism by cox and lipoxygenase. cox products are the prostaglandins (pgs), prostacyclin (pgi ) and thromboxanes, and the lipoxygenase produces the leukotrienes. the innate immune response to lipopolysaccharide is an efficient defense mechanism that provides maintenance of homeostasis and therefore health in the face of an almost continuous exposure to microorganisms and their products. detrimental consequences of this immune response only occur if excessive and uncontrolled mediator output results in endothelial damage, neutrophil-mediated tissue damage, and uncontrolled activation of the coagulation and fibrinolytic cascades and the complement system. ultimately, the combination of these events culminates in cardiovascular instability, impaired hemostasis, organ failure, shock, and death. the following discussion addresses the various pathophysiologic events in the development of endotoxemia and shock and the role of inflammatory mediators. normal endothelium plays an important role in regulating blood pressure and regional tissue perfusion and provides an anticoagulant surface. endothelial dysfunction and damage result in a decreased responsiveness to vasoactive agents (vasoplegia), increased vascular permeability, and a tendency for clot formation in the microvasculature. if the basement membrane and underlying matrix are compromised, further microvascular hemorrhage can occur. endothelial cell damage is primarily neutrophilmediated. more specifically, damage is caused by oxygen-derived free radicals, which are produced within endothelial cells via reactions involving neutrophil-derived elastase and hydrogen peroxide molecules, endothelial cell enzymes such as xanthine oxidase, and endothelial cytosolic iron. the hypochloric anion radical (ho˙) is thought to be responsible most directly for endothelial cell cytotoxicity. no˙produced by constitutively expressed nitric oxide synthase in endothelial cells may afford protection from oxygen radical-induced endothelial cell damage. no is able to scavenge superoxide radicals and react with them to form peroxynitrite. variations in the ability to produce no may explain why vascular beds in different organs vary in their susceptibility to neutrophilmediated damage. excessive production of no by an inducible form of nitric oxide synthase (i nos), however, contributes to tissue damage, and increased peroxynitrite concentrations may be responsible in part for paf-induced increases in vascular permeability. in addition to oxygen-derived free radicals, activated neutrophils release matrix metalloproteinases, which contribute to tissue damage. vascular endothelial cells are further susceptible to direct effects of various cytokines, most prominently tnf-α and il- . these cytokines are thought to cause damage via the induction of cox activity and production of prostanoids and through generation of free radicals. neutrophil activation by lipopolysaccharide and cytokines results in stimulation of phagocytosis and the respiratory burst, release of lysosomal enzymes and inflammatory mediators, and expression of adhesion molecules. perhaps the single most specific clinicopathologic indicator of endotoxemia is pronounced neutropenia, which temporally correlates with peak plasma concentrations of tnf. neutropenia is caused primarily by margination of neutrophils in the vasculature, whereas significant loss through active migration into peripheral tissues likely is limited to the presence of a localized source of infection. margination is made possible by adhesion molecules on endothelial cells and leukocytes that interact and allow sticking of leukocytes to the endothelial lining of blood vessels. endotoxin or cytokines such as tnf and il- can initiate expression of adhesion molecules. subsequent transmigration of cells into tissue spaces is aided by the production of leukocyte collagenase, which allows enzymatic destruction of the vascular basement membrane. margination and transmigration of neutrophils occurs in three phases. , in the first phase of tethering and rolling, endothelial cells are stimulated to express p-selectin and e-selectin, which bind to p-selectin glycoprotein ligand- and sialylated lewis-x-like structures on leukocytes, respectively. whereas p-selectin is stored preformed in weibel-palade bodies of endothelial cells, e-selectin is expressed only following stimulation by cytokines. additionally, constitutively expressed l-selectin on neutrophils can bind to endothelial glycoproteins and glycolipids. during the second phase, firm adhesion is mediated by binding of neutrophil integrins (lfa- and mac- , also known as cd a/cd and cd b/cd ) to intercellular adhesion molecule (an immunoglobulin structure) on endothelial cells. although integrins are expressed constitutively on the leukocyte surface, activation signals are necessary to induce a high-affinity state and interaction with the endothelial surface. transmigration finally requires the expression of yet another adhesion molecule, namely platelet/endothelial cell adhesion molecule , which is located at the intercellular junction of endothelial cells. chemotactic factors including activated complement factor c a and the cxc chemokines control transmigration. the latter group includes il- , which is expressed by endothelial cells in response to activation. rebound neutrophilia, which is observed frequently following episodes of endotoxemia, is caused by neutrophil release from the bone marrow reserve pool and by stimulation of myeloid cell proliferation via granulocyte-macrophage colony-stimulating factor and is mediated primarily by tnf and il- . in health, coagulation and fibrinolysis underlie stringent control mechanisms that allow appropriate clot formation and their resolution. coagulopathies frequently are observed in horses with colic , , and foals with sepsis and are likely attributable to endotoxemia. disseminated intravascular coagulation (dic) results from a widespread activation of the coagulation and fibrinolytic systems and failure of their control mechanisms. ultimately, this leads to disseminated fibrin deposition in the microvasculature, consumption of platelets and clotting factors, and accumulation of fibrin degradation products (fdps). depending on the underlying disease process and the impairment of the systems, dic can manifest as a diffuse thrombotic syndrome leading to ischemic organ failure, a fibrinolytic syndrome with uncontrolled hemorrhage, or a combination of both. a procoagulant state in which one can detect clinicopathologic abnormalities precedes dic. activation of the coagulation cascade culminates in the cleavage of fibrinogen to fibrin by the serine protease thrombin. thrombin deposition on endothelial cell surfaces leads to platelet adherence and their activation by surface-bound paf. the intrinsic and extrinsic arms of the coagulation cascade are activated in endotoxemia. the intrinsic pathway is initiated by activation of coagulation factor xii (hageman factor), prekallikrein, and highmolecular-weight kininogen, which compose the contact system. although direct activation of coagulation factor xii by endotoxin has been demonstrated, the extrinsic pathway likely is more important for the development of coagulopathy in endotoxemia and sepsis. activation of the extrinsic pathway depends on the interaction of coagulation factor vii with tissue factor, which is the only coagulation factor not constitutively present in blood. tissue factor is present in subendothelial tissues and is exposed on vascular injury but also is expressed on endothelial cells and mononuclear phagocytes in response to lipopolysaccharide. , increased expression of monocyte tissue factor (also described as increased procoagulant activity) was found to be associated significantly with coagulopathy and poor prognosis in horses with colic. furthermore, lipopolysaccharide-induced tissue factor expression by equine peritoneal macrophages may be associated with the development of intraabdominal adhesions. regulatory mechanisms of the coagulation cascade include tissue factor pathway inhibitor, antithrombin iii (at iii), and the protein c system. protein c acts as an anticoagulant by inactivating clotting factors v and viii and promotes fibrinolysis by inactivating plasminogen activator inhibitor (pai). protein c activation by thrombin-thrombomodulin complexes is important for the anticoagulative properties of normal endothelium, and downregulation of endothelial thrombomodulin expression by tnf and il- and decreased expression of at iii and tissue factor pathway inhibitor by damaged endothelial cells contribute to the procoagulant state in endotoxemia and sepsis. [ ] [ ] [ ] in addition, activation of vascular endothelial cells leads to a loss of prostacyclin and no production and an increased release of thromboxane a (txa ). as a result, platelets are stimulated to aggregate and release txa and paf, thereby further promoting clot formation. the crucial step in the fibrinolytic cascade is the conversion of plasminogen to plasmin, a fibrin-degrading enzyme. tissue-type (tpa) and urokinase-type (upa) plasminogen activator are the major initiators of fibrinolysis, whereas pai and α -antiplasmin are the main regulatory components. , tnf and il- have been shown to induce the release of upa and tpa and the synthesis of pai. activation of fibrinolysis leads to consumption of α -antiplasmin and accumulation of fdps, which if present in high concentrations can interfere with platelet aggregation, fibrin polymerization, and thrombin formation and can promote bleeding. additionally, fdps mediate an increase in vascular permeability. lipopolysaccharide infusion in rabbits and human beings resulted in an early increase in plasma tpa activity, followed by a later profound rise in pai activity and fall in tpa activity. increased plasma pai concentrations also were found in horses with colic compared with controls. , thus although fibrinolysis may compensate initially for accelerated coagulation, its subsequent inhibition contributes to clot formation. activation of the complement system in endotoxemia occurs via the alternative pathway through interaction with lipopolysaccharide. increased concentrations of plasmin and kallikrein (caused by activation of the fibrinolytic and contact system) further promote this pathway by directly activating complement factors c a and c a. aside from being key molecules in the complement cascade, c a and c a are anaphylatoxins and cause an increase in vascular permeability via mast cell degranulation. c a further activates the lipoxygenase pathway in neutrophils and monocytes, acts as a chemotaxin for leukocytes and monocytes, and promotes neutrophil adhesion to endothelial cells. in response to acute inflammation, synthesis and secretion of a number of proteins called the acute phase proteins increases in hepatocytes, whereas synthesis of albumin decreases. the primary function of this acute phase response may be to suppress and contain inflammatory responses. il- and il- are the most important cytokines that induce the acute phase response, which typically begins within a few hours of the insult and subsides within to hours, unless the initiating cause persists. in horses, fibrinogen (the most commonly evaluated), haptoglobin, transferrin, ferritin, ceruloplasmin, coagulation factor viii:c, serum amyloid a protein, c-reactive protein, α -acid glycoprotein, and phospholipase a are considered part of the acute phase response. one must consider the effect of acute inflammation on the serum concentration of several coagulation factors when evaluating coagulation profiles. serum fibrinogen concentration is determined primarily by the acute phase response, although fibrinogen is consumed increasingly on activation of the clotting cascade. shock is characterized by a loss of homeostasis attributable to breakdown of hemodynamic control mechanisms, decreases in cardiac output and the effective circulating volume, and inadequate perfusion of vital organs. shock caused by endotoxemia is classified as distributive shock and is largely initiated by vascular dysfunction in the periphery. peripheral vascular beds are of major importance for the regulation of local tissue perfusion and affect systemic blood pressure by regulating total peripheral resistance. normally, vascular smooth muscle tone is regulated by endothelin- (vasoconstriction), no, and prostacyclin (vasodilation) released from vascular endothelial cells. as mentioned before, detrimental effects of no are attributable to induction of i nos in macrophages and other cell types, rather than endothelialderived no. peripheral vasomotor effects of endotoxin manifest as vasodilation and vasoplegia and are mediated by pgi , no, and mediators such as bradykinin. widespread vasodilation leads to vascular blood pooling, decentralization of blood flow, decreased venous return, and in effect decreased effective circulating volume and cardiac output. compensatory responses in the form of an initial hyperdynamic phase include tachycardia, increased cardiac output and central venous pressure, pulmonary hypertension, peripheral vasoconstriction, and increased peripheral vascular resistance. , , the early vasoconstrictive phase corresponds to an increased serum concentration of txa , but additional vasoconstrictors such as arginine vasopressin, angiotensin ii, serotonin, endothelin, and norepinephrine likely are implicated in the pathogenesis of shock and organ failure. with progression of disease, the animal enters a stage of decompensated shock and progressive systemic hypotension, which correspond to increased plasma concentrations of prostacyclin, pge and bradykinin. , inadequate blood flow and oxygen delivery to tissues caused by hypotension is confounded by direct myocardial suppression via no, increased vascular permeability, intravascular microthrombosis, and impaired tissue oxygen extraction and results in progressive metabolic acidosis and inhibition of normal cellular metabolism. quantification of endotoxin in plasma samples is possible. the limulus amebocyte lysate assay is an activity assay based on the endotoxin-sensitive hemolymph coagulation cascade in the horseshoe crab limulus polyphemus. in limulus this reaction is thought to be a defense mechanism against gram-negative infection. although frequently used as a research tool, the assay is not convenient enough to become a routine clinical test. the clinician therefore must appreciate the primary disease processes associated with a high risk of endotoxemia and rely on clinical signs and clinicopathologic data to achieve a diagnosis. in some cases, endotoxemia may be the first indication of disease or may be the most overt of otherwise subtle clinical manifestations. with colitis or proximal enteritis, for example, one may detect signs of endotoxemia before the development of colic, diarrhea, or gastric reflux, which more specifically indicate the nature of the primary illness. diseases such as peritonitis or pleuritis, however, may show nonspecific clinical findings including fever, anorexia, and depression. findings such as neutropenia, which indicate endotoxemia, should urge the clinician to search for a septic process. in vivo experiments in horses clearly show that many of the clinical signs associated with acute gastrointestinal disease and sepsis are attributable to the activities of lipopolysaccharide and cytokines such as tnf. on administration of sublethal doses of lipopolysaccharide the clinical response can be divided into the early hyperdynamic and the later hypodynamic or shock phases. clinical signs during the first phase, which begins within to minutes after lipopolysaccharide administration, include anorexia, yawning, sweating, depression, evidence of abdominal discomfort, muscle fasciculation, and recumbency. heart and respiratory rates increase, and decreased borborygmi suggest ileus. hyperemia of the mucous membranes and an accelerated capillary refill time indicate the hyperdynamic state. if one administers large amounts of lipopolysaccharide or if exposure is ongoing, depression worsens progressively, anorexia persists, and feces develop diarrheic character. signs of colic typically abate after the initial stage. fever develops as a result of direct action of tnf on the thermoregulatory center and il- -induced local production of pge in or near the hypothalamus. , because of compromised peripheral perfusion, mucous membrane color changes to brick red or purple, a dark "toxic" line appears, and capillary refill time is prolonged. inadequate peripheral perfusion and compromised organ function finally characterize the hypodynamic shock phase. body temperature may become subnormal and the skin, especially on extremities, is cool to the touch. the arterial pulse weakens and venous fill is decreased. vascular endothelial damage and increased capillary permeability result in a muddy mucous membrane color and diffuse scleral reddening. hemostatic abnormalities can manifest in the form of thrombosis, such as of the jugular vein, or increased bleeding tendency with mucosal petechiation or ecchymoses and prolonged bleeding from venipuncture sites. bleeding also may occur in spontaneous epistaxis or prolonged hemorrhage after nasogastric intubation. additional clinical signs typically reflect the development of organ failure. renal failure and laminitis appear to be common complications of endotoxemia in horses. renal failure results from ischemic cortical necrosis and acute tubular necrosis caused by coagulopathy-induced afferent arteriolar obstruction. clinical signs may include oliguria, anuria, or hematuria caused by renal infarction. laminitis may lead to lameness, increased digital arterial pulsation, increased warmth of the hoof wall, and sensitivity to hoof tester pressure. other signs of organ failure include icterus, anorexia and depression caused by liver failure, tachypnea and dyspnea caused by pulmonary failure, colic and bleeding caused by ischemia-induced gastrointestinal ulceration and abnormal motility patterns, and persistent tachycardia or cardiac arrhythmia in cases of cardiac failure. in pregnant mares, fetal death and abortion can occur because of increased production of pgf α and decreased serum progesterone concentrations. , alterations in the hemogram and serum biochemical profile are nonspecific and mainly reflect the underlying disease process and the occurrence of organ failure. leukopenia caused by neutropenia may be the most specific indicator of acute bacterial sepsis or endotoxemia. in prolonged cases, an increased proportion of immature neutrophil forms (bands) and toxic changes are observable. toxic changes resulting from neutrophil activation include vacuolation, cytoplasmic granulation, basophilic cytoplasm, and döhle's bodies. because neutropenia occurs early in the development of endotoxemia, it also may be a useful parameter for monitoring horses at risk. on recovery, neutropenia typically is followed by a pronounced rebound neutrophilia. an elevated hematocrit and total serum protein concentration are evidence of dehydration; however, splenic contraction caused by increased sympathetic stimulation and protein losses also influence these parameters. a normal or only slightly decreased serum protein and albumin concentration in the face of an elevated hematocrit and clinical signs of dehydration should alert the clinician to the possibility of protein loss. hypoproteinemia and hypoalbuminemia can occur because of loss via the gastrointestinal or urinary tract or with pleural or peritoneal cavity effusion. increased vascular permeability and edema formation contribute to hypoproteinemia. serum electrolyte abnormalities primarily depend on the nature and duration of underlying disease processes and need to be evaluated individually. common sources of electrolyte loss are gastrointestinal secretions, urine, and sweat; however, severe effusion into body cavities may contribute. in anorexic patients, lack of dietary intake is a confounding factor that warrants consideration. in human patients, gram-negative sepsis frequently is associated with hypocalcemia, more specifically a decrease in serum ionized calcium concentration. endotoxin is thought to be a causative factor, and proposed mechanisms include acquired parathyroid gland insufficiency, dietary vitamin d deficiency, impaired calcium mobilization, and renal α-hydroxylase insufficiency leading to decreased , -hydroxylation of vitamin d. hypocalcemia in septic human patients was found to be associated with hypotension and poor outcome. in horses with surgically managed gastrointestinal disease, decreased serum ionized calcium concentration was a common finding and was most severe in patients with strangulating or nonstrangulating infarctions. in some horses, ionized calcium concentration decreased further throughout surgery. treatment with calcium gluconate resulted in normalization of serum ionized calcium concentrations in all cases. septic neonatal patients are frequently hypoglycemic. aside from decreased oral intake and generally increased metabolism, glucose use by the infecting bacteria, inhibition of gluconeogenesis by endotoxin, and insulinlike activity produced by macrophages are responsible for hypoglycemia. interestingly, experimental endotoxin administration results in transient hyperglycemia in adult horses, whereas profound hypoglycemia occurs in foals. one should evaluate coagulation parameters if coagulopathy is suspected. the most significant changes can be expected with severe inflammatory disease such as colitis, , devitalized intestine as with strangulating obstruction, , and with increased duration of disease. in horses with acute gastrointestinal disease, coagulation profiles were considered normal in only horses. although coagulation times may be shortened during the procoagulant state, commonly observed abnormalities with developing dic include an increased concentration of fdps and soluble fibrin monomer, prolonged prothrombin time indicative of factor vii consumption, prolonged activated partial thromboplastin time indicative of factor viii:c and ix consumption, prolonged thrombin time, decreased at iii activity, thrombocytopenia, and decreased protein c and plasminogen activities. fibrinogen concentration frequently increases and reflects the acute phase response rather than coagulation abnormalities. some clinicians make a diagnosis of dic if three or more coagulation parameters (specifically at iii, fdps, platelet count, prothrombin time, and activated partial thromboplastin time) are abnormal, whereas others require overt clinical signs of hemorrhage and concomitant thrombosis in addition to classic laboratory findings. the prognostic value of coagulation parameters has been evaluated. , , overall, persistence or worsening of abnormalities in the face of treatment appears to be more indicative of poor outcome than alterations in any specific parameter. in one study, decreased serum at iii concentration was the parameter most commonly associated with fatal outcome in mature horses with colic. one should further evaluate the serum biochemical profile regarding compromise or failure of specific organ systems. increases in serum creatinine and urea nitrogen concentration can have prerenal, renal, or postrenal causes. in cases of endotoxemia and sepsis, prerenal azotemia caused by dehydration and decreased renal blood flow and renal azotemia caused by organ failure are most likely to occur. one can use urine specific gravity and the response to fluid therapy to differentiate renal from prerenal causes of azotemia. although ideally one should perform urinalysis before initiating fluid therapy, one should never delay treatment to obtain a sample and instead should use the first available urine sample. with prerenal azotemia, urine specific gravity is increased, urinalysis is normal in other respects, and azotemia resolves with adequate fluid therapy. azotemia in the face of normal or decreased urine specific gravity, however, indicates compromised renal function. depending on the extent of renal damage, proteinuria and hematuria also may be present. bacteriuria and an elevated urine leukocyte count may occur if urinary tract infection is the underlying cause for the development of endotoxemia. in these cases, urine culture and sensitivity testing are indicated to aid appropriate antimicrobial therapy. increased serum activity of liver enzymes (aspartate aminotransferase, γ-glutamyltransferase, sorbitol dehydrogenase, alkaline phosphatase) are common in endotoxemic patients; however, liver failure caused by endotoxemia is rare. sorbitol dehydrogenase is the most liver-specific of the enzymes and a sensitive indicator of acute hepatocellular necrosis; however, sorbitol is unstable and routine assays may not be available. one should evaluate liver enzymes and function tests (serum indirect and direct bilirubin concentration, serum bile acids and blood ammonia) in cases of prolonged and profound depression to rule out hepatoencephalopathy. one should evaluate arterial blood gases in patients with primary respiratory disease or with clinical evidence of respiratory failure and in profoundly depressed, recumbent patients, especially neonates. hypoxemia observed in response to endotoxin infusion is thought to be caused by an increase in ventilation-perfusion mismatch rather than pulmonary edema as occurs in human patients with acute respiratory distress syndrome. the lung is not a major shock organ in horses; however, pulmonary edema may occur in patients with associated sepsis or complications such as dic. the ideal treatment for endotoxemia is prevention. if one possibly can recognize and closely monitor patients at risk, one can provide treatment proactively and may reverse the effects of endotoxin before the inflammatory response has developed a dynamic of its own. unfortunately, endotoxemia can develop rapidly, and horses are exquisitely sensitive to the effects of endotoxin; therefore, many equine patients are not evaluated until reaching more severe stages of endotoxemia or shock. prognosis and patient outcome then frequently depend on the severity of complications associated with endotoxemia. treatment of endotoxemia involves multiple aspects, and the following strategies have been proposed : • inhibition of endotoxin release into the circulation • scavenging of lipopolysaccharide molecules to prevent direct effects and interaction with inflammatory cells • inhibition of cellular activation by lipopolysaccharide • inhibition of mediator synthesis • interference with the effects of inflammatory mediators • general supportive care in addition, complications such as renal failure, one also must address liver failure, cardiac failure, laminitis, and abortion in pregnant mares. when evaluating reports concerning the efficacy of any one treatment, one should keep in mind differences in underlying disease processes and the complexity of the inflammatory cascade. a "one for all" treatment most likely will not be found, and similarly, any one treatment can only address one or few pathophysiologic aspects of endotoxemia. understanding the rationale for different treatment strategies is important to be able to tailor treatment to the needs of the patient. inhibition of endotoxin release requires identification and removal of its source. therefore whenever endotoxemia is evident, the clinician should strive to reach a diagnosis of the underlying disease and ascertain whether ischemic or inflamed bowel or a gram-negative septic process is present. aside from history, physical examination, and routine laboratory tests, evaluation may include exploratory laparotomy in colic patients, roentgenologic and ultrasonographic evaluation of the pleural and peritoneal cavity and organs, ultrasonographic evaluation of umbilical remnants in neonatal foals, evaluation of passive transfer and calculation of a sepsis score in foals, and repeated culturing of blood or other specimens. if one suspects an infectious process, one should pursue identification of the responsible microorganisms and their antimicrobial sensitivity spectrum; however, one should not delay treatment to obtain culture results. specimen containers with removal devices for antimicrobials are available and are useful in cases for which one has initiated treatment before specimen collection. once one reaches a diagnosis, one must take appropriate measures to correct the primary disease process. examples are removal of devitalized sections of bowel or infected umbilical remnants, drainage of infected pleural or peritoneal fluid, and gastric lavage followed by administration of mineral oil and/or activated charcoal in cases of grain overload to prevent further absorption of endotoxin. one must address any septic process with appropriate antimicrobial therapy. initially, broad-spectrum coverage of the most likely organisms is recommended; one then should specify therapy according to results of culture and sensitivity testing. sepsis in foals is caused predominantly by gram-negative organisms, of which e. coli, actinobacillus spp., klebsiella spp., salmonella spp. and pasteurella spp. frequently are identified. the reader is referred to other texts for review of general principles of antimicrobial therapy. regarding endotoxemia specifically, antimicrobial therapy has been suggested to increase the amount of circulating endotoxin by inducing endotoxin release on cell death of gram-negative bacteria. a recent in vitro study compared endotoxin release and inflammatory mediator activity between antimicrobials commonly used to treat e. coli septicemia in foals and specifically evaluated amikacin, ampicillin, amikacin plus ampicillin, ceftiofur, and imipenem. although these antimicrobials showed no difference in the ability to kill bacteria, amikacin and the amikacin/ampicillin combination resulted in the lowest and ceftiofur in the greatest release of endotoxin. endotoxin release appeared to be dose-dependent in that lesser amounts were released at higher antimicrobial concentrations. based on these results and clinical experience, combining antimicrobial therapy with endotoxin-binding agents such as polymyxin b may be beneficial, especially when using β-lactam antimicrobials. antimicrobials frequently are given perioperatively to colic patients to lower the risk of peritonitis, incisional infection, and generalized sepsis and endotoxemia. because antimicrobial therapy has been implicated in the development of colitis, the duration of treatment should be minimal. unless evidence of sepsis, such as fever or changes in the leukogram, is present, perioperative administration of antimicrobials should not exceed a to hours. conversely, antimicrobial therapy frequently is used in cases of infectious colitis to treat the inciting cause and to prevent sepsis from translocation of bacteria. endotoxin typically has a short plasma half-life and is removed rapidly by mononuclear phagocytes or neutralized by binding to serum proteins and lipoproteins. many conditions responsible for the development of endotoxemia in horses, however, are associated with an ongoing release of endotoxin. examples include severe gastrointestinal inflammation as in proximal enteritis or colitis, grain overload, or uncontrolled sepsis. therapy directed against endotoxin itself may be able to interrupt the continuous activation of the inflammatory cascade in these cases. further benefits of antiendotoxin treatment may be derived if large amounts of endotoxin have been released before the inciting cause can be addressed. an important consideration regarding the efficacy of immunotherapy is the region of the lipopolysaccharide molecule against which antibodies are raised. the o-chain of lipopolysaccharide acts as a potent antigen on infection with gram-negative bacteria ; however, antibodies directed against the o-chain are serotype specific and cannot afford significant cross-protection against heterologous bacterial strains. the core and lipid a region, both of which show a much higher degree of homology between lipopolysaccharide derived from different bacterial strains, offer a more promising target for immunotherapy. active immunization against endotoxin has been reported for horses. vaccination with a bacterin/ toxoid vaccine prepared from rough mutants of salmonella typhimurium or s. enteritidis protected horses against homologous and heterologous endotoxin challenge , and carbohydrate overload. despite these encouraging results and the current availability of a vaccine for use in horses (endovac-equi, immvac inc., columbus, missouri), active immunization against endotoxin does not appear to be a common practice. in comparison, passive immunization with antilipopolysaccharide antibodies is used widely. rough bacterial mutants, most commonly j of e. coli o :b and s. minnesota re , are used to immunize donor horses and subsequently prepare serum or plasma products. proposed mechanisms of action after binding of the antibodies to lipopolysaccharide include steric blockade of lipid a interaction with cellular receptors and enhanced bacterial clearance by opsonization. [ ] [ ] [ ] studies concerning the efficacy of antibody administration in equine patients vary in their results. beneficial effects have been described in experimental models of endotoxemia, acute gastrointestinal disease, and neonates with sepsis, , [ ] [ ] [ ] whereas in other studies, antibodies failed to protect foals and horses against endotoxin effects. - furthermore, administration of a s. typhimurium antiserum to foals was associated with an increased respiratory rate and higher serum activities of il- and tnf. various equine serum and plasma products are currently commercially available. an antiserum raised against the lipopolysaccharide-core of s. typhimurium (endoserum) is available for administration to endotoxemic horses at a recommended dose of . ml/kg body mass. diluting the serum ten-to twentyfold in crystalloid intravenous solutions, administering it slowly over to hours, and monitoring the patient for adverse reactions is advisable. although the product is marketed for use in foals with failure of passive transfer, adverse effects have been reported, and one should use caution when administering it to neonates. plasma from donors inoculated with j (e. coli) and s. typhimurium (re mutant) is available under a restricted license (polymune-j, vet dynamics, inc., san louis obispo, california). the manufacturer recommends administration of at least to l in cases of endotoxemia. in addition, hyperimmune plasma, which has a guaranteed minimum immunoglobulin g content but does not contain specific antiendotoxin antibodies (hi-gamm equi, lake immunogenics, inc., ontario, new york; polymune and polymune-plus, vet dynamics, inc.), is marketed for treatment of failure of passive transfer, and many clinicians use it to treat endotoxemia and sepsis. in addition to antibodies and protein, plasma contains active constituents such as complement components, fibronectin, clotting factors, and at iii and therefore may be particularly useful in patients with endotoxemia-induced coagulopathy. volumes of to ml/kg body mass of hyperimmune plasma have been recommended for use in endotoxemic patients. , polymyxin b polymyxin b is a cationic polypeptide antibiotic that binds to the anionic lipid a portion of lipopolysaccharide and neutralizes its endotoxin capacity. at dosages required for antimicrobial activity, polymyxin b carries the risk of respiratory paralysis and ototoxic, nephrotoxic, and neurotoxic side effects; however, a much lower dose is required for endotoxin-binding activity. the effects of polymyxin b in horses have been evaluated in different experimental models. , , in an in vivo study in foals, treatment with polymyxin b at a dosage of u/kg body mass before infusion with s. typhimurium lipopolysaccharide resulted in significantly less severe elevations of body temperature, respiratory rate, and serum activities of tnf and il- compared with untreated controls. similarly, polymyxin b treatment of adult horses given endotoxin significantly ameliorated clinical signs and decreased plasma tnf activity. in the latter study, benefits of treatment were also evident at lower dosages of polymyxin b ( and u/kg body mass) and administration of polymyxin b hour after the start of endotoxin infusion. conversely, polymyxin b failed to ameliorate clinical signs of endotoxemia or prevent the development of coagulopathy, acidosis, lameness, and shock in experimental carbohydrate overload. side effects suggestive of neurotoxicity appeared after repeated administration of mg/kg body mass ( , u/kg) and in milder form, . mg/kg body mass ( , u/kg) polymyxin b. nephrotoxicity was not observed. currently, use of polymyxin b in equine patients is recommended at dosages of to u/kg body mass every to hours. one should initiate treatment as early in the disease process as possible, because the beneficial effects of lipopolysaccharide scavenging are limited to the first to hours after the onset of endotoxemia, before tolerance develops. side effects in the form of neuromuscular blockade and apnea, which necessitate slow infusion of the drug in human patients, have not been observed in horses. one therefore can administer the entire dose as a slow bolus. if one uses polymyxin b in horses with hypovolemia, dehydration, or azotemia, one should attempt to improve peripheral tissue perfusion, minimize the polymyxin b dose, and closely monitor patients for nephrotoxicity. close monitoring is also important if one administers medications such as aminoglycoside antibiotics, which share a similar spectrum of potential side effects, concurrently. azotemic neonates have been reported to be more susceptible to the nephrotoxic effects of polymyxin b than adult horses. in an attempt to decrease the risk for adverse effects while preserving lipopolysaccharide-neutralizing ability, a conjugate of polymyxin b with dextran has been developed. in conjugated form, polymyxin b is prevented from extravasation into tissues, where it exerts toxic effects by interaction with cell membranes. in addition, conjugation increases the residence time of polymyxin b in the circulation and therefore should prolong the antiendotoxic effect. the polymyxin b-dextran combination was evaluated at a total dose of mg/kg body mass of polymyxin b in . g/kg body mass dextran given minutes before administration of endotoxin in horses. treatment was found to block the development of tachycardia, tachypnea, fever, and neutropenia completely and to prevent increases in serum concentrations of tnf, il- , txb (a txa metabolite), and the prostacyclin metabolite -keto-pgf α . although mild adverse effects in the form of tachypnea, sweating, and increased systolic blood pressure were observed, these were transient and could be prevented by pretreatment with ketoprofen. the polymyxin b-dextran combination is not commercially available at this time. natural endotoxin-binding proteins such as lbp, lipoproteins, and scd have been evaluated experimentally. results of these studies are controversial, and detrimental effects occurred in some cases. a protein receiving much attention regarding potential therapeutic efficacy is the bactericidal permeability-increasing protein (bpi). this protein is structurally similar to lbp but is expressed exclusively in myeloid precursors of polymorphonuclear leukocytes. bpi is stored in primary granules of mature neutrophils and during inflammation is expressed on their cell membranes and secreted into the extracellular environment. bpi has an even higher affinity for lipopolysaccharide than lbp and shows antibacterial activity specific for gram-negative bacteria. binding of bpi to the gram-negative bacterial membrane results in growth arrest and is an important factor in the antibacterial activity of intact neutrophils. furthermore, bpi binding disrupts normal membrane organization and makes bacteria more susceptible to hydrophobic substances, including antimicrobials. experimentally, recombinant bpi has been shown to protect against the toxic and lethal effects of isolated lipopolysaccharide and intact gram-negative bacteria, and clinical trials in human patients show promising results concerning its therapeutic use. the biology and potential use of bpi in horses has not been evaluated. treatments aimed at inhibiting lipopolysaccharide interaction with cells or turning off intracellular signaling pathways are under investigation. nontoxic lipopolysaccharide or lipid a structures can act as endotoxin antagonists, if they competitively inhibit binding to lbp or cellular receptors or inhibit cellular activation by other mechanisms. of the potential antagonists that have been evaluated experimentally, lipopolysaccharide and lipid a from the phototrophic bacterium rhodobacter sphaeroides, and a synthetic compound (e ) the structure of which is based on r. sphaeroides lipopolysaccharide, have been most promising. [ ] [ ] [ ] [ ] [ ] unfortunately, species differences exist regarding cellular response to these structures, and r. sphaeroides lipopolysaccharide acts as a potent inducer of cytokine expression in equine cells. based on results of receptor transfection studies in other species, , tlr is likely responsible for this phenotypic variation. additional compounds including lipopolysaccharide derived from nitrogen-fixing plant bacteria of the species rhizobium are being evaluated to reveal further insight into structural requirements for endotoxin antagonists in horses. nonsteroidal antiinflammatory drugs (nsaids) are probably the most commonly used drugs to treat endotoxemia. the rationale for their use is inhibition of prostaglandin endoperoxide h synthase, that is, cox, and thereby inhibition of prostanoid production (see figure . - ). additional beneficial effects may include scavenging of oxygen-derived free radicals and iron chelation; however, side effects may occur at dosages required to achieve these effects. prostanoids have been identified as important mediators in the inflammatory response in a number of studies, and inhibition of their synthesis is associated with beneficial effects. two cox isoforms are recognized: constitutively expressed cox- and inducible cox- . upregulation of cox- expression results from various proinflammatory stimuli, including lipopolysaccharide, tnf-α, and il- . constitutively expressed cox products are likely important for maintenance of homeostasis, whereas increased production of prostanoids by cox- is thought to be responsible for detrimental effects during inflammation and shock. research has focused on the development of selective cox- inhibitors; however, none of these products are currently available for use in horses. in horses the most commonly used nsaid to treat endotoxemia is flunixin meglumine. beneficial effects of flunixin meglumine have been described in experimental models of endotoxemia [ ] [ ] [ ] and in clinical cases. in equine colic patients, treatment with flunixin meglumine before exploratory surgery resulted in reduced plasma concentrations of txb and pge and had a favorable effect on cardiovascular parameters. flunixin meglumine was shown further to maintain cardiac output and systemic arterial blood pressure, improve blood flow to vital organs, reduce pulmonary endothelial damage, and improve survival on endotoxin challenge. [ ] [ ] [ ] [ ] nsaid use in horses carries the risk of side effects, most importantly the development of gastrointestinal ulceration and renal papillary necrosis (renal crest necrosis). in a study comparing the side effects of flunixin meglumine ( . mg/kg body mass), phenylbutazone ( . mg/kg body mass), and ketoprofen ( . mg/kg body mass) given times daily for days, lesions of the gastric glandular mucosa occurred most commonly. phenylbutazone resulted in the most severe side effects, which included small intestinal edema, erosions, and ulcers in the large colon and decreased serum albumin concentration. renal crest necrosis occurred more frequently in horses treated with phenylbutazone but also occurred with flunixin meglumine treatment. despite the higher risk of side effects, use of phenylbutazone has been suggested for certain cases. in colic patients, phenylbutazone may provide analgesia and ameliorate endotoxin-induced ileus without masking cardiovascular effects of endotoxin, which are used to determine the necessity of surgical exploration. for similar reasons and to minimize side effects a reduced dose of flunixin meglumine ( . mg/kg body mass) has been suggested and is used widely in horses. at this dosage, flunixin meglumine was shown to inhibit eicosanoid synthesis efficiently in an in vivo model of endotoxemia. reduction of clinical signs, however, was dose dependent, and therefore one should choose the appropriate dose based on the circumstances of each case. ketoprofen has been suggested to have superior effects because of a proposed dual inhibitory effect on cox and lipoxygenase and may carry a decreased risk of side effects compared with flunixin meglumine and phenylbutazone. a comparison of cytokine and eicosanoid production by lipopolysaccharide-stimulated isolated monocytes in vitro, however, showed no significant difference between horses pretreated with flunixin meglumine ( . mg/kg body mass) or ketoprofen ( . mg/kg body mass), respectively. eltenac has been evaluated in an experimental endotoxemia model in horses. given minutes before lipopolysaccharide infusion, eltenac at a dose of . mg/kg protected against changes in clinical, hemodynamic, and hematologic parameters and blunted the lipopolysaccharide-induced rise in plasma cytokine concentrations in comparison with controls. some parameters, however, including heart rate, leukocyte count, lactate concentration, and plasma tnf activity, were not improved. ibuprofen may have beneficial effects superior to the other nsaids, because it may be possible to achieve tissue concentrations safely that allow iron chelation to occur. according to a study in healthy foals, dosages of ibuprofen up to mg/kg every hours can be given safely for up to days. the use of corticosteroids for antiinflammatory therapy in sepsis and endotoxemia has been controversial in human and equine patients, and beneficial effects superior to the ones achieved by nsaids have not been demonstrated consistently overall. corticosteroids inhibit the activity of phospholipase a and the release of arachidonic acid from cell membrane phospholipids, as well as the production of tnf, il- , and il- in response to a lipopolysaccharide stimulus. experimentally, beneficial effects of dexamethasone in equine endotoxemia have been demonstrated. , to inhibit tnf production by equine peritoneal macrophages, however, the required concentration of dexamethasone was high and corresponded to an in vivo dosage (approximately mg/kg body mass) greatly exceeding current recommendations. although single doses of corticosteroids are unlikely to carry a disproportionate risk of side effects, one should consider the suggested association of laminitis with corticosteroid use in horses. in cases of sepsis, further immunosuppressive effects could be detrimental. in human patients with certain types of septic shock, dysfunction of the hypothalamic-pituitary-adrenal axis has been recognized and successfully treated with hydrocortisone replacement therapy. use of corticosteroids for this indication has not been evaluated in horses. pentoxifylline, a methylxanthine derivative and phosphodiesterase inhibitor, has been suggested for use in endotoxemia because of its effects on neutrophil function and its ability to inhibit the production of various cytokines, interferons, and thromboplastin. decreased production of tnf, il- , txb , and thromboplastin in response to endotoxin was shown in an equine ex vivo model. in horses given endotoxin followed by treatment with pentoxifylline ( . mg/kg body mass followed by continuous infusion of mg/kg/hr for hours), however, only minimal beneficial effects were observed. treatment significantly improved body temperature, respiratory rate, and whole blood recalcification time, but no effect was observed regarding heart rate, blood pressure, leukocyte count, plasma fibrinogen concentration, and serum cytokine concentrations. the conclusion was that benefits of treatment with pentoxifylline might be restricted to administration of high bolus doses or continuous infusion early in the pathophysiologic process. in an in vivo endotoxemia model in horses, combination of pentoxifylline ( mg/kg body mass) and flunixin meglumine ( . mg/kg body mass) was found to have greater benefit than each treatment on its own. the currently recommended dosage for oral administration of pentoxifylline is mg/kg every hours. because of its rheologic properties, that is, the ability to increase erythrocyte deformability and microvascular blood flow, pentoxifylline may be particularly useful in endotoxemic patients showing evidence of laminitis. an intravenous preparation of pentoxifylline is not commercially available. dimethyl sulfoxide (dmso) is used frequently in an attempt to scavenge oxygen-derived radicals. the treatment may be most appropriate in cases of ischemia-induced intestinal damage and associated reperfusion injury. however, dmso failed to show beneficial effects in an experimental model of intestinal ischemia when administered on reperfusion of the ischemic intestine. dmso at the commonly used dose of g/kg body mass was shown to increase mucosal loss after ischemia and reperfusion of the large colon, and hence a reduced dose of . g/kg body mass has been proposed for cases of intestinal ischemia. for intravenous administration, dmso needs to be diluted in polyionic solutions to a concentration not exceeding %. oral administration of a % to % solution via nasogastric intubation is also possible. aside from dmso the xanthine oxidase inhibitor allopurinol has been suggested as a treatment to prevent oxygen radical-induced tissue damage. during periods of ischemia, tissue xanthine dehydrogenase is converted to xanthine oxidase, which on reperfusion catalyzes the generation of superoxide radicals. , evaluation in horses showed beneficial effects of mg allopurinol per kilogram body mass administered hours before endotoxin challenge. in another study, mucosal damage attributable to oxygen-derived free radicals was not attenuated by allopurinol in an experimental ischemia-reperfusion model. lidocaine given intravenously has been suggested as an antiinflammatory, analgesic, and prokinetic agent, and some clinicians use it to treat colic and laminitis in horses. in an experimental endotoxemia model in rabbits, lidocaine was found to inhibit hemodynamic and cytokine responses to endotoxin profoundly if given immediately following lipopolysaccharide infusion. use of lidocaine therefore may have additional merit in endotoxemic patients. a common regimen for lidocaine use in horses is administration of an initial bolus ( . mg/kg body mass) section . endotoxemia followed by continuous infusion at a rate of . mg/ kg/min. one should monitor patients for toxic neurologic effects associated with a lidocaine overdose. high concentrations of ω- fatty acids can alter the phospholipid composition of cellular membranes toward a decreased ratio of ω- to ω- and thereby can affect membrane functions such as phagocytosis, receptor binding, and activities of membrane-bound enzymes. most importantly for the treatment of endotoxemia, ω- fatty acid incorporation into cell membranes decreases the availability of arachidonic acid (an ω- fatty acid) for eicosanoid synthesis and provides alternative substrates. metabolism of ω- fatty acids via the cox and lipoxygenase pathway leads to the production of -series prostaglandins and -series leukotrienes, which have less biologic activity than their -series and -series counterparts derived from arachidonic acid. aside from these mechanisms, ω- fatty acids prevent lipopolysaccharideinduced upregulation of cd in monocytic cells and therefore may be able to block transmembrane signaling of lipopolysaccharide. cells from horses given linseed oil (high in ω- fatty acids) for weeks before blood collection showed significantly decreased expression of procoagulant activity, txb , and tnf in response to lipopolysaccharide stimulation. , in an in vivo experimental model of endotoxemia in horses, treatment resulted in prolonged activated partial thromboplastin time and whole blood recalcification time, suggesting an anticoagulant effect; however, a significant beneficial effect on clinical response and serum eicosanoid concentrations was not observed. because dietary addition of ω- fatty acids requires several weeks of treatment, intravenous infusion was evaluated and shown to alter the composition of cell membrane phospholipids rapidly. further evaluation of this treatment for use in horses is necessary before dosage recommendations can be made. monoclonal and polyclonal antibodies against equine tnf have been evaluated. [ ] [ ] [ ] administration of a monoclonal antibody preparation before lipopolysaccharide infusion resulted in significantly reduced plasma tnf-activity, improved clinical abnormality scores, lower heart rate, and higher leukocyte count compared with controls. furthermore, plasma concentrations of lactate and -keto-pgf α were reduced significantly, whereas txa production was not affected. in another study, administration of a rabbit polyclonal antibody against recombinant human tnf was unable to improve clinical and hematologic parameters when given shortly ( minutes) after lipopolysaccharide infusion, although inhibition of tnf activity was present in vitro. , findings in horses are in agreement with studies in other species and suggest that beneficial effects of tnf inhibition may be limited to administration before lipopolysaccharide exposure. widespread clinical use therefore is unlikely to become feasible. clinical trials in human patients have not shown significant benefits of tnf antibody treatment. , the effects of selective paf receptor antagonists have been evaluated. paf is implicated in the development of systemic hypotension, lipopolysaccharide-induced platelet aggregation, ileus, and increased vascular permeability and may mediate recruitment of leukocytes to inflamed tissues. , a study in horses using the paf receptor antagonist sri - before lipopolysaccharide infusion showed significant decreases in heart rate and shorter elevation of lactate concentrations in response to the treatment. although not statistically significant, additional beneficial effects included delayed onset of fever, a shortened period of neutropenia, and reduced maximal platelet aggregation. whenever possible, the clinician should correct volume and electrolyte deficits, or at least improve them, before anesthetizing a patient for a surgical procedure. for initial resuscitation, polyionic solutions such as lactated ringer's solution given at rates of to ml/kg/hr are appropriate. patients with severe hypovolemia and shock may require higher fluid volumes. a viable alternative to large-volume resuscitation with isotonic fluids is the use of small volumes of hypertonic solutions, which transiently raise plasma osmolality, thereby causing a fluid shift from the interstitial space into the vasculature and rapidly restoring circulating volume. hypertonic saline solution ( . % sodium chloride) is the most commonly used hypertonic solution and has been shown to have beneficial effects in endotoxemic horses. a dose of ml/kg is recommended, which one should give as a bolus infusion over to minutes, followed by administration of an isotonic solution to restore total body fluid volume. one should use hypertonic saline with caution in patients with sodium and/or chloride derangements and should monitor serum electrolyte concentrations in the case of repeated administration. improvement of the cardiovascular status in response to fluid therapy is indicated by normalization of heart rate, mucous membrane color, and capillary refill time. failure of urination to occur despite appropriate fluid resuscitation should result in critical evaluation of renal function. once one has stabilized the patient, one should choose a maintenance fluid rate to maintain adequate hydration and plasma volume. for adult horses, the maintenance fluid rate is approximately ml/kg/hr, whereas neonatal foals that are not nursing may require larger volumes ( ml/kg/hr). one should monitor fluid administration carefully in endotoxemic patients, because lowered plasma oncotic pressure caused by hypoproteinemia along with an increased vascular permeability increase the risk of tissue edema formation. furthermore, a rapid increase in total body fluid volume may be detrimental in patients with compromised cardiac and peripheral vasomotor function and may increase the severity of vascular pooling in peripheral organs. in these patients, hypertonic saline or colloids may be more appropriate means of stabilization than large volumes of crystalloid solutions. plasma is an ideal colloid and should be administered to maintain a serum total protein concentration above . g/dl. to raise plasma protein concentration and colloid osmotic pressure significantly, however, horses often require large volumes of plasma ( to l or more in a -kg horse), and one should consider alternative colloids. furthermore, high-molecular-weight polymers are thought to provide superior oncotic effects in cases of sepsis and endotoxemia, when vascular permeability is increased. hetastarch, or hydroxyethyl starch, (hespan) is commercially available as a % solution in . % sodium chloride. hetastarch molecules have very high molecular weight, and degradation must occur before renal excretion. these properties result in a longer plasma half-life and prolonged oncotic effects compared with other colloids; persistence of the oncotic effect for hours was found in hypoproteinemic horses. a dosage of to ml/kg given by slow intravenous infusion along with an equal or greater volume of crystalloid fluids is recommended. , in human patients, prolonged activated partial thromboplastin time, decreased factor viii activity, and decreased serum fibrinogen concentration have been described in association with hetastarch use. in the limited number of equine studies, bleeding times were not affected , ; however, one should monitor patients treated with hetastarch for coagulopathy. one should base correction of serum electrolyte concentrations on the results of laboratory evaluation. ideally, one should evaluate serum electrolyte concentrations of patients receiving fluid therapy daily. one should take ongoing losses and lack of dietary intake into account, especially when serum concentrations, as in the case of potassium, poorly reflect total body electrolyte stores. potassium supplementation is recommended in patients experiencing prolonged (greater than hours) periods of anorexia. one can add calcium in the form of calcium gluconate, which is available as a % solution. based on a study in healthy horses, rates of administration for calcium gluconate in the range of . to . mg/kg/min are recommended, and as a guideline, one should administer . to ml/kg body mass per day of a % solution. one can add potassium in the form of potassium chloride or potassium gluconate to intravenous solutions at a dose of to meq/l given at a maintenance rate. administration of potassium should not exceed a rate of . to meq/kg/hr. metabolic acidosis in endotoxic shock is attributable to lactic acidemia and inadequate tissue perfusion. acid-base balance often improves considerably after fluid resuscitation (preferably with alkalinizing solutions such as lactated ringer's solution) alone; however, additional sodium bicarbonate may be required in cases in which serum bicarbonate concentration remains below meq/l. for adult horses, the bicarbonate deficit (in meq hco ) is calculated as . × body mass (kg) × base deficit, whereas for foals one should use a factor of . . as a general rule, one should administer half the required amount as a bolus followed by the remaining half over to hours. because endotoxemia is a dynamic process and losses are ongoing, one should reevaluate acid-base status at least once daily. foals with sepsis are frequently hypoglycemic, and % dextrose solutions are useful as initial resuscitation fluids. one should reduce the glucose concentration of intravenous solutions according to the blood glucose concentration to avoid prolonged hyperglycemia. administration of hyperimmune plasma ( to ml/kg body mass) is highly recommended in foals with evidence of partial or complete failure of passive transfer. one should consider positive inotropic and vasomotor agents in patients with persistently inadequate tissue perfusion. lower dosages of dopamine ( . to µg/kg/min) result in vasodilation of the renal, mesenteric, coronary, and intracerebral vasculature via dopaminergic effects, whereas higher dosages (up to µg/kg/min) also exert stimulation of β -adrenergic receptors, resulting in increased myocardial contractility and heart rate. dobutamine is a direct β -adrenergic agonist and does not appear to have significant vasodilator properties. dosages for dobutamine of to µg/kg/min as continuous intravenous infusion have been recommended for use in horses. in addition, norepinephrine was evaluated in hypotensive critically ill foals that were refractory to the effects of dopamine and dobutamine. at dosages up to . µg/kg/min administered concurrently with dobutamine, six out of seven foals showed an increase in mean arterial pressure, and all foals had increased urine output. because of the risk of cardiac side effects, close monitoring of heart rate and rhythm should accompany infusion of inotropes. indirect blood pressure measurements using a tail cuff may be used to monitor the effects of treatment. more frequently than overt thrombosis or bleeding attributable to dic, hemostatic abnormalities occur in the form of alterations in the coagulation profile. a procoagulant state with shortened bleeding times or prolonged bleeding times caused by consumption of clotting factors may be evident. one should address abnormalities in the coagulation profile as early as possible but especially if they persist more than hours after initiation of therapy. because of the complex interactions of coagulation and fibrinolysis during endotoxemia, one should combine anticoagulant therapy with the administration of fresh frozen plasma to replace clotting and fibrinolytic factors. heparin acts as an anticoagulant by activation of at iii and subsequent inhibition of thrombin, release of tissue factor pathway inhibitor from endothelial cells, and inhibition of platelet aggregation. because endogenous at iii levels frequently are decreased in patients with coagulopathy, addition of heparin to fresh frozen plasma may be the most effective route of administration. an initial dose of iu/kg body mass followed by to iu/kg body mass times daily has been recommended. anemia caused by erythrocyte agglutination occurs in some patients during therapy with unfractionated heparin , but typically resolves within hours if therapy is discontinued. because of the risk of microthrombosis associated with erythrocyte agglutination, use of low-molecular-weight heparin ( iu/kg body mass subcutaneously every hours) has been recommended but may be cost-prohibitive. one may give aspirin orally ( to mg/kg body mass, every hours), which irreversibly inhibits platelet cox activity, to inhibit platelet aggregation and microthrombosis. platelet hyperaggregability has been implicated in the pathogenesis of carbohydrate-induced laminitis, and heparin and aspirin have been recommended to prevent development of laminitis. in an in vitro study, however, aspirin did not inhibit endotoxin-induced platelet aggregation. luteolysis caused by increased concentrations of pgf α leads to pregnancy loss in endotoxemic mares before day of pregnancy. daily administration of altrenogest (regu-mate, hoechst-roussel agri-vet, somerville, new jersey) at a dose of mg orally consistently prevented fetal loss in mares if administered until day of pregnancy. treatment with flunixin meglumine, by blockade of pgf α release, also may contribute to the maintenance of pregnancy in endotoxemic mares. the pathogenesis of fetal loss and abortion caused by endotoxemia, surgery, or systemic disease later in gestation is not understood completely. proposed mechanisms include direct effects on the fetus, placental function, or placental progesterone production. the pathophysiology of laminitis caused by endotoxemia is understood incompletely; however, decreased digital blood flow , and intravascular microthrombosis have been implicated. decreased no production by vascular endothelial cells in response to endotoxin has been suggested as a mechanism for vasoconstriction and decreased blood flow ; however, use of no donors remains controversial. maintenance of adequate peripheral perfusion and anticoagulant and antiinflammatory therapy may be helpful in preventing and treating laminitis caused by endotoxemia. although the innate immune response to endotoxin (lipopolysaccharide) is crucially important for the preservation of homeostasis and health, large amounts of endotoxin can evoke an excessive and uncontrolled inflammatory response and result in a dysfunction of hemostatic and circulatory control mechanisms, loss of vascular integrity, and finally tissue damage. conditions commonly associated with the development of endotoxemia in horses are acute gastrointestinal diseases, especially of ischemic and severe inflammatory nature, and localized or generalized infections. although measuring endotoxin concentrations in equine plasma is possible, this is not feasible in a clinical setting, and one typically reaches a diagnosis of endotoxemia based on clinical signs and clinicopathologic data. successful treatment of endotoxemia requires resolution of the primary disease process in addition to neutralization of circulating endotoxin, interference with the activities of inflammatory mediators, and general supportive care. newer treatments, such as blockade of endotoxin-interaction with cells or interruption of cell signaling pathways, are under investigation. possible sequelae of endotoxemia include dic, multiple organ failure, circulatory failure, and death. frequently, the outcome of conditions associated with endotoxemia in horses depends on the severity of associated complications; for example, renal compromise, laminitis, and abortion. resting state the lateral margins of the vesicle, that is, the buccal mucosa, are in close contact with the cheek teeth. caudally, the external space communicates with the pharynx through the aditus pharyngis. the mucous membrane of the mouth is continuous at the margin of the lips with the skin and during life is chiefly pink but can be more or less pigmented, depending on the skin color and the breed type. the lips are two muscular membranous folds that unite at angles close to the first cheek teeth. each lip presents an outer and an inner surface. the upper lip has a shallow median furrow (philtrum); the lower lip has a rounded prominence or chin (mentum). the internal surface is covered with a thick mucous membrane that contains small, pitted surfaces that are the openings of the ducts of the labial glands. small folds of the mucous membrane called the frenula labii pass from the lips to the gum. the free border of the lip is dense and bears short, stiff hairs. the arteries of the mouth are derived from the maxillary, mandibular, labial, and sphenopalatine arteries of the major palatine artery. the veins drain chiefly to the lingual facial vein. sensory nerves originate from the trigeminal nerve (cranial nerve v) and the motor nerves from the facial nerve (vii). the cheeks spread back from the lips and form both sides of the mouth and are attached to the alveolar borders of the bones of the jaws. the cheeks are composed of skin and muscular and glandular layers and then the internal mucous membrane. the skin is thin and pliable. in contrast, the oral mucous membrane is dense and in many areas of the oral cavity is attached firmly to the periosteum so that construction of oral mucosal flaps can be achieved only by horizontal division of the periosteal attachment. such a feature is important in reconstructive techniques applied to the oral cavity. the blood supply to the cheeks comes from the facial and buccal arteries and the sensory nerves from the trigeminal and motor nerves from the facial nerve. the hard palate (palatum durum) is bounded rostrally and laterally by the alveolar arches and is continuous with the soft palate caudally. the hard palate has a central raphe that divides the surface into two equal portions. the word mouth is used commonly to signify the first part of the alimentary canal or the entrance to it. the mouth is bounded laterally by the cheeks, dorsally by the palate, and ventrally by the body of the mandible and by the mylohyoideus muscles. the caudal margin is the soft palate. the mouth of the horse is long and cylindric, and when the lips are closed, the contained structures almost fill the cavity. a small space remains between the root of the tongue and the epiglottis and is termed the oropharynx. the cavity of the mouth is subdivided into sections by the teeth. the space external to the teeth and enclosed by the lips is termed the vesicle of the mouth, and in the enter the nose from the glands of the vomeronasal duct. to what extent these secretions aid in pheromone reception is not known. that portion of the palatine mucosa immediately behind the incisor teeth frequently is swollen (lampas) during eruption of the permanent teeth. this swelling is physiologic and not pathologic. the tongue is situated on the floor of the mouth between the bodies of the mandible and is supported by the sling formed by the mylohyoideus muscles. the root of the tongue is attached to the hyoid bone, soft palate, and pharynx. the upper surface and the rostral portion of the tongue are free; the body of the tongue has three surfaces. the apex of the tongue is spatulate and has a rounded border. the mucous membrane adheres intimately to the adjacent structure and on the dorsum is dense and thick. the lingual and sublingual arteries supply the tongue from the linguofacial trunk and matching veins. the linguofacial trunk drains into the linguofacial vein. the lingual muscles are innervated by the hypoglossal nerve (xii) and the sensory supply is from the lingual and glossopharyngeal (ix) nerves. the formula for the deciduous teeth of the horse is times i - c - p - for a total of . the permanent dental formula is times i - c - p - or p - m - for a total of or . in the mare the canine teeth are usually small or do not erupt, hence reducing the number to or . the first premolar tooth (wolf tooth) is often absent and has been reported as occurring in only % of the upper dentition of thoroughbred horses. the teeth of the horse are complex in shape and are compounded of different materials (dentin, cementum, and enamel). they function as grinding blades to masticate and macerate cellulose food in the important first stage of the digestive process. the cheek teeth in the horse are a well-documented feature of the evolution of equus caballus. the first incisor is present at birth or the first week of life. the second incisor erupts at to weeks of age; the third incisor, at to months of age; the first and second premolars, at birth to weeks of age; and the third premolar, months of age. the eruption times for the permanent teeth are as follows: first incisor, / years of age; second incisor, / years of age; third incisor, / years of age; the canine tooth, to years of age; the first premolar (wolf tooth), to months of age; the second premolar, / years of age; the third premolar, years of age; the fourth premolar, years of age; the first molar, to months of age; the second molar, years of age; and the third molar, / to years of age. this eruption sequence clearly indicates that the eruption of the second and third permanent premolar teeth give the potential for dental impaction. the modern horse has six incisor teeth in each jaw that are placed close together so that the labile edges form a semicircle. the occlusal surface has a deep enamel invagination (infundibulum) that is filled only partially with cementum. as the incisor teeth wear, a characteristic pattern forms in which the infundibulum is surrounded by rings of enamel, dentin, enamel, and crown cementum in a concentric pattern. each incisor tooth tapers from a broad crown to a narrow root so that as the midportion of the incisor is exposed to wear, the cross-sectional diameters are about equal; that is, at years of age, the central incisor tooth of the horse has an occlusal surface that is an equilateral triangle. observations on the state of eruption, the angles of incidence of the incisor teeth, and the pattern of the occlusal surfaces are used as guides for aging of horses. the canine teeth are simple teeth without complex crowns and are curved. the crown is compressed and is smooth on its labial aspect but carries two ridges on its lingual aspect. no occlusal contact occurs between the upper and lower canine teeth. when erupted, the six cheek teeth of the horse function as a single unit in the mastication of food. each arcade consists of three premolar and three molar teeth. the maxillary arcade is slightly curved, and the teeth have a square occlusal surface. the occlusal surfaces of the mandibular teeth are more oblong, and each arcade is straighter. the horse is anisognathic, that is, the distance between the mandibular teeth is narrower (one-third) than the distance between the upper cheek teeth. this anatomic arrangement affects the inclination of the dental arcade as the jaws slide across each other in the food preparation process. the unworn upper cheek tooth presents a surface with two undulating and narrow ridges, one of which is lateral and the other medial. on the rostral and lingual side of the medial style is an extra hillock. the central portion of these surfaces is indented by two depressions that are comparable with, but much deeper than, the infundibula of the incisor teeth. when the teeth have been subjected to wear, the enamel that closed the ridges is worn through and the underlying dentin appears on the surface. thus after a time the chewing surface displays a complicated pattern that may be likened to the outline of an ornate letter b, the upright stroke of the b being on the lingual aspect. dentin supports the enamel internally, cementum supports the enamel lakes, and the peripheral cementum fills in the spaces between the teeth so that all six teeth may function as a single unit, that is, the dental arcade. transverse ridges cross each tooth so that the whole maxillary arcade consists of a serrated edge. the serrations are formed so that a valley is present at the area of contact with adjacent teeth. these serrations match fitting serrations on the mandibular arcade. the true roots of the cheek teeth are short compared with the total length of the tooth. cheek teeth have three roots: two small lateral roots and one large medial root. by custom, that portion of the crown embedded within the dental alveolus is referred to as the reserve crown, and the term root is confined to that area of the tooth that is comparatively short and enamel free. wear on the tooth gradually exposes the reserve crown, and the roots lengthen. in an adult -lb horse the maxillary cheek teeth are between . and . cm in length. dental wear accounts for erosion and loss of tooth substance at a rate of mm/yr. the pulp chambers of the teeth are also complex. the incisors and canines have a single pulp chamber. the mandibular cheek teeth have two roots and two separate pulp chambers. the maxillary cheek teeth, although they have three roots, have in fact five pulp chambers. as occlusal wear proceeds, deposition of secondary dentin within the pulp chambers protects the chambers (e.g., the dental star, medial to the infundibulum on the incisor teeth). in the mandibular cheek teeth the transverse folding of the enamel anlage (during morphogenesis of the tooth) does not take place, and the occlusal surface is a simple surface of central dentin surrounded by enamel. each tooth then is conformed to a single arcade by the presence of peripheral crown cementum. the oral cavity and oropharynx are subject to a variety of diseases. however, many conditions affecting the first portion of the alimentary system produce the same clinical signs, regardless of their cause. the clinical signs may include inappetance or reluctance to eat, pain on eating or swallowing, oral swelling, oral discharge, and fetid breath. affected animals may show some interest in food but hesitate to eat it. salivation may be excessive and may be contaminated with purulent exudate or blood. the occurrence of bruxism (i.e., grinding of teeth) can indicate discomfort in other areas of the alimentary tract; for example, bruxism and frothing oral saliva are characteristic features of gastric ulceration in the horse. the clinician needs to be aware that considerable weight loss can occur rapidly with inability to feed and swallow. diseases that result in denervation of the pharynx and inappropriate swallowing can have the complication of inhalation pneumonia. after a complete physical examination and ascertaining the history, the clinician should approach examination of the mouth systematically in all cases. one can examine a considerable portion of the mouth and teeth from the outside by palpation of the structures through the folds of the cheek. most horses allow an oral examination without sedation or the use of an oral speculum. in many cases, however, one best achieves the detailed oral examination by sedation and the use of an oral speculum and a light source. one should irrigate the mouth to wash out retained food material so as to be able to inspect and palpate the lips, cheeks, teeth, and gums. the classic signs of dental disease in the horse include difficulty and slowness in feeding, together with a progressive unthriftiness and loss of body condition. in some instances, the horse may quid, that is, it may drop poorly masticated food boluses from the mouth, and halitosis may be obvious. additional problems reported by owners include bitting and riding problems and headshaking or head shyness. facial or mandibular swelling may occur. nasal discharge can result from dental disease associated with maxillary sinus empyema. mandibular fistulae frequently are caused by lower cheek tooth apical infections. some correlation exists between the age of the animal and clinical signs (table . - ). ancillary aids for a complete examination of the oral cavity of the horse may include radiology, endoscopic examination, fluoroscopy, biopsy, and culture. one should take care always during endoscopic evaluation of the oral cavity using a flexible endoscope. the author recommends sedation and the use of an oral speculum to prevent inadvertent mastication of the endoscope. if one uses general anesthesia as part of the diagnostic workup, then endoscopic evaluation of the oral cavity is much easier. in selected cases, advanced imaging technologies such as computed tomography, magnetic resonance imaging, or nuclear scintigraphy may be beneficial. the lips of the horse are mobile and prehensile. in many ways they function like the tip of the elephant's trunk in that they test, manipulate, and sample the environment for potential nutritive value. consequently, loss of motor function (e.g., facial palsy) affects the efficiency of the prehensile system. the lips grasp food in grazing or browsing, and the incisor teeth section the food. with mastication and lubrication with saliva, the bolus of food forms and is manipulated from side to side across the mouth, assisted by the tight cheeks of the horse and the palatine ridges. swallowing begins as the food bolus contacts the base of the tongue and the pharyngeal walls. during swallowing, the soft palate elevates to close the nasopharynx, the base of the tongue elevates, and the hyoid bone and the larynx move rostrally following contraction of the hyoid muscles. during this process, the rima glottidis closes and the epiglottis tilts dorsally and caudally to protect the airway so that food is swept through lateral food channels around the sides of the larynx into the laryngoesophagus. fluoroscopic studies in nursing foals in the dorsoventral view showed that contact occurs between the lateral food channels in the midline so that in outline the food bolus achieves a bow tie shape. dysphagia is defined as a difficulty or inability to swallow. anatomic classifications for dysphagia include prepharyngeal, pharyngeal, and esophageal (postpharyngeal) dysphagias. the site of the cause for dysphagia influences the clinical signs. prepharyngeal dysphagia is characterized by dropping food (quidding) or water from the mouth, reluctance to chew, hypersalivation, or abnormalities in prehension. pharyngeal and esophageal dysphagias are characterized by coughing; nasal discharge containing saliva, water, or food material; gagging; anxiousness; and neck extension during attempts to swallow. the following section describes esophageal dysphagia in more detail. causes of dysphagia can be divided into four types: painful, muscular, neurologic, or obstructive (table . - ). pain and obstruction cause dysphagia by interfering with the mechanics of prehension, bolus formation and transfer to the pharynx, and deglutition. muscular and neurologic causes of dysphagia impede prehension and swallowing by affecting the motor function of the lingual or buccal musculature, muscles of mastication (temporal and masseters), and pharyngeal and cranial esophageal muscles. sensory loss to the lips, buccal mucous membranes, pharynx, or tongue also may cause dysphagia. neurologic causes of dysphagia may affect the forebrain, brainstem, or peripheral nerves that control prehension (cranial nerves vm, vs, vii, and xii), transfer of the food bolus to the pharynx (cranial nerves vs and xii) and swallowing (cranial nerves ix and x). diagnosis of the cause of dysphagia is based on physical examination including a careful oral examination, neurologic examination, clinical signs, and endoscopy of the pharynx, esophagus, and guttural pouches. radiology may be useful to assess the bony structures of the head and throat. ultrasonography is valuable for examining the retropharyngeal space and esophagus to detect and evaluate masses. one may detect pharyngeal or esophageal causes of dysphagia with routine endoscopic examination or with contrast radiography. although one also can use endoscopy to assess deglutition, one must remember that sedation adversely affects the deglutition mechanism. one may assess deglutition using fluoroscopy or manometry, but these techniques require specialized equipment. specific diagnostic procedures for nonalimentary causes of dysphagia are covered elsewhere in this text (see chapter ). specific treatments aimed at resolving the underlying disorder causing dysphagia are discussed in detail elsewhere. one should avoid feeding roughage with long fiber length (hay or grass) to most horses with dysphagia. dietary modifications that promote swallowing such as feeding slurries made from complete pelleted feeds may be sufficient to manage some cases of partial dysphagia. one must take care to prevent or avoid aspiration pneumonia in horses with pharyngeal or esophageal dysphagia. one can manage foals by feeding mare's milk or a suitable substitute through a nasogastric tube. one also may administer pellet slurries or formulated liquid diets via nasogastric tubes to older horses. prolonged nutritional management of dysphagic horses may require extraoral feeding using a tube placed through an esophagostomy. formulated pelleted diets are often easy to administer through a tube as slurry and are balanced to meet the nutritional requirements for healthy horses. one must feed sufficient quantities to deliver adequate calories ( to mcal/day for a -kg horse). adjustments may be necessary for horses that are cachectic or have extra metabolic demand (such as pregnancy). adding corn oil to the ration ( cup every or hours) is a common method of increasing fed calories. liquid diets also have been used for enteral feeding but may not be tolerated as well as pelleted diets. regardless of the method of nutritional management, one must monitor and replace salivary losses of electrolytes. saliva contains high concentrations of na, k, and cl. a group of ponies with experimental esophagostomies and a horse with esophageal squamous cell carcinoma were fed a complete pelleted diet through esophagostomy tubes but developed metabolic acidosis, hyponatremia, and hypochloremia apparently because of salivary losses. surprisingly, salivary losses of potassium did not result in hypokalemia in these cases, presumably because of replacement in the diet. however, if the diet is deficient in potassium, hypokalemia may result. one often can accomplish electrolyte replacement by adding nacl and kcl to the diet. one can maintain horses for months with frequent feedings through an esophagostomy tube. parenteral nutrition (total or partial) may be useful in the short term but is not often feasible for long-term management. tooth eruption is a complex phenomenon involving the interplay of dental morphogenesis and those vascular forces responsible for creating the eruption pathway. these changes are responsible for osteitis and bone remodeling within the maxilla and mandible. young horses frequently show symmetric bony swelling resulting from these eruption cysts. in some cases, additional clinical signs of nasal obstruction with respiratory stridor or nasal discharges may be apparent. pathologic problems associated with maleruption include a variety of dental diseases. oral trauma can displace or damage erupting teeth or the permanent tooth buds. as a result, teeth may be displaced and erupt in abnormal positions or may have abnormal shapes. supernumerary teeth, incisors and molars, can develop, as well as palatal displacement of impacted teeth (maxillary p - , or third cheek tooth). in almost all of these conditions some form of surgical treatment is necessary. significant evidence from the location of apical osteitis in diseased teeth (table . - ) confirms that dental impaction is a major cause of dental disease in the horse. in a series of extracted teeth, were p - or p - (cheek tooth or , respectively). early observations had indicated that the first molar (m , or cheek tooth ) was the most commonly diseased tooth, and an "open infundibulum" in this tooth has been suggested as the cause. studies on cementogenesis of the maxillary cheek teeth have shown, however, that in fact most maxillary cheek teeth have a greater or lesser degree of hypoplasia of cementum within the enamel lakes and that this "lesion" rarely expands into the pulp. the central infundibular hole is the site of its vascular supply to the unerupted cement lake. on those occasions in which cases one can use apicoectomy and retrograde endodontic techniques to save the diseased tooth. one must take care, however, in selection of patients. in most cases of apical osteitis in the horse that result from dental impaction, immature root structures make achieving an apical seal of the exposed pulp difficult. gingival hyperemia and inflammation occur during the eruption of the permanent teeth and are common causes of a sore mouth in young horses (particularly -year-olds as the first dental caps loosen). such periodontal changes usually resolve as the permanent dental arcade is established. during normal mastication, the shearing forces generated by the occlusal contact of the cheek teeth essentially clean the teeth of plaque and effectively inhibit deposition of dental calculus. wherever occlusal contact is ineffective, periodontal changes and calculus buildup occur; for example, the deposition of calculus on the canine teeth of mature geldings and stallions is common. routine dental prophylaxis forms an important component of maintaining normal occlusal contact, and for this reason one should remove arcade irregularities that result in enamel point formation on the buccal edges of the maxillary cheek teeth and the lingual edges of the mandibular cheek teeth. one should remove these edges annually in horses that are at grass and twice yearly in young horses, aged horses, and stabled horses. horses at grass have been shown to have a greater range of occlusal contact and therefore better periodontal hygiene than stabled horses. in stabled horses the range of occlusal contact is narrower and the formation of enamel points occurs more frequently with subsequent buccal ulceration and the initiation of a cycle of altered occlusal contact and hence irregular arcade formation. this process leads to severe forms of periodontal disease and wave mouth formation. periodontal disease occurs with abnormal occlusal contact and initiation of the cycle of irregular wear and abnormal contact. such changes progress to loss of alveolar bone, gross periodontal sepsis, and loss of tooth support. in this sense periodontal disease truly is the scourge of the equine mouth and results in tooth loss. palatine clefts may result from an inherited defect and are caused by failure of the transverse palatal folds to fuse in the oral cavity. harelip accompanies few palatine clefts in the horse. the degree of palatine clefting depends on the stage at which interruption in the fusion of the part ii disorders of specific body systems caries of cementum occurs, that is, secondary inflammatory disease and acid necrosis of the cementum, apical osteitis may develop. pulpitis is key to the pathogenesis of dental decay in the horse. the initiation of inflammatory pulp changes may be a sequela to dental impaction or dental caries or may result from fracture of a tooth. if the onset of the inflammatory process is slow, then formation of secondary dentin within the pulp chambers may protect the pulp and the tooth. secondary dentin formation occurs from stimulation of odontoblasts within the pulp chamber. such changes are the normal process of protection during dental wear and attrition as crown substances wear away and the reserve crown comes into wear. in acute disease, however, this defense mechanism is ineffective, and the changes that occur and that are sequelae to pulpitis reflect the location of each affected tooth. for example, pulpitis and apical osteitis of the third mandibular cheek tooth most commonly results in the development of a mandibular dental fistula. pulpitis of the third maxillary cheek tooth, however, results in an inflammatory disease within the rostral maxillary sinus and in development of chronic maxillary sinus empyema (figure . - ) . oblique radiographs greatly assist the diagnosis of dental decay by demonstrating sinus tract formation, sequestration of bone, mandibular osteitis, hyperplasia of cementum, and new bone formation (so-called alveolar periosteitis). the management of dental decay in the horse usually involves surgical extraction of the diseased tooth. in some palatopalatal folds occurs. toxic or teratogenic effects are documented in other species, but little data are available in the horse. in recent years, treatment for repair of uncomplicated palatine defects has been recommended but prognosis is generally poor because of the considerable nursing care required and the high incidence of surgical failures. one should emphasize early surgery and the use of mandibular symphysiotomy in affording surgical exposure. the combination of mandibular symphysiotomy and transhyoid pharyngotomy to approach the caudal margins of the soft palate affords surgical access, and one can construct mucosal flaps to repair the defects. however, the incidence of surgical breakdown is high, and healing by first intention is the exception rather than the rule. a recent surgical report documented the successful closure of a median cleft of the lower lip and mandible in a donkey. foals born with a severely deviated premaxilla and palate have a wry nose. one can achieve surgical correction of the deviated premaxilla by submucosal division of the premaxilla across the nose at the line of the first cheek tooth. circumstantial evidence indicates that such a defect has a genetic cause, and the defect occurs most frequently in the arabian breed. other developmental abnormalities are subepiglottic cysts resulting from cystic distortion of remnants of the thyroglossal duct, which may cause dyspnea and choking in foals. surgical removal of these cysts results in normal function. the most significant developmental defect of dental origin is a maxilla that is longer than the mandible, that is, the horse is parrot-mouthed. an overbite of cm in the incisor arcade may be present in a horse with a mismatch of less than cm between the first upper and lower cheek teeth. parrot mouth and monkey or sow mouth are thought to be inherited conditions. some correction of minor incisor malocclusion occurs up to years of age. recognition and detection of parrot mouth are important in the examination of potential breeding stock. surgical attempts to inhibit overgrowth of the premaxilla by wiring or by the application of dental bite plate procedures have been documented in recent years. as has been indicated, the horse is by nature a curious animal and uses its lips as a means of exploring a variety of objects. wounds of the lips, incisive bone, and the mandibular incisor area occur commonly in the horse and usually result from the horse getting the lips, jaw, or teeth caught in feeding buckets, in fence posts, or in halters or having a segment of tongue encircled with hair in tail chewing. as the horse panics and pulls away from its oral entrapment, considerable trauma can occur to the lips, teeth, and gums. most wounds repair satisfactorily, provided one finds them early and observes the basic principles of wound hygiene, excision of necrotic tissue, and wound closure. one must ensure that oral mucosal defects are closed and that effective oral seals are made before external wounds are closed. in some cases, offering specially constructed diets or even feeding the horse by nasogastric tube or esophagostomy during the healing processes may be necessary. foreign body penetration of the tongue, cheek, or palate has been reported in grazing and browsing horses and in particular in horses that have certain hay sources that contain desiccated barley awns or yellow bristle grass. other plant material and grass awns also occasionally may penetrate the tongue, gingiva, or cheek, causing inflammation or abscesses. ulcerative stomatitis also results from the toxicity of phenylbutazone therapy. vesicular stomatitis is a highly contagious viral blistering disease described in more detail elsewhere. treatment of glossitis and stomatitis primarily aims at removing the inciting cause. actinobacillus lignieresii, the causative agent of actinobacillosis, has been isolated and identified from ulcers on the free border of the soft palate and oral and laryngeal granulomata. the bacterium also was reported in a sublingual caruncle in a horse with a greatly swollen tongue. therapy with ml of % sodium iodide and g of ampicillin every to hours effected a clinical cure. saliva is important for lubricating and softening food material. the horse has paired parotid, mandibular, and polystomatic sublingual salivary glands. the parotid gland is the largest of the salivary glands in the horse and is situated in the space between the ramus of the mandible and the wing of the atlas. the parotid duct is formed at the ventral part of the gland near the facial crest by the union of three or four smaller ducts. the duct leaves the gland above the linguofacial vein, crosses the tendon of the sternocephalicus muscle, and enters the mouth obliquely in the cheek opposite the third upper cheek tooth. the parotic duct orifice is small, but some dilation of the duct and a circular mucous fold (the parotid papillae) exist at this point. the mandibular gland is smaller than the parotid gland and extends from the atlantal fossa to the basihyoid bone. for the most part, the mandibular gland is covered by the parotid gland and by the lower jaw. the mandibular duct is formed by union of a number of small duct radicles that emerge along the concave edge of the gland and run rostral to the border of the mouth opposite the canine tooth. the orifice is at the end of a sublingual caruncle. the mandibular gland possesses serous, mucous, and mixed alveolar glandular components. the parotid gland is a compound alveolar serous gland. the parotid salivary gland can secrete saliva to yield rates of ml/min, and a total daily parotid secretion can be as much as l in a -kg horse. parotid secretion only occurs during mastication, and administration of atropine or anesthesia of the oral mucosa can block secretion. parotid saliva is hypotonic compared with plasma, but at high rates of flow, concentrations of sodium, chloride, and bicarbonate ions increase. parotid saliva of the horse has a high concentration of calcium, and occasionally calculi (sialoliths) form within the duct radicles of the parotid salivary gland. congenital parotid duct atresia, acquired stricture from trauma to the duct, or obstruction by plant material (sticks or foxtails and other seeds) also may occur. the clinical signs of sialolithiasis or other forms of ductule obstruction include a fluid swelling in the form of a mucocele proximal to the stone and occasionally inflammation of the parotid gland. ultrasonography is useful to diagnose salivary mucoceles and to detect foreign bodies or sialoliths. measurement of electrolyte concentrations in aspirates from suspected mucoceles might be helpful to distinguish them from hematomas. salivary potassium and calcium concentrations are higher than plasma. treatment may require surgical removal of the stone or plant material in the case of sialolithiasis or foreign body obstructions. other causes of obstruction may require resection of the affected portion of the duct or chemical ablation of the gland. primary sialoadenitis is unusual but can occur in one or both glands. the condition is painful and may be associated with a fever and anorexia. secondary sialoadenitis is more common and usually is associated with trauma. infectious sialoadenitis from corynebacterium pseudotuberculosis or other bacterial pathogens also may occur. diagnosis is by physical examination and by finding an enlarged edematous parotid gland tissue on ultrasonographic examination. culture and cytologic examination of aspirates may be useful for diagnostic purposes. treatment in usually palliative, consisting of nonsteroidal antiinflammatory drugs. appropriate antibiotic therapy is indicated as directed by culture and sensitivity results. chemical irritation, glossitis, stomatitis, or other causes of prepharyngeal dysphagia cause ptyalism or excessive salivation in horses. specific therapy for the ptyalism usually is not required as long as salivary losses are not excessive, resulting in dehydration and electrolyte imbalances. ingestion of the fungal toxin slaframine also causes hypersalivation in horses. the fungus rhizoctonia leguminicola, which produces slaframine, causes black patch disease in red clover. slaframine is a parasympathomimetic compound that stimulates exocrine secretion in the parotid gland. slaframine toxicosis most commonly occurs in the spring or early summer and rarely requires treatment other than removal from the pasture. mowing removes the source in most cases because regrowth in pastures often has less fungal contamination. . the esophagus has no digestive or absorptive functions and serves as a conduit to the stomach for food, water, and salivary secretions. the esophageal mucosa is a keratinized stratified squamous epithelium. the submucosa contains elastic fibers that contribute to the longitudinal folds of the esophagus and confer elasticity to the esophageal wall. a transition occurs in the muscle type composing the tunica muscularis from striated skeletal muscle in the proximal two thirds of the esophagus to smooth muscle in the distal third. in the proximal esophagus the skeletal muscle layers spiral across one another at angles. within the smooth muscle layers of the distal esophagus the outer layer becomes more longitudinal, whereas the inner layer thickens and becomes circular. the wall of the terminal esophagus can be to cm thick. deep cervical fascia, pleura, and peritoneum contribute to the thin fibrous tunica adventitia of the esophagus. motor innervation to the striated skeletal muscle of the esophagus includes the pharyngeal and esophageal branches of the vagus nerve, which originate in the nucleus ambiguus of the medulla oblongata. parasympathetic fibers of the vagus nerve supply the smooth muscle of the distal esophagus. sympathetic innervation of the esophagus is minimal. passage of ingesta through the esophagus can be considered part of the swallowing process, which consists of oral, pharyngeal, and esophageal stages. the oral stage is voluntary and involves transport of the food bolus from the mouth into the oropharynx. during the involuntary pharyngeal stage the food bolus is forced through the momentarily relaxed upper esophageal sphincter by simultaneous contractions of the pharyngeal muscles. in the esophageal phase of swallowing the upper esophageal sphincter closes immediately, the lower esophageal sphincter opens, and esophageal peristalsis propels the bolus into the stomach. unlike a food bolus, liquids do not require peristalsis to reach the lower esophageal sphincter and may precede the food bolus during swallowing. the upper esophageal sphincter prevents esophagopharyngeal reflux during swallowing and air distention of the esophagus during inspiration. upper esophageal pressure increases in response to pressure from a food bolus and to increased intraluminal acidity, as would occur with gastroesophageal reflux. the lower esophageal sphincter is a smooth muscle located at the gastroesophageal junction that is morphologically ill defined but forms an effective functional barrier. normally the lower esophageal sphincter is closed in response to gastric distention to restrict gastroesophageal reflux. relaxation of the lower esophageal sphincter permits passage of ingested material from the esophagus to the stomach. distention of the stomach with ingesta mechanically constricts the lower esophageal sphincter. gastric distention also triggers a the esophagus is a musculomembranous tube that originates from the pharynx dorsal to the larynx and terminates at the cardia of the stomach. in adult thoroughbred horses the esophagus is approximately cm long. the cervical portion is approximately cm long; the thoracic portion, approximately cm long; and the short abdominal portion, only approximately cm long. the cervical esophagus generally lies dorsal and to the left of the trachea in the cervical region. in the thorax the esophagus courses through the mediastinum lying dorsal to the trachea and crosses to the right of the aortic arch dorsal to the heart base. impactions. intramural causes of esophageal obstruction include tumors (squamous cell carcinoma), strictures, diverticula, and cysts. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mediastinal or cervical masses (tumors or abscesses) may cause extramural obstructions. congenital anomalies are covered in detail later. the clinician must perform a thorough physical examination, including complete oral and neurologic examination, to help rule out causes of dysphagia and nasal discharge other than esophageal obstruction. the clinical signs associated with esophageal obstructions are related primarily to regurgitation of food, water, and saliva caused by esophageal (postpharyngeal) dysphagia. horses with esophageal obstruction are often anxious and stand with their neck extended. one may note gagging or retching, particularly with acute proximal obstructions. bilateral frothy nasal discharge containing saliva, water, and food material; coughing; odynophagia; and ptyalism are characteristic clinical signs, the severity of which varies with the degree and location of the obstruction. distention in the jugular furrow may be evident at the site of obstruction. one may observe other clinical signs related to regurgitation of saliva, water, and food material, such as dehydration, electrolyte or acid-base imbalances, weight loss, and aspiration pneumonia. in extreme cases, pressure necrosis from the impaction or trauma to the esophagus may cause esophageal rupture. if the rupture is in the cervical esophagus, crepitus or cellulitis may be evident along with signs of systemic inflammation. thoracic auscultation is important to determine whether aspiration pneumonia is present. intrathoracic esophageal rupture may result in pleuritis and its associated clinical signs. passage of a nasogastric tube is an effective way to detect and localize an obstruction but provides little information about the nature of the obstruction or the condition of the esophagus. the most direct method for diagnosis of esophageal obstructions is endoscopic examination. most cases of esophageal obstruction occur at sites of natural narrowing of the esophageal lumen, such as the cervical esophagus, the thoracic inlet, base of the heart, or the terminal esophagus, thus one may need an endoscope longer than m for complete evaluation. endoscopic evaluation is useful before relief of an impaction to localize the obstruction and to investigate the nature of the impaction if one suspects a foreign body. foreign bodies may be retrievable via transendoscopic tethering. one can obtain critical diagnostic and prognostic information following resolution of the impaction. assessing the affected esophagus for mucosal ulceration, rupture, masses, strictures, diverticula, and signs of functional abnormalities is important (figure . - ) . ultrasonography of the cervical region is useful not only to confirm a cervical esophageal impaction but also part ii disorders of specific body systems vagal reflex that increases lower esophageal sphincter tone, a safety mechanism against gastroesophageal reflux. the mechanical and vagal mechanisms that promote lower esophageal sphincter tone prevent spontaneous decompression of the stomach, which along with a lack of a vomiting reflex in the horse, increases the risk of gastric rupture during episodes of severe distention. esophageal obstruction has many causes (table . - ) and most often is manifested clinically by impaction of food material and resulting esophageal dysphagia. esophageal obstruction may be caused by primary impactions (simple choke) of roughage, particularly leafy alfalfa hay, coarse grass hay, bedding, and even grass. prior esophageal trauma or poor mastication caused by dental abnormalities may predispose horses to primary esophageal impaction. wolfing or gulping food may precipitate primary impactions, particularly if the horse is exhausted or mildly dehydrated after a long ride or is weakened from chronic debilitation. impactions also may result from disorders that physically impede the passage of food material and fluid by narrowing the luminal diameter, reduce the compliance of the esophageal wall, or alter the conformation of the esophageal wall such that food material accumulates in a pocket or diverticulum. foreign bodies, intra-or extramural masses, or acquired or congenital anomalies cause these so-called secondary to provide critical information about the location and extent of the impaction and esophageal wall thickness and integrity. ultrasonography may provide information about the cause. radiographic assessment of the esophagus can confirm the presence of esophageal obstruction in cases in which one cannot view the affected area adequately using endoscopy. one can detect impacted food material in the esophagus by a typical granular pattern and often can observe gas accumulation proximal to the obstruction. air or barium contrast radiographic studies are most useful for evaluating the esophagus following relief of the impaction if one suspects a stricture. one often can detect esophageal dilation, diverticula, rupture, functional disorder (megaesophagus), or luminal narrowing caused by extraluminal compression more easily using contrast radiographic studies instead of endoscopy ( figure . - ). - one should take care when interpreting radiographic studies in sedated horses, particularly after passage of a nasogastric tube or other esophageal manipulations that may contribute to esophageal dilation. the primary goal of treatment for esophageal impaction is to relieve the obstruction. parenteral administration of acepromazine ( . mg/kg intravenously), xylazine ( . to . mg/kg intravenously) or detomidine ( . to . mg/kg intravenously), oxytocin ( . to . iu/kg intramuscularly), and/or esophageal instillation of lidocaine ( to ml of % lidocaine) may reduce esophageal spasms caused by pain or may decrease esophageal tone. [ ] [ ] [ ] [ ] some clinicians advocate parasympatholytic drugs such as atropine ( . mg/kg intravenously) to reduce salivary secretions and lessen the risk of aspiration. however, undesirable effects of atropine including excessive drying of the impaction and inhibition of distal gastrointestinal motility may preclude its use. resolution of an impaction may require physical dispersal of the material. one can use a nasogastric tube to displace the impacted material along with external massage if the obstruction is in the cervical region. often, carefully lavaging the esophagus with water via an uncuffed or a cuffed nasogastric tube while the head is lowered is necessary to aid in breaking up the impaction. some clinicians advocate a dual tube method whereby a tube is placed through each nasal passage into the esophagus for ingress and egress of the lavage fluid. because of the risk of aspiration of water and food material, esophageal lavage sometimes is done under general anesthesia with a cuffed nasotracheal tube. in refractory cases, intravenous administration of isotonic fluid containing . % nacl and kcl ( to meq/l) for hours at a rate of to ml/kg/ day along with esophageal relaxants such as oxytocin may promote hydration and softening of the impaction and help prevent or alleviate any electrolyte or acid-base imbalances resulting from salivary losses of chloride, sodium, and potassium. one should note that the effects of oxytocin on esophageal tone occur in the proximal two thirds of the esophagus and may not be effective for mucosa has recovered as assessed by endoscopy, one can feed the horse soft food (moistened pellets and bran mashes). one can return the patient gradually to a highquality roughage diet over to days, depending on the degree of esophageal damage induced by the impaction and the nature of any underlying disease. the prognosis for survival is good ( %), but some horses may require permanent dietary modification if persistent chronic obstruction is a problem. aspiration pneumonia and perforation are potential complications of severe or prolonged esophageal obstructions. if aspiration is suspected, administration of broad-spectrum antibiotics that are effective against gram-positive and gram-negative organisms, including metronidazole ( mg/kg orally every hours) for anaerobes is advisable. a subsequent section describes treatment of esophageal perforation or rupture. esophagitis refers to a clinical syndrome of esophageal inflammation that may or may not be ulcerative. the major protective mechanisms of the esophageal mucosa include salivary and food material buffers, normal peristaltic motility, and the barrier formed by the gastroesophageal sphincter. reflux esophagitis is caused by repeated episodes of gastric fluid regurgitation into the distal esophagus and subsequent chemical injury to the mucosa ( figure distal obstructions. , rarely, esophageal obstruction ultimately may require esophagotomy to relieve the impaction. one must enforce strict restriction of food and water, including access to bedding material, until the obstruction is resolved and the esophagus has regained function. systemic effects of dysphagia associated with esophageal impaction include dehydration, hyponatremia, hypochloremia, and metabolic alkalosis from prolonged loss of salivary free water and electrolytes. if the duration of a complete esophageal obstruction is hours or longer, one should correct dehydration and electrolyte and acid-base imbalances. one can restore fluid and electrolyte balance with oral electrolyte solutions if the patient is less than % to % dehydrated and the esophageal obstruction is resolved. horses that are greater than % to % dehydrated or those that have a refractory obstruction or moderate to severe electrolyte imbalances may require intravenous fluid therapy with solutions containing . % nacl and kcl ( to meq/l). one should perform esophageal endoscopy after relief of the impaction to determine whether any complications of the impaction have developed or if a primary cause of the obstruction is present. endoscopic examination is critical to determine the postobstruction treatment plan and for follow-up evaluation of esophageal healing. one should reevaluate the horse every to weeks following resolution of the impaction if one notes esophageal dilation or mucosal injury. additional evaluation via radiography may be warranted to assess motility and transit times. dilation proximal to the site of obstruction, mucosal injury from trauma, stricture formation, formation of a diverticulum, megaesophagus, and esophagitis are sequelae to esophageal obstruction that predispose patients to reobstruction. the rate of reobstruction may be as high as %. depending on the duration of the obstruction and the degree of trauma or dilation, the risk of reobstruction is high for to hours or longer, thus one should withhold food for at least to hours after resolution of the obstruction. sucralfate ( mg/kg orally every hours) may hasten healing if esophageal ulceration is evident, but the efficacy of sucralfate for this purpose is not established. some clinicians suggest that administration of a nonsteroidal antiinflammatory drug (nsaid) such as flunixin meglumine ( mg/kg orally or intravenously every hours) or phenylbutazone ( to mg/kg orally or intravenously every to hours) for to weeks after resolution of the impaction may reduce the development of strictures. judicious use of nsaids is recommended to prevent nsaid-induced worsening of esophageal mucosal injury. one should avoid orally administered nsaids if esophagitis is present. after to hours or when the esophageal duodenal strictures caused by chronic ulceration commonly have reflux esophagitis. diagnosis requires endoscopic examination of the esophagus. one may note diffuse, patchy, linear, or coalescing erosion or ulcerations (see figures . - and . - ). one also may observe significant edema or hyperemia. determining whether an underlying disease, such as infection, neoplasia, esophageal strictures, or diverticula, is present is important. in addition, one must examine the stomach to determine whether the esophagitis is associated with gastritis, gastric obstruction, or gastric ulcer disease. contrast radiography may be helpful to detect esophageal ulceration and is useful to assess esophageal motility and transit time. the principles of therapy for reflux esophagitis include control of gastric acidity, mucosal protection, and correction of any underlying disorder contributing to gastroesophageal reflux. reduction of gastric acid production with h histamine receptor blockers such as ranitidine or proton pump antagonists such as omeprazole is critical for resolution of the esophagitis. some clinicians advocate using sucralfate to promote healing of ulcerated esophageal mucosa. however, the ability of sucralfate to bind ulcerated esophageal mucosa is not proven, nor is the efficacy of sucralfate for hastening esophageal ulcer healing. horses with reflux esophagitis following delayed gastric outflow caused by gastroduodenal ulcer disease, gastric paresis, or proximal enteritis may benefit from prokinetic drugs that act on the proximal gastrointestinal tract. metoclopramide ( . to . mg/kg subcutaneously every to hours) reduces gastroesophageal reflux by increasing lower esophageal sphincter tone, gastric emptying, and gastroduodenal coordination. one should exercise caution when giving metoclopramide to horses because they are prone to extrapyramidal neurologic side effects of the drug. cholinergic drugs such as bethanechol ( . to . mg/kg subcutaneously every to hours or . to . mg/kg orally every to hours) may improve gastric emptying and are effective for treating reflux esophagitis. for esophagitis from trauma or pressure injury after esophageal impaction, judicious use of nsaids may be warranted to reduce esophageal inflammation and pain. dietary modification may be necessary for patients with esophagitis, depending on the degree of ulceration or if motility is impaired. one should feed horses with mild esophagitis frequent small meals of moistened pellets and fresh grass. severe esophagitis may necessitate withholding food and complete esophageal rest for several days. although the prognosis for esophagitis is good in the absence of underlying disease, the risk of esophagus is delayed, such as in functional disorders of the esophagus. like ulceration of the squamous portion of the stomach in horses, gastric acid and bile salt chemical injury is a major mechanism of esophageal squamous epithelial ulceration. , reflux esophagitis may occur along with gastric ulcer disease, motility disorders, increased gastric volume from gastric outflow obstructions, gastric paresis, intestinal ileus, or impaired lower esophageal sphincter function. , other causes of esophagitis in horses include trauma (foreign bodies, food impactions, nasogastric tubes), infection (mural abscesses), or chemical injury (pharmaceuticals, cantharidin) ( figure . - ). [ ] [ ] [ ] [ ] the clinical signs of esophagitis are nonspecific and similar to esophageal obstruction and gastric ulcer diseases. gagging or discomfort when swallowing may be evident, and hypersalivation and bruxism are signs of esophageal pain. esophageal (postpharyngeal) dysphagia may be evident. one may note partial or complete anorexia such that horses with chronic esophagitis may have significant weight loss. esophageal hypomotility dysfunction caused by the inflammatory process may result in esophageal impaction. clinical signs of underlying diseases that predispose to esophagitis may predominate or mask the signs of esophagitis. horses with gastrointestinal motility disorders such as proximal enteritis or gastric outflow obstruction are at a high risk of developing reflux esophagitis because of the presence of gastric acid and bile salts in the fluid reflux. foals with gastric, pyloric, or stricture formation is high if severe circumferential or coalescing ulcerations are present. esophagitis from severe trauma or infection may be prone to stricture formation. motility dysfunction of the equine esophagus is caused most commonly by hypomotility resulting in esophageal dilation (ectasia) or megaesophagus. although megaesophagus in horses most commonly is acquired, reports indicate idiopathic megaesophagus in young horses may be congenital. [ ] [ ] [ ] [ ] acquired megaesophagus in horses may be a consequence of chronic or recurrent esophageal obstruction. , esophageal impactions of a short duration cause a proximal dilation of the esophagus that is generally reversible. however, if the duration of the obstruction is long enough, the motility of the esophagus proximal to the site of obstruction may be impaired permanently. other causes of acquired megaesophagus include extraesophageal obstruction by tumors or abscesses, pleuropneumonia, and vascular ring anomalies. , acquired megaesophagus also may result from neurologic, neuromuscular, and muscular disorders. neurologic diseases that cause vagal neuropathy-such as equine protozoal myeloencephalitis, equine herpesvirus myeloencephalitis, and idiopathic vagal neuropathy-have been associated with megaesophagus in horses. pleuropneumonia may be associated with a vagal neuropathy resulting in megaesophagus. megaesophagus is an early sign of equine dysautonomia and may be observable in patients with botulism. myasthenia gravis is a well-known cause of megaesophagus in nonequine species but has not been reported in horses. also in other species, electrolyte disorders, cachexia, primary myopathies, myositis, and addison's disease may affect esophageal motility but have not been associated with megaesophagus in horses. one can induce iatrogenic megaesophagus by the α adrenergic agonist detomidine, but this is transient and reversible. , nonetheless, the use of this drug may complicate clinical evaluation of esophageal motility. esophageal inflammation, particularly reflux esophagitis, may affect motility and cause megaesophagus. however, because esophageal hypomotility affects the tone and function of the lower esophageal sphincter, reflux esophagitis also may be a complication of a primary functional disorder. thus assessing esophageal motility in horses with esophagitis that is not responding appropriately to treatment is important. along with a complete physical examination one should include a careful neurologic examination to help rule out primary neurologic causes of megaesophagus. because esophageal hypomotility is a functional obstruction, the clinical signs of esophageal hypomotility or megaesophagus are similar to esophageal obstruction. unlike mechanical obstruction the onset of clinical signs is insidious rather than acute. the clinical signs include those associated with esophageal dysphagia. , , [ ] [ ] [ ] [ ] the cervical esophagus may be dilated enough to be evident externally. weight loss is a common sign. signs attributable to an underlying disease may be evident. diagnosis of esophageal hypomotility requires transit studies. one can measure the transit time of a bolus from the cervical esophagus to the stomach by fluoroscopy or contrast radiography. , other signs of esophageal hypomotility and megaesophagus include pooling of contrast material and an absence of peristaltic constrictions. , , , endoscopy may reveal a dilated esophagus and an absence of peristaltic waves. , one may observe evidence of underlying disease causing obstruction or esophageal dilation. , one should evaluate the esophagus for evidence of esophagitis that is causing esophageal motility dysfunction or is a result of impaired esophageal clearance of gastric fluid. esophageal manometry may be useful to document abnormal postdeglutition contraction pressures, contraction time, and propagation times but is not often available for routine clinical application. , one should perform other diagnostic tests such as a complete blood count and chemistry to help determine a possible underlying cause. cerebral spinal fluid analysis may be indicated to rule out neurologic disorders. specialized testing such as electromyography to detect neuromuscular disorders may also be indicated. treatment of esophageal hypomotility or megaesophagus should aim at treating the underlying cause. dietary modification should aim at improving esophageal transit of food. one should feed the horse slurries of pellets, and feeding from an elevated position to promote transit may be beneficial. metoclopramide or bethanechol may benefit patients with reflux esophagitis associated with megaesophagus by increasing lower esophageal tone, gastric emptying, and reducing gastroesophageal reflux. the prognosis depends on the underlying cause and the degree of dilation. although many cases of megaesophagus associated with reflux esophagitis respond well to treatment, many other forms of megaesophagus including congenital megaesophagus have a poor prognosis. strictures most commonly are caused by pressure necrosis from esophageal impactions that induce circumferential erosion or ulceration of the esophageal mucosa, although esophageal injury caused by oral administration of corrosive medicinal agents and trauma to the neck may also result in stricture formation. congenital strictures also have been reported. strictures caused by mucosal and submucosal trauma are termed esophageal webs or rings. strictures may also originate in the muscular layers and adventitia of the esophagus (mural strictures) or in all of the layers of the esophagus (annular stenosis). , horses with these lesions have a presentation similar to those with simple obstructions, because strictures result in partial obstruction and impaction of food material in the lumen. one can detect esophageal webs or rings with endoscopy (see figure . - ), whereas identification of mural strictures or annular stenosis may require a double-contrast esophogram (see figure . - ). in a retrospective study of horses with esophageal stricture following simple obstruction, maximal reduction in esophageal lumen diameter occurred within days of the esophageal obstruction. although surgery has been used to relieve such strictures, initial medical management is warranted because strictures may resolve with conservative therapy, and the esophagus continues to remodel for up to days following ulceration. in one report, seven horses with esophageal obstruction-induced stricture were treated conservatively by feeding a slurry diet and administering antiinflammatory and antimicrobial medications, and five of seven were clinically normal within days. one of the five successfully treated horses had a -cm area of circumferential ulceration, suggesting that the potential exists for extensive mucosal injury to resolve without permanent stricture formation. if resolution of strictures within days is insufficient, one should investigate other methods to increase esophageal diameter. bougienage has been used successfully in small animal patients and human beings. the technique involves passage of a tubular dilatable instrument down the esophagus and stretching of the stricture. one may perform the technique by passing a nasogastric tube with an inflatable cuff. however, one has to perform the procedure frequently to have any success, and horses do not tolerate it well. alternatively, a number of surgical techniques have been used to resolve strictures, including resection and anastomosis, , temporary esophagostomy with fenestration of the stricture, esophagomyotomy for strictures of the muscularis and adventitia, , or patch grafting with local musculature. however, such surgeries are fraught with complications, largely because of the propensity of the traumatized esophagus to restricture. , the esophagus lacks a serosal layer and does not rapidly form a fibrin seal as does the remainder of the intestinal tract, so anastomoses tend to leak. in addition, tension on the esophagus during swallowing and movement of the neck impairs healing of anastomoses. , in spite of these difficulties, the long-term prognosis for horses with chronic esophageal strictures treated surgically is better than for those treated nonsurgically. two types of diverticula are traction (true) diverticula and pulsion (false) diverticula. traction diverticula result from wounding and subsequent contraction of periesophageal tissues, with resultant tenting of the wall of the esophagus. pulsion diverticula arise from protrusion of esophageal mucosa through defects in the muscular wall of the esophagus and usually result from trauma or acute changes in intraluminal pressure. traction diverticula appear as a dilation with a broad neck on contrast esophagography, whereas pulsion diverticula typically have a flask shape with a small neck on an esophagram (see figure . - ). , although traction diverticula are usually asymptomatic and of little clinical significance, pulsion diverticula may fill with feed material, ultimately leading to esophageal obstruction. [ ] [ ] [ ] a movable mass in the midcervical region may be noticeable before onset of complete obstruction. pulsion diverticula may be corrected surgically by inverting or resecting prolapsed mucosa and closing the defect in the wall of the esophagus. , , inversion of excessive mucosa may reduce the diameter of the esophageal lumen and predispose horses to esophageal obstruction and therefore should be reserved for small diverticula. congenital disorders of the esophagus are rare. reported congenital abnormalities include congenital stenosis, persistent right aortic arch, esophageal duplication cysts, [ ] [ ] [ ] intramural inclusion cysts, , and idiopathic megaesophagus. , , in the one report of congenital stenosis, double-contrast radiography revealed concentric narrowing of the thoracic esophagus in the absence of any vascular abnormalities at the base of the heart. successful treatment included having the foal stand with the forelimbs elevated off the ground following each feeding. persistent right aortic arch is a congenital anomaly in which the right fourth aortic arch becomes the definitive aorta instead of the left aortic arch, which results in constriction of the esophagus by the ligamentum arteriosum as it extends between the anomalous right aorta and the left pulmonary artery. clinical signs may include those associated with esophageal (postpharyngeal) dysphagia, drooling, and distention of the cervical esophagus resulting from partial obstruction of the thoracic esophagus. , endoscopic examination typically reveals dilation of the esophagus cranial to the obstruction section . esophageal diseases with evidence of diffuse esophagitis. successful surgical treatment of persistent right aortic arch has been reported in one foal. esophageal duplication cysts and intramural inclusion cysts cause typical signs of esophageal obstruction, including salivation, esophageal dysphagia, and swelling of the cervical esophagus as the cysts enlarge. , , such signs can make them difficult to differentiate from other forms of esophageal obstruction (choke). endoscopic examination may reveal compression of the esophageal lumen and communication with the esophageal lumen if it exists. ultrasonographic examination may be the most useful method of antemortem diagnosis if the cyst is in the cervical esophagus. examination of an aspirate of the mass may aid in the diagnosis by revealing the presence of keratinized squamous cells. , surgical treatments have included complete surgical resection and surgical marsupialization. , , the latter appears to be more successful and results in fewer complications. , complications of surgical resection have included laryngeal hemiplegia following surgical trauma to the recurrent laryngeal nerve in the region of the esophagus and esophageal fistula formation. perforation typically occurs in the cervical region in response to external trauma, necrosis of the esophageal wall caused by a food impaction, or rupture of an esophageal lesion such as an impacted diverticulum. the esophagus is particularly vulnerable to external trauma in the distal third of the neck because only a thin layer of muscle covers it at this point. iatrogenic perforation may occur in response to excessive force with a stomach tube against an obstruction or a compromised region of the esophagus. esophageal perforations may be open or closed and tend to cause extensive cellulitis and necrosis of tissues surrounding the wound because of drainage of saliva and feed material within fascial planes. systemic inflammation associated with endotoxemia from septic cellulitis may occur. closed perforations of the esophagus are particularly troublesome because food material, water, saliva, and air may migrate to the mediastinum and pleural space via fascial planes. , because of the leakage of air into the tissues surrounding the rupture, extensive subcutaneous and fascial emphysema frequently develops and is usually evident clinically and on cervical radiographs. pneumomediastinum and pneumothorax are potentially fatal complications of esophageal ruptures. treatment should include converting closed perforations to open perforations if possible, extensive debridement and lavage of affected tissues, broadspectrum antibiotics, tetanus prophylaxis, and esophageal rest. the clinician may achieve the latter by placing a feeding tube into the esophagus via the wound. alternatively, one may place a nasogastric tube using a small tube ( -f diameter) . for open perforations, once the wound has granulated and contracted to a small size, one may attempt peroral feeding. extensive loss of saliva via esophageal wounds may lead to hyponatremia and hypochloremia. in addition, transient metabolic acidosis occurs because of salivary bicarbonate loss, followed by progressive metabolic alkalosis. although reports of esophageal wounds healing well by second intention exist, healing takes a prolonged time. in addition, some perforations never completely heal and form permanent esophagocutaneous fistulae that may require surgical correction. the development of esophageal strictures is not common because wounds are usually linear and not circumferential. however, traction diverticula may develop. other complications of esophageal wounds include horner's syndrome and left laryngeal hemiplegia. in a retrospective study on esophageal disorders, only of horses with esophageal perforations survived long-term ; in a report of esophageal trauma following nasogastric intubation, of horses were euthanized. the prognosis is therefore poor in horses with esophageal perforations, largely because of the extent of cellulitis, tissue necrosis, shock, and local wound complications. specialized endoscopic equipment allowing visual inspection of the entire adult equine stomach has become increasingly available to veterinarians in academia and private practice. thus gastric disease in horses recently section . diseases of the stomach has gained increasing awareness among veterinarians, owners, and trainers. peptic ulcer disease is defined as erosions or ulcers of any portion of the gastrointestinal tract normally exposed to acid. mucosal damage can include inflammation, erosion (disruption of the superficial mucosa), or ulceration (penetration of the submucosa). in severe cases, fullthickness ulceration can occur, resulting in perforation. the proximal (orad) portion of the equine stomach is lined by stratified squamous mucosa similar to the esophageal lining. the distal (aborad) portion of the stomach is lined with glandular mucosa, and the distinct junction between the two regions is deemed the margo plicatus. ulceration can occur in either or both gastric regions, although different clinical syndromes and pathophysiologic mechanisms apply. as a result, the broad term equine gastric ulcer syndrome (egus) has been used to encompass the wide array of associated clinical syndromes. egus develops in horses of all ages and continues to be of major clinical and economical importance. the prevalence of gastric ulceration has been reported for a variety of breeds and types of horses; however, most current data involve thoroughbreds in race training. the prevalence of squamous ulceration in horses in race training varies from % to % - and can be as high as % when limited to animals actively racing. in a survey of active show horses, % had gastric ulceration, with only horse having ulceration of the glandular fundus. in one large retrospective study ( adult horses from to ) evaluating incidence of gastric ulceration identified at necropsy, an overall prevalence of . % was found. the highest prevalence was found in thoroughbreds (including arabians) and standardbred trotters, and cold-blooded horses were affected significantly less. lesions were located most commonly in the squamous mucosa along the margo plicatus, followed by the glandular body, proximal squamous mucosa, and antrum. many studies investigating prevalence of gastric ulceration do not differentiate between squamous and glandular lesions or evaluate only squamous disease. in a recent study in which the gastric antrum and pylorus were evaluated in horses in a hospital setting, % had antral or pyloric erosions or ulcerations, % had squamous mucosal lesions, and % had lesions involving the glandular body. a correlation between the presence or severity of squamous disease and antral/pyloric disease was not identified. the reported prevalence of gastric ulceration in foals varies from % to %. [ ] [ ] [ ] an imbalance between inciting and protective factors in the mucosal environment can result in ulcer formation. , the major intrinsic factors promoting ulcer formation include hydrochloric acid, bile acids, and pepsin, with hydrochloric acid being the predominant factor. various intrinsic factors protect against ulcer formation such as the mucus-bicarbonate layer, maintenance of adequate mucosal blood flow, mucosal prostaglandin e and epidermal growth factor production, and gastroduodenal motility. in human beings, extrinsic ulcerogenic factors include nonsteroidal antiinflammatory drugs, helicobacter pylori, stress, changes in diet, or gastrointestinal disorders, especially those resulting in delayed gastric emptying. in human neonates, physiologic stress associated with a major primary illness seems to be associated strongly with gastric ulcers. many of the other factors mentioned previously are believed to be important in horses, but clear evidence of an infectious agent has not yet been identified in horses or foals with egus. , recently, the possibility of helicobacter infection in horses has reemerged with the identification of polymerase chain reaction products from urel, a protongated urea channel unique to gastric-dwelling helicobacter species, in the squamous epithelium of three horses, two of which had squamous erosions. the specific factors involved in injury and the protective mechanisms vary between regions of the proximal gastrointestinal tract. the pathophysiology of squamous mucosal ulceration in the horse appears similar to that in gastroesophageal reflux disease in human beings and ulceration of the nonglandular mucosa in pigs. excess acid exposure is the predominant mechanism responsible for squamous mucosal ulceration, although many details remain unclear. hydrochloric acid is secreted by parietal cells in the gastric glands via a hydrogen-potassium adenosine triphosphatase (h + ,k + -atpase) pump on the luminal side. horses secrete acid continuously, and measured ph of equine gastric contents varies from less than to greater than depending on the dietary state of the horse (fed or fasted). , a protocol of repeated -hour periods of fasting and feeding has been shown to induce squamous erosion and ulceration. because this protocol results in periods of prolonged gastric acidity (ph < . ) and because concurrent administration of the histamine (h ) receptor antagonist ranitidine reduces lesion severity, the protocol supports the role of acid exposure in the pathogenesis of squamous ulcer disease. several peptides can stimulate or inhibit parietal cell secretion of acid. the predominant stimuli for hydrochloric acid secretion are gastrin, histamine, and acetylcholine via the vagus nerve. g cells release gastrin within the antral mucosa, whereas mast cells and enterochromaffin-like cells release histamine in the gastric gland. histamine binds to type receptors on the parietal cell membrane, causing an increase in cyclic adenosine monophosphate and resulting in phosphorylation of enzymes that activate the proton pump. gastrin and acetylcholine can act via calcium-mediated intracellular pathways and also stimulate histamine release directly. isolated equine parietal cells respond maximally to histamine stimulation and only minimally to carbachol and pentagastrin. gastrin release is controlled primarily by gastrin-releasing peptide, which is stimulated by gastric distention and increased luminal ph, but the interaction between gastrin and histamine has not been elucidated fully in the horse. somatostatin, released by fundic and antral d cells, is the primary inhibitor of gastric acid secretion by parietal cells. the inhibitory effect of somatostatin is primarily paracrine, but plasma levels of somatostatin negatively correlate with gastric luminal acidity. epidermal growth factor, a peptide produced in saliva, also inhibits gastric acid secretion. foals can produce significant amounts of gastric acid by the second day of life, with consistent periods of acidity (ph < . ) in clinically normal animals. , in one study, foals tended to have a high gastric ph at day of age, but in a study of critically ill foals, some foals demonstrated periods of gastric acidity on the first day of life. suckling was associated with an immediate rise in gastric ph, whereas periods of rest in which foals did not suck for more than minutes were associated with prolonged periods of acidity. whereas premature human infants are capable of gastric acid production at weeks of gestation, only of premature foals demonstrated an acidic ph recording in a study of gastric ph profiles in critically ill foals. however, multiple factors likely were involved in critically ill foals of this study, and the true ontogeny of gastric acid production in foals is currently unknown. equine squamous mucosa is thin at birth but becomes hyperplastic and parakeratotic within days. the parallel between decreasing ph and proliferation of squamous epithelium correlates with that observed in other species. the combination of a thin gastric epithelium with a high acid output may leave neonatal foals susceptible to ulcer formation at a young age. in addition, one must remember the difference in normal appearance of the squamous mucosa when interpreting gastric endoscopy in a neonatal population. in esophageal squamous mucosa, intercellular tight junctions and bicarbonate secretion are the major factors involved in protection against acid injury in other species, although squamous bicarbonate secretion had not been documented in the horse. [ ] [ ] [ ] the principal barrier is a glycoconjugate substance secreted by cells in the stratum spinosum, with a contribution from the tight junctions in the stratum corneum. this barrier function is considered weak at best, and thus a functioning lower esophageal sphincter, normal salivary flow, and salivary mucins contribute to the prevention of acid injury in human gastroesophageal reflux disease. in horses a mechanical barrier like the lower esophageal sphincter is not available to protect the gastric squamous mucosa from acid exposure. the normal gastric fill line rests just below the cardia, so only the squamous mucosa along the lesser curvature adjacent to the margo plicatus should receive exposure to acidic gastric contents regularly. not surprisingly, this correlates with the most common location of squamous mucosal ulceration. bile salts and pepsin have been implicated as contributing factors to ulcer disease in many species. in rabbit esophageal mucosa, bile salt absorption occurs and is correlated directly with mucosal barrier disruption. the unconjugated bile salts cholate and deoxycholate have a pk a (negative logarithm of the ionization constant of an acid) of and . , respectively, and therefore cannot remain in solution and cause mucosal damage in the presence of acid. alternatively, the conjugated bile salt taurocholate (pk a . ) can cause mucosal injury in the ionized salt form at ph or the un-ionized acid form at ph to . in the pig, bile salts or acid alone cause squamous mucosal damage, whereas a combination of the two result in extensive damage in vitro. in the horse a similar synergistically damaging effect was found with the addition of bile salts and acid (ph . ) to stratified squamous mucosa in vitro in one study. in addition, the investigators were able to document levels of bile salts and acid sufficient to cause mucosal damage in gastric contents within hours of feed deprivation. this is not surprising, given that duodenogastric reflux occurs normally in the horse. in a separate in vitro study of equine squamous mucosa, prolonged exposure to acid alone (ph . ) had a damaging effect, and synergism with exposure to a combination of acid and pepsin or taurocholate was not found. the lack of synergism likely is caused by the lower ph used in this study and stresses the importance of acid exposure in squamous ulcer disease. pepsinogens are secreted primarily by chief cells, although secretion by neck cells, cardiac glands, and antral pyloric glands also occurs. in an acidic environment (ph < . ), pepsinogen is converted to the active pepsin. although the proteolytic activity of pepsin normally is directed toward dietary protein, it also can act on the gastric mucosa. thus acid remains the major contributing factor to squamous mucosal damage, although other factors such as pepsin and bile salts may play an important role as well in the initiation or perpetuation of disease. several mechanisms help protect the glandular mucosa from acid injury. the mucus-bicarbonate layer serves to titrate h + ion from the gastric lumen to co and h o. cellular restitution and prostaglandins of the e series, which enhance mucosal blood flow and secretion of mucus and bicarbonate in the glandular mucosa have not been documented in squamous epithelium. , of these mechanisms, mucosal blood flow is likely the most important contributor to overall gastric mucosal health. nitric oxide is a key regulator of mucosal blood flow and prostaglandin synthesis and thus may play a role in mucosal protection. dietary factors also have been implicated in ulcer disease. horses in race training have a high incidence of gastric ulceration and frequently are fed high-concentrate, low-roughage diets. in one study, higher volatile fatty acid (acetic, propionic, and isovaleric acid) concentrations, higher gastric juice ph, and lower number and severity of nonglandular ulceration were documented after feeding an alfalfa hay-grain diet compared with a bromegrass hay diet. however, many factors differed between the diets, such as digestible energy, bulk, crude protein, and mineral content (especially calcium). thus dietary factors represent an important area of further investigation in the pathophysiology of egus, particularly squamous ulceration. the pathophysiologic correlation between exercise and squamous ulcer disease has not yet been defined despite the high prevalence of ulceration in performance horses. preliminary work suggests that gastric compression occurs during treadmill exercise, presumably because of an increase in intraabdominal pressure. such contracture could result in increased acid exposure to the squamous mucosa by raising the fill line of gastric contents. further studies in this laboratory have provided support for this theory by demonstrating a high ph in the proximal stomach, immediately distal to the lower esophageal sphincter, during resting conditions that decreases during treadmill exercise (m. lorenzo-figueras and a.m. merritt, personal communication, ) . risk factors associated with gastric ulceration include gender and age, and the reported prevalence of gastric ulcers has increased over time. in one study, ulcers were found more commonly in stallions, and the prevalence of gastric ulceration decreased with age, independent of gender, although this trend was only significant in the population of standardbred trotters. interestingly, the frequency of gastric ulceration increased from less than % before to approximately % after . in a study of thoroughbred horses in race training, an increase in squamous ulcer severity was noted in horses years old or older and in those horses that had raced. in the same study, severity of glandular lesions did not change between examinations, and age (> years) was the only factor associated with glandular lesion severity. several studies have failed to document a correlation between nonsteroidal antiinflammatory drug (nsaid) administration and naturally occurring ulcer disease. , , , , however, nsaid administration is a well-known cause of gastric ulceration under experimental conditions. [ ] [ ] [ ] [ ] [ ] nsaid-related ulceration typically is described as predominantly glandular, although nonglandular ulceration also can occur by a mechanism that has not yet been characterized fully. nsaids cause a decrease in prostaglandin e synthesis because of inhibition of the cyclooxygenase pathway. therefore a resultant decrease in glandular mucosal protection, most notably via decreased mucosal blood flow and mucus production, is the most likely mechanism of action. in one study, however, phenylbutazone administration resulted in ulceration of the glandular mucosa at the pyloric antrum but did not alter mucosal prostaglandin e concentration significantly. clinical signs typically associated with gastric ulceration in foals include poor appetite, diarrhea, and colic. many foals probably never exhibit clinical signs, and some do not exhibit clinical signs until ulceration is severe or fatal perforation has occurred. glandular ulceration typically is considered the most clinically significant type of disease in this population. the physiologic stress of a concurrent illness has been associated with gastric ulceration in foals. retrospectively, ( %) of foals up to days of age with a clinical disorder were found to have lesions in the gastric glandular mucosa, and prospectively ( %) of foals up to days of age with a clinical disorder had glandular ulceration. by contrast, only % to % of clinically normal foals examined in endoscopic surveys had lesions observed in the gastric glandular mucosa. , critically ill neonatal foals can have a greatly different ph profile compared with that in clinically normal foals, potentially because of alterations in gastric motility and acid secretion. gastric ulceration was not identified in any animals at necropsy in that study; however, ulceration has been documented in a similar population. thus factors other than acid exposure, most notably mucosal blood flow, may play an important role in the stressrelated ulceration in neonates. subjectively, gastric ulceration and rupture in the hospitalized neonatal population occurs less commonly now than in previous reports. advances in overall neonatal care, especially supportive care, likely have contributed to this decline. in suckling foals less than days old, lesions typically originate in the squamous mucosa adjacent to the margo plicatus along the greater curvature. such lesions can occur in foals as young as days of age and have been observed in % of foals less than days old. histologic examination of these lesions has revealed disruption of the epithelial layers of the mucosa and a neutrophilic infiltration. another phenomenon that occurs in young foals is the shedding, or desquamation, of squamous epithelium, which appears as flakes or sheets of epithelium. desquamation occurs without ulceration in up to % of foals less than days of age, and this process typically is not associated with clinical signs. , , in older foals, lesions become more prevalent in the squamous mucosa, particularly along the lesser curvature. lesions also are found in the squamous mucosa of the fundus and adjacent to the margo plicatus. these lesions can be severe and often are associated with clinical signs such as diarrhea, poor appetite, and poor growth and body condition. diarrhea is the most frequent sign in symptomatic foals with squamous mucosal lesions and is associated with more diffuse erosion or ulceration of the squamous mucosa than that which occurs in asymptomatic foals. in some foals, poor growth, rough hair coat, a potbelly appearance, or all of those occur along with moderate to severe squamous mucosal ulceration. in horses with severe or diffuse squamous ulceration, bruxism or colic may occur. gastroduodenal ulcer disease occurs almost exclusively in suckling and early weanling foals. clinical signs of duodenal ulceration are similar to those described for gastric ulceration (bruxism, colic, salivation, diarrhea), but the consequences are often more severe. lesions occur primarily in the proximal duodenum and range from diffuse inflammation to severe ulceration. foals with duodenal ulceration often have delayed gastric emptying and may have gastroesophageal reflux. complications can include gastric or duodenal rupture, duodenal stricture, and ascending cholangitis. severe squamous and esophageal ulceration and aspiration pneumonia can occur following gastroesophageal reflux. , [ ] [ ] [ ] [ ] the gastroduodenal ulcer disease syndrome can occur in outbreaks and most commonly is identified in intensive breeding operations. the cause of duodenal lesions in foals is not known. one theory is that the problem begins with diffuse duodenal inflammation that can coalesce down to a focal area of ulceration (g.d. lester and a.m. merritt, personal communication, ) . a temporal relationship between gastroduodenal ulcer disease and rotaviral diarrhea has been suggested, but an infectious cause remains unproven. although lesion location and severity associated with rotaviral infection varies among species, duodenal ulceration has not been reported. clinical signs attributable to egus in older horses vary and classically include anorexia and chronic or intermittent colic of varying severity. many horses with endoscopic evidence of disease may appear to be clinically normal or have vague signs that include decreased consumption of concentrates, postprandial episodes of colic, poor performance or failure to train up to expectations, poorquality hair coat, and decreased condition or failure to thrive. diarrhea typically is not associated with gastric ulceration in adult horses, although ulceration can occur concurrently with other causes of diarrhea. horses actively racing are more likely to have squamous ulceration than those solely in training. lesions occur predominantly in the squamous mucosa, particularly adjacent to the margo plicatus ( figure . - ) . in more severe cases, lesions can extend dorsally into the squamous fundus. clinically relevant lesions typically affect a greater portion of the squamous mucosa and can be deep enough to cause bleeding. however, bleeding from ulcers in the gastric squamous mucosa typically is not associated with anemia or hypoproteinemia. according to a recent study, the incidence of glandular lesions, particularly within the pyloric region, may be higher than previously reported, which emphasizes the importance of a thorough endoscopic examination and proper documentation of lesion location when reporting or discussing egus, especially the differentiation between squamous and glandular disease. although one may suspect a diagnosis of egus based on clinical signs and response to treatment, the only current method of confirmation is via gastroendoscopy, which one can perform easily in the standing horse or foal with mild sedation. in adult horses a -m endoscope allows for visual inspection of the entire stomach, pylorus, and proximal duodenum. shorter scopes permit examination of the gastric body and fundus, but not the pyloric antrum in most cases. one should use an endoscope with a maximum external diameter of mm for neonatal foals. numerous scoring systems for lesion severity have been described, but a recent consensus has been published by the equine gastric ulcer council (table . - ). duodenal ulceration can be difficult to confirm. duodenoscopy is the most specific means of diagnosis, although the procedure is more difficult than gastroscopy. additionally, an endoscope at least cm in length is needed for foals up to to months old, and a longer endoscope usually is required for older animals. diffuse reddening or inflammation may be the only recognizable lesion in cases of early duodenal disease. excessive enterogastric reflux of bile through the pylorus suggests duodenal dysfunction. however, the pylorus frequently appears open, and some degree of enterogastric reflux is common under normal conditions. ulceration at the pylorus or pyloric antrum also suggests the presence of a duodenal lesion. if one can perform gastroendoscopy, but not duodenoscopy, the severity of lesions, particularly in the glandular mucosa and in the squamous mucosa of the lesser curvature dorsal to the pyloric antrum, usually will be severe when duodenal ulcers are present. multiple pharmacologic treatments have been suggested for treating egus. because acid has been implicated as the most important pathophysiologic component of squamous ulcer disease, most antiulcer therapy centers on suppression or neutralization of gastric acid. severity and location of gastric lesions and severity and duration of clinical signs, as well as medication cost, can play a role in the therapeutic management of egus (table . - ). if gastroendoscopy is unavailable, some guidelines to therapy can be used, but the efficacy of the treatment is based on clinical signs, which are often vague or nonspecific. signs of colic or diarrhea that result from gastric ulcers often resolve within hours. one can note improvements in appetite, bodily condition, and attitude within to weeks. if one does not observe improvement in clinical signs, treatment has not been effective or gastric ulceration was not the primary problem. the principal therapeutic options for ulcer treatment include h antagonists (cimetidine, ranitidine, famotidine, nizatidine), proton pump blockers (omeprazole, pantoprazole, rabeprazole, esomeprazole), the mucosal adherent sucralfate, and antacids. the h antagonists suppress hydrochloric acid secretion through competitive inhibition of the parietal cell histamine receptor that can be overcome partially with exogenous pentagastrin. use of h antagonists has been successful in raising gastric ph and resolving gastric lesions in foals and adult horses. , , clinical and experimental evidence has demonstrated greater individual variability with lower dosages of h antagonists. thus dosage recommendations are based on levels necessary to increase gastric ph and promote ulcer healing in a majority of horses. commonly recommended dosages are to mg/kg orally every hours or . mg/kg intravenously every hours for cimetidine and . mg/kg orally every hours or . to mg/kg intravenously every hours for ranitidine. famotidine has been used less extensively in the horse, but a dose of to mg/kg/day has been recommended. because gastric perforation caused by glandular ulcer disease has been reported in hospitalized neonates, many clinicians routinely use prophylactic antiulcer therapy in this population. although clinically normal foals respond predictably to ranitidine, sick neonates have shown variability in ph response to intravenously administered ranitidine, with a much shorter duration of action and in some cases no noticeable response. thus currently used dosing schedules for hospitalized foals may be inadequate. because some critically ill foals have a predominantly alkaline gastric ph profile and because gastric acidity may be protective against bacterial translocation in neonates, the need for prophylactic ulcer therapy is controversial. in critically ill human neonates, intravenous administration of ranitidine raises gastric ph and gastric bacterial colonization but does not increase the risk of sepsis. in a retrospective study of hospitalized foals less than days of age, no difference in the frequency of gastric ulceration at necropsy was found between those foals that received prophylactic treatment for gastric ulcers and those that did not. because the study was retrospective, specific details regarding lesion location and severity were not available; however, none of the foals in the study died because of gastric ulcer disease. h antagonist therapy should continue for to days, but complete ulcer healing may take to days. if an animal is kept in race training during therapy, clinical signs may resolve but the lesions may not. currently, cimetidine and ranitidine are available in injectable, tablet, and liquid forms. famotidine and nizatidine are available in tablets. proton pump inhibitors block secretion of h + at the parietal cell membrane by irreversibly binding to the h + ,k + -atpase proton pump of the cell. these agents have a prolonged antisecretory effect, which allows for once-daily dosing. omeprazole, the first proton pump inhibitor to be developed, is the only currently approved agent for the treatment of egus. several studies have documented the safety of orally administered omeprazole in foals and adult horses. , omeprazole has demonstrated efficacy in the healing of nsaid-induced ulcers in horses and in naturally occurring cases of egus. , more importantly, omeprazole has been shown to eliminate or reduce the severity of gastric ulcers in thoroughbreds maintained in race training. the available equine preparation of omeprazole (gastrogard, merial, ltd., duluth, georgia) is recommended at a dose of mg/kg orally every hours. initial reports suggested that to days of omeprazole therapy were necessary to achieve maximum acid suppression; however, an increase in gastric ph and a decrease in acid output are evident to hours after omeprazole paste administration. after initial treatment ( days), treatment with or mg/kg every hours has been shown to decrease or prevent the recurrence of disease in animals maintained in training. the powder form of omeprazole degrades rapidly in an acidic environment, thus one must use an enteric-coated capsule (as used in the human preparation) or a specially formulated paste (such as gastrogard) to allow delivery of the active drug to the small intestine for absorption. many compounding pharmacies prepare omeprazole in liquid or paste formulation for use in horses, but their efficacy has not been evaluated to date. other proton pump inhibitors have been developed recently for use in human beings, including rabeprazole, lansoprazole, esomeprazole, and pantoprazole. in gastroesophageal reflux disease treatment in human beings, esomeprazole has demonstrated a higher rate of healing at and weeks compared with omeprazole, but rabeprazole, lansoprazole, and pantoprazole have similar efficacy. an intravenous formulation of pantoprazole recently became available commercially and may prove beneficial for patients in need of antiulcer therapy that cannot be treated orally. research regarding the pharmacokinetics and efficacy of other proton pump inhibitors in horses is not currently available. sucralfate is effective in treating peptic ulcers and preventing stress-induced ulcers in human beings. the mechanism of action likely involves adherence to ulcerated mucosa, stimulation of mucus secretion, enhanced prostaglandin e synthesis, and increased concentration of growth factor at the site of ulceration, although the prostaglandin effects may not play an important role in ulcer healing. these are factors relevant to glandular mucosa, and the efficacy of sucralfate in treating ulcers in the equine gastric squamous mucosa remains undetermined. sucralfate may be effective in preventing stress-induced ulcers in neonatal foals, because these occur in the glandular mucosa, although no clinical evidence directly supports this concept. in human beings, sucralfate provides protection against stress-induced ulcers with a decreased risk of pathogenic gastric colonization. one should give sucralfate at a dosage of to mg/kg every to hours. the efficacy of sucralfate in an alkaline ph is controversial but appears likely. [ ] [ ] [ ] moreover, at the time of administration of an section . diseases of the stomach h antagonist, the gastric ph likely will have returned to an acidic ph since the last dosage and will remain so for to minutes depending on the route of administration; thus one likely can administer the agents simultaneously if so desired. the use of antacids to treat gastric ulcers has not been examined critically in the horse. research in horses has shown that g aluminum hydroxide per g magnesium hydroxide results in an increase in gastric ph above for approximately hours. thus although antacids may be useful for treating ulcers in horses, a dose of approximately to ml at least every hours is necessary for a standard adult horse. the use of synthetic prostaglandin e analogs, such as misoprostol, has been effective in treating gastric and duodenal ulcers in human beings, and the proposed mechanism of action involves inhibition of gastric acid secretion and mucosal cytoprotection. frequently reported adverse effects of intestinal cramping and diarrhea in human beings have precluded the use of misoprostol in horses. one should consider prokinetic drugs in foals with duodenal disease and gastroesophageal reflux and when one suspects delayed gastric emptying without a physical obstruction. the cholinergic drug bethanechol has been shown to increase the rate of gastric emptying in horses. in cases of acute gastric atony, bethanechol . to . mg/kg administered subcutaneously every to hours has been effective in promoting gastric motility and emptying, followed by oral maintenance dosages of . to . mg/kg to times daily. adverse effects can include diarrhea, inappetance, salivation, and colic, but at the dosages stated, adverse effects have been infrequent and mild. a complete review of ileus and prokinetic therapy is available in chapter . . for foals with severe gastroduodenal ulcer disease that have developed duodenal stricture, surgical therapy is necessary. , these animals require a serious financial commitment because intensive perioperative medical therapy is critical for a successful outcome. even with surgical therapy, these foals often warrant a guarded prognosis. pyloric stenosis is a structural resistance to gastric outflow. congenital pyloric stenosis has been reported in foals and one yearling and results from hypertrophy of the pyloric musculature. [ ] [ ] [ ] acquired pyloric stenosis can result from neoplasia or duodenal ulceration. [ ] [ ] [ ] [ ] clinical signs depend on the degree of obstruction and include abdominal pain, salivation, and teeth grinding. complete or near complete obstruction can result in gastric reflux and reflux esophagitis. in foals with congenital pyloric hypertrophy, clinical signs may begin with the consumption of solid feed. in foals one can make a presumptive diagnosis via gastric endoscopy and radiography (plain and contrast studies). depending on the cause and severity of disease, gastric endoscopy may provide a presumptive diagnosis in the adult horse. measurement of gastric emptying can aid the diagnosis. several methods of measurement are currently available, including nuclear scintigraphy, acetaminophen absorption, and postconsumption [ c] octanoic acid blood or breath testing. , , exploratory laparotomy shows a distended stomach and thickened pylorus accompanied by a relatively empty intestinal tract. if complete obstruction is not present, medical therapy with a prokinetic such as bethanechol can increase the rate of gastric emptying. phenylbutazone and cisapride also have been shown to attenuate the delay in gastric emptying caused by endotoxin administration. , surgical repair is necessary for definitive treatment of complete or near-complete obstruction and consists of gastroenterostomy or pyloroplasty. , gastric dilation can be classified as primary, secondary, or idiopathic. causes of primary gastric dilation include gastric impaction, grain engorgement, excessive water intake after exercise, aerophagia, and parasitism. , secondary gastric dilation occurs more commonly and can result from primary intestinal ileus or small or large intestinal obstruction. time to development of gastric reflux is proportional to the distance to the intestinal segment involved, with duodenal obstruction resulting in reflux within hours. clinical signs of gastric dilation include those associated with acute colic and in severe cases, ingesta appearing at the nares. associated laboratory abnormalities include hemoconcentration, hypokalemia, and hypochloremia. the most common reported cause of gastric rupture in horses varies between reports. in a retrospective study of horses, gastric rupture occurred most commonly as a secondary phenomenon ( %), usually because of small intestinal obstruction, with primary gastric dilation and idiopathic rupture occurring almost equally ( % and %, respectively). in another retrospective study of horses in combination with a search of the veterinary medical database (vmdb), % of the gastric rupture cases were classified as idiopathic. risk factors for gastric rupture include feeding grass hay, not feeding grain, gelding, and a nonautomatic water source. , nasogastric intubation does not preclude the possibility of gastric rupture, and the amount of reflux obtained before rupture varies greatly. because these reports were retrospective, one cannot rule out confounding factors with certainty. regardless of the initiating cause, gastric rupture usually occurs along the greater curvature. in horses with rupture caused by gastric dilation, tears in the seromuscular layer are frequently larger than the corresponding tears in the mucosal layer, indicating that the seromuscularis likely weakens and tears before the mucosa. , in contrast, horses with gastric rupture following gastric ulceration usually demonstrate full-thickness tears of equal size in all layers. gastric rupture is usually fatal because of widespread contamination of the peritoneal cavity, septic peritonitis, and septic shock. initial clinical signs vary with the primary disease; however, when rupture occurs, a previously painful animal can exhibit signs of relief. subsequent signs are consistent with peritonitis and shock, including tachypnea, tachycardia, sweating, and muscle fasciculations. surgical repair is thus limited but has been reported for partial-thickness tears, and in one case of a combined tear of the mucosa and muscularis with only a focal serosal tear, a full-thickness repair was performed with a favorable outcome. gastric impaction can result in acute or chronic signs of colic in the horse. although a specific cause is not always evident, ingestion of coarse roughage (straw bedding, poor-quality forage), foreign objects (rubber fencing material), and feed that may swell after ingestion or improper mastication (persimmon seeds, mesquite beans, wheat, barley, sugar beet pulp) have been implicated. possible predisposing factors include poor dentition, poor mastication and rapid consumption of feedstuffs, and inadequate water consumption. clinical signs can vary from anorexia and weight loss to those consistent with severe abdominal pain. in severe cases, spontaneous reflux may occur, with gastric contents visible at the nares. in cases of acute severe abdominal pain, one often makes a diagnosis during exploratory celiotomy. in animals not exhibiting signs of colic warranting surgical intervention, an endoscopic finding of a full stomach after a normally adequate fast ( to hours) often can confirm the diagnosis. abdominal radiographs are reserved for smaller horses and ponies. in addition to pain management, specific treatment consists of gastric lavage via nasogastric intubation or massage and injection of fluid to soften the impaction during laparotomy. [ ] [ ] [ ] nonulcerative gastritis rarely occurs in the horse; however, a single case of emphysematous gastritis caused by clostridium perfringens has been reported. mimic that of a strangulating or nonstrangulating small intestinal obstruction, so distinguishing between the two syndromes is important because appropriate treatment of small intestinal obstruction usually requires surgical intervention. studies suggest that the survival rate for horses with dpj that endured surgical exploratory laparotomy was poor compared with those treated medically, although differences in disease severity may have accounted for the results in these early reports. , the clinical syndrome of dpj was well described in the s, and although recognized by its classical presentation, varying degrees of focal intestinal and systemic illness may occur. - dpj usually occurs alone but can occur along with gastritis, ileitis, typhlitis, and or colitis. typical pathologic findings in horses with dpj include involvement of the duodenum and usually the proximal jejunum. the ileum and large colon usually are determined to be grossly normal. gastric distention is a common finding and is thought to be caused by hypersecretory mechanisms in the proximal small intestine and a functional ileus of affected enteric segments. the small intestine may be to cm in diameter because of fluid distention with malodorous, red to brown-red intralumenal fluid accumulation. duodenal (and jejunal) serosal surfaces may have varying degrees and distribution of bright-red to dark-red petechial and ecchymotic hemorrhages and yellow to white streaks. the enteric mucosal surfaces are usually hyperemic and have varying degrees of petechiation and ulceration. microscopically, the most severe lesions have been located in the duodenum and proximal jejunum but may extend proximally to the gastric mucosa and aborally to the large intestinal mucosa and submucosa. microscopic lesions consist of varying degrees of mucosal and submucosal hyperemia and edema. more severe lesions include villus degeneration with necrosis and more severely, sloughing of villous epithelium. the lamina propria, mucosa, and submucosa may have varying degrees of granulocyte infiltration (predominantly neutrophils), and the muscular layers and serosal surfaces contain small hemorrhages. proximal small intestinal serosal fibrinopurulent exudate is a common finding in the more severe cases; therefore the term hemorrhagic fibrinonecrotic duodenitis-proximal jejunitis has been suggested as a more descriptive name for this syndrome. horses with dpj often have evidence of multiple organ involvement such as hepatic changes including congestion and varying degrees of biliary duct hyperplasia. additional systemic involvement likely is caused by endotoxin absorption, metabolic imbalances such as acidemia, and circulatory changes. the cause of this syndrome remains an enigma (much like the cause of other inflammatory conditions affecting the intestinal tract). several microorganisms have been implicated as playing a role in triggering dpj, including clostridium spp., salmonella spp., and some mycotoxins, but efforts to reproduce the syndrome experimentally have been futile. a recent dietary change with an abrupt increase in dietary concentrate level has been suggested to predispose a horse to developing dpj because of intraluminal microbial imbalances. two intracellular processes control intestinal secretion, the cyclic nucleotide (cyclic adenosine monophosphate and cyclic guanosine monophosphate) and the calcium systems. agents (inflammatory mediators, microorganisms, toxic agents) can activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e ) or guanyl cyclase (bacterial enterotoxins) and induce increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate, respectively. this reaction causes phosphorylation of specific protein kinases, which induce the actual mucosal membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotidedependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. calcium may act through calmodulin, which then can activate membrane-phosphorylating protein kinases. the net effect is increased movement of sodium and chloride into the mucosal cell from the interstitium, with secretion of sodium and chloride into the intestinal lumen. water follows the directional flux of sodium and chloride through highly permeable intercellular spaces. several bacterial toxins and endogenous mediators can cause active secretion and contribute to a synergistic mucosal secretory response. passive secretion of protein-rich fluid into the lumen occurs following damage to the mucosal epithelium, capillary endothelium, and submucosal inflammation in the proximal small intestine. the clinically relevant events that result from active and passive fluid secretion are proximal small intestinal distention and nasogastric reflux, dehydration, and circulatory shock. the concentration of protein in the peritoneal fluid from horses with dpj is usually higher than in horses with small intestinal obstruction. a disproportionate increase in total protein concentration relative to nucleated cell count occurs probably by leakage of blood or plasma into the peritoneal cavity without a significant stimulus for leukocyte chemotaxis. suggested mechanisms for increased abdominal fluid protein concentration include serositis associated with inflamed intestine and small intestinal distention causing passive congestion and increased capillary hydrostatic pressure of visceral peritoneal vessels. small intestinal ileus is another hallmark sign of dpj and the pathophysiology is complicated, involving primary and secondary dysfunction of the central, autonomic, and enteric nervous systems and their purported roles in governing intestinal motility. primary role-players in dpj-associated ileus include peritoneal inflammation, inflammatory cell migration/activation within the muscularis, small intestinal mechanical distention, and effects of endotoxin absorption. the use of prokinetic agents for treating ileus and gastric/small intestinal distention in horses with dpj is becoming more common, but veterinarians should realize that a potential restriction on their use is the need for normal intestinal integrity. in spite of that, one may use motility modifiers judiciously. the veterinarian has the challenge of differentiating horses with dpj from horses with small intestinal obstructive lesions so as to avoid surgical intervention (table . - ). horses with dpj typically show signs of acute abdominal pain initially, and then after gastric decompression, volume replacement, and analgesic therapy, the colic signs subside, but signs of lethargy and malaise become more apparent. in contrast, horses with obstructive lesions of the small intestine usually show signs of abdominal pain until the affected viscus is repaired via surgical intervention or the viscus ruptures. another differentiating characteristic is the large volume (> to l with each decompressive effort) of nasogastric reflux that is often malodorous and orange-brown or red-brown. dpj-affected horses have moderate to severe small intestinal distention palpated on rectal examination, temperature of . °to . °c ( . °to . °f), dehydration, brick-red mucous membranes, lethargy and absent borborygmi, prolonged capillary refill time, tachycardia (> beats/min), and tachypnea. although the signs of abdominal pain usually resolve after gastric decompression, most horses remain severely lethargic. without periodic removal of the fluid that accumulates in the proximal intestinal tract, signs of abdominal pain usually recur. horses with dpj often require gastric decompression at -hour intervals, with to l of fluid recovered each time. nasogastric tubes left in place for long periods of time cause varying degrees of pharyngitis, laryngitis, and esophagitis. typical clinical laboratory findings include an increased packed cell volume and total plasma protein reflective of volume depletion, a metabolic acidosis (with elevated anion gap) in longstanding or severe cases, an increased peritoneal fluid protein concentration (often > . g/dl), and a mild to moderate elevation of the peritoneal white blood cell count, although the count usually is less than , cells per microliter. , the peritoneal fluid is usually yellow and turbid, but in severe cases diapedesis occurs resulting in a serosanguinous color. the white blood cell count in the peripheral blood may be normal, decreased, or increased. in addition, hyponatremia, hypochloremia, hypokalemia, and acid-base alterations (elevated anion gap) are often evident. the loss of enteric bicarbonate through evacuation of enterogastric reflux and poor tissue perfusion from hypovolemia can lead to metabolic acidosis. one makes a definitive diagnosis of dpj in most cases by gross examination of the duodenum and proximal jejunum at surgery or at necropsy. some equine practitioners have observed an apparent geographic relationship in the incidence and severity of the syndrome, with more cases occurring in the southeastern united states. horses with dpj appear to share a common characteristic clinical presentation, and the mechanisms leading to electrolyte imbalances, fluid loss, ileus, and endotoxemia and septicemia are similar. treatment regimens are supportive and aim at plasma volume replacement (usually in the form of crystalloid fluid replacement), analgesia and antiinflammatory therapy, gastric decompression, antiendotoxin therapy, antimicrobial therapy if indicated, nutritional support, and nursing care. one should institute aggressive intravenous polyionic fluid therapy immediately in a horse with dpj. one should calculate the total fluid deficit based on clinical assessment of dehydration (e.g., for % or moderate dehydration, . × kg body mass = l) and should administer replacement fluids rapidly (up to to l per hour per -kg adult horse). administering intravenous hypertonic saline ( %) may be useful to treat hypovolemic shock in horses with severe circulatory shock. the use of to l of hypertonic saline ( % nacl) improved systemic blood pressure and cardiac output in horses with hemorrhagic shock and in a model of equine endotoxemia. if one chooses this treatment option, intravenous administration of replacement isotonic fluids must follow immediately to maintain tissue integrity. one should not allow horses with significant volumes of gastric reflux to ingest foodstuffs or liquids orally. once one has administered replacement fluids and the horse is well hydrated, one should administer maintenance fluid amounts, which may be as high as ml/kg/day. unfortunately, the intravenous fluid therapy itself may accelerate the flux of fluid from the vasculature into the intestinal lumen because of a reduction in intravascular oncotic pressure and an increased capillary perfusion pressure, which can result in an increased volume of gastrointestinal reflux. however, the veterinarian should not consider reducing the volume of intravenous fluid therapy because excessive fluid losses continue to occur. one should monitor plasma protein concentration, overall hydration, and the volume of reflux and then determine the rate of intravenous fluid administration. during the initial hours of therapy, even aggressive intravenous fluid administration results in only moderate clinical improvement. the clinical response, as evidenced by improved hydration status, decreased nasogastric reflux, improved attitude, and improvement in values reflecting kidney function (decreased blood urea nitrogen and creatinine), correlates with improvement of intestinal damage. horses with dpj that continue to reflux large volumes of enterogastric fluid frequently for more than to hours most likely will experience protein loss from the inflamed and disrupted intestinal mucosal barrier and from systemic protein catabolism. decreased colloid oncotic pressure leads to decreased effective circulating fluid volume and edema. total plasma protein may decline to below g/dl and the albumin may decrease to below . g/dl. fresh or thawed frozen plasma is ideal for replacement of functional proteins. one should consider treatment with intravenous plasma therapy or a combination of plasma and synthetic colloid (e.g., synthetic amylopectin) as soon as one sees evidence of a consistent decline in total plasma protein or albumin (< . g/dl) or if the horse is developing dependent edema. fresh plasma (preferred) or fresh frozen plasma is the treatment of choice if coagulation disorders accompany protein loss. an average-size horse ( kg) requires to l of plasma (albumin . g/dl) or synthetic colloid to improve plasma oncotic pressure. administration of additional aliquots of to l of a balanced colloidal solution may be necessary if the dpj crisis continues. in addition to albumin (the major colloid component), plasma contains other components that provide overall systemic support (e.g., fibronectins, complement inhibitors, elastase and proteinase inhibitors, antithrombin iii). one may administer a % solution of hydroxyethyl starch (hetastarch ( %), abbott laboratories, north chicago, illinois), a synthetic colloid, at to ml/kg. because of the large size of the starch molecules, this solution is an effective plasma volume expander, resulting in sustained dosedependent decreases in packed cell volume and plasma protein concentration with increased oncotic pressure. the cost of an appropriate amount of commercial plasma or synthetic colloid solution for treatment of adult horses with dpj may be prohibitive but can be life-saving. horses with enteritis frequently absorb large amounts of endotoxin from the disrupted intestinal mucosal barrier, therefore putting these horses at a high risk for laminitis. one should monitor digital pulses every to hours until systemic signs of enteritis have abated (fever, leukopenia, etc.). treatment to combat endotoxemia is critical, and several therapeutic approaches are available. choice of treatment options is based on severity of disease, renal function, hydration status, and economics. the reader is referred to chapter . for a thorough discussion of endotoxemia pathophysiology, treatment, and prevention. nonsteroidal antiinflammatory drugs are the most frequently used group of drugs for treatment of abdominal pain in horses (flunixin meglumine . mg/kg intravenously every hours or phenylbutazone . mg/kg orally or intravenously every hours). the clinician must weigh the benefit of the analgesic effect of nonsteroidal antiinflammatory drugs with the possibility of further damage to the intestine by potentially blocking the protective effects of intestinal mucosal prostaglandins. one should consider other classes of drugs for treating colic associated with dpj. butorphanol (torbugesic; an opioid analgesic) at . to . mg/kg with detomidine (dormosedan; an α-agonist) at . to . mg/kg given intramuscularly every to hours is a useful combination that has minimal effects on gastrointestinal motility. because clostridium spp. are suspected as a causative agent of dpj, penicillin often is administered to affected horses. however, one should consider broad-spectrum antimicrobial coverage for horses with dpj. one can add an aminoglycoside (gentamicin, amikacin) or thirdgeneration cephalosporin (ceftiofur [naxcel], upjohn co., kalamazoo, michigan) to the penicillin therapy, keeping in mind the potential adverse effects of these drugs on renal function. effective antisecretory medications targeting the equine small intestine have not been identified. one should consider the nutritional needs of horses with dpj. most horses have a total body protein loss from cachexia and a protein-losing enteropathy. total parenteral nutrition may be indicated in horses that remain anorectic for more than to days. parenterally administered solutions containing glucose, balanced amino acid solutions, lipid emulsions, balanced electrolyte and trace minerals, and vitamins have been administered to adult horses with small intestinal ileus or enterocolitis. based on a small number of horses, this therapy has proved promising in terms of minimizing protein losses and decreasing the duration of illness. providing for part of the nutritional requirements of the horse ( to , kcal/day) is possible with glucose-amino acid solutions, which are of moderate cost. one may suppose reasonably that providing nutritional support to an anorectic, severely ill horse will facilitate the healing process and even shorten the duration of illness. thus the overall cost of providing parenteral nutritional supplementation to horses with dpj may well be offset by quicker recovery and diminished requirements for other, expensive treatments. normal (healthy) intestine is necessary for optimum performance of prokinetic agents in horses. many motilitymodifying agents likely are ineffective in cases of dpj. however, some benefit may come of the judicious use of prokinetic agents in inflammatory conditions of the equine intestine, particularly if the agent provides additional effects such as analgesia. for example, lidocaine infusion has several actions that may be beneficial in the treatment of ileus, including suppression of primary afferent firing, antiinflammatory properties, an observed analgesic effect, and direct stimulation of smooth muscle. an infusion dose of to mg/min over to hours has been recommended. the reader is referred to chapters . and . for a complete description of motility modifying agents. medical therapy is sufficient in most cases of dpj, but in those cases in which the horse continues to produce copious enterogastric reflux, one may consider surgery as an option. refractory cases have been observed to improve with surgical intervention; however, some horses with refractory dpj have been observed to recover with supportive medical care alone even after days of refluxing large amounts of fluid every to hours (personal observation). the decision of when to intervene surgically often is difficult. one may elect surgery to determine the extent of gross pathologic condition and intestinal distention and to perform intestinal bypass so as to direct enterogastric reflux toward the cecum and colon, where the fluid can be reabsorbed. allen and clark have described two approaches for surgical therapy in such cases. a standing right flank laparotomy with resection of the last rib has been used to approach the duodenum and cecal base. using this approach, one makes a small stoma between the duodenum and cecum using a handsewn . -to . -cm side-to-side anastomosis. the stoma may act as a shunt to decompress the proximal small intestine and deliver the small intestinal fluid to the cecum for reabsorption. following recovery, the stoma likely will close. when a veterinarian is confronted with a horse exhibiting abdominal discomfort, with small intestinal distention palpable per rectum, and greater than l of gastric reflux, the veterinarian should recommend referral of the horse to a facility capable of performing abdominal surgery. the chance that such a horse has an intestinal obstruction is too great to decide to treat it as if it may have dpj. surgery on such horses is not unusual, even though dpj is possible, to rule out an obstruction. at present, the survival of horses with dpj that undergo surgery is much greater than previously described, and certainly greater than that of horses with small intestinal obstruction that do not have surgery. horses with dpj that receive appropriate therapy have a reasonably good chance of making a full recovery. horses that continue to have frequent episodes of voluminous nasogastric reflux and systemic signs of endotoxemia and septicemia have a poorer prognosis for recovery. frequent complications of dpj include laminitis, thrombophlebitis, and weight loss. the clinical signs of chronic wasting and poor body condition, although nonspecific for a diagnosis of malabsorption antemortem, can be attributed to proliferative or inflammatory intestinal disorders, often collectively referred to as chronic inflammatory bowel diseases. clinical signs include alimentary lymphosarcoma, granulomatous enteritis, multisystemic eosinophilic epitheliotropic disease (meed), and lymphocyticplasmacytic enterocolitis-conditions affecting young and adult horses. proliferative enterocolitis, a transmissible disease of foals to months of age characterized by significant small intestinal pathologic changes, will be included in this group. however, several other primarily small intestinal conditions described from a morphologic perspective, such as chronic postinfarctive inflammation and mycobacterial infections, will not be discussed. in addition, a single case of aa amyloid-associated gastroenteropathy in an -year-old morgan stallion that had evidence of severe malabsorption based on poor d-xylose absorption is included. for comparative purposes, table . - lists the clinical and clinicopathologic features of the diseases, and tables . - and . - present the gross morphologic and histopathologic findings, respectively. the extent of small intestinal disease is the key to determine whether one can demonstrate malabsorption based on abnormal carbohydrate absorption. as described in chapter . , this is not an all-or-nothing situation. in the same animal the staging of the pathologic changes differs in different regions of the small and large intestines, thus influencing severity of clinical signs and absorption findings. furthermore, the extent of pathologic changes in different animals with ultimately the same morphologic diagnosis affects absorption studies and progress of the disease. early diagnosis remains a challenge, and even multiple intestinal biopsies taken at exploratory laparotomy may prove unhelpful. by contrast, intestinal infiltration with the predominant cell types can be found in grossly normal appearing intestinal tissue. alimentary lymphosarcoma of the horse may represent a primary neoplasia of the gut associated lymphoid tissue with significant cellular infiltration of the small intestine and associated lymph nodes with minimal large intestinal or systemic involvement. case series and pathology reports indicate that young horses to years of age primarily are affected, although the age range can be broad. [ ] [ ] [ ] no breed or sex predilection exists. prevalence is unknown. despite the progressive nature of lymphomata, onset of clinical signs can be rapid and the animal may become acutely ill. as with all adult cases of chronic inflammatory bowel disease, antemortem diagnosis is by a process of exclusion and usually is confirmed post mortem. frequently, the horse has anemia, thrombocytopenia, neutrophilia or neutropenia, hypoalbuminemia, normal serum protein or hyperproteinemia, and hypergammaglobulinemia. lymphocytosis is rare. one may palpate intraabdominal masses, mainly enlarged mesenteric lymph nodes, rectally. abdominocentesis has been of diagnostic value. carbohydrate absorption tests usually reveal partial to total malabsorption indicative of the severely reduced surface area resulting from significant villous atrophy and the extensive mucosal or transmural infiltration. rectal biopsy has aided diagnosis. early confirmation of a suspected diagnosis necessitates exploratory laparotomy to obtain multiple intestinal and lymph node biopsies. in the future, markers of cancer cells may become available and may be cost-effective to aid diagnosis. prognosis is poor. natural progress of the disease is unknown. most horses are presented in an advanced state of disease. immunosuppressive drugs or chemotherapy may afford temporary improvement. however, outcome is unaffected. the chronic wasting condition granulomatous enteritis was first described in ; of horses were young standardbreds. most affected horses are to years of age. case reports from many countries revealed a predominance of standardbred over thoroughbred horses by three to one. , some of the standardbreds were related, implicating a genetic predisposition. prevalence is low. the condition is sporadic and has an insidious onset, and the course can be protracted. significant diagnostic features include anemia, slight increases or decreases in white blood cell counts, hypoalbuminemia, normal serum protein or hypoproteinemia, occasional increases in serum alkaline phosphatase activity, normal serum γ-glutamyltransferase activity, and enlarged mesenteric lymph nodes on rectal palpation. reduced carbohydrate absorption to the level of partial to total malabsorption is reported frequently, consistent with the severe morphologic changes throughout the small intestine. one can attribute the low proportion of horses exhibiting diarrhea , to the preferential distribution of inflammatory infiltration in the small intestine, with lesser involvement of the large intestine. rectal biopsy can be a useful aid to diagnosis. treatment of horses with granulomatous enteritis with a variety of drugs, particularly corticosteroids, has not affected the outcome except in the short term. one successful response has been reported. prolonged corticosteroid administration produced clinical remission in a -year-old standardbred gelding based on improvement in clinical signs and in d-xylose absorption. five months after cessation of approximately months therapy, d-xylose absorption was normal and the horse was bright, alert, and resumed a level of athletic performance. parenteral administration of dexamethasone sodium phosphate was tapered to achieve a minimal effective dose to reduce intestinal inflammation and abolish clinical signs. adverse effects were not reported. the outcome of this single case is encouraging. surgery may be indicated if the disease is localized. two young horses underwent resection of the thickened terminal small intestine to confirm a diagnosis and provide a means of treatment; one horse died months after surgery, and the other has remained clinically normal for at least years. the cause of granulomatous enteritis is unknown. several infectious agents have been implicated, including mycobacterium avium. the condition may represent a granulomatous hypersensitivity reaction. immunemediated responses to dietary, parasitic, or bacterial antigens may be important initiating factors. recently, six cases purported to represent granulomatous enteritis were linked to environmental contamination with aluminum. although the case definition was flawed and problems existed with the data and interpretation, the report nevertheless raised the possibility that a toxicologic basis may exist for some equine inflammatory bowel disorders. lumen from parasitic, bacterial, or dietary sources. infectious agents have not been identified. , widespread use of the avermectins has tended to reduce parasite loads and composition to favor small strongyles (cyathostomes). eosinophilia is a feature of parasitism in the equine intestinal tract, although nematodes rarely have been identified in any lesions of meed. , however, failure to detect larval structures in these lesions may be attributable to chronicity of the disease and destruction of the parasites in tissue. biopsies of the rectal mucosa or of the skin, liver, intestinal tract, and lymph nodes may assist in diagnosis. treatment has been attempted with a variety of drugs, including antibiotics, corticosteroids, and anthelmintics with larvicidal activity. immediate improvement has not been borne out in the long term. prognosis is poor. the clinical objective is to reach a tentative diagnosis early in the course of the disease for intervention to be more than transient. unlike the other conditions (see table . - ), meed has definitive liver and pancreatic involvement, and thus maldigestion may make a significant contribution to the wasting disease. for example, the lowered albumin and protein could result in part from impaired pancreatic enzyme digestion, and the effects of inflammatory lesions in the liver and ileum may decrease bile salt concentrations. the morphologic findings in lymphocytic-plasmacytic enterocolitis reflect the predominant infiltrative cellular elements of this rarely encountered condition. a retrospective study of horses provided the information presented in the tables. no specific clinical or clinicopathologic features differentiate this condition antemortem from other inflammatory diseases of adult horses. carbohydrate absorption was abnormal or delayed in of horses, consistent with the predominance of small intestinal pathologic changes. rectal biopsies were abnormal in of horses, two of which were reported as having lymphocytic-plasmacytic proctitis. prognosis is poor. treatment has been unsuccessful, probably because of the advanced nature of the condition at the beginning of treatment. proliferative enteropathy has not been associated with abnormal carbohydrate absorption based on three horses subjected to carbohydrate absorption tests. however, the florid mucosal lesions in the jejunum and ileum undoubtedly contribute to impaired digestive function and potential malabsorption of vitamins, minerals, and amino acids in the distal small intestine. the condition meed encompasses disorders characterized by a predominant eosinophilic infiltrate in the gastrointestinal tract, associated lymph nodes, liver, pancreas, skin, and other structures and accompanied by some degree of malabsorption and enteric protein loss. the disorders include chronic eosinophilic gastroenteritis, eosinophilic granulomatosis, chronic eosinophilic dermatitis, and probably basophilic enterocolitis. the condition differs from idiopathic eosinophilic enterocolitis, in which segmental lesions in the small or large intestine induce signs of colic requiring surgical intervention , without evidence of malabsorption or multisystem involvement. although prevalence is low, meed appears to be more common than granulomatous enteritis based on the accumulated published reports and personal experience in australia and the united states. most affected horses are to years of age, and standardbreds and thoroughbred are reported to predominate. the condition is sporadic and has an insidious onset, and the course is protracted with a duration of to months. diarrhea is common in contrast to granulomatous enteritis. severe skin lesions with exudative dermatitis and ulcerative coronitis are prominent and frequently are the principal reason for veterinary attention being sought. despite extensive tissue eosinophilia, systemic eosinophilia is rare. hematologic values are usually unremarkable. notable features include hypoalbuminemia and normal serum protein or hypoproteinemia, and because of liver involvement, serum γ-glutamyltransferase and alkaline phosphatase activities may be increased. most reports of carbohydrate absorption test findings (glucose or d-xylose) indicate retarded absorption and a reduced or normal peak concentration delayed to at least minutes. one can interpret this pattern as the existence of sufficient small intestinal absorptive capacity to enable moderate absorption with possibly delayed gastric emptying or ileocecal ejection. morphologic changes are less pronounced in the small intestine than in the large intestine, and small intestinal lesions predominate segmentally in the proximal duodenum and distal ileum. furthermore, significant hyperkeratosis of the fundic region may contribute to gastric muscle contractile disruption. diarrhea can be a consequence of the severe segmental or multifocal granulomatous lesions in the large intestine with mucosal and transmural thickening and extensive ulceration. abundant fibrosis is a feature of all affected tissues (see table . - ). the cause of meed is unknown and could represent a chronic ongoing immediate hypersensitivity reaction against undefined antigens ingested or excreted into the affects foals to months of age, particularly those that have been weaned recently. the disease is caused by lawsonia intracellulare, an obligate intracellular bacterium found in the cytoplasm of proliferative crypt epithelial cells of the intestine. the condition in a foal was described first as intestinal adenomatosis, because of similarity to the swine disorder of the same name. later, molecular studies showed that intestines from an affected foal contained l. intracellulare sequences as determined by polymerase chain reaction analysis and confirmed by southern blot hybridization. recently, studies of a cluster of affected foals on three breeding farms in canada provided much information on the clinical syndrome, laboratory investigations, and response to treatment. two of the three farms bred arabians, hence a demographic predominance of arabian foals exists. clinical signs included depression, rapid and significant weight loss, edema, diarrhea, and colic. poor body condition, a rough hair coat, and potbelly appearance were common findings. other problems often were concurrent, including respiratory tract infection, dermatitis, intestinal parasitism, and gastric ulceration. significant laboratory findings were anemia, transient leucocytosis, hypoalbuminemia, hypoproteinemia, and elevated serum creatine kinase concentrations. diagnosis was confirmed by identifying characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells using silver stains and by results of polymerase chain reaction analysis and immunohistochemical testing. antemortem diagnosis relied on clinical signs, hypoproteinemia, and exclusion of common enteric infections. one can confirm diagnosis in live animals by fecal polymerase chain reaction analysis (positive in of foals tested) and serologic testing; foals with proliferative enteropathy were evaluated serologically and had antibodies against lawsonia intracellulare. treatment is effective. most foals received erythromycin estolate ( to mg/kg per os every to hours), alone or with rifampin ( to mg/kg per os every hours) for to weeks. foals frequently needed supportive therapy at the outset for stabilization. response to therapy has been excellent. rapid improvement in clinical signs even within hours preceded the rise in plasma protein concentration. the source of the infection was undetermined. no apparent link existed between the three farms and a swine operation or solid and liquid waste disposal on pasture. however, one cannot exclude airborne spread of dried fecal material over distances. comparisons of epidemiologic findings from the swine disease indicated that overcrowding, feed changes, antibiotic usage, and mixing and transportation were potential risk factors at two of the farms. recent weaning appeared to be a key element in the pathogenesis. samuel l. jones acute diarrhea caused by colitis in adult or young horses is a potentially life-threatening disorder of a variety of causes (table . - ) characterized by hypersecretion of fluid, motility disturbances, altered microbial flora in the colon, and an impaired mucosal barrier caused by direct injury or inflammation. many of the clinical and clinicopathologic features are similar regardless of the cause. severe dehydration with profound electrolyte abnormalities is common, as is systemic inflammation from absorption of endotoxin or other bacterial products through the compromised mucosa. gastrointestinal protein loss may result in reduced colloid oncotic pressure from hypoproteinemia, leading to tissue edema. colitis is a highly catabolic disorder, and weight loss may be rapid and severe. some cases of colitis may be complicated by extensive mucosal ulceration, serosal inflammation, or mural ischemia/infarction extending from the inflammation or resulting from coagulopathies. thus diagnostic measures aimed at determining the cause necessarily must be accompanied by clinical and laboratory assessment of hydration, electrolyte and acid-base balance, plasma protein concentration and colloid oncotic pressure, organ function, and evaluation of the degree of systemic inflammation and of the integrity of the intestinal wall. although therapeutic strategies are similar for many causes of colitis, consisting primarily of control of local and systemic inflammation, maintenance of fluid and electrolyte balance, promotion of tissue perfusion, replacement of plasma protein, preservation of colloid oncotic pressure, promotion of mucosal repair, restoration of the microbial ecology of the colon, and nutritional management, some causes of acute colitis have specific therapies aimed at eliminating the cause. a variety of infectious organisms has been identified as causes of acute colitis in adult horses. the clinical syndromes associated with these infections are indistinguishable in most horses. however, appropriate diagnostic tests including fecal bacterial culture, fecal bacterial toxin analysis, pcr, and/or serology may identify specific infectious organisms. salmonella is a genus of gram-negative facultatively anaerobic bacteria that are common gastrointestinal pathogens in horses. many serotypes of salmonella have been reported to infect horses, but those classified in group b appear to be associated more commonly with disease than those in other groups. group b includes s. typhimurium and s. agona, two of the species most frequently isolated from horses. - s. typhimurium is the most pathogenic serotype in horses and is associated with a higher case fatality rate than other species of salmonella. the number of horses that are infected inapparently with and actively shed salmonella in their feces has been reported to be as high as % to %, but actual prevalence of salmonella-shedding in the general horse population is likely to be much lower, less than % to %. horses shedding salmonellae are a potential source of infection to susceptible horses, , as are environmental reservoirs. [ ] [ ] [ ] for these reasons, salmonellosis is one of the most common nosocomial diseases in horses. nosocomial salmonellosis significantly affects morbidity and mortality in hospitalized horses. the emergence of multidrug resistance in equine salmonella isolates has been a cause of concern because of the importance of salmonellosis as a nosocomial disease and because salmonella represents a significant zoonotic pathogen. , [ ] [ ] [ ] the virulence of the bacteria varies tremendously with serotype and even among strains of the same serotype in part because of the important role of host susceptibility in the pathogenicity of particular organisms. the infective dose is generally millions of organisms inoculated orally, but various environmental and host factors can reduce the infective dose to a few thousand or even hundreds of organisms. [ ] [ ] [ ] environmental factors or stresses that increase susceptibility to salmonella infection are not well defined, but high ambient temperature, for example, is known to increase the prevalence of salmonellosis in horses greatly. indeed, the peak incidence of salmonellosis in horses occurs in late summer and fall. , , other environmental and host factors that increase the risk of salmonella infection include transportation, antibiotic administration, gastrointestinal surgery, general anesthesia, preexisting gastrointestinal disease, change in diet, and immunosuppression. , , host factors that restrict gastrointestinal colonization and invasion by pathogens include gastric ph, commensal gastrointestinal flora, gastrointestinal motility, the mucosal barrier and mucosal immunity. , gastric acidity is an important defense mechanism preventing live organisms from reaching the intestine. altering the gastric ph with histamine receptor antagonists, for example, may increase susceptibility to infection. gastrointestinal flora inhibits the proliferation and colonization of salmonella by secreting bacteriocins, short-chain fatty acids, and other substances that are toxic to salmonella. in addition, elements of the normal flora compete for nutrients and space, especially on the mucosa. being predominantly anaerobic, the normal flora maintain a low oxidationreduction potential in the environment of the large intestine, which inhibits the growth of many bacterial pathogens. the importance of normal host gastrointestinal ecology is illustrated by the fact that disturbances of the colonic flora with antibiotics, changes in feed, ileus, or other underlying gastrointestinal disease greatly increase the susceptibility of the host to infection by salmonella, often resulting in serious disease. the immune status of the host may be one of the most important factors determining not only the susceptibility to salmonella infections but also the degree of invasion and subsequent outcome of the infection. local immunity, such as mucosal antibody secretion and enterocyte-derived cationic peptides, prevents colonization of the mucosa. , , opsonizing antibodies and activation of the complement cascade are important in fighting systemic invasion by salmonella by increasing the efficiency of phagocytosis and by direct bactericidal activity. humoral immunity, however, is often ineffective in preventing disease and dissemination once invasion occurs and salmonella has established in its intracellular niche. following invasion, salmonella is capable of surviving and multiplying within macrophages, rendering humoral (noncellular) immune systems ineffective. , specific cellular immunity may be the most effective defense mechanism in the host arsenal against dissemination and systemic infection by salmonella. , oral inoculation with small numbers of virulent organisms may induce protective immunity in horses and calves, but the duration of the immunity is not known. , oral and parenteral vaccines using killed or attenuated organisms and bacterial products have been promising but are effective only against homologous organisms and are usually not cross-protective among different serogroups. [ ] [ ] [ ] in adult horses, salmonella primarily infects the cecum and proximal colon, causing enterocolitis, and the ability to disseminate beyond the intestine and cause enteric fever is limited. in foals, however, salmonellosis often is associated with septicemia. the ability of salmonella to cause enterocolitis depends on the ability of the bacteria to invade the gastrointestinal mucosa. , invasion of the gastrointestinal mucosa occurs preferentially through specialized enterocytes called m cells that overlay intestinal lymphoid tissues such as peyer's patches in nonequine species. a variety of enteric pathogens exploit m cells during infection of intestinal tissue. invasion of the epithelium occurs by self-induced uptake via the apical membrane of the m cell, often killing the cell in the process. salmonellae then invade neighboring cells via the basolateral membrane, eventually spreading the destruction of the epithelium beyond the principle area of attack. virulent salmonellae have a well-developed invasion mechanism involving generation of an apparatus called a type iii secretory system that enables virulence gene products to be injected directly into enterocytes. virulence proteins injected by salmonellae into enterocytes engage the cellular machinery and induce the cell to engulf the bacteria by macropinocytosis. salmonella virulence gene products also induce enterocyte chloride and fluid secretion and upregulate enterocyte transcription of inflammatory cytokines (tumor necrosis factor α and interleukin- β) and chemokines that trigger a mucosal inflammatory response. , , after salmonellae invade the mucosa, they are phagocytosed quickly by macrophages and dendritic cells in the lamina propria and lymphoid tissues. the ability of salmonellae to disseminate systemically and cause enteric fever is associated with the ability to survive and proliferate in macrophages. indeed, phagocytes have an important role in dissemination of the pathogen to blood, lymph nodes, liver, and spleen. most salmonellae in the blood and tissues of infected animals that are competent to cause enteric fever are within phagocytic cells. in adult horses with salmonellosis, dissemination appears to be limited to the intestine and mesenteric lymph nodes, and salmonella rarely is cultured from blood. however, in foals and in some adults, salmonella causes an enteric feverlike disease with dissemination to mesenteric lymph nodes, liver, spleen, and blood. salmonella organisms require specific virulence gene clusters encoded on the chromosome or on plasmids for intracellular survival in macrophages. some of these genes are sensors that signal the bacteria that it has entered an intracellular environment and turn on genes required for intracellular survival. others, like invasion genes, are transported from the bacteria and injected into macrophage cytosol by a type iii secretory system to prevent phagosome/lysosome fusion and subvert other essential macrophage killing mechanisms. salmonellae also possess multiple genes that confer resistance to reactive oxygen and nitrogen metabolites, perhaps the most lethal antimicrobial mechanisms of macrophages. diarrhea associated with salmonellosis has multiple causes. a salmonella cytotoxin inhibits protein synthesis in mucosal cells, causing morphologic damage and altered permeability. virulent salmonellae also produce an enterotoxin similar to the heat-labile toxin (lt) produced by escherichia coli. , the enterotoxin contributes to but is not required in the pathogenesis of diarrhea. , salmonella enterotoxin increases secretion of chloride and water by colonic mucosal cells in many species, including horses, by increasing intracellular cyclic adenosine monophosphate concentrations. the ability of virulent salmonellae to cause diarrhea appears to be associated most closely with the ability to invade enterocytes and to trigger an inflammatory reaction in the intestinal tissue. , gene products injected into enterocyte cytosol by the type iii secretory system of invading salmonellae stimulate chloride and fluid secretion. salmonella invasion of enterocytes is also a potent activator of inflammatory chemokine and cytokine production, resulting in the recruitment of leukocytes, particularly neutrophils, and activation of resident macrophages and mast cells. products of these activated leukocytes, including prostaglandins, leukotrienes, reactive oxygen metabolites, and histamine, are potent stimulators of chloride secretion in the colon of many species. , [ ] [ ] [ ] the enteric nervous system integrates the diverse processes of pathogen recognition, triggering of the inflammatory response, and induction of enterocyte fluid secretion. many of the inflammatory mediators studied stimulate colonic secretion by prostaglandin-dependent mechanisms, resulting in increased intracellular cyclic adenosine monophosphate or calcium concentrations or both in mucosal cells. in addition, these mediators and the enteric nervous system may stimulate secretion by prostaglandin-independent mechanisms, inhibit sodium and water absorption, cause motility disturbances, and potentiate tissue injury, all of which enhance the pathogenicity and dissemination of salmonella and contribute to the pathogenesis of diarrhea. , neutrophils recruited to the mucosa by signals generated by the infected enterocytes physically contribute to mucosal injury by producing a variety of products that are lethal to pathogens but are also toxic to host cells. , moreover, neutrophils attracted to infected epithelial cells accumulate beneath the monolayer, lifting it off the basement membrane in sheets. neutrophils also migrate across the epithelial monolayer in potentially massive numbers. transepithelial migration of neutrophils increases the permeability to macromolecules, bacterial products, and even bacteria. potentially massive losses of electrolytes, water, and protein can occur depending on bacterial and host factors. perhaps most devastatingly, mucosal injury and altered permeability allow systemic absorption of bacterial products and dissemination of bacteria, resulting in systemic inflammatory responses such as occur with endotoxemia and septicemia. four syndromes of salmonella infection have been described clinically and reproduced experimentally in horses: ( ) inapparent infections with latent or active carrier states; ( ) depression, fever, anorexia, and neutropenia without diarrhea or colic; ( ) fulminant or peracute enterocolitis with diarrhea; and ( ) septicemia (enteric fever) with or without diarrhea. inapparent infections can be activated to clinical disease in compromised horses, such as horses with colic or horses being treated with antibiotics, causing mild to severe enterocolitis. in addition, latent infections (nonshedding) can become active infections (shedding) under certain conditions, such as transportation stress and antibiotic treatment. horses with depression, anorexia, fever, and neutropenia without diarrhea generally have a good prognosis and recover in several days without specific treatment. the septicemic form is restricted mostly to neonatal foals and is uncommon in adult horses. this discussion focuses on acute enterocolitis. acute enterocolitis is characterized by severe fibrinonecrotic typhlocolitis, with interstitial edema and variable degrees of intramural vascular thrombosis that may progress to infarction. severe ulceration of the large intestinal mucosa may occur with serosal ecchymoses and congestion. the earliest signs of enterocolitis are usually fever and anorexia. , signs of colic may be apparent early in the course of the disease, especially if ileus is present. clinical signs of endotoxemia are common and range from fever, elevated heart and respiratory rates, poor peripheral perfusion, and ileus to fulminant and rapidly progressive signs of endotoxemic shock. oral mucous membranes are often pale with perigingival hyperemia (a toxic rim) but may be brick red or cyanotic, with prolonged capillary refill time. one may note weakness, muscle fasciculations, cold extremities, and other signs suggestive of hypotensive shock; synchronous diaphragmatic flutter; abdominal pain; and significant metabolic and electrolyte abnormalities in severe cases of enterocolitis. one also may note signs of mild dehydration before diarrhea is apparent. once diarrhea is evident, dehydration may become severe rapidly. occasionally, horses die peracutely, without developing diarrhea. diarrhea may not be apparent for several days but usually occurs by to hours after the fever begins. , the duration of the diarrhea may be days to weeks. the character of the first diarrheal feces is usually watery with particles of roughage but may become fluid rapidly without solid material. finding frank blood and fibrin in the feces is unusual. the volume of feces is often large, with frequent defecation. one may note straining or signs of colic when the patient is defecating, and rectal prolapse may occur occasionally. persistent straining and rectal prolapse may be a sign of colonic infarction. abdominal borborygmi are often absent early in the course of the disease because of ileus but become evident later, usually when diarrhea begins. fluid and gas sounds are commonly audible, but normal progressive motility is less frequently audible than normally. transrectal palpation may reveal edematous rectal mucosa and colon and fluid-filled colon and cecum. one may obtain gastric reflux, especially early in the course when ileus is evident. hematologic abnormalities early in the course of the disease include moderate to severe neutropenia, lymphopenia, and leukopenia, a mild to moderate left shift, and toxic changes in the neutrophils. , thrombocytopenia, moderate to severe hemoconcentration, and hyperfibrinogenemia are also common. neutropenia is an early but nonspecific indicator of salmonellosis, often occurring concurrently with the onset of fever. later in the course of disease, one may see neutrophilic leukocytosis, indicating recovery. a degenerative left shift, with metamyelocytes and myelocytes in the peripheral blood, is a poor prognostic sign. serum biochemical analysis may reveal azotemia, elevations in serum sorbitol dehydrogenase and γglutamine aminotransferase activity, and elevated serum lactic acid concentration. azotemia is often prerenal, but acute hemodynamic renal failure may occur in severely dehydrated, endotoxemic, or septicemic patients. indeed, elevation of creatinine concentration is a poor prognostic indicator in horses with acute colitis. hemodynamic renal disease may be complicated by toxic injury caused by administration of nephrotoxic drugs. hyponatremia may also contribute to prerenal azotemia. elevations in hepatocellular enzymes are usually mild and reflect damage to the hepatocytes from absorbed toxins such as endotoxin and from poor perfusion caused by hypotensive shock or dehydration. lactic acidemia may be present, reflecting poor tissue perfusion. plasma protein rapidly drops as protein is lost in the gastrointestinal tract, causing moderate to severe hypoalbuminemia and hypoglobulinemia. peripheral or organ edema (vascular leak syndrome) may occur if hypoproteinemia is severe, coupled with increases in endothelial permeability induced by systemic inflammation. hypokalemia, hyponatremia, hypochloridemia, and hypocalcemia are common electrolyte abnormalities in patients with enterocolitis. metabolic acidosis also may be present. coagulopathies, such as decreased antithrombin iii activity and disseminated intravascular coagulation, may occur. urinalysis may reveal isosthenuria, proteinuria, hematuria, cylindruria, and glucosuria if hemodynamic or toxic renal injury is present. the number of leukocytes in the feces usually is elevated, and occult blood may be detectable. peritoneal fluid is usually normal except when severe mural inflammation or colonic infarction occurs. routine detection of salmonellae in feces involves five daily cultures of large samples ( to g) of feces using enrichment techniques. , , however, the sensitivity of fecal culture can be as low as % to %, even if one cultures several fecal samples collected daily. concurrent culture of rectal biopsy specimens and feces increases the sensitivity of culture techniques to % to %. currently, the polymerase chain reaction (pcr) is the most sensitive and rapid test for detecting salmonella in feces. a single pcr test applied early in the course of disease is a more sensitive test for the presence of salmonella than repeated fecal cultures. , detection of salmonellae in feces does not prove a diagnosis of salmonellosis, but the positive predictive value of a positive pcr or culture results is high in horses with compatible clinical signs. culture of peripheral blood may allow isolation of the organism if bacteremia or septicemia is present, but blood cultures are not a sensitive test for salmonellosis in adult horses. although foals are more likely than adults to become septicemic, culture of blood is recommended in all cases with signs suggestive of septicemia. increased numbers of fecal leukocytes suggest an invasive process in the colon but are not specific for salmonellosis. early in the course of the disease, dehydration, electrolyte and acid-base imbalances, endotoxemia, and sepsis may be life threatening. aggressive treatment during the acute stages to replace fluids lost in the diarrhea and to control sepsis and endotoxemia is often effective in controlling the primary disease. weight loss and hypoproteinemia are often severe. complications such as multiorgan dysfunction, vascular leak syndrome with peripheral and organ edema, laminitis, acute renal failure, venous thrombosis and septic phlebitis, irreversible protein-losing enteropathy or chronic malabsorption, pulmonary aspergillosis, and gastrointestinal infarction can occur. in many instances, horses recover from acute salmonellosis with aggressive treatment, only to succumb to complications of the disease, partially explaining the high fatality rate of equine salmonellosis compared with human salmonellosis. chronic, mild to moderate diarrhea occasionally occurs in horses after a bout of severe salmonellosis, usually with protein-losing enteropathy. if the chronic diarrhea persists beyond to weeks after the onset of signs, the prognosis for recovery is poor. potomac horse fever (equine monocytic ehrlichiosis) is caused by the obligate intracellular rickettsial organism neorickettsia risticii. [ ] [ ] [ ] [ ] the disease is most common from late summer to early fall, with a peak incidence in july and august. potomac horse fever was described first in the northeast but since has been diagnosed in most areas of the continental united states, with a particularly high prevalence in the northeast and midwest. the geographic distribution is characterized by a significantly higher percentage of cases found along waterways and rivers. , the disease occurs sporadically, temporally and geographically, and can affect any age group of horses. the case fatality rate is % to %. transmission of n. risticii has been reproduced experimentally by oral, intramuscular, intradermal, subcutaneous, and intravenous routes. , however, the natural route of infection has remained a mystery until recently. the epidemiologic data, the fact that many other rickettsial diseases are transmitted by insect vectors, and the finding that the disease can be transmitted via whole blood have implicated insect vectors in the natural transmission of the organism. attempts to transmit the disease experimentally with ticks (dermacentor variablis) or biting flies (stomoxys calcitrans) have been unsuccessful. , recently, n. risticii has been found to infect virgulate cercariae larval stages of trematodes that use operculate freshwater snails (juga spp.) as part of their life cycle in northern california. infected virgulate cercariae have been identified in aquatic snails collected in other parts of the country as well. virgulate cercariae are part of the life cycle of trematodes that are common parasites of many species and use freshwater snails and aquatic insects as intermediate hosts. although the trematode species infected with n. risticii remains to be identified definitively, at least two species are considered potential vectors. during the trematode life cycle, aquatic snails release large numbers of infected cercariae into water, where they seek their next intermediate host. infected metacercaria have been identified in a variety of aquatic insects. preliminary studies suggest that n. risticii in fact may be transmitted via ingestion of mature caddis flies containing infected metacercariae. possibly horses are infected by drinking water containing infected cercaria released from snails or by ingesting infected metacercariae in other aquatic insects. the number of pcr-positive snails in endemic regions corresponds to the seasonal incidence of potomac horse fever and may be as high as %. the pathogenesis of n. risticii is not understood completely. the organism infects and survives in monocytes and monocyte-derived leukocytes and can be found in blood monocytes during natural infections, but the sequence of events resulting in enterocolitis remains speculative. the organism appears first to infect blood monocytes in experimentally infected horses, which may be the vehicle of organ infection. , however, in naturally infected horses, whether leukocytes of monocytic lineage or epithelial cells are infected first is unclear. the target organ is the gastrointestinal mucosa, with the most severe lesions found in the large intestine. , infection of human colonic cells in vitro does not cause major cytopathologic effects for several days. disruption of the microvilli in the region of the plasma membrane where sodium chloride channels are located has been observed in human colonic cell cultures. infection in horses is associated with variable degrees of morphologic damage. , mild morphologic damage and mononuclear cell infiltration of the lamina propria occur early during the infection, but fibrinous, necrotizing typhlocolitis with severe mucosal ulceration and inflammation of the lamina propria may occur later in the disease. vasculitis and intravascular coagulation are consistent features in the large intestine, with perivascular edema. one can observe n. risticii in mucosal cells and macrophages and mast cells of the lamina propria. , n. risticii can survive and multiply in macrophages by inhibiting the production of reactive oxygen intermediates and by avoiding lysosomal digestion by blocking phagosome-lysosome fusion. [ ] [ ] [ ] evidence of impaired sodium chloride absorption in the colon has been suggested to contribute to diarrhea in infected horses and may be related to destruction of the enterocyte membrane structure in the region of sodium chloride channels. , direct injury to the mucosa by n. risticii and colonic inflammation are likely to be prominent features leading to diarrhea, especially later in the disease. fluid, protein, and electrolyte loss likely is caused by mucosal injury and effects on enterocyte fluid secretion caused by the inflammatory response. like other inflammatory conditions of the colon, systemic inflammation caused by absorption of bacteria and bacterial products is a potential complication of n. risticii infections if mucosal injury is severe, which contributes to the clinical signs seen during the disease. n. risticii infection is clinically similar to other forms of enterocolitis and is characterized by anorexia, depression, and fever. , , experimental infections produce a biphasic fever occurring to days apart. decreased gastrointestinal motility, manifested as reduced borborygmi, occurs during the early stages before the onset of diarrhea. diarrhea occurs in % of cases and occurs days after the second fever episode during experimental infections. , the diarrhea can be moderate to severe and dehydrating. ileus can develop at any stage of the disease and can cause signs of moderate to severe colic. systemic signs of endotoxemia, shock, and peripheral edema may occur and are similar to those described for salmonellosis. experimental and natural infection with n. risticii can cause abortion of infected fetuses in pregnant mares. , laminitis is a complication in % to % of naturally occurring cases and is often severe. other complications include protein-losing enteropathy, thrombosis, and renal failure, as described for salmonellosis. hematologic abnormalities reflect endotoxemia, dehydration, and sepsis and are essentially identical to those described for salmonellosis. neutropenia with a left shift is a consistent feature and occurs concurrently with or soon after the onset of diarrhea. thrombocytopenia is common and often severe. neutrophilic leukocytosis occurs later in the course of the disease. hyperfibrinogenemia is usually more pronounced than that with salmonellosis. serum electrolyte, acid-base, and biochemical abnormalities are also similar to those described for salmonellosis. coagulopathies are common during n. risticii infection and reflect activation of coagulation pathways. disseminated intravascular coagulation is common and may be related to the high frequency of laminitis associated with n. risticii infection. one cannot base diagnosis of n. risticii infection solely on clinical signs because the disease is clinically similar to other forms of enterocolitis. however, in endemic areas, acute colitis is likely to be caused by n. risiticii, and thus the clinical signs of acute inflammatory colitis in fact may have a high predictive value. serologic evidence of infection, such as rising antibody titers to n. risticii detected by indirect immunofluorescence or enzymelinked immunosorbent assay in paired serum samples may be helpful in establishing a diagnosis. , one should take care when interpreting the indirect immunofluorescence serologic test for n. risticii because the test appears to have a high false-positive rate. culture of the organism from blood is possible but is difficult and is generally useful only in the research laboratory. recently developed polymerase chain pcr tests for n. risticii dna are rapid, highly sensitive (as sensitive as culture), and specific for n. risticii infection and can be applied to blood or feces. [ ] [ ] [ ] prevention prevention of the disease by reducing exposure to the causative organism is difficult because the mode of transmission is not known. a killed vaccine has been developed that is effective in preventing clinical illness other than fever in % of experimentally challenged horses using the vaccine strain. however, field studies suggest the vaccine has limited benefit for prevention or decreasing the severity of natural infection. vaccine failures have been attributed to strain differences in antigenicity or to poor antibody responses to the vaccine. , clostridiosis is an important cause of acute enterocolitis in foals and adult horses. clostridium perfringens and c. difficile are associated most commonly with intestinal clostridiosis in horses, but other clostridial species, including c. septicum, c. cadaveris, and c. sordellii also have been isolated from horses with enterocolitis. [ ] [ ] [ ] [ ] [ ] [ ] in horses of all ages, clostridial enterocolitis appears to be a common antibiotic-associated and nosocomial cause of enterocolitis. , , however, clostridiosis in neonatal foals is a distinct clinical entity and is discussed in more detail elsewhere. this chapter focuses on adult intestinal clostridiosis. clostridia are obligate anaerobic to aerotolerant spore-forming gram-positive rods that are ubiquitous in the environment in the spore form. clostridia are elements of the normal flora of horses of all ages and are among the first bacteria acquired after birth. however, clostridia inhabiting the gastrointestinal tract normally are found in low numbers and do not produce enterotoxins. clostridiosis is associated with an increase in the number of a particular species of clostridia in the gastrointestinal tract and, perhaps most importantly, exotoxin production. although the conditions resulting in exotoxin production are not understood fully, several factors increase clostridial numbers in the gastrointestinal tract. dietary factors are known to affect the numbers of clostridium species shed in the horse feces. experimental induction of colic increases fecal shedding of clostridium species in the absence of diarrhea. antibiotics, particularly administered orally or recycled via the enterohepatic system, are well documented to increase the recovery of clostridia colony-forming units (cfus) in equine feces and may result in clinical clostridiosis. , , [ ] [ ] [ ] [ ] indeed, clostridiosis associated with c. perfringens or c. difficile is likely to be the most important cause of antibiotic-induced enterocolitis in the horse. clostridium perfringens c. perfringens is made up of many genetically distinct strains of variable virulence that produce one or more of a large group of exotoxins. the pattern of exotoxin production is used to classify c. perfringens into five types, a, b, c, d, and e. c. perfringens type a is the most common clostridium isolate from healthy horses of all ages and adults and foals with diarrhea worldwide. c. perfringens types a, b, c, and d have been associated with hemorrhagic enteritis in foals less than days of age, with type c being the most common cause in north america. the primary toxin produced by c. perfringens type a is α-toxin (phospholipase c), which interferes with glucose uptake and energy production and activates arachidonic acid metabolism and signaling pathways in enterocytes. oral administration of α-toxin does not cause tissue necrosis but causes increased secretion by small intestinal mucosal cells in human beings. , the β-toxin of types b and c is a cytotoxin that causes enterocyte necrosis, ulceration, and ultimately severe intestinal inflammation and hemorrhage. a novel toxin designated β may also have a role in c. perfringens enterocolitis. the biologic activity of the β -toxin is similar to β-toxin, but β -toxin is not related to β-toxin in sequence. the β -toxin was prevalent in two groups of horses with acute enterocolitis but not in healthy horses. the β -toxin appears to be associated predominantly with c. perfringens that would have been classified otherwise as type a but that in fact may represent a previously undescribed type. virulent strains of c. perfringens type a and to a lesser extent type c also may produce enterotoxin. enterotoxin is a cytotoxin that inserts into cell membranes to form pores, which alter permeability to water and macromolecules and ultimately lead to cellular necrosis. massive desquamation of the intestinal mucosa resulting from enterotoxin cytotoxicity triggers an inflammatory response, intestinal edema, mural hemorrhage, and systemic inflammation. enterotoxin also alters tight junction integrity, resulting in increased paracellular permeability by a noncytotoxic mechanism. clostridium difficile c. difficile produces several toxins, but only two, toxin a and toxin b have been studied in detail. toxin b is a potent cytotoxin in vitro, but its role in enterocolitis is less clear than the role of toxin a. toxin b does not induce fluid secretion, inflammation, or characteristic alterations in intestinal morphology. c. difficile induces an inflammatory response with hypersecretory diarrhea that is induced in large part by the enterotoxin toxin a. toxin a induces neutrophil influx into intestinal tissue, mast cell degranulation, and secretion of prostaglandins, histamine, cytokines, and -hydroxytryptamine by these activated leukocytes. , the products of neutrophils and mast cells have a prominent role in the vasodilatory and secretory responses in the intestine during c. difficile infection. the enteric nervous system is central to the induction of intestinal inflammation and mucosal secretion by toxin a. a model for toxin a-induced secretory diarrhea has emerged in which toxin a stimulates substance p-containing afferent sensory nerve fibers, which in turn stimulate mast cell degranulation, recruitment and activation of polymorphonuclear leukocytes, and vasodilation. [ ] [ ] [ ] toxin a-induced stimulation of enterocyte secretion can occur via secretomotor neuronal stimulation by substance p-containing sensory neurons or products of mast cells and polymorphonuclear leukocytes. neural blockade or depletion of substance p abolishes mast cell degranulation, polymorphonuclear leukocyte influx, and enterocyte secretion. how toxin a triggers the sensory component of the enteric nervous system is not known yet, but toxin a-induced necrosis of enterocytes likely exposes afferent neurons to the noxious milieu of the intestinal contents. equine intestinal clostridiosis is clinically similar to other forms of acute enterocolitis in horses. , although the clinical course is usually acute, peracute colitis with rapid death may occur. occasionally, a milder, more prolonged clinical course occurs. one may note fever, anorexia, and depression before the onset of gastrointestinal signs, but more commonly no prodromal signs are apparent. signs of endotoxemia and shock may accompany acute signs of colic and severe, dehydrating diarrhea. diarrhea may not be profuse but is usually dark and foul smelling. like the clinical signs, hematologic and serum biochemical abnormalities are similar to those associated with other forms of enterocolitis and reflect fluid, protein, and electrolyte loss and systemic inflammation from endotoxemia. neutropenia, leukopenia, and hemoconcentration are common. hypoproteinemia may be profound. one often may note hyponatremia, hypokalemia, hypochloremia, hypocalcemia, and a mixed prerenal/renal azotemia, as well as metabolic acidosis and coagulopathies. serum concentrations of hepatocellular enzymes such as sorbitol dehydrogenase may be elevated, and liver function may be reduced. preliminary diagnosis of equine intestinal clostridiosis caused by c. perfringens is based on the isolation of greater than cfus of c. perfringens type a per gram of feces from patients with diarrhea and signs suggestive of toxemia. similar criteria are used to screen human patients for c. perfringens type a infection. normal horses shed fewer than cfus/g of feces, and usually horses with intestinal clostridiosis shed greater than × cfus/g. , however, identification of increased numbers of clostridium organisms in the feces does not prove infection. detection of c. perfringens toxins in feces or intestinal contents in horses with increased numbers of fecal cfus and clinical signs of enterocolitis is more conclusive evidence of an enterotoxigenic infection than culture alone. immunoassays are available that are primarily designed to detect c. perfringens enterotoxin. however, the reliability (specificity) of some immunoassays for diagnosis of c. perfringens infection has come into question. recently, pcr multiplex and gene probe assays have been developed to detect the major lethal toxins in isolates or fecal samples to determine the pattern or toxin production and are currently the preferred methods of detection. [ ] [ ] [ ] as for c. perfringens, diagnosis of c. difficile infection consists of culture of the organism from feces and identification of toxins in the feces. bacterial culture of c. difficile may be difficult and may be an insensitive test in horses. [ ] [ ] detection may require enrichment techniques and culture of multiple fecal samples. , detection of toxins a and b in feces is regarded as the preferred test for diagnosis of c. difficile infection in human beings. commercial enzyme-linked immunosorbent assays are available for toxins a and b that are specific and appear to be more sensitive than a single culture for identifying c. difficile infection in adult horses. , one also may induce toxin production by c. difficile isolates. sensitive pcr methods also have been developed for application to fecal samples and isolates to detect the genes for toxins a and b. proliferative enteropathy is a chronic hyperplastic disorder of the small intestine and has been described in a variety of mammalian and avian species. , the only causative agent identified to date that induces proliferative enteropathy is the obligate intracellular pathogen lawsonia intracellulare. , the pig is the most frequently naturally affected species. however, the reports of equine proliferative enteropathy associated with l. intracellulare have increased in recent years. [ ] [ ] [ ] [ ] the relatedness of the strains of l. intracellulare causing proliferative enteropathy in pigs and horses or even among other affected species is not known. no host restriction is apparent because hamsters and other rodents can be infected with porcine strains of l. intracellulare. before the year , reports of proliferative enteropathy in the literature describing isolated cases were sporadic. [ ] [ ] [ ] however, since , outbreaks on breeding farms have been documented on farms in canada, suggesting that a new strain has emerged. the mode of infection is fecal-oral, and infected animals can shed large numbers of organisms in feces. affected animals shedding the organism in the feces serve as a source of infection for herdmates. possibly nonequine species serve as reservoirs contributing to outbreaks on horse farms. factors that increase the risk of proliferative enteropathy in pigs include overcrowding, ration changes, transport, and weaning. , like pigs, horses are affected as weanlings. factors associated with weaning and other stresses may affect immunity and increase susceptibility to infection. the incubation period is to weeks in nonequine species and is presumed to be similar in horses. experimental l. intracellulare infection produces characteristic pathologic lesions in pigs and hamsters that are identical to lesions in horses with proliferative enteropathy. , a profound hyperplasia of the mucosa predominantly caused by proliferation of crypt epithelium and crypt hyperplasia is induced locally in infected islands of tissue and eventually extends to the entire distal jejunum and ileum. l. intracellulare preferentially infects proliferating cells, thus the tropism for the crypt epithelium. infected cells proliferate far more rapidly than uninfected cells, suggesting that l. intracellulare directly induced the proliferative response. however, the molecular basis for the enhanced proliferation is not known. l. intracellulare penetrates epithelial cells in a membrane-bound vesicle but eventually escapes the vacuole and is found free in the cytoplasm concentrated at the apical pole of the cell. the gross pathologic lesions found in equine proliferative enteropathy are characteristic. [ ] [ ] [ ] [ ] lesions may be segmental and typically are found in the ileum and terminal jejunum in horses. however, lesions also may affect the duodenum. severe mucosal hypertrophy often occurs but may wane during the chronic stages of the disease. the mucosa may become corrugated with focal erosions or ulcers. one often can identify submucosal edema easily on cut sections of affected segments. moderate to severe crypt hyperplasia with atrophy of the intestinal villi is a consistent feature. the hyperplastic crypts are branched and may herniate into the submucosa. necrosis, edema of the submucosal and lamina propria, hemorrhage, mononuclear inflammation and muscular hypertrophy have been reported in affected intestinal segments but are not consistent. special stains such as silver stain are required to detect intracellular organisms. the organisms are curved or comma-shaped rods found clustered in the apical cytoplasm of hyperplastic crypt epithelium. the proliferative response of the intestinal mucosa alters absorption of nutrients and fluid secretion by disrupting the architecture of the villi and by altering the maturation of epithelial cells into absorptive cells, accounting for the secretory diarrhea and often severe weight loss. , the combined effects of the inflammatory part ii disorders of specific body systems response and malabsorption may account for the protein-losing enteropathy. proliferative enteritis most commonly affects weanling foals ages to months. the clinical signs of proliferative enteropathy include ill thrift, weight loss, peripheral edema, diarrhea, and colic. [ ] [ ] [ ] [ ] the diarrhea is usually in the form of soft feces but may be profuse and watery. some foals with mild diarrhea have black, tarry feces. secondary complications such as gastric ulceration, bronchopneumonia, or parasitism may occur concurrently with the proliferative enteropathy. clinicopathologic features include mild to moderate anemia, moderate to severe hypoalbuminemia (often < g/dl), hypoglobulinemia, neutrophilic leukocytosis, and hyperfibrinogenemia. creatine kinase activities may be elevated in affected foals. prerenal azotemia and electrolyte imbalances such as hyponatremia may be associated with diarrhea. peritoneal fluid analysis is usually unremarkable. ultrasonographic examination of the small intestine often reveals significant thickening of the intestinal wall ( figure . - ). intestinal edema may be evident as a hypoechoic appearance to one or more layers of the intestinal wall. methods for antemortem diagnosis include serologic analysis of l. intracellulare antibodies and pcr analysis of feces. serologic analysis using an indirect immunofluorescent antibody test may be the most useful single test available. the pcr test is specific but the sensitivity may be low. by the time clinical signs appear, % of pigs are serologically positive for anti-lawsonia intracellulare immunoglobulin g (igg). in contrast, only % of pigs had positive fecal pcr tests. of the seven foals tested in an outbreak of equine proliferative enteropathy, four foals with confirmed disease and three with suspected proliferative enteropathy had serologic titers against l. intracellulare of : or greater. in contrast, serum samples collected from foals before the outbreak were negative for l. intracellulare antibodies. fecal pcr for l. intracellulare was positive in of foals tested, and half of the serologically positive foals had negative fecal pcr tests. many clinicians combine serologic analysis with fecal pcr testing to increase the sensitivity and specificity of these diagnostic methods. isolation and culture of the organism requires cell culture techniques that are not widely available. thus no practical method exists for culturing the organism from feces or tissues that is available for clinical use. definitive diagnosis requires histopathologic examination of affected tissues. diagnosis is based on typical histopathologic findings of mucosal hypertrophy and submucosal edema and identification of small, curved, rod-shaped intracellular bacteria at the apical pole of epithelial cells in affected segments of intestine. special stains such as warthin-starry silver stain are required to detect the bacteria in histopathologic specimens. pcr analysis of affected intestinal tissue is a specific test for the presence of l. intracellulare and, unlike fecal pcr analysis, appears to be sensitive. strongyle infections in horses are caused by two groups of nematodes: large and small strongyles (see the section cyathostomiasis). large strongyles that are pathogenic in horses include strongylus vulgaris, s. edentatus, and s. equinus. of these species, s. vulgaris is by far the most important cause of disease in the large intestine and in fact is the most pathogenic parasitic infection in horses. s. vulgaris infection in horses is manifested clinically by two forms: acute and chronic disease. the age and resistance of the host, the infective dose, and the size and function of the affected arteries influence the type and degree of disease that occurs. sudden ingestion of large numbers of infective larvae by a naïve host causes acute strongylosis, whereas ingestion of fewer infective larvae over a long period of time by an older, more resistant host causes chronic strongylosis. acute strongylosis is more likely to cause colic than diarrhea and may be rapidly fatal. chronic strongylosis tends to cause debilitation and signs of colic but may also cause diarrhea. diarrhea associated with acute strongylosis occurs within several days of infection and is likely to be caused by migration of the larvae through the intestinal wall. fourth-stage larvae migrate through the mucosa and submucosa into the arterioles of the intestine, causing mural edema, hemorrhage, and infiltration with inflammatory cells into the intestinal wall. , increased secretion and decreased absorption of fluid and electrolytes, stimulated by inflammatory mediators such as prostaglandins and histamine, may play a role in the diarrhea induced by s. vulgaris. interstitial edema and damage to the interstitial matrix and mucosa may result from inflammation and migration of the parasites, causing increased secretion of fluid and albumin loss. abnormal gastrointestinal motility also may play a role in the development of diarrhea. migration of larvae through the intestinal wall early in the course of infection affects myoelectric activity and motility in the large intestine and may affect retention of ingesta and absorption of fluid. , the cause of death in acute strongylosis has not been addressed but may be related to massive migration through the vasculature, causing thrombosis with ischemia and infarction of the intestine. chronic strongylosis causes typical verminous arteritis and is associated more commonly with natural infections in horses than with acute strongylosis. lesions of the large intestinal vasculature caused by migration of larvae through the intima are characterized by thrombus formation, narrowing of the arterial lumen, fibrosis, and thickening of the arterial wall. , embolization may occur, causing acute segmental infarction of the large intestine, but more commonly reduced blood flow without embolization causes ischemia and occasionally infarction. , postmortem examination of horses with colonic infarction failed to reveal embolization as the cause in most cases. reduced blood flow in the tissues of the intestine usually results from narrowing of the arterial lumen by the thrombus and formation of microthrombi at sites independent of the parasites. release of vasoconstrictive inflammatory mediators such as leukotrienes from platelets, neutrophils, and eosinophils and elaboration of parasitic antigens or toxins may cause vasoconstriction and ischemia. horses with experimental strongylosis were found to have a % reduction of blood flow in the colonic vasculature. clearly, reduced blood flow is an important effect of chronic strongylosis, but the relationship between blood flow and diarrhea is unclear. disrupted motility resulting from ischemia may lead to diarrhea by reducing the retention of ingesta and absorption of fluid. acute infarction and mucosal ulceration have been found to cause severe chronic diarrhea in naturally infected horses. release of inflammatory mediators such as prostaglandins, histamine, and kinins from inflammatory cells associated with thrombi and inflamed intestine also may affect secretion, absorption, and motility, leading to diarrhea. the clinical signs of acute strongylosis caused by s. vulgaris infection are characterized by depression, moderate to severe colic, and fever. diarrhea is less often a feature of acute strongylosis than is colic. most cases of acute strongylosis occur in young, naïve horses that are introduced to an infested environment or are inoculated experimentally with infective larvae. this form of strongylosis often is not recognized naturally. chronic strongylosis, however, occurs most commonly as a natural syndrome. weight loss or poor weight gain; chronic, intermittent colic; fever; poor appetite; and diarrhea are frequent signs. , diarrhea may be profuse and watery, or the feces may be soft but of normal volume. transrectal palpation may reveal thickening and fremitus in the cranial mesenteric artery. young horses are most commonly affected, but older horses also may be affected. horses with acute infarction or large intestinal ulceration following chronic strongylosis may have signs of severe abdominal pain, sepsis, and endotoxemia, and profuse, watery diarrhea is common. hematologic abnormalities associated with strongylosis include neutrophilic leukocytosis and eosinophilia. [ ] [ ] [ ] neutrophilia appears to be an early event during the course of the disease, and eosinophilia tends to appear later. , hyperfibrinogenemia also may occur, especially later in the course of the disease. serum αand β-globulin and igg(t) concentrations are characteristically elevated. [ ] [ ] [ ] horses with chronic ulcerative colitis following strongylosis may exhibit severe hypoalbuminemia. peritoneal fluid analysis may reveal an elevated protein concentration and eosinophilia. , tentative diagnosis is based on clinical signs, hematologic abnormalities, and peritoneal fluid analysis. elevated serum αand β-globulin concentrations and igg(t) concentration support the diagnosis. fecal analysis may reveal strongyle eggs, but fecal egg counts are often unreliable because nonpatent larvae cause the disease. appropriate preventive measures are important in controlling this disease, including management procedures such as preventing overcrowding, reducing exposure of susceptible individuals, and instituting proper deworming schedules. ivermectin is the preferred anthelmintic used to control strongylosis in horses. monitoring fecal egg counts as a means of evaluating the efficacy of parasite control measures is recommended. infection with small strongyles (cyathostomiasis) is well recognized as a cause of diarrhea and large intestinal disease in horses of all ages. [ ] [ ] [ ] [ ] [ ] [ ] clinical disease is caused by intramural larval stages. the cyathostome life cycle requires migration by fourth-stage larvae through the mucosa of the large intestine and may include a period of hypobiosis during which the larvae remain encysted within the mucosal layer of the large intestine. after a period of hypobiosis the larvae emerge in response to a largely unknown stimulus. most cases occur when larval emergence takes place, classically in the late winter and spring in the northern temperate zones when larvae are expected to emerge and in the late fall or winter months in the southeastern united states and subtropical regions. sudden emergence of encysted larvae causes the mucosal injury, ulceration, and inflammatory reaction responsible in large part for the clinical disease. , however, migration of the larvae as they penetrate the mucosa affects motility patterns and can cause inflammation that may contribute to diarrhea. chronic, eosinophilic, granulomatous colitis and diarrhea with histopathologic evidence of hypobiotic cyathostome larvae in the large intestine have been reported in two horses during a period in which emergence of larvae would not be expected to occur (early winter). natural emergence of cyathostome larvae causes fibrinous inflammation of the large intestine, focal necrosis, mural hemorrhage, and ulceration of the large intestinal mucosa, which even may result in bleeding into the lumen. mild to moderate eosinophilic and mononuclear inflammation of the lamina propria occurs, and moderate to severe interstitial edema frequently occurs. , colonic inflammation and interstitial edema may contribute to the diarrhea, along with the loss of the mucosal barrier, by causing increased active and passive secretion of fluid, electrolytes, and protein. protein loss is often significant, resulting in profound hypoalbuminemia and interstitial edema of skin and other organs. chronic, granulomatous colitis has been reported to occur in response to encysted larvae and may cause diarrhea by increased secretion following granulomatous inflammation or disruption of the interstitium by granulomatous infiltration. administration of an anthelmintic to horses with a heavy load of encysted larvae also may cause rapid larval death and acute and often severe inflammation similar to natural emergence. cyathostomiasis may be the most commonly identified cause of chronic diarrhea in the horse. [ ] [ ] [ ] however, an acute syndrome also has been associated with cyathostomiasis. clinical signs of cyathostomiasis are characterized by moderate to severe weight loss or poor weight gain, ill thrift, ventral edema, intermittent fever, and intermittent, mild colic. acute onset of diarrhea is typically profuse and progresses to chronic diarrhea that is often mild, is the consistency of bovine feces, and may be intermittent. [ ] [ ] [ ] [ ] [ ] [ ] appetite is usually normal, but affected horses occasionally have a ravenous appetite. transrectal palpation usually does not reveal any abnormalities. cyathostomiasis may affect any age of horse, and clinical signs are more common during periods of emergence of larvae, corresponding to late winter and spring in northern temperate zones. the deworming history may appear to be adequate. neutrophilic leukocytosis is typically evident, but the white blood cell count may be normal. [ ] [ ] [ ] [ ] [ ] [ ] profound hypoalbuminemia is a characteristic feature of cyathostomiasis, manifested clinically by ventral edema. plasma αand β-globulin concentrations may be elevated, which can result in a normal total plasma protein concentration in spite of hypoalbuminemia. [ ] [ ] [ ] the serum igg(t) concentration, however, has been reported to be normal, which may help distinguish cyathostomiasis from s. vulgaris infection. , , in addition, peritoneal fluid analysis does not usually reveal any abnormalities, in contrast to horses with s. vulgaris infection. fecal analysis may be unrewarding because the infection is often not patent when clinical signs are apparent. measurement of plasma fructosamine may provide a measure of protein catabolism or protein loss in the absence of hypoalbuminemia. plasma fructosamine concentrations are significantly lower in horses with experimental cyathostomiasis than in normal controls, , suggesting that this test may be a useful diagnostic tool. however, the test has not yet been validated in naturally occurring cases, and neither the specificity nor the sensitivity is known. rectal scrapings or rectal mucosal biopsies may reveal evidence of cyathostome larvae. , definitive diagnosis usually requires microscopic examination of biopsy specimens of the cecum and ascending colon, collected by laparotomy. examination of biopsy specimens collected from the small intestine is recommended to rule out other causes of weight loss and diarrhea. one should include appropriate diagnostic tests, such as culture of feces for pathogenic bacteria, in the workup to rule out other causes. preventive measures are appropriate for other horses on premises known to have a problem with cyathostomiasis, particularly frequent deworming (every weeks) during times of high infectivity (spring and summer in the north and fall, winter, and early spring in the south) to eliminate parasites before they become patent. because of high levels of resistance to benzimidazoles, avermectins (ivermectin or moxidectin) are the drugs of choice. [ ] [ ] [ ] resistance to ivermectin has been demonstrated, but the prevalence of ivermectin resistance appears to remain low. although daily pyrantel pamoate administration also has been reported to reduce worm burdens and pasture infectivity in young and mature horses effectively, cyathostome resistance has been reported and is a concern for the use of this drug as a routine preventive anthelmintic. , diarrhea in adult horses may also occur secondary to administration of antimicrobial or antiinflammatory medications or after ingestion of toxic compounds. affected horses exhibit clinical signs that may be indistinguishable from signs exhibited by horses with diarrhea of infectious etiology. antibiotic-associated diarrhea has been reported in many species, including horses. certain antibiotics-such as trimethoprim-sulfonamide combinations, erythromycin, penicillins, tetracyclines, clindamycin, and lincomycinare associated with naturally occurring and experimental enterocolitis syndromes in horses. , [ ] [ ] [ ] [ ] in some cases, such as with trimethoprim-sulfonamide combinations, the geographic incidence of antibiotic-associated diarrhea appears to differ considerably. clostridium perfringens, c. difficile, and salmonella spp. are apparently the most common causes of antibioticassociated diarrhea in horses. outbreaks of c. difficile have been reported in hospitalized horses being treated with antibiotics. , in sweden, accidental erythromycin ingestion has been associated with c. difficile enterocolitis in mares in which their foals were being treated for rhodococcus equi. , , interestingly, this phenomenon has not been reported in other areas of the world. foals being treated with erythromycin are at a higher risk for diarrhea than foals being treated with other antibiotics. tetracycline administration has been shown to be associated with an increase in the numbers of gram-negative enteric bacteria and c. perfringens in the feces of horses and reactivation of salmonellosis and prolongation of fecal shedding of salmonella. , the most common mechanism by which antibiotics cause diarrhea is by disrupting the gastrointestinal flora. the normal large intestinal flora, comprised of mainly obligate anaerobes and streptococci, protects the host from pathogenic bacteria by colonization resistance. ecologic factors play an important role in colonization resistance. for example, surface bacteria in the large intestine interact with receptors on the mucosal cells, facilitating adherence to the mucosa. , in doing so, the normal organisms compete more successfully for this important niche. competition for space and nutrients is an important means of preventing colonization and proliferation of pathogenic bacteria. in addition, anaerobic bacteria produce short-chain fatty acids (scfas) and other metabolites that are toxic to facultative anaerobic bacteria, especially in the conditions of the large intestine. , , organisms of the normal flora also produce bacteriocins that inhibit growth of potential pathogens. antibiotics that deplete the population of obligate anaerobes and streptococci efficiently decrease colonization resistance. production of fatty acids diminishes, thus reducing competition for space and nutrients. as a result, gram-negative enteric bacteria such as salmonella are able to proliferate. in addition, pathogenic anaerobes normally found in low numbers can proliferate. antibiotic-resistant strains of bacteria, especially gram-negative enteric bacteria and possibly clostridia, may be selected by antibiotic administration, allowing proliferation of pathogenic bacteria resistant to many antibiotics. obligate anaerobic commensal organisms, perhaps the most critical group of microbes for maintaining colonization resistance, are usually susceptible to macrolides, tetracyclines, β-lactams, and lincosamides, perhaps explaining the high incidence of diarrhea associated with the administration of these antibiotics. in addition to reduction of colonization resistance, depletion of the normal anaerobic microbial population in the intestine decreases carbohydrate fermentation and production of scfas, which contributes to the pathogenesis of antibiotic-associated diarrhea by decreasing absorption of sodium and water by the colonic mucosa. ampicillin decreases colonic fermentation of carbohydrates in human beings. human patients with antibioticassociated diarrhea have greatly impaired colonic fermentation and low production of scfas. erythromycin, ampicillin, or metronidazole treatment is associated with decreased production of scfas in patients with and without diarrhea. absorption of sodium and water is stimulated by absorption of scfas in the equine colon, suggesting that reduction of colonic scfa content by antibiotic-induced depletion of anaerobic flora has similar effects in horses as in human beings. broad-spectrum antibiotics exert a more profound effect on the gastrointestinal flora than narrow-spectrum antibiotics. antibiotics administered orally, especially those that are poorly absorbed, are more likely to cause diarrhea than are parenterally administered antibiotics. for instance, clindamycin is less likely to cause diarrhea in human beings when administered intravenously than when administered orally. antibiotics with extensive enterohepatic circulation, such as tetracyclines and erythromycin, are excreted in high concentrations in the bile and are associated more commonly with diarrhea than antibiotics that do not undergo enterohepatic circulation. antibiotics may cause diarrhea by means other than by disrupting the normal flora. direct toxic effects may play a role in producing irritation, increasing secretion, and disrupting motility patterns. tetracyclines are irritating to the gastrointestinal mucosa and may cause inflammation and increase secretion. erythromycin has been shown to interact with smooth muscle cells, stimulating gastrointestinal motility. , normal peristalsis plays an important role in suppressing the population size of potentially pathogenic bacteria. normally, bacteria that are prevented from adhering to the mucosa by colonization resistance are swept aborally by peristalsis and are excreted in the feces. disruption of normal motility patterns may prevent clearance of pathogenic bacteria, contributing to the colonization of mucosal surfaces. diarrhea induced by antibiotics usually occurs within days of antibiotic administration or can occur several days after cessation of antibiotic treatment. the clinical syndrome of antibiotic-associated diarrhea can vary from mild diarrhea to fulminant enterocolitis with severe diarrhea. mild cases of diarrhea are common, especially in foals receiving erythromycin, trimethoprim-sulfonamide combinations, or rifampin. , mild cases of diarrhea are usually not clinically significant. however, acute, severe enterocolitis can occur in all ages of horses receiving antibiotics and can be life threatening. clinical signs are identical to other causes of acute enterocolitis. severe, dehydrating diarrhea, endotoxemia, sepsis, and shock may occur. hemoconcentration, neutropenia, hypoproteinemia, and electrolyte and acid-base imbalances are common. severe hyponatremia may occur in foals with antibiotic-associated diarrhea, especially if trimethoprimsulfonamide and rifampin combinations are the cause. more detailed descriptions of the clinical and laboratory findings were given previously. diagnosis is presumptive, because definitive diagnosis of antibiotic-associated diarrhea is impossible. fecal culture and pcr testing may reveal salmonella or clostridium infection. toxicity resulting from nonsteroidal antiinflammatory drug (nsaid) administration has been well documented in several species, including horses. [ ] [ ] [ ] [ ] [ ] [ ] [ ] in horses and human beings, nsaid toxicity is manifested by renal and gastrointestinal disease. elderly human patients are more susceptible to nsaid toxicity, but the effects of age on nsaid toxicity in horses are less well defined. foals are considered to be more susceptible than adult horses to gastrointestinal disease following nsaid administration, and ponies may be more susceptible than horses. all nsaids are capable of inducing gastrointestinal and renal damage at toxic concentrations, and the toxicity is not significantly different among products. aspirin is potentially more toxic than other nsaids because it irreversibly inactivates cyclooxygenase by acetylation, whereas other nsaids reversibly inhibit cyclooxygenase. however, phenylbutazone is the drug most commonly reported to cause toxicity in horses, perhaps because of its widespread use by veterinarians and horse owners. phenylbutazone toxicity in horses is characterized by mucosal ulceration throughout the gastrointestinal tract, oral ulceration, renal papillary necrosis, vasculopathy, thrombosis, and protein-losing enteropathy with hypoalbuminemia. [ ] [ ] [ ] this discussion focuses on the toxic effects of nsaids on the large intestine but necessarily includes elements of upper gastrointestinal and renal disease. horses with large intestinal disease resulting from nsaid toxicity generally are receiving inappropriately large doses. the dosage regimen recommended for phenylbutazone ( . mg/kg twice in day and then . mg/kg twice daily) is considered to be safe. experimental studies in horses, however, have shown toxicity to occur when greater than the recommended dosage ( . mg/kg/day) is administered for several days. , in most reported cases of phenylbutazone toxicosis horses were receiving higher than recommended dosages. , , regardless, administration of phenylbutazone at the recommended dosage has been reported to cause a significant decrease in plasma protein concentration and gastrointestinal disease. , moreover, signs of nsaid toxicity have been reported in normovolemic horses treated with appropriate doses of phenylbutazone. , dehydration, sepsis, and endotoxemia exacerbate the renal and gastrointestinal toxicity of nsaids. clearly, the margin of safety is narrow for phenylbutazone and probably for other nsaids used in horses as well. gastrointestinal disease induced by nsaids is manifested by mucosal ulceration, inflammation, bleeding, and protein-losing enteropathy. , , , in addition to direct effects on the mucosal barrier, nsaid administration has been shown to cause an acute relapse of preexisting colonic inflammatory disease and worsen colonic inflammation in human beings. , , whether this occurs in horses is not clear. the mechanism by which nsaids induce mucosal damage is probably multifactorial. direct irritation may play a role in oral and gastric irritation and ulceration; however, parenteral administration of nsaids produces oral and gastric ulceration as well. inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (the constitutive cox) and cyclooxygenase (the inducible cox) appears to be the most important mechanism of mucosal injury. prostaglandins, particularly pge and pgi , are critical for mucosal health. , pge has been shown to increase mucosal blood flow; increase secretion of mucus, water, and bicarbonate; increase mucosal cell turnover rate and migration; stimulate adenyl cyclase activity; and exert other protective effects in the gastric mucosa of several species. , , perhaps most importantly, pge and pgi have a role in maintaining epithelial tight junction integrity, which is indispensable for mucosal barrier function and repair after mucosal injury. in spite of the overwhelming amount of information about the role of prostaglandins in maintaining the mucosal barrier in other species and clear clinical and experimental evidence that nsaids injure the equine colonic mucosa, the role of prostaglandins in mucosal protection in the equine colon is not yet well defined. inhibition of cox- and cox- in equine colonic mucosa with flunixin meglumine resulted in reduced electric resistance of the mucosa and increased permeability to macromolecules in vitro (a.t. blikslager and s.l. jones, ) , suggesting that flunixin treatment disrupts the epithelial tight junctions in the equine colon. mucosal changes were correlated with a profound inhibition of pge and pgi concentrations in the treated tissues. in other studies, administration of a pge analog prevented the gastrointestinal manifestations of phenylbutazone toxicosis in ponies. recent development of nsaids specific for cox- have greatly reduced the frequency and severity of gastrointestinal side effects in human beings taking nsaids for chronic musculoskeletal conditions. thus cox- -specific nsaids hold promise for use in horses to treat arthritis and reduce the incidence of toxicity. for example, the cox- -specific inhibitor etodolac was less harmful to equine colonic mucosa than flunixin meglumine in vitro (a.t. blikslager and s.l. jones, ) . moreover, etodolac was significantly more permissive than flunixin for recovery of the mucosa in equine ischemic-injured intestinal tissues, and in fact, recovery was no different than control tissues. however, their use is at present limited because the specificity of the so-called cox- selective inhibitors and their efficacy as analgesics have not been demonstrated in the horse. nsaid-induced mucosal injury is associated with a significant inflammatory response to microbial products exposed to the lamina propria. this inflammation exacerbates mucosal dysfunction and injury associated with nsaid toxicity. for example, depletion of neutrophils or blockade of neutrophil influx into gastrointestinal tissues or inhibition of neutrophil activation and release of toxic products prevents many of the pathophysiologic effects of nsaid toxicity in the gastrointestinal tract. [ ] [ ] [ ] [ ] the inflammatory response alone may result in moderate to severe gastrointestinal ulceration, mural vascular thrombosis and edema, fluid secretion, protein-losing enteropathy, and mucosal hemorrhage. nsaid colitis manifests as two clinical syndromes: right dorsal colitis (rdc) and generalized nsaid toxicity. rdc is an ulcerative disorder isolated to the right dorsal segment of the large intestine. , , the most prominent clinical signs of rdc are anorexia, lethargy, and colic. anorexia, depression, diarrhea, fever, and signs of section . inflammatory diseases of the gastrointestinal tract causing diarrhea endotoxemia also may be features. if the rdc is chronic, weight loss, intermittent colic, lethargy, anorexia, and ventral edema are common clinical signs, along with soft and unformed feces. severe ulceration of the right dorsal colonic mucosa results in proteinlosing enteropathy and significant hypoproteinemia attributable mainly to hypoalbuminemia. hypoproteinemia may be severe enough to cause peripheral (usually ventral) edema.in some cases, one may note dehydration, electrolyte abnormalities, neutropenia or anemia, azotemia, and biochemical abnormalities if the ulceration and diarrhea are severe or if systemic inflammation is present. clinical signs of generalized nsaid toxicity may vary from mild diarrhea with no systemic signs to severe dehydrating diarrhea with anorexia, fever, depression, peripheral edema, oral ulceration, and colic. , , clinical signs of systemic inflammation caused by endotoxemia may occur, manifested as poor peripheral perfusion, tachycardia, tachypnea, weakness, trembling, and cyanotic or hyperemic oral mucous membranes. hematuria or oliguria may be present if renal involvement is present. complications associated with other forms of severe enterocolitis, such as laminitis, thrombophlebitis, and severe weight loss, may occur. hematologic abnormalities of generalized nsaid toxicity are nonspecific and include neutropenia with a left shift or leukocytosis and hemoconcentration. serum biochemical analysis is characterized by profound hypoproteinemia, hyponatremia, and metabolic acidosis. , hypocalcemia, hypokalemia, hypochloremia, and elevated hepatocellular enzyme activities also may occur. hypoproteinemia may occur without signs of diarrhea. azotemia may be prerenal from dehydration but frequently is caused by renal failure resulting from a combination of hemodynamic and toxic renal injury. urinalysis frequently reveals hematuria, proteinuria, cylindruria, and isosthenuria. fecal occult blood is frequently detectable. diagnosis of either form of nsaid colitis is often presumptive, with a history of overdose of nsaids being strong evidence of nsaid toxicity. but as discussed earlier, toxicity may occur with dosage regimens that are not considered inappropriate, particularly if the horse experiences a concurrent period of dehydration. one can use ultrasonographic examination of the right dorsal colon to confirm a diagnosis of rdc, but the sensitivity of this method is questionable. ultrasonography ( . -to -mhz transducer at the right twelfth to fifteenth intercostal spaces below the margin of the lung axial to the liver) may reveal a thickened right dorsal colon (> . cm) and evidence of colonic edema in horses with rdc. however, the sensitivity of this method of diagnosis is questionable. one can use nuclear scintigraphy of horses after infusion with technetium -labeled white blood cells to document inflammation of the right dorsal colon. diagnosis of rdc may require one to perform laparotomy or laparoscopic examination of the right dorsal colon. one must rule out other causes of enterocolitis, such as salmonellosis, potomac horse fever, clostridiosis, and antibiotic-associated diarrhea. cantharidin is the toxic principle found in beetles of the genus epicauta, commonly known as blister beetles. [ ] [ ] [ ] ingestion of the beetles in contaminated alfalfa hay causes release of the toxin from the tissues of the beetle and absorption through the gastrointestinal tract. transcutaneous absorption may occur but appears to be rare in horses. blister beetles feed on the flowers of alfalfa and may be incorporated into processed alfalfa hay if the hay is cut and processed simultaneously, as by crimping. [ ] [ ] [ ] the beetles often swarm, and one may find large numbers of beetles in small portions of hay. the lethal dose of cantharidin is less than mg/kg, but the concentration of cantharidin varies from species to species of blister beetles and between sexes. , as many as to as few as to beetles may be lethal. usually, only one or a few horses fed contaminated hay ingest beetles because the beetles are concentrated in a small portion of the hay. however, outbreaks involving many horses on a farm have occurred. most cases occur in texas and oklahoma, but horses in other states may be affected as well, especially if hay is imported from states where blister beetles are common. peak incidence is in late summer and fall. the fatality rate may be % or greater, , but if the patient survives several days, recovery is probable. cantharidin is absorbed from the gastrointestinal tract and excreted via the kidney. cantharidin is a potent irritant, causing acantholysis and vesicle formation when applied topically. , , the chemical is thought to disrupt oxidative metabolism in the mitochondria, causing mitochondrial swelling, plasma membrane damage, and changes in membrane permeability. the mucosa of the gastrointestinal tract is affected most commonly in horses because they ingest the toxin. cell swelling and necrosis occur, resulting in mucosal ulceration. oral, esophageal, gastric, and small and large intestinal ulceration have been observed in natural and experimental canthariasis. , , severe fibrinous to pseudomembranous inflammation and submucosal edema of the intestine also have been reported. diarrhea probably results from the severe ulceration and inflammation of the large intestine, causing increased secretion of water, electrolytes, and protein and decreased absorption of fluid. large volumes of fluid and protein are lost in the gastrointestinal tract, causing hemoconcentration and profound hypoalbuminemia in some cases. , , cystitis and myocarditis occur in natural and experimentally produced cases of cantharidin toxicity. , , cystitis occurs because renal excretion of cantharidin results in high concentrations in urine. occasionally, hemorrhagic cystitis may occur, with hematuria or frank hemorrhage into the bladder. the cause of the myocarditis and myocardial necrosis is unknown but also may be a direct effect of the toxin on the myocardium. elevated plasma creatine kinase activity often occurs and has been postulated to arise from the damaged myocardium. , horses have a characteristically stiff gait, but histopathologic evidence of skeletal muscle injury that explains the elevated plasma creatine kinase activity has not been observed. the kidneys are often pale, swollen, and moist, with occasional infarcts. hypocalcemia and hypomagnesemia are biochemical features of cantharidin toxicity in horses that have not been explained. , , hypocalcemia may occur from hypoalbuminemia, but the ionized calcium concentration often is decreased along with the total calcium concentration, indicating that hypoalbuminemia is not responsible for the hypocalcemia. in addition, clinical signs of hypocalcemia, such as synchronous diaphragmatic flutter, are often associated with hypocalcemia from cantharidin toxicity. hypocalcemia associated with hypoalbuminemia alone does not produce clinical signs. cantharidin toxicity can cause a range of clinical signs from mild depression and abdominal discomfort to fulminant signs of toxemia and rapid death, depending on the ingested dose of toxin. most commonly, clinical signs include depression, sweating, irritability, abdominal pain, elevated heart and respiratory rates, fever, polyuria, polydypsia, and profuse diarrhea. , , blood is rarely visible in the feces. affected horses frequently posture to urinate; indeed, stranguria and pollakiuria are characteristic of cantharidin toxicity. signs of hypocalcemia include synchronous diaphragmatic flutter and tremors. a stiff and stilted gait may be evident. one may note neurologic signs such as head pressing, swaying, and disorientation. signs of systemic inflammation from endotoxemia may be apparent in severe cases. some horses develop severe depression and toxemia and may die within hours after ingestion of cantharidin without developing diarrhea. , hematologic abnormalities include hemoconcentration and neutrophilic leukocytosis. occasionally, neutropenia and leukopenia may accompany endotoxemia. serum biochemical analysis usually reveals elevated creatine kinase activity, hypocalcemia, and hypoalbuminemia. , biochemical abnormalities include hypocalcemia (ionized and total calcium concentrations), hypomagnesemia, and azotemia. , , urine specific gravity is characteristically in the hyposthenuric range. , microscopic hematuria and mild proteinuria may be evident. fecal occult blood is often present, but hematochezia is unusual. one can make a tentative diagnosis based on clinical signs and the finding of blister beetles in the hay. determining the species of the insects may be necessary to estimate the amount of cantharidin ingested. all species of epicauta contain cantharidin, but some have small amounts. definitive diagnosis requires the measurement of the cantharidin concentration in gastric or intestinal contents and urine. , measurement of cantharidin concentration in the beetles is often done but is not necessary. arsenic toxicosis is an unusual cause of diarrhea in horses, resulting from ingestion of arsenic-containing herbicides, insecticides, and other pest control products contaminating water or roughage used as a food source. the toxicity of arsenic depends on the valence of the element. , arsenate may be reduced to arsenite in mammalian systems, and arsenite is thought to be more toxic than arsenate and less rapidly excreted in urine. arsenate and arsenite uncouple oxidative phosphorylation, leading to breakdown of energy metabolism in the cells of many tissues. widespread cellular injury and death occur rapidly during acute arsenic toxicosis. multiorgan failure usually results. in fact, cardiomyopathy and pulmonary disease are common causes of death in human beings. damage to the large intestine is probably caused in part by direct cellular toxicity and corrosion by the compound. however, vasculitis is a hallmark of the disease in human beings and horses and is thought to be the most important mechanism of large intestinal disease in human beings. , acute hemorrhagic colitis is a feature of arsenic toxicosis, with severe mural edema and mucosal ulceration. profuse, hemorrhagic diarrhea and abdominal pain result. chronic arsenic toxicity can occur but appears to be rare in horses. acute depression, weakness, abdominal pain, hemorrhagic diarrhea, and shock are characteristic of acute arsenic toxicosis in horses. death may occur before diarrhea is evident. initial clinical signs may be difficult to distinguish from other peracute forms of colitis and are related to endotoxic shock, metabolic disturbances, and dehydration. later, cardiac arrhythmias, pulmonary edema, acute renal failure, and neurologic deficits (ataxia and stupor) may develop. one may observe anuria or polyuria. hemolytic anemia caused by preferential binding of arsenic compounds to red blood cells is a feature of arsenic poisoning in human beings. hematologic abnormalities may be apparent after the peracute stages from injury to bone marrow cells and ongoing hemolysis. leukopenia and thrombocytopenia have been described in human patients. serum biochemical analysis may reveal azotemia, hepatocellular enzyme activities higher than generally attributed to endotoxemia, and elevated creatine kinase activity. urine specific gravity may be in the isosthenuric range, with hematuria, cylindruria, and proteinuria evident by urinalysis. diagnosis may be possible by measuring blood and urine arsenic concentration, but these tests may not be diagnostic. postmortem diagnosis is by measuring the arsenic concentration in liver and kidney samples. history of exposure and clinical signs remain the primary means of diagnosis. other disorders associated with diarrhea in adult horses include anaphylaxis, carbohydrate overload, and sand enteropathy. careful evaluation of history, environment, and management will assist the clinician in arriving at an accurate diagnosis. severe intestinal anaphylaxis is a syndrome in horses characterized by peracute, rapidly fatal colitis. the severe syndrome is clinically and pathologically similar to other known causes of peracute colitis, such as salmonellosis, clostridiosis, and antibiotic-associated diarrhea. some cases are less severe and manifest as mild to moderate diarrhea or colic. an ige-mediated type i hypersensitivity or an ige-independent anaphylactoid reaction can produce the syndrome of intestinal anaphylaxis. , local gastrointestinal exposure to a food, environmental contaminant, drug, or other allergen usually induces intestinal anaphylaxis, , but anaphylaxis also may occur with systemic exposure to an allergen. [ ] [ ] [ ] [ ] massive mast cell degranulation, secretion of inflammatory mediators, and activation of enteric neural reflexes in the intestine causes profound alterations in blood flow, increased vascular permeability and interstitial edema, recruitment of neutrophils, altered motility, mucosal injury, absorption of microbial products, and mucosal hypersecretion. [ ] [ ] [ ] [ ] [ ] systemic signs may be caused by the anaphylactic reaction or may be associated with systemic inflammation triggered by microbial products (endotoxin) absorbed through the injured and hyperpermeable mucosa. intestinal anaphylaxis in horses may be a peracute, fulminant enterocolitis with endotoxemia that may be fatal. , this form is characterized by severe intramural edema and hemorrhagic inflammation of the large intestine, often producing submucosal thickening on the order of many centimeters. vascular thrombosis may be widespread with mucosal and serosal petechia and ecchymoses. less severe forms of intestinal anaphylaxis may manifest as patchy areas of intestinal edema and congestion. diarrhea results from intestinal inflammation initiated by the type i hypersensitivity response. many of the mediators of type i hypersensitivity, such as histamine and -hydroxytryptamine, have well-documented stimulatory effects on mucosal secretory activity, vascular and epithelial permeability, and motility [ ] [ ] [ ] in the intestine. systemic inflammation from endotoxemia may be overwhelming once the mucosal barrier breaks down. infarction of intestinal segments and other organs may occur from intravascular coagulation. ileus, abdominal distention, and moderate to severe abdominal pain may result from motility disturbances and infarction of the large intestine. the clinical signs are similar to those described for other forms of peracute colitis. however, the severity may vary, manifesting as colic or moderate diarrhea. characteristically, severe shock, signs of systemic inflammation from endotoxemia, and severe metabolic disturbances are observable. , heart and respiratory rates may be elevated greatly, with other signs of cardiovascular collapse such as weak and thready peripheral pulses and peripheral vasoconstriction. however, peripheral vasodilation may occur later in the course of disease. dark red, muddy, or cyanotic mucous membranes with a prolonged capillary refill time signify sepsis. borborygmi are usually absent, and abdominal tympany may be heard on percussion, following ileus. moderate to severe colic may accompany ileus. severe diarrhea may occur, but death may occur before diarrhea is evident. multiorgan failure from disseminated intravascular coagulation is not unusual. rapid onset of weakness, staggering, and trembling commonly precedes death. the syndrome may cause death in to hours. hematologic abnormalities include severe neutropenia and leukopenia, thrombocytopenia, and hemoconcentration. serum biochemical alterations include hyponatremia, hypokalemia, hypocalcemia, and severe metabolic acidosis. blood urea nitrogen and creatinine may be elevated from prerenal or renal azotemia. if acute renal failure accompanies the colitis, hyperkalemia may result. hepatocellular enzyme activity may be elevated in the serum from endotoxemia. severe coagulopathies are common, resulting in prolonged coagulation times, elevated fibrinogen, decreased antithrombin iii activity, and elevated plasma concentration of fibrin degradation products. analysis of peritoneal fluid may be valuable because infarction of the large intestine is not unusual. protein concentration and the white blood cell count may be elevated. red blood cell counts are less likely to be elevated, because infarction and not strangulation of the intestine occurs. diagnosis is based on clinical signs, postmortem findings, and exclusion of other causes. cultures and toxicologic analysis of fecal samples and gastrointestinal tissues fail to demonstrate a clear cause. other diagnostic tests are also inconclusive. if an antigen is suspected as the trigger of the anaphylaxis, a prausnitz-küstner passive cutaneous anaphylaxis sensitization test can confirm the presence of antigen-specific ige in the patient serum. overeating of soluble carbohydrates, especially so-called hot grains such as corn, overwhelms the digestive capability of the small intestine, resulting in a high percentage of the soluble carbohydrates entering the large intestine. the amount of soluble carbohydrates that produce diarrhea varies according to the previous dietary history of the individual. horses fed diets higher in soluble carbohydrates are more resistant to the deleterious effects of carbohydrate overload. gradual accommodation to a diet high in carbohydrates can be accomplished over several weeks. however, horses fed an unusually large amount of grains or other form of soluble carbohydrates often develop diarrhea and may, depending on the amount ingested, develop severe colitis, systemic inflammation from endotoxemia, metabolic acidosis, and laminitis. [ ] [ ] [ ] [ ] the pathogenesis of colitis from carbohydrate overload is caused primarily by the toxic effects on the microbial flora in the large intestine. a sudden delivery of soluble carbohydrates to the large intestine causes rapid fermentation by gram-positive lactic acid-producing bacteria and a sudden increase in organic acid production. the cecal ph rapidly decreases, and the lactic acid concentration rapidly increases. rapid organic acid production overwhelms the buffering capacity of the large intestine not only by directly depleting the buffers found in the contents but also by reducing the efficiency of buffer secretion. bicarbonate secretion is linked to absorption of volatile fatty acids, which are produced in low amounts by fermentation of soluble carbohydrates. the contents of the large intestine become profoundly acidic, resulting in unfavorable conditions for the microbial flora. lactic acid-producing bacteria flourish, while the gram-negative bacteria, especially the enterobacteriaceae, are killed in large numbers by the acids. large quantities of endotoxin are released from the dying bacteria. the osmotic load from the lactic acid produced in the large intestine is an important factor in the development of diarrhea because organic acids such as lactic acid are absorbed poorly. mild cases of carbohydrate overload may result purely from osmotic diarrhea. in more severe cases, the acidic contents of the large intestine are toxic to the mucosa, causing necrosis of the mucosal tissues, similar to that occurring in ruminal acidosis. mucosal ulceration allows absorption of large quantities of endotoxin and lactic acid produced by the massive die-off of acid-intolerant microbes and fermentation of soluble carbohydrates, normally poorly absorbed by intact mucosa. systemic inflammation from endotoxemia may be overwhelming, and profound metabolic acidosis may occur. secretory diarrhea caused by the direct effects of acid luminal contents on the mucosa, as well as the effects of inflammatory mediators on enterocyte secretion, worsens the acidosis and dehydration. systemic inflammation from endotoxemia, along with intestinal inflammation, adversely affects intestinal motility, and ileus develops. ileus and gas production from fermentation of the carbohydrates may cause severe distention of the large intestine and signs of abdominal pain. laminitis is a frequent complication of endotoxemia and lactic acidosis. in fact, carbohydrate overload is used to induce laminitis as an experimental model because of the consistency of the laminitis produced. [ ] [ ] [ ] clinical signs of colitis from carbohydrate overload can vary according to the amount of carbohydrates ingested and accommodation of the flora to a high-carbohydrate diet. mild cases may result in a transient osmotic diarrhea with no systemic effects. more severe cases are characterized by signs similar to those described for other forms of colitis, including abdominal pain, moderate to severe diarrhea, and dehydration. signs of endotoxemia and sepsis are frequently present in severe cases. elevated heart and respiratory rates are common, with peripheral vasoconstriction early in the disease, followed by peripheral vasodilation as the disease progresses. depression may be profound from metabolic acidosis and endotoxemia. abdominal auscultation and percussion may reveal ileus and intestinal tympany. nasogastric intubation may yield significant gastric acidic reflux. one may note particles of grain in the gastric reflux and the feces, if grain overload is the source of the carbohydrate overload. laminitis may complicate mild and severe cases of carbohydrate overload, especially if the animal has had previous bouts of laminitis. hematologic abnormalities include neutropenia and leukopenia. severe dehydration may result in profound hemoconcentration. protein loss later in the course of disease may result in hypoproteinemia. serum biochemical abnormalities include azotemia, elevated hepatocellular enzyme activity, hyponatremia, and hypokalemia. severe hypocalcemia and metabolic acidosis are characteristic of the disease. serum lactate concentrations are elevated in the absence of evidence of intestinal strangulation or infarction. peritoneal fluid analysis often reveals no abnormalities. sand enteropathy is described in more detail under the heading of obstructive diseases, because acute obstruction is often associated with abnormally large amounts of sand in the large intestine. however, chronic sand-induced diarrhea is a distinct syndrome that can occur at any age from abnormal accumulation of sand in the large intestine. , chronic diarrhea and signs of colic may occur without obstruction. the pathogenesis of sand accumulation in individual horses, other than simple ingestion of large quantities, is unclear. presumably the sand causes irritation and may disrupt motility, leading to diarrhea. the diarrhea is usually not severe and dehydrating and may be intermittent. weight loss is characteristic and can be severe in some cases. complications may occur such as peritonitis and acute obstruction. diagnosis usually is based on finding abnormal amounts of sand in the feces. because sand-induced chronic diarrhea is associated primarily with sand accumulation in the ventral colon, auscultation of the ventral abdomen immediately behind the xiphoid process may reveal characteristic sand sounds. this technique is only sensitive if peristalsis is present. ultrasonography also may be useful to identify sand in the ventral colon but is not useful to quantitate the amount of sand. occasionally, radiography may be required to detect sand in the colon. the principles of therapy of acute diarrhea from colitis are similar regardless of the cause and include replacement of fluid and electrolyte losses, control of colonic inflammation and reduction of fluid secretion, promotion of mucosal repair, control of endotoxemia and sepsis, and reestablishment of normal flora. this section focuses on a review the principles of therapy with references to specific therapies for particular causes as they arise. replacement of fluid and electrolyte losses is of primary concern in treating horses with salmonellosis. depending on the severity of the disease, fluid losses may be minimal or massive. one can administer fluid and electrolytes orally or intravenously. some horses with mild to moderate diarrhea may maintain hydration and electrolyte balance by consuming water and electrolytes voluntarily. freshwater and water containing electrolytes should be available in all cases. in many instances, periodic nasogastric intubation and administration of water and electrolytes via the tube may be sufficient to maintain hydration. in more severe cases, one can maintain indwelling nasogastric tubes and can administer up to to l of fluid by the tube every to minutes, if ileus is not evident. however, intravenous administration of fluids is preferred in most cases, requiring significant quantities of fluid to replace and maintain hydration and electrolyte balance. for patients with severe diarrhea to require large volumes ( to l/day) of intravenous fluids to maintain hydration is not unusual. frequent monitoring of packed cell volume, serum electrolyte concentration, venous blood gases or total serum carbon dioxide, blood urea nitrogen and creatinine, urine protein and cytologic findings, and body weight is important to monitor hydration, electrolyte and acid-base balance, and renal function. isotonic sodium chloride or lactated ringer's solution frequently is used to restore and maintain fluid and electrolyte balance. one can add potassium chloride to the fluids and administer it at a rate up to . to . meq/ kg/hr. generally, a rate of less than . meq/kg/hr is used. hypertonic nacl solutions ( to l of % to % nacl) have been used in horses that are severely hyponatremic (< meq/dl). one should not administer hypertonic solutions to severely dehydrated horses, but such solutions have been used clinically without complication and with considerable beneficial effect in patients with endotoxemia. the beneficial effects of hypertonic nacl are short-lived ( to minutes). one should administer isotonic solutions concurrently or immediately following administration of hypertonic nacl solutions. isotonic ( . %) or hypertonic ( . %) sodium bicarbonate solutions are used to correct metabolic acidosis. prolonged administration of sodium-containing fluids may promote diuresis and renal water loss or accumulation of peripheral edema and should be used conservatively when one notes a free water loss. administration of isotonic dextrose ( %) or . % dextrose/ . % nacl solutions may be beneficial when free water loss (sodium excess) is evident. many horses with acute colitis are concurrently hypoproteinemic because of gastrointestinal losses and are absorbing bacterial products that induce a systemic inflammatory response. thus plasma oncotic pressures are abnormally low in the face of increased vascular permeability. interstitial edema formation is a clinical problem in these patients and contributes to organ dysfunction. crystalloid fluids, although critical for replacing water and electrolyte losses from diarrhea, actually may contribute to a drop in plasma oncotic pressure because of hemodilution. , administration of colloid solutions are important for volume expansion and to maintain plasma oncotic pressures, which improve tissue perfusion and oxygenation and organ function in hypovolemic, hypotensive, and hypoproteinemic patients with or without systemic inflammatory response syndrome. colloids are more effective than crystalloid fluids at expanding plasma volume and thus require smaller volumes. moreover, the effect of colloid volume expansion is longer lasting than crystalloid fluid volume expansion, because colloids are retained in the vasculature better. , natural colloids, such as plasma and purified albumin are used commonly. in addition to its beneficial colloidal properties, plasma harvested from donor horses immunized with rough mutants of escherichia coli (j ) or salmonella typhimurium may have other benefits for treatment of endotoxemia from gastrointestinal disease. , the horse may require large volumes ( to l/day) to increase and maintain plasma protein concentration significantly. synthetic colloids such as dextrans, starches, or polymerized hemoglobin are also available for use in the horse. hetastarch ( to ml/kg of a % solution) increases colloidal oncotic pressures for up to hours in hypoproteinemic horses and has beneficial effects on cardiac output and other cardiorespiratory parameters, vascular permeability, interstitial fluid content, and tissue perfusion in models of hypoproteinemia and systemic inflammatory response syndrome. when one administers synthetic or even natural colloids, monitoring plasma oncotic pressure may be more relevant than monitoring plasma protein concentrations as a means of assessing the need for plasma or other colloid administration. hetastarch may prolong bleeding times by altering von willebrand's factor function; thus one should use this synthetic colloid cautiously in horses with suspected coagulopathies, active hemorrhage, or other bleeding problems. control of colonic inflammation and secretion is a difficult and poorly studied aspect of equine acute colitis. the role of inflammation and mediators such as prostaglandins as causes of fluid loss is well known for salmonella and clostridium infections. cox inhibitors (nsaids) have antisecretory effects in the equine colon and in models of salmonellosis that appear to extend to clinical management of salmonellosis. , , [ ] [ ] [ ] indeed, nsaids commonly are administered to horses with salmonellosis. however, prostaglandins such as pge and pgi are also cytoprotective to gastrointestinal mucosa and critical for mucosal repair. the doses of nsaids used pharmacologically to inhibit colonic inflammation and secretion in fact may be detrimental to the mucosa if not used judiciously. nsaids have been shown to exacerbate colonic inflammation in human beings with inflammatory colitis, impede mucosal healing in several models of mucosal injury, and have well-documented detrimental effects on colonic mucosa in horses. , , in addition to toxicity to the colonic mucosa, gastric ulceration is not unusual in horses with enterocolitis and may be related to treatment with nsaids. in addition to nsaids, other drugs occasionally are used as antiinflammatory or antisecretory therapy. metronidazole has beneficial effects in experimental models of gastrointestinal inflammation, including nsaid toxicity and may be useful for treating horses with colitis, but evidence supporting its use is lacking. bismuth subsalicylate solutions administered orally often are used to decrease inflammation and secretion in the colon. in adult horses the volume of solution necessary to be beneficial is large ( to l every to hours). often the solution is administered twice daily instead of to times daily. if one does not achieve a beneficial effect within to days of treatment, one should discontinue administration of bismuth subsalicylate solution. one can administer the treatment more frequently in foals, and clinical improvement occurs more often in foals than in adult horses. in light of the role of reactive oxygen metabolites in colonic inflammation, free radical scavengers have been advocated to reduce the effects of these molecules. sulfasalazine metabolites have been shown to reduce reactive oxygen metabolite-induced colonic inflammation in other species, and sulfasalazine has been used to treat chronic inflammatory disease in horses but has not been used to treat acute colitis. the only free radical scavenger used commonly in horses with colitis is dimethyl sulfoxide, which at a dosage of . to . g/kg intravenously every to hours in a % solution has been used in clinical cases of colitis, but evidence of efficacy has not been established. systemic inflammatory response syndrome associated with endotoxemia frequently occurs in patients with salmonellosis. the principles of therapy for endotoxemia are covered in detail elsewhere in this chapter. oral administration of activated charcoal and mineral oil is used commonly to reduce absorption of endotoxin in horses with colitis. low doses of nsaids (such as flunixin meglumine at . to . mg/kg intravenously every to hours) inhibit eicosanoid synthesis induced by endotoxin. in addition, administration of nsaids prevents laminitis from endotoxemia, a devastating complication of salmonellosis. one must remember that prostaglandins are important for mucosal healing and may worsen mucosal injury in colitis. although the benefits of low doses of nsaids administered to horses with systemic inflammatory response syndrome are believed to outweigh the risks of worsening gastrointestinal damage, judicious use is recommended. sucralfate ( mg/kg orally every hours) has been advocated to aid in healing the colonic mucosa, but the efficacy in the large intestine is questionable. misoprostol ( µg/kg orally to times daily) and other synthetic pge analogs have been shown in several species including horses to enhance mucosal healing in the intestine and promote recovery in experimental models of colitis. misoprostol may be particularly useful for treating nsaid toxicity, the generalized form or rdc. however, the efficacy of misoprostil for hastening mucosal healing is clinically unproven in equine colitis. the primary drawbacks of prostaglandin analogs such as misoprostol are the side effects of the drug, including abdominal cramping, diarrhea, sweating, and abortion in pregnant mares. one can add psyllium mucilloid to the diet ( tablespoons once or twice daily) to increase the production of scfas in the colon. amylase-resistant fermentable fiber such as psyllium is hydrolyzed by colonic bacteria to scfas such as butyrate, which represent a major energy source for colonocytes. butyrate and other scfas hasten epithelial maturation and stimulate salt (and thus fluid) absorption in the colon, improve the clinical course of ulcerative colitis, and hasten colon healing. psyllium is itself a source of butyrate in the colon and also promotes the movement of amylase sensitive carbohydrates into the distal colon, which then are fermented to scfas. thus psyllium is thought to be clinically useful for promoting mucosal healing in colitis. many horses with salmonellosis or other forms of colitis have mild to severe signs of abdominal pain from gas and fluid distention of the colon, colonic ischemia, or infarction. one can accomplish analgesia with nsaids such as flunixin, but the potential for worsening mucosal injury or nephrotoxicity may prevent the use of analgesic doses, especially in horses with suspected nsaid toxicity. newer nsaids that specifically target cox- (the inducible cox) but have little activity against cox- (the constitutive cox) may be useful analgesics that spare the gastrointestinal mucosa. for example, etodolac ( to mg/kg intravenously or orally once daily) has analgesic properties in horses and may spare the intestinal mucosa from the detrimental effects associated with nonselective cox inhibitors (a.t. blikslager, personal communication, ) . however, the specificity for cox- in horses is unproven. thus avoiding the use of any nsaids in horses with rdc or other forms of nsaid toxicity is advisable. xylazine or detomidine may provide temporary relief of pain. butorphanol is a useful analgesic that one can administer intramuscularly ( . mg/kg every hours) or as a continuous infusion. an infusion of . µg/kg/hr in isotonic crystalloid fluid such as lactated ringer's solution has been suggested. continuous lidocaine infusions ( . mg/kg intravenous loading dose administered slowly over minutes and followed by mg/kg/hr infusion in isotonic crystalloid fluids) can provide profound visceral analgesia and may have added prokinetic benefits if ileus is present. broad-spectrum antibiotic treatment often is recommended in neutropenic horses or horses with signs of septicemia. neutropenia is associated with an increased risk of septicemia and septic complications such as septic phlebitis and infection of surgical site. septicemia is a potentially life-threatening complication of enterocolitis and may be caused directly by salmonella, clostridium, other invasive enteric bacteria, or indirectly by toxic injury to the colonic mucosa that breaks down the barrier to luminal microbes. neutropenia possibly may weaken host defenses enough to render horses susceptible to organisms that breach the mucosal barrier. although most attempts to culture bacteria from the blood of adult horses with colitis fail to isolate organisms, no detailed studies have been undertaken to determine the prevalence of bacteremia or septicemia in these patients. disseminated aspergillosis has been reported in horses as a complication of acute colitis, demonstrating the potential for systemic infections with rarely pathogenic organisms stemming from colonic mucosal injury in the face of potential immunosuppression from neutropenia. , broad-spectrum antibiotics lessen septic complications in human patients. however, evidence supporting this principle in horses with colitis is lacking. treatment with antibiotics is controversial in horses with salmonellosis and is not thought to alter the course of the enterocolitis. antibiotics directly targeted at the salmonella are reserved for patients with the enteric fever (septicemia) form of salmonellosis, documented with positive blood cultures. lipid-soluble antibiotics are suited ideally for salmonella infections, because the bacteria persist intracellularly. trimethoprim-sulfadiazine or other potentiated sulfa drugs, enrofloxacin, and chloramphenicol are preferred antibiotics for the enteric fever form of salmonellosis for this reason. as with other causes of enterocolitis, the use of antibiotics for equine monocytic ehrlichiosis is controversial. fear of inducing salmonellosis or other forms of antibiotic-induced diarrhea and the difficulty of diagnosing the disease early have caused most authors to recommend judicious use of antibiotics. however, in patients with a high suspicion of neorickettsia risticii infection, treatment with antibiotics often is indicated before definitive diagnosis. lipid-soluble drugs are desirable because the organism can live within cells. oxytetracycline ( . mg/kg intravenously every hours), doxycycline ( mg/kg orally every hours), trimethoprim-sulfadiazine ( mg/kg trimethoprim orally or intravenously every to hours and mg/kg sulfadiazine every to hours), or erythromycinrifampin ( mg/kg and mg/kg, respectively, orally every hours) have been used effectively to treat clinical cases. , [ ] [ ] [ ] the tetracyclines appear to be the most effective antibiotics for treatment of potomac horse fever. treatment is most successful if initiated before the onset of diarrhea. , clostridiosis if one has administered antibiotics since the onset of enterocolitis, one should discontinue administration as soon as possible. specific treatment with metronidazole ( to mg/kg orally every hours) is effective for treating clostridiosis in human beings and appears to be effective in horses. , metronidazole resistance in clinical isolates of clostridium difficile has been reported in one outbreak but appears to be rare in most human and equine cases. metronidazole-resistant isolates were sensitive to vancomycin, which may be effective for treating clinical cases if one suspects metronidazole resistance. however, metronidazole remains the treatment of choice. some authors describe the off-label use of c. perfringens type c antitoxin in cases of neonatal clostridiosis, described in more detail elsewhere. antitoxin preparations generally are not advocated for use in adult horses with clostridiosis. lawsonia intracellulare is susceptible to a variety of antibiotics in vitro, including chlortetracycline, erythromycin, penicillin, difloxacin, and ampicillin. lipid-soluble antibiotics with a large volume of distribution usually are chosen to treat proliferative enteropathy because l. intracellulare is an intracellular organism. erythromycin estolate ( to mg/kg orally every to hours) alone or with rifampin ( mg/kg orally every hours) is the most commonly reported efficacious treatment for proliferative enteropathy. chloramphenicol ( mg/kg orally every hours) has also been reported to be effective if erythromycin worsens the diarrhea. anecdotal reports suggest that oxytetracycline and doxycycline also may be effective. supportive care including maintenance of hydration and electrolyte balance and plasma or colloid administration to increase colloid oncotic pressure in hypoalbuminemic patients is also indicated. one should treat affected foals until clinical signs, hypoproteinemia, and ultrasonographic evidence of intestinal thickening resolve. the prognosis depends on the duration of the disease and the degree of fibrosis and destruction of the intestinal architecture. hypercoagulability is a common complication of enterocolitis, associated with systemic inflammation from endotoxemia. administration of heparin ( to iu/kg subcutaneously or intravenously every to hours) may prevent thrombosis in these patients, provided antithrombin iii concentrations are adequate in the plasma. concentrated sources of antithrombin iii are not available for use in horses, but whole plasma may provide an important source. treatment with heparin is thought to decrease thrombosis, especially of the jugular vein, a serious complication of salmonellosis. low-dose aspirin treatment ( mg/kg orally every to hours) along with heparin treatment may provide added benefit by irreversibly inhibiting platelet function. heparin and aspirin may have protective effects on the digital lamina. , heparin also may enhance the phagocytic activity of the reticuloendothelial system by enhancing the efficiency of opsonins such as fibronectin and immunoglobulin, thereby stimulating phagocytosis of products of coagulation and possibly other particles, including bacteria. , maintenance of the bacterial flora and antagonism of pathogenic bacteria such as salmonella in the gastrointestinal tract are important defense mechanisms preventing colonization by pathogenic bacteria. the use of probiotic preparations containing beneficial bacteria has been shown to prevent colonization of pathogenic bacteria, including salmonella, in poultry. little work has been done to investigate the efficacy of these products in preventing salmonellosis in horses, but ongoing studies may provide important information. probiotic and other preparations designed to restore normal flora to the gastrointestinal tract, such as fecal suspensions, sour milk, and yogurt, have been used clinically to shorten the course of salmonellosis, with variable results. therefore prevention of infection by using probiotic agents and other means is important. exposure of susceptible horses to salmonella should be avoided, but the task is difficult, especially because asymptomatic infections are common and the bacteria are ubiquitous in the environment. prophylactic use of probiotic preparations, judicious use of antibiotics in susceptible horses, control of environmental conditions such as temperature, and restricted exposure to pathogenic bacteria are important for control of salmonellosis. because altered large intestinal flora appears to play an important role in the pathogenesis of equine intestinal clostridiosis or any antibiotic-associated diarrhea, probiotic preparations have been advocated to treat affected horses. sour milk, a product containing lactose-producing streptococcus species, appears to improve the clinical course greatly in horses suspected of having clostridium perfringens type a infection. sour milk may benefit the patient by altering the flora and antagonizing enterotoxigenic c. perfringens type a but also is reported to be bactericidal against c. perfringens type a. preparations of saccharomyces boulardi are effective for reducing diarrhea and the frequency of c. difficile recurrence in human beings. however, whether relapse is a problem in horses with c. difficile colitis is not clear. lactobacillus preparations have a protective effect in human beings and decrease the severity and duration of antibioticassociated diarrhea. , however, evidence of their clinical usefulness in horses is lacking. good nursing care and adequate nutrition are vital to the treatment of horses with salmonellosis. salmonellosis is a severely catabolic disease, increasing caloric requirements greatly. normal intake of roughage to provide energy may be inadequate; however, one should avoid feeding of grains to prevent carbohydrate overload. dietary management usually consists of restricting or eliminating long-stem roughage (hay) from the diet and feeding exclusively a complete pelleted diet (at least % dietary fiber). the rationale behind this recommendation is to reduce the mechanical load on the colon. frequent meals ( to times a day) are recommended. one can add corn oil ( cup every to hours) to the pellets to increase the caloric intake without adding roughage or grain. one should note that if a horse with colitis refuses to eat pelleted feed, then one should feed good-quality grass hay. in anorectic or severely catabolic patients, enteral and parenteral nutrition (total and partial) has been used successfully to provide calories and nutritional support. strongylus vulgaris infection requires treatment of the migrating parasite larvae and the lesions produced by the parasite. fenbendazole ( mg/kg orally every hours for days or mg/kg orally every hours for days) and ivermectin ( mg/kg orally) are effective in killing fourth-stage larvae. other anthelmintics also may be part ii disorders of specific body systems effective when given at higher doses than those required to kill adult worms. the efficacy of these anthelmintics against larvae within thrombi is not known. thrombolytic and antithrombotic therapy has been advocated in horses with suspected strongylosis. , heparin ( to iu intravenously or subcutaneously every to hours) is often administered as an anticoagulant. aspirin ( to mg/kg orally every to hours) is usually combined with heparin to inhibit platelet adhesion. aspirin also may inhibit release of platelet products such as thromboxane that affect the motility of the large intestine. low-molecular-weight dextrans have been advocated as antithrombotics that act by inhibiting platelet function and coagulation. , the clinical efficacy of dextran administration appears to be good, but no controlled studies have been performed. anthelmintic administration is usually the only treatment necessary for mild to moderate cases of cyathostomiasis treated early in the course of the disease (within to weeks of onset). fenbendazole is effective against many larval stages, but resistance is increasing. although the reported efficacy of ivermectin varies against certain stages, one study reported an overall efficacy of %. currently, fenbendazole ( . to mg/kg orally every hours for days) followed on day by ivermectin ( mg/kg orally) is the most commonly advocated treatment regimen. , moxidectin ( µg/kg orally once daily) also may be effective against adults and l and l larval stages and may be useful for treating cyathostomiasis. antiinflammatory therapy also may be beneficial, especially in severe or refractory cases. nsaid administration may have limited value, but dexamethasone appears to be efficacious in refractory cases when used with larvicidal anthelmintics. , pretreatment with dexamethasone or prednisolone is indicated before anthelmintic administration if heavy larval loads are suspected to prevent an acute exacerbation of the disease by rapid death of encysted larvae. bismuth subsalicylate often is administered orally as an antisecretory agent in young animals. supportive care may be necessary in severe cases, particularly if hypoproteinemia is severe. horses occasionally require administration of intravenous crystalloid fluids and plasma or other colloids. proper nutritional support is also important. supportive care is the most important principle of therapy for cantharidin toxicity. intravenous fluid administration; maintenance of electrolyte balance, especially calcium; and prevention of further renal and urinary tract damage is important. , diuresis by intravenous fluid administration is often sufficient to prevent renal failure. furosemide often is administered after rehydration of the patient to further promote diuresis and to decrease the concentration of the toxin in the urine, which may ameliorate some of the effects on the urinary tract mucosa. diuresis also has been suggested to increase clearance of the toxin, but no evidence for this has been found. judicious use of nsaids may be necessary to control abdominal pain but should be reserved until the patient is rehydrated and renal failure has been ruled out. cantharidin is lipid-soluble; therefore oral administration of mineral oil may prevent further absorption of the toxin. activated charcoal often is administered with the mineral oil. to reduce arsenic absorption, one should initiate administration of cathartics such as mineral oil and magnesium sulfate slurries and activated charcoal by nasogastric tube immediately. chelation therapy with sodium thiosulfate to g in ml of water orally and dimercaprol (bal) mg/kg intramuscularly every hours is indicated. dimercaprol is a specific antidote for trivalent arsenicals, but its efficacy in horses is questionable. intravenous fluid administration may help treat shock, replace fluid lost in feces, and promote diuresis but should be monitored carefully because pulmonary edema is a frequent complication. the horse may require more specific treatment of renal, cardiac, pulmonary, or neurologic disease. treatment of intestinal anaphylaxis is in principle similar to treatment of other forms of colitis but is often unsuccessful because of the rapidly progressive nature of the syndrome. inclusion of heparin in intravenous fluids ( to iu/kg intravenously every to hours) may help prevent vascular thrombosis. administration of hypertonic saline solutions or colloids may prove to be useful during initial periods of shock. early treatment with prednisolone succinate ( to mg/kg intravenously) or dexamethasone ( . to . mg/kg intravenously) may be essential for successful treatment. mild cases of carbohydrate overload may not require treatment other than exclusion of grains from the diet for several days to weeks and gradual reintroduction of grain into the diet later if the horse needs the extra energy. patients showing signs of colic or diarrhea without other systemic signs may benefit from administration of mineral oil, charcoal, and fluids via nasogastric tube. one also may lavage residual carbohydrates from the stomach with the nasogastric tube. nsaids such as phenylbutazone ( . to . mg/kg/day intravenously) or flunixin meglumine ( mg/kg intravenously every hours) often are administered to prevent laminitis. phenoxybenzamine and heparin given before the onset of laminitis may prevent or decrease the severity of laminitis. , more severe cases with dehydrating diarrhea, systemic signs of endotoxemia, or metabolic acidosis require intravenous fluid support to maintain water, electrolyte, and acid-base balance in addition to the previously mentioned treatments. large amounts of bicarbonatecontaining solutions may be required. one should take care when administering hypertonic bicarbonate solutions, because many patients already may be hyperosmotic from lactic acidemia. isotonic sodium bicarbonate . % may be useful in the hyperosmotic patient. careful attention to calcium balance is also important, because severe hypocalcemia may occur. one should institute aggressive therapy for systemic inflammation from endotoxemia. one should administer broad-spectrum antibiotics intravenously to combat bacteremia and septicemia, which frequently complicate colitis induced by carbohydrate overload. in extreme cases, especially if the patient has ingested a large quantity of grain, surgical removal of the grain from the large intestine may be indicated, especially if one can accomplish surgery before the onset of severe clinical signs. however, administration of oral cathartics, such as magnesium sulfate slurries or mineral oil, or a combination of these, is often sufficient to clear the carbohydrates from the large intestine before fermentation, mucosal damage, and absorption of endotoxin and lactic acid occur. oral administration of activated charcoal may prevent absorption of endotoxin by binding the molecules in the lumen of the bowel. in any case, one should discontinue feeding of the source of the soluble carbohydrates, such as grains. one should feed the horse low-carbohydrate and low-protein roughage such as grass or oat hays until the microbial flora recovers. oral administration of probiotic preparations containing lactobacillus is contraindicated; however, other sources of normal equine large intestinal microbial flora, such as fecal extracts from normal feces, may be useful to reintroduce appropriate microorganisms. complications from laminitis and sepsis are common and often cause death. treatment of sand enteropathy requires removal of the sand from the gastrointestinal tract using psyllium products and magnesium sulfate slurries administered orally. analgesics may be required initially to relieve pain and stimulate appetite. a diet high in roughage often stimulates further passage of sand. treatment may require several weeks to remove as much sand as possible. prevention of the disease is important, and recurrence is not unusual. lumen results in clinical signs similar to those of simple obstruction, occlusion of the blood supply results in a more rapid deterioration of the intestinal mucosa and subsequent onset of endotoxemic shock. although a great deal of interest in the relevance and treatment of intestinal reperfusion injury has arisen recently, - the lesion that develops during strangulation is often severe, leaving little viable bowel for further injury during reperfusion. although extensive lengths of strangulated small intestine may be resected, strangulation of the large colon presents a much greater treatment dilemma because strangulated intestine usually extends beyond the limits of surgical resection. therefore horses with large intestinal strangulation often recover with extensive intestinal injury left in place. thus subtle degrees of reperfusion injury may be important in horses with large colon disease, warranting further work in this area in an attempt to reduce mortality. strangulating obstruction may be divided into hemorrhagic and ischemic forms. , hemorrhagic strangulating obstruction, which is most common, involves initial occlusion of veins before occlusion of arteries because of the greater stiffness of arterial walls. this lesion is characterized by a darkened appearance to affected bowel and increased thickness as blood is pumped into the lesion. ischemic strangulating obstruction occurs if the intestine is twisted tightly enough to occlude arteries and veins simultaneously. in the case of the colon, such strangulation has been suggested to be determined by how much ingesta is in the colon, because intestinal contents may prevent the intestine from twisting tightly. tissue involved in ischemic strangulating obstruction appears pale and of normal or reduced thickness because of a complete lack of blood flow ( figure . - ). bowel peripheral to strangulating lesions also may become injured because of distention, which reduces mural blood flow once it reaches critical levels. furthermore, as this intestine is decompressed, it also may undergo reperfusion injury. [ ] [ ] [ ] small intestinal strangulation horses with small intestinal strangulating obstruction typically have moderate to severe signs of abdominal pain that are only intermittently responsive to analgesic medications. during the latter stages of the disease process, horses may become profoundly depressed rather than painful as affected intestine necroses. horses have progressive signs of endotoxemia, including congested mucous membranes, delayed capillary refill time, and an elevated heart rate (> beats/min in most cases). in addition, one typically obtains reflux following passage of a stomach tube, and one usually can detect loops of distended small intestine on rectal palpation of the abdomen. however, these latter findings vary depending on the duration and location of the obstruction. for example, horses with ileal obstructions tend to reflux later in the course of the disease process than horses with a jejunal obstruction. furthermore, a horse that has an entrapment of small intestine in the epiploic foramen may not have palpable loops of small intestine because of the cranial location of these structures. abdominocentesis can provide critical information on the integrity of the intestine and is indicated in horses in which one suspects strangulation of the small intestine. a horse that has signs compatible with a small intestinal obstruction and additionally has serosanguinous abdominal fluid with an elevated protein level (> . mg/dl) is likely to require surgery, although one must differentiate these a b figure . - ischemic strangulating obstruction of the small colon by a mesenteric lipoma. a, the lipoma (arrow) has encircled a segment of small colon tightly. b, following resection of the lipoma, a pale area of strangulated small colon clearly is demarcated (arrows), the appearance of which is consistent with ischemic strangulating obstruction. cases from proximal enteritis. in general, horses with small intestinal strangulation show continued signs of abdominal pain, whereas horses with proximal enteritis tend to be depressed after initial episodes of mild abdominal pain. in addition, horses with small intestinal strangulation continue to deteriorate clinically despite appropriate medical therapy and will likely begin to show an increased white blood cell count (> , cells/µl) in the abdominal fluid as the duration of strangulation increases. however, cases occur in which the differentiation between small intestinal strangulation and proximal enteritis is not clear, at which point one may elect surgery rather than risking delay of abdominal exploration of a horse with a potential strangulating lesion. the prognosis for survival in horses with small intestinal strangulating lesions is generally lower than for most forms of colic. however, recent studies indicate that in excess of % of horses with small intestinal strangulating lesions are discharged from the hospital. nonetheless, veterinarians should warn owners that the long-term survival rates are reduced substantially to below %, in part because of long-term complications such as adhesions. , in addition, the prognosis is particularly low for some forms of strangulation, including entrapment of small intestine within a mesenteric rent. the epiploic foramen is a potential opening (because the walls of the foramen are usually in contact) to the omental bursa located within the right cranial quadrant of the abdomen. the foramen thus is bounded dorsally by the caudate process of the liver and caudal vena cava and ventrally by the pancreas, hepatoduodenal ligament, and portal vein. intestine may enter the foramen from the visceral surface of the liver toward the right body wall or the opposite direction. studies differ as to which is the most common form. , in the case of entrapments that enter the foramen in a left-to-right direction, the omental bursa ruptures as the intestine migrates through the epiploic foramen, which may contribute to intraabdominal hemorrhage often seen with this condition. clinical signs include acute onset of severe colic with examination findings compatible with small intestinal obstruction. the condition tends to be more prevalent in older horses, possibly because of enlargement of the epiploic foramen as the right lobe of the liver undergoes ageassociated atrophy. however, the disease also has been recognized in foals as young as months of age. one makes a definitive diagnosis at surgery, although ultrasonographic findings of distended loops of edematous small intestine adjacent to the right middle body wall suggest epiploic foramen entrapment. in general, thickened, amotile intestine on ultrasonographic examination is highly predictive for small intestinal strangulating obstruction. small intestine entrapped in the epiploic foramen may be limited to a portion of the intestinal wall (parietal hernia), and the large colon may become entrapped within the epiploic foramen. in treating epiploic foramen entrapment, one must not enlarge the epiploic foramen by blunt force or with a sharp instrument, because rupture of the vena cava or portal vein and fatal hemorrhage may occur. prognosis has improved substantially over the last decade, with current short-term survival rates (discharge from the hospital) ranging from % to %. preoperative abdominocentesis has been found consistently to be the most predictive test of postoperative survival. , lipomata form between the leaves of the mesentery as horses age and develop mesenteric stalks as the weight of the lipoma tugs on the mesentery. the stalk of the lipoma subsequently may wrap around a loop of small intestine or small colon causing strangulation. one should suspect strangulating lipomata in aged (> years old) geldings with acute colic referable to the small intestinal tract. , ponies also appear to be at risk of developing disease, suggesting alterations in fat metabolism may predispose certain horses to development of mesenteric lipomata. one usually makes the diagnosis at surgery, although on rare occasions one can palpate a lipoma per rectum. treatment involves surgical resection of the lipoma and strangulated bowel, although strangulated intestine is not always nonviable. studies indicate that approximately % to % of horses are discharged from the hospital following surgical treatment. a volvulus is a twist along the axis of the mesentery, whereas torsion is a twist along the longitudinal axis of the intestine. small intestinal volvulus theoretically is initiated by a change in local peristalsis or the occurrence of a lesion around which the intestine and its mesentery may twist (such as an ascarid impaction). volvulus is reportedly one of the most commonly diagnosed causes of small intestinal obstruction in foals. , the theory is that young foals may be at risk of small intestinal volvulus because of changing feed habits and adaptation to a bulkier adult diet. onset of acute, severe colic, a distended abdomen, and radiographic evidence of multiple loops of distended small intestine in a young foal suggest small intestinal volvulus. however, one cannot differentiate volvulus from other causes of small intestinal obstruction preoperatively. in adult horses, volvulus frequently occurs in association with another disease process, during which small intestinal obstruction results in distention and subsequent rotation of the small intestine around the root of the mesentery. although any segment of the small intestine may be involved, the distal jejunum and ileum are affected most frequently because of their longer mesenteries. one makes the diagnosis at surgery by palpating a twist at the origin of the cranial mesenteric artery. treatment includes resection of devitalized bowel, which may not be an option because of the extent of small intestinal involvement (similar to large colon volvulus). prognosis is based on the extent of small intestine involved and its appearance following surgical correction of the lesion. in general, horses with greater than % of the small intestine devitalized are considered to have a grave prognosis. a number of structures, when torn, may incarcerate a segment of intestine (typically the small intestine), including intestinal mesentery, the gastrosplenic ligament, the broad ligament, and the cecocolic ligament. horses with such incarcerations have signs typical of a horse with strangulating small intestine, including moderate to severe signs of abdominal pain, endotoxemia, absent gastrointestinal sounds, distended small intestine on per rectal palpation, nasogastric reflux, and serosanguinous abdominal fluid. however, the prognosis for many of these horses appears to be lower than for horses with other types of small intestinal strangulations. for example, in horses with small intestine entrapped in a mesenteric rent, only of horses were discharged from the hospital, and only of horses for which follow-up information was available survived long term (> months). poor outcome may result from the difficulty in unentrapping incarcerated intestine, the degree of hemorrhage, and the length of intestine affected. inguinal herniae are more common in standardbred and tennessee walking horses that tend to have congenitally large inguinal canals. inguinal herniae also may occur in neonatal foals but differ from herniae in mature horses in that they are typically nonstrangulating. the nature of the hernia (direct versus indirect) is based on the integrity of the parietal vaginal tunic. in horses in which the bowel remains within the parietal vaginal tunic, the hernia is referred to as indirect, because strictly speaking the bowel remains within the peritoneal cavity. direct herniae are those in which strangulated bowel ruptures through the parietal vaginal tunic and occupies a subcutaneous location. these direct herniae most commonly occur in foals and should be suspected when a congenital inguinal hernia is associated with colic, swelling that extends from the inguinal region or the prepuce, and intestine that may be palpated subcutaneously. , although most congenital indirect inguinal herniae resolve with repeated manual reduction or application of a diaper, surgical intervention is recommended for congenial direct herniae. historical findings in horses with strangulating inguinal herniae include acute onset of colic in a stallion that recently had been used for breeding. a cardinal sign of inguinal herniation is a cool, enlarged testicle on one side of the scrotum (figure . - ). , however, inguinal herniae also have been reported in geldings. one also can detect inguinal herniae on rectal palpation, and one can use manipulation of herniated bowel per rectum to reduce a hernia, but this is generally not recommended because of the risk of rectal tears. in many cases, the short segment of herniated intestine greatly improves in appearance after reduction and in some cases can be left unresected. the affected testicle will be congested because of vascular compromise within the spermatic cord, and although the testicle may remain viable, resection generally is recommended. the prognosis in adult horses is good, with up to % of horses surviving to months. horses that have been treated for inguinal herniae may be used for breeding. in these horses, the remaining testicle will have increased sperm production, although an increased number of sperm abnormalities will be noticeable following surgery because of edema and increased temperature of the scrotum. although umbilical herniae are common in foals, strangulation of herniated bowel is rare. in one study, of ( %) horses with umbilical herniae had incarcerated intestine. clinical signs include a warm, swollen, firm, and painful hernia sac associated with signs of colic. the affected segment of bowel is usually small intestine, but herniation of cecum or large colon also has been reported. in rare cases, one may find a hernia that involves only part of the intestinal wall, called a richter's hernia. in foals that have a richter's hernia, an enterocutaneous fistula may develop. in one study, of foals with strangulating umbilical herniae survived to discharge, although at least died of long-term complications. an intussusception involves a segment of bowel (intussusceptum) that invaginates into an adjacent aboral segment of bowel (intussuscipiens). the reason for such invagination is not always clear but may involve a lesion at the leading edge of the intussusception, including small masses, foreign bodies, or parasites. in particular, tapeworms (anoplocephala perfoliata) have been implicated. ileocecal intussusceptions are the most common intestinal intussusceptions in the horse and typically affect young animals. in one study evaluating cases of ileocecal intussusception, the median age of the horses was year old. acute ileocecal intussusceptions are those in which the horses has a duration of colic of less than hours and involve variable lengths of intestine that ranged in one study from to cm long. in acute cases the involved segment of ileum typically has a compromised blood supply. chronic ileocecal intussusceptions typically involve short segments of ileum (up to cm long), and the ileal blood supply is frequently intact. abdominocentesis results vary because strangulated bowel is contained within the adjacent bowel. obstruction of the small intestine often is evident, including nasogastric reflux and multiple distended loops of small intestine on rectal palpation. horses with chronic ileocecal intussusceptions have mild, intermittent colic, often without evidence of small intestinal obstruction. in one study, a mass was palpated in the region of the cecal base in approximately % of cases. transabdominal ultrasound may be helpful in discerning the nature of the mass. the intussusception has a characteristic target appearance on cross section. other segments of the small intestine also may be intussuscepted, including the jejunum (figure . - ) . in one study of jejunojejunal intussusceptions, the length of bowel involved ranged from . to . m. attempts to reduce intussusceptions at surgery are usually futile because of intramural swelling of affected bowel. one should resect jejunojejunal intussusceptions. for acute ileocecal intussusceptions, one should transect the small intestine as far distally as possible and perform a jejunocecal anastomosis. in horses with particularly long intussusceptions (up to m has been reported), one may attempt an intracecal resection. for horses with chronic ileocecal intussusceptions, one should perform a jejunocecal bypass without small intestinal transection. the prognosis is good for horses with chronic ileocecal intussusceptions and guarded to poor for horses with acute ileocecal intussusceptions, depending on the length of bowel involved. herniation of intestine through a rent in the diaphragm is rare in the horse and may involve any segment of bowel, although small intestine is herniated most frequently. diaphragmatic rents may be congenital or acquired, but acquired herniae are more common. congenital rents may result from incomplete fusion of any of the four embryonic components of the diaphragm: pleuroperitoneal membranes, transverse septum, and esophageal mesentery. in addition, abdominal compression of the foal at parturition may result in a congenital hernia. acquired herniae are presumed to result from trauma to the chest or a sudden increase in intraabdominal pressure, such as might occur during parturition, distention of the abdomen, a sudden fall, or strenuous exercise. herniae have been found in a number of different locations, although large congenital herniae are typically present at the ventral most aspect of the diaphragm, and most acquired herniae are located at the junction of the muscular and tendinous portions of the diaphragm. a peritoneopericardial hernia has been documented in at least one horse. figure . - jejunojejunal intussusception in a horse presented for colic. the intussusceptum has become ischemic because of invagination of intestine and its mesenteric blood supply into the intussuscipiens. clinical signs usually are associated with intestinal obstruction rather than respiratory embarrassment. however, careful auscultation may reveal an area of decreased lung sounds associated with obstructed intestine and increased fluid within the chest cavity. such signs may prompt thoracic radiography or ultrasound, both of which one can use to make a diagnosis. auscultation also may reveal thoracic intestinal sounds, but differentiating these from sounds referred from the abdomen typically is not possible. in one report, two of three horses diagnosed with small intestinal strangulation by diaphragmatic hernia had respiratory acidemia attributable to decreased ventilation. treatment of horses with diaphragmatic hernia is fraught with complications because of the need to reduce and resect strangulated bowel and the need to repair the defect in the diaphragm. , because dorsal defects in the diaphragm are among the common forms of diaphragmatic defect, closing the diaphragmatic hernia via the approach used for abdominal exploration may not be possible. however, because herniation is likely to recur, scheduling a second surgery using an appropriate approach to resolve the diaphragmatic defect is appropriate. horses with large colon volvulus have rapid onset of severe, unrelenting abdominal pain, most often in postpartum broodmares. once the large colon strangulates (≥ -degree volvulus), gas distention is significant, leading to gross distention of the abdomen, compromised respiration as the distended bowel presses up against the diaphragm, and visceral pooling of blood as the caudal vena cava is compressed. horses with this condition are frequently refractory even to the most potent of analgesics. these horses may prefer to lie in dorsal recumbency, presumably to take weight off the strangulated colon. an abbreviated physical examination is warranted in these cases, because the time elapsed from the onset of strangulation to surgical correction is critical. under experimental conditions, the colon is irreversibly damaged within to hours of a -degree volvulus of the entire colon. despite severe pain and hypovolemia, horses may have a paradoxically low heart rate, possibly related to increased vagal tone. in addition, results of abdominocentesis often do not indicate the degree of colonic compromise , and in many cases are not worth attempting because of extreme colonic distention. palpation per rectum reveals severe gas distention of the large colon, often restricting access to the abdomen beyond the pelvic brim. one may make the diagnosis tentatively based on signalment, severity of pain, and degree of distention. at surgery, the volvulus typically is located at the mesenteric attachment of the colon to the dorsal body wall and the most common direction of the twist is dorsomedial using the right ventral colon as a reference point. however, the colon may twist in the opposite direction, twist greater than degrees (up to degrees has been reported) or twist at the level of the diaphragmatic and sternal flexures. in all cases, one should decompress the colon as much as possible, and in many cases a colonic evacuation via a pelvic flexure enterotomy greatly aids correction of the volvulus. one must determine after correction of the volvulus whether the colon has been injured irreversibly and should base the determination on mucosal color and bleeding (if an enterotomy has been performed), palpation of a pulse in the colonic arteries, serosal color, and appearance of muscular motility. if one judges the colon to be damaged irreversibly, one can consider the feasibility of a large colon resection. although % of the colon can be resected (that part of the colon distal to the level of the cecocolic fold), damage from the volvulus usually exceeds that which can be resected. in these cases, surgeons may elect to resect as much damaged bowel as possible or may advise euthanasia. the prognosis is guarded to poor because of the rapid onset of this disease. in one study the survival rate was %. in a more recent report the survival rate was % for horses with -degree volvulus of the large colon compared with % for horses with -degree volvulus. however, one study in central kentucky documented a high success rate, possibly because of early recognition of the disease and the proximity of the hospital to the surgical caseload. postoperative complications include hypovolemic and endotoxic shock, extensive loss of circulating protein, disseminated intravascular coagulation, and laminitis. in addition, large colon volvulus has a propensity to recur. although one study documented a recurrence rate of less than %, some authors believe recurrence may be as high as %. therefore one should consider methods to prevent recurrence in patients at risk of recurrence, particularly broodmares that tend to suffer from the disease recurrently during the foaling season. , the most common intussusceptions of the large intestine are cecocecal and cecocolic intussusceptions. , both are likely attributable to the same disease process, with variable inversion of the cecum. these conditions doughnut-shaped prolapse of rectal mucosa and submucosa. type ii prolapses involve full-thickness rectal tissue, whereas type iii prolapses additionally have invagination of small colon into the rectum. type iv prolapses involve intussusception of proximal rectum or small colon through the anus in the absence of prolapse of tissue at the mucocutaneous junction at the anus. one can differentiate type iv from other forms of prolapse by their appearance and a palpable trench between prolapsed tissue and the anus. type i prolapses occur most frequently in horses with diarrhea, in which the rectal mucosa becomes irritated and protrudes intermittently during episodes of tenesmus. if tenesmus persists, rectal mucosa can remain prolapsed. rectal mucosa rapidly becomes congested and edematous under these conditions, which one should treat with osmotic agents such as glycerin or magnesium sulfate and by massaging and reducing the prolapse. a purse-string suture may be required to keep the mucosa inside the rectum. topical application of lidocaine solution or jelly, epidural anesthesia, and sedation may help reduce tenesmus that incites and exacerbates rectal prolapse. one can apply similar treatments to type ii rectal prolapses. however, these more severe prolapses may not be reducible without surgical resection of mucosa and submucosa from the prolapsed bowel. , type iii and iv rectal prolapses are more serious injuries because of involvement of small colon. in horses with type iii prolapses, one should perform an abdominocentesis to determine if injured small colon has resulted in peritonitis. one should reduce the small colon component manually if possible, although prolapsed rectal tissue typically requires mucosal/ submucosal resection. one should perform surgical exploration of the abdomen to determine the status of the small colon, although one can use serial abdominocenteses in lieu of surgery to detect progressive necrosis of bowel. type iv prolapses occur most commonly in horses with dystocia. these prolapses are almost always fatal because of stretching and tearing of mesenteric vasculature, with subsequent infarction of affected bowel. therefore euthanasia usually is warranted tend to occur in young horses ( % were less than years old in one study) and may be associated with intestinal tapeworms. horses show highly variable clinical signs, including acute severe colic, intermittent pain over a number of days, or chronic weight loss. these variable presentations likely relate to the degree to which the cecum has intussuscepted. initially, the cecal tip inverts, creating a cecocecal intussusception, which does not obstruct flow of ingesta. as the intussusception progresses, the cecum inverts into the right ventral colon (cecocolic intussusception), obstructs flow of ingesta, and often causes severe colic. the cause of abdominal pain is often difficult to differentiate in these cases, although detecting a mass on the right side of the abdomen by per rectal palpation or ultrasound examination sometimes is possible. , treatment involves manual surgical reduction by retracting the intussusceptum directly or via an enterotomy in the right ventral colon. however, a number of cases occur in which one cannot reduce the cecum readily because of severe thickening or in which surgical procedures result in fatal contamination. for example, one report stated that of horses were euthanized in the perioperative period because of complications, and another report stated that of horses were euthanized before or during surgery. the latter included all of the horses with chronic disease because of irreversible changes to the cecum. however, one recent report on cecocolic intussusceptions indicated that seven of eight horses that underwent right ventral colon enterotomy and cecal resection survived long-term, suggesting that continued improvements in surgical techniques may improve the prognosis. colocolic intussusceptions are rare but have been reported to affect the pelvic flexure and the left colons. [ ] [ ] [ ] [ ] although the condition is reported to be more common in young horses, - the condition may affect older horses. clinical findings may include a palpable mass on the left side of the abdomen. ultrasonography also may be useful. treatment requires manual reduction of the intussusception at surgery, , or resection of affected bowel. because the left colons may be exteriorized extensively and manipulated at surgery, - the prognosis is fair. rectal prolapse may occur following any disease that causes tenesmus, including diarrhea, rectal neoplasia, and parasitism, or prolapse can occur following elevations in intraabdominal pressure during parturition or episodes of coughing. , rectal prolapses are classified into four categories (table . - ) based on the extent of tissue prolapsed and the severity. type i rectal prolapse is most common and is characterized by a intussusception of rectum and poor small colon through the anus based on physical examination findings. however, confirmation of severe small colonic injury requires abdominal exploration via a midline approach or laparoscopy. a horse with compromised small colon conceivably could undergo a colostomy of the proximal small colon, but the compromised small colon typically necroses beyond that which can be resected via a midline abdominal approach. nonstrangulating infarction occurs following cranial mesenteric arteritis caused by migration of strongylus vulgaris and has become a rare disorder since the advent of broad-spectrum anthelmintics. although thromboemboli have been implicated in the pathogenesis of this disease, careful dissection of naturally occurring lesions has not revealed the presence of thrombi at the site of intestinal infarctions in most cases. these findings suggest that vasospasm plays an important role in this disease. clinical signs vary greatly depending on the extent to which arterial flow is reduced and the segment of intestine affected. any segment of intestine supplied by the cranial mesenteric artery or one of its major branches may be affected, but the distal small intestine and large colon are more commonly involved. no clinical variables exist that one can use to differentiate this disease from strangulating obstruction reliably. in some cases, massive infarction results in acute, severe colic. occasionally, one may detect an abnormal mass and fremitus on palpation of the root of the cranial mesenteric artery per rectum. one should consider this disease a differential diagnosis in horses with a history of inadequate anthelmintic treatment and the presence of intermittent colic that is difficult to localize. although one should perform fecal parasite egg counts, they are not indicative of the degree of parasitic infestation. in addition to routine treatment of colic, dehydration, and endotoxemia, medical treatment may include aspirin ( mg/kg every hours) to decrease thrombosis. definitive diagnosis requires surgical exploration. however, these cases are difficult to treat because of the patchy distribution of the lesions and the possibility of lesions extending beyond the limits of surgical resection. in addition, further infarction may occur following surgery. the prognosis is fair for horses with intermittent mild episodes of colic that may be amenable to medical therapy but is poor in horses that require surgical intervention. , surgical exploration of a horse with on-going intestinal injury exacerbates shock induced largely by endotoxin traversing damaged mucosa, and this in turn correlates with mortality. the initial clinical step in the workup of horses with colic is taking a thorough history. however, one may have to delay taking a complete history until after the physical examination and initial treatment, because management of abdominal pain may take precedence. if possible, one should obtain the vital components of the history before examination and treatment: the duration and severity of colic symptoms, analgesics already administered, and a history of any adverse drug reactions. the two most critical factors from a history that would support a decision to explore a horse with colic surgically are the duration of signs and the extent of pain. one deduces the latter from asking the owner about the presence and frequency of pawing, looking at the flanks, rolling, repeatedly going down and getting back up, posturing as if to lie down or urinate, among other clinical evidence of pain. table . - lists other important components of the history one should obtain to try to ascertain why colic has occurred. just as the history necessarily may need to be brief to allow rapid treatment of colic, so the clinician must be able to alter the extent of the physical examination to treat the horse in a timely fashion. the most critical examination finding is the heart rate of the horse, because it provides an excellent assessment of the cardiovascular status of the horse. the heart rate is likely the single most reliable predictor of the need for surgery and survival. , because analgesics can alter the heart rate dramatically, if possible, one should obtain the heart rate before administering analgesics. other components of the examination are designed specifically to gather information about the cardiopulmonary status of the horse (quality of the pulse, mucous membrane color, capillary refill time, respiratory rate, and full auscultation of the chest), and the nature of the intestinal obstruction (auscultation of gastrointestinal sounds, per rectal palpation of the abdomen, and presence of nasogastric reflux). although classic presentations exist for horses with obstructions of the small or large intestine (table . clinical management of colic is distinctly different from management of many other clinical syndromes because the initial focus is often not on defining the definitive diagnosis but rather on deciding whether a horse requires surgical exploration. therefore the clinician must collect historical, physical examination, and clinicopathologic information and make a decision whether these findings warrant medical management or whether to perform surgical exploration of the abdomen because of a suspected obstructive or ischemic lesion. for example, one may examine a horse with signs of severe abdominal pain, poor cardiovascular status, and abdominal distention that may be compatible with an extensive list of differential diagnoses but that more importantly indicate the need for abdominal exploration to minimize the extent of intestinal injury. the speed with which one can make this clinical decision has a tremendous effect on the well-being of the patient, , because delaying gastric fluid accumulation because of direct compression of the small intestine by distended colon or via tension on the duodenocolic ligament. the most useful diagnostic test for determining the type of intestinal obstruction is rectal palpation of the abdomen. however, one can reach only approximately one third of the abdomen via the rectum, and this percentage may be substantially less in large horses or heavily pregnant horses. nonetheless, attempting to determine the type of obstruction present (small intestine versus large intestine, and simple obstruction versus strangulating obstruction) is worthwhile; this information directly affects prognosis. in one study, interns and residents at a veterinary teaching hospital were able to predict the type of lesion with a specificity exceeding %. findings from palpation are helpful in educating the client about the potential findings in surgery and the likelihood of survival for the horse. before considering how to manage signs of colic, one should remember that such signs are poorly localized. therefore although colic is most frequently associated with intestinal disease, one should consider dysfunction of other organ systems, including urinary obstruction, , biliary obstruction, uterine torsion or tears, , ovarian artery hemorrhage, and neurologic disease as differential diagnoses. however, the duration and severity of colic the most immediately useful clinicopathologic information in horses with colic are the packed cell volume and total protein, because one can use them to substantiate clinical estimates of dehydration and they correlate strongly with prognosis. , a serum biochemical profile is useful for assessing electrolyte imbalances, tissue perfusion (anion gap or lactate), and kidney and liver function. one can use serum biochemical or blood gas analysis to assess acid-base status. horses with colic most frequently show evidence of metabolic acidosis associated with poor tissue perfusion caused by hypovolemia or endotoxemia, but one may note other abnormalities such as metabolic alkalosis in association with extensive loss or sequestration of stomach chloride. metabolic acidosis has been investigated further in horses with colic by measuring blood lactate, although this test is not offered routinely in many laboratories. lactate levels also have been inferred from measurement of the anion gap, although one study noted that lactate in horses with colic did not account for the entire anion gap. lactate levels and anion gap closely correlate with prognosis for survival. , , other key components of assessment of the horse with colic are abdominocentesis and complete blood count. the total white blood cell count and differential can provide crucial evidence of systemic inflammation associated with endotoxemia stemming from colic attributable to colitis (leukopenia, neutropenia, and a left shift) rather than an obstruction (highly variable complete blood count findings). peritoneal fluid may be helpful in determining the integrity of the intestine. specifically, as the intestine becomes progressively devitalized, the peritoneal fluid becomes serosanguinous as red blood cells leak into the abdomen, followed by an elevation in the total protein (> . g/dl) and progressive increases in total nucleated cell count (> , cells/µl). however, these findings do not always correlate well with the condition of the intestine, particularly in horses with large colon volvulus. for example, in a study of horses with large colon volvulus, the average total protein ( . g/dl) and total nucleated cell count ( cells/µl) were normal despite the fact that only % with a -degree volvulus survived. , these measures may appear normal because the development of severe mucosal injury following large colon volvulus is rapid and may not allow enough time for protein and leukocytes to equilibrate with the abdominal fluid. investigators have taken all the variables routinely assessed during evaluation of horses for colic and have attempted to develop models to predict accurately the need for surgery and the prognosis for life. [ ] [ ] [ ] [ ] none of these predictor models has taken the place of clinical decision making, although these studies have added part ii disorders of specific body systems signs are excellent predictors of whether a horse requires surgical exploration of the abdomen. in fact, refractory pain supersedes all other predictors of the need for surgery in the colic patient. once signs of colic have been recognized and categorized as to their severity, rapidly and effectively relieving the pain is critical for the well-being of the horse and to reduce the owner's anxiety. in addition, pain is best managed before it becomes severe. several classes of analgesics are readily available to treat horses with colic (table . - ), including α -agonists (xylazine, detomidine), opiates (butorphanol), and nonsteroidal antiinflammatory drugs (nsaids, such as flunixin meglumine). although much of this information is familiar to most practitioners, several principles deserve emphasis. the short-duration drugs xylazine and butorphanol, which provide analgesia for to minutes, allow the veterinarian to determine if pain is recurrent within the time period of the typical examination. in contrast, flunixin meglumine is not as potent as an analgesic but has a much longer duration of action. to avoid deleterious effects on gastrointestinal mucosa and the kidneys, one should not administer flunixin meglumine more frequently than recommended. , the recent discovery of two isoforms of cyclooxygenase (cox), the enzyme inhibited by nsaids, has resulted in discovery of drugs that can more selectively inhibit proinflammatory cox- while permitting continued constitutive production of prostanoids. such specificity may be advantageous in horses with colic, particularly when one considers recent evidence of reduced intestinal recovery from an ischemic event with flunixin compared with a drug that is more selective for cox- . one should reserve the α agonist detomidine for horses with severe, unrelenting pain because of its tremendous potency. in addition, one should remember that α -agonists reduce the heart rate associated with a transient increase in blood pressure, , thereby reducing the predictive value of the heart rate and pulse pressure. tremendously to understanding of the importance of some prognostic factors, particularly those reflecting cardiovascular function. simple obstruction involves intestinal obstruction of the lumen without obstruction of vascular flow. however, because a tremendous volume of fluid enters the small intestinal lumen daily, , the obstructed intestine tends to become distended, which in turn may reduce mural blood flow. ultimately, such distention may result in necrosis of tissues, particularly in the immediate vicinity of the obstruction. few are the causes of simple obstruction in the small intestine, and the incidence of these obstructions is low (approximately % of all referred horses in one large hospital-based study). however, in some geographic regions, this type of obstruction is prevalent. for example, in the southeastern united states, ileal impactions are common. ileal impactions most commonly occur in adult horses in the southeastern united states. although feeding of coastal bermuda hay has been implicated in the regional distribution of the disease, separating geographic location from regional hay sources as risk factors has been difficult. nonetheless, feeding coastal bermuda hay likely places horses at risk of ileal impaction, particularly if the coarse fiber content of the hay is high. furthermore, sudden changes in feed from an alternate type of hay to coastal bermuda hay likely places a horse at risk of ileal impaction. studies in england have revealed tapeworm infection as another important risk factor for ileal impaction. based on risk analysis, the data suggested that in excess of % of the ileal impaction cases studied were associated with serologic or fecal evidence of tapeworm infection. because of the poor sensitivity of fecal analysis for tapeworms, proudman and trees have developed a serologic test (enzyme-linked immunosorbent assay) with a sensitivity of approximately % and a specificity of %. clinical signs of horses with ileal impaction are typical for a horse with small intestinal obstruction, including onset of moderate to severe colic and loops of distended small intestine palpable per rectum as the condition progresses. because the ileum is the distal most aspect of the small intestinal tract, nasogastric reflux may take a considerable time to develop and is found in approximately % of horses requiring surgical correction of impacted ileum. , one usually makes the diagnosis at surgery, although on occasion one may palpate an impacted ileum per rectum. multiple loops of distended small intestine frequently make the impaction difficult to palpate. ileal impactions may resolve with medical treatment but frequently require surgical intervention ( figure . - ) . at surgery, one can infuse fluids directly figure . - appearance of roundworms that have been retrieved within the nasogastric reflux from a foal with an ascarid impaction. the large size of these ascarids (bar = cm) contributes to the risk of impaction following sudden kills of these parasites by broad-spectrum anthelmintics. into the mass, allowing the surgeon to breakdown the impaction. the surgeon may include dioctyl sodium sulfosuccinate in the infused fluid to aid in disruption of the mass. extensive small intestinal distention and intraoperative manipulation of the ileum may lead to postoperative ileus, but recent studies indicate that this complication is less frequent as the duration of disease before admission decreases. recent studies indicate that the prognosis for survival is good. , ileal hypertrophy is a disorder in which the muscular layers (circular and longitudinal) of the ileum hypertrophy for unknown reasons (idiopathic) or following an incomplete or functional obstruction. for idiopathic cases, proposed mechanisms include parasympathetic neural dysfunction resulting in chronically increased muscle tone and subsequent hypertrophy of the muscular layers of the ileal wall. such neural dysfunction possibly could result from parasite migration. alternative hypotheses include chronic increases in the muscular tone of the ileocecal valve, leading to muscular hypertrophy of the ileum as it contracts against a partially occluded ileocecal valve. the jejunum also may be hypertrophied, alone or with the ileum. clinical signs include chronic intermittent colic as the ileum hypertrophies and gradually narrows the lumen diameter. in one study, partial anorexia and chronic weight loss ( to months) were documented in % of the horses, most likely because of intermittent colic and reduced appetite. because hypertrophy does not affect the ileal mucosa, no reason exists to believe that these horses experience malabsorption of nutrients. one usually makes the diagnosis at surgery, although one may palpate the hypertophied ileum per rectum in some cases. for treatment, one performs an ileocecal or jejunocecal anastomosis to bypass the hypertrophied ileum. without surgical bypass, intermittent colic persists and the thickened ileum ultimately may rupture. the prognosis is fair with surgical treatment. secondary ileal hypertrophy is most commonly notable in horses that previously have had colic surgery and that may have a partial or functional obstruction at an anastomotic site. for example, in one case report, a horse developed ileal hypertrophy after surgical correction of an ileocecal intussusception. ileal hypertrophy also was noted in a horse with cecal impaction in which an ileocolic anastomosis was oriented incorrectly. horses are typically re-presented for recurrence of colic in these cases. surgical therapy is directed at addressing the cause of small intestinal obstruction and resecting hypertrophied intestine. meckel's diverticulum is an embryonic remnant of the vitelloumbilical duct, which fails to atrophy completely and becomes a blind pouch projecting from the antimesenteric border of the ileum. , however, similar diverticula also have been noted in the jejunum. these diverticula may become impacted, resulting in partial luminal obstruction, or may wrap around an adjacent segment of intestine, causing strangulation. occasionally, an associated mesodiverticular band may course from the diverticulum to the umbilical remnant and serve as a point around which small intestine may become strangulated. mesodiverticular bands also may originate from the embryonic ventral mesentery and attach to the antimesenteric surface of the bowel, thereby forming a potential space within which intestine may become entrapped. clinical signs range from chronic colic for an impacted meckel's diverticulum to acute severe colic for intestine strangulated by a mesodiverticular band. one makes the diagnosis at surgery, and treatment requires resection of the diverticulum and any associated bands. the prognosis is good for horses with simple impaction of a meckel's diverticulum and is guarded for horses with an associated small intestinal strangulation. adhesions of one segment of bowel to another or of a segment of intestine to other organs and the body wall most typically occur following abdominal surgery and may be clinically silent, cause chronic colic attributable to partial obstruction, or result in acute obstruction. these differing clinical syndromes are attributable to the type of adhesions that develop. for example, a fibrous adhesion that does not by itself obstruct the intestinal lumen might serve as the pivot point for a volvulus, whereas an adhesion between adjacent segments of the intestinal tract may create a hairpin turn that causes chronic partial obstruction. the number of adhesions that develop also may vary greatly from horse to horse. some horses may develop a single adhesion adjacent to an anastomotic site or a discrete segment of injured intestine, whereas other horses may develop diffuse adhesions involving multiple segments of intestine, likely because of widespread inflammation of the peritoneum at the time of the original surgery. the mechanism whereby adhesions develop is complex but likely involves initial injury to the serosa initiated by intestinal ischemia, reperfusion injury, and luminal distention. importantly, such injury involves infiltration of neutrophils into the serosa accompanied by loss of mesothelial cells. in one study assessing the margins of resected small intestine, extensive neutrophil infiltration was documented in the serosa, particularly in the proximal resection margin that had been distended before correction of a variety of strangulating lesions. regions of serosal injury and inflammation subsequently undergo reparative events similar to any wound, including local production of fibrin, de novo synthesis of collagen by infiltrating fibroblasts, and ultimately maturation and remodeling of fibrous tissue. unfortunately, during this process, fibrin may result in injured intestinal surfaces adhering to adjacent injured bowel or an adjacent organ. once a fibrinous adhesion has developed, new collagen synthesis may result in a permanent fibrous adhesion. alternatively, proteases released by local phagocytes may lyse fibrinous exudate, thereby reversing the adhesive process. thus one can view formation of adhesions as an imbalance of fibrin deposition and fibrinolysis. prevention of adhesions depends on inhibition of the mechanisms involved in adhesion formation, including reduction of serosal injury with early intervention and good surgical technique, reduction of inflammation by administration of antiinflammatory medications, physical separation of inflamed serosal surfaces (e.g., carboxymethylcellulose and hyaluronan), [ ] [ ] [ ] and pharmacologic modulation of fibrinous adhesion formation (e.g., heparin). in addition, early return of motility in the small intestine after surgery may reduce contact time between inflamed surfaces of intestine, thereby reducing the chances of adhesion formation. horses at greatest risk of developing adhesions after colic surgery appear to be those that have small intestinal disease. , in one study of horses undergoing surgical correction of small intestinal obstruction, % developed a surgical lesion associated with adhesions. foals appear to have an increased incidence of adhesions compared with mature horses regardless of the nature of the abdominal surgery. one study indicated that % of foals developed lesions attributable to adhesions regardless of the type of initial surgery. studies conflict as to whether the degree of surgical intervention influences adhesion formation, but in one study, horses that require enterotomy or resection and anastomosis were at greatest risk of developing adhesions. as an indication of the importance of postoperative adhesion formation, adhesions were among the most common reasons for repeat laparotomy in postoperative colic patients. , clinical signs of horses with adhesions vary greatly depending on whether the adhesion is causing partial obstruction or complete luminal obstruction or involves intestinal vasculature. adhesions would be an important differential diagnosis for intermittent colic in the postoperative period, particularly if such colic was not relieved by nasogastric decompression of the stomach. continued intermittent colic should prompt abdominocentesis to determine if septic peritonitis is present, which may contribute to adhesion formation. placement of a large bore drain and peritoneal lavage ( figure surgery should prompt immediate nasogastric intubation to decompress the stomach. treatment should include attempts at obtaining reflux from the horse at frequent intervals rather than relying on passive flow of reflux. in addition, administration of intravenous fluids should account for the maintenance requirement ( ml/kg/day, about l/hr in the average horse) and fluid losses via reflux. in practice, this requires frequent monitoring of packed cell volume and total protein to ensure that the horse remains well hydrated. although concerns have arisen that overhydrating horses may contribute to increased nasogastric reflux, keeping horses well-hydrated to avoid hypovolemic shock is critical. additionally, one should monitor electrolytes frequently, particularly considering their potential role in smooth muscle contraction and nerve excitability. because of the important role of inflammation in postoperative ileus, including elaboration of cox- -produced prostanoids, administration of nsaids is indicated. nsaid administration is particularly necessary if postoperative ileus is associated with endotoxemia, because lipopolysaccharide-induced prostanoid production disrupts propulsive motility in horses. , interestingly, phenylbutazone is more effective than flunixin meglumine at reducing the deleterious actions of lipopolysaccharide on intestinal motility. however, one should use caution when administering nsaids to patients with postoperative ileus in light of research suggesting that complete inhibition of prostanoid production can alter motility patterns in normal equine intestine. the advent of selective cox- inhibitors may provide optimal antiinflammatory treatment in the future. other treatments aimed at specifically modulating intestinal motility include lidocaine (bolus of . mg/kg followed by . mg/kg/min for hours), erythromycin ( . to . mg/kg slow intravenous infusion in l saline every hours), and metoclopramide ( . mg/kg/hr). , , the mechanism of lidocaine is presumed to be inhibition of sensory nerve activity within the wall of the intestine, thereby reducing reflex sympathetic inhibitory activity. in addition, intravenously administered lidocaine appears to be an effective analgesic. thus an important feature of intravenous lidocaine therapy may be to control postoperative pain-induced reduction of gastrointestinal motility and mucosal secretory activity. metoclopramide may stimulate intestinal motility by several mechanisms, including dopamine receptor blockade, cholinergic stimulation, and adrenergic blockade. although metoclopramide has been shown to be beneficial for reversing postoperative ileus in clinical patients and research animals, it has central nervous system excitatory side effects in the horse that make its use difficult. nonetheless, administration of metoclopramide to horses with postoperative ileus resulted in elect repeat laparotomy or laparoscopy. in one study of adhesions, % of repeat laparotomies were performed within days, suggesting that surgical colic attributable to adhesions typically occurs within months of an initial surgical procedure. unfortunately, the prognosis for horses with colic attributable to adhesions is low, with only % of horses in one study surviving from adhesion-induced colic. the definition of ileus is intestinal obstruction, including physical and functional obstructions. however, in veterinary medicine, the term typically is used to designate a lack of progressive aboral propulsion of ingesta resulting in functional obstruction. one typically bases the diagnosis of postoperative ileus on the presence of excessive gastric fluid accumulation (reflected as excessive nasogastric reflux). postoperative ileus may occur following any abdominal exploratory procedure. however, horses undergoing surgery for strangulating small intestinal lesions or small intestinal obstructive lesions such as an ileal impaction are at greatest risk. recently, the syndrome of postoperative ileus in horses has been broadened to include those horses that may have delayed transit of ingesta through the large intestine following surgery. this large intestinal ileus may follow any type of surgery, particularly horses that have had orthopedic surgery, and is characterized by reduced fecal output (fewer than three piles of manure per day) rather than excessive nasogastric reflux. however, horses with excessive nasogastric reflux are unlikely to have normal fecal output, so the distinction between these two manifestations of ileus is not absolute. mechanisms involved in precipitating postoperative ileus characterized by small intestinal dysfunction likely involve local inflammation, reduced coordination of progressive motility, and increased sympathetic tone. a recent series of studies in the rat has shown that surgical manipulation of intestine results in delayed transit time associated with infiltration of neutrophils into intestinal longitudinal muscle [ ] [ ] [ ] and upregulation of inducible nitric oxide synthase and cox- . the mechanisms in the horse may be similar in that extensive manipulation of the intestine resulted in abnormal intestinal motility in ponies, and prostanoids and nitric oxide alter or reduce intestinal motility in horses. [ ] [ ] [ ] clinical signs of postoperative ileus following colic surgery include evidence of abdominal pain, increased heart rate, reduced gastrointestinal sounds, and reflux of gastric fluid via a nasogastric tube. of these signs, heart rate is critical because it appears to be a more sensitive indicator of pain in the postoperative period than overt evidence of colic. therefore a sudden increase in the heart rate of a postoperative patient following colic a significantly reduced duration of reflux and shorter postoperative hospital stays compared with horses not receiving this drug. in the same study, constant infusion of metoclopramide was superior to intermittent infusion. recent in vitro studies indicate that metoclopramide effectively increases smooth muscle contractile activity throughout the small intestine. similarly, the motilin agonist erythromycin had stimulatory effects on equine small intestine, although the results were not uniform throughout the small intestine. erythromycin stimulates contractile activity in the longitudinal muscle of the pyloric antrum but inhibits contractile activity in circular smooth muscle in this segment of the gastrointestinal tract. the latter may be attributable to activation of motilin receptors on inhibitory nerves and may result in enhanced gastric emptying. in vivo studies on erythromycin confirmed the stimulatory action of this drug on the distal small intestine and indicated this drug also stimulates contractile activity in the cecum and pelvic flexure. however, the stimulation depends on the temporal association with surgery. erythromycin stimulated contractile activity in the postoperative period in the ileum and pelvic flexure but not the cecum, suggesting this drug may be useful for treating select cases of postoperative ileus. for horses with presumed ileus of the large colon, signs included reduced fecal output (fewer than three piles of manure per day), reduced gastrointestinal sounds, variable presence of colic, and on occasion a palpable impaction of the cecum or large colon. risk factors for this syndrome include orthopedic surgery, length of the operative period, and most importantly inadequate treatment with phenylbutazone, presumably resulting from insufficient control of postoperative pain. although treatment of large colon impaction in the postoperative period typically is uncomplicated, onset of cecal impaction is fatal in many cases because of the difficulty in recognizing horses that have cecal dysfunction. therefore one should pay close attention to fecal production and optimal analgesic treatment in any horse following an orthopedic procedure. other painful procedures, including ophthalmologic procedures, also likely place horses at risk of developing ileus of the large intestine. simple obstructions of the large intestine such as impaction tend to have a more gradual onset than those of the small intestine, although horses may become acutely and severely painful with some forms of colon displacement. in fact, some of these cases mimic and may progress toward large colon volvulus. medical therapy is frequently successful in correcting large colon impactions. however, cecal impactions present much more of a dilemma because of the greater propensity of this organ to rupture, the relative difficulty of surgically manipulating the cecum, and the onset of cecal dysfunction that may prevent the cecum from emptying following surgical resolution of impaction. cecal impaction may be divided into two syndromes: primary cecal impactions that result from excessive accumulation of ingesta in the cecum and secondary cecal impactions that develop while a horse is being treated for a separate problem. , although primary impactions typically consist of impacted, relatively dry fecal material and secondary cecal impactions tend to have fluid contents, considerable overlap exists between the two syndromes, and one must approach each case carefully. in horses with primary cecal impactions, onset of abdominal pain occurs over a number of days, reminiscent of the development of a large colon impaction. one should differentiate cecal impactions from large colon impactions on the basis of rectal palpation findings. cecal impactions have a propensity to rupture before the development of severe abdominal pain or systemic deterioration and therefore must be monitored closely. secondary cecal impactions typically develop following unrelated surgical procedures that result in postoperative pain (particularly orthopedic surgeries). secondary cecal impactions may be even more difficult to detect because one may attribute postoperative depression and decreased fecal output to the operative procedure rather than to colic. by the time horses with secondary cecal impactions show noticeable signs of colic, the cecum may be close to rupture. in many cases, no signs of impending rupture are evident. therefore all horses that undergo surgeries in which considerable postoperative pain may develop should have feed intake and manure production closely monitored. a recent study indicated that horses that produce fewer than three piles of manure daily in the postoperative period are at risk of developing a large intestinal impaction. furthermore, horses that underwent prolonged (> hour) orthopedic surgery that received inadequate treatment with phenylbutazone were at considerable risk of reduced postoperative fecal output. these results are in contrast to statements indicating that nsaids may place horses at risk of impaction, statements that appear to be based largely on clinical impressions rather than on risk analysis. the diagnosis of primary cecal impaction is based on palpation of a firm, impacted cecum per rectum. in some cases, cecal impactions may be difficult to differentiate from large colon impactions. however, careful palpation reveals the inability to move the hand completely dorsal to the impacted viscus because of the attachment of the cecum to the dorsal body wall. treatment for horses with primary cecal impactions may include initial medical therapy, including aggressive administration of intravenous fluids and judicious use of analgesics. however, if the cecum is distended grossly or if medical therapy hasno effect within a reasonable period of time, surgical evacuation of the cecum via a typhlotomy is indicated. in addition, performing an ileocolostomy to bypass the cecum is advisable, because postoperative cecal motility dysfunction with recurrence of the impaction is common. , in horses that develop secondary cecal impactions, diagnosis is based on palpation of a greatly distended cecum filled with semifluid intestinal contents. the nature of the contents likely is related to the more rapid progression of this disease compared with primary cecal impaction. one should not delay surgery because of the risk of cecal rupture. however, if the cecum appears healthy following typhlotomy and evacuation, bypass of the cecum is not as critical as it is for primary impactions as long as one can control the inciting cause of the impaction (such as orthopedic pain). the prognosis is guarded for surgical treatment of all cecal impactions because of the potential for the cecum to rupture during prolonged medical treatment or during surgical manipulation, the possibility of abdominal contamination during surgery, and the extensive surgical procedures required. in a recent report, seven of nine horses for which cecal impaction was treated by typhlotomy and ileocolostomy or jejunocolostomy lived long term. however, a separate report indicated that all horses with cecal impaction following another disease process had cecal rupture without any signs of impending rupture. ingesta impactions of the large colon occur at sites of anatomic reductions in luminal diameter, particularly the pelvic flexure and the right dorsal colon. although a number of risk factors have been reported, most have not been proved. however, a sudden restriction in exercise associated with musculoskeletal injury appears frequently to be associated with onset of impaction. another consideration is equine feeding regimens, which usually entail twice daily feeding of concentrate. such regimens are associated with large fluxes of fluid into and out of the colon, associated with readily fermentable carbohydrate in the colon and subsequent increases in serum aldosterone, respectively. one may prevent these fluid fluxes, which may cause dehydration of ingesta during aldosterone-stimulated net fluid flux out of the colon, with frequent small feedings. amitraz, an acaricide associated with clinical cases of colon impaction, can induce impaction of the ascending colon. , this effect may provide some clues as to the pathogenesis of large colon impaction. in particular, amitraz appears to alter pelvic flexure pacemaker activity, resulting in uncoordinated motility patterns between the left ventral and left dorsal colon and excessive retention of ingesta. absorption of water from the ingesta increases with retention time, dehydrates the contents of the colon, and results in impaction. conceivably, parasite migration in the region of a pacemaker may have a similar action. other factors implicated in large colon impaction include limited exercise, poor dentition, coarse roughage, or dehydration. clinical signs of large colon impaction include slow onset of mild to moderate colic. fecal production decreases, and the feces are often hard, dry, and mucuscovered because of delayed transit time. the heart rate may be elevated mildly during episodes of pain but is often normal. signs of abdominal pain are typically well controlled with administration of analgesics but become increasingly more severe and refractory if the impaction does not resolve. the diagnosis is based on palpation of a firm mass in the large colon per rectum. however, one may underestimate the extent of the impaction by rectal palpation alone because much of the colon is out of reach. adjacent colon may be distended if the impaction has resulted in complete obstruction. one should attempt initial medical treatment. administration of analgesics (e.g., flunixin meglumine at . to . mg/kg intravenously every to hours; butorphanol at . to . mg/kg intramuscularly every to hours; or xylazine at . to . mg/kg intravenously as needed) controls intermittent abdominal pain. administration of oral laxatives such as mineral oil ( to l by nasogastric tube every to hours) and the anionic surfactant dioctyl sodium sulfosuccinate ( to g/ kg diluted in to l of water by nasogastric tube every to hours) are used commonly to soften the impaction. saline cathartics such as magnesium sulfate ( . mg/kg in to l by nasogastric tube) also may be useful. one should not permit access to feed. for impactions that persist, one should institute aggressive oral and intravenous fluid therapy ( to times the maintenance fluid requirement). if the impaction remains unresolved, the horse becomes uncontrollably painful, or extensive gas distention of the colon occurs, surgery is indicated. in addition, one can monitor abdominal fluid serially to determine the onset of intestinal compromise. at surgery, one evacuates the contents of the colon via a pelvic flexure enterotomy. the prognosis is good for those horses in which impactions resolve medically ( % long-term survival in one study) and fair in horses that require surgical intervention ( % long-term survival in the same study). enteroliths are mineralized masses typically composed of magnesium ammonium phosphate (struvite). however, magnesium vivianite also has been identified in enteroliths, along with variable quantities of sodium, sulfur, potassium, and calcium. the formation of magnesiumbased minerals is puzzling because of the relative abundance of calcium in colonic fluids, which would favor the formation of calcium phosphates (apatite) rather than struvite. however, elevated dietary intake of magnesium and protein may play a role. many horses that develop enteroliths are located in california and are fed a diet consisting mainly of alfalfa hay. analysis of this hay has revealed a concentration of magnesium approximately times the daily requirements of the horse. furthermore, the high protein concentration in alfalfa hay may contribute to calculi formation by increasing the ammonia nitrogen load in the large intestine. enteroliths most commonly form around a nucleus of silicon dioxide (a flintlike stone), but nidi have included ingested nails, rope, and hair. enteroliths usually are found in the right dorsal and transverse colons. although enterolithiasis has a wide geographic distribution, horses in california have the highest incidence. in one california study, horses with enterolithiasis represented % of the surgical colic population, and arabians, morgans, american saddlebreds, and donkeys were at greatest risk of this disease. in a study of enterolithiasis in texas, risk factors also included feeding of alfalfa hay and arabian breed. however, in that study, miniature horses were also at risk. horses with enteroliths are rarely under years old, although an enterolith in an -month-old miniature horse has been reported recently. enterolithiasis is characterized by episodic, mild to moderate, intermittent abdominal pain. progressive anorexia and depression may develop. the amount of pain depends on the degree of obstruction and amount of distention. partial luminal obstruction allows the passage of scant, pasty feces. heart rate varies and depends on the degree of pain. in some cases, an enterolith is forced into the small colon, where it causes acute small colon obstruction. one may diagnose enteroliths by abdominal radiography or at surgery. on rare occasions, one may palpate an enterolith per rectum, particularly if it is present in the distal small colon. in general, these cases require surgery, although enteroliths being retrieved per rectum have been reported. in fact in one study, % of horses presented for treatment of enterolithiasis had a history of passing an enterolith in the feces. however, enteroliths typically are located in the right dorsal colon, transverse colon, or small colon. at surgery, one gently pushes the enterolith toward a pelvic flexure enterotomy, but removal frequently requires a separate right dorsal colon enterotomy to prevent rupture of the colon. following removal of an enterolith, one must conduct further exploration to determine if other enteroliths are present. solitary enteroliths are usually round, whereas multiple enteroliths have flat sides. the prognosis is good ( % -year survival in one study of cases), unless the colon ruptures during removal of an enterolith. in one recent study, rupture occurred in % of cases. sand impactions are common in horses with access to sandy soils, particularly horses eating feed placed on the ground. some horses, especially foals, deliberately eat sand. fine sand tends to accumulate in the ventral colon, whereas coarse sand may accumulate in the dorsal colon. , however, individual differences in colonic function may contribute to accumulation of sand, because some horses can clear consumed sand, whereas others cannot. distention from the impaction itself, or gas proximal to the impaction, causes abdominal pain. in addition, sand may trigger diarrhea, presumably because of irritation of the colonic mucosa. in horses with sand impactions, clinical signs are similar to those of horses with large colon impactions. one may find sand in the feces, and auscultation of the ventral abdomen may reveal sounds of sand moving within the large colon. however, unlike sand-induced diarrhea, one may not hear sand impactions easily because of the lack of colonic motility. to determine the presence of fecal sand, one places several fecal balls in a rectal palpation sleeve or other container, which subsequently is filled with water. if sand is present, it accumulates at the bottom of the container. in addition, one may detect mineral opacity within the colon on abdominal radiographs, particularly in foals, ponies, and small horses. abdominal paracentesis typically yields normal fluid and poses some risk because large quantities of sand in the ventral colon make inadvertent perforation of the colon more likely. peritoneal fluid is often normal but may have an elevated protein concentration. initially, medical therapy is warranted. administration of psyllium hydrophilic mucilloid ( . to . kg/ kg in to l of water by stomach tube) may facilitate passage of sand. one should administer the solution rapidly because it will form a viscous gel. an alternative method of administration is to mix psyllium with l of mineral oil, which will not form a gel and can be pumped through a nasogastric tube easily. one then pumps to l of water through the tube. the psyllium separates from the oil phase and mixes with the water, forming a gel within the gastrointestinal tract. psyllium is thought to act by stimulating motility or by agglutinating the sand. however, a recent experimental study failed to show a benefit of this treatment for clearing sand from the colons of otherwise normal horses. if a severe impaction is present, one should not give the psyllium until softening the impaction by administrating intravenous or oral fluids and other laxatives. perforation is a potential complication in horses with sand impactions because the sand stretches and irritates the intestinal wall and causes inflammation. therefore if colic becomes intractable, one should perform surgical evacuation of the large colon. the prognosis is generally good. , displacement of the ascending colon is a common cause of large intestinal obstruction. the ascending colon is freely movable except for the right dorsal and ventral colons. contact with adjacent viscera and the abdominal wall tends to inhibit movement of the ascending colon from a normal position; however, accumulation of gas and fluid or ingesta may cause the colon to migrate. feeding practices, including feeding of large concentrate meals, likely plays a role in initiating displacement of the large colon. large concentrate meals increase the rate of passage of ingesta, allowing a greater percentage of soluble carbohydrates to reach the large intestine, which in turn increases the rate of fermentation and the amount of gas and volatile fatty acids produced. the production of large amounts of volatile fatty acids stimulates the secretion of large volumes of fluid into the colon. the association between feeding concentrate and development of displacements of the large colon is illustrated by studies indicating that ascending colon displacement is more prevalent in horses fed a highconcentrate, low-roughage diet. abnormal motility patterns of the ascending colon also have been suggested to contribute to the development of colonic displacement. feeding stimulates colonic motility via the gastrocolic reflex, but large meals may alter normal motility patterns and concurrently allow rapid accumulation of gas and fluid from fermentation. , migration of parasite larvae (strongyles) through the intestinal wall also has been shown to alter colonic motility patterns. other experimental studies also have shown that strongylus vulgaris infection results in reduced blood flow to segments of the large intestine without necessarily causing infarction. electric activity of the colon and cecocolic junction increases after infection with s. vulgaris and cyathostome larvae, probably reflecting a direct effect of migration through the intestine and an early response to reduced blood flow. displacements of the ascending colon generally are divided into three types: left dorsal displacement, right dorsal displacement, and retroflexion. left dorsal displacement is characterized by entrapment of the ascending colon in the renosplenic space. the colon often is twisted degrees such that the left ventral colon is situated in a dorsal position relative to the left dorsal colon. the entrapped portion may be only the pelvic flexure or may involve a large portion of the ascending colon, with the pelvic flexure situated near the diaphragm. the colon may become entrapped by migrating dorsally between the left abdominal wall and the spleen or may migrate in a caudodorsal direction over the nephrosplenic ligament. occasionally, one can palpate the ascending colon between the spleen and abdominal wall, lending support to the first mechanism of displacement. gastric distention is thought to predispose horses to left dorsal displacement of the ascending colon by displacing the spleen medially, allowing the colon room to migrate along the abdominal wall. right dorsal displacement begins by movement of the colon cranially, medial (medial flexion) or lateral (lateral flexion) to the cecum. according to one author, the proportion of right dorsal displacements with medial versus lateral flexion is approximately : . in either case the pelvic flexure ends up adjacent to the diaphragm. retroflexion of the ascending colon occurs by movement of the pelvic flexure cranially without movement of the sternal or diaphragmatic flexures. displacement of the ascending colon partially obstructs the lumen, resulting in accumulation of gas or ingesta and causing distention. secretion of fluid in response may exacerbate the distention. tension and stretch of the visceral wall is an important source of the pain associated with colonic displacement. tension on mesenteric attachments and the root of the mesentery by the enlarged colon also may cause pain. ischemia rarely is associated with nonstrangulating displacement of the colon. however, vascular congestion and edema often occur in the displaced segments of colon, resulting from increased hydrostatic pressure from reduced venous outflow. morphologic damage to the tissues is usually minor. clinically, displacement of the ascending colon is characterized by intermittent signs of mild to moderate abdominal pain of acute onset. however, one also may note an insidious onset of colic. one may note dehydration if the duration of the displacement is prolonged. the heart rate may be elevated during periods of abdominal pain but is often normal. abdominal distention may be present if the colon is enlarged by gas, fluid, or ingesta. fecal production is reduced because progressive motility of the large intestine is absent. one often diagnoses left dorsal displacements by palpation per rectum. one can feel the left ventral colon in a dorsal position; it often is filled with gas. one can trace the ascending colon to the nephrosplenic space, and the spleen may be displaced medially. alternatively, one can reach a tentative diagnosis using abdominal ultrasonography. the spleen is visible on the left side of the abdomen, but the gasdistended bowel obscures the left kidney. evaluation of this technique indicates that false positives occur in few instances, although false negatives occasionally may occur. a definitive diagnosis therefore may require surgery. right dorsal displacements are characterized by the presence of the distended ventral colon running across the pelvic inlet and may be felt between the cecum and the body wall if a lateral flexion is present. the pelvic flexure is usually not palpable. retroflexion of the ascending colon may produce a palpable kink in the colon. if the displaced colons are not distended by gas in the instance of right dorsal displacement and retroflexion, the ascending colon may not be palpable and is conspicuous by its absence from a normal position. peritoneal fluid may increase in amount, but the color, protein concentration, and white blood cell count are usually normal. however, as the displaced segment becomes edematous, fluid leaking through the serosa into the peritoneal fluid increases the protein concentration. surgical correction of colon displacement is the most effective means of resolving this disorder. however, nonsurgical intervention has been successful in select cases of nephrosplenic entrapment of the large colon. [ ] [ ] [ ] before attempting such manipulations, the clinician must be certain of a diagnosis. one anesthetizes the horse and places it in right lateral recumbency, rotates the horse up to dorsal recumbency, rocking it back and forth for to minutes, and then rolls the horse down into left lateral recumbency. one should palpate the nephrosplenic space per rectum to determine whether the entrapment has been relieved before recovering the horse from anesthesia. one may administer phenylephrine ( - µg/kg/min over minutes) to decrease the size of the spleen. more recently, phenylephrine has been used successfully with to minutes of exercise to reduce nephrosplenic entrapments in four of six horses. the authors suggested that the technique be used on horses with mild to moderate colonic distention, particularly when financial constraints are severe. a number of cases occur in which nonsurgical interventions do not correct the problem and others in which nonsurgical manipulations correct the entrapment but result in large colon volvulus or displacement. one should take horses in such condition to surgery promptly. the prognosis for horses with large colon displacement is good. in one study on horses with nephrosplenic entrapment of the large colon, survival exceeded %. the horse, particularly young horses, may ingest foreign material that can cause obstruction, such as bedding, rope, plastic, fence material, and feedbags. these foreign bodies may result in impaction with ingesta and distention of the intestine, typically in the transverse or descending colon. young horses usually are affected. in one study the obstructing mass could be palpated per rectum in three of six horses. fecaliths are common in ponies, miniature horses, and foals. older horses with poor dentition also may be predisposed to fecaliths because of the inability to masticate fibrous feed material fully. fecaliths commonly cause obstruction in the descending colon and may cause tenesmus. other clinical signs are similar to those of enterolithiasis. abdominal radiography may be useful in smaller patients to identify the obstruction, especially if gas distention around the foreign body or fecalith provides contrast. the horse usually requires surgical treatment. mural masses such as abscesses, tumors (adenocarcinoma, lymphosarcoma), granulomata, and hematomas ( figure . - ) can cause luminal obstruction and impaction, typically in older horses. impaction may result from obstruction of the lumen or impaired motility in the segment of intestine with the mass. abscesses may originate from the lumen of the intestine or may extend from the mesentery or mesenteric lymph nodes. intramural hematomas form most commonly in the descending colon and cause acute abdominal pain. once the acute pain from the hematoma subsides, impaction proximal to the hematoma develops because of impaired motility through the affected portion of the colon. trauma, ulceration of the mucosa, and parasitic damage are speculated causes of intramural hematomas. , stricture of the large intestine occurs when fibrous tissue forms in a circular pattern around or within the intestine, reducing the luminal diameter and the ability of the wall to stretch. strictures may be congenital or may follow peritonitis, previous abdominal surgery, or inflammatory bowel disease. in a report of horses with inflammatory bowel disease, horses had strictures, four of which were in the small intestine and two of which were in the large colon. clinical signs vary according to the degree of luminal obstruction. partial obstruction and impaction tend to produce mild to moderate abdominal pain of insidious onset. mural hematomas tend to produce signs of acute abdominal pain. , per rectal palpation of the abdomen may reveal the presence of a mass or simply the impacted segment but not the mass itself. one may note fever, weight loss, and anorexia if an abscess or tumor is the cause. an elevated white blood cell count; hyperfibrinogenemia; hyperglobulinemia; or normocytic, normochromic anemia may occur with abscesses or tumors. peritoneal fluid may reflect the cause of the mass. tumor cells may occur infrequently. one may note evidence of inflammation with bacteria if the cause of colic is an abscess or granuloma, in which case one should culture the fluid. hematomas may cause hemorrhage into the peritoneal fluid. treatment usually requires surgical resection of the mass. one may treat abscesses with appropriate antibiotics if the impaction can be resolved medically with oral or intravenous analgesics and laxatives. streptococcus spp, actinomyces pyogenes, corynebacterium pseudotuberculosis, rhodococcus equi, anaerobic bacteria, and gram-negative enteric organisms commonly are involved in abscesses. atresia of a segment of the colon is a rare congenital abnormality in horses. the heritability and causes of the condition are unknown. one potential mechanism for development of the lesion is intestinal ischemia during fetal life, which results in necrosis of a segment of intestine. clinical signs include a failure to pass meconium and colic within the first to hours of life. secondary abdominal distention results from complete intestinal obstruction, and abdominal radiographs may reveal gas-distended colon. one makes the diagnosis at surgery. any portion of the colon may be absent, but the distal segment of the large colon or the proximal small colon usually is affected most severely. if sufficient tissue is present, one may attempt anastomosis to the proximal blind end of the colon. the prognosis depends on which segment of the colon is absent but is usually poor because of an absence of distal colon. neoplasia in the alimentary tract of the horse is uncommon. primary and metastatic neoplasia can affect multiple locations within the oral cavity and gastrointestinal tract (boxes . - and . - ). neoplasia is not limited to older horses. the average age of horses with squamous cell carcinoma is . to . years. , the alimentary form of lymphoma occurs most commonly in horses less than years of age. identification of benign versus malignant tumors is imperative to justify treatment and predict survival. clinical signs associated with alimentary neoplasia are related to the tumor location. clinical signs of oral neoplasia can include enlargement or ulceration of the mandible or maxilla. neoplasia of the tongue results in weight loss, quidding, prepharyngeal dysphagia, halitosis, and nasal discharge containing feed material if the oropharynx is involved. [ ] [ ] [ ] tumors of the esophagus cause signs typical of esophageal dysphagia, ptyalism, choke, intermittent colic, fever, weight loss, and halitosis. , , gastric neoplasia can be associated with abnormal chewing and swallowing behavior, anorexia, weight loss, chronic intermittent colic, abdominal distention, and intermittent fever. abdominal neoplasia has been implicated in % of horses with intermittent or chronic colic. , altered stool character, weight loss, ventral edema, and recurrent fever have been associated with intestinal neoplasia. acute signs of abdominal discomfort can occur in intestinal obstructions from malignant and benign neoplastic disease. paraneoplastic syndromes may occur in the horse. the most common syndromes are cancer cachexia, ectopic hormone production, anemia, leukocytosis, thrombocytopenia, hypergammaglobulinemia, fever, and neurologic abnormalities. horses with cancer cachexia have profound weight loss despite adequate consumption of calories. diagnosis of alimentary neoplasia can be challenging. data collected from a complete blood count, biochemistry panel, and urinalysis may support the diagnosis of neoplasia but rarely confirms it. normocytic normochromic anemia indicates chronic disease and is the most likely cause of anemia associated with neoplasia. blood loss anemia (via gastrointestinal tract) and immune-mediated hemolytic anemia (lymphoma) are less frequent causes of anemia associated with abdominal neoplasia. peripheral eosinophilia has been reported in association with multisystemic eosinophilic, epitheliotropic disease with lymphoma. leukocytosis and hyperfibrinogenemia are common findings. serum chemistry can confirm hypoalbuminemia caused by inflammation of the bowel wall. hyperglobulinemia can be characterized with serum electrophoresis, which is nonspecific and can reveal chronic inflammation. a few cases of lymphoma have been identified with monoclonal hypergammaglobulinemia. ectopic hormone production may result in hypercalcemia (calcium > mg/dl), which is associated with alimentary neoplasia such as lymphoma, multiple myeloma, carcinomata, and ameloblastoma. , hypoglycemia (blood glucose < mg/dl) can occur with neoplasia of the pancreas or liver. rectal examination may detect an abdominal mass, thickening of the intestinal wall, lymph node enlargement, or a gritty texture in horses with carcinomatosis. rectal biopsy can reveal lymphoma in some cases. fecal occult blood test is nonspecific for neoplastic disease but can reveal blood loss through the gastrointestinal tract. occasionally, abdominocentesis can identify neoplasia if the tumor exfoliates cells into the abdomen. one can diagnose squamous cell carcinoma, adenocarcinoma, and mesothelioma from peritoneal fluid. , , , characterization of peritoneal fluid as an inflammatory exudate or modified transudate without any neoplastic cells present is common. cytologic analysis of peritoneal fluid samples collected by abdominocentesis accurately predicted the presence of neoplasia in of cases in one study. cytologic examination of two or more peritoneal fluid samples increases the sensitivity of this test for detecting abdominal neoplasia. measurement of peritoneal fluid glucose concentration and ph is valuable to differentiate inflammation in the peritoneum caused by neoplasia from bacterial peritonitis. abdominal neoplasia typically is associated with peritoneal glucose concentrations similar to blood and ph higher than . . d-xylose absorption tests can reveal malabsorptive diseases that include lymphoma. , immunoglobulin m deficiency is associated with lymphoma in some young adult horses, but the prevalence of immunoglobulin m deficiency in horses with lymphoma and the value of measuring serum immunoglobulin m concentrations for the diagnosis of lymphoma have not been evaluated. dna cell cycle analysis of suspect neoplastic cells has been used to detect lymphoma in equine patients confirmed with the disease. this method of evaluating fluid or tissues aspirates may increase the accuracy for diagnosing neoplasia in the future. a complete evaluation of the oral cavity may include using a full-mouth speculum, radiographs, and endoscopy of the pharynx. evaluation of the esophagus and stomach with a -m endoscope can reveal intralumenal masses. pleuroscopy has been used to obtain biopsy samples of extralumenal masses surrounding the esophagus. contrast radiography can assist in the diagnosis of neoplasia within the wall or outside of the esophagus. , ultrasonography of the stomach, small intestine, cecum, and large colon is useful in detecting intestinal wall thickness, abdominal masses, and excessive peritoneal fluid. identification of neoplasia in the liver, kidney or spleen may support metastasis to other parts of the gastrointestinal tract or lymph nodes. laparoscopy and exploratory laparotomy often are required to obtain a final diagnosis. lymphoma is the most common neoplasia in the horse and has been divided into four categories. this section covers only the intestinal/alimentary form. in the past, lymphoma has been called lymphosarcoma, but the preferred term by oncologists is lymphoma because no benign form of this disease exists. lymphoma originates from lymphoid tissue and predominantly affects the small intestine and intestinal lymph nodes. chronic weight loss from malabsorption, intermittent colic, and fever are the most common clinical findings. , chronic diarrhea has been reported in some cases. paraneoplastic pruritus and alopecia have been identified in one case of diffuse lymphoma. one generally does not note peripheral lymphadenopathy, but one may palpate enlargement of the intestinal lymph nodes on rectal examination. large colon resection for treatment of lymphoma in horses has increased short-term survival in two horses. chemotherapy in two mares that were pregnant extended their lives long enough to foal normally. long-term prognosis for intestinal lymphoma is poor. squamous cell carcinoma (scc) is a malignant tumor of the gastrointestinal epithelium. scc is the second most common neoplasia in the horse and is the most common oral neoplasia. however, the incidence of scc is rare. , , in the oral cavity scc may affect the lips, tongue, hard palate, pharynx, and mucosa. , treatment for scc in the oral cavity may involve surgical resection, iridium- brachytherapy, -fluorouracil, or intralesional cisplatin. , [ ] [ ] [ ] the prognosis for survival is good if complete removal of the tumor is possible. metastasis beyond the regional lymph nodes is rare for oral scc. squamous cell carcinoma is the most common tumor of the stomach and esophagus , and can invade these areas and metastasize to the lymph nodes and lungs. abnormal masses were palpated on rectal examination in four of five cases of gastric scc. treatment by surgical resection is not possible in most cases and the horses die or are euthanized. adenocarcinoma is a malignant tumor that can occur in the small intestine, cecum, and large colon. the tumor arises from the glandular crypts of the gastrointestinal tract and has been reported in middle-aged and older horses. metastasis to the lymph nodes, liver, and lungs can occur. intestinal adenocarcinoma has been reported to metastasize to the bone and was diagnosed using nuclear scintigraphy following injection of technetium- m hydroxymethylene diphosphate. , no reports of successful surgical resection have been published. leiomyosarcoma is a malignant tumor of the smooth muscle lining the gastrointestinal tract and has been reported in the stomach, small intestine, and rectum. , , , , in one case report, gastroscopy could not identify the mural mass in the stomach that was found during exploratory surgery. another report describes a favorable outcome for surgical resection of a leiomyosarcoma that was protruding from the anal sphincter in a -year-old quarter horse. prognosis for survival is favorable if surgical resection is possible. leiomyoma is a benign tumor of the smooth muscle of the gastrointestinal tract that can occur in the stomach, small intestine, and small colon. , clinical signs are consistent with intestinal obstruction. surgical resection and anastomosis of the affected portion of the intestine have been performed without complications. lipoma is a benign tumor that occurs in older horses ( to years) and arises from mesenteric adipocytes. the tumor grows on a stalk that wraps around the intestine, causing a strangulating lesion manifested clinically by acute obstructive colic. intestinal injury caused by pedunculated lipomata may occur in the small intestine, small colon, and rectum. long-term survival with surgical resection and anastomosis of the affected segment has been reported to be % to %. , oral neoplasia oral cavity neoplasia may involve the dental tissue (odontogenic tumors), bone (osteogenic tumors), or soft tissues (see box . - ). ameloblastoma occurs in horses greater than years old and mainly affects the mandible. ameloblastic odontoma affects younger horses and usually involves the maxilla. both are benign but locally invasive. radiographs may distinguish the difference between an ameloblastoma (radiolucent lesion) and ameloblastic odontoma (radiolucent lesion with partially mineralized density). the best treatment option is surgical resection and radiation therapy regardless of the type. juvenile mandibular ossifying fibroma occurs in the rostral mandible of young horses between the ages of months and years. the fibroma may cause significant distortion of the bone. with early diagnosis and surgical excision of the mass, the horse has a good prognosis. melanomas, sarcoids, and oral papilloma occur on the mouth and lips. melanomas rarely metastasize, but they commonly are found in the parotid salivary glands and lymph nodes. sarcoids are the most common skin tumor that can involve the mouth. ulcerations of the buccal mucosa are difficult to treat. intralesional cisplatin, cryosurgery, radiation, and laser excision have been tried with limited success. equine papilloma virus is responsible for the common skin wart found on the lips and muzzle of young horses. these lesions are usually selflimiting but may be removed successfully by cryosurgery or excision. a number of detailed and informative reviews are available describing the anatomy, physiology, and pathophysiology of the equine peritoneum. [ ] [ ] [ ] [ ] [ ] the peritoneum consists of a single layer of mesothelial cells lining the peritoneal cavity and serosal surfaces of the intraabdominal viscera. the mesothelial lining of the diaphragm, abdominal walls, and pelvic cavity is termed parietal peritoneum. the visceral peritoneum includes the serosal surfaces of the intraabdominal organs. the parietal and visceral portions of the peritoneum are contiguous with each other through the omentum, mesenteries, and ligaments. caudally, the peritoneum reflects over the surfaces of the pelvic organs (portions of the urogenital tract and rectum), excluding them from the peritoneal space, and thus much of the pelvic cavity and contents are described as retroperitoneal. the peritoneal space communicates with the lumen of the uterus (and thus the external environment) via the fallopian tubes in females. in males the peritoneum forms a true blind sac. the vascular supply and nervous innervation of the visceral peritoneum are supplied by the splanchnic vessels and visceral autonomic nerves, respectively. branches of the intercostal, lumbar, and iliac arteries supply the parietal peritoneum, and the phrenic and intercostal nerves provide nervous innervation. the clinical relevance is that inflammation of the parietal peritoneum is perceived as somatic pain, resulting in a splinted abdominal wall, pain on external palpation, and reluctance to move. visceral pain is mediated by small type c sensory fibers, which are believed to be stimulated by bowel distention, smooth muscle spasms, tension on the mesentery, and ischemia. the peritoneal lining functions as a semipermeable barrier to the diffusion of water and low-molecular weight solutes between the blood and the abdominal cavity. the peritoneum secretes a serous fluid that lubricates the abdominal cavity, inhibits adhesion formation, and has minor antibacterial properties. , macrophages, mast cells, mesothelial cells, and lymphocytes provide immune function within the peritoneum. , peritoneal macrophages impart antibacterial activity via complement receptors, phagocytic activity, interaction with t lymphocytes, neutrophil chemotaxis, and fibroblast activation. the peritoneal surface maintains a high level of fibrinolytic activity through the production of plasminogen activators by mesothelial cells. this function, together with the lubricant properties of the peritoneal fluid, helps to maintain gliding surfaces within the peritoneum and prevent adhesion formation. in quadrupeds, peritoneal fluid produced by the mesothelium tends to move ventrally and cranially, aided largely by diaphragmatic movement. peritoneal fluid, waste products, and foreign material (including bacteria) exit the peritoneal cavity to enter the lymphatic system through diffusely distributed subendothelial pores or via the large diaphragmatic stomata, depending on particle size. large molecules and particles greater than approximately , mw (such as bacteria) exit through the diaphragmatic stomata and ultimately enter the thoracic duct. peritonitis is inflammation of the mesothelial lining of the peritoneal cavity and is characterized by desquamation and transformation of mesothelial cells; chemotaxis of neutrophils; release of several soluble mediators of inflammation; exudation of serum, fibrin, and protein into the peritoneal cavity; and depression of fibrinolytic activity. peritonitis occurs in association with a variety of disorders that result in mechanical, chemical, or infectious insult to the peritoneal lining. [ ] [ ] [ ] [ ] any process resulting in disruption or irritation of the peritoneal lining, inflammation or infection of abdominal organs, or compromise of the intestinal wall can result in peritonitis (box . - ). common mechanical injuries include blunt or perforating trauma to the abdominal wall, breeding and foaling accidents, and abdominal surgery. a variety of traumatic insults of iatrogenic origin can cause peritonitis, such as the pathogenesis of peritonitis as a series of stages, as reviewed and described by trent, is useful. the contamination stage, lasting to hours, involves introduction of bacteria into the peritoneum and initiation of the acute inflammatory response previously described. if the organisms are not eliminated and infection is established, the process evolves to the stage of acute diffuse peritonitis. although the overall movement of contaminants is toward the diaphragmatic stomata and into the thoracic duct, the nature of the peritoneal circulation is such that regardless of the location of the initial contamination, bacteria spread throughout the peritoneum within several hours. the stage of acute diffuse peritonitis lasts up to days. the inflammatory response persists and escalates with continued exudation of proteinaceous fluid and influx of inflammatory cells. offending organisms are delivered to the lymphatic system and may be eliminated by the immune system. organisms, however, may gain access to the systemic circulation in sufficient numbers to result in clinically relevant septicemia. in human beings and laboratory animals having undergone polymicrobial contamination of the peritoneum, the organisms causing septicemia at this stage are usually coliforms, e. coli in particular. this stage of the disease process has the highest mortality because of the effects of severe peritoneal inflammation, endotoxemia, and septicemia. if the animal survives this stage but fails to eliminate the infection in the peritoneal cavity, the disease enters a transitional phase referred to as the acute adhesive (or localizing) stage. this stage occupies a time frame of perhaps to days after the initial insult. neutrophils are still active, macrophages are increasing in numbers, and fibrin aggregates are being organized or lysed. in human beings and laboratory animals, selective reduction and synergism continue such that anaerobes and gram-negative aerobes predominate. if infection persists beyond this point, organization of fibrin proceeds and organisms become isolated from host defenses. at this point, the disease process enters the stage of chronic abscessation. this stage can begin as early as days after inoculation and persists indefinitely. clinical signs of peritonitis depend on the primary disease process, the duration of the problem, and the extent of peritoneal inflammation. localized peritonitis may have few or no systemic manifestations, whereas severe localized or generalized peritonitis often is accompanied by severe toxemia or septicemia or both. septic peritonitis usually causes more severe clinical signs because of the inflammatory mediators released in response to bacterial toxins and because of the presence of endotoxin when gram-negative organisms are involved. most clinical signs are nonspecific and include fever, depression, inappetance, decreased borborygmi, and dehydration. additional signs, reported in horses (ages months to years) with peritonitis, were colic, ileus, weight loss, and diarrhea. horses with peracute peritonitis, as occurs with rupture of the bowel or rectal tear, have severe toxemia, weakness, depression or severe colic, tachycardia, tachypnea, and circulatory failure. fever may not be present depending on the degree of shock. typical clinical findings include sweating, pawing, muscle fasciculations, weak peripheral pulses, red to purple mucous membranes, prolonged capillary refill time, and decreased skin elasticity. parietal pain, characterized by reluctance to move, splinting of the abdominal wall, and sensitivity to external abdominal pressure occur in some acute cases. urination or defecation may be painful for the horse, and urine and fecal retention may be evident on rectal examination. palpation of the abdomen externally may elicit flinching, aversion movements, or groaning. with extensive abdominal fecal contamination, rectal examination may reveal a gritty feeling of the serosal and parietal surface of the peritoneum because of fibrin deposition and a dry texture of the peritoneum. in horses with more chronic peritonitis, rectal examination findings can include pain on palpation of fibrinous or fibrous adhesions, intestinal impaction or distention following ileus and dehydration, an abdominal mass (abscess or neoplasia), or an impression of bowel floating in fluid. in many cases, one can detect no abnormalities on rectal examination. horses with localized, subacute, or chronic peritonitis may have signs of chronic or intermittent colic, depression, anorexia, weight loss, intermittent fever, ventral edema, exercise intolerance, decreased or absent intestinal sounds, and mild dehydration. heart rate and respiratory rate may be normal. fecal output may be normal; however, horses with chronic diarrhea and weight loss have been reported. foals with peritonitis usually exhibit signs of colic (acute or chronic) and are febrile, depressed, and inappetant. in some foals with primary peritonitis, pleural effusion occurs. in young foals, peritonitis can cause rapid metabolic deterioration, and determination and correction of the primary problem requires immediate attention. in older foals, peritonitis may occur insidiously, as occurs following s. equi or r. equi infections. clinicopathologic abnormalities vary depending on the time of onset and severity of peritonitis. horses with acute, septic peritonitis can have leukopenia, hemoconcentration, metabolic acidemia, azotemia, and electrolyte imbalances reflective of systemic inflammation from endotoxemia and hypovolemia. horses with peritonitis of a few days' duration may have leukocytosis and hyperfibrinogenemia. plasma protein levels vary depending on the hydration status, degree of exudation into the peritoneum, and type of underlying problem. in chronic peritonitis, hyperproteinemia with hyperglobulinemia may be present. neonates with uroperitoneum caused by urinary bladder rupture or urachal disease tend to develop azotemia, hyponatremia, hypochloremia, hyperkalemia, and acidosis. foals with peritonitis following septicemia, severe enterocolitis, severe meconium impaction, intussusception, small intestinal volvulus, gastric or duodenal rupture, or ascarid impactions usually have clinicopathologic findings reflective of systemic inflammation, such as inflammatory leukogram or leukopenia, hemoconcentration, and acidosis. chronic abscessation, as occurs in foals with r. equi and streptococcal infections, results in clinicopathologic findings reflecting chronic inflammation (anemia, hyperfibrinogenemia, hyperglobulinemia). abnormalities in the composition of peritoneal fluid occur with peritoneal inflammation, and peritoneal fluid analysis is principal to the diagnosis of peritonitis. one collects peritoneal fluid through puncture of the abdomen on the ventral midline. one should clip and prepare an area aseptically. usually, the lowest point of the abdomen, to cm caudal to the xiphoid cartilage, is prepared for puncture; although in some cases one may perform paracentesis more caudally, particularly when one suspects a specific area of sequestered fluid or abscessation. in addition, one may choose a site to the right of midline in an attempt to avoid the spleen. one can perform peritoneal puncture using a / -inch, -gauge needle or, following local anesthesia and a stab incision with a no. scalpel blade, using a sterile cannula. one collects fluid by gravity flow and should collect fluid in a tube containing anticoagulant, preferably edta for cytologic examination, and in a sterile tube without anticoagulant for visual inspection and, if desired, for culture. one should fill the edta tube to half its volume, because the edta will alter the refractive index of the fluid, resulting in a falsely elevated value for total solids when one collects only a small volume and tests it with a refractometer. one should evaluate peritoneal fluid routinely as to color, turbidity, total protein, white blood cell (wbc) count and differential, and the presence of bacteria as determined by gram stain. normal peritoneal fluid is clear and straw-colored and does not coagulate spontaneously. peritoneal fluid becomes turbid when increased numbers of white blood cells and concentration of protein are present. pink or red fluid indicates free hemoglobin or hemorrhage. blood introduced into the peritoneal fluid iatrogenically in some cases may be differentiated from blood from internal hemorrhage based on the presence of platelets and hematocrit. fluid with iatrogenic blood contamination contains platelets, whereas fluid with blood following internal hemorrhage or diapedesis often does not have platelets. blood contamination resulting from splenic puncture often results in the packed cell volume of the sample being greater than that of the blood. large volumes of dark brown or green fluid with a fetid odor obtained from several sites strongly suggest bowel rupture, but one should perform cytologic examination for confirmation. the distribution of polymorphonuclear and mononuclear cells varies widely, and one should interpret the results of cell counts and differentials as supporting a number of disorders rather than a specific diagnosis. normal equine peritoneal fluid contains fewer than nucleated cells per microliter. , wbc counts in acute peritonitis (> , /µl) are reported to be higher than those in chronic peritonitis ( , to , /µl) [ ] [ ] [ ] ; however, this is not always the case, and the wbc count depends most on the cause of the peritonitis. the wbc level does not always correlate with severity of peritonitis or the prognosis. the peritoneal fluid wbc count can be greater than , /µl following enterocentesis, with no clinical signs or problem. conversely, peritoneal wbc counts of fewer than , /µl may be found in foals or horses with intraabdominal abscesses. the peritoneal wbc count can increase to greater than , /µl following celiotomy and can be higher if an enterotomy is done. postoperatively, the wbc count normally continues to decline and returns to near normal by to days. failure of the wbc count to decrease suggests peritonitis resulting from a postoperative complication. finally, peritoneal fluid wbc counts greater than , /µl indicate severe focal or generalized peritoneal sepsis. the distribution of polymorphonuclear and mononuclear cells varies in normal peritoneal fluid, , but polymorphonuclear cells usually predominate. with acute peritonitis, polymorphonuclear cells typically increase to a greater degree than mononuclear cells, but this depends on the cause. in horses that have bowel disease accompanied by endotoxemia, the number of peritoneal mononuclear cells increases, as does transformation of mesothelial cells to macrophages. in chronic cases, one easily may mistake transforming mesothelial cells for neoplastic cells, which can make diagnosis difficult, particularly when the presenting problem is compatible with a neoplastic process. in such cases, consultation with a clinical pathologist regarding cytologic findings is prudent. factor, and interleukin- have been measured in the peritoneal fluid of horses with abdominal disorders, but the diagnostic and prognostic implications of the presence or absence of these enzymes and analytes is limited. [ ] [ ] [ ] one should submit peritoneal fluid samples in appropriate media (port-a-cul vial, bbl microbiology system) for aerobic and anaerobic cultures in an attempt to identify the pathogenic organism(s). obligate anaerobic bacteria such as bacteroides are difficult to culture, because one must collect, transport, and culture the sample under strict anaerobic conditions. frequently, bacterial cultures are negative when bacteria are present in peritoneal fluid. to enhance recovery of bacteria, one can inoculate peritoneal fluid into blood culture medium (septi-chek columbia, hoffmann-laroche inc., nutley, new jersey), and if the horse has received antimicrobial treatment, one first should pass fluid through an antimicrobial removal device (a.r.d., becton dickinson & co., cockeysville, maryland). early and aggressive therapy is required if treatment of peritonitis is to be successful. the goals of treatment are to resolve the primary problem, minimize the inflammatory response, and prevent long-term complications. in the acute phase, one gives primary consideration to the arrest of endotoxic, septic, or hypovolemic shock; correction of metabolic and electrolyte abnormalities and dehydration; and management of pain. in the absence of blood gas and electrolyte determinations, adequate volumes of a balanced electrolyte solution are required to correct dehydration and support the cardiovascular system. if the plasma protein concentration of the horse is less than . g/dl, one should consider administration of plasma or synthetic colloids. one should administer flunixin meglumine (banamine) for its local and systemic antiinflammatory effects. dosages vary depending on the severity of peritonitis, degree of toxemia, severity of pain, and hydration status of the horse and range from . mg/kg intramuscularly or intravenously every to hours to . mg/kg intramuscularly or intravenously every hours. the higher dosage provides greater visceral analgesia, whereas the lower dosage is effective in modifying the effects of experimental endotoxemia. in addition to analgesic and general antiinflammatory effect, flunixin meglumine may be effective in retarding adhesion formation when administered early in the acute, diffuse stage of septic peritonitis. heparin therapy has been recommended to prevent adhesion formation and to render bacteria more susceptible to cellular and noncellular clearing mechanisms. in experimental models using laboratory animals, normal peritoneal fluid protein concentration is less than . g/dl. protein levels between . g/dl and . g/dl can be difficult to interpret, but one should consider levels greater than . g/dl to be elevated abnormally. fibrinogen concentration increases with inflammation, and levels greater than mg/dl in the peritoneal fluid suggest that an acute inflammatory process is present. fibrinogen content will also increase from blood contamination. the presence of free and phagocytosed bacteria in peritoneal fluid indicates generalized suppuration, abscessation, or compromised bowel. if one observes numerous microorganisms of mixed types free in the peritoneal fluid or if one observes plant material, massive bacterial contamination of the abdomen following bowel rupture likely has occurred. the presence of toxic or degenerate neutrophils and bacteria within polymorphonuclear cells helps to distinguish peritoneal fluid from intestinal contents in such cases. enterocentesis yields discolored fluid containing mixed microorganisms and plant material and that is largely devoid of white blood cells. bacterial contamination of a sample can occur during collection of the sample, and iatrogenic contamination of a sample can result in free and intracellular bacteria in peritoneal fluid, particularly if processing is delayed. in such cases the bacterial numbers are few and the neutrophils appear healthy. in some cases of gastrointestinal perforation the luminal material, inflammatory cells, and protein may be sequestered by the omentum and further contained by fibrinous adhesions. abdominal fluid obtained via standard ventral paracentesis may have low cellularity and protein content but large numbers of mixed bacteria indicating bowel rupture. examples include gastric rupture along the greater curvature of the stomach between the omental layers (omental bursa) and perforated gastric or duodenal ulcers in foals. correlating all cytologic findings with clinical and clinicopathologic findings is important for interpreting the results of peritoneal fluid cytologic examination. biochemical analysis of peritoneal fluid may be useful in detecting sepsis when cytologic examination and culture are negative or otherwise unavailable. in a prospective study by van hoogmoed, rodger, spier, et al., peritoneal fluid ph and glucose concentrations from horses with septic peritonitis were significantly lower than horses with nonseptic peritonitis and healthy horses. peritoneal fluid ph less than . , glucose less than mg/dl, and fibrinogen concentration greater than mg/dl were considered highly predictive of septic peritonitis. serum to peritoneal glucose concentration differences of greater than mg/dl was considered the most diagnostically useful test for septic peritonitis in the study. increased activities of alkaline phosphatase, lactic dehydrogenase, creatine kinase, aspartate aminotransferase, tumor necrosis heparin therapy was associated with decreased adhesions in septic peritonitis. heparin has not yet been demonstrated clearly to have similar efficacy in horses, although it may. suggested dosages range from to iu subcutaneously every hours for hours to to iu/kg subcutaneously every hours. one should note that heparin induces red blood cell aggregation in horses, which may adversely affect capillary blood flow. one should initiate antimicrobial therapy after making a diagnosis of peritonitis and before the results of peritoneal culture are available, because isolating an organism may take several days and often culture fails to isolate the organism(s). intravenous administration of antimicrobials is preferred over oral or intramuscular routes in acute, diffuse, septic peritonitis because more reliable levels of drug are achieved in the tissues and peritoneal fluid than otherwise would be obtained in horses with hypovolemia or decreased intestinal motility. the combination of a β-lactam antibiotic with an aminoglycoside is appropriate in most circumstances and certainly in the acute diffuse stage of septic peritonitis. these drugs act synergistically to provide a broad spectrum of activity against a variety of gram-positive and gramnegative aerobic and anaerobic bacteria. potassium penicillin ( , to , iu/kg intravenously every hours) combined with gentamicin ( . mg/kg every hours) is an appropriate regimen. in most cases, peritonitis will have resulted from bowel contamination, and thus one should presume a mixed infection with gram-negative aerobic bacteria and gram-positive and gram-negative anaerobic bacteria. one also should presume the same in many cases of traumatic peritonitis, as occurs with foreign body puncture, breeding trauma, or foaling trauma. therefore a strong possibility exists of infection involving penicillin resistant bacteroides fragilis, so that adding metronidazole ( mg/kg orally every to hours) to the regimen is prudent. combination therapy with β-lactam and aminoglycoside antibiotics (and metronidazole when indicated) is a standard and generally effective protocol. one can modify this antimicrobial regimen when culture and antimicrobial sensitivity results become available. aminoglycosides and nonsteroidal antiinflammatory drugs have the potential to induce acute renal tubular damage, particularly when dehydration and decreased renal perfusion are present. therefore adequately rehydrating the patient and ensuring that renal function is intact before initiating treatment with these drugs are important. furthermore, maintaining hydration and monitoring renal function during the course of treatment are important. monitoring serum creatinine concentration; performing serial uninalysis observing for pigment, red blood cells; and casts; determining the ratio of γ-glutamyltransferase to creatinine in the urine; and therapeutic drug monitoring of aminoglycoside levels are useful in this regard. sodium ampicillin and ceftiofur sodium are β-lactam antibiotics that may be useful in combination therapy. these drugs have an extended gram-negative spectrum compared with penicillin. however, as a third-generation cephalosporin, ceftiofur is less effective against anaerobes than penicillin. one also may consider ceftiofur, trimethoprim-potentiated sulfonamides, amikacin, and enrofloxacin for treatment of gram-negative infection based on culture and sensitivity results. enrofloxacin is a quinolone drug with excellent activity against gramnegative pathogens, including pseudomonas, and also can be effective against resistant staphylococci (personal observation). such staphylococci may be involved in infections caused by traumatic puncture of the abdominal wall. enrofloxacin has a variety of potential toxic effects, including cartilage damage in young growing animals. however, a recent study concluded the drug was safe when administered to adult horses intravenously at mg/kg every hours for weeks. one probably should avoid using the drug in young, growing animals until the issue of cartilage damage is resolved. administration of enrofloxacin to horses constitutes off-label usage. one should treat horses with acute, diffuse, septic peritonitis with antibiotics until the white blood cell count, plasma fibrinogen, and abdominal fluid analysis are normal. in horses that respond to therapy, this process takes a variable amount of time depending on the offending organisms and stage of disease at the time treatment is initiated. horses with abdominal abscessation resulting from polymicrobial infection may require many months of antibiotic treatment. abdominal abscesses caused by streptococci and corynebacterium pseudotuberculosis also may require long-term treatment (weeks to months). long-term antibiotic treatment generally necessitates the use of oral antibiotics, and the options are limited. trimethoprim-potentiated sulfonamides are administered orally and are effective against a variety of gram-positive and gram-negative organisms, although some streptococci are resistant. metronidazole is an orally administered drug effective against anaerobic bacteria, as previously discussed. other orally administered antimicrobials one may consider for long-term use include doxycycline (broad spectrum), erythromycin (gram-positive spectrum), and enrofloxacin (mostly gram-negative spectrum). importantly, rifampin, when used with other drugs, can be effective in penetrating and resolving abscesses. combination therapy with erythromycin and rifampin is the standard treatment for rhodococcus equi infection in foals. peritonitis caused by actinobacillus equuli usually is manifested as a diffuse, supporative peritoneal exudate. the same is true for some cases involving streptococci (personal observation). these infections generally respond well to combination therapy with penicillin and gentamicin. if streptococci are involved as the sole pathogen, then penicillin alone should be effective. streptococci potentially can be involved in mixed, synergistic peritoneal infections in horses. drainage or lavage of the peritoneal cavity may be of benefit in removing toxic bacterial by-products and products of inflammatory cells. high numbers of inflammatory cells and release of their mediators can persist even after the primary stimulus of the inflammatory response has resolved. infusing large volumes of isotonic, warmed fluid into the peritoneal cavity also dilutes the inflammatory mediators, possibly reducing their deleterious effects. when successful, peritoneal lavage decreases the peritoneal fluid wbc count and total protein, potentially reflecting a decrease in diffuse inflammation. the benefits of peritoneal lavage are controversial, and a positive effect may be more likely during the acute, diffuse stage of disease. , some studies suggest peritoneal lavage, along with heparin therapy, may reduce the incidence of adhesions. one should perform peritoneal drainage and lavage using a drain of no less than f diameter. foley-type catheters can be used, but "mushroom" drains provide a larger area for fluid to enter the drain. two approaches to peritoneal lavage are ( ) retrograde irrigation through a ventrally placed ingress-egress drain and ( ) placement of ingress catheter(s) in the paralumbar fossa(e) for infusion of fluids, with a drain placed ventrally for removal of infused fluid. one must recognize that thorough peritoneal lavage can be achieved only via ventral midline laparotomy. complications associated with the use of abdominal drains or repeated peritoneal penetration to drain fluid include retrograde infection, local irritation, pneumoperitoneum, and subcutaneous seepage around the drain and resultant cellulitis. if the patient is hypovolemic or hypoproteinemic, one should consider volume replacement and administration of plasma before removing large quantities of fluid from the abdomen. in horses with suspected parasite involvement, such as verminous arteritis, one should give larvicidal doses of an anthelmintic once the condition of the horse is stabilized. ivermectin, fenbendazole, and thiabendazole have been recommended as larvacidal therapies. visualization of auscultation sounds of the large intestine small intestinal betagalactosidase activity in the horse rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases equine lymphocyticplasmacytic enterocolitis: a retrospective study of cases eosinophilic enterocolitis and dermatitis in two horses breath hydrogen measurements in ponies: a preliminary study the oral glucose tolerance test in the horse small intestinal malabsorption in the horse: an assessment of the specificity of the oral glucose tolerance test transient glucose malabsorption in two horses: fact or artifact? chronic diarrhoea in adult horses: a review of referred cases effect of food deprivation on d-xylose absorption test results in mares malabsorption syndromes in the horse the diagnostic value of the d-xylose absorption test in horses with unexplained chronic weight loss small intestinal malabsorption in horses effects of extensive small intestinal resection in the pony effects of extensive resection of the small intestine in the pony clinical remission of granulomatous enteritis in a standardbred gelding following long term dexamethasone administration clinical and pathophysiological features of granulomatous enteritis and eosinophilic granulomatosis in the horse inflammatory bowel disease in horses: cases segmental eosinophilic colitis: a review of cases chronic idiopathic inflammatory bowel diseases of the horse section . malabsorption syndromes and maldigestion: pathophysiology, assessment, management, and outcome references of critical care medicine consensus conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis sisson and grossman's the anatomy of domestic animals observational study of "urine testing" in the horse and donkey stallion oral examination and diagnosis: management of oral disease fluoroscopic investigations of swallowing in the horse esophageal dysfunction in a weanling thoroughbred cervical esophagostomy to permit extraoral feeding of the horse use of a liquid diet as the sole source of nutrition in six dysphagic horses and as a dietary supplement in seven hypophagic horses acid-base and electrolyte alterations associated with salivary loss in the pony dysphagia caused by squamous cell carcinoma in horses diseases of the teeth comparative dental pathology (with particular reference to caries and paradental disease in the horse and the dog) some aspects of equine dental radiology some aspects of equine dental decay the median cleft of the lower lip and mandible and its surgical correction in a donkey sisson and grossman's the anatomy of domestic animals esophageal disorders of the horse esophageal disorders in horses: results of nonsurgical and surgical management recurrent esophageal obstruction due to squamous cell carcinoma in a horse squamous cell carcinoma of the lower cervical oesophagus in a pony squamous cell carcinoma: an unusual cause of choke in a horse megaesophagus and aspiration pneumonia secondary to gastric ulceration in a foal persistent right aortic arch in a yearling horse intramural esophageal cyst in a horse surgical correction of esophageal diverticulum in a horse on the true definition of dysphagia retrieval of an esophageal foreign body in a horse dysphagia caused by squamous cell carcinoma in two horses peptic ulcer pathophysiology recommendations for the diagnosis and treatment of equine gastric ulcer syndrome (egus) gastric ulcers in horses: a comparison of endoscopic findings in horses with and without clinical signs pipers fs et al: factors associated with gastric lesions in thoroughbred racehorses cross-sectional study of gastric ulcers of the squamous mucosa in thoroughbred racehorses gastric ulceration in mature thoroughbred horses epidemiological study of gastric ulceration in the thoroughbred racehorse: horses - endoscopic evaluation of the relationship between training, racing, and gastric ulcers prevalence of gastric ulcers in show horses postmortem findings of gastric ulcers in swedish horses older than age one year: a retrospective study of horses ( - ) endoscopic findings of the gastric antrum and pylorus in horses: cases gastric and duodenal ulcers in foals: a retrospective study endoscopic appearance of gastric lesions in foals: cases prevalence of gastric lesions in foals without signs of gastric disease: an endoscopic survey gastroduodenal ulceration in foals regional gastric ph measurement in horses and foals peptic ulcer disease in the pediatric population is helicobacter (campylobacter) pylori associated with gastritis/ulcer disease in asymptomatic foals? aetiopathogenesis and treatment of peptic ulcer in the horse: a comparative review evidence of helicobacter infection in the horse. proceedings of the society for microbiologists comparative pathophysiology of nonglandular ulcer disease: a review of experimental studies section . diseases of the stomach references . blackwell rb, white na: duodenitis-proximal jejunitis in the horse hemorrhagic fibrinonecrotic duodenitis-proximal jejunitis in horses: cases comparison of duodenitis-proximal jejunitis and small intestinal obstruction in horses: cases ( - ) prognostic indicators for horses with duodenitis-proximal jejunitis: horses ( - ) duodenitis-proximal jejunitis integrative immunophysiology in the intestinal mucosa immunophysiology of intestinal electrolyte transport peritoneal fluid constituents in horse with colic due to small intestinal disease modification of gastrointestinal motility in horses. proceedings of the eighteenth annual forum of the american college of veterinary internal medicine haemodynamic effects of small volume hypertonic saline in experimentally induced haemorrhagic shock equine proliferative enteropathy: a cause of weight loss, colic, diarrhea and hypoproteinemia in foals on three breeding farms in canada chronic inflammatory and lymphoproliferative lesions of the equine small intestine aa amyloid-associated gastroenteropathy in a horse malabsorption in the horse associated with alimentary lymphosarcoma clinical aspects of lymphosarcoma in the horse: a clinical report of cases alimentary lymphosarcoma in the horse equine granulomatous enteritis clinical and pathophysiological features of granulomatous enteritis and eosinophilic granulomatosis in the horse chronic idiopathic inflammatory bowel diseases of the horse pathology of equine granulomatous enteritis rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases chronic enteritis associated with malabsorption and protein-losing enteropathy in the horse clinical remission of granulomatous enteritis in a standardbred gelding following long term dexamethasone administration granulomatous enteritis in nine horses a cluster of granulomatous enteritis cases: the link with aluminum equine granulomatous enteritis linked with aluminum chronic eosinophilic gastroenteritis in the horse chronic eosinophilic dermatitis: a manifestion of a multisystemic, eosinophilic, epitheliotropic disease in five horses basophilic enterocolitis in a horse segmental eosinophilic colitis: a review of cases inflammatory bowel disease in horses: cases equine lymphocyticplasmacytic enterocolitis: a retrospective study of cases proliferative and inflammatory intestinal diseases associated with malabsorption and maldigestion references . smith bp: salmonella infection in horses prevalence and epizootiology of equine salmonellosis emergence of antibiotic-resistant salmonella agona in horses in kentucky fecal shedding of salmonella spp by horses in the united states during and and detection of salmonella spp in grain and concentrate sources on equine operations epidemiologic study of salmonellae shedding in the feces of horses and potential risk factors for development of the infection in hospitalized horses risk factors for nosocomial salmonella infection among hospitalized horses an outbreak of salmonellosis among horses at a veterinary teaching hospital outbreak of salmonella infantis infection in a large animal veterinary teaching hospital influence of fecal shedding of salmonella organisms on mortality in hospitalized horses control of an outbreak of salmonellosis caused by drug-resistant salmonella anatum in horses at a veterinary hospital and measures to prevent future infections antimicrobial resistance in salmonella and escherichia coli isolated from horses utilization of both phenotypic and molecular analyses to investigate an outbreak of multidrugresistant salmonella anatum in horses understanding the role of endotoxins in gramnegative sepsis salmonellosis in hospitalized horses: seasonality and case fatality rates salmonellosis in equidae: a study of cases pathogenesis of acute bacterial diarrheal disorders the alimentary canal as a microbial habitat enteric defensins: antibiotic peptide components of intestinal host defense regional specialization in the mucosal immune system: what happens in the microcompartments? salmonella: a model for bacterial pathogenesis pathogenesis of bacterial infections in animals review of veterinary microbiology a preliminary evaluation of some preparations of salmonella typhimurium vaccines in horses aromatic-dependent salmonella typhimurium as modified live vaccines for calves intranasal immunogenicity of a delta cya delta crp-paba mutant of salmonella enterica serotype typhimurium for the horse m cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process type iii secretion machines: bacterial devices for protein delivery into host cells epithelial cells as sensors for microbial infection extraintestinal dissemination of salmonella by cd -expressing phagocytes oxygen-dependent anti-salmonella activity of macrophages pathogenesis of experimental salmonellosis: inhibition of protein synthesis by cytotoxin pathogenesis of salmonella-mediated intestinal fluid secretion: activation of adenylate cyclase and inhibition by indomethacin elevated camp in intestinal epithelial cells during experimental cholera and salmonellosis role of salmonella enterotoxin in overall virulence of the organism mutation of invh, but not stn, reduces salmonella-induced enteritis in cattle importance of the intestinal inflammatory reaction in salmonella-mediated intestinal secretion pathophysiology of infectious diarrhea: changes in intestinal structure and function digestive physiology of the large intestine in adult horses. . pathophysiology of colitis neuroimmunophysiology of the gastrointestinal mucosa: implications for inflammatory diseases transepithelial signaling to neutrophils by salmonellae: a novel virulence mechanism for gastroenteritis review article: pathobiology of neutrophil interactions with intestinal epithelia equine salmonellosis: experimental production of four syndromes characteristics and risk factors for failure to survive of horses with acute diarrhea: cases ( - ) diagnosing salmonellosis in horses: culturing of multiple versus single faecal samples comparison of rectal mucosal cultures and fecal cultures in detecting salmonella infection in horses and cattle comparison of polymerase chain reaction and microbiological culture for detection of salmonellae in equine feces and environmental samples rapid pcr detection of salmonella in horse faecal samples potomac horse fever equine monocytic ehrlichiosis (potomac horse fever): a review experimental reproduction of potomac horse fever in horses with a newly isolated ehrlichia organism causative ehrlichial organisms in potomac horse fever ehrlichial diseases attempted ehrlichia risticii transmission with dermacentor variabilis (acari: ixodidae) attempted transmission of ehrlichia risticii (rickettsiaceae) with stomoxys calcitrans (diptera: muscidae) production and characterization of ehrlichia risticii, the agent of potomac horse fever, from snails (pleuroceridae: juga spp.) in aquarium culture and genetic comparison to equine strains infection of aquatic insects with trematode metacercariae carrying ehrlichia risticii, the cause of potomac horse fever transmission of ehrlichia risticii, the agent of potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report infection rate of ehrlichia risticii, the agent of potomac horse fever, in freshwater stream snails (juga yrekaensis) from northern california ultrastructural study of ehrlichial organisms in the large colons of ponies infected with potomac horse fever enterocolitis caused by ehrlichia sp. in the horse (potomac horse fever) growth of ehrlichia risticii in human colonic epithelial cells respiratory burst activity associated with phagocytosis of ehrlichia risticii by mouse peritoneal macrophages in vitro killing of ehrlichia risticii by activated and immune mouse peritoneal macrophages lack of lysosomal fusion with phagosomes containing ehrlichia risticii in p d cells: abrogation of inhibition with oxytetracycline pathophysiological changes in the large colon of horses infected with ehrlichia risticii disease features in horses with induced equine monocytic ehrlichiosis (potomac horse fever) clinical and hematologic variables in ponies with experimentally induced equine ehrlichial colitis (potomac horse fever) evaluation of fetal infection and abortion in pregnant ponies experimentally infected with ehrlichia risticii identification of ehrlichia risticii as the causative agent of two equine abortions following natural maternal infection effect of equine ehrlichial colitis on the hemostatic system in ponies detection of serum antibodies against ehrlichia risticii in potomac horse fever by enzyme-linked immunosorbent assay evidence for a high rate of false-positive results with the indirect fluorescent antibody test for ehrlichia risticii antibody in horses detection and quantitation of ehrlichia risticii genomic dna in infected horses and snails by real-time pcr comparison of pcr and culture to the indirect fluorescent-antibody test for diagnosis of potomac horse fever diagnostic application of polymerase chain reaction for detection of ehrlichia risticii in equine monocytic ehrlichiosis (potomac horse fever) evaluation of vaccination of horses as a strategy to control equine monocytic ehrlichiosis association of deficiency in antibody response to vaccine and heterogeneity of ehrlichia risticii strains with potomac horse fever vaccine failure in horses equine intestinal clostridiosis: an acute disease in horses associated with high intestinal counts of clostridium perfringens type a a method for reproducing fatal idiopathic colitis (colitis x) in ponies and isolation of a clostridium as a possible agent hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals apparent outbreaks of clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital a prospective study of the roles of clostridium difficile and enterotoxigenic clostridium perfringens in equine diarrhoea clostridial enterocolitis prevalence of clostridium perfringens enterotoxin and clostridium difficile toxin a in feces of horses with diarrhea and colic clostridium difficile associated with acute colitis in mature horses treated with antibiotics the effect of colic on the microbial activity of the equine intestine comparative effects of oral administration of trimethoprim/sulphadiazine or oxytetracycline on the faecal flora of horses colonic fermentation to short-chain fatty acids is decreased in antibiotic-associated diarrhea clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia lincomycin-induced severe colitis in ponies: association with clostridium cadaveris an investigation into clostridium perfringens enterotoxin-associated diarrhoea enterotoxigenic clostridium perfringens esophageal duplication cyst as a cause of choke in the horse beta toxin, a novel toxin produced by clostridium perfringens prevalence of beta -toxigenic clostridium perfringens in horses with intestinal disorders an overview of clostridium perfringens enterotoxin the effects of clostridium perfringens type a enterotoxin in shetland ponies: clinical, morphologic and clinicopathologic changes an update on clostridium perfringens enterotoxin clostridium difficile infection direct evidence of mast cell involvement in clostridium difficile toxin a-induced enteritis in mice neutrophil recruitment in clostridium difficile toxin a enteritis in the rabbit neuronal involvement in the intestinal effects of clostridium difficile toxin a and vibrio cholerae enterotoxin in rat ileum increased substance p responses in dorsal root ganglia and intestinal macrophages during clostridium difficile toxin a enteritis in rats cp- , , a substance p antagonist, inhibits rat intestinal responses to intestinal infarction associated with mesenteric vascular thrombotic disease in the horse the role of parasites in colic retropulsion-propulsion in equine large colon diarrhoea in horses associated with ulceration of the colon and caecum resulting from s. vulgaris larval migration clinical response of pony foals experimentally infected with strongylus vulgaris host response to experimentally induced infections of strongylus vulgaris in parasite-free and naturally infected ponies morphologic and clinicopathologic changes following strongylus vulgaris infections of immune and nonimmune ponies increase of immunoglobulin t concentration in ponies as a response to experimental infection with the nematode strongylus vulgaris larval cyathostomiasis immature stages of trichonema spp as a cause of diarrhoea in adult horses in spring larval cyathostomiasis (immature trichonema-induced enteropathy): a report of clinical cases diagnosis and successful treatment of diarrhoea in horses caused by immature small strongyles apparently insusceptible to anthelmintics recurrent diarrhoea in aged ponies associated with larval cyathostomiasis outbreak of larval cyathostomiasis among a group of yearling and two-year-old horses pathogenicity of cyathostome infection chronic diarrhoea in adult horses: a review of referred cases diarrhoea in adult horses: a survey of clinical cases and an assessment of some prognostic indices the pathogenic effects of experimental cyathostome infections in ponies fructosamine measurement in ponies: validation and response following experimental cyathostome infection re-evaluation of ivermectin efficacy against equine gastrointestinal parasites prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses equine cyathostome infection: suppression of faecal egg output with moxidectin experimental cyathostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: comparison of parasite burdens, immunity and colonic pathology identification and characterization of a pyrantel pamoate resistant cyathostome population lethal complications following administration of oxytetracycline in the horse lincomycin-associated colitis in horses risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: cases ( - ) case control and historical cohort study of diarrhea associated with administration of trimethoprim-potentiated sulphonamides to horses and ponies the association of erythromycin ethylsuccinate with acute colitis in horses in sweden effects of transportation, surgery, and antibiotic therapy in ponies infected with salmonella the influence of the normal flora on clostridium difficile colonisation of the gut studies on experimental enteric salmonellosis in ponies physiology of diarrhea: large intestine impaired colonic fermentation of carbohydrate after ampicillin the relationship of absorption characteristics and gastrointestinal side effects of oral antimicrobial agents prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period hypovolemic hyponatremia and signs of neurologic disease associated with diarrhea in a foal toxicology of nonsteroidal antiinflammatory drugs colitis induced by nonsteroidal anti-inflammatory drugs: report of four cases and review of the literature the effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study experimentally induced phenylbutazone toxicosis in ponies: description of the syndrome and its prevention with synthetic prostaglandin e phenylbutazone toxicosis in the horse: a clinical study ulceration and stricture of the right dorsal colon after phenylbutazone administration in four horses phenylbutazone toxicity in a horse biochemical and haematological effects of phenylbutazone in horses medical management of right dorsal colitis in horses: a retrospective study mechanisms of intestinal mucosal repair prostaglandins in the gut and their relationship to non-steroidal anti-inflammatory drugs the future of antiinflammatory therapy the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse mechanisms of acute and chronic intestinal inflammation induced by indomethacin mechanisms of nsaid-induced gastrointestinal injury defined using mutant mice pathogenesis of nsaid gastropathy: are neutrophils the culprits? icam- and p-selectin expression in a model of nsaid-induced gastropathy gastric ulceration induced by nonsteroidal anti-inflammatory drugs is a neutrophil-dependent process the recognition and medical management of right dorsal colitis in horses the application of technetium- m hexamethylpropyleneamine oxime ( mtc-hmpao) labeled white blood cells for the diagnosis of right dorsal ulcerative colitis in two horses cantharidin toxicosis in horses experimental cantharidiasis in the horse pathology of blister beetle (epicauta) poisoning in horses etiologic agents, incidence, and improved diagnostic methods of cantharidin toxicosis in horses clinical features of blister beetle poisoning in equids: cases clinical and diagnostic veterinary toxicology environmental biochemistry of arsenic trace metal poisoning gee, honey, why does the iced tea have a garlic taste? arsenic intoxication acute diarrheal disease in the horse the mucosal barrier, ige-mediated gastrointestinal events, and eosinophilic gastroenteritis ige-mediated (and food-induced) intestinal disease induction of anaphylaxis in mouse intestine by orally administered antigen and its prevention with soluble high affinity receptor for ige vaccine-associated anaphylactic-like reaction in a horse equine anaphylaxis equine anaphylaxis pathologic changes in experimental equine anaphylaxis role of intestinal mast cells in modulating gastrointestinal pathophysiology role of -hydroxytryptamine in intestinal water and electrolyte movement during gut anaphylaxis intestinal anaphylaxis in the rat: mediators responsible for the ion transport abnormalities motility effects of intestinal anaphylaxis in the rat intestinal anaphylaxis: rapid changes in mucosal ion transport and morphology exhaustion shock in the horse lactic acidosis: a factor associated with equine laminitis changes in the caecal flora associated with the onset of laminitis intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report plasma endotoxin levels in horses subjected to carbohydrate induced laminitis surgical treatment of sand colic: results in horses diarrhea associated with sand in the gastrointestinal tract of horses sand-induced diarrhea in a foal abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation nasogastric electrolyte replacement in horses acute colitis in horses. . initial management effect of hydroxyethyl starch infusion on colloid oncotic pressure in hypoproteinemic horses oncotic, hemodilutional, and hemostatic effects of isotonic saline and hydroxyethyl starch solutions in clinically normal ponies colloid volume expanders: problems, pitfalls and possibilities immunity targeting common core antigens of gram-negative bacteria protection against clinical endotoxemia in horses by using plasma containing antibody to an rc mutant e. coli (j ) enterotoxin activity of a salmonella typhimurium of equine origin in vivo in rabbits and the effect of salmonella culture lysates and cholera toxin on equine colonic mucosa in vitro enterotoxin-induced fluid accumulation during experimental salmonellosis and cholera: involvement of prostaglandin synthesis by intestinal cells nacl transport across equine proximal colon and the effect of endogenous prostanoids nonsteroidal anti-inflammatory drugs activate quiescent inflammatory bowel disease misoprostol provides a colonic mucosal protective effect during acetic acidinduced colitis in rats rationale for the luminal provision of butyrate in intestinal diseases pharmacokinetics and adverse effects of butorphanol administered by single intravenous injection or continuous intravenous infusion in horses necrotizing mycotic vasculitis with cerebral infarction caused by aspergillus niger in a horse with acute typholocolitis pulmonary aspergillosis in horses: cases ( - ) effect of antibiotics on clinical, pathologic and immunologic responses in murine potomac horse fever: protective effects of doxycycline effect of treatment with oxytetracycline during the acute stages of experimentally induced equine ehrlichial colitis in ponie effect of treatment with erythromycin and rifampin during the acute stages of experimentally induced equine ehrlichial colitis in ponies use of metronidazole in equine acute idiopathic toxaemic colitis antimicrobial susceptibilities of equine isolates of clostridium difficile and molecular characterization of metronidazole-resistant strains equine neonatal clostridiosis: treatment and prevention antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy antithrombotic actions of aspirin in the horse evaluation of heparin for prophylaxis of equine laminitis: cases ( - ) phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin cold insoluble globulin and heparin interactions in phagocytosis by macrophage monolayers: mechanism of heparin enhancement probiotics in man and animals double-blind report on the efficacy of lactic acid-producing enterococcus sf in the prevention of antibiotic-associated diarrhoea and in the treatment of acute diarrhoea effect of lactobacillus gg yoghurt in prevention of antibiotic associated diarrhoea comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites efficacy of oral ivermectin paste against mucosal stages of cyathostomes elimination of mucosal cyathostome larvae by five daily treatments with fenbendazole moxidectin: spectrum of activity and uses in an equine anthelmintic program references . laws eg, freeman de: significance of reperfusion injury after venous strangulation obstruction of equine jejunum how important is intestinal reperfusion injury in horses? clinical relevance of intestinal reperfusion injury in horses strangulating volvulus of the ascending colon in horses mucosal alterations in experimentally induced small intestinal strangulation obstruction in ponies histologic findings in the gastrointestinal tract of horses with colic large colon resection evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal diseases of the small intestine small intestinal herniation through the epiploic foramen: cases ( - ) determining the diagnosis and prognosis of the acute abdomen duodenitis-proximal jejunitis risk factors and clinical signs associated with cases of equine colic short-term survival and prevalence of postoperative ileus after small intestinal surgery in horses short-and longterm survival and prevalence of postoperative ileus after small intestinal surgery in the horse survival after small intestine resection and anastomosis in horses abdominal adhesions after small intestinal surgery in the horse mesenteric rents as a source of small intestinal strangulation in horses: cases ( - ) small intestine incarceration through the epiploic foramen of the horse right hepatic lobe atrophy in horses: cases incarceration of the jejunum in the epiploic foramen of a four month old foal use of diagnostic ultrasonography in horses with signs of acute abdominal pain parietal hernia of the small intestine into the epiploic foramen of a horse transection of the pelvic flexure to reduce incarceration of the large colon through the epiploic foramen in a horse incarceration of the small intestine in the epiploic foramen: report of cases pedunculated lipomas as a cause of intestinal obstruction in horses: cases ( - ) an analysis of cases of intestinal obstruction caused by pedunculated lipomas strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma abdominal surgery in foals intestinal surgery in the foal effects of extensive resection of the small intestine in the pony incarceration of small intestine through rents in the gastrosplenic ligament in the horse jejunal displacement through the mesometrium in a pregnant mare strangulating obstruction caused by intestinal herniation through the proximal aspect of the cecocolic fold in horses congenital inguinal hernias associated with a rent in the common vaginal tunic in five foals ruptured inguinal hernia in new-born colt foals: a review of cases acquired inguinal hernia in the horse: a review of cases surgical treatment of acquired inguinal hernia in the horse: a review of cases different types of inguinal herniation in two stallions and a gelding surgery of the small intestine complications of umbilical hernias in horses: cases strangulated umbilical hernias in horses: cases ( - ) surgical management of intussusception in the horse ileocecal intussusception in horses: cases ultrasonographic diagnosis of small-intestinal intussusception in three foals jejunal intussusception in adult horses: cases ileocecal intussusception corrected by resection within the cecum in two horses diaphragmatic hernias in horses and cattle diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases peritoneopericardial hernia in a horse diaphragmatic herniation as a cause of lethargy and exercise intolerance in a mare diaphragmatic hernia repair in three young horses surgical repair of a diaphragmatic hernia in a racehorse diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases morphologic alterations observed during experimental ischemia of the equine large colon equine large intestinal volvulus: a review of cases diseases and surgery of the large colon large colon volvulus: surgical treatment of horses colopexy in broodmares: cases cecocolic intussusception in horses: cases ( - ) cecocolic and cecocecal intussusception in horses: cases ( - ) cecal amputation via a right ventral colon enterotomy for correction of nonreducible cecocolic intussusception in horses resection of intussuscepted large colon in a horse intussusception of the large colon in a horse intussusception of the left dorsal colon in a horse intussusception of the colon in a filly rectal prolapse in the horse rectal prolapse and cystic calculus in a burro rectal prolapse in a foaling mare management of rectal injuries rectum and anus disruption to the blood supply to the small colon following rectal prolapse and small colon intussusception in a mare laparoscopic diagnosis of ischemic necrosis of the descending colon after rectal prolapse and rupture of the mesocolon in two postpartum mares intestinal infarction associated with mesenteric vascular thrombotic disease in the horse diseases of the large colon diagnostic and prognostic procedures for equine colic surgery examination of the horse with colic detection of endotoxin in cases of equine colic determining the diagnosis and prognosis of the acute abdomen risk factors and clinical signs associated with cases of equine colic accuracy of clinicians in predicting site and type of lesion as well as outcome in horses with colic struvite urethral calculus in a three-month-old thoroughbred colt urolithiasis in horses cholelithiasis in horses: ten cases review of cases of colic in the pregnant mare surgical management of uterine torsion in the mare: a review of cases rabies in horses: cases mechanisms of pain and their therapeutic implications gastrointestinal pharmacology nonsteroidal anti-inflammatory drugs the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse alpha adrenoceptor agonists in the horse: a review cardiovascular effects of medetomidine, detomidine and xylazine in horses cardiovascular effects of xylazine and detomidine in horses prognosis in equine colic: a study of individual variables used in case assessment a: study of variables commonly used in examination of equine colic cases to assess prognostic value clinical evaluation of blood lactate levels in equine colic use of clinical pathology in evaluation of horses with colic the anion gap as a prognostic indicator in horses with abdominal pain strangulating volvulus of the ascending colon in horses diseases and surgery of the large colon multivariable prediction model for the need for surgery in horses with colic prognosis in equine colic patients using multivariable analysis prognosis in equine colic: a comparative study of variables used to assess individual cases prognostic index for acute abdominal crisis (colic) in horses feeding and digestive problems in horses: physiologic responses to a concentrated meal effect of meal feeding on plasma volume and urinary electrolyte clearance in ponies evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression morphologic effects of experimental distention of equine small intestine surgical reduction of ileal impactions in the horse: cases medical treatment of horses with ileal impactions: cases ascarids: recent advances the purported role of coastal bermuda hay in the etiology of ileal impactions: results of a questionnaire (abstract) tapeworm infection is a significant risk factor for spasmodic colic and ileal impaction colic in the horse use of excretory/secretory antigens for the serodiagnosis of anoplocephala perfoliata cestodosis ileal impaction in the horse: cases evaluation of factors associated with postoperative ileus in horses: cases idiopathic muscular hypertrophy of the equine small intestine: cases ( - ) obstruction of the ileum in the horse: a report of clinical cases ileal muscular hypertrophy and rupture in a pony three years after surgery for ileocaecal intussusception jejunocolostomy or ileocolostomy for treatment of cecal impaction in horses: nine cases ( - ) small intestinal strangulation caused by meckel's diverticulum in a horse volvulus associated with meckel's diverticulum in the horse congenital jejunal diverticulum in a foal mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse abdominal adhesions after small intestinal surgery in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal the characteristics of intestinal injury peripheral to strangulating obstruction lesions in the equine small intestine current concepts in management of abdominal adhesions one percent sodium carboxymethylcellulose prevents experimentally induced abdominal adhesions in horses effect of carboxymethylcellulose and a hyaluronate-carboxymethylcellulose membrane on healing of intestinal anastomoses in horses intraperitoneal use of sodium carboxymethylcellulose in horses undergoing exploratory celiotomy prevention of intraabdominal adhesions in ponies by low-dose heparin therapy retrospective analysis of the results of exploratory laparotomies in horses with gastrointestinal disease surgical treatment for colic in the foal retrospective evaluation of repeat celiotomy in horses with acute gastrointestinal disease risk factors for reduced postoperative fecal output in horses: cases ( - ) role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model the effect of prostaglandin e on motility of the equine gut in vitro investigation of the effect of prostaglandins and nonsteroidal antiinflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure evaluation of nitric oxide as an inhibitory neurotransmitter in the equine ventral colon prostanoid production via cox- as a causative mechanism of rodent postoperative ileus the action of low dose endotoxin on equine bowel motility sir frederick hobday memorial lecture: all wind and water-some progress in the study of equine gut motility antagonism of endotoxin-induced disruption of equine bowel motility by flunixin and phenylbutazone cyclooxygenase inhibitors in equine practice in vitro effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: cases ( - ) role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period cecal impaction in the horse caecal disease in equids ileocolostomy: a technique for surgical management of equine cecal impaction diseases and surgery of the cecum treatment of impaction colics large colon impaction in horses: cases ( - ) effects of amitraz, several opiate derivatives and anticholinergic agents on intestinal transit in ponies experimental studies of druginduced impaction colic in the horse retropulsion-propulsion in equine large colon clinical and structural features of equine enteroliths petrographic and geochemic evaluation of equine enteroliths enteroliths in horses evaluation of enterolithiasis in equids: cases risk factors for enterolithiasis among horses in texas obstructive enterolith in an -month-old miniature horse surgical treatment of sand colic in equids: cases ( - ) surgical treatment of sand colic: results in horses diarrhea associated with sand in the gastrointestinal tract of horses abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation failure of psyllium mucilloid to hasten evaluation of sand from the equine large intestine nonstrangulated colonic displacement in horses functions of the equine large intestine and their interrelationship in disease comparisons of age, breed, history and management in horses with colic motor functions of the intestine the effect of strongylus vulgaris larvae on equine intestinal myoelectrical activity displacement of the large colon reversal of colonic net absorption to net secretion with increased intraluminal pressure use of ultrasound in horses for diagnosis of left dorsal displacement of the large colon and monitoring its nonsurgical correction renosplenic entrapment of the large colon in horses: cases renosplenic entrapment of the large colon in horses: cases further experiences with non-surgical correction of nephrosplenic entrapment of the left colon in the horse effect of phenylephrine on hemodynamics and splenic dimensions in horses displacement of the large colon associated with nonsurgical correction of large-colon entrapment in the renosplenic space in a mare foreign body obstruction of the small colon in six horses fecalith impaction in four miniature foals obstruction of the small colon by intramural haematoma in three horses submucosal haematoma as a cause of obstruction of the small colon in the horse: a review of four cases inflammatory bowel disease in horses: cases intestinal atresia in horses clinical survey of tumours and tumourlike lesions in horses in south east queensland abdominal neoplasia (excluding urogenital tract) clinical manifestation of squamous cell carcinoma in horses lymphoma (lymphosarcoma) in horses oral and dental tumors in equine denistry t cell-rich b cell lymphosarcoma in the tongue of a horse multiple myeloma in a horse rhabdomyosarcoma of the tongue in a horse paraneoplastic bullous stomatitis in a horse gastric squamous cell carcinoma in three horses use of esophagoscopy in the diagnosis of esophageal squamous cell carcinoma in a horse gastric hyperplastic polyp in a horse gastric leiomyosarcoma in a horse multisystemic, eosinophilic, epitheliotropic disease with intestinal lymphosarcoma in a horse an immunohistochemical study of equine b-cell lymphoma squamous cell carcinoma of the equine stomach: a report of five cases six cases of squamous cell carcinoma of the stomach of the horse small intestinal adenocarcinoma in a horse ganglioneuroma as a cause of small intestinal obstruction in the horse: a case report intestinal carcinoid in a mare: an etiologic consideration for chronic colic in horses leiomyoma of the small intestine in a horse jejunal intussusception associated with leiomyoma in an aged horse duodenal leiomyoma associated with colic in a two-year-old horse leiomyosarcoma of the duodenum in two horses pedunculated lipomas as a cause of intestinal obstruction in horses: cases ( - ) clinical aspects of lymphosarcoma in the horse: a clinical report of cases multiple peripheral nerve sheath tumors in the small intestine of a horse equine adenocarcinomas of the large intestine with osseous metaplasia intestinal myxosarcoma in a thoroughbred mare gastrointestinal stromal tumors of the equine cecum colonic adenocarcinoma with osseous metaplasia in a horse intestinal adenocarcinoma causing recurrent colic in the horse equine colonic lipomatosis large colon resection for treatment of lymphosarcoma in two horses colic in a mare caused by a colonic neurofibroma small colon intussusception associated with an intralumenal leiomyoma in a pony leiomyoma of the small colon in a horse an analysis of cases of intestinal obstruction caused by pedunculated lipomas rectal leiomyosarcoma in a horse strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases standing rectal and tail surgery disseminated peritoneal leiomyomatosis in a horse ascites as a result of peritoneal mesotheliomas in a horse a case of peritoneal mesothelioma in a thoroughbred mare omental fibrosarcoma in a horse neoplasia of the mouth and surrounding structure pleuroscopic diagnosis of gastroesophageal squamous cell carcinoma in a horse recurrent esophageal obstruction due to squamous cell carcinoma in a horse chronic colic in the mature horse: a retrospective review of cases recurrent colic in the mature horse: a retrospective review of cases paraneoplastic syndromes lymphosarcoma and associated immune-mediated hemolytic anemia and thrombocytopenia in horses differentiation of chronic lymphocytic leukemia in the horse: a report of two cases hypercalcemia associated with malignancy in a horse adenocarcinoma of intestinal origin in a horse: diagnosis by abdominocentesis and laparoscopy differentiation between intra-abdominal neoplasms and abscesses in horses, using clinical and laboratory data: cases ( - ) malabsorption in the horse associated with alimentary lymphosarcoma immunodeficiency associated with lymphosarcoma in a horse flow cytometric methods to diagnose selected equine immune-mediated disorders pleural effusion associated with squamous cell carcinoma of the stomach of a horse use of diagnostic ultrasonography in horses with signs of acute abdominal pain the indications for equine laparotomy: an analysis of cases equine lymphosarcoma diarrhoea in the horse as a result of alimentary lymphosarcoma paraneoplastic pruritus and alopecia in a horse with diffuse lymphoma lymphoma in the horse. proceedings of the twelfth annual meeting of the american college of veterinary internal medicine squamous cell carcinoma of the pharyngeal wall in a horse squamous cell carcinoma of the oral, pharyngeal and nasal mucosa in the horse treatment of superficial ulcerative squamous cell carcinoma in three horses with topical -fluorouracil intratumoral chemotherapy with cisplatin in oily emulsion in horses excision of oral squamous cell carcinoma in a horse extensive resection and anastomosis of the descending (small) colon in a mare following strangulation by a mesenteric lipoma section . peritonitis references . hosgood g: peritonitis. . a review of the pathophysiology and diagnosis the peritoneum and peritoneal cavity large animal internal medicine diseases of the peritoneum and mesentery equine internal medicine peritonitis associated with actinobacillus equuli in horses: cases veterinary gastroenterology peritonitis and other intra-abdominal infection antibiotic susceptibility of bacterial pathogens from horses peritonitis in horses: cases biologic reactions to endotoxin effect of dietary alpha-linoleic acid on equine monocyte procoagulant activity and eicosanoid synthesis review of cases of peritonitis in the horse reference values for equine peritoneal fluid diagnostic cytology in the equine species: overview of effusions (peritoneal, pleural, and synovial joint) and transtracheal wash effects of enterocentesis on peritoneal fluid constituents in the horse internal abdominal abscesses in the horse: a study of cases equine peritoneal fluid analysis following celiotomy analysis of equine peritoneal fluid evaluation of peritoneal fluid ph, glucose concentration, and lactate dehydrogenase activity for detection of septic peritonitis in horses tumor necrosis factor and interleukin- activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease low dose flunixin meglumine: effects on eicosanoid production and clinical signs induced by experimental endotoxemia in horses heparin in the treatment of experimental peritonitis erythrocyte agglutination associated with heparin treatment in three horses therapeutic strategies involving antimicrobial treatment of large animals with peritonitis effect of long-term administration of injectable enrofloxacin solution on physical and musculoskeletal variables in adult horses peritoneal lavage in the horse pharmacologic principles therapeutic drug monitoring: a tool for rational drug therapy. proceedings of the seventh american college of veterinary internal medicine forum toxicity of quinolones although a number of potential causes of peritonitis exist, sepsis is a common and serious complication, and the identification and control of bacterial sepsis is critical for a successful outcome. bowel leakage (as well as external trauma) results in contamination of the peritoneum with large numbers of many types of bacteria. the intestinal tract contains a mixed population of bacteria, and the quantity of bacteria and prevalence of anaerobic species increase in the distal segments. [ ] [ ] [ ] [ ] [ ] [ ] [ ] there are approximately × anaerobic and × aerobic bacteria per milliliter of cecal and colonic fluid, thus the potential for bacterial contamination of the peritoneum is great. high mortality is associated with contamination from the lower bowel because of the large numbers of bacteria present. hirsch and jang reported isolation of an infective agent from equine peritoneal fluid in approximately % of attempts. obligate anaerobic bacteria were cultured most frequently, followed by members of the enterobacteriaceae family (escherichia coli). penicillin-resistant bacteroides fragilis was isolated from % to % of cases. in another study in which bacteria were identified in equine abdominal fluid by cytologic examination or culture, e. coli was the organism most commonly isolated. in human beings and laboratory animals the well-established fact is that despite the variety of organisms initially introduced subsequent to these events, established infections are characterized by only a few types of bacteria, which are often gram-negative aerobes and anaerobic bacteria. this selectivity occurs through the processes of selective reduction of bacterial populations and bacterial synergism. a well-known example of synergism in human beings and laboratory animals is peritonitis involving e. coli and b. fragilus. the presence of each organism is beneficial to the survival of the other, and each is important in the overall pathogenesis of the disease. e. coli is associated with septicemia and early mortality, whereas b. fragilis infection tends to result in chronic abscessation with delayed morbidity and mortality. some evidence suggests that in horses, in addition to coliforms and anaerobes, streptococci and perhaps c. psuedotuberculosis may survive selective reduction and participate in synergistic infection following polymicrobial contamination.biologic events resulting from contamination of the abdomen or injury to the mesothelial cells have been described [ ] [ ] [ ] [ ] and include release of catecholamines, histamine, and serotonin from peritoneal mast cells; vasodilation and hyperemia; increase in peritoneal vascular permeability; secretion of protein-rich fluid into the peritoneum; transformation of mesothelial cells into macrophages; and influx of polymorphonuclear cells, humoral opsonins, natural antibodies, and serum complement into the peritoneal cavity. additionally, depression of the peritoneal fibrinolytic activity, fibrin deposits on the peritoneal surface, and sympathetic-mediated ileus of the gastrointestinal tract can occur.these processes benefit the animal by confining contamination and infection, and indeed, with clean, minimally invasive procedures such as enterocentesis or trocharization, this is effective. however, with greater severity of peritoneal contamination or irritation, these processes are magnified and become deleterious, resulting in problems such as hypovolemia, hypoproteinemia, ileus with resultant bowel distention, ischemia of the bowel wall with subsequent absorption of bacteria and toxins, and ultimately adhesion and abscess formation. additionally, systemic responses to bacterial toxins, particularly lipopolysaccharide, , can compromise the metabolic condition of the patient further. equine peritoneal macrophages release several mediators when exposed to bacterial lipopolysaccharide, undoubtedly an important component of septic peritonitis.pathologic description of peritonitis includes origin (primary or secondary), onset (peracute, acute, chronic), distribution (localized versus diffuse), and presence of bacteria (septic versus nonseptic). , clinically, viewing key: cord- - xg v h authors: beikzadeh, samira; ghorbani, marjan; shahbazi, nayyer; izadi, farzaneh; pilevar, zahra; mortazavian, amir mohammad title: the effects of novel thermal and nonthermal technologies on the properties of edible food packaging date: - - journal: food eng rev doi: . /s - - -y sha: doc_id: cord_uid: xg v h edible packaging is influenced by factors such as formulation, production technology, and solvent and additive properties. with the increase in the request for coating and film quality, appropriate form, and high product safety and storage period, various technologies such as high hydrostatic pressure, irradiation, ultrasound, high-pressure homogenization, cold plasma, and microwave have been reviewed. the present study states definitions and mechanisms of novel technologies. the experimental condition, packaging matrix, and the results pertaining to the effects of these technologies on various types of edible packaging is also discussed. the most of the matrix used for packaging was whey protein, soy protein isolate, chitosan, and gelatin. the technologies conditions such as power, frequency, time, temperature, dose, pressure, and voltage can have a significant influence on the application of them in film and coating. therefore, finding the optimum point for the features of the technologies is important because improper use of them reduces the properties of the edible packaging. nowadays, researchers have studied the use of environmentally friendly packaging which includes natural polymers [ ] . natural polymers such as carbohydrates (chitosan, starch, alginate, celluloses, and hydrocolloids like gellan gum and seed-mucilages), lipids (waxes such as candelilla, bee, carnauba, glycerols, and fatty acids), proteins (gluten, collagen, soybean, zein, and milk protein), and composites are utilized to generate novel packaging and reduce the consumption of synthetic polymers [ , ] . natural polymers are further applied to various coatings and films. coatings are spread in liquid form on the food and eaten directly while films are used in solid forms [ ] . polysaccharides and protein films have appropriate mechanical characteristics and appearances. protein films have more impressive gas barrier properties and better mechanical characteristics owing to their unique structure and higher intermolecular connection capability compared to other films [ ] . carbohydrate films are resistant toward gas, oil, and fat, and both protein and carbohydrate films have high water vapor permeability owing to their hydrophilic behavior [ ] . lipid films, on the other hand, are resistant to moisture loss. despite the advantages of natural polymers, they have not yet been widely applied in food manufacturing due to their relatively high cost regarding this type of packaging and weak mechanical, sensory, and barrier properties (oxygen, carbon dioxide, aroma, and water vapor) of the produced films [ ] . the difference in edible film formation is related to material and additive types and properties, formulation, and film production technology. in general, materials are dissolved in acetic acid, water, alcohol, and their mixture with other solvents, and the solution is then cast and dried under optimal conditions. additives such as plasticizers (for brittleness reduction and increased flexibility and toughness), antimicrobial materials (for increasing the product's shelf-life), flavor, and color agents (for increased sensory properties and overall acceptability) can be used in the production of films [ , ] . novel technologies such as microwave, high hydrostatic pressure, irradiation, cold plasma, ultrasound, and highpressure homogenization are applied with the purpose of quality improvement, thermal treatment, energy efficiency, preservation, texture and surface modification, analysis, and extraction to name a few [ , ] . therefore, this review attempts to discuss and address the definition and mechanism of novel technologies, the optimum conditions for the application of these technologies on the edible packaging, and the influences of the mentioned technologies on properties of edible packaging. microwave, high hydrostatic pressure, irradiation, cold plasma, ultrasound, and high-pressure homogenization are considered to be the most commonly applied packaging technologies. the influences of the mentioned technologies on some of the food edible packaging properties are summarized in fig. . electromagnetic radiation frequencies ranging from mhz to ghz create the effect of polarization and cause microwave heating. microwave heating has advantages over traditional heating such as reducing the processing time and maintaining the quality of food. food materials have different dielectric properties which they store and spread when exposed to an electromagnetic field. microwave is usually used in the food industry for pre-cooking, frozen material tempering, and drying [ ] . from an industrial point of view, during film production and after spreading it over the plate, it is important to select the most appropriate method such as microwave for rapid drying. in the traditional approach, the film is dried under the air, taking about h [ ] . microwave can heat the components based on the transference of mass and heat through the interaction between polar material and microwave energy [ ] . because of the electromagnetic field generated by microwave, only the surface of the component is not influenced, and due to the high penetration depth, the inside parts of the materials are sufficiently heated. furthermore, various food components have the divergence dielectric behavior and absorb heat differently and selectively [ ] . kaya and kaya [ ] formed films containing whey protein isolate about ( and % (w/w)) and dried by microwave. to do this, films were spread onto the plate and dried ( °c , h and rh) and microwave condition (power w, mhz, and min). the result indicated that transfer rate of water vapor was directly related to the temperature and increased with it (from to °c). the films dried with both methods showed no main difference in terms of water vapor permeability (wvp) and modulus of elasticity (me). microwave method enhanced tensile strength (ts) and fig. the influence of technologies on the properties of edible food packaging food eng rev sides "" elongation at break (eab) in film samples compared to room condition. in addition, dried samples by microwave had better haze and gloss values compered to others. cárdenas et al. [ ] dried chitosan film at convection oven ( h at °c) and microwave ( mhz, min). they dried chitosan films using two methods; microwave (full power, min, mhz) and convention oven ( °c for h) and reported that microwave increased uv-vis light barrier property of films, although had no significant effect on their thermal and structural characteristic. in addition, cárdenas et al. [ ] reported that cu nanoparticle in chitosan composite film due to connection to matrix filled binding points of film against water and accelerated the removal of non-bounded water during drying by microwave (full power, min, mhz). the results of studies on microwave effects on edible packaging are summarized in table . food irradiation is a physical treatment used to improve the shelf-life through reducing the spoilage of various vegetables, and animal and seafoods. this process consists of exposing foods to ionizing radiation chiefly electron beam (e-beam), uv, and γ where high-energy radiation expels electrons from atoms and ionizes the molecules. the international unit of measurement for the radiation e-beam and the gamma dose absorbed by the product is kilogray (kgy) and uv radiation is measured in nanometers (nm) [ , , , ] . the use of radiation technique in food products was authorized in [ ] . with radiation technique, packaging with desired functional characteristics can be obtained [ , ] . in addition, packaging improves the physical and chemical quality of food products but does not significantly inhibit microbial growth. irradiation is able to inhibit microbial growth and/or produce cross-linking films by covalently linking the protein molecules [ , , ] . food protein alteration is an important approach to improving the properties of the biopolymer applied to edible film production. the irradiation process on the protein crosslinking can result in the formation of free radicals, hydrogen abstraction, and novel connection inside chains, which improves the final structure properties. furthermore, this process occures rapidly whitout the need for catalyst and temperature rise [ ] . e-beam irradiation ( - kgy) was investigated on the quercetin release level of film including gelatin and chitosan in hydroethanolic medium ( % ethanol) at room temperature. the induced irradiation created novel interactions and connections between chitosan and gelatin with the antioxidant material, resulting in the increased quercetin level residual within the film following kgy irradiation dose. however, the lag-time (the period needed previously the release begins) increased ten times. therefore, the e-beam irradiation led to the entrapment of a significant amount of antioxidants [ ] . irradiation by new interactions between biopolymers has been used for packaging disinfection. gamma irradiation ( kgy) was used to generate cross-linked proteins and prepare proteins/glycerol and protein/glycerol/polysaccharide films. structure analysis was performed by ftir [ ] . the second derivative ftir spectrum showed an increase in the amount of β-sheet phase and a reduction in a-helix phase in γirradiated cross-linked proteins in comparison with no irradiation sample. methylcellulose addition and γ-irradiation of proteins enhanced the puncture strength and had no significant effects on wvp. irradiation induced % decay in the proteins/methylcellulose/peppermint formulation on day while the control underwent % decay. irradiation (γ) improves film characteristics through producing cross-connected protein. the changes in structural parameters and the release levels of ferulic acid and tyrosol in film including gelatin and chitosan irradiated with e-beam at kgy doses were studied. ferulic acid can cause some connections with the polymer networks; non-irradiated films showed higher remaining ferulic acid compared with tyrosol. e-beam induced a loss of about % in tyrosol and % in ferulic acid residual trapped within the matrix of polymer while all tyrosol was released. moreover, the ts value significantly increased and water vapor permeability decreased [ ] . through some covalent bindings with the polymer network, irradiation can reduce the additive diffusivity from packaging to outside. the chitosan coating with ultraviolet ray were applied for the preservation of jujube during storage. uv irradiation restrained the rate of respiration, electrolyte leakage, and malonaldehyde level. in addition, combined treatment for days displayed . % weight loss lower compared to that in the untreated and uncoated type. in addition, the enzyme avtivities of treated jujubes were kept at a higher level, and the reduction in chlorophyll and vitamin c was controlled [ ] . after absorbing the ultraviolet, the microorganism died due to the alteration of the molecular structure of the nucleoprotein and inhibition of metabolism. gamma irradiation ( kgy) process was applied together with cinnamaldehyde and thyme oil in whey and soy protein coatings for shrimp. changes in aerobic plate counts, bacterial growth, and sensory characteristics were investigated by ouattara et al. [ ] . the obtained results revealed that coating and γ-irradiation treatments had synergistic effects on the reduction in aerobic bacteria including pseudomonas putida and aerobic plate counts (apcs). without irradiation, an improvement of preventive influences was observed in the coating containing antimicrobial agents applied to the shrimp related to the level of antimicrobial materials; with irradiation, on the other hand, the preventive influence was enhanced owing to the antimicrobial material interaction influence. irradiation had no detrimental influences on the organoleptic parameters. irradiation with coating showed synergistic influence in initial solution without additives. in another research, gamma irradiation ( - kgy) decreased the apcs in three groups of beef samples containing and . % of ascorbic acid and a sample including ascorbic acid ( . %) and packed with calcium caseinate coating. also, in all samples, thiobarbituric acid reactive substances (tbars) and −sh radical production were stabilized during storage in comparison with the control sample [ ] . gamma irradiation improved lipid oxidation and -sh radicals resulted in reduced bacterial growth. several food products coated with different solutions were treated with gamma ray up to kgy. irradiation combined with a coating completely inhibited the microbial growth in shrimp, decreased - . log unit in the apcs of ready-to-eat pizzas, and significantly decreased the mold growth of strawberries. contrary to uncoated/untreated sample, the shelf-life improved in shrimp and pizza. gamma irradiation had no significant influence on the organoleptic parameters [ ] . edible coatings containing biodegradable materials combined with γ-irradiation prevent the microbial attack on the hyani date fruits (considered to have high moisture content). this goal was achieved by preparing triple blend solutions (polyvinyl alcohol, chitosan, and tannic acids) using γirradiation with doses varying between and kgy. the results showed that with the increase in the irritation dose, antimicrobial properties and the ts of edible coatings were improved. moreover, γ-irradiation with titanic acid was able to increase the shelf-life of fruit in condition ( °c and % rh) from to days [ ] . methylcellulose coating combined with citrus extract (ce), lactic acid bacteria metabolites (lab), rosemary extract (re), and organic acids (oa) was studied on the sensitivity of salmonella typhimurium, escherichia coli, and listeria monocytogenes in broccoli florets. for this propose, florets were separately inoculated with each pathogen microorganism and packed with the coating and dipped in baths containing antimicrobial agents. samples were irradiated with γirradiation at doses of to . kgy. following γ-irradiation, methylcellulose coating increased the radio sensitivity of food pathogens. the oa + ce, oa + lab, and all irradiated coatings significantly reduced e. coli, s. typhimurium, and l. monocytogenes, respectively [ ] . in a study by abad et al. [ ] , the tamarillo fruits were irradiated at doses of to gy and packed with the stafresh ™ coating (the yellowish wax covering palm leaves with a thick layer and applied in coating fresh fruit). the irradiated and coated fruits were preserved under better conditions compared with the control samples. weight loss and respiration of the fruits were and % lower than the control during its shelf life. firmness and appearance increased up to and % compared to the control, respectively. furthermore, radiation had no influence on fruit flavor. the results of the studies on irradiation effects on edible packaging are summarized in table . recently, plasma technology has been employed as an alternative to other physical and chemical methods for food processing with high quality [ , ] . plasma is produced by applying energy to a gas blend, and it is as the fourth state of matter which is electrified gas along with ultraviolet photons, electrons, positive and negative ions, free radicals, atoms, and excited or non-excited gas molecules. these chemically reactive particles have a significant impact on food processing. generally, there are two types of plasma, namely thermal and nonthermal (cold plasma). the thermal plasma is produced at extreme pressures (≥ pa) and requires up to mw of power; also, the temperature of the electrons and ions of the plasma approach thermal equilibrium. cold plasma, on the other hand, is generated by electrical discharge instead of heating the entire gas at atmospheric pressure and low levels of power [ , ] . cold plasma can be applied in the decontamination of food material with inactive microbial agents such as bacteria, bacterial spores, fungi, and viruses. therefore, cold plasma treatment can be used as an alternative heat-based technique for sterilizing sensitive materials such as vitamins, proteins, volatile compounds, and polycarbonate and polythene [ , , ] . the cold plasma has been reported to interact with ingredients of edible films such as proteins, lipids, carbohydrates (seed-mucilage), water, and phenolic compounds, through the addition or removal of the specific functional group. these interactions resulted in the uniformity of treatment, no use of and showed no effect on wvp and me. [ ] chitosan film microwave full power, min, mhz microwave increased uv-vis light barrier and had no effect on thermal properties. [ ] food eng rev sides "" hazardous solvents, increased mechanical strength and flexibility, and improved hydrophobic properties; they also helped maintain the sealing properties of polymers, reduce gas permeation and undesirable contaminants without any traces, maintain atmospheric air, and increase elongation [ , , , , ] . chen et al. [ ] produced film containing chitosan/ciprofloxacin/zein by plasma at p = w for s. the result of ftir showed that films were well coated with zein, and xrd patterns confirmed that the cold plasma had no influence on crystal type. no significant changes were observed in thickness and encapsulation efficiency while surface free energy, thermal stability, and wet ability and bacterial growth were increased and reduced, respectively. cold plasma improved the function of drug release in film. cold plasma was done on carboxymethyl cellulose (cmc)-coated polypropylene film including essential oils. first, polypropylene films were modified with cold plasma at v = kv and f = khz for min. cmc films including essential oils were then coated with plasma-treated polypropylene. the results showed that atmospheric cold plasma treatment was able to create c=o and o−h groups on the polypropylene films. subsequently, contact angle, indicative of surface wettability, was reduced from . °to . °. cold plasma increased the mechanical and antimicrobial characteristics. in addition, plasma increased wvp while essential oil decreased it [ ] . in general, plasma can be used as a pretreatment in the production of bilayer film because it makes better connection of natural film to synthetic polymers. the effect of plasma on melon packed in polypropylene film was investigated [ ] . polypropylene film was subjected to cold plasma at kv, . khz, rh = % for to min for each side. the findings showed that dry matter and titratable acidity increased, although insoluble solid content was reduced. furthermore, microbial shelf life significantly increased in -min treatment while the effect of cold plasma on microbial shelf life after + min was negligible. in general, cold plasma is an efficient method in the decontamination of fresh-cut-fruit. s i f u e n t e s -n i e v e s e t a l . [ ] r e p o r t e d t h a t hexamethyldisiloxane (hmdso) cold plasma treatment (p = w, et = min) enhanced hydrophobic characteristics in starch film and increased ts and young's modulus. however, a reduction from to % was observed in eab value. these results suggested that the hmdso plasma treatment is capable of producing starch-based films as a suitable packaging material. cold plasma (p = w, et = min) was applied on defatted soybean meal film [ ] . the films were treated with different types of plasma-forming gases including ar, dry air, o , he, and n . no significant changes were observed in elastic modulus, ts and wvp. lightness value and stretch kgy e-beam decreased the release rate of acid and tyrosol, and increased ts [ ] γ-irradiation soy and whey protein isolates coating pre-cooked shrimps, pizzas, and strawberries and kgy irradiation had effect on microbial growth and no effect on sensory characteristics [ ] γ-irradiation calcium caseinate, and whey protein isolate film strawberries kgy gamma irradiation of proteins increased the puncture strength [ ] γ-irradiation polyvinyl alcohol/chitosan/tannic acid hayani date fruit to kgy irradiation enhanced the mechanical properties of fruit [ ] γ-irradiation methylcellulose coating broccoli florets - . kgy gamma irradiation eliminated the growth of foodborne pathogens [ ] γ-irradiation stafresh mt coating tamarillo fruits . - kgy irradiation reduced weight loss up to % and respiration rate and increased firmness, and appearance [ ] ability increased in the case of all gases except for air, n and ar. the effect of cold plasma on smoked salmon packed with dsm film was further investigated. cold plasma indicated no influence on the color of salmon while it delayed lipid oxidation and reduced the hardness [ ] . pankaj et al. [ ] treated sodium caseinate film by plasma (v = and kv, et = - min). plasma was able to enhance hydrophilic property in films owing to the enhance in o−h and c=o groups. in addition, roughness and surface oxygen content and glass transition temperature increased and decreased, respectively. the effect of cold plasma on film and coating are summarized in table . edible films have weak mechanical properties in comparison with synthetic samples. therefore, various physical and chemical technologies need to be done to overcome these problems. ultrasound had beneficial effects on the processing and preservation of food. the ultrasound technology is divided into low and high intensities. in low-intensity technology, high frequency ( - mhz) and low power ( mw/cm to < / w ) are used. this type as an analytical method examines information about quality properties of food [ ] . but in high-intensity ultrasound, typically low frequencies range ( - khz) and high power range ( - w/cm ) are used. this method had several application in food systems inducing filtration, fogging, crystallization, emulsification, cutting, degassing, oxidation and reduction reactions, drying, extraction, tenderization, cleaning, and sterilization [ , ] . high-intensity type transmitted via a fluid environment create strings or cloudy bubbles. the bubble grows at critical sizes and will quickly disappear. the general process is called cavitation phenomenon, which involves the creation, growth, and breakdown of bubbles. the growth and breakdown of bubbles will result in the production of high temperatures and pressures in the matrix [ ] . in addition, ultrasound can be used for increasing the mass transfer owing to the great temperature and pressure changes. the combination of microwave and ultrasound can accelerate the extraction technique for target materials from their matrix [ , ] . ahmadi et al. [ ] studied ultrasound treatment ( khz) for , , , and min on methylcellulose-forming solutions. the time of ultrasound exposure had an inverse relationship with the amount of wvp, meaning an ultrasound process of min produced the lowest wvp. the highest eab and ts were obtained at and min, respectively. following min of ultrasound, mechanical properties were improved, although with the increase in time to min, ts and eab values of films were reduced. these results can be ascribed to the cavitation phenomenon. the reduction in ts and eab after min can be the result of the increased cavitation size and number of bubbles. growing bubbles create spaces between molecules and reduce film resistance. increases in these parameters after min of sonication can be due to the breakdown in chemical connections in the main chain of methylcellulose films and the formation of new chemical bonds, forming a denser network. under the influence of high pressure and temperature due to solvent decomposition and dissolved molecules inside the system of bubbles, several reactive radicals are produced. according to the cavitation mechanism, the contact between the methylcellulose film solution and ultrasonic waves can cause the dissolved water and ethanol molecules to collapse, thereby creating hydrogen and hydroxyl radicals. ultrasound can decrease wvp because of the creation of covalent connection between steam and water molecules and radicals. this reaction can possibly reduce wvp through trapping the water molecules inside the network. furthermore, increasing the time of exposure with ultrasound waves can increase the number of radicals and covalent bonds and decrease wvp, respectively. fan et al. [ ] used ultrasonic pretreatment ( w, min at room temperature) to modulate the interaction between the kidney bean protein isolate (kbpi) and chitosan and prepare composite films. the ultrasonic increased blending between kbpi and chitosan generated flexible films. moreover, ultrasound pretreatment had no effect on the transmission of light, thereby increasing the opacity of the film. borah et al. [ ] applied ultrasound treatment ( ± khz, w, t = , and min) to composite film forming solutions of lime pomace and potato peel powder. ultrasound treatment improved film properties with the increase in sonicated duration time, and decreased wvp and film moisture absorption in sample via -min sonication. in addition, ultrasound treatment increased the stretching properties of the composite films. sun et al. [ ] confirmed that ultrasound treatment (up to s, khz) is an appropriate method for solutions of soy protein films and positively affects the number and dispersion of soybean protein seeds. the size of the seeds decreased with the increase in the time of ultrasound exposure, resulting in a uniform appearance of the film. also, the soy protein film structure of the ultrasound treatment was more uniform and its mechanical properties significantly improved, which ultimately caused a significant difference in ts and eab. marcet manrique et al. [ ] applied ultrasound ( khz at several temperatures and times) in the egg yolk film-forming solution. optimum ultrasound treatment was °c for min for film preparation. optimum conditions for ultrasound treatments improved the resolution of casting solutions, solubility, thickness, and mechanical characteristics of the prepared films. the results (lower particle size, lower poly dispersity index, and more film compression) showed that ultrasound food eng rev sides "" was able to degrade protein aggregation by modifying the protein structure from the breaking point of covalent and non-covalent bonds. liu et al. [ ] applied ultrasound treatment for , , , , and min at khz frequency and w to produce composite film solutions using tea polyphenol/polyvinyl alcohol by stripping method. ultrasound treatment decreased the ts. the best overall film properties were achieved at : volume ratio of tea polyphenol/polyvinyl alcohol and -min ultrasound duration. zhong et al. [ ] determined the effects of different ultrasound treatments ( °c, - min, khz, and , , , and w) on the blending film of methylcellulose/stearic acid. exacerbation of the treatment condition, including ultrasound exposure time, resulted in reduced viscosity of the methylcellulose/stearic acid composite emulsions and increased the homogenization of fat distribution and internal microstructure of films. ultrasound treatment improved mechanical and superficial hydrophobic properties. the treated sample showed the lowest wvp for min and w. however, excessive exposure to ultrasound reduced mechanical properties and moisture inhibition and created enormous spaces in the polymer matrix, leading to a negative effect on film performance. furthermore, ultrasound improved the hydrophobic surface characteristics of composite films in the appropriate range. in general, ultrasound, as a modification form of film-forming emulsions, significantly enhances the compositional properties of films. cheng et al. [ ] made use of sonication ( khz for , . , , , , and min at ± °c) to measure the dispersions of gelatinized maize starch film-forming solutions. ultrasound treatment improved transparency, moisture resistance, and film structure. furthermore, this treatment enhanced the starch amylose solubility and the free mobility of polymers in starch dispersion, resulting in a sharp reduction in viscosity and increased solubility even for % dispersion. marcuzzo et al. [ ] applied ultrasound treatment at hz for ( - ) min on gluten-based film-forming solutions because protein films obtained from gluten in acidic conditions exhibited some of the problems in protein dispersion. sonication process is considered as a physical method for improving protein dispersion and the appearance of this film without adding a chemical additive. gluten had a high tolerance to ultrasound treatment, a useful approach to improving the hydrophilic surface properties and appearance of films. rodriguez-turienzo et al. [ ] applied sonication ( w, khz for , , and min) on the whey protein coatingforming solutions for frozen salmon fish. the results showed a reduction in drip loss in thawing and after cold storage in coated fish treated for min than those sonicated for min. the ultrasound treatment had no effect on the color of frozen fish, but enhanced the whiteness of the salmons during cooking compared to samples coated with un-sonicated whey protein. these coatings had no influence on sensory characteristics of fish. ultrasound treatment significantly inhibited the lipid oxidation of salmon fillet compared to uncoated and un-sonicated samples. therefore, ultrasound treatment of whey protein solutions can reduce the oxidation of frozen fish fats without adding chemicals and enhanced the shelf-life of products. banerjee et al. [ ] used sonication (acoustic power, khz and khz for . and h) to produce sodium caseinate and whey protein concentrate (wpc) films. sonication process enhanced the ts of films. the average of ts in treated film was % compared to the untreated film. the ultrasound process in sodium caseinate films was more effective compared with wpc film. the intermediate power with low-frequency levels led to a much better ts in sodium caseinate film. increasing the time of ultrasound exposure resulted in more powerful films. wang et al. [ ] produced modified microcrystalline wheat bran cellulose (mwbc) by acid hydrolysis of wheat-bran increased surface free energy, thermal stability, wvp, and drug release function and reduced bacterial growth. [ ] starch-based film -p = w, et = min increased wvp, ts, young´s modulus, and hydrophobic property and decreased eab. [ ] defatted soybean meal film smoked salmon p = w, et = min increased stretch ability and lightness and decreased lipid oxidation and hardness. [ ] (cmc)-coated polypropylene film -v = kv, f = khz, et = min increased wvp, antimicrobial and mechanical properties. [ ] sodium caseinate film -v = and kv, et = - min increased the roughness, surface oxygen content, and hydrophilic property and decreased glass transition temperature. [ ] food eng rev cellulose (wbc) using microwave/ultrasonic system ( w, °c, and min). then they formed a film of it. the result indicated that ultrasonic/microwave system enhanced tensile strength (ts) and water holding capacity (whc) and reduced oxygen permeability (op) and water vapor permeability (wvp) contents in films containing mmwbc due to decreasing of particle size and increasing of free hydroxyl numbers and interactions of carboxyls with amino components. in general, this system varied mechanical characteristics due to effect on size, packing style, distribution, and shape of filler particles. also, ultrasonic/microwave system improved thermal properties such as tg and tc due to breaking of hydrogen links and happening of acid hydrolysis in wheat-bran cellulose amorphous part that maintain stable of crystalline part. wang et al. [ ] determined the effect of microwave/ ultrasonic system ( °c, w, , , , , and min) on film containing soy protein and titanium dioxide. with the increase of microwave/ultrasonic system time up to min increased ts, homogenous of structure and whc and reduced wvp and op. this system produced films with compact and homogeneous surfaces due to reducing of particle size, increasing of particle surface, free hydroxyls, and interaction of particles together, although longer time of treatment, could destroy matrix and produce a weak film. wang et al. [ ] determined the effects of ultrasound/ microwave treatment ( °c to avoid of oleic acid oxidation, min and w) on soybean protein isolate (spi) edible films with different proportions from oleic acid to stearic acid. in summary, variations in stearic acid and oleic acid ratios and microwave/ultrasound treatment had important impact on wvp and angular contact angle. film containing : ratio of stearic acid to oleic acid prepared under microwave/ultrasound condition ( °c, min, and w) indicated the lowest wvp and the highest angle ( °). the lower temperature and period of microwave may rely on activating and facilitating the influence of radiation on the solid phase emission [ ] . however, the effect of ultrasound causes cavitation (creation, growth and breakdown of bubbles) in the solution [ ] . consequently, ultrasound/microwave process ( min and w) resulted in the creation of compressed-films, followed by improved contact angles. also, the surface of treated films is fairly uniform because ultrasound/ microwave treatment improves the mixing of films, resulting in a relatively smooth and continuous surface [ ] . the results pertaining to studies on ultrasound and ultrasound/microwave effects on edible packaging are summarized in table . high-pressure homogenization (hph) develops the structural and physicochemical characteristics of packaging thus it can be applied in film production [ , , , ] . hph treatment has been employed in the food industry to process various food products, including meat, fruits, juices, and vegetables with a pressure range of to mpa depending on the application purpose [ ] . the influence of hph process depends on the applied pressure, temperature, storage time, and molecular size [ ] . several studies have reported that the hph process reduces the internal viscosity, increases the power of inflation [ ] , forms a weak gel [ ] , decreases temperature and enthalpy gelatinization [ ] , and affects the crystalline structure [ , ] . saricaoglu et al. [ ] used hph treatment at a pressure between and mpa on meat protein solution for film forming. by enhancing pressure to mpa in suspensions treated, the particle size decreased and then increased due to protein accumulation. the reduction in particle size decreased water solubility and increased wvp and ts. the reduction in the size of the particle protein resulting from the increase in the pressure up to mpa led to the homogeneous distribution of their particles. all samples were homogeneous except the sample homogenized with mpa. the non-porous and smooth film resulted in a higher film clarity. additionally, the eab values increased by the increase in pressure to mpa, but reduced with a pressure to mpa. in general, hph treatments to mpa improved the solution characteristics and the preventive and mechanical properties of film. shahbazi et al. [ ] evaluated starch under the influence of hph at and mpa for production of film including starch and κ-carrageenan. it can be concluded that this method is safe for starch, inexpensive, and fast. in addition, the hph process produced smooth hydrogels with stronger gel nets, and reduced the degree of crystallization in starch grains and the amount of crystallization relative. the hph process resulted in a better dispersion of starch in the k-carrageenan matrix, which ultimately increased water resistance and film obstruction properties. the produced film under hph had the highest hydrophobic level and ts value. these changes were more pronounced when applied with a high level of homogenization pressure. hph had no influence on starch granules integrity. however, the many points and cavities appearing outside the granule were increased with the increase in process intensity. in addition, the pressure produced during hph can cause physical damage to the starch granule. the probable changes in the shape and appearance of granules were due to the partial gelatinization caused by the increase in the temperature during homogenization [ ] . changes and cavities created at the cross section of wheat starch were due to the high pressure and shear of sonication, but the integrity of granules did not change [ ] . the films produced in optimal conditions showed no significant influence on thermal and mechanical characteristics and water vapor transmission rate. the transmission rate value was reduced at pressures above mpa. oxygen transmission rate decreased when a high pressure was applied to a gelatin-based film solution for a long time at room temperature may be due to the presence of a dense polymer matrix affecting oxygen transmission rate that blocks the release of oxygen molecules [ ] . longer high pressure produced films with compact polymer matrix and decreased oxygen transmission rate. high-pressure treatment influenced the structure of gelatin films and prevented the creation of semi-crystalline region, hence the reduction in the crystalline phase. heremans and smeller [ ] reported that the pressure from to mpa was sufficient to make compatible variations in protein. in addition, the reduction in the enthalpy showed the amount of crystalline inside the system due to the highpressure mechanical forces. high pressure increased mechanical properties in films. the mechanical forces during high-pressure treatment increased rigidity in the gelatin gel due to the improvement of hydrogen bonding. optimum conditions had a significant increase in water-proofing properties of film [ ] . the higher density in the gelatinbased film structure reduced the rate of water vapor transmission value due to the increase in the resistance of films to the water releases [ ] . films produced under highpressure treatments were lighter, redder, and more rigorous than control films. the enhance in lightness is be due to the increase in light diffusion from denatured proteins [ ] . a summary of studies on hph effects in packaging are reported in table . as a nonthermal process, high hydrostatic pressure (hhp) has been applied to the structural and physical modification of biopolymers including protein and polysaccharide. the reaction balance of the system moves toward to the reduction of exterior forces under high-pressure situation; consequently, with decreasing volume, the hhp leads to chemical reaction and modifications in physical structure [ ] . additionally, hhp can prevent the loss of moisture during processing and cause physical modification in the film properties [ , ] . spi + oleic and stearic acids microwave/ ultrasonic system: °c, min, and w ultrasound/ microwave improved the mixing of film resulted in uniformity of surface. [ ] food eng rev high hydrostatic pressure (hhp) can be used for physical modification of starch. the application of this method induces a unique starch gelatinization at room condition compared to samples treated under thermal condition [ , , , ] . starch granules were partially decomposed by high pressure, and the gel texture became harder and had less amount of amylose release compared with samples treated by the thermal method [ , , , ] . kim et al. [ ] gelatinized starch by hhp to produce buckwheat and tapioca starch films. films prepared via hhp had better physical properties, water resistance, and thermal properties compared to those prepared by heat treatment process regardless of the starch type. consequently, hhp can be applied as a cold gelatinization method in the preparation of starch films. a high degree of hydrostatic pressure processing at mpa ( °c, min) and thermal processing ( °c, min) was used for starch gelatinization. as a result, films prepared under hhp had higher ts and eab compared with those under thermal conditions, regardless of the starch type. buckwheat starch films produced by hhp had lower wvp and higher water solubility in comparison with heat treatment films. starch films prepared using hhp are able to maintain a higher moisture content in the film matrix compared to thermal processing films, and water can act as a plasticizer in starch-based films [ ] . in addition, film solutions prepared using hhp have less viscosity compared to those treated by thermal conditions mainly because of the crystalline structure residue that prevents the melting of amylopectin molecules by amylose until some are stabilized [ ] . hhp in starchy films results in higher formation enthalpy and improves the thermal stability of films [ ] . in general, the optical properties of films prepared using hhp can be the result of various reasons such as sample type and process condition (pressure, time, and temperature). edible films and coatings are placed on/or within foods and have special functions such as mechanical protection of food, inhibition of material transfer (water, gas, fat), preservation, and transfer of food components and additives such as colors and flavors and prevention of microorganism growth. widespread researches have been studied for novel thermal and nonthermal technologies to improve edible film and coating properties and their application in food industry. as a result, to improve the novel thermal and nonthermal effects on packaging, different parameters such as technology properties, types of edible film and coating, type of food matrix, and packaging storage condition must be considered. as a suggestion, the combination of different technology effects on edible film and coating features can be considered. studying the effect of combining two nonconventional treatments, gamma irradiation and the application of an edible coating, on the postharvest quality of tamarillo the effect of ultrasound treatment on some properties of methylcellulose films effect of high hydrostatic pressure processing on microbiological shelf-life and quality of fruits pretreated with ascorbic acid or sncl modification of food ingredients by ultrasound to improve functionality: a preliminary study on a model system cold plasma: a new technology to modify wheat flour functionality milk protein-based edible film mechanical strength changes due to ultrasound process effect of psyllium husk on physical, nutritional, sensory and staling properties of dietary prebiotic sponge cake effects of psyllium and marve seed mucilages on physical, sensory and staling properties of sponge cake seed mucilages as the functional ingredients for biodegradable films and edible coatings in the food industry controlled release of tyrosol and ferulic acid encapsulated in chitosan-gelatin films after electron beam irradiation impact of electron beam irradiation on fish gelatin film properties pressureinduced changes in the structure of corn starches with different amylose content ultrasound treated potato peel and sweet lime pomace based biopolymer film development the potential of cold plasma for safe and sustainable food production edible films and coatings: characteristics and properties edible protein films: properties enhancement pressure −temperature phase diagrams of maize starches with different amylose contents combined effects of antimicrobial coating, modified atmosphere packaging, and gamma irradiation on listeria innocua present in ready-to-use carrots (daucus carota) physicochemical properties of edible films from chitosan composites obtained by microwave heating colloidal cu nanoparticles/chitosan composite film obtained by microwave heating for food package applications effect of high-pressure homogenization on the structure of cassava starch preparation, characterization and functional evaluation of chitosan-based films with zein coatings produced by cold plasma impact of ultrasonic treatment on properties of starch film-forming dispersion and the resulting films ultrasonic/microwave assisted extraction and diagnostic ion filtering strategy by liquid chromatography-quadrupole time-of-flight mass spectrometry for rapid characterization of flavonoids in spatholobus suberectus processing chocolate milk drink by low-pressure cold plasma technology edible films and coatings in seafood preservation: a review effect of edible co-polymers coatings using γ-irradiation on hyani date fruit behavior during marketing edible films and coatings: structures, active functions and trends in their use preparation and characterization of kidney bean protein isolate (kpi)-chitosan (ch) composite films prepared by ultrasonic pretreatment effect of using cold plasma treatment on the surface and physicochemical properties of starch-chitosan composite film effects of high-pressure homogenization on the properties of starchplasticizer dispersions and their films edible films and coatings: a review, innovations in food packaging food bioprocessing by nonthermal plasma technology protein structure and dynamics at high pressure application of cold plasma to develop carboxymethyl cellulose-coated polypropylene films containing essential oil plasticization and moisture sensitivity of potato peel-based biopolymer films effect of high-pressure microfluidization on the structure of cassava starch granule state diagram of potato starch-water mixtures treated with high hydrostatic pressure microwave drying effects on properties of whey protein isolate edible films high hydrostatic pressure processing for the preparation of buckwheat and tapioca starch films substances with antibacterial activity in edible films-a review radiation processing of food proteins-a review on the recent developments protein quality and microbiological changes in aerobicallyor vacuum-packaged, irradiated fresh pork loins use of γ-irradiation to produce films from whey, casein and soya proteins: structure and functionals characteristics fabrication of ordered flower-like zno nanostructures by a microwave and ultrasonic combined technique and their enhanced photocatalytic activity effect of high hydrostatic pressure on physicochemical and structural properties of rice starch nano-tio particles and high hydrostatic pressure treatment for improving functionality of polyvinyl alcohol and chitosan composite films and nano-tio migration from film matrix in food simulants development of ultrasound treated polyvinyl alcohol/tea polyphenol composite films and their physicochemical properties effect of simultaneous ultrasonic/microwave assisted extraction on the antioxidant and antibacterial activities of burdock leaves edible coating and irradiation of sapota fruit: a concise review effects of high pressure homogenization on the physicochemical properties of corn starch effects of sonication on physical properties of native and crosslinked wheat starches transparent and edible films from ultrasound-treated egg yolk granules effect of ultrasound treatment on properties of gluten-based film power ultrasound in food processing-the way advances in the application of ultrasound in food analysis and processing in-package atmospheric pressure cold plasma treatment of strawberries combination of high-pressure treatment, mild heating and holding time effects as a means of improving the barrier properties of gelatin-based packaging films using response surface modeling disposal of spent tributylphosphate by gliding arc plasma edible protein films: sources and behavior cold plasma treatments for improvement of the applicability of defatted soybean meal-based edible film in food packaging combined effect of antimicrobial coating and gamma irradiation on shelf life extension of precooked shrimp (penaeus spp.) microbiological and biochemical characteristics of ground beef as affected by gamma irradiation, food additives and edible coating film use of gammairradiation technology in combination with edible coating to produce shelf-stable foods cold plasma: background, applications and current trends physicochemical characterization of plasmatreated sodium caseinate film migration of styrene monomer from polystyrene packaging materials into foods: characterization and safety evaluation effect of radiation losses on hotspot formation and propagation in microwave heating effects of edible coatings based on ultrasound-treated whey proteins in quality attributes of frozen atlantic salmon (salmo salar) apple peel-based edible film development using a high-pressure homogenization high pressure homogenization of mechanically deboned chicken meat protein suspensions to improve mechanical and barrier properties of edible films nonthermal plasma-a tool for decontamination and disinfection physical modification of starch by high-pressure homogenization for improving functional properties of κ-carrageenan/starch blend film functional, thermal, and antimicrobial properties of soluble soybean polysaccharide biocomposites reinforced by nano tio advanced synthesis of materials for intermediate-temperature solid oxide fuel cells physicochemical characterization of argon plasma-treated starch film hexamethyldisiloxane cold plasma treatment and amylose content determine the structural, barrier and mechanical properties of starch-based films effect of highpressure homogenization on particle size and film properties of soy protein isolate effect of high pressure on the physical properties of barley starch effect of ultrasound treatment on properties of soy proteins film sodium chloride levels in comminuted chicken muscle in relation to processing characteristics and fresnel reflectance detected with a polarimetric probe effects of combined treatments of irradiation and antimicrobial coatings on reduction of food pathogens in broccoli florets microwave heating in food processing cold plasma treatment for fresh-cut melon stabilization biopolymer interactions affect the functional properties of edible films based on agar, cassava starch and arabinoxylan blends understanding high pressureinduced changes in wheat flour-water suspensions using starchgluten mixtures as model systems natural-based plasticizers and biopolymer films: a review high pressure-induced tapioca starch gels: physico-chemical characterization and stability development of bioactive coatings based on γ-irradiated proteins to preserve strawberries effect of high-pressure homogenization on the structure and thermal properties of maize starch effect of highpressure homogenization on microstructure and rheological properties of alkali-treated high-amylose maize starch the effects of ultrasonic/microwave assisted treatment on the properties of soy protein isolate/microcrystalline wheat-bran cellulose film the effects of ultrasonic/microwave assisted treatment on the properties of soy protein isolate/titanium dioxide films the effects of ultrasonic/microwave assisted treatment on the water vapor barrier properties of soybean protein isolate-based oleic acid/stearic acid blend edible films report of a joint fao/iaea/who expert committee effect of ultraviolet irradiation combined with chitosan coating on preservation of jujube under ambient temperature effects of ultrasound treatment on lipid self-association and properties of methylcellulose/stearic acid blending films publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -z rc ubj authors: wilkins, pamela a. title: disorders of foals date: - - journal: equine internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: z rc ubj nan before the s, intensive management of the compromised neonate was unusual and little was known regarding many of the problems of this special patient population. although some specific conditions had been described by astute clinician-researchers, most notably the "dummy" foal syndrome and respiratory distress syndrome caused by primary surfactant deficiency, little information regarding the diagnosis and management of conditions of the foal during the neonatal period was available, although at least one active group was investigating fetal and neonatal physiology of the horse in great britain. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] when treatment of compromised foals was undertaken, the approach most commonly resembled treating them as small adults with little understanding of the different physiology of the equine neonate. the advent of improved management of reproductive efficiency of mares led naturally to increased interest in preservation of the conceptus to parturition and the foal thereafter. interested clinicians, taking their lessons from the field of human perinatology/neonatology and sometimes working hand-in-hand with their counterparts in the human field, pioneered investigations into these small patients and created the fields of equine perinatology and equine neonatal intensive care. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] because of the foresight and energy of these early investigators, the field of veterinary perinatology/neonatology exploded in the s, leading to the creation of equine neonatal intensive care units throughout the united sates and the world. from these units information about the normal and abnormal physiology of foals, the medical conditions affecting them, and methods for treatment and management of these problems has been developed through observational, retrospective, and prospective studies. this veritable explosion of information over the last years has improved greatly the ability of all practitioners to provide appropriate care for these patients, whether in the field or at an equine neonatal intensive care unit. the ability not only to save the lives of these patients but also to treat them in such a manner as to allow them to fulfill their purposes, whether as pleasure animals or racing athletes, has improved almost exponentially from those early days. [ ] [ ] [ ] [ ] this chapter aims to provide the clinician with some of the most current information regarding the management of these patients, recognizing that much still remains unknown and that advances will continue to be made in this dynamic field. the reader is cautioned that much of this chapter is flavored by the experiences of the author and that variation in approach and treatment of specific problems exists between neonatal intensive care units (nicus) and between clinicians in the same nicu and that each year results in change. in some cases, information that is presented has been gleaned from human nicu studies, essentially using the critically ill infant as the experimental model. many of the problems of the newborn foal have their genesis in utero. identification of high-risk pregnancies is an important component of prenatal care of the foal, and some of the most commonly encountered problems of the dam resulting in abnormal foals include previous or concurrent disease, poor reproductive history, poor perineal or pelvic conformation, poor general health, poor nutritional condition, prolonged transport, history of previous abnormal foals, placental abnormalities, and twins. some of the more common causes of abortion can result in the birth of severely compromised foals of variable gestation lengths (box - ). these include pa m e l a a . wi l k i n s infectious causes such as equine herpesvirus (ehv) types (most commonly) and (rarely), equine infectious anemia, equine arteritis virus, bacterial and fungal placentitis, leptospirosis, equine ehrlichiosis, and gram-negative septicemia/endotoxemia. [ ] [ ] [ ] noninfectious causes of abortion include twinning and noninfectious placental abnormalities such as extensive endometrial fibrosis, body pregnancy, and abnormal length (long or short) of the umbilical cord. , to the equine neonatologist opportunities for intervention may appear limited, and in the case of many of the aforementioned causes of fetal loss, this is true. however, one can do much in an attempt to preserve the pregnancy and in effect treat the fetus. when one is faced with a threatened pregnancy, one has various ways of evaluating the fetus and its environment and may use many potential therapies. once one identifies a pregnancy as high risk, one should evaluate the fetus for viability. evaluation should include as thorough an evaluation as possible of the reproductive tract, placenta, and fetal fluids. prepartum disorders in the mare usually are readily recognizable, but disorders of the fetus and placenta can be more subtle and difficult to determine. the first step is to take a thorough history of the mare. of particular interest is any history of previous abnormal foals, but the history taking should include questions regarding transportation; establishment of an accurate breeding date (sometimes more difficult than one would suspect); any pertinent medical history including any diagnostic testing performed for this pregnancy such as culture, endometrial biopsy, and cytologic results; and any rectal and ultrasound examination results. additionally, one should obtain information regarding possible ingestion of endophyte-infected fescue or exposure to potential infectious causes of abortion. , a complete vaccination and deworming history is requisite, as is a complete history of any medications and supplements administered during pregnancy. after obtaining a history, one examines the mare per rectum. this examination should include palpation of the cervix, uterus, fetus, and all palpable abdominal contents. one should note any abnormalities. the cervix should be tight throughout gestation; the late gestation uterus will be large and distended with fluid and usually pulled craniad in the abdomen. palpation of the fetus frequently results in some fetal movement; however, one should interpret lack of movement with caution, for some normal fetuses do not respond. ultrasonographic evaluation of the uterus and conceptus per rectum can provide valuable information, particularly regarding placental thickness if placentitis is a concern. one may evaluate fetal fluids and estimate fetal size from the size of the eye later in gestation. in the author's hospital the practitioners choose not to perform vaginal examinations or speculum examinations because of an association between these examinations and the subsequent development of placentitis. unless placentitis is recognized with ultrasonograhic evaluation per rectum and culture is desirable, these types of examinations are generally not necessary. following examination per rectum, one performs transabdominal ultrasonographic evaluation of the uterus and conceptus. one can generate a biophysical profile of the fetus from this examination in the late-term fetus and readily determine viability. , one also readily can determine the presence or absence of twins in the late pregnant mare in this manner. one performs the sonogram through the acoustic window from the udder to the xiphoid ventrally and laterally to the skinfolds of the flank. imaging of the fetus usually requires a lowfrequency ( . -mhz) probe, whereas examination of the placenta and endometrium requires a higher-frequency ( . -mhz) probe. a complete description of this examination is beyond the scope of this chapter, but the reader will find several complete descriptions of the technique and normal values for specific gestation lengths within the relevant veterinary literature. the utility of this examination lies in its repeatability and low risk to the dam and fetus. sequential examinations over time allow the clinician to follow the pregnancy and to identify changes as they occur. a companion to transabdominal ultrasongraphy is evaluation of the fetal electrocardiogram (ecg). one can measure fetal ecgs continuously using telemetry or can obtain them using more conventional techniques several times throughout the day. , , one places electrodes on the skin of the mare in locations aimed at maximizing the magnitude of the fetal ecg. because the fetus frequently changes position, multiple sites may be needed in any -hour period. to begin, one places an electrode dorsally in the area of the sacral prominence with two electrodes placed bilaterally in a transverse plane in the region of the flank. the fetal ecg maximal amplitude is low, usually . to . mv, and can be lost in artifact or background noise, so one commonly must move electrodes to new positions to maximize the appearance of the fetal ecg. the normal fetal heart rate during the last months of gestation ranges from to beats/min, a fairly wide distribution. the range of heart rate of an individual fetus can be narrow, however. bradycardia in the fetus is an adaptation to in utero stress, most commonly thought to be hypoxia. by slowing the heart rate, the fetus prolongs exposure of fetal blood to maternal blood, increasing the time for equilibration of dissolved gas across the placenta and improving the oxygen content of the fetal blood. the fetus also has altered the distribution of its cardiac output in response to hypoxia, centralizing blood distribution. , tachycardia in the fetus can be associated with fetal movement, and brief periods of tachycardia should occur in the fetus in any -hour period. persistent tachycardia is a sign of fetal distress and represents more severe fetal compromise than bradycardia. the author has recognized dysrhythmias in the challenged fetus, most commonly as atrial fibrillation but also apparent runs of ventricular tachycardia. the ability to monitor the fetus in a high-risk pregnancy inevitably has led to questions of whether, how, and when to intervene. most equine neonatologists would agree that removal of the fetus from the uterus before its attainment of readiness for birth is not desirable. one of the difficulties in determining fetal preparedness for birth is that prediction of parturition is difficult in these mares. many of the parameters used in normal mares are unreliable in the high-risk pregnant mare. one must have an accurate history of any previous gestation length in terms of days for the specific mare in question to allow a more accurate estimate of her usual gestational length. evaluation of the usual mammary gland parameters, including size, the presence of "wax," and alteration of electrolyte concentrations, is not generally predictive in the high-risk mare, for in the author's experience many of these mares have changes predictive of parturition for weeks before actual parturition. , this circumstance may be related to the observation that many high-risk pregnant mares, particularly those with placentitis, are presented for a primary complaint of early onset lactation. although pulmonary system maturity in human beings can be assessed with some degree of accuracy using measurement of lecithin/sphingomyelin ratios, this measurement-along with sphingomyelin, cortisol, and creatinine concentrations in the amnionic fluid-has proved to be of no benefit in the horse. [ ] [ ] [ ] amniocentesis carries a high risk of abortion in the horse, even with ultrasound guidance, and is not a clinically useful technique at this time. currently, no clear-cut guidelines are available as to when to intervene, but the presence of persistent fetal tachycardia or prolonged absence of fetal movements, including breathing movements, as determined by transabdominal ultrasound evaluation, should initiate discussion regarding the appropriateness of induction of parturition or elective cesarean section. the goal of induction or cesarean section is to remove a pregnancy that is threatening the survival of the dam with no thought to fetal survival or to remove the fetus from a threatening environment to improve its likelihood for survival. preterm induction is ill advised if fetal survival is desirable because of the limited ability to treat severely immature neonates. timing of intervention in these circumstances remains an art, not a science. the approach to management of the high-risk pregnancy is dictated to some degree by the exact cause for concern, but for many mares therapy is similar. many high-risk mares have placentitis, primarily caused by ascending bacterial or fungal infections originating in the region of the cervix. these infections can cause in utero sepsis or compromise the fetus by local elucidation of inflammatory mediators or altered placental function. , premature udder development and vaginal discharge are common clinical signs. treatment consists of administration of broad-spectrum antimicrobial agents and nonsteroidal antiinflammatory drugs (table - ). in the author's clinic, trimethoprim-sulfonamide drugs have been the antimicrobial of choice based on unpublished studies performed at the facility demonstrating increased concentration of these agents in the fetal fluids compared with penicillin and gentamicin. however, if culture and sensitivity results are available, one should institute directed therapy. nonsteroidal antiinflammatory agents such as flunixin meglumine are useful to combat alterations in prostaglandin balance that may be associated with infection and inflammation. although the efficacy of these agents is best when administered before the development of clinical signs, to date no detrimental effects have been reported in the fetus or dam when chronically used at low doses in well-hydrated patients. tocolytic agents and agents that promote uterine quiescence have been used and include altrenogest, isoxuprine, and clenbuterol. [ ] [ ] [ ] [ ] [ ] altrenogest usually is administered, although its need in late gestation has been challenged. the efficacy of isoxuprine as a tocolytic in the horse is unproven, and bioavailability of orally administered isoxuprine appears to be highly variable. the long-term use of clenbuterol is inadvisable because of receptor population changes associated with chronic use and its unknown effects on the fetus at this time. clenbuterol may be indicated during management of dystocia in preparation for assisted delivery or cesarean section. the intravenous form of clenbuterol is not currently available in the united states. one can use three additional strategies in managing high-risk pregnancy patients. in mares with evidence of placental dysfunction, with or without signs of fetal distress, the author provides intranasal oxygen supplementation in the hope of improving oxygen delivery to the fetus. intranasal oxygen insufflation of to l/min to the mare significantly increases pao and percent oxygen saturation of hemoglobin. because of the placental vessel arrangement of the horse, improvement of these two arterial blood gas parameters should result in improved oxygen delivery to the fetus. blood gas transport is largely independent of diffusion distance in the equine placenta, particularly in late gestation, and depends more on blood flow. information from other species cannot be extrapolated to the equine placenta because of its diffuse epitheliochorial nature and the arrangement of the maternal and fetal blood vessels within the microcotyledons. , umbilical venous po is to mm hg in the horse fetus, compared with to mm hg in the sheep, whereas the maternal uterine vein to umbilical vein po difference is near . also unlike the sheep, the umbilical venous po values decrease to mm hg in response to maternal hypoxemia and increase in response to maternal hyperoxia. [ ] [ ] [ ] vitamin e (tocopherol) is administered orally to some high-risk mares as an antioxidant. administration of large doses of vitamin e before traumatic brain injury improves neurologic outcome in experimental models and has been examined as possible prophylaxis for human neonatal encephalopathy. [ ] [ ] [ ] extrapolation of that information to the compromised equine fetus suggests that increased antioxidant concentrations in the fetus may mitigate some of the consequences of uterine and birth hypoxia, but no evidence is available to date demonstrating that protection occurs or that vitamin e accumulates in the fetus in response to supplementation of the mare. finally, many high-risk mares are anorectic or held off feed because of their medical condition. these mares are at particularly great risk for fetal loss because of their lack of feed intake, which alters prostaglandin metabolism. therefore one should administer . % to % dextrose in . % saline or water ( % dextrose) intravenously at maintenance fluid rates to these patients. perhaps the most important aspect of managing high-risk pregnancy mares is frequent observation and development of a plan. one should observe mares at least hourly for evidence of early-stage labor and should put them under constant video surveillance if possible. depending on the primary problem, the team managing the mare should develop a plan for handling the parturition once labor begins and for fetal resuscitation following delivery. any equipment that might be needed should be readily available stallside, and a call sheet, listing contact numbers for all involved, should be posted on or near the stall. the plan should include a decision as to how to handle a complicated dystocia, should it occur, with permission for general anesthesia and cesarean section obtained before the event so that time is not wasted. an important question to be posed to the owner at the outset is which is most important to the owner, the mare or the foal, for the answer may dictate the direction of the decision tree once labor begins. early recognition of abnormalities is of utmost importance for successful management of critically ill foals. to recognize the abnormal, one must know the normal. immediately following birth, foals effect several important physiologic and behavioral changes. chief among these changes is the adaptation of the cardiovascular and respiratory systems to extrauterine life. the normal transition of the respiratory tract involves opening closed part ii disorders of specific body systems alveoli and absorption of fluid from the airway, accomplished by a combination of breathing efforts, expiration against a closed glottis (grunting), and a change in sodium flux across the respiratory membrane from net secretion to net absorption. [ ] [ ] [ ] [ ] [ ] the transition from fetal to neonatal circulatory patterns requires resolution of the pulmonary hypertension present in the fetus, normally shunting blood flow through the lower resistance ductus arteriosus in the fetal state, to direct cardiac output to the pulmonary vasculature for participation in gas exchange. this change is achieved by the opening of alveoli, decreasing airway resistance and providing radial support for pulmonary vessels, functional closure of the ductus arteriosus, and increasing the oxygen tension in the lung, reversing pulmonary vasoconstriction mediated by hypoxia. , pulmonary tree vasodilators (prostacyclin, nitric oxide [no] ) and vasoconstrictors (endothelin- , leukotrienes) play apparently well-coordinated, but as yet not fully elucidated, roles. in the normal newborn this change is smooth and rapid. these critical events are undermined by factors such as inadequate lung development, surfactant deficiency (primary or secondary), viral or bacterial infection, placental abnormalities, in utero hypoxia, and meconium aspiration. spontaneous breathing should begin in the neonate within minute of birth, many foals attempt to breathe as their thorax clears the pelvic canal. during the first hour of life, the respiratory rate of a healthy foal can be as high as breaths per minute but should decrease to to breaths per minute within a few hours. similarly, the heart rate of a healthy newborn foal has a regular rhythm and should be at least beats/min at the first minute. , one usually can auscultate a continuous murmur over the left side of the heart, although its loudness may vary with position. this murmur is thought to be associated with some shunting through the ductus arteriosus. one may auscultate variable systolic murmurs, thought to be flow murmurs, during the first week of life. one should investigate more thoroughly murmurs that persist beyond the first week of life in an otherwise healthy foal, along with any murmur associated with persistent hypoxia. auscultation of the thorax shortly after birth reveals a cacophony of sounds as airways open and fluid is cleared. end-expiratory crackles are consistently audible in the dependent lung during and following lateral recumbency. for a normal newborn foal to appear slightly cyanotic during this initial adaptation period is not unusual, but this should resolve within minutes of birth. the equine fetus, as do all fetuses, exists in a moderately hypoxic environment, but the equine fetus has a greater partial pressure of oxygen, around mm hg. because the fetus is well adapted to low oxygen tensions, cyanosis is rarely present in newborn foals once adaption occurs, even those with low oxygen tensions. although in many species the fetal blood oxygen affinity is greater than the maternal blood, in the equine fetus the oxygen affinity of its hemoglobin is only about mm hg greater than the maternal blood because of decreased levels of , -diphosphoglycerate compared with other species. the result is enhanced oxygen unloading in the equine fetus compared with others. , -diphosphoglycerate concentration increases after birth in the foal and reaches mature levels by to days of age. the major blood adaptation of the equine fetus to chronic hypoxia is an increase in packed cell volume of up to %, increasing the oxygen content of the blood as compensation for decreased oxygen delivery at the placenta. a larger than expected packed cell volume in any newborn foal should alert the clinician for possible sequelae from chronic hypoxia. the presence of significant cyanosis that persists should prompt the clinician to evaluate the foal thoroughly for cardiac anomalies resulting in significant right-to-left shunting or separated circulations, such as transposition of the great vessels. the chest wall of the foal is compliant, facilitating passage through the pelvic canal during parturition. this compliance requires that the foal actively participate in inspiration and expiration with several potential consequences. first, restriction of the thorax or the abdomen can result in impaired ventilation, which can occur easily when one restrains a foal and may result in spuriously abnormal arterial blood gas values (see the discussion on arterial blood gas evaluation, respiratory diseases associated with hypoxemia in the neonate). second, foals with primary pulmonary parenchymal disease resulting in poorly compliant lungs develop paradoxical chest wall motion, with the thorax moving inward during inspiration. [ ] [ ] [ ] [ ] the work of breathing can increase greatly, resulting in respiratory failure because of respiratory muscle fatigue. a foal that appears suddenly to improve a previously abnormal respiratory rate and pattern may in fact be in greater respiratory difficulty because of fatigue. one can observe a reduction in respiratory rate or abnormal breathing pattern in premature/dysmature foals or foals subjected to peripartum hypoxia/asphxia. although the genesis of these patterns is not understood fully, cheyne-stokes (lengthy periods of apnea interrupted by short breaths that wax and wane in depth), cluster (short periods of apnea interspersed with long periods of breathing), and biot's breathing (periods of apnea and breathing with no discernible pattern) may occur in these cases. foals attempting to maintain an adequate lung volume expire against a partially closed glottis, called valsalva's maneuver, producing an audible grunt. foals are normally nonresponsive while in the birth canal but should respond to stimulation immediately after birth. the lack of responsiveness while in the birth canal has lead to presumption of fetal death during dystocia. because of this, one should attempt other tests before determining that a foal is dead intrapartum. one possibly may detect pulses in the tongue, neck, or any presented limbs or palpate the thorax for a heartbeat. in the author's facility, nasotracheal intubation of the foal combined with measurement of co tensions in the exhaled gas aids practitioners in cases where they can reach the nose. nasotracheal intubation of foals under these circumstances actually can be performed readily with minimal practice. having long endotracheal tubes available of several different diameters ( to mm outer diameter) with an inflatable cuff is important. one can pass the tube blindly using a finger in one nostril for guidance and can check the position frequently by palpation of the throatlatch region. one inflates the cuff and begins manual ventilation with % oxygen or room air using an ambu-bag or equivalent. one can obtain continuous measurement of co tension using a capnograph or single-use disposable end-tidal co monitor attached to the ambu-bag or the nasotracheal tube. in a dead foal the end-tidal co measurement will be negligible after the first to breaths. one must ensure tube placement and seal integrity and allow for multiple breaths. some co will "wash out" with the first few breaths and can result in false hope initially. end-tidal co varies in living intrapartum foals, depending on cardiac output and ventilation frequency, but should be consistently greater than mm hg and is usually closer to mm hg. once one establishes manual ventilation of a living foal, one must continue ventilation until the foal is delivered satisfactorily. the author has resuscitated and maintained many foals successfully in this manner throughout induction of general anesthesia in the mare and cesarean section delivery of the foal. the nasotracheal tube also provides a convenient site for administration of intratracheal medications such as epinephrine used for extrauterine intrapartum resuscitation of the foal. the reader is cautioned that intratracheal epinephrine increases endtidal co measurements transiently, even in a dead foal, because of local actions on tissues. one should allow a washout period after intratracheal administration of epinephrine. the righting reflex is present as the foal exits the birth canal, as is the withdrawal reflex. cranial nerve responses are intact at birth, but the menace response may take as long as weeks to develop fully. one should not consider lack of a menace reflex diagnostic of visual deficits in the newborn foal. within an hour of birth the normal foal will demonstrate auditory orientation with unilateral pinna control. the normal pupillary angle is ventromedial in the newborn foal; this angle gradually becomes dorsomedial over the first month of life. foals should begin attempting to stand shortly after birth and should be able to achieve this on their own within hours of birth. the normal newborn foal has a suck reflex shortly after birth and should be searching for an udder even before it stands. the expectation is that a normal foal will be sucking from the dam unaided by hours post partum; many foals are overachievers and will be sucking well before this time. the normal foal may defecate shortly after standing but may not attempt defecation until after it first successfully sucks from the dam. urination varies more, with filly foals usually urinating before colt foals, but both usually do not urinate for several hours following birth, up to hours for some colts. for colt foals to fail to drop their penises when urinating over the first few days of life is not unusual. the gait of the newborn foal is hypermetric and the stance is base wide. extreme hypermetria of the forelimbs, usually bilateral but occasionally unilateral, has been observed in some foals and is associated with perinatal hypoxic/ischemic insults, but this gait abnormality usually resolves without specific therapy within a few days. spinal reflexes tend to be exaggerated, whereas the crossed extensor reflex may not be fully present until weeks of age. foals also exhibit an exaggerated response to external stimuli (noise, sudden visual changes, touch) for the first few weeks of life. foals are not bonded strongly to their mother for the first few weeks of life and will follow any large moving object, including other horses and human beings. orphan foals bond with surrogate mothers until they are several months of age; their primary motivation appears to be appetite. conversely, mares strongly bond with their foals shortly after parturition; the process begins once the chorioallantois ruptures and is driven more by olfaction and taste than by vision or hearing. interference with this process, by medical intervention or excessive owner manipulation of the foal, can disrupt normal bonding and result in foal rejection by the dam. most newborn foals make the transition to extrauterine life easily. however, for those in difficulty, recognition of the condition immediately and institution of appropriate resuscitation is of utmost importance. a modified apgar scoring system has been developed as a guide for initiating resuscitation and assessing probable level of fetal compromise (table - ). one also must at least perform a cursory physical examination before initiating resuscitation, for issues of humaneness are associated with with serious problems such as severe limb contracture, microophthalmia, and hydrocephalus, among others. the initial assessment begins during presentation of the fetus. although the following applies primarily to attending the birth of a foal from a high-risk pregnancy, one can perform quiet and rapid evaluation during any attended birth. the goal in a normal birth with a normal foal is to disturb the bonding process minimally. this goal also applies to high-risk parturitions, but some disruption of normal bonding is inevitable. the lead clinician should control tightly the number of persons attending, and the degree of activity surrounding, the birth. one should evaluate the strength and rate of any palpable peripheral pulse and should evaluate the apical pulse as soon as the chest clears the birth canal. bradycardia (pulse < beats/min) is expected during forceful contractions, and the pulse rate should increase rapidly once the chest clears the birth canal. persistent bradycardia is an indication for rapid intervention. the fetus is normally hypoxemic compared with the newborn foal, and this hypoxemia is largely responsible for the maintenance of fetal circulation by generation of pulmonary hypertension. the fetus responds to conditions producing more severe in utero hypoxia by strengthening the fetal circulatory pattern, and the neonate responds to hypoxia by reverting to the fetal circulatory pattern. during a normal parturition, mild asphyxia occurs and results in fetal responses that pave the way for a successful transition to extrauterine life. if more than mild transient asphyxia occurs, the fetus is stimulated to breathe in utero; this is known as primary asphyxia. if the initial breathing effort resulting from the primary asphyxia does not correct the asphyxia, a second gasping period occurs in several minutes, known as the secondary asphyxia response. if no improvement in asphyxia occurs during this period, the foal enters secondary apnea, a state that is irreversible except with resuscitation. therefore the first priority of neonatal resuscitation is establishing an airway and breathing pattern. one should assume that foals not spontaneously breathing are in secondary apnea and should clear the airway of membranes as soon as the nose is presented. if meconium staining is present, one should suction the airway before delivery of the foal is completed and before the foal breathes spontaneously. one should continue to the trachea if aspiration of the nasopharynx is productive. overzealous suctioning worsens bradycardia as it worsens hypoxia. one should stop suctioning once the foal begins breathing spontaneously, as hypoxia will worsen with continued suction. if the foal does not breathe or move spontaneously within seconds of birth, one should begin tactile stimulation. if tactile stimulation fails to result in spontaneous breathing, one immediately should intubate the foal and manually ventilate the foal using an ambu-bag or equivalent. one can use mouth-to-nose ventilation if nasotracheal tubes and an ambu-bag are not available. the goal of this therapy is to reverse fetal circulation, and hyperventilation with % oxygen is the best choice for this purpose. however, recent evidence suggests that no clinical disadvantages are apparent in using room air for ventilation of asphyxiated human neonates rather than % oxygen. , human infants resuscitated with room air recovered more quickly than those resuscitated with % oxygen in one study as assessed by apgar scores, time to the first cry, and the sustained pattern of breathing. in addition, neonates resuscitated with % oxygen exhibited biochemical findings reflecting prolonged oxidative stress, present even after weeks of postnatal life, which did not appear in the group resuscitated with room air. thus the current accepted recommendations for using % oxygen in the resuscitation of asphyxiated neonates needs further discussion and investigation. , almost % of foals requiring resuscitation respond to hyperventilation alone and require no additional therapy. one can initiate nasotracheal intubation while the foal is in the birth canal if the foal will not be delivered rapidly, such as with a difficult dystocia. this technique is "blind" and requires some practice but may be beneficial and lifesaving. once spontaneous breathing is present, one apgar score in the foal should provide humidified oxygen via nasal insufflation at to l/min. one should initiate cardiovascular support in the form of chest compression if the foal remains bradycardic despite ventilation and a nonperfusing rhythm is present. one should make sure the foal is on a hard surface in right lateral recumbency with the topline against a wall or other support. approximately % of foals are born with fractured ribs and an assessment for the presence of rib fractures is in order before initiating chest compressions. palpation of the ribs identifies many of these fractures, which usually are multiple and consecutive on one side of the thorax and located in a relatively straight line along the part of the rib with the greatest curvature dorsal to the costochondral junction. unfortunately, ribs to frequently are involved, and their location over the heart can make chest compression a potentially fatal exercise. auscultation over the ribs during breathing results in a recognizable click, identifying rib fractures that may have escaped detection by palpation. one should initiate drug therapy if a nonperfusing rhythm persists for more than to seconds in the face of chest compression. epinephrine is the first drug of choice (table - ) . practitioners pose various arguments regarding the best dose and the best frequency of administration for resuscitation. however, most of the data are acquired from human cardiac arrest studies and are not strictly applicable to the equine neonate because the genesis of the cardiovascular failure is different. , vasopressin is gaining attention as a cardiovascular resuscitation drug, and although the author has used this drug in resuscitation and as a pressor, experience is limited at this time. the author does not use atropine in bradycardic newborn foals because the bradycardia usually is caused by hypoxia, and if the hypoxia is not corrected, atropine can increase myocardial oxygen debt. the author also does not use doxapram because it does not reverse secondary apnea, the most common apnea in newborns. because birthing areas are generally cold, one should dry the foal and place it on dry bedding once resuscitation is complete. the fetus has some homeothermic mechanisms, but its size in relation to its mother and its position within her body means that it is in effect a poikilotherm. the body temperature of the foal generally reflects that of its environment, namely its mother, although the human fetal temperature directly measured at cesarean section, induction of labor, or during labor is approximately . °c higher than the mothers. , adaptation from poikilothermy to homeothermy normally takes place rapidly following birth. the fetus is capable of nonshivering thermogenesis, primarily through the oxidation of brown fat reserves, but this type of thermogenesis is inhibited in utero, probably by placental prostaglandin e and adenosine. , immediately after birth the foal must adapt to independent thermoregulation. local physical factors, including ambient temperature and humidity, act to induce cold stress, and the newborn must produce heat by metabolic activity. in response to the catecholamine surge associated with birth, uncoupling of oxidative phosphorylation occurs within mitochondria, releasing energy as heat. this nonshivering thermogenesis is impaired in newborns undergoing hypoxia or asphyxiation and in those that are ill at birth. infants born to mothers sedated with benzodiazepines are affected similarly, a consideration in the choice of sedative and preanesthetic medications in mares suffering dystocia or part ii disorders of specific body systems undergoing cesarean section. [ ] [ ] [ ] heat losses by convection, radiation, and evaporation are high in most areas where foals are delivered, resuscitated ,and managed, and one must take care to minimize cold stress in the newborn and the critically ill foal. supplementary heat, in the form of radiant heat lamps or warm air circulating blankets, may be required. one should use fluid therapy conservatively during postpartum resuscitation, for the neonate is not volume depleted unless excessive bleeding has occurred. some compromised newborn foals are actually hypervolemic. fluid therapy of the neonate is discussed in more detail later in this chapter. because the renal function of the equine neonate is substantially different from the adult, one cannot simply scale down fluid therapy from adult therapy. [ ] [ ] [ ] if intravenous fluids are required for resuscitation and blood loss is identified, administration of ml/kg of a non-glucose-containing polyionic isotonic fluid over minutes (about l for a -kg foal) once intravenous access is established can be effective. the author stresses non-glucose-containing polyionic intravenous fluids because hyperglycemia, but not hypoglycemia, immediately after fetal or neonatal asphyxia interfered with the recovery of brain cell membrane function and energy metabolism in neonatal piglets in one recent study. these findings suggest that post-hypoxic-ischemic hyperglycemia is not beneficial and might even be harmful in neonatal hypoxic-ischemic encephalopathy. indications for this shock bolus therapy include poor mentation, poorly palpable peripheral pulses, and the development of cold distal extremities, compatible with hemorrhagic shock. one should reassess the patient after the initial bolus and administer additional boluses as necessary. ideally, one should follow up on blood pressures and ecg readings and initiate appropriate pressor therapy if needed. again, these procedures are discussed in detail later in the chapter. one can administer glucose-containing fluids after resuscitation at a rate of to mg/kg/min (about ml/hr of % dextrose or ml/hr of % dextrose) to the average -kg foal, particularly in the obviously compromised foal. this therapy is indicated to help resolve metabolic acidosis, to support cardiac output because myocardial glycogen stores likely have been depleted, and to prevent postasphyxial hypoglycemia. under normal conditions, the fetal-to-maternal blood glucose concentration gradient is % to % in the horse, and glucose is the predominant source of energy during fetal development. , glucose transport across the placenta is facilitated by carrier receptors (glucose transporter [glut] receptors), and a direct relationship exists between maternal and fetal blood glucose concentration when maternal glucose is in the normal range. the glut receptors in the placenta are stereospecific, saturable, and energy independent. although the enzyme kinetics for glut isoform suggest that they are not saturable under conditions of euglycemia, equine maternal hyperglycemia results in increased fetal glucose concentration to a plateau point, likely caused by glut saturation. at term, the net umbilical uptake of glucose is to mg/kg/min, with most of the glucose being used by the brain and skeletal muscle. [ ] [ ] [ ] the fetus only develops gluconeogenesis under conditions of severe maternal starvation. a certain percentage of the delivered glucose is used to develop large glycogen stores in the fetal liver and cardiac muscle in preparation for birth, and at birth the foal liver produces glucose at a rate of to mg/ kg/min by using these stores. fetal glycogen stores also are built using the substrates lactate, pyruvate, and alanine; fetal uptake of lactate across the placenta is about half that of glucose. , the transition to gluconeogenesis, stimulated by increased circulating catecholamine concentration from birth and by stimulation of glucagon release at the time the umbilical cord breaks takes to hours in the normal foal, and glycogenolysis supplies needed glucose until feeding and glucose production are accomplished. in the challenged foal, glycogen stores may have been depleted and gluconeogenesis delayed, so provision of glucose at rates similar to what the liver would normally produce during this period is requisite. persistent pulmonary hypertension (pph) also is known as reversion to fetal circulation or persistent fetal circulation, and its genesis lies in the failure of the fetus to make the respiratory and cardiac transition to extrauterine life successfully or reversion of the newborn to fetal circulatory patterns in response to hypoxia or acidosis. differentiating this problem from other causes of hypoxemia in the newborn requires some investigation, and multiple serial arterial blood gas analyses are necessary to confirm suspicion of this problem (see the section on arterial blood gas analysis, respiratory diseases associated with hypoxemia in the neonate). however, one should suspect the condition in any neonate with hypercapnic hypoxemia that persists and worsens; these foals are in hypoxemic respiratory failure. the fetal circulatory pattern, with pulmonary hypertension and right-to-left shunting of blood through the patent foramen ovale and ductus arteriosus, is maintained in these cases. pulmonary vascular resistance falls at delivery to about % of fetal values, while pulmonary blood flow increases accordingly. early in the postnatal period these two changes balance each other, and mean pulmonary and systolic pressures remain increased for several hours. systolic pulmonary pressures can remain equivalent to systemic pressure for up to hours of age in human infants, although diastolic pulmonary pressures are well below systemic diastolic pressures by hour. mean pulmonary artery pressures fall gradually over the first hours. the direct effects of lung expansion and increasing alveolar oxygen tension probably provide the initial stimulus for pulmonary arteriolar dilation and partly result from direct physical effects, but vasoactive substances are released in response to physical forces associated with ventilation, for example prostacyclin. other vasoactive mediators thought to play a role in regulating pulmonary arteriolar tone include no, prostaglandins d and e , bradykinin, histamine, endothelin- , angiotensin ii, and atrial natriuretic peptide. the increase in alveolar and arterial oxygen tensions at birth is required for completion of resolution of pulmonary hypertension. much of this increase is thought to be mediated by no, evidence for this being the parallel increase during gestation of the pulmonary vasodilation response to hyperoxia and the increase in no synthesis. however, inhibition of no synthesis does not eliminate the initial decrease in pulmonary artery resistance occurring because of opening of the airways. when these mechanisms fail, one can recognize pph. right-to-left shunting within the lungs and through patent fetal conduits occurs and can result from many factors, including asphyxia and meconium aspiration, but in many cases the precipitating trigger is unknown. inappropriately decreased levels of vasodilators (no) and inappropriately increased levels of vasoconstrictors (endothelin- ) currently are being examined as potential mechanisms. chronic in utero hypoxia and acidosis may result in hypertrophy of the pulmonary arteriolar smooth muscle. in these cases, reversal of pph can be difficult and cannot be achieved rapidly. treatment of pph is twofold: abolishment of hypoxia and correction of the acidosis, for both abnormalities only bolster the fetal circulatory pattern. initial therapy is provision of oxygen intranasally at to l/min. some foals respond to this therapy and establish neonatal circulatory patterns within a few hours. failure to improve or worsening of hypoxemic respiratory failure following intranasal oxygen administration should prompt intubation and mechanical ventilation with % oxygen. this serves two purposes, one diagnostic and one therapeutic. ventilation with % oxygen may resolve pph and, if intrapulmonary shunt and altered ventilation-perfusion relationships are causing the hypoxic respiratory failure, arterial oxygen tension (pao ) should exceed mm hg under these conditions. failure to improve pao suggests pph or large right-to-left extrapulmonary shunt caused by congenital cardiac anomaly. the vasodilators prostacyclin and telazoline (an α-blocking vasodilator) cause pulmonary vasodilation in human infants with pph, but the effects on oxygenation vary and the sideeffects (tachycardia, severe systemic hypotension) are unacceptable. recognition of no as a potent dilator of pulmonary vessels has created a significant step forward in the treatment of these patients, for inhaled no dilates vessels in ventilated portions of the lung while having minimal effects on the systemic circulation. based on evidence presently available, use of inhaled no in an initial concentration of about ppm in the ventilatory gas seems reasonable for term and near-term foals with hypoxic respiratory failure and pph that fails to respond to mechanical ventilation using % oxygen alone. , the author has used this approach in the clinic, administering a range of to ppm no with success. hypoxic ischemic encephalopathy (hie), currently referred to as neonatal encephalopathy in the human literature, is one systemic manifestation of a broader syndrome of perinatal asphyxia syndrome (pas), and management of foals with signs consistent with a diagnosis of hie requires the clinician to examine other body systems fully and to provide therapy directed at treating other involved systems. although pas primarily manifests as hie, the gastrointestinal tract and kidneys frequently are affected by peripartum hypoxia/ischemia/ asphyxia, and one should expect complications associated with these systems. hypoxic ischemic encephalopathy also may affect the cardiovascular and respiratory systems, and one also may encounter endocrine disorders in these patients. hypoxic ischemic encephalopathy has been recognized as one of the most common diseases of the equine neonate for generations. , , in the past hie has been known as dummy foal syndrome and as neonatal maladjustment syndrome. the designation hie, although not perfect, attempts to describe the syndrome in terms of the suspected underlying pathophysiology. a wide spectrum of clinical signs is associated with hie and can range from mild depression with loss of the suck reflex to grand mal seizure activity. typically, affected foals are normal at birth but show signs of central nervous system abnormalities within a few hours after birth. some foals are obviously abnormal at birth, and some do not show signs until hours of age. hypoxic ischemic encephalopathy commonly is associated with adverse peripartum events, including dystocia and premature placental separation, but a fair number of foals have no known peripartum period of hypoxia, suggesting that these foals result from unrecognized in utero hypoxia (box - ). severe maternal illness also may result in foals born with pas. in human beings, ascending placental infection now is suspected of being a major contributor to neonatal encephalopathy in infants, and the incidence of neonatal encephalopathy increases with the presence of maternal fever, suggesting a role for maternal inflammatory mediators. the underlying pathophysiologic details of hie in the foal are unknown, and to date accurate experimental models of hie and pas in the foal have not been described. however, a great deal of attention has been paid to peripartum hypoxia/asphyxia by human counterparts because the effects of adverse peripartum events in the human neonate have far ranging implications for the affected human neonate and for society. therefore equine neonatologists have long looked to human studies and models of the human disease for understanding of the syndrome in the equine neonate. perinatal brain damage in the mature fetus usually results from severe uterine asphyxia caused by an acute reduction of uterine or umbilical circulation. the fetus responds to this challenge by activation of the sympathetic adrenergic nervous system, causing a redistribution of cardiac output that favors the central organs: brain, heart, and adrenal glands. , if the hypoxic insult continues, the fetus reaches a point beyond which it cannot maintain this centralization of circulation, cardiac output falls, and cerebral circulation diminishes. the loss of oxygen results in a substantial decrease in oxidative phosphorylation in the brain with concomitant decreased energy production. the na + /k + pump at the cell membrane cannot maintain the ionic gradients, and the membrane potential is lost in the brain cells. in the absence of the membrane potential, calcium flows down its large extracellular/intracellular concentration gradient through voltage-dependent ion channels into the cell. this calcium overload of the neuron leads to cell damage by activation of calcium-dependent proteases, lipases, and endonucleases. protein biosynthesis is halted. calcium also enters the cells by glutamate-regulated ion channels as glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles following anoxic cellular depolarization. once the anoxic event is over, protein synthesis remains inhibited in specific areas of the brain and returns to normal in less vulnerable areas of the brain. loss of protein synthesis appears to be an early indicator of cell death caused by the primary hypoxic/anoxic event. a second wave of neuronal cell death occurs during the reperfusion phase and is thought to be similar to classically described postischemic reperfusion injury in that damage is caused by production of and release of oxygen radicals, synthesis of no, and inflammatory reactions. additionally, an imbalance between excitatory and inhibitory neurotransmitters occurs. part of the secondary cell death that occurs is thought to be caused by apoptosis, a type of programmed cell death termed cellular suicide. secondary cell death also is thought be caused by the neurotoxicity of glutamate and aspartate resulting again from increased intracellular calcium levels. , in human infants the distribution of lesions with hypoxic-ischemic brain damage following prenatal, perinatal, or postnatal asphyxia falls into distinct patterns depending on the type of hypoxia-ischemia rather than on postconceptual age at which the asphyxial event occurs. periventricular leukomalacia was associated with chronic hypoxia-ischemia, whereas the basal ganglia and thalamus were affected primarily in patients experiencing acute profound asphyxia, providing direct evidence that the nature of the event determines the severity and distribution of neurologic damage in human beings. these remarkably selective patterns of injury in children, with differential variability in the damage caused to regions anatomically located within millimeters of each other, resulted in the hypothesis that location within neurotransmitter-specific circuitry loops is important. this hypothesis has important implications in the design of neuroprotective strategies and therapies for neonates experiencing hypoxic-ischemic-asphyxial events. now the evidence is overwhelming that the excitotoxic cascade that evolves during hie extends over several days from the time of insult and is modifiable. , in brain injury, traumatic or hypoxic, the mechanisms underlying delayed tissue injury still are understood poorly. many believe that neurochemical changes, including excessive neurotransmitter release, are pivotal in the pathophysiology of secondary neuronal death. excitatory amino acid neurotransmitters and magnesium are known to play at least a minimal role in secondary cell death following brain injury; a fair body of literature regarding these factors has been generated over the last years. the activation of the n-methyl-d-aspartate (nmda) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury and is suspected to play a role in hie. [ ] [ ] [ ] mechanically injured neurons demonstrate a reduction of voltage-dependent mg + blockade of nmda current that can be restored partially by increasing extracellular mg + concentration or by pretreatment with calphostin c, a protein kinase c inhibitor. this finding suggested that administration of mg + to patients with brain injury could lead to improved outcome. subsequently, magnesium sulfate solution was shown to improve dramatically the immediate recovery of rats from hypoxia. however, although pretreatment with magnesium sulfate protected against hypoxic ischemic brain injury, postasphyxial treatment worsened brain damage in -day-old rats, suggesting an age-related response in the rat. delayed magnesium treatment of mature rats following severe traumatic axonal brain injury improved motor outcome when administered up to hours after injury, with early treatments providing the most benefit. maternal seizure in rats is associated with fetal histopathologic changes that are abolished by administration of magnesium sulfate to the mother, and magnesium sulfate has been demonstrated to protect the fetal brain from severe maternal hypoxia. clinical trials investigating the efficacy of magnesium treatment following hypoxia in infants are under way, with few reports currently in the medical literature. magnesium sulfate was used to treat nine infants after perinatal asphyxia in one study (no control group), and all children were neurologically normal at year of age. seizures did not occur in any of these children, nor were any adverse side effects noted. magnesium sulfate administration failed to delay the global impairment in energy metabolism after hypoxia ischemia, characteristic of severe brain damage, in newborn piglets; at hours after hypoxia ischemia, no difference could be found in the severity of injury in piglets treated with magnesium compared with piglets treated with placebo, suggesting magnesium may not be protective with severe acute injury. in developing countries, birth hypoxia frequently is associated with hie, and although this finding is attributed most frequently to inadequate obstetric care, poor nutrition also may play a role. red blood cell magnesium levels were measured in more than women in labor at a teaching hospital in south africa. fifty five of the women delivered infants with hie and had significantly lower levels of magnesium than controls; the infants with hie also had significantly lower magnesium levels than controls. the large majority ( of ) of the women giving birth to hie infants were from poor social circumstances, suggesting nutrition might play a role in some cases of hie, with maternal magnesium levels affecting outcome in the infants. the authors suggested an early pregnancy intervention study may help determine the role of magnesium in the pathogenesis of hie in human infants born to at-risk mothers. therapy for the various manifestations of hypoxiaischemia involves control of seizures, general cerebral support, correction of metabolic abnormalities, maintenance of normal arterial blood gas values, maintenance of tissue perfusion, maintenance of renal function, treatment of gastrointestinal dysfunction, prevention and recognition and early treatment of secondary infections, and general supportive care. control of seizures is important because cerebral oxygen consumption increases fivefold during seizures. one can use diazepam for emergency control of seizures (table - ). if diazepam does not stop seizures readily or one recognizes more than two seizures, then one should replace diazepam with phenobarbital given to effect. the half-life of phenobarbital can be long in the foal ( hours), and one should keep this in mind when monitoring neurologic function in these cases after phenobarbital administration (j.e. palmer, personal communication, ). earlystage, preseizure administration of phenobarbital has been advocated by some investigators for prevention of neonatal encephalopathy. however, one recent study in asphyxiated human infants demonstrated that early phenobarbital treatment was associated with a threefold increase in the incidence of subsequent seizures and consequently a trend toward increased mortality. seizures per se were associated with almost a twentyfold increase in mortality. their findings suggest that early phenobarbital administration may produce adverse rather than beneficial effects following asphyxia. because this was an observational study; the results need to be confirmed by appropriate randomized trials in similar clinical settings. if phenobarbital fails to control seizures, one may attempt phenytoin therapy. in cases of hie, one should avoid ketamine and xylazine because of their association with increased intracranial pressure. one must protect the foal from injury during a seizure and also ensure the patency of the airway to prevent the onset of negative pressure pulmonary edema or aspiration pneumonia. probably the most important therapeutic interventions are aimed at maintaining cerebral perfusion, which is achieved by careful titration of intravenous fluid support, neither too much nor too little (see fluid therapy in neonates) and judicious administration of inotropes and pressors to maintain adequate perfusion pressures (see pressor and inotrope therapy in neonates). cerebral interstitial edema is only truly present in the most severe cases , ; in most cases the lesion is intracellular edema and most of the classic agents used to treat cerebral interstitial edema (e.g., mannitol) are minimally effective treating cellular edema. occasionally the author uses thiamine supplementation in the intravenous fluids to support metabolic processes, specifically mitochondrial metabolism and membrane na + ,k + -atpases, involved in maintaining cellular fluid balance. , this therapy is rational and inexpensive but unproven in efficacy. only if cellular necrosis and vasogenic edema are present are drugs such as mannitol and dimethyl sulfoxide indicated, and again these cases are usually the most severely affected. in the author's clinic, practitioners rarely have used dimethyl sulfoxide in neonates for the last several years and have recognized no change in outcome by discontinuing its use. when the practitioners use intravenously administered dimethyl sulfoxide, they do so within the first hour after an acute asphyxial insult and use it primarily for its hydroxyl radical scavenging effects and its theoretical modulation of postischemic reperfusion injury. naloxone has been advocated for treating hie in human beings and in foals, [ ] [ ] [ ] perhaps based on a study suggesting that postasphyxia blood-brain barrier disruption was related causally to poor neurologic outcome in a lamb model of hie and that naloxone prevented disruption and neurologic dysfunction among those survivors with an intact blood-brain barrier. however, other studies have demonstrated that naloxone exacerbates hypoxic-ischemic brain injury in -day-old rats subjected to unilateral common carotid artery ligation and hypoxia. moreover, systemic acidosis and cellular edema were no different in naloxone-treated animals compared with animals treated with saline solution. the authors concluded that high doses of naloxone in fact may reduce the resistance of the fetus to hypoxic stress. the use of naloxone in human neonatal resuscitation remains controversial, for whether the contradictory effects are related to a reduction in acute neuronal swelling by osmotic effects or by a more direct receptor-mediated mechanism is currently unknown. naloxone is most effective in resuscitation of compromised human infants born to mothers addicted to drugs. some practitioners are using γ-aminobutyric acid adrenergic agonists to manage hie in foals, based on evidence showing neuroprotection when used in ischemia alone and combined with nmda antagonists. [ ] [ ] [ ] the author currently has no experience with these compounds and cannot comment regarding their efficacy in foals. regional hypothermia also is being investigated as a potential therapy for global hypoxia/ischemia; published data are consistent with the theory that cooling must be continued throughout the entire secondary phase of injury (about days) to be effective. experimentally, this approach has resulted in dramatic decreases in cellular edema and neuronal loss; its practical application remains to be demonstrated. despite a lack of consensus regarding the use of magnesium to treat infants with hie, the author has used magnesium sulfate infusion as part of the therapy for selected foals with hie for the past several years. the rationale is based primarily on the evidence demonstrating protection in some studies and a failure of any one study to demonstrate significant detrimental effects. the clinical impressions of the author to date suggest that the therapy is safe and may decrease the incidence of seizure in patients. the author administers magnesium sulfate as a constant rate infusion over hour after giving a loading dose. the author has continued the infusion for up to days without demonstrable negative effect beyond some possible trembling. given the current evidence, a -hour course of treatment may be effective and all that is necessary. postasphyxial treatment certainly may be beneficial in foals with hie, and maternal magnesium therapy may be beneficial in certain high-risk pregnancy patients. foals with pas often have a variety of metabolic problems including hypo-or hyperglycemia, hypo-or hypercalcemia, hypo-or hyperkalemia, hypo-or hyperchloremia, and varying degrees of metabolic acidosis. although one needs to address these problems, one should not forget the normal period of hypoglycemia that occurs postpartum and should not treat aggressively so as to avoid worsening the neurologic injury. foals suffering from pas also have frequent recurrent bouts of hypoxemia and occasional bouts of hypercapnia. intranasally administered oxygen is generally needed in these cases as a preventative therapy and as direct treatment, for the appearance of the abnormalities can be sporadic and unpredictable. additional respiratory support, particularly in those foals with centrally mediated hypoventilation and periods of apnea or abnormal breathing patterns, include caffeine (per os or per rectum) and positive pressure ventilation. caffeine is a central respiratory stimulant and has minimal side effects at the dosages used ( mg/kg loading dose; . mg/kg as needed). the author purchases whatever oral form of caffeine is available at the local convenience store or drug store and administers it dissolved in warm water per rectum. foals treated with caffeine have an increased level of arousal and are more reactive to the environment. adverse effects generally are limited to restlessness, hyperactivity, and mild to moderate tachycardia. mechanical ventilation of these patients can be rewarding and generally is required for less than hours. one must monitor and maintain blood ph within the normal range. metabolic alkalosis can develop in some of these foals and requires clinician tolerance of some degree of hypercapnia. ph is important in evaluation and consideration of alternatives for treatment. if the respiratory acidosis is not so severe as to affect the patient adversely (generally > mm hg), and the ph is within normal limits, the foal may tolerate hypercapnia. the goal is to normalize ph. foals with respiratory acidosis as compensation for metabolic alkalosis do not respond to caffeine. metabolic alkalosis in critically ill foals frequently is associated with electrolyte abnormalities, creating differences in strong ion balance. one handles this ph perturbation best by correcting the underlying electrolyte problem. maintaining tissue perfusion and oxygen delivery to tissues is a cornerstone of therapy for pas to avoid additional injury. one should maintain the oxygen-carrying capacity of the blood; some foals require transfusions to maintain a packed cell volume greater than %. adequate vascular volume is important, but one should take care to avoid fluid overload in the foal. early evidence of fluid overload is subtle accumulation of ventral edema between the front legs and over the distal limbs. fluid overload can result in cerebral edema, pulmonary edema, and edema of other tissues, including the gastrointestinal tract. this edema interferes with normal organ function and worsens the condition of the patient. one maintains perfusion by supporting cardiac output and blood pressure by judicious use of intravenous fluid support and inotrope/pressor support. the author does not target therapy to a specific systolic, mean, or diastolic pressure but monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for these patients to require pressor therapy is not unusual, but in some cases the hypoxic damage is sufficiently severe to blunt the response of the patient to the drugs. the kidney is a target for injury in patients with pph, and for renal compromise to play a significant role in the demise of these foals is not unusual. clinical signs of renal disease are generally referable to disruption of normal control of renal blood flow and tubular edema leading to tubular necrosis and renal failure. these foals have signs of fluid overload and generalized edema. one must balance urine output and fluid therapy in these cases to prevent additional organ dysfunction associated with edema. although evidence has accumulated that neither dopamine nor furosemide play a role in protecting the kidney or reversing acute renal failure, these agents can be useful in managing volume overload in these cases. [ ] [ ] [ ] the aim is not to drive oliguric renal failure into a highoutput condition but rather to enhance urine output. overzealous use of diuretics and pressors in these cases can result in diuresis requiring increased intravenous fluid support and can be counterproductive. the author's approach is more conservative. low doses of dopamine administered as a constant rate infusion of to µg/kg/min are usually effective in establishing diuresis by natriuresis. one should avoid large doses of dopamine (> µg/kg/min) because high doses can produce systemic and pulmonary vasoconstriction, potentially exacerbating pph. one can administer a bolus ( . to . mg/kg) or constant rate infusion ( . to . mg/kg/hr) of furosemide, but once furosemide diuresis is established, one must evaluate electrolyte concentrations and blood gas tensions frequently because potassium, chloride, and calcium losses can be considerable and because significant metabolic alkalosis can develop from strong ion imbalances. the author does not aim for urine production rates of ml/hr, as has been presented by other authors as a urine output goal for critically ill equine neonates. rather the author looks for urine output that is appropriate for fluid intake and does not attempt to drive urine output to an arbitrary goal by excessive fluid administration or pressor use. although the average urine output for a normal equine neonate is about ml/kg/hr (~ ml/hr for a -kg foal), these values were obtained from normal foals drinking a milk diet with a large free water component. [ ] [ ] [ ] the urine of normal newborn foals is dilute, reflecting the large free water load they incur by their diet. expecting critically ill foals to produce such large volumes of urine, particularly those on restricted diets or receiving total parenteral nutrition, is an exercise in futility, and manipulating fluid, pressor, or diuretic therapy in attempt to meet an artificial goal is inappropriate. fluid therapy in the critically ill neonate is discussed later in this chapter. one final caveat regarding renal dysfunction in pas is that one should perform therapeutic drug monitoring when it is available. many antimicrobial agents used to manage these cases, most notably the aminoglycosides, depend on renal clearance. aminoglycoside toxicity occurs in the equine neonate and exacerbates or complicates the management of renal failure originally resulting from primary hemodynamic causes. the author monitors aminoglycoside concentrations for -minute peak and -to -hour trough values in these cases and adjusts dosage and frequency of drug administration based on these results. the author considers a trough value of less than µg/dl as desirable for gentamicin and amikacin. foals with pas suffer from a variety of problems associated with abnormalities within the gastrointestinal tract. commonly they have ileus, recurrent excessive gastric reflux, and gas distention. these problems are exacerbated by constant feeding in the face of continued dysfunction and continued hypoxia. frequently, enteral feeding cannot meet their nutritional requirements, and partial or total parenteral nutrition is required. one must give special attention to passive transfer of immunity (see failure of passive transfer) and glucose homeostasis in these cases. although some practitioners use prokinetic agents as therapy for ileus in these cases, the author's approach is again more conservative. appearance of damage to the gastrointestinal tract can be subtle and lag behind other clinical abnormalities for days to weeks. low-grade colic, decreased gastrointestinal motility, decreased fecal output, and low weight gain are among the most common clinical signs of gastrointestinal dysfunction in these case, but more severe problems, including necrotizing enterocolitis and intussusception, have been associated with these cases. the return to enteral feeding must be slow in many of these cases. a currently debated topic is constant versus pulsed enteral feeding. [ ] [ ] [ ] the author uses pulsed feeding through an indwelling small-gauge feeding tube. in many foals these tubes stay in place for weeks and cause no problems as the foals are returned to their dams for sucking or are trained to drink from a bottle or bucket. foals with pas are also susceptible to secondary infection. treatment of recognized infection is covered under sepsis in this chapter. if infection is recognized in these patients after hospitalization, one should give attention to the likelihood of nosocomial infection and should direct antimicrobial therapy based on known nosocomial pathogens in the nicu and their susceptibility patterns until culture and sensitivity results become available. one should make repeat determinations of immunoglobulin g (igg) concentration; additional intravenous plasma therapy may be required. nosocomial infections are often rapidly overwhelming, and acute deterioration in the condition of a foal with pas should prompt a search for nosocomial infection. the prognosis for foals with pas is good to excellent when the condition is recognized early and aggressively treated in term foals. up to % of these neonates survive and go on to lead productive and useful athletic lives. [ ] [ ] [ ] [ ] the prognosis decreases with delayed or insufficient treatment and concurrent problems such as prematurity and sepsis. in human nicus the survival rates of low-gestationlength infants has increased dramatically since the s concurrent with improvements in obstetric and neonatal care. the now routine, well-validated use of antenatal steroid and artificial surfactant therapies has contributed greatly to the enhanced survival of this patient population, although the use of these particular therapies is not common or frequently indicated in the equine nicu. , however, with improved care, outcomes in the equine nicu population have improved also, with survival of premature patients in many nicus exceeding %. in the equine population, gestation length is much more flexible than in the human population; however, the definition of the term prematurity needs reexamination. traditionally, prematurity is defined as a preterm birth of less than days of gestation in the horse. given the variability of gestation length in the horse, ranging from days to more than days in some mares, a mare with a usual gestation length of days possibly could have a term foal at days, whereas a mare with a usual gestation length of days may have a premature foal at days, considered the normal gestation length. foals that are born postterm but are small are termed dysmature; a postmature foal is a postterm foal that has a normal axial skeletal size but is thin to emaciated. dysmature foals may have been classified in the past as small for gestational age and are thought to have suffered placental insufficiency, whereas postmature foals are usually normal foals that have been retained too long in utero, perhaps because of an abnormal signaling of readiness for birth, and have outgrown their somewhat aged placenta. postmature foals become more abnormal the longer they are maintained, also may suffer from placental insufficiency, and are represented best by the classic foal born to a mare ingesting endophyteinfested fescue. box - compares the characteristics of premature/dysmature foals with those of postmature foals. the causes of prematurity/dysmaturity/postmaturity include the causes of high-risk pregnancy presented in box - . additional causes include iatrogenic causes such as early elective induction of labor based on inaccurate breeding dates or misinterpretation of late-term colic or uterine bleeding as ineffective labor. most causes remain in the category of idiopathic, with no discernible precipitating factor. despite lack of an obvious cause, premature labor and delivery does not just happen, and even if undetermined, the cause may continue to affect the foal in the postparturient period. all body systems may be affected by prematurity, dysmaturity, and postmaturity, and thorough evaluation of all body systems is necessary. respiratory failure is common in these foals, although the cause usually is not surfactant deficiency. immaturity of the respiratory tract, poor control of respiratory vessel tone, and weak respiratory muscles combined with poorly compliant lungs and a greatly compliant chest wall contribute to respiratory failure in these cases. most require oxygen supplementation and positional support for optimal oxygenation and ventilation. one must extend effort to maintain these "floppy foals" in sternal recumbency. some foals may require mechanical ventilation. these foals also require cardiovascular support but are frequently unresponsive to commonly used pressors and inotropes: dopamine, dobutamine, epinephrine, and vasopressin. careful use of these drugs and judicious intravenous fluid therapy are necessary. the goal should not be one of achieving specific pressure values (e.g., mean arterial pressure of mm hg) but of adequate perfusion. renal function, reflected in low urine output, is frequently poor initially in these cases because of delay in making the transition from fetal to neonatal glomerular filtration rates. the delay can result from true failure of transition or from hypoxic/ischemic insult. one should approach fluid therapy cautiously in these cases; initial fluid restriction may be in order to avoid fluid overload. many premature/dysmature/postmature foals have suffered a hypoxic insult and have all of the disorders associated with pas, including hie. treatment is similar to that of term foals with these problems. these foals also are predisposed to secondary bacterial infection and must be examined frequently for signs consistent with early sepsis or nosocomial infection. the gastrointestinal system of these foals is not usually functionally mature, which may result from a primary lack of maturity or from hypoxia. dysmotility and varying degrees of necrotizing enterocolitis are common. one commonly encounters hyperglycemia and hypoglycemia. hyperglycemia generally is related to stress, increased levels of circulating catecholamines, and rapid progression to gluconeogenesis, whereas hypoglycemia is associated with diminished glycogen stores, inability to engage gluconeongenesis, sepsis, and hypoxic damage. immature endocrine function is present in many of these foals, particularly regarding the hypothalamic-pituitary-adrenal axis, and contributes to metabolic derangements. , one should delay enteral feeding when possible until the foal is stable regarding metabolic and cardiorespiratory parameters. on intiating enteral feeding, one should provide small volumes initially and slowly increase the volume over several days. one frequently encounters musculoskeletal problems, particularly in premature foals, that include significant flexor laxity and decreased muscle tone. postmature foals frequently are affected by flexure contracture deformities, most likely because of decreased intrauterine movement as they increase in size. premature foals frequently exhibit decreased cuboidal bone ossification that predisposes them to crush injury of the carpal and tarsal bones if weight bearing is not strictly controlled. physical therapy in the form of standing and exercise is indicated in the management of all these problems, but one should take care to ensure that the patient does not fatigue or stand in abnormal positions. bandaging of the limbs is contraindicated because this only increases laxity, although light bandages over the fetlock may be necessary to prevent injury to that area if flexor laxity is severe. the foals are predisposed to angular limb deformity and must be observed closely and frequently for this problem as they mature. the overall prognosis for premature/dysmature/ postmature foals remains good with intensive care and good attention to detail. many of these foals (up to %) survive and become productive athletes. complications associated with sepsis and musculoskeletal abnormalities are the most significant indicators of poor athletic outcome. the last years have seen an explosion of new therapeutic agents purportedly useful for treating sepsis. unfortunately, clinical trials investigating these new therapies have failed to demonstrate a positive effect, have shown negative results, or have resulted in diametrically opposed study results, one showing a benefit and another showing no benefit or a detrimental effect. on a positive note, the survival rate of foals being treated for sepsis has improved. work was done regarding foal diseases and their treatment in the s, but the field did not attract much serious attention until the s. since that time almost every major veterinary college and many large private referral practices have constructed nicus or their equivalent. next to hypoxic ischemic asphyxial syndromes, sepsis is the number one reason for presentation and treatment at these facilities. neonatal septicemia of the horse has been the subject of three international workshops, - and a perinatology lecture covering some aspect of neonatal sepsis has been presented at almost every large continuing education meeting attended by equine veterinarians. concensus criteria conferences in the early s defined sepsis and septic shock for human beings. , sepsis was defined as the systemic response to infection manifested by two or more of the following conditions as a result of infection: a) temperature > °c or < °; b) heart rate > beats/min; c) respiratory rate > breaths per minute or paco < torr; and d) white blood cell count > , cell/µl, < , cell/µl, or > % immature (band) forms. septic shock was defined as sepsis induced hypotension or the requirement for vasopressors/ionotropes to maintain blood pressure despite adequate fluid resuscitation along with the presence of perfusion abnormalities that may include lactic acidosis, oliguria, or acute alteration in mental status. these definitions are broadly acceptable and applicable to neonatal sepsis in foals, and many of the treatment modalities in human medicine have been applied in some manner to the equine neonatal patient. additional definitions that have come into vogue that are actually useful at times, include the following: sirs, the systemic inflammatory response system; mods, multiple organ system dysfuction; and mofs, multiple organ failure syndrome. (sirs is sick, mods is sicker, and mofs is dying.) the compensatory response syndrome (cars) ideally balances sirs and keeps it from becoming detrimental. if balance is achieved, recovery is possible. imbalance progresses to septic shock, mods, and mofs. in horses, mods is manifested most commonly as renal failure, hepatic failure, central nervous system dysfunction, and disseminated intravascular coagulation. managing the septic patient involves early recognition of all the potential alphabet combinations and supporting the patient or intervening in the face of multiple clinical consequences, termed chaos (cardiovacular compromise; homeostasis; apoptosis; organ dysfunction; suppression of the immune system). inflammatory mediators are involved in all these processes and can be beneficial or detrimental, depending on timing and opposing responses. neutrophils, platelets, lymphocytes, macrophages, and endothelial cells are involved, and the implicated inflammatory molecules grow daily in numbers. sepsis in the foal initially can be subtle, and the onset of clinical signs varies depending on the pathogen involved and the immune status of the foal. for the purposes here, the discussion is limited to bacterial sepsis, but the foal also is susceptible to viral and fungal sepsis, which can appear similar to bacterial sepsis. failure of passive transfer (fpt) of immunity can contribute to the development of sepsis in a foal at risk. , testing for and treating fpt has received attention in the veterinary literature. it remains true, however, that foals presented to nicus that have an ultimate diagnosis of sepsis have fpt. , the current recommendation is that foals have igg levels greater than or equal to mg/dl for passive transfer to be considered adequate. other risk factors for the development of sepsis include any adverse advents at the time of birth, maternal illness, or any abnormalities in the foal. although the umbilicus frequently is implicated as a major portal of entry for infectious organisms in the foal, the gastrointestinal tract may be the primary site of entry. other possible portals of entry include the respiratory tract and wounds. early signs of sepsis include depression, decreased suck reflex, increased recumbency, fever, hypothermia, weakness, dysphagia, failure to gain weight, increased respiratory rate, tachycardia, bradycardia, injected mucous membranes, decreased capillary refill time, shivering, lameness, aural petechia, and coronitis. if sepsis is recognized early, patients with sepsis may have a good outcome, depending on the pathogen involved. gram-negative sepsis remains the most commonly diagnosed, but increasingly gram-positive septicemia is being recognized. foals in intensive care units and at referral hospitals have an additional risk of nosocomial infection. an attempt to isolate the organim involved early in the course of the disease becomes important. if possible, one should obtain blood cultures, and if localizing signs are present, one should obtain samples as deemed appropriate. cultures should be aerobic and anaerobic. recently, work has been done evaluating real-time polymerase chain reaction technology in sepsis in the foal as a means of identifying causative organisms. , until one obtains antimicrobial sensitivity patterns for the pathogen involved, one should initiate broad-spectrum antimicrobial therapy (table - ). intravenously administered amikacin and penicillin are good first-line choices, but one should monitor renal function closely. other first-line antimicrobial choices might include high-dose ceftiofur sodium or ticarcillin/clavulanic acid. one should treat failure of passive transfer if present. one should provide intranasal oxygen insufflation at to l/min even if hypoxemia is not present to decrease the work of breathing and provide support for the increased oxygen demands associated with sepsis. should arterial blood gas analysis reveal significant hypoventilation, one may administer caffeine orally or per rectum to increase central respiratory drive. mechanical ventilation may be necessary in cases of severe respiratory involvement such as with acute lung injury or acute respiratory distress syndrome. if the foal is hypotensive, one may administer pressor agents or inotropes by constant rate infusion (table - ) . inotrope and pressor therapy generally is restricted to referral centers where these drugs can be given as constant rate infusions and blood pressure can be monitored closely. some practitioners use nonsteroidal antiinflammatory agents and, in specific circumstances, corticosteroids. use of these drugs should be judicious because they may have several negative consequences for the foal including renal failure and gastric/dunodenal ulceration. [ ] [ ] [ ] nursing care is one of the most important aspects of treating septic foals. foals should be kept warm and dry. they should be turned at -hour intervals if they are recumbent. feeding septic foals can be a challenge if gastrointestinal function is abnormal, and total parenteral nutrition may be needed. if at all possible, foals should be weighed daily and blood glucose levels monitored frequently. some foals become persistently hyperglycemic on small glucose infusion rates. these foals may benefit from constant rate low-dose insulin infusions (table - ) . recumbent foals must be examined frequently for decubital sore development, the appearance of corneal ulcers, and for heat and swelling associated with joints and physis. the prognosis for foals in the early stages of sepsis is fair to good. once the disease has progressed to septic shock the prognosis decreases, although short-term botulism is a neuromuscular disease of foals characterized by flaccid paralysis. although the disease is discussed in detail elsewhere in this text, the form most commonly observed in foals, the toxicoinfectious form, deserves some specific comments. the causative organism is clostridium botulinum, an anaerobic organism. although affected adults usually acquire the disease by ingestion of preformed toxin elucidated from the organism, in the foal less than months of age the organism can survive and multiply in the gastointestinal tract and produce necrotic foci within the liver, giving the foal constant exposure to newly formed toxin. the horse is exquisitely sensitive to the toxin, and only small quantities of toxin are required to produce clinical signs and death in affected animals. the ε-toxin of c. botulinum binds to the presynaptic membrane of motor neurons and prevents transmission of impulses by blocking the release of acetylcholine from the presynaptic vessicles. this block produces the clinical signs of muscle weakness, manifested in foals as trembling (shaker foals) or acute recumbency. pupillary dilation, dysphagia, tremors, recumbency, and terminal respiratory distress caused by respiratory muscle paralysis occur. foals can be found acutely dead. in endemic areas (the northeast and mid-atlantic regions of united states), for these foals to be evaluated first as having colic is not unusual. treatment aims to neutralize the toxin by administration of botulinum antitoxin and to provide antimicrobial treatment of the infection with penicillin, metronidazole, and/or oxytetracycline. , at a minimum, feeding of milk replacer via indwelling nasogastric tube at % of the body weight of the foal per day divided into every -hour meals is required. many of these foals require respiratory support (in the form of intranasal oxygen insufflation), because of respiratory muscle paralysis. respiratory acidosis is present on arterial blood gas analysis in most of these foals because of hypoventilation and lateral recumbency, but they can tolerate some degree of hypercapnia (paco ~ mm hg) if the ph is normal and oxygenation (pao > mm hg; percent oxygen saturation of hemoglobin, > %) is adequate. metabolic alkalosis can accompany the respiratory acidosis, but this is a compensatory change and resolves once gas exchange is normalized. some of these patients require mechanical ventilation, which may be lifesaving. one may discontinue mechanical ventilation as clinical signs resolve and the respiratory muscles gain strength. nursing care is important, and these foals should be turned every hours. they should be maintained in sternal recumbency if possible and kept warm and dry. with good nursing care, good nutritional support, and adequate respiratory support, the prognosis for these foals is good. the limiting factor in the prognosis for life is often financial. foals that recover from the acute stage of this disease eventually fully recover. botulism is an expensive disease to treat and is also an entirely preventable disease. , all pregnant mares in endemic areas should be vaccinated against c. botulinum. vaccination does not prevent all cases of botulism, particularly if the foal has failure of passive transfer or acquires the disease after maternal immunity wanes and before its own vaccination. nutritional muscular dystrophy or white muscle disease is a vitamin e/selenium-responsive muscle disease of horses of all ages probably caused by a dietary deficiency of selenium and vitamin e. the condition occurs most commonly in geographic areas with low selenium levels in the soil, generally the northeastern, northwestern, great lakes and mid-atlantic regions of the united states. two forms of the disease are described in foals: the fulminant form, in which the foal is found acutely dead, and the subacute form. in the fulminant form, death usually is attributed to myocardial lesions resulting in cardiovascular collapse. the subacute form is characterized by dysphagia and gait abnormalities primarily caused by stiffness of the muscles of locomotion. paralysis, if present, is not flaccid as in botulism. abnormal function of respiratory muscles may complicate the clinical situation. aspiration pneumonia may be present following problems associated with swallowing; the tongue and pharyngeal muscles frequently are affected in the early stages of disease. foals with severe disease may have widespread muscle necrosis leading to hyperkalemia, which can be severe and result in death of the foal. serum activities of the muscle enzymes creatine kinase and aspartate aminotransferase may be greatly increased. diagnosis is confirmed at necropsy or ante mortem by determination of decreased vitamin e, selenium, and glutathione peroxidase concentrations in the blood of the foal before supplementation. myoglobinuria and acute renal failure are not uncommon in these foals. treatment of foals with nutritional muscular dystrophy is primarily supportive. one should address all metabolic abnormalities. some foals require intranasal oxygen insufflation. affected foals are unable to suck effectively, and one should provide enteral (via an indwelling nasogastric tube) or parenteral nutritional support. because of the high likelihood of aspiration pneumonia, one should administer broad-spectrum antimicrobial therapy parenterally. the patient should be kept quiet and should be stimulated minimally. affected foals should receive parenteral (intramuscular) vitamin e and selenium supplementation. selenium is toxic in large doses. the prognosis for severely affected foals is guarded. for less severely affected foals the prognosis is good with appropriate treatment. the disease is preventable by ensuring that mares receive sufficient vitamin e and selenium while pregnant and by supplementing foals with parenteral injections of vitamin e and selenium at birth in endemic areas. a more complete discussion of the pathophysiology of this disease and the nutritional management is presented elsewhere in this text. primary liver disease is uncommon in the foal and occurs primarily as a sequela to sepsis. clinical signs of severe liver disease may include depression, ataxia, and seizures. in affected foals, increases in serum liver enzyme activities and concentrations of ammonia and bile acids frequently can be identified. the mechanism(s) underlying hepatoencephalopathy are not delineated clearly, although increased excitatory neurotransmitters, or compounds that mimic their activity, are implicated. hepatoencephalopathy is discussed in more detail elsewhere in this text. tyzzer's disease (clostridium piliformis infection) rarely causes primary liver disease in foals from to about days of age. this disease is almost uniformly fatal. the incubation period is short, and the mare is thought to be the carrier. [ ] [ ] [ ] [ ] [ ] clinical signs range from acute death to depression, fever, and pronounced icterus. the feces of affected foals may appear white to grey because of the lack of bile. clinicopathologic abnormalities include leukopenia, hyperfibrinogenemia, metabolic acidosis, and hypoglycemia. , liver lesions at postmortem are characterized microscopically by multiple foci of necrosis. one usually can demonstrate variable numbers of elongated, slender intracytoplasmic bacilli within hepatocytes bordering the necrotic foci. infiltration of the portal triads with inflammatory cells and biliary duct hyperplasia and degeneration are observable. the bacillus also occurs in association with myocardial lesions. lesions in the intestine are characterised by mucosal necrosis with inflammatory cell infiltration, increased mucus production, submucosal lymphoid hyperplasia, and submucosal hemorrhage. necrosis of lymphoid follicles, congestion, and hemorrhage can be present in the spleen and mesenteric lymph nodes. affected foals may have a profound metabolic acidosis that is unresponsive to treatment. the clinical course is short, and most affected foals die within a few hours of developing neurologic signs. primary liver disease has been reported in association with ferrous sulfate administration in a probiotic compound. the lesion was massive hepatocellular necrosis and liver failure. the product is no longer commercially available. portosystemic shunt is rare in the foal but has been reported in foals as young as months of age. [ ] [ ] [ ] most infectious causes of neurologic abnormalities in foals are associated with sepsis. although rarely reported, halicephalobus gingivalis (deletrix) infection has been reported in three foals; in one case the foal was weeks of age. , possibly transmission in these cases was transmammary; the dam in one case died year later with confirmed h. deletrix infestation of her udder. listeria monocytogenes has been reported as a cause of neurologic disease in foals. recently, sarcocystis neurona was identified as the causative agent of central nervous system disease in a foal, and equine herpes myeloencephalitis has been diagnosed in individual foals and in herd outbreaks involving foals. , neospora also was reported in one foal recently. rhodococcus equi abscesses can form in the central nervous system or cause neurologic signs associated with compression, as with vertebral body abscesses. [ ] [ ] [ ] cerebellar hypolasia, occipitoatlantoaxial malformation, and agenesis of the corpus callosum with cerebellar vermian hypoplasia have been reported in foals. [ ] [ ] [ ] [ ] [ ] [ ] ivermectin toxicity and moxidectin toxicity have been reported. , electrolyte abnormalities such as extreme hypo-or hypernatremia may result in neurologic manifestations of disease. , cervical stenotic myelopathy and degenerative myelopathy also have been reported in foals, although the age at onset is usually more than months. idiopathic epilepsy of arabian foals usually is associated with another infectious disease and is thought to be temporary and self-limiting. causes, diagnosis, and treatment of fpt of immunity are covered in detail elsewhere in this text. failure of passive transfer occurs when a foal fails to ingest a significant quantity of good-quality colostrum. failure of passive transfer may occur by several mechanisms: failure of the foal to suck from the dam for any reason and failure of the dam to produce sufficient quantity of quality colostrum. box - presents causes of fpt. several methods are available for measuring igg concentration in blood; the most reliable are enzyme-linked immunosorbent assay and single radial immunodiffusion technology-based tests. [ ] [ ] [ ] [ ] [ ] [ ] [ ] foals usually are tested at hours of age, but one may test the foal earlier if colostrum ingestion has occurred and a concern exists regarding the passive transfer of immunity status of the foal, recognizing that additional increases in igg concentration may occur with additional time. , the concentration of igg in the blood of the foal has been used as an indicator of the adequacy of passive transfer, but the actual blood concentration at which fpt is diagnosed has been challenged in recent years. [ ] [ ] [ ] foals with sepsis commonly have a serum igg concentration of less than mg/dl. , foals with fpt are more likely to die from sepsis. , , [ ] [ ] [ ] one should consider the igg concentration only as a marker for adequacy of colostral absorption. all the measured igg is unlikely to be directed against the specific pathogen affecting any particular neonate, and igg is not the only immune protection afforded the foal by colostrum. many factors that confer local and more general immunity to the newborn are present in colostrum; these include growth factors, cytokines, lactoferrins, cd , leukocytes, and other yet to be described proteins. [ ] [ ] [ ] [ ] [ ] by considering igg a marker of adequacy for passive transfer, similar to γ-glutamyltransferase in calves, the clinician can make choices for replacement that are more beneficial to the patient. after one identifies fpt in a foal, treatment depends on the current condition of the foal and its local environment. foals not presently ill and on well-managed farms with low population density and low prevalence of disease may not require treatment if their igg concentration is between and mg/dl. critically ill neonates with fpt in an equine nicu are by definition ill and in an environment with high disease prevalence. these patients require immediate treatment of fpt and frequent reassessment of their passive immunity status. critically ill foals often fail to demonstrate the expected increase in blood igg concentration based on grams of igg administered per kilogram of body mass compared with healthy, colostrum-deprived foals. , , sick foals also demonstrate a more rapid decline in igg concentration than do healthy foals because they use and catabolize available protein. one may treat foals with fpt by oral or intravenous administration of various products containing igg. one can attempt oral administration of additional colostrum or igg-containing products such as plasma, serum, or lyophilized colostrum in foals less than to hours of age. [ ] [ ] [ ] depending on the age of the foal and the maturity and function of the gastrointestinal tract, this treatment may be effective. many nicus and large breeding farms maintain colostrum banks for this purpose. one should administer plasma intravenously if the foal is not expected to absorb additional colostrum or if the enteral route is unavailable. commercially available hyperimmune plasma products designed for use in foals are available and can be stored frozen. plasma and banked colostrum should be stored in a non-frost-free freezer to minimize protein loss associated with freeze-thaw cycling. one should administer plasma through special tubing with an in-line filter and should monitor patients closely for transfusion reactions. one may use serum and concentrated igg products, but the practitioner should be aware that many of these products focus on igg retention and not on other factors associated with passive transfer of immunity. one should measure igg concentration after transfusion and provide additional plasma as necessary. administration of plasma to critically ill foals without fpt may be beneficial through provision of other factors present in the plasma. in these situations, fresh frozen plasma or fresh plasma may be best, particularly if transfusion of clotting proteins is desired. neonatal isoerythrolysis is a hemolytic syndrome in newborn foals caused by a blood group incompatibility between the foal and dam and is mediated by maternal antibodies against foal erythrocytes (alloantibodies) absorbed from the colostrum. the disease most often affects foals born to multiparous mares and should be suspected in foals less than days of age with clinical signs of icterus, weakness, and tachycardia. a primiparous mare can produce a foal with neonatal isoerythrolysis if she has received a prior sensitizing blood transfusion or has developed placental abnormalities in early gestation that allowed leakage of fetal red blood cells into her circulation. many are the causes of jaundice in newborn foals, including sepsis, meconium impaction, and liver failure, but these usually can be differentiated readily from neonatal isoerythrolysis by measuring the packed cell volume, which is usually less than % in foals with neonatal isoerythrolysis. foals with neonatal isoerythrolysis are born clinically normal then become depressed and weak and have a reduced suckle response within to hours of birth. the rapidity of onset and severity of disease are determined by the quantity and activity of absorbed alloantibodies. affected foals have tachycardia, tachypnea, and dyspnea. the oral mucosa is initially pale and then becomes icteric in foals that survive to hours. hemoglobinuria may occur. seizures caused by cerebral hypoxia are a preterminal event. the salient laboratory findings are anemia and hyperbilirubinemia. most of the increased bilirubin is unconjugated, although the absolute concentration of conjugated bilirubin generally is increased well above normal. urine may be red to brown and is positive for occult blood. the natural development of neonatal isoerythrolysis has several prerequisites. first, the foal must inherit from the sire and express an erythrocyte antigen (alloantigen) that is not possessed by the mare. blood group incompatibility between the foal and dam is not particularly uncommon, but most blood group factors are not strongly antigenic under the conditions of exposure through previous parturition or placental leakage. factor aa of the a system and factor qa of the q system are highly immunogenic, however, and nearly all cases of neonatal isoerythrolysis are caused by antibodies to these alloantigens. the exception is in the case of mule foals in which a specific donkey factor has been implicated. [ ] [ ] [ ] mares that are negative for aa or qa or both are considered to be at risk for producing a foal with neonatal isoerythrolysis. the risk involves approximately % and % of thoroughbred and standardbred mares, respectively. second, and perhaps most important, the mare must become sensitized to the incompatible alloantigen and produce antibodies to it. the mechanism for this is not known in many instances but generally is believed to result from transplacental hemorrhage during a previous pregnancy involving a foal with the same incompatible blood factor. sensitization via transplacental contamination with fetal erythrocytes earlier in the current pregnancy is possible, but an anamnestic response is generally necessary to induce a pathogenic quantity of alloantibodies. ten percent of thoroughbred mares and % of standardbred mares have antibodies to the ca blood group antigen without known exposure to erythrocytes. some common environmental antigen is postulated possibly to lead to production of anti-ca antibodies. data suggest that these natural antibodies may suppress an immune response to other blood group antigens because mares negative for aa that have anti-ca antibodies often do not produce antibodies to aa of the erythrocytes in their foals that also contain ca antigen. this antibodymediated immunosuppression is thought to result from the destruction of fetal cells before the dam mounts an immune response to other cell surface antigens. natural alloantibodies have not been associated with neonatal isoerythrolysis in horses. after the mare becomes sensitized to the erythrocytes of her foal, alloantibodies are concentrated in the colostrum during the last month of gestation. unlike the human neonate, which acquires alloantibodies in utero and thus is born with hemolytic disease, the foal is protected from these antibodies before birth by the complex epitheliochorial placentation of the mare. thus the final criterion for foal development of neonatal isoerythrolysis is ingestion in the first hours of life of colostrumcontaining alloantibodies specific for foal alloantigens. immunoglobulin-coated foal erythrocytes are removed prematurely from circulation by the mononuclear phagocyte system or are lysed intravascularly via complement. the rapidity of development and severity of clinical signs are determined by the amount of alloantibodies that was absorbed and their innate activity. alloantibodies against aa are potent hemolysins and generally are associated with a more severe clinical syndrome than antibodies against qa or other alloantigens. the highest alloantibody titers are likely to be produced by mares that were sensitized in a previous pregnancy and then subsequently reexposed to the same erythrocyte antigen during the last trimester of the current pregnancy. prior sensitization of a mare by blood transfusion or other exposure to equine blood products may predispose to neonatal isoerythrolysis. one can make a tentative diagnosis of neonatal isoerythrolysis in any foal that has lethargy, anemia, and icterus during the first days of life. blood loss anemia caused by birth trauma is attended by pallor. icterus caused by sepsis or liver dysfunction would not be associated with anemia. one must base the definitive diagnosis of neonatal isoerythrolysis on demonstration of alloantibodies in the serum or colostrum of the dam that are directed against foal erythrocytes. the most reliable serodiagnostic test for neonatal isoerythrolysis is the hemolytic cross-match using washed foal erythrocytes, mare serum, and an exogenous source of absorbed complement (usually from rabbits). although this test is impractical in a practice setting, a number of qualified laboratories routinely perform this diagnostic service. the direct antiglobulin test (coombs' test) may demonstrate the presence of antibodies on foal erythrocytes; however, false negatives occur frequently. most human or veterinary hematology laboratories can perform routine saline agglutination cross-match between mare serum and foal cells. because some equine alloantibodies act only as hemolysins, agglutination tests may be falsely negative. most field screening tests of colostrum have not proved to be reliable enough for practical use. if one recognizes neonatal isoerythrolysis when the foal is less than hours old, one must withhold the dam's milk and feed the foal an alternative source of milk during the first day of life. one can accomplish this by muzzling the foal and feeding it via nasogastric tube. the minimum necessary amount of milk is % of body mass every hours (e.g., a -kg foal should receive ml or pint of mare's milk or milk replacer every hours). the udder of the mare should be stripped regularly (at least every hours) and the milk discarded. in most instances, clinical signs are not apparent until after the foal is hours old, when colostral antibodies have been depleted or the absorptive capacity of the foal's intestine for immunoglobulin has diminished. withholding milk at this point is of minimal benefit. supportive care to ensure adequate warmth and hydration is paramount. the foal should not be stressed and exercise must be restricted. confining the mare and foal to a box stall is a best. intravenous fluids are indicated to promote and minimize the nephrotoxic effects of hemoglobin and to correct any fluid deficits and electrolyte and acid-base imbalances. antimicrobials may be necessary to prevent secondary infections. one should monitor foals carefully for the necessity of blood transfusion, although transfusion should be used only as a lifesaving measure. when the packed cell volume drops below %, blood transfusion is warranted to prevent life-threatening cerebral hypoxia. erythrocytes from the dam are perfect in terms of nonreactivity with the blood of the foal; however, the fluid portion of the blood of the mare has to be removed completely from the cells to prevent administration of additional harmful alloantibodies to the foal. one can pellet the erythrocytes of the dam from blood collected in acid-citrate-dextrose solution by centrifugation or gravity and then aseptically draw off the plasma by suction apparatus or syringe and replace it with sterile isotonic ( . %) saline. one thoroughly mixes the cells with the saline and then repeats the centrifugation or sedimentation, followed by aspiration and discarding of the saline. one should perform this washing process at least three times. one then can suspend the packed erythrocytes in an equal volume of isotonic saline for administration. erythrocyte washing by centrifugation is more desirable than gravity sedimentation because antibody removal is more complete and packed cell preparations can be prepared more quickly (each gravity sedimentation requires to hours). packed red blood cells are advantageous in overcoming the problem of volume overload. when equipment or conditions do not allow the safe use of dam erythrocytes, an alternative donor is necessary. because the alloantibodies absorbed by the foal generally are directed against aa or qa and because the latter are highly prevalent among most breeds of horses, a compatible blood donor is difficult to identify. the odds of finding a donor without aa or qa are higher in quarter horses, morgans, and standardbreds than in thoroughbreds and arabians. previously blood-typed individuals negative for aa and qa and free of alloantibodies are optimal. one should give to l of blood or to l of packed erythrocytes over to hours. these allogeneic cells have a short life span and represent a large burden to the neonatal mononuclear phagocyte system, which may cause increased susceptibility to infection. in addition, these cells sensitize the foal to future transfusion reactions. one must measure all potential harm against the benefit in each situation. if a mule foal is the patient, one should not use blood from a female previously bred to a donkey. in cases in which transfusion will be delayed, one cannot identify a compatible donor, or the packed cell volume is so low as to be life-threatening (hemoglobin < mg/dl), one may administer polymerized bovine hemoglobin products at a dose of to ml/kg. one may use dexamethasone ( . mg/kg) to treat peracute neonatal isoerythrolysis if the packed cell volume is less than % and transfusion may be delayed or is not fully compatible, but dexamethasone has detrimental effects on blood glucose regulation in the neonate, and because the antibody in question is of maternal origin, corticosteroid therapy in immunosuppressive doses probably is not indicated. intranasal oxygen insufflation ( to l/min) may be beneficial. most foals with neonatal isoerythrolysis have adequate passive transfer of immunity, but antimicrobial therapy is indicated to protect against secondary sepsis resulting from the compromised condition of the foal. supportive care and good nursing care, including keeping the foal warm and quiet are essential. one should expect the packed cell volume to decline again to days after transfusion. the prognosis for neonatal isoerythrolysis in foals depends on the quantity and activity of absorbed antibodies and is indirectly proportional to the rate of onset of signs. in peracute cases the foal may die before the problem is recognized, whereas foals with slowly progressive signs often live with appropriate supportive care. like most diseases, neonatal isoerythrolysis is much more effectively prevented than treated. any mare that has produced a foal with neonatal isoerythrolysis should be suspect for the production of another affected foal; thus one should provide all subsequent foals with an alternative colostrum source and discard the colostrum of the dam unless she is bred to a stallion with known blood type compatibility. mares negative for aa and qa alloantigens are most at risk of producing affected foals, thus they should be identified by blood-typing. subsequently, breeding of these mares may be restricted to aa-and qa-negative stallions, thus eliminating the possibility of producing an affected foal. in breeds with a high prevalence of aa or qa alloantigens (e.g., thoroughbreds and arabians), a stallion negative for these and suitable based on other criteria may be difficult to identify. if these "at risk" mares are bred as desired, their serum should be screened in the last month of pregnancy for the presence of erythrocyte alloantibodies. one must test mares with low or equivocal titers closer to the time of parturition. if one detects alloantibodies, the colostrum of the dam should be withheld and the foal then should be provided with an alternative colostrum source. maternal alloantibodies to ca do not appear to mediate neonatal isoerythrolysis in foals and actually may be preventive by removing potentially sensitizing cells from the circulation ; therefore one should not deprive foals of colostrum from mares possessing anti-ca antibodies, even when ca is present on their erythrocytes. rarely, the antigens de, ua, pa, and ab have been associated with neonatal isoerythrolysis in foals; however, to consider mares without these alloantigens to be at risk for neonatal isoerythrolysis is not practical. these syndromes recently have been recognized and described within the veterinary literature, although they have been recognized widely in human neonatology for many years. [ ] [ ] [ ] [ ] affected foals demonstrate these hematologic abnormalities within the first week of life, and the mechanism is similar to neonatal isoerythrolysis following ingestion of maternal antibody directed against the platelet or the neutrophil. in general, affected foals are healthy but may demonstrate bleeding tendencies if thrombocytopenia is severe or they may be more susceptible to sepsis. one confirms the diagnosis by appropriate testing for platelet-and neutrophil-associated antibody. one must rule out other causes of neonatal thrombocytopenia and neutropenia, particularly sepsis. foals born to the mare in the future seem likely to be at risk for developing similar problems, and one should treat future foals as one treats neonatal isoerythrolysis foals: prevent sucking from the dam and provide an alternate source of passive immunity in the form of banked colostrum or intravenous plasma. one should provide an alternative nutritional source, such as foal milk replacer, to the foal for the first hours of life and should muzzle the foal while it is in the company of its dam for that period of time. treatment is primarily supportive, but in the case of severe thrombocytopenia, transfusion of platelet-enriched fresh plasma may be indicated. granulocyte colony-stimulating factor has been used in foals with neutropenia, but substantial efficacy has yet to be demonstrated. broad-spectrum antimicrobial therapy may be prudent in cases of alloantibody-associated neutropenia. treatment with immunosuppressive doses of corticosteroids is probably unwarranted, given the increased risk of infection, because the antibody in question is of maternal origin. other specific diseases of the immune system of foals, severe combined immunodeficiency, selective igm deficiency, transient hypogammaglobulinemia, agammaglobulinemia, and other unclassified immunodeficincies are covered in detail elsewhere in this text. the neonate can experience respiratory distress immediately after birth because of several congenital respiratory tract or cardiac anomalies. chief among these causes are bilateral choanal atresia, stenotic nares, dorsal displacement of the soft palate caused by anatomic deformity or neurologic impairment, accessory or ectopic lung lobes, lung lobe hypertrophy, lung lobe dysplasia, cardiac anomalies with right-to-left shunting, and miscellaneous causes such as subepiglotic cysts and severe edema of the larynx. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] one must evaluate and treat these situations immediately and should consider them true emergencies. one readily can recognize foals with airway occlusion by the lack of airflow through the nostrils despite obvious attempts to breathe and by respiratory stridor. these foals may demonstrate open-mouth breathing and their cheeks may puff outward when they exhale. one foal with congenital bilateral choanal atresia was recognized during extrauterine intrapartum resusucitation because of an inability to pass a nasotrancheal tube. one can establish an effective airway by orotracheal intubation in these cases under most circumstances, but some foals require an emergency tracheostomy. one diagnoses the underlying problem by endoscopy or radiography in most cases. treatment of choanal atresia and cystic structures is surgical, whereas severe laryngeal edema and laryngeal paralysis frequently respond to medical management. until the underlying problem is resolved in these cases, one should administer broad-spectrum antimicrobial therapy and feed the foal by intubation or total parenteral nutrition. one can give colostrum, but these foals frequently develop aspiration pneumonia if allowed to suck from their dams, so intravenously administered plasma also may be necessary to provide sufficient passive immunity. arterial blood gas determinations are the most sensitive indicator of respiratory function readily available to the clinician. the most readily available arteries for sampling are the metatarsal arteries and the brachial arteries. portable arterial/venous blood gas analyzers now are making arterial blood gas analysis more practical in the field, and the technique is no longer reserved for large referral practices. managing a critically ill equine neonate without knowledge of arterial blood gas parameters is veritably impossible. pulse oximetry is useful, but these monitors only measure oxygen saturation of hemoglobin. desaturation can occur rapidly in critically ill neonates. the utility of these monitors in the foal has yet to be demonstrated clearly, particularly in cases of poor peripheral perfusion. the most common abnormalities recognized with arterial blood gas analysis are hypoxemia with normo-or hypocapnia and hypoxemia with hypercapnia. hypoxemia is defined as decreased oxygen tension of the arterial blood (decrease pao ), and hypoxia is defined as decreased oxygen concentration at the level of the tissue, with or without hypoxemia. hypoxia results from hypoxemia, decreased perfusion of the tissue bed in question, or decreased oxygen-carrying capacity of the blood resulting from anemia or hemoglobin alteration. five primary means by which hypoxemia may develop are ( ) low concentration of oxygen in the inspired air such as in high altitude or in an error mixing ventilator gas; ( ) hypoventilation; ( ) ventilation/perfusion mismatch; ( ) diffusion limitation; and ( ) intrapulmonary or intracardiac right-to-left shunting of blood. hypoxemia is not an uncommon finding in neonates but must be evaluated in terms of the current age of the foal and its position. , [ ] [ ] [ ] [ ] one also must consider the difficulty encountered in obtaining the sample because severe struggling can affect the arterial blood gas results. table - presents normal arterial blood gas parameters for varying ages of foals. the normal foal has a small shunt fraction (~ %) that persists for the first few days of life and contributes slightly to a blunted response to breathing % oxygen compared with the adult. hypoxemia frequently occurs in foals with prematurity, pas, and sepsis, although other conditions also result in hypoxemia in the neonate. in the early stage of sepsis associated hypoxemia, paco may be within normal limits or decreased if the foal is hyperventilating for any reason. if the lung is involved significantly in the underlying pathologic condition, such as with severe pneumonia, acute lung injury, or acute respiratory distress syndrome, increased paco may well be present, representing respiratory failure. hypoxemia usually is treated with intranasal humidified oxygen insufflation at to l/min. hypercapnia is not a simple matter to treat. one must try to distinguish between acute and chronic hypercapnia. acute hypercapnia usually is accompanied by a dramatic decrease in blood ph of . ph units for each mm hg increase in paco . this acidemia can promote circulatory collapse, particularly in the concurrently hypoxemic and/or hypovolemic patient. the effects of more chronic co retention are less obvious because the time course allows for adaptation. the ph change is less, about . ph units per mm hg increase in paco , because it is balanced by enhanced renal absorption of bicarbonate by the proximal renal tubule. most foals with acute respiratory distress are in the acute stages of respiratory failure, but chronic adaptation begins to occur within to hours and is maximal in to days. one will note an increase in bicarbonate, particularly if the acidemia is primarily respiratory in origin. intravenous administration of sodium bicarbonate to correct respiratory acidosis/ acidemia should be done cautiously in these foals because co retention may only be increased. also, one should remember that meq of sodium is administered with each meq of bicarbonate and hypernatremia has been seen in foals treated exuberantly with sodium bicarbonate. foals with hypercapnia of several days' duration also may develop a blunted respiratory drive to increased co . in these foals, oxygen administration, although essential to treat hypoxemia, may further depress ventilation and further decrease ph. this effect is caused by a loss of hypoxic drive following oxygen therapy. one should consider these foals candidates for mechanical ventilation if the paco is greater than mm hg or is contributing to the poor condition of the foal, such as causing significant ph changes. if hypercapnia is caused by central depression of ventilation, as frequently occurs in foals with pas, one can administer caffeine ( mg/kg loading dose; then . mg/kg as needed) per rectum or orally in foals with normal gastrointestinal function. other clinicians may recommend continuous rate infusions of doxapram hydrochoride (dopram; mg/total dose at . mg/ kg/min) for these foals. if this therapy fails, one should consider mechanical ventilation. mechanical ventilation of foals with central respiratory depression is rewarding and may be necessary only for a few hours to days. a special category is the foal with botulism exhibiting respiratory failure caused by respiratory muscle paralysis. these foals do well with mechanical ventilation, although the duration of mechanical ventilation is more prolonged, frequently more than week. foals with primary metabolic alkalosis usually have compensatory respiratory acidosis. treatment of hypercapnia is not necessary in these cases because it is in response to the metabolic condition. these foals do not respond to caffeine, and they should not be ventilated mechanically if this is the only disorder present. in the neonate, bacterial pneumonia usually results from sepsis or aspiration during sucking. foals with sepsis can develop acute lung injury or acute respiratory distress syndrome as part of the systemic response to sepsis, and this is frequently a contributor to the demise of foals in septic shock. the best way to diagnose bacterial pneumonia is by cytologic examination and culture of a transtracheal aspirate, but blood culture may aid in early identification of the causative organism and allow for early institution of directed antimicrobial therapy. a second frequent cause of bacterial pneumonia in the neonate is aspiration caused by a poor suck reflex or dysphagia associated with pas, sepsis, or weakness. one must take care to ensure that aspiration is not iatrogenic in foals being bottle fed. auscultation over the trachea while the foal is sucking helps identify occult aspiration. one should suspect occult aspiration pneumonia in any critically ill neonate that is being bottle fed or is sucking on its own that has unexplained fever, fails to gain weight, or has a persistently increased fibrinogen level. older foals develop bacterial pneumonia, frequently following an earlier viral infection. bacterial pneumonia is discussed in depth elsewhere in this text, but a few comments specific to the foal are necessary. one should auscultate and percuss the thorax of the foal, but results may not correlate closely with the severity of disease. the most commonly isolated bacterial organism in foal pneumonia is streptococcus zooepidemicus, and one may isolate it alone or as a component of a mixed infection. [ ] [ ] [ ] transtracheal aspirate for culture and cytologic examination is recommended because mixed gram-positive and gram-negative infections are common, and antimicrobial susceptibility patterns can be unpredictable. one should split the obtained aspirate and submit samples for bacterial culture, virus isolation, and cytologic examination. additional diagnostics include radiography, ultrasonography, and serial determination of white blood cell counts (with differential) and blood fibrinogen concentrations. treatment includes administration of appropriate antimicrobial therapy. some foals may benefit from nebulization with saline or other local products. ascarid larval migration through the lung can mimic bacterial pneumonia. in these cases the foal may not respond to antimicrobial therapy and should be dewormed with ivermectin. deworming the mare within month of parturition and frequent deworming of the foal prevent ascarid migration pneumonia in most foals. a special category of bacterial pneumonia in foals is rhodococcus equi bronchopneumonia. this pneumonia of young foals was described first in . the organism originally was known as corynebacterium equi and is a gram-positive pleomorphic coccobacillus usually less than µm in diameter and µm in length. the organisms frequently are associated in l-and v-shaped clusters that have been termed chinese character formations. r. equi has an acid-fast staining characteristic under some growing circumstances because of the presence of mycolic acid in its cell wall, similar to mycobacterium and nocardia species. mycolic acid promotes granuloma formation. the organism is able to multiply in and destroy macrophages as it prevents phagosome lysosome fusion. , much attention has been paid to this organism in recent years, given its propensity to produce enzootic and epizootic outbreaks of disease. the organism is thought to be primarily an opportunistic pathogen, and it lives in the soil of most geographic areas. foals are affected most frequently between the ages of and months, when maternally derived immunity has begun to wane. the disease is insidious, and foals may have significant pulmonary involvement before developing noticeable clinical signs. phagocytosis of r. equi by equine macrophages is not associated with a functional respiratory burst and, at least in human beings, the l-arginine-no pathway is not required for intracellular killing of this organism. , optimal binding of r. equi to mouse macrophages in vitro requires complement and is mediated by mac- , a leukocyte complement receptor type (cr , cd b/ cd ). opsonisation of r. equi with specific antibody is associated with increased phagosome-lysosome fusion and enhanced killing of r. equi, suggesting that the mechanism of cellular entry is important. neutrophils from foals and adult horses are fully bactericidal, and killing of r. equi is enhanced considerably by specific opsonizing antibody. the ability of r. equi to induce disease in foals likely depends on host and microbial factors. knowledge of the virulence mechanisms of r. equi was speculative until the discovery of the virulence plasmid. as opposed to most environmental r. equi organisms, isolates from clinically affected foals typically contain -to -kb plasmids encoding an immunogenic virulence-associated protein (vapa) that is expressed on the bacterial surface in a temperature-regulated manner. plasmid-cured bacteria lose their ability to replicate and survive in macrophages and are cleared from the lungs within weeks of intrabronchial challenge without producing pneumonia. however, expression of vapa alone is not sufficient to restore the virulence phenotype. six other genes have approximately % overall amino acid identity with vapa, and the identification of multiple genes with considerable homology suggests these genes constitute a virulence-associated gene family in r. equi. other candidates for virulence factors include capsular polysaccharides and cholesterol oxidase, choline phosphohydrolase, and phospholipase c exoenzymes ("equi factors"), but their roles have not been defined clearly. the primary manifestation of disease caused by r. equi infection is severe bronchopneumonia with granuloma, abscess formation, or both. up to % of foals diagnosed with bronchopneumonia also have extrapulmonary sites of infection. as the pneumonia progresses, clinical signs may include decreased appetite, lethargy, fever, tachypnea, and increased effort of breathing characterized by nostril flaring and increased abdominal effort. cough and bilateral nasal discharge are inconsistent findings. a smaller percentage of affected foals may have a more devastating, subacute form. these foals may be found dead or have acute respiratory distress with a high fever and no previous history of clinical respiratory disease. hyperfibrinogenemia is the most consistent laboratory abnormality in foals with r. equi pneumonia. neutrophilic leukocytosis (> , cells/µl), with or without monocytosis, is common. thoracic radiography is a useful diagnostic aid, frequently revealing a prominent alveolar pattern with poorly defined regional consolidation and/or abscessation. ultrasonography is a helpful diagnostic tool when the disease involves peripheral lung tissue. although a number of serologic tests have been described, serologic diagnosis of r. equi infections is controversial and difficult because exposure of foals to this organism at a young age leads to production of antibody without necessarily producing clinical disease. , serologic tests may be more useful at the farm level to detect overall exposure than at the individual level. bacteriologic culture combined with cytologic examination of a tracheobronchial aspirate remains the most definitive method for accurate diagnosis of r. equi pneumonia. however, foals without clinical disease exposed to contaminated environments may have r. equi in their tracheae from inhalation of contaminated dust; therefore one should interpret culture results in the context of the overall case presentation. culture results in one study were as sensitive as polymerase chain reaction-based assays and offered the advantage of allowing in vitro antimicrobial susceptibility testing. however, polymerase chain reaction is likely to be a useful tool, and results from a second trial suggest the assay is more sensitive and specific than culture of tracheobronchial aspirates for diagnosis. the combination of erythromycin and rifampin has become the treatment of choice for r. equi infections in foals, and the combination reduces the likelihood of resistance to either drug. the recommended dosage regimen for rifampin is mg/kg every hours or mg/kg every hours orally. the recommended dose of estolate or ethylsuccinate esters of erythromycin is mg/kg every or hours orally. recently, azithromycin has been recommended for treatment of r. equi infection at a dosage of mg/kg orally every hours for to days and then every other day. alternatively, clarithromycin at . mg/kg every hours orally, in combination with rifampin, may be therapeutically effective. severely affected foals may require intranasal oxygen insufflation, intravenous fluid support, and nutritional support. treatment generally continues for to weeks until all clinical and laboratory evidence of infection is resolved. although well tolerated by most foals, erythromycin can result in soft feces. this diarrhea is generally self-limiting and does not require cessation of therapy, but one should monitor affected foals carefully. an idiosyncratic reaction characterized by severe hyperthermia and tachypnea has been described in foals treated with erythromycin during periods of hot weather. affected foals should be moved to a colder environment and treated with antipyretic drugs and alcohol baths if necessary. clostridium difficile enterocolitis has been reported in the dams of nursing foals treated with erythromycin given orally. the dam is exposed to active erythromycin by coprophagy or by drinking from a communal water source where the foal has "rinsed" its mouth. prevention of r. equi pneumonia on farms with recurrent problems is problematic. the most clearly demonstrated prophylactic measure to date has been the administration of plasma that is hyperimmune to r. equi to foals within the first week of life and then again when maternal immunity begins to wane at around days of age. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] no effective vaccination protocols for the dam or foal have been described to date. farm management is important in preventing disease, and control measures include frequent manure removal, avoidance of overcrowded conditions, and planting of dusty or sandy soils. the prognosis for r. equi bronchopneumonia is fair to good in foals with the more chronic form of the disease. foals with acute respiratory distress have a more guarded prognosis, as do foals with sites of significant extrapulmonary infection. the long-term prognosis for survival for foals with r. equi bronchopneumonia is good, and many foals perform as expected as athletes. the most commonly identified causes of viral pneumonia in foals are equine herpesviruses and (ehv- and ehv- ), equine influenza, and equine arteritis virus (eva). equine herpesvirus is probably the most clinically important, but outbreaks of eva in neonates have occurred and are devastating. , [ ] [ ] [ ] [ ] [ ] [ ] adenovirus is reported sporadically and as a problem in arabian foals with severe combined immunodeficiency. [ ] [ ] [ ] in the neonate, infection with ehv- or eva is almost uniformly fatal and antemortem diagnosis is difficult, even once an outbreak on a particular farm is identified. several factors appear common to foals with ehv- , including icterus, leukopenia, neutropenia, and petechial hemorrhage, but these problems also are identified in foals with severe sepsis. , , the antiviral drug acyclovir ( to mg/kg orally or per rectum to times per day) has been used in cases of ehv- in neonates, with some evidence of efficacy in mildly affected foals or foals affected after birth. if viral pneumonia is a possibility, one should collect blood and tracheal aspirates at presentation for bacterial and virus isolation. the lungs of foals with ehv- or eva are noncompliant, and pulmonary edema may be present. mechanical ventilation of these cases may prolong life, but death is generally inevitable because of the magnitude of damage to the lungs. foals suspected of having ehv- or eva should be isolated because they may be shedding large quantities of virus and pose a threat to other neonates and pregnant mares. foals with eva generally are born to seronegative mares, and intravenous treatment with plasma with a high titer against eva may prove beneficial because passive immunity appears to have a large role in protection against this disease in neonates. , older foals and weanlings may be affected by herpesviruses. disease is usually mild, although a fatal pulmonary vasculotropic form of the disease has been described recently in young horses. , the clinical signs of disease are indistinguishable from influenza and include a dry cough, fever, and serous to mucopurulent nasal discharge, particularly if secondary bacterial infection occurs. rhinitis, pharyngitis, and tracheitis may be present. treatment of affected foals is primarily supportive. foals also may become infected with ehv- . the predominant clinical signs are fever and lymphoid hyperplasia with pharyngitis. , diagnosis is by virus isolation. rib fractures have been recognized in % to % of all neonatal foals and can be associated with respiratory distress. potential complications of rib fractures include fatal myocardial puncture, hemothorax, and pneumothorax. rib fractures frequently are found during physical examination by palpation of the ribs or by auscultation over the fracture sites. one can confirm the diagnosis by radiographic and ultrasonographic evaluation. often multiple ribs are affected on one side of the chest. specific treatment is generally unnecessary, but direct pressure on the thorax should be avoided in all cases. some specific patients may benefit from surgical stabilization of some fractures, particularly those fractures overlying the heart. pneumothorax can occur spontaneously or following excessive positive pressure ventilation or following tracheostomy surgery or trauma. any foal being ventilated mechanically that suddenly has respiratory distress and hypoxemia should be evaluated for pneumothorax. diagnosis is by auscultation and percussion of the thorax, but one can confirm the diagnosis with radiographic and ultrasonographic evaluation of the thorax. needle aspiration of air from the pleural space also confirms the diagnosis. treatment is required in cases in which clinical signs are moderate to severe or progressive and involves closed suction of the pleural space. subcutaneous emphysema can complicate treatment of this problem. idiopathic or transient tachypnea has been observed in clydesdale, thoroughbred, and arabian breed foals. in human infants, transient tachypnea can be related to delayed absorption of fluid from the lung, perhaps because of immature sodium channels. in foals, tachypnea generally occurs when conditions are warm and humid and is thought to result from immature or dysfunctional thermoregulatory mechanisms. clinical signs of increased respiratory rate and rectal temperature develop within a few days of birth and may persist for several weeks. treatment involves moving the foal to a cooler environment, body clipping, and provision of cool water or alcohol baths. these foals frequently are treated with broad-spectrum antimicrobial drugs until infectious pneumonia can be ruled out. a syndrome of bronchointerstitial pneumonia and acute respiratory distress has been described in older foals and appears to be a distinct entity from acute respiratory distress syndrome in neonatal foals in association with sepsis. the underlying cause has not been identified, but the genesis is probably multifactorial with several potential pathogens being implicated. affected foals have acute respiratory distress with significant tachypnea, dyspnea, nostril flare, and increased inspiratory and expiratory effort. auscultation reveals a cacophony of abnormal sounds including crackles and polyphonic wheezes in all lung fields. loud bronchial sounds are audible over central airways, and bronchovesicular sounds are lost peripherally. affected foals are cyanotic, febrile, and unwilling to move or eat. foals may be found acutely dead. laboratory abnormalities include leukocytosis, hyperfibrinogenemia, and hypoxemia with hypercapneic acidosis. foals can be dehydrated severely and have coagulation changes consistent with disseminated intravascular coagulation. hypoxic injury to other organs, primarily the kidneys and liver, can occur. chest radiographs reveal a prominent interstitial pattern overlying a bronchoalveolar pattern that is distributed diffusely throughout the lung. this syndrome is a respiratory emergency. treatment is broad-based and includes administration of oxygen, nonsteroidal antiinflammatory agents, broad-spectrum antimicrobial therapy, nebulization, judicious intravenous fluid therapy, nutritional support, and corticosteroid therapy. one must manage hyperthermia in the foal. corticosteroid therapy appears to have been lifesaving in most of the reported surviving foals. because this syndrome is associated with high environmental temperatures in some areas, prevention involves control of ambient temperatures, not transporting foals during hot weather, and keeping foals out of direct sun on hot days, particularly foals being treated with erythromycin for suspected or confirmed r. equi infection. uroperitoneum has been recognized as a syndrome in foals for more than years. , classically, affected foals are to hours old at the time clinical signs first are recognized. [ ] [ ] [ ] previous reports had a proportionately larger affected male than female population. , , the hypothesis was that colts were more at risk because their long, narrow, high-resistance urethra was less likely to allow bladder emptying, resulting in rupture of a full bladder during parturition when high pressures were applied focally or circumferentially around the bladder. more recent reports suggest that such extreme sex bias may have been an artifact of small case numbers in the early reports. rupture or disruption of any structure of the urinary tract can occur. the dorsal wall of the bladder has been reported to be a frequent disruption site, with the ventral wall less likely to be involved. the urachus appears to be the next most commonly affected structure. a few cases of ureteral and urethral defects have been reported. , sepsis does not appear to favor one site over the others. the pathophysiology of uroperitoneum is not yet understood fully. the high pressure exerted on a full bladder during parturition once was thought to be the main cause. full bladder and obstruction caused by a partial umbilical cord at parturition, strenuous exercise, and external trauma have been reported as causes. a few reports describe smooth and noninflamed edges of torn tissue, suggesting the possibility of congenital bladder wall defects. , , sepsis leading to urinary tract rupture and uroperitoneum may occur in foals hospitalized for a variety of unrelated problems. the onset of clinical signs of uroperitoneum may be insidious in these foals, and diagnosis may be less obvious. clinical signs associated with uroperitoneum in the neonatal foal typically include straining to urinate, dribbling urine, and a stretched-out stance. weakness, tachycardia, tachypnea, and not sucking well are also common. a distended abdomen may be evident, and one may feel a fluid wave on ballottement of the abdomen. occasionally, urine accumulates in the scrotum and should not be confused with hernia. foals also may show signs of sepsis, including fever, injected mucous membranes, diarrhea, and disease of other body systems. laboratory findings vary depending on the duration of the uroperitoneum and on the presence and severity of sepsis. classic findings include hyperkalemia, hyponatremia, and hypochloremia arising from equilibration of urine electrolytes and water with blood across the peritoneal membrane. [ ] [ ] [ ] the usual foal diet of milk, which is high in potassium and low in sodium, promotes the electrolyte abnormalities. foals that develop uroperitoneum while receiving intravenous fluids may not have classic electrolyte imbalances at the time clinical signs are recognized. increased serum creatinine concentration is often present, whereas blood urea nitrogen concentrations occasionally, but not consistently, are increased. [ ] [ ] [ ] metabolic acidosis and hypoxemia may be present. some patients also have serum hypoosmolality. one should test foals for failure of passive transfer. one of the most sensitive laboratory tests for uroperitoneum is the ratio of peritoneal to serum creatinine. a ratio greater than or equal to : is considered diagnostic of uroperitoneum. one should collect peritoneal fluid and test it for creatinine concentration, as well as for cytologic findings, culture, and sensitivity. cytologic evaluation of peritoneal fluid is necessary to identify concurrent peritonitis or other gastrointestinal compromise. one should perform an electrocardiogram on initial evaluation of a foal with suspected uroperitoneum because hyperkalemia may result in bradycardia, increased duration of the qrs complex, a shortened q-t interval, increased p-wave duration, prolonged p-r interval, or atrioventricular conduction disturbances. other possible cardiac sequelae to hyperkalemia include cardiac arrest, third-degree atrioventricular block, ventricular premature contractions, and ventricular fibrillation. , for any foal exhibiting signs of dypsnea, tachypnea, or hypoxemia, one should have thoracic radiographs taken before induction of anesthesia to rule out pleural effusion, pneumonia, or acute respiratory distress syndrome, which could complicate ventilation and oxygenation during anesthesia and the postoperative period. ultrasonography has become the tool of choice in the diagnosis of uroperitoneum and is a useful tool available to the practitioner. one can image free peritoneal fluid readily, and tears within the bladder are readily visible. the empty bladder with a significant defect, in a fluid-filled abdomen, will collapse on itself and often have a u shape. one also can visualize urachal and urethral lesions. six of eight foals in one study had urinary tract lesions identified sonographically, and all foals of another study underwent sonographic evaluation, and a significant correlation between ultrasonographic findings and location of the lesion at surgery existed. , initial treatment aims to stabilize the patient and correct any electrolyte and acid-base abnormalities and provide fluid volume replacement. one should use . % or . % saline with % dextrose until laboratory data are available. a potassium concentration of greater than . meq/l can be life threatening. one can manage hyperkalemia by peritoneal drainage to decrease whole-body potassium stores using teat cannulae, foley catheters, large-gauge ( or ) intravenous catheters, or human peritoneal dialysis catheters. fluid replacement at least should equal the amount of fluid removed from the abdomen to prevent acute hypotension caused by expansion of previously collapsed capillary beds. abdominal drainage also helps ventilation and decreases the work of breathing by decreasing pressure on the diaphragm. one may administer calcium gluconate, glucose, sodium bicarbonate, or insulin intravenously to decrease serum potassium concentrations. these maneuvers do not correct the whole-body potassium overload, however, and once therapy is discontinued, hyperkalemia can reappear until the urine is removed from the abdomen. one should correct hyponatremia slowly. because of the real possibility of concurrent sepsis, one should obtain blood cultures before preoperative administration of antimicrobials. broad-spectrum coverage (penicillin and amikacin or ceftiofur sodium) is recommended until culture results become available. one should perform therapeutic drug monitoring when using aminoglycoside therapy. however, the peak value may be depressed because of the increased volume of distribution represented by the volume of urine in the abdomen, so one should not make dose adjustment based on a low peak until obtaining a new peak after surgical correction of the uroperitoneum. one should treat foals with failure of passive transfer with adequate volumes of intravenously administered plasma. after one has addressed the metabolic abnormalities, one may consider surgical management. medical management using an indwelling foley catheter has been described. preoperative medical stabilization reduces anesthetic risk. safer inhalant agents such as isoflurane also have decreased risk. removal of the internal umbilical remnant at the time of surgery is usual. one should consider culturing any removed umbilical remnant and submitting the remnant for histopathologic evaluation. recurrence of urinary tract rupture can occur. sepsis, hypoxemia, pneumonia, peritonitis, and acute respiratory distress syndrome complicate the management of uroperitoneum. many affected foals are persistently oxygen dependent for several days following surgical correction, and one should perform serial arterial blood gas analyses before discontinuing intranasal oxygen supplementation. prognosis is associated closely with concurrent illness, especially septicemia. uncomplicated uroperitoneum from a defect in the bladder has a good prognosis. if the location of the lesion is other than the bladder, the prognosis is not as favorable. foals with septicemia have a much poorer prognosis. , acute renal failure most often occurs as a complication of prenatal asphyxial syndrome, sepsis, or aminoglycoside therapy. acute renal failure also has been reported following oxytetracycline administration in foals. the dose of oxytetracycline commonly used to treat flexural deformities in foals is approximately times the antimicrobial dose. many foals treated in this manner also have suffered some degree of perinatal asphxia, which also damages the kidney, because of prolonged parturition precipitated in part by the flexural deformity. evaluation of renal function in these foals before the administration of the first dose of oxytetracycline and continued monitoring of serum creatinine concentrations before administering subsequent doses of this nephrotoxic compound would seem reasonable. hemodialysis has been used as therapy in one of these cases, but prevention is important because these foals may fail to respond to usual therapy for oliguric renal failure and are euthanized. the most commonly reported congenital deformity of the kidney of the foal is renal hypoplasia and dysplasia, which may have a heritable component. , renal arteriovenous malformations have been reported also. ectopic ureters and fenestrated ureters have been described in the foal. [ ] [ ] [ ] congenital renal defects, among others, were reported in three weak, recumbent neonatal foals born to mares being treated for equine protozoal myeloencephalitis. mares received sulfadiazine or sulfamethoxazoletrimethoprim, pyrimethamine, folic acid, and vitamin e orally. the foals were anemic, leukopenic, azotemic, hyponatremic, and hyperkalemic. serum folate concentrations were lower than those reported in the literature for clinically normal brood mares. treatment was unsuccessful. necropsy revealed lobulated kidneys with thin cortices and a pale medulla. the authors postulated that oral administration of sulfonamides, , -diaminopyrimidines (pyrimethamine with or without trimethoprim), and folic acid to mares during pregnancy is related to congenital defects in newborn foals. the umbilicus serves as the conduit for nutrition and gas exchange between the dam and the fetal foal. the urine from the foal is expelled via this structure into the allantoic cavity. the author has recognized cases of in utero bladder distention in the fetus that were associated with multiple twists decreasing urine flow or focal stenosis creating the same effect. foals born with this condition did not have bladder rupture associated with parturition but did have other severe abnormalities that eventually resulted in their demise, primarily premature delivery with failure to adapt to extrauterine life (p.a. wilkins, j.e. palmer, and f.t. bain, unpublished data). at birth the umbilicus breaks, leaving a small external remnant and a large internal remnant. the umbilicus long has been regarded as the primary site of entry of pathogens into the neonate, although this has been challenged recently. treatment of the umbilicus after birth involves dipping it (preferably just the most distal component) with various caustic compounds. the most current recommendation is to treat the umbilicus with dilute chlorhexidine, povidone-iodine, or dilute iodine solutions for just a few times following birth. exhuberant treatment of the umbilical stump with caustic solutions can lead to scalding of the ventral abdomen and may promote patency of the urachus. the ultrasonographic appearance and measurements of the umbilical arteries, urachus, and umbilical vein of foals from hours to weeks of age have been described in detail. a . -mhz sector scanner transducer placed across the midline of the ventral portion of the abdominal wall of the foal works best because of the superficial location of these structures. the mean (± sd) diameter of the umbilical vein was . ± . cm immediately cranial to the umbilical stalk, . ± . cm midway between the umbilicus and liver, and . ± . cm at the liver. the urachus and umbilical arteries of normal foals have a mean total diameter of . ± . cm at the bladder apex. the umbilical arteries scanned along either side of the bladder have a mean diameter of . ± . cm. one can use these measurements and the ultrasonographic appearance of the internal umbilical structures from clinically normal foals as references to diagnose abnormalities of the umbilical structures in neonatal foals. , the most common abnormalities of these structures are focal abscess formation, hematoma, and urachal tear. herniae traditionally have been thought to develop from failure of closure at the umbilical stump after birth. however, the closure of the body wall defect at the umbilicus was studied in relation to the development of umbilical herniae in a large group of normal foals followed from birth until months of age or from birth until months of age. at birth, approximately half of these foals had a defect in the body wall at the umbilicus that was termed a palpable umbilical ring. in foals this defect disappeared within days, but in one foal the ring did not close and a hernial sac with abdominal contents was palpable. this foal was considered to be the only foal to have a truly congenital umbilical hernia. twelve foals developed an umbilical hernia between and weeks of age. the prevalence of umbilical herniae was much higher than in other studies, possibly because of the prospective nature of the study. based on this study, the large majority of umbilical herniae would appear not to result from failure of closure but rather to be acquired after birth. one should consider the palpable ring structure within the body wall at the umbilicus a variant of normal in the foal and should not call it a hernia until the foal is at least month of age. in one study of horses treated for umbilical herniae over a / -year period, only . % developed complications in association with umbilical defects. six horses had intestinal incarceration; the incarceration was reduced manually in horses before admission and resolved without treatment in others. the hernia was surgically reduced in horse. herniorrhaphy was performed on of the horses in which the incarceration did not require surgical reduction, and the fifth was managed conservatively. the study confirmed that complications of umbilical herniae are rare in horses; however, when they do develop, they may be one of various forms, some of which are insidious in onset. the primary differential diagnosis for an external swelling in the umbilical stump region is an external abdominal abscess, which will be firm, variably painful, warm, and nonreducible. ultrasonographic evaluation readily can confirm either possibility. one report describes a -day-old foal that died from intestinal strangulation caused by a remnant of vitelline vein that extended between the umbilicus and the portal vein. patent urachus frequently is recognized in the abnormal neonate, probably because of the increased recumbency and decreased movement of these patients. cauterization of a patent urachus is no longer recommended except in cases that persist for long periods of time (> month) after the foal becomes more active. surgical resection may provide relief in some foals, but most cases resolve without treatment if given enough time. foals with a patent urachus may posture and strain frequently to urinate, some of this may be associated with irritation or local infection of the urachus. one can alleviate this by administration of broad-spectrum antimicrobial therapy such that the drug has a high concentration in the urine (e.g., trimethoprim-sulfa drug combinations) and by oral administration of phenazopyridine hydrochloride (pyridium), a dye that anesthetizes the urinary tract epithelial surfaces (see table - ). this dye turns the urine orange and stains everything yellow-orange that it or the urine touches but can provide a great deal of relief to foals with this problem. the umbilicus has been considered the traditional point of entry of bacteria into the septic neonate, and septic foals have been referred to as having "navel ill" and "joint ill" in the past. although current thought suggests that the gastrointestinal tract may be the route of entry in most septic neonates, infection of the umbilicus-termed omphalitis, or omphalophlebitis if the vessels are involved-still occurs as a single focus of infection or along with more generalized infection. external signs, such as swelling, heat, pain, ventral edema, or purulent discharge may be present in some foals, but more usually external signs are minimal and one suspects infection because of infection in another site (e.g., an infected joint), fever, or otherwise unexplained increased blood fibrinogen concentration. one confirms the diagnosis by ultrasonographic evaluation of the internal umbilical remnant. any of the umbilical structures may be involved. a complete description of the evaluation is available within the relevant veterinary literature, but the examination is performed best with the foal standing using a . -mhz probe with a standoff. the usual finding is that the affected structure is larger than expected. a fluid-filled core and echogeneic shadows consistent with gas may be apparent in some cases. interpretation requires some experience, and the examiner should be familiar with variants of normal, such as gas shadows associated with a patent urachus and enlarged vessels caused by hematoma formation, so that treatment is not initiated inappropriately. two options for treatment are surgical and medical. medical treatment is preferable in cases in which the lesion is well localized and small and in foals with a medical condition that is not amenable to anesthesia and surgical intervention. one should institute broad-spectrum antimicrobial therapy, and one may need to continue therapy for to weeks. most affected foals respond to medical therapy. frequent reevaluation of the abnormality is necessary, every to days initially, and one should measure blood fibrinogen concentrations at reevaluation because they should stabilize and decrease with effective treatment. failure to respond to therapy within days to weeks suggests that an empiric change in the antimicrobial used may be necessary. in foals that are refractory to medical management or where the lesion is large, surgical excision of the entire umbilical remnant may be desirable. colic in the foal can be difficult to diagnose accurately because one cannot perform an examination per rectum. however, many diagnostic aids, most importantly ultrasonography, are available to help differentiate medical from surgical causes of abdominal discomfort in the foal. intestinal accidents of all types described in adult horses, with the possible exception of enteroliths, occur in foals. intussusception, volvulus, displacement, diaphragmatic hernia, and intra-and extraluminal obstruction have been reported in foals. abdominal ultrasonographic and radiographic evaluation greatly aids diagnosis. treatment is primarily surgical. foals with pas and intestinal dysmotility are at increased risk of intussusception and displacement, and miniature breed foals appear to be at increased risk for fecolith and enterolith formation. meconium retention or impaction is a common cause of abdominal discomfort in newborn foals. most foals defecate shortly after their first meal. the usual practice for most owners or veterinarians attending the birth of a foal is to administer an enema to aid this process. in the past, phosphate-based commercially available enemata (fleet) were used frequently, but if used excessively these types of enemata can create problems of their own, including rectal irritation and hyperphosphatemia. the best enema is warm soapy water made with a mild soap such as liquid ivory soap that can be administered through soft rubber tubing using gravity flow. foals with significant meconium retention become colicky within the first few hours of life as gas accumulates within their bowel. frequently, one can palpate the meconium through the abdominal wall. additional diagnostics can include abdominal ultrasonography and radiography, particularly if one must rule out other, more serious types of colic. these foals assume a classic stance with an arched back. one must differentiate this stance from the stance assumed by foals with uroperitoneum, which is more extended. foals with meconium retention have had simultaneous ruptured bladder, however, so the clinician must be sure to evaluate the foal fully for both problems. foals that do not respond rapidly to enema administration need additional treatment, which can include giving mineral oil ( to ounces) by nasogastric tube. one can treat persistent meconium retention resulting in significant abdominal distention by muzzling the foal to prevent further milk intake and administering intravenous fluids at an appropriate maintenance rate. if continuous rate infusion is possible, % to % dextrose is the preferred fluid to use to provide calories to the foal. one should not use dextrose as a bolus fluid. more aggressive treatment would include administration of retention enemata made using acetylcysteine, which serves as an irritant and increases secretion. extreme cases of meconium retention may require surgical intervention, but this is usually not necessary and most cases resolve with medical management alone within to hours. some foals require pain managment. one should avoid nonsteroidal antiinflammatory drugs in the neonate because of their effects on renal function and gastric mucosal blood flow (see gastric ulcers). many foals respond well to butorphanol administered intramuscularly at a dose of to mg to an average -kg foal. intranasal oxygen insufflation is beneficial in foals with significant abdominal distention. one should evaluate foals with meconium impaction/ retention for evidence of pas because intestinal dysmotility is common in pas. colostrum is a laxative, and these foals also may suffer from failure of passive transfer, with meconium retention resulting from the lack of adequate colostrum. these foals are also at risk of sepsis because the mucosal intestinal barrier probably has been disrupted and translocation of bacteria can occur. one should obtain blood cultures on these foals and should monitor them closely for signs of sepsis. atresia within the gastrointestinal system of the foal occurs infrequently, but clinical signs are characteristic. acute colic occurs within the first few hours and is accompanied by abdominal distention similar to meconium retention. three primary types of atresia are described in the foal: membrane atresia, cord atresia, and blind-end atresia. antemortem diagnosis of atresia, short of abdominal exploratory surgery, is aided by the lack of meconium staining of the rectum or any administered enema fluids. additional diagnostic tests may include administration of a barium enema for a radiographic study, colonoscopy, and abdominal ultrasonography. abdominocentesis is usually normal until bowel rupture is imminent or has occurred. one can make affected foals more comfortable by muzzling them to prevent further milk intake and by supplying them with fluids and nutrition intravenously. if one attempts surgical correction, one first should initiate broad-spectrum antimicrobial therapy and determine passive transfer status. frequently, these foals are hypoxemic because of the abdominal distention, and oxygen supplementation is desirable. solid white foals born to overo-overo matings of american paint horses may suffer from congential aganglionosis of the ileum, cecum, and colon. these foals present similarly to foals with meconium impaction or atresia in that colic develops shortly after birth and involves progressive abdominal distention with feeding. the inherited defect is in the endothelin receptor gene. [ ] [ ] [ ] [ ] no effective treatment exists, but the clinician should be aware that not all white foals of this mating are affected, and some simply may have meconium retention, so a short period of treatment may be warranted. necrotizing enterocolitis is considered the most common acquired gastrointestinal emergency of human infants. , the to infants that die every year from this disease in the united states and the large number of infants who develop short gut syndrome from this disease only represent the tip of the iceberg of the problems necrotizing enterocolitis causes. the widespread fear of necrotizing enterocolitis among neonatologists and pediatric surgeons has contributed in large part to the use of the intravenous route rather than the gastrointestinal tract for nourishing these infants for long periods. the pathogenesis of necrotizing enterocolitis is unknown but may result from a disturbance of the delicate balance among gastrointestinal perfusion, enteric organisms, and enteral feeding. risk factors for necrotizing enterocolitis in human infants include prematurity, hypoxic-ischemic insult, and formula or breast milk feedings. the clinical spectrum of necrotizing enterocolitis is multifactoral and ranges from temperature instability, apnea, lethargy, abdominal distention, bilious residuals, septic shock, disseminated intravascular coagulation, and death. medical management is usually adequate treatment for necrotizing enterocolitis. in the neonatal foal, necrotizing enterocolitis is probably one of the most underrecognized causes of gastrointestinal dysfunction and in the past has been attributed only to infection with anaerobic organisms including clostridium perfringens type c and c. difficile. although a specific form of enteritis is associated with intestinal infection by these organisms, most necrotizing enterocolitis is associated with prematurity or pas in the infant and the foal. one should suspect necrotizing enterocolitis in any foal that is having difficulty tolerating oral feeding, demonstrating signs of ileus, or having episodes of colic and in any foal with occult blood or frank blood in the stool. foals exhibiting any of these clinical signs should not be fed orally if possible and should receive parenteral nutrition until gastrointestinal function returns to near normal. the mucosal barrier of the intestine is unlikely to be fully intact, and these foals are at risk for sepsis from bacterial translocation. one should institute broadspectrum antimicrobial therapy in these foals and, if any evidence of coordinated gastrointestinal motility is apparent, should administer sucralfate orally as a protectant. gastric ulcer disease has been recognized in foals, and lesions vary in anatomic distribution, severity, and cause. in clinically normal neonatal foals (< days of age), gastric ulcers and mucosal desquamation have been documented. [ ] [ ] [ ] [ ] because of these reports and other early reports of death following ruptured clinically silent ulcers in neonatal foals, for years many clinicians felt it necessary to treat critically ill neonates with antiulcer medication prophylactically. [ ] [ ] [ ] recently, this paradigm has been challenged. the pathophysiology of gastric ulcer disease is described most reasonably as an imbalance in protective and aggressive factors. [ ] [ ] [ ] these protective factors are responsible for maintaining a healthy gastrointestinal tract by promoting adequate mucosal blood flow, adequate mucus and bicarbonate production, prostaglandin e production, epithelial growth factor production, gastric afferent innervation, epithelial cell restitution, and gastroduodenal motility. probably the most important factor is maintenance of mucosal blood flow. hypoxia, no, prostaglandins, and gastric afferent innervation influence mucosal blood flow. the aggressive factors include gastric acid, bile salts, pepsin, and enzymes. few specific causes have been found for gastric ulcer disease in foals. excessive administration of nonsteroidal antiinflammatory drugs can result in ulceration of the glandular and squamous epithelium because of an inhibition of prostaglandin production, which leads to a decrease in mucosal blood flow and an increase in acid production. nonsteroidal antiinflammatory drugs also can impair the healing of lesions and rarely are indicated in neonatal equine medicine. , in the critically ill neonate the suspected cause of gastric ulcers has shifted away from an excessive amount of intraluminal gastric acid toward gastric mucosal ischemia caused by hypoxia, low blood flow conditions, or both. perforating gastric ulcers are more likely a manifestation of necrotizing enterocolitis than of excessive gastric acid. shock, sepsis, or trauma can result in gastric mucosal ischemia, allowing for the disruption of epithelial cell integrity and permitting damage by aggressive factors or providing an environment suitable for the establishment of bacteria colonization. , impairment of mucosal blood flow also may result in reperfusion injury, allowing the formation of gastric ulcers. in the sick neonatal foal (< days of age) a wide variability in the intragastric ph has been documented depending on the type of disease, severity, and milk intake frequency and volume, suggesting that in the critically ill equine neonate, ulcer prophylaxis using histamine antagonists or proton pump inhibitors is not only unnecessary but unlikely to work. clinically significant gastric ulcers can occur in the squamous, glandular, or both portions of the stomach as a primary problem or resulting from another problem. clinical signs include diarrhea, abdominal pain, restlessness, rolling, lying in dorsal recumbency, excessive salivation, and bruxism. in the neonatal foal the only clinical signs present may be depression or partial anorexia until a more catastrophic event, such as perforation, occurs. some lesions in the gastric mucosa extend from the pylorus into the proximal duodenum and can result in stricture of the pylorus and proximal duodenum. these foals are usually older (> month of age) and have a greater volume of reflux. bruxism and ptyalism are also more prominent in these older foals. the most sensitive and specific method for diagnosing gastric ulcers is visualization by endoscopic examination. unfortunately, the use of gastric endoscopy has led to recognition of relative nonlesions and ulcers resulting from other problems and of clinically significant disease states. the clinician should not stop simply when ulceration of the stomach is recognized with endoscopy but should examine that patient fully for other potential sources of the clinical signs. other diagnostic tests may help in determining the severity of the ulcers, including fecal occult blood or gastric blood assessments, contrast radiography, abdominal ultrasound, and abdominocentesis. endoscopy of the foal stomach carries an additional risk of exacerbating colic in the short term, unless the examiner ensures that as much introduced air as possible is evacuated from the stomach at the end of the procedure. the presence of a brown gastric reflux fluid may indicate the presence of bleeding ulcers. blood in the feces of the neonate is more consistent with a diagnosis of necrotizing enterocolitis, which can be associated with gastric ulcers. contrast radiography is useful if one suspsects delayed gastric emptying or pyloric or duodenal stricture in older foals. if a stricture has occurred, one will note a delay in complete emptying of barium from the stomach (> hours). abdominal ultrasound may be useful to visualize free abdominal fluid and gastric or small intestinal distention if one suspects a perforation. one can visualize portions of the descending duodenum, and a thickened duodenum should increase the index of suspicion for duondenal stricture. abdominocentesis also may confirm perforation. traditional therapy for gastric ulceration includes mucosal adherents, histamine type receptor antagonists, proton pump inhibitors, and antacids. the most widely used mucosal adherent is sucralfate, which is a hydroxy aluminum salt of sucrose. the main therapeutic action of sucralfate is to bind to the negatively charged particles in the ulcer crater. , at a ph less than , sucralfate is converted to a sticky viscous gel, which adheres to the ulcer crater and remains adhered for hours, but at a higher ph, sucralfate remains in a suspension. sucralfate is still effective because it inhibits pepsin and buffers hydrogen ions. other important actions of sucralfate include stimulating production of prostaglandin e, which maintains mucosal blood flow; increasing bicarbonate secretion; stimulating mucous secretion; decreasing peptic activity; and binding epidermal growth factor. the histamine type receptor antagonists include cimetidine, ranitidine, and famotidine. these compounds block the interaction of histamine with the histamine type receptor on the parietal cell, resulting in inhibition of gastric acid secretion. clinically normal neonatal foals have a highly acidic gastric fluid that is influenced by sucking. intravenous and oral administration of ranitidine increases intragastric ph in normal foals but critically ill neonatal foals have a blunted response to ranitidine administration. , one possible conclusion reached from these studies is that in critically ill neonatal foals, gastric ulcers may not be caused by an increased intraluminal gastric acidity. the most commonly used proton pump inhibitor is omeprazole. this drug has not as yet been approved for use in foals under days of age. omeprazole inhibits the secretion of hydrogen ions at the parietal cell by irreversibly binding to the h + ,k + -atpase proton pump of the cell. most of the lesions in older foals were healed after daily administration of omeprazole for days according to one report. table - summarizes the therapeutic agents for treating gastric ulcers in foals. prophylactic treatment of critically ill neonates for gastric ulcers has been standard therapy for years because of the evidence of clinically silent ulcers. this approach may not be appropriate for several reasons. an increased incidence of nosocomial pneumonia and systemic sepsis is associated with high gastric ph in human patients in intensive care. [ ] [ ] [ ] patients in intensive care units treated prophylactically with histamine type receptor antagonists are more likely to develop pneumonia during ventilation therapy and gastric colonization with potentially pathogenic bacteria or yeast. , an acidic environment appears to protect against airway colonization by bacteria of intestinal origin and bacteria translocated across the gastrointestinal tract. pathogenesis of ulcers in the neonatal foal most likely does not involve increased intraluminal gastric acid but instead may be caused by decreased mucosal perfusion associated with shock, hypoxia, and hypoxic/ischemic insult to the gastric mucosa. a recent report revealed that gastric ulcer disease in equine nicu patients is independent of pharmacologic prophylaxis. in this study, despite decreased treatment, the incidence of gastric ulcers found in these foals at necropsy had decreased significantly. the decrease was attributed to overall improvement in management of these cases. similarly, in a human intensive care unit, the incidence of stress ulcers decreased independent of the use of prophylaxis. , early treatment of sepsis, sufficient oxygenation, improved monitoring, institution of enteral feedings, and improved nursing care may contribute to the reduction in gastric ulcers in the neonatal patient. use of histamine type receptor antagonist and proton pump inhibitors apparently may not be necessary; however, in some instances sucralfate may be useful. sucralfate reduced the rate of bacterial translocation in a rat model during hemorrhagic shock and also may prohibit the generation of acute gastric mucosal injury and progression to ulcer formation induced by ischemia-reperfusion. , in a human medical intensive care unit, airway colonization by new pathogens occurred more frequently in patients receiving agents that increased gastric ph than in those receiving sucralfate. , in the critically ill neonatal foal, risk factors for gastric ulceration have not been identified clearly, although foals treated routinely with nonsteroidal antiinflammatory drugs may be at increased risk for gastric lesions. prophylactic treatment for gastric ulcers in critically ill neonates may not be necessary, and one should consider carefully the pros and cons of their use before their administration. foal heat diarrhea is a mild, self-limiting form of diarrhea that occurs in foals between and days of age, about the time of the "foal heat" in the dam. the definitive cause of foal heat diarrhea has yet to be described, but the condition may be associated with dietary changes or changes in gastrointestinal function that occur around that time. this form of diarrhea is not caused by stongyloides westeri infestation as previously thought. foals with foal heat diarrhea are not systemically ill and should not require therapy. one should evaluate fully any foals with diarrhea at this time for other possible causes of diarrhea, particularly if they are unwell or exhibit anorexia or dehydration. viral diarrhea occurs most commonly in large groups of mares and foals that are housed together. rotavirus is an isolate from the feces of up to % of foals with diarrhea worldwide, alone or with another pathogen. , the virus infects and denudes the microvilli, resulting in increased secretion combined with decreased absorption. the virus interferes with disaccharidase function and alters the function of the intestinal sodium-glucose cotransport proteins. the initial clinical signs are anorexia and depression, with profuse watery diarrhea occurring shortly thereafter. severely affected foals may become significantly dehydrated and have electrolyte abnormalities, primarily hyponatremia and hypochloremia with metabolic acidosis. these foals generally require intravenous fluid support, whereas less severely affected foals may require only symptomatic therapy. definitive diagnosis is by detection of the virus in the feces of foals with diarrhea. however, none of the available tests are particularly sensitive, and the virus also may be found with other intestinal pathogens. recently, vaccination of pregnant mares has been suggested as a means of prevention, with preliminary results suggesting efficacy. , although a definitive role for adenovirus has not been established in the foal, adenovirus is a common co-isolate from foals with rotaviral diarrhea. a specific equine coronavirus recently has been identified from an immunocompetent foal with diarrhea, and a second report of cornavirus diarrhea was published recently. , one case report suggests a parvovirus caused diarrhea in the foal. treatment of viral diarrhea in foals is primarily supportive. intravenous fluid and parenteral nutritional support may be necessary in severe cases. very young foals may benefit from intravenous plasma administration and broad-spectrum antimicrobial coverage to limit bacterial translocation. one can administer sucralfate orally in these cases as a gastrointestinal protectant and to discourage bacterial translocation. foals with moderate to severe metabolic acidosis may benefit from sodium bicarbonate administration if their ventilatory function is normal. one administers sodium bicarbonate at half the calculated deficit ( . × standard base excess × body mass in kilograms) as an isotonic solution at the maintenance fluid rate. one should reevaluate sodium and bicarbonate (or standard base excess) concentrations regularly. nonspecific therapy of diarrhea is discussed elsewhere in this text. diarrhea is frequently the primary presenting complaint in foals with sepsis, so one should rule out this differential diagnosis in foals less than week of age. one should evaluate all neonatal foals with diarrhea for possible sepsis and should include a blood culture whenever possible. clostridium perfringens and c. difficile are recognized increasingly as serious pathogens of the foal. - foals with either pathogen generally have abdominal pain, dehydration, and profuse watery diarrhea. some foals may have red-tinged or frankly bloody feces, which carries a poorer prognosis. most foals with this type of diarrhea require intensive care or, at the minimum, intravenous fluid administration. outbreaks of this type of diarrhea on farms occasionally occur, and the suggestion is that the dam has a role in transmission of the bacteria. diagnosis is by recognition of the offending organism by gram stain of the feces, by bacterial isolation from the feces, and by detecting the presence of toxins associated with the organisms. specific treatment includes oral administration of metronidazole and broad-spectrum antimicrobial coverage as prophylaxis for bacterial translocation associated sepsis in younger foals. foals with severe blood loss in their feces may require transfusion of whole blood. salmonella spp., escherichia coli, bacteroides fragilis, and aeromonas hydrophila have been implicated in diarrhea in foals. salmonella generally is associated with septicemia in foals, and although some convincing evidence exists for a role for e. coli in foal diarrheal disease, the extent of e. coli as a pathogen of the gastrointestinal tract in foals has yet to be described fully. , [ ] [ ] [ ] [ ] proliferative enteropathy is a transmissible enteric disease caused by lawsonia intracellulare. , most foals have been weaned before the appearance of clinical signs of depression, rapid and significant weight loss, subcutaneous edema, diarrhea, and colic. poor body condition with a rough hair coat and a pot-bellied appearance are common in affected foals. clinicopathologic abnormalities included hypoproteinemia, leukocytosis, anemia, and increased serum creatine kinase concentration. postmortem reveals characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa. antemortem diagnosis of equine proliferative enteropathy is based on clinical signs, hypoproteinemia, and the exclusion of other common enteric pathogens. fecal polymerase chain reaction analysis may be positive for the presence of l. intracellulare, and affected foals develop antibodies against l. intracellulare. treatment with erythromycin estolate alone or combined with rifampin for a minimum of days is recommended with additional symptomatic treatment when indicated. cryptosporidium spp. cause gastroenteritis and diarrhea in many animal species and are not host-specific. cryptosporidium has been implicated as the casual agent of diarrhea in foals, but the organism is isolated from the feces of diarrheic foals and normal foals with the same frequency and concentration, making a clear role for the organism difficult to elucidate. - diarrhea caused by cryptosporidium in other species and that described for foals is generally self-limiting, with a clinical course of between to days. immunosuppressed patients, including foals compromised by concurrent disease, are thought to be at increased risk for complications resulting from infection with this organism. , treatment is symptomatic. cryptosporidiosis is a disease with zoonotic potential, and one should take appropriate precautions, including use of gloves and frequent hand washing, if organisms are identified in the feces of any patients so as to prevent spread to other patients and personnel. eimeria leukarti, trichomonas equi, and giardia equi have been identified in the feces of normal horses and horses with diarrhea. transmission studies have failed to produce reliable clinical signs, and the prevalence and significance of these organisms in the genesis of foal diarrhea remain unknown. strongyloides westeri is a common parasitic infection of foals. , transmission is transmammary, and patent infection is recognizable in the foal by to days of age. this nematode previously was associated anecdotally with foal heat diarrhea, but the association has not been demonstrated clearly. the diarrhea is generally mild and is treated effectively by deworming with benzimidazole or ivermectin anthelmintics. strongylus vulgaris fourth-stage larvae cause diarrhea in young foals during migration through the arterioles of the cecum and descending colon. clinical signs may resemble thromboembolic colic. the prepatent period is about months, and diagnosis is based on clinical examination, clinicopathologic changes, and farm deworming history. patients with diarrhea associated with this parasite may have peripheral leukocytosis, neutrophilia, eosinophilia, and hypoproteinemia. appropriate deworming with ivermectin (label dose), fenbendazole ( mg/kg/day orally for days), or thiabendazole ( mg/kg/day orally for days) is recommended, with the last two drug dosages being larger than the label dose. cyathostomiasis, or diarrhea resulting from the sudden emergence of encysted cyathostome larvae, is an unusual cause of diarrhea in the foal. the clinician managing critically ill neonates must recognize that intravenous fluid therapy simply cannot be scaled down from adult management approaches. fluid management of the ill neonate, particularly over the first few days of life, must take into consideration that the neonate is undergoing a large transition from the fetal to the neonatal state and that important physiologic changes are taking place. these transitions include shifts in renal handling of free water and sodium and increased insensible losses because of evaporation from the body surface area and the respiratory tract. the newborn kidney has a limited ability to excrete excess free water and sodium, and the barrier between the vascular and interstitial space is more porous than that of adults. water and sodium overload, particularly in the first few days of life, can have disastrous long-term consequences for the neonate. , in the equine neonate, excess fluid administration frequently manifests as generalized edema formation and excessive weight gain, frequently equivalent to the volume of excess fluid administered intravenously. in cases in which antidiuretic hormone secretion is inappropriate, as in some foals with pas, generalized edema may not form, but the excess free water is maintained in the vascular space. this syndrome of inappropriate antidiuretic hormone secretion is recognized in the foal that gains excessive weight not manifested as edema generally, with decreased urine output and electrolyte abnormalities such as hyponatremia and hypochloremia. the foal manifests neurologic abnormalities associated with hyponatremia. the serum creatinine concentration varies in these cases, but urine always is concentrated compared with the normally dilute, copious amounts of urine produced by foals more than hours of age on a milk diet. if measured, serum osmolarity is less than urine osmolarity. the treatment for this disorder is fluid restriction until weight loss occurs, electrolyte abnormalities normalize, and urine concentration decreases. if the clinician is unaware of this differential diagnosis, the neonate can be assumed mistakenly to be in renal failure, and the condition can be exacerbated by excessive intravenous fluid administration in an attempt to produce diuresis. the problem of appropriate fluid management in critically ill neonates has been recognized by medical physicians for years and has resulted in changes in fluid management of these patients. the approach taken has been one of fluid restriction, in particular sodium restriction but also free water restriction, and has resulted in improved outcome and fewer complications, such as patent ductus arteriosus and necrotizing enterocolitis. , the calculations used for maintenance intravenous fluid support in these patients takes into consideration the ratio of surface area to volume and partially compensates for insensible water losses. maintenance fluids are provided as % dextrose to limit sodium overload and provide sufficient free water to restore intracellular and interstitial requirements. the calculation for maintenance fluid administration is as follows: first kg body mass ml/kg/day second kg body mass ml/kg/day all additional kilograms of body mass ml/kg/day as an example, the average -kg foal would receive ml/day for the first kg of body mass, ml/day for the next kg of body mass, and ml/day for the remaining kg of body mass for a total of ml/day. this translates to an hourly fluid rate of about ml/hr. one should adjust the fluid and sodium requirements for ongoing losses exceeding the maintenance requirements. these losses can take the form of diarrheal losses and excessive urine output, such as those with glucose diuresis and renal damage resulting in an increased fractional excretion of sodium. the normal fractional excretion of sodium in neonatal foals is less than that of adult horses, usually less than % (j.e. palmer, unpublished data). in the critically ill foal the sodium requirement can be met with as little as meq of sodium per day, about that administered in a single liter of normal equine plasma. one can address sodium deficits by separate infusion of sodium-containing fluids, although this may not be necessary if one considers the sodium being administered in other forms, including drugs administered as sodium salts and any constant rate infusions (pressors, inotropes, etc.) that are being provided as solutions made with . % sodium chloride. the author has used this approach to fluid therapy in her nicu for the last few years and believes that the percentage of foals suffering from generalized edema and related problems has decreased. if one takes this approach to fluid therapy, one should take the weight of the patient once daily, or even twice daily, and monitor the fluid intake and output as closely as practical. one should evaluate any larger than anticipated weight gains or losses. one should not expect urine output to approach the reported normal of ml/hr for a -kg foal because the free water administered is limited, unless the patient is experiencing diuresis (glucosuria, resolution of the syndrome of inappropriate antidiuretic hormone secretion, resolution of previous edematous state, renal disease). one should obtain the urine specific gravity several times daily and should determine fractional excretion of sodium at regular intervals. if the volume of urine produced by the patient is measured accurately, one can determine sodium losses accurately and can obtain creatinine clearance values. one should obtain blood pressure measurements at regular intervals throughout the day because hypotension can be a problem in these patients, particularly in septic foals and foals suffering from pas, and one may need to increase fluid therapy to maintain adequate vascular volume. patients with hypotension may need inotrope and pressor support. inotrope and pressor therapy generally is restricted to referral centers where these drugs can be administered as constant rate infusions and blood pressure can be monitored closely. blood pressure can be monitored directly or indirectly by the use of cuffs placed on the base of the tail. both techniques have advantages and disadvantages. although direct blood pressure measurements are considered the gold standard and are generally more accurate, the difficulty in placing and maintaining arterial catheters and lines in these patients severely restricts the utility of this method. indirect techniques can be inaccurate and are affected by cuff size and placement. however, indirect techniques are easier to use in the nicu and can be useful if trained staff are using the equipment. in the author's nicu, once practitioners identify the appropriate cuff size, they dedicate that cuff to that patient for the duration of the hospitalization to decrease variability caused by using different cuffs. one should monitor the blood pressure of all recumbent patients at regular intervals, and trends upward or downward should prompt the clinician to make necessary adjustments. foals suffering from pas and sepsis are the patients most at risk for significant hypotension and perfusion abnormalities. perfusion is maintained by supporting cardiac output and blood pressure with judicious use of intravenous fluid support and inotrope/pressor support. the author does not aim for any specific target systolic, mean, or diastolic pressure. instead the author monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for nicu patients to require inotrope and pressor therapy is not unusual, but in some cases hypoxic and septic damage is sufficiently severe to blunt the response of the patient to the drugs. one must approach each patient as an individual, and no single inotrope/pressor protocol will suffice for all patients. dobutamine is a β-adrenergic inotrope that is frequently used as first choice therapy in nicu patients. its effects are β at the lower dose range. neonates have a limited ability to increase stroke volume in an effort to maintain cardiac output, and one may observe tachycardia in these patients as heart rate increases to maintain cardiac output and vascular pressure. dobutamine is useful after patients are volume replete for support of cardiac output. the dose range is between to µg/kg/min provided as a constant rate infusion. dopamine has dopaminergic activity at low doses, β and β activity at moderate doses, and α activity at high doses. dopamine causes norepinephrine release, which has lead to the suggestion that this is its major mode of action at higher doses. at doses greater than µg/kg/min, intrapulmonary shunting, pulmonary venous vasoconstriction, and reduced splanchic perfusion may occur. dopamine also produces natriuresis at lower doses through a direct effect on renal tubules. for these reasons, dopamine has fallen out of favor at some referral institutions. norepinephrine has α and β activity but variable β activity, resulting in potent vasopressor effects; it has inotropic and chronotropic effects, but its chronotropic effect usually is blunted by vagal reflexes slowing the heart rate induced by the increase in blood pressure. in many critical care units, norepinephrine has become a pressor of choice and frequently is used along with dobutamine. evidence suggests that splanchic perfusion is maintained better with norepinephrine than with some other pressors. the dose range is . to . µg/kg/min, although larger doses have been used when necessary in certain patients. epinephrine has α , α , β , and β activity; β activity predominates and results in increased cardiac output and decreased peripheral resistance at low doses. epinephrine has been associated with hyperglycemia, hypokalemia, lipolysis, increased lactate concentration, and increased platelet aggregation. the effect on renal function is controversial. use of epinephrine usually is limited to those patients not responding to other pressors. vasopressin (antidiuretic hormone) is a pressor gaining a great deal of attention in the critical care literature. vasopressin appears to be depleted from the neurohypophysis in septic shock, and short-term administration of vasopressin spares conventional vasopressor use, in addition to improving some measures of renal function. low-dose vasopressin infusion increases mean arterial pressure, systemic vascular resistance, and urine output in patients with vasodilatory septic shock that are hyporesponsive to catecholamines. these data indicate that low-dose vasopressin infusions may be useful in treating hypotension in patients with septic shock. the author has been using low-dose vasopressin in patients in her nicu for the past few years and has the clinical impression that blood pressure is defended more readily using this agent in concert with other management strategies. the author commonly uses low-dose vasopressin constant rate infusion with dobutamine constant rate infusion as the initial inotrope/pressor therapy in cases requiring pressure defense, although no prospective studies are yet available regarding this drug in veterinary medicine. for quality health care for animals, advances in medical science, and in some breeds the increasing value of the juvenile equine athlete. equine veterinarians that encounter pediatric orthopedic problems are only beginning to get the information needed to make appropriate treatment decisions. the equine neonate has specific differences in structure and physiology from adults that one must consider when designing an optimal therapeutic or management strategy. few investigations have focused on the equine neonatal musculoskeletal system, - but a large body of clinical information exists, and one can make cautious extrapolations from work in other species. neonatal equine bones have accelerated modeling and remodeling processes that result in accelerated fracture healing and an increased susceptibility to deformation caused by excessive loading. contralateral limb varus deformities of the growth centers (most commonly distal radius and metacarpus/ metatarsus) are common in overloaded limbs. the increased plasticity of the skeletal structure also is mirrored in the soft tissue support system, for these units become flaccid within weeks of immobilization. this laxity is important, because it further compromises the use of the fractured limb and can last as long as the coaptation was in place. additional divergences from adult physiology include musculoskeletal immaturity (generalized or focal) and immune system differences. finally, foals are lighter and can tolerate and will assume recumbency more readily than adults. the net results of these differences are that one must consider the use of external coaptation carefully, fractures heal quickly, one must consider damage to the contralateral limb from overstress, reducing weight bearing is possible, and infection is always lurking. stresses can affect the musculoskeletal system of the foal at any time, including in utero. although rare, reports describe in utero fractures (k. sprayberry, personal communication, ) that result in foal locomotor problems and even maternal uterine damage from sharp bone ends. the cause is presumably from vigorous muscular activity of the foal, but one cannot rule out direct trauma. the fractures result in foal lameness and can increase the likelihood of dystocia and caused colic in one mare when the broken bones damaged the uterus. treatment depends on how long the fracture has been present and on the fracture location and configuration, but if the fracture is repairable, internal fixation probably is necessary. fractures occurring during foaling result from aggressive obstetric manipulation (mandibles) or the advances in medical care of equine neonates in the last years have resulted in the survival of many foals that previously would have died from sepsis, asphyxia, and prematurity; and the successful management of their musculoskeletal system can be a major challenge. major factors adding to the challenge are the immaturity of components of the musculoskeletal system and the demands placed on them by a growing and active foal. additional pressures to treat orthopedic conditions in foals have come from an overall increase in the demand chest compression. one should stabilize unstable mandibular fractures. appendicular fractures usually do not occur during parturition because of the robust character of the bones of the foal. after birth, foals are susceptible to external trauma from many sources. the dilemma is that younger foals with fractures are more likely to heal but also are more likely to develop contralateral limb problems because of excessive weight bearing and affected limb flexor tendon laxity if the limb is immobilized fully. as a result, internal fixation is often the best choice for neonatal fractures to keep the fractured limb in use. proximal sesamoid bone fractures result from hyperextension of the fetlock joint. foals are lame after the fracture, but the lameness can be mild and often diminishes quickly. soft tissue swelling occurs over the sesamoids. fractures are usually simple, can occur uniaxially or biaxially, and can be apical, midbody, or basilar. fractures can occur in any joint and can affect multiple sesamoids in one foal. however, they most commonly are single forelimb fractures and in thoroughbreds are most frequent in the left front medial proximal sesamoid (j.p. morehead, personal communication, ). of particular interest to neonatologists is that proximal sesamoids fractures often occur in recovered neonatal patients that are allowed too much exercise too soon. foals from the nicu need a gradual introduction to pasture turnout to allow their musculoskeletal system to adjust. mares are often in need of turnout, but in the interest of their foals, they must wait. treatment of proximal sesamoid fractures in foals is stall confinement with support bandaging. healing occurs, albeit with some distortion of the shape of the sesamoid. severely displaced fragments result in large and misshapen sesamoids, and surgery may be considered for these foals, because restriction of fetlock flexion can occur after conservative therapy. third phalangeal fractures are also common in foals. these foals have a lameness that worsens with hoof compression. hoof abscesses are uncommon in young foals but should be considered. most commonly, radiographs reveal nonarticular small fractures on the wings on the third phalanx. the fractures are associated with hard ground and exercise. the fractures heal with stall confinement, and unlike adults, leave no discernable radiographic fibrous union. avulsion fractures of the proximal insertion of the peroneus tertius and the origin of the long digital extensor tendon have been reported. , both soft tissue structures attach to the extensor fossa of the distal femur. the two affected foals had lameness of a hindlimb associated with swelling, pain, and crepitation. radiographs revealed multiple avulsion fractures of the extensor fossa. because of the intraarticular fragments in the femoropatellar joint, and the fear of later degenerative joint disease, fragments were removed arthroscopically. both foals were juveniles at last follow-up; one foal was considered normal, and one had a mild residual lameness. tendon and ligament damage is uncommon in neonates probably because of their low body weight. extensor tendon damage following flexural deformities is the most common tendon problem and is discussed in congential flexural deformities of foals. gastrocnemius ruptures are one of the most devastating problems and have occurred after forced extraction because of a breech presentation, severe flexor tendon laxity, and tarsal contracture. loss of gastrocnemius function usually results in a non-weight-bearing limb, although an intact superficial digital flexor tendon may make some weight bearing possible. complete loss of support is difficult to treat successfully. coaptation of the limb is logical but difficult to obtain. schroeder-thomas splints have been used but are difficult to manage. tube casts also are used but must be changed frequently, and cast sores are inevitable (l.r. bramlage, personal communication, ) . the prognosis for athletic function is guarded. treatment for ligamentous injuries is usually some form of coaptation, although surgical repairs have been performed when coaptation was unworkable. coaptation in proper limb alignment allows the ligaments to heal and should be used if the injury will destabilize a joint and cause damage to growing epiphyses or cuboidal bones. one can achieve coaptation with casts or splints under a bandage. casts are initially a greater expense, and cast sores and their resulting white hairs are a risk, but the rigid immobilization and the lack of the requirement for daily adjustment makes them preferable. important to musculotendinous health is some measure of weight bearing to avoid laxity after coaptation removal, which one can achieve by using tube casts and splints that allow weight bearing. following coaptation, bandaging and a gradual return to exercise are recommended for ligamentous injuries. patellar luxation can affect foals in one or both hindlimbs, and the luxation can vary from a laxity in the medial attachments to complete luxations that cannot be replaced in the patellar groove of the distal femur. , medial luxations have not been reported. clinical signs vary from a slight discontinuous motion during stifle flexion to an inability to stand. many foals have a crouching stance on the affected limb because of an inability to extend the stifle. the pathophysiology of patellar luxations is unknown. congenital bilateral luxations are common in miniature horse foals and are believed to be genetic. luxations are rarer in other breeds and are occasionally traumatic. the affected limbs are usually not grossly abnormal except for effusion of the femoropatellar joint and the luxation. a shallow trochlear groove has been reported to be a cause of patellar luxation, but objective evidence is lacking. one should evaluate foals for the ability to stand. once the appropriate supportive care is provided, if a foal cannot stand, euthanasia is recommended. most bilateral luxations in horses fit in this category. however, miniature horse foals often can stand sufficiently to nurse despite bilateral luxations, and one may consider treatment. treatment consists of replacing and stabilizing the patella and sometimes surgically deepening the patellar groove. delaying surgical repair until the foal is approximately days old is recommended to avoid neonatal problems, allow the musculoskeletal system to mature, and provide good anchors for suture. some surgeons worry that delay may cause further femoropatellar developmental abnormalities, but in a small number of cases, this has not been an issue. the prognosis for miniature horse foals appears to be good because of their low body weights and modest performance expectations. too few reports about the correction of unilateral luxations in light horses exist to make a definitive statement about prognosis except that success and failure have been experienced. , congenital flexural deformities in foals can be classified as severe (rarely correctable), moderate (correctable with therapy), or mild (self-correctable). examples of severe flexural deformities include arthrogryposis (deformities of multiple limbs and often the head and neck), severe carpal deformities (flexor angle of the carpus less than degrees), and tarsal contractures (rare). extraordinary methods have been used to correct severe deformities but are often unsuccessful. mild flexural deformities are those that result in an upright conformation to the limb, but the foal can bear weight on the limb and load the flexor structures. these foals require no specific treatment and will self-correct with controlled exercise. moderate flexural deformities are those that make bearing weight on the limb and loading the flexor structures and ligaments difficult for the foal. when these deformities occur bilaterally (most common), the foals cannot rise to suckle or does so with great difficulty, and the lack of weight bearing worsens the flexural deformity. examples of moderate flexural deformities include carpal and forelimb fetlock flexural deformities that usually occur together, hindlimb fetlock flexural deformities with coronopedal flexion or hyperextension, and the uncommon coronopedal flexural deformity alone. treatment of moderate flexural deformities aims to place the solar surface of the foot on the ground so that the weight of the foal can stretch the flexor structures. splints are useful for restoring the limb to normal orientation but require attention to detail because the splints often exert an extreme amount of tension on the soft tissues, and the skin of the foal is thin. pressure sores are easy to create and at a minimum result in an extended convalescence. the first step in splint application is to apply a separate heavy bandage to the limb, which should be reapplied as necessary because the bandage can slip and cause focal constriction. commercial gauze over cotton bandage material works better than sheet cotton as a bandage. the splint is made of polyvinyl chloride pipe cut in half or thirds. using % of the diameter of the pipe results in less splint rotation but is bulkier and leaves more splint exposed to cause trauma. one cuts off the corners of the splint and pads the ends with gauze or roll cotton covered with tape. palmar or plantar placement of the splint is preferable, but severe deformities may require initial dorsal placement. as the limb straightens, one can bend the splint to tape the fetlock into the bend to extend it. one can tape the splint tightly to the limb over the bandage with nonelastic (white or duct) tape. this procedure requires at least two persons, one to extend the limb firmly and hold the limb and one to tape. one should leave the splint on for to hours and then remove it for to hours. one can reapply splints as necessary. in addition to splints, some medications are of value for treating flexural deformities. oxytetracycline ( to mg/kg) given intravenously appears to relax the soft tissues. the mechanism of action is unknown, and the drug is most efficacious when given in the first days of life. this dose is high but appears to be safe for healthy foals and can be repeated at -hour intervals. foals should be normovolemic during tetracycline administration. one should use the drug with caution in foals with renal impairment. foals should be urinating and have reasonable urinary parameters (serum urea nitrogen, creatinine, and urinalysis) before tetracycline use. diarrhea is an uncommon sequela to tetracycline use. one should monitor the unaffected limbs closely because all limbs experience a relaxation of the palmar/plantar support. , discontinuation of tetracycline therapy before affected limbs are normal but after they can bear weight is common because of worsening laxity in the "normal" limbs. one also can use phenylbutazone ( mg/kg) for a short time when the splints are used. some analgesia appears to help the foals use the limbs and stretch the soft tissues. one should not use phenylbutazone for long periods of time because of the potential of inducing gastric ulcers. surgical treatment of congenital flexural deformities rarely is indicated. severely affected foals rarely respond favorably to surgery, and mildly affected foals do not need it. surgery is most appropriate for foals with moderate flexural deformities that are neglected or have not responded to splinting and tetracycline. the most common surgical therapy performed for congenital flexural deformities is the inferior check ligament desmotomy for fetlock or coronopedal flexural deformities. ruptures of the extensor tendons commonly occur with congenital flexural deformities and result from the foal overloading the extensor tendons. no specific therapy for the ruptures is necessary. if the rupture is extensive, it can interfere with the ability to extend the fetlock and to place the foot flat. these foals then tend to knuckle over, even after correction of the flexural deformity. a firm fetlock bandage extends the digit and assists in foot placement until the extensor tendons heal. foals commonly are born with hyperextension deformities of the fetlock of varying degrees of severity. all but the worst deformities self-correct as muscle tone improves. a deeply bedded stall is all that is usually necessary to protect the soft tissues, but one can apply a light bandage to the coronary band and pastern if trauma is a problem. severe deformities are more problematic but rare, so therapeutic recommendations are not available. hyperextension of the carpus occasionally occurs and usually is treated conservatively. however, a tube cast to align the limb may be necessary to protect the dorsal surface of developing carpal bones. neonatal foals exhibit three categories of forelimb conformational deviations: angulation, rotation, and carpal offset. angular deviations most commonly are centered in the metaphysis and epiphysis, but their location is described by the closest joint, usually the carpus and fetlock. when the deviation of the distal limb is lateral to the long axis, the deviation is valgus, and when the deviation is medial, the deviation is varus. more than one joint can be affected, and although rare in neonates, valgus and varus can occur in different joints in one limb. rotational deformities appear to originate most commonly in the diaphysis or metaphysis of the radius or the metacarpus. in neonates the direction of rotation of the distal limb at both sites is almost exclusively outward. associated angular and rotational deviations occur. in neonates, limb deviations occur in foals with narrower chests and less developed pectoral muscles than in straight foals, and they appear to have an initial greater overall weakness in the musculoskeletal system because it first interacts with gravity, body mass, and ground reaction forces. however, after the first few days of life, the asymmetric loading of the growth centers does affect limb deviations. angulation results from a compressive load that is asymmetric in a frontal plane but is uniform in the sagittal plane, and rotation occurs when the compressive load is asymmetric in both planes and the limb develops around an overloaded axis point. considered this way, valgus and outward rotation deviations in young foals are coupled, as are varus and inward rotation in older foals. the loading asymmetry for valgus/outward rotation foals is accentuated as foals assume a base-wide posture that is more stable side-to-side but promotes a lateralization of the limb load. the specific effects of intermittent versus static loads, strain magnitude versus strain rate, and shear and hydrostatic stress on growing bones is only beginning to be understood. however, clinical experience supports the general observation that excessive cartilage compression is deleterious to bone growth. offset carpal conformation describes a joint that appears to deviate outwardly and then inwardly, all within the carpus. the deformity is thought to be centered at the radiocarpal joint, but the specific structural cause of offset has not been determined. this conformation is more common in older foals but occasionally occurs in neonates. the deviation is particularly common when incomplete ossification of the carpal bones is present. the causes of conformational deviations are a matter of some debate. as always, the major factors are genetics or environment. genetic influences include the assortment of alleles that controls bone form and growth and the assortment that modulates bone remodeling. many in the horse industry believe that genetics is a strong determiner of limb conformation. environmental influences are many and include the intrauterine environment, the postnatal limb load, nutrition, and bad luck. suffice to say, the situation is complex, but one must consider biologic and mechanicobiologic influences when evaluating the growth of long bones. several factors may contribute to the common occurrence of deviations in the carpus. first, the carpus is in the middle of the limb and is subject to the greatest bending forces. second, the carpal anatomy is complex and perhaps is not understood completely. the carpus has seven cuboidal bones, two long bones, and two epiphyses (distal radial and lateral styloid); and cartilage surrounds all. the ligamentous support includes collateral ligaments, innumerable intracarpal ligaments, and a palmar carpal soft tissue ligament. the distal radial physis is not flat transversely, but undulates in the frontal and sagittal planes. a separate center of ossification for the lateral styloid process is found at its palmar-lateral aspect. because of this separate center of ossification, more cartilage and less bone are in the lateral aspect of the distal radial growth center, suggesting it may be more susceptible to growth alterations from load. less common conformation deformities in young foals include hindlimb deformities, windswept conformation, diaphyseal deviations (usually of the metacarpus/ metatarsus), gross congenital malformations such as agenesis and polydactyly, and acquired varus deformities of the carpus and fetlock. hindlimb conformational deviations can manifest as tarsal and fetlock angular deformities and external limb rotation, usually centered above the tarsus. windswept foals have limbs (usually both forelimb or both hindlimbs) that are curved in the same direction in the frontal plane. diaphyseal deviations, agenesis, and polydactyly are rare and have various presentations. acquired varus deformities are caused by excessive loading, which appears to be focused medially on the growth plates. one should evaluate the limbs to determine the location, extent, and potential cause of the deviation. evaluation consists of observation and then palpation for heat, swelling, or ligament laxity. ligamentous laxity of the medial carpal ligaments is an important cause of carpal valgus and should be evaluated carefully. lameness is not a characteristic of uncomplicated angular limb deformities and suggests further evaluations are necessary. radiography is indicated for foals with severe deviations (all tarsal valgus), ligamentous laxity, lameness, or joint effusions. ultrasonography may be valuable for selected soft tissue evaluations. conservative therapy is by far the most commonly used therapy in foals less than days of age. mild to moderate carpal valgus and external rotation of the carpus and fetlock are common and normal in neonates, particularly light breed horses. most congenital limb deviations improve with age, if the developing musculoskeletal system is protected from overuse and abnormal loads. approximately % of thoroughbred foals with congenital carpal valgus self-correct. those foals that do not most often have abnormal bone (incomplete ossification) with normal stress or normal bone with abnormal stress (ligamentous laxity or contralateral limb lameness). correction continues for several months, and on average, foals reach their straightest conformation (regarding angulation) at approximately months of age (e.m. santschi, unpublished data). determination of the appropriate treatment for foals with angular limb deformities is based on the age of the foal, the severity and location of the deviation, and its causes. one must evaluate the entire foal and the affected limb. if the carpal collateral ligaments have no laxity and carpal incomplete ossification is not suspected, one may use an exercise program such as in table - , assuming that the foal has no contradicting additional problems. exercise is essential for the robust development of almost every body system for neonates, and fresh air and good ventilation reduce the occurrence of respiratory disease. appropriate limb loading along with growth and maturity is what straightens limbs, but excessive amounts of loading can be deleterious. for example, one should use exercise cautiously in foals with very asymmetric deviations. when one limb is much more deviated than the other, it appears to be loaded excessively and compromised more than if both limbs were affected similarly. and finally, limb deviations are additive. foals with external rotation and carpal valgus improve more slowly than those with one type of deviation. incomplete ossification of cuboidal bones and focal ligamentous laxity are complicating matters of great potential impact on adult conformation. they generally manifest as a moderate to severe limb deviation. physical examination indicates laxity because angular limb deviations are reducible. radiographs are the best way to evaluate the extent of carpal bone ossification. incomplete ossification of the cuboidal bones can be focal or widespread. focal immaturity is not common but can result in severe angulation. generalized immaturity is more frequent and initially often manifests as an offset conformation with valgus angulation. when the foal becomes heavier, assumes a base-wide stance, and is allowed exercise, crushing of the bones of the lateral carpus (usually the lateral styloid process of the radius, the ulnar, the fourth and the intermediate facet of the third carpal bone) results in a permanent intracarpal valgus deviation. the same result occurs when significant medial carpal ligament laxity goes untreated. in the forelimb, foals with collateral ligamentous laxity and moderate to severely immature cuboidal bones should have external coaptation placed on the affected limb to maintain axial orientation. tube casts that allow weight bearing on the digit are preferred to splints. ligamentous laxity in the carpus usually responds to tube casting for to days followed by bandaging and cautious exercise. the duration of similar coaptation necessary for immature carpal bones depends on the degree of immaturity and the speed with which the bones mature. because casts cannot be left on neonatal limbs for more than to days because of their fast growth, more than one cast may be necessary. treatment of tarsal valgus and rotational deformities is much less common than in the forelimbs because deviations are less common than in the forelimb, because some breeds prefer an outward position to the hindlimb, and perhaps because owners recognize it less frequently. hindlimbs generally are unaffected by ligamentous laxity, but tarsal incomplete ossification is common and often is associated with tarsal valgus. treatment of tarsal incomplete ossification is important because tarsal crushing results in an unfavorable prognosis for athletic performance. , hindlimbs require a slightly different approach to coaptation than forelimbs because of their anatomy. foals can rise to stand if their forelimbs are fixed in extension but cannot do so if their hindlimbs are extended. the multiple bony protuberances of the hock make cast sores more likely than in the forelimb, so casts are problematic. gutter splints are not useful because of the angle of the hock. severely limiting exercise is part of allowing the tarsus to mature without cartilage crushing, but foals cannot always be recumbent. extra small articulated anterior cruciate ligament splints for human beings (playmaker wraparound, dj orthopedics, vista, california) have given the best results. for small foals, a padded bandage is necessary under the splint, which is reversed to conform to the angle of the hock. the splints allow enough flexion in the hock for the foal to rise but appear sufficient when combined with stall rest to protect the cartilage from crushing. splints are left on the hocks until the cuboidal bones have ossified as shown by radiography. fetlock conformational deviations in neonates that are treated best conservatively are rare. outward rotation is the most common deviation but is thought to have minimal effect on the performance and improves with maturity. the only therapy used is to rasp the toe square to promote central breakover. severe outward rotation can promote a fetlock valgus conformation, so one can use a medial hoof wall extension of epoxy to bring the limb load medially. the most commonly treated fetlock deviations are inward but usually occur in foals older than days. however, if the deviation is noticed in neonates, one can use small lateral hoof wall extensions that generally are made of epoxy with fiberglass cloth embedded to prevent chipping. windswept foals are born with multiple deviations. evaluating the foal as a whole is best rather than focusing on individual joints. most of these foals become straight over time with conservative therapy. no surgical procedures are commonly accepted for direct treatment of rotational or carpal offset deviations, so angular deviations are described. surgical procedures to correct carpal and fetlock valgus include periosteal transection and elevation and transphyseal bridging. periosteal elevation is thought to accelerate growth on the concave side of the metaphysis, and transphyseal bridging is used to restrict the growth on the convex side of the physis. studies indicate an approximately % improvement of carpal valgus foals after periosteal transection and elevation, but unfortunately they do not compare foals that had surgery with controls that did not. , recently, some have suggested that most of the correction was unrelated to the surgery, and one experimental study supports that conclusion. as a result, at this time making firm recommendations about the indications for periosteal transection and elevation is difficult. however, periosteal transection and elevation has a low likelihood of complications and may be effective. the procedure is inexpensive and can be done in the field and therefore may be an option for clients with foals with carpal valgus in which a transphyseal bridging is undesirable or unnecessary. one indication is the very young foal born with a notably asymmetric epiphysis that results in a severe carpal valgus. this distal radial appearance is not particularly common, but the lack of ossification in the epiphysis can make a firm hold with a transphyseal bridging difficult to achieve. however, one can use distolateral radial periosteal elevation at an early age in an attempt to accelerate correction of the valgus and protect developing carpal bones. often a degree of anxiety exists about correction of fetlock angulations because of the much shorter time period for physeal growth. most fetlocks are in their final conformation by days of age, so correction is best accomplished with earlier treatment, usually by weeks of age. one can perform periosteal elevation on the medial (for varus deviations) or lateral (for valgus deviations) aspect of the distal metacarpus/metatarsus. the definitive treatment of limb angulation at a growth plate is transphyseal bridging. one should consider using the procedure at about weeks of age for all moderate to severe fetlock deviations, at about weeks for severe carpal deviations, and to weeks for mild fetlock deviations, moderate carpal deviations, and any worsening angular deformities. one must perform bridge removal when the limb straightens to prevent overcorrection. diaphyseal deviations are rare but can occur in varying degrees of severity. if the foal can bear weight on the limb, a conservative approach is indicated. one can consider periosteal elevation of the length of the concave surface of the long bone. if the foal cannot bear weight on the limb because of the severity of deviation, euthanasia is probably the best option. however, a revision osteotomy and internal fixation may be appropriate for selected foals. polydactyly is also rare and sometimes can be corrected surgically. the outcome is based on the degree of articular involvement. bacteria may invade the foal musculoskeletal system and cause orthopedic infection after delivery by the circulation, by direct extension from another system, or by direct inoculation. hematogenous delivery is by far the most common and results in infection of synovial structures (joints, tendon sheaths, bursae) and bone. extension from another site without hematogenous delivery is rare. direct inoculation almost exclusively results from traumatic rather than surgical wounds. much is still to be learned about the pathophysiology of orthopedic infection, including the source of the infecting bacteria. the umbilicus commonly is accepted as a possible source of bacteria, but many believe that the gastrointestinal and respiratory tracts are at least equally responsible. associated conditions in foals with septic arthritis include failure of passive transfer, pneumonia, and enteritis. the classification of orthopedic sepsis in foals into infection of bones and joints is probably irrelevant because most foals with septic arthritis also have infectious osteitis or osteomyelitis. , septic arthritis is more readily recognizable because the reactivity of the synovium to the bacteria causes joint effusion and lameness and because early radiographic signs of bone infection in foals are equivocal. also unclear are the reasons for the apparent site predilection for orthopedic infection in foals. the femoropatellar joint and the tarsocrural joint are affected most frequently, followed by the carpal and fetlock joints, and finally an assortment of miscellaneous joints such as the elbow, shoulder, and hip. the common association of osteomyelitis of the distal femoral, tibial, and metacarpal/metatarsal physes with a newly recognized septic arthritis suggests that the infection in that area started at the growth center (epiphysis, physis, or metaphysis). the localization of the apparent initial site of infection to the growth center has been suggested to result from "looping" metaphyseal vessels with sluggish blood flow that allow pathogens more time to escape the circulation. , however, transmission electron microscopy indicates that osteogenic cells and the vascular endothelium are a continuous network in developing embryos, indicating that the relationship between circulation and bone is more intimate than previously suspected. a possible association between osteomyelitis and thickened or traumatized cartilage exists. focal osteomyelitis lesions occur commonly at the bone cartilage junction , and particularly in areas where cartilage is attached at an angle to the long axis or where thickened. an association also exists between incomplete ossification of the central and third tarsal bones and osteomyelitis. trauma to the metaphysis is a known predisposing cause of osteomyelitis in young bacteremic rabbits. a trend exists for foals with more than one joint affected to be affected bilaterally in the same joint, rather than in random joints. this trend suggests that a "window" exists when a joint may be more susceptible to infection and that trauma to the developing cartilage may be a contributing factor. in neonates, cartilage is vascular, and possibly small traumatic cartilage lesions with associated hemorrhage and exposure of bacterial binding sites might be the inciting cause for the location of infection. the pathogens most commonly associated with septic arthritis in young foals are also those that frequently are implicated in neonatal sepsis. the most commonly isolated gram-negative organisms are escherichia coli and other enterobacteriaceae, actinobacillus equuli, and salmonella spp. frequently isolated gram-positive organisms include streptococcus spp., staphylococcus spp., and rhodococcus equi. anaerobic bacteria and fungi are rare but should be considered in refractory cases. the diagnosis of orthopedic sepsis can be challenging. the most common clinical sign is lameness, followed by swelling around a joint or metaphysis. joint effusion alone may cause the swelling, but edema is also common, especially if metaphyseal osteomyelitis is present. but effusion and edema can be difficult to detect because of the tissue surrounding the focus of infection in the shoulder, elbow, hip, and coffin joints. one should evaluate lame foals carefully by palpation to localize pain and swelling. if one can find no pain or swelling, one should obtain a complete blood count and fibrinogen level. although a complete blood count is not always abnormal in foals with septic arthritis, abnormalities should raise the index of suspicion of infection. elevations in fibrinogen are fairly common in septic arthritis, and fibrinogen almost always is elevated if the infection involves bone. if hematologic values are normal, the lameness could be caused by trauma, but the foal should be monitored closely for improvement, and closer evaluation is indicated if improvement is not rapid. an arthrocentesis is the diagnostic test of choice for confirmation of septic arthritis. one should perform joint puncture in a sterile fashion, and sedation is indicated to get an atraumatic tap. short-term anesthesia is preferable when joints have effusion because one may perform joint lavage at the same time. normal joint fluid should be clear to slightly yellow, should be viscous, and should contain less than nucleated cells per deciliter. the cell ratio should be roughly : polymorphonuclear and mononuclear. the total protein content should be less than . mg/dl. one should consider joints to be infected if the nucleated cell count is greater than , cells/dl. for joints falling between and , cells/dl, if the polymorphonuclear cell count is > %, one should consider the joints infected. cytologists are often reluctant to diagnose infection when nuclear degeneration or bacteria are not visible. this is overly conservative and results in delay in treating infections because bacteria and nuclear degeneration are rare in early cases of joint infection. out of an abundance of caution, one should treat lame foals with suspicious joints as infected unless they are clearly normal. one should always culture joint fluid in an attempt to identify the offending organisms, but because of difficulties in culturing pathogens from joint fluid samples, absence of growth does not mean absence of infection. one obtains the best culture results if the foal has not been treated with antimicrobial agents beforehand. one should obtain as much joint fluid as possible for culture and should incubate it overnight in blood culture media before plate inoculation. as always in potentially septic foals, blood culture may assist in the isolation of the organism. other orthopedic infections that do not involve the joint may be more difficult to detect. often these are not apparent until infection breeches the joint and causes lameness. however, astute caretakers may notice early clinical signs such as mild lameness, fever, or edema centered at a growth center. radiography and advanced imaging modalities such as magnetic resonance imaging are the best diagnostic tools for the localization of areas of osteitis and osteomyelitis. one should examine the area of concern carefully, giving particular attention to the growth centers and subchondral bone. interpretation of radiographs may be difficult because these areas are complex and normally have irregular bone margins in the growing foal. if a normal contralateral joint is available, comparison radiographs may be useful. because of the high metabolic turnover in growing foal bone, changes occur faster than with adults, so radiographs at the earliest sign of potential infection of bone and joint are recommended. if evidence of osteolysis is clear, aspiration of the area may yield material for culture. the goals of treatment are to eliminate infection immediately and then resolve inflammation. bacteria and products of inflammation elicited by infection are responsible for destruction of bone and cartilage. the ultimate aim of treatment is to protect the structures critical to athletic performance such as subchondral bone and cartilage in weight-bearing areas. advances in the treatment of sepsis have resulted in hospital discharge rates of % for foals with septic arthritis, but their rate of high performers is %, indicating a need for improvement. equine veterinarians cannot replace what has been destroyed, so early identification and aggressive therapies are presently the best methods to improve performance rates. one achieves the goals of treatment by physical removal of bacteria, products of inflammation, and debris and by medications to kill the bacteria and reduce inflammation. one should optimize the physiology and general health of the foal to assist this process; one should include other treatments and supportive therapies for septic foals, especially treatment of failure of passive transfer, in the therapeutic plan. intravenous administration of antimicrobials (see chapter ) is the cornerstone of treatment of orthopedic infection, and if the drug is administered early in the course of infection and bacteria are susceptible, intravenous administration may be sufficient to eliminate the organisms. however, treatment of many foals does not begin until disease is advanced. if treatment begins after bacteria have had a chance to establish themselves, one should bring all appropriate methods to bear to end the infection. additional therapies for septic arthritis include joint lavage, arthrotomy (for drainage), , debridement (arthroscopically or arthrotomy), intraarticular administration of antimicrobials, intravenous regional perfusion, and antimicrobial beads. , one can use any sterile isotonic solution to flush a joint, and additives do not appear to give significant additional benefit. if radiographs do not indicate osteomyelitis, lavage, intraarticular antibiotics, and if possible, regional perfusion are recommended. if osteitis or osteomyelitis is present, debridement is indicated arthroscopically or via arthrotomy (one should culture the debris if the pathogen is unknown). if the joint is closed, one may use antibiotics intraarticularly. if the joint is left open to drain, regional perfusion is useful. antimicrobial beads theoretically are best to use if the wound is closed, but they appear to give benefit even if the wound is open under a bandage. because of concerns about the use of beads in a joint, beads often are used in tissue defects and the surrounding tissues. the major goal is to remove material that is compromising healthy tissues and to obtain high concentrations of antimicrobials in infected tissues. high antimicrobial concentrations are necessary because adhered bacteria are difficult to kill and may require many times the in vitro bacterial minimum inhibitory concentration. intraarticular administration of antimicrobials has been used for many years and has great value. regional perfusion of diluted antimicrobials recently has come into use and may be administered intraosseously or intravenously. intravenous perfusion is preferable because no special equipment is needed, but intraosseous perfusion may be valuable where intravenous access is impossible. the concept behind both procedures is to fill the venous vasculature in the area of the infection with antimicrobials diluted by a sterile balanced electrolyte solution. one isolates the anatomic area of interest using one or two tourniquets. the perfusate diffuses into all tissues and achieves much higher concentrations than are possible using intravenous therapy. this technique has shown excellent results as an adjunct therapy for orthopedic infection. for foals, to ml total of perfusate containing mg amikacin is useful for most single joint sites. amikacin has given consistently good results without complication and is a good choice based on its concentration-dependent activity. one may use a higher volume for the stifle, but the thigh musculature makes an effective tourniquet difficult to achieve. because of concerns that perfusion might dislodge bacteria and renew systemic sepsis, high concentrations of systemic antimicrobials are recommended at the time of the perfusion. if joint lavage and intraarticular administration of antimicrobials are not sufficient to resolve infection, one may perform arthrotomy to assist the joint to drain. passive and active drains add foreign material and so are not useful. maintaining the joint under a sterile bandage is critical and can be difficult to do in proximal joints such as the stifle and elbow. tie-over bandages can be useful in this application. the best measure of success is the resolution of lameness and local inflammation. radiographs may be helpful, but the most common sign of success is a failure of the infection to progress, rather than radiographic healing. one should continue intravenously administered antimicrobials for at least week after the resolution of lameness. if an appropriate drug is available, one should give foals antimicrobials orally for at least weeks more. a total of at least weeks of antimicrobials is recommended for most foals with orthopedic infection. treatment failures usually result from an inability to kill bacteria adhered to isolated tissue (usually dead bone). sometimes this failure is caused by incomplete debridement or an inability to access a known site of infection, but more frequently it is because infection has flourished in an unknown site. for this reason, multiple imaging modalities (radiographs, ultrasound, computed tomography, and magnetic resonance imaging) used multiple times are recommended for all refractory cases of septic arthritis. osteomyelitis not associated with a joint still involves a growth center. the ideal treatment for these infections is surgical debridement, systemic antimicrobial therapy, and some form of local antibiotic delivery. , even in the face of large initial osseous defects, infection may resolve, the defect may heal, and the foal may regain normal limb anatomy and function with appropriate therapy. clinical studies on newborn throughbred foals suffering from convulsions with special reference to blood gas chemistry and pulmonary ventilation respiratory distress in a newborn foal with failure to form lung lining film clinical studies on the newborn thoroughbred foal. . heart rate, auscultation and electrocardiogram blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following h measurements of pulmonary ventilation in normal newborn thoroughbred foals during the first three days of life some parameters of respiratory function in normal and abnormal newborn foals with special reference to levels of pao during air and oxygen inhalation modern concepts of neonatal disease in foals studies on fetal, neonatal and maternal cortisol metabolism in the mare surfactant studies in the fetal and neonatal foal neuropathology of the convulsive foal syndrome metabolic profiles of newborn foals neuropathological changes associated with the neonatal maladjustment syndrome in the thoroughbred foal blood gas and acid-base status in spontaneously delivered, term-induced and induced premature foals developments in management of the newborn foal in respiratory distress. . evaluation clinical and clinicopathological characteristics of the septicaemic neonatal foal: review of cases intensive care of the neonatal foal nutritional support of the foal during intensive care comparison of empirically developed sepsis score with a computer generated and weighted scoring system for the identification of sepsis in the equine neonate ion transport properties of fetal sheep alveolar epithelial cells in monolayer culture pulmonary vascular biology during neonatal transition regulation of vasodilator synthesis during lung development morin fc rd: persistent pulmonary hypertension of the newborn: role of nitric oxide and endothelin in pathophysiology and treatment clinical studies on the newborn thoroughbred foal. . perinatal behavior the adaptive processes of the newborn foal blood pressure, electrocardiogram and echocardiogram measurements in the growing pony foal transfer of gases and metabolites in the equine placenta: a comparison with other species a comparative study of blood gas tensions, oxygen affinity and red cell , dpg concentrations in foetal and maternal blood in the mare, cow and sow neonatal polycythemia and hyperviscosity respiratory mechanics and breathing pattern in neonatal foals mechanics of ventilation: compliance respiratory studies in foals from birth to seven days old the distribution of ventilation-perfusion ratios in the lungs of newborn foals neurological examination of newborn foals maternal behavior equine medicine and surgery umbilical cord compression produces pulmonary hypertension in newborn lambs: a model to study the pathophysiology of persistent pulmonary hypertension in the newborn the sequence of events in neonatal apnoea resuscitation with room-air or oxygen supplementation resuscitation with room air instead of % oxygen prevents oxidative stress in moderately asphyxiated term neonates six years of experience with the use of room air for the resuscitation of asphyxiated newly born term infants resuscitation of newborn infants with room air or oxygen thoracic trauma in newborn foals an advisory statement from the pediatric working group of the international liaison committee on resuscitation pharmacology of pediatric resuscitation vasopressin and epinephrine for cardiac arrest temperature of the human fetus continuous monitoring of fetal temperature by noninvasive probe and its relationship to maternal temperature, fetal heart rate, and cord arterial oxygen and ph suppressive action of endogenous adenosine on ovine fetal nonshivering thermogenesis perinatal thermogenesis factors influencing the initiation of nonshivering thermogenesis reversible umbilical cord occlusion: effects on thermogenesis in utero diazepam in labour: its metabolism and effect on the clinical condition and thermogenesis of the newborn renal clearance, urinary excretion of endogenous substances, and urinary diagnostic indices in healthy neonatal foals indices of renal function: values in eight normal foals from birth to days a comparison of inulin, para-aminohippuric acid, and endogenous creatinine clearances as measures of renal function in neonatal foals effects of hyperglycemia or hypoglycemia on brain cell membrane function and energy metabolism during the immediate reoxygenation-reperfusion period after acute transient global hypoxia-ischemia in the newborn piglet equine uteroplacental metabolism at mid-and late gestation glucose and oxygen metabolism in the fetal foal during late gestation mechanism of glucose transport across the human and rat placental barrier: a review glucose production in pregnant women at term gestation: sources of glucose for human fetus estimation of glucose turnover and c recycling in the human newborn by simultaneous [ - c]glucose and [ , - h ]glucose tracers glucose disposal of low birth weight infants: steady state hyperglycemia produced by constant intravenous glucose infusion precursors to glycogen in ovine fetuses activation of glycogenolysis in neonatal liver the onset of breathing at birth stimulates pulmonary vascular prostacyclin synthesis pulmonary arterial pressure changes in human newborn infants from birth to days of age postnatal circulatory adaptation in healthy term and preterm neonates pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn regulation of pulmonary vascular tone in the perinatal period the structural basis of persistent pulmonary hypertension of the newborn infant tolazoline hcl (priscoline) nitric oxide for respiratory failure in infants born at or near term effect of inhaled nitric oxide on experimentally induced pulmonary hypertension in neonatal foals systematic review of therapy after hypoxic-ischaemic brain injury in the perinatal period infection, inflammation and the risk of cerebral palsy the fetal circulation and its response to stress fetal adaptations to spontaneous hypoxemia and responses to maternal oxygen breathing pathophysiology of perinatal brain damage enhanced calcium uptake by ca pyramidal cell dendrites in the postischemic phase despite subnormal evoked field potentials: excitatory amino acid receptor dependency and relationship to neuronal damage activation of synaptic nmda receptors by action potential-dependent release of transmitter during hypoxia impairs recovery of synaptic transmission on reoxygenation molecular and biochemical mechanisms of perinatal brain injury glycine site of the excitatory amino acid n-methyl-d-aspartate receptor in neonatal and adult brain reduction of voltagedependent mg + blockade of nmda current in mechanically injured neurons magnesium attenuates a striatal dopamine increase induced by anoxia in the neonatal rat brain: an in vivo microdialysis study pretreatment with magnesium sulfate protects against hypoxic-ischemic brain injury but postasphyxial treatment worsens brain damage in seven-day-old rats improved motor outcome in response to magnesium therapy received up to hours after traumatic diffuse axonal brain injury in rats fetal rat brain damage caused by maternal seizure activity: prevention by magnesium sulfate can magnesium sulfate reduce the risk of cerebral injury after perinatal asphyxia magnesium sulfate treatment after transient hypoxia-ischemia in the newborn piglet does not protect against cerebral damage concentrations of magnesium and ionized calcium in umbilical cord blood in distressed term newborn infants with hypoxic-ischemic encephalopathy preliminary study on the pharmacokinetics of phenobarbital in the neonatal foal adverse effects of early phenobarbital administration in term newborns with perinatal asphyxia negative pressure pulmonary edema as a post-anesthetic complication associated with upper airway obstruction in a horse cerebral oedema and cerebellar herniation in four equine neonates cerebral edema edematous necrosis in thiamine-deficient encephalopathy of the mouse a herd outbreak of equine leukoencephalomalacia dimethyl sulfoxide (dmso): a review naloxone reverses neonatal depression caused by fetal asphyxia the effects of naloxone on the post-asphyxic cerebral pathophysiology of newborn lambs naloxone exacerbates hypoxic-ischemic brain injury in the neonatal rat resuscitation of the newly born infant: an advisory statement from the pediatric working group of the international liaison committee on resuscitation neurologic disorders in foals other than hypoxicischemic encephalopathy combination therapy protects ischemic brain in rats: a glutamate antagonist plus a gamma-aminobutyric acid agonist effect of gamma-aminobutyric acid modulation on neuronal ischemia in rabbits cerebral hypothermia for prevention of brain injury following perinatal asphyxia current options in the management of apnea of prematurity hypocapnia and hypercapnia in respiratory management of newborn infants acute renal failure in children: aetiology and management low-dose dopamine in the intensive care unit: dnr or dnrx? use of dopamine in acute renal failure: a meta-analysis the effects of dopamine and epinephrine on hemodynamics and oxygen metabolism in hypoxic anesthetized piglets initial experience with norepinephrine infusion in hypotensive critically ill foal multiple organ involvement in perinatal asphyxia continuous enteral feeding impairs gallbladder emptying in infants trophic feeding of the preterm infant continuous nasogastric milk feeding versus intermittent bolus milk feeding for premature infants less than grams functional residual capacity and passive compliance measurements after antenatal steroid therapy in preterm infants current surfactant use in premature infants effects of the fungal endophyte acremonium coenophialum in fescue on pregnant mares and foal viability postnatal renal adaptation in preterm and term lambs risk factors of hypoglycemia in premature infants studies on equine prematurity. . effect of salt and water loss on the reninangiotensin-aldosterone system in the newborn foal studies on equine prematurity. . histology of the adrenal cortex of the premature newborn foal angular limb deformities in premature/ dysmature foals neonatal septicemia workshop , dorothy havemeyer foundation dorothy havemeyer foundation american college of chest physicians/society of critical care medicine consensus conference definitions of the systemic inflammatory response syndrome and allied disorders in relation to critically injured patient definition of sepsis to sirs with love failure of passive transfer in foals: incidence and outcome on four studs in new south wales a prospective study of septicaemia in colostrum-deprived foals care of the gut in the surgical intensive care unit: fact or fashion? bacterial isolates from blood and their susceptibility patterns in critically ill foals: cases ( - ) development of real-time taqman pcr systems to facilitate the diagnosis and research of septicemia in foals expressed sequence tags (ests) isolated from blood of a septic thoroughbred foal early goal-directed therapy in the treatment of severe sepsis and septic shock chronic flunixin meglumine therapy in foals clinical and pathological effects of flunixin meglumine administration to neonatal foals gastric ulcers in foals experimentally induced toxicoinfectious botulism in horses and foals toxicoinfectious botulism in foals and adult horses botulism in the horse white muscle disease of foals naturally-occurring tyzzer's disease (bacillus piliformis infection) in horse foals suspected tyzzer's disease in two foals four cases of tyzzer's disease in foals in england bacillus piliformis infection (tyzzer's disease) in foals in northwestern united states: a retrospective study of cases tyzzer's disease in a foal: light-and electron-microscopic observations clinical and clinicopathologic findings in two foals infected with bacillus piliformis serum biochemical and haematological findings in two foals with focal bacterial hepatitis (tyzzer's disease) toxic hepatopathy in neonatal foals the diagnosis and surgical correction of congenital portosystemic vascular anomalies in two calves and two foals clinical signs and radiographic diagnosis of a portosystemic shunt in a foal clinical and diagnostic features of portosystemic shunt in a foal evidence for transmission of halicephalobus deletrix (h gingivalis) from dam to foal halicephalobus (micronema) deletrix infection in two half-sibling foals listeriosis in an arabian foal with combined immunodeficiency suspected protozoal myeloencephalitis in a two-month-old colt pathological findings in horses dying during an outbreak of the paralytic form of equid herpesvirus type (ehv- ) infection central nervous system neosporosis in a foal cauda equina syndrome, diskospondylitis, and a paravertebral abscess caused by rhodococcus equi in a foal vertebral body osteomyelitis due to rhodococcus equi in two arabian foals rhodococcus equi vertebral osteomyelitis in quarter horse colts agenesis of the corpus callosum with cerebellar vermian hypoplasia in a foal resembling the dandy-walker syndrome: pre-mortem diagnosis by clinical evaluation and ct scanning cerebellar hypoplasia and degeneration in a foal cerebellar hypoplasia and degeneration in the young arab horse: clinical and neuropathological features imaging diagnosis: occipitoatlantoaxial malformation in a miniature horse foal occipitoatlantoaxial malformation with duplication of the atlas and axis in a half arabian foal occipitoatlantoaxial malformation in two non-arabian horses ivermectin toxicosis in a neonatal foal presumed moxidectin toxicosis in three foals hypovolemic hyponatremia and signs of neurologic disease associated with diarrhea in a foal extrapontine myelinolysis with involvement of the hippocampus in three children with severe hypernatremia relationships between radiography of cervical vertebrae and histopathology of the cervical cord in wobbling foals assessment of colostral transfer and systemic availability of immunoglobulin g in new-born foals using a newly developed enzyme-linked immunosorbent assay (elisa) system evaluation of a test kit for determination of serum immunoglobulin g concentration in foals relationships among serum immunoglobulin concentration in foals, colostral specific gravity, and colostral immunoglobulin concentration measurement of igg in equine blood by immunoturbidimetry and latex agglutination a rapid, specific test for detecting absorption of colostral igg by the neonatal foal practical methods of determining serum immunoglobulin m and immunoglobulin g concentrations in foals passive immunity in the foal: measurement of immunoglobulin classes and specific antibody gammaglobulin and antibody variations associated with the maternal transfer of immunity and the onset of active immunity immunoglobulin metabolism in the neonatal foal prevalence (treatment days) and severity of illness in hypogammaglobulinemic and normogammaglobulinemic foals factors associated with failure of passive transfer of colostral antibodies in standardbred foals failure of passive transfer in foals failure of passive transfer of colostral immunity in the foal: incidence, and the effect of stud management and plasma transfusions the incidence and consequences of failure of passive transfer of immunity on a thoroughbred breeding farm failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals secretion and composition of colostrum and milk immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses interleukin- in human milk improved recovery of insulin-like growth factors (igfs) from bovine colostrum using alkaline diafiltration cytokine-inducing activity of a proline-rich polypeptide complex (prp) from ovine colostrum and its active nonapeptide fragment analogs measurement of betacellulin levels in bovine serum, colostrum and milk in vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence activities of gammaglutamyltransferase, alkaline phosphatase and aspartateaminotransferase in colostrum, milk and blood plasma of calves fed first colostrum at - , - , - and - h after birth efficacy of intravenous plasma to transfer passive immunity in clinically healthy and clinically ill equine neonates with failure of passive transfer evaluation of intravenous administration of concentrated immunoglobulin g to colostrumdeprived foals lyophilized hyperimmune equine serum as a source of antibodies for neonatal foals absorption of bovine colostral immunoglobulins g and m in newborn foals comparison of freezing and lyophilizing for preservation of colostrum as a source of immunoglobulins for calves a comparison of the reduction in immunoglobulin (igg) concentration of frozen equine plasma treated by three thawing techniques use of blood and blood products neonatal isoerythrolysis in mule foals characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis prevalence of anti-red blood cell antibodies in the serum and colostrum of mares and its relationship to neonatal isoerythrolysis polymerized hemoglobin therapy in a foal with neonatal isoerythrolysis post-transfusion survival of cr-labeled erythrocytes in neonatal foals strategies for prevention of neonatal isoerythrolysis in horses and mules detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia neonatal alloimmune thrombocytopenia in a quarter horse foal neonatal thrombocytopenia: new insights into pathogenesis and implications for clinical management thrombocytopenia in horses detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia reef vb: cardiovascular disease in the equine neonate congenital polyalveolar lobe in three foals critical pulmonary stenosis in a newborn foal bilateral hypoplasia of the soft palate in a foal pulmonary lobar hypertrophy in a foal equine congenital defects congenital bilateral choanal atresia in a standardbred foal evaluation of pulse oximetry in anaesthetised foals using multiple combinations of transducer type and transducer attachment site electrocardiographic findings during parturition and blood gas tensions immediately after birth in thoroughbred foals blood gas and acid-base changes in the neonatal foal blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following h mixed venous blood gases in recumbent and upright positions in foals from birth to days of age ventilatory support of the neonatal foal report of foal pneumonia panel association of microbiologic flora with clinical, endoscopic, and pulmonary cytologic findings in foals with distal respiratory tract infection microbiologic changes during antimicrobial treatment and rate of relapse of distal respiratory tract infections in foals pathologic changes and pathogenesis of parascaris equorum infection in parasite-free pony foals spezifische infektiose pneumonie beim fohlen. ein neuer eiterreger beim pferd interaction of rhodococcus equi with phagocytic cells from rhodococcus equi-exposed and non-exposed foals electron microscopic investigation of intracellular events after ingestion of rhodococcus equi by foal alveolar macrophages influence of rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses rhodococcus equi infection of monocytes/macrophages from human immunodeficiency (hiv)-infected patients and healthy individuals: evaluation of intracellular killing and nitric oxide production the intracellular bacterium rhodococcus equi requires mac- to bind to mammalian cells rhodococcus (corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses identification of -to -kilodalton antigens associated with virulent rhodococcus equi virulence-associated -to kilodalton antigens in rhodococcus equi: temperature-dependent expression and location of the antigens role of the -kilobase plasmid and plasmid-encoded virulence-associated protein a in intracellular survival and virulence of rhodococcus equi characterization of avirulence-associated gene family in rhodococcus equi corynebacterium equi infections in horses, - : a review of cases clinical manifestations, diagnosis, treatment, and prevention of rhodococcus equi infections in foals detection of corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay rhodococcus equi pneumonia in foals: comparison of elisa and agid serology on a commercial thoroughbred breeding farm studies of naturally occuring and experimental rhodococcus equi (corynebacterium equi) pneumonia in foals detection of virulent rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of r. equi pneumonia in foals comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals use of erythromycin-rifampin combination in treatment of rhodococcus equi pneumonia pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals hyperthermia in foals treated with erythromycin alone or in combination with rifampin for respiratory disease during hot environmental conditions clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia strategies for the control of rhodococcus equi infections on enzootic farms immunoprophylaxis of rhodococcus equi pneumonia in foals failure of hyperimmune plasma to prevent pneumonia caused by rhodococcus equi in foals rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals protection against naturally acquired rhodococcus equi pneumonia in foals by administration of hyperimmune plasma methods of implementation of an immunoprophylaxis program for the prevention of rhodococcus equi pneumonia: results of a -year field study effect of prophylactic administration of hyperimmune plasma to prevent rhodococcus equi infection on foals from endemically affected farms valuation of equine immunoglobulin specific for rhodococcus equi virulenceassociated proteins a and c for use in protecting foals against rhodococcus equi-induced pneumonia associations between physical examination, laboratory, and radiographic findings and outcome and subsequent racing performance of foals with rhodococcus equi infection: cases ( - ) endoscopic and virological observations on respiratory disease in a group of young thoroughbred horses in training epidemiology of ehv- and ehv- in the mare and foal populations on a hunter valley stud farm: are mares the source of ehv- for unweaned foals? clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus- on a stud farm foetal and neonatal foal losses on equine herpesvirus type (ehv- ) infected farms before and after ehv- vaccination was introduced an outbreak of foal perinatal mortality due to equid herpesvirus type : pathological observations equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations involvement of adenovirus in pneumonia in a thoroughbred foal adenoviral infection of arab foals with respiratory tract disease isolation of an adenovirus from an arab foal with a combined immunodeficiency disease clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus- infection compared with septic and premature foals neonatal equine herpesvirus type infection on a thoroughbred breeding farm passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a standardbred herd with high seroprevalence fatal nonneurological ehv- infection in a yearling filly pulmonary vasculotropic ehv- infection in equids virological and molecular biological investigations into equine herpes virus type (ehv- ) experimental infections equine herpesvirus type : prevalence and seroepidemiology in foals subcutaneous emphysema in a neonatal foal immature epithelial na+ channel expression is one of the pathogenetic mechanisms leading to human neonatal respiratory distress syndrome bronchointerstitial pneumonia and respiratory distress in young horses: clinical, clinicopathologic, radiographic, and pathological findings in cases ( - ) risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: cases ( - ) disease of foals du plessis jl: rupture of the bladder in the newborn foal and its surgical correction metabolic abnormalities associated with rupture of the urinary bladder in neonatal foals exploratory celiotomy for suspected urinary tract disruption in neonatal foals: a review of cases uroperitoneum in the foal uroperitoneum in the hospitalized equine neonate: retrospective study of cases repair of a defect in the bladder of a foal rupture of the urinary bladder in neonatal foals bladder defects in newborn foals ultrasound of the urinary tract nonsurgical management of ruptured urinary bladder in a critically ill foal hemodialysis for treatment of oxytetracycline-induced acute renal failure in a neonatal foal bilateral renal hypoplasia in four young horses bilateral renal dysplasia and hypoplasia in a foal with an imperforate anus clinical vignette: renal arteriovenous malformation in a quarter horse foal percutaneous ultrasound-guided pyelography aided diagnosis of ectopic ureter and hydronephrosis in a -week-old filly bilateral ureteral tears in a foal multiple ureteral defects in a belgian foal congenital defects in newborn foals of mares treated for equine protozoal myeloencephalitis during pregnancy ultrasonography of umbilical structures in clinically normal foals clinical, ultrasonographic, and surgical findings in foals with umbilical remnant infections closure of the abdominal wall at the umbilicus and the development of umbilical hernias in a group of foals from birth to months of age complications of umbilical hernias in horses: cases persistent vitelline vein in a foal atresia coli in the foal: a review of six cases endothelin receptor b polymorphism associated with lethal white foal syndrome in horses a dinucleotide mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome (lwfs): a horse variant of hirschsprung disease a missense mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome: an equine version of hirschsprung disease incidence of the endothelin receptor b mutation that causes lethal white foal syndrome in white-patterned horses necrotizing enterocolitis presenting in the emergency department: case report and review of differential considerations for vomiting in the neonate new concepts in necrotizing enterocolitis hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals endoscopic appearance of gastric lesions in foals: cases endoscopic evaluation of changes in gastric lesions of thoroughbred foals gastroduodenal ulceration in foals prevalence of gastric lesions in foals without signs of gastric disease: an endoscopic survey gastric ulcers in foals exsanguination due to gastric ulceration in a foal gastrointestinal diseases of foals pathophysiology of peptic disorders in foals and horses: a review clinical syndromes of gastric ulceration in foals and mature horses gastroduodenal ulceration in foals stress ulcer: is routine prophylaxis necessary? peptic ulcer pathophysiology intragastric ph in critically ill neonatal foals and the effect of ranitidine a review of medical treatment for peptic ulcer disease effect of sucralfate on healing of subclinical gastric ulcers in foals effect of ranitidine on intragastric ph in clinically normal neonatal foals effects of omeprazole paste on healing of spontaneous gastric ulcers in horses and foals: a field trial incidence of pneumonia in mechanically ventilated patients treated with sucralfate or cimetidine as prophylaxis for stress bleeding: bacterial colonization of the stomach the protective role of gastric acidity in neonatal bacterial translocation upper gastrointestinal tract bleeding in critically ill pediatric patients gastric colonization as a consequence of stress ulcer prophylaxis: a prospective, randomized trial is prophylaxis for gastric ulcers necessary in critically ill equine neonates? a retrospective study of necropsy cases - stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration influence of stress ulcer prophylaxis on translocation of bacteria from the intestinal tract in rats effects of sucralfate on acute gastric mucosal injury and gastric ulcer induced by ischemiareperfusion in rats stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration efficacy of ivermectin in controlling strongyloides westeri infections in foals foal diarrhoea between and in the united kingdom associated with prevalence of and risk factors for fecal shedding of cryptosporidium parvum oocysts in horses overwhelming strongyloidosis in a foal verminous arteritis in a -month-old thoroughbred foal randomised trial of fluid restriction in ventilated very low birthweight infants restricted versus liberal water intake for preventing morbidity and mortality in preterm infants hyponatraemia in children with acute cns disease: siadh or cerebral salt wasting? comparison of norepinephrinedobutamine to dopamine alone for splanchnic perfusion in sheep with septic shock depletion of neurohypophyseal content of vasopressin in septic shock hemodynamic and metabolic effects of low-dose vasopressin infusions in vasodilatory septic shock references . firth ec, poulos pw: blood vessels in the developing growth plate of the equine distal radius and metacarpus microangiographic studies of metaphyseal vessels in young foals physeal form of the longbones of the foal comparison of splinting and casting on the degree of laxity induced in thoracic limbs in young horses histological features of the dorsal cortex of the third metacarpal bone mid-diaphysis during postnatal growth in thoroughbred horses effect of oxytetracycline on metacarpophalangeal and distal interphalageal joint angles in newborn foals the blood supply of the growth plate and the epiphysis: a comparative scanning electron microscopy and histological experimental study in growing sheep fractures of the proximal sesamoid bones in thoroughbred foals avulsion of the origin of the peroneus tertius tendon in a foal avulsion fracture of the origin of the extensor digitorum longus muscle in a foal surgical reconstruction of a ruptured medial collateral ligament in a foal correction of patellar luxation by recession sulcoplasty in three foals lateral patellar luxation in miniature horse foals anatomy and therapeutic resection of the peroneus tertius in a foal preliminary observations on oxytetracycline treatment of congenital flexural deformities in foals effect of oxytetracycline on metacarpophalangeal and distal interphalangal join angles in newborn foals torsion in quadrapeds and its impact on mammalian joints computer model of endochondral growth and ossification in long bones: biological and mechanobiological influences observations on the evaluation and selection of foal limb deformities for surgical treatment treatment response and athletic outcome of foals with tarsal valgus deformities: cases incomplete ossification of the tarsal bones in foals: cases periosteal transection and periosteal stripping for correction of angular limb deformities in foals periosteal transection and stripping for treatment of angular limb deformities in foals: clinical observations restricted exercise and transphyseal bridging for correction of angular limb deformities effect of hemicircumferential periosteal transection and elevation in foals with experimentally induced angular limb deformities diaphyseal angular limb deformities in three foals current concepts of infectious polyarthritis in foals factors associated with prognosis for survival and athletic use in foals with septic arthritis: cases ( - ) the site of focal osteomyelitis lesions in foals pathological features of multiple bone infection in the foal stromal cell culture and relationships in perimedullary spaces of chick embryo shaft bones tarsal osteomyelitis in foals cartilage canals in equine articular/epiphyseal growth cartilage and a possible association with dyschondroplasia a histological study of acute hematogenous osteomyelitis following physeal injuries in rabbits open drainage, intra-articular and systemic antibiotics in the treatment of septic arthritis/tenosynovitis in horses comparison of various treatments for experimentally induced equine infectious arthritis a retrospective study of horses affected with septic arthritis/tenosynovitis regional intravenous perfusion of the distal limbs of horses with amikacin sulfate treatment of sepsis in the small tarsal joints of horses with gentamicin-impregnated polymethylmethacrylate beads use of antibioticimpregnated polymethyl-methacrylate in horses with open or infected fractures or joints: cases ( - ) the effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse regional perfusion of the equine carpus for antibiotic delivery how to perform equine digital intravascular perfusion surgical management of rhodococcus equi metaphysitis in a foal intraosseous regional perfusion for treatment of septic physitis in a -week-old foal key: cord- - if qquj authors: nan title: perspectives on the economics of the environment in the shadow of coronavirus date: - - journal: environ resour econ (dordr) doi: . /s - - - sha: doc_id: cord_uid: if qquj nan the environmental and resource economics special issue "economics of the environment in the shadow of coronavirus" comes at a hugely critical time for environmental economists and policy makers alike. we are in a situation of significant social change, a change that could potentially lay the foundation for mankind's future in the years to come. as part of this special issue, ere is trialling a novel, experimental form of article, drawing together short, focussed pieces from a wide group of authors addressing the plethora of issues which such a fundamental challenge as the coronavirus pandemic generates. these provide critical and reflective perspectives on the environmental, socio-economic and policy paths that may be taken in the near and further future-strategies that could lead mankind either on roads to a much more sustainable development, or along paths that could bring about more instability, inequality and further environmental pressures. this innovative article combines short, policy-relevant and less technical papers that deal with specific aspects and provide clear recommendations for policy makers and suggestions for future research alike. the target audiences are policy makers and companies, but also researchers who want quick yet sufficiently detailed knowledge about particular analyses relating to covid- and issues in environmental economics. we hope that the articles contained within this perspectives collection provide the necessary information for policy makers to take wise decisions for our future, and for researchers the knowledge to help guide policy makers in their decisions. humankind has been very fortunate to have lived through a period of sustained economic growth pretty much since the agricultural revolution, with especially high rates of growth starting from the second half of the twentieth century. this economic progress has allowed us to make unprecedented improvements in consumption, in health, in education and in addressing inequality. many of us have been fortunate enough to have lived without a war for the past years, which is widely believed to be due to the development of international institutions and a deepening of international trade that led to widespread cooperation and, with it, it brought a new era of global stability. at the same time, the rapid increases in humankind's population, from around billion in to . billion in , coupled with an increase in real-world gdp by a factor of roughly during the same period, have led mankind to progressively push closer to the boundaries of planet earth. to provide additional food for the surge in population, agricultural land use has increased by %; to provide goods and services for the surge in demand, the material footprint of our production increased by an estimated factor of ; and to provide energy for our lifestyles, our use of non-renewable and polluting resources (coal, oil, gas) increased by a factor of . this increase in consumption coupled with a similar increase in input use has transformed the face of the planet earth and has given rise to unwanted side effects and new challenges. some of these challenges are well known, such as local and global pollution, problems of waste and certainly climate change. another, often neglected challenge, has been a consistent pressure on biodiversity due to our increase in land use. a mixture of burgeoning population and increasing resource use that carved deeply in nature's pristine areas has led to species conflict manifested not only in the rapid loss of other species, but also in a much ignored yet increasingly visible negative feedback in the form of viral crossovers (smith et al. ). the linkages between economic development, viral crossovers in the form of communicable diseases and environmental issues in particular have, up to now, seen little attention from environmental economists. as we have now seen, it was worryingly neglectful on our part to not consider these feedbacks more seriously. the greater interconnectedness via global trade and international migration, air travel for both tourism and business purposes, as well as the ongoing growth of large city hubs, have made it easy for communicable diseases to transcend local spaces and quickly make their appearance in even remote corners of the world. while the black death and the spanish flu have been among the worst communicable disease outbreaks in recent history, in late a new virus was detected in wuhan, china. identified as a new member of the coronavirus family and subsequently called covid- , within the course of half a year this virus has spread out from the huanan wet market in wuhan across the whole world. even inhabitants from otherwise remote places such as villages in timbuktu, the korubo and yanomami tribes of the amazon, the navajo nation of north america and the arctic inuit have already tested positive for due to initial uncertainty surrounding both the impact of covid- and its spread through society, many policy makers quickly decided to shut down interactions among individuals by restricting local, national and international mobility. these "lockdowns" had pervasive impacts on economic activity across the globe, with significant reductions in production, increases in unemployment, falls in international migration, diminished levels of international trade, significant increases in bankruptcy filings and large ripple effects down supply chains. relative impacts between developed and developing countries are still very much developing. globally, herd immunity is expected to take some time to develop if indeed it ever does. a vaccine that has the potential to be potent and widely available may need at least another or years for development and broad deployment. some countries are already close to a second wave-this pandemic is here to stay for a while. the question is as to how we shall deal with it. while reducing physical contacts to "flatten the curve" of disease and death has been the preferred policy to slow down the spread of the were advocating for. it is at this point where the contributions selected for this special issue provide first thoughts, first answers and first suggestions for policy makers from the cuttingedge research of environmental economists. in particular, the arguments forwarded support a strengthened focus on economic recovery that, first and foremost, should not undermine the green transition, while also, if possible, provide measures to advance the green transition. the articles then discuss the approaches and potential difficulties that policy makers will be faced with when being confronted with the precise means to implement these green recoveries. as a first step, due to unprecedented levels of unemployment in places such as the usa and significant contractions to economic growth in most countries of the world, an important consideration is that the focus of the stimuli packages should be the economic recovery, i.e. to predominantly deal with the direct impact of the lockdowns on economic activity. once the virus is contained and the short-run recoveries are under way, then it is, however, important to quickly integrate longer-term factors into policy making (borghesi and co-authors). here, it is vital that, in contrast to the stimuli in the aftermath of the financial crisis, policy makers also address inequality (koundouri and co-authors). a more specific focus on furthering a green transition should only be placed once a certain level of economic recovery has been achieved. this is especially vital as the disruptions to supply chains can have fundamental and unpredictable consequences, as often even companies themselves are not fully aware of their complete supply chains. cazcarro and co-authors estimate some of the impacts of these trade-related supply chains and, for example, show that the european demand changes due to covid- have, in total, larger impacts on the rest of the world than on europe itself. requirements for successful green stimuli are that these policies are implemented in a clear and transparent manner (rickels and peterson). in this regard, ing and nicolai argue that companies are likely to prefer stimuli packages that are tight to some environmental efforts rather than to new environmental regulations. on the converse, linking stimuli with environmental efforts is more costly for policy makers and likely to be less efficient. several of the articles in this special issue draw particular attention to the fact that the green stimuli are not enough to successfully further a green transition. what is also necessary is to couple this with a price on carbon and a restructuring of the subsidies paying attention to both the green and fossil industry (gawel and lehmann). a stronger social contract with a higher degree of citizen involvement will furthermore help gain public support but also strengthen social norms and thus decentralized internalization of externalities. lopez-feldman and co-authors discuss the policy responses to covid- with a focus on latin america and argue that, to minimize the likely rebound effect, policies need to be much better coordinated. we have seen that international cooperation quickly breaks down when a crisis looms, so that it would make sense to design international institutions with binding laws and penalties in case of non-compliance. what we have seen so far is that covid- has the potential to become a game changer when it comes to combining stimulus packages with the green transition. that this is a sensible strategy derives from the observation that restricting global warming to . °c requires efforts that go beyond what countries were willing to do so far, and that the stimuli provide the needed opportunity. while the articles contained in this special issue already provide many reasons for policy makers to push for green stimuli, they also clearly point out the difficulties associated with implementing these well. some articles in this special issue also show limitations of current policies or research approaches. for example, borghesi and co-authors discuss that during the covid- crisis the market stability reserve helped to stabilize the eu ets price, but imperfectly. it is, therefore, important to consider ways in which these imperfections can be redesigned. on a different topic, laude explains how the covid- crisis has brought to light both advantages and problems with having local, short supply chains for food, and that there is a substantial lack of research directed towards the impact of crises on the agricultural sector. another example is a more cautionary tale and deals with the covid- cases data. here, cohen and co-authors show very clearly that researchers must be careful with simply using these data as there are many problems in the data collection processes, which differ across countries and also time. as a final remark, we would like to observe that this special issue not only comes at a very turbulent time for mankind in general, but it also comes at a special time for environmental economists in particular. the covid- crisis gives the opportunity to invest significant amounts of money towards aiding the green transition, and the widespread public support is there. we need to now be able to advise policy makers on efficient, reasonable and relevant policies that they may implement as part of the green stimuli packages. however, these policies also need to be well structured and grounded in good research. the peer-reviewed articles in this special issue provide suggestions and articles with these features and will thus, hopefully, serve as a first benchmark in this endeavour. in this paper, we use a multisectoral and multiregional model of the world economy to evaluate the short-term effects that the covid- crisis may have on environmental pressures and resource consumption (measured in terms of water, air emissions and materials extraction). specifically, we focus on the relationship between current and forecast changes in demand and mobility patterns in the eu + uk for and its effect on global resources through global supply chains. this integrated analysis could answer urgent research questions: what are the short-term impacts on emissions and resource consumption of the current and predicted declines in final demand? do the impacts differ among environmental pressures? how elastic are environmental responses to demand drops? on the basis of these short-term responses, what is the relationship between economic growth and environmental pressure? do these effects go beyond european countries through global supply chains? in order to evaluate the short-term effects of changes in aggregated sectoral consumption and demand on environmental pressures, and their diffusion through global supply chains, we develop an environmentally extended multiregional input-output model (see miller and blair , or recently hubacek et al. ; guan et al. ), using the exiobase . database (stadler et al. ) . we focus on water consumption (blue and green), mineral extraction and emissions (co eq , sox, nox, nh and co). we rely on the estimates from the eurostat spring forecast (ec a) which estimate for the eu + uk a change of − % in consumer expenditure, . % in government expenditure and − . % in investment for . sectoral changes in private consumption are estimated based on ec ( b) and oecd ( ), assuming different sectoral sensitivities to the covid- crisis. specific details and the complete matching process can be found in online appendix. our results are as follows: figure shows the eu + uk (light blue bars) and global (dark blue bars) change in different environmental pressures associated with the shock to final demand and household mobility in eu + uk, as a consequence of the covid- lockdown. the expected percentage impacts are much larger in the eu + uk, as the simulation of final demand and mobility directly affects these countries, given the high level of intra-eu dependence. however, the european lockdown also impacts foreign resources due to the interlinkages throughout the production chain. in general, the largest decline happens to gas emissions, both in eu + uk and worldwide. the average . % fall in the aggregate of the three cited components of european final demand (households, government expenditure and investment, representing a . % fall in total final demand) that globally represents a . % decline, would involve a . % decline in global water consumption and . % in mineral extraction. the global fall in emissions from the slowdown in economic activity and european mobility restrictions would be around . % (co eq ). comparing the effect in eu + uk with the global impact, the percentage change runs from four times larger in europe, in the case of water, to times greater for co eq emissions. we find a drop of more than % for mineral extraction and emissions in eu + uk. emissions are reduced, and by more than water and minerals. also, even when no direct electricity demand change is assumed, given the reduction in sector activity, indirect energy demand diminishes and so do emissions. specifically, our model indicates that the production and supply of electricity explain approximately % of the greenhouse emissions fall worldwide and % in the eu + uk. in line with the results of le quéré et al. ( ) , our estimates confirm that the reduction of emissions associated with private mobility restrictions would account for % of the total fall in emissions in europe, whereas the reduction linked to air travel reaches . %. worldwide, these drops would be % and %, respectively. the european lockdown affects the iron and steel industries globally, representing % of the global fall in greenhouse emissions. several other resources are less dependent on the most-affected sectors. the supply chain of food clearly depends on the supply of water. it is difficult to estimate the reductions in the horeca sector based on the available data. that sector, as well as many others that indirectly require these natural factors throughout their supply chains, exhibits significant reductions in resource use. our data show that the largest decline in water consumption (both blue and green) is associated with the primary sectors, which, as expected, experience slight negative growth rates around − . %, given the relatively stable and anticyclical nature of food demand. analysing the changes induced by the different components of final demand, the decrease in household consumption and investment drives environmental impacts to a lesser extent. worldwide, the fall in investment mainly affects environmental impacts (except for water consumption). reductions in investment have a larger role in reducing emissions in small countries like croatia and malta, and also in eastern europe, such as in hungary, latvia, and slovenia. in the eu + uk, household consumption is the most significant element of final demand driving the changes in environmental pressures (with the exception of mineral extraction), explaining % of the european greenhouse gas emissions fall. as for minerals, their strong dependency on gross capital formation destinations, such as construction, manufacture of machinery and equipment, computer and related activities, explains that the . % fall in investment worldwide (- . % in eu + uk) triggers a . % drop in their global extraction (− . % in europe). these changes are distinct by country, with implications for different areas of the world. the intra-eu trade is revealed to be highly important, showing that the main changes occur within the eu + uk countries (even if the individual shock of a given country is not so significant). figure displays the percentage falls in co eq , so x , no x and nh emissions by eu + uk country. according to the projected changes, the greatest cuts are expected in co eq emissions. almost a quarter of the estimated reduction in co eq emissions occur in germany (figure si ) . the reductions in co eq emissions in smaller economies, such as ireland, austria, greece, and cyprus exhibit the largest percentage falls. reductions in mediterranean and eastern countries are high, mainly in large countries like france, romania, portugal, and spain. we note the potential reduction in no x emissions as a consequence of the production shutdown in france and germany (as it occurs with nh emissions), but the largest percentage drops occur in smaller economies, like austria, ireland, and greece. france also shows the highest reductions in so x emissions, together with poland and germany, ireland and smaller countries. our model also allows us to evaluate how the reduction in the eu + uk demand modifies the pressures on environmental resources outside europe, by considering transmission effects through global supply chains. the largest percentage reduction in domestic impacts arising from the lockdown and subsequent situations in europe occurs in non-european developed countries, with smaller effects in developing economies (fig. ) . we find considerable indirect declines in mineral extraction (larger than %) and gas emissions (between − . and − . %) in the usa and japan. these are mostly associated with linkages with italy, germany, uk and france. in countries such as china, brazil and india, none of the falls in environmental pressure exceeds . % (with the exception of a % fall in mineral extraction in india). again, we find important declines related to the commercial linkages of developing countries with european powers, such as france, germany and the uk, and with other european countries severely affected by the pandemic (and thus their final demand), such as italy and spain. these results support the statements of baldwin and mauro ( ) on the existence of a "supply-chain contagion" related to the covid- lockdown (in europe in this study). however, the "environmental supply-chain contagion" is modest outside the eu + uk. one of the main implications of our work is that the changes occurring in in the eu + uk are not, in and of themselves, able to sufficiently reduce global environmental pressures. these changes have entailed reductions in domestic activities, which have also affected other eu partners, given the high level of intra-eu trade. although some of those changes do not have strong impacts on domestic environmental pressures, transport restrictions within the eu have notably reduced co eq emissions and, even more positively, reduced other pollution-induced health damage. changes outside europe occur due to spillover effects, being relatively more notable for minerals. however, the demand for goods, which ultimately depends on resource use and pressures external to the eu + uk, has not fallen so clearly, and the lion's share of the pressures has not been reduced as much as final demand. in short, we have shown the importance of intersectoral relationships and multipliers to understand demand changes, which are uncertain along , and which we anastasios xepapadeas on december , the european commission presented the european green deal (egd )-a roadmap for making the eu's economy sustainable by turning climate and environmental challenges into opportunities across all policy areas, and making the transition just and inclusive for all. the central objective of the egd is to attain a climate neutral eu by , which means that the eu will aim to reach net-zero greenhouse gas emissions by that year. the actions required to reach this target include decarbonizing the energy sector, which accounts for more than % of the eu's greenhouse gas emissions; renovating buildings to help reduce energy use which currently accounts for % of the eu's energy consumption; supporting industry so it can innovate and become a global leader in the green economy; and promoting cleaner transport, which constitutes an important source of the eu's emissions. in terms of resources needed, at least € trillion (european commission ) are projected to be necessary over the next decade according to the european commission, with sources including the eu budget, national budgets and the private sector. furthermore, the egd encompasses the so-called just transition mechanism whose objective is to help reduce the negative impacts on coal mines or steel factories associated with decarbonization. the roadmap for the egd includes actions related to climate ambition, clean energy, circular economy, smart mobility, greening the common agricultural policy, preserving and protecting biodiversity, and attaining a toxic-free environment and was planned to commence during the spring of . the covid- pandemic which appeared in europe in early , and the containment measures taken in order to control the pandemic and reduce the transmissibility of the virusthe r -below , have had a profound impact on the economy. in terms of macroeconomics, the covid- shock on the economy can be regarded as a keynesian supply shock in a multi-sector economy which triggers shortages in aggregate demand larger than the shocks themselves (guerrieri et al. ) . policies to deal with immediate impacts of covid- are aimed at a fast recovery from the recession, so that the policies are designed and implemented in a short-run context. the question which this note seeks to explore is whether the policies undertaken as a response to covid- could have long-run implications-positive or negative-in terms of sustainability and the objective of carbon neutrality. arrow et al. ( ) state that economic development is sustained at a given point in time if intergenerational well-being is non-declining at this point in time. intergenerational well-being is non-declining if the comprehensive wealth of the economy is non-declining. comprehensive wealth is the value of the assets of an economy, with the asset base or productive base consisting of reproducible capital, natural capital, human capital and health capital. social capital can also be included in the productive base. thus, the issue of sustainability can be analysed in terms of non-declining comprehensive wealth or productive base. the implications of the covid- shock on sustainability should therefore be examined in the context of its impacts on the productive base of an economy. the impact of covid- on the productive base is realized directly through morbidity and mortality and indirectly through the recession that is induced by policies implemented to contain the pandemic and the positive effects of public spending aimed at recovery. r is the basic reproduction number which is defined as the average number of secondary infections produced when one infected individual is introduced into a host population where everyone is susceptible. in a fully susceptible population, an infection can get started if and only if r > . if r < , a typical infective replaces itself with less than one infective, and the number of infectives tends to zero with the passage of time (e.g. hethcote ) . see, for example, the world economic outlook (imf, ) in which the projection for the percentage change in output in the euro area in is − . % with a rebound to + . % in . it should be noted, however, that in the current world of deep uncertainty, these predictions could be inaccurate. greenstone and nigam ( ) indicate that moderate distancing policies have substantial economic benefits in terms of mortality benefits and avoided hospital intensive care unit costs. the valuation is performed in shadow prices, with the shadow price for an asset being the present value of the contribution to well-being from one additional unit of the given asset. sustainability can also be defined in a similar way in terms of comprehensive investment. thus, the virus does not destroy reproducible capital per se, but it reduces its utilization through containment policies. however, the pandemic could affect capital accumulation if the recession slows down investment in reproducible capital. covid- seems to be beneficial for natural capital, at least in the short run. coal use fell by % at china's six largest power plants between the last quarter of and march , while in europe satellite images showed nitrogen dioxide (no ) emissions fading away over northern italy, with a similar picture in spain and the uk (henriques ) . according to a recent international energy agency ( ) report, global co emissions are expected to decline during to . gtco , which is . % lower than in ( . gtco ) (global carbon project ). this would be the lowest level since , and six times larger than the previous record reduction of . gt in due to the financial crisis. however, the international energy agency ( ) report also indicates that if efforts to contain the virus and restart economies are more successful, the decrease in energy demand could be limited to less than %. since the international energy agency's ( ) april report, data show signs of a recovery in carbon emissions. a very recent reportin june -states that new data show a v-shaped recovery in carbon emissions, with carbon emissions declining from february , reaching a minimum in april and then recovering slowly towards the february levels (domjan ) . regarding human capital, defined in terms of changes in the work force and education level, the pandemic is having a short-term negative effect on work force, but in the long term it might change educational patterns and the geographical structure of the supply of education if extensive on-line teaching is established. health capital is, of course, negatively affected through the value of statistical life. the above discussion suggests that the pandemic has a profound impact on the factors characterizing sustainability. thus, policies to start up the economy after appropriate containment of the pandemic and return to long-term desired growth paths will be beneficial for productive-based sustainability components such as health, human and social capital and reproducible capital through the increase in its utilization. the issue of primary interest is the impact of recovery-related policies on natural capital and climate change. this issue relates to the way in which the egd might be adjusted during the start-up period and whether the current beneficial impact of the virus-induced recession on emissions signals a long-term impact. recent results in climate science have established an approximately proportional relationship between the change in the global average surface temperature relative to the preindustrial period (the temperature anomaly) and cumulative co emissions relative to the same base period (e.g. matthews et al. ). with cumulative emissions since being approximately ± gtco (friedlingtein et al. ) , the expected reduction of approximately . gtco relative to due to the covid-related recession will have negligible effects on the temperature anomaly. furthermore, if the recovery projections in are realized, then it is reasonable to expect that the pre-pandemic situation will reemerge with regard to greenhouse gas emissions, unless the pandemic continues in strong recurring waves which make extended and persistent lockdowns necessary. this situation, however, cannot be regarded as the most likely scenario. if emissions recover in the short or even medium term, the projected time period for crossing the . °c threshold-according to the business-as-usual or alternative emissions paths-will not be affected in a significant way (ipcc ) . in this context, the covid- event is expected to have a negative impact on the global productive base (health, human, social and reproducible capital) and therefore on the global sustainability conditions, while the seemingly beneficial impact on natural capital, and especially climate, will be temporary and a return to pre-pandemic paths is most likely. this means that in the post-pandemic world, risks related to climate change damages, including risks from tipping elements and crossing of climate thresholds, are not expected to change. how does this picture fit with the european response to covid- in the context of the egd? the eu is currently developing and implementing a number of policies and stimulus packages to address the recession. the purpose of this note is not to analyse these measures, but rather to explore induced adjustments to the egd. it has been reported that there will be some reprioritization of edg initiatives as a result of the eu response to the pandemic (involved in europe ). some of the initiatives such as the renewed sustainable financial strategy, which aims to increase private investment in sustainable projects, or the "renovation wave", will remain since they are expected to stimulate economic activity; others such as "offshore renewable energy" or "the biodiversity strategy for " might be delayed, but initiatives such as the new eu strategy on adaptation to climate change and the new eu forest strategy will be delayed to . it is clear that the covid- shock and the need to start up the economy will make substantial policy changes necessary. looking at sustainability in terms of natural capital and the environment, it should be clear that any short-run improvement is transient, while looking at sustainability in terms of climate change, it is most likely that there will be no change in the long-term trends. what is important is that, after the shock, the start-up will be based on environmentally friendly policies. thus, maintaining initiatives such as the renovation wave, or promoting the pillar of cleaner transport, is important. on the other hand, delaying initiatives like the strategy on adaptation to climate change may need to be reconsidered. this is because the rationale behind delaying such strategies seems to be that recovery from the recession is expected to be rapid and therefore the delays will be of short duration and no significant time will be lost. however, recovery might be impeded or delayed by issues such as new, possibly weaker waves of the pandemic or more technical issues such as fiscal multipliers. fiscal multipliers during a recession when shocks are concentrated in certain sectors are not expected to be operational, with the multiplier for government spending being around one and the multiplier for transfers likely less than one. these factors might result in the delays-initially projected to be of short duration-extending for a much longer period. the important point here is that the covid- shock will have a negligible effect on the evolution of the temperature anomaly. thus, if adaptation activities and decarbonization do not proceed rapidly, the risks of a climate shock will not be sufficiently mitigated. the need for strong action now is exemplified by the fact that the emissions gap in between current policies and the emissions necessary to keep the temperature anomaly below . °c in is approximately - gtco (unep ). the covid impact on emissions in is expected to reduce the gap by approximately gtco , which falls very far short of the gap that needs to be closed. the important aspect of a climate shock is that in addition to the negative impacts on the productive base of the economy-human, health and social capital-it will have a much more serious negative impact on the reproducible capital relative to the pandemic. this is because the climate shock will not just reduce the utilization of this type of capital, it will destroy part of the capital stock, since it will affect infrastructure, equipment, buildings and so on. recovery in such a case will clearly be slower and more difficult. this creates a serious argument against delaying adaptation programmes which could provide substantial benefits in the presence of climate shocks. typical adaptation projects (e.g. early warning systems, water resource and flood-risk management, sustainable agriculture, strengthening the resilience of existing infrastructure making new infrastructure resilient) are characterized by high benefit-cost ratios (fankhauser ; gca ) . under the resource constraints imposed by pandemic containment policies and the deep structural uncertainty characterizing the situation, the prioritization of these adaptation policies could necessitate the use of max-min expected utility criteria in decision making. as second-round benefits, adaptation programmes which involve investment could stimulate the economy and provide new jobs. to summarize, three important points should be taken into account: (i) that the indications thus far suggest that the benefits associated with the reduction of greenhouse gas emissions because of the covid- are transient, which means that the long-term trends associated with climate change are not expected to change, and therefore, mitigation and adaptation strategies should be strongly pursued in the post-covid- era ; (ii) that there is a need for an ambitious and comprehensive european economic recovery plan from the covid- crisis (european economic and social committee b); (iii) that climate-change-related investments, in particular in adaptation, are expected to deliver significant economic benefits. based on these points, it becomes clear that a green recovery plan with resources directed towards achieving the combined objective of both providing the necessary economic stimuli for recovery and also promoting the transition to a low-carbon economy and adaptation to climate change along with investment in natural capital and increase in comprehensive savings could be a feasible and efficient plan. the egd is an important strategy for securing the sustained development of the eu and protecting climate as a global public good. at the same time, it is clear that addressing the pandemic requires action now in the form of new policies and changes in priorities. however, although covid- is of necessity in the spotlight at present as a major global threat, it should not displace action aimed at an equal or greater global threatthat of climate change-under the misconception that the temporary drop in emissions during the pandemic allows us to delay climate change action now. a green recovery plan could realistically provide the double dividend of helping the eu economies to recover from the covid- crisis and, at the same time, promote the attainment of a climate-neutral eu. according to the gca ( ) study, adaptation investment of $ . trillion in the areas mentioned will provide total net benefits of $ . trillion by , with benefit-cost ratios ranging between and . see, for example, the recent eu european economic and social committee ( a) opinion about the transition to a low-carbon eu and the financing of adaptation to climate change. involved in europe ( ), leaked: full list of delayed european green deal initiatives-euractiv.com. available at https ://europ e.vivia nedeb eaufo rt.fr/leake d-full-list-ofdelay ed-europ ean-green -deal-initi ative s-eurac tiv-com/. ipcc ( ), global warming of . °c. an ipcc special report on the impacts of global warming of . °c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty. masson-delmotte, v., zhai, p. rebound effect studies have been generally focused on energy use (sorrell ) , although some studies for other natural resources have recently emerged (freire-gonzález and font vivanco ). rebound effect occurs when the use of resources is not reduced as expected after a resource efficiency policy or a specific behaviour. empirical rebound studies aim at capturing the secondary effects of policies and behaviours in order to obtain more adjusted assessments of policies and actions. it is well known in the rebound literature that, counterintuitively, resource efficiency may not reduce the use of these resources, but the contrary. this extreme case is known as backfire, khazzoom-brookes postulate, or jevons' paradox. rebound effects are not usually observed by policy-makers, as it requires different perspectives and approaches coming from social, behavioural and environmental sciences. environmental and social sciences show us that human-environment systems are deeply interconnected. this way of thinking has, however, still not fully permeated in mainstream policy decision circles, which are largely rooted in old intellectual paradigms and other short-term interests. the pandemic has caused many abrupt changes in production and consumption, transport patterns, working conditions, social interaction and many other aspects. most of these changes have been triggered by the policies implemented to contain the pandemic. overall, they have translated into improvements in most environmental indicators, such as carbon emissions, air quality and biodiversity loss (saadat et al. ) . while some authors claim that such changes will not have a lasting impact when the epidemic subsides (mccloskey and heymann ), others argue that aspects related to urban planning, micro-mobility, sharing economy, public transportation, teleworking, tourism, etc., may change for good (honey-rosés et al. ). an important question is, thus, whether covid- will reduce environmental impacts in the future, when economic activity returns to "normality" (in terms of pre-covid conditions). rebound literature shows the importance of considering behavioural and systemic responses to answer this question. beyond other considerations, the pandemic has accelerated some already observed trends, like the pace of implementation and use of digital technologies. one of the most remarkable changes are those related to the impulse of information and communications technologies (ict), due to imposed social distancing rules. there already was a tendency towards an increased use of ict, but its use has been dramatically accelerated due to the pandemic. this acceleration can be observed in many areas, such as teleworking, e-commerce, remote social relationships, virtual sightseeing, surveillance technologies, and other online areas and events (cultural, academic, leisure, educational, etc.) . for instance, in many countries, nonessential workers have been legally obliged to be confined during the pandemic to stop the contagion of the virus, thus promoting telework. despite the potential advantages of teleworking in increasing labour productivity in many industries (harker martin and mcdonnell ) , rigidities in corporate culture and other legal and cultural restraints were hindering and adjourning its consolidation. the use of ict is thought to be environmentally beneficial, largely due to decreased transport, but this premise has been challenged by rebound effect studies. gossart ( ) shows that existing evidence suggests that ict are subject to important rebound effects, mainly because it is a general-purpose technology, and so prone to backfire (sorrell ) . takahashi et al. ( ) calculated the rebound effect of ict services in a case study on videoconferences and found that rebound can reduce up to % of carbon savings. joyce et al. ( ) recently found for sweden strong environmental rebound effects associated with ict use, in most cases far above % (more resources use than before). this backfire effect is strongest for energy use and total material footprint, both close to %. another change may take place in land use and the housing sector, as initial evidence suggests that attributes such as floor space and outdoor space will have elevated importance (mikolai et al. ) . the potential re-distribution of time and expenditures towards resource-intensive sectors, such as construction, water and energy services, will likely cause material, water and energy rebounds. however, the expansion of teleworking can, at the same time, reallocate space and incomes in office rental market. city centres will not need to concentrate workspaces, changing mobility patterns and urban structures in the long term (elldér ). public transport may also be negatively impacted in the short and mid-term, leading to increased private transport (honey-rosés et al. ), another resource-intensive activity. other structural changes may also take place, such as changes in sufficiency measures and broader productivity, leading to macro-economic rebound effects (lemoine ). the pandemic may increase the social acceptance of sufficiency measures such as working less time, spending more time with family and friends, or connecting with nature. these measures have long been proposed to reduce consumption and associated environmental impacts (hayden and shandra ). these measures have, however, been associated with macro-economic price rebound effects as the decreased demand for some products can lower their price and induce additional demand (sorrell et al. ) . moreover, the postpandemic society may likely be a more productive one in labour and capital terms. for example, teleworking (harker martin and macdonnell ) and increased spending in research and development have been associated with productivity growth, which boosts economic growth and resource use. the covid- pandemic will likely cause a range of changes in society, but their permanence and effect on the environment are unclear, especially if we contemplate the secondary effects of behaviour, measures and policies. a key question is whether they will acquire a certain level of permanence, even modifying the mindsets of agents. given the high uncertainty around this aspect, its real dimension could only be assessed ex-post. however, due to confinements, the pandemic has greatly accelerated the expansion and use of general-purpose technologies, like ict. as this has been a long-observed trend, before the irruption of the virus, they have probably come to stay to a large degree. the pandemic offers a great potential to improving (and consolidating) environmental conditions. but beyond what conventional environmental indicators show, additional measures would be needed to counteract hidden rebound effects and, therefore, take full advantage of potential improvements. recent literature shows that different economic instruments like environmental taxation, resource pricing or setting limits to resource use, can be effective for this purpose. this is particularly necessary in this case, given the high risk of backfire due to the high expansion of general-purpose technologies observed. the global financial and commodity markets are facing economic distortions caused by the coronavirus (covid- ) pandemic (fernandes ; irwin ). covid- acts as a negative shock to overall demand of goods and services, resulting in aggravated shortrun volatility in prices (albulescu ). among these products, oil has been dramatically affected due to community lockdown regulations, shutdown of car factories, decline in energy use and increase in unemployment (reed a; the associated press ). however, a decrease in oil consumption may lead to reductions in carbon dioxide emissions (peña-lévano et al. ). data recorded by epa ( ) during march-april show an improvement in overall air quality, especially in high-density populated cities (regan ). air pollution is considered by many scientists as a negative contributor in the coronavirus situation by worsening the susceptibility of infection. a decline in emissions somehow may help prevent mortality temporarily, especially among more vulnerable individuals with underlying health conditions, such as heart and respiratory diseases (conticini et al. ; dutheil et al. ; mooney ; ogen ). thus, in this short article, we discuss the interaction between fossil fuels, air pollution and health risk under the coronavirus pandemic and the lockdown regulations during the period of march-may . the oil market has been dramatically affected by several exogenous factors during the pandemic period: ( ) "shelter in place" mandates aimed at curbing the spread of the coronavirus have decreased people's social mobility and transportation activities. households leave their houses just for short travels for some essential errands, such as shopping groceries and/ or medicine. only essential personnel are required to be physically present at their jobs, whereas the majority are working from home (reed a); ( ) commercial flight demand plummeted globally (reuters ). in the usa, the passenger volume dropped % from a year ago (compared to april ) as estimated by airlines for america, with an overall reduced accommodation of passengers per carrier (rappeport and chokshi ); ( ) tourism activity slumped as many governments banned international travel, heightened border and immigration controls and barred the entrance of foreign visitors to decrease risk of infection. these policies further decreased the demand for taxis and cruise services. for regions such as the caribbean islands (where tourism is a primary economic staple good), this also represents a significant decline in their gross domestic products (semple ); ( ) unemployment has pervaded among the national economies (mazzei and tavernise ). more than million people have filed for unemployment in the usa in a -week period (u.s. department of labor ). this condition is mirrored in other countries. worsening unemployment conditions have substantially reduced consumers' purchasing power. among its consequences is an overall decline in car sales (reuters ; the associated press ); ( ) many industries, restaurants and buildings have been shut down during the lockdown. these closures reduced energy consumption, especially petroleum use, which is a large energy input in the usa and other countries (eia ). meanwhile, global oil supply responded slowly to the decline in demand as refineries cannot abruptly halt production (caldara et al. ; peña-lévano ). the russia-saudi arabia oil price war in march worsened the situation by oversupplying the market and consequently dropping oil prices (irwin ; reed a). in april, the major petroleum exporting countries agreed to decrease the world oil output by % during may-july, which is equivalent to . million barrels a day (krauss ; reed b). two recent studies published in geophysical research letters validate the realized reduction in nitrogen dioxide (no ) pollution in several regions. bauwens et al. ( ) compared no levels in the atmosphere recorded in january to april and for the same period in . notably, no , which is produced by emissions from vehicles and industrial operations, can cause serious lung ailments. their estimates indicate a significant reduction of % in china and - % drop in the usa and western europe. shi and brasseur ( ) estimate a % reduction in no pollution in northern china in january and february . in the same period, they also found a % reduction in particulate matter pollution (particles smaller than . µm). while such cleaner air conditions may persist only temporarily, these trends indicate that the desired environmental gains are feasible and realizable if stringent emission regulations, perhaps mirroring to some extent the pandemic's enforced limits on social mobility and industrial activities, are enforced in the future. several studies relate ambient air quality to mortality and morbidity conditions caused by covid- . this contention echoes an earlier correlation applied to the sars virus outbreak in china in the early s. researchers from ucla's school of public health analysed air pollution levels and sars fatality rates among chinese residents. their results indicate that sars patients' probability of dying would be doubled among residents in areas with high air pollution indexes (cui et al. ) . as applied to the current pandemic, the center for disease control and prevention (cdc) explains that covid- causes a respiratory illness with a heightened risk among people who are years and older as well as those with certain underlying health conditions. latest cdc statistics on the pandemic's severity and fatality indicate that persons with heart ailment, diabetes and chronic respiratory diseases could be at a higher risk of being severely infected by the virus (vogel ). wu et al. ( ) further clarify that pre-existing health conditions identified as relatively more susceptible to contracting covid- are similar to those normally affected by air pollution in the usa. their study estimates that an increase of g m − in long-term exposure to particular matter (pm . ) increases the coronavirus mortality rate by %. isaifan ( ) presents corroborating evidence indicating that % of covid- -related deaths were cases with pre-existing illnesses, with majority of the victims over years of age. conticini et al. ( ) , however, warn that even young and healthy individuals could also be at risk as prolonged exposure to dangerous air pollutants causing chronic respiratory issues could be an additional co-factor that helps increase their vulnerability to being infected by the virus. ogen ( ) establishes that exposure to no may be an important instigator of covid- fatalities according to his research involving four european union countries. these findings are supported by the findings of a study published by the italian society of environmental medicine (setti et al. ) on virus infections in northern italy associated with air pollutants tagged as carriers and boosters. poor air quality has been cited as an aggravating factor in virus transmission. using data from italian province capitals, coccia ( ) notes an accelerated transmission dynamics of covid- leading to his conclusion that the spread of this virus can be considered more as following an "air pollution-to-human transmission" mechanism instead of an interpersonal transmission mode. moreover, fattorini and regoli ( ) analyse long-term air quality data in northern italy and suggest that chronic exposure to a contaminated atmosphere may have created conducive conditions for the spread of the virus. several studies relate the time frame of exposure to toxic air pollutants to mortality and morbidity conditions. lim et al. ( ) establish a significant association between longterm ozone (o ) exposure and elevated mortality risk of certain respiratory diseases. given limited data on the current pandemic, hoang and jones ( ) present emerging evidence on the severity of covid- infection attributed to persistent air pollution conditions, thus suggesting that longer exposure to a polluted atmosphere could aggravate virus infection. zhu et al. ( ) , however, provide concrete evidence suggesting that even short-term exposure to air pollution could increase probability of virus infection. their study analysed daily confirmed cases in cities in china recorded from january to february and found significant relationships between the levels of certain air pollutants and the number of newly identified covid- -infected cases. specifically, their results indicate that a -μg/m increase in the air pollutants' levels was associated with about . % to . % increases in daily new covid- cases. conversely, the health benefits of cleaner air resulting from reduced emissions from fossil fuel during the pandemic's lockdown period deserve attention. several studies recognize that improved air quality during the pandemic temporarily mitigated health risks associated with respiratory illnesses. cole et al. ( ) employed a two-step analytical approach using machine learning techniques and the augmented synthetic control method to estimate possible reductions in death rates in certain regions in china and for the whole country that may be attributed to actual reductions in no concentrations during the lockdown period. isaifan ( ) analyse air quality conditions prior to and during the lockdown period. his results indicate that lives may have been saved due to diminished ambient pollution levels, although eluding possible virus infection still is not necessarily guaranteed. this contention is corroborated by another china-based study conducted by dutheil et al. ( ) . these studies' assertions imply that air quality improvements realized even in such a shorter period of time (spanning less than half of a year) already could have some health benefit potentials, especially in relation to respiratory ailments. the health benefits of the current pandemic's notable environmental gain in air quality, however, will be optimized only if such favourable conditions are sustained over the longer term. the recent lockdown has been short-lived as some communities nowadays have started to revert to normal social routines and regular users of fossil fuels among industries have resumed operations. a brief respite from a usual contaminated atmosphere does not ensure an effective permanent eradication of chronic health conditions. the pandemic experience, however, demonstrates that better health is maintained and ensured not only through medical remedies and but also through more favourable environmental conditions, if sustained over a much longer period. current restrictions on social mobility and economic flexibility under covid- pandemic conditions have actually produced important economic and environmental repercussions that are interestingly contrasting. a general economic slowdown overtly reflected in, among others, reduced consumer demand, spiralling unemployment figures and significant drop in oil consumption causes heightened fears of an imminent economic recession. however, in spite of all the negativity surrounding the pandemic, its environmental consequence of improved air quality is a highly positive note. interestingly, the global community has been trying to accomplish such feat of attaining better air quality over many years of discussions, policy making and policing each other. unexpectedly, it took a serious pandemic to realize such feat. this article traces the interplay of reduced oil consumption with economic issues as well as environmental consequences under pandemic conditions. the more imperative issues now lie on the severity of a looming recession and the global economy's resiliency in transcending the difficult challenges it may bring. should that happen, will the economic cost burdens be outweighed by the realized environmental gains? experts may be quick to assert that improved environmental conditions actually may be short-lived as expected resurgence of resumed economic activities may only quickly bring back pre-covid air conditions. however, proponents of a cleaner world can always draw some inspiration from recent successes in air quality control, especially with the assurance that cleaner air is not necessarily a lofty goal. the challenge in the future lies in achieving such environmental benefit without the need to sacrifice the economic health of the global community. on february , , the brazilian ministry of health confirmed that a -year-old man was positive for sars-cov- : covid- had arrived to latin america. as of july , there have been . million confirmed cases in latin america, compared to . million in the european union and . million in the usa (jh-csse ). furthermore, so far there have been more than , deaths in the region and the trends show that the first wave of the pandemic is far from over (ibid.). there is of course no suitable time for a pandemic to arrive, but these are especially complicated times for latin america. the region is in the midst of a difficult economic situation accompanied by rising social discontent (eclac ; oecd ). moreover, it is characterized by high rates of informality, health systems with limited and unequal capacity, and most of the countries have high levels of debt (oecd ). under these circumstances, covid- is having major short-run socio-economic effects with possible serious long-run consequences, including several potential implications for the environment and the management of natural resources. restrictions of free movement and circulation within and across urban areas of latin america have reduced economic activity as well as the use of motorized vehicles. as a result, many latin american megacities have experienced a short-run decrease in air pollution. concentrations of no have decreased considerably in cities all over the region compared to the levels observed prior to the lockdown measures (iadb ). levels of pm , pm . and co have decreased in bogota, buenos aires and quito (bogota's district secretary of environment, personal communication, june ; roa ; rocha ). nevertheless, the pandemia has not had the same effect on air quality in all the major cities in the region. in mexico city, the reductions in so , pm . and pm concentrations have been modest, and there has been no reduction in ozone. in rio de janeiro, ozone concentrations have increased (dantas et al. ) . furthermore, as the virus and its negative consequences spread across rural areas and make its way through the southernmost part of the region, outdoor and indoor pollution might actually increase. in mexico, as well as in other countries in the region, the use of firewood is likely to rise as rural households try to deal with income reductions (masera et al. ). meanwhile, as winter hits central and southern chile, urban households might increase their use of firewood for heating given that, due to the lockdowns, they have to spend more time inside dwellings (encinas et al. ) . this rise in air pollution could arguably increase the risks associated with covid- . it is too soon to do a formal evaluation of the effects of the pandemic on deforestation and land use change in the region. nevertheless, the available information suggests that covid- is likely to have negative effects on forest cover across the region. early deforestation warnings from peru show that, although deforestation decreased between march and april , since then it has increased surpassing the levels observed during the same period in . according to data from the brazilian national institute for space research (inpe), the first quarter of already evidenced a rise of % in deforested hectares compared to last year's figures. the figures for april reinforce this pattern, with a % increase with respect to april (manzano ). from january to april , deforestation alerts in indigenous territory increased % when compared to the same period of the previous year (greenpeace brasil ). although at this point it cannot be claimed that the pandemic caused the observed increase in deforestation, it certainly does not seem to have provided incentives to halt it. in colombia, contrary to other countries in the amazon region, the trend in showed a reduction in deforestation compared to . however, started with an increasing tendency and the quarantine seems to have worsened the situation (fcds ). the absence of environmental monitoring during the pandemic seems to have encouraged illegal armed groups and regional mafias to take advantage of the situation, exacerbating deforestation with the possible intensification of illegal activities from which these actors derive income, such as illegal mining, land grabbing and illicit crops (bbc ). according to the pan american health organization, in may there were already , covid- -confirmed cases in the amazon basin (martín ). the impact of the pandemic in forest-based indigenous communities is an important source of concern. the spread of the virus in these communities could imply a tragedy that, in addition to the human losses, could affect the traditional knowledge, having negative impacts on the governance of natural resources in the region. this could lead to even more deforestation processes in the future. covid- has caused a disruption in the national and international trade of nature-based goods and services. tourism has come to a halt, affecting the economy of almost all of the countries in the region (mooney and zegarra ). in countries like costa rica, where the touristic industry is intertwined with nature, the shock to the sector could have negative effects for biodiversity and forests. without income from tourism, and given that as a slow recovery process is anticipated, the incentives to protect forests are expected to decrease in the short and medium run. fishing and aquaculture are other industries that have been negatively affected. information for the case of the chilean salmon aquaculture industry suggests that there has been a reduction in demand from international markets (chávez et al. ) . the effect of the shock is being transmitted through the value chain, affecting processing plants and farming facilities. the economic crisis can end up having long-run negative consequences for the environment if, as a result, regulations and environmental policies are relaxed or if institutions are weakened. although at this point there is no evidence of any country in the region purposefully relaxing environmental regulations to promote growth, it is certainly a possibility. what has been observed is that, in order to fund measures to reduce the economic and social impacts of the pandemic, some countries have decided to reallocate funds across the public administration. ecuador, for example, announced cuts affecting the ministry responsible for enforcing environmental regulations (bbc news ). something similar is happening in mexico, where the plan announced by the president is to reduce the operational budget of almost all government entities by % (d.o.f. ). even if countries have a relatively strong environmental legal framework, without a budget to monitor and enforce the regulations, this framework is worthless. countries in the region will very likely incur fiscal deficits and increase their debts in order to fight the crisis. it remains to be seen how the service of the increased debt will impact economic growth and the environment. in the meantime, it seems that the short-run legitimate demands to recover employment levels and improve the health systems might very well push aside the necessary investments to successfully tackle climate change and biodiversity loss. if this indeed happens, it could be the most serious effect of the covid- pandemic in both environmental and social terms. the economic projections suggest that the region will experience a crisis whose magnitude has no precedent in modern history (eclac ). in order to overcome this apparently insurmountable challenge, latin american countries will need well-designed policies that should reconcile economic objectives with social and environmental goals. the social unrest manifested recently in the social mobilizations in the region, should make clear that the apparent trade-off between economic, social and environmental objectives is the result of a false dichotomy between shortand long-run objectives. if environmental objectives are put aside, as has so often happened with social objectives, the economy might recover in the short run but at a very high price. the lockdown measures seem to be having a temporal positive effect on reducing urban pollution in some latin american cities. the challenge now is how to intervene to prevent a return to the same or even higher pre-quarantine emission levels. this is an opportunity to rethink the urban environmental policies while trying to recover from an unprecedented social crisis. at the same time, the observed increase in deforestation reopens political and academic debates about the role of national parks, indigenous reserves and other protection categories in a context of deteriorated livelihoods, illegal economies and a lack of state presence. latin american countries could see this moment as an opportunity to improve regional cooperation in order to design and implement coordinated policy responses not only to the economic crisis but also to the challenges of mitigation and adaptation to climate change. furthermore, countries should coordinate efforts to increase monitoring and presence in the region to effectively reduce deforestation. we have presented an account of some of the most evident environmental effects that the covid- pandemic is having in latin america at this point. considering that we are in the midst of the health crisis and in the beginning of an economic one, it is natural to expect that the trends that we see now will change in the near future and that other environmental impacts will become evident. research that contributes to a better understanding of the environmental impacts and the effectiveness of different policy responses to the pandemic in latin america will be invaluable. there are many potential paths for future research; here, we mention just a few. the consequences of the interactions between poor air quality and covid- on human health are clearly worth studying. this is particularly relevant for the latin american context, characterized by health systems with very limited capacity and high numbers of population without formal employment. results from studies in this area could help us provide better guides to set environmental quality goals, as well as to implement policy interventions that can reduce pollution in the region's context of income inequality and spatial segregation. the short-run environmental effects of covid- show early warnings of an increase in the pressure on forest and other ecosystems across latin america. understanding the impacts of the pandemic on terrestrial and marine ecosystems, as well as on livelihood opportunities for local communities, has the potential to contribute to the design of policies which can improve management and conservation. the pandemic is opening new research questions regarding the impacts of global shocks on natural resource-based industries that participate in international markets. furthermore, the paths that different countries take to get out of the economic crisis might have profound impacts on international trade. if, for example, the world transitions to more reliance upon local production, or if emissions-related tariffs are imposed, large exporters of commodities in the region will be highly affected. the impacts that these potential trade changes could have on the environment are unknown. furthermore, if developed countries implement recovery plans that include provisions to reduce emissions in significant ways, as has been discussed in the european union, will latin american countries be able to respond in the same way? in any case, latin american countries are highly vulnerable to the effects of climate change and some of these effects (e.g. migration) could result in future health crises. a better understanding of the ways in which individuals might adapt to a changing climate, as well as of the barriers that they face to adopt adaptation measures, will be a valuable tool for the design of adaptation policies that prevent future health crises in the region and elsewhere. the distributional and gender-differentiated impacts of the pandemic, and the related environmental policy responses, is another area that deserves attention, especially because early evidence shows that the more vulnerable segments of the population in the region are the ones that are being hit hardest. finally, as has been recently pointed out by , the experience of the pandemic might lead to changes in behaviour and personal choices. it remains to be seen if this is in fact the case, and if so, how are these changes in behaviour modulated by the local context. an even more important issue to consider is what would these changes imply for the design of behaviour-based policy instruments aiming to change consumption and production patterns, as well as transport and land use decisions in latin america. bbc ( ) the place where nature isn´t healing. https ://www.bbc.com/reel/video / p bd wfc/the-forgo tten-peopl e-of-the-globa l-pande mic accessed june bbc news ( ) coronavirus en ecuador: las multitudinarias protestas por las drásticas medidas económicas y recortes de lenín moreno. https ://www.bbc.com/mundo / notic ias-ameri ca-latin a- accessed june chávez c, salazar c, simon j ( ) efectos socioeconómicos y respuestas públicoprivadas de corto plazo ante la crisis del covid- en el sector salmonicultor, una fotografía de la experiencia internacional. interdisciplinary center for aquaculture research-incar, fondap-anid, center, chile. https ://www.incar .cl/wp-conte nt/ uploa ds/ / /pb .pdf accessed june dantas g, siciliano b, frança b. b, da silva c. m, arbilla g ( ) the impact of covid- partial lockdown on the air quality of the city of rio de janeiro, brazil. sci total environ . https ://doi.org/ . /j.scito tenv. . . d.o.f. ( ) decreto por el que se establecen las medidas de austeridad que deberán observar las dependencias y entidades de la administración pública federal bajo los criterios que en el mismo se indican. de abril de . presidencia de la república de méxico. eclac ( ) report on the economic impact of coronavirus disease (covid- ) on latin america and the caribbean: study prepared by the eclac, santiago, p encinas f, truffello r, urquiza a, valdés m ( ) covid- , pobreza energética y contaminación: redefiniendo la vulnerabilidad en el centro-sur de chile. centro de investigación e información periodística. https ://ciper chile .cl/ / / /covid - pobre za-energ etica -y-conta minac ion-redefi nien do-la-vulne rabil idad-en-el-centr o-surde-chile /. accessed june fcds. ( ). cifras deforestación en el bioma amazónico, enero-abril .https :// fcds.org.co/site/wp-conte nt/uploa ds/ / /defor estac ion_ .pdf accessed june greenpeace brasil ( ). desmatamento em terras indígenas aumenta % em . https ://www.green peace .org/brasi l/press /desma tamen to-em-terra s-indig enas-aumen ta- -duran te-a-pande mia-da-covid - / accessed june helm d ( ) . contributing to a global effort. oecd. roa s ( ) medidas para enfrentar al covid- mejoran calidad del aire en dos ciudades ecuatorianas. mongabay-latam.https ://es.monga bay.com/ / /menor -conta minac ion-del-aire-por-coron aviru s-en-quito -y-cuenc a-ecuad or/ accessed jun rocha l ( ) por la cuarentena, la contaminación del aire bajó a la mitad en la ciudad de buenos aires. infobae. https ://www.infob ae.com/socie dad/ / / /por-lacuare ntena -la-conta minac ion-del-aire-bajo-a-la-mitad -en-la-ciuda d-de-bueno s-aires / accessed june resource exporters are now facing new urgent economic policy challenges due to covid- . these challenges are aggravated due to their dependence on finite commodities with volatile prices and demand. in response to the pandemic, resource exporters (such as botswana and saudi arabia) announced cuts in expenditures along with large fiscal (tax relief) and consumption-focused macroeconomic stimulus packages. critically, the novel issues raised by the coronavirus pandemic bring new trade-offs of energy policy between shortterm gains and long-term sustainability, creating an urgent need for critical, quantitative, policy-focused research in the resource exporters-energy policy nexus. a few novel issues emerged in resource exporters during the pandemic that give rise to short-term economic challenges. first, price shocks of unprecedented magnitude for commodities (deutsche bank ; world bank ), from coffee (hernandez et al. ) to hydrocarbons (iea b), causing a large drop in energy investment (iea a). for oil, prices dropped initially due to travel bans and economic activities hiatus, and further with oil price wars following the collapse of opec + agreement. by april , with oversupply, the rise of stockpiles, and the saturation of available oil storage, oil prices reached the lowest level in more than years, and west texas intermediate (wti) reached negative levels for the first time. prices have subsequently partially recovered but are expected to remain low with continuous fears of new covid- waves and uncertain demand. second, unprecedented economic contraction, because the pandemic-triggered decline in economic activity was significantly exacerbated by declines in export revenue. third, unprecedented fiscal pressure, resulting from costs of fiscal and economic stimulus packages plus a simultaneous rise in domestic expenses (especially healthcare and unemployment benefits) and sharp declines in resource export revenue. the effects have been so colossal that states like kuwait are considering halting legally-mandated contributions to the future generations sovereign wealth fund (swf) to ease fiscal pressures (al-zo'bi ). fourth, record and continuous withdrawals from swfs to fund post-pandemic recovery along with reallocation of funds and increased government debt (examples in arabian business ; holter and bloomberg ). even states with the largest swfs such as norway and kuwait have been affected, with the latter expecting depletion of its fiscal stabilization swf (al-zo'bi ). fifth, in oil-exporting gulf states, the unusual stay of millions of citizens and guest workers in the upcoming scorching summer in lieu of usual tourism travel or home-country visits, pressuring existing energy capacity. critically, in oil exporters, these novel issues and policy responses to them further expose existing economic fragilities and challenges, threatening long-term economic and environmental sustainability. for distorted oil economies in urgent need for economic diversification, said diversification and reverse dutch disease are impeded by their existing high distortions in labour, fiscal, industrial and energy markets (shehabi ) . oil exporters suffer from economic inefficiencies resulting from pervasive oligopolies (shehabi ) and market failure in long-run contracts for exploration and development of natural resources (ruta and venables ). fossil fuels are the primary source of energy in most oil exporters. in developing oil exporters, the dominant political economy undergirding policy making is a welfare rentier state, in which maintaining a political equilibrium is central. balancing spending-saving decisions for sustainable resource rents management depends on the sustainability of windfall expectations (gelb and grassmann ). reforming energy policy-which drives fiscal, industrial and environmental policies-is critical for these states' long-term development. yet the novel issues create new trade-offs of post-pandemic energy policy: achieving short-term gains will be at the expense of longterm gains in resource economies. thus, it is critical for fast policy-focused research to address the resource exporters-energy policy nexus, especially in the following two areas. although investments in renewables have been more resilient than in fossil fuels, and while we have seen an increased share of global energy spending on clean energy technologies in (iea a), the reality in hydrocarbon-exporting developing states is different. global oil market supply and demand dynamics have raised their opportunity cost for transitioning away from fossil fuels and for investing in green technology. accordingly, post-pandemic economic stimuli might achieve short-term recovery but harm long-term energy transition. first, the rise in opportunity costs of energy transition will facilitate the reallocation of funds away from renewables projects towards post-pandemic economic stimuli (similar to "green tape" cuts in australia and canada). second, recovering lost investments in energy transition projects is unlikely because the resource rents that fund them are likely to continue to be low in the future, given low resource prices and demand pressures of climate change mitigation. third, continuous withdrawals of diminishing swfs will entail limited resources for future energy transition projects. this is especially so as recovering swfs' withdrawals is unlikely in the light of expected low resource export revenue and the collapse in financial and commodity markets accompanying the unprecedented global recession. finally, economic stimuli expand consumption and welfare redistributive measures, which increase greenhouse gas (ghg) emissions ) and exacerbate existing distortions that have been shown to prevent economic and energy diversification (shehabi ) . new research should investigate the design and implementation of new economic solutions that have at their forefront long-term energy transition goals along with short-term economic recovery. three policy solutions are suggested at the consumer, the energy industry and economy-wide levels. a policy solution to target consumers is technological advancements (both private and public funded) in energy efficiency coupled with economic incentives to rationalize energy consumption. this policy combination can achieve energy transition goals despite expected delays in renewables and without requiring large multiyear investments. it is especially important in gulf oil states where skyrocketing cooling and desalination needs in the summer will be met using fossil fuels (renewables contribute less than % of power generation). a policy suggestion at the sectoral level is investing in clean energy technologies to decarbonize the energy sector itself, namely through carbon capture and storage as well as hydrogen. beyond short-term economic and environmental benefits, these investments can keep oil exporters relevant in a future with a diminishing role of hydrocarbons and collapsing commodity prices. the final policy suggestion is microeconomic and energy policy reform that can moderate effects of export price declines on the economy without the need for additional cuts in renewables investments or further withdrawals from swfs. examples include microeconomic reforms of labour and human capital to increase long-term productive capacity and oligopoly regulation in non-tradables and energy sectors which can increase efficiency and welfare gains that translate economywide (shehabi ) . consequently, resource rents could be salvaged for swfs resources or investments necessary for future development. in the light of the aforementioned novel issues in resource exporters, the postpandemic energy policy and economic stimulus packages are likely to have negative long-term effects on the environment. the reasons are as follows. first, while lockdown measures that minimized transportation and human activity reduced short-term emissions, they also increased power demand which in resource exporters is met mostly through fossil fuels, especially as international demand and prices remain low. second, short-term improvements will be negated upon the resumption of human activity, absent changes in energy policy regimes. importantly, as domestic energy prices remain low and highly subsidized in developing resource exporters, the extent to which covid- restrictions would shift energy consumption habits and behaviours of agents (households and institutions) remains very doubtful. third, there will be limited resources in the future to dedicate towards environmental regulation and ghg emissions reduction. the reason is that funding the post-pandemic recovery will reallocate funds away from environmental projects and savings in swfs. fourth, ghg emissions will increase due to consumption-focused economic stimulus packages and expanded use of fossil fuels to meet rising energy demand. this is particularly problematic because even prior to the advent of the pandemic, resource exporters were among the highest energy consumers and carbon emitters globally. indeed, the ten highest per capita carbon emitters are all resource exporters, with emissions ranging from tonnes (t) per capita (qatar) to . t per capita (kazakhstan), more than . times the global average of . t per capita (ritchie and roser ) . finally, and most importantly, the implementation of policy instruments-namely energy subsidy reform and carbon taxes-to achieve resource exporters' intended nationally determined contributions (indcs), is rendered significantly more difficult post-pandemic. it is a consequence of the magnitude of novel economic contraction and fiscal pressures in welfare-based states. these additional political constraints exacerbate environmental laws' enforcement, for which there already was a widespread failure in resource exporters (unep ). therefore, new research must quantify effects of proposed economic stimuli and of rising use of fossil fuels on resource exporters' environment and economy and accordingly evaluate and design new alternative policies that can achieve short-term recovery and national climate target goals. to that end, four policy suggestions are offered. first, reducing emissions in ways other than the politically difficult tax instruments, mainly through enhancing energy efficiency that reduces emissions at a given consumption level coupled with economic incentives that reduce energy consumption. second, implementing carbon tax instruments in ways that do not harm the most vulnerable of populations and reduce resource exporters' fiscal distortions, thus achieving a "double dividend". third, in the light of the novel issues, a key policy solution is adopting "green welfare expansion": including green options in the typical post-pandemic welfare packages at lower governmental budgetary requirements. examples include expanding subsidies for green technologies (such as solar panels, public transportation or electric cars), as well as excluding from subsidies non-essential high-carbon-emitting products for households above a certain income level. fourth, increasing the political viability of green recovery packages (such as clean physical infrastructure and natural capital investments), including engaging the private sector in initiatives that are typically public-led-such as climate finance, renewables expansion and resource mobilization. this policy is critical because, although green recovery packages may boost economic growth while helping climate change , they are politically contentious in resource exporters where they compete with welfare distribution. critically, applicable to the aforesaid two research areas, the pandemic offers resource exporters an opportunity to engage in a comprehensive reform agenda that addresses short-term pandemic effects while advancing long-term energy transition, economic and resource sustainability. the challenge for policy makers is to avoid implementing policies in haste. properly coordinated policy reforms offer an avenue to address inefficiencies and underlying structural distortions in a way to realize mutual gains and multiple policy objectives at the lowest cost. in designing these reforms and addressing the two areas above, the most suitable research methods are economy-wide general equilibrium models that can quantify effects of shocks and policy solutions in a "second-best" environment. this feature is necessary given the large existing economic distortions in resource economies. these models represent economic linkages and agents and include a wide range of policies (energy, carbon, fiscal, labour or industrial). as such, they can inform evidence-based policy making that accounts for political economic considerations. this research will be critical for filling gaps in the literature on long-term economic and resource sustainability in resource exporters. the current global greenhouse gas (ghg) emissions trajectory indicates that the world is likely to experience catastrophic consequences due to climate change, unless swift action is taken towards funding green solutions and the defunding of fossil fuel activities ( given the ambition of the european union to become a net zero-carbon economy by and the numerous calls to avoid the bailout and stimulus packages towards fossil fuel companies , we examine whether the features of the european central bank's (ecb) € billion pandemic emergency purchase programme (pepp) encourages the resilience of the incumbent fossil fuel sector, or whether it promotes the growth of the emerging low-carbon energy sector during the covid- pandemic and beyond. we draw on a novel dataset of corporate bonds issued in the european energy sector between january and june in combination with the european central bank's (ecb) purchases under the pandemic emergency purchase programme in response to covid- . we show that the likelihood of an energy company bond to be bought as part of the ecb's programme increases with the ghg intensity of the bond issuing firm. we also find weaker evidence that the ecb's pepp portfolio during the pandemic is likely to become tilted towards companies with anti-climate lobbying activities and companies with less transparent ghg emissions disclosure in the event of increased euro-denominated bond issuances in the following months, or re-denominations of non-euro bonds already issued by european energy companies. kainen et al. ). that does not mean, however, that the aim of central banks to remain sector neutral is achievable in practice, as the implementation of ecb's post- quantitative easing shows that assets purchased by central banks to stimulate overall economic growth are benefitting more from the policy than assets which are not purchased by the bank (haldane et al. ; matikainen et al. ) . this means that the choice of asset class through which asset purchasing programs are implemented matters. this is particularly important in the low-carbon economy context, as the fossil fuel energy sector is largely financed through bonds and syndicated bank loans (cojoianu et al. ) , whereas much of the emerging clean technology companies are financed through private equity, equity issuances and asset financing (cojoianu et al. ; gaddy et al. ) . given that the ecb has chosen to enact its asset purchasing program post- crisis predominantly through bonds, this has been shown to favour the incumbent fossil fuel industry (battiston and monasterolo ; matikainen et al. ) , as % of ecb's corporate bond purchases (out of a total of € billion) are in ghg intensive sectorsthough they make up only % of the eurozone area economy and produce % of ghg emissions. the criteria for the corporate bonds bought under the pepp are that: (i) the company must be incorporated in the eurozone and its bond issuance denominated in euro, (ii) the firm cannot be a financial corporation (or a credit institution supervised by the ecb), (iii) it cannot be a public entity, (iv) the bond issuance has to be endorsed by one positive credit rating by an external credit assessment institution accepted within the eurosystem credit assessment framework and (v) they have a maximum maturity of up to years and a minimum maturity of months. in order to understand whether the ecb's bond buying activity during the covid- pandemic has been tilted towards less transparent, more fossil fuel intensive as well as anticlimate lobbying european energy companies, we undertake the following steps. first, we collect all the bonds issued by european energy companies during the period january to june from bloomberg. these span the following energy subsectors as classified by bloomberg industry classification system (bics): power generation, renewable energy, integrated oil and gas companies, oil and gas exploration and production, oil and gas services and utilities. this results in bonds. we then match each bond with ecb's bondholding portfolio, the borrower's record on pro/anti-climate lobbying from influencemap, the ghg intensity of the borrower (collected from bloomberg and measured as thousands tco -e/million eur revenue) and the ghg reporting completeness of the borrower (which is assessed by bloomberg and quantified as if the company is transparent about the organisational boundary it chooses to quantify its ghg emissions and otherwise, bloomberg terminal code es ). we further collect the borrower's revenue (million eur), bond amount issued (million eur) and coupon rate for each bond, also from bloomberg. our resulting dataset with complete data across all variables of interest is comprised of a cross-section of bonds issued across several currencies, and eurodenominated bonds. our dependent variable quantifies the likelihood that the bond of a european energy company is bought by the ecb during the first months of and coded as if it has been bought by the ecb, and if it has not. for our model, we employ a binary logistic regression model with robust standard errors. the full model specification is the following, where ε i is the stochastic error: we show that after controlling for the revenue of the issuer, the bond amount raised and the rate of the coupon, the ecb is statistically significantly more likely to buy the bonds of more ghg intensive european energy companies (models - , table ). on average, a one standard deviation increase in the ghg intensity of an energy company results in a % increase in the likelihood that its bonds are bought by the ecb (β = . , p < . , odds ratio: . , model ). when we consider only euro-denominated bonds (models and ), which are directly under the remit of the ecb, ghg disclosure completeness and pro-climate lobbying are statistically insignificant, yet negative, which suggests that the ecb may be likely to tilt its portfolio towards companies with poorer ghg emission disclosures and less responsible climate lobbying activities. subsequently, we include the bonds issued by european energy companies in denominations other than euro, to account for potential sample selection bias due to the choice of energy companies to abstain from issuing euro-denominated bonds as they may have received discouraging signals from the ecb. in other words, analysing the bond issuance of european energy companies in all currencies considers signals that the ecb may have given to the energy companies prior to issuance, while analysing only euro-denominated bonds only considers the observable decision of the ecb to purchase the bonds of specific energy companies post-issuance. when we do so (model ), it emerges that considering the entire universe of bonds issued by european energy companies, the ecb's portfolio is tilted not only to those energy companies that are more ghg intensive, but also to companies which are less transparent on their ghg performance as well as those companies who are more likely to oppose progressive climate action. having established statistical significance, we investigate the economic and statistical relevance (brooks et al. ) . inspecting the economic relevance of the ghg intensity variable in model , we find ghg intensity to have the largest marginal effects. in terms of statistical relevance, we find ghg intensity to have by far the largest shapley r-squared value, contributing more than % to the overall explanatory power of model . in conclusion, the importance of ghg intensity is underlined by its marginal economic effects and its statistical relevance, as it explains more variation in the dependent variable on its own than all other variables taken together. we also conduct further robustness tests controlling for ecb bond i = + * pro-climate lobbying activities score i + * ghg emissions intensity i + * ghg reporting completeness i + * borrower revenue i + * bond issuance amount i + * bond coupon rate i + i . bond maturity, bond rating and interactions of key variables and find our results to remain statistically significant. in conclusion, drawing on a novel dataset of corporate bonds issued in the european energy sector since january and the database of ecb's purchases under the pepp in response to covid- , we find evidence that the likelihood for a bond to be bought by the ecb increases with the ghg intensity of the bond issuing firm. we also find weaker evidence that the ecb's pepp portfolio during the pandemic is likely to become tilted towards companies with anti-climate lobbying activities and companies with less transparent ghg emissions disclosure. our findings imply that, at later stages of the covid- recovery, an in-depth analysis may be necessary to understand if, and if yes, why the ecb fuelled the climate crisis. even if one accepts that fossil fuel companies were eligible for pepp, then our preliminary evidence still raises the significant question, why the ecb was more likely to directly finance those fossil fuel firms that are likely more harmful to the planet (i.e. have a higher ghg intensity)? the coronavirus covid- pandemic is the defining global health crisis of our time, causing over half a million deaths to date ( july ). but covid- is much more than a health crisis, it has tremendous socioeconomic impact, the scale of which is still hard to assess. measures to address the health crisis generate economic impacts (and vice versa). social isolation measures to "flatten the curve of the pandemic" buy time to increase capacity in the healthcare sector but inevitably deepen the macroeconomic recession. the world bank expect the global economy contract by . % in , approximately three times the size of the - great financial crisis (gfc) and far more widespread. emerging markets and developing economies (emdes) have been severely hit by massive capital outflows, reducing debt servicing abilities (especially for dollardenominated debt) and generating long-term challenges; "with more than % of emdes expected to experience contractions in per capita incomes this year, many millions are likely to fall back into poverty" (world bank ). the economic consequences of the adverse coronavirus shock are: (i) an elevation of uncertainty, which increases precautionary savings, thus reducing consumption and also curtails the appetite for productive investments; (ii) a rise in unemployment, part of which is likely to be permanent; (iii) a decline in the volume of international trade and disruptions in global supply chains; (iv) falls in commodity prices (especially the price of oil), making current account financing of traditional commodity exports challenging; (v) a sharp increase in the required risk premia for holding risky assets. this initially resulted in a plunge in prices of risky assets (e.g. stocks or high-yield bonds) and a sharp increase in financial volatility. however, central bank interventions (especially by the federal reserve of the united states of america-us fed) have seen recoveries from march . extraordinary macroeconomic policy response, on both the fiscal and monetary fronts, has slowed the rates of economic decline. in many countries, fiscal measures have replaced a proportion of lost incomes and mitigated default risk, loan guarantees have helped keep businesses afloat, and liquidity provision by central banks have kept the financial system functional. fiscal authorities in european and the usa took measures that can be classified within three broad categories: immediate fiscal measures and direct transfers to households; tax deferrals and liquidity measures; and loan guarantees. the scale of intervention is truly unprecedented reaching almost % of gdp in germany and % of gdp in the usa. on the monetary front, the fed cut interest rates to zero, announced unlimited purchases of treasuries and mortgage backed securities and started buying corporate debt. moreover, the fed opened debt swap lines with foreign central banks to provide dollar liquidity to the international financial system. the value of fed measures to date exceeds . trillion dollars. the european central bank ecb "has offered low-interest loans to banks, significantly boosted asset purchases, and allayed fears of member-country defaults by lifting distributional restrictions on its bond-buying program" (world bank ). moreover, the eu's recovery plan (proposed may ) mobilizes investments through a recovery instrument of € bn for the period - and a reinforced a long-term budget of € . trillion for the period - . despite these unprecedented measures, the course of the pandemic and its developing economic impact remains uncertain. how can long-term economic dislocation be avoided? a first priority should be to ensure that the work force remains employed. second, governments should channel financial support to public and private institutions that support vulnerable citizen groups. third, small medium enterprises (smes) should be safeguarded against bankruptcy. (the need for taxpayer money to support large nonfinancial corporations is much less obvious.) fourth, policies will be needed to support the financial system as nonperforming loans mount. fifth, fiscal packages, comparable to the loss of gdp, will have to be financed by national debt. while this should be structured to avoid another debt crisis, finance should be directed to investments with positive social, economic and environmentally sustainable profiles, as discussed in the following. there is widespread scientific speculation that economic growth has pushed humanity into new ecological niches wherein humans and animals exchange novel, infectious viruses of which covid- is just the latest in a considerable list of examples. furthermore, the intergovernmental panel on climate change (ipcc) has warned that global warming will likely accelerate the emergence of new viruses. overall, climate change has the potential to end up killing more people than covid- , although this is obtusely referred to as an "increased frequency and severity of natural disasters". such language, the delayed, cumulative nature of the threat and the necessity of coordinated international response, all mitigate against the urgent action that is required. timing is also important. as ipcc ( ) reports, the level and speed of change needed to successfully tackle the climate crisis, is unprecedented; incremental changes will not be enough. that said, there are aspects of the climate change crisis which are less challenging than the covid- pandemic. as sterner ( ) notes, the climate crisis requires policy changes that are less disruptive, economically, socially and culturally, than the measures being taken right now to tackle covid- . for the climate, we do not need to close down the economy. on the contrary, we need a transition to a low-carbon economy that supports public and private investments in renewables, energy efficient and circular, technologies and infrastructure. these technologies exist (wind, solar, etc.) and are becoming consistently cheaper than fossil fuels, while energy storage installations are increasing exponentially. there is growing evidence that green stimulus policies have advantages over traditional fiscal stimulus and that climate-positive policies also offer superior economic characteristics. for example, hepburn et al. run a global survey to assess stimulatory fiscal recovery policies implemented in response to gfc. the economists from countries were asked to ascertain their perspectives on covid- fiscal recovery packages according to: "speed of implementation", "long-run economic multiplier", "climate impact potential" and "overall desirability" according to social, political and personal factors. the responses indicate that green stimulus policies deliver higher multiples due to reduced long-term energy costs and flow-on effects to the wider economy. "relaunching the economy does not mean going back to the status quo before the crisis but bouncing forward. we must repair the short-term damage from the covid- crisis in a intergovernmental panel on climate change, . way that also invests in our long-term future". this code explains the strategy of the eu recovery plan based on three axis: the european green deal, adaptation to the digital age, and a fair and inclusive recovery for all. we must start investing in what makes our socio-economic system resilient to crisis. now is the time to usher in systemic economic change and the good news is that we have our blueprint: it is the combination of un agenda ( sdg) and european commission's european green deal. the european green deal (egd) announced december is the new growth strategy of the european union and is based on four principles: (a) climate neutrality by , (b) protection of human life and biodiversity by cutting pollution, (c) world leadership in clean technology, (d) leave no one behind. the egd investment plan amounts to eur billion per year by . the egd just transition mechanism will help mobilize at least € billion over the period - in the most affected regions. the - multiannual financial framework (mff) allocates an overall target of % for climate mainstreaming across all eu programs. the proposed european climate law aims to turn the egd's political commitment of climate neutrality, into a legal obligation. based in un environment program (unep) emissions gap report , global emissions need to be reduced by % by . unfortunately, the proposed law does not include an ambitious goal with regards emissions, nor does it address the legislative interventions required to achieve climate neutrality by . together with the egd, the sustainable development goals (sdgs) and the paris agreement call for deep transformations. while significant progress is being made on some goals, no country is currently on track towards achieving all sdgs. sachs et al. ( ) identify the major interventions needed to achieve each sdg and group them in six sdg transformations, which operationalize the sdgs at government level and can prove instrumental for egd implementation. the success of the join implementation of the sdgs, the egd and the eu recovery plan will depend on the eu's capacity to engage with its citizens in codesigning the pathways that will allow them to reach the vision, hence the introduction of the european commission climate pact. https ://ec.europ a.eu/info/live-work-trave l-eu/healt h/coron aviru s-respo nse/recov ery-plan-europ e_en. european parliament, committee on environment, public health and food safety draft report on the proposal for a regulation of the european parliament and of the council establishing the framework for achieving climate neutrality and amending regulation (eu) / (european climate law), / (cod). the review of the climate law summarizes the review of the climate change committee of the greek ministry of energy and the environment, the first author of this paper is a member of this committee and co-author of this greek review. united nations environment programme ( ). emissions gap report . unep, nairobi. the proposed climate law does not allow the european commission to impose sanctions on member states (ms), while there is lack of reference to the financial mechanisms that will be required to achieve the goal of climate neutrality. moreover, the law does not consider (a) the well-documented heterogeneity in area-specific climate vulnerability among ms, (b) an eu wide carbon tax and (c) strengthening the european ets. sachs et al. ( ) : the sustainable development goals and covid- . sustainable development report . cambridge: cambridge university press. ( ) education, gender and inequality; ( ) health, well-being and demography; ( ) energy decarbonization and sustainable industry; ( ) sustainable food, land, water and oceans; ( ) sustainable cities and communities; and ( ) digital revolution for sustainable development. https ://ec.europ a.eu/clima /polic ies/eu-clima te-actio n/pact_en. recent generations, including the present, have experienced at least three global crises: the financial crisis - , the ongoing covid- pandemic and the developing climate crisis. if we continue attempting to address the latter two with the same socio-economic model that gave rise to the former crisis, we will fail to find a sustainable and resilient socio-economic-environmental pathway. we believe that we can even do better than just react to crises by adapting to the new crisis-born reality. we can use the integration of scientific, economic and socio-political knowledge to design policies which not only address the immediate impacts of the covid- pandemic, but mitigate the existential threats of future pandemics and the ongoing and unfolding disasters of climate change, biodiversity loss and planetary boundary exceedance. what is needed now is a fundamental transformation of economic, social and financial systems that will trigger exponential change in strengthening social, economic, health and environmental resilience. we need big thinking and big changes. system innovation and transitions thinking can help but calls for intense public participation. now is the time, in addition to directing funds to the control of the epidemic and relevant biomedical research, as well as investing in border security, safe travel and safe trade, now is the time for financial institutions and governments to embrace eu taxonomy for sustainable investments ( ) to phase out fossil fuels by deploying existing renewable energy technologies, eliminate fossil fuel subsidies and redirect them to green and smart climate mitigation and adaptation infrastructural projects, invest in circular and low-carbon economies, shift from industrial to regenerative agriculture and invest in food security, promote european supply chains, reduce transportation needs and exploit the limits of the digital revolution, while ensuring secure information and communication technology networks. a decisive march along this sustainable pathway will enhance economic and environmental resilience, create jobs, and improve health and well-being. the transition should be inclusive and "leave no one behind", hence the need of transforming citizens into codesigners and co-owners of the sustainability transition pathways. kiel institute for the world economy, kiellinie , kiel, germany. wilfried. committing to rigid shutdown measures to contain the spreading of the corona virus has been undertaken on the tacit assumption that these measures will be temporary and can be loosened when the covid- infection rates decrease and discontinued altogether once vaccines are available. mitigating climate change and achieving ambitious temperature targets as set out in the paris agreement require a long-term structural change taking us away from our current carbon-intensive economy to a zero-carbon and then net-negative carbon economy. as current research holds out little hope that a "perfect" vaccine in the form of solar climate engineering will be available in the future, the measures and efforts required must translate into a permanent, ongoing form of commitment. while progressive climate change and the spread of the coronavirus operate on very different timescales, impatience about the duration of corona lockdown has indicated once more a fundamental problem for (long-term) environmental concerns. clearly, the economic and social costs associated with the emergence of the virus and the shutdown are significant , oecd . but any serious cost-benefit analysis would need to take into account not only the fact that different degrees of lockdown are available but also that the overall cost is affected by the expectations of agents regarding possible future re-lockdowns due to insufficient containment of the virus. seen thus, it is anything but clear at which point in time the actual cost of lockdown would have exceeded the economic cost of the virus spreading in an unmitigated (or insufficiently mitigated) way. during the course of lockdown measures, voices calling for a "green" recovery stimulus package centring around low-carbon investments in the aftermath of the corona crisis have make themselves heard. by contrast, advocates of postponing climate mitigation-related taxes, levies, and regulations have also entered the fray, claiming that timely recovery should not be jeopardized by any additional economic burdens. the debate on the relation between (economic) recovery and climate policies has been conducted from three major perspectives. the first of these is largely notable for general statements of intent recommending that the recovery should be "green" and sustainable, that eu climate targets should be supported, and that other environmental targets (maintaining biodiversity, etc.) need to be taken into account when designing recovery measures. such well-meant counsels as the statement issued by the german national academy of sciences leopoldina ( ) are useful in reminding us that recovery from the corona crisis should not come at the expense of neglecting other objectives and that climate policy should not be backburnered, as was the case after the financial crisis in . otherwise, they are of little practical value. the second approach has involved rather detailed proposals calling either for a "greening" of recovery by foregrounding measures to support renewable energies, public transport, energy efficiency, etc. or for a "blackening" of recovery by postponing and/or abandoning climate measures and environmental regulations. predominantly, these proposals are representing the positions of the various interest groups involved. for example, representatives of the aviation industry try to prevent the harmonization of carbon prices on fuels with respect to kerosene and argue against the introduction of kerosene taxes. this idea resurfaces in the discussion on recovery measures by, say, the austrian aviation association ( ). on the other hand, in its comprehensive list of (recovery) demands, the ngo german environment action ( ) urges for example for the abandonment of blue hydrogen projects (though not explaining why this is likely to stimulate economic recovery). various other interest groups are in favour of postponing, suspending or even abandoning existing environmental and climate regulations. for example, janusz kowlaski, the polish deputy minister of state assets urges "…[that] the ets [european emissions trading scheme] should be removed from january , or at least poland should be excluded from the system". clearly, there is no point in discussing nonsensical ideas of this kind. but some of these proposals also make sensible suggestions like adjusting the german cap on renewable energy installations or abandoning the eu average fleet-consumption regulation because the former contradicts german renewable energy targets and the latter is an inefficient instrument for regulating vehicle emissions. however, these suggestions do nothing to provide stimulus for a quick recovery. while specific processes and regulation timelines for regulations may need to be adjusted in the context of the corona crisis, sensible measures of this kind should be discussed and decided upon in the regular political process. confining potential stimulus and recovery measures to their proper purpose does not mean imposing a ban on meaningful (climate or environmental) policies that are not associated with the corona crisis. the third and most sensible perspective replaces specific proposals with (sustainability) assessment guidelines like those suggested by the world bank ( ). while hardly any possible recovery measure would perform well against the comprehensive list of criteria provided by the world bank, such guidelines are helpful in arguing against interest group driven proposals. the world bank has suggested that potential measures up for consideration as part of a recovery strategy need to be assessed against both, short-and long-term criteria, an example for the former being the expected economic multiplier associated with certain measures. bayer et al. ( ) suggest that income transfers (as planned under the us cares package) perform well against this specific short-term criteria: they could help to stabilize private-sector spending and the multiplier could increase to in the case of transfers being conditional-but not related to emissions but to the propensity to consume, i.e. conditional on being unemployed. however, privatesector spending like this should not imply any unintended adverse effects on essential long-term structural change that might arise from such things as (temporarily) adjusted risk preferences. once postponed or stimulated demand and investment take place during the recovery process, carbon-price signals are vital in providing technology-neutral incentives for low-co purchasing and production decisions. overloading stimulus or recovery packages with too many (emission-related) conditions performs poorly against the short-term criteria with respect to a timely recovery. even worse, the inclusion in recovery packages of various detailed suggestions from the various interest groups usually results in a non-transparent, rent-seeking, and political bargaining process in which it remains unclear whether (sensible) individual emissionrelated decisions are being prioritized at the expense of a more challenging long-term climate policy. accordingly, accounting for the long-term criteria requires that existing or planned climate policies providing incentives for emission reductions and technological innovation should remain in place and not be postponed, let alone weakened. otherwise, uncertain (short-term) recovery impulses most likely come at the cost of less efficient emission-reduction paths in the long term. the coronavirus pandemic is having a serious impact on the economy. eurostat estimates that seasonally adjusted gdp decreased by . % in the eu during the first quarter of , compared with the previous quarter. in certain sectors, the covid- crisis has led to the temporary or even permanent closure of sites, a collapse in demand and an increase in production costs linked to the fight against the spread of the virus. this decline in activity can trigger bankruptcies, which result in unemployment and the destruction of physical and human capital, leading in turn to a loss of specific knowledge and skills and to market concentration and the partial relocation of production activities. from an environmental point of view, bankruptcies and relocation may generate higher emissions by increasing production in more polluting sites abroad and by increasing the transportation of goods. in addition, the decline in economic activity may have a negative effect on research and development spending, which is crucial for future growth and the development of more environmentally friendly technologies. many measures have been put in place to limit the economic effects of the covid- crisis. at the european union level, a number of measures such as direct subsidies, selective tax benefits, advance payments, state guarantees for loans and subsidized public loans to companies have been implemented. in addition, many large companies are being bailed out and there is a debate on whether conditions should be attached to this state aid. these conditions could relate to various commitments such as relocating activities, paying taxes, safeguarding employment, and protecting the environment. on may , the eu confirmed that large companies receiving emergency cash during the covid- crisis will not be obliged to devote funds to "greening" their operations. rescued firms will only have to report on their use of the aid and in some cases the aid could be attached to conditions such as a ban on dividends and management bonus payments. nevertheless, member states are free to design national measures in line with additional policy objectives, such as further enabling the green transformation of their economies. many stakeholders are still pushing for conditions to be attached to the bailouts. for instance, the eg think tank proposes making the rescue of airlines conditional on the use of less polluting fuels and tying aid to car manufacturers to the development of electric vehicles. a group of german companies has also requested that state aid be linked to climate actions. this article examines the merits of making aid conditional on environmental efforts. we focus on the rescue plans for companies rather than on recovery plans or the european green deal, which was launched before the covid- crisis. we show that tying aid to environmental efforts is difficult to implement and requires both controls and sanctions, as well as a large amount of information. we also discuss the merits of tying bailouts to environmental efforts compared with implementing more stringent environmental policies (presuming that companies will be bailed out in any case). since environmental efforts can lead to higher product prices and lower profits, two dimensions of acceptability must be taken into account: acceptability from the point of view of companies and social acceptability. acceptability of environmental policies by companies is a necessary condition for their implementation. in the midst of the current crisis, many polluting industries are lobbying to weaken and delay environmental regulations. for instance, the environmental protection agency issued a sweeping suspension of its enforcement of environmental laws, allowing companies to breach environmental standards during the coronavirus outbreak. in indonesia, the trade ministry revoked rules requiring basic certification that wood exports were legally produced, in response to lobbying from the furniture and logging industries. lobbyists may also encourage legislators to compensate companies for losses related to environmental regulation. the implementation of rescue plans, by providing aid and guarantees, could be a tool to make environmental efforts more acceptable for firms. acceptability by society is also central. it depends on the redistributive effects of environmental policies, especially since they are often regressive. in france, the "red caps" and "yellow vests" movements illustrate the difficulty of implementing such policies. both of these movements led to the cancellation and delay of environmental policies (eco-tax applied to heavy vehicles and carbon tax, respectively). the covid- crisis may affect societies' support for more ambitious environmental policies. several observations can be made. first, it appears that there is a confirmation bias, as countries and ngos that had already committed to fight against global warming want to intensify efforts, while countries that were already recalcitrant are calling for a decrease in efforts in the light of the economic crisis. second, the covid- crisis makes the risk of disaster more salient and vivid and may therefore increase the demand for stronger environmental protection. as explained by sunstein ( ) , if a particular risk is cognitively "available" then people will have an increased fear of the risk in question. finally, the crisis has revealed that populations will accept drastic measures (lockdown, wearing face masks) and a rapid change in social norms (social-distancing, for instance), which shows the ease with which individuals could adapt to more ambitious environmental policies. see oates and portney ( ) for a literature review of interest groups and environmental regulation. "polluter bailouts and lobbying during covid- pandemic", the guardian, april , . burkey and durden ( ) and joskow and schmalensee ( ) have detailed how firms can influence the regulator's decisions in the context of pollution rights markets, while bovenberg and goulder ( ) , hepburn et al. ( ) and nicolaï ( ) have shown that few permits are sufficient to neutralize the losses in profits and make the implementation of pollution permits acceptable. on march , the czech prime minister, andrej babis said that the european green deal should be put to one side. during the same period, the green coalition of environmental organizations organized an appeal urging lawmakers to design a green, healthy and just recovery. we study two possible ways of increasing environmental efforts: making aid to companies conditional on environmental efforts or making existing environmental policy more stringent. making aid conditional on efforts to protect the environment can take different forms. commitments may relate, for example, to reducing pollutant emissions, using less polluting production processes, increasing expenditure on research and development, and producing less polluting products. for instance, in france, renault's bailout requires it to increase the share of electric vehicles, while the billion euro air france bailout imposes the use of at least % of alternative jet fuel by , a target of % emissions cuts by , and a % decrease in domestic flights by , especially those that compete with high-speed trains. conditional aid as currently envisaged is equivalent to negotiating and implementing individualized standards on technology, final products, research and development efforts, emissions and performance for each company. the first question that arises is how to determine the conditions. conditional bailouts allow the regulator to individualize the standards applied to a particular firm, making it possible to set the most appropriate instrument and the optimal level of severity for each firm. when making its decisions, the regulator should consider the possibility of a rebound effect as explained by saunders ( ) . an emissions standard may avoid such an effect, but a standard on technology may increase emissions. furthermore, the regulator should take into account its access to information. if the firm commits to finance research and development in green technology in exchange for being bailed out, it will be hard for the government to determine whether the state aid was actually allocated to research. for example, since the results of research are uncertain, a lack of innovation may be explained either by the firm's characteristics (anti-selection problem) or by the misallocation of public funds (moral hazard problem). finally, the regulator should take into account competition, since firms receiving bailouts usually have market power. a government will mainly intervene to save a firm if its bankruptcy would have a significant effect on the economy. the government should hence ensure that the conditions do not further distort competition. a firm with a competitive advantage, for example with cleaner technology, may have an interest in advocating more ambitious environmental efforts for itself but also for its competitors in order to increase its market share by preventing the entry or inducing the exit of such competitors. the effectiveness of conditions will therefore depend on the type of commitment, competition in the market, and the demand elasticity of the final good. another question relates to the duration of the contract. some actions are reversible and after a certain period of time companies can cancel or amend them. for example, the use of cleaner but more expensive inputs is an easily reversible strategy, while a shift in production, for example from internal combustion vehicles to electric vehicles, is a more expensive choice to change. in addition, research and development efforts produce long-term effects with the creation of innovations and possible technological spillovers. the duration must therefore be individualized in function of the company's characteristics. it should also be noted that conditional aid can lead to windfall gains. some companies had already planned emission reductions and changes in strategy. these decisions were aligned with the companies' interests. if the company benefits from the environmental efforts put in place, these can no longer be considered as a counterpart to the company's bailout and the company should then make greater efforts. it seems clear that the success of such schemes lies in companies' compliance with the commitments. companies may nonetheless have an interest in not complying with them. it is therefore necessary to monitor companies' implementation of the commitments and to sanction those that fail to honour them. supervision costs will thus be incurred. one possibility for alleviating control costs is to publicly announce the commitments, which facilitates the monitoring of companies' commitments by society (e.g. by journalists, politicians and non-governmental organizations). this also gives the firm a further incentive to respect the agreement in order to maintain its reputation and to retain its customer base (heyes et al. ( ) and heyes and kapur ( ) ). we now focus on the sanctions to be applied in the event of non-compliance with the commitments. various penalties could be considered, such as financial penalties, a ban on applying for the attribution of public contracts and the state's participation in the company. the sanctions must be explicit, credible and sufficiently significant to discourage companies from breaking their contracts. however, given the current economic situation and the fragility of companies, governments should be cautious when using financial penalties or bans from participating in public contracts since, if effective, they could be fatal to companies. an alternative could be for the state to take a share in the company in the event of non-compliance with commitments. such shareholdings would make it possible to influence companies' strategic choices, but they raise efficiency problems. cavaliere and scabrosetti ( ) , schmidt ( ) and de fraja ( ) , for instance, highlight two effects: production is more efficient in privatized firms because better incentives can be given to managers and employees (productive efficiency), while public firms are more socially efficient because the government cares about social welfare and internalizes externalities associated with firm liquidations (allocative efficiency). existing environmental policies inevitably generate monitoring costs; however, these costs will be borne in any case. increasing the stringency of environmental policy involves determining new levels for existing instruments, which generates costs, but there will be no additional monitoring costs. moreover, opting for a more stringent environmental policy means that market-based instruments can be employed, which is not feasible in the first strategy. due to the principle of non-discrimination, it is unlikely that individualized taxes could be introduced under the negotiated bailout conditions. environmental economists agree that these instruments are more effective than command-and-control instruments since they have the potential to generate revenue and allow for a double dividend, provide incentives to invest in clean technology and require less information to be designed. nevertheless, making aid conditional on environmental efforts makes it possible to individualize the regulatory instruments for each firm, which is not possible in the context of marketbased instruments. the positive effect of the instrumental individualization induced by conditionality only materializes if the existing environmental policy is based on commandand-control instruments. with market-based instruments, individualization is not required to achieve an efficient outcome, whereas it is necessary in the case of command-and-control instruments. companies' negotiation power is an additional argument for increasing the severity of environmental policies rather than conditioning bailouts on environmental efforts. regardless of the policy, firms may use their bargaining power to mitigate the stringency of the regulation or the effort they commit to make. it is clearly easier for a firm to influence its specific negotiated conditions, which are one-on-one relationships, than to influence environmental policies, which would require coordination and a common interest among firms. furthermore, making environmental policy more stringent will affect all companies and not only those receiving bailouts, which increases the attractiveness of this strategy. since they apply to a larger number of companies, these more restrictive policies will have a greater effect on the environment. to conclude, this note analyses the advantages and disadvantages of making bailouts conditional on environmental efforts. we show that such a system could be beneficial for the environment and would be a counterpart to bailing out companies. however, it is costly, requires a large amount of information, and is less efficient than increasing the stringency of existing environmental policies. on the other hand, making aid conditional on environmental effort is more acceptable from the companies' point of view than increasing the severity of environmental policies. companies would rather commit themselves to environmental efforts than pay taxes or buy permits in order to minimize compliance costs. to succeed in making environmental policy more ambitious, the regulator needs the support of society to compensate for companies' reluctance. if there is strong public support, governments should not negotiate conditional aid but should increase the severity of environmental policies. they can integrate such actions into recovery plans or, in the case of the european union, into the revision of the green deal. it is therefore particularly important to study how the covid- crisis has changed society's perception of the need for more stringent environmental policies. for instance, a recent survey by ipsos shows that three out of four people in major countries expect their government to make protection of the environment a priority when planning a recovery from the coronavirus pandemic, although there is considerable heterogeneity across countries. the post-covid- reality is changing the context of most policies, including those in the fields of energy and the environment. the eu green deal objectives should be maintained but concrete policies may have to be adjusted to this new reality. a major energy transition is under way, shaped by political will to tackle climate change. policies have been defined under the paris agreement and geared to a number of targets. the eu has decided to reduce its greenhouse gas emissions by by at least % compared to and has agreed to continue the path towards climate neutrality by . the immediate recession following the covid- crisis drastically reduced energy consumption and greenhouse gas emissions (le quéré et al. ) . the sudden decline in greenhouse gas emissions is the opposite of what a meaningful response to climate change should be, in terms of both quality of life and economic efficiency. the challenge is to structurally decouple economic growth from emissions, not to have them both going down. the lockdown may have some lasting effect in changing some of our habits ), e.g. a generalized familiarity with telework. one can also expect a more general reflection on whether all business and leisure travel, not least by air, is necessary. nevertheless, many changes may not be as extensive and lasting as some would wish. for this reason, it is important to integrate a climate check into the stimulus packages that governments and european institutions are currently designing. the current health crisis and the likely economic downturn could be seen as an unsolicited-and much regrettableopportunity towards a carbon-neutral future koundouri ) . the agreements reached in the context of the eurogroup, the latest turn in the european budgetary discussions, as well as the statements by the commission president all point into this promising direction. the eu green deal has confirmed the eu emissions trading system (ets) as a key element and the price of eu allowances serves as a key indicator worldwide. as could and should be expected, carbon prices have fallen during the lockdown reflecting reduced demand for allowances in line with the drop in power and industrial production. this temporary drop from the € to a € - range, acts as an important automatic stabilizer for businesses in distress. a repetition of the experience of a sharp fall in the see on this issue the important draft report prepared by the european parliament committee on the environment, public health and food safety (european parliament, ). https ://lifed icetp rojec t.eui.eu/ / / /covid - -clima te-polic y-and-carbo n-marke ts/. https ://www.consi lium.europ a.eu/en/press /press -relea ses/ / / /repor t-on-the-compr ehens iveecono mic-polic y-respo nse-to-the-covid - -pande mic/-point : "work is ongoing on a broader roadmap and an action plan to support the recovery of the european economy through high quality job creation and reforms to strengthen resilience and competitiveness, in line with a sustainable growth strategy". https ://ember -clima te.org/carbo n-price -viewe r/. price of eu ets allowances after the recession and a long period of very lowcarbon price levels should be avoided. there are two reasons to believe that this could happen: (i) the eu ets now has a market stability reserve (msr) operating in the short-medium term and (ii) long-term market expectations could be shaped by the carbon neutrality objective. the msr started to operate last year and absorbs a potential oversupply in the market in the short and medium term. if the allowances market is "long" beyond a threshold set in legislation ( million tons, which is approximately % of ets emissions in ), the msr intervenes by withdrawing allowances equal to a percentage ( % up to , % thereafter) of the excess. at the end of , the allowance market was . billion tons "long" and, therefore, the supply of allowances for the period september to august has been reduced by million tons (these allowances are placed in the msr). some question whether the msr, with its delayed adjustment mechanism, will be sufficient to avoid a sharp fall in the allowance price (cf. flachsland et al. ) . in fact, any reduction in the supply of allowances through the msr for the period september -august will still be based on the excess supply of allowances in , i.e. before the current crisis started. in case the msr is unable to absorb the surplus, the eu commission should propose changes in the planned review of the ets in . long-term expectations might overtake this short-term risk. in its european green deal, the commission already indicated its intention to tighten the greenhouse gas emission reduction target from % to %, or even % (with respect to levels). today, the ets sectors face a mandatory emission reduction of % by compared to , and revised targets will likely increase that obligation by at least another or %. such a reduction in the supply of allowances would likely push their prices up, or at least reduce the extent of their decline, in the face of lower demand due to the economic downturn. therefore, carbon market participants as well as those developing innovative clean technologies and products of the future are likely to expect significantly higher carbon prices in the future. this raises the question about a possible loss of competitiveness in europe's traditional industries vis-à-vis competitors located in countries where no similar burden is imposed. some proposed a border adjustment, which would imply both a levy on imports and possibly a rebate on exports. however, such mechanisms are not easy to implement and are subject to criticism on both analytical and political grounds. therefore, it is of crucial importance to realize that a wto-compatible border adjustment mechanism will take some time before it can be implemented. meanwhile, at least equal attention needs to go to domestic policy reinforcement, such as support for innovation and a less rigid interpretation of the state aid rules. governments and european institutions are currently developing a major investment stimulus package. the commission's impact assessments on climate and energy policy serve as useful guidance on where a major surge in investments is needed. we know that energy efficiency requires a major push in the construction sector, both for new buildings and renovations in social housing, hospitals and schools. we know that the energy transition requires more investments in renewable energy, digitised grid infrastructure and energy storage. the significantly reduced electricity demand of today indicates that much more attention should go towards managing flexibility in real time instead of increasing baseload capacity. in transport, electrification is on its way, but investment in charging facilities, traffic management, clean public transport and long-distance rail needs to be ramped up. in industry, not least in power generation and carbon intensive industrial sectors, major efforts are being undertaken to develop new technologies based on hydrogen and carbon capture, use and storage, for example. such investments can create the jobs we need in the post-covid- era and allow to realise the eu green deal objectives at the same time. apart from carbon prices and investment support schemes, it remains of equal importance to maintain a consistent energy price signal throughout the economy. however, the recent fall in the prices of fossil fuels is upsetting the incentives that should support a transition towards sustainability. a reference criterion for economic decisions has been lost and the consequence may be a paralysis, or even a "comeback" of fossil fuels, mothballing or abandonment of investment in renewables. consequently, the business case of the green transition may appear to be weakened in the near term, but a clear policy response may be politically more acceptable because of the price drop in fossil fuels. this raises the question about the other half of europe's emissions, namely those not covered by the ets, in particular the sectors of transport and buildings. the pricing of these emissions can be much improved through the planned review of the eu's energy tax directive. this offers a major opportunity to remove the all too generous exemptions, such as on maritime and aviation fuels, or by adding a co element to the harmonized minimum tax rates. in such a manner, prices of fossil fuels could be stabilized at the pre-covid levels via temporary taxes, whose revenue could help finance the various income-support policies adopted and/ or an acceleration of investment projects for sustainability. in this way the eu should better prepare for a long period of low oil and gas prices and seize the opportunities these can offer. public measures to combat the covid- pandemic have led to a severe economic crisis. in order to cope with this crisis, comprehensive government aid is being requested. accordingly, governments across the world have pledged billions of euros for extensive recovery programs. one of the main questions debated in this context at the moment is how "green" these recovery programs should be ). the expectation of huge amounts of public money being distributed at short notice brings interest groups of every shade to the scene-preferably with old wish lists on hand. consequently, there is a big risk that recovery programs will be captured by interest groups (for an overview of the literature on regulatory capture, see dal bo ). on the one hand, climate change mitigation is put under pressure as being an "extra burden" for industries. for example, european car manufacturers have called for postponing the upcoming tightening of eu emission standards for car fleets (topham and harvey ) . some eu member states call for stalling the eu commission's plan of a european green deal (simon ). on the other hand, many recommend spending the public money mainly on measures that also help mitigating climate change-among them frans timmermans, executive vice-president of the european commission (schulz ), or fatih birol, head of the international energy agency (birol ) . there is one thing that must not be overlooked in this politico-economic competition: public funds are still short and must be used reasonably. otherwise ill-designed (green) subsidies can quickly turn into a part of the problem instead of being the solution. previous "cash for clunkers" programs warn as an example of a misguided recovery measure. these programs were introduced in many countries after the financial crisis and provided financial incentives to trade old, less fuel-efficient cars for new, more efficient ones. empirical analyses have shown very mixed results regarding both the economic and the environmental stimulus effects of these measures (grigolon et al. ; li et al. ; mian and sufi ) . at the beginning of every discussion about (green) recovery programmes, it is therefore important to develop transparent and sensible criteria based on which public aid should be allocated. after the initial bail-out programmes, public recovery programmes to stabilize the economy are now debated politically. certainly, this generates an unprecedented window of opportunity for structural transformation. moreover, the distribution of public aid may also justify committing beneficiaries to public interests to a certain extent. consequently, the currently available political degrees of freedom should be used to promote the transition of society towards sustainability. subsidies to branches like tourism, aviation and agriculture-which are particularly hit by the crisis and are lagging behind in terms of sustainability-should be paid conditional on meeting minimum environmental standards. new investments into long-lived, fossil-fuelled assets must be avoided. a recovery program cannot only be about re-establishing the status quo ante by assigning large public funds, possibly creating new barriers for sustainability transitions. in this respect, it makes sense to implement recovery programs that are in line with the objective of mitigating climate change-as called for by many at the moment. however, such green recovery programmes must not be arbitrary. green recovery programmes must go beyond green subsidies. first of all, it is also important to reduce unnecessary barriers for green investments, for example by revising legal constraints to the expansion of renewable energies like solar photovoltaics of wind power. moreover, any green recovery program can only effectively and efficiently spur decarbonization if it combines with a carbon price and the abolition of environmentally harmful subsidies. the direction of recovery must be crystal clear. otherwise green subsidies risk being ineffective and costly approaches to mitigating climate change (kalkuhl et al. ; palmer and burtraw ) , while imposing additional burdens on public budgets and reducing political degrees of freedom in the future. for subsidies to be economically justified, they need to meet clear criteria. for green recovery programs to succeed in the competition for public funds with other important policy fields (such as health or digitalization), they must help stabilize the economy. moreover, policy makers need to be aware that some of the currently observed economic problems might even resolve without any government aid. it can expected, for example, that global supply chains will resume and that people will catch up on purchasing durable goods like cars, at least partly. it is exactly (the maintenance of) environmental regulation that may help steer this consumption towards more sustainable modes. government interventions must take effect where permanent disruptions are looming. one example: innovative green business models may particularly be at risk if banks limit loans in the presence of the current uncertainties (lehmann and söderholm ) . in this case, government loans may provide direct assistance. in contrast, attempts to lower prices for goods and services-e.g. for cars (vat reduction, purchase premiums) or electricity (reduction of energy levies)-are rather inappropriate means to stabilize the economy. such measures fail to address the actual sources of insufficient investments or reduced purchasing power, and are therefore inefficient ways of spending public budgets. furthermore, it is unclear whether and to what extent such discounts will be passed through to final consumers by market prices (peltzman ) . green recovery programs should focus on government interventions that would also have been economically reasonable without the covid- crisis-for example, to correct market failures next to the co externality (bennear and stavins ; fischer and newell ; lehmann )-and that have the highest priority for climate policy. moreover, those measures should be implemented for which rational concepts have been drafted already and that can be realized promptly. positive examples of such "no-regret measures" can be found in the transport sector, for instance. this sector is severely lagging behind in terms of climate change mitigation, and economic rationales for public expenditures exist at least partly (briggs et al. ; low and astle ). in addition to that, numerous actors have already developed elaborated programmes of measures. those measures that can be implemented quickly, should now be launched-for instance to electrify the transport sector or to strengthen public transport. (green) recovery programmes must not only address the expenditure side. a currently still disregarded issue is the question how the required billions of euro could be raised. public expenditures for a green recovery program should at least partly be funded by polluters by implementing a carbon price and abandoning ecological harmful subsidies. such policies internalizing environmental costs would not be an extra sacrifice-but rather part of the solution both for revenue problems and for the redirection towards sustainability (for a review of the double-dividend hypothesis, see goulder ). the coronavirus crisis has opened up a window of opportunity for transformation. this should be used without getting off the regulatory track. green recovery programs must not be reduced to a mere competition for green subsidies. abandoning barriers to green investments and imposing a carbon price are equally important. where economically sensible, green subsidies should contribute to both stabilizing the economy and mitigating climate change. moreover, smart green recovery programs may contribute to raising revenues for the additionally necessary public expenditures. the world is currently facing the largest pandemic since spanish flu in . this has led to a lockdown policy on an unprecedented scale and measures of social distancing that are expected to continue. in france, as elsewhere, the "great lockdown" has disrupted food production chains through simultaneous shocks to demand and supply. populations have seen their food consumption habits be severely modified (e.g. closure of restaurants and markets). fearing food shortages, consumers have often stockpiled basic necessities, which has led them to actually provoke shortages, albeit temporarily. food production has also been disturbed, most notably by the reduction in the available workforce, whether domestic (due to the lockdown, illness or childcare) or foreign (temporary closure of borders). the issues at stakes are numerous, including deglobalization and environmental impacts, in both the long and short terms . the pandemic will lead to unprecedented uncertainties in food supply chains. in addition to the health crisis, a dramatic drop in worldwide gdp is anticipatedapproximately %-with a partial catch-up in , according to the international monetary fund (imf ). the reduction in income will soon impact food consumption. some authors are already pointing to an increase in social inequalities. the most fragile populations could slide into severe food insecurity, including in developed countries, as shown by deaton and deaton ( ) for canada and power et al. ( ) for the uk. however, the fao is rather reassuring about the total volume of food (cereal) at the global level. it also notes that the world price index has fallen in recent months. this does not necessarily mean that this decline is effective regardless of the type of agricultural production. in particular, fruits and vegetables could become more expensive. agriculture is by nature a risky activity, and the adoption of eco-environmental practices can only increase this risk. as a consequence, organic farmers, for instance, could be particularly affected by the combination of health and economic crises. the cost of organic production is therefore structurally higher, which could pose a problem in the event of an income shock. to add value to their production, many small farmers combine organic farming with short food supply chains. this makes it possible to regain a more comfortable margin than that allowed by mass distribution. however, here again, the health crisis has profoundly changed marketing channels. while supermarkets were stormed with shoppers at the beginning of the crisis, some short channels were closed, such as markets and restaurants, to preserve social distancing. this article therefore considers the consequences of covid- for sustainable agricultural practices, particularly for farmers who have chosen organic farming in short supply chains. to do so, we use the multi-level perspective (geels ) approach to conceptualize socio-economic transitions. the multi-level perspective (mlp) approach analyses transitions as mutation processes from one socio-technical regime to another under the pressure of macrolevel forces and the emergence of market niches that could provide the basis for a new regime (geels ) . we show here why local food supply chains are perceived by some farmers-especially the smallest-as a way to enhance the economic value of eco-friendly agricultural practices, such as organic farming. in france, the current dominant socio-technical regime based on conventional agriculture emerged gradually in the s. conventional agriculture diffuses slowly at first because of the high investment costs. however, the french state and farmers' unions support it as a means of increasing production and improving living and working conditions. farmers are then inclined to take on more debt and become dependent on their suppliers (phytosanitary products, seeds, tools, etc.), the food industry and supermarkets. as a result, this modernization of agriculture has been controversial from the outset, at least with regard to farmer autonomy and the country's food sovereignty (levidow et al. ). the early development of organic farming in france began in the s due to the effects of three negative effects of conventional agriculture, namely pollution, soil impoverishment and the lack of autonomy mentioned above. the organic label has become a marketing argument that is profitable for large farms, since they achieve significant scale effects on these standardized products. large scale organic farming is therefore less demanding and, by its nature, allows to benefit from the effects of scale. this is new competition for small farmers struggling to differentiate themselves. the organic label itself does not allow farmers to free themselves from the pressures upstream and downstream in the production chain. to differentiate themselves from large organic farms, some farmers are starting to sell in open-air markets again. at the same time, the idea that fair trade can concern north-north relations and not only north-south relations is beginning to emerge, which favours the development of community-supported agriculture. a new initiative is gradually emerging: collective farmers' shops. these initiatives began, for example, in the south of france in the mid- s and represent a more important restructuring of the farmers' market. in the medium term, the drop in income could increase the consumption of basic necessities (giffen goods) to the detriment of organic products. everything will depend on the elasticity of the demand for these products. until now, organic products have been extremely popular. it is possible that "industrial organic" will take market share from a "more artisanal organic" if consumers still want to consume organic products but cannot afford to spend a large part of their budget on them. farmers in short supply chains often have several distribution channels simultaneously, especially market gardening farmers. this allows them to be more responsive to demand. thus, the closure of farmers' markets and restaurants can be compensated for by farm shops, community-supported agriculture (csa), fixed point or home delivery, or collective farmers' shops. regarding hygiene measures, farmers are faced with two main types of strategies: receiving customers in the original locations or digitizing the process, including the more marginal case of automatic food dispensers (e.g. for eggs). selling in physical places must respect social distancing, which discourages consumption. however, conserving this method makes it possible to maintain the relational proximity between farmers and consumers. initially, this relational proximity is one of the main arguments in favour of short supply chains, as it is supposed to allow better traceability of products and to fight against social isolation (especially of the elderly). the other strategy, therefore, is to limit direct contact between human beings by means of computer tools. internet platforms already existed before the crisis and are expected to develop. some were public, such as those set up to supply school canteens. the pandemic has accelerated this process, and some regions have launched their own platforms. this solution is very time-consuming and seems difficult to sustain, unlike the others, which should have an impact in the long term. another practice has been reinforced: farm shops. during the lockdown, some consumers presumably had more time to cook and go to farms. others fled from supermarkets, considering that the products were handled by a large number of people and that there was too much traffic. it is difficult to predict whether these changes in behaviour will have a long-term impact. in addition, the pandemic has raised concerns about the reliability of international distribution channels. it is therefore possible that some policies may be sensitive to the has been growing at a very fast pace (see an indicative list of references in supplementary material ). however, little attention has been paid to the reliability of this type of epidemiological data to make statistical inferences. our initial aim was to produce a detailed statistical analysis of the relationship between weather conditions and the spread of covid- . this question has attracted significant attention from the media (e.g. ravilious ; clive cookson ) and the research community (e.g. araujo and naimi ; carleton et al. ; see a wider list in supplementary material ) due to the possibility that summer weather might slow the spread of the virus. after going through all the steps of such an analysis, we reached the unexpected conclusion that the limitations of the available covid- data are so severe that we would not be able to make any reliable statistical inference. this applies, for example, to the data provided by the john hopkins university (dong et al. ) and the data collated by xu et al. ( ) . this is a concerning and very important finding considering that such data are being widely used to make crucial policy decisions on a wide range of topics. since invalid causal inferences could be made with the publicly available covid- data, and then enter policy-making discourse, there is an urgent need to raise awareness among the scientific community and decision makers regarding the limitations of the information at their disposal. the elements discussed in this paper are also likely to be applicable to other epidemiological datasets obtained with insufficient testing and monitoring, either during exceptional epidemics or seasonal outbreaks. several challenges could undermine any causal statistical analysis of the influence of a potential determinant, such as the weather, on the spread of covid- . to start, confounding variables are likely to pose a significant problem: many factors (e.g. changes in policy or social interactions) are simultaneously influencing how the disease spreads. in addition, significant challenges come from the limitations of the covid- case count data itself. firstly, testing capacity has been a major issue in most countries. before march , very few countries had sufficient testing capacity. by april , highincome countries had significantly increased their testing capacity, but testing remained critically infrequent in most low-and middle-income countries. figure , panel a, illustrates the effect that insufficient testing capacity has on the number of confirmed cases. it distinguishes between three phases of limited (i), intermediate (ii) and widespread (iii) testing. in phases i and ii, there is a risk that the number of confirmed cases depends more on the number of tests available than on the actual number of people who have covid- , questioning the validity of any analysis relying too heavily on these data. moreover, there have been numerous concerns regarding the accuracy of the covid- tests performed so far (ai et al. ; apostolopoulos and tzani ; hu ; hall et al. ) . figure , panel b , illustrates the effects of both false-negative and false-positive test results on the number of confirmed cases. false-negative results would imply that the number of confirmed covid- cases is underestimated. false-positive results would imply that people who do not have covid- are included in the number of confirmed covid- cases. concerns regarding test accuracy create an additional problem of measurement that might affect statistical analyses. the two above-mentioned challenges are inherent in all current datasets of covid- confirmed case count and mortality. in addition, specific datasets may have imperfect geographical or time coverage. to look at the impact of the weather on the spread of covid- , we initially used a well-established approach, similar to the ones used previously to look at the impact of the weather on other diseases (e.g. deschenes and enrico ; gasparrini et al. ) (see details in supplementary material ). however, the fundamental measurement issues associated with the covid- case count data cannot be corrected by statistical techniques, as we outline in the following. the main problem is that the weather could be influencing the number of tests carried out and the segment of the population tested. for example, other respiratory diseases are often similar to covid- in their symptoms (e.g. who ) and are more common during cold weather (e.g. deschenes and enrico ; gasparrini et al. ) , which could influence the number of tests performed on people displaying symptoms of respiratory infection. therefore, even if the model correctly identified the impact of the weather on covid- case counts, it cannot distinguish between the impact of the weather on the spread of the disease and its impact on testing. table provides a non-exhaustive list of elements that could undermine any analysis of the impact of the weather on the spread of covid- using data on confirmed cases. the evidence suggests that the weather may correlate with the number of tests conducted and who gets tested. we have not been able to find any specific covid- -related evidence that the weather could impact test accuracy (e.g. the weather affecting the nasopharyngeal or oropharyngeal swabs used in the pcr analysis), even though this could be possible. other points of concern include: the fact that there may be indirect effects of weather conditions on other factors that could have an impact on the spread of covid- (such as social interactions or air pollution); the heterogeneity of impacts across populations and subgroups within a population; and the fact that some people may have travelled and therefore been infected in a different place from where the cases are reported. we ran our model (as detailed in supplementary material ) and provide results and robustness checks in supplementary material . the model would technically suggest a negative correlation (e.g. colder days would be associated with more confirmed covid- cases, and hotter days with fewer cases). yet, these results could be highly misleading since these estimates are likely to be substantially biased because of the aforementioned reasons. figure , panel a, provides an illustration of how we could have obtained a negative correlation even if temperature had no impact or a positive impact on the spread of covid- in our sample. the total number of estimated cases is given by the size of the circles as a function of temperature (x-axis). the circles in green correspond to the effects we are interested in-those that explain the influence of temperature on the spread of covid- . if temperature has no effect on the spread of covid- , then the green circles should be the same size at low and high temperatures. the pink circles represent the possible effect of temperature on testing (as reported in table ) under the illustrative assumption that high temperatures reduce testing frequency. in this case, the overall result is a negative correlation between temperature and confirmed covid- cases, even if temperature has no effect on the spread of the disease. in practice, we naturally do not know the direction of the bias caused by the effect of temperature on testing when using standard statistical methods. there is also no way for us to evaluate the contribution of each of these effects (green table non-exhaustive list of reasons why weather conditions could affect the number of covid- tests carried out and who gets tested potential reason potential implication unrelated respiratory diseases are weather sensitive (e.g. deschenes and enrico ; gasparrini et al. ) and can be confused with covid- (e.g. ai et al. ; chen et al. ) more patients with symptoms of unrelated respiratory diseases could be tested during cold weather the prevalence of other weather-sensitive respiratory diseases might make false-positive results more likely, especially if only radiographic imaging is used, since it is possible to confuse these diseases for covid- (e.g. ai et al. ; chen et al. ) the incidence of other pathologies (e.g. cardiovascular diseases) is influenced by the weather (e.g. deschenes and enrico ; gasparrini et al. ) hospital capacity and the workload of medical staff and testing structures are affected by weather conditions, with potential implications on the number of tests conducted at-risk individuals suffering from unrelated conditions are more likely to be tested for covid- , even if they only have mild symptoms for covid- people may be more inclined to seek medical attention depending on the weather (e.g. norris et al. ) due to weather conditions, people may or may not decide to seek medical attention, affecting the number of patients going to the hospital with covid- , and the workload of medical staff or pink) in our estimate. we arrive at the final size of the circles and cannot be sure if the association that we are interested in is either negative, null or positive. figure , panel b, focuses on the risk that effects could be different across different samples. the circles in blue capture other underlying factors that are influenced by temperature (such as acclimatization or the level of social interactions in the population), as well as other socioeconomic factors (such as the demographic characteristics of a population). these factors could be radically different in different regions but may also evolve over time (e.g. between winter and summer seasons). there are strong reasons to be concerned with the scenario illustrated in figure , panel b. in our sample, for example, we only have data from the start of the pandemic until end of april ; some countries (e.g. china) may be over-represented in the dataset; and the average daily temperature is relatively low at . °c. furthermore, many countries have implemented a stringent containment policy during the period covered by the sample. containment policies may have heightened (or lowered) the sensitivity of the spread of the disease to the weather because social interactions are limited. we are not able to observe how for example, sars-cov- is more infecƟous; the weather affects people's immune systems; people-to-people transmission is higher. for example, changes in the number of people with respiratory diseases other than covid- , affecƟng the number of tests performed. for example, social interacƟons evolve; or samples are for different countries. example temperature (a) esƟmates are likely to be biased because the weather influences data collecƟon (b) esƟmates could vary between samples fig. effects potentially captured by our estimate. the size of the circles represents the estimated number of cases at different temperatures. these are examples that do not correspond to actual data. in these examples, we assume no correlation between temperature and the effects in green (see legend), a negative correlation with the effects in pink (example ) and a positive correlation with those in blue. (color figure online) the impact of the weather on covid- might change at different gradients of social interaction. finally, our estimate is based on small, observed changes in temperatures, and not on radical increases or reductions in temperatures. the spread of covid- may respond differently to large variations in temperature, e.g. by °c or °c across seasons, making seasonal predictions even more unreliable. strong precautions need to be taken before using covid- case count datasets for inference. the results of our model using existing covid- data would seemingly imply a negative association between temperature and confirmed covid- cases. any projection of covid- cases with such estimates could conclude that, during the upcoming months of june to september , southern hemisphere countries would be exposed to higher risks of covid- spread and northern hemisphere countries to lower risks. these types of unsubstantiated results could be used as a misinformed justification for an early relaxation of effective social distancing measures in the northern hemisphere. these findings have equally strong implications for statistical analyses focusing on other questions that rely on covid- confirmed case count and/or mortality count data. even though the exact nature of the effects may change, such studies are also at risk of capturing the effect that their parameters of interest have on tests and test results. for example, studies interested in the effect of containment policies may have to consider that these policies substantially affect testing because they change the awareness of the disease in the population, political demands for more testing or the risk of contracting other respiratory diseases. other studies may also produce estimates that are very specific to the current circumstances in the development of the pandemic and are, therefore, not suitable to use for forecasts of what could happen in the coming months. in the medium term, more reliable data need to be gathered, for example through experimental studies that randomly test a sample of the population for covid- . in the short term, we are in a situation of fundamental uncertainty about how different factors affect or are affected by the widespread societal changes we see with the covid- pandemic. therefore, scientists, policy makers, journalists and the general public need to be very cautious when discussing how the spread of covid- correlates with the weather or any other factor. in the long term, this paper suggests that more attention should be given to how epidemiological data is recorded and used during exceptional epidemics and seasonal outbreaks, since insufficient testing and monitoring can undermine essential statistical analyses. this article calls for the complementary use of different methods for data collection, such as random testing in samples of the population. sars to novel coronavirus-old lessons and new lessons global rise in human infectious disease outbreaks carbon emissions come roaring back: will the economy, too? tellimer the european green deal sets out how to make europe the first climate-neutral continent by , boosting the economy, improving people's health and quality of life, caring for nature, and leaving no one behind. european neighbourhood policy and enlargement negotiations the eu's response to the covid- outbreak and the need for unprecedented solidarity amongst member states adaptation to climate change global carbon budget adapt now: a global call for leadership on climate resilience. the global commission on adaptation does social distancing matter? wp - macroeconomic implications of covid- : can negative supply shocks cause demand shortages? wp - will covid- have a lasting impact on the environment? bbc global energy and co emissions in does telework weaken urban structure-travel relationships? the influence of energy efficiency on other natural resources use: an input-output perspective rebound effects and ict: a review of the literature energy efficiency and consumptionthe rebound effect-a survey is telework effective for organizations? a meta-analysis of empirical research on perceptions of telework and organizational outcomes hours of work and the ecological footprint of nations: an exploratory analysis the impact of covid- on public space: a review of the emerging questions al-hukoma al-kuwaitiyya tuhathir min nafath al-ihtiyati khilal ash'hur [the kuwaiti government warns of depletion of the reserves gulf sovereign wealth funds could shed $ bn amid covid- chaos will covid- fiscal recovery packages accelerate or retard progress on climate change? oxford review of economic policy volatile coffee prices: covid- and market fundamentals (ico coffee break series no. ; ed.). international food policy research institute (ifpri) and international coffee organization (ico) oil-rich norway will withdraw a record $ billion from wealth fund as covid- batters economy world energy investment international trade in natural resources: practice and policy commodity markets outlook: implications of covid- for commodities path dependency and directed technical change: evidence from the auto industry the porter hypothesis at : can environmental regulation enhance innovation and competitiveness? how could the ecb's monetary policy support the sustainable finance transition? mimeo financial data science: the birth of a new financial research paradigm complementing econometrics? does the fossil fuel divestment movement impact new oil & gas fundraising? available at ssrn entrepreneurs for a low carbon world: how environmental knowledge and policy shape the creation and financing of green start-ups venture capital and cleantech: the wrong model for energy innovation ita-per-un-esper iment o-di-massa -never -waste -a-good-crisi s/ european economic and social committee (eesc, dg regio) opinion on "financing the transition to a low-carbon economy and the challenges in financing climate change adaptation letter: let's not waste this chance to transition to an environmentally sustainable future. financial times °c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development and efforts to eradicate poverty report prepared by the world in initiative. international institute for applied systems analysis (iiasa), laxenburg, austria. www.twi .org sachs the ages of globalization: geography, technology, and institutions the sustainable development goals and covid- . sustainable development report six transformations to achieve the sustainable development goals e-confe rence -the-epide miolo gy-and-econo mics-of-coron aviru s visual toolbox for system innovation the coronavirus stimulus package: how large is the transfer multiplier? centre for economic policy research discussion paper dp mit klimafreundlichen investitionen raus aus der wirtschaftskrise the environmental impacts of the coronavirus world bank group ( ) proposed sustainability checklist for assessing economic recovery interventions the survey is published on the ipsos website and covers , people from may to . behavioral and distributional effects of environmental policy the political economy of clean air act legislation: an analysis of voting in the us senate on amendments to the privatization and efficiency: from principals and agents to political economy multiple receptor ambient monitoring and firm compliance with environmental taxes under budget and target driven regulatory missions productive efficiency in public and private firms emissions trading with profit-neutral permit allocations salience games: private politics when public attention is limited community pressure for green behavior is environmental regulation bad for competition? a survey the political economy of market-based environmental policy: the u.s. acid rain program regulating pollution with endogenous monitoring emission reduction and profit-neutral allowances the strategic use of innovation to influence regulatory standards the khazzoom-brookes postulate and neoclassical growth incomplete contracts and privatization the availability heuristic, intuitive cost-benefit analysis, and climate change draft report on the proposal for a regulation of the european parliament and of the council establishing the framework for achieving climate neutrality and amending regulation (eu) / (european climate law will covid- fiscal recovery packages accelerate or retard progress on climate change? never waste a good crisis: for a sustainable recovery from covid- , sustainable development solutions network the use of multiple policy instruments for environmental protection: an economic perspective automotive modal lock-in: the role of path dependence and large socio-economic regimes in market failure regulatory capture: a environmental and technology policies for climate change mitigation climate change policy's interactions with the tax system scrapping subsidies during the financial crisis -evidence from europe the environmental impacts of the coronavirus will covid- fiscal recovery packages accelerate or retard progress on climate change? renewable energy subsidies: secondbest policy or fatal aberration for mitigation? resour justifying a policy mix for pollution control: a review of economic literature can technology-specific deployment policies be cost-effective? the case of renewable energy support schemes evaluating "cash-for-clunkers": program effects on auto sales and the environment path dependence in urban transport: an institutional analysis of urban passenger transport in the effects of fiscal stimulus: evidence from the cash for clunkers program cost-effectiveness of renewable electricity policies prices rise faster than they fall timmermans promises green recovery to eu lawmakers green deal facing delays due to coronavirus, eu admits issue of food sovereignty and seek to reinforce the relocation of part of the food supply chains. of course, it is not a question of valuing protectionist policies at all costs. in contrast, in addition to raising prices (french labour is expensive), this would make the national food system more vulnerable in the event of poor harvests (droughts, floods, etc.). however, to be truly effective, relocation would require profound changes to the french food system, for example, reducing the production of meat and cheese, which are heavily dependent on soybeans. both environmental (greenhouse gases and biodiversity) and social (food sovereignty, profitability of small farms) objectives are compatible here. finally, the short food supply chains therefore represent a radical change to consumption that food security and canada's agricultural system challenged by covid- technological transitions as evolutionary reconfiguration processes: a multi-level perspective and a case-study international monetary fund's ( ) world economic outlook agroecological research: conforming or transforming the dominant agro-food regime? how covid- has exposed inequalities in the uk food system: the case of uk food and poverty the challenge of using epidemiological case count data: the example of confirmed covid- cases and the weather university of oxford; and institute for new economic thinking at the oxford martin school the publicly available datasets on confirmed covid- cases and deaths provide a key opportunity to better understand the drivers of the pandemic will spring slow spread of coronavirus in northern hemisphere? the guardian scientists hopeful warmer weather can slow spread of coronavirus. the financial times spread of sars-cov- coronavirus likely to be constrained by climate ultraviolet radiation decreases covid- growth rates: global causal estimates and seasonal implications an interactive web-based dashboard to track covid- in real time epidemiological data from the covid- outbreak, real-time case information correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases covid- : automatic detection from x-ray images utilizing transfer learning with convolutional neural networks covid- testing: challenges, limitations and suggestions for improvement finding covid- from chest x-rays using deep learning on a small dataset extreme weather events, mortality, and migration mortality risk attributable to high and low ambient temperature: a multicountry observational study q&a: similarities and differences-covid- and influenza". ( ) consulted on th april use of radiographic features in covid- diagnosis: challenges and perspectives an empirical investigation into factors affecting patient cancellations and no-shows at outpatient clinics cohen is the first author. he had the original idea, wrote most of the paper and the code to produce the econometric analysis. he also coordinated the team. jani ensured the material was consistent with the epidemiological evidence. li produced the required climate data for the statistical analysis. lu helped on literature review, on coding the econometric analysis and on producing the tables. schwarz helped on data coding and matching and created the projections. all authors contributed to the text. data and materials availability all data and software are publicly available at https :// githu b.com/morit zpsch warz/covid - -weath er-oxfor d. key: cord- - vr y authors: guo, siyu; wu, jiarui; ni, mengwei; jia, shanshan; zhang, jingyuan; zhou, wei; liu, xinkui; wang, miaomiao; zhang, xiaomeng title: comparative efficacy of danshen class injections for treating acute coronary syndrome: a multidimensional bayesian network meta-analysis of randomized controlled trials date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: vr y background: acute coronary syndrome, that is a common and serious cardiovascular disease, imposes a huge economic burden on global public health. and danshen class injections are commonly used in the treatment of acute coronary syndrome in china. thus, the bayesian network meta-analysis was devised to investigate the efficacy of different danshen class injections against acute coronary syndrome. methods: eligible inclusion and exclusion criteria were established in advance. then, a systematic literature search was performed in several databases from inception to february . further, the included randomized controlled trials data were adopted to calculation, prepare graphs and multidimensional cluster analysis by winbugs . . , stata v. . and r . . software, respectively. results: a total of eligible randomized controlled trial studies with patients were obtained that evaluated the clinical effectiveness rate, the level of hypersensitive c-reactive protein, c-reactive protein, interleukin- , fibrinogen, and adverse reactions after the application of danshen class injections plus western medicine. compared with western medicine alone, danshen class injections combined with western medicine therapy were associated with significantly improved the therapeutic effect. in addition, the results of the multidimensional cluster analysis demonstrated that danhong injection + western medicine and danshen injection + western medicine had better therapeutic effects. however, since most eligible randomized controlled trial studies did not focus on the monitoring of adverse reactions, the safety of these chinese herbal injections needs to be further explored. conclusion: based on this bayesian network meta-analysis results, danhong injection + western medicine and danshen injection + western medicine might have a better impact on acute coronary syndrome patients. nevertheless, more large samples, high-quality clinical and multicenter randomized controlled trial studies should be tested and verified in the future. acute coronary syndrome (acs) is a medical emergency (kosuge and kimura, ) . the underlying mechanism of acs is triggered by atheromatous plaque enlargement, instability, and rupture or erosion (amsterdam et al., ; saremi, ) . acs includes st elevation myocardial infarction (stemi), unstable angina (ua) and non-st elevation myocardial infarction (nstemi) (hedayati et al., ) . in , it is estimated that in the united states, , persons were diagnosed with acute coronary syndrome (virani et al., ) . additionally, the risk of acs is greater with age (timmis, ) . presently, the treatments for acs mainly rely on anti-platelet, anti-coagulant, anti-ischemic, thrombolytic and percutaneous coronary intervention (pci) (wu et al., ) . however, although clopidogrel is an effective antiplatelet drug in patients with acs, it has the limitations of high bleeding risk and stent thrombosis . previous studies reported that pci may be related to vascular endothelial cell dysfunction, platelet aggregation and neutrophil obstruction, which will lead to restenosis of the stent or some major adverse cardiac events (mace) (farooq et al., ; kirtane et al., ) . to find more effective and promising therapeutic approaches to manage acs, traditional chinese medicine (tcm) combined with western medicine (wm) have received widespread attention from clinicians in china . in the theory of tcm, the pathogenesis of acs is closely correlated to stagnant blood block (wu et al., ) . furthermore, chinese herbal injections (chis) are a new type of tcm formulation with high bioavailability and fast action . according to accumulated evidenced-based data, the combination of chis and wm is associated with considerable efficacy in treating patients with acs (zhao et al., ) . for example, a systematic review reported that danhong injection (dh) could improve the therapeutic efficacy and reduce the incidence of mace events in acs patients after pci (zou et al., ) . moreover, systematic reviews and meta-analysis studies shown that sodium tanshinone iia sulfonate injection (sts) and danshenchuanxiongqin injection (dscxq) combined with wm appeared to be efficacious in the treatment ua (zhang et al., ; tan et al., ) . in china and japan, danshen (radix salviae miltiorrhizae) is the dried root or rhizome of salvia miltiorrhiza bunge, commonly used to treat cardiovascular-related diseases (meim et al., ; yuan et al., ) . the relative studies validated that danshen possesses the characteristics of relaxing coronary artery muscle, improving microcirculation, reducing blood viscosity and inhibiting thrombogenesis (cheng, ) . the chis containing the extract of danshen are termed danshen class injections (dscis) . in this context, this study included dscis, namely, danshen injection (ds), fufang danshen injection (ffds), danhong injection (dh), dansenduofensuanyan injection (dsdfsy), danshen salvianolic acids injection (dssa), danshenchuanxiongqin injection (dscxq), sodium tanshinone iia sulfonate injection (sts) and guanxinning injection (gxn). compared with the traditional double-arm meta-analysis, network meta-analysis (nma) can synthesize different interventions for the same disease and perform direct or indirect comparisons (grant, ; sun et al., ; zhang et al., ) . moreover, nma can assist assessing and ranking the therapeutic effects of different treatments (rouse et al., ; pan et al., ) . however, at present, there are many systematic review studies on the adjuvant treatment of acs with a certain type of dscis, lacking of comparison of efficacy between different types of dsci (wu et al., ; zhang et al., ; wu et al., ; tan et al., ; zou et al., ) . therefore, the present research adopted nma to explore the comparative effectiveness and safety between different dscis plus wm against acs, providing a reference for clinical practice. the procedure of the current nma research was conducted based on the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines (hutton et al., ) (supplementary file ). the eligibility criteria for this study conformed the picos framework, namely, participants, interventions, comparisons, outcomes and study design. so, randomized controlled trials (rcts) that met the following criteria were included in this study: ( ) study design. only rcts mentioned in articles were enrolled. ( ) participants: the current nma included patients were diagnosed with acs, and without limitations on race, age, gender, or nationality. ( ) interventions and comparisons: the experiment group was treated by chis (ds, ffds, dh, dsdfsy, dssa, dscxq, sts, and gxn) combined with wm. patients in the control group were both received wm treatment or wm plus another chis, excluding pci. the commonly used wm drugs were primarily nitrate, low molecular weight heparin, aspirin, nifedipine, statins, b-receptor blocker, and so on. there are no restrictions on dosage and duration of treatment. the corresponding treatment measures should be taken for patients with other complications. ( ) outcomes. the primary outcomes of this study were the clinical effectiveness rate. according to clinical symptoms and objective indicators, the effectiveness status was divided into effective, effective, and ineffective. the clinical effectiveness rate = (number of total patients − number of invalid patients)/number of total patients * %. it was regarded as invalidation when the clinical symptoms and electrocardiogram didn't change or worsen, or the frequency of chest pain wasn't decreased or increased (ministry of health of people's republic of china, ) . additionally, the secondary outcomes were hypersensitive c-reactive protein (hs-crp), c-reactive protein (crp), interleukin- (il- ), fibrinogen (fib) and adverse reactions (adrs). previous research evidences suggested that local and systemic inflammation play a pivotal role in the pathogenesis of acs (liuzzo, ; elhajj et al., ) . for example, the increase of il- , hs-cpr and cpr concentration can be observed in patients with systemic signs of systemic inflammation (stefanadis et al., ; centurioń, ; nanchen et al., ) . in addition, fib is the main determinant of thrombus formation, and its increase is related to the degree of coronary atherosclerosis, especially in patients with acs (de moerloose et al., ; kurtul et al., ; buljubasic et al., ) . rcts were excluded if any of the following conditions were accord: ( ) the therapy in rcts includes pci, or other tcm preparations. ( ) the article data is wrong or incomplete. ( ) the article could not be obtained full text. ( ) data duplicates. ( ) no related outcome. studies concerning rcts published from inception to february were identified through a systematic electronic search of the following seven databases: the china national knowledge infrastructure database (cnki), the chinese scientific journals full-text database (vip), the wan-fang database, the chinese biomedical literature database(sinomed),thecochranelibrary,pubmedandembase.for this purpose, the free-text keywords and mesh (medical subject heading) terms were utilized, including "acute coronary syndrome [mesh terms]", "acute coronary syndromes", "coronary syndrome, acute", "coronary syndromes, acute", "syndrome, acute coronary", "syndromes, acute coronary", "danshen", "compound danshen", "xiangdan", "fufangdanshen", "danhong", "beitong", "salvianolate", "salvianolic acid", "danshenduofensuan", "danshenchuanxiongqin", "sodium tanshinone iia sulfonate", "guanxinning" and "randomized controlled trial [publication type]". in addition, there was no restrictions on blinding methods, publication year and language. furthermore, the references of related articles were manually reviewed to identify further studies. the detailed and specific retrieval strategies are shown in supplementary file . data of eligible studies were independently extracted by two reviewers (syg and mwn) by using microsoft excel (microsoft corp, redmond, wa). the specifically designed form captured information on the study characteristics, including publication data (publication date, title and authors' names), details of patients' characteristics (sample sizes, age and sex), intervention (the types of chis, dose and duration), outcomes (the primary and secondary outcomes) and factors to evaluate risk of bias. according to the cochrane risk of bias tool , two investigators (sj and jz) independently conducted the quality assessment of all included rcts. each trial was scored as low, high, or unclear risk of bias on quality evaluation items, including random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias) and other bias. when discordance occurred in the process of literature selection, data extraction and quality assessment, the final results were resolved by adjudication with a third reviewer (wz) or consensus. in consideration of the clinical and within-study and betweenstudy heterogeneity among the included rcts, the randomeffect model was selected. moreover, according to the bayesian hierarchical model and markov chain monte carlo algorithm, winbugs . . (mrc biostatistics unit, cambridge, uk) software was adopted to analyze data. for dichotomous outcomes, the combined results were presented as odds ratios (ors) with % confidence intervals ( % cis). for continuous outcomes, the results were calculated as the mean differences (mds) with % cis. if % cis of ors did not include and % cis of mds did not contain , the differences between the groups would be considered statistically significant (huang et al., ) . in the winbugs program, we set , iterations, and the first iterations were regarded as burn-in for annealing to eliminate the influence of the initial value (crainiceanu and goldsmith, ) . furthermore, the stata v. . software was utilized (stata corporation) to generate graphs in order to compare the relationship between different treatment measures. the surface under the cumulative ranking (sucra) area was employed to rank the probabilities for different interventions under each outcome. the sucra ranges from to %, assigning to the worst and best therapeutic measures, respectively (cope and jansen, ; trinquart et al., ) . in addition, the publication bias of included rcts were checked though a comparison adjusted funnel plot (chaimani et al., ) . the multidimensional cluster analysis was performed to assess comprehensive efficacy of competing treatments in any three outcomes based on sucra value. hence, the "scatterplot d" package in r . . software (mathsoft, cambridge,usa) was used for multidimensional clustering analysis in this study. these interventions were clustered by using the k-means method, and the number of clusters was modified according to the actual situation (ball and hall, ; haldar et al., ; ahlqvist et al., ) . the steps of clustering were as follows: ( ) the included interventions were randomly divided into k initial categories, and the average of the outcome indicators of these k categories were regarded as the initial aggregation points. ( ) an intervention was classified as the closest aggregation point category, and then the aggregation point of that category was updated to the mean of the present outcome indicators. all interventions were recategorized and classified and repeat step ( ) until they have been assigned. finally, the ranking of treatment interventions was visually displayed through a three-dimensional stereogram. the different colors dots in the figure represent different types of interventions. a total of articles were obtained by searching databases. after removing the duplicates, articles need to be screened for the title and abstract. then, studies were excluded because of reviews, meta-analysis, system reviews, animal experiment and other irrelevant literature. ultimately, rcts studies conducted in china from to were eligible in this nma. the details of the articles screening process are depicted in figure . seven types of dscis were incorporated, including ds, ffds, dh, dsdfsy, dscxq, sts and gxn. the network graphs of the above chis with different outcomes are described in figure . the detailed information on chis shown in supplementary file . overall, the eligible rcts studies involved , patients ( , in the control group, and , in the experiment group), and most of them were middle-aged and elderly. with the exception of five studies that did not report gender composition, male patients accounted for approximately . % of the total study population. ffds + wm (n = ). the therapies of the control group were wm, primarily including nitrate, low molecular weight heparin, aspirin, nifedipine, statins, b-receptor blocker, etc. moreover, the duration of treatment reported by most studies was days. the details of the included study characteristics are shown in table . in terms of random sequence generation (selection bias), a total of rcts used a random number table to generate random sequences, so they were considered to be low risk. regarding performance bias, rct study mentioned the use of the single blind method, so it was evaluated as low risk. in terms of attrition bias, all studies had no incomplete data, so the evaluation was "low risk". regarding reporting bias, studies reported incomplete outcome indicators mentioned in the design plan, so they were regarded as high risk. in terms of other bias, five studies did not report whether the experiment group and the control group were comparable at baseline, which might affect the study results, so they were rated as high risk. in addition, the risk bias entries of the remaining studies were considered "unclear" due to insufficient information. in general, the quality of included studies was not high. the risk of bias for each eligible rcts is depicted in figure . apart from ds and ffds injection, other chis combined with wm were superior to wm alone, and the difference was statistically significant. according to the ranking of sucra probabilities (figure , table ), dh + wm ( . %) was the most likely to become the best intervention for improving the clinical effectiveness rate. moreover, the other chis were ranked as follows: dsdfsy+wm ( . %) > ds+wm ( . %) > sts+wm ( . %) > gxn+wm ( . %) > dscxq+wm ( . %) > ffds+wm ( . %). a total of rcts involving interventions investigated hs-crp [ds + wm vs. wm (n = ), dscxq + wm vs. wm (n = ), dh + wm vs. wm (n = ) and gxn + wm vs. wm (n = )]. the specific outcomes are described in table . dh+wm (md= − . , % cis: − . ~− . ) could achieve a better effect in hs-crp than wm alone, and the difference was statistically significant. simultaneously, there was no statistically significant difference between other interventions. base on the data of sucra probabilities (figure , table ), the chis were ranked as follows: dscxq+wm ( . %) > ds+wm ( . %) > dh+wm ( . %) > gxn+wm ( . %). in total, rcts with chis observed data regarding crp [ds + wm vs. wm (n = ), dscxq + wm vs. wm (n = ), dh + wm vs. wm (n = ), ffds+wm (n = ), gxn+wm (n = ) and dh + wm vs. ffds + wm (n = )]. the results demonstrated that no significant differences, which are shown in table . according to the sucra values ( figure , table ), the chis were ranked as follows: dh+wm ( . %) > ds+wm ( . %) > ffds+wm ( %) > gxn+wm ( . %) > dsdfsy+wm ( . %). a total of rcts with chis presented data about il- [dsdfsy + wm vs. wm (n = ), dh + wm vs. wm (n = ) and ffds+wm (n = )]. additionally, the results demonstrated that no significant differences in il- was observed among the above chis, which are shown in table . in terms of the sucra values ( figure , table ), the chis were ranked as follows: ffds+wm ( . %) > dsdfsy+wm ( %) > dh+wm ( . %). the data on the fib were available for rcts including types of chis [ds + wm vs. wm (n= ), dsdfsy + wm vs. wm (n = ), dh + wm vs. wm (n = ), gxn+wm vs. wm (n= ) and ffds+wm (n = )]. simultaneously, the results showed that no significant differences among above chis groups for fib ( table ) . according to the sucra values ( figure , table ), the chis were ranked as follows: ds+wm ( %) > gxn+wm ( . %) > dh+wm ( . %) > ffds+wm ( . %) > dsdfsy+wm ( . %). in terms of safety, studies did not address adrs, and studies indicated that there were no significant adrs. meanwhile, study (li y m, ) only reported monitoring adrs related to liver and kidney dysfunction and bleeding tendency, but did not report the corresponding results. in addition, study (yang and zhang, ) only described cases of adrs in the experiment group (sts+wm) and cases in the control group (dh+wm), but none of them affected the treatment. in yang's research (lin and yu, ) , cases (allergic rash, vomiting and bloating) and cases (vomiting) of adrs occurred in the experiment group (dsdfsy+wm) and the control group (wm), respectively, and the symptoms were mild. in lao's study (lao et al., ) , one patient in the control group (wm) had elevated transaminase and returned to normal levels after hepatoprotective treatment; none of the experiment group (dh+wm) had abnormal liver and kidney test indicators during the treatment process, and there were no serious adrs. in ruan's study (ruan et al., ) , adrs occurred in the experiment group (ffds+wm), including drowsiness, fever and mild gastrointestinal reactions; adrs occurred in the control group (wm), containing dizziness, drowsiness and mild gastrointestinal reactions. in the research of xia and whang ( ) , patient in the experiment group (gxn+wm) presented with facial redness and mild headache, and had rashes.; the control group (wm) developed cases of flushing and mild headache, case of rash, and case of local swelling by injection. in the study zhao ( ) , some patients in the experiment group (gxn+wm) and the control group (wm) developed headaches, which were relieved by reducing nitrate dosage. in the research of xiao ( ), patient in the experimental group (dh+wm) developed facial redness and mild headache, while patients in the control group (ffds +wm) developed a sense of local swelling from the injection. however, since most eligible rcts studies did not focus on the monitoring of adrs, the safety of these chis needs to be further explored. when cluster analysis was conducted to interventions that reported the hs-crp, clinical effectiveness rate and crp, dh +wm was superior to the other regimens, and wm only was evaluated the worst ( figure a ). in terms of the clinical effectiveness rate, hs-crp and fib, ds + wm and dh + wm were similarly superior ( figure b ). furthermore, regarding the clinical effectiveness rate, crp and il- , dh+wm was similarly preferred ( figure c ). moreover, ds + wm and dh + wm were dominant in the comprehensive ranking of the clinical effectiveness rate, crp and fib ( figure d ). with regard to the comprehensive ranking of the clinical effectiveness rate, fib and il- , the results indicated that dh + wm had the potential to be the best intervention ( figure e ). in summary, the results of the multidimensional cluster analysis demonstrated that dh+wm and ds+wm might have better therapeutic effects. this study drew a comparison-adjusted funnel plot for clinical effectiveness rate to test publication bias. when the distribution points in the funnel chart are symmetrical, it means that there is no publication bias (riley et al., ) . as depicted in figure , the points in the funnel chart were asymmetric based on the position of the center line, and the angle between the adjusted auxiliary line and the center line was large, indicating a potential publication bias in this study. as depicted in figure , there were three-side rings. the results indicated that the % cis contained , and if was between . and . . consequently, there was some inconsistency in this nma study. we performed the approach of current nma to evaluate the comparative efficacy and safety of reported dscis combination with wm in the treatment of acs. this study included eligible rcts with chis that evaluated the clinical effectiveness rate, the level of hs-crp, crp, il- , fib and adrs after the application of dscis plus wm. according to nma results, compared with wm alone, the types of chis combined with wm therapy were associated with significantly improved the therapeutic effect. in addition, the results of the multidimensional cluster analysis demonstrated that dh+wm and ds+wm had better therapeutic effects. in terms of the ranking of sucra probabilities, dh + wm was superior to other chis for improving the clinical effectiveness rate. however, since different chis have different effects and reactions on patients, the clinical selection of chis should better improve the efficacy according to the patient's condition. dh is a tcm compound preparation extracted from two famous herbs, danshen (radix salviae miltiorrhizae, salvia miltiorrhiza bunge) and honghua (carthami flos, carthamus tinctorius l.) (feng et al., ) . since dh launch in , it has been widely used to prevent and treat acs (yao et al., ) . the main active ingredients of dh are flavonoids and phenolic compounds, for example, danshensu, salvianolic acid b, protocatechuic aldehyde, tanshinone iia, rosmarinic acid and hydroxysafflor yellow a (xie et al., ; wang et al., ) . in addition, previous pharmacological studies have demonstrated that dh have the effects of antioxidant, anti-inflammatory, vasodilation and protect cardiac muscle due to its main pharmacological active components (wan et al., ; jiang et al., ) . for example, previous studies have demonstrated that salvianolic acid b could strongly inhibit tumor necrosis factor-a-induced nuclear factor-kb (nf-kb) activation in human aortic endothelial cells (chen et al., ) . furthermore, dh combined with conventional therapy for weeks can significantly reduce the plasma levels of plasma fibrinogen c (fib-c), glucose protein ii b/iii a receptor complex and hs-crp in acs patients after pci zhao and jiang, ) . in one study, the results indicated that dh + wm not only improved symptoms in patients with ua, but also improved some laboratory indicators, such as crp, n-terminal brain natriuretic peptide and homocysteine (sun et al., ) . currently, one published nma showed that dh + wm had advantages in improving the total efficacy and electrocardiography of ua . ds is one of most commonly used tcm preparation for treating coronary heart disease, heart-stroke and cerebrovascular diseases. phenolic acids, including danshensu, salvianolic acid a/b, lithospermic acid b and rosmarinic acid, are the main active contents of ds, extracted from danshen (zhang et al., ) . besides, the most notable effects of phenolic acid in danshen are antioxidant, anticoagulant, anti-thrombotic and cell protection (wang et al., ; orgah et al., ) . a systematic review revealed that ds as one adjuvant treatment of wm for ua could significantly improve the total clinical effectiveness rate. furthermore, it also could significantly correct t-wave inversion, reduce the level of fib and adjust blood lipid level . apart from efficacy, the safety of chis in the treatment of acs is also an important issue worthy of consideration by clinicians. in this study, studies did not address adrs, and studies indicated that there were no significant adrs. thus, we could not draw detailed conclusions about the safety information of chis. according to the results, the adrs mainly included allergic rash, vomiting, drowsiness, flushing, mild gastrointestinal reactions and headache. previous studies had shown that most of the adrs linked to dhi therapy were mild and moderately severe, with the primary disposition of discontinuation and without other treatment . in addition, it is noteworthy to monitor the adrs of patients minutes after chis injection. since few studies in this nma had focused on adrs. therefore, more experiments and clinical evidence are needed to verify the safety of these chis. in terms of the design and contents, this nma study has some particular merits as follows. first, this is the first nma to evaluate the efficacy and safety of dscis plus wm in the treatment of acs. second, it strictly established inclusion and exclusion criteria and a comprehensive literature search. finally, the rankings of eligible chis regarding the different outcome indicators provide evidence and recommendation for the clinical selection of medication. nevertheless, several potential limitations of the present nma should be considered. first, all the enrolled rcts were only performed in chinese patients, so it is not clear whether the conclusions of this current study are applicable to acs patients in other countries. second, the quality of included studies was not high, mainly because most trials did not report detailed information of allocation concealment and blinding. in addition, due to the diversity of wm and the different doses and courses of chis, there may have been clinical heterogeneity. for this reason, we believe that the methodological quality of clinical trials should be valued and improved, in order to promote the appropriate use of chis. for instance, rcts should be registered in advance to ensure transparency, and secondly, the test process should be reported in as much detail as possible in the literature. despite the above limitations, the results of present nma demonstrated a complete evaluation of the clinical effect in multiple aspects and provided several clinical suggestions of different chis for acs patients. in summary, this nma results showed that dscis combined with wm therapy could have a positive influence on patients with acs. what's more, compared with wm alone, dscis combined with wm therapy might be associated with significantly improved the therapeutic effect. in addition, dh+wm and ds+wm therapies are potentially the preferred treatments for acs. however, since most eligible rcts studies did not focus on the monitoring of adrs, the safety of these chis needs to be further explored. due to several limitations, more large samples, high-quality clinical and multicenter rcts studies should be tested and verified in the future. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. novel subgroups of adult-onset diabetes and their association with outcomes: a data-driven cluster analysis of six variables aha/acc guideline for the management of patients with non-st-elevation acute coronary syndromes: a report of the american college of cardiology/american heart association task force on practice guidelines a clustering technique for summarizing multivariate data fibrinogen in relation to degree and composition of coronary plaque on intravascular ultrasound in patients undergoing coronary angiography effect of danhong injection on plasma endothelin and c-reactive protein in patients with acute coronary syndrome the effect of danhong injection in hemorheology in old patients with acute coronary syndrome serum biomarkers and source of inflammation in acute coronary syndromes and percutaneous coronary interventions graphical tools for network meta-analysis in stata clinical research on treating acute coronary syndrome by danhong injection clinical observation of danhong injection in the treatment of senile acute coronary syndrome. chin salvianolic acid b attenuates vcam- and icam- expression in tnf-alphatreated human aortic endothelial cells effect of danhong injection on platelet activation and inflammatory factors in patients of acute coronary syndrome after intervention therapy danshen: a versatile chinese herbal drug for the treatment of coronary heart disease quantitative summaries of treatment effect estimates obtained with network meta-analysis of survival curves to inform decision-making bayesian functional data analysis using winbugs clinical observation of cases of acute coronary syndrome treated by danhong injection effect of danhong injection on plasma brain natriuretic peptide in patients with acute coronary syndrome intervention of danhong injection on serum mmp- in patients with acute coronary syndrome. china health ind clinical observation of danhong injection in the treatment of acute coronary syndrome the role of inflammation in acute coronary syndromes: review of the literature restenosis: delineating the numerous causes of drug-eluting stent restenosis effect of danhong injection and atorvastatin on the levels of circulating il - , scd l and apn in acute coronary syndrome neuroprotective effect of danhong injection on cerebral ischemia-reperfusion injury in rats by activation of the pi k-akt pathway the clinical study of danhong injection on the levels of serum hs-crp and mmp- in patients with acute coronary syndromes. china health ind the uptake of bayesian methods in biomedical meta-analyses: a scoping revie- ) the effect of danhong injection on serum high sensitivity c-reaction protein in patients with acute coronary syndrome effect of danhong injection on circulating inflammatory factors and txb in acute coronary syndrome cluster analysis and clinical asthma phenotypes clinical analysis of danhong injection applied to treat acute coronary artery syndrome non-st-segment acute coronary syndromes the cochrane collaboration's tool for assessing risk of bias in randomised trials effects of xiangdan injection in acute coronary syndrome and inflammatory markers comparative observation of the efficacy of danhong injection and tanshinone iia sulfonate injection in the treatment of cases of acute coronary syndrome comparative efficacy of chinese herbal injections for the treatment of community-acquired pneumonia: a bayesian network meta-analysis of randomized controlled trials the prisma extension statement for reporting of systematic reviews incorporating network meta-analyses of health care interventions: checklist and explanations bioactivityintegrated uplc/q-tof-ms of danhong injection to identify nf-kb inhibitors and anti-inflammatory targets based on endothelial cell culture and network pharmacology association between intraprocedural thrombotic events and adverse outcomes after primary percutaneous coronary intervention for st-segment elevation myocardial infarction (a harmonizing outcomes with revascularization and stents in acute myocardial infarction [horizons-ami] substudy) implications of using the cabrera sequence for diagnosing acute coronary syndrome the association of plasma fibrinogen with the extent and complexity of coronary lesions in patients with acute coronary syndrome lipid-lowering effect of danhong injection combined with low-dose simvastatin on acute coronary syndrome chinese herbal injections for acs clinical effect of danshen polyphenolate injection combined with ticagrelor in the treatment of patients with acute coronary syndrome effect of danhong injection on plasma tissue factors in patients with acute coronary syndrome salvia miltiorrhiza polyphenolate injection combined with low molecular weight heparin calcium in the treatment of cases of acute coronary syndrome a comprehensive strategy to evaluate compatible stability of chinese medicine injection and infusion solutions based on chemical analysis and bioactivity assay effect of guanxining injection on serum ykl- and hs-crp levels in patients with non-st-segment elevation acute coronary syndrome clinical observation of danhong injection in the treatment of senile acute coronary syndrome effect of danhong injection on the high-sensitivity c-reactive protein, fibrinogen and von willebrand factor in patients with acute coronary syndrome postmarketing safety surveillance and reevaluation of danhong injection: clinical study of cases observation on treatment of acute coronary syndrome by danhong injection danhong injection was used to treat cases of acute coronary syndrome clinical study of danshen injection in treating acute coronary syndrome effect of danhong injection on serum hypersensitive c-reactive protein in patients with acute coronary syndrome effects of danhong injection on inflammatory factors vascular endothelial function and platelet activation in patients with acute coronary syndrome effect of guanxining injection on plasma fibrinogen and c-reactive protein in patients with acute coronary syndrome effect of salvia miltiorrhiza polyphenolate on the level of modified albumin in angina pectoris influence and its efficacy of salvianolate adjuvant therapy on vascular endothelial function in patients with acute coronary syndrome china mod efficacy of danshen class injection in the treatment of acute cerebral infarction: a bayesian network meta-analysis of randomized controlled trials what are the best salvia miltiorrhiza injection classes for treatment of unstable angina pectoris? a systematic review and network meta-analysis clinical analysis of cases of acute coronary syndrome treated by danhong injection atherosclerosis: an inflammatory disease study on the clinical efficacy of danshen injection in the treatment of acute coronary syndrome. chin effect of danshen injection on serum homocysteine, folic acid and c-reactive protein levels in patients with stsegment elevation acute myocardial infarction -x ministry of health of people's republic of china inflammation during acute coronary syndromes -risk of cardiovascular events and bleeding clinical observations of patients regarding the effect of danhong injection on hs-crp of patients with acute coronary syndrome pharmacological potential of the combination of salvia miltiorrhiza (danshen) and carthamus tinctorius (honghua) for diabetes mellitus and its cardiovascular complications the impact of major dietary patterns on glycemic control, cardiovascular risk factors, and weight loss in patients with type diabetes: a network meta-analysis interpretation of random effects meta-analyses network meta-analysis: an introduction for clinicians effects of xiangdan injection on serum levels of c-reactive protein, amino terminal brain natriuretic peptide and cardiac enzymes of patients with acute coronary syndrome cardiac mr imaging in acute coronary syndrome: application and image interpretation treating cute coronary syndrome with danshen heat production of atherosclerotic plaques and inflammation assessed by the acute phase proteins in acute coronary syndromes effect of danshen injection on serum homocysteine and c reactive protein in patients with st segment elevation acute myocardial infarction effect of danhong injection on serum high sensitivity c-reactive protein in patients with acute coronary syndrome the index and improvement effect of using danhong injection to patients with atherosclerosis symptoms of coronary heart disease (chd) efficacy and safety of anti-egfr monoclonal antibodies combined with different chemotherapy regimens in patients with ras wild-type metastatic colorectal cancer: a meta-analysis sodium tanshinone ii a sulfonate injection as adjuvant treatment for unstable angina pectoris: a meta-analysis of randomized controlled trials acute coronary syndromes uncertainty in treatment rankings: reanalysis of network meta-analyses of randomized trials heart disease and stroke statistics- update: a report from the chinese herbal injections for acs frontiers in pharmacology | www protective effects of carthamus tinctorius injection on isoprenaline-induced myocardial injury in rats compared efficacy of clopidogrel and ticagrelor in treating acute coronary syndrome: a meta-analysis clinical observation of cases of elderly patients with acute coronary syndrome treated by danhong injection danhong injection protects hemorrhagic brain by increasing peroxiredoxin in aged rats clinical observation of danhong injection in treating senile acute coronary syndrome new developments in the chemistry and biology of the bioactive constituents of tanshen effect of danhong injection on plasma endothelin- and high sensitivity c-reactive protein in patients with acute coronary syndrome influence of danshen ligustrazin for injection on serum high-sensitivity c-reactive protein and cardiac function of patients with acute coronary syndrome danhong injection in the treatment of acute coronary syndrome: a systematic review and meta-analysis danshen injection as adjuvant treatment for unstable angina pectoris: a systematic review and meta-analysis clinical observation on curative effect of acute coronary syndrome treated with salvianolate injection and its influences on serum hs-crp, il- levels effects of guanxinning injection on non-stsegment elevation acute coronary syndrome comparision of the curative effects of danhong injection vs compound danshen injection in the treatment for the elderly patients with non-st-elevation acute coronary syndrome integrating qualitative and quantitative characterization of traditional chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry clinical observation of cases of acute coronary syndrome treated by danhong injection combined with western medicine effect of danhong injection on plasma endothelin in patients with acute coronary syndrome cost-effectiveness analysis of two regimens for acute coronary syndrome successful treatment with danhong injection for hepatic veno-occlusive disease screening of cardioprotective diseases bioactive components from danshen extracts and lc-ms analysis network meta-analysis of chinese herbal injections plus the folfox regimen for the treatment of colorectal cancer in china the efficacy of chinese herbal medicines on acute coronary syndrome with renal insufficiency after percutaneous coronary intervention chemical fingerprint and metabolic fingerprint analysis of danshen injection by hplc-uv and hplc-ms methods evaluation of danshen ligustrazine on improving the clinical symptoms of non-st elevation acute coronary syndrome (middle and low risk group) clinical observation on efficacy and safety of traditional chinese medicine for activating blood circulation and removing blood stasis in acute coronary syndrome danshenchuanxiongqin injection in the treatment of unstable angina pectoris: a systematic review and meta-analysis effects of danhong injection on hs-crp, il , tc, tg and ldl-c in old patients with acute coronary syndrome effect of danhong injection on et- , sp-sel, and hs-crp in patients with acute coronary syndrome undergoing percutaneous coronary intervention observation on the curative effect of guanxining injection in treating acute coronary syndrome treatment of danhong injection combined with naoxintong capsule in acute coronary syndrome patients undergoing pci operation: study for a randomized controlled and double-blind trial the effect of danhong injection on the level of serum hs-crp in patients with acute coronary syndromes. china health ind the therapeutic efficacy of danhong injection combined with percutaneous coronary intervention in acute coronary syndrome: a systematic review and meta-analysis sg and jw conceived and designed the study. sg, mn, sj, and jz conducted the systematic review and extracted and analyzed the data. wz and xl performed interpretation of results. sg drafted the initial manuscript. mw and xz critically reviewed the manuscript for important intellectual content. all authors contributed to the article and approved the submitted version. the authors declare that the research was conducted in the key: cord- -p szd k authors: mann, jaclyn kelly; ndung'u, thumbi title: the potential of lactoferrin, ovotransferrin and lysozyme as antiviral and immune-modulating agents in covid- date: - - journal: nan doi: . /fvl- - sha: doc_id: cord_uid: p szd k coronavirus disease (covid- ), caused by sars coronavirus (sars-cov- ), is spreading rapidly with no established effective treatments. while most cases are mild, others experience uncontrolled inflammatory responses with oxidative stress, dysregulation of iron and coagulation as features. lactoferrin, ovotransferrin and lysozyme are abundant, safe antimicrobials that have wide antiviral as well as immunomodulatory properties. in particular, lactoferrin restores iron homeostasis and inhibits replication of sars-cov, which is closely related to sars-cov- . ovotransferrin has antiviral peptides and activities that are shared with lactoferrin. both lactoferrin and lysozyme are ‘immune sensing’ as they may stimulate immune responses or resolve inflammation. mechanisms by which these antimicrobials may treat or prevent covid- , as well as sources and forms of these, are reviewed. of this review is to consider the potential of specific antimicrobial proteins that are abundant in nature to act as therapeutics in covid- (as antivirals and/or counteracting the pathology) and to stimulate further research in this avenue. recently it was reported that tear lactoferrin and lysozyme are relevant biomarkers of mucosal immune competence and that the levels of these predict the risk of acquiring upper respiratory tract infections [ ] . lactoferrin and lysozyme concentrations decrease with age [ , ] , potentially increasing risk for respiratory infections. lactoferrin and lysozyme are among the most abundant antimicrobials found in nature that are widely distributed in animal tissues and secretions [ ] [ ] [ ] , and are considered among the most promising antimicrobials to become medicines for clinical use [ , ] . they both act widely against bacteria, viruses and fungi, as well as having positive stimulatory effects on the immune system yet dampening the pathological effects of an overreacting immune system. lactoferrin and lysozyme are found in markedly high concentrations in tears compared with any other body fluid, and lactoferrin is found in similarly high concentration in breast milk and colostrum -this indicates the important role of these proteins in defense [ , , ] . however, the usual concentrations are only just adequate, and lower than normal levels in these secretions increase susceptibility to infection [ ] . while sars-cov- is readily detected in throat swabs, nasal swabs, saliva and sputum, and in a third of patients in feces [ ] [ ] [ ] , the virus is only infrequently detected in tears in a similar timeframe [ ] [ ] [ ] . when detected in tears, this has been in patients that had conjunctivitis symptoms [ , ] . a similar scenario is reported for sars-cov [ , ] . the potential activity of lactoferrin and lysozyme against sars-cov- and against the immune-mediated pathology in covid- (summarized in figure and table ) is considered. since ovotransferrin is more abundantly available than lactoferrin and can substitute lactoferrin in many applications [ ] , its potential as a covid- therapeutic is also reviewed. lactoferrin sequesters free iron, removing a substrate required for bacterial growth; however, it also has antimicrobial effects independent of iron sequestration [ ] . lactoferrin is cationic (highly positively charged) and this enables interaction with various negatively charged microbial and viral surfaces, dna, as well as with cell surfaces that are required for bacterial and viral adhesion or for early interactions required for viral entry [ ] . lactoferrin may also exert antiviral effects intracellularly [ ] . potent antiviral effects of both human and bovine lactoferrin have been shown against both enveloped and naked viruses such as cytomegalovirus, herpes simplex virus and hepatitis b and c virus among others, whether in the metal saturated or apo form [ , ] . bovine lactoferrin may have higher antiviral activity than human lactoferrin -they are highly similar and possess identical multifunctions [ ] . therefore, bovine lactoferrin is a good equivalent for human lactoferrin, especially since it is recognized by the european food safety authority as a safe dietary supplement with medicinal properties and no contraindications [ ] . importantly, bovine lactoferrin inhibits sars-cov cell entry by binding to heparan sulphate proteoglycans (hspgs) [ ] . hspgs on [ , ] intact and peptides antibacterial iron sequestration direct interaction with bacterial surface [ , ] intact and peptides antifungal iron sequestration direct interaction with fungal surface [ , ] intact immune enhancement, immune restoration in immunosuppression model enhanced phagocytic activity as well as cytokine production of macrophages enhanced intestinal immune responses: dendritic cell maturation, th /th balance restored and humoral immunity promoted [ , ] peptides anti-inflammatory downregulates il- and tnf-␣ and myeloperoxidase activity in peritonitis binds to angiotensin ii receptor type to inhibit angiotensin ii pro-inflammatory activity ace inhibitory activity (antihypertensive) [ ] [ ] [ ] [ ] intact iron-binding activity* sequestering free iron [ ] intact and peptides antioxidant* sequestering free iron free radical scavenging [ , ] lysozyme intact and peptides antiviral inhibits viral entry by binding to cell receptors or virus -cationic and hydrophobic nature is required rather than enzymatic activity binds nucleic acids inhibits virus-induced cell fusion affects cell signaling, including nf-b pathway, to influence susceptibility to infection [ ] [ ] [ ] [ ] intact and/or peptides antibacterial hydrolyzes cell wall of gram-positive bacteria (enzyme activity) insert into and form pores in negatively charged bacterial membranes [ ] † specific anticoronavirus activity has been demonstrated: inhibits sars-cov cell entry by binding to hspgs; inhibits entry and postentry steps of sars-cov- replication and elevates interferon-stimulated genes in sars-cov- -infected cells. ‡ the action is of immune homeostasis -its action is appropriate in the context of the immune environment [ ] . *iron sequestration has an antioxidant effect, and, in turn, these activities of lactoferrin/ovotransferrin have an anti-inflammatory effect -they limit immune pathology. ace: angiotensin-converting enzyme; age: advanced glycation end product; at : at is angiotensin ii receptor type ; cov: coronavirus; hspg: heparan sulphate proteoglycan; m : anti-inflammatory macrophages; sars-cov: severe acute respiratory syndrome coronavirus. interferon-stimulated genes in sars-cov- -infected cells. ‡ the action is of immune homeostasis -its action is appropriate in the context of the immune environment [ ] . *iron sequestration has an antioxidant effect, and, in turn, these activities of lactoferrin/ovotransferrin have an anti-inflammatory effect -they limit immune pathology. ace: angiotensin-converting enzyme; age: advanced glycation end product; at : at is angiotensin ii receptor type ; cov: coronavirus; hspg: heparan sulphate proteoglycan; m : anti-inflammatory macrophages; sars-cov: severe acute respiratory syndrome coronavirus. the cell surface provide an anchoring site on the cell surface and many viruses, including sars-cov, employ hspgs for adhesion to susceptible cells. sars-cov- entry is highly similar to that of sars-cov [ ] and was recently shown to be susceptible to lactoferrin-mediated inhibition of entry [ ] . lactoferrin inhibited both entry and postentry steps of sars-cov- replication, and elevated interferon-stimulated genes [ ] . besides its direct antimicrobial effect, through sequestering free iron and restoring iron homeostasis, lactoferrin reduces oxidative stress and inflammation, which is pertinent to the covid- pathology. lactoferrin counteracts iron dysregulation through sequestering free iron and restoring levels of various proteins (ferroportin, ceruloplasmin, transferrin receptor and ferritin) that are altered during inflammation [ , ] . lactoferrin reduces intracellular levels of reactive oxygen species as well as reducing oxidative stress-induced apoptosis [ ] , and short-term oral administration of bovine lactoferrin improves antioxidant capacity [ ] . importantly, lactoferrin can 'sense' the immune activation status and respond accordingly [ ] . for example, in individuals with high baseline immune activation bovine lactoferrin downregulates il- and tnf-α production by peripheral blood mononuclear cells (after days of mg per day oral administration) while in those with low immune activation, bovine lactoferrin upregulated these cytokines [ ] . lactoferrin suppresses extracellular traps released by neutrophils during inflammation [ ] , and has also been shown to stimulate pro-inflammatory macrophages (m ) to change to the anti-inflammatory macrophages (m ) type [ , ] . similarly, pasteurized whole cow's milk has been shown to polarize macrophages from m toward a proresolving m phenotype [ ] . in addition, lactoferrin-derived peptides inhibit angiotensin ii pro-inflammatory activity through binding to the angiotensin ii receptor type [ ] , and lactoferrin as well as other peptides in cow's milk have an antithrombotic effect [ ] . these effects of counteracting iron dysregulation, oxidative stress, neutrophil and macrophage-induced inflammation, ras-induced inflammation and thrombosis are highly relevant to covid- . further, lactoferrin shows potential benefit in alzheimer's disease through decreasing amyloid-beta aggregation (which leads to inflammation and neuron degeneration) [ , ] . this aggregation may be induced by microbes and this has been suggested for sars-cov- [ ] -a potential neuroprotective role of lactoferrin in covid- is a hypothesis requiring further investigation. oral administration of lactoferrin, usually bovine lactoferrin, in human and animal studies of various inflammatory disease states shows safety [ ] . in animal studies, oral bovine lactoferrin was shown to decrease inflammation and myeloperoxidase (a marker of neutrophil infiltration) in inflammatory bowel disease [ , ] . in animal models of sepsis, a single oral dose of lactoferrin prior to insult protected against progression of insult-induced systemic inflammatory responses [ ] and when orally administered after sepsis-induced lung injury, bovine lactoferrin was an effective therapeutic [ ] . further showing positive effects of lactoferrin in lung pathology, oral doses of human or mouse lactoferrin reduced mycobacterium tuberculosis-induced lung pathology in a mouse model [ ] and aerosolized bovine lactoferrin administered in a mouse model of cystic fibrosis with a pseudomonas aeruginosa lung infection resulted in decreased bacterial load, decreased infiltrated leukocytes and reduced iron overload [ ] . in human studies, lactoferrin decreased late onset sepsis and necrotizing enterocolitis in preterm infants [ ] , and oral bovine lactoferrin ( mg/day for months) decreased serum il- and increased il- as well as improved antioxidant activity in alzheimer's disease [ , ] . in pregnant women suffering from anemia and/or thrombophilia, mg of bovine lactoferrin taken orally twice a day improved hematological parameters, including serum iron, serum ferritin, hemoglobin and il- levels, more effectively than the standard of care [ ] . clinical effect has also been observed following lactoferrin administration in viral diseases. in hepatitis c-infected patients who responded to bovine lactoferrin monotherapy, when bovine lactoferrin was then combined with ribavirin and interferon, there was a sustained virologic response in % of individuals compared with a sustained virologic response in % of individuals who were treated with a combination of ribavirin and interferon alone [ ] . long-term oral consumption of bovine lactoferrin-containing products including yoghurt and milk (in the range of to mg lactoferrin per day) either reduces incidence or ameliorates symptoms of common viral infections, such as norovirus, likely though direct antiviral activity as well as the enhancement of systemic immunity (increased natural killer cell activity and th cytokine responses) achieved by bovine lactoferrin consumption [ ] . importantly, serrano et al., reported that a liposomal bovine lactoferrin nutritional syrup administered at - mg lactoferrin/day resolved symptoms of covid- patients within - days and considering their contacts who received half this daily dose, none developed symptoms of the infection [ ] . ovotransferrin shares many of the same activities as human/bovine lactoferrin and is more abundant than the latter [ ] . ovotransferrin combines the iron transport and defense functions of mammalian serum transferrin and lactoferrin, respectively, and shares about % sequence homology with each protein [ ] . however, the structural analogy between ovotransferrin and lactoferrin is much closer than the sequence homology [ ] and similar clusters of positively charged residues responsible for antiviral activity are found in the n-lobes of these proteins [ ] . ovotransferrin not only has antifungal activity [ ] and a wide range of antibacterial activity through sequestration of iron and through binding to bacterial surfaces via cationic peptides [ , ] , but the antiviral activity of intact ovotransferrin was greater than that of intact bovine lactoferrin when studying marek's disease virus [ ] . peptides in ovotransferrin that have high sequence homology with these bovine lactoferrin and human lactoferrin peptides acting against herpes simplex virus, human cytomegalovirus and adenovirus, were shown to have double the antiviral activity compared with the bovine lactoferrin peptides [ ] . recently, it was also reported that ovotransferrin upregulates antiviral interferon i in virus-infected macrophages [ ] . ovotransferrin has immunomodulatory, antioxidant and anti-inflammatory properties, and due to these properties it is being investigated as a therapeutic for cancer and cardiovascular disease [ , ] . ovotransferrin, as well as hydrolyzates, are able to scavenge free radicals, with higher activity than other known antioxidants such as ascorbate (vitamin c) -ovotransferrin showed protective effects against oxidative stress-induced dna damage, that was occurring via reaction of iron with hydrogen peroxide, in human leukocytes [ , ] . specifically, antioxidant peptides are derived from egg white hydrolyzate, where ovotransferrin peptides are in one of the most active fractions [ ] . an ovotransferrin peptide attenuates tnf-α-induced inflammation and superoxide generation in endothelial cells [ ] . hydrolyzates of ovotransferrin, as well as other egg white peptides, have also shown potent ace inhibitory activity [ , ] and as is the case for bovine lactoferrin, an ovotransferrin peptide blocked angiotensin ii effects via the angiotensin ii receptor type [ ] , thereby reducing inflammation potentiated by ras activation. in an animal model study of peritonitis, ingestion of mg/kg feed of an ovotransferrin peptide significantly attenuated the inflammatory responses: serum levels of tnf-α, il- and myeloperoxidase activity were significantly reduced [ ] . as is the case for bovine lactoferrin, these described activities of ovotransferrin, are highly pertinent to covid- pathology. lysozyme kills gram-positive bacteria through hydrolyzing the β- , glycosidic bond between n-acetylglucosamine and n-acetylmuramic acid in the bacterial cell wall [ ] . however, besides its enzymatic activity, it exerts antimicrobial effects through its cationic nature which enables it to bind to negatively charged surfaces (as in the case of lactoferrin), thereby expanding its activity well beyond that of gram-positive bacteria [ , , ] . the immunomodulatory function of lysozyme has only recently been appreciated [ ] . although lysozyme acting on microbes within neutrophils and macrophages increases their proinflammatory response, when it is released extracellularly by these cells as well as epithelial cells, it limits inflammation: it decreases the oxidative burst and chemotaxis in neutrophils [ ] , it significantly suppresses tnf-α and il- production by macrophages [ ] , it binds and decreases circulating levels of ages (which are pro-oxidative) as well as enhancing their renal excretion [ ] , and exogenous lysozyme disrupts the ability of peptidoglycan to bind complement factors that act as anaphylotoxins [ ] . furthermore, when subjected to simulated gastrointestinal digestion, the hydrolyzate of hen egg white lysozyme (hewl) showed marked antioxidant and ace-inhibitory activity [ ] . as described earlier, the oxidative stress (including involvement of ages), inflammation induced by neutrophils and macrophages, the tnf-α and il- cytokines, and an activated ras system are features in ards and/or severe covid- . it is noteworthy that, as is the case for lactoferrin, lysozyme has a neuroprotective function in alzheimer's disease through preventing amyloid-beta aggregation [ ] , which may have implications for the neurological manifestations in severe covid- . from mouse and porcine models it is clear that lysozyme plays an important role in limiting inflammation systemically, resulting in decreased immune-driven pathology [ , , ] . human clinical trials with lysozyme are limited [ , ] , but have shown, mostly through oral administration of hewl, antiviral effects against herpes (through oral administration of hewl at g/day [ , ] ), measles [ , ] and hepatitis ( - mg/day of lysozyme chloride for - weeks significantly reduced post-transfusion hepatitis incidence to % compared with % [ ] ), successful treatment of gum infections ( mg/day [ , ] ) and skin ulcers [ , ] , improvement of immune responses in cancer patients with immune suppression [ ] , and rapid resolution of inflammatory foci and stabilization of lysozyme levels in serum and stool of premature infants with diseases following mg/l supplementation in milk for - weeks [ , ] . human lysozyme in combination with bovine lactoferrin ( . g lysozyme and . g bovine lactoferrin per day) reduced enteric dysfunction in malawian children [ ] . no local or systemic unfavorable effects have been reported in these human trials. regarding the investigation of lysozyme in lung diseases, in eastern europe, hewl has been used successfully in combination with antibiotics to treat bronchitis and pneumonia in humans with no respiratory or systemic toxicity [ , ] . administration of lysozyme through aerosols to treat pneumonia has been investigated in animal models [ , ] . a % solution of aerosolized human lysozyme in hamsters with p. aeruginosa-induced pneumonia resulted in decreased lung histopathological changes, alveolar septal apoptosis, neutrophils and other leukocytes in the bronchiolar lavage fluid as well as increased activity of lysozyme in that fluid [ ] . however, it should be noted that lysozyme impairs the ability of hyaluronan to prevent elastase injury to elastic fibers through binding of the lysozyme to the elastic fibers, and thus on inhalation of lysozyme in an animal model of emphysema, airspaces further increased [ ] , which cautions against the inhalation route of administration of lysozyme in similar disease states. cow's milk is the most readily available source of lactoferrin, with an average concentration of . g/l in low heat pasteurized cow's milk (and . g/kg in semihard cheese produced from that milk) [ ] , which is in good agreement with other studies [ ] [ ] [ ] , though the range experienced ( . - . g/l) is dependent on several factors [ ] . the concentration in colostrum is higher, but varies greatly between breeds and may be anything between . and g/l, and is typically at the lower end of the range [ ] [ ] [ ] . the degradation of bovine lactoferrin in milk with low heat pasteurization ( • c for s) is minimal [ , ] , while ultrahigh temperature (uht) processes significantly denature the protein [ ] . large-scale isolation of bovine lactoferrin is performed from cheese whey [ ] where only % of the total bovine lactoferrin in milk is found [ ] , and the cost of purified bovine lactoferrin remains high -hence methods to achieve large-scale production of lactoferrin are being developed [ , ] . bovine lactoferrin is sold in bulk powder form, capsules (typically - mg), liposomal syrups ( mg/ ml) or as a liposomal lactoferrin nebulizer [ , , ] . the majority of bovine lactoferrin taken orally can be considered to survive gastric transit ( % for the apo form and % for the more stable iron-bound form [ ] ) and thereafter enter the intestine from where it is absorbed into the circulation, but liposomalization or encapsulation has been shown to enhance availability and effect [ , ] . it is also important to note that digestion with enzymes in the gi tract (pepsin, trypsin or chymotrypsin) yields lactoferrin fragments that are still able to bind iron [ ] , and that fragments of lactoferrin have antimicrobial activity [ ] , which may be stronger than that of the intact protein [ , ] . peptides of lactoferrin are considered promising antivirals, but isolation costs and stability pose challenges to reach the clinical phase [ ] , thus, at present the whole intact protein or food products/supplements with high content of lactoferrin are more accessible. a hindrance for use in medicine is the classification of lactoferrin products (as well as egg white powder and lysozyme discussed below) as food supplements, where these are not intended to treat disease, there is no controlled system for reporting effectiveness, and the active ingredient is not always of the same quality or integrity [ ] . ovotransferrin is abundant in hen egg white ( g/l egg white) [ ] . methods to pasteurize egg whites use temperatures that minimize damage to heat sensitive proteins in the egg white, such as lysozyme and ovotransferrin [ ] [ ] [ ] . dried egg white powder, where egg whites are equivalent to kg powder, sold as a supplement is a compressed source of these proteins. although . - . % of children have an allergy to egg white, about % outgrow the allergy -nevertheless, many medicines and vaccines have ingredients derived from egg [ ] . iron-bound ovotransferrin is more resistant to gastrointestinal digestion [ , ] , with iron-bound ovotransferrin well absorbed after ingestion [ ] . ovotransferrin is more readily digested by pepsin in the stomach compared with lysozyme [ ] ; however, the bioactive peptides (antimicrobial, antioxidant, anti-inflammatory and ace-inhibitory) of ovotransferrin, as well as other egg white proteins, described earlier have mostly been produced by digestion that simulates that occurring in the gi tract [ ] , and these peptides resist further digestion [ ] and are readily transported into human intestinal cells [ ] . ovotransferrin does however lose iron-binding activity after hydrolysis [ ] . simpler protocols with better yield and purity as well as low cost, will enable the use of isolated egg white proteins such as ovotransferrin or their peptides as pharmaceuticals [ ] , while presently the most readily available source is egg white powder. egg white is also the most readily available source of lysozyme ( . g/l egg white) [ ] . isolated hewl is commercially available and is labeled as a food supplement by the european commission. it is however available as lysozyme hydrochloride tablets ( , or mg), granules ( or %) and syrup ( . %) in japan, and is prescribed by doctors to improve expectoration in bronchitis, bronchial asthma and bronchiectasis [ ] . it is also sold as a food or pharmaceutical grade powder, and is widely used in the food industry as well as in pharmaceutical products (e.g., eye drops, wound healing creams, oral health products and over-the-counter drugs). widerspread use is hindered somewhat by the isolation cost ($ . /g) and there is significant effort being made around the world for commercial production of lysozyme, especially human lysozyme [ , ] . methods to produce human lysozyme, which has higher enzymatic activity than hewl and is therefore preferred, are under development (e.g., using transgenic rice), although these have a higher production cost than for hewl [ ] . however, the antiviral and immunomodulatory effects do not derive from the enzymatic activity, and the most available form of lysozyme currently is hewl, whether isolated or in egg white. oral administration of hewl results in systemic effects -after oral administration of - mg hewl in human subjects peak plasma concentrations are reached within an hour (with overnight fasting increasing absorption by sevenfold) and return to undetectable levels after days [ , ] . while hewl is fairly resistant to digestion in the stomach and partially resistant to digestion in the duodenum [ , ] , enzymatic hydrolysis does produce antimicrobial fragments and broadens the antimicrobial spectrum [ ] . susceptibility of proteins to proteolytic digestion is very strongly related to protein stability [ ] , and polyols or their derivatives are commonly used to enhance protein stability in formulations [ ] . an easily accessible and safe polyol may therefore be considered to improve stability of lactoferrin, ovotransferrin and hewl following ingestion, and here it is suggested that glycerol may be a particularly suitable supportive solvent for the powdered sources of lactoferrin, ovotransferrin and hewl. glycerol is a low cost, readily available, sweet-tasting polyol, with excellent solvent and emulsifying properties, which is safe for ingestion and widely used in pharmaceutical applications (such as cough syrups) [ ] [ ] [ ] . it is known to effectively stabilize proteins as well as refold denatured proteins [ , ] , thereby restoring activities of enzymes that were inactivated by diverse processes [ ] . in particular, glycerol was already shown to protect ovotransferrin and lysozyme when these proteins were subjected to stresses [ , ] , and to partially restore the structure/activities of these proteins after denaturation [ , ] . other properties may add further benefit, including anti-inflammatory [ ] and antiviral effects [ , ] , as well as the ability to inhibit ace activity and decrease angiotensin ii [ ] . in view of the direct antiviral effects of lactoferrin, ovotransferrin and lysozyme against a wide range of viruses (including sars-cov- for lactoferrin) and their antimicrobial effects against a wide range of bacteria and fungi that may cause secondary infections in covid- patients [ ] ; their immunomodulatory properties which stimulate antimicrobial responses yet promote resolution of inflammation, and in particular their previously shown beneficial effects in counteracting pathological neutrophil infiltration, macrophage activation, free iron overload, oxidative stress, age effects, excessive proinflammatory cytokine production (il- and tnf-α in particular), and thrombus formation, which all feature in severe covid- ; and their abundance and good safety profile -further testing of their potential role in prevention of sars-cov- infection or prevention of severe covid- is suggested. the main suggestion is to use these antimicrobials upon presentation of symptoms to prevent noncritical cases from progressing to critical cases, although they may also be considered as a preventative for those at high risk of infection where lower quantities could be taken as a means of lowering risk of infection. since the number of sars-cov- infection cases is growing so rapidly, the most expedient way to achieve this is through oral administration, which is suitable in the case of lactoferrin, ovotransferrin and lysozyme as these substances have systemic effects following ingestion. it is further suggested that, in the current circumstances of the covid- pandemic, good quality nonisolated forms of these (such as egg white powder, bovine colostrum powder and other nonultrahigh temperature milk products with appreciable lactoferrin content) should also be tested while ensuring the desired concentrations of each antimicrobial are met, especially in settings where the isolated forms may not be as readily accessible. in favor of this suggestion, studies using lactoferrin-containing milk or lactoferrin-supplemented yoghurt have shown clinical efficacy in viral diseases [ , ] , pasteurized whole milk has shown the effect of switching macrophages from m to m [ ] , several peptides in milk are antithrombotic [ ] , and several peptides in egg white besides those in ovotransferrin show supportive antioxidant as well as ace-inhibitory effects [ , ] . however, in those individuals who are already critically ill and on ventilators, more care may need to be taken with the approach. here, perhaps lactoferrin and lysozyme could be considered for intravenous administration or nebulization -a liposomal bovine lactoferrin nebulizer product is available. the accessibility and reasonable cost (in comparison to some of the other drugs -such as remdesivir and tocilizumab -under investigation to treat covid- ) make these antimicrobials attractive as a therapeutic option and we therefore call for their rapid testing in clinical trials. • severe covid- is reminiscent of hyperferritinemic syndrome. • ferritin contributes to the inflammation by directly activating macrophages, and free iron may be liberated from ferritin by free radicals. • free iron reacts with oxygen or hydrogen peroxide to form free radicals, thereby driving oxidative stress and leading to tissue injury. • there is no established effective treatment for covid- , and some treatment options being explored are unlikely to be widely available soon, especially in resource-limited settings. • an abundant and safe antimicrobial that could act via oral ingestion to lower the risk of infection or prevent mild cases from progressing to severe disease would be ideal. • tear lactoferrin and lysozyme levels predict the risk of acquiring upper respiratory tract infections, and these antimicrobials are abundant in natural secretions. • ovotransferrin is more abundant than lactoferrin and can substitute lactoferrin in many applications. • bovine lactoferrin has been shown to inhibit sars-cov and sars-cov- cell entry. • lactoferrin restores iron homeostasis through sequestering free iron and modulating levels of proteins involved in controlling iron balance between blood and tissues. • lactoferrin reduces oxidative stress and inflammation, and it is immune 'sensing' with its effect dependent on the environment. • oral administration of lactoferrin in animal models and human studies of viral diseases, as well as various inflammatory disease states, shows beneficial effects and safety. • ovotransferrin has antiviral peptides that are conserved with those found in human and bovine lactoferrin, and ovotransferrin may have a more potent antiviral effect. • ovotransferrin has immunomodulatory, antioxidant, anti-inflammatory and angiotensin-converting enzyme-inhibitory activities. • lysozyme exhibits antiviral activity via its cationic peptides and has immunomodulatory, antioxidant and angiotensin-converting enzyme-inhibitory properties. • oral administration of lysozyme in animal models and human studies shows its ability to limit inflammation systemically, resulting in decreased immune-driven pathology. • lactoferrin is abundant in cow's milk, while ovotransferrin and lysozyme are abundant in hen egg white. • high costs of isolation have limited widerspread use of purified forms of these antimicrobials. • isolation costs and stability pose challenges for bioactive peptides of these antimicrobials to reach the clinical phase. future perspective • these antimicrobials could be used upon presentation of symptoms to prevent noncritical cases from progressing to critical cases, and lower quantities could be taken to lower risk of infection in those at high risk. • good quality nonisolated forms of these should also be tested while ensuring the desired concentrations of each antimicrobial are met, especially in settings where the isolated forms may not be as readily accessible. • the accessibility and reasonable cost make these antimicrobials attractive as a therapeutic option and we therefore call for their rapid testing in clinical trials. potential of large 'first generation' human-to-human transmission of -ncov a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding virology, epidemiology, pathogenesis, and control of covid- a review of sars-cov- and the ongoing clinical trials remdesivir for the treatment of covid- : preliminary report structural and functional basis of sars-cov- entry by using human ace estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship covid- : immunopathology and its implications for therapy hypothesis for potential pathogenesis of sarscov- infection-a review of immune changes in patients with viral pneumonia implications of gastrointestinal manifestations of covid- renal complications in covid- : a systematic review and meta-analysis rhabdomyolysis and acute renal failure in an adolescent with coronavirus disease neurologic manifestations of hospitalized patients with coronavirus disease in wuhan, china covid- gone bad: a new character in the spectrum of the hyperferritinemic syndrome? • the similarities of covid- with hyperferritinemic syndrome, and the underlying pathology covid- ) time musings: our involvement in covid- pathogenesis, diagnosis, treatment and vaccine planning epub ahead of print) the hyperferritinemic syndrome: macrophage activation syndrome, still's disease, septic shock and catastrophic antiphospholipid syndrome the role of cytokines including interleukin- in covid- induced pneumonia and macrophage activation syndrome-like disease covid- : consider cytokine storm syndromes and immunosuppression hemophagocytosis causes a consumptive anemia of inflammation macrophages: a trojan horse in covid- ? diagnostic morphology: biophysical indicators for iron-driven inflammatory diseases importance of iron chelation in free radical-induced oxidative stress and human disease iron and iron-related proteins in the lower respiratory tract of patients with acute respiratory distress syndrome the role of iron metabolism in lung inflammation and injury the role of iron in pulmonary pathology serum ferritin as a predictor of the acute respiratory distress syndrome epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study molecular strategies to prevent, inhibit, and degrade advanced glycoxidation and advanced lipoxidation end products all the 'rage' in lung disease: the receptor for advanced glycation end products (rage) is a major mediator of pulmonary inflammatory responses role of receptor for advanced glycation end products in regulating lung fluid balance in lipopolysaccharide-induced acute lung injury and infection-related acute respiratory distress syndrome diagnosis, prevention, and treatment of thromboembolic complications in covid- : report of the national institute for public health of the netherlands inflammation as a regulator of the renin-angiotensin system and blood pressure clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury tear lactoferrin and lysozyme as clinically relevant biomarkers of mucosal immune competence dietary lactoferrin alleviates age-related lacrimal gland dysfunction in mice normal tear protein profiles and age-related changes antiviral properties of lactoferrin: a natural immunity molecule the mechanisms of the antiviral activity of lactoferrin are explained, and its effect against a range of different viruses is reviewed from bacterial killing to immune modulation: recent insights into the functions of lysozyme the immunomodulatory actions of lysozyme are described in detail pharmacological aspects and therapeutic applications of lysozymes antiviral peptides as promising therapeutic drugs lactoferrin in aseptic and septic inflammation the ability of lactoferrin to act as an immune 'sensing' agent is described, and the evidence for reduction of immune pathology in a wide range of disease models following lactoferrin administration is reviewed lysozyme: an antibacterial body present in great concentration in tears, and its relation to infection of the human eye lysozyme in human body fluids consistent detection of novel coronavirus in saliva comparison of throat swabs and sputum specimens for viral nucleic acid detection in cases of novel coronavirus (sars-cov- ) infected pneumonia (covid- ) detection of sars-cov- in different types of clinical specimens characteristics of ocular findings of patients with coronavirus disease (covid- ) in hubei province assessing viral shedding and infectivity of tears in coronavirus disease (covid- ) patients evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov- infection tears and conjunctival scrapings for coronavirus in patients with sars the severe acute respiratory syndrome coronavirus in tears pc ovotransferrin: characterization and alternative immunotherapeutic activity the intracellular inhibition of hcv replication represents a novel mechanism of action by the innate immune lactoferrin protein inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans bovine lactoferrin is shown to inhibit sars coronavirus cell entry by binding to heparan sulphate proteoglycans, which the virus uses as an anchoring site on the cell surface morphological cell profiling of sars-cov- infection identifies drug repurposing candidates for covid- . biorxiv ( ) bovine lactoferrin is shown to inhibit sars coronavirus replication in vitro, inhibiting both entry and postentry steps in replication the protective effects of lactoferrin against murine norovirus infection through inhibition of both viral attachment and replication physicochemical and antibacterial properties of lactoferrin and its hydrolysate produced by heat treatment at acidic ph identification of the bactericidal domain of lactoferrin la force fm. damage of the outer membrane of enteric gram negative bacteria by lactoferrin and transferrin the antifungal activity of lactoferrin and its derived peptides: mechanisms of action and synergy with drugs against fungal pathogens lactoferrin as a natural immune modulator bovine lactoferrin supplementation supports immune and antioxidant status in healthy human males lactoferrin accelerates reconstitution of the humoral and cellular immune response during chemotherapy-induced immunosuppression and bone marrow transplant in mice human lactoferrin attenuates the proinflammatory response of neonatal monocyte-derived macrophages lactoferrin in a context of inflammation-induced pathology lactoferrin suppresses neutrophil extracellular traps release in inflammation antihypertensive effects of lactoferrin hydrolyzates: inhibition of angiotensin-and endothelin-converting enzymes lactoferrin, ovotransferrin & lysozyme in covid- review aerosolized bovine lactoferrin counteracts infection, inflammation and iron dysbalance in a cystic fibrosis mouse model of pseudomonas aeruginosa chronic lung infection peptides affecting coagulation antiviral activity of ovotransferrin derived peptides ovotransferrin peptides that are conserved with peptides from bovine lactoferrin (which are also active against herpes simplex virus) were shown to be more potent than bovine lactoferrin peptides in their effect against marek's disease virus antiviral effect of ovotransferrin in mouse peritoneal macrophages by up-regulating type i interferon expression ovotransferrin antimicrobial peptide (otap- ) kills bacteria through a membrane damage mechanism antifungal activity of ovotransferrin towards genus candida interaction between lactoferrin and ovotransferrin and candida cells in vitro immune-enhancing activity of ovotransferrin from egg white via mapk signaling pathways in raw . macrophages ovotransferrin enhances intestinal immune response in cyclophosphamide-induced immunosuppressed mice antioxidant, angiotensin-converting enzyme inhibitory activity and other functional properties of egg white proteins and their derived peptides: a review in vitro cytotoxic and ace-inhibitory activities of promod p hydrolysate of ovotransferrin from chicken egg white egg white-derived antihypertensive peptide irw (ile-arg-trp) inhibits angiotensin ii-stimulated migration of vascular smooth muscle cells via angiotensin type i receptor the ovotransferrin-derived peptide irw attenuates lipopolysaccharide-induced inflammatory responses antiviral activity of ovotransferrin discloses an evolutionary strategy for the defensive activities of lactoferrin antioxidant effects of ovotransferrin and its hydrolysates lysozyme association with nucleic acids inhibition of human immunodeficiency type virus (hiv- ) life cycle by different egg white lysozymes structural and functional modeling of human lysozyme reveals a unique nonapeptide, hl , with anti-hiv activity enhancement of anti-herpetic activity of glycyrrhizic acid by physiological proteins human lysozyme has fungicidal activity against nasal fungi dose-dependent effect of lysozyme upon candida albicans biofilm human lysozyme possesses novel antimicrobial peptides within its n-terminal domain that target bacterial respiration modulation of neutrophil function by lysozyme. potential negative feedback system of inflammation anti-inflammatory effect of lysozyme from hen egg white on mouse peritoneal macrophages ace inhibitory peptides and antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white lysozyme liposomal lactoferrin as potential preventative and cure for covid- the ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin milk modulates macrophage polarization in vitro intranasal lactoferrin enhances alpha-secretase-dependent amyloid precursor protein processing via the erk / -creb and hif- pathways in an alzheimer's disease mouse model potential novel role of covid- in alzheimer's disease and preventative mitigation strategies oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance effect of bovine lactoferrin as a novel therapeutic agent in a rat model of sepsis-induced acute lung injury oral recombinant human or mouse lactoferrin reduces mycobacterium tuberculosis tdm induced granulomatous lung pathology oral lactoferrin for the prevention of sepsis and necrotizing enterocolitis in preterm infants a pilot study on the effect of lactoferrin on alzheimer's disease pathological sequelae: impact of the p-akt/pten pathway lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis effect of lactoferrin in patients with chronic hepatitis c: combination therapy with interferon and ribavirin lactoferrin for prevention of common viral infections crystal structure of hen apo-ovotransferrin. both lobes adopt an open conformation upon loss of iron the utility of ovotransferrin and ovotransferrin-derived peptides as possible candidates in the clinical treatment of cardiovascular disease the golden egg: nutritional value, bioactivities, and emerging benefits for human health the potential of egg white proteins, including lysozyme and ovotransferrin, and their bioactive peptides to be used in preventing/curing diseases is reviewed purification and characterization of antioxidant peptides from enzymatically hydrolyzed chicken egg white structure and activity study of egg protein ovotransferrin derived peptides (irw and iqw) on endothelial inflammatory response and oxidative stress protective properties of lysozyme on β-amyloid pathology: implications for alzheimer disease increased inflammation in lysozyme m-deficient mice in response to micrococcus luteus and its peptidoglycan hen egg lysozyme attenuates inflammation and modulates local gene expression in a porcine model of dextran sodium sulfate (dss)-induced colitis milk-derived proteins and peptides in clinical trials on lysozyme therapy. i. lysozyme tablets in treatment of some localized and generalized viral skin diseases the antimicrobial effect of lysozyme on nasal mucosa a random controlled study of the prophylactic effect of lysozyme chloride on post-transfusion hepatitis use of lysozyme in parodontopathy clinical trial of lysozyme treatment of crural ulcers in humans lysozyme in the feeding of premature infants with mixed pathology supplementation with lactoferrin and lysozyme ameliorates environmental enteric dysfunction: a double-blind, randomized, placebo-controlled trial lysozyme in the overall treatment of children with an influenza infection and pneumonia the characteristics of lysozyme and carbenicillin action on the clinico-immunological status of patients with chronic bronchitis aerosolized recombinant human lysozyme ameliorates pseudomonas aeruginosa-induced pneumonia in hamsters bioengineered lysozyme in combination therapies for pseudomonas aeruginosa lung infection the effect of lysozyme on elastase-mediated injury determination of bovine lactoferrin concentrations in cheese with specific monoclonal antibodies lactoferrin concentrations in milk from normal and subclinical mastitic cows detection of lactoferrin in bovine and goat milk by enzyme-linked immunosorbent assay determination of lactoferrin and immunoglobulin g in animal milks by new immunosensors factors affecting the lactoferrin concentration in bovine milk composition and properties of bovine colostrum: a review comparison of lactoferrin content in colostrum between different cattle breeds separation of lactoferrin-a and -b from bovine colostrum heat stability of bovine lactoferrin at acidic ph effect of processing intensity on immunologically active bovine milk serum proteins studying lactoferrin n-glycosylation large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows matrix-immobilized yeast for large-scale production of recombinant human lactoferrin physico-chemical properties influence the functions and efficacy of commercial bovine lactoferrins gastric digestion of bovine lactoferrin in vivo in adults formulation for oral delivery of lactoferrin based on bovine serum albumin and tannic acid multilayer microcapsules liposomalization of lactoferrin enhanced its anti-inflammatory effects via oral administration iron-binding fragments from the n-terminal and c-terminal regions of human lactoferrin egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents: a review international egg pasteurization manual thermal stability of high concentration lysozyme across varying ph: a fourier transform infrared study polyols (glycerol and ethylene glycol) mediated amorphous aggregate inhibition and secondary structure restoration of metalloproteinase-conalbumin (ovotransferrin) current understanding of egg allergy the effect of trypsin on bovine transferrin and lactoferrin resistance of metal complexes of conalbumin and transferrin to proteolysis and to thermal denaturation iron-ovotransferrin preparation does not interfere with ciprofloxacin absorption in vitro digestions and ige binding of proteins from white and whole hen's egg food-derived bioactive peptides in human health: challenges and opportunities purification and characterisation of angiotensin i converting enzyme (ace) inhibitory peptides derived from enzymatic hydrolysate of ovotransferrin transport of antihypertensive peptide rvpsl, ovotransferrin - , in human intestinal caco- cell monolayers concentration of egg white lysozyme in the serum of healthy subjects after oral administration recent advances for the production and recovery methods of lysozyme what is new in lysozyme research and its application in food industry? a review studies on biotransformation of lysozyme. iv. radioimmunoassay of lysozyme and its evaluation immunoreactivity of hen egg allergens: influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis effect of polyols on the conformational stability and biological activity of a model protein lysozyme the effect of glycerol supplements on aerobic and anaerobic performance of athletes and sedentary subjects in: the mak-collection part i, mak value documentations soothing properties of glycerol in cough syrups for acute cough due to common cold trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation correct protein folding in glycerol anti-irritant and anti-inflammatory effects of glycerol and xylitol in sodium lauryl sulphate-induced acute irritation virucidal effect of glycerol as used in donor skin preservation effect of glycerol on intracellular virus survival: implications for the clinical use of glycerol-preserved cadaver skin antihypertensive effect of a combination of uracil and glycerol derived from lactobacillus plantarum strain twk -fermented soy milk co-infections: potentially lethal and unexplored in covid- influence of camel milk on the hepatitis c virus burden of infected patients acknowledgments e allen is acknowledged for her valuable contribution in the form of discussions on this topic. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord- -bsmqqi j authors: bajraktari, saranda; sandlund, marlene; zingmark, magnus title: health-promoting and preventive interventions for community-dwelling older people published from inception to : a scoping review to guide decision making in a swedish municipality context date: - - journal: arch public health doi: . /s - - - sha: doc_id: cord_uid: bsmqqi j background: despite the promising evidence of health-promoting and preventive interventions for maintaining health among older people, not all interventions can be implemented due to limited resources. due to the variation of content in the interventions and the breadth of outcomes used to evaluate effects in such interventions, comparisons are difficult and the choice of which interventions to implement is challenging. therefore, more information, beyond effects, is needed to guide decision-makers. the aim of this review was to investigate, to what degree factors important for decision-making have been reported in the existing health-promoting and preventive interventions literature for community-dwelling older people in the nordic countries. methods: this review was guided by the prisma-scr checklist (preferred reporting items for systematic reviews and meta-analysis extension for scoping reviews), the methodological steps for scoping reviews described in the arksey and o′malley’s framework, and the medical research council’s (mrc) guidance on complex interventions. eligible studies for inclusion were randomised controlled trials (rcts) concerning health promotion or primary prevention for community-dwelling older people implemented in the nordic countries. additionally, all included rcts were searched for related papers that were reporting on additional factors. eligible studies were searched in seven databases: pubmed, scopus, cinahl, academic search elite, psycinfo, socindex, and sportdiscus. results: eighty-two studies met the inclusion criteria (twenty-seven unique studies and fifty-five related studies). twelve studies focused on fall prevention, eleven had a health-promoting approach, and four studies focused on preventing disability. all interventions, besides one, reported positive effects on at least one health outcome. three studies reported data on cost-effectiveness, three on experiences of participants and two conducted feasibility studies. only one intervention, reported information on all seven factors. conclusions: all identified studies on health-promoting and preventive interventions for older people evaluated in the nordic countries report positive effects although the magnitude of effects and number of follow-ups differed substantially. overall, there was a general lack of studies on feasibility, cost-effectiveness, and experiences of participants, thus, limiting the basis for decision making. considering all reported factors, promising candidates to be recommended for implementation in a nordic municipality context are ‘senior meetings’, ‘preventive home visits’ and ‘exercise interventions’ on its own or combined with other components. the population across the world is growing older which calls for effective health-promoting and preventive interventions in order to help older people maintain a good quality of life. in accordance with the world health organisation (who), health promotion is defined as the process of enabling the population/individual to increase control over and improve their health, while disease prevention is defined as measures taken to prevent the occurrence of disease or limit its development [ , ] . the implementation of health promotion and prevention is imperative given that increased levels of dependency in managing activities of daily living (adls) is related to a reduction in self-rated health [ ] as well as higher societal costs [ ] . in sweden, municipalities have a responsibility to address health concerns and social care needs among older people ultimately aiming to optimize the person's quality of life by promoting independence and opportunities to participate in society [ ] . therefore, municipalities need to consider health promoting and preventive interventions besides, and to complement, the provision of social care. such interventions can promote various aspects of the health and well-being of older people by strengthening the person's opportunities to be active and participate in society [ ] . simultaneously, a more health promoting approach to the provision of municipality services for older people could reduce the expected increase in health and social care costs. several studies show that health promotion and prevention in different forms have resulted in a range of positive effects such as maintenance of ability to perform adls [ ] , enhanced quality of life [ , ] , prevention of functional decline [ , ] , and reduced falls [ ] . in addition, some interventions have shown to be cost-effective [ , ] . in all, examples in the previous literature indicates that positive effects can be achieved from both multi-professional and single-professional interventions [ , ] , from both short and long-term interventions [ , ] and both group-based and individual interventions [ , ] . even though the existing evidence is promising in improving health outcomes among older people, the range of interventions have varied considerably regarding their content, design and outcomes used, making them hard to compare [ ] . since resources (e.g. staff) are limited, not all promising health-promoting or preventive interventions can be implemented. thus, more information than mere evidence on effects, based on single trials, is needed to provide sufficient guidance for decision-makers on what type of intervention to implement [ ] . the question of which interventions to implement needs to be guided by a systematic decision-making process based on the best available evidence [ ] . in this systematic process, a range of factors need to be considered, e.g. intervention design, effects, cost-effectiveness, feasibility of recruitment and intervention procedures as well as an understanding of how participants experience the intervention. the challenge with this task is that many health-promoting interventions often miss to report all such information relevant for decision making [ , ] . in addition, the issue of context should be considered when assessing how evidence can be transferred from controlled trials to clinical settings [ ] . in this study, the context is focused on the nordic countries, because these countries, to a large extent, share similar welfare systems characterized by publicly funded health and social care. a scoping review design has been proposed as an effective tool to disseminate research findings and provide an overview of evidence for decision-makers and policymakers [ ] , and is especially appropriate when exploring a heterogeneous or complex body of literature [ ] . given the potentially positive effects on older peoplesh ealth and the cost-effective use of societal resources, a comprehensive overview of the existing evidence on health promoting and preventive interventions is needed. therefore, the aim of this review was to investigate to what degree factors important for decision-making have been reported in the existing health-promoting and preventive interventions literature for community-dwelling older people in the nordic countries. this scoping review follows the prisma-scr checklist (preferred reporting items for systematic reviews and meta-analysis extension for scoping reviews) [ ] as well as the methodological steps for scoping reviews described in the arksey and o′malley's framework [ ] . the arksey and o′malley's framework consists of five stages: ) identifying the research question; ) identifying relevant studies; ) selecting studies; ) charting the data; ) collating, summarizing and reporting the results [ ] . this scoping review has been conducted following an unpublished work plan. health promotion and prevention often include several interacting components and can, therefore, be considered as complex interventions. the medical research council's (mrc) guidance for the process of developing, evaluating and implementing complex interventions was used to identify the research questions of this scoping review [ ] . according to the mrc guidelines, this process includes several phases in which evaluations of feasibility, effectiveness and cost-effectiveness provide essential knowledge. in addition, the pico framework (population, intervention, comparison, outcome) which is recommended to frame the research question but also to guide the whole process in a review, was used as an additional source in guiding the formulation of the research questions regarding the population, intervention/control and effects on possible outcomes [ ] . hence, the research questions were: . in which contexts have interventions been conducted? . for which populations have interventions been conducted? . how have the interventions been designed (e.g., which components, duration of interventions and mode of delivery)? . which feasibility aspects have been described? . how have the participants experienced the interventions? . were interventions effective, and on which outcomes? . were interventions cost-effective? the eligibility criteria were defined in advance but were modified with increased familiarity with the literature. eligible studies were: ) interventions categorised as health promotion (hp) or primary prevention (pp) following the who's definition [ , ] and addressing behavioural risk factors, injury prevention, physical health, social and mental health, ) including populations of community-living older people + as of it being the lowest retirement age in the nordic countries, hence exclude the risk of missing relevant studies due to the age limitation, ) implemented in a nordic country (denmark, finland, iceland, norway, sweden and faroe islands), ) studies applying a randomized controlled trial design (rct) for the evaluation of effects (research question six), ) studies related to the identified rcts addressing the remaining research question, e.g. experiences of participants, feasibility as well as studies on cost-effectiveness. only studies written in english were included and to decrease the risk of missing relevant articles, no year limit was applied. the exclusion criteria were: secondary prevention programmes related to a specific disease or diagnosis e.g. interventions implemented for participants with a neurological condition such as stroke or alzheimer's disease, tertiary prevention programmes (e.g. rehabilitation, hospital discharge) as well as studies in populations with extensive needs for support in adls. furthermore, interventions focusing on dental health promotion; interventions targeting older people with cognitive malfunction; programmes assessing effects of medication or evaluations of effects only focused on specific body structures [ ] , were also excluded. seven online databases were searched: pubmed, sco-pus, cinahl, academic search elite, psycinfo, socindex, and sportdiscus. in designing the most suitable search strategy, a librarian at umeå university was consulted on several occasions. the search strategy was based on a combination of words to capture key terms related to the purpose of this study: "health promotion", "prevention", "old people", "community-dwelling", "nordic countries", "randomised controlled trial" and their synonyms/alternative words. a detailed outline of the search strategy, including the full syntaxes to screen the databases and numbers of search results, is available in additional file . the initial search strategy was piloted and refined in the light of early findings. the search for literature was conducted from inception to january , (last date searched). identification of studies, relevant to this review, was done in two stages. at the first stage, we identified rcts in the field of health promoting and preventive interventions for community dwelling older people conducted in the nordic countries. to decrease the risk of missing relevant studies during the first stage of identifying studies, we did not limit our search to only primary prevention programmes. we applied this inclusion criterion when screening titles and abstracts for study selection. in the second stage, reference lists of identified and selected studies from the first stage (the rcts) were examined for the purpose of identifying related studies, i.e. studies evaluating the same intervention but at different follow-ups, looking at different outcomes, or addressing the other research questions. search results were exported in endnote reference manager, which was used to remove duplicates. in the next step, the endnote reference manager was used to ease the process of identifying and excluding irrelevant studies through searching for key exclusion terms (hospital discharge, cognitive malfunction, dementia etc.). titles and abstracts of the remaining studies were organised in an excel document and read independently by all authors. studies that all authors agreed did not meet all of the eligibility criteria were removed. in cases of uncertainty, disagreement was resolved by reading the whole study and discussion among the three authors. after screening titles and abstracts and excluding studies not meeting the inclusion criteria, the remaining studies were read in full text. in line with the process of identifying research questions, the mrc framework and the pico framework were used to guide the process of data extraction. the included studies were distributed between authors sb and mz who independently charted the data for summarizing information related to the research questions, each question targeting one of the seven factors: context, population, intervention content, feasibility, experiences of participants, effects and cost-effectiveness. disagreement was resolved through discussion between all authors. all authors read the extracted data and discussed the results. main results are presented in the text under a specific heading for each of the research questions. results are presented and described by referring to either the original study/ study (at first-hand study protocol, if available. if no study protocol was identified we referred to the first published rct), related studies (other publications related to the original study) or intervention (referring to the specific interventions evaluated in each study). in the section below there is a description of the factors (data items) extracted to address the research questions. to the extent available, data on context, population, intervention content, feasibility, experiences of participants, effects and cost-effectiveness have been extracted from the included studies. the extraction of data regarding intervention context focused on identifying the setting (e.g. primary care, clinical, home, physical activities facilities) in which the specific intervention was evaluated as well as the country, and if available, the municipality in which the study was conducted. data extracted on population concerned how the target population was defined in age, frailty/morbidities, gender, and socio-economic status. the data extracted concerning feasibility was specifically focused on identifying participation rates and retention. if a pilot or feasibility study was published, the aim and main results of the study were also extracted. information on experiences of participants was extracted from related qualitative studies, and main results on experiences of participants were summarised. effects were examined by extracting effects on specific health outcomes at different time-points as reported in each study. in general, the data extracted regarding effects included effect sizes if reported, confidence intervals and p-values for outcomes for which a statistically significant difference was reported. no effect sizes, confidence intervals or p-values were extracted for outcomes upon which no significant difference was reported, they are mentioned in text however. the first step in exploring cost-effectiveness was to identify if such studies had been conducted. the primary objective when looking at identified studies on costeffectiveness was to examine if evaluated interventions were found to be cost-effective and in relation to which outcomes cost-effectiveness was established. furthermore, if available, data concerning methodological aspects of such studies were extracted, e.g. perspective used (health provider/payer or social perspective), outcome-and cost measures and how they were affected by the specific intervention, comparator (e.g. no intervention, alternative intervention) and time horizon (over which time horizon costs and effects were measured) [ ] . the search yielded a total of studies. after removing duplicates, titles and abstracts were screened and studies obviously not meeting the inclusion criteria were excluded. all remaining studies were read in full text (n = ) and studies which did not meet the eligibility criteria were removed (n = ). all original studies, identified in stage , were in stage reference checked resulting in related studies being identified and included. in all, a total of studies were included for analysis, original studies and related studies. the search process is presented in a prisma flowchart in fig. . the total number of participants in the included studies (extracted primarily from the original studies, if available) was , . one municipality-based study included a very large sample (n = , ) [ ] . considering all studies except the one by poulstrup and jeune [ ] , sample sizes varied from participants [ ] to participants [ ] . the duration of interventions varied from a one-session discussion group [ ] to three weekly group exercise sessions over a period of one year [ ] . of the original studies, focused specifically on fall prevention (looking primarily at fall-related parameters and fall risk factors, e.g. falls, fear of falling, balance performance, bone mineral density) [ , - , - , ] . eight fall prevention interventions were single component and included only exercise [ , - , , ] , while five combined an exercise component with one or more different components, e.g. preventive home visits (phv), discussion groups, nutrition, medication review [ , , , , ] . eleven studies had a health promoting approach. five of these studies focused on promoting general health (interventions which in addition to focusing on functional status also focused on health-related quality of life and/or social support aspects) [ , , , , ] , four promoted exercising [ ] [ ] [ ] ] , and two focused on promoting mental wellbeing [ , ] . the four remaining studies focused on preventing disability [ , [ ] [ ] [ ] . findings on intervention type, intervention aim, context, and population are presented below in table . these findings are also described in the text, separately for each factor, in the sections below. there were no related studies identified for of the original studies, so all related studies found were linked to only of the original studies. of the original studies: one study reported results in nine related studies [ ] , two reported in seven related studies [ , ] , and one reported in six related studies [ ] . the remaining interventions reported results in one to five related studies. for further details, see table below. among the related studies, included evaluations of effects, eight were qualitative studies analysing experiences of participants, four were health economic evaluations, three were study protocols, and two were pilot studies. findings on intervention content, effects and feasibility aspects are also described separately in the sections below, while detailed information on these factors is presented in table . geographically, the studies were conducted in finland (n = ), sweden (n = ), denmark (n = ) and norway (n = ). no studies were identified from iceland or faroe islands. interventions were implemented either at home (n = ) or in other settings (n = ), e.g. gyms and exercise halls [ , , , ] , clinics/hospitals [ , , , , ] or research centres [ , ] . the remaining interventions were implemented in a combination of settings (n = ). for further details, see table , "context" column. the population targeted in the included studies varied regarding age and health-related conditions. in six studies, the target population was defined in relation to age and location of residence [ , ] , four of these studies were municipality-based and targeted a broad population of older people from several municipalities [ , , , ] . the remaining studies defined the target population in relation to age and location of residence/municipality in [ ] , whereas two applied a narrow age span - [ ] , - [ ] . one study reported only the mean age of the participants [ ] . five studies had samples consisting only of female participants [ , table detailed results concerning intervention content, effects on health outcomes, and feasibility aspects of included studies in the field of health-promoting and preventive interventions for community dwelling older people in the nordic countries from inception to (continued) original study intervention content effects (significant between-group differences) feasibility aspects month after the intervention period. control: counselling session on fall prevention at baseline irr = . *, % ci = . - . [ ] . no sig. difference in hand grip strength, knee flexion (right/left) [ ] , incidence of falls overall [ ] or in the incidence of falls requiring medical treatment [ ] , depressive symptoms [ ] , dynamic balance [ ] . -year and -year: no sig. difference between i vs control in the incidence of falls requiring medical treatment [ ] .. walking duration increased* for combined (t and t + n) vs n and c [ ] . no sig. differences in balance, mobility, nutritional measures (e.g. body weight, energy intake) [ , ] , aerobic capacity (maximal work-load or work time) [ ] . -month: only effects in physical activity level preserved in t vs c and n [ ] . no effects were preserved on: rmr, leg press, dips, step test, muscle strength [ ] , aerobic capacity (maximal work-load or work time) [ ] , adl [ ] . , , , ] . for further details, see table , "population" column. given the broad range of intervention types, interventions varied by content, modes of delivery, duration and professionals involved. in most of the studies, the intervention content included a physical activity component (n = ). in twelve of these studies, exercise was the only component and included different exercise forms such as resistance/ strength [ ] , balance [ ] , rocking-chair training [ ] , nintendo wii exercise [ ] , or a combination of different exercise forms [ , - , , , , ] . the remaining seven studies included different components, e.g. exercise and multidisciplinary check-ups [ ] , exercise and comprehensive information on, e.g. medication, nutrition, removing home hazards [ , , ] , exercise and a social activity programme [ ] , exercise and nutrition [ ] , and exercise and vitamin d [ ] . the eight remaining studies did not include any practical exercise component. these studies included, senior meetings or discussion groups and home visits [ , , ] , a discussion group, activity groups and an individual intervention [ ] , case-management [ ] , anonymous self-care telephone calls [ ] , physical activity counselling [ ] , or an education programme for home-visitors [ ] . regarding modes of delivery, six studies were individually based [ , , , , , ] , seven were group-based [ , , , , , , ] , and studies included group and individual interventions [ , , , , , , - , - , ] . studies including only individually based interventions were provided at home and were either self-managed [ ] , supervised [ , , ] , telephone-based [ ] or digital [ ] . studies including only group-based interventions were delivered in the format of exercise groups [ , , , , , ] or an educational group [ ] . studies including both group formats and individual interventions included group formats and home visits [ , , , , , ] , group formats and home training [ , , , , ] group formats and individual counselling on health [ , , , , ] . the number of sessions included in the interventions varied, as did the duration. for individually-based interventions, the number and duration of sessions ranged from one single home visit [ , ] or one personal counselling session on nutrition [ ] to daily independently performed exercise sessions ( - repetitions) over a period of months [ ] . group-based components ranged from one single discussion group [ ] to three min exercise session a week for over one year [ ] , while the education programme for home visitors included regular education over a period of three years [ ] . studies combining group and individually-based components ranged from one single home visit and four discussion groups [ , ] to two weekly exercise sessions over one year in combination with monthly lectures on various themes and psychosocial activities combined with a single individual geriatric assessment and counselling on fall prevention [ ] . in studies, the interventions were delivered by a multiprofessional team [ - , , , , , - , , - ] including, e.g. physiotherapist, occupational therapists, nurses, dietitian, dentist and healthcare students. in twelve studies, the interventions were implemented by one profession, of which seven interventions were delivered by physiotherapists [ , , , , , , ] , one by occupational therapists [ ] , three by exercise instructors/leaders [ , , ] , and one by unspecified trained personnel [ ] . feasibility aspects were reported sporadically across studies. all interventions reported on methodological aspects table detailed results concerning intervention content, effects on health outcomes, and feasibility aspects of included studies in the field of health-promoting and preventive interventions for community dwelling older people in the nordic countries from inception to (continued) original study intervention content effects (significant between-group differences) duration: regular education ( municipal meetings) for home visitors during years and one education programme ( h) for gps in the first year control: no intervention (education program) for home visitors in another control municipalities. increased risk for catastrophic functional decline rr . ***, % ci . - . [ ] . fewer persons ( -year-olds) in the intervention group had moved to a nursing home hr . *, % ci . - . [ ] . effects on functional ability in women were preserved or . *, % ci . - . . no sig. difference in functional ability for men [ ] . no sig. difference in functional decline or mortality in both man and women [ ] . notes: *p ≤ . , **p ≤ . , ***p ≤ . . a maximum score for fes- = , higher score implies higher concern for falling, lower score implies lower concern for falling, b maximum score for bbs = , higher score implies higher degree of functional balance and vice versa, c maximum score for -item of feasibility such as recruitment and retention/dropout numbers. with recruitment numbers, we refer to the total number of eligible participants (meeting inclusion criteria) who agreed to participate in the study. the mean recruitment rate (eligible participating population/total eligible population) in all the studies included in this review was %, varying from % [ ] to % [ , , , , ] . however, there was some inconsistency regarding how the eligible population was defined. for instance, in one study the total eligible population consisted of only those who volunteered [ ] , or of the population receiving an invitation [ ] or the whole population in a specific community [ ] . thus, participation rates are not consistent among included interventions and this inconsistency should be taken into consideration when interpreting the mean recruitment rate. mean retention rate in the total number of original studies included in this literature search was %. retention rate varied from % [ ] to % [ ] . beside the information related to recruitment and retention rates, only two feasibility/pilot studies were identified [ , ] . kristensson et al., investigated the feasibility of a case management intervention by specifically assessing sampling and sample characteristics as well as possible effects on perceived health [ ] . lood et al., ( ) investigated the feasibility of evaluating senior meetings in the "elderly in the risk zone" intervention [ ] among a specific group of older people (foreign-born) by specifically assessing recruitment and retention rates, questionnaire administration, and variability of data [ ] . in relation to five of the original studies, eight related studies explored the experiences of participants [ , , , ] or both the experiences of participants and professionals delivering the intervention [ , , , ] . based on qualitative methods and interviews, participants' experiences were described related to i) a single preventive home visit (phv) [ ] , ii) senior meetings [ , , , ] , iii) multidisciplinary fall prevention programmes [ , ] , and iv) case management intervention [ ] . findings from interviews on phvs showed that home visits contributed to empowerment and increased selfesteem by making participants feel in control over their health. however, for some, it did not come at the right time, either because they felt too healthy to benefit from it or because they felt too ill to be able to participate [ ] . findings on senior meetings revealed that although independent older people may find it difficult to accept or act upon health-promoting information, the discussion groups, provided in a multi-dimensional approach, could motivate acting upon such information, and thus, senior meetings were perceived as a "key to action" [ ] . these findings were in line with experiences of foreign-born older people who felt empowered by the opportunities gained, such as the possibility to meet other people, discuss experiences, as well as become acquainted with possibilities to make everyday life better and safer [ ] . however, their capabilities to adhere and act upon knowledge in the long-term (six months to one year after their participation in the programme) was dependent on personal and environmental resources [ ] . furthermore, professionals delivering the interventions, revealed that for a senior meeting intervention to succeed in reaching out to the target group, it is necessary to recognise the person's resources and empower their capabilities in maintaining health [ ] . empowerment and raised awareness were also emphasized in a group-based multidisciplinary fall prevention program delivered through a client-centered approach. the involved professionals observed that building trust and a safe atmosphere within the group increased participants' engagement in discussions which contributed to the success of the intervention. a contributing factor for creating this sort of atmosphere was the role-shifting negotiated by the group leaders from being the expert to being a facilitator of the discussion [ , ] . however, it was noticed that for a group format to be successful, group composition should be taken into consideration for the participants to feel fellowship [ , ] . furthermore, in a home-based case-management intervention, participants experienced case managers as a helping hand in navigating within the health system, and thus, contributed to feelings of control and safety [ ] . additionally, experiences of participants were explored as secondary outcomes through a survey related to a nintendo wii training fall prevention intervention [ ] , or through a single open-ended question related to a telephone-based health-promoting intervention [ ] . findings from the survey showed that training with a digital device (wii) was experienced positively and did not lead to any adverse effect [ ] . a self-care telephone intervention influenced participant's attitudes positively, e.g. towards self-care [ ] . for several interventions, effects were evaluated in relation to a wide range of outcomes, and all, besides one intervention on nutritional counselling [ ] , reported a positive effect on at least one health outcome evaluated in comparison to a control group. however, the magnitude of effects and follow-ups at which interventions were evaluated, varied substantially and therefore, should be taken into consideration when evaluating effects. to summarise intervention effects, we classified health outcomes in broader categories (table ) . for example, balance confidence, balance performance, dynamic balance, impaired balance, postural balance, postural sway, velocity moment in standing balance, are categorised under "balance". details on effects are found in table . four studies presented a health-economic evaluation. three studies adopted a cost-effectiveness analysis method [ , , ] and one a cost-utility analysis method [ ] . two studies provided an economic evaluation of single interventions; a case-management intervention [ ] and an education programme for home visitors [ ] . the other two studies compared different interventions focused on health promotion [ ] , and falls prevention [ ] . in these four studies, a societal perspective was chosen including cost from different sectors e.g., health care and social care. the time horizon used varied from three months [ ] , one year [ , ] , two years [ ] and up to three years [ ] . all studies based their estimates of costs on intervention costs, healthcare costs and municipality costs. in addition, the value of informal care was included in one study [ ] . costeffectiveness was evaluated in relation to active life-years gained [ ] , quality-adjusted life-years (qalys) [ , ] and number of injurious falls prevented [ ] . findings from the economic analysis showed that two interventions were considered cost effective [ , ] whilst two were not [ , ] . a one-session discussion group was found to be more cost-effective when compared to an individual intervention or an activity group in an intervention comparing three different occupation-focused healthpromoting interventions to a control group [ ] . the discussion group showed significant effects on qalys gained at and month follow up's and lower total costs [ ] . furthermore, an exercise intervention showed high probability to be cost-effective in preventing falls in relation to a threshold of euro per injurious fall prevented when compared to three other fall preventive interventions focusing on exercise and vitamin d supplements [ ] . in contrast, no significant difference was observed in total costs or qalys gained when comparing a case management intervention to no intervention in a cost-utility analysis. nevertheless, the case management intervention led to lower levels of informal care and need for help with instrumental adls [ ] . neither did a training programme for home visitors result in significant differences in total cost or active life-years gained in comparison with usual practice of performing preventive home visits [ ] . this scoping review provides a comprehensive overview of health-promoting and preventive interventions for community-dwelling older people in the nordic countries that to some extent, can guide decision-making in a swedish municipality context. however, while all included studies report some positive effects, not all potentially effective interventions can be implemented since resources are limited. thus, the evidence on effects needs to be critically reflected upon, but several other factors need to be considered as well. our study exposes gaps in knowledge regarding cost-effectiveness, experiences of participants and feasibility of the interventions, knowledge that could broaden the understanding of which interventions seem most promising and feasible to implement from a decision-makers´perspective. while the scope of this review includes interventions with different foci, the summary of findings on the seven evaluated factors, show that some interventions such as senior meetings, preventive home visits (phv) and exercise interventions alone or combined with other components, seem to be strong candidates for implementation, e.g. [ , , ] . in all, the total evidence for these interventions included positive effects on a range of outcomes, in some cases confirmed by evaluations at different follow-ups, with established cost-effectiveness, and supported by qualitative findings based on the experiences of participants. in the section below we provide a deeper discussion about the previously mentioned intervention examples and argument how the findings from this review could guide decision making and how additional knowledge, generally missing across the different interventions, is needed to better guide decisions on which interventions to implement. senior meetings, one type of intervention investigated in four different studies, seems potentially effective in promoting general health and wellbeing among communitydwelling older people [ , , , ] . the study which provides the broadest evidence base is the "elderly persons in the risk zone"-study conducted in gothenburg [ ] , which evaluated a four-sessions senior meeting intervention combined with a home visit. several related studies support the implementation of senior meetings given the positive results on a range of health outcomes, e.g., physical function [ ] and adls [ ] , outcomes for which effects were established at different follow-ups ( months to -year follow-ups). qualitative findings on the experiences of participants also provide an understanding of why the intervention was effective by concluding that senior meetings were experienced as a "key to action" in empowering participants to engage in preventive approaches to improve health [ ] . the benefits of senior meetings, albeit with other content, were also verified in the studies by zingmark et al., [ ] and johansson et al., [ ] . in the study by zingmark et al., [ ] two group-based formats of interventions (a discussion group and an activity group) were implemented by occupational therapists which both resulted in positive effects. in our results, evidence on costeffectiveness regarding senior meetings was limited to the study by zingmark et al., who found a one-session discussion group to be the most cost-effective intervention format [ ] . recently, however, a publication based on data from the "elderly persons in the risk zone" supports the cost-effectiveness of senior meetings as well, even in the long term (over four years) [ ] . thus, senior meetings seem to be a strong candidate for implementation in a swedish municipality context. yet, the exact format can be further discussed given the variation in the number of sessions and the specific content, e.g. one session discussion [ ] , four sessions combined with a home visit [ ] , twelve sessions combined with two home visits [ ] . in addition, feasibility aspects related to recruitment during implementation in a municipality context seem to be a critical feature to improve reach in the intended population, thus requiring specific contextual knowledge [ ] . our results show that phvs have the potential to improve general health by preventing deterioration in health in community dwelling older people. however, phvs have varied regarding the specific format e.g. from one visit [ ] to twelve visits [ ] and have shown positive effects on several outcomes e.g. limiting progression in morbidity [ ] , reducing the number of emergency department visits [ ] , maintaining adl ability [ ] reducing lower extremity fractures [ ] . positive effects were also reported for an education programme for the home visitors conducting the phvs, in terms of lower admission rates to nursing homes for those receiving two home visits per year [ ] . the most promising results on phvs were established in the "elderly persons in the risk zone" study where a single home visit was evaluated and showed positive effects adls [ ] , frailty and fear of falling [ ] , life satisfaction and morbidity [ ] . this study was the only one, among phv interventions, to conduct a -year follow up at which some effects persisted and thus validates post-intervention effects [ ] . the positive effects of phvs in the "elderly persons in the risk zone" study are partly explained by the experiences of participants, who felt empowered and in control as a result of the information given and having the opportunity to discuss health-related matters with a qualified professional [ ] . however, these findings on long-term effects are in contrast to a previous phv trial that indicated that intervention effects remained only for as long as the home visits were ongoing [ ] , and thus, highlights the importance of long term follow-ups over. conflicting results regarding specific effects of pvhs and their health-economic effects have been reported also in a recent report from sbu enquiry service (swedish agency for health technology assessment and assessment of social services) about preventive home visits, also referred to from the swedish national board on health and welfare [ ] . in some studies, though, phvs have shown to be cost-effective while annual follow-up visits can be potentially even more costeffective. such findings have been established when conducting health economic analysis based on data from the elderly persons in the risk zone [ ] as well as in a previous swedish study including twice-annual home visits over a period for two years [ ] . despite the conflicting results on some outcome effects of phvs [ ] , they still can be considered a good alternative to group-based interventions, e.g. senior meetings, since not all potential participants can or like to engage in a group format. interventions including exercise or combining exercise with other components (e.g. medication review, guidance on nutrition, cessation of alcohol and smoking, home hazard assessment and modifications) showed to be promising for preventing falls. findings on these interventions showed improvements in different factors related to falls risk and physical functioning, e.g. muscle strength, mobility, balance or self-rated health [ , , , ] which could indirectly lead to fall reduction [ ] . positive effects were observed for both home-based [ ] and group-based interventions [ ] , regardless of whether they were shorter ( months) [ ] or longer ( year) in duration [ ] . furthermore, interventions including more frequent group sessions reported additional effects, such as improvement in motivation to continue with physical activity [ , ] , and perhaps consequently a reduction in injurious falls and fractures, as reported in two fall prevention interventions [ , ] . both interventions included balance exercise in combination with resistance/strength exercise provided over one year or longer, but varied in terms of content, number of sessions, and delivery approaches used e.g. multifactorial [ ] and multiple components [ ] . in line with evidence from a recent systematic review and metaanalysis, exercise-based interventions, aiming to improve balance and strength, are one of the most feasible and cost-effective approaches to prevent falls among older people living in the community [ ] . this approach has also been integrated into some current swedish guidance, on physical training, balance and more, issued from the national board on health and welfare in the form of training for professionals working with older people and fall prevention [ ] . however, effectiveness of exercisebased interventions is dependent on the uptake and longterm adherence [ ] . groups sessions led by professionals over a longer period ( year or more) seems to affect this aspect positively but can be costly, foremost in terms of human resources needed if provided to a large population of older people. since group training might not be the solution for all, other effective alternatives such as multifactorial interventions could work in these cases. also, multifactorial interventions have shown positive effects on preventing falls [ ] and could be considered an alternative to exercise-based interventions. nonetheless, no health-economic evaluation was identified for these interventions, and thus, still makes them less robust in terms of cost-effectiveness. while our results, indicate that there are several healthpromoting and preventive interventions that could improve health and well-being among community-dwelling older people, implementation needs to be considered, not only in relation to effects but also concerning the resources available, i.e. how limited resources can be used in a way that yields the largest health benefits [ , ] and other feasibility aspects such as reach in the population; a key factor for successful implemtation of research in practice. health economic evaluations, including evaluation of both costs and effects, can provide such important information. however, in this scoping review, only four health economic evaluations were identified, indicating a general lack of information to guide decision making. however, information regarding intervention content, e.g. duration and intensity of interventions, can at least provide some information about the resources required. regarding individual interventions, the study by dahlin-ivanoff et al. included one single preventive home visit requiring one and a half to two hours of a professional's time [ ] in contrast to the study by möller et al. in which a case management intervention, required at least one hour per month during a -month intervention of professional's time [ ] . similarly, for group-based interventions, the span for the time required was two hours for a one session discussion group [ ] , to two and a half hours per week over the course of one year [ ] . while these examples all include interventions with some positive effect, the time for which staff need to be allocated differs substantially. even though these examples lack information on other types of costs that can be affected by interventions (e.g. social care consumption), they provide some guidance on which resources are needed and the magnitude of staffing which is a central cost of a healthpromoting or preventive intervention [ ] . despite a growing literature of health-promoting and preventive interventions that have shown positive effects in well-controlled trials, the translation of such trials to practice has proven to be challenging [ ] . evidence has shown that feasibility or pilot studies are important to ensure effective practical implementation and to decrease threats to validity of health outcomes [ ] . however, in our literature search, there was a lack of piloting and feasibility studies. in the absence of feasibility or pilot studies, other reported aspects such as information on study participation rates and adherence could indicate the degree to which an intervention reaches out to the target population, and thus, increase chances of a successful translation of research evidence into clinical practice [ ] . reaching older people with health promotion is crucial for achieving a health impact for the whole population, but has also been shown to be challenging [ , ] . findings from all original studies, in this review, showed that approximately a third of the persons eligible declined to participate due to different reasons, i.e. being too sick or too healthy [ ] . qualitative data on experiences of participants could to some extent reveal why an intervention is or is not appealing to larger groups of older people, however, only a few studies on experiences of participants were identified in this review. while this review provides some guidance on which interventions have shown positive health effects in a nordic context, future research is needed on how to translate evidence into practice, e.g. through exploring alternative ways of reaching out to a larger population and incorporating support for behaviour change and adherence in the long-term. some examples of new promising approaches explored in this review were wii training [ ] and physical activity counselling [ ] . the digital approaches used through video training or self-care telephone calls are potentially feasible to be implemented considering the more limited resources required to implement them, e.g. the smaller number of direct personal contacts needed with providers of health care for older people while still resulting in positive effects. in light of the ongoing coronavirus pandemic and related measures of social distancing, the importance of addressing loneliness and isolation among older people is accentuated. digital approaches to delivering effective interventions could complement the challenge of isolation and the need to reach out to a higher number of older people. for example, using smartphones and tablets may be a potentially cost-effective way to increase reach in the population. at present, there is a big supply of smartphone applications for exercise, however, most lack evidence regarding their scientific and implementation validity in the older population. research in the area is, however, developing and one example is an ongoing large clinical trial on digital fall prevention in sweden [ ] . finally, in discussing the results of this study, it is notable that some important aspects of healthy ageing, were less frequently evaluated. only two studies focussed on mental wellbeing and social participation, one showed some effects in reducing loneliness [ ] and the other in improving general mental health [ ] . this gap in research has also been supported in other reviews, where promoting wellbeing and mental health have shown to be both effective and potentially cost-effective [ , ] , and should, therefore, be further researched. the scope of this review was broad. it included information on several factors extracted from all identified original and their related studies, and therefore provides an overview of the knowledge base in the field of healthpromoting and preventive interventions in the nordic countries. given the broad scope of this review, we choose to not include some information, e.g. data concerning when studies were performed or adverse events, which could be seen as a limitation of the study. data concerning when studies were performed would enrich information on the context and content of the interventions. however, the description of the study period, e.g. the period for the recruitment of participants, have not been reported consistently among all studies, therefore might not have produced many data. although a wide range of outcome effects was extracted, important information on adverse events was not extracted and beyond the scope of this study, guided primarily by the mrc guidelines. additionally, recent systematic reviews show that adverse events, for example, concerning fall prevention programmes seem to be rather poorly reported hence, would probably not make a significant difference in our conclusions, if included in the analysis [ , ] . another important factor to consider, which may lead to better developed and evaluated interventions, is if the studies have a theoretical foundation that may explain the causal link between intervention and outcomes [ ] . however, considering the already broad focus of this review, we choose to limit the presentation of results and not include data on the theoretical foundations for each intervention. furthermore, the quality of the included studies has not been evaluated the same way it would be assessed in a systematic review, meaning that the quality can differ between the studies. it is, however, in line with prisma guidelines on scoping reviews considering this step optional [ ] . yet a quality assessment of the included studies or grading of evidence might have led to stronger conclusions as a result of a reduction in uncertainty related to outcome effects. finally, this review did not include studies from the rest of the world, albeit such studies could have provided relevant information. the choice to do so was due to the importance of contextual factors concerning complex interventions [ ] . limiting the inclusion of interventions deriving from countries with similar welfare models and cultural context might increase chances of effective implementations of promising interventions. furthermore, research shows that there is is often a lack of information regarding the influence of the context when conducting and evaluating complex interventions [ ] . thus, more research on the influence of contextual factors in the effectiveness of certain interventions would add to the knowledgebase important for decision-makers. this scoping review, following the mrc guidelines, provides an overview of the evidence and evidence gaps of health-promoting and preventive intervention studies for community-dwelling older people in nordic countries hence, of importance for decision-makers, research councils and researchers. all interventions, besides one, showed positive effects on at least one health outcome, although the magnitude of effects and number of follow-ups differed substantially. given that evidence on effects alone are not enough information for decision-makers, information on other factors is needed. overall, there was a general lack of studies related to costeffectiveness, experiences of participants and feasibility. therefore, such studies are strongly warranted. in all, based on the evidence presented, senior meetings, preventive home visits and exercise interventions alone or combined with other components seem to be strong candidates for implementation in a swedish municipality context. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . search strategies and numbers of records identified in each database. additional file . preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews (prisma-scr) checklist. world health organization (who). health promotion glossary. geneva: who the ottawa charter for health promotion satisfaction in everyday life for frail -year-old adults: a danish population study costs explained by function rather than diagnosis--results from the snac nordanstig elderly cohort in sweden socialtjänstlag (sfs : ). the swedish parliament world report on ageing and health health-promoting interventions for persons aged and older are successful in the short term--results from the randomized and three-armed elderly persons in the risk zone study occupation-focused interventions for well older people: an exploratory randomized controlled trial health promotion interventions for older people living alone: a systematic review a multi-component exercise regimen to prevent functional decline and bone fragility in home-dwelling elderly women: randomized, controlled trial. osteoporosis int home visits to prevent nursing home admission and functional decline in elderly people: systematic review and meta-regression analysis exercise for preventing falls in older people living in the community. cochrane database syst rev occupation-focused health promotion for well older people -a cost-effectiveness analysis economic evaluation of health promotion and primary prevention actions for older people-a systematic review health promotion can postpone frailty: results from the rct elderly persons in the risk zone self-care telephone talks as a health-promotion intervention in urban home-living persons + years of age: a randomized controlled study effectiveness of the chaos falls clinic in preventing falls and injuries of home-dwelling older adults: a randomised controlled trial effects of a oneyear home-based case management intervention on falls in older people: a randomized controlled trial scoping review of health promotion and disease prevention interventions addressed to elderly people why don't we see more translation of health promotion research to practice? rethinking the efficacy-toeffectiveness transition decision making in health and medicine: integrating evidence and values missing data: a special challenge in aging research health promotion interventions for community-dwelling older people with mild or pre-frailty: a systematic review and meta-analysis criteria for evaluating evidence on public health interventions scoping studies: towards a methodological framework guidance for conducting systematic scoping reviews prisma extension for scoping reviews (prisma-scr): checklist and explanation developing and evaluating complex interventions: the new medical research council guidance cochrane handbook for systematic reviews of interventions international classification of functioning, disability and health (icf) world health organization methods for the economic evaluation of health care programmes old women with a recent fall history show improved muscle strength and function sustained for six months after finishing training a -year combined weight-bearing training program is beneficial for bone mineral density and neuromuscular function in older women fall prevention by nursing assistants among community-living elderly people. a randomised controlled trial effects of new, individually adjusted, progressive balance group training for elderly people with fear of falling and tend to fall: a randomized controlled trial effects of home exercises and group training on functional abilities in home-dwelling older persons with mobility and balance problems. a randomized study does a falls prevention program impact perceived participation in everyday occupations? a pilot randomized controlled trial efficacy of nintendo wii training on mechanical leg muscle function and postural balance in community-dwelling older adults: a randomized controlled trial the otago exercise program performed as group training versus home training in fall-prone older people: a randomized controlled trial prevention of fall injuries requiring hospital treatment among community-dwelling elderly a multifactorial fall prevention programme in home-dwelling elderly people: a randomized-controlled trial study protocol for prevention of falls: a randomized controlled trial of effects of vitamin d and exercise on falls prevention design of a multidimensional, health-promoting, randomised three-armed controlled trial for "prefrail" people of + years living at home a person-centred approach to health promotion for persons + who have migrated to sweden: promoting aging migrants' capabilities implementation and rct study protocol effect of a social intervention of choice vs. control on depressive symptoms, melancholy, feeling of loneliness, and perceived togetherness in older finnish people: a randomized controlled trial effects of a physical and nutritional intervention program for frail elderly people over age . a randomized controlled pilot treatment trial motivational characteristics and resistance training in older adults: a randomized controlled trial and -year follow-up benefits of home-based rocking-chair exercise for physical performance in community-dwelling elderly women: a randomized controlled trial home-based video exercise intervention for community-dwelling frail older women: a randomized controlled trial effect of physical activity counseling on disability in older people: a -year randomized controlled trial effects of comprehensive geriatric intervention on physical performance among people aged years and over prevention of disability by exercise among the elderly: a population-based, randomized, controlled trial preventive home visits to older people in denmark: methodology of a randomized controlled study effects of exercise on health-related quality of life and fear of falling in home-dwelling older women exercise and vitamin d in fall prevention among older women a randomized clinical trial effects of different strength training frequencies on maximum strength, body composition and functional capacity in healthy older individuals task-specific balance training improves self-assessed function in community-dwelling older adults with balance deficits and fear of falling: a randomized controlled trial long-term effects of new progressive group balance training for elderly people with increased risk of falling -a randomized controlled trial home training with and without additional group training in physically frail old people living at home: effect on health-related quality of life and ambulation the efficacy of a multifactorial falls-prevention programme, implemented in primary health care thinking and acting in a new way: influences of a falls-prevention program on participants' everyday life. physical occup ther geriatr letting go of an old habit: group leaders' experiences of a client-centred multidisciplinary falls-prevention programme maintenance of exercise-induced benefits in physical functioning and bone among elderly women combined resistance and balance-jumping exercise reduces older women's injurious falls and fractures: -year follow-up study effects of risk-based multifactorial fall prevention on postural balance in the community-dwelling aged: a randomized controlled trial effects of risk-based multifactorial fall prevention on health-related quality of life among the community-dwelling aged: a randomized controlled trial. health qual life outcomes increased muscle strength improves managing in activities of daily living in fall-prone communitydwelling older women effects of risk-based multifactorial fall prevention program on maximal isometric muscle strength in community-dwelling aged: a randomized controlled trial the long-term effect of a multifactorial fall prevention programme on the incidence of falls requiring medical treatment the effects of multifactorial fall prevention on depressive symptoms among the aged at increased risk of falling effect of a riskbased multifactorial fall prevention program on the incidence of falls costeffectiveness of vitamin d supplementation and exercise in preventing injurious falls among older home-dwelling women: findings from an rct a -year follow-up after a -year rct with vitamin d and exercise: effects on falls, injurious falls and physical functioning among older women effects of a multimodal exercise program on physical function, falls, and injuries in older women: a -year community-based, randomized controlled trial preventive home visits and health--experiences among very old people multi-professional and multi-dimensional group education -a key to action in elderly persons physical function and fear of falling years after the health-promoting randomized controlled trial: elderly persons in the risk zone positive health outcomes following health-promoting and disease-preventive interventions for independent very old persons: long-term results of the three-armed rct elderly persons in the risk zone minor positive effects of health-promoting senior meetings for older community-dwelling persons on loneliness, social network, and social support for whom is a health-promoting intervention effective? predictive factors for performing activities of daily living independently long-term outcome for adl following the health-promoting rctelderly persons in the risk zone understanding the "black box" of a health-promotion program: keys to enable health among older persons aging in the context of migration bridging barriers to health promotion: a feasibility pilot study of the 'promoting aging migrants' capabilities study promoting aging migrants' capabilities: a randomized controlled trial concerning activities of daily living and self-rated health supporting decisionmaking by a health promotion programme: experiences of persons ageing in the context of migration distribution and evaluation of sense of coherence among older immigrants before and after a health promotion intervention -results from the rct study promoting aging migrants' capability viewing oneself as a capable person -experiences of professionals working with older finnish immigrants the use of case management for community-dwelling older people: the effects on loneliness, symptoms of depression and life satisfaction in a randomised controlled trial effects on leisure activities and social participation of a case management intervention for frail older people living at home: a randomised controlled trial cost-utility analysis of case management for frail older people: effects of a randomised controlled trial case management for frail older people -a qualitative study of receivers' and providers' experiences of a complex intervention case managers for frail older people: a randomised controlled pilot study effects on healthcare utilization of case management for frail older people: a randomized controlled trial (rct) effects of a physical training and nutritional intervention program in frail elderly people regarding habitual physical activity level and activities of daily living-a randomized controlled pilot study effects of physical training on aerobic capacity in frail elderly people ( + years). influence of lung capacity, cardiovascular disease and medical drug treatment: a randomized controlled pilot trial effects of nutritional intervention and physical training on energy intake, resting metabolic rate and body composition in frail elderly. a randomised, controlled pilot study long-term effect of physical activity counseling on mobility limitation among older people: a randomized controlled study customer-oriented counseling for physical activity in older people: study protocol and selected baseline results of a randomized-controlled trial (isrctn ) effects of comprehensive health assessment and targeted intervention on chair rise capacity in active and inactive community-dwelling older people effects of comprehensive geriatric assessment-based individually targeted interventions on mobility of pre-frail and frail community-dwelling older people pragmatic exercise-oriented prevention of falls among the elderly: a population-based, randomized, controlled trial cost effectiveness of preventive home visits to the elderly: economic evaluation alongside randomized controlled study a feasible model for prevention of functional decline in older home-dwelling people-the gp role. a municipality-randomized intervention trial educational intervention toward preventive home visitors reduced functional decline in communityliving older women structured home visits to older people. are they only of benefit for women? a randomised controlled trial preventive home visits to older home-dwelling people and different functional decline patterns effects of comprehensive geriatric assessment and targeted intervention on mobility in persons aged years and over: a randomized controlled trial assessment of postural balance in community-dwelling older adults -methodological aspects and effects of biofeedback-based nintendo wii training modelling long-term cost-effectiveness of health promotion for communitydwelling older people exploring reach and experiences of participation in health-promoting senior meetings in a municipality context preventive home visits to older people are cost-effective preventive home visits to older people preventive home visits for mortality, morbidity, and institutionalization in older adults: a systematic review and meta-analysis exercise to prevent falls in older adults: an updated systematic review and meta-analysis reporting of complex interventions in clinical trials: development of a taxonomy to classify and describe fall-prevention interventions ett fall för teameten utbildning om att förebygga fallolyckor preventing falls among older fallers: study protocol for a two-phase pilot study of the multicomponent live life program multifactorial and multiple component interventions for preventing falls in older people living in the community nuts and bolts of conducting feasibility studies evaluating the public health impact of health promotion interventions: the re-aim framework effectiveness of a self-managed digital exercise programme to prevent falls in older community-dwelling adults: study protocol for the safe step randomized controlled trial mental health interventions among older adults: a systematic review mental wellbeing of older people: making an economic case. australian e-journal for the advancement of mental health taking account of context in population health intervention research: guidance for producers, users and funders of research publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations our thanks to umeå university library for assisting with advice in performing the search strategy for the literature. we also thank shion gosrani (public health support officer at north tyneside council) for proofreading the manuscript for english language. authors' contributions sb was involved in designing the search strategy, executing the search strategy, assessing studies for inclusion, extracting, classifying and presenting the data, writing and editing the manuscript. mz was involved in assessing studies for inclusion, extracting, classifying, and presenting the data, writing, revising and commenting the manuscript. ms was involved in assessing studies for inclusion, contributing in presenting the data, revising and commenting the manuscript. sb, mz, ms read and approved the final version of the manuscript. work with this study was included in the ordinary work of the three authors. salary of the doctoral student is partially financed by umeå university's industrial doctoral school for research and innovation (ids). open access funding provided by university of umea. all data analysed during this study are included in this published article and its additional files. the search strategy is available in additional file . prisma extensions for scoping reviews-checklist is included in additional file .ethics approval and consent to participate not applicable. not applicable. author details key: cord- -qj zu authors: nan title: spezielle arzneimitteltherapie in der schwangerschaft date: - - journal: arzneiverordnung in schwangerschaft und stillzeit doi: . /b - - . - sha: doc_id: cord_uid: qj zu nan wie die meisten anderen medikamente ist auch paracetamol plazentagängig. anfänglich wurde aufgrund einzelner fallberichte ein teratogenes potenzial beim menschen vermutet. auch in den vergangenen jahren wurden toxische auswirkungen auf das ungeborene diskutiert: eine assoziation von gastroschisis mit einer mütterlichen kombinationsmedikation aus paracetamol und pseudoephedrin im . trimenon fanden werler und mitarbeiter ( ) bei retrospektiver auswertung von erkrankten säuglingen. kein zusammenhang konnte zwischen mütterlicher paracetamoleinnahme und ventrikelseptumdefekten festgestellt werden (cleves ) . eine neuere studie diskutiert ein möglicherweise erhöhtes risiko von paracetamol in der spätschwangerschaft für asthma bronchiale erhöhte ige-spiegel im vorschulalter (shaheen , shaheen . abgesehen von methodischen mängeln der studie erscheint dieser zusammenhang biologisch wenig plausibel. alle vorliegenden daten zusammengefasst, gibt es beim menschen keine ernsthaften hinweise auf teratogenität (Übersicht in . zur Überdosis bei suizidversuchen siehe kapitel . . . . die an lymphozyten beobachteten diskreten genotoxischen effekte (hongslo ) scheinen keine klinische relevanz zu besitzen. empfehlung für die praxis: paracetamol ist das analgetikum und antipyretikum der wahl. es kann in jeder phase der schwangerschaft innerhalb des üblichen dosisbereichs eingesetzt werden. die thrombembolieprophylaxe genutzt. die analgetische, antipyretische und antiphlogistische wirkung erfolgt über eine hemmung der prostaglandinsynthese bei einzeldosen ab mg. aufgrund der geringen therapeutischen breite im antiphlogistischen bereich (tagesdosen von . mg und darüber) wurde acetylsalicylsäure als antirheumatikum weitgehend durch die neueren, nichtsteroidalen antirheumatika (nsar) verdrängt. salicylate sind lipophil, sie werden nach oraler gabe rasch resorbiert und gelangen leicht über die plazenta zum fetus. die metabolisierung und eliminierung durch kopplung an glucuronsäure in der leber erfolgt beim fetus und beim neugeborenen nur langsam wegen der noch verminderten enzymaktivität und der geringen glomerulären filtrationsrate. "low-dose"-therapie. niedrig dosiert mit - mg pro tag wird acetylsalicylsäure als thrombozytenaggregationshemmer zur thrombembolie-prophylaxe eingesetzt und in manchen fällen zur prävention einer präeklampsie verordnet. außerdem diskutiert man den nutzen niedriger dosen zur prävention von abortneigung und anderen schwangerschaftskomplikationen bei frauen mit anti-kardiolipin-oder anti-phospholipid-antikörpern mit oder ohne systemischem lupus erythematodes (backos ) . in einer prospektiven studie mit jeweils schwangeren in fall-und kontrollgruppe, mit mindestens aborten in der anamnese bzw. nachweisbaren autoantikörpern konnte keine verringerung der abortrate unter acetylsalicylsäure in kombination mit prednison im vergleich zu placebo festgestellt werden. es fand sich jedoch in der behandlungsgruppe ein signifikant höheres risiko für eine frühgeburt (laskin ) . eine meta-analyse fand gegenüber placebo ebenfalls keine reduzierung des abortrisikos, aber ein signifikant geringeres risiko für eine frühgeburt (kozer , kozer . eine erhöhung der schwangerschaftsrate durch eine kombinierte therapie mit prednison und acetylsalicylsäure konnte bei kinderwunschpatientinnen mit nachweisbaren autoantikörpern und wiederholt erfolglosen in-vitro-fertilisationen (ivf) erzielt werden (geva , geva . die untersuchung einer vergleichbaren therapie bei patientinnen mit intrauteriner insemination (iui) konnte dieses ergebnis beim vergleich mit einer nicht behandelten gruppe bestätigen (hsieh ) . viele untersuchungen haben sich mit dem nutzen einer "low-dose"-behandlung zur prävention einer plazentationsstörung mit daraus resultierendem schwangerschaftshochdruck und intrauteriner wachstumsverzögerung beschäftigt. umfassend wurde dieses thema von der "collaborative low-dose aspirin in pregnancy study" (clasp ) an insgesamt . frauen untersucht. im gegensatz zu früheren ergebnissen sind eindeutige vorteile wohl nur bei schwangeren mit einer sich früh entwickelnden präeklampsie -vor schwangerschaftswochen (ssw) -und mit pathologischer vorgeschichte zu erwarten. ren und wurden durch paracetamol und andere analgetika verdrängt. pyrazolon-und phenylbutazonverbindungen besitzen eine prostaglandinantagonistische wirkung, die ab schwangerschaftswoche - einen vorzeitigen ductus-arteriosus-verschluss beim fetus auslösen kann. ein fallbericht beschreibt eine schwangere, die aufgrund einer nierenkolik kurz vor ende der schwangerschaft hoch dosiert mit metamizol behandelt wurde und darunter ein oligohydramnion entwickelte (catalan ) . eine brasilianische studie berichtet über einen von anderen autoren bisher nicht bestätigten zusammenhang zwischen metamizol-einnahme durch die mutter und dem vermehrten auftreten von wilms-tumoren bei den kindern (sharpe ) . in einer retrospektiven studie hatte bei kindern mit akuter leukämie im alter von x monaten ein höherer anteil der mütter metamizol in der schwangerschaft eingenommen als in der gesunden kontrollgruppe. daraus wurde ein signifikant erhöhtes risiko für eine frühkindliche leukämie nach metamizol-therapie in der schwangerschaft abgeleitet (alexander ) . diese schlussfolgerung sollte jedoch aufgrund der geringen fallzahl und der unzureichenden informationen über den zeitpunkt der exposition sehr kritisch bewertet werden. im Übrigen liegen keine hinweise auf embryotoxische eigenschaften beim menschen vor. eine prospektive untersuchung von im . trimenon mit metamizol behandelten schwangeren fand im vergleich zur kontrolle kein signifikant erhöhtes risiko für große fehlbildungen (bar-oz ) . dieses ergebnis kann nach sichtung von eigenen prospektiv erfassten metamizol-expositionen im . trimenon bestätigt werden. zu propyphenazon liegen in unserer eigenen datenbank prospektiv erfasste fälle mit mütterlicher therapie im . trimenon vor. von diesen schwangerschaften endeten mit abbruch, mit einem spontanabort und mit einer lebendgeburt. es fand sich eine große fehlbildung (neuralrohrdefekt) bei einem abortierten fetus. insgesamt ergibt sich daraus kein anhalt für ein teratogenes risiko. zu phenazon und propyphenazon liegen keine ausreichend dokumentierten erfahrungen zur anwendung in der schwangerschaft vor. phenylbutazon (z. b. ambene ® ) und verwandte verbindungen wie famprofazon, kebuzon, mofebutazon (z. b. mofesal ® ) und oxyphenbutazon sind schwache analgetika und antipyretika, die über eine hemmung der prostaglandinsynthese stark antiphlogistisch wirken. phenylbutazon ist bei morbus bechterew indiziert. phenylbutazonverbindungen können die blutbildung schädigen, sie führen zu einer flüssigkeitsretention und kumulieren stark (halbwertszeit - stunden). diese eigenschaften sind in der schwangerschaft unerwünscht. im tierversuch wirkt phenylbutazon teratogen. zur beurteilung embryotoxischer effekte beim menschen liegen keine ausreichenden daten vor, ein erhebliches teratogenes potenzial erscheint unwahrscheinlich. durch den prostaglandinantagonismus können phenylbutazonverbindungen ebenso wie acetylsalicylsäure und andere nsar einen vorzeitigen verschluss des ductus arteriosus verursachen, wenn nach woche - behandelt wird (Überblick in . empfehlung für die praxis: auf den einsatz von metamizol, phenazon und propyphenazon sollte verzichtet werden. analgetikum der wahl ist paracetamol, in bestimmten fällen auch in kombination mit codein. nach heutiger kenntnislage ergibt sich aus einer dennoch erfolgten exposition mit einem der genannten mittel im . trimenon keine risikosituation, die weitergehende diagnostik erfordert oder in der ein risikobegründeter abbruch einer gewünschten und intakten schwangerschaft erwogen werden müsste (siehe kapitel . ). zusätzliche untersuchungen wie dopplersonographische kontrollen des ductus arteriosus sollten eingeplant werden, wenn mit diesen medikamenten nach woche behandelt wurde. g . . opiate sind zentral wirksame, starke analgetika, die in ihrer wirkung dem morphin, dem hauptalkaloid des opiums, vergleichbar sind und ebenfalls zur abhängigkeit und beim neugeborenen zu atemdepression und entzugssymptomatik führen können. bei den opiaten unterscheidet man reine agonisten (endorphine, morphin und therapeutisch ähnliche opiate) von reinen antagonisten (naloxon) und stoffen, die sowohl agonistische als auch antagonistische eigenschaften besitzen (pentazocin). hinsichtlich des toxischen potenzials in der schwangerschaft ist die kurzfristige therapeutische gabe von opiaten, z. b. in der perinatalphase, anders zu bewerten als der opiatabusus (siehe auch abschnitt . . ). körpereigene endorphine reagieren spezifisch mit opiatrezeptoren und können morphinwirkungen auslösen. systematische untersuchungen zur teratogenität von morphin (z. b. capros ® ) oder hydromorphon (z. b. dilaudid ® ) liegen nicht vor. es gibt jedoch bislang keine hinweise, dass diese substanzen fehlbildungen beim menschen verursachen. ein fallbericht mit intrathekaler langzeitbehandlung mit morphin wegen chronischer schmerzen beschreibt ein gesundes neugeborenes mit normalen apgar-werten, ohne entzugssymptomatik und normaler entwicklung im alter von monaten (oberlander ) . bei prospektiv erfassten fällen mit langzeitbehandlung aus dem eigenen datenbestand finden sich frühgeborene sowie ein reif geborenes kind mit entzugssymptomatik. fehlbildungen waren nicht nachweisbar. die einmalige intramuskuläre applikation von - mg morphin nach schwangerschaftswochen führte zu einer reduzierung der fetalen atembewegungen bei insgesamt nicht reduzierten kindsbewegungen. es wurde ein fetal-mütterlicher plasmaquotient von , ermittelt ( kopecky ) . appliziert. in der nabelschnur finden sich - % der mütterlichen plasmakonzentration. bei ausreichendem zeitlichem abstand zur entbindung scheint das risiko einer neonatalen atemdepression gering zu sein. im vergleich zu einer kontrollgruppe fanden sich bei den kindern von mit fentanyl behandelten müttern keine unterschiede bei atemdepression, apgar-score, naloxonbedarf sowie verschiedenen neurologischen parametern bis zu stunden nach geburt (rayburn ) . die letzte fentanyldosis wurde in dieser untersuchung im durchschnitt minuten vor der entbindung gegeben. in einer weiteren publikation waren in einer gruppe von neugeborenen ebenfalls weder atemdepression noch neurologische abweichungen in den ersten stunden nach der geburt zu beobachten. die applikation von fentanyl erfolgte jeweils etwa minuten vor der schnittentbindung (eisele ) . die reif geborenen, gesunden neugeborenen von frauen, die unter der geburt fentanyl epidural erhielten, zeigten keine atemdepression. die autoren diskutieren, dass eine epiduralanästhesie mit fentanyl im hinblick auf den stillerfolg der kinder wahrscheinlich günstiger ist als eine i.v.-applikation im rahmen einer allgemeinnarkose (jordan ) . andere untersucher beobachteten, dass eine patientenkontrollierte analgesie mit fentanyl i.v. unter der geburt gleich gut verträglich für das neugeborene ist wie eine epiduralanästhesie (nikkola ) . ein fallbericht beschreibt die anwendung von fentanylpflastern mit einer transdermalen dosis von ca. ? g/stunde während der gesamten schwangerschaft. das gesunde reif geborene kind wies normale apgar-werte auf. nach stunden entwickelte sich jedoch eine leichte entzugssymptomatik mit Übererregbarkeit und schreiattacken, die nach tagen abgeklungen war. die kindlichen blutspiegel wiesen unmittelbar nach der geburt ein drittel der mütterlichen werte auf und einen tag nach entbindung nur noch % ( regan ) . weder unsere eigenen daten zu frauen mit fentanylexposition im . trimenon noch beobachtungen anderer autoren ergeben hinweise auf teratogenität. fentanyl wurde in fetalen organen in der frühschwangerschaft nachgewiesen (cooper ) . mehrere veröffentlichungen beschreiben die intravenöse und epidurale anwendung von alfentanil (rapifen ® ) in der geburtshilfe (Übersicht bei briggs , gin ) . die verträglichkeit für das neugeborene scheint der des fentanyls zu gleichen. ein untersucher hat geringe neuromuskuläre funktionsabweichungen in den ersten minuten nach geburt ermittelt, in der nabelschnur betrug die konzentration ca. % der mütterlichen werte. in einigen neueren studien wurde remifentanil zur schmerzreduktion unter der geburt eingesetzt. dabei wurde in einer der studien über häufige therapieabbrüche wegen starker nebenwirkungen bei der mutter berichtet, wie z. b. Übelkeit, erbrechen, atemdepression oder juckreiz. nebenwirkungen beim neugeborenen fanden sich in keiner der studien (Übersicht in . die anwendung von sufentanil zur analgesie bei frauen unter der geburt ergab eine deutlich geringere rate an hypotonien im vergleich zu anderen analgesieverfahren, aber signifikant mehr schwankungen der fetalen herzfrequenz bzw. eine häufiger auftretende fetale bradykardie (van de velde ) . berichte über teratogene effekte liegen weder für alfentanil vor noch für remifentanil (ultiva ® ) und sufentanil (sufenta ® ). für eine endgültige bewertung des teratogenen risikos sind die vorliegenden daten jedoch unzureichend. empfehlung für die praxis: bei gegebener indikation dürfen fentanyl und ggf. auch die anderen präparate in jeder phase der schwangerschaft eingesetzt werden. bei verabreichung kurz vor der entbindung muss wie bei allen analgetika vom opiattyp mit einer atemdepressiven wirkung beim neugeborenen gerechnet werden. bei rückenmark-nahen analgesieverfahren sind auswirkungen auf den kreislauf der mutter (hypotonie) zu vermeiden. g . . andere narkoanalgetika und zentral wirksame analgetika pharmakologie und toxikologie. pentazocin (fortral ® ) wurde (in den usa) in kombination mit dem antihistaminikum tripelenamin unter dem namen t's and blues als intravenös injizierbare droge gehandelt. tierexperimentell erwies sich diese kombination als nicht teratogen. intrauterine wachstumsverzögerung und verhaltensauffälligkeiten sind jedoch bei ratten nach pränataler applikation gehäuft aufgetreten. vergleichbare effekte haben sich auch beim menschen nach gebrauch dieser droge gezeigt. berichte zur therapeutischen anwendung fehlen. bei wiederholter einnahme bis zum ende der schwangerschaft muss mit opiattypischen entzugssymptomen gerechnet werden, wie z. b. unruhe, zittrigkeit, muskelhypertonus, diarrhö und erbrechen. pentazocin kann den uterustonus erhöhen (Übersicht in . pentazocin hat sich ebensowenig wie tilidin (in valoron n ® ) gegenüber pethidin in der geburtshilfe durchsetzen können. hinweise auf ein teratogenes potenzial beim menschen gibt es zu beiden substanzen bisher nicht. tramadol (z. b . tramal ® ) ist in deutschland eines der meistverschriebenen opioidanalgetika. seine analgetische wirkung entspricht der von codein und liegt damit bei einem zehntel der wirkstärke vom morphin. im gegensatz zu morphin hat es in äquianalgetischen dosen jedoch keine deutliche atemdepressive wirkung. tramadol wird von drogenabhängigen missbraucht. bei der anwendung unter der geburt war tramadol sowohl hinsichtlich der schmerzreduktion als auch der mütterlichen nebenwirkungen dem pethidin unterlegen. unterschiede in der neonatalen entwicklung fanden sich jedoch nicht (keskin ) . in einer meta-analyse der cochrane database gab es keinen unterschied in der wirksamkeit beider substanzen ( elbourne ) . in unserer datenbank befinden sich prospektiv erfasste schwangerschaften mit tramadoltherapie im . trimenon. unter den lebendgeborenen hatten kinder große fehlbildungen ( , %), davon vorhofseptumdefekte, eine transposition der großen gefäße, eine meningomyelozele mit hydrozephalus und spina bifida und ein kind mit angeborenem katarakt und mikrophthalmus. in mindestens einem fall hatte die mutter zusätzlich teratogene medikamente (u. a. carbamazepin) eingenommen. ein verdacht auf embryotoxische effekte lässt sich mit diesen daten nicht untermauern, zumal von anderer seite bisher weder zu tramadol noch zu anderen opioidanalgetika substantielle hinweise auf teratogenität beim menschen geäußert wurden. Über embryotoxizität wurde bisher auch nicht im zusammenhang mit buprenorphin (temgesic ® ; siehe kapitel . . ), dextropropoxyphen, flupirtin (katadolon ® ), meptazinol (meptid ® ), nalbuphin (nubain ® ), nefopam (z. b. silentan ® ) und piritramid (dipidolor ® ) berichtet. systematische untersuchungen zur teratogenität fehlen jedoch. alle morphinähnlichen opiate können abhängig von behandlungsintervall und dosis zur atemdepression beim neugeborenen und zu entzugserscheinungen führen. dies gilt insbesondere für die substitution nach drogenabusus (siehe auch abschnitt . . ). neonatale entzugserscheinungen können ebenso wie beim heroin ggf. erst verzögert einsetzen. empfehlung für die praxis: bei entsprechender indikation kann mit erprobten vertretern aus dieser arzneigruppe wie tramadol oder auch buprenorphin in der schwangerschaft behandelt werden. als schmerzmittel sollten jedoch paracetamol (ggf. mit codein) oder (bis woche ) ibuprofen bevorzugt werden. nach heutiger kenntnislage ergibt sich aus einer exposition mit anderen in diesem abschnitt genannten mitteln keine risikosituation, die weitergehende diagnostik erfordert oder einen risikobegründeten abbruch einer gewünschten und intakten schwangerschaft (siehe kapitel . ). g . . naloxon pharmakologie und toxikologie. naloxon (z. b. narcanti ® ) ist in der lage, die atemdepressorische wirkung von opiaten aufzuheben. bei kindern, deren mütter in der schwangerschaft opiatabusus betrieben haben, kann naloxon entzugserscheinungen verursachen. ein teratogenes potenzial wurde beim menschen bisher nicht beschrieben. den sich große fehlbildungen, darunter vorhofseptumdefekte, einmal kombiniert mit einer pulmonalklappenstenose, sowie eine spina bifida, eine lippen-gaumen-spalte und eine komplexe skelettfehlbildung. zusammenfassend ergibt sich daraus kein anhalt für ein erhöhtes abort-oder gesamtfehlbildungsrisiko. systematische studien zur anwendung von indometacin im . trimenon liegen nicht vor. eigene daten umfassen prospektiv erfasste schwangerschaften mit diclofenac-therapie im . trimenon, von denen mit einem abbruch, mit einem spontanabort und mit einer lebendgeburt endeten. große fehlbildungen fanden sich in fällen, darunter vorhofseptumdefekte, ein hydrozephalus, eine doppelanlage der niere, eine nierenagenesie und beidseitiger klumpfuß. daraus lässt sich kein anhalt für ein erhöhtes fehlbildungsrisiko ableiten. für naproxen im . trimenon fand sich in retrospektiven analysen mit daten des schwedischen geburtsregisters ein leicht erhöhtes risiko für spaltbildungen bei nicht erhöhtem gesamtfehlbildungsrisiko (ericson ) und ein ebenfalls leicht erhöhtes risiko für kardiovaskuläre fehlbildungen (källén ) . ein fallbericht beschreibt ein neugeborenes mit großer beidseitiger lippen-und gaumen-spalte, hypertelorismus, breiter nasenwurzel, tiefem ohransatz sowie asymmetrischem fehlen von endphalangen beider füße nach mütterlicher therapie mit bisoprolol, naproxen und sumatriptan bis woche (kajantie ). der expositionszeitraum spricht gegen einen ursächlichen zusammenhang. von den durch uns prospektiv erfassten fällen mit naproxentherapie im . trimenon endeten schwangerschaften mit einem spontanabort, die lebendgeborenen waren gesund. spontanaborte. zwei studien beschreiben ein erhöhtes abortrisiko durch die einnahme nichtsteroidaler antiphlogistika , nielsen . die geringe fallzahl beider studien lässt deren aussage jedoch fraglich erscheinen. des weiteren werden in einer dieser arbeiten lediglich rezeptierungen registriert ohne angabe zur tatsächlichen einnahme des medikamentes. eine genaue angabe der substanzen fehlt in beiden publikationen. auswirkungen auf den kreislauf und andere organsysteme beim fetus. im letzten drittel der schwangerschaft können nichtsteroidale antirheumatika (nsar) zum vorzeitigen verschluss des ductus arteriosus beim fetus führen (mas ) . eine mekoniumanalyse bei neugeborenen zur klärung eines möglichen zusammenhanges zwischen nsar und persistierendem pulmonalen hypertonus (pphn) fand bei neugeborenen mit pphn mehr als doppelt so häufig nsar im mekonium (ibuprofen, naproxen, indometacin sowie acetylsalicylsäure) als bei gesunden kindern (alano ) . je reifer der fetus, desto größer ist die wahrscheinlichkeit, dass sich sein ductus arteriosus unter der antiphlogistischen therapie schließt (rasanen ) . schon von schwangerschaftswoche an wurde dieser effekt beobachtet (bivins ) . vor woche soll der fetale kreislauf nur in - % der fälle ansprechen, mit wochen in % und ab woche in % der fälle (moise (moise , . auch ein scheinbar paradoxer effekt wurde nach pränataler exposition mit nsar bei neugeborenen beobachtet: ein persistierender ductus arteriosus. dieser musste operativ verschlossen werden (norton ) . von den autoren wurde postuliert, dass indometacin in diesem fall die intima des ductus geschädigt und damit den spontanverschluss verhindert hat. aus einem vorzeitigen ductusverschluss kann sich ein pulmonaler hypertonus beim neugeborenen entwickeln, wie z. b. bei einem reifen kind, dessen mutter wochen vor entbindung wegen einer thrombophlebitis außer heparin täglich mg diclofenac für tage erhalten hatte. der pulmonale hypertonus persistierte und musste tage lang mit hohen dosen no-inhalation behandelt werden. ein offenbar ischämisch verursachter trikuspidalklappenreflux blieb auch danach bestehen (zenker ) . ein weiterer fall mit pulmonalem hypertonus wurde bei einem neugeborenen mit geschlossenem ductus arteriosus beschrieben, das in woche auf grund einer fetaler bradykardie per sectio entbunden wurde. die mutter war drei tage zuvor mit diclofenac therapiert worden (siu ) . in einem weiteren fall mit zweimal täglich mg naproxen innerhalb der letzten tage vor entbindung entwickelte ein reif geborenes kind stunden nach geburt einen pulmonalen hypertonus bei rechtsherzhypertrophie und geschlossenem ductus arteriosus. die symptomatik normalisierte sich unter sauerstofftherapie bis zum . lebenstag. nach monaten war das kind klinisch gesund, bei leichter echokardiographisch noch nachweisbarer rechtsherzhypertrophie (talati ) . auch die fetale und neonatale nierenfunktion kann bis zur anurie gehemmt werden, wenn im letzten drittel der schwangerschaft mit einem nsar behandelt wurde. dieser effekt wird auf eine minderperfusion der niere und einen anstieg des zirkulierenden vasopressin zurückgeführt (van der heijden , walker . ebenfalls durch minderperfusion beim fetus werden die bei neugeborenen nach pränataler nsar-exposition beobachteten fälle von nekrotisierender enterokolitis (nec) erklärt (ojala , parilla , major , norton . nierenfunktionsstörungen und nec traten auch bei neugeborenen auf, bei denen man den persistierenden ductus arteriosus nach der geburt mit indometacin verschließen wollte. schließlich wurden auch intrakranielle blutungen besonders bei frühgeborenen beschrieben, möglicherweise als folge einer durch indometacin induzierten hemmung der thrombozytenaggregation (norton ) . es ist anzunehmen, dass die exemplarisch beschriebenen organstörungen beim fetus nach gabe aller nsar auftreten können (z. b. ductusverschluss bei ketoprofen und nifluminsäure; radi , llanas . beim vorwiegend als cox- -hemmstoff wirkenden nimesulid wurde in zwei kasuistiken über (dialysepflichtiges) nierenversagen beim kind berichtet, nachdem die mutter in der spätschwangerschaft behandelt worden war. im zweiten fall waren es mg/tag zur tokolyse von woche bis ( balasubramaniam , peruzzi . bei einem in woche geborenen kind wurde ein akutes nierenversagen beschrieben nach feststellung eines oligohydramnions in woche und bereits im . trimenon, also vor der "sensiblen phase" erfolgter therapie mit nimesulid, diclofenac und paracetamol (benini ) . eine weitere kasuistik beschreibt ein kind mit chronischer nierenschädigung nach vierwöchiger nimesulidtherapie der mutter ab woche , bei der bereits wochen nach therapiebeginn ein oligohydramnion diagnostiziert wurde. eine konservative therapie war noch im alter von monaten erforderlich (magnani ) . holmes und mitarbeiter ( ) berichten ebenfalls über ein oligohydramnion, welches wochen nach einer in woche begonnenen nimesulidtherapie zur wehenprophylaxe auffiel und sich nach ende der therapie wieder normalisierte. das reif geborene kind war gesund. ein ähnliches ergebnis fand sich in einem bericht über schwangerschaften mit nimesulidtherapie wegen vorzeitiger wehen. alle frauen entwickelten ca. tage nach therapiebeginn ein oligohydramnion, welches sich nach behandlungsende zurückbildete. keines der kinder wies eine manifeste nierenschädigung auf (locatelli ) . in einer prospektiven studie entwickelte rund die hälfte der frauen, die eine nimesulidtherapie zur prophylaxe vorzeitiger wehen erhielten, nach ca. vierwöchiger therapie ein oligohydramnion, welches sich nach therapieende wieder zurückbildete. in einem fall mit akutem nierenversagen des frühgeborenen hatte die mutter die vorgeschriebenen kontrolluntersuchungen nicht wahrgenommen. in keinem fall war eine beendigung der therapie wegen verschluss des ductus arteriosus erforderlich (sawdy ) . paladini ( ) beschreibt fälle mit verschluss des ductus ateriosus beim neugeborenen nach einnahme von maximal analgetischen einzeldosen kurz vor der geburt (paladini ) . andere untersucher fanden keine nebenwirkungen beim neugeborenen nach tokolyse mit sulindac bei schwangeren in woche - (sawdy ) . sulindac soll aufgrund der geringen plazentagängigkeit seines aktiven (sulfid-)metaboliten keine dopplersonographisch feststellbaren veränderungen auf den fetalen kreislauf besitzen (carlan und , kramer . dieser vorzug gegenüber anderen nsar wird in anderen publikationen nicht bestätigt (kramer ) . schwangerschaften wurde eine abgebrochen und endeten mit der geburt eines gesunden kindes (eigene daten). eine kleine randomisierte studie fand keine unterschiede beim tokolytischen effekt zwischen celecoxib und indometacin. im gegensatz zu indometacin waren unter celecoxib kein partieller vorzeitiger verschluss des ductus arteriosus und auch keine verminderung der amnionflüssigkeit nachweisbar (stika ) . beim randomisierten vergleich von rofecoxib und magnesiumsulfat war weder in der effektivität der tokolyse noch hinsichtlich neonataler nebenwirkungen ein unterschied feststellbar (mcworther ) . für eine endgültige aussage sind die fallzahlen zu gering. man kann davon ausgehen, dass die bei den klassischen nsar und acetylsalicylsäure beschriebenen fetotoxischen wirkungen in der spätschwangerschaft auch bei den cox- -inhibitoren zu erwarten sind. in einer kleinen randomisierten studie fand man nach rofecoxib-einnahme im vergleich zu placebo eine verspätete follikelruptur. das könnte ein hinweis für eine mögliche herabsetzung der fertilität bei einnahme von rofecoxib zum konzeptionszeitpunkt sein -ein effekt, der auch im zusammenhang mit anderen nsar schon erörtert wurde (pall ) . die vorliegenden daten zu coxiben in der schwangerschaft erlauben keine differenzierte risikobewertung. empfehlung für die praxis: selektive cox- -inhibitoren sind aufgrund mangelnder erfahrung in der schwangerschaft zu meiden. nach heutiger kenntnis ergibt sich aus einer dennoch erfolgten exposition im . trimenon keine risikosituation, die eine invasive diagnostik erfordert oder einen risikobegründeten abbruch einer gewünschten und intakten schwangerschaft (siehe kapitel . ). eine ultraschallfeinuntersuchung sollte zur bestätigung der normalen entwicklung des fetus angeboten werden. g . man unterscheidet zwischen der medikamentösen migräneprophylaxe und der behandlung einer migräneattacke. die pathophysiologie der attacke verläuft in drei phasen: prodromalstadium mit vasokonstriktion der gefäße der betroffenen hirnhälfte, schmerzstadium mit vasodilatation, Ödemstadium, das mit einer erhöhten gefäßpermeabilität einhergeht und lange anhalten kann. zur medikamentösen therapie gibt es unterschiedliche ansätze. die im folgenden angeführten mittel werden zum teil an anderer stelle in diesem buch detailliert erörtert. generell werden zur medikamentösen prophylaxe und therapie der migräne die folgenden, mit einschränkungen auch in der schwangerschaft akzeptablen mittel empfohlen (göbel ) . keines der angegebenen medikamente steht in verdacht, beim menschen teratogen zu wirken. allerdings sind beispielsweise cyclandelat und flunarizin bisher unzureichend untersucht. die zur migräneprophylaxe empfohlenen dosen von betarezeptorenblockern (metoprolol und propranolol) können auch beim fetus zu einer relativen bradykardie führen. diese ist nicht bedrohlich, kann aber falsch interpretiert werden, wenn die medikation nicht bekannt ist. metoprolol, propranolol, cyclandelat, flunarizin, acetylsalicylsäure, magnesium, amitriptylin, naproxen (nicht nach woche ) baclofen während der gesamten schwangerschaft verabreicht. alle kinder waren gesund und wiesen keine entzugssymptomatik auf (roberts , munoz ) . zur baclofen-therapie per os über den gesamten zeitraum der schwangerschaft, bei der wesentlich höhere dosierungen benötigt werden, gibt es kasusistiken (dosis - mg/ tag). beide neugeborenen wiesen keine fehlbildungen, aber eine entzugssymptomatik auf. diese äußerte sich in einem fall in krampfanfällen am . lebenstag (ratnayaka ) . beim anderen neugeborenen wurden kurz nach der entbindung Übererregbarkeit und atembeschwerden beschrieben (moran ) . aus eigener beobachtung können wir über gesunde neugeborene nach oraler baclofen-therapie im . trimenon berichten. nach behandlung mit chlormezanon in der schwangerschaft wird über eine fulminant verlaufende hepatitis mit lebertransplantation und der anschließenden geburt eines gesunden kindes berichtet (bourliere ) . eigene daten zur chlormezanon-therapie im . trimenon umfassen fälle. von den lebend geborenen kindern wiesen große fehlbildungen auf (arthrogryposis, klumpfuß). die analyse von prospektiv erfassten schwangerschaften mit tetrazepam-exposition im . trimenon ergab keinen hinweis auf ein erhöhtes risiko für große fehlbildungen (eigene daten). zu tolperison (z. b. mydocalm ® ) können wir über prospektiv erfasste schwangerschaften berichten. davon wies kind multiple skelettfehlbildungen auf. ausreichende erfahrungen über die anwendung in der schwangerschaft liegen zu keinem dieser teilweise recht alten und therapeutisch überholten mittel vor. empfehlung für die praxis: abgesehen von der notfallbehandlung mit dantrolen bei maligner hyperthermie sind myotonolytika in der schwangerschaft ausnahmesituationen vorbehalten. physiotherapeutische maßnahmen und antiphlogistika/antirheumatika sind vorzuziehen. falls erforderlich, kann kurzzeitig die spannungslösende wirkung des besser untersuchten diazepam genutzt werden. eine exposition mit den genannten myotonolytika rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). g . . gicht-anfallsbehandlung, colchicin pharmakologie und toxikologie. neben den nichtsteroidalen säureantiphlogistika (nsar) wie ibuprofen ist colchicin das klassische mittel für den akuten gichtanfall. colchicin passiert die plazenta, besitzt als mitosehemmstoff mutagene und genotoxische eigenschaften und wirkt tierexperimentell in verschiedenen spezies embryotoxisch. bei dauertherapie mit täglich mg wurde bei der geburt ein mütterlicher plasmaspiegel von , ng/ml gemessen, im nabelschnurblut waren es , ng/ml (amoura ) . bei patienten, die mit colchicin behandelt wurden, sind mutagene effekte an den lymphocyten beschrieben worden. colchicin ist die einzige wirksame behandlung zur vorbeugung von attacken beim familiärem mittelmeerfieber (fmf) und der sich bei fmf-patienten chronisch entwickelnden amyloidose der niere. interessant ist, dass das abortrisiko bei unbehandelten frauen mit fmf fast doppelt so hoch ist wie bei patientinnen mit colchicin-therapie (rabinovitch ) . teratogene schäden wurden auch nach länger dauernder behandlung des fmf nicht beobachtet (Übersicht in . eine kürzlich publizierte große studie einer israelischen arbeitsgruppe, die offenbar früher veröffentlichte arbeiten ( barkei , rabinovitch ) einschließt, umfasst schwangerschaften mit colchicin-exposition der mutter und , bei denen der vater behandelt wurde. insgesamt wurden lebendgeborene registriert (berkenstadt ) . wie bereits in älteren arbeiten diskutiert ( barkei , rabinovitch , fand sich bei nicht erhöhter gesamtfehlbildungsrate ein statistisch nicht signifikanter anstieg der häufigkeit chromosomaler anomalien ( aneuploidien (davon u. a. trisomien , klinefelter-syndrom) sowie balancierte y-chromosom-translokation bei mütterlicher erkrankung). einige arbeiten (ben-chetrit , ben-chetrit zitieren eine publikation von rabinovitch und mitarbeitern ( ) mit . geburten nach colchicin-exposition und fällen einer trisomie bei den neugeborenen. dieses zitat war jedoch weder in der originalpublikation nachvollziehbar, noch ließ es sich nach persönlicher kommunikation mit dem autor bestätigen. es führte jedoch zu der anhaltenden diskussion über die notwendigkeit einer amniozentese bei mütterlicher colchicin-therapie. ein leicht erhöhtes risiko für chromosomenaberrationen und aneuploidien wurde zwar immer wieder diskutiert, war aber bisher nicht eindeutig belegbar. aus unserer sicht ist deshalb eine invasive diagnostik, wie z. b. eine amniozentese, primär nicht indiziert. eine neuere arbeit beschäftigt sich explizit mit dem risiko der väterlichen erkrankung an fmf und dem risiko einer therapie des vaters mit colchicin zum zeitpunkt der befruchtung. bei frauen von toxikologie. umfangreiche untersuchungen haben für keines der schon lange gebräuchlichen antihistaminika wie brompheniramin, chlorphenamin, chlorphenoxamin, clemastin, dexchlorpheniramin, dimetinden, diphenhydramin, hydroxyzin, mebhydrolin und pheniramin den früher geäußerten verdacht auf teratogene effekte beim menschen bestätigt (källén , Übersicht in , lione . bei insgesamt behandlungen mit acrivastin und mit alimemazin in der frühschwangerschaft wurden keine fehlbildungen beobachtet (källén , wilton . epidemiologische untersuchungen an insgesamt im . trimenon mit astemizol exponierten schwangeren ergaben weder eine erhöhte fehlbildungsrate noch andere abweichungen im schwangerschaftsverlauf , pastuszak . daten zur einnahme von cetirizin im . trimenon finden sich in drei prospektiven studien mit über und einer retrospektiven studie mit schwangerschaften. hinweise für ein erhöhtes fehlbildungsrisiko ergeben sich daraus nicht (weber-schöndorfer , paulus , källén . cyproheptadin (peritol ® ) hat tierexperimentell eine diabetogene wirkung auf die inselzellen des fetalen pankreas. hinweise auf vergleichbare wirkungen beim menschen gibt es bisher nicht. im schwedischen geburtsregister finden sich bei fällen nach behandlung mit cyproheptadin im . trimenon keine hinweise auf eine fruchtschädigende wirkung (källén ) . bei schwangeren mit ebastin und , die fexofenadin im . trimenon eingenommen hatten, fanden sich keine fehlbildungen (källén ) . eigene fälle mit fexofenadinbehandlung geben ebenfalls keine hinweise auf teratogenität. zur therapie mit hydroxyzin liegen zwei studien mit insgesamt schwangerschaften vor, ohne auffälligkeiten im schwangerschaftsverlauf oder beim neugeborenen . in einem fallbericht zur anxiolytischen therapie mit täglich mg hydroxyzin am ende der schwangerschaft entwickelte das in woche geborene kind stunden nach geburt tonisch-klonische anfälle. die plasmakonzentration stunden nach geburt war identisch mit den mütterlichen werten. die anfälle wurden als entzugssymptomatik gewertet. nach monaten war die neurologische entwicklung des kindes normal (serreau ) . nach der anwendung von loratadin in der frühschwangerschaft wurde im schwedischen geburtsregister nach auswertung von . fällen der verdacht auf ein erhöhtes risiko für das auftreten von hypospadien bei männlichen nachkommen geäußert. das gesamtfehlbildungsrisiko war nicht erhöht (källén , källén . dabei handelte es sich meist um leichte formen der hypospadie (grad ). bei späterer analyse der daten von inzwischen . loratadinexpositio-nen wurde der ursprüngliche verdacht nicht erhärtet (pers. mitteilung källén ) . in anderen studien fanden sich ebenfalls keine hinweise auf einen möglichen zusammenhang zwischen loratadintherapie in der frühschwangerschaft und hypospadien. moretti und mitarbeiter ( ) berichten über schwangerschaften mit loratadinexposition im . trimenon und fanden kein erhöhtes fehlbildungsrisiko gegenüber einer kontrollgruppe. in einer weiteren prospektiven studie mit frauen mit loratadintherapie, davon im . trimenon, gab es ebenfalls keinen hinweis auf teratogene effekte . hypospadien wurden in beiden studien nicht beobachtet. in einer neuen retrospektiven studie wurden kinder mit hypospadien und . männliche kontroll-kinder ohne fehlbildungen auf eine mütterliche loratadineinnahme im . trimenon verglichen (werler ) . ein zusammenhang konnte auch hier nicht nachgewiesen werden. bei weiteren prospektiv erfassten schwangerschaften mit loratadinexposition zwischen schwangerschaftswoche und wurden keine hypospadien gefunden (unveröffentlichte daten des european network of teratology information services -entis ) . die analyse eigener daten von schwangerschaften mit loratadintherapie, davon im . trimenon behandelt, ergab keine hinweise auf teratogene effekte. keiner der männlichen nachkommen hatte eine hypospadie. im zusammenhang mit meclozintherapie im . trimenon bei . schwangeren des schwedischen geburtsregisters war kein signifikant erhöhtes fehlbildungsrisiko nachweisbar (källén ) . es fanden sich jedoch deutlich häufiger mehrlingsschwangerschaften. bei eigenen fällen mit mizolastinexposition gab es keine hinweise auf teratogenität. die analysen von insgesamt schwangeren mit terfenadinbehandlung , loebstein , schick ) und weiteren im . trimenon exponierten schwangeren des schwedischen geburtsregisters (källén ) ließen kein erhöhtes fehlbildungsrisiko erkennen. in einer untersuchung wurde beobachtet, dass sich nach anwendung von antihistaminika in den letzten beiden schwangerschaftswochen die häufigkeit schwerwiegender augenhintergrundsveränderungen bei frühgeborenen, der so genannten retrolentalen fibroplasie, verdoppelte (zierler ) . andere untersucher konnten diesen effekt nicht bestätigen. bei neugeborenen wurden entzugssymptome wie zittrigkeit und diarrhö nach langfristiger antihistaminikabehandlung bis zur geburt (z. b. mit diphenhydramin und hydroxyzin) beschrieben. bei fällen mit lokaler anwendung von levocabastin fanden sich keine fehlbildungen (eigene daten). zu den anderen antihistaminika einschließlich desloratadin (aeri-us ® ) und levocetirizin (xusal ® ) sowie zu den neueren lokaltherapeutika epinastin (relestat ® augentropfen) und olopatadin (opata-nol ® augentropfen) liegen keine erfahrungen in der schwangerschaft vor. empfehlung für die praxis: in der schwangerschaft können h -antihistaminika zur behandlung allergischer erkrankungen eingesetzt werden. Ältere präparate mit umfangreicher markterprobung, wie z. b. clemastin und dimetinden, stellen kein problem dar. von den neueren, nicht sedierenden antihistaminika sollten loratadin und cetirizin bevorzugt werden. die einnahme weniger gut dokumentierter medikamente erfordert weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . pharmakologie und toxikologie. bei der hyposensibilisierung appliziert man einschleichend und kontinuierlich ansteigend dosen des allergens subkutan. das immunsystem bildet daraufhin blockierende antikörper, die das allergen binden sollen, noch bevor es mit sensibilisierten mastzellen reagieren kann. kommt es nach abschluss der behandlung zu einer exposition mit dem allergen, ist die histaminausschüttung der mastzellen deutlich verringert und damit die allergische reaktion schwächer. die hyposensibilisierung hat sich bei heuschnupfen und insektenstichallergien gut bewährt; bei ausgeprägtem asthma ist sie weniger erfolgreich. spezifische embryo-oder fetotoxische effekte sind nicht zu erwarten (metzger ) . eine seltene anaphylaktische reaktion kann jedoch den embryo/fetus mittelbar schädigen (luciano ) . empfehlung für die praxis: eine vor eintritt der schwangerschaft begonnene hyposensibilisierung kann bei guter verträglichkeit fortgesetzt werden. auf dosissteigerungen sollte wegen möglicher unverträglichkeiten verzichtet werden. eine hyposensibilisierung sollte aus demselben grund nicht während der schwangerschaft begonnen werden, es sei denn, die situation, z. b. bedrohliche reaktionen auf insektenstiche, erfordert diese therapie. ein risikobegründeter schwangerschaftsabbruch ist aufgrund einer hyposensibilisierung ebenso wenig indiziert wie zusätzliche diagnostische maßnahmen (siehe kapitel . ). zierler s, purohit d. prenatal antihistamine exposure and retrolental fibroplasia. am j epidemiol ; : - . asthma bronchiale muss auch bei schwangeren ausreichend therapiert werden, um neben dem wohlergehen der mutter eine ausreichende oxygenierung im fetoplazentaren bereich zu gewährleisten. schweres, unzureichend therapiertes asthma ist mit einem höheren risiko für frühgeburtlichkeit, intrauterine wachstumsverzögerung und präeklampsie assoziiert (beckmann , bracken , johnson , olesen . alle bisher untersuchten antiasthmatika haben sich als verträglich für den embryo bzw. fetus erwiesen (schatz ) . dieser abschnitt orientiert sich an der aktuellen, vom schweregrad abhängigen stufentherapie des asthmas, geht aber auch auf nicht mehr aktuelle arzneimittel ein. am ende des abschnitts werden expektoranzien und antitussiva besprochen. schweregrad : leichte intermittierende symptomatik, wenn symptome seltener als ein-oder zweimal pro woche auftreten, wird ein inhalierbares, kurzwirksames g -sympathomimetikum bei bedarf eingenommen. schweregrad : persistierendes leichtes asthma, definiert durch gelegentliche nächtliche symptome oder symptome tagsüber, die aber noch nicht täglich auftreten, wird mit einer basistherapie behandelt. dies sind in der regel ein inhalatives corticoid oder bei allergischem asthma cromoglicinsäure. bedarfsweise kann zusätzlich ein inhalierbares, kurzwirksames g -sympathomimetikum genommen werden. schweregrad : persistierendes mittelgradiges asthma mit täglichen beschwerden und nächtlichen symptomen einmal pro woche und häufiger sollte mit einem langwirksamen g -sympathomimetikum zusätzlich zum inhalativen corticoid (ggf. dosissteigerung gegenüber schweregrad !) therapiert werden. bedarfsweise kann wieder ein kurzwirksames g -sympathomimetikum eingenommen werden. sollte das nicht ausreichen, kommen auch theophyllin und/oder anticholinergika in frage, außerhalb der schwangerschaft auch leukotrien-rezeptor-antagonisten. schweregrad : persistierendes schweres asthma mit ständigen symptomen wird oral mit glucocorticoiden (z. b. prednisolon) therapiert oder mit einer kombination aus drei oder mehr substanzen aus stufe ; selten sind orale glucocorticoide in kombination mit mehreren anderen substanzen erforderlich. g . . selektiv wirkende g -sympathomimetika pharmakologie. beim vegetativen nervensystem unterscheidet man im bereich des sympathikus § -und g -rezeptoren, letztere werden in g und g -rezeptoren unterteilt. stimulierung der g -rezeptoren bewirkt am herzen eine aktivitätssteigerung. g -rezeptoren vermitteln hingegen ein erschlaffen der glatten muskulatur an den gefäßen (vasodilatation), an den bronchien (bronchodilatation) und am uterus (tokolyse) und führen zum anstieg der konzentration von glucose, fettsäuren und ketonkörpern im blut. darüber hinaus fördern g -agonisten an den bronchien die mukoziliäre clearance und reduzieren die gefäßpermeabilität. ein sympathomimetikum, das ausschließlich g -sympathomimetisch wirkt, ohne gleichzeitig andere adrenerge rezeptoren zu aktivieren, gibt es bisher nicht. sympathomimetika mit vorwiegender g -wirksamkeit haben in der asthmatherapie aber inzwischen solche mittel ersetzt, die noch deutliche g -aktivität aufwiesen. g -sympathomimetika wirken nach inhalation ebenso schnell wie nach intravenöser injektion und erreichen ihr wirkungsmaximum nach - minuten. durch die depotfunktion der bronchialschleimhaut verlängert sich zudem der therapeutische effekt gegenüber einer parenteralen anwendung, obwohl die plasmakonzentration nur bei % liegt. entsprechend geringer fallen die unerwünschten wirkungen im vergleich zur systemischen applikation aus. nach oraler gabe von g -sympathomimetika werden - % resorbiert. unabhängig von der applikationsart werden diese mittel nach metabolisierung in der leber und kopplung an sulfat mit dem urin ausgeschieden. fenoterol (z. b. berotec ® ) , reproterol (in bronchospasmin ® ), salbutamol (z. b. sultanol ® ) und terbutalin (z. b. bricanyl ® ) sind pharmaka, die ein günstiges verhältnis zwischen g -und g -stimulierung aufweisen und daher seit vielen jahren mit erfolg zur behebung der bronchokonstriktion bei asthma eingesetzt werden. sie gehören zu den kurzwirksamen vertretern dieser arzneimittelgruppe. ihre wirkung ist auf - stunden begrenzt. einige untersuchungen legen nahe, dass die anwendung von fenoterol mit einem erhöhten risiko für bedrohliche kardiopulmonale nebenwirkungen verbunden ist. demgegenüber sind formoterol (z. b. oxis ® ) und salmeterol (z. b. serevent ® ) länger als stunden wirksam. sie sind nicht zur behandlung akuter asthmasymptome geeignet und stellen keinen ersatz für eine basistherapie dar, vielmehr sollten sie nur kombiniert mit z. b. inhalierbaren glucocorticoiden gegeben werden (bekanntmachung des bundesinstituts für arzneimittel und medizinprodukte / ). einer noch unveröffentlichten studie des herstellers glaxosmithkline zufolge kann die langfristige einnahme von salmeterol zu einem gerin-gen, aber signifikanten anstieg der durch asthma bedingten todesfälle führen (arzneimittelbrief ) . sympathomimetika hemmen im . und . trimenon die kontraktilität der uterusmuskulatur. sie werden daher auch als tokolytika eingesetzt. toxikologie. es gibt keine hinweise darauf, dass g -sympathomimetika fehlbildungen hervorrufen oder das fetale wachstum beeinträchtigen (z. b. bakhireva ) . alle sympathomimetika können jedoch in entsprechend hoher dosis nicht nur bei der mutter, sondern auch beim fetus eine tachykardie oder andere rhythmusstörungen verursachen. eine kasuistik beschreibt eine fetale tachykardie mit vorhofflattern in woche , nachdem die mutter versehentlich über stunden inhalativ Überdosen von albuterol erhalten hatte und selbst eine herzfrequenz von - /min aufwies. die symptomatik sistierte spontan nach absetzen der medikation (baker ) . g -sympathomimetika können die kohlenhydrattoleranz beeinträchtigen. das ist bei einer diabetogenen stoffwechsellage der schwangeren zu berücksichtigen (källén ) . für clenbuterol (z. b. spiropent ® ), pirbuterol und tulobuterol (z. b. brelomax ® ), bambuterol (bambec ® ) liegen keine für eine spezifische risikobewertung ausreichenden erfahrungen zur anwendung im . trimenon vor. es gibt jedoch auch bei diesen wirkstoffen bislang keine hinweise auf teratogene effekte beim menschen. empfehlung für die praxis: sympathomimetika gehören auch in der schwangerschaft zur asthmatherapie, sie dürfen gemäß den empfehlungen des asthmastufenplans angewendet werden. salbutamol ist mittel der . wahl unter den kurz wirksamen sympathomimetika. am ende der schwangerschaft müssen wehenhemmung und g -spezifische effekte beim fetus bedacht werden (siehe oben). inhalierbare corticosteroide sind mittel der wahl in der asthmabasis-therapie der schweregrade bis . sie wirken antiinflammatorisch, d. h. antiphlogistisch, antiallergisch und immunsuppressiv. außerdem haben sie einen günstigen effekt auf die ansprechbarkeit der betarezeptoren an den bronchien. in der asthma-therapie werden als inhalierbare glucocorticoide budesonid (pulmicort ® ) und folgende halogenierte derivate bevorzugt verwendet: beclometason (z. b. beconase ® , sanasthmyl ® ), flunisolid (syntaris ® ), fluticason (z. b. atemur ® ) und mometason (z. b. asmanex ® ). seit ist der wirkstoff ciclesonid (alvesco ® ) in deutschland als inhalatives corticoid zur asthma-therapie bei erwachsenen zugelassen. theoretische bedenken gegen die anwendung von inhalierbaren glucocorticoiden in der schwangerschaft beruhten auf ergebnissen einiger studien bei systemischer anwendung, in denen eine erhöhte rate an lippen-gaumen-spalten bei den kindern diskutiert wird, sowie auf der beobachtung, dass eine dauerhafte cortisontherapie in der schwangerschaft bei entsprechender dosierung zur fetalen wachstumsverzögerung führen kann. studien zur anwendung von inhalierbaren corticosteroiden in der schwangerschaft konnten diese zweifel beseitigen (martel , bakhireva , schatz . budesonid ist mit mehr als schwangeren (gluck ) das am besten untersuchte mittel, gefolgt von beclometason (ca. schwangere) und fluticason (ca. ). auch bei triamcinolon (ca. ) und flunisolid (ca. ) gibt es bisher keinen hinweis auf ein erhöhtes fehlbildungsrisiko oder auf unerwünschte fetale effekte (namazy , norjavaara , källén . das gilt auch für das am schlechtesten untersuchte mometason (eigene beobachtungen). bei schwerem asthma oder zur therapie des asthmaanfalls dürfen glucocorticoide auch systemisch appliziert werden. empfehlung für die praxis: inhalierbare glucocorticoide sind gemäß asthmastufenplan auch in der schwangerschaft mittel der wahl, wobei besser untersuchte substanzen zu bevorzugen sind. bei systemischer anwendung von glucocorticoiden (siehe kapitel . . ) ist die dosis, sofern es klinisch möglich ist, rasch zu reduzieren, um unerwünschte wirkungen (auch auf die mutter) zu verhindern. wird bis zur geburt systemisch behandelt, müssen geburtshelfer und pädiater über die medikation informiert werden, um mögliche stoffwechselauswirkungen beim neugeborenen zu beachten. es gibt keine wissenschaftlich belegte corticosteroiddosis, die einen risikobegründeten schwangerschaftsabbruch erforderlich macht (siehe kapitel . zur prävention des allergischen asthmas und des belastungsasthmas wird cromoglicinsäure als pulver oder lösung vorbeugend inhaliert. nur - mg einer -mg-dosis erreichen die alveolen, der rest wird verschluckt. ein prozent hiervon wird im darm resorbiert und unverändert mit dem urin ausgeschieden. die halbwertszeit beträgt - minuten. die wirkung tritt erst nach - tagen ein. cromoglicinsäure wirkt nicht embryotoxisch, wie sich bei einer großen zahl behandelter schwangerer bestätigt hat (Überblick bei . nedocromil (z. b . tilade ® ), dessen wirkungsmechanismus dem von cromoglicinsäure ähnelt, ist in der schwangerschaft noch nicht ausreichend untersucht. hinweise auf unverträglichkeiten beim ungeborenen liegen nicht vor. empfehlung für die praxis: cromoglicinsäure kann zur prävention eines allergisch bedingten asthmas auch schwangeren gegeben werden. es darf auch als augen-und nasentropfen verwendet werden. eine anwendung von nedocromil erfordert keine konsequenzen. pharmakologie und toxikologie. substanzen wie ketotifen (z. b. zaditen ® ) und oxatomid sind bezüglich ihrer pränatalen verträglichkeit bisher nicht ausreichend untersucht. hinweise auf embryotoxische wirkungen beim menschen liegen nicht vor. empfehlung für die praxis: ketotifen und oxatomid gehören nicht zum standardtherapieschema bei asthma und sollten in der schwangerschaft nicht angewendet werden. eine dennoch erfolgte applikation stellt weder eine indikation zum risikobegründeten abbruch der schwangerschaft noch für zusätzliche diagnostik dar (siehe kapitel . ). die daraus resultierende erhöhung der intrazellulären camp-konzentration bewirkt eine erschlaffung der bronchialmuskulatur. die plasmakonzentration des theophyllins korreliert gut mit der bronchodilatation, aber auch mit den unerwünschten wirkungen. bei mäßiggradiger obstruktion wirkt theophyllin weniger bronchodilatatorisch als g -sympathomimetika. theophyllin wirkt am herzen gering positiv inotrop und stimuliert verschiedene abschnitte des zentralnervensystems. es steigert die empfindlichkeit des atemzentrums gegenüber co und verursacht auf diese weise eine zunahme der atemfrequenz und -tiefe. diesen effekt nutzt man zur behandlung der apnoeneigung bei frühgeborenen. wie andere methylxanthine wird theophyllin nach oraler gabe rasch resorbiert und in der leber demethyliert und oxidiert. nur etwa % werden unverändert über die nieren ausgeschieden. theophyllin ist plazentagängig (arwood ) . seine halbwertszeit beträgt etwa stunden. bei schwangeren ist sie mit etwa stunden verlängert (sutton ) . aufgrund der veränderten proteinbindung sind bei schwangeren in der regel serumspiegel von - ? g/ml therapeutisch ausreichend und führen außerdem zu einer geringeren nebenwirkungsrate bei den neugeborenen (schatz ) . kinder metabolisieren theophyllin rascher als erwachsene, bei frühgeborenen sind jedoch halbwertszeiten bis zu stunden gemessen worden (aranda ). toxikologie. obwohl theophyllin im tierversuch in hohen konzentrationen teratogen wirkt, wurden beim menschen keine embryotoxischen effekte beobachtet (Überblick bei . während der spätschwangerschaft wurde unter theophyllin eine zunahme der fetalen atembewegungen (ohne pathologische relevanz) beobachtet (ishikawa ) . früher behauptete zusammenhänge zwischen mütterlicher therapie und erhöhtem risiko für eine nekrotisierende enterokolitis (nec) beim neugeborenen wurden unter anderem in einer studie an schwangeren widerlegt, die theophyllin als tokolytikum oder zur surfactantbildung vor woche erhalten hatten (zanardo ) . nach zwei neueren arbeiten (dombrowski , schatz ) gab es keine nennenswerten unterschiede hinsichtlich der geburtsparameter bei inhalierbaren g -sympathomimetika, inhalativen corticoiden und theophyllin. nur bei den schwangeren selbst war die nebenwirkungsrate unter theophyllin höher. beschrieben sind vor allem zittrigkeit, tachykardie und erbrechen. empfehlung für die praxis: theophyllin kann in der gesamten schwangerschaft bei asthma gemäß stufenplan angewendet werden. die niedrigste therapeutisch sinnvolle serumkonzentration sollte angestrebt werden, um nebenwirkungen bei mutter und neugeborenem zu minimieren. g . . anticholinergika ipratropiumbromid und oxitropiumbromid pharmakologie und toxikologie. da eine bronchokonstriktion auch über den nervus vagus induzierbar ist, können anticholinerg wirkende substanzen therapeutisch wirksam sein. ipratropiumbromid (z. b. atrovent ® ) ist in der lage, eine vollständige pulmonale vagolyse zu bewirken. seine bronchodilatatorische aktivität kann zwei drittel der aktivität von g -sympathomimetika erreichen. es wird allein (z. b. atrovent ® ) oder in kombination mit fenoterol (berodual ® ) angeboten. hinweise auf pränatale toxizität liegen nicht vor. oxitropiumbromid und tiotropiumbromid (spiriva ® ) sind bezüglich einer anwendung in der schwangerschaft unzureichend untersucht. empfehlung für die praxis: ipratropiumbromid darf in der schwangerschaft zur bronchodilatation verwendet werden. die anwendung von oxitropiumbromid rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch zusätzliche diagnostik (siehe kapitel . ). g . . antileukotriene pharmakologie und toxikologie. die leukotrien-rezeptor-antagonisten montelukast (singulair ® ), zafirlukast und pranlukast sowie der lipoxygenase-inhibitor zileuton werden zur vorbeugung asthmatischer beschwerden eingesetzt. der hersteller merck berichtet über abgeschlossene prospektiv erfasste schwangerschaften unter montelukast. von den lebendgeborenen waren im . trimenon exponiert, es fanden sich kinder mit anomalien, darunter ein mädchen mit fehlender linker hand. diese als "amnionband-syndrom" klassifizierte anomalie wurde nicht dem medikament zugeschrieben. eine weitere extremitätenanomalie (hypoplasie des rechten daumens) wurde bei prospektiv dokumentierten schwangerschaften des schwedischen medizinischen geburtsregisters beobachtet. unter den retrospektiven fallberichten des herstellers befand sich ein neugeborenes mit fehlender anlage von unterarm und hand. unsere eigenen daten umfassen prospektiv erfasste schwangerschaften mit exposition im . trimenon ohne hinweise auf extremitätenfehlbildungen. auch die ergebnisse von in nordamerikanischen teratologischen zentren beobachtete schwangerschaften (montelukast = , zafirlukast = ; bakhireva ) sprechen nicht für ein substantiell erhöhtes gesamtfehlbildungsrisiko, reichen aber nicht für eine differenzierte risikoeinschätzung. weder können daraus teratogene eigenschaften noch eine unbedenklichkeit für die anwendung schwangerer gefolgert werden. pharmakologie und toxikologie. iodsalze, z. b. iodkalium (kalium iodatum) können in der schwangerschaft in sekretolytischer dosis beim fetus und neugeborenen die schilddrüsenfunktion beeinträchtigen (therapeutische einzeldosis beim erwachsenen - mg). dies ist nicht zu verwechseln mit der für schwangere empfohlenen iodsubstitution ( ? g/tag). eine versehentliche gabe von kalium iodatum zur sekretolyse in der schwangerschaft führt bei kurzfristiger anwendung nicht zur strumaentwicklung . die reifung des zentralnervensystems, die von einer ausreichenden versorgung mit schilddrüsenhormons abhängig ist, kann jedoch gestört werden, wenn ab ende des . trimenon eine hohe ioddosis die dann bereits aktive schilddrüse des fetus supprimiert. empfehlung für die praxis: die gabe von iodsalzen als expektorans ist in der schwangerschaft kontraindiziert. eine versehentliche sekretolyse mit iod erfordert aber weder einen risikobegründeten schwangerschaftsabbruch noch zusätzliche vorgeburtliche diagnostik (siehe kapitel . ). dextromethorphan (z. b . neotussan ® ) hat keine analgetische komponente und ein offenbar geringeres (aber dennoch vorhandenes!) suchtpotenzial. der antitussive effekt ist vergleichbar mit dem von codein (reynolds ) . ausgehend von tierexperimentellen befunden wurde dextromethorphan ende der er jahre ein teratogenes potenzial unterstellt. aufgrund der erfahrungen an über schwangerschaften hat sich dieser verdacht für den menschen nicht bestätigt (martinez-frias , einarson , andaloro . andere antitussiva wie benproperin (tussafug ® ), clobutinol (z. b. silomat ® ), dropropizin (larylin ® ), eprazinon (eftapan ® ), isoaminil, noscapin (z. b. capval ® ) , pentoxyverin (sedotussin ® ) und pipazetat sind hinsichtlich pränataler risiken beim menschen unzureichend untersucht. empfehlung für die praxis: in begründeten fällen darf codein bei hartnäckigem, trockenem husten oder in kombination mit paracetamol als analgetikum in allen phasen der schwangerschaft verordnet werden. auch dextromethorphan kann als antitussivum eingesetzt werden. falls codein präpartal oder regelmäßig in hoher dosis als suchtmittel oder zur substitutionsbehandlung heroinabhängiger genommen wird, muss beim neugeborenen mit atemdepression und im fall hoher dosen auch mit bedrohlichen entzugserscheinungen gerechnet werden. wurden die nicht empfohlenen mittel verwendet, erfordert dies weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). g . . unspezifisch wirkende sympathomimetika die im folgenden besprochenen arzneimittel gehören nicht zum aktuellen asthma-therapiestufenplan. einige der sympathomimetika finden sich in kombination mit anderen wirkstoffen in rezeptfreien mitteln gegen erkältungen, deren anwendung nicht erwünscht ist. pharmakologie und toxikologie. orciprenalin (alupent ® ) wirkt stimulierend auf g -rezeptoren, allerdings nicht so selektiv wie die g -sympathomimetika. entsprechend stärker fallen die unerwünschten wirkungen am herzen und im stoffwechsel aus. das gilt auch für die stimulierende wirkung auf das zentralnervensystem und die verminderung von tonus und motilität im magen-darm-trakt. hinweise auf spezifische embryo-oder fetotoxische wirkungen liegen weder zu orciprenalin noch zu hexoprenalin vor. empfehlung für die praxis: orciprenalin und hexoprenalin gehören nicht zur standardtherapie des asthmas. spezifische g -sympathomimetika sind vorzuziehen. eine dennoch erfolgte therapie rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch zusätzliche diagnostik (siehe kapitel . ). pharmakologie und toxikologie. adrenalin (epinephrin; z. b. suprarenin ® ) ist ein katecholamin, das natürlicherweise im körper vorkommt und sowohl § -als auch g -adrenerge wirkung besitzt. bei asthma bronchiale trägt zwar die schwache stimulierung der § -rezeptoren über eine vasokonstriktion zur verminderung des Ödems der bronchialschleimhaut bei, allerdings sind die § -sympathomimetischen herz-kreislauf-wirkungen (tachykardie, extrasystolen, hypertonie) so stark, dass selektive g -sympathomimetika vorgezogen werden. adrenalin ist notfallsituationen vorbehalten und wird dann i.v. oder endotracheal appliziert bzw. inhalativ als aerosol gegeben (z. b. zum abschwellen der oberen atemwege). nach oraler gabe ist es unwirksam, weil es im magen-darm-trakt inaktiviert wird. katecholamine sind plazentagängig, sie werden dort aber teilweise enzymatisch inaktiviert (morgan ) . im gegensatz zu tierexperimentellen ergebnissen haben sich beim menschen keine hinweise auf teratogene effekte ergeben . eine systemische anwendung kann die durchblutung von uterus und plazenta beeinträchtigen und zur fetalen hypoxie führen. empfehlung für die praxis: adrenalin ist vitalen indikationen vorbehalten. es gehört nicht zur asthma-standardtherapie. eine exposition rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). die einigen lokalanästhetika beigefügten mengen sind als unbedenklich anzusehen. pharmakologie und toxikologie. isoprenalin weist keine § -sympathomimetische wirkung, aber eine etwa gleich starke g -und g -aktivität auf. die g -wirkung am herzen schränkt die verwendung auf notfallsituationen ein, z. b. zur abschwellung der oberen atemwege. spezifische embryo-oder fetotoxische wirkungen wurden bisher nicht bekannt. die systemische gabe von isoprenalin könnte, ähnlich wie bei adrenalin, eine verminderte utero-plazentare durchblutung mit fetaler hypoxie verursachen. bei kurzfristiger inhalativer anwendung oder in dermatologischen präparaten ist dies aber nicht zu erwarten. pharmakologie und toxikologie. ephedrin ist das älteste bronchodilatatorisch wirkende asthmamittel. es gehört zu den indirekten sympathomimetika, die über eine vermehrte ausschüttung der körpereigenen katecholamine wirken. ephedrin hat sowohl § -als auch g -aktivität mit entsprechenden unerwünschten wirkungen, so dass es inzwischen als ungeeignet für die asthmatherapie erachtet wird. heute findet man ephedrin und andere (indirekte) sympathomimetika wie pseudoephedrin, phenylephrin, phenylpropanolamin in kombinationsmitteln gegen erkältungen. eine unveröffentlichte studie aus schweden untersuchte erkältungspräparate auf mögliche teratogene effekte: im . trimenon nahmen mehr als . schwangere phenylpropanolamin ein, ca. phenylpropanolamin plus cinnarizin und mehr als pseudoephedrin. die fehlbildunsgrate war in keiner der gruppen erhöht (källén , persönliche kommunikation) . auch in der bundesrepublik deutschland werden produkte, die zusätzlich dextromethorphan, doxylamin etc. enthalten, noch angeboten (z. b. wick medinait ® ). zumindest bei unkontrolliertem gebrauch und höheren dosen dieser mittel sind embryotoxische wirkungen durch sympathomimetika nicht auszuschließen, wie publizierte fallberichte über extremitätendefekte in erinnerung rufen (gilbert- barness ) . empfehlung für die praxis: ephedrin gehört nicht zur asthma-standardtherapie. mittel gegen erkältungen, die ephedrin und andere sympathomimetika enthalten, sollen nicht genommen werden. eine (versehentliche) exposition rechtfertigt jedoch weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . morgendliche Übelkeit und erbrechen gehören zu den unangenehmen begleiterscheinungen vieler schwangerschaften. man schätzt, dass - % aller schwangeren in den ersten wochen unter morgendlicher Übelkeit leiden und die hälfte von ihnen zusätzlich unter erbrechen (emesis gravidarum). nur in sehr seltenen fällen ( , - %) ist das erbrechen so stark, dass ein klinikaufenthalt wegen störungen des energie-und elektrolythaushaltes erforderlich ist (hyperemesis gravidarum). während früher darüber diskutiert wurde, ob Übelkeit und erbrechen prognostisch ungünstige faktoren für die schwangerschaft darstellen, gibt es heute eine debatte darüber, ob das fehlen von morgendlicher Übelkeit ein prognostisch ungünstiges zeichen für die entwicklung des kindes ist. ging in einer kontrollierten prospektiven studie dieser frage nach und verglich kinder von schwangeren ohne Übelkeit und erbrechen mit solchen, die daran litten und eine standarddosis doxylamin-pyridoxin (kontrollgruppe ) bzw. eine höhere dosis doxylamin-pyridoxin (kontrollgruppe ) erhielten. es gab keine unterschiede zwischen den kindern der verschiedenen gruppen. bei jüngeren schwangeren und denen, die schon mindestens ein kind haben, sowie jenen, die während der schwangerschaft zu hause bleiben, ermittelte källén ( ) ein höheres risiko für Übelkeit und erbrechen. Über den wert von antiemetika wurde intensiv diskutiert, weil dieser arzneimittelgruppe ursprünglich ein embryotoxisches potenzial unterstellt wurde und gleichzeitig die wirksamkeit der therapie umstritten war. umfangreiche untersuchungen ergaben keinen anhalt für ein erhöhtes fehlbildungsrisiko der klassischen antiemetika (asker , mazotta , seto , brent . auch der von einer untersuchergruppe beschriebene zusammenhang von retrolentaler fibroplasie bei frühgeborenen und antihistaminika-anwendung in den letzten beiden schwangerschaftswochen (zierler ) mazotta , seto , lione . untersuchungsergebnisse berichten über eine besserung der symptomatik bei % der behandelten, diese war bei % komplett (Übersicht in broussard ) . dimenhydrinat soll ebenso wie diphenhydramin einen wehen fördernden effekt besitzen (broussard ) . hinweise auf ein teratogenes risiko beim menschen liegen nicht vor, dies wurde kürzlich in einer retrospektiven fall-kontroll-studie bestätigt (czeizel ) . empfehlung für die praxis: dimenhydrinat ist akzeptabel, wenn kein risiko für eine frühgeburt vorliegt. meclozin ist jedoch antiemetikum der wahl in der schwangerschaft. ten. daher wird es heute als beruhigungsmittel und bei schlafstörungen verwendet. in den er jahren wurde neben anderen antihistaminika auch diphenhydramin ein teratogenes potenzial unterstellt. dies ließ sich in späteren untersuchungen nicht bestätigen (mazotta , seto , lione . bei ca. im . trimenon behandelten schwangeren wurde eine fehlbildungsrate von , % gefunden (asker ) . diphenhydramin hat, ebenso wie dimenhydrinat, einen oxytocinartigen, wehenfördernden effekt, der in den er und er jahren unter der geburt genutzt wurde (broussard , brost , smithells , der sich in ausführlichen prospektiven und retrospektiven studien jedoch nicht bestätigen ließ (zusammenfassung in brent , mckeigue . aufgrund der kritik von verbraucherverbänden im in-und ausland wurde lenotan ® auch in der bundesrepublik deutschland vom markt genommen. vor einigen jahren wurde dann das risiko nochmals bewertet, dabei gab es definitiv keinen anhalt für einen teratogenen effekt (brent , kutcher . auch eine therapie mit höherer dosierung hat nach einer vergleichenden studie keine negativen auswirkungen auf die schwangerschaft (atanackovic (källén , seto , lione (bsat , einarson (mazotta , broussard . dies bestätigt eine neuere schwedische studie, die unter ande-phenothiazin-antiemetika rem . im . trimenon mit promethazin behandelte schwangere untersuchte (asker ) . auch triflupromazin (das frühere psyquil ® ) scheint nicht embryotoxisch zu wirken (mcelhatton ) . thiethylperazin wird vor allem in der schweiz und in osteuropa als antiemetikum genutzt, ohne dass sich bisher hinweise auf risiken für den fetus ergaben (einarson ) . livan ) . fallberichte beschreiben die erfolgreiche intravenöse anwendung nach versagen anderer arzneimittel bei schwerer hyperemesis gravidarum zwischen den schwangerschaftswochen und . die neugeborenen waren unauffällig (siu , world , guikontes . in einer prospektiven studie wurden mit ondansetron behandelte schwangere mit zwei unterschiedlichen kontrollgruppen verglichen (vutyavanich ) . eine weitere untersuchung ergab nur eine wirksamkeit bei starker Übelkeit, bei leichter und mittlerer symptomatik war pyridoxin placebo nicht überlegen (sahakian ) . scopolamin (scopoderm ® tts) ist ein parasympatholytikum, das äußerlich als pflaster zur antiemetischen behandlung eingesetzt wird. bei im . trimenon behandelten schwangeren wurde keine erhöhte fehlbildungsrate festgestellt alginsäure oder alginat führen in anwesenheit von magensäure zu einem viskösen gel, das auf dem mageninhalt "schwimmt", wie eine mechanische barriere wirkt und damit den gastroösophagealen reflux reduziert. vor kurzem wurde eine studie an schwangeren veröffentlicht, die die wirksamkeit und sicherheit im ./ . trimenon untersucht hat . calciumcarbonat neutralisiert die salzsäure unter bildung von calciumchlorid, kohlendioxid und wasser. etwa - % der oral aufgenommenen dosis werden resorbiert. bei patienten mit normaler nierenfunktion besteht nach einnahme von calciumcarbonathaltigen präparaten in therapeutischer dosierung keine gefahr einer hypercalcämie. exzessive calciumzufuhr kann jedoch bei schwangeren zu dem äußerst seltenen milch-alkali-syndrom führen (gordon ) , so dass nicht mehr als , g elementares calcium (entsprechend , g calciumcarbonat) täglich eingenommen werden sollten. bei einer schwangeren, die einen monat lang täglich rennie und ca. ml milch zu sich nahm, litt das neugeborene an einer vorübergehenden hypercalcämie. eine andere kasuistik beschreibt krämpfe als folge einer hypocalcämie beim neugeborenen, dessen mutter exzessiv während der gesamten schwangerschaft antacida eingenommen hatte (robertson ) . im gegensatz zu der o.g. fallbeschreibung von gordon ( ) (ruigomez , källén . anzahl und muster der fehlbildungen waren gegenüber der jeweiligen kontrollgruppe nicht auffällig. auch frühgeburten und intrauterine wachstumsretardierung traten nicht häufiger auf. zu vergleichbaren ergebnissen kommt eine neuere multizentrische prospektive studie des european network of teratology information services (entis) mit schwangeren, davon wurden im . trimenon behandelt. bei ranitidin (n= ), cimetidin (n= ), famotidin (n= ), nizatidin (n= ) und roxatidin (n= ) zeigte sich keine erhöhte fehlbildungsrate. allerdings war die frühgeburtenrate in der behandelten gruppe höher, ohne dass sich dafür eine erklärung fand. die bei famotidin beobachteten neuralrohrdefekte sind eher als zufällig zu betrachten in einem kanadischen bericht zu anwendungen von pinaverium in der schwangerschaft werden unauffällige kinder und ein spontanabort genannt. fünf mütter hatten das mittel im . trimenon, fünf zwischen schwangerschaftswoche und eingenommen. in all diesen fällen war die einnahme unbeabsichtigt, es war mit dem antiemetikum diclectin verwechselt worden (einarson ) . darifenacin (emselex ® ) ist ein selektiver m -rezeptorantagonist, der zur behandlung der überaktiven harnblase eingesetzt wird. erfahrungen in der schwangerschaft gibt es noch nicht. spezifische embryotoxische effekte beim menschen sind bei anwendung der genannten belladonna-alkaloide bisher nicht beobachtet worden. auch die anwendung unter der geburt wird, soweit dokumentiert (z. b. für das anticholinergikum glycopyrronium) offenbar gut vom fetus vertragen (ure mesalazin wird sehr häufig in der schwangerschaft verordnet, ohne dass sich bisher hinweise auf teratogene wirkungen ergeben haben (habel , diav-citrin , marteau ursodeoxycholsäure wirkt bei hepatozellulären schäden, die durch gallensäuren induziert sind, also vor allem bei cholestatischen erkrankungen, wie der primär biliären zirrhose. aufgrund der symptomatischen wirkung ist eine dauerbehandlung erforderlich. aussagefähige untersuchungen zur anwendung bei schwangeren gibt es bisher nur für ursodeoxycholsäure in der zweiten schwangerschaftshälfte, vor allem zur gut wirksamen behandlung bei schwangerschaftscholestase (roncaglia , mazella , die mit juckreiz, ikterus sowie erhöhter alkalischer phosphatase (ap) und + -glutamyltranspeptidase ( + -gt) einhergeht. unter dieser therapie wurde nicht nur eine besserung mütterlicher symptome und laborparameter beob-achtet, sondern auch eine verringerung der erkrankungsbedingten frühgeburtlichkeit (palma ) . es wurde keine zunahme toxischer ursodeoxycholsäuremetaboliten im mekonium gefunden. der gallensäuregehalt des mekoniums kann sich durch die mütterliche erkrankung verändern, nicht aber durch die medikation (rodrigues (robert ) . eine studie des european network of teratology information services (entis) mit vornehmlich dexfenfluramin exponierten schwangerschaften erbrachte keine hinweise auf teratogene eigenschaften der appetitzügler (vial ) . publizierte erfahrungen zu sibutramin, das strukurell amphetamin ähnelt und ein wiederaufnahmehemmstoff von serotonin, noradrenalin und (in geringerem maß) dopamin ist, liegen nur in form von bzw. fallberichten vor , kardioglu , aus denen sich kein spezifisches risiko ergibt. wir überblicken zurzeit im . trimenon exponierte schwangere: von lebend geborenen kindern hatten zwei fehlbildungen, eines eine einseitige nierenagenesie, das andere einen vorhofseptumdefekt. orlistat wird aus dem gastrointestinaltrakt kaum resorbiert, so dass teratogene effekte unwahrscheinlich erscheinen. zahlreiche publikationen beschäftigen sich mit dem risiko für schwangerschaftskomplikationen einschließlich fehlbildungen durch mütterliche adipositas (scialli penicilline gehen ungehindert auf den fetus über und lassen sich in der amnionflüssigkeit nachweisen. es gibt bei mehreren tausend ausgewerteten schwangerschaften keine anzeichen dafür, dass die therapie mit penicillinen embryo-oder fetotoxisch wirkt (berkovitch , dencker a, larsen da die clearance von cephalosporinen in der schwangerschaft erhöht ist, müssen ggf. korrekturen von dosis oder dosisintervall vorgenommen werden (heikkilä ) . cephalosporine sind plazentagängig und in der amnionflüssigkeit in bakteriziden konzentrationen nachweisbar. im zusammenhang mit cephalosporinen der zweiten und dritten generation, insbesondere cefotetan, wurden immunhämolytische ereignisse bei den behandelten patientinnen beobachtet (garratty ) . nach bisherigen beobachtungen, z. b. zu cefuroxim im . trimenon (berkovitch ) , verursachen cephalosporine in therapeutischer dosis keine teratogenen schäden (czeizel b da die clearance von lactam-antibiotika und g -lactamase-inhibitoren in der schwangerschaft erhöht ist, müssen ggf. korrekturen von dosis oder dosisintervall vorgenommen werden (heikkilä ) . soweit untersucht, passieren g -lactam-antibiotika oder g -lactamase-inhibitoren die plazenta und erreichen den fetus in relevanten mengen. fehlbildungen oder andere unerwünschte effekte sind bisher weder im tierversuch noch beim menschen beobachtet worden (lewis a, sigg . pivmecillinam hat bei weit über . schwangeren, über davon im . trimenon exponiert, weder eine erhöhte fehlbildungsrate noch andere auffälligkeiten beim neugeborenen gezeigt (vinther sriver toxikologie. weder zu erythromycin a) noch zu azithromycin, clarithromycin (einarson , josamycin, roxithromycin und spiramycin (Übersicht bei briggs , d) hat sich bisher ein ernsthafter verdacht auf teratogene eigenschaften beim menschen ergeben. allerdings wurde kürzlich anhand der daten des schwedischen medizinischen geburtsregisters für erythromycin eine gegenüber phenoxymethylpenicillin schwach signifikant erhöhte fehlbildungsrate bei . kindern mit einer exposition in der frühschwangerschaft beschrieben (källén e, mickal . auch zu clindamycin liegen keine entsprechenden hinweise vor. bedrohlich ist die nach mehrwöchiger behandlung bei - % der patientinnen auftretende pseudomembranöse colitis, die auch nach vaginaler clindamycinanwendung beobachtet wurde (trexler ). schwangerschaftskomplikationen infolge bakterieller vaginosen lassen sich durch eine vaginale clindamycintherapie nicht ausreichend verhüten (joesoef die menschliche folsäurereduktase ist sehr viel weniger empfindlich gegenüber trimethoprim als das bakterielle enzym. dies könnte erklären, dass teratogene schäden durch folsäureantagonistische antibiotika beim menschen bisher nicht nachgewiesen wurden. in einer neueren retrospektiven fall-kontroll-untersuchung wird jedoch wieder eine kausale assoziation diskutiert zwischen der therapie mit trimethoprim und anderen ebenfalls nicht onkologischen folsäureantagonisten wie carbamazepin, phenytoin, phenobarbital, primidon, triamteren und neuralrohrdefekten, kardiovaskulären fehlbildungen, lippen-gaumen-spalten und anomalien der harnwege. die autoren erörtern eine präventive gabe von multivitamin-und folsäurepräparaten (hernandez-diaz ) . tatsächlich hat sich der vorschlag, folsäure während einer antibiotikatherapie mit den hier besprochenen mitteln zu verabreichen, bisher aber nicht überzeugend begründen lassen. von den umfangreichen, eher beruhigenden erfahrungen der anwendung von co-trimoxazol bei banalen harnwegsinfektionen in der schwangerschaft kann nicht auf eine generelle sicherheit der therapie mit einer vielfach höheren dosis bei opportunistischen infektionen wie pneumocystis-carinii-pneumonie (pcp) im rahmen einer hiv-infektion geschlossen werden. bisher wurde nach einer solchen behandlung von schwangeren nicht über ein erhöhtes fehlbildungsrisiko berichtet. zu tetroxoprim liegen noch keine ausreichenden erfahrungen vor. (schaefer ) . zum gleichen ergebnis kamen auch drei andere publikationen mit kleineren fallzahlen (larsen , loebstein , berkovitch . lediglich in einem fall wurde eine neonatale hepatitis mit inkompletter intrahepatischer cholestase nach ofloxacinbehandlung der mutter in woche beobachtet (wiedenhöft ) . die im tierversuch bei jungen hunden nach postnataler (!) behandlung beobachteten irreversiblen gelenkknorpelschäden (gough ) wurden bei präpartal exponierten kindern bisher nicht gesehen. pipemidsäure (deblaston ® ) und nalidixinsäure gehören zu den älteren gyrasehemmstoffen. sie erreichen nur in den ableitenden harnwegen wirksame konzentrationen. gegenüber anderen standardantibiotika haben sich diese mittel jedoch nicht durchsetzen können. hinweise auf teratogene effekte liegen nicht vor. allerdings wurde in einer retrospektiven studie mit den daten des ungarischen fehlbildungsregisters ein möglicherweise zufälliger zusammenhang zwischen pylorusstenose und der behandlung mit nalidixinsäure in der spätschwangerschaft diskutiert. das fehlbildungsrisiko war insgesamt nicht erhöht (czeizel d) . eine weitere publikation zu nalidixinsäure berichtet über den anstieg des intrakraniellen druckes beim fetus (Übersicht bei . empfehlung für die praxis: gyrasehemmstoffe sind in der schwangerschaft kontraindiziert. in wohl begründeten fällen (unkomplizierte harnwegs-und atemwegsinfektionen gehören nicht dazu!), in denen besser erprobte antibiotika nicht wirksam sind, sollten nur gyrasehemmstoffe eingesetzt werden, zu denen erfahrungen an einer größeren zahl von schwangeren vorliegen, z. b. norfloxacin oder ciprofloxacin. die einnahme eines gyrasehemmstoffes rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). nach exposition mit einem der weniger gut untersuchten mittel dieser gruppe im . trimenon wird ein hoch auflösender ultraschall empfohlen. g . . nitrofurantoin und andere harnwegstherapeutika metronidazol besitzt ein experimentell ermitteltes mutagenes und kanzerogenes potenzial (Übersicht bei dobias ) . es bestand deshalb die befürchtung, es könne auch beim menschen mutagen oder kanzerogen wirken. bisher ließen sich derartige effekte nicht bestätigen (burtin , piper . in einer retrospektiven untersuchung wurde nach vorgeburtlicher metronidazol-exposition eine statistisch nicht signifikante assoziation mit neuroblastomen im kindesalter beobachtet (thapa ). eine andere über mehr als jahre laufende untersuchung ergab keinen anhalt für ein erhöhtes malignomrisiko nach metronidazol-behandlung (beard ) . auf der grundlage von über . analysierten schwangerschaften besitzt metronidazol beim menschen kein teratogenes potenzial (diav-citrin b, caro-paton , burtin , piper . hinweise aus dem ungarischen fehlbildungsregister auf einen zusammenhang zwischen vaginaler behandlung mit metronidazol und miconazol im . und . schwangerschaftsmonat und einem vermehrten auftreten von syndaktylien und hexadaktylien haben andere untersucher bisher nicht beobachtet (kazy ) . die zur systemischen behandlung von trichomonaden, amöben und bakterieller vaginose benutzten mittel nimorazol (esclama ® ), ornidazol und tinidazol (simplotan ® ) sind aufgrund der spärlichen datenlage nicht ausreichend zu bewerten. bisher liegen keine hinweise auf teratogenität beim menschen vor (Übersicht in . empfehlung für die praxis: metronidazol darf bei entsprechender indikation auch in der schwangerschaft angewendet werden. dies betrifft auch die systemische therapie, zumal zweifel an der wirksamkeit der vaginalen applikation bestehen. die parenterale gabe ist nur bei bedrohlichen anaerobierinfektionen angezeigt. metronidazol sollte bei der behandlung nimorazol und tinidazol vorgezogen werden. eine behandlung mit nimorazol oder tinidazol rechtfertigt jedoch weder einen risikobegründeten abbruch der schwangerschaft noch invasive diagnostik (siehe kapitel . ). aminoglykoside wirken ototoxisch. pränatale streptomycin-oder kanamycin-injektionen haben zu gehörschäden bei den betroffenen kindern geführt (Übersicht bei . die besonders sensible phase dauert bis zum vierten schwangerschaftsmonat. auch im zusammenhang mit gentamycin wurde ein solcher fall beschrieben (sanchez-sainz-trapaga ). tierexperimentelle ergebnisse sprechen außerdem für eine nephrotoxizität der aminoglykoside, die sich in der fetalen niere anreichern. ein fallbericht über eine konnatale nierendysplasie nach mütterlicher therapie (hulton ) beweist allerdings noch kein klinisch relevantes risiko beim menschen, ebenso wenig der fall einer letal verlaufenden hydronephrose bei verdacht auf hochgradige ureterabgangsstenose nach gentamycin-therapie in schwangerschaftswoche bis und vorangegangener ciprofloxacin-behandlung einer harnwegsinfektion . in einer retrospektiven studie mit daten des ungarischen fehlbildungsregisters wurden keine hinweise auf teratogene effekte der aminoglykoside gefunden. in dieser studie wurden schwangere ausgewertet, die mehrheitlich oral mit neomycin oder parenteral mit gentamycin behandelt worden waren (czeizel c) . empfehlung für die praxis: eine parenterale aminoglykosid-therapie darf nur bei vital bedrohlichen infektionen mit gramnegativen problemkeimen und bei versagen der für die schwangerschaft primär empfohlenen antibiotika erfolgen. die serumkonzentration muss während der therapie regelmäßig kontrolliert werden. eine aminoglykosid-behandlung rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). je nach umfang einer parenteralen therapie muss die hörleistung des kindes frühzeitig kontrolliert werden. da aminoglykoside nach lokaler und oraler applikation praktisch nicht resorbiert werden, ist diese form der anwendung bei entsprechender indikation während der gesamten schwangerschaft zulässig. g . . tuberkulostatische behandlung in der schwangerschaft eine aktive tuberkulose (tbc) muss auch in der schwangerschaft behandelt werden, weil die erkrankung nicht nur die mutter, sondern auch den fetus gefährdet. im gegensatz zu früheren mutmaßungen verschlechtert eine schwangerschaft den verlauf einer tbc nicht (davidson ) . zu den gebräuchlichen tuberkulostatika in der schwangerschaft zählen isoniazid (inh), rifampicin und ethambutol sowie pyrazinamid als reservemittel (american thoracic society ). diese mittel haben sich bisher nicht als teratogen oder fetotoxisch beim menschen gezeigt (bothamley . selbst die therapie einer multiresistenten erkrankung hat sich als gut verträglich für das ungeborene erwiesen (shin (schlagenhauf ) . aufgrund der zunehmenden resistenzen werden bei der malariatherapie zahlreiche kombinationen eingesetzt, vor allem mit artemisininderivaten. empfehlung für die praxis: aufgrund des umfangs an erfahrungen ist chloroquin mittel der wahl zur malariaprophylaxe in der schwangerschaft ggf. in kombination mit proguanil. falls, was zunehmend vorkommt, von beiden mitteln keine ausreichende wirksamkeit zu erwarten ist, sind die anderen mittel je nach resistenzlage anzuwenden. generell muss der beratende arzt mit der patientin erörtern, ob die reise in tropische regionen verschoben werden kann (siehe auch abschnitt . . ). g . . artemisininderivate pharmakologie und toxikologie. in einer studie zu den artemisininderivaten artesunat und artemether wurden bzw. dokumentierte schwangerschaftsverläufe ausgewertet, bei denen eine akute plasmodium falciparum-malaria behandelt wurde. nur bei insgesamt dieser fälle fand die therapie im . trimenon statt (mcgready ) . diese und eine zweite studie mit einmaliger gabe von artesunat plus pyrimethamin-sulfadoxin bei schwangeren fanden keine hinweise auf teratogenität. beim vergleich mit einer nicht exponierten kontroll-gruppe in der zweiten studie waren spontanabortrate und totgeburtenrate nicht signifikant unterschiedlich. bei im . trimenon behandelten war das geburtsgewicht der kinder signifikant höher als in der kontrollgruppe und wurde als erfolg der antiparasitären therapie interpretiert (dean empfehlung für die praxis: chinin darf in der schwangerschaft zur therapie der chloroquinresistenten malaria tropica verwendet werden. in dieser situation ist das potenzielle behandlungsrisiko für den fetus weit geringer als die gefährdung durch die schwere mütterliche erkrankung. auf hypoglykämien muss bei der mutter geachtet werden. auch wenn embryotoxische wirkungen von chinin in analgetischen mischpräparaten nicht zu erwarten sind, sollten derartige mittel gemieden werden, da sie keiner rationalen therapie entsprechen. gleiches gilt für regelmäßigen oder exzessiven konsum von chininhaltigen getränken. g . . chloroquin pharmakologie und toxikologie. chloroquin (z. b. resochin ® , weimerquin ® ), ein malariamittel aus der gruppe der -aminochinoline, verfügt über eine gute und rasche schizontozide wirkung gegen die erythrozytären formen aller plasmodienarten. in vielen malariaendemiegebieten treten zunehmend resistenzen gegen dieses recht gut verträgliche, seit vielen jahrzehnten gebräuchliche medikament auf. diese resistenzen betreffen überwiegend den erreger der schwer und häufig auch tödlich verlaufenden malaria tropica (plasmodium falciparum). aber auch bei plasmodium vivax, dem erreger der weniger schwer verlaufenden malaria tertiana, wurden resistenzen gegen chloroquin beobachtet. in der für die malariaprophylaxe üblichen dosierung und bei der tägigen behandlung des akuten malariaanfalls wirkt chloroquin weder embryo-noch fetotoxisch (phillips-howard ) . zur chloroquinbehandlung rheumatischer erkrankungen siehe abschnitt . . . empfehlung für die praxis: chloroquin kann in allen stadien der schwangerschaft zur infektionsprophylaxe und zur therapie der malaria angewendet werden, sofern eine ausreichende wirksamkeit anzunehmen ist. bei der therapie von pilzinfektionen ist die lokale therapie mit den länger gebräuchlichen substanzen auch in der schwangerschaft als ungefährlich zu betrachten. bei mykosen im genitalbereich soll gleichzeitig der partner behandelt werden. ist eine systemische therapie erforderlich, muss sorgfältig ein mittel aus dieser zumindest hypothetisch riskanten arzneimittelgruppe ausgesucht werden (sobel ) . in letzter zeit ist es "modern" geworden, harmlose pilzbefunde im stuhl wegen vermeintlich damit zusammenhängender, unspezifischer symptome zu behandeln. dies sollte, vor allem in der schwangerschaft, unterbleiben. g . . clotrimazol pharmakologie und toxikologie. clotrimazol (z. b. canesten ® , canifug ® ) ist ein antimykotikum aus der gruppe der imidazolderivate. diese beeinträchtigen die ergosterolbiosynthese und führen damit bei pilzen zu störungen der zellmembranpermeabilität. clotrimazol wird prak-tisch nicht resorbiert und nur zur lokalen therapie von mykosen an haut und schleimhäuten verwendet. es gibt umfangreiche untersuchungen zur behandlung vaginaler mykosen in der schwangerschaft, aus denen sich kein embryotoxisches potenzial ableiten lässt b, king . eine verringerung der frühgeburtenrate durch lokale therapie von vaginosen mit clotrimazol wurde ebenfalls beobachtet (czeizel c) . empfehlung für die praxis: clotrimazol gehört nach nystatin zu den antimykotika der wahl in der schwangerschaft. g . . nystatin pharmakologie und toxikologie. nystatin (z. b. candio-hermal ® , moronal ® ) ist ein bei candidainfektionen (soor) von haut und schleimhäuten wirksames antimykotikum, das nicht resorbiert wird. es wird an ergosterol in der zellmembran von pilzen gebunden und bewirkt dort eine störung der zellmembranpermeabilität. trotz breiter anwendung wurden keine hinweise auf embryo-oder fetotoxische wirkungen beobachtet (king b, czeizel b, king . der erprobungsgrad dieser mittel ist aber geringer als der des clotrimazols. hinweise aus dem ungarischen fehlbildungsregister auf einen zusammenhang zwischen vaginaler behandlung mit miconazol plus metronidazol im . und . schwangerschaftsmonat und einem vermehrten auftreten von syndaktylien und hexadaktylien wurden von anderen untersuchern bisher nicht bestätigt (kazy ) . empfehlung für die praxis: bifonazol, croconazol, econazol, fenticonazol, isoconazol, ketoconazol, miconazol, omoconazol, oxiconazol, sertaconazol und tioconazol sind antimykotika der zweiten wahl für eine lokale therapie. nystatin und clotrimazol sind, wo immer möglich, vorzuziehen. g . . weitere lokal wirksame antimykotika pharmakologie und toxikologie. amorolfin (loceryl ® ), ciclopirox (batrafen ® ), naftifin (exoderil ® ), terbinafin (lamisil ® ), tolciclat und tolnaftat (z. b. tinatox ® ) sind bezüglich ihrer pränatalen toxizität beim menschen nur unzureichend untersucht. substantielle hinweise auf ein erhöhtes fehlbildungsrisiko nach lokaler anwendung liegen bisher nicht vor (sarkar ) . empfehlung für die praxis: eine äußerliche behandlung mit amorolfin, ciclopirox, naftifin, terbinafin, tolciclat und tolnaftat sollte in der schwangerschaft möglichst vermieden werden. eine dennoch erfolgte behandlung rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). nystatin und clotrimazol sollen, wo immer möglich, bevorzugt werden. g . . "conazol-antimykotika" zur systemischen anwendung pharmakologie und toxikologie. itraconazol (z. b. sempera ® , siros ® ) und miconazol sind imidazolderivate. sie beeinträchtigen die ergosterolbiosynthese und führen zu störungen der zellmembranpermeabilität bei pilzen. im tierversuch passieren sie die fetoplazentare schranke gut. fluconazol (z. b. diflucan ® ) und ketoconazol (nizoral ® ) sind triazolderivate, deren wirkung den strukturell verwandten imidazolderivaten entspricht. im tierversuch wurden nach verabreichung sehr hoher dosen teratogene effekte beobachtet. es liegen berichte über sechs kinder mit multiplen fehlbildungen an schädel, skelett und herz vor, deren mütter wegen einer meningitis parallel zu antiretroviralen medikamenten bei hiv-infektion langfristig und hoch dosiert ( - mg/tag) fluconazol erhalten hatten (lopez-rangel , aleck , pursley , lee . die symptome einiger dieser kinder ähnelten dem so genannten antley-bixler-syndrom. kein anhalt für ein erhöhtes fehlbildungsrisiko fand sich hingegen in anderen prospektiven kohortenstudien und retrospektiven untersuchungen mit über frauen, die in den monaten vor oder während einer schwangerschaft meist wegen vaginalmykose niedrig dosiert ( mg/tag) mit fluconazol behandelten wurden (jick , sørensen , campomori , mastroiacovo , inman . eine vom european network of teratology information services (entis) durchgeführte prospektivstudie zu den neueren "conazol-antimykotika" ergab unter schwangeren, die im . trimenon durchschnittlich tage lang wegen vaginal-, haut-oder anderer mykosen mit fluconazol systemisch behandelt worden waren, ebenfalls keine hinweise auf ein teratogenes potenzial (vial, pers. kommunikation ) . die zu itraconazol vorliegenden erfahrungen mit der systemischen behandlung von vaginal-, haut-oder anderen mykosen im . trimenon umfassen drei prospektive kohortenstudien mit , und schwangeren (paulus, pers. mitteilung , vial, pers. kommunikation , bar-oz , aus denen sich keine hinweise auf teratogenität ergeben. das gilt auch für eine kleinere fallzahl aus einer arzneimittelverordnungsstudie (jick ) . die durchschnittliche behandlungsdauer in der entis-studie (vial, pers. kommunikation ) betrug tage. ketoconazol hemmt die steroidsynthese und wird auch zur behandlung des cushing-syndroms eingesetzt. da es außerdem der bildung von testosteron entgegenwirkt, könnte die geschlechtsentwicklung männlicher feten gestört werden. derartiges wurde außerdem bisher aber nicht beobachtet. zwei schwangere mit cushing-behandlung im . und . trimenon wurden von gesunden kindern (ein junge und ein mädchen) entbunden, die auch keine nebennierenrindenanomalien aufwiesen (berwaerts , amado fallberichte beschreiben aborte und pränatal dystrophe frühgeborene. jedoch darf der mögliche einfluss der zugrunde liegenden, z. t. schweren infektionen nicht übersehen werden. auch über normale schwangerschafts-und geburtsverläufe wird berichtet. hinweise auf ein erhöhtes fehlbildungsrisiko liegen bisher nicht vor. für eine differenzierte risikobewertung der parenteralen applikation reichen die vorliegenden fallzahlen nicht aus (ely , Übersicht in dean . die vereinzelt beobachteten vorübergehenden nierenfunktionsstörungen beim neugeborenen könnten durch protrahierte wirkung von arzneimitteldepots in der plazenta und im fetus erklärt werden (dean ) . zur neueren liposomzubereitung des amphotericin b liegt erst ein bericht mit unauffälligem ausgang vor, in dem eine schwangere im . trimenon wegen viszeraler leishmaniose behandelt worden war (king ) . empfehlung für die praxis: amphotericin b darf in der schwangerschaft nur bei bedrohlichen, generalisierten mykosen parenteral eingesetzt werden. wurde während der organogenese behandelt, rechtfertigt dies keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). in einem solchen fall sollte jedoch zur bestätigung der normalen entwicklung des fetus ein hoch auflösender ultraschall angeboten werden. die lokale anwendung ist unbedenklich. g . . flucytosin pharmakologie und toxikologie. flucytosin (ancotil ® ) hat eine gute wirkung gegen cryptococcus neoformans und viele candidaspezies. es wird bei systemischen infektionen mit diesen erregern eingesetzt und wirkt durch hemmung der dna-synthese. in der pilzzelle wird flucytosin unter anderem zu dem zytostatikum -fluorouracil metabolisiert. diese reaktion ist in geringerem ausmaß auch im menschlichen organismus zu erwarten. im tierversuch wirkt flucytosin in dosen teratogen, die niedriger sind als die humantherapeutisch üblichen. fehlbildungen wurden beim menschen bisher nicht beschrieben, allerdings gibt es praktisch keine publizierten erfahrungen zur verabreichung von flucytosin im . trimenon. wenige erfahrungen zur anwendung im . und . trimenon bei bedrohlicher disseminierter cryptococcose erbrachten keine hinweise auf fetale störungen (ely (reuvers , cowden , ausreichende erfahrungen liegen jedoch nicht vor. in endemiegebieten wurde eine vorteilhafte wirkung auf mütterliche anämie, geburtsgewicht und neonatale sterblichkeit bei verabreichung im . und . trimenon beobachtet (christian . ein nennenswertes embryotoxisches risiko beim menschen ist nach zwei neueren studien mit und im . trimenon exponierten schwangeren nicht zu erkennen (reuvers , de silva . in einer weiteren studie wurden kinder mit fehlbildungen unter ausgetragenen schwangerschaften beobachtet. art und zahl der fehlbildungen weckten keinen verdacht auf teratogene effekte . tierexperimentell hat mebendazol bei der maus teratogene effekte erkennen lassen. flubendazol, dem mebendazol strukturell ähnlich, zeigte bei im . trimenon exponierten kindern keine teratogenen eigenschaften (reuvers (chippaux , pacque , erlauben jedoch keine differenzierte risikobetrachtung. empfehlung für die praxis: ivermectin darf bei zwingender indikation auch in der schwangerschaft eingesetzt werden. g . . aciclovir und andere herpes-virustatika pharmakologie und toxikologie. aciclovir (z. b. zovirax ® ) wirkt über eine hemmung der viralen dna-polymerase bei varizellen sowie bei herpesviren typ und typ . bisherige erfahrungen mit mehreren tausend vom hersteller gesammelten und in der fachliteratur publizierten prospektiv oder retrospektiv erfassten verläufen, davon etwa die hälfte im . trimenon, lassen kein embryo-oder fetotoxisches risiko von aciclovir bei systemischer anwendung erkennen (stone , ratanajamit . dies gilt vorbehaltlich der methodischen schwächen von schwangerschaftsregistern beim arzneimittelhersteller. nach äußerlicher acicloviranwendung werden nur geringe substanzmengen resorbiert. zu famciclovir (famvir ® ), einem prodrug des penciclovir, liegen keine dokumentierten erfahrungen beim menschen vor. ganciclovir (cymeven ® ) ist tierexperimentell in dosen embryotoxisch, die den therapeutischen beim menschen entsprechen. einzelne fallberichte beschreiben einen normalen schwangerschaftsausgang nach therapie in der frühschwangerschaft (pescovitz ). eine fallsammlung des herstellers mit mit valaciclovir (valtrex ® ) exponierten schwangeren, davon im . trimenon, und eine fallserie mit zehn frauen, die ab woche bis zur geburt behandelt wurden, zeigten keine hinweise auf embryo-oder fetotoxische schäden (glaxo- wellcome , kimberlin . zusammenfassend sind famciclovir, ganciclovir sowie valaciclovir und valganciclovir (ester bzw. prodrugs von aciclovir und ganciclovir) hinsichtlich ihrer verträglichkeit in der schwangerschaft unzureichend untersucht. empfehlung für die praxis: die äußerliche anwendung von aciclovir ist unproblematisch. die systemische gabe ist nur dann indiziert, wenn beispielsweise bei disseminierter herpes-oder varizellenerkrankung die mutter gefährdet ist oder wenn davon auszugehen ist, dass der fetus durch die therapie vor einer intrauterinen infektion geschützt wird. die anderen virustatika sind nur bei infektionen indiziert, bei denen sie eine therapeutische Überlegenheit gegenüber aciclovir gezeigt haben. eine therapie im . trimenon rechtfertigt weder einen risikobegründeten abbruch der schwangerschaft noch invasive diagnostik (siehe kapitel . ). ein hoch auflösender ultraschall ist -mit ausnahme von aciclovir -nach anwendung im . trimenon zu empfehlen. g . . amantadin pharmakologie und toxikologie. amantadin (z. b. amanta ® ) verstärkt die dopaminaktivität am rezeptor und wird deshalb als antiparkinsonmit-tel eingesetzt. als virustatikum wirkt es in erster linie gegen influenza-a-viren. im tierversuch ist amantadin in hohen dosen teratogen. beim menschen wurden verschiedene fehlbildungen nach behandlung mit amantadin beschrieben, die allerdings kein typisches muster erkennen lassen (Übersicht in . andererseits erlauben die ebenfalls dokumentierten unauffälligen verläufe keinen ausschluss eines teratogenen risikos. empfehlung für die praxis: amantadin ist in der schwangerschaft kontraindiziert. wurde während der organogenese behandelt, rechtfertigt dies keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). in einem solchen fall sollte jedoch zur bestätigung der normalen entwicklung des fetus ein hoch auflösender ultraschall angeboten werden. g . . ribavirin pharmakologie und toxikologie. ribavirin (z. b. rebetol ® , virazole ® ) hat experimentell ein breites antivirales spektrum, die klinische relevanz ist jedoch begrenzt. es wird u. a. bei rs-viruserkrankungen im säuglingsalter und in kombination mit § -interferonen bei hepatitis c eingesetzt. im tierversuch ist ribavirin bei mehreren spezies teratogen und wirkt experimentell mutagen. die kinder von schwangeren, die in der zweiten schwangerschaftshälfte wegen schwerer masernverläufe behandelt worden waren, wiesen keine fetotoxischen effekte auf (atmar ). Über weitere fälle mit einer behandlung in der spätschwangerschaft und einen fall mit prophylaktischer intramuskulärer verabreichung von injektionen im . trimenon wegen sars (severe acute respiratory syndrome) wird berichtet (rezvani g . . hiv-therapie und prophylaxe in der schwangerschaft das ziel einer antiretroviralen therapie (art) in der schwangerschaft ist einerseits die verhinderung der mutter-kind-Übertragung von hiv, andererseits die optimale behandlung der schwangeren bei möglichst geringen unerwünschten wirkungen der medikamente auf die schwangere und das ungeborene kind. unumstritten ist der protektive wert einer perinatalen prophylaxe mit dem nucleosidanalogen reverse-transkriptase-inhibitor (nrti) zidovudin zur vermeidung der vorwiegend unter der geburt möglichen mutter-kind-Übertragung (vertikale transmission) von hiv (connor pharmakologie und toxikologie. zidovudin (retrovir ® ), auch als azidothymidin (azt) bezeichnet, ist das älteste zur antiretroviralen therapie (art) eingesetzte virustatikum. es hemmt als nukleosidanaloger reverse-transkriptase-inhibitor (nrti) kompetitiv die vermehrung von hiv- -und hiv- -viren. zidovudin ist gut plazentagängig, die nachgewiesene metabolisierung in der plazenta erklärt möglicherweise den transmissionshemmenden effekt der substanz. die bisher vorliegenden erfahrungen mit mehreren . dokumentierten schwangerschaftsverläufen sprechen gegen ein nennenswertes teratogenes potenzial (watts , mofenson ) . nach den daten des antiretroviral pregnancy registry war die fehlbildungsrate mit , % nicht erhöht. es wird diskutiert, ob zidovudin und andere nrtis mitochondriale dysfunktionen verursachen können. blanche und mitarbeiter ( ) berichten über nicht hivinfizierte kinder mit solchen auffälligkeiten, die intrauterin und nach der geburt mit zidovudin allein oder zusammen mit lamivudin exponiert waren. zwei der kinder starben noch im säuglingsalter, weitere wiesen eine neurologische symptomatik auf (blanche ) . andere kohortenstudien konnten diese medikamenteninduzierten mitochondriopathien nicht bestätigen. tierexperimentelle befunde an affen ergaben strukturelle und funktionelle störungen der mitochondrienfunktion in herz-und skelettmuskelzellen der jungtiere, nachdem die muttertiere beim menschen übliche zidovudindosen erhalten hatten. ent-sprechende herzanomalien konnten bei pränatal exponierten kindern nicht nachgewiesen werden. in einer prospektiven studie fand sich bei mit zidovudin exponierten kindern, die bis zum alter von jahren echokardiographisch untersucht wurden, kein hinweis auf kardiale toxizität (lipshultz ) . die einzige wiederholt beobachtete auffälligkeit bei neugeborenen nach intrauteriner zidovudinexposition ist das auftreten einer vorübergehenden anämie, die sich innerhalb der ersten lebenswochen normalisiert (sperling , connor . in einer nachsorgestudie von kindern des pediatric aids clinical trials group protocol (pactg ) zeigten kinder mit einer intrauterinen zidovudinexposition keine auffälligkeiten ihrer körperlichen, immunologischen und kognitiven parameter bis zum . lebensjahr (curlane ). es fanden sich auch keine hinweise auf tumorbildung bei über prä-und perinatal exponierten kindern (curlane , hanson (graham , edwards . auch beim menschen wurde dies immer wieder erörtert. es gibt hinweise darauf, dass verschiedene anomalien, wie z. b. neuralrohrdefekte (suarez , shaw ), aber auch nieren-, herz-und bauchwandfehlbildungen (abe , chambers nach fieberhaften infekten in der frühschwangerschaft häufiger auftreten. moretti und mitarbeiter ( ) haben in einer meta-analyse zum risiko von neuralrohrdefekten nach hyperthermie insgesamt studien mit . fällen ausgewertet und sowohl in den einbezogenen fall-kontroll-studien als auch in kohorten-studien signifikante assoziationen mit einem odds ratio von etwa , ermittelt. abe und mitarbeiter ( ) haben in einer retrospektiven studie mit wenigen dutzend betroffenen schwangeren schwache signifikanzen sowohl für fieber als auch generell für infekte im . trimenon ermittelt. chambers und mitarbeiter ( ) diskutiert wird u. a., dass hohe körpertemperaturen vaskuläre störungen verursachen können, so dass die sich entwickelnden organe vorübergehend nicht ausreichend durchblutet werden (graham ). suarez und mitarbeiter ( ) haben in ihrer retrospektiven studie, die von neuralrohrdefekten betroffene familien sowie gesunde kontrollen einschließt, ein geringeres risiko bei einnahme fiebersenkender medikamente beobachtet. es fanden sich auch schwache signifikanzen bei anderen potenziell zur Überwärmung führenden faktoren, wie z. b. heizdecken, sauna und kochen in überwärmter küche während des . andere untersuchungen belegen kein erhöhtes risiko durch saunen, obwohl schon nach - minuten die körpertemperatur auf über , °c steigen kann. in finnland, wo dieser frage nachgegangen wurde, ist häufiges saunen auch während der schwangerschaft üblich. auch der gebrauch elektrischer heizdecken und geheizter wasserbetten hat bei anderen untersuchern kein signifikant erhöhtes fehlbildungsrisiko erkennen lassen. eine weitere studie hat bei kindern im alter von und jahren häufiger defizite im emotionalen und kognitiven bereich beobachtet, wenn im . oder . trimenon fieber berichtet wurde (dombrowski die durch impfstoffe angeregte spezifische immunität der mutter schützt auch das kind durch diaplazentaren Übergang der mütterlichen igg-antikörper. bisher haben sich für keinen impfstoff entwicklungstoxische eigenschaften gezeigt und bei keinem lebendimpfstoff hinweise auf eine infektionsbedingte schädigung des ungeborenen. der umfang an dokumentierten erfahrungen ist jedoch für die einzelnen impfstoffe sehr unterschiedlich. generell sollten routineimpfungen in der schwangerschaft, insbesondere solche mit lebendimpfstoffen, unterbleiben. besteht aber ein erkennbares expositionsrisiko und liegt kein impfschutz vor, kann und muss ggf. auch während der schwangerschaft im interesse von mutter und kind geimpft werden (siehe unter den jeweiligen impfstoffen). mehr oder weniger detailliert wird in verschiedenen impfempfehlungen auf schwangere eingegangen (z. b. empfehlungen der ständigen impfkommission -stiko). bei fragestellungen zum risiko von tropischen impfungen und malariaprophylaxe in der schwangerschaft sollten auch die allgemeinen risiken von fernreisen mit der schwangeren erörtert werden (siehe abschnitt . . ). wenn eine impfung tatsächlich indiziert ist, muss diese selbstverständlich auch in der schwangerschaft durchgeführt werden. , silveira . auch konnte kein zusammenhang mit fehlgeburten beobachtet werden. catindig und mitarbeiter ( ) nach impfung mit dem fsme-impfstoff haben sich bisher keine hinweise auf entwicklungstoxische effekte beim menschen gezeigt. empfehlung für die praxis: da dieser impfstoff nicht systematisch untersucht ist, sollte während einer schwangerschaft die impfindikation kritisch geprüft werden. g . . gelbfieberimpfung der gelbfieberimpfstoff enthält einen abgeschwächten lebendimpfstoff. ein fallbericht beschreibt eine gelbfieberinfektion beim neugeborenen im zusammenhang mit einer impfung im . trimenon (tsai ) . dieser befund wurde von anderer seite nicht bestätigt. in einer untersuchung an geimpften schwangeren, davon vier im . und im . trimenon, erbrachte keine entwicklungsauffälligkeiten bei den kindern bis zum alter von - jahren (nasidi ) . eine leicht erhöhte spontanabortrate wird in einer anderen, allerdings sehr kleinen retrospektiven studie mit schwangeren beschrieben (nishioka ) . unter im . trimenon geimpften wurde keine konnatale infektion und kein hinweis auf teratogene effekte gefunden . bisherige erfahrungen sprechen also gegen ein nennenswertes entwicklungstoxisches risiko der impfung. empfehlung für die praxis: da gelbfieber im erkrankungsfall lebensbedrohlich sein kann, muss eine schwangere bei unaufschiebbarer reise in ein endemiegebiet auch im . trimenon geimpft werden (american college of obstetricians and gynecologists ). g . . bisher publizierte fallserien mit mehr als . frauen, die kurz vor oder während der schwangerschaft geimpft wurden, ergeben keine hinweise auf eine entwicklungstoxische wirkung dieses inaktivierten impfstoffs (munoz , deinhard . der fall einer zns-fehlbildung nach impfung in woche (sarnat ) wurde von anderen untersuchern nicht bestätigt. das us-amerikanische "advisory committee on immunization practices of the centers for disease control" empfahl , schwangere während der grippesaison aufgrund möglicher influenzabedingter komplikationen unabhängig vom trimenon zu impfen. empfehlung für die praxis: wenn andere beeinträchtigende faktoren, wie z. b. asthma, vorliegen oder in der vorgeschichte wiederholt von influenzainfektionen berichtet wird, sollten schwangere geimpft werden, ggf. auch im . trimenon. g . . systematische untersuchungen zur pränataltoxizität dieses inaktivierten impfstoffs liegen nicht vor. ein passiver schutz vor einer möglicherweise bedrohlichen infektion im säuglingsalter ist durch diaplazentaren Übertritt mütterlicher antikörper nach impfung der mutter im . trimenon möglich (glezen ) . empfehlung für die praxis: eine impfempfehlung für schwangere wird zurzeit noch diskutiert. g . . als hepatitis-b-impfstoff wird ein biotechnologisch hergestelltes, nicht vermehrungsfähiges oberflächenantigen eingesetzt. die bisher publizierten verläufe nach impfungen von über schwangeren zeigten keine unerwünschten wirkungen beim fetus (ingardia , reddy , grosheide . bei nahezu % der schwangeren mit serokonversion nach einer impfung während der schwangerschaft wurden auch im nabelschnurblut protektive antikörperkonzentrationen gefunden (ingardia ) . systematische untersuchungen zum hepatitis-a-impfstoff in der schwangerschaft liegen nicht vor. wahrscheinlich ist dieser totimpfstoff ähnlich zu beurteilen wie der hepatitis-b-impfstoff. (boettiger ) . die rötelnimpfung wird mit dem zurzeit gebräuchlichen abgeschwächten lebendimpfstoff ra / durchgeführt. dieser ist auch in den kombinationsimpfstoffen mit masern und mumps (mmr) enthalten. der rötelnimpfvirus ist plazentagängig und kann den fetus infizieren. bei , % bis , % (durchschnitt , %) in der frühschwangerschaft geimpfter fällt später der nachweis von rötelnspezifischen igm-antikörpern im nabelschnurblut beim neugeborenen positiv aus (enders ) . in einem fall wurde eine persistierende subklinische infektion beobachtet (hofmann ) . in ca. - % konnte das impfvirus nach impfung aus abortmaterial isoliert werden , center for disease control . die einzelfalldarstellung eines angeborenen katarakts nach mütterlicher impfung konnte nicht durch andere untersucher bestätigt werden (fleet ) . berichte über insgesamt schwangerschaften aus deutschland, schweden, england und den usa wurden bisher publiziert, bei denen seronegative frauen im zeitraum von monaten vor konzeption bis in die schwangerschaft hinein versehentlich mit unterschiedlichen rötelnlebendimpfstoffen geimpft wurden (enders . im zeitraum mit theoretisch hohem risiko, also ein bis wochen vor bis wochen nach konzeption, wurden der schwangeren geimpft. es wurde dabei keine rötelnembryopathie beobachtet. dies spricht dafür, dass eine impfbedingte schädigung sehr unwahrscheinlich ist. rein statistisch kann man mit einer solchen fallzahl jedoch nur ausschließen, dass das risiko größer als , % ( % konfidenzintervall) ist. empfehlung für die praxis: eine rötelnimpfung sollte unmittelbar vor und während der schwangerschaft nicht durchgeführt werden. bisherige erfahrungen sprechen gegen ein risiko für rötelnembryopathie durch impfung. daher ergeben sich aus einer versehentlichen impfung keine konsequenzen wie schwangerschaftsabbruch oder invasive diagnostik. ob eine seronegative schwangere mit hohem expositionsrisiko gegenüber röteln auch während der schwangerschaft geimpft werden sollte, muss individuell entschieden werden. g . . tollwutimpfung der tollwutimpfstoff enthält einen abgeschwächten lebendimpfstoff, der aus menschlichen zellkulturen gewonnen wird. der heute zur verfügung stehende impfstoff ist im gegensatz zu früheren tollwutimpfstoffen gut verträglich. fallberichte zur aktiven und/oder passiven impfung bei über schwangeren zeigen keine auffälligkeiten (chutivongse , chabala , fescharek ). die mütterlichen antikörper scheinen die plazenta zu überwinden. empfehlung für die praxis: da tollwut eine tödlich verlaufende erkrankung ist, muss eine schwangere nach einem tollwutverdächtigen tierbiss immer simultan (aktiv und passiv) geimpft werden. g . . typhusimpfung es gibt zwei typhusimpfstoffe: die parenteral zu verabreichende inaktivierte typhusvakzine und den oralen typhus-lebendimpfstoff mit salmonella typhi typ a. der lebendimpfstoff schützt nicht gegen paratyphus a und b, weist aber eine geringere nebenwirkungsrate auf als die inaktivierte vakzine. bei einer typhuserkrankung in der schwangerschaft ist durch die typhöse septikämie das abortrisiko erhöht. deshalb ist auch für schwangere der schutz vor einer infektion ratsam, vor allem bei einem längeren aufenthalt in entsprechenden ländern. eine untersuchung mit rund schwangeren, die den lebendimpfstoff im . trimenon erhielten, erbrachte keine spezifischen auffälligkeiten (mazzone ) . bei entsprechender indikation darf eine schwangere geimpft werden. die erstinfektion mit varizellen in der schwangerschaft kann in etwa % der fälle zu schäden beim embryo bzw. fetus führen. dieser verdacht hat sich bisher aber nicht nach impfung mit diesem lebendimpfstoff ergeben. bei vom hersteller prospektiv dokumentierten schwangerschaften (merck/cdc pregnancy registry ) wurden insgesamt fehlbildungen unter lebendgeborenen beobachtet ( , %), unter den seronegativen waren es ( , %) und begrenzt auf die seronegativen mit impfung im . oder . trimenon / ( , %). diese fehlbildungsraten wurden als nicht statistisch signifikant erhöht bewertet. außerdem war keine varizellenembryopathie unter den fehlbildungen. empfehlung für die praxis: während einer schwangerschaft sollte nicht geimpft werden. bei dennoch erfolgter anwendung sind keine konsequenzen erforderlich. g . . immunglobuline immunglobulinlösungen enthalten hauptsächlich immunglobulin-g-(igg-)antikörper und werden aus gepooltem menschlichem plasma hergestellt. das ausmaß der plazentapassage von igg-antikörpern ist abhängig vom gestationsalter, der dosierung, der dauer der behand-lung und der art des verabreichten präparates. immunglobuline kommen bei sehr unterschiedlichen mütterlichen oder fetalen indikationen zum einsatz, z. b. bei antikörpermangel, bei infektionserkrankungen (insbesondere zur prävention), bei autoimmunkrankheiten zur besserung der symptome bei der mutter oder bei der behandlung fetaler krankheitssymptome, wie z. b. dem fetalen herzblock bei mütterlichem lupus erythematodes. sowohl immunglobuline als auch hyperimmunseren gegen spezifische infektionen wirken nach heutiger erkenntnis nicht embryotoxisch. unspezifische risiken durch menschliche blutprodukte wie die Übertragung von virusinfektionen und anaphylaxie sind nicht völlig auszuschließen und könnten mittelbar auch den fetus gefährden. eine untersuchung an kindern von müttern, die gammaglobulin zur hepatitisprophylaxe während der schwangerschaft erhalten hatten, beschreibt signifikant gehäufte veränderungen der hautlinien an den fingerkuppen der pränatal exponierten kinder (ross ) . diese kaum als fehlbildungen zu bewertenden effekte traten nur dann auf, wenn die exposition in den ersten tagen der schwangerschaft erfolgte. dieser bericht ist eher als anekdotisch zu betrachten. empfehlung für die praxis: standard-gammaglobulin und hyperimmunseren dürfen bei gegebener indikation auch während der schwangerschaft verabreicht werden. g . . thiomersalhaltige impfstoffe die früher in manchen impfstoffen als konservierungsstoff enthaltenen mengen an thiomersal bzw. ethylquecksilber (ca. ? g) sind kürzlich als gefahrenpotenzial diskutiert worden (bigham , clements ). bei genauerer betrachtung ergab sich, dass die mengen an ethylquecksilber, zumal nach einmaliger verabreichung, sehr gering sind. ein vergleich mit dem für den menschen riskanteren methylquecksilber muss berücksichtigen, dass dieses die blut-hirn-schranke leichter überwindet. bisher liegen keine fallberichte vor, die den verdacht einer pränatalen schädigung durch eine thiomersalhaltige impfung begründen. dies erscheint plausibel, wenn man andere situationen mit erhöhter quecksilberbelastung zum vergleich heranzieht, wie die in manchen ländern höhere "hintergrundbelastung" mit quecksilber durch regelmäßigen verzehr kontaminierter fische. die who empfiehlt thiomersalhaltige impfstoffe für die so genannte dritte welt, da sie dort leichter verfügbar, billiger, sicherer und wirksamer sind (bigham ). während der schwangerschaft kommt es zu gravierenden hämodynamischen veränderungen. ab schwangerschaftswoche nimmt das blutvolumen zu, am ende der schwangerschaft beträgt die steigerung %. sowohl gefäßwiderstand als auch blutdruck sinken, und der ruhepuls steigt um - schläge pro minute. daraus resultiert ein - %iger anstieg herzminutenvolumens. während der geburt kommt es zu einer weiteren zunahme des auswurfvolumens und der blutdruck steigt. im allgemeinen werden ein bis drei tage nach geburt, manchmal auch erst nach einer woche, die hämodynamischen ausgangswerte erreicht (oakley ) . während herzkrankheiten in der schwangerschaft selten sind (unter %), kommen behandlungsbedürftige hypertone und hypotone regulationsstörungen häufiger vor. g . . bei den hochdruckkrankheiten schwangerer unterscheidet man folgende formen: chronische hypertonie (mit oder ohne proteinurie), die vor, während oder nach der schwangerschaft diagnostiziert wird. präeklampsie, eklampsie: proteinurie ( g mg/ h) und neu aufgetretene hypertonie (fakultativ: Ödeme). pfropfgestose: präeklampsie bei schwangeren mit chronischer hypertonie (häufigkeit: bei - % der schwangeren mit chronischer hypertonie). schwangerschaftshochdruck: eine nach schwangerschaftswochen entstehende hypertonie ohne proteinurie, die sich spätestens wochen nach der entbindung zurückbildet. ungefähr die hälfte dieser schwangeren entwickelt eine präeklampsie. ein blutdruckwert von / mmhg gilt als grenzwert für eine hypertonie in der schwangerschaft. patientinnen haben ein niedriges risiko, wenn die werte im grenzbereich liegen, wenn sie keine auffälligkeiten bei der körperlichen untersuchung bieten, ein normales ekg und echokardiogramm aufweisen und keine proteinurie besteht. eine antihypertensive therapie von schwangeren mit blutdruckwerten bis / mmhg stellt keinen vorteil für den schwangerschaftsverlauf und das befinden der mutter dar, so dass eine medikamentöse behandlung nicht indiziert ist. komplikationen schwerer hypertoner zustände sind hirnblutungen der mutter oder kardiale probleme. vor allem über eine plazentadysfunktion sind abruptio der plazenta, frühgeburt, wachstumsretardierung und perinataler tod assoziiert. das risiko für eine schwangere und das werdende kind ist bei einer hypertonie mit gefäß-bzw. organschäden, mit kardiovaskulären begleiterkrankungen und/oder mit proteinurie, das heißt bei jeder form der präeklampsie, als hoch einzuschätzen. man nimmt an, dass die präeklampsie auf einer gestörten interaktion zwischen trophoblastinvasion und dezidua beruht. die daraus folgende mangelnde dilatation der spiralarterien kann zur plazentaren hypoperfusion führen. eine kausale therapie außer der geburt gibt es nicht. blutdrucksenkung bei diastolischen werten g mmhg und die gabe von mg/tag acetylsalicysäure sind möglichkeiten einer konservativen therapie, die nur unter strikter kontrolle der fetoplazentaren einheit erfolgreich sein kann. das hellp-syndrom (hämolyse, erhöhte leberwerte, erniedrigte thrombozytenzahl) beinhaltet zusätzliche risiken für mutter und fetus. eine große prospektive studie mit fast . hypertensiven schwangeren bestätigt, dass das fetale risiko bei präeklampsie und pfropfgestose deutlich höher ist als bei den beiden anderen hypertonieformen (ray ) . welche klinische bedeutung der in-vitro-untersuchung von houlihan ( ) zukommt, muss offen bleiben: labetalol, hydralazin, nifedipin und magnesiumsulfat haben einen signifikant relaxierenden effekt auf die nabelarterie, dagegen führt methyldopa zu keiner Änderung des gefäßwiderstandes. eine metaanalyse zu möglichen Änderungen der fetalen und neonatalen herzfrequenz bei mütterlicher antihypertensiver medikation kommt zu dem schluss, dass die vorliegenden daten zu nifedipin, hydralazin, labetalol und methyldopa zu ungenau für eine endgültige aussage sind (waterman ). die auswahl antihypertensiver medikamente unterscheidet sich von einer behandlung außerhalb der schwangerschaft. trotz vielfältiger untersuchungen und erfahrungen gibt es jedoch nach wie vor keine einheitlichen empfehlungen für schwangere. systematische kontrollierte studien mit großer fallzahl und exposition im . trimenon sind rar. als langzeitantihypertensivum bei chronischer hypertonie kommt in erster linie methyldopa infrage. mittel der zweiten wahl sind metoprolol, dihydralazin/hydralazin und nifedipin. bei den mutter und fetus mehr gefährdenden, durch präeklampsie bedingten hochdruckformen haben sich dihydralazin, nifedipin und urapidil bewährt. auch g -rezeptorenblocker können gegeben werden, von denen das in deutschland nicht zur verfügung stehende labetalol am besten untersucht ist. hypertensive erkrankungen in der schwangerschaft erfordern vor allem eine spezialisierte diagnostik, die dann eine ggf. indizierte therapie begleiten bzw. steuern kann. pindolol (visken ® ) neben einer g -auch eine g -blockierende wirkung. gute erfahrungen werden auch mit labetalol beschrieben, das eine zusätzliche § -rezeptor-blockierende komponente besitzt. alle g -rezeptorenblocker passieren die plazenta. nach heutiger erkenntnis haben sie keine teratogenen eigenschaften. zwar gibt es einen bericht zu neugeborenen, die im . trimenon atenolol exponiert waren und von denen kinder große fehlbildungen aufwiesen. die uneinheitlichkeit dieser fehlbildungen sowie die ergebnisse anderer untersuchungen sprechen aber gegen einen ursächlichen zusammenhang . in unserem pharmakovigilanzzentrum konnten wir bisher mehr als schwangerschaften prospektiv nachverfolgen, die im . trimenon metoprolol exponiert waren. unter lebend geborenen kindern wiesen grobstrukturelle fehlbildungen auf ( %): je zwei fälle mit gaumenspalte und vorhofseptumdefekt, je ein fall mit einer stenose der a. pulmonalis, einer zwerchfellhernie und einer polyzystischen niere. atenolol kann zu einem geringeren gewicht der plazenta, einer intrauterinen wachstumsverzögerung und einem geringeren geburtsgewicht führen (tabacova ). diskutiert wird, dass andere g -rezeptorenblocker dieselben symptome verursachen können (magee ) . es ist nicht endgültig geklärt, welchen anteil an der möglicherweise zugrunde liegenden plazentaren perfusionsminderung atenolol (oder ein anderer g -rezeptorenblocker) oder der behandlungsbedürftige hypertonus hat. g -rezeptorenblocker erhöhen -wenn auch nur sublim -den tonus des uterus und können auf diese weise die perfusion reduzieren. auch die blutzuckersenkende wirkung der betablocker wird als ursache diskutiert. das postnatale wachstum im ersten lebensjahr ist offenbar ebenso wenig beeinträchtigt wie die übrige entwicklung der kinder (reynolds ). bayliss ( ) untersuchte schwangerschaften mit hypertonie, dabei wurde in fällen mindestens ein antihypertensivum eingenommen. unbehandelte schwangere dienten als kontrollgruppe. das ergebnis ist interessant: nur die neugeborenen, deren mütter atenolol zur konzeption oder ab dem . trimenon (n= ) bis zur geburt eingenommen hatten, wiesen ein statistisch signifikant niedrigeres geburtsgewicht auf. atenolol im . trimenon führte nicht zu diesem effekt. unabhängig vom eingenommenen antihypertensivum stand eine pfropfgestose (im / . trimenon) in zusammenhang mit einem geringeren geburtsgewicht. bei einem kind wird eine retroperitoneale fibromatose mit medullärer kompression und später resultierender skoliose in zusammenhang mit der mütterlichen atenololtherapie beschrieben. die autoren halten diese assoziation für erwähnenswert, da analoge befunde bei erwachsenen nach atenololexposition beschrieben wurden (satgé ). zu sotalol siehe abschnitt . . . es gibt keine ausreichenden erfahrungen zu alprenolol, betaxolol . ein nennenswertes teratogenes risiko ist auch bei diesen g -rezeptorenblockern unwahrscheinlich. eine neonatale g -rezeptorenblockade infolge mütterlicher therapie ist theoretisch bei jeder substanz zu erwarten und kann sich in erniedrigter herzfrequenz und hypoglykämie äußern. ein fallbericht beschreibt sogar unter augentropfenanwendung von , %igem timolol eine fetale bradykardie und arrhythmie bei schwangerschaftswochen, die sich unter dosishalbierung und anschließendem absetzen besserte (wagenvoort ). weitere fälle wurden trotz häufiger glaukombehandlung mit diesen mitteln in der schwangerschaft nicht berichtet. atemdepression bei neugeborenen wurde unter intravenöser gabe von propranolol kurz vor der schnittentbindung beobachtet (Überblick in , ist aber eher eine ausnahmeerscheinung. ein absetzen der medikation - stunden vor der entbindung wird von manchen autoren erörtert. dieses vorgehen ist kaum zu rechtfertigen. die meist nur milden symptome einer g -rezeptorenblockade bessern sich beim neugeborenen innerhalb von stunden folgenlos. dennoch sollten sich geburtshelfer und pädiater über die mütterliche medikation informieren. die verstärkung vorzeitiger wehentätigkeit durch g -rezeptorenblocker ist theoretisch denkbar. es wurde jedoch bei gabe von g -rezeptorenblockern während einer tokolyse mit g -sympathomimetika kein negativer einfluss auf die wehenhemmung beschrieben (trolp ). insgesamt muss davon ausgegangen werden, dass ein den blutzucker senkender effekt einerseits und eine leicht erhöhte uteruswandspannung andererseits basis des immer wieder beobachteten geringeren geburtsgewichts sind. empfehlung für die praxis: g -rezeptorenblocker gehören zu den antihypertensiva der wahl in der schwangerschaft, wobei erprobte mittel wie metoprolol zu bevorzugen sind. atenolol sollte eher nicht verordnet werden. timolol darf zur glaukombehandlung in der gesamten schwangerschaft angewendet werden. eine therapie mit atenolol oder wenig untersuchten g -rezeptorenblockern rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). eine gewichtsrestriktion des fetus durch einnahme von g -rezeptorenblockern durch die mutter ist möglich. mit perinatalen auswirkungen wie herzfrequenzabnahme und hypoglykämie muss bei allen g -rezeptorenblockern gerechnet werden, wenn bis zur geburt behandelt wurde. in einzelnen fällen wurden nach gabe von § -methyldopa während der schwangerschaft hepatotoxische effekte beobachtet (smith ) . einer weiteren untersuchung zufolge kann bei neugeborenen nach präpartaler behandlung der mutter in den ersten beiden lebenstagen ein um - mmhg erniedrigter blutdruck auftreten, der jedoch klinisch keine relevanz besitzt (whitelaw ). § -methyldopa hatte in einer in-vitro-untersuchung keinen einfluss auf den gefäßwidersand der nabelarterie (houlihan ). günenç ( ) analysierte mit hilfe der dopplersonographie den effekt von methyldopa bei schwangeren mit präeklampsie. der gefäßwiderstand der arteria uterina wurde durch die therapie herabgesetzt, jedoch nicht von nabelarterien oder fetaler arteria cerebri media. empfehlung für die praxis: § -methyldopa ist eines der ältesten antihypertensiva, das auch in der schwangerschaft für mutter und ungeborenes gut verträglich ist. es ist das mittel der . wahl bei chronischer hypertonie in der schwangerschaft. signifikante unterschiede zur kontrollgruppe ergaben sich bei der frühgeburtsrate. ferner ließ sich sowohl bei den früh geborenen als auch bei den reif geborenen kindern in einigen der zentren eine tendenz zu einem geringeren geburtsgewicht feststellen. diese effekte sind am ehesten durch die art und schwere der meist zugrunde liegenden plazentaren störungen zu erklären und nicht durch die medikamentenexposition (weber-schöndorfer ) . nifedipin sollte nicht in kombination mit magnesium i.v. gegeben werden (vetter , waismann , da dies zu gravierendem blutdruckabfall mit fetaler hypoxie oder bradykardie führen kann. nifedipin kann auch nach sublingualer anwendung zur rapiden blutdrucksenkung führen (hata ). gute erfahrungen wurden mit nifedipin als tokolytikum gemacht (siehe abschnitt . . ). khandelwal ( ) berichtet über schwangere mit chronischer nierenerkrankung und proteinurie, von denen diltiazem im ./ . trimenon einnahmen. die autoren diskutieren diltiazem als alternative zu den in der schwangerschaft kontraindizierten angiotensin-ii-rezeptor-antagonisten. verapamil, mit dem auch fetale supraventrikuläre tachykardien behandelt werden, kann hyperprolaktinämie und galaktorrhö verursachen (siehe auch unter antiarrhythmika abschnitt . . ). zusammenfassend ergeben sich aus den bisherigen publikationen keine hinweise auf ein nennenswertes teratogenes risiko beim menschen. empfehlung für die praxis: calciumantagonisten gehören zu den antihypertensiva der . wahl in der schwangerschaft, wobei erprobte mittel wie nifedipin und als antiarrhythmikum verapamil (siehe dort) zu bevorzugen sind. eine therapie mit einem weniger gut untersuchten calciumantagonisten im . trimenon rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). eine hoch auflösende ultraschallfeindiagnostik kann aber zur bestätigung der normalen entwicklung des fetus angeboten werden. sind antihypertensiva, die das angiotensin konvertierende enzymsystem hemmen (ace-hemmstoffe). sie haben inzwischen eine starke verbreitung bei der behandlung des bluthochdrucks erfahren. zu captopril und enalapril liegen die meisten erfahrungen vor. es gibt nur wenige fallberichte zu anderen ace-hemmstoffen wie lisinopril (tomlinson ) . in den bisher publizierten fallserien mit über im . trimenon behandelten schwangeren und weiteren über von uns und anderen teratologischen zentren in europa dokumentierten schwangerschaften zeigten sich keine eindeutigen hinweise auf teratogene effekte beim menschen , burrows , bar . eine methodisch kritisch zu bewertende verschreibungsstudie fand ein von anderen untersuchern bisher nicht bestätigtes erhöhtes fehlbildungsrisiko u. a. für herzseptumdefekte (cooper seit längerem ist bekannt, dass ace-hemmstoffe in der zweiten schwangerschaftshälfte zur mangeldurchblutung der plazenta (de moura ), zu fetaler hypotonie, oligohydramnion und dialysepflichtiger anurie beim neugeborenen führen können (murki , filler , lavoratti . der pathomechanismus ist folgender: die fetale nieren-und urinproduktion beginnt ende des . trimenons. ace-inhibitoren setzen den gefäßtonus der nierengefäße herab, so dass es zu einer reduzierten urinproduktion kommt. daraus resultiert ein oligohydramnion, da nach schwangerschaftswoche die fetale urinproduktion die hauptquelle für die amnionflüssigkeit ist. eine hypoxämisch bedingte dysgenesie der nierentubuli wurde beobachtet (prasad ) . eine hypoplasie der schädelknochen kann als folge einer minderperfusion und des durch das oligohydramnion bedingten erhöhten druckes auf den schädel beobachtet werden (barr ) . derartige entwicklungsstörungen wurden auch tierexperimentell unter hoher dosis beobachtet. es gibt fallbeschreibungen zur rückbildung eines oligohydramnions nach absetzen des ace-hemmstoffes (muller ) . inwieweit die nach gabe von ace-hemmstoffen beobachteten spontanaborte, intrauterinen fruchttode und frühgeburten mit atemnotsyndrom medikamentenbedingt oder dem behandelten schweren hypertonus zuzuordnen sind, ist nicht geklärt. das gilt auch für die fälle des persistierenden ductus arteriosus, der theoretisch mit durch arzneimittel verursachte erhöhte bradykininkonzentrationen erklärt werden könnte. empfehlung für die praxis: ace-hemmstoffe sind in der gesamten schwangerschaft kontraindiziert bzw. nur der therapie schwerer, nicht anders behandelbarer erkrankungen vorbehalten. da es keine hinweise auf ein nennenswertes teratogenes potenzial in der frühschwangerschaft gibt, rechtfertigt eine exposition im . trimenon keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). umgehend sollte aber auf eines der empfohlenen antihypertensiven mittel umgestellt werden. die fetale entwicklung sollte per ultraschallfeindiagnostik kontrolliert und bei längerfristiger therapie in der spätschwangerschaft ein oligohydramnion ausgeschlossen werden. beim neugeborenen muss auf die nierenfunktion und eine mögliche hypotonie geachtet werden, wenn im letzten drittel der schwangerschaft (versehentlich) mit ace-hemmstoffen behandelt wurde. g . . angiotensin-ii-rezeptor-antagonisten zu den anderen peripheren § -rezeptorenblockern bunazosin (andante ® ), doxazosin (z. b. diblocin ® ), indoramin (wydora ® ), terazosin (z. b. heitrin ® ) und urapidil (z. b. ebrantil ® ) liegen ebenfalls keine ausreichenden erfahrungen zur abschätzung des embryotoxischen potenzials vor. urapidil, intravenös injiziert, wird allerdings von der deutschen sektion der "international society for the study of hypertension in pregnancy" als alternative zu dihydralazin bei der präeklampsiebehandlung empfohlen. es soll gegenüber dihydralazin den vorteil besitzen, dass der intrazerebrale druck nicht ansteigt. schulz ( ) kommt in einer klinischen vergleichsstudie zu dem ergebnis, dass urapidil eine gleichwertige antihypertensive alternative zu dihydralazin bei präeklampsie darstellt. guanabenz, guanethidin (z. z. nur in augentropfen thilodigon ® ), guanfacin und moxonidin (z. b. cynt ® ) gehören zur gruppe der zentral wirksamen § -rezeptoragonisten, zu denen eine fundierte risikobewertung mangels dokumentierter erfahrungen nicht möglich ist. minoxidil (z. b. lonolox ® ), ein vasodilatator, der in lokaler anwendung zur förderung des haarwuchses benutzt wird, kann einzelberichten zufolge eine hypertrichosis beim fetus verursachen, die sich aber in den ersten lebensmonaten wieder verliert. einzelne fallberichte zu neugeborenen mit verschiedenen fehlbildungen lassen keine differenzierte risikobewertung zu. auch zu dem vasodilatator diisopropylamin liegen keine ausreichenden erfahrungen vor. gleiches gilt für cicletanin (justar ® ). phenoxybenzamin (dibenzyran ® ), ein § -adrenerger blocker, wird zur behandlung des phäochromozytoms und bei neurogenen blasenentleerungsstörungen eingesetzt. publizierte erfahrungen zur anwendung im . trimenon gibt es nicht. in den kasuistiken mit exposition in der späteren schwangerschaft wurden keine entwicklungsstörungen beschrieben. bosentan (tracleer ® ) ist ein endothelin-rezeptor-antagonist, der bei pulmonaler arterieller hypertonie zur verbesserung der körperlichen belastbarkeit eingesetzt wird. es gibt nur einen fallbericht zu einer kompliziert verlaufenden schwangerschaft mit versehentlicher einnahme von bosentan und sildenafil bis zur entbindung in schwangerschaftswoche . das wachstumsretardierte mädchen hatte keine fehlbildungen. nach anfänglich gutem gedeihen verstarb es im alter von monaten an einer rs-virus-infektion (molelekwa ) . sildenafil (viagra ® ) wurde in einer experimentellen vergleichstudie an menschlichen plazenten getestet: plazenten aus normalen schwangerschaften und aus solchen mit fetaler wachstumshemmung wurden mit und ohne sildenafil untersucht. sildenafil verbesserte die plazentare durchblutung bei den plazenten, die aus schwan-gerschaften mit intrauteriner wachstumsverzögerung stammten (wareing ) . in den usa soll sildenafil für die indikation der pulmonalen hypertonie zugelassen werden. ein kürzlich publizierter fallbericht (siehe bosentan) hat keine fehlbildungen nach sildenafil beobachtet. der serotoninantagonist ketanserin wird unter anderem auch bei arterieller hypertonie eingesetzt. bisherige erfahrungen bei der behandlung der präeklampsie haben keine spezifischen fetotoxischen effekte gezeigt. nesiritide ist ein neuer wirkstoff zur behandlung der herzinsuffizienz. erfahrungen in der schwangerschaft gibt es nicht. empfehlungen für die praxis: urapidil kann in der spätschwangerschaft als alternative zu dihydralazin bei der präeklampsie eingesetzt werden. prazosin kommt im ./ . trimenon nur bei versagen der primär empfohlenen antihypertensiva infrage. phenoxybenzamin kann beim phäochromozytom eingesetzt werden. die anderen genannten substanzen sind mangels ausreichender erfahrung in der schwangerschaft zu meiden. besser erprobte, in den vorangehenden abschnitten besprochene mittel sind vorzuziehen. eine dennoch erfolgte einnahme stellt keine indikation zum risikobegründeten schwangerschaftsabbruch oder zu invasiver diagnostik dar (siehe kapitel . ). g . . hypotonie und antihypotonika eine hypotonie ist im prinzip ohne klinische bedeutung für den schwangerschaftsverlauf. sie sollte abgegrenzt werden von einer in der schwangerschaft nicht seltenen kreislaufdysregulation. bei deren therapie stehen physikalische maßnahmen, wie das tragen von kompressionsstrümpfen, beingymnastik vor dem aufstehen, kaltwasseranwendungen und bürstenmassage im vordergrund. auch kaffee ist in maßen erlaubt. eine medikamentöse therapie ist gewöhnlich nicht indiziert. in den er jahren wurde, überwiegend auf den deutschsprachigen raum begrenzt, den folgen einer chronischen hypotonie in der schwangerschaft besondere aufmerksamkeit gewidmet. eine bis auf % erhöhte frühgeburtsrate schrieb man der unbehandelten hypotonie zu (goeschen ), und es wurde für eine medikamentöse behandlung plädiert. dabei wurde das wirkungsprofil (z. b. tonisierung auch des venösen systems) bei dihydroergotamin günstiger als bei den adrenergen substanzen beurteilt (goeschen ) . andere autoren widersprachen einer therapieempfehlung aus "fetaler" indikation (wolff ). in der englischsprachigen literatur finden sich zu diesem thema und zur risikobewertung der betreffenden arzneimittel praktisch keine publikationen, da man dort die hypotonie in der schwangerschaft nicht als therapiepflichtige erkrankung betrachtet. (oudijk ) . als zweite wahl, in kombination mit und ohne digitalis, kommen sotalol und/oder flecainid infrage (doherty , oudijk . bei flecainid dauert es ca. stunden (maximal tage), bis der umschlag in einen sinusrhythmus zu erwarten ist (krapp ) . einige autoren diskutieren verapamil als zweite therapieoption (athanssiadis ), andere halten es für kontraindiziert (oudijk ) . falls diese medikamente nicht zum sinusrhythmus führen, kann adenosin direkt in die vena umbilicalis appliziert werden. es gibt beispiele dafür, dass der hydrops sich allmählich nach erfolgreicher kardioversion zurückbildet (d' souza ), das kann tage (porat ) oder auch - wochen dauern. berichtet wird auch über einen fetus mit einem hydrops als folge einer tachykarden rhythmusstörung, der über die mutter mit flecainid behandelt wurde. ein sinusrhythmus stellte sich dennoch nicht ein. es kam lediglich zur abnahme der herzfrequenz, die aber ausreichte, um zu einer rückbildung des hydrops zu führen (krapp ) . bei nicht erfolgreicher therapie wird ggf. auch über eine vorzeitige entbindung diskutiert, um z. b. postnatal elektrisch kardiovertieren zu können. im allgemeinen wird eine antiarrhythmische therapie bei herzgesunden schwangeren gut vertragen. als fetale nebenwirkung kann es selten zur bradykardie kommen, die bei direkter adenosinapplikation in die nabelvene wahrscheinlicher ist als bei einer diaplazentaren therapie. nicht auszuschließen ist auch ein arrhythmogener effekt des antiarrhytmikums, der zum kammerflimmern beim fetus und zum intrauterinen fruchttod führen kann. eine fetale bradykardie kann zunächst durch ein kompensatorisch größeres schlagvolumen ausgeglichen werden. eine fetale herzfrequenz von /minute wird als hämodynamisch nicht mehr ausreichend beschrieben (eronen ). daraus kann sich eine herzinsuffizienz entwickeln, die bis zum hydrops führen kann. die ursache ist meist ein av-block iii°, der durch diaplazentare autoantikörper der mutter (meist anti-ro-antikörper) verursacht wird. als therapieoption kommen die gabe von halogenierten steroiden (solange der av-block noch nicht komplett ist) oder ggf. eine vorzeitige entbindung infrage, um postnatal einen schrittmacher implantieren zu können. auch sympathomimetika wurden versuchsweise eingesetzt. (tambocor ® ) und propafenon (z. b. rytmonorm ® ) sowie encainid und lorcainid. die klasse-ii-antiarrhythmika umfassen die g -rezeptorenblocker. zu den klasse-iii-antiarrhythmika gehören amiodaron (corda- . das nukleosid adenosin wird keiner der klassischen antiarrhythmikagruppen zugeordnet. klasse-ia-antiarrhythmika: chinidin wird nach oraler zufuhr fast vollständig resorbiert und erreicht in - stunden seine maximale serumkonzentration. etwa % werden über die nieren, % über die leber ausgeschieden. als vagusantagonist kann es trotz depressorischer wirkung auf die schrittmacherzellen die herzfrequenz leicht erhöhen. chinidin als eines der ältesten antiarrhythmika hat offenbar kein nennenswertes teratogenes potenzial. es ist plazentagängig und erreicht beim fetus ähnlich hohe konzentrationen wie bei der mutter. sowohl bei schwangeren als auch bei feten wurde es erfolgreich eingesetzt. der beschriebene wehenfördernde effekt des chinidins ist bei antiarrhythmischer dosierung nicht zu erwarten. auch disopyramid soll eine wehenfördernde wirkung besitzen . fallberichte in zusammenhang mit fehlbildungen nach disopyramid oder procainamid wurden bisher nicht publiziert. beide substanzen sind plazentagängig. procainamid wurde auch erfolgreich bei fetaler tachykardie eingesetzt. zu ajmalin, detajmium und prajmalium liegen keine ausreichenden erfahrungen zur pränatalen verträglichkeit vor. klasse-ib-antiarrhythmika: die meisten der umfangreichen erfahrungen mit lidocain in der schwangerschaft liegen zur anästhetischen anwendung vor. zur antiarrhythmischen behandlung wird es parenteral appliziert, da es oral nicht ausreichend wirkt. cuneo ( ) beschreibt einen fetus mit qt-verlängerung im ekg, der ventrikuläre tachykardien und einen intermittierenden av-block ii°hatte und erfolgreich mit lidocain therapiert wurde. ein teratogener effekt beim menschen ist nicht beschrieben. lidocain ist gut plazentagängig und kann bei hohen konzentrationen beim neugeborenen zur zns-depression führen, zur anwendung unter der geburt siehe kapitel . . . Über eine völlig andere indikation wird in einer studie aus frankreich berichtet: die anwendung von lidocain zur herbeiführung des fetozids in fällen. feten (zwischen schwangerschaftswoche und ) mit verschiedenen fehlbildungen erhielten über die nabelvene zunächst sufentanil ( ? g), dann bis ml lidocain ( %) und erlitten dadurch eine kardiale asystolie (senat ) . phenytoin ist ein teratogenes antikonvulsivum (siehe abschnitt . . ). mexiletin ist plazentagängig und hat sich in wenigen fallberichten bisher als nicht bedenklich erwiesen. zu aprindin und tocainid gibt es keine für eine bewertung ausreichenden erfahrungen. klasse-ic-antiarrhythmika: eine vielzahl an fallberichten beschreibt die gute wirksamkeit von flecainid bei der behandlung von fetalen tachykardien (krapp ) . besonders bei feten, die schon einen hydrops entwickelt haben, ist es digitalisglykosiden überlegen. eine engmaschige kontrolle der mütterlichen serumkonzentration als hinweis auf die fetale konzentration wird empfohlen (rasheed ) , damit nebenwirkungen minimiert werden können. in einer kasuistik ist eine hyperbilirubinämie beim neugeborenen als nebenwirkung beschrieben (nach athanassiadis ) . bisher ist im gegensatz zu tierexperimentellen erfahrungen kein teratogener oder fetotoxischer effekt beim menschen erkennbar; allerdings gibt es kaum fallberichte zur anwendung im . trimenon. propafenon ist bisher unzureichend in der schwangerschaft untersucht. der hersteller berichtet über mehr als schwangerschaften unter propafenon, aus denen kein nennenswertes vorgeburtliches risiko abgeleitet werden kann. in unserer datenbank gibt es im . trimenon exponierte schwangerschaften, darunter einen spontanabort und zwei abbrüche wegen der mütterlichen erkrankung. die lebend geborenen kinder waren gesund. klasse-ii-antiarrhythmika: zu g -rezeptorenblockern siehe abschnitt . . . klasse-iii-antiarrhythmika: amiodaron hat eine sehr lange eliminationshalbwertszeit von - tagen. wenn eine fetale exposition vermieden werden soll, müsste das medikament einige monate vor der konzeption abgesetzt werden. folgende fetale nebenwirkungen sind gehäuft aufgetreten: fetale bradykardien sowie konnatale hypothyreosen, ausgelöst durch den jodanteil von % (lomenick , grosso (magee ) . die erfahrungen mit amiodaron im . trimenon sind auf etwa exponierte schwangerschaften, die im wesentlichen unauffällig waren, begrenzt . sotalol ist aufgrund seines guten plazentaren Übergangs ein potentes antiarrhythmikum, das zur behandlung von fetalen tachykardien infrage kommt. in einer fallserie von feten mit tachykardie wurde eine akkumulation im fruchtwasser, nicht aber im fetus selbst festgestellt. von den feten, die sotalol als monotherapie erhielten, konnte bei ein sinusrhythmus wiederhergestellt werden, zwei wurden rückfällig, in einem fall kam es zum intrauterinen fruchttod. bei zwei der vier feten, die zusätzlich digoxin erhielten, war die behandlung erfolgreich (oudijk (danielsson ) . klasse-iv-antiarrhythmika: zu den bereits seit längerem eingeführten calciumantagonisten verapamil und diltiazem siehe abschnitt . . . tierexperimentelle ergebnisse zeigten zwar teratogene entwicklungsstörungen, z. b. im bereich der distalen phalangen, die bisherigen erfahrungen beim menschen erbrachten aber keine entsprechenden hinweise. adenosin hat eine sehr kurze halbwertszeit von weniger als sekunden und muss i.v. injiziert werden. die bisherigen erfahrungen bei schwangeren und mit der behandlung fetaler arrhythmien ergaben keine fetotoxischen effekte (hubinont ). das gleiche gilt für die elektrokardioversion einschließlich der implantierten defibrillatoren. die reizschwelle beim fetalen herzen liegt relativ hoch, außerdem befindet sich der fetus außerhalb des direkten spannungsfeldes bzw. stromflusses (joglar ). empfehlung für die praxis: da antiarrhythmika selbst arrhythmien verursachen können, ist die indikation einer behandlung kritisch zu prüfen. mittel der wahl für die therapie der schwangeren sind in der gruppe ia chinidin, in ib lidocain, in ic kommen sowohl propafenon als auch im . und . trimenon flecainid infrage. in gruppe ii sollten lang eingeführte g -rezeptorenblocker bevorzugt werden. ist ein klasse-iii-antiarrhythmikum erforderlich, sollte sotalol gewählt werden. in der gruppe iv sind verapamil und diltiazem akzeptabel. wegen erwiesener teratogenität ist phenytoin kontraindiziert. wurde mit einem der primär nicht empfohlenen mittel behandelt oder sind diese aus mütterlicher oder fetaler indikation zwingend erforderlich, rechtfertigt dies keinen risikobegründeten abbruch der schwangerschaft. abgesehen von gut untersuchten g -rezeptorenblockern und calciumantagonisten sollte bei einer exposition im . benzothiadiazide werden gut im magen-darm-trakt resorbiert und unverändert mit dem urin ausgeschieden. sie passieren die plazenta und können, sub partu gegeben, zu elektrolytveränderungen (hyponatriämie, hypokaliämie), zu thrombozytopenie und reaktiver hypoglykämie (infolge eines diabetogenen effekts auf die mutter) beim neugeborenen führen. außerdem wurde eine geburtsverzögerung durch die hemmende wirkung auf die glatte muskulatur beschrieben. bei patientinnen mit schwerer präeklampsie ist das intravasale volumen in den meisten fällen vermindert; benzothiadiazidderivate würden es noch zusätzlich reduzieren (sibai ) . außerdem wurde eine herabsetzung der plazentaperfusion beobachtet, die über eine beeinträchtigung der fetalen versorgung zu vermindertem intrauterinen wachstum führt. klinisch gibt es bisher keinen anhalt für teratogene wirkungen dieser saluretika, dies haben publizierte erfahrungen an insgesamt über . behandelten schwangeren ergeben. am besten untersucht ist hydrochlorothiazid. in einer gruppe von im . trimenon behandelten schwangeren wurde weder eine häufung spezieller anomalien noch eine erhöhte gesamtfehlbildungsrate gefunden (Übersicht in . auch bei neugeborenen mit indapamid-exposition im . trimenon waren weder häufigkeit noch art der anomalien auffällig (Übersicht in . anhand dänischer und schottischer register wurden bzw. schwangerschaften analysiert, in denen mindestens einmal diuretika verschrieben worden waren (olesen . insbesondere bei kombination mit aminoglykosiden wird eine ototoxische wirkung beschrieben (brown , salamy . in zwei fallberichten wurden nach behandlung mit etacrynsäure im . trimenon eine schädigung des innenohres (jones ) und störungen des säure-basen-haushaltes (fort ) beschrieben. bisher gibt es keine anderen substantiellen hinweise auf teratogene schäden beim menschen. der umfang an dokumentierten erfahrungen ist jedoch unzureichend für eine differenzierte risikobewertung. nach therapie mit bumetanid im . trimenon wurden in einer gruppe von schwangeren zwei kinder mit fehlbildungen des herzens geboren (zitiert in . zu den anderen schleifendiuretika azosemid, etozolin, piretanid und torasemid, liegen keine erfahrungen in ausreichendem umfang vor. eine spezifische teratogene wirkung ist bisher bei keinem der genannten mittel zu erkennen. anhand dänischer und schottischer register wurden bzw. schwangerschaften analysiert, in denen mindestens einmal diuretika verschrieben worden waren (olesen bar , sørensen , chan , sanson , schneider , dulitzki . eher anekdotischen charakter hat ein fallbericht zur aplasia cutis bei tinzaparin-therapie ab woche (sharif ). daher können im bedarfsfall die vorzüge niedermolekularer heparine auch in der schwangerschaft genutzt werden, so beispielsweise bei schwerer, früh manifester präeklampsie, abortneigung und intrauteriner wachstumsretardierung infolge erworbener oder angeborener thrombophilie ( bar , kupferminc ). obwohl niedermolekulare heparine die menschliche plazenta nicht oder kaum überwinden (greer , sanson , wurden im tierexperiment dennoch auswirkungen auf die fetale gerinnung beobachtet. (hall ) . besonders gefürchtet sind größere zerebrale blutungen in der spätschwangerschaft und unter der geburt. die cumarinembryopathie ähnelt der chondrodysplasia punctata conradi-hünermann (savarirayan , becker . im zusammenhang mit diesem krankheitsbild wurde die ätiologisch relevante mutation der arylsulfatase e (arse) beschrieben, die zum (völligen) aktivitätsverlust dieses enzyms führt. die cumarinembryopathie als identischer phänotyp wird mit einer cumarinbedingten arse-hemmung in zusammenhang gebracht (savarirayan ). (schaefer ) . in dieser, von uns durchgeführten, multizentrischen studie wurden schwangere mit acenocoumarol therapiert, mit phenprocoumon, mit fluindion, mit phenindion und mit warfarin. vier der patientinnen erhielten vitamin-k-antagonisten. im vergleich zu einer nichtbehandelten kontrollgruppe fand sich ein mit , % vs. , % (or , ) signifikant erhöhtes risiko für große fehlbildungen nach exposition im .trimenon. die beobachteten fehlbildungen waren jedoch heterogen, nur zwei cumarinembryopathien unter insgesamt lebendgeborenen wurden beobachtet ( , %). andere auswirkungen einer cumarintherapie. weitere ergebnisse der multizentrischen kohortenstudie (schaefer ) sind ein geringeres geburtsgewicht, das nur zum teil durch die erhöhte frühgeburtenrate erklärt wurde. spontanaborte traten -mal häufiger unter cumarintherapie auf, am höchsten war die rate bei phenprocoumon mit % gegenüber % in der kontrollgruppe. natürlich kann auch die grunderkrankung der mütter, wie herzklappendefekte, embolien, verschiedene koagulopathien, zum ungünstigeren abschneiden der cumaringruppe beigetragen haben. die mütterliche erkrankung war in einigen fällen auch der grund für den medizinisch indizierten schwangerschaftsabbruch. ein problem stellen die undifferenzierten, das teratogene risiko übertreibenden warnhinweise auf packungsbeilagen dar, die häufig zu einer erheblichen verunsicherung von patientinnen und Ärzten führen. sensible phase. weder in den früher publizierten fallberichten noch bei den von uns beobachteten, ausschließlich vor schwangerschaftswoche exponierten lebendgeborenen ergeben sich hinweise darauf, dass bis schwangerschaftswoche p.m. ein nennenswertes risiko für eine cumarinembryopathie besteht. es wird gelegentlich von einer sensiblen phase in den wochen bis gesprochen. eine kritische analyse derjenigen fallberichte, die als beleg für ein embryopathierisiko vor woche p.m. interpretiert werden könnten (hall , balde , ruthnum , lapiedra , cox , lässt zweifel an der richtigkeit dieser hypothese aufkommen. es ist zumindest nicht eindeutig, dass in diesen fallberichten ausschließlich vor woche p.m. behandelt wurde, dass es sich um cumarinspezifische anomalien handelte und dass nicht weitere teratogene faktoren im spiel waren. die zwei in unserer studie erfassten typischen embryopathien ereigneten sich bei schwangeren, die deutlich länger als bis woche bzw. ausschließlich danach behandelt wurden. andererseits könnte das erhöhte spontanabortrisiko ergebnis einer embryotoxischen schädigung sein. es kann aber auch ebenso wie die erhöhte rate an frühgeburten, folge der grunderkrankung sein. mentale entwicklung. die spätere entwicklung im alter von bis jahren wurde in einer studie an etwa kindern mit pränataler cumarinexposition untersucht (van driel , wesseling . nur zwei kinder in dieser gruppe wiesen bei der geburt typische zeichen einer cumarinembryopathie auf. diese waren im alter von bzw. jahren normal entwickelt (van driel ) . die durchschnittliche größe der exponierten kinder unterschied sich nicht von einer kontrollgruppe. keines der exponierten kinder war hinsichtlich seiner neurologischen entwicklung deutlich auffällig. lediglich leichte neurologische abweichungen traten etwas häufiger auf, wenn die mutter im . oder . trimenon behandelt wurde. der durchschnittliche iq unterschied sich nicht signifikant von der kontrollgruppe. allerdings wurden in der cumaringruppe mit gegenüber mehr kinder mit einem iq x gezählt. diese kinder wiesen keine typischen dysmorphiezeichen auf. auch hinsichtlich der verhaltensentwicklung gab es keine häufung von problematischen entwicklungen, allenfalls leichte differenzen bei einzelnen tests. drei andere studien mit insgesamt kindern fanden ebenfalls keine signifikanten unterschiede hinsichtlich körperlicher und mentaler entwicklung (olthof , wong , chong die diagnose der dysmorphiezeichen ist nicht immer einfach, sie unterliegt subjektiven bewertungsunterschieden und ist z. t. nur radiologisch nachweisbar (harvey , lu . gewöhnlich treten nur einige und nicht alle beschriebenen fehlbildungen bzw. dysmorphien auf. g . funktionsstörungen des zns kommen häufiger bei kindern mit mittelgesichtshypoplasie vor; die angaben bewegen sich im zweistelligen prozentbereich. moore und mitarbeiter ( ) untersuchten kinder mit einem antiepileptika-syndrom und fanden bei etwa % verhaltensauffälligkeiten, sprachentwicklungsstörungen, lernstörungen und bei % zwei oder mehr autistische symptome. im vergleich der verschiedenen antiepileptika finden sich diese entwicklungsauffälligkeiten vor allem nach vorgeburtlicher exposition mit valproinsäure (Übersicht in schmitz , adab . g . . wie spezifisch wirken die einzelnen antiepileptika? eine spezifische zuordnung von fehlbildungsmustern zu den einzelnen antiepileptika ist, abgesehen von einigen ausnahmen, nicht möglich (morrow ( ) haben kinder untersucht, deren mütter eine epilepsie in der vorgeschichte angaben und die während der schwangerschaft weder unter krampfanfällen litten noch antiepileptisch behandelt wurden. diese kinder zeigten gegenüber einer kontrollgruppe weder einschränkungen der intelligenzentwicklung noch die nach antikonvulsiver behandlung in der schwangerschaft gehäuft beschriebenen dysmorphien des gesichts oder der finger. adab und mitarbeiter ( ) stellten hingegen einen niedrigen sprach-iq ( x ) signifikant häufiger fest, wenn während der schwangerschaft -unabhängig von einer antikonvulsiven therapiemehr als generalisiert tonisch-klonische anfälle auftraten. g . . folsäure und antiepileptika eine substitution mit folsäure bei therapie mit folsäure-antagonistischen antiepileptika in der schwangerschaft wird verschiedentlich empfohlen, der nachweis protektiver wirksamkeit wurde bisher jedoch nicht erbracht (hernandez- diaz ) . da heute generell für alle frauen mindestens bis schwangerschaftswoche eine folsäureprophylaxe empfohlen wird (siehe kapitel . vitamine), sollten selbstverständlich auch an epilepsie erkrankte frauen mit kinderwunsch eine folsäuresubstitution durchführen, und zwar mit einer dosis von mg/ tag, wie sie auch zur minderung des wiederholungsrisikos von neuralrohrdefekten empfohlen wird. dabei ist zu beachten, dass folsäure den arzneimittelmetabolismus der hydroxylasen in der leber anregt, so dass die konzentrationen von antiepileptika im blut der mutter erniedrigt sein können. g . . vitamin k und antiepileptika unabhängig von einer medikation der mutter weisen neugeborene und insbesondere frühgeborene einen vitamin-k-mangel auf, der zur verhütung von blutungskomplikationen unmittelbar nach geburt durch substitution behoben werden muss. darüber hinaus gehören carbamazepin, ethosuximid, oxcarbazepin, phenytoin, phenobarbital, topiramat, vigabatrin und zonisamid zu den enzym induzierenden arzneimitteln, die eine verminderung der vitamin-k-abhängigen gerinnungsfaktoren induzieren können. als indirekter marker kann die prothrombinvorstufe pivka ii (protein induced by vitamin k abscence of factor ii) beim neugeborenen erhöht sein. es wurde vielfach empfohlen, dass bei einer therapie mit vitamin-kantagonisierenden medikamenten die mutter in den letzten vier schwangerschaftswochen täglich vitamin k (z. b. konakion ® ) einnimmt, zunächst mg am tag und während der letzten beiden wochen mg. die wirksamkeit dieses vorgehens ist umstritten (hey ). kaaja und mitarbeiter ( ) vitamin k wird zwar oral ähnlich gut aufgenommen wie parenteral, aber unmittelbar nach der geburt kann diese verabreichungsform aufgrund der situation im kreissaal unzuverlässig sein, so dass eine intramuskuläre applikation von , - mg vitamin k zu empfehlen ist. diese soll vor allem zur prävention von spätblutungen (ab wochen) der oralen verabreichung überlegen sein (american academy of pediatrics ). wird die orale prophylaxe gewählt, ist gewissenhaft darauf zu achten, dass das neugeborene die dosis auch tatsächlich herunterschluckt und in den ersten beiden wochen alle tage eine zusätzliche orale dosis erhält. g . . die neueren antiepileptika zu den neueren antiepileptika zählen felbamat, gabapentin, lamotrigin, levetiracetam, oxcarbazepin, pregabalin, tiagabin, topiramat, vigabatrin, zonisamid. sie wurden in den er jahren zunächst als so genannte add-on-antiepileptika eingeführt als zusatz zu klassischen antiepileptika bei fokalen epilepsien. die proteinbindung ist bei den neuen antiepileptika meist niedriger als bei den klassischen antiepileptika: felbamat %, gabapentin %, lamotrigin %, oxcarbazepin %, tiagabin %, topiramat %, vigabatrin %, zonisamid - %. die neuen antiepileptika induzieren das cytochrom-p -enzymsystem gar nicht oder, wie oxcarbazepin und topiramat, in geringerem umfang und sie bilden keine potenziell teratogenen arenoxid-bzw. epoxidmetaboliten (bruno ) . im gegensatz zu den klassischen antiepileptika besitzen sie keine nennenswerte antifolatwirkung und zeigen eine geringere interaktion mit sexualhormonen. tierexperimentell haben felbamat, gabapentin und lamotrigin bisher keine hinweise auf teratogenität erbracht, während alle klassischen antiepileptika im tierversuch teratogen wirken. die mit ausnahme von lamotrigin noch recht spärlich vorliegenden klinischen verlaufsbeobachtungen zur schwangerschaft deuten bei monotherapie nicht auf eine spezifische teratogenität hin. möglicherweise ist das teratogene risiko dieser gruppe bei monotherapie geringer als bei den klassischen antiepileptika. eine abschließende beurteilung der neuen antiepileptika ist jedoch noch nicht möglich. typische fehlbildungen. carbamazepin wirkt wie die anderen klassischen antiepileptika nicht nur im tierversuch, sondern auch beim menschen teratogen. ein spezifisches carbamazepin-syndrom wurde ende der er-jahre postuliert, das epikanthus, antimongoloide lidachse, kurze nase, langes philtrum, hypoplasie der endphalangen, mikrozephalie und entwicklungsretardierung umfasste (jones ) . andere untersucher konnten die spezifität dieser auffälligkeiten nicht bestätigen oder fanden keine häufung von hypoplasien der distalen phalangen. fehlbildungen, die im zusammenhang mit carbamazepin vermehrt beschrieben wurden, betreffen herz und extremitäten, hüftanomalien, inguinalhernien, gaumenspalten und hypospadien (ornoy toxikologie. es gibt nur wenige berichte über die therapie mit ethosuximid in der schwangerschaft. typische fehlbildungsmuster wurden bei den kindern von behandelten frauen nicht beobachtet (lindhout ) . eine andere fallsammlung mit im . trimenon exponierten schwangeren ergab keinen anhalt für fehlbildungen (rosa, zitiert in briggs ) . auch wenn die vorliegenden berichte für eine differenzierte risikobewertung nicht ausreichen, scheint kein erhebliches teratogenes potenzial vorzuliegen. Über eine erhöhte neonatale blutungsbereitschaft durch einen vitamin-k-antagonismus wurde berichtet (siehe kapitel . . ). zu den anderen succinimiden mesuximid (petinutin ® ) und phensuccimid liegen keine für eine beurteilung ausreichenden erfahrungen vor. (janz ) und gilt als erwiesen, auch wenn dies nicht in allen studien bestätigt werden konnte (samrén ). im vordergrund stehen herzfehlbildungen, lippen-kiefer-gaumen-spalten und urogenitale fehlbildungen. ursprünglich wurden diese anomalien als "fetales hydantoin-syndrom" bezeichnet (siehe kapitel . . und . . ). häufigkeit großer fehlbildungen. kaaja und mitarbeiter ( ) einschränkungen der kognitiven entwicklung wurden unter phenytointherapie gehäuft beobachtet (scolnik , vanoverloop , hättig . eine gesichtsdysmorphie kann auf ein erhöhtes risiko mentaler entwicklungseinschränkungen hinweisen (orup ) . koch und mitarbeiter ( ) vpa wird nach oraler gabe gut resorbiert und liegt im plasma zu % an eiweiß gebunden vor. die lipophilie erklärt, dass vpa die blut-hirn-schranke und die plazenta leicht überwindet. gegen ende der schwangerschaft wird vpa in stärkerem umfang in der leber metabolisiert, gleichzeitig nimmt der ungebundene anteil im plasma zu. beide effekte können sich aufheben, so dass die verfügbare aktive substanz in etwa gleich bleibt (nau ) . die konzentration im nabelvenenblut ist bei geburt mit dem , - , fachen deutlich höher als im mütterlichen plasma (nau ) . neugeborene scheiden vpa aufgrund der noch nicht ausgereiften leberenzyme verzögert aus. die halbwertszeit kann von - auf - stunden verlängert sein. vpa verstärkt möglicherweise zyklusunregelmäßigkeiten und wird im zusammenhang mit dem polycystischen ovar-syndrom (pcos) diskutiert, das mit einer herabgesetzten fertilität und einem erhöhtem testosteronspiegel einhergeht (isojärvi ) . eine nennenswerte beeinträchtigung oraler kontrazeptiva durch enzyminduktion ist nicht bekannt. typische fehlbildungen. ein valproinsäure-syndrom wurde in den er jahren definiert, das dysmorphe entwicklungen an augenlidern, nase und mund umfasste, wie z. b. epikanthus, flache nasenwurzel, flaches philtrum sowie schmale sich überkreuzende finger und zehen und hyperkonvexe nägel (kozma ) . außerdem wurde über eine als trigonozephalie imponierende auffälligkeit der schädelform berichtet. weitere fallberichte beschreiben verschiedene präaxiale extremitätenanomalien (rodriguez-pinella , sharony , robert speziell für fehlende oder hypoplastische extremitätenanlagen errechneten rodriguez-pinella und mitarbeiter ( ) gegenüber einer kontrollgruppe ein etwa fach erhöhtes risiko, danach sind , % der exponierten kinder von einer derartigen entwicklungsstörung betroffen. dosis-wirkungs-beziehung. einige studien untersuchten für vpa die dosisabhängigkeit des fehlbildungsrisikos. mehr als . mg/tag oder eine serumkonzentration über ? g/ml ergab ein signifikant höheres risiko (kaneko , samrén . morrow und mitarbeiter ( ) (kaaja ) . andere organanomalien. dean und mitarbeiter ( ) verglichen pränatal exponierte kinder mit (älteren) geschwistern, bei denen die mütter keine antiepileptika in der schwangerschaft genommen hatten. bei monotherapie mit vpa ergab sich ein signifikant höheres risiko für gesichtsdysmorphien ( % versus %). erkrankungen in der späteren kindheit (sehstörungen, otitis media, gelenkprobleme) wurden ebenfalls häufiger beobachtet. die ergebnisse dieser studie sind aufgrund der kleinen fallzahlen vorsichtig zu bewerten, bemerkenswert ist der hohe anteil auffälliger kinder bei den pränatal exponierten und den kontrollen. die autoren einer anderen studie warnen von der Überbewertung der zeichen einer gesichtsdysmorphie (kini ) . sie fanden auffälligkeiten am häufigsten bei vpa, allerdings wiesen auch % der kinder nicht behandelter, an epilepsie erkrankter mütter einige der dysmorphiezeichen auf. neonatale auffälligkeiten. zu weiteren auffälligkeiten der therapie mit vpa gehören fetale hypoxie mit niedrigen apgar-werten, mikrozephalie und ein vermindertes postnatales wachstum. auch leberzellnekrosen wurden bei einzelnen kindern nach vpa-behandlung der mutter beschrieben (legius ) , ebenso hämorrhagien infolge von fibrinogenmangel und gestörter thrombozytenfunktion (bavoux ) und eine hypoglykämie des neugeborenen (ebbesen ). mentale entwicklungsauffälligkeiten. koch und mitarbeiter ( ) zeigten, dass Übererregbarkeit und andere neurologische auffälligkeiten im verlauf der kindheit mit der vpa-konzentration im nabelschnurblut bei geburt korrelieren. eine beeinträchtigung der kognitiven entwicklung wird von einigen autoren diskutiert (ornoy ) in einer untersuchung an kindern mit antiepileptika-syndrom - waren vpa-exponiert -zeigten % der kinder sprachentwicklungsstörungen, lernstörungen und verhaltensauffälligkeiten. bei % fanden sich mindestens autistische symptome, bei kindern wurde regelrecht die diagnose autismus gestellt, bei zweien ein asperger syndrom diagnostiziert (moore ) . eine andere untersuchung an kindern, deren mütter mit einem antikonvulsivum (mono)therapiert wurden, fand man bei einer nachuntersuchung die stärksten auffälligkeiten in der vpa-gruppe. gaily und mitarbeiter ( ) (morrow ) . unter prospektiv (monotherapie) und retrospektiv von uns dokumentierten schwangerschaften sahen wir keine große fehlbildung. chambers und mitarbeiter ( ) neurotransmitter, antikonvulsiv wirkt und bei fokaler epilepsie verwendet wird. von in einer publikation erwähnten schwangerschaften wurden ausgetragen. darunter befand sich ein kind mit hüftluxation bei steißlage (leppik ) . langzeitwirkungen psychotroper substanzen in der schwangerschaft sind bis auf wenige ausnahmen kaum untersucht, z. b. bei einigen antidepressiva (mattson , casper , nulman , klassischen antiepileptika und bei harten drogen (details siehe bei den entsprechenden substanzen). besonders psychopharmaka könnte man unterstellen, dass sie das fetale und kindliche zns in seiner funktionellen entwicklung stören und zu verhaltensauffälligkeiten, feinmotorischen störungen oder intellektuellen defiziten führen. (steyn , bolte . trizyklische antidepressiva treten aufgrund ihrer hohen lipidlöslichkeit rasch diaplazentar über. in manchen tierspezies erwiesen sich trizyklische antidepressiva als teratogen. in den er und er jahren wurden den klassischen antidepressiva auch beim menschen fehlbildungen zugeordnet, darunter extremitätenfehlbildungen, herzfehler, polydaktylie und hypospadie. jedoch konnte bei keinem der seit längerem gebräuchlichen präparate der verdacht auf teratogene effekte bestätigt werden (mcelhatton entis studie pers. mitteilung , ericson , mcelhatton , brunel , patuszak . speziell zu dosulepin, doxepin, lofepramin, opipramol und trimipramin liegen keine für eine fundierte risikobewertung ausreichenden fallzahlen zur anwendung in der schwangerschaft vor. allerdings ist auch bei diesen schon lange eingeführten mitteln nicht mit einem nennenswerten teratogenen risiko beim menschen zu rechnen. nach lang andauernder intrauteriner exposition (bis zur geburt) wurden bei neugeborenen entzugssymptome wie zittrigkeit, Übererregbarkeit, atemnotsyndrom und vereinzelt auch krämpfe beobachtet a, bromiker , schimmel . sieht im gegensatz zu anderen untersuchern ein höheres risiko für neonatale anpassungsstörungen bei trizyklischen antidepressiva als bei den selektiven serotonin-wiederaufnahme-hemmstoffen (ssri). er hat in seiner studie mit annähernd . schwangeren in der trizyklika-gruppe vor allem clomipramin (n = ) untersucht. diese und andere studien (ericson ) haben auch ein etwas höheres geburtsgewicht nach trizyklika-exposition beobachtet als bei den ssri. dieses phänomen wird mit einem diabetogenen effekt und intrauteriner hyperglykämie erklärt. bei kindern, die vorwiegend im . trimenon mit trizyklika exponiert waren, zeigten sich im vorschulalter gegenüber einer kontrollgruppe keine abweichungen bei intelligenzentwicklung, verhalten und sprachentwicklung (nulman ) . eine spätere prospektive untersuchung derselben autorengruppe findet auch bei jenen kindern, deren mütter während der gesamten schwangerschaft trizyklika ( mutter-kind-paare; davon bereits in der studie von erfasst) genommen hatten, keine arzneispezifischen auffälligkeiten in der entwicklung der kinder im alter zwischen und monaten. die dauer der depression hatte jedoch auswirkungen auf den intelligenzquotienten und die häufigkeit depressiver episoden auf die sprachentwicklung. die autoren leiten daraus die notwendigkeit einer therapie bei schwangeren ab (nulman ) . empfehlung für die praxis: trizyklische antidepressiva gehören immer noch zu den mitteln der wahl bei therapiebedürftiger depression in der schwangerschaft. eine monotherapie ist anzustreben, gut dokumentierte präparate wie amitriptylin, clomipramin, desipramin, imipramin und nortriptylin sind zu bevorzugen. die anderen der o. g. antidepressiva sind aufgrund der geringeren erprobung reservemittel in der schwangerschaft. eine unter therapie stabile patientin sollte während einer schwangerschaft diese medikation unverändert fortsetzen, um keine für mutter und kind bedrohlichen krisen zu provozieren. zur dosisanpassung sollten die mütterlichen serumkonzentrationen während der schwangerschaft und nach entbindung untersucht werden. die anwendung unzureichend erprobter substanzen rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ) noch invasive diagnostik. entzugssymptome beim neugeborenen sind möglich. deshalb sollte in den ersten beiden lebenstagen auf symptome beim kind geachtet werden. reaktive depressionen oder angstzustände sind nicht zwangsläufig eine indikation für die behandlung mit antidepressiva; auch psychotherapeutische optionen sollten ausgeschöpft werden. unter den ssri geht citalopram am stärksten plazentar über, gefolgt von fluoxetin. den geringsten Übergang fand man bei sertralin, gefolgt von paroxetin (hendrick ) . es ist bekannt, dass serotonin schon während der embryogenese, also bevor es als neurotransmitter funktioniert, an der regulation der zellmigration, des axonwachstums und der anlage der synaptischen kommunikation beteiligt ist. tierexperimentelle ergebnisse zeigen, dass erhöhte serotoninspiegel neuroanatomische abweichungen verursachen mit verringerter anzahl von betaadrenergen-und serotoninrezeptoren sowie abnormer serotoninrezeptorbindung im zentralnervensystem (zns). bei mehreren tausend schwangerschaften unter ssri-behandlung im . trimenon sind teratogene effekte i.s. großer fehlbildungen beim menschen bislang nicht eindeutig nachzuweisen , auch wenn einige neuere publikationen vor allem bei paroxetin ein etwas höheres risiko für herzfehlbildungen diskutieren (siehe dort). bei behandlung mit ssri am ende der schwangerschaft fanden chambers und mitarbeiter ( ) cohen , chambers ) zusammen und errechnen ein relatives risiko von , ( % ki , - , ) für das auftreten einer neonatalen symptomatik nach ssri-exposition in der spätschwangerschaft gegenüber einer ausschließlich in der frühschwangerschaft exponierten und einer unbehandelten kontrollgruppe. soweit klinisch vertretbar, kann mit der patientin eine reduktion bzw. auch das vorübergehende absetzen des antidepressivums vor der geburt vereinbart werden, um den nicht selten beobachteten anpassungsstörungen des neugeborenen entgegen zu wirken. das notwendige zeitintervall richtet sich dabei nach der halbwertszeit des medikamentes. langzeitentwicklung. eine studie an müttern mit ssri im vergleich zu ebenfalls erheblich depressiven frauen ohne medikation beobachtete bei den exponierten kindern signifikant geringere werte beim apgar-score, bei einigen psychomotorischen parametern sowie bei feinmotorischen kontrollfunktionen. die übrigen mentalen entwicklungsparameter unterschieden sich nicht. untersucht wurden kinder im alter zwischen und monaten (casper ). auch mattson und mitarbeiter ( ) verglichen bis monate alte, pränatal ssri exponierte kinder mit einer kontrollgruppe, die unverdächtigen medikamenten ausgesetzt war. dabei zeigten die mit ssri exponierten etwas häufiger psychomotorische entwicklungsrückstände, jedoch keine vermehrten auffälligkeiten bei den mentalen entwicklungsparametern des bayley-tests. oberlander und mitarbeiter ( ) fanden in einer kleinen studie eine eingeschränkte mimik und herzfrequenzvariabilität nach schmerzreiz im alter von monaten bei kindern mit ssri-exposition. bei anderen untersuchungen wurden keine eindeutigen entwicklungsunterschiede festgestellt, z. b. bei psychomotorischen tests im alter von und monaten (oberlander ) . ein weiterer vergleich zwischen pränatal ssri-exponierten ( fluoxetin, paroxetin, sertralin) und kontroll-kindern ( trizyklische antidepressiva, unbehandelt) im alter von jahren fand ebenfalls keine psychomotorischen entwicklungsunterschiede (simon ) . heikkinen und mitarbeiter ( ) beobachteten keine neurologischen differenzen im alter von einem jahr bei mit citalopram exponierten kindern gegenüber nicht exponierten. nulman und mitarbeiter ( ) sahen keine auffälligkeiten bei fluoxetin exponierten kindern im alter zwischen und monaten. es ergaben sich keine hinweise auf teratogenität bei mehreren hundert ausgewerteten schwangerschaften einer studie der europäischen teratologischen arbeitsgruppe entis sowie in anderen kleinen fallserien (review in hallberg und sjöblom ) und bei über . im schwedischen medizinischen geburtsregister erfassten schwangerschaften (hallberg und sjöblom , ericson escitalopram ist ein aktives isomer des citaloprams. von uns wurden exponierte schwangere prospektiv erfasst. unter den lebendgeborenen gab es keine fehlbildung. zwei schwangerschaften endeten als spontanabort, eine wurde aus persönlichen gründen abgebrochen. die datenlage ist für eine differenzierte beurteilung noch unzureichend. tierexperimentell wurde keine teratogenität beobachtet. fehlbildungsrisiko. mehrere studien mit insgesamt über . schwangerschaften und eine ähnlich große zahl vom hersteller registrierter fälle ergaben keine hinweise auf ein substantiell erhöhtes fehlbildungsrisiko , hallberg , hines b, pastuszak . ein gering erhöhtes risiko für herzfehlbildungen wurde jedoch kürzlich diskutiert (diav-citrin ) . auch eine leicht erhöhte abortrate wurde beschrieben, ohne die ursachen -grunderkrankung oder medikation -klären zu können (chambers ) . außerdem wurden ein vermehrtes auftreten kleinerer fehlbildungen, sowie bei behandlung im letzten trimenon von frühgeburten und anpassungsstörungen der neugeborenen beobachtet (Übersicht in hines , cohen , chambers . bei kritischer sicht erscheint eine kausale assoziation bei den kleinen fehlbildungen wenig wahrscheinlich und wird auch von anderen autoren bezweifelt (robert ) . anpassungsstörungen nach der geburt. bei manchen neugeborenen wurden wenige tage dauernde, als entzug oder serotonerge Überstimulation (laine ; siehe auch einleitung zu den ssri) interpretierte symptome wie zittrigkeit, Übererregbarkeit und erhöhter muskeltonus beobachtet, wenn bis zum ende der schwangerschaft behandelt wurde (zusammenfassung in hines , mhanna , chambers , spencer . bei einem von uns beobachteten fall traten bei dem frühgeborenen kind ausgeprägte extrapyramidale symptome auf. fluoxetin hat einschließlich seiner aktiven metaboliten mit bis zu tagen die längste halbwertszeit unter den ssri und ist daher in hinsicht auf die neonatalperiode problematischer als andere ssri. auf eine möglicherweise erhöhte blutungsbereitschaft postpartal wird ebenfalls hingewiesen (mhanna ) . langzeitentwicklung. bei einer nachuntersuchung im vorschulalter von vorwiegend im . trimenon exponierten kindern ergaben sich keine abweichungen bezüglich intelligenzentwicklung, verhalten und sprachentwicklung gegenüber zwei vergleichsgruppen mit amitriptylin oder ohne medikation (nulman ) . eine spätere prospektive untersuchung derselben autorengruppe findet auch bei kindern, deren mütter während der gesamten schwangerschaft fluoxetin ( mutter-kind-paare; davon bereits in der studie von erfasst) genommen hatten, keine arzneispezifischen auffälligkeiten in der entwicklung im alter zwischen und monaten. die dauer der depression hatte jedoch auswirkungen auf den intelligenzquotienten und die häufigkeit depressiver episoden auf die sprachentwicklung. die autoren leiten daraus die notwendigkeit einer therapie bei schwangeren ab (nulman ) . keine substantiellen hinweise auf spezifische teratogene effekte haben sich bisher bei mehreren hundert schwangerschaften unter fluvoxamin gezeigt (hallberg ein erhöhtes risiko für eine omphalocele (or , ; % ki , - , ), nicht aber für andere fehlbildungen, fanden alwan und mitarbeiter ( ) auf der basis von geburts-bzw. fehlbildungsregisterdaten. tierexperimentell ergab sich bei paroxetin bisher keine teratogenität. insgesamt sind die schwachen hinweise auf das häufigere auftreten von (herz-)fehlbildungen unter paroxetin zurückhaltend zu bewerten, da sie nur in einigen analysen beobachtet wurden, und die gesamtrate aller fehlbildungen nicht erhöht zu sein scheint. anpassungsstörungen nach der geburt. bei behandlung bis zur geburt wurden wiederholt symptome beschrieben, u. a. Übererregbarkeit, schlafund trinkstörungen, tremor, erhöhter muskeltonus, atemnotsyndrom und hypoglykämie, die eine stationäre Überwachung erforderlich machten (Übersicht in moses-kolko , sanz , jaiswal ). herbst und gortner ( ) empfehlen, bei symptomen einer neonatalen enzephalopathie differentialdiagnostisch auch an eine paroxetinbehandlung der mutter zu denken. die symptome beginnen in den ersten lebenstagen und dauern im extremfall einen monat, meist aber nicht länger als - wochen. in einer studie mit kindern waren betroffen (costei ) . sanz ( ) berichtete über spontanmeldungen an das internationale who-drug-monitoring-zentrum in uppsala, schweden. von insgesamt fällen mit einer durch ssri induzierten symptomatik bei neugeborenen (davon mit krampfanfällen) war paroxetin mit überproportional häufig betroffen. neben rezeptorspezifischen unterschieden zwischen den ssri erörtern die autoren als ursache für eine entzugssymptomatik die verkürzte halbwertszeit des mittels, nachdem die hemmung des cytochrom-p - d -enzyms durch das arzneimittel selbst nach der geburt entfällt. in einem weiteren fallbericht wies ein lethargisches reifes neugeborenes als einziges symptom eine fehlende schmerzreaktion in den ersten beiden lebenswochen auf (morag ) . andere autoren fanden keine häufung neonataler probleme bei paroxetin im vergleich zu anderen ssri oder trizyklika . zwei fallberichte diskutieren den zusammenhang einer paroxetinbehandlung in der spätschwangerschaft mit einer thrombozytenfunktionsstörung, die zu subarachnoidalblutung bzw. ventrikelblutung und krampfanfällen beim (reifen) neugeborenen führte (duijvestijn , salvia-roiges . zur langzeitentwicklung nach pränataler ssri-exposition siehe einleitung dieses abschnitts. weit über dokumentierte schwangerschaftsverläufe haben keine hinweise auf teratogene effekte erbracht , Übersicht in hallberg , hendrick , chambers , ericson , kulin , eigene erfahrungen), und auch tierexperimentell gibt es keinen anhalt für teratogenität. nach gabe von sertralin wurden zittrigkeit, unruhe, Übererregbarkeit, erhöhter muskeltonus und schrilles schreien beobachtet und zunächst als neonataler entzug interpretiert (chambers , kent . wie bei den anderen ssri können diese symptome auch zeichen einer serotonintoxizität sein, dazu gehört auch der fall eines nystagmus beim neugeborenen (oca und donn ; siehe auch einleitung zu den ssri). empfehlung für die praxis: ssri gehören zu den mitteln der wahl bei therapiebedürftigen depressionen in der schwangerschaft. die gut untersuchten und in der schwangerschaft i.a. gut verträglichen mittel sertralin und citalopram sollten bei einer neueinstellung bevorzugt werden. eine unter therapie mit einem anderen ssri stabile patientin sollte -zumal während einer schwangerschaftdiese medikation unverändert fortsetzen, um keine für mutter und kind bedrohlichen krisen zu provozieren. die exposition mit einem weniger gut erprobten ssri im . trimenon rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel ). eine ultraschallfeindiagnostik sollte jedoch angeboten werden. bei gabe von ssri bis zur geburt müssen anpassungsstörungen und möglicherweise auch eine erhöhte blutungsbereitschaft beim neugeborenen bedacht werden. daher sollte in den ersten lebenstagen die beobachtung des neugeborenen gewährleistet sein, am besten durch entbindung in einem perinatologischen zentrum. soweit klinisch vertretbar, sollte mit der patientin eine reduktion bzw. auch das vorübergehende absetzen des antidepressivums vor der entbindung vereinbart werden; dabei richtet sich das erforderliche zeitintervall nach der halbwertszeit des medikamentes. aufgrund der sehr langen halbwertszeit ist dieses vorgehen besonders bei fluoxetin anzuraten. inaktiviert. monoaminooxidase-hemmstoffe sind strukturell dem amphetamin verwandt. therapeutisch wird heute vorwiegend moclobemid und wegen der dabei erforderlichen strengen diät nur noch selten tranylcypromin (jatrosom ® ) eingesetzt. unter mao-hemmern kann ein hypertonus in der schwangerschaft verstärkt und die plazentaperfusion gemindert werden mit negativen auswirkungen auf die fetale entwicklung. außerdem können mao-hemmer eine tokolyse mit betarezeptorenblockern aufheben und unter der geburt mit narkotika interagieren. eine ältere fallsammlung von schwangeren, die im . trimenon mit mao-hemmstoffen behandelt wurden, davon mit tranylcypromin , sowie ein bericht über kinder mit anomalien und über plazentainfarkte (kennedy ) atomoxetin ist ein selektiver noradrenalin-wiederaufnahme-hemmer, der kürzlich zur behandlung der aufmerksamkeitsdefizit-hyperaktivitätsstörung zugelassen wurde. im gegensatz zu methylphenidat gehört es nicht zu den psychostimulanzien. die gefahr einer Überdosierung mit dem risiko von krampfanfällen wurde im zusammenhang mit gleichzeitig gegebenen und ebenfalls über das cytochrom-p -isoenzym- d verstoffwechselten antidepressiva (paroxetin, fluoxetin, bupropion) erörtert. erfahrungen in der schwangerschaft liegen nicht vor. der wirkmechanismus von bupropion, das vor allem zur raucherentwöhnung verwendet wird und in der bundesrepublik als antidepressivum nicht zugelassen ist, ist nicht genau bekannt. zu duloxetin, einem serotonin-noradrenalin-wiederaufnahme-hemmstoff (snri), das zunächst als urologikum (yentreve ® ) zur behandlung der belastungsharninkontinenz zugelassen wurde, liegen bisher keine ausreichenden erfahrungen beim menschen vor, die aussagen zum teratogenen potenzial erlauben. tierexperimentell wurden bei kaninchen kardiovaskuläre und skelettfehlbildungen bei dosen beobachtet, die unterhalb des maximalen klinischen bereichs lagen. wie bei anderen serotonerg wirkenden antidepressiva muss auch bei duloxetin mit toxischen symptomen wie Übererregbarkeit beim neugeborenen in den ersten lebenstagen gerechnet werden. zu mirtazapin, einem noradrenerg und serotonerg wirkenden antidepressivum, finden sich bei rund veröffentlichten , biswas , kesim , saks bzw. von uns dokumentierten schwangerschaftsverläufen mit behandlung vorwiegend im . trimenon keine hinweise auf teratogenität. auch tierexperimentell liegen keine hinweise auf teratogenität vor. mirtazapin wird auch bei hyperemesis gravidarum eingesetzt (guclu , rohde , dorn , saks . zu nefazodon, das die wiederaufnahme von noradrenalin und serotonin hemmt, liegen fast publizierte, vorwiegend im . trimenon exponierte schwangerschaften vor , einarson . weder diese daten (fehlbildungsrate , %) noch tierexperimentelle ergebnisse deuten bisher auf teratogenität hin. oxitriptan ist die physiologische vorstufe des neurotransmitters serotonin, das dessen konzentration im zns erhöht. ausreichende erfahrungen zur schwangerschaft liegen nicht vor. dies gilt auch für tianeptin, das im gehirn die aufnahme des serotonins erhöht. trazodon ist strukturell dem nefazodon verwandt und besitzt sedative eigenschaften. es wird auch als hypnotikum verschrieben und hat sich bei etwa publizierten schwangerschaftsverläufenmeistens im . trimenon angewendet -bisher nicht als teratogen erwiesen (einarson , mcelhatton . gleiches gilt für tierexperimentelle ergebnisse. trazodon ist strukturell dem nefazodon verwandt und besitzt sedative eigenschaften. es wird auch als hypnotikum verschrieben und hat sich bei etwa publizierten schwangerschaftsverläufen -meistens im . trimenon -bisher nicht als teratogen erwiesen (einarson , mcelhatton . gleiches gilt für tierexperimentelle ergebnisse. zu venlafaxin, einem so genannten bizyklischen antidepressivum, das die wiederaufnahme von noradrenalin und serotonin hemmt, liegen einschließlich einer kontrollierten studie mit exponierten schwangeren publizierte erfahrungen an etwa schwangeren vor und aus unserer eigenen datenbank, die bisher keine hinweise auf teratogene effekte geben , ellingrod . auch im tierversuch wurde keine teratogenität beobachtet. entzugserscheinungen nach der geburt sind nicht auszuschließen. die wenigen bis einige dutzend dokumentierten schwangerschaften zu amineptin, amoxapin, medifoxamin, dem noradrenalin-wiederaufnahme-hemmstoff reboxetin (eigene beobachtungen) und viloxazin (mcelhatton , brunel ) ergeben keine spezifischen teratogenen wirkungen, reichen aber für eine differenzierte risikobewertung nicht aus. das gleiche gilt für das heute auch von schwangeren häufig eingenommene, aber formal unzureichend untersuchte, pflanzliche antidepressivum johanniskraut (hypericin; z. b. esbericum ® ; siehe auch kapitel . ). empfehlung für die praxis: die hier genannten antidepressiva sollten -mit ausnahme von hypericin und ggf. mirtazapin und venlafaxin -in der schwangerschaft primär nicht verschrieben werden, da keine ausreichenden erfahrungen beim menschen vorliegen. andererseits sollte eine stabil eingestellte patientin während der schwangerschaft nicht auf andere medikamente umgesetzt werden, um für mutter und kind keine bedrohlichen krisen zu provozieren. die einnahme im . trimenon rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). eine ultraschallfeindiagnostik kann jedoch angeboten werden, um eine normalentwicklung des fetus zu bestätigen. entzugserscheinungen nach geburt sind nicht auszuschließen. daher ist in den ersten beiden lebenstagen eine zuverlässige beobachtung des neugeborenen zu gewährleisten und eine entbindung in einem perinatologischen zentrum anzustreben. g . . antipsychotische behandlung neuroleptika rufen eine psycho-physiologische umstimmung bei gesunden und psychisch kranken hervor, bei der die intellektuellen fähigkeiten erhalten bleiben. sie wirken wahrscheinlich über eine blockade zerebraler dopaminrezeptoren. zur gruppe der neuroleptika zählen (schwach und stark wirksame) phenothiazine, thioxanthene, butyrophenone als erste generation und die so genannten atypischen zu amisulprid (z. b. solian ® ), einem benzamidderivat und selektiven dopamin-d -und -d -rezeptorantagonisten, liegen nur einzelfallberichte vor, die bisher keine spezifische teratogenität erkennen lassen. wir überblicken prospektiv dokumentierte schwangerschaften, von denen eine als spontanabort endete. von den lebend geborenen kindern wies keines eine fehlbildung auf. allerdings reicht diese geringe zahl für eine differenzierte risikobewertung nicht aus. zur perinatalen dosisanpassung siehe "empfehlung für die praxis" weiter unten. aripiprazol (abilify ® ) wird als erstes atypisches neuroleptikum der . generation bezeichnet. es zeichnet sich aus durch gemischten dopaminergen agonismus (in regionen verminderter dopaminerger aktivität, beispielsweise im mesokortikalen bereich) und antagonismus (in regionen mit erhöhter dopaminkonzentration, z. b. im mesolimbischen bereich). im gegensatz zu den klassischen neuroleptika und manchen atypika wie dem risperidon bewirkt es keine erhöhung, sondern tendentiell sogar eher eine erniedrigung des prolaktins. besonders beim umsetzen von einem klassischen neuroleptikum (phenothiazine, butyrophenone wie haloperidol) muss mit einer ungeplanten schwangerschaft gerechnet werden, da sich durch den geringeren einfluss auf den prolaktinspiegel im vergleich zu den klassischen neuroleptika die fertilität verbessern kann. daher muss eine wirksame kontrazeption sichergestellt werden. bisher sind keine wechselwirkungen mit kontrazeptiva bekannt. für eine bewertung des risikos in der schwangerschaft liegen weder ausreichende daten noch einzelfallhinweise auf spezifische teratogene ereignisse vor. tierexperimentell fanden sich bei ratten vermehrt zwerchfellhernien und bei kaninchen skelettauffälligkeiten. zur perinatalen dosisanpassung siehe "empfehlung für die praxis" weiter unten. im gegensatz zu den klassischen neuroleptika sind menstruationszyklus und fertilität bei olanzapin (zyprexa ® ) mit seinem kombinierten d / -ht a -rezeptorantagonismus kaum beeinträchtigt. der prolaktinspiegel ist nur in geringem maße und nur vorübergehend erhöht. besonders beim umsetzen von einem klassischen neuroleptikum (phenothiazine, butyrophenone wie haloperidol) muss mit einer ungeplanten schwangerschaft gerechnet werden, da sich durch den geringeren einfluss auf den prolaktinspiegel im vergleich zu den klassischen neuroleptika die fertilität verbessern kann. daher muss eine wirksame kontrazeption sichergestellt werden. bisher sind keine wechselwirkungen mit kontrazeptiva bekannt. olanzapin hat sich tierexperimentell bei ratten und kaninchen nicht als teratogen erwiesen. etwa schwangerschaften wurden inzwischen ausgewertet, die z. t. mit den rund vom hersteller gesammelten fallberichten identisch sind. diese daten ergeben bisher keine hinweise auf embryo-oder fetotoxische effekte , levinson , ernst , mendhekar , biswas , malek-ahmadi , nagy , neumann , goldstein , kirchheiner . auch die von uns prospektiv erfassten schwangerschaften mit nur einer großen fehlbildung ergeben keinen hinweis auf teratogenität. nach anwendung bis zum ende der schwangerschaft wurden sedierung und anhaltender ikterus bei einem säugling im zusammenhang mit der mütterlichen olanzapineinnahme diskutiert (goldstein ) . bei drei retrospektiv erfassten kindern, deren mütter bis zur geburt behandelt wurden, traten in der neonatalzeit krampfanfälle auf (goldstein , eigene erfahrungen). da unter den prospektiv dokumentierten fällen keine krampfanfälle beobachtet wurden, ist ein hohes krampfrisiko nicht gegeben, ein ursächlicher zusammenhang ist aber nicht auszuschließen. die lange halbwertszeit von etwa stunden und die noch nicht voll entwickelte exkretionsleistung beim neugeborenen könnten derartige toxische wirkungen begünstigen. von lebend geborenen kindern in unserer prospektiven datenbank wiesen ( %) vorübergehende, nicht durch frühgeburtlichkeit bedingte symptome (vorwiegend zittrigkeit) auf. zur perinatalen dosisanpassung siehe "empfehlung für die praxis" weiter unten. beim umsetzen von einem klassischen neuroleptikum (phenothiazine, butyrophenone wie haloperidol) auf quetiapin (seroquel ® ) mit seinem kombiniertem d / -ht a -rezeptorantagonismus muss mit einer ungeplanten schwangerschaft gerechnet werden, da durch den geringeren einfluss auf den prolaktinspiegel im vergleich zu den klassischen neuroleptika die fertilität ansteigen kann. daher muss eine wirksame kontrazeption sichergestellt werden. bisher sind keine wechselwirkungen mit kontrazeptiva bekannt. etwa bisher veröffentlichte schwangerschaftsverläufe und vom hersteller gesammelte fallberichte lassen keine spezifische teratogenität oder -soweit untersucht -bleibende funktionsstörungen erkennen, sie sind aber unzureichend für eine differenzierte risikobewertung (mckenna , pace zitiert in gentile , taylor , tényi . von weiteren in unserer datenbank prospektiv erfassten schwangerschaften wiesen (statistisch nicht signifikant gegenüber einer nicht behandelten kontrollgruppe) eine große fehlbildung auf: vorhofseptumdefekt, lippen-gaumen-spalte. die mutter des kindes mit der spaltbildung rauchte zigaretten täglich und war außerdem mit olanzapin behandelt worden. rauchen wird bei frauen mit einem gleichzeitig vorliegenden defekt des enzyms tgf-alpha als risikofaktor für spaltbildungen diskutiert. im tierversuch ist keine teratogenität bekannt. von den lebend geborenen kindern in unserer prospektiven datenbank wiesen ( %) vorübergehende, nicht durch frühgeburtlichkeit bedingte anpassungssymptome (vorwiegend zittrigkeit) auf. zur perinatalen dosisanpassung siehe "empfehlung für die praxis" weiter unten. beim umsetzen von einem klassischen neuroleptikum (phenothiazine, butyrophenone wie haloperidol) auf risperidon (risperdal ® ), einem benzisoxazolderivat und kombiniertem d / -ht a -rezeptorantagonisten, muss mit einer ungeplanten schwangerschaft gerechnet werden, da sich durch den geringeren einfluss auf den prolaktinspiegel im vergleich zu den klassischen neuroleptika die fertilität normalisieren kann. daher muss eine wirksame kontrazeption sichergestellt werden. bisher sind keine wechselwirkungen mit kontrazeptiva bekannt. zu risperidon gibt es etwa publizierte fallberichte , ratnayake , mackay ) und rund vom hersteller gesammelte datensätze sowie von uns prospektiv erfasste schwangerschaftsverläufe, die bisher keine spezifische teratogenität erkennen lassen. von den in unserer datenbank prospektiv erfassten schwangerschaften wies kind eine große fehlbildung auf. dies ist gegenüber der kontrollgruppe statistisch nicht signifikant. allerdings erlaubt die geringe fallzahl keine differenzierte risikobewertung. im tierversuch ist keine teratogenität bekannt. zur perinatalen dosisanpassung siehe "empfehlung für die praxis" weiter unten. beim umsetzen von einem klassischen neuroleptikum (phenothiazine, butyrophenone wie haloperidol) auf ziprasidon (zeldox ® ), einem kombiniertem d / -ht a -rezeptorantagonisten, muss mit einer ungeplanten schwangerschaft gerechnet werden, da durch den geringeren einfluss auf den prolaktinspiegel im vergleich zu den klassischen neu-roleptika die fertilität steigen kann. daher muss eine wirksame kontrazeption sichergestellt werden. bisher sind keine wechselwirkungen mit kontrazeptiva bekannt. zu ziprasidon gibt es nur wenige publizierte fallberichte. von den in unserer datenbank prospektiv erfassten schwangerschaften wies ein kind (von lebendgeborenen) eine große fehlbildung auf (vorhofseptumdefekt). dies ist gegenüber einer gesunden kontrollgruppe statistisch nicht signifikant und lässt keine spezifische teratogenität erkennen, ist aber unzureichend für eine differenzierte risikobewertung. im tierversuch hat sich ziprasidon beim kaninchen als teratogen erwiesen (herzfehlbildungen). zur perinatalen situation und dosisanpassung um die geburt siehe abschnitt "empfehlung für die praxis". empfehlung für die praxis: ein atypisches neuroleptikum kann bei entsprechender indikation in der schwangerschaft eingesetzt werden, wobei möglichst länger eingeführte präparate bevorzugt werden sollten. olanzapin ist das bisher am umfangreichsten in der schwangerschaft dokumentierte mittel aus dieser gruppe. da olanzapin den blutzuckerspiegel erhöhen kann, muss ein gestationsdiabetes ausgeschlossen werden. die anderen genannten atypischen neuroleptika sind aufgrund der geringeren erprobung reservemittel in der schwangerschaft, die unter stabiler therapie während einer schwangerschaft jedoch nicht umgesetzt werden sollten, um keine für mutter und kind bedrohliche krise zu provozieren. wegen allgemeiner nebenwirkungen sollte eine neueinstellung von clozapin in der schwangerschaft unterbleiben, eine gut auf clozapin eingestellte patientin muss jedoch nicht umgestellt werden. generell sind sorgfältige schwangerschaftsüberwachung und engmaschige psychiatrische kontakte unerlässlich, um rechtzeitig krisen bei der mutter und entwicklungskomplikationen beim fetus (frühgeburtsbestrebungen, wachstumsretardierung) begegnen zu können. nach exposition im . trimenon sollte die normale entwicklung des fetus mittels ultraschallfeindiagnostik bestätigt werden. die einnahme eines atypischen neuroleptikums rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). bei therapie im letzten schwangerschaftsdrittel sollte, wenn möglich, die dosis in den tagen vor der geburt reduziert werden, um symptome beim neugeborenen zu vermeiden. in den ersten beiden lebenstagen muss eine zuverlässige beobachtung des neugeborenen gewährleistet sein, daher sollte die entbindung möglichst in einem perinatalzentrum erfolgen. bei erkrankungen mit hoher rezidivgefahr (wie etwa bipolare störungen) sollte bedacht werden, dass eine zu rasche dosisreduktion vor geburt in dieser für die mutter vulnerablen phase probelmatisch sein kann und postpartal das höchste risiko für ein rezidiv besteht. die notwendigkeit einer postpartalen dosiserhöhung in therapeutische bzw. hochtherapeutische bereiche muss frühzeitig besprochen werden. lithiumsalze werden nach oraler gabe gut resorbiert und zu mehr als % unverändert mit dem urin ausgeschieden, ihre halbwertszeit beträgt stunden. bei schwangeren ist die lithiumausscheidung durch die niere um - % gesteigert. lithium ist plazentagängig und erreicht im fetus ebenso hohe konzentrationen wie im mütterlichen serum. in den er jahren wurde lithium eine erhebliche teratogenität unterstellt und herzfehlbildungen als folge der behandlung im . trimenon angesehen, insbesondere die sonst seltene ebstein-anomalie mit einer fehlanlage der trikuspidalklappe. zur dokumentation exponierter feten wurde das so genannte "lithium-baby-register" in dänemark eingerichtet und dann international ausgeweitet. als es geschlossen wurde, lagen berichte über kinder vor, davon hatten ( %) fehlbildungen, in fällen am herzen und den großen gefäßen. andere anomalien betrafen die äußeren ohranlagen, das gehirn, die ureter und das endokrine system (Übersicht bei kozma ) . der hohe anteil von kindern mit fehlbildungen erklärt sich aus der retro-spektiven fallerfassung, bei der wie üblich auffällige verläufe überrepräsentiert sind. spätere prospektive kohorten-und retrospektive fall-kontroll-untersuchungen ergaben nur zum teil erhöhte fehlbildungsraten, u. a. auch für herzfehler. doch scheint das teratogene risiko deutlich geringer zu sein, als früher angenommen (kozma , cohen , jacobson , källén , zalzstein (pinelli ) , krampfanfälle und hypothyreose wurden beschrieben (malzacher , zegers , frassetto , llewellyn . diese toxischen effekte des lithiums besserten sich meist innerhalb von - wochen nach der geburt. bei neugeborenen mit ausgeprägter hypothyreose und konnataler struma wurde jedoch auch über eine wochenlang erforderliche thyroxinsubstitution berichtet (frassetto ) . länger anhalten kann auch ein so genanntes "floppy-infant-syndrom" mit lethargie, trinkschwäche, tachypnoe, tachykardie, zyanose, temperaturregulationsstörung und muskelhypotonie. die spätere entwicklung der kinder verläuft anscheinend normal (kozma ) . empfehlung für die praxis: ist eine lithiumtherapie in der schwangerschaft erforderlich, sollten gleich bleibend niedrige serumkonzentrationen angestrebt werden, insbesondere im . trimenon. die mütterliche tagesdosis (üblicherweise bis höchstens mg/tag) sollte auf - einzeldosen verteilt werden. eine ultraschallfeindiagnostik bzw. eine fetale echokardiographie sind nach exposition im . trimenon zu empfehlen. die schwangere soll keine salzarme diät einhalten und keine diuretika einnehmen. letztere können einen paradoxen antidiuretischen effekt bei gleichzeitiger lithiumtherapie entfalten. wegen veränderungen im flüssigkeitshaushalt während der schwangerschaft sollte die serumkonzentration ( , - , meq/l) monat-lich kontrolliert und die dosis dann ggf. erhöht werden. im letzten schwangerschaftsmonat sind wöchentliche kontrollen erforderlich und vor der geburt alle tage. in der woche vor der geburt sollte, wenn möglich, die dosis um - % herabgesetzt werden. eine dehydratation ist ggf. mit parenteraler flüssigkeitssubstitution zu therapieren. nach der geburt kann die vor der schwangerschaft übliche dosis wieder eingenommen werden. dabei muss zunächst weiter engmaschig der spiegel kontrolliert werden und wegen der erneuten umstellung des flüssigkeitshaushalts die dosis ggf. vorübergehend nach unten angepasst werden. aufgrund der unreifen renalen elimination insbesondere in den ersten lebenstagen ist auf toxische symptome beim kind zu achten. außerdem muss eine hypothyreose ausgeschlossen werden. die entbindung sollte in einem perinatalzentrum erfolgen. gerade bei bekannter bipolarer störung muss bei der planung der medikation für die mutter das hohe postpartale rezidivrisiko bedacht werden und zwar sowohl für postpartale depressionen als auch für manien. . tierexperimentell finden sich hinweise auf teratogenität. kavain ist zusammen mit anderen kavalactonen einer der hauptinhaltsstoffe aus der kava-kava-wurzel (piper methysticum, rauschpfeffer). diesem wirkstoff mit antidopaminerger und daher auch prolaktinerger wirkung, werden psychostabilisierende eigenschaften zugeschrieben. untersuchungen zur anwendung bei schwangeren liegen nicht vor, allerdings gibt es bisher auch keine hinweise auf teratogene effekte. aufgrund seiner hepatotoxizität hat es in den vergangenen jahren an bedeutung verloren. meprobamat ist einer der ältesten tranquilizer. seit der einführung der benzodiazepine hat meprobamat therapeutisch keine große bedeutung mehr. in einer studie mit frauen, die meprobamat im . trimenon erhalten hatten, war die häufigkeit von herzfehlbildungen erhöht (milkovich diazepam wird nach oraler gabe rasch resorbiert und im blut überwiegend an plasmaproteine gebunden transportiert. in der leber erfolgen eine hydroxylierung und die metabolisierung zu dem noch aktiven desmethyldiazepam, das nach glucuronidierung über die nieren ausgeschieden wird. die halbwertszeit beträgt - tage, beim neugeborenen ist sie aufgrund verminderter clearance erheblich verlängert. orale kontrazeptiva können durch hemmung des metabolismus die alprazolam-oder diazepamkonzentration erhöhen. außerdem können sie die enterale absorption von benzodiazepinen verändern und zu einer wirkungsabschwächung von lorazepam durch einwirkung auf dessen kinetik führen. andererseits ist eine beeinträchtigung oraler kontrazeptiva bei benzodiazepintherapie durch cytochrom-p -enzyminduktion möglich . diazepam ist gut plazentagängig. unter der geburt ist die konzentration im nabelvenenblut bis zu fach höher als im mütterlichen blut. fehlbildungsrisiko. für benzodiazepine besteht nach heutigem wissen kein nennenswertes teratogenes risiko, obwohl die vorliegenden studien ein teilweise widersprüchliches bild ergeben. die meisten erfahrungen liegen zu diazepam vor. im zusammenhang mit einer benzodiazepintherapie im . trimenon wurden herzfehlbildungen, lippen-kiefer-gaumen-spalten, inguinalhernien und komplexe andere fehlbildungen beschrieben (Übersicht bei mcelhatton ). andere studien konnten teratogene effekte nicht bestätigen , patuszak . in einer metaanalyse zeigten die gesammelten daten von kohortenstudien mit schwangeren, die mit benzodiazepinen behandelt wurden, keine auffälligkeiten. die zusammenfassende analyse der verfügbaren retrospektiven fall-kontroll-studien erbrachte hingegen ein erhöhtes risiko für große fehlbildungen bzw. für isolierte mundspaltbildungen nach behandlung der mütter mit benzodiazepinen (dolovitch ). auch rodriguez-pinilla ( ) erörtert im zusammenhang mit zwei retrospektiven fall-kontroll-untersuchungen mit daten aus fehlbildungsregistern schwache, aber statistisch signifikante assoziationen zwischen benzodiazepin-exposition im . trimenon und spaltbildungen, intestinalen atresien und mikrozephalie. zu alprazolam ergaben sich bislang keine hinweise auf teratogenität (schick-boschetto , st clair . zwei retrospektive fall-kontroll-untersuchungen mit rund mit chlordiazepoxid exponierten schwangeren , sowie patientinnen mit alprazolam, etwa mit clonazepam (details siehe kapitel . antiepileptika), mit medazepam, mit nitrazepam und mit tofisopam ergaben ebenfalls keine hinweise auf nennenswerte teratogene effekte , eros . bonnot und mitarbeiter ( ) fanden kein erhöhtes risiko für spezielle fehlbildungen bei benzodiazepinen insgesamt, jedoch eine assoziation zwischen analatresie und lorazepam: von kindern mit anal-atresie, deren mütter benzodiazepine genommen hatten, waren pränatal mit lorazepam exponiert. anpassungsstörungen nach der geburt. laegreid und mitarbeiter ( ) berichteten über acht kinder, deren mütter während der gesamten schwangerschaft arzneimittelabusus mit täglich mindestens mg diazepam und mindestens mg oxazepam betrieben hatten. alle kinder wiesen gesichtsdysmorphien auf, einige außerdem eine mikrozephalie sowie postpartal toxische symptome (apnoe) und entzugserscheinungen. später wurden unterschiedlich ausgeprägte mentale retardierungen, konzentrationsstörungen und hyperkinesien beobachtet. diesen falldarstellungen ist jedoch vorgehalten worden, art und umfang der exposition nicht ausreichend abgesichert und in einem fall ein zellweger-syndrom nicht ausgeschlossen zu haben. in nachfolgeuntersuchungen wurde bei den etwa monate alten kindern eine besserung der symptomatik festgestellt (laegreid ) . als gesichert wird hingegen das risiko funktioneller störungen beim neugeborenen angesehen, wenn unter der geburt benzodiazepine hoch dosiert verabreicht wurden oder wenn über längere zeiträume, das letzte schwangerschaftsdrittel inbegriffen, regelmäßig diazepam oder andere benzodiazepine eingenommen wurden. einerseits muss nach hohen dosen sub partu mit atemdepression gerechnet werden, wie z. b. bei therapie einer eklampsie. andererseits kann nach andauernder exposition eine entzugssymptomatik mit unruhe, tremor, muskelhypertonus, erbrechen, durchfall auftreten, wie z. b. nach opiaten. auch zerebrale krampfanfälle in der neonatalphase sind möglich und ein wochen bis monate anhaltendes "floppy-infant-syndrom" mit muskelschlaffheit, lethargie, temperaturregulationsstörungen und trinkschwäche. aufgrund der akkumulation im fetus können im einzelfall schon geringe dosen diazepam (unter mg) beim neugeborenen zu klinischen symptomen führen (peinemann ) . das neugeborene metabolisiert benzodiazepine wesentlich langsamer als der erwachsene. langzeitwirkungen einer pränatalen exposition auf die spätere entwicklung des kindes sind nicht abschließend geklärt. besorgnis erregende hinweise gibt es bisher aber nicht. benzodiazepine können in der peripartalphase bilirubin aus der albuminbindung im blut verdrängen und zumindest theoretisch einen icterus neonatorum verstärken. empfehlung für die praxis: bei strenger indikationsstellung sind benzodiazepine mittel der wahl zur behandlung einer angstsymptomatik und in bestimmten fällen auch von schlafstörungen in der schwangerschaft. sie sollten, auch nach ausschöpfung aller nicht medikamentöser behandlungsmöglichkeiten und medikamentöser alternativen (z. b. niedrig dosierte antidepressiva, wie etwa amitriptylin), nur kurzzeitig verordnet werden. eine dauertherapie im letzten tri-menon, z. b. als zusatzmedikation zur wehenhemmung oder eine behandlung am geburtstermin ist wegen möglicher neonataler komplikationen (siehe oben) besonders kritisch zu prüfen. in den ersten lebenstagen muss verstärkt auf symptome beim kind geachtet werden. nach möglichkeit sollte mit der werdenden mutter eine dosisreduktion rechtzeitig vor dem erwarteten geburtstermin besprochen werden (abhängig von der halbwertszeit). im fall einer therapie mit benzodiazepinen empfiehlt es sich, aufgrund der durch enzyminduktion verursachten unsicherheit hormoneller kontrazeption keine systemische hormontherapie, also auch keine oralen kontrazeptiva, vorzusehen, da selbst die gelegentlich empfohlene verdopplung der dosis nicht die gewünschte sicherheit garantiert. ein intrauterinsystem mit lokaler gestagenabgabe (mirena ® ) wäre zu bevorzugen oder bei etwas geringerer sicherheit ein intrauterinpessar (iud). nur wenn diese methoden nicht vertragen werden, ist eine höher dosierte hormonelle kontrazeption -ggf. mit einschränkungen der verlässlichkeit -in betracht zu ziehen. hierfür kommt eine durchgehende einnahme von täglich dosen eines niedrig dosierten monophasischen präparates infrage und zwar im langzyklus durchgehend für - monate. g . . zaleplon, zolpidem und zopiclon zaleplon (sonata ® ), zolpidem (z. b. stilnox ® ) und zopiclon (ximovan ® ) sind neuentwickelte hypnotika mit agonistischer wirkung am benzodiazepinrezeptor. sie sind chemisch nicht mit der gruppe der benzodiazepine verwandt. wegen des geringeren suchtpotenzials finden sie heute zunehmende verbreitung. untersuchungen an verschiedenen tierspezies lassen nach angaben der hersteller keine teratogenen effekte erkennen. zur anwendung von zaleplon gibt es keine ausreichenden erfahrungen für eine differenzierte risikobeurteilung in der schwangerschaft. zu zolpidem liegen auf der basis von einem dutzend im . trimenon exponierten schwangerschaften keine hinweise für teratogene eigenschaften vor (wilton ). für eine differenzierte risikobewertung reichen die daten jedoch nicht aus. in einer prospektiven studie mit im . trimenon mit zopiclon behandelten schwangeren fanden sich keine auffälligkeiten gegenüber einer kontrollgruppe (diav-citrin ). zu eszopiclon (lunesta ® , estorra ® ), dem s-enantiomer des zopiclon, gibt es keine erfahrungen in der schwangerschaft. empfehlung für die praxis: die hier genannten arzneimittel sollten während der schwangerschaft nicht als hypnotika verordnet werden. eine dennoch erfolgte einnahme im . trimenon rechtfertigt keinen risikobegründeten schwan- (arai , nomoto , von graevenitz . die mit ausnahme der älteren ergotaminabkömmlinge liegen zu den meisten dieser mittel keine ausreichenden erfahrungen in der schwangerschaft vor. hinweise auf ein erhebliches teratogenes potenzial beim menschen sind bisher nicht erkennbar. in gut begründeten einzelfällen ist die behandlung mit parkinsonmitteln auch im . trimenon akzeptabel, z. b. bei therapie eines prolaktinoms mit ergotaminderivaten oder von extrapyramidalen nebenwirkungen einer neuroleptika-therapie mit biperiden. bei restless-legs-symptomen sollten möglichst andere therapieoptionen genutzt werden. keines der hier erörterten mittel rechtfertigt, wenn es im . trimenon eingenommen wurde, einen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). eine ultraschallfeindiagnostik sollte jedoch angeboten werden, wenn eines der unzureichend untersuchten produkte im . trimenon eingenommen wurde. steyn dw, odendaal hj. the effect of oral ketanserin on fetal heart rate tumor-nekrose-faktor-(tnf-) § -antikörper: adalimumab, etanercept, infliximab. auch corticosteroide sind zu den immunsuppressiva zu rechnen (siehe kapitel . ). zu den immunstimulanzien gehören interferone, kolonie-stimulierende faktoren (csf) und glatiramer. klinische erfahrungen liegen vor allem zu schwangerschaften nach nieren-und lebertransplantation und zur langzeittherapie mit azathioprin, ciclosporin und tacrolimus in kombination mit einem niedrig dosierten glucocorticoid (prednisolon) vor. insbesondere dann, wenn keine abstoßungsreaktion auftritt und die transplantation mindestens - jahre zurückliegt, ist die prognose für eine schwangerschaft gut. zwar wird häufiger per sectio entbunden, und es kommt vermehrt zu frühgeburten und zu intrauteriner wachstumsretardierung (small for gestational age) sowie zur passageren nierenfunktionsstörung beim neugeborenen. ein erhöhtes fehlbildungsrisiko oder bleibende funktionsdefizite sind aber aufgrund der vorliegenden erfahrungen nicht zu erwarten. bei einem vergleich von schwangerschaften organtransplantierter frauen einige jahre vor und nach transplantation zeigte sich, dass verschiedene befunde in beiden gruppen ähnlich häufig auftraten. das betrifft die rate an präeklampsie ( %), an frühgeburten ( %), niedrigerem geburtsgewicht ( %), small for gestational age ( %) und die säuglingssterblichkeit ( %). eine erhöhte fehlbildungsrate wurde in keiner der beiden gruppen festgestellt (källén ) . diese studie legt nahe, dass die geschilderten komplikationen in erster linie durch die schwere der mütterlichen erkrankung verursacht werden und nicht durch die transplantation oder die immunsuppressive therapie. die abortrate war in dem kollektiv vor transplantation höher als danach. zur antirheumatischen therapie siehe kapitel . . . g . azathioprin (aza) (z. b. imurek ® ) ist ein zur immunsuppression genutzter antimetabolit, der zu mehr als % zu -mercaptopurin ( -mp) metabolisiert wird. von aza werden nach oraler gabe ca. % resorbiert, während es bei -mp durchschnittlich nur % sind . von vielen autoren wird das risiko für beide substanzen bei immunsuppressiven dosierungen als vergleichbar angesehen. aza wirkt in bakterientestsystemen mutagen und im tierexperiment teratogen. annähernd studien bzw. fallserien mit insgesamt mehr als . schwangeren sowie weitere fallberichte zu schwangerschaften lassen kein nennenswert erhöhtes fehlbildungsrisiko erkennen (berkovitch , moskovitz , armenti . nur fand anhand der daten aus dem dänischen geburtsregister bei einer fallzahl von nur frauen eine signifikante erhöhung an grobstrukturellen fehlbildungen, perinataler sterblichkeit und frühgeburtlichkeit. wie andere zytotoxische substanzen vermag aza das intrauterine wachstum zu hemmen, so dass verschiedentlich ein erniedrigtes geburtsgewicht beobachtet wurde. dies kann allerdings auch folge der grunderkrankung oder der häufig gleichzeitig erfolgten glucocorticoidtherapie sein. eine leukopenie der mutter am ende der schwangerschaft kann auf eine neonatale hämatopoesehemmung und immunsuppression hinweisen (davison ) . bei bar-oz , lamarque . intrauterine wachstumsverzögerung und eine erhöhte rate an frühgeburtlichkeit wurden beschrieben, jedoch sind diese befunde wahrscheinlich durch die mütterliche grunderkrankung bedingt. bei sechs im ersten lebensjahr untersuchten kindern fanden sich veränderungen bei den b-und t-lymphozyten sowie den natural-killer-zell-funktionen (nk), die aber offenbar klinisch nicht relevant waren (dipaolo ) . in zwei anderen untersuchungen zeigte sich keine veränderte immunreaktion bei kindern bis zum alter von jahren (baarsma ) bzw. bis zum alter von jahren (rieder ) . weder die untersuchung von rieder ( ) an kindern, noch eine andere von an kindern erbrachte hinweise auf neurologische störungen bzw. auf einschränkungen der intellektuellen entwicklung. ein einzelner fallbericht beschreibt ein -jähriges kind mit hepatoblastom nach mütterlicher behandlung mit ciclosporin während der gesamten schwangerschaft (roll (jain ) . wie bei anderen immunsuppressiva wurde ein vermehrtes auftreten von präeklampsie, frühgeburten, geringerem geburtsgewicht und schnittentbindungen beobachtet. die erfahrungen in der schwangerschaft beruhen auf retrospektiven fallberichten, fallserien (jain , rayes , einer kleinen prospektiven studie und auf der von arzneimittelherstellern eingerichteten "national transplantation registry" (armenti ). etwa schwangerschaften sind unter tacrolimus dokumentiert, das in der regel zusammen mit prednison, aber auch mit anderen immunsuppressiva gegeben wird. ein nennenswertes teratogenes risiko konnte bisher nicht festgestellt werden. die oben erwähnten schwangerschaftskomplikationen und vorübergehenden nebenwirkungen beim neugeborenen wurden von den meisten autoren beobachtet. in der prospektiven studie von jain ( ) lebendgeborenen fanden sich vier kinder mit verschiedenen fehlbildungen (kainz ) . in einem retrospektiven fallbericht findet sich ein frühgeborenes zwillingspaar mit cardiomyopathie, an der eines der beiden kinder verstarb (vyas ). nagy ( ) berichtet über u. a. mit tacrolimus exponierten schwangerschaften, darunter sind zwei kinder mit einem kleinen ventrikelseptumdefekt. hypoplastische nägel und beidseits kurze fünfte finger wurden bei einem sonst gesunden frühgeborenen nach immunsuppressiver kombinationstherapie mit tacrolimus und mmf (siehe unten) beschrieben (pergola die bisherigen erfahrungen mit ungefähr mehrheitlich wegen morbus crohn behandelten schwangeren wurden retrospektiv, z. b. aus krankenakten, gewonnen (z. b. mahadevan , katz ). sie lassen kein teratogenes risiko erkennen. aus berichten über schwangerschaften mit anwendung von etanercept (enbrel ® ), davon einmal bis zur . schwangerschaftswoche, lässt sich kein teratogenes risiko erkennen , wallace , sills . die von uns prospektiv erfassten schwangerschaften mit exposition im . trimenon erbrachten zwei gesunde kinder, einen spontanabort und zwei schwangerschaftsabbrüche aus persönlichen gründen. zu den monoklonalen antikörpern adalimumab (humira ® ), basiliximab, (simulect ® ), daclizumab (zenapax ® ), efalizumab (raptiva ® ), muromonab-cd (orthoclone ® okt ), palivizumab (synagis ® ) liegen keine ausreichenden erfahrungen in der schwangerschaft vor. weitere monoklonale antikörper siehe kapitel . . interferon- § -arzneimittel werden therapeutisch bei chronisch aktiver hepatitis b und c sowie bei chronisch myeloischer leukämie, haarzell-leukämie und anderen malignomen eingesetzt. hierzu zählen interferon alfa- a (roferon ® ), interferon alfa- b (intron a ® ), interferon alfacon- (inferax ® ) sowie peginterferon alfa- a (pegasys ® ) und peginterferon alfa- b (pegintron ® ). auch bei essentieller thrombozythämie werden interferon- § -arzneimittel verabreicht. interferon- § ist nicht plazentagängig. zahlreiche fallberichte haben bisher keine hinweise auf spezifische schädigungen der vorgeburtlichen entwicklung erbracht (Übersicht bei . eine -jährige schwangere, die durchgängig wegen thrombozythämie interferon alfa- a erhielt, brachte ein gesundes, wachstumsretardiertes weibliches frühgeborenes zur welt. mutter und kind zeigten klinische und laborchemische charakteristika eines (neonatalen) lupus-syndroms (fritz ) . interferon beta human (fiblaferon ® ) ist für schwer verlaufende viruserkrankungen zugelassen, interferon beta- a (avonex tm , rebif ® ) und interferon beta- b (betaferon ® ) bei multipler sklerose. boskovic ( ) führte eine kleine prospektive studie mit an multipler sklerose erkrankten schwangeren durch, die g -interferon erhielten und verglich sie mit zwei kontrollgruppen, eine umfasste unbehandelte schwangere mit multipler sklerose und eine weitere gesunde schwangere. in der behandelten gruppe war die spontanabort-und totgeburtenrate erhöht. in unserer datenbank gibt es unter mit interferon beta- a exponierten schwangeren zwei spontanaborte und fünf schwangerschaftsabbrüche aus persönlichen gründen. unter lebendgeborenen befand sich ein kind mit einer schweren hüftdysplasie. von mit interferon beta- b exponierten schwangerschaften endeten in einem spontanabort, zwei wurden aus persönlichen gründen abgebrochen und die fünf lebend geborenen kinder waren gesund. es ist zurzeit ungeklärt, ob g -interferone ursächlich zu einer höheren abortrate führen. interferon gamma- b (imukin ® ) wird zur verhinderung schwerwiegender infektionen bei septischer granulomatose eingesetzt. generell in dosierungen, wie sie zur therapie chronisch-entzündlicher prozesse erforderlich sind, können hydroxychloroquin und chloroquin möglicherweise abortiv wirken. im tierversuch reicherte sich chloroquin in der fetalen retina und im zns an. immer wieder wird der fall einer mutter mit lupus erythematodes zitiert, die unter dauertherapie mit chloroquin drei geschädigte und ein gesundes kind zur welt brachte. bei zwei dieser kinder wurde eine cochleovestibularisparese diagnostiziert und bei einem kind wurde im alter von jahren ein wilms-tumor festgestellt (hart ) . dokumentierte erfahrungen zu etwa schwangeren konnten jedoch kein spezifisches risiko belegen: bei kindern von müttern, die in der schwangerschaft wegen lupus erythematodes oder rheumatoider arthritis durchschnittlich monate hoch dosiert mit hydroxychloroquin oder chloroquin behandelt wurden, waren im mittleren alter von jahren keine ophthalmologischen auffälligkeiten nachweisbar (klinger ) . motta ( ) konnte dies bei der untersuchung weiterer in schwangerschaft und stillzeit hydroxychloroquin exponierter kinder bestätigen. andere kleinere untersuchungen an insgesamt mehr als erkrankten schwangeren mit systemischem lupus erythematodes (sle) oder rheumatoider arthritis geben keine hinweise auf ein teratogenes potenzial (motta , buchanan , parke . eine größere prospektive studie mit schwangeren konnte im alter von durchschnittlich jahren bei den lebendgeborenen weder visuelle, akustische noch wachstums-oder sonstige entwicklungsdefizite finden (costedoat-chalumeu ) . einige autoren empfehlen ausdrücklich eine fortsetzung der therapie beim lupus erythematodes während der gesamten schwangerschaft, weil sie ein größeres risiko in einer sonst eher möglichen exazerbation der erkrankung sehen (costedoat-chalumeu , khamashta ). (pinter , rosa ); diesen stehen mehr als publizierte unauffällige verläufe gegenüber (sinha , tarnacka , messner , schaefer , dupont trientin wird ebenfalls bei morbus wilson eingesetzt. bei während der (gesamten) schwangerschaft behandelten frauen fanden sich keine hinweise auf spezifische auffälligkeiten im schwangerschaftsverlauf und bei den neugeborenen (eigene beobachtungen, Überblick in devesa ) . da als nebenwirkung der behandlung häufig eine eisen-mangelanämie beobachtet wurde, wird eine therapiebegleitende eisensupplementierung empfohlen (schmidt leflunomid (arava ® ), ein pyrimidinsynthesehemmstoff, war im tierversuch bei serumkonzentrationen teratogen, die den therapeutischen werten beim menschen entsprechen. auffällig waren anophthalmie bzw. mikrophthalmie und hydrozephalus. allerdings zeigten sich gleichzeitig toxische effekte bei den muttertieren, so dass der teratogene charakter der schädigung kontrovers diskutiert wird. die halbwertszeit von leflunomid beträgt wochen und länger. ausreichende erfahrungen beim menschen liegen nicht vor (brent ) . der autor diskutiert eine mehrtägige behandlung mit x g colestyramin zur verkürzung der eliminationshalbwertszeit auf etwa tag, wenn der empfohlene zeitliche abstand zu einer schwangerschaft nicht eingehalten werden kann. bei der planung einer schwangerschaft wird ein therapiefreies intervall von ca. monat vor konzeption empfohlen. bisher gibt es keine fallberichte, die einen teratogenen effekt beim menschen belegen. von publizierten schwangerschaftverläufen wurden abgebrochen, eine endete als spontanabort und mit der geburt eines frühgeborenen und zweier reifgeborener kinder ohne fehlbildungen . wir beobachteten gesunde kinder nach mütterlicher leflunomidexposition wochen vor konzeption und weitere gesunde kinder nach behandlung im . trimenon, davon eines nachweislich bis woche . zu anakinra (kineret ® ), einem anti-interleukin- g -therapeutikum, und abatacept (orencia ® ), einem selektiven co-stimulans-blocker bei rheumatoider arthritis, gibt es bisher keine erfahrungen in der schwangerschaft. zu ademetionin (gumbaral ® ), hyaluronsäurepräparaten (z. b. hyaject ® ) und oxaceprol (ahp ® ) liegen keine systematischen untersuchungen zum nutzen und zur verträglichkeit in der schwangerschaft vor. bei schwangeren mit glucosamin(dona ® )-therapie, das zur schmerzlinderung bei rheumatoider arthritis und gonarthrose eingesetzt wird, waren keine grobstrukurellen fehlbildungen nachweisbar (sivojelezova ) . bei weiteren prospektiv nachverfolgten schwangerschaften mit exposition im . trimenon fanden sich ebenfalls keine anomalien (eigene daten). empfehlung für die praxis: zur antirheumatischen basistherapie (dmard) in der schwangerschaft kommt als erstes sulfasalazin infrage. azathioprin, ciclosporin, (hydroxy-)chloroquin, aber auch goldpräparate und d-penicillamin sind akzeptabel. methotrexat, leflunomid, anakinra und biologica sind kontraindiziert. nichtsteroidale antirheumatika können bis woche und prednison/prednisolon in der gesamten schwangerschaft bei bedarf eingesetzt werden. soll penicillamin als chelatbildner z. b. bei morbus wilson eingesetzt werden, muss die dosis so niedrig wie möglich gewählt werden. eine begleitende gabe von kupfer in präventiver absicht wird nicht empfohlen, da allenfalls die penicillaminwirksamkeit beeinträchtigt würde. empfehlenswert ist allerdings eine ergänzende therapie mit pyridoxin (vitamin b ). ob der chelatbildner trientin eine alternative für die morbus-wilson-behandlung in der schwangerschaft darstellt, kann mangels erfahrung noch nicht entschieden werden. keines der in diesem abschnitt besprochenen mittel rechtfertigt nach (versehentlicher) anwendung im . trimenon einen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). zusätzliche untersuchungen wie eine ultraschallfeindiagnostik sollten jedoch eingeplant werden. g . . thalidomid thalidomid (ehemals contergan ® ) ist seit in den usa für die behandlung des erythema nodosum leprosum, eines schweren, entzündlichen verlaufs bei lepra, unter dem namen thalomid tm zugelassen. im rahmen des so genannten steps-programms (system for thalidomide education and prescribing safety; Übersicht in neiger ) sollen in dieser form bisher nicht praktizierte einschränkungen und kontrollen bei verordnungsbefugten Ärztinnen und patientinnen teratogene ereignisse weitgehend ausschließen. es wird jedoch befürchtet, dass dieses wirksame medikament auch (unkontrolliert) bei anderen entzündlichen und immunologischen erkrankungen eingesetzt wird, wie bei ulzerierenden hiv-assoziierten hauterkrankungen, anderen aids-begleitenden krankheiten, morbus behçet, chronischen graftversus-host-erkrankungen nach transplantation, therapierefraktären arthritiden etc. (teratology society ) . am teratogenen risiko der substanz hat sich seit der rücknahme vom markt in den frühen er jahren nichts geändert. laut einer publikation wurden "contergan-kinder" in lateinamerika seit registriert, vorwiegend in solchen regionen brasiliens, in denen lepra endemisch ist. dort war das medikament weiter frei erhältlich. die dunkelziffer geschädigter kinder liegt wahrscheinlich um ein vielfaches höher (castilla ) . wie kürzlich vom teratologischen zentrum in porto allegre berichtet (schüler, pers. mitteilung ) , ist es keine ausnahme, dass frühschwangere frauen in unkenntnis des risikos das thalidomid eines an lepra erkrankten familienmitglieds für andere beschwerden einnehmen. besonders von analphabetismus betroffene familien sind mit verordnungsprotokollen wie dem oben genannten kaum zu erreichen. thalidomid kann neben der bekannten phokomelie und amelie (vorwiegend der arme) auch weniger ausgeprägte muskel-und skelett-fehlanlagen an den extremitäten verursachen. häufig wurden daumenanomalien (z. b. dreigliedrigkeit) beobachtet. außerdem sind anomalien der ohrmuschel, des gehörgangs und mittelohrs mit oder ohne taubheit möglich. ferner treten auf: hirnnervenparesen (nervus facialis), fehlbildungen am herzen und anderen organen. mentale entwicklungsauffälligkeiten, auch autismusartige symptome, abnorme tränensekretion (krokodilstränen) oder andere augenanomalien wie kolobom, glaukom, mikrophthalmie und ptosis ) sind selten. der schädigungsmechanismus des thalidomids wird bis heute diskutiert, z. b. über eine transkriptionsstörung jener gene, die die blutgefäßbildung (angiogenese) in sich entwickelnden organen steuern (stephens ) . auch die nachkommen von "contergan-patienten" wurden untersucht. eine hypothese besagte, dass diese ebenfalls ein höheres fehlbildungsrisiko, z. b. aufgrund einer zusätzlichen mutagenen wirkung des thalidomids, haben. diese vermutung konnte widerlegt werden (z. b. strömland ) . in einzelnen fällen lag offenbar eine genetisch definierte, vererbbare anomalie mit gleichem erscheinungsbild vor, die mit "contergan" verwechselt wurde. zum risiko bei einnahme des vaters siehe kapitel . . empfehlung für die praxis: thalidomid ist als das für den menschen stärkste teratogen absolut kontraindiziert, wenn eine schwangerschaft nicht mit sicherheit ausgeschlossen werden kann. g . . sonstige immunmodulatoren glatiramer (copaxone ® ) wird bei der multiplen sklerose eingesetzt. chemisch ist es ein synthetisches polypeptid aus vier aminosäuren. der hersteller berichtet, dass es auch bei einer sehr hohen dosierung keine fehlbildungen im tierversuch bei ratten und kaninchen gegeben habe. die erfahrungen beim menschen beschränken sich auf wenige unauffällige fallberichte: unter lebendgeborenen fand sich kein kind mit einer fehlbildung (paulus , johnson . granulozyten-kolonien-stimulierende faktoren wie lenograstim (granocyte ® ), pegfilgrastrim (neulasta ® ) und nartograstim sind unzureichend untersucht. diese substanzen werden auch physiologisch in plazenta und dezidua gebildet und finden sich im nabelschnurblut. ein teratogenes risiko erscheint deshalb wenig wahrscheinlich. zu dem auch als immunstimulator und als hämatopoetischer wachstumsfaktor eingesetzten zytokin filgrastim (neupogen ® ) liegen erfahrungen im . und . trimenon vor (sangalli ) , jedoch nur wenige einzelfälle im . trimenon. die meisten kinder wurden gesund geboren. filgrastrim passiert die plazenta und wird in der schwangerschaft auch zur prophylaxe von neonatalen infektionen bei frühgeborenen eingesetzt (calhoun ) . zu inosin (isoprinosine ® ) liegen keine ausreichenden erfahrungen vor. levamisol wird als immunmodulator in kombination mit fluorouracil bei kolorektalen karzinomen gegeben oder als anthelmintikum in der tiermedizin. es bestehen Ähnlichkeiten zu metronidazol. bei schwangeren, davon waren im . trimenon exponiert, gab es keine besonderheiten ( ) welche konsequenzen erfordert bzw. erlaubt eine in der schwangerschaft diagnostizierte maligne erkrankung. nach heutigem wissensstand führt eine zurückliegende polychemotherapie bei einem großteil der exponierten weder zur dauerhaften infertilität, noch zu einer signifikant erhöhten abortrate oder einem geringeren geburtsgewicht bei den später entstehenden schwangerschaften (falconer ) . außer dauer, dosis und art der applizierten zytostatika hat das alter der frau bei der chemotherapie einen wichtigen einfluss auf die spätere fertilität (minton ) . eine neuere arbeit untersuchte die ovarielle funktion nach einer intensivierten therapie bei non-hodgkin-lymphomen mit cyclophosphamid, doxorubicin und vincristin (mega-chop). nur eine von patientinnen, die mit jahren älteste, hatte auch noch monate nach therapieende eine ovarielle dysfunktion. acht patientinnen wurden schwanger und bekamen insgesamt kinder. sieben frauen hatten prophylaktisch monatlich gnrh-analoga erhalten, darunter nicht die -jährige, jedoch der frauen, die später schwanger wurden (dann ) . eine zurückliegende zytostatische behandlung stellt auch kein nennenswertes risiko für fehlbildungen dar. trotz des mutagenen und zytotoxischen potenzials vieler antineoplastischer substanzen sind bisher nicht vermehrt klinisch relevante chromosomenstörungen oder gendefekte beschrieben worden, so dass eine amniozentese oder chorionzottenbiopsie nicht routinemäßig durchgeführt werden muss. in einer untersuchung von etwa . schwangerschaften, bei denen der vater eine krebsbehandlung hinter sich hatte, war das geschlechtsverhältnis der kinder mit , : , (kontrollgruppe: , : , ) zugunsten der mädchen verändert (green ) . auch eine länger zurückliegende radiotherapie führt i.a. weder beim mann noch bei der frau zu bleibender infertilität, wenn ovarien bzw. testes nicht gezielt bestrahlt wurden (stovall ) , und hat nach heutigem kenntnisstand keine relevanten auswirkungen auf eine spätere schwangerschaft. in einer untersuchung war die wahrscheinlichkeit, kinder mit einem gewicht unter . g zu gebären etwas höher, wenn früher im bereich des beckens bestrahlt wurde (green ) . boice ( ) untersuchte in einer multizentrischen studie mehr als . kinder von eltern, die in ihrer kindheit mit einer radiotherapie behandelt wurden: bei % der eltern betrug die gonadendosis mehr als msv, bei % mehr als . msv. es fanden sich keine hinweise für eine erhebliche zunahme genetischer auffälligkeiten. eine maligne erkrankung während der schwangerschaft ist mit , - pro . schwangerschaften selten. am häufigsten treten lymphome, leukämien, brustkrebs, zervix-und ovarialkarzinome, melanome, sowie schilddrüsen-und colonkarzinome auf. es gibt keine substantiellen hinweise dafür, dass eine schwangerschaft per se die prognose beeinflusst. dies gilt auch für das mammakarzinom (merlob ) , das allerdings in der gravidität häufig erst in einem späteren stadium erkannt wird. abgesehen vom melanom gibt es keine fallbeschreibungen über metastasierungen der plazenta oder des fetus. wenn im . trimenon eine maligne erkrankung diagnostiziert wird, entscheiden sich viele paare aufgrund des potenziellen teratogenen risikos der zu erwartenden therapie für einen abbruch der schwangerschaft, so dass hierüber die wenigsten erfahrungen vorliegen. die dokumentierten fälle zeigen jedoch, dass fehlbildungen keineswegs obligatorisch auftreten. eine polychemotherapie im . oder . trimenon kann je nach substanz und verabreichter dosis zu fetaler wachstumsverzögerung und/oder zur passageren knochenmarksdepression mit fetaler anämie, leukopenie und thrombozytopenie führen. ein intrauteriner fruchttod wurde nur selten beschrieben. Überraschenderweise wird die chemotherapie vom fetus meistens ohne bleibende schäden toleriert. bisher konnte auch keine beeinträchtigung der intellektuellen entwicklung festgestellt werden (nulman ) . im . trimenon wird häufig eine vorzeitige entbindung diskutiert, um dann "freie hand" für die therapie zu haben und den fetus nicht weiter mit potenziell toxischen substanzen zu belasten. empfehlung für die praxis: im allgemeinen wird eine frist von jahren bei der frau und von sechs monaten beim mann nach einer antineoplastischen therapie empfohlen. sollte vorher eine schwangerschaft eintreten, ist dies kein grund, eine intakte und gewollte schwangerschaft abzubrechen. eine maligne erkrankung während einer schwangerschaft ist selten und erfordert größtmögliche interdisziplinäre fachärztliche und psychosoziale unterstützung. die entscheidung eines paares über eine antineoplastische therapie in der schwangerschaft sollte nach sorgfältiger aufklärung über die jeweiligen risiken von ärztlicher seite mitgetragen werden. jede krebserkrankung in der schwangerschaft verlangt eine individuelle beratung und behandlung. in der regel werden neoplastische erkrankungen heute nach optimierten therapieschemata behandelt, die auch in der schwangerschaft beibehalten werden sollten, um der schwangeren bestmögliche chancen für ihr Überleben zu gewährleisten. deshalb werden in diesem abschnitt im gegensatz zu den übrigen kapiteln keine therapievorschläge aus embryonaltoxikologischer perspektive gegeben. hoch auflösende ultraschalldiagnostik sollte diesen frauen zur bestätigung einer normalen entwicklung des fetus angeboten werden. (mulvihill ) . beschrieben wird auch der spontanabort eines männlichen fetus in schwangerschaftswoche mit jeweils nur zehen und einer syndaktylie (garrett ) , sowie ein . g schweres neugeborenes, das einen vorhofseptumdefekt hatte und an einem atemnotsyndrom starb (thomas schardein und www.motherisk.org) . publiziert wurden jedoch auch ein abortierter fetus mit nierenagenesie nach einer kombinationstherapie (mennuti ) , ein . g schweres neugeborenes mit vorhofseptumdefekt, das an einem atemnotsyndrom starb und dessen mutter mit vincristin und vinblastin behandelt worden war (thomas ) , ein kind mit gaumenspalte nach vincristin bis woche , gefolgt von vinblastin und anderen zytostatika (mulvihill ) sowie eine frau mit einer kombinationstherapie wegen morbus hodgkin im . trimenon, deren kind mit hydrozephalus vier stunden nach geburt verstarb (zemlicki ) . ferner gibt es berichte zu unauffälligen schwangerschaften nach exposition im . und/oder . trimenon, jedoch auch über eine neonatale panzytopenie (pizzuto ) und intrauterine wachstumsretardierung. drei kinder, deren mütter wegen brustkrebs im . bzw. . trimenon mit vinorelbin (navelbine ® ) plus -fluorouracil behandelt wurden, waren im alter von bzw. jahren normal entwickelt (cuvier rodriguez , horbelt , arango , brunet vor, darunter auch mit exposition im . trimenon (aviles ) , bei denen gesunde kinder zur welt kamen. die weitere entwicklung dieser beiden kinder verlief bis zum alter von bzw. jahren normal. bei der exposition im ./ . trimenon wurde bei einigen kindern eine vorübergehende panzytopenie beschrieben (hsu , murray (elit ) . ein anderes frühgeborenes der schwangerschaftswoche , dessen mutter eine polychemotherapie in woche erhielt, zeigte ab dem . lebenstag eine schwere leukopenie und anämie und verlor ab dem . lebenstag kopf-und lanugobehaarung. nach wochen erholte sich das haarwachstum. eine nachuntersuchung nach einem jahr zeigte bis auf einen moderaten beidseitigen hörverlust (sensorineural) eine normale entwicklung (raffles ) . teniposid (vm -bristol ® ) ist ein semisynthetisches derivat des podophyllotoxins. es hemmt die topo-isomerase, verhindert die dna-synthese und die zelltransformation in die prophase. nur ein fallbericht zu einem gesunden neugeborenen liegt vor, bei dem die mutter während des . trimenons gleichzeitig mit anderen chemotherapeutika behandelt worden war (lowenthal (steege , shotton , wobei einer einen offenbar gesunden zwilling hatte. der dritte fetus zeigte retinale defekte (rugh ) . auch berichte mit unauffälligem schwangerschaftsausgang liegen vor (jacobs ) . cyclophosphamid (endoxan ® ) ist eine alkylierende substanz, die man zur behandlung einer vielzahl von malignen erkrankungen und bei autoimmunerkrankungen, wie z. b. dem systemischen lupus erythematodes, einsetzt. tierexperimente bei ratten, mäusen, kaninchen, affen und hühnern führten zu zns-, kraniofazialen und skelettfehlbildungen (zitiert nach enns ) . die erfahrungen zur behandlung schwangerer mit cyclophosphamid im . trimenon beruhen auf einer kleinen fallserie (aviles ) und auf retrospektiven kasuistiken. insgesamt gibt es berichte über mehr als im . trimenon behandelte frauen, darunter eine zwillingsschwangerschaft: kinder waren gesund (peres , aviles , pizzuto , feten bzw. kinder hatten große oder kleine fehlbildungen (paskulin , paladini , vaux , giannakopoulou , enns , mutchinick , kirshon , murray , toledo , greenberg , zwei schwangerschaften endeten als spontanabort (clowse ) und bei weiteren kam es in woche / zum fruchttod (peres , ba-thike . ein weiterer junge wurde mit multiplen fehlbildungen geboren und entwickelte mit jahren ein schilddrüsenkarzinom, mit ein neuroblastom und im alter von jahren wurde ein metastasiertes papilläres schilddrüsenkarzinom diagnostiziert. seine zwillingsschwester war gesund (zemlickis ) . wir konnten bisher im . trimenon exponierte schwangerschaften prospektiv auswerten, davon endete eine als spontanabort, eine wurde im . trimenon trotz unauffälligem hoch auflösendem ultraschallbefund aus mütterlicher indikation abgebrochen, und es wurden drei gesunde kinder geboren. neuerdings wird eine cyclophosphamid-embryopathie (enns ) bzw. eine cyclophosphamid-methotrexat-cytarabin-embryopathie (vaux ) diskutiert: kennzeichen sind zns-auffälligkeiten, faziale dysmorphien, distale extremitätenreduktionsdefekte sowie augenund ohrfehlbildungen und eine wachstumsretardierung. neun der oben genannten fallbeschreibungen entsprechen zumindest teilweise diesem muster, darunter auch solche, bei denen die mütter im rahmen eines systemischen lupus erythematodes als einziges zytostatikum cyclophosphamid erhalten hatten. durch therapie mit cyclophosphamid im . und . trimenon kann eine panzytopenie und ein verringertes geburtsgewicht beim neugeborenen verursacht werden. auch frühgeburten treten häufiger auf (kerr ) . zahlreiche fallbeschreibungen schildern einen unauffälligen ausgang der schwangerschaft, auch bei malignen erkrankungen (ring , köseoglu , luisiri , oates ). bei zwei lupus-patientinnen, die wegen der schwere der erkrankung im . trimenon cyclophosphamid erhielten, endete die schwangerschaft mit spätabort (clowse ) . ifosfamid (z. b. holoxan ® ) und trofosfamid (ixoten ® ) sind dem cyclophosphamid strukturell ähnlich. es gibt einen fallbericht über ein gesundes kind, dessen mutter wegen ewing-sarkom des beckens im . trimenon u. a. mit ifosfamid behandelt wurde (merimsky ) . chromosomale anomalien und malignomentstehung wurden bei nicht schwangeren patienten beobachtet, die mit melphalan (alkeran ® ) behandelt worden waren. entsprechende auswirkungen bei pränatal exponierten kindern sind nicht bekannt. zur anwendung in der schwangerschaft liegt lediglich ein bericht über eine fehlgeburt unter monotherapie im . trimenon vor (zemlickis ) . zu bendamustin (ribamustin ® ) wird über ein gesundes kind berichtet, dessen mutter im . trimenon behandelt worden war . zu estramustin (z. b. cellmustin ® ) liegen keine erfahrungen in der schwangerschaft vor. g . . andere alkylanzien busulfan (z. b. myleran ® ) wirkt speziell auf das knochenmark alkylierend und wird deshalb bei leukämien und bei der vorbereitung zur knochenmarktransplantation eingesetzt. mindestens schwangerschaften, darunter mit anwendung im . trimenon wurden publiziert, von denen wiesen kinder bzw. feten unterschiedliche fehlbildungen auf . vier von mit dacarbazin (detimedac ® ) behandelte schwangere waren im . trimenon exponiert. alle neugeborenen waren unauffällig (dipaola , aviles . in von fallberichten mit einer mechlorethamin-therapie im . trimenon (zusammen mit anderen zytotoxischen substanzen) wurden folgende anomalien beschrieben: oligodaktylie, hirnblutungen, hydrozephalus und nierenanomalien (zemlickis , mennuti . in fällen kam es zum abort und zweimal zum schwangerschaftsabbruch, andere schwangerschaften verliefen unauffällig (aviles ) . procarbazin (natulan ® ) ist bestandteil einer kombinationstherapie bei morbus hodgkin und anderen lymphomen. von im . trimenon exponierten neugeborenen waren lediglich gesund (aviles , schapira . folgende anomalien wurden beschrieben: multiple hämangiome, nieren-und extremitätenanomalien, lippen-, kiefer-, gaumen-spalten, vorhofseptumdefekt sowie intrauterine wachstumsretardierung. eine frau, die tage lang versehentlich mg täglich im . trimenon einnahm, wurde von einem gesunden kind entbunden (daw in der Übersicht von werden schwangere beschrieben, vier davon waren im . trimenon exponiert (feliu , alegre . die lebend geborenen kinder wiesen keine fehlbildungen auf. bei dieser kinder kam es im alter von monaten zur vorübergehenden neutropenie. nachfolgeuntersuchungen an kindern dieser gruppe im alter zwischen monaten und jahren zeigten eine normale entwicklung. zuazu ( ) (garcia , blatt , hassenstein . ein kind, dessen mutter gleichzeitig cyclophosphamid und eine kobaltbestrahlung der linken axilla und supraklavikularregion von woche - erhalten hatte, wies eine anal-atresie mit rektovaginaler fistel auf (murray ) . kim ( ) ein fallbericht (nakajima ) zeigte erneut, dass eine zytostatische therapie (doxorubicin und ifosfamid) im ./ . trimenon zu gesunden, aber wachstumsretardierten kindern führen kann. bei doxorubicin sind jedoch kardiotoxische nebenwirkungen bekannt: drei fallberichte über junge schwangere, die in ihrer kindheit bzw. jugend doxorubicin erhalten hatten und kardial unauffällig waren, dekompensierten am ende der schwangerschaft (pan ) . zu epirubicin (z. b. farmorubicin ® ) liegen mindestens fallberichte über kombinationstherapien vor, davon expositionen im . trimenon, die mit spontanabort endeten. bei den übrigen schwangerschaften gab es einen abort, eine totgeburt und ein kind starb kurz nach der geburt. beschrieben wurden ferner intrauterine wachstumsretardierung, frühgeburten und eine vorübergehende leukopenie (ring , gadducci , giacalone , müller , goldwasser . der plazentare Übergang von epirubicin ist gering und liegt etwas über dem von doxorubicin (gaillard ) . fünf falldokumentationen berichten über eine kombinationstherapie mit idarubicin (zavedos ® ) nach dem . trimenon (claahsen , reynoso . im ersten fall kam es nach therapiebeginn zum intrauterinen fruchttod, im zweiten wurde ein wachstumsretardiertes, im Übrigen aber gesundes neugeborenes beschrieben. das dritte kind wurde in woche geboren und fiel mit einer drei tage andauernden reversiblen herzinsuffizienz auf, die von den autoren auf idarubicin zurückgeführt wurde (achtari ) . in weiteren fallberichten wird von einer reversiblen rechtsherzvergrößerung beim kind berichtet (niedermeier , siu ). in einem dieser beiden fälle wurden außerdem ein ventrikelseptumdefekt, kurze finger mit dysplastischen nägeln, kurze extremitäten und faziale auffälligkeiten diagnostiziert, die sich mit der ausschließlich im . und . trimenon erfolgten exposition mit idarubicin und cytarabin nicht erklären lassen (niedermeier ) . idarubicin hat zwar eine geringere kardiotoxizität als die traditionellen anthrazykline, die größere lipophilie begünstigt jedoch den plazentaren Übergang. dies könnte erklären, warum es in den wenigen fallbeschreibungen so häufig zu kardialen komplikationen beim fetus kam. zu mitoxantron (z. b. onkotrone ® ) liegen fallbeschreibungen vor. die eine betrifft eine kombinationsbehandlung u. a. mit idarubicin, mit folgendem fruchttod (reynoso ) . außerdem wurde ein unauffälliges neugeborenes nach polychemotherapie in woche - beobachtet (azuno ) sowie ein gesundes und ein wachstumsretardiertes kind nach therapie im . trimenon (giacalone ) . mitoxantron besitzt immunmodulatorische eigenschaften und wird bei bestimmten formen der multiplen sklerose eingesetzt. zur behandlung schwangerer mit aclarubicin und pirarubicin liegen keine informationen vor. g . . andere zytotoxische antibiotika bleomycin (z. b. bleo-cell ® ) ist ein zytotoxisches glycopeptid-antibiotikum, das mit anderen chemotherapeutika bei morbus hodgkin, non-hodgkin-lymphomen und teratomen eingesetzt wird. es zeigten sich weder fetopathien noch chromosomenstörungen (lowenthal ) . ein kind, dessen mutter außerdem mit etoposid und cisplatin bis eine woche vor der entbindung behandelt worden war, wies eine neutropenie und leukopenie auf (raffles ) . zwei andere kinder, die den gleichen substanzen im ./ . trimenon ausgesetzt waren, waren unauffällig (han ). weitere kinder, darunter mit beginn der polychemotherapie im . trimenon, zeigten ebenfalls keine anomalien (aviles ) . unauffällig waren auch kinder, die im . und . trimenon mit dactinomycin (lyovac-cosmegen ® ) exponiert waren . zu mitomycin (z. b. mito-medac ® ) gibt es keine erfahrungen in der schwangerschaft. g . . folsäure-analoge antimetabolite (folsäure-antagonisten) aminopterin nach behandlung mit aminopterin, einer dem methotrexat verwandten substanz, wurden schon in den er jahren fehlbildungen beschrieben (warkany , meltzer , thiersch methotrexat (ein methylderivat des aminopterins, im englischen auch amethopterin genannt; z. b. mtx hexal ® ) hat zwar nur eine halbwertszeit von - stunden, aber ca. - % werden als polyglutamat-derivat in hepatozyten und erythrozyten über mehrere monate gespeichert (hendel ) . es wird bei einem breitem spektrum von indikationen eingesetzt: zur beendigung von ektopen oder unerwünschten schwangerschaften, bei autoimmunerkrankungen, bei chronisch entzündlichen krankheiten und zur behandlung von neoplasien. methotrexat besitzt ein teratogenes risiko mit einem ähnlichen muster an fehlbildungen wie aminopterin (siehe oben), so dass verschiedentlich von einem aminopterin/methotrexat-syndrom gesprochen wird (bawle ) . betrachtet man die Übereinstimmung der bei methotrexat, cyclophosphamid und cytarabin beschriebenen fehlbildungen (siehe unter cyclophosphamid . . ), dann scheint der begriff aminopterin/methotrexat-embryopathie nicht gerechtfertigt. ob diese arzneimittel einen gemeinsamen embryotoxischen wirkungsmechanismus besitzen, wie vaux ( ) vermutet, ist unklar. ob noch weitere antimetabolite vergleichbare fehlbildungen hervorrufen, ist derzeit nicht zu beurteilen. in über veröffentlichungen fanden sich mehr als im . trimenon behandelte schwangere. da es sich in der regel um retrospektive fallberichte handelt und nicht um eine prospektive studie, verbieten sich berechnungen eines fehlbildungsrisikos. bei einigen dieser publikationen fehlen zudem angaben zur dosis und indikation, wie z. b. bei mcelhatton ( ) , die über exponierte feten berichtet, davon im . trimenon. zwölf feten mit nicht genanntem expositionszeitraum waren auffällig, darunter schädelanomalien und andere skelettfehlbildungen. im folgenden werden nur die arbeiten mit genaueren angaben vorgestellt. zehn veröffentlichungen beschreiben schwangere, die im rahmen einer polychemotherapie im . trimenon methotrexat erhalten hatten. interessanterweise fanden sich in dieser hochrisikogruppe gesunde kinder (zemlickis , aviles , feliu , dara , pizzuto , ein kind mit einer inguinalhernie (giannakopulou ) , ein spontanabort (giacalone ) , eine totgeburt ohne fehlbildungen (peres ) (z. b. seidahmed , milunsky ). die gesamtdosis lag zwischen und mg methotrexat. in dieser schwangerschaften ( kinder) wurde zusätzlich misoprostol (yedlinski , adam , wheeler einige tage nach methotrexat verabreicht. in einem fall wurde zuvor erfolglos eine kürettage durchgeführt (bawle ) . andere berichte thematisieren pränatal diagnostizierte schädigungen exponierter feten, die zum abbruch der schwangerschaft führen (chapa , krähenmann . antirheumatische therapie mit mtx. zehn veröffentlichungen mit mehr als im . trimenon exponierten schwangerschaften beziehen sich auf die als "low-dose" bezeichnete therapie bei erkrankungen aus dem rheumatischen formenkreis. doch abgesehen von der kürzlich veröffentlichten kleinen prospektiven studie aus frankreich (lewden ) und unseren eigenen noch unveröffentlichten daten handelt es sich um retrospektive fallbeschreibungen oder allenfalls um kleine prospektive fallserien mit maximal schwangerschaften (Østensen (Østensen , donnenfeld . insgesamt fanden sich kinder mit typischen fehlbildungen, von denen allerdings zwei mütter (del campo , powell ) mit × , mg/woche (bis woche ) bzw. mg/tag (bis woche ) mehr mtx einnahmen als bei "low-dose" (maximal mg/woche) üblich ist. eine weitere erhielt , mg/tag für zwei tage in woche (nguyen ) und die vierte schwangere , mg/woche bis woche in kombination mit täglich mg folsäure (buckley ) . dem stehen fallbeschreibungen mit gesunden kindern mit einer dosis zwischen , und mg/woche gegenüber (Østensen , donnenfeld , feldkamp , kozlowski ), spontanaborte (Østensen (Østensen , kozlowski ) und schwangerschaftsabbrüche ohne embryopathischen hintergrund. berichtet über retrospektive schwangerschaften unter "low-dose"-mtx, wobei genauer einnahmezeitraum und dosis nicht bekannt sind. es kamen gesunde kinder zur welt, bei schwangerschaften wurden fehlbildungen beschrieben keine spezifizierung, endeten als spontanabort und schwangerschaftsabbrüche wurden durchgeführt. in einer prospektiven französischen studie (lewden ) mit fällen und einer wöchentlichen mittleren mtx-dosis von , mg wurden spontanaborte, schwangerschaftsabbrüche und lebendgeburten registriert, darunter keine mtx-embryopathie. diese erfahrungen decken sich mit unseren eigenen: von prospektiv dokumentierten im . trimenon exponierten schwangerschaften (wöchentliche dosis bis mg mtx) wurden bei unauffälligem ultraschallbefund abgebrochen, endeten als spontanabort und mit der geburt eines gesunden kindes ( frühgeborenes mit wochen). ein kind, dessen mutter zusätzlich phenprocoumon und andere medikamente einnahm, war mit . g am geburtstermin wachstumsretardiert, wies eine inguinalhernie auf und war tage lang hyperirritabel. bei kindern mit typischen fehlbildungen scheint eine begleitende kleinwüchsigkeit bestehen zu bleiben. in der weiteren entwicklung wurden sowohl eine normale intelligenzentwicklung als auch mentale retardierung beobachtet. die dosierungsbereiche für mtx bei polychemotherapie, abortversuch und bei rheumatischen indikationen ("low-dose") überschneiden sich. daher ist die schlussfolgerung, es gibt für mtx sichere und riskante indikationen, unzulässig. zwar fand sich bei mg wöchentlich bisher nur ein fallbericht mit verdächtiger symptomatik, so dass die von feldkamp ( ) geäußerte these plausibel erscheint, dass mtx erst ab einer wöchentlichen dosis von mg teratogen wirkt. die autorin postuliert ferner eine sensible phase zwischen woche und . für eine definitive schlussfolgerung sind die vorliegenden daten jedoch noch unzureichend. empfehlung für die praxis: nach behandlung mit dem teratogenen methotrexat wurden bei einer reihe von schwangerschaften entwicklungsanomalien beobachtet, die im wesentlichen aus einer pränatal beginnenden wachstumsretardierung, einem schweren ossifikationsdefekt des calvariums, fazialen dysmorphien, zns-störungen mit oder ohne intelligenzminderung und extremitätendefekten bestehen. eine unbedenkliche dosis kann nicht angegeben werden, allerdings gibt es bisher keine hinweise auf teratogene effekte unterhalb einer wöchentlichen dosis von mg. eine (versehentlich) ins . trimenon hinein weiter geführte antirheumatische "low-dose"-therapie scheint nur ein gering erhöhtes fehlbildungsrisiko zu besitzen. generell sind bei einer exposition im . trimenon fehlbildungen keineswegs obligat, auch nicht bei der behandlung von bösartigen erkrankungen. dass kinderwunschpatientinnen nach einer (antirheumatischen) methotrexat-therapie mindestens monate pause bis zur konzeption einhalten, kann mit den jetzt vorliegenden daten nicht begründet werden. jeder schwangeren, die im . trimenon mit mtx exponiert war, sollte eine hoch auflösende ultraschalldiagnostik zur bestätigung einer normalen entwicklung des fetus angeboten werden. g . . purin-analoge antimetabolite (purin-antagonisten) -mercaptopurin ( -mp; puri-nethol ® ) ist ein purin-analogon, das über eine hemmung der nukleinsäuresynthese wirkt (siehe auch azathioprin = aza, prodrug von -mp). es wurde bisher kein spezifisches fehlbildungssyndrom beschrieben. -mp wird auch als immunsuppressivum eingesetzt, z. b. bei chronisch entzündlichen darmerkrankungen ("inflammatory bowel disease" = ibd). auch wenn die plasma-hwz von -mp und aza mit - stunden sehr kurz ist, beträgt die halbwertszeit des zytostatisch aktiven metaboliten, der tioguanin-nukleotide, bis tage. wirkungsweise und metabolisierung von -mp sind interindividuell variabel. dabei spielt die thiopurin-methyltransferase (tpmt) eine rolle, deren aktivität genetisch determiniert ist. aza und -mp können die plazenta passieren . von mehr als im . trimenon exponierten schwangeren nahmen über das -mp im rahmen einer ibd ein , häufig in kombination mit prednisolon und eine kleine gruppe sogar fortlaufend während der gesamten schwangerschaft. die meisten kinder der mit -mp therapierten schwangeren in dieser und anderen publikationen wurden ohne anomalien geboren , aviles , dara , pizzuto . einige kinder bzw. feten wiesen fehlbildungen auf, wie z. b. polydaktylie (mulvihill ) , hypospadie (sosa ), hydrozephalus , lungenhypoplasie, sowie harnblasen-und urethrafehlbildungen , gaumenspalte und gesichtsdysmorphien (tegay ) . ein nennenswertes teratogenes potenzial lässt sich aus diesen berichten nicht ableiten. die dosierungsbereiche bei den beiden wichtigsten behandlungsindikationen ibd und leukämie überschneiden sich. daher ist die indikation primär kein unterscheidungsmerkmal hinsichtlich potentieller teratogenität. tioguanin (thioguanin-gsk ® ) ist ein purin-analogon, das dna-brüche in säugetierzellen verursacht. es ist dem -mp hinsichtlich struktur, wirkungsweise, nebenwirkungen und interaktionen ähnlich. bei im . trimenon behandelten schwangeren fand sich kein auffälliges neugeborenes (Übersicht in . eine weitere kasuistik beschreibt kind mit kraniosynostose, fingeranomalien und radiusaplasie, dessen mutter in woche gleichzeitig auch mit cytarabin, das vermutlich die fehlbildungen verursacht hat, behandelt wurde (schafer ) . auch artlich ( ) berichtet über fehlgebildetes kind nach exposition im . trimenon (siehe unter cytarabin). das kind einer wegen haarzell-leukämie bis woche mit cladribin (z. b.leustatin ® ) behandelten schwangeren zeigte keine teratogenen effekte (alothman ) . zu fludarabin (fludara ® ) gibt es bisher keine informationen. (aviles (aviles & , ein spontanabort ereignete sich tage nach ende der zytostase (zuazu ) , zwei schwangerschaftsabbrüche wurden registriert, einer davon mit wochen und unauffälligem befund beim fetus (zemlickis , lilleyman . drei kinder wiesen extremitätenanomalien auf, eines von ihnen hatte zusätzlich eine bilaterale mikrotie und atresie des äußeren gehörgangs (wagner (peres , veneri , requena , aviles und , blatt . auch über spätaborte bzw. totgeburten wurde berichtet (greenlund , zitiert nach zuazu , des weiteren über frühgeborene mit schwerer, aber reversibler panzytopenie (hsu , murray , engert ). reynoso ( ) berichtet über gesunde kinder und ein frühgeborenes der schwangerschaftswoche , dessen mutter wegen aml ab schwangerschaftswoche zytostatisch behandelt wurde. im alter von jahren wurde eine angeborene adhärenz der iris an der kornea des linken auges diagnostiziert. bis zum . lebensjahr entwickelte sich der junge im Übrigen unauffällig. -fluorouracil ( -fu; z. b. -fu hexal ® ) greift durch verdrängen von uracil ebenfalls in die dna-und rna-synthese ein. zur lokalen vaginalen anwendung von -fluorouracil im . trimenon liegen berichte über fünf gesunde kinder vor ). kasuistiken zur anwendung im . trimenon zusammen mit anderen chemotherapeutika beschreiben gesunde kinder , peres , zemlicki , zwei spontanaborte (giacalone ) und eine komplexe fehlbildung nach exposition in woche und (stephens ) . paskulin ( ) beschreibt ein kind, das intrauterin bis woche mit cyclophosphamid, fluorouracil und doxorubicin exponiert war und mit wachstumsretardierung, fazialen dysmorphien und verschiedenen distalen extremitätenfehlbildungen geboren wurde. die meisten der mehr als kinder mit intrauteriner -fu-exposition im . und . trimenon waren gesund (ginopoulos , berry . selten wurde eine intrauterine wachstumsretardierung beschrieben (zemlickis ) . dreicer ( ) berichtet über einen jungen, der im ./ . trimenon hohen dosen -fu ausgesetzt war (insgesamt g), bei der geburt in schwangerschaftswoche mit . g ein unterdurchschnittliches gewicht aufwies und im alter von zwei jahren normal entwickelt war. zur topischen anwendung von fluorouracil siehe abschnitt . . zu gemcitabin (gemzar ® ) und capecitabin (xeloda ® ) gibt es keine informationen zur verträglichkeit in der schwangerschaft. g . . taxane taxane wirken antineoplastisch über eine hemmung der synthese der mikrotubuli. sie werden beim mammakarzinom eingesetzt. erfahrungen zur anwendung im . trimenon liegen nicht vor. sechs kasuistiken zur exposition mit paclitaxel (taxol ® ) im ./ . trimenon berichten über gesunde kinder (v.a. gonzales-angulo , cardonick , gaducci , mendez , sood (raffles ) . elit ( ) berichtete über ein mit bleomycin, cisplatin und etoposid exponiertes kind, das bis zum alter von monaten untersucht wurde. dabei bestätigte sich die bereits intrauterin abzeichnende ventrikulomegalie, die zu einer hirnatrophie geführt hatte. in einer fallserie von peres ( ) wird über eine totgeburt mit schwangerschaftswochen ohne fehlbildungen berichtet. die mutter hatte wegen eines non-hodgkin-lymphoms cisplatin und etoposid in woche erhalten. außer der in fällen beschriebenen intrauterinen wachstumsretardierung waren die übrigen kinder gesund (ohara , dipaola , giacalone , hoffmann . ob die tierexperimentell beobachtete erhöhte rate an tumoren, z. b. der haut, nach pränataler exposition mit cisplatin für den menschen relevant ist, kann nicht beurteilt werden. carboplatin (z. b. ribocarbo ® ) ist dem cisplatin verwandt. fetotoxische wirkungen wurden bei einer zwischen woche und behandelten schwangeren nicht beobachtet (mendez (consoli , carradice , fadilah . ein weibliches reifgeborenes, das intrauterin ab woche mit atra und idarubicin exponiert war, wies einen vorhofseptumdefekt und eine milde rechtsventrikuläre dilatative kardiomyopathie auf, die sich nach ein bis zwei monaten vollständig zurückbildete. der hämodynamisch unbedeutende vorhofseptumdefekt war weiter nachweisbar (siu ) . zu amsacrin (amsidyl ® ), miltefosin (miltex ® ), das auch bei leishmaniose eingesetzt wird, sowie zu pentostatin (nipent ® ) und mitoguazon gibt es keine informationen zur verträglichkeit in der schwangerschaft. gleiches gilt für die topo-isomerase-inhibitoren irinotecan (campto ® ) und topotecan (hycamtin ® ), sowie für die photosensitizer temoporfin (foscan ® ) und porfimer-natrium (photofrin ® ) und für bexaroten (targretin ® ), einem agonisten am retinoid-x-rezeptor. auch zu pemetrexed. (alimta ® ), einem thymidylat-synthese-inhibitor, und zu mitotan (lysodren ® ) liegen keine erfahrungen in der schwangerschaft vor. imatinib, ein protein-tyrosinkinase-inhibitor (glivec ® ) wird u. a. zur therapie der chronisch myeloischen leukämie eingesetzt. wenige fallberichte existieren zur anwendung in der schwangerschaft. eine -jährige gebar ein gesundes . g schweres mädchen, das intrauterin bis woche mit imatinib exponiert war (ali ) . hensley ( ) beschreibt einen jungen mit hypospadie und zwei schwangerschaftsabbrüche wegen fehlbildungen der feten (hydrozephalus, herzfehler). in einem weiteren fallbericht mit imatinib-exposition bis woche geht es um ein mädchen mit pylorusstenose (heartin ) . erlotinib (tarceva ® ) ist ein neu zugelassenes zytostatikum zur behandlung des nicht-kleinzelligen lungenkarzinoms. erfahrungen zur schwangerschaft liegen nicht vor. g . . weitere antineoplastisch wirkende enzyme und antikörper asparaginase (asparaginase ® ) ist ein pflanzliches enzym, das die verfügbarkeit der aminosäure asparagin für das tumorwachstum reduziert. es wird mit anderen chemotherapeutika bei akuter leukämie kombiniert. bei im . trimenon exponierten kindern fanden sich keine fehlbildungen, kinder wiesen jedoch eine knochenmarkhypoplasie auf und kind chromosomenanomalien (turchi , scheuning . alemtuzumab (mabcampath ® ), ibritumomab-tiuxetan (zevalin ® ), cetuximab (erbitux ® ), edrecolomab und bortezomib (velcade ® ) und tositumomab (bexxar ® ) sind monoklonale antikörper, für die es keine erfahrungen zur verträglichkeit in der schwangerschaft gibt. zu rituximab (mabthera ® ) liegen erfahrungsberichte mit unauffälligem schwangerschaftsausgang vor. zwei behandlungen fanden aus versehen im . trimenon statt, die andere ab woche (ojeda-uribe , kimby , herold . trastuzumab (herceptin ® ) ist ein monoklonaler antikörper, der das "human epidermal growth factor receptor "-protein blockiert und eine geschätzte hwz von tagen hat. es gibt einen fallbericht über eine versehentliche exposition einer -jährigen mit brustkrebs, die bis woche alle drei wochen das medikament bekam. als in woche die schwangerschaft bemerkt wurde, zeigte sich ein anhydramnion bei einem gesunden weiblichen fetus. allmählich regenerierte sich die fruchtwassermenge. in woche wurde ein gesundes mädchen entbunden, das auch im alter von monaten eine normale nierenfunktion zeigte und keinen anhalt für eine pulmonale hypoplasie aufwies (watson ) . noch in der erprobung ist gefitinib beim kleinzelligen bronchialkarzinom, zu dem es noch keine erfahrungen in der schwangerschaft gibt. gleiches gilt für lapatinib und für bevacizumab (avastin ® ), das zusammen mit -fluorouracil beim metastasierten colon-oder rektumkarzinom angewendet wird. aldesleukin ( (cullins ) und ein weibliches neugeborenes mit indifferenter genitalentwicklung (tewari ) . bei einem mädchen, dessen mutter bis zum . schwangerschaftsmonat tamoxifen nahm, wurde im alter von jahren ein adenom der vagina diagnostiziert. auch über unauffällige verläufe wird berichtet , isaacs , lai (bond ) . fallbeschreibungen über fehlgeschlagene abortversuche und Überdosierungen unter der geburt beschreiben derartige seltene vorkommnisse. eine untersuchung in brasilien spricht für eine kausale beziehung zwischen fehlgeschlagenem abortversuch mit dem auch als ulkusmittel verwendeten misoprostol (cytotec ® ) und einer in wenigen fällen beobachteten möbius-sequenz (u. a. hirnnervenaplasie und extremitätendefekte) bei den kindern. auch andere fehlbildungen, wie schädelknochendefekte, omphalocele und gastroschisis, wurden beobachtet (orioli , gonzalez , hofmeyr , castilla , schüler . die zum abortversuch oral oder manchmal zusätzlich auch vaginal genommene dosis von misoprostol variierte zwischen und . ? g und betrug durchschnittlich ? g. die hohen dosen waren zeitlich über bis zu tage verteilt angewendet worden. in einer retrospektiven brasilianischen fall-kontroll-studie ergab die medikamentenanamnese der mütter von kindern mit möbius-sequenz, dass nahezu die hälfte misoprostol angewendet hatte. in einer kontrollgruppe von kindern mit neuralrohrdefekten waren es lediglich % der mütter. zwei prospektive kohortenstudien zu misoprostol fanden keine auffälligkeiten bei schwangerschaftsverlauf und befinden der neugeborenen, allerdings betrug die anzahl exponierter mütter nur bzw. und schließt bei derart selten auftretenden defekten kein risiko aus (bellemin , schüler ). ein teil der mütter hatte misoprostol als schutz vor magengeschwüren bei einer behandlung mit nichtsteroidalen antirheumatika eingenommen. schon ? g misoprostol können den arteriellen strömungswiderstand in den aa. uterinae dopplersonographisch nachweisbar heraufsetzen. dieser effekt kann die beobachteten disruptionsfehlbildungen als folge einer perfusionsstörung erklären (yip ) . zusammenfassend muss nach (versehentlicher) misoprostolanwendung ein geringes, teratogenes risiko angenommen werden. obwohl für keine der indikationen in der schwangerschaft zugelassen, ist ein trend zu beobachten, dass misoprostol vermehrt eingesetzt wird, sei es zur abortinduktion, zur geburtseinleitung oder in der postpartalperiode. sowohl die einfache applikation per os als auch der preis spielen dabei eine rolle. so ist misoprostol bei der geburtseinleitung nach blasensprung das wirkungsvollste medikament, das ohne zusätzliche applikationsbedingte infektgefährdung eingesetzt werden kann. es wird in der who-liste der essentiellen medikamente geführt (world health organization ) . eine mögliche Überstimulation und pathologische ctg-muster geben allerdings anlass, vor einem unkritischen einsatz zu warnen. bei zustand nach sectio oder anderen transmuralen eingriffen ist misoprostol wegen deutlich erhöhter gefahr einer uterusruptur kontraindiziert. wirkorte sind vor allem der uterusmuskel und die milchdrüsenausführungsgänge. voraussetzung für die oxytocinwirkung am schwangeren uterus ist ein sehr komplexes geschehen. dazu gehören eine abnahme der estrogen-und progesteronkonzentration im blut mit einer verminderung der § -und g -adrenergen sicherung der uterusmuskulatur. die wehenanregende wirkung soll durch eine depolarisierung der muskelzellmembran ausgelöst werden. während der schwangerschaft erhöht sich die oxytocinkonzentration im blut nur gering. erst am ende steigen sowohl die konzentration als auch die zahl der oxytocinrezeptoren im myometrium deutlich an. während der verschiedenen geburtsphasen bis zum pressen kann ein anstieg der oxytocinkonzentration um das -bis fache beobachtet werden. diese konzentrationserhöhung wird beispielsweise während der austreibungsphase durch den so genannten ferguson-reflex bewirkt. dabei wird der druckreiz von dem in der kreuzbeinhöhle gelegenen frankenhäuserschen ganglion über rückenmarksbahnen zum hypophysenhinterlappen geleitet. (higby , katz . eine wirkliche prävention von frühgeburten muss weiter greifen. kein langzeittokolyse-schema hat eindeutig zur verringerung von kindlicher morbidität und mortalität beigetragen (higby ) . als wehenhemmer wurden bzw. werden g -adrenerge substanzen, calciumantagonisten, magnesium, prostaglandin-und oxytocinantagonisten und nitroglycerin verwendet. am weitesten verbreitet sind verschiedene g -selektive sympathomimetika. diese auch in der asthmatherapie bewährten pharmaka haben weniger kardiovaskuläre nebenwirkungen als die nichtspezifischen g -sympathomimetika. g . . g -sympathomimetika pharmakologie und toxikologie. fenoterol (partusisten ® ) ist das in deutschland am meisten zur wehenhemmung verwendete g -sympathomimetikum. auch clenbuterol, ritodrin, salbutamol, terbutalin und das nicht g -spezifische hexoprenalin gehören zu den in der tokolyse gebräuchlichen g -sympathomimetika. bei intravenöser applikation begrenzen kardiovaskuläre wirkungen die anwendung von g -sympathomimetika. seit langem und kontrovers wird die wirksamkeit der oralen behandlung erörtert (baumgarten ) . obwohl vergleichbare konzentrationen wie bei der intravenösen behandlung erreicht werden (von mandach ), wird eine effektive tokolytische wirkung angezweifelt. tierexperimentell und in einzelnen fallberichten beim menschen wurden myokardnekrosen beim fetus und myokardinfarkte bei der mutter nach tokolyse mit g -sympathomimetika beobachtet. ein erhöhter sauerstoffbedarf des myokards zusammen mit dem durch das tokolytikum bedingten intrazellulären calciumeinstrom wird als ursache diskutiert. ein vermuteter kardioprotektiver einfluss von verapamil führte zeitweise zur kombination beider pharmaka (weidinger ) , bis berichte über lungenödeme unter dieser kombination bei gleichzeitiger Überwässerung erschienen (grospietsch ) . lungenödeme wurden auch bei einer tokolyse mit ritodrin alleine wiederholt beschrieben. insbesondere bei kombination mit corticosteroiden steigt die glucosekonzentration. das kann bei insulinabhängigen diabetikerinnen zum abrupten anstieg des insulinbedarfs führen. wiederholt wurden hyperkinetische verhaltensauffälligkeiten im kindesalter als folge wochenlanger g -sympathomimetischer tokolyse diskutiert. auch über passagere neurologische abweichungen in den ersten lebenstagen, die durch entsprechende tests im vergleich zu nicht exponierten neugeborenen ermittelt wurden, ist berichtet worden (thayer ) . eine abschließende beurteilung ist hierzu bisher nicht möglich. empfehlung für die praxis: eine kurzzeittokolyse mit g -sympathomimetika, kontinuierlich intravenös oder als bolusgabe (spätling (higby , morales . higby und mitarbeiter ( ) sahen die prostaglandinantagonisten wie indometacin sogar als die einzig effektiven tokolytika an. sulindac hat entgegen anders lautender mitteilungen die gleichen nebenwirkungen wie andere prostaglandinantagonisten (kramer ) . diese mittel können zum vorzeitigen verschluss des ductus arteriosus botalli und über eine herabgesetzte nierenfunktion des fetus zum oligohydramnion führen. dies ist offenbar bei kurzzeitiger tokolyse (höchstens stunden) und vor woche kaum problematisch (norton ) . andererseits sind generelle vorteile dieser wirkstoffgruppe gegenüber den g -sympathomimetika und calciumantagonisten nicht erwiesen. indometacin wurde auch zur behandlung des polyhydramnions angewendet (nordstrom ) . wenn die letzte indometacin-applikation nicht länger als stunden zurücklag, war bei sonst vergleichbarem befinden des neugeborenen eine surfactant-therapie etwas häufiger erforderlich als bei nicht exponierten kindern (abbasi ) . beim vergleich von frühgeborenen mit intrakranieller hämorrhagie fand sich gegenüber kontrollen kein signifikanter zusammenhang mit einer indometacin-tokolyse (suarez ) . von frühgeborenen, bei denen eine chirurgische intervention wegen persistierendem ductus arteriosus erforderlich wurde, war ein größerer anteil intrauterin mit indometacin exponiert als bei konservativ therapierten kindern (suarez ) . als ursache hierfür wurde eine schädigung der intima des ductus diskutiert, die den spontanverschluss verhindert hat (siehe auch kapitel . . ). in einer metaanalyse beschreiben cuzzolin und mitarbeiter ( ) fälle mit renalen komplikationen als ausdruck möglicher nephrotoxizität nach tokolytischer applikation von prostaglandinantagonisten, weisen aber darauf hin, dass diese eher selten sind. beim vergleich der verträglichkeit dieser mittel ergaben sich widersprüchliche ergebnisse. sawdy und mitarbeiter ( ) fanden bei indometacin, sulindac und nimesulid keinen unterschied in der wirksamkeit der tokolyse sowie bei maternalen oder neonatalen nebenwirkungen. sciscione und mitarbeiter ( ) wiesen jedoch auf ein unter indometacin erhöhtes risiko für bronchopulmonale dysplasien hin. empfehlung für die praxis: eine tokolyse mit prostaglandinantagonisten ist möglich. auswirkungen auf den kreislauf und die nierenfunktion des fetus mit resultierendem oligohydramnion müssen bedacht werden (siehe auch kapitel . . ). g . . oxytocinantagonisten pharmakologie und toxikologie. seit dem jahr ist der oxytocinantagonist atosiban für die wehenhemmung zugelassen. es ist ein parenteral wirksames spezifisches tokolytikum mit wenigen nebenwirkungen. aus preisgründen wird es in deutschland überwiegend bei problemsituationen eingesetzt, wie z. b. diabetes mellitus. fetotoxische wirkungen wurden bisher nicht beschrieben. empfehlung für die praxis: der klinische wert von atosiban zur wehenhemmung ist gesichert. g . . andere tokolytika früher wurde ethylalkohol erfolgreich als tokolytikum eingesetzt. seine wirkung beruht auf der hemmung der oxytocinausschüttung. per intravenöser zufuhr wurden ‰ und mehr im mütterlichen blut angestrebt. da die schädigende wirkung von alkohol auf die kindliche entwicklung nachgewiesen ist, gehört diese therapie nicht mehr zu den akzeptablen tokolytischen maßnahmen. nitroglyzerin als pflaster und i.v. verabreicht hat sich in kleineren untersuchungen als wirksames tokolytikum erwiesen. kopfschmerzen waren ein häufiges therapiebedingtes symptom, das seine weitere verbreitung verhinderte. negative auswirkungen auf den kreislauf des neugeborenen sind aufgrund der kurzen halbwertszeit nicht wahrscheinlich (black . der oral verfügbare vasopressin-v a -rezeptor-hemmstoff relcovaptan hat sich in einer kleinen randomisierten studie gegenüber plazebo als wirksam bei der wehenhemmung erwiesen (steinwall ) . empfehlung für die praxis: alkohol ist kontraindiziert. nitroglyzerin kann, wenn eine entsprechende indikation vorliegt, unter berücksichtigung der nebenwirkungen als tokolytikum eingesetzt werden. g . . vaginaltherapeutika es gibt hinweise dafür, dass die behandlung bakterieller vaginosen frühgeburten verhindern kann (hoyme , donders . bei risikoschwangerschaften scheint eine protektive wirkung möglich durch eine systemische antibiotische behandlung, die eher als eine vaginale applikation von beispielsweise clindamycin (z. b. sobelin ® ) oder metronidazol (z. b. arilin ® ) eine aszendierende infektion vermeidet bzw. heilt (donders , joesoef . eine systemische (orale) antiinfektive therapie führt bei berücksichtigung der für die schwangerschaft empfohlenen mittel zu keinem entwicklungstoxischen risiko (siehe kapitel . ). problematisch sind povidon-iod als vaginal-suppositorien und iodspülungen der scheide wegen der möglichen passageren beeinträchtigung der fetalen schilddrüsenfunktion ab woche (siehe abschnitt . . ). die behandlung mit anderen vaginaltherapeutika, die desinfizienzien enthalten, z. b. dequaliniumchlorid (fluomycin ® ), hexetidin (vagi-hex ® ), policresulen (albothyl ® ) oder mit estrogenen steht bisher nicht im verdacht, teratogen zu wirken. im bemühen um eine rationale therapie sollte man jedoch veraltete und in ihrer wirksamkeit umstrittene mittel meiden. auch der einsatz von nitrofuranen wie furazolidon und nifuratel (inimur ® ) sowie vom antimykotikum chlorphenesin ist kritisch zu prüfen. g . . spermizide kontrazeptiva frei verkäufliche vaginale kontrazeptiva, die als creme, gel, tabletten oder schaumovula angeboten werden (z. b. patentex ® ), enthalten nonoxinol als spermizid wirksame substanz. diese form der kontrazeption galt jahrelang als völlig ungefährlich, bis in den usa in einer studie an kindern von müttern, die trotz anwendung vaginaler kontrazeptiva schwanger wurden, über einen geringen anstieg der fehlbildungsrate berichtet wurde (jick ) . eine metaanalyse mehrerer, z. t. erheblich umfangreicherer untersuchungen konnte diesen verdacht entkräften (einarson ). in zahlreichen publikationen wurde erörtert, dass der gebrauch dieses spermizids über eine schädigung der vaginalschleimhaut und eine störung der physiologischen bakterienflora eine hiv-infektion bei entsprechendem kontakt begünstigen könne (rosenstein , stafford . empfehlung für die praxis: eine konzeption trotz anwendung eines nonoxinol- -haltigen vaginalen kontrazeptivums stellt nach heutigem wissen kein risiko dar. g . . intrauterinpessare die kupferkonzentration im eileitergewebe ist bei frauen mit kupferhaltigen iucds (intrauterine contraceptive devices) erhöht. im serum finden sich jedoch keine erhöhten coeruloplasmin-und kupferkonzentrationen (wollen ) . einige schwangerschaften mit liegendem (und verbleibendem) iucd sind bisher beschrieben worden. abort-und frühgeburtsraten sind im vergleich zu frauen erhöht, die sich das iucd entfernen ließen. sichere hinweise auf ein spezifisches fehlbildungsrisiko haben sich nicht ergeben (Übersicht in . dies ist auch für das als intrauterines system (ius) bezeichnete produkt mit levonorgestrel (mirena ® ) zu erwarten. empfehlung für die praxis: ein verbleibendes iucd rechtfertigt aus embryotoxikologischer sicht weder einen risikobegründeten abbruch einer schwangerschaft noch erfordert es invasive diagnostik (siehe kapitel . ). hormone hormone sind körpereigene stoffe, die physiologische prozesse steuern. ihre regulation erfolgt auf drei ebenen, der zwischenhirn-hypothalamus-ebene (vorwiegend releasing-funktion), der stimulatorebene in der hypophyse und der drüsenebene in den jeweiligen organen. die ausschüttung der hormone wird über regelkreise zwischen den drei ebenen gesteuert. wenn die mutter mit hormonen behandelt wird, sind auch beim fetus auswirkungen auf den verschiedenen ebenen möglich. die in diesem abschnitt besprochenen klassischen hormone sind von den gewebshormonen oder mediatoren zu unterscheiden, zu denen u. a. auch die prostaglandine (siehe abschnitt . . ) und leukotriene gehören. trh steuert die schilddrüsenfunktion und regt die prolaktinsekretion an. mit seinem analogon protirelin konnte man den effekt pränatal verabreichter corticoide auf die lungenreifung des fetus verstärken, eine günstige wirkung auf das neonatale atemnotsyndrom ließ sich jedoch nach auswertung von über . fällen nicht nachweisen (ballard , collaborative , actobat . glucocorticoide allein waren genauso wirksam. es wurde kontrovers diskutiert, ob die in der exponierten gruppe beobachtete leichte entwicklungsverzögerung im alter von jahr durch trh bedingt ist (crowther , mccormick . ghrh (growth hormone releasing hormone). synthetische analoga sind sermorelin und somatorelin (ghrh ferring ® ). ghrh und seine analoga wirken durchblutungsmindernd im uterusbereich und hemmen die proliferation des endometriums. daher werden sie präoperativ zur verkleinerung von myomen eingesetzt. bei versehentlicher anwendung während der schwangerschaft sind abort und intrauterine wachstumsretardierung denkbar. diese effekte wurden bisher aber ebenso wenig beobachtet wie eine hormonwirkung auf den fetus (Übersicht bei . gnrh (gonadotropin releasing hormone) bzw. lhrh (luteinizing hormone releasing hormone). synthetische analoga sind buserelin (z. b. profact ® ), gonadorelin (z. b. kryptocur ® ), goserelin (zola-dex ® ), leuprorelin (z. b. enantone ® ), nafarelin (synarela ® ) und triptorelin (z. b. decapeptyl ® ) . cetrorelix (cetrotide ® ) und ganirelix (orgalutran ® ) sind antagonisten der gnrh. therapeutisch werden gnrh-analoga bei hypothalamischer ovarialinsuffizienz und in der onkologie eingesetzt und ebenso wie die gnrh-antagonisten zur vermeidung eines vorzeitigen anstiegs des luteinisierenden hormons (lh) und damit eines vorzeitigen eisprungs im rahmen einer assistierten reproduktion. bei über im . trimenon versehentlich mit gnrh-analoga behandelten schwangeren fanden sich weder eine häufung angeborener anomalien oder fehlgeburten noch eine hemmende wirkung auf das intrauterine wachstum (Übersicht in cahill . in einer nur kinder umfassenden untersuchung wurden bei kindern im alter von durchschnittlich jahren entwicklungsauffälligkeiten diagnostiziert, wie z. b. aufmerksamkeitsdefizite, motorische und sprachstörungen sowie bei einem kind eine epilepsie. die autoren führen dies auf einen möglichen entwicklungstoxischen effekt der gnrh-analoga zurück (lahat ) . klinische erfahrungen mit der versehentlichen gabe von gnrh-antagonisten bei einer bereits bestehenden schwangerschaft sind unzureichend für eine risikobewertung, bisher liegen jedoch keine verdachtsmomente für eine schädigung des embryos vor. bei der üblichen anwendung in der reproduktionsmedizin scheinen gnrh-agonisten und -antagonisten hinsichtlich wirksamkeit und schwangerschaftsverlauf vergleichbar zu sein. somatostatin (somatostatin ® ) und octreotid (sandostatin ® ), ein synthetisches octapeptidderivat des somatostatins, hemmen sowohl die freisetzung des somatotropins (sth) wie auch des thyreoidea stimulierenden hormons (tsh). unter den hypothalamushormonen nimmt somatostatin daher eine sonderstellung ein. therapeutisch wird es als hämostyptikum, bei karzinoiden und zur senkung der wachstumshormonkonzentration bei akromegalie verwendet. in einigen fallberichten wird über die behandlung schwangerer mit octreotid berichtet, ohne dass nebenwirkungen beobachtet wurden (boulanger , blackhurst , takeuchi , colao . lanreotid (somatuline autogel ® ), ein analogon des somatostatins, wird seit zur therapie der akromegalie eingesetzt. pegvisomant (somavert ® ) ist ein somatotropin-rezeptorantagonist, der bei akromegalie eingesetzt wird und -verglichen mit den bisherigen therapieoptionen -den insulin-like-growth-factor- (igf- ) und damit die symptome der akromegalie am effektivsten senkt (stewart ) . erfahrungen zur anwendung in der schwangerschaft liegen noch nicht vor. empfehlung für die praxis: für den einsatz der hypothalamischen releasinghormone gibt es während der schwangerschaft kaum eine indikation. eine versehentliche applikation erfordert weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). g . . hypophysenvorderlappenhormone im hypophysenvorderlappen (hvl) werden hormone gebildet, die endokrine körperdrüsen stimulieren oder regulieren. die freisetzung der hvl-hormone wird durch hypothalamische releasinghormone gesteuert. hypophysäre hormone sind aufgrund ihrer hohen molekularmasse nicht plazentagängig, eine direkte beeinflussung des fetus ist daher nicht zu erwarten. die folgenden hormone gehören zu den hvl-hormonen. adrenocorticotropes hormon (acth), als medikament tetracosactid (synacthen ® ), stimuliert die synthese der gluco-und mineralocorticoide in der nebennierenrinde. thyreotropin oder thyreoidea stimulierendes hormon (tsh) stimuliert die synthese der schilddrüsenhormone. somatotropin (sth) oder wachstumshormon (gh) (z. b. genotropin ® , norditropin ® ), ein dem somatotropin strukturell und funktionell ähnliches hormon, wird von der plazenta mit fortschreiten der schwangerschaft in zunehmender menge gebildet. es wird auch als humanes plazentares laktogen (hpl) oder seltener als humanes choriales somatomammotropin (hcs) bezeichnet. funktionell hat es Ähnlichkeit mit prolaktin (siehe unten). zu den gonadotropinen zählen das follikelstimulierende hormon (fsh) (urofollitropin, follitropin alpha, follitropin beta; z. b. gonalf ® , puregon ® ) und das luteinisierungshormon (lh). während der schwangerschaft wird das dem lh analog wirkende hcg (humanes choriongonadotropin) in der plazenta synthetisiert. prolaktin fördert zusammen mit einigen anderen hormonen das wachstum der milchgänge und die synthese der milchproteine, außerdem beeinflusst es den flüssigkeitshaushalt der mutter. es hat keine therapeutische bedeutung. von den hypophysenvorderlappenhormonen werden fsh und gemische aus fsh und lh therapeutisch eingesetzt, dazu gehören plazentares hcg (humanes choriongonadotropin; z. b. choragon ® ) und hmg (humanes menopausengonadotropin). analoga sind menotropin (menogon ® ) bzw. urogonadotropin. indikationen für diese hormonbehandlung sind ovulationsinduktion und erhaltung des corpus luteum. eine stimulierung der ovulation mit gonadotropinen kann zu mehrlingsschwangerschaften führen, darunter in - % zu drillingen (scialli ). zwei publikationen beschreiben eine komplexe fehlbildung und vier neuroblastomfälle im ersten lebensjahr nach gonadotropinstimulation (mandel , litwin . diese befunde wurden durch andere untersuchungen ebenso wenig bestätigt wie andere risiken für den verlauf der schwangerschaft oder die spätere kindesentwicklung. es gibt auch keine nennenswerten hinweise auf eine schädigung, wenn hormone des hypophysenvorderlappens versehentlich während einer schwangerschaft appliziert wurden. melatonin steuert periphere, dem biorhythmus unterworfene vorgänge im organismus. es stimuliert die progesteronsekretion, hemmt die prostaglandinsynthese und hat (experimentell) einen tokolytischen effekt. es liegen keine ausreichenden erfahrungen zur therapeutischen anwendung von melatonin in der schwangerschaft vor, z. b. zur vermeidung des jetlags bei interkontinentalflügen. empfehlung für die praxis: für die gabe von hypophysenvorderlappenhormonen gibt es in der schwangerschaft keine indikation. eine versehentliche applikation rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). eine untersuchung von . schwangerschaften, in denen bromocriptin während der ersten wochen gegeben wurde, zeigte zwar einen geringen anstieg der frühabortrate, jedoch keine hinweise auf teratogene effekte (krupp ) . da die meisten frauen die therapie nach feststellung der schwangerschaft abgesetzt hatten, bestätigt das ergebnis der untersuchung gleichzeitig die unschädlichkeit der weiter bestehenden hyperprolaktinämie für den sich entwickelnden fetus. eine studie mit schwangerschaften bewies ebenfalls verträglichkeit und wirksamkeit einer therapie von mikro-und makroprolaktinomen mit bromocriptin oder lisurid, selbst wenn sie bis in die frühschwangerschaft hinein erfolgte. treten bei makroprolaktinomen im verlauf der schwangerschaft ophthalmologische probleme auf, wird die wiederaufnahme der therapie empfohlen (ventz ) . in einzelfällen empfiehlt sich eine dauertherapie während der gesamten schwangerschaft. cabergolin, das aufgrund seiner längeren wirkdauer nur ein-bis zweimal pro woche eingenommen werden muss, hat in über unter dieser therapie entstandenen schwangerschaften keinen anhalt für teratogene effekte gezeigt (ricci , robert , selbst wenn in einzelnen fällen durchgehend behandelt wurde (de turris , jones . bei schwangerschaften, in denen die frauen wegen einer bromocriptinresistenz mit quinagolid therapiert wurden, zeigten die neugeborenen keine auffälligkeiten. in fällen war eine therapie bis zur geburt erforderlich (morange ) . weitere vom hersteller gesammelte schwangerschaftsverläufe, bei denen durchschnittlich tage in die schwangerschaft hinein behandelt wurde, geben ebenfalls keine hinweise auf entwicklungstoxische effekte (zitiert in webster ) . metergolin ist wahrscheinlich ähnlich wie die übrigen dopaminagonisten zu bewerten. die wenigen erfahrungen reichen für eine differenzierte risikobewertung nicht aus. empfehlung für die praxis: bromocriptin und cabergolin sind bei hyperprolaktinämischer amenorrhö aufgrund der umfangreichen erprobung dopaminagonisten der wahl. nach der konzeption sollte das mittel in der regel abgesetzt werden. eine weiterbehandlung rechtfertigt jedoch weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). dies gilt auch für die anwendung von lisurid, metergolin und quinagolid. g . . hypophysenhinterlappenhormone pharmakologie und toxikologie. von der neurohypophyse, dem hypophysenhinterlappen (hhl), werden oxytocin und vasopressin (adiuretin) sezerniert. strukturell ähneln diese oktapeptidhormone den hypothalamischen hormonen. oxytocin (z. b. syntocinon ® ) ist das klassische wehenhormon. während der schwangerschaft wird es in zunehmender menge gebildet und gleichzeitig durch die ebenfalls gesteigerte synthese des enzyms schwangerschaftsoxytocinase inaktiviert. erst bei fetaler reife wird dieses gleichgewicht zugunsten des oxytocins verändert oder vorzeitig bei einer plazentafunktionsstörung, so dass kontraktionen des uterus über § -rezeptoren induziert werden. eine hypoxie des fetus kann als folge einer oxytocinüberdosierung und Überstimulation des uterus im rahmen der geburtseinleitung auftreten (siehe auch kapitel . ). vasopressin oder antidiuretisches hormon (adh) spielt therapeutisch auch in der schwangerschaft eine rolle bei der behandlung des diabetes insipidus. Über teratogene wirkungen wurde bisher nicht berichtet (ray ) . das enzym schwangerschaftsoxytocinase inaktiviert neben oxytocin auch vasopressin. von den synthetischen analoga argipressin, desmopressin (z. b. minirin ® ), lypressin, ornipressin und terlipressin (z. b. glycylpressin ® ) wurde zur behandlung des schwangerschaftsbedingten diabetes insipidus am häufigsten desmopressin verschrieben. die tierexperimentell induzierbaren, offenbar durch vasokonstriktion hervorgerufenen peripheren extremitätenanomalien wurden beim menschen bisher ebenso wenig beobachtet wie andere spezifische schwangerschaftsstörungen. allerdings ist die zahl dokumentierter verläufe mit rund für eine differenzierte risikobeurteilung zu klein (siristatidis , ray . in fällen mit thrombozytenfunktionsstörung, wie z. b. nach einer therapie mit acetylsalicylsäure (ass), vermag desmopressin die aktivität der thrombozyten zu stimulieren. der einsatz erfolgt meist kurzfristig peripartal. empfehlung für die praxis: oxytocin darf in der geburtshilfe zur einleitung und verstärkung von wehen eingesetzt werden. schwere fälle von adh-mangel (diabetes insipidus) rechtfertigen die gabe von vasopressin bzw. desmopressin in der schwangerschaft. dabei sind jedoch genaue kontrollen der kreislauf-und nierenfunktion unerlässlich. auch bei einer thrombozytenfunktionsstörung kann desmopressin gegeben werden. eine behandlung mit den anderen vasopressinanaloga rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . ). die hormonalen veränderungen und der geänderte stoffwechselbedarf während der schwangerschaft gehen bei jeder gesunden frau mit einer physiologischen anpassung der schilddrüsenfunktion einher. dies ist eine wichtige voraussetzung für die normale embryonale und fetale entwicklung sowie für eine ungestörte schwangerschaft. die fetale schilddrüse nimmt ihre aktivität am ende des dritten schwangerschaftsmonats auf (burrow ) , vorher ist der embryo ganz auf die thyroxinversorgung durch die mutter angewiesen. in der schwangerschaft steigt der mütterliche bedarf an iodid. sowohl die mütterliche als auch die fetale schilddrüsenfunktion sind von einer ausreichenden iodzufuhr abhängig. in iodmangelregionen muss daher möglichst schon vor der schwangerschaft eine ausreichende iodversorgung sichergestellt werden. eine substitution erst nach dem . trimenon kann bei gravierendem iodmangel-reifungsstö-rungen des zentralnervensystems nicht mehr verbessern (xue-yi , pharoah . der tägliche iodbedarf während der schwangerschaft beträgt ? g. auch in der bundesrepublik deutschland ist die iodaufnahme häufig unzureichend. da eine zufuhr durch iodsalz, iodierte nahrungsmittel und seefische unzuverlässig erscheint, sollten während der schwangerschaft täglich ? g mit tabletten substituiert werden. g . . hypothyreose, triiodthyronin (t ) und thyroxin (t ) hypothyreote schwangere haben ein höheres risiko für komplikationen (glinoer ) und außerdem kann eine hypothyreose die geistige entwicklung des kindes beeinträchtigen. dies ist seit langem insbesondere im zusammenhang mit iodmangel bekannt. eine neuere studie an über hypothyreoten frauen (nach schwangerschaftswochen diagnostiziert) ergab, dass ihre bis zum alter von jahren nachverfolgten kinder mental und motorisch schlechtere testergebnisse zeigten als kinder von euthyreoten oder leicht hyperthyreoten schwangeren (pop ) . auch haddow ( ) kam bei einer untersuchung an ca. sieben-bis neunjährigen kindern zu ähnlichen ergebnissen; die mütter litten in der schwangerschaft an einer diskreten hypothyreose. daher sollten unterfunktionen der mütterlichen schilddrüse gerade auch im interesse des werdenden kindes diagnostiziert und behandelt werden. zum risiko einer hypothyreose des neugeborenen nach thyreostatischer therapie der mutter siehe auch abschnitt . . . pharmakologie und toxikologie. die hormonal wirksamen schilddrüsenhormone sind die l-formen von triiodthyronin (t ) und thyroxin (t ), die nur in freier, nicht-proteingebundener form stoffwechselaktiv sind. t ist dabei das biologisch wirksame hormon, das relativ schnell anflutet und eine kürzere wirkdauer hat, während t als ein weniger wirksames prohormon oder hormondepot anzusehen ist, das bedarfsgesteuert zu t deiodiert wird. die plazenta benötigt für ihre entwicklung schilddrüsenhormone, sie deiodiert t zu rt (reverses t ) und t zu t . die plazenta lässt schilddrüsenhormone nur eingeschränkt passieren (burrow ) . jedoch kommt bei fetaler schilddrüsenagenesie ein quantitativer transfer aufgrund des dann bestehenden hohen konzentrationsgradienten zustande. an arzneimitteln stehen levothyroxin (z. b. eferox ® ) und liothyronin (z. b. thybon ® ) oder kombinationspräparate (z. b. novothyral ® ) zur verfügung. teratogene oder fetotoxische wirkungen sind bei den üblichen dosierungen, die physiologische verhältnisse herstellen, nicht zu erwarten. in der schwangerschaft steigt der bedarf an schilddrüsenhormon, so dass hypothyreote frauen ihre dosis entsprechend anpas-sen müssen. als kontrollparameter für die richtige therapeutische einstellung dient der tsh-wert (alexander ) . empfehlung für die praxis: im bedarfsfall sollten präparate mit levothyroxin verordnet werden, da der mütterliche organismus durch die konversion zu triiodthyronin die kontrolle über die tatsächliche hormonaktivität behält. falls erforderlich, ist auch iod zu substituieren. zu beginn einer schwangerschaft (ab schwangerschaftswochen) sollte die t -dosis um ca. % gesteigert werden. als faustregel gilt die empfehlung, dass schwangere nach feststellung der schwangerschaft die thyroxindosis um - ? g erhöhen sollen. im . trimenon ist eine weitere dosiserhöhung erforderlich und zwar auf - % über der ausgangsdosis vor der schwangerschaft. mit dem tsh-wert lässt sich die therapeutische einstellung kontrollieren. schilddrüsenhormone sollen nicht parallel zu einer thyreostatischen therapie gegeben werden, da dies den bedarf an plazentagängigen thyreostatika erhöht. g . . hyperthyreose und thyreostatika eine unbehandelte, manifeste hyperthyreose der mutter stellt ein risiko für die schwangerschaft und den fetus dar. beschrieben sind fetale wachstumsretardierung, präeklampsie, frühgeburt und intrauteriner fruchttod bzw. totgeburt (glinoer ) . bei der basedow-krankheit wie auch bei der hashimoto-thyreoiditis, die in der regel zur hypothyreose führt, sollten zu beginn der schwangerschaft und am anfang des . trimenons die schilddrüsen-autoantikörper bei der schwangeren bestimmt werden. hohe werte, besonders von tsh-r-immunglobulinen (= tsi) sind ein hinweis darauf, dass diese antikörper diaplazentar übergehen könnten. man schätzt, dass es auf diese weise bei - % der schwangeren mit basedow-krankheit zu einer vorübergehenden hyperthyreose beim fetus bzw. neugeborenen kommt (carrol ) . eine kürzlich veröffentlichte prospektive studie an frauen berichtet über eine wesentlich höhere rate von , %. vier neugeborene mit gesteigerter schilddrüsenfunktion hatten eine struma (rosenfeld . bei der schilddrüsenfunktion der neugeborenen wurden keine signifikanten unterschiede zwischen den verschiedenen thyreostatika gefunden. unter einer mütterlichen erhaltungsdosis von bis zu mg ptu oder bis zu mg methimazol pro tag zeigten bzw. % der kinder neonatal erhöhte tsh-werte (momotani ) . in der oben erwähnten studie von rosenfeld ( ) hatten , % der neugeborenen nach intrauteriner ptu-exposition eine hypothyreose und , % gleichzeitig eine struma. nicht bei allen dieser kinder war die schilddrüsenfunktion unmittelbar nach der geburt supprimiert, sondern zum teil erst bei der kontrolluntersuchung nach zwei wochen. fallbeschreibungen führten zu der hypothese, methimazol könne beim fetus hautdefekte (aplasia cutis), choanalatresie, Ösophagusatresie, tracheo-ösophageale fisteln, hypoplastische brustwarzen, faziale dysmorphien und eine mentale sowie motorische entwicklungsverzögerung verursachen (barbero , karg , ferraris , karlsson , clementi , wilson , hall , johnsson , vogt . foulds ( ) kommt bei der sichtung aller kasuistiken zu dem schluss, dass es inzwischen fallberichte von kindern oder feten gibt, die im . trimenon methimazol/carbimazol exponiert waren und die ein fehlbildungsmuster aufweisen, das als embryopathie zu werten ist. auf der anderen seite haben mehrere fallsammlungen weder nach behandlung mit ptu noch bei carbimazol/methimazol morphologische entwicklungsstörungen (wing ) oder auswirkungen auf größe und funktion der schilddrüse und auf die physische und intellektuelle entwicklung der kinder erkennen lassen (eisenstein , messer ). in einer multizentrischen prospektiven fall-kontroll-studie an methimazol exponierten schwangerschaften fand sich kein erhöhtes gesamtfehlbildungsrisiko. allerdings wies unter den kindern mit fehlbildungen eines eine choanalatresie und ein anderes eine Ösophagusatresie auf (di gianantonio ) . aufgrund der jetzigen datenlage kann man feststellen, dass thyreostatika nicht zu einer nennenswerten zunahme der gesamtfehlbildungsrate führen. allerdings kann methimazol mit einer häufigkeit von / . bis / . exponierte feten zu o.g. organentwicklungsstörungen führen . eine sorgfältig eingestellte thyreostatische therapie führt heute kaum noch zu einer schweren angeborenen struma. früher wurden strumabedingte atemwegsobstruktion und behinderung des geburtsvorgangs als folgen der therapie mit thyreostatika, zum teil in kombination mit hoch dosiertem iod oder mit schilddrüsenhormonen, beschrieben (Übersicht bei . insgesamt sollte sich die therapie mit thyreostatika eher an klinischen befunden, wie der herzfrequenz der mutter, als an laborwerten orientieren. natriumperchlorat (irenat ® ) ist nur selten bei übermäßiger iodaufnahme indiziert. in der schwangerschaft kann es den iodtransfer zum fetus beeinträchtigen. bei schwerer thyreotoxikose der mutter kann eine operative strumaresektion auch während der schwangerschaft indiziert sein. empfehlung für die praxis: eine manifeste hyperthyreose muss auch in der schwangerschaft behandelt werden. propylthiouracil ist, insbesondere im . trimenon, thyreostatikum der wahl, thiamazol (methimazol) und carbimazol sind als reservemittel zu betrachten. thyreostatika sind so niedrig wie möglich zu dosieren. die thyreostatische therapie soll nicht mit einer thyroxinsupplementierung kombiniert werden, da diese den thyreostatikabedarf der mutter erhöht. sowohl fetale hypothyreosen als auch eine hyperthyreose wurden gelegentlich nach mütterlicher thyreostatikatherapie infolge des plazentaren Übergangs mütterlicher autoantikörper beschrieben. daher sollte die schilddrüse des fetus sonographisch kontrolliert werden. unverzichtbar ist das screening der schilddrüsenlaborparameter beim neugeborenen, das in vielen ländern routinemäßig durchgeführt wird. zu erwägen ist eine zweite kontrolle nach tagen. leichte symptome einer hyperthyreose mit grenzwertigen laborparametern können in der schwangerschaft symptomatisch ohne thyreostatika behandelt werden, z. b. mit g -rezeptorenblockern wie propranolol oder metoprolol. nach therapie mit thiamazol (methimazol) und carbimazol im . trimenon sollte eine ultraschallfeindiagnostik zur bestätigung der normalen entwicklung des fetus angeboten werden. g . . glucocorticoide pharmakologie. die nebennierenrinde (nnr) bildet verschiedene hormongruppen, die gluco-und die mineralocorticoide, die u. a. den kohlenhydrat-und mineralstoffwechsel regeln. während der schwangerschaft treten veränderungen im hormonhaushalt der nnr auf. etwa vom . monat an erhöht sich die konzentration des cortisols im serum und die ausscheidung steigt zum ende der schwangerschaft an. therapeutisch sind vor allem glucocorticoide von bedeutung. man unterscheidet die nicht halogenierten von den halogenierten corticoiden. die ausschließlich lokal, dermal oder inhalativ verwendeten derivate werden an anderer stelle besprochen (siehe unter asthma, dermatika, augen-, nasen-und ohrentropfen). in der plazenta werden cortisol und prednisolon, nicht aber betamethason und dexamethason enzymatisch inaktiviert. perinatal finden sich im fetalen blut nur % der mütterlichen konzentration von prednison und prednisolon; bei betamethason sind es % und bei dexamethason nahezu %. hauptindikationen für glucocorticoide. glucocorticoide sind in der therapie allergischer, entzündlicher und proliferativer erkrankungen wirk-sam. dabei werden unphysiologisch hohe dosierungen eingesetzt. außerdem werden sie in der substitutionstherapie bei nebennierenrindenversagen verabreicht und zur induktion der lungenreife des fetus. in tabelle . sind für die verschiedenen glucocorticoide die dosierungen zusammengefasst, deren wirksamkeit mg prednisolon entsprechen. (pradat . eine meta-analyse aller bisher publizierten kohorten-und fall-kontroll-studien (carmichael , fraser ) ergibt ein signifikant erhöhtes risiko für spaltbildungen (odds ratio , ) bei nicht erhöhter gesamtfehlbildungsrate (park- wyllie ) . eine neue prospektive kontrollierte studie mit im . trimenon exponierten müttern fand weder ein erhöhtes gesamtfehlbildungsrisiko, noch einen einzigen fall von lippen-gaumen-spalten (gur ) . auch hardy ( ) fand keine assoziation zwischen oraler steroidmedikation und spaltbildungen. zusammenfassend ist ein geringes risiko für gaumenspalten mit oder ohne lippenbeteiligung nicht auszuschließen, wenn während der sensiblen phase zwischen woche bis mit glucocorticoiden behandelt wird. eine sichere dosis lässt sich zwar nicht angeben, aber bei bis mg prednisolon/tag ist das individuelle risiko extrem gering. in abhängigkeit von der therapiedauer, dosis und indikation kann es bei behandlung mit glucocorticoiden zur intrauterinen wachstumsretardierung (iugr), zur frühgeburt sowie zu vorübergehender hypoglykämie, hypotonie und elektrolytstörungen beim neugeborenen kommen. in einer neueren arbeit konnten weder beim geburtsgewicht noch bei der basiskonzentration von cortisol und bei cortisolwerten nach stressinduktion durch impfungen unterschiede zwischen kindern mit längerer prednisolon-exposition in der schwangerschaft und solchen gesunder mütter gefunden werden. die kinder wurden mindestens bis zum alter von monaten untersucht (miller ) . recht gut untersucht sind die effekte von pränatal verabreichten, plazentagängigen, halogenierten glucocorticoiden, um die lungenreifung zu fördern und ein respiratory-distress-syndrom (rds) beim neugeborenen zu verhindern. die Überlebensrate der frühgeborenen steigt durch diese therapie, und hirnblutungen treten seltener auf. eine betamethason-oder dexamethason-therapie zur fetalen lungenreifung zwischen schwangerschaftswoche und führte in zwei untersuchungen an über schwangeren in den tagen nach applikation bei den betamethason exponierten feten zu ausgeprägten, als stresssymptome interpretierten reaktionen, wie z. b. herabgesetzte atem-und körperbewegungen sowie eine eingeschränkte variabilität der herzfrequenz. das befinden der neugeborenen war letztlich unbeeinträchtigt (senat , mulder . in einer retrospektiven studie wurde ein vermehrtes auftreten von gastrointestinalem reflux bei neugeborenen gefunden, die vorgeburtlich mit steroiden behandelt wurden (chin ) . der wehenfördernde effekt sowie ein vorzeitiger verschluss des fetalen ductus arteriosus nach gabe von glucocorticoiden in der spätschwangerschaft scheinen klinisch nicht relevant zu sein. entgegen einzelner mitteilungen tritt eine neugeborenensepsis nach induktion der lungenreife mit glucocorticoiden nicht gehäuft auf. langzeitbeobachtungen bis zum alter von bis jahren zeigten überdies keine körperlichen, intellektuellen und psychosozialen auffälligkeiten nach glucocorticoidanwendung zur lungenreifung (dalziel , dessens , french , rotmensch . bei drohender frühgeburt nach schwangerschaftswoche wird heute eine einmalige applikation von glucocorticoiden für ausreichend gehalten. wenn schwangere dexamethason bzw. betamethason zur lungenreifung des fetus vor woche erhalten haben und später erneut eine frühgeburt droht, kann eine zweite gabe sinnvoll sein (rcog , nih . nach schwangerschaftswoche ist eine medikamentöse unterstützung der lungenreifung in der regel nicht notwendig. empfehlung für die praxis: eine substitution mit corticoiden ist auch in der schwangerschaft selbstverständlich weiterzuführen. die induktion der lungenreifung bei drohender frühgeburt wird einmalig zwischen schwangerschaftswoche und durchgeführt. eine systemische behandlung der mutter mit glucocorticoiden darf bei entsprechender indikationsstellung auch in der schwangerschaft durchgeführt werden. prednison und prednisolon sind hierfür mittel der wahl. die erhaltungsdosis sollte zwischen woche und möglichst mg/tag nicht überschreiten. notfallbehandlungen unterliegen selbstverständlich keinen dosisbeschränkungen. bei einer selten erforderlichen, höher dosierten behandlung über viele wochen sollte das fetale wachstum sonographisch beobachtet werden. dauert diese therapie bis zur geburt, muss eine nebenniereninsuffizienz des neugeborenen bedacht und ggf. behandelt werden. g . . diabetes mellitus und schwangerschaft diabetes mellitus ist der sammelbegriff für heterogene störungen des stoffwechsels, deren leitsymptom die chronische hyperglykämie ist. man unterscheidet im wesentlichen drei typen. während typ i auf einer gestörten insulinsekretion beruht, sind typ ii und der gestationsdiabetes (gdm) durch eine gestörte insulinwirkung gekennzeichnet. beide ursachen können auch gleichzeitig vorkommen. ein vor oder zu beginn einer schwangerschaft bestehender mütterlicher diabetes mit ungenügender blutzuckerkontrolle (hba c g , %) korreliert mit einer erhöhten rate an fehlbildungen. hba markiert als "blutzuckergedächtnis" die blutzuckerstoffwechsellage der patientin für die dauer der erythrozytenüberlebenszeit ( tage). je höher der hba c ist, desto höher ist das risiko. bei einem hba c von , % wird ein fehlbildungsrisiko von % angegeben; liegt das hba c bei , %, steigt es auf % an. zu den häufigsten fehlbildungen bei kindern diabetischer mütter zählen anomalien an wirbelsäule und extremitäten, am herz-kreislauf-system sowie neuralrohrdefekte, seltener sind urogenitale entwicklungsstörungen, gastrointestinale fisteln und atresien (Übersicht bei ). bei diabetischen schwangeren ist die abortrate erhöht, die perinatale mortalität liegt deutlich über dem durchschnitt und die frühgeburtenrate beträgt fast % (arbeitsgemeinschaft , gamson ) . die neonatale morbidität ist gekennzeichnet durch makrosome neugeborene mit ungenügender organreife, mangelentwicklung und postpartalen stoffwechselstörungen, insbesondere von hypoglykämien. bei allen neugeborenen diabetischer mütter muss eine hypoglykämie ausgeschlossen werden. ein manifester diabetes mellitus kann in der schwangerschaft zu uteroplazentaren versorgungsproblemen und daraus resultierenden erkrankungen der mutter führen, wie z. b. präeklampsie. die überwiegende mehrheit der diabeteserkrankungen des typs ii und auch von erstmals in der schwangerschaft auftretendem gestationsdiabetes (gdm) entwickelt sich auf dem boden eines metabolischen syndroms ("wohlstands"-adipositas mit hyperlipidämie, hypertonie und glucosetoleranzstörung). am anfang besteht eine insulinresistenz der insulinabhängigen gewebe, so dass erhöhte insulinspiegel zur verwertung von glucose in den geweben notwendig sind. durch die hyperinsulinämie wird das hungergefühl erhöht, das wiederum zur erhöhten nahrungsaufnahme, weiterer adipositas etc. führt -ein circulus vitiosus. abnehmen führt zu sinkenden insulinspiegeln und zu einer erhöhten sensibilität und dichte der insulinrezeptoren. eine gewichtsreduktion auf einen body-mass-index (bmi) möglichst von p kg/m sollte vor einer schwangerschaft erreicht werden! zum risiko von vorbestehender adipositas für die schwangerschaft siehe auch abschnitt . . . eine gute stoffwechseleinstellung mit normoglykämie ist das ziel jeder diabetestherapie in der schwangerschaft, denn die diabetische fetopathie geht auf hyperglykämien der mutter zurück, die auch beim fetus zur hyperglykämie führt. dieser reagiert mit einer gesteigerten insulinproduktion, die zu einer beta-zell-hypertrophie/-hyperplasie führt. eine fetale hyperinsulinämie begünstigt auch die entwicklung eines respiratory-distress-syndroms (rds) durch ausbildung hyaliner membranen und beeinträchtigung der surfactantbildung in den fetalen pneumozyten durch eingriff in enzymatische vorgänge. bei kindern von müttern mit unzureichender blutzuckereinstellung in der schwangerschaft (meist beim unerkannten oder unzureichend behandelten gdm) ist das risiko erhöht, bereits in der pubertät oder im jungen erwachsenenalter adipös zu werden oder einen diabetes mellitus bzw. eine glukosetoleranzstörung zu entwickeln. Übergewicht und gestationsdiabetes nehmen weltweit in den industrienationen zu, so dass inzwischen von einer häufigkeit des gdm von bis zu % ausgegangen wird. die awmf-leitlinien empfehlen, bei jeder schwangeren (mindestens) einen glucosetoleranztest durchzuführen (arbeitsgemeinschaft ). während der schwangerschaft ändert sich die insulinempfindlichkeit: in woche - besteht eine erhöhte insulinempfindlichkeit mit höherer hypoglykämiegefahr, während in der zweiten schwangerschaftshälfte die insulinempfindlichkeit abnimmt, so dass oft eine dosissteigerung notwendig wird. sofort nach der entbindung kehrt die ursprüngliche insulinempfindlicheit zurück. empfehlung für die praxis: bei diabetes mellitus ist die exakte einhaltung der normoglykämie die beste voraussetzung für eine ungestörte prä-und postnatale entwicklung des kindes und eine geringe mütterliche morbidität. dieses ziel sollte möglichst schon vor einer geplanten schwangerschaft erreicht werden. jede schwangere diabetikerin sollte unabhängig vom typ des diabetes fachgerecht interdisziplinär betreut werden und möglichst in einem perinatalzentrum entbinden. g . . insulin pharmakologie und toxikologie. das inselorgan, der endokrine anteil des pankreas, bildet und sezerniert insulin, glucagon und somatostatin. klinische bedeutung besitzt vor allem die störung der insulinproduktion, glucagon ist für die gegenregulation bei hypoglykämien wichtig. humaninsulin ist im gegensatz zu oralen antidiabetika nicht plazentagängig. eine bessere blutzuckerkontrolle und vorteile für das befinden des neugeborenen können erreicht werden, wenn in form einer intensivierten insulintherapie täglich mindestens dreimal präprandial insulin ein kurzwirksames insulin appliziert wird, eventuell ergänzt durch ein langzeitinsulin zur nacht, anstelle der zweimaligen applikation eines langzeitinsulins. die substitutionstherapie mit humanem insulin bei schwangeren diabetikerinnen hat nach den sehr umfangreichen erfahrungen keine embryotoxischen wirkungen. das ideale insulin für die behandlung schwangerer sollte ähnlich wie die natürliche insulinsekretion des pankreas zu einer guten glykämischen kontrolle bei der mutter führen und nicht die plazenta passieren. außerdem sollte es keine oder kaum antikörperbildung auslösen ("not immunogenic"), denn es gibt hinweise darauf, dass anti-insulin-antikörper im gegensatz zu insulin die plazenta passieren können. die mütterliche morbiditätsrate soll durch ein neues insulin nicht erhöht werden und eine bestehende diabetische retinopathie sollte sich nicht verschlimmern (minimale igf-i-aktivität). in aller regel erfolgt die einstellung schwangerer mit humanem normalinsulin und verzögerungsinsulin. seit einigen jahren gibt es insulinanaloga: kurzwirksames insulinlispro (humalog ® ), insulinaspart (novorapid ® , novomix ® ), insulinglusilin (z. b. apidra ® ) und die langwirksamen präparate insulinglargin (lantus ® ) und insulindetemir (levemir ® ). insulinlispro ist in vielen, meist kleineren retrospektiven und prospektiven studien an insgesamt mehr als schwangeren nach den oben genannten kriterien am besten untersucht (wyatt , cypryk , garg , masson , scherbaum , persson , bhattacharyya . bisher wurde unter insulinlispro keine erhöhte fehlbildungsrate beobachtet, das blutzuckertagesprofil gleicht dem von humaninsulin, der blutglucosewert stunde postprandial ist bei insulinlispro niedriger (mecacci ) . daraus ist jedoch nicht generell abzuleiten, dass die neugeborenenparameter besser ausfallen. in einer niederländischen studie an schwangeren mit typ-i-diabetes wurde festgestellt, dass im vergleich zu normalgewichtigen kindern insulinlispro in der gruppe makrosomer neugeborener mit % vs. % überrepräsentiert war (evers ) . ein fortschreiten der diabetischen retinopathie unter insulinlispro wurde bisher nicht beobachtet, ist aber auch noch nicht ausreichend untersucht (loukovaara , buchbinder . nach dem stand der heutigen erfahrungen ist die bildung von insulinantikörpern bei behandlung mit insulinlispro und humaninsulin ähnlich niedrig (gamson ) . insulinaspart ist bisher weniger untersucht. eine multinationale europäische studie vergleicht zz. die mütterlichen und fetalen komplikationen bei typ-i-diabetikerinnen bei einer therapie mit insulinaspart und mit humaninsulin (actrapid ® ). insulinglusilin und die langwirksamen insulinanaloga wie insulindetemir sind fast gar nicht untersucht. aufgrund der hinweise, dass sich eine retinopathie unter insulinglargin verschlechtern könnte, sollten die langwirksamen insu-linanaloga spätestens bei feststellung der schwangerschaft abgesetzt bzw. umgestellt werden (Übersicht gamson ) . einzelne fälle (devlin ) berichten über schwere nächtliche hypoglykämien unter normalinsulin, die nach umstellung auf insulinglargin nicht mehr auftraten. in der erprobung befindet sich das kurzwirksame inhalierbare insulin pramlintid. empfehlung für die praxis: ein typ-i-diabetes-mellitus muss schon vor einer schwangerschaft mit insulin gut eingestellt sein. humaninsuline sind mittel der ersten wahl. eine gut auf insulinlispro eingestellte frau muss in der gravidität nicht zwangsläufig umgestellt werden. langzeitanaloga sollten jedoch abgesetzt werden. schwangere mit einem typ-ii-diabetes oder einem gestationsdiabetes, der diätetisch allein nicht ausreichend therapiert ist, sollten humaninsulin erhalten. auch bei grenzwertig erhöhten blutglukosewerten und dem vorliegen einer fetalen makrosomie sollte mit einer insulintherapie begonnen werden. insulin tierischer herkunft sollte während der schwangerschaft wegen möglicher antikörperbildung nicht verwendet werden. bei schwangeren diabetikerinnen, die bereits insulinpflichtig waren, kann der insulinbedarf stark ansteigen. zur therapiekontrolle ist die ultraschallbiometrie des wachsenden fetus heranzuziehen. da glucocorticoide und tokolytika die kohlenhydrattoleranz der mutter verringern, sind bei gabe dieser medikamente besonders sorgfältige stoffwechselkontrollen anzuraten. g . . orale antidiabetika pharmakologie und toxikologie. orale antidiabetika sind keine hormone und wirken nicht substitutiv wie insulin. die überwiegend verwendeten sulfonylharnstoffderivate stimulieren die noch funktionsfähigen g -zellen des pankreas. zu ihnen gehören als mittel der zweiten generation glibenclamid (= glyburid; z. b. euglucon ® n), glibornurid (glutril ® ), gliclazid (diamicron uno ® ), glimepirid (z. b. amaryl ® ), glipizid und gliquidon (glurenorm ® ). zu den sulfonylharnstoffen der ersten generation zählen acetohexamid, chlorpropamid, tolazamid und tolbutamid (orabet ® ). die biguanidderivate metformin (z. b. glucophage ® ) und phenformin vermindern die glucosesynthese in der leber, führen zu einer verzögerten glucoseresorption aus dem darm und zur verstärkten glucoseaufnahme in die muskulatur. acarbose (glucobay ® ) und miglitol (diastabol ® ) verringern als § -glucosidase-hemmstoffe die kohlenhydratresorption im darm. dies ist ein umstrittener weg der diabetestherapie. die glinide nateglinid (starlix ® ) und repaglinid (novonorm ® ) sind postprandiale glucoseregulatoren, die zu einer kurzfristigen insulinsekretion aus den g -zellen führen. sowohl bei diesen medikamenten wie auch bei den glitazonen pioglitazon (actos ® ) und rosiglitazon (z. b. avandia ® ), die als so genannte "insulin-sensitizer" die empfindlichkeit der peripheren zellen für insulin verbessern, fehlen wirksamkeitsbelege bezüglich der diabetesspezifischen spätfolgen. evidenzbasierte, endpunktbezogene, positive ergebnisse liegen nur für insulin, metformin und für sulfonylharnstoffpräparate vor. muraglitazar (pargluva ® ) ist wie die anderen glitazone auch ein aktivator von peroxisomen-proliferator-aktivierten-rezeptoren (ppar). die studienergebnisse zeigten ein erhöhtes risiko kardiovaskulärer ereignisse und todesfälle, so dass es von der us-amerikanischen federal drug administration (fda) bislang nicht zugelassen wurde. sitagliptin ist ein inkretin-mimetikum: im darm vorkommende hormone (inkretine) steigern bei nahrungsaufnahme bedarfsgerecht die insulinsekretion. bei diabetes werden weniger inkretine als bei gesunden produziert. sitagliptin blockiert den normalerweise raschen enzymabbau der inkretine. es ist noch nicht zugelassen. da orale antidiabetika den blutzucker nicht so zuverlässig regulieren wie insulin, sind sie wenig geeignet für die behandlung des diabetes in der schwangerschaft. studien zur anwendung in der gravidität gibt es zu glibenclamid und zu metformin. beim neugeborenen begünstigt es hypoglykämien, wenn bis zum ende der schwangerschaft behandelt wird. einige ältere untersuchungen beobachteten erhöhte fehlbildungsraten (piacquadio ) , die zunächst als hinweise für ein teratogenes risiko der oralen antidiabetika interpretiert wurden (towner ) . heute wird vermutet, dass die unter oralen antidiabetika auftretenden hyperglykämien selbst ein teratogenes potenzial besitzen. insofern sind auch substanzspezifische unterschiede in der plazentagängigkeit von untergeordneter relevanz, z. b. ist tolbutamid besser plazentagängig als glipizid (elliott ) und glibenclamid geht nur minimal über (koren ) . neuere fallberichte beobachteten kein erhöhtes fehlbildungsrisiko, eine differenzierte risikobeurteilung des teratogenen potenzials erlauben sie jedoch nicht. randomisierte untersuchungen fanden keine unterschiede im schwangerschaftsverlauf und beim status der neugeborenen bei mehreren mit glibenclamid behandelten frauen mit gestationsdiabetes im vergleich zu insulin. die therapie wurde jeweils nach der embryogenese begonnen. im nabelschnurblut konnte glibenclamid nicht nachgewiesen werden, die insulinkonzentration war dort in beiden gruppen gleich. auch die anzahl hypoglykämischer kinder und das durchschnittliche geburtsgewicht unterschieden sich nicht signifi-kant (jacobson , langer , kremer ). jacobson ( ) beobachtete bei glibenclamid signifikant häufiger eine präeklampsie. ob diese befunde ausreichen, die bisherigen empfehlungen für die insulintherapie bei gestationsdiabetes infrage zu stellen, erscheint fraglich (greene ) . metformin stimuliert im gegensatz zu glibenclamid nicht die insulinsekretion und führt auch nicht zur hypoglykämie bei der schwangeren. bei übergewichtigen diabetikern ist die gabe eines wirkstoffs, der zu einer erhöhten insulinempfindlichkeit und zu einem verminderten insulinbedarf führt, sinnvoller als die gabe von glibenclamid. metformin wird nicht nur bei typ-ii-diabetikerinnen eingesetzt, sondern auch bei frauen mit einem pcos (polyzystisches ovar-syndrom) im rahmen der sterilitätsbehandlung, zur senkung der erhöhten abortrate und zur vermeidung bzw. therapie eines gestationsdiabetes. studien zur verträglichkeit im . trimenon sind rar. glueck ( ) fand bei schwangerschaften keine hinweise auf ein teratogenes risiko. in mehreren studien konnte eine senkung der abortrate bei frauen mit einem pcos nachgewiesen werden (palomba , jakubowicz ). kontrovers diskutiert wird, wie lange metformin zur "stabilisierung der schwangerschaft" bei pcos gegeben werden sollte. bisher gibt es keine eindeutigen belege dafür, dass eine gabe über schwangerschaftswoche bis hinaus zu besseren ergebnissen führt. die von glueck ( & postulierte präventive wirkung hinsichtlich gestationsdiabetes durch eine über das . trimenon hinaus fortgesetzte therapie konnte in einer randomisierten prospektiven untersuchung nicht bestätigt werden (vanky ) . hier sind weitere studien notwendig. zu rosiglitazon und acarbose gibt es nur wenige einzelfallbeschreibungen (kalyoncu ), zu den übrigen antidiabetika liegen keine erfahrungen in der schwangerschaft vor. empfehlung für die praxis: auch eine typ-ii-diabetikerin sollte schon bei planung einer schwangerschaft mit insulin eingestellt werden. eine dennoch weitergeführte therapie mit oralen antidiabetika rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). mit ultraschallfeindiagnostik sollte die morphologische entwicklung des fetus kontrolliert werden. es ist bisher nicht erwiesen, ob beim gestationsdiabetes nach dem . trimenon glibenclamid oder metformin eine alternative zum insulin darstellen. insulin ist nach wie vor die therapie der wahl. wurde metformin bei pcos zur unterstützung der schwangerschaft gegeben, sollte es um woche bis abgesetzt werden. g . . estrogene pharmakologie und toxikologie. nur während der schwangerschaft wird außer estron und estradiol auch estriol synthetisiert, das sonst nur als metabolit auftritt. physiologisch und auch pharmakologisch wirken estrogene stimulierend auf das wachstum von uterus, eileitern sowie besonders auf das wachstum des endometriums. weiterhin bewirken sie eine proliferation des vaginalepithels, eine zunahme der zervixsekretion und eine weitstellung des zervikalkanals. die früher manchmal übliche gabe zur verbesserung der wehenbereitschaft am termin wurde durch wirksamere pharmaka abgelöst. therapeutisch werden estrogene heute zur hormonellen kontrazeption, zur substitution im klimakterium und zur malignombehandlung verwendet. zu den verfügbaren substanzen gehören estradiol (z. b. estrifam ® ) und seine derivate ethinylestradiol (bestandteil der meisten estrogenhaltigen "pillen"), estriol (z. b. estriol jena-pharm ® ) und mestranol (in esticia ® ). polyestradiol sowie die estrogene fosfestrol, chlorotrianisen und epimestrol sind derzeit nicht zugelassen. die relativ niedrig dosierten zubereitungen zur hormonalen kontrazeption (kombinationspräparate aus estrogen und gestagen) einschließlich der notfallkontrazeption ("pille danach") und zubereitungen zur behandlung einer amenorrhö sind aufgrund ihrer häufigen (versehentlichen) anwendung in der frühschwangerschaft recht gut untersucht. sie bergen offenbar kein nennenswertes risiko (ahn , raman-wilms , källén ), auch nicht für geschlechtsdifferenzierungsstörungen, wenn während des sensiblen zeitraumes nach schwangerschaftswoche behandelt wurde. allerdings gibt es nach fallberichten aus den er jahren über herzfehlbildungen, vacterl-syndrom u. a. im zusammenhang mit der einnahme oraler kontrazeptiva in der schwangerschaft vereinzelt auch neuere publikationen, die eine erhöhte rate von (harnwegs-)anomalien diskutieren (li ) . auswirkungen einer intrauterinen exposition mit estrogenen auf die spätere fertilität konnten bisher nicht bestätigt werden. in einer Übersichtsarbeit wurden alle bisherigen studien zu störungen der männlichen reproduktion infolge einer intrauterinen estrogeneinwirkung analysiert. hier wurden sowohl medikamente der mutter, physiologisch erhöhter estrogenspiegel (z. b. bei zwillingsschwangerschaften), vegetarische (soja-)diät (soja enthält nichtsteroidale phytoestrogene; siehe auch west ) und umweltschadstoffe mit estrogenartiger wirkung (organochlorverbindungen wie pcb oder dioxine; siehe auch kapitel . ) berücksichtigt. allenfalls beim hodenkrebs, nicht jedoch bei hypospadien, hodenhochstand oder spermienzahlen ließ sich eine gewisse assoziation erkennen . eine ältere publikation berichtet von abweichender psychosexueller entwicklung pränatal exponierter jungen, deren diabetische mütter mit estradiol und progesteron behandelt worden waren (yalom seit etwa jahren werden progesteron (z. b. utrogest ® ) sowie halboder vollsynthetische derivate (z. b. -hydroxyprogesteron) zur behandlung des drohenden abortes eingesetzt. bis heute gibt es jedoch keinen wirkungsnachweis, deshalb ist das behandlungskonzept überholt (acog ) . bessere erfolgsquoten nach progesteronbehandlung können vorgetäuscht sein, da die patientinnen oft auch intensivärztlich und pflegerisch betreut werden. ein symposium der who über arzneimittelbehandlung während der schwangerschaft hat die nutzlosigkeit dieser therapie festgestellt, die in der bundesrepublik deutschland, in frankreich und italien verbreitet war, in skandinavien hingegen nicht praktiziert wurde (who-report ) . dennoch wird immer wieder beim drohenden abort die therapie mit gestagenen vorgeschlagen, heute meistens mit dem natürlichen progesteron. eine aktuell noch diskutierte indikation zur hormonellen verhinderung eines aborts ist die hcg-therapie bei der seltenen corpus-luteum-insuffizienz. der zusammenhang zwischen hormontherapie und vermehrtem auftreten von hypospadien wird kontrovers diskutiert (carmichael , källén . wenn überhaupt führen gestagene nur äußerst selten zu dieser häufig auch spontan vorkommenden und meist geringfügigen anomalie. die relativ niedrig dosierten zubereitungen zur hormonalen kontrazeption einschließlich der notfallkontrazeption ("pille danach") und produkte zur behandlung einer amenorrhö sind aufgrund ihrer häufigen (versehentlichen) anwendung in der schwangerschaft recht gut untersucht. sie bergen insbesondere hinsichtlich extragenitaler fehlbildungen nach heutigem wissen kein erkennbares risiko (ahn , brent , raman-wilms , källén . allerdings gibt es nach fallberichten aus den er jahren über herzfehlbildungen, vacterl-syndrom u. a. im zusammenhang mit der einnahme von oralen kontrazeptiva in der schwangerschaft vereinzelt auch neuere publikationen, die eine erhöhte rate von (harnwegs-)-anomalien diskutieren (li ) . die notfallkontrazeption wird heute als reine gestagentherapie mit × , mg bzw. × , mg levonorgestrel (duofem ® , levogynon ® ) durchgeführt. sicherheit, verträglichkeit und nebenwirkungen haben ein so günstiges profil, dass in einigen ländern, wie z. b. der schweiz, eine verschreibungsfreie abgabe erfolgt. bei dieser therapie wird der eisprung verhindert und kein abort induziert. embryotoxische wirkungen wurden, falls die schwangerschaft doch weiter besteht, bisher nicht beschrieben (american academy of pediatrics , food and drug administration ). geschlechtsdifferenzierungsstörungen durch gestagene in kontrazeptiver dosis während des sensiblen zeitraumes ab schwangerschaftswoche wurden nicht beobachtet. wenn jedoch wiederholt deutlich höhere dosen der -nor-gestagene mit ihrem androgenisierenden potenzial eingenommen wurden, kann eine vorübergehende klitorisvergrößerung auftreten (Übersicht bei . negative auswirkungen dieser intrauterinen exposition auf die fertilität im erwachsenenalter wurden bisher nicht vermehrt beobachtet. die entwicklung bis ins jugendalter scheint nach großen langzeituntersuchungen, z. b. zu depotpräparaten mit medroxyprogesteron ("dreimonatsspritze"), altersgemäß zu verlaufen (pardthaisong ) . früher wurde nach gabe androgener gestagene (norethisteronabkömmlinge), in höherer dosis als zur kontrazeption heute üblich, eine auswirkung auf das spätere geschlechtsspezifische verhalten der kinder angenommen. zur hoch dosierten anwendung von gestagenen, wie z. b. in der malignomtherapie, liegen keine ausreichenden erfahrungen vor. empfehlung für die praxis: während einer schwangerschaft gibt es keine stichhaltige indikation für die therapie mit gestagenen. dies gilt auch für das überholte behandlungskonzept mit progesteron bei drohendem abort. doch weder eine solche therapie noch in der frühschwangerschaft versehentlich eingenommene kontrazeptiva erfordern einen risikobegründeten schwangerschaftsabbruch oder zusätzliche diagnostik (siehe kapitel . ). das gilt für die heute üblichen niedrig dosierten ein-oder mehrphasenpräparate, die notfallkontrazeption mit levonorgestrel und die behandlung einer amenorrhö mit norethisteronacetat und ethinylestradiol. die (versehentliche) applikation hoch dosierter präparate für andere indikationen rechtfertigt ebenfalls keinen risikobegründeten abbruch der schwangerschaft. in einem solchen fall kann mit ultraschallfeindiagnostik die normale organentwicklung dokumentiert werden. g . . diethylstilbestrol pharmakologie und toxikologie. diethylstilbestrol (des) ist ein synthetisches nichtsteroidales estrogenaktives arzneimittel, das in den er jahren in den usa zur therapie des drohenden abortes und außerdem zur begrenzung des längenwachstums bei heranwachsenden mädchen (venn ) verordnet wurde. großes internationales aufsehen erregte die entdeckung, dass bei töchtern, deren mütter während der schwangerschaft des erhalten hatten, im adoleszentenalter vermehrt adenokarzinome der vagina auftraten (herbst ) . dies ist der einzige beim menschen nachgewiesene fall für vorgeburtlich ausgelöste karzinome ("transplazentare karzinogenese"). das risiko für diese bei jungen frauen sonst seltene erkrankung wird mit bis zu , % angegeben. andere krebsrisiken, wie z. b. für brustkrebs, ließen sich nicht eindeutig nachweisen (hatch ) . mindestens % der im . trimenon pränatal exponierten jungen frauen wiesen außerdem anomalien an scheide, uterus oder eileitern auf (mittendorf ) . andere untersucher konnten kein erhöhtes risiko für leiomyome und ovarialzysten erkennen, sahen aber häufiger parovarialzysten . bei männlichen nachkommen besteht offenbar ein erhöhtes risiko für kryptorchismus, testikuläre hypoplasie und abnorme samenzellmorphologie (mittendorf ) . ein erhöhtes hypospadierisiko bei söhnen, deren mütter als embryo selbst pränatal exponiert waren, wird kontrovers diskutiert , klip ). des weiteren gibt es hinweise darauf, dass solche frauen ein erhöhtes risiko für frühgeburten und andere schwangerschaftskomplikationen haben (papiernik zu den anabolika gehören z. b. clostebol, metenolon (z. b. in anti-focal ® ), nandrolon (z. b. deca-durabolin ® ) und tibolon (liviella ® ). für diese medikamente gibt es in der schwangerschaft ebenfalls keine behandlungsindikation. im zusammenhang mit kraftsport und bodybuilding werden jedoch "schwarz" importierte präparate verwendet, die auch ohne entsprechende deklaration androgene bzw. anabolika enthalten können. sie wurden schon "versehentlich" während einer schwangerschaft weiter genommen. die praktischen erfahrungen zur pränatalen verträglichkeit von androgen-und anabolika-präparaten reichen für eine differenzierte risikobewertung, auch bezüglich einer androgenisierenden wirkung, nicht aus. empfehlung für die praxis: androgene und anabolika sind während der schwangerschaft absolut kontraindiziert. eine versehentliche anwendung erzwingt jedoch keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). insbesondere bei wiederholter anwendung sollte mit ultraschallfeindiagnostik die organentwicklung kontrolliert werden. cyproteronacetat ist das im reproduktionsfähigen alter am häufigsten verschriebene antiandrogen. in kombination mit ethinylestradiol wird es als kontrazeptive pille (diane ® ) angeboten. dieses präparat wird besonders bei gleichzeitig bestehender akne verschrieben. das bundesinstitut für arzneimittel und medizinprodukte (bfarm) hatte wegen des verdachts auf lebertumoren die anwendung von diane ® drastisch eingeschränkt. das präparat sollte nur noch bei androgenisierungserscheinungen und akne mit narbenbildung verschrieben werden. die antiandrogene wirkung von cyproteronacetat kann theoretisch zur feminisierung männlicher feten führen. doch selbst bei versehentlicher fortführung der behandlung mit täglich mg (in diane ® ) bis in die sensible phase über schwangerschaftswoche hinaus, ist eine feminisierung nicht beobachtet worden. vom hersteller wurden schwangere mit männlichen feten registriert, die während der (nahezu) gesamten genitalentwicklungsphase mg täglich eingenommen hatten und weitere schwangere, die sogar - mg cyproteron täglich eingenommen hatten. die lebend geborenen knaben waren normal entwickelt. bei einem spätabort wurden ebenfalls keine entwicklungsstörungen festgestellt. weitere fallserien deuten ebenfalls nicht auf teratogene effekte beim menschen hin (eigene daten und die des european network of teratology information services, entis). allerdings reicht der umfang an erfahrungen für eine differenzierte risikobewertung nicht aus. danazol, ein synthetisches modifiziertes androgen, leitet sich von ethisteron ab und ist ein antiestrogener hemmstoff. danazol wurde zur behandlung der endometriose, bei benigner knotenbildung in der brust, hereditärem angioneurotischem Ödem und auch als kontrazeptivum eingesetzt. zahlreiche publikationen mit über exponierten schwangeren offenbaren ein erhebliches virilisierendes risiko für weibliche feten, wenn täglich mit mg noch nach schwangerschaftswoche (funktionsaufnahme der androgenrezeptoren) behandelt wurde. bei normalem innerem genitale zeigten bis über % der pränatal exponierten mädchen eine klitorisvergrößerung oder das vollbild eines weiblichen pseudohermaphroditismus. bei der späteren entwicklung fanden sich keine weiteren auffälligkeiten, wie z. b. virilisierung oder störungen des sexualverhaltens (Übersicht bei . eine erhöhte abortneigung nach gabe von danazol könnte auch durch die endometriose als grunderkrankung verursacht sein. die wirkung der übrigen in diesem abschnitt angesprochenen antiestrogene und antiandrogene ist in der schwangerschaft nicht untersucht, so dass eine differenzierte risikobewertung nicht möglich ist. empfehlung für die praxis: antiestrogene, antiandrogene und danazol sind in der schwangerschaft absolut kontraindiziert. eine versehentliche applikation rechtfertigt jedoch keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). durch ultraschallfeindiagnostik sollte die ungestörte organentwicklung zumindest bei höher dosierten produkten bestätigt werden. g . . mifepriston (ru ) pharmakologie und toxikologie. mifepriston ist ein progesteron-und glucocorticoid-antagonist. es wurde als abortivum in deutschland zugelassen. eine dosis von mg ist für den abbruch einer frühschwangerschaft erforderlich, in kombination mit einem prostaglandinpräparat sind mg jedoch ebenso effektiv (peyron ) . zu den pharmakologischen effekten des mifepristons zählen unter anderem die senkung der lh-(luteinisierungshormon-)sekretion, eine beschleunigte gelbkörperregression und die zunahme der kontraktilität der uterusmuskulatur. auswirkungen auf die plazentare produktion von progesteron, choriongonadotropin und humanem plazentaren laktogen wurden ebenfalls beobachtet. mifepriston wurde wegen seines progesteronantagonismus auch als monatlich einzunehmendes "interzeptivum" (wirksamkeit im gegensatz zum kontrazeptivum erst nach einer konzeption) versucht. es hat sich dabei allerdings als ebenso wenig zuverlässig erwiesen wie zur medikamentösen beendigung extrauteriner schwangerschaften. außerdem wird der einsatz zur zervixreifung, geburtseinleitung sowie bei endometriose und uterus myomatosus diskutiert. mifepriston überschreitet die plazenta und beeinflusst tierexperimentell nicht die konzentration an fetalem progesteron, estradiol oder cortisol. nur die aldosteronkonzentration scheint anzusteigen. bezüglich teratogenese sind die tierexperimentellen ergebnisse widersprüchlich. in einer fallserie mit etwa schwangerschaften, die nach abbruchversuchen mit mifepriston ausgetragen wurden, beobachtete man verschiedene fehlbildungen, darunter kinder mit klumpfuß (sitruk-ware ). eine spezifische teratogene wirkung lässt sich aus dieser publikation und aus anderen fallbeschreibungen, die mehrheitlich gesunde neugeborene umfassen (pons , lim ), nicht eindeutig ableiten. generell kann jedoch der missglückte versuch eines schwangerschaftsabbruchs die fetale entwicklung gefährden. empfehlung für die praxis: falls eine schwangerschaft nach vergeblicher anwendung von mifepriston ausgetragen wird, sollte eine ultraschallfeindiagnostik die normale organentwicklung bestätigen. ein risikobegründeter schwangerschaftsabbruch ist durch einen missglückten abortversuch nicht zwingend indiziert (siehe kapitel . ). g . . clomifen pharmakologie und toxikologie. bei fehlender ovulation ohne hyperprolaktinämie wird seit über zwei jahrzehnten der estrogenantagonist clomifen (z. b. clomhexal ® , dyneric ® ) zur ovulationsauslösung eingesetzt. eine Überdosierung, insbesondere in kombination mit hcg (humanes choriongonadotropin), kann zur Überstimulierung der ovarien führen. zu den unerwünschten wirkungen zählen eine erhöhte rate an mehrlingsschwangerschaften und die vergrößerung der ovarien. die wirkung beruht offenbar auf einer kompetitiven besetzung der estrogenrezeptoren im hypothalamusbereich, die zu vermehrter lh-(luteinisierungshormon-)freisetzung führt. es gibt eine anhaltende diskussion darüber, ob clomifen fehlbildungen, wie z. b. neuralrohrdefekte, verursacht (van loon ). ein fallbericht beschreibt eine glaskörperanomalie bei einem kind, dessen mutter mg clomifen bis woche eingenommen hatte (bishai ) . in japan wurden . durch clomifen induzierte schwangerschaften über einen zeitraums von jahren beobachtet. von den lebend geborenen kindern wiesen , % fehlbildungen auf, eine gegenüber der kontrollgruppe nicht erhöhte rate (kurachi ) . allerdings wurde nicht differenziert, ob nur vor oder auch nach eintritt der schwangerschaft mit clomifen behandelt wurde. die fallsammlung eines herstellers ergab bei . clomifen-patientinnen fehlbildungen ( , %). bei frauen fand die clomifeneinnahme (auch) nach der konzeption statt, in dieser gruppe hatten kinder ( , %) fehlbildungen. eine studie mit daten eines fehlbildungsregisters fand eine erhöhte inzidenz für kraniosynostosen bei schwangeren, die clomifen vor oder während der schwangerschaft eingenommen hatten (reefhuis ) . eine weitere ähnliche konzipierte arbeit mit unbekannter fallzahl stellte signifikant häufiger penoskrotale hypospadien nach clomifen fest (meijer ) . ein erhöhtes individuelles risiko lässt sich jedoch mit den derzeit vorliegenden studienergebnissen nicht belegen. eythropoietine werden bei schwerer anämie, z. b. bei chronischen nierenerkrankungen und nach nierentransplantation, eingesetzt, aber auch bei krebs-und hiv-therapie, sowie bei thalassämie und bei therapieresistenter anämie in der schwangerschaft. rekombinantes humanes erythropoietin ist nicht plazentagängig und hat sich in einer reihe von berichten und fallserien als gut verträglich in der schwangerschaft erwiesen. ein nennenswertes risiko für den embryo/fetus besteht nicht. ob die in schwangerschaften beschriebene schwere mütterliche hypertonie und verschlechterung der nierenfunktion der schwangeren auf die gabe von erythropoietin zurückzuführen ist, konnte nicht abschließend geklärt werden . zu darbepoetin alfa liegen weniger erfahrungen in der schwangerschaft vor als zu epoetin, jedoch wurden negative effekte bisher nicht beschrieben (goshorn (ebi , duncan , brodsky . einige untersucher beobachteten eine erhöhte abortrate nach narkosen, es ist aber schwierig, dies den narkosemitteln zuzuordnen. tierexperimentelle ergebnisse zu einzelnen substanzen sind aufgrund der hohen dosen und wiederholten anwendungen nicht auf klinische situationen einer anästhesie übertragbar. lokalanästhetika, die entweder gespritzt oder aufgesprüht werden, galten lange als mittel der wahl in der schwangerschaft, weil man annahm, dass sie am ort der applikation verbleiben und nicht zum fetus übergehen. doch auch diese form der anästhesie schließt komplikationen nicht aus, da auch lokalanästhetika je nach ort und durchblutung der injektionsstelle den fetus über den mütterlichen kreislauf erreichen können. im zusammenhang mit operativen eingriffen verwendete muskelrelaxanzien sind quartäre ammoniumpräparate, die unter physiologischen bedingungen stark ionisiert vorliegen und daher nur langsam die plazenta überschreiten. trotzdem kommt es zu einem nachweisbaren Übergang auf den fetus. g . . halogenierte inhalationsnarkotika pharmakologie und toxikologie. desfluran (suprane ® ), enfluran, halothan, isofluran (z. b. forene ® ) und sevofluran (sevorane ® ) gehören zu den halogenierten inhalationsnarkotika. in der perinatalphase ist zum einen ihre relaxierende wirkung am uterus zu beachten, die zur minderung der wehentätigkeit führen kann, und zum anderen ihr atemdepressiver effekt, vor allem bei risikogeburten. halothan ist eines der ältesten und am weitesten verbreiteten halogenierten inhalationsnarkotika. teratogene wirkungen beim menschen sind nicht bekannt. im tierversuch haben sich dagegen skelett-und andere anomalien, minderwuchs, verhaltensabweichungen und absterben der frucht gezeigt. diese auffälligkeiten wurden bei der üblichen anwendung beim menschen nicht beobachtet. bei gabe von halothan um den geburtstermin (z. b. sectionarkosen) muss mit einer verstärkten uterusrelaxation, einer erhöhten blutungsgefahr sowie mit einer atemdepression des neugeborenen gerechnet werden. unter den volatilen anästhetika hat halothan die stärkste kreislaufdepressorische wirkung. hohe dosen können herzrhythmusstörungen und herzstillstand verursachen, besonders wenn zusätzlich § -sympathomimetische tokolytika oder katecholamine eingesetzt werden. Über lebertoxizität wurde bei wiederholungsnarkosen berichtet. enfluran ist ein fluorierter ether, der mit einer metabolisierungsrate von - % nur zu geringen teilen verstoffwechselt wird. die anwendung bei sectionarkosen wird vom neugeborenen gut vertragen (tunstall , abboud . tierexperimentell gibt es unter versuchsbedingungen, die sich grundlegend von der klinischen situation unterscheiden, teilweise embryotoxische effekte. teratogene wirkungen beim menschen sind nicht bekannt. enfluran wird wegen der gegenüber isofluran in fast allen bereichen ungünstigeren eigenschaften (anflutung, abflutung, kreislaufdepression, metabolisierungsrate) nur noch in wenigen zentren für narkosen benutzt. isofluran ist das strukturisomer von enfluran. es gehört mit einer metabolisierungsrate von nur , %, genau wie desfluran, zu den am wenigsten verstoffwechselten halogenierten inhalationsnarkotika. beilin und mitarbeiter ( ) desfluran weist von allen anästhesiemitteln den niedrigsten blut/gasund gewebe/blut-verteilungskoeffizienten auf sowie die geringste löslichkeit. es ist das am schwächsten wirksame narkosegas. desfluran wird von allen halogenierten inhalationsnarkotika am wenigsten verstoffwechselt, daher ist das toxische potenzial gering. wegen des schnellen einschlafens und angenehmen aufwachens der patienten wird desfluran häufig für sectionarkosen benutzt, ohne dass nachteile für die neugeborenen oder die mutter bekannt wurden. es gibt zwei fallberichte über maligne hyperthermie. teratogene wirkungen beim menschen sind nicht bekannt. die uterusrelaxierende wirkung ist abhängig von der anästhesietiefe, ähnlich wie bei den übrigen halogenierten inhalationsnarkotika. trotz des schnellen anflutens kann desfluran aufgrund seiner schwachen wirksamkeit nicht als einleitungsnarkotikum gewählt werden. die für eine narkose erforderlichen konzentrationen von mehr als % würden die atemwege reizen und könnten dadurch zu atemanhalten führen. sevofluran ist ein weiteres halogeniertes anästhetikum, das als halogen nur fluorid enthält. aufgrund der physikalischen eigenschaften ist der anstieg im blut etwas langsamer als bei desfluran, aber schneller als bei allen anderen halogenierten inhalationsnarkotika. im gegensatz zu desfluran ist sevofluran als einleitungsnarkotikum gut geeignet. die metabolisierungsrate liegt bei - %. dadurch kommt es im blut zum anstieg von anorganischem fluorid und im atemkalk zur bildung von so genanntem compound a. beide substanzen haben zwar ein nephrotoxisches potenzial, erreichen aber keine schädigenden konzentrationen. sevofluran wird heute in vielen geburtshilflichen zentren als standardnarkotikum bei der sectio angewandt, ohne dass negative einflüsse auf das neugeborene beschrieben werden und auch teratogene wirkungen sind beim menschen nicht bekannt. empfehlung für die praxis: die halogenierten inhalationsnarkotika gehören in der geburtshilfe zu den standardnarkotika. sie können bei beachtung der möglichen charakteristischen nebenwirkungen während der gesamten schwangerschaft eingesetzt werden. bei anwendung unter der geburt sind uterusrelaxation und depressorische auswirkungen auf das neugeborene zu beachten. lachgas ist gegenüber den halogenierten inhalationsnarkotika ein gut verträgliches narkotikum, das weder deren negative wirkung auf das herz-kreislauf-system noch auf den uterus besitzt. unter der geburt kann durch die inhalation eines lachgas-sauerstoff-gemisches eine schnelle und einfache analgetische wirkung erzielt werden, die sich außerdem sehr gut steuern lässt. in seltenen fällen kann auch lachgas beim neugeborenen eine atemdepression hervorrufen, die eine beatmung erforderlich macht (langanke ) . umfangreiche studien über die anwendung von lachgas lassen bei über . schwangeren keine teratogenen effekte erkennen (crawford . andere studien thematisieren risiken bei der anwendung während der geburt. taylor und mitarbeiter ( ) stellten fest, dass höhere dosen von lachgas zu einem verzögerten geburtsverlauf führen. polvi und mitarbeiter ( ) fanden veränderungen im zerebralen gefäßwiderstand des kindes. diese beiden studien wurden jedoch kritisiert, weil sie nicht auf mögliche andere auslösende umstände eingehen. (herman , cordier ). aus diesem grunde gibt es obergrenzen für die belastung am arbeitsplatz. in den usa ist für lachgas eine maximal zulässige raumluftkonzentation von ppm festgesetzt, in deutschland gelten noch ppm als unbedenklich. eine korrekte einhaltung der maximal zulässigen belastung durch narkosegase ist in der praxis nur schwer möglich. kontrollmessungen müssen deshalb wiederholt durchgeführt werden. bei beruflich exponiertem anästhesie-und op-personal wurde über erhöhte abortraten berichtet, die auf die chronische exposition mit inhalationsnarkotika zurückgeführt wurden (hemminki , vessay . später ließ sich der verdacht in ausführlichen epidemiologischen studien nicht eindeutig bestätigen und begleitende faktoren wie stress, kaffeekonsum, rauchen und angespannte körperhaltung sowie vorbestehende abortneigung wurden als auslösende ursachen angenommen (rowland (rowland & . in verschiedenen studien wurden ein geringeres geburtsgewicht und ein kürzeres gestationsalter festgestellt (ericson , rosenberg , pharoah , cohen . die langzeitentwicklung im alter von bis jahren wurde von ratzon ( ) (z. b. trapanal ® ) . nach intravenöser injektion erreicht die konzentration eines injektionsnarkotikums sofort ihren maximalspiegel im blut, der dann wegen der rasch einsetzenden umverteilung und ausscheidung schnell wieder abfällt. die wirkung im gehirn setzt wegen des starken blutflusses kurzfristig ein und klingt mit der umverteilung schnell wieder ab. alle injektionsnarkotika passieren wegen ihrer hohen lipidlöslichkeit rasch die plazenta, werden aber vor erreichen des fetalen gehirns zunehmend im fetalen blut verdünnt und zum teil auch in der fetalen leber aufgenommen. daher bewirkt eine einmalige bolusinjektion eines nicht zu hoch dosierten injektionsnarkotikums keine anästhesie des fetus bzw. des neugeborenen, während nach wiederholten dosen mit einer depressorischen wirkung beim fetus gerechnet werden muss. die konzentration im neugeborenen ist bei anwendung unter der geburt umso geringer, je mehr zeit zwischen injektion des narkotikums und der geburt des kindes verstreicht. alle genannten injektionsnarkotika können während der schwangerschaft benutzt werden, sie werden nachfolgend einzeln vorgestellt. etomidat ist ein imidazolderivat, das durch unspezifische esterasen abgebaut wird. es führt zu einem extrem schnellen wirkungseintritt mit sehr raschem abklingen (halbwertszeit im serum minuten). die kurze wirkdauer beruht ähnlich wie bei den barbituraten auf umverteilung vom gut durchbluteten gehirn in schlechter durchblutete große gefäßgebiete, nämlich muskel-und fettgewebe (lipophilie). etomidat besitzt keine kardiodepressorischen eigenschaften, kann aber zu myo-kloni und dyskinesien führen und wirkt hemmend auf die nebennierenrindenfunktion. ketamin ist ein rasch wirkendes injektionsnarkotikum, das eine gute analgetische wirkung besitzt und die atmung kaum beeinflusst. es gibt keine studien zur beeinflussung der fetalen entwicklung beim menschen. aufgrund einer verstärkung der empfindlichkeit gegenüber sympathikomimetika führt es zu deutlichen kardiovaskulären effekten, wie z. b. anstieg von herzfrequenz und blutdruck. ketamin stimuliert dosisabhängig den tonus und die wehenfrequenz des uterus und darf bei uteriner hyperaktivität und drohender fetaler hypoxie nicht eingesetzt werden. hohe dosen können fetale funktionen beeinträchtigen und ein erweitertes monitoring unter der geburt erforderlich machen (barak , reich . ketamin führt bei kaiserschnittentbindungen zu teilweise therapiebedürftigen angstzuständen in folge von horrorvisionen, was den einsatz trotz seiner guten analgetischen eigenschaften stark limitiert. ketamin s wird eine in dieser hinsicht bessere verträglichkeit zugeschrieben. propofol ist in nordamerika heute das neben thiopental am meisten angewandte injektionsnarkotikum in der schwangerschaft. als einleitungssubstanz stellt es eine geeignete alternative zu thiopental dar (richardson . für die intubation zeichnet sich propofol durch einen raschen bewusstseinsverlust aus. die kurze aufwachzeit und die geringen nebenwirkungen sind von großem vorteil für die schwangere patientin. nach injektion überwindet propofol die plazenta rasch, die fetalen blutkonzentrationen entsprechen etwa % der mütterlichen werte (jauniaux , dailland . propofol wird rasch aus dem kreislauf des neugeborenen entfernt (dailland , moore , valtonen . in klinischen untersuchungen fand sich bei einleitung zur sectio mit propofol mit - , mg/kg kg im vergleich mit - mg/kg kg thiopental kein unterschied in den apgar-werten, den säure-basen-parametern und dem neurologischen zustand des neugeborenen (d' alessio ). in einer studie, die den enns (early neonatal neurobehavioral scale) benutzte (d'alessio , celleno , wurde bei neugeborenen nach sectionarkose mit propofol im vergleich zu thiopental ein ungünstigeres ergebnis bei einigen neurologischen funktionen nachgewiesen. diese effekte zeigen sich aber nur zeitlich begrenzt. gin und mitarbeiter ( b) fanden dagegen, dass propofol dem thiopental als einleitungsnarkotikum zur sectio überlegen war. blutdruckabfälle waren nicht häufiger zu beobachten als nach thiopental. wird die gewinnung von eizellen bei der in-vitro-fertilisation (ivf) in propofol-narkose vorgenommen, so hat dies keinen negativen effekt auf den schwangerschaftserfolg (beilin , christiaens ). thiopental-natrium ist ein thiobarbiturat, das sich durch schnellen wirkungseintritt auszeichnet. die kurze wirkdauer ist durch umverteilung bedingt. anfangs reichert sich das medikament wegen der guten durchblutung im gehirn an. die anschließende umverteilung in das muskel-und fettgewebe lässt die konzentration im gehirn rasch unter die narkotisch wirksame schwelle abfallen. da thiobarbiturate den uterustonus und die wehentätigkeit nicht beeinflussen, bleibt nach der geburt die kontraktionsfähigkeit des uterus erhalten. außerdem wurden keine interaktionen mit g -sympathikomimetika beschrieben. thiobarbiturate lassen sich bereits eine minute nach injektion im fetalen blut nachweisen. die konzentration liegt dabei nur gering unter der im mütterlichen blut. während der geburt ist bei niedriger dosierung (i.v. bis mg/kg kg) keine beeinträchtigung des fetus zu erwarten. bei höherer dosierung muss mit einer atemdepression beim neugeborenen gerechnet werden (langanke generell werden lokalanästhetika in allen phasen der schwangerschaft jedoch gut vertragen. sie scheinen keine anhaltenden auswirkungen auf die neurophysiologie des neugeborenen zu haben. es wurden keine teratogenen schäden nach einsatz im . trimenon beobachtet. lidocain, das am häufigsten eingesetzte lokalanästhetikum, hat wegen des niedrigen pka-wertes ( , bis , ) einen schnellen wirkungseintritt und überschreitet leicht die plazenta. ein negativer einfluss auf die schwangerschaft ist nicht bekannt. in einer studie mit mehr als . schwangeren fand sich keine zunahme der fehlbildungsrate . lidocain wird auch in der geburtshilflichen periduralanästhesie eingesetzt. es lindert den geburtsschmerz, ohne die wehenstärke oder die mitarbeit der gebärenden wesentlich zu beeinträchtigen. auch bei dieser anwendung gibt es einige wenige berichte über negative auswirkungen. sie betreffen zeitlich begrenzte veränderungen der fetalen kardiopulmonalen anpassung (bozynski ) , veränderungen von evozierten potenzialen im stammhirn (bozynski ) und temperaturregulationsstörungen mit hyperthermie nach mehrstündiger epiduralanalgesie (macaulay ) . in einigen studien wurde die periduralanästhesie mit veränderungen des verhaltens beim neugeborenen in zusammenhang gebracht. neuere studien haben jedoch gezeigt, dass solche veränderungen selten und nur vorübergehend sind (decocq , fernando . bupivacain ist das zurzeit am häufigsten verwendete lokalanästhetikum in der geburtshilfe. es hat eine starke wirkung und ausgeprägte zentralnervöse und kardiotoxische nebenwirkungen, die in der schwangerschaft zusätzlich durch progesteron verstärkt werden. bupivacain kann zu einem reentry-phänomen mit auslösung ventrikulärer tachykardien und kammerflimmern führen. die rate an toxischen wirkungen konnte deutlich gesenkt werden, nachdem das , %ige bupivacain in der geburtshilfe nicht mehr angewandt wurde. der hauptvorteil dieser substanz liegt in der langen wirkdauer ( - stunden). besonders in geringen konzentrationen kommt es eher zur sensorischen als zur motorischen blockade. wegen der hohen eiweißbindung ist die plazentapassage gering. ropivacain ist ein lokalanästhetikum vom amidtyp mit einer dem bupivacain vergleichbaren pharmakokinetik und pharmakodynamik. die vorzugsweise sensorische blockade ist noch ausgeprägter als bei bupivacain. dennoch hat sich bei ropivacain im vergleich zu bupivacain bisher kein vorteil hinsichtlich geringerer inzidenz motorischer blockaden (die eine instrumentelle entbindung notwendig machen) bei gleicher anästhesiequalität nachweisen lassen (eddleson ) . toxische und zentralnervöse nebenwirkungen treten bei ropivacain im vergleich mit bupivacain erst bei einer höheren gesamtmenge an verabreichtem lokalanästhetikum auf (santos ) . die austestung einer minimal lokalanästhetischen konzentration (mlac) konnte nachweisen, dass die anästhetische potenz von ropivacain um % geringer ist als die von bupivacain. ein zusatz von opioiden zu epidural verabreichten lokalanästhetika bietet den vorteil eines schnelleren wirkungseintritts, einer verbesserten analgesie und einer reduktion des bedarfs an lokalanästhetika. dadurch kann die rate an motorischen blockaden und instrumentellen entbindungen gesenkt werden. bei rückenmarksnahen regionalanästhesieverfahren werden lipophile opioide wie sufentanil und fentanyl bevorzugt, da sie schnell am applikationsort aufgenommen werden, und dadurch die analgesie regional begrenzt bleibt. das risiko einer späteren atemdepression kann aufgrund einer kürzeren liquorverweildauer gesenkt werden. andere opioidbedingte nebenwirkungen, wie Übelkeit und erbrechen, atemdepression und pruritus, treten seltener auf (gogarten ) . lipophile opioide wie sufentanil werden jedoch leichter in das gefäßsystem aufgenommen und können in signifikanten konzentrationen im plasma nachgewiesen werden. beim vergleich von fentanyl mit sufentanil führt letzteres zur besseren schmerzausschaltung und reichert sich trotz einer nachweisbaren plazentapassage weniger ausgeprägt im neugeborenen an (loftus ) . eine gesamtdosis von ? g sufentanil epidural führt nicht zu einer klinisch relevanten neonatalen beeinträchtigung (siehe auch kapitel . ). empfehlungen für die praxis: lokalanästhetika dürfen auch in der schwangerschaft zur infiltrations-und leitungsanästhesie eingesetzt werden. das gilt auch für präparate mit adrenalinzusatz. bewährte vertreter dieser gruppe, wie z. b. in der geburtshilfe bupivacain, sind zu bevorzugen. prilocain ist wegen des vergleichsweise hohen risikos der methämoglobinbildung zu meiden. g . . muskelrelaxanzien pharmakologie und toxikologie. muskelrelaxanzien werden immer dann in der narkose eingesetzt, wenn sich durch narkotika allein keine ausreichende erschlaffung der skelettmuskulatur erreichen lässt. alcuronium (alloferin ® ), atracurium (tracrium ® ), cisatracurium (nimbex ® ), mivacurium (mivacron ® ), pancuronium (pancuronium duplex ® ), rocuronium (esmeron ® ) und vecuronium (norcuron ® ) gehören wie tubocurarin zu den kompetitiv hemmenden muskelrelaxanzien. im gegensatz zu den narkotika und lokalanästhetika passieren muskelrelaxanzien wegen ihres hohen dissoziationsgrades und ihrer geringen lipidlöslichkeit die blut-hirn-schranke und auch die plazenta nur in geringem ausmaß. im nabelschnurblut bzw. im fetalen gewebe erreichen sie deshalb nur etwa % der bei der mutter gemessenen konzentration. die übertragenen konzentrationen reichen nicht aus, um beim fetus eine relaxierende wirkung hervorzurufen. es gibt jedoch einen fallbericht über eine zehn stunden anhaltende neugeborenenparalyse nach gabe von mg d-tubocurarin zur behandlung eines status epilepticus bei der mutter (lusso ) und über eine an tierexperimentelle beobachtungen erinnernde arthrogrypose beim kind nach behandlung eines mütterlichen tetanus mit tubucurarin über , wochen am ende des . trimenons (jago ) . teratogene eigenschaften wurden bisher nicht beobachtet. insbesondere pancuronium hat sich in der geburtshilfe bewährt. bei einer dosierung von , mg/kg wurden bei schnittentbindungen keine nebenwirkungen an den neugeborenen beobachtet (langanke ) . es kann auch zur relaxierung des fetus bei intrauteriner transfusion benutzt werden (moise ) . atracurium soll pancuronium bei der direkten relaxation des fetus überlegen sein, wenn dieser wegen einer anämie für eine intrauterine transfusion vorbereitet werden soll (mouw ) . in einer anderen untersuchung werden vorteile des vecuroniums bei dieser anwendung beschrieben. einschränkungen der fetalen herzfrequenz sollen geringer als bei pancuronium sein (watson ) . suxamethonium (succinylbischolin; lysthenon ® , pantolax ® ) ist ein depolarisierendes muskelrelaxans, das durch die plasmacholinesterase schnell abgebaut wird. fand bei kindern, deren mütter während der schwangerschaft mit succinylcholin behandelt worden waren, keine anomalien. vorübergehende atemdepression bei neugeborenen wurde vereinzelt nach succinylcholinbehandlung unter der geburt beschrieben. bei ca. - % der bevölkerung ist die plasmacholinesterase erniedrigt. zusätzlich verringert sich die aktivität dieses enzyms am ende der schwangerschaft um bis zu %. bei diesen patientinnen kann es nach succinylcholingabe zur verlängerung der wirkung und auch zur apnoe beim neugeborenen kommen (cherala ) . solche komplikationen sollten durch anwendung der niedrigsten effektiven dosis vermieden werden. succinylcholin kann bereits bei dosen von mg/kg den tonus des uterus erhöhen oder die wehentätigkeit stimulieren. dieser unerwünschte effekt ist bei drohender fetaler hypoxie zu beachten. mivacurium (mivacron ® ) ist ein kurz wirkendes, nicht depolarisierendes muskelrelaxans, das ebenso wie succinylcholin durch plasmacholinesterase abgebaut wird. wegen der häufig kurzen op-zeiten bei kaiserschnitt bietet es sich als muskelrelaxans an, führt aber bei einem mangel an plasmacholinesterase ebenso wie succinylcholin zur verlängerung der wirkdauer. völlig andere indikationen haben präparate mit clostridium botulinum toxin (botox ® , dysport ® ), die beim blepharospasmus und anderen fokalen spastizitäten sowie bei primärer hyperhidrosis appliziert werden. systematische untersuchungen zur schwangerschaft liegen nicht vor. anaphylaktoide reaktionen sind nicht auszuschließen (siehe auch botulismus in abschnitt . ). empfehlung für die praxis: im rahmen der narkose dürfen die üblichen muskelrelaxanzien (pancuronium, suxamethonium) in der schwangerschaft eingesetzt werden, dabei sollten möglichst niedrige dosierungen gewählt werden. clostridium botulinum toxin sollte in der schwangerschaft nicht verabreicht werden, da es sich nicht um eine vitale indikation handelt. eine dennoch erfolgte exposition erfordert keine konsequenzen, wenn die mutter keine nennenswerten nebenwirkungen hatte. in diesem abschnitt werden die wichtigsten arzneimittel in der dermatologie sowie andere häufig verwendete lokaltherapeutika behandelt. weitergehende informationen zu einzelnen arzneimitteln finden sich unter den substanzbegriffen in anderen abschnitten. vaginaltherapeutika siehe kapitel . . . g . . schwangerschaftstypische veränderungen an der haut die umstellung des organismus in der schwangerschaft führt an der haut zu typischen morphologischen und funktionellen veränderungen. sie sind völlig normal und müssen nicht behandelt werden. dazu gehören: zur lokalen therapie mit neomycin gibt es prospektiv erfasste fälle, davon mit exposition im . trimenon. Über fehlbildungen wurde nicht berichtet. zu den ausschließlich lokal angewandten antibiotika framycetin (leukase ® ), meclocyclin (meclosorb ® creme), mupirocin (z. b. turixin ® salbe), nadifloxacin (nadixa ® creme), neomycin (z. b. nebacetin ® ) und tyrothricin (z. b. tyrosur ® gel) liegen keine ausreichenden erfahrungen vor. für kein antibiotikum in äußerlicher anwendung hat sich bisher ein verdacht auf teratogene effekte ergeben. mittel, die systemisch unbedenklich sind, können, falls sinnvoll, auch für die lokale therapie eingesetzt werden (siehe auch kapitel . ). siehe abschnitt . . und folgende. bei topischer anwendung hat sich bisher für kein virustatikum ein verdacht auf teratogene effekte ergeben. dies gilt auch für das virustatikum aciclovir (z. b. zovirax ® ). generell ist die lokale antivirale therapie von herpes-simplex-infektionen wegen möglicher resistenzentwicklungen nicht zu empfehlen. besser geeignet scheinen austrocknende maßnahmen oder falls indiziert, eine systemische therapie. im eigenen datenbestand finden sich bei prospektiv erfassten schwangerschaften mit lokaler aciclovir-therapie, davon mit exposition im . trimenon, keine hinweise auf entwicklungstoxische schäden. auch die wesentlich umfangreicheren erfahrungen mit der systemischen anwendung von aciclovir haben kein risiko für das ungeborene erkennen lassen (siehe abschnitt . . ). auch die lokale behandlung von condylomata acuminata (feigwarzen) mit podophyllotoxin (z. b. condulox ® , wartec ® ), einem pflanzlichen mitosehemmstoff , bargman , hat keine hinweise auf teratogenität ergeben (eigene daten). systematische untersuchungen liegen allerdings nicht vor. Über insgesamt schwangere mit einer äußerlichen therapie mit dem immunmodulator bzw. virustatikum imiquimod (aldara ® ) wegen condylomata acuminata oder anderer warzen, davon im . trimenon, wird berichtet. alle wurden von gesunden kindern entbunden (einarson , maw . von eigenen prospektiv ausgewerteten schwangerschaften mit lokaler imiquimodtherapie endeten mit einem spontanabort. bei den lebendgeborenen wurden keine fehlbildungen beobachtet. zu den vorwiegend lokal eingesetzten virustatika foscarnet-natrium (z. b. triapten ® ), idoxuridin (z. b. virunguent ® ), penciclovir (vectavir ® ), tromantadin (viru-merz ® ) und vidarabin liegen keine ausreichenden erfahrungen vor. virustatika, die in der schwangerschaft systemisch angewendet werden dürfen, sind in der regel auch als lokaltherapeutika unproblematisch (siehe kapitel . . bis . . ). (weber ) . hinweise auf teratogene effekte bei vaginaler anwendung in der schwangerschaft haben sich in einer retrospektiven vergleichenden untersuchung von fehlgebildeten und gesunden kindern nach mütterlicher povidon-iod-exposition nicht ergeben sagrotan ® ; triclosan, z. b. in sicorten plus ® ) sind in der schwangerschaft als relativ sicher anzusehen. sie sollten in einer konzentration von nicht mehr als % und nur an der unverletzten haut angewendet werden. bei höheren konzentrationen muss mit relevanter resorption gerechnet werden. chlorhexidin (z. b. lemocin cx ® ) ist bei schwangeren zur desinfektion von haut und schleimhäuten geeignet. es hat sich zur desinfektion der scheide bei geburten und des abdomens beim kaiserschnitt bewährt (Übersicht in . mit dem neurotoxischen phenolderivat hexachlorophen ist dagegen zurückhaltung in der schwangerschaft angezeigt, da bei behandlung größerer flächen und mit konzentrationen von mehr als % resorptive vergiftungsbilder mit zns-symptomatik bei den behandelten patienten beobachtet wurden. in einigen tierexperimentellen studien hat sich hexachlorophen als teratogen gezeigt. in den letzten jahrzehnten wurde in mehreren publikationen der berufliche kontakt mit hexachlorophen kontrovers bezüglich möglicher fetotoxischer wirkungen diskutiert. eine ältere untersuchung an . gewerblich exponierten schwangeren fand keine auffälligkeiten (baltzar ), eine weitere retrospektive untersuchung postulierte einen zusammenhang zwischen mentaler entwicklungsretardierung und beruflicher exposition im letzten schwangerschaftsdrittel (roeleveld pharmakologie und toxikologie. quecksilber kann aus zubereitungen zur äußeren anwendung (früher in mercuchrom ® ) quantitativ resorbiert werden und ist potenziell entwicklungstoxisch (lauwerys pharmakologie und toxikologie. calcipotriol (z. b. psorcutan ® ) ist ein vitamin-d -derivat, das zur therapie der psoriasis sowohl extern als auch intern angewendet wird. es führt zur reduktion der keratinozytenproliferation und hat auch immunmodulatorische funktionen. grundsätzlich ist in der schwangerschaft eine d-hypervitaminose zu vermeiden. anwendungen im empfohlenen dosisbereich ( p g/woche einer , %igen zubereitung) führen aber nach heutigem wissen nicht zu einer störung des calcium-stoffwechsels. systematische untersuchungen zur pränataltoxizität beim menschen fehlen zu calcipotriol ebenso wie zu dithranol (z. b. micanol ® ), das als antimitotische substanz in der schwangerschaft theoretisch suspekt ist, obwohl eine quantitative resorption der üblicherweise - %igen zubereitungen nicht wahrscheinlich ist. zu dem ausschließlich lokal angewendeten vitamin-d-derivat tacalcitol (z. b. curatoderm salbe ® ) liegen keine ausreichenden erfahrungen zur anwendung in der schwangerschaft vor. es ist ähnlich zu bewerten wie calcipotriol. g . . retinoide zur akne-und psoriasistherapie pharmakologie. isotretinoin ( -cis-retinsäure; roaccutan ® , isotrex-gel ® ) und tretinoin (all-trans-retinsäure; z. b. cordes ® vas, vesanoid ® ) sind natürliche derivate des vitamin a (retinol). sie werden bei äußerlicher und systemischer anwendung seit über jahren mit großem erfolg bei akne eingesetzt. tretinoin ist außerdem in systemischer zubereitung zur behandlung der promyelozytären leukämie zugelassen. retinsäure ist ein körpereigener wachstumsfaktor, der in allen zellen vorkommt und an spezifische retinoidrezeptoren gebunden wird. eine besonders wichtige funktion hat die retinsäure während der embryonalphase, da sie u. a. die entwicklung von gehirn und wirbelsäule steuert. retinoide stimulieren die proliferation epidermaler zellen, an der haut lockern sie die hornschicht auf und begünstigen auf diese weise die hautabschilferung. isotretinoin führt zusätzlich zur atrophie der talgdrüsen. diese eigenschaften erklären die wirksamkeit in der aknetherapie. die halbwertszeit von isotretinoin und seinem metaboliten -oxo-isotretinoin beträgt durchschnittlich bzw. stunden, im extremfall bis zu einer woche (nulman ) . bei der behandlung der psoriasis haben sich acitretin (neotigason ® ) und das inzwischen aus dem handel genommene etretinat (tigason ® ) bewährt. beide führen zu lang anhaltend hohen retinoidkonzentrationen im körper. dabei wird acitretin zu etretinat metabolisiert, dessen halbwertszeit - tage beträgt. alkoholgenuss steigert die umwandlung zu etretinat (larsen ) . zu den synthetischen, polyaromatischen, rezeptorselektiven retinoiden gehören adapalen (differin ® ), das zur therapie einer schweren acne vulgaris eingesetzt wird und tazaroten (zorac ® ) zur behandlung der psoriasis. für die topische behandlung eines aids-assoziierten kaposi-sarkoms steht als neue substanz , %iges alitretinoin-gel (panretin ® ) zur verfügung, das durch aktivierung von retinoidrezeptoren das wachstum von tumorzellen hemmen soll. toxikologie. die ausgeprägten teratogenen eigenschaften der retinoide waren vor der markteinführung tierexperimentell bekannt. retinoide sind heute die beim menschen am stärksten teratogen wirksamen arzneimittel seit thalidomid (contergan ® ). ihre anwendung in der schwangerschaft erhöht das spontanabortrisiko und führt zum charakteristischen retinoid-syndrom: fehlanlage der ohren einschließlich agenesie oder stenose des gehörgangs, störungen der gesichts-und gaumenbildung, mikrognathie, kardiovaskuläre defekte und entwicklungsstörungen im bereich des thymus und des zns, die von neurologischen schäden mit beteiligung von augen und innenohr bis zum hydrozephalus reichen (lammer (lammer und . intelligenzdefizite wurden auch bei kindern ohne erkennbare fehlbildungen beobachtet (adams ). prospektive studien zur mütterlichen einnahme von isotretinoin in der schwangerschaft ergaben bis zu % spontanaborte, vermehrt frühgeburten und bis zu % große fehlbildungen. nachuntersuchungen der intrauterin mit retinoid exponierten kinder im alter von - jahren fanden eine hohe rate an mentalen retardierungen und speziellen schwächen bei der visuell-räumlichen verarbeitung. bei % der kinder mit intelligenzdefekten wurden keine großen fehlbildungen diagnostiziert (adams ). vor allem in nordamerika wurden durch isotretinoin geschädigte kinder geboren, obwohl wissenschaftliche fachgesellschaften eindringlich auf das teratogene risiko hingewiesen hatten, wie z. b. die teratology society der usa ( ). offenbar funktionierte die vorgeschriebene aufklärung in vielen fällen nicht ausreichend . an hersteller und fda wurden bis zum jahre über fälle gemeldet. zahlreiche publikationen berichten über einzelfälle oder kleine fallserien, wie z. b. in kalifornien erfasste schwangerschaften, von denen mit einem schwangerschaftsabbruch endeten, mit einem spontanabort und mit lebendgeburten. eines der kinder wies die bekannten fehlbildungen auf, bei den anderen kindern waren keine nachweisbar . moericke ( ) beschreibt feten nach schwangerschaftsabbruch, die zwar keine äußeren fehlbildungen aufwiesen, jedoch mittel-und innenohranomalien. neben früheren fallberichten zu multiplen fehlbildungen bei acitretin (z. b. de die-smulders ) wurde kürzlich ein weiterer fallbe-richt mit täglich mg bis woche und typischer embryopathie (mikrozephalie, fasziale dysmorphien, vorhofseptumdefekt, bilaterale sensorineurale taubheit) publiziert. im alter von monaten hatte das kind eine persistierende mikrozephalie sowie eine neurologische entwicklungsretardierung (barbero ) . geiger und mitarbeiter ( ) berichteten über insgesamt schwangerschaften mit acitretin, von denen mit schwangerschaftsabbruch, mit spontanabort und weitere mit einer lebendgeburt endeten. einer der abortierten feten wies typische fehlbildungen auf. die beiden lebendgeborenen waren gesund, lediglich bei einem der kinder waren hörstörungen bei hohen frequenzen auffällig. von schwangerschaften mit präkonzeptioneller acitretin-behandlung (im mittel monate vor der konzeption) endeten mit spontanabort, mit schwangerschaftsabbruch und mit einer lebendgeburt. vier kinder wiesen unspezifische fehlbildungen auf. bei frauen mit etretinat-therapie in der schwangerschaft wurde über lebendgeborene berichtet, von denen retinoidtypische und unspezifische fehlbildungen aufwiesen. unter den schwangerschaftsabbrüchen fanden sich feten mit retinoidspezifischen und mit anderweitigen fehlbildungen, weitere schwangerschaften endeten mit einem spontanabort. unter lebendgeborenen von insgesamt fällen mit einer etretinat-therapie vor der schwangerschaft (im mittel monate vor konzeption) waren bei kindern typische, bei weiteren unspezifische fehlbildungen nachweisbar. auch bei schwangerschaftsabbrüchen wurden retinoidspezifische fehlbildungen diagnostiziert (geiger ) . in postmarketing-studien wurden fälle einer acitretin-behandlung zum zeitpunkt der konzeption beim vater erfasst. fünf schwangerschaften endeten mit der geburt gesunder kinder, mit einem spontanabort und mit einem schwangerschaftsabbruch (geiger ) . Äußerliche anwendung. fünf fallbeschreibungen haben in den letzten jahren den verdacht aufkommen lassen, dass auch nach topischer applikation von tretinoin vitamin-a-säure-typische fehlbildungen nicht sicher auszuschließen sind (selcen , colley , navarre-belhassen , lipson , camera . zwei kontrollierte studien mit insgesamt etwa schwangeren erbrachten hingegen keine hinweise auf teratogene effekte (shapiro , jick . die größere dieser studien beruht jedoch auf verordnungsprotokollen, von denen nicht zwingend auf eine tatsächlich erfolgte anwendung der mutter geschlossen werden kann. außerdem erlauben design und fallzahlen dieser studien noch nicht die annahme einer unbedenklichkeit (martinez-frias ) . eine neuere prospektive studie mit im . trimenon äußerlich mit tretinoin behandelten frauen ergab weder ein erhöhtes abort-noch ein erhöhtes fehlbildungsrisiko. es fanden sich auch keine hinweise auf eine erhöhte inzidenz von retinoidverdächtigen kleinen anomalien im vergleich zu einer kontrollgruppe. die studie gibt allerdings keine angaben zur dosis und häufigkeit der lokalen tretinoinbehandlung (loureiro ) . im eigenen datenbestand haben wir schwangerschaften mit lokaler tretinointherapie im . trimenon prospektiv erfasst. bis auf einen abort endeten alle schwangerschaften mit einer lebendgeburt. grobstrukturelle fehlbildungen wurden nicht beobachtet. auch aufgrund pharmakokinetischer daten ist ein nennenswertes teratogenes risiko bei äußerlicher anwendung nicht wahrscheinlich, wenn die behandelte fläche nicht allzu groß ist: die resorptionsquote beträgt durchschnittlich % und maximal etwa % (van hoogdalem ), die konzentration der topischen retinoidpräparate liegt bei , %, ein nennenswerter anstieg der endogenen retinoidkonzentrationen im plasma ( - ? g/l) nach äußerer anwendung wurde nicht beobachtet. Übliche tägliche dosen sind maximal g salbe, die mg wirkstoff enthalten ( , %ig). allerdings muss bedacht werden, dass stark entzündete haut oder zusätzliche (desinfizierende) anwendungen (z. b. mit benzoylperoxid; siehe dort) die resorptionsquote erhöhen können. das bfarm (bundesinstitut für arzneimittel und medizinprodukte) warnt vor der äußerlichen applikation von tretinoin in der schwangerschaft (bfarm ). eine topische anwendung von isotretinoin (isotrex-gel ® ) ist genauso wie tretinoin zu bewerten. eigene daten zur lokalen isotretinoin-anwendung umfassen prospektiv erfasste schwangerschaften. nur eines von lebendgeborenen wies eine fehlbildung (gaumenspalte) auf. zur topischen anwendung von adapalen (differin ® ) gibt es einen fallbericht mit therapie bis woche , bei dem die schwangerschaft nach sonographischer diagnose zerebraler und okulärer fehlbildungen, die als nicht retinoidtypisch bewertet wurden, abgebrochen wurde (autret ). im eigenen datenbestand haben wir prospektiv dokumentierte schwangerschaften mit adapalentherapie im . trimenon, die alle mit der geburt eines gesunden kindes endeten. in einer französischen prospektiven studie wurden schwangerschaften mit topischer retinoidtherapie (tretinoin, isotretinoin oder adapalen) ausgewertet, dabei zeigte sich weder ein hinweis auf ein erhöhtes abortrisiko, noch war ein teratogenes risiko nachweisbar (carlier ) . allerdings wird weder nach substanzen differenziert, noch gibt es angaben zu zeitpunkt und dauer der therapie. bei lokaler anwendung von tazaroten (zorac ® ) werden % der applizierten dosis perkutan resorbiert. es hat eine halbwertszeit von - stunden. seine metaboliten sind hydrophil, so dass keine anreicherung im fettgewebe stattfindet. nach behandlung in der schwangerschaft wurde über gesunde kinder berichtet, allerdings ohne angaben zu therapiedauer und dosis (menter ) . wir überblicken prospektiv erfasste schwangerschaften mit therapie im . trimenon, von denen mit einem spontanabort und mit der geburt eines lebenden kindes endeten, ein neugeborenes wies ein kleines hämangiom auf. bei den anderen beiden fanden sich keine auffälligkeiten (eigene daten). zu alitretinoin-gel (panretin ® ) liegen keine erfahrungen in der schwangerschaft vor. empfehlung für die praxis: die systemische therapie mit den retinoiden acitretin, etretinat, isotretinoin und tretinoin ist in der schwangerschaft absolut kontraindiziert. bei frauen im gebärfähigen alter ist eine behandlung nur bei ausreichendem kontrazeptivem schutz und nach ausschluss einer schwangerschaft erlaubt, wenn andere therapieansätze wirkungslos waren. eine sichere kontrazeption muss nach absetzen von acitretin und etretinat noch zwei jahre weitergeführt werden und nach absetzen von isotretinoin noch einen monat. bei deutlichem unterschreiten dieser zeitvorgaben, insbesondere bei behandlung in die frühschwangerschaft hinein, ist eine erhebliche schädigung der embryonalen entwicklung möglich. nach eingehender analyse im einzelfall muss eventuell ein schwangerschaftsabbruch erörtert werden. die äußerliche anwendung von retinoiden ist während der schwangerschaft ebenfalls kontraindiziert. im fall einer solchen therapie in der frühschwangerschaft ist ein risikobegründeter schwangerschaftsabbruch (siehe kapitel . ) aufgrund des offenbar nur geringen, wenn überhaupt vorhandenen teratogenen risikos nicht inidiziert. eine ultraschallfeindiagnostik sollte jedoch angeboten werden. g . . photochemotherapie und fumarsäure-präparate pharmakologie und toxikologie. die photochemotherapie (puva-therapie) der schwer verlaufenden psoriasis erfolgt mit oraler gabe oderheute bevorzugt -äußerer anwendung von -methoxy-psoralen (methoxsalen; meladinine ® ) und anschließender langwelliger uva-bestrahlung. durch das uv-licht wird das psoralen chemisch aktiviert, bindet stärker an dns und schädigt die zellen. der zytotoxische effekt der puva-behandlung ist aufgrund der geringen eindringtiefe des uv-lichtes auf die haut beschränkt. das european network of teratology information services entis hat schwangerschaften analysiert, bei denen die systemische puva-therapie mit -methoxypsoralen durchgeführt wurde (garbis ) . in dieser studie, in der sich die puva-therapie auf das . trimenon beschränkte, fanden sich ebenso wie in einer skandinavischen studie (gunnarskog ) keinerlei hinweise auf embryotoxische effekte. fumarsäure wird in geringen mengen auch bei der nahrungsherstellung z. b. als antioxidanz verwendet, in der psoriasis-therapie sind hingegen nach einschleichen dosierungen von täglich einigen mg üblich, die als nebenwirkungen u. a. leuko-und lymphopenien verursachen können. die verträglichkeit für den fetus wurde nicht untersucht. wir überblicken schwangerschaften, bei denen in das . trimenon hinein eine psoriasis mit fumarsäure (dimethylfumarat + ethylhydrogenfumarat; fumaderm ® ) behandelt wurde. hinweise auf embryotoxische oder teratogene effekte haben sich dabei nicht ergeben ( spontanabort, totgeburt, bei den lebend geborenen kindern fanden sich keine großen fehlbildungen). empfehlung für die praxis: die photochemotherapie mit -methoxypsoralen und uva-bestrahlung ist in der schwangerschaft wegen möglicher mutagener wirkungen kontraindiziert, auch eine fumarsäure-behandlung sollte unterbleiben. eine dennoch erfolgte anwendung rechtfertigt weder einen risikobegründeten schwangerschaftsabbruch noch invasive diagnostik (siehe kapitel . , gleiches gilt für die behandlung von warzen (eigene daten). diese erfahrungen beruhen allerdings auf geringen fallzahlen. empfehlung für die praxis: die lokale behandlung mit -fluorouracil ist, mit ausnahme einzelner warzen (verrucae vulgares), in der schwangerschaft kontraindiziert, salicylsäure sollte als alternativ-therapie geprüft werden. die behandlung von condylomata sollte bis nach der geburt verschoben werden oder andere vorgehensweisen wie kryotherapie und trichloressigsäure gewählt werden. die lokale anwendung dieses zytostatikums stellt jedoch keine indikation für einen risikobegründeten abbruch der schwangerschaft oder invasive diagnostik dar (siehe kapitel . ). g . . lithium pharmakologie und toxikologie. lithium wird neben der oralen therapie von bipolaren störungen (siehe abschnitt . . ) auch zur lokalen therapie einer seborrhoischen dermatitis eingesetzt (z. b. efadermin salbe ® ). es besitzt eine antientzündliche wirkung. die perkutane penetration ist sehr gering und plasmakonzentrationen sind wesentlich geringer als nach oraler aufnahme (sparsa aufgrund letal verlaufender intoxikationen in den usa ("gasping-syndrom" mit progressiver enzephalopathie und schwerer metabolischer azidose, knochenmarkdepression und multiplem organversagen) ist benzylbenzoat in der neonatologie in verruf geraten. allerdings wurde in den o.g. fällen benzylbenzoat als spüllösung bei zentralen kathetern verwendet. nach äußerlicher applikation ergaben sich bisher, abgesehen von reizwirkungen auf haut und schleimhaut, weder im tierversuch noch bei der anwendung beim menschen hinweise auf nennenswerte toxizität (fölster-holst ) . crotamiton wird zu weniger als % der applizierten dosis perkutan resorbiert. eine kumulation der substanz war bisher nicht nachweisbar. im vergleich zu anderen antiskabiosa soll es etwas weniger wirksam sein (fölster-holst ) . bei kokosöl ist ein toxisches potenzial nicht anzunehmen (richter ) . lindan hat ein neurotoxisches potenzial. lindan , %ig wird zu % perkutan resorbiert (fölster-holst ; siehe auch abschnitt . . ). im tierversuch konnte gezeigt werden, dass sich lindan nicht nur im fettgewebe, sondern auch im hoden anreichern und zur zerstörung von leydig-zellen führen kann (suwalsky ) . nach europäischen umweltrichtlinien darf es nur noch bis ende verwendet werden. die synthetischen pyrethroide allethrin i, permethrin und pyrethrin haben längere halbwertszeiten als das "natürliche" pyrethrum, weshalb dieses bei der therapie einer pedikulose bevorzugt werden sollte. permethrin wird zu etwa % perkutan resorbiert (fölster-holst ) . aufgrund seiner langzeitwirkung wird es als effektiver angesehen als pyrethrum, obwohl keine systematischen studien zum vergleich beider substanzen vorliegen. weltweit ist eine erhebliche zunahme von resistenzen gegen pyrethroidderivate, einschließlich permethrin festzustellen (richter ) . die prospektive untersuchung von schwangeren mit anwendung von permethrin-shampoo, im . trimenon, ergab keinen hinweis auf ein embryotoxisches risiko (kennedy , kennedy (sudakin ) . eine mutter, die in afrika während der gesamten schwangerschaft neben einer malariaprophylaxe mit chloroquin ihre arme und beine täglich mit einer %igen deet-lotion eingerieben hatte, brachte ein in seiner geistigen entwicklung retardiertes kind zur welt (schaefer ) . da deet neurotoxische eigenschaften besitzt und über die haut resorbiert wird, schließen die autoren einen kausalen zusammenhang nicht völlig aus. es gibt allerdings keine weiteren berichte zu entwicklungstoxischen schäden beim menschen. in einer prospektiven randomisierten doppelblindstudie fanden sich bei schwangeren, die im . und . trimenon durchschnittlich , g/tag deet anwendeten, im vergleich zur kontrollgruppe keine unterschiede in der entwicklung der neugeborenen. bei % der behandelten frauen war deet im nabelschnurblut nachweisbar. unterschiede in der entwicklung der kinder bis zum . lebensjahr zeigten sich nicht (mcgready ) . zur anwendung von deet im . trimenon liegen keine systematischen untersuchungen vor, nur einzelfallbeobachtungen mit gesund geborenen kindern (eigene daten). icaridin hat ein geringeres toxisches potenzial als deet, systematische untersuchungen zur schwangerschaft liegen allerdings nicht vor. empfehlung für die praxis: von der bedenkenlosen, großflächigen anwendung von insektenrepellents vom typ des deet über längere zeit ist in der schwangerschaft abzuraten. in gebieten mit hohem malariarisiko, die während einer schwangerschaft nur aus zwingenden gründen besucht werden sollten, ist das mit der anwendung von deet verbundene risiko für mutter und kind als eindeutig geringer einzuschätzen als das risiko durch eine malariainfektion. pyrethroidhaltige repellents sind zu meiden. wo immer möglich, sollten in der schwangerschaft andere repellents einschließlich icaridin bevorzugt werden. weder die anwendung von deet im . trimenon noch von pyrethroidhaltigen repellents erfordert einen risikobegründeten abbruch der schwangerschaft oder invasive diagnostik (siehe kapitel . ). g . . augen-, nasen-und ohrentropfen augen-, nasen-und ohrentropfen dürfen bei entsprechender indikation generell auch in der schwangerschaft angewendet werden. allerdings gilt auch hier, dass eine wohl begründete medikamentenwahl erfolgen sollte und sowohl fragwürdige kombinationspräparate als auch (pseudo-)innovationen während der schwangerschaft zu meiden sind. im zweifelsfall kann man sich an empfehlungen zur systemischen therapie in den entsprechenden abschnitten orientieren. insbesondere bei augentropfen ist mit einer quantitativen arzneimittelresorption über die konjunktiven zu rechnen. daher ist nicht auszuschließen und teilweise beobachtet worden, dass beispielsweise atropinartige substanzen und betarezeptorenblocker (siehe abschnitt . . ) als augentropfen die fetale herzfrequenz erhöhen bzw. senken können. bedrohliche situationen sind bei üblichen dosen zum diagnostischen weittropfen oder zur glaukombehandlung nicht zu erwarten. die ebenfalls zur glaukomtherapie verwendeten carboanhydrasehemmer brinzolamid (azopt ® ), dorzolamid (z. b. trusopt ® ) und zur systemischen anwendung acetazolamid (z. b. diamox ® ) sind zwar nicht systematisch untersucht, bisher haben sich jedoch bei den länger eingeführten präparaten keine negativen auswirkungen auf den fetus gezeigt. eigene daten zu insgesamt ca. prospektiv dokumentierten schwangerschaften mit mütterlicher brinzolamid-oder dorzolamidtherapie deuten nicht auf ein embryotoxisches risiko hin. die mütterliche therapie mit mg/tag acetazolamid (diamox ® ) in den letzten tagen vor entbindung führte bei einem mit wochen geborenen kind zu tachypnoe, respiratorisch-metabolischer azidose, hypoglykämie und hypokaliämie. die serumkonzentration stunden nach der geburt betrug , ? g/ml, das entspricht beinahe der therapeutischen konzentration bei erwachsenen ( - ? g/ml). nach normalisierung des ph-wertes besserten sich die klinischen symptome spontan. am . tag war kein acetazolamid mehr nachweisbar, die weitere entwicklung des kindes verlief normal (ozawa ) . bei den neugeborenen von frauen, die wegen eines idiopathischen, erhöhten intrakraniellen drucks mit durchschnittlich mg/tag acetazolamid behandelt wurden, davon im . trimenon, waren keine fehlbildungen oder andere auffälligkeiten nachweisbar (lee ) . zu latanoprost (z. b. xalatan ® ) wurde über prospektiv dokumentierte behandlungen berichtet, davon im . trimenon. eine schwangerschaft endete mit einem spontanabort. die reifgeborenen kinder wiesen keine fehlbildungen auf minoxidil findet oral als antihypertensivum anwendung. es hat eine vasodilatatorische wirkung und wird lokal bei androgener alopezie und anderen arten von haarausfall angewendet (z. b. regaine ® frauen lösung). die substanz ist lipophil, ihre perkutane resorption soll - % betragen. dabei werden serumkonzentrationen erreicht, die weit unterhalb einer therapeutischen, antihypertensiv wirksamen konzentration für erwachsene liegen. in einer prospektiven studie wurden schwangere mit minoxidil-lösung behandelt, eines von lebend geborenen kindern wies eine herzfehlbildung auf (shapiro ) . zwei eigene prospektiv erfasste schwangerschaften mit exposition im . trimenon endeten mit der geburt gesunder kinder. bei einer frau, die seit einigen jahren mindestens zweimal täglich minoxidil lokal auf ihre kopfhaut applizierte, diagnostizierte man beim fetus hirn-, herz-und vaskuläre fehlbildungen. der pathologische befund zeigte eine deutliche herzvergrößerung mit einer distalen stenose der aorta, ein erheblich verlängertes colon sigmoideum, eine hirnventrikelerweiterung, zerebrale hämorrhagien sowie ischämische areale in der plazenta (smorlesi ) . in einer weiteren publikation wird nach einer mehrjährigen lokalen %igen minoxidil-behandlung über eine ausgeprägte kaudale hypotrophie des fetus berichtet mit aplasie der unteren wirbelsäule, fehlanlage der unteren extremitäten und des harnableitenden systems, kompletter nierenagenesie und Ösophagusatresie (rojansky (biesalski ) . der tagesbedarf beträgt etwa mg retinol oder mg g -carotin. vitamin a wird ähnlich wie vitamin c im embryo angereichert. die konzentration von vitamin-a-metaboliten im serum ist bei schwangeren im . trimenon vermindert und beträgt zwischen , und , ? g/l, in der zweiten schwangerschaftshälfte steigt sie auf etwa % des wertes nichtschwangerer frauen an (malone ) . selbst nach -wöchiger gabe von täglich . ie vitamin a liegen die spitzenwerte der metaboliten retinsäure und isotretinoin höchstens geringfügig über den vorher gemessenen konzentrationen (wiegand ). eckloff und mitarbeiter ( ) wiesen bei einer tagesdosis von . ie vitamin a einen anstieg der plasmakonzentrationen von all-trans-retinoinsäure nach, die zu isotretinoin metabolisiert wird. toxikologie. in kapitel . "dermatika und lokaltherapeutika" wird ausführlich beschrieben, dass vitamin-a-derivate, wie die retinoide isotretinoin und acitretin, die zur therapie schwerer formen von akne und psoriasis eingesetzt werden, beim menschen teratogen wirken und daher in der schwangerschaft absolut kontraindiziert sind. vor etwa jahren wurde erstmals diskutiert, dass vitamin-a-präparate in dosen über . ie pro tag ähnlich wie die retinoide beim menschen teratogen wirken und das charakteristische "retinoidsyndrom" auslösen können (rosa ). in deutschland haben aufgrund einer empfehlung des bundesgesundheitsamtes die hersteller von multivitaminpräparaten die zusammensetzung ihrer produkte so geändert, dass sie nicht mehr als . ie pro tagesdosis enthalten. in anderen ländern wurde ähnlich verfahren (bundesgesundheitsamt , laschinski , teratology society . die unbedenklichkeit solch niedriger dosen wurde verschiedentlich, u. a. durch die studie von an schwangeren in ungarn, bestätigt. eine untersuchung des european network of teratology information services (entis) ergibt erstaunlicherweise keine hinweise auf eine teratogene wirksamkeit selbst hoher, im . trimenon genommener vitamin-a-dosen ( . - . , mittelwert . ie/tag). insbesondere können die in einer anderen studie gemachten beobachtungen nicht bestätigt werden, dass hohe dosen, insbesondere solche über . ie/tag, einen bestimmten typ von zns-anomalien verursachen (rothman ) . die entis-studie ist mit schwangeren die bisher größte vitamin a (retinol) vitamin-a-studie (mastroiacovo (botto ) . man muss dazu kritisch anmerken, dass die zahl der mütter mit einer hohen dosis in beiden gruppen sehr gering war. daher kann dieses ergebnis allenfalls als hypothese gewertet werden, die durch andere studien bisher nicht bestätigt wurde. eine weitere fall-kontroll-studie fand keinen zusammenhang zwischen einer im normbereich liegenden vitamin-a-aufnahme aus leber oder multivitaminprodukten und dem auftreten von spaltbildungen (mitchell ) . generell wird in der schwangerschaft vor dem verzehr von leber gewarnt, da eine portion ( g), auch gebraten, bis zu . ie retinol enthalten kann. es gibt jedoch kaum klinische hinweise darauf, dass der verzehr von leber zu fehlbildungen beim menschen führt. das könnte daran liegen, dass vitamin a bzw. der teratogene metabolit all-trans-retinolsäure nach leberverzehr nur / jener konzentrationsspitzen im serum erreicht, die nach einnahme standardisierter vitamin-a-dosen in tablettenform gemessen wurden (buss obwohl der zusammenhang zwischen folsäuremangel und neuralrohrdefekt nicht genau geklärt ist, bestätigen die meisten epidemiologischen untersuchungen bis heute einen protektiven effekt der folsäuresubstitution (wald . auswirkungen auf den methioninstoffwechsel spielen neben dem eventuell ebenfalls relevanten methioningehalt der mütterlichen ernährung offenbar eine rolle . ein protektiver effekt wird auch bei anderen fehlbildungen diskutiert (bailey , koletzko , wie z. b. kardialen defekten , botto oder analatresie (myers ) sowie bei aborten (gindler . man ist sich heute weitgehend einig, dass alle frauen in der frühschwangerschaft, möglichst schon ab planung einer schwangerschaft, bis zur woche täglich ? g folsäure zusätzlich einnehmen sollen. frauen mit risikoanamnese (bereits neuralrohrdefekte in der familie aufgetreten) oder bei folatantagonistischer therapie, z. b. mit bestimmten antiepileptika, werden - mg/tag empfohlen. eine Überdosierung der folsäure schädigt nach bisherigen erfahrungen die embryonale entwicklung nicht. die maskierung einer seltenen vitamin-b -mangelanämie durch eine folsäureeinnahme ist zwar möglich, hat aber angesichts der zeitlich begrenzten einnahme keine bedeutung. in manchen ländern, wie z. b. kanada und den usa, wird eine allgemeine anreicherung von nahrungsmitteln (getreideprodukten) mit folsäure vorgeschrieben und zwar mit etwa , mg/kg. in den usa wurde seit der folsäureanreicherung über eine abnahme der neuralrohrdefekte um % ) und % (mills ) , in kanada um % (persad ) berichtet. auch in der bundesrepublik deutschland und anderen europäischen ländern wird die folsäureanreicherung von nahrungsmitteln erörtert, da die substitution mit tabletten nur von wenigen schwangeren praktiziert wird und die durchschnittliche ernährung in deutschland nur ? g/tag statt der erforderlichen ? g enthält (bgvv ) . in deutschland befolgen nur % der frauen, die eine schwangerschaft planen, und bei nicht geplanten schwangerschaften nur % den rat einer perikonzeptionellen folsäureeinnahme. in den niederlanden sind es hingegen über % (gärtner ) . häufig beobachtet man infolge unzureichender ärztlicher beratung, dass folsäure erst im späten . trimenon und danach eingenommen wird. die hochempfindliche phase der neuralrohrentwicklung ist aber schon mit wochen abgeschlossen. gelegentlich wird diskutiert, ob eine ausgewogene ernährung für die folsäureversorgung ausreicht. epidemiologische daten und untersuchungen zum intraerythrozytären folsäurestatus unter berücksichtigung verschiedener zufuhrbedingungen sprechen dagegen. einerseits gibt es hinweise darauf, dass mütter von kindern mit neuralrohrdefekten einen pathologisch erhöhten bedarf an folsäure aufweisen, der deutlich über der mit "gesunder" ernährung zugeführten menge liegt. andererseits ergab sich aus einer vergleichenden untersuchung, dass nur tabletten und angereicherte nahrungsmittel, nicht aber diätetisch aufgenommene folsäure zu einer signifikanten verbesserung des folsäurestatus führen (cuskelly ) . schließlich soll nicht unerwähnt bleiben, dass nach kritischer auswertung aller vorliegenden daten auch heute noch zweifel an der protektiven wirkung einer zusätzlichen verabreichung von folsäure in der schwangerschaft geäußert werden (källén , kalter . empfehlung für die praxis: um die protektive wirkung der folsäure bei neuralrohrdefekten zu nutzen, sollten möglichst schon bei der planung einer schwangerschaft sowie während der ersten wochen , mg/tag eingenommen werden. die substitutionsdosis ist auf - mg/ tag g . . vitamin c (ascorbinsäure) pharmakologie und toxikologie. vitamin c (z. b. cebion ® , ctebe ® ) ist im zellulären stoffwechsel zur aufrechterhaltung des oxidations-reduktions-gleichgewichts wichtig. der tagesbedarf an vitamin c beträgt etwa mg. vitamin-c-mangel führt zu skorbut mit störungen des kollagenstoffwechsels und zur blutungsneigung. die vitamin-c-konzentration im fetalen blut ist -mal so hoch wie im mütterlichen blut, da sich nach dem plazentaren Übergang von dehydro-ascorbinsäure vitamin c im fetus anreichert (malone ) . es ist nicht bekannt, ob gaben von vitamin c das reduktions-oxidations-gleichgewicht des fetus beeinflussen. diskutiert werden der zusammenhang zwischen mütterlichem vitamin-c-mangel und einem erhöhten risiko für gestationsdiabetes (zhang a und sowie eine assoziation zwischen mütterlichem vitamin-c-spiegel bzw. einer protektiven vitamin-c-substitution im . und . trimenon und vorzeitigem blasensprung (casanueva , tejero pharmakologie. eisen(ii)-salze (z. b. eisendragees-ratiopharm ® ) werden nach oraler gabe gut resorbiert und sind für die eisensubstitution in der schwangerschaft geeignet. der zusatz von vitaminen und spurenelementen zu oralen eisen(ii)-präparaten hat keinen erwiesenen therapeutischen nutzen. kombinationspräparate mit folsäure sind nicht zu empfehlen, da die eisenresorption aus diesen zubereitungen um bis zu % reduziert ist (pietrzik ) . etwa - % der patientinnen, die eisen-(ii-)präparate einnehmen, klagen über gastrointestinale beschwerden, die bei vorbestehender morgendlicher Übelkeit zum wechsel auf ein anderes präparat oder zur beendigung der eisensubstitution zwingen können (letzky ) . die parenterale applikation von eisenpräparaten (singh ) wie eisen(iii)-gluconat-komplex (ferrlecit ® ) ist nur bei ausgeprägter anämie indiziert und macht in kombination mit anderen antianämika eine transfusionstherapie in der schwangerschaft weitgehend überflüssig. toxikologie. der verdacht, dass nach eisensubstitution in der schwangerschaft die fehlbildungsrate gering ansteigen könnte (nelson ) , hat sich in umfangreichen prospektiven untersuchungen nicht bestätigt (dfg , royal college ein fallbericht beschreibt die mütterliche calciumtherapie wegen osteoporose mit einer tagesdosis von . mg bis woche und die weitere therapie mit täglich . mg, kombiniert mit einer colecalciferolbehandlung bis zum ende der (drillings-)schwangerschaft. in woche kam es zum spontanabort eines fetus, die beiden anderen kinder wurden gesund geboren (harsch in einer neueren prospektiven studie wurden die schwangerschaften von frauen mit biphosphonat-therapie, davon mit exposition im . trimenon ausgewertet (alendronsäure: , etidronsäure: , pamidronsäure: , risedronsäure: ). die schwangerschaften endeten mit lebendgeborenen und einem spontanabort. hinweise auf embryotoxische effekte fanden sich nicht (levy . in einer kleinen retrospektiven untersuchung wurde ein protektiver effekt für eine neurodermitis des kindes bei frauen mit atopieneigung festgestellt (dunstan ) . empfehlung für die praxis: eine überzeugende notwendigkeit zur substitution mit omega- -fettsäuren besteht bei ausgewogener ernährung nicht. alternative heilmittel und phytotherapeutika die verträglichkeit alternativer heilmittel in der schwangerschaft ist bisher nicht systematisch untersucht. es liegen auch keine fallberichte über teratogene schädigungen bei einhalten der empfohlenen dosierungen vor, sie sind zumindest bei homöopathika auch nicht zu erwarten. gegen akupunktur ist ebenfalls nichts einzuwenden, wenn sie fachkundig in der schwangerschaft praktiziert wird. nicht alle pflanzlichen präparate sind harmlos: bei phytotherapeutika sollten therapeutische dosen eingehalten und tees nicht exzessiv genossen werden. die herkunft sollte deklariert sein, da kontaminationen mit unerwünschten pflanzlichen bestandteilen, schwermetallen, wie z. b. blei (tait ) (gut ) . allerdings sind auch hier systematische untersuchungen zur teratogenität und embryotoxizität rar. trotz der breiten anwendung von pflanzlichen therapeutika auch in der schwangerschaft sind hinweise oder fallberichte zu teratogenen effekten selten und zumindest bei einhaltung der empfohlenen dosierungen auch kaum zu erwarten. eine retrospektive studie zur anwendung von phytotherapeutika konnte beim vergleich mit "schulmedizinischen therapeutika" kein erhöhtes fehlbildungsrisiko durch pflanzliche substanzen nachweisen (leung ) . eigene daten umfassen prospektiv dokumentierte schwangerschaften, davon mit anwendung im . trimenon. ein teratogenes risiko oder hinweise auf vermehrte spontanaborte lassen sich nicht beobachten. bei der anwendung pflanzlicher zubereitungen und teedrogen sollte stets darauf geachtet werden, dass ihre herkunft bekannt und die inhaltsstoffe eindeutig deklariert sind. vor einer kontamination mit pflanzenschutzmitteln oder toxischen schwermetallen sowie mikrobieller verunreinigung nicht zertifizierter ware wird gewarnt (ihrig ) . auf alkoholische zubereitungen sollte zumindest bei längerfristiger therapie verzichtet werden. ein fallbericht beschreibt exzessiven gebrauch von phytopharmaka mit % alkoholgehalt in der frühschwangerschaft, angeblich ohne weitere alkoholeinnahme. das kind wies typische zeichen eines fetalen alkoholsyndroms auf (ernst ) . diese kasuistik sollte eher als anekdotisch betrachtet werden, vergleichbare berichte gibt es bisher nicht. g . . aloe vera pharmakologie und toxikologie. aloe vera wird extern eingesetzt zur förderung der wundheilung oder bei hautproblemen und intern als immunstimulans. systematische untersuchungen zur oralen anwendung in der schwangerschaft fehlen bisher. aloe soll eine stimulierende wirkung auf die uterusmuskulatur haben, so dass theoretisch das risiko für einen spontanabort erhöht sein könnte (ernst ) . die lokale anwendung in der schwangerschaft ist wahrscheinlich unproblematisch (nordeng in einer großen retrospektiven studie wurden . frauen zu ihrem lakritzkonsum (starke "skandinavische lakritze") in der schwangerschaft, dem geburtsgewicht der kinder sowie dem schwangerschaftsalter bei entbindung befragt. bei starkem wöchentlichen lakritzkonsum ab mg glyzyrrhizin war zwar keine signifikante reduzierung des geburtsgewichtes nachweisbar, es fand sich jedoch ein leicht erhöhtes risiko für eine geburt bereits vor wochen (strandberg ) . in einer retrospektiven finnischen studie wurden frauen mit frühgeborenen kindern mit frauen mit reifgeborenen hinsichtlich ihres lakritzkonsums ("skandinavische lakritze") verglichen. es wurde festgestellt, dass bei starkem lakritzkonsum ab wöchentlich mg glyzyrrhizin ein - fach erhöhtes frühgeburtsrisiko besteht. es wird vermutet, dass glyzyrrhizin einen lokalen anstieg des prostaglandinspiegels im uterus bewirken und dadurch vorzeitige wehen auslösen könne (strandberg ) . die methodik der studie wurde jedoch wegen fehlender adjustierung auf weitere faktoren des lebensstils kritisiert (hughes (ernst ) . bei gabe von himbeerblättertee zur erleichterung und verkürzung der geburt bei frauen waren keine nebenwirkungen nachweisbar. eine verkürzung der geburt konnte in der ersten phase nicht, in der zweiten phase um min registriert werden. weiterhin fanden sich signifikant weniger forceps-entbindungen als in der kontrollgruppe (ernst (portnoi ) . in anderen randomisierten plazebokontrollierten doppelblindstudien zeigte sich jedoch, dass sowohl die Übelkeit als auch die häufigkeit des erbrechens mit ingwer signifikant reduziert wurden (willetts , keating , vutyavanich , fischer-rassmusen . bis auf eine arbeit ohne hinweise zur entwicklung des neugeborenen (keating ) , fanden sich in den anderen studien keine hinweise auf ein erhöhtes abort-oder fehlbildungsrisiko. eine neuere randomisiert-kontrollierte studie ermittelte keine unterschiede in der wirksamkeit und bei der entwicklung der neugeborenen gegenüber vitamin b (pyridoxin). ein erhöhtes fehlbildungsrisiko war nicht erkennbar (smith ) . auch andere substanzspezifische nebenwirkungen wurden bisher nicht beobachtet (betz ) . da ingwer in vitro eine hemmung der thromboxansynthese bewirkt, nahmen einige autoren an, dass er die testosteron-rezeptor-bindungen beim fetus beeinflussen und somit auf die geschlechtsdifferenzierung im kindlichen hirn einwirken könnte (backon ) . dieser effekt erscheint jedoch im üblichen dosisbereich unwahrscheinlich. im tierversuch wurden in studien keine entsprechenden effekte beobachtet (weidner , wilkinson . empfehlung für die praxis: die anwendung von ingwer bei schwangerschaftsübelkeit und -erbrechen in üblicher dosierung stellt kein problem dar. g . . johanniskraut (hypericum perforatum) pharmakologie und toxikologie. johanniskraut (z. b. esbericum ® ) wird bei leichten depressiven verstimmungen, psychovegetativen störungen und nervöser unruhe mit erfolg eingesetzt (nordeng ) . durch eine induktion von isoenzymen von cytochrom p (cyp ) können interaktionen mit anderen arzneimitteln auftreten, z. b. eine beeinträchtigung der wirkung oraler kontrazeptiva (siehe abschnitt . ). weiterhin wurden unter der johanniskraut-therapie zyklusstörungen (zwischenblutungen, menstruationsunregelmäßigkeiten) beobachtet (yue ) . im tierversuch fanden sich keine hinweise für teratogene effekte (jurgens (leuschner ) . beim menschen gibt es bisher keine hinweise darauf, dass therapeutische dosen teratogen sind, allerdings wurde dies nicht systematisch untersucht. theroretisch kann kampfer spontanaborte auslösen. es ist plazentagängig und der fetus bildet noch keine enzyme zur hydroxylierung und glucuronidierung von kampfer (rabl publiziert wurden fälle, in denen eine längere einnahme von pyrrolizidinalkaloidhaltigen zubereitungen in der schwangerschaft zu einer leberschädigung mit venöser verschlusskrankheit (veno occlusive disease) und zum tod des kindes führte. im ersten fall trank die mutter während der gesamten schwangerschaft einen pflanzentee, bei dem später festgestellt wurde, dass er pyrrolizidinalkaloide enthielt (ernst ) . zunächst war der huflattich im tee angeschuldigt worden, schließlich stellte sich heraus, dass die ursache bei einer verunreinigung mit pestwurz lag. im zweiten fall hatte die schwangere zum täglichen kochen eine türkische gewürzmischung verwendet, die diverse pyrrolizidinalkaloide enthielt (rasenack ) . eine südafrikanische veröffentlichung soll über kinder mit veno occlusive disease nach mütterlicher einnahme pyrrolizidinalkaloidhaltiger phytotherapeutika und hoher mortalitätsrate beobachtet haben. bei überlebenden kindern entwickelte sich eine leberzirrhose mit portaler hypertension (ernst ) . msv, jedoch meist unter msv, wenn sich der uterus im strahlengang befand. dies schließt zwei untersuchungsgänge unter einbeziehung des unterbauchs einschließlich Übersichtsaufnahme (scout) ein. zur dosisberechnung gehören u. a. die röhrenspannung in kv, die anzahl der rotationen, die oberste und unterste schicht mit angabe des korrespondierenden wirbelkörpers oder in cm oberhalb des rumpfendes, die mas (milliamperesekunden) pro rotation oder als summenangabe für die gesamte untersuchung, die schichtdicke, der vorschub und der gerätetypische so genannte kerma-wert, der die dosis auf der rotationsachse in freier luft angibt und mit ctdi luft bezeichnet wird. die streustrahlung bei untersuchung anderer körperregionen wie oberbauch, thorax, extremitäten oder zahnröntgen ist zu vernachlässigen, weil sie weit unter msv liegt. auswirkungen von röntgenstrahlung. röntgenstrahlen können in abhängigkeit von der dosis und vom entwicklungsstadium des embryos fruchttod, fehlbildungen verschiedener organsysteme, vor allem der augen, allgemeine wachstumsretardierung, mikrozephalie und mentale retardierung hervorrufen. dies ist sowohl tierexperimentell als auch empirisch beim menschen belegt . in den ersten tagen nach konzeption (also noch während der "alles-oder-nichts-phase") wird die niedrigste letaldosis mit rad ( mgy) angegeben. während der eigentlichen embryogenese wird dieser wert mit - rad, später mit über rad ( gy) beziffert . schwere zns-fehlbildungen während der frühen embryogenese ( - tage nach konzeption) sollen erst ab rad ( mgy) zu erwarten sein. mit bleibender wachstumsretardierung rechnet man bei - rad. mikrozephalie und mentale retardierung wurden besonders nach dosen oberhalb rad zwischen woche und beobachtet. die meisten untersuchungen kommen zu dem schluss, dass unterhalb einer strahlendosis von mgy, entsprechend rad, mit keinem nennenswerten anstieg des fehlbildungsrisikos beim menschen zu rechnen ist a, sternberg . eine kürzlich veröffentlichte studie beobachtet ein geringeres geburtsgewicht im zusammenhang mit zahnröntgen in der schwangerschaft und interpretiert dies als folge einer funktionsstörung der schilddrüse, die beim zahnröntgen ebenfalls getroffen werde (hujoel ) . andere autoren widersprechen dieser hypothese und halten eher die zugrunde liegende zahnerkrankung für ursächlich (lockhart ). weitaus schwieriger als die beurteilung des teratogenen strahlenrisikos ist die frage nach mutagenen und krebsauslösenden effekten zu beantworten. für mutagene effekte gibt es keine schwellendosis, unterhalb derer wie bei der teratogenese kein effekt zu erwarten ist. punktmutationen ereignen sich bekanntermaßen auch spontan. die zur verdopplung der punktmutationsrate führende strahlendosis wird mit - rad ( - gy) angegeben a, neel ). einerseits bedeutet eine verdopplung der mutationsrate eines bestimmten gens noch keine häufigkeitsverdopplung einer daran gekoppelten erkrankung. andererseits sollten die völlig unzureichenden kenntnisse zu den auswirkungen auf spätere generationen zu großer zurückhaltung bei der definition unbedenklicher expositionsgrenzwerte für die gesamtbevölkerung führen . bei den eltern von etwa an neuroblastom erkrankten kindern wurden röntgenanwendungen vor der schwangerschaft nicht häufiger durchgeführt als bei einer gesunden kontrollgruppe (patton ) . in einer studie an zwillingsschwangerschaften ermittelten harvey und mitarbeiter ( ) bei einer fetaldosis von , sv einen anstieg des leukämierisikos um den faktor , . lengfelder ( ) zieht bereits ein erhöhtes leukämierisiko in erwägung, wenn die zusätzliche pränatale strahlenexposition des embryos im bereich der natürlichen hintergrundbelastung von etwa , sv liegt. dagegen nehmen andere autoren bei exposition mit , - , sv noch kein risiko für den embryo an (boice ) . wakeford und mitarbeiter ( ) haben für kinder unter jahren das relative und absolute risiko errechnet, nach intrauteriner strahlenexposition an einem karzinom zu erkranken. das absolute risiko geben sie mit % pro gray an. ihre detaillierte berechnung basiert auf der weltweit größten datensammlung zum karzinomrisiko durch intrauterine röntgenexposition, vorwiegend pelvimetrie, dem oxford survey of childhood cancers (oscc). die autoren leiten vergleichbare risiko-koeffizienten aus den japanischen daten von atombombenopfern ab und fassen zusammen, dass selbst für eine vergleichsweise niedrige fetaldosis von msv, die in den er jahren bei einer röntgenaufnahme des beckens erreicht wurde, bereits ein erhöhtes risiko vorliegt. andere autoren halten solche risikoannahmen für zu hoch. sie berufen sich ebenfalls auf die nicht einmal . Überlebende umfassende gruppe intrauterin exponierter hiroshimaopfer und auf verlaufsdaten von exponierten kindern in hiroshima. diese nicht selten als beleg für ein vergleichsweise niedriges krebsrisiko nach radioaktiver exposition zitierten untersuchungen sind jedoch angesichts methodischer mängel und der damaligen politischen interessenlage der amerikanischen untersucher kritisch zu bewerten. empfehlung für die praxis: bei anwendung bildgebender diagnostischer verfahren im bereich des unterbauches sollte bei frauen im gebärfähigen alter primär auf röntgenverfahren verzichtet werden, insbesondere wenn eine schwangerschaft nicht sicher auszuschließen ist. die mit "nein" beantwortete frage nach einer vorliegenden schwangerschaft schließt eine solche bekanntermaßen nicht aus! jede röntgenuntersuchung des unterbauchs, von deren ergebnis nicht unmittelbar vital indizierte therapiemaßnahmen abhängen, sollte sicher-heitshalber nur in der ersten zyklushälfte durchgeführt werden. falls röntgenuntersuchungen unverzichtbar sind, darf nur mit den modernsten geräten und unter optimalem schutz der fruchthöhle gearbeitet werden. röntgenaufnahmen außerhalb der genitalregion und der fruchthöhle stellen weder eine indikation für einen risikobegründeten abbruch der schwangerschaft noch für weitere vorsorgemaßnahmen dar (siehe kapitel . (bal , chow , read , schlumberger . von zahlreichen kindern dieser studien liegen berichte zu ihrer entwicklung bis ins erwachsenenalter vor, ohne dass hinweise auf spätfolgen wie karzinomentstehung oder genetische defekte daraus erkennbar werden. schlumberger und mitarbeiter ( ) beobachten jedoch eine erhöhte abortrate, wenn die behandlung innerhalb eines jahres vor der schwangerschaft stattfand. die autoren diskutieren sowohl die exposition der gonaden als auch eine ungenügende schilddrüsenhormoneinstellung nach der nuklear-medizinischen therapie als ursachen. read und mitarbeiter ( ) (newnham , visser , ließen sich diese auswirkungen nicht bestätigen. nachfolgeuntersuchungen an etwa . kindern im alter von - jahren, deren mütter -mal per ultraschall zwischen woche und untersucht worden waren, ergaben hinsichtlich gewichtszunahme und anderer entwicklungsparameter keine auffälligkeiten im vergleich zu einer kontrollgruppe mit nur einer ultraschalluntersuchung (newnham ) . gepulste doppleruntersuchungen, flowmessungen und untersuchungen im . trimenon erfordern eine höhere energiedosis und können bei längerer fokussierung eines bereichs theoretisch eher zur Überwärmung embryonalen gewebes und zu entwicklungsschäden führen. daher wird weiterhin empfohlen, ultraschall nur medizinisch indiziert anzuwenden (bly ) . empfehlung für die praxis: ultraschalluntersuchungen im medizinisch notwendigen umfang sind in der schwangerschaft akzeptabel. video-und einzelbilddarstellungen fürs familienalbum gehören nicht dazu. bei der mrt werden magnetfelder erzeugt, die sich nicht von anderen elektrischen anwendungen einschließlich radiowellen unterscheiden. die magnetfeldstärke wird für die patienten mit , bis tesla (t) und für das untersuchungspersonal mit - mt angegeben. die mrt wird seit rund jahren auch in der schwangerschaft angewendet. mittels mrt wurde z. b. die plazenta lokalisiert, fetale diagnostik betrieben und geprüft, ob die beckenmaße eine vaginale entbindung zulassen (de wilde ). die überwiegend im . und . trimenon gemachten erfahrungen haben bislang keine negativen auswirkungen der dabei erzeugten elektromagnetischen felder und des gerätelärms auf den fetus erbracht (kok b, Übersicht in robert , brent . dies betrifft auch nachuntersuchungen von kindern im alter von bzw. - jahren einschließlich hör-und sehtests (kok , baker . untersuchungen an mrt-personal ergaben keinen anhalt für ein reproduktionstoxisches risiko (gauthier , Übersicht in cragan . vor allem indigokarmin, aber auch evans-blau sind in zahlreichen fällen mit guter verträglichkeit zur markierung bei amniozentese verwendet worden. indigokarmin ist dem serotonin ähnlich, daher ist eine indirekt vasoaktive wirkung nicht auszuschließen. dennoch sind keine dem methylenblau vergleichbaren effekte bei über dokumentierten schwangerschaften beobachtet worden (cragan (halperin ). tierexperimente zeigten ebenfalls keine teratogenen effekte. die substanz wurde nach applikation am auge in der amnionflüssigkeit einer schwangeren nachgewiesen. auch zur retina-angiographie und zur messung des hepatischen blutflusses mit indocyaningrün (mit natriumiodid in icg-pulsion ® ) liegen keine hinweise auf unverträglichkeit für das ungeborene vor. indocyaningrün konnte nicht im nabelvenenblut nachgewiesen werden (fineman ethanol hemmt die ausschüttung der hormone oxytozin und vasopressin aus dem hypophysenhinterlappen. bei gesteigerter wehentätigkeit führt ethanol in hoher dosis ( g ‰) sowohl nach intravenöser als auch nach oraler gabe bei zwei drittel der schwangeren zur wehenhemmung. die pränatale schädigung durch chronischen alkoholkonsum beruht auf der direkten teratogenen wirkung von ethanol und seines abbauproduktes acetaldehyd auf den fetus. das fas und seine varianten treten nur bei chronischer mütterlicher alkoholkrankheit auf, wobei eine korrelation zwischen fas-risiko und fortschreiten der mütterlichen alkohol-krankheit besteht (majewski ) . der eigentliche schädigungsmechanismus ist auch heute trotz intensiver klinischer und tierexperimenteller forschung noch nicht bekannt. die in einigen tierversuchen beobachteten entwicklungstoxischen auswirkungen paternaler alkoholexposition ließen sich beim menschen bisher nicht belegen (passar (day , olson . das so genannte "binge-drinking" oder das "saturday-night drinking", also das gelegentliche heftige trinken ist in der schwangerschaft sicher gefährlicher für das kind als das regelmäßige soziale trinken geringer mengen. so wiesen bailey und mitarbeiter ( ) nach, dass bei alkohol trinkenden schwangeren nicht nur die absolute menge, sondern auch das trinkmuster für die schädigung des ungeborenen kindes ausschlaggebend ist. in einer kontrollierten prospektiven studie fand dosiskorrelierte verhaltensstörungen noch im vorschulalter bei sonst kognitiv nicht beeinträchtigten kindern, deren mütter während der schwangerschaft "binge-drinking" praktizierten. spectrum disorder (fasd) werden heute vor allem in den usa alle formen der kindlichen schädigung durch den chronischen alkoholabusus während der schwangerschaft zusammengefasst: das klassische fetale alkoholsyndrom (fas) als schwerste ausprägung, die fetalen alkohol-effekte (fae) ohne die typische kraniofaziale dysmorphie, die durch alkohol bedingten funktionellen entwicklungsneurologischen störungen (alcohol related neurodevelopmental disorders, zeitpunkt noch an diese diagnose zu denken ist (streissguth , spohr . bis ins erwachsenenalter reichen die folgen der intrauterinen alkoholschädigung mit körperlichem minderwuchs, mentalen entwicklungproblemen und anpassungsstörungen besonders in der arbeitswelt (autti-ramo diese xanthinderivate werden als lipophile substanzen gut aus dem magen-darm-trakt resorbiert, sie passieren die plazenta und können bei stärkerem coffeinkonsum eine vermehrte aktivität des fetus mit zunahme der atembewegungen und veränderungen seiner herzfrequenz einschließlich arrhythmien hervorrufen. nach bisheriger erfahrung sind jedoch keine negativen folgen für das neugeborene und die weitere entwicklung im kindesalter zu erwarten (castellanos ) . im tierversuch führt coffein in extrem hohen dosen ( mg/kg/tag) zu geringfügigen entwicklungsstörungen an den phalangen. in den usa wurde daher mit unterstützung der gesundheitsbehörden, der verbraucherverbände und der kaffee-und cola-produzenten untersucht, ob coffeinhaltige getränke auch bei menschen fehlbildungen hervorrufen können. im gegensatz zu den genannten tierversuchen nehmen erwachsene durchschnittlich nicht mehr als - mg/kg/tag an coffein zu sich. ausführliche epidemiologische studien in verschiedenen ländern erbrachten keine hinweise auf embryotoxische effekte unter diesen bedingungen (christian ) . eine neue prospektive dänische untersuchung fand eine leicht erhöhte totgeburtenrate, wenn die schwangere mehr als tassen kaffee getrunken hatte (wisborg ) . zahlreiche publikationen befassen sich mit einer möglicherweise erhöhten abortrate und intrauteriner wachstumsretardierung bei coffeingenuss (signorello , leviton . eine metaanalyse unter einbeziehung von rund . schwangeren ergab hinweise auf eine leicht erhöhte rate an spontanaborten und wachstumsretardierten kindern (iugr), wenn die mutter mehr als mg coffein pro tag zu sich nahm (fernandes ) . bis heute sind derartige auswirkungen bei durchschnittlichem konsum nicht eindeutig allein dem kaffee zuzuschreiben und von anderen einflüssen wie z. b. rauchen und alkohol zu trennen. in einer untersuchung wurde nur bei männlichen neugeborenen eine reduktion des geburtsgewichts beobachtet (vik ) . der insgesamt schwache zusammenhang zwischen kaffeekonsum und fehlgeburten wurde auch damit begründet, dass schwangerschaftsübelkeit ohnehin mit einem geringeren fehlgeburtsrisko assoziiert ist und gleichzeitig das kaffeetrinken verleidet. barr und mitarbeiter ( ) konnten bei den kindern von schwangeren keinen effekt auf somatische entwicklungsparameter und iq bis zum alter von , jahren finden. eine beeinträchtigung der weiblichen fertilität durch regelmäßigen genuss größerer mengen von coffein wurde ebenfalls diskutiert. . statistisch signifikant waren die jeweiligen konzentrationsunterschiede zwischen kindern von aktiven und passiven raucherinnen sowie von frauen aus nichtraucherhaushalten. fehlbildungen. rauchen ist embryo-und fetotoxisch, birgt aber offenbar kein erhebliches fehlbildungsrisiko. allerdings wird von zahlreichen autoren ein zusammenhang zwischen rauchen während der frühschwangerschaft und lippen-und gaumenspalten diskutiert a & b, zeiger , chung , romitti ), insbesondere bei gleichzeitigem vorliegen eines transforming-growth-factor- § (tgf- § )-polymorphismus als beispiel für das zusammenspiel von genetischen und umweltfaktoren bei der teratogenese. in einer metaanalyse von internationalen publikationen ließ sich nachweisen, dass mütterliches rauchen in der schwangerschaft mit einem erhöhten risiko für nichtsyndromale orofaziale spalten verbunden ist, wobei der effekt konstanter und deutlicher bei lippenspalten mit und ohne gaumenbeteiligung war als bei isolierten gaumenspalten (deacon ) . das risiko für kinder von raucherinnen mit genetischer disposition (siehe oben) wird mit maximal : angegeben gegenüber einer prävalenz in der gesamtbevölkerung von etwa : (chung ) . in anderen untersuchungen werden leicht erhöhte risiken für kraniosynostose , källén ), gastroschisis (martinez-frias ), harnwegsanomalien (li ) , herzfehlbildungen (wasserman ) , extremitätendefekten (källén , wasserman und klumpfuß (skelly ) erörtert, die bislang aber nicht als eindeutig erwiesen gelten. (chanoine ) . morbidität und mortalität in der kindheit sind im zusammenhang mit rauchen schwierig zu beurteilen, weil fast immer sowohl eine pränatale als auch eine postnatale exposition besteht. soweit bekannt, scheint rauchen in der schwangerschaft keine langfristigen auswirkungen auf das postnatale wachstum zu haben. eine untersuchung an neugeborenen, die noch nicht direkt rauch exponiert waren, hat gezeigt, dass kinder von raucherinnen häufiger einschränkungen respiratorischer funktionen aufwiesen. ein kombinierter effekt von prä-und postnataler exposition auf die entstehung von nahrungsmittelallergien in den ersten lebensjahren wurde von einer untersuchergruppe beobachtet (kulig ) . eine weitere publikation betont den prädiktiven wert der konzentration des metaboliten cotinin im mekonium für das risiko frühkindlicher atemwegsinfektionen (nuesslein ) . in einer prospektiven "follow-up"-studie konnte noch im alter von jahren ein erhöhtes risiko für Übergewicht nachgewiesen werden (chen ) . eine vermehrte infektneigung, besonders von otitiden unter den kleinkindern rauchender mütter ist inzwischen allgemein akzeptiert. auch koliken kommen häufiger vor (shenassa ) . im vergleich zu nicht rauchenden müttern führt tabakkonsum während der schwangerschaft zu einem fach höherem risiko, dass die kinder an sids (sudden infants death syndrome) sterben , alm . in einer englischen fallserie mit prospektiv erfassten ecstasy exponierten schwangeren wurden kinder mit entwicklungsanomalien bei insgesamt lebendgeborenen beschrieben. es handelt sich dabei jedoch z. t. um kleine anomalien (z. b. fußdeformitäten), ein typisches muster war nicht zu erkennen. knapp die hälfte der mütter hatte zusätzlich alkohol oder andere drogen in nicht näher bezeichneter menge zu sich genommen (mcelhatton ) . in einer weiteren untersuchung an schwangeren wurde eine doppelt so hohe rate kleiner entwicklungsanomalien im vergleich zu einer nicht exponierten kontrollgruppe beobachtet. hier zeigten sich in der neugeborenenzeit gehäuft neurologische auffälligkeiten einschließlich störungen des muskeltonus und Übererregbarkeit. die spontanabortrate war nicht erhöht, aber es ereigneten sich drei totgeburten in der exponierten gruppe (felix ) . auch in dieser untersuchung wurden neben rauchen und alkohol z. t. noch andere drogen genommen. niedrigeres geburtsgewicht und entzugserscheinungen wurden auch von weiteren untersuchern beschrieben (smith ) . unter bis zum . lebensjahr nachuntersuchten kindern wurden signifikant häufiger lernschwierigkeiten in der schule beobachtet. allerdings betrieb ein großteil der mütter während der schwangerschaft nicht nur amphetaminabusus, sondern konsumierte zusätzlich opiate und alkohol, rauchte mehr als zigaretten täglich und befand sich in einer problematischen psychosozialen lage. nur % der kinder lebten mit jahren noch bei ihren müttern (cernerud ) . empfehlung für die praxis: schwangere sollen amphetamine unter allen umständen meiden. eine dennoch erfolgte exposition rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). nach ausgeprägtem konsum im . trimenon sollte die normale entwicklung des fetus per ultraschallfeindiagnostik bestätigt werden. g . . cannabis pharmakologie und toxikologie. marihuana, die blätter der cannabispflanze (indischer hanf) mit dem harzartigen extrakt haschisch gehört außer alkohol, nikotin und ecstasy zu den häufig in der schwangerschaft konsumierten drogen. beim rauchen sollen im vergleich zu tabak eine fach höhere kohlenmonoxidkonzentration und ein fach höherer teergehalt im blut erreicht werden. delta- -tetrahydrocannabinol (thc), der wichtigste von mehreren wirkstoffen des marihuanas, passiert die plazenta und kann zur abnahme der kindlichen herzfrequenz führen. die fehlbildungsrate ist nach genuss von marihuana in der schwangerschaft nicht höher; aber ein regelmäßiger konsum erhöht möglicherweise die perinatale sterblichkeit. eine metaanalyse ergab keine schlüssigen hinweise auf eine erniedrigung des geburtsgewichts, zumindest bei moderatem, nur gelegentlichem cannabisgenuss (english ) . wie bei anderen drogen sind embryotoxische effekte hier häufig nicht von zusätzlichen einwirkungen wie z. b. zigarettenrauchen zu unterscheiden. es gibt bisher auch keine hinweise, dass die in früheren tierexperimentellen untersuchungen marihuana zugeordneten chromosomenbrüche klinische relevanz besitzen. neugeborene können entzugserscheinungen mit zittrigkeit und unruhe zeigen. die daten zur weiteren entwicklung im kindesalter sind uneinheitlich. eine langzeitstudie fand bei kindern, deren mütter während der schwangerschaft regelmäßig, d. h. mehrfach pro woche bis täglich marihuana konsumiert hatten, im alter von jahren eine signifikant beeinträchtigte sprach-und gedächtnisleistung (fried ) sowie einen signifikant kleineren kopfumfang auch bei älteren kindern, obwohl die geburtsmaße nicht auffällig waren (fried ) . insgesamt werden die abweichungen der kognitiven entwicklung dieser kohorte als subtil beschrieben a) und keine auswirkungen auf das spätere wachstum und die pubertätsentwicklung gesehen (fried b) . eine andere langzeitstudie hat die entwicklung von kindern, deren mütter während der schwangerschaft alkohol oder cannabis konsumiert hatten, im alter von jahren anhand verschiedener tests und der beurteilung durch die lehrer bewertet. hatte die mutter im . oder . trimenon täglich marihuana geraucht, fanden sich häufiger einschränkungen bei den kognitiven leistungen (goldschmidt ) . auch diese autoren bezeichnen die ergebnisse insgesamt als subtil und bewerten die Übertragbarkeit auf andere cannabis exponierte schwangere zurückhaltend. empfehlung für die praxis: schwangere sollen marihuana unter allen umständen meiden. dennoch erfolgter konsum rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). sporadischer genuss begründet auch keine zusätzliche diagnostik. g . . kokain pharmakologie. kokain (koks, schnee) ist das alkaloid benzoylekgoninmethylester des coca-strauches (erythroxylon coca), der hauptsächlich in den anden wächst. die blätter enthalten etwa % kokain. in europa ist die stimulierende droge seit mitte des . jahrhunderts bekannt. wurde kokain als anästhetikum eingeführt. es ist den lokalanästhetika chemisch verwandt und hat sich nur zur äußerlichen anwendung in der augen-und hno-heilkunde durchgesetzt. crack ist die freie base (free base) des kokains und kann geraucht werden. kokain blockiert die wiederaufnahme von noradrenalin und dopamin an der synapse und erhöht auf diese weise die katecholaminkonzentration. dies führt zu einem sympathikomimetischen und zentral stimulierenden effekt. bei oraler aufnahme wird kokain wegen seiner vasokonstriktorischen wirkung und der hydrolytischen spaltung im magen nur langsam resorbiert. in der leber wird es innerhalb von stunden zum unwirksamen hauptmetaboliten benzoylekgonin metabolisiert. etwa % werden unverändert über die niere ausgeschieden. die resorption erfolgt intranasal innerhalb von minuten (verzögerung durch vasokonstriktion). intravenöse applikation oder rauchen von crack führen innerhalb weniger minuten zum wirkungseintritt. kokain findet sich in relativ hoher konzentration in der amnionflüssigkeit und die konzentration fällt aufgrund der geringen clearance nur langsam ab. daher kann der fetus auch über seine bis zur schwangerschaftswoche gut durchlässige haut aus der amnionflüssigkeit kokain aufnehmen (woods ) . toxikologie. in den usa wurde bei bis % aller schwangeren ein kokainkonsum ermittelt (fantel ) . bis anfang der er jahre hielt man kokain für eine pränatal nicht toxische droge. dann wurden zahlreiche entwicklungsstörungen dem wiederholten kokain-oder "crackgenuss" in der schwangerschaft angelastet. sporadischer gebrauch in der frühschwangerschaft bei intakten lebensverhältnissen und ohne weitere schädigende faktoren wie alkohol, andere drogen, infektionen, mangelernährung und traumata scheint nach den bisher vorliegenden erfahrungen das fehlbildungsrisiko nicht nennenswert zu erhöhen. erwiesene folgen des ausgeprägten abusus sind eine erhöhte abortrate, frühgeburten, totgeburten, intrauterine wachstumsverzögerung und mikrozephalie. außerdem wurde über zerebrale infarkte, nekrotisierende enterokolitis beim neugeborenen, fehlbildungen von urogenital-und skelettsystem sowie über intestinale atresien und infarkte berichtet a, hoyme , schaefer , mercado , chasnoff ). das weite spektrum der morphologischen veränderungen kann durch eine vasokonstriktion mit minderdurchblutung der plazenta und in fetalen organen erklärt werden. während der gesamten schwangerschaft kann es infolgedessen zu (fokalen) differenzierungsund wachstumsstörungen kommen. trotz der vielzahl publizierter einzelschädigungen exponierter kinder von kokain abhängigen müttern ließ sich bis heute kein typisches kokain-syndrom definieren, wie z. b. das "coke-baby" mit charakteristischen persistierenden morphologischen und psychomentalen folgen (little ) . kokain und "crack" rufen bei schwangeren stärkere herz-kreislaufund neurologische wirkungen hervor als bei nichtschwangeren. es wird diskutiert, ob die schädigung des embryos nach minderperfusion eine direkte folge des sauerstoffmangels ist oder eher durch hochreaktive, toxische sauerstoffradikale nach reperfusion des ischämischen gewebes verursacht wird, denn im . trimenon verfügt die fetoplazentare einheit noch nicht über genügend schützende antioxidantien. postnale entwicklung. die akuten symptome beim neugeborenen sind weniger ausgeprägt als nach einem heroinentzug: schlafstörungen, tremor, trinkschwäche, erbrechen, schrilles schreien, niesen, tachypnoe, weiche stühle und fieber. darüber hinaus wurden in verschiedenen studien auffälligkeiten in neurologischen tests bei neugeborenen sowie spätere verhaltensabweichungen, entwicklungsstörungen, eeg-veränderungen und vereinzelt plötzlicher säuglingstod beobachtet (eyler b) . die auffälligkeiten in der neonatalzeit sind in der regel nach einem jahr nicht mehr nachweisbar. zwar finden sich in sorgfältigen prospektiven studien diskrete physiologische und entwicklungsneurologische effekte, ihr ausmaß auf die kindliche entwicklung ist jedoch nicht ausreichend zu bewerten (schiller ) . bandstra und mitarbeiter ( fanden in einer prospektiven longitudinalstudie an kindern bis zum . lebensjahr, dass auch schwere kokain-exposition während der schwangerschaft nicht eindeutig als unabhängiger risikofaktor für die mentale und psychomotorische entwicklung sowie für die entwicklung des verhaltens der kinder nachzuweisen war. zu einem ähnlichen ergebnis kommen beeghly und mitarbeiter ( ) in einer untersuchung zur sprachentwicklung im alter von und , jahren. eine literaturanalyse der von - publizierten arbeiten ermittelt in prospektiven studien zu kindern bis zum . lebensjahr keinen überzeugenden beweis dafür, dass pränatale kokainexposition mit einer entwicklungsschädigenden störung assoziiert ist. viele der symptome, die für kokainspezifisch gehalten wurden, waren eher mit anderen pränatalen faktoren korreliert, wie tabak, marihuana, alkohol und der qualität der kindlichen lebensbedingungen (frank ) . richardson ( ) und messinger ( stellten fest, dass frauen, die während der schwangerschaft kokain einnahmen, eher an stress und ernährungsstörungen litten, öfter alleinstehend waren und dazu neigten, zusätzlich marihuana, tabak, alkohol und tabletten zu konsumieren. zusammengefasst muss man davon ausgehen, dass kokainkonsum eine hochrisikoschwangerschaft bedingt. die beim kind beobachteten mentalen und motorischen defizite sind nicht nur direkte folge dieser "chemischen" exposition sondern assoziiert mit den anderen o.g. risikofaktoren und dem häufig resultierenden niedrigen geburtsgewicht. empfehlung für die praxis: da kokain potenziell entwicklungstoxisch ist, darf es während der gesamten schwangerschaft nicht konsumiert werden. kokainkonsum rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). bei wiederholter anwendung, vor allem unter problematischen lebensbedingungen, sollte durch eine ultraschallfeindiagnostik die normale entwicklung des fetus überprüft werden. g . . lsd pharmakologie und toxikologie. in älteren arbeiten wurde der verdacht geäußert, das halluzinogen lsd (lysergsäurediethylamid) könne fehlbildungen an augen, gehirn und skelett verursachen (Übersicht in . auch über chromosomenbrüche wurde berichtet. die im wesentlichen in einzelfallberichten beschriebenen klinischen auffälligkeiten können nicht als erwiesen angesehen werden. allerdings sind die vorliegenden daten für eine differenzierte risikobewertung unzureichend. empfehlung für die praxis: schwangere sollen lsd unter allen umständen meiden. ein dennoch erfolgter konsum rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . bei den neugeborenen können die schweren, meist - stunden nach geburt auftretenden entzugssymptome mit atemnotsyndrom, hyperirritabilität, tremor, diarrhö, erbrechen, störungen des schlaf-wach-rhythmus und z. t. therapierefraktäre zerebrale krampfanfälle ohne behandlung zum tod führen. bei % der kinder treten symptome bis hin zu zerebralen krampfanfällen erst verzögert nach bis wochen auf. das risiko für lebensbedrohliche entzugssymptome ist besonders hoch, wenn die abhängigkeit der mutter nicht bekannt ist und ein zuverlässiges monitoring sowie die rechtzeitige medikamentöse prophylaxe mit opiaten (morphin) nicht eingeleitet werden. nach erfolgreicher therapie der entzugssymptome sind bleibende defekte offenbar nicht zu erwarten. jedoch scheint der plötzliche kindstod (sids) bei pränatal opiat exponierten kindern häufiger aufzutreten als in kontrollgruppen nicht exponierter kinder. ein akuter opiatentzug während der schwangerschaft kann fruchttod und vorzeitige wehen auslösen. gute erfolge wurden mit der umstellung der schwangeren auf die ersatzdrogen methadon bzw. levomethadon (l-polamidon ® ) (halbwertszeit - stunden) erzielt. neonatale atemdepression und entzugssymptome treten auch unter methadon und anderen ersatzdrogen auf. es gibt hinweise dafür, dass die entzugssymptomatik nach methadon sogar schwerer und länger verläuft als nach intrauteriner heroin-exposition. am wirksamsten und verträglichsten wird der säugling mit einer oralen opiatzufuhr therapiert (arlettaz , siddappa . obwohl es plausibel erscheint, dass die entzugserscheinungen mit der mütterlichen dosis am ende der schwangerschaft korrelieren, konnten berghella und mitarbeiter ( ) keine signifikanten unterschiede zwischen methadon substituierten schwangeren mit täglich , oder mg erkennen. zu den in den letzten jahren vermehrt diskutierten alternativen ersatzdrogen gehört buprenorphin (halbwertszeit - stunden). nach den erfahrungen an über schwangeren scheint die entzugssymptomatik im vergleich zum methadon milder zu verlaufen (Übersicht bei , kayemba-kay's , schindler . als ursache dafür wird ein geringerer plazentarer transfer diskutiert (nanovskaya , rohrmeister . andere therapiekonzepte bei heroin-abhängigen schwangeren betreffen naltrexon-implantate (hulse ) . anders als bei alkoholgeschädigten kindern ist die neurologische und kognitive entwicklung offenbar stärker durch das soziale umfeld in den ersten lebensjahren beeinflusst als durch den umfang der pränatalen opiat-exposition. intakte familienverhältnisse z. b. durch adoption nach der geburt erlauben offenbar eine weitgehend normale intellektuelle entwicklung der kinder (ornoy , coles . nur aufmerksamkeitsdefizit und hyperaktivität (adhd) traten auch bei adoptierten kindern noch häufiger auf als bei unbelasteten kontrollkindern, allerdings mit % deutlich seltener als bei den im drogenumfeld verbliebenen kindern ( %). dies ergab eine nachuntersuchung von -bis -jährigen kindern (ornoy ) . empfehlung für die praxis: akuter opiatentzug ist während der schwangerschaft zu vermeiden. bei heroinabhängigkeit ist eine einstellung auf methadon oder buprenorphin zu empfehlen. die substitution erfordert eine genaue dosistitrierung und sollte nur von erfahrenen Ärzten vorgenommen werden. die tägliche methadon-oder buprenorphindosis muss sich am vorangegangenen drogenkonsum und an der stärke der entzugssymptome orientieren. zusätzlicher drogenkonsum kann durch screening im urin nachgewiesen werden. durch umfangreiche soziale hilfestellung muss versucht werden, die beschaffungskriminalität zu beenden. in aussichtslosen fällen ist rechtzeitig auf eine adoption bzw. auf eine pflegefamilie hinzuarbeiten (siehe oben). neugeborene müssen ggf. über mehrere wochen beobachtet werden, damit auch verzögert auftretende schwere entzugserscheinungen mit opiaten behandelt werden können. in einzelnen fällen wurden im zusammenhang mit phencyclidinabusus mikrozephalie, gesichtsasymmetrie und ein komplexes intraund extrakraniales fehlbildungssyndrom beschrieben, ohne dass sich bisher eine kausale beziehung belegen ließ. intrauterine wachstumsretardierung und postnatale interaktionsdefizite sowie andere neurologische abweichungen wurden ebenso beobachtet wie opiattypische entzugserscheinungen. nachuntersuchungen an kindern im alter von einem jahr erbrachten keine auffälligkeiten gegenüber einer kontrollgruppe (wachsmann ). tierexperimentell wurde eine degeneration fetaler kortexneurone beschrieben (Übersicht in . empfehlung für die praxis: schwangere sollen phencyclidin unter allen umständen meiden. ein dennoch erfolgter gebrauch rechtfertigt keinen risikobegründeten schwangerschaftsabbruch (siehe kapitel . ). eine ultraschallfeindiagnostik sollte die normale entwicklung des fetus bestätigen. g . . psilocybin pharmakologie und toxikologie. psilocybin ist ein halluzinogen aus pilzen ("magic mushrooms"). es gibt keine ausreichenden erfahrungen in der schwangerschaft, die eine differenzierte risikobewertung erlauben. auf der anderen seite wurden bisher keine reproduzierbaren anomalien im zusammenhang mit psilocybin-einnahme beschrieben. lungsparameter war der mit , % erhöhte anteil geistig retardierter kinder der einzig signifikante befund, der aber aufgrund der kleinen fallzahl nicht verallgemeinert werden sollte. in einer späteren, deutlich erweiterten untersuchung desselben autors findet sich ebenfalls keine signifikant erhöhte fehlbildungsrate. das gilt auch für die schwangeren, die zwischen woche und hohe medikamentendosen in suizidaler absicht eingenommen hatten . in einer dänischen publikation zu schwangeren fand man zwar eine verdoppelte rate an spontanaborten aber kein erhöhtes fehlbildungsrisiko und keine zunahme der frühgeburten (flint ). häufig wird die frage gestellt, wie eine schwangere nach suizidversuch behandelt werden sollte. dabei spielen sowohl die sorge um eine spezifische embryotoxizität der eingenommenen noxe eine rolle als auch die unbedenklichkeit der indizierten antidotbehandlung. bisherige erfahrungen belegen, dass eine gefährdung des fetus primär von der noxe und nicht von der antidot-behandlung ausgeht. dies wurde z. b. bei methanol-intoxikation ebenso beobachtet wie bei Überdosen von paracetamol und eisenpräparaten. andererseits gibt es praktisch zu keinem antidot epidemiologische studien im . trimenon, die die verträglichkeit der therapie für den embryo belegen. die vorliegenden fallberichte und fallserien geben bislang jedoch keinerlei hinweise auf teratogenität oder fetotoxizität, abgesehen vom chelatbildner penicillamin, der ethanoltherapie und methylenblau (nach injektion in die amnionhöhle). zum chelatbildner dimercaprol ( , -dimercaptopropanol, synonym: british anti-lewisite = bal) liegen mehrere fallberichte zur anwendung bei arsen-und bleivergiftung vor, die keine hinweise für ein embryotoxisches risiko geben (bailey ) . der chemisch verwandte, in der bundesrepublik deutschland zugelassene chelatbildner , -dimercapto- -propansulfonsäure (dmps; dimaval ® ) ist analog zu bewerten. allerdings ist bei jeder länger dauernden chelattherapie zu bedenken, dass auch essentielle nahrungsbestandteile, wie z. b. zink eliminiert werden und sich daraus potenziell riskante mangelzustände für den fetus ergeben können. umfangreichere erfahrungen gibt es zu antidotsubstanzen, die vor allem bei anderen indikationen eingesetzt werden (z. b. atropin, pyridoxin) . bei der auch in der schwangerschaft häufiger beschriebenen paracetamolvergiftung in suizidaler absicht besteht das risiko der mütterlichen und fetalen leberschädigung. die therapie mit dem antidot acetylcystein (fluimucil antidot ® ) richtet sich wie bei nichtschwangeren nach der eingenommenen menge paracetamol oder der paracetamol-konzentration im serum der mutter (mcelhatton ) . acetylcystein überwindet die plazenta und ist auch beim fetus als antidot wirksam (horowitz ) . auch bei eisenvergiftungen in suizidaler absicht würde das unterlassen einer antidottherapie mit deferoxamin (desferal ® ) mutter und fetus gefährden (mcelhatton , olenmark . empfehlung für die praxis: grundsätzlich muss jede schwangere mit einer intoxikation so behandelt werden wie eine nichtschwangere, d. h. alle therapeutischen maßnahmen, die aus klinisch toxikologischer sicht indiziert sind, sollten anwendung finden. allerdings sollte die entgiftungsbehandlung aktuellen richtlinien folgen. die in den folgenden kasuistiken beschriebenen therapiemaßnahmen sind nicht immer aktuell, da es sich z. t. um "historische" berichte handelt. aufgrund neuer erkenntnisse haben sich die therapieempfehlungen bei vergiftungen in den vergangenen jahren teilweise grundlegend geändert. da es den rahmen dieses buches sprengen würde, darauf einzugehen, sollten im bedarfsfall kompetente giftinformationszentren gefragt oder fachbücher (z. b. mühlendahl ) konsultiert werden. mehrere fallberichte beschreiben arsenvergiftungen bei schwangeren nach dem . trimenon. in den meisten fällen waren die neugeborenen gesund, sogar bei intoxikationsbedingten enzephalopathien der mutter. es wurden jedoch auch letale verläufe berichtet und frühgeburten mit kurz darauf verstorbenem neugeborenen (bollinger , daya , lugo , kantor . co überwindet die plazenta und kann im fetalen blut zu vergleichbaren konzentrationen wie im mütterlichen führen. empirische beobachtungen, tierexperimentelle ergebnisse und theoretische berechnungsmodelle zeigen, dass im fetus mit einer mehrstündigen verzögerung sowohl beim anfluten als auch beim abbau des co zu rechnen ist. erst nach etwa - stunden wird ein gleichgewicht erreicht, die eliminationshalbwertszeit ist beim fetus -bis -mal länger als bei der mutter (Übersicht in barlow und sullivan ) . zns-schäden beim fetus werden insbesondere dann beschrieben, wenn die mutter bewusstseinseingeschränkt war bzw. eine grad- oder - -symptomatik aufwies, auch dann, wenn sie sich rasch wieder erholte. zu den möglichen späteren klinischen auffälligkeiten beim kind zählen mentale und motorische entwicklungsretardierungen, aber auch schwere zerebralparetische schädigungen. der reife fetus reagiert empfindlicher auf die co-intoxikation als der embryo während der organogenese. eine geringgradige akute exposition der mutter mit vorübergehenden, leichten symptomen wie kopfschmerzen und Übelkeit (entsprechend grad - ) oder die chronische co-exposition, z. b. im rahmen der beim rauchen üblichen belastungen ( packung zigaretten/tag oder bis etwa ppm raum-bzw. stadtluft aufgrund gewerblicheroder umwelt-exposition), resultiert in mütterlichen cohb-konzentrationen von - % und ist offenbar nicht mit fetalen schäden assoziiert , Übersicht in barlow . der fetus einer raucherin toleriert eine zusätzliche co-exposition keineswegs besser, weil er bereits daran gewöhnt ist, denn seine kompensationsfähigkeit ist möglicherweise schon ausgeschöpft. seit über jahren (maresch ) gibt es berichte über co-vergiftungen in der schwangerschaft, die sowohl unauffällige verläufe, als auch fruchttod und zns-defekte beschreiben (aubard , kopelman . abgesehen von den zns-schäden sind teratogene, also fehlbildungsauslösende wirkungen des co unwahrscheinlich. bedenken zur fetalen verträglichkeit der therapie der co-vergiftung mit der hyperbaren oxygenierung wegen möglicher retinaschädigung oder vorzeitigem verschluss des ductus arteriosus wurden geäußert, aber nicht bestätigt (silverman ) . auf jeden fall ist eine unterbehandelte schwere co-intoxikation das größere fetotoxische risiko. empfehlung für die praxis: aufgrund der stark verzögerten kinetik des co im fetalen organismus und dem daraus resultierenden erhöhten risiko hypoxischer zns-schädigung beim kind muss die indikation zur hyperbaren oxygenierung bei schwangeren mit co-bedingten bewusstseinseinschränkungen großzügig gestellt werden. die therapie sollte länger durchgeführt werden, als es symptome und co-konzentrationsverlauf bei der mutter nahe legen. jede schwangere mit bewusstseinseinschränkung durch co, mit einer über % liegenden cohb-konzentration oder mit abweichungen der fetalen herzfrequenz (dezelerationen, tachykardie, silente herzfrequenz) muss so rasch wie möglich hyperbar behandelt werden und bis zum beginn der therapie % sauerstoff erhalten. da co den fetus stark verzögert erreicht und nur sehr langsam wieder abgebaut wird, ist auch ein um viele stunden verzögerter behandlungsbeginn bei bereits einsetzender spontanbesserung mütterlicher symptome noch sinnvoll und indiziert! eine methanol-vergiftung in der schwangerschaft kann den fetus bei länger bestehender azidose sekundär schädigen. obwohl methanol plazentagängig ist, scheint der fetus zunächst durch seine langsamere verstoffwechselung des methanols zu dessen toxischen metaboliten wie formaldehyd relativ geschützt zu sein. die klassische therapie mit ethanol i.v. exponiert natürlich auch den fetus mit alkohol und ist aufgrund der nicht auszuschließenden neurologischen folgen, die vom "binge-drinking" und von der tokolyse mit alkohol bekannt sind, nicht als völlig unbedenklich zu bewerten. daher wird neuerdings auch fomepizol als alternatives antidot vorgeschlagen (velez ) . auf jeden fall darf weder bei methanol noch bei ethylenglykol eine (alkohol-)therapie aus falscher rücksicht auf den embryo unterbleiben (tenenbein ) . ein fallbericht mit methanol-intoxikation in der spätschwangerschaft beschreibt ein gesundes neugeborenes nach behandlung der mutter mit ethanol, hämodialyse und alkalisierung (hantson ) . in einem weiteren fall verstarben die mutter und das per sectio in woche entbundene kind einige tage nach der geburt. bei der azidotischen mutter (ph , ) wurde nach stunden eine alkohol-therapie begonnen und erst am . tag mit fomepizol behandelt. im blut des azidotischen neugeborenen (ph , ) fanden sich mit , mg/dl methanol ähnliche konzentrationen wie bei der mutter (belson ). einige fallberichte schildern akzidentelle und suizidale Überdosierungen mit unterschiedlichem ausgang. eine mutter in schwangerschaftswoche berichtete, zwei stunden nach aufnahme von chlorpyrifos in suizidaler absicht keine kindsbewegungen mehr gespürt zu haben. nach anfänglicher magenspülung wurde erst stunden später eine intensivtherapie begonnen. inzwischen war der fetus verstorben. außer niedrigen mütterlichen pseudocholinesterase-spiegeln fanden sich hohe konzentrationen an chlorpyrifos im fetalen blut. einige weitere fälle einer organophosphat-intoxikation bei schwangeren endeten mit der geburt gesunder kinder. in diesen fällen erfolgte eine rasche therapie, u. a. mit atropin und pralidoxim (kamha ). eines dieser kinder entwickelte sich bis zum alter von jahren unauffällig. in berichten über schwangere, die in suizidaler absicht größere mengen des herbizids paraquat eingenommen hatten, wurde geschildert, dass kein fetus und nur zwei mütter die intoxikation überlebten. die paraquat-konzentrationen waren im fetus höher als im mütterlichen serum (talbot ) . ein weiterer fallbericht beschreibt die einnahme von - ml paraquat in suizidaler absicht in schwangerschaftswoche . die mutter wurde erfolgreich u. a. mit hämodialyse behandelt. die schwangerschaft schien sich unbeeinträchtigt weiter zu entwickeln, wurde aber in woche abgebrochen. im embryonalen gewebe fanden sich , ? g/g und in der amnionflüssigkeit , ? g/ml paraquat. die mütterlichen serumwerte sollen zu diesem zeitpunkt deutlich darunter gelegen haben (initial waren es , ? g/ml). die autoren diskutieren einen größeren schutz des embryos gegenüber paraquat im vergleich zum reifen fetus. sie weisen darauf hin, dass insbesondere bei intoxikationen in der späteren schwangerschaft der dann ohnehin stärker gefährdete fetus ein für die mutter riskantes reservoir für rückflutendes paraquat darstelle und unter diesem aspekt ein schwangerschaftsabbruch erörtert werden müsse (tsatsakis ) . eine ausnahme ist der bericht über die geburt eines reifen, gesunden und sich bis zum alter von jahren normal entwickelten mädchens, dessen mutter in schwangerschaftswoche eine Überdosis paraquat zu sich nahm und anschließend mit kohle-hämoperfusion, hochdosis-cyclophosphamid und methylprednisolon behandelt wurde (jenq ) . Über rund fälle von thalliumingestion in suizidaler absicht oder zur provokation eines aborts wird berichtet, sowie kürzlich über einen fall mit chronischer intoxikation durch ein thalliumhaltiges rodentizid am arbeitsplatz. die meisten kinder überlebten die mütterliche vergiftung bei adäquater therapie der mutter. außer alopezie scheinen frühgeburt und intrauterine wachstumsretardierung, nicht aber fehlbildungen mögliche folgen einer pränatalen exposition -auch im . trimenon -zu sein (hoffmann ) . vereinzelt gibt es berichte zur wasserintoxikation unter der geburt, z. b. den fall eines stunden alten neugeborenen, das durch krämpfe und eine hyponatriämie mit mmol/l auffiel (mutter: mmol/l). in diesem fall hatte die mutter wenige stunden vor der geburt liter wasser getrunken. die weitere entwicklung des kindes war unauffällig (mcelhatton ) . die in manchen fällen bei der mutter gemessenen ass-spiegel lagen über denen, die im tierversuch bereits teratogene schäden induzieren. entwickelte die mutter keine schweren toxischen symptome, so traten weder fetale blutungen noch spontanaborte oder intrauteriner fruchttod auf. diese befunde stehen im gegensatz zu der in anderen studien beobachteten zunahme der spontanabortrate nach therapeutischer anwendung von nichtsteroidalen antiphlogistika (nsaid) wie ass, ibuprofen etc. , nielsen . palatnick ( ) eine neonatale bromid-intoxikation mit hypotonie und späterer normaler entwicklung nach einnahme einer hohen dosis durch die mutter am ende der schwangerschaft belegt die anreicherung dieser substanz im fetus (pleasure ). eine carbamazepin-intoxikation in schwangerschaftswoche in suizidaler absicht führte zum koma der patientin und wurde mit kohle und plasmapherese behandelt. hinweise auf eine beeinträchtigung des fetus zeigten sich nach der geburt nicht, apgar und nabelarterien-ph waren normal (saygan-karamursel ) . in schwangerschaftswoche nahm eine frau mg/kg colchicin ein. das kind wurde stunden später per sectio geboren, war gesund und wies nur einen sehr niedrigen colchicinspiegel im serum auf ( x ng/ ml). trotz intensivmedizinischer maßnahmen verstarb die mutter (blache ). fallsammlungen zu diazepam-intoxikationen haben bisher kein spezifisches entwicklungstoxisches risiko erkennen lassen (cerqueira ) . ein fallbericht beschreibt eine schwangere in woche , die ca. mg eines benzodiazepins, wahrscheinlich diazepam eingenommen hatte. in ihrem serum fanden sich ? g/l des benzodiazepins, im urin ? g/l. in der kinetokardiotokographie etwa stunden nach ingestion sah man erwartungsgemäß phasen silenter bis eingeschränkt undulatorischer oszillation der fetalen herzfrequenz. darüber hinaus fanden sich unmittelbar nach klinikaufnahme dezelerationen, die nicht mit uteruskontraktionen einhergingen, sondern mit phasen gesteigerter kindsbewegungen. die basalfrequenz war dabei nicht besonders auffällig. nach etwa stunden hatte sich dies, als normalisierung gedeutet, wieder umgekehrt, d. h. es folgten akzelerationen auf die kindsbewegungen. dieses von der lage der schwangeren unabhängige phänomen wurde als passagere hypoxämie infolge der intoxikation gedeutet (heinrich ) . ein fallbericht beschreibt eine digitalis-intoxikation mit , mg digitoxin im . schwangerschaftsmonat. nach spontangeburt in woche verstarb das kind am . lebenstag. beidseits fanden sich hämorrhagische infarkte der nieren und degenerative neuronale veränderungen im zns, die als hypoxische folge der anhaltenden bradykardie gewertet wurden (sherman ). es gibt mehrere publikationen zur eisen-Überdosierung in der schwangerschaft (tran , mcelhatton , la-coste , dugdale . in einer fallserie wurden schwangere mit Überdosis nachverfolgt. sechs waren im . trimenon exponiert, im . und im . insgesamt gab es neugeborene ohne fehlbildungen, davon waren frühgeborene, eines hatte einen angeborenen genitalherpes und ein anderes -nach mütterlicher eisen-intoxikation in schwangerschaftswoche / -einen ausgeprägten neugeborenenikterus. fünf kinder wiesen unterschiedliche fehlbildungen auf, alle waren im . oder . trimenon exponiert. zwei spontanaborte in woche und wurden beobachtet, einer nach unmittelbar vorangehender vergiftung, ein anderer nach einem abdominaltrauma. fünf schwangerschaften wurden abgebrochen. serumeisenspiegel wurden in fällen ermittelt, davon lagen im mittleren toxischen bereich ( - ? mol/l) und im hochtoxischen ( g ? mol/l) (mcelhatton ) . eine chelattherapie mit intravenös verabreichtem deferoxamin ist indiziert, wenn der serumeisenspiegel über ? mol/l liegt, oder wenn eine Überdosis anzunehmen ist und die schwangere krampft, bewusstlos oder im schock ist. in diesen fällen ist keine serumeisenbestimmung abzuwarten. in der o.g. fallserie erhielten frauen deferoxamin und eine andere entgiftungsbehandlung (ipecac , magenspülung , aktivkohle , bikarbonat ). alle mütter überlebten. es wurden keine toxischen effekte durch deferoxamin beobachtet. Ähnliche ergebnisse wurden von anderen autoren beschrieben (khoury , turk . empfehlung für die praxis: ein erhebliches risiko ist für den fetus nicht gegeben, wenn die mutter wie eine nichtschwangere nach einer eisenintoxikation adäquat behandelt wird. allerdings sind aufgrund der geringen fallzahlen zum . trimenon keine abschließenden aussagen zur teratogenität zu machen. ein schwangerschaftsabbruch aus furcht vor einer schädigung der frucht ist nicht gerechtfertigt. nach einer Überdosis von mg haloperidol in schwangerschaftswoche wurden für einige tage verminderte kindsbewegungen im ultraschall beobachtet. das in woche geborene kind entwickelte sich bis zum . lebensmonat normal (hansen beim erwachsenen wird paracetamol zu einem aktiven metaboliten verstoffwechselt, der in hohen konzentrationen hepatotoxisch wirkt und nur begrenzt durch konjugation mit glutathion entgiftet werden kann. diese konjugationsleistung scheint der fetus mit fortschreitender schwangerschaft besser zu bewältigen. die metabolisierung des paracetamols erfolgt in der fetalen leber -mal langsamer als in der des erwachsenen. hierdurch bildet der fetus weniger toxische metabolite und ist dadurch relativ geschützt. vom teratologischen beratungszentrum newcastle in großbritannien wurden schwangere mit paracetamol-Überdosis erfasst und nachverfolgt (mcelhatton ) , davon mit kombinationspräparaten, die zusätzlich dextropropoxyphen enthielten. in fällen erfolgte die einnahme im . trimenon. insgesamt kinder wiesen verschiedenartige fehlbildungen auf, die nicht für eine kausalbeziehung zwischen einnahme und auffälligkeit sprachen, zumal die exposition jenseits des . trimenons lag. die spontanabortrate war mit - % nicht erhöht. keines der neugeborenen oder der untersuchten abortierten feten wies zeichen einer leber-oder nierenschädigung auf. dies trifft auch auf ein kind zu, dessen mutter in schwangerschaftswoche / zweimal so hohe paracetamoldosen eingenommen hatte, dass eine lebertransplantation erwogen wurde (rosevaer ) . soweit daten zu acetylcystein als antidot vorliegen, deuten diese nicht auf eine spezifische entwicklungstoxische eigenschaft hin. empfehlung für die praxis: wie auch außerhalb einer schwangerschaft muss in abhängigkeit von der serumkonzentration des paracetamols unverzüglich mit einer antidottherapie begonnen werden, und zwar im interesse von mutter und fetus. ein aufschieben dieser therapie hat in einzelnen fällen zum absterben des fetus bzw. zum tod der mutter geführt. andererseits gibt es keine hinweise auf fetotoxizität, wenn toxische symptome bei der mutter ausbleiben oder toxische serumspiegel nicht erreicht werden. daher ist in den weitaus meisten fällen einer paracetamol-Überdosis ein schwangerschaftsabbruch aus furcht vor einer schädigung der frucht nicht gerechtfertigt. podophyllotoxin, in hoher dosis äußerlich aufgetragen, hat bei einzelnen schwangeren zu psychiatrischer symptomatik geführt, zu einem mütterlichen todesfall, einem intrauterinen fruchttod (stoudemire , slater , montaldi , chamberlaine , ward ) und einer fehlbildung mit beteiligung von extremitäten, herz und ohr nach exposition zwischen schwangerschaftswoche und ). g . Über etwa fälle von schlangenbissen bei schwangeren wird in der literatur berichtet, nur in einem teil davon wird der verlauf detailliert beschrieben (sebe b, langley , nasu , dao , pantanowitz . außerdem gibt es einige wenige kasuistiken zu spinnenbissen (pantanowitz ) . genaueres zur wirksamkeit der verschiedenen speziesabhängigen neurotoxine, zytotoxine und hämatotoxine auf den fetus ist nicht bekannt. berichtet wird z. b. über vier frauen in sri lanka, von denen in schwangerschaftswoche bis je zwei von kobras und vipern gebissen wurden (james ) . drei der frauen zeigten keine vergiftungssymptome, sie bemerkten jedoch übereinstimmend eine starke abnahme der kindsbewegungen. auch die fetale herzfrequenz sank. nach gabe spezifischer antiseren normalisierten sich kindsbewegungen und herzfrequenz innerhalb von stunden. diese drei mütter brachten termingerecht gesunde kinder zur welt. die vierte schwangere bemerkte ebenfalls innerhalb der ersten stunden eine verlangsamung der kindsbewegungen, sie wurde jedoch erst mit antiserum behandelt, nachdem sich ein schweres vergiftungsbild mit hämolyse und nierenversagen entwickelt hatte. kurz darauf kam es zu einer totgeburt. die von den schwangeren übereinstimmend beobachtete verminderung der kindsbewegungen zeigt, dass schlangengift den fetus anscheinend schon bei niedrigen dosen erreicht, selbst wenn . zur umwelt gehören die belebte und unbelebte umgebung des menschen. umwelteinflüsse auf die schwangere umfassen fremdstoffe in der atemluft, in der nahrung und auf der haut, im häuslichen umfeld oder am arbeitsplatz. zu den umwelteinflüssen gehören ferner physikalische einwirkungen, wie ionisierende strahlung, elektromagnetische felder, lärm, ernährungs-, freizeit-und arbeitsgewohnheiten. da die wohnung formal betrachtet in deutschland zur umwelt gehört, werden belastungen durch schadstoffe in raumluft durch die umweltgesetzgebung geregelt. g . . schadstoffe im umfeld der schwangeren für schadstoffe in der umwelt und chemische substanzen am arbeitsplatz sind auswirkungen auf die vorgeburtliche entwicklung viel schlechter untersucht als für arzneimittel. eine unterscheidung zwischen umwelt-und arbeitsplatzschadstoffen ist nicht sinnvoll, da viele umweltschadstoffe aus industriellen prozessen freigesetzt werden. bei der risikobewertung von umweltchemikalien kann deshalb zunächst auf die informationen über gesundheitliche risiken von industriechemikalien zurückgegriffen werden. diese sind nach dem arbeitsschutzgesetz vorgeschrieben und werden im so genannten "sicherheitsdatenblatt" zusammengefasst. toxikologische informationen beruhen überwiegend auf ergebnissen von tierexperimenten, nur in einigen fällen liegen zusätzlich ergebnisse von retrospektiven, epidemiologischen studien vor, bei denen meistens aussagefähige daten zum ausmaß der exposition fehlen (Übersicht in , spielmann . da nach der gesetzlichen regelung für industriechemikalien nur bei einem begründeten verdacht reproduktionstoxikologische tierexperimente durchgeführt werden müssen, fehlen nach schätzungen des bundesinstitutes für risikobewertung (bfr) diese daten bei % der ca. . industriechemikalien, die in der eu in einer menge von jährlich mehr als tonne produziert werden (höfer ) . grundsätzlich ist es beruhigend, dass mit den verfügbaren epidemiologischen methoden kein erhöhtes fehlbildungsrisikos bei der in mitteleuropa durchschnittlich vorkommenden exposition mit chemikalien am arbeitsplatz und in der umwelt feststellbar ist. kritischer zu sehen sind allerdings industriegebiete, in denen beispielsweise schwermetalle, persistierende organochlorverbindungen oder organische lösungsmittel verarbeitet werden mit potenziellen auswirkungen auf die fertilität, die abortrate sowie die entwicklung der hirnleistung und des immunsystems, symptome, die teilweise erst im kindes-oder erwachsenenalter nachweisbar sind. in einzelfällen wurde bei vergiftungen der mutter mit solchen stoffen auch das ungeborene kind schwer geschädigt. deshalb sind der sorglose umgang mit schadstoffen und die unkritische weiterbeschäftigung einer schwangeren an potentiell belasteten arbeitsplätzen weder aus medizinischer noch aus arbeitsrechtlicher sicht akzeptabel. in deutschland werden die maximalen arbeitsplatzkonzentrationen (mak-werte) für industriechemikalien von der mak-werte-kommission der deutschen forschungsgemeinschaft (dfg) geregelt (dfg ) . seit gibt die mak-werte-kommission auch empfehlungen für den umgang mit industriechemikalien in der schwangerschaft am arbeitsplatz, die kontinuierlich überarbeitet werden, und die wir auch als grundlage der bewertung ansehen. in diesem kapitel werden einleitend die wichtigsten schadstoffe mit ihren wirkungen in der gravidität vorgestellt und anschließend werden die mak-werte in der schwangerschaft, soweit sie vergeben wurden, diskutiert. g . . quecksilber (siehe auch abschnitt . . ) im gegensatz zu den in der bundesrepublik deutschland durchschnittlich gemessenen konzentrationen von x ? g/l quecksilber (hg) im blut, finden sich in schweden und japan aufgrund häufigeren verzehrs belasteter meerestiere höhere durchschnittswerte, bei den inuit (eskimos) wurden sogar über ? g/l hg im mütterlichen blut und über ? g/l im nabelschnurblut gemessen (bjerregard und hansen ) . anorganisches hg kann die plazenta kaum überwinden, reichert sich aber in ihr an. hingegen gelangt organisches hg fast ungehindert durch die plazenta. hg findet sich vorwiegend in gehirn, leber und niere. die höhe der hg-konzentration in den organen des fetus und neugeborenen korreliert mit der zahl der amalgam-füllungen der mutter (schiele ) . eine neuere untersuchung zum hg-gehalt im haar von neugeborenen und ihren müttern findet zwar erhöhte konzentrationen bei amalgam-füllungen, jedoch keine unterschiede zwischen frauen mit alten füllungen und frauen, die während der schwangerschaft neue amalgam-füllungen erhielten . schwere vorgeburtliche schädigungen durch organisches hg wurden in den er jahren in minamata, japan beobachtet. stark hg-haltige industrieabwässer führten nach methylierung des hg durch bakterien zur anreicherung in fischen. mütter, die vor und besonders während der schwangerschaft hochgradig belastete fische verzehrt hatten, bemerkten meist nur leichte parästhesien. nach überwiegend normalem schwangerschaftsverlauf waren die kinder bei der geburt unauffällig, aber im alter von etwa sechs monaten entwickelten sie zeichen einer beginnenden, z. t. letal verlaufenden zerebralparese. neben diesen symptomen der fetotoxischen eine so genannte "amalgam-entgiftung" mit chelatbildnern (siehe kapitel . ) ist in der schwangerschaft zu unterlassen, weil amalgam-plomben keine quecksilber-vergiftung verursachen. außerdem ist der häufig empfohlene chelatbildner dmps beim fetus nicht wirksam, weil die besonders problematischen zns-depots nicht erreicht werden. (sterling ) . mit wesentlich größerem forschungsaufwand wurde dem verdacht nachgegangen, dass amerikanische vietnamkriegsveteranen unter fruchtbarkeitsstörungen durch das hantieren mit agent orange litten. ein beweis hierfür konnte nicht erbracht werden. in seveso (italien) wurden bei einem unfall in einer chemiefabrik dioxine, u. a. das später als "sevesogift" bezeichnete tcdd ( , , , -tetrachlor-p-dibenzodioxin) freigesetzt. mehrere untersuchungen an den damals geborenen kindern kamen zu widersprüchlichen ergebnissen. dabei ist zu berücksichtigen, dass viele schwangerschaften aus angst vor fehlbildungen abgebrochen wurden. eine tera-retardierung, mikrozephalie, dem fetalen alkoholsyndrom ähnelnde kraniofaziale dysmorphien, zns-funktionsstörungen und auch fälle von partieller schädel/hirnagenesie wurden in diesem zusammenhang beobachtet. in den meisten fällen handelte es sich um einen abusus mit toluol (Übersicht in . eine häufung von zns-fehlbildungen wurde auch bei kindern von müttern beschrieben, die beruflich mit lösungsmitteln exponiert waren. außerdem wurde über ein leicht erhöhtes risiko für kardiovaskuläre anomalien und inguinalhernien nach beruflicher exposition der mutter mit kohlendisulfid berichtet und über ein erhöhtes risiko für gaumenspalten, kardiovaskuläre, intestinale und multiple fehlbildungen bei nicht spezifiziertem gewerblichem lösungsmittelkontakt (laumont ) . andere entwicklungsstörungen sowie früh-und fehlgeburten wurden ebenfalls im zusammenhang mit gewerblicher exposition publiziert. mehrere untersuchungen beschäftigen sich mit der tätigkeit in chemischen reinigungen (z. b. doyle , zielhuis . die ergebnisse sind widersprüchlich, z. b. scheint die abortrate bei frauen, die mit chlorierten lösungsmitteln arbeiten, erhöht zu sein (mcmartin , kyyrönen . eine erhöhte fehlbildungsrate oder ein typisches fehlbildungsmuster ließen sich bisher jedoch nicht belegen (mcmartin ) . in kalifornien wurden wiederholt auswirkungen einer trinkwasserkontamination mit trichlorethen auf die vorgeburtliche entwicklung untersucht. entgegen anfänglichen verdachtsmeldungen gab es keinen signifikanten anstieg pränataler entwicklungsstörungen. einschränkungen in der sprachentwicklung im zusammenhang mit beruflicher lösungsmittelexposition der mutter beschreibt eine studie mit kindern im alter von bis jahren (siambani ) . eine metaanalyse von studien aus den jahren - , bei der . schwangerschaften erfasst wurden, ergab hinweise darauf, dass kinder von vätern mit beruflichem kontakt zu organischen lösungsmitteln ein gering erhöhtes risiko für neuralrohrdefekte haben, auch wurden tendenziell mehr fehlgeburten beobachtet (logman ) . empfehlung für die praxis: die daten zur pränatalen toxizität von organischen lösungsmitteln lassen keine abschließende beurteilung zu. bei konsequenter einhaltung arbeitshygienischer vorgaben sowie bei gelegentlichen arbeiten mit lösungsmitteln im haushalt (reinigung, renovierung) ist mit einem messbaren anstieg des fehlbildungsrisikos nicht zu rechnen. weil schwangerschaftskomplikationen und geringe funktionsdefizite unzureichend oder gar nicht untersucht sind oder zu widersprüchlichen ergebnissen führten, sollen schwangere nicht mit organischen lösemitteln arbeiten, wenn wiederholt mit "quantitativem" kontakt gerechnet werden muss. im gewerblichen bereich sollte eine individuelle expositionsabschätzung veranlasst werden. (dabrowski ) . mehrere untersuchungen haben sich mit der auswirkung von trinkwasserchlorierung beschäftigt, die zur kontamination des wassers mit trihalomethanen (chloroform, bromoform u. a.) führen kann. als mögliche folge wurden sowohl eine erhöhte fehlbildungsrate, speziell neuralrohrdefekte, ein verringertes geburtsgewicht, verringerte körperlänge und kopfumfang sowie frühgeburtlichkeit und höhere abortraten diskutiert. in diesen retrospektiven studien fehlen meistens informationen über die tatsächliche exposition der schwangeren und im vergleich zu nicht exponierten kontrollgruppen waren die relativen risiken nur gering erhöht (källén , nieuwenhuijsen . umweltbelastungen mit arsen-und bor-verbindungen sowie mit phthalaten, bei denen estrogenartige nebenwirkungen vermutet werden, haben zur reproduktionstoxizität beim menschen keine klinisch relevanten ergebnisse erbracht , moore , desesso , fail . ein tendenziell verringertes geburtsgewicht wurde mit zunehmender kohlenmonoxid-konzentration als indikator für luftverschmutzung festgestellt. eine retrospektive studie hat die geburtsdaten von über . kindern im raum los angeles und messdaten von monitorstationen in wohnortnähe ausgewertet (ritz ) . obwohl der beobachtete trend biologisch plausibel erscheint, wurden in dieser studie weitere relevante faktoren wie (passiv-)rauchen unzureichend dokumentiert. acrylamid ist weit verbreitet in industriellen prozessen. generell kann es in hohen dosen neurotoxisch wirken. acrylamid findet sich im zigarettenrauch und lässt sich im blut von rauchern in erhöhtem maße nachweisen. außerdem wird es in verschiedenen nahrungsprodukten wie z. b. kartoffelchips und pommes frites gefunden. studien zu auswirkungen acrylamidhaltiger nahrung in der schwangerschaft beim menschen gibt es bislang nicht. es gibt auch keine fallberichte, die schädigungen neugeborener beschreiben. in tierversuchen bzw. zellkulturexperimenten hat sich acrylamid als mutagen, karzinogen und neurotoxisch erwiesen. widersprüchlich sind die ergebnisse von studien, die den einfluss von chemischem giftmüll auf den schwangerschaftsverlauf untersuchen. eine studie aus kalifornien berichtet darüber, dass neuralrohrdefekte und herzanomalien häufiger bei kindern von frauen auftraten, die in der nähe von giftmülldeponien wohnten, und dass mit der ent-schwellendosen nicht definieren, daher werden keine mak-werte festgesetzt. für schwangere gibt es keine eigenen mak-werte, stattdessen wird für einzelne chemische stoffe angegeben, ob bei einhaltung des mak-wertes ein fruchtschädigendes risiko besteht oder nicht. der begriff "fruchtschädigend" wird dabei sehr weit definiert und umfasst jeden effekt eines stoffes, der ein abweichen von der normalentwicklung hervorruft und "prä-oder postnatal zum tod oder zu permanenten morphologischen oder funktionellen schädigungen der leibesfrucht führt." die bewerteten substanzen werden in vier kategorien (a-d) eingestuft. mit krebserzeugenden stoffen muss in der schwangerschaft jeglicher kontakt gemieden werden. wie weiter oben angesprochen, gibt es nur für wenige arbeits-bzw. schadstoffe ausreichende erfahrungen beim menschen, die eine differenzierte risikobewertung ermöglichen. die anzahl der in tabelle . wiedergegebenen stoffe ist, gemessen an der gesamtzahl der weltweit produzierten arbeits-und schadstoffe sehr klein und die humantoxikologischen daten sind in den meisten fällen unzureichend. das muss besonderes bei den substanzen der gruppe c berücksichtigt werden, bei denen bei einhaltung des mak-wertes ein fruchtschädigendes risiko ausgeschlossen wird. die orientierung am so genannten no observed adverse effect level (noael), der in tierversuchen ermittelt wird, ist in den meisten fällen für die beim menschen übliche komplexe expositionssituation nicht ausreichend, insbesondere weil noael-werte nur in ausnahmefällen reproduktionstoxikologisch fundiert sind. die einstufung von in der schwangerschaft unbedenklichen stoffen in die gruppe c (tabelle . ) ist bemerkenswert, denn experten in anderen ländern haben sich bisher gescheut, aufgrund von tierexperimenten und den meist unvollständigen epidemiologischen daten ein risiko für die schwangerschaft auszuschließen. bei genauer analyse dieser gruppe überrascht, dass im jahr auch stoffe mit krebserzeugenden eigenschaften in gruppe c eingestuft wurden, für die ein nicht genotoxischer wirkungsmechanismus angenommen wird. dazu gehören u. a. formaldehyd und das "sevesogift" dioxin bzw. tcdd. weitere probleme bei der einstufung in gruppe c werden dadurch belegt, dass von den stoffen, die vor jahren in die gruppe c eingestuft waren, mehr als inzwischen anderen gruppen zugeordnet wurden, und zwar in die gruppe der krebserzeugenden bzw. krebsverdächtigen arbeitsstoffe, wie z. b. , -dichlorethan, malathion und parathion. auch toluol wird der gruppe c zugeordnet, obwohl es embryotoxisch wirkt, wenn es von abhängigen frauen missbräuchlich in konzentrationen inhaliert wird ("sniffing"), die den mak-wert in manchen fällen nur um das fache überschritten haben . es ist daher problematisch, diesem stoff aufgrund der beim menschen sporadisch erhobenen daten eine unbedenklichkeit bei einhalten des mak-wertes zu attestieren. organische quecksilber-verbindungen (methylquecksilber) waren die einzige stoffgruppe, die ursprünglich in schwangerschaftsgruppe a der mak-werte-liste eingestuft wurde. inzwischen werden sie den krebserzeugenden stoffen zugeordnet (siehe oben). es mag überraschen oder beruhigend wirken, dass bis heute nur industriechemikalien in schwangerschaftsgruppe b eingestuft wurden (tabelle . ). diese stoffe werden auch bei einhaltung der üblichen arbeitsschutzbedingungen als potenziell fruchtschädigend angesehen. ein risiko ist auch bei exposition außerhalb des arbeitsplatzes gegeben, wenn überdurchschnittliche mengen über die nahrung aufgenommen werden. zu dieser gruppe gehören u. a. polychlorierte biphenyle (pcb), deren toxische wirkung in der schwangerschaft weiter oben beschrieben wird. schwangere dürfen nach ansicht der mak-werte-kommission nicht mit krebserzeugenden stoffen in berührung kommen. eine ausnahme bilden seit die nicht genotoxisch wirkenden karzinogene, für die eine schwellendosis angenommen wird. sie werden schwangerschaftsgruppe c zugeordnet, für die "ein risiko der fruchtschädigung bei einhaltung des mak-wertes und des bat-wertes nicht befürchtet werden muss." (dfg ) . zu diesen stoffen mit nicht genotoxischen, krebserzeugenden eigenschaften gehören u. a. das dioxin tccd und formaldehyd. insbesondere die erfahrungen mit dem hormonpräparat diethylstilbestrol, das nach vorgeburtlicher exposition bei den töchtern nach der pubertät u. a. zu scheidenkarzinomen führte, hat die aufmerksam-keit auf das risiko einer "transplazentaren karzinogenese" gelenkt. mit mehr als stoffen ist die gruppe der krebserzeugenden und krebsverdächtigen arbeitsstoffe die umfangreichste risikogruppe im abschnitt mak-werte. zu den chemikalien, die beim menschen nicht nur fruchtschädigende, sondern möglicherweise auch krebserzeugende oder tumorfördernde wirkungen haben, gehören u. a. organisches quecksilber und die organischen lösungsmittel trichlorethen (tri) und tetrachlorethen (per). allerdings sind beim menschen in der schwangerschaft vermittelte karzinogene effekte wie beim diethylstilbestrol bisher bei keiner anderen substanz nachgewiesen worden. die auswirkungen von keimzellmutationen umfassen sowohl genetisch bedingte variationen ohne krankheitswert als auch fruchtbarkeitsstörungen, fruchttod, fehlbildungen und erbkrankheiten. aufgrund der zufälligkeit der verteilung von mutationsereignissen im erbgut (genom) ist nicht zu erwarten, dass ein mutagener stoff eine substanzspezifische fehlbildung hervorruft. deshalb ist der nachweis zwischen einer exposition und dem auftreten von erbkrankheiten beim menschen kaum zu erbringen. in dieser situation kann man keimzellmutagene bisher nur aufgrund erhöhter mutationsraten bei den nachkommen exponierter versuchstiere erkennen. die von der mak-werte-kommission benannten substanzen sind in anlehnung an krebserzeugende stoffe in fünf kategorien eingeteilt (tabelle . ). obwohl bisher der nachweis fehlt, dass durch keimzellmutagene beim menschen genauso wie bei versuchstieren die nachfolgenden generationen geschädigt werden können, sollten schwangere und frauen im gebärfähigen alter jeden kontakt mit solchen stoffen vermeiden. die keimzellmutagene werden in weitgehender analogie zu den kategorien für krebserzeugende arbeitsstoffe in folgende kategorien eingeteilt: keimzellmutagene, deren wirkung anhand einer erhöhten mutationsrate unter den nachkommen exponierter personen nachgewiesen wurde. bisher wurde noch kein chemischer stoff dieser kategorie zugeordnet. keimzellmutagene, deren wirkung anhand einer erhöhten mutationsrate unter den nachkommen exponierter säugetiere nachgewiesen wurde. empfehlung für die praxis: die übliche exposition mit ionisierender strahlung in der umwelt erfordert keine konsequenzen während der schwangerschaft. dies betrifft auch die höhenstrahlung bei flugreisen und die regional unterschiedliche radon-und andere hintergrundstrahlung. nahrungsmittel, von denen eine anreicherung radioaktiver nuklide bekannt ist, sollten selbstverständlich gemieden werden. g . . elektromagnetische felder mögliche auswirkungen elektromagnetischer felder auf die schwangerschaft wurden wiederholt diskutiert. methodische schwierigkeiten mit der definition von exposition und potenziellen effekten erschweren jedoch schlussfolgerungen aus den bisher vorliegenden, meist unbedenklichen ergebnissen. elektromagnetische felder wurden beispielsweise im zusammenhang mit elektrisch beheizten wasserbetten, elektrischen heizdecken und anderen geräten untersucht. sowohl eine leicht erhöhte abortrate als auch harnwegsanomalien wurden in einzelnen untersuchungen beobachtet, allerdings ist die einwirkung anderer begleitfaktoren nicht auszuschließen (Übersicht in . speziell mit dem gebrauch von heizdecken beschäftigt sich eine studie an schwangeren . entwicklungstoxische effekte wurden nicht nachgewiesen. auch bei einer wohnung in der nähe von hochspannungsleitungen konnten bisher keine störungen des schwangerschaftsverlaufs nachgewiesen werden (blaasaas . zu den auswirkungen von mobiltelefonnutzung und den digitalen mobiltelefonsendern in wohnraumnähe gibt es bisher keine aussagefähigen studien (celik ) . bei den bisher in der literatur erfassten schwangeren, die vom blitzschlag getroffen wurden, überlebten alle mütter und die hälfte der kinder ohne jeden schaden, während die anderen sechs kinder in utero bzw. kurz nach der geburt starben. anatomische entwicklungsstörungen wurden nicht beobachtet (vatter ) . der tod der feten wird anscheinend durch herzstillstand (asystolie) und der im vergleich zum erwachsenen begrenzten fähigkeit zur ausbildung eines stabilen kammerersatzrhythmus verursacht. auch andere stromschlagereignisse können, wenn der stromfluss über die uterusregion erfolgt, kardiale störungen des fetus bis zum fruchttod verursachen. darüber hinaus wurde eine vorübergehende reduktion der fetalen spontanmotorik beobachtet. in den meisten fällen ist jedoch eine unbeeinträchtigte entwicklung des kindes zu erwarten . pregnancy outcome after exposure to ranitidine and other h -blockers. a collaborative study of the european network of teratology information services statin drugs and congenital anomalies life-threatening milk-alkali syndrome resulting from antacid ingestion during pregnancy oral -aminosalicylic acid for inflammatory bowel disease in pregnancy: safety and clinical course subfertility and prepregnancy overweight/obesity: possible interaction between these risk factors in the etiology of congenital renal anomalies delivery outcome after the use of acid-suppressing drugs in early pregnancy with special reference to omeprazole use of omeprazole during pregnancy -no hazard demonstrated in infants exposed during pregnancy sibutramine use in pregnancy: report of two cases placental transmission of atropine at full term pregnancy anticholinergics induce eclamptic seizures efficacy and safety of ursodeoxycholic acid versus cholestyramine in intrahepatic cholestasis of pregnancy outcome of pregnancy after first trimester exposure to h receptor antagonists primary sclerosing cholangitis and pregnancy an open-label, multicentre study to assess the safety and efficacy of a novel reflux suppressant (gaviscon advance) in the treatment of heartburn during pregnancy safety of first trimester exposure to histamine h -blockers. a prospective cohort study foetal outcome in women with inflammatory bowel disease treated during pregnancy with oral mesalazine microgranules ursodeoxycholic acid administration in patients with cholestasis of pregnancy: effects on primary bile acids in babies and mothers preliminary data on exposure to statins during pregnancy use of proton pump inhibitors in pregnancy and rates of major malformations: a meta-analysis risk of congenital abnormalities in children born to women with ulcerative colitis: a population-based, case-control study birth outcome in women exposed to -aminosalicyclic acid during pregnancy: a danish cohort study ursodeoxycholic acid in the treatment of cholestasis of pregnancy: a randomized, double-blind study controlled with placebo pregnancy outcome after medication with hmc-coa reductase inhibitors in the first trimester pregnancy outcomes after maternal exposure to simvastatin and lovastatin handling surveillance types of data on birth defects and exposures during pregnancy calcium carbonate consumption during pregnancy: an unusual cause of neonatal hypocalcemia bile acid patterns in meconium are influenced by cholestasis of pregnancy and not altered by ursodeoxycholic acid treatment a randomised controlled trial of ursodeoxycholic acid and s-adenosyl-l-methionine in the treatment of gestational cholestasis use of cimetidine, omeprazole, and ranitidine in pregnant women and pregnancy outcomes severe fetal haemorrhage during treatment with cholestyramine for intrahepatic cholestasis of pregnancy teratology public affairs committee position paper: maternal obesity and pregnancy isolated neonatal hypomagnesemia associated with maternal overuse of stool softener spina bifida phenotypes in infants or fetuses of obese mothers lowered weight gain during pregnancy and risk of neural tube defects among offspring (abstract) glycopyrrolate reduces nausea during spinal anaesthesia for caesarean section without affecting neonatal outcome first-trimester in utero exposure to anorectics: a french collaborative study with special reference to dexfenfluramine does maternal obesity increase the risk of congenital anomalies? cushing's syndrome in pregnancy treated by ketoconazole: case report and review of the literature antileprosy drugs, pregnancy and fetal outcome persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues drug treatment for tuberculosis during pregnancy: safety considerations drugs in pregnancy and lactation randomized trial of artesunate and mefloquine in comparison with quinine sulfate to treat p. falciparum malaria pregnant women safety of metronidazole in pregnancy: a meta-analysis fluconazole treatment for vulvovaginal candidiasis during pregnancy is metronidazole teratogenic? a meta-analysis maternal fever and birth outcome: a prospective study absence of any adverse effect of inadvertent ivermectin treatment during pregnancy antenatal anthelmintic treatment, birthweight, and infant survival in rural nepal reduction of maternal-infant transmission of human immunodeficiency virus type with zidovudine treatment. pediatric aids clinical trials group protocol study group prenatal prescription of macrolide antibiotics and infantile hypertrophic pyloric stenosis risk of birth defects associated with nelfinavir exposure during pregnancy mebendazole and albendazole treatment of geohelminth infections in children and pregnant women lack of long-term effects of in utero exposure to zidovudine among uninfected children born to hiv-infected women. pediatric aids clinical trials group protocol / teams a population-based case-control study of oral griseofulvin treatment during pregnancy population-based case-control teratologic study of topical miconazole a population-based case-control teratological study of oral nystatin treatment during pregnancy a population-based case-control teratological study of vaginal econazole treatment during pregnancy augmentin treatment during pregnancy and the prevalence of congenital abnormalities: a population-based case-control teratologic study use of cephalosporins during pregnancy and in the presence of congenital abnormalities: a population-based, case-control study nitrofurantoin and congenital abnormalities a population-based casecontrol teratologic study of nalidixic acid a population-based case-control study of the safety of oral anti-tuberculosis drug treatment during pregnancy oral phenoxymethylpenicillin treatment during pregnancy. results of a population-based hungarian casecontrol study a population-based case-control study of oral oxytetracycline treatment during pregnancy a teratological study of aminoglycoside antibiotic treatment during pregnancy a case-control teratological study of spiramycin, roxithromycin, oleandomycin and josamycin a teratological study of lincosamides a population-based casecontrol teratologic study of oral chloramphenicol treatment during pregnancy a population-based casecontrol teratologic study of oral erythromycin treatment during pregnancy no teratogenic effect after clotrimazole therapy during pregnancy a lower rate of preterm birth after clotrimazole therapy during pregnancy use of antibiotics during pregnancy a population based case-control teratologic study of oral metronidazole treatment during pregnancy teratogenic study of doxycycline a case-control analysis of the teratogenic effects of co-trimoxazole managing tuberculosis during pregnancy use of amphotericin b during pregnancy: case report and review the safety of the combination artesunate and pyrimethamine-sulfadoxine given during pregnancy birth outcome of pregnancies after exposure to phenoxymethylpenicillin in utero periconceptional exposure to efavirenz and neural tube defects hemangiomas and other congenital malformations in infants exposed to antiretroviral therapy in utero effect of mebendazole therapy during pregnancy on birth outcome pregnancy outcome following gestational exposure to metronidazole: a prospective controlled cohort study pregnancy outcome after gestational exposure to mebendazole: a prospective controlled cohort study genotoxicity and carcinogenicity of metronidazole association between maternal fever and psychological/behavior outcomes: a hypothesis a prospective controlled multicentre study of clarithromycin in pregnancy cryptococcal pneumonia complicating pregnancy european collaborative study. exposure to antiretroviral therapy in utero or early life: the health of uninfected children born to hiv-infected women myelomeningocele in a child with intrauterine exposure to efavirenz severe immune hemolytic anemia associated with prophylactic use of cefotetan in obstetric and gynecologic procedures acyclovir pregnancy registry and valacyclovir pregnancy registry quinolone arthropathy -acute toxicity to immature articular cartilage discoverer of maternal hyperthermia as a human teratogen the safety of antimalarial drugs in pregnancy prenatal use of metronidazole and birth defects: no association fluconazole-induced congenital anomalies in three infants adverse pregnancy outcome in women exposed to acyclovir during pregnancy: a population-based observational study treatment of bacteriuria in pregnancy with single dose fosfomycin trometamol: a review entis-study on anthelmintics during pregnancy. persönliche mitteilung vancomycin during pregnancy: does it cause hearing loss or nephrotoxicity in the infant? pregnancy outcome after exposure to injectable ribavirin during embryogenesis fluconazole teratogenicity relationship between a case of severe hearing loss and use of gentamycin in the pregnant mother pregnancy outcome following gestational exposure to terbinafine: a prospective comparative study acute onset lactic acidosis and pancreatitis in the third trimester of pregnancy in hiv- positive women taking antiretroviral medication pregnancy outcome after prenatal quinolone exposure chemically induced birth defects pregnancy outcome following exposure to clarithromycin mefloquine for malaria chemoprophylaxis - : a review tolerability of malaria chemoprophylaxis in non-immune travellers to sub-saharan africa: multicentre, randomised, double blind, four arm study maternal illness, including fever, and medication use as risk factors for neural tube defects treatment of multidrug-resistant tuberculosis during pregnancy: a report of cases inadvertent intrauterine infusion of ampicillin-sulbactam initial multicenter experience with double nucleoside therapy for human immunodeficiency virus infection during pregnancy the effects of inadvertent exposure of mefloquine chemoprophylaxis on pregnancy-outcomes and infants of us army service women literatur american college of obstetricians and gynecologists. immunization during pregnancy. technical bulletin okt. ; nr. thiomersal in vaccines: balancing the risk of adverse effects with the risk of vaccine-preventable disease twenty years' experience of rubella vaccination in sweden: years selective vaccination (of -year-old girls and of women postpartum) and years of a general two-dose vaccination tetanus toxoid and spontaneous abortions: is there epidemiological evidence of an association? confirmed rabies exposure during pregnancy and human diploid cell vaccine neonatal tetanus incidence in china, - , and risk factors for neonatal tetanus, guangxi province, china pneumococcal vaccination during pregnancy for preventing infant infection. cochrane database syst rev postexposure rabies vaccination during pregnancy: effect on women and their infants the evidence for the safety of thiomersal in newborn and infant vaccines tetanus toxoid and congenital anomalies a/nj/ / influenza vaccination program: effects on maternal health and pregnancy outcome mmr vaccine and autism: a review of the evidence for a causal association akzidentelle rötelnschutzimpfungen um den zeitpunkt der konzeption und in der frühschwangerschaft infektionen und impfungen in der schwangerschaft postexposure rabies vaccination during pregnancy: experience from postmarketing surveillance with patients fetal consequences of maternal rubella immunization maternal immunization immune response to hepatitis b vaccine in pregnant women receiving post-exposure prophylaxis neonatal tetanus: a continuing challenge in the southeast of turkey: risk factors, clinical features and prognostic factors oral poliovirus vaccination and pregnancy complications birth defects and drugs in pregnancy. littleton/usa: publishing sciences group persistent fetal rubella vaccine virus infection following inadvertent vaccination during early pregnancy correlation of maternal and fetal hepatitis b antibody titers following maternal vaccination in pregnancy missed opportunities for tetanus vaccination in pregnant women, and factors associated with seropositivity meningococcal vaccine in pregnancy: an assessment of infant risk hepatitis b vaccine in pregnancy: maternal and fetal safety a population-based study of measles, mumps, and rubella vaccination and autism oral typhoid vaccine and pregnancy safety of influenza vaccination during pregnancy yellow fever vaccination and pregnancy: a four-year prospective study yellow fever vaccination during pregnancy and spontaneous abortion: a case-control study spontaneous abortions following oral poliovirus vaccination in first trimester congenital anomalies after oral poliovirus vaccination during pregnancy hepatitis-b vaccination in pregnancy: safety and immunogenic response in mothers and antibody transfer to neonates exposure to yellow fever vaccine in early pregnancy dermatoglyphics in offspring of women given gamma globulin prophylaxis during pregnancy cerebral embryopathy in late first trimester: possible association with swine influenza vaccine placental and breast transfer of antibodies after maternal immunization with polysaccharide meningococcal vaccine: a randomized, controlled evaluation safety of tetanus toxoid in pregnant women: a hospital-based case-control study of congenital anomalies congenital yellow fever virus infection after immunization in pregnancy first-trimester drug use and congenital disorders fetal tachycardia: is digitalis still the first-line therapy? angiotensin-converting enzyme inhibitors use in the first trimester of pregnancy teratogen update: angiotensin-converting enzyme inhibitors effects of amiodarone administration during pregnancy on neonatal thyroid function and subsequent neurodevelopment anti-hypertensive drugs in pregnancy and fetal growth: evidence for "pharmacological programming" in the first trimester? fetal toxicity of valsartan and possible reversible adverse side effects radiofrequency catheter ablation in drug refractory maternal supraventricular tachycardias in advanced pregnancy fetal toxic effects of angiotensin ii receptor antagonists: case report and follow-up after birth clonidine: placental transfer and neonatal adaption drugs in pregnancy and lactation neonatal sensorineural hearing loss associated with furosemide: a case-control study assessing the teratogenic potential of angiotensinconverting enzyme inhibitors in pregnancy felodipine use in pregnancy prenatal diagnosis and in utero treatment of torsades de pointes associated with congenital long qt syndrome major congenital malformations after first-trimester exposure to ace inhibitors class iii antiarrhythmics and phenytoin: teratogenicity due to embryonic cardiac dysrhythmia and reoxygenation damage maternal bartter's syndrome in pregnancy treated by amiloride fetal hydrops due to supraventricular tachycardiasuccessful outcome in a difficult case arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor neonatal anuria by ace inhibitors during pregnancy amiodarone-induced neonatal hypothyreoidism: a unique form of transient early-onset hypothyreoidism hydralazine for treatment of severe hypertension in pregnancy: meta-analysis neurodevelopment after in utero amiodarone exposure the safety of calcium channel blockers in human pregnancy: a prospective, multicenter cohort study effects of fetal exposure to diazoxide in man neonatal head circumference and the treatment of maternal hypertension eisenmenger's syndrome in a week pregnancy -management with bosentan and sildenafil effects of captopril on the human foetal placental circulation: an interaction with bradykinin and angiotensin i pregnancy with prolonged fetal exposure to an angiotensin-converting enzyme inhibitor fatal neonatal renal failure due to maternal enalapril ingestion expert consensus document on management of cardiovascular diseases during pregnancy effect of diuretics on fetal growth: a drug effect or confounding by indication? pooled danish and scottish cohort data drug treatment of fetal tachycardias treatment of fetal tachycardia with sotalol: transplacental pharmacokinetics and pharmacodynamics fetal supraventricular tachycardia diagnosed and treated at weeks of gestation: a case report an unusual case of neonatal anuria neonatal ecg changes caused by supratherapeutic flecainide following treatment for fetal supraventricular tachycardia use of antihypertensive medications in pregnancy and the risk of adverse perinatal outcomes: mcmaster outcome study of hypertension in pregnancy (moship ) prothrombin and pivka-ii levels in cord blood from newborns exposed to anticonvulsants during pregnancy minor anomalies accompanying prenatal exposure to topiramate polycystic ovaries and hyperandrogenism in women taking valproate for epilepsy valproate, lamotrigine, and insulin-mediated risks in women with epilepsy are anti-epileptic drugs harmful when given during pregnancy pregnancy outcome in women treated with phenobarbital monotherapy pattern of malformations in the children of women treated with carbamazepine during pregnancy maternal carbamazepine and infant spina bifida major malformations in offspring of women with epilepsy enzyme-inducing antiepileptic drugs in pregnancy and the risk of bleeding in the neonate congenital malformations due to antiepileptic drugs pharmacokinetics of zonisamide in perinatal period dysmorphic features: an important clue to the diagnosis and severity of fetal anticonvulsant syndromes antiepileptic drug treatment in pregnancy: drug side effects in the neonate and neurological outcome long-term neuropsychological consequences of maternal epilepsy and anticonvulsant treatment during pregnancy for school-age children and adolescents preliminary report on teratogenic effects of zonisamide in the offspring of treated women with epilepsy valproic acid embryopathy: report of two siblings with further expansion of the phenotypic abnormalities and a review of the literature wirksamkeit und verträglichkeit bei epilepsien im erwachsenenalter einfluss von psychopharmaka auf reproduktion und kontrazeption sodium valproate, pregnancy, and infantile fatal liver failure safety of tiagabine: summary of trials clonazepam use in pregnancy and the risk of malformations teratogenic effects of antiepileptic drugs: implication for the management of epilepsy in women of childbearing age pregnancy and the risk of teratogenicity levetiracetam monotherapy during pregnancy: a case series digit effects produced by prenatal exposure to antiepileptic drugs vortrag jahrestagung des international clearinghouse of birth defects surveillance and research. malta, . epilepsy and anticonvulsants during pregnancy. entis study, preliminary results the teratogenic effect of carbamazepine: a meta-analysis of exposures oxcarbazepine in pregnancy: clinical experience in argentina maternal administration of thalidomide or valproic acid causes abnormal serotonergic neurons in the offspring: implication for pathogenesis of autism safety of the newer antiepileptic drug oxcarbazepine during pregnancy gabapentin exposure in human pregnancy: results from the gabapentin pregnancy registry a clinical study of children with fetal anticonvulsant syndrome the new antiepileptic drugs and women: efficacy, reproductive health, pregnancy and fetal outcome anti-epileptic drugs in pregnancy: current safety and other issues malformation risk of anti-epileptic drugs in pregnancy: a prospective study from the uk epilepsy and pregnancy register preliminary evidence of phenytoin-induced alteration in embryonic gene expression in a mouse model valproic acid and its metabolites: placental transfer, neonatal pharmacokinetics, transfer via mother's milk and clinical status in neonates of epileptic mother's topiramate kinetics during delivery, lactation, and in the neonate: preliminary observations -epoxide- , -diol pathway of carbamazepine in early pregnancy in maternal serum, urine, and amniotic fluid: effect of dose, co-medication, and relation to outcome of pregnancy perinatal outcome following third trimester exposure to paroxetine short-term oral diazepam treatment during pregnancy: a population-based teratological case-control study a population-based casecontrol study of oral chlordiazepoxide use during pregnancy and risk of congenital abnormalities dose response and dose equivalence of antipsychotics intravenous pentazocine and methylphenidate abuse during pregnancy. maternal lifestyle and infant outcome pregnancy outcome following firsttrimester exposure to zopiclone: a prospective controlled cohort study pregnancy outcome after gestational exposure to loratadine or other antihistamines: a prospective controlled cohort study the safety of haloperidol and penfluridol in pregnancy: a multicenter, prospective, controlled study pregnancy outcome after gestational exposure to paroxetine: a prospective controlled cohort study increased risk for cardiovascular anomalies after pregnancy exposure to paroxetine or fluoxetine: a prospective, multicenter, controlled, observational study (manuskript feriggestellt) benzodiazepine use in pregnancy and major malformations or oral cleft: meta-analysis of cohort and case-control studies remergil ® ): behandlungsoption bei therapieresistenter hyperemesis gravidarum? -ein fallbericht neonatal intraventricular haemorrhage associated with maternal use of paroxetine prospective controlled study of hydroxyzine and cetirizine in pregnancy pregnancy and antidepressant counseling a multicentre prospective controlled study to determine the safety of trazodone and nefazodone use during pregnancy pregnancy outcome following gestational exposure to venlafaxine: a multicentre prospective controlled study abrupt discontinuation of psychotropic drugs during pregnancy: fear of teratogenic risk and impact of counselling venflaxine: a hetercyclic antidepressant delivery outcome after the use of antidepressants in early pregnancy the reproductive safety profile of mood stabilizers, atypical antipsychotics, and broad-spectrum psychotropics a population-based case-control teratologic study of nitrazepam, medazepam, tofisopam, alprazolam and clonazepam treatment during pregnancy goiter in a newborn exposed to lithium in utero entis collaborative study of selective serotonin reuptake inhibitors during pregnancy clinical utilization of atypical antipsychotics in pregnancy and lactation olanzapine-exposed pregnancies and lactation: early experience ntp-cerhr expert panel report on the reproductive and developmental toxicity of methylphenidate levodopa in pregnancy mirtazapine use in resistant hyperemesis gravidarum: report of three cases and review of the literature the use of selective serotonin reuptake inhibitors during pregnancy and breastfeeding: a review and clinical aspects serious life events and congenital malformations: a national study with complete follow-up citalopram in pregnancy and lactation birth defects and drugs in pregnancy placental passage of antidepressant medications aktuelle arzneitherapie der schizophrenie: empfehlungen für den allgemeinarzt. arzneiverordnung in der paroxetin-entzugs-syndrom als differenzialdiagnose der akuten neonatalen enzephalitis? ntp-cerhr expert panel report on reproductive and developmental toxicity of fluoxetine prospective multicentre study of pregnancy outcome after lithium exposure during first trimester paroxetine withdrawal in a neonate with historical and laboratory confirmation a] neonate characteristics after maternal use of antidepressants in late pregnancy fluoxetine use in early pregnancy lithium therapy and congenital malformations fetal abnormalities associated with highdose tranylcypromine in two consecutive pregnancies (abstract) suspected congenital sertraline dependence mirtazapine use in two pregnant women: is it safe? healthy outcome under olanzapine-treatment in a pregnant woman neonatal toxicity and transient neurodevelopmental deficits following prenatal exposure to lithium: another clinical report and a review of the literature einfluss von psychopharmaka auf reproduktion und kontrazeption pregnancy outcome following maternal use of the new selective serotonin reuptake inhibitors neurodevelopment in late infancy after prenatal exposure to benzodiazepines -a prospective study teratogenic effects of benzodiazepine use during pregnancy effects of exposure to selective serotonin reuptake inhibitors during pregnancy on serotonergic symptoms in newborns and cord blood monoamine and prolactin concentrations antipsychotics and the treatment of women with psychosis clinical antipsychotic trials of intervention effectiveness (catie) investigators. effectiveness of antipsychotic drugs in patients with chronic schizophrenia clonazepam use in pregnancy and the risk of malformations the use of lithium and management of women with bipolar disorder during pregnancy and lactation the safety of risperidone: a post-marketing study on patients olanzapine in pregnancy risks associated with selective serotonin reuptake inhibitors in pregnancy lethargy in a newborn: lithium toxicity or lab error? neurodevelopmental outcome of infants and toddlers exposed prenatally to selective serotonin reuptake inhibitors the effects of benzodiazepine use during pregnancy and lactation the use of phenothiazines during pregnancy and lactation the outcome of pregnancy in women exposed to therapeutic doses of antidepressants. a collaborative study of the european network of significant changes in antipsychotic drug use during pregnancy pregnancy outcome of women using atypical antipsychotic drugs: a prospective comparative study necrotizing enterocolitis in a newborn: maternal psychotropic drugs suspected potenzial fluoxetine chloride (prozac) toxicity in a newborn effects of prenatal meprobamate and chlordiazepoxide hydrochloride on human embryonic and fetal development paroxetine use throughout pregnancy: does it pose any risk to the neonate? neonatal signs after late in utero exposure to serotonin reuptake inhibitors olanzapine and pregnancy child development following exposure to tricyclic antidepressants or fluoxetine throughout fetal life: a prospective, controlled study neurodevelopment of children exposed in utero to antidepressant drugs pain reactivity in -month-old infants after prenatal and postnatal selective serotonin reuptake inhibitor medication exposure pharmacologic factors associated with transient neonatal symptoms following prenatal psychotropic medication exposure association of maternal sertraline (zoloft) therapy and transient neonatal nystagmus pregnancy outcome following gestational exposure to venlafaxine: a multicenter prospective controlled study is benzodiazepine use during pregnancy really teratogenic? prospective assessment of pregnancy outcome following first trimester exposure to benzodiazepines pregnancy outcome following first-trimester exposure to fluoxetine (prozac) severe and prolonged sedation in five neonates due to persistence of active diazepam metabolites case report and review of the perinatal implications of maternal lithium use no complications with risperidone treatment before and throughout pregnancy and during the nursing period treating depression in pregnancy prenatal exposure to benzodiazepines: a case-control study. vortrag . jahreskonferenz des european network of teratology information services mirtazapine (remergil) for treatment resistant hyperemesis gravidarum: rescue of a twin pregnancy effectiveness and cost of olanzapine and haloperidol treatment of schizophrenia mirtazapine: treatment of depression, anxiety, and hyperemesis gravidarum in the pregnant patient. a report of cases neonatal convulsions and subarachnoidal hemorrhage after in utero exposure to paroxetine selective serotonin reuptake inhibitors in pregnant women and neonatal withdrawal syndrome: a database analysis alprazolam exposure during early human pregnancy toxic neonatal effects following maternal clomipramine therapy update on new developments in the study of human teratogens outcomes of prenatal antidepressant exposure chloral hydrate, methylmercury hydroxide and ethidium bromide affect chromosome segregation during meiosis of saccharomyces cerevisiae fluoxetine hydrochloride (prozac) toxicity in a neonate first-trimester exposure to alprazolam pregnancy outcome of women exposed to azathioprine during pregnancy: a prospective multicenter study. (abstract) the reproductive effects of beta interferon therapy for multiple sclerosis teratogen update: reproductive risks of leflunomide (arava tm ); a pyrimidine synthesis inhibitor: counseling women taking leflunomide before or during pregnancy and men taking leflunomide who are contemplating fathering a child drugs in pregnancy and lactation hydroxychloroquine and lupus pregnancy: review of a series of cases transplacental passage of recombinant human granulocyte colony-stimulating factor in women with an imminent preterm delivery the use of disease modifying antirheumatic drugs in women with rheumatoid arthritis of childbearing age: a survey of practice patterns and pregnancy outcomes huong du lt. safety of hydroxychloroquine in pregnant patients with connective tissue diseases safety of hydroxychloroquine in pregnant patients with connective tissue diseases maternal azathioprine therapy and depressed haematopoiesis in the babies of renal allograft patients paternal and maternal exposure to leflunomide: pregnancy and neonatal outcome wilson's disease treated with trientine during pregnancy immunologic evaluation during the first year of life of infants born to cyclosporine-treated female kidney transplant recipients: analysis of lymphocyte subpopulations and immunoglobulin serum levels pregnancy and wilson's disease the safety of -mercatopurine for childbearing patients with inflammatory bowel disease: a retrospective cohort study neonatal lupus and iugr following alpha-interferon therapy during pregnancy the ototoxicity of chloroquine phosphate pregnancy after liver transplantation under tacrolimus pregnancy after liver transplantation with tacrolimus immunosuppression: a singel center's experiencee update at years pregnancy after kidney and kidneypancreas transplantation under tacrolimus: a single center's experience copolymer reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind, placebo-controlled trial otterblad olausson p. pregnancy outcome after maternal organ transplantation in sweden outcome of pregnancy in women receiving infliximab for the treatment of crohn's disease and rheumatoid arthritis review of the course and outcome of pregnancies in women treated with tacrolimus levamisole (decartis ® ) treatment during pregnancy systemic lupus erythematosus flares during pregnancy ocular toxicity and antenatal exposure to chloroquine or hydroxychloroquine for rheumatic diseases analysis of pregnancy outcomes in transplant recipients treated with sandimmun mycophenolate mofetil in pregnancy after renal transplantation: a case of major fetal malformations pregnancy outcome following first trimester exposure to chloroquine intentional infliximab use during pregnancy for induction of maintenance of remission in crohn's disease. aliment prenatal exposure to penicillamine and oral clefts: case report morbus wilson und schwangerschaft. literaturübersicht und kasuistische mitteilung teratogen update: thalidomide: a review, with a focus on ocular findings and new potential uses the effect on the fetus of medications used to treat pregnant inflammatory bowel-disease patients follow-up of infants exposed to hydroxychloroquine given to mothers during pregnancy and lactation pregnancy outcome in liver transplant recipients the re-emergence of thalidomide: results of a scientific conference azathioprine, mercaptopurine and birth outcome: a population-based cohort study neurodevelopment in children exposed in utero to cyclosporine and azathioprine following maternal renal transplant: preliminary results hydroxychloroquine in pregnant patients with systemic lupus erythematosus immunmodulatoren zur therapie der multiplen sklerose bei frauen im fertilen alter kidney transplantation during the first trimester of pregnancy: immunosuppression with mycophenolate mofetil, tacrolimus, and prednisone infant with severe penicillamine embryopathy born to a women with wilson disease teratogen uptake: azathioprine and -mercaptopurine pregnancies following liver transplantation -how safe are they? a report of cases under cyclosporine a and tacrolimus long-term follow-up of children with in utero exposure to immunosuppressives hepatoblastoma in a -year-old child of a liver-transplanted mother teratogen update: penicillamine prophylactic granulocyte colony-stimulating factor treatment for acquired chronic severe neutropenia in pregnancy kupfer und penicillamin in der schwangerschaft successful ovulation induction, conception, and normal delivery after chronic therapy with etanercept: a recombinant fusion anti-cytokine treatment for rheumatoid arthritis successful pregnancies and abortions in symptomatic and asymptomatic wilson's disease hypothesis: thalidomide embryopathy-proposed mechanism of action offspring of male and female parents with thalidomide embryopathy: birth defects and functional anomalies cardiotoxic tranplacental effect of idarubicin administered during the second trimester of pregnancy methotrexate/misoprostol embryopathy: report of four cases resulting from failed medical abortion successful pregnancy in acute promyelocytic leukemia pregnancy under treatment of imatinib and successful labor in a patient with chronic myelogenous leukemia (cml) successful outcome with anagrelide in pregnancy managing hairy cell leukemia in pregnancy combined chemotherapy and radiotherapy during conception and two trimesters of gestation in a woman with metastatic breast cancer management of chemotherapy in a pregnancy complicated by a large neuroblastoma teratogenic effects in a case of maternal treatment for acute myelocytic leukaemia -neonatal und infantile course growth and development of children of mothers treated with chemotherapy during pregnancy: current status of children non-hodgkin's lymphomas and pregnancy: presentation of cases mitoxantrone and etoposide in breast milk non-hodgkin's lymphoma in pregnancy adult and two children with fetal methotrexate syndrome management of breast cancer during pregnancy using a standardized protocol pregnancy outcome following cancer chemotherapy genetic effects of radiotherapy for childhood cancer drugs in pregnancy and lactation full-term pregnancy in a patient diagnosed with acute leukemia treated with a protocol including vp- multiple congenital anomalies associated with weekly low-dose methotrexate treatment of mother absence of birth defects in offspring of women treated with dactinomycin use of chemotherapy during human pregnancy successful treatment of acute promyelocytic leukaemia during pregnancy the use of disease modifying antirheumatic drugs in women with rheumatoid arthritis of childbearing age: a survey of practice patterns and pregnancy outcomes prenatal diagnosis of methotrexate embryopathy successful fetal outcome after exposure to idarubicin and cytosine-arabinoside during the second trimester of pregnancy -a case report cyclophosphamide for lupus during pregnancy acute promyelocytic leukemia during pregnancy: report of cases goldenhar's syndrome associated with tamoxifen given to the mother during gestation (letter) vinorelbin in pregnancy fertility and ovarian function are preserved in women treated with an intensified regimen of cyclophosphamide, adriamycin, vincristine and prednisone (mega-chop) for non-hodgkin lymphoma successful pregnancy during chemotherapy for acute leukemia procarbazine in pregnancy metastatic breast cancer in pregnancy: first case of chemotherapy with docetaxel developmental delay in fetal aminopterin/methotrexate syndrome hydroxyurea use during pregnancy: a case report in sickle cell disease and review of the literature chemotherapy for metastatic melanoma during pregnancy methotrexate exposure prior to and during pregnancy anagrelide therapy in pregnancy: report of a case of essential thrombocythemia high total dose -fluorouracil treatment during pregnancy an endodermal sinus tumor diagnosed in pregnancy: case report and review of the literature apparent cyclophosphamide (cytoxan) embryopathy: a distinct phenotype? non-hodgkin-lymphom und schwangerschaft successful treatment of acute promyelocytic leukemia in pregnancy with all-trans retinoic acid pregnancy outcomes following treatment of cancer clinical teratology counseling and consultation case report: low dose methotrexate exposure in the early weeks of pregnancy acute leukemia and pregnancy management of an advanced ovarian cancer at weeks of gestation: case report and literature review the safety of -mercatopurine for childbearing patients with inflammatory bowel disease: a retrospective cohort study chemotherapy with epirubicin and paclitaxel for breast cancer during pregnancy: case report and review of the literature passage transplacentaire de l'épirubincine doxorubicin in the first trimester of pregnancy teratogenic effects of combination chemotherapy anthracyclines during pregnancy: embryo-fetal outcome in patients cis-platinum neoadjuvant chemotherapy in a pregnant woman with invasive carcinoma of the uterine cervix chemotherapy for breast carcinoma during pregnancy multimodal cancer chemotherapy during the first and second trimester of pregnancy: a case report pregnancy associated breast cancer: a case report successful chemotherapy including epirubicin in a pregnant non-hodgkin's lymphoma patient paclitaxel chemotherapy in a pregnant patient with bilateral breast cancer congenital anomalies in children of patients who received chemotherapy for cancer in childhood and adolescence pregnancy outcome of female survivors of childhood cancer: a report from the childhood cancer survivor study pregnancy outcome of partners of male survivors of childhood cancer survivor study congenital anomalies probably induced by cyclophosphamide acute leukemia during pregnancy: a single institutional experiencee with cases pregnancy outcome after prenatal exposure to bleomycin, etoposide and cisplatin for malignant ovarian germ cell tumors: report of cases teratogenicity of adriamycin successful outcome of pregnancy in chronic myeloid leukaemia treated with imatinib pharmakokinetics of methotrexate in erythrocytes in psoriasis imatinib treatment: specific issues related to safety, fertility, and pregnancy efficacy and safety of a combined rituximab chemotherapy during pregnancy primary ovarian carcinoma during pregnancy mixed germ cell malignancy of the ovary concurrent with pregnancy sinusoidal fetal heart rate pattern during chemotherapy in a pregnant woman with acute myelogenous leukemia tamoxifen as systemic treatment of advanced breast cancer during pregnancy -case report and literature review oat cell carcinoma of the uterine cervix in a pregnant woman treated with cis-diamminedichloroplatinum management of the pregnant patient with hodgkin's disease neonatal effects of breast cancer chemotherapy administered during pregnancy a woman with a balanced autosomal translocation who received chemotherapy while pregnant safety of rituximab therapy during the first trimester of pregnancy: a case history teratogenic effects of first-trimester cyclophosphamide therapy cyclophosphamide therapy in a serious case of lupus nephritis during pregnancy inadvertent -fluorouracil treatment in early pregnancy: a report of three cases stage of pregnancy-dependent transplacental passage of mpt after cis-platinum treatment outcome of first-trimester exposure to low-dose methotrexate in eight patients with rheumatic disease in utero first trimester exposure to low-dose methotrexate with increased fetal nuchal translucency and associated malformations successful pregnancy after tamoxifen and megestrol acetate therapy for endometrial carcinoma low dose methotrexate in the first trimester of pregnancy: result of a french collaborative study consequences of acute myelogenous leukemia in early pregnancy normal infant after combination chemotherapy including teniposide for burkitt's lymphoma in pregnancy wegener's granulomatosis in pregnancy mcelhatton a review of the reproductive toxicity of methotrexate in human pregnancy congenital anomalies due to attempted abortion with -amino pteroylglutamic acid paclitaxel and carboplatin chemotherapy administered during pregnancy for advanced epithelial ovarian cancer fetal renal malformation following treatment of hodgkin's disease during pregnancy management of cancer in pregnancy: a case of ewing's sarcoma of the pelvis in the third trimester infant or mother with malignant disease hydroxyurea in pregnant women with polycythemia vera methotrexate-induced congenital malformations chemotherapy-induced amenorrhea and fertility in women undergoing adjuvant treatment for breast cancer pregnancy outcome after the use of an aromatase inhibitor for ovarian stimulation the effect on the fetus of medications used to treat pregnant inflammatory bowel-disease patients eclampsia after polychemotherapy for nodalpositive breast cancer during pregnancy pregnancy outcome in cancer patients: experience in a large cooperative group fetal marrow suppression after maternal chemotherapy for leukaemia multimodal cancer therapy for breast cancer in first trimester of pregnancy the human teratogenic effect of cyclophosphamide good outcome for infant of mother treated with chemotherapy for ewing sarcoma at to weeks' gestation multiple anomalies in a fetus exposed to low-dose methotrexate in the first trimester treatment of acute myeloid leukemia during the second and third trimesters of pregnancy azathioprine, mercaptopurine and birth outcome: a population-based cohort study neurodevelopment of children exposed in utero to treatment of maternal malignancy non-hodgkin's lymphoma in pregnancy -fluorouracil exposure during the period of conception: report on two cases successful treatment of an advanced cystadenocarcinoma in pregnancy with cisplatin, adriamycin and cyclophosphamid (cap) regimen. case report administration of rituximab during the first trimester of pregnancy without consequences for the newborn low dose weekly methotrexate in early pregnancy. a case series and review of literature prenatal detection of multiple fetal anomalies following inadvertent exposure to cyclophosphamide in the first trimester of pregnancy doxorubicin-induced cardiomyopathy during pregnancy: three case reports of anesthetic management for cesarean and vaginal delivery in two kyphoscoliotic patients combined chemotherapy and teratogenicity polycythemia vera and pregnancy: a case report with the use of hydroxyurea in the first trimester assessment of fetal risk associated with exposure to cancer chemotherapy during pregnancy: a multicenter study treatment of acute leukemia during pregnancy: presentation of nine cases teratogen uptake: azathioprine and -mercaptopurine methotrexate-induced congenital malformations transplacental effects of maternal cancer chemotherapy. case report acute leukemia during pregnancy: obstetrics management and perinatal outcome of two cases acute leukemia during pregnancy: the toronto leukemia study group experience with long-term followup of children exposed in utero to chemotherapeutic agents acute leukemia and pregnancy -fatal fetal outcome after exposure to idarubicin during the second trimester chemotherapy for breast cancer during pregnancy: an -year experience from five london teaching hospitals vacop-b chemotherapy for high grade non-hodgkin's lymphoma in pregnancy radiation and radiomimetic chlorambucil and the fetal retina chemical inducers of ovulation: comparative results teratogenic effects of antileukemic chemotherapy successful pregnancy following continuous treatment with combination chemotherapy before conception and throughout pregnancy chemically induced birth defects chromosomal aberrations in a newborn whose mother received cytotoxic treatment during pregnancy a case of methotrexate embryopathy with holoprosencephaly, expanding the phenotype possible teratogenic effect of chlorambucil on a human fetus transient dilated cardiomyopathy in a newborn exposed to idarubicin and all-trans-retinoic acid (atra) early in the second trimester of pregnancy paclitaxel and platinum chemotherapy for ovarian carcinoma during pregnancy acute leukemia and pregnancy renal agenesis after first trimester exposure to chlorambucil multiple con-genital anomalies in a fetus exposed to -fluorouracil during the first trimester genetic effects of radiotherapy for childhood cancer: gonadal dose reconstruction risk of spontaneous abortion among nurses handling antineoplastic drugs -mercaptopurine teratogenicity ambiguous genitalia in infant exposed to tamoxifen in utero exposure to hydroxyurea during pregnancy: a case series therapeutic abortion with a folic acid antagonist, -amino pteroylglutamic acid, administerd by the oral route the investigation and management of hodgkin's disease in the pregnant patient fetal effects during cyclophosphamide and irradiation therapy platinum based chemotherapy to treat recurrent sertoli-leydig cell ovarian carcinoma during pregnancy anthracyclines in the treatment of malignancy in pregnancy cyclophosphamide, methotrexate and cytarabine embryopathy: is apotosis the common pathway? acute leucemia and pregnancy congenital abnormalities in baby born to cytarabine treated mother attempted abortion with aminopterin herceptin (trastuzumab) therapy during pregnancy: association with reversible anhydramnios fetal methotrexate and misoprostol exposure: the past revisied a single institutional experience with pregnancies in essential thrombocythemia anomalies associated with failed methotrexate and misoprostol termination fetal outcome after in utero exposure to cancer chemotherapy teratogenicity and carcinogenicity in a twin exposed in utero to cyclophosphamide pregnancy outcome in hematologic malignancies neonatal outcome after exposure to indomethacin in utero: a retrospective case cohort study der unwert der oralen tokolyse. vortrag . dtsch. kongr. f. perinatale medizin misoprostol exposure during pregnancy: a french collaborative study maternal and fetal cardiovascular effects of transdermal glyceryl trinitrate and intravenous ritodrine overdosage of misoprostol in pregnancy teratogenicity of misoprostol: data from the latin-american collaborative study of congenital malformations (eclamc) effectiveness of nifedipine versus atosiban for tocolysis in preterm labour: a meta-analysis with an indirect comparison of randomised trials nsaid-induced nephrotoxicity from the fetus to the child tokolyse mit einem nitroglycerinpflaster treatment of sexually transmitted bacterial diseases in pregnant women bacterial vaginosis during pregnancy: screen and treat? maternal spermicide use and adverse reproductive outcome: a meta analysis diltiazem for maintenance tocolysis of preterm labor: comparison to nifedipine in a randomized trial congenital abnormalities in brazilian children associated with misoprostol misuse in first trimester of pregnancy pathophysiologie der lungenödementstehung bei der tokolytischen a risk-benefit assessment of therapies for premature labour do tocolytic agents stop preterm labor? a critical and comprehensive review of efficacy and safety limb reduction anomaly after failed misoprostol abortion aktuelle aspekte der thüringer frühgeburtenvermeidungsaktion nicardipine versus salbutamol in the treatment of premature labor. a prospective randomized study vaginal spermicides and congenital disorders bacterial vaginosis: review of treatment -options and potential clinical indications for therapy controversies in tocolytic therapy calcium channel blockers for inhibiting preterm labour (cochrane review) a randomized double-blind study comparing the fetal effects of sulindac to terbutaline during the management of preterm labor efficacy and safety of indomethacin compared with magnesium sulfate in the management of preterm labor: a randomized study indomethacin treatment for polyhydramnios. effective but potentially dangerous? neonatal complications after the administration of indomethacin for preterm labor myocardial infarction during nifedipine therapy for preterm labor epidemiological assessment of misoprostol teratogenicity nifedipine and ritrodine in the management of preterm labor: a randomized multicenter trial effect on normal vaginal flora of three intravaginal microbicidal agents potentially active against human immunodeficiency virus type a double-blind randomized study of fetal side effects during and after short-term maternal administration of indomethacin, sulindac, and nimesulide for the treatment of preterm labour chemically induced birth defects teratogenicity of misoprostol pregnancy outcome after exposure to misoprostol in brazil: a prospective, controlled study neonatal effects after antenatal treatment with indomethacin vs. sulindac (abstract) bolustokolyse in theorie und praxis safety study of nonoxynol- as a vaginal micro-bicide: evidence of adverse effects the effect of relcovaptan (sr ), an orally active vasopressin v a receptor antagonist, on uterine contractions in preterm labor indomethacin tocolysis and itraventricular hemorrhage the effect of in utero exposure to indomethacin on the need for surgical closure of a patent ductus arteriosus in the neonate in utero exposure to terbutaline. effects on infant behavior and maternal selfesteem severe hypotension and fetal death due to tocolysis with nifedipine pharmacokinetics of fenoterol in pregnant women die behandlung des spätabortes und der drohenden frühgeburt mit th a in kombination mit isoptin who model list of essential medicines the localization and concentration of copper in the fallopian tube in women with or without an intrauterine contraceptive device misoprostol's effect on uterine arterial blood flow and fetal heart rate in early pregnancy timing and magnitude of increases in levothyroxine requirements during pregnancy in women with hypothyroidism american college of obstetricians and gynecologists. acog committee opinion. use of progesterone to reduce preterm birth arbeitsgemeinschaft der wissenschaftlichen medizinischen fachgesellschaften die ärztliche betreuung der schwangeren diabetikerin arbeitsgemeinschaft der wissenschaftlichen medizinischen fachgesellschaften empfehlungen zu diagnostik und therapie des gestationsdiabetes (gdm) antenatal thyrotropin-releasing -hormone to prevent lung disease in preterm infants. north american -thyrotropin-releasing hormone study group choanal atresia associated with prenatal methimazole exposure: three new patients insulin lispro and regular insulin in pregnancy intrauterine exposure to clomiphene and neonatal persistent hyperplastic primary vitreous the treatment of a thyrotropin-secreting pituitary macroadenoma with octreotide in twin pregnancy normal pregnancy in a woman with nesidioblastosis treated with somatostatin analog octreotide nongenital malformations and exposure to progestational drugs during pregnancy: the final chapter of an erroneous allegation (abstract) drugs in pregnancy and lactation is insulin lispro associated with the development or progression of diabetic retinopathy during pregnancy? maternal and fetal thyroid function risk of gnrh agonist administration in early pregnancy in ovulation induction, update maternal corticosteroid use and risk of selected congenital anomalies hypospadias and maternal intake of progestins and oral contraceptives congenital viral infection? diethylstilbestrol (des) (www.cdc.gov/des/) antenatal steroid use is associated with increased gastroesophageal reflux in neonates methimazole embryopathy: delineation of the phenotype collaborative trial of prenatal thyrotropin-releasing hormone and corticosteroids for prevention of respiratory distress syndrome author's response: severe embryopathy and exposure to methimazole in early pregnancy australian collaborative trial of antenatal thyrotropin-releasing hormone: adverse effects at -month follow-up pregnancy complications and perinatal outcome in diabetic woman treated with humalog (insulin lispro) or regular human insulin during pregnancy population-based case-control study of teratogenic potential of corticosteroids cardiovascular risk factors after antenatal exposure to betamethasone: -year follow-up of a randomised controlled trial twenty-year follow-up of antenatal corticosteroid treatment long-term treatment with cabergoline in pregnancy and neonatal outcome: report of a clinical case use of insulin glargine during pregnancy in a type diabetic woman (letter) teratogen update: antithyroid drugs -methimazole, carbimazole and propylthiouracil adverse effects of prenatal methimazole exposure intellectual capacity of subjects exposed to methimazole or propylthiouracil in utero administration of a gonadotropinreleasing hormone agonist during pregnancy, follow-up of pregnancies exposed to triptorelin comparative placental transport of oral hypoglycemic agents in humans: a model of human placental transfer macrosomia despite good glycaemic control in type i diabetic pregnancy; results of a nationwide study in the netherlands malformations following methimazole exposure in utero: an open issue nonprescription drugs advisory committee and the advisory committee for reproductive health drugs. december carbimazole embryopathy: an emerging phenotype teratogenic potential of corticosteroids in humans repeated antenatal corticosteroids: size at birth and subsequent development the safety and efficacy of insulin analogs in pregnancy insulin lispro therapy in pregnancies complicated by type diabetes: glycemic control and maternal and fetal outcomes the regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology height, weight, and motor-social development during first months of life in infants born to mothers with polycystic ovary syndrome who conceived on and continued metformin through pregnancy pregnancy outcomes among women with polycystic ovary syndrome treated with metformin metformin therapy throughout pregnancy reduces the development of gestational diabetes in women with polycystic ovary syndrome darbepoetin alfa treatment for post-renal transplantation anemia during pregnancy oral hypoglycemic drugs for gestational diabetes (editorial) pregnancy outcome after first trimester exposure to corticosteroids: a prospective controlled study maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child methimazole as a teratogenic etiology of choanal atresia/multiple congenital anomaly syndrome cancer risk in women exposed to diethylstilbestrol in utero prenatal exposure to stilbestrol comparison of glyburide and insulin for the management of gestational diabetes in a large managed care organization effects of metformin on early pregnancy loss in the polycystic ovary syndrome severe malformations in infant born to hyperthyroid woman on methimazole cabergoline treatment for a large macroprolactinoma throughout pregnancy hormone therapy during pregnancy and isolated hypospadias: an international case-control study oral contraceptives in the etiology of isolated hypospadias a case of rosiglitazone exposure in the second trimester of pregnancy severe embryopathy and exposure to methimazole in early pregnancy hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study glyburide and fetal safety; transplacental pharmacokinetic considerations aplasia cutis congenita after methimazole exposure in utero glyburide for the treatment of gestational diabetes bromocriptine in pregnancy: safety aspects congenital malformations of newborn infants after clomiphen-induced ovulation long-term follow-up of children born after inadvertent administration of a gonadotrophin-releasing hormone agonist in early pregnancy insulin and glyburide therapy: dosage, severity level of gestational diabetes, and pregnancy outcome oral contraceptive use after conception in relation to the risk of congenital urinary tract anomalies normal development after exposure to mifepristone in early pregnancy limb-body wall complex with complete absence of external genitalia after in vitro fertilization maternal diabetes: an independent risk factor for major malformations with increased mortality of affected infants progression of retinopathy during pregnancy in type diabetic women treated with insulin lispro hormonal treatment in pregnancy: a possible risk factor for neuroblastoma prenatal exposure to sex hormones: a case-control study pregnancy outcome in type diabetes mellitus treated with insulin lispro (humalog) the credibility of the actobat follow-up study maternal metabolic control and perinatal outcome in women with gestational diabetes treated with regular or lispro insulin: comparison with non-diabetic pregnant women clomiphene and hypospadias: the necessity to investigate on a detailed level (abstract) antithyroid drug treatment of graves' disease in pregnancy: long term effects on somatic growth, intellectual development and thyroid function of the offspring infant cortisol response after prolonged antenatal prednisolone treatment teratogen update: carcinogenesis and teratogenesis associated with exposure to diethylstilbestrol (des) in utero effects of propylthiouracil and methimazole on fetal thyroid status in mothers with graves' hyperthyroidism prolactinomas resistant to bromocriptine: long-term efficacy of quinagolide and outcome of pregnancy national institutes of health consensus development panel. antenatal corticosteroids revisited: repeat courses -national institutes of health consensus development conference statement should we continue or stop insulin sensitizing drugs during pregnancy? hypospadias in sons of women exposed to diethylstilbestrol in utero prospective parallel randomized, doubleblind, double-dummy controlled clinical trial comparing clomiphene citrate and metformin as the first-line treatment for ovulation induction in nonobese anovulatory women with polycystic ovary syndrome obstetrical outcome in women exposed to diethylstilbestrol during their fetal life: a case-control analysis the long-term growth and development of children exposed to depo-provera during pregnancy and lactation birth defects after maternal exposure to corticosteroids: prospective cohort study and meta-analysis of epidemiological studies insulin lispro therapy in pregnancies complicated by type diabetes mellitus early termination of pregnancy with mifepristone (ru ) and the orally active prostaglandin misoprostol maternal thyroid hormone levels in pregnancy and the subsequent cognitive and motor performance of the children effects of in-utero exposure to oral hypoglycemic drugs development after exposure to mifepristone in early pregnancy maternal hypothyroxinaemia during early pregnancy and subsequent child development: a -year follow-up study first trimester exposure to corticosteroids and oral clefts fetal genital effects of firsttrimester sex hormone exposure: a meta-analysis ddavp use during pregnancy: an analysis of its safety for mother and child ovulation stimulation, assisted reproduktive techniques, and craniosynostosis pregnancy outcome after cabergoline treatment in early weeks of gestation pregnancy outcome after treatment with the ergot derivative, cabergoline malformation surveillance and maternal drug exposure: the madre project corticosteroids during pregnancy and oral defects: a case-control study impaired thyroid function in offspring of propylthiouracil treated women: a prospective controlled study on pregnancies longterm outcomes of infants exposed to multiple courses of betamethasone in utero antenatal corticosteroids to prevent respiratory distress syndrome insulin lispro in pregnancy -retrospective analysis of cases and matched controls the reproductive toxicity of ovulation induction effect of dexamethasone and betamethasone on fetal heart rate variability in preterm labour: a randomised study three cases of diabetes insipidus complicating pregnancy fetal malformations and failed medical termination of pregnancy pegvisomant: an advance in clinical efficacy in acromegaly. review male reproductive disorders in humans and prenatal indicators of estrogen exposure: a review of published epidemiological studies successful pregnancy in an acromegalic woman treated with octreotide congenital malformations in pregnancies complicated by niddm neural tube defects after infertility treatment: a review placental abnormalities associated with misoprostol administration (abstract) metformin reduces pregnancy complications without affecting androgen levels in pregnant polycystic ovary syndrome women: results of a randomized study oestrogen treatment to reduce the adult height of tall girls: long-term effects on fertility schwangerschaften bei hyperprolaktinämischen patientinnen aplasia cutis congenita after exposure to methimazole: a causal relationship? a comparative review of the tolerability profiles of dopamine agonists in the treatment of hyperprolactinaemia and inhibition of pregnancy dietary oestrogens and male fertility potential choanal atresia and hypothelia following methimazole exposure in utero: a second report drugs in pregnancy and delivery. report on the th european symposium on clinical pharmacological evaluation in drug control. copenhagen: provis edit comparison of propylthiouracil versus methimazole in the treatment of hyperthyroidism in pregnancy risk of benign gynecologic tumors in relation to prenatal diethylstilbestrol exposure congenital anomaly rate in offspring of mothers with diabetes treated with insulin lispro during pregnancy timing of vulnerability of the brain to iodine deficiency in endemic cretinism prenatal exposure to female hormones. effect of psychosexual development in boys normal pregnancy outcome following inadvertent exposure to rosiglitazone, gliclazide, and atorvastatin in a diabetic and hypertensive woman comparative maternal and neonatal effects of halothane and enflurane for cesarean section maternal awareness and neonatal outcome after ketamine induction for anesthesia for cesarean section the use of propofol, nitrous oxide or isoflurane does not affect the reproductive success rate following gamete intrafalopian transfer (gift) lidocaine toxicity after maternal pudendal anesthesia in a term infant with fetal distress effect of prenatal lignocaine on auditory brain stem evoked response surgery during pregnancy and fetal outcome neurobehavioural effects of propofol on the neonate following elective caesarean section placental transfer of succinylcholine causing transient respiratory depression in the newborn comparison of assisted reproductive technology performance after oocyte retrieval under general anaesthesia (propofol) versus paracervical local anaesthetic block. a case controlled study anaesthesia, pregnancy, and miscarriage: a study of operating room nurses and anaesthetists maternal occupational exposure and congenital malformations nitrous oxide in early human pregnancy intravenous propofol during cesarean section: placental transfer, concentrations in breast milk, and neonatal effects. a preliminary study effects of maternal anesthesia in the neonate can anesthesiologic strategies for cesarean section influence newborn jaundice? a retrospective and prospective study serum bupivacaine concentrations and transplacental transfer following repeated epidural administrations in term parturients during labour the safety of anesthesia and surgery during pregnancy reproductive and developmental toxicity of anesthetics in humans a double-blind comparison of , % ropivacaine and , % bupivacaine for extradural analgesia in labour survey of infants born in or to swedish women working in operating rooms during their pregnancy neonatal welfare and placental transfer of fentanyl and bupivacaine during ambulatory combined spinal epidural analgesia for labour maternal and fetal levels of propofol at caesarean section the hemodynamic effects of propofol and thiopentone for induction of caesarean section birth defects and drugs in pregnancy transfer of methohexital across the perfused human placenta extrapolation of the evidence on teratogenicity of chemicals between humans and experimental animals: chemicals other than drugs arthrogryposis following treatment of maternal tetanus with muscle relaxants placental propofol transfer and fetal sedaton during maternal general anaesthesia in early pregnancy arzneimittelanwendung in schwangerschaft und stillperiode long term effects of developmental halothane exposure on radial arm maze performance in rats placental transfer and neonatal effects of epidural sufentanil and fentanyl administration with bupivacaine during labor anaesthesia for caesarean delivery. part iii: general anaesthesia epidural analgesia in labor and fetal hyperthermia the use of fetal neuromuscular blockade during intrauterine procedures a comparison between propofol and thiopentone as induction agents in obstetric anesthesia effect of atracurium or pancuronium on the anemic fetus during and directly after intravascular intrauterine transfusion. a double blind randomized study outcome of pregnancy among women in anesthetic practice nitrous oxide inhalation: effects on maternal and fetal circulation at term ketamine: an update on the first twenty-five years of clinical experience developmental evaluation of children born to mothers occupationally exposed to waste anesthetic gases propofol as an induction and maintenance agent for caesarean section: maternal und neonatal effects (abstract) occupational hazards to reproduction and health in anaesthetists and peadiatricians reduced fertility among women employed as dental assistants exposed to high levels of nitrous oxide nitrous oxide and spontaneous abortion in female dental assistants comparative systemic toxicity of ropivacaine and bupivacaine in nonpregnant und pregnant ewes neurodevelopmental disability: a sibling-control study comparison of . % enflurane with . % isoflurane in oxygen for caesarean section: avoidance of awareness without nitrous oxide comparison of propofol and thiopentone for induction of anaesthesia for elective caesarean section occupational hazards of anesthesia comparison of pancuronium and vecuronium for fetal neuromuscular blockade during invasive procedures bei einer patientin wurde die therapie mit brimonidin und in beiden fällen mit timolol kombiniert da prostaglandine den uterustonus erhöhen und eine minderperfusion des fetus verursachen können, ist generell zurückhaltung geboten. falls ein schweres glaukomleiden die lokale behandlung mit prostaglandinderivaten unbedingt erfordert, sollte die dosis so niedrig wie möglich gewählt werden. einem fallbericht zufolge wurde nach mütterlicher pilocarpin-behandlung (z. b. borocarpin ® ) über die gesamte schwangerschaft ein gesundes kind geboren glaucothil ® ) sind zwar nicht systematisch untersucht, haben aber bisher keine negativen auswirkungen auf den fetus gezeigt es haben sich auch in der schwangerschaft sehr häufig verwendete präparate mit xylometazolin (z. b. olynth ® ) und oxymetazolin (z. b. nasivin ® ) bisher nicht als riskant für den fetus erwiesen, obwohl theoretisch (in hohen dosen) eine vasokonstriktion zur versorgungsstörung beim fetus führen könnte um schäden an der nasenschleimhaut zu vermeiden, sollten "entzugsstrategien" angeboten werden rhinospray ® ) liegen keine ausreichenden erfahrungen in der schwangerschaft vor latanoprost exposure in pregnancy the use of topical % imiquimod during pregnancy: a case series fluticasone propionate aqueous nasal spray in pregnancy rhinitis die skabiestherapie unter besonderer berücksichtigung des frühen kindesalters, der schwangerschaft und stillzeit a retrospective study of the teratogenicity of dermatological coal tar products pregnancy outcome after periconceptional and first-trimester exposure to methoxsalen photochemotherapy teratogenic risk with etretinate and acitretine treatment is there a reproductive safety risk in male patients treated with acitretine (neotigason ® /soriatane ® )? psoralen photochemotherapy (puva) and pregnancy accutane ® -exposed pregnancies continued occurence of accutane ® exposed pregnancies teratogenicity of isotretinoin and etretinate first trimester topical tretinoin management of glaucoma in pregnancy and lactation maternal drug use in early pregnancy and infant cardiovascular defect podophyllum: suspected teratogenicity from topical application outcome of pregnancy following exposure to permethrin head lice shampoo pregnancy outcome following exposure to permethrin and use of teratogen information inadvertent -fluorouracil treatment in early pregnancy: a report of three cases retinoic acid embryopathy unusually high risk for adverse outcomes of pregnancy following fetal isotretinoin exposure acitretin is converted to etretinate only during concomitant alcohol intake prenatal and early postnatal intoxication by inorganic mercury resulting from maternal use of mercury containing soap the use of acetazolamide in idiopathic intracranial hypertension during pregnancy multiple congenital defects associated with maternal use of topical tretinoin minor malformations characteristic of the retinoic acid embryopathy and other birth outcomes in children of women exposed to topical tretinoin during early pregnancy first-trimester exposure to topical tretinoin: its safety is not warranted [letter treatment of external genital warts with % imiquiod cream during pregnancy: a case report safety of the insect repelent n,n,-diethyl-m-toluamide (deet) in pregnancy pharmacokinetics and safety of tazaroten temporal bone pathology in fetuses exposed to isotretinoin risk of intrauterine growth retardation, malformations and other birth outcomes in children after topical use of corticosteroid in pregnancy multiple congenital malformations associated with topical tretinoin steady-state pharmacokinetics of isotretinoin and its -oxo metabolite: implications for fetal safety -fluorouracil exposure during the period of conception: a report of two cases transient renal tubular acidosis in a neonate following transplacental acetazolamide kopfläuse -umgang mit einer wieder auflebenden parasitose topical medications during pregnancy mental retardation and parental occupation: a study on the applicability of job exposure matrices extreme caudal agenesis intrauterine diethyltoluamide exposure and fetal outcome otocerebral anomalies associated with topical tretinoin use safety of topical minoxidil solution: a one-year, prospective, observational study safety of first-trimester exposure to topical tretinoin: prospective cohort study topically applied minoxidil may cause fetal malformation: a case report deet: a review and update of safety and risk in the general population plasma absorption and ultrastructural changes of rat testicular cells induced by lindane recommendations for isotretinoin use in women of childbearing potential transdermal absorption of topical anti-acne agents in man; review of clinical pharmacokinetic data neonatal transient hypothyroidism: aetiological study. italian collaborative study on transient hypothyroisism wissenschaftlicher beirat der bundesärztekammer. zur anwendung von polyvinylpyrrolidon-jod-komplexen (povidonjod: pvp-jod) bei ausgewogener und vielseitiger ernährung ist eine zusätzliche substitution von vitaminen oder mineralien nicht routinemäßig erforderlich, mit ausnahme von folsäure in der frühschwangerschaft und iodid. es wird kontrovers diskutiert, ob eine substitution weitere vitamine das risiko von fehlbildungen senkt combined chemotherapy and radiotherapy during conception and first two trimesters of gestation in a woman with metastatic breast cancer the safety of higher than standard dose of doxylamine-pyridoxine for nausea and vomiting of pregnancy folic acid supplementation and the occurrence of congenital heart defects, orofacial clefts, multiple births, and miscarriage bundesinstitut für gesundheitlichen verbraucherschutz und veterinärmedizin). versorgung der deutschen bevölkerung mit folsäure noch immer ungenügend bedeutung von vitamin a für die lungenentwicklung high doses of vitamin e and pregnancy outcome pregnancy outcome following high doses of vitamin e supplementation; a prospective controlled study vitamin a and cardiac outflow tract defects do multivitamin or folic acid supplements reduce the risk for congenital heart defects? evidence and gaps dietary folate as a risk factor for neural-tube defects: evidence from a case-control study in western australia treatment of wilson's disease with zinc. xvii: treatment during pregnancy comparison of three outpatient regimens in the management of nausea and vomiting in pregnancy teratogenität von retinoiden (isotretinoin, etretinat) und vitamin a the teratogenic metabolites of vitamin a in women following supplements and liver vitmin c supplementation to prevent premature rupture of the chorioamniotic maternal and fetal plasma levels of pyridoxal phosphate at term: adequacy of vitamin b supplementation during pregnancy elevated plasma homocysteine in early pregnancy: a risk factor for the development of severe preeclampsia effect of increasing dietary folate on red-cell folate: implications for prevention of neural tube defects prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation hungarian cohort-controled trial of periconceptional multivitamin supplementation shows a reduction in certain congenital abnormalities use of , iu vitamin a during early pregnancy without teratogenic effect fish oil supplementation in pregnancy modifies neonatal allergen-specific immune responses and clinical outcomes in infants at high risk of atopy: a randomized controlled trial human plasma all-trans-, -cis and -cis oxoretinoic acid profiles during subchronic vitamin a supplementation prävention von neuralrohrdefekten. weiterhin nur geringe akzeptanz der empfehlungen zur folsäureprophylaxe folic acid supplements during pregnancy and the risk of miscarriage low maternal dietary intake of iron, magnesium, and niacin are associated with spina bifida in the offspring osteoporose und mehrlingsgraviditätein erfahrungsbericht mit positivem ausgang riboflavin status in pregnancy impact of folic acid fortification of the us food supply on the occurrence of neural tube defects folic acid metabolism and human embryopathy maternal vitamin d status during pregnancy and childhood bone mass at age years: a longitudinal study use of folic acid and delivery outcome: a prospective registry study folic acid and human malformation: a summary and evaluation maternal nutritional status and the risk for orofascial cleft in humans empfehlungen zur gabe von vitamin a in der schwangerschaft double-blind randomised controlled trial of folate treatment before conception to prevent recurrence of neuraltube defects anaemia and haematinics in pregnancy fetal outcome after intrauterine exposure to biphosphonates preventing neural tube defects with periconceptional folic acid supplementation: a population-based intervention program in the people's republic of china vitamin passage across the placenta biotin plasma levels of the human fetus high vitamin a intake in early pregnancy and major malformations: a multicenter prospective controlled study accelerated folate-breakdown in pregnancy medical research council (mrc) vitamin study research group. prevention of neural tube defects: results of the mrc vitamin study neural tube defect rates before and after food fortification with folic acid retinoic acid receptor alpha gene variants, multivitamin use, and liver intake as risk factors for oral clefts: a population-based case-control stud in denmark periconceptional use of multi-vitamins and the occurrence of neural tube defects folic acid supplementation and risk for imperforate anus in china hyperhomocysteinaemia and recurrent early pregnancy loss: a meta-analysis homocysteine and folate levels as risk factors for recurrent early pregnancy loss empfehlungen der-: perikonzeptionelle folsäuresubstitution. richtlinien zur prävention von neuralrohrdefekten randomised clinical trials of fish oil supplementation in high risk pregnancies the transplacental effects of alendronate on the fetal skeleton in rats folic acid fortification: a populationbased study of its effect on the incidence of open neural tube defects (ontds) zur biokinetik von mikronährstoffen und deren interaktionen. dialog ernährung und vitamins and minerals in pregnancy clinical teratology counseling and consultation report: high-dose g -carotene use during early pregnancy low vitamin b level as a risk factor for early recurrent abortion vitamin a congeners folic acid and neural-tube defects -time for action? teratogenicity of high vitamin a intake royal college of general practitioners. morbidity and drugs in pregnancy no effects of biphosphonates on the human fetus women and omega- fatty acids plasma folate, vitamine b , and homocyst(e)ine concentrations in preeclamptic and normotensive peruvian women is dietary intake of methionine associated with a reduction in risk for neural tube defect-affected pregnancies? periconceptional intake of vitamin supplements and risk of multiple congenital anomalies letter to the editor: intravenous iron polymaltose comlex for treatment of iron deficiency anaemia in pregnancy resistant to oral iron therapy possible prevention of neutraltube defects by periconceptional vitamin supplementation collagen synthesis during pregnancy, vitamin c availability, and risk of premature rupture of fetal membranes summary of the teratology society public affairs committee symposium: folic acid prevention of neural tube defects-public policy issues position paper: recommendations for vitamin a use during pregnancy primary prevention of neural tube defects with folic acid supplementation: cuban experience riboflavin deficiency and preeclampsia quantifying the effect of folic acid safety of vitamin a: recent results marginal biotin deficiency is teratogenic maternal plasma ascorbic acid (vitamin c) and risk of gestational diabetes mellitus vitamin c and the risk of gestational diabetes mellitus: a case-control study empfehlung für die praxis: pflanzentees und anderen zubereitungen mit ungeklärter herkunft, bei denen pyrrolizidinalkaloide enthalten sein könnten, sollten in der schwangerschaft strikt gemieden werden letter to the editor: ginger in preventing nausea and vomiting of pregnancy; a caveat due to its thromboxane synthetase activity and effect on testosterone binding ist ingwer ein klinisch relevantes antiemetikum? eine systematische Übersicht randomisierter kontrollierter studien drugs in pregnancy and lactation effect of viscum album l. on rapidly proliferating amniotic fluid cells. sister chromatid exchange frequency and proliferation index multiple follicular development associated with herbal medicine schmerzbehandlung mit pflanzlichen antirheumatika oral evening primrose oil: its effect of length of pregnancy and selected intrapartum outcomes in low-risk nulliparous women herbal medicinal products during pregnancy: are they safe seroius adverse effects of unconventional therapies for children and adolescents: a systematic review of recent evidence ginger treatment of hyperemesis gravidarum can herbal products be used safely during pregnancy? focus on echinacea homöopathie und schulmedizin in der schwangerschafts-und geburtsbetreuung re: preterm birth and licorice consumption during pregnancy weisen teedrogen der traditionellen chinesischen medizin qualitätsmängel auf? surveillance study of sinupret in comparison with data of the mainz birth registry potenzial toxicities of herbal therapies in the developing fetus ginger syrup as an antiemetic in early pregnancy cinnamon improves glucose and lipids of people with type diabetes are herbal medicinal products less teratogenic than western pharmaceutical products? reproductive toxicicity studies of d-camphor in rats and rabbits use of herbal drugs in pregnancy: a survey among norwegian women prospective comparitive study of the safety and effectiveness of ginger for the treatment of nausea and vomiting in pregnancy camphor ingestion for abortion (case report) veno-occlusive disease in a fetus caused by pyrrolizidine alkaloids of food origin uterotonic action of extracts from a group of medicinal plants a randomized controlled trial of ginger to treat nausea and vomiting in pregnancy homeopathy for induction of labour preterm birth and licorice consumption during pregnancy birth outcome in relation to licorice consumption during pregnancy severe congenital lead poisoning in a preterm infant due to a herbal remedy an evaluation of echinacea angustifolia in experimental rhinovirus infections ginger for nausea and vomiting in pregnancy: randomized double-masked, placebo-controlled trial investigation of the teratogenic potential of a zingiber officinale extract in the rat wissenschaftliche verlagsgesellschaft mbh stuttgart effect of ginger tea on the fetal development of sprague-dawley rats effect of ginger extract on pregnancy-induced nausea: a randomized controlled trial john's wort (hypericum perforatum) administered radionuclides in pregnancy a three-year follow-up of children imaged in utero with echo-planar magnetic resonance high-dose radioiodine treatment for differentiated thyroid carcinoma is not associated with change in female fertility or any genetic risk to the offspring diagnostic imaging committee, society of obstetricians and gynaecologists of canada. obstetric ultrasound biological effects and safety childhood and adult cancer after intrauterine exposure to ionizing radiation utilization of developmental basic science principles in the evaluation of reproductive risks from pre-and postconception environmental radiation exposures reproductive and teratologic effects of low-frequency electromagnetic fields: a review of in vivo and in vitro studies using animal models reproductive and teratogenic effects of electromagnetic fields pregnancy outcome after diagnosis of differentiated thyroid carcinoma: no deleterious effect after radioactive iodine treatment teratogen update: methylene blue dye use during amniocentesis and birth defects a review of the current use of magnetic resonance imaging in pregnancy and safety implications for the fetus deutsche gesellschaft für medizinische physik und deutsche röntgengesellschaft. pränatale strahlenexposition aus medizinischer indikation. dosisermittlung, folgerungen für arzt und schwangere infertility and pregnancy outcome among magnetic resonance imaging workers safety of indocyanine green angiography during pregnancy: a survey of the retina, macula, and vitreous societies methylene blue-induced hyperbilirubinemia in neonatal glucose- -phosphate dehydrogenase (g pd) deficiency safety of fluorescein angiography during pregnancy prenatal x-ray exposure and childhood cancer in twins antepartum dental radiography and infant low birth weight intrauterine effects of ultrasound: animal studies absence of harmful effects of magnetic resonance exposure at . t in utero during the third trimester of pregnancy: a follow-up study antepartum dental radiography and low birth weight normal placenta: gadoliniumenhanced, dynamic mr imaging changing perspectives on the genetic doubling dose of ionizing radiation for humans, mice, and drosophila effects of repeated prenatal ultrasound examinations on childhood outcome up to years of age: follow-up of a randomised controlled trial effects of frequent ultrasound during pregnancy: a randomized controlled trial parental exposure to medical radiation and neuroblastoma in offspring a -year retrospective analysis of the efficacy and safety of radioactive iodine in treating young graves' patients intrauterine effects of electromagnetic fields-(low frequency, mid frequency rf, and microwave): review of epidemiologic studies exposure to radioactive iodine- for scintigraphy or therapy does not preclude pregnancy in thyroid cancer patients embryotoxicity of stable isotopes and use of stable isotopes in studies of teratogenic mechanisms radiation risk in pregnancy effects of frequent ultrasound during pregnancy risk coefficients for childhood cancer after intrauterine irradiation: a review members of contrast media safety committee of european society of urogenital radiology (esur). the use of iodinated and gadolinium contrast media during pregnancy and lactation fetal thyrotrophin: the best indicator of long term thyroid function after in utero exposure to iodine- intrauterine effects of ultrasound: human epidemiology sudden infant death syndrome and prenatal maternal smoking: rising attributed risk in the back to sleep era methadone maintenance in a swiss perinatal center: ii. neonatal outcome and social resources fetal alcohol spectrum disorders in finland: clinical delineation of older children and adolescents prenatal exposure to binge drinking and cognitive and behavioral outcomes at age years longitudinal influence of prenatal cocaine exposure on child language functioning severity of prenatal cocaine exposure and child language functioning through age seven years: a longitudinal latent growth curve analysis caffeine use during pregnancy and child outcome: a -year prospective study prenatal cocaine exposure and children's language functioning at and . years: moderating effects of child age, birthweight, and gender effects of caffeine consumption on pregnancy outcome: a review maternal methadone dose and neonatal withdrawal maternal smoking during pregnancy and offspring iq parental cigarette smoking and the risk of acute leukemia in children maternal smoking during pregnancy and risk of brain tumors in offspring review of the epidemiological evidence relating toluene to reproductive outcomes effects of caffeine on development and behavior in infancy and childhood: a review of the published literature amphetamine addiction during pregnancy: year follow-up of growth and school performance smoking during pregnancy: a significant cause of neonatal thyroid enlargement maternal cocaine use and genitourinary tract malformations maternal smoking during pregnancy in relation to child overweight: follow-up to age years teratogen update: evaluation of the reproductive and developmental risks of caffeine maternal cigarette smoking during pregnancy and the risk of having a child with cleft lip/palate behavioral development in children prenatally exposed to drugs and alcohol prenatal tabacco effects on neuropsychological outcomes among preadolescents prenatal alcohol exposure predicts continued deficits in offspring size at years of age maternal smoking during pregnancy is associated with a higher risk of non-syndromic orofacial clefts in infants maternal cannabis use and birth weight: a meta-analysis european concerned action: maternal alcohol consumption and its relation to the outcome of pregnancy and child development at months birth outcome from a prospective, matched study of prenatal crack/cocaine use: i. interactive and dose effects on health and growth birth outcome from a prospective, matched study of prenatal crack/cocaine use: ii. interactive and dose effects on neurobehavioral assessment prenatal cocaine exposure prospective pregnancy outcome in women exposed to amphetamines maternal smoking before and after pregnancy: effect on behavioral outcomes in middle childhood moderate to heavy caffeine consumption during pregnancy and relationship to spontaneous abortion and abnormal fetal growth: a meta-analysis growth, development, and behavior in early childhood following prenatal cocaine exposure: a systematic review growth and pubertal milestones during adolescence in offspring prenatally exposed to cigarettes and marihuana a literature review of the consequences of prenatal marihuana exposure. an emerging theme of a deficiency in aspects of executive function growth from birth to early adolescence in offspring prenatally exposed to cigarettes and marijuana -and -month neurobehavioral follow-up of children prenatally exposed to marijuana, cigarettes and alcohol. develop behavioral prenatal marijuana and alcohol exposure and academic achievement at age ntp-cerhr expert panel report on the reproductive and developmental toxicity of amphetamine and methamphetamine passive smoking and pregnancy outcome in central poland further evidence for an association between -maternal smoking and craniosyostosis prenatal cocaine exposure and fetal vascular disruption methadone maintenance vs. implantable naltrexone treatment in the pregnant heroin user a randomised controlled trial of morphine versus phenobarbitone for neonatal abstinence syndrome drinking moderately and pregnancy. effects on child development use of buprenorphine in pregnancy: patient management and effects on the neonate inhalant abuse in pregnancy recognition of the fetal alcohol syndrome in early infancy maternal smoking and craniosynostosis maternal smoking during pregnancy and limb reduction malformations in sweden buprenorphine withdrawal syndrome in newborns: a report of cases multicenter allergy study group, germany. the association between pre-and postnatal tobacco smoke exposure and allergic sensitization during early childhood parental smoking and neonatal serum levels of polychlorinated biphenyls and hexachlorobenzene tabakspezifische transplazentare kanzerogene, nikotin und cotinin im urin von neugeborenen rauchender mütter a review of the literature relating coffeine consumption by women to their risks of reproductive hazards maternal smoking during pregnancy and the risk of congenital urinary tract anomalies smoking during pregnancy and the risk for hyperkinetic disorder in offspring united kingdom-based case-control study. smoking and orofacial clefts: a united kingdom-based case-control study tobacco smoking and oral clefts: a meta-analysis failure to recognize fetal alcohol syndrome in newborn infants is there a cocaine syndrome? dysmorphic and anthropometric assessment of infants exposed to cocaine zur frage der interruption bei alkoholkranken frauen prenatal exposure to salicylates and gastroschisis: a case-control study congenital anomalies after prenatal ecstasy exposure cocaine, pregnancy and postpartum intracerebral hemorrhage the maternal lifestyle study: cognitive, motor and behavioral outcomes of cocaine-exposed and opiate-exposed infants through three years of age transplacental transfer and metabolism of buprenorphine maternal passive smoking during pregnancy and fetal developmental toxicology. part : gross morphological effects maternal passive smoking during pregnancy and fetal developmental toxicology. part : histological changes cotinine in meconium indicates risk for early respiratory tract infections binge alcohol consumption by non-alcohol dependent women during pregnancy affects child behavior, but not general intellectual functioning; a prospective controlled study smoking during pregnancy and babbling abilities of the -month-old infant association of prenatal alcohol exposure with behavioral and learning problems in early adolescence developmental outcome of schoolage children born to mothers with heroin dependency: importance of environmental factors effect of paternal alcohol consumption before conception on infant birth weight moderate alcohol consumption during pregnnancy and the incidence of fetal malformations: a meta-analysis prenatal cocaine exposure: effects on the development of school-age children opiate addiction in gravidity -consequences for the newborn. results of an interdisciplinary treatment concept candidate genes for nonsyndromic cleft lip and palate and maternal cigarette smoking and alcohol consumption: evaluation of genotype-environment interactions from a population-based case-control study of orofacial clefts from in utero and childhood exposure to parental smoking to childhood cancer: a possible link and the need for action. human & experimental kokain in der schwangerschaft: ein zweites contergan? chemically induced birth defects follow-up of infants prenatally exposed to cocaine neonatal outcome following buprenorphine maintenance during conception and throughout pregnancy alkoholkonsum und intrauterine dystrophie maternal smoking and infantile gastrointestinal dysregulation: the case of colic methadone dosage for prevention of opioid withdrawal in children maternal coffeine consumption and spontaneous abortion: a review of the epidemiologic evidence talipes equinovarus and maternal smoking: a population-based case-control study in washington state effects of prenatal methamphetamine exposure on fetal growth and drug withdrawal symptoms in infants born at term teratogene effekte von nikotin prenatal alcohol exposure and long-term developmental consequences the fetal alcohol syndrome in adolescence die berliner verlaufsstudie von kindern mit einem fetalem alkoholsyndrom (fas). . pädiatrische befunde longterm outcome of children with fetal alcohol syndrome: psychopathology, behavior and intelligence correlates of psychopathology and intelligence in children with fetal alcohol syndrome die berliner verlaufsstudie von kindern mit einem fetalem alkoholsyndrom (fas). . psychiatrische und psychologische befunde fetal alcohol syndrome in adolescents and adults maternal drinking during pregnancy: attention and short term memory in -year old offspring: a longitudinal prospective study a dose-response-study of the enduring effects of prenatal alcohol exposure: birth to years laboratory work and pregnancy outcome high caffeine consumption in the third trimester of pregnancy: gender-specific effects on fetal growth what happens to babies exposed to phencyclidine (pcp) in utero? parental cigarette smoking and risk for congenital anomalies of the heart, neural tube, or limb teratogen update: smoking and reproductive outcomes teratogen update: toluene maternal consumption of coffee during pregnancy and stillbirth and infant death in first year of life: prospective study maternal and transplacental effects of cocaine oral clefts, maternal smoking, and tgfa: a meta-analysis of gene-environment interaction eine epidemiologische studie aus ungarn untersucht den schwangerschaftsverlauf von frauen, die wegen akuter vergiftungen während verschiedener phasen der schwangerschaft im krankenhaus behandelt wurden aflatoxine und cytochalasin b und d, gibt es bisher keine sicheren anhaltspunkte dafür, dass diese giftstoffe auch beim menschen fehlbildungen hervorrufen (Übersicht in schardein ) lässt erahnen, dass ggf. auch pflanzliche gesundheitsprodukte kritisch beobachtet werden müssen. dafür spricht auch eine publikation über leberschäden bei einem neugeborenen, dessen mutter große mengen pflanzentees zu sich genommen hatte, die pyrrolizidinalkaloide enthielten wie langwierig die klärung hypothetischer assoziationen zwischen fehlbildungen und giften in nahrung oder heilmitteln pflanzlicher herkunft sein kann, wird an dem von renwick ( ) vermuteten zusammenhang zwischen neuralrohrdefekten (exenzephalie, spina bifida) und dem verzehr von bräunlich verfärbten kartoffeln deutlich. es dauerte fast ein jahrzehnt, bis zweifelsfrei gezeigt werden konnte, dass die genannten fehlbildungen nicht folge des verzehrs von (verdorbenen) kartoffeln waren diphtherie) gibt es berichte über spezielle embryotoxische auswirkungen nach erkrankung der mutter in der schwangerschaft aspirin overdose in mother and fetus carbon monoxide poisoning in pregnancy are there teratogenic risks associated with antidotes used in the acute management of poisoned pregnant women reproductive hazards of industrial chemicals amatoxins do not cross the placental-barrier methanol toxicity in a newborn fatal colchicine poisoning. two particular cases. (abstract) multiple organ failure with the adult respiratory distress syndrome in homicidal arsenic poisoning intoxication by benzodiazepines during pregnancy toxic effect of podophylline application in pregnancy a study of adverse effects on the progeny after intoxication during pregnancy teratologic evaluation of infants born to mothers who attempted suicide by drugs during pregnancy morsures de serpents au cours de la grossesse arsenic ingestion in pregnancy acute iron poisoning: its effects and treatment pregnancy outcome after suicide attempt by drug use: a danish population-based study haloperidol overdose during pregnancy methanol poisoning during late pregnancy kctg-verlaufsbeobachtung nach diazepam-intoxikation thallium poisoning during pregnancy: a case report and comprehensive literature review placental transfer of n-acetylcysteine following human maternal acetaminophen toxicity snake bite in pregnancy mother and fetus both survive from severe paraquat intoxication organophosphate poisoning in pregnancy: a case report arsenic encephalopathy in pregnancy with recovery podophyllum: suspected teratogenicity from topical application fetal damage due to mushroom poisoning with amanita phalloides during the first trimester of pregnancy deferoxamine treatment for acute iron intoxication in pregnancy fetal compromise caused by maternal carbon monoxide poisoning maternal ginseng use associated with neonatal androgenization a multicenter, prospective study of fetal outcome following accidental carbon monoxide poisoning in pregnancy acute iron intoxication in pregnancy: case report and review of the literature a review of venomous animal bites and stings in pregnant patients exposure to non-steroidal anti-inflammatory drugs during pregnancy and risk of miscarriage: population-based cohort study drug overdoses during pregnancy acute maternal arsenic intoxication with neonatal death Über einen fall von kohlenoxydgasschädigung des kindes in der gebärmutter the outcome of pregnancy following iron overdose by the mother drugs during pregnancy and lactation the consequences of iron overdose and its treatment with desferrioxamine in pregnancy outcome of pregnancy following deliberate iron overdose by the mother paracetamol overdose in pregnancy: analysis of the outcomes of cases referred to the teratology information service podophyllin poisoning associated with the treatment of condyloma accuminatum. a case report intrauterine fetal death caused by pit viper venom poisoning in early pregnancy risk of adverse birth outcome and miscarriage in pregnant users of non-steroidal anti-inflammatory drugs: population based observational study and case-control study fatal iron intoxication in late pregnancy aspirin poising during pregnancy: increased fetal sensitivity management of snake and spider bite in pregnancy neonatal bromide intoxication: prenatal ingestion of a large quantity of bromides with transplacental accumulation in the fetus botulism and pregnancy spina bifida, anencephaly, and potato blight botulism in a pregnant woman favourable neonatal outcome following maternal paracetamol overdose and severe fetal distress. case report hepatic veno-occlusive disease in newborn infant of a woman drinking herbal tea mega-dose carbamazepine complicating third trimester of pregnancy chemically induced birth defects amanita poisoning during pregnancy mort néonatale par malformations multiplesà la suite de l'action du poison d'abeilles snakebite during pregnancy organophosphate poisoning associated with fetal death: a case study transplacental neonatal digitalis intoxication hyperbaric oxygen teatment during pregnancy in acute carbon monoxide poisoning podophllin poisoning: systemic toxicity following cutaneous application observations of an infant born to a mother with botulism delirium induced by topical application of podophyllin: a case report paraquat intoxication during pregnancy: a report of cases poisoning in pregnancy. maternal & fetal toxicology nd edition s. - methanol poisoning during pregnancy -prediction of risk and suggestions for management birth weight and congenital anomalies following poisonous mushroom intoxication during pregnancy intentional iron overdose in pregnancymanagement and outcome acute intentional iron overdose in pregnancy experience with acute paraquat poisoning in crete successful therapy of iron intoxication in pregnancy with intravenous deferoxamine and whole bowel irrigation inhalational methanol toxicity in pregnancy treated twice with fomepizole foetal and neonatal exposure to aflatoxins fatal systemic poisoning following podophylline treatment of condyloma accuminatum maternal water intoxication as a cause of neonatal seizures leichten fällen nur durch mäßig ausgeprägte mentale retardierung auffiel, kamen bei manchen kindern noch herz-, skelett-, augen-und ohrfehlbildungen hinzu in der europäischen eurohazcon-studie war das risiko bei frauen, ein fehlgebildetes kind zur welt zu bringen, erhöht, wenn sie im umkreis von km zu einer giftmülldeponie wohnten. außerdem gab es hinweise auf vermehrtes auftreten chromosomaler störungen (vrijheid deutsche forschungsgemeinschaft, jährliche aktualisierung) für chemische arbeitsstoffe bzw. industriechemikalien aufgenommen. mit zustimmung des bundesarbeitsministers von der dfg herausgegeben hat die liste nicht nur arbeitsrechtliche konsequenzen für betroffene frauen und für die berufsgenossenschaften, sondern sie ist bis heute die einzige "amtliche" grundlage für die ärztliche beratung von schwangeren, die am arbeitsplatz oder auch außerhalb ihrer arbeit mit industrie-und umweltchemikalien in kontakt kommen. in tabelle . sind die stoffe aufgelistet, die die mak-werte-kommission hinsichtlich ihrer fruchtschädigenden eigenschaften verschiedenen risikogruppen zugeordnet hat zu den auswirkungen von mobiltelefonnutzung und den digitalen mobiltelefonsendern in wohnraumnähe gibt es bisher keine aussagefähigen studien zu potenziellen auswirkungen auf eine schwangerschaft. nach einem stromschlag während der schwangerschaft sollte das fetale befinden per ultraschall kontrolliert werden. andere regelmäßige applikationen von erheblichen elektromagnetischen feldern sollten vermieden werden chernobyl fallout and outcome of pregnancy in finland environmental exposure to lead and childrens intelligence at the age of seven years reproductive hazards of industrial chemicals a preliminary assessment of the consequences for inhabitants of the uk of the chernobyl accident während der schwangerschaft verzehr bestimmter fischarten einschränken organochlorines and heavy metals in pregnant women from the disko bay area in greenland risk of selected borth defects by maternal residence close to power lines during pregnancy chernobyl, childhood cancer, and chromosome effect of magnetic field emitted by cellular phones on fetal heart rate maternal residental proximity to hazardous waste sites and risk for selected congenital malformations an assessment of the developmental toxicity of inorganic arsenic health status at birth of inuit newborns prenatally exposed to organochlorines risk of congenital anomalies near hazardous-waste landfill sites in europe: the eurohazcon study pesticide exposure and birth weight: an epidemiological study in central poland fetal death and congenital malformation in babies born to nuclear industry employees: report from the nuclear industry family study spontaneous abortion in dry cleaning workers potentially exposed to perchloroethylene maternal blood lead effects on infant intelligence at age months accidental electrical shock in pregnancy: a prospective cohort study european food safety authority (efsa). risikobewertung für quecksilber in fisch für besonders gefährdete bevölkerungsgruppen hydroxylated pcb metabolites and pcbs in serum from pregnant faroese women general, reproductive, developmental, and endocrine toxicity of boronated compounds follow up study of children born to mothers resident in seascale, west cumbria persistent organochlorine compounds and birth weight cognitive deficit in -year-old children with prenatal exposure to methylmercury animal testing and alternative approaches for the human health risk assessment under the proposed new european chemicals regulation teratogen update: polychlorinated biphenyls intellectual impairment in children exposed to polychlorinated biphenyls in utero inhalant abuse in pregnancy relationships of maternal blood lead and disorders of pregnancy to neonatal birthweight drinking water chlorination and delivery outcome -a registry-based study in sweden effects of dioxins and polychlorinated biphenyls on thyroid hormone status of pregnant women and their infants spontaneous abortions and congenital malformations among women exposed to tetrachlorethylene in dry cleaning effect of polychlorinated biphenyls on psychodevelopment parental smoking and neonatal serum levels of polychlorinated biphenyls and hexachlorobenzene exposure to organic solvents during pregnancy and oral clefts: as case control study maternal and neonatal hair mercury concentrations: the effect of dental amalgam paternal organic solvent exposure and adverse pregnancy ourtcomes: a meta-analysis preganancy outcome following maternal organic solvent exposure: a meta-analysis of epidemiologic studies intellectual function of children exposed to polychlorinated biphenyls in utero the oestrogenic potential of the phthalate esters prenatal methyl mercury exposure from ocean fish consumption in the seychelles child development study chlorination disinfection by products in water and their association with adverse reproductive outcomes: a review stillbirths among offspring of male radiation workers at sellafield nuclear processing plant effects of environmental exposure to polychlorinated biphenyls and dioxins on cognitive abilities in dutch children at months of age the effect of ambient carbon monoxide on low birth weight among children in southern california between and . environ health perspect intrauterine effects of electromagnetic fields-(low frequency, mid frequency rf, and microwave): review of epidemiologic studies sex ratio after exposure to dioxinlike chemicals in taiwan chemically induced birth defects untersuchungen zur quecksilberbelastung fetaler und frühkindlicher organe infolge mütterlicher exposition durch zahnamalgam maternal periconceptional use of electric bed-heating devices and risk for neural tube defects and orofacial clefts maternal occupational exposure to organic solvents during pregnancy and subsequent cognitive and visual functioning in the child: a prospective controlled study women in dental surgeries: reproductive hazards in environmental exposure to metallic mercury significant increase in trisomy in berlin nine months after the chernobyl reactor accident: temporal correlation or causal relation? bewertung des embryotoxischen risikos von industriechemikalien in der schwangerschaft review of recent vietnamese studies on the carcinogenic and teratogenic effects of phenoxy herbicide exposure prenatal pcb exposure and neonatal behavioral assessment scale (nbas) performance cognitive development of preschool children prenatally exposed to pcbs and mehg maternal seafood diet, methylmercury exposure and neonatal neurologic function male reproductive disorders in humans and prenatal indicators of estrogen exposure. a review of published epidemiological studies lifetime exposure to environmental lead and children's intelligence at - years -the port pirie cohort study blitzschlag -tödliche schädigung des fetus in der spätschwangerschaft chromosomal congenital anomalies and residence near hazardous waste landfill sites lead exposure and motor functioning in / -year-old children: the yugoslavia prospective study teratogen update: toluene effects of low-level lead exposure in utero menstrual disorders among drycleaning workers eine substitution ist selten indiziert, z. b. bei addison-krankheit. die erforderlichen dosen an glucocorticoiden und mineralocorticoiden helfen, wieder physiologische verhältnisse zu erreichen, und haben weder für die mutter noch für den exponierten fetus nebenwirkungen. eine langzeitbehandlung mit hohen therapeutischen dosen bei allergischen, entzündlichen oder proliferativen erkrankungen führt zu gravie-andere augen-, nasen-und ohren-präparate glucocorticoide, cromoglicinsäure, antihistaminika, antibiotika und aciclovir sowie filmbildner ("künstliche tränenflüssigkeit"), wie z. b. povidon (z. b. arufil ® ), dürfen indikationsgerecht angewendet werden. aus grundsätzlichen erwägungen sollte auf chloramphenicol verzichtet werden.die nasale oder inhalative anwendung von budesonid und anderer corticosteroide hat keine nennenwerte teratogenität gezeigt (källén ) .bei frauen, die im rahmen einer randomisierten doppelblindstudie fluticason-nasenspray benutzten, fand sich gegenüber der placebogruppe kein unterschied in der entwicklung der neugeborenen (ellegard ) .zu dem neueren ophthalmologikum loteprednol (lotemax ® ) gibt es bisher keine erfahrungen zur anwendung in der schwangerschaft. es ist ähnlich zu bewerten wie andere lokale glucocorticoide und für eine kurzzeitanwendung akzeptabel, wenn keine anderen therapiemöglichkeiten zur verfügung stehen. g . . hämorrhoidenmittel pharmakologie und toxikologie. hämorrhoidenmittel (hämorrhoidensalben und suppositorien) sind lokaltherapeutika, die als einzelstoffe oder in kombination meistens lokalanästhetika, glucocorticoide, antibiotika und desinfizienzien enthalten. diese präparate werden auch zur nachbehandlung operativer eingriffe im rekto-analen bereich eingesetzt.empfehlung für die praxis: die üblichen hämorrhoidenmittel haben sich in der schwangerschaft als unbedenklich erwiesen. g . . venentherapeutika aescin-präparate (rosskastanienextrakt) bei venenbeschwerden sind in der schwangerschaft bisher nicht als problematisch aufgefallen, aber nicht systematisch untersucht.eine venenverödung bei krampfadern, z. b. mit polidocanol (macrogollaurylether; z. b. aethoxysklerol ® ), darf -falls zwingend erforderlich -auch während der schwangerschaft durchgeführt werden.da ausreichende erfahrungen auch für die lokale anwendung von minoxidil in der schwangerschaft nicht vorliegen, sollte auf eine längerfristige anwendung verzichtet werden.gleiches gilt für eflornithin (vaniqa ® ), das zur äußerlichen behandlung des hirsutismus angeboten wird. g . . kosmetika kosmetika, auch haarkosmetika einschließlich färben und dauerwelle, dürfen, wenn es die befindlichkeit der schwangeren fördert, im üblichen rahmen angewendet werden. adams j, lammer e. relationship between dysmorphology and neuropsychological functions in children exposed to isotretinoin (in utero). in: fujii t, boer gj (ed (hubbard ) . ließen erste studien vermuten, dass sich diese schwerwiegenden angeborenen fehlbildungen durch gabe von multivitaminpräparaten (smithells ) bzw. folsäure (laurence ) verhindern lassen. umfangreiche untersuchungen in den usa (mulinare ) , australien (bower ) , kuba (vergel ), england (medical research council und ungarn bestätigten eine protektive wirkung der folsäuresubstitution. in der ungarischen studie lag die häufigkeit von neuralrohrdefekten bei kindern von frauen, die vor und während der schwangerschaft ein multivitaminpräparat eingenommen hatten, das eine tägliche dosis von , mg folsäure enthielt, um % bzw. % ) niedriger als in der kontrollgruppe, die nur spurenelemente eingenommen hatte g . . ultraschall-und magnetresonanz-kontrastmittel pharmakologie und toxikologie. als kontrastmittel wird bei der ultraschalldiagnostik d-galaktose (echovist- ® , echovist- ® ) eingesetzt, von dem kein pränatal toxisches risiko zu erwarten ist.gadopentetsäure (magnevist ® ), gadobensäure (multihance ® ), gadodiamid (omniscan ® ), gadoteridol (prohance ® ), gadotersäure (dotarem ® ) und gadoxetsäure (primovist ® ) sind ionische, paramagnetische kontrastmittel, die bei der magnetresonanzdarstellung (mrt) benutzt werden. soweit untersucht, ergaben tierversuche keine hin-arnd) und die als alcohol related birth defects (arbd) bezeichneten fehlbildungen.das fetale schädigungsmuster hängt von der zeitlichen intensität des mütterlichen alkoholkonsums in der schwangerschaft ab. so führt ein intensiver alkoholmissbrauch in der frühschwangerschaft eher zu den typischen kraniofazialen dysmorphien und organschädigungen, während heftiges trinken in der späteren fetalen phase der schwangerschaft zu einer ausgeprägten neuronalen schädigung des rasch wachsenden gehirns mit der folge psychomentaler und kognitiver störungen sowie ausgeprägter veränderungen des verhaltens (fae) führt. schwere alkoholikerinnen trinken in der regel während der gesamten schwangerschaft.die häufigkeit des auftretens eines klassischen fetalen alkohol-syndroms bei einer alkoholkranken schwangeren liegt bei - % (abel , majewski , die inzidenz eines fas bei : geburten (abel ). diese zahlen sind schätzwerte und hängen stark vom sozialen umfeld des jeweils untersuchten kollektivs ab. die diagnose fas wird bei geburt selten und die diagnose fae in der neugeborenzeit praktisch nie gestellt (little ). die häufigkeit der weniger ausgeprägten fae-kinder ist sicher deutlich höher und liegt etwa bei - : geburten (schöneck ) , dazu gibt es bisher keine größeren studien. das bild eines voll ausgeprägten fas ist gekennzeichnet durch eine prä-und postnatale dystrophie, mikrozephalie, mentale retardierung und minderwuchs. dazu gehören: eine typische kraniofaziale dysmorphie mit schmalen lidspalten (blepharophimose), schmalem lippenrot, kurzem breiten nasenrücken, langem unmodelliertem philtrum und geringgradig dysmorphen ohren sowie verschiedene fakultative organschädigungen, insbesondere herzfehler, nierenfehlbildungen und gaumenspalte sowie kleinere hautveränderungen. die diagnose kann prima vista gestellt werden.kinder mit fae weisen nur geringe dysmorphe störungen auf, außerdem können ein mikrozephalus, minderwuchs, eine diskrete mentale retardierung, aufmerksamkeitsstörungen (adhs) und oft ausgeprägte psychische verhaltensauffälligkeiten beobachtet werden. die diagnose gelingt nur bei bekanntem mütterlichem alkoholabusus.langzeituntersuchungen von kindern mit fas zeigen eine unerwartete persistenz des klinischen bildes. so bildet sich zwar die kraniofaziale dysmorphie langsam zurück, aber mikrozephalie, minderwuchs, aufmerksamkeitsstörungen und kognitive defizite bleiben. die schulleistungen verschlechterten sich trotz erheblicher förderung der kinder durch ihre pflegeeltern in einer -jahres-"follow-up"-studie ebenso wie ihre soziale integration. während der pubertät verstärken sich in der regel die probleme der betroffenen kinder, so dass auch zu diesem sicht in sasco ) . andere untersuchungen finden keine hinweise auf transplazentare karzinogenese (brondum ) . in einer schwedischen prospektiven studie, die insgesamt , millionen geburten einschloss, wurde der zusammenhang von mütterlichem rauchen in der schwangerschaft und kindlichem risiko für hirntumoren untersucht. die autoren fanden bei den raucherinnen einen signifikanten anstieg der häufigkeit von hirntumoren, jedoch keinen unterschied zwischen benignen und malignen tumoren. besonders betroffen waren - -jährige kinder. die autoren interpretieren eine mögliche kausalität sehr vorsichtig (brooks , hersh . außerdem wurden bei über der hälfte von untersuchten schwangeren vorzeitige wehen und frühgeburten beobachtet . toluol und andere organische lösungsmittel wurden auch hinsichtlich ihrer auswirkungen am arbeitsplatz untersucht. dort fand man z. t. hinweise auf erhöhte spontanabortraten (bukowski , taskinen , die bei toluol schnüffelnden schwangeren so nicht beobachtet wurden (bukowski pantanowitz ) . antiseren stehen bislang nicht im verdacht, entwicklungstoxisch zu wirken. sie können jedoch im falle einer mütterlichen anaphylaxie mittelbar auch den fetus gefährden.ein fallbericht über ein kind mit multiplen fehlbildungen, dessen mutter im . schwangerschaftsmonat von einer biene gestochen wurde (schneegans ) , hat anekdotischen charakter und belegt selbstverständlich keinen kausalzusammenhang.empfehlung für die praxis: die behandlung mit antiseren nach schlangenoder giftspinnenbissen darf nicht wegen der schwangerschaft unterbleiben. sie kann auch bei fehlen von vergiftungssymptomen der mutter indiziert sein, wenn unregelmäßigkeiten der fetalen herzaktion oder eine abnahme der kindsbewegungen beobachtet werden. g . . pilze nach pilzvergiftung mit dem hochgiftigen knollenblätterpilz (amanita phalloides) erlitt eine patientin im ersten schwangerschaftsdrittel einen abort (kaufmann ) . das zyklische oktapeptidtoxin alpha-amanitin hemmt die proteinsynthese und kann über die plazenta hinweg die fetale leber schädigen. in einem weiteren fall brachte eine patientin nach vergiftung im . monat und erfolgreicher behandlung mit plas-berufliche exposition. zahnärztliches personal hat beruflich kontakt mit hg, daher wurden störungen der fruchtbarkeit vermutet. eine kleine studie mit messung der individuellen hg-belastung fand eine signifikante zunahme der abortrate (sikorski ) , andere untersuchungen konnten diesen effekt nicht bestätigen. eine häufung von fehlbildungen, mentaler retardierung und anderen funktionsstörungen ließ sich in keiner dieser arbeiten nachweisen . schwangeren auf den färöer inseln zeigten, dass der verzehr von seefischen und stark belastetem fleisch und fett von meeressäugern bei säuglingen zu einer konzentrationsabhängigen verschlechterung neurologischer testergebnisse führte (steuerwald ) . auch über kognitive defizite bei -jährigen kindern wurde im zusammenhang mit mütterlichem verzehr belasteter fische berichtet (grandjean ) . eine untersuchung an "normal exponierten" mutter-kind-paaren auf den seychellen erbrachte hingegen keine mit der methylquecksilber-exposition korrelierenden entwicklungsdefizite bis zum alter von jahren (myers (emory ) . ein um ? g/dl höherer wert soll zu einem um - punkte schlechteren ergebnis im bayley-test führen (Überblick bei wong ) . in einer mit blei belasteten region jugoslawiens wurden feinmotorische entwicklungseinschränkungen bei kindern im alter von ⁄ jahren festgestellt, die mit den postnatal ermittelten bleikonzentrationen im blut korrelierten (wasserman ) . die exposition mit blei nach der geburt soll den ergebnissen der so genannten port-pirie-studie zufolge für einschränkungen der intelligenzentwicklung entscheidender sein als eine exposition während der schwangerschaft (baghurst , tong (gladen ) . bei den inuit (eskimos) fand man eine verringerung der körperlänge bei neugeborenen in abhängigkeit von der kontamination mit persistierenden organochlorverbindungen bei den müttern (dewailly (rogan ; siehe auch abschnitt . . zu estrogenen und kapitel . zur exposition des vaters). neurologische auffälligkeiten bei "normaler" belastung. bei neugeborenen von müttern, die mit pcbs belastete fische aus dem ontariosee (usa) verzehrt hatten, korrelierten abweichungen in verhaltenstests und bei autonomen reflexen mit einer erhöhten pcb-konzentration ( g ng/g fett) im nabelschnurblut (stewart ) . eine kontamination der verzehrten fische mit anderen organischen umweltschadstoffen führte hingegen nicht zur beeinträchtigung der reaktionen beim neugeborenen. auch andere publikationen beschreiben abweichungen beim muskeltonus, beim visuellen erkennen und bei verschiedenen psychomotorischen eigenschaften bei neugeborenen und älteren kindern, deren mütter in der schwangerschaft vermehrt mit pcbs exponiert waren (Übersicht in jacobson ) . es wird diskutiert, dass pcbs und dioxine durch die beeinträchtigung der fetalen schilddrüsenfunktion die zns-reifung stören und zu entwicklungsauffälligkeiten führen (koopman-esseboom ) . insgesamt vermitteln diese studien den eindruck, dass die vorgeburtliche exposition mit pcbs die entwicklung des kindes stärker beeinflusst als jene über die muttermilch (patandin ) .jacobsen und jacobson ( ) beschrieben psychomentale auswirkungen nach perinataler pcb-exposition und das fortbestehen intel-lektueller defizite bis zum alter von jahren nach leicht erhöhten pcb-konzentration während der schwangerschaft, die von anderen autoren nicht bestätigt wurden (stewart , lackmann , middaugh .bei kindern, deren mütter während der schwangerschaft mit polybromierten biphenylen (pbbs) kontaminiertes fleisch gegessen hatten, wurde in michigan (usa) eine neuro-psychologische entwicklungsverzögerung festgestellt, die im alter von - jahren nicht mehr nachzuweisen war. das flammschutzmittel pbb war mit tierfutter verwechselt worden.empfehlung für die praxis: die vorliegenden erfahrungen sind unzureichend für eine differenzierte risikobeurteilung. es wird vermutet, dass persistierende halogenierte kohlenwasserstoffe in höherer dosis aborte, früh-und totgeburten und in sehr hoher dosis auch fehlbildungen verursachen können. die heute bei uns übliche umweltbelastung mit diesen stoffen führt offenbar nicht zu störungen des schwangerschaftsverlaufes oder einem erhöhten fehlbildungsrisiko. mögliche psychomotorische auswirkungen bei mäßig erhöhter exposition in der schwangerschaft sowie der geringe sicherheitsabstand zum tierexperimentell ermittelten noael bei polychlorierten dioxinen und furanen erfordern weiterhin energische präventivmaßnahmen. g . unter organischen lösungsmitteln versteht man zahlreiche -auch chlorierte -kohlenwasserstoffe, die leicht flüchtig und lipophil sind. dazu gehören aceton, benzol, ethylether, n-hexan, methyl-ethyl-keton, tetrachlorethen (per) , toluol, trichlorethen (tri), xylol. lösungsmittel werden farben und klebstoffen zugesetzt, zur chemischen reinigung benutzt und in großen mengen bei verschiedenen industriellen prozessen (entfettung, lederverarbeitung, nahrungsmittelherstellung etc.) eingesetzt. organische lösungsmittel können durch inhalation und über die haut aufgenommen werden. ihre biologische halbwertszeit kann -wie bei tetrachlorethen -mehr als stunden betragen.besonderheiten in der schwangerschaft. für die meisten lösungsmittel wurde ein plazentarer Übergang experimentell nachgewiesen.zur pränatalen exposition beim menschen gibt es einige falldarstellungen geschädigter kinder und retrospektive arbeiten, bei denen die auswirkungen von gemischen verschiedener lösungsmittel am arbeitsplatz untersucht wurden (Überblick bei .mehrere berichte beschreiben kinder, deren mütter während der schwangerschaft lösungsmittel schnüffelten. intrauterine wachstums- (boice , baverstock ). einen hinweis darauf, dass radioaktive nuklide bereits präkonzeptionell durch paternal-mutagene wirkung, d. h. nach strahlenexposition der väter, das malignomrisiko von kindern erhöhen können, ergaben studien an vätern mit einem arbeitsplatz in der kernbrennstoffaufbereitungsanlage sellafield in england (gardner ) . eine umfangreiche analyse von ca. . geburten in der umgebung von sellafield in der zeit von - zeigte auch eine erhöhte rate von totgeburten bei solchen vätern (parker ) . eine andere untersuchung an über . in der britischen atomindustrie beschäftigten männern und knapp . beschäftigten frauen ergab nur für die präkonzeptionell exponierten frauen ein erhöhtes fehlgeburtsrisiko, totgeburten waren jedoch nicht häufiger, auch nicht die fehlbildungen (doyle ) . key: cord- -f v cih authors: paul, aneesh mathews; susanthomas, sinnu title: multifaceted covid- outbreak date: - - journal: nan doi: nan sha: doc_id: cord_uid: f v cih the time when everyone is struggling in the cruel hands of covid , we present the holistic view on the effects of this pandemic in certain aspects of life. a lot of literature exists in covid- , but most of them talk about the social and psychological side of the covid problems. covid- has affected our day-to-day life and its effects are extensive. most of the literature presents the adverse effect of the pandemic, but there are very few state-of-the-art approaches that discuss its beneficial effects. we see the multiple faces of the pandemic in this paper. to the best of our knowledge, this is the first review that presents the pros and cons of the pandemic. we present a survey that surrounds over effects on education, environment, and religion. the positive side of covid- raises an alarm for us to wake up and work in that direction. digital transformation in the marketplace. devaux et al. [ ] investigated the effects of hydroxychloroquine against sars-cov- virus. faridi [ ] has studied the effect of middle east respiratory syndrome coronavirus (mers-cov) that has caused havoc in saudi arabia in . the author has seen the effect of mers-cov on male and female in riyadh. the authors in [ ] , [ ] , [ ] , [ ] assessed the psy-chological stress of covid- on health workers. xiang et al. [ ] reported an overview of infected healthcare workers in china and italy during the early periods of the covid- . the authors in [ ] , [ ] , [ ] studied some social impacts of covid- . chakraborty and maity [ ] studied the covid- effect on the economy and global environment. ivanov [ ] predicted the impact of covid- on global supply chains. xu et al. [ ] studied the air quality index to see the effects of covid- on the environment. chinazzi et al. [ ] studied the effect of travel and quarantine influence on the dynamics of the spread of covid- . braun [ ] narrated examples of the situations of the poor during covid- . ahmed et al. [ ] highlighted the precarious position of postdoctoral fellows in academic positions due to covid- . staniscuaski et al. [ ] projected out the problems faced by academic mothers having many difficulties working at home during covid- . bouillon et al. [ ] discussed the positive side effect of coronavirus on air pollution. suicide rate has increased during the pandemic time [ ] , [ ] .the situation of covid- has diverse effects in india [ ] . in this paper, we study the multi-faceted effects of covid- on our planet. our contribution in this paper is threefold. ) the pandemic has affected the entire education sys-tem and a new era of distance learning has emerged. a review on various education systems during the pandemic is looked out. ) covid- has benefits in certain areas such as the environment. the environmental effects are discussed. ) overall change in religious practices has changed and we review these aspects in this paper. the remainder of this paper is organized as follows. section ii highlights the overall change in the education system during the covid- season, and discusses the social and psychological impacts of the pandemic. section iii presents the religious and environmental effects of coronavirus. section iv presents the conclusions of this paper. education system is one of the prime pillars in developing a nation. it constitutes an important ingredient in deter-mining the growth of a country. human development is an important determinant in a person's health and trade. the education system is severely interrupted in most of the countries since the outbreak of this pandemic across the globe. the schools, colleges, and universities are in the total closure mode. billions of academic learners became devoid of their knowledge acquisition during this pandemic. the teachers, students, schools, and families -all became a victim of this bitter truth. the world has gone under complete reorganization during this period be it any sector, the education sector is not left apart. the speed of the pandemic and the closure of schools was so fast that it was difficult to come up with a solution with all facilities. the closure of educational institutes will not only have short term impact, but leave a footprint on economic and societal components. there are number of areas in education that is affected by the pandemic: the landscape of higher education across the world is defined by the cross border movements of the students. globally every year there is an increase of % in the number of students studying abroad as shown in fig. . as per the unesco [ ] , the students enrolled for higher education for a period of typically a year to seven years. according to the statistics given by organization for economic cooperation and development (oecd), the in-ternational student population with demographic changes is likely to reach million by [ ] . most of the international students prefer either the united states, the united kingdom, germany, france, or australia for their higher education [ ] as shown in fig. . as per the statistics in , the top host countries involved in sending students to other countries include china, india, south korea, and france [ ] as shown in fig. . the pandemic has brought a sluggish impact on the movement of students across the border. the travel restrictions during lockdown and the fear of pandemic will affect the cash flow at the universities. parents are afraid to send their ward across any border in this situation. the universities in these countries are undergoing extreme pressure on student admission. if this problem persists, there is a possibility of decline in international higher education in the com-ing years. the pandemic has brought a devastating effect on the global education system. the pandemic has shrinked the world under their own home and hometown and cross border movements seem to be a threat to the life of an individual. ) online learning active learning is not only a source of fun but also a source of formation of cognitive social skills. carlsson et al. [ ] emphasized on the increase in cognitive skills with the total number of school days attended. the study carried out in sweden showed that crystallized intelligence can be aug-mented significantly by % of a standard devia-tion while attending ten days of extra schooling. the closure of schools for almost a month at the beginning of this pandemic can cause a trivial loss of % of the standard deviation. the pandemic has left the learning systems with no options other than embracing a distant or online learning. as per the statistics released by unesco [ ] , the pandemic has affected nearly . billion learners around the world. the recovery of the disruption of the learning process is essential to facilitate the continuity of the education system. when physical presence is a risky situation, an alternative has to be taken at various levels of learning. online learning is a new strategy embraced by the education system in this time of pandemic. the transition from active learning to passive learning was very rapid during this pandemic. the curriculum was not designed for passive learning, so the viewers are losing their interest in the content. the shift in developed countries to the online learning system does not pose any problem, but for developing and under-developed countries -it is a challenging situation. the rural areas of these countries do not have the basic infrastructure to facilitate the online learning. the pandemic has posed a threat to the overall development of the underprivileged in these countries resulting in shattering their economies. the video telephony softwares is being used for distant learning. the concept of keeping the electronic gadgets far from the children has been loosened even for a primary school going child during this pandemic. the online learning has removed the commuting time for the learners but on the other hand, made them addicted to electronics devices leading to many social, psychological, and physical disabilities. online learning brought a paradigm shift in one's own comfort zone. the hassle of traffic jams, pollution, queues, health problems, allergies is halted in this course of time. most of the learners are happy with the online learning system since environmental problems do not leave them void of attending classes. online learning has brought an end to the centuries old practice of chalk and talk. due to the sudden change to the online learning in the education system, the preparedness of the tutors was a concern. an inhibition of this sudden change was found in the tutors during the beginning phase. the course curriculum was not made for passive learning. the sudden shift in the teaching system with inadequate preparation from the learner side was also noticeable. teaching is a knack that everybody is not gifted with, so many tutors are not so effective in an online mode. in countries like india, where there is a huge shortage of technology savvy tutors, this model of learning would not work out. lack of infrastructure and resources in the rural parts of these countries is an obstacle for teachers for a complete preparation of imparting the knowledge. an unavailability of dedicated online platforms is posing a threat for outcome based education. the tutors are adjusting the platforms with the video telephony platforms. if the problem of pandemic persists, there is a need for creating dedicated learn-ing platforms. most of the schools and universities undergo the ad-mission process during the month of may-june for fall semester. due to the severity of the pandemic in many parts of the world, the admission process is hindered. the situation in the admission process is becoming alarming in the foreseeable future with the pandemic situation. traditional admission procedures would not take place in this season. new procedural strategies for admissions should be considered in order to fill the gap in this pandemic. some universities are not con-sidering taking any students the current academic year, while some are luring people with discounts. it is a crucial task for the students to decide which school they would like to attend without visiting respective campuses. the pandemic has forced people to create a virtual world of working at home. the virtual world cre-ates effortless paths to collaborate across the globe. the conferences, academic meetings, classes, and seminars have gone online leaving a space for academic collaborations. we see a lot of unprecedented collaborative work globally among the educators [ ] during this pandemic leading to a loss in the travel economy. the cancellation of universityfunded international travel for conferences, blanket bans on any international travel for spring break, canceling study-abroad programs [ ] made different academicians closer virtually. collaborations serve a larger purpose as an individual and also as an organization [ ] . there are lots of scope for online conferencing platform business. the concept of education will be reformed envisaging the global collaboration. globally, the collaboration has brought a new direction to certification courses and degrees. these collaborations fulfills the need of each other while dividing the work in chunks. the pandemic has brought a halt to the organizational structure making a scarcity in the manpower. the universities are facing challenges to recruit new students, and faculty during this pandemic. the retention is also questionable. the recruitment for the faculty is a worrisome issue for the administration when the risk of losing students is hovering around them. when survival of many institutes is a burden for them, the recruitment of new faculty members increases their load. due to the recession in the corporate sector, the recruitment process for the students is a great disaster. the job offers have been withdrawn creating a havoc in the student community. the global outlook of the pandemic would massively devastate the livelihoods in the entire world. fig. the consequences of a sudden shift in the learning system brought a slowdown to the world economy. the international students from china and india constitute . % and . % of the total international students in the usa higher education sector. the travel restrictions during the pandemic would cut down the admission process leading to an economic burstdown. the conveyance to the institutes are at a halt causing recession in the travel sector. all the learners cannot afford to stay near their institutes, so they stay far and face a time-consuming and costly commute [ ] . students spend approximately £ a month for commutation to their academic institutes. pandemic has saved the pocket of students in higher education. in countries like india, private schools and private vehicles charge a heavy conveyance fare for the commutation. the pandemic has given relief to the parents. same time, the train services and the road services are hit badly. cashflow in these services reduced leading to an economic crunch in these sectors. the students use to take long commutes to the institutes taking away their well-being [ ] . they are deprived of their sleep and exercises. to commute long distances, students get up early and the daily routine is hampered. lack of daily exercises make them obese which is a major cause of concern among the youngsters. students carry a heavy load of bags on their backs to the school in countries like india. carrying school bags are back breaking work to the students. heavy loads of school bags have deleterious effects on the spine of children [ ] . many measures are taken to reduce the amount of school baggage, but it was all at a minuscule level. the online learning during the pandemic season turned out to be a heavy relief to the students carrying heavy school bags. being in a well-being state is an important aspect of human being. we tend to give rest to the body if it is not in a position to commute. the learners refrain from going to class if they are not well. the pandemic situation takes off all the health issues and helps in smooth learning of classes. the students are free to learn from their home in any physical condition. the structure of the learning system is based on various assessment procedures. the students are assessed based on the merit system. the pandemic hit the world during the key assessment period cancelling many exams. the cancellation of exams would have a long-term consequence on the ca-reer of the student. first, the internal assessment and then the public examinations were cancelled. the grades at the end of the academic year were predicted according to some undefined rules influ-encing the privileged students. education system is shifting to an online assessment system that can cre-ate measurement errors. these errors in an abrupt assessment would increase the differences between the privileged and under-privileged students in the future. the labour market would face the dire consequences of inefficient assessment scores. the entrance exam in higher education is a worst hit in assessment procedures. the entrance exams to top universities are either postponed or cancelled. the exam agencies are coming up with alternative solutions in consultation with the international in-stitutes. ) strike free education education system is at stake be it teachers strike or any other political strike. these strikes prevent students from attending classes. according to the study at argentina [ ], days of teachers strike there is a decline of years of education by : % in an academic year. the teacher strike has a negative effect on student learning and their overall achievement [ ]. frequent political strikes or hartals impact the overall education system. according to the statistics in kerala, india [ ], there would be one hartal in every four days leading to disruption in the holistic coverage of prescribed syllabus. the online education system is not affected by any sort of socio-political disruptions. education system in the virtual platform eased out the disturbances due to the strike. the education aspects during the pandemic impacted the family in many ways. a) the education system comes with mid day meals for the underprivileged in countries like india and the pandemic situation has taken the bread out of the mouth of some children. children from poor families would come to school with the greed for getting a one time meal. if the pandemic persists, then there is a high chance of drop-outs from the school. moreover, it would be a tremendous challenge to keep up the motivation of the underprivileged children after the pandemic. b) most of the parents in the pandemic era are working from home. it is difficult for most of the parents to handle domestic pressure and work pressure at home. working parents are juggling with children and working at home. global home schooling would pro-duce disparities depending on the ability of the family members to help their children learn. the inequality in each student skill set would overall affect human capital growth. c) the unprecedented learning system needs assistance of basic infrastructure for its smooth conduct. power supply and internet connectivity are the essentials needed without disruption. to avail these resources at home and keep the student without stress is a burdensome work for the parents. in developing countries, it is a difficult situation to maintain the resources around the clock. d) women take care of the children and rela-tives at home when compared to men. they are more insecure in their jobs. women are struggling with their household obligations and work during the pandemic. the juggling between children at home and work would reduce their opportunities and earnings at the workplace. women have to work harder in order to compensate for the workload and at an increased stress during this period. the study says that many women have left their job during the pandemic due to the imbal-ance in the worklife. covid is a disaster that would widen the gender inequalities. studies reveal that there is an increase of % in usage of electronic gadgets by the impressionable minds. gadget addiction is one of the major drawbacks of the online learning system. irritational behavioural patterns are observed in the students during this pandemic. the long time exposure to electronic gadgets are making them obese. an attachment towards gadgets creates a space for emo-tional imbalances in their personality. students have confined themselves into their own territory keeping them away from the societal component of life. studying and living together with their companions under one roof increases their social abilities but lockdown has created a void space for problem solving and decision making skills. social unawareness and lack of cognitive skills would be more visible. these skills improve their employability, productivity, health, and well-being in the future, and ensure the overall progress of the nation. people around the world are worried about the undergoing changes in the climate. the global temperature is a major concern for many environmen-tal changes. the last five years ( - ) were recorded as the hottest years. globally °c temperature has increased since the last century. an increase in per capita gross domestic product (gdp) is proportional to global warming. a study conducted by [ ] shows the environmental degradation and co emission has increased with the economic growth and more production [ ] . according to the census in , the countries with the highest co emis-sion in the world is shown in fig. . we see that the environmental degradation increases with the increase in production for economic growth. a lot of measures were taken to reduce the hazardous emis-sion, but a substantial decrement was not possible. the co or greenhouse gas disturbs the natural regulation of temperature in the atmosphere and leads to global warming and climate change. humans manipulated nature according to his whims and fancies that resulted in paradoxical im-reduced by % or . gigatonnes (gt) which is equivalent to a decade earlier data. there was an average decline of % energy demand per week during full lockdown and an average of % decline in partial lockdown countries. an unprecedented decline in demand for various fuels is seen during the pandemic as shown in fig. . the crisis of pandemic is paving a way for clean energy transitions. this decline in co emission is unprecedented and would be temporary, unless there is a resilient effort to change the structure. balances. humans are responsible for the emission of the greenhouse gas in the atmosphere over the last years. covid is the only disaster that has come as a boom to the environment. the major sources of co emission are energy, agricultural processing, land use changes, industrial processing, and other waste. electricity and heat is generated by burning fossil fuel, coal, and natural gas. a total of . % greenhouse gases are emitted while burning these fuels and are the leading cause for tempera-ture regulation. industries emit . %, transporta-tion - . %, agriculture processing - . %, land use change - . %, and industrial processes- . %. distribution of different sources of greenhouse gas are shown in fig. . before the arrival of pandemic, it was difficult to control the industrial and transportation emission. an impossible action of putting a halt on these hazardous sources was done overnight. accord-ing to estimates published by international energy ) vibration in the earth crust high frequency seismic waves are propagated into the earth mainly due to the activities of the human. the seismic noise renders the real time estimate of population dynamics. the covid pandemic period is the longest seismic noise quiet period ever recorded. according to the royal observatory of belgium [ ], the seismic noise of the earth during the pandemic is not prevalent, reducing the vibration of the earth by %. the vibrations are reduced by one-third of the normal activity during the lockdown. it becomes easy for seismologists to detect the movement in the earth crust without much of an expedition. the construction projects in some countries were at complete hold during the initial stages of the lockdown. the availability of the workforce and the site constraints halted some of the projects. construction activities create an adverse impact on the environment. the burning of fossil fuel, noise, and the waste of the construction contribute to the regulation of the temperature in the environment. the halt in construction reduced the amount of pm by three times in the month of april . air pollution is recorded highest in many cities of india. the annual average pm . concentration during the lockdown was much better than the safer limit [ ], [ ], [ ] , [ ] as shown in fig. . under the banner of economic growth, entire industrial and other waste is dumped into the rivers making it difficult to breathe. the aquatic species are becoming extinct due to the pollutants in the river. india is at the top of river pollution. ganges river is the most populated river in the world. the present pandemic has come as a blessing in disguise for rivers. the water pollution has decreased con-siderably during covid period. the waters from the rivers in india are tested during covid and the results provoke us to take measures to clean the rivers. the ph levels, the conductivity level, dissolved oxygen (do), and the biological oxygen demand (bod) of the water is reduced during the lockdown period [ ] . a betterment in standards of drinking water was seen during the lockdown period as shown in fig. . pandemic season was a lockdown for mankind, but on the contrary animals were liberalized. humans were away but animals took over the deserted cities and towns. animals took the advantage of the drop in human activity and came out to explore and play in the public places. scavengers are not around to shoo them away giving a space for wildlife to thrive. mallard ducks, wild deers, herd of goats, troop of monkeys, kangaroos, gangs of turkeys, and many others are taking human spaces. road mortality was a threat to the wildlife population [ ] . the mortality has reduced to % due to less traffic on roads in the usa. less roadkill reduces the ecological imbalances. some animals have successfully adapted to live alongside humans and their survival is dependent on them. an absence of human activity endangers some wildlife species. some governments mobilize funds to feed and preserve these animals, and the lockdown hindered their progress. according to the livestock census of , there are around million stray dogs in india. these dogs are fed by ngos or leftovers from restaurants. the closed restaurants and the restrictions in the movement made these stray dogs starve. the sustenance of the people in rural places of poor countries became difficult during the pandemic. people are driven to take extreme steps for their livelihood through poaching. the illegal hunting of endangered species in african continent is a threat for the wildlife society. according to study conducted by traffic, the wildlife poaching in india has increased twice during the pandemic pe-riod. it has increased from % to % during the lockdown period. it may turn disastrous and pave a way for another pandemic. humans struggled from recent pandemics such as aids, ebola, mers, and sars that came as an effect of consumption of animal meat [ ] , [ ] . it becomes the responsibility of the wildlife conservation society to prevent any pandemic in the future. due to the clean air and lockdown, non-covid diseases are at steep decline in countries prone to all pollution. the behavioural changes during the lockdown has brought a decline in insurance claims by % in india. waterborne infectious diseases and respiratory related diseases are being recorded as lowest during the pandemic time. the claims on deadly diseases such as cancer has turned down by % as per the statistics of the insurance companies in india [ ] . due to decrease in vaccination [ ] and disruption in the hospital services, there is a possibility of an outbreak of other diseases. religion makes people follow different practices and form socio-cultural groups. each culture recorded in human his- ) wildlife effects tory practised some organized system of beliefs and prac-tices. we tend to see very few people practicing faith in normal life. for some it seems absolutely mandatory but for some these are obnoxious practices. religion and faith is an integral part of people's lives worldwide, even though it is increasing. religious practices were hampered during lockdown. various aspects of religion during lockdown are discussed in detail: religion is a predominant factor for satisfaction in life, on the contrary the religious tensions can be annoying [ ] and affect the economic growth of the country. religious fervency is vigorous in most secularized countries [ ] . the polarization towards targeted groups increased in many countries during the earlier stage of the pandemic [ ] . since the cases of the virus were aggravated by the religious gathering in some countries, we could see religious bigotries coupled with the pandemic. the virus has morphed itself into an anti-community virus [ ] , [ ] . the bigotries and xenophobia towards different sects of people can be seen in different countries as shown in fig. . prayer meeting in france, and many more [ ] , [ ] , [ ] . the pandemic spread in various countries was sparked by religious gatherings as shown in fig. religion and politics are a crucial part of life and covid- has acquainted the human life without these jargon words. the places that culminated religious polarization at the earlier stages of the pandemic were felt at peace in the later stages of the pandemic. everybody came out in unison to curb this pandemic through their services. charity works and social commitment was seen at large during the pandemic. the role of religious practices in spreading covid- was predominant [ ] , [ ] . the religious lead-ers surpassing the mass gathering orders became a source of virus carriers in the entire nation. some of the early covid outbreaks were traced back to religious gatherings such as daegu church in south korea, bnei-brak in israel, oom in iran, tablighi-jamaat in india, tabligh-e-jamaat in malaysia, many people are fervent in religious practices such as visiting places of worship, mass gatherings, religious celebrations, and many more. all these practices are hindered during the pandemic. entire paradigm shift was seen in the religious fraternity. the religious holidays and celebrations were practiced at home. the key moments of rituals were experienced in their own home. religious leaders were bound to ask their followers to stay at home during pandemics. they started releasing double the amount of messages for the community to cope up with the stress during the pandemic. the religious organizations started doing more charita-ble services. people started living with faith rather than religious places. social distancing would be the most tricky in places of worship. the survey concludes that the public has become comfortable staying at home and practising their faith till the resumption of the normal situation [ ] . religious leaders are challenged to foster and to bring their services and communities together in these trying times from a distance. the online platforms were used to connect to the community during religious ceremonies. during the pandemic time, the searches for prayer have skyrocketed in google search engines. many spiritual and therapeutic activities, such as yoga, meditation, martial arts, and conscious dance classes have gone online during this pandemic. these temporary solutions are not sustainable solutions as they need physical relationships with people. the places of worship is a source of income for many religious leaders and the common man. these sources of income are hindered by the pandemic. life without religious practices also hit livelihoods of businesses around the places of worship. a loneliness during the pandemic times created furore among the individuals. people were compla-cent in their comfort zone but they were kicked out of that with hopelessness and despair. adapting to a new environment with a u-turn in an individual's life was a difficult task. life is fragile during pan-demic time but increase in spirituality and faith be-came a vital part of their life. religion is considered as a source of solace in terms of pain and scepticism. the role of prayer in the current pandemic situation among the general public is noteworthy [ ] . there was an increased interest ever recorded in search of prayer as per the daily data recorded from google for countries. according to tearfund covid prayer public omnibus research [ ] conducted in the uk during the lockdown period gauged the responses to spiritual practices. the statistics was conducted on , uk adults aged + and shows that nearly half ( %) of uk adults pray regularly and a quarter ( %) of uk adults attended online religious service during lockdown. one in twenty uk adults ( %) who attended religious service have never gone to church and twothirds ( %) of uk adults agree that prayer changes the world. generally, religion is more appealing to the older generation, but during the lockdown period the reli-gious revival was seen in younger ones. the highest number of quran apps from google playstore was downloaded during pandemic [ ] . irrespective of any religion, everybody started seeking hope in their faith and started praying for various topics as shown in fig. . we humans have gone through multiple virus pandemics in different times. pandemic came with human devastation but with times we came over it. covid- is a disaster in many aspects of life, but in some it has proved a blessing. this paper describes the multiple faces of virus outbreak. we have looked upon a few possible areas of life which have been affected by covid- such as the educational sector, environmental sector, and religious sector. the areas where it is a boom leaves a space to ponder on the living standard of human beings. lot of effort was taken with respect to some serious problems on the earth, but everything was in vain and it was noticed that there was a sudden break in these problems during a pandemic. once the pandemic is over, there is a call by the earth to make it a better healthy living place. comparative pathogenesis of covid- , mers, and sars in a nonhuman primate model effects of covid pandemic in daily life rolling updates on coronavirus disease (covid- ) predicting covid- in china using hybrid ai model a weakly-supervised framework for covid- classification and lesion localization from chest ct deep learning covid- features on cxr using limited training data sets accurate screening of covid- using attention based deep d multiple instance learning dual-sampling attention network for diagnosis of covid- from community acquired pneumonia diagnosis of coronavirus disease (covid- ) with structured latent multi-view representa-tion learning wearable sensing and telehealth technology with potential applications in the coronavirus pandemic easyband: a wearable for safety-aware mobility during pandemic outbreak the impact of covid- on consumers: preparing for digital sales a comprehensive review of the covid- pandemic and the role of iot, drones, ai, blockchain, and g in managing its impact new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? middle east respiratory syndrome coronavirus (mers-cov): impact on saudi arabia the psychological im-pact of covid- pandemic on health care workers in a mers-cov endemic country occurrence, prevention, and management of the psychological effects of emerging virus outbreaks on healthcare workers: rapid review and meta-analysis epidemic on medical staff in china: a cross-sectional study mental health impact of covid- pandemic on spanish healthcare workers the impact of novel coronavirus sars-cov- among healthcare workers in hospitals: an aerial overview covid- : disease, management, treatment, and social impact mitigating the psychological effects of social isolation during the covid- pandemic loneliness and social isolation during the covid- pandemic covid- outbreak: migration, effects on society, global environment and prevention predicting the impacts of epidemic outbreaks on global supply chains: a simulation-based analysis on the coronavirus outbreak possible environmental effects on the spread of covid- in china the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak the moment to see the poor the precarious position of postdocs during covid- impact of covid- on academic mothers coronavirus and exceptional health situations: the first disaster with benefits on air pollution can we expect an increased suicide rate due to covid- ? uncomfortably numb: suicide and the psychological undercurrent of covid- covid- pandemic: mental health and beyond -the indian perspective facts and figures: mobility in higher education study abroad statistics: convincing facts and figures online learning during the covid- pandemic centre for monitoring indian economy long school commutes are terrible for kids the effects of teacher strike activity on student learning in south african primary schools kerala suffers hartal every each one burns rs -crore hole in state's economy economic growth and carbon dioxide emissions? quieting of high-frequency seismic noise due to covid- pandemic lockdown measures air pollution dropped % in bengaluru during lockdown air quality in chennai during lockdown -do we have clues to mitigate air pollution reduction in water pollution in yamuna river due to lockdown under covid- pandemic impact of covid- mitigation on wildlife-vehicle conflict prioritizing zoonoses for global health capacity building-themes from one health zoonotic disease workshops in countries anthropogenic drivers of emerging infectious diseases insurers see up to % fall in non-covid medical claims who and unicef warn of a decline in vaccinations during covid- religious polarization, religious conflicts and individual financial satisfaction: evidence from india religious polarization: contesting religion in secularized western european countries india: infections, islamophobia, and intensifying societal polarization new center for public integrity/ipsos poll finds most americans say the coronavirus pandemic is a natural disaster statca of increase in anti-asian sentiment, attacks covid- and religious congregations: implications for spread of novel pathogens religion and the covid- pandemic god and covid- public health response to the initiation and spread of pandemic covid- in the united states high covid- attack rate among attendees at events at a church-arkansas have prayers changed in lockdown? people of faith answer in crisis, we pray: religiosity and the covid- pandemic tearfund covid prayer public omnibus research how coronavirus is leading to a religious revival key: cord- -nwbmxepi authors: margină, denisa; ungurianu, anca; purdel, carmen; tsoukalas, dimitris; sarandi, evangelia; thanasoula, maria; tekos, fotios; mesnage, robin; kouretas, demetrios; tsatsakis, aristidis title: chronic inflammation in the context of everyday life: dietary changes as mitigating factors date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: nwbmxepi the lifestyle adopted by most people in western societies has an important impact on the propensity to metabolic disorders (e.g., diabetes, cancer, cardiovascular disease, neurodegenerative diseases). this is often accompanied by chronic low-grade inflammation, driven by the activation of various molecular pathways such as stat (signal transducer and activator of transcription ), ikk (iκb kinase), mmp (matrix metallopeptidase ), mapk (mitogen-activated protein kinases), cox (cyclooxigenase ), and nf-kβ (nuclear factor kappa-light-chain-enhancer of activated b cells). multiple intervention studies have demonstrated that lifestyle changes can lead to reduced inflammation and improved health. this can be linked to the concept of real-life risk simulation, since humans are continuously exposed to dietary factors in small doses and complex combinations (e.g., polyphenols, fibers, polyunsaturated fatty acids, etc.). inflammation biomarkers improve in patients who consume a certain amount of fiber per day; some even losing weight. fasting in combination with calorie restriction modulates molecular mechanisms such as m-tor, foxo, nrf , ampk, and sirtuins, ultimately leads to significantly reduced inflammatory marker levels, as well as improved metabolic markers. moving toward healthier dietary habits at the individual level and in publicly-funded institutions, such as schools or hospitals, could help improving public health, reducing healthcare costs and improving community resilience to epidemics (such as covid- ), which predominantly affects individuals with metabolic diseases. chronic inflammation is a central process involved in a high number of metabolic disorders (e.g., obesity, metabolic syndrome, diabetes, dyslipidemia, etc.), including neurodegenerative (alzheimer), malignant diseases, and autoimmune diseases. in most if not all chronic inflammatory conditions, there is an extensively failed resolution of inflammation with high influx of leukocytes, which in their effort to resolve inflammation stimulate the synthesis of pro-inflammatory molecules and establish a highly inflammatory micro-environment, leading to extensive fibrosis and tissue damage [ ] . chronic low-grade inflammation has been shown to either induce or aggravate metabolic disturbances, including insulin resistance and dyslipidemia, which contributes to the development of other complications [ ] . there is accumulating evidence that, in the case of autoimmune diseases, when the immune system loses self-tolerance and attacks the body's cells and tissues, metabolic disturbances are key contributors to disease progression. results from the type diabetes mellitus (t dm) prediction and prevention studies on t dm showed that metabolic disturbances preceded the seroconversion to positive autoantibodies by several months or years in type diabetes mellitus [ , ] . many chronic inflammatory diseases originate or have their development promoted by an unbalanced diet. although the exact mechanism remains unclear, de rosa et al. suggest that metabolic pressure, as a result of increased caloric intake, leads to an altered adipose tissue homeostasis. this results in the synthesis of adipokines and facilitates the overactivation of nutrient-sensing mechanisms, altering the balance between pro-inflammatory and regulatory t-cells, ultimately resulting in the loss of immunotolerance [ ] [ ] [ ] . in addition, dietary components have the ability to influence the immune response through the modulation of gut bacteria metabolism, impacting the risk of developing chronic diseases either directly in the gastrointestinal tract, or in other more distant organs that impact general metabolism [ ] [ ] [ ] [ ] . recent studies have investigated long term exposure to low doses of chemical mixtures that can be a part of modern lifestyles, such as pesticides, food additives, or additives contained in food coating materials, proving that different disturbances appeared from minor biochemical disturbances. these early alterations are generally followed by oxidative stress induction and organ damage depending on the period of exposure [ ] [ ] [ ] [ ] [ ] [ ] . recently, it has been shown that long term exposure to stressors might also have a positive association with increased vulnerability of the population to the microbial and viral infections [ ] . metabolomics are an emerging biological field that allow for the identification and simultaneous measurement of a large number of small molecules called metabolites in biological matrixes. it has become the most accurate method to detect metabolic imbalances and is useful for prevention and early detection of diseases. moreover, metabolomics have vast applications in clinical practice [ ] . targeted metabolomic analysis provides insights regarding the normal function of endogenous metabolism, dietary intake, microbiota, drug metabolism, and nutrient adequacy [ ] . the challenge of chronic inflammatory diseases with respect to early diagnosis can be tackled with metabolomics through the identification of biomarkers that can discriminate high-risk populations. in a group of autoimmune patients, it was found that their fatty acid-based metabolic profile and lifestyle factors including physical activity and alcohol consumption were valuable predictive markers of autoimmune diseases [ ] . humans are exposed to a large number of substances from food, water, cosmetics, air, and so forth, each at low levels of exposure, and are able to induce cumulative/synergistic effects. many studies have focused on the effects induced by administering a single substance at medium-high doses to laboratory animals. recently, the concept of real-life risk simulation has emerged, since there is growing evidence that the effects of chemical mixtures at concentrations for which individual components failed to elicit have adverse effects when tested individually [ ] . the concept of real-life risk simulation can also incorporate dietary interventions because, in our diets, we expose the human body to myriad substances in diverse doses [ , , ] . the discovery of inflammation regulators opened a new window in therapeutics to clear low-grade chronic inflammation. a large number of physiological processes promote the physiological process of regulating inflammation. the development of such an approach targets the stimulation of endogenous processes that naturally occur during inflammation, which are hampered mainly by the lack of suitable human models and the heterogeneity of inflammatory disorders. another limitation includes the lack of sensitive measurements able to capture the different stages of inflammation and metabolites [ ] [ ] [ ] [ ] [ ] . the present paper aims to evaluate the impact diet might have on immune response, with special attention as to how lifestyle changes can help mitigate low-grade inflammation. real-life risk simulation (rlrs) concept. this analysis can be highly relevant in the context of the present viral spread of sars-cov- , since the inflammation is once again in the front line of an acute pathological response. identifying strategies to modulate the immune response might prove useful for reducing the virus's impact on the respiratory tract and thus diminishing its impact on each patient, as well as on the general medical system. the majority of studies that assess dietary habits, metabolism, and nutrient intake are based on food frequency questionnaires. however, food frequency questionnaires (ffq) have several limitations, including inconsistent responses on food choices, mostly because answers depend on responders' memory. moreover, ffq that are filled out by the responder instead of a trained healthcare professional only provide an overview of the macronutrients' intake while not fully capturing the micronutrient status of the person [ ] . micronutrient deficiencies are common in both developing and affluent countries, affecting two billion people worldwide, according to the world health organization (who) [ ] . the primary cause of micronutrient deficiencies or "hidden hunger" is poor dietary intake of micronutrients while other socioeconomic factors play an important role as well [ ] . several diet and nutrients assessment tools have been developed to evaluate inflammation status. the dietary inflammation index (dii) is based on literature data and aims to evaluate if a responder follows a proor anti-inflammatory diet. since its development, there has been an increasing interest in dii, although other indexes are being developed with similar efficacy [ ] . an important limitation of these indexes is the lack of causality and direct association to a person's symptoms. thus, the application of these indexes in clinical practice is hampered. a novel empirical, close-ended, and self-administered questionnaire developed by the european institute of nutritional medicine provides an inflammation status score that captures the interaction between the autonomic nervous system and inflammation [ ] . there is growing evidence that imbalances in the autonomic nervous system reflect local or systemic inflammation found in various diseases and that diet and lifestyle factors can act as regulators of sympathetic and parasympathetic activity [ ] [ ] [ ] [ ] . through a -question series, responders provide data on the presence/absence and status of inflammatory response in different body systems. overall, the nutritional medicine exam (numex) consists of questions and assesses the nutritional deficiencies status in seven categories: inflammation, nutrition, perceived stress, oxidation, sugar metabolism, amino acids metabolism, and gut microbiome. designed by medical doctors and nutritionists, the aim of this empirical test is to assist the individual and the healthcare professional to evaluate the overall inflammatory status based on autonomic nervous system changes and track its progression after targeted lifestyle changes. at a molecular level, metabolomics is the only method that can capture small, time-dependent fluctuations in the metabolism, thus indicating ncd-related metabolic imbalances [ ] . overall, traditional and well-established diet and nutrient assessment methods including ffq and dii have provided valuable information on the role of specific foods on health and disease, as discussed in the present review. with the advent of advanced tools, metabolomics is complementary to the standard approach to provide tailor-made recommendations depending on an individual's specific needs at a given time. recent literature considers bmi cut-off values to not fully depict metabolic disturbances associated with obesity. the bmi is a mathematical approximation and does not reflect the percentage of total body fat between body fat and total body muscle or bone mass. as such, bmi does not reflect cardiometabolic risk. a more comprehensive classification describes four phenotypes for obese individuals: normal weight obese (nwo), metabolically obese normal weight (monw), metabolically healthy obese (mho), and metabolically unhealthy obese (muo), or "at risk" obese with ms. this classification takes into account bmi, fat mass, and waist circumference, but also general biochemical parameters (e.g., fasting plasma glucose, total cholesterol, ldl, hdl, triglycerides) [ ] [ ] [ ] [ ] [ ] [ ] . all four classes are characterized by impairments of different severity of inflammatory pathways [ , ] . lifestyle and nutrition are modifiable factors that interact with genetics in regulating chronic inflammation, leading to aforementioned complications. the changes in nutritional patterns in western societies-caused by a high intake of fat and energy-dense, processed foods, as well as a low intake of fibers, fruits, and vegetables-are associated with a rising prevalence of asthma, allergies, and autoimmune diseases involving inflammatory mechanisms [ , ] . high fat diets determine, among other things: intestinal inflammation, favoring lipopolysaccharides (lps) absorption from gram-negative gut bacteria, and increasing lipoperoxidation that induces insulin resistance and inflammation. saturated fatty acids and lps activate toll-like receptor (tlr ) signaling pathways further contribute to promoting systemic inflammation and consequent metabolic disorders [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( figure ). lifestyle-and diet-induced inflammation affects several cellular pathways, which stimulates the synthesis and secretion of various pro-inflammatory molecules. this ultimately maintains the low-grade inflammation state. interestingly, populations that consume a diet rich in fruits, vegetables, and fibers have lower incidences of inflammatory diseases compared to western populations [ , ] . the mediterranean diet-based on olive oil, fish, vegetables, and fruits, in addition to incorporating myriad beneficial phytochemicals-discourages cardiovascular diseases [ ] [ ] [ ] [ ] . sourcing food from organic agriculture could further improve the beneficial health effects of a mediterranean diet, as suggested in a study comparing an organic and nonorganic mediterranean diet on male patients with chronic kidney disease [ ] . this was hypothesized to be due to a decreased exposure to pesticides, since animal studies have repeatedly found that exposure to pesticide mixtures can be a source of toxicity [ ] . however, although most studies have found that organic food consumers are healthier, it is not clear whether health benefits can be attributed to a decreased exposure to synthetic pesticides [ ] . dietary changes that include specific metabolites can modulate gene expression via epigenetic modifications, such as dna methylation or chromatin remodeling (e.g., histone acetylation or deacetylation). for example, a diet rich in folate and methionine can shape the host epigenome with a direct impact on molecular pathways associated with obesity-related inflammation. moreover, global dna hypermethylation in adipocytes derived from obese subjects is correlated with the expression of genes involved in proinflammatory interactions [ , ] . for example, hypermethylation at kb upstream of the adiponectin gene's promoter site was observed in adipocytes of obese mice fed a high-fat diet, but also in human adipocytes. dna methyltransferase (dnmt ) expression is correlated with the methylation of the adiponectin gene, resulting in decreased expression of adiponectin in obese mice and increased expression in healthy mice. studies on human adipocytes show a correlation between dnmt expression and bmi, suggesting that obesity is a cause or cofactor of hypermethylation of adiponectin gene [ , ] . another factor inducing epigenetic changes is ros overproduction in expanded adipose tissue, influencing histone acetylation/deacetylation equilibrium, thus inducing nfκb activation [ ] [ ] [ ] . on the other hand, nutrient restriction decreases akt (protein kinase b) activity and stimulates foxo (a forkhead box o transcription factor) activity, thus stimulating the expression of proteins involved in cell metabolism, autophagy, and stress-response, contributing to the resolution of inflammation [ , ] . fasting regimens are correlated with increased insulin sensitivity, improvement of blood pressure, and inflammatory status, regardless if they are associated with weight reduction. for example, days of intermittent fasting induced an increase of glucose uptake rates and a significant increase of anti-inflammatory adiponectin in lean young men (bmi of kg/m ) without a significant decrease in body weight. these results were consistent with data from animal studies [ , ] . an important causal factor for low grade inflammation influenced mainly by lifestyle is the impairment of gut microbiota. bacteroidetes and firmicutes constitute approximately % of the intestinal population, but the equilibrium is fundamentally changing with ageing and depending on diet composition. a decline of microbiota diversity occurs during ageing and obese individuals. gut dysbiosis has been found in several inflammatory pathologies such as obesity, diabetes, cardiovascular, and neurodegenerative diseases. this can be connected to the induction of chronic low-grade inflammation since the gut microbiome is intimately connected to innate immune responses [ ] [ ] [ ] . the relationship between gut microbiome and the host immune system are influenced by lifestyle interventions. for instance, secretory iga levels increase after periodic fasting. this can be linked to changes in gut microbiome composition [ , ] , with proteobacteria modulating the adaptive humoral local response. some studies showed that microbiota composition and diversity has a great impact on a population's general health status. for example, when comparing the fecal microbiota of european and rural african children (burkina faso), a higher proportion of prevotella and xylanibacter (involved in the digestion of fibers and generation of short chain fatty acids (scfas)) was found in the latter group, which lacked european subjects. these observations could be correlated with the higher prevalence of inflammatory diseases in european populations compared to rural african ones [ , ] . chronic exposure to environmental pollutants or food additives could also predispose one to chronic pathologies, which promotes inflammation [ ] . xenobiotics promotes chronic inflammation, which is thought to be the generation of lipotoxic conditions, i.e., in the development non-alcoholic fatty liver disease [ ] . this can be mitigated by lifestyle interventions such as periodic fasting [ ] . the exposure to xenobiotics such as heavy metals, pesticides, nanoparticles, polycyclic aromatic hydrocarbons, dioxins, furans, polychlorinated biphenyls, or non-caloric artificial sweeteners can also promote chronic inflammation by disturbing the gut microbiota [ , , ] . decreasing inflammatory burden is more important than ever during the covid- pandemic. this can be accomplished through everyday actions (e.g., lifestyle, diet, smoking cessation, weight decrease, sport, etc.). there is a lot of information available in the scientific community regarding the risk of covid- complications; even the likelihood of death is highly increased by some chronic diseases, mostly associated with an impaired inflammatory profile (e.g., obesity, type ii diabetes, hypertension, chronic pulmonary disease, etc.) [ , ] . the literature data shows that people without comorbidities have a much lower risk of severe symptoms as a result of the sars-cov- infection [ ] . on the other hand, increased levels of inflammatory markers cytokines with pro-inflammatory outcomes constitute predictors of adverse outcome in covid- patients [ ] . evidence proves that some dietary elements such as zinc or vitamin d might provide protective effects against viral load [ ] . as such, this reduces the inflammatory burden through a healthy diet, associating (based on rlrs principles) several protective components (e.g., fiber, polyphenols, pufas, vitamins, etc.) that constantly increase our chance of being better protected against different immune challenges. fermentable dietary fiber are not enzymatically digested in the small intestine; they pass into the colon and are transformed by gut bacteria into scfas [ , ] . the systemic distribution and generation of scfas-acetate, propionate, and butyrate (the most abundant)-in the distal colon is important to inhibit inflammatory signals. germ-free animal models were characterized by inflammatory flairs, due to the absence of tissue/blood scfas [ ] [ ] [ ] . butyrate is a representative member of scfas and has a high affinity for different g-protein-coupled receptors (gpcrs) found throughout the body: gpr is found in adipose tissues and immune cells and gpr is found in immune cells. however, gpr a is present in colonic cells and gpr and gpr are activated by butyrate, which favors the production of peptide yy (pyy). this contributes to gastric emptying and intestinal transit inhibition, which thereby reduces appetite and promotes glucagon-like peptide (glp- ). these outcomes indirectly stimulate insulin secretion. gpr a activates the inflammation-associated pathway in colonic macrophages and dendritic cells, inducing the differentiation of il- -producing t-cells and release of il- from intestinal epithelial cells [ , , ] . the presence of fiber in the diet is extremely important ( table ) as it generates scfas and promotes the proliferation of commensal bacteria, which limits the access of pathogenic bacteria to the gut epithelium. moreover, scfas favor epithelial mucus secretion that increases the protective effect on the intestinal surface and the proper maintenance of the barrier function [ , ] . scfas have anti-inflammatory effects that bind to the nuclear transcription factor pparγ (peroxisome proliferator-activated receptor γ) and, consequently, inhibit the nf-kb pathway [ , ] . this ultimately lowers the expression of vcam- (vascular adhesion molecule ) and icam- (intracellular adhesion molecule ), as well as the synthesis of tnfα, il- , and ifn-γ (interferon γ) [ ] . the main physiological role of histones is to interact with dna and stabilize its structure. when they are acetylated, this loosens the contact between histones and dna, uncoiling the dna structure that thus becomes transcriptionally active. the histone acetylation process is a result of the balance between the induction of histone acetyl transferases (hats) and the inhibition of histone deacetylase (hdacs). the same acetylation process causes dna to bind to transcription factors, such as stat (signal transducer and activator of transcription ), nf-kβ (nuclear factor kappa-light-chain-enhancer of activated b cells, and foxp . consequently, this regulates gene expression, including inflammation proteins [ , ] . scfas (with butyrate and acetate being the most and least effective, respectively) act as inhibitors for histone deacetylase (hdacs), thus contributing to the inhibition of the transcription for inflammatory proteins [ , ] . fasting is a process that has been known for thousands of years. it was quite frequent in ancient times, because access to food was difficult and as a result, individuals were obliged to survive without food until it was available again [ ] . fasting is a survival mechanism in both animals [ , ] and humans, especially in countries where food conservation is not widespread [ ] . in the rest of the world, fasting has been employed either due to religious convictions or in wellness centers. since , one of the methods used to address morbid obesity and related diseases has been the "zero calorie diet", thus translating into clinical practice the scientific data generated by centuries of fasting. fasting can be divided into three broad categories: • periodic fasting, which lasts from days to a few weeks; • intermittent fasting, which lasts from to h and can be done daily or every second day or twice a week, and • fasting-mimicking diet, the diet that mimics fasting to achieve its beneficial effects, in which restriction of calories and specific foods is necessary (e.g., fat) [ ] [ ] [ ] . there is a lot of research that shows the beneficial effects of fasting on health and also on different pathological conditions. fasting increased lifespan in prokaryotic organisms such as yeast s. cerevisiae and nematode c. elegans [ , [ ] [ ] [ ] [ ] , but also on animal models that performed fasting for long periods (e.g., the royal penguin) [ ] . other models have shown better brain function [ , ] , increased lifespan and longevity [ ] [ ] [ ] , and improved maintenance of muscle mass after fasting [ ] . studies on animal models reveal the beneficial effect fasting has on cancer as a complementary disease management strategy in concert with drug treatments [ ] [ ] [ ] [ ] . studies on animal models show an improvement in neurodegenerative diseases after fasting, while other studies prove that intermittent fasting diets boost the levels of antioxidant defense, neurotrophic factors (bdnfs, h- and fgf ), proteins involved in adaptive response (hsp- and grp- ), and reduce pro-inflammatory cytokines levels (tnfa, il- β, and il- ) [ ] [ ] [ ] [ ] [ ] . it has been found that intermittent fasting can prevent and reverse all aspects of metabolic syndrome in rodents: body fat, inflammation, and blood pressure are reduced; insulin sensitivity is increased; and the functional capacity of the neuromuscular and cardiovascular systems are improved [ ] [ ] [ ] . an intermittent fasting diet has also been found to improve hyper-glycaemia in diabetic rodent models [ ] and in myocardial infarction models, as the heart is protected from ischemic damage by this type of regimen [ ] . elevated leptin levels usually predict a pre-inflammatory condition, while adiponectin and ghrelin may suppress inflammation and increase insulin sensitivity [ , ] . fasting can reverse every major abnormality caused by metabolic syndrome, by increasing insulin and leptin sensitivity, suppressing inflammation and stimulating autophagy [ , ] . there are several studies that show an increased use of fat and ketone bodies for energy [ , ] , as well as an increase of growth hormone and glucagon secretion [ ] [ ] [ ] , with a decrease in blood sugar, insulin, and igf- levels. after intermittent fasting, total fat, abdominal fat, and blood pressure are decreased, while glucose metabolism is improved in obese individuals [ , [ ] [ ] [ ] [ ] . in addition, periodic fasting significantly changes the composition of the human gut microbiota [ ] . finally, studies of utmost importance show the effect of intermittent fasting mainly in the fight against cancer as a supplement along with the classic treatment, with promising results [ , ] . below, we analyze the effects of the interrelation between fasting and inflammation as well as the relevant molecular mechanisms. fasting not only results in weight loss but it is a survival mechanism that impacts many metabolic pathways [ , , ] . fasting's many benefits are related to the regulation of key molecular pathways. initially, during fasting, the downregulation of insulin-like growth-factor- (igf- ) and mammalian target of rapamycin (mtor) occur. these pathways are upregulated in the presence of food excess as they sense nutrients and therefore activate anabolic metabolism. when there is a lack of food for several hours, catabolic processes are activated. aging appears modulated by changes in the insulin-like growth-factor- receptor signaling system, as longevity is enhanced by a decrease in igf- signaling [ ] [ ] [ ] . the igf- signal induces mtor activation. reduced mtor activity is related to extended lifespan in different organisms [ ] , as mtor induces activation of foxo proteins. foxo proteins are transported to the nucleus and activate genes associated with autophagy [ ] , which emphasizes the link between autophagy and foxo proteins. when the amp/atp ratio is high, the ampk path is activated [ ] ; this results in increased energy production and reduced atp utilization. in addition, the mitochondrial biogenesis and mitophagy repair and replace damaged mitochondria. as a result, the cells have "younger" and more efficient mitochondria. in addition to the aforementioned condition, activation of this path has been associated with increased lifespan in various studies in both c. elegans and drosophila melanogaster [ , ] . in mammals, fasting does not appear to affect ampk activation, but further studies are needed to be able to draw surer conclusions [ ] . like ampk, sirtuins are associated with life [ ] and autophagy [ ] . some sirtuins are found in the cytoplasm (sirt ), some (sirt ) in the nucleus having dna repair action, and others in mitochondria. in general, sirtuins are associated with mitochondrial biogenesis and mitophagy for damaged mitochondria, thereby enhancing mitochondrial cells without problems and are thus more efficient in energy production [ , ] . sirt is modulated by nad + level and is increased in energy depletion states (such as fasting or exercise) for which nad + is a sensor, which contributes to the reduction of inflammation through nf-kβ down-regulation and related transcription factors [ , , ] . a study on rats showed that inflammation decreased with fasting [ ] . other work has shown nf-kβ inhibition and the modulation of nrf , sirtuins, sod , and increased lifespan [ ] [ ] [ ] [ ] . in a study, intermittent fasting appears to significantly reduce corticosterone (cort), interleukin (il- ), and tumor necrosis factor-alpha (tnf-α) levels [ ] . nrf plays a key role in oxidative stress and toxicity; the right balance in ros levels is very important so that mitochondrial and all other pathways can function properly. the absence of ros, however, does not activate nrf , which in turn does not activate are (antioxidant response). thus, a critical amount of ros ("hormesis hypothesis") is necessary for the upregulation of are, which allows cells and mitochondria to be able to deal with oxidative stress and different kinds of toxins [ ] , which consequently increases their lifespan [ ] (figure ). during the last decades, many studies have been conducted on the effect of fasting on several markers related to metabolism. most of them determine the effect of fasting on weight. however, there are several studies that identify changes in lipid and carbohydrate metabolism as well as key hormones that affect the above (e.g., insulin). recently, some studies have focused on fasting's effect on inflammatory markers such as tnf-α, interleukins, crp, and bdnf, as well as the hormones adiponectin and leptin. the largest study on fasting's effects is an observational study including subjects that describes metabolic changes after a -to -day fasting period [ , ] . all the participants fasted according to the buchinger wilhelmi fasting guidelines, which include a daily caloric intake of - kcal together with a variety of lifestyle changes (e.g., dietary advices, physical exercise). a beneficial modulating effect of fasting was observed on blood lipids, glucoregulation, and altogether general health-related blood parameters. additionally, it was associated with a reduction in weight, abdominal circumference, and blood pressure. in another study, which used the same fasting guidelines, improved metabolic markers were observed after periodic fasting, including a decrease in blood glucose levels associated with changes in gut microbiome composition [ ] . in this study, the analysis of the gut microbiome after days of periodic fasting showed that fasting caused a decrease in the abundance of bacteria known to degrade dietary polysaccharides such as lachnospiraceae and ruminococcaceae, concomitant to an increase in bacteroidetes and proteobacteria known to use host-derived energy substrates. a study of eight healthy non-obese men discovered that in days of fasting every other day, with h fasting on fasting days, adiponectin increased and leptin decreased, while no changes in il- or tnf-α were observed. protocol allowed them to maintain normal exercise but also to consume food in order to keep their weight stable [ ] . redman et al., in a two-year study of people who followed a reduced calorie intake diet ( %) observed a reduction in leptin. these individuals lost an average of . kg while the control group gained an average of . kg in the same period [ ] . another study including eight women and two men, all overweight with asthma, showed that fasting every other day and reducing calories to less than % of their normal intake on the days of fasting, for eight weeks, resulted in a reduction in tnf-α and bdnf, but no change in crp. in this study, patients lost % of their initial weight during the study. asthma symptoms also improved as well as some indicators of oxidative stress ( -protein carbonyls, isoprostane, nitrotyrosine, and -hydroxynonenal adducts) [ ] . in , another study [ ] found that weeks of reduced calorie intake every other day resulted in reduced crp levels, increased adiponectin levels, and reduced leptin levels in adults who were either overweight or normal weight. on fasting days, they consumed only % of the calories they normally consumed each day. in addition, their weight was significantly reduced by . ± . kg and the coronary heart disease risk was improved as the concentration of tg reduced. another study [ ] found reduced high sensitivity-c-reactive protein (hs-crp) of women with polycystic ovary syndrome (pcos) during the ramadan period in iran and in which participants aged to years old with an average of age . , followed everyday . h fasting, isocaloric diet, for days. an important study involving men (resistance-trained) who fasted every day h, followed an isocaloric diet for eight weeks, and consumed % of their energy needs in the -h eating window, showed that there was an increase in adiponectin and a reduction of leptin of il- and il- β [ ] . faris' study on healthy volunteers ( men and women) fasting for - h each day for days, showed a reduction in il- , il- β, tnf-α, total leukocytes, granulocytes, lymphocytes, and monocytes [ ] . another study [ ] involving patients aged to years old with nonalcoholic fatty liver disease, following ramadan fasting (every day -h fasting for days), showed a reduction in il- and hs-crp, compared to volunteers who did not fast. these and other studies are presented in detail in table . collectively, an increasing number of studies show that fasting has numerous health benefits and could be used to prevent or manage the development of cardiometabolic disorders, metabolic diseases, and immune diseases. although extended periods of fasting can be challenging without medical advices, recent studies also showed that time-restricted eating can be practiced safely as a routine. for instance, a recent study showed that h of time-restricted eating for weeks improved cardiometabolic health for patients with metabolic syndrome [ ] . [ ] ns-non-significant; hs-crp-highly sensitive crp; ldl-low density lipoproteins; hdl-high density lipoproteins; gsh-glutathione; tac-total antioxidant capacity; t -triiodotironine; tg-triglycerides; bmi-body mass index; bf-body fat; sbp-systolic blood pressure; dbp-diastolic blood pressure; , -dinor-ipf( α)-iii - , -dinor- -iso prostaglandin f α; hba c-glycated hemoglobin; igf- -insulin growth factor . given that a large part of the global population suffers from various metabolic disorders, it is important to look for non-pharmacological ways to deal with these conditions. targeted changes in lifestyle and especially diet can be economical tools to mitigate the development of metabolic disorders when they are at an early stage. these changes include increased fiber and polyphenol intake compared to the current western diets, but also well-structured, personalized fasting protocols, which can reduce the risk of metabolic disorders (figure ) . this could be implemented in various institutions by improving the nutritional quality of foods served in schools, hospitals, prisons, government buildings, or senior centers. moving toward healthier food options in hospitals might ultimately help improve patient health and reduce healthcare costs. improving diet can have a positive effect on the immune response as a hallmark of rlrs. since inflammation is associated with the acute pathological response to covid- and other infectious diseases, improvement of the immune response and inflammatory markers may lead to an improved physiological resilience to disturbances by infectious agents such as viruses and bacteria, and possibly milder symptoms. inflammation in atherosclerosis metabolic syndrome is an inflammatory disorder: a conspiracy between adipose tissue and phagocytes dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children who later progress to type diabetes circulating metabolites in progression to islet autoimmunity and type diabetes metabolic pressure and the breach of immunological self-tolerance insulin-leptin axis, cardiometabolic risk and oxidative stress in elderly with metabolic syndrome adiponectin: possible link between metabolic stress and oxidative stress in the elderly diet and inflammation inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric crohn's disease gut microbiome: profound implications for diet and disease cross-regulatory circuits linking inflammation, high-fat diet, and the circadian clock a mixture of routinely encountered xenobiotics induces both redox adaptations and perturbations in blood and tissues of rats after a long-term low-dose exposure regimen: the time and dose issue endocrine disruptors leading to obesity and related diseases overview of the effects of chemical mixtures with endocrine disrupting activity in the context of real-life risk simulation: an integrative approach (review) genotoxic, cytotoxic, and cytopathological effects in rats exposed for months to a mixture of chemicals in doses below noael levels six months exposure to a real life mixture of chemicals' below individual noaels induced non monotonic sex-dependent biochemical and redox status changes in rats covid- , an opportunity to reevaluate the correlation between long-term effects of anthropogenic pollutants on viral epidemic/pandemic events and prevalence obesity a risk factor for increased covid prevalence, severity and lethality (review) metabolomics: an emerging but powerful tool for precision medicine. cold spring harb. mol. case stud. , , a application of metabolomics: focus on the quantification of organic acids in healthy adults metabolomic analysis of serum fatty acids for the prediction of the effect of chronic vitamin deficiency and long term very low dose exposure to pesticides mixture on neurological outcomes-a real-life risk simulation approach application of novel technologies and mechanistic data for risk assessment under the real-life risk simulation (rlrs) approach resolution of inflammation: a new therapeutic frontier endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators immunohistochemical evaluation of autogenous mandibular bone grafts integration: an experimental study the routine and specialised staining for the histologic evaluation of autogenous mandibular bone grafts an experimental study the human metabolic profile reflects macro-and micronutrient intake distinctly according to fasting time preventing and controlling micronutrient deficiencies in populations affected by an emergency the epidemiology of global micronutrient deficiencies an empirical dietary inflammatory pattern score enhances prediction of circulating inflammatory biomarkers in adults the sympathetic nervous response in inflammation inflammation, immunity and the autonomic nervous system nutritional stimulation of the autonomic nervous system effects of weight changes in the autonomic nervous system: a systematic review and meta-analysis the metabolic phenotype in obesity: fat mass, body fat distribution, and adipose tissue function normal weight obese (nwo) women: an evaluation of a candidate new syndrome new obesity classification criteria as a tool for bariatric surgery indication the metabolically healthy but obese individual presents a favorable inflammation profile metabolic and body composition factors in subgroups of obesity: what do we know? obesity: preventing and managing the global epidemic role of "western diet' in inflammatory autoimmune diseases the impact of western diet and nutrients on the microbiota and immune response at mucosal interfaces. front increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis ceramides-lipotoxic inducers of metabolic disorders role of intestinal inflammation as an early event in obesity and insulin resistance chylomicrons promote intestinal absorption of lipopolysaccharides lps/tlr signal transduction pathway preclinical and clinical results regarding the effects of a plant-based antidiabetic formulation versus well established antidiabetic molecules lipoprotein redox status evaluation as a marker of cardiovascular disease risk in patients with inflammatory disease the asthma epidemic dietary fiber intake and mortality in the nih-aarp diet and health study investigation of the association between dietary intake, disease severity and airway inflammation in asthma primary prevention of cardiovascular disease with a mediterranean diet effect of diet on asthma and allergic sensitisation in the international study on allergies and asthma in childhood (isaac) phase two quercetin and epigallocatechin gallate induce in vitro a dose-dependent stiffening and hyperpolarizing effect on the cell membrane of human mononuclear blood cells the effects of italian mediterranean organic diet (imod) on health status limitations in the evidential basis supporting health benefits from a decreased exposure to pesticides through organic food consumption folate and one-carbon metabolism and its impact on aberrant dna methylation in cancer cell-and tissue-specific epigenetic changes associated with chronic inflammation in insulin resistance and type diabetes mellitus obesity-induced dna hypermethylation of the adiponectin gene mediates insulin resistance increased oxidative stress in obesity and its impact on metabolic syndrome dietary polyphenols mediated regulation of oxidative stress and chromatin remodeling in inflammation epigenetic modifications in adipose tissue-relation to obesity and diabetes the spleen in local and systemic regulation of immunity intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake effect of intermittent fasting and refeeding on insulin action in healthy men probiotics and neurodegenerative diseases: deciphering the gut brain axis long live foxo: unraveling the role of foxo proteins in aging and longevity intestinal microbiota, diet and health changes in human gut microbiota composition are linked to the energy metabolic switch during d of buchinger fasting diet, microbiota, and microbial metabolites in colon cancer risk in rural africans and african americans impact of diet in shaping gut microbiota revealed by a comparative study in children from europe and rural africa toxicology for real-life risk simulation-editorial preface to this special issue integrated transcriptomics and metabolomics reveal signatures of lipid metabolism dysregulation in heparg liver cells exposed to pcb effects of periodic fasting on fatty liver index-a prospective observational study effects of single and combined toxic exposures on the gut microbiome: current knowledge and future directions the role of gut microbiota in intestinal inflammation with respect to diet and extrinsic stressors. microorganisms nicotine and sars-cov- : covid- may be a disease of the nicotinic cholinergic system covid in northern italy: an integrative overview of factors possibly influencing the sharp increase of the outbreak (review) zinc and respiratory tract infections: perspectives for covid (review) impact of diet-modulated butyrate production on intestinal barrier function and inflammation the impact of nutrition on the human microbiome dysregulation of allergic airway inflammation in the absence of microbial colonization regulation of inflammatory responses by gut microbiota and chemoattractant receptor gpr the role of short-chain fatty acids in health and disease gpr /ffa : physiopathological relevance and therapeutic prospects metabolite-sensing receptors gpr and gpr a facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals diet, metabolites, and "western-lifestyle" inflammatory diseases short-chain fatty acids stimulate angiopoietin-like synthesis in human colon adenocarcinoma cells by activating peroxisome proliferator-activated receptor gamma butyrate reduces colonic paracellular permeability by enhancing ppargamma activation regulation of inflammation by short chain fatty acids molecular mechanisms of insulin resistance that impact cardiovascular biology immunomodulatory effects of deacetylase inhibitors: therapeutic targeting of foxp + regulatory t cells the microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial a mixture of trans-galactooligosaccharides reduces markers of metabolic syndrome and modulates the fecal microbiota and immune function of overweight adults effects of rye and whole wheat versus refined cereal foods on metabolic risk factors: a randomised controlled two-centre intervention study effects of arabinoxylan and resistant starch on intestinal microbiota and short-chain fatty acids in subjects with metabolic syndrome: a randomised crossover study a high intake of dietary fiber influences c-reactive protein and fibrinogen, but not glucose and lipid metabolism, in mildly hypercholesterolemic subjects effect of wk of resistant starch supplementation on cardiometabolic risk factors in adults with prediabetes: a randomized controlled trial effects of an isocaloric healthy nordic diet on insulin sensitivity, lipid profile and inflammation markers in metabolic syndrome-a randomized study (sysdiet) decreased insulin secretion and incretin concentrations and increased glucagon concentrations after a high-fat meal when compared with a high-fruit and -fiber meal fiber-rich diet with brown rice improves endothelial function in type diabetes mellitus: a randomized controlled trial a diet high in fatty fish, bilberries and wholegrain products improves markers of endothelial function and inflammation in individuals with impaired glucose metabolism in a randomised controlled trial: the sysdimet study dietary supplementation with inulin-propionate ester or inulin improves insulin sensitivity in adults with overweight and obesity with distinct effects on the gut microbiota, plasma metabolome and systemic inflammatory responses: a randomised cross-over trial greater expression of postprandial inflammatory genes in humans after intervention with saturated when compared to unsaturated fatty acids a short-term diet and exercise intervention ameliorates inflammation and markers of metabolic health in overweight/obese children could resistant starch supplementation improve inflammatory and oxidative stress biomarkers and uremic toxins levels in hemodialysis patients? a pilot randomized controlled trial resistant dextrin, as a prebiotic, improves insulin resistance and inflammation in women with type diabetes: a randomised controlled clinical trial strawberries decrease circulating levels of tumor necrosis factor and lipid peroxides in obese adults with knee osteoarthritis low-energy diets differing in fibre, red meat and coffee intake equally improve insulin sensitivity in type diabetes: a randomised feasibility trial effects of increased wholegrain consumption on immune and inflammatory markers in healthy low habitual wholegrain consumers postprandial glucose and nf-kappab responses are regulated differently by monounsaturated fatty acid and dietary fiber in impaired fasting glucose subjects is there any place for resistant starch, as alimentary prebiotic, for patients with type diabetes? inulin controls inflammation and metabolic endotoxemia in women with type diabetes mellitus: a randomized-controlled clinical trial oligofructose-enriched inulin improves some inflammatory markers and metabolic endotoxemia in women with type diabetes mellitus: a randomized controlled clinical trial whole-grain intake favorably affects markers of systemic inflammation in obese children: a randomized controlled crossover clinical trial a time to fast the safety limits of an extended fast: lessons from a non-model organism comparative physiology of fasting, starvation, and food limitation long-term energy balance in child-bearing gambian women fasting: molecular mechanisms and clinical applications effects of intermittent fasting on health impact of intermittent fasting on health and disease processes human bcl- reverses survival defects in yeast lacking superoxide dismutase and delays death of wild-type yeast the sir / / complex and sir alone promote longevity in saccharomyces cerevisiae by two different mechanisms replicative and chronological aging in saccharomyces cerevisiae dietary deprivation extends lifespan in caenorhabditis elegans heterothermy in growing king penguins caloric restriction increases learning consolidation and facilitates synaptic plasticity through mechanisms dependent on nr b subunits of the nmda receptor late-onset intermittent fasting dietary restriction as a potential intervention to retard age-associated brain function impairments in male rats effects of intermittent feeding upon body weight and lifespan in inbred mice: interaction of genotype and age apparent prolongation of the life span of rats by intermittent fasting the effects of infantile stimulation and intermittent fasting and feeding on life span in the black-hooded rat beneficial effects of exercise on growth of rats during intermittent fasting fasting cycles retard growth of tumors and sensitize a range of cancer cell types to chemotherapy starvation-induced activation of atm/chk /p signaling sensitizes cancer cells to cisplatin mitochondrial production of reactive oxygen species and incidence of age-associated lymphoma in of mice: effect of alternate-day fasting starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy intermittent fasting and caloric restriction ameliorate age-related behavioral deficits in the triple-transgenic mouse model of alzheimer's disease aberrant heart rate and brainstem brain-derived neurotrophic factor (bdnf) signaling in a mouse model of huntington's disease dietary restriction normalizes glucose metabolism and bdnf levels, slows disease progression, and increases survival in huntingtin mutant mice age and energy intake interact to modify cell stress pathways and stroke outcome the fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity caloric restriction alternate-day fasting protects the rat heart against age-induced inflammation and fibrosis by inhibiting oxidative damage and nf-kb activation. free radic intermittent fasting and dietary supplementation with -deoxy-d-glucose improve functional and metabolic cardiovascular risk factors in rats intermittent feeding and fasting reduces diabetes incidence in bb rats cardioprotection by intermittent fasting in rats the effects of ghrelin on inflammation and the immune system autophagy regulates lipid metabolism cardioprotective effect of intermittent fasting is associated with an elevation of adiponectin levels in rats fasting therapy-an expert panel update of the consensus guidelines effects of intermittent fasting on glucose and lipid metabolism intermittent metabolic switching, neuroplasticity and brain health flipping the metabolic switch: understanding and applying the health benefits of fasting fasting imparts a switch to alternative daily pathways in liver and muscle alternate day fasting (adf) with a high-fat diet produces similar weight loss and cardio-protection as adf with a low-fat diet short-term modified alternate-day fasting: a novel dietary strategy for weight loss and cardioprotection in obese adults the effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women alternate-day fasting in nonobese subjects: effects on body weight, body composition, and energy metabolism fasting and cancer treatment in humans: a case series report fasting and cancer: molecular mechanisms and clinical application safety, health improvement and well-being during a to -day fasting period in an observational study including subjects the endocrine regulation of aging by insulin-like signals evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate null mice the first long-lived mutants: discovery of the insulin/igf- pathway for ageing mtor is a key modulator of ageing and age-related disease the genetics of ageing sensing of energy and nutrients by amp-activated protein kinase the amp-activated protein kinase aak- links energy levels and insulin-like signals to lifespan in c. elegans a critical role of snf a/dampkalpha (drosophila amp-activated protein kinase alpha) in muscle on longevity and stress resistance in drosophila melanogaster metabolic adaptations to fasting and chronic caloric restriction in heart, muscle, and liver do not include changes in ampk activity linking sirtuins, igf-i signaling, and starvation deacetylation of nuclear lc drives autophagy initiation under starvation sirtuins at a glance regulation of pgc- alpha, a nodal regulator of mitochondrial biogenesis age-associated development of inflammation in wistar rats: effects of caloric restriction molecular inflammation as an underlying mechanism of the aging process and age-related diseases extending healthy life span-from yeast to humans the hallmarks of aging ramadan diurnal intermittent fasting modulates sod , tfam, nrf , and sirtuins (sirt , sirt ) gene expressions in subjects with overweight and obesity intermittent fasting could ameliorate cognitive function against distress by regulation of inflammatory response pathway role of nrf in oxidative stress and toxicity extending life span by increasing oxidative stress. free radic metabolic slowing and reduced oxidative damage with sustained caloric restriction support the rate of living and oxidative damage theories of aging alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. free radic alternate day fasting for weight loss in normal weight and overweight subjects: a randomized controlled trial effects of ramadan fasting on glucose homeostasis, lipid profiles, inflammation and oxidative stress in women with polycystic ovary syndrome in kashan effects of eight weeks of time-restricted feeding ( / ) on basal metabolism, maximal strength, body composition, inflammation, and cardiovascular risk factors in resistance-trained males intermittent fasting during ramadan attenuates proinflammatory cytokines and immune cells in healthy subjects the effects of ramadan fasting on body composition, blood pressure, glucose metabolism, and markers of inflammation in nafld patients: an observational trial randomized cross-over trial of short-term water-only fasting: metabolic and cardiovascular consequences the effect of short periods of caloric restriction on weight loss and glycemic control in type diabetes alternate day fasting and endurance exercise combine to reduce body weight and favorably alter plasma lipids in obese humans long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans the effects of modified alternate-day fasting diet on weight loss and cad risk factors in overweight and obese women the effect of intermittent energy and carbohydrate restriction v. daily energy restriction on weight loss and metabolic disease risk markers in overweight women improvement in coronary heart disease risk factors during an intermittent fasting/calorie restriction regimen: relationship to adipokine modulations key: cord- - lbrfsrh authors: adam, kirsten c.s.; chang, lillian; rangan, nicole; serences, john t. title: steady-state visually evoked potentials and feature-based attention: pre-registered null results and a focused review of methodological considerations date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: lbrfsrh feature-based attention is the ability to selectively attend to a particular feature (e.g., attend to red but not green items while looking for the ketchup bottle in your refrigerator), and steady-state visually evoked potentials (ssveps) measured from the human electroencephalogram (eeg) signal have been used to track the neural deployment of feature-based attention. although many published studies suggest that we can use trial-by-trial cues to enhance relevant feature information (i.e., greater ssvep response to the cued color), there is ongoing debate about whether participants may likewise use trial-bytrial cues to voluntarily ignore a particular feature. here, we report the results of a preregistered study in which participants either were cued to attend or to ignore a color. counter to prior work, we found no attention-related modulation of the ssvep response in either cue condition. however, positive control analyses revealed that participants paid some degree of attention to the cued color (i.e., we observed a greater p component to targets in the attended versus the unattended color). in light of these unexpected null results, we conducted a focused review of methodological considerations for studies of feature-based attention using ssveps. in the review, we quantify potentially important stimulus parameters that have been used in the past (e.g., stimulation frequency; trial counts) and we discuss the potential importance of these and other task factors (e.g., feature-based priming) for ssvep studies. attending to a specific feature leads to systematic changes in the firing rates of neurons that encode the relevant feature space. for example, when looking for a ripe tomato, the firing rate of neurons tuned to red will be enhanced and the firing rate of neurons tuned to other colors will be suppressed (e.g. responses to green; ipata et al., ; martinez-trujillo & treue, ; störmer & alvarez, ) . although there is broad agreement that participants may learn to suppress irrelevant distractors with sufficient experience, there is disagreement about whether these behavioral suppression effects may be volitionally implemented on a trial-by-trial basis in response to an abstract cue (i.e., a "volitional account"), or if they instead are solely implemented via implicit or statistical learning mechanisms (i.e., a "priming-based" account). consistent with a volitional account, some work has found that participants can learn to use a trial-by-trial cue to ignore a particular color ( suppress a color on a trial-by-trial basis independent of target enhancement (i.e., a strong version of a volitional suppression account), we predicted that the time-course of enhancement vs. suppression of the ssvep signal would be reduced or reversed (i.e., that suppression of the cued, to-be-ignored color may happen even prior to enhancement of the other color). alternatively, if participants recode the "ignore" cue to serve as an indirect "attend" cue (e.g., "since i'm cued to ignore blue, that means i should attend red"; beck & hollingworth, ; becker et al., ) , then we predicted that target enhancement would always precede distractor suppression regardless of whether participants were cued to attend or ignore a particular color. to preview the results, we were unable to fully test our hypotheses about the time- course of feature-based enhancement and suppression because we did not find evidence for an overall attention effect with our task procedures. despite robust ssvep amplitude (cohen's d > ), we observed no credible evidence that the ssvep response was higher for an attended versus unattended color in either cue condition. positive control analyses revealed that our lack of ssvep effect was not due to a complete lack of attention to the attended color: erp responses (p ) to the targets were modulated by attention as light of our inconclusive results, we also performed a focused methodological review of key potential task differences between our work and prior work that may have resulted in our failure to detect the effect of feature-based attention on ssvep amplitude. we considered whether task factors such as stimulus flicker frequency, sample size, stimulus duration, and stimulus color might have impacted our ability to observe an attention effect. no single methodological factor that we considered neatly explains our lack of effect. given our results and literature review, we propose that future work is needed to systematically explore two key factors: ( ) variation in feature-based attention effects across stimulus flicker frequencies and ( ) the extent to which feature-based priming modulates ssvep attention effects. we published a pre-registered research plan on the open science framework prior to data collection (https://osf.io/kfg h/). our raw data and analysis code will be made available online on the open science framework at https://osf.io/ew dv/ upon acceptance for publication. healthy volunteers (n = ; gender = female, male; mean age = . years [sd = . , min = , max = ]; handedness not recorded; corrected-to-normal visual acuity; normal color vision) participated in one . to hour experimental session at the university of california san diego (ucsd) campus, and were compensated $ /hr. procedures were approved by the ucsd institutional review board, and all participants provided written informed consent. inclusion criteria included normal or corrected-to- normal visual acuity, normal color vision, age between and years old, and no self- reported history of major neurological disorders (e.g., epilepsy, stroke). data were excluded from analysis if there were fewer than trials in either cue condition (either due to leaving the study early or after artifact rejection). a sample size of was pre- registered, and artifact rejection criteria were pre-registered (see section "eeg preprocessing" below for more details). after running each participant, we checked whether the data were usable (i.e., sufficient number of artifact-free trials) so that we would know when to stop data collection. to reach our final sample size (n = participants with usable data), we ran a total of participants. nine participants' data were not used for the following reason: subjects with an error in the task code (n = ), subjects who stopped the study early due to technical issues or to participants' preferences (n = ), subjects with too many artifacts (n = ). note, we were one subject short of our pre-registered target sample size of because data collection was suspended due to covid- . however, as our later power analyses will show, we do not believe the addition of further subject would have meaningfully altered our conclusions. heterochromatic flicker photometry task. we chose perceptually equiluminant colors for each participant using a heterochromatic flicker photometry task. participants viewed a large circular, flickering stimulus ( º radius) on a black screen ( . cd/m ). we generated circular color spaces in cielab-space with varying luminance (circles centered on: l = - , a = , b = ; colors equally spaced around circle with radius = ) for use in the task. participants matched each of the colors to a medium-gray reference color (rgb = . . . ). on each trial, the circular background was flickered between two different colors. one color was always medium-gray, and the other color was the to-be-matched color on that trial. the colors of circular background were phase reversed at a rate of hz, giving the appearance of a fast flicker when the subjective luminance values were not matched. on top of the flickering circular stimulus small oriented bars were drawn in the medium- gray reference color (the bars changed locations at a rate of hz). the oriented bars served no purpose other than subjectively making it easier to discriminate fine-grained differences in luminance between the flickering colors (i.e., these bars gave secondary visual cues about equiluminance via the "minimally distinct border" phenomenon, kaiser, ) . participants increased or decreased the luminance of the to-be-matched color (using up and down arrow keys) until the amount of perceived flicker was minimized -the point of perceptual equiluminance. the luminance starting value of the to-be-matched color was chosen at random on each trial. once satisfied with their response, the participant pressed spacebar to continue to the next trial. each to-be-matched color was repeated times ( trials total). feature-based attention task. all stimuli were viewed on a luminance calibrated crt monitor ( x resolution, hz refresh rate) from a distance of ~ cm in a dimly lit room. stimuli were generated using matlab a and the psychophysics toolbox (brainard, ; kleiner et al., ; pelli, ) . participants rested their chin on a chin- rest and fixated a central dot ( . º radius) throughout the experiment. the stimulus was a circular aperture (radius = ~ . º) filled with oriented bars (each bar ~ . º long and ~. º wide). bars were centered on a grid and separated by ~ bar length such that they never overlapped with one another. on each individual frame (~ . ms) this grid was randomly phase shifted ( : π in x and y coordinates) and rotated ( : º), thus giving the appearance of random flicker. to achieve steady state visually evoked potentials (ssvep) half of the bars flickered at hz ( frames on, frames off) and the other half flickered at hz ( frames on, frames off). due to the jittered rotation of bar positions and to the random assignment of colors to bars on each "on" frame, this means that the individual pixels that were "on" for each color varied from frame to frame. the unpredictable nature of each bar's exact position is thus quite similar to unpredictable stimuli that have been used in past work (e.g., andersen et al., ) . for each "off frame" no bars of that color were shown (e.g., if hz had an "on" frame and hz had an "off" frame, then only out of bars would be shown on the black background). if both the hz and hz bars were "off", then a black screen would be shown on that frame. see figure s for an illustration of some example frame-by-frame screenshots of the stimuli. on each trial (figure ), the participants viewed the stimulus array of flickering, randomly oriented bars presented on a black background ( . cd/m ). half of these bars were shown in one color (randomly chosen from the possible colors) and the other half were in another randomly chosen color (with the constraint that the two sets of bars must be two different colors). during an initial baseline ( , ms), participants viewed the flickering dots while they did not yet know which color to attend; during this baseline, the fixation point was a medium gray color (same as the reference color in the flicker photometry task). after the baseline, the fixation dot changed color, cuing the participants about which color to attend or ignore. in the "attend cue" condition, the color of the fixation point indicated which color should be attended. in the "ignore cue" condition, the color of the fixation point indicated which color should be ignored. these two conditions were blocked, and the order was counterbalanced across participants (further details below). during the stimulus presentation ( , ms), participants monitored the relevant color for a brief "target event" ( ms). during this brief target event, a percentage of lines in the relevant color will be coherent (iso-oriented). critically, the orientation of each coherent target or distractor event was completely unpredictable (randomly chosen between - degrees); thus, participants could not attend to a particular orientation in advance in order to perform the task. a target event occurred on % of trials, and participants were instructed to press the spacebar as quickly as possible if they detected a target event. importantly, physically identical events (iso-oriented lines in a random orientation, ms) could also appear in the distractor color ( % of trials). participants were instructed that they should only respond to target events; if they erroneously responded to the distractor event, the trial was scored as incorrect. the target and/or distractor events could begin as early as cue onset ( ms) and no later than , ms after stimulus onset). participants could make responses up to second into the inter-trial interval. if both a target and distractor event were present, their onset times were separated by at least ms. target events, and distractor events in the main conditions. in the 'attend cue' condition, participants made a response when the iso-oriented lines were the same color as the cue (target event) and did not respond if the iso-oriented lines occur on the uncued color (distractor event). in the 'ignore cue' condition, participants made a response when the iso-oriented lines occurred on the uncued color (target event), and they did not respond if the iso-oriented lines occurred on the cued color (distractor event). note, all lines were of equal size in the real experiment; lines are shown at different widths here for easier visualization of the target and distractor colors. here, the iso-oriented lines are drawn at vertical in all examples. in the actual task, the iso-oriented lines could be any orientation ( - ). to ensure that the task was effortful for participants, the coherence of the lines in the target stimulus was adapted at the end of each block if behavior was outside the range of - % correct. at the beginning of the session, the target had % coherent iso- oriented lines. if accuracy over the block of trials was > %, coherency decreased by %. if block accuracy was < %, coherency increased by %. the maximum allowed coherence was % iso-oriented lines (so that participants would not be able to simply individuate and attend a single position to perform the task) and the minimum allowed coherence was %. the presence and absence of target and distractor events was balanced within each block yielding a total of sub-conditions within each cue type ( % each): ( ) target event + no distractor event, t d ( ) no target event + distractor event, t d ( ) target event + distractor event, t d ( ) no target event + no distractor event, t d . participants completed both task conditions (attend cue and ignore cue). the two conditions were blocked and counterbalanced within a session (i.e., half of participants performed the "attend cue" task for the first half of the session and the "ignore cue" task for the second half of the session.) each block of trials took approximately min sec. participants completed blocks ( per condition) for a total of trials per cue condition. note, we originally planned for blocks ( per condition) in the pre- registration, but the block number was reduced to after the first few participants did not finish all blocks. summary of deviations from the registered procedures. as described in-line above, there were some minor deviations from the pre-registration: ( ) we made changes to the pre-registered task code to fix errors that we discovered while running the first subjects (e.g., incorrect cues and behavioral feedback in the 'ignore cue' condition). ( ) we included code for eye-tracking, which allowed us to give participants automated real- time feedback if they blinked when they were not supposed to, i.e., during the stimulus period. ( ) we reduced the total number of experimental blocks from ( per cue condition) to ( per cue condition) due to time constraints. ( ) we had to prematurely stop data collection at n = out of due to covid- . ( ) we forgot to specify a specific statistical test for quantifying the robustness of overall ssveps in section "checking that an ssvep is elicited at the expected frequencies before collecting the full sample", so we have described our justification for the statistical tests we present here. ( ) due to unanticipated failure to detect an overall attention effect, we performed additional non- pre-registered control analyses to attempt to rule out possible explanations of this null effect (see section: "non pre-registered control analyses" below). eeg pre-processing continuous eeg data were collected online from ag/agcl active electrodes mounted in an elastic cap using a biosemi activetwo amplifier (cortech solutions, wilmington, nc). an additional external electrodes were placed on the left and right mastoids, above and below each eye (vertical eog), and lateral to each eye (horizontal eog). continuous gaze-position data were collected from an sr eyelink + eye- tracker (sampling rate: , hz; sr research, ottawa, ontario). we also measured stimulus timing with a photodiode affixed to the upper left-hand corner of the monitor (a white dot flickered at the to-be-attended color's frequency; the photodiode and this corner of the screen were covered with opaque black tape to ensure it was not visible). data were collected with a sampling rate of hz and were not downsampled offline. data were saved unfiltered and unreferenced (see: kappenman & luck, ) , then referenced offline to the algebraic average of the left and right mastoids, low-pass filtered (< hz) and high-pass filtered (>. hz). artifacts were detected using automatic criteria described below, and the data were visually inspected to confirm that the artifact rejection criteria worked as expected. we excluded subjects with fewer than trials remaining per cue condition. eye movements and blinks. we used the eye-tracking data and the heog/veog traces to detect blinks and eye movements. blinks were detected on-line during the task using the eye tracker. if a blink was detected (i.e., missing gaze position returned from the eye tracker), the trial was immediately terminated and the participant was given feedback that they had blinked (i.e., the word "blink" was written in white text in the center of the screen). if eye-tracking data could not be successfully collected (e.g., calibration issues), the veog trace was used to detect blinks and/or eye movements during offline artifact rejection. to do so, we used a split-half sliding window step function (luck, ; window size = ms, step size = ms, threshold = microvolts.) we also used a split-half sliding-window step function to check for eye-movements in the gaze-coordinate data from the eye-tracker (window size = ms, step size = ms, threshold = º) and in the horizontal electrooculogram (heog), window size = ms, step size = ms, threshold = microvolts, and to detect blinks and/or eye movements we also pre-registered an analysis plan for examining the time-course of ssvep amplitude. however, because our data failed to satisfy pre-registered pre-requisite analyses, we do not report these time-course effects here (for completeness, we show the time-course of snr in figure s ). checking that an ssvep is elicited at the expected frequencies before collecting the full sample. at n = , we planned to confirm that our task procedure successfully produced reliable ssvep responses (i.e., check that we observed peaks at the correct stimulus flicker frequencies). if our task procedures failed to elicit an ssvep at the expected frequencies, we had planned to stop data collection and alter the task to troubleshoot the problem (e.g., optimize timing, choose different flicker frequencies, make stimuli brighter, etc.). we planned to begin data collection over again if we failed this trouble-shooting step. note, at this early stage we only verified if the basic method worked (ssvep frequencies were robust): we did not test whether any hypothesized attention effects were present, as this could inflate our false discovery rate (kravitz & mitroff, ) . note, in the original pre-registration we failed to specify what test we would run to determine if ssvep frequencies were robustly represented in the eeg signal. theoretical chance for snr would be , so the simplest test would be to compare the snr for our stimulation frequencies ( and hz) to using a t-test, which we report. however, it is often is better to compare to an empirical baseline with a reasonable amount of noise (combrisson & jerbi, ) . as such, we opted to also compute an effect size comparing the snr for our stimulation frequencies to all other frequencies (with the exception that we did not use frequencies +/- hz of or hz as baseline values, since snr was calculated as the power at frequency f divided by the power in the adjacent -hz bins). checking achieved power for the basic attention effect. without we did not anticipate our failure to find an overall attention effect with these task procedures and set of pre-registered "sanity check" analyses described above. to further understand the lack of ssvep attention effect, we performed additional non-pre- registered control analyses. we first confirmed that our ssvep procedure was effective at eliciting robust, frequency-specific modulations of the eeg signal. after collecting the first participants, we checked that overall ssvep amplitudes for our two target frequencies ( and hz) were robust when collapsed across conditions (fig a) before proceeding with data collection. we indeed found that the ssvep signal was robust during the stimulus period even with n= for both the hz frequency (mean snr = . , sd = . , snr > : p < . ) and for the hz frequency (mean snr = . , sd = . , snr > : p < . ). these values were similar for the full n= sample (fig b) . to compute an effect size, we compared snr values for each target frequency ( hz and hz) to the snr values for each baseline frequency (frequencies from - hz not within +/- hz of or hz). snr values for the target frequencies were significantly higher than baseline, mean cohen's d = . (sd = . ) and . (sd = . ), respectively (see figure s ) . as planned, we also confirmed that the electrodes we selected a priori (o , oz, and o ) were reasonable given the topography of overall ssvep amplitudes (i.e., they fell approximately centrally within the brightest portion of the heat map; figure experimental conditions. color scale indicates snr. as expected, the a priori electrodes o , o , and oz (magenta circles) showed robust snr during the stimulus period. next, we checked for a basic attention effect, defined as a larger amplitude response evoked by the attended frequency compared to the ignored frequency). note, for the sake of clarity, all conditions are translated into "attend" terminology. that is, if a participant was cued to "ignore blue" ( hz) during the "ignore cue" condition (and the other color was red and hz), this will instead be plotted as "attend red" ( hz). figure shows the gaussian wavelet-derived frequency spectra during the stimulus period ( - ms) as a function of cue type (attend versus ignore) and attended frequency (attend hz or attend hz). we found a main effect of measured frequency, whereby snr was overall higher for versus hz, f( , ) = . , p < . , η p = . . however, we found no main effect of attended frequency (p = . ) or cue type (p = . ), and we found no significant interactions (p >= . ). collapsed down to a paired t-test, the observed effect size for attended versus unattended snr values was cohen's d = . . to detect an effect of this size with % power ( -β = . ; α = . ) would require a sample size n > , * . given that we did not find an overall attention effect, we did not analyze or interpret analysis of the ssvep time-course. however, for completeness we have shown the time course in figure s . frequency spectra in the attend cue (a) and ignore cue (b) conditions during the stimulus period. although we observe expected peaks at hz and hz, this ssvep response is not modulated by the attention manipulation. (c-d). violin plots of the signal-to-noise ratio at the ssvep frequencies in the attend cue (c) and ignore cue (d) conditions. although we pre-registered that we would analyze all trials (those with and without target/distractor events), most prior studies have included only trials without any target or distractor events in the main ssvep analysis (e.g., andersen et al., ; müller et al., ) . to ensure that our null result was not due to this analysis choice, we also planned in our pre-registration to examine the ssvep attention effect for trials with and without target and distractor events. when restricting our analysis to only trials without targets or distractors ( % of the trials, or trials total before artifact rejection), we likewise found no attention effect. as before, we found a main effect of measured frequency ( > hz), p < . , but no effect of cue condition (p = . ) or attended frequency (p = . ), and, most critically, we found no interaction between measured frequency and attended frequency (p = . ). frequency spectra for all combinations of target and distractor presence are shown in figures s and s . finally, we also pre-registered that we would check whether the similarity of the target and distractor colors ( versus degrees apart on a circular color wheel; figure b) would modulate the ssvep attention effect. we likewise found that the similarity of the distractor colors did not significantly modulate the ssvep response, and we found no attention effect (interaction of measured frequency and attended frequency) in either color distance condition (p >= . ; figure s ). we conducted additional control analyses to rule out possible sources of our failure to find an attention effect. first, we examined the photodiode recording to rule out any failures due to trial indexing. the photodiode measured the luminance of a white dot that flickered at the attended frequency on each trial. as expected, performing an fft on the photodiode time-course thus yielded near-perfect tracking of the attended frequency ( figure a-b, p < . ) . on the other hand, we again found null results for the main attention manipulation ( figure c -f) when using an fft analysis that more closely followed prior work. we ran a repeated measures anova on the signal to noise ratio values during the stimulus period, including the factors measured frequency ( hz, hz), attended frequency ( hz, hz), and cue type (attend, ignore). we found no main effect of measured frequency (p = . ), attended frequency (p = . ), or cue type (p = . ), and we found no significant interactions (p >= . ). however, the average signal- to-noise ratio of the stimulus frequencies was overall robust (m = . , sd = . , greater than chance value of : p < x - ), so our inability to observe the attention effect was not due to lack of overall ssvep signal. given that some work has reported significant effects only for the second harmonic figures s and s ) . we found no significant attention effects for either second harmonic frequency. we also re-ran the fft analysis with other electrode-selection methods to ensure our a priori choice of electrodes did not impede our ability to observe an effect (m. x. cohen & gulbinaite, ). we found no evidence that electrode choice led to our null effect, as exploiting information from all electrodes by implementing rhythmic entrainment source separation (ress) likewise yielded null effects ( figure s -s ). to ensure that inconsistent task performance did not lead to null effects, we repeated the main fft analysis on only accurate trials. we likewise found null attention effects when analyzing only accurate trials ( figure s ) . finally, we tested whether phase consistency, rather than power, may track attention in our task (e.g., nunez et al., ; tallon-baudry et al., ) . to do so, we performed an fft on single trials rather than on condition-averaged waveforms, and we extracted single-trial phase values. we calculated a phase-locking index by computing mean-resultant vector length on each condition's histogram of single-trial phase values. mean-resultant vector length ranges from (fully random values) to (perfectly identical values), for reference, see zar ( ) . we found no effect of attention on this phase- locking index ( figure s ). was not due to using gaussian wavelets rather than an fft, we repeated the main analysis with an fft. frequency spectra for the attend cue condition (c) and ignore cue condition (d) reveal an overall robust ssvep signal at hz and hz, but no modulation by attention. likewise, violin plots of signal-to-noise ratios again show robust signal but no modulation by attention in either the attend cue condition (e) or the ignore cue condition (f). positive control: analysis of event-related potential (p b) for an attention effect. consistent with prior work, we found a significantly larger p component for target onsets compared to distractor onsets ( figure ) . a repeated measures anova with within-subjects factors cue type (attend cue or ignore cue) and event type (target or distractor onset) revealed a robust main effect of event type (target > distractor), f( , ) = . , p < x - , η p = . , and a main event of cue type (attend > ignore), f( , ) = . , p = . , η p = . , but no interaction between event type and cue type (p = . ). control analyses confirmed this p modulation was not due to differential rates of making a motor response for targets and distractors ( figure s ). the main effect of event type (target > distractor) remained when analyzing only trials where participants made a motor response (p < . ). thus, the p was overall larger for target than distractor events, consistent with prior work that found this erp attention effect alongside an ssvep attention effect. we defined "feature-based attention manipulation" as having the following characteristics: ( ) participants were cued to attend a feature(s) within a feature dimension (e.g., attend red, ignore blue) rather than across a feature dimension (e.g. attend contrast, ignore orientation), ( ) the attended and ignored feature were both frequency-tagged in the same trials (rather than only feature tagged per trial), ( ) each frequency was both "attended" and "ignored" on different trials, so that the amplitude of a given frequency could be examined as a function of attention, ( ) the task could not be performed by adopting a strategy of splitting spatial attention to separate spatial locations. after applying these screening criteria, some of the studies that we initially we identified a total of experiments from unique papers (tables s -s ) meeting our inclusion criteria. from these experiments, we quantified variables such as the number of subjects, number of trials, frequencies used, and the presence or absence of an attention effect in the expected direction (attended > ignored). if more than one group of participants was used (e.g., an older adults group) then we included the study but only quantified results for the healthy young adult group (quigley et al., ; quigley & müller, ) . the tasks used in these studies fell broadly into one of categories: ( ) a competing gratings task, ( ) a whole-field flicker task, ( ) a hemifield flicker task and ( ) a central task with peripheral flicker. in the competing gratings task (table s ), participants viewed a stream of centrally-presented, superimposed gratings (e.g., a red horizontal grating and a green vertical grating). because colored, oriented gratings were typically used, participants could thus generally choose to attend based on either one or both features (color and/or orientation). each grating flickered at its own frequency (e.g. green grating shown at . hz, red grating shown at . hz, as in chen et al., ) . because the gratings were superimposed, on any given frame only one of the two gratings was shown. on frames where both gratings should be presented according to their flicker frequencies, a hybrid "plaid" stimulus was shown. studies using a competing gratings task include: (allison et in the whole-field flicker task (table s ) , participants viewed a spatially global stimulus comprised of small, intermingled dots or lines. typically, half of the dots or lines were presented in one feature (e.g., red) and the other half of the lines were presented in another (e.g., blue); each set of dots flickered at a unique frequency. although the most common attended feature was color, some task variants included ( ) attending high or low contrast stimuli ( ) attending a particular orientation or ( ) attending a particular conjunction of color and orientation. the whole-field flicker task was the most common task variant, and it is also most similar to the task performed here. studies using a whole- first, we examined whether insufficient power (e.g., fewer subjects and/or trials relative to prior work) could have led to our failure to detect an attention effect. the number of studies employing each task variant is plotted in figure a , the number of subjects per experiment is plotted in figure b , the number of trials per experiment is plotted in figure c , and stimulus duration is plotted in figure d next, we examined the percentage of trials where the attended feature was repeated (e.g., if the attended color was red on trial n, what was the chance that red would also be attended on trial n+ ?). the priming-based account of feature-based attention posits that participants cannot use trial-by-trial cues to enhance a particular feature, but rather, feature-based enhancement happens automatically when a particular feature is repeated (theeuwes, ) . thus, if there is a substantial proportion of trials where the repeated color was attended (e.g. with possible colors, both the attended and ignored color will be repeated on % of trials), then the observed attentional enhancement effects might be driven primarily by incidental repetitions of attended features. in our study, we used different colors to reduce the potential effect of inter-trial priming on the observed ssvep attention effects ( % repeats of the attended color, % repeats of the attended color and the ignored color). we quantified the approximate percentage of trials on which an attended feature on one trial is repeated on the next trial (within a given block of trials). in some studies, participants were cued to attend more than one feature on a given trial, or they sometimes attended to a conjunction of features. in these cases, we in the studies with % repeats might be equally attributed to their low trial counts (median of repeats as the present study (störmer & alvarez, ). störmer and alvarez found a significant attention effect while using unique colors (intermixed randomly from trial to trial). the findings by störmer and alvarez provide evidence against the feature-based priming account, and suggest the task factor "number of colors" cannot definitively explain our inability to observe an attention effect. however, given the lack of extant work using unpredictable color cues, we think future, systematic work is needed to determine the degree to which inter-trial priming effects may modulate the size and reliability of feature- based attention effects. ssvep frequencies. we examined frequencies that have been most commonly used in the literature. in our study, we chose relatively high frequencies ( and hz) in order to have increased temporal resolution for detecting potential time-course effects. in addition, some have argued that using higher frequencies as advantages for driving a more finally, we examined whether the type of task and task difficulty may have influenced our ability to detect an attention effect. in particular, the specific targets that we used may differ slightly from prior work. in our experiment, participants detected a brief period ( ms) of an on average ~ % coherent orientation (the coherent line orientation was a random, unpredictable direction, from - degrees). in this task, participants performed well above chance, but the task was still fairly challenging overall (d' = . ). this raises the possibility that, compared to prior ssvep studies, subjects were giving up on some percentage of the trials and that this contributed to the lack of attention effects. for the reviewed papers in which participants detected a target within the flickering stimulus ("whole-field flicker task" and "hemifield flicker task"), we compiled information about participants' accuracy, the duration of the target, the type of target, and the percentage of dots/lines that comprised the target (table s ) . we found that our particular task (detect a coherent orientation in the cued color) was slightly different from the other tasks that have been used. two other prior studies did not use a behavioral task at all: participants were simply instructed to monitor a particular feature without making any overt response (pei et although the particulars of the luminance and motion tasks subtly differ from our orientation task, it is not clear why ssveps would track attention when the target is a coherent luminance value or motion direction, but not when the target is a coherent condition. thus, because we found no overall ssvep attention effect, we were unable to test our hypotheses about how this attention effect was modulated by being cued to attend versus cued to ignore. despite the lack of an ssvep attention effect, positive control analyses indicated that that participants did successfully select the cued target color (i.e., we observed a significantly larger p component for target events in the attended color than in the ignored color). given our failure to observe an effect of attention on ssvep amplitude with our task procedures, we performed a focused review of the literature to quantify key methodological aspects of prior studies using ssveps to study feature-based attention. based on this review, we concluded that sample size and trial counts likely did not explain our failure to find an effect; our sample size and trial counts were near the maximum values found in the surveyed literature. likewise, the range of accuracy values found in the literature suggests that task difficulty does not explain our failure to find an attention effect. however, two key, intentional design differences may have hampered our ability to find an effect: ( ) the number of colors in our stimulus set and ( ) the frequencies used to generate the ssvep. the first key design difference in our study was the number colors in our stimulus set. we purposefully minimized the influence of inter-trial priming on our estimates of feature-based attention (theeuwes, ) by using unique colors and randomly chose target and distractor colors on each trial. according to a priming account of feature-based attention, a relatively high proportion of trials where the attended color is repeated back- to-back could inflate or even entirely drive apparent feature-based attention effects. using colors somewhat protects against this possibility, because it ensures that the attended color is repeated on % of trials, and both the attended/ignored colors are repeated on only % of trials. in the literature, we found that most studies had back-to-back color repeats on at least % of trials. it is thus plausible that inter-trial priming could contribute to observed attention differences in these studies. contrary to a priming account, however, one study found robust feature-based attention effects using a set of unique colors (störmer & alvarez, ) , suggesting that participants can use a cue to direct feature-based attention even when the proportion of repeated trials is relatively low. to date, however, no study has directly manipulated the proportion of repeated trials or the number of possible stimulus colors in an ssvep study. given emerging evidence that history-driven effects play an important role in shaping both spatial and feature-based attentional selection ( the second key design difference in our study was the chosen set of frequencies. to ensure adequate temporal resolution to characterize time-course effects, we chose to use slightly higher frequencies ( and hz). we believed these values would be reasonable, because an initial study of the time-course of spatial attention used ssvep it is perhaps puzzling that frequencies above hz have been commonly used in the spatial attention literature but have not been used in the feature-based attention literature. the truncation of the frequency distribution in the reviewed literature could be a piece of the "file drawer" in action. it is possible that other researchers likewise discovered that they were unable to track feature-based attention using certain frequencies, but that these null results were never published due to journals' and authors' biases toward publishing positive results (ferguson & heene, ; rosenthal, ) and biases against publishing negative results (i.e., "censoring of null results", guan & vandekerckhove, ; sterling, ; sterling et al., ) . thus, our results highlight the practical and theoretical importance of regularly publishing null results. on the practical side, if prior null results had been published, we may have better known which frequencies to use or avoid, and we would have been able to test our key hypotheses. on the theoretical side, our results highlight how seemingly unimportant null results can have implications for theory when viewed in the context of the broader literature. for example, if certain frequencies track spatial but not feature-based attention, this may inform our understanding of the brain networks and cognitive processes differentially modulated by flicker frequency (ding et al., ; srinivasan et al., ) . in short, we found no evidence that ssveps track the deployment of feature-based attention with our procedures, and future methodological work is needed to determine constraints on generalizability of the ssvep method for tracking feature-based attention. we performed a focused review of prior studies using ssveps to study feature-based attention, and from this review we identified two key factors (frequencies used; likelihood of inter-trial feature priming) that should be systematically investigated in future work. history-driven modulations of population codes in early visual cortex during visual search top-down attention is limited within but not between feature dimensions the berger rhythm: potential changes from the towards an independent brain-computer interface using steady state visual evoked potentials effects of feature-selective and spatial attention at different stages of visual processing attention facilitates multiple stimulus features in parallel in human visual cortex global facilitation of attended features is obligatory and restricts divided attention behavioral performance follows the time course of neural facilitation and suppression during cued shifts of feature-selective attention color-selective attention need not be mediated by spatial attention tracking the allocation of attention in visual scenes with steady-state evoked potentials attentional selection of feature conjunctions is accomplished by parallel and independent selection of single features bottom-up biases in feature-selective too little, too late, and in the wrong place: alpha band activity does not reflect an active mechanism of selective attention attentive and pre-attentive aspects of figural processing templates for rejection: configuring attention to ignore task-irrelevant features top-down versus bottom-up attentional control: a failed theoretical dichotomy evidence for negative feature guidance in visual search is explained by spatial recoding no templates for rejection: a failure to configure attention to ignore task-irrelevant features how many trials does it take to get a significant erp effect? it depends attention to a threat-related feature does not interfere with concurrent attentive feature selection the psychophysics toolbox distinct attention networks for feature enhancement and suppression in vision high gamma power is phase-locked to theta oscillations in human neocortex location-based explanations do not account for active attentional suppression feature-based attention resolves differences in target-distractor similarity through multiple mechanisms the power of human brain magnetoencephalographic signals can be modulated up or down by changes in an attentive visual task tracking feature-based attention normal electrocortical facilitation but abnormal target identification during visual sustained attention in schizophrenia using neuronal populations to study the mechanisms underlying spatial and feature attention rhythmic entrainment source separation: optimizing analyses of neural responses to rhythmic sensory stimulation exceeding chance level by chance: the caveat of theoretical chance levels in brain signal classification and statistical assessment of decoding accuracy feature guidance by negative attentional templates depends on search difficulty taming the white bear: initial costs and eventual benefits of distractor inhibition attentional modulation of ssvep power depends on the network tagged by the flicker frequency cortical mechanisms of prioritizing selection for rejection in visual search statistical regularities induce spatial as well as feature-specific suppression a vast graveyard of undead theories: publication bias and psychological science's aversion to the null global enhancement but local suppression in feature-based attention near-real-time feature-selective feature-based attention is constrained to attended locations in older adults a bayesian approach to mitigation of publication bias attention differentially modulates the amplitude of resonance frequencies in the visual cortex feature-based attentional tuning during biological motion detection measured with ssvep the functional organization of human extrastriate cortex: a pet-rcbf study of selective attention to faces and locations human eeg responses to ? hz flicker: resonance phenomena in visual cortex and their potential correlation to cognitive phenomena lip responses to a popout stimulus are reduced if it is overtly ignored when conflict cannot be avoided relative contributions of early selection and frontal executive control in mitigating stroop conflict temporal dynamics of divided spatial attention a category-specific top-down attentional set can affect the neural responses outside the current focus of attention sensation luminance: a new name to distinguish cie luminance from luminance dependent on an individual's spectral sensitivity the effects of electrode impedance on data quality and statistical significance in erp recordings time courses of attentional modulation in neural amplification and synchronization measured with steady-state visual-evoked potentials audio-visual synchrony and feature-selective attention co- amplify early visual processing attention induces synchronization-based response gain in steady-state visual evoked potentials what's new in psychtoolbox- ? estimates of a priori power and false discovery rates induced by post-hoc changes from thousands of independent replications large-scale network-level processes during entrainment an introduction to the event-related potential technique feature-based attention increases the selectivity of population responses in primate visual cortex cortical summation and attentional modulation of combined chromatic and luminance signals neural mechanisms of divided feature-selective attention to colour the ignoring paradox: cueing distractor features leads first to selection, then to inhibition of to-be-ignored items. attention, perception feature-based attention selective attention to stimulus location modulates the steady-state visual evoked potential feature-selective attention enhances color signals in early visual areas of the human brain it takes two to tango: suppression of task-irrelevant features requires (spatial) competition concurrent recording of steady-state and transient event- related potentials as indices of visual-spatial selective attention effects of spatial selective attention on the steady-state visual evoked potential in the - hz range the time course of cortical facilitation during cued shifts of spatial attention the steady-state visual evoked potential in vision research: a review individual differences in attention influence perceptual decision making memory for object features versus memory for object location: a positron-emission tomography study of encoding and retrieval processes causal involvement of visual area mt in global feature-based enhancement but not contingent attentional capture neural responses to target features outside a search array are enhanced during conjunction but not unique- neural correlates of object-based attention the videotoolbox software for visual psychophysics: transforming numbers into movies feature- selective attention: evidence for a decline in old age feature-selective attention in healthy old age: a selective decline in selective attention cortical evidence for negative search templates steady-state evoked potentials the file drawer problem and tolerance for null results expectations do not alter early sensory processing during perceptual decision-making capture versus suppression of attention by salient singletons: electrophysiological evidence for an automatic attend-to-me signal steady-state visual evoked potentials distributed local sources and wave-like dynamics are sensitive to flicker frequency rapid adaptive adjustments of selective attention following errors revealed by the time course of steady-state visual evoked potentials publication decisions and their possible effects on inferences drawn from tests of significance-or vice versa publication decisions revisited the effect of the outcome of statistical tests on the decision to publish and vice versa feature-based attention elicits surround suppression in feature space stimulus specificity of phase-locked and non-phase-locked hz visual responses in human. the journal of neuroscience attentional capacity for processing concurrent stimuli is larger across sensory modalities than within a modality feature-based attention: it is all bottom-up priming selection of visual objects in perception and working memory one at a time using frequency tagging to quantify attentional deployment in a visual divided attention task inhibition in selective attention experience-dependent attentional tuning of distractor rejection attention selects informative neural populations in human v steady-state visually evoked potentials: focus on essential paradigms and future perspectives protecting visual short- term memory during maintenance: attentional modulation of target and distractor representations statistical regularities modulate attentional capture how to inhibit a distractor location? statistical learning versus active, top-down suppression the neural correlates of feature-based selective attention when viewing spatially and temporally overlapping images effect of higher frequency on the classification of steady-state visual evoked potentials biostatistical analysis an independent brain- computer interface using covert non-spatial visual selective attention feature-based attention modulates feedforward visual processing orientation. for example, just like in the coherent motion direction tasks used by others, the angle of the coherent orientation in our task was completely unpredictable. thus, participants in our task and in other tasks could not form a template of an orientation or motion direction they should attend in advance and instead had to attend to an orthogonal feature dimension such as color. in addition, in both prior tasks and the current task there were an equal number of coherent events in the cued and uncued color. if participants failed to attend to the cued color and instead responded to any orientation event, their performance in the task would be at chance. behavioral performance in the reviewed studies ranged from d' = . to d' = . (table s ). in many studies, performance was quite high (d' > or accuracy > %) relative to performance in our study (adamian et in this pre-registered study, we sought to test whether cuing participants to ignore however, we failed to replicate this basic overall attention effect; we found no difference in ssvep amplitude as a function of attention in either the attend cue or the ignore cue key: cord- -pmufsvg authors: nieuwland, mante s.; arkhipova, yana; rodríguez-gómez, pablo title: anticipating words during spoken discourse comprehension: a large-scale, pre-registered replication study using brain potentials() date: - - journal: cortex doi: . /j.cortex. . . sha: doc_id: cord_uid: pmufsvg numerous studies report brain potential evidence for the anticipation of specific words during language comprehension. in the most convincing demonstrations, highly predictable nouns exert an influence on processing even before they appear to a reader or listener, as indicated by the brain’s neural response to a prenominal adjective or article when it mismatches the expectations about the upcoming noun. however, recent studies suggest that some well-known demonstrations of prediction may be hard to replicate. this could signal the use of data-contingent analysis, but might also mean that readers and listeners do not always use prediction-relevant information in the way that psycholinguistic theories typically suggest. to shed light on this issue, we performed a close replication of one of the best-cited erp studies on word anticipation (van berkum, brown, zwitserlood, kooijman & hagoort, ; experiment ), in which participants listened to dutch spoken mini-stories. in the original study, the marking of grammatical gender on pre-nominal adjectives (‘groot/grote’) elicited an early positivity when mismatching the gender of an unseen, highly predictable noun, compared to matching gender. the current pre-registered study involved that same manipulation, but used a novel set of materials twice the size of the original set, an increased sample size (n= ), and bayesian mixed-effects model analyses that better accounted for known sources of variance than the original. in our study, mismatching gender elicited more negative voltage than matching gender at posterior electrodes. however, this n -like effect was small in size and lacked support from bayes factors. in contrast, we successfully replicated the original’s noun effects. while our results yielded some support for prediction, they do not support the van berkum et al. effect and highlight the risks associated with commonly employed data-contingent analyses and small sample sizes. our results also raise the question whether dutch listeners reliably or consistently use adjectival inflection information to inform their noun predictions. according to current theories of language comprehension, people implicitly and routinely anticipate upcoming words by activating their meaning and possibly other features in advance (e.g., altmann & mirkovic, ; dell & chang, ; levy, ; pickering & gambi, ; pickering & garrod, ) . the most convincing evidence for word anticipation or prediction is when a neural effect of a predictable word is obtained before that word is presented, for example, on a preceding article or adjective. when readers or listeners predict a specific noun, pre-nominal words that mismatch the predicted word elicit an enhanced event-related potential (erp) response compared to matching words (for a review, see kutas, delong & smith, ) . several studies have provided such evidence in the past decades (e.g., delong, urbach & kutas, ; van berkum, brown, zwitserlood & hagoort, ; otten & van berkum, , wicha, moreno & kutas, ) . however, the replicability and consistency of the observed patterns have recently come into question (ito, martin & nieuwland, a,b; kochari & flecken, ; nieuwland, politzer-ahles, heyselaar, segaert, darley, kazanina, et al., ) , which has highlighted the need for replication of key findings. the current study aims to replicate one such highly influential erp study on linguistic prediction (experiment of van berkum et al., , henceforth vb ), which tested for effects of prediction on pre-nominal adjectives during comprehension of spoken dutch mini-stories. it has long been known that the language comprehension system works highly incrementally by incorporating novel input into the unfolding interpretation as soon as possible (e.g., marslen-wilson, ; marslen-wilson & tyler, ) . rather than waiting for a word or sentence to finish, listeners can use the initial sound of a word to identify the potential meaning or reference of a word (e.g., connolly & phillips, j o u r n a l p r e -p r o o f but an amplitude reduction of the noun-elicited n alone does not provide clear evidence that a specific word was predicted ('lexical prediction'), because it is also compatible with a more passive pre-activation of related semantic content (which may emerge naturally from comprehension of the preceding context; for discussion, see baggio, ; van berkum, ). in addition, the observed effect could mean that the predictable noun is easier to integrate with the sentence than an unpredictable word, simply because it is a more plausible sentence continuation (for discussion, see federmeier & kutas, ) , regardless of whether it was actually predicted. for these reasons, researchers typically argue that evidence for lexical prediction is strongest when it is observed before the predicted noun is heard or read, and is obtained by comparing erps to words that themselves have little semantic meaning (e.g., the english articles 'a/an') and/or do not differ in meaning (e.g., the dutch adjectives 'groot/grote', which have the same meaning but differ in the presence of the inflectional suffix '-e' to mark grammatical gender). differential effects elicited by these prenominal critical words cannot be due to a difference in the meaning of the words themselves, and are therefore thought to arise from the phonological or grammatical relationship between the prenominal word and the predicted noun. erp evidence for lexical prediction has been reported from various prenominal manipulations in spanish, english and dutch. in a pioneering study by wicha, bates, moreno and kutas ( ) , native spanish speakers listened to sentence pairs in which a relatively predictable (i.e., moderate-to-high cloze probability) or an unexpected and incongruent noun was replaced with a drawing. crucially, gender-marked prenominal articles elicited an enhanced n erp when their gender did not match that of the predictable nouns. in j o u r n a l p r e -p r o o f a follow-up experiment with written spanish sentences, found a very similar n effect of gender-mismatch with a predictable noun. in a subsequent experiment with written spanish sentences but without accompanying drawings, wicha et al. ( ) found that gender-mismatching articles elicited a different pattern, namely a positive erp effect (p ) compared to matching articles. in more recent studies on comprehension of written sentences, gender-mismatch on prenominal articles was associated with n -like effects, i.e. an enhanced negativity in the typical n time window (dutch: fleur, flecken, rommers & nieuwland, ; otten & van berkum, ; spanish: foucart, martin, moreno & costa, ; martin, branzi & bar, ; molinaro, gianelle, caffarra & martin, ) , although sometimes with a time course or scalp distribution unlike the typical n effects elicited by nouns. in a series of studies on comprehension of dutch mini-stories, vb reported evidence for prediction from a different manipulation also involving grammatical gender. these studies capitalized on the dutch grammatical rule by which adjectives are marked with an inflectional suffix when they modify an indefinite noun of common gender but not of neuter gender. for example, the suffix on 'grote' in 'een grote boekenkast' (a large bookcase) agrees with the common gender of 'boekenkast', whereas lack of the inflectional suffix '-e' in 'groot' in 'een groot schilderij' (a large painting) agrees with the neuter gender of 'schilderij'. in experiment of vb , participants listened to a two-sentence context that presumably led people to predict a specific noun (e.g., schilderij -painting). this context was followed by an adjective phrase that contained two gender-marked adjectives and either the predictable noun (e.g., 'groot maar onopvallend schilderij' -big but unobtrusive painting) or a less predictable noun of a different gender (e.g., 'grote maar onopvallende boekenkast' -j o u r n a l p r e -p r o o f big but unobtrusive bookcase). vb found that a mismatch between the inflectional suffix (or lack of thereof) on the first adjective and the gender of a predictable noun (e.g., 'grote' when the predictable noun was 'schilderij') elicited a more positive erp than a match. this positive erp effect had a very early onset, namely about - ms after the first acoustic difference between words with and without inflection, although when erps were time-locked to adjective onset the erp difference had a later time-course ( - ms). in experiment , participants listened to only the target sentences, which by themselves presumably did not lead to a specific noun prediction. consistent with this hypothesis, no statistically significant erp effect was obtained for the adjectives. in experiment , participants read a subset of the materials from experiment in a self-paced reading study, where participants press a button to make each next word appear on the screen. the participants slowed down when the second adjective mismatched the predictable noun, compared to a match. although no such effect was obtained on the first adjective (which would be a behavioral equivalent of the erp results obtained in experiment ), these reading time results nevertheless supported the hypothesis that people anticipated the predictable noun (but see guerra, nicenboim & helo, , for a recent failure to find prediction-related reading time results for spanish sentence comprehension). in two follow-up studies (otten, nieuwland & van berkum, ; otten & van berkum, ) , the same suffix-based manipulation elicited erp effects that were different from those obtained in experiment of vb . in a study with spoken materials (otten et al., ; henceforth ot ) , prediction-mismatching adjectives elicited a negative erp effect at right-frontal electrodes that started at about ms and lasted until ms (based on the associated scalp distribution, the authors were reluctant to interpret this effect as an n modulation), time-locked to the adjective j o u r n a l p r e -p r o o f onset. in a study with written materials (otten & van berkum, ) , predictioninconsistent adjectives elicited a negative erp effect that appeared as late as - ms after adjective-onset. the gender-effects reported by wicha and colleagues, by ot and vb , as well as by various others, indeed suggest prediction of a specific noun, but various questions about the functional significance of these effects remain. for example, it is unclear whether pre-activated information (which is presumably already available before an article or adjective is presented) includes grammatical gender (e.g., pickering & gambi, ; wicha et al., ) . it is possible that the initial prediction is limited to word meaning, and that people use gender information to evaluate whether the specific word can still appear. the second question is whether effects of gender-mismatch reflect the detection of a prediction mismatch or (also) the updating or revision of a prediction (for discussion, see nieuwland et al., b) . importantly, aside from these questions about interpretation, a major obstacle to any unitary interpretation of the available results is that qualitatively different types of effects have been obtained with (sometimes very) similar gender-based manipulations (for discussion, see ito et al., b; kochari & flecken, ) . this could signal something meaningful, namely that different processes are engaged in each of these studies. however, it could also signal the problem with statistically significant effects obtained in noisy, small-sample settings, which are associated with increased probability of overestimated or wrong-sign effect estimates (e.g., gelman & carlin, ; vasishth, mertzen, jäger & gelman, ) . this problem with small-sample effects has also surfaced in another wellknown demonstration of prediction, a study on english sentence comprehension by delong, urbach and kutas ( ) , who capitalized on the phonological rule for j o u r n a l p r e -p r o o f indefinite articles (i.e., 'a/an' signals that the next word will start with a consonant or a vowel, respectively). indefinite articles that mismatched a predictable noun in terms of phonology (e.g., 'an' if the predictable word was 'kite') elicited an enhanced n compared to matching articles. this effect is therefore sometimes taken to demonstrate phonological prediction (pickering & garrod, ) . however, the delong et al. results have proven controversial. a study by martin, thierry, kuipers, boutonnet and costa ( ) reported a similar effect for mismatching articles, but differences in the analysis complicated a quantitative and qualitative comparison to the delong et al. results (for discussion, see ito, martin & nieuwland, a,b) . another study with this manipulation (ito et al., ) did not obtain a reliable effect of gender mismatch. moreover, a recent, large-scale (n= ) direct replication study (nieuwland et al., b) failed to replicate the result of delong et al. in an analysis that duplicated the original, and found no statistically significant effect in an additional analysis that took into account subject-and itemlevel variance. nieuwland et al. concluded that the 'a/an' article-effect may indeed be non-zero, but that it is likely far smaller than originally reported and too small to observe without very large sample sizes. nieuwland et al. further speculated that the a/an manipulation does not elicit reliable or strong prediction effects because these articles are diagnostic of the next word, which need not be a noun (e.g., 'an old kite'). unexpected articles thus do not actually refute the upcoming noun altogether, but signal that the noun cannot appear immediately after the article. stronger or more reliable effects might therefore be obtained with gender-marked articles or adjectives, j o u r n a l p r e -p r o o f which can disconfirm the predicted noun because they agree with that noun in gender irrespective of intervening words . given the strength of gender agreement relationships, the apparent lack of consistent patterns across studies with gender-based manipulations may seem disconcerting. establishing the nature and timing of such effects is critical to developing hypotheses about the mechanisms that underlie the generation and evaluation of predictions. for example, prediction-mismatching suffixes elicited an early onset, positive erp effect in vb , who did not commit to a specific functional interpretation of this effect beyond the conclusion that the effect demonstrated lexical prediction (see also van berkum, ) . however, this positive erp response could be related to p effects seen for syntactically unexpected information (e.g., osterhout, holcomb & swinney, ) and for morphosyntax agreement mismatch (e.g., tanner, grey & hell, ; wicha et al., ) . such p effects are thought to reflect a reanalysis or syntactic integration process (e.g., kaan, harris, gibson & holcomb, ; kaan & swaab, ) . in contrast, studies reporting n or n like effects suggest that predictions impact the activation of word meaning (lexical access), and have led some authors to argue that people even predict the specific form of the prenominal article itself along with the noun (delong et al., ) . however, before attempting to explain the different effects of prediction and their association with specific linguistic manipulations or experimental procedures, the field needs to establish which of the key findings can be replicated with similar methods and materials, in a sufficiently large sample to obtain a sufficiently reliable and plausible effect estimate. this is not a trivial issue in a research field where it has long been and still is rather common to select a dependent variable based on visual inspection of low-sample erp data (kilner, ; luck & gaspelin, ) , a practice that leads to over-estimated effect sizes and higher rates of false positives (e.g., gelman & loken, ; gelman & carlin, ; vul, harris, winkielman & pashle, ). moving away from erp analysis based on visual inspection, recent erp studies on language comprehension have pre-registered data processing steps and statistical analyses, and explicitly distinguish between confirmatory and exploratory analyses (e.g., fleur et al., ; nieuwland et al., b; sassenhagen & bornkessel-schlesewsky, ) . the current study tries to replicate the main result obtained in experiment of vb , along with that of ot , as it used the same manipulation and similar materials. like delong et al. ( ) , vb is an influential and highly cited erp study (at time of writing, citations on google scholar) that features in major theoretical reviews on linguistic prediction (e.g., altmann & mirkovic, ; kutas & federmeier, ; pickering & garrod, ) . however, like delong et al., vb has yet to be successfully replicated. the only available study with the same genderbased inflection-manipulation found an effect in the opposite direction (ot ). moreover, the key evidence reported by vb came from an analysis in a time window that was based on visual inspection of the grand-average erp waveforms (p. ). this procedure has long been, and probably still is common (see kilner, ; luck & gaspelin, ; see also nieuwland, , for related discussion), although it is not always explicitly mentioned in methods sections (for example, in some work by j o u r n a l p r e -p r o o f the first author of this paper, see nieuwland, ditman & kuperberg, ; nieuwland & kuperberg, ) . selection of a spatial and/or temporal region-of-interest is an appropriate method to sidestep the requirement for multiple comparison correction. however, it is only robust and valid when the selection is independent of the data, whereas it has an increased risk of false positives if the selection is based on where an effect looks strongest in grand-average erps (e.g., kilner, ; luck & gaspelin, ; see also http://deevybee.blogspot.com/ / /interpreting-unexpectedsignificant.html). therefore, we deem it important to perform a pre-registered attempt to replicate the effect of key results of vb in a sufficiently powered study (see methods for power analyses). the erp effect of prediction-mismatching inflections is also important because this manipulation tests for online prediction in a unique, possibly quite subtle way. dutch adjective-suffix inflection is a better cue to noun gender than definite articles. in dutch, 'het' is also used for diminutive nouns, whereas 'de' is used for plural nouns, irrespective of noun gender. this pattern is different for adjectives following the indefinite article 'een' that rules out a plural noun. the absence of a suffix is compatible with a diminutive noun irrespective of gender ('een leuk boekje/tafeltje', a nice little book/table), but suffix-presence is only consistent with a common gender noun. absence and presence of the suffix therefore have different repercussions for whether the general semantic meaning of the predicted noun can still follow: if one predicted 'boek', then 'een leuke' disconfirms that lexical meaning; if one predicted 'tafel, then 'een leuk' does not entirely disconfirm that meaning, as the diminutive 'tafeltje' could follow. even in an experiment where no such diminutives appear, participants may be sensitive to the possibility of the expected j o u r n a l p r e -p r o o f word appearing in diminutive form and therefore do not take 'missing' inflection as a cue that the predicted meaning is wrong (see also nieuwland et al., b) . aside from this issue of 'cue reliability', gender-mismatching inflections might be relatively hard to detect compared to prediction-mismatching gender-marked articles ('el/la' in spanish, 'de/het' in dutch), and therefore be less likely to yield prediction effects. the inflection manipulation relies on the detection of an absent or present inflection within a short time frame, whereas the article manipulation relies on detecting two entirely different lexical items. in addition, it involves the detection of prediction-relevant information from an adjective that itself is relatively unexpected and contains novel semantic content. this differs from detection of a gender-marked article that itself might be predicted along with the noun and therefore generate stronger effects. for these reasons, the inflection-based gender manipulation might pose a stronger test of the predictive use of gender-relevant information during language comprehension than the more commonly used article-based gender manipulation (e.g., foucart et al., ; martin et al., ; wicha et al., ) . the current study aims to replicate previously observed patterns for prediction-mismatching adjective inflections (vb ; ot ). we use a novel set of experimental materials that is twice the size of that in vb , and based on materials from a recent erp study on lexical prediction (fleur et al., ) . fleur et al. constructed two-sentence mini-stories that either suggested a definite noun phrase (e.g., 'het boek', the book) or an indefinite noun phrase ('een boek', a book) as its most likely continuation. following these contexts, participants saw a definite noun phrase with either the expected noun ('het boek') or an unexpected, different-gender noun ('de roman'). using pre-registered data preprocessing procedures and statistical analyses, fleur et al. found that gender-mismatching articles elicited an enhanced n compared to gender-matching articles (see also otten & van berkum, , for similar results). these findings are relevant for the current study, because they show that we use materials that have already demonstrated relevant erp effects of prediction. in the current study, we only used a subset of the story contexts of fleur et al., namely those in which an indefinite noun phrase was the expected continuation, with a minimum cloze value of % for both articles and nouns (the cut-off used for the main analysis in vb ). to match the manipulation of vb , we added two adjectives to each target noun phrase (see table for an example story). using bayesian mixed-effects model analyses, we take a spatiotemporal region of interest approach to test for effects of prediction-match on average voltage values for each trial. the choice of rois and time windows was based on vb and ot . these analyses aim to answer the question of whether previous neural evidence of lexical prediction from gender-marked, pre-nominal adjectives can be replicated. we preregister further exploratory analyses to test the effect of prediction-match with traditional anovas (on average values per condition per subject, following vb and ot ), and to test whether the prediction-match effect is similar for common and neuter gender nouns. an initial, minimum sample size was determined by a mixed-effects a priori power analysis with the simr package (green & macleod, ) . because no single-trial data were readily available from vb and ot , single-trial data were adapted from a previously published study by ito et al. ( a,b (nieuwland et al., ) . for the simulation, the number of items for the model was extended to to match the number used in the current study. power analysis by simulation (number of simulations = ) showed that subjects was sufficient to detect an effect at a significance level of alpha = . with % power. we set the initial sample size slightly higher at n= , which is more than times the sample size of vb , and refers to the participants ultimately used for statistical analysis, and thus to the minimum number of participants that were tested. participants were excluded from the statistical analysis using pre-defined criteria (a response accuracy under %, or insufficient trials after artefact rejection, described in the data preprocessing section). each excluded participant was replaced by another participant, and the number of excluded participants will be reported. however, this sample size was set as a minimum, because the simulation did not take into account the potential effects of the absence/presence of the inflection, and does not guarantee the bayes factor evidence strength required by this journal. in the original studies, which used anovas, the absence/presence of inflection was approximately balanced across items. in the current study, however, this factor is explicitly accounted for in the model (see sassenhagen & alday, ) , using a more powerful analysis that simultaneously takes into account sources of variance (subjects, items, presence of inflection) that were not included in the original study's anovas. this is important because, not only may the effect of match differ for different-gender adjectives (see the pre-registered exploratory analysis), unaccountedfor variation that is orthogonal to the effect of interest (e.g., random intercept variation) can reduce power, while unaccounted-for variation that is confounded with our effect of interest (e.g., random slope variation) can drive differences between means, with increased risk of false positives and overestimation of effect size (for discussion, see barr et al., ) . therefore, our final sample size was not based on the a priori power analysis and we continued to increase our sample size from to in steps of participants until we reached the bayes factor evidence strength required by this journal (see statistical analysis). however, our laboratory was shut down due to the unfolding covid- /coronavirus pandemic when we reached . because we had no view of a continuation of testing in the foreseeable future, and because we were confident that the remaining to-be-tested participants would not change our conclusions, we were granted an early sampling finish by the editor. instead of the materials used in the original study (vb ), we used a suitable set of stimuli readily available from a previous study (fleur et al., ) . this set of materials was created following a similar procedure as that of the original, was larger than that of the original, and had already been normed for cloze probability. moreover, as stated in the introduction, fleur et al. had already demonstrated a prediction-consistency n effect on pre-nominal articles using these stimuli (see also otten & van berkum, ). the critical stimuli for the current study consisted of mini-stories, each of which had one context sentence and two possible target sentences. each mini-story was written to suggest a specific combination of an indefinite article plus predictable j o u r n a l p r e -p r o o f noun in the target sentence. in a cloze test (n= ), each mini-story was truncated before the article , and participants were asked to complete each story. the stories in the current study were completed by at least % of the respondents using the same combination of the expected indefinite article and noun . the average cloze probability of the indefinite articles was . % (sd = . %, range - %), and . % for the nouns (sd = . %, range - %). these values are numerically higher than those of the original studies . of the predictable nouns, were common gender 'de' words, and were neuter-gender 'het' words . after the norming, we added two adjectives (separated by a function word, e.g., 'groot en sterk' or 'grote en sterke', which both mean 'big and strong') in materials in the cloze test of vb were truncated after the indefinite articles. our procedure differed in where the materials were truncated because we also wanted to obtain cloze values for the prenominal articles (fleur et al., ) . different spellings of the same word were permitted and counted towards the same response, such as 'tattoo/tatoeage' or 'tv/televisie'. for comparison, the expected nouns in vb had an average cloze of % (sd = %, minimum = %) while the unexpected nouns had an average of % (sd = %). the expected nouns of ot had an average of % (sd = %, range - %), whereas the unexpected nouns had an average of % (sd = %). although the difference in noun cloze-values between the current study and the original study is small, it is unlikely to make it harder to find a prediction-effect in the current study, given that higher noun-cloze is associated with more, not less predictive processing (e.g., kutas & federmeier, ) . between the indefinite article and the critical noun. if one or more participants in the cloze test had used an adjective to complete a sentence fragment (which was rare), that adjective was not used for that item in the eeg experiment. for each mini-story, we created a prediction-matching and -mismatching condition. in the matching condition, the absence or presence of an overtly realized suffix '-e' on both adjectives matched the gender of the predictable noun ('dik en spannend' when the predictable noun is 'boek'), and the second adjective was followed by the predictable noun. in the mismatching condition, the absence or presence of the suffix mismatched the gender of the predictable noun ('dikke en spannende' when the predictable noun is 'boek'), and the second adjective was followed by a different-gender noun that was semantically possible but less predictable (e.g., 'roman'). these alternative nouns had appeared at most only once in the cloze responses (average cloze value . %, sd = . %, range - %). like in the materials of vb and ot , there was no overlap in the set of predictable nouns and alternative nouns (which means that a direct comparison between these nouns may be confounded by lexical variables). all sentences were grammatically correct. the critical nouns were never sentence-final, and all subsequent words were identical for the matching and mismatching condition of a given story. an additional set of filler mini-stories of sentences each was added to the materials. sixty fillers were similar to the prediction-match condition: they also contained high cloze nouns (average cloze . %, sd = . %, range . - %), preceded by pre-nominal adjectives. due to these fillers, highly-constraining stories in the entire experiment were almost twice as likely to end in a predictable than an unpredictable noun. we added these fillers to counter the argument that participants will adapt to unexpected syntactic information (i.e., start expecting unexpected j o u r n a l p r e -p r o o f information) and therefore not show prediction-consistency effects. such adaptationeffects have been reported, albeit only for frequent repetition of an unexpected syntactic structure (see fine, jaeger, farmer, & qian, ; but see also a recent failure to replicate this type of adaptation effect, stack, james & watson, ), not for varied sentences structures as used in the current experiment. we have also added relatively non-constraining stories to increase the variability of our materials and to make the ratio between the experimental and filler stories more similar to that of vb . these were adapted from a subset of low-constraint materials used by otten and van berkum ( ; 'prime control' stories), and they did not contain nouns preceded by two adjectives in the second sentences. the current study thus used different fillers and a slightly different ratio between experimental and filler items ( / ) than vb and ot ( / and / , respectively), although it was similar to these previous studies in using only grammatically correct and semantically coherent/plausible mini-stories as fillers. although the possible effect of the number and type of fillers on comprehension is not precisely known, some erp studies suggest that a high proportion of predictionlicensing materials actually boost predictive processing (e.g., brothers, swaab & traxler, ; lau, holcomb & kuperberg, ) . in addition, fleur et al. ( ) obtained n evidence for prediction on prenominal articles in a study that only included high-constraint sentences, of which % contained a critical 'de/het' article. for these reasons, we do not think there is a convincing a priori argument that our materials will elicit less predictive processing than those of vb and ot . the mini-stories were recorded with a normal speaking rate and intonation by a female native speaker . recordings followed the procedure described in vb . target sentences of the critical stories were recorded in both conditions. the context sentence was recorded once, together with either the prediction-matching target sentence or the prediction-mismatching target sentence (counterbalanced over stories). we ensured that the critical inflections were always clearly distinguishable from the subsequent word. the recordings of context and target sentences were stored separately. from the target sentence recordings, we identified the acoustic onset of the critical adjectives, of the critical inflections therein, and of the critical noun. inflection onset was determined as the moment at which adjectives begin to differ between conditions in terms of their respective phonemes, following vb . unlike vb and ot , we included comprehension questions to encourage participants to pay attention to the meaning of the stories, and as a means to exclude participants who did not pay sufficient attention. a potential null effect of prediction is then unlikely to result from participants not paying attention to the meaning of the stories. in our view, the importance of ruling out such a 'lack of attention' account balances out the slight deviation from the original studies. it is not known whether and how comprehension questions -that are orthogonal to the manipulation of interest -change the way that people process the meaning of linguistic stimuli. to the best of our knowledge, there is no evidence to suggest that comprehension questions cause participants to process stimuli less predictively compared to when there are no comprehension questions. moreover, our materials including the comprehension questions largely overlap with and are highly similar to those of fleur et al. ( ) , who obtained relevant erp evidence for prediction on pre-nominal, gender-marked articles, as has been also reported in experiments without comprehension questions (otten & van berkum, ). in our study, of the stories ( %) were followed by a yes/no comprehension question ( follow a filler story, follow a critical story; questions were designed to elicit a 'yes' response, and the other to elicit a 'no' response). each question was answerable from the preceding mini-story, irrespective of the critical manipulation when it followed a critical story. participants who answered fewer than from the questions correctly ( %) would have been replaced by new participants who are presented with the same materials. however, none of our participants met this exclusion criterion, and they had on average % correct answers (sd = , range = - ). materials were organized into two trial lists. in one list, one half of the critical stories was presented in the matching condition and the other half in the mismatching condition, and vice versa in the second list. conditions within each list were pseudorandomized such that no more than filler stories were presented successively and no more than matching or mismatching critical stories were presented successively. both lists start with practice stories, including two practice comprehension questions. procedure after electrode application, subjects sat in a sound-attenuating booth and listened to the stories from a speaker placed in front of them. although participants in vb listened to the stories over earphones, we decided to present the stories over speakers, which was also done in later work by van berkum and colleagues, including ot . this method of delivery is more comfortable for participants and, unlike a headphone setup, does not introduce additional artefacts. our participants were asked to listen attentively to the stories and answer the comprehension questions. after the practice stories, the stories were presented in blocks of items, separated by rest periods. participants self-paced through the experiment by button press. upon pressing, each trial started with a ms auditory tone, followed by a ms silence, the spoken context sentence, a ms of silence, and the spoken target sentence. participants were instructed to sit still and to refrain from eye-movements and blinks during the second, target sentence. to signal to subjects when to sit still and refrain from eye-blinks and eye-movements, an asterisk was displayed from ms before the onset of the target sentence to ms after the sentence offset. if a trial was followed by a comprehension question, the question appeared in its entirety on the screen upon disappearance of the asterisk. used by some groups of speakers but that are considered ungrammatical by others). the third test was the peabody vocabulary test (dunn & dunn, ; schlichting, ) , in which participants hear words and have to select matching pictures from sets of four options. the fourth test was the dutch version of stairs words, an adaptive test for assessing receptive vocabulary size (hintz, jongman, dijkhuis, van 't hoff, damian, schröder et al., ) . on each trial, the participant saw a word or a non-word foil (ratio : ) and indicated whether or not they knew the item. the eeg was recorded from active electrodes (fz, fcz, cz, pz, oz, f / , f / , fc / , fc / , t / , c / , cp / , cp / , p / , p / , o / ) relative to a leftmastoid reference electrode, along with activity at a right-mastoid reference channel and eog channels. the electrode locations were similar but not identical to those of vb and ot , however they still allowed for a similar quadrant-based roi analysis as reported in these earlier studies. data was recorded with a brainamp dc j o u r n a l p r e -p r o o f amplifier, at a sampling rate of hz, using a time constant of s ( . hz) and high cut-off of hz in the hardware filter (this high cut-off differed from the hz used in vb but matched that of ot and allowed for later analysis in the - hz gamma frequency band), with an additional high cut-off of hz in the recording software . electrode impedance was kept below kΩ where possible, which differed from the procedure in vb (who used passive electrodes), but it is under the guidelines of the hardware manufacturer, and lowering impedances to under kΩ takes prohibitively long and could cause too much discomfort to participants. because we had a large number of trials and participants, and because the recordings took place in an air-conditioned room to ensure a cool and dry environment, such impedance differences were very unlikely to meaningfully impact statistical power (see kappenman & luck, ) . in addition, our sample size calculation was based on high impedance (biosemi) data and we had already demonstrated prediction erp effects in our lab with a less restrictive impedance threshold (< kΩ, fleur et al., ) . regardless, impedance values were stored before and after the experiment for potential checks. data pre-processing was performed using brainvision analyzer. first, bad eeg channels were identified through visual inspection (as electrodes showing poor signal for at least half of the experiment due to blocking, faulty connectivity or other large-amplitude artefact) and interpolated through spherical splines. our pre- because the brainrecorder software could only achieve the pre-registered filteroutcome by a combination of hardware and software filtering, we deviated from the pre-registered protocol in the filter settings. to err on the safe side, we furthermore doubled the pre-registered sampling rate. we matched the precise vb hardware filter settings during offline filtering. j o u r n a l p r e -p r o o f registration stipulated interpolation of maximally eeg channels per participant. we ended up interpolating channel from participants, channels from participants, channels from participants, and channels from participant. an interpolation procedure was not used or mentioned in vb but we use it to avoid unnecessary data loss. then, the continuous data were filtered with a . - hz ( db) butterworth iir band-pass filter (we also included a hz notch filter, which was not preregistered) to match the hardware filter of vb . we then segmented the data into epochs from before to ms after the onset of the first critical adjective. however, because this epoch did not always extend ms beyond the later noun as in vb , we created separate segments for the nouns ranging from ms before to ms after noun onset. we used separate segments instead of longer segments that included all the critical adjective and noun data, because longer segments would have also included artefact-rich post-sentence data in many of the trials. each segment was then screened for large muscle artifacts, electrode drift, and amplifier blocking. we corrected for artefacts in the segments (due to eye-movements, blink, cardiac or steady muscle activity ) using independent component analysis (trained on segments extracted from continuous data that was filtered with a . - hz zero phase shift butterworth band-pass filter plus notch filter). this procedure was not included in vb but was used in ot and avoids unnecessary data loss, which is important because participants might find it hard or distracting to avoid blinking and eyemovements. we subsequently extracted smaller segments running from ms before to ms after the onset of adjectives, inflections and nouns. for each segment, baseline correction was performed by subtracting average voltage in the relevant (of three) ms prestimulus interval from the entire segment. we then applied an automated artefact rejection procedure that rejects epochs with values exceeding +/- μv. although no such rejection procedure was applied in vb , we felt it was important to apply one objective artefact criterion instead of only relying on visual inspection, also to remove segments with artefacts that had been overlooked during visual inspection. in vb , no participant exclusion criteria were mentioned, but it was stated that, on average, . % of all trials were rejected (based on visual inspection of the data), and that there were no asymmetries between conditions. here, participants were excluded if their comprehension question accuracy was under %, if they had fewer than remaining trials from the initial trials ( %) in any of the conditions (matching/mismatching adjectives time-locked to onset or inflection, matching/mismatching nouns), or if they had fewer than remaining trials ( . %) on average across all conditions. two participants were excluded, which left us with a total of participants, who had, on average, match and mismatch trials time-locked to inflection, match and mismatch trials time-locked to onset, and match and mismatch noun-trials (corresponding to a trial rejection rate of approximately % for inflection and adjective onset trials, and % for noun trials). all raw data and pre-processed data are available on https://osf.io/jqhpz. before statistical analysis, we downsampled the data segments to hz, matching that of vb . spatiotemporal roi, so that we did not miss effects at rois where vb /ot did not observe statistically significant effects. we defined three spatiotemporal rois for the nouns (based on vb ), such that we averaged activity within a - ms time window after noun onset within the left-posterior quadrant, the right-posterior quadrant, and the midline. using the trial-level data from these rois, we performed bayesian linear mixed effects model analysis using the 'brms' package (bürkner, ) in the r software (r core team, ), which fits bayesian multilevel models in the stan programming language (stan development team, ) with formula syntax that is similar to that of the 'lme ' package (bates, maechler, bolker & walker, ) . in addition to bayes factor hypothesis testing, this analysis allows for bayesian (match/mismatch with the predictable word) and the deviation-coded fixed factor 'gender' (common/neuter suffix). the factor 'gender' captures the potential effect of the absence or presence of the inflectional suffix '-e', which is not manipulated within each item (and therefore not included as a random slope for 'item'). voltage ~ match + gender + (match + gender | subject) + (match | item) we followed the suggestions for replication studies by dienes ( ) and dienes and mclatchie ( ) , namely to use, for the effect of interest, a prior with a zero mean and a standard deviation that is the previously reported effect size. the previous effect sizes went in both directions (i.e. a positivity in vb , a negativity in ot ) and corresponded to roughly a . μv difference. to perform replication tests of those studies, the prior on the effect of match was a normal distribution with a zero mean and sd = . μv (here and elsewhere, the normal distribution was used lacking an obvious reason to assume non-normality). in other words, there is a % prior probability that the parameter lies between - . and . μv (of note, these are twosided tests, but we make up for the associated overall lower prior density in our sampling plan described below). we also included a prior for the effect of gender, which centered on mean zero with a normal distribution because the effect could be positive or negative, and a prior sd that was the same as for match (assuming the effect of gender is unlikely to be bigger than that of match), such that there is a % probability that the parameter lies between - . and . μv. we included an intercept prior with a normal distribution, mean zero and sd of . , such that there is a % probability that the intercept parameter lies between - and μv. this decision was informed by intercept parameters in previous studies j o u r n a l p r e -p r o o f (fleur et al., ; ot ; vb ) , and appeared suitable for analyses time-locked to inflections, adjectives or nouns. we did not include priors for the standard deviations of group-level ('random') effects, but used the corresponding default priors, which "are used (a) to be only very weakly informative in order to influence results as little as possible, while (b) providing at least some regularization to considerably improve convergence and sampling efficiency" (https://rdrr.io/cran/brms/man/set_prior.html; bürkner, ) . likewise, we also did not include a prior for the standard deviation of the residual error. we did include a prior for the correlations of group-level ('random') effects using as the lkj ( ) to sum up, we performed prediction-match replication tests ( using inflection-onset time-locked data, involving the adjective-onset time-locked data), after having collected data from participants who met the inclusion criteria. the required strength of the evidence for statistical inference was set at a bayes factor of at least , either for the alternative hypothesis or the null-hypothesis. this was double the evidence strength required by this journal, which we used to 'make up' for the lower prior density of our normal prior (i.e., a two-sided test) compared to a halfnormal prior (i.e. a one-sided test). we took sufficiently strong evidence (bayes factor > ) for the alternative hypothesis at any roi selection as a successful replication in the sense that it demonstrates the use of inflection information. in that scenario, the observed effect was either positive or negative, which we would take as a replication of either vb or ot , respectively. based on vb , we expected the effect polarity to be the same in the two time windows. if this was not the case, this could signal a problem with the inflection-onset time-locked analysis, perhaps due to application of a baseline correction in an already unfolding effect (for example, because participants pick up on relevant acoustic differences between conditions before inflection onset, see discussion in vb ). the adjective-timelocked effect then receives priority in guiding our conclusions, since baseline differences are less likely to be a problem for that analysis. importantly, we took sufficiently strong evidence for the null hypothesis at all these selections as a failure to replicate both vb and ot . factors for the alternative hypothesis reached or all obtained bayes factors for the null hypothesis stayed below , we followed the guidelines of this journal and tested additional participants until that evidence strength is reached (one bayes factor that j o u r n a l p r e -p r o o f sufficiently supports the alternative hypothesis, or all bayes factors sufficiently supporting the null hypothesis). sample size was increased in steps of participants, to be capped at a maximum of participants for practical considerations. however, we were forced to halt testing before reaching that number because of a covid- pandemic related lockdown of our institute, and we therefore report analyses on a total sample size of only participants. after completion of data collection, we performed additional analyses with different priors for the effect size of 'match' to investigate the robustness of the obtained results (normal( , ) and normal( ,. ) to cover a wider/narrower range of plausible values). the noun analyses were performed once the data collection had stopped, and served as positive controls because n effects of predictable versus unpredictable words are highly common throughout the psycholinguistic literature (for review, see kutas & federmeier, ) and typically of a relatively large effect size compared to prenominal manipulations. if no effect is observed for the adjectives and no n effect of match is observed for the nouns, no valid inference about predictive processing can be drawn from the adjective data (see also delong et al., ; nieuwland et al., b; vb ) . for the three noun-rois, we tested the following model: voltage ~ match + (match | subject) + (match | item). similar to the adjective analyses, the prior on the estimated effect size had a normal distribution and a zero mean (although unexpected nouns were expected to elicit more negative voltage than expected nouns). the prior standard deviation of the match parameter was set at μv, corresponding to the j o u r n a l p r e -p r o o f approximate effect sizes reported by vb and ot . the intercept prior was the same as for the adjective analysis, as were the other priors (but no prior is included for 'gender'). like the nouns in vb and ot , there was no overlap between predictable and unpredictable nouns, which means that the noun-comparison was confounded by lexical variables (e.g., frequency) and contextual variables (e.g., plausibility) that are known to influence n amplitude (e.g., kutas & federmeier, ; nieuwland et al., b) . in addition, vb , ot , and this study used different nouns altogether. despite these caveats about between-study differences, an additional model was tested with a stronger noun prior, to test whether the obtained n effect had changed the support for the specific noun effect-size reported by vb . for this analysis, we used a normal prior for 'match' with a mean at - . μv and a standard deviation of . μv, corresponding to the strongest effect reported in vb (at the left-posterior quadrant). this prior defines a % probability that the 'match' parameter lies between - . and - . μv. the factor 'gender' was included in the analyses because, in principle, it was considered a nuisance variable. instead of only counterbalancing gender across items, our confirmatory analyses explicitly accounted for the associated variance when testing the effect of prediction-match (see also sassenhagen & alday, ) . however, we further considered an effect of 'gender' in an exploratory analysis, because the effect of prediction-match could depend on gender (see also loerts, wieling, & schmid, ) . for example, the prediction effect could be greater when the predictable noun has common gender compared to neuter gender, perhaps because j o u r n a l p r e -p r o o f it is easier to detect a mismatch on an overt suffix than on the absence of a suffix, or perhaps because an overt suffix rules out the expected noun meaning, whereas the absence of the suffix does not rule out entirely the expected noun meaning (it could signal an upcoming diminutive noun irrespective of gender; see also loerts et al., , for relevant discussion). an alternative scenario is also possible, namely that the prediction-match effect is greater when the predictable noun has neuter gender as opposed to common gender, perhaps because people find it harder to detect a mismatch on an overt suffix. this could be related to the fact that language learners tend to add the suffix incorrectly more often than they omit it incorrectly; both young and old language learners tend to overgeneralize the suffix like in 'een moeilijke boek' (weerman, bisschop & punt, ; for a review on dutch adjectival inflection, see van de velde & weerman, ) . it is possible that such overgeneralizations by l learners change inflection processing even in l speakers, or that overgeneralizations in childhood continue to impact inflection processing later in life, even if only very subtly. in both these hypothetical scenarios, one could expect to observe an interaction between 'match' and 'gender' on inflection-elicited erps. there was no strong a priori reason to assume that this interaction term would yield a strong effect, given that previous studies approximately balanced the 'gender' factor across items. nevertheless, we considered this possible interaction effect and performed exploratory tests that included the interaction term as a fixed effect and included a bysubject random slope in the brms model. voltage ~ match * gender + (match * gender | subject) + (match | item) for this analysis, we add one prior to the brms model for the adjective and inflection analyses, namely a normal distribution prior with a mean zero and sd of j o u r n a l p r e -p r o o f for the slope of the interaction parameter. this prior defines a % probability that the parameter for the interaction term, i.e. the difference in the match effect for common and neuter gender adjectives, lies between - and μv. at the request of the reviewers, we conducted repeated-measures anovas that closely follow the analyses of vb and ot . however, we note that the primary basis for our conclusions is the bayes mixed-effects analysis approach described previously. the anova analyses were performed after data collection had ended, using the function 'aov_car' from the 'afex' r package (singmann, bolker, westfall, aust, & ben-shachar, ; in our pre-registration we planned on using jasp, ). averaging over items, we analyzed mean amplitude values per condition per subject in the - ms time window after adjective inflection onset (vb ), and in the - ms time window after adjective onset (ot ). for the n effects at the noun, only the - ms time window was analysed. we conducted repeated measures anovas on the five rois defined above. in the midline region, prediction-match (matching vs. mismatching) was fully crossed with using g*power with 'spss standards' (faul, erdfelder, lang, & buchner, ) , we established that our minimum sample size of n= also yielded sufficient a priori power for these anova analyses. for the lowest relevant f-value from vb (f ( , ) = . ), ie. the most conservative estimate, the partial eta-squared measure of effect size was η p = . (see lakens, ) . the lowest f-value in ot was (f ( , ) = . ), which yielded η p = . . to detect this latter, more conservative effect size with a power of . at an alpha level of . , the required sample size is n = . this would be the required sample size if we used only about half of our items. j o u r n a l p r e -p r o o f the five electrodes (fz, fcz, cz, pz, oz) . the analysis at the four quadrants was carried out by crossing prediction-match with hemisphere (left vs. right) and anteriority (anterior vs posterior). in all analyses, greenhouse-geisser correction was applied to f-tests with more than one degree of freedom (greenhouse & geisser, ) . we report the mean voltage and sd for each condition, the estimated difference between conditions with the % ci, and cohen's d measure of effect size. j o u r n a l p r e -p r o o f figure . inflection effects. the graphs show the grand-average erps elicited by gender-matching (solid blue lines) and gender-mismatching inflection (dotted red lines) at the pre-registered rois. in these and following figures, color-shaded areas show the within-subject standard error of the condition mean (cousineau, ; morey, ; calculated with the 'rmisc' package in r). we emphasize that these erp plots do not directly correspond to the results of our statistical analyses, which account for variance associated with different items and with common/neuter gender. time-locked to inflection onset and to adjective onset, the mismatch estimate is negative at all rois and a majority of the posterior distribution falls under zero. this is also visible in figure j o u r n a l p r e -p r o o f figure . results from the bayesian hypothesis tests for the gender-mismatch effect time-locked to inflection onset. graphs depict the prior (light blue) and posterior (dark blue) density, with prior and posterior density at zero marked by a yellow and red dot, respectively. the ratio of the density values at zero, the bayes factor, is labelled on each graph, here showing the bayes factor evidence in support of the null hypothesis (bf null ), with higher values corresponding to the increased belief in the nullhypothesis given our data. results from the bayesian hypothesis tests for the noun mismatch effects at the three pre-registered rois. although the graphs highlight posterior density at zero with a red dot, the posterior samples did not contain the value zero, which is why bf null is labelled as zero. we also computed bayes factors with different priors to investigate the robustness of the obtained results (table ) . for effects time-locked to the inflection or adjective onset, use of a wider prior (sd = ) increased the obtained bf null at each roi, with half of the rois yielding moderate evidence for the null hypothesis and the other half yielding anecdotal evidence. with a narrower prior (sd= . ), the tests yielded anecdotal evidence. for comparison, we also performed exploratory analyses on erps time-locked to inflection with a prior mean and sd roughly based on vb (m= . , sd= . ) and ot (m=- . , sd= . ), reported in appendix table a . . with this 'stronger' vb prior, we found strong evidence for the null hypothesis (bfs null ranging from . to . for the rois). with the ot priors, we found anecdotal/moderate evidence for the null hypothesis (bfs null ranging from . to . ), which suggests that while the obtained effect is likely to be a negativity, it is probably much smaller than the effect reported by ot . for the noun effects, using a prior that corresponded to the strongest effect reported in vb did not impact the results because our posterior samples never included zero. hence, even though the obtained n effects had mean estimates that were lower than those reported in vb , our results nevertheless yielded extreme evidence against the null due to the high precision of our estimates. j o u r n a l p r e -p r o o f table . bayes factor results (bfs null ) from analyses with different priors. for effects time-locked to inflection or adjective onset, the new prior for the standard deviation for the mismatch effect was either wider ( ) or narrower ( . ) than in the main analyses. for noun effects, the prior corresponded to the strongest effect reported in vb . inflection onset prior adjective onset prior noun prior roi sd = sd = . sd = sd = . as shown in figure , mismatching inflection elicited a more pronounced negativity for common gender than for neuter gender, at least at frontal rois (see appendix figures a. and a. for effects at individual erp channels, and see figure for scalp distributions of the mismatch effects). at the left-anterior roi, this negativity started as early as ms and lasted for ms, whereas the other rois showed a negativity mostly in the - ms time window. for neuter nouns, the negativity was also visible at posterior rois, but anterior rois showed a positivity, at least from approximately ms onwards. the corresponding adjective onset effects are shown in figure (see also gender. these results only lend some support to the interaction pattern, albeit weak. in all analyses, the credible interval included zero and the bf null only yielded anecdotal evidence, although the posterior probability of the effect being negative was high for inflection-locked effects at anterior rois. figure shows the pairwise mismatch j o u r n a l p r e -p r o o f effects for common and neuter nouns separately. not reported in detail here, estimates for the mismatch effect were very similar to those from the models without interaction term. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f table . results from the pre-registered exploratory analysis of the interaction between gender and gender-mismatch. each cell gives the corresponding estimate (b) in μv for the interaction term (negative values correspond to a more negative mismatch effect for common gender than for neuter gender), the associated credible interval (cri), and the posterior probability of the effect being negative (p(b)< , the percentage of posterior samples under zero), and the bf null . j o u r n a l p r e -p r o o f figure . results from the pre-registered exploratory analyses. the graphs show the mismatch effects (mismatch minus match) at each roi, time-locked to inflection and adjective onset, for common gender (purple) and neuter gender (orange). dots represent the marginal mean, whiskers represent the % credible interval. for erps time-locked to inflection onset, repeated measures anovas on the four quadrants revealed a marginally significant prediction-mismatch effect (f( , ) = . , p = . , mean difference = - . µv, % ci our pre-registered analyses averaged activity from selected electrodes and time points within spatiotemporal rois based on vb /ot . to better characterize the effects of interest inside and outside of the rois, we performed exploratory mass regression analyses. first, we downsampled the pre-processed, segmented adjective onset and inflection data to hz (i.e., one sample for every ms) to speed up the analysis. then, we performed a mixed-effects model analysis using the 'lme ' package (bates et al., ) for each channel, and for each sample between - to ms relative to inflection onset (this shorter window minimized distortion from effects associated with noun onset) and between - to ms relative to adjective onset. we first tried analyses with the same fixed and random effects as in the preregistered exploratory analysis, but because all models failed to converge, even after removing random correlations, we opted for simpler models, also to further speed up the analysis. we here report results from a model with the main effect and interaction between match and gender as fixed effects, a by-subjects random slope for match, and here too, the effect fluctuated at an alpha-range frequency, especially between and ms after onset. corresponding results for the interaction terms are available on our osf page. taken together, these results suggest that the effect of gender-mismatch was strongest towards the end or after the pre-registered time windows, and had a posterior scalp distribution. however, we emphasize that the results of these exploratory analyses only have a descriptive purpose. using a rather conservative method to control the false discovery rate that does not take into account spatiotemporal contingencies in the data (benjamini and hochberg, ) , the tested samples did not survive correction for multiple comparisons. we performed a pre-registered, close replication of van berkum et al. ( , experiment ), a canonical erp study on lexical prediction during spoken discourse comprehension. in the original study, the marking of grammatical gender on prenominal adjectives ('groot/grote') elicited an early positivity when it mismatched the gender of an unseen, highly predictable noun, compared to matching gender. in our large-scale (n= ) replication effort, we did not obtain this pattern of effects, but, if anything, a reverse pattern: mismatching gender elicited enhanced negativity j o u r n a l p r e -p r o o f compared to matching gender, reminiscent of the effects reported by otten and colleagues ( ) . we observed enhanced negativity at all spatiotemporal rois, whether time-locked to onset of the inflection or the adjective. however, this enhanced negativity was generally very small (approximately between - . and - . μv at the different rois), and our bayes factor hypothesis tests either anecdotally or moderately favoured the null hypothesis. in contrast, we successfully replicated vb 's prediction-mismatch n effect for the nouns, observing extreme evidence against the null hypothesis even when our prior corresponded to the strongest nounelicited effect reported in vb . pre-registered exploratory analyses showed that, at the anterior and midline rois, the negativity obtained in the inflection time-locked analysis was primarily generated by common gender adjectives ('grote') and close to zero for neuter gender adjectives ('groot'). however, like the main effect of gender-mismatch, the observed gender by mismatch interaction effect was weak and not supported by our bayes factor tests. further exploratory analyses suggested that the main effect of gendermismatch was most pronounced at posterior electrodes, where it was similar for common and neuter gender, and strongest near the end or even after the pre-registered time windows. taken together, these results do not support the effect reported by vb . however, the results did not yield clear evidence against lexical prediction more generally, and in fact yielded some evidence in support of prediction. in the sections below, we discuss our results in more detail and briefly consider their implications for theory and research on predictive language comprehension. interpreting our pre-nominal results is not entirely straightforward because different sources of evidence point in different directions. as our primary and preregistered source, the obtained bayes factors weakly favoured the null hypothesis. from the bfs null (quantifying support for the null-hypothesis at each of the rois, time-locked to inflection or adjective), were over , and were over ('moderate evidence'). however, these values were generally low, and nowhere near the preregistered threshold (bf= ) that would have allowed us to halt sampling and claim replication success or failure. at the same time, the gender mismatch effect estimates themselves were clearly suggestive of a negativity. this was evident from the posterior probabilities of the effect being negative, which were consistently higher than % across all pre-registered tests, and from the exploratory analyses. this discrepancy is primarily caused by the prior, which influences the bayes factor much more strongly than the estimate, as also demonstrated by our results obtained with varying priors. with pre-registered, widened and narrowed zero-mean priors, bayes factor support for the null hypothesis increased and decreased, respectively, without noticeable effect on the obtained estimates. with exploratory priors centered on the estimates reported by vb , we obtained strong bayes factor support for the null hypothesis while the estimate became less negative by only about . µv. for this reason, it is generally advisable to pre-register a range of informative and plausible priors. the influence of the prior can be considered either a bug or a feature of bayesian null-hypothesis testing, depending on your perspective (for discussion, see kruschke, ; kruschke & liddell, ; rouder, haaf, vandekerckhove, ; van ravenzwaaij & wagenmakers, ) . weighing the two sources of evidence is ultimately a judgment call. despite insufficient bayes factor support to claim a replication failure, we are fairly confident that our results do not replicate vb 's positivity. however, we are much less confident regarding a replication of ot 's negativity, because our effect appears quantitatively and qualitatively different. our effect was approximately only one-third of the ot effect size. whereas the ot effect had a clear right-anterior maximum, our effect was not particularly lateralized and most prominent at posterior channels (and therefore not unlike an n effect in terms of scalp distribution and timing). nevertheless, and despite the bayes factor evidence supporting the null hypothesis, our results do suggest that if a true population-level effect exists at all, it is likely small and negative. although defining a close or exact replication remains controversial (e.g., simons, ; zwaan, etz, lucas, donnellan, ), we consider our study to be a close replication of vb and ot , not an exact one. readers might therefore be tempted to attribute the difference in results to a difference in methods. while influences of methodological differences cannot be ruled out, we consider it unlikely that they are the primary cause of the different results. for example, we used a different and larger set of prediction-inducing stories, but our stories were constructed in the same way as those of vb /ot , and had an equally strong, if not stronger cloze probability manipulation as the originals. moreover, our items were based on a set of items that have twice demonstrated a prenominal prediction effect on gendermarked articles ('de/het') with a much smaller sample size (n= and n= ; fleur et al., ) . we also used different filler items, but retained a similar experimental to filler ratio as vb /ot . we used a different speaker, but this speaker was not j o u r n a l p r e -p r o o f faster than that of vb . differences between our study and vb /ot are detailed and justified in the methods section, and also summarized in the online supplementary table . we also summarize differences between our study and our pre-registration in supplementary table . perhaps the strongest argument against the role of methodological differences does not involve a comparison between our study and vb /ot , but between vb and ot . these studies were highly similar to each other, but nevertheless yielded two different types of effects. this discrepancy has previously been discussed in terms of as-yet-unidentified differences. for example, when discussing vb , ot and other studies, otten and van berkum ( , p. ) note that "a systematic inventarization across all studies shows that this variability cannot be accounted for by differences in language, stimulus modality, type of prediction probe, or differences in working memory capacity of participants. one possibility is that perhaps the broader context in which stimuli are presented (i.e. the type of filler that is used,the length of the experiment) matters more than commonly assumed, but we refrain from speculating about specific other factors that could critically influence the way people make predictions, or process prediction-inconsistent data". otten and van berkum thus discussed the discrepant effects as two meaningful demonstrations of lexical prediction (i.e., as two 'true' effects), as is typical for the broader psycholinguistic literature (e.g., ito, corley, pickering, martin & nieuwland, ; pickering & gambi, ) . the current study, however, was premised on the assumption that only one of the original effects can be a 'true' effect, and that the other effect therefore is likely a false positive. false positives and wrong-sign estimates are to be expected in noisy, small-sample settings (gelman & carlin, ) , especially when analysis choices are j o u r n a l p r e -p r o o f contingent on the data (e.g., based on visual inspection of erp waveforms, as was the case in vb and ot ). our results suggest that the positivity reported by vb is more likely to be a false positive finding than the negativity reported by ot . we anticipated a potential role of adjective-gender and the concomitant inflection in shaping the neural response to gender-mismatch. we considered two potential scenarios. the mismatch effect could be greater for common gender than for neuter gender, either because people find it easier to detect mismatch on overt suffixes than on absent suffixes, or because overt suffixes have a bigger impact than absent ones because only overt suffixes rule out the expected noun entirely. alternatively, the mismatch effect could be greater for neuter gender than for common gender, possibly because detection of a mismatch on overt suffixes is more difficult for language developmental reasons (e.g., weerman et al., ) . the results from our pre-registered exploratory analyses did not conclusively favour one scenario over the other. erps time-locked to inflection suggested a stronger mismatch effect at the left-anterior roi for common gender than for neuter gender. however, this pattern was very weak and its significance remains unclear. one obstacle to interpretation is the early positive erp effect that was visible right after adjective onset and therefore not elicited by the inflections (whose onset occurred at least ms after adjective onset). this positive effect may have shown up as a negativity when we time-locked to inflection onset, as an artefact introduced by the baselining procedure . this brings us to two general caveats. first, our design was not optimized for this interaction analysis. because predictable common and neuter gender nouns were preceded by different contexts and adjectives, the mismatch effects for each gender involve a comparison between different sets of adjectives. how this impacted the results is not known, but it could have generated patterns such as the early positivity for common gender adjectives. second, while our sample size is much larger than in typical erp experiments on language comprehension, it was also not optimized (and probably too small) to reliably detect a gender by mismatch interaction. one additional relevant observation pertains to the left-posterior roi. from all rois, the gender-mismatch effect there was strongest, whereas the interaction effect there was weakest and near zero, reflecting similar gender-mismatch effects for common and neuter gender. we conclude, therefore, that our results are most consistent with a gender-mismatch effect for common and neuter gender adjectives. while our gender-mismatch effect may appear surprisingly weak, a weaker effect than those reported by vb and ot was to be expected. the original effects were both just statistically significant at the α = . level. in small-sample, noisy data sets, such effects already tend to have an overestimated effect size and increased chance of a wrong sign (gelman & carlin, ) . moreover, their effects were based on data selected via visual inspection, a procedure that further overestimates the effect size. in the current study, weakness of the observed effect was partly the result of the pre-registered time windows based on vb /ot ; the effect appeared strongest towards the end or even after the pre-registered time windows. beyond the comparison to vb /ot , the pre-nominal prediction effect on dutch adjectival inflection may be generally smaller than other pre-nominal prediction effects for several potential reasons. one reason is the unexpectedness of the gender-marked adjectives themselves, as suggested by recent results from our laboratory on written language comprehension (fleur et al., ) . in fleur et al., gender-mismatching definite articles elicited enhanced negativity in the n time window compared to matching articles when the context presumably led participants to expect a particular, gender-marked article-noun combination (e.g., 'de' when they expected 'het boek'). this effect was found in two identical experiments with preregistered analyses and much smaller sizes (n= and n= ) than the current one. however, when participants expected an indefinite article-noun combination that lacks gender-marking (e.g., 'een boek'), there was only a small gender-mismatch effect on definite articles in the n time window. in other words, gender-mismatch effects may be relatively small, and therefore harder to detect, when participants do not expect a gender-marked word in the first place, as may have been the case in our study. that said, there is no principled reason why prediction-effects cannot be obtained on adjectives at all, even when they are unexpected. a highly predictive language comprehension system should be able to make do . unless perhaps the meaning of the adjective is incompatible with the predicted noun or changes the noun prediction. in the current study, vb and ot , the critical adjectives were selected for being semantically compatible or congruent with the high-cloze noun, and it is assumed that the meaning of the adjective does not change the noun prediction. whether gender-mismatch effects can be obtained on semantically incongruent adjectives (e.g., 'blue' if the predicted noun is 'banana') is an open question, but we think this is unlikely. another reason, one that we find more plausible, could be the difficulty with detecting mismatch on fleeting, relatively subtle information in the spoken signal. we emphasize that we do not claim people predict less when listening than when reading. however, our manipulation on word-final inflections is arguably more subtle than a comparison between two entirely different words (e.g., 'de/het', 'el/la'), because our participants needed to distinguish between a schwa sound or an inter-word 'silent' period. this relatively small acoustic/phonetic difference might be hard to discern (e.g., bailey & hahn, ) , and is sometimes further distorted by coarticulation effects (influences on pronunciation associated with preceding or subsequent sounds) . we would expect a large pre-nominal prediction effect when the mismatching condition differs more strongly acoustically from the matching condition (e.g., a spoken version of the 'de/het' manipulation). people typically need only one or two phonemes to detect a deviation from a predicted noun (e.g., van berkum et al., ; van petten et al., ) . this was also demonstrated by our noun results; prediction-mismatching nouns elicited strong n effects starting as early as ms after noun onset. the weak nature of our pre-nominal prediction effect should not be taken as evidence against lexical prediction more generally. it does raise the question, however, whether listeners reliably or consistently use adjectival inflection information to inform their noun predictions. when a misprediction is evident, people may use the available gender information to revise their initial noun prediction, and perhaps even change their initial prediction to a new noun (as demonstrated by in some items, coarticulation might make the conditions phonemically more dissimilar (e.g., /d/ sounds different in 'verkleed' versus 'verklede', see also vb and our methods section). j o u r n a l p r e -p r o o f concomitant effects on noun-elicited n s, e.g., fleur et al., ; szewczyk, & wodniecka, ) . however, when evidence for misprediction is less compelling or ambiguous, people might be 'reluctant' to let go of their initial noun prediction (e.g., nieuwland et al., ) . such reluctance could make sense because our comprehension system must deal with or compensate for coarticulation effects, disfluencies and noisy real-world environments (e.g., corley & stewart, ; mattys, davis, bradlow & scott, ; norris, mcqueen & cutler, ) . future research efforts should elucidate which pre-nominal manipulations elicit more reliable spoken language prediction effects than others. especially when combined with computational modelling (e.g., norris et al., ) , such efforts can reveal the speech processing mechanisms involved in evaluating discourse-based lexical predictions. furthermore, it remains to be established whether the adjectival inflection manipulation has different effects on predictive processing during reading and listening (e.g., otten et al., ; otten & van berkum, ). in vb 's self-paced reading experiment (experiment ), readers slowed down upon encountering gendermismatching adjectives compared to matching adjectives. however, this effect did not occur at the first of two gender-mismatching adjectives but on the second one appearing words downstream (e.g., 'onopvallende' in 'grote maar nogal onopvallende', english translation: 'unobtrusive' in 'big but rather unobtrusive'). in a written language version of ot , otten and van berkum ( ) observed enhanced negativity for gender-mismatching adjectives compared to matching adjectives, but this effect occurred as late as - ms after word onset. in sum, while gendermismatching adjectives elicited rather weak effects in the current spoken language study, their effects during reading may be even weaker or less consistent. to dutch spoken mini-stories. in the original study, the marking of grammatical gender on pre-nominal adjectives ('groot/grote') elicited an early positivity when mismatching the gender of an unseen, highly predictable noun, compared to matching gender. in our large-scale, pre-registered replication effort, we did not obtain such a positivity, but found enhanced negativity instead. however, this negativity was small and our pre-registered bayes factor analyses generally favoured the null-hypothesis. although reminiscent of the right-anterior negativity reported in a similar study by otten et al. ( ) , the current negativity was much smaller and had a posterior scalp distribution. our results highlight the risks associated with data-contingent analysis. given that data-contingent analysis has been and still is common in the psycholinguistic literature, especially in eeg research, some key findings in this literature may prove hard to replicate (e.g., nieuwland et al., ; nieuwland, ) . the weak nature of our pre-nominal prediction effect should not be taken as evidence against lexical prediction more generally. recent work from our laboratory, for example, observed strong pre-nominal prediction effects on gender-marked articles during reading (fleur et al. ) , with pre-registered analyses but smaller sample sizes. the weak nature of the current effect may reflect the difficulty in detecting gender-mismatch from fleeting, relatively subtle information in the spoken signal. our results therefore raise the question whether dutch listeners reliably or consistently use adjectival inflection information to inform their noun predictions. j o u r n a l p r e -p r o o f effect of gender-mismatch (mismatch minus match) on erp time-locked to inflection onset, plotted as the voltage estimate and corresponding % confidence interval (gray area) at each timepoint and channels. dots underneath the voltage estimates indicate statistically significant samples (not corrected for multiple comparisons). n.b. samples occurring ms after inflection may be distorted by effects associated with noun onset. figure a. . results from the mass regression analyses. effect of gender-mismatch (mismatch minus match) on erp time-locked to adjectives onset, plotted as the voltage estimate and corresponding % confidence interval (gray area) at each timepoint and channels. dots underneath the voltage estimates indicate statistically significant samples (not corrected for multiple comparisons). n.b. samples occurring ms after adjective onset may be distorted by effects associated with noun onset. new and updated tests of print exposure and reading abilities in college students incrementality and prediction in human sentence processing mixed-effects modeling with crossed random effects for subjects and items meaning in the brain phoneme similarity and confusability random effects structure for confirmatory hypothesis testing: keep it maximal fitting linear mixed-effects models using lme controlling the false discovery rate: a practical and powerful approach to multiple testing goals and strategies influence lexical prediction during sentence comprehension brms: an r package for bayesian multilevel models using stan event-related potential components reflect phonological and semantic processing of the terminal word of spoken sentences hesitation disfluencies in spontaneous speech: the meaning of um the p-chain: relating sentence production and its disorders to comprehension and acquisition praat script to detect syllable nuclei and measure speech rate automatically probabilistic word pre-activation during language comprehension inferred from electrical brain activity using bayes to get the most out of non-significant results four reasons to prefer bayesian analyses over significance testing ppvt-iii: peabody picture vocabulary test g*power : a flexible statistical power analysis program for the social, behavioral, and biomedical sciences does reading ability predict individual differences in the syntactic processing of spoken language? poster presented at the international meeting of the a rose by any other name: long-term memory structure and sentence processing rapid expectation adaptation during syntactic comprehension definitely saw it coming? the dual nature of the pre-nominal prediction effect can bilinguals see it coming? word anticipation in l sentence reading beyond power calculations: assessing type s (sign) and type m (magnitude) errors simr: an r package for power analysis of generalized linear mixed models by simulation the garden of forking paths: why multiple comparisons can be a problem, even when there is no "fishing expedition" or "p-hacking" and the research hypothesis was posited ahead of time a crack in the crystal ball: evidence against pre-activation of gender features in sentence comprehension taking perspective: personal pronouns affect experiential aspects of literary reading stairs words: a new adaptive test for assessing receptive vocabulary size in english, dutch, and german. poster presented at architectures and mechanisms of language processing rmisc: rmisc: ryan miscellaneous. r package version how the brain processes violations of the grammatical norm: an fmri study predicting form and meaning: evidence from brain potentials how robust are prediction effects in language comprehension? failure to replicate article-elicited n effects why the a/an prediction effect may be hard to replicate: a rebuttal to delong jasp (version . theory of probability the p as an index of syntactic integration difficulty repair, revision, and complexity in syntactic analysis: an electrophysiological differentiation the effects of electrode impedance on data quality and statistical significance in erp recordings bias in a common eeg and meg statistical analysis and how to avoid it lexical prediction in language comprehension: a replication study of grammatical gender effects in dutch. language bayesian assessment of null values via parameter estimation and model comparison a look around at what lies ahead: prediction and predictability in language processing thirty years and counting: finding meaning in the n component of the event-related brain potential (erp) reading senseless sentences: brain potentials reflect semantic incongruity brain potentials during reading reflect word expectancy and semantic association calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and anovas a lexical basis for n context effects: evidence from meg dissociating n effects of prediction from association in single-word contexts a cortical network for semantics: (de)constructing the n expectation-based syntactic comprehension generating random correlation matrices based on vines and extended onion method neuter is not common in dutch: eye movements reveal asymmetrical gender processing how to get statistically significant effects in any erp experiment (and why you shouldn't) vocabulary knowledge predicts lexical processing: evidence from a group of participants with diverse educational backgrounds the temporal structure of spoken language understanding sentence perception as an interactive parallel process prediction is production: the missing link between language production and comprehension bilinguals reading in their second language do not predict upcoming words as native readers do speech recognition in adverse conditions: a review. language and cognitive processes hierarchical levels of representation in language prediction: the influence of first language acquisition in highly proficient bilinguals the peer reviewers' openness initiative: incentivizing open research practices through peer review do 'early' brain responses reveal word form prediction during language comprehension? a critical review dissociable effects of prediction and integration during language comprehension: evidence from a large-scale study using brain potentials on the incrementality of pragmatic processing: an erp investigation of informativeness and pragmatic abilities when the truth is not too hard to handle: an event-related potential study on the pragmatics of negation large-scale replication study reveals a limit on probabilistic prediction in language comprehension prediction, bayesian inference and feedback in speech recognition. language, cognition and neuroscience brain potentials elicited by garden-path sentences: evidence of the application of verb information during parsing great expectations: specific lexical anticipation influences the processing of spoken language discourse-based word anticipation during language processing: prediction or priming? discourse processes does working memory capacity affect the ability to predict upcoming words in discourse? predicting while comprehending language: a theory and review an integrated theory of language production and comprehension r: a language and environment for statistical computing. r foundation for statistical computing bayesian inference for psychology, part iv: parameter estimation and bayes factors a common misapplication of statistical inference: nuisance control with null-hypothesis significance tests the p as a correlate of ventral attention network reorientation peabody picture vocabulary test-iii-nl afex: analysis of factorial experiments. r package version a failure to replicate rapid syntactic adaptation in comprehension rstan: the r interface to stan exposure to print and orthographic processing the mechanisms of prediction updating that impact the processing of upcoming word: an event-related potential study on sentence comprehension dissociating retrieval interference and reanalysis in the p during sentence comprehension how inappropriate high-pass filters can produce artifactual effects and incorrect conclusions in erp studies of language and cognition sentence comprehension in a wider discourse: can we use erps to keep track of things the neuropragmatics of 'simple' utterance comprehension: an erp review anticipating upcoming words in discourse: evidence from erps and reading times studying texts in a second language: the importance of test type the resilient nature of adjectival inflection in dutch time course of word identification and semantic integration in spoken language advantages masquerading as 'issues' in bayesian hypothesis testing: a commentary on tendeiro and kiers bayesian data analysis in the phonetic sciences: a tutorial introduction the statistical significance filter leads to overoptimistic expectations of replicability puzzlingly high correlations in fmri studies of emotion, personality, and social cognition bayesian hypothesis testing for psychologists: a tutorial on the savage-dickey method l and l acquisition of dutch adjectival inflection anticipating words and their gender: an event-related brain potential study of semantic integration, gender expectancy, and gender agreement in spanish sentence reading potato not pope: human brain potentials to gender expectation and agreement in spanish spoken sentences expecting gender: an event related brain potential study on the role of grammatical gender in comprehending a line drawing within a written sentence in spanish reshaping data with the reshape package ggplot : elegant graphics for data analysis stringr: simple, consistent wrappers for common string operations forcats: tools for working with categorical variables (factors) cowplot: streamlined plot theme and plot annotations for 'ggplot making replication mainstream for help with creating the stimulus materials and/or data collection, we thank j o u r n a l p r e -p r o o f key: cord- -rwy n l authors: taheri, yasaman; suleria, hafiz ansar rasul; martins, natália; sytar, oksana; beyatli, ahmet; yeskaliyeva, balakyz; seitimova, gulnaz; salehi, bahare; semwal, prabhakar; painuli, sakshi; kumar, anuj; azzini, elena; martorell, miquel; setzer, william n.; maroyi, alfred; sharifi-rad, javad title: myricetin bioactive effects: moving from preclinical evidence to potential clinical applications date: - - journal: bmc complement med ther doi: . /s - - -z sha: doc_id: cord_uid: rwy n l several flavonoids have been recognized as nutraceuticals, and myricetin is a good example. myricetin is commonly found in plants and their antimicrobial and antioxidant activities is well demonstrated. one of its beneficial biological effects is the neuroprotective activity, showing preclinical activities on alzheimer, parkinson, and huntington diseases, and even in amyotrophic lateral sclerosis. also, myricetin has revealed other biological activities, among them as antidiabetic, anticancer, immunomodulatory, cardiovascular, analgesic and antihypertensive. however, few clinical trials have been performed using myricetin as nutraceutical. thus, this review provides new insights on myricetin preclinical pharmacological activities, and role in selected clinical trials. polyphenols are a wide group of plant-derived molecules resulting from secondary metabolism, ubiquitously distributed in vegetable kingdom where they display different activities such as protective effect against uv rays, bacteria, virus and fungi infections, modulation of plant hormones, enzyme inhibition and pollinator attraction [ ] . in nature, there are a plethora of different polyphenols that can be classified in the following main classes: simple phenolic acids (e.g. gallic, vanillic, syringic, p-hydroxybenzoic), hydroxycinnamic acid derivatives (such as caffeic acid, p-coumaric, ferulic, sinapic), flavonoids, stilbenes and lignans. the largest common class of polyphenols present in human diet is represented by flavonoids [ , ] . chemically flavonoids are classified in flavans, flavones, flavonols, and anthocyanidins [ ] . among the flavonols, myricetin, a , ′, ′, , ′, -hexahydroxyflavone, possess one of the most hydroxylated structures (fig. ) . the solubility of myricetin in water is poor ( . μg/ml) but increases when deprotonated in basic aqueous media and in some organic solvents (dimethylformamide, dimethylacetamide, tetrahydrofuran and acetone) [ ] . the chemical stability of myricetin is ph and temperature dependent [ ] . depending on the environment conditions, myricetin can exert, in vitro, both a potent antioxidant and a pro-oxidant effect. buchter et al. [ ] attributed its direct antioxidant action to several structural elements. on the other hand, chobot and hadacek [ ] demonstrated the pro-oxidative properties of myricetin to molecular oxygen reduction to reactive oxygen species (ros) and iron (iii) to iron (ii) and also highlighted the ability of myricetin to serve as a substitute for ascorbic acid, albeit less efficiently. myricetin is mainly present in the glycoside form (o-glycosides), in vegetables, fruits, nuts, berries, herbs, plants together with beverages, such as tea, wine, fruit and medicinal plants [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there are numerous factors that can influence myricetin levels in plant foods such as genetic and environmental factors, germination, and ripeness degree, variety, seasonal variation, and storage, processing and cooking. the estimate of total flavonoid intake is difficult to calculate, as appropriate tables of food composition are not yet available. however, reliable data on daily flavonoid intake in a population are needed to develop proper dietary recommendations and even for correct data interpretation from intervention studies. the flemish dietetic association database determined an average daily intake of myricetin of . ± . mg mullie et al. [ ] . in a korean adult population, jun et al. [ ] estimated an average intake of . mg/day representing about - % of flavonol subclass, while a mean intake of myricetin mg/day ranged from to mg/day in adults ( to years) in the european union was reported by vogiatzoglou et al. [ ] . the knowledge on habitual flavonoids consumption is also crucial to determine their possible impact on human health. myricetin exhibited antioxidant properties and free radical-scavenging effects [ ] . these activities seem to support a wide range of beneficial outcomes including, anti-platelet aggregation, antihypertensive, immunomodulatory, anti-inflammatory, anti-allergic, analgesic, anticancer actions and so on [ , [ ] [ ] [ ] [ ] [ ] [ ] . the main goal of the present review is to provide new insights on myricetin preclinical pharmacological activities, and its role in selected clinical trials. [ ] . the first time myricetin was identified was in plants of the myricaceae, comptonia peregrina (l.) coult. and later morella cerifera (l.) small [ , ] . the myricetin concentration in the plants such as rosa canina l. (rosa hip), urtica dioica l. (nettle), and portulaca oleracea l. (purslane) found between and mg/kg [ ] . myricetin was isolated from polygonum bellardii all. (polygonaceae) as yellow needles ( mg) from aerial parts using meoh extract [ ] . previously, a prescreening of leaves of polygonaceous plants was estimated that myricetin glycosides were relatively rare consituents [ ] . trigonella foenum-graecum l. gemmo-modified extract had the richest content in myricetin ( mg/kg), followed by euphorbia tirucalli l. ( mg/kg), rhizomes of cyperus rotundus l. ( mg/kg) and seed extract of t. foenum-graecum ( mg/kg). c. rotundus gemmomodified extracts contained mg/kg myricetin [ ] . the highest level of myricetin content has been identified in the strawberry and spinach [ ] . species of anacardium and mangifera (anacardiaceae) found to have high levels of hydroxylated compounds like myricetin, gallic acid, proanthocyanidins and flavonols. in marantodes pumilum (blume) kuntze (primulaceae) were identified quercetin, myricetin, kaempferol, catechin and epigallocatechin [ ] . the most common sources of myricetin are vegetables, fruits, nuts, berries and tea [ ] . myricetin-rich foods are listed in table based on the usda food database (compiled data from all fruits and vegetables that contain information on myricetin concentration) [ ] . in black fruits the quantities varied between and mg/kg [ ] . myricetin is the most abundant flavonol of black currant, and its quantity varied significantly among black currant cultivars [ ] . at the same time, honey is also a source of flavonoids, especially myricetin. the hplc analyses of honeys from australian eucalyptus have shown that the flavonoids myricetin, quercetin, tricetin, kaempferol and luteolin exist in all honeys. myricetin was found in range from . - . μg/ g honey [ ] . in grapes, flavonol glycosides from the following aglycons have been identified: myricetin ( ′, ′, ′-trioh), laricitrin ( ′-meo analog of myricetin) and syringetin ( ′, ′-dimeo analog of myricetin), quercetin and kaempferol [ ] . the simultaneous presence of these aglycons was detected in different types of red wine vitis vinifera l. grapes [ ] , while in white wine, only quercetin, kaempferol and isorhamnetin were detected [ ] . myricetin displays multiple preclinical biological effects [ ] . thus, in the following subsections, the antimicrobial, antioxidant, neuroprotective, antidiabetic, anticancer, immunomodulatory, cardioprotective, analgesic, anti-hypertensive and wound healing potential of myricetin are briefly discussed and summarized. antimicrobial mechanism of flavonoids may involve membrane disruption, inhibition of cell envelope synthesis, inhibition of nucleic acid synthesis, inhibition of bacterial virulence and quorum sensing, which impairs their ability to form biofilms, inhibition of efflux pumps, and inhibition of nadh-cytochrome c reductase activity and atp synthase [ , ] . myricetin inhibited escherichia coli dna gyrase (ic . mg/dl) [ ] , and dnab helicase (ic . μm) [ ] , and cellular dna and rna polymerases [ ] . myricetin showed a significant antimicrobial activity against foodborne pathogens in terms of minimum inhibitory concentration (mic, mg/ml) < . , < . , < . , < . at h and < . , < . , < . , < . at h incubation for escherichia coli, salmonella paratyphi, salmonella cholerasuis, and salmonella enteritidis, respectively [ ] . the compound myricetin revealed curlidependent e. coli biofilm formation inhibition (ic = . μm), curli contributes to the robustness of e. coli biofilms [ ] . yadav et al. [ ] demonstrated the anti-tubercular activity of selected flavonoids including myricetin and their structure-activity relationships were evaluated against mycobacterium tuberculosis h rv strain radiometrically. myricetin was found to be active against m. tuberculosis, with a mic of μg/ml, and structure-activity relationships authenticated their anti-tubercular potential due to the presence of hydroxy groups in their structure. the inhibitory activity of the compounds were evaluated against dna gyrase from e. coli by dna supercoiling. mean antibacterial activity in terms of mic and ic were μg/ml and . mg/ml respectively. the structureactivity relationship analysis suggests that, the presence of hydroxyl and substitution in the ring a and b position are essential for the best inhibitory effects [ ] . the inhibitory effect of myricetin on severe acute respiratory syndrome-coronavirus (sars-cov) helicase, nsp , and hepatitis c virus (hcv) helicase, ns h was also assessed [ ] . myricetin was found to inhibit sars-cov helicase protein by affecting the atpase activity (ic . μm), however, it failed to affect the atpase activity of the hcv ns helicase. desouza and wahidullah [ ] reported the antimicrobial activity on e. coli, klebsiella pneumoniae, proteus mirabilis, pseudomonas aeruginosa, salmonella typhi, shigella flexneri, staphylococcus aureus, vibrio cholerae and myricetin showed the best activity against p. aeruginosa (mic . μg/ml). gendaram et al. [ ] reported the myricetin antibacterial effect against s. aureus by the disc diffusion method ( μg/disc, inhibition zone mm) but reported no antibacterial activities against p. aeruginosa, e. coli, enterococcus faecalis, or micrococcus luteus. however, at μm concentration, myricetin did not exhibit antimicrobial activity on gram-positive bacteria but showed inhibitory activity against sortase a (srta) from s. aureus ( %; ic . μm) [ ] . in vitro antimicrobial activity of six natural phytochemicals including myricetin (alone and with combination) were evaluated against five strains of p. aeruginosa by using a time-kill assay. the compound showed the mic as μg/ml against all five strains of p. aeruginosa [ ] . other reports of the compound based on antimicrobial and antiviral studies are presented in table . plant-based compounds considered as natural antioxidants have attracted a large number of communities of scientist, researchers, industries and traditional healers for their health-promoting characteristics. the antioxidant table myricetin (mg/ g) rich foods [ ] cranberry dock sweet potato leaves chard, swiss broadbeans, immature seeds rutabagas garlic blueberry peppers, hot chili, green blackberry lotus root lemon source: usda food database (compiled data from all fruits and vegetables that contain information on myricetin concentration) potential of myricetin has been reported by several authors in the last few decades. hou et al. [ ] studied the antioxidant effect of hs -myr micelles and independent myricetin by using frap (ferric reducing antioxidant power) and abts ( , ′azino-bis( -ethylbenzothiazoline- -sulphonic acid) assays. the abts assay displayed an improved value from . to . % in hs -myr micelles and to . % in independent myricetin at two different concentrations and incubation periods. the frap assay also presented an improved value from . to . mm fe + /g in hs -myr micelles and . to . mm fe + /g in independent myricetin at two different concentrations and incubation periods. myricetin in hs -myr micelles exhibited in both assays stronger antioxidant effects when compared to independent myricetin. barzegar [ ] reported the ros-protection efficiency of the compound myricetin in a cell-free and cell-based system. a low concentration of compound significantly inhibited intracellular ros production and also protected cells against toxicity induced by peroxide compounds. guitard et al. [ ] reported that, myricetin is more efficient than α-tocopherol and synthetic antioxidants on preservation of omega- oils. other studies on antioxidant potential of the compound are presented in table . natural flavonoids have exerted positive impacts on body through affecting multiple cell systems and modulating the activity of various pathways to reduce cognitive decline and neuronal dysfunction [ ] . myricetin is one of such flavonoids, and multiple studies have been conducted to assess the neuroprotective effects of this compound and its interaction with brain receptors ( table ). the main mechanisms are shown in fig. . myricetin antidiabetic activity has been reported by several authors in the last few years and limited reports are also available on its anti-obesity activity but in this review, we focused on only its antidiabetic potential. karunakaran et al. [ ] reported the in vitro effect of myricetin on high glucose-induced β-cell apoptosis, possibly via cyclin-dependent kinase (cdk ) inhibition. data revealed that myricetin ( μm) significantly protect β-cells reducing apoptosis in ins- cells and rat islets that were incubated with glucose at the concentration of mm for and h, respectively. docking studies predicted myricetin inhibited activation of cdk . the effect of myricetin was evaluated in diabetes mellitus-associated kidney injuries and dysfunction in an experimental mouse model with diabetes mellitus induced by consecutive injections of low-dose streptozotocin (stz) [ ] . the data revealed that myricetin (orally twice a day, mg/kg/day, for moths) inhibited the iκbα/nf-κb pathway, with this pathway being independent of nuclear factor erythroid related factor (nrf ) regulation. it was also reported that myricetin activates glucagon-like peptide receptor (glp- r) and its long-term oral administration ( mg/kg, for days) validates its glucoregulatory effects [ ] . insulin's metabolic action is mediated via the activation of phosphatidylinositol -kinase (pi k) and its downstream effectors, the protein kinase b (pkb/akt) kinases [ ] . in contrast, amp-activated protein kinase (ampk) signal pathway is likely to mediate the effect of insulin-independent stimuli for glucose uptake in muscle [ ] . in an in vitro study, myricetin enhanced akt and ampk protein activity, encouraged glucose uptake and reduced insulin resistance [ ] . the mechanisms of myricetin for improving insulin-sensitive tissue might be the amelioration of impaired signaling intermediates downstream of insulin receptors through enhancing the secretion of β-endorphin, which in turn led to the activation of peripheral μ-opioid receptors [ , ] . then, myricetin affects insulin receptor phosphorylation, insulin receptor substrate- (irs- ), the p regulatory subunit of pi k, akt and akt substrate of kd, with subsequent effects on glucose transporter (glut ) translocation [ ] . other previous studies on antidiabetic potential of the compound are shown in table . cancer is responsible for second highest cause of death across the globe [ , ] . it has been reported that number of death due to this devastating disease would expand to over million by [ , ] . laboratory and clinical studies have reported that myricetin from natural sources exerts promising effects against various types of cancer [ , ] . the dietary compound myricetin also has the potential to inhibit key enzymes involved in cancer initiation and growth. myricetin has presented cytotoxic activity in human colon cancer cells. kim et al. [ ] demonstrated that myricetin significantly induces the bcl -associated x dose-dependent reduction in lithium-induced head twitches and anxiolytic activity by altering -hydroxytryptamine transmission. [ ] in vitro pro-oxidant agent and reduced the formation of ordered amyloid beta (aβ) aggregation. [ ] in silico destabilizes the β-sheet ordered amyloid oligomers formed by the undecapeptide aβ ( - ) model. [ ] in vitro marked modulation of metal-induced aβ aggregation, more than metal-free aβ aggregation. increase cell survival rate of aβ (with metal ions). [ ] in vitro increases α-secretase (adam ) enzyme activity and decreases of β-secretase (bace- ). it also exerts neuroprotective activity against aβ ( - ) with multifunctional role in counteracting ad progress. [ ] in vitro dose-dependent inhibition of α-synuclein fibrils formation and destabilization (ec = . - . μm). [ ] in vitro dose-dependent inhibition of aβ fibrils formation from fresh aβ ( - ) and aβ . the ec value for formation, extension and destabilization aβ fibrils ranges from . - . μm. [ ] in vivo increases the number of hippocampal ca pyramidal neurons and survival in a rat model ( mg/kg). improved learning and memory in a rat model with ad. [ ] reduces the aggregation of different abnormal proteins and eliminates various toxic proteins related to neurodegenerative diseases. improves physiological functions of hsp molecular chaperone and reduces mis-folded proteins. [ ] in vitro and in vivo increases gaba receptor activity via calcium channel/ camk-ii dependent mechanism, which is distinctively different from that of most existing benzodiazepine binding site agonists of gaba receptor. [ ] in vivo increases mrna for brain-derived neurotrophic factor (bdnf) in the hippocampus of male c bl/ mice at and mg/ kg ( days). [ ] in vivo increases bdnf concentrations in the hippocampus of male c bl/ mice at mg/kg ( days). [ ] in vivo enhances expression and activity of erk / -creb pathway and na + , k + -atpase while reduces oxidative stress level in hippocampus. improves learning and memory when compared with d-galactose. [ ] in vivo reduces seizure severity and mortality rates in mouse models and signaling pathways (bdnf-trkb) and regulates gad / gaba with mmp- expression. [ ] in vivo interacts with rna, especially cag motif, and decreases the huntingtin protein translation and sequestration. reduces cytotoxicity in hd and other polyq disease models. [ ] in vitro suppresses intracellular ros production, re-establishes mitochondrial trans-membrane potential, and inhibits mkk and jnk activation. [ ] in vitro and in vivo inhibits activation of microglia (neuroinflammation), expression of pro-inflammatory mediators and reduces the number of dopaminergic neurons. [ ] in vivo dose-dependent delay in climbing ability loss, but increases the life span of flies expressing human α-synuclein in brain. [ ] in vivo prevents the loss of dopaminergic neurons and dopamine content in brain of parkinson flies. [ ] in vivo dose-dependent inhibitory activity on α-synuclein aggregation. [ ] in vivo diminishes dopamine neuron degeneration, which is induced by -hydroxydopamine and -methyl- -phenyl-pyridinium in substantia nigra-striatum. [ ] aβ amyloid beta, cns central nervous system, bdnf brain-derived neurotrophic factor taheri this study suggested that myricetin can be utilized for the design of therapeutic agents against human colon cancer. myricetin also acts as a potent inhibitor of human flap endonuclease (hfen ) protein (ic nm), based on inhibitory mechanisms, molecular docking, and cancer cell-based assays [ ] . the hfen protein is a functional member of the ′-nuclease superfamily. by chemical nature, hfen is a metal iondependent and structure-specific nuclease and also instrumental in dna replication and repairing processes. molecular docking studies revealed that ring a of myricetin compound, including -keto and -oh, was found stretched towards the two divalent metal ions. both metal ions are critical as they seem to interact with arg and lys amino acids through hydrogen bonds. these interacted residues are well known for their critical interplay in hfen 's activity during human colon cancer. myricetin has also been shown to protect against ovarian cancer through suppressing ovarian cancer cell angiogenesis [ ] . anti-angiogenic effects of myricetin ( to μm) assessed through in vitro (huvec) and in vivo (cam) models revealed that this compound significantly inhibits angiogenesis induced by ovcar- cells. in skov human ovarian cancer cells, myricetin inhibited viability and induced apoptosis ( μg/ml, time-dependent manner) through endoplasmic reticulum stress and dna double-strand breaks [ ] . zheng et al. [ ] stated that in a and ovcar ovarian cancer cells, the dietary flavonoid myricetin induced significant cytotoxicity (ic = μm). in a recent study, tavsan and kayali [ ] reported that myricetin suppressed ovarian cancer cell growth, induced apoptosis, arrested cell cycle and also had the potential to inhibit cell invasion in a significant manner (ic = μm a , μm ovcar- , . μm skov , and > μm osf). thus, it can be concluded that myricetin has enough potential to cope with ovarian cancer in a significant manner. myricetin has potent anticancer-promoting activity against skin cancer. it was found capable of inhibiting neoplastic cell transformation and mitogen-activated protein kinase (mek ) activity (myricetin or μm) [ ] . molecular interaction between myricetin and mek suppressed mek activity leading to downstream signaling to the erk/p rsk/ap- pathway. in another study, myricetin has been presented as a potent chemoprotective agent against skin cancer [ ] . myricetin can bind directly to central kinases including pi -k, akt, jak , raf , mek , mkk , and fyn, which regulate multiple cell signaling pathways in cancer cells. myricetin inhibited -o-tetradecanoylphorbol- -acetate (tpa)and epidermal growth factor (egf)-induced cell transformation by and %, respectively at μm concentration. sun et al. [ ] recently reported that myricetin has anticancer activity against skin cancer a cell lines, by inducing apoptosis and cell cycle arrest and exhibited low toxicity. an earlier in vitro study demonstrated the antimetastatic effect of myricetin in human lung adenocarcinoma a cells [ ] . this study revealed that myricetin ( to μm) suppresses adenocarcinoma a cell invasion and migration through inhibition of the erk pathway in a time-dependent manner. along with a combination of radiotherapy, myricetin was found responsible to enhance the tumor radio-sensitivity of lung cancer a and h cells through significant suppression of cell-surviving fraction and proliferation [ ] . wang et al. [ ] found that the combination of myricetin with -fluorouracil chemotherapy has the potential to enhance tumor chemo-sensitivity of esophageal cancer ec cells. sun et al. [ ] investigated the function of myricetin phytochemical against human t bladder cancer in a dose-and time-dependent fashion, and stated that myricetin significantly inhibits both t cancer cells viability and proliferation (ic = μm). the preclinical immunomodulatory effects of myricetin have also been increasingly reported. ghassemi-rad et al. [ ] concluded that myricetin has the potential to inhibit t-lymphocyte activation in a mouse model through bead-immobilized anti-cd and anti-cd monoclonal antibodies. this study clarified the mechanism of action and reported the suppressive effect of myricetin on t lymphocytes mediated through extracellular h o generation. in mouse primary macrophages and raw . monocytic cell-line, this phenolic compound was found to inhibit the lipopolysaccharide (lps)-induced interleukin (il)- production in a significant manner through down-regulation of nf-κb binding activity [ ] . in isolated rat aortic rings, myricetin induced endothelium-dependent contractile responses at μm. earlier, jiménez et al. [ ] reported that, in cultured bovine endothelial cells, this compound is responsible for stimulating the production of cytosolic free calcium. in a myricetin in vivo enhanced enzymatic and non-enzymatic antioxidant defense system and showed protective effects against oxidative damage in liver and kidney of streptozotocin-cadmium-induced diabetic model. [ ] myricetin in vivo inhibitory activity against α-glucosidase (ic = μm) in dose dependent manner. [ ] myricetin in vivo anti-hyperglycemic and renoprotective effects at . mg/kg. [ ] myricetin in vivo improved and re-established renal functions and activities of the glutathione peroxidase and xanthine oxidase enzymes in diabetic rat model. [ ] myricetin in vivo antidiabetic activity against t-bhp-induced oxidative stress. [ ] myricetin in vivo reduced glycemia in diabetic rats up to % after days of treatment at mg/ h. [ ] myricetin in vivo stimulated lipogenesis in rat adipocytes and enhanced the stimulatory effect of insulin (ec = μm). [ ] myricetin in vitro inhibited intestinal α-glucosidase ( %) and porcine α-amylase ( %) with ic vale of . mm. [ ] abelmoschus moschatus medik. (aerial part) in vivo improved insulin sensitivity in rats. [ ] ampelopsis grossedentata (hand.-mazz.) w.t. wang (leaves) in vivo inhibitory activity against α-glucosidase (ic = . μm). [ ] azadirachta indica a.juss. (leaves) in vivo enhanced insulin signaling pathway and glucose utilization in skeletal muscle. [ ] hovenia dulcis thunb. (seeds) in vitro inhibited intestinal α-glucosidase with ic = μg/ml and α-amylase with ic = μg/ml. [ ] myrtus communis l. (leaves) in vivo significant antidiabetic activity in diabetic models. [ ] syzygium cumini (l.) skeels (seeds) in vitro inhibitory activity against α-glucosidase (ic = . μg/ml) and α-amylase (ic = . μg/ml). dose-dependent manner, myricetin inhibited the secretion of a potent t cell growth factor, namely il- protein from mouse el- t cells, activated with phorbol myristate -acetate (pma) plus ionomycin [ ] . in vitro evidence demonstrated that at - μm, myricetin inhibits cd expression and lymphocytes proliferation in a mouse model. moreover, an in vitro investigation revealed that myricetin significantly effects il- expression. however, further in vitro and in vivo investigations are required to explore myricetin as an immunomodulatory agent. previous studies have demonstrated that myricetin also has beneficial effects on the human vascular system [ ] . in human umbilical vein endothelial cells, myricetin ( μm), revealed vasculoprotective effects through changes at the transcriptional level [ ] . myricetin has been presented as a functional agent towards preventing atherosclerosis through inhibition of cd cell surface protein and mrna expression in a significant manner [ ] . in isolated and langendorff-perfused rat hearts, without affecting contractility and relaxation, myricetin elicited coronary dilation [ ] . in triton-treated hyperlipidemic rats, evidence from an in vivo investigation demonstrated that myricetin exerts lipid-lowering activity and suggests that myricetin can be utilized in the treatment of hyperlipidemia and cardiovascular diseases (cvd) [ ] . in wistar rats, myricetin significantly inhibited the effects of histopathological changes of isoproterenol on heart rate, the levels of different cardiac marker enzymes, including lactate dehydrogenase (ldh), creatine kinase (ck), aspartate aminotransferase (ast), superoxide dismutase (sod) and catalase (cat), as well changes in vascular reactivity and electrocardiographic patterns [ ] . a mechanism-based study by scarabelli et al. [ ] demonstrated that myricetin exerts strong inhibitory activity against signal transducer and activator of transcription (stat ) activation, and also protects the heart from ischemia/reperfusion-injury. the available genomic and genetics data from preclinical experiments have shown that myricetin is likely to confer the first line of defense against cardiovascular and other associated diseases. in acetic acid-induced writhing response, formalininduced paw licking, sedative activity and hot plate test models, myricetin revealed potent analgesic effects, closely related with peripheral analgesia, but not with the opioid system [ ] . the compound also produced a significant analgesic effects in a rat model of neuropathic pain, by decreasing spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia lasting for several hours ( . - mg/kg i.p.) [ ] . the antihypertensive effects of myricetin were evaluated in the deoxycorticosterone acetate (doca)-salt-hypertensive rat model. myricetin reduced systolic blood pressure, vascular reactivity changes and reversed the increased heart rate induced by doca. at oral doses of and mg myricetin/kg b.w., the compound displayed antihypertensive propertie in the doca rat model of hypertension [ ] . in another study, the compound lowered the high blood pressure that was induced by fructose doses of and mg/kg p.o. in rats and reversed sugar-triggered metabolic changes [ ] . the wound-healing effects of myricetin- -o-β-rhamnoside were investigated on three different types of cells, keratinocytes, fibroblasts, and endothelial cells. the compound exhibited significant wound healing activity at μg/ml [ ] . although the number of clinical studies reporting myricetin health benefits in ailments and disorders is low, the increasing data from preclinical studies have supported its beneficial effects [ , ] . in a -week randomized placebo-controlled clinical trial the effect of mg blueberin ( mg blueberry leaves, vaccinium arctostaphylos l., and mg myricetin, three times per day) on fasting plasma glucose and some other biochemical parameters has been investigated in female volunteers ( ± years; body mass index, bmi, ± kg/m ) with diabetes type . the blueberin treatment significantly reduced fasting plasma glucose from ± . mg/l to ± . mg/l. in addition to antidiabetic effects, results showed that blueberin also possessed pharmacologically relevant antiinflammatory properties, reduced plasma enzyme levels of alanine aminotransferases (alt), ast, glutamyltransferase (ggt), and reduced serum c-reactive proteins (crp) [ ] . emulin™ ( mg of patented blend of chlorogenic acid, myricetin, and quercetin), when regularly consumed, was able not only to lower the acute glycemic impact of foods, but also to chronically decrease blood glucose levels in type diabetic humans (reductions between and %) [ ] . this study was performed in male and female with fasting glucose range between to mg/ml and a bmi ≥ kg/m . data from different studies also indicate the importance of myricetin as a chemopreventive agent, acting on cell proliferation, signaling mechanisms, apoptosis, angiogenesis, and tumor metastasis [ ] . through the analysis of habitual food consumption of , participants of finnish mobile clinic health examination survey developed during - , knekt et al. [ ] estimated that higher myricetin intakes in men led to lower prostate cancer risk. in a prospective study, gates et al. [ ] analyzed the association between the common dietary flavonoids (myricetin, kaempferol, quercetin, luteolin and apigenin) intake and epithelial ovarian cancer incidence in [ ] . the association between flavonoids and flavonoid-rich foods intake and exocrine pancreatic cancer development within the α-tocopherol, β-carotene cancer prevention study cohort were also examined [ ] . of the , male smokers with pancreatic cancers, the data obtained suggests that a flavonoid-rich diet may decrease pancreatic cancer risk in male smokers not consuming supplemental α-tocopherol and/ or β-carotene. tang et al. [ ] showed that high/increased flavonoids (e.g., myricetin) intake is associated with lower lung cancer risk in their studied population (meta-analysis of prospective studies and casecontrol studies involving lung cancer cases and , non-cases). the intake of g lyophilized grape powder (rich in flavans, anthocyanins, quercetin, myricetin, kaempferol, and resveratrol) also had a great impact in key risk factors for coronary heart disease (lowered levels of triglyceride, lowdensity lipoproteins, apolipoproteins b and e) in both preand post-menopausal women [ ] . the study was performed on pre-and post-menopausal women for weeks. however, wide ranges of clinical studies are still needed on the potential activities of myricetin which have been already indicated through in vitro and in vivo experiments. myricetin is a flavonoid present in many foods that has shown biological activities in numerous studies and has a potential use as a nutraceutical. its antimicrobial and antioxidant role is widely studied, and numerous studies have shown neurobiological activities and a potential beneficial impact on ad, pd, hd and als. also, preclinical studies have revealed antidiabetic, anticancer, immunomodulatory, anti-cardiovascular, analgesic and antihypertensive activities. these studies investigated the effect of myricetin, pure compound or plant extract rich in this compound. in plant studies, the extracts rich in myricetin always have other flavonoids that have also shown antioxidant activity alone. nevertheless, new well-designed studies have to be performed to study all of the biological effects described before, as well as preclinical studies comparing the effect of myricetin compared to other flavonoids and phytochemicals. in the case of neurological diseases, more in-depth studies have to be designed to show the pre-clinical results. chapter -metabolic responses of plants upon different plant-pathogen interactions kaempferol: a key emphasis to its anticancer potential in vitro and in vivo assessment of free radical scavenging and antioxidant activities of veronica persica poir nomenclature of flavonoids (iupac recommendations ) preformulation studies of myricetin: a natural antioxidant flavonoid myricetin: biological activity related to human health myricetin-mediated lifespan extension in caenorhabditis elegans is modulated by daf- exploration of pro-oxidant and antioxidant activities of the flavonoid myricetin flavonols (kaempeferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants phenolic acid and flavonol contents of gemmo-modified and native extracts of some indigenous medicinal plants total phenolic compounds, flavonoids, and radical scavenging activity of selected tropical plants content of the flavonols quercetin, myricetin, and kaempferol in edible berries determination of myricetin in medicinal plants by highperformance liquid chromatography antioxidant constituents of three selected red and green color amaranthus leafy vegetable characterization of bioactive compounds and antioxidant activity of fruit beers estimation of daily human intake of food flavonoids estimation of dietary flavonoid intake and major food sources of korean adults flavonoid intake in european adults ( to years) myricetin: a dietary molecule with diverse biological activities myricetin attenuated diabetes-associated kidney injuries and dysfunction via regulating nuclear factor (erythroid derived )-like and nuclear factor-κb signaling myricetin induces cell death of human colon cancer cells via bax/bcl -dependent pathway inhibition of interleukin- production in mouse macrophagesvia decreased nuclear factor-κb dna binding activity by myricetin, a naturally occurring flavonoid polyphenols: potential use in the prevention and treatment of cardiovascular diseases analgesic activity of myricetin isolated from myrica rubra sieb effect of myricetin on deoxycorticosterone acetate (doca)-salt-hypertensive rats antiproliferative activity of pteleopsis suberosa leaf extract and its flavonoid components in human prostate carcinoma cells two new flavonoid glycosides from the halophyte limonium franchetii flavonoids from comptonia peregrina the diarylheptanoid (+)-ar, s-myricanol and two flavones from bayberry (myrica cerifera) destabilize the microtubule-associated protein tau polyphenols from aerial parts of polygonum bellardii and their biological activities flavonoids in the leaves of twentyeight polygonaceous plants flavonoids and phenolic acids from labisia pumila (kacip fatimah) dietary flavonoids: bioavailability, metabolic effects, and safety department of agriculture (usda) food database flavonol content varies among black currant cultivars flavonoids in monospecific eucalyptus honeys from australia wine and grape polyphenols -a chemical perspective syringetin, a flavonoid derivative in grape and wine, induces human osteoblast differentiation through bone morphogenetic protein- /extracellular signal-regulated kinase / pathway comprehensive review of antimicrobial activities of plant flavonoids phytochemicals in helicobacter pylori infections: what are we doing now? structure-activity relationship of flavonoids on their anti-escherichia coli activity and inhibition of dna gyrase myricetin inhibits escherichia coli dnab helicase but not primase differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular dna and rna polymerases antimicrobial efficacy of plant phenolic compounds against salmonella and escherichia coli novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone dnak anti-hiv- activity of flavonoid myricetin on hiv- infection in a dual-chamber in vitro model screening of flavonoids for antitubercular activity and their structure-activity relationships identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp antibacterial phenolics from the mangrove lumnitzera racemosa anti-oxidative and antibacterial constituents from sedum hybridum molecular docking and screening studies of new natural sortase a inhibitors activity and interactions of antibiotic and phytochemical combinations against pseudomonas aeruginosa in vitro inhibitory effects of flavonoids on moloney murine leukemia virus reverse transcriptase activity compounds from syzygium aromaticum possessing growth inhibitory activity against oral pathogens activity of plant flavonoids against antibiotic-resistant bacteria antibacterial activity directed isolation of compounds from punica granatum flavonols inhibit sortases and sortasemediated staphylococcus aureus clumping to fibrinogen structure elucidation, conformational analysis and thermal effects on membrane bilayers of an antimicrobial myricetin ether derivative antimicrobial activity of antibiotics in combination with natural flavonoids against clinical extended-spectrum β-lactamase (esbl)-producing klebsiella pneumoniae inhibitory effect of dietary phenolic compounds on chlamydia pneumoniae in cell cultures ultra-small micelles based on polyoxyl hydroxystearate for ocular delivery of myricetin: optimization, in vitro, and in vivo evaluation antioxidant activity of polyphenolic myricetin in vitro cell-free and cell-based systems rosmarinic and carnosic acids as superior natural antioxidant alternatives to α-tocopherol for the preservation of omega- oils antioxidant behavior of mearnsetin and myricetin flavonoid compounds-a dft study microarray and pathway analysis highlight nrf /are-mediated expression profiling by polyphenolic myricetin development of a myricetin/ hydroxypropyl-β-cyclodextrin inclusion complex: preparation, characterization, and evaluation antiinflammatory and antioxidant activities of some extracts and pure natural products isolated from rhus tripartitum (ucria) ferric reducing and radical scavenging activities of selected important polyphenols present in foods antioxidant capacity and vasodilatory properties of mediterranean food: the case of cannonau wine, myrtle berries liqueur and strawberry-tree honey structural elucidation and antioxidant activities of proanthocyanidins from chinese bayberry (myrica rubra sieb. et zucc.) leaves activity-guided isolation of antioxidant principles from limoniastrum feei (girard) batt effect of myricetin, pyrogallol, and phloroglucinol on yeast resistance to oxidative stress myricetin suppresses oxidative stress-induced cell damage via both direct and indirect antioxidant action nitric oxide scavenging rates of solubilized resveratrol and flavonoids myricetin affords protection against peroxynitrite-mediated dna damage and hydroxyl radical formation comparative study on antioxidant capacity of flavonoids and their inhibitory effects on oleic acid-induced hepatic steatosis in vitro myricetin protects cells against oxidative stress-induced apoptosis via regulation of pi k/akt and mapk signaling pathways protective effect of flavonoids against reactive oxygen species production in sickle cell anemia patients treated with hydroxyurea the neuroprotective potential of flavonoids: a multiplicity of effects effect of myricetin on behavioral paradigms of anxiety disclosure of a fundamental clue for the elucidation of the myricetin mechanism of action as amyloid aggregation inhibitor by mass spectrometry amyloid beta-peptide - selfassembly and its inhibition: a model undecapeptide system to gain atomistic and secondary structure details of the alzheimer's disease process and treatment myricetin: a naturally occurring regulator of metal-induced amyloid-β aggregation and neurotoxicity multifunction of myricetin on aβ: neuroprotection via a conformational change of aβ and reduction of aβ via the interference of secretases antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for α-synuclein fibrils in vitro potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of alzheimer's disease myricetin protects hippocampal ca pyramidal neurons and improves learning and memory impairments in rats with alzheimer's disease polyphenolic flavonoid (myricetin) upregulated proteasomal degradation mechanisms: eliminates neurodegenerative proteins aggregation flavonoid myricetin modulates receptor activity through activation of channels and camk-ii pathway dihydromyricetin exerts a rapid antidepressant-like effect in association with enhancement of bdnf expression and inhibition of neuroinflammation myricetin attenuates depressant-like behavior in mice subjected to repeated restraint stress in vivo investigation on the potential of galangin, kaempferol and myricetin for protection of d-galactose-induced cognitive impairment myricetin attenuates the severity of seizures and neuroapoptosis in pentylenetetrazole kindled mice by regulating the of bdnf-trkb signaling pathway and modulating matrix metalloproteinase- and gabaa myricetin reduces toxic level of cag repeats rna in huntington's disease (hd) and spino cerebellar ataxia (scas) myricetin attenuated mpp+-induced cytotoxicity by anti-oxidation and inhibition of mkk and jnk activation in mes . cells myricetin prevents dopaminergic neurons from undergoing neuroinflammation-mediated degeneration in a lipopolysaccharide-induced parkinson's disease model effect of myricetin on the transgenic drosophila model of parkinson's disease effect of myricetin on the loss of dopaminergic neurons in the transgenic drosophila model of parkinson's disease inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds myricetin reduces -hydroxydopamine-induced dopamine neuron degeneration in rats myricetin protects against high glucose-induced β-cell apoptosis by attenuating endoplasmic reticulum stress via inactivation of cyclin-dependent kinase myricetin: a potent approach for the treatment of type diabetes as a natural class b gpcr agonist insulin signalling and the regulation of glucose and lipid metabolism ampactivated protein kinase and muscle insulin resistance. front biosci (landmark ed) myricetin attenuates hyperinsulinemia-induced insulin resistance in skeletal muscle cells myricetin ameliorates defective post-receptor insulin signaling via β-endorphin signaling in the skeletal muscles of fructose-fed rats minireview: therapeutic potential of myricetin in diabetes mellitus myricetin ameliorates defective post-receptor insulin signaling via beta-endorphin signaling in the skeletal muscles of fructose-fed rats myricetin modulates streptozotocincadmium induced oxidative stress in long term experimental diabetic nephrotoxic rats α-glucosidase inhibitory activities of myricetin in animal models of diabetes mellitus myricetin, a natural flavonoid, normalizes hyperglycemia in streptozotocin-cadmium-induced experimental diabetic nephrotoxic rats beneficial effect of myricetin on renal functions in streptozotocin-induced diabetes myricetin may provide protection against oxidative stress in type diabetic erythrocytes effects of myricetin on glycemia and glycogen metabolism in diabetic rats insulinomimetic effects of myricetin on lipogenesis and glucose transport in rat adipocytes but not glucose transporter translocation inhibition of α-glucosidase and α-amylase by flavonoids improvement of insulin sensitivity in obese zucker rats by myricetin extracted from abelmoschus moschatus α-glucosidase inhibition and antihyperglycemic activity of flavonoids from ampelopsis grossedentata and the flavonoid derivatives molecular approach to identify antidiabetic potential of azadirachta indica evaluation of total flavonoids, myricetin, and quercetin from hovenia dulcis thunb. as inhibitors of α-amylase and α-glucosidase biochemical studies on the effect of phenolic compounds extracted from myrtus communis in diabetic rats syzygium cumini seed exhibits antidiabetic potential via multiple pathways involving inhibition of αglucosidase, dpp-iv, glycation, and ameliorating glucose uptake in l cell lines potential antihyperglycaemic effect of myricetin derivatives from syzygium malaccense assessment, origin, and implementation of breath volatile cancer markers vocc: a database of volatile organic compounds in cancer age and cancer risk: a potentially modifiable relationship programmed cell death, from a cancer perspective: an overview discovery of myricetin as a potent inhibitor of human flap endonuclease , which potentially can be used as sensitizing agent against ht- human colon cancer cells myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p -dependent apoptotic pathway myricetin induces apoptosis via endoplasmic reticulum stress and dna double-strand breaks in human ovarian cancer cells myricetin induces apoptosis and enhances chemosensitivity in ovarian cancer cells flavonoids showed anticancer effects on the ovarian cancer cells: involvement of reactive oxygen species, apoptosis, cell cycle and invasion myricetin is a novel natural inhibitor of neoplastic cell transformation and mek myricetin is a potent chemopreventive phytochemical in skin carcinogenesis myricetin exerts potent anticancer effects on human skin tumor cells myricetin suppresses invasion and migration of human lung adenocarcinoma a cells: possible mediation by blocking the erk signaling pathway enhancement of recombinant myricetin on the radiosensitivity of lung cancer a and h cells myricetin enhance chemosensitivity of -fluorouracil on esophageal carcinoma in vitro and in vivo potential anticancer activity of myricetin in human t bladder cancer cells both in vitro and in vivo myricetin-induced oxidative stress suppresses murine t lymphocyte activation involvement of thromboxane a in the endothelium-dependent contractions induced by myricetin in rat isolated aorta inhibition of interleukin- production by myricetin in mouse el- t cells gene expression profiling of human umbilical vein endothelial cells exposed to myricetin morin and myricetin attenuate cd expression and oxldl uptake in u -derived macrophages distinct signalling mechanisms are involved in the dissimilar myocardial and coronary effects elicited by quercetin and myricetin, two red wine flavonols lipid lowering and antioxidant activity of flavones in triton treated hyperlipidemic rats cardioprotective potential of myricetin in isoproterenol-induced myocardial infarction in wistar rats targeting stat by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury antiallodynic effect of the flavonoid myricetin in a rat model of neuropathic pain: involvement of p and protein kinase c mediated modulation of ca + channels effect of myricetin on blood pressure and metabolic alterations in fructose hypertensive rats wound healing potential of chlorogenic acid and myricetin- -o-β-rhamnoside isolated from parrotia persica protective functions of myricetin in lps-induced cardiomyocytes h c cells injury by regulation of malat development of m , myricetin- -o-beta-d-lactose sodium salt, a derivative of myricetin as a potent agent of anti-chronic colonic inflammation effect of blueberin on fasting glucose, c-reactive protein and plasma aminotransferases, in female volunteers with diabetes type : double-blind, placebo controlled clinical study effect of emulin on blood glucose in type diabetics molecular mechanisms underlying anticancer effects of myricetin flavonoid intake and risk of chronic diseases a prospective study of dietary flavonoid intake and incidence of epithelial ovarian cancer flavonoid intake and risk of pancreatic cancer in male smokers (finland) flavonoids intake and risk of lung cancer: a meta-analysis grape polyphenols exert a cardioprotective effect in preand postmenopausal women by lowering plasma lipids and reducing oxidative stress publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -cdo y ob authors: fthenakis, g. c.; mavrogianni, v. s.; gallidis, e.; papadopoulos, e. title: interactions between parasitic infections and reproductive efficiency in sheep date: - - journal: veterinary parasitology doi: . /j.vetpar. . . sha: doc_id: cord_uid: cdo y ob abstract this review article summarises the many reports in the literature, confirming that, in sheep, parasitic infections can adversely affect reproductive efficiency; examples, which refer to all parts of the reproductive cycle of sheep, are as follows: trichostrongylosis in ewe-lambs (which can lead to delayed attainment of puberty), myiosis of the prepuce (which can cause impediment of mating), chorioptic mange or trypanosomosis in rams (which can lead to testicular degeneration or azoospermia, respectively), trypanosomosis or sarcoptic mange in pre-conceptual ewes (which can lead to poor conception rates or reduced number of ovulations, respectively), toxoplasmosis or neosporosis in pregnant ewes (which are causes of abortion), trichostrongylosis or trematode infections in lactating ewes (which can cause reduction of milk yield and can be a risk factor for mastitis, respectively), cryptosporidiosis in newborn lambs (which can be a cause of deaths), coccidiosis in growing pre-weaned lambs (which can cause suboptimal growth rate). in other cases, the reproductive status of the animal can influence the parasitic infection; examples are as follows: the increase in faecal parasitic output during the peri-parturient period (as a consequence of the peri-parturient relaxation of immunity), the heavier trichostrongylid infections of twin lambs compared to lambs from single parities (as a consequence of developmental origin issues in twin lambs). all the above examples support the idea of presence of interactions between parasitic infections and reproductive efficiency in sheep. in sheep, 'reproductive efficiency' includes the ability of ewes to ovulate, be mated, conceive with semen from fertile rams, carry foetuses to term and, finally, lamb live-born lambs, which will be weaned in the appropriate time at an optimal bodyweight. the term implies efficient conception through active gametes, uninterrupted pregnancy, normal delivery of the newborn(s) ('eutocia'), unimpaired lactation within this frame, trichostrongylid gastrointestinal infections currently are among the major challenges in sheep health management, due to the widespread anthelmintic resistance in many parts of the world torres-acosta et al., ) , which increases potential adverse effects in health and welfare of animals. the present paper reviews and discusses interactions between parasitic infections and reproductive efficiency in sheep. the article focuses only on direct effects of parasites on the reproductive efficiency of sheep. nevertheless, it is noteworthy that many arthropods (flies, midges, ticks, etc.) can transmit various infective agents, which may adversely affect the reproductive efficiency of sheep. examples of such agents include anaplasma phagocytophilum (stuen and longbottom, ) , as well as various viruses, e.g., the bluetongue virus (osburn, ) , the rift valley fever virus (bath, ) and the schmallenberg virus (lievaart-peterson et al., ) , which have a foetopathic and/or abortifacient effect. in this article, the topics are organised in a pattern according to the reproductive cycle of small ruminants. puberty is the end point of a series of events affecting the development of the 'hypothalamo-pituitary-gonadal' axis leading to reproductive competence. from a practical point of view, puberty for females is the age at which the animal can support pregnancy to term. puberty is a complex mechanism involving primarily the reactivation of the gonadotropin-releasing hormone (gnrh) secretory system, affected by various factors (senger, ; ebling, ) . among those, energy-deprived animals have been found to have a delayed puberty that has been attributed to a lesser frequency of gnrh pulses and accordingly of luteinizing hormone (lh) pulses i'anson et al., ; polkowska et al., ) . the age at which ewe-lambs are mated, is crucial for productivity of a flock, since cost is high for maintaining unbred animals in a flock. management of ewe-lambs for enhanced reproductive performance requires increased energy availability. this can be achieved by either increasing energy intake by the animal (i.e., availability of high-energy ration or increased quantity of feed) or by reducing its energy drains (e.g., anthelmintic treatment) . possibly therefore, parasitic infections can have an effect in attainment of puberty through interaction with energy intake as discussed herebelow. there is evidence that parasitic infections, specifically gastrointestinal trichostrongylid or trypanosoma congolense (osaer et al., ) infections, can adversely affect onset of puberty and age at first lambing by depressing weight gain of affected animals. administration of a long-acting nematocide anthelmintic, which effectively protected treated ewe-lambs for up to days post-ram introduction, allowed treated animals to exhibit their full reproductive potential during that time . treated ewe-lambs showed earlier reproductive activity, as expressed by short 'interval to first mating after ram introduction' and increased 'cycling rate'; this resulted in significantly younger 'age at first mating' . anthelmintic-treated ewe-lambs reached heavier bodyweight, which is a determinant for puberty in sheep ; thus, they were mated and conceived earlier than untreated controls. there are also similar findings in cattle, showing that nematode-infected heifers reach puberty with a delay compared to uninfected animals (díaz-torga et al., ) . genital myiosis caused by various dipteran insects, can lead to vulvar oedema and subcutaneous fistulae around the vulva in ewes and to difficult/partial exteriorising of penis and markedly thickened prepuce with subcutaneous fistulae along the tissue in rams (fragkou et al., ) . these lesions can impede mating. development of myiosis lesions takes place during the summer months (wall, ) . at para-mediterranean areas and other locations of similar (north hemisphere) or of respective (south hemisphere) geographical latitude, summer months coincide with the sheep reproductive season; at more northern (north hemisphere) or more southern (south hemisphere) latitudes, they precede that period (abecia et al., (abecia et al., , . tick predilection for the genital organs (vulvar mucosa, scrotum) of sheep has been reported (fourie and kok, ; mbuh et al., ) and may be responsible for local nuisance and/or inflammatory reaction, which may also adversely affect mating. one may also suggest that ectoparasitic infections with intense pruritus (e.g., heavy lice infestation, mange) could lead to reduced mating activity of rams, as these animals would be busy scratching rather than being sexually active. ridler et al. ( ) have proposed that parasitic diseases can affect the scrotal circumference of rams and that parasite control is important for keeping rams sound for breeding. scrotal circumference in rams has been associated with their reproductive performance (kafi et al., ; gouletsou and fthenakis, ) . however, gaglio et al. ( ) have not identified a significant effect of gastrointestinal trichostrongylid infection to semen parametres in rams. (rhodes, (rhodes, , has presented evidence that extensive chorioptic mange in the scrotum of rams led to reduced quality of their semen, due to seminal degeneration, as a consequence of long-standing increased intra-scrotal temperature due to the inflammation; semen quality was restored after cure of the skin lesions. lopes et al. ( ) have identified that toxoplasma gondii-infected rams have produced smaller volumes of ejaculate than healthy animals, whilst sangare et al. ( ) have reported azoospermia in tr. congolense-infected rams. finally, sarasa et al. ( ) have reported that sarcoptic mange can cause reduction of the testicular mass in capra pyrenaica (a wild small ruminant); these findings may have implications for sheep, in which species sarcoptic mange is a significant health problem (doukas et al., ) . moreover, parasitic agents may be transmitted through the semen of rams. t. gondii shedding has been identified in the semen of rams (de moraes et al., a,b) and the possibility for transmission of the parasite to ewes during mating has been described (de moraes et al., a,b) depending on the tachyzoite content of semen. in a recent study, dna of neospora caninum was detected in the semen of experimentally infected rams (syed-hussain et al., ) , although none of the ewes mated with those rams developed the disease. no relevant studies are available regarding besnoitia infection of rams. nevertheless, experimental work performed in bucks has indicated that besnoitia caprae can affect their genital system, with cysts of the protozoon identified in the testicular parenchyma and the scrotum (oryan et al., ) . other clinical studies have indicated that besnoitia besnoiti can affect the testicular parenchyma and scrotum of bulls (sekoni et al., ; dubey et al., ) , leading to reduced libido and suboptimal semen quality, although in subsequent studies the protozoon could not be detected in the semen of affected bulls (esteban-gil et al., ) . in general, there is still little published evidence to corroborate an adverse association between parasitism and testicular function (which is reflected in semen quality), although available results point out to that direction. possibly, parasitic infections can directly affect semen quality of rams and, hence, influence conception rates in a flock. alternatively, changes in testicular function can lead to reduced production of testosterone, which is a determinant of a ram's sexual behaviour and social ranking within a flock (parkinson, ) ; this may lead to changes in social interactions and behaviours during mating period. all the above can affect conception rates in a flock, especially if the ram:ewe ratio is at the lower acceptable level or if reproductive management techniques are applied in the flock. this is an area where further work will elucidate mechanisms and potential interactions between parasitism and reproduction. the period immediately before and around conception is a potentially vulnerable period. during that period, adverse developmental origin of the foetus might initiate, as the result of reduced availability of nutrients to the female animal (fleming et al., ) . the significance of increased energy available to ewes in the pre-conception period has been recognised for a long time (clark, ; walkden-brown et al., ; dobson et al., ) . increased energy is provided to ewes by means of modifying the nutritional regime in the period before ewes would be put with rams for mating. at the start of the mating season, ewes should have a body-condition score of ' ' to ' . ' on the five-point scale (lovatt, ) . 'flushing' consists of administration of an additional quantity of concentrate feed mixture, on top of the ration administered to cover maintenance requirements of the animals and should commence at least days before start of the mating period; that interval is equivalent to the length of two full oestrous cycles of sheep. in animals with appropriate body-condition score, this increased energy feeding aims to producing higher ovulation numbers, leading to greater number of lambs born per ewe. in animals at lower body condition score, there is a benefit to other reproductive parametres (e.g., earlier start of the annual reproductive activity, improved cycling and lambing rate), but no significant improvement in their fecundity (heasman et al., ; fthenakis et al., ) . the converse is also true. poor body condition of ewes is associated with reduced fertility, characterised by delayed oestrus development and reduced 'cycling rate' in a flock. ovulation rates decrease as body condition score of the animals deteriorates (dobson et al., ) , whilst embryo mortality at the early stages of pregnancy is higher in undernourished animals (gunn and doney, ) . in general, decreased energy availability around the peri-conceptional period, depresses reproductive performance of extensively (hill farming research organisation, ) or intensively (orskov, ) managed sheep and leads to reduced cyclic activity, reduced ovulation rates and suboptimal ova survival, as well as increased risk of early embryonic deaths (gunn, ) . decreased energy availability at the post-conception period leads to compensatory changes in the gravid uterus later in pregnancy, e.g., increased vascular density of the placentomes (zhu et al., ) , which in turn can lead to higher risk of foetal infection with parasitic abortifacient agents (section . ), especially in multiple pregnancies; health of the lamb(s) after birth is also adversely affected (fleming et al., ) . lambs from multiple parities were found to be more heavily infected with trichostrongylids than lambs from single parities (hayward et al., ) . twin foetuses develop a between-them competition for nutrients, are enveloped by a smaller placenta than single foetuses and live in a restricted physical space . the findings of hayward et al. ( ) are in line with the 'developmental origins of health and disease' concept (formerly known as foetal programming or the barker hypothesis), which implicates early in utero development and the maternal environment experienced during that period as being of significance to development of disease in adulthood (fleming et al., ) . the most significant energy-drain of clinically healthy sheep is parasitism by gastrointestinal helminths. the effects of these parasites in reducing energy availability for their hosts have been well documented (coop and kyriazakis, ) . these authors have proposed that gastrointestinal nematodes can reduce nutrient availability to the host, through a reduction in voluntary feed intake and/or a reduction in the efficiency of absorbed nutrients; the relative contribution of each of these two mechanisms depend on the species of parasite and its location in the gastrointestinal tract (coop and kyriazakis, ) . with regard to trematode infections, reduced feed conversion efficiency, present even in low burden infections (hawkins and morris, ) , as well as depressed appetite and feed intake, also occurring in these helminthoses (taylor et al., ; rojo-vázquez et al., ) , contribute to decreased energy availability for affected sheep. it thus becomes evident that parasitism by the above helminths may affect reproductive efficiency in ewes during the peri-conception period, mostly through the decreased energy availability for the animals. the precise adverse effects can vary depending on the level of parasitism and the general condition of the animal. it is also noteworthy that reduced feed intake occurring in such cases, would reduce the benefits of supplementation with high-energy feed before mating ('flushing'), as animals would not benefit from all energy provided. similar results have been published in cases of t. congolense infection (osaer et al., ) . affected animals showed lower rates of conception and pregnancy and had lower concentrations of progesterone, which is a major factor in establishment of pregnancy, with a function to synchronise development of the maternal endometrium with intrauterine arrival of the embryo (wilmut and sales, ; lawson and cahill, ) . as an association between luteal phase progesterone blood concentrations and embryo survival has been suggested (noakes, ) , perhaps the reduced progesterone levels may be responsible for the conception problems in affected ewes. fthenakis et al. ( ) reported that sarcoptes scabieiinfested ewes had fewer ovulations than uninfested animals and attributed that to nutrient availability at the pre-conception period. nevertheless, when progesterone and equine chorionic gonadotrophin were administered exogenously, no differences were evident between infested and healthy animals. this indirectly suggests reduced hormone concentrations in the parasitised animals during the peri-conceptional period, which lends further support to the idea of sarcoptic mange influencing embryo implantation and survival. in sheep, there are no reports directly associating quality of female gametes (ova) with parasitism. nevertheless, some relevant results have been published in cattle. tritrichomonas foetus has been reported to damage oocytes (benchimol et al., ) . also, n. caninum has been detected in bovine follicles during assisted reproduction manipulations (silva et al., ) , as well as in bovine foetuses (marques et al., ) , although there are reports suggesting that embryo transfer is a safe method to avoid vertical transmission of these protozoon (landmann et al., ; moskwa et al., ) . these implications should be borne in mind, as assisted reproductive technologies for sheep are developing and being applied in larger numbers of animals (amiridis and cseh, ) . in view of the above, one should consider the strategic administration of anthelmintic drugs before the start of the mating period, with a view to improve reproductive efficiency. in fact, mavrogianni et al. ( ) have reported that ewes given a broad-spectrum long-acting anthelmintic before the start of the mating period had a higher 'cycling rate' reflecting better functionality of the genital system of the treated animals and higher 'total lambs born per ewe' and 'liveborn lambs per ewe' indicating increased number of ovulations during the peri-conceptional period. in dairytype production systems, anthelmintic administration at the pre-conception period (which coincides with the end of a lactation period) would also contribute to maintaining a longer persistency of lactation (another energy-dependent function), although drug withdrawal periods (athanasiou et al., ) should be taken into account when designing strategic treatments. finally, anthelmintic administration before the mating season has the added advantage of avoiding the inadverted administration of albendazole or netobimin, broad-spectrum anthelmintics with confirmed embryotoxic properties (delatour et al., ; navarro et al., ) , to ewes at the first stage of pregnancy. as sheep have a seasonal pattern of reproductive activity, depending on the geographical latitude, the pre-mating period would also differ according to location. hence, administration of anthelmintics at that period would have differing effects from a parasitological viewpoint, resulting from the difference in season. this should be taken into account when strategic treatments are carried out at the pre-mating period. furthermore, one should have in mind the possibility of promoting anthelmintic resistance that way and should consider an appropriate cost-benefit analysis. one should always take into account that in para-mediterranean areas and locations of similar latitude (in the north hemisphere), as well as in locations of respective latitude in the south hemisphere, reproductive activity of sheep would start at the beginning of the summer (hence, anthelmintic administration should be planned for late spring). moreover, in more northern (north hemisphere) or more southern (south hemisphere) areas, reproductive activity of sheep would start in the autumn (hence, anthelmintic administration should be planned for late summer). foetopathies and abortions are significant problems in pregnant ewes and major sources of financial losses in sheep flocks (menzies, ) . various protozoa can cause abortion in ewes. the principal problem by parasites in pregnant ewes is toxoplasmosis, caused by the intracellular protozoon t. gondii, which is a confirmed abortifacient agent (buxton and rodger, ) . ewes often become infected through consumption of oocyst-contaminated concentrate feed, given to the animals as an energy-supplement (buxton and rodger, ) . if infection takes place before the st- th day of gestation, embryonic death occurs, followed by absorption or expulsion of small embryos, rarely being noticed. if infection takes place later, up to the th- th day of gestation, then abortion takes place. finally, if infection takes place after that, congenitally infected lambs are born. in embryos, the organism causes coagulative necrosis in the placental cotyledons, as well as microglial foci, representing an immune response (buxton and rodger, ) . toxoplasmosis has been well-studied around the world, with many scientific references describing all aspects of the disease (buxton and rodger, ; dubey, ) . neosporosis, caused by the protozoon n. caninum, is an emerging reproductive problem in ewes; many facets of the disease still remain unclear (dubey and schares, ) . in the initial literature, the infection had not been always associated with abortion (otter et al., ; chanton-greutmann et al., ) , despite evidence showing the abortifacient role of the organism in experimental infections (buxton et al., , . progressively, however, reports from various parts of the world have associated the parasite with abortion in ewes (masala et al., ; spilovská et al., ; howe et al., ; moreno et al., ) , although frequency and clinical significance of the problem require further elucidation (dubey and schares, ) . the organism establishes itself in the maternal caruncular septum, before crossing to the foetal placental villi. a direct foetopathic effect of the organism has been described to be the cause of abortion (innes, ; gibney et al., ) , although further studies are necessary to fully clarify the pathogenesis of the infection. other protozoa that have been reported with an abortifacient role, include sarcocystis ovicanis (s. tenella), s. arieticanis, trypanosoma brucei subsp. brucei, t. congolense, t. vivax and theileria spp. (buxton, ; heckeroth and tenter, ; bawa et al., ; nagore et al., ; batista et al., ). the relaxation of acquired immunity to parasites around lambing and its consequences have been well documented (armour, ; gibbs, ; barger, ) . this is manifested with a rise in faecal parasitic output and had initially, for nematode infections, been associated with increased prolactin concentrations. fleming, fleming, , investigated the potential role of prolactin and prostaglandin f ␣ (two hormones, the concentrations of which increase at the end of pregnancy); they found that total egg production by haemonchus contortus in infected sheep increased after administration of prolactin, but not after 'administration of prostaglandin f ␣ , to those animals. prolactin is a peptide hormone, responsible for initiating and sustaining lactation in ewes (castro et al., ) . it acts in a cytokine-like manner and as an important regulator of the immune system (rovensky et al., ) . blood concentrations of prolactin in pregnant ewes start to increase from the th- th day of gestation (banchero et al., ) . beasley et al. ( a,b) reported that, in ewes experimentally infected with trichostrongylus colubriformis, the rise in faecal egg counts at the end of pregnancy had been preceded by a decrease in the immunological competence of the ewes; this was shown by reduced numbers of circulating eosinophils and by decreased total antibody and igg titres. the changes coincided with increased prolactin concentrations at the end of pregnancy, but, nevertheless, the authors considered that they were unrelated to hormonal effects; this confirmed a similar earlier hypothesis by coop et al. ( ) . as significant differences have been reported in blood concentrations of prolactin according to season (gomez brunet and lopez sebastian, ) , the peri-parturient rise in faecal parasitic output should have differed in accord with time of the year. according to coop and kyriazakis ( ) , this relaxation of immunity and the ensuing increase in faecal egg counts appear to have a nutrition-based background. under the conditions of high metabolic demand, which occur at the end of pregnancy, susceptibility of ewes to gastrointestinal parasites is increased (kahn et al., ) . finally, coop and kyriazakis ( ) proposed a nutritional, rather than an endocrinological, involvement in the relaxation of immunity during that period and presented the following arguments: (a) grade of immunity expression in pregnant ewes is consistent with the reproductive effort, i.e., the number of foetuses borne, (b) termination of pregnancy leads to abrupt restoration of immunity and (c) nutritional management of pregnant ewes can alter the time of first observation of the relaxation. beasley et al. ( ) reported that feeding ewes a low quality diet resulted in a peri-parturient rise in faecal parasitic output starting days before lambing and increasing substantially thereafter, whilst in ewes fed a high quality diet there was only a short rise of small magnitude; these findings lend further support to the above theory. a peri-parturient increase of oocyst/cyst output has also been recorded in cryptosporidium (xiao et al., ; ortega-mora et al., ) and giardia (xiao et al., ) infections of pregnant ewes. in both cases, the authors recorded an increase of oocyst/cyst numbers in faeces of pregnant ewes, as well as an increase in the number of ewes, which excreted oocysts/cysts in their faeces. perhaps, a combination of all above factors may determine the whole process. the parasites can also play a role in increasing the metabolic pressure in pregnant ewes. the increased parasitic output during the pre-partum period has significant consequences for the epidemiology of the respective diseases. lambs are born in a contaminated environment and, thus, are exposed to the infective forms of the various parasites at a very young age. in sheep, pregnancy is a metabolically demanding period. during the first days of pregnancy, there is a slow foetal growth (blanchart and sauvant, ; economides and louca, ) ; during the second month of pregnancy, when foetal attachment has been established and placental growth has been completed, foetus(es) can acquire up to - % of their future birth bodyweight; finally, at the last stage of pregnancy, the ovine foetus(es) can develop rapidly, to acquire up to - % of their future birth bodyweight . hence, energy requirements of pregnant ewes increase, as the end of pregnancy is approaching. in the final month of gestation, protein requirements of pregnant ewes also increase, due to foetal requirements and the need to prepare colostrum in the mammary glands . the situation may be aggravated in cases of heavy parasitic infections, as parasites increase the energy requirements of their hosts (coop et al., ; dakkak, ) , as well as protein synthesis by the host, and consequently protein requirements, due to tissue invasion and damage (solomons, ) . recently, papadopoulos et al. ( ) have shown that trematode infections (fasciola hepatica and dicrocoelium dendriticum) in pregnant adult ewes led to increased ␤hydroxybutyrate concentrations in blood, thus indicating a potential association between trematode infections and pregnancy toxaemia. the authors attributed that on the direct effects that trematodes exert on the liver of affected sheep, as well as on the general energy drain effects of parasitism on the pregnant animals; they suggested that in flocks where many risk factors for pregnancy toxaemia would accumulate (e.g., suboptimal feeding), synergistic effect of those, coupled with trematode infection, could lead to clinical cases of pregnancy toxaemia. valderrábano et al. ( ) took the opposite approach and reported that increased feeding allowance during pregnancy resulted in improved response of pregnant sheep against h. contortus infection. the findings support the idea that response of pregnant ewes to parasitic infections during pregnancy may be enhanced by increased nutrition planes in the earlier stages (valderrabano and uriarte, ) . potential metabolic problems caused by parasitic infections are expressed, ultimately, in the birth bodyweight of lambs born. osaer et al. ( ) have reported that lambs born from t. congolense-infected ewes were of smaller bodyweight than those born from healthy animals. moreover, gatongi et al. ( ) and fthenakis et al. ( ) have administered a nematocidal treatment to ewes at the end of pregnancy and found that birthweight of lambs from treated ewes was higher than that of lambs from untreated animals. albendazole and the respective pro-benzimidazole, netobimin, have a confirmed teratogenic effect to sheep embryos, causing skeletal, renal and/or vascular malformations (navarro et al., ) , when administered to ewes during the first stage of gestation. active metabolites of these drugs can cross the placenta and reach the foetal blood circulation (capece et al., ) . often, the affected foetuses are absorbed or expelled, so ewes will be seen as barren animals at the end of the lambing season. consequently, if there is a need for administration of these drugs, they should be given before start of the mating period, as pre-conception administration of the drug does not appear to cause a foetopathic effect during the subsequent pregnancy (teruel et al., ) . otherwise, anthelmintic drugs with no foetopathic effects must be used. the precaution should extend to later stages of the breeding season, if rams remain with ewes for a long period of time, as there is always the possibility for some ewes to have been mated later in the season . levamisole has also been reported to potentially cause abortion if administered in late pregnancy (braun, ) , hence, it should better be avoided at that period. tissue lesions caused by genital myiosis may result to development of connective tissue in the vaginal and vulvar regions; these lesions can cause dystocia at lambing, due to possible foetomaternal disproportion, as a result of the lesions narrowing the birth canal (arthur and bee, ) . also, presence of connective tissue can lead to difficulties in dilatation of the birth canal, which may also lead to dystocia. dystocia may also occur in cases of births of malformed embryos, formed consequently to albendazole/netobimin administration at the first stage of pregnancy (section . ). finally, leontides et al. ( ) have postulated that d. dendriticum-infection may act as a risk factor for retention of foetal membranes in ewes during the subsequent lambing; they attributed the effect to the reduced energy availability of parasitised ewes, which may affect leucocyte function of ewes, a determinant of placental retention (azawi, ) , that way potentially associating the parasitic infection with the increased incidence of the reproductive disorder in ewes. . . peri-parturient rise in faecal parasitic output: post-partum period the increase in faecal parasitic output ('peri-parturient egg rise') continues after lambing and contributes to lambs for acquiring the infective forms of the various parasites at a young age. beasley et al. ( ) found that increased parasitic output from infected ewes was evident for up to . months after lambing, but, again, suggested that an association with endocrinological factors was unlikely (bar, possibly, a potential role for cortisol), concluding that some other factor(s) would be contributing to the relaxation of immunity to nematodes and the consequent increase in faecal parasitic output during the post-partum period (beasley et al., ) . suarez et al. ( ) and cruz-rojo et al. ( ) have documented that gastrointestinal nematode parasitism can cause - % reduction in milk yield of affected ewes, as well as shorter persistency of lactation. anthelmintic treatment has also been found to increase milk production (rinaldi et al., ) . more specifically, fthenakis et al. ( ) and cringoli et al. ( ) have reported that administration of an anthelmintic with a long persistent activity at the final stage of pregnancy, resulted in a significant (up to %) increase in total milk production throughout the subsequent lactation period. finally, fthenakis et al. ( ) have identified a lower milk production in ewes with sarcoptic mange: up to % compared to healthy animals. the situation regarding potential effects on milk composition is not clear. cruz-rojo et al. ( ) have described that milk from ewes with gastrointestinal nematode parasitism had lower fat and total protein content during the last stage of lactation, but sechi et al. ( ) have not found a significant effect of parasitism on milk composition. it is clear that parasitism leads to reduction in milk production of affected animals. the above studies have been carried out in dairy breeds, with a view to estimate milk production and financial effects of parasitism for dairy farmers. results of direct studies of potential milk yield reduction due to parasitism, in the growth of lambs of the affected ewes are not available and can only inferred from the above reports. reduced milk yield of ewes leads to suboptimal growth rate of lambs (fthenakis and jones, ) and, during the neonatal period, even to increased death rate of lambs (dwyer, ; brozos et al., ) . in this context, it is noteworthy the report by juste jordán and garcía pérez ( ) , who found that adverse effects of parasitism in milk yield were more pronounced at the final stage of lactation, when, however, there is little dependence of lambs on maternal milk, as they consume solid feed. the nutritive value of milk is also dependent upon its composition. nevertheless, a variety of factors can influence milk composition (e.g., nutrition, genetics, stage of lactation, mammary health), which may be difficult to control in order to test potential adverse effects of parasitism; that may explain the unclear results among the respective studies. the major defence mechanism against bacteria invading into the mammary gland is phagocytosis (craven and williams, ) . the process is regulated through a variety of factors, among them energy resources of the host (greenberg and grinstein, ; stuart and ezekowitz, ) , which may indicate a potential adverse role for parasites. in two recent publications, mavrogianni et al. ( mavrogianni et al. ( , have shown the effects of gastrointestinal parasitic infections to development of mastitis in ewes. in a field study, trematode infections (f. hepatica and d. dendriticum) in lactating multiparous ewes have led to increased incidence of clinical or subclinical mastitis during the first two weeks post-partum (mavrogianni et al., ) . in an experimental study, deposition of mannheimia haemolytica into the teat duct of trichostrongylid-infected ewes resulted to development of clinical mastitis, whilst healthy controls developed only subclinical disease after challenge . the above studies were the first to confirm that parasitic infections predispose ewes to mastitis, both diseases being significant health and welfare problems in sheep flocks. it is interesting to note that in one of these studies (mavrogianni et al., ) , the association between trematode infection and mastitis was shown in the immediately post-partum period, when relaxation of immunity (sections . and . ) would be present. in the other one of the above papers , the authors have presented evidence of impaired local defence mechanisms in the mammary glands of parasitised ewes, which might explain pathways for the association observed. during infections with the various abortifacient parasitic agents, foetuses may survive depending upon their age at infection (section . ). in such cases, weak lambs, usually congenitally infected with the respective agent, may be born. such lambs may die soon after birth, from attacks of predators or from hypothermia, as often they are unable to stand up on time, suck and respond to external stimuli (wilsmore, ) . a significant and well documented (de graaf et al., ; fayer, ; taylor et al., ; shahiduzzaman and daugschies, ) health problem in newborn and young lambs is cryptosporidiosis. the disease is an infectious enteritis that causes high morbidity and mortality of affected animals. it is caused by the enteric protozoa cryptosporidium spp., which can affect newborns alone or in mixed infection with escherichia coli or viruses affecting the intestinal tract (e.g., rotavirus, coronavirus) (chatzopoulos et al., ) . cryptosporidium spp. are located at the microvilli in the intestine of affected lambs and impair intestinal function. the disease causes suboptimal growth rate and often death of affected animals, leading to heavy economic losses in the sheep industry. lambs which survive infection at a young age, remain asymptomatic carriers and shed oocysts, contributing to increased environmental contamination and infection of newborn lambs. giardia spp. is an intestinal protozoon affecting lambs at the end of the neonatal period (o'handley and olson, ) . infection is often asymptomatic, although it can cause diarrhoea, which becomes severe and life-threatening in cases of co-infection with other enteric pathogens (andrew thompson et al., ) . specific works in lambs have not been reported. in calves, infection with giardia duodenalis can lead to loss of the epithelial barrier function, villus atrophy and crypt hyperplasia in the small intestine, changes which may result in clinical disease usually characterised by intermittent diarrhoea (ruest et al., ) . in any case, the lesions would cause malabsorption, leading to suboptimal weight gain, reduced feed-efficiency and ill-thriftiness of the affected animals (olson et al., ; buret, ; sweeny et al., ) . in general, the significance of this infection has not yet been fully elucidated and further studies are needed. eimeria spp. are well-described enteric protozoa (taylor et al., ; andrews, ; taylor, ) , which can affect growing lambs from the age of days onwards, causing coccidiosis. up to different species have been reported to affect lambs; e. crandallis and e. ovinoidalis are considered to be the most pathogenic, perhaps because they cause extensive damage in both the small and the large intestine. infections usually remain inapparent, although affected lambs may not be thriving as expected. watery, haemorrhagic diarrhoea can occur and may result to death, if the infection is not properly managed. the onset of intense infection of lambs with gastrointestinal helminths (tapeworms and nematodes) coincides with the start of consumption of solid feed by these animals, specifically grazing. tapeworms (moniezia spp., avitellina spp., stilesia spp., thysaniezia spp.) are regarded of little pathogenic significance, only in heavy infections causing suboptimal growth rate and possibly clinical signs (taylor et al., ) . the effects of gastrointestinal nematode parasitism on the growth of unweaned lambs have been well documented. these parasites are of importance in mutton-type production systems, where lambs remain with their dams for over days of age (sargison, ) . in contrast to that, in dairy-type production systems, lambs are weaned at a younger age (gelasakis et al., ) and, usually, are taken for slaughter; hence, chances for building up a significant production-limiting parasitic burden are minimal. gastrointestinal nematode infections cause significant growth retardation or delay in age of slaughter, which have been documented repeatedly in the older to the more recent scientific literature (coop et al., ; sweeny et al., ) . the significant financial losses associated with the growth retardation of unweaned lambs have led to the need for frequent anthelmintic treatments in these animals, which, in turn, have led to development of widespread anthelmintic resistance in sheep flocks around the world torres-acosta et al., ) . currently, many strategies for anthelmintic treatment of lambs in mutton-type production systems have been advocated, with emphasis given to strategic administration of the drugs (sargison, (sargison, , . the review has covered many aspects of interaction between parasitic infections and reproduction in sheep. in the majority of cases, parasitic infections lead to reduced reproductive efficiency, although there are a few cases where the reproductive stage of the animal influences the parasitic infection. further collaboration of parasitologists with obstetricians, endocrinologists and immunologists will contribute to deeper investigations into these topics and the elucidation of potential relationships, in order to improve health, welfare and production of sheep. pharmaceutical control of reproduction in sheep and goats hormonal control of reproduction in small ruminants assisted reproductive technologies in the reproductive management of small ruminants reproductive health management of sheep and goats the public health and clinical significance of giardia and cryptosporidium in domestic animals some aspects of coccidiosis in sheep and goats epidemiology of helminth disease in farm animals maternal dystocia: treatment proposals for withdrawal period of sheep milk for some commonly used veterinary medicinal products: a review postpartum uterine infection in cattle endocrine and metabolic factors involved in the effect of nutrition on the production of colostrum in female sheep influence of sex and reproductive status on susceptibility of ruminants to nematode parasitism rift valley fever infection by trypanosoma vivax in goats and sheep in the brazilian semiarid region: from acute disease outbreak to chronic cryptic infection effects of trypanosoma vivax on pregnancy of yankasa sheep and the results of homidium chloride chemotherapy the periparturient relaxation of immunity in merino ewes infected with trichostrongylus colubriformis: endocrine and body compositional responses. vet. parasitol the periparturient relaxation of immunity in merino ewes infected with trichostrongylus colubriformis: parasitological and immunological responses. vet. parasitol the influence of reproductive physiology and nutrient supply on the periparturient relaxation of immunity to the gastrointestinal nematode trichostrongylus colubriformis in merino ewes tritrichomonas foetus damages bovine oocytes in vitro consequences of pregnancy on the evolution of lactation non-infectious prenatal pregnancy loss in the doe treatment and control of peri-parturient metabolic diseases: pregnancy toxemia, hypocalcemia, hypomagnesemia mechanisms of epithelial dysfunction in giardiasis protozoan infections (toxoplasma gondii, neospora caninum and sarcocystis spp.) in sheep and goats: recent advances the pathogenesis of experimental neosporosis in pregnant sheep toxoplasmosis and neosporosis immunity to experimental neosporosis in pregnant sheep placental transfer of albendazole sulphoxide enantiomers in sheep management effects on colostrogenesis in small ruminants: a review abortion in small ruminants in switzerland: investigations during two lambing seasons ( - ) with special regard to chlamydial abortions rotavirus infections in domestic animals studies of reproduction in sheep. . the ovulation rate of the ewe as affected by the plane of nutrition effect of anthelmintic treatment on the productivity of lambs infected with the intestinal nematode nutrition-parasite interaction teladorsagia circumcincta egg output at the outset of natural and induced lactation in ewes the effect of a daily intake of ostertagia circumcincta larvae on body weight, food intake and concentration of serum constituents in sheep defences of the bovine mammary gland against infection and prospects for their enhancement the effect of moxidectin . % vs ivermectin . % on milk production in sheep naturally infected by gastrointestinal nematodes effect of infection with teladorsagia circumcincta on milk production and composition in assaf dairy sheep. vet. parasitol gastrointestinal strongylosis and malabsorption of nutrients a review of the importance of cryptosporidiosis in farm animals experimental infection by toxoplasma gondii using contaminated semen containing different doses of tachyzoites in sheep characterization of reproductive disorders in ewes given an intrauterine dose of toxoplasma gondii tachyzoites during the intrauterine insemination albendazole: a comparison of relay embryotoxicity with embryotoxicity of individual metabolites metabolic cues for puberty onset in free grazing holstein heifers naturally infected with nematodes effects of stress on reproduction in ewes ovine sarcoptic mange: clinical features and economic impact toxoplasmosis in sheep-the last years neosporosis in animals-the last five years development of early tissue cysts and associated pathology of besnoitia besnoiti in a naturally infected bull (bos taurus) in south africa the welfare of the neonatal lamb the neuroendocrine timing of puberty the effects of the quantity and quality of feed on the performance of pregnant and lactating goats no detection of besnoitia besnoiti dna in the semen of chronically infected bulls cryptosporidium: a water-borne zoonotic parasite acute or chronic administration of prolactin alters ovine infections of haemonchus contortus effects of exogenous reproductive hormones on haemonchus contortus populations in lambs nutrition of females during the peri-conceptional period and effects on foetal programming and health of offspring attachment preferences of hyalomma truncatum and hyalomma marginatum rufipes ticks (acari: ixodidae) on two sheep breeds effect of restricted nutrition on puberty in the lamb: patterns of tonic luteinizing hormone (lh) secretion and competency of the lh surge system internal and external determinants of the timing of puberty in the female observations in ovine myiosis in greece, with special reference to clinical findings and treatment of genital myiosis health management of ewes during pregnancy the effect of experimentally induced subclinical mastitis on milk yield of ewes and on the growth of lambs effects of sarcoptic mange on the reproductive performance of ewes and transmission of sarcoptes scabiei to newborn lambs effects of three anthelmintic regimes on milk yield of ewes and growth of lambs efficacy of moxidectin against sarcoptic mange and effects on milk yield of ewes and growth of lambs influence of gastrointestinal trichostrongylidosis on ram fertility effects of three nematode anthelmintic treatment regimes on flock performance of sheep and goats under extensive management in semi-arid kenya farm conditions and production methods in chios sheep flocks hypobiosis and the periparturient rise in sheep the extent of parasite-associated necrosis in the placenta and foetal tissues of cattle following neospora caninum infection in early and late gestation correlates with foetal death effect of season on plasma concentrations of prolactin and cortisol in pregnant, non-pregnant and lactating ewes clinical evaluation of reproductive ability of rams phagocytosis and innate immunity dietary energy and ovulation rate in sheep the interaction of nutrition and body condition at mating on ovulation rate and early embryo mortality in scottish blackface ewes depression of productivity in sheep infected with fasciola hepatica maternal effects and early-life performance are associated with parasite resistance across life in free-living soay sheep influence of restricted maternal nutrition in early to mid gestation on placental and fetal development at term in sheep comparison of immunological and molecular methods for the diagnosis of infections with pathogenic sarcocystis species in sheep science and hill farming hfro - potential involvement of neospora caninum in naturally occurring ovine abortions in new zealand the host-parasite relationship in pregnant cattle infected with neospora caninum effect of treatment with netobimin on milk production of sheep regional differences in the distribution of gonadotropin-releasing hormone cells between rapidly growing and growth-restricted prepubertal female sheep seasonal variation in semen characteristics, scrotal circumference and libido of persian karakul enhancing immunity to nematode parasites in single-bearing merino ewes through nutrition and genetic selection confirmation of the prevention of vertical transmission of neospora caninum in cattle by the use of embryo transfer modification of the embryo-maternal relationship in ewes by progesterone treatment early in the oestrous cycle a matched case-control study of factors associated with retention of fetal membranes in dairy ewes in southern greece schmallenberg virus infection in small ruminants-first review of the situation and prospects in northern europe aspects of toxoplasma infection on the reproductive system of experimentally infected rams (ovis aries) clinical examination of sheep neospora caninum: evaluation of vertical transmission in slaughtered beef cows (bos indicus) administration of a long-acting antiparasitic to pre-pubertal ewe-lambs in greece results in earlier reproductive activity and improved reproductive performance pre-existing gastrointestinal trichostrongylosis predisposes ewes to clinical mastitis after experimental mammary infection trematode infections in pregnant ewes can predispose to mastitis during the subsequent lactation period vaccination programs for reproductive disorders of small ruminants parasites of sheep and goats and their prevalence in bokova, a rural area of buea sub division occurrence of neospora caninum and toxoplasma gondii infections in ovine and caprine abortions studies on neospora caninum dna detection in the oocytes and embryos collected from infected cows identification, genetic diversity and prevalence of theileria and babesia species in a sheep population from northern spain anthelmintic induced congenital malformations in sheep embryos using netobimin infertility in the cow: general considerations, anatomical, functional and management causes giardiasis and cryptosporidiosis in ruminants effects of giardiasis on production in a domestic ruminant (lamb) very intensive systems role of adult sheep in transmission of infection by cryptosporidium parvum to lambs: confirmation of periparturient rise histopathologic and ultrastructural studies on experimental caprine besnoitiosis effects of trypanosoma congolense and nutritional supplements on establishment and outcome of pregnancy in trypanotolerant djallonké ewes effects of trypanosoma congolense infection and diet on puberty, age at first lambing and haematology changes in djallonké ewe lambs bluetongue results of a survey to determine whether neospora is a significant cause of ovine abortion in england and wales update on parasitic diseases of sheep anthelmintic resistance in sheep in europe: a selected review potential association between trematode infections and development of pregnancy toxaemia in sheep normal reproduction in male animals the effect of dietary protein restriction on the secretion of lh and fsh in pre-pubertal female lambs seminal degeneration associated with chorioptic mange of the scrotum of rams the effect of extensive chorioptic mange of the scrotum on reproductive function of the ram ram and buck management dairy goat production and the importance of gastrointestinal strongyle parasitism update on trematode infections in sheep evidence for immunomodulatory properties of prolactin in selected in vitro and in vivo situations morphological changes in the jejunum of calves naturally infected with giardia spp and cryptosporidium spp influence of an experimental trypanosoma congolense infection on the reproductive function of djallonke and sahelian rams in subhumid zone negative effect of the arthropod parasite, sarcoptes scabiei, on testes mass in iberian ibex, capra pyrenaica pharmaceutical control of endoparasitic helminth infections in sheep pharmaceutical treatments of gastrointestinal nematode infections of sheep-future of anthelmintic drugs effects of anthelmintic treatment on milk production in sarda dairy ewes naturally infected by gastrointestinal nematodes loss of libido and terminal sterility in a friesian bull naturally infected with besnoitia besnoiti in northern nigeria. a case report puberty therapy and prevention of cryptosporidiosis in animals increased incidence of dna amplification in follicular than in uterine and blood samples indicates possible tropism of neospora caninum to the ovarian follicle pathways to the impairment of human nutritional status by gastrointestinal pathogens the first finding of neospora caninum and the occurrence of other abortifacient agents in sheep in slovakia phagocytosis: elegant complexity treatment and control of chlamydial and rickettsial infections in sheep and goats epidemiology and effects of gastrointestinal nematode infection on milk productions of dairy ewes impacts of naturally acquired protozoa and strongylid nematode infections on growth and faecal attributes in lambs transmission of neospora caninum to sheep by transfer through semen emerging parasitic diseases of sheep veterinary parasitology albendazole sulphoxide administered prior to mating and its relation with fertilization and mouse embryo development anthelmintic resistance in sheep farms: update of the situation in the american continent management of pre-pubertal small ruminants: physiological basis and clinical approach effect of nutritional status and fat reserves on the periparturient immune response to haemonchus contortus infection in sheep effect of nutrition in early pregnancy on the periparturient relaxation of immunity to gastro-intestinal parasitism in prolific ewes role of male-female interaction in regulating reproduction in sheep and goats ovine cutaneous myiasis: effects on production and control effect of an asynchronous environment on embryonic development in sheep perinatal mortality in sheep with special reference to ovine enzootic abortion. the royal veterinary college periparturient rise in the excretion of giardia sp. cysts and cryptosporidium parvum oocysts as a source of infection for lambs periconceptional nutrient restriction in the ewe alters mapk/erk / and pi k/akt growth signaling pathways and vascularity in the placentome the authors have nothing to disclose. key: cord- -r j yzfc authors: mcdonagh, phillip; sheehy, paul a; norris, jacqueline m title: identification and characterisation of small molecule inhibitors of feline coronavirus replication date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: r j yzfc feline infectious peritonitis (fip), a feline coronavirus (fcov) induced disease, is almost invariably fatal with median life expectancy measured in days. current treatment options are, at best, palliative. the objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against fcov in vitro to determine viable candidates for therapy. a resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against fcov. plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and cpe inhibition and ifa-based time of addition assays. three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced cpe at low micromolar concentrations. orthogonal assays confirmed inhibition of cpe was associated with significant reductions in viral replication. selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were , , and for chloroquine, mefloquine, and hexamethylene amiloride respectively. preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. these results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with fip. feline infectious peritonitis (fip), a feline coronavirus (fcov) induced disease, is almost invariably fatal with median life expectancy measured in days. current treatment options are, at best, palliative. the objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against fcov in vitro to determine viable candidates for therapy. a resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against fcov. plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and cpe inhibition and ifa-based time of addition assays. three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced cpe at low micromolar concentrations. orthogonal assays confirmed inhibition of cpe was associated with significant reductions in viral replication. selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were , , and for chloroquine, mefloquine, and hexamethylene amiloride respectively. preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. these results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with fip. ß elsevier b.v. all rights reserved. a number of compounds have demonstrated an inhibitory effect on the virus in vitro (barlough and shacklett, ; hsieh et al., ; keyaerts et al., ) , but there is little or no published data regarding their use in treating fip. the broad spectrum antiviral ribavirin demonstrated in vitro efficacy but provided limited clinical benefit and produced toxicity in cats (weiss et al., ) . more recently in a small study involving experimentally infected cats treatment with chloroquine, a drug with demonstrated in vitro antiviral efficacy, was associated with mild improvements in clinical signs, however there was no statistically significant difference in survival time compared to untreated cats (takano et al., ) . efficacious and safe antiviral therapeutics are desperately needed for fip treatment. modern antiviral drug discovery often involves high throughput screening of vast chemical libraries. these large scale unfocused screens are expensive and beyond the reach of companion animal medicine. an alternative approach is to utilise a more focused screening strategy, enriching the screening library with compounds considered likely to have an antiviral effect based on a prior knowledge of their pharmacodynamics and the viral life cycle. focused screening panels may consist of compounds related to those demonstrated effective against the challenge virus or those demonstrated effective against closely related viruses. in the current study we screened compounds with previously demonstrated antiviral activity against coronaviruses or other rna viruses, for antiviral activity against fcov using an optimised resazurin-based cpe inhibition assay. cytotoxicity of compounds was determined prior to screening using sequential resazurin-and srb-based assays to determine the optimal minimally toxic test concentration and to enable calculation of selectivity indices. the antiviral effects of compounds identified during screening were confirmed with plaque reduction and virus yield reduction assays. virucidal suspension assays and time of addition assays provided initial information on the stage of viral replication targeted and the potential mechanism of action. crandell rees feline kidney (crfk) cell line was propagated in dulbecco's modified eagle's medium (dmem; sigma-aldrich, castle hill, nsw, australia) supplemented with % fbs (sigma-aldrich) (dmem- ) in a humidified incubator at c in % co in air. two strains of fcov, fipv wsu - (fipv ) and fecv wsu - (fecv ), acquired from the american type culture collection (virginia, usa), were used. fcov fecv was originally isolated from mesenteric lymph nodes and intestinal washes of a . year old female domestic shorthaired cat that died of acute haemorrhagic gastroenteritis while fcov fipv was originally isolated from the liver, spleen, and lungs from a case of neonatal death in a -day-old male persian kitten (mckeirnan et al., ) . pathogenicity studies of these two isolates have shown that fipv is highly virulent and reliably causes signs of classic fip following oronasal inoculation, while fecv causes a low grade fever and mild enteritis, but no signs of fip (pedersen, ) . despite the dissimilar in vivo biological properties of the two isolates, the two have similar in vitro properties in immortalised cell lines. compounds were selected for the test panel based on their reported in vitro antiviral properties against coronaviruses or other rna viruses (see supplementary material for details). the compounds tested and their screening concentrations are shown in table . stock solutions were prepared by dissolving compounds in ultrapure water or dmso (sigma-aldrich). compounds were sterile filtered with a . mm regenerated cellulose filter (corning inc., corning, ny, usa), aliquoted into sterile single use microtubes (sarstedt, numbrecht, germany), and stored for a maximum of months at À c until use. to determine an appropriate screening concentration, cytotoxicity of test compounds was determined using sequential resazurin and sulforhodamine b assays. the resazurin-based assay was performed as for the antiviral screening assay except compounds were added in ml volume and there was no infection step. to perform the srb assay, cells were immediately fixed post fluorescent data acquisition by decanting culture media by inverting plates and adding % trichloroacetic acid for h at c. srb staining was as previously described by (vichai and kirtikara, ) except that . % srb was used for staining. following solubilisation of bound dye, od was measured using the fluostar omega microplate reader (bmg labtech, mornington, australia). viability was compared to untreated controls. test compound concentrations selected for subsequent antiviral screening were those resulting in cell viability of % or greater. compounds showing marked, moderate, or mild antiviral effects were defined as those showing - %, - %, and - % inhibition of cpe respectively. compounds demonstrating marked cpe inhibition were classified as candidate compounds and were selected for further characterisation. using the resazurin-based cpe inhibition assay a concentration-response experiment was conducted with serial dilutions of identified candidate compounds (nine concentrations per compound). to enable calculation of the selectivity index, a repeat cytotoxicity screen was performed concurrently. each treatment was performed in triplicate and repeated in three independent experiments. data were exported to microsoft excel for calculation of cell viability and cpe inhibition according to the formulae described above. data analysis were conducted in graphpad prism, with the % inhibitory concentration (ic ) and % cytotoxic concentration (cc ) values calculated using the inbuilt non-linear curve fitting functions following log transformation of compound concentrations. the selectivity index (si) for each compound was calculated according to the following formula: . . confirmatory assays plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified using the cpe inhibition assay. virus yield reduction assays were performed in -well plates (sarstedt). wells were seeded with .  cells well À in ml dmem- . plates were kept at room temperature for min and then at c in % co in air for h prior to the addition of test compounds. compounds were diluted in dmem to the required concentrations with ml added to each well. cells were incubated at c in % co in air for an additional h prior to infection with fcov fipv at moi . in ml dmem. cells were incubated for a further h at c in % co . at and h post-infection (hpi) cell monolayers were visually assessed for cpe using an olympus ckx inverted phase-contrast microscope (olympus, melville, ny, usa) and culture media was collected and stored at À c for virus titration. untreated infected cells, untreated uninfected cells, and treated uninfected cells were included as controls. this latter control was included to allow assessment of morphological changes to cells due to compound treatment. titration of extracellular virus harvested at and hpi was performed using the tcid method as described by mcdonagh et al. ( ) . each treatment and time point was performed in triplicate and repeated in two independent experiments, with results representing mean ae se. plaque reduction assays were performed in -well plates (corning). cells seeded at a density of  cells well À in ml dmem- were held at room temperature for min prior to incubation at c in % co in air for h, by which time monolayers were approximately % confluent. culture media was discarded and replaced with ml dmem supplemented with % fbs plus ml of various concentrations of test compounds in dmem (or ml dmem only for control wells) using five or six concentrations per compound. after exposure to the compound for h, cells were infected with pfu well À fcov fipv in ml dmem. virus was allowed to adsorb for min with plates rocked every min to ensure an even distribution of inoculum. culture media was discarded after min and cells overlaid with ml . % carboxymethylcellulose, % fbs in dmem containing the same concentration of compound as present prior to and during infection. cells were fixed and stained with . % (w/v) crystal violet hpi prior to manual plaque counting. the relative plaque number was calculated for each treatment, with the value of untreated control defined as %. each treatment was performed in duplicate, and repeated in three independent experiments, with data representing mean ae se. a virucidal suspension assay was performed to assess virucidal effects of test compounds. the assay was performed as above with the exception that virus was mixed and incubated with test compounds prior to infection. stock fcov fipv , diluted in dmem to  pfu ml À , was mixed with an equal volume of test compound diluted in dmem to  the test concentration used during screening. the control virus suspension was mixed with dmem containing an equal concentration of dmso as the test samples. virus suspensions were incubated for h at room temperature before serial dilution in dmem to infect cells with pfu well À in ml. following serial dilution of the virus, cells were exposed to test compounds at concentrations greater than log lower than concentrations previously shown to have no antiviral effect. the experiment was performed in triplicate and repeated in two independent experiments. data represent mean ae se. a modification of the resazurin-based cpe inhibition assay was performed to assess the effect of time of compound addition on the antiviral efficacy of identified compounds. the cpe inhibition assay was performed as previously described with the exception that test compounds were added at various time points before and after infection. the selected time points were h prior to infection, concurrent with infection, and , , or h postinfection. treatments were performed in triplicate and repeated in three independent experiments. data represent mean ae se. to further elucidate the stage of viral replication affected by each compound the effect of time of addition on viral antigen expression was examined. cells were seeded at a density of .  cells well À in ml dmem- in -well plates (mclear , greiner bio-one). after seeding plates were kept at room temperature for min and then incubated at c in % co in air for h prior to the first time-point of compound addition. compounds were added in ml to duplicate wells at different time points prior to, concurrent with, or postinfection. cells were infected with fcov fipv at moi . in ml or mock infected with ml dmem for an infection period of h. an infection period of h was selected based on the reported one step growth curve of fcov (rottier et al., ) . at hpi (measured from the end of the infection period) cells were fixed in % formaldehyde in pbs and permeabilised in ice cold methanol. viral antigen was detected with a biotinylated anti-fcov antibody (ccv - ; custom monoclonals international, sacramento, ca, usa) and visualised with streptavidin-conjugated alexafluor (life technologies, mulgrave, vic, australia). to enable accurate segmentation, cells were stained with the whole cell stain hcs cell mask blue (life technologies) and dapi (life technologies) to enhance nuclear visualisation. fluorescent imaging was performed using the bd pathway bioimager (bd bioscience, franklin lakes, nj, usa). images of wells were acquired using a  objective (na . ) using a  montage with laser autofocus performed for each montage frame. hcs cell mask blue/dapi, images were acquired with ex / bp and em lp filters, and alexa fluor images acquired with ex / bp and em lp filters. image analysis was performed using the free opensource image analysis software cellprofiler (r , www.cellprofiler.org) with data exported to fcs express image cytometry (version . . , de novo software, los angeles, ca, usa) for analysis. each treatment was performed in duplicate, and data represents mean ae sd. to assess efficacy against different fcov strains, identified candidate compounds were tested against fcov fecv using the resazurin-based cpe inhibition assay. the assay was performed as described, except that cells were infected with either fcov fipv or fecv at moi . . each treatment was performed in triplicate and repeated in three independent experiments, with data representing mean ae se. three of nineteen tested compounds showed marked inhibition of virus induced cpe (fig. ) and were selected for further characterisation. pre-treatment with chloroquine at mm, mefloquine at mm, and hexamethylene amiloride at mm resulted in . %, . %, and . % inhibition of cpe respectively. a further two compounds, glycyrrhizic acid at mm and cinanserin at mm displayed a mild antiviral effect with a . % and . % reduction in cpe respectively. all other compounds demonstrated limited or no inhibitory effect on cpe. included among these ineffective compounds was ribavirin, a broad spectrum antiviral compound that had previously shown in vitro (barlough and scott, ; weiss and oostrom-ram, ) , and to a limited extent in vivo efficacy against fcov (weiss et al., ) , as well as rfeifn-v which had previously shown in vitro efficacy against fcov (mochizuki et al., ; truyen et al., ) . a concentration-response study was conducted with chloroquine, mefloquine, and hexamethylene amiloride. a repeat cytotoxicity screen was concurrently performed for these compounds to allow calculation of selectivity indices. all compounds demonstrated a clear concentration-response effect over the tested range (fig. ) . calculated ic , cc , and si values for the compounds are shown in table . virus yield reduction assays confirmed the cpe inhibition identified during screening was associated with a marked reduction in extracellular viral titre. determination of extracellular virus titre was performed at and hpi with results shown in fig. . for chloroquine and mefloquine there was a considerable difference in the resulting concentration-response curves at and hpi, while for hexamethylene amiloride the shape of the curve was similar at both time points. differences in concentration-response curves between the two time points is reflected in the ic values, with increased ic values for chloroquine and mefloquine at hpi compared to hpi, while for hexamethylene amiloride ic values were similar at both time points (table ) . plaque reduction assays confirmed the findings of the cpe inhibition and virus yield reduction assays. pre-treatment with chloroquine, mefloquine, or hexamethylene amiloride resulted in a concentration-dependent decrease in plaque number, with high concentrations completely inhibiting macroscopic plaque formation. for all compounds plaque morphology was similar between treated and untreated wells however plaque size was smaller in treated versus untreated wells. during the virus yield reduction assay cells were monitored for the development of cpe using phase contrast microscopy. it was noted that infected and uninfected cells treated with chloroquine, mefloquine, or hexamethylene amiloride displayed characteristic morphological changes. these changes consisted of a large number of variably sized cytoplasmic (predominantly perinuclear) inclusions in addition to the presence, in some cells, of an increased number of cytoplasmic vacuoles. to investigate the nature of these inclusions, separate wells were stained with mg ml À neutral red in dmem for h. these inclusions appeared to accumulate the vital dye neutral red following suggesting they were likely dilated endosomes/lysosomes (fig. ) . using a virucidal suspension assay no virucidal effects were seen for chloroquine, mefloquine, or hexamethylene amiloride, with the infectivity of virus suspensions exposed to the compounds not significantly different from virus incubated with media alone. the effect of time of addition on the antiviral activity of selected compounds was assessed using a modification of the resazurin-based cpe inhibition assay and through ifa of viral protein expression. based on the cpe inhibition assay maximum antiviral effect was seen when compounds were added prior to or concurrent with infection, following which there was a time-dependent reduction in cpe inhibition (fig. ) . for all tested compounds cpe inhibition remained greater than % when compounds were added at the latest tested time point of h postinfection. the cpe inhibition assay encompasses multiple rounds of viral replication. to further elucidate the stage of viral replication affected by test compounds a single replication cycle ifa-based assay was conducted which confirmed that, based on viral antigen expression, all three compounds possess antiviral properties when added prior to, or at the time of infection. furthermore all compounds displayed a time of addition dependent reduction in antiviral effect; however the extent and timing of this reduction varied. the inhibitory effect of chloroquine was reduced, based on an increase in the percentage of fcov antigen positive cells, when added at any time postinfection (fig. ) . a similar result was seen for hexamethylene amiloride, although in this case a significant increase in the number of infected cells was not seen until compound addition was delayed until hpi. in contrast, mefloquine remained effective when added up to hpi suggesting it may act at a later stage of viral replication than chloroquine and hexamethylene amiloride. the efficacy of the three identified candidate compounds was tested against fcov fecv , a serotype ii enteric biotype fcov. comparison of the virus control (no treatment) wells showed fcov fipv infection resulted in more pronounced cpe over the h infection period compared to fcov fecv . pre-treatment with chloroquine, mefloquine, or hexamethylene amiloride provided a degree of protection against strain fcov fecv . pretreatment with hexamethylene amiloride provided protection against virus induced cpe that was similar for the two strains, with a reduction in cpe of . % and . % for fcov fipv and fecv respectively. both chloroquine and mefloquine however were more effective against fcov fipv than fecv , with cpe inhibition for chloroquine of . % for versus . %, and for mefloquine . % versus . % for strains fipv and fecv respectively. this study identifies three compounds (chloroquine, mefloquine, and hexamethylene amiloride) demonstrating a marked inhibitory effect on fcov replication in vitro by significant reductions in virus induced cpe and viral titres at low micromolar concentrations when present during the early stages of viral replication. an antiviral effect of chloroquine had previously been demonstrated against fcov, and hexamethylene amiloride had previously demonstrated efficacy against other coronaviruses, however this is the first demonstration of antiviral efficacy of mefloquine against a coronavirus. initial compound screening was performed using a cpe inhibition assay, with subsequent virus yield reduction assays and plaque reduction assays used for confirmatory testing. for the effective compounds the ic values, and corresponding selectivity index, varied with the assay method utilised. this is not unexpected given the assays measure different endpoints, and has been reported for other antiviral drugs such as the retroviral protease inhibitor saquinavir where the reported ic calculated based on production of viral p antigen is approximately -fold lower than that based on production of mature virions (buss and cammack, ) . similarly variation in assay conditions may result in the calculation of significantly different ic values. the concentration-response curve of chloroquine against sars-cov determined using a pcr based virus yield reduction assay was shown to shift considerably to the right when viral genome copies were assayed days post-infection compared to day postinfection (keyaerts et al., ) . a similar finding was noted in the current study for both chloroquine and mefloquine, with differences in potency reported with the tcid based virus yield reduction assay performed at and hpi, however this was not seen for hexamethylene amiloride. two compounds, ribavirin and rfeifn-v, which had previously demonstrated in vitro efficacy against fcov, failed to demonstrate significant inhibition of cpe during screening. for both compounds these discordant results are likely attributable to testing at concentrations below their useful therapeutic range and variations in assay conditions and sensitivity compared with previous work. the screening concentration of compounds used in this study was determined based on cytotoxicity testing to achieve cell viability greater than %. previous studies with ribavirin demonstrated ic values of . mg ml À ( mm) (barlough and scott, ) based on a visual assessment of protection from cytopathic effect and . mg ml À ( . mm), based on the reduction of extracellular viral titre (weiss and oostrom-ram, ) . the concentration used for screening ( . mm) was therefore more than times lower than the ic previously calculated based on a similar assay endpoint. from the results of the current study, virus yield reduction assays appear to provide a more sensitive assessment of antiviral efficacy than cpe inhibition assays, with the ic values calculated based on viral titre reduction significantly lower than those calculated based on cpe inhibition for all compounds. a small antiviral effect of ribavirin cannot therefore be ruled out based on the current findings, as although the tested concentrations did not provide protection against virus induced cpe, it may have been associated with a reduction in extracellular viral titre. the practical relevance of such a small antiviral effect is questionable, particularly given the known toxicity profile of this compound in cats. for rfeifn-v reductions in viral titres of . - . logs have been reported when crfk cells were treated with , u ml À h post-infection (truyen et al., ) and . - . logs when fcwf cells were pre-treated with - , u rfeinf-v (mochizuki et al., ) . protection from cpe was not seen in the current study when cells were pre-treated with rfeinf-v at u ml À , a concentration significantly lower than that previously shown to be effective using the same virus strain and cell line (truyen et al., ) . the tested concentration was however similar to that used by mochizuki et al. ( ) . this apparent lack of efficacy in this case may reflect differences in the drug exposure and infection conditions, the viral isolate tested, or an intrinsic enhanced susceptibility to the antiviral effects of interferon in fcwf cells compared to crfk cells as used in this study (weiss and toivio-kinnucan, ) . alternatively it may be that, as suggested for ribavirin, virus yield reduction assays provide a more sensitive assessment of antiviral effects than cpe inhibition assays, and that the screening method utilised failed to identify mild antiviral effects. a number of different mechanisms of action have been suggested to account for the antiviral properties of the compounds identified in this study against other viruses. for chloroquine antiviral effects have been ascribed to inhibition of glycosylation of viral proteins (savarino et al., ) or cellular receptors for viral attachment (vincent et al., ) , inhibition of glycoprotein expression (dille and johnson, ) , or inhibition of endosome mediated viral entry (savarino et al., ) . the antiviral effect of mefloquine against jc virus has been postulated to be due to its action as an adenosine mimetic (brickelmaier et al., ) , while for hexamethylene amiloride it has been suggested antiviral properties against different viruses may arise through competitive inhibition of viral rna polymerase (gazina et al., ) , an indirect mutagenic effect (levi et al., ) , or inhibition of viroporins (wilson et al., ) . interestingly all three compounds showing marked antiviral efficacy against fcov in this study resulted in similar morphological changes in cells exposed to sub-toxic concentrations. increased numbers of variably size cytoplasmic inclusions that accumulate the viral dye neutral red suggests these compounds result in a perturbation of the normal endocytic pathway in crfk cells. alterations in the endocytic pathway have previously been reported for chloroquine (dean et al., ) , mefloquine (labro and babin-chevaye, ) , and for amiloride and some of its derivatives (dutta and donaldson, ). this suggests a common physiological effect on treated cells for all three candidate antivirals and possibly a shared mechanism of action. viruses are known to usurp a variety of host endocytic pathways for cell entry and intracellular movement and inhibition of these pathways may be a useful therapeutic approach. although targeting a cellular pathway may be associated with an increased risk of toxicity, if that pathway is critical for viral replication this approach may slow or limit the development of resistance. time of addition studies demonstrated all compounds were most effective when added prior to infection, suggesting a mechanism of action involving early stages of viral replication. the cpe inhibition based time of addition assay involved infection at low moi with a h infection period, allowing for multiple rounds of viral replication. as a result of this, even with the delayed addition of compounds, cells uninfected by the original inoculum are effectively pre-treated prior to challenge with progeny virions produced during the primary replication cycle. using an ifa-based time of addition study involving a single replication cycle we were able to further clarify of the effect of time of addition, and refine the possible stage of the viral life cycle targeted by each compound. based on the ifa results chloroquine was effective only if present at the time of infection, supporting the hypothesis that chloroquine acts during cell entry for fcov fipv , possibly through inhibition of endosomal ph (takano et al., ) . hexamethylene amiloride and mefloquine provided significant antiviral effects when compound addition was delayed for up to and hpi respectively, suggesting that if the antiviral effects of these compounds arise through perturbation of endosomal function, the effects occur at different stages of the viral life cycle. alternatively distinct mechanisms of action may account for the observed effects of these compounds, as suggested for other viruses. there is limited published pharmacokinetic or safety data to inform the potential therapeutic application of the identified compounds in cats and given the relatively low si of all three compounds consideration must be given to their in vivo safety in this species. the human approved pharmaceuticals chloroquine and mefloquine are generally considered well-tolerated drugs, albeit with a narrow therapeutic index, while the clinical use of hexamethylene amiloride has not been reported. pharmacokinetic data available for chloroquine and mefloquine in humans would suggest that effective plasma concentrations could be achieved at standard therapeutic doses (pussard and verdier, ; simpson et al., ) . chloroquine has been shown to accumulate in leukocytes, where the concentration may be two orders of magnitude greater than that of plasma (mackenzie, ) , with the highest concentration reported in monocytes (french et al., ) . thus therapeutic concentrations may be attained in the target cells of virulent biotype fcovs at relatively low plasma concentrations, minimising the risk of dose-dependent adverse effects. mefloquine is known to accumulate within brain parenchyma at concentrations approximately - times higher than found in serum, with tissue concentrations of up to mm reported (nevin, ; pham et al., ) . mefloquine may therefore be useful in the treatment of dry (non-effusive) fip, where cns lesions are common (pedersen, ) although the potential for neurotoxicity must be considered. although the concentration of mefloquine achieved in the cns is greater than the cc of this compound in immortalised feline kidney cells, in humans this tissue concentration is achievable at therapeutic doses, despite in vitro data in human cells showing a cc approximately equal to that determined in the current study (brickelmaier et al., ) . it may be therefore that the more static cell population of the cns is more refractory to the toxic effects of mefloquine than mitotically active immortalised cells. this study has identified three compounds demonstrating marked in vitro inhibition of fcov in an immortalised cell line at low micromolar concentrations, including the first demonstration of antiviral effects of mefloquine against a coronavirus. although the low si of the three compounds may limit their therapeutic utility, these preliminary studies open the way for further investigation and potential optimisation of these compounds as antiviral agents. effectiveness of three antiviral agents against fip virus in vitro antiviral studies of feline infectious peritonitis virus in vitro identification and characterization of mefloquine efficacy against jc virus in vitro. antimicrob measuring the effectiveness of antiretroviral agents effects of exogenous amines on mammalian cells, with particular reference to membrane flow inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine search for inhibitors of endocytosis: intended specificity and unintended consequences randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis uptake of chloroquine and hydroxychloroquine by human blood leucocytes in vitro: relation to cellular concentrations during antirheumatic therapy amiloride is a competitive inhibitor of coxsackievirus b rna polymerase treatment of cats with feline infectious peritonitis synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle effects of amodiaquine, chloroquine, and mefloquine on human polymorphonuclear neutrophil function in vitro fidelity variants of rna dependent rna polymerases uncover an indirect, mutagenic activity of amiloride compounds pharmacologic actions of -aminoquinoline compounds in vitro inhibition of feline coronavirus replication by small interfering rnas isolation of feline coronaviruses from two cats with diverse disease manifestations inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro epileptogenic potential of mefloquine chemoprophylaxis: a pathogenic hypothesis a review of feline infectious peritonitis virus infection: - cerebral uptake of mefloquine enantiomers in fatal cerebral malaria antimalarial -aminoquinolines: mode of action and pharmacokinetics effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein effects of chloroquine on viral infections: an old drug against today's diseases anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors population pharmacokinetics of mefloquine in patients with acute falciparum malaria&ast analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase n is not important and a process of acidification of the endosome is necessary effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo antiviral potency of interferon-omega (ifn-omega) against selected canine and feline viruses sulforhodamine b colorimetric assay for cytotoxicity screening chloroquine is a potent inhibitor of sars coronavirus infection and spread evaluation of free or liposomeencapsulated ribavirin for antiviral therapy of experimentally induced feline infectious peritonitis inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication this study was supported by donations from the rex cat club (especially sharon barton and tracey gleeson), participants of the national annual feline health seminars, christine atkins, ruth thurling, cat fanciers association of nsw, and the cat protection society of nsw. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . / j.vetmic. . . . key: cord- -gw xnb x authors: yang, mengling; dong, yinmiao; he, qingnan; zhu, ping; zhuang, quan; shen, jie; zhang, xueyan; zhao, mingyi title: hydrogen: a novel option in human disease treatment date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: gw xnb x h( ) has shown anti-inflammatory and antioxidant ability in many clinical trials, and its application is recommended in the latest chinese novel coronavirus pneumonia (ncp) treatment guidelines. clinical experiments have revealed the surprising finding that h( ) gas may protect the lungs and extrapulmonary organs from pathological stimuli in ncp patients. the potential mechanisms underlying the action of h( ) gas are not clear. h( ) gas may regulate the anti-inflammatory and antioxidant activity, mitochondrial energy metabolism, endoplasmic reticulum stress, the immune system, and cell death (apoptosis, autophagy, pyroptosis, ferroptosis, and circadian clock, among others) and has therapeutic potential for many systemic diseases. this paper reviews the basic research and the latest clinical applications of h( ) gas in multiorgan system diseases to establish strategies for the clinical treatment for various diseases. molecular hydrogen (h ) is the lightest chemical element in the earth's atmosphere. h is often mixed in gas cylinders for deep-sea divers to breathe, to prevent decompression and nitrogen sickness [ ] . in mammals, h is spontaneously produced by intestinal bacteria in the process of anaerobic metabolism to produce energy and is enzymatically catabolized by hydrogenases to provide electrons. therapeutic applications of h were first described in . dole et al. reported that hyperbaric hydrogen caused marked regression of tumors in mice with skin squamous carcinoma [ ] . however, hyperbaric h is not a clinically feasible option, and h is a physiologically inert gas that seems not to react with any active substances, including oxygen gas, in mammalian cells. thus, h was perceived as being nonfunctional and was disregarded clinically. in , the potential therapeutic benefits of h were described. ohsawa et al. discovered that h has selective antioxidant properties that protect the brain against ischemia/reperfusion (i/r) injury and stroke by specifically neutralizing hydroxyl radicals ( ⋅ oh) and peroxynitrite (onoo-) but not superoxide anion radical ( ⋅ o -), hydrogen peroxide (h o ), and nitric oxide (no) [ ] . the report generated worldwide attention and thrust h into the spotlight of therapeutic medical gas research. many studies using cellular, animal, and clinical experiments in a variety of biomedical fields have explored the therapeutic and preventive effects of h . the collective data have indicated that h is an important pathophysiological regulatory factor with antioxidative, anti-inflammatory, and antiapoptotic effects on cells and organs [ ] [ ] [ ] . it is so convenient to use that h can be easily administered in various ways, including inhalation, injection of h -rich saline (hrs), drinking h -rich water (hw), bathing in hw, and using hrs eyedrops. as well, the production of intestinal h by bacteria can be increased via oral administration of acarbose and lactulose. liu and his colleagues demonstrated that the hydrogen concentration reached a peak of min after oral and intraperitoneal administration, and in only min following intravenous administration [ ] . beginning on december in wuhan, china, illness and pneumonia named coronavirus disease- (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has spread to become a pandemic. the seventh edition of chinese clinical guidance for covid- pneumonia diagnosis and treatment ( th edition) issued by china national health commission recommended the inhalation of oxygen mixed with hydrogen gas ( . % o and . % h ), bringing h to the forefront of contemporary therapeutic medical gas research. the preventive and therapeutic effects of h have been intensively investigated for various pathological processes. in this review, we summarize the most recently published literature concerning the use of h in respiratory, cardiovascular, nervous, digestive, reproductive, urinary, motor, and sensory system diseases, as well as for the treatment of metabolic syndrome and cancer. we also briefly discuss some known mechanisms underlying the action of h . we hope that this information will increase the understanding of the therapeutic activities of h and inform future h -based therapies. to fully explain the preventive and therapeutic effects of h , biological effects and possible mechanisms are summarized in figures and . . . anti-inflammatory effect of h . the anti-inflammatory effect of h has already been reported in many studies. in the early stage of inflammation, h can reduce the infiltration of neutrophils and m macrophages, and the release of proinflammatory factors by downregulating the expression of intercellular cell adhesion molecule- (icam- ), granulocyte-macrophage colony-stimulating factor (gm-csf), and granulocyte colony-stimulating factor (g-csf) [ ] . h can also inhibit the expression of proinflammatory cytokines during the progress of inflammation and has been revealed in many animal models to decrease the overexpression of early proinflammatory cytokines, such as interleukin-(il-) β, il- , il- , il- , tumor necrosis factor-alpha (tnfα) [ ] , interferon-gamma (inf-γ), and late proinflammatory cytokines, such as high-mobility group box- protein (hmgb ) [ ] . tanaka and colleagues [ ] conducted a gene array analysis of lung grafts from donor rats pretreated with hydrogen ventilation. the authors described that pretreatment with h obviously elevated the expression of two stress-response proteins: heat shock protein a (hspa ) and dual-specificity phosphatase (dusp ). hsp protein nrf- ho- tnf-nf-b inf-fas fasl il- il- il- il- il- encoded by hspa has anti-inflammatory and antiapoptotic properties. dusp can suppress excessive autophagy by inactivating mitogen-activated protein kinases (mapks) to alleviate the inflammatory response. therefore, we speculate that h can positively regulate the expression of stressresponse proteins and improve the anti-inflammatory ability of the organs. another study [ ] demonstrated ventilation with % h in the air significantly reduced the mrna levels of the early growth responsive gene- (egr- ) and chemokine (c-c motif) ligand (ccl ), suggesting that h can affect the progress of inflammation by regulating the transcription of proinflammatory regulatory factors and chemokines. in many diseases, such as airway inflammation and cerebral ischemia, type i hypersensitivity caused by mast cell activation will aggravate the tissue congestion and edema [ , ] . h inhibits the body's inflammatory response by inhibiting th reaction to reduce mast cell activation as well [ ] . h can also regulate inflammation by regulating the physiological pathway of t cells. for example, hydrogen treatment can inhibit the overactivation of the immune system by restoring the loss of regulatory t cells (tregs) [ ] and alleviates inflammation by preventing activation-regulated chemokine-mediated t-cell chemotaxis. the regulation effects of h on programmed cell death, oxidative stress, and mitochondrial function are closely related to the inflammatory response. however, the antiinflammatory effect of hydrogen cannot be overstated. it would be better to be used as a supplementary therapy. many studies have established the antioxidant activity of h . however, recently, a study [ ] has shown that hydrogen can rapidly and slightly increase -hydroxy- ′ -deoxyguanosine ( -ohdg) in urine, a marker for oxidative stress, to a similar level as the increase caused by exercise. exercise-induced generation of reactive oxygen species (ros) is crucial in cell adaptation, and short-term exposure to low levels of ros can protect neurons from oxidative stress that would otherwise be lethal [ ] . h may act as a mitohormetic effector to mediate the beneficial effects on the body through hormetic mechanisms [ ] . the antioxidant effect of h is mainly reflected in several aspects. h was first found to directly eliminate hydroxyl radicals and peroxynitrite. compared with traditional antioxidants, such as superoxide dismutase (sod), catalase, oxidative medicine and cellular longevity and α-tocopherol, h can easily penetrate biofilms and does not affect the normal metabolic redox reaction due to its small molecular weight and antioxidative activity which selectively affects only the strongest oxidant [ ] . h can enhance the expression of the heme oxygenase- (ho- ) antioxidant by activating nuclear factor erythroid -related factor (nrf- ), an upstream regulating molecule of ho- . h can also downregulate ros directly or as a regulator of a gas-mediated signal. further, by upregulating the expression of sod and glutathione (gsh), and downregulating the expression of nadph oxidase (nox ) [ ] , h can significantly reduce ros. another study [ ] showed that h mainly blocks the phosphorylation of ask and its downstream signal molecules p mapk or c-jun n-terminal kinase (jnk), but not the production of ros by nadph oxidase. the effects on the free radical chain reaction of lipid peroxidation may be another important mechanism of hydrogen antioxidation. since otha et al. reported at the th symposium of medical molecular hydrogen at nagoya, japan, in that exposure to a low concentration of hydrogen causes abnormal oxidation of phospholipids [ ] , many studies have established that h can protect cells from lipid and fatty acid peroxidation [ , ] . additionally, h can also reduce the expression of myeloperoxidase (mpo) [ ] or decrease mitochondrial oxidoreductase activity [ ] and stabilize mitochondrial membrane potential [ ] , so as to improve the tissue damage caused by oxidative stress. stress. the accumulation of unfolded protein in the endoplasmic reticulum (er) caused by pathological stress can trigger er stress. zhao et al. [ ] observed that hydrogen inhalation significantly reduced the er stress-related protein and alleviated tissue damage in myocardial i/r injury and later found that a mixture of h and o can block endoplasmic reticulum stress via the pkr-like er-localized eif α kinase-eukaryotic initiation factor alpha-activating transcription factor (perk-eif α-atf ), inositol-requiring enzyme -x-box binding protein (ire -xbp ), and atf pathways. a study of the relationship between h and er stress in rats with i/r injury found that h induced the decrease of grp and tnf receptorassociated factor (traf ) expression [ ] , suggesting that the protective effects of h on myocardial i/r injury are related to decreased er stress. the accumulation of ros can trigger the release of calcium from the er, which results in the depolarization of mitochondria and the loss of the mitochondrial membrane potential [ ] . the negative regulation of ros and the inhibition of programmed cell death by h help maintain the structure and function of mitochondria. it was reported [ ] that hydrogen treatment can block the opening of mitochondrial permeability transition pores in neurons during neurodegenerative disease. however, whether h can indirectly block these pores by reducing the production of ros or acts directly is unclear. early studies [ ] showed that hrs moderated mitochondrial structural damage and simultaneously reduced microrna-(mir-) in hypoxic-reperfusion nerve tissue. however, recent studies have shown that the increase of mir- in injured tissues may be a compensatory action to maintain cell function and reduce ros production [ , ] . whether h can directly inhibit mir- or indirectly reduce it by alleviating inflammation also remains unclear. mitochondrial damage caused by oxidative stress is an important cause of many neurodegenerative diseases. early clinical experiments on parkinson's disease [ ] showed that h can significantly improve neurodegenerative symptoms with a therapeutic effect comparable to that of nonergot dopamine therapy. this cannot be explained by the antioxidative effect of h , and thus, it was suggested that h may target mitochondria to improve the energy metabolism of cells. the observation that h treatment significantly improved the level of sh-sy y atp and Δψm in neuroblastoma [ ] is an indication that h treatment can elevate energy metabolism in mitochondria by activating oxidative phosphorylation. the observations of the conserved structural features shared between hydrogenases and the energy-converting complex i prompted the suggestion that h might serve as both a reductant and oxidant [ ] . the hypothetical function of h in rectifying mitochondrial electron flow can explain the scavenging effect on ros and the ability to slightly improve oxidative stress. . . the effects of hydrogen on the immune system. the main effect of h on the immune system is to reduce the production of immune active substances. evidence suggests that h relieves inflammation in some autoimmune disorders, including rheumatoid arthritis (ra) [ , ] , dermatomyositis [ ] , and psoriasis [ ] . however, whether h directly influences immune cells or organs remains unclear. recent studies have found that h can relieve the dysregulated th /th balance and can influence the number of tregulatory cells (tregs). h was first reported to restore treg loss in a rat model of chronic pancreatitis [ ] and was later proven to increase cd +cd +foxp +treg cells and significantly reduce nasal mucosa damage in animals with allergic rhinitis, which may be secondary to the restoration of th /th balance [ ] . upregulation of tregs has been reported in cerebral i/r models [ ] . this may be caused by the upregulation of tumor necrosis factor-beta (tnf-β ) and downregulation of mir- or mir- . h can also activate peroxisome proliferator-activated receptor-gamma coactivator- alpha (pgc- α) to influence some kinds of t cells [ ] . the specific mechanisms underlying the effects of h on immune cells remain to be defined. upregulates the antiapoptotic factors b-cell lymphoma- (bcl- ) and b-cell lymphoma-extra-large (bcl-xl) [ ] . additionally, terasaki and colleagues reported that h can downregulate the gene expression of proapoptotic bax and inhibit its translocation to mitochondria by an unknown mechanism [ ] . h can also inhibit apoptosis by activating the phosphatidylinositol- -kinase/protein kinase b (pi k/akt) and the janus kinase /signal transducer and activator of transcription (jak -stat ) signaling pathways in rats with myocardial ischemia-reperfusion injury (miri) [ , ] , as well as downregulating the p mapk signaling pathway in rat models with lipopolysaccharide-(lps-) induced acute lung dysfunction [ ] and cerebral ischemia-reperfusion injury (ciri) [ ] . interestingly, wang et al. recently discovered that h inhibited the growth, migration, and invasion of the a and h lung cancer cell lines and promoted cell apoptosis, suggesting that h might play crucial roles in the treatment of lung cancer [ ] . li et al. also revealed the apoptosis-inducing effect of h on kyse- human esophageal squamous cell carcinoma cells [ ] . thus, h may protect normal cells from damage and suppressing cancer cells. although the activation of autophagy can maintain the energy balance of cells through the degradation of macromolecular substances, excessive autophagy or autophagy-related stress triggered by stress stimuli will aggravate the inflammatory damage in tissues and organs. h plays a dual role in the regulation of autophagy. under the regulation of h , autophagy can be activated when protein aggregation becomes toxic and blocked once excessive autophagy causes damage to tissues. zhuang et al. [ ] showed that h treatment downregulated the expression of phosphomammalian target of rapamycin (p-mtor)/mtor and p in lps-treated neuroglial cells and increased the expression of phospho-amp-activated protein kinase (p-ampk)/ampk, light chain (lc ) ii/lc i, triggering receptor expressed on myeloid cells (trem- ), and beclin- to activate autophagy and attenuate neuroinflammation in sepsis. guan et al. [ ] revealed that h can ameliorate chronic intermittent hypoxia-induced kidney injury by decreasing er stress and inducing autophagy by inactivating oxidative stress-dependent p and jnk mapks. h can also inhibit autophagy by downregulating nf-κb, beclin- , and mapk and upregulating the ho- , mtor, and lc b signaling pathways. zhang et al. [ ] found that saturated hydrogen saline alleviated lps-treated lung injury and significantly reduced the expression of autophagy-related proteins, including lc and beclin- (p < : ), suggesting that hydrogen saline can protect lung tissue against excessive autophagy. saturated hydrogen saline can prevent excessive autophagy by eliminating excessive free radicals, reducing the concentration of free radicals in lung tissue, and promoting the expression of mtor. ho- can function as an endogenous cytoprotective protein to assist in the prevention of oxidative stress and excessive cell autophagy. h can increase the tissue expression of ho- by promoting the expression of nuclear erythroid -related factor (nrf ) [ ] . toll-like receptors (tlrs) could be a potential target for h in autophagy regulation. tlr , a key factor in the recog-nition of viral and bacterial factors, can be activated by lps to induce autophagy of macrophages [ ] . the inhibition of an lps-induced inflammatory response by h supports the speculation that tlr may be a potential pathway of hydrogen-induced autophagy. . . . pyroptosis. pyroptosis is an inflammatory programmed cell death pathway that protects multicellular hosts from invasive pathogens, including microbial infections [ ] . human and mouse caspase- , human caspase- and caspase- , and mouse caspase- act as essential activators of pyroptosis. while pyroptosis is normally beneficial for the host, excessive pyroptosis can result in sepsis and septic shock. although there is no experimental data to explain the relationship between h and the pyroptosis pathway, it is conceivable that the regulation of some inflammatory factors and nuclear factors by h will interfere with the triggering of pyroptosis, or at least reduce pyroptosis-related inflammation. in one study [ ] , inhalation of % h reduced the expression of caspase- , a key factor for pyroptosis activation. physical rupture of cells caused by pyroptosis leads to the release of the proinflammatory cytokines il- β and il- , while hydrogen pretreatment can significantly reduce the level of these cytokines [ ] . h has also been shown to regulate the expression of atg , which inhibits pyroptosis [ ] . it has been proposed that hmgb [ ] and ifn-γ [ ] are necessary for caspase- -dependent pyroptosis activation. the negative effect of h on the expression of these two factors may protect cells from pyroptosis. h is able to block the expression of caspase- [ ] , which serves both as the activator of apoptosis, and also blocks pyroptosis by cleaving gasdermin d [ ] . bidirectional crosstalk exists between the caspase- produced in apoptosis and the caspase in pyroptosis. the mechanism of this crosstalk remains unclear. human immunodeficiency virus (hiv) can accelerate the depletion of cd + t cells via interferon-gamma inducible protein -(ifi -) triggered pyroptosis [ ] . thus, the regulation of pyroptosis by h may be a potential mechanism to treat hiv affection. ferroptosis is a form of regulated cell death proposed by dixon et al. [ ] in . ferroptosis is accompanied by a lethal iron-dependent accumulation of lipid hydroperoxides. although laboratory verification has not been forthcoming, we can still speculate that hydrogen can interfere with ferroptosis to alleviate inflammatory necrosis of tissues and organs in the pathological state, given the great deal of overlap between hydrogen regulation and ferroptosis pathways. it has been well-established that hydrogen has a negative regulatory effect on ros. we speculate that the most critical redox imbalance in the process of ferroptosis will be eliminated by hydrogen; thus, ferroptosis will be blocked. in addition, h is able to block mapk pathways, which are to prevent the depletion of reducing substances caused by iron ions and ros [ ] . a recent study [ ] showed that hmgb , which can be downregulated by hydrogen [ ] , can act as a positive regulator of ferroptosis via the ras-jnk/p pathway. oxidative medicine and cellular longevity ho- activity can be increased by hydrogen. ho- is a potential source of intracellular iron, and a recent study [ ] demonstrated that ho- -deficient renal epithelial cells were more sensitive to ferroptosis, indicating that free iron produced by ho- does not promote ferroptosis itself, and that ho- has an antiferroptotic effect. however, the effects of hydrogen on ferroptosis may not always be inhibitory. for example, mir- , an inflammatory mirna that is downregulated by hydrogen, can reduce the occurrence of ferroptosis [ ] . the mechanism underlying the action of hydrogen on ferroptosis is yet to be fully clarified. some of the anti-inflammatory and antioxidation mechanisms of hydrogen are similar to those of gpx [ ] . both molecules have negative effects on the formation of lipid peroxide and nf-κb. the combination of hydrogen and gpx activator may provide a new solution for the treatment of inflammation and other lipid peroxidationmediated diseases. . . . circadian clock. the circadian clock refers to an endogenous oscillator that controls h physiological, metabolic, and behavioral processes. this clock is particularly crucial in maintaining homeostasis [ ] . intestinal microbiota, which regularly produce hydrogen gas in the process of the energy-producing anaerobic fermentation [ ] , undergo diurnal oscillations in composition and function [ ] . in humans, the amount of hydrogen produced varies depending on the individual and the time of the day. wilking et al. suggested that the circadian regulation of protein expression plays an important role in the cellular response to oxidative stress; they concluded that levels of byproducts of oxidative stress, such as protein damage, or lipid peroxidation, also oscillate with circadian rhythmicity, indicating circadian oscillations of oxidative stress responses. thus, this rhythmicity of antioxidant levels can be exploited for a more precise targeting of ros to offer better protection for the cells [ ] . as the antioxidant activity of h has been widely verified, we suggest that h may exert a negative regulatory effect on ros by regulating circadian rhythm, but there is yet no evidence regarding how h is involved in the regulation of circadian rhythm. applications of h h has preventive and therapeutic effects on different system diseases ( due to its small molecular weight, hydrogen in the inhaled gas mixture can reduce airway resistance, increase oxygen dispersion, and increase oxygen flow. covid- can provoke an inflammatory storm by excessively activating the immune system, causing severe inflammatory damage to the lungs and extrapulmonary tissue, which is also the main cause of death [ ] . a study involving patients with ncp showed that patients in the intensive care unit displayed a significantly higher level of inflammatory factors that included il- , il- , il- , and tnf-α and that most of these factors could be downregulated by hydrogen [ ] . we speculate that the application of hydrogen may reduce the risk of inflammatory storm and thus prevent severe effects. many ncp patients need ventilator-assisted therapy. inflammatory changes in the respiratory system make lung tissue prone to ventilatorinduced lung injury (vili), even with low tidal volume [ ] . ventilation with % h was proved to be able to downregulate the mrnas for proinflammatory mediators such as tnf-α, il- β, egr- , and ccl and induced antiapoptotic genes including bcl- and bcl-xl. it is consistent with the histopathological results which showed that inflammatory cell infiltration and bronchial epithelial apoptosis were improved in vili mice after h treatment. h has a potential to protect human lung tissues from vili as well via its anti-inflammatory, antioxidant, and antiapoptotic effects [ ] . moreover, hydrogen can also increase surfactant proteins to prevent further lung injury [ ] . one study reported that chest computed tomography scans performed at the early stage in patients that ultimately developed in severe infection revealed multiple small flake shadows and interstitial changes, suggesting that pulmonary fibrosis affects the prognosis of the disease. hrs has been reported to reverse epithelial-mesenchymal transition (emt) and prevent pulmonary fibrosis by inhibiting oxidative stress and increasing the expression of e-cadherin [ ] . hydrogen also has the potential to protect lung tissues from pulmonary hypertension [ ] , sepsis [ ] , smoke inhalation injury [ ] , hemorrhagic shock and resuscitation [ ] , and other toxic substances/events. in animal models, hydrogen also affects asthma and chronic obstructive pulmonary disease [ ] . system. myriad forms of irreversible damage that occur in nervous system diseases are often caused by neuroinflammation, excessive oxidative stress, mitochondrial dysfunction, and cell death. the therapeutic effects of hydrogen on the nervous system have been verified in animal models and clinical trials. hydrogen can reportedly reduce the loss of dopaminergic neurons and can improve nigrostriatal degeneration a mouse model of parkinson's disease (pd) following treatment with -hydroxydopamine [ ] and methyl- -phenyl- , , , -tetrahydropyridine [ ] . a recent oxidative medicine and cellular longevity clinical trial showed that hw can improve the total unified parkinson's disease rating scale score of pd [ ] . early clinical trials revealed that hydrogen improves pd through antioxidation. more recent research found that hydrogen may slightly increase oxidative stress or act as the rectifier of the mitochondrial electron flow and improve pd by regulating mitochondrial energy [ ] or via hormetic mechanisms. by hormetic mechanisms, h will simulate strenuous exercise to generate a mild increase of ros which will evoke hormesis and then activate the nrf , nf-κb pathways, and heat shock responses to protect neurons and other tissues [ ] . hydrogen also improved cognitive function in alzheimer's patients in a clinical trial [ ] . the collective findings indicate the safety and effectiveness of hydrogen in the treatment of neurodegenerative diseases. hydrogen has also been shown to protect the nervous system of a fetus or newborn. in two case studies, pregnant women who had become infected with covid- displayed a high delivery rate of fetuses with intrauterine distress leading to a higher incidence of perinatal hypoxic-ischemic brain damage [ , ] . the findings indicate the necessity of measures to prevent neonatal encephalopathy or reduce neonatal neurological deficit for pregnant women infected with covid- . h can inhibit the activation of proinflammatory cytokines, microglia [ ] , and -hydroxy dehydrogenase ( -ohdg) [ ] to reduce oxidative damage and neuroinflam-mation in the fetal brain in animal models. the protective effects have been verified by clinical trials. hippocampal damage caused by i/r injury in the uterus on day after birth was reportedly improved for pregnant women who had been treated with hw and was associated with reductions of hydroxy ketone and -ohdg [ ] . the available data support the possibility that hydrogen therapy could protect fetuses of pregnant women infected with covid- . in addition, hydrogen can also protect against spinal cord injury and a variety of brain injuries caused by ischemia, hypoxia, trauma, and hemorrhage. a clinical study of patients suffering from cerebral infarction reported that hydrogen inhalation improved imaging results, national institutes of health stroke scale scores used to quantify the severity of stroke, and physical therapy evaluations based on the barthel index [ ] . a rat model of subarachnoid hemorrhage revealed the influence of hydrogen in lessening neurological deficits [ ] and endothelial cell injury [ ] . in rats, hydrogen can also relieve neuropathic pain [ ] after spinal cord injury and can ameliorate neurotoxicity [ ] . with the acceleration of urbanization and aging of global societies, the risk of cardiovascular diseases (cvd) has increased. the world health organization ranks cvd as the leading cause [ ] sensory system reduces wound area and levels of proinflammatory cytokines [ ] improves auditory brainstem response [ ] metabolic syndrome decreases ldl and increases high-density lipoprotein [ ] decreases glucose and insulin levels [ ] stimulates energy metabolism [ ] cancer controls cancer progression and improves quality-of-life [ ] reduces proportion of terminal pd- + cd + t cells [ ] oxidative medicine and cellular longevity of death globally, accounting for . million deaths annually. two of every five deaths in china are attributed to cvd, which exceeds the death rate due to cancer or other diseases [ ] . however, most clinical trials to date have failed to effectively prevent and treat cvd. thus, novel therapies are urgently required. during the past decade, many basic and clinical studies have provided evidence supporting the view that h treatment protects against cvd and improves cardiac function. ohsawa et al. discovered that consumption of hw for months reduced the oxidative stress level and the volume of atherosclerotic lesions derived from macrophage accumulation in apolipoprotein e knockout mice (apoe-/-mice) [ ] . iketani et al. recently revealed that continuous administration of hw in low-density lipoprotein (ldl) receptordeficient mice decreased the numbers of endothelial cells in the atheroma expressing the senescence factors p ink a and p , as well as suppressing macrophage infiltration and tnf-α expression in the atheroma, suggesting that vascular aging can be suppressed by hw [ ] . another study demonstrated increased flow-mediated dilation in the high-h group in which eight males and females drank hw containing ppm h , indicating that h may protect the vasculature from shear stress-derived ros that is detrimental [ ] . in addition to treating atherosclerosis, h reduces miri, which refers to a heart lesion that develops upon the resumption of the flow of oxygen-rich blood after a period of ischemia and which usually occurs during acute myocardial infarction or open-heart surgery [ , ] . a recent series of studies by li et al. found that hw inhibited cardiomyocyte apoptosis by activating the pi k/akt and jak -stat signaling pathways and can also reduce the level of oxidative stress in myocardial tissue by upregulating the expression of the nuclear erythroid -related factor /antioxidant response element (nrf /are) signaling pathway, which alleviated i/r injury in isolated rat hearts [ , , ] . other studies demonstrated that intraperitoneal injection of hw before reperfusion significantly decreased the concentration of malondialdehyde (mda) and infarct size, as well as reducing myocardial -ohdg and the levels of tnf-α and il- β in an area at risk zones [ , ] . moreover, qian et al. described the hydrogen-mediated protection of myocardium degeneration due to radiation-induced injury in rats [ ] . treatment with hrs was shown to ameliorate vascular dysfunction, including aortic hypertrophy and endothelial function, in spontaneously hypertensive rats by abating oxidative stress, restoring baroreflex function, preserving mitochondrial function, and enhancing nitric oxide bioavailability [ ] . in another study, the intraperitoneal administration of hrs improved hemodynamics and reversed right ventricular hypertrophy in male sprague-dawley rats with pulmonary hypertension induced by monocrotaline [ ] . h inhalation is also a favorable strategy to mitigate mortality and functional outcome of postcardiac arrest syndrome in a rat model [ ] . these collective findings indicate the potential of h in novel therapeutic approaches for the clinical treatment of cvd. diseases. hepatic ischemia-reperfusion injury (hiri) is common in liver sur-gery and liver transplantation. h treatment ameliorated hiri in a mouse fatty liver model by reducing hepatocyte apoptosis, inhibiting macrophage activation and inflammatory cytokines, and inducing ho- and sirt expression [ ] . inhalation of high concentrations of hydrogen significantly improved liver function in a mouse hiri model by activating the a a receptor-mediated pi k-akt pathway [ ] . a recent series of studies also discovered that portal vein injection of hrs in miniature pigs with laparoscopic hiri promoted liver function recovery and liver regeneration by reducing hepatocyte autophagy and apoptosis and inhibited er stress, with significant hepatoprotective effects observed [ ] [ ] [ ] [ ] . hydrogen flush after cold storage refers to an end-ischemic h flush directly to donor organs ex vivo. this approach can significantly protect liver grafts from iri [ ] , providing a potentially effective strategy for organ preservation. other studies demonstrated the protective effects of h in liver damage induced by parasites [ ] , obstructive jaundice [ ] , shock and resuscitation [ ] , sepsis [ ] , doxorubicin [ ] , and aflatoxin b [ ] . in a mouse model of nonalcoholic fatty liver disease (nafld), hw downregulated nrf -mediated mir- expression by targeting the maternally expressed long noncoding rna gene [ ] , providing a rationale for further clinical trials. in a human study, hw also significantly reduced liver fat accumulation in twelve overweight outpatients with nafld [ ] . another in vivo study revealed that oral hw significantly attenuated oxidative stress in patients with chronic hepatitis b [ ] . in recent years, it has become widely accepted that bile acids are a nutrient signaling hormone [ ] . molecular hydrogen was recently demonstrated to participate in the regulation of bile acid metabolism, particularly in the inhibition of bile acid oxidation, in some gut bacteria [ ] . intestinal i/r injury is a multifactorial pathophysiological process with high morbidity and mortality. i/r injury occurs in a variety of clinical settings that include major cardiovascular surgery, trauma, shock, and small intestinal transplantation [ ] . yao et al. recently observed that intraperitoneal injection of h attenuated i/r-induced mucosal injury and apoptosis of epithelial cells in mice by regulating mirnas, in particular by regulating mir- a- p [ ] . furthermore, hw reportedly inhibited intestinal i/r-induced oxidative stress, apoptosis, and inflammation and promoted epithelial cell proliferation in rats, which protected against intestinal contractile dysfunction and damage induced by intestinal i/r [ ] [ ] [ ] . most gastrointestinal microbial species encode the genetic capacity to metabolize h , meaning that h might affect the gut bacterial composition [ ] , and bacterial translocation is an important cause of multiple organ dysfunction syndromes in critical illness. ikeda et al. described that the luminal administration of hw prevented intestinal dysbiosis, hyperpermeability, and bacterial translocation in a murine model of sepsis [ ] . in another study, the inhalation of % h also attenuated intestinal injuries caused by severe sepsis in male nrf ko mice by regulating ho- and hmgb release [ ] . moreover, an in vivo study revealed that h inhalation improved the prognosis in patients with stage iv colorectal cancer by activating pgc- α and restoring exhausted cd + t cells [ ] . oxidative medicine and cellular longevity wang et al. interestingly discovered that cytotoxinassociated gene a cytotoxin, a virulence factor of helicobacter pylori that augments the risk of gastric cancer, can be delivered into host cells by the h -utilizing respiratory chain of the bacterium, extending the roles of h oxidation to include transport of a carcinogenic toxin [ ] . although this study indicated that h may play a role in increasing gastric cancer risk, abundant studies also demonstrated that h is protective in gastric damage induced by oxidative stress [ ] and aspirin [ ] . franceschelli et al. found that electrolyzed reduced water, which is rich in molecular hydrogen, rapidly improved symptoms in patients with gastroesophageal reflux disease [ ] . h treatment also controlled the severity of chronic pancreatitis [ ] and acute necrotizing pancreatitis [ ] . yang et al. recently demonstrated using a mouse model of human endometrial tumor xenograft that hw has an antitumor effect that was sufficient to inhibit xenograft volume and weight of endometrial tumors via the ros/nlrp /caspase- /gsdmd-mediated pyroptotic pathway, indicating a biphasic effect of h on cancer that involves the promotion of tumor cell death and protection of normal cells [ ] . other authors reported that h inhalation reduced the size of the endometrial explants, inhibited cell proliferation, improved sod, and regulated the expression of matrix metalloproteinase and cyclooxygenase in an endometriosis rat model [ ] . hrs is effective in attenuating ovary injury induced by i/r [ ] and cisplatin [ ] . hw improved serum anti-müllerian hormone levels and reduced ovarian granulosa cell apoptosis in a mouse immune premature ovarian failure model induced by zona pellucida glycoprotein [ ] . in a hemisectioned spinal cord injury rat model, hrs inhibited the injury-induced ultrastructural changes in gonadotrophs, ameliorated the abnormal regulation of the hypothalamicpituitary-testis axis, and thereby promoted the recovery of testicular injury [ ] . in irradiated rats, hrs improved testis weight, testis dimensions, sperm count, sperm motility, and serum testosterone levels [ ] . hw stimulated spermatogenesis as well as increased sperm production and sperm motility in mice of different ages [ ] . based on previous studies, begum et al. hypothesized that h may modulate intracellular mapk camp and ca + signals involved in testosterone hormone production to improve male fertility caused by redox imbalance [ ] . finally, h decreased the percentage of sperm abnormalities and improved sperm morphology following the prolonged exposure of mouse low doses of radiation [ ] . the collective data indicate that hydrogen can protect both female and male fertility. . . effects of hydrogen on urinary system diseases. acute kidney injury (aki) is an important risk factor for the development of chronic kidney disease. wu et al. recently found that saturated hydrogen alleviates ccl -induced aki via jak /stat /p signaling [ ] . inhalation of a hydrogen-rich aerosol appears to be very useful for renal protection and inflammation reduction in septic aki, based on the observations of increased anti-inflammatory cytokine (il- and il- ) mrna levels in renal tissues and increased macrophage polarization to the m type, which generates additional anti-inflammatory cytokines (il- and transforming growth factor-beta, tgf-β) [ ] . additionally, h can alleviate aki induced by i/r [ ] , liver transplantation [ ] , burns [ ] , and sodium taurocholate-induced acute pancreatitis [ ] . recently, lu et al. demonstrated that hw can restore a balanced redox status and alleviate cyclosporine a-induced nephrotoxicity by activating the keap /nrf signaling pathway [ ] . h can ameliorate kidney injury induced by chronic intermittent hypoxia by decreasing er stress and activating autophagy through the inhibition of oxidative stress-dependent p and jnk mapk activation [ ] . hw also reportedly can inhibit the development of renal fibrosis and prevent hk- cells from undergoing emt mediated through sirt , a downstream molecule of tgf-β . hrs markedly reduced interstitial congestion, edema, and hemorrhage in renal tissue, prevented renal injury, and promoted renal function recovery after i/r injury in rats through antiapoptotic and antiinflammatory actions in kidney cells [ ] . other authors described that hw significantly reduced the increased postvoid residual volume in obstructed rats and ameliorated bladder dysfunction secondary to bladder outlet obstruction by attenuating oxidative stress [ ] . metabolic syndrome is associated with excess calorie intake and encompasses a range of medical conditions that include obesity, insulin resistance, and dyslipidemia. many studies have demonstrated the protective effects of h in metabolic syndrome. qiu et al. reported that saturated hydrogen decreased total cholesterol, total glyceride, and ldl, increased highdensity lipoprotein in the peripheral blood, and alleviated the activity of isocitrate lyase, suggesting that h could improve lipid metabolism disorders by inhibiting the glyoxylic acid cycle [ ] . glucose and insulin levels in the serum were also significantly lower in h -treated mice, which markedly improved type diabetes mellitus and diabetic nephropathy-related outcomes [ ] . moreover, gut-derived hydrogen production induced by l-arabinose reportedly had beneficial effects on metabolic syndrome in c bl/ j mice fed a high-fat diet [ ] and reduced oxidative stress and the peripheral blood il- β mrna level in sixteen type diabetic patients [ ] . h treatment has also shown positive effects on energy metabolism. in , kamimura et al. reported that prolonged consumption of hw significantly controlled fat and body weights in db/db obese mice by stimulating energy metabolism [ ] . a recent study revealed that h attenuated allergic inflammation in a mouse model of allergic airway inflammation by reversing an energy metabolic pathway switch from oxidative phosphorylation to aerobic glycolysis [ ] . oxidative medicine and cellular longevity the serum -ohdg levels in h -treated race horses were significantly suppressed, strongly suggesting a protective effect of h in exercise-induced, ros-mediated detrimental tissue damage [ ] . additionally, hydrogen bathing attenuated exercise-induced muscle damage and delayed-onset muscle soreness but had no effects on the peripheral neutrophil count and both dynamics and functions of neutrophils [ ] . these findings highlight that further studies are needed to clarify the mechanisms of h . inhalation of h significantly decreased infarct zone and area with loss of tissue structure, attenuated muscle damage, and enhanced functional recovery in a mouse hindlimb i/r injury model [ ] . finally, hasegawa et al. revealed that h improved muscular dystrophy in the mdx mouse model for duchenne muscular dystrophy [ ] . . . effects of hydrogen on sensory system diseases. hydrogen has a therapeutic role in alleviating the damage to some sensory organs, mainly through antioxidation. hydrogen promotes wound healing in tissue or protective barriers including skin and mucosa. for example, preinhalation of hydrogen-containing gas decreased wound healing time in a rat model of radiation-induced skin injury [ ] . other authors reported that h inhalation reduced the wound area and levels of proinflammatory cytokines in pressure ulcers [ ] . moreover, hydrogen can improve skin lesions in some immune disorders by interfering with the immune system or ros removal [ ] . hw also benefits the wound healing process of the oral palate. hydrogen also may protect hearing and vision. kurioka et al. [ ] demonstrated that hydrogen inhalation significantly reduced outer hair cell loss and improved auditory brainstem response after noise exposure, indicating a protective effect for noise-induced hearing loss. hydrogen has been shown to be effective in treating cornea injury caused by alkali [ ] , fluoride, chloropicrin [ ] , and ultraviolet b radiation [ ] . . . effects of hydrogen on cancer. many animal models have established the efficacy of hydrogen against cancers. the attributes of hydrogen include blocking of the regulator for chromosome condensation [ ] , some crucial molecules in stemness [ ] , proliferation [ ] , and angiogenesis [ ] , and the alleviation of oxidative stress. the combination therapy of hydrogen and other novel antineoplastic drugs, such as ly [ ] , which is an inhibitor of pi k, has demonstrated great potential and efficacy. an increasing number of clinical trials are being carried out. a recent survey [ ] on advanced cancer patients exemplified that hydrogen can control cancer progression and improve the quality-of-life. akagi [ ] treated stage iv colorectal carcinoma patients using hydrogen inhalation and documented enhanced mitochondrial activity due to the activation of pgc- α to reduce the proportion of terminal pd- + cd + t cells. the depletion of these cells is associated with improved cancer prognosis [ , ] . this therapeutic effect has also been confirmed in another trial conducted in one patient with metastatic gallbladder cancer [ ] . in a case report in , hydrogen gas therapy resulted in the disappearance of the metastatic brain tumors in a woman diagnosed with lung cancer [ ] . finally, hydrogen can also reduce the side effects of cisplatin [ ] and radiotherapy [ ] . though growing evidences have shown the effects of h on alleviating both cancer progression and side effects of chemotherapeutics, the h therapy applied in cancer is just in a nascent stage. at present, the published researches on the anticancer effects of h mainly focus on lung cancer [ ] , colorectal cancer [ ] , and glioma [ ] . it remains unclear how many cancers can effectively be alleviated by h and how many can not be. at present, patients with covid- pneumonia are usually treated with high flow pure oxygen (without adding h ), although the effect of o when associated with h may give better results [ ] . the production of mucus in these patients reduces the absorption of o , while with a mixture of o and h , the bronchioles and the alveoli of lungs are further expanded, optimizing the absorption of o [ ] . h is used as a catalyst to accelerate the binding of hemoglobin with o and the release of hemoglobin with carbon dioxide [ ] . hydrogen has great potential in the regulation of oxidative stress, inflammation, energy metabolism of organelles, and programmed cell death. many animal experiments and clinical trials have established the protective effects of hydrogen on many organs and systems. research in this area has increased over the past years. however, the details of the specific molecular mechanisms of the therapeutic effects of hydrogen remain unclear. for example, whether hydrogen can truly be used to regulate ferroptosis, pyroptosis, or the circadian clock is not known. since h is not something like rapamycin or leucine only going to have one direction (opposite) effects on autophagy, is it possible to regulate autophagy or apoptosis in a specific direction? previous studies have clearly explained the antioxidative stress effect of hydrogen. however, some recent clinical trials have shown that h can induce oxidative stress in some cases as well. ventilation with h can induce a mild increase of ros to activate the nrf , nf-κb pathways, and heat shock responses. h -induced ros production can also be observed in cancer cells. the specific mechanism underlying the hydrogen-induced increase of oxidative stress should be explained by more experiments. these and other questions concerning the mechanism of hydrogen should be further explored. there are many factors that limit the clinical use of hydrogen. firstly, hydrogen is considered unsafe at concentrations above % because such a high level of h is explosive and might bring cytotoxic effects. previous studies have indicated that the concentration of hydrogen should be stabilized beyond % to enable protection from acute oxidative stress. however, even % of hydrogen is not absolutely safe. most clinical ventilators are equipped with platinum hot manometers, because h and o can overheat the platinum surface at room temperature. secondly, there is a lack of specialized devices that enable the administration of effective hydrogen concentrations, while ensuring that they are safe. thirdly, oxidative medicine and cellular longevity there have been few large-scale controlled human studies on the effects of hydrogen. fourthly, liu and his colleagues demonstrated that the inhalation of h resulted in a slower elevation of the h concentration than that achieved with intraperitoneal, intravenous, or oral administration. however, the elevated h concentrations were maintained for at least minutes after inhalation. thus, it should be deliberated to choose the administration of h [ ] . as a result, the dose-specific effects or side effects of hydrogen in humans remain unclear. the data regarding the known mechanisms underlying the action of hydrogen indicate that hydrogen can alleviate the damage in multiple organs in ncp patients. the comparisons of the different modalities of hydrogen indicate the value of hw in the effective treatment of such patients. hydrogen is inexpensive and safe and can be administered through many ways. we anticipate that as large-scale clinical trials confirm the therapeutic efficacy and safety of hydrogen, its full clinical potential will be realized. novel coronavirus pneumonia h : hydrogen i/r: ischemia/reperfusion hrs: h -rich saline hw: h -rich water ros: reactive oxygen species er: endoplasmic reticulum emt: epithelial-mesenchymal transition cvd: cardiovascular diseases nafld: nonalcoholic fatty liver disease aki: acute kidney injury mda: malondialdehyde nrf : nuclear erythroid -related factor pgc- α: peroxisome proliferator-activated receptorgamma coactivator- alpha. the authors declare no conflict of interest. psychophysiological reactions in humans during an open sea dive to m with a hydrogenhelium-oxygen mixture hyperbaric hydrogen therapy: a possible treatment for cancer hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals hydrogen protects lung from hypoxia/re-oxygenation injury by reducing hydroxyl radical production and inhibiting inflammatory responses effect of shikonin on spinal cord injury in rats via regulation of hmgb /tlr /nf-kb signaling pathway hydrogen-rich saline attenuates cardiac and hepatic injury in doxorubicin rat model by inhibiting inflammation and apoptosis estimation of the hydrogen concentration in rat tissue using an airtight tube following the administration of hydrogen via various routes combined early fluid resuscitation and hydrogen inhalation attenuates lung and intestine injury hydrogen gas reduces hmgb release in lung tissues of septic mice in an nrf /ho- -dependent pathway profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement hydrogen inhalation ameliorates ventilator-induced lung injury hydrogen inhalation ameliorated mast cell-mediated brain injury after intracerebral hemorrhage in mice allergic airway inflammation induces migration of mast cell populations into the mouse airway hydrogen water ameliorates the severity of atopic dermatitis-like lesions and decreases interleukin- β, interleukin- , and mast cell infiltration in nc/nga mice hydrogen treatment protects mice against chronic pancreatitis by restoring regulatory t cells loss inhalation of hydrogen gas elevates urinary -hydroxy- ′-deoxyguanine in parkinson's disease consumption of hydrogen water reduces paraquat-induced acute lung injury in rats hydrogen and oxygen mixture to improve cardiac dysfunction and myocardial pathological changes induced by intermittent hypoxia in rats molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases beneficial biological effects and the underlying mechanisms of molecular hydrogen -comprehensive review of original articles molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction hydrogen inhalation protects against acute lung injury induced by hemorrhagic shock and resuscitation hydrogen gas inhalation attenuates seawater instillation-induced acute lung injury via the nrf pathway in rabbits therapeutic efficacy of molecular hydrogen: a new mechanistic insight hydrogen gas attenuates myocardial ischemia reperfusion injury independent of postconditioning in rats by attenuating endoplasmic reticulum stress-induced autophagy propofol inhibits parthanatos via ros-er-calcium-mitochondria signal pathway in vivo and vitro inhalation of hydrogen of different concentrations ameliorates spinal cord injury in mice by protecting spinal cord neurons from apoptosis, oxidative injury and mitochondrial structure damages neuroprotective effect of hydrogen-rich saline in global cerebral ischemia/reperfusion rats: up-regulated tregs and down-regulated mir- , mir- and nf-κb expression loading mir- in endothelial progenitor cells derived exosomes boosts their beneficial effects on hypoxia/reoxygeneation-injured human endothelial cells via protecting mitochondrial function microrna- is upregulated in hypoxic cardiomyocytes through akt-and p -dependent pathways and exerts cytoprotective effects pilot study of h therapy in parkinson's disease: a randomized double-blind placebo-controlled trial molecular hydrogen protects against oxidative stress-induced sh-sy y neuroblastoma cell death through the process of mitohormesis molecular hydrogen decelerates rheumatoid arthritis progression through inhibition of oxidative stress molecular hydrogen: new antioxidant and antiinflammatory therapy for rheumatoid arthritis and related diseases open-label trial and randomized, double-blind, placebocontrolled, crossover trial of hydrogen-enriched water for mitochondrial and inflammatory myopathies positive effects of hydrogen-water bathing in patients of psoriasis and parapsoriasis en plaques hydrogen-rich saline ameliorates allergic rhinitis by reversing the imbalance of th /th and up-regulation of cd +cd +foxp +regulatory t cells, interleukin- , and membrane-bound transforming growth factor-β in guinea pigs immunological effect of hydrogen gas-hydrogen gas improves clinical outcomes of cancer patients hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress effects of hydrogen-rich water on the pi k/akt signaling pathway in rats with myocardial oxidative medicine and cellular longevity ischemia-reperfusion injury attenuation of cardiac ischaemiareperfusion injury by treatment with hydrogen-rich water saturated hydrogen saline attenuates endotoxin-induced lung dysfunction hydrogen saline suppresses neuronal cell apoptosis and inhibits the p mitogen-activated protein kinase-caspase- signaling pathway following cerebral ischemia-reperfusion injury hydrogen gas inhibits lung cancer progression through targeting smc influence of hydrogenoccluding-silica on migration and apoptosis in human esophageal cells in vitro molecular hydrogen attenuates sepsis-induced neuroinflammation through regulation of microglia polarization through an mtor-autophagydependent pathway hydrogen protects against chronic intermittent hypoxia induced renal dysfunction by promoting autophagy and alleviating apoptosis effects of hydrogen rich water on the expression of nrf and the oxidative stress in rats with traumatic brain injury tlr mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases hydrogen gas inhalation attenuates sepsis-induced liver injury in a fundc -dependent manner h protects against lipopolysaccharide-induced cardiac dysfunction via blocking tlr -mediated cytokines expression atg deficiency intensifies inflammasome activation and pyroptosis in pseudomonas sepsis the endotoxin delivery protein hmgb mediates caspase- -dependent lethality in sepsis hydrogen-rich saline attenuates isoflurane-induced caspase- activation and cognitive impairment via inhibition of isoflurane-induced oxidative stress, mitochondrial dysfunction, and reduction in atp levels pyroptosis and apoptosis pathways engage in bidirectional crosstalk in monocytes and macrophages ferroptosis: an iron-dependent form of nonapoptotic cell death disulfiram/copper induces antitumor activity against both nasopharyngeal cancer cells and cancer-associated fibroblasts through ros/mapk and ferroptosis pathways hmgb regulates erastininduced ferroptosis via ras-jnk/p signaling in hl- /nras q l cells heme oxygenase- mitigates ferroptosis in renal proximal tubule cells activation of glutathione peroxidase as a novel anti-inflammatory strategy genomics of circadian rhythms in health and disease microbiota diurnal rhythmicity programs host transcriptome oscillations the clinical application of hydrogen as a medical treatment circadian rhythm connections to oxidative stress: implications for human health an overview of sars-cov- (covid- ) infection and the importance of molecular hydrogen as an adjunctive therapy hydrogen/oxygen mixed gas inhalation improves disease severity and dyspnea in patients with coronavirus disease in a recent multicenter, open-label clinical trial clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical features of patients infected with novel coronavirus in wuhan, china regional physiology of ards protective effects of hydrogen-rich saline against lipopolysaccharide-induced alveolar epithelial-to-mesenchymal transition and pulmonary fibrosis oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats saturated hydrogen saline ameliorates lipopolysaccharide-induced acute lung injury by reducing excessive autophagy protective effects of hydrogen-rich saline on rats with smoke inhalation injury hydrogen gas inhalation ameliorates lung injury after hemorrhagic shock and resuscitation hydrogen-rich saline inhibits tobacco smoke-induced chronic obstructive pulmonary disease by alleviating airway inflammation and mucus hypersecretion in rats molecular hydrogen is protective against -hydroxydopamine-induced nigrostriatal degeneration in a rat model of parkinson's disease hydrogen in drinking water reduces dopaminergic neuronal loss in the -methyl- -phenyl- , , , -tetrahydropyridine mouse model of parkinson's disease effects of molecular hydrogen assessed by an animal model and a randomized clinical study on mild cognitive impairment clinical characteristics and intrauterine vertical transmission potential of covid- infection in nine pregnant women: a retrospective review of medical records clinical analysis of neonates born to mothers with -ncov pneumonia administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model molecular hydrogen affords neuroprotection in a translational piglet model of hypoxic-ischemic encephalopathy maternal molecular hydrogen administration ameliorates rat fetal hippocampal damage caused by in utero ischemia-reperfusion hydrogen gas inhalation treatment in acute cerebral infarction: a randomized controlled clinical study on safety and neuroprotection hydrogen gas therapy improves survival rate and neurological deficits in subarachnoid hemorrhage rats: a pilot study hydrogen inhalation attenuates oxidative stress related endothelial cells injury after subarachnoid hemorrhage in rats hydrogen-rich saline alleviated the hyperpathia and microglia activation via autophagy mediated inflammasome inactivation in neuropathic pain rats china cardiovascular diseases report : an updated summary consumption of hydrogen water prevents atherosclerosis in apolipoprotein e knockout mice administration of hydrogen-rich water prevents vascular aging of the aorta in ldl receptor-deficient mice consumption of water containing over . mg of dissolved hydrogen could improve vascular endothelial function mitochondrial aldehyde dehydrogenase in myocardial ischemic and ischemiareperfusion injury effect of hydrogen-rich water on the nrf /are signaling pathway in rats with myocardial ischemia-reperfusion injury antiinflammatory effect of hydrogen-rich saline in a rat model of regional myocardial ischemia and reperfusion hydrogen-rich saline protects myocardium against ischemia/reperfusion injury in rats the potential cardioprotective effects of hydrogen in irradiated mice chronic hydrogen-rich saline treatment attenuates vascular dysfunction in spontaneous hypertensive rats protective effects of hydrogen-rich saline on monocrotaline-induced pulmonary hypertension in a rat model h gas improves functional outcome after cardiac arrest to an extent comparable to therapeutic hypothermia in a rat model the clinical pathology of severe acute respiratory syndrome (sars): a report from china prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis covid- and cardiovascular disease cardiovascular considerations for patients, health care workers, and health systems during the covid- pandemic prevalence and impact of cardiovascular metabolic diseases on covid- in china molecular hydrogen protects against ischemia-reperfusion injury in a mouse fatty liver model via regulating ho- and sirt expression inhalation of high concentrations of hydrogen ameliorates liver ischemia/reperfusion injury through a a receptor mediated pi k-akt pathway comparative study on protective effect of hydrogen rich saline and adipose-derived stem cells on hepatic ischemia-reperfusion and hepatectomy injury in swine effect of hydrogen-rich saline on apoptosis induced by hepatic ischemia reperfusion upon laparoscopic hepatectomy in miniature pigs influence of hydrogen-rich saline on hepatocyte autophagy during laparoscopic liver ischaemia-reperfusion combined resection injury in miniature pigs hydrogen-rich saline protects against small-scale liver ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress hydrogen flush after cold storage as a new end-ischemic ex vivo treatment for liver grafts against ischemia/reperfusion injury anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver inflammation hydrogen-rich saline protects against liver injury in rats with obstructive jaundice hyperoxygenated hydrogenrich solution suppresses shock-and resuscitation-induced liver injury preadministration of hydrogen-rich water protects against lipopolysaccharide-induced sepsis and attenuates liver injury anti-injury effect of hydrogen-enriched water in a rat model of liver injury induced by aflatoxin b high-content hydrogen waterinduced downregulation of mir- alleviates non-alcoholic fatty liver disease by regulating nrf via targeting meg hydrogen-rich water reduces liver fat accumulation and improves liver enzyme profiles in patients with non-alcoholic fatty liver disease: a randomized controlled pilot trial effect of hydrogen-rich water on oxidative stress, liver function, and viral load in patients with chronic hepatitis b bile acid signaling in metabolic disease and drug therapy bile acid oxidation by eggerthella lenta strains c and dsm t therapeutic roles of carbon monoxide in intestinal ischemia-reperfusion injury microrna files in the prevention of intestinal ischemia/reperfusion injury by hydrogen rich saline the effects of hydrogenrich saline on the contractile and structural changes of intestine induced by ischemia-reperfusion in rats luminal injection of hydrogen-rich solution attenuates intestinal ischemia-reperfusion injury in rats the effects of hydrogen-rich saline solution on intestinal anastomosis performed after intestinal ischemia reperfusion injury effects of molecular hydrogen-dissolved alkaline electrolyzed water on intestinal environment in mice hydrogen-rich saline regulates intestinal barrier dysfunction, dysbiosis, and bacterial translocation in a murine model of sepsis hydrogen gas protects against intestinal injury in wild type but not nrf knockout mice oxidative medicine and cellular longevity with severe sepsis by regulating ho- and hmgb release hydrogen gas restores exhausted cd + t cells in patients with advanced colorectal cancer to improve prognosis hydrogen metabolism inhelicobacter pyloriplays a role in gastric carcinogenesis through facilitating caga translocation the protective of hydrogen on stress-induced gastric ulceration protective role of hydrogen-rich water on aspirin-induced gastric mucosal damage in rats modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of rich water molecular hydrogen: a new biological vision hydrogen-rich saline attenuates acute hepatic injury in acute necrotizing pancreatitis by inhibiting inflammation and apoptosis, involving jnk and p mitogen-activated protein kinase-dependent reactive oxygen species hydrogen inhibits endometrial cancer growth via a ros/nlrp /caspase- /gsdmd-mediated pyroptotic pathway effects of hydrogen gas inhalation on endometriosis in rats protective effect of hydrogen rich saline solution on experimental ovarian ischemia reperfusion model in rats hydrogenrich saline attenuates chemotherapy-induced ovarian injury via regulation of oxidative stress hydrogen-rich water exerting a protective effect on ovarian reserve function in a mouse model of immune premature ovarian failure induced by zona pellucida hydrogen-rich saline attenuates spinal cord hemisection-induced testicular injury in rats protection by hydrogen against gamma ray-induced testicular damage in rats combination of korean red ginseng extract and hydrogenrich water improves spermatogenesis and sperm motility in male mice molecular hydrogen may enhance the production of testosterone hormone in male infertility through hormone signal modulation and redox balance protective effects of hydrogen against low-dose long-term radiation-induced damage to the behavioral performances, hematopoietic system, genital system, and splenic lymphocytes in mice saturated hydrogen alleviates ccl -induced acute kidney injury via jak /stat /p signaling aerosol inhalation of a hydrogen-rich solution restored septic renal function hydrogen-rich saline alleviates kidney fibrosis following aki and retains klotho expression hydrogen-rich saline attenuates acute kidney injury after liver transplantation via activating p -mediated autophagy effects of hydrogen-rich saline on early acute kidney injury in severely burned rats by suppressing oxidative stress induced apoptosis and inflammation hydrogen-rich saline attenuates acute renal injury in sodium taurocholate-induced severe acute pancreatitis by inhibiting ros and nf-κb pathway hydrogen-rich water alleviates cyclosporine a-induced nephrotoxicity via the keap /nrf signaling pathway hydrogen rich water attenuates renal injury and fibrosis by regulation transforming growth factor-β induced sirt hydrogen-rich saline promotes the recovery of renal function after ischemia/reperfusion injury in rats via anti-apoptosis and anti-inflammation preventive effect of hydrogen water on the development of detrusor overactivity in a rat model of bladder outlet obstruction saturated hydrogen improves lipid metabolism disorders and dysbacteriosis induced by a high-fat diet subcutaneous injection of hydrogen gas is a novel effective treatment for type diabetes l-arabinose elicits gut-derived hydrogen production and oxidative medicine and cellular longevity ameliorates metabolic syndrome in c bl/ j mice on highfat-diet hydrogen gas production is associated with reduced interleukin- β mrna in peripheral blood after a single dose of acarbose in japanese type diabetic patients molecular hydrogen improves obesity and diabetes by inducing hepatic fgf and stimulating energy metabolism in db/db mice hydrogen attenuates allergic inflammation by reversing energy metabolic pathway switch pilot study: effects of drinking hydrogen-rich water on muscle fatigue caused by acute exercise in elite athletes intravenous infusion of h -saline suppresses oxidative stress and elevates antioxidant potential in thoroughbred horses after racing exercise involvement of neutrophil dynamics and function in exercise-induced muscle damage and delayed-onset muscle soreness: effect of hydrogen bath protective effect of hydrogen gas inhalation on muscular damage using a mouse hindlimb ischemia-reperfusion injury model molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice protective effect of inhalation of hydrogen gas on radiation-induced dermatitis and skin injury in rats hydrogen gas inhalation protects against cutaneous ischaemia/reperfusion injury in a mouse model of pressure ulcer improvement of psoriasis-associated arthritis and skin lesions by treatment with molecular hydrogen: a report of three cases inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species molecular hydrogen effectively heals alkali-injured cornea via suppression of oxidative stress high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury therapeutic effect of molecular hydrogen in corneal uvb-induced oxidative stress and corneal photodamage molecular hydrogen suppresses glioblastoma growth via inducing the glioma stemlike cell differentiation therapeutic efficacy of hydrogen-rich saline alone and in combination with pi k inhibitor in non-small cell lung cancer a gallbladder carcinoma patient with pseudo-progressive remission after hydrogen inhalation brain metastases completely disappear in non-small cell lung cancer using hydrogen gas inhalation: a case report molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice hydrogen protects rats from dermatitis caused by local radiation pathological findings of covid- associated with acute respiratory distress syndrome strategies for the prevention and management of coronavirus disease key: cord- -xtsnlu f authors: drago-serrano, maria elisa; campos-rodríguez, rafael; carrero, julio césar; de la garza, mireya title: lactoferrin: balancing ups and downs of inflammation due to microbial infections date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: xtsnlu f lactoferrin (lf) is a glycoprotein of the primary innate immune-defense system of mammals present in milk and other mucosal secretions. this protein of the transferrin family has broad antimicrobial properties by depriving pathogens from iron, or disrupting their plasma membranes through its highly cationic charge. noteworthy, lf also exhibits immunomodulatory activities performing up- and down-regulation of innate and adaptive immune cells, contributing to the homeostasis in mucosal surfaces exposed to myriad of microbial agents, such as the gastrointestinal and respiratory tracts. although the inflammatory process is essential for the control of invasive infectious agents, the development of an exacerbated or chronic inflammation results in tissue damage with life-threatening consequences. in this review, we highlight recent findings in in vitro and in vivo models of the gut, lung, oral cavity, mammary gland, and liver infections that provide experimental evidence supporting the therapeutic role of human and bovine lf in promoting some parameters of inflammation and protecting against the deleterious effects of bacterial, viral, fungal and protozoan-associated inflammation. thus, this new knowledge of lf immunomodulation paves the way to more effective design of treatments that include native or synthetic lf derivatives, which may be useful to reduce immune-mediated tissue damage in infectious diseases. lactoferrin (lf) is a conserved iron-binding mammalian glycoprotein with antimicrobial activity, present in secretions that recover mucosal sites regarded as portals of entry and/or invasion of pathogenic agents [ ] . antimicrobial activity has been mostly characterized in lf of bovine and human origin isolated from milk [ , ] . mechanisms underlying the antimicrobial action of lf result from both direct (microbiostatic and/or microbicidal) and indirect (immunomodulatory) effects [ ] [ ] [ ] . at present, the therapeutic and prophylactic treatments for microbial infections that ameliorate both the antibiotic multiresistance and the inflammatory response have prompted the searching of agents that display both antimicrobial and modulatory properties such as lf. this review is focused on the modulatory impact of lf on the inflammatory response induced by infectious microorganisms, mainly in the lf was initially named lactotransferrin, due to being a milk glycoprotein that chelates iron. this protein belongs to the transferrin family, which includes the avian egg ovotransferrin (ovotf) and the mammalian serum and lymph transferrin (tf), but differs from other members of the family in its higher affinity for iron. lf is synthesized by the mammary gland and then it is abundant in colostrum and milk, through which it has been suggested to participate in the initial protection in newborns [ ] [ ] [ ] [ ] [ ] [ ] . regarding the content, human mature milk is highly enriched in lf ( . mg/ml) in comparison with bovine milk ( . mg/ml) [ ] [ ] [ ] . the amino acid sequences of both proteins exhibits approximately % identity [ , ] . lf is also present in many fluids and exocrine secretions, such as tears, saliva, and mucosal surfaces of the respiratory, urinary-reproductive and intestinal tracts; in these sites, lf contributes to the primary innate-immune defense system of mammals that exerts antimicrobial activity against an extensive variety of pathogens [ , , [ ] [ ] [ ] [ ] [ ] . lf is also synthesized during the natural cellular development of promyelocytes to myelocytes, and was early recognized as an important component of the secondary granules of polymorphonuclear (pmn) neutrophils [ , ] . these cells store lf ( - μg/ neutrophils) and release it at the sites of infection, which are acidic due to the activity of pathogens [ , , , ] . in plasma, lf derives from neutrophils and its concentration is very low ( . - μg/ml) [ ] ; nevertheless, in patients with sepsis the degranulation of activated neutrophils leads to secretion of significant levels of lf (~ . mg/ml) into the bloodstream [ ] . neutrophils also release lf in feces whose concentrations markedly increase during inflammatory processes such as inflammatory bowel disease (ibd), ulcerative colitis and crohn's disease, due to the response against pathogenic bacteria [ ] . lf was initially named lactotransferrin, due to being a milk glycoprotein that chelates iron. this protein belongs to the transferrin family, which includes the avian egg ovotransferrin (ovotf) and the mammalian serum and lymph transferrin (tf), but differs from other members of the family in its higher affinity for iron. lf is synthesized by the mammary gland and then it is abundant in colostrum and milk, through which it has been suggested to participate in the initial protection in newborns [ ] [ ] [ ] [ ] [ ] [ ] . regarding the content, human mature milk is highly enriched in lf ( . mg/ml) in comparison with bovine milk ( . mg/ml) [ ] [ ] [ ] . the amino acid sequences of both proteins exhibits approximately % identity [ , ] . lf is also present in many fluids and exocrine secretions, such as tears, saliva, and mucosal surfaces of the respiratory, urinary-reproductive and intestinal tracts; in these sites, lf contributes to the primary innate-immune defense system of mammals that exerts antimicrobial activity against an extensive variety of pathogens [ , , [ ] [ ] [ ] [ ] [ ] . lf is also synthesized during the natural cellular development of promyelocytes to myelocytes, and was early recognized as an important component of the secondary granules of polymorphonuclear (pmn) neutrophils [ , ] . these cells store lf ( - µg/ neutrophils) and release it at the sites of infection, which are acidic due to the activity of pathogens [ , , , ] . in plasma, lf derives from neutrophils and its concentration is very low ( . - µg/ml) [ ] ; nevertheless, in patients with sepsis the degranulation of activated neutrophils leads to secretion of significant levels of lf (~ . mg/ml) into the bloodstream [ ] . neutrophils also release lf in feces whose concentrations markedly increase during inflammatory processes such as inflammatory bowel disease (ibd), ulcerative colitis and crohn's disease, due to the response against pathogenic bacteria [ ] . physiologically, lf can be found as a fully iron-loaded (holo-lf) or iron-free protein (apo-lf) [ , [ ] [ ] [ ] . the holo-lf is conformationally more rigid and is more resistant to denaturation and proteolysis than the apo-lf, but instead, apo-lf is generally more effective against bacteria than holo-lf [ , [ ] [ ] [ ] [ ] . in this regard, it has been reported that holo-lf can be utilized as an iron source by several groups of microorganisms [ , [ ] [ ] [ ] . however, this is not always the case because studies on intestinal epithelial-barrier function and mucosal inflammation carried out in a caco- cells model and macrophages activated with lipopolysaccharide (lps) showed that both lf forms effectively inhibited the pro-inflammatory response. nevertheless, apo-lf was more effective in downregulating inflammation, probably due to its ability to bind and neutralize lps, as well as to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect [ ] . much evidence exists of the successful experimental use of lf from different origins (human, bovine, porcine, caprine, camelid, and buffalo) against the growth of diverse pathogens. as mentioned above, most results indicate that lf from different origins can exert bacteriostatic effects due to its iron-chelating activity, but it can also be bactericidal due to its interaction with lps and porins in gram-negative bacteria, or with teichoic acids in gram-positive bacteria. these interactions lead to membrane damage and bacterial death [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, the antimicrobial activity of lf is also highly dependent on its cationic properties, because the addition of positive charges to lf via amidation enhances its antibacterial and antiviral properties and, in contrast, the addition of negative charges by acylation abolishes them [ ] . as mentioned, lf displays antiviral properties against common virus infections. these antiviral properties are related to its ability to block the cellular attachment or replication of virus by inducing type i interferons (α/β) with antiviral action [ ] . thus, lf from diverse mammals shows a potent activity against replication of human immunodeficiency virus, cytomegalovirus, and hepatitis c virus [ , ] . less information exists about the microbicidal action of lf against fungi and protozoa [ ] [ ] [ ] [ ] . very important is the finding that lf synergizes with antibiotics and drugs, and even with other proteins of the innate immune system such as lysozyme and natural secretory iga (siga) antibodies, potentiating the antimicrobial effect [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the largest external source of lf is milk consumption. when consumed, lf can be enzymatically cleaved by pepsin in the stomach and by trypsin in the small intestine. in adults, whereas hlf is completely degraded, about % of blf resists proteolytic digestion mediated by pepsin [ ] . noteworthy to mention is that digestive tract of babies and infants has a relatively high ph and secretes low levels of pepsin, which allows for the innocuous transit of hlf and blf into epithelial cells, which can be extremely important at this stage of life [ , ] . nevertheless, native hlf derived from pancreatic juices and neutrophils is discharged into exocrine secretions of mucosal surfaces in adults, and thus it acts protecting those sites from invaders. in addition, diverse biological activities of lf including the facilitation of iron absorption, modulation of mucosal immunity and stimulation of mucosal differentiation result of its interaction with lf receptors (lfrs) expressed in the gastrointestinal cells [ , ] . one reason why lf can be used as a pharmaceutical is because its activity is maintained in some of its component peptides after being cleaved by proteolytic enzymes, e.g., the peptides derived from the n terminus of lf by pepsin, so-called lactoferricins (lfcins), lack the iron-chelating activity, and are characterized by their strong cationic charge. remarkably, lfcins often show a higher microbicidal activity than the parental lf as well as synergistically act with drugs and antibiotics against microbes [ , , ] . moreover, several lfcins have been synthesized and experimented against microbes [ ] . on the other hand, synthetic lfcin - , lactoferrampin (lfampin - ), and a fusion peptide of both called lfchimera, have been successfully assayed against multiresistant bacteria, and also against bacteria that typically form biofilms [ , ] . this synthetic lfchimera has also been shown to be effective against parasitic protozoa [ ] [ ] [ ] . another synthetic lfchimera prepared by the fusion between lfcin - and lfampin - , was effective against pseudomonas aeruginosa by down-regulating pyocyanin, elastase and biofilm formation [ ] . the presence of lf in secretions and its various mechanisms of action allow this glycoprotein to combat all types of microbes that colonize mucosae in the different bodily regions. however, depending on the site, microbes can be exposed to different concentrations of lf, to complexes of lf with other proteins, or to diverse levels of lf derivatives [ ] . at the same time, lf can help against the inflammatory process produced by strong immune reaction in infections. therefore, all findings on lf activities suggest that lf and lfcins can be of potential use as antimicrobial and anti-inflammatory compounds, either alone or as adjuncts to conventional antibiotics and drugs. in this sense, lf is one of the most studied proteins since the commercial point of view, being highly appreciated as a nutraceutical in some countries, promoted as a supplement in diarrheic diseases, cancer, increasing immunity, improvement of memory, and several other conditions. human lf has been cloned in different vectors and expressed as recombinant (r-hlf) overall in eukaryotic systems which can glycosylate it, such as yeasts and fungi [ , ] ; however, the best product is obtained from transgenic cows and plants [ ] [ ] [ ] . interestingly, r-hlf expressed in the cow mammary gland, enhanced systematic and intestinal immune responses in piglets used as a model of infants [ ] . in addition, when the meat from the progeny of hlf transgenic cows was analyzed, no abnormalities of its nutrient composition were found [ ] . thus, the wide use of lf in human health care is promissory. next, we will review the effects of lf as an anti-inflammatory protein in a number of infectious diseases in which it has been studied, mainly of gastrointestinal and respiratory tracts. inflammatory response is elicited by germ-line encoded pattern-recognition receptors (prrs) expressed in many cell types that interact with their ligands from exogenous or endogenous origin, namely pathogen-associated molecular patterns (pamps), or danger-associated molecular patterns (damps), respectively. some prrs comprise a large family of receptors such as toll-like receptors (tlrs) [ ] [ ] [ ] . upon ligand binding, tlrs lead to signaling pathways resulting in the activation and translocation of the nuclear factor (nf)-κb to the nucleus. nf-κb modulates the expression of pro-inflammatory cytokines such as interleukin (il)- , il- , type-i interferon (ifn-α, and ifn-β), tumor necrosis factor (tnf) α, as well as chemoattractant cytokines (chemokines). another class of prrs includes nod-like receptors (nlrs), some of which, such as nlrp , nlrp and nlrp , function as sensors or adaptors forming the "inflammasomes" [ ] . activation of inflammasomes by pamps and/or damps induces signal pathways resulting in the activation of caspase- that cleaves the inactive pro-forms of cytokines (il- , and il- ) to generate their active forms. besides to generate active pro-inflammatory cytokines, some inflammasomes regulate cell death in response to microbial and endogenous danger signals [ ] [ ] [ ] [ ] . although lf displays direct microbiostatic and/or microbicidal activities, indirect antimicrobial mechanisms have also been ascribed to its capability of modulating a wide array of humoral and cellular components of the innate and adaptive immunity [ , ] . immunomodulatory role of lf is due, in part, to its interactions with cell surface receptors that favor either elicitation of signal pathways, or lf translocation into nucleus and gene targeting [ ] [ ] [ ] . a summary of the modulatory effects of lf on inflammation due to microbial infections is shown in table . gastrointestinal infections blf treatment of cultured cells infected with e. coli lf and biopsies from patients with crohn's disease ↓il- , ↓il- and ↓tnfα mrna expression [ ] blf treatment of intestinal cell cultures infected with e. coli lf isolated from crohn's disease patients ↑ferroportin (fpn) in infected cells suggesting that blf action on inflammatory response in epithelial cells involves the iron homeostasis [ ] blf-nanoparticles (blf-nano) administration to balb/c mice infected with s. enterica serovar typhimurium ↑tnf α, ↑interferon (ifn) β and ↑ifniii levels (proinflammatory cytokines) [ ] blf administration to c h/hej mice infected with entamoeba histolytica (e. histolytica) ↑il- (th ), ↑il- , ↑iga ↓damage and ↓inflammation [ ] blf treatment to balb/c mice infected with helicobacter pylori (h. pylori) ↓gastric colonization and ↓inflammation (histopathology score) [ ] blf treatment of rotavirus infection children ↔ifnγ, ↔il- and ↔rotavirus incidence in children whether fed or unfed with blf [ ] gut-related systemic infections (sepsis) ↓lung infection, ↑ifnγ, ↑il- in spleen cell cultures, ↓tnfα and ↓il- β correlated with ↓lung pathology. ↑lymphocytic recall response towards bcg [ , ] recombinant human lf mixed with bcg vaccine in mice infected with m. tuberculosis early↑ and late↓ of pro-inflammatory cytokines that correlated with the ↓lung pathology [ ] blf effect in enhancing bcg vaccine by oral route in mice infected with m. tuberculosis ↓colony forming units (cfu) and ↓inflammation in the lungs, ↑ifnγ producing t cd and cd cells and ↑il- lymphocytes [ ] blf effects on cystic fibrosis and bronchial ib - cell cultures infected with burkholderia cenocepacia (b. cenocepacia) ↓il- β (pro-inflammatory cytokine), ↓il- (anti-inflammatory cytokine) [ ] blf administration to a murine model of lung injury by lps ↓bronchioalveolar leukocytes, ↓tnf-α, ↓myeloperoxidase (mpo) activity, ↑il- , ↓lung edema and inflammation [ ] blf administration to a murine model of respiratory syncytial virus infection ↔viral loads and ↔lung inflammation [ , ] blf administration to a murine model of influenza ↔viral load and ↔ifnγ, il- and il- in the lungs [ ] other mucosal and systemic sites blf effects on mammary gland in cows with staphylococcous aureus (s. aureus) mastitis ↓bacterial load, ↑c levels, ↓tnfα mrna expression via nuclear factor κb (nfκb) inhibition, ↑curation, ↑proinflammatory cytokines is correlated with ↑peptides derived from blf-elastase proteolysis [ ] [ ] [ ] [ ] blf effects on oral candidiasis in immunosuppressed mice infected with candida albicans (c. albicans) blf blocked the suppressive effects of candidiasis in polymorphonuclear (pmn) neutrophils; ↑ifnγ and tnfα production in cervical lymph nodes [ ] blf effects on hamsters with amoebic liver abscess by e. histolytica no damage or inflammation in the liver [ ] human lf (hlf) effects on balb/c mice infected with listeria monocytogenes (l. monocytogenes) ↓bacterial load and ↓necrotic foci in the liver, ↔necrotic foci in the spleen, ↓tnfα, il- β and ifnγ mrna [ ] hlf and peptide-hlf derivatives administration to c h/tif mice infected with e. coli o k uropathogenic strain ↓bacterial load in the bladder and kidneys, ↓leukocyte in urine, ↓urinary il- levels at h and systemic il- levels at h post-infection [ ] hlf expressing transgenic mice infected with s. aureus ↓bacterial growth, ↓septicemia, ↓mortality than congenic litter mates. ↑th polarization in the spleen, given that: ↑tnfα and ↑ifnγ, ↓il- and ↓il- upon stimulation ex vivo with exotoxin toxic shock syndrome toxin- compared with congenic controls [ ] ↓ decrease; ↑ increase; ↔ no changes. throughout the gastrointestinal tract, lf is present as an iron-binding multifunctional glycoprotein regarded as a natural compound able to inhibit the pathogens growth. lf is also able to up-and down-modulate both humoral and cellular components of immunity involved in the regulation of the inflammatory response having a key role in maintaining gut homeostasis [ ] . balance of homeostasis results from the tight regulation of several events, since too little inflammation disrupts the process of tissue repairing and remodeling, whereas too much inflammation entails collateral impact by causing tissue damage with life-threatening consequences [ ] . mucosal compartment of the small intestine is a scenario where takes place a physiologic inflammatory response orchestrated by innate and adaptive mechanisms mediated by intestinal epithelial cells and by a wide array of immunocompetent cells at lamina propria, such as dendritic cells, macrophages and tγδ lymphocytes, all with a key role in maintaining the gut homeostasis and combating infections [ ] . however, in some infectious clinical conditions of the large intestine, such as ibd, inflammation has a double-edged sword role by either enabling or inhibiting cancer development and progression [ , ] . as it was commented before, antimicrobial activity of blf against a wide array of pathogens has been profusely evidenced. in contrast, data about the regulatory role of blf on the parameters of gut inflammation caused by enteropathogenic microorganisms have been provided by a limited number of articles. findings from in vitro and in vivo models of infection show that blf displays up-and down-modulatory effects on pro-inflammatory th cytokine profile. the role of blf on the resolution of infections by modulating mediators of inflammation has been documented in models of infection caused by several strains of enteropathogenic bacteria [ ] [ ] [ ] [ ] [ ] , and parasites [ ] . additionally, it has been found that lf promotes the development of bifidobacterium, one of the major genera of bacteria of the colon flora used as probiotics, in a manner independent of the iron saturation level of lf [ , ] . this effect is believed to help maintaining the gut homeostasis. regarding to the gastric inflammation, the treatment with blf or hlf as single agents or in combination with antimicrobial drugs, was found to favor eradication of bacteria and to protect against gastritis caused by helicobacter pylori or helicobacter felis, as described in murine models [ , ] . however, other human and murine trials did not support this finding, and even more, bacterial growth and gastric inflammation seemed to be enhanced by blf or hlf administration [ , ] . these controversial data may reflect experimental or clinical settings of lf treatment. interestingly, lf as a single component failed to eradicate the h. pylori infection but in combination with triple esomeprazole, clarithromycin and amoxicillin therapy and with probiotics, favored the resolution of infection and ameliorated the inflammatory response more effectively than in combination with two-antibiotic treatment, as described in experimental mice treated with r-hlf from transgenic goats, and also in trails of patients treated with native blf. interestingly, r-hlf not only inhibited the growth of h. pylori, but also suppressed the expression of two of its major virulence factors [ , ] . on the other hand, several studies have demonstrated the effect of lf on modulating inflammation in the small intestine. for example, the anti-inflammatory activities of both r-hlf and native blf have been tested in models of bacterial infection by shigella flexneri in rabbits [ ] , and salmonella enterica serovar typhimurium in susceptible balb/c mice [ ] . unlike with lf-untreated animals, macroscopic and microscopic observations evidenced that both lf treatments favored the resolution of infection and protected mice from tissue damage caused by the intestinal inflammation [ , ] . mechanisms accounting for the anti-inflammatory role of blf may result, in part, from the elicitation of siga response with a key role in luminal clearance of pathogens and in down-modulation of intestinal inflammation [ , , , , ] . although lf may modulate inflammation by inhibiting the growth of pathogens through the iron chelating ability of apo-lf, the iron-free form of lf used in most studies, assays based on the murine typhoid model showed that pharmaceutical formulation of iron-saturated blf (holo-blf) enclosed in nanocapsules displayed both antimicrobial activity and modulatory properties on the inflammation. the latter was evidenced by up-and down-modulation of cytokines involved in innate and adaptive immune responses as well on hematopoietic cytokines, with a key role in the generation of both granulocyte (pmn neutrophils) or agranulocyte (monocytes/macrophages) phagocytes [ ] . thus, iron-loaded blf nanocapsules seem to evoke the convergence of innate and adaptive immune responses of pro-and anti-inflammatory cytokines resulting in the protection toward typhoid infection and concomitant intestinal inflammation. down-modulatory effects of blf on pro-inflammatory cytokines has also been documented in cultures of caco- monolayer cells infected with recombinant escherichia coli invasive strain harboring inv gene from yersinia pestis; this strain is able to accomplish invasion but not intracellular multiplication within epithelial cells [ ] . in this model, apo-blf as well as holo-blf decreased levels of il- elicited by non-invasive e. coli wild type strain, and il- , il- and tnfα by e. coli invasive. in addition, both apo-and holo-blf inhibited an il- increased response caused by e. coli invasive strain, but levels of this cytokine remained elevated. these findings suggested that the effect of blf toward inflammatory mediators was iron-independent and that constant high il- levels provided protection by inducing recruitment of phagocytes to combat the infection [ ] . in the large intestine, the regulatory impact of blf toward inflammatory response has been described in in vitro models of infection by adherent invasive e. coli (aiec) strains with a presumable role in the pathogenesis of crohn's disease. this disease, along with ulcerative colitis, are two clinical entities of ibd characterized by an abnormal response to commensal bacteria colonizing the intestinal lumen [ , ] . aiec lf strain is found in lesions of inflamed colon tissue in children suffering crohn's disease with a preponderant th pro-inflammatory response [ ] . findings in the model of infection by aiec lf indicated that blf inhibited the bacterial invasion and the pro-inflammatory cytokine response of tnfα, il- and il- in epithelial monolayers and in cultures from colonic biopsies from patients with crohn's disease, suggesting a potential therapeutic role for blf as antibacterial and anti-inflammatory agent [ ] . up-modulatory effects of blf on inflammatory cytokines in response to bacterial infections have been found in assays of infection with aiec lf in caco- monolayers stimulated with ifn-γ to mimic the preponderant response of th associated cytokines found in crohn's disease patients [ ] . data from these assays showed that in unstimulated infected cells, blf inhibited both bacterial invasion and survival, while in infected cells primed with ifn-γ, blf increased il- production whereas counteracted the inhibitory effect of aiec infection on ferroportin protein expression. ferroportin is an iron exporter protein regulated by the inflammation that determines the survival of intracellular pathogens by reducing the intracellular iron levels. apparent conflicting data described in the infection model with aiec lf strain regarding the anti-inflammatory [ ] versus pro-inflammatory [ ] role of blf seem to evidence two sides of the same coin, i.e., the ability of blf to up-and down-modulate the inflammatory response and iron availability resulting in the resolution of infection. in parasitic infections of the large intestine, we previously developed a model of intracecal infection by the protozoan entamoeba histolytica in susceptible c h/hej mice (a strain with a spontaneous mutation in the tlr gene) to simulate the intestinal infection caused by this parasite in humans [ ] . in this model, the oral therapeutic administration of blf resolved the intestinal infection with amoebae at high efficiency, effect that was shown to be associated to an increased local expression of il- and il- and the elicitation of siga in cecum [ ] , as happened with the salmonella infection. both il- and il- are th interleukins involved in the up-regulation of siga and il- , and also is a secondary inflammatory cytokine that exhibits pro-and anti-inflammatory properties [ , ] . thus, oral blf administration led to the anti-inflammatory response of mediators such as th cytokines and iga that collaborate in the protection against the parasite and maintenance of homeostasis with protective impact on tissue integrity. on the other hand, in experimental assays conducted in mice infected with enterovirus, blf treatment displayed protective action resulting from hampering the viral interaction with host cells [ ] . however, in vitro assays indicated that blf did not exhibit any suppressive activity against rotavirus [ ] . in addition, assessment of rotavirus infection incidence in children, either treated or not with blf, showed that blf neither prevented the viral infection nor had an effect on the levels of ifn-γ or il- as markers of pro-and anti-inflammatory interleukins, respectively [ ] . thus, therapeutic action of blf by itself on viral enteritis is unclear; however, assays in suckling rats showed that administration of whey protein concentrate containing blf as well as other bioactive components, favored the resolution of acute gastroenteritis caused by rotavirus infection [ ] . this result suggests a synergic effect from the bulk rather than each single bioactive component that provided protection to enterovirus. sepsis is a life-threatening syndrome that results by the harmful effects of inflammatory response associated to gut systemic infections [ , , ] . as in the case of infectious intestinal diseases, assessment of inflammatory parameters regulated by blf in response to systemic infections have been analyzed in a limited number of infection models, induced by e. coli strains administered by enteral or parenteral routes [ , , ] . in most cases, models mimic sepsis conditions found in human systemic infections by invasive enteropathogenic bacteria in immunocompetent hosts, or by microbiota members causing opportunistic nosocomial infections in neonatal or adult patients underwent solely parenteral nutrition, post-surgery antibiotic treatment, or immunosuppression, among other conditions [ , ] . therapeutic action of blf and the synthetic peptide lfchimera was tested in balb/c mice infected by intragastric route with enterohemorrhagic e. coli (ehec); the latter is a food borne pathogen causing mild diarrhea, hemorrhagic colitis and, in some patients, hemolytic uremic syndrome characterized by anemia, thrombocytopenia and kidney injury [ ] . in infected mice that underwent both lf and lfchimera treatments, fecal bacterial output and kidney colonization were found reduced, while lfchimera significantly decreased mortality rate. histological analysis in hematoxylin-eosin stained tissue slices to assess the inflammatory response reveled that both lf and lfchimera treatments decreased kidneys damage in comparison with mice without blf treatments that showed tubular necrosis, glomerular injury and intratubular hyaline cast. furthermore, in vivo and in vitro assays have documented the role of r-hlf against gut-related systemic infection by e. coli strain ec causing meningitis in orogastrically infected neonatal rats [ ] . this model mimics clinical observations in neonates which evidence that parenteral feeding as sole route of nutrition is a risk factor of sepsis, resulting from translocation of microbiota members, e.g., gram-negative enterobacteria, from intestinal tract to systemic organs via bloodstream, with potentially fatal consequences. treatment with r-hlf decreased the clinical sepsis illness and bacterial loads in the kidney and blood while in vitro assays in macrophage cultures showed that levels of nitric oxide, tnfα and nf-κb expression elicited by lps were even higher following the addition of r-hlf. these findings suggest that protective action of r-hlf resulted from an optimal activation of macrophages via pro-inflammatory cytokine elicitation to enhance their bacterial killing activity [ ] . effect of blf against sepsis caused by e. coli and staphylococcus aureus has been demonstrated in cyclophosphamide immunosuppressed mice, as a model to mimic immunosuppressive therapy for autoimmune and neoplastic diseases. antisepsis action of blf evidenced by the reduction of bacterial load in the spleen and liver was associated with a rise of circulating leukocytes induced by the elicitation of il- in the spleen as well as peritoneal macrophages in immunosuppressed mice treated with blf [ ] . studies in mice systemically infected with e. coli evidenced that blf enhanced the killing activity and recruitment of neutrophils [ , , ] . action of blf against e. coli sepsis resulted from the elicitation of il- involved in the production of acute phase proteins, as well as from the amelioration on tnfα levels increased in response to the infection [ ] . since high levels of circulating pro-inflammatory cytokines such as tnfα have lethal consequence, the results described above suggest that the ability of blf to control the tnfα increase may underlie its protective action to sepsis, as found in in vitro and in vivo assays [ , ] . clinical trials have confirmed the potential application of blf to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates, diseases that were associated with the up-modulation of t regulatory cells (foxp + cd +, and cd hi) involved in the control of intestinal immune response against pathogens, strengthening the essential role of blf in the control of the intestinal homeostasis [ ] . pro-and anti-inflammatory properties of lf in bacterial infections have been ascribed to its ability to act as a lps-binding protein (lbp). modulatory effects of either hlf or blf, including derived lfcins, on parameters associated with inflammation, has been evidenced in models of intestinal and systemic-related endotoxemia induced by lps (also known as endotoxin) from gram-negative enterobacteria [ , ] . these experimental models are intended to assess the impact of lf at intestinal and systemic levels on inflammatory markers elicited by lps that affect the gut barrier, bacterial translocation, diarrhea, tissue damage and endotoxic shock, among others [ , [ ] [ ] [ ] [ ] . in some cases, experimental assays with lps administration encompass the use of zymosan, d-galactosamine (d-galn) or carrageenan to increase the sensibility of animals toward the toxic effects of low lps doses [ , ] . porcine lf (plf) bioactive derivatives such as plf peptide (lfp ) have been proved to confer protection in mice against lps damage to colon, associated with its down-modulatory action on pro-inflammatory cytokines tnfα, il- and ifnγ. lfp elicited the expression of tight junction proteins involved in the regulation of intestinal permeability, i.e., zonula occludens- , occludin and claudin- , as well as decreased colonic apoptosis [ , [ ] [ ] [ ] [ ] [ ] [ ] . protective action of hlf on the gut barrier function against the lps-induced intestinal damage has also been described for hlf in in vitro cultures of caco- cells and jejune segments of mice underwent lps-endotoxemia [ , ] . iron status of blf had no impact on parameters of the gut barrier function, as documented in caco- cell cultures incubated with murine (j a. ) macrophages; however, apo-blf displayed stronger neutralizing effects than holo-blf against the pro-inflammatory cytokine generation in response to lps and thermally inactivated e. coli cm antigens, suggesting that iron content may determine the protective role of lf toward the inflammation caused by gut endotoxemia and/or sepsis [ ] . at intestinal level, neutralizing activity of blf or hlf has been tested in models of endotoxemia in mice induced by the intraperitoneal administration of lps [ , ] . other models of lps neutralization by blf include enterogenic endotoxemia in rats injected with carrageenan by intraperitoneal route, and also infection with e. coli in rats treated with nebacetin by intraduodenal via to enable the lps release [ ] . in the model of mice endotoxemia, intraperitoneal administration of hlf provided protection against deleterious effects of lps on the intestinal integrity [ ] . moreover, blf administered by intraperitoneal route in mice attenuated the lps-induced diarrhea by decreasing the production of pro-inflammatory mediators with powerful diarrheagenic activity, i.e., prostaglandin e (pge) by enterocytes in the small intestine and nitric oxide in plasma [ ] . in the enterogenic endotoxemia model in rats, blf decreased the endotoxic activity of lps, as measured in plasma on a dose-dependent manner, and also decreased the bacterial loads in the mesenteric lymph nodes [ ] . some experimental assays in neonatal mice indicated that feeding with blf and/or bifidobacteria decreased the intestinal levels of lps without changes in cell populations producing tnfα, ifnγ and il- in peyer's patches [ ] . at the systemic level, the neutralizing activity of hlf-derived lf peptide has been tested in models of endotoxemia in mice, induced by intravenous administration of lps and d-galn. in this assays, lf exhibited protective activity against lethal intravenous lps challenge by decreasing circulating levels of tnfα [ , ] . the dual action of lf on circulating tnfα seems to underlie its protective role, since down-modulation of tnfα provided protection against the deleterious impact of endotoxemia, whereas the modulatory role of lf in the control of tnfα elicitation is critical for eradication of gut-related systemic infections [ , , ] . in another example, blf displayed prophylactic action against lethal shock occurring upon intravenous injection of lps in germ-free piglets; this is a valuable model to study the primary toxicity of endotoxin portion of lps, i.e., lipid a, rather than the secondary toxicity of o and r polysaccharide portions [ ] . in cultures of monocytes from lps-treated piglets, blf inhibited the interaction of lps with cd , an antigen surface marker expressed by mononuclear phagocytes. priming of these phagocytic cells by lps via cd ligation, resulted in the elicitation of powerful pro-inflammatory cytokines including tnfα, il- and il- with lethal outcome for host in endotoxemic conditions [ ] . moreover, oral administration of blf prior to an intravenous lps challenge in piglets provided protection against the mortality caused by lps-induced hypothermia [ ] . like blf, protective activity against hypothermia has been described with hlf in mice underwent lps-endotoxemia [ ] . effect of blf on lps-neutralization and e. coli bacteremia has been explored in lps toxicity susceptible c h/hecr mice carrying a defective tlr- gene as well as in cba mice resistant to lps [ , ] . unlike its protective activity in cba mice, blf failed to confer protection against endotoxemia and e. coli bacteremia in c h/hecr mice due to its inability to ameliorate the elicitation of tnf-α and ifnγ, and to prevent il- decrease. thus, an unbalanced cytokine response may be responsible of the high susceptibility to lps endotoxemia in c h/hecr mice [ ] . differential impact of concurrent, prophylactic, and therapeutic effects of intraperitoneal administration of hlf on endotoxemia were analyzed in mice by intravenous administration of lps [ ] . in the concurrent scheme, hlf administered prior to the lps challenge decreased the serum levels of tnfα, nitric oxide, il- and il- . in the prophylactic and therapeutic protocols, hlf significantly down-modulated serum tnfα and nitric oxide, but no significant fluctuations were seen in the levels of il- and il- . in a mice model of hepatitis induced by intraperitoneal co-administration of lps and zymosan, orally administered blf decreased the serum aspartate aminotransferase activity (a marker of liver inflammation), and increased in the small intestine the production of il- , an anti-inflammatory cytokine with a role in the amelioration of inflammatory response [ , ] . the findings indicate that the up-modulation of il- levels by blf seems to provide therapeutic action in the small intestine induced by lps-zymosan in this model of hepatitis. treatment with r-hlf has also been analyzed in the same mice models of induced hepatitis [ ] . in the experimental scenario, r-hlf provided protection against hepatitis development as determined by the decrease in alanine transaminase activity as the marker of liver damage, which was associated with down-modulation of serum tnfα levels. moreover, the protective effect of r-hlf was not found in mice pre-treated with gadolinium chloride that destroys kupffer cells, suggesting that these cells are the source of tnfα and the targets of r-hlf [ ] . the mechanisms of lf action on the inflammatory response have been analyzed in regarding to the ability of lf to interact with lps and block its interaction with tlr in in vitro cultures of intestinal cell lines and murine peritoneal or cell line macrophages [ , , [ ] [ ] [ ] [ ] [ ] . these assays have evidenced that lf down-or up-modulates lps-mediated inflammatory response via dependent or independent tlr /nf-κb signal pathway. down-modulatory impact on lps-mediated inflammation was found in assays of intestinal porcine epithelial cell line (ipec- ) treated with the plf-derivative peptide lfp , effect that resulted from its ability to inhibit myd /nf-κb and myd /mapk signaling pathways [ , ] . similarly, in human monocyte cell lines (thp- and mono mac ), hlf also displayed down-modulatory activity on lps-associated cytokine response by blocking the binding of nf-κb to the tnfα promoter [ ] . assays in cultures of human peripheral blood mononuclear cells showed that blf displayed an anti-inflammatory response by driving the differentiation of lps-treated monocytes toward dendritic cells with low capacity of both differentiation and elicitation of th response by counteracting the tlr mediated activation signals [ ] . on the other hand, up-modulatory impact of lf on inflammatory effects of lps was documented in assays of raw . macrophage cell line and peritoneal macrophages, indicating that blf, in the form of complex with lps, enhanced cytokine response of tnfα, il- and il- via tlr -nf-κb signal pathway [ ] . however, another study suggests that the up-modulatory effect of lf on lps-mediated inflammation may involve pathways other than tlr signaling. thus, assays on raw . macrophage cell line treated with lps showed that elicitation of il- levels by blf was tlr -independent [ ] . therefore, lf displayed a dual role on the lps-mediated response of pro-inflammatory cytokines that encompasses alternative routes of tlr signalization. other presumable mechanism of lps-mediated inflammation includes the ability of lf to control the expression of iron-regulating proteins as found in thp- monocyte/macrophage cultures; in these cells, lf prevented the lps-induced decreased of ferroportin by reducing the il- levels [ ] . as we mentioned before, ferroportin is an iron binding protein involved in the control of iron levels during inflammatory response. in summary, the findings described above provide the experimental evidence that support the protective role of lf against the deleterious effects of lps-induced pro-inflammatory cytokine response on the gut-barrier function, diarrhea, bacterial translocation, and tissue damage. having in mind the antimicrobial and lps-binding protein activities of lf, its application either alone or in combination with probiotics, or as an adjunctive compound of antibiotics, may represent a very promising strategy for the treatment and prevention of sepsis and endotoxic shock. the human respiratory tract (rt) is responsible for the mobilization of millions of liters of gases throughout life. delivery of life-requiring oxygen to the systemic circulation and organs implies the potential incorporation of countless particles, toxicants and microbes, which are countered by local innate and adaptive immune responses that avoid their entry into the lung tissue and circulation and protect the lung structure and function [ ] . infections in the rt are very frequent in the population and represent a considerable cause of worldwide morbidity and mortality [ ] . infections of the upper rt such as common cold, laryngitis, pharyngitis, epiglottitis, otitis and sinusitis are typically caused by virus, bacteria, and, at less extension, by fungi. as an example, the common cold is a viral disease considered as the most frequent infection in humans, which can be caused by rhinoviruses, coronavirus, parainfluenza and adenovirus, and less frequently by respiratory syncytial virus and enterovirus. however, the influenza virus, a common cause of seasonal flu, can simultaneously affect other parts of the rt, including the lower tract. infections of the lower rt are mainly caused by bacteria, but also by virus, fungi and even parasites. they include bronchitis, pneumonia and pulmonary abscesses, among others. tuberculosis, caused by the bacterium mycobacterium tuberculosis, is among the most prevalent infections in the lower rt, causing mainly pneumonia, but also affecting other organs [ ] . all rt infections are accompanied with an inflammatory process whose intensity depends on many variables, including the type of pathogen and its virulence, the inoculum, the affected tissue, the host immunological status and whether the infection is acute or chronic. although the inflammatory process is essential for the control of invasive infectious agents, the development of an exacerbated or chronic inflammation results in alterations of the respiratory capacity due to the lung tissue damage, including edema, increased airway resistance and mucus production, such as in the infection with influenza virus [ ] . understanding how inflammation alters the respiratory system is indispensable for the development of better therapeutic interventions to support breathing and lung plasticity as a clinical treatment. in this regard, evidence of the modulation of rt inflammation by blf associated to certain microbes has been reported using several in vitro and in vivo models. moreover, lf is considered the second most important antimicrobial and anti-inflammatory peptide after lysozyme in the upper rt [ ] . tuberculosis is by far the most studied in vivo model of microbial pulmonary infection. this bacterial infection affecting nearly a third of the world's population and having a rate for new infections of approximately . % per year [ ] , is targeted by the bacillus calmette-guerin (bcg) vaccine, the most widely used vaccine in the world which has remained almost unchanged since [ ] . a study carried out in showed that a single subcutaneous immunization of mice with a mix of bcg and blf emulsified with freund's adjuvant, followed by a challenge with m. tuberculosis in aerosol, resulted in decreased mycobacterial loads in the lungs and spleen [ ] . splenocyte proliferative response to heat-killed bcg showed increased il- and ifnγ production. in a subsequent study by the same group, it was showed that blf admixed to the bcg vaccine, in incomplete freund's adjuvant or pbs, increased mice protection against a m. tuberculosis challenge when compared with mice that received bcg alone [ ] . in addition, there was a significant reduction of lung bacterial load associated to increased production of ifnγ and il- by splenocytes, as mentioned before; in this study, blf addition to the vaccine also resulted in a clear reduction in lung pathology, concomitant with down-regulation of pro-inflammatory mediators tnfα or il- β, suggesting that the main action of lf to enhance the bcg vaccine relied on its immunomodulatory properties reducing the immune-related tissue pathology, in part, by modulating macrophages and dendritic cells ability to present antigens and stimulate t-cells [ ] . noteworthy, the lymphocytic recall response towards bcg antigens two months after infection was higher in the mice that received lf as adjuvant, suggesting that lf improved the specific t-cell th response as determined by the increase in infγ production [ ] . the potential of r-hlf to reduce the m. tuberculosis tissue damage and pulmonary histopathology was also demonstrated in ulterior studies of the same group. they showed that r-hlf produced in the yeast pichia pastoris expression system with a glycosylation pattern similar to its natural human neutrophil counterpart, in contrast to the non-glycosylated r-hlf, was able to improve the efficacy of the bcg vaccine in protecting against the challenge with m. tuberculosis in aerosol, as manifested primarily in a significant reduction in the associated pulmonary pathology [ ] . in this case, the mycobacterial loads in the lung and spleen were not significantly reduced in the bcg-r-hlf group compared with the controls treated with bcg alone, but rather, the protection was associated with changes in the pathological manifestation of the lung disease; this was probably due to the notable immunomodulatory function of the granulocytic lf used in this study, when compared with the lf form from secretions, which is more microbicidal [ ] . recently, a r-hlf expressed in chinese hamster ovary (cho) cells was also used in the mixture with the bcg vaccine, showing a slight decrease over the time in the lung pathology after aerosol challenge with m. tuberculosis, which correlated with an initial increase in the secretion of inflammatory cytokines followed by their posterior decrease [ ] . the efficacy of blf in enhancing the bcg vaccine action was more recently analyzed using a more amenable route of administration. in this study, mice that received drinking water containing . % blf at day or post-infection had lower colony forming units (cfu) and lower inflammation in the lungs, with increased numbers of ifnγ producing t cd and cd cells and il- producing lymphocytes when compared with animals vaccinated with bcg alone [ ] . noteworthy, blf did not affect the in vitro replication of m. tuberculosis but instead enhanced the killing of bacteria by macrophages in a nitric oxide dependent way [ ] . these studies using the models of pulmonary infection with m. tuberculosis suggest that lf promotes certain up-regulation of pro-inflammatory response, while down-regulating overall tissue immunopathology. the role of lf in the inflammation in other pulmonary infections has been less addressed. homeostatic effect of blf on inflammation was reported in in vitro cultures of cystic fibrosis (cf) bronchial cells (ib - ) infected with burkholderia cenocepacia, a gram-negative opportunistic bacterium that recurrently infects patients with cf forming biofilms and is usually highly resistant to currently available broad-spectrum antibiotics. thus, even though the addition of blf did not reduce the rates of bacterial invasion, it decreased the release of pro-inflammatory il- β, while augmented the secretion of anti-inflammatory il- , suggesting a role for blf in protecting cf bronchial infected cells from the inflammation-associated damage [ ] . noteworthy, this study correlated with a previous one addressed on sputum samples from patients with cf which showed an inverse association between the levels of lf in the secretions and the inflammation burden [ ] . another study showed that the decrease of lf levels in patients with cf was due to its cleavage by the increased cathepsin activity in pseudomonas aeruginosa-positive sputum samples, another biofilm-forming opportunistic pathogen of these patients. a similar result with lf and tf undergoing proteolysis was previously reported in bronchioalveolar lavage of p. aeruginosa infected cf patients [ ] . these results suggest that the proteolytic cleavage of lf in patients with cf can contribute to b. cenocepacia and p. aeruginosa-associated lung damage, and that infection-associated lung damage can be improved by the exogenous therapeutic administration of lf, due to its potent immunomodulatory properties. similar protective effect of blf has been found in a murine model of lung injury induced by intraperitoneally administered lps [ ] . in this study, the intraperitoneal injection of blf ( mg/mouse) h before (prophylactic effect) or h after (therapeutic effect) lps challenge, were associated with significant reduction of the total number of leukocytes in bronchioalveolar lavage samples, increased il- , and decreased tnfα concentrations and myeloperoxidase activity [ ] . these changes paralleled attenuation of lung edema and inflammatory infiltration, suggesting a protective role of blf by avoiding the damage caused by the lps-induced acute inflammatory response. however, the protective role of lf based on the regulation of inflammation is not observed in all cases of rt infections. there are several viral infection models where the immunomodulatory effect of lf has not been documented. thus, although lf has in vitro antiviral activity against the respiratory syncytial virus (rsv) [ ] , as well as an immunomodulatory effect reducing the release of il- by hep- cells infected with rsv [ ] , the oral or intraperitoneal administration of different doses ( to mg/animal/day) of blf to mice from h before until h post-rsv infection did not have any effect on viral loads, pulmonary airflow resistance or obstruction, degree or type of pulmonary inflammation and serum t cellular responses, evaluated on day post-rsv infection [ ] . similar result was observed in a study carried out in mice treated by intranasal route with blf on days - , and evaluated on day post-rsv infection [ ] . another example is a mouse model of influenza infection, where the daily oral administration of . mg of blf from h before infection, did not have any effect on viral load and concentration of ifnγ, il- and il- cytokines evaluated at six days post-infection, when compared with untreated infected mice [ ] . the reason for the lack of blf protecting effect in these viral infection mice models is unknown, but possible explanations could be related to timing, dosing, and route of blf administration. in contrast, oral blf and curcumin supplementation to children with recurrent viral rt infections resulted in immune modulation by modifying the lymphocyte population and cytokine responses that reduce the rate of infections [ ] . a recent study aimed to determine the effect of three months supplementation with blf-fortified formula on respiratory tract infections and diarrhea in chinese weaned infants ( - months age), showed similar results, with a reduction in the incidence rate of respiratory-related illnesses when compared with a placebo group [ ] . moreover, a study in patients with chronic rhinosinusitis has established an association between the genetic deficiency of lf synthesis in the upper rt and the increased susceptibility of certain individuals to bacterial colonization, biofilm development, and recalcitrant sinus disease [ ] . this new knowledge of lf immunomodulation paves the way to more general design of t cell-dependent vaccines that incorporate naturally occurring granulocytic components, which may be useful in infectious diseases to reduce immune-mediated tissue damage. the role of blf on inflammatory response associated to infection has been described in other mucosal sites, such as the mammary gland of cows suffering staphylococcal mastitis [ ] . assessment of mammary gland secretions showed that, in sick cows, intramammary infusion of blf decreased the numbers of staphylococci and increased c levels, whereas in healthy animals blf infusion increased the numbers of pmn leukocytes expressing cd b, an integrin when complexed with cd (cr ) acts as receptor for the ic b complement fragment [ ] . according to these findings, up-modulatory effect of blf on pro-inflammatory components of innate immunity may underlie its therapeutic action toward mastitis; in fact, alternative approaches have also been tested to decrease the deleterious effect of inflammation on tissue integrity. combination of blf and antibiotics was found to be effective to control the staphylococcal mastitis and to attenuate the mrna expression of tnfα via the inhibition of nf-κb activation [ ] . thus, this approach may contribute to decrease the effects of inflammation on tissue damage and also to reduce the antibiotic dosage for the eradication of staphylococcal infection by multiresistant strains. inflammatory response in staphylococcal mastitis seems to be correlated with the elicitation of peptides derived from blf-elastase proteolysis that display low concanavalin a and low iron-binding affinities, as well as antibacterial properties, but induce the expression of pro-inflammatory cytokines il- and tnfα leading to neutrophil infiltration [ , ] . like staphylococcal mastitis, elicitation of low cona affinity lf-peptide derivatives in parotid saliva was correlated with the severity of symptoms of periodontitis patients [ ] . in oral cavity, the modulatory action of blf on infection-associated inflammatory response has been documented in mice infected with candida albicans [ ] . experimental settings of oral candidiasis in immunosuppressed mice showed that oral administration of blf displayed therapeutic effect by inhibiting the suppressive effects of infection on inflammatory parameters of innate response, such as circulating pmn neutrophils and cervical lymph-node cells; moreover, generation of ifnγ and tnfα was found increased in cervical lymph-nodes cultures primed with heat-killed c. albicans from blf-treated mice [ ] . according to in vitro assays testing full length blf and blf-derived peptides, therapeutic effect of parental blf relies on the n-terminal portion associated with its ability to up-modulate the killing action of pmn neutrophils by increasing the superoxide generation, protein kinase c, p mapk activity, and the expression of p phox [ ] . these findings suggest that, in the murine model of candidiasis, the up-modulatory effect of blf on parameters of inflammation provides protection to c. albicans infection. data from in vivo and in vitro assays support the role of blf and hlf in up-and down-modulation of inflammatory response to extra-intestinal infections, such as hepatic amoebiasis [ ] , listeriosis [ , ] , urinary tract infections [ ] , legionellosis [ ] , and staphylococcal septicemia [ ] . in experimental amoebic liver abscess in hamsters, blf treatment by gavage had protective action against the hepatic lesions by e. histolytica and favored the normalization of the liver function [ ] . having in mind its intrinsic anti-inflammatory and therapeutic action, as well as its low toxicity, the use of blf as adjunct of conventional drugs such as metronidazole, may contribute to decrease the drug dosage in the treatment of amoebiasis and even decrease the drug resistance of the parasite. in vitro experiments on listeria monocytogenes infection in ifnγ primed thp cell cultures indicated that blf displayed a protective action against cell death by necrosis, whereas bovine lfcin b diverted the death cell from necrosis to apoptosis [ ] . findings indicated a protective role of blf and lfcin b by reducing the inflammatory response associated to necrosis caused by the intracellular infection of the pathogenic bacteria in macrophages, and favored the anti-inflammatory conditions of cell death by apoptosis on these cells. data from studies of infection with l. monocytogenes in mice indicate that treatment with hlf displayed significant effects on one main organ target of this pathogen in regards to bacterial colonization, necrosis, and mrna expression of pro-inflammatory cytokines tnfα, il- β and ifnγ [ ] . although the majority of the experimental data on lf properties has been obtained with blf, some experimental studies indicate that hlf displays even more potent antibacterial and anti-inflammatory action. murine model of infection of urinary bladder with the uropathogenic e. coli o k strain showed that perorally administered hlf decreased the bacterial load in the kidneys and urinary bladder as well as the inflammatory response, as evidenced by reduced il- levels in urine at h post-infection, and in plasma, at h post-infection [ ] . thus, hlf administered by peroral route provides therapeutic action against infection and inflammation in remote sites such as the urinary tract. in vitro assays of legionella pneumophila infection in cultures of monocytes from healthy volunteers indicated that unlike holo-hlf that promoted the bacterial growing, apo-hlf inhibited the intracellular multiplication of the pathogen in both inactivated and ifnγ activated monocytes [ ] . these findings suggest a synergy between the antibacterial effect of iron-free human lactoferrin and the stimulating killer effects of the pro-inflammatory cytokine ifnγ on infected cells, leading to conditions for control of bacterial multiplication inside cells. a model of s. aureus infection in hlf-transgenic mice showed the pivotal role of hlf in the elicitation of a th profile of cytokines, which determined the resolution of systemic infection. polarization of the immune response to the th profile in hlf-transgenic mice was evidenced by up-modulation of tnfα and ifnγ levels and down-modulation of th cytokines il- and il- in culture supernatants of spleen cells [ ] . in summary, the studies mentioned above show that lf and its derivative peptides display bimodal effects that provide conditions for the up-and down-regulation of inflammation leading to the resolution of extra-intestinal infections. the biotechnological development of formulations of lf as nanoparticles for potential clinical use, in addition to lf-hydrolysate, and native lf alone or in combination with probiotics, may have application for the control of infections and inflammation [ , , , , , ] . evidence from the basic studies in animals of experimentation about the prophylactic and therapeutic activity of lf as antimicrobial and modulatory agent on inflammatory response, have promoted this glycoprotein from the innate immune system as a focus of interest for the biotechnological development of nanoparticle-based formulations for potential clinical use. in addition, lf-hydrolysate, and native lf alone or in combination with antibiotics and probiotics, may have potential application in the control of neonatal infections, and in inflammation. more studies are necessary to support the generalized practical application of lf, mainly in the control of inflammation associated to infections. the authors thank to the mexican council of science and technology (conacyt) for the supporting grants (mireya de la garza) and (julio césar carrero). julio césar carrero also thanks to programa de apoyo a proyectos de investigación e innovación tecnológica (papiit-unam) for grant no. in and to the institutional program "nuatei" for financial support. thanks also to carlos villasana and luisa samaniego-barrón for their technical assistance and pavel petrosyan for the english editing of the manuscript. the authors declare no conflict of interest. lactoferrin: a multifunctional glycoprotein lactoferrin from milk: nutraceutical and pharmacological properties overview of lactoferrin as a natural immune modulator lactoferrin-a multifunctional protein with antimicrobial properties immunomodulatory effects of lactoferrin on antigen presenting cells nanocapsules loaded with iron-saturated bovine lactoferrin have antimicrobial therapeutic potential and maintain calcium, zinc and iron metabolism the isolation of a red protein from milk preparation and properties of lactosiderophilin (lactotransferrin) of human milk a structural framework for understanding the multifunctional character of lactoferrin relative molecular mass, isoelectric point, iron-binding properties and uptake by the liver occurrence, structure, biochemical properties and technological characteristics of lactoferrin metal-combining properties of human lactoferrin (red milk protein). . the involvement of bicarbonate in the reaction proteins of iron metabolism the physiology of lactoferrin lactoferrin in milk from different species an iron-binding protein common to many external secretions lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties biological role of lactoferrin lactoferrin concentration during involution of the bovine mammary gland concentrations of lactoferrin and iron in human milk at different stages of lactation concentration of lactoferrin and transferrin throughout lactation in cow's colostrum and milk molecular evolution of the transferrin family and associated receptors swiss-prot database the significance of iron in infection the involvement of lactoferrin in the hyposideremia of acute inflammation lactoferrin: a general review antimicrobial properties of lactoferrin the therapeutic potential of lactoferrin lactoferrin content of peripheral blood cells lactoferrin biosynthesis during granulocytopoiesis lactoferrin turnover in man lactoferrin, an iron-binding protein in neutrophilic leukocytes crohn's disease activity assessed by fecal calprotectin and lactoferrin: correlation with crohn's disease activity index and endoscopic findings structure of human lactoferrin at . -a resolution three-dimensional structure of diferric bovine lactoferrin at . a resolution heterogeneity of bovine lactotransferrin glycans the effects of differences in pspa alleles and capsular types on the resistance of streptococcus pneumoniae to killing by apolactoferrin bactericidal activity of human lactoferrin: differentiation from the stasis of iron deprivation in vivo and in vitro effects of lactoferrin on yersinia pseudotuberculosis antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment human hololactoferrin: endocytosis and use as an iron source by the parasite entamoeba histolytica iron-saturated lactoferrin and pathogenic protozoa: could this protein be an iron source for their parasitic style of life? ability of neisseria gonorrhoeae, neisseria meningitidis, and commensal neisseria species to obtain iron from lactoferrin the impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation lactoferrin: mechanism of action, clinical significance and therapeutic relevance une protéine multifonctionnelle molecular structure and biological function elucidation of the antistaphylococcal action of lactoferrin and lysozyme in vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence lactoferrin is a lipid a-binding protein lactoferrin inhibits the endotoxin interaction with cd by competition with the lipopolysaccharide-binding protein two outer membrane proteins are bovine lactoferrin-binding proteins in mannheimia haemolytica a in vitro studies of the digestion of caprine whey proteins by human gastric and duodenal juice and the effects on selected microorganisms antibacterial and antiviral activity of camel milk protective proteins comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin lactoferrin for prevention of common viral infections antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro effectiveness of human, camel, bovine and sheep lactoferrin on the hepatitis c virus cellular infectivity: comparison study lactoferrin: a protein of the innate immune system capable of killing parasitic protozoa twenty-five years of research on bovine lactoferrin applications microbicidal action of lactoferrin and lactoferricin and their synergistic effect with metronidazole in entamoeba histolytica effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis bactericidal synergy of lysostaphin in combination with antimicrobial peptides synergy and antagonism between iron chelators and antifungal drugs in cryptococcus effector mechanisms of iga amoebicidal activity of milk, apo-lactoferrin, slga and lysozyme enhancement of the activity of novobiocin against escherichia coli by lactoferrin effect of bovine apo-lactoferrin on the growth and virulence of actinobacillus pleuropneumoniae antifungal activities of natural and synthetic iron chelators alone and in combination with azole and polyene antibiotics against aspergillus fumigatus gastric digestion of bovine lactoferrin in vivo in adults characterization of mammalian receptors for lactoferrin bioactive proteins/peptides and functional properties evaluation of synergistic activity of bovine lactoferricin with antibiotics in corneal infection effects of bovine lactoferrin hydrolysate on the in vitro antimicrobial susceptibility of escherichia coli strains isolated from baby pigs structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against pseudomonas aeruginosa in vitro and in vivo bactericidal activity of lfchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides bactericidal effect of bovine lactoferrin, lfcin, lfampin and lfchimera on antibiotic-resistant staphylococcus aureus and escherichia coli microbicidal effect of the lactoferrin peptides lactoferricin - , lactoferrampin - , and lactoferrin chimera on the parasite entamoeba histolytica reduction of the infectivity of toxoplasma gondii and eimeria stiedai sporozoites by treatment with bovine lactoferricin parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasite giardia intestinalis lactoferrin-derived peptides and lactoferricin chimera inhibit virulence factor production and biofilm formation in pseudomonas aeruginosa perspectives on interactions between lactoferrin and bacteria expression of cloned human lactoferrin in baby-hamster kidney cells research progress in physicochemical characteristics of lactoferrin and its recombinant expression systems expression, characterization, and biologic activity of recombinant human lactoferrin in rice recombinant human milk proteins plant-based biopharming of recombinant human lactoferrin supplementation transgenic cow's milk containing recombinant human lactoferrin enhances systematic and intestinal immune responses in piglets nutritional composition analysis of meat from human lactoferrin transgenic bulls mechanisms and pathways of innate immune activation and regulation in health and cancer innate immunity gone awry: linking microbial infections to chronic inflammation and cancer recognition of bacterial pathogens and mucosal immunity converging roles of caspases in inflammasome activation, cell death and innate immunity interactions of lactoferrin with cells involved in immune functionthis paper is one of a selection of papers published in this special issue bovine lactoferrin can be taken up by the human intestinal lactoferrin receptor and exert bioactivities the n domain of human lactoferrin is required for internalization by caco- cells and targeting to the nucleus mammalian lactoferrin receptors: structure and function lactoferrin protects rabbits from shigella flexneri-induced inflammatory enteritis effect of bovine lactoferrin in salmonella ser. typhimurium infection in mice lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive escherichia coli strains lactoferrin prevents invasion and inflammatory response following e. coli strain lf infection in experimental model of crohn's disease. dig. liver dis lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases oral lactoferrin treatment resolves amoebic intracecal infection in c h/hej mice inhibition of helicobacter pylori infection by bovine milk glycoconjugates in a balb/ca mouse model effects of lactoferrin-containing formula in the prevention of enterovirus and rotavirus infection and impact on serum cytokine levels: a randomized trial protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic escherichia coli o :h infection protective effects of lactoferrin in escherichia coli-induced bacteremia in mice: relationship to reduced serum tnf α level and increased turnover of neutrophils enhanced clearance of escherichia coli and staphylococcus aureus in mice treated with cyclophosphamide and lactoferrin differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on lps-induced inflammatory responses in mice neutralization of endotoxin in vitro and in vivo by a human lactoferrin-derived peptide lactoferrin augments bcg vaccine efficacy to generate t helper response and subsequent protection against challenge with virulent mycobacterium tuberculosis lactoferrin enhanced efficacy of the bcg vaccine to generate host protective responses against challenge with virulent mycobacterium tuberculosis cho expressed recombinant human lactoferrin as an adjuvant for bcg influence of oral lactoferrin on mycobacterium tuberculosis induced immunopathology lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with burkholderia cenocepacia iron-modulated biofilm lactoferrin protects against lipopolysaccharide-induced acute lung injury in mice lack of effect of bovine lactoferrin in respiratory syncytial virus replication and clinical disease severity in the mouse model lactoferrin reverses respiratory abnormalities in respiratory syncytial virus (rsv) infection of mice effects of orally administered bovine lactoferrin and lactoperoxidase on influenza virus infection in mice effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis effect of orally administered bovine lactoferrin on the immune response in the oral candidiasis murine model potential antimicrobial effects of human lactoferrin against oral infection with listeria monocytogenes in mice human lactoferrin and peptides derived from a surface-exposed helical region reduce experimental escherichia coli urinary tract infection in mice enhanced th response to staphylococcus aureus infection in human lactoferrin-transgenic mice intestinal inflammation and colorectal cancer: a double-edged sword? ability of lactoferrin to promote the growth of bifidobacterium spp. in vitro is independent of receptor binding capacity and iron saturation level effect of lactoferrin on helicobacter felis induced gastritis lactoferrin and desferrioxamine are ineffective in the treatment of helicobacter pylori infection and may enhance h. pylori growth and gastric inflammation in mice human recombinant lactoferrin is ineffective in the treatment of human helicobacter pylori infection recombinant human lactoferrin enhances the efficacy of triple therapy in mice infected with helicobacter pylori multi-faceted functions of secretory iga at mucosal surfaces. front. immunol. , , lactoferrin increases both resistance to salmonella typhimurium infection and the production of antibodies in mice characterization of adherent-invasive escherichia coli isolated from pediatric patients with inflammatory bowel disease protection against murine intestinal amoebiasis induced by oral immunization with the kda antigen of entamoeba histolytica and cholera toxin transcriptional regulation of the mucosal iga system cytokines in sepsis: potent immunoregulators and potential therapeutic targets-an updated view lactoferrin inhibits enterovirus infection by binding to vp protein and host cells inhibitory effects of human and bovine milk constituents on rotavirus infections supplementing suckling rats with whey protein concentrate modulates the immune response and ameliorates rat rotavirus-induced diarrhea sepsis: current dogma and new perspectives the blessings and curses of intestinal inflammation lactoferrin protects neonatal rats from gut-related systemic infection lactoferrin can protect mice against a lethal dose of escherichia coli in experimental infection in vivo antibacterial system generated by lactoferrin in mice in vivo is primarily a killing system oral lactoferrin to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates and effect on t-regulatory cells lactoferrin-lipopolysaccharide (lps) binding as key to antibacterial and antiendotoxic effects reciprocal interactions between lactoferrin and bacterial endotoxins and their role in the regulation of the immune response bovine lactoferrin protects lipopolysaccharide-induced diarrhea modulating nitric oxide and prostaglandin e in mice. can bovine lactoferrin ingestion protects against inflammation via il- induction in the small intestine of mice with hepatitis treatment of enterogenic endotoxinemia with lactoferrin in rats lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice porcine lactoferrin-derived peptide lfp- protects intestinal barrier by maintaining tight junction complex and modulating inflammatory response lfp- , a porcine lactoferrin peptide, ameliorates lps-induced inflammation via the myd /nf-κb and myd /mapk signaling pathways protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal caco- cells in vivo effects of bifidobacteria and lactoferrin on gut endotoxin concentration and mucosal immunity in balb/c mice the protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets gastrointestinal and hepatic mechanisms limiting entry and dissemination of lipopolysaccharide into the systemic circulation variations in susceptibility to proteoglycan-induced arthritis and spondylitis among c h substrains of mice: evidence of genetically acquired resistance to autoimmune disease lethality in lps-induced endotoxemia in c h/hecr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin bovine lactoferrin induces interleukin- production in a hepatitis mouse model and human intestinal myofibroblasts lactoferrin protects against development of hepatitis caused by sensitization of kupffer cells by lipopolysaccharide lactoferrin prevents lps-induced decrease of the iron exporter ferroportin in human monocytes/macrophages bovine lactoferrin counteracts toll-like receptor mediated activation signals in antigen presenting cells lactoferrin works as a new lps-binding protein in inflammatory activation of macrophages lactoferrin activates macrophages via tlr -dependent and -independent signaling pathways mattsby-baltzer, i. lactoferrin down-regulates the lps-induced cytokine production in monocytic cells via nf-κb respiratory epithelial cells orchestrate pulmonary innate immunity infecciones respiratorias. in temas de bacteriología y virología médica; oficina del libro fefmur the inflammatory response triggered by influenza virus: a two edged sword the effects of corticosteroid on tissue lactoferrin in patients with nasal polyposis global epidemiology of tuberculosis bcg-different strains, different vaccines? influence of bovine lactoferrin on expression of presentation molecules on bcg-infected bone marrow derived macrophages a novel recombinant human lactoferrin augments the bcg vaccine and protects alveolar integrity upon infection with mycobacterium tuberculosis in mice loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis transferrin and lactoferrin undergo proteolytic cleavage in the pseudomonas aeruginosa-infected lungs of patients with cystic fibrosis effect of compounds with antibacterial activities in human milk on respiratory syncytial virus and cytomegalovirus in vitro lactoferrin and surfactant protein a exhibit distinct binding specificity to f protein and differently modulate respiratory syncytial virus infection immune modulation by lactoferrin and curcumin in children with recurrent respiratory infections effect of bovine lactoferrin from iron-fortified formulas on diarrhea and respiratory tract infections of weaned infants in a randomized controlled trial reduced levels of lactoferrin in biofilm-associated chronic rhinosinusitis cleaved inflammatory lactoferrin peptides in parotid saliva of periodontitis patients a novel bovine lactoferrin peptide, fkcrrwqwrm, suppresses candida cell growth and activates neutrophils apoptotic death of listeria monocytogenes-infected human macrophages induced by lactoferricin b, a bovine lactoferrin-derived peptide lactoferrin inhibits or promotes legionella pneumophila intracellular multiplication in nonactivated and interferon γ-activated human monocytes depending upon its degree of iron saturation. iron-lactoferrin and nonphysiologic iron chelates reverse mon biological activity of lactoferrin-functionalized biomimetic hydroxyapatite nanocrystals anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns key: cord- - sejcqda authors: heindel, jerrold j.; belcher, scott; flaws, jodi a.; prins, gail s.; ho, shuk-mei; mao, jiude; patisaul, heather b.; ricke, william; rosenfeld, cheryl s.; soto, ana m.; vom saal, frederick s.; zoeller, r. thomas title: data integration, analysis, and interpretation of eight academic clarity-bpa studies date: - - journal: reprod toxicol doi: . /j.reprotox. . . sha: doc_id: cord_uid: sejcqda “consortium linking academic and regulatory insights on bpa toxicity” (clarity-bpa) was a comprehensive “industry-standard” good laboratory practice (glp)-compliant -year chronic exposure study of bisphenol a (bpa) toxicity that was supplemented by hypothesis-driven independent investigator-initiated studies. the investigator-initiated studies were focused on integrating disease-associated, molecular, and physiological endpoints previously found by academic scientists into an industry standard guideline-compliant toxicity study. thus, the goal of this collaboration was to provide a more comprehensive dataset upon which to base safety standards and to determine whether industry-standard tests are as sensitive and predictive as molecular and disease-associated endpoints. the goal of this report is to integrate the findings from the investigator-initiated studies into a comprehensive overview of the observed impacts of bpa across the multiple organs and systems analyzed. for each organ system, we provide the rationale for the study, an overview of methodology, and summarize major findings. we then compare the results of the clarity-bpa studies across organ systems with the results of previous peer-reviewed studies from independent labs. finally, we discuss potential influences that contributed to differences between studies. developmental exposure to bpa can lead to adverse effects in multiple organs systems, including the brain, prostate gland, urinary tract, ovary, mammary gland, and heart. as published previously, many effects were at the lowest dose tested, . μg/kg /day, and many of the responses were non-monotonic. because the low dose of bpa affected endpoints in the same animals across organs evaluated in different labs, we conclude that these are biologically – and toxicologically – relevant. the consortium linking regulatory and academic insights on the toxicity of bisphenol a (bpa), known as clarity-bpa, is a novel toxicity study designed to integrate the strength of a us food and drug administration (fda) industry-standard "guideline" study with investigator-initiated studies that focused on disease-associated and molecular endpoints. the hypothesis underlying this design was that the published results from the independent studies would be apparent in a guideline-compliant study and would thereby provide a stronger dataset for regulatory agencies as well as to test whether industry standard endpoints are sensitive, specific and predictive for agents that interfere with hormone systems. details of the study design are published [ ] and will only be briefly summarized here. in clarity-bpa, pregnant nctr-sprague dawley rats were gavaged daily with vehicle (carboxymethyl cellulose), ethinyl estradiol (ee; . or . μg/kg/day) as the positive control or bpa ( . , , , , or , μg/kg/day) from gestational day (gd) to birth at the fda national center for toxicological research (nctr) facility employing guideline protocols for toxicity testing. offspring were gavaged with the same doses starting on postnatal day (pnd) . daily dosing was continued for - years (continuous-dose) or stopped at pnd (stop-dose). the core guideline study included rats/dose/sex/age. rats were euthanized and blood and tissues isolated for analysis at and years. the independent labs that participated in the clarity-bpa study assessed a variety of endpoints related to disease/dysfunction and a variety of molecular endpoints. endpoints were assessed at pnd , , , , age months, and age year depending on study design. importantly, all independent lab experiments were blinded to control and exposure groups, with data decoded only after completion of all studies. results of the core guideline study were published in a single report [ ] , and results of several academic studies were published independently in peer-reviewed journals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . assessments of the overall study design [ , ] and a summary of available datasets from multiple clarity-bpa studies [ ] are also published in peer-reviewed journals. importantly, there has not been a single publication to integrate the results from the independent studies. to address this critical gap, the present report represents a synthesis and integration of the results of of the independent studies by principal investigators willing to participate in an effort to coalesce the findings and assess patterns of bpa exposures across the various end organs examined in the same animal cohorts and, frequently, in the same animals. for each organ system, we provide the rationale for the study, overview of methodology, summary of major findings, and discussion of results of the clarity-bpa study in comparison to previous results from the independent labs along with plausible reasons for the differences. additionally, we applied an integrative analysis approach by using rand circos-plots generated with the mixomics [ ] program to identify correlations in bpa responses across organs collected from the same or comparable individual rats within this same consortium study, thus expanding our findings to a systems biology level to reveal strong organismal relationships at three different timepoints: days of age (weaning), - days of age (young adult) and months of age (older adult). additionally, relationships between investigator findings within this consortium study were tested at three different dosages: lowest dose ( . μg/kg/day), middle dose ( μg/kg/day), and highest dose ( μg/kg/day). in this integrative correlation analyses section, we emphasize first those associations identified in males and females at the lowest dose and at months of age, as evidence of such persistent or developmental origins of health and disease (dohad) effects would suggest that we need to rethink the lowest safe dose of bpa. rosenfeld's laboratory has used this mixomics analyses approach in previous bpa studies to integrate various 'omics and phenotypic data generated in her lab together [ ] [ ] [ ] . however, to our knowledge, this is the first time such a program has been used to perform integrative correlation analyses with comprehensive datasets spanning different ages and dosages and generated in the laboratories of multiple investigators. the clarity-bpa program consisted of independent investigators. table shows a listing of all the independent clarity-bpa investigators and the endpoints assessed. four investigators have not published their data, one investigator has a manuscript in preparation. ten investigators have published their data and eight of those chose to be a part of this manuscript. a description of those studies and their results are described below. the prins lab has established that the developing prostate has heightened sensitivity to estrogenic exposure that can reprogram the gland to have elevated disease risk in adulthood [ , [ ] [ ] [ ] [ ] . together with dr. shuk-mei ho and using sprague dawley rats, we determined that while developmental exposure to bpa at environmentally relevant doses alone is not sufficient to drive prostate pathology, such early-life exposure reprograms the rat prostate epigenome and increases susceptibility to estrogen-driven carcinogenesis with aging [ , [ ] [ ] [ ] [ ] [ ] . in addition, using a humanized prostate model containing normal human prostate stem and progenitor cells, our lab found similar results wherein low-dose in vivo bpa exposure increases susceptibility to estrogen carcinogenicity, implicating direct relevance of the rodent model to human disease [ , , [ ] [ ] [ ] . most recently, a detailed dose-response study in sprague dawley rats (zivic miller laboratories, pittsburgh, pa) that included internal free bpa and bpa-glucuronide (bpa-g) dosimetry demonstrated a nonmonotonic response to brief neonatal bpa exposures in a rat prostate lobe-specific manner [ ] . significantly more lateral lobe high-grade prostate intraepithelial neoplasia (pin) lesions-the precursor to prostate cancer-as well as progression to adenocarcinoma were found in rats developmentally exposed to low-dose bpa (≤ μg/kg/day) and given testosterone plus estradiol (t + e) implants in adulthood that doubled circulating estradiol (e ) levels. this finding is biologically relevant because e levels increase in aging men [ ] and, together with testosterone, induce prostate cancer in rat and human epithelia [ ] and accelerate prostate cancer progression [ , , ] . further, estrogenic activity is amplified in metastatic prostate cancer in humans [ , ] . a separate laboratory independently analyzed neonatal bpa exposures (both oral and subcutaneous depot) to sprague dawley rat pups with adult t + e treatments and similarly determined that low-dose bpa exposures increase susceptibility to estrogen-driven high-grade pin in the dorsolateral lobe with aging [ , ] . taken together, we propose that a combination of developmental bpa exposures with rising adult estrogen levels may augment prostate cancer risk. listing of independent clarity-bpa investigators and endpoints assessed. the goals of clarity-bpa studies on the prostate gland were to ) examine whether developmental and/or chronic bpa exposures are sufficient to drive pathology in separate regions of the prostate gland in rats supplied by the fda; ) test the hypothesis that early-life bpa exposures increase susceptibility to later-life neoplasia and adenocarcinoma in response to elevated e levels, as occurs in aging men; and ) assess whether chronic bpa exposures modify stem cell homeostasis within the dorsolateral prostate lobes. for goal , nctr sprague dawley rats (clarity-bpa study) were gavaged daily with vehicle, ee ( . μg/kg/day), or bpa ( . , , , , or , μg/kg/day) from gd to year (continuous-dose) or from gd to pnd (stop-dose). for goal , rats were gavaged daily from gd to pnd (stop-dose) and were given t + e implants at pnd to drive carcinogenesis with aging. prostates were collected at -year necropsy at fda labs, coded, and shipped to the university of illinois at chicago, where they were processed and analyzed for histopathology by dr. maarten bosland, who was blinded to treatments and controls. for goal , nctr sprague dawley rats were gavaged daily with vehicle, ee ( . μg/kg/day), or bpa ( . , , or μg/kg/day) from gd to months (continuous-dose), at which time prostates were removed and shipped on ice overnight to the prins laboratory for stem cell isolation and culture. dorsolateral lobe epithelial stem cells were isolated by direct prostasphere d culture and passaged three times to enhance stem cell purification. bpa was absent during this -week culture period. we measured spheroid numbers and size as well as gene expression by qrt-pcr to determine whether in vivo exposure to bpa altered stem cell self-renewal, progenitor cell proliferation, and lineage commitment. prostate findings generated in this clarity-bpa study are published [ ] . developmental or continuous exposure to bpa alone at any dose did not produce prostate pathology that differed from vehicle controls, similar to findings reported in the core studies. however, we confirmed our prior reports that developmental bpa exposure sensitizes the prostate to later-life e -driven carcinogenesis, an apical adverse outcome (fig. a,b) . specifically, compared to vehicle controls, perinatal exposure of rats to bpa at low ( . μg/kg/day), medium ( μg/ kg/day), and high ( , μg/kg/day) doses resulted in more severe pin lesions, shifting from low-grade pin in controls to high-grade pin with the highest severity score at the lowest tested bpa dose [ ] . notably, high-grade pin in humans is a precursor to prostate cancer, while low-grade pin is not considered clinically relevant. importantly, the . μg/kg/day bpa exposure led to a four-fold increase in adenocarcinoma fig. . dorsolateral prostate pathology and prostasphere numbers in rats treated with vehicle or increasing doses of bpa. aeb) prostate pathology in -year-old rats treated with bisphenol a (bpa) during gestation to weaning (stop-dose) and given implants of testosterone + estradiol (t + e) at postnatal day to elevate circulating estradiol levels. a) severity scores of lateral lobe prostate (lp) intraepithelial neoplasia (pin) lesions were significantly elevated in rats given ethinyl estradiol (ee) or . , , or , μg/kg/day bpa during development as compared to vehicle controls. *p < . , **p < . vs controls. b) multiplicity of dorsolateral prostate (dlp) ductal adenocarcinoma was significantly increased in rats treated with . μg/kg/day bpa during early life as compared to vehicle controls. **p < . vs controls. cde) prostaspheres in -month old rats treated continuously with vehicle or bpa from gestational day through time of tissue collection. number of prostaspheres cultured from dlps of rats exposed to bpa from gestation through months of age. daily exposure to ee or . μg/ kg/day bpa doubled the spheroid numbers as compared to vehicle controls (anova = . ; multiplicity in the dorsolateral prostate ducts, an effect not seen at higher bpa doses. dose-specific responses to chronic bpa exposures were observed for stem and progenitor cells harvested from dorsolateral prostates at months of age. in vivo continuous exposure to . μg/kg/day bpa doubled the total prostasphere number, reflecting increased stem cell numbers in adult prostates (fig. c) . prostasphere size, a marker of progenitor cell proliferation in cultured spheroids, increased steeply in response to μg bpa and to a lesser degree to μg bpa compared to vehicle-treated controls (fig. d ). tightly paralleling prostasphere size effects, exposure to ee or μg/kg/day bpa significantly increased ck , sox , and hoxb expression, while ee or or μg/kg/day bpa suppressed ck , trop , and tbx mrna [ ] . this indicates that chronic bpa exposure permanently modifies the lineage commitment of prostate stem cell progeny, increasing basal progenitors and suppressing luminal progenitor cells. together, these results show that chronic low-dose bpa exposure alters adult prostate stem cell homeostasis in a dose-dependent manner, increasing stem cell numbers at the lowest dose and elevating progenitor cell proliferation, while also shifting lineage commitment to favor basal progenitor cells at -and -fold higher doses. the dosespecific responses observed over a -fold bpa range are likely due to differential engagement of estrogen receptor (er) populations and membrane versus nuclear signaling pathways. reprogramming of adult rat prostate stem cell homeostasis by chronic low-dose bpa may underpin an increased carcinogenic risk in the prostate with aging. collectively, the results provide unbiased evidence that bpa exposures at human-relevant doses result in adverse effects on the rat prostate gland. overall, the clarity-bpa study on prostate endpoints confirmed previous studies that developmental exposure to bpa at environmentally relevant low doses markedly increases prostate cancer susceptibility to aging-related elevations in circulating estrogens. further, low-dose bpa exposure alone was confirmed as sufficient to increase prostate stem cell numbers and reprogram the epithelial progenitor cell lineage. nonetheless, there are also several differences between these results and previously reported findings using a similar model. first, few ventral or dorsal lobe lesions were noted in the clarity-bpa study using nctr-sprague dawley rats derived from > years of breeding at the fda facility. this contrasts with our previous dose-response study using zivic-miller sprague dawley rats, where inverted ushaped dose-response curves were observed in pin severity in those prostate regions [ , , ] . further, the incidence of lateral lobe pin and dorsolateral prostate ductal adenocarcinomas was not affected by perinatal bpa or ee exposure with adult t + e treatment in the present study, whereas our previous findings found elevated pin and carcinoma incidence in the lateral lobe at months and year, respectively, in rats treated neonatally with μg/kg/day bpa plus adult t + e [ , ] . these divergent findings likely result from multiple variations in experimental designs between studies, including differences in sprague dawley rat sub-strains, diet compositions, exposure periods (gd to pnd vs pnd , , and used previously), and exposure routes (daily gavage vs subcutaneous oil depot used previously), as well as the lack of t + e tube replacement every weeks in the present study as done previously. notably, the chronic high incidence of lateral prostate inflammation found in all rats in the present studies, including %- % penetrance in control rats and the elevated mortality in t + e treated rats, phenomena not observed in our prior work with sprague dawley rats, is possibly related to housing conditions and treatment protocols. despite these divergent design details that may account for differences in histopathology findings, the overall conclusions regarding bpa effects on the prostate are consistent between studies. the use of rats as a model for prostate cancer in clarity-bpa is also a confounding variable. unlike men, who develop prostate adenocarcinoma at high rates with aging [ ] , most rat strains including sprague dawley do not spontaneously develop prostate cancer, highlighting fundamental biological differences in prostate carcinogenesis between the two species. the use of rats as a model for human prostate carcinogenesis requires either potent chemical carcinogens and/or extended exposure to natural sex steroids with high receptor affinity [ ] . of the compounds used to induce prostate cancer in rats, extended exposure to testosterone at physiological levels with two-fold elevated e is a physiologically relevant model because e levels rise in aging men. as such, developmental bpa exposure combined with adult t + e is the most relevant experimental regime for testing bpa effects on prostate carcinogenesis in a rat model, with potential for direct applicability to humans. a major limitation of our prostate clarity-bpa studies was sample size, which was severely underpowered to detect statistical differences for all carcinogenic endpoints for the rats treated with t + e. power calculations based on our prior studies were presented in our animal plan, showing the need for animals per dose in the aging study. however, that number was only achieved in vehicle and ee control groups, whereas bpa groups had - animals per dose. the very low number of animals in the and μg/kg/day bpa groups given adult t + e (n = ) resulted in non-normality of data distributions, which prevented inclusion in statistical analysis. low numbers for the remaining doses may account for lack of significance in several endpoints. the underpowering of this study at nctr labs defies the fda's own recommendations of animals/group for carcinogenicity studies [ , ] which was the number used for the parallel clarity-bpa core study. we had not previously examined stem cell homeostasis in the rat prostate gland, so the clarity-bpa dataset on that endpoint is novel. we have examined bpa effects previously on adult human prostate stem cells and human embryonic stem cells, and both of those studies observed similar stimulatory effects of bpa on stem and progenitor cell proliferation, among other endpoints [ , ] . the vom saal and ricke labs have demonstrated that low bpa doses alter the testes, epididymis, seminal vesicles, preputial glands, and prostate in male mice [ ] [ ] [ ] . we have also found statistically significant effects of endogenous and exogenous estrogens on the urogenital sinus (ugs) of fetal mice and rats [ , [ ] [ ] [ ] [ ] [ ] . these studies involved examination of males during fetal life as well as later in adulthood to determine if there were long-term effects. we found that bpa increases estrogen receptor alpha gene expression (esr ) in ugs mesenchyme in male mouse fetuses in both primary culture [ , ] and in vivo; this latter experiment suggested epigenetic mechanisms as we found changes in dna methyltransferases [ ] . bpa exposure during fetal life also caused an increase in androgen receptor gene expression in the fetal ug s mesenchyme, and subsequently, an increase in androgen receptor protein in adult male mouse prostate [ , , ] . an indication of the very high sensitivity of the ugs to estrogens was shown in a study in which a . pg/ml increase in fetal serum estradiol (e ) administered via a silastic capsule implanted in the mother significantly decreased the size of the urethral lumen based on d reconstructions [ ] . it also significantly increased the number of prostatic glandular buds, size of the glands, and overall prostate size, particularly in the dorsal (colliculus) region of the ugs when mice are examined at birth using a d computer assisted reconstruction technique (fig. ) ; these effects persisted and enlarged prostates were found later in adulthood. the malformation of the colliculus was associated with enlargement of the utriculus, which is the remnant of the portion of the mullerian duct that differentiates into the cranial region of the vagina in a female and persists within the prostate tissue near the colliculus. the size of the utricle increases as a function of estrogenic chemicals present during the critical period of ugs differentiation, which occurs near the end of the first trimester in human pregnancy and shortly before birth in rats and mice [ ] . in summary, malformation of the ugs, particularly in the collicular region, as well as decreased size of the urethra (fig. ) have been observed in all of our prior studies involving elevated estrogen in mice and rats exposed as fetuses via the pregnant dam. consistent with several studies, high doses of estrogens have the opposite effect on the prostate, resulting in non-monotonic dose-response relationships [ , ] . the finding that increased estrogen decreases the size of the urethra led us to conduct additional studies in adult mice treated with testosterone (t) and e capsules to determine whether adverse effects of estrogens on the bladder-urethra were restricted to initial development of the ugs in males or continued throughout life. the estrogen treatment mimics the gradual increase in free serum estradiol in men as they age. adult exposure to t + e induced obstructive voiding disorder, associated with droplet voiding pattern and an inability to exhibit sustained voids, bladder hypertrophy, diverticula, calculi, and eventual decompensation with hydronephrosis [ ] . administration of bpa and testosterone (bpa + t) produces similar results in adult male mice [ ] . taken together, these findings suggest that the male urogenital system is subject to disruption by estrogenic chemicals, including bpa, during both fetal and adult life. our primary objective was to examine the impact of bpa and ee on development of the urethra and morphology of the ugs. we examined pnd male rats as described previously. [ ] . briefly, nctr sprague dawley female rats were assigned to one of eight treatment groups: vehicle control; . , , , , or , μg/kg/ day bpa; or . or . μg/kg/day ee. starting at gd and continuing until parturition, dams were administered chemicals daily by gavage. there were no naïve (not gavaged) controls included, although a pre-clarity study showed that gavage significantly alters fetal development [ ] . to eliminate the possibility of litter effects, one pup per litter was randomly selected by fda personnel and coded. we were thus blind to treatment group. from each pup, the ugs was removed, fixed, and sectioned. serial tissue sections were imaged (distance between each image = μm), and all images were imported into a three-dimensional reconstruction software program (biovis d; montevideo, uruguay). biovis d was used to trace the urethra and other structures in each image to render a d image. immunohistochemistry was completed using different antibodies focusing on the urethral epithelial area located within the widest part of the urethra and surrounding tissue. all results were analyzed relative to the body weight of the animal. we replicated our prior finding that the colliculus region of the ugs showed significant changes in structure and morphology (fig. a) . the changes were observed at all bpa doses except the μg dose group, which only had animals, all of which were selected by nctr personnel from only one of the replicate breedings that occurred over months (this is not standard procedure). the only significant effect at the highest dose of bpa ( , μg/kg/day) was on the shape of the colliculus, which is associated with the remnant of the mullerian duct that forms the cranial region of the vagina in females. unlike all other outcomes, there was a monotonic dose-response to estrogens (fig. b) . the low ee dose significantly differed from vehicle controls, while the higher ee dose only tended to differ from vehicle controls (p = . ). in addition, the dose of bpa and . dose of ee resulted in a significantly enlarged colliculus (fig. d) . the second major finding was that bpa reduced the length and width of the urethra (fig. ) . these effects occurred as a result of maternal exposure to low doses of bpa ( . , μg/kg/day). the . dose fig. . d serial section reconstruction of the urogenital sinus (ugs) from gestation day (gd) male cd- mice exposed to low doses of bisphenol a (bpa) and ethinyl estradiol (ee) from gestation day - via feeding the pregnant dam. ugs depicted for each treatment was closest to the group mean. there was a marked alteration in urethra shape, particularly at the junction of the bladder and urethra, which is constricted (*) in mice exposed to estrogenic chemicals compared to controls. in addition, the ugs region (prostatic sulcus or colliculus, arrow) is significantly enlarged by bpa compared to controls, based on data published in timms et al. [ ] . of ee also significantly reduced the length of the urethra. collectively, these findings suggest that low-dose effects of bpa on the colliculus and urethra are via an estrogenic mode of action. an exception was our finding that males treated with , , or μg/ kg/day bpa showed a significant decrease in thickness of the urothelium, which was not affected by either dose of ee. immunohistochemistry results using antibodies were highly variable, and there were no consistent histological localization changes associated with treatment of bpa or ee, in contradiction to prior results. in contrast to our findings from studies with various strains of mice, bpa increased body weight at birth, but only in the low-dose bpa . , and μg/kg/day groups. in addition, both doses of ee were significantly heavier than vehicle gavaged controls (fig. b) . adding a quadradic term in our statistical analysis revealed a significant nonmonotonic effect of bpa on body weight at birth. other similar findings that show an increase in body weight at birth due to fetal exposure to a low dose of bpa in rats has been reported [ ] . variability among the collected pup body weights was higher than expected ( fig. a) , which was perhaps due to chronic stress associated with daily gavage [ ] . for example, rat pups selected for morphological and structural analyses of the pnd urogenital system ranged in body mass from . to . g, a > % range in body mass. however, even with this and other limitations [ ] we observed consistent findings that supported our prior studies of ugs malformations, decrease in the size of the urethra, and enlargement of the colliculus as a result of exposure to bpa and ee. the effects we found to be significantly different from controls occurred at specific doses of bpa and ee, consistent with findings that through a variety of mechanisms, hormones, hormonal drugs, such as ee, and hormonally active chemicals, such as bpa, result in nonmonotonic dose response curves, with results at one dose being different from results at other doses when there is as much as a -fold difference between doses [ ] . the interesting exception was the one finding of a high ( , μg/kg/day) dose of bpa impacting the shape fig. . colliculus measurements. colliculus angle (cao) was defined as the angle the colliculus makes at the juncture with the cranial urethra (a) and analyzed by treatment (b). the colliculus size (cs), shaded blue was determined by measuring the colliculus distance between the lowest caudal point of the colliculus and the lowest caudal point drawn on the urethra and taking the reciprocal (c) and analyzed by treatment (d). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) fig. . c. urethra d reconstruction using biovis d demonstrating how this software can isolate and quantify individual regions of the ugs to calculate length and width (μm), surface area (μm ), and volume (μm ) of the urethra. shown is urethral distance measured straight across from the dorsal (most cranial point of urethra on most dorsal section) to ventral aspect: dorsal-ventral distance (i); cranial-caudal distance (j); urethral volume (k); and surface area (l). a. the width of the urethral lumen was significantly smaller in the bpa and ee . groups relative to vehicle controls. b. the length of the urethra was significantly shorter in the bpa . and ee . groups relative to negative controls. of the colliculus, which contains the remnant of the mullerian duct. this is consistent with our prior finding that a high ( μg/kg/day) oral dose of des in mice blocked the development of prostate ducts but resulted in the lack of regression of the mullerian ducts that would normally regress in male fetuses in response to mullerian inhibiting hormone [ ] . no other statistically significant difference was found between the highest dose of bpa and vehicle controls. finally, a finding that was not previously examined was measurement of the thickness of the urothelium, which in this study was significantly decreased by bpa at , and μg/kg/day doses, while neither dose of ee produced this effect, suggesting that unlike our other findings, this effect may not have been mediated by estrogenic mechanisms. the soto laboratory has conducted studies on the effects of perinatal bpa exposure at several endpoints spanning reproduction, neuroendocrine development, behavior, obesity, and mammary gland development and carcinogenesis [ ] [ ] [ ] [ ] [ ] . in these studies, exposure started at gd and most of them ended at pnd . additionally, even the lowest doses ( ng/kg /day) had significant effects. regarding the mammary gland, developmental studies in mice reveal altered mammary gland morphogenesis (from gd to months of age), increased sensitivity to e , and development of intraductal hyperplasia after months of age [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . additionally, bpa exposure alters mammary gland development in fetuses of non-human primates [ ] . in wistar [ ] and wistar-furth [ ] rat models, we found that bpa induces intraductal hyperplasia in the mammary gland and changes in the dna methylome, which manifest at all ages studied. at days of age, methylome and transcriptome results are consistent with histological findings (i.e., intraductal hyperplasia) [ ] . additionally, carcinomas in situ are found in bpa-treated animals [ ] . sprague dawley rat model studies also reveal that, regardless of whether animals are exposed gestationally or gestationally and lactationally, preneoplastic lesions develop in bpa-exposed female offspring across all doses as early as pnd . further, mammary gland adenocarcinomas develop in bpa-exposed offspring by pnd , and carcinomas are present in bpatreated animals but absent in controls [ ] . both mouse and rat models indicate a developmental effect of bpa that predisposes the mammary gland to neoplastic development. the fact that carcinomas appear in rats exposed solely to bpa suggests that bpa is a complete mammary gland carcinogen [ ] . additionally, early developmental effects revealed by altered morphometric parameters indicate that the mammary gland is a sensitive bpa target, as effects are detected at doses as low as ng/kg /day. this suggests that the mammary gland could be used as an endpoint to assess developmental toxicity and as an indicator of increased propensity to neoplastic development [ , ] . several studies indicate that bpa is a mammary gland toxicant [ , ] . further, several studies have found that bpa exposure at diverse developmental stages (fetal, neonatal, adult) increases the propensity of developing mammary cancer in rodent models [ ] [ ] [ ] [ ] . diverse studies spanning different endpoints ranging from development of the ductal system of the fetal mammary gland to mammary carcinogenesis reveal a non-monotonic dose-response curve [ , ] . our overarching goal was to obtain morphological markers of altered mammary gland development that could be used as indicators of an increased propensity for cancer and to provide a quantitative assessment of mammary gland development. we explored the following hypotheses: ( ) pre-pubertal mammary gland morphology at pnd is an excellent predictor of pathological outcomes that manifest during adulthood, based on data obtained independently in our laboratory (mostly in mouse models) and that of dr. s. fenton (national toxicology program) (mostly using rat models); ( ) dna methylation profiles and concomitant alterations of gene expression at pnd are predictors of pathological outcomes that manifest during adulthood, which was done in collaboration with dr. shioda, mass general hospital); ( ) perinatal exposure to bpa induces abnormal post-pubertal/adult development of the mammary gland; and ( ) bpa generates non-monotonic dose-response curves. we obtained mammary glands from clarity-bpa rats treated with vehicle control, bpa, or ee. female rats were dosed with vehicle, ee ( . or . μg/kg/day), or bpa ( . , , , , or , μg/kg/ day) from gd to pnd (stop-dose) or until the end of the experiment ( years; continuous-dose). assessing mammary gland morphogenesis: in contrast to mouse models there is a paucity of reports on the effect of fetal bpa exposure on rat mammary gland morphogenesis. this is in part due to the florid structure of the ductal tree that grows more conspicuously into the third dimension and makes quantitative assessment beyond weaning challenging. this feature of the rat mammary gland hinders the use of standard morphometric tools for the analysis of the rat mammary ductal system. instead, conventional scoring methods are used. they are called semi-quantitative because they construct a score from qualitative and countable morphological features, such as terminal end buds; the higher the score the more developed the gland is. we compared a semi-quantitative method (the scoring method blind for our urethra study by staff at the nctr ( fig. -b ). there were blocks of matings, but males exposed to μg/kg/day bpa were all selected from treatment block , which tended to be lighter than pups from other blocks. using the criteria reported in davis and fenton [ ] modified for the present study per montévil et al., [ ] with a quantitative method consisting of confocal microscopy, d reconstruction, and analysis using a software tool we developed within the clarity study [ ] . the analysis included quantities such as the aspect ratio (length/width), the epithelial area, and the fractal dimension of the epithelium in d (the projection of its d image), an evaluation of the surface of the epithelium, of its volume, and of its d fractal dimension. additionally, several plugins from imagej were also used to count d objects, assess d shapes and to skeletonize the epithelium. the skeletonized epithelium was used for counting the number of branches, measuring average branch length, etc. we developed a plugin that reconstructed the mammary tree for analysis. this reconstruction was then used as the basis for evaluating characteristics such as branching (branching angles and the tortuosity of the branches), local duct thickness, etc. overall our method assesses structural features of mammary glands. all these measurements were performed in whole-mounted mammary glands, with the exception of methylome and transcriptome studies which were done on frozen tissues. first and foremost, our computer-assisted unsupervised analysis that performs distinct measurements demonstrated that the doseresponse curve to bpa in pnd mammary glands is non-monotonic, with a breaking point between and μg/kg/day doses (fig. ). similar to the analysis of pnd glands non-monotonic dose-response curve were observed in all quantitative studies (at pnd and months) as well as semi-quantitative studies (at pnd and pnd ). these results show that various bpa effects are different from those of ee, while some are similar (fig. ) . this is not surprising, since not all estrogenic substances produce the same effects [ , , ] . consistent with these data showing non-monotonic and more pronounced effects at low doses, the clarity-bpa core study found a significant increase in mammary gland adenocarcinomas and adenomas with the lowest dose of bpa ( . μg/kg/day) when exposure stopped at pnd . we compared automated quantitative measurements of the glands (method briefly described above) with the semiquantitative developmental scores for the pnd gland (assessed using the davis and fenton method) [ ] and found correlations between this score and numerous morphological features. highest correlations with the score were for d fractal dimension of the gland (cc: . ; p = . e- ) and number of branches (cc: . ; p = . e- ). additionally, we also compared developmental scores with dimensions from principal component analysis (pca). the scoring captured aspects of the two first dimensions of pca (size and thickness of glands) and was not correlated to the third (length of ducts) or to any additional dimensions. this relationship between developmental score and pca dimensions is meaningful because it corresponds to the directionality of developmental characteristics observed between control and . μg/kg/ day ee-treated glands. indeed, the developmental scoring criterion was optimized to detect effects resulting from ee exposure, the positive control for comparison with bpa-exposed mammary glands. because the effects of ee and bpa were not similar in all studied endpoints, this comparison was insufficient to detect significant non-linear responses in ductal length and several other morphological features that were shown to be affected by other analyses. nevertheless, semi-quantitative scoring did show a non-significant non-monotonic response in morphological development between glands exposed to or μg/kg/day bpa. bpa and ee resulted in different responses-while ee accelerated gland development, bpa led to abnormal development when assessed at pnd . eight lesions were identified in whole-mounts and histological sections from eight pnd mammary glands across both continuousdose and stop-dose treatment groups. no lesions manifested in vehicletreated animals, and all lesions were diagnosed as benign or malignant, ranging from lobular hyperplasia, fibroadenoma, periductular fibrosis, or ductal epithelial necrosis with lymphocytic infiltration to ductal carcinoma in situ. we identified total lesions in whole-mounts and excised from -month-old mammary glands across both continuousdose and stop-dose treatment groups. three malignant tumors (adenocarcinomas) were classified from continuous-dose and stop-dose . μg ee-treated females, and the remaining lesions/benign tumors were found in vehicle and . , , and , μg/kg/day bpa-treated females. benign lesions included lobular or ductular alveolar dilatations (with and without secretions), periductular fibrosis (with and without lymphocytic infiltration), fibroadenomas, and adenomas. notably, the number of animals per group in this experiment was ∼ , whereas our previous study (acevedo et al.) had - animals per group. this could explain why there were neoplastic lesions in bpa groups (frequency: ∼ per group) and none in controls. the most salient results of these experiments are that i) developmental exposure to bpa reveals a non-monotonic dose-response curve on mammary gland development at all ages examined, showing a break between the ug/kg/day and ug/kg/day doses, ii) perinatal bpa exposure alters mammary gland development at all ages studied, and iii) pnd mammary glands provide a very sensitive end point to assess developmental toxicity. as noted above, our previous experiments in rats show neoplastic outcomes from developmental bpa exposures. data reported in the clarity-bpa core study agree with our previous work regarding neoplastic outcomes in the mammary gland, thus confirming our observation that bpa is a complete mammary gland carcinogen. the experiments detailed above were aimed at obtaining early morphological markers of altered mammary gland development that could be used as in both graph the x axis represents bpa doses in ug/kg/day. non-linear regression illustrates a breaking point between and μg/kg/day doses. graphs represent mean and standard deviation for each dose, and fit with a combination of linear and step functions. this pattern was observed for the majority of endpoints measured. indicators of an increased propensity for cancer and to provide a quantitative assessment of mammary gland development. our previous experience with effects of bpa on mammary gland development were done in mice, so the results described here cannot be directly compared to those mouse studies. points assessed in previous rat experiments (intraductal hyperplasia, carcinoma in situ) were observed at pnd and in wistar-furth rats [ ] and in our sprague dawley model [ ] , although we did not examine pnd in the experiments described in this study. regarding transcriptome and methylome studies, most of our animals were co-housed with the highest-dosed animals ( , μg bpa), which has been shown to transmit detectable circulating bpa and bpa-g in some non bpa-exposed controls and thus raises the possibility of contamination [ , ] . additionally, a majority of rna samples had significantly damaged rna with no detectable s and s peaks. this level of degradation precludes generation of rna integrity numbers. we have previous evidence of significantly different gene expression patterns at pnd in sprague dawley rats exposed perinatally in our laboratory to , . , or μg bpa (not part of clarity-bpa). we are now comparing data of both experiments and hope to submit the paper by the end of the year. our most salient result is non-monotonicity of the bpa effect. there is little literature on the effect of bpa on rat mammary gland development, which generally parallels our far more extensive findings in mice. the comparison of experiments conducted in our lab with clarity-bpa studies regarding the transcriptome are likely to reveal in detail whether clarity-bpa data match our previous results regardless of protocol differences. in summary, there are no obvious discrepancies overall with our previous and present findings. the most important and novel finding of the present mammary gland study is the clear non-monotonicity of the bpa dose-response curve observed in pnd mammary glands in a double-blinded experiment analyzed with a non-supervised computer-based technology involving different measurements. the fact that non monotonic effects of similar shape and characteristics were also demonstrated using a simpler set of end points in pnd and -month-old specimens is a promising finding regarding the feasibility of introducing these end points in toxicological studies. these results show the importance of applying statistical methods appropriate for non-monotonic responses. linear models are a powerful tool to provide evidence of a causal relationship because they quantitatively relate the changes of a putative cause with the one of the effects. hence, exhibiting a linear response provides empirical evidence of a causal relationship. however; this method does not apply to nonmonotonic responses, which are common in endocrinology because the putative causes are involved in multilevel, complex regulatory processes resulting from the evolutionary history of hormone functions. in this complex context, an appropriate way to show the presence of causation is to demonstrate the prevalence of a specific non-monotonic pattern, here a breaking point between μg/kg/day bpa and μg/ kg/day bpa. hence, we conclude that the non-monotonic dose response curve (nmdrc) we documented reveals the presence of a causal relationship. finally, given the extensive number of distinct measurements used in this study and the fact that bpa and ee do not induce identical changes, this study is informing us against dismissing end points that are not modified by ovarian estrogens or their analogues when testing the endocrine disrupting effects of "xenoestrogens". the flaws laboratory has conducted several studies on the effects of bpa on ovarian function in mice. collectively, those studies show that bpa may cause infertility in mice by destroying ovarian follicles, the key structures required to maintain fertility in females. further, bpa may destroy mouse ovarian follicles by inhibiting their growth to the antral stage, which is required for normal ovulation and thus fertility [ , ] ; data also indicate that bpa may destroy mouse ovarian follicles by inducing follicle death via apoptosis, known as atresia [ ] . finally, bpa may cause infertility in mice by reducing the ability of the ovary to synthesize e levels [ , ] . these previous findings are published in several peer-reviewed journals [ ] [ ] [ ] [ ] [ ] and review articles [ , ] . several studies on the effects of bpa on the ovary and female reproductive outcomes indicate that bpa is an ovarian toxicant that significantly reduces fertility [ ] [ ] [ ] [ ] [ ] . further, some recent studies indicate that prenatal exposure to bpa causes adverse transgenerational effects on ovarian function and fertility in female offspring [ , ] . we tested the hypothesis that bpa exposure inhibits ovarian follicle growth and induces atresia, leading to low e levels. we obtained ovaries and sera from rats treated by the fda with vehicle control, bpa ( . , , , , or , μg/kg/day), or ee ( . or . μg/kg/day) from gd until year. ovaries and sera were collected on pnd , , and and at months and year and shipped to the flaws laboratory at the university of illinois. ovaries were histologically evaluated to determine the effect of bpa and ee on the numbers and health of primordial, primary, preantral, and antral follicles. we also measured e and progesterone levels in sera. exposure to bpa ( . and μg/kg/day) decreased numbers of primordial, primary, preantral, and total healthy follicle numbers at pnd (fig. ). exposure to ee ( . μg) decreased preantral follicles (pnd , months) and antral follicles (pnd and months) and increased primary follicles ( year) compared to controls. additionally, both bpa ( and , μg/kg/day) and ee ( . and . μg/kg/ day) exposure decreased e levels in animals dosed for year (fig. ). collectively, these results indicate that ee and bpa exposures at some doses and timepoints affect ovarian follicle numbers and sex steroid levels in rats [ ] . our previous data indicate that bpa exposure increases atresia and reduces antral follicle growth in mice [ , , ] . however, bpa exposure did not increase atresia or reduce antral follicle growth in the clarity-bpa study. we did observe some effects of bpa on ovarian follicles that were similar to studies in other species, such as lambs and mice. in our study, bpa exposure at . and μg/kg/day decreased follicle numbers at pnd . in a study of lambs, rivera et al. showed that subcutaneous exposure to μg bpa from pnd - decreases the primordial follicle pool in pnd ovaries [ ] . in a study of mice, prenatal exposure to . or μg/kg/day bpa decreases the number of primordial follicles present in pnd ovaries [ ] . these studies indicate that several species are susceptible to bpa-induced reductions in follicle numbers. contrary to mouse and lamb studies, however, we did not examine follicle populations at pnd or . thus, we may have missed effects of bpa on pnd and pnd ovaries. it is also possible that effects of bpa exposure on the rat ovary may occur at later timepoints than in the mouse or at earlier timepoints than in the lamb. similar to our study, other studies with rats show that exposure to bpa affects follicle numbers. one particular study found that bpa exposure ( . or μg/kg/day from gd to pnd ) decreases the number of primary follicles in female wistar rats [ ] . another study found that exposure to bpa ( μg) from gd to pnd increases the number of primary, secondary, antral, and total follicles in the ovary compared to control wistar rats [ ] . although the effects of bpa on specific follicle populations differed between our study and other rat studies, they collectively indicate that prenatal and prepubertal exposure to bpa can affect follicle numbers in the ovary. any differences in the effects of bpa on specific follicle populations likely stem from different doses and timing of exposure. our previous studies indicate that bpa exposure significantly inhibits ovarian steroidogenesis, leading to reduced e levels , so we also expected that bpa exposure would significantly decrease sex steroid hormone levels in rats. although some doses of bpa significantly decreased e levels, not all bpa doses affected e levels in rats. however, we may not have statistical power to observe significant effects of bpa exposure on hormone levels at some doses and timepoints. we initially planned to collect and analyze serum samples from each treatment group and timepoint. however, cycling females were euthanized when predicted to be in estrus based on a vaginal smear from the previous day, and this method is not always successful in predicting estrous cyclicity on the collection day. because it is important to measure hormone levels from animals on the same day of the estrous cycle, the sample size was < in some treatment groups used for hormone analysis. this could have reduced our statistical power to observe differences between treatment groups. in fact, we noticed that several bpa treatments reduced e levels compared to controls at year, but this reduction was not always statistically significant (p > . ). more than a decade of work by the patisaul laboratory and others using multiple rodent models has repeatedly shown that developmental exposure to bpa can alter the structure and sexual differentiation of many brain regions, including the anteroventral periventricular nucleus (avpv), amygdala, medial preoptic area, and mediobasal hypothalamus, resulting in altered sexually dimorphic behaviors, particularly anxiety [ ] [ ] [ ] [ ] [ ] [ ] . available human data corroborate animal data and link prenatal exposure to heightened risk of deleterious childhood behaviors including anxiety [ ] [ ] [ ] [ ] . further, our work shows that the avpv is particularly sensitive, with perinatal bpa altering its physical size and its sex-specific dimorphism of dopaminergic and kisspeptin neurons, among other on pnd , rats from each group were euthanized, and one ovary from each animal was fixed for histological evaluation of ovarian follicle types. graph represents mean ± sem of number of follicles. *significant difference between control group and bpa or ee groups (n = - ; p ≤ . ). outcomes [ , , ] . of most relevance to clarity-bpa, work in multiple rat strains shows that developmental bpa exposure can alter expression of ers in multiple brain regions, including the avpv, amygdala, and surrounding structures, that coordinate reproductive and other sexually dimorphic behaviors [ , ] . two of these studies were conducted in the same animal strain and in the same facility as clarity-bpa, as a prelude to clarity-bpa studies under nearly identical conditions and using a similar dose range. the results of these three studies are summarized in fig. . the first of these studies (study a [ ] ) was initiated by sherry ferguson and colleagues at nctr and used two doses of bpa ( . and μg/kg/day), two doses of ee ( and μg/kg/day), a vehicle control (carboxy methylcellulose, cmc), and, uniquely, a naïve control (which underwent the same handling as gavage without inserting the gavage needle). exposure was entirely prenatal, with dams exposed from gd through the day of birth. heads of pnd offspring were rapidly frozen and shipped to the patisaul lab for quantification of erα and erβ expression in the hypothalamus and amygdala via in situ hybridization. as expected, bpa-related effects were region-, dose-, and sex-specific, with some known sex differences in er expression eliminated at the lowest bpa dose of . μg/kg/day [ ] . bpa-and ee-related effects were directionally similar, with exposure resulting in upregulation of er (α or β) in most circumstances. the most striking result, however, was a substantial difference in er levels between vehicle and naïve controls. er levels were markedly lower in gavaged controls, particularly in the amygdala, a region integral to stress-and fear-related responses. erβ expression was especially responsive. these results suggest a suppressive effect of gavage on er expression, a finding that has significant implications because gavage is traditionally the dosing method of choice for regulatory-compliant toxicity studies. additionally, bpa and ee exposure-related increases in er expression generally returned expression levels to a range typical of naïve animals. thus, it was concluded that increased expression levels in exposed animals likely reflect an interaction of exposure and stress. because naïve animals did not consume the vehicle, however, effect of the vehicle itself cannot be ruled out. the second study conducted as a prelude to clarity-bpa (study b [ ] ) was a -day subchronic study designed and carried out by nctr and similar in scale to clarity-bpa [ , ] . the patisaul laboratory's portion of the project examined only females but included vehicle controls of both sexes to ensure known sex differences could be reliably detected and to establish the degree to which bpa and ee could "masculinize" the female brain. four doses of bpa ( . , , , and μg/kg/day) and two doses of ee ( . and μg) were used. because it was initiated before completion of the study described above, no naïve controls were included. two exposure windows were used: gd through pnd , and gd through pnd . offspring brains were isolated and frozen on pnd or pnd and analyzed for er expression in the preoptic area via in situ hybridization, as done in the prior study. concordant with the pnd study, effects were region-and sexspecific. in this case, however, bpa exposure generally decreased er expression, which is opposite of what was found on pnd . this is not surprising given the dramatic age-dependent differences in baseline er expression levels observed across the rodent brain [ , , ] . as in the pnd study, erβ appeared to be more sensitive. effects of bpa and ee were, again, generally concordant in direction but not necessarily dose. low-dose bpa effects were not always recapitulated by the lowest dose of ee, with similar results at higher doses. many prior studies have shown that bpa and other endocrine disrupting chemical-related effects are not always linear [ , ] , with some effects observable at low but not high doses for reasons that remain elusive. whether these nonmonotonic effects are reproducible in brain and other tissues was of interest in the clarity-bpa study. we hypothesized that bpa would alter sexually dimorphic and steroid hormone sensitive brain morphology, gene expression and behavior. we tested this hypothesis by ) assessing brain transcriptomics in the hypothalamus, hippocampus and amygdala on pnd ; b) quantifying the size of multiple sexually dimorphic brain areas (the avpv, sexually dimorphic nucleus (sdn), posterior dorsal portion of the medial amygdala (mepd), and locus coeruleus (lc)) in juveniles and; c) testing for behavioral changes in juveniles and adults. clarity-bpa studies used two groups of animals. the first group was for behavioral analyses by the patisaul lab and the laboratory of cheryl rosenfeld from the university of missouri, in collaboration with sherry ferguson and her research team at nctr. exposure spanned gd to pnd and, given the laborious nature of the studies, included only a subset of dose groups available in the clarity-bpa study (vehicle; . , , and μg/kg/day bpa; . μg/kg/day ee). rats designated for this study were transferred from the main facility to a separate building at weaning to assess anxiety-related behaviors, exploratory behavior, and spatial navigation (rosenfeld laboratory). one group of animals was tested as juveniles; another was tested as adults. the brains of juvenile animals were collected and analyzed by unbiased stereology for evidence of abrogated volumetric sex differences in the avpv, sexually dimorphic nucleus (sdn), posteriodorsal portion of the medial amygdala (mepd), and locus coeruleus (lc) [ ] . . effects of continuous exposure to ee and bpa at year. at year, rats from each group were euthanized and serum was collected from the blood to measure sex steroid hormones. graph represents means ± sem of the amount of estradiol present in serum. *significant difference between control group and bpa or ee groups (n = - ; p ≤ . ); p > . . the second group of clarity-bpa animals analyzed was similar to the first set of the two pre-clarity-bpa studies and, accordingly, er expression on pnd was of primary interest. these rats were exposed prenatally to one of five doses of bpa ( . , , , , or , μg/ kg/day), vehicle, or two doses of ee ( . or . μg/kg/day) and collected on pnd . pre-clarity-bpa studies used in situ hybridization, but while this technique allows exceptionally high anatomical resolution, it is limited to identification of only one or two genes per section at a time. because identification of other previously unidentified gene pathways was also considered an important goal, clarity-bpa brains were analyzed by a combination of targeted and untargeted transcriptomics assays, with the hypothesis that exposure would alter er expression levels and other targets in the er signaling cascade. three regions of interest (hypothalamus, hippocampus, and amygdala) were isolated by microdissection, and rna was analyzed by rna sequencing and qrt-pcr to obtain a richer picture of the genes impacted by exposure. despite numerous prior studies showing robust and reproducible effects of developmental bpa exposure on anxiety and exploratory behaviors [ , ] , effects in this case were subtle and sporadic. for example, in the open field test, which assesses exploratory behavior and anxiety, juveniles exposed to . and μg/kg/day bpa had statistically significant effects at a few interval endpoints, such as time resting in the second five minutes of the test. however, overall evidence for bpa-related effects was minimal and inconsistent and thus not interpreted to be indicative of a biologically meaningful effect [ ] . in unexposed controls, some anticipated sex differences were either not detected or the opposite of expected effects, leading to the conclusion that some behavioral sex differences may be uniquely different in the nctr-sprague dawley strain compared to other sprague dawley strains [ ] , a finding not atypical for in-house rodent strains. vehicle controls showed expected volumetric sex differences (avpv, sdn, and mepd) and no exposure eliminated those differences. although one group has previously reported a volumetric sex difference in the lc that is sensitive to neonatal steroid manipulation, and another has reported sensitivity to developmental bpa exposure [ , ] , neither phenomenon was observed in clarity-bpa animals. this dimorphism may be strain-specific and thus not a universally applicable endpoint for endocrine disruption. as in prior studies, however, the avpv was particularly sensitive to bpa. all doses of bpa enlarged the female avpv, and a similar enlargement was observed in males at and μg/kg/day doses. because endogenous estrogen, via action of erα, reduces (masculinizes) avpv volume [ , ] , the effect of bpa observed in clarity-bpa animals was consistent with anti-estrogenic activity. bpa also increased mepd volume, but only in the right mepd of males exposed to μg/ kg/day bpa. the mepd has numerous structural and functional asymmetries, some of which are maintained by circulating androgens [ , ] , thus an effect on only one side is biologically plausible. however, the functional significance of the bpa-related effect is not clear. the mepd integrates olfactory and pheromonal information with hormonal, social, and other cues to facilitate appropriate reproductive behaviors in adulthood [ ] . fig. . concordance of bpa-induced er mrna expression changes in the amygdala and hypothalamus across nctr-based studies. a. estrogen receptor beta (esr ) expression in the medial amygdala (mepd) is sexually dimorphic at birth, with higher levels in females, but switches at approximately pnd , demonstrating how expression can change across development. significant differences in expression compared to pnd levels are represented by **p < . ; significant sex differences are represented by † < . . b. a representative autoradiogram depicting the sex-specific expression of esr in the mepd and the neighboring central portion of the ventrolateral region of the ventromedial nucleus (cvmnvl) on pnd . as the sex difference in mepd expression diminishes, the one in the cvmnvl remains pronounced demonstrating that sex differences in er expression are age and regionspecific. c. an example from study revealing how dramatically different esr expression differed between the gavaged (vehicle) and naïve controls. bpa exposure elevated esr expression in both sexes but not to the level of the naïve controls. ***p < . ; ### < . compared to male vehicle controls; § § § < . compared to female vehicle controls. the direction of er (esr and esr ) expression changes by dose and study are summarized in table for the hypothalamus and amygdala. images adapted and compiled from prior patisaul publications [ , , ] . in the pnd animals overall, the greatest number of differentially expressed genes were in the male hypothalamus and female amygdala [ , ] . in the hypothalamus, elevated erα and erβ expression was observed in both sexes at . , , and μg bpa. in the hippocampus, the only evidence of er disruption was heightened erβ expression in males at the , μg/kg/day bpa dose. similarly, only erβ was altered in the amygdala, with expression levels non-monotonically heightened in both sexes. pathway analysis in the amygdala of both sexes revealed enrichment for corticotropin releasing hormone signaling, an outcome concordant with extensive prior data suggesting bpa-related effects on anxiety and other stress-related behaviors. similarly, gonadotropin releasing hormone (gnrh) signaling was also identified as a perturbed pathway, consistent with prior work by patisaul and others showing bpa-related disruption of the avpv and hypothalamic-pituitary-gonadal axis even at low doses. significantly, clarity-bpa transcriptome data were consistent with data obtained in the first study by the patisaul laboratory in conjunction with nctr showing heightened er expression in bpa-exposed animals. this reproducibility is particularly remarkable because the two studies used different techniques. the clarity-bpa study used microisolated tissue containing the entire region of interest, which allowed assessment of the entire transcriptome but lacked the anatomical resolution of in situ hybridization. nevertheless, both studies showed that prenatal bpa exposure disrupts neonatal er expression in the hypothalamus and amygdala. transcriptomics analysis was also confirmatory for bpa-related effects in other hormone-sensitive pathways critical for sociosexual behaviors. additional genes altered by bpa included oxytocin and gaba vesicular transporter (slc a ) in the hypothalamus, oxytocin in the hippocampus, and androgen receptor, oxytocin, and vasopressin receptors in the amygdala. numerous genes involved in glutamate signaling were also disrupted in the amygdala. further, disruption of oxytocin and vasopressin signaling has been identified previously by patisaul and colleagues as sensitive to bpa exposure [ , ] . the clarity-bpa studies and two preceding collaborative nctr studies unequivocally show that er expression in the rat brain is altered by developmental exposure to bpa at doses as low as . μg/kg/day. in neonates, er expression is generally heightened, which likely sensitizes the brain to endogenous estrogen. this may explain why bpa is so often observed to be "estrogenic" in vivo, despite its limited binding affinity for ers in vitro [ , ] . this is particularly significant given that the brain can synthesize its own estrogen and is thus not necessarily dependent on circulating levels [ , ] . disruption of brain er is one of the most consistently observed outcomes of developmental bpa exposure. additionally, clarity-bpa studies provide further compelling evidence that developmental bpa exposure alters oxytocin-and vasopressin-related signaling pathways and avpv volume. significantly, clarity-bpa gene expression data from the rosenfeld lab with older animals are highly concordant. using clarity-bpa rats that were tested on the barnes maze to assess spatial navigation abilities and euthanized at months of age, the rosenfeld lab found evidence for disruption of hippocampal oxytocin and vasopressin gene expression in animals dosed at μg/kg/day bpa from gd through pnd [ ] . hypothalamic erα was downregulated in males exposed to bpa or . μg ee, while hypothalamic erβ was only reduced in ee-exposed males. directionally, these effects are consistent with those found by patisaul's team in pnd animals from the subchronic exposure pre-clarity-bpa study [ ] . collectively, these data are consistent with robust literature by patisaul the rosenfeld lab previously showed that developmental exposure to varying doses of bpa that are considered environmentally relevant affects spatial navigational learning and memory in polygynous deer mice (peromyscus maniculatus bairdii) [ , ] . male deer mice show enhanced spatial navigational ability compared to males of related peromyscus spp. [ ] . this behavior in deer mice is considered a sexually selected trait because it confers an advantage in locating females that are likely widely dispersed throughout the habitat [ ] . however, male deer mice developmentally exposed to bpa show reduced spatial navigational learning and memory [ , ] , suggesting that they would be at a disadvantage in locating potential female breeding partners. we also showed that even if they are able to locate potential reproductive partners, females prefer control males : over males with early bpa exposure [ ] . further, female deer mice developmentally exposed to bpa or ee show masculinized or improved spatial navigational learning and memory. these previous studies also show that the dietary exposure dose provided to dams results in similar serum concentrations identified in pregnant women unknowingly exposed to this chemical [ ] . follow-up studies in the related species of california mice (peromyscus californicus), who are monogamous and biparental, show that developmental exposure to bpa and ee does not affect spatial navigational learning and memory in males or females. however, bpa reduces socio-communicative behaviors and affects biparental care [ ] . examination of the global transcriptomic profile in the hypothalamus revealed several genes that are differentially expressed in male and female california mice developmentally exposed to bpa [ ] . gene expression differences also persist in the hypothalamus of male and female california mice engaged in parental behaviors [ ] . such gene expression differences are likely due to dna methylation and potentially other epigenetic changes [ ] [ ] [ ] [ ] . our previous collective findings indicate that, in a rodent model, bpa can disrupt transcriptomic profiles in the hypothalamus that might be epigenetic in origin. spatial navigational learning and memory can be affected by early exposure to bpa, especially in those species in which it is considered a sexually selected trait. other rodent and human studies suggest that males typically tend to exhibit enhanced spatial navigational learning and memory compared to females [ ] [ ] [ ] . several other rodent studies, including in rats, indicate that developmental exposure to bpa can affect spatial navigational learning and memory, with males typically being more vulnerable [ ] [ ] [ ] [ ] [ ] [ ] [ ] . boys developmentally exposed to a stronger estrogen, diethylstilbestrol (des), during gestation also show impairments in this behavioral response compared to unexposed agematched boys [ ] . past non-human primate models, rodent and zebrafish (danio rerio) animal models, and in vitro cell culture studies strongly indicate bpa alters individual candidate genes in the hippocampus, hypothalamus, or isolated neurons from these and associated brain regions [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . some implicated genes and their protein products include erα (esr and transcript variants), erβ (esr ), dna methyltransferases (dnmt , a, b) , androgen receptor (ar), brain derived neural factor (bdnf), vasopression (avp), and oxytocin receptor (otr).a this study tested the following hypotheses. study : female and male rats developmentally exposed to bpa show later spatial navigational learning and memory impairments. study : developmental exposure of rats to bpa or ee induces behavior-relevant gene expression and dna methylation changes in the hippocampus and hypothalamus at adulthood. in study , pregnant nctr-sprague dawley rats were orally dosed from gd to parturition, and offspring were directly orally dosed until weaning (pnd ). treatment groups included vehicle control, three bpa doses ( . , , or μg/kg/day), and a . μg/kg/day ee reference estrogen dose. at adulthood, one animal/sex/litter was tested for days in the barnes maze. after completion of this and other behavioral tests, animals were humanely euthanized and measured for serum testosterone concentrations. in study , rna and dna were isolated from hypothalamus and hippocampus to examine expression of genes (dnmt , dnmt a, dnmt b, esr , esr , avp, ar, ot, otr, and bdnf) potentially affected by early-life bpa ( μg/kg/day) or . μg/kg/day ee exposure. three genes (bdnf, dnmt b, and esr ) were examined for dna methylation changes in their putative ′ promoter regions. molecular changes in the hippocampus were correlated to prior barnes maze performance (measured in study ), including sniffing correct holes, distance traveled, and velocity. the μg/kg/day bpa group sniffed more incorrect holes on day than those in control, . μg/kg/day bpa, and ee groups. notably, μg/kg/day bpa females were less likely than control females to locate the escape box in the allotted time (p = . ; fig. a ). similarly, . μg/kg/day bpa females showed a trend for prolonged latency to locate the escape hole during the -minute time period. paradoxically, . μg/kg/day bpa males showed improved latency to locate the correct escape hole relative to control males (p = . ; fig. b ). the significance of this finding remains uncertain. no differences in serum testosterone concentration were detected in any male or female treatment groups. these results suggest that developmental exposure of rats to bpa may disrupt aspects of spatial navigational learning and memory. study : exposure to bpa and/or ee ablated normal profiles of sexually dimorphic gene expression/promoter dna methylation that should have otherwise been observed in the hippocampus and hypothalamus. bpa exposure led to hypermethylation of the putative ′ promoter region of hippocampal bdnf, whereas in this same brain region, ee-exposure resulted in hypomethylation of bdnf in female rats ( fig. a-c) . bdnf methylation was weakly associated with bdnf expression in hippocampi of male and of female rats (fig. a) . bdnf methylation tended to correlate with its gene expression pattern in female hippocampi. hippocampal bdnf expression in females showed a trending negative association with sniffing the correct hole in the barnes maze (fig. b) . hippocampal expression of avp, esr , oxt, and otr were strongly and positively associated with velocity of control rats in the barnes maze, but such correlations were absent in bpa-and eeexposed rats (fig. c-f) . these findings suggest bpa exposure induces unique gene expression and epigenetic changes in hypothalamus and hippocampus of adult rats, with the latter brain region governing spatial learning and memory ability (fig. ) . there are likely several explanations why the current clarity-bpa study results differed in relation to the effects of bpa on spatial learning and memory behaviors from our past studies with deer mice where developmental exposure to bpa had greater effects in males by impairing spatial learning and memory [ , ] . in contrast, data obtained from the clarity-bpa study suggests that females were more vulnerable to the effects of bpa as developmental exposure to this chemical caused spatial learning and memory deficits in females, whereas, developmental exposure to bpa either did not alter this behavior in males or inexplicably at the lowest dose tested ( . μg/kg/day bpa) enhanced this trait in males. the first is species-as detailed above, spatial navigational learning and memory is an important behavioral response for polygynous deer mice. it is not clear the extent that this behavior has undergone evolutionary selection in male or female nctr-sprague dawley rats. the fact that the response was greater in female than male rats indicates it is not a male sexually selected trait in this species. another major reason for conflicting findings is the dose and route of exposure. in our previous studies, bpa was incorporated into the diet to replicate the primary and chronic route of exposure in most humans [ , ] . additionally, this method is considered non-invasive and induces minimal stress to pregnant dams and neonates. instead, clarity-bpa studies used oral gavage to dose pregnant dams and neonates. while this might replicate daily oral exposure in humans, it could also introduce a degree of stress. while internal serum concentrations of bpa were not measured in the current studies, an analogous fda study did measure such concentrations following similar exposure doses [ ] . that previous study reported internal dosimetry data collected in the preceding nctr day subchronic bpa study, which used the same animal model, dosing regimen (i.e., daily oral gavage of dams from gd through start of fig. . overall hazard ratio for (a) females and (b) males in each treatment group to locate the escape hole in a barnes maze. note that increasing ratio equates to shorter latency. for both graphs, upper, middle, and lower bars represent ratio of locating the correct escape hole at % upper confidence limit, mean, and % lower confidence limit, respectively, for each group. hazard ratio was used to account for those individuals who did not locate the escape hole in the allotted time ( min). comparisons of the significant two-way interaction for treatment * sex are shown. the bpa doses chosen for clarity-bpa were selected to provide low, middle, and upper levels of exposure and to be below the no-observed-adverse-effect level (noael) of mg/kg/day bpa as detailed previously [ , ] . however, it is not clear if these doses actually replicate internal circulating concentrations in pregnant women and those unknowingly exposed to the chemical. in our past studies, we also exposed dams to bpa-treated diets from weeks before conception through lactation, as studies suggest that bpa can be transferred across the placenta and via milk [ ] [ ] [ ] [ ] . however, current studies directly exposed neonates to bpa via oral gavage, which could have stressed the animals. other potential explanations to account for disparate results between the clarity-bpa and past studies include differences in animal husbandry, such as phytoestrogen contamination in the diet and bpa contamination from cages and other housing equipment, and age of individuals at the time of behavior and biomolecular assessments. even so, the experiments done as part of the clarity-bpa project suggest that bpa can affect aspects of spatial learning and memory and lead to associated changes in dna methylation and gene expression in the hippocampus and hypothalamus. the belcher lab previously reported the effects of bpa on contractile function in isolated myocytes and in the hearts of both rats and mice. those studies demonstrate that very low concentrations of bpa and α-estradiol sex-specifically alter rapid estrogen signaling in females by mechanisms that involve activation of erα and erβ [ ] . in those studies, bpa exposure caused abnormal ca + handling, altered excitation-contraction coupling, and increased arrhythmias in females [ ] . additional in vivo studies demonstrate that bpa exposure sexspecifically alters collagen content, modifies the extracellular matrix of hearts of both males and females, alters fatty acid and glycolytic metabolism, and increases sensitivity of the female heart to ischemic damage [ ] . several additional studies demonstrate that bpa impacts the heart and cardiac function and indicate that the heart is a target for effects of bpa [ ] [ ] [ ] . the finding that bpa, like endogenous estrogen, can impact cardiac function is not surprising-ers are expressed in the heart of both males and females [ ] , but the impacts of bpa in the heart are often sex-specifically regulated and can differ in males and females [ ] [ ] [ ] [ ] [ ] . the human relevance of these experimental studies is supported by numerous epidemiological studies and systematic reviews that support an association between higher bpa exposures and increased risk for cardiovascular disease, obesity, type diabetes, insulin resistance, and hypertension in adults as well as obesity in children [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this study tested the central hypothesis that bpa has harmful disruptive effects on the heart, in a dose-dependent fashion (possibly nonmonotonic), that result in cardiac pathology. with early-life exposure to vehicle control (white), . μg/kg/day ee (orange), or μg/kg/day bpa (violet). data expressed as mean ± sem. each circle represents average percent methylation of each corresponding cg site from - individual colonies (n = males and n = females per group). for methylation analysis, *p < . , ***p < . , and ****p < . vs vehicle by two-way anova and tukey's multiple test comparison. for gene expression analysis, ***p < . vs vehicle by one-way anova and tukey test. gene expression levels are expressed as gene expression level in treatment group relative to control group using the −ΔΔct method. reproduced with permission from the epigenetics. quantitative morphometry and histopathology analysis were performed on hearts isolated at pnd and and at months of the clarity-bpa study to determine the effects of bpa and ee. at each analyzed timepoint, animals were weighed and euthanized. hearts were harvested and their weights were recorded at nctr. isolated hearts from vehicle ( . % cmc), bpa ( . , , , , or , μg/kg/day), and ee ( . or . μg/kg/day) groups were fixed for h in % formalin, post-fixed in fresh neutral buffered formalin for an additional h, transferred to % ethanol, and shipped to the belcher laboratory at the university of cincinnati. there, hearts were prepared, sectioned, and stained with: ) hematoxylin and eosin for histological evaluation of tissue structure, cellular morphology, and pathology; or ) picrosirius red to quantify total collagen. total left ventricle (lv) area, diameter, wall thickness, and collagen staining were measured and calculated [ , ] . cardiomyopathy, late stage cardiomyopathy (focal fibrosis), diffuse degeneration, and inflammatory infiltration phenotypes were each scored according to the standardized system of nomenclature and diagnostic criteria [ ] , with cardiomyopathy and lv pathology assessed using a standardized four-point severity scale [ , ] . the largest observed effects in the study animals analyzed were due to treatment duration (stop-dose vs continuous-dose) and linked to increased stress resulting from daily gavage of offspring. mean body weight of stop-dose control males at pnd was . % greater than in the continuously exposed control group. observed differences in body weights were indicative of effects due to post-weaning dosing procedures and/or vehicle in continuously dosed animals. the sex-specific decreased weight found only in males was consistent with previous studies showing that prolonged postnatal stress in males can decrease weight gain over time, whereas body weight of female sprague dawley rats is resistant to the effects of stress [ ] [ ] [ ] . evidence of bpa or ee alterations on gross cardiac endpoints related to cardiac hypertrophy were limited. alterations in lv wall thickness were not observed at any dose in either sex. a significant decrease in heart weight and heart weight normalized to body weight was observed in females exposed to . μg/kg/day bpa, consistent with our previous findings in mice [ ] . in males, collagen accumulation was increased in the highest ee dose group at pnd . in female hearts, decreased collagen content was observed in rats treated with , μg/kg/day bpa at pnd and . μg/kg/day ee at months. in the clarity-bpa study, abundant early progressive cardiomyopathy lesions were found in the hearts of most control and exposed animals analyzed at pnd [ ] . lesion incidence and severity were greater in control males than in females. in bpa-or ee-treated females at pnd , cardiomyopathy incidence was increased compared to control females, with significant increases in severity detected in . , , or , μg/kg/day bpa groups and in both ee groups. in a male exposed to μg/kg/day bpa and female from each of the two lowest bpa dose groups ( . and μg/kg/day), a diffuse degeneration phenotype involving much of the myocardium was also observed [ ] . at pnd and months, cardiomyopathy in both males and females was observed in all control samples from both stop-dose and continuous-dose arms of the clarity-bpa study [ ] . at pnd , the diffuse degeneration phenotype was again observed in males and females from continuous-dose (males: , , , μg/kg/day bpa and . μg/kg/day ee; females: . , μg/kg/day bpa and . , . μg/kg/day ee) and stop-dose (males: , , μg kg/day bpa and . , . μg/kg/day ee; females: μg/kg/day bpa) groups. this degree of extensive cardiac pathology is an indication of exposure-related cardiotoxicity [ ] . bpa significantly decreased heart weight and heart weight normalized to body weight in females exposed to . μg/kg/day bpa, consistent with our previous findings in mice [ ] . in female hearts, decreased collagen content was observed in rats treated with , μg/kg/day bpa at pnd and . μg/kg/day ee at months. cardiomyopathy incidence was increased compared to control females in . , , or , μg/kg/day bpa groups and in both ee groups. at pnd and months, cardiomyopathy in both males and females was observed in all control samples from both stop-dose and continuous-dose arms of the clarity-bpa study, precluding detection of bpa-specific effects. the lack of overt morphology phenotypes in clarity-bpa or eeexposed hearts was expected. pathology associated with the majority of cardiac insults, including toxicants, typically becomes evident only after adverse cardiovascular events, such as cardiac ischemia or velocity is expressed as mean from seven observations. p < . was considered statistically significant. p < . was considered statistically significant. only samples with detectable ct values were used in correlation analysis. reproduced with permission from the epigenetics. myocardial infarction [ , ] . for experimental studies with rodents, it is well-accepted that an intervention resulting in increased βadrenergic stimulation, ischemic injury, or genetic manipulation is required to elevate cardiac fibrosis, hypertrophy, and phenotypes resulting in overt cardiac pathology [ ] . such manipulations were not possible in the clarity-bpa study and limit any interpretations resulting from negative data. additionally, compared to control mice, the hearts of control nctr-sprague dawley rats had relatively higher levels of collagen due to known species-specific differences in the proportions of myocytes and fibroblasts present in mouse and rat hearts [ ] . based on increased severity and incidence of cardiomyopathy lesions and the diffuse cardiac degeneration phenotypes observed, the noael for bpa in the heart found in clarity-bpa study was < . ug/ kg/day. cardiac lesions were also observed more often, and at lower doses in females, findings that support previously reported sex difference for the adverse effects of bpa in the heart. previous studies indicated that bpa may interfere with thyroid hormone action. moriyama et al. [ ] reported that bpa is an indirect antagonist on the two major forms of thyroid hormone receptor (tr), trα and trβ . others have shown that bpa can interfere with thyroid hormone-dependent processes in frogs [ ] and zebrafish [ ] . the zoeller lab reported that bpa exposure increases total serum thyroxine t in -week-old male and female rat pups [ ] , consistent with an inhibitory effect of bpa on trβ in the pituitary that mediates negative feedback of t on thyroid stimulating hormone (tsh) [ ] . however, bpa exposure increases hippocampal rc /neurogranin mrna in male pups, which is directly regulated by thyroid hormone through trα [ , ] , consistent with elevated serum t [ ] . these data indicate that bpa may selectively antagonize trβ compared to trα, producing a hormonal profile similar to that of thyroid resistance syndrome [ ] . however, literature about the potential action(s) of bpa on thyroid hormone signaling is complex. lee et al. [ ] reported that bpa could reduce expression of several genes in rat gh cells related to controlling thyroid hormone levels, but only at μm. no effects of bpa were observed in frtl- cells. sheng et al. [ ] reported that − m bpa could suppress t -induced gene expression in cv- cells, but through a non-genomic mechanism. kitamura et al. [ ] reported that bpa essentially does not bind to mammalian tr. likewise, xu et al. [ ] reported that bpa does not affect thyroid hormone signaling in perinatal rats, and kobayashi et al. [ ] reported that bpa exposure of rat dams from gd to pnd does not affect serum t levels in offspring at weeks of age. in humans, park et al. [ ] reported that bpa is negatively associated with serum tsh, an observation similar to that of aung et al. [ ] . these data indicate that the effect of bpa on thyroid hormone signaling is dependent upon the context, including the cell type, receptor isoform, and species. table tabulates animal studies published to date that have evaluated the effect of bpa on thyroid hormone. these data present a complex picture, but variability in experimental designs suggests that timing of analysis may be a key factor. this clarity-bpa study on the thyroid aimed to determine whether: ) bpa exposure reduces serum thyroid hormone levels, and ) effects on serum thyroid hormone levels affect specific thyroid-dependent endpoints in the developing brain. we received serum, brain, liver, pituitary, and heart tissues from all doses of bpa in the clarity-bpa animals on pnd as well as a separate set of controls and rats of the same age treated with the drug propylthiouracil (ptu). ptu was used because it is known to reduce serum t and this would allow us to ensure that our techniques were capable of measuring endpoints sensitive to thyroid hormone. we measured serum thyroid hormones and several thyroid-dependent endpoints in clarity-bpa and ptu-treated animals and did follow-up studies on ptu-treated animals. our findings indicate that nctr-sprague dawley rats are remarkably insensitive to low thyroid hormones for unknown reasons and are therefore not appropriate to study thyroid "disruption" in general. specifically, while ptu treatment reduced serum t and increased serum tsh in a predicable manner, there were virtually no effects on endpoints of thyroid hormone action in the developing brain. this observation is not consistent with any other published study of low thyroid hormone on brain development. bpa did not affect serum thyroid hormones in these nctr sprague-dawley rats, in contrast to all other studies using different strains of rats. it would be incredibly complex to unravel why clarity-bpa data were not consistent with our previous findings or published literature. at this stage, our best guess is that nctr-sprague dawley rats simply do not respond to low thyroid hormone in a manner that has been published before. with the same individual male and female rats tested for multiple experiments that span various systems, the current studies provide a unique opportunity to integrate these interdependent datasets together to determine how developmental exposure to bpa affects not just one independent system but may induce systemic and inter-related effects. moreover, using the same rats across study designs may permit us to gain insights into the biology of how various bodily systems relate and may influence each other, whether through hormonal or other regulatory factors. to achieve these lofty goals, we used the mixomics r package [ ] , as we have done in previous bpa studies performed in the rosenfeld laboratory [ ] [ ] [ ] . in this case, we used the program to correlate data obtained by independent clarity-bpa investigators. independent datasets obtained from the master chemical effects in biological systems (cebs) database at niehs were matched based on individual animal id, such that data from the same individual used across various study designs were linked together. however, not all investigators used the same sets of animals. thus, to increase the amount of data integration that could be achieved, we also linked those results for animals from the same litter but that were used in different studies. rats used in patisaul's and rosenfeld's behavioral studies were generated from different litters than those used by other clarity-bpa investigators. to be able to link the young adult ∼ days of age behavioral studies with results from around this same time, animals used for these studies were pair-matched to similar counterparts at around this age based on the similar birth date and animal body weight. in the initial analyses, we considered effects of the low dose bpa ( . μg/kg) at days of age, set , where several investigators used the same set of animals. in follow-up analyses, we considered the effects of dose ( . , , and μg/kg/day) and age ( days of age-weaning, young adult- - days of age, and older adult days of age), and the integrative analyses for these additional dose and ages where done by using the methods described above. even so, there were still individual investigator data because of sample replicates and other issues that could not be integrated with the data from other investigators. herein, we present the r plot results for females and males at days of age who were developmentally exposed to bpa at . μg/kg/day. the r plots based on these other ages, doses, and sexes, which were used to generate tables and and supplementary tables - , are included in supplementary file . we conducted sparse discriminant analysis with partial least square regression with function 'block.splsda'. circos plots were generated using the 'circosplot' function, with correlations calculated as described by gonzález et al. [ ] . this analysis provides several diagrams to show data relationships. r plots shows overall correlation between collective categories, such as behavioral, cardiovascular, uterine, mammary, etc results. in contrast, the circos plots shows individual parameters measured within the broad categories, such as uterine assessments is one broad category and within this group, apoptosis and pcna analyses were performed, relate to each other. the program developers recommend examining the circus plots at a correlation of at least . [ ] . we tested several correlations spanning from . to . and found that . provided sufficient stringency and at the same time likely provided meaningful correlations. for females at this age, integrative correlation analyses were performed for adipose tissue (measured by dr. ben-jonathan's group), ovarian follicle (dr. flaw's group), heart (dr. belcher's group), uterine (dr. ho's group), splenic (dr. kaminski's group), and mammary gland (dr. soto's group) assessments. r-plot analyses revealed that several parameters significantly correlated with each other (fig. ) . these data suggest that developmental exposure to bpa can simultaneously affect more than a single system, which could be due to direct targeting of the various organs and/or changes in one system lead to downstream affects in other organs. this type of data analysis, however, does not permit us to tease apart these possibilities, which may not be mutually exclusive, or determine directionality. follow-up studies assessing the same organs at different time points might help to decipher how bpainduced changes in one organ lead to downstream affects in other organs. this fig. circos plot analyses revealed several positive correlations between individual parameters within these groups (fig. a) . for example, serum e concentrations positively correlated with primordial follicles. serum prolactin (prl) concentrations positively correlated with primordial follicles and ovarian fat macrophages. serum leptin concentrations positively correlated with select cd +, cd +/cd +, and cd + t lymphocytes within the spleen. mammary gland epithelial area positively correlated with cd l cells within the spleen. cd l, cd +/cd +, and other cd + splenic cells also positively associated with subcutaneous and gonadal fat pad weight. apoptosis within the uterus positively correlated with heart luminal area and ovarian fat macrophages. uterine weight positively correlated with mammary gland epithelial area and cd + and cd + t lymphocytes within the spleen. serum leptin positively correlated with mammary gland fat pad area, subcutaneous fat pad weight, gonad adipocyte size, and ovarian fat macrophages. ovarian fat macrophages positively correlated with heart weight and heart collagen area. mammary gland epithelial area positively correlated with heart collagen area. several negative correlations were also evident for these females across categories (fig. b) . serum e concentrations negatively correlated with subcutaneous fat pad weight, mammary gland fat pad area and coverage, uterine necropsy weight, and cd +, cd +, and cd +/cd + t cells within the spleen. total progesterone concentrations negatively associated with cd + cells within the spleen. serum prl was inversely associated with cd + and cd +/cd + cells within the spleen. mammary gland coverage negatively correlated with uterine apoptosis and heart luminal area. preantral ovarian follicles negatively correlated with ovarian fat macrophages and subcutaneous fat pad weight. primordial ovarian follicles inversely correlated with subcutaneous adipose size, gonadal fat weight, and cd+ and cd +/cd + t lymphocytes within the spleen. total healthy ovarian follicles negatively correlated with subcutaneous adipose tissue size and mammary gland weight. total unhealthy ovarian follicles negatively associated with serum leptin concentrations. as detailed above, we extended these analyses to examine the effects of dose and age for those datasets that could be integrated together with one of the above methods. we will first consider the effects of different dosages in female rats at months of age. as shown in table , which summarizes the r plots for these different dosages, several broad categories correlated strongly with each other at the lowest dosage of bpa ( . μg/kg/day), which is also shown in fig. . this table though also reveals that several of these correlations were also evident at the middle ( μg/kg/day) and highest dosage ( μg/kg/day). a few examples of such correlations that spanned all three dosages at months of age include: white adipose tissue weight to heart, mammary gland peptide hormone steroid hormone results; ovarian follicle and mammary gland results; mammary gland to heart, peptide hormone, and steroid hormone results. in tracing the correlations back to those present at − days of age (supplementary table ). the correlations that extended all three dosages ( . , , and μg/kg/day) were mammary gland histology and uterine results and peptide hormones and splenic function results. we next considered those correlations only observed at the low dose. for instance, mammary gland morphometrics correlated with periovarian adipose tissue qpcr (r = . ); mammary gland histology correlated with ovarian follicle results (r = . ) and uterine results (r = . ); ovarian follicle results correlated with peptide hormones (r = . ) and uterine results (r = . ); peptide hormones were associated with heart results (r = . ), behavior results (r = . ), and splenic function results (r = . ); periovarian and subcutaneous adipose tissue qpcr correlated with behavior results (r = . and . , respectively); steroid hormones correlated strongly with uterine results but surprisingly not with the behavioral results (r = . and . , table mixomics data integration comparison at . , and gμ/kg/day of bpa at pnd in females. respectively); heart and behavioral results correlated with splenic function results (r = . and . , respectively). in considering those correlations present in females at days of age, the ones extend across all dosages relate to mammary gland changes (supplementary table ). for example, mammary gland morphometrics strongly correlated table mixomics data integration comparison at . , and μg/kg/day of bpa at pnd in males. nd: not determined. fig. . r plot correlations across -month-old female data for low-dose bpa exposure ( . μg/kg/day) and controls. this analysis compares white adipose tissue, ovarian follicle, heart, mammary gland, and uterine assessments with peptide and steroid hormone data, and splenic function results. values with strong correlation (r ≥ . ) are highlighted. representative pca diagrams with corresponding r-values are also delineated. in the pca diagrams, each individual replicate is represented with a circle, and the blue and orange circles represent those exposed to bpa or vehicle control, respectively. n = for bpa and vehicle control, respectively. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) across all dosages with mammary gland histology and prolactin results. at the lowest and highest dosages of bpa, mammary gland morphometrics also correlated with ovarian follicle results (r = . and . , respectively), and in turn ovarian follicle results were linked to uterine results at these two dosages (r = . and . , respectively). at the low dose of bpa, mammary gland morphometrics and prolactin and steroid hormones were strongly correlated (r = . and . , respectively). prolactin (prl) and steroid hormone results were strongly linked (r = . ), and brain and steroid function results were closely associated (r = . ). for males at months of age, integrative correlation analyses were performed for adipose tissue/peptide hormones (dr. ben-jonathan's group), heart (dr. belcher's group), prostate (dr. prins' group), and splenic function (dr. kaminsky's group) measurements. r-plot analyses revealed correlations for adipose tissue weight and heart results (r = . ) (fig. ). macrophages within adipose tissue correlated with prostate and urinary bladder weight (r = . ). adipose tissue weight and splenic function results were strongly correlated (r = . ). peptide hormones and splenic function results showed strong correlation (r = . ). prostate to heart and prostate to splenic function results had some associations (r = and . and . , respectively). several positive correlations were evident in the circos plot (fig. a) . examples include heart luminal area positively correlated with prostate necropsy weight and subcutaneous adipose tissue size. prl and adiponectin positively correlated with subcutaneous adipose tissue macrophages. gonadal fat weight and subcutaneous (sq) fat pad weight positively correlated with adipose tissue macrophages. splenic nkt (cd +) and cd + t lymphocytes positively correlated with sq macrophages. ventral prostate weight and gonadal fat weight positively correlated with splenic cd + cells. several negative correlations were evident for males at this age (fig. b ). for instance, prl concentrations were inversely associated with heart wall length. adiponectin was negatively associated with cd + and cd l cells within the spleen. sq fat pad weight was inversely linked to cd + t lymphocytes in the spleen. total ventral prostate weight was negatively linked to gonadal fat macrophages. prostate necropsy weight was in turn inversely correlated with splenic cd + t lymphocytes. gonadal fat macrophages negatively associated with several splenic cells, including total nkt cd +, cd +/cd +, cd +, cd +, and cd l+/cd + t cells. as with the female results, additional integrative correlation analyses were performed for males with the methods detailed above to examine relationships that spanned various dosages and ages. at months of age, the only correlations that spanned all three dosages include adipose tissue weight strongly correlated with adipose tissue size, which is to be expected, and the heart results ( table ). the surprising correlation between peptide hormones and prostate results evident at the lowest dosage ( . μg/kg/day) was absent at the other two dosages. at the middle dosage μg/kg/day), adipose size, adipose tissue weight, and macrophages in adipose tissue correlated with prostate results (r = . to . ). several other correlations were evident at this dosage for adipose tissue weight including to heart results and peptide hormones (r = . and . , respectively). finally, at this same dosage, heart and prostate results were strongly correlated (r = . ). several strong correlations were evident at - day old males exposed to all three doses of bpa (supplementary table ). for instance, peptide hormones correlated strongly (r ≥ . ) at all three dosages for adipose tissue size and weight. subcutaneous adipose tissue qpcr correlated with epididymal adipose tissue qpcr and macrophages in adipose tissue. qpcr for both types of adipose tissue correlated at all three dosages with splenic function results. similar to female results at this age, strong correlations (r > . ) were evident at the low dosage for various adipose tissue measurements, peptide hormones, and behavioral results, but many of these were absent at the highest dosage tested. interestingly, splenic function results showed robust correlation with behavioral results at the low and high dose of bpa (r = . and . , respectively). for males at days of age, there were limited datasets that could be combined. of those that could be integrated, the same dosages as above were not tested in all studies. as shown in supplementary table , heart and splenic function results correlated at the middle dosage but not the lowest dosage (r = . and . , respectively). brain and splenic function results correlated at the highest dosage but not the lowest dosage (r = . and . , respectively). this integrative approach has assuredly provided important clues about how various systems relate to each other. however, the main fig. . a) positive and (b) negative circos plot correlations between white adipose tissue, ovarian follicle, heart, mammary gland, and uterine assessments with peptide and steroid hormone data in -month-old females exposed to low-dose bpa ( . μg/kg/day) or controls. results for bpa-exposed females are indicated with a blue line outside of the circle; orange line indicates results for control females. color of the line further from the circle indicates treatment group where these results are greater. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) limitation of this analysis is that it only reveals correlation and not causation, thus the potential sequential order of pathological changes cannot be determined. mixomics diagrams reveal that in females exposed to low-dose bpa ( . μg/kg/day) considered safe by the fda, there are strong inter-relationships among white adipose tissue, behavior, ovarian follicle, heart, mammary gland, and uterine assessments with peptide and steroid hormone measurements. in males, this same bpa dose results in strong correlations between white adipose tissue, heart, and prostate gland measurements and peptide hormones. at all three doses of bpa, in both sexes, and multiple ages, clear associations between adipose tissue measurements, splenic function in terms of various white blood cells, and peptide hormones were evident. the circus plots (figs. and ) representing the low dose of bpa ( . μg/kg/day) in males and females further revealed that adipose tissue measurements, including macrophages within this tissue, were strongly linked to t lymphocyte populations in the spleen but the dynamics showed sex-dependent differences. for instance, in male at this low dosage an increased in gonadal fat macrophages was negatively associated with total nkt cd +, cd +/cd +, cd +, cd + cells, and cd l+/cd t lymphocytes. thus, the findings suggest that obesity may affect t lymphocyte cells, including regulatory t cells (treg-cd +) and nk-natural killer cells, present to fight various infections, including viruses. as detailed above though this approach only reveals potential associations. however, it is clear from human studies that obese individuals have reductions in these key t lymphocyte lineages [ ] [ ] [ ] [ ] . thus, the findings suggest that by acting as an obesogen [ ] , bpa may thereby also compromise immune function and render individuals susceptible to pathogens, such as covid- . the findings broadly indicate that there is strong and potentially even unrecognized inter-connectedness between organ systems, and thus, by targeting even a single tissue or organ, bpa can lead to downstream and widespread pathological changes. conceivably, bpa might also induce systemic effects on multiple tissues and organs and on hormone production. notwithstanding, these integrative analyses reveal that the effects of low-dose bpa are not confined to a single tissue, organ, or system. instead, complex changes occur in several systems in male and female rats developmentally exposed to bpa. by analyzing multiple ages, bpa doses, and results in males and females with this mixomics analysis approach, we have identified key associations across body systems. this large-scale approach was possible because of the unique design of these consortium studies, in which several parameters were measured in individual male and female rats. thus, this analysis makes it impossible to disregard effects at all three doses on health outcomes in this experiment. the fact that several associations between multiple organ systems in males and females were observed at the lowest dose tested, . μg bpa/kg/day provides convincing evidence that even this low dose, which is considered safe by most regulatory agencies, can lead to systemic health consequences. at the outset of these studies in , such integrative correlation analyses programs did not exist. consequently, it was not envisioned at the time to use the same animals for all of the investigator studies to allow for such analyses to be done. several of the animals were used across multiple studies and by using approaches detailed above, we were able to pair-match animals across studies to allow for these assessments to be done. yet, there were still individual investigator results that could not be integrated with this mixomics approach. the fact though that we were able to integrate several investigators findings together and come up with meaningful biological correlations is important, and we are not aware of a comparable study that has used such an approach to inter-relate multi-investigator data across a range of organ systems/biological endpoints, bpa doses, ages, and sexes. overall, many of the independent laboratories confirmed bpa responses in a variety of organ systems in clarity-bpa, including results in the brain, prostate, urinary tract, ovary, mammary gland, and heart (summarized in table ). bpa effects were not observed on thyroid endpoints or on sperm and immune system parameters, the latter two of which were previously published [ , , ] . but the failure of the nctr sprague-dawley rats to exhibit significant responses to low t is unique to this strain and should disqualify it as a model of thyroid disruption. the absence of any effect on sperm at any dose is problematic because the authors did not include either of the positive control (ee) dose groups which are needed to provide verification of system sensitivity [ ] . thus, the 'no effect' conclusions drawn by the authors are difficult to interpret because the sensitivity of their study to detect effects of bpa is unknown. all other independent investigators included examination of the positive control. clarity-bpa also assessed immune function although the published literature on the effects of bpa on the immune system is contradictory with little consensus as noted in [ ] . in the clarity-bpa study which examined the effect of bpa on splenocytes / measurements were statistically different from controls. the most significant effect was the augmentation of lymphoproliferation in response to pokeweed mitogen stimulations in one-year old rats. this effect was also observed in the ee group. the other positive effects were not dose dependent. the authors did not examine the data for non-monotonic dose responses and concluded that the observed changes were unlikely to compromise immune competence in adults [ , ] . toxicity of bpa was observed across a range of doses that differed in the examined organs. such organ/endpoint differences in sensitivity is expected due to the diversity of cell types, hormone receptor expression levels, vascularity, metabolism, and complexity of signaling pathways across organs. nonetheless, patterns did emerge when findings across organ systems were integrated and examined together. in many instances, the greatest, and in some cases only, statistically significant effects were observed at the lowest exposure dose used in clarity-bpa ( . μg/kg/day), as delineated previously [ ] . this demonstrates that . μg/kg-day is not a "no adverse effect level" [ ] . toxicity evaluations for a compound require determination of the lowest observable adverse effect levels (loael). a strength of the clarity-bpa consortium approach is that we were able to conduct a novel mixomics statistical analysis utilizing multiple data sets from clarity that identified statistically significant associations in . μg/ kg/day outcomes reported by multiple investigators across multiple organ systems. this analytical approach provided clear, quantitative findings demonstrating that the lowest dose tested by clarity investigators ( . μg/kg/day) led to consistent statistically significant adverse effects that cannot be dismissed as occurring randomly and are "not biologically plausible. our mixomics statistical analysis thus provides convincing evidence that . μg/kg/day is the new oral loael for bpa. in several of these studies, a non-monotonic bpa dose-response was observed. for some organs such as the mammary gland, there was a break between and μg/kg/day doses in all datasets at all ages, which was analyzed by specific statistical methods appropriate for nonmonotonicity. the fact that this break is present in all time points analyzed and in the vast majority of the specific measurements clearly indicate that the nmdrc is not spurious [ ] : on the contrary, it shows that there is an underlying causal relationship. for others a u-shaped or inverted u-shaped response (e.g., prostate stem cell numbers) was reported [ , ] or a w-shaped response (e.g. ovary [ ] ) was observed. given the variety of these non-linear dose-responses, it is reasonable to question whether the results reflect tissue, heart, and prostate assessments with peptide hormone data in -month-old males exposed to low-dose bpa ( . μg/kg/day) or controls. results for bpa-exposed males are indicated with a blue line outside the circle; orange line indicates results for control males. color of the line further from the circle indicates treatment group where these results are greater. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) consistent results between clarity-bpa and prior studies with mice and rats indicating that bpa exposure impairs steroidogenesis and causes low e levels. neurobehavior -spatial learning escape tasks in barnes maze, increased latency in μg/kg/day bpa females, and trend for increased latency in . μg/kg/day bpa females at pnd ; males exposed to . μg/kg/day bpa show trend for improved latency, but relevance of this finding is uncertain. male deer mice developmentally exposed to bpa through maternal diet ( mg or mg/ kg feed weight) show reduced spatial navigational learning and memory. clarity-bpa studies differ from our current findings that show males are more vulnerable to bpa-induced cognitive disruptions. exposure to μg/kg/day bpa disrupts patterns of sexually dimorphic gene expression/ promoter. female deer mice exposed to bpa through the maternal diet ( mg or mg/kg feed weight) either show no affect or a trend for a masculinized response with improved latency. conflicting findings may be due to different rodent models (nctr sprague-dawley rats vs. deer mice), route of exposure (oral gavage vs. dietary), dose and duration of bpa exposure, and other potential factors. hippocampus of /kg/day μg bpa female offspring have hypermethylated putative ' promoter regions of bdnf. bdnf methylation weakly associated with bdnf expression in hippocampi of female rats. neurobehavior -anxiety and juvenile anatomy no systematic effects of bpa observed on any endpoint related to anxiety and exploration in juveniles or adults. expansive literature by us and others shows heightened anxiety in multiple species, including rats, mice, deer mice, and prairie voles. in clarity-bpa, not all expected behavioral sex differences were observed in nctr-sprague dawley rats, suggesting unique strain differences. in juveniles, statistically significant effects of . and μg/kg/day bpa identified on few endpoints in the interval open field analysis, but overall evidence for bpa-related effects minimal and inconsistent. changes in avpv volume. no effect of ee on behavior or avpv volume in clarity-bpa raises some question about the sensitivity of this strain to the masculinizing influence of estrogen and/or efficacy of ee in the brain. enlarged female avpv at all bpa dose levels; male avpv enlargement at and μg/kg/ day bpa doses. changes in avpv neuron numbers, th, and kisspeptin. clarity-bpa results on avpv and mepd volume are consistent with our prior work in rats showing bpa-related effects on avpv volume and sexual differentiation and gene expression changes in the juvenile amygdala. enlarged mepd (right side only) in males exposed to μg/kg/day bpa. disruption of gene expression in juvenile rat amygdala, including downregulation of erβ, and sex-specific disruption of mc r, mc r, and tac . disrupted er expression in female juveniles of the same strain examined as part of the subchronic study that preceded clarity-bpa. (continued on next page) true biological effects of bpa exposure, or are spurious and not toxicologically relevant. this can be addressed in two ways. first, this was expected because non-monotonicity is a hallmark feature of many endocrine responses and is well described for bpa [ , ] . for example, dose-specific responses across a -fold bpa dose range could be due to differential engagement of multiple receptors that mediate bpa actions (e.g., erα, erβ, gper, errs, ar, tr) through both genomic and non-genomic (i.e., membrane-initiated) pathways, each with differing threshold responses. it is well-known that increasing bpa doses (in this experiment, , -fold from . to μg/kg/day) activate the expression of entirely different genes, as would be the case for any endogenous or exogenous hormone [ ] . additionally, during organogenesis and tissue repair different cell types with different receptors interact to form organs, increasing the complexity of interactions and thus the likelihood of non-monotonic dose-response curves [ ] . but, perhaps more importantly, we applied a rigorous mixomics approach [ ] , as done in previous bpa studies performed in the rosenfeld laboratory [ ] [ ] [ ] , to ascertain whether the low dose of bpa simultaneously affected several endpoints in different laboratories. as such, if low-dose effects in the same animals are identified across multiple systems, it seems unlikely that these findings are not important. this approach confirmed that the low dose effects of bpa were observed in many organs and tissues at once while shedding light on the interconnectedness of these low dose effects on the different organs and tissues. these results show the irreversible deviation from normal development that occurs after exposure to bpa. it was also expected that the results of the clarity-bpa study would not exactly match the previously published findings of the investigator-initiated studies due to important differences in experimental designs. as highlighted in table many study design aspects in clarity-bpa, such as species (rat vs mouse), strain of rat model (nctr-sprague dawley; not used in any independent labs), route of exposure (direct gavage including gavage in newborn animals not used in any independent labs), use of cmc vehicle (not used in any independent labs), and dosing throughout the lifetime (not typically done in any independent labs), are different from previously published investigator-initiated studies. these protocol differences can have major implications for the results including gavage [ , , ] . importantly, prior work conducted by the patisaul lab in collaboration with nctr under conditions similar to clarity-bpa produced strong evidence that maternal gavage can induce effects in the newborn offspring brain [ ] . gavage has been shown to induce stress [ , ] , which can affect disruption of erβ expression across postnatal rat brain. we have also found evidence of disrupted oxytocin and vasopressin signaling in adult rats and prairie voles. disruption of ot and avp or its receptors in hippocampus, hypothalamus, and amygdala. interference with gabaergic and glutamatergic signaling has also been shown in several different capacities. disruption of genes and pathways related to gaba and glutamate signaling in the amygdala. heart decreased collagen in hearts at pnd and age mo. altered rapid estrogen signaling in females that involves activation of er. same results in clarity-bpa as published previously for same endpoint examined. myocardial degeneration in males and females at pnd and . female cardiomyopathy incidence and severity at pnd at bpa doses of . , and , μg/kg/day. in vivo studies demonstrate that bpa exposure sex-specifically alters collagen content, modifies the extracellular matrix of hearts of both males and females, alters fatty acid and glycolytic metabolism, and increases sensitivity of the female heart to ischemic damage. increased sensitivity of heart to ischemic damage. bpa significantly decreased heart weight and heart weight normalized to body weight in females exposed to . μg/kg/day bpa. thyroid no effect on body weight. body weight of dams reduced in dosedependent manner. clarity-bpa is the only published study that does not report an effect of bpa on serum t . no effect on serum t . serum t in pups increased on pnd but not on pnd , or (dosing stopped at weaning). no effect on brain endpoints. tsh not affected in pups. ptu decreased serum t as expected. rc /neurogranin mrna increased in dentate gyrus of pnd brain. ptu did not affect brain endpoints known to be affected by thyroid hormone. mammary gland altered morphology of the mammary gland; nonmonotonic dose-response curves to bpa with a breaking point between and μg/kg/day bpa doses. altered mammary gland morphology, nonmonotonic dose response curves, intraductal hyperplasia and neoplasms, changes in methylome and transcriptome resulting from developmental bpa exposure, at doses as low as ug/kg/day. different effects of bpa and ee. morphological results expand our previous work demonstrating non-monotonic dose-responses to bpa exposure. in the core study, significant increase of adenocarcinomas and adenomas in the lowest dose ( . μg) when exposure stopped at pnd is consistent with our early findings. although for some endpoints bpa and ee had similar results, for other endpoints the results were different and even opposite. data on methylome and transcriptome are highly suspect of contamination as a result of co-housing of the animals with those receiving the highest bpa dose. neoplastic results found in core study, but this part of study had too few animals to measure incidence. all studied endpoints. much has been written about how factors including animal model, diet, vehicle, housing conditions can produce variability across studies [ , , , , , ] , and thus will not be exhaustively described here. some specifically identified differences were the result of design issues of guideline studies per se, and some were due to situations that occurred during the study. these limitations include direct gavage of dams and pups, lack of a non-gavage control to assess possible effects of gavage, use of a block design in which each block did not contain the same controls and doses (an example is that all the μg/ kg/day bpa animals in the pnd urogenital sinus studies [ ] which were selected from block , abnormal variance in animal weights (see fig. )), failure to euthanize on the same day of estrus, and inadequate steps to preserve rna quality for analysis of the mammary gland studies. none of the independent researcher used gavage thus they did not use the cmc vehicle which has possible side effect of causing inflammation of the gut altering the gut microbiome [ ] . any extrinsic factor that alters gut microbiota can lead to pathophysiological changes in the host through various axes, including the microbiota-gut-brain axis [ , [ ] [ ] [ ] [ ] . the large differences in animal weights within a group and the failure to euthanize animals on the same day of the estrous cycle can table comparison of clarity-bpa study design with prior academic studies. (for interpretation of the references to colour in this table legend, the reader is referred to the web version of this article.) red color indicates that aspect of the published independent studies was different from that of the clarity-bpa study design. for example, no published independent studies used the sprague-dawley/cd /nctr br, the cmc vehicle. green color indicates that aspect of the published independent studies was similar to that of the clarity-bpa study design. for example, all published independent studies analyzed their results blinded, as did the clarity-bpa study. the numbers explain some subtle differences between the published independent studies and the clarity-bpa study. . mice and rats used. . no prior studies have used gavage, although many have used oral exposure routes. . sprague dawley from zivic miller. . sprague dawley, holzman. . small number of prior studies have examined rat mammary glands (wistar/furth and sprague dawley), but more data from the soto lab has come from mouse models. . bpa administered via diet. . diet used in some studies; other diets also used. . no studies dosed pups directly. . for studies in the rosenfeld laboratory, females were exposed weeks before gestation and throughout the postnatal period to replicate real-world exposure. . polypropylene cages and glass water bottles. . animals in the rosenfeld laboratory were housed in polypropylene or polystyrene cages. . polyethylene cages and water bottles. . sample size in clarity-bpa less than requested. . variable sample sizes, some larger than clarity-bpa. diminish the ability to detect significant effects on numerous sensitive endpoints. the decision to exclude a negative control group that was not gavaged is particularly surprising because in a pre-clarity study it was reported that the non-gavaged negative controls were significantly different from gavaged negative controls [ ] . importantly, animal numbers were severely limited for several independent studies, leaving them underpowered for appropriate statistical analyses despite uniform requests from the labs for appropriate numbers (see for example the prins study [ ] ). this compromised data analysis for many endpoints (see flaws and prins above). although clarity-bpa animals displayed phenotypes similar to those shown in prior academic studies, they were not always statistically significant while independent studies with appropriate power found significant effects. another important variable is the reported possibility of contamination of control and lowest-dose exposure animals due to cohousing of several cohorts with a high-dose group ( , μg/kg/day bpa) that was not used in common clarity-bpa studies. this was further complicated by lack of assessment of blood levels of free bpa and bpa-g across the lifespan at the fda-nctr facility to confirm or rule-out possible cross-contamination of animals. the fda previously published the recommendation that no bpa study should be conducted without biomonitoring for the possibility of contamination of vehicle controls with bpa, which they reported to occur in the pre-clarity-bpa study [ ] . nonetheless, every independent study reported here, except for the thyroid study, showed effects of bpa on a variety of health-predictive or apical endpoints. all studies showed effects of bpa at the lowest dose, . ug/kg/day. taken together the findings presented here provide strong evidence that the previous bpa results in publications from independent labs could be repeated in, and effectively augment, a guideline study even with many significant differences in study design. in addition, mixomics analysis of the data indicates that bpa's effects in one tissue were correlated with effects in another tissue, providing strong evidence that the data are revealing true effects of a low dose of bpa in this guideline, glp compliant study and that by targeting one organ system, bpa may induce systemic effects. while the underlying mechanisms accounting for the different dose-response shapes is unknown, this knowledge is not required to conclude that they are biologically and toxicologically relevant. even if the dose-response shape were linear, the mechanism would not be known; thus, this requirement represents a fundamental bias in the interpretation of toxicological data. it is important, therefore, to focus on what is known rather than what is not known. we agree, as recently published [ ] , that even in the light of the limitations of study design, that clarity-bpa was an important and powerful exercise. it demonstrated that independent scientists focusing on hypothesis-driven disease focused endpoints at relevant levels of biological organization and federal scientists (fda, ntp) focusing on a guideline compliant study could work together to design and perform a complex experiment with endpoints ranging from epigenetic to organ to whole animal. even with major study design differences across studies, the clarity-bpa study and related, previously published data, show that bpa affects multiple organ systems is reproducible, even in a guideline compliant study. therefore, the current data from the investigator-initiated studies should be considered for regulatory purposes. the authors report no declarations of interest. niehs/fda clarity-bpa research program update a two-year toxicology study of bisphenol a (bpa) in sprague-dawley rats: clarity-bpa core study results evaluation of bisphenol a (bpa) exposures on prostate stem cell homeostasis and prostate cancer risk in the nctr-sprague-dawley rat: an niehs/fda clarity-bpa consortium study effects of perinatal bisphenol a exposure on the volume of sexually-dimorphic nuclei of juvenile rats: a clarity-bpa consortium study prenatal bisphenol a (bpa) exposure alters the transcriptome of the neonate rat amygdala in a sex-specific manner: a clarity-bpa consortium study gene expression and dna methylation changes in the hypothalamus and hippocampus of adult rats developmentally exposed to bisphenol a or ethinyl estradiol: a clarity-bpa consortium study effects of continuous bisphenol a exposure from early gestation on day old rat testes function and sperm molecular profiles: a clarity-bpa consortium study effects of bisphenol a on incidence and severity of cardiac lesions in the nctr-sprague-dawley rat: a clarity-bpa study effects of developmental exposure to bisphenol a on spatial navigational learning and memory in rats: a clarity-bpa study clarity-bpa: effects of chronic bisphenol a exposure on the immune system: part -quantification of the relative number and proportion of leukocyte populations in the spleen and thymus clarity-bpa: effects of chronic bisphenol a exposure on the immune system: part -characterization of lymphoproliferative and immune effector responses by splenic leukocytes impact of low-dose oral exposure to bisphenol a (bpa) on juvenile and adult rat exploratory and anxiety behavior: a clarity-bpa consortium study bisphenol a exposure, ovarian follicle numbers, and female sex steroid hormone levels: results from a clarity-bpa study clarity-bpa: bisphenol a or propylthiouracil on thyroid function and effects in the developing male and female rat brain fetal bisphenol a and ethinylestradiol exposure alters male rat urogenital tract morphology at birth: confirmation of prior low-dose findings in clarity-bpa endocrine disruption and reproductive pathology a combined morphometric and statistical approach to assess nonmonotonicity in the developing mammary gland of rats in the clarity-bpa study reproductive toxicology xxx (xxxx) xxx-xxx toxicology testing -lessons from clarity-bpa flaws in design, execution and interpretation limit clarity-bpa's value for risk assessments of bisphenol a clarity-bpa academic laboratory studies identify consistent low-dose bisphenol a effects on multiple organ systems mixomics: an r package for' omics feature selection and multiple data integration early genistein exposure of california mice and gut microbiota-brain axis effects bisphenol a and bisphenol s disruptions of the mouse placenta and potential effects on the placenta-brain axis endocrine disruption of gene expression and microrna profiles in hippocampus and hypothalamus of california mice: association of gene expression changes with behavioural outcomes estrogen imprinting of the developing prostate gland is mediated through stromal estrogen receptor alpha: studies with alphaerko and betaerko mice the role of wnt a in prostate gland development sonic hedgehog-patched gli signaling in the developing rat prostate gland: lobe-specific suppression by neonatal estrogens reduces ductal growth and branching estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells developmental exposure to estradiol and bisphenol a increases susceptibility to prostate carcinogenesis and epigenetically regulates phosphodiesterase type variant the role of estrogens in normal and abnormal development of the prostate gland serum bisphenol a pharmacokinetics and prostate neoplastic responses following oral and subcutaneous exposures in neonatal sprague-dawley rats neonatal exposure to estradiol/bisphenol a alters promoter methylation and expression of nsbp and hpcal genes and transcriptional programs of dnmt a/b and mbd / in the rat prostate gland throughout life identification of secretaglobin scgb a as a target for developmental reprogramming by bpa in the rat prostate directed differentiation of human embryonic stem cells into prostate organoids in vitro and its perturbation by low-dose bisphenol a exposure exposure of human prostaspheres to bisphenol a epigenetically regulates snord family noncoding rnas via histone modification bisphenol a promotes human prostate stem-progenitor cell self-renewal and increases in vivo carcinogenesis in human prostate epithelium prostate cancer risk and dna methylation signatures in aging rats following developmental bpa exposure: a dose-response analysis estradiol in elderly men induction at high incidence of ductal prostate adenocarcinomas in nbl/cr and sprague-dawley hsd:sd rats treated with a combination of testosterone and estradiol- beta or diethylstilbestrol the oestrogen receptor alpha-regulated lncrna neat is a critical modulator of prostate cancer estrogen-dependent signaling in a molecularly distinct subclass of aggressive prostate cancer estradiol suppresses tissue androgens and prostate cancer growth in castration resistant prostate cancer reprogramming of the epigenome by mll links early-life environmental exposures to prostate cancer risk dna methylome changes by estradiol benzoate and bisphenol a links early-life environmental exposures to prostate cancer risk prostate cancer incidence in populations worldwide: an analysis of time trends overall and by age group chemical and hormonal induction of prostate cancer in animal models review of low dose studies memorandum, us food and drug administration, us food and drug administration prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses a physiologically based approach to the study of bisphenol a and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior endocrine disruptor bisphenol a is implicated in urinary voiding dysfunction in male mice low-dose bioactivity of xenoestrogens in animals: fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice altered prostate growth and daily sperm production in male mice exposed prenatally to subclinical doses of alpha-ethinyl oestradiol estradiol and bisphenol a stimulate androgen receptor and estrogen receptor gene expression in fetal mouse prostate mesenchyme cells dose-related estrogen effects on gene expression in fetal mouse prostate mesenchymal cells estrogen receptor expression and methylation of esr promoter in mouse fetal prostate mesenchymal cells induced by gestational exposure to bisphenol a or ethinylestradiol intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice reproductive malformation of the male offspring following maternal exposure to estrogenic chemicals reproductive tract lesions in male mice exposed prenatally to diethylstilbestrol estrogenic chemicals in plastic and oral contraceptives disrupt development of the fetal mouse prostate and urethra testosterone and beta-estradiol induce glandular prostatic growth, bladder outlet obstruction, and voiding dysfunction in male mice prenatal bisphenol a exposure alters sex-specific estrogen receptor expression in the neonatal rat hypothalamus and amygdala perinatal exposure to bisphenol a alters early adipogenesis in the rat should oral gavage be abandoned in toxicity testing of endocrine disruptors? hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses effects of low doses of bisphenol a on the metabolome of perinatally exposed cd- mice perinatal exposure to environmentally relevant levels of bisphenol-a decreases fertility and fecundity in cd- mice mammalian development in a changing environment: exposure to endocrine disruptors reveals the developmental plasticity of steroid-hormone target organs perinatal bpa exposure alters body weight and composition in a dose specific and sex specific manner: the addition of peripubertal exposure exacerbates adverse effects in female mice bisphenol a exposure disrupts neurotransmitters through modulation of transaminase activity in the brain of rodents perinatally administered bisphenol a as a potential mammary gland carcinogen in rats in utero exposure to bisphenol a alters the development and tissue organization of the mouse mammary gland does cancer start in the womb? altered mammary gland development and predisposition to breast cancer due to in utero exposure to endocrine disruptors new insights into fetal mammary gland morphogenesis: differential effects of natural and environmental estrogens bisphenol a alters the development of the rhesus monkey mammary gland perinatal exposure to the xenoestrogen bisphenol-a induces mammary intraductal hyperplasias in adult cd- mice exposure to environmentally relevant doses of the xenoestrogen bisphenol-a alters development of the fetal mouse mammary gland the male mammary gland: a target for the xenoestrogen bisphenol a perinatal bisphenol-a exposure increases estrogen sensitivity of the mammary gland in diverse mouse strains perinatal exposure to bisphenol-a alters peripubertal mammary gland development in mice prenatal bisphenol a exposure induces preneoplastic lesions in the mammary gland in wistar rats induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol a exposure prenatal exposure to bpa alters the epigenome of the rat mammary gland and increases the propensity to neoplastic development estrogens in the wrong place at the wrong time: fetal bpa exposure and mammary cancer oral exposure to bisphenol a increases dimethylbenzanthracene-induced mammary cancer in rats chronic oral exposure to bisphenol a results in a non-monotonic dose response in mammary carcinogenesis and metastasis in mmtv-erbb mice in utero exposure to bisphenol a shifts the window of susceptibility for mammary carcinogenesis in the rat exposure to the endocrine disruptor bisphenol a alters susceptibility for mammary cancer haschek and rousseaux's handbook of toxicologic pathology importance of dosage standardization for interpreting transcriptomal signature profiles: evidence from studies of xenoestrogens low-dose bpa exposure alters the mesenchymal and epithelial transcriptomes of the mouse fetal mammary gland comparison of life-stage-dependent internal dosimetry for bisphenol a, ethinyl estradiol, a reference estrogen, and endogenous estradiol to test an estrogenic mode of action in sprague dawley rats toxicity evaluation of bisphenol a administered by gavage to sprague dawley rats from gestation day through postnatal day bisphenol a inhibits follicle growth and induces atresia in cultured mouse antral follicles independently of the genomic estrogenic pathway bisphenol a down-regulates rate-limiting cyp a to acutely inhibit steroidogenesis in cultured mouse antral follicles bisphenol a impairs follicle growth, inhibits steroidogenesis, and downregulates rate-limiting enzymes in the estradiol biosynthesis pathway mouse strain does not influence the overall effects of bisphenol a-induced toxicity in adult antral follicles bisphenol a inhibits cultured mouse ovarian follicle growth partially via the aryl hydrocarbon receptor signaling pathway evidence for bisphenol a-induced female infertility: a review flaws, bisphenol a and reproductive health: update of experimental and human evidence developmental programming: impact of prenatal exposure to bisphenol-a and methoxychlor on steroid feedbacks in sheep neonatal bisphenol-a exposure alters rat reproductive development and ovarian morphology without impairing activation of gonadotropin-releasing hormone neurons the effects of in utero bisphenol a exposure on the ovaries in multiple generations of mice the effects of in utero bisphenol a exposure on reproductive capacity in several generations of mice bisphenol a exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries neonatal exposure to bisphenol a or diethylstilbestrol alters the ovarian follicular dynamics in the lamb in utero bisphenol a exposure disrupts germ cell nest breakdown and reduces fertility with age in the mouse ovarian dysfunctions in adult female rat offspring born to mothers perinatally exposed to low doses of bisphenol a exposure to a low dose of bisphenol a impairs pituitary-ovarian axis in prepubertal rats: effects on early folliculogenesis sex specific impact of perinatal bisphenol a (bpa) exposure over a range of orally administered doses on rat hypothalamic sexual differentiation neonatal genistein or bisphenol-a exposure alters sexual differentiation of the avpv anxiogenic effects of developmental bisphenol a exposure are associated with gene expression changes in the juvenile rat amygdala and mitigated by soy the role of bisphenol a in shaping the brain, epigenome and behavior opening the black box of endocrine disruption of brain development: lessons from the characterization of bisphenol a sex-specific esr mrna expression in the rat hypothalamus and amygdala is altered by neonatal bisphenol a exposure impact of early-life bisphenol a exposure on behavior and executive function in children prenatal and early childhood bisphenol a concentrations and behavior in school-aged children bisphenol a exposure and symptoms of anxiety and depression among inner city children at - years of age prenatal bisphenol a exposure and child behavior in an inner-city cohort effects of environmental endocrine disruptors and phytoestrogens on the kisspeptin system neonatal bisphenol a exposure alters sexually dimorphic gene expression in the postnatal rat hypothalamus investigation of the effects of subchronic low dose oral exposure to bisphenol a (bpa) and ethinyl estradiol (ee) on estrogen receptor expression in the juvenile and adult female rat hypothalamus sexually dimorphic expression of hypothalamic estrogen receptors alpha and beta and kiss in neonatal male and female rats sex specific expression of estrogen receptors alpha and beta and kiss in the postnatal rat amygdala non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment effects of sex steroids on the development of the locus coeruleus in the rat exposure to bisphenol a during the fetal and suckling periods disrupts sexual differentiation of the locus coeruleus and of behavior in the rat estradiol and the developing brain wired for reproduction: organization and development of sexually dimorphic circuits in the mammalian forebrain sexual dimorphism and steroid responsiveness of the posterodorsal medial amygdala in adult mice sexual dimorphism in neuronal number of the posterodorsal medial amygdala is independent of circulating androgens and regional volume in adult rats post-weaning social isolation of male rats reduces the volume of the medial amygdala and leads to deficits in adult sexual behavior impact of low dose oral exposure to bisphenol a (bpa) on the neonatal rat hypothalamic and hippocampal transcriptome: a clarity-bpa consortium study endocrine disruption of vasopressin systems and related behaviors in vitro and in vivo interactions of bisphenol a and its metabolite, bisphenol a glucuronide, with estrogen receptors alpha and beta edc- : the endocrine society's second scientific statement on endocrine-disrupting chemicals steroid metabolism in the brain: from bird watching to molecular biology, a personal journey extra-gonadal sites of estrogen biosynthesis and function the impact of neonatal bisphenol-a exposure on sexually dimorphic hypothalamic nuclei in the female rat a novel model for neuroendocrine toxicology: neurobehavioral effects of bpa exposure in a prosocial species, the prairie vole (microtus ochrogaster) gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression disruption of adult expression of sexually selected traits by developmental exposure to bisphenol a sex and dose-dependent effects of developmental exposure to bisphenol a on anxiety and spatial learning in deer mice (peromyscus maniculatus bairdii) offspring spatial navigation strategies in peromyscus: a comparative study sexually selected traits: a fundamental framework for studies on behavioral epigenetics effects of developmental bisphenol a exposure on reproductive-related behaviors in california mice (peromyscus californicus): a monogamous animal model hypothalamic transcriptomic alterations in male and female california mice (peromyscus californicus) developmentally exposed to bisphenol a or ethinyl estradiol hypothalamic gene expression changes in f california mice (peromyscus californicus) parents developmentally exposed to bisphenol a or ethinyl estradiol animal models to study environmental epigenetics environmental health factors and sexually dimorphic differences in behavioral disruptions chapter -animal models of transgenerational epigenetic effects bisphenol a and phthalate endocrine disruption of parental and social behaviors gonadal hormone levels and spatial learning performance in the morris water maze in male and female meadow voles, microtus pennsylvanicus sex differences in spatial ability and activity in two vole species (microtus ochrogaster and m. pennsylvanicus) evolution of sex differences in spatial ability effect of neonatal rat bisphenol a exposure on performance in the morris water maze adolescent exposure to bisphenol-a increases anxiety and sucrose preference but impairs spatial memory in rats independent of sex exposure to bisphenol a appears to impair hippocampal neurogenesis and spatial learning and memory perinatal exposure to bisphenol-a impairs spatial memory through upregulation of neurexin and neuroligin expression in male mouse brain perinatal exposure to low-dose bisphenol a impairs spatial learning and memory in male rats impairment of learning and memory performances induced by bpa: evidences from the literature of a moa mediated through an ed perinatal exposure to bisphenol-a impairs learning-memory by concomitant down-regulation of n-methyl-d-aspartate receptors of hippocampus in male offspring mice effects of prenatal exposure to diethylstilbestrol (des) on hemispheric laterality and spatial ability in human males sex differences in the adult hpa axis and affective behaviors are altered by perinatal exposure to a low dose of bisphenol a sex-specific epigenetic disruption and behavioral changes following low-dose in utero bisphenol a exposure estrogenic chemicals at puberty change eralpha in the hypothalamus of male and female rats bisphenol a induces transforming growth factor-beta mrna in the preoptic area: a cdna expression array and northern blot study gestational exposure to low dose bisphenol a alters social behavior in juvenile mice dna methylation of bdnf as a biomarker of early-life adversity prenatal exposure to bisphenol a impacts midbrain dopamine neurons and hippocampal spine synapses in non-human primates comparison of serum bisphenol a concentrations in mice exposed to bisphenol a through the diet versus oral bolus exposure daily bisphenol a excretion and associations with sex hormone concentrations: results from the inchianti adult population study two-generation reproductive toxicity study of dietary bisphenol a in cd- (swiss) mice three-generation reproductive toxicity study of dietary bisphenol a in cd sprague-dawley rats disposition of low doses of c-bisphenol a in male, female, pregnant, fetal, and neonatal rats bisphenol a concentration in breast milk following consumption of a canned coffee drink determination of bisphenol a and related substitutes/ analogues in human breast milk using gas chromatography-tandem mass spectrometry determination of free bisphenol a (bpa) concentrations in breast milk of u.s. women using a sensitive lc/ms/ms method rapid estrogen receptor-mediated mechanisms determine the sexually dimorphic sensitivity of ventricular myocytes to beta-estradiol and the environmental endocrine disruptor bisphenol a bisphenol a and beta-estradiol promote arrhythmia in the female heart via alteration of calcium handling bisphenol a alters autonomic tone and extracellular matrix structure and induces sex-specific effects on cardiovascular function in male and female cd- mice lifelong exposure to bisphenol a alters cardiac structure/function, protein expression, and dna methylation in adult mice chronic exposure to bisphenol a reduces successful cardiac remodeling after an experimental myocardial infarction in male c bl/ n mice altered heart proteome in fructose-fed fisher rats exposed to bisphenol a expression of oestrogen receptor alpha and beta in rat heart: role of local oestrogen synthesis bisphenol a and the risk of cardiometabolic disorders: a systematic review with meta-analysis of the epidemiological evidence relationship between urinary bisphenol a levels and diabetes mellitus urinary bisphenol a levels and measures of obesity: results from the national health and nutrition examination survey urinary bisphenol a and obesity: nhanes endocrine disruptive compounds and cardio-metabolic risk factors in children association of urinary bisphenol a concentration with medical disorders and laboratory abnormalities in adults association of urinary bisphenol a concentration with heart disease: evidence from nhanes urinary bisphenol a and type- diabetes in u.s. adults: data from nhanes accumulation and endocrine disrupting effects of the flame retardant mixture firemaster(r) in rats: an exploratory assessment nonpproliferative lesions of the heart and vasculature in rats, guides for toxicologic pathology characterization of spontaneous and chemically induced cardiac lesions in rodent model systems: the national toxicology program experience morphologic aspects of rodent cardiotoxicity in a retrospective evaluation of national toxicology program studies morphological changes induced in rats following prolonged exposure to stress effects of gestational stress: . evaluation of male and female adult offspring effects of gestational stress: . evaluation of maternal and juvenile offspring behavior relevance of mouse models of cardiac fibrosis and hypertrophy in cardiac research determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse thyroid hormone action is disrupted by bisphenol a as an antagonist the xenoestrogen bisphenol a inhibits postembryonic vertebrate development by antagonizing gene regulation by thyroid hormone generation of fluorescent zebrafish to study endocrine disruption and potential crosstalk between thyroid hormone and corticosteroids bisphenol-a, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters rc /neurogranin expression in the developing rat brain insight into the physiological actions of thyroid hormone receptors from genetically modified mice transcriptional induction of rc /neurogranin by thyroid hormone: differential neuronal sensitivity is not correlated with thyroid hormone receptor distribution in the brain cell-specific effects of thyroid hormone on rc /neurogranin expression in rat brain thyroid hormone receptors and resistance to thyroid hormone disorders thyroid hormone disrupting potentials of bisphenol a and its analogues -in vitro comparison study employing rat pituitary (gh ) and thyroid follicular (frtl- ) cells low concentrations of bisphenol a suppress thyroid hormone receptor transcription through a nongenomic mechanism thyroid hormonal activity of the flame retardants tetrabromobisphenol a and tetrachlorobisphenol a perinatal bisphenol a affects the behavior and src- expression of male pups but does not influence on the thyroid hormone receptors and its responsive gene effects of in utero and lactational exposure to bisphenol a on thyroid status in f rat offspring associations between urinary phthalate metabolites and bisphenol a levels, and serum thyroid hormones among the korean adult population -korean national environmental health survey (konehs) thyroid hormone parameters during pregnancy in relation to urinary bisphenol a concentrations: a repeated measures study suppressive effects of neonatal bisphenol a on the neuroendocrine system neonatal exposure to bisphenol a alters the hypothalamic-pituitarythyroid axis in female rats developmental treatment with bisphenol a or ethinyl estradiol causes few alterations on early preweaning measures maternal and fetal exposure to bisphenol a is associated with alterations of thyroid function in pregnant ewes and their newborn lambs visualising associations between paired' omics' data sets lipodystrophy and obesity are associated with decreased number of t cells with regulatory function and proinflammatory macrophage phenotype reproductive toxicology xxx (xxxx) xxx-xxx impaired treg and nk cells profile in overweight women with gestational diabetes mellitus foxp (+) t regulatory cell levels in obese, asthmatic, asthmatic obese, and healthy children circulating regulatory t cells are reduced in obesity and may identify subjects at increased metabolic and cardiovascular risk environmental obesogens: mechanisms and controversies effects of continuous bisphenol a exposure from early gestation on day old rat testes function and sperm molecular profiles: a clarity-bpa consortium study large effects from small exposures. ii. the importance of positive controls in low-dose research on bisphenol a varients of the human prostat lncapcell line as tools for study discrete components of the androgen-mediated proliferative response the mammary gland response to estradiol:monotonic at the cellular level, nonmonotonic at the tissue-level of organization? molecular mechanisms involved in the non-monotonic effect of bisphenol-a on ca + entry in mouse pancreatic β-cells global analysis of ligand sensitivity of estrogen inducible and suppressible genes in mcf /bus breast cancer cells by dna microarray meeting report: batch-to-batch variability in estrogenic activity in commercial animal diets-importance and approaches for laboratory animal research animal models of endocrine disruption the estrogenic content of rodent diets, bedding, cages, and water bottles and its effect on bisphenol a studies the role of carrageenan and carboxymethylcellulose in the development of intestinal inflammation effects of exposure to bisphenol a and ethinyl estradiol on the gut microbiota of parents and their offspring in a rodent model microbiome disturbances and autism spectrum disorders gut dysbiosis in animals due to environmental chemical exposures effects of phytoestrogens on the developing brain, gut microbiota, and risk for neurobehavioral disorders this work was supported by national institutes of health grants u es (jaf), u es (ams), u es (rtz), u es (csr), r es (smb), u es (hbp), u es (gsp), u es (fvs). supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.reprotox. . . . key: cord- -elyn fdh authors: Żółkiewicz, jakub; marzec, aleksandra; ruszczyński, marek; feleszko, wojciech title: postbiotics—a step beyond pre- and probiotics date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: elyn fdh as an imbalance in the intestinal microbiota can lead to the development of several diseases (e.g., type diabetes, cancer, among others), the use of prebiotics, probiotics, and postbiotics to alter the gut microbiome has attracted recent interest. postbiotics include any substance released by or produced through the metabolic activity of the microorganism, which exerts a beneficial effect on the host, directly or indirectly. as postbiotics do not contain live microorganisms, the risks associated with their intake are minimized. here, we provided a critical review of postbiotics described in the literature, including their mechanisms of action, clinical characteristics, and potential therapeutic applications. we detailed the pleiotropic effects of postbiotics, including their immunomodulatory, anti-inflammatory, antioxidant, and anti-cancer properties. although the use of postbiotics is an attractive strategy for altering the microbiome, further study into its efficacy and safety is warranted. the assemblage of microorganisms that inhabit the human body, their genomes and metabolites, as well as the environment in which they live, is called the microbiota. microorganisms that are part of the microbiome can be isolated from all areas in constant contact with the external environment (e.g., the skin, upper respiratory tract, or urogenital tract). however, they are most abundant in the gastrointestinal tract. our interdependent relationship with the intestinal microbiota is established during the first three years of life [ ] . the human body provides a stable, nutrient-rich environment for the inhabiting microorganisms, and in return, receives a number of benefits. these benefits include stimulation of the immune system, improved digestion and absorption of food, reduced growth of pathogenic flora, and maintenance of intestinal barrier integrity. these beneficial effects of the interaction between the microbiota and the gastrointestinal tract can be observed not only locally, but also in distant organs, due to systemic distribution of substances and cells produced in the intestine. this phenomenon is called the gut-organ axis, according to which we can distinguish the gut-brain, gut-skin, gut-lung axis, and so on. several factors can affect the composition of the microbiota starting from the perinatal period, including the composition of the maternal gut microbiota, the mode of delivery and type of food the mother consumes, antibiotic therapy, and stress [ ] . moreover, many studies have shown that an imbalance in the intestinal microbiota-dysbiosis-can lead to the development of allergic or autoimmune diseases (e.g., inflammatory bowel disease, type diabetes, among others), cancer, and psychiatric disorders [ ] . as such, therapeutic strategies and preparations that affect the composition of the microbiota, and thus, the patient's well-being, have become increasingly popular. as summarized in figure , there are currently three main ways in which the microbiota can be modulated, i.e., through the use of prebiotics, probiotics, synbiotics, or postbiotics. prebiotics are figure . optimal microbiome composition as a token of human wellbeing. microbiome composition and structure is one of the factors determining proper human development and health. the roof that represents microbiome may be impermeable and reliable, only provided structures below are solid. a suitable diet and physical activity form the basis for the construction. metaphorical foundation, which is represented by diet and exercise, underlines principal role of healthy lifestyle in sustaining human health and wellbeing. it is a lifestyle modification one should implement first when commencing the process of building human welfare. the connectors (pillars) between "roof" and "foundation" that cement the construction are pre-, pro-, and postbiotics. a lot of research is currently focused on determining the ideal proportion and shape of each pillar, so all of the construction's elements depicted in this figure would be in the state of harmony. it is important to note that the composition of the microbiome is also affected by other factors that are not found in this figure, e.g., route of labor, use of medicines, or having siblings. the concept of postbiotics is based on the observation that the beneficial effects of the microbiota are mediated by the secretion of various metabolites. however, its precise definition remains under discussion. according to tsilingiri et al., postbiotics include any substance released by or produced through the metabolic activity of the microorganism, which exerts a beneficial effect on the host, directly or indirectly [ ] . for the purposes of this article, we assume that postbiotics optimal microbiome composition as a token of human wellbeing. microbiome composition and structure is one of the factors determining proper human development and health. the roof that represents microbiome may be impermeable and reliable, only provided structures below are solid. a suitable diet and physical activity form the basis for the construction. metaphorical foundation, which is represented by diet and exercise, underlines principal role of healthy lifestyle in sustaining human health and wellbeing. it is a lifestyle modification one should implement first when commencing the process of building human welfare. the connectors (pillars) between "roof" and "foundation" that cement the construction are pre-, pro-, and postbiotics. a lot of research is currently focused on determining the ideal proportion and shape of each pillar, so all of the construction's elements depicted in this figure would be in the state of harmony. it is important to note that the composition of the microbiome is also affected by other factors that are not found in this figure, e.g., route of labor, use of medicines, or having siblings. the concept of postbiotics is based on the observation that the beneficial effects of the microbiota are mediated by the secretion of various metabolites. however, its precise definition remains under discussion. according to tsilingiri et al., postbiotics include any substance released by or produced through the metabolic activity of the microorganism, which exerts a beneficial effect on the host, directly or indirectly [ ] . for the purposes of this article, we assume that postbiotics include all substances of bacterial or fungal origin that confer beneficial effect to the host and do not meet the definition of a probiotic and are not exclusively of a prebiotic nature ( figure ). include all substances of bacterial or fungal origin that confer beneficial effect to the host and do not meet the definition of a probiotic and are not exclusively of a prebiotic nature ( figure acquisition. lysis of bacterial cells may be achieved by chemical and mechanical techniques. these methods include enzymatic extraction, solvent extraction, sonication, and heat. extraction, dialysis, and chromatography are used to isolate and identify desired molecules. scfa, short-chain fatty acids according to the current literature, postbiotics are not considered as synbiotics. synbiotics are a combination of prebiotics and probiotics that are claimed to have a beneficial impact on gut microbiome. however, it is believed that postbiotics may also strengthen the intestinal microbiome [ ] , so we believe that term "synbiotics" should be reviewed and postbiotics should be incorporated in its definition. although postbiotics do not contain live microorganisms, they show a beneficial health effect through similar mechanisms that are characteristic of probiotics while minimizing the risks associated with their intake. therefore, like prebiotics, postbiotics appear to lack serious side effects while maintaining similar effectiveness to probiotics (although currently there are no studies directly comparing substances belonging to both groups). here, we provided a critical review of the postbiotic drugs described in the literature, including their mechanisms of action, clinical characteristics, and potential therapeutic applications. cell-free supernatants containing biologically active metabolites secreted by bacteria and yeast into the surrounding liquid can be obtained directly from cell cultures. after an incubation period, the microbes are centrifuged and then removed. finally, the resulting mixture is filtered to ensure sterility. supernatants produced from cultures of different microorganisms show differing activities. lactobacillus acidophilus and lactobacillus casei supernatants have anti-inflammatory and antioxidant effects on intestinal epithelial cells, macrophages, and neutrophils by reducing the secretion of the pro-inflammatory tumor necrosis factor α (tnf-α) cytokine and increasing secretion of the according to the current literature, postbiotics are not considered as synbiotics. synbiotics are a combination of prebiotics and probiotics that are claimed to have a beneficial impact on gut microbiome. however, it is believed that postbiotics may also strengthen the intestinal microbiome [ ] , so we believe that term "synbiotics" should be reviewed and postbiotics should be incorporated in its definition. although postbiotics do not contain live microorganisms, they show a beneficial health effect through similar mechanisms that are characteristic of probiotics while minimizing the risks associated with their intake. therefore, like prebiotics, postbiotics appear to lack serious side effects while maintaining similar effectiveness to probiotics (although currently there are no studies directly comparing substances belonging to both groups). here, we provided a critical review of the postbiotic drugs described in the literature, including their mechanisms of action, clinical characteristics, and potential therapeutic applications. cell-free supernatants containing biologically active metabolites secreted by bacteria and yeast into the surrounding liquid can be obtained directly from cell cultures. after an incubation period, the microbes are centrifuged and then removed. finally, the resulting mixture is filtered to ensure sterility. supernatants produced from cultures of different microorganisms show differing activities. lactobacillus acidophilus and lactobacillus casei supernatants have anti-inflammatory and antioxidant effects on intestinal epithelial cells, macrophages, and neutrophils by reducing the secretion of the pro-inflammatory tumor necrosis factor α (tnf-α) cytokine and increasing secretion of the anti-inflammatory cytokine interleukin (il- ) [ ] . meanwhile, supernatants derived from l. casei and lactobacillus rhamnosus gg cultures can prevent the invasion of colon cancer cells [ ] . as cell-free supernatants can reduce oxidative stress in vivo [ ] and provide direct antitumor activity, they may be clinically useful in the prevention of cancer. supernatants derived from bacterial cultures of the genera lactobacillus and bifidobacterium were also recently shown to display antibacterial activity by preventing the invasion of enteroinvasive e. coli strains into enterocytes in vitro [ ] . although these antibacterial properties may result from the inhibition of adhesion of the pathogenic bacterial strains (due to competition for receptor sites), the cell supernatants could also have a local effect on the intestinal environment, cell barrier, and expression of protective genes [ ] . therefore, cell-free bacterial supernatants are promising anti-infectious agents, for example, for the treatment of diarrhea. meanwhile, lactobacillus plantarum supernatants were found to have a positive effect on the maturation and morphological structure of the intestinal barrier [ ] . administering these supernatants to lambs early in their life was associated with an increase in the absorption surface of the intestine and a decrease in the population of intestinal pathogens [ ] . the concentration of inflammatory markers (il- β and tnf-α) in the intestinal mucosa also decreased (p < . ) [ ] . there is also evidence of the beneficial effects of supernatants derived from yeast cultures. in particular, supernatants from saccharomyces cerevisiae and saccharomyces boulardii reversed the state of disturbed intestinal peristalsis caused by stress stimuli [ ] . s. boulardii supernatants also show anti-inflammatory and antioxidant activity [ ] , similar to bacterial cell supernatants, and can accelerate wound healing and regeneration of the intestinal barrier [ ] . during their growth, microorganisms produce biopolymers with different chemical properties. these biopolymers can be released outside the bacterial cell wall, forming a heterogeneous group of substances called exopolysaccharides (epss). epss are currently used in the food industry as stabilizing, emulsifying, and water-binding agents [ ] , although their biological function is not entirely clear. nonetheless, the use of epss in pharmaceutical products and functional foods has attracted recent interest. epss may modulate the immune response by interacting with dendritic cells (dcs) and macrophages and enhancing the proliferation of t and nk lymphocytes [ ] . in addition, an eps isolated from tofu, which is a product of l. plantarum, induced nitric oxide (no) secretion and enhanced the phagocytic potential of macrophages in an in vitro model [ ] . this eps also increased iga concentrations in the intestinal mucosa (p < . ) and stimulated lymphocyte proliferation (p < . ) [ ] . meanwhile, using an eps derived from l. casei as an adjuvant increased the effectiveness of the foot-and-mouth disease vaccine [ ] . some epss produced by lactobacillus strains isolated from fermented durian fruit possess antimicrobial and antioxidant properties [ ] . the ability to bind iron ions was shown to account for the antioxidant potential of an eps obtained from lactobacillus helveticus called uronic acid, which, notably, is also responsible for the antioxidant properties of green tea [ ] . epss can also have a positive effect on lipid metabolism by inhibiting cholesterol absorption [ ] . indeed, the consumption of kefiran (an eps produced by lactobacillus kefiranofaciens) delayed the development of atherosclerosis in a preclinical animal (rabbit) model (p < . ) [ ] . kefiran also prevented blood pressure increases and stabilized blood glucose levels in rats who consumed excessive cholesterol [ ] . thus, epss such as kefiran are potential candidates for preventing cardiovascular diseases. β-glucans, another class of epss, can interact with dectin- receptors on the surface of macrophages and activate them [ ] . as a result, β-glucans may enhance the cellular immune response against bacteria, viruses, parasites, and cancer cells [ , ] . β-glucans may also have a positive effect on probiotics' efficacy, for example, by facilitating the adhesion of lactobacilli to the intestinal epithelium [ ] . they can also increase the bioavailability and absorption of carotenoids (compounds with antioxidant and anti-inflammatory properties) in the gastrointestinal tract [ ] . moreover, topically applied β-glucans (in the form of a cream) may help patients suffering from atopic dermatitis, nutrients , , of by reducing the number and severity of exacerbations (p = . ) while maintaining a good safety profile [ ] . microorganisms have evolved defense mechanisms against the harmful effects of reactive oxygen species (ros), which can damage lipids, proteins, carbohydrates, and nucleic acids. in particular, antioxidant enzymes, such as glutathione peroxidase (gpx), peroxide dismutase (sod), catalase, and nadh-oxidase, play key roles in combating ros. indeed, two strains of l. fermentum were found to have a high content of gpx [ ] , and were later documented to possess potent antioxidant properties in vitro [ ] . antioxidant properties of postbiotics derived from lactobacillus plantarum were demonstrated in the study conducted by izuddin et al. [ ] . this effect was observed thanks to increased gpx concentration in serum (p < . ). moreover, genetically modified lactobacillus strains that synthesize sod or catalase showed superiority in relieving symptoms in a mouse model of crohn's disease relative to their unmodified counterparts [ ] . furthermore, lactobacillus strains with increased catalase activity were more effective in relieving inflammation in a mouse model of inflammatory bowel disease than strains of the same bacterium producing sod (both strains decreased the body temperature comparing to the controls with p < . ) [ ] . this trial revealed that anti-inflammatory activity of lactobacillus strains is dependent on the antioxidative enzyme expression profile of each strain. besides, genetically modified lactobacillus lactis expressing catalase was shown to prevent chemically induced colon cancer in mice [ ] . currently, we lack data regarding the use of sole antioxidant enzymes in vivo. many components of the bacterial cell wall are immunogenic (i.e., elicit a specific immune response), including bacterial lipoteichoic acid (lta). lta is found in the cell walls of gram-positive bacteria and can be spontaneously released into the environment [ ] . although lta has been shown to exhibit immunostimulatory effects [ ] , data on its activity are ambiguous. some reports indicate that lta reduces il- production and induces the production of cytokines with immunoregulatory activity (e.g., il- ) [ ] . in contrast, others have shown lta does not alleviate inflammatory processes and actually causes damage to tissues in the intestine [ ] . the use of lta in dermatological diseases is slightly less controversial. the topical application of lta enhances non-specific defense mechanisms, leading to the release of anti-infectious peptides, including human β-defensin and cathelicidin [ ] . in fact, bacteria of the genera lactobacillus and bifidobacteria, which produce significant amounts of lta, stimulate the skin mast cell response against some bacterial and viral infections [ ] . this data suggests lta may be useful for treating a wide range of skin infections. moreover, based on its anti-inflammatory and anti-cancer activity, lta may have broader utility [ , ] . despite these beneficial activities, lta may exert side effects in living organisms and cause an excessive inflammatory response. therefore, further safety evaluation for lta is warranted. short-chain fatty acids (scfas) are a product of fermentation of plant polysaccharides by intestinal microbiota. well-known scfas include acetic, propionic, and butyric acids, which can form the corresponding fatty acid salts (i.e., acetate, propionate, and butyrate). butyrate is one of the most important energy sources for enterocytes, as it helps to renew the intestinal epithelium and can also modulate gene expression by incompetently inhibiting histone deacetylases. butyrate also shows immunosuppressive effects [ ] . for example, butyrate has been shown to induce food tolerance by increasing the expression of immunosuppressive cytokines (e.g., type interferons [ifns], il- , tgf-β) and downregulating several cytokines and proinflammatory receptors (e.g., toll like receptor (tlr) / , caspase- , nlrp , il- β, il- , il- , il- , mapk). these immunosuppressive effects of butyrate result from the inhibition of nf-κb transcription factor activity and its intracellular pathways [ ] . indeed, rectal administration of butyrate caused a significant regression of inflammatory changes in the large intestine of patients with ulcerative colitis relative to patients receiving placebo [ ] . intestinal colonization with roseburia intestinalis, which produces significant amounts of butyrate, was shown to inhibit atherogenesis in a mouse model of atherosclerosis [ ] . the same study also noted a significant reduction in endotoxemia (likely resulting from sealing of the intestinal barrier) and inflammatory markers in the serum and aorta (including lipopolysaccharide and tnf-α) [ ] . it is worth highlighting that intestinal administration of tributyrin results in analogous effects as observed in abovementioned part of the trial, implying the beneficial effect of r. intestinalis is mediated, at least in part, by butyrate. in terms of its potential mechanism, scfas can affect energy management by stimulating g-protein coupled receptors (gpcrs) and secretion of the glucagon-like peptide (glp- ). indeed, an increase in serum and fecal acetate was associated with an increase in insulin sensitivity and a reduction in body fat in vivo, likely through increased glp- levels [ ] . acetate can also directly regulate appetite in the central nervous system [ ] , indicating a potential application in preventing cardiovascular diseases. furthermore, using a diet high in acetate significantly increased resistance to enterohaemorrhagic e. coli o :h infection in a mouse model [ ] . this phenomenon is likely a result of the sealing properties of acetate on the intestinal barrier, which prevents lethal toxins from entering the general circulation [ ] . propionate is another scfa that is one of the main substrates of gluconeogenesis in the liver. in addition to its role in carbohydrate metabolism, propionate has a statin-like effect, inhibiting the cholesterol synthesis pathway [ ] . propionate also shows an anti-inflammatory activity effect in vivo that is comparable to that of butyrate [ ] . indeed, many studies are currently underway regarding the therapeutic use of scfas in medicine. for example, a recent report showed that scfas may provide symptom relief using an animal model of inflammation and demyelination that occurs in the brain during multiple sclerosis [ ] . bacterial lysates (bls) are obtained by the chemical or mechanical degradation of gram-positive and gram-negative bacteria commonly found in the environment. their clinical use is based on the concept of the gut-lung axis, i.e., the functional connection between the immune system of the intestine and the respiratory system [ ] . in particular, studies have shown that orally administered lyophilized bls reach the peyer's patches in the small intestine, where they stimulate dcs, and subsequently activate t and b lymphocytes [ ] . mature lymphocytes then migrate to the mucous membrane of the respiratory tract and initially stimulate the innate immune system and promoting iga secretion [ ] . indeed, the safety of bls use has been confirmed during many clinical studies on various diseases, including recurrent upper respiratory tract infections in children [ ] . a causal relationship between a decrease in the incidence of infections in highly developed countries and an increase in allergic diseases has been proposed, potentially due to the so-called hygiene hypothesis. therefore, using bls, which mimic the presence of bacteria, to stimulate the immune system, is an attractive option in the case of insufficient exposure to microorganisms. indeed, a meta-analysis including over children showed a significantly lower incidence of respiratory infections in those receiving a commercially available bl preparation compared to the control group [ ] . similarly, a systematic review proved the effectiveness of bl add-on therapy in reducing the frequency of wheezing episodes and asthma exacerbations in children (p < . for both endpoints) [ ] . infection prevention is one, though not the only, reason for the positive effects of bls on reducing episodes of exacerbation of asthma in children [ ] and chronic obstructive pulmonary disease in adults [ ] . bls can also reduce the frequency of allergic rhinitis episodes [ ] and alleviate the symptoms of atopic dermatitis [ ] . finally, ingesting heat-killed lactobacillus paracasei may be applicable in reducing the symptoms of dry eye syndrome, which primarily arises from the long-term, repetitive exposure to blue light emitted by led screens [ ] . the gut microbiota produces an array of molecules, including vitamins, phenolic-derived metabolites, and aromatic amino acids. due to high bioavailability, antioxidative features, and signaling properties, these substances are considered to be important contributors in host-microbiome crosstalk. it has been demonstrated that in situ-produced bacterial foliate can be absorbed in the colon and incorporated into host's tissues. folate plays an important role in dna synthesis, reparation, and methylation, and is also considered as an antioxidative agent. therefore, intestinal-produced folate may exert systemic function. citizens of countries with mandatory folate food fortification were reported to have lower risk of stroke compared to the controls (rr, . ; p = . ) [ ] . however, the impact of on colorectal cancer risk takes the form of a u-shaped relationship, and the optimum folate status has not been defined [ ] . nevertheless, the issue of intestinal bacteria producing folate and their potential clinical application in maintaining optimal folate status deserves further consideration. in vitro, both folate-producing lactobacillus helveticus cd and the intracellular cell-free extract of this strain demonstrated antioxidative activity [ ] . several bacterial strains have been shown to synthesize vitamin b de novo [ ] . supplementation of lactobacillus acidophilus in yogurt matrix was associated with elevated vitamin b and folate serum levels (p < . ) and reduced prevalence of anemia (p < . ) [ ] . vitamin k is a cofactor required for the synthesis of clotting factors. whereas the contribution of microbiome-derived to vitamin k resources has been established, locally produced vitamin k appears to have underpinning mechanisms. vitamin k concentration in the human gut has been associated with microbiome structure. however, it did not result in the alteration of inflammation biomarkers (il- and tnf-α; both p > . ) [ ] . gut microbiota is postulated to be actively involved in aromatic amino acids (aaa) metabolism. aaa, as bioactive molecules, may act on distant organs, such as the kidneys, brain, and cardiovascular system [ ] . for example, the genetic modification of gut microbiota metabolism enabled to control indoxyl sulfate plasma levels. indoxyl sulfate contributes to the progression of chronic kidney disease, implying the possible role of targeting aaa metabolism in renal disorders [ ] . the interplay between dietary polyphenols and gut microbiota has drawn a great deal of attention. polyphenols modulate the structure and are concurrently metabolized by gut microbiota. hosts' responses to the dietary interventions may differ and this observation formed the basis for developing term called "metabotype" [ ] . metabotyping investigates the relationship between a metabolic phenotype and gut microbiome-derived metabolites that characterize the metabolism of the parent compound, thus providing a rationale for developing "personalized nutrition" [ ] . postbiotics derived from dietary polyphenols include, i.e., urolithin a (ua), equol, and -prenylnaringenin . mice treated with ua for weeks weighed . % less than controls. apart from anti-obesity effects, ua improved the insulin resistance score (homa-ir) in a statistically significant manner (p < . ) [ ] . the first human trial demonstrated the safety profile of orally administered ua, along with improvement in fatty acid oxidation rate, systemic mitochondrial health, and serum acylcarnitinen concentration (p < . ) [ ] . one year of equol supplementation in middle-aged japanese women resulted in arterial stiffness reduction (p < . ) and elevation of lipid parameters (hdl, ldl, and total cholesterol concentrations-all p < . ) [ ] . moreover, one year of equol intake caused a significant increase in whole body bone mineral density among postmenopausal women ( . g/cm vs. . g/cm ; p = . ) [ ] . due to the high heterogeneity of substances classified as postbiotics and the limited framework of this study, this section summarizes only the most important mechanisms of action that are characteristic of postbiotics (figure ). it should be emphasized that there is currently insufficient data available to understand the complex effects of postbiotics in their entirety. however, postbiotics will likely have pleiotropic effects on the human body. figure . mechanisms of action of postbiotics. postbiotics display pleiotropic properties. due to the induction of differentiation of t regulatory lymphocytes and synthesis of anti-inflammatory cytokines, postbiotics restore the imbalance between two major arms of immune system represented by th and th lymphocytes. the balance between th and th lymphocytes is vital for immunoregulation, and its disturbance causes various immune diseases, including atopic disorders. antibacterial activity is probably mediated by postbiotics' impact on the molecular structure of enterocytes, which results in sealing the intestinal barrier. "statin-like" activity of postbiotics and its future therapeutic application in metabolic and related diseases is highly anticipated. the immunomodulatory effects of the gut microbiome have long been suggested [ ] . for example, butyrate (a scfa) induces the differentiation of regulatory t cells (tregs) in the intestine [ ] . in addition, propionate (another scfa) enhances the formation of peripheral tregs [ ] . various fractions of postbiotics isolated from bacillus coagulans culture (supernatant, cell wall fragments) also induce anti-inflammatory cytokine production and promote t helper (th) -dependent immune responses [ ] . moreover, numerous in vitro experiments have shown that the supernatant from a bifidobacterium breve culture induces the maturation and survival of dcs, and consequently, increases il- secretion and inhibits tnf-α secretion [ ] . these properties may be responsible for limiting the th -mediated responses and enhancing th -mediated responses [ ] , as is often observed in those prone to atopic diseases. in a mice model, postbiotics derived from streptococcus thermophilus were shown to enhance th lymphocyte response in mesenteric lymph nodes compared to controls (p < . ) [ ] . as inflammation is inextricably linked to carcinogenesis, any substance that inhibits inflammation may also have anti-cancer potential. indeed, the scfa propionate (produced by propionibacterium freudenreichii) was shown to selectively induce apoptosis in gastric cancer cells [ ] . scfas also influence the regulation of oncogenes and suppressor genes through epigenetic modifications. l. rhamnosus gg supernatant increased zo- expression (responsible for the correct structure of tight junctions between cells and cell adhesion) and decreased mmp- expression (which helps degrade the intercellular matrix, thus facilitating cancer cell penetration) [ ] . indeed, changes in zo- and mmp- level caused by exposure to the l. rhamnosus gg supernatant helped reduce colorectal tumor cell invasion in an in vitro model (p = . ) [ ] . due to the induction of differentiation of t regulatory lymphocytes and synthesis of anti-inflammatory cytokines, postbiotics restore the imbalance between two major arms of immune system represented by th and th lymphocytes. the balance between th and th lymphocytes is vital for immunoregulation, and its disturbance causes various immune diseases, including atopic disorders. antibacterial activity is probably mediated by postbiotics' impact on the molecular structure of enterocytes, which results in sealing the intestinal barrier. "statin-like" activity of postbiotics and its future therapeutic application in metabolic and related diseases is highly anticipated. the immunomodulatory effects of the gut microbiome have long been suggested [ ] . for example, butyrate (a scfa) induces the differentiation of regulatory t cells (tregs) in the intestine [ ] . in addition, propionate (another scfa) enhances the formation of peripheral tregs [ ] . various fractions of postbiotics isolated from bacillus coagulans culture (supernatant, cell wall fragments) also induce anti-inflammatory cytokine production and promote t helper (th) -dependent immune responses [ ] . moreover, numerous in vitro experiments have shown that the supernatant from a bifidobacterium breve culture induces the maturation and survival of dcs, and consequently, increases il- secretion and inhibits tnf-α secretion [ ] . these properties may be responsible for limiting the th -mediated responses and enhancing th -mediated responses [ ] , as is often observed in those prone to atopic diseases. in a mice model, postbiotics derived from streptococcus thermophilus were shown to enhance th lymphocyte response in mesenteric lymph nodes compared to controls (p < . ) [ ] . as inflammation is inextricably linked to carcinogenesis, any substance that inhibits inflammation may also have anti-cancer potential. indeed, the scfa propionate (produced by propionibacterium freudenreichii) was shown to selectively induce apoptosis in gastric cancer cells [ ] . scfas also influence the regulation of oncogenes and suppressor genes through epigenetic modifications. l. rhamnosus gg supernatant increased zo- expression (responsible for the correct structure of tight junctions between cells and cell adhesion) and decreased mmp- expression (which helps degrade the intercellular matrix, thus facilitating cancer cell penetration) [ ] . indeed, changes in zo- and mmp- level caused by exposure to the l. rhamnosus gg supernatant helped reduce colorectal tumor cell invasion in an in vitro model (p = . ) [ ] . some postbiotics can have direct antimicrobial effects by sealing the intestinal barrier, competitively binding to receptors required by some pathogenic bacteria, changing the expression of host genes, or modulating the local environment [ ] . indeed, combining postbiotics and probiotics effectively prevented rotavirus-associated diarrhea in a preclinical model [ ] . furthermore, randomized clinical trials conducted in a group of children aged - months showed that daily intake of products containing l. paracasei postbiotic led to a reduction in the incidence of diarrhea [ , ] , acute gastroenteritis, pharyngitis, laryngitis, and tracheitis [ , ] . butyrate (a scfa), was found to support the regeneration of the intestinal epithelium [ ] . meanwhile, supernatant obtained from one of the most common probiotic strains, l. rhamnosus gg, helps to protect human intestinal smooth muscle cells from damage [ ] . the use of bacterial lysates in children was associated with significant reduction of respiratory tract infection compared to controls (md = − . ; p < . ) [ ] . postbiotics may also play a role in lipid metabolism and could reduce the risk of cardiovascular incidents. for example, the scfa propionate can inhibit condensation of cholesterol precursors, leading to statin-like effects [ ] . kefiran also has antiatherogenic properties, which may result from the reduction of inflammation, prevention of cholesterol accumulation in macrophages, and reduction of lipid concentration [ ] . moreover, lactobacillus bls were found to reduce the levels of triglycerides and ldl cholesterol while increasing the level of beneficial hdl cholesterol in an obese mouse model [ ] . this beneficial effect of fragmented lactobacillus bacteria on the lipid profile was caused by activation of the peroxisome proliferator-activated receptor (ppar)α, which is also the therapeutic target of the fibrate class of lipid-lowering drugs [ ] . moreover, propionate stimulated the release of peptide yy and glp- in human colonic cells and resulted in a significant reduction of total adipose content (p = . ) and intrahepatocellular lipid content (p = . ) in vivo [ ] . autophagy is a homeostatic mechanism through which damaged organelles and proteins are cleaned out. this self-degradative process can act as a response to various stress stimuli, including nutrient stress. intracellular receptor nod detects bacterial peptidoglycan and promotes autophagy and inflammatory signaling. irving et al. revealed that this effect was mediated by outer membrane vesicles-spherical membrane structures naturally shed by all gram-negative bacteria as part of their normal growth in vitro and in vivo [ ] . postbiotic-obtained lactobacillus fermentum triggers autophagy in hepatic cells hepg . autophagy inductive potential of l. fermentum displayed protective effects in pharmacologically induced liver toxicity [ ] . mitophagy is a specific autophagy elimination of damaged mitochondria. in humans, urolithin a inhibits mitophagy, and may therefore prevent or delay the development of an age-related decline in muscle health [ ] . oxytocin is a multidirectional neuropeptide that plays a dominant role in stimulating uterine contractions during labor, modulating behavior, and creating an emotional bond. in addition, oxytocin can stimulate and accelerate wound healing. the administration of bls obtained by sonication of lactobacillus reuteri increased the number of oxytocin-producing cells in the hypothalamic periventricular nuclei, resulting in an elevated oxytocin concentration in blood serum in animal models (p < . ) [ ] . comparable results were obtained by the administration of l. reuteri probiotics in both animal and human models, suggesting that the use of bls is sufficient to achieve satisfactory results, with a significantly improved safety profile [ ] . bacterial culture and the production of probiotics are somewhat unpredictable in nature. the problem of dose standardization, which is a significant issue in the production of probiotics, does not exist in the case of postbiotics. from an economic standpoint, the benefits of postbiotics include longer shelf life, easier storage, transport, and a reduced need to maintain a low temperature in comparison to probiotics. the use of a repetitive production process and the possibility of more precise quantitative control (except for bls) are additional advantages of postbiotics compared to probiotics. when discussing the therapeutic benefits, attention should be paid to the superiority of postbiotics over postbiotics in the context of safety. the undoubted advantage of postbiotics is bypassing the problem of acquiring antibiotic resistance genes and virulence factors, which may occur in vivo when probiotics are used [ ] . postbiotics eliminate the need for exposure to live microorganisms, which is particularly important in children with an immature immune system and a leaky intestinal barrier. functional foods can be defined as dietary items with additional health benefits besides their nutritional value. physiologic profits of functional foods are provided by adding new (e.g., probiotics or postbiotics) or already present ingredients. the favorable safety profile of postbiotics makes them rational candidates for use in functional foods. when discussing the clinical use of postbiotics, one cannot ignore galactosyllactose ( '-gl), which is formed as a result of fermentation of human milk oligosaccharides (hmos) and can therefore be classified as postbiotic. in addition to immunomodulatory activity, '-gl also has natural anti-inflammatory properties and improves intestinal barrier integrity [ ] . as bacteria, along with their fragments and metabolites, are passed to the baby in the mother's milk, such a complex mixture cannot obviously be replaced by a single substance. however, combining prebiotics, postbiotics, and hmos into one preparation is a tempting concept for mapping the composition and properties of natural human milk. in addition, functional foods could be enriched with postbiotics to increase the host's immune activity. for example, the cell-free fraction of fermented milk prevented salmonella infection in a mouse model [ ] . it is worth mentioning that the effectiveness of the postbiotics used in the abovementioned study was equivalent for preparations produced on both laboratory and industrial scales. postbiotics from b. breve and streptococcus thermophilus are currently used in the production of functional foods (particularly for modified milk), and their efficacy assessed in randomized clinical trials. for example, b. breve and s. thermophilus postbiotics reduced the incidence of symptoms suggestive of food or inhalation allergy in the first months of life in children with a positive history of atopy, and the effect persisted after discontinuation of the preparation [ ] . the use of the above postbiotics was also associated with a milder course of acute diarrhea in infants [ ] . notably, one of the active metabolites of s. thermophilus is the abovementioned '-gl [ ] . postbiotics are considered may be a viable therapeutic option for allergic diseases, as they can restore the balance of th /th -mediated immune responses and support maturation of the immune system. indeed, available data support the use of postbiotics in preventing asthma/wheezing exacerbations in children [ , ] . in addition, the severity of atopic dermatitis symptoms was inversely proportional to the number of butyrate-producing bacteria in the intestine [ ] , and the oral intake of bls was associated with better results of atopic dermatitis treatment in children [ ] . finally, postbiotics may have beneficial effects in food allergies. a clinical study of over children showed that the presence of a rich butyrate-producing bacterial microbiota was associated with an earlier resolution of cow's milk allergy [ ] . postbiotics play a vital role in the maturation of the immune system, affect barrier tightness and the intestinal ecosystem, and indirectly shape the structure of the microbiota. as such, postbiotics may be useful in treating or preventing many disease entities, including those for which effective causal therapy has not yet been found (e.g., alzheimer's disease, inflammatory bowel disease, or multiple sclerosis). indeed, clinical trials aimed at modifying the microbiota of patients suffering from the abovementioned diseases are currently underway, and the first results are promising [ , ] . postbiotics may be particularly useful in infants, as the first months of life are critical for developing the proper structure of the microbiota. as the microbiota "matures" up to about three years of age [ ] , any abnormalities can be associated with short-and long-term consequences (e.g., necrotizing enterocolitis and asthma, respectively) [ ] . creating the appropriate environment for the formation of the correct microbiota appears crucial for the proper development and preservation of the child's future well-being, and postbiotics can provide such conditions. postbiotics may also be useful in the prevention and treatment of sars-cov- infection, as the structure and metabolic activity of the intestinal microbiome may be related to the occurrence of biomarkers predicting the severe coronavirus disease (covid- ) course [ ] . the potential value of postbiotics is not limited to therapeutic applications. indeed, the emergence of biological doping (and its detection) is an area of interest. a recent study in mice showed that the presence of bacteria of the genus veillonella in the gut, which can metabolize lactic acid to propionate, significantly increased the animals' physical performance [ ] . a similar result was obtained by the enteral administration of propionic acid, indicating the possibility of using postbiotics to modify physical fitness and the independence of the observed effect from the presence of bacteria [ ] . the use of metabolites or fragments derived from microorganisms (i.e., "postbiotics") is an attractive therapeutic and preventive strategy in modern medicine. according to current data, such postbiotics have pleiotropic effects, including immunomodulatory, anti-inflammatory, antioxidant, and anti-cancer properties. some of these properties are even in clinical use. the boundary between probiotics and postbiotics is blurred in some trials, as their impact on the results is often not evaluated separately. we expect further research into the biological activities of these metabolites will unveil novel uses for postbiotics in medicine and beyond. funding: the financial support for the language assistance was provided by nutricia poland. the authors thank proper medical writing, warsaw, poland, for the language assistance provided in the preparation of this paper. conflicts of interest: m.r. received speaker's honoraria from nutricia, bayer ag. w.f. received speaker's honoraria from nutricia, bayer ag, vifor, ranbaxy. j.Ż. and a.m. declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. the composition of the gut microbiota throughout life, with an emphasis on early life the maternal gut microbiome during pregnancy. mcn am making sense of the microbiome in psychiatry systematic review with meta-analysis: the efficacy of probiotics in inflammatory bowel disease systematic review with meta-analysis: lactobacillus rhamnosus gg for treating acute gastroenteritis in children-a update probiotic supplements might not be universally-effective and safe: a review what else? benef. microbes rational identification of diet-derived postbiotics for improving intestinal microbiota function probiotic cell-free supernatants exhibited anti-inflammatory and antioxidant activity on human gut epithelial cells and macrophages stimulated with lps cell-free supernatants from probiotic lactobacillus casei and lactobacillus rhamnosus gg decrease colon cancer cell invasion in vitro antioxidant properties of potentially probiotic bacteria: in vitro and in vivo activities probiotic bacteria and their supernatants protect enterocyte cell lines from enteroinvasive escherichia coli (eiec) invasion postbiotic l. plantarum rg improves ruminal epithelium growth, immune status and upregulates the intestinal barrier function in post-weaning lambs effects of saccharomyces cerevisiae or boulardii yeasts on acute stress induced intestinal dysmotility saccharomyces boulardii improves intestinal cell restitution through activation of the alpha beta integrin collagen receptor food and health potentials of exopolysaccharides derived from lactobacilli enhanced natural killer cell activation by exopolysaccharides derived from yogurt fermented with lactobacillus delbrueckii ssp. bulgaricus oll r- characterization and immunomodulatory activity of an exopolysaccharide produced by lactobacillus plantarum jlk isolated from fermented dairy tofu immunostimulatory activity of exopolysaccharides from probiotic lactobacillus casei wxd strain as a novel adjuvant in vitro and in vivo probiotic properties of exopolysaccharide-producing lactobacillus strains isolated from tempoyak structural elucidation and antioxidant activities of exopolysaccharides from lactobacillus helveticus mb - kefiran reduces atherosclerosis in rabbits fed a high cholesterol diet effects of an exopolysaccharide (kefiran) on lipids, blood pressure, blood glucose, and constipation dectin- is a major beta-glucan receptor on macrophages orally administered particulate beta-glucan modulates tumor-capturing dendritic cells and improves antitumor t-cell responses in cancer glucan supplementation enhances the immune response against an influenza challenge in mice naturally occurring -substituted ( , )-β-d-glucan producing lactobacillus suebicus and pediococcus parvulus strains with potential utility in the production of functional foods exopolysaccharides from milk fermented by lactic acid bacteria enhance dietary carotenoid bioavailability in humans in a randomized crossover trial and in rats β-glucan-based cream (containing pleuran isolated from pleurotus ostreatus) in supportive treatment of mild-to-moderate atopic dermatitis two antioxidative lactobacilli strains as promising probiotics dietary postbiotic lactobacillus plantarum improves serum and ruminal antioxidant activity and upregulates hepatic antioxidant enzymes and ruminal barrier function in post-weaning lambs use of superoxide dismutase and catalase producing lactic acid bacteria in tnbs induced crohn's disease in mice lactobacilli with superoxide dismutase-like or catalase activity are more effective in alleviating inflammation in an inflammatory bowel disease mouse model. drug des oral administration of a catalase-producing lactococcus lactis can prevent a chemically induced colon cancer in mice antibiotic-induced release of lipoteichoic acid and peptidoglycan from staphylococcus aureus: quantitative measurements and biological reactivities lipoteichoic acid isolated from lactobacillus plantarum inhibits lipopolysaccharide-induced tnf-alpha production in thp- cells and endotoxin shock in mice bacterial teichoic acids reverse predominant il- production induced by certain lactobacillus strains into predominant il- production via tlr -dependent erk activation in macrophages induction of intestinal pro-inflammatory immune responses by lipoteichoic acid antimicrobial peptides and skin immune defense system commensal bacteria lipoteichoic acid increases skin mast cell antimicrobial activity against vaccinia viruses enhancement of anti-cancer immunity by a lipoteichoic-acid-related molecule isolated from a penicillin-killed group a streptococcus anti-inflammatory potential of probiotics: lipoteichoic acid makes a difference sodium butyrate inhibits the nf-kappa b signaling pathway and histone deacetylation, and attenuates experimental colitis in an il- independent manner butyrate inhibits nf-kappa b activation in lamina propria macrophages of patients with ulcerative colitis interactions between roseburia intestinalis and diet modulate atherogenesis in a murine model a proliferative probiotic bifidobacterium strain in the gut ameliorates progression of metabolic disorders via microbiota modulation and acetate elevation the short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism acetate-producing bifidobacteria protect the hose for enteropathogenic infection via carbohydrate transporters study of the mechanism of inhibition of ketogenesis by propionate in bovine liver. can anti-inflammatory properties of the short-chain fatty acids acetate and propionate: a study with relevance to inflammatory bowel disease bang oturai, a. short-chain fatty acids and gut microbiota in multiple sclerosis probiotic-induced suppression of allergic sensitization and airway inflammation is associated with an increase of t regulatory-dependent mechanisms in a murine model of asthma immunoregulatory and immunostimulatory responses of bacterial lysates in respiratory infections and asthma immunostimulation with om- in children with recurrent infections of the upper respiratory tract: a double-blind, placebo-controlled multicenter study broncho-vaxom in pediatric recurrent respiratory tract infections: a systematic review and meta-analysis bacterial lysate add-on therapy for the prevention of wheezing episodes and asthma exacerbations: a systematic review and meta-analysis mechanical bacterial lysate administration prevents exacerbation in allergic asthmatic children-the eolia study bacterial lysates as a potentially effective approach in preventing acute exacerbation of copd clinical and immunological benefits of om- bacterial lysate in patients with allergic rhinitis, asthma, and copd and recurrent respiratory infections adjuvant treatment with the bacterial lysate (om- ) improves management of atopic dermatitis: a randomized study effect of heat-killed lactobacillus paracasei kw ingestion on ocular disorders caused by visual display terminal (vdt) loads: a randomized, double-blind, placebo-controlled parallel-group study folic acid in stroke prevention in countries without mandatory folic acid food fortification: a meta-analysis of randomized controlled trials bacterial folate biosynthesis and colorectal cancer risk: more than just a gut feeling antioxidative potential of folate producing probiotic lactobacillus helveticus cd genomic analysis reveals lactobacillus sanfranciscensis as stable element in traditional sourdoughs plasma cobalamin and folate and their metabolic markers methylmalonic acid and total homocysteine among egyptian children before and after nutritional supplementation with the probiotic bacteria lactobacillus acidophilus in yoghurt matrix fecal concentrations of bacterially derived vitamin k forms are associated with gut microbiota composition but not plasma or fecal cytokine concentrations in healthy adults gut microbial metabolites of aromatic amino acids as signals in host-microbe interplay modulation of a circulating uremic solute via rational genetic manipulation of the gut microbiota where to look into the puzzle of polyphenols and health? the postbiotics and gut microbiota associated with human metabotypes the gut microbiota: a key factor in the therapeutic effects of (poly)phenols urolithin a exerts antiobesity effects through enhancing adipose tissue thermogenesis in mice the mitophagy activator urolithin a is safe and induces a molecular signature of improved mitochondrial and cellular health in humans effects of equol supplement on bone and cardiovascular parameters in middle-aged japanese women: a prospective observational study natural s-equol decreases bone resorption in postmenopausal, non-equol-producing japanese women: a pilot randomized commensal microbe-derived butyrate induces the differentiation of colonic regulatory t cells metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation ganedenbc ™ cell wall and metabolites: anti-inflammatory and immune modulating effects in vitro supernatant from bifidobacterium differentially modulates transduction signaling pathways for biological functions of human dendritic cells bifidobacterium breve and streptococcus thermophilus secretion products enhance t helper immune response and intestinal barrier in mice milk fermented by propionibacterium freudenreichii induces apoptosis of hgt- human gastric cancer cells prevention of rotavirus diarrhea in suckling rats by a specific fermented milk concentrate with prebiotic mixture cow's milk and rice fermented with lactobacillus paracasei cba l prevent infectious diseases in children: a randomized controlled trial postbiotics for preventing and treating common infectious diseases in children: a systematic review preventive effect of cow's milk fermented with lactobacillus paracasei cba l on common infectious diseases in children: a multicenter randomized controlled trial modulation of distal colonic epithelial barrier function by dietary fibre in normal rats protective role of postbiotic mediators secreted by lactobacillus rhamnosus gg versus lipopolysaccharide-induced damage in human colonic smooth muscle cells fragmented lactic acid bacterial cells activate peroxisome proliferator-activated receptors and ameliorate dyslipidemia in obese mice effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults the immune receptor nod and kinase rip interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling lactobacillus fermentum postbiotic-induced autophagy as potential approach for treatment of acetaminophen hepatotoxicity microbial lysate upregulates host oxytocin addressing the antibiotic resistance problem with probiotics: reducing the risk of its double-edged sword effect human milk oligosaccharides and synthetic galactosyloligosaccharides contain '-, -, and '-galactosyllactose and attenuate inflammation in human t , ncm- , and h cells and intestinal tissue ex vivo postbiotics produced at laboratory and industrial level as potential functional food ingredients with the capacity to protect mice against salmonella infection a non-hydrolyzed, fermented milk formula reduces digestive and respiratory events in infants at high risk of allergy effects of long-term consumption of a fermented infant formula (with bifidobacterium breve c and streptococcus thermophilus ) on acute diarrhea in healthy infants dung ta, c. identification and synthesis of a trisaccharide produced from lactose by transgalactosylation nonspecific immunomodulators for recurrent respiratory tract infections, wheezing and asthma in children: a systematic review of mechanistic and clinical evidence severity of atopic disease inversely correlates with intestinal microbiota diversity and butyrate-producing bacteria early-life gut microbiome composition and milk allergy resolution alteration of gut microbiota in inflammatory bowel disease (ibd): cause or consequence? ibd treatment targeting the gut microbiome how baby's first microbes could be crucial to future health gut microbiota may underlie the predisposition of healthy individuals to covid- meta-omics analysis of elite athletes identifies a performance-enhancing microbe that functions via lactate metabolism this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -vcm v ix authors: pollmann, michael title: causal inference for spatial treatments date: - - journal: nan doi: nan sha: doc_id: cord_uid: vcm v ix i propose a framework, estimators, and inference procedures for the analysis of causal effects in a setting with spatial treatments. many events and policies (treatments), such as opening of businesses, building of hospitals, and sources of pollution, occur at specific spatial locations, with researchers interested in their effects on nearby individuals or businesses (outcome units). however, the existing treatment effects literature primarily considers treatments that could be assigned directly at the level of the outcome units, potentially with spillover effects. i approach the spatial treatment setting from a similar experimental perspective: what ideal experiment would we design to estimate the causal effects of spatial treatments? this perspective motivates a comparison between individuals near realized treatment locations and individuals near unrealized candidate locations, which is distinct from current empirical practice. furthermore, i show how to find such candidate locations and apply the proposed methods with observational data. i apply the proposed methods to study the causal effects of grocery stores on foot traffic to nearby businesses during covid- lockdowns. how can we do causal inference with spatial treatments? in the setting of this paper, a spatial treatment, such as the opening of a "million dollar plant" (greenstone and moretti, ; greenstone et al., ) occurs at a geographic location, and the outcome of interest, such as earnings, is measured for separate individuals who are located nearby. this distinction between units of treatment assignment and outcome units has received little attention in theoretical work in causal inference. in the absence of guidance from theoretical work, most recent empirical studies using highly-detailed location data rely on adaptations of the familiar difference-in-differences method. unfortunately, these adaptations to the spatial treatment setting implicitly either rely heavily on functional form assumptions or on partly incongruent nonparametric assumptions to identify causal effects. this is in stark contrast to settings with individual-level treatments, where many researchers prefer causal inference based on quasi-experimental methods with simpler, more transparent assumptions, that obtain credibility by emulating an "ideal experiment" the researcher wished to have run. in this paper, i propose (quasi-) experimental methods for spatial treatments that are motivated by an ideal experiment where the spatial locations of treatments are random. these methods are based on a simple insight: suppose the ideal experiment randomly chooses some locations from a larger set of candidate locations. then quasi-experimental methods should compare individuals near locations that are chosen to individuals near locations that were not chosen for treatment. for a formal characterization of estimands and estimators, i extend the potential outcomes framework for individual-level treatments to allow treatments to be randomized across space and to directly affect nearby individuals. within this framework, i derive finite sample design-based standard errors similar to those of neyman ( neyman ( , for randomized experiments with individual-level treatment assignment for a fixed population. in the "million dollar plant" example, my proposals using micro location data are analogous to the approach greenstone and moretti ( ) take with aggregate data, while most current empirical work takes a conceptually distinct approach. suppose we want to estimate the average effect of a million dollar plant on individuals who are, say, mile away. the method employed by most recent empirical work compares individuals on an "inner ring" around the million dollar plant with radius mile to individuals on an "outer ring," who are, say, miles away from the same million dollar plant. since many observable and unobservable characteristics correlate with distance from any one point in space (lee and ogburn, ; kelly, ) , this comparison of inner and outer ring is often unattractive: if treatment always occurs in the city center, the inner ring, or treated, individuals are urban individuals, while the outer ring, or control, individuals are suburban and rural individuals. researchers attempt to ameliorate this issue by adding a pre vs. post comparison in a difference-in-differences approach, where outcomes for urban and suburban individuals are allowed to be on different levels, but must evolve along parallel trends. in contrast, greenstone and moretti ( ) take a different approach with data aggregated at the county level. they compare counties that "won" the bidding war for a million dollar plant to "runners-up" counties that were also very seriously considered as locations for million dollar plants, but ultimately "lost" (greenstone and moretti, ) . in short, the methods i propose compare individuals who are mile away from the million dollar plants to individuals who are mile away from locations that would have been chosen for the plants in the losing counties. since these counterfactual locations are rarely known in observational studies, i show how to find suitable candidate locations in practice. with micro location data, the methods then estimate the same detailed estimands targeted by the difference-in-differences approach. they have an attractive quasi-experimental interpretation and are valid by design if the choice of treatment location is as good as random within a set of plausible candidate locations. the difference-in-differences approach of current empirical practice relies on either partly incongruent nonparametric or functional form assumptions that are not guaranteed to be satisfied even in a true randomized experiment. the comparison of individuals on an inner ring to those on an outer ring inherently makes two assumptions: first, the treatment must not affect individuals on the outer ring directly. this is most easily achieved by choosing an outer ring with large radius, such that these "control" individuals are far away. second, the individuals on inner and outer rings must be comparable. this is most easily achieved by choosing an outer ring close to the inner ring, in conflict with the first assumption. even when the differences in levels between inner and outer ring are differenced out with individual fixed effects in panel data, the parallel trends assumption is particularly strong in spatial treatment settings. suppose, for instance, that treatment only occurs in city centers. then the assumption may require individuals living in downtown areas to be on parallel trends to those on the outskirts of the city. furthermore, researchers typically estimate the effect not just at one distance but at multiple distances, typically using the same outer ring control group. this effectively requires that individuals at all distances up to the outer ring are on the same parallel trend, with additively separable time fixed effects. these assumptions are not just approximations to make finite sample analysis feasible where asymptotically an analogous nonparametric specification identifies treatment effects: identification in this approach rests upon the functional form assumptions even asymptotically, and even with experimental data. instead, i recommend estimators that are formally valid under the quasi-experimental variation in treatment location sometimes used to informally justify the assumptions of the difference-in-differences approach. the difference-in-differences approach generally yields the most credible estimates if the treatment is known not to have an effect past a short, known, distance. then individuals on the outer ring are likely to be comparable. sometimes, these comparisons are then justified by the fact that the exact location of the treatment was as good as random. for instance, (linden and rockoff, , p. ) , referencing bayer et al. ( ) , argue that for their treatment, sex offenders moving into neighborhoods, "the nature of the search for housing is also a largely random process at the local level. individuals may choose neighborhoods with specific characteristics, but, within a fraction of a mile, the exact locations available at the time individuals seek to move into a neighborhood are arguably exogenous." the estimators proposed in this paper allow researchers to make use of such credible identifying variation directly, rather than relying on an ultimately arbitrary outer ring. i demonstrate the quasi-experimental methods i propose in an application studying the causal effects of grocery stores on foot-traffic to nearby restaurants during covid- lockdowns in april of . in this application, i observe the exact spatial locations of grocery stores as well as other businesses in the san francisco bay area. i show how to find "control" neighborhoods that are similar to neighborhoods of actual grocery stores except for the absence of one marginal grocery store. the outcome of interest, foot-traffic to restaurants, is measured as the number of customers whose smarthphone location is shared with safegraph. i find that restaurants at distances of less than . miles from a grocery store have substantially more weekly customers than restaurants near counterfactual grocery store locations in comparable neighborhoods lacking the marginal grocery store. this suggests a positive externality of grocery stores on nearby businesses, akin to anchor stores in shopping malls, at least when customer mobility is reduced as during the covid- pandemic. while i argue in favor of a design-based, quasi-experimental approach in this paper, the difference-in-differences approach has its own advantages, such that both approaches are complementary. specifically, the comparison with an "outer ring" effectively removes time-specific noise that is shared within a larger region but distinct across regions. in contrast, the methods proposed in this paper focus mostly on eliminating confounding due to differences in the spatial neighborhoods, such as population density, of treated and control individuals. whether spatial variation, temporal variation, or functional form assumptions yield the most credible estimates of causal effects depends on the particular empirical setting. researchers may find studies particularly credible if several distinct identification strategies lead to similar conclusions. doubly-robust estimators (e.g. robins and rotnitzky, ; belloni et al., ) , which model both the outcome (conditional expectation) and assignment process (propensity score), may offer an attractive bridge between approaches. the framework developed in this paper allows me to extend the proposed methods to settings where multiple treatment locations are close to one another, as in the application to foot-traffic caused by grocery stores, which are often near other grocery stores. the existing difference-in-differences approach, in contrast, is not applicable when treatment locations are too close to one another. in the framework of this paper, i can allow for such interference between spatial treatments and illustrate the complications it causes. in recent work, zigler and papadogeorgou ( ) and aronow et al. ( ) specifically study such interference in a spatial treatment setting. they derive average effect estimands that are identified despite interference. in the present paper, i instead define the estimands of interest based on an ideal experiment that rules out interference by design. in extensions that complement the work by zigler and papadogeorgou ( ) and aronow et al. ( ) , i then discuss assumptions under which these estimands are identified even when there is interference. in addition, i demonstrate how to find additional candidate treatment locations, where treatment could have occurred but did not, with observational data, increasing the number of settings the proposed methods can be applied to. furthermore, i view the framework developed in this paper as particularly helpful for deriving standard errors of estimators of the effects of spatial treatments. by providing formulas for finite sample design-based standard errors, i sidestep the often difficult decisions regarding clustering and "spatially correlated errors" (e.g. conley, ) that arise in practice for virtually any application using spatial relationships between observations. aronow et al. ( ) also provide some design-based standard errors, but focus on asymptotic normality and sampling-based variances in the style of conley ( ) for the estimator most similar to the ones proposed in this paper. the results in their work therefore complement those in this paper. the interpretation of the standard errors i propose is simple: they reflect the variation in the estimator that arises from randomizing treatment locations, holding the individuals in the sample fixed. this is the same variation that is needed for internal validity of the causal effect estimates (abadie et al., ) . in the baseline setting, the variance estimators i derive are similar to clustering at the level of treatment assignment (abadie et al., ) . the approach i take in this paper, generalizes straightforwardly to settings with a contiguous region or multiple treatments close to one another. clustering, in contrast, is based on sharp, sometimes arbitrary, boundaries and the absence of interference between clusters. finally, the framework highlights nuances in interpretation that have received little attention in the literature thus far. most recent empirical work estimates the effects of spatial treatments at multiple distances. however, the average effects at different distances are not generally comparable. since some individuals are often more likely -before realization of treatment assignment -to be close to treatment locations, their treatment effects typically get more weight in average effect estimands at shorter distances, and less weight in average effect estimands at longer distances. in other words, we cannot generally interpret effect-by-distance curves or the change in effect between distances as average within-individual effects. even the aggregate weight placed on individuals near any one treatment location varies with distance. both of these effects can lead to estimates of average treatment effects that increase in distance, even though individual-level treatment effects are decreasing in distance for every individual. the framework in this paper allows me to characterize estimators with alternative weights on individuals to mitigate such issues. in addition, in this framework i can show how to aggregate individual-level treatment effects to estimate the aggregate effects of treatment at a location on all nearby individuals. the framework and methods discussed in this paper may also prove useful for causal inference questions not directly related to spatial treatments. first, other non-spatial settings also feature "treatments" that are not directly assigned to individuals but affect them based on some measure of distance. in this paper, i briefly discuss bartik ( ) -, or shift-share, instruments, where for instance industry-level shocks affect all cities depending on industry composition. the perspective taken in this paper resembles that of borusyak and hull ( ) , with non-random distances from candidate treatment locations but random variation in which candidate locations are realized. second, i develop an approach to finding suitable unrealized candidate locations in observational data based on flexible machine learning methods. this approach may extend to other settings with dependency between observations where it is sometimes challenging to find (good) control observations, such as event studies and other time series settings. third, separating treatment assignment and outcome individuals in this framework further clarifies distinctions between design-based and sampling-based inference (abadie et al., ) . while design-based inference captures variation in treatment locations, sampling-based inference can reflect sampling of individuals at fixed locations, within fixed regions (infill asymptotics (cressie, ) in the spatial statistics literature), of a growing contiguous space (expanding domain asymptotics (cressie, ) ), or of independent regions (clustering). the present paper focuses on design-based inference specifically; in-depth comparisons of different modes of inference are beyond its scope. my current analysis is limited in at least three important ways. first, i assume that outcome individuals have fixed locations. this is problematic if individuals move, or migrate, strategically in response to the treatment. second, the framework is not directly applicable to settings where we are interested in the causal effects of spatially correlated characteristics of places, such as in the literature on social mobility (e.g. chetty et al., ) . instead, the present paper focuses on treatments that occur at discrete locations in space. while the ideal experiment of randomizing treatment locations also creates a spatially correlated covariate of interest (distance from treatment), the randomization distribution it induces is much simpler to characterize. third, alternative estimators that are more robust or more efficient in certain settings may exist. while i attempt to offer theory and estimators for a variety of spatial treatment settings, the primary focus of this paper lies in developing a coherent conceptual framework that allows me to characterize, discuss, and exploit the ideal experiment with spatial treatments. in particular, the present paper provides no formal justification for the use of methods from the literature on sample splitting and double robustness (e.g. chernozhukov et al., ) . the need to consider many relative spatial locations for finding suitable unrealized candidate locations makes this a high-dimensional estimation problem in observational settings, suggesting the importance of methods and insights from that literature. the remainder of this paper is organized as follows. the final part of the introduction highlights the wide range of empirical applications for which this work is relevant, as well as connections to the theoretical literature. section develops a potential outcomes framework for spatial treatments. section contains the main results on identification, estimation, and inference under the ideal experiment. section discusses how to extend these results to additional settings of empirical relevance. section shifts the focus from experimental to observational data, proposing assumptions and methods that allow researchers to emulate the ideal experiment. section shows how to apply these methods in practice. in the conclusion, i discuss limitations of the present paper and fruitful directions for future research on causal inference for spatial treatments. empirical relevance the methods i propose are relevant for a diverse range of questions from many applied fields in economics and other social sciences. recent studies estimating the effects of spatial treatments using individual-level outcome and location data include stock ( stock ( , ; linden and rockoff ( ) ; currie et al. ( ) ; aliprantis and hartley ( ) ; sandler ( ); diamond and mcquade ( ) ; chalfin et al. ( ) ; rossin-slater et al. ( ) . notably, dell and olken ( ) explicitly consider counterfactual treatment locations in a quasi-experimental setting, as well as the permutation distribution based on counterfactual assignments. much more existing empirical work studying spatial treatments is limited to aggregated outcome data. if micro location data had been available for these studies at the time, researchers would likely have asked questions that can be answered using the methods i propose in this paper. experimental and observational studies of spatial treatments in economics using aggregate data fitting into the framework of this paper include duflo ( ) ; miguel and kremer ( ) ; cohen and dupas ( ) in development economics, greenstone et al. ( ); feyrer et al. ( ) public and labor economics, (jia, ) in industrial organization, and environmental economics (keiser and shapiro, ) . furthermore, a recent literature has documented large geographic variation in a diverse range of outcomes (for instance chetty et al., ; chetty and hendren, ; finkelstein et al., finkelstein et al., , bilal, ) . many potential sources or causes of this inequality, as well as many potential remedies such as place-based policies, involve spatial treatments. related theoretical literature this paper sits at the intersection of the literatures on causal inference, spatial statistics and econometrics. a small number of recent theoretical papers has similarly studied spatial treatments, albeit with a different focus. most closely related zigler and papadogeorgou ( ) , aronow et al. ( ), and imai et al. ( ) focus on settings with interference between treatment locations and show that only some average treatment effects are identified without additional semiparametric assumptions. in contrast, i define estimands of interest in a setting without interference, and discuss application-specific assumptions to retain identification of these estimands under interference. furthermore, i discuss a broader range of estimands and estimators, in particular for observational data where unrealized candidate treatment locations are rarely known. the simpler baseline setting also highlights interpretation and weighting issues that are obscured in the presence of interference. papadogeorgou et al. ( ) take a conceptually different approach. they develop a framework based on spatial point patterns (cf. cressie, ) rather than fixed units of observation to answer a distinct question. for them, the locations of outcome units vary with the treatment assignment, and the number of outcome units is the object of interest. instead of contrasting different treatment assignments to define effects, their estimand contrasts entire assignment mechanisms (stochastic interventions, muñoz and van der laan, ) . mcintosh ( ) proposes an estimator for settings where individuals known to be unaffected by the treatment exist as a natural group group. pouliot ( ) also studies a setting where the locations of outcomes and covariates are spatially misaligned, but not in the context of spatial treatments and causal inference. within the causal inference literature, the setting of this paper most closely relates to work on interference and networks. some work in causal inference explicitly considers spatially correlated treatments (delgado and florax, ; druckenmiller and hsiang, ) , but is not directly applicable to the patterns generated by spatial treatments. the literature on interference is concerned with spillover, or indirect, effects of treatments assigned to individuals in violation of the stable unit treatment value assumption (rosenbaum, ; hudgens and halloran, ; tchetgen tchetgen and vanderweele, ; aronow and samii, ; vazquez-bare, ; sävje et al., ; sävje, ; basse et al., ) . treatment effects in network settings typically originate from individual-level treatment assignment and propagate through the network (e.g. basse et al., ). in contrast to the interference and networks literatures, the present paper is concerned with a setting where the units of treatment are separate from outcome units. while the effect "spills over" to the outcome units, there is no interference between different treatment units if they are few and far apart, as in the baseline setting of this paper. consequently, the estimands and estimators of interest in spatial treatment settings generally differ from those in interference and network settings. for spatial treatment settings with interference, the spatial relationships between observations allow me to make semiparametric functional form assumptions to limit interference; see section . for details. similar assumptions may sometimes also be plausible if treatments are directly assigned to individuals but have spillover effects on other individuals. the design-based finite sample inference developed in this paper complements samplingbased large sample asymptotic theory developed in the spatial statistics and econometrics literature. conley ( ) proposed standard errors taking into account cross-sectional (spatial) dependence in a gmm framework; see also case ( ) ; lahiri et al. ( ) ; lee ( ) ; andrews ( ) ; kelejian and prucha ( ) ; bester et al. ( ); lahiri and robinson ( ) ; kuersteiner and prucha ( ) and references therein for alternative results. spatial proximity is also commonly used to motivate cross-sectional dependence in the literature on clustered sampling (moulton, (moulton, , moulton and randolph, ; hansen, ; donald and lang, ; barrios et al., ; cameron and miller, ; abadie et al., ) . the spatial statistics and econometrics literature is primarily concerned with descriptive estimands, modeling the spatial correlations existing in outcome data even in the absence of spatial treatments. textbook treatments of such models in spatial statistics and econometrics include cressie ( ) ; cressie and wikle ( ); anselin ( ) ; anselin et al. ( ) ; anselin and rey ( ) ; lesage and pace ( ) ; arbia ( ) . since treatment assignment (or distance from treatment) does not vary within location, "increasing domain asymptotics" (cressie, ) (asymptotics in the number or size of regions or clusters) are likely needed for consistency of causal effect estimates. spatial treatment applications, however, typically feature a large number of individuals near a smaller number of treatment locations, such that alternative "infill asymptotics" (cressie, ) may offer better approximations. the primary contribution of this paper to this literature is a focus on the estimation of causal effects and design-based in-sample inference, rather than descriptive estimands and sampling-based inference. this paper also connects to the literature on estimation of treatment effects under unconfoundedness and doubly-robust estimation. specifically, i propose a formal notion of unconfoundedness (cf. rosenbaum and rubin, ; imbens and rubin, ) that is appropriate for spatial treatments. with individuals and treatment locations distributed across space, a large number of covariates, such as population density or average income at different distances, are predictive of both outcomes and treatment assignment probabilities. doublyrobust estimators are particularly promising in observational settings with spatial treatments: they have attractive consistency and efficiency properties based on the combination of outcome and treatment (propensity score) modeling. recent work has adapted these estimators to high-dimensional settings (belloni et al., (belloni et al., , farrell, ; chernozhukov et al., ; . initial results suggest that such estimators may also perform well in spatial treatment settings. finally, this paper contributes to the recent literature illustrating creative uses of modern machine learning methods for economic analyses (see mullainathan and spiess, ; glaeser et al., ; athey, ; gentzkow et al., ; , for recent reviews). i propose a method for finding unrealized candidate treatment locations based on an adversarial task: finding unrealized locations that are indistinguishable (to the algorithm) from realized treatment locations. most closely related, use generative adversarial networks (goodfellow et al., ) to create samples for simulation studies. kaji et al. ( ) similarly propose "adversarial estimation" to estimate structural models using generative adversarial networks. in each of these applications, the aim is to generate synthetic samples which look indistinguishable from the real data. in the application of this paper, only the unrealized candidate treatment locations are synthetic, while the outcome and covariate data around it are real. in this paper, i argue in favor of convolutional neural networks in particular, based on the similarity between spatial data and image data, which sparked more recent developments in this method (krizhevsky et al., ) . relative spatial positions are similar to relative positions of pixels, and different covariates at each location correspond to the different color channels of images. for economic applications using satellite data (see donaldson and storeygard, , for a review), convolutional neural networks have also shown promise (e.g. jean et al., ; engstrom et al., ) . convolutional neural networks are particularly attractive for spatial settings because they build on relevant economic intuition for regularization: while the geographic space might be large and high-dimensional, the immediate spatial neighborhood often matters the most, and relative distances matter similarly at different absolute locations. through careful design decisions, the methods i propose for the spatial treatment setting retain some interpretability in addition to the good performance commonly associated with "black box" machine learning algorithms. in this section, i propose an extension of the potential outcomes notation (cf. imbens and rubin, ) that treats the level of treatment assignment as conceptually distinct from the level at which we measure outcomes. this distinction separates the intervention that is the cause of the effect from the individuals for whom the effect is measured. it allows me to formally characterize estimands of interest, and to derive estimators and their properties in the following sections. with spatial treatments, potential outcomes of individuals are functions not of an individual-level binary or continuous treatment, but of a set of candidate treatment locations. we are interested in the effects of spatial treatments. let s denote the set of candidate treatment locations, shown as triangles in figure . th set of candidate treatment locations is assumed to be finite; in the example of figure just two locations in the region shown. this reflects an inherent scarcity that is common to most applications: only a small number of locations are ultimately realized, and most locations are infeasible, unsuitable, implausible, or unlikely for the treatment. in spatial settings, the candidate locations are typically given by latitude and longitude or other (relative) coordinates, such that s ⊂ r . throughout this paper, the set s is finite, as virtually any practical application will be based on some discretized, or rounded, locations. one can, however, take s as defining a finely spaced grid over r . this is convenient to figure : illustration of the setup. while typically only relative locations matter, locations are often given by their "gps coordinates" as latitude and longitude. in the figure, the candidate treatment locations at which the treatment may occur are given by triangles. the small circles indicate the locations of individuals. the researcher typically estimates the treatment effects, caused by treatment at one of the candidate locations and experienced by the individuals, conditional on distance from treatment. when the (weighted) euclidean distance function is used, individuals within a narrow distance bin from a candidate location are located on a ring, here displayed as an area shaded gray. if driving time is used instead to measure distance, individuals at a given distance need not be located on a circular ring. the figure shows data from a single region. in the baseline setting of this paper, the researcher has data from multiple such regions, with treatment realized only in some of them. if treatment is realized at multiple (both) candidate locations (triangles) within the same region, there is potential interference between them, complicating estimation and inference. in the baseline setting, the probability of treatment at locations and in regions describes a two-stage process. in the first stage, a fixed number of regions are chosen randomly for treatment somewhere in the region. in the second stage, a single candidate location in each chosen region is chosen randomly to receive treatment. conceptualize situations where treatment could be realized anywhere with some positive probability. the random variable ⊂ s denotes the set of the realized treatment locations. we measure the outcome of interest for units indexed by . for the remainder of this paper, i will refer to these outcome units as individuals, but in some settings may be a business, census tract, or similar, typically small, unit with fixed geographic location. denote the set of all individuals by i. individual has spatial location, or residence, , shown as small circles in figure . throughout this paper, i assume that the locations of individuals are fixed; there is no migration. in some applications, corresponds to, for instance, the workplace of individual rather than their residence. the location of is in the same space as the candidate treatment locations, such that typically ∈ r are latitude and longitude. define potential outcomes for each individual ∈ i potential outcomes: as the outcome for individual if treatment is realized in locations ⊂ s. to simplify notation, and consistent with standard potential outcomes notation, let the potential outcome of individual in the absence of any realized treatment be ( ) ≡ (∅). the treatment effects of primary interest contrast some treatment vector ⊂ s with the absence of realized treatments, ′ = ∅. specifically, i define the effect of on an individual ∈ i as treatment effects: oftentimes, the treatment vector of interest, , is a singleton, = { } for a single candidate location ∈ s. with slight abuse of notation, define treatment effects: i define meaningful average treatment effects in section . these average treatment effects average across both individuals and treatment vectors . distances distances between treatment locations and individuals are central to defining interesting average treatment effects in section . for instance, the researcher may estimate the average effect of a treatment at a distance of mile. in figure , the areas shaded gray highlight all locations approximately mile away from any candidate treatment location. the distance between treatment location ∈ s and individual ∈ i is given by a distance function distance function: ( , ) ≥ importantly, the distance between two locations must be observable (to the researcher) and must not be affected by treatment assignment, ruling out migration in response to the treatment. the distance function is used for two purposes. first, to estimate heterogeneous average treatment effects by distance from treatment. second, to assume distances at which treatments have no effect to limit interference and to thereby aid in estimation and inference. when locations are given as cartesian coordinates, we can use the euclidean distance in r : euclidean distance: when locations are given by latitude and longitude, the great circle distance is more accurate than a euclidean distance with fixed weights on latitude and longitude. for some applications in social sciences, driving distances are arguably more relevant. suppose the spatial treatment corresponds to an employer opening a new location. then an individual's access to the treatment, and hence treatment effect, likely depends on driving time rather than straight line distance. however, computing driving times between many locations may be computationally and financially expensive. when using straight line distances instead, some interpretability, but not validity, is lost. we can also study the effects of state-wide policies and other clustered assignments in this framework. in this setting, each candidate treatment location ∈ s corresponds to one cluster, or state. the appropriate distance function for this setting is in the simplest case of state-wide policies, we use this distance function to estimate the treatment effect at a distance of . this corresponds to estimating the treatment effect of the policy by comparing individuals in treated states to individuals in untreated states. we can generalize the cluster membership function to be smooth in distance to a treated state: for individuals in treated states, this distance is . for individuals in untreated states, the distance is smallest if they are most exposed to treated states. exposure may measure, for instance, distance to the state border, shared media markets, number or cost of flights between airports, or the relevance of the industries of treated state to 's occupation. regions in many applications, it is convenient to group individuals and treatment locations into regions. for instance, in a sample of data from different cities, individuals and treatment locations of each city may form a separate region. when regions are not directly coded in the data, one can sometimes define regions based on geographic proximity such that treatment locations only have effects within their own regions. that is, no individual is close enough to candidate treatment locations from two or more distinct regions to be affected by both of them. figure shows data from one such region. in the baseline setting of this paper, the researcher has access to data from multiple such regions, but this requirement is relaxed in section . . throughout, i denote regions by subscripts = , . . . , . let s ⊂ s be the set of candidate treatment locations within region . the set of realized treatment locations within region is . if treatment is realized within region , ̸ = ∅, let = , and otherwise, = ∅, let = . if = , i say that region "is treated" or "is a treated region." analogously, if = , i say that region "is a control region." let i ⊂ i be the set of individuals with residence in region . the region where individual resides is given by ( ), such that ∈ i ( ) . interference the notation in this paper can be seen as an extension of the notation of the literature on interference (cf. aronow and samii, ) . consider first a setting with individual-level treatments. let be the treatment assigned to an individual = , . . . , , and ∈ { , } be the vector stacking all of the . in the absence of interference, that is, under the stable unit treatment value assumption (cf. imbens and rubin, ) , the observed outcome of individual is = ( ). with interference, the outcome of individual may depend not only on her own treatment assignment, but also on the treatment assignment of other individuals. that is, the potential outcomes of are function a function of the entire rather than only her own , and her observed outcome is = ( ). notationally, spatial treatments generalize this setting by allowing to have a dimension other than , the number of individuals. for the closest analogy, enumerate the candidate treatment locations by = , , . . . , where is the finite number of candidate treatment locations. the random variable of realized treatment locations takes on values ∈ { , } , such that ≡ whenever the kℎ candidate location is treated, and ≡ otherwise. the realized outcome for individual is then = ( ), where is rather than dimensional. consider the example given in figure . some individuals are at a distance of mile from both treatment locations. if the treatment has an effect at that distance, the treatment states of both candidate locations jointly determine the observed outcome. the two candidate locations can interfere because conditional on the treatment state of just one of them, the outcome for some individuals still varies depending on the treatment state of the other candidate location. the literature on interference is typically interested in answering (at least) one of two questions. first, what is the effect of changing 's treatment status, holding the treatment status of 's neighbors fixed? second, what is the effect of changing the treatment status of 's neighbors, holding the treatment status of fixed? with spatial treatments, neither of these questions is of primary interest. if is mile away from a realized treatment location, then a neighbor of , say ′ , is also approximately mile away from the same realized treatment location. a counterfactual where is mile away from a realized treatment location, while her neighbor ′ is not, is typically not feasible or relevant in practice. the treatment does not spill over from to ′ , it affects both of them directly, such that decompositions into direct and indirect effects (cf. hudgens and halloran, ) are not well defined. interference in the spatial treatment setting refers to multiple treatment locations affecting the same individual, rather than the treatment or effect of one individual spilling over to another individual. formally, a treatment location affects an individual if for some set of treatment locations ⊂ s, the outcome of changes when is included or excluded: . two treatment locations , ′ ∈ s interfere with one another if there is an individual affected by treatment at both locations, that is in spatial treatment settings, it is often natural to assume that treatment locations that are far away from an individual do not affect her. formally, assume that whenever ( , ) > max for some sufficiently large distance max , ( ∪ { }) = ( ∖ { }) for all ⊂ s. assumption formally states that there is no interference across regions. assumption (no interference across regions). individuals in region are unaffected by treatment locations in regions ′ ̸ = . that is, for ∈ i and ⊂ s, regions are sufficiently far apart that individuals in one region are unaffected by treatment locations in another region. the results in this paper, however, fundamentally rely on the absence of interference between treatment locations that are far apart, not on separate regions. section . discusses a setting where all data available to the researcher comes from a single large contiguous region. if the region is sufficiently large and realized treatment locations are sufficiently scarce, it is still possible to estimate causal effects without strong additional assumptions. the separate region framework, however, helps clarify key concepts by simplifying estimators, and it is applicable to a large number of empirical studies. the assumption that treatment locations only affect individuals within the same region is similar in spirit to assumptions that interference or spillovers are limited to family members, classrooms, or other subgroups in settings with individual-level treatments (e.g. vazquez-bare, ). the assignment mechanism (imbens and rubin, ) determines the probabilities with which treatment is realized at each of the candidate treatment locations. the marginal probability that treatment is realized at a location ∈ s is given by pr( ∈ ). in the main part of the paper, i consider a two-stage assignment mechanism that imposes structure on pr( ∈ ) as well as on the conditional probabilities pr( ∈ | ′ ∈ ) and pr( ∈ | ′ ̸ ∈ ). in the first stage, either a fixed number of regions is chosen to receive treatment, or assignment is through independent bernoulli trials (coin flips). in the second stage, a single location receives treatment in each treated region. i discuss methods for some observational settings that deviate from this assignment mechanism in sections . . suppose the randomization of treatments across regions takes the form a completely randomized experiment with a fixed number of treated regions. assumption formalizes this design together with an assumption that each region is equally likely to be treated. define ≡ pr( = ) for = , . . . , to be the probability that a region receives treatment. note that the completely randomized design differs from experiments that are paired or stratified at the region-level. results for stratified experiments are generally similar and can be obtained by substituting the appropriate covariances of treatment indicators in the proofs. estimating the variance of estimators under paired designs is often difficult (e.g. bai et al., , for individual-level treatment assignment), but does not contribute conceptually to our understanding of the spatial treatment setting. assumption (completely randomized experiment). regions are chosen for treatment according to a completely randomized design (e.g. imbens and rubin, , ch. . ) where each region has equal marginal probability of receiving treatment somewhere, = for all regions . that is, all assignment vectors ∈ { , } with ∑︀ = ≡ are equally likely, and assignments with ∑︀ ̸ = have zero probability: as an alternative to completely randomized designs with fixed probability of treatment, i also consider designs where treatment is decided by independent coin flip for each region, potentially with different probabilities. assumption below formalizes this assumption. assumption (bernoulli trial). regions are chosen for treatment according to a bernoulli trial (e.g. imbens and rubin, , ch. . ) where region has marginal probability of receiving treatment somewhere and assignment is independent across regions. that is, the probability of assignment ∈ { , } is such that the number of treated regions varies. in the main part of the paper, i consider a setting with exactly one treated location in each treated region. this restriction of the assignment mechanism rules out interference by design under the minimal assumption that treatments have no effects across regions. for each candidate treatment location in a region, ∈ s , define the probability of treatment conditional on the region receiving treatment as ( ) ≡ pr( ∈ | = ). then, by the definition of conditional probabilities, pr( ∈ ) = pr( ∈ | = ) pr( = ) = ( ) . the notational distinction between treatment of regions and treatment of particular locations within regions is motivated by an asymmetry in which potential outcomes are observed: in control regions, the control potential outcomes are observed for all individuals near each (unrealized) candidate treatment location. in treated regions, in contrast, only the treated potential outcomes corresponding to one particular treatment location are observed for all individuals. this asymmetry is apparent in the estimators and variances throughout section . individual-level effects express the average effects of treatment locations on individuals. the most intuitive estimator of the average effect of a spatial treatment on nearby individuals takes the simple average of individuals near realized treatment and subtracts from it the average outcome of properly chosen control individuals. in this section, i first show who the proper control individuals are under the ideal experiment of random variation in treatment locations. then i present properties of this estimator and discuss its interpretation as the average treatment effect on the treated. the average of individuals who are treated at a distance ± ℎ from a treated location is where ( ) = if and only if individual is in a region ( ) that is treated. the indicator function equals if and only if the distance between individual and the realized treatment location in her region, ( ) , is within the distance bin of distances between − ℎ and + ℎ. for instance, to estimate the average outcome for individuals who are between and miles from treatment, calculate¯( . ) with ℎ = . . the choice of control individuals to compare this average of treated individuals to is less obvious. recent empirical studies compare the treated to controls on an outer ring; that is, to individuals ′ in treated regions ( ( ′ ) = ) who are farther away from treatment. effectively, this estimates the treatment effect at distance as¯( ) −¯( ′ ) where ′ ≪ . in analogy to individual-level randomized experiments, one might also consider taking the simple average of individuals in control regions, ∑︀ ( − ( ) ) / ∑︀ ( − ( ) . while either of these strategies is valid under further assumptions or in particular settings, below i argue in favor of a different strategy that is justified by the experimental design. one particular choice of (weighted) control average is, however, justified by the experimental design of the ideal experiment considered in this paper: most importantly, the estimator¯( ) only averages over individuals who are at approximately distance from some candidate location ∈ s ( ) . the remaining weighting is similar to inverse probability weighting estimators of the average effect of the treatment on the treated (att) in settings with individual-level treatments (cf. imbens, ) . to see that the control average¯( ) provides the appropriate counterfactual for the simple average of the treated ( ), consider the expected value of the terms in the numerator of the latter. it is straightforward to show that see appendix a. for the details. the difference between the expression above and the terms of the estimator¯( ) is that the latter can only average over individuals in control regions, with ( ) = , requiring the additional inverse probability weight ( ) in¯( ). the estimator¯( ) therefore aligns, in expectation, the weights placed on each control potential outcome ( ) with those placed on the corresponding treated potential outcome ( ) bȳ ( ). consequently, the estimator^( estimates a weighted average of the differences ( ) − ( ), which are the individual-level treatment effects ( ) defined in section above. the particular inverse probability weights make^( ) an estimate of the average treatment effect on the treated at a distance of ± ℎ. theorem states approximate finite sample properties of this estimator. (i) unbiasedness for the att: is the average potential outcome of individuals at distance from location corresponding to treatment at location ,˜( ) averages the˜( , ) within region , with weights proportional to the probability of treatment at location .˜( ) is the analogous withinregion average potential outcome for the same individuals but in the absence of treatment. ( ) and˜( ) similarly average the within-region averages across regions. the number of individuals at distance from location is ( , ),¯( ) when averaged within region , while¯( ) is the expected number of individuals at distance from realized treatment across regions. the theorem is a special case of theorem below with weight remark . the approximation in theorem arises because the denominators of the estimator ( ) are stochastic. the proof proceeds by deriving the finite sample properties of an infeasible demeaned estimator˜( ) with non-stochastic denominators that satisfies^( )−˜( ) = ( − ), where is the number of regions; details are given in the appendix. even with relatively few regions, the approximation is likely to perform well in practice. similar issues arise with individual-level treatments if treatment is decided by successive coin flips, rather than by fixing the number of treated. in spatial treatment settings, however, it is rarely feasible to hold the number of individuals near treatment fixed when randomizing the assignment of treatment locations. when all candidate locations have equal numbers of individuals in the distance bin, the approximations in the theorem above hold with equality. remark . the expected value given by theorem is also relevant for other estimators usinḡ ( ) as the mean of the treated but relying on a different control comparison group and auxiliary assumptions to justify the comparison. when researchers argue that randomization in the spatial locations for treatments allows them to estimate the treatment effect usinḡ ( ) (or close analogs), they therefore implicitly estimate the average treatment effect on the treated. i believe there is value in making the estimation target explicit: as i argue in section . . below, the att as defined above does not necessarily allow the most meaningful comparisons of the effects at different distances. remark . the control average¯( ) used by the proposed estimator^( ) simplifies to the simple average over all individuals in control regions, if each individual is equally likely to be distance from realized treatment. this typically requires that treatment can be realized at any location within a region with equal probability ( ( ) is constant within ), and the probability that a region is selected for treatment ( ) must be proportional to its area. then the unconditional treatment probability ( ) is constant for all locations , not just for a small, finite, set of candidate locations. figure illustrates and contrasts this with the more common setting where only a small number of candidate locations have positive probability of receiving treatment. remark . the variance given in the theorem is the design-based variance (abadie et al., ) of the estimator. it expresses the variation in the estimate arising from assigning treatment randomly to one candidate location in a fixed number of randomly chosen regions. the individuals whose outcomes are measured are held fixed across these repeated samples; the only difference between samples lies in which potential outcome is observed for each individual. the thought experiment behind the variance above is therefore similar to performing a permutation, or placebo, test. aronow et al. ( ) also suggest permutation tests as an alternative basis for inference in the spatial treatment setting. remark . the first three terms in the variance expression are similar to the variance of the difference in means estimator in a completely randomized experiment with individual-level treatments (cf. imbens and rubin, , ch. ). in the ideal spatial experiment considered in this section, treatment is randomized similar to a completely randomized experiment across regions with outcomes aggregated within regions (and distance bins).˜( ) ( ) is the variance of aggregated treated potential outcomes,˜( ) ( ) is the variance of aggregated control potential outcomes, and ( ) ( ) resembles a variance of treatment effects, such that˜( ) ( ) +˜( ) ( ) − ( ) ( ) resembles the variance of the difference in means under repeated sampling of fixed individuals but varying treatment assignment, the framework of this paper. remark . there is a distinct asymmetry between treated and control outcomes in the expressions for the variance: there are two terms capturing different variances of treated potential outcomes, but only one variance of control potential outcomes. in a treated region, ( ) only averages over potential outcomes corresponding to the realized treatment location, but not those of other, unrealized, candidate treatment locations. the variance of this estimator therefore depends both on how treated potential outcomes vary across regions and within region across candidate locations. if most of the variance is across regions, the final term, −˜( ) is large (negative), reducing the overall variance of the estimator that is due to the variance of treated potential outcomes,˜( ) ( ). since most of the variance is across regions, little is lost by only observing outcomes corresponding to one treatment location in regions with treatment. in a control region, in contrast, we observe the control potential outcomes ( ) that are the counterfactual to all candidate treatment locations s ( ) in the region.¯( ) therefore averages over potential outcomes for all candidate locations within each region, and˜( ) ( ) is the variance of such averages of ( ) within region, across candidate locations. remark . the last two terms in the variance expression arise due to the two-stage randomization in the ideal experiment. after randomizing between regions, the ideal experiment also randomizes between the candidate treatment locations within each treated region. when each region only has a single candidate treatment location, the variance can be simplified to only use the first three terms (scaled), as there is no second stage randomization in that case. estimation of variance without further assumptions, one can only estimate the first two terms of the variance,˜( ) ( ) and˜( ) ( ), to form a conservative estimator of the approximate finite sample variance of^( ). if there is a single candidate treatment location per region, the fifth term can be combined and estimated along with the first (and second) term. the third and fourth term are (approximately) variances of treatment effects, which are unidentified. however, the third term is larger in absolute value than the fourth term (see appendix a. . ), such that − ( ) ( ) + ( ) ≤ . intuitively, ( ) ( ) is approximately the unconditional variance of treatment effects, while ( ) is the variance of the conditional expectation (conditional on region) of treatment effects. by the law of total variance, the difference is expectation of the conditional variance, which is necessarily non-negative. hence, dropping both terms yields a conservative estimate of the variance. if there are multiple candidate treatment locations per region, one can still estimate the fifth term under semiparametric assumption on potential outcomes, such as constant treatment effects. specifically, note that the fifth term consists of the variance of regionaverage potential outcomes, ≈ var( (¯( , )| ∈ s )). one can readily estimate the variance of ≈ var( (¯( , ))), as in the estimation of ≈˜( ) ( ). one can also estimate both types of variances for control potential outcomes because in control regions, the relevant control potential outcome for individuals at distance from any candidate treatment location are observed. if treatment effects are constant, one can estimate¯by scaling^¯( ) by the ratio of the average within region variance to the across region variance of average control potential outcomes. the estimand ( ) is not generally appropriate when the researcher is interested in how the effect of the treatment changes with distance from treatment. as an alternative, i propose the estimand − ( ) with a more attractive interpretation when comparing effects at the figures show regions with individuals (small circles) and candidate treatment locations (triangles), highlighting areas that are distance away from a candidate treatment location in gray. suppose each candidate treatment location is equally likely to be realized. in panel a, all individuals who are distance away from a candidate treatment location receive equal weight in the estimand ( ). in estimation, if the region is in the control group, we take the simple average of outcomes of the highlighted individuals. in panel b, some individuals are distance away from both candidate treatment locations, so these individuals receive greater weight in the estimand ( ). in estimation, if the region is in the control group, we take the average of outcomes of the highlighted individuals, but individuals who are located in both gray rings receive twice the weight. in panel c, candidate treatment locations are everywhere (for illustration, only candidate treatment locations along a grid are displayed). if we assume that candidate treatment locations extend past the boundaries of the region, then all individuals in the region receive equal weight in the estimand ( ). in estimation, if the region is in the control group, we take the simple average of outcomes of all individuals. distance from treatment different distances. additional, one can interpret − ( ) as the expected average effect at distance of a new treatment location. figure illustrates the problem of interpreting the difference between the estimands ( ) and ( ′ ) as the pattern of treatment effects across distance from treatment . suppose the researcher is interested in comparing the average treatment effect at a short distance = short and long distance ′ = long . suppose further that there are two types of candidate treatment locations, each type equally likely to be realized. the first type of candidate treatment locations has many individuals located at the short distance and few individuals at the long distance. these first candidate locations all have relatively small treatment effect at both distances, but decreasing in distance from treatment. the second type of candidate treatment locations has few individuals located at the short distance, and many individuals at the long distance. these second candidate locations all have relatively large treatment effect at both distances, but also decreasing in distance from treatment. in the example in figure , the estimand ( ) is increasing in distance even though the treatment effect of any single treatment location is decreasing in distance from treatment. the estimand ( short ) places most weight on the first type of candidate locations because most individuals at the short distance from treatment are near this type of location. in contrast, the estimand (long) places most weight on the second type of candidate locations. hence, (long) > (short). this inequality states that the average treatment effect at a long distance for the average individual at the long distance from a candidate treatment location is larger than the average effect at a short distance for the average individual at the short distance from candidate treatment locations. it does not imply that the average effect of any single treatment is increasing in distance from treatment. instead, the average individual at a long distance may simply be both a different type of individual (in terms of observables and unobservables) and also be exposed to a different treatment location on average. an alternative estimand, − ( ) defined below and also shown in figure , avoids such issues in interpretation by placing the same aggregate weight on each candidate treatment location irrespective of the distance . the estimand − ( ) first separately averages the potential outcomes of nearby individuals for each candidate treatment location. these averages are then averaged again, with weights proportional only to the probability of treatment at the location. in contrast, the estimand ( ) uses weights proportional to the product of the treatment probability and the number of individuals near the treatment location. formally, where¯( , ) is the average effect of a given candidate location on individuals at distance ± ℎ from it. these average effects are then averaged with weights ( ), which do not depend on distance from treatment. hence, the weight placed on the average effect of a given location does not depend on the distance from treatment. to also non-parametrically control for observable differences in pre-treatment variables , one can estimate − ( ) separately using only individuals with covariate values falling into groups defined by . the comparison of − ( ) and − ( ′ ) then compares individuals with the same average exposure to the different candidate locations and similar individual characteristics . holding the aggregate weight per treatment location constant across distance from treatment is attractive when the treatment effects are expected to be heterogeneous by region or location. such heterogeneity is particularly plausible in many spatial treatment settings: oftentimes, the exact implementation of the treatment differs substantially from location to location. for instance, the million dollar plants in the study of greenstone and moretti ( ) are each operated by distinct companies which may differ in their labor demand and wage setting. hence, heterogeneous treatment effects arise not only due to differences between individuals, but also due to differences in the implementation of the treatments. since spatial treatments are often larger, rarer, and more complex, their implementation tends to vary more than, say, the administration of a drug in medical trials to different patients, or the content of a job training program across training sites or cohorts. additionally, the estimand − ( ) has an attractive interpretation as the expected effect at distance of a new treatment location. consider the following hierarchical model. first, when treatment is realized at location , its average effect at distance is drawn as ( ) ∼ . second, the individual-level effect of location on individual is given by where ( ) is a mean zero individual-specific component. then, as the width of the distance bin, ℎ, goes to , the estimand − ( ) approaches the mean of the distribution . hence, one can interpret − ( ) as the expected average individual-level treatment effect of a new treatment location drawn in the same way as existing realized treatment locations. i propose the following estimator to estimate − ( ): theorem gives the approximate properties of the finite sample distribution of − ( ). under assumptions (no interference across regions) and (completely randomized design), the estimator^− ( ) has an approximate finite sample distribution over the assignment distribution with (ii) and variance as given by theorem with the theorem is a special case of theorem below with weight as specified above. remark . the estimator here is exactly unbiased because under a completely randomized design, the sum of weights is constant across assignment realizations. this is different from theorem above, where the number of treated individuals varies. here, treated individuals are averaged by candidate treatment location, and the number of treated locations is constant across assignment realizations by assumption . more generally, the same ideas allow estimation of any weighted average of individual-level treatment effects that places non-zero weights only on the effects of candidate treatment locations with positive probability of realization. write these estimands of individual-level average effects of the spatial treatment on individuals at a distance from treatment as: where ( ) is the effect of treatment at location on individual , and ( , ) are weights specified by the researcher. the estimand here therefore can be any weighted average of the effect of single treatments on individuals with weights as specified by the researcher. for the average effect of the treatment at distance , weights ( , ) are non-zero only when location and individual are (approximately) distance apart. while i define the att estimands ( ) and − ( ) for distance bins ± ℎ above by using the rectangular, or uniform, kernel function {| ( , ) − | ≤ ℎ}, the weights ( , ) can generally use any kernel function in place of distance bins to estimate the effects at distance . the average effect of the treatment on the treated estimands in equations (i) and are special case of . for att the estimand corresponding to^( ), choose weights for att the estimand corresponding to^− ( ), choose weights i propose an inverse probability weighting estimator (cf. imbens, ) to estimate the weighted average treatment effect in equation above. in short, the estimator is the difference between weighted average outcomes of individuals near realized treatment locations and weighted average outcomes of individuals in regions without treatments. the weights here need to account for two aspects: first, the researcher specifies the desired weights ( , ) in the estimand. second, individuals near locations with high treatment probability are relatively more likely, across samples of repeated treatment assignment, to appear in the sum of "treated" individuals than in the sum of "control" individuals due to the experimental design. the estimator cancels out the probability weighting induced by averaging over individuals (not) near realized treatment for the treated (control) average. to estimate the average effect of the treatment on the treated, the treatment probabilities ( ) ( ) ( ) can be included in the weights ( , ). the proposed estimator for the weighted average treatment effect in iŝ the weights ( , ) are fixed and specified by the researcher. the weights ( ) and , in contrast, are stochastic due to their dependence on the treatment assignment random variables and . specifically, ( ) = unless treatment is realized at location , in which case it is equal to the inverse of the probability of this event. similarly, = unless there is no treatment in the region of individual , in which case it is equal to the inverse of the probability of no treatment in the region. consequently, the stochastic weights are equal to in expectation. the estimator divides each term by the sum of realized weights, ( ) ( , ) and ( , ), such that it is the difference between a convex combination of treated outcomes and a convex combination of control outcomes. theorem gives the approximate finite sample properties of the estimator^( ). under assumption proof: see appendix a. . remark . the variance in theorem can be estimated analogously to that of theorem , also see appendix a. . . remark . the approximate finite sample variance is smaller under bernoulli trials than under a completely randomized design. this is due to the nature of the approximation, which does not penalize the variance as heavily when for instance few treated regions are available under an imbalanced assignment. in practice, the difference between both designs is negligible due to the factor − in the denominator of , such that √ → and there is no difference between the two designs under standard asymptotics in the number of regions. the aggregate effects of a single treatment on all affected individuals is of importance for cost-benefit and welfare analysis. in this section, i propose estimators of aggregate effects that build on estimators of individual-level effects. in experiments with spatial treatments, there are two units of observation: outcome individuals and spatial treatments. the individual-level treatment effects of the previous section are average effects per outcome individual. the aggregate treatment effects of this section are average effects per spatial treatment. suppose the researcher is interested in the aggregate effect that a single treatment location has on all affected individuals. define the estimand where, as before, ( ) = ( ) − ( ) is the effect of treatment location on individual . the aggregate treatment effect sums these effects across individuals and averages them across candidate treatment locations , with weights ( ). in this section, i focus on the average aggregate treatment effect on the treated, , with weights ( ) ≡ pr( = | = ) pr( = ) these weights place larger weight on the effects of treatment locations that are more likely to be realized. the estimand therefore answers the question: what is the expected aggregate effect of a treatment location under the observed policy of assigning treatments to locations? one can estimate the aggregate effect by aggregating outcomes at the region-level: . this is the propensity score weighting estimator of an average treatment effect on the treated, where the outcome variable of interest is the sum over the outcomes of all individuals in a region. when there is a single candidate treatment location per region, standard results from the literature on experiments with individual-level treatments apply (cf. imbens, ) , with regions taking the role of individuals. estimators based on region-aggregate outcomes are likely to have very large variance. each region-aggregate outcome is the sum of outcomes of individuals in the region. if there is substantial variance in the number of individuals per region and outcomes are positive, the aggregate outcome of regions with many individuals can be substantially larger than the aggregate outcome of smaller regions. for instance, suppose that the number of individuals per region is poisson distributed with mean , and individual-level outcomes are i.i.d. within and across regions, with mean and variance . then region-aggregate outcomes have variance · ( + ) by the law of total variance. hence, aggregate potential outcomes have large variance, which leads to large variance of the estimator (cf. imbens, ) . variation in region sizes generates a large variance of the region-aggregate estimator , in two ways. first, if there is variance in the number of individuals per region, then in finite samples, some treatment assignments will be such that there are more individuals in treated regions than in control regions. suppose outcomes are positive and constant, for instance all individuals have the exact same value for the outcome. then the treatment effect estimate^, in such a sample is positive and sensitive to the scale of the outcome value. hence, the estimator^, can have large variance even when there is no variance in potential outcomes. second, variation in region sizes increases the variance in a sampling-ofregions thought experiment. even if the average individual-level treatment effect was known, needing to estimate the number of times the effect is realized on average per region can create substantial variance. the design-based variances considered in this paper condition on the individuals in the sample. with known number of individuals and known individual-level average treatment effect, it is possible to form an estimator of aggregate treatment effects with a design-based variance equal to zero, in contrast to the variance results for the estimator , above. i therefore recommend an estimator of average aggregate effects that reduces the variance by building on the estimator of the average individual-level effect at a distance . let where¯( ) is the average number of individuals at distance from candidate treatment locations: using the same distance bins (uniform kernel and bandwidth equal to bin width) for botĥ ( ) and¯( ). the set of distances d contains the midpoints of the bins that partition the full space into distance bins. for instance, if one uses distance bins [ , ], ( , ], . . . , ( , ] for a treatment that is known not to have effects past a distance of miles, then d = { . , . , . . . , . }. the theoretical properties of the estimator^, follow from those of^( ) in theorem above. theorem . under the ideal experiment, the estimator^, has an approximate finite sample distribution over the assignment distribution with remark . for approximate unbiasedness, the estimator^, must be based on^( ), not^− ( ). intuitively, when "integrating" the effect^( ) against the number of individuals at this distance, one needs to ensure that^( ) is an unbiased estimate of the effect for these particular¯( ) individuals. remark . the variance follows from theorem and theorem . the covariances can be derived analogously. since^, is a sum, its variance is a sum of the covariances of the terms. in the design-based perspective, the analysis is conditional on the individuals in the sample. hence, the number of individuals in each bin,¯( ), is fixed. the estimators^( ) for distances ∈ d are therefore the only stochastic components. remark . the optimal choice of distance bins (and bandwidths) remains an open question. if individuals are spread uniformly across space, equal-width rings with larger radii have larger area and hence contain more individuals. more generally, in densely populated areas, smaller bins may be preferable, and under suitable sequences of populations (infill asymptotics or growing number of regions), it may be possible to allow ℎ → and |d| → ∞. generally, in the formula above, additional distance bins decrease the (squared) weights¯( ) at the cost of increasing variances var i discuss issues in imposing parametric assumptions on the decay of treatment effects over distance from treatment and estimation by least squares regression. first, i show how to impose a parametric model on the individual-level effects at different distances. second, i show how to estimate aggregate effects based on such a model. most simple linear parametric models for the decay of average treatment effects over distance from treatment take the form ( ) = ∑︁˜( ) where˜are known functions of distance, and are coefficients to be estimated. in many settings, one needs to impose a distance after which the treatment has no effect, even within region, to obtain reasonable estimates from parametric models. assumption below formalizes this assumption. assumption . the treatment has no effect after a distance max if, for any individual ∈ i, set of treatment locations ⊂ s, and location ∈ such that ( , ) > max , without such a restriction, any simple functional form for˜will typically offer a poor approximation for at least some distances from treatment. one can improve the approximation to the treatment effect at short distances by using functions that only fit the treatment effect pattern up to the maximum distance max : relatively simple functions may well approximate the average treatment effects at distances ∈ ( , max ). this imposes contextual knowledge that average treatment effects are negligible at large distances from treatment. it also resembles a "bet on sparsity" (hastie et al., ) : if treatment effects really are negligible at distances longer than max , the estimators proposed below will likely perform well. if treatment effects are not negligible even at long distances, then no (parametric) estimator will perform well. for instance, one can impose a linear functional form on the treatment effect decay by choosing = , = . the coefficient then estimates the rate of decay. a quadratic functional form is imposed by = , = , = . in principle, the analysis in this section can be extended also to functional forms that are non-linear in the parameters, such as an exponential decay of treatment effects with unknown rate of decay, ( ) = exp( (− )). to estimate the parameters , suppose initially that there is only a single candidate treatment location in each region. this allows the definition of the distance of individual from the candidate treatment location uniquely as , irrespective of realized treatment. then estimate the weighted linear regression with weights reflecting those in section . . depending on the estimand, such as ( ) or − ( ) for different versions of the average effect of the treatment on the treated. the function ℎ models the average control potential outcomes at each distance from candidate treatment locations. for semiparametric estimation, specify the treatment effect decay ( ) parametrically, and estimate ℎ nonparametrically, as a partially linear model (e.g. robinson, ) . in this paper, i instead focus on parametric linear estimation, which imposes known parametric functions and ℎ and estimates their coefficients: the same caveat about setting a maximum distance applies also to ℎ. since there is no interest in effects at distances larger than max , the constant captures the mean outcome for individuals at such larger distances. in practice, one typically not only want to impose a zero treatment effect after distance max (assumption ), but a treatment effect that tends to zero continuously at max . to this end, estimate the linear regression with transformed covariates figure illustrates what it means to impose this restriction. in panel (a), without the restriction, the estimated treatment effect will jump to discontinuously at max . imposing the restriction in panel (b), the estimated treatment effect is continuous also at max . the restriction generally reduces the variance of the estimator, in particular for estimating aggregate effects, as discussed below. in practice, most functional forms for imply not just a zero effect after distance max , but also a non-zero effect at distances slightly shorter than max . the figure shows a scatter plot of outcomes against distance from treatment. both regression estimators use a quadratic in distance that is set to at a distance of max . the restricted estimator further restricts the regression coefficients such that function is continuous at max . the same parametric functional form can be imposed to estimate the average aggregate effects of the treatment. under the parametric model, the average aggregate treatment effect on the treated is solving for plugging this into the regression specification above, obtain the one-step regression specification where the coefficient on the first (transformed) covariate is the estimate of the average aggregate treatment effect. the transformed covariates are readily computed by realizing they are equal to the original covariates multiplied or shifted by average covariates. the average here is taken across all regions, both treated and untreated, such that this estimates has similarly attractive properties as the nonparametric estimator^, above, in leveraging that the number of individuals near candidate treatment locations are available irrespective of assignment. when there is more than one candidate treatment location per region, augment the regression approach as follows. the variable is not uniquely defined, since there are multiple "distances from candidate treatment locations" for individuals. suppose individual in a control region ( ( ) = ) is mile away from one candidate treatment location and miles away from a different candidate treatment location. then should be used to estimate the control mean ℎ( ) for the two distances = and = . one can therefore duplicate observation . specifically, if individual is in a region with |s ( ) | candidate treatment locations, then include |s ( ) | times in the regression. each version of uses the corresponding to a different candidate treatment location. this ensures ( | = , = ) = and hence results in consistent linear regression estimates. the framework and estimators proposed in the previous sections can readily be adapted to variations in the setting that are of empirical relevance, such as panel data and settings with interference. panel data can serve two distinct purposes in settings with spatial treatments. first, one can use pre-treatment outcomes to reduce the variance of treatment effect estimators. second, with panel data one can base identification of causal effects on a "parallel trends" assumption that is familiar from difference-in-differences methods. i show that existing empirical work relies on a version of this assumption that is not justified by the ideal experiment, discussed in section above, or other (quasi-) random variation in the location of spatial treatments. reducing variance under the ideal experiment, the nonparametric estimators proposed above are (approximately) unbiased, but may have large variance. the variance may be particularly large if potential outcomes of individuals in different regions are on substantially different levels. then, for some treatment assignment realizations, treatment is predominantly realized in the regions with large potential outcomes, such that the estimate ex-post overstates the true average treatment effect. symmetrically, under the inverse of this assignment, treatment is predominantly realized in the regions with small potential outcomes, and the estimate understates the true effect. ex-ante (on average across treatment assignments) the estimator is unbiased. the large differences between estimates for different treatment assignments imply, however, a large design-based variance of the estimator. if the researcher has a pre-treatment outcome for each individual, she can difference out the different levels of the potential outcomes in different regions. to implement this, simply take the difference between post-treatment and pre-treatment outcome for each individual, − pre , and then use the same estimators as before. this does not substantively affect the approximate unbiasedness of the nonparametric estimators. denote, for instance, the estimator^( ) with differenced outcomes as^d iff ( ). it follows immediately that where the pre-treatment outcomes, pre , are fixed across treatment assignments. the treated and control weights, ( ( ) , ( ) , ) and ( ( ) , ) mirror the weights in¯( ) and¯( ), respectively. hence, they are (approximately) equal in expectation by the arguments for unbiasedness of^( ) itself (appendix a. . ). hence, the second term is equal to zero in expectation. subtracting the pre-treatment outcomes from the potential outcomes within the estimand ( ) does not change the estimand at all because the pre-treatment outcomes cancel between the treated potential outcome and the control potential outcome. for the regression estimators, one can alternatively use pre-and post-outcomes as separate observations and include individual fixed effects in the regression for a similar effect. differencing out the levels of the potential outcomes greatly reduces the variance of the estimator if the temporal persistence in potential outcomes is large. this is most easily seen in the case with one candidate treatment location per region. then subtracting the pre-treatment outcomes affects the marginal variances¯( ) ( ) and¯( ) ( ). i recommend using the same formula for the variance estimator as before, but applied to the differenced outcomes, to obtain an estimate of the variance of^d iff ( ). loosely speaking, the variance of^d iff ( ) is smaller if the differenced outcomes have smaller marginal variance: var( ( ) − pre ) < var( ( )) and var( ( ) − pre ) < var( ( )). hence, using the differenced outcomes is likely to reduce the variance if the coefficients in (population) regressions of post-treatment potential outcomes on pre-treatment outcomes is at least . . one can also incorporate multiple pre-periods into this approach for variance reduction. relying on randomized treatment assignment for identification, additional pre-periods can be useful for differencing out the levels of the potential outcomes more precisely. specifically, if there are period specific unobservable components affecting outcomes, averaging over outcomes from multiple pre-periods may provide a more precise estimate of the level of control potential outcomes in the post-period. since the target of estimation is the postperiod effect, however, it is attractive to give greater weight to pre-periods that are closer in time to the post-period. intuitively, the goal is to use the pre-period outcomes to make a one-step-ahead forecast of post ( ). small adjustments have to be made if the pre-period data is for different individuals than those observed in the post-period. since the individuals are distinct, there is no single pre for a post-period individual . instead, the goal is to deterministically construct an estimate^p re based on pre-period outcomes of individuals with locations near the location of , . one can then use the same estimators as before with the transformed outcomes −^p re . if the construction of^p re does not depend on treatment assignment and post-period outcomes, formal results, such as theorem , continue to hold for the differenced outcomes. averaging over pre-period outcomes for different individuals estimates the expected outcome conditional on location. however, it fails to remove individual-specific fixed effects that are not correlated with the location of the individual. intuitively, the loss due to only having pre-period outcomes for different individuals is greater if individual-specific components are large and individual-time specific "noise" is small. in practice, pre-period outcomes for different individuals remain useful as long as neighbors' outcomes are sufficiently predictive of own outcomes, and neighborhood-level outcomes are sufficiently stable across time. identification based on parallel trends one can alternatively use the panel structure of the data to rely on a "parallel trends" assumption for identification of causal effects in a difference-in-differences approach. in practice, such an approach uses the same estimators as proposed for variance reduction. under the ideal experiment, treatment assignment is independent both of the post-period potential outcomes and of trends between the pre-period and the post-period, conditional on the known randomization probabilities. hence, the experimental setting allows the use of the pre-period data but does not require it, as discussed before. in observational settings, discussed in section , using pre-period data augments the assumption underlying identification. whether treatment assignment is more plausibly conditionally independent of levels or of trends depends on the setting. existing empirical work oftentimes uses panel data without control regions in which no treatment occurred (for instance linden and rockoff, ; currie et al., ; diamond and mcquade, ) . instead, these papers compare individuals near a treated location to individuals farther away from the same treated locations. these farther-away individuals are the control group in a difference-in-difference setup. figure illustrates which individuals these estimators are based on. when estimating the treatment effect ( ) at distance , individuals in an "inner ring" at radius from realized treatment constitute the treatment group. individuals who are substantially farther away in an "outer ring" around the same realized treatment location constitute the control group. typically, the same outer ring individuals serve as control units irrespective of the distance at which the treatment effect is estimated. in a difference-in-differences setup, estimators used in much existing empirical work hence rely on a different parallel trends assumption. specifically, individuals on the inner ring need to be on the same trend as individuals on the outer ring. for each distance for which the researcher estimates an effect, she obtains a different set of inner ring individuals. when the effect at each distance is estimated using the same outer ring individuals, she therefore needs to assume that individuals at any distance from treatment (up to the farther distance) are on parallel trends. effectively, this is the semi-parametric functional form assumption that control potential outcomes in all neighborhoods within a region share the same additively separable time-specific component. for these existing estimator, one additionally needs to assume that the farther-way individuals are unaffected by the treatment. if individuals on the outer ring were directly affected, their outcomes in the post-period would not generally reflect the control potential outcomes of individuals on inner rings even when the parallel trend assumption holds. researchers therefore typically restrict the control group to individuals who are substantially farther away from treatment than the treated individuals. however, this assumption is partly incongruent with the parallel trends assumption: the farther the control individuals are figure : existing estimators focus only on regions that received treatment. in this figure, the realized treatment location is shown as filled-in triangle. the treatment group consists of individuals in an "inner ring" at a given distance of interest from treatment, here displayed as small filled-in circles. the control group consists of individuals in an "outer ring" who are farther away from realized treatment, here displayed as hollow squares. existing estimators use pre-and post-treatment data for both groups in a difference-in-differences setup. typically, when researchers estimate the effect at multiple distances, the same control group is used for all distances. panel a shows the estimator^( short ) for a short distance short . panel b shows the estimator^( med ) for a medium distance med . away from treatment, the less likely the parallel trends assumption is to hold. the choice of distance for the outer ring needs to carefully balance these two competing assumptions. as with other difference-in-differences estimators, demonstrating an absence of pre-trends can strengthen the credibility of the parallel trends assumption. for inner vs. outer ring estimators, researchers need to argue that the absence of pre-trends suggests parallel trends even into the post-treatment period based on randomness of timing, not randomness of treatment locations. when treatment effects at multiple distances are estimated, control potential outcomes at each distance from treatment must be on parallel trends with one another and with the outer ring control individuals. for instance, (diamond and mcquade, , figures , , ) illustrate the absence of pre-trends in plots of three-dimensional data (time since treatment, geographic distance from treatment, outcome). in contrast to the more familiar two-dimensional plots from non-spatial settings, it is unfortunately challenging to include standard errors in such figures. it is therefore oftentimes difficult to accurately assess the magnitude and sometimes even direction of possible pre-trends visually. i therefore recommend formal sensitivity analysis and estimation of the partially identified set of treatment effects under small violations of the parallel trends assumption. recent theoretical work has proposed promising approaches to this problem for non-spatial settings that likely extend to the setting of spatial treatments (for instance manski and pepper, ; freyaldenhoven et al., ; rambachan and roth, ) . in settings with panel data, existing estimators and the estimators proposed in this paper both compare individuals near realized treatment to individuals farther away. for existing estimators, far-away individuals are in an outer ring around the realized treatment locations. for the estimators proposed in this paper, far-away individuals are in other, untreated, regions, near candidate treatment locations that appear similar to real treatment locations. however, the assumption for estimators using individuals on an outer ring as a control group is not generally justified by an ideal experiment of randomizing treatment locations. suppose the researcher is interested in the effect of the treatment at some distance . if individuals on the outer ring were the proper control individuals under an ideal experiment, then for each individual on the outer ring, there must be at least one candidate treatment location at distance from the individual. similarly, there must be candidate treatment locations such that individuals who are at distance from the realized treatment would be in the outer ring relative to these locations. this suggests that candidate treatment locations are everywhere and realized with equal probability, as in panel (c) of figure . this assumption is, in general, testable and typically violated in treatments that are of interest to social scientist; an example is given below. hence, these estimators are generally based on functional form assumptions such as additive separability of time-specific effects, rather than on an ideal experiment that involves randomized treatment locations. a first example illustrates when existing estimators based on an outer ring are relatively more attractive. suppose the researcher is interested in the effect of a spatial treatment at a distance of . miles, and knows that individuals at a distance of . miles are unaffected by it. in this setting, individuals at either distance from treatment are likely similar. they live in the same neighborhood and experience the same conditions except for exposure to treatment. the parallel trends assumptions between inner and outer ring individuals is plausible. instead, one should primarily focus on supporting the argument that the treatment has no effect after a distance of . miles. a second example illustrates when the estimators proposed in this paper based on untreated regions are relatively more attractive. suppose the researcher is interested in the effect of a spatial treatment that is typically realized in the city center. she is only willing to assume that the treatment has no effect after a distance of more than miles. then a comparison of individuals close to the treatment to individuals farther than miles from treatment may compare individuals who live close to the city center to individuals living in suburban neighborhoods. the parallel trends assumption between these individuals is less plausible. since treatment is typically realized in city centers, simple tests are likely to reject pr(treatment at | in rural area). instead, it may be more attractive to compare the individuals near treatment to individuals who live close to the city center of other, untreated, cities. with panel data, one can then assume that the inner city neighborhoods of treated and untreated cities are on parallel trends. if one can argue that the location of the treatment was chosen (quasi-) randomly from a set of candidate treatment locations across multiple cities, the assumption is satisfied by design. the estimators proposed in this paper allow this information to be used directly for identification and estimation of causal effects. in this section, i discuss two assumptions on how realized treatment locations that are close to one another interfere. under either of these assumptions, average treatment effects very similar to those in section are identified and readily estimated. the two assumptions i focus on in this section are: (i) treatment locations have additively separable effects; and (ii) only the nearest realized treatment location matters. additively separable treatment effects are an appropriate specification if the effect of each treatment is independent of the realization of other treatments. for instance, the effects of toxic waste plants (cf. currie et al., ) or air-polluting power plants (cf. zigler and papadogeorgou, ) on exposure to pollution are likely approximately additive. typically, only the nearest realized treatment location matters if individuals only access, or visit, a single realized treatment location. for instance, if a developing country quasi-randomly chooses locations to construct new schools (cf. duflo, ) , it may be plausible to assume that only the nearest school matters to an individual. for the effects of infrastructure projects, such as additional bus or subway stops, on commute times and real estate prices (cf. gupta et al., ) , the appropriate assumption may depend on the type of stops that are added. an additive effects specification for bus or subway stops may be a good approximation if each stop gives access to a different transit line. a specification where only the nearest stop matters may be more appropriate for stops of the same line. in contrast, if the treatments interact in some way leading to diminishing or increasing returns in the number of nearby treatment locations, different parametric assumptions on the functional form of these returns may be necessary. this section serves as an example for how to incorporate such assumptions on interference into the analysis of causal effects. i focus one two settings: a first setting where if a region is treated, a fixed number of candidate locations in the region are realized (completely randomized design within region), and data from untreated regions are available. a second setting where treatment assignment to candidate locations is independent (bernoulli trials), but all data come from a single (contiguous) region. suppose that if region receives treatment, exactly˜of the |s | candidate treatment locations are realized, each with equal marginal probability. assuming a completely randomized experiment between the candidate treatment locations within a region greatly simplifies the formulas in this section without mechanically resolving key conceptual issues. in practice, it is sometimes more plausible to assume that the assignment mechanism guarantees some minimum distance between realized treatment locations. it may be possible to obtain analogous results for such more complicated assignment mechanisms. continue to consider a setting with some regions with no realized treatment location. the presence of regions without realized treatment locations is a crucial simplification because it allows identification of control potential outcomes. within this setting, one can see how the assumptions on treatment effects limit interference and allow estimation average treatment effects similar to those in section . to give an example, suppose a company operating chain stores (quasi-) randomly chooses which cities to enter, and opens multiple stores in chosen cities. then there are multiple realized treatment locations close to one another (in the same city), but also control regions with (unrealized) candidate treatment locations. i discuss a settings without untreated regions further below. even in settings with control regions, one needs to make an assumption on interference to identify and estimate the treatment effects as defined in section . . . suppose one makes no such assumption. if some treated regions have multiple, for instance two, realized treatment figure : an example of a region with three candidate treatment locations (panel a): (blue), (red), (yellow). suppose exactly two of these treatment locations are realized whenever the region is treated, such that there is interference. under the assumption that only the nearest realized treatment location matters, panel b illustrates the locations for which we can estimate effects for individuals in each area. for individuals in the orange area, we can estimate the effects of the red and yellow locations. for individuals in the green area, we can estimate the effects of the blue and yellow locations. for individuals in the purple area, we can estimate the effects of the blue and read locations. locations, then it is impossible to identify the average (across all candidate locations) causal effect of implementing one treatment location. but even the effect of implementing multiple treatment locations at once is difficult to estimate in the detail of interest. presumably, one would be interested in the average effect of implementing two treatment locations at distance and , respectively. a non-parametric estimate of this effect is likely based on very few individuals, since few treated individuals are at distance from one treatment and at distance from another treatment. for a given pair of realized treatment locations, there are at most two locations where circles around them with radii and intersect. limiting estimation to only individuals residing close to such intersection points is therefore oftentimes impractical. it implies a dramatic reduction in sample size relative to estimating the effect of a single treatment at a given distance, based on all individuals around this distance ring. if there are more than two realized treatment locations, or treated regions vary in the number of realized treatment locations, this estimation issue worsens. a simple example, illustrated by figure a, helps to build intuition for the estimators proposed below. suppose the researcher has data from multiple regions . each region has three candidate treatment locations; , , , , and , . if region receives treatment, the assignment mechanism randomly chooses exactly two of the three candidate locations to be realized. hence, each candidate location has marginal conditional probability of / of being realized. the set of realized treatment locations in region , , satisfies ∈ i present and discuss two assumptions on interference and how to estimate effects under them in turn. assumption (additive separability of treatment effects). let ⊂ s be an arbitrary subset of the candidate treatment locations, and let ∈ be an arbitrary location in this subset. the effects of spatial treatments are additively separable if, for all individuals ∈ i, ( ) = ( ∖ ) + ( ). assumption formally states that the effects of all treatment locations are additively separable. intuitively, the assumption requires that there are no diminishing (or increasing) returns to having additional treatment locations nearby. under assumption , one can still identify the average treatment effects defined in section . these estimands are weighted averages of individual-level treatment effects ( ) of individual and candidate treatment location which are distance apart. for exposition, i focus on the example with three candidate treatment locations, two of which are realized in treated regions. under the additive separability, assumption , the potential outcomes satisfy hence the treatment effect of interest is where each of the potential outcomes has positive probability of realization. one can therefore estimate ( ) aŝ additive ( , ) = each term in the sum is an unbiased estimator of the the corresponding potential outcome, such that (^a dditive ( , )) = ( , ). one can then average such estimators to estimate, for instance, the att estimand, ( ). see appendix a. for a generalization. assumption (only nearest realized treatment location matters). let ⊂ s be an arbitrary subset of the candidate treatment locations, and ∈ i an arbitrary individual. only the nearest realized treatment location matters if whenever ∈ satisfies ( , ) ≤ ( ′ , ) for all ′ ∈ , we have, for ′ ∈ ∖ { }, assumption states that if ′ ∈ is not the nearest realized location to individual , it does not affect her. the assumption also implies that if individual is at equal distance to two treatment locations and , then both have the same effect on her: under assumption , only some of the average treatment effects of section are nonparametrically identified in general. specifically, it is impossible to identify the effect of a candidate treatment location on an individual if the treatment location is never the nearest realized location for the individual. consider the example of three candidate locations with two realized locations. the effect of location , is unidentified for individuals nearer to both locations , and , . panel b of figure highlights areas in which each candidate treatment location is nearest with positive probability before realization of treatment assignment. generally, the estimand ( ) from section is identified nonparametrically under assumption if it only places weight on individual level effects ( ) if is the nearest realized location to individual with positive probability. formally, write this as where the probability is taken over draws from the assignment distribution of for fixed , , , and . in the example illustrated in figure , one can estimate ( , ) for individuals in the purple and green shaded areas. under assumption , the potential outcomes satisfy an unbiased estimator of ( , ) iŝ one can then average estimates^n earest ( ) across individuals and locations to estimate average treatment effects similar to those in section . however, the estimator^n earest ( , ) is undefined for individuals in the orange shaded area (last line of the definition). since location , is never the nearest realized treatment location for these individuals, it is impossible to estimate its effect on individuals in that area. that is, only average treatment effects that place weight ( , , ) = on individuals in the orange area are identified nonparametrically. see appendix a. for examples of identified estimands and the general setting. suppose the researcher has data on a single contiguous region with individuals , outcomes and candidate treatment locations s. the realized treatment locations are ⊆ s, with assignment to locations , ′ ∈ s independent when ̸ = ′ . assumption formalizes this assumption, which is a straightforward extension of assumption above. assumption (independent treatment assignment -single region). assignment of treatment to locations is independent: for ∈ s,˜⊆ s with ̸ ∈˜⊆ s, as before, the researcher is interested in the weighted average treatment effect with known weights ( , ), for instance for a distance-bin estimator of the average effect of the treatment on the treated. to estimate the average this average treatment effect, consider the estimator which is the difference in average outcomes for individuals near realized candidate locations, remark . while all treatment probabilities are known to the experimenter in experimental analyses, they typically need to be estimated in observational studies. to this end, first note that one can generally write, for any potential assignment ∈ s , second, pr( ⊂ ) pr( = ) cancels between numerator and denominator. hence, to estimate pr( ∈ | ⊂ ) in practice, it is convenient to parameterize this conditional probability and estimate where ( ) are other (spatial) covariates specific to treatment location (and its neighborhood). the framework, estimators, and analysis of this paper are applicable more generally to settings where treatment assignment is separate from the units of observation, and the effect of treatment is moderated by some observable, not necessarily geographic, distance from treatment. i give two examples in this section: firm entry in markets with differentiated products, and shift-share designs based on randomness of the shifts. for the first example, suppose the researcher is interested in the effects of firm entry on competition in markets with differentiated products. she has data for several markets on prices charged by firms ∈ i for products with horizontal or vertical locations in characteristics space. in some markets, a new firm enters with a product with characteristics . here, the estimand ( ) measures the average effect an entrant has on the price of a product at distance in characteristics space. for short distances , it captures competitive effects or deterrence behavior by firms selling products very similar to the product of the entrant. for longer distances , it captures ripple effects that arise if in equilibrium firms with more different products react to the price changes of firms with products similar to the entrant's. these estimands are therefore informative about the nature of competition. firm entry, however, is not generally random. theoretical models of competition and profits may therefore help to determine the probability of firm entry at any given point in characteristics space, conditional on the locations of existing competitors in characteristic space. for instance, expected profits of the entrant may come from a structural model based on distance to competitors in characteristics space (cf. hotelling, ) , perhaps calibrated to pre-treatment data. intuitively, validity of the estimator then requires that firm entry is random conditional on the expected profitability in the model. the structural model provides a baseline to enhance the credibility of the quasi-experimental analysis, but does not directly restrict the estimated pattern of competition. for a second example, suppose the researcher is interested in the causal effects of exogenous shocks to individual industries on employment outcomes in cities based on their industry mixes (cf. autor et al., ) . the framework of this paper is useful in this setting if the claims of causal identification are based on randomness in which industries are shocked, rather than on randomness in industry composition. importantly, the analysis in this paper reflects that cities with similar industry mixes are shocked similarly, in a way that is difficult to capture accurately with existing clustered standard errors. adao et al. ( ) and borusyak et al. ( ) develop alternative approaches based on the same idea, and show how it relates to bartik ( ) and shift-share instruments more generally. a benefit of the framework of this paper is that its results are not specific to linear (or other) functional form and that it allows for very transparent estimation of aggregate effects. the setting fits into the framework of this paper as follows. data are available for time periods = , . . . , . in some time periods, a single industry ∈ s = { , . . . , } receives an exogenous shock, potentially with different industries shocked in different time periods. assume that the time periods are chosen such that the shock only affects outcomes within the same time period. define the indicator = if an industry in period is shocked, and = otherwise. the researcher observes employment outcomes for cities ∈ i in time period . city has industry shares ∈ r , satisfying , ∈ [ , ] and ∑︀ = , = . here, the distance function captures exposure to the shock. city is heavily exposed to shocks of sector if industry has large share , , such that the "distance" ( , ) = − , is small between industry and city . the estimands ( ) and measure the effects of the exogenous industry shocks. for = , the estimand ( ) measures the average effect of shocking an industry on employment in cities with employment only in the shocked industry. for = . , the estimand measures the average effect of the exogenous shock on cities with % of their employment in the shocked industry. the estimand measures the aggregate effect of the exogenous shock across all cities, averaged across shocks to different sectors. the estimators and inference procedures of section are valid if it is random in which time periods and sectors an exogenous shock occurs. in principle, one can augment the variance calculations to allow, for instance, dependence structure in the shocked industry across time periods. the results in section . are relevant for settings where shocks occur to multiple industries in the same time period. while the previous sections presumed that the researcher designed the experiment for assignment of the spatial treatment, much empirical work relies on observational data. the primary challenge to observational studies in this setting is that researchers typically do not observe the exact locations of unrealized candidate treatment locations. to emulate the analysis of the ideal experiment with observational data, researchers need to estimate candidate treatment locations and their treatment probabilities. estimation is then based on an unconfoundedness assumption stating that among individuals near candidate treatment locations, whether their treatment location is realized is as good as random, conditional on characteristics of the individuals and the neighborhood of the candidate treatment location. suppose that there are multiple regions = , . . . , , defined such that any treatment location only affects individuals within the same region. define the location-specific treatment indicator ( ) to equal if location in region is treated, and − if the location is no treated: where = if treatment occurs somewhere in region , and is the realized treatment location in region as in section . in treatment effect settings with individual-level randomized experiments, unconfoundedness is often written as ⊥ ⊥ ( ), ( ) | = which is equivalent to an assumption on densities i similarly define unconfoundedness of spatial treatments at distance from location as here, = means that the sets of individual covariates are the same up to permutation. in practice, it is rarely feasible to find two candidate locations with equal number of individuals and equal covariates. instead, one can assume that treatment is unconfounded conditional on, for instance, average characteristics of individuals in the neighborhoods of candidate locations. such an assumption greatly simplifies estimation in practice. alternatively, one can make an individual-level unconfoundedness assumption for spatial treatments as a conditional mean equality, where, for the control potential outcome, there is no conditioning on distance from candidate treatment locations. in other words, individuals with the same covariates , potentially including neighborhood characteristics of , in control regions offer a valid comparison for the individuals treated at distance . such an assumption simplifies estimation, but is not justified by experimental design or arguments that the location of the treatment is as good as random. in this section, i outline a general strategy for finding unrealized candidate treatment locations with observational data. these counterfactual locations for the treatment are necessary for the quasi-experimental methods i propose in this paper. consider first the example of linden and rockoff ( ) given in the introduction, where the choice of candidate locations is relatively straightforward. they argue that the exact houses where sex offenders move in are as good as random due to random availability of houses within neighborhoods. here, the candidate treatment locations are houses in these neighborhoods. hence, the candidate locations are known, but their probabilities of treatment need to be estimated. see section . for propensity score estimation. when there are no (or insufficiently many) known unrealized candidate locations, however, the problem of choosing candidate locations from continuous space is hard. in principle, one could imagine estimating the probability of treatment at any location in a region conditional on all the features of the region. this is akin to estimating the spatial distribution of treatment locations ∼ ( ), where are the characteristics of region , potentially relative locations of all individuals in the region as well as moments of their covariates. one could then use the estimated^to inform the treatment probabilities at each point in the region as inputs in the estimators proposed in this paper. in practice, it is typically sufficient to find a finite number of candidate treatment locations that offer a plausible counterfactual to the realized treatment locations. computationally, it is often infeasible to use a continuous distribution , since the weight of each individual when estimating effects at distance would depend on the integral of^along a ring with radius around her location, . instead, i recommend finding a finite number of candidate locations. the average across these finitely many candidate locations approximates the strategy based on the complete distribution . i propose taking draws ∼^( ) to obtain candidate treatment locations. perhaps surprisingly, recent machine learning methods achieve good results at this task, despite the difficulty of estimating itself. specifically, i recommend a formulation similar to generative adversarial networks (goodfellow et al., ) ; see liang ( ) and singh et al. ( ) on the relationship between generative adversarial networks and density estimation. most closely related to this paper, use generative adversarial networks to draw artificial observations from the distribution that generated the (real) sample, for use in monte carlo simulations. generative adversarial methods for drawing ∼ ( ) are based on iteration between two steps. first, a generator generates draws˜∼˜( ), where˜is an implicit estimate of the density maintained by the generator in the current iteration. second, a discriminator receives as input either counterfactual locations proposed by the generator,˜| , or real treatment locations, | , and guesses whether its input is real. both the generator and the discriminator are highly flexible parametric models for their given tasks. the discriminator is trained by taking (stochastic) gradient descent steps in the direction that improves discrimination between real and counterfactual locations. the generator is trained by taking (stochastic) gradient descent steps in the direction that leads to fooling the discriminator into classifying counterfactual locations as real. effectively, the output of such models are counterfactual candidate treatment locations | that are indistinguishable (to the discriminator) from real treatment locations | . with a sufficiently flexible discriminator, the process is therefore similar to matching. if a proposed candidate location˜is noticeably different from all real treatment locations , a flexible discriminator will learn to reject˜. in contrast, synthetic control-type methods (abadie et al., ) would average multiple candidate locations, for instance˜and˜, to create a synthetic counterfactual for a real treatment location . if˜and˜individually differ from all real treatment locations , the discriminator will reject them despite their average resembling . the goal therefore is to find "false positives:" occasions when the classifier suspects a missing realized location even though there is no such missing realized location. typical classifier networks do not directly make binary predictions, but give a continuous activation score that indicates how likely each location (or the "no missing location" category, see below) is. in practice, i recommend looking for high activation scores for a particular location and low activation score for "no missing location." alternatively, one can look directly for activation scores resembling the activation scores of real treatment locations. such locations are likely to be decent matches for the real treatment locations, since they must share features of realized locations in order to achieve high activation scores. in the remainder of this section, i discuss how to tune generic machine learning methods to find suitable candidate treatment locations in social science applications. i recommend four high-level implementation decisions in adapting these methods. first, discretization of geographic space into a fine grid for tractability. second, convolutional neural networks capture the idea that spatial neighborhoods matter in a parsimonious way. third, incorporating the adversarial task of the discriminator into a classification task for the generator greatly simplifies training. fourth, data augmentation (rotation, mirroring, shifting) for settings where absolute locations and orientation are irrelevant. discretization to tractably summarize the relative spatial locations of individuals and treatment locations, i recommend discretizing geographic space into a fine grid. discretization provides an approximation that is particularly tractable for the convolutional neural networks recommended below. in principle, future improvements to, for instance, capsule neural networks (hinton et al., ) or other novel methods, may replace this as the preferred architecture and eliminate the need for discretization. for each grid cell, one can include a count of individuals with residence in the cell, potentially separately for individuals with different values of covariates, as well as average covariate values of the individuals in the cell or other moments of their covariates. if the grid is very fine, this discretization retains almost all meaningful information about relative locations. for instance, in the application of this paper, each grid cell has size . mi × . mi (approximately m × m). the discretized grid creates a three-dimensional array: the first two dimensions determine spatial location, and the third dimension enumerates the different covariates that are summarized. rather than taking the spatial dimensions to be entire regions, i recommend using square cutouts of regions such that the probability of treatment in the center of the cutout is only affected by individuals and covariates within the cutout. convolutional neural networks convolutional neural networks (cf. krizhevsky et al., ) have been particularly successful at image recognition tasks. in image recognition tasks, the input is a d array: a d grid of pixels, with multiple layers corresponding to the rgb color channels. for spatial treatments, the input also is a d array: the d spatial grid with layers corresponding to different covariates as described above. convolutional steps in neural networks generally retain the shape of the d grid, but the value of each neuron is a function of the covariates (or neurons) of the previous step not just at the same grid cell, but also the covariates (or neurons) at neighboring grid cells. figure illustrates this aspect of the convolution operation. however, the particular way in which the neighborhood of a grid cell is averaged is the same for any point in the grid. this makes convolutional layers substantially more parsimonious than fully connected layers, and allows the neural network to capture neighborhood patterns appearing in different parts of a region in a unified way. in particular, i recommend using at least two convolutions with reasonably large spatial reach. consider the application in this paper, where grocery stores are spatial treatments and restaurants are individuals with foot-traffic to the restaurants as the outcome. the first convolution allows each grid cell to see what other cells are around it. in the example, the output of the first convolution for a particular grid cell may be: "there are grocery stores nearby, competing restaurants very close, and restaurants within walking distance." the second convolution then uses the information on such neighborhoods to determine whether treatment is likely in a grid cell: "if there are many grid cells nearby (in all directions) containing restaurants or grocery stores facing much competition, this location is probably in center of a shopping area and reasonably likely to contain another grocery store." intuitively, the first convolution may measure what is important to the restaurants, while the second figure : convolutions in a neural network allow the prediction of a candidate location in a grid cell to depend on the characteristics of neighboring grid cells (up to a user-specified distance). these models remain parsimonious by requiring the same "neighborhood scan" to be performed for each grid cell. convolution translates how that is important for the treatment location choice, mirroring the unconfoundedness assumption (equation ) of the previous section. adversarial classification generative adversarial networks are oftentimes difficult to train despite recent advances such as networks with wasserstein-type criterion function . the difficulty arises because the training of generator and discriminator needs to be sufficiently balanced such that both improve. in contrast, convolutional neural networks for image classification are much easier to train. i therefore recommend to set up the problem of finding candidate treatment locations as a classification task. specifically, the convolutional neural network takes a given input array and "classifies" it into, say, categories, where each category corresponds to a grid cell and signifies that there should be an additional treatment location at that point in the grid. to retain the adversarial nature of the task, train the classification on three sets of data: first, regions with at least one real treatment location, but with one treatment location removed. the correct classification of such region data is into the category corresponding to the grid cell from which the treatment location was removed. second, regions with at least one real treatment location, but without any treatment location removed. the correct classification of such region data is into a specially added category signifying no missing treatment location. third, regions without treatment locations. these are also classified as not missing any treatment location. the neural network then balances two tasks: a generative task of picking the correct location if a treatment location is missing, and a discriminatory task of deciding whether a treatment location is missing at all. this structure retains the attractive interpretation of generative adversarial networks, but is substantially easier to train. technically, it resembles denoising autoencoders (cf. vincent et al., ) . data augmentation data augmentation serves two closely related purposes. first, rotating, mirroring, and shifting regions, while maintaining relative distances, produces additional, albeit dependent, observations. this is helpful since training neural networks requires large numbers of training samples. second, these transformations effectively regularize the parameters of the estimated model. one can choose transformation that induce equivariance to rotation, mirroring, and shifts as appropriate for the particular setting. for instance, in many applications in the social sciences, north-south and east-west orientation is irrelevant on a small scale; only the relative distances matter. in particular, suppose an individual who visits a business to the north of her home because it is on the way to work in the north. if the whole space was rotated, the individual equally visits the same business now to the west as it is still on the way to work, now also rotated to be to the west of her home. in image classification, the use of similar data augmentation is common and often associated with greater generalizability of the learned models. shifting the entire grid has two further desirable effects: first, if one imposes a continuous shift of relative coordinates in combination with a fixed grid, the exact discretization becomes less relevant. the average (across draws from the shift distribution) distance in grid cells between two observations becomes directly proportional to their actual distance. second, the location of an observation within a grid cell is no longer fixed. this is attractive because the classification is not actually informative of whether the candidate treatment location is at the center or towards the edge of a grid cell. with a continuous shift of the observations, the center of the grid cell points to different absolute locations depending on the shift. one can then average over several realizations of the shift to reduce the influence of the particular translation of grid cell to absolute location. there are at least two notable alternatives or complements to data augmentation in the machine learning literature. first, spatial transformer networks (jaderberg et al., ) attempt to estimate a rotation or other transformation that makes the subsequent classification task as easy as possible. second, some recent work considers imposing the desired in-and equivariance properties on the convolution kernel. similarly, penalization of deviations from in-or equivariance serves as a less strict regularization of the model parameters. ultimately, current implementations of these methods are less computationally efficient than data augmentation and standard convolutional neural networks. furthermore, simulation evidence suggests that data augmentation achieves the first order gains implied by these properties. one can also inspect the models to assess the implied degree of invariance, and consider averaging parameters as implied by invariance. suppose candidate treatment locations s are known (in all regions), for instance as output of the convolutional neural network classification tast described in the previous section. the remaining challenge in implementing the methods proposed in this paper is the estimation of the "propensity score" pr( ∈ ). i briefly sketch propensity score estimation in two canonical settings: a fixed number of realized treatment locations per treated region (often just one realized location), and independent bernoulli trials determining realization of treatment at candidate locations. fixed number of realized treatment locations suppose there are a fixed number of realized treatment locations per treated region. then the problem of propensity score estimation resembles discrete choice modeling: there are |s | discrete alternatives in region , a fixed number of which is realized. see, for instance, greene ( ) for an overview of estimation methods. when treatment assignment is independent across locations, propensity score estimation for spatial treatments is similar to propensity score estimation for individual-level treatments. logistic regression is a simple option. each candidate treatment location ∈ s is a separate observation. with logistic regression, regress the indicator { ∈ } on covariates ( ) that describe the neighborhood of candidate location as well as (moments of) the characteristics of individuals near location , ( ) : ( , )= for all distances of interest . adjusting for the true propensity score is likely sufficient for unconfoundedness in equation , similar to the setting with individual-level treatments (cf. rosenbaum and rubin, ) . using estimated propensity scores in observational studies, the propensity score is typically estimated by the methods above rather than known. even when the propensity score is known, there may be benefits from using estimated propensity scores for parts of the analysis as in experiments with individual-level treatments (cf. hahn, ; hirano et al., ; frölich, b) . when estimated propensity scores are close to or , the inverse propensity score weighting estimators proposed in this paper may perform poorly (cf. frölich, a; busso et al., ) because small estimation errors in the propensity scores have large effects on the weights when denominators are close to zero. to reduce the effect of estimation error from this first-stage estimation, i also use cross-fitting and a doubly-robust moment condition (e.g. chernozhukov et al., ) in the application of this paper. while existing results assuming i.i.d. data are not directly applicable to the spatial treatment setting, doubly-robust moments likely still substantively reduce the effect of error due to propensity score (and outcome model) estimation. treatments, restaurants are the (outcome) individuals, and foot-traffic (the number of customers) is the outcome of interest. i argue that the inner ring vs. outer ring comparison used in many recent empirical studies is unattractive in this setting: its identifying assumption is not credible, and it requires discarding the majority of the sample for practical reasons. i show how to implement the methods proposed in this paper, and argue that the control groups these methods are based on are preferable to outer ring control groups. the average treatment effect of interest is identified by an ideal experiment where some grocery store locations are randomly closed during covid- lockdowns. specifically, take a restaurant near a grocery store at location . what is the difference between the number of customers of restaurant during the covid- lockdown when there is a grocery store at location , and the number of customers of restaurant if there was no grocery store at location , holding fixed the locations of all other businesses and grocery stores. in the notation of this paper, if are the locations of other grocery stores, the treatment effect of interest is ( ) = ( ∪ { }) − ( ). this effect is distinct from fixing a spatial location near a grocery store, and considering the difference in the outcome (during covid- ) of the business that exists at this point in space when there is a grocery store nearby, and the outcome (also during covid- ) of the, possibly different, business that would have been at the same location, had there never been a grocery store nearby. grocery stores may have causal effects on the number of customers to nearby restaurants if they draw customers into the shopping and business area. in particular during the first few weeks of the covid- lockdowns, when individual mobility was greatly reduced, getting groceries may have been one of the few trips still made. if grocery store customers are more likely to stop by coffee shops or restaurants for pick-up orders right before or after getting groceries, restaurants and similar businesses may receive more foot-traffic if there is a grocery store nearby. large department stores serving as "anchor stores" of shopping malls may play a similar role in normal times. relatedly, jia ( ) studies the effects of new wal-mart stores on existing businesses. study the effect of restaurant closings on nearby restaurants. the effects of grocery stores on nearby restaurants are informative about several questions. do grocery stores have (positive) externalities on other businesses? if so, should mall operators subsidize grocery stores through lower rent such that they internalize these externalities, to support other businesses in the mall? in the context of pandemics, are grocery stores likely choke points leading to bunching of customers at nearby restaurants instead of spreading out across all restaurants, increasing the risk of infections? alternatively, grocery stores may resolve a coordination problem: suppose that the overall reduced number of restaurant customers is insufficient to operate restaurants profitably or with reduced loss when spread across all restaurants. grocery stores may then help to resolve a coordination problem between restaurants, by focusing potential restaurant customers on the nearby restaurants. i use safegraph data on the number of customers of each business in the week starting april , . i restrict the sample to businesses in the area between san francisco and san jose in the san francisco bay area, as highlighted in figure . restricting to businesses with the outcome of interest is the inverse hyperbolic sine of visits to restaurants, with visits as measured by safegraph. to interpret the percentage point effect on the number of safegraphtracked customers as the overall effect, assume that safegraph's sample selection is orthogonal to the presence and absence of grocery stores. otherwise, the estimates retain internal validity as the effects on the number of safegraph-tracked customers to these restaurants. the inverse hyperbolic sine allows for zero visits, and effects on it can be transformed into elasticity estimates similar to log( ) or log( + ) specifications (see bellemare and wichman, , for a discussion). businesses with fewer customers may also be open. however, grocery stores with few if any customers tracked by safegraph are unlikely to have effects on the number of safegraph-tracked customers to nearby restaurants. safegraph ( ) describes the algorithm used for attributing visits to businesses. generally, pick-up orders as well as outside dining are likely picked up by the algorithm as long as a customer's smartphone sends location data at the point of interest for more than one minute. for errors in attribution to matter in the application of this paper, they need to correlate with the presence or absence of nearby grocery stores. the inverse hyperbolic sine is defined as arcsinh( ) ≡ ln( + √︀ + ). hence arcsinh( ) = , arcsinh( ) ≈ . , arcsinh( ) = . , and arcsinh( ) ≈ ln( ) + . if ≥ . figure : the comparison of businesses on an inner vs. outer ring around a particular grocery. the grocery store is marked by an orange triangle in the center of the figure. other businesses are small blue circles. businesses on the gray inner ring, at a distance of . ± . miles, are primarily in strip malls, while businesses on the gray outer ring, at a distance of . ± . miles, are away from these main shopping areas. figure illustrates why comparisons between observations on an inner ring and observations on an outer ring around a strategically chosen location are often not attractive. here, businesses (blue circles) on the inner ring are at a distance of . ± . miles from the grocery store (orange triangle), while businesses on the outer ring are at a distance of . ± . miles from the same grocery store. while inner ring businesses are part of the same strip mall, outer ring businesses are outside of the primary shopping areas. interpreting differences in outcomes for these two groups of businesses as causal effects requires assuming that outer ring businesses are unaffected by treatment and have similar outcomes as inner ring businesses in the absence of treatment. generally, distance from treatment often correlates with many other variables (kelly, ) . with small numbers of grocery stores (see below), the mode of average treatment effect estimates may not be close to the true average effect, even if the locations of grocery stores were random. this arises due to spatial correlations in outcomes even in the absence of treatments (cf. lee and ogburn, , in a network setting). while panel data can in principle relax one of the underlying assumptions, the common (visual) test for the absence of pre-trends carries little information about the validity of the identifying assumption in this setting. with panel data, the assumption of comparability of inner and outer ring businesses is relaxed slightly to an assumption of parallel trends. businesses on inner and outer rings are allowed to have different average levels of customers, but trends in the inverse hyperbolic sine of the number of customers must be parallel. however, even if panel data suggested that trends between inner and outer ring businesses were indeed parallel pre covid- , one may question whether this is informative about changes in (potential) outcomes during covid- lockdowns. given the dramatic decrease in customers for all businesses, it is questionable that this decrease would have occurred in parallel with only an additive shift (in the inverse hyperbolic sine) for inner and outer ring businesses in the absence of treatment. additionally, the estimand of a difference in differences estimator in this setting is the additional effect of grocery stores on nearby businesses during covid- on top of any effects that may have already existed pre covid- . even if the parallel trends assumption was credible, this estimand differs from the estimand of interest described above. the difference in differences estimand can be negative even though the effect of grocery stores on nearby businesses is positive during covid- if the effect of grocery stores pre covid- was also positive but larger in magnitude, for instance due to overall difference in the scale of the number of customers. finally, in most instances, businesses on the outer ring around a grocery store are not actually far away from grocery stores ("untreated"), as illustrated by panel (a) of figure . here, some of the businesses on the outer ring centered around the grocery store in the center of the figure are very close to a second grocery store to the north. applying the inner vs. outer ring estimator in this setting therefore requires restricting the sample to the neighborhoods of the few grocery stores that are sufficiently far away from other grocery stores. specifically, to guarantee the absence of interfering grocery stores for an outer ring "no effect" distance of . miles, only grocery stores with no other grocery store within × . miles can be used. panel (b) of figure shows the locations of the remaining grocery stores. compared to figure , these grocery stores are in more remote, less (sub-) urban neighborhoods. while the average treatment effect of grocery stores in such locations may continue to be of interest, it is plausibly distinct from the treatment effect in areas with higher population or business density. figure shows the comparison of means resulting from the inner vs. outer ring estimation. the average outcome of any distance to treatment (blue curve) is differenced with the average outcome of the outer ring (horizontal gray line), here chosen to be businesses between . and . miles from real grocery store locations. using grocery store fixed effects improves upon these estimates slightly by allowing the weights on the outer ring of each grocery store to vary by distance from treatment. intuitively, if % of all inner ring businesses are at distance from grocery store a, then the outer ring businesses around grocery store a should receive % of the aggregate weight of all outer ring businesses for estimating the effect at distance . if the fraction of inner ring businesses that are near grocery store a is different at distance , then also the businesses on the outer ring of grocery store a should on aggregate receive the different weight. estimates from this fixed effect specification are shown in row of table . for row , the aggregate weight for businesses near each grocery store are constant at / (weighting each of the grocery store locations equally), irrespective of the number of businesses near each grocery store, resembling the weighting of the estimand att-eq ( ) and facilitating a comparison of the effect at different distances from treatment. note that, for the inner ring vs. outer ring estimation, i cannot estimate the effect at a distance of larger than . miles because i have to assume that there was no treatment effect at that distance to be able to define an outer ring that is not near any grocery store. the spatial experiment estimator based on the ideas proposed in this paper, also shown in table , suggests that there indeed likely is no treatment effect past that distance. however, : panel (a) shows an example of a grocery store (triangle in the center) with a second "interfering" grocery store (triangle towards the top) nearby. some businesses on the outer ring are close to (treated by) this second grocery store and therefore not a valid control group. panel (b) shows that restricting the sample to the (out of ) grocery stores without interference leads to a sample selected heavily towards less business-dense areas compared to the overall sample shown in figure . the inner ring vs. outer ring estimator additionally requires that the average outcome at those longer distances is informative about the average outcome at shorter distances. as argued above, figure suggests this assumption is not a particularly good approximation. this application is covered by the framework of section . for a single contiguous region with independent treatment assignment. the key idea behind identification for the proposed methods is that the location of a grocery store is as good as random between candidate locations with similar numbers and industries of nearby businesses. figure shows an example of an ideal comparison where the only difference between the (parts of the) regions is the absence of the bottom-most grocery store, and all other relative distances are the same. the approach i propose for observational data proceeds in two steps: first, it finds good "matches" for each grocery store; that is, locations without a grocery store that are similar in terms of the number, types, and relative locations of other businesses and grocery stores. second, assume the matched data resemble the ideal experiment of randomizing grocery stores between the real and counterfactual candidate treatment locations. i recommend inverse propensity score weighting estimators based on the results of sections and . . conceptually similar combinations of matching or stratification and propensity score weighting or regression adjustments have been advocated for by abadie and imbens ( ) , (imbens and rubin, , ch. ) , and kellogg et al. ( ) , among others. the grocery store location prediction following section . discretizes the south bay region into a fine grid and aggregates characteristics of businesses in each grid cell. figure illustrates the discretization for the surroundings of an example grocery store, see panel (a). for each grid cell, record the number of grocery stores as in panel (b) . other characteristics of each grid cell, for instance the number of businesses by industry are recorded in similar grids as in panel (c). figure : the propensity score model can still distinguish between some of the false positives / counterfactual locations and real grocery store location, resulting in many candidate locations with low propensity score. after a propensity score matching step and re-estimation of the propensity score, overlap is better. real and counterfactual grocery store locations have similar (estimated) propensity score. based on this discretization, i use the method as described in section . to find counterfactual candidate grocery store locations that are indistinguishable from the real grocery store locations. since the method can find a very large number of counterfactual grocery store locations, i use propensity score matching to narrow the sample down to a smaller but more balanced sample of real and counterfactual grocery store locations. panel (a) of figure shows the limited overlap in propensity scores before this second matching step, while panel (b) shows good overlap for the final set of candidate locations. to estimate propensity scores in this setting, i assume that grocery store openings are independent decisions at each location, assumption . in practice, this assumption is primarily relevant at the margin of opening (or closing) additional grocery stores relative to the existing grocery stores. since there are neighborhoods similar in other businesses but differing in the number of grocery stores, this assumption may offer a reasonable approximation. the inverse probability weighted real and counterfactual grocery store locations are similar in everything except their exposure to real grocery stores, which differs by one additional grocery store. figure shows that the exposure to treatment is as intended: the number of grocery stores at distance between . and . miles from a business is the same between businesses at any distance from real and counterfactual grocery store locations, except businesses at that distance from a candidate grocery store location. businesses at distance from a real grocery store have exactly one additional real grocery store at distance on average, compared to businesses at distance from counterfactual grocery store locations. furthermore, the composition of nearby businesses is similar between real and counterfactual grocery store locations at any distance. figure shows that the fraction of restaurants among businesses at distance from counterfactual grocery store locations is comparable to the fraction of restaurants among businesses at distance from real grocery store locations. this lends credibility to the treatment effect estimates below. treated and control businesses are alike, except for a single additional grocery at the intended distance. distance from potential grocery store location in miles share of businesses that are restaurants realized false true figure : the composition of businesses near real and counterfactual grocery store locations is similar. it is encouraging that counterfactual grocery store locations mimic the business composition pattern across distance of real grocery store locations. since the fraction of restaurants decreases meaningfully from short distances to longer distances, inner vs. outer ring comparisons of all businesses would compare businesses in different industries. inner ring vs. outer ring comparisons of restaurants would compare restaurants in different (business) neighborhoods. figure : weighted mean of inverse hyperbolic sine of visits for businesses near real grocery store locations (blue line) and for businesses near counterfactual grocery store locations (red line). the difference between the two lines at a given distance is the estimate of the average treatment effect at that distance. panel (a) includes all businesses, while panel (b) restricts the sample to restaurants. there is a substantial estimated treatment effect at very short distances of up to . miles, and no meaningful difference between treated and control businesses at larger distances. given candidate treatment locations and propensity scores, i estimate treatment effects with the estimators of section . . to interpret the estimated effect as the average effect of opening single grocery stores, rather than the marginal of adding a grocery store to existing exposure, one can make the additivity assumption . additivity may be plausible if each additional grocery store brings new customers into an area. during covid- , customers may reduce the number of different grocery stores they shop at to limit their exposure. furthermore, there is differentiation in the grocery store market: the customers at discount grocery outlets may be distinct from the customers at whole foods. figure shows the average outcome of all businesses (panel a) and restricted to restaurants (panel b) by distance from candidate treatment location, contrasting real grocery store locations (blue line) and counterfactual grocery store locations (red line). at very short distances, businesses (including restaurants) on average have more customers if a (real) grocery store is nearby. if the grocery store is . or more miles away, it has no more effect on the businesses. table shows the spatial experiment estimator, which is the same as the difference between the curves at each distance for restaurants (corresponding to panel b of figure ). i also report estimates for the alternative estimator^a tt-eq ( ), which holds the aggregate weight placed on each grocery store constant across distances. i recommend this estimator for comparisons of effects across distances. since the grocery stores causing the effects are heterogeneous in their numbers of customers, their effects on foot-traffic to nearby restaurants is likely to be heterogeneous as well. i also estimate the att using a doubly-robust moment (e.g. chernozhukov et al., ) . the natural extension of the att-moment to the spatial treatment setting with interference table : estimated effects on the inverse hyperbolic sine of number of visits to restaurants using different estimators. the first panel uses the inner vs. outer ring comparison. the second panel uses the inverse probability weighting estimators for spatial experiments proposed in this paper. the third and final panel uses a doubly-robust version of the spatial experiment estimator. for each method, i implement to estimators: the average effect of the treatment on the treated (^( )), and the equal weighted att estimator (^a tt-eq ( )) that has a more attractive interpretation for comparing the effect at different distances. standard errors for the inner vs. outer ring estimators are clustered by grocery store. standard errors for the spatial experiment estimators will be reported in future version. note that the inner ring vs. outer ring comparison uses substantially fewer treatment locations because it requires restricting the sample to isolated grocery stores. where ( ( ) ) averages over all combinations of candidate grocery store locations and individuals satisfying ( , ) ≈ . { ∈ } plays the role of the "treatment indicator." the function ( , ) gives the expected outcome (inverse hyperbolic sine of number of visits) for a business with covariates , including neighborhood characteristics, when there are grocery stores at locations . for a business near a real grocery store, the conditional mean function is evaluated in the absence of the nearby grocery store , ∖ { }, with the parameter of interest, ( ), capturing the difference between actual outcome and expected outcome in the absence of the nearby grocery store. for businesses near an unrealized candidate location , the conditional mean function is evaluated at the background treatment exposure level . the propensity score ( ) gives the probability that there is a real grocery store at candidate location , conditional on characteristics of the neighborhood of . this moment function satisfies the neyman orthogonality condition of chernozhukov et al. ( ) . relative to the spatial experiment estimator, which treats the propensity score as known, this estimator has the advantage of reducing the impact of small errors in the estimated propensity score through orthogonalization. overall, the inverse propensity score weighting estimator and the doubly-robust estimator yield similar results as shown in table above. grocery stores have an economically large positive effect during covid- lockdowns only at short distances of less than . miles. intuitively, grocery store customers do visit nearby restaurants and coffee shops, but are unlikely to walk for more than a couple of minutes from the grocery store location. for instance, at the (control) average inverse hyperbolic sine of visits of approximately . , an increase of . points implies a % increase in the number of customers. the aim of this paper is to argue that leveraging quasi-random variation in the location of spatial treatments is both conceptually attractive and feasible in many settings in practice. i propose a framework and experimental approach for estimating the effects of spatial treatments. this approach uses random variation in the realized locations of the spatial treatments for causal identification. i argue that an alternative estimator commonly used in practice is not justified by the same random variation, but instead identifies causal effects only under sometimes questionable functional form assumptions. to operationalize the (quasi-) experimental approach with observational data, i propose a machine learning method to find counterfactual locations where the treatment could have occurred but did not. the proposed method specifically leverages that neighborhood characteristics are predictive of both the location of treatments and the outcomes of individuals. convolutional neural networks learn this rich spatial dependence structure encoding relevant institutional features from the data. i incorporate the appealing properties of generative adversarial networks in a classification problem that leads to much simpler training in practice, similar to denoising autoencoders. i illustrate the proposed methods in an application studying the causal effects of grocery stores on foot-traffic to nearby restaurants during covid- lockdowns. several key questions remain for future research. in some settings, the spatial treatment is endogenous, but geographic characteristics which are continuous in space are available as plausibly exogenous instruments (cf. feyrer et al., feyrer et al., , james and smith, ) . it is unclear how to construct powerful instruments from such geographic characteristics and incorporate them in the causal framework of this paper. in this paper, i also assume that there is no migration response to the treatment. to allow for migration, one could either focus on outcomes at fixed geographic locations instead of outcomes of fixed individuals or embrace a local average treatment effect (angrist et al., ) with a large number of compliance types if individuals move to different distances from treatment. the analysis in this paper is focused on estimating (potentially weighted) average treatment effects. in practice, decision makers may often be more interested in the optimal location for the spatial treatment. here, consider the expectation of the term in the numerator corresponding to individual , the first step uses that the realized outcome is the potential outcome corresponding to the realized treatment. the second step rewrites the potential outcome and distance bin indicator function in terms of non-stochastic candidate locations by summing over all possible treatment locations in the region, ∑︀ ∈s ( ) { = ( ) }. the third step moves the expectation into the summation, and the non-stochastic distance bin indicator function and potential outcome out. the final step resolves the expectation in terms of the probabilities determined by the experimental design, defined in section . the general estimator of interest in the setting without interference can be written aŝ where index denotes regions, = if region is treated at some location, and is the single treatment location chosen in region (if any). the weight function ( , ) is chosen by the user to weight individuals and treatment locations as desired and primarily place weight on pairs that are distance apart. for instance, for the att estimator with distance bin, choose ( , ) = ( ) { ( , ) ≤ ℎ}. the probabilities of treatment in regions and locations are given by ≡ pr( = ) and ( ) ≡ pr( = { }| = ). the first term averages over individuals at distance from a realized treatment location. the second term averages over individuals at distance from unrealized candidate treatment locations. the estimator estimates the weighted average treatment effect: with user-specified weights . the experiment considered here is a completely randomized experiment at the region level, where a fixed number of regions receive treatment at exactly one location each, and treatment in a region is assumed to have no effect on outcomes in other regions -regions are "far apart." the estimator^( ) is hard to analyze (in finite samples) because the denominators are random. this arises because, depending on treatment assignment, there may be more or fewer individuals near realized / unrealized locations. the same problem exists in standard randomized experiments when the treatment is randomized by an independent coin flip for each individual, such that the number of treated varies from assignment to assignment. in that setting, we can instead analyze the experiment with number of treated fixed at the value observed in the realized sample. conditioning on the number of realized treatment locations is not sufficient in the spatial setting because the number of individuals would still vary since some locations have more individuals near them than other locations. conditioning on the number of individuals restricts the assignment distribution asymmetrically -inverting an assignment generally changes the number of individuals near treatment -such that standard estimators are no longer unbiased by design. the theoretical analysis of^( ) therefore relies on an approximate estimator that fixes the denominators (at their expected values), and centers the numerators in a way that minimizes the difference between^( ) and its approximation. the approximate estimator is where ( ) and ( ) are average potential outcomes: here, i show that the estimators^( ) and˜( ) are very close in large enough samples. this motivates the use of exact finite sample results for the mean and variance of the infeasible estimator˜( ) for inference with the feasible estimator^( ). the analysis uses the mean value theorem to derive the difference^( ) −˜( ) and argues that this difference is small in large enough samples. as a practical matter, a sample is large enough if the number of individuals near treatment and control are close to their expected values. the approximation of^( ) by˜( ) is particular close when also the average outcomes are close to their expected values. to simplify notation, define the following shorthands: where˜, ( ) and˜, ( ) replicate^, ( ) and^, ( ) but with expected values rather than sample averages in the denominators. the sample average denominators are^, ( ) and , ( ) (scaled such that they converge under suitable conditions when grows), and the expected value of the denominators is ( ) (similarly scaled). without loss of generality, i fix the distance and weighting of interest and suppress the dependence on and in the following derivations for ease of presentation. the feasible estimator written in terms of the shorthand notation iŝ where˙is some convex combination of^and . it is straightforward to see thatΔ( , , , ) = . hence the left-hand-side of the equality above is justΔ(^,^,˜,˜), such that the right-hand-side is an expression for^−˜. hencê each of the four terms is a product with each factor close to zero under appropriate asymptotics. for instance, with independent regions and bounded outcomes and number of individuals per region, one can get √ (^− ) → . that is, the difference between the estimators^( ) and˜( ) is negligible under standard asymptotic frameworks. since the difference between estimators is very small for large samples, exact finite sample results for ( ) likely provide decent approximations for^( ) in smaller samples. consider the expected value of the estimator˜( ). to show: (˜( )) = ( ). since ( ) is the first term of˜( ), i proceed by showing that (˜( ) − ( )) = . since the denominators are non-stochastic, it suffices to show that the expectations of the numerators are equal to zero. the "first term" and "second term" designations below therefore refer to the first and second term of˜( ) − ( ). the expectation of the numerator of the first term is: the first equality rewrites the observed outcome = ( ( ) ) in terms of potential outcome ( ) = ( ) for = . the second equality moves all non-stochastic terms out of the expectation. the third equality rewrites the expectation of indicators as probabilities. the fourth equality distributes the difference ( ) − , ( ) and replaces , ( ) by its definition. for the second term, the factor multiplying the ratio cancels with the denominator, and the numerator is equal to the first term, such that the difference is equal to zero. analogously, the expectation of the numerator of the second term is: hence (˜( )) = ( ). the approximate estimator˜( ) in equation is the sum of three terms. since the first term, ( ) is fixed, the variance only depends on the last two terms. for the third equality, distribute out the − term of −( − ( ∑︀ ( ))) . . ., which is non-stochastic and hence does not contribute to the variance, such that only +( ∑︀ ( )) . . . remains of the second term. the fourth and final equality above distributes out the ( ) of the second term and then combines the first and second term by factoring out ( ). for ease of notation, definē the only stochastic terms left are the ( ); they represent the design-based variation that is due to random treatment assignment. the average¯+ , ( , ) consists only of a sum of potential outcomes, which are non-stochastic in the design-based perspective, in the numerator and the expected number of individuals near treatment, which is also non-stochastic, in the denominator. the where pr( ′ = | = ) is determined by the completely randomized design. let be the (fixed) number of treated regions in a completely randomized design. then pr( ′ = | = ) = − − since, under assumption , if region receives treatment, − of the remaining − regions receive treatment, each with equal probability. so )︂ the first equality combines the added term with the first summation and the subtracted term with the second summation. the second equality simplifies the factor of the first term, factors the second term into and ′ , and notices that both summations are the same, yielding the square in the second term. here, the third summation is "missing" the terms where = ′ . adding and subtracting by combining the added = ′ term into the second term and the subtracted = ′ term into the third term. note that dropping the (unidentified) variances of treatment effects (terms four and five) unambiguously leads to a conservative estimator of the variance. the absolute value of the factor in the fourth term, , is larger than the factor in the fifth term, − , and the numerator of the ratio in the fourth term is larger than the numerator of the ratio in the fifth term by jensen's inequality (while the denominators are identical). hence the absolute value of the fourth term is larger than the fifth terms, such that dropping both terms increases the expression, leading to a conservative estimator of the variance. to estimate the first term, takê to estimate the second term, takê { ̸ ∈ } pr( = ) pr( ̸ ∈ | = ) the estimator^a dditive ( ) here generalizes the estimator in section the main text to allow for an arbitrary number of candidate locations in each region, |s |. the formula is specific to completely randomized designs within treated regions with fixed number of realized locations, and equal probability for each of the when should you adjust standard errors for clustering? sampling-based vs. design-based uncertainty in regression analysis synthetic control methods for comparative case studies: estimating the effect of california's tobacco control program bias-corrected matching estimators for average treatment effects shift-share designs: theory and inference blowing it up and knocking it down: the local and city-wide effects of demolishing high concentration public housing on crime cross-section regression with common shocks identification of causal effects using instrumental variables spatial econometrics: methods and models. studies in operational regional science advances in spatial econometrics: methodology, tools and applications. new directions in spatial econometrics perspectives on spatial data analysis. advances in spatial science a primer for spatial econometrics. palgrave texts in econometrics towards principled methods for training generative adversarial networks estimating average causal effects under general interference, with application to a social network experiment design-based inference for spatial experiments with interference the impact of machine learning on economics exact p-values for network interference experienced segregation machine learning methods that economists should know about using wasserstein generative adversarial networks for the design of monte carlo simulations approximate residual balancing: debiased inference of average treatment effects in high dimensions the china syndrome: local labor market effects of import competition in the united states inference in experiments with matched pairs clustering, spatial correlations, and randomization inference who benefits from state and local economic development policies? randomization tests of causal effects under interference place of work and place of residence: informal hiring networks and labor market outcomes elasticities and the inverse hyperbolic sine transformation program evaluation and causal inference with high-dimensional data inference on treatment effects after selection among high-dimensional controls inference with dependent data using cluster covariance estimators the geography of unemployment non-random exposure to exogenous shocks: theory and applications quasi-experimental shift-share research designs new evidence on the finite sample properties of propensity score reweighting and matching estimators a practitioner's guide to cluster-robust inference spatial patterns in household demand reducing crime through environmental design: evidence from a randomized experiment of street lighting in new york city double/debiased machine learning for treatment and structural parameters the impacts of neighborhoods on intergenerational mobility i: childhood exposure effects where is the land of opportunity? the geography of intergenerational mobility in the united states free distribution or cost-sharing? evidence from a randomized malaria prevention experiment gmm estimation with cross sectional dependence statistics for spatio-temporal data statistics for spatial data (rev environmental health risks and housing values: evidence from , toxic plant openings and closings difference-in-differences techniques for spatial data: local autocorrelation and spatial interaction the development effects of the extractive colonial economy: the dutch cultivation system in java who wants affordable housing in their backyard? an equilibrium analysis of low-income property development inference with difference-in-differences and other panel data. the review of the view from above: applications of satellite data in economics accounting for unobservable heterogeneity in cross section using spatial first differences schooling and labor market consequences of school construction in indonesia: evidence from an unusual policy experiment poverty from space: using high-resolution satellite imagery for estimating economic well-being robust inference on average treatment effects with possibly more covariates than observations geographic dispersion of economic shocks: evidence from the fracking revolution: reply geographic dispersion of economic shocks: evidence from the fracking revolution sources of geographic variation in health care: evidence from patient migration place-based drivers of mortality: evidence from migration pre-event trends in the panel event-study design finite-sample properties of propensity-score matching and weighting estimators a note on the role of the propensity score for estimating average treatment effects text as data big data and big cities: the promises and limitations of improved measures of urban life bartik instruments: what, when, why, and how generative adversarial nets discrete choice modeling identifying agglomeration spillovers: evidence from winners and losers of large plant openings bidding for industrial plants: does winning a 'million dollar plant' increase welfare? take the q train: value capture of public infrastructure projects on the role of the propensity score in efficient semiparametric estimation of average treatment effects generalized least squares inference in panel and multilevel models with serial correlation and fixed effects the elements of statistical learning: data mining, inference and prediction transforming auto-encoders efficient estimation of average treatment effects using the estimated propensity score latent space approaches to social network analysis stability in competition toward causal inference with interference estimating spatial treatment effects: an application to base closures and aid delivery in afghanistan nonparametric estimation of average treatment effects under exogeneity: a review causal inference for statistics, social, and biomedical sciences: an introduction spatial transformer networks. advances in neural information processing systems geographic dispersion of economic shocks: evidence from the fracking revolution: comment combining satellite imagery and machine learning to predict poverty what happens when wal-mart comes to town: an empirical analysis of the discount retailing industry an adversarial approach to structural estimation consequences of the clean water act and the demand for water quality hac estimation in a spatial framework combining matching and synthetic controls to trade off biases from extrapolation and interpolation the standard errors of persistence imagenet classification with deep convolutional neural networks dynamic spatial panel models: networks, common shocks, and sequential exogeneity on asymptotic distribution and asymptotic efficiency of least squares estimators of spatial variogram parameters central limit theorems for long range dependent spatial linear processes asymptotic distributions of quasi-maximum likelihood estimators for spatial autoregressive models network dependence can lead to spurious associations and invalid inference on how well generative adversarial networks learn densities: nonparametric and parametric results estimates of the impact of crime risk on property values from megan's laws how do right-to-carry laws affect crime rates? coping with ambiguity using bounded-variation assumptions estimating treatment effects from spatial policy experiments: an application to ugandan microfinance worms: identifying impacts on education and health in the presence of treatment externalities random group effects and the precision of regression estimates an illustration of a pitfall in estimating the effects of aggregate variables on micro units alternative tests of the error components model machine learning: an applied econometric approach population intervention causal effects based on stochastic interventions on the application of probability theory to agricultural experiments. essay on principles. section on the application of probability theory to agricultural experiments. essay on principles. section causal inference with spatio-temporal data: estimating the effects of airstrikes on insurgent violence in iraq spatial econometrics for misaligned data an honest approach to parallel trends semiparametric efficiency in multivariate regression models with missing data root-n-consistent semiparametric regression interference between units in randomized experiments the central role of the propensity score in observational studies for causal effects local exposure to school shootings and youth antidepressant use determining point-of-interest visits from location data: a technical guide to visit attribution externalities of public housing: the effect of public housing demolitions on local crime causal inference with misspecified exposure mappings average treatment effects in the presence of unknown interference nonparametric density estimation under adversarial losses nonparametric policy analysis nonparametric policy analysis: an application to estimating hazardous waste cleanup benefits on causal inference in the presence of interference identification and estimation of spillover effects in randomized experiments extracting and composing robust features with denoising autoencoders bipartite causal inference with interference the average of treated individuals at distance ± ℎ from realized treatment locations isthe term inside the square equals zero:the first equality substitutes the definition of¯+ , ( , ). the second equality splits the ratio into two separate sums, one of treated, the other of control potential outcomes. for the first term, the third equality factors out = , which is constant across regions by assumption , and cancels ( ) ( ) . for the second term, the third equality factors out − = − , which is constant across regions by assumption , and notes that the sum of conditional probabilities is equal to in each region, ∑︀ ( ) = . both terms are equal to zero by the definitions of , ( ) and , ( ).hence, under assumption (completely randomized experiment), equation becomes var (︂˜()︂ where = = by assumption .bernoulli trial under assumption (bernoulli trial), equation becomesto treat the variances under assumptions and jointly, define both under assumption and under assumption , the variance of˜( ) depends on squares of (sums of) the potential outcome sums¯+ , ( , ).consider the first square of potential outcomes above. by applying the binomial theorem twice, rewrite the squared sum of potential outcomes as the difference between estimable marginal variances and an inestimable (approximate) treatment effect variance. by dropping the inestimable variance of treatment effects, one obtains a conservative estimate of the variance.since ( + ) = + + and ( − ) = − + , ( + ) = + − ( − ) . similarly for the second square of potential outcomes above:substituting these expressions into equation , the variance in equation consists of five terms. the first and third terms resemble a variance of outcomes of treated individuals. the second term resembles a variance of outcomes of control individuals. the fourth and fifth terms resemble variances of treatment effects. it is only possible to identify the effect of a candidate treatment location on individual , ( ), if is the closest realized treatment location to with positive probability. one can take^,the estimator^n earest ( ) is equal to if the region of individual is treated but location is not the closest realized treatment location to . this happens both when is not realized itself, and when another realized treatment location ′ is closer to . if is the closest realized location to ,^n earest ( ) is equal to the outcome of scaled by the inverse of the probability of this event. if the region is not treated,^n earest ( ) is equal to the outcome of scaled by the inverse of the probability of region not being treated. clearly,^n earest ( ) is an unbiased inverse probability weighting estimator of ( ) ≡ ( ) − ( ) under the assumption that only the nearest realized treatment matters. key: cord- - rzibpbl authors: eng, yi shin; lee, chien hsing; lee, wei chang; huang, ching chun; chang, jung san title: unraveling the molecular mechanism of traditional chinese medicine: formulas against acute airway viral infections as examples date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: rzibpbl herbal medicine, including traditional chinese medicine (tcm), is widely used worldwide. herbs and tcm formulas contain numerous active molecules. basically, they are a kind of cocktail therapy. herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease interactions are complex. there is potential for both benefit and harm, so only after understanding more of their mechanisms and clinical effects can herbal medicine and tcm be helpful to users. many pharmacologic studies have been performed to unravel the molecular mechanisms; however, basic and clinical studies of good validity are still not enough to translate experimental results into clinical understanding and to provide tough evidence for better use of herbal medicines. there are still issues regarding the conflicting pharmacologic effects, pharmacokinetics, drug interactions, adverse and clinical effects of herbal medicine and tcm. understanding study validation, pharmacologic effects, drug interactions, indications and clinical effects, adverse effects and limitations, can all help clinicians in providing adequate suggestions to patients. at present, it would be better to use herbs and tcm formulas according to their traditional indications matching the disease pathophysiology and their molecular mechanisms. to unravel the molecular mechanisms and understand the benefits and harms of herbal medicine and tcm, there is still much work to be done. it is common to initiate the therapy of orthodox western medicine by fitting the pharmacologic characteristics of a drug, including pharmacokinetic and pharmacodynamic effects, to the disease pathophysiology. physicians further validate the clinical application according to evidence-based medicine (ebm); however, this is not the case for tcm. tcm developed in ancient china, and at that time, physicians managed diseases with herbs only by clinical experience, without any knowledge of disease pathophysiology, nor the pharmacologic activities of herbs, to say nothing of the molecular mechanisms of herbs. to unravel the molecular mechanism of tcm formulas, it would be better to understand how physicians prescribe them first. tcm includes herbal therapy, acupuncture, massage, and dietary therapy. in the current work, tcm will be simply defined as herbal and dietary therapies. tcm is widely popular in east asia and forms the kampo medicine in japan, as well as traditional korean medicine; importantly, traditional medicines form the mainstream of healthcare in these countries. in ancient china, several famous tcm textbooks summarized the clinical experiences of using tcm formulas against various diseases, including endemic diseases, and each formula has its indication, including specific symptoms of patients. this is quite different from using tcm formulas based on the yin-yang theory (two opposite, but complementary forces) and five elements theories (everything in the world can be classified into the natural five elements. these elements promote as well as feedback each other to keep everything in balance). kampo medicine in japan classified tcm theories into ancient formula sect and recent formula sect for prescribing tcm formulas. the physicians of the ancient formula sect (a-physicians) use the indications of formula formed before formula can have active molecules that are pharmacologically different from that ingredient or the tcm formula. obversely, the pharmacological activities of a tcm formula may differ from those of their active ingredient or active molecules of ingredients. as a consequence, unraveling the molecular mechanism of a tcm formula needs comparison of the pharmacological activities between the formula, its ingredients, and the active molecules in the ingredients. the amount of most bioactive compounds in the herbs is very low. combination of herbs to form a tcm formula can further decrease their concentrations. is it possible that herbs and tcm formulas can be effective in this low concentration of bioactive compounds? is it possible that little amount of bioactive molecules can cause interactions? in orthodox western medicine, vitamins of little amount can show their clinical effects. the molecular mechanisms are the key, instead of their amount. in the real world practice, herbs and tcm formulas are bioactive. several side effects of tcm formulas have been reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] that raise the safety issue of tcm formula. natural products and tcm formulas might not be safe; however, some a-physicians suppose that the side effects may come from the misuse of tcm formulas without fulfilling their indications, while r-physicians consider the side effects developing from the misinterpretation of the disharmony. most tcm physicians do not agree that these side effects come from tcm formulas themselves, although with the same indications or disharmony, it is common to find that some patients respond well while others do not-while some patients develop side effects, some show responses opposite to the in vitro pharmacological effects. for example, panax quinquefolius prolongs thromboplastin time, prothrombin time (pt) and thrombin time in vitro [ ] . however, in combination therapy with warfarin, panax quinquefolius actually decreases seral concentration of warfarin and has shortened inr in a clinical trial [ ] . with therapy using panax ginseng, one can develop thrombosis [ ] , bleed [ , ] , or remain without any particular response [ ] . several factors may affect the molecular mechanisms and subsequent clinical effects of tcm formulas, including individual gene-based response, composition and amount of active molecules in tcm formulas, complex interactions, and appropriateness of use of tcm formulas. individual genetic basis is unique to metabolize tcm formulas and produces different responses. from the results of pharmacokinetic study of gan-lu-siao-du-yin, a tcm formula (submitted data), the blood concentrations of structurally-related index molecules, baicalin and baicalein, wogonin, and wogonoside, are highly variable between participants. the different patterns of blood concentrations support the unique pharmacokinetic profile based on individual genes. different concentrations of active molecules may affect the pharmacologic activities. therefore, individual gene-based metabolism could be one of the major factors affecting the molecular mechanisms and subsequent clinical effects of tcm formulas. to provide insights into action mechanisms of tcm formulas, metabolomic technologies might be helpful. a metabolomics integrative approach accepts a 'top-down' strategy to express the function of organisms through terminal symptoms of metabolic network and will gain a revolution in understanding of the holistic concept of tcm [ , ] . such technologies have been used to investigate the biological mechanisms of different syndromes of patients by studying the functional activities of the human body from a system-wide perspective. for example, the overall biological characterization of the urine of psoriasis patients with tcm blood stasis syndrome was performed to investigate the therapeutic metabolomic mechanism of the optimized yinxieling formula [ ] . in addition, metabolomics have been considered a powerful tool in diagnosis and treatment of primary dysmenorrhea by supporting information on changes of metabolites and changes in endocrinal, neural, and immune pathways [ ] . the xiang-fu-si-wu formula has been demonstrated to affect some significant perturbations in sphingolipid and glycerophospholipid metabolism as well as steroid hormone biosynthesis to make the metabolic discrepancy return to the normal level [ ] . tcm formulas are complex with numerous active chemical molecules in variable amounts. among these molecules with different pharmacologic activities, it is unclear which one mainly accounts for the clinical effect of a tcm formula, as the most abundant molecule might not be the most important one for a specific activity. the amount of an active molecule can be easily changed in different batches of product, or by different agriculture and collection of medicinal plants, therefore, understanding the molecular mechanisms of a tcm formula requires analysis not only of the mechanisms of the tcm formula as a whole, but those of individual active molecules and ingredient respectively as well. understanding the molecular mechanisms of an active molecule can facilitate its development into an investigational new drug (ind). meanwhile, unraveling the molecular mechanisms of a tcm formula can help to validate its traditional use and avoid its misuse and side effects. to keep a relatively constant amount of active molecules and pharmacologic activities, several things should be paid attention to, including use of right specie, use of right part of a plant, and use of a plant harvested in the right season. both use of closely related but wrong species and use of wrong part of herbs might lead to different active molecules with different pharmacologic activities, and various clinical effects and side effects. plants harvested in different seasons might contain variable amounts of active molecules thereby affecting their activities. some active molecules are secondary metabolites of plants against physical, chemical, or biological stimulants. active molecules of some plants can vary from year to year and place to place. therefore, confirmation of its authenticity is the cornerstone. in addition to this, fingerprints of the active molecules are needed to confirm the authenticity of a plant or a formula and to confirm the amount of active molecules via high-performance liquid chromatography (hplc) or liquid chromatography coupled with mass spectrometry (lcms). this is highly necessary for quality control and efficacy assessment. to have quality control of the products of tcm formulas, good manufacturing practice (gmp) procedure should be followed to avoid (a) inadequate processing that might lead to different chemical compositions of the final product; (b) inadequate storage conditions or prolonged storage that might lead to microbial contamination and early decay of the active molecules; and (c) adulteration of formulas and accidental contamination creating serious uncertainty in quality. adulteration is a plant or formula containing active pharmaceuticals or other bioactive agents for the purpose of claiming better efficacy or broader indications. accidental contamination is the plant raw materials containing heavy metals or other toxic substances from the manufacturing process due to ecological collapse. lead, mercury, and arsenic contamination in traditional chinese herbs has been reported [ ] [ ] [ ] [ ] . . complex interplays between herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease in the clinical practice of orthodox medicine, the more drugs used, the more adverse drug reaction (adr) occurred [ ] . this is commonly caused by drug-drug interactions. tcm formulas are mixtures of several ingredients. each ingredient has several bioactive compounds, so a tcm formula has numerous bioactive compounds. thinking of dozens or even hundreds of active molecules in a tcm formula been taken at once implies that the probability of drug interaction could be high. such interactions may be found between herb and drug, herb and food, herb and herb, herb and microbiome, and even between herb and disease. for example, in herb-drug interaction, scutellariae baicalensis is a common ingredient in tcm formulas. s. baicalensis contains baicalin, a flavonoid, as one of its major molecules. interactions between s. baicalensis and drugs are found due to baicalin affecting metabolic enzymes of drugs, displacing plasma protein binding, and regulating various transporters involved in the pharmacokinetics [ ] . baicalin may inhibit the expression and hydroxylation activity of cyp a in the liver to change the pharmacokinetics of drugs [ ] . co-administration of extract of s. baicalensis, and mefenamic acid, a kind of nsaid, can potentiate its anti-inflammatory effect [ ] . co-administration of baicalin and rosuvastatin, a hmg-coa reductase inhibitor commonly used to reduce serum cholesterol level, might find reduced plasma concentration of rosuvastatin in certain patients with certain genomes [ ] . as for herb-food and herb-food-drug interactions, baicalin can potentiate the antioxidant activity of β-carotene, which is a terpenoid of red-orange color, abundant in plants and fruits [ ] . the intakes of flavonoid-rich foods and beverages, containing baicalin and rutin, might compete with the binding site of calcium channel blockers on human serum albumin to affect their clinical effects [ , ] . by contrast, baicalin and rutin will increase the binding affinity of curcumin on human serum albumin to change its bioavailability [ ] . herbs may also interact with each other. for example, baicalin and berberine are important coexisting molecules of the combination of s. baicalensis and coptidis chinensis. berberine, but not baicalin, can increase glucose consumption. co-administration of berberine and baicalin had a synergetic effect on glucose utilization [ ] . additionally, tcm herbs are commonly used as food supplements and dietary therapy. foods have been reported to modify the intestinal microbiome [ ] . commensal microbiota have been thought to be involved in the development of the innate and adaptive immunity, nutrient metabolism of humans, and protection from the overgrowth of intestinal pathogens [ ] . herbs can change pharmacokinetics of drugs by intestinal microbiota [ ] . the intestinal microbiome is metabolically active to play an important role in the absorption of certain active molecules and change their bio-availabilities, particularly in those containing glycosidic linkages [ , ] . therefore, change of intestinal microbiome by herbs-containing foods may affect human health care and drug therapy. as for interactions between disease and herb, more absorption of active molecules of maxing shigan decoction (mxgst), including liquiritin, glycyrrhizin, amygdalin, prunasin, ephedrine, pseudoephedrine, and methylephedrine, can be found in rsv pneumonia-infected rats vis-à-vis normal rats by reducing the clearance rates of these active molecules [ ] . there are highly complex interactions between herbs and drugs, foods, herbs, microbiome, and diseases, and most of these complex interactions are not completely discovered or remain unseen, just like the submerged part of an iceberg. therefore, there are still insufficient data to completely understand the molecular mechanisms, pharmacokinetics, pharmacodynamics, and interactions of tcm formulas. acute airway infections, including acute bronchitis, viral pneumonia, and acute exacerbation of chronic obstructive pulmonary disease (copd), are commonly caused by viruses of different families, including rhinovirus, influenza, and parainfluenza virus, enteroviruses, coronavirus, adenovirus, respiratory syncytial virus, etc. [ , ] . these viruses infect epithelia, produce inflammation, induce immune response, and cause symptoms. from the viewpoint of pathophysiology, tcm formulas used to manage airway viral infections need to have antiviral activity against such viruses listed above, and/or to induce antiviral cytokines, and/or anti-inflammatory effect, and/or to relieve symptoms commonly presented in airway infections ( figure ). to simplify the molecular mechanisms and to correlate the pharmacologic activities with their clinical effects, five formulas of a-physicians will be used as examples against airway infections: ge-gen-tang (ggt; table ) [ ] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [ ] . ggt has antiviral activities against respiratory syncytial virus (rsv) [ ] and influenza virus [ ] . ggt can reduce the mortality of influenza virus-infected mice [ ] . among its active molecules, uralsaponins from glycyrrhiza uralensis [ ] and procyanidin from cinnamomum cassia [ ] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [ ] . paeonol and , , , , -penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [ ] . of its anti-inflammatory effects, ggt can suppress the interleukin- α (il- α) production induced by interferons (ifn) in influenza [ ] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il- , tnf-α, inos, and cox- ) [ ] . ggt can stimulate il- and ifn-β to counteract viral infection [ ] , and can also enhance the phagocytic activity of macrophages [ ] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th /th [ ] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, ge-gen-tang (ggt; table ) [ ] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [ ] . ggt has antiviral activities against respiratory syncytial virus (rsv) [ ] and influenza virus [ ] . ggt can reduce the mortality of influenza virus-infected mice [ ] . among its active molecules, uralsaponins from glycyrrhiza uralensis [ ] and procyanidin from cinnamomum cassia [ ] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [ ] . paeonol and , , , , -penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [ ] . of its anti-inflammatory effects, ggt can suppress the interleukin- α (il- α) production induced by interferons (ifn) in influenza [ ] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il- , tnf-α, inos, and cox- ) [ ] . ggt can stimulate il- and ifn-β to counteract viral infection [ ] , and can also enhance the phagocytic activity of macrophages [ ] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th /th [ ] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, suppresses nuclear factor-kappa b (nf-κb) via the phosphoinositide -kinase (pi k) pathway, inhibits the production of nitric oxides (no), prostaglandin e (pge ), and reactive oxygen species (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase- (cox- ) [ ]( figure ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [ ] . however, this study design uses intraperitoneal injection to show the induction of no [ ] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [ ] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge , il- β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the anti-inflammatory effect [ ] [ ] [ ] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde down-regulate no and tnf-α production to show anti-inflammatory activity [ ] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [ ] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [ ] . -gingerol ( -g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il- , but decreases il- and transforming growth factor-β (tgf-β ) levels [ ] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [ ] (figure ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase- (cox- ) [ ] (figure ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [ ] . however, this study design uses intraperitoneal injection to show the induction of no [ ] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [ ] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge , il- β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the antiinflammatory effect [ ] [ ] [ ] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde downregulate no and tnf-α production to show anti-inflammatory activity [ ] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [ ] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [ ] . -gingerol ( -g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il- , but decreases il- and transforming growth factor-β (tgf-β ) levels [ ] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [ ] (figure ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. ma-huang- tang (mht; table ) [ ] have been reported to be effective in the treatment of influenza, upper respiratory tract infection, acute and chronic bronchitis, and asthma [ , ] . the indication to use mht is patients with chills, fever without sweating, headache, shortness of breath, and joint pain. clinically, mht can effectively reduce fever and flu symptoms, including myalgia, headache, arthralgia, fatigue and cough, in patients with seasonal influenza type a [ ] . among its active molecules, l-ephedrine from ephedra sinica and amygdalin from prunus armeniacae, possess the antitussive effect [ ] , so co-administration of ephedra sinica and prunus armeniacae shows a better antitussive effect than use singly [ ] . mht clearly shows anti-inflammatory activity via suppressing the no/pge pathway [ ] , reducing inflammatory cells infiltration and reducing pro-inflammatory cytokine, including tnf-α, il- β, and il- , in lung experiments [ ] . in an acute bronchial asthma mice model, mht can also mitigate the pathological changes of acute asthma-like syndrome through inhibition of the toll-like receptor (tlr ) pathway [ ] . mht can modulate th /th cytokines via decreasing il- & il- and increasing ifn-γ levels. mht can inhibit th cells [ ] , and decreases il- , il- , tnf-α, cd +, cd + t cell levels (th response), but increases il- , ifn-γ, and cd + t cell levels (th response) to increase cd +/cd + ratio [ ] . among its active ingredients, ephedra sinica inhibits pge biosynthesis, reduces ige-mediated histamine release, reduces the mrna or protein levels of il- β, il- , tnf-α, cox , and nf-κb [ ] and inhibits complement activation of both classical and alternative pathways [ ] . additionally, ephedra sinica can directly activate both alpha-and beta-adrenergic receptors to reduce bronchial mucosal edema and to dilate the bronchus respectively [ , ] . to understand the molecular mechanism, several active molecules have been identified. ephedrannin a and b, from ephedra sinica, effectively suppressed the transcription of tnf-α, il- β, and nf-κb, and the phosphorylation of p mitogen-activated protein (map) kinase to exert their anti-inflammatory actions on lps-stimulated macrophages [ ] . glycyrrhiza uralensis and cinnamomum cassia contain active molecules mediating anti-inflammatory and immunomodulatory effects mentioned in the ggt section. for its antiviral activity, mht was initially thought to inhibit airway viruses through inducing antiviral ifn. however, herbacetin from ephedrine alkaloid-free extract of ephedra sinica might have anti-influenza activity similar to its extract containing ephedrine and pseudoephedrine [ ] . the study of ephedra sinica is relative rare owing to it containing ephedrine and pseudoephedrine, which are illegal in several countries. glycyrrhiza uralensis and cinnamomum cassia also have active antiviral constituents mentioned in the ggt section ( figure ). ma-xing-gan-shi- tang (mxgst; table ) [ ] , a similar formula to ma-huang-tang, is effective against influenza virus infection [ ] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge level in the hypothalamus [ ] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [ ] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev ), forced vital capacity (fvc), and fev /fvc in patients with acute exacerbation of copd [ ] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [ ] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and anti-pyretic effects in an lps-induced hyperthermia rat model [ ] . mxgst has bronchodilation effect mediated by stimulation of β -adrenoceptors in pigs [ ] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [ ] , reduce neutrophilic inflammation [ ] , and in a copd rat model, can decrease il- , il- , and tnf-α, but increase ifn-γ [ ] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure ). molecules , , x; doi: www.mdpi.com/journal/molecules ma-xing-gan-shi- tang (mxgst; table ) [ ] , a similar formula to ma-huang-tang, is effective against influenza virus infection [ ] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge level in the hypothalamus [ ] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [ ] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev ), forced vital capacity (fvc), and fev /fvc in patients with acute exacerbation of copd [ ] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [ ] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and antipyretic effects in an lps-induced hyperthermia rat model [ ] . mxgst has bronchodilation effect mediated by stimulation of β -adrenoceptors in pigs [ ] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [ ] , reduce neutrophilic inflammation [ ] , and in a copd rat model, can decrease il- , il- , and tnf-α, but increase ifn-γ [ ] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure ). xiao-qing-long- tang (xqlt; table ) [ ] is one of the most common prescriptions used against allergic rhinitis [ ] . xqlt, at higher cumulative doses, might reduce the incidence of pneumonia and protect against admission [ ] . xqlt with/without ma-xing-gan-shi-tang (mxgst) is the most frequently prescribed tcm formula for copd [ ] , and is also commonly used in the treatment of patients with respiratory diseases, such as common cold, flu, bronchitis, asthma, bronchiectasis, and emphysema. the indication to use xqlt is patients with cough, watery rhinorrhea or watery sputum, but without thirst. to manage airway viral infection, xqlt is effective against human respiratory syncytial virus (rsv) infection by preventing viral attachment, internalization, syncytial formation, and by stimulating ifn-β secretion [ ] , and has been proven beneficial against influenza virus in vivo through the augmentation of antiviral iga antibody [ , ] . xqlt can reduce the airway inflammation with the decrease of eosinophils count, the ovalbumin (ova)-specific immunoglobulin e (ige) antibody, and histamine release [ ] [ ] [ ] , can also modulate th /th balance thereby reducing il- and restoring ifn-γ levels [ , ] . among its active molecules, schisandrin a, a bioactive lignin of schisandra sphenanthera, inhibits the il β-induced inflammation via suppression of mitogen-activated protein kinase (mapk) and nf-κb signal pathways [ ] , and can also inhibit the nf-κb, mapk and pi k/akt pathways, partially mediated by the activation of the nuclear factor erythroid -related factor /heme oxygenase- (nrf /ho- ) pathway to manage inflammatory and oxidative disorders caused by over-activation of macrophages [ ] . schisandrin b, another bioactive lignin of schisandra sphenanthera, increases the expression of nrf and ho- and blocks the activation of nf-κb induced by lps to suppress the production of vascular cell adhesion molecule (vcam- ), intercellular adhesion molecule (icam- ), tnf-α, and il- expressions in human umbilical vein endothelial cells (huvecs) [ ] . α-cubebenoate, isolated from schisandra chinensis, can block the increase of il- β and il- during inflammation [ ] , and inhibit lps-induced expression of inos and cox- [ ] . oral polysaccharide from schisandra chinensis showed antitussive effect in a guinea pig model [ ] . the active molecules of common ingredients, including ephedra sinica, cinnamomum cassia, paeonia lactiflora, glycyrrhiza uralensis, and zingiber officinale, have several pharmacologic activities mentioned in the above sections. most of these activities aim at inhibiting inflammation induced by airway infection, however, lectin from pinellia ternate may activate macrophages, induce neutrophil migration, cytokine release, ros overproduction and the activation of the nf-κb signaling pathway to produce pro-inflammatory activity [ ] (figure ). therefore, interactions, including both synergistic and antagonistic between active molecules in a tcm formula could be so complex as to affect the final effects. from more than two thousand years ago in china and japan, ye-gan-ma-huang- tang (ygmht; table ) [ ] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev , and asthma control test (act) score of refractory asthmatic patients [ ] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev , and fev % predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [ ] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [ ] and ev [ ] . it can regulate the serum levels of tnf-α, il- , and il- to show clinical effect in management of cough and variant asthmatic symptoms in children [ ] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [ ] . slpcd can significantly down-regulate the mrna expression levels of th cytokines (il- , il- , il- , and il- ) and up-regulate those of th cytokines (il- and ifn-γ) in lungs of asthmatic mice [ ] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il- β, il- , tnf-α), and attenuating the pulmonary edema [ ] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge production by decreasing the mrna and protein expression of inos and cox- , respectively, as well as by suppressing nf-κb activation [ ] (figure ). molecules , , x; doi: www.mdpi.com/journal/molecules from more than two thousand years ago in china and japan, ye-gan-ma-huang- tang (ygmht; table ) [ ] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev , and asthma control test (act) score of refractory asthmatic patients [ ] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev , and fev % predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [ ] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [ ] and ev [ ] . it can regulate the serum levels of tnf-α, il- , and il- to show clinical effect in management of cough and variant asthmatic symptoms in children [ ] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [ ] . slpcd can significantly down-regulate the mrna expression levels of th cytokines (il- , il- , il- , and il- ) and up-regulate those of th cytokines (il- and ifn-) in lungs of asthmatic mice [ ] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il- β, il- , tnf-α), and attenuating the pulmonary edema [ ] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge production by decreasing the mrna and protein expression of inos and cox- , respectively, as well as by suppressing nf-κb activation [ ] (figure ). to unravel the molecular mechanism of tcm formulas, several issues need to be solved. many pharmacologic activities are obtained from in vitro and animal studies. could these activities be extrapolated into humans? there are so many active molecules with various pharmacologic activities in a tcm formula or herbs. are these molecules specific for that specific activity of a tcm formula or herbs? could they reach a minimal serum level to exert that particular pharmacologic activity in humans? active molecules may have the same activity with different potency. the most abundant one may not be the most important one for a particular activity. is it possible that there might be unidentified active molecules that actually account for a particular pharmacologic activity? several pharmacologic activities of a tcm formula cannot find a corresponding active molecule. is it possible that interactions between active molecules, e.g., synergism, but not active molecules themselves, account for a specific activity? is it possible that new active molecules are generated during preparation of the tcm formula? the genetic basis will affect the drug pharmacokinetics. is there a specific gene or single nucleotide polymorphism (snp) largely affecting the pharmacokinetic profile? does publication bias exist so that undesired pharmacologic effects are not published? is it possible that a particular gene is prone to a specific adr of a tcm formula or herbs? could a specific adr come from interactions between specific active molecules? several tcm formulas clearly show that some ingredients are not active in managing their traditional applications. could these inactive ingredients be omitted? all these questions need many studies to determine valid answers. at this moment, it would be better to use herbs and tcm formulas according to their traditional indications and the disease pathophysiology by matching their molecular mechanisms. as the world population has continued to age, the elderly have become associated with chronic diseases requiring multiple medications. increasing dissatisfaction with orthodox medicine and/or preference for alternative therapists and/or naturopathy has meant people continue to seek alternatives to maintain or improve their health, and this has spurred the growth of complementary and alternative medicine (cam) therapies. the validity of pharmacologic studies, safety issues, and validation of clinical effects of herbal medicine and tcm are limitations that should be highly considered; however, most clinicians are not familiar with herbal medicine and tcm so they do not recommend herbal therapies. more effort should be placed on unraveling the molecular mechanisms in order to solve the above issues. the reasons that limit clinicians from becoming more familiar with herbal medicine and for not recommending herbal therapies are explored below. herbal medicine and tcm form a part of complementary and alternative medicine (cam). however, evidence-based research in the field of cam therapies is still limited. there is a wrong perception that a naturally derived product is relatively safe. it is highly important to identify both usefulness and safety of cam and integrate these health approaches with orthodox medicine through rigorous scientific investigation to improve health care. it is also important for medical educators and providers to recognize the trend, the evidence, the benefits, and the risks of herbal medicine and tcm to educate clinicians for appropriate patient management and education. tcm studies published in chinese are usually not translated into english. the terminology of tcm is also difficult to translate, particularly those used by r-physicians, so medical education of herbal medicine and tcm has been neglected worldwide, except in germany and china. with their increasing use since the s, understanding the molecular mechanisms, benefits, and limitations by physicians has become increasingly important to monitor their benefits and harmfulness. most of the evidence supporting clinical claims of herbs and natural products come from studies of inadequate design that do not provide tough evidence of the effects. relatively few well-designed studies support their clinical claims. sometimes, adequately powered, double-blinded, placebo-controlled clinical trials come to conclusions against previous reports, such as echinacea for upper respiratory infection [ ] , or ginkgo for dementia or mild cognitive impairment [ ] . additionally, the amounts of most bioactive compounds in herbs and tcm formulas are very low. is it possible that the clinical effects of herbs and tcm formulas can be effective with such a small amount of bioactive compounds? one study discussed the clinical effect of ma-huang-tang (mht) against seasonal influenza [ ] . mht showed an equivalent clinical effect as neuraminidase inhibitors. however, not every tcm formula can provide such evidence. several tcm formulas, such as ma-xing-gan-shi-tang (mxgst), ge-gen-tang (ggt), and xiao-qing-long-tang (xqlt), are among the top ten most common tcm prescriptions for patients with upper respiratory tract infections (urtis) in taiwan [ ] . they are commonly used for urtis, but more research is required to validate their clinical effects and mechanisms. without tough clinical evidence and clear molecular mechanisms, physicians tend to avoid herbal therapies. most clinical claims of herbal therapies are based on bench studies that do not possess external validity to support their conclusions; for example, using animal-derived cancer cell lines to study antiviral effects in humans; using cancer cell lines to study physical changes; using intraperitoneal injection to study oral medications; and using high-dose pharmacological designs to study physiological responses, etc. therefore, there is still much space for improvement of our understanding of the mechanisms of herbal medicine. herbs are pharmacologically active and can positively or negatively affect patient's health. with increasing use of herbs as dietary supplements and alternative therapy, there is an increased risk of negative impacts, such as adverse effects and interactions. herbal medicine is among the most common causes of drug-induced liver injury (dili) [ ] . additionally, several adverse effects of commonly used herbs have been reviewed, including hypoglycemic or hyperglycemic effect, hypolipidemic or hyperlipidemic effect, hormonal activities, hypertensive, cardioactive [ ] , and hepatotoxic effects [ ] . some of them are serious even in recommended dosage, such as ephedra alkaloids (derived from ephedra sinica or ma huang) [ ] [ ] [ ] . however, most of the molecular mechanisms causing side effects are unknown. plants containing pyrrolizidine alkaloids can lead to hepatotoxicity and venoocclusive disease, possibly by the accumulation of toxic metabolites produced via the cytochrome system [ ] . some herbal treatments containing aristolochic acid (aa), including aristolochia fangchi, aristolochia debilis sieb. et zucc., aristolochia manshuriensis, aristolochia debilis, can cause aa nephropathy requiring renal replacement therapy [ , ] ; additionally, aa is associated with urothelial cancers [ ] . although most of the above issues have been identified, the unrecognized issues are just the tip of the iceberg. tcm formulas have numerous bioactive compounds to form a kind of cocktail therapy. however, the amounts of each bioactive compound in herbs and tcm formulas are very low. this may raise questions about the likelihood of their interactions with others during administration in a decoction. ginseng, a common natural product used among adults [ ] , has about ginsenosides as its major bioactive compounds [ ] , although the actual amount of each ginsenoside is small. after oral administration, ginsenosides are metabolized and transformed by intestinal microbiota. diet can markedly influence the transformation of ginsenosides. they exert pharmacologic effects in animals, and also show various clinical effects in randomized controlled trials, including several side effects and drug interactions [ ] , so small amounts of bioactive compounds do have clinical effects and side effects, which might come from the individual pharmacological activity of bioactive compounds or their synergism. the side effects may also come from individual unfavorable bioactivity or from interactions. additionally, herbal medicine and tcm therapy are commonly used in combination with orthodox medicine by patients, sometimes unknown to their doctors. the complex and unknown interactions, including herb-drug, herb-herb, herb-food, herb-microbiome, and herb-disease interactions, make use of herbal medicine more complicated and requiring frequent monitoring. the mechanisms of these interactions can be divided into molecular mimicry and pharmacologic interactions, such as pharmacodynamic and pharmacokinetic interactions. for example, with molecular mimicry, several herbs naturally has coumarin or salicylate analogue that may potentiate the bleeding risk of warfarin and salicylate, respectively. for pharmacodynamic interactions, ephedra sinica (ma huang) should not be used with sedative or anti-hypertensive agents. for pharmacokinetic interactions, st john's wort and several tcm formulas have been noted to affect the cyp system of liver, which may increase or decrease the effects of other drugs [ ] . additionally, the herb-drug interaction may be individualized, e.g., in combination with warfarin, panax ginseng may cause thrombotic event [ ] , bleeding [ , ] , or neither [ ] . currently, most of the molecular mechanisms of these identified interactions are not fully understood, to say nothing of the unrecognized interactions. quality uncertainty impacts the reproducibility of clinical efficacy and safety of commercially available natural products. variability of the quality of natural products may come from a lack of standardization of manufacturing, including misidentification of authenticity, inadequate processing, inadequate storage, adulteration of formulas, and accidental contamination [ ] . most of those quality problems can be gradually solved by following the who guidelines on good agriculture and collection practices for medicinal plants [ ] and botanical drug development guidance for industry of fda [ ] . several health benefits of herbal medicine and tcm are claimed; for example, herbs and tcm formulas, including those discussed above, are believed to have anti-oxidative activities helpful against several diseases. this idea is based on reactive oxygen and nitrosative species (ros/rns) as metabolic byproducts that can cause damage to cellular macromolecules; thus, many diseases can be triggered by oxidative stress under high levels of ros/rns. these diseases include cancer, inflammation, and degenerative diseases. oxidative stress causes damage either with an overwhelming production of ros/rns or under insufficient levels of antioxidants or repair mechanisms, so blocking the generation of ros/rns might prevent and/or manage these diseases [ ] . however, ros/rns are also signaling molecules for several physiological functions, including regulation of vascular tone, control of ventilation, and erythropoietin production, etc. actually, ros-mediated responses may protect against oxidative stress [ ] . additionally, ros/rns may play a dual role in different diseases, i.e., ros/rns might contribute to, or counteract, the disease progression. it remains unclear that more dosage of antioxidants is not better and may even worsen a medical condition [ ] . there are insufficient data to establish the ability of tcm to decrease ros/rns levels and establish its effects on the disease, and this affects the interpretation of any claims of benefit. to validate such claims of the benefits of herbal medicine and tcm, much work remains to be done. only when these limitations can be minimized, can the molecular mechanisms of herbs and tcm formulas be understood. consequently, clinicians can help patients by giving adequate prescriptions and suggestions to minimize the harmfulness and maximize the benefits to healthcare. herbal medicine, including tcm, is commonly used worldwide. herbal remedies contain numerous active molecules to form a kind of cocktail therapy. these active molecules may interact with each other to affect the therapeutic effects and produce side effects. understanding their pharmacological effects, interactions, side effects, clinical effects, and the underlying molecular mechanisms is very important to provide benefits, but avoid harm, to the patient. to unravel the molecular mechanisms, much work remains to be done. the abc clinical guide to herbs trends in alternative medicine use in the united states, - : results of a follow-up national survey trends in the use of complementary health approaches among adults: united states complementary and alternative medicine use among adults and children: united states current state of phytotherapy in germany glycyrrhizic acid inhibits virus growth and inactivates virus particles antiviral effect of cimicifugin from cimicifuga foetida against human respiratory syncytial virus pterodontic acid isolated from laggera pterodonta inhibits viral replication and inflammation induced by influenza a virus a possible mechanism of interstitial pneumonia during interferon therapy with sho-saiko-to a case of acute eosinophilic pneumonia due to sho-saiko-to autoimmune hepatitis with drug-induced pneumonia due to sho-saiko-to two cases of pneumonia caused by sho-saiko-to acute lymphoblastic leukemia complicated by type c hepatitis during treatment and further by acute interstitial pneumonia due to sho-saiko-to in -year-old an autopsy case of interstitial pneumonia probably induced by sho-saiko-to a case of interstitial pneumonia with chronic hepatitis c following interferon-alfa and sho-saiko-to therapy a comparative study on anticoagulant activities of three chinese herbal medicines from the genus panax and anticoagulant activities of ginsenosides rg and rg brief communication: american ginseng reduces warfarin's effect in healthy patients: a randomized thrombosis of a prosthetic aortic valve disclosing a hazardous interaction between warfarin and a commercial ginseng product ginseng and vaginal bleeding ginseng face cream and unexplained vaginal bleeding interaction between warfarin and panax ginseng in ischemic stroke patients systems biology: metabonomics potential role of metabolomics apporoaches in the area of traditional chinese medicine: as pillars of the bridge between chinese and western medicine application of metabolomics on diagnosis and treatment of patients with psoriasis in traditional chinese medicine biomarkers of primary dysmenorrhea and herbal formula intervention: an exploratory metabonomics study of blood plasma and urine plasma metabolic profiling of normal and dysmenorrhea syndrome rats and the effects of xiang-fu-si-wu decoction intervention adulterants in asian patent medicines heavy metal content of ayurvedic herbal medicine products arsenic and mercury intoxication due to indian ethnic remedies lead, mercury, and arsenic in us-and indian-manufactured ayurvedic medicines sold via the internet risk factors for adverse drug events among nursing home residents role of intestinal microbiota in baicalin-induced drug interaction and its pharmacokinetics inhibitory effects of baicalin on the expression and activity of cyp a induce the pharmacokinetic changes of midazolam in rats herb-drug interactions between scutellariae radix and mefenamic acid: simultaneous investigation of pharmacokinetics, anti-inflammatory effect and gastric damage in rats the effect of herbal medicine baicalin on pharmacokinetics of rosuvastatin, substrate of organic anion-transporting polypeptide b baicalin in radical scavenging and its synergistic effect with beta-carotene in antilipoxidation studies on the competitive binding of cleviprex and flavonoids to plasma protein by multi-spectroscopic methods: a prediction of food-drug interaction spectroscopic investigation on the food components-drug interaction: the influence of flavonoids on the affinity of nifedipine to human serum albumin the increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: a potential for drug delivery system interaction of baicalin with berberine for glucose uptake in t -l adipocytes and hepg hepatocytes could the gut microbiota reconcile the oral bioavailability conundrum of traditional herbs? role of metabolism by intestinal microbiota in pharmacokinetics of oral baicalin pharmacokinetics of maxing shigan decoction in normal rats and rsv pneumonia model rats by hplc-ms/ms upper respiratory infections common viral respiratory infections. in harrison's principles of ministry of health and welfare. taiwan herbal pharmacopeia a pharmacologic study on the mechanism of action of kakkon-to: body temperature elevation and phagocytic activation of macrophages in dogs ge-gen-tang has anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines mechanism of action of the suppression of influenza virus replication by ko-ken tang through inhibition of the phosphatidylinositol -kinase/akt signaling pathway and viral rnp nuclear export effect of interleukin- level augmented by kakkon-to, a herbal medicine, on the early stage of influenza infection in mice uralsaponins m-y, antiviral triterpenoid saponins from the roots of glycyrrhiza uralensis high-throughput screening for anti-influenza a virus drugs and study of the mechanism of procyanidin on influenza a virus-induced autophagy identification of suitable natural inhibitor against influenza a (h n ) neuraminidase protein by molecular docking antiviral activity and possible mechanism of action of constituents identified in paeonia lactiflora root toward human rhinoviruses kakkon-to suppressed interleukin-la production responsive galgeun-tang attenuates cigarette smoke and lipopolysaccharide induced pulmonary inflammation via ikappabalpha/nf-kappab signaling anti-inflammatory and immunomodulatory effects of paeonia lactiflora pall. a traditional chinese herbal medicine the pharmacological activities of licorice nitrite oxide and inducible nitric oxide synthase were regulated by polysaccharides isolated from glycyrrhiza uralensis fisch isoliquiritigenin attenuates adipose tissue inflammation in vitro and adipose tissue fibrosis through inhibition of innate immune responses in mice cinnamaldehyde reduces il- beta-induced cyclooxygenase- activity in rat cerebral microvascular endothelial cells cinnamaldehyde inhibits pro-inflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling cinnamaldehyde and -methoxycinnamaldehyde as nf-kappab inhibitors from cinnamomum cassia anti-inflammatory activity of cinnamon (c. zeylanicum and c. cassia) extracts-identification of e-cinnamaldehyde and o-methoxy cinnamaldehyde as the most potent bioactive compounds cellular immunity and lung injury in respiratory virus infection puerarin attenuates airway inflammation by regulation of eotaxin- -gingerol as an arginase inhibitor prevents urethane-induced lung carcinogenesis by reprogramming tumor supporting m macrophages to m phenotype differential inhibition of t lymphocyte proliferation and cytokine synthesis by [ ]-gingerol antipyretic effect of mao-to, a japanese herbal medicine, for treatment of type a influenza infection in children prescription pattern of chinese herbal products for adult-onset asthma in taiwan: a population-based study the efficacy of ma-huang-tang (maoto) against influenza antitussive effects of l-ephedrine, amygdalin, and makyokansekito (chinese traditional medicine) using a cough model induced by sulfur dioxide gas in mice effect of ephedra with semen armeniacae amarum by compatibility of different ratio in acute toxicity test and antiasthmatic simultaneous determination and anti-inflammatory effects of traditional herbal medicine, mahwang-tang post-treatment with ma-huang-tang ameliorates cold-warm-cycles induced rat lung injury huang tang ameliorates bronchial asthma symptoms through the tlr pathway huang tang ameliorates asthma though modulation of th /th cytokines and inhibition of th cells in ovalbumin-sensitized mice antiviral effects of ma huang tang against h n influenza virus infection in vitro and in an icr pneumonia mouse model phytochemistry and pharmacology of genus ephedra. chin a component of the medicinal herb ephedra blocks activation in the classical and alternative pathways of complement ephedra in perspective-a current review ephedrannin a and b from roots of ephedra sinica inhibit lipopolysaccharide-induced inflammatory mediators by suppressing nuclear factor-kappab activation in raw . macrophages the pharmacological actions of ephedrine alkaloids-free ephreda herb extract and preparation for clinical application anti-influenza agents from plants and traditional chinese medicine an study on gypsum compounds and their antipyretic function and anti-inflammatory mechanisms antipyretic and anti-asthmatic activities of traditional chinese herb-pairs, ephedra and gypsum maxingshigan decoction for treating aecopd in cases and nursing measures. china pharm traditional chinese medicine therapy decreases the pneumonia risk in patients with dementia antitussive, anti-pyretic and toxicological evaluation of ma-xing-gan-shi-tang in rodents the effects of ma-xing-gan-shi-tang on respiratory resistance and airway leukocyte infiltration in asthmatic guinea pigs changes in the level of cytokine in rats with chronic obstructive pulmonary disease of phlegm heat cumber lung type after treatment of maxing shigan decoction. chin the prescriptions frequencies and patterns of chinese herbal medicine for allergic rhinitis in taiwan the use of chinese herbal medicine in the treatment of chronic obstructive pulmonary disease (copd) xiao-qing-long-tang (sho-seiryu-to) inhibited cytopathic effect of human respiratory syncytial virus in cell lines of human respiratory tract in vivo anti-influenza virus activity of kampo (japanese herbal) medicine "sho-seiryu-to" to indicate a break in thought or interpretation and its mode of action in vivo anti-influenza virus activity of kampo (japanese herbal) medicine "sho-seiryu-to"-effects on aged mice, against subtypes of a viruses and b virus, and therapeutic effect proteomic analysis of anti-inflammatory effects of a kampo (japanese herbal) medicine "shoseiryuto (xiao-qing-long-tang)" on airway inflammation in a mouse model xiao-qing-long-tang attenuates allergic airway inflammation and remodeling in repetitive dermatogoides pteronyssinus challenged chronic asthmatic mice model effects of xiaoqiongtang decoction on airway inflammation and airway remodeling in patients with copd anti-allergic activity of a kampo (japanese herbal) medicine "sho-seiryu-to (xiao-qing-long-tang)" on airway inflammation in a mouse model schisandrin a inhibits the il- beta-induced inflammation and cartilage degradation via suppression of mapk and nf-kappab signal pathways in rat chondrocytes schisandrin a suppresses lipopolysaccharide-induced inflammation and oxidative stress in raw . macrophages by suppressing the nf-kappab, mapks and pi k/akt pathways and activating nrf /ho- signaling schisandrin b inhibits lps-induced inflammatory response in human umbilical vein endothelial cells by activating nrf anti-septic activity of alpha-cubebenoate isolated from schisandra chinensis identification of a novel anti-inflammatory compound, alpha-cubebenoate from schisandra chinensis antitussive activity of the schisandra chinensis fruit polysaccharide (scfp- ) in guinea pigs models pinellia ternata lectin exerts a pro-inflammatory effect on macrophages by inducing the release of pro-inflammatory cytokines, the activation of the nuclear factor-kappab signaling pathway and the overproduction of reactive oxygen species meta-analysis on shegan mahuang tang for refractory asthma clinical observation on therapeutic effect of traditional chinese medicine granules made by formula of shegan mahuang decoction for patients with asthma yakammaoto inhibited human coxsackievirus b (cvb )-induced airway and renal tubular injuries by preventing viral attachment, internalization, and replication yakammaoto inhibits enterovirus infection by reducing viral attachment, internalization, replication, and translation effect of modified shegan mahuang decoction on cytokines in children patients with cough and variant asthma antiasthmatic effects of sanglong pingchuan decoction through inducing a balanced th /th immune response network pharmacology-based investigation of protective mechanism of aster tataricus on lipopolysaccharide-induced acute lung injury inhibitory effects of irigenin from the rhizomes of belamcanda chinensis on nitric oxide and prostaglandin e( ) production in murine macrophage raw . cells echinacea for preventing and treating the common cold ginkgo biloba for preventing cognitive decline in older adults: a randomized trial a randomized, controlled trial comparing traditional herbal medicine and neuraminidase inhibitors in the treatment of seasonal influenza traditional chinese medicine treatments for upper respiratory tract infections/common colds in taiwan acg clinical guideline: the diagnosis and management of idiosyncratic drug-induced liver injury herbal medicines: a guide for health-care professionals hepatotoxicity of drug used in the treatment of gastrointestinal disorders adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids is there an association between ephedra and heart failure? a case series efficacy and safety of ephedra and ephedrine for weight loss and athletic performance: a meta-analysis hepatotoxicity of herbal remedies rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including chinese herbs chinese herbs nephropathy: a clue to balkan endemic nephropathy? kidney int aristolochic acid as a probable human cancer hazard in herbal remedies: a review panax ginseng and panax quinquefolius: from pharmacology to toxicology progress in cytochrome p enzyme in toxicity of traditional chinese medicines. chin the state pharmacopoeia commission of the people's republic of china. pharmacopoeia of the people's republic of china who guidelines on good agricultural and collection practices (gacp) for medicinal plants drug development guidance for industry positive or negative actors? biomolecules free radicals in the physiological control of cell function the authors would like to thank teachers of the center for languages and culture of kaohsiung medical university for the technical support. the authors declare no conflict of interest. key: cord- -i h f authors: kandala, ngianga-bakwin; magadi, monica akinyi; madise, nyovani janet title: an investigation of district spatial variations of childhood diarrhoea and fever morbidity in malawi date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: i h f although diarrhoea and malaria are among the leading causes of child mortality and morbidity in sub-saharan africa, few detailed studies have examined the patterns and determinants of these ailments in the most affected communities. in this paper, we investigate the spatial distribution of observed diarrhoea and fever prevalence in malawi using individual data for , children from the malawi demographic and health survey. we highlight inequalities in child health by mapping the residual district spatial effects using a geo-additive probit model that simultaneously controls for spatial dependence in the data and potential nonlinear effects of covariates. the residual spatial effects were modelled via a bayesian approach. for both ailments, we were able to identify a distinct district pattern of childhood morbidity. in particular, the results suggest that children living in the capital city are less affected by fever, although this is not true for diarrhoea, where some urban agglomerations are associated with a higher childhood morbidity risk. the spatial patterns emphasize the role of remoteness as well as climatic, environmental, and geographic factors on morbidity. the fixed effects show that for diarrhoea, the risk of child morbidity appears to be lower among infants who are exclusively breastfed than among those who are mixed-fed. however, exclusive breastfeeding was not found to have a protective effect on fever. an important socio-economic factor for both diarrhoea and fever morbidity was parental education, especially maternal educational attainment. diarrhoea and fever were both observed to show an interesting association with child's age. we were able to discern the continuous worsening of the child morbidity up to – months of age. this deterioration set in right after birth and continues, more or less linearly until – months, before beginning to decline thereafter. independent of other factors, a separate spatial process produces district inequalities in child's health. the success of any policy or health care intervention depends on a broader and accurate understanding of the socio-economic, environmental and cultural factors that determine the occurrence of disease and death. until recently, available information on childhood morbidity was derived from clinics and hospital records. however, information obtained from hospitals represents only a small proportion of all cases, since many other cases do not seek medical attention (black, ) . thus, the hospital records may not be appropriate for estimating the prevalence of diarrhoea for program developments (woldermicael, ) . policy-makers and researchers often want to know the distribution of a disease prevalence by geographical region, or association with environmental factors (diggle, moyeed, & thomson, ; thomson, connor, milligan, & flasse, ) . in this regard, mapping risk variations in child morbidity is an invaluable tool. further, the mapping of variation in risk of childhood morbidity can help improve the targeting of scarce resources for public health interventions. therefore, geographic information system (gis) is a powerful tool for public health practitioners that can easily allow them to assess patterns, trends and relationships between health events and environmental, socio-economic and other geographic factors (see for instance the case of disease surveillance, preparedness and response coordination to combat health threats such as west nile virus, anthrax, severe acute respiratory syndrome (sars) and bioterrorism (gardner & harrington, ) ). gis further helps us to understand childhood disease prevalence at the community level (see for instance garg, omwomo, witte, lee, & deming, ; mbonye, mbonye, , ) and helps to identify underserved populations, and can help public health agencies to efficiently allocate scarce program resources to appropriate locations. for example, the communicable disease control division of the boston public health commission (bphc) uses gis to help identify at-risk populations and determine where to focus efforts to vaccinate residents against influenza. this paper is based on a study of the spatial distribution of childhood diarrhoea and fever in malawi. the study applied bayesian statistical and geo-statistical techniques to the demographic and health survey (dhs) data of malawi with location (district) attributes and other information to answer specific questions about geographic inequalities in childhood disease prevalence. the dhs in malawi conducted in is a valuable resource for population-based morbidity data, although we recognize its limitation, like other dhss elsewhere, in that it relates only to reported child morbidity during the last weeks before the survey. thus, our results might be influenced by the effects of seasonality in diseases prevalence. to gain an understanding of the geographic variation or patterns based on the observed morbidity prevalence, a bayesian hierarchical model was fitted, with the inclusion of spatial (district) and nonlinear metrical (mother's and child's age) covariates. of particular interest in this study, was whether a significant geographic variation in childhood diarrhoea and fever existed; and if so, what potential risk factors could explain such variation? background: characteristics of the study area malawi is an african country in the south-east region of the continent with a population of about million and a population growth rate of about . percent/year (national statistical office [malawi] and orc macro, ; who, ) . with a gross national product (gnp) of us$ /person/year, malawi is among the least economically developed countries in the world (national statistical office [malawi] and orc macro, ; who, ) . agriculture accounts for over a third of the gnp, about percent of export earnings and approximately three-quarters of total employment (unicef and government of malawi, ) . despite economic difficulties malawi has invested a lot in health. according to unicef statistics there is a reasonably good network of health facilities in malawi (the median distance from home to the nearest facility is km). for percent of births, mothers have received at least one antenatal care from a trained health worker, and percent of children aged - months possess a health card indicating a high under-five clinic attendance and about percent of children are reported to have been vaccinated against the most common illnesses. however, improvements in health indicators have been smaller than expected. currently, the maternal mortality rate is estimated at - / , live births. life expectancy at birth is only years, largely because of high mortality amongst the children. for every live births, about die before the age of year and before the age of years (national statistical office [malawi] and orc macro ( ) . findings of the mdhs point to important changes in malawi's health and demographic profile. mortality of children under age has declined since the early s. during the period - , the under-five mortality rate was deaths/ live births, compared with / between - and orc macro ( ) . although this represents important progress, the rate of the downward trend is modest and childhood mortality remains at a very high level. a comparison of the and malawi dhs shows that the prevalence of diarrhea decreased slightly from % to % between the two periods. however, the percentage of ill children who received treatment reduced. all these point to the fact that malawian children are growing up in an environment of high morbidity, low utilization of health care, and consequently high mortality risks. the census results also indicate that there is geographic variation in the rates of infant and under-five mortality with highest mortality rates in the southern regions followed by the central regions and the least in the northern regions. household socio-economic status is associated with child survival because it determines the amount of resources (such as food, good sanitation and health care) that are available to infants (millard, ) . measures of socio-economic status that are thought to be associated with infant heath include: maternal and paternal education; household wealth; household size; parental occupation; and rural or urban residence. kandala and madise ( ) , who used the dhs data from malawi and zambia, in their study of childhood morbidity, found that the level of maternal education was highly significant in the two countries. they also found that childhood morbidity was lower among educated women, and that although this effect attenuated with the inclusion of other socio-economic factors in the models, maternal education remained significant. lower morbidity was also reported in households with large number of adult members (kandala, ; kandala & madise, ) . the impact of the household's size should, however, not be over-interpreted, since to some extent it directly mirrors infant mortality. for instance, a household with high mortality risk will remain small. in contrast, a household's size might also reflect its wealth, as a rich household will attract occupants. again, in a large household, a child might benefit from the help of several adults. large households may benefit from scale economies in time for childcare as well as in expenditures. alternatively, they may have become better at raising children through accumulated experience (christianensen & alderman, ) . child-level demographic factors such as birth order, the length of preceding birth interval, and the survival status of the preceding child have been shown to be strongly associated with infant mortality and health in africa as well as asia (cleland & sathar, ; kandala, ; kandala & madise, ; koenig, phillips, campbell, & d'souza, ; madise & diamond, ; whitworth & stephenson, ) . first and higher order births, those born after birth intervals of less than years, and those whose previous sibling have died appear to have high risks of morbidity and of dying in infancy. some researchers have documented evidence of a u-shape pattern in the association between maternal age and infant mortality and morbidity, with teenage and older mothers having elevated risk of child loss (bicego & ahmad, ; geronimus & korenman, ; kandala, ; kandala & madise, ; manda, ) . sex differentials in infant health and mortality have been observed universally. in the majority of the world regions, girls have lower mortality, at least for the first few months of life (curtis & steele, ; kandala, ; sastry, ) . exceptions have been noted in some asian countries. in india, girls are more than percent likely to die before their fifth birthday than boys and this is thought to be the result of son preference, which is manifest in lower spending on health for girls and higher prevalence of immunization among boys (claeson, bos, mawji, & pathmanthan, ; timaeus, harris, & fairbarn, ) . historically, variations in incidence and prevalence of diarrhoea and fever have been related to family socioeconomic factors and neglected temporal and geographical gradients and other variations in risk, in order to generate hypotheses towards the causation of disease. in this paper, we take advantage of advances in giss and how the technology provides opportunities to study associations between environmental exposure and the spatial distribution of diseases. jacquez ( ) discusses how gis can be used to monitor disease outcomes, identify health risks, and design and implement intervention plans. the epidemiological approach has not yielded all the answers, but it holds great merit and much potential to further contribute to the knowledge of disease etiology. this study enhances our understanding of diarrhoea and fever prevalence in a dimension that could not have been possible prior to the availability of gis. the results will help us making further decisions in planning for diarrhoea and fever research. individual data record was constructed for , children for diarrhoea and , children for fever. each record consisted of morbidity information and a list of covariates as shown in appendix a. geo-additive logistic models were used (on the probability of a child having diarrhoea and fever during the reference period) to determine the socio-economic and demographic variables that are associated with the ailments while simultaneously controlling for spatial dependence in the data and possible nonlinear effects of covariates. the dhs data have been collected hierarchically at the family and community levels which are inter-related. standard analysis of the fixed effects covariates for child morbidity neglects this correlation structure and dependence in the data. this neglect leads to underestimation of standard errors of the fixed effects that inflates the apparent significance of the estimates (bolstad & manda, ) . our analysis includes this correlation structure and account for the dependence of community in the model. the model also permitted borrowing strength from neighbouring areas to obtain estimates for areas that may, on their own, have inadequate sample sizes. this gives more reliable estimates of the fixed effect standard error. the response variable in this application is defined as y i ¼ if child i had diarrhoea or fever during the reference period t, and y i ¼ otherwise. the commonly adopted model for the analysis of this data is the probit or logistic model, and the standard measure of effects is the odds ratio (or) (mbonye, (mbonye, , woldermicael, ; yoannes, streat-eld, & bost, ) . because of the geographical nature of our data and the presence of nonlinear effects for some covariates, the assumption of a strictly linear predictor may not be appropriate, however. we use semi-parametric models to flexibly model the effects of selected socio-economic factors, continuous (metrical) and spatial covariates. our analysis is based on a flexible geo-additive model using the district as the geographic unit of analysis, which allows separating smooth structured spatial effects from random effect and estimate the effect of continuous covariates nonlinearly without assuming a linear functional form. a probit model with dynamic and spatial effects pr was used instead; where h is a known response function with a probit link function, and f . . . ; f p are nonlinear smooth effects of the nonlinear covariates and f spat is the effect of district s i f ; . . . ; sg where child i lives. in a further step, we split up the spatial effect f spat into a spatially correlated (structured) and an uncorrelated (unstructured) effect: the rationale is that a spatial effect is usually a surrogate of many unobserved influences, some of them may obey a strong spatial structure and others may be present only locally. models with a predictor that contains a spatial effect are also called geo-additive models (see kammann & wand, ) . more detailed description of these models is available elsewhere (for example, kandala, lang, klasen, & fahrmeir, ; kandala & madise, ) . this model is an extension of the probit or logistic model with a strictly linear predictor z i ¼ a þ w i g, the two main differences are the use of a flexible predictor to model the effects of covariates that clearly have nonlinear effects on diarrhoea and fever and the use flexible methods to introduce the spatial dimension on determinants of diarrhoea or fever and allocate these spatial effects to structured and unstructured (random) components. this is done jointly in one estimation procedure that simultaneously identifies socio-economic determinants, and the spatial effects that are not explained by these socio-economic determinants. in this way, we are able to identify district patterns of prevalence of diarrhoea and fever that are either related to socio-economic variables that are not in the model and that have a clear spatial pattern, or point to spatial (possibly epidemiological or environmental) processes that account for these spatial patterns. identifying spatial patterns of disease prevalence beyond the known family socio-economic determinants should also assist in poverty mapping and associated district targeting of resources (elbers et al., ) . the standard measure of effect is still the or for logistic model and mean for probit model (but because of the use of a fully bayesian approach that relies on prior assumption to make posterior inference, instead of 'or' or mean, we have 'posterior or' or posterior mean), and tests for significance, linear trends and interactions are not carried out as usual using likelihood ratio tests but the deviance information criteria (dic) (spiegelhalter, best, carlin, & van der line, ) is used instead for model fit and comparison. to account for possible departures from the assumed distribution, % confidence intervals (cis) for the posterior ors and probability maps (the equivalent of cis for the spatial effects) are calculated using robust standard errors estimated via markov chain monte carlo simulation techniques. the estimated coefficients follow the same interpretation as those of ordinary logistic regression: y ¼ , y ¼ , and exp (b i ) is the or that y ¼ when x i increases by . note that the probit model corresponds to a logistic model with the cumulative distribution function replaced by the standard normal distribution. the coefficient of covariates (say, child's place of delivery: hospital, antenatal visit, marital status, etc.) represent the difference in posterior log odds between the various categories and they are not easy to interpret apart from the sign. taking the exponential of the coefficients gives the posterior or and exponentiating the % confidence limits gives the confidence interval for the or. although the estimation process is complex, the estimated posterior coefficients (posterior mean) should be interpreted as those of ordinary probit models. we estimated models where either a structured or an unstructured effect was included as well as a model where both effects were included. based on these results, markov random field (mrf) priors were assumed for the structured effect (fstr(s)) and penalized spline (p-spline) prior for the nonlinear effects of metrical covariates f ; . . . ; f p . the analysis was carried out using bayesx-version . (brezger, kneib, & lang, ) , a software for bayesian inference based on markov chain monte carlo simulation techniques. we investigated the sensibility to the choice of different priors for the nonlinear and spatial and we noticed that results for this application are not sensitive to the choice of different priors. visual inspection of the maps of the observed diarrhoea and fever prevalence by regions and districts (figs. and ) suggest that regional classification conceal district variations. table shows that overall, the highest prevalence of both diarrhoea and fever were observed in the central region, followed by the southern region. although the northern region reported the lowest overall prevalence of both fever and diarrhoea, it had the district with the highest fever incidence of percent (nkhata bay), further confirming that regional classifications do mask important district variations. the geographical variation was apparent for the district maps for the two morbidity conditions, but from the region maps it was not apparent. the hypothesis that regional classifications conceal district variations was investigated for both levels separately using geo-additive probit models. prevalence of disease for each household were related to the distance of the household from the nearest next district and region. the region variables were categorized into dummies. there was a clear spatial pattern as observed in the distribution of diarrhoea and fever prevalence at the district level (table ) . for example, the aggregate regional levels of diarrhoea (table or left panel of fig. ) in the central and northern regions of malawi mask large district variability. the geographical information given in table (or right panel of fig. ) is highly aggregated and conceals local and district specific effects. on the other hand, diarrhoea prevalence by districts in table (or fig. ) strongly depended on the sample size and may be rather unstable. smoothing techniques were used to stabilize the observed prevalence in the sample as shown in figs. and . the bivariate distributions of fixed effects included in the analysis by the outcome variables are given in appendix a, also showing significance based on chisquare (w ) tests. in the bivariate analysis based on the w tests, factors that where significantly associated with diarrhoea are type of breastfeeding, child's age, parental education (both mother's and father's education), number of under-five children in the household, household size, place of residence (rural-urban), antenatal visit during pregnancy, child's place of delivery and the ethnicity. for fever, these factors are type of breastfeeding, child's size at birth, child's age, parental education (both mother's and father's education), number of under-five children in the household, household size, place of residence, antenatal visit during pregnancy, child's place of delivery and ethnicity. the results for diarrhoea presented in figs. and suggest considerable spatial auto-correlation in the underlying posterior means. the left panel of fig. reveals high-risk clusters mainly in the central districts of malawi. the result of the nonlinear effect of child's age (figs. and ) suggests that there is continuous worsening of the diarrhoea morbidity up to about months of age. shown are the estimated posterior means together with the % cis. for comparison a regression line obtained by assuming a linear fit is added to the plot. this deterioration set in right after birth and continues, more or less linearly, until months and decreases thereafter. we find the influence of the mother's age (fig. ) on diarrhoea to be nonlinear. there is a general tendency for diarrhoea morbidity to decline with increasing maternal age, but the patterns for older age are inconclusive. in particular, the interpretation of results at the end of the observation (wide confidence interval) is less reliable due to few observations. with regard to the fixed parameters, table shows that the prevalence of diarrhoea in malawi is lower among infants who are exclusively breastfed (but higher for those who are mixed fed), whose mothers are well educated, with a father having up to primary education (posterior mean either strictly negative or positive indicating, respectively, low risk and higher risk of diarrhoea). in general, lower parental education is associated with higher risk of diarrhoea. we did not find a statistically significant association between the risk of diarrhoea and child's sex, preceding birth interval, multiple birth (twin or singleton birth), the antenatal visits during pregnancy, birth order of the child, father's education, vaccination status, child's place of delivery (whether hospital or home), mother's marital status, child's place of residence, household size, the economic status of the household and child's size at birth. the right panel of fig. reveals a strong north-south gradient in the district spatial effects in malawi with a fairly sharp dividing line that runs through the centre (the capital city lilongwe) of the country. over and above the impact of the fixed effects, there appear to be negative influences on fever in the north and centralwest that are spread and affect most of the districts there. the right panel of fig. reveals also lower risk of fever in the capital lilogwe in spite of being surrounded by some of the high-risk districts. the result of the nonlinear effect of child's age (fig. ) suggests that there is continuous worsening of the child morbidity rate up to about months of age. fig. shows the influence of the mother's age on fever to be nonlinear, but with a general tendency to decline with age. the results for the youngest and oldest maternal ages are less reliable as shown by the wide cis. the fixed parameters show that the prevalence of fever (table ) is higher among infants of small size at birth and low parental education (incomplete primary education). children living in urban areas are associated with lower risk of fever. the variables child's sex, family size, mother's marital status, the number of antenatal visits during pregnancy, the type of feeding, preceding birth interval, multiple birth (twin or singleton birth), birth order of the child, vaccination status, child's place of delivery (whether hospital or home), the economic status of the household and child's size at birth were not statistically significant. this study has shown significant district-specific geographical variation in childhood diarrhoea and fever in malawi. the posterior mean estimates of the residual smooth spatial district effects (shades of white coloured ¼ low risk morbidity, shades of black coloured ¼ high risk morbidity, and shades of grey coloured ¼ not significant risk) are shown in the left panel of maps of figs. and . in addition, posterior probability maps (right panel of figs. and ) indicate significance of the spatial effects (negative/positive effect on diarrhoea, grey coloured ¼ non-significant). note that the residual spatial effects are centered about zero, i.e. the average over all districts is zero, while the overall level is estimated through the intercept term in eq. ( ). over and above the impact of the fixed effects, there appear to be widespread negative influences on child morbidity in the central districts. the central districts are at a lower altitude than other parts of the country. it is likely that climatic factors and associated diseases are responsible for this pronounced district pattern. food insecurity associated with drought and flooding in the shire valley, which is a result of hazardous effect of climatic variation are among possible explanations for these negative effects. furthermore, the central districts are among the high-density population areas and this environment tends to increase the child's exposure to disease. for fever, it appears that children living in northern and central-west districts are at lower risk compared with children living in the central-east and south. in general, children living in provincial capitals are at significantly lower risk compared with children in the rural areas. the negative spatial effects on child morbidity in southern districts correspond to districts that are among densely populated areas in the province; therefore, their share of disease spread may be one of the major factors of this negative impact on child morbidity. from the analysis, it also appears that living in the capital cities such as lilongwe is associated with significantly lower prevalence of fever, despite being surrounded by areas with negative district effects. living in the capital is likely to provide access to health care that is superior in ways that have not been captured adequately in the fixed effects. the same is, however, not true for diarrhoea, where some urban agglomerations, such as lilongwe, are associated with higher risk of diarrhoea. possibly because of the high density of population associated with the phenomenon of slums in urban areas. in malawi, childhood diarrhoea and fever are associated with child's age and mother's age at birth of the child. figs. and show the effect of child's age on diarrhoea and fever in malawi and figs. and show the effect of mother's age on the two ailments. shown are the posterior means together with % cis. for comparison a regression line obtained by a linear fit is added to the plot. while the effect of the variable ''mother's age'' is almost linear for both ailments, its effects on the variable ''child's age'' are clearly nonlinear. the linear model assumes a negative relationship between mother's age at child's birth and risk of diarrhoea or fever, and between child's age and risk of diarrhoea and fever. as we show in figs this glosses over important nonlinearity in the effects. the data suggest deterioration in child diarrhoea that sets in right after birth and continues, more or less linearly, until months of age. this immediate deterioration in child morbidity was not expected, as the literature commonly associates such deterioration with weaning at around - months. in a kenyan study, children aged - years were the most vulnerable (magadi, ) . one reason for this unexpected finding could be that, according to the dhs surveys, most parents gave their children liquids other than breast milk shortly after birth, a factor which might contribute to infections. this is due to the influence of poor-quality nutrition that is replacing breast milk as well as the onset of infectious diseases. these diseases are often related to unclean water and food which is replacing the breast milk, and the child no longer benefits from the mother's antibodies that are transmitted through the breast milk (stephenson, ) . initially, the worsening health status shows up as acute under-nutrition. but then childhood morbidity develops and worsens until about age . at that time, the body has developed its immune system to fight the impact of infectious diseases more effectively (moradi & klasen, ; who, ) . the influence of mother's age on child diarrhoea and fever show a general tendency for child morbidity to decline with increasing maternal age. part of the explanation for the observed association of morbidity risk and younger mother's age may be attributed to the tendency for young mothers to be socially and economically disadvantaged (world bank, ) , and the fact that younger mothers do not often use health services as much as older mothers (magadi, madise, & rodrigues, ) . these results are indicative of general trends and may be of use to planners for targeting policy. tables and ) after we controlled for the spatial dependence in the data, the fixed effects show the importance of parental education, breastfeeding, ethnicity, size of child, and rural-urban residence on child morbidity. the findings are generally as expected and consistent with the literature. children of highly educated mothers or living in urban areas are at lower risk of fever than other children (cleland & sathar, ; curtis & steele, ; hobcraft, mcdonald, & rutsein, ; kandala, ; madise, banda, & benaya, ) . the higher rural fever risk is possibly due to the fact that rural areas in sub-saharan african are under-developed and have less public service per capita compared with urban areas (brockerhoff, ; kuate defo, ) . as a result, living in rural areas provides no access to beds with mosquito nets or better health care and increases the risk of malaria and fever. after we controlled for child, household, and districts characteristics the residential location (rural versus urban) does not affect child diarrhoea. the urban-rural effect may be captured by the district effects. furthermore, the lack of corresponding urban advantage with respect to diarrhoea may be partly attributed to growing urban poverty in many parts of sub-saharan africa, which has been associated with poor sanitation in the densely populated slum settlements leading to increased incidence of diarrhoea among children of the urban poor (magadi, ) . we have established in this analysis that diarrhoea and fever especially during the early months of life is sensitive to low levels of parental education. similarly, in a study of the variation in african mortality, blacker ( ) , cites the much lower levels of female education in each country. studies using wfs and dhs data have shown that about half of the education-mortality association is accounted for by the economic condition of the household (bicego & boerma, ; cleland & van ginneken, ) . we find that, maternal education rather than paternal education matters a lot in reducing diarrhoea risk, whereas both low maternal and paternal education influence fever risk. there are also some ethnic differences in terms of diarrhoea and fever risk where, for example children from the sena ethnic group are more likely to have both diarrhoea and fever compared to other children. this suggests the need for in-depth studies in these communities to understand cultural child rearing practices that may put children at an increased risk of diarrhoea and fever. it should be noted that in the dhs data set, questions regarding fever control apply also to malaria control although there is a net clinical difference between the two diseases. malaria-relevant indicators include the reported treatment and care (whether antimalarials were given and facilities attended) to under- s who had fever in the weeks preceding the interview. the interpretation of fever in this report should take into account the fact that the dhs surveys are (for logistical reasons) mostly conducted in the dry, least malarious season (africa malaria report, who, ) . in addition, the observed prevalence of diarrhoea can be considered as incidence rate of diarrhea. because of the short followup period of weeks before the interview, we have ignored the role of competing risk, although diarrhoea may be present in different forms: persistent diarrhoea (more than days), acute watery diarrhoea, or dysentery (blood in stool). other diseases that can occur concurrently with diarrhoea include measles and malaria. malnutrition also often accompanies diarrhoea. it is important to point out that some of the factors observed to be significantly associated with prevalence of both fever and diarrhoea in the bivariate analysis, such as antenatal care and delivery care, turned out not to be significant in the multivariate analysis that simultaneously controlled for spatial effects as well as the effects of other covariates. it is possible that health care utilization is a reflection of accessibility of health care services and has been captured by the spatial effects in the multivariate analysis. also vaccination status turns out to be statistically insignificant, although other studies in rural sub-saharan africa have reported immunization status of a child as an important factor associated with diarrhoea (see for instance mbonye, mbonye, , . it should be noted that in the dhs, the reliability of parental recall of vaccination status for vaccines given to infants has not been studied and it is therefore unknown and may affect these results. possible reasons for the lack of association between childhood disease and the economic status may be because, many of the household wealth indices use assets that are more likely to be found in urban areas than in rural areas. thus, most of the rural households will be in the lowest wealth category even if they have other indicators of wealth (e.g. livestock or farm machinery). the consequence of this misclassification would be to lower the risk of childhood diseases of rural households. another limitation with household wealth indices derived from dhs is that they are based on current status data so that they might not capture the true level of household wealth during the infancy of children born several years before the survey. however, since these analyses are restricted to births within years of the survey, this bias will not be substantial. to explain further these inequalities in childhood diseases prevalence at district level in malawi, further research is needed to scrutinize the spatial pattern of occurrence of water-borne illnesses to specific locations. this will enable us to draw a picture of where and whether events are concentrated, which will help in turn to guide local public health response or investigation to help identify at-risk populations and determine where to focus efforts to vaccinate residents against malaria. moreover, the measure of disease prevalence used here, recall of whether a child had been ill with diarrhoea or fever in the past weeks is less perfect as it is quite subjective, based on a short-term recall. future work needs to address the question of disease environment more closely. in conclusion, the study findings carry some important general pointers to policy directions. for instance, the age effect suggests the need to pay attention to child feeding practices, particularly during the first months after birth. second, the nonlinear influence of mother's age indicates that childcare promotion messages should be targeted particularly to younger parents. of high significance are the district influences on child morbidity. in particular, they suggest that in malawi, some urban agglomerations are associated with higher risk of diarrhoea. also, more emphasis must be placed upon the role of remoteness as well as climatic and geographic factors on childhood morbidity. it would be of value to investigate district-level factors not included in our models, such as environmental, socio-economic, cultural and human behavioural factors involved in the etiology of the disease. the north-central divide in malawi highlights the importance of such considerations. demographic and health surveys, comparative studies no maternal education and child survival: a comparative study of survey data from countries diarrhoeal diseases and child morbidity and mortality infant and child mortality: development, environment, and custom investigating child mortality in malawi using family and community random effects: a bayesian analysis bayesx-software for bayesian inference based on markov chain monte carlo simulation techniques child survival in big cities: are the poor disadvantaged? working paper child malnutrition in ethiopia: can maternal knowledge augment the role of income? reducing child mortality in india in the new millenium the effect of birth spacing on childhood mortality in pakistan maternal education and child survival in developing countries: the search for pathways of influence variations in familial neaonatal mortality risks in four countries childhood malaria in the gambia: a case-study in model-based geostatistics welfare in villages and towns: micro-level estimation of poverty and inequality bayesian inference for generalized additive mixed models based on markov random field priors putting health on the map care seeking during fatal childhood illnesses: siaya district the socio-economic costs of teenage childbearing: evidence and interpretation demographic determinants of infant and early child mortality: a comparative analysis spatial analysis in epidemiology: nascent science or a failure of gis? geoadditive models spatial modelling of socio-economic and demographic determinants of childhood undernutrition and mortality in africa semiparametric analysis of the socio-demographic determinants of undernutrition in two african countries spatial epidemiology of childhood diseases in malawi and zambia. african population studies birth intervals and child hood mortality in rural bangladesh areal and socioeconomic differentials in infant and child mortality in cameroon infant mortality in zambia: socio-economic and demographic correlates determinants of infant mortality in malawi: an analyssis to control for death clustering within families status of women and infant/child health in kenya with particular reference to the high mortality zone in nyanza province maternal and child health among the urban poor in frequency and timing of antenatal care in kenya: explaining the variations between women of different communities unobserved family and community effects on infant mortality in malawi prevalence of childhood illnesses and care-seeking practices in rural uganda risk factors for diarrhoea and upper respiratory tract infections among children in a rural area of uganda: integrating immunisation and childcare programmes key to reducing child mortality a causal model of high rates of child mortality the nutritional status of elites in india, kenya, and zambia: an appropriate guide for developing international reference standards for undernutrition? sfb discussion paper no. . university of munich. national statistical office [malawi] and orc macro family-level clustering of childhood mortality risk in north-east brazil bayesian measures of models complexity and fit burden of infection on growth failure the ecology of malaria as seen from earth observation satellites can use of health care ex-plain sex differentials in child mortality in the developing world? situation analysis of poverty in malawi birth spacing, sibling rivalry and child mortality in india diarrhoeal morbidity among young children in eritrea: environmental and socioeconomic determinants poverty analysis of the malawian integrated household survey, - malaria: the current situation who global database on child growth and malnutrition africa malaria report. who child morbidity patterns in ethiopia key: cord- - uz v k authors: schneiderová, kristýna; Šmejkal, karel title: phytochemical profile of paulownia tomentosa (thunb). steud. date: - - journal: phytochem rev doi: . /s - - -y sha: doc_id: cord_uid: uz v k paulownia tomentosa, a member of the plant family paulowniaceae and a rich source of biologically active secondary metabolites, is traditionally used in chinese herbal medicine. flavonoids, lignans, phenolic glycosides, quinones, terpenoids, glycerides, phenolic acids, and miscellaneous other compounds have been isolated from different parts of p. tomentosa plant. recent interest in this species has focused on isolating and identifying of prenylated flavonoids, that exhibit potent antioxidant, antibacterial, and antiphlogistic activities and inhibit severe acute respiratory syndrome coronavirus papain-like protease. they show cytotoxic activity against various human cancer cell lines and inhibit the effects of human cholinesterase, butyrylcholinesterase, and bacterial neuraminidases. most of the compounds considered here have never been isolated from any other species of plant. this review summarizes the information about the isolated compounds that are active, their bioactivities, and the structure–activity relationships that have been worked out for them. the plant paulownia tomentosa (thunb.) siebold & zucc. ex steud. is a very adaptable and extremely fastgrowing timber tree native to central and western china traditionally used in chinese medicine. this deciduous tree is now grown in many areas worldwide, mostly as a decorative ornamental tree (erbar and g} ulden ; zhu et al. ) . two related varieties of p. tomentosa have been described-var. tsinlingensis has a round to shallowly cordate leaf base and a glabrous or sparsely hairy lower leaf surface, whereas var. tomentosa is characterized by a cordate leaf blade base and an abaxial surface that is densely hairy when mature (hong et al. ) . paulownia was named paulownia in honour of anna paulowna ( paulowna ( - , queen consort of the netherlands and a daughter of tsar paul i of russia. nowadays, it is commonly known under its synonym bignonia tomentosa (zhu et al. ) or as the princess-tree, empress-tree, foxglove tree, royal paulownia, kiri, or mao pao tong (yuan bian zhong) (erbar and g} ulden ; hong et al. ) . the genus paulownia was first assigned to family bignoniaceae by swiss botanist thunberg ( ). it was then transferred to the scrophulariaceae family by the dutch scholars zuccarini and siebold ( ) (zhu et al. ). at last, paulownia has been categorized as a family of its own, paulowniaceae, based on data from the latest molecular phylogenetic studies. this table non-prenylated flavonoid aglycones isolated from p. tomentosa leaves (zhu et al. ) weak cytotoxic activity in vitro against human leukaemia (hl- ) and human hepatoma (smmc- ) cell lines ; no a-glucosidase inhibitory effect (zhao et al. ); weak aldose-reductase inhibitory activity (kadota et al. ) ; moderate inhibitory effect on human immunodeficiency virus- protease (lee et al. ) ; no activity against lipopolysaccharide (lps)induced no production in raw . macrophages (li et ( ) leaves , flowers jiang et al. ) antioxidant (prince vijeya singh et al. ); antibacterial, anti-inflammatory, antispasmodic, antidiarrhoic, antiproliferative, vasorelaxant (jiang et al. ); neuroprotective (losi et al. ); cardioprotective (psotová et al. ); chemopreventive activity against skin cancer ); activity reviewed by shukla and gupta ( ) and patel et al. ( ) h h oh h oh kaempferol ( ) leaves (si et al. a ) cytotoxic, pro-apoptic (suppresses cell metastasis via inhibition of the erk-p -jnk and ap- signaling pathways in u- os human osteosarcoma cells) ; antioxidant (e.g., attenuates bladder hyperactivity caused by potassium chloride after protamine sulphate-induced bladder injury) (huang et al. ) ; impact on human health and cancer chemoprevention reviewed by , luteolin ( ) leaves (si et al. a; li ) memory-improving (yoo et al. ); inhibition of aamylase activity (funke and melzig ) ; cytotoxic (bgc- gastric carcinoma xenografts in nude mice) ; vascular protective (si et al. ( ) bark (si et al. b) , leaves (si et al. a ) antifibrotic (yoon et al. ) ; antioxidant, antiproliferative, anti-inflammatory, cardioprotective ; neuroprotective (ghosh et al. ); anti-diabetic [dose-dependent inhibition of both na ? atpase and sodium hydrogen exchanger in type diabetic erythrocytes (mishra and rizvi ) ; inhibition of pi k (koch et al. ) ]; antiviral [anti hcv (khachatoorian et al. ) ; hcmv (cotin et al. ) ]; no anti-hiv activity in vitro tested on h cells in the absence of toxicity (tang et al. ); review of bioactivities by russo et al. ( ) oh h oh oh oh , -dimethylquercetin (syn. rhamnazin) leaves, green immature fruit (wollenweber et al. ) antimicrobial activity, poor antifungal effect (omosa et al. ); low antimicrobial activity, low toxicity against human lymphocytes and monocytes, antioxidant/antiinflammatory activity (martini et al. ; pelzer et al. ); low affinity to acetylcholinesterase (remya et al. ) ; low inhibitory effect on no production in raw . cells (sudsai et al. ) ; no activity against hsv- , low toxicity against vero cells (tian et al. ); no antioxidant activity (takamatsu et al. ) ; no trypanocidal activity (grael et al. ) ; activity against lipid peroxidation in rat liver microsomes (yun et al. ) ; cytotoxicity against tk- , mcf- and uacc- cells (lopez-lazaro et al. ) ; no inhibition of glycolysis in various tumor cells (suolinna et al. , -trimethylquercetin ( ) thrombin inhibition (shi et al. ); inhibition of il- synthesis in basophils (hirano et al. ) ; weak trypanocidal activity (jordao et al. ); weak inhibition of no production in lps-activated mouse peritoneal macrophages (matsuda et al. ) ; weak inhibition of degranulation of rbl- h cells (mastuda et al. ) ; inhibition of pgp activity (scambia et al. ) ; no inhibition of glycolysis in a variety of tumor cells (suolinna et al. flowers jiang et al. ; kim et al. a , b) inactive against glutamate-induced neurotoxicity studied in primary-cultured rat cortical cells (kim et al. a, b) ; no trypanocidal activity (grael et al. ) h h oh ome ome -hydroxy- , flowers (kim et al. a , b) inactive against glutamate-induced neurotoxicity studied in primary-cultured rat cortical cells (kim et al. a, b) ; inhibition of inflammation by induction of synovial apoptosis of fibroblast-like synoviocytes through caspase activation in rats with adjuvant arthritis (li et al. ; inhibition of jak -stat signal pathway in rats ); inhibition of phosphodiesterase (yang et al. ) ; low toxicity on b f and sk-mel- melanoma cells (rodriguez et al. ) ; antimutagenic activity (miyazawa et al. ) h family includes only one genus and between six and ten species. it was originally introduced in by the research of nakai (erbar and g} ulden ) . biological activity and traditional uses of p. tomentosa extracts according to both legends and records, people were already using paulownia for various purposes about , years ago. chinese herbal medicine has used p. tomentosa traditionally to relieve bronchitis, especially by reducing coughing, asthma, and phlegm (zhu et al. ). it has also been used to treat conjunctivitis, dysentery, enteritis, erysipelas, gonorrhea, hemorrhoids, parotitis, traumatic bleeding, and tonsillitis (jiang et al. ; si et al. ). solutions prepared from the leaves and fruit extracted in water have been used in daily applications to promote the healthy growth of hair and turn grey hair dark. an extract prepared from the wood and leaves may relieve swollen feet. pharmacological experiments have shown that extracts from the fruit can reduce blood pressure. nowadays, injections and tablets prepared from paulownia flowers and fruit are used for the herbal treatment of chronic bronchitis and other kinds of inflammation (zhu et al. ). more than physiologically active constituents have been isolated from different parts of the paulownia plant. their biological activity has been tested using both the isolated compounds and different types of extracts. for example, n-butanol, etoac, and meoh extracts obtained from the fruit have displayed antiradical activity in anti-dpph and peroxynitrite assays, due to mainly the presence of flavonoids and phenolic glycosides, but not of all compounds present in these extracts have been identified) (Š mejkal et al. b ). an meoh extract obtained from the fruit inhibited hache (ic = . mg/ml) and bche (ic = . mg/ml) significantly more strongly than chcl and water extracts (possibly due to the content of phenolic glycosides and c-prenylated dihydroflavonols and flavanones) (cho et al. ). significant concentration-dependent anti-inflammatory properties of etoh extracts of the bark of the tree have also been observed recently using a lipopolysaccharide (lps)induced nitric oxide production inhibition model in the murine macrophages cell line raw . (si et al. a ). kim et al. ( a, b) showed potential of aqueous extract of p. tomentosa to suppress glutamate induced toxicity in primary cultured rat cortical cells (with possible sesquiterpene lactone as active substance). the bio-activities of individual compounds and, where possible, the structure-activity relationships are in tables , , and and discussed in separate chapters. flavonoids represent the most numerous group of secondary metabolites isolated from p. tomentosa. they can be divided into simple non-prenylated flavonoids - (table ) , c-prenylated and c-geranylated flavonoids - (tables , , respectively), and flavonoid glycosides - . compounds , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] have never been isolated from any other species. most of the isolated flavanones are characterized by a s configuration in contrast to the dihydroflavonols, for which r, r isomer is often observed. some of the flavonoid compounds found in p. tomentosa have been categorized as dietary flavonoids. consuming these compounds is believed to deliver health benefits. the activities of these flavonoids are frequently been reviewed, for example in the papers of romano et al. ( ) , marzocchella et al. ( ) , ross and kasum ( ) , and havsteen ( ) . some simple flavonoid substances isolated from fruit of p. tomentosa are commonly observed as the lipophilic components of different plant exudates. flavonoids - show broad-spectrum pharmacological activities (table ) . many papers report their potential role as anticancer compounds for use against human cervical and breast (bulzomi et al. (bulzomi et al. , , hepatoma, leukemic , osteosarcoma , gastric carcinoma , colon adenocarcinoma (sánchez-tena et al. ) , or prostatic (zhang and coultas ) cancer cell lines. their antioxidant properties could explain their promising cardioprotective (paneerselvam et al. ; psotová et al. ) and neuroprotective effects (losi et al. ). antimicrobial (betts et al. ; jiang et al. ; mankovskaia et al. ) and antiviral (khachatoorian et al. ) bio-activities against different pathogens have also been discovered. in some cases, no specific biological activity has been observed (kim et al. a (kim et al. , b tang et al. ) . paulownia tomentosa is also a rich source of prenylated and geranylated flavonoids ( - ), whose occurrence is limited to relatively a few plant families (yazaki et al. ). there are sometimes misunderstandings in the naming of these compounds. a compound containing both a phenolic skeleton and a terpenoid side chain is often designated as a ''prenylated'' phenolic substance, even though it contains not a prenyl, but rather a geranyl or other type of terpenoid side-chain. prenylated flavonoids are biosynthesized by a combination of several pathways: the acetate, shikimate, and mevalonate ( fig. ) . it is now known that the prenyl and geranyl moieties are biosynthesized via mevalonate or deoxyxylulose pathway. the connection of terpenoid and flavonoid biosynthetic antioxidant ; cytotoxic (a human ovarian cancer cell line) (murphy et al. (murphy et al. , oh bark (sticher and lahloub ; zhu et al. ) antiphlogistic (choi et al. ), low cox- / and -lox inhibition (wang et al. ; diaz lanza et al. kuzuyama et al. ) . the side chain can later be converted into different moieties by several reactions. it is unclear, whether the described modifications of the prenyl side-chain are natural-sunlight, the presence of oxygen and an elevated temperature can all affect the terpenoid metabolism-or are artefacts of isolation. some of the changes in the prenyl moiety have been observed after treatment of an extract, or during the separation of a mixture in the acidic environment of silica gel (navrátilová et al. ; Š mejkal ) (fig. ) . most of the prenylated flavonoids isolated from p. tomentosa belong to c-geranylated group of flavonoids ( - ). some of them have their sidechain further modified by hydroxylation ( - , , , - ), methoxylation ( - , , ), oxidation ( , , ), cyclization ( , - ), or reduction ( , , - ) . interestingly, these compounds have been isolated from the leaves, flowers and fruit, but they are most commonly isolated from the roots, root bark, or bark of different plants (botta et al. ) . compounds , , and have been found in the yellow dendritic trichomes on the adaxial side of the p. tomentosa leaves. on the other hand, no significant detected concentration of secondary metabolites has been detected in the white dendritic trichomes on the adaxial side of the leaves or the brown dendritic trichomes on the flower buds ). glandular hairs found on the young reproductive organs of p. tomentosa are rich in flavonoids, with concentrations over , times greater than those on the surfaces of the young leaves . some seasonal variations in the appropriate time for harvesting the fruit have been discovered. autumn is the best time to obtain high concentrations of flavonoids whereas early summer is better for phenylpropanoid glycosides (holubová and Š mejkal ) . the antioxidant Š mejkal et al. a, b; zima et al. ) , antibacterial (navrátilová et al. ; Š mejkal et al. b) , antiphlogistic , cytotoxic (kollár et al. ; Š mejkal et al. a , and severe acute respiratory syndrome coronavirus papain-like protease (sars-cov plpro) activities (cho et al. ) of isolated geranylated flavonoids have been described recently, together with their inhibitory effect on both human acetylcholinesterase and butyrylcholinesterase (cho et al. ) . the great ability of several geranylated flavanones to interact with bacterial sialidase isolated from clostridum perfringens (cp-nani), a bacterium causing various gastrointestinal diseases, has recently been reported (lee et al. ) . possible relationships between structure and activity have been proposed for each of these biological activities. nevertheless, it is difficult to evaluate real structure-activity relationships only by comparing the published studies, because the study conditions and the assays employed are often different. generally, the addition of an isoprenoid chain renders the derivate molecule more pharmacologically effective than the parent compound, probably because the prenyl group increases the lipophilicity and confers a strong affinity for biological membranes (botta et al. ; epifano et al. ; chen et al. ) . interestingly, neither the geranyl side-chain nor its substitution affects the antioxidant activity of flavonoids. the spatial arrangement of the substitution of the flavanone skeleton is more important. for example, the antiradical activity is increased by -hydroxyl substitution of the ring b, whereas -methoxyl substitution diminishes it. the general rules postulated for the antioxidant activity of flavonoids in vitro are applicable (havsteen ; chen et al. ) , there are many review publications that touch on this topic, and it is not aim of this paper to delve deeply into this (plaza et al. ) . the type of antioxidant assays used for the experiment could also be a factor that significantly affects this activity (zima et al. ) . numerous reports about structure related antimicrobial activity have been published, but comparison shows the results to be widely conflicting (cushnie and lamb ) . based on several studies, it has been postulated that hydroxylation at position c- or c- of ring a and positions c- or c- of ring b increases the antibacterial activity (Š mejkal et al. b; navrátilová et al. ) . however, contrary to this assumption, and had no significant antibacterial activity (navrátilová et al. ) . interestingly, some c-geranylated flavonoids do not able inhibit the growth of gram-negative bacteria ( , , - , , , , - , , , and ) . resistance to these compounds is probably due to the more complex structure and hydrophilic nature of g-cell walls (navrátilová et al. ; Š mejkal et al. b) . furthermore, substitution of the geranyl side-chain with carbonyl, hydroxyl or methoxyl groups diminishes the antibacterial activity in a manner similar to what is seen when the geranyl substituent at c- forms a ring by reacting with the hydroxyl group at c- . compounds , , , , , and exhibit some degree of activity in the range of the concentrations tested on the gram-positive bacteria staphyloccocus aureus and various methicillin resistant strains of s. aureus. the level of activity varied in depending on both the structure and the bacterial strain used in the assay. flavonoid structures like are more effective in protecting plants from water loss because of their reduced polarity navrátilová et al. ). compounds , , , , and were also tested for their ability to affect the initiation of the eukaryotic translation via dual-luciferase reported assay (firefly and renilla), but only showed a modest activity in comparison with anisomycin (navrátilová et al. ) . the cytotoxicity of the prenylated flavonoids obtained from p. tomentosa has been tested in more than different cell lines. the type of prenylation strongly affects the cytotoxicity of a flavonoid in the traditional p- murine leukemia model. the unmodified -prenyl group and the presence of corresponding ortho-dihydroxy or trihydroxy substitution of the flavonoid ring b are crucial to its activity against p- as compared with other prenyl substituents (hakim et al. (hakim et al. , (hakim et al. , . similar findings have been observed for modified c- geranyl groups and the substitution of the ring b for other cell lines tested (Š mejkal et al. ) . however, replacing the parahydroxy group of the ring b of a c- prenylated flavanone with several different acyl substituents resulted in greater cytotoxicity (aniol et al. ; Š mejkal ) . it has also been found that cytotoxic activity is diminished by the presence of a c- hydroxyl substituent on the ring c, -methoxy substitution of the ring b or a para-hydroxy substituted ring b (Š mejkal et al. a) . for this reason, it is important to emphasize that the relative importance of the substitution of ring b can differ according to the cell line used. modification of a prenyl or geranyl sidechain can not only change the direct cytotoxicity, but it may also affect the proliferation cells or trigger apoptosis (kollár et al. ; Šmejkal et al. a Š mejkal ) . prenylated flavonoids - , modified with an unusual , -dihydro- h-pyran moiety, have been found to inhibit the severe acute respiratory syndrome corona virus plpro enzyme more effectively than their parent compounds, the precursors from which were they derived (cho et al. ) . the presence of a geranyl group at the c- position ( , , - , , , , and ) seems to be crucial for the hache and bche inhibitory effects. the most effective inhibitor was . all of the geranylated flavonoids, apart from , inhibited hache dosedependently. it appears that greater inhibition is observed when ring b of the flavanone bears free hydroxyl groups (cho et al. ). phytochem rev ( the crystal structure of the bacterial sialidase cp-nani catalytic domain in a complex with the geranylated flavonoid diplacone ( ) provides structural insights into the binding mode of natural flavonoidbased inhibitors at atomic resolution. it shows how the geranyl and c- hydroxyl groups of contribute to the stability of the enzyme-inhibitor complex. time-dependent competitive inhibition patterns have been observed. structural comparison of the human sialidases neu -neu with the cp-nani-diplacone ( ) complex suggests that the interaction between human sialidases and is likely to be unfavourable because of polar or ionic repulsion (lee et al. ) . six flavonoid glycosides have been extracted from the stem bark - (si et al. ) and flowers - (scogin ) of p. tomentosa. apigenin- -o-b-d-glucopyranoside (synonym: cosmosiin) (kurkina et al. ) ( ) shows anti-hiv activity in vitro on h cells (tang et al. ) and enhances the secretion of adiponectin, the phosphorylation of the tyrosine residue of insulin receptor-b, and the translocation of glut (rao et al. ) . compound shows no significant hepatic glc- -phosphatase inhibitory activity in vitro (kumar et al. ). apigenin- -o-b-d-glucuronopyranoside ( ) was more potent than apigenin ( ) and omeprazole in an assay analysing the inhibition of reflux esophagitis and gastritis promoted surgically and by application of indomethacine in rats (min et al. ) . no information about the biological activity of apigenin- -o-[b-d-glucuronopyranosyl ( ? )-o-b-d-glucuronopyranoside] ( ) has been found in the literature. delphinidin- -o-glucoside (synonym: myrtillin) ( ) protects microglia from inflammationinduced stress signaling (carey et al. ) , is cytotoxic against the mcf- (breast) cancer cell line (vareed et al. ) , and has an the affinity for the estrogenic erb receptor (hidalgo et al. ). delphinidin- , -di-o-glucoside ( ) and cyanidin- , -di-o-glucoside ( ) are potent antioxidants tanaka et al. ) . compounds and are potent ace inhibitors in vitro (hidalgo et al. ; persson et al. ). only three lignans: (?)-paulownin ( ), (?)-sesamin ( ), (?)-piperitol ( ) have been isolated from the wood of p. tomentosa (ina et al. ; takahashi and nakagawa ; zhu et al. ) (fig. ). several reports have described their pharmacological effects pan et al. ). compound is a promising antifungal agent that acts against the basidiomycetes fomitopsis palustris and trametes versicolor (kawamura et al. ) . sesamin ( ) has been previously isolated from the wood of p. tomentosa, and p. kawakamii and the hybrid of p. elongata p. fortunei (takahashi and nakagawa ; zhu et al. ). compound is the major lignan in sesame, and has various biological activities, such as antioxidant, angiogenic (chung et al. ) , antiphlogistic (chatrattanakunchai et al. ) , antihypertensive (nakano et al. ) , insecticidal (nascimento et al. ) , neuroprotective , and anticarcinogenic ( . however, this compound showed no significant ability to reduce the formation of atherosclerotic lesions in the apoe (-/-) gene-knockout mouse, whereas specific dietary polyphenols, especially quercetin and theaflavin were more active (loke et al. ) . it has been suggested that some of the biological effects attributed to sezamine ( ) may, in fact, be caused by its metabolites and that may be acting as a proactive substance in the body (yasuda and sakaki ) . the metabolism of sesamim ( ) by cytochrome p and udp-glucuronosyltransferase has been found remarkably different in humans as compared to other animals. further investigation into the safety of taking sesamin ( ) with therapeutic drugs that are metabolised by cyp c is also needed (yasuda and sakaki ) . a total of thirteen phenylpropanoid glycosides ( - ) with multifarious bioactivities, natural compounds derived biosynthetically from the amino acid phenylalanine, have been isolated from different parts of the p. tomentosa plant (table ) (damtoft and jensen ; kang et al. ; ota et al. ; schilling et al. ; si et al. b , d, si et al. b sticher and lahloub ; Šmejkal et al. b; zhu et al. ). phenylpropanoids are of great interest for fabricating effective tonic, anticancer, hepatoprotective, immunostimulating, antimicrobial, and anti-inflammatory phytopreparations (kurkin ; galvez et al. ; panossian and wagner ; fu et al. ; pan et al. ) . a new furanoquinone, methyl- -hydroxy-dinaphthol [ , - , ]furan- , -dione- -carboxylate (mhddc) ( ) (fig. ) identified in p. tomentosa stem bark (meoh extract) has been shown to possess antiviral activity against poliovirus types and , using hela cells in vitro (kang et al. ). its cathepsin k and l inhibitory activities in vitro have recently been discovered. the -oh functional group may have a favourable effect on this reduction potential which prevents the degradation of bone the matrix carried out by osteoclasts (park et al. ). the naphtoquinone plumbagin ( ) has been detected in the leaves and fruit of p. tomentosa (babula et al. ) . it has been used in traditional systems of medicine since ancient times (pile et al. ) . it has exhibited promising antimalarial activity in vitro with ic of ( - ) nm and ( - ) nm, respectively, against d chloroquine-sensitive p. falciparum and k chloroquineresistant p. falciparum clones, using an assay based on sybr green i. toxicity testing indicated relatively low toxicity at dose levels up to mg/kg body weight (for a single oral dose) and mg/kg (for daily dose given for consecutive days) for acute and subacute toxicity, respectively. based on the results of in vivo antimalarial testing, plumbagin administered at a the dose of mg/kg of body weight for consecutive days exhibited moderate to weak antimalarial activity with regard to its ability to reduce parasitaemia and prolong survival time (sumsakul et al. ) . other published studies of plumbagin ( ) described effects such as antifungal, antibacterial, cytotoxic (krishnaswamy and purushothaman ) , anticoagulant (santhakumari et al. ) , anthelmintic (antischistosomal) (zhang and coultas ) and an anti-inflammatory effect on the amelioration of experimental ulcerative colitis in mice at a dose of - mg/ kg (pile et al. ) . six iridoids: -b-hydroxyharpagide ( ), paulownioside ( ), catalpol ( ), aucubin ( ), tomentoside ( ) and -hydroxytomentoside ( ) have been isolated from the leaves of p. tomentosa ( - ) (adriani et al. ; franzyk et al. ) , the bark of the trunk and roots ( ) (plouvier ) and parts of the young plant ( , , , and ) (damtoft and jensen ) (fig. ) . compound shows neuroprotective activity (li et al. ), a cardioprotective effect against myocardial infarction-specifically reperfusion damage (huang et al. a, b) , and radioprotective effects . it also increases glucose utilization by increasing the secretion of b-endorphin from the adrenal gland (shieh et al. ) . compounds and possess antispasmodic activity (deurbina et al. ) . aucubin ( ) had antioxidant and pancreasprotective effects on streptozotocin-induced diabetes in rats (jin et al. ) and it may improve obesityinduced atherosclerosis by attenuating the tnf-ainduced inflammatory response (park ) . a weak antibacterial effect (against streptococcus pneumoniae and mg-hemolytic streptococcus, mic . mg/ml) (zheng et al. ) and neuroprotective effects of on diabetes and diabetic encephalopathy (xue et al. ) have also been observed. the sesquiterpenic lacton isoatriplicolide tiglate ( ) has been isolated from p. tomentosa flowers. it has neuroprotective effects against glutamate-induced neurotoxicity (kim et al. a (kim et al. , b and cytotoxic activity against several cancer cell lines: a lung carcinoma; sk-ov- adenocarcinoma; sk-mel- malignant melanoma; xf central nervous system tumors; hct colon adenocarcinoma, against which its effect is comparable to that of reference substance adriamycin (moon and zee ) ; human breast cancers mda-mb- , mcf , hs t, and t d; and the hela, siha and c a cervical cancer cell lines (jung et al. ) . seven phytosterols have been isolated from p. tomentosa leaves: ursolic acid ( ) (zhu et al. ; zhang and li ) , -epiursolic acid ( ), pomolic acid ( ), corosolic acid ( ), maslinic acid ( ), b-sitosterol ( ), and daucosterol ( ) (zhang and li ) . most of these show various biological activities, e.g., is a potentially useful for treating alzheimer's disease (because of its ability to block the interactions of the amyloid b-cd ) (wilkinson et al. ) . it can prevent the recruitment of the monocytes that accelerate atherosclerosis, a major complication of diabetes, in mice (ullevig et al. ) , and it also possesses antibacterial (wong et al. ) , anti-trypanosomal, and anti-leishmanial properties (bero et al. ) . compound has also shown cytotoxic effects against k and k /adr human chronic myelogenous leukaemia, hl and hl /adr human acute myelocytic leukemia cancer cells, and the human colon cancer cell lines sw and sw (shan et al. ) . compounds - also show promising cytotoxic potential, e.g., is effective against the human chronic myelogenous leukaemia cell line and also against cells derived from chronic myeloid leukaemia patients (vasconcelos et al. ). compound shows cytotoxic effects against osteosarcoma mg- cells (cai et al. ) and immunosuppressive activity on myeloid-derived suppressor cells in the murine sarcoma model (horland et al. (deepak and handa ) , and (schinella et al. ). an immunomodulatory effect of in protecting mice against candidiasis disseminated by the cd ? th immune response has been seen (lee et al. ). other recently discovered pharmacological effects include antimalarial ( ) (moneriz et al. ) , hypotensive and endothelium-dependent vasorelaxant effects ( ) (estrada et al. ). compound is potentially useful for treating cerebral ischemic injuries (guan et al. ) , and has promising antidiabetic effects (sivakumar et al. ). ( ). the relatively most abundant constituents were ( % of the total glycerides), ( %), and and ( %) (asai et al. ). another analysis of the secretions of the glandular hairs on the leaves of both bud flushes and adult trees as well as the flowers of adult trees showed that these secretions contain ten glycerides ( , - , , , - ) . these compounds showed sticky character, but they were not toxic to several insects. therefore, the glandular hairs on the leaves and flowers may serve only to physically deter herbivores (fig. ) . six known phenolic acids, namely p-hydroxybenzoic acid ( ), vanillic acid ( ), gallic acid ( ), cinnamic acid ( ), p-coumaric acid ( ), and caffeic acid ( ) have been identified in the leaves of p. tomentosa ( - ), bark ( and ) and wood ( ) (ota et al. ; si et al. c si et al. , b , and p-ethoxybenzaldehyde ( ) is present in flowers (yuan et al. ). the , -dihydroxy- -geranylchromone ( ) isolated from fruits shows only moderate cytotoxic activity against a suspension culture of nicotiana tabacum cv. bright yellow (by- ), which confirms that ring b of the flavonoid skeleton is important for this activity (Š mejkal et al. a) . a large group of essential oil substances found in the flowers of p. tomentosa have also been identified (oprea et al. ; ibrahim et al. ) (fig. ) . plants are still highly esteemed all over the world as rich sources of therapeutic agents. in this context, traditional chinese herbal medicines, such as paulownia continue to influence a modern healthcare. it has been estimated that approximately , plant species exist on earth, but little is known about the phytochemical or therapeutic qualities of most of them. thanks to the development of the spectral and other analytical methods used in modern phytochemistry, many of the principal physiologically active secondary metabolites have been identified and researched in detail. the accumulated knowledge of traditional medicine is therefore, playing an important role in enhancing the success of drug discovery in herbal medicine. approximately % of the currently known antimicrobial, cardiovascular, immunosuppressive, and anticancer drugs are of plant origin (pan et al. ) . as mentioned in this review, p. tomentosa is a rich source of multifarious secondary metabolites, mainly prenylated flavonoids. as of today compounds, including flavonoids, lignans, phenolic glycosides, quinones, terpenoids, glycerides, phenolic acids, and other miscellaneous compounds have been isolated from various extracts of this plant. of increasing interest are the isolation and identification of p. tomentosa prenylated flavonoids, as they have shown promising pharmacological effects. in the first experiments, their antioxidant, antibacterial, antiphlogistic, cytotoxic, and sars-cov pl activities have been discovered along with inhibitory effects on human acetylcholinesterase, butyrylcholinesterase, and bacterial neuraminidases. more than compounds with modified prenyl or geranyl side-chains attached at c- of the flavonoid skeleton have been isolated from the flowers, fruit, and leaves of p. tomentosa. only two of these compounds have shown the presence of a five-carbon side-chain; for the others a ten-carbon side-chain is typical. further, only a few compounds have shown a geranyl moiety modified by hydroxylation at c- or c- . some compounds have a geranyl group modified by formating a heterocyclic moiety, which is also unusual. most of them have never been isolated from any other plant species. however, further in vivo pharmacology studies are needed to precisely elucidate biological mechanism of action, efficacy, and toxicity of these promising therapeutic agents. elucidation of the structure-activity relationships is also crucial for their further total syntheses and introduction into medical practice. for these reasons, the study of p. tomentosa as a source of biologically active metabolites is significant and future interest in this plant is ensured. isolation and characterization of paulownioside, a new highly oxygenated iridoid glucoside from paulownia tomentosa acteoside: a new antihypertensive drug in vitro immunomodulatory activity of verbascoside from nepeta ucrainica l antiproliferative activity and synthesis of -prenylnaringenin derivatives by demethylation of -o-and -o-substituted isoxanthohumols effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor and dexamethasone geranylated flavanones from the secretion on the surface of the immature fruits of paulownia tomentosa acylglycerols (=gly-cerides) from the glandular trichome exudate on the leaves of paulownia tomentosa chromatografické stanovení naftochinonů v rostlinách (chromatographic evaluation of naphtochinones in plants) active components from siberian ginseng (eleutherococcus senticosus) for protection of amyloid b( - )-induced neuritic atrophy in cultured rat cortical neurons catechin prodrugs and analogs: a new array of chemical entities with improved pharmacological and pharmacokinetic properties in vitro antitrypanosomal and antileishmanial activity of plants used in benin in traditional medicine and bio-guided fractionation of the most active extract antifungal synergy of theaflavin and epicatechin combinations against candida albicans prenylated flavonoids: pharmacology and biotechnology antiproliferative effect of catechin in grx cells naringenin and b-estradiol coadministration prevents hormone-induced human cancer cell growth the naringenininduced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background corosolic acid triggers mitochondria and caspase-dependent apoptotic cell death in osteosarcoma mg- cells stilbenes and anthocyanins reduce stress signaling in bv- mouse microglia investigation of two flacourtiaceae plants: bennettiodendron leprosipes and flacourtia ramontchi cardiovascular protective flavonolignans and flavonoids from calamus quiquesetinervius sesamin inhibits lysophosphatidylcholine acyltransferase in mortierella alpine a review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention structure-activity relationship of natural flavonoids in hydroxyl radicalscavenging effects propolin c from propolis induces apoptosis through activating caspases, bid and cytochrome c release in human melanoma cells determination of flavonoids in the flowers of paulownia tomentosa by high-performance liquid chromatography chemical modification and anticancer effect of prenylated flavanones from taiwanese propolis radio-protective effect of catalpol in cultured cells and mice a systematic review on biological activities of prenylated flavonoids cholinesterase inhibitory effects of geranylated flavonoids from paulownia tomentosa fruits geranylated flavonoids displaying sars-cov papain-like protease inhibition from the fruits of paulownia tomentosa anti-inflammatory and antinociceptive effects of sinapyl alcohol and its glucoside syringin angiogenic activity of sesamin through the activation of multiple signal pathways eight flavonoids and their potential as inhibitors of human cytomegalovirus replication antimicrobial activity of flavonoids tomentoside and -hydroxytomentoside, two new iridoid glucosides from paulownia tomentosa antimicrobial activity of brazilian propolis extracts against rumen bacteria in vitro antiinflammatory activity and chemical composition of extracts of verbena officinalis studies on the vascular effects of the fractions and phenolic compounds isolated from viscum album ssp. album in vitro antispasmodic activity of peracetylated penstemoniside, aucubin and catalpol lignan and phenylpropanoid glycosides from phillyrea latifolia and their in vitro anti-inflammatory activity phenolic compounds from artemisia iwayomogi and their effects on osteoblastic mc t -e cells chemistry and pharmacology of oxyprenylated secondary plant metabolites ontogeny of the flowers in paulownia tomentosa-a contribution to the recognition of the resurrected monogeneric family paulowniaceae pomolic acid of licania pittieri elicits endotheliumdependent relaxation in rat aortic rings dietary flavonoids: role of (-)-epicatechin and related procyanidins in cell signaling halohydrins of antirrhinoside-the correct structures of muralioside and epimuralioside naturally occurring phenylethanoid glycosides: potential leads for new therapeutics dietary flavonol epicatechin prevents the onset of type diabetes in nonobese diabetic mice effect of different phenolic compounds on a-amylase activity: screening by microplatereader based kinetic assay pharmacological activities of phenylpropanoids glycosides neuroprotective role of nanoencapsulated quercetin in combating ischemiareperfusion induced neuronal damage in young and aged rats hepatoprotective effects of syringin on fulminant hepatic failure induced by d-galactosamine and lipopolysaccharide in mice a study of the trypanocidal and analgesic properties [of substances] from lychnophora granmongolense (duarte) semir & leitao filho maslinic acid, a natural inhibitor of glycogen phosphorylase, reduces cerebral ischemic injury in hyperglycaemic rats by glt- up-regulation studies on b-sitosterol and ceramideinduced alterations in the properties of cholesterol/sphingomyelin/ganglioside monolayers artoindonesianins a and b, two new prenylated flavones from the root of artocarpus champeden artoindonesianin p, a new prenylated flavone with cytotoxic activity from artocarpus lanceifolius prenylated flavonoids and related compounds of the indonesian artocarpus (moraceae) the biochemistry and medical significance of the flavonoids advanced research on acteoside for chemistry and bioactivities potential anti-inflammatory, anti-adhesive, anti/estrogenic, and angiotensin-converting enzyme inhibitory activities of anthocyanins and their gut metabolites luteolin, a flavonoid, inhibits ap- activation by basophils changes in the level of bioactive compounds in paulownia tomentosa fruits corosolic acid impairs tumor development and lung metastasis by inhibiting the immunosuppressive activity of myeloid-derived suppressor cells effect of diplacone on lps-induced inflammatory gene expression in macrophages prenylated and geranylated flavonoids increase production of reactive oxygen species in mouse macrophages but inhibit the inflammatory response catalpol decreases peroxynitrite formation and consequently exerts cardioprotective effects against ischemia/reperfusion insult eleutheroside b or e enhances learning and memory in experimentally aged rats anti-oxidant activity and attenuation of bladder hyperactivity by the flavonoid compound kaempferol chemical composition, antimicrobial activity of the essential oil of the flowers of paulownia tomentosa (thunb.) steud. growing in egypt antiplatelet aggregatory effects of the constituents isolated from the flower of carthamus tinctorius piperitol from paulownia tomentosa antiinflammatory role of naringenin in rats with ethanol induced liver injury determination of flavonoids from paulownia tomentosa (thunb) steud. by micellar electrokinetic capillary electrophoresis phenylethanoid glycosides in plants: structure and biological activity antioxidant and pancreasprotective effect of aucubin on rats with streptozotocininduced diabetes trypanocidal activity of chemical constituents from lychnophora salicifolia mart inhibitory effect and mechanism on antiproliferation of isoatriplicolide tiglate (pcac) from paulownia coreana matteuorienate a and b, two new and potent aldose reductase inhibitors from matteuccia orientalis (hook.) trev antibacterial phenylpropanoid glycosides from paulownia tomentosa steud an antiviral furanoquinone from paulownia tomentosa steud angiotensin converting enzyme inhibitory phenylpropanoid glycosides from clerodendron trichotomum antifungal activity of constituents from the heartwood of gmelina arborea: part . sensitive antifungal assay against basidiomycetes divergent antiviral effects of bioflavonoids on the hepatitis c virus life cycle a comprehensive review on flavanones, the major citrus polyphenols sesamin, attenuates behavioural, biochemical and histological alterations induced by reversible middle cerebral artery occlusion in the rats hiv- integrase inhibitory phenylpropanoid glycosides from clerodendron trichotomum peroxyl radical scavenging capacity of extracts and isolated components from selected medicinal plants neuroprotective effects of a sesquiterpene lactone and flavanones from paulownia tomentosa steud against glutamate-induced neurotoxicity in primary cultured rat cortical cells inhibition of aldose reductase by phenylethanoid glycoside isolated from the seeds of paulownia coreana anti-herbivore structures of paulownia tomentosa: morphology, distribution, chemical constituents and changes during shoot and leaf development the dietary flavonoids naringenin and quercetin acutely impair glucose metabolism in rodents possibly via inhibition of hypothalamic insulin signalling geranylated flavanone tomentodiplacone b inhibits proliferation of human monocytic leukaemia (thp- ) cells xanthine oxidase inhibitors from brandisia hancei in vitro neuroprotective activities of phenylethanoid glycosides from callicarpa dichotoma plumbagin: a study of its anticancer, antibacterial and antifungal properties phenolic glycosides from dodecadenia grandiflora and their glucose- -phosphatase inhibitory activity antioxidant prenylated flavonoids from propolis collected in okinawa phenylpropanoids from medicinal plants: distribution, classification, structural analysis, and biological activity antidepressant activity of some phytopharmaceuticals and phenylpropanoids flavonoids from tanacetum vulgare flowers structural basis for the promiscuous biosynthetic prenylation of aromatic natural products immunoregulatory activity of daucosterol, a beta-sitosterol glycoside, induces protective th immune response against disseminated candidiasis in mice two new triterpenes from the rhizome of dryopteris crassirhizoma, and inhibitory activities of its constituents on human immunodeficiency virus- protease anti-inflammatory effect and hplc analysis of extract from edible cirsium setidens anthocyanin compositions and biological activities from the red petals of korean edible rose (rosa hybrid cv. noblered) structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors neuroprotection of catalpol in transient global ischemia in gerbils -dimethoxy hesperetin induces apoptosis of fibroblast-like synoviocytes in rats with adjuvant arthritis through caspase activation phenolic compounds of abies nephrolepis and their no production inhibitory activities therapeutic effect of , -dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting jak -stat signal pathway -dimethoxy hesperetin inhibits inflammation by inducing synovial apoptosis in rats with adjuvant-induced arthritis the inhibitory effect of phenylpropanoid glycosides and iridoid glucosides on free radical production and b -integrin expression in human leucocytes anti-inflammatory effect of the , , -trihydroxy- -geranylflavanone isolated from the fruit of artocarpus communis in s b-induced human monocytes release of acetylcholine by syringin, an active principle of eleutherococcus senticosus, to raise insulin secretion in wistar rats specific dietary polyphenols attenuate atherosclerosis in apolipoprotein e-knockout mice by alleviating inflammation and endothelial dysfunction distribution and biological activities of the flavonoid luteolin cytotoxic activity of flavonoids and extracts from retama sphaerocarpa boissier apigenin modulates gabaergic and glutamatergic transmission in cultured cortical neurons inhibitory effects of luteolin on human gastric carcinoma xenografts in nude mice and its mechanism catechinincorporated dental copolymers inhibit growth of streptococcus mutans biological activity of five antibacterial flavonoids from erythrophyllum (combretaceae) dietary flavonoids: molecular mechanisms of action as anti-inflammatory agents structural requirements of flavonoids for inhibition of antigeninduced degranulation, tnf-a and il- production from rbl- h cells studies on physiologically active substances in citrus fruit peel. part xx. structure and physiological activity of phenyl propanoid glycosides in lemon (citrus limon burm. f.) peel structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action the endocrine activities of -prenylnaringenin and related hop (humulus lupulus l.) flavonoids the effects of apigenin- -o-b-d-glucuronopyranoside on reflux oesophagitis and gastritis in rats quercetin modulates na ? /k ? atpase and sodium hydrogen exchanger in type diabetic erythrocytes antimutagenic activity of flavonoids from pogostemon cablin multi-targeted activity of maslinic acid as an antimalarial natural compound anticancer compound of paulownia tomentosa cytotoxic flavanones of schizolaena hystrix from the madagascar rainforest cytotoxic compounds of schizolaena hystrix from the madagascar rainforest sesamin metabolites induce an endothelial nitric oxide-dependent vasorelaxation through their antioxidative property-independent mechanisms: possible involvement of the metabolites in the antihypertensive effect of sesamin insecticidal activity of chemical constituents from aristolochia pubescens against anticarsia gemmatalis larvae minor cgeranylated flavanones from paulownia tomentosa fruits with mrsa antibacterial activity increase of b-endorphin secretion by syringin, an active principle of eleutherococcus senticosus, to produce antihyperglycemic action in type -like diabetic rats role of sympathetic tone in the loss of syringin-induced plasma glucose lowering action in conscious wistar rats hypoglycemic effect of syringin from eleutherococcus senticosus in streptozotocin-induced diabetic rats antileishmanial phytochemical phenolics: molecular docking to potential protein targets antimicrobial flavonoids and diterpenoids from dodonaea angustifolia the analysis of the volatile and semi-volatile compounds of the paulownia tomentosa flowers by gas chromatography coupled with mass spectrometry the chemistry of color changes in kiri wood (paulownia tomentosa steud.) iii. a new caffeic acid sugar ester from kiri wood pharmacological activities and mechanisms of natural phenylpropanoid glycosides an update on lignans: natural products and synthesis bioactive constituents from chinese natural medicines. xxxvi. four new acylated phenylethanoid oligoglycosides, kankanosides j , j , k , and k , from stems of cistanche tubulosa new perspectives on how to discover drugs from herbal medicines: cam's outstanding contribution to modern therapeutics effect of epicatechin and naloxone on cardioprotective phenotype stimulating effect of adaptogens: an overview with particular reference to their efficacy following single dose administration pharmacological activity of phenylpropanoids of the mistletoe, viscum album, host. pyrus caucasica acteoside and martynoside exhibit estrogenic/antiestrogenic properties aucubin, a naturally occurring iridoid glycoside inhibits tnf-a-induced inflammatory responses through suppression of nf-jb activation in t -l adipocytes a furanquinone from paulownia tomentosa stem for a new cathepsin k inhibitor apigenin and cancer chemoprevention: progress, potential and promise (review) xanthohumol and related prenylated flavonoids inhibit inflammatory cytokine production in lps-activated thp- monocytes: structure-activity relationships and in silico binding to myeloid differentiation protein- (md- ) acute and chronic antiinflammatory effects of plant flavonoids effect of vaccinium myrtillus and its polyphenols on angiotensin-converting enzyme activity in human endothelial cells constituents of the leaves of macaranga tanarius interventional effects of plumbagin on experimental ulcerative colitis in mice substituent effects on in vitro antioxidizing properties, stability, and solubility in flavonoids the heterosides of catalpa bignonioides walt. (bignoniaceae) protective role of apigenin on the status of lipid peroxidation and antioxidant defense against hepatocarcinogenesis in wistar albino rats chemoprotective effect of plant phenolics against anthracyclineinduced toxicity on rat cardiomyocytes. part iii. apigenin, baicalelin, kaempferol, luteolin and quercetin antimethicillinresistant staphylococcus aureus (mrsa) activity of ''pacific propolis'' and isolated prenylflavanones insulin-mimetic action of rhoifolin and cosmosiin isolated from citrus grandis (l.) osbeck leaves: enhanced adiponectin secretion and insulin receptor phosphorylation in t -l cells design of potent inhibitors of acetylcholinesterase using morin as the starting compound the natural triterpene maslinic acid induces apoptosis in ht colon cancer cells by a jnk-p -dependent mechanism effects of several flavonoids on the growth of b f and sk-mel- melanoma cell lines: relationship between structure and activity novel insights into the pharmacology of flavonoids dietary flavonoids: bioavailability, metabolic effects, and safety cytotoxic geranylflavonoids from bonannia graeca the flavonoid quercetin in disease prevention and therapy: facts and fancies antiparasitic activity of c-geranyl flavonoids from mimulus bigelovii epicatechin gallate impairs colon cancer cell metabolic productivity angicoagulant activity of plumbagin site-specific inhibitory mechanism for amyloid b aggregation by catecholtype flavonoids targeting the lys residues quercetin potentiates the effect of adriamycin in a multidrug-resistant mcf- human breast-cancer cell line: p-glycoprotein as a possible target verbascoside and isoverbascoside from paulownia tomentosa steud anti-inflammatory and apoptotic activities of pomolic acid isolated from cecropia pachystachya tomentomimulol and mimulone b: two new c-geranylated flavonoids from paulownia tomentosa fruits anthocyanins of the bignoniaceae proliferation-inhibiting and apoptosis-inducing effects of ursolic acid and oleanolic acid on multi-drug resistance cancer cells in vitro immunomodulatory active compounds from tinospora cordifolia a new phenolic constituent and a cyanogenic glycoside from balanophora involucrata (balanophoraceae) metabolism-based synthesis, biologic evaluation and sars analysis of o-methylated analogs of quercetin as thrombin inhibitors plasma glucose lowering mechanisms of catalpol, an active principle from roots of rehmannia glutinosa, in streptozotocin-induced diabetic rats role of apigenin in human health and disease. in: preedy vr (ed) beer in health and disease prevention structure and activity relationship of antioxidant flavonoids from leaves of paulownia tomentosa var. tomentosa. in: nd international papermaking and environment conference studies on the phenylethanoid glycosides with anti-complement activity from paulownia tomentosa var. tomentosa wood characterization of phenolic acids and antioxidant activities of paulownia tomentosa var. tomentosa leaves structure elucidation of phenylethanoid glycosides from paulownia tomentosa steud. var. tomentosa wood apigenin derivates from paulownia tomentosa steud. var. tomentosa stem barks evaluation of total phenolics, flavonoids and anti-inflammatory property of ethanolic extracts of paulownia tomentosa var. tomentosa bark phenolic extractives with chemotaxonomic significance from the bark of paulownia tomentosa var antioxidant properties and neuroprotective effects of isocampneoside ii on hydrogen peroxide-induced oxidative injury in pc cells plant-based corosolic acid: future anti-diabetic drug? cytotoxic potential of c-prenylated flavonoids c-geranyl compounds from paulownia tomentosa fruits antiradical activity of paulownia tomentosa (scrophulariaceae) extracts cytotoxic activity of c-geranyl compounds from paulownia tomentosa fruits antibacterial cgeranylflavonoids from paulownia tomentosa (scrophulariaceae) fruits cytotoxic activities of several geranyl-substituted flavanones phenolic glycosides of paulownia tomentosa bark inhibition of nitric oxide production by compounds from boesenbergia longiflora using lipopolysaccharide-stimulated raw . macrophage cells antimalarial activity of plumbagin in vitro and in animal models identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay effect of flavonoids on aerobic glycolysis and growth of tumor cells studies on constituents of medicinal plants. vii. the stereochemistry of paulownin and isopaulownin antioxidant effect of flavonoids on dcf production in hl- cells maqui berry (aristotelia chilensis) and the constituent delphinidin glycoside inhibit photoreceptor cell death induced by visible light apigenin- -o-b-dglucopyranoside, an anti-hiv principle from kummerowia striata alterations in skeletal muscle indicators of mitochondrial structure and biogenesis in patients with type diabetes and heart failure: effects of epicatechin rich cocoa catechin attenuates behavioral neurotoxicity induced by -ohda in rats -o-methylkaempferol and -quercetin glycosides from the whole plant of nervilia fordii inhibitory effects of eleutherococcus senticosus extracts on amyloid b( - )-induced neuritic atrophy and synaptic loss synthesis of dihydroxyphenacyl glycosides for biological and medicinal study: b-oxoacteoside from paulownia tomentosa indonesian propolis: chemical composition, biological activity and botanical origin ursolic acid protects diabetic mice against monocyte dysfunction and accelerated atherosclerosis anthocyanins in cornus alternifolia, cornus controversa, cornus kousa and cornus florida fruits with healt benefits pomolic acid-induced apoptosis in cells from patients with chronic myeloid leukemia exhibiting different drug resistance profile protective effect of naringenin against lead-inuced oxidative stress in rats anti-inflammatory compounds of ''qin-jiao'', the roots of gentiana dahurica (gentianaceae) catechin stimulates osteogenesis by enhancing pp a activity in human mesenchymal stem cells dihydroquercetin: more than just an impurity? a high-content drug screen identifies ursolic acid as an inhibitor of amyloid-b interactions with its receptor cd surface flavonoids in catalpa ovata, greyia sutherlandii and paulownia tomentosa chemical constituents and antibacterial activity of melastoma malabathricum l spectroscopic study on interaction between cistanoside f and bovine serum albumin antioxidative effects of phenylethanoids from cistanche deserticola hepatoprotective activity of phenylethanoids from cistanche deserticola neuroprotective properties of aucubin in diabetic rats and diabetic encephalopathy rats hesperetin- , -odimethylether selectively inhibits phosphodiesterase and effectively suppresses ovalbumin-induced airway hyperresponsiveness with a high therapeutic ratio cytotoxic phenolic glycosides from boschniakia himalaica how is sesamin metabolised in the human liver to show its biological effects? prenylation of aromatic compounds, a key diversification of plant secondary metabolites antiproliferative prenylated stilbenes and flavonoids from macaranga alnifolia from the madagascar rainforest effects of luteolin on spatial memory, cell proliferation, and neuroblast differentiation in the hippocampal dentate gyrus in a scopolamine-induced amnesia model antifibrotic effects of quercetin in primary orbital fibroblasts and orbital fat tissue cultures of graves' orbitopathy phenylethanoid oligoglycosides and acylated oligosugars with vasorelaxant activity from cistanche tubulosa isolation and bioassay of herbicidal active ingredient from paulownia tomentosa lipid peroxidation inhibitory activity of some constituents isolated from the stem bark of eucalyptus globulus taxifolin glycoside blocks human ether-a-go-go related gene k(?) channels identification of plumbagin and sanquinarine as effective chemotherapeutic agents for treatment of schistosomiasis studies on the chemical constituents from the leave of paulownia tomentosa antitumor activities of extracts and compounds from the roots of daphne tangutica maxim new lignans and their biological activities glucosidase inhibitory constituents from toona sinensis isolation, modification and cytotoxic evaluation of flavonoids from rhododendron hainanense enzymatic extraction and antibacterial activity from eucommia ulmoides leaves paulownia in china: cultivation and utilization. asian network for biological science and international development research centre, chinese academy of forestry antiradical and cytoprotective activities of several c-geranyl-substituted flavanones from paulownia tomentosa fruit key: cord- -i bucuma authors: natarajan, pavithra; tomich, john m. title: understanding the influence of experimental factors on bio-interactions of nanoparticles: towards improving correlation between in vitro and in vivo studies date: - - journal: arch biochem biophys doi: . /j.abb. . sha: doc_id: cord_uid: i bucuma bionanotechnology has developed rapidly over the past two decades, owing to the extensive and versatile, functionalities and applicability of nanoparticles (nps). fifty-one nanomedicines have been approved by fda since , out of the many nps based formulations developed to date. the general conformation of nps consists of a core with ligands coating their surface, that stabilizes them and provides them with added functionalities. the physicochemical properties, especially the surface composition of nps influence their bio-interactions to a large extent. this review discusses recent studies that help understand the nano-bio interactions of iron oxide and gold nps with different surface compositions. we discuss the influence of the experimental factors on the outcome of the studies and, thus, the importance of standardization in the field of nanotechnology. recent studies suggest that with careful selection of experimental parameters, it is possible to improve the positive correlation between in vitro and in vivo studies. this provides a fundamental understanding of the nps which helps in assessing their potential toxic side effects and may aid in manipulating them further to improve their biocompatibility and biosafety. the term nanotechnology was coined by prof. norio taniguchi in and is defined as the science, engineering and technology conducted at the nanoscale i.e. to nm. the nanoscale materials generally referred to as nanoparticles (nps) are highly desirable because of their small size, optical properties, high surface area to volume ratio and their multifunctional nature. bionanotechnology comprises research at the interface of nanotechnology and biology that has established a niche in biomedical sciences. liposomes - , peptide-based [ ] [ ] [ ] and synthetic polymer-based [ ] [ ] [ ] , three-dimensional macromolecular assemblies and nanocages [ ] [ ] [ ] are examples of hollow/porous core nps. solid core nps may be composed of inorganic metals such as iron oxide, gold, silver, platinum, silicon, quantum dots, titanium dioxide, gadolinium, selenium, copper oxide, zinc oxide or metallic hybrids, or organic carbon nanoparticles. the surfaces of inorganic nps are generally modified with synthetic or naturally occurring polymers and/or monomers which may be of biological origin such as peptides, proteins, carbohydrates, lipids, dna, rna, pna, aptamers, hybrid bio-synthetic molecules and others. these relatively flexible capping ligands improve the stability, biocompatibility and functionalize the nps for various applications or for further modifications. fig. , depicts the various components and configurations of nanoparticle-bioconjugates. drugs that have poor pharmacodynamics can be delivered using nps that may overcome these shortcomings by improving their half-lives, stabilities and bioavailabilities. however, their use is not limited to drug delivery systems (dds). their other applications include use as optical imaging agents and analytical probes/biosensors, thus making them suitable theranostics agents. [ ] [ ] [ ] [ ] fifty-one nanomedicines have been approved since by fda for clinical use j o u r n a l p r e -p r o o f with ~ products in clinical trials as of . owing to their potential, nanomaterials are being utilized in the recent fight against sars-cov- . , gold nanoparticles based immunoassays have been developed that enable rapid detection of sars-cov- infected asymptomatic patients or individuals showing mild symptoms. , an mrna vaccine which went into phase clinical trial in march , codes for the prefusion stabilized spike protein of sars-cov- and it is encapsulated in lipid nanoparticles which serve as effective delivery agents . the focus of this review is on gold and iron oxide nps which are the top inorganic nps in clinical trials (fig. d) . iron oxide nps are the only metal-containing nps that have received approval to date for clinical use and most of them are mri contrast agents. gold nanoparticles (aunps) exhibit plasmon resonance which can be followed using uv-vis spectrophotometric detection assays , , surface-enhanced raman spectroscopy (sers) and confocal/ luminescence microscopy. , the magnetic iron oxide nanoparticles (feonps), also commonly called superparamagnetic iron oxide nanoparticles (spions) are used as contrast agents for magnetic resonance imaging (mri) , , for bio-detection such as tracking the implanted stem cells in vitro , in binding assays and hyperthermia , and magnetic field guided drug delivery in cancer treatment. besides, the electron dense gold and iron nps are used widely in electron microscopy analyses. au-magnetite composites used in (sers) analyses improve the intracellular signal intensity essential to studying interactions of nps with biomolecules. delivery systems must be non-toxic by themselves, should not be cleared quickly from the body and trigger adverse immunological responses. it, therefore, becomes vital to understand j o u r n a l p r e -p r o o f their interactions at a molecular level, to determine how suitable they are for delivery and determine the applications for which they are best suited. the review is divided into four sections which discuss the (i) synthesis and functionalizing of nps, (ii) the discrepancies observed between the effects of nps in vitro and in vivo, followed by a detailed review of (iii) in vitro and (iv) in vivo studies of gold and iron oxide nps, which demonstrate the need to carefully consider experimental factors to improve the correlation between in vitro and in vivo studies. this review also presents recent in vitro and in vivo studies that assess the biosafety/toxicity of nps and the influence of surface ligands on nano-bio interactions such as uptake and immune response. we will emphasize the importance of standardization in nanotechnology with a focus on the experimental parameters since they have a significant impact on the outcome of studies. standardization is essential to make valid comparisons between studies and to prevent redundancy in research which help develop the field of nanomedicine. , j o u r n a l p r e -p r o o f the basic principle of nps syntheses is to promote nucleation of the monomeric element (e.g. lipids for liposomes and metal ions for inorganic metal nps), facilitating their assembly in a controlled manner to form stable and well-structured entities with narrow size distributions. multiple routes and techniques used in np syntheses have been established that are broadly categorized as chemical, physical and biological. most chemical and biological methods use facile synthesis techniques that are easily controlled and reproducible, yet low in cost and scalable. functionalization of nps has proved essential as they affect stability in the presence of salts and prevent aggregation over time, thereby increasing their shelf-life. they may also have other purposes including-promoting cellular uptake, co-functionalization to promote the delivery of drugs and nucleic acids, use in biochemical assays serving as binding partners, and provide additional functionalities to the delivery system. there are a wide range of biocompatible molecules used to functionalize the nps for use in nanomedicine which have been divided into major categories in this review. ( ligand exchange by direct substitution of surface ligands is one of the commonly used method for functionalizing nps. aunps form gold-thiol bonds facilitating exchange of smaller ligands such as citrate molecules with larger molecules by direct binding of ligands to nps via au-s bond formation (fig c.) . biodegradable polymers are widely used as surface coatings, as they are easy to synthesize, widely studied, allow for precise chemical binding of molecules or can be modified with functional groups to bind other molecules using facile chemistries like edc-nhs and disulfide conjugations . they have been recognized to increase the circulation time of the nanoparticles by preventing opsonization by phagocytes in vivo. therefore, a wide range of fda-approved nanoparticles and in vivo devices are coated with one or more of the abovementioned polymers. a recent report also suggests that peg-like polymers may not be as inert as currently believed. their oxidative degradation in vivo can lead to detrimental effects on the cell membrane and affect signal transduction pathways. therefore, recent emphasis has been on j o u r n a l p r e -p r o o f the use of natural or synthetic biocompatible surface coatings which display minimal adverse effects. lipid amphiphiles comprised of one acyl chain generally form micelles while those with two acyl chains assemble into bilayer-like membrane vesicles called liposomes. commonly, lipid formulations yield self-assembled structures that are greater than nm. the first liposomal formulation to be approved by the fda was doxil in , subsequently additional liposomal formulations with active ingredients (ais) have been approved. single chained lipid amphiphiles such as lysophosphatidylcholine and two-chained dopc, popc , as well as cholesterol and/or their mixtures have been incorporated into liposomes , polymeric liposomes (polymer modified lipid components) and to functionalize inorganic core nps. a reverse phase evaporation method that involves exchanging the existing surface ligands with lipids in an organic solvent followed by transfer to an aqueous solvent, is commonly used for lipid membrane assembly on nps. this technique has been employed in the synthesis of hybrid lipid bilayer coatings on nps where inner and outer layer have different compositions. , another common technique involves adsorption of liposomes , on the nps where the charged head moieties interact with the surface and encapsulate the nps within liposomes. however, lipid membranes often have low stability in solution due to fusion, leading to increases in the particle size. this can be remedied by increasing the surface charges that promote repulsion between particles or by incorporating spacers such as peg that sterically hinder particle association. these methods improve colloidal stability. utilized to functionalize nps with peptides (fig c.) . although amines bind to the gold surfaces, the strength of au-n (~ kj/mol) bond is much weaker than the au-thiol bond ( kj/mol) that is commonly used to bind cysteine containing peptides under appropriate conditions. , , cell penetrating peptides like hiv- derived tat peptide promote the uptake of molecules or complexes that cannot penetrate the cell membrane efficiently by themselves. they are therefore used to co-functionalize the surface of nanoparticles and are widely explored for delivery of nanoparticles in radiation therapy specific proteins can be used to functionalize nps for targeted delivery or to serve as binding partners in assays. abraxane® is an fda-approved chemotherapeutic drug that consists of nanoparticle albumin bound (nab)-paclitaxel. albumin is an abundant serum protein used as surface coating for nps as it improves bioavailability, has low immunogenicity and good biocompatibility. , nab-paclitaxel and its variations comprise a major percentage of the protein based nanomedicines in clinical trials (fig. d) . this success has fostered the use of albumin as a surface coating for additional nps delivery systems. [ ] [ ] [ ] antibodies/immunoglobulins are widely used due to their high specificity in detecting and binding to specific antigens and have been successfully employed for disease treatments as antibody drug conjugates (adcs), four of which are commercially available. since protein structure defines function, any structural alterations due to temperature transitions or ph, limit the chemistries available for attachment to nps. general strategies for binding antibodies and proteins to inorganic surfaces therefore include covalent binding to a modified surface , , or by physical adsorption promoted by electrostatic interactions. the orientation of the antibody is more important for its functioning than its coverage on the surface and hence orienting covalent binding strategies are more widely employed. j o u r n a l p r e -p r o o f finetti et al. used "click" chemistry to immobilize anti cd- and anti-rabbit-igg on the surface of aunps. thus, using the benefits of click chemistry, antibodies immobilized nps can be produced for a wide range of applications. antibody immobilized aunps are also widely used in immunostaining for analysis using electron microscopy, and plasmon resonance mediated confocal imaging. , , antibodies tagged with fluorescence molecule on aunps allows for dual imaging, reducing cost and time. nps are commonly coated with nucleic acids such as dna, dsrna, ssrna, sirna, mrna, and microrna, as they facilitate the delivery of the nucleic acids into cells or for use in binding assays. dna grafted polymers such as poly(acrylic acid) embedded dna are also used for functionalizing nanoparticles as they facilitate polyvalent dna nanostructure formation. a common strategy for functionalizing nps with nucleic acids is to utilize the electrostatic interactions between the negatively charged nucleic acids and cationic nps which mediates their adsorption to nps. , this does not require extensive modification of the nucleic acids. recently nucleic acids have also been identified as templates that control and facilitate inorganic nps synthesis. aptamers that bind with high affinity and specificity to proteins and peptides are commonly conjugated to aunps and feonps for detection of molecules using colorimetric binding assays , and magnetic isolation , respectively. j o u r n a l p r e -p r o o f in vitro studies are often indicators of potential outcomes in animal studies and provide mechanistic information at the cellular level. they allow researchers to explore the effect of different doses, chemicals at relatively lower cost and reduced time. they also allow for probing popc, -palmitoyl- -oleoyl-sn-glycero- -phosphocholine; popg, -palmitoyl- -oleoyl-sn-glycero- -phosphoglycerol; dope, dioleoylphosphatidylethanolamine; oqlcs, octadecyl-quaternized lysine modified chitosan; tat, transactivator of transcription; cpp, cell penetrating peptide; scfvegfr, short chain variable, anti-efgr the underlying mechanisms leading to toxicity, immunogenicity, metabolic changes and analyzing gene expression profiles. these cell culture studies reduce the number and cost of animals required to statistically assess the effect of nps. nps on the other hand encounter a very complex environment in vivo which cannot be mimicked accurately in vitro. and therefore, there are obvious discrepancies due to these inherent differences between in vitro and in vivo environments. khlebtsov et al. have examined the lack of correlation between in vitro and in vivo behavior of nps. they emphasize on the need for systematization of data obtained from various studies on nps, to gain a fundamental understanding of factors affecting their biointeractions. the inconsistencies observed between their effects in vitro and in vivo is also due to differences in experimental factors. [ ] [ ] [ ] [ ] [ ] for example, one basic consideration is to use the cell lines/ primary cell types for in vitro studies that belong to the same species that is being investigated in vivo. surprisingly, this is overlooked often. there are hundreds of reports on the toxicity of nps in vitro 'or' in vivo but very few recent studies have compared their effects in vitro 'and' in vivo. table summarizes the studies belonging to latter group. recent reviews by foroozandeh et al. and behzadi et al. discussed the effect of nanoparticle physicochemical properties such as size, shape, surface composition on their uptake and intracellular trafficking. unfortunately, few articles discuss the effect of experimental parameters on cellular uptake. in the following section the importance of carefully selecting cell lines, determining effect of dosage, time and media type in understanding np interactions will be j o u r n a l p r e -p r o o f discussed. we also review recent studies that explore cellular uptake routes, immune responses and toxicity induced by aunps and feonps with different surface compositions. a. influence of various experimental parameters cell lines used to study nps are commonly selected based on availability; they should be chosen based on the applications of nps and the expected in vivo exposure. several studies have shown that nanoparticle uptake and toxicity profiles vary between cell lines, cell sub-types and to some extent between species. [ ] [ ] [ ] [ ] [ ] the uptake of nps is also dependent on cell-specific functions. although immortalized cell lines are easier to maintain, readily accessible and widely studied, they differ from cells in vivo due to repeated in vitro manipulations and the initial immortalization itself. joris et al. facilitates co-culturing of multiple cell types simultaneously, to evaluate the effect of nps treatment on the crosstalk between the cell types or to study transcytosis. [ ] [ ] [ ] three-d cell cultures that make use of a scaffold increase the surface area of exposure, while only ~ % area is available in a d cell culture. the md -mb breast cancer cells in d culture, in comparison to their d counterpart, had increased viability and showed a lesser change in the cytoplasmic actin network that plays a major role in intracellular processes. thus, the toxicity of the nps could be underestimated by testing their effect in just d cell cultures and immortalized cell lines. in vivo, nps and drugs have a tendency to accumulate in the liver generally, which clears foreign materials and thus, the liver is an important tissue to consider for studying nps. the sandwich hepatocyte culture model uses primary hepatocytes, grown between two layers of collagen that keeps them competent and polarized with functional j o u r n a l p r e -p r o o f bile networks and helps to assess the hepatotoxicity of drugs and nps accurately. while -d cultures mimic the in vivo environment more closely not all labs have transitioned to this approach. traditional d cultures still predominate in the current literature. cell culture media composition varies depending upon the requirements of each cell line. observed a similar effect where aunps pre-exposed to protein poor medium had a higher tendency to aggregate than in protein rich medium. interestingly, nm aunps exerted more adverse effects on cells in rpmi in comparison to dmem. hence, while designing and implementing studies, we should consider the choice of cell culture media which is crucial. another non-trivial factor to be considered is the method by which nps are administered as documented by moore et al. when poly(vinylpyrrolidone) (pvp) coated aunps were administered as a concentrated bolus directly to j a. mouse macrophages, the protein corona formation was -fold higher than aunps pre-mixed with media. the macrophages also phagocytosed more aunps administered as a concentrated dose in comparison to the pre-mixed aunps. this study emphasizes how a minor detail such as the initial administration of nps can affect the outcome of the study. thus, to be able to compare studies between research groups, we should consider every minor detail and develop a robust analytical method. due to a lack of standardized/universal methods of testing nps, it is difficult to compare and obtain a better understanding of nps bio-interactions. the hence, more studies are needed to assess the effect of repeated exposure to nps at prescribed intervals. gokduman et al. cell viability tests are widely used to assess the toxicity of nps. this typically involves a single dose of nps followed by short-term evaluations of viability. whereas, in vivo studies focus on studying the systemic effects and accumulation of nps. therefore, there is an apparent disconnect between most in vitro and in vivo studies. reactive oxygen species produced by cells in response to nps is a potent early marker for nanoparticle toxicity. oxidative stress exerted by nps may be inevitable in some cases and can be ameliorated by the naturally occurring antioxidants or by supplementation with antioxidants such as thymoquinone to reduce these effects. feng et al. observed that cationic pei coated feonps were endocytosed in high numbers compared to peg-feonps and were more toxic to cells as they dramatically reduced cell viability in a concentration dependent manner. increased ros generation that disrupted the cell cycle by arresting cells in g -phase cell cycle, led to apoptosis. genotoxicity induced by the pei-feonps was observed to be an indirect effect and not due to direct interaction with the dna. in contrast nm 'naked' feonps intercalated with dna base pairs in primary lymphocytes and generated high levels of ros that reduced the cell viability. cells may use an active, energy dependent endocytic pathway or energy independent passive diffusion to internalize nps. table summarizes the uptake pathways used by nanoparticles with different surface chemistries in various cell types. endocytosis is broadly classified as -clathrin mediated endocytosis (cme), caveolae mediated endocytosis (cvme), macropinocytosis and clathrin and caveolae independent endocytosis. phagocytosis is a type of endocytic pathway which is only employed by immune cells such as macrophages, neutrophils and dendritic cells. cargo is transported intracellularly in endocytic vesicles formed by cell membrane invaginations. endocytic vesicles can be classified based on the protein markers on the vesicle membrane associated with the endocytic pathway, further influencing the cargo's intracellular sorting. (fig. ) cme and macropinocytosis promote the fusion of endocytic vesicles with the highly acidic lysosomes (~ ph ) that can cause degradation of the functionalizing ligands and nps themselves. while the cargo transported in the caveosomes, enter the golgi and endoplasmic reticulum, bypassing the lysosomes. cvme also favor transcytosis like in the case of nab-paclitaxel. [ ] [ ] [ ] [ ] some oncology and viral medications such as trastuzumab emtansine (t-dmi) and chloroquine , respectively, target the endocytosis pathways. hence, studying the mechanism of uptake is important for the fundamental understanding of nano-bio interactions and drug delivery. chemical inhibitors of endocytosis are commonly used to study the endocytic uptake pathways. some inhibitors may have a generalized inhibitory effect while some are relatively j o u r n a l p r e -p r o o f more specific. methyl-β-cyclodextrin although commonly used as an inhibitor of cvme, it can also inhibit cholesterol dependent clathrin and caveolin independent pathways. similarly, dynasore may inhibit dynamin independent endocytic pathways as well. therefore, the chemical inhibitors should be selected wisely and the results should be interpreted appropriately. sirna mediated knockdown of proteins, essential to specific endocytic routes on the other hand is less ambiguous than chemical inhibitors. in some cases other endocytic pathways may be upregulated to compensate for inhibition of one pathway. although the net uptake of nps may seemingly be unaffected, one should not discount changes in the uptake mechanism. , , endocytosis of nps is time dependent. he et al. observed that although the uptake of cationic calrrrrrrrr (r ) peptide functionalized aunps was slower in comparison to the hydrophobic calnnpfvyli (pfv) peptide coated aunps, in the initial one hour, their net uptake was higher at the end of h of incubation. iec- epithelial cells also seemed to use different endocytosis pathways to internalize peptide bilayer coated feonps in a time dependent manner. the surface composition plays a crucial role since they may also help in endosomal escape as observed for highly cationic nps. , different cell types may use different endocytic pathways for the uptake of the same nps and a single cell type may use multiple pathways for the uptake of nps. srijampa et al. identified that monocytes and macrophages generally studied for their phagocytic response may also use other endocytosis pathways alongside phagocytosis for nps uptake. b.end endothelial cells internalized more of the negatively charged feonps in comparison to epithelial cells, using cvme, which was enhanced in the endothelial cells since they overexpressed the caveolin- j o u r n a l p r e -p r o o f nps can elicit an immune response by interfering and interacting with intracellular signaling pathways directly or indirectly via the reactive oxygen and nitrogen species produced. for example, the transition metals on the surface of nps or in spions generate ros as described previously, which triggers a pro-inflammatory response. the nps bio-interactions in the complex in vivo environment are dependent on their physicochemical properties, contributing to their translocation to the different organs and tissues and ultimate clearance. , therefore, it is vital to discern the relationship between the nps and the interactions with endogenous molecules that influence their biodistribution. in this section, the route of administration has an obvious role to play on the tissue distribution which is generally chosen based on the end application of nps. however, i.v. injections are used more commonly since they can provide a near instantaneous response and is suitable for delivery of materials that cannot be absorbed efficiently or that can undergo proteolytic or ph disruption. another major advantage of i.v. injections is the increased bioavailability of drugs. the animal model selected for a particular study may influence the administration route. intramuscular delivery in mice is generally not recommended as their muscles are small, making it difficult to get reproducible results. the genetic background of animals will also show variations in nps interactions due to differences in their response to foreign molecules. the c bl/ and the balb/c mice, for example, fundamentally exhibit different immune responses that could affect j o u r n a l p r e -p r o o f their adaptive immunity. c bl/ and balb/c are prototypical, th and th type mouse strains, respectively, and therefore can have an altered response to nps. when nps are administered, they have to cross various hurdles before they reach the there has been an increase in studies exploring intradermal delivery using microneedles as it is minimally invasive. dur et al. in vitro studies can guide one in explaining the effects of nps in vivo. for instance, ros and rns generated in response to nps activates the cells and induces secretion of cytokines/chemokines. (fig. ) this leads further to the infiltration of immune cells, which may cause tissue necrosis or induce apoptosis of cells causing organ damage. thus, the immune response to the nps can lead to a cascade of events that induces toxicity. in vivo, toxicity is determined by assessing ultrastructural changes in the tissues (fig. ) , comparing cytokine levels and other molecular markers in serum and analyzing blood cell counts (hematology) injection. pei-feonps at and . mg/kg doses were highly toxic to mice leading to death, but a dose of . mg/kg was well tolerated. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f this may also lead to metabolic changes. reactive nitrate species (rns) produced mainly by immune cells such as macrophages and neutrophils along with the ros are considered to be indicators of cellular activation. altogether, the intracellular changes may cause cellular toxicity (iv) and cause an immune response by inducing changes in cytokine and chemokines secretion (v). nps can also be exocytosed in vesicles called exosomes which may be inherently targeted to different tissues. therefore, a cascade of events determines the bioavailability, clearance, toxicity profile and thus, the net effect of nps. analyzing nanomaterial bioconjugates: a review of current and emerging purification and characterization techniques functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology doxorubicin/gold nanoparticles coated with liposomes for chemo-photothermal synergetic antitumor therapy cell-membrane-mimicking lipid-coated nanoparticles confer raman enhancement to membrane proteins and reveal membrane-attached amyloid-beta conformation cationic lipid-coated gold nanoparticles as efficient and non-cytotoxic intracellular sirna delivery vehicles polymeric liposomes-coated superparamagnetic iron oxide nanoparticles as contrast agent for targeted magnetic resonance imaging of cancer cells biodegradable drug-delivery peptide nanocapsules self-assembly mechanism of a peptide-based drug delivery vehicle stimuli-responsive polypeptide vesicles by conformation-specific assembly synthesis of hollow polymer nanocapsules exploiting gold nanoparticles as sacrificial templates differentially charged hollow core/shell lipid-polymer-lipid hybrid nanoparticles for small interfering rna delivery bioreducible cationic polymer-based nanoparticles for efficient and environmentally triggered cytoplasmic sirna delivery to primary human brain cancer cells pretreated macrophage-membrane-coated gold nanocages for precise drug delivery for treatment of bacterial infections designed formation through a metal organic framework route of zno/znco o hollow core-shell nanocages with enhanced gas sensing properties mesoporous hollow cu-ni alloy nanocage from core-shell cu@ni nanocube for efficient hydrogen evolution reaction nano based drug delivery systems: recent developments and future prospects transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors enhanced accumulation of theranostic nanoparticles in brain tumor by external magnetic field mediated in situ clustering of magnetic nanoparticles erlotinib-conjugated iron oxide nanoparticles as a smart cancer-targeted theranostic probe for mri mri guided magnetochemotherapy with high-magnetic-moment iron oxide nanoparticles for cancer theranostics nanoparticle-based medicines: a review of fda-approved materials and clinical trials to date nano research for covid- rapid covid- test based on new biolabeling technology called plasmonic-fluor ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays identifier nct , safety and immunogenicity study of -ncov vaccine (mrna- ) for prophylaxis of sars-cov- infection analysis of complexes formed by small gold nanoparticles in low concentration in cell culture media tracing size and surface chemistry-dependent endosomal uptake of gold nanoparticles using surface-enhanced raman scattering quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy cellular uptake and nanoscale localization of gold nanoparticles in cancer using label-free confocal raman microscopy exceedingly small iron oxide nanoparticles as positive mri contrast agents differentiating between cancer and inflammation: a metabolic-based method for functional computed tomography imaging potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts go-functionalized large magnetic iron oxide nanoparticles with enhanced colloidal stability and hyperthermia performance water-dispersible sugar-coated iron oxide nanoparticles. an evaluation of their relaxometric and magnetic hyperthermia properties have nanoscience and nanotechnology delivered? progress towards standardized and validated characterizations for measuring physicochemical properties of manufactured nanomaterials relevant to nano health and safety risks synthesis, characterization, applications, and challenges of iron oxide nanoparticles size-controlled synthesis of sub- -nanometer citrate-stabilized gold nanoparticles and related optical properties digestive ripening, nanophase segregation and superlattice formation in gold nanocrystal colloids optimal design and characterization of superparamagnetic iron oxide nanoparticles coated with polyvinyl alcohol for targeted delivery and imaging synthesis and characterization of nanometer-size fe o and gamma-fe o particles quantitative measurement of ligand exchange with small-molecule ligands on iron oxide nanoparticles via radioanalytical techniques current trends in using polymer coated gold nanoparticles for cancer therapy facile and general method for synthesis of sugar coated gold nanoparticles hydroxide assisted synthesis of monodisperse and biocompatible gold nanoparticles with dextran a simple and efficient method for polymer coating of iron oxide nanoparticles colloidal stability of gold nanoparticles modified with thiol compounds: bioconjugation and application in cancer cell imaging synthesis and characterization of superparamagnetic iron oxide nanoparticles as calcium-responsive mri contrast agents smart nanoparticles for drug delivery application: development of versatile nanocarrier platforms in biotechnology and nanomedicine approved drug products with therapeutic equivalence evaluations understanding the nano-bio interfaces: lipid-coatings for inorganic nanoparticles as promising strategy for biomedical applications. frontiers in chemistry poly(ethylene glycol)-lipid conjugates regulate the calciuminduced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine silicon quantum dots metal-enhanced photoluminescence by gold nanoparticles in colloidal ensembles: effect of surface coating cell-penetrating peptide-modified gold nanoparticles for the delivery of doxorubicin to brain metastatic breast cancer synthesis and in vitro evaluation of fluorescent and magnetic nanoparticles functionalized with a cell penetrating peptide for cancer theranosis penetratin peptide-functionalized gold nanostars: enhanced bbb permeability and nir photothermal treatment of alzheimer's disease using ultralow irradiance biocompatible peptide-coated ultrasmall superparamagnetic iron oxide nanoparticles for in vivo contrast-enhanced magnetic resonance imaging a study of the cellular uptake of magnetic branched amphiphilic peptide capsules protein coated gold nanoparticles as template for the directed synthesis of highly fluorescent gold nanoclusters bovine serum albumin (bsa) coated iron oxide magnetic nanoparticles as biocompatible carriers for curcumin-anticancer drug increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine multiresponsive albumin-lonidamine conjugated hybridized gold nanoparticle as a combined photothermal-chemotherapy for synergistic tumor ablation acquired resistance to antibody-drug conjugates anti-her antibody and scfvegfr-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer impact of conjugation strategies for targeting of antibodies in gold nanoparticles for ultrasensitive detection of beta-estradiol click chemistry immobilization of antibodies on polymer coated gold nanoparticles a novel immuno-gold labeling protocol for nanobody-based detection of her in breast cancer cells using immuno-electron microscopy surface plasmon resonance scattering and absorption of anti-egfr antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer dna-grafted poly(acrylic acid) for one-step dna functionalization of iron oxide nanoparticles binding of single stranded nucleic acids to cationic ligand functionalized gold nanoparticles nanoparticles enable efficient delivery of therapeutic sirna targeting rest into glioblastoma cells superparamagnetic iron oxide nanoparticles functionalized by peptide nucleic acids nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles gold nanoparticle-aptamer-based lspr sensing of ochratoxin a at a widened detection range by double calibration curve method aptamer-modified gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors disposable aptamer-sensor aided by magnetic nanoparticle enrichment for detection of salivary cortisol variations in obstructive sleep apnea patients dose-, treatment-and time-dependent toxicity of superparamagnetic iron oxide nanoparticles on primary rat hepatocytes biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies gold nanoparticles: distribution, bioaccumulation and toxicity. in vitro and in vivo studies assessing cell-nanoparticle interactions by high content imaging of biocompatible iron oxide nanoparticles as potential contrast agents for magnetic resonance imaging natural tripeptide capped ph-sensitive gold nanoparticles for efficacious doxorubicin delivery both in vitro and in vivo effect of surface chemistry and associated protein corona on the long-term biodegradation of iron oxide nanoparticles in vivo a systematic comparison of in vitro cell uptake and in vivo biodistribution for three classes of gold nanoparticles with saturated peg coatings uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings cellular uptake of nanoparticles: journey inside the cell parametrizing the exposure of superparamagnetic iron oxide nanoparticles in cell cultures at different in vitro environments effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response functionalized gold nanoparticles as biosensors for monitoring cellular uptake and localization in normal and tumor prostatic cells selection of potential iron oxide nanoparticles for breast cancer treatment based on in vitro cytotoxicity and cellular uptake nanoparticle administration method in cell culture alters particle-cell interaction evaluating the effect of assay preparation on the uptake of gold nanoparticles by raw . cells iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues gold quantum dots impair the tumorigenic potential of glioma stem-like cells via beta-catenin downregulation in vitro it takes more than a coating to get nanoparticles through the intestinal barrier in vitro sandwich-cultured hepatocytes as a tool to study drug disposition and drug-induced liver injury is it time to reinvent basic cell culture medium? protein corona composition of superparamagnetic iron oxide nanoparticles with various physico-chemical properties and coatings effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (spions): colloidal stability, cytotoxicity, and cellular uptake studies how entanglement of different physicochemical properties complicates the prediction of in vitro and in vivo interactions of gold nanoparticles intracellular accumulation of gold nanoparticles leads to inhibition of macropinocytosis to reduce the endoplasmic reticulum stress evaluation of dna interaction, genotoxicity and oxidative stress induced by iron oxide nanoparticles both in vitro and in vivo: attenuation by thymoquinone cytotoxicity and cell cycle effects of bare and poly(vinyl alcohol)-coated iron oxide nanoparticles in mouse fibroblasts bare surface of gold nanoparticle induces inflammation through unfolding of plasma fibrinogen functionalized gold nanoparticles for topical delivery of methotrexate for the possible treatment of psoriasis endocytosis of nanomedicines physical principles of nanoparticle cellular endocytosis uptake mechanisms of non-viral gene delivery uptake mechanism of metabolic-targeted gold nanoparticles extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles dynasore -not just a dynamin inhibitor caveolin- and cdc mediated endocytosis of silica-coated iron oxide nanoparticles in hela cells increased cellular uptake of peptide-modified pegylated gold nanoparticles. biochemical and biophysical research communications mechanisms for cellular uptake of nanosized clinical mri contrast agents effects of gold nanoparticles with different surface charges on cellular internalization and cytokine responses in monocytes differential internalization of brick shaped iron oxide nanoparticles by endothelial cells interactions at the cell membrane and pathways of internalization of nano-sized materials for nanomedicine the gene transfection and endocytic uptake pathways mediated by pegylated pei-entrapped gold nanoparticles folic acid-capped pegylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis mechanisms of nanoparticle-induced oxidative stress and toxicity iron-mediated lipid peroxidation and lipid raft disruption in low-dose silica-induced macrophage cytokine production. free radical biology and medicine gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo gold nanoparticles as an adjuvant: influence of size, shape, and technique of combination with cpg on antibody production iron oxide nanoparticles-based vaccine delivery for cancer treatment physical characterization and in vivo organ distribution of coated iron oxide nanoparticles administration of substances to laboratory animals: routes of administration and factors to consider routes of administration innate immune response in th -and th -dominant mouse strains evaluating the potential of gold, silver, and silica nanoparticles to saturate mononuclear phagocytic system tissues under repeat dosing conditions effect of surface-modified superparamagnetic iron oxide nanoparticles (spions) on mast cell infiltration: an acute in vivo study effect of removing kupffer cells on nanoparticle tumor delivery biodistribution, clearance and morphological alterations of intravenously administered iron oxide nanoparticles in male wistar rats peg-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney mri contrast agents biodegradable gold nanoclusters with improved excretion due to ph-triggered hydrophobic-to-hydrophilic transition effect of gold nanoparticle size on their properties as contrast agents for computed tomography comparisons of the biodistribution and toxicological examinations after repeated intravenous administration of silver and gold nanoparticles in mice effects of core size and peg coating layer of iron oxide nanoparticles on the distribution and metabolism in mice intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy the toxicity and distribution of iron oxide-zinc oxide core-shell nanoparticles in c bl/ mice after repeated subcutaneous administration accumulation of biosynthesized gold nanoparticles and its impact on various organs of sprague dawley rats: a systematic study nanoparticle-based vaccines: opportunities and limitations the physiological sources of, clinical significance of, and laboratory-testing methods for determining enzyme levels. labmedicine size-and cell type-dependent cellular uptake, cytotoxicity and in vivo distribution of gold nanoparticles biodistribution, pharmacokinetics, and toxicity of dendrimer-coated iron oxide nanoparticles in balb/c mice this manuscript is contribution number - -j from the kansas agricultural experiment key: cord- -pmtecdko authors: hao, da cheng; gu, xiao-jie; xiao, pei gen title: phytochemical and biological research of polygoneae medicinal resources date: - - journal: medicinal plants doi: . /b - - - - . - sha: doc_id: cord_uid: pmtecdko this chapter synthesizes current knowledge on phytochemistry, bioactivity, molecular phylogeny, and omics of polygoneae medicinal resources. many species in the seven genera of polygoneae are traditional and popular medicinal herbs. anthraquinones and stilbenes are characteristic medicinal compounds of polygoneae, while other useful constituents, such as alkaloids, glycosides, flavonoids, volatile oils, tannins, resins, coumarins, and lignans, have also been isolated from polygoneae species. among versatile bioactivities of polygoneae compounds, their effects on the cardiovascular and nervous systems and their anti-inflammatory, antimicrobial, antiparasitic, insecticidal, anticancer, and antioxidant activities are highlighted. interspecific hybridization and the following polyploidization play a major role in polygoneae diversification. nuclear its and chloroplast dna sequences are retrieved to resolve the interspecific relationship and reassign some taxa to the appropriate genus. omics study of polygoneae is at the budding stage, which will revolutionize the sustainable utilization of polygoneae medicinal resources. no. r . % of the total organic ester compounds. -phenyl- -pentanone and acetophenone were . % and . % of the total volatiles, respectively. the liverwort (porella vernicosa) complex produces a very hot tasting polygodial, a drimane-type sesquiterpene dialdehyde. the same compound has been isolated from two ferns, thelypteris hispidula and blechnum fluviatile, as well as from the higher plants polygonum hydropiper and p. hydropiper f. purpurascens (polygonaceae); cinnamosma, caspicodendron, canella, and warburgia species (canellaceae); and pseudowintera colorata, tasmannia lanceolata, drimys, and zygogynum species (winteraceae) (asakawa et al., ) . in addition, the liverworts and higher plants that elaborate polygodial and its related pungent drimane dials contain a small amount of a-tocopherol, g-tocopherol, or d-tocotrienol. the drimane-type sesquiterpenoids and tocopherols might be useful chemotaxonomic markers in some lower and higher plants, including polygonaceae. -furanmethanol, ( h)-furanone, -hydroxy- -cyclopenten- -one, , -dihydroxy- , -dimethyl- ( h)-furan- -one, h-pyran- , ( h)-dione, -hydroxy-gammabutyrolactone, , -dimethyl- -hydroxy- ( h)-furanone, , -furandicarboxaldehyde, , -dihydro- , -dihydroxy- -methyl h-pyran- -one, (s)-(+)- , -dideoxyribonolactone, -(hydroxymethyl)-furancarboxaldehyde, -deoxy-d-mannoic lactone, etc. were found in the callus of p. minus (vikram et al., ) . tantry resveratrol ( , , -trihydroxy-trans-stilbene) is a natural polyphenolic compound that exists in p. cuspidatum, grapes, peanuts, and berries, as well as their manufactured products. resveratrol is a pharmacologically active compound that interacts with multiple targets in a variety of cardiovascular disease models to exert protective effects or induce a reduction in cardiovascular risk parameters . endothelial hyperpermeability induced by hyperglycemia is the initial step in the development of atherosclerosis, one of the most serious cardiovascular complications in diabetes. resveratrol ameliorates high-glucose-induced hyperpermeability mediated by caveolae via vegf/kdr pathway (tian et al., ) . polydatin, a resveratrol glucoside extracted from p. cuspidatum, attenuates cardiac hypertrophy through modulation of cardiac ca + handling and calcineurin-nfat signaling pathway (ding et al., ) . , , , -tetrahydroxystilbene- -o-b-d-glucoside (tsg), extracted from the root of p. multiflorum (he shou wu), had inhibitory effects on angiotensin ii-induced proliferation of vascular smooth muscle cells (xu et al., a) . its antiproliferative effect might be associated with the downregulation of intracellular reactive oxygen species (ros), followed by the suppression of the src-mek / -erk / signaling pathway, hence blocking cell cycle progression. tsg could prevent cardiac remodeling induced by pressure overload in rats (xu et al., b) , which may be related to a decreasing angiotensin ii level, an antioxidant effect, suppression of transforming growth factor-b expression, and inhibition of extracellular signal-regulated kinase / and p mitogen-activated protein kinase activation. proteomic analysis was used to investigate the molecular events occurring in the atherosclerotic rats after tsg treatment (yao et al., ) . tsg treatment suppresses atherosclerosis by altering the expression of different proteins. calreticulin, vimentin, hsp , lipocortin , and apo a-i are key proteins that may be novel molecular targets responsible for atherogenesis suppression induced by tsg treatment. dysregulated tonic tension and calcium sensitization in blood vessels have frequently been observed in many cardiovascular diseases. emodin of p. multiflorum inhibits tonic tension through suppressing pkcd-mediated inhibition of myosin phosphatase in rat isolated thoracic aorta (lim et al., ) . p. viviparum (pv) is a perennial herb and widely distributed in high-elevation mountain regions, such as the tibetan plateau. in tibetan traditional medicine, pv is usually used to boost the blood circulation to dissipate blood stasis. pv induces vasorelaxation in the rat thoracic aorta via activation of nitric oxide (no) synthase in endothelial cells (chang et al., ) . , , , -tetrahydroxystilbene- -o-b-d-glucoside (tsg) of p. multiflorum could raise the content of cyp (cytochrome p ) a and then promote the lipolysis of cholesterol (wang et al., a) . tsg also showed the best ldl-reducing effect. emodin could inhibit hmg-coa reductase and dgat , which were key enzymes in the synthesis of tc and tg. physcion increased the content of htgl and then could boost the lipolysis of triglyceride. physcion showed the best vldl-reducing effect. the lipid regulation activity might be due to an overall synergy of tsg, emodin, and physcion. tsg attenuates human platelet aggregation, secretion, and spreading in vitro (xiang et al., ) . antiatherosclerotic effects of p. aviculare l. ethanol extract in apoe knockout mice fed a western diet are mediated via the mapk pathway (haeng park et al., ) . extracts of p. persicaria inhibit g protein-activated inwardly rectifying k + channels (lajter et al., a) . the electrophysiologically active agents, not the new flavonoids from the chloroform extract, are among the minor compounds of hplc eluates. resveratroloside and catechin-( a ! )-catechin, the newly found constituents in the invasive variety, have similar no inhibition potency as that of piceid (the major constituent of p. cuspidatum) (fan et al., b) , but the newly found major constituent, that is, piceatannol glucoside, showed no apparent effect. the total content of resveratrol measured in the root extracts of the swiss sample was about . times less than that of the chinese one. when the invasive variety of p. cuspidatum is used in traditional medicine, the chemical difference should be kept in mind. p. tinctorium, a traditional medicine used in china and korea, improves , -dinitrofluorobenzene-induced atopic dermatitis-like lesional skin (han et al., ) . p. tinctorium inhibited il- and tslp production through the attenuation of caspase- activation in an animal model of allergic rhinitis (jeong et al., ) . flavonol glucuronides at physiologically achievable concentrations within the range of . - mm significantly inhibited the production of ros as well as elastase release in human neutrophils model and should be considered as responsible for the anti-inflammatory activity of the p. aviculare (granica et al., ) . p. chinense aqueous leaf extract has gastroprotective activity on ethanol-induced hemorrhagic mucosal lesions in rats (ismail et al., ) . p. viviparum inhibits the lipopolysaccharide-induced inflammatory response in raw . macrophages through heme oxygenase- induction and activation of the nrf pathway (cheng et al., ) . guizhou miao medicine p. capitatum has antibacterial activity and can be used in bladder infection (hu et al., ) . methanolic crude extract of aerial parts of p. maritimum showed a high antibacterial activity against gram-positive bacterial strains: bacillus cereus, bacillus subtilis, and staphylococcus aureus with a highest mic of mg/ml (el-haci et al., ) . p. orientale extracts had protective effect against clavibacter michiganense subsp. sepedonicum, the causal agent of bacterial ring rot of potato (cai et al., ) . ethanol extracts of p. cuspidatum, resveratrol, (+)-catechin, and emodin -o-b-dglucopyranoside inhibit hiv- -induced syncytium formation ). an ethyl acetate subfraction separated from p. cuspidatum root and its major component, emodin, inhibited epstein-barr virus lytic cycle (yiu et al., ) . in vitro and in vivo studies suggested the inhibitory effects of emodin isolated from p. cuspidatum on coxsackievirus b₄ . resveratrol, (e)- , , -trihydroxystilbene- -o-beta-d-glucopyranoside- -( , , -trihydroxybenzoate), and catechin- -ogallate, isolated from p. cuspidatum, had inhibitory effect against neuraminidase (na) activity , with ic values of . , . , and . mmol/ l, respectively. (e)- , , -trihydroxystilbene- -o-beta-d-glucopyranoside- -( , , -trihydroxybenzoate) and catechin- -o-gallate had significant inhibitory effect against h n influenza virus (ec ¼ . , . mmol/l, respectively), with very low cytotoxicity to the host cells; their therapeutic selective index (si) in mdck cells ranged from to . emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction (ho et al., ) . p. chinense had antidiarrheal activity (xiao et al., ) . ellagic acid and corilagin are two components contributing to this effect. cinnamoylphenethyl amides from p. hyrcanicum possess antitrypanosomal activity (moradi-afrapoli et al., b) . the essential oil of p. hydropiper exhibited lc values of . and . and confertifolin exhibited lc values of . and . against the second and fourth instar larvae of aedes albopictus (dengue vector mosquito), respectively (maheswaran and ignacimuthu, ) . the ovicidal activity of % on - h old eggs, repellent activity of . min, oviposition deterrent activity of . %, and adulticidal activity of % at ppm concentration of confertifolin were recorded. trans-piceid (t-pc) is abundant in p. cuspidatum and in grapes and grape products such as wine. piceid presents antiproliferative effects in intestinal epithelial caco- cells, which is unrelated to resveratrol release (storniolo et al., ) . -methoxystypandrone of p. cuspidatum inhibits signal transducer and activator of transcription and nuclear factor-kb signaling by inhibiting janus kinase and ikb kinase (kuang et al., ) . resveratrol inhibits invasion and metastasis of colorectal cancer cells via malat -mediated wnt/b-catenin signal pathway (ji et al., ) . constitutively activated stat plays a pivotal role in oncogenesis and metastasis in many human cancers, and stat has been validated as a novel anticancer drug target. -methoxystypandrone of p. cuspidatum demonstrated a potent inhibitory effect on stat activation and significantly inhibited cell proliferation of human breast cancer cells (liu et al., b) , especially those with constitutively activated stat (ic . - . mm). the sar analysis of quinone analogs suggested that the phenolic and carbonyl groups are the key structures contributing to their inhibitory activities against the stat signaling. aqueous and organic extracts of selected species from five genera (fallopia, oxyria, persicaria, polygonum, and rumex) of the family polygonaceae occurring in the carpathian basin were screened in vitro for antiproliferative activity against hela (cervix epithelial adenocarcinoma), a (skin epidermoid carcinoma), and mcf (breast epithelial adenocarcinoma) cells (lajter et al., b) , using the -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide assay. a total of n-hexane, chloroform, % methanol, and water extracts of different plant parts were investigated. extracts of p. hydropiper, rumex acetosa, r. alpinus, r. aquaticus, r. scutatus, and r. thyrsiflorus at or mg/ml demonstrated substantial cell growth inhibitory activity (at least % inhibition of cell proliferation) against one or more cell lines. r. acetosa and r. thyrsiflorus proved to be the most active and are considered worthy of activity-guided phytochemical investigations. , -dihydroxybenzyl alcohol of reynoutria japonica (p. cuspidatum) showed more potent inhibitory activity against topoisomerase i (ic mm) than camptothecin (hwangbo et al., ) , the positive control (ic mm). , -dihydroxybenzyl alcohol, citreorosein, cis-resveratrol, trans-resveratrol, and trans-resveratrol- -o-b-d-glucopyranoside showed stronger inhibitory activities toward dna topoisomerase ii (ic . , , , . , and mm, respectively) than the positive control, etoposide (ic mm). emodin and citreorosein displayed weak cytotoxicities against human lung cancer (a ), ovarian cancer (sk-ov- ), human liver hepatoblastoma (hepg ), and colon adenocarcinoma (ht- ) cell lines. flavonoid glucuronides of p. amphibium had antileukemic activity (smolarz et al., ) . a potential antitumor ellagitannin of p. capitatum, davidiin, inhibited hepatocellular tumor growth by targeting ezh (wang et al., b) . the n-butanol extract of p. bellardii has the highest cytotoxicity in hela, mcf- , and hepg- cells, with ic values of . , . , and . mg/ml, respectively (abd el-kader et al., b). myricetin- -o-( -acetyl-a-arabinofuranoside) exhibited a marked cytotoxicity in hela (ic . mg/ml) and hepg- ( . mg/ml) cells. (À)-loliolide of p. aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (yang et al., a) . quercetin- -o-b-d-glucuronide isolated from p. aviculare inhibits cellular senescence in human primary cells (yang et al., b) . apoptosis induction by p. minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and s-phase cell cycle arrest in hepg cell line (mohd ghazali et al., ) . resveratrol- -o-d-( -galloyl)-glucopyranoside isolated from p. cuspidatum exhibits anti-hepatocellular carcinoma viability by inducing apoptosis via the jnk and erk pathway . polydatin inhibits growth of lung cancer cells by inducing apoptosis and causing cell cycle arrest . -methyltryptanthrin of p. tinctorium induced differentiation of p cl embryonal carcinoma cells into spontaneously beating cardiomyocyte-like cells (seya et al., ) . polydatin from p. cuspidatum exhibited protective effects against carbon tetrachlorideinduced liver injury in mice . p. cuspidatum contained many more contributing antioxidants other than resveratrol (kurita et al., ) . tetrahydroxystilbene glucoside of p. multiflorum exhibited protective effect against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic mc t -e cells . compared with p. multiflorum polysaccharide pmp- , pmp- exhibited a much stronger antioxidant capacity against free radicals, lipid oxidation, and protein glycation (lv et al., ) . the ic₅₀ values of pmp- were . , . , and . mg/ml for superoxide anion scavenging, hydroxyl radical scavenging, and hydroxyl peroxide scavenging, respectively. the inhibitory ability of pmp- on lipid oxidation marked in the rat liver, followed by the heart and kidney. pmp- also showed satisfactory suppression of ages formation. the etoac fraction of p. aviculare displayed the highest content of flavonoids (sum, . mg/g) with the strongest peroxynitrite scavenging activity (ic , . mg/ml) (nugroho et al., ) . the activities of the eight compounds (myricitrin, isoquercitrin, avicularin, quercitrin, myricetin, desmanthin- , quercetin, and kaempferol) were comparable to that of the positive control (l-penicillamine; ic : . mg/ml). the folkloric medicinal uses of p. aviculare are mainly attributed to flavonoids, such as particularly highly contained myricetin, myricitrin, and desmanthin- . xanthone and lignan glycosides from the aerial parts of p. bellardii showed significant antioxidant potential by dpph(Á) scavenging activity test (abd el-kader et al., a). , -dihydropyranobenzopyrone, amplexicine, catechin, rutin, quercetin- -o-b-d-galactopyranoside, chlorogenic acid, galloyl glucose, caffeic acid, gallic acid, and scopoletin, isolated from p. amplexicaule, exhibited considerable antioxidant activity in a dpph radical scavenging assay (tantry et al., ) . resveratrol reverses the effects of chronic unpredictable mild stress on behavior, serum corticosterone levels, and bdnf expression in rats . resveratrol attenuates oxidative damage and ameliorates cognitive impairment in the brain of senescence-accelerated mice (liu et al., a) . p. minus possesses antioxidant and anticholinesterase activity and demonstrated enhanced cognition in vivo . the data suggest neuroprotective properties of the extract. hexane extracts of p. multiflorum improve tissue and functional outcome following focal cerebral ischemia in mice . tetrahydroxystilbene glucoside of p. multiflorum attenuates neuroinflammation through the inhibition of microglia activation (zhang et al., a) . -hydroxycalamenene of reynoutria elliptica (p. ellipticum) attenuated the cell death of transformed rgc- cells (jo et al., ) . neurons rely on the release and subsequent cleavage of gsh to cysteinylglycine (cys-gly) by astrocytes in order to maintain optimal intracellular gsh levels. in neurodegenerative diseases characterized by oxidative stress, neurons need an optimal gsh supply to defend themselves against free radicals released from activated microglia and astroglia. the rate of gsh synthesis is controlled largely by the activity of g-glutamyl cysteine ligase. expression of g-glutamyl cysteine ligase and of the xc-system, which facilitates cystine uptake, is regulated by the redox-sensitive transcription factor, nuclear factor erythroid- -related factor (nrf ). resveratrol and p. multiflorum were all identified as potent nrf activators and "gsh and cys-gly boosters" (steele et al., ) . the antidepressant-like effects of resveratrol in the fst (forced swim test) and tst (tail suspension test) are mediated, at least in part, by modulating hypothalamic-pituitary-adrenal axis, bdnf expression, and erk phosphorylation in the brain region of mice . the antimelanogenic activity of , , , -tetrahydroxystilbene- -o-b-d-glucopyranoside, isolated from p. multiflorum, is likely mediated through a noncompetitive inhibition on tyrosinase, downregulation of the expression of melanogenic proteins, and reduction of tyrosinase/trp- complex formation (cheung et al., ) . supplementation for weeks with p. minus and the proprietary eurycoma longifolia extract, physta, was well tolerated and more effective than placebo in enhancing sexual performance in healthy volunteers (udani et al., ) . p. cuspidatum inhibits pancreatic lipase activity and adipogenesis via attenuation of lipid accumulation , thus having antiobesity effect. constituents from the root of p. multiflorum have promotion effect on hair growth (sun et al., ) . the semialcoholic extract of p. senegalensis possesses a-glucosidase inhibitory activity and antioxidant potency (bothon et al., ) . proanthocyanidins, isolated from stems of p. multiflorum, strongly inhibit a-amylase with an acarbose equivalence (ae) value of . mmol ae/g and inhibit a-glucosidase with an ae value of . mmol ae/g (wang et al., b) . they have potential as functional ingredients in reducing postprandial hyperglycemia. phenolic constituents from aerial parts of p. hyrcanicum had in vitro a-glucosidase inhibitory activity (moradi-afrapoli et al., a). adverse reactions induced by p. multiflorum are common, with patients developing drug-induced liver injury (dili) and even liver failure . although the mechanism is unknown, many studies have suggested that an idiosyncratic reaction occurs, which is related to genetic polymorphisms of cyp a . the influence of stilbene glucoside on the pharmacokinetics of emodin may be attributed to the inhibition of ugt a mrna expression (ma et al., ) . p. cuspidatum markedly increased the systemic exposure and brain concentration of carbamazepine (cbz) and cbze through inhibiting the activities of cyp a and mrp (chi et al., ) . molecular markers can help elucidate how neutral evolutionary forces and introduction history contribute to genetic variation in invaders. genetic diversity, population structure, and colonization patterns in the invasive p. cespitosum, a highly selfing, tetraploid asian annual introduced to north america, were examined using nine diploidized polymorphic microsatellite markers (ssr; matesanz et al., ) . low heterozygosity was found in all populations, consistent with the selfing mating system of p. cespitosum. despite the high selfing levels, substantial genetic variation within and among p. cespitosum populations was revealed, based on the percentage of polymorphic loci, allelic richness, and expected heterozygosity. inferences from individual assignment tests and pairwise fst values indicated high among-population differentiation, which indicates that the effects of gene flow are limited relative to those of genetic drift, probably due to the high selfing rates and the limited seed dispersal ability of p. cespitosum. population structure appears to be the result of the random movement of propagules across the introduced range, possibly associated with human dispersal. the high population differentiation, genetic diversity, and fine-scale genetic structure (populations founded by individuals from different genetic sources) in the introduced range suggest that multiple introductions to this region may have occurred. high genetic diversity may further contribute to the invasive success of p. cespitosum in its introduced range. among rapd primers, only c primer had two specific bands, which could distinguish p. capitatum from p. nepalense (zhou et al., ) . four pairs of specific primers were designed based on the two sequences of rapd marker bands, and only one pair primer (z - ) was successfully converted into scar marker, which could be used as an effective scar mark to identify z dna for p. capitatum. aflp was used to identify the knotgrass (p. aviculare) population at the crime site as the most likely origin of the botanical evidence (koopman et al., ) . the genetic diversity of representational populations of p. capitatum including individuals was investigated by issr marker (zhou et al., ) . srap (sequence-related amplified polymorphism) was used to detect the polymorphisms of radix polygoni multiflori in chongqing (cheng et al., ) . srap might be superior to rapd in genetic diversity studies. analysis of nuclear internal transcribed spacer (nrits) sequences reveals polymorphism among five p. barbata populations belonging to five geographic locations in india (choudhary et al., ) . upgma analysis based on the its datasets shows that the sampled populations are grouped according to their geographic locations and are supposed to evolve under reproductive isolation, which most likely are due to the long-distance distribution and population fragmentation. dna bar codes are used to discriminate the polygonaceae in china pharmacopoeia and their adulterants (song et al., ) . the amplification efficiency of six candidate dna bar codes (rbcl, trnh-psba, ndhj, rpob, rpoc , and accd) was %, while the efficiency of ycf and nrits was % and %, respectively. the interspecific divergence was highest for the trnh-psba ( . %), followed by the nrits ( . %) across all species pairs, while intraspecific variation both within populations and between populations was absent. the trnh-psba can not only distinguish species of polygonaceae in china pharmacopoeia but also recognize eight other species of polygonaceae including their adulterants. p. multiflorum (fallopia multiflora) is often confused and substituted with the roots of fallopia multiflora var. ciliinervis, pteroxygonum giraldii, cynanchum auriculatum, and stephania cepharantha. the nrits regions of six fallopia species were sequenced and analyzed (zheng et al., ) . the diagnostic primers pmits and pmits , which amplified an expected bp its fragment from f. multiflora, were designed. no amplified product was observed when other species was used. based on s rrna gene sequence analysis, p. multiflorum could be easily distinguished from adulterants and other herbs with similar medicinal components (yan et al., ) . all p. multiflorum samples were divided into four clades based on matk sequence. permutations of matk were related to the geographic distributions of the samples. these markers can be used to authenticate the botanical origin of p. multiflorum. the best bar code of p. multiflorum is psba-trnh, with significant interpopulation variability (sun et al., ) . the combination of loci gave better performance for distinguishing populations than a single locus. it could be good to use matk + rbcl + psba-trnh + its or psba-trnh alone for p. multiflorum in geoherbalism identification. cdna-amplified fragment length polymorphism (cdna-aflp) transcript profiling was applied to generate the expression profiles of p. minus in response to salicylic acid (sa) and methyl jasmonate (meja) elicitations (ee et al., ) . two sets of genes were induced by sa and meja, respectively. stress-related genes were proved to lead to the expression of genes involved in secondary metabolite biosynthetic pathways. a total of transcript-derived fragments (tdfs) were upregulated, including from sa-treated and from meja-treated samples. the cdna-aflp transcripts generated using different mse /taq primer combinations showed that treatments with sa and meja induced genes mostly involved in scavenging ros, including zeaxanthin epoxidase, cytosolic ascorbate peroxidase , and peroxidase. of these stress-related genes, % of other annotated tdfs are involved mainly in secondary metabolic processes, and two genes encoding (+)-delta cadinene synthase and cinnamoyl-coa reductase were highlighted. the leaf of an aromatic plant p. minus is widely used as a food additive and in the perfume industry. the leaf also accumulates secondary metabolites such as flavonoid. a standard cdna library of p. minus leaves was constructed, and two normalized fulllength enriched cdna libraries were constructed from stem and root in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis (roslan et al., ) . large-scale sequencing of p. minus cdna libraries identified expressed sequence tags (ests). from the three cdna libraries, ests encoding seven genes were mapped to the flavonoid biosynthetic pathway. three flavonoid biosynthetic pathway-related ests, namely, chalcone synthase, chs (jg ); flavonol synthase, fls (jg ; figure . ); and leucoanthocyanidin dioxygenase, ldox (jg ), were selected for further examination by quantitative rt-pcr (qrt-pcr) in different p. minus organs. expression was detected in leaf, stem, and root. based on transcriptome data, gene expression studies can be initiated to better understand the underlying physiological processes. various active components have been extracted from the root of p. cuspidatum. however, the genetic basis for their activity is little known. , , short reads ( . gb) of p. cuspidatum root transcriptome were obtained via illumina hiseq sequencing (hao et al., ) . a total of , unigenes were assembled de novo and annotated. , , , and unigenes were, respectively, mapped to the mevalonic acid (mva), methyl-d-erythritol -phosphate (mep), shikimate ( figure . ), and resveratrol biosynthesis pathways, suggesting that they are (hao et al., ) , which is responsible for biosynthesis of anthraquinone and stilbene. involved in the biosynthesis of pharmaceutically important anthraquinone and resveratrol. eighteen potential udp-glycosyltransferase unigenes were identified as the candidates most likely to be involved in the biosynthesis of glycosides of secondary metabolites. identification of relevant genes could be important in eventually increasing the yields of the medicinally useful constituents of the p. cuspidatum root. from the previously published transcriptome data of nonmodel plant taxa, shared orthologs were identified and characterized. this information will be very useful for future functional, phylogenetic, and evolutionary studies of these plants. the buckwheat family polygonaceae is a diverse group of plants and is a good model for investigating biogeography, breeding systems, coevolution with symbionts such as ants and fungi, functional trait evolution, hybridization, invasiveness, morphological plasticity, pollen morphology, and wood anatomy. age estimates for polygonaceae were obtained by calibrating a bayesian phylogenetic analysis (schuster et al., ) , using a relaxed molecular clock with fossil data. eighty-one species of polygonaceae were analyzed with mrbayes to infer species relationships. one nuclear (nrits) and three chloroplast (cp) markers (the trnl-f spacer region and matk and ndhf genes) were used. seven calibration points including fossil pollen and a leaf fossil of muehlenbeckia (a southern hemisphere group) were used to infer node ages. results of the beast analyses indicate an age of . / . million years (my) with an uncertainty interval of . - . my for the stem age of polygonaceae. this age is older than previously thought (approximately . - . my). the estimated divergence time for muehlenbeckia is . / . ( . - . ) my and its crown clade is . / . ( . - . ) my. because the breakup of gondwana occurred from to my ago, diversification of muehlenbeckia is best explained by oceanic longdistance and maybe stepping-stone dispersal rather than vicariance. interspecific hybridization and the following polyploidization play a major role in plant diversification, but quantifying the contribution of this mechanism to diversification within taxonomically complex clades remains difficult. incongruence among gene trees can provide critical insights, especially when combined with data on chromosome numbers, morphology, and geography. molecular phylogenetic studies using three cpdna regions and nrits sequences were performed to explore the hybrid speciation in persicaria (polygonum, polygonaceae; figure . ) (kim and donoghue, ) , with an emphasis on sampling within section eupersicaria. there are major conflicts between the combined cpdna tree and the nrits tree; a variety of incongruence tests rejected stochastic error as the cause of incongruence in most cases. both the tree incongruence results and information on chromosome numbers suggest that the origin of polyploid species involved interspecific hybridization. the recognition of several previously named species that have been treated as belonging within other species was supported. repeated allotetraploidy (as distinct from radiation at the tetraploid level) might be the key mechanism governing the diversification of this taxonomically challenging group. multiple instances of allopolyploid speciation were also shown in persicaria using a low-copy nuclear gene region (leafy second intron) (kim et al., b) , which belong to polygonum in flora of china. fifteen species seem to be allopolyploids, which is higher than the number found in previous comparisons of cp dna (tamura et al., ) . b, matk + rbcl + trnl-f tree inferred from neighbor joining method. the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. the analysis involved nucleotide sequences. all ambiguous positions were removed for each sequence pair. there were a total of positions in the final dataset. phylogenies (kim and donoghue, ) . this underestimation of the extent of allopolyploidy is due in at least three cases to homogenization of nrits toward the maternal lineage. the diploid species, p. lapathifolia, has been involved in at least six cases of allopolyploid speciation. of the diploids, this species is the most widespread geographically and ecologically and also bears more numerous and conspicuous flowers, illustrating ecological factors that may influence hybridization frequency. with a few exceptions, the allopolyploid species also are widespread, plastic, ecological generalists. hybridization events fostered by human introductions may be fueling the production of new species that have the potential to become aggressive weeds. its sequences from indian polygonum taxa were examined to investigate relationships among various sections proposed previously (choudhary et al., ) . the maximum parsimony trees obtained from its sequences suggested eight major groups of the indian polygonum spp. the relationships among different sections were largely congruent with those inferred from morphological characters as described by hooker. the treatment of the persicaria suggested by haraldson on the basis of anatomical characters proved to be nearly in line with that based on its data. a high resolution of phylogeny of the himalayan polygonum (e.g., p. microcephalum, p. assamicum, p. recumbens, and p. effusum) was provided and merger of the section amblygonon in the section persicaria was supported. molecular differences were detected among persicaria barbata collected from different geographic locations of india, although these were not differentiated at the morphological level. to examine the phylogenetic relationships of koenigia (polygonaceae), samples representing all species of koenigia and closely related taxa (aconogonon, bistorta, and persicaria) were sequenced for nr its and four cp regions (trnl-f, atpb-rbcl, rbcl, and rpl -trnl(uag); fan et al., a,b) . trib. polygoneae and trib. rumiceae are recovered on both cp and its trees (figure . ) , while trib. atraphaxideae is not. the placement of p. bistorta is uncertain due to conflict between cp and its trees (figure . ) . it was proposed that the genus koenigia be circumscribed to include five species, that is, perennials (k. forrestii and k. nummularifolia) and annuals (k. islandica, k. pilosa, and k. nepalensis). however, on both cp and its trees, k. nepalensis (p. nepalense) is more closely related to p. capitatum and p. chinense (figure . ) , instead of koenigia. k. islandica and k. fertilis (p. fertile, figure . a), both of which are from the himalayan region, can be merged into a single species. p. delicatulum (k. delicatula), p. campanulatum, and p. lichiangense occupy an isolated position at the base of the polygonaceae (figure . a) , which might be reassigned to a new genus. the tetraploid p. minor (p. minus), which is sister to the p. hydropiperoides complex in the nrits tree (kim and donoghue, ) , may have arisen through hybridization between an unknown diploid lineage or possibly a tetraploid in the p. hydropiperoides complex and the diploid p. hydropiper. it is not entirely clear that p. hydropiper served as the maternal parent, since the relationship between p. hydropiper and p. minor is weakly supported in the cpdna tree (figure . b) . morphologically, p. minor is more similar to the diploid p. foliosa (p. foliosum), which should also be considered as a possible diploid maternal lineage for p. minor (kim and donoghue, ) . the pollen apertures likely evolved in parallel in the aconogonon-koenigia-bistorta clade and persicaria clade (fan et al., a,b) , and tricolpate pollen is probably the ancestral one. quincuncial aestivation likely evolved during the early evolution of koenigia and its close relatives. the uplift of the himalayas has played a vital role in promoting species diversification of koenigia. k. islandica expanded its range during pleistocene glacial cycles by tracking changes in newly available habitats. there is little study addressing chemotaxonomy and metabolomics of polygonum and related genera, although such studies can provide vital information for sustainable utilization of polygonaceae medicinal resources. the family polygonaceae has been supported as monophyletic, but the circumscription of some subgroups, especially polygonum and related taxa, has been controversial. the use of population genetic approaches, sampling from multiple populations, would shed light on this issue. more extensive sampling inside and outside the himalayas and hengduan mountains is indispensable to explore the phylogenetic hypothesis and facilitate species diversity conservation. xanthone and lignan glycosides from the aerial parts of polygonum bellardii all growing in egypt polyphenols from aerial parts of polygonum bellardii and their biological activities distribution of drimane sesquiterpenoids and tocopherols in liverworts, ferns and higher plants: polygonaceae, canellaceae and winteraceae species in vitro biological effects of two anti-diabetic medicinal plants used in benin as folk medicine protective effect of polygonum orientale l. extracts against clavibater michiganense subsp. sepedonicum, the causal agent of bacterial ring rot of potato polygonum viviparum l. induces vasorelaxation in the rat thoracic aorta via activation of nitric oxide synthase in endothelial cells active neuraminidase constituents of polygonum cuspidatum against influenza a(h n ) influenza virus polygonum viviparum l. inhibits the lipopolysaccharide-induced inflammatory response in raw . macrophages through haem oxygenase- induction and activation of the nrf pathway tyrosinase inhibitory activity of a glucosylated hydroxystilbene in mouse melan-a melanocytes a new herb-drug interaction of polygonum cuspidatum, a resveratrol-rich nutraceutical, with carbamazepine in rats studies on genetic diversity among populations of persicaria barbata (l.) h. hara from india based on internal transcribed spacer sequences of nuclear ribosomal dna phylogeny and systematics of indian polygonum sensu lato in the subfamily polygonoideae based on its sequences of nuclear ribosomal dna quercetin -o-( -galloyl)-beta-d-galactoside from polygonum viscosum (polygonaceae) polydatin attenuates cardiac hypertrophy through modulation of cardiac ca + handling and calcineurin-nfat signaling pathway transcriptome profiling of genes induced by salicylic acid and methyl jasmonate in polygonum minus screening of biological activities of polygonum maritimum l. from algerian coast. asian pac molecular phylogeny of koenigia l. (polygonaceae: persicarieae): implications for classification, character evolution and biogeography anti-inflammatory activity of the invasive neophyte polygonum cuspidatum sieb. and zucc. (polygonaceae) and the chemical comparison of the invasive and native varieties with regard to resveratrol in vitro and ex-vivo cellular antioxidant protection and cognitive enhancing effects of an extract of polygonum minus huds (line-minus™) demonstrated in a barnes maze animal model for memory and learning antioxidant and antiinflammatory flavonol glucuronides from polygonum aviculare l anti-atherosclerotic effects of polygonum aviculare l. ethanol extract in apoe knock-out mice fed a western diet mediated via the mapk pathway genuine traditional korean medicine, naju jjok (chung-dae, polygonum tinctorium) improves , -dinitrofluorobenzene-induced atopic dermatitis-like lesional skin de novo characterization of the root transcriptome of a traditional chinese medicinal plant polygonum cuspidatum emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction prescription parsing of miao medicine polygonum capitatum and kelintong capsule phytochemical and biological research of polygoneae medicinal resources inhibition of dna topoisomerases i and ii of compounds from reynoutria japonica gastroprotective activity of polygonum chinense aqueous leaf extract on ethanol-induced hemorrhagic mucosal lesions in rats inhibition of il- and tslp production through the attenuation of caspase- activation in an animal model of allergic rhinitis by naju jjok (polygonum tinctorium) resveratrol inhibits invasion and metastasis of colorectal cancer cells via malat mediated wnt/b-catenin signal pathway -hydroxycalamenene isolated from the rhizomes of reynoutria elliptica exerts neuroprotective effects both in vitro and in vivo incongruence between cpdna and nrits trees indicates extensive hybridization within eupersicaria (polygonaceae) a new stilbene glucoside gallate from the roots of polygonum multiflorum allopolyploid speciation in persicaria (polygonaceae): insights from a low-copy nuclear region polygonum cuspidatum inhibits pancreatic lipase activity and adipogenesis via attenuation of lipid accumulation botanical dna evidence in criminal cases: knotgrass (polygonum aviculare l.) as a model species -methoxystypandrone inhibits signal transducer and activator of transcription and nuclear factor-kb signaling by inhibiting janus kinase and ikb kinase content of resveratrol and glycoside and its contribution to the antioxidative capacity of polygonum cuspidatum (itadori) harvested in kochi inhibition of g protein-activated inwardly rectifying k + channels by extracts of polygonum persicaria and isolation of new flavonoids from the chloroform extract of the herb antiproliferative activity of polygonaceae species from the carpathian basin against human cancer cell lines hexane extracts of polygonum multiflorum improve tissue and functional outcome following focal cerebral ischemia in mice polyflavanostilbene a, a new flavanol-fused stilbene glycoside from polygonum cuspidatum five new stilbene glycosides from the roots of polygonum multiflorum emodin inhibits tonic tension through suppressing pkcd-mediated inhibition of myosin phosphatase in rat isolated thoracic aorta chemical constituents in herbs of polygonum jucundum resveratrol attenuates oxidative damage and ameliorates cognitive impairment in the brain of senescence-accelerated mice small-molecule stat signaling pathway modulators from polygonum cuspidatum in vitro and in vivo studies of the inhibitory effects of emodin isolated from polygonum cuspidatum on coxsackievirus b₄ resveratrol reverses the effects of chronic unpredictable mild stress on behavior, serum corticosterone levels and bdnf expression in rats purification, antioxidant activity and antiglycation of polysaccharides from polygonum multiflorum thunb stilbene glucoside inhibits the glucuronidation of emodin in rats through the down-regulation of udp-glucuronosyltransferases a : application to a drug-drug interaction study in radix polygoni multiflori cyp a polymorphism in chinese patients with acute liver injury induced by polygonum multiflorum effect of polygonum hydropiper l. against dengue vector mosquito aedes albopictus l genetic diversity and population structure in polygonum cespitosum: insights to an ongoing plant invasion apoptosis induction by polygonum minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and s-phase cell cycle arrest in hepg cell line in vitro a-glucosidase inhibitory activity of phenolic constituents from aerial parts of polygonum hyrcanicum simultaneous quantification and peroxynitrite-scavenging activities of flavonoids in polygonum aviculare l. herb flavonoid biosynthesis genes putatively identified in the aromatic plant polygonum minus via expressed sequences tag (est) analysis age estimates for the buckwheat family polygonaceae based on sequence data calibrated by fossils and with a focus on the amphi-pacific muehlenbeckia -methyltryptanthrin-induced differentiation of p cl embryonal carcinoma cells into spontaneously beating cardiomyocyte-like cells flavonoid glucuronides with anti-leukaemic activity from polygonum amphibium l phytochemical and biological research of polygoneae medicinal resources authentication of the family polygonaceae in chinese pharmacopoeia by dna barcoding technique effect of nrf activators on release of glutathione, cysteinylglycine and homocysteine by human u astroglial cells piceid presents antiproliferative effects in intestinal epithelial caco- cells, effects unrelated to resveratrol release analysis of head volatile constituents of polyonum cuspidatum flower by hs-gc/ms promotion effect of constituents from the root of polygonum multiflorum on hair growth mega : molecular evolutionary genetics analysis version . resveratrol and cardiovascular health -promising therapeutic or hopeless illusion? -dihydropyranobenzopyrone: a previously undetermined antioxidant isolated from polygonum amplexicaule. chin resveratrol ameliorates high-glucose-induced hyperpermeability mediated by caveolae via vegf/kdr pathway effects of a proprietary freeze-dried water extract of eurycoma longifolia (physta) and polygonum minus on sexual performance and well-being in men: a randomized, double-blind, placebo-controlled study a recent review on phytochemical constituents and medicinal properties of kesum (polygonum minus huds.). asian pac characterization of proanthocyanidins in stems of polygonum multiflorum thunb as strong starch hydrolase inhibitors antidepressant-like activity of resveratrol treatment in the forced swim test and tail suspension test in mice: the hpa axis, bdnf expression and phosphorylation of erk in vitro effects of active components of polygonum multiflorum radix on enzymes involved in the lipid metabolism a potential antitumor ellagitannin, davidiin, inhibited hepatocellular tumor growth by targeting ezh study on chemical composition of ethylacetate fraction from polygonum amplexicaule var. sinense -tetrahydroxystilbene- -o-b-d-glucoside (thsg) attenuates human platelet aggregation, secretion and spreading in vitro a bioactivity-guided study on the anti-diarrheal activity of polygonum chinense linn resveratrol- -o-d-( -galloyl)-glucopyranoside isolated from polygonum cuspidatum exhibits anti-hepatocellular carcinoma viability by inducing apoptosis via the jnk and erk pathway a new stilbene glucoside from the roots of polygonum multiflorum thunb inhibitory effects of , , , -tetrahydroxystilbene- -o-b-d-glucoside on angiotensin ii-induced proliferation of vascular smooth muscle cells the effect of , , , -tetrahydroxystilbene- -o-b-d-glucoside on pressure overload-induced cardiac remodeling in rats and its possible mechanism genetic variation and identification of cultivated fallopia multiflora and its wild relatives by using chloroplast matk and s rrna gene sequences polygonumosides a-d, stilbene derivatives from processed roots of polygonum multiflorum inhibitory effects of (À)-loliolide on cellular senescence in human dermal fibroblasts quercetin- -o-b-d-glucuronide isolated from polygonum aviculare inhibits cellular senescence in human primary cells proteomic analysis for anti-atherosclerotic effect of tetrahydroxystilbene glucoside in rats inhibition of epstein-barr virus lytic cycle by an ethyl acetate subfraction separated from polygonum cuspidatum root and its major component, emodin protective effects of polydatin from polygonum cuspidatum against carbon tetrachloride-induced liver injury in mice protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic mc t -e cells study on the chemical constituents of the active fraction of polygonum capitatum tetrahydroxystilbene glucoside attenuates neuroinflammation through the inhibition of microglia activation a review of the pharmacological effects of the dried root of polygonum cuspidatum (hu zhang) and its constituents polydatin inhibits growth of lung cancer cells by inducing apoptosis and causing cell cycle arrest a new chromone glycoside from roots of polygonum multiflorum molecular authentication of the traditional medicinal plant fallopia multiflora genetic diversity of polygonum capitatum from guizhou populations by issr markers study on sequence characterized amplified region (scar) markers of polygonum capitatum key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- - ddfsc authors: ceylan, rahmiye figen; ozkan, burhan; mulazimogullari, esra title: historical evidence for economic effects of covid- date: - - journal: eur j health econ doi: . /s - - - sha: doc_id: cord_uid: ddfsc like wars and socio-politic shifts, contagious diseases have changed the economics and politics of the world throughout history. in , the world faced covid- , a globally effective virus leading to mass losses and socio-economic panic. due to apparent psycho-social conditions, analyzing the potential economic effects of the covid- pandemic was inevitable. thus, discussing economic effects of previous global and regional epidemics is considered beneficial. this research evaluated most of the known epidemics and their effects on economics and socio-politics by reviewing scientific literature. in addition to the vast literature and observations on the ongoing process, we assessed the potential impacts of covid- and potential ways to overcome these impacts. the most urgent socio-economic measures needed to combat the negative effects of a contagious disease are related to unemployment with its income effects and security of all sectors. to prevent persistent unemployment, service, retail, and even industrial sectors need to be supported. additionally, we discussed the need for re-organizing the funding and managerial sustainability of healthcare services to be prepared for future. the world has witnessed many challenging situations throughout history. these challenges were wars sometimes or revolutions that reshaped the socio-politics completely. yet, another challenge has been contagious diseases. the world had witnessed many epidemics. the plague of the medieval and its variations and the great influenza of - were the most devastating ones. yet, the contagious diseases having global effects had forgotten long time ago even if there appeared some recent encounters in the past the differentiating features of covid- or sars-cov from the recent encounters are its geographical dispersion in terms of contagion and its causalities. however, its apparent size and global effects were underestimated at the beginning. it is sound to mention the scope of previous epidemics to differentiate covid- . sars-cov (severe acute respiratory syndrome) had started in china and affected taiwan, singapore, usa and canada. during its influential period, around , cases were reported and more than people had died with an % fatality rate [ ] . avian influenza-h n that spread from poultry to human resulted in deaths as of [ ] . the most recent epidemic of mers-cov (middle east respiratory syndrome) mostly affected the middle east in with more than deaths. the only out of region case was south korea, where deaths were reported in . what makes covid- more devastating is its speed of contagion mostly in europe and the americas, although it started in the east asia. by the end of april, number of reported cases and deaths were more than million and . consecutively around the world [ ] . with its % fatality rate, this global disease was announced as a pandemic by the who on th of march . the fatal process has been continuing, while the attempts for vaccination and cure have gained attention. however, considering the endurance of previous epidemics, nobody is sure apparently whether it will be completely controlled soon. even if the health dimension is controlled soon, it is expected that its economic impacts would be long lasting in the societies. therefore, it is important to project the possible economic and leading policy level outcomes of this pandemic. accordingly, it was considered as contributory to evaluate the effects of previous epidemics with a historical perspective, overview the existing situation on the covid- and to provide some policy insights within the scope of this research. the plague or with its well-known name the black death of the medieval had changed flow of history, even if it is hard to measure its effects statistically. the black death changed the role of the working class, led the refurbishment of capital accumulation structures and welfare distribution [ ] . the destructed labour and land resources in the medieval led to the discovery of a new continent, the americas. besides, the change of labour endowments or declining labour supplies even led to the shift from feudalism to centralisation of governments [ ] . the production structures shifted from labour to capital based and productive centres shifted from rural to urban during the medieval, and plague had significant effects on these challenges [ ] . yet, these effects took place within centuries and the exact impact of plague is hard to measure. however, the effects of recent epidemics were more measurable. the furthest encounter, spanish flu was not only seriously fatal but also it had significant effects on politics and economics of the twentieth century as it coincided with the wwi. spanish flu took place between and , resulted in around million deaths in countries and the loss of young and middle-aged group led to a path to the wwii [ ] . the relationship of the war and the flu in italy was portrayed in a population study that . of total . flu deaths encountered were of soldiers, meaning the loss of war power [ ] . barro and ursua [ ] focused on the effects of disasters experienced since on gdp and consumption. they found that the spanish flu was the fourth economic shock on income and consumption following wwii, wwi and the great depression. they also indicated that these sorts of disasters led to around % per capita income loss. in an earlier attempt to comment on prospective covid- effects, barro and his friends estimated growth of national income and consumption expenditures of countries between and on human capital loss due to the wwi. the impacts of flu concerning the gdp and consumption loss were around % and % respectively. accordingly, they concluded that the great influenza had affected the society and economics seriously as well [ ] . the effects of the spanish flu on earnings, capital returns and poverty were estimated for sweden between and using official datasets [ ] . it was understood that the pandemic affected poverty and returns on capital negatively, while effects on earnings and effective income were ambiguous. with a different perspective, natural and manmade disasters like wars were compared for sweden between and [ ] . it was understood that female labour replaced male labour during and after the great influenza. in a consumer research based on general social survey between and , it was seen that societal trust loss appeared during and after spanish flu affected forthcoming generations negatively as well and consumption preferences changed significantly [ ] . due to recent experiences, it can be said that epidemics affect societies within a limited time frame. but there are continuous and cyclical disasters affecting societies. the impact of spanish flu was compared with spring frosts in norway. it was understood that the psycho-social impact of the epidemic and loss of middle-aged labour led to an economic breakdown. however, the losses encountered due to frosts were long lasting that they affected the society till the end of s until technological advancements purified the losses [ ] . therefore, it can be noted that morality effect is a major reason in longer recovery after an epidemic and people tend to develop technological solutions for cyclical and natural disasters. due to changing labour market composition and economic conditions during and after the influenza, both productivity and overall income had declined and savings and investment potential were affected negatively. influenza contributed to losses of the wwi and was one of the factors changing economics and politics of the world via centralisation of governments until the end of s [ ] . while the recent diseases were limited geographically, they affected the economics of disease centres anyway. with changing economic outlay in terms of globalisation and growing services sector, the effects of these diseases were measured in a multidimensional way. demand-oriented sectors had changed and affected other sectors due to changing income levels and unemployment. the economic impact of sars-cov was analysed for hong kong [ ] . demandoriented sectors, as tourism and transportation, downsized critically. consumption demand and foreign trade declined in the short term and the depression in these sectors resulted in unemployment. yet the productivity in industrial sectors was unaffected and sars-cov was considered as a demand shock and the revival did not take much time. taiwanese tourism was overviewed [ ] and it was noted that number of incoming tourists fell from . million in to . million in . consumption expenditures and gdp fell accordingly and labour demand for tourism and retailing had shrunk between and % within a year. accordingly, it was concluded that the economic impact of the disease was visible even in the short term. the effects of immediate quarantine enforcements and costs of post-syndrome health care services provided due to sars-cov were compared for canada [ ] . it was found that if the contagion process is followed up strictly, immediate quarantine would be less costly than health care services provided afterwards. potential economic effects of sars were evaluated for china and hong kong [ ] . the analyses indicated that taking precautions are more efficient and less costly than posterior healthcare services in terms of loss of labour force and foregone income. in a macroeconomic perspective, consumption and services demand and their supplies are directly attached that a fall in overall demand led to declination in the overall supplies. in addition, the declination in demand and supply referred to % fall in services income on average. fan [ ] also noted that transparency in data collection and information of the society is essential departing from sars data. she concluded that getting prepared is much more important than utilisation of post-epidemic health services. this research was a projection and warning for future contagious diseases. in a comparative research, rising female labour in the job market in accordance with declination in real per capita gdp during and after spanish flu was notified [ ] . this significance of spanish flu was confirmed later as well [ ] . hiv mostly affected underdeveloped african countries with negative supply shifts in production and labour market. yet, sars-cov appeared as a services market destroyer. public policies were suggested for epidemic management to control the losses. confirming previous research on sars, lionello [ ] indicated that rising social fear and reduction in social contact resulted in reduced supplies and reduced labour demand specifically in the services sector between and %. following sars-cov, avian influenza-h n was also analysed in the scope of poultry sector. the computable general equilibrium (cge) simulations determined the need for environmental disinfection and promotion of personal hygiene among the workers for philippines [ ] . the disease control was tested for the taiwanese economy through macro simulations. the findings indicated that if the spread from poultry to human could be avoided, the loss would be kept within the sector and the economic loss would range between . and . %. yet, if the human transition cannot be prevented, estimated real gdp loss would be between . and . % and labour supply would decline by around . - . % [ ] . in addition, the global gdp was estimated to decline between . and . % due to downsizing in the poultry sector as a result of h n . however, the average downsizing effect of spread over human was estimated to range between . and . %. it was emphasized once more that precautionary actions are required [ ] . economic vulnerability to epidemics was assessed by sands and his friends [ ] . they indicated that underestimation of epidemic impacts on people and societies have led to underinvestment in epidemic control and management around the world. therefore, economic structures have become vulnerable to epidemic shocks. to cope with this vulnerability, they have suggested better public management and global support to countries by the who. they also noted that private health systems and attached financial organisations are not ready to bear responsibility of the combat with potential results of epidemics. especially, shrinking services and industries facing lower labour supplies and reducing demand are expected to downsize all economic structures. following this historical review of epidemics, it is important to note that some projections have already been made on potential effects of covid- . subsequently, it would be beneficial to consider the recent literature on prospective effects of covid- . recent research has focused on evaluating initial effects of the pandemic regarding consumption, services, finance and investments and industries partly. due to lockdown enforcements and voluntary social distancing, services sector, travel, tourism, catering and leisure, got affected critically. china, where the novel virus showed up first, the foregone tourism revenue was forecasted by luxurious travel agencies as % and this would mean almost $ billion in referring to data [ ] . due to iata's (the international air transportation association) recent projections, the aviation industry's expected passenger revenue loss for is $ billion and this counts more than % of the overall projected revenue [ ] . the change in offline consumption expenditures was evaluated for cities of china [ ] . there reported a % reduction in expenditures for retailing and services. the estimated rate for wuhan district was even more than %. therefore, the researchers projected at least . % fall in overall gdp in due to the fall in consumption. the shock on retail and services sector for india was estimated by kasare [ ] . the findings indicated that loss of hotels, restaurants and aviation would be % in , if the lockdown continues for months. these findings referred to declination in national income and rising budget deficit in . the bank transactions were evaluated to estimate spending and saving attitudes in the usa and results showed that consumer spending for necessities rose by % due stockpiling from the end of february to the mid of march [ ] . besides, expenditures in grocery stores and supermarkets had risen by % between the th of february and nd of march in the usa, compared to records [ ] . this research also confirmed the rising risk perceptions and social and economic fear everywhere. however, the consumption and purchasing attitudes had changed in the meanwhile. most of the spending started to take place online after the stockpiling phase and during temporary and permanent lockdowns. even in countries having moderate and lower income per capita, the internet users started to shift to online shopping and these shopping experiences incorporate some basic industry products like textiles and leisure tools as well as consumer goods [ ] . this shifting consumption demand was observed in japan as well at the beginning of the pandemic [ ] . yet, as most of the consumption spending has already been maintained online in japan, higher devastation has been awaited for services demand due to covid- . a utilitarian welfare maximization approach was used to forecast the trade of between death toll, life expectancy and consumption differences [ ] using mortality rate estimates taken from an earlier study conducted in the imperial college of london [ ] . if the mortality rate appears as . %, around % of consumption's share in national income will be lost in . if the average rate reduces to . %, the expected loss of consumption will be %. on the other hand, the loss in public resilience and rising security concerns are and will be the major distractors of demandoriented industries even after the pandemic. while people focused on their daily consumption, manufacturing industries got affected negatively even in these few months. demand loss accompanied by excess supply led to lower prices as an instance in household appliances, all sort of vehicles, textiles, etc. most of the manufacturing industries, like car manufacturing in europe, those purchase intermediaries are in loss apparently as well and tendency to save in exchange for spending has been rising all over the world [ ] . therefore, as awaited, the lockdown enforcements and declining demand are apparently leading to downsizing in all these industries. so, reducing prices are expected to lead stagnation and reduce the economic worth everywhere. in accordance, declining prices, including the global oil prices which fell down $ per barrel, will not mean more industrial gains. yet due to shrank services demand, incorporating retailing, tourism, aviation and leisure, unemployment came into agenda mainly in services dependent economies irrespective of governmental or municipal supports. unfortunately, the cyclical impact of rising unemployment seems to reduce all interior and international trade opportunities due to falling income and negatively shifting demand for most products [ , ] . finally, the macroeconomic effects should be considered shortly. mckibbin and fernando [ ] ran different scenarios regarding mortality and contagion of the disease to estimate the potential income effects of covid- . they inferred that the global income will reduce by . % in based on figures. besides, social welfare spending, which is a must, seems to affect fiscal balances and national budgets. the programmes announced to support by many countries referred to % of national budgets on average [ ] . besides, some projections made by international organisations are overwhelming as well. ilo (international labour organisation) estimated a . % job deterioration for the second quarter of due to covid- , meaning loss of million full-time jobs. the previous quarterly estimate was million and the estimated unemployment rose by almost % by the mid of april [ ] . besides, it was noted that % of employers and % of self-employed enterprises got affected by the lockdown measures. the imf (international monetary fund) projected a % reduction in global output for in january. yet, in the mid of april , the expected negative growth rate shrank to − . %, which is a huge declination in a quite short time period [ ] . in the same report, per capita income is expected to downsize by . % on average, with the most severe declination in the advanced economies by . %. accordingly, many economic policies should be developed in a short time and should be incorporated against the crash induced by covid- . the ongoing covid- pandemic, will most likely have many devastating global effects. besides, even if the mortality rate seems lower, thousands of lives were lost already at the time of this research. recent research on covid- confirmed previous findings that precautionary actions might be much less costly than recovery expenses. besides, the effects of immediate lockdown for weeks were perceived as much less harming than prolonging the control process [ ] . many restrictions have been imposed on mobility and economic activities, especially in europe and the usa. the economic impact is bi-directional for covid- . it has both supply and demand effects. with regards to consumption, we have been facing changing consumer attitudes and marketing channels. at the beginning of the covid- process, rising consumption demand has been encountered attached to stockpiling. but, both the demand dynamics and consumption and purchasing attitudes have changed. webbased online shopping tools have long been used all over the world. however, due to the quarantine enforcements and voluntary social distancing, there is a tendency to leave physical retail channels and shift to online shopping became visible. therefore, much of the consumption started to take place online and mostly restricted to consumables. while demand for necessities grows, aggregate demand has been falling. this rising online retailing tendency may induce growth in delivery sectors. but it also means unemployment in medium and large scale retail outlets. accordingly, the excess labour supply of retailing is a matter of fact to be considered. in addition to changing consumption and purchasing behaviours, there have been devastating effects on the services sector, which is also demand driven. specifically, the service sectors were shut down almost in every place where the disease has spread. the lack of operation in leisure, tourism, transportation and related services means reducing national income and unemployment of masses. unemployment would cyclically lead to reduced income, declining demand and a further reduction in all productive fields. also, having affected from the process is inevitable for agriculture and food industries as they are both attached to the services sector demand as well as direct consumption. in other words, agro-food demand of hotels or restaurants has shrunk due to lockdowns. therefore, excess food and agriculture supplies are expected to lead to declining prices. the downsizing of the services sector, when health care was left aside, seems to continue. besides, it seems that outdoor services demand will not recover soon checking by psychological effects of past epidemics. consequently, even maintaining agricultural labour and productivity seems to be problematic. there are endowment-related problems in term of industries. as mentioned above, consumption demand is almost restricted to non-durables. demand for durables and technology-endowed products have shrunk as well. besides, lockdown and reduced participation of employees to industrial production led to operational revenue loss in most countries, while closed borders meant the inability to reach inputs, energy and technological intermediaries. however, it is not definite when the pandemic ends or whether a second wave is on the way. if the process prolongs, the loss in industrial fields would also mean huge unemployment and income loss. if more and more people get unemployed in every sector, how can countries maintain rising consumption demand? the economies of many countries will derogate for sure and there will be global effects, some of which are already visible. the labour market contractions are very overwhelming. existing disequilibria in labour markets can get worsened within the process and it should be seriously taken care. this can be achieved by supporting employers rather than providing compensation payments to employees. the retailing outlets and market chains have faced with a challenge. the stores can utilise online channels, but declining offline shopping will still destroy labour market dynamics. accordingly, either employment in the retail sector needs to be secured or alternative ways should be applied. for underqualified labour, most of the countries need to arrange the attachment between agriculture and food industries and the retail sector. this also refers to an opportunity for developed courier services. in addition, smes involved in industrial production needs to be supported to prevent employee layouts and closures and interventions need to be timely checking out labour market estimates. when the services sector is considered, we cannot set health services apart this time. almost in all of the developed world, private health services and public investment calls to healthcare are being discussed. even though there have been warnings on contagious diseases, it was understood that precautionary acts were neglected. it is not possible to offer full expropriation of health care services. however, coordination between the public and private sector seems to be an obligation for future residing in this experience. how it could be achieved is another question. what should be avoided? the dilemma for most governments lays between the decision on fiscal deficits or health losses accompanied by economic and social devastation. in a macroeconomic perspective, fiscal and monetary actions need to be considered jointly. we have noted earlier that many countries allocated % of their budgets to fund early relief measures. some countries also have imposed different sorts of monetary expansion to keep macroeconomic indicators stable. simultaneous monetary and fiscal expansion or double deficit would most possibly lead to rising inflation and further poverty. however, again there we face another question. should we focus on the sustainability of the existing economic system or should we focus on maintenance of income levels not to distract economic activities? besides, many countries persuaded their banking systems to offer cheaper credits for urgent needs of households and smes. but these credits can be achievable only if the banking sector trusts in the financial system. therefore, the decision between lower interest rates and higher inflation, which might deteriorate macroeconomic indicators in the close future, is an additional dilemma specifically for developing countries. regarding this general overview, we can conclude that governments and financial management authorities should focus on three issues in the short term. in terms of economics, controlling labour market impurities and backward support to industries and services sector by financial tools are essential. morality assistance to individuals and societies is also required for economic and social trust. without trust, none of the sectoral impositions will be effective in economic recovery both with demand and supply aspects. besides, the covid- experience showed the need for reorganisation of health care services in a collaboratively. it is not hard to say that covid- experience will change many economic and social dynamics. relieving the pain needs proper time management and financial planning, and we learned the urge to get prepared for the future. epidemics and trust: the case of the spanish flu a simple planning problem for covid- lockdown. nber working paper series no: how does household spending respond to an epidemic? consumption during the covid- pandemic. nber working paper series no: thinking ahead about the trade impact of covid- macroeconomic crises since the coronavirus and the great influenza epidemic lessons from the "spanish flu" for the coronavirus's potential effects on mortality and economic activity the economic implications of epidemics old and new the growth economics of epidemics evaluating the economic consequences of avian influenza the potential economic impact of avian flu pandemic on taiwan the impact of the covid- pandemic on consumption: learning from high frequency transaction data microbes and markets: was the black death an economic revolution economics in the time of covid- . centre for economic policy research e-book sars: economic impacts and implications. erd policy brief of asian development bank spanish flu in italy: new data, new questions impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand the economic impact of quarantine: sars in toronto as a case study trading off consumption and covid- deaths the effect of coronavirus (covid- ) in the tourism industry in china international labour organisation monitor: covid- and the world of work world economic outlook what doesn't kill you makes you stronger? the impact of the spanish flu epidemic on economic performance in sweden effects of coronavirus disease (covid- ) on tourism industry of india. stud. indian place names globalization and disease: the case of sars economics and epidemics: an historical analysis on the effects of infectious diseases on economic development of four major outbreaks. bsc thesis on macroeconomics retail markets get a boost during covid- short term effects of covid- on us soybean and wheat exports the economic impact of covid- impacts of avian influenza virus on animal production in developing countries. cab reviews: perspectives in agriculture, veterinary science spillover of covid- : impact on the global economy the black death and the origins of the great divergence across europe, - european review of economic history disasters and community resilience: spanish flu and the formation of retail cooperatives in norway can trade policies soften the economic impacts of an avian influenza outbreak? assessment of economic vulnerability to infectious disease crises economic impact of sars: the case of hong kong the responses of consumption and prices in japan to the covid- crisis and the tohoku earthquake mitigating the covid economic crisis. centre for economic policy research e-book a general equilibrium analysis of the economic impact of a tourism crisis: a case study of the sars epidemic in taiwan publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -y vg frb authors: montané, xavier; kowalczyk, oliwia; reig-vano, belen; bajek, anna; roszkowski, krzysztof; tomczyk, remigiusz; pawliszak, wojciech; giamberini, marta; mocek-płóciniak, agnieszka; tylkowski, bartosz title: current perspectives of the applications of polyphenols and flavonoids in cancer therapy date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: y vg frb the development of anticancer therapies that involve natural drugs has undergone exponential growth in recent years. among the natural compounds that produce beneficial effects on human health, polyphenols have shown potential therapeutic applications in cancer due to their protective functions in plants, their use as food additives, and their excellent antioxidant properties. the possibility of combining conventional drugs—which are usually more aggressive than natural compounds—with polyphenols offers very valuable advantages such as the building of more efficient anticancer therapies with less side effects on human health. this review shows a wide range of trials in which polyphenolic compounds play a crucial role as anticancer medicines alone or in combination with other drugs at different stages of cancer: cancer initiation, promotion, and growth or progression. moreover, the future directions in applications of various polyphenols in cancer therapy are emphasized. the appearance of the severe acute respiratory syndrome coronavirus (sars-cov- ) in december last year and its very rapid spread around the world in early , known to cause covid- disease, has evidenced, among other things, the importance of investing in research to improve the people's quality of life or eradicate diseases that still do not have an effective treatment. as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , ′-trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cellsevents that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , -trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cells-events that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . curcuminoids are natural polyphenols that contain two phenol units joined through a linear diarylheptanoid. the presence of curcuminoids gives a yellow color to plants that contain these kinds of natural structures. the phenolic rings of curcuminoids are chemically modified with other chemical groups with the aim of overcoming some drawbacks of natural curcuminoids in clinical applications such as their poor solubility, low absorption, and bioavailability [ ] . among the curcuminoids, curcumin is one of the most known and studied structures with a high potential as medicine to treat different cancers, apart from also being useful in treating other types of diseases. nonetheless, the poor solubility of curcumin in water of acidic and physiological ph requires the use of diverse alternatives to avoid losing the effectiveness of curcumin as a medicine, such as the synthesis of other curcumin derivatives or the combination of curcuminoids with surfactants or co-surfactants. curcumin is a natural compound and the principal curcuminoid of turmeric plants, which is responsible for turmeric's yellow color [ ] . in addition to its applications in medicine, the use of curcumin has reached other fields. in the food industry, it has been used as a dietary supplement (it is sold as herbal supplement) or a food additive. additionally, it is used in cosmetics and other products. curcumin is commonly used in cancer therapies of different types of cancer: lung, cervix, prostate, breast, bone, and liver [ ] . nevertheless, the administration of free curcumin presents some drawbacks: poor solubility in water, instability in aqueous conditions, low bioavailability, and poor cellular uptake. to overcome these problems, two different solutions were attempted: - the synthesis of curcumin derivatives [ ] , and - the encapsulation of curcumin in different nanostructures ranging from liposomes to natural biopolymeric nanoparticles [ , ] . one of the curcumin derivatives used in breast and renal cancer therapies is dimethoxy curcumin. chen et al. recently proved that this curcumin derivative can be effective in the therapy of colon cancer cells due to causing the reduction of survivin expression and the enhancement of e-cadherin, a cell adhesion molecule, whose loss contributes to the formation of epithelial types of cancers such as carcinomas [ ] . recently, various research groups have reported that the combination of both curcumin and resveratrol can reduce the incidence of lung and prostate cancer [ , ] . lignans are diphenolic compounds found in a wide variety of plants including broccoli, beans, soybeans, rye, sesame seeds, pumpkin seeds, flax seeds, and some berries in very small amounts (µg of lignans per g of dry product) [ ] . their structure consists of two c -c units linked by β,β' bonds. lignans are one of the two main groups of phytoestrogens, which are well known for their good antioxidant properties. in fact, some antioxidant phytochemical compounds could be used as anticancer drugs as they are mimicking the functions of human hormones. some studies on rats showed that lignans prevent the growth of breast and prostate tumors [ , ] . numerous lignans could be considered as possible anticancer medicines due to their large pharmacologically valuable properties. among all of them, arctigenin, magnolol, and honokiol are the main lignans investigated in medicine. nonetheless, etoposide is a commercial lignin belonging to the podophilotoxin subfamily that is used in the treatment of different types of cancer such as lung cancer and breast cancer [ , ] . however, etoposide chemotherapy presents several side effects: low blood cell counts, vomiting, diarrhea, fever, loss of appetite, and alopecia. certain plants belonging to the family known as compositae produce arctigenin, especially the seeds of greater burdock (arctium lappa). some studies revealed that arctigenin inhibits the growth of various cancer cells: stomach, lung, liver, and colon, as well as leukocytes [ ] . at the same time, the addition of arctigenin intensifies the activity of caspase- , which is a protein that plays a crucial role in the death of carcinogenic cells. as a matter of fact, huang et al. demonstrated that the treatment of ovcar and skov ovarian cancers with arctigenin causes the apoptosis of cancer cells in vitro [ ] . one of the most used conventional anticancer drugs is doxorubicin, which is a medicine that belongs to the anthracycline family applied in the treatment of, among other cancers, bladder, stomach, ovaries, lung and thyroid cancers. however, doxorubicin exhibits side effects among which the most frequent are severe nauseas, vomiting, and alopecia [ ] . studies were conducted by lee et al. on adding natural products such as arctigenin to doxorubicin and determining the efficiency of both drugs in improving breast cancer treatment and reducing the side effects provoked by doxorubicin [ ] . the work concludes that the combination of arctigenin and doxorubicin induced the apoptosis of mda-mb- human breast cancer cells in vitro. the addition of arctigenin ameliorates the cellular uptake of doxorubicin, which causes the death of carcinogenic cells. another lignan that was tested in some studies on cancer therapy is magnolol. as its name indicates, magnolol is an isomer of honokiol found in magnolia bark [ ] . since ancient times, extracts from the bark of magnolia have been used in traditional chinese, korean, and japanese medicine. in the last decades, the research on the use of natural products in various cancer treatments has been focused on attempts of understanding mechanisms that induce the antitumor agents' response in the tumor cells [ ] . this year, su and co-workers elucidated the mechanism that reduces the endogen activity of nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), which is a protein complex that controls dna transcription and cell survival. therefore, the cells that do not have regulated nf-κb can contribute to the onset and growth of various types of cancers. moreover, magnolol used in the treatment of colorectal cancer reduces the phosphorylation of protein kinase c delta type (pkcδ) and nf-κb, which are two proteins that are involved in tumour progression in vitro and in vivo [ ] . following the methodology used with other drugs, magnolol was co-encapsulated with trastuzumab, an anticancer drug commonly used in stomach or throat cancer therapies, and gold nanoparticles, building a nanocarrier cluster. the synthesized nanocarriers induced a specific photothermal near-ir response combined with targeted anticancer activity resulting in an improvement of magnolol cytotoxicity to breast cancer cells [ ] . as mentioned before, honokiol (also known as houpa or hnk) is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus of magnolias, which includes around species. honokiol, which has been used in traditional eastern herbal medicines as an analgesic and together with magnolol, obovatol, and -o-methylhonokiol in the treatment of anxiety and mood disorders, has a spicy odor [ ] . honokiol is most frequently taken orally. in nature, honokiol and magnolol isomers are found together. usually, the separation and purification of both compounds had always been complexed, and it is commonly limited to hplc. in , amblard and co-workers developed a method in which the authors protect the near hydroxyl groups in magnolol to produce a magnolol acetonide that can be simply separated from honokiol via flash chromatography over silica [ ] . recent studies suggest that honokiol could be an effective agent in cancer treatment due to its physical properties-honokiol's ability to easily cross the blood-brain barrier and the bloodcerebrospinal fluid barrier-and its high bioavailability. many research studies have shown that honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that p-coumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that pcoumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids have a wide spectrum of functions in plants: -flavonoids attract pollinating insects through the color or smell that they give to the plant or its flowers, -filtration of uv light, -protection against herbivorous predators, -protection against fungi, -they are involved in the hormone auxin transport, -regulation of the cell cycle, -pigmented blue colors given by anthocyanins are responsible for the resistance of plants to the photooxidation of uv light from the sun, and - in carnivorous plants, they attract prey. usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c ′ in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : -in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. -in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. - in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, -by stopping the progression and proliferation of cancer cells, and -by inducing cancer cells apoptosis. one of the latest investigations to clarify the mechanism of kaempferol as an anticancer drug against breast tumors was carried out by zhu et al. the authors mentioned that kaempferol induced apoptosis and dna damage in mda-mb- cancer cells by the upregulation of the phosphorylated form of the h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm) [ ] . da and co-workers tested kaempferol in prostate cancer cells [ ] . the authors concluded that the use of kaempferol against lncap prostate cancer cell lines led to cancer cells death and impeded cancer cell proliferation and invasion in a dose-dependent manner. quercetin is the most common flavonoid in human diet with an average daily consumption of - milligrams [ ] . quercetin is mainly found in red onions, kale, apples, grapes, broccoli, and tea. in red onions, quercetin represents around % of its dry weight. various in vitro and in vivo studies showed that quercetin is one of the most potent antioxidants of the flavonoid family [ ] , which makes it an ideal candidate for an anticancer drug. indeed, quercetin is the active ingredient of yang-yin-qing-fei-tang, which is a traditional chinese medicine. furthermore, quercetin exhibited cytotoxicity in various tumor cells, in breast, cervical, colon, liver, lung, gastric, prostate cancers, and in leukemia [ , ] . making use of the anticancer effects of quercetin, the most recent studies combined quercetin with other anticancer drugs with the aim of increasing the efficiency of cancer therapies. some examples are summarized below. one of the natural compounds that lately has been combined with quercetin in cancer therapy studies is curcumin. srivastavaa et al. showed that the mixture of quercetin and curcumin improved the inhibition of cancer cell proliferation by regulating the wnt/β-catenin signaling and promoting the carcinogenic cells death by distinct pathways [ ] . furthermore, sunoqrot and co-workers combined both curcumin and quercetin by preparing nanoparticles with encapsulated curcumin and a shell of quercetin covalently bonded with polyethylene glycol (peg) prepared in a one-pot procedure [ ] . once tested in vivo, these nanocarriers exhibited a controlled drug delivery of curcumin in physiological conditions, which makes it a potentially powerful tool in cancer therapy. it has also been observed that the addition of quercetin to docetaxel therapy in prostate cancer reduces the docetaxel resistance of carcinogenic cells. that increases the efficacy of cancer therapy resulting from an intensification of the apoptosis of cancer cells and the reduction of tumor proliferation and migration [ ] . flavones are a class of flavonoids with a chemical structure very similar to flavonols, from which they only differ in the non-hydroxyl substitution at the carbon -position of flavones ( figure ). flavones are basically found in herbs (parsley, thyme, chamomile, mint, chrysanthemum flowers) and red or purple plants and vegetables (apple skins, broccoli, cabbages, celery, onion leaves, carrots, and red peppers) [ ] . in plants, flavones usually act as defense mechanisms against diseases originated by pathogens. some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . to enhance the anticancer effects of luteolin, the flavone is usually used together with other anticancer drugs. ren and co-workers demonstrated that the application of luteolin in combination with oxalipatlin, a conventional anticancer drug used to inhibit the development of cancer cells, stopped the proliferation of gastric cancer cells in vitro by the upregulation of the activity of caspase- and bax proteins [ ] . the construction of nanocarriers containing anticancer drugs allows obtaining controlled drug delivery systems. by the encapsulation of luteolin in polymeric micelles, hu et al. developed a thermosensitive nanocarrier that demonstrated an improved apoptosis of colorectal cancer cells compared to the administration of free luteolin [ ] . flavanones are colorless ketones derived from flavone. flavanones are found in a wide variety of foods included in our daily diet and in herbs [ , ] in citrus fruits, flavanones are usually glycosylated by a disaccharide in position ( figure ). they present different functions in plants: taste-modifying properties (eriodictyol, homoeriodictyol and sterubin), and -they are responsible for the bitter taste in citrus fruits (naringin). in the last decades, flavanones have gained a lot of importance in medicine for their antioxidant activity, radical scavenging, cardiovascular, anti-inflammatory, antiviral, and anticancer effects [ ] . naringenin and hesperetin are the most often investigated for being anticancer drugs. nevertheless, some tests were carried out using other flavanones such as didymin and alpinetin [ , ] . naringenin is a flavanone predominating in oranges and grapefruits. it is also found in bergamot, sour orange, tomatoes, cocoa, water mint, beans, etc. [ , ] . in some of these fruits, narigenin is present in its glycosidic form: naringin (which has attached a disaccharide neohesperidose via a glycosidic linkage at carbon ). as it has been proven in several studies, naringenin induces cytotoxicity in various carcinogenic cells of breast, stomach, liver, cervix, pancreas, colon cancers, and in leukemia [ ] . nevertheless, its poor solubility and instability in physiological medium limits the medical applications of naringenin. to solve these drawbacks, akhter et al. reported the encapsulation of naringenin in plga (poly(lactide-co-glycolid acid)) nanoparticles. moreover, they suggested that the encapsulated naringenin showed higher cytotoxicity when compared with free naringenin due to a more controlled drug release [ ] . another option that could enhance the anticancer properties of naringenin involves the synthesis of naringenin derivatives [ ] . an alternative recent study demonstrated naringenin's effectivity as an anticancer drug in breast cancer treatment is due to the activation of the caspase- protein and caspase- enzymes [ ] , while kumar and co-workers showed in vivo that naringenin showed antitumor effects on skin cancer [ ] . hesperetin and hesperetin's -o-glycoside (also known as hesperidin) are the main flavonoids found in lemons and sweet oranges [ ] . hesperetin's anticancer properties against specific tumors are well documented in numerous research publications: -it inhibits glucose uptake in various cancer cell lines [ , ] , -reduces the nf-κb activity, which leads to a decrease in tumor progression [ ] , and -upgrades the apoptosis via the induction of intracellular ros formation [ ] . in a more recent study, the addition of hesperetin improves the activity of cisplatin, which is an anticancer drug that is commonly used to treat lung cancer [ ] . it was observed that hesperetin inhibits mdr protein (multidrug resistance protein ), which is associated with the resistance to cisplatin developed in a great number of patients subjected to cancer therapy. curiously, the administration of both naringenin and hesperetin were tested in vitro and in vivo trials to analyze the anticancer effects in human pancreatic cancer [ ] . for the first time, the authors reported that the combination of both naringenin and hesperetin could be used as a potential non-toxic cancer therapy system that stops pancreatic cancer development. flavanols or flavan- -ols are another group of monomeric flavonoids. catechin and its derivatives are included in this group. natural sources of flavan- -ols are mainly the "tea plant" (camellia sinensis), and some cocoas. therefore, they are highly present in the human diet in both beverages (tea) and solid foods (chocolates) [ , ] . since studies of flavanols have started in the course of the last century, it has been found that these compounds provide resistance against dangerous trespassers, including microbes, fungi, insects, and herbivorous animals [ ] . thereby, the flavanols' health benefits have been broadly studied in humans. some investigations suggest that the intake of cocoa flavanols could help in the prevention of cardiovascular and metabolic diseases. indeed, the european food safety authority approved cocoa products containing mg of flavanols because they "help to maintain the elasticity of blood vessels, which contributes to normal blood flow" [ ] . epigallocatechin gallate (epigallocatechin- -gallate or egcg) is a catechin that is mostly found in tea and one of the polyphenolic compounds most commonly found in nature; it is also the ester of epigallocatechin and gallic acid [ ] . the objective of finding a correlation between green tea intake and the risk of cancer onset has been a well-studied topic [ ] . as an obvious example, the study presented by guo et al. [ ] validated that the consumption of green tea-and therefore catechins-up to seven cups a day provided a small reduction in the prostate cancer risk. moreover, egcg has been tested against certain cancer cell lines. in ht- colorectal cell lines, egcg upregulated the activity of tfr (transferrin receptor), which is a carrier protein for transferrin, and inhibited the activity of the ferritin-h protein via the iron chelation activity in ht- colorectal cancer cells [ ] . in another example, the synergistic effect of egcg and trail (tumor necrosis factor (tnf)-related apoptosis-inducing ligand), a protein that causes cell death, intensifies the activity of both caspase and the death receptor , causing the death of sw and hct colon cancer cells [ ] . despite the fact that egcg is commonly found in nature, this flavanol shows some drawbacks that limit its applications in cancer therapy (poor stability, low absorption, and hepatotoxicity) [ ] . so, the encapsulation of egcg can be a promising solution to minimize the limitations of the egcg use [ ] . the (−)-epicatechin molecule is a flavonoid of which large quantities are found in cocoa [ ] . the use of epicatechin in cancer therapy has been emerging over the last decade in the attempt to overcome some of the drawbacks of egcg [ , ] . pereyra-vergara and co-workers studied the effects and mechanism of (−)-epicatechin in breast cancer cells [ ] . it was shown that the addition of (−)-epicatechin to carcinogenic cells results in the apoptosis of the two tested breast cancer cell lines (mda-mb- and mcf- ). moreover, the authors proved that (−)-epicatechin increased the intracellular ros production and intensified the activity of bcl associated agonist of cell death (bad) and bcl- -like protein (bax), proteins that are associated with cell apoptosis. isoflavones are another type of biological active flavonoids. isoflavones are mostly found in plants of the leguminosea family. this family includes many species that are of great importance in the human diet (peas, lentils, licorice, beans, chickpeas, and carob), in animal fodder (alfalfa, clover, and carob) and as ornamental plants (mimosa and false acacia) [ , ] . since isoflavones present estrogenic properties, plants use these kinds of compounds as part of their natural defense system against the overpopulation of herbivores by controlling their male fertility [ ] . moreover, these properties make isoflavones good complementary therapeutic options in treating menopause and its symptoms such as osteoporosis, anxiety, emotional instability, and headaches. genistein and daidzein are the most studied compounds of this subgroup in terms of medical applications. nevertheless, other isoflavones such as glabridin and alpinumisoflavone have raised interest as potential cancer medicines in various types of cancer such as breast, liver, or thyroid cancers [ , ] . the isoflavone most reported in medicine is genistein, which is a phytoestrogen compound produced in soybeans. genistein was for the first time isolated in . however, it was not until the end of the last century that researchers started to explore its potential beneficial effects on human health and its possible applications as a medical compound in a wide range of diseases, including cardiovascular diseases, osteoporosis prevention, diabetes, and some types of cancers [ ] . it has been proven that genistein is involved in the regulation of different genes that are associated with the onset of cancers by various mechanisms [ ] . in a recent research, hsiao et al. studied the effects and mechanisms of genistein against leukemia cell lines. in fact, the application of genistein to hl- leukemia cells revealed that this natural medicine kills the carcinogenic cells via two different pathways (endoplasmatic reticulum stress and mitochondria-dependent pathway) in vitro and in mouse xenograft models in vivo [ ] . furthermore, different authors studied the effects of genistein when it is combined with other anticancer drugs [ ] . in a recent investigation, liu et al. tested mixtures of genistein and cisplatin in varied concentrations as a plausible anticancer agent in the treatment of cervical cancer cells [ ] . the authors proved that the addition of genistein improved the chemotherapeutic activity of cisplatin, requiring a lower dose of the drug in cancer treatment, which led to a reduction in the therapy side effects. the second isoflavone most commonly found in nature, which similar to genistein is also isolated from soybeans, is daidzein [ ] . the chemical structure of daidzein is very similar to genistein, without the hydroxyl group at position (table ) . rigalli et al. studied in vitro the effects of daidzein use in breast cancer therapy [ ] . in one of those studies, they proved that daidzein downregulated the expression of multidrug resistance-associated protein (mrp ) in both michigan cancer foundation- (mcf- ) and mda-mb- breast cancer cell lines. the reduction of this protein's activity is very important because mrp is involved in transporting many of the chemotherapeutic drugs out of the cells (for example, doxorubicin or mitoxantrone). in another study in vivo, mice were inoculated with t breast cancer cells and then treated with daidzein administered orally for days. in this case, the highest dose of daidzein ( mg/kg) was required to observe a considerable decrease in tumor size. at the same time, the authors reported that the combination of daidzein with regular exercise promotes the breast cancer cells apoptosis via the fas/fasl-mediated mechanism [ ] . chalcones are a class of polyphenolic compounds that are characterized by the presence of an aromatic ketone and an enone in their central core. many fruits such as citrus and apples, vegetables such as tomatoes, potatoes, shallots, and bean sprouts, and some edible plants such as licorice contain chalcones [ ] . besides, chalcones can be synthesized in the form of base-catalyzed aldol condensation of benzaldehydes with acetophenones (for example, sodium hydroxide) [ ] . the most studied chalcone in the field of medicine is ellagic acid, which has been investigated as a potential antitumor agent [ , ] . ellagic acid is an antioxidant that is found in various natural resources: in oak species such as white oak (quercus alba) and european red oak (quercus robur) or in medicinal fungi (phellinus linteus). peaches, pomegranates, grapes, strawberries, raspberries, pecans, walnuts, and raw chestnuts also contain a considerable amount of ellagic acid [ ] . the anti-proliferative and antioxidative properties of ellagic acid have encouraged researchers to study the health benefits of this natural compound. for years, the effects of treating tumors with ellagic acid have been studied by the evaluation of various alternatives (chemical modifications of ellagic acid or its encapsulation among other options) [ ] . one of the last studies that examined the breast cancer treatment with ellagic acid was published by yousuf et al. [ ] . this work evaluated the capacity of numerous phytochemicals in addition to ellagic acid (capsaicin, tocopherol, rosmarinic acid, ursolic acid, limonene, caffeic acid, and ferulic acid) to inhibit the activity of cyclin-dependent kinase (cdk ), which is an important gene associated with cancer progression. among all the tested natural compounds, ellagic acid showed the highest binding affinity for cdk , decreasing the tumor proliferation. however, the encapsulation of ellagic acid to enhance its poor solubility combined with an improvement of its controlled delivery was attempted by some research groups [ , ] . in a recent work, pirzadeh-naeeni et al. reported the nanoencapsulation of ellagic acid in two different biopolymers (schizophyllan and chitin), which were then tested against mcf- breast cancer cells [ ] . in this case, the controlled release of ellagic acid improved the cytotoxicity when compared with non-encapsulated ellagic acid. it also reduced the progression of tumor cells. anthocyanidins are water-soluble pigments found in plants. they are responsible for leaves, flowers, and fruit colors. some fruits included in the human diet are rich in anthocyanins: blueberries, raspberries, black rice, and black soybeans (normally known as dark fruit). the term anthocyanin was coined in by ludwig clamor marquart, a german pharmacist, to denote the blue pigment of red cabbage (brassica oleracea) [ ] . table . summary of various polyphenols, their chemical structures, and their anticancer effects. resveratrol dna protection against reactive oxygen species (ros), trap the hydroxyl and superoxide groups and the free radicals produced into the cells. inhibition of a lung cancer cells with the activation of caspase- . u. s. department of health and human services public health service food and drug administration status: bulk ingredient for human prescription compounding. [ , ] other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). [ [ ] [ ] [ ] , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). improves the cellular uptake of doxorubicin and reduces its side effects. apoptosis of mda-mb- breast cancer cells. [ , ] of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). kappa-light-chain-enhancer of activated b cells (nf-κb) signaling through protein kinase c delta type (pkcδ) inactivation. upgrades the cytotoxicity of trastuzumab and increases the specificity to breast cancer cells. synergistic effects of honokiol and doxorubicin in breast cancer by suppressing the metastasis of carcinogenic cells and apoptosis induction. apoptosis of multiple myeloma cancer cells. [ , ] p-coumaric acid apoptosis of hct- colon cancer cells through ros mitochondrial pathway. inhibits the growth of snu- gastric cancer cells. [ [ ] [ ] [ ] kaempferol induces the apoptosis and dna damage in mda-mb- breast cancer cells by the upregulation of h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm). induces the apoptosis of lncap prostate cancer cells. impedes the proliferation of cancer cells. luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] u. s. department of health and human services public health service food and drug administration status: drug for further processing. promotes apoptosis of pancreatic cancer cells by increasing intracellular ros. damages dna of hela cervical cancer cells. inhibits the growth of cancer cells and induces its apoptosis. [ , ] luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] naringenin [ , ] naringenin apoptosis of breast cancer cells by the increase of the activity of caspase- and caspase- . suppression of skin cancer cells. [ , ] molecules , in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used anthocyanidins have varied functions in plants: attracting pollinating insects, preventing the freezing of fruits such as grapes, and protecting plants against harmful uv radiation [ ] . moreover, these kinds of compounds are widely used in the food industry (preparation of food coloring, a parameter for determining wine quality) and in medical industry (decreased risk of contracting various diseases such as obesity, improved memory and age-related deficiencies, or improvement of the immunological system) due to their chemical and physical properties [ ] . some anticancer properties of anthocyanidins extracted from the plant cyanomorium coccineum have been recently described by rescigno et al., which demonstrated the antiproliferative effect of anthocyanidins against different leukemia cell lines [ ] . delphinidin, which can be found in red cabbage, grapes, berries, and sweet potatoes among other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used as chemotherapy adjuvant agents in cancer therapies. however, the process of discovering the polyphenols' mechanisms of action as anticancer drugs and their interactions with tumors requires further studies in order for those natural compounds to improve the actual therapeutic strategies. the authors declare no conflict of interest. cancer nanotherapy: concept for design of new drug cancer statistics molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal advances in cancer therapy with plant based natural products new aspects of natural-killer-cell surveillance and therapy of cancer natural polymers: a source of inspiration discovery, development, and regulation of natural products. in using old solutions to new problems-natural drug discovery in the st century discovery, development, and regulation of natural products cancer chemoprevention with dietary phytochemicals dietary phytochemicals and human health phytochemicals modulate cancer aggressiveness: a review depicting the anticancer efficacy of dietary polyphenols and their combinations polyphenols as antimicrobial agents plant polyphenols: chemical properties, biological activities, and synthesis the pharmacology of avenanthramides: polyphenols ultrasound-assisted extraction of biologically active compounds and their successive concentration by using membrane processes stability and anti-proliferative properties of biologically active compounds extracted from cistus l. after sterilization treatments minireview-biological effects of resveratrol resveratrol and cancer: challenges for clinical translation resveratrol in cancer patients: from bench to bedside resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses in vitro and in vivo evaluation of the antitumor efficiency of resveratrol against lung cancer. asian pac phase i randomized, double-blind pilot study of micronized resveratrol (srt ) in patients with hepatic metastases-safety, pharmacokinetics, and pharmacodynamics a phase study of srt (resveratrol) with bortezomib for patients with relapsed and or refractory multiple myeloma resveratrol reduces the levels of circulating androgen precursors but has no effect on, testosterone, dihydrotestosterone, psa levels or prostate volume. a -month randomised trial in middle-aged men biological activities of curcuminoids, other biomolecules from turmeric and their derivatives-a review compound from natural sources, a true scientific challenge-a review synthesis of curcumin derivatives and analysis of their antitumor effects in triple negative breast cancer (tnbc) cell lines photo-triggered capsules based on lanthanide-doped upconverting nanoparticles for medical applications encapsulation for cancer therapy dimethoxy curcumin induces apoptosis by suppressing survivin and inhibits invasion by enhancing e-cadherin in colon cancer cells potential of curcumin and resveratrol as biochemical and biophysical modulators during lung cancer in rats evaluation of biophysical as well as biochemical potential of curcumin and resveratrol during prostate cancer lignans in food and nutrition flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis anticancer effects of a plant lignan -hydroxymatairesinol on a prostate cancer model in vivo irinotecan compared with etoposide in combination with platinum in previously untreated extensive stage small cell lung cancer: an updated systemic review a systematic review and pooled analysis of studies of oral etoposide in metastatic breast cancer molecular mechanisms of the action of arctigenin in cancer arctigenin promotes apoptosis in ovarian cancer cells via the inos/no/stat /survivin signalling nanoparticle delivery strategies to target doxorubicin to tumor cells and reduce side effects arctigenin enhances the cytotoxic effect of doxorubicin in mda-mb- breast cancer cells therapeutic applications of compounds in the magnolia family anticancer activity of natural compounds from plant and marine environment suppression of pkcdelta/nf-kappab signaling and apoptosis induction through extrinsic/intrinsic pathways are associated magnolol-inhibited tumor progression in colorectal cancer in vitro and in vivo near ir responsive targeted integrated lipid polymer nanoconstruct for enhanced magnolol cytotoxicity in breast cancer facile purification of honokiol and its antiviral and cytotoxic properties honokiol for cancer therapeutics: a traditional medicine that can modulate multiple oncogenic targets honokiol: a review of its anticancer potential and mechanisms synergistically enhanced antimetastasis effects by honokiol-loaded ph-sensitive polymer−doxorubicin conjugate micelles honokiol induces apoptosis in human lymphoid leukemia molt b cells the natural product honokiol induces caspase-dependent apoptosis in b-cell chronic lymphocytic leukemia (b-cll) cells honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis bioactivity of phenolic acids: metabolites versus parent compounds: a review dietary hydroxybenzoic acid derivatives-nature, occurrence and dietary burden in situ antioxidant and antimicrobial activities of naturally occurring caffeic acid, p-coumaric acid and rutin, using food systems events associated with apoptotic effect of p-coumaric acid in hct- colon cancer cells effects of p-coumaric acid on microrna expression profiles in snu- human gastric cancer cells anti-proliferative properties of p-coumaric acid in snu- gastric cancer cells anthocyanins and other flavonoids research trends in flavonoids and health phenol content in sprouted grains tautomerism of flavonol glucosides: relevance to plant uv protection and flower colour enhancement of oxidative and drought tolerance in arabidopsis by overaccumulation of antioxidant flavonoids dietary flavonoids: bioavailability, metabolic effects, and safety role of fisetin in chemosensitization health-promoting components of fruits and vegetables in the diet the mechanism of anticancer action and potential clinical use of kaempferol in the treatment of breast cancer kaempferol suppresses proliferation and induces cell cycle arrest, apoptosis, and dna damage in breast cancer cells kaempferol promotes apoptosis while inhibiting cell proliferation via androgen-dependent pathway and suppressing vasculogenic mimicry and invasion in prostate cancer review of the biology of quercetin and related bioflavonoids fruits and vegetables in the prevention of cellular oxidative damage quercetin is the active component of yang-yin-qing-fei-tang to induce apoptosis in non-small cell lung cancer pharmacological basis and new insights of quercetin action in respect to its anti-cancer effects curcumin and quercetin synergistically inhibit cancer cell proliferation in multiple cancer cells and modulate wnt/β-catenin signaling and apoptotic pathways in a cells bioinspired polymerization of quercetin to produce a curcumin-loaded nanomedicine with potent cytotoxicity and cancer-targeting potential in vivo quercetin reverses docetaxel resistance in prostate cancer via androgen receptor and pi k/akt signaling pathways the herbal medicine sho-saiko-to inhibits proliferation of cancer cell-lines by inducing apoptosis and arrest at the g( )-g( )-phase medicinal importance, pharmacological activities, and analytical aspects of hispidulin: a concise report curcumin and apigenin-novel and promising therapeutics against chronic neuroinflammation in alzheimer's disease in vitro anti-inflammatory effect of apigenin in the helicobacter pylori-infected gastric adenocarcinoma cells antal, i. study on the pulmonary delivery system of apigenin-loaded albumin nanocarriers with antioxidant activity apigenin and cancer chemoprevention a plant flavone playing noble roles in cancer prevention via modulation of key cell signaling networks. recent pat. anti-cancer drug discov apigenin in cancer therapy: anti-cancer effects and mechanisms of action mutant p , stabilized by its interplay with hsp , activates a positive feed-back loop between nrf and p that induces chemo-resistance to apigenin in pancreatic cancer cells the stability and activity changes of apigenin and luteolin in human cervical cancer hela cells in response to heat treatment and fe + /cu + addition intestinal absorption of luteolin and luteolin -o-beta-glucoside in rats and humans molecular targets of luteolin in cancer apoptosis induced by luteolin in breast cancer: mechanistic and therapeutic perspectives luteolin suppresses the proliferation of gastric cancer cells and acts in synergy with oxaliplatin thermosensitive in situ gel containing luteolin micelles is a promising efficient agent for colorectal cancer peritoneal metastasis treatment polyphenols: a concise overview on the chemistry, occurrence, and human health a comprehensive review on flavanones, the major citrus polyphenols didymin, a dietary flavonoid glycoside from citrus fruits, induces fas-mediated apoptotic pathway in human non-small-cell lung cancer cells in vitro and in vivo antiproliferative effect of alpinetin in bxpc- pancreatic cancer cells antiatherogenic properties of naringenin, a citrus flavonoid review of the flavonoids quercetin, hesperetin naringenin. dietary sources, bioactivities, and epidemiology inhibitory effects of naringenin on tumor growth in human cancer cell lines and sarcoma s- -implanted mice sonication tailored enhance cytotoxicity of naringenin nanoparticle in pancreatic cancer: design, optimization, and in vitro studies optimizing the flavanone core toward new selective nitrogen-containing modulators of abc transporters naringenin has a chemoprotective effect in mda-mb- breast cancer cells via inhibition of caspase- and - activities naringenin suppresses chemically induced skin cancer in two-stage skin carcinogenesis mouse model therapeutic potential of hesperidin and its aglycone hesperetin: cell cycle regulation and apoptosis induction in cancer models hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells natural compounds regulate glycolysis in hypoxic tumor microenvironment molecular mechanisms behind the biological effects of hesperidin and hesperetin for the prevention of cancer and cardiovascular diseases hesperidin: a promising anticancer agent from nature hesperetin reverses p-glycoprotein-mediated cisplatin resistance in ddp-resistant human lung cancer cells via modulation of the nuclear factor-κb signaling pathway combined administration of naringenin and hesperetin with optimal ratio maximizes the anti-cancer effect in human pancreatic cancer via down regulation of fak and p signaling pathway cocoa flavanols: natural agents with attenuating effects on metabolic syndrome risk factors scientific opinion on the modification of the authorisation of a health claim related to cocoa flavanols and maintenance of normal endotheliumdependent vasodilation pursuant to article ( ) of regulation (ec) no / following a request in accordance with article of regulation (ec) no epigallocatechin- -gallate (egcg): chemical and biomedical perspectives cancer prevention by tea: animal studies, molecular mechanisms and human relevance green tea and the risk of prostate cancer a systematic review and meta-analysis iron chelation properties of green tea epigallocatechin- -gallate (egcg) in colorectal cancer cells: analysis on tfr/fth regulations and molecular docking. evid-based complementary altern epigallocatechin- -gallate induces apoptosis as a trail sensitizer via activation of caspase and death receptor in human colon cancer cells the safety of green tea and green tea extract consumption in adults-results of a systematic review cancer therapeutics with epigallocatechin- -gallate encapsulated in biopolymeric nanoparticles flavor chemistry of cocoa and cocoa products-an overview antileukemic action of (−)-epicatechin in the spleen of rats with acute myeloid leukemia induction of apoptosis of sw human colon cancer cells by (−)-epicatechin isolated from bulnesia sarmienti apoptosis induced by (−)-epicatechin in human breast cancer cells is mediated by reactive oxygen species phenolic compounds as functional ingredients in beverages phytochemical mimicry of reproductive hormones and modulation of herbivore fertility by phytoestrogens. environ. health persp glabridin inhibits migration and invasion by transcriptional inhibition of matrix metalloproteinase through modulation of nf-kappa b and ap- activity in human liver cancer cells alpinumisoflavone inhibits tumor growth and metastasis in papillary thyroid cancer via upregulating mir- - p recent progress on biocompatible nanocarrierbased genistein delivery systems in cancer therapy the role of genistein and synthetic derivatives of isoflavone in cancer prevention and therapy genistein induces apoptosis in vitro and has antitumor activity against human leukemia hl- cancer cell xenograft growth in vivo anti-inflammatory and anticarcinogenic effect of genistein alone or in combination with capsaicin in tpa-treated rat mammary glands or mammary cancer cell line effects of genistein on anti-tumor activity of cisplatin in human cervical cancer cell lines nutritional epigenetic regulators in the field of cancer the phytoestrogens daidzein and equol inhibit the drug transporter bcrp/abcg in breast cancer cells: potential chemosensitizing effect synergetic inhibition of daidzein and regular exercise on breast cancer in bearing- t mice by regulating nk cells and apoptosis pathway a review on natural chalcones an update synthesis and anti-inflammatory activity of three nitro chalcones novel -amino chalcones: design, synthesis and biological evaluation antiproliferative and pro-apoptotic activities of -and -aminochalcones against tumor canine cells ellagic acid induces cell cycle arrest and apoptosis through tgf-β/smad signaling pathway in human breast cancer mcf- cells ellagic acid, sulforaphane, and ursolic acid in the prevention and therapy of breast cancer: current evidence and future perspectives ellagic acid controls cell proliferation and induces apoptosis in breast cancer cells via inhibition of cyclin-dependent kinase development of biodegradable nanoparticles for oral delivery of ellagic acid and evaluation of their antioxidant efficacy against cyclosporine a-induced nephrotoxicity in rats preparation of β-cd-ellagic acid microspheres and their effects on hepg cell proliferation a comparative study on schizophyllan and chitin nanoparticles for ellagic acid delivery in treating breast cancer anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits anthocyanin biosynthesis and degradation mechanisms in solanaceous vegetables: a review food applications and physiological effects of anthocyanins as functional food ingredients antiproliferative and antiviral extracts from sardinian maltese mushroom (cynomorium coccineum l.) updating the research on prodelphinidins from dietary sources influence of fruit juice processing on anthocyanin stability delphinidin induces apoptosis via cleaved hdac -mediated p acetylation and oligomerization in prostate cancer cells delphinidin inhibits bdnf-induced migration and invasion in skov ovarian cancer cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -v pu authors: tootee, ali; larijani, bagher title: ramadan fasting during covid- pandemic date: - - journal: j diabetes metab disord doi: . /s - - -z sha: doc_id: cord_uid: v pu nan intermittent fasting) can be attributed to reduced free-radical production. [ ] recently, it was brought to light that fasting can activate certain adaptive cellular responses which can improve glucose regulation, increase resistance to stress, and suppress inflammatory reactions. [ ] today, it is demonstrated that fasting can activate different cellular pathways which foster intrinsic defense mechanisms against metabolic and oxidative stress, and this can lead to removal of damaged cells and molecules. [ ] decisive evidence emerged from numerous in-depth studies have clearly demonstrated that fasting can produce therapeutic effects in many noncommunicable disorders such as obesity, diabetes, cardiovascular disease, cancers, and neurodegenerative diseases. [ ] fasting can also play a protective role against aging. a comprehensive meta-analysis study which was recently conducted has demonstrated that the average life expectancy can significantly increase by fasting. [ ] moreover, fasting can improve physical composition, function, and endurance. [ , ] fasting can also enhance cognitive functions in terms of spatial memory, associative memory and working memory, and can reverse neurotoxic effects of obesity, diabetes, and neuroinflammation. [ ] this dietary intervention can even facilitate amelioration of cognitive deficits in traumatic brain injury. [ ] moreover, results of an study we conducted in demonstrated that fasting has a remarkable influence in promotion of psychologic health and regulation of sleep pattern. [ ] these evidence and similar findings indicate that the effects of ramadan fasting are not merely confined those related to refraining from eating and drinking, and they include a diverse range of physical, psychological, and spiritual health. considering diverse benefits of fasting, this ancient dietary alternation has been recently bought to light as a therapeutic clinical intervention. in clinical settings, caloric restriction and fasting has been documented to improve several cardiometabolic risk factors in nonobese humans. [ ] this healing effect is medicated through a wide range of mechanisms such as modification of blood pressure, resting heart rate, levels of high-density and low-density lipoprotein (hdl and ldl) cholesterols, triglycerides, glucose, and insulin concentration, and insulin resistance. [ ] [ ] [ ] besides, atherosclerosisassociated systemic inflammation and oxidative stress markers can be significantly reduced in fasting. [ , ] fasting can also protect individuals against malignancies. several detailed studies have demonstrated that different fasting regimens can decrease the rate of occurrence of spontaneous tumors in aged animals. moreover, growth of induced tumors may be suppressed in fasting while their sensitivity to chemotherapy and radiotherapy is markedly increased. [ ] [ ] [ ] it is also well-documented that excessive energy intake can increase the risks of stroke, alzheimer's disease, and parkinson's disease. astonishingly, fasting can delay or even reverse development and progress of such neurological defects. [ ] this can be attributed to several mechanisms such as strengthening mitochondrial function, stimulation of autophagy, increasing production of neurotrophic-factor, activation of antioxidant defense mechanisms, and acceleration of dna repair processes. [ ] by means of the aforementioned mechanisms, fasting can also alleviate symptoms of asthma, multiple sclerosis, and arthritis. [ ] [ ] [ ] notwithstanding all documented health benefits of fasting, in this ramadan, there has emerged some uncertainty as to whether fasting is safe during the current outbreak. millions of people have contracted covid- , hundreds of thousands have develop severe illness, and tens of thousands have perished. [ ] this time, arguably for the first time in the modern era, jurisprudence scholars, academics, and medical practitioners all seem perplexed as to whether temporary starvation and dehydration might increase the risk of contracting the circulating corona virus. unfortunately, there is a paucity of academic literature on the effects of ramadan fasting. although there exists a considerable body of literature on the benefits of intermittent fasting, [ ] most such research have focused on mere calorie restriction, and have not taken the potential effects of simultaneous dehydration and dryness of airways into consideration. [ ] this makes a substantial difference, and, therefore, the findings of studies on different patterns of intermittent fasting cannot be simply generalized to ramadan fasting. moreover, it is sometimes claimed that dry mouth and throat caused by ramadan fasting may increase the risk of contracting the virus. although this belief has been refuted by major health and religious authorities, the evidence produced in this regard are neither reliable nor incontrovertible. [ ] therefore, it appears that scientific studies exclusively carried on ramadan fasting constitute the only available source of information for jurisprudence scholars, academics, and medical practitioners. as regards immunological changes, it is documented that ramadan fasting does not lead to any immunological malfunction. [ ] on the contrary, there exist a considerable body of evidence that suggest ramadan fasting can strengthen the immune system against infections. this immunoprotective effect, in part, may be attributed to increased serum levels certain immunoglobulins such as iga. [ ] moreover, levels of some elements of the complement system (such as c ) are demonstrated to increase during ramadan fasting, thereby protecting individuals against a wide range of microorganisms. [ ] many such immunoprotective effects are also confirmed by clinical evidence. for instance, lahdimawan et al. have demonstrated that ramadan fasting can play a protective role against tuberculosis, and can minimize the risk of the infection in healthy individuals. [ ] nonetheless, one should be very cautious in interpretation of such results. in fact, several studies have brought some unfavorable consequences of fasting into light. for instance there is some evidence suggesting that ramadan fasting can decrease igg levels. [ ] moreover, in patients with diabetes, it is demonstrated that ramadan fasting can lead to an increased risk of infection. [ ] notwithstanding all aforementioned evidence in favor or against the practice of ramadan fasting in terms of infection risk, there is no conclusive evidence to acknowledge or refute increased susceptibility of fasting individuals to contracting covid- infection. similarly, no evidence has emerged indicating that drinking water during daytime can prevent corona virus infection in healthy individuals. [ ] inconclusive as the situation may appear, the current pandemic has led to formation of a individual-based approach to jurisprudence dilemmas in some schools of islam. accordingly, many academics, physicians, and jurisprudence scholars have arrived at the conclusion that the risk of infection (and development of possible life-threatening consequences) should be considered on an individualized basis. accordingly, the elderly and the frail should refrain from fasting lest potential deadly consequences. nonetheless, old persons in quarantine who are confident that they are not exposed to the virus can fast provided that they consume a balanced diet and have adequate rest. [ ] in terms of healthcare professionals, it is advised that they should assess their own situation on a daily basis, and they could break their fasting if they cannot perform their duties or perceive themselves at a great risk. all aforementioned recommendations and advises need to be taken into consideration by both the persons intending to fast and the physicians in charge. in this regard, an official declaration has recently been published by the academy of medical sciences (ams) of islamic republic of iran. in this official announcements, it is recommended that healthy individuals younger than can observe ramadan fasting provided that they follow certain precautions. [ ] these precautions include: strict adherence to all infection control guidelines and procedures, proper hydration in non-fasting hours, humidification of indoor air, consumption of a balanced diet rich in fruits and vegetables, and avoiding dry and hot environments. this assembly which was consisted of a great number of high-ranking physicians, scientists, theologists, and, jurisprudence scholars, has also recommended that considering high incidence of hypertension, diabetes, and cardiovascular disease in persons older than , they should refrain from fasting lest possible dire consequences. however, ultimately, this decision should be made by any individual patient himself according to his physicians' advise. in regards with patients with diabetes who are younger than , according to this announcement, they can only observe fasting provided that their diabetes is perfectly controlled (with hba c < ) through proper lifestyle modifications and medications. this official document has also decided that fasting should be prohibited in patients with history of organ transplantation (with the exception of corneal transplantation). in regards with those infected with the virus, the assembly announced that they should refrain from fasting for several weeks after complete recovery. it is noteworthy that these recommendations are all in complete accordance with the highly credible scientific evidence ams members had previously published on the subject. for instance, in a comprehensive research on the subject which was published by members on the assembly in , authors had contended that although ramadan fasting is generally safe and wholesome for healthy individuals, those with certain diseases and adverse health conditions should seek their physicians' advice in this regard. [ ] in conclusion, this year, it appears that there exists a widespread confusion surrounding the practice of ramadan fasting amid covid- pandemic, and jurisprudence scholars, scientists, and physicians are all similarly perplexed as to whether or not fasting is safe. in some schools of islam, it is even contended that fear and risk of contracting the infection does not justify non-adherence of followers to fasting which is considered as one of the four fundamental pillars of faith in islam. [ ] accordingly, several different (and sometimes contradictory) guidelines, fatwas, and recommendations have emerged on the subject. therefore, it would be feasible to contend that the ultimate decision in this regard should be made by each individual person (according to the fatwas) based on the recommendations of the physicians in charge. respiratory disease and ramadan. the lancet respiratory medicine, , respiratory disease and ramadan the effect of ramadan fasting on fasting serum glucose in healthy adults ramadan major dietary patterns ramadan fasting and diabetes. payesh (health monitor) islamic principles and decision making in bioethics caloric intake and aging fasting: molecular mechanisms and clinical applications effects of intermittent fasting on health, aging, and disease intermittent metabolic switching, neuroplasticity and brain health dietary restriction in rats and mice: a meta-analysis and review of the evidence for genotype-dependent effects on lifespan time-restricted feeding is a preventative and therapeutic intervention against diverse nutritional challenges effects of eight weeks of time-restricted feeding ( / ) on basal metabolism, maximal strength, body composition, inflammation, and cardiovascular risk factors in resistance-trained males cognitive and behavioral evaluation of nutritional interventions in rodent models of brain aging and dementia shortterm caloric restriction exerts neuroprotective effects following mild traumatic brain injury by promoting autophagy and inhibiting astrocyte activation ramadan fasting, mental health and sleep-wake pattern significant improvement in cardiometabolic health in healthy nonobese individuals during caloric restriction-induced weight loss and weight loss maintenance intermittent food deprivation improves cardiovascular and neuroendocrine responses to stress in rats long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma the effect of intermittent energy and carbohydrate restriction v. daily energy restriction on weight loss and metabolic disease risk markers in overweight women caloric restriction improves health and survival of rhesus monkeys caloric restriction and cancer: molecular mechanisms and clinical implications fasting and cancer: molecular mechanisms and clinical application brain insulin resistance in type diabetes and alzheimer disease: concepts and conundrums autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities fasting followed by vegetarian diet in patients with rheumatoid arthritis: a systematic review diet-induced weight loss in obese children with asthma: a randomized controlled trial a diet mimicking fasting promotes regeneration and reduces autoimmunity and multiple sclerosis symptoms covid- -looking beyond tomorrow for health care and society ramadan during covid- : what clerics and health experts say, in gulf news effects of ramadan fasting on serum immunoglobulin g and m, and salivary immunoglobulin a concentrations the effect of fasting on the immune system of athletes during holly ramadan the effect of ramadan fasting on blood biochemical factors related to immune system in summer effect of ramadan fasting on the ability of serum, pbmc and macrophages from healthy subjects to kill m. tuberculosis ramadan fasting and infectious diseases: a systematic review ayatollah sistani's fatwa on fasting during ramadan in the shadow of coronavirus ramadan fasting amid corona virus pandemic in academy of medical sciences islamic fasting and health publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -ak pq authors: nan title: th european congress of intensive care medicine athens - greece, october – , abstracts date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: ak pq nan objectives: evaluate the levels of tnf, il- and pai-i in different moments of the ards and the possible relationships among them. methods: septic patients with ards were studied. also significant differences for: tnf, pai-i and il- in septic patients and both evaluations of ards with control gropup; pai- between septics and nd evaluation in ards, and between the ist and nd evaluation in ards; il- between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il- , with clinical signs, are suggestive of infection; ) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; ) due to its own kynetics, il- takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution ( % hes, mean molecular weight (mw) , daltons, degree of substitution (ds) . ) or human albumin % (ha) for volume therapy for days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule- [selam -i] , intercellular adhesion molecule- [sicam -i] , vascular cell adhesion molecule- [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from • to • during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from • to , • sgmp- increased significatly only in the ha-group ( • to • only pao /fio was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the month period , patients were hospitalized on our intensive care unit. central venous catheters were examined for microbiological culture. specimens appeared to be possitive ( %). patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was / ( %). the incidence of sepsis due to the presence of all central venous lines was / ( %). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n= ), escherichia colt (n= ), others (n= ). in the epic study the percentage for sepsis on the icu was . % for the netherlands and . % for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than , ] ] ). results: five patients ( , %) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( . + . and . + . , respectively, p< , , mann-whitney u test). no patient with a saps greater than survived. mortality among the patients with saps from to was , %o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=- , , p= . ). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than , failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of neutropenic(wbc < , / )patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in from patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within minutes up to , hours after administration of antipyretics. three patients received , g of metamisol and one , g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level ( . + . ~ accompanied'by hypotension. but in - hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required + ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by other methods used in icu patients. methods: we prospectively studied adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution "d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and % limits of agreement (l of a = d_+ sd . td-fick - . - . to . fick-echo . - . to . there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n ; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il- , il- , il- , tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from . % to . % in patients with abscessed pneumonia and lung abscesses and from % to % if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il- . intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in % and mortality was decreased from % to %. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of ) rbct ; ) number of units transfused; and ) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from to and excluded patients who died in the first days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first days after onset of sepsis. overall -day mortality was %. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco (pico ) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico > mmhg. pentaspan| ( ml) was infused in rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico were performed before and repeated miff and hr after pentaspun| infusion. we calculated the pico -arterial pco' difference (pico -paco ) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was : : mmhg and lactate . : : . mmol/l. pentaspan| increased ci by % (p< . ) but did not change pico ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between and hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion , nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: . moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem : / . . moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med ; : - ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita mg/kg (tb), or varied doses of ec, followed hours later by bolus mg/kg iv lps. -day survival is shown below: group: s tb ec( . gmikgi ec( .sgm/kg) ec(i. gm/kg) alive (n) t ~ total (n) s s "signiflcant;y different from all other groups, p< . s / rats given lps followed hours later by ec ( . gm/kg) also died. additional rats were treated with s (n= ) or gm/kg ec (n= ) followed by mg/kg lps, then sacrificed at hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl ~/mm ), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, - scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = hz impedance (z; {dyn.sec.cm " }); zl = first harmonic z; zc = characteristic z; z ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < . between fio . and . , fio . and fio . &no - . _+ . - . _+ . # - . + . m - . + . * - . + . * - . + . * - . _+ . * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa ( + rnmhg versus + mmhg; p < . ). hypoxia increased (ppa-ppao) at all levels of q by an average of mmi-ig in minipigs and mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: ) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. ) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august to january , thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from to mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from . mm hg + . to . _+ . s.e.m., p= . , systemic vascular resistance ( + to + dyne.sec/ cm s, p=. ), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output ( . _+ . to . _+ . i/min p=. ) and increased pulmonary artery pressure ( . _+ . to . _+ . mm hg; p=. ) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of patients survived for more than days, patients died of cancer complications (all patients had maintained blood pressure for h on nma) and patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included mongrel dogs ( • pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of mg/kg of e. coli endotoxin, the dogs were randomized to receive a ml/kg infusion in rain of a hypertonic ( mosm]l) solution iucludiug either a mixture of glucose % with u insulin and meq kcl/l (glk-group ) or hydroxyethyl starch . % in naci . % (hes-group ). in each dog, a . % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and miu, rain, rain, and nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table ) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. , for group , for group ) were drawn and immediately analysed at ~ using the abl radiometer for po , pco and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the ps at ph= . , pco = mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps "in vivo" (i.e. the ps at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in patients aged from to with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of nm(mmi) & nm (mm ), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic ) and also some clinical and biochemical findings on the , , day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from . ~ . to . +. on the -th day after the operation. true decrease of the level mm from . ~. to . +. un & optimal density and increase of distribution index from . to . are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from . +. g/i to i . +. g/i on the -th day after the operation simultaneously with the decrease of cic from . ~ to . ~ . (p . ) were founded. some stages of the investigation true increase of lymphocytes from . + . % to . + . % was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=. -. in p<. ) was discovered and it also showed positive dynamics of the course of the disease. a year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin (il ) and (il ), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n= ), ards (n= ) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a rain resting period (baseline), then min after dopamine ( mcg/kg/min) infusion, and finally rain after the end of dopamine infusion (recovery gmbf _+ (perfusion units) gmbf ~a% vs baseline) * p < . vs "baseline" and "recovery". conclusions : ) despite a slight increase in co (+ %), the dramatical increase in gmbf (+ %) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. ) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and , -dihydroneopterin (nh ) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n ( pm), nh ( ilm), lipopolysaccharide (lps, ~g/ml), and interferone-~/(ifn-~/, u/ml) for h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn- seem to have a more than additive effect nh did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in patients, with sepsis, with severe sepsis and with septic shock, as well as in healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in anesthetized rats, catheters were placed in the femoral vein and artery. rats were given an iv bolus of lipopolysaccharide (lps, mg/kg), at baseline (to). after h (t ), rats received at random an infusion of either can ( mg/kg/h; can group, n=l ) or an equivalent volume of . % naci ( cc/kg/h; nac group, n= ), giyen over h (t -t ). a third group (sham group, n= ) received . % nac in place of lps, and then was treated like the nac group. mean blood pressure (mbp), blood lactate and nitrates (no ) were measured each h. glucose, creatinine and asat were also measured in rats (n= in each group). the can _+ * + "t . + . "~ . +_ . "t + " + " *p< . can vs naci ?p< . vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no , confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), patients with non septic shock (nss), patients with bacterial infection without shock ( nf) and icu patients without shock and without infection (control) were compared for proct levels at dayl, , , , . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p= . ), infection ( . +_ vs _+ ,p= . ), shock ( _+ vs +.- ,p= . ), death at day ( _+ vs _+ ,p= . ). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day proct levels ( = . ) in patients with shock, dayl proct levels are correlated with saps, infection and death at day , but not with arterial lactate levels (p= . ), white blood calls (p= . ) or fever (p= . ). proct levels remain higher i~i septic shock patients at day , and ( figure) . i c edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin ( gg/kg). the blood flow to the mucosal and the muscular layer was measured in regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map ( . _+ . vs . -+ . , p< . ), and phi ( . !-_ . vs . _+ . , p< . ), with a constant co ( _+ vs _+ , p= . ) and qst, aa ( . _+ . vs . _+ . , p= . ). the results of regional pertusion are presented in the table. (flow in ml/rain g of tissue; mean _+ sem ; * p< . vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a % increase in flow. acute increase in wbc count (from a mean of lo.oo mm a to o /mm~), between the rd and the th day of therapy. there was a decline of the wbc count to an average of about . mm a after decreasing the daily dose of the medication to mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c complement between . to . g/i. normal values . to . g/i there was no change in c values. conclusions : an early increase in wbc count was observed ( rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin- receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, h, h, h, h, and h after the administration of mg/kg (n= ) or mg/kg (n= ) of i - ra or the corresponding placebo (n = ). / patients ( %) were treated with adrenergie agents and / ( %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l beck from to . results: within the observation period s. was isolated from the stool cultures of patients. in patients (g m, f, median age yrs) s. could be detected in blood cultures ( s. enteritidis, s. typhimurium). in addition, in of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least up to days, of the patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids ( ), immunosuppressive drugs ( ), chronic alcoholism ( ), malignancies ( ), none ( ). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) + + death (n) duration of shock (h) + + noradrenaline (rag/h) , _+ + temperature (~ , + , + pvr (dynxsecxcm - ) + + co (ljmin) , _+ , , + , lactate (mmol/l) + , , + interleukin- (pg/ml) _+ + interleukin- (pg/ml) , _+ , , + , tnf-alpha (pg/ml) , + , + neopterin (nmol/l) , + , + crp (rag/l) _+ +_ pro-ct (ng/ml) , + , , + there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within h did not. however, in late cardiogenic shock (> h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine ( . mg/kg base in h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from / to / , pts with sfm (who ) treated with iv quinine for less than h were included prospectively. blood samples were collected at o, , , , , and h viability was assessed by culturing parasitized red blood cells in the presence of h-hypoxanthine, and radioactivity was determined at h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/ )xlo /icso for quinine (nmo]/]) was called the parasiticida/ index. results: pts were included, • saps . -+ . . the initial parasitemia was t. + . %. complications of malaria were coma ( pts), shock ( pts), renal failure ( pts) and acute lung injury ( pts). all strains were sensitive to quinine (icso -- nmol/ ). in pts who were not given a loading dose, parasite viability increased by and %, with concomitantly low quinine levels ( and #mow] at h); pt died. in pts that received a loading dose (serum quinine at h = . -- . ~mol/]) a marked decrease of parasite viability (by +_ % at h) was shown. viability was inversely correlated with plasma quinine (r=. , p-.o ) and parasiticidal index (r=. , p-.o ). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in consecutive admission to a university hospital icu in - and the icu and hospital survival of the bacteremia patients. during these years patients had positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of . % (vs. , % in all icu patients) and six month survival of . % (vs. . % in all icu patients). the most common bacteria were enterobacteriaceae ( , %), staphylococcus aureus ( , %) , coagulase negative staphylococci ( . %), pseudomonas ( . %) and slieptococci ( . %). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from - to - , records of consecutivepts with septic shock (crit care med , : - ) admitted to the icu were reviewed retrospectively. results: among pts with septic shock admitted during the study period, had hiv infection- of whom had aids-(gr. i) and were hiv-negative (gr. ill. ten gr. ii pts ( %) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in % gr. i and % gr. ii pts in gr . i pts ( %) a multivariate analysis demonstrated that hiv infection and sap i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il- and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il- , the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied critically ill patients ( men, women; - years old) in three diferents groups. group i: patients without evidence of infection, group i : patients with sepsis and with septic shock (group iii). we measured plasma il-lo, tnf and il- levels in the first hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il- were higher in group iii (median, pg/ml; range, - pg/ml) than in group ii (median, pg/ml; range, - pg/ml; p <. ) and group i (median, pg/ml; range, - pg/ml; p <. ). median il- concentrations did not differ among patients who survived (median pg/ml; range, - pg/ml) and those who died during the overall follow-up period ( days) (median, ; range, - pg/ml); but patients who died in short-term (< hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, pg/ml; range, - pg/ml). in patients with bacteriemia ( %), levels of il- were higher (median, pg/ml; range, - pg/ml) than in those with negative blood culture (median, , pg/ml; range - . pg/ml; p< . ). there was a good correlation between plasma il-io concentration and levels of tnf (r= . ; p < . ) and il- (r= . ; p < . ). the correlation between levels of il- and the apache ii score was significant only in the septic shock group (r= . ; p <. ). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il- levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital - lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : patients (age : • years ; saps ii : • were prospectively investigated (septic shock : bone criteria). following parameters were collected during hours : systemic hemodynamic parameters (swan ganz catheter a h -ref computer -baxter lab.), calculated systemic oxygen transport (do ), oxygen consumption (vo ) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco (pco ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter , cold z computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp- ) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < . ) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam- concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il- , il- and increased expressior of adhession molecules (selectin e, p, icam- ) measure of icam- and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi = . ). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/ o g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx ( . b , difco) in saline at mg/kg or h. both interventions produced % reduction in map within min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:( )baseline, ( ) mid-point of map reduction; ( ) nadir of hypotension, ( )midpoint of map recovery, & ( ) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o o:oc ., objectives : evaluate the relationship between il- , a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il and ils in severe sepsis and septic shock. methods : twenty-eight icu patients ( m, f, mean age + y) were studied as soon as they developped a severe sepsis (n = ) or a septic shock episode (n= ) as defined by a conference consensus in ( ). tnf~, il , il s and il- plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il- plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < . ) between il- and tnf a (r= . ), il- and il~ (r = . ) and il- and il s (r = . ) as well as between il- and apache n score (r= . ). patients who died (n = ) had il- levels higher than patients who survived but this difference was not statistically significant ( pg/ml vs . pg/ml; p> . ). conclusions : during severe sepsis and sepsis shock, il- seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : ( ) crit care med ; : - . objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin ( . mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least mmhg and/or a decrease in the necessary vasoactive medication of at least % within hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than nmol/l. results: of patients were found to respond to steroid medication, did not. mean cortisol levels before and after corticotropin were • and • nmol/l in the responder group (rg) and • and • nmol/l in the non responder group (nrg). in the rg out of ( %) were found to have an adrenal insufficiency, in the nrg out of ( %). in the rg -weeks mortality was . % (l out of ), the overall mortality % ( out of ). mortality in the nrg was % ( out of ) (p < . ) and % ( out of ) (p < . ) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam- and s-tnt levels continuously starting at the beginning of sepsis. methods: patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within hrs following the beginning of sepsis. blood was drawn every hrs in the first ;~ hrs, after hrs, followed once per day for days. s-tnt, icam- , elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) . ( . - . ), . ( . - ), . ( . - . ) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. . ( . - . ) . patients had s-tnt levels > . pg/l. of these died, whereas only of patients died with s-tnt values < . pg/l (p= . ). all patients that died had elevated sjcam- levels ( ilg/l:cut-off ) whereas in the survivor group only % had elevated icam- levels (p= , ). conclusions: increased slcam- and s-tnt levels were found during early sepsis in the majority of patients, a high sicam- and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on critically ill postoperative septic patients (their multiple organ failure score was - /with icg monitor. in cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during - hours in every minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on patients using previously defined criteria. methods: normotensive patients with fever (temperature > . ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients ( %) had or developed sirs within the first three days, patients ( %) did not. sepsis was present in % at inclusion. in patients with sirs, % did not progress to sepsis or septic shock, % progressed to sepsis (mean interval . • . days), and patient (< %) directly progressed from sirs to septic shock. in patients with sepsis, % progressed to septic shock (mean interval . • . days). sepsis was preceded by sirs in %. septic shock was preceded by sepsis in % and by sirs in %. conclusions: % of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. patients with double pneumonia were subjected to the evaluation. sh in the concentration of mg/l in the volume of - m / hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence . - times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, ' , h ) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by % in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. , goris et al arch. surg. , kuaus et al ann. surg. colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il- with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for - hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, o ng/ml lps was required to increase cgmp levels, whereas the presence of % fbs shifted the lps concentration curve i times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (> ~ min) but not removal of small molecules (< , d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin -~ (il-i). % fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed > hours following admission to our icu, during a year period ( january through january ) were reviewed. patients (pts) were included, mean age , y (sd , ), saps , (sd , ), mac cabe ( and ) %, mortality directly due to sepsis %. pts had mrsa bacteremia and methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age ( , + , vs , + , ) , saps ( , + , vs , + , ), use of vancomycin ( % vs %), mechanical ventilation ( % vs %), number of days (d) before the drawing of the first positive blood culture (median d, range - d vs median d, range - d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) ( % vs % p< , ), central venous catheter infection due to staph.aureus ( , % vs % p< , ), but previous use of antibiotics was not significantly different ( , % vs %). the outcome of the bacteremic pts was not statistically different: saps at the first day of bacteremia ( , +_. , vs , + , ), severe sepsis and septic shock ( % vs %), persistence of the bacteremia ( % vs %), mortality directly due to bacteremia ( % vs %). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio ; : - ) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of dead cases had been analized. results: in cases ( , %) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in cases ( %) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in cases ( %) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of dead cases ( %) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs ( kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a minute intravenous infusion )f escherichia colt (serotype h , sigma, st. louis, mo) was begun ~t a rate of pg/kg. an infusion of either saline at . ml/kg/min (group ; n= ) or saline and dobutamine at a rate of pg/kg/min (group ii; n= ) vas begun mn after the end of the endotoxin infusion. tesults : to td t ~ fl w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: patients with sepsis (criteria according to accp/sccm, ) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output< ml/ h, cr> . mg/dl and bun> mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on , and hours. cytokines tnfa, i - and ii- were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and h solute mass transfer of tnf and i - were calculated (v h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included pts and group b pts with mean+sd age ( _+ vs _+ , ns) and apache scores( _+ vs -+ . , ns). the mean ta-+ sd was . + vs -+ , p< . . the mean_+se morbidity period of group b vs group c was _+ vs _+ . p< . . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was . and for i - was . . the solute mass tranfer was -fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, delivery (do ) and consumption (c ). objectives: the main purpose of this article is to determine the relationship betwee~ delivery an consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d and v . we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after hours. high levels of do and v may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january and december . specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of patient there were ( , ~) adults and ( , ( ~) children. almost two thirds of the patients ( - , ~) were males. the predominantly cause ( , ~) of children's burns was scalding b~y hot liquids and flame burns ~ , ~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa ( " in adult patients) and staphyloccocus epidermidis ( , % in children). in only five patients ( , ~ the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in , % of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was , ~, but the mortality rate caused of sepsis was low (i %) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do (vigilance ~ baxter), v (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t ) na infusion ( , to , hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp ) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il and il were determined every hours for days and on day after fulfilling the criteria of ss and severe sepsis. results : in pts, sepsis was caused by a gram (-) microorganism, in pts by a gram (+) and in pts no microorganism was identified. there were survivors ( %) (s) and non-survivors ( %) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il- were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl ( c ) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion >_ .i ]tg/kg/min with a map _< mmhg. cardiovascular support was limited to na _+ dobutamine (db), c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t = ); and at the end of treatment (t = ) with c . conclusions: c can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. c is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t ); vl, dobu and na. results: in patients with treated ss, tests were performed (vl n= ; dobu n= ; na n= method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion ~> . ~g/kg/min with a map _< mmhg. cardiovascular support was limited to na + dobutamine (db), c was administered for up to h at a fixed dose-rate of either i, . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at h from the start of treatment (t = ); and at the end of treatment (t - ) with c . conclusions: c is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. c is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc ( c ) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . [xg/kg/ min with a map _< mmirlg. cardiovascular support was limited to na + dobutamine. c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = ); at the end of treatment (t = ); and h after treatment (t = ) with c . results. -median values (* assessment made at h or when c discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to c . the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of c in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc ( cg ) given by continuous infusion for h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . ~tg/kg/min with a map _< mmhg. cardiovascular support was limited to na • dobutamine. c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. plasma was collected from each patient over a h period and analysed for c . pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/ -plasma elimination halflife). conclusion: the pharmacokinetics of c in patients with septic shock are dose-independent at infusion rates up to . mg/kg/h. at higher rates, clearance of c decreases without any marked change in volume of distribution. c metabolism may be partially saturable at dose-rates above . mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc ( c ) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd- mice ( - g) were instrumented under gaseous anaesthesia (isofluorane, %) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after h recovery, endotoxin administration (e. col• :b , - . mgkg - i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from + to + mmhg (n= , p< . ) at h, with a survival rate at h, h and h of %, % and % respectively. c administered as a h continuous infusion ( mgkg-th -t i.v., n= ), h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from + to + mmhg (n= ) at h, with an improved survival rate at h, h and h of %, % and % respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using c represents a novel and promising treatment for septic shock. cultures of e.coli ( , %) and candida( , %) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia ( , %cultures) and common e.coli( , %)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in . objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of months all surgical patients on icu were registrated prospectively, more than parameters for each of them were documented'daily in a paradox file. results (see table ): of patients fulfilled the criterion of a septic shock (r. bone, ) , of them died at the lth day, while the surviving group of patients stayed almost days at icu. obiectives: to compare the effects of and % pentastarch solutions to a human albumin solution on oxygen delivery (do ) in septic patients. methods: this stud}, included septic patients with fever (t > ~ tachycardia flqr > /rain), tachypnea (rr > /min) or mechanical ventilation, leukocytosis (wbc> /mm ) or leukopcnla (wbc< ()/mm ) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < mmhg. patients were randomized to receive ml of % albunun (n:i ), hydroxyethyl starch (hes -mw /d.s. . ) % (n: ) or t % (n=i ); patients were also treated with adrenergic agents. results cardiac index (c ) increased significantly only in % lies (table) hemoglobin (hb) decreased significantly at min in the same group. there was not significant change in oxygen delivery ( do ). baseline ci alb . :: . (l'min/m ) hes % . = . hes % . polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after days ( day = onset of septic shock ) in % of the patients pci was demonstrable. after days % of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. . pci develops early in the course of illness ( % after days ). . pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: ) can serial measurements of vapco and avph also reflect the onset of tissue hypoxia during endotoxemia? ) are whole body changes in vapco and avph associated with parallel changes in regional circulation? methods: in anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo ). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n= ), group (n-- ) received mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do . results: systemic do crit was higher ( . _+ . vs . + . ml/kg.min, p < . ) and critical oxygen extraction ratio (o ererit) was lower ( . +_ . vs . +_ . %, p< . ) in the endotoxie than in the control group. meslenteric and femoral do crit were higher in the endotoxic than in the control group ( . _+ . vs . _+ . mlll g tissue.min and . _+ . vs . _+ . ml/min, respectively, both p< . ). regional o ercrit were lower in we endotoxic than in the control group (mesenteric: . _+ . vs .i_+ . %, renal: . + . vs . -+ . % and femorah . -+ . vs . _+ . %, all p< . ). vapco and avph followed a similar pattern in both groups, increasing slightly before do crit was reached, but dramatically wh,en do fell below do crit. in the presence like in the absence of endotoxin, systemic and regional do crit calculated from vo , from vapco , or from avph were similar; do crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. ) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: anesthetized dogs received endotoxin ( mg/kg iv), followed min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo ) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n= ) maintained low arterial pressure and systemic vascular resistance. the dogs receiving a continuous infusion of , , ~ug/kg.min of sin- starting min following initial fluid resuscitation (each dose for h), had a higher cardiac index ( . -+ . vs . _+ . l/min.kg, p< . ) and lower systemic and pulmonary vascular resistance than the control group ( -+ vs -+ and -+ vs -+ dyne.sec/cm , respectively, both p< . ). arterial pressure and pulmonary artery pressure were not influenced. sin- significantly increased the mesenteric blood flow from -+ to + % of baseline levels and renal blood flow from -+ to -+ % (both p< . ) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo , oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin- increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv- g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) - il- , and soluble (s) e-selectin were measured in both tcv- and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv versus placebo (takeda chemical industries ltd., osaka, japan). tcv- was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock ( . mg/kg body weight intravenously over hrs, twice a day for one week). day mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining patients patients were randomised to receive tcv- and to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il- and il- , considered to reflect severity of disease, tended to be higher in tcv treated patients: . . vs . ng/ml for il- (p. ) and . vs . ng/ml for il- (p. ) in tcv- and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day i.e. the end of tcv treatment (mortality: out of in the tcv group and out of patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il- and plasma il- levels tended to decrease more rapidly in tcv treated patients compared to controls (p=. and p=. , for il- and /l- respectively) conclusions: tcv- is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv- may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged - y.) with verified septicemia had been included in this study. beginning from day oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days - . patients, f and m died @u= ring their stay at the icu, of them in less than h~urs: after admission. at the begin surviving patients showed olab values between - %, non-survivors between - %, com= pared to neopterin values of - m~ol/l in survivors and - ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in consecutive children on day after uncomplicated open heart surgery was compared to gtt in children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o, g/kg in a % aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within minutes in / . after closed heart surgery gtt was delayed (+- ) min vs. (+- ) compared to controls, but transit was achieved in all aptients within rain. mean dopamine dose was , mcg/kgmin after cpb and , meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge ', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only newborns, b.w. - g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were + , and aado , -+ , kpa. results. newborns ( %) died shortly alter" admission or during transport without possibity to further is newborns ( , %) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in newborns ( , %) and of them survived the mortality rate of group mentioned was %. conclusion. the fact that in the czech republic ( mil. population with natality rate of about . newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, - , ltb and thromboxane b release in whole blood: each of . ml hepariaised blood of healthy adult volunteers was iucubated lbr hears with eillier ill ug/ml lps, mg/ml b&,ine smfactant (alveulhct/themae), rag/nil artificial surfactant (exosurf/wellcome), og lps + i mg/ml bovine surfactant or i ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. ' e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of . ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr hours with eilher aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of - but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with . ~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b ) and the lipooxygenase (lencotrieae b ) pathways. conclusion: we cooclude: ) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. ) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. ) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope , tcpco or san leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo and lb content, capillary flow rate and oxygen uptake rate. aim of the study: ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. ) to investigate the possibility of obtainiug infonnations about the local nptake and the venous as well as arterial satnration. patients and methods:we investigaled non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope , tcpco and san were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded spectres within minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo vahms were sorted by a software package. on the assumption that the low values correspond to the tibo value of the veaons ends of the capillaries and the high values to those of the arterial ends we ) it is possible to gel infonnations about the local o -uptake and arterial saturation ia the peripheral tissue by photoslg'ctroscopy. ) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from( - )ws.continnous positive air way pressure was applied to six cases at peep level from ( - )cm h o through nasal pronge,(group i),the other cases were managed as routine,(group ii).blood gases, tcpo ,tcco ,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among ( . %)of cases,while ( . %)of cases need imv.the cases of group ii need imv among ( %)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel , the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all ( ) pediatric intensive care units (icus) in the netherlands ( tertairy and non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality aenee, standardized mortality rate). efficiency was determined by objective criteria (mortality risk > % or administration of at least icu-dependent therapy)'. consecutive admissions aged < years old were included. overall, observed and expected mortality were in good agreement (p > . ). between hospitals, crude mortality showed wide variations (mean . %, range - %). however, in each center, observed and expected mortality were similar (mean ratio . , range . - . ). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was %, however large variations between units were found (range: - %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the -bed neonatal intensive care unit of a university hospital material and method: a total of consecutive outbem newborns admitted to nlod from , . to ., . were enrolled to the study. babies who died or were discharged from the unit within hours of treatment were excluded from the study, mean birth weight was g (+/- r), mean gestatational age was weeks (+/- . wks), mean age at the admission was h (+/- hi. multiple (~_ j organ system failure occurred jn . % of babies at the admission./~tertal lactates were measure/at the admission, among - hour and - hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was . %, mean length of nicu stay for survivors was . days (+/- . day). we found high lactate levels at the admission in . % babies (~ . % with levels above . retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of years was recorded in patients ( . %), patients ( . %) reported a - year remission and patients ( . %) had a remission lasting less than months before they became pregnant. results: seven patients ( . %) developed medium attacks in the second half of pregnancy, four patients ( . %) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first - weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during - weeks. a significant portion of pregnant women with b.a. ( %) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight ( - gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about % of newborns weighing below g who are born before weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i -ml and -ml pump volume. the right sided pumps are % smaller for biventricular use. between february and may we implanted this assist system in children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in infant. patient n.l: kg, lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl mcg/kg/h, hypocapnia of mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at ppm, and the gas aoalyser for no and no (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/ of systemic was obtained with a dramalic improvement. no was continued at ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu days after. patient n. : . kg, monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was % wilh a pap at % of systemic. two hours of c wenlional therapy failed o improve ihc patient and no administration was slarled at ppm. so dramatically incrcased to %, but the pap dropped only to % of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at ppm in the following days. she was extubaled days after surgery and discharged from the icu days after. patient n. : kg, 'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after d;b's and disclmrged from the icu after days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case : severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after days ( hours) -the patient was extubated weeks later. case : tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis ( days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: ) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. ) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to near term neonates with severe persistent pphn, oxygenation index > and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the patients studied responded to the initial trial of no ( ppm x minutes), as defined by a greater than percent improvement in pad as well as a fall in the el to < . these patients were continued on ino therapy, with patterns of response emerging: pattern babies (n= ) continued to show a sustained response to ino and were successfully weaned from it within days -all survived. pattern babies (n= ) failed to sustain their response to ino over hours, as definded by a rise in the el > . six survived, five with ecmo. pattern babies (n= ) had a sustained dependence on ino for - weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern ) not only required ino for longer periods of time than did the sustained responders (pattern ), but also required significantly higher doses of ino we report on the air transport of paediatric intensive care patients. these transports fall into three categories: ) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = ). one patient was transferred on ind. mean transfer time . hours (se + . hrs). ) long distance international transport using chartered aircraft (n = ). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time . hours (se + . hrs) ) long distance international transport using commercial aircraft (n = ). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time hours (se _+ .fhrs, longest hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; ) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication ) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. ) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials ( hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_. children,asa i-if,median age years,range - who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( children,age - yrs,ketamine , mg/kg, diazepam - mg total dose ) or per os ( children,ketamine - mg/kg,diazepam , mg/kg or midazclam , - , mg/kg ) _resu_l_t_s. - minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted - minutes and allowed adequate needle emg and nerve conduction investigation. children recieved additional , - , vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was neonates admitted during the years to , with gestational age _< weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo ) < . ), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo before the administration of the st dose of surfeclant, delivery, apgar score at min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was . %. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: . , % c : . , . ). for every . on average decrease of a/apo before surfactant administration the risk of p-ivh in the neonates was . times higher ( % ci: . , . ). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: . , % ci: . , . ). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of ( boys, girls) accidental ingestions of foreign bodies in children (mean age : . years, range : months- years). no child had ingested more than foreign object. the majority of the ingested foreign bodies were : coins (n : ), toy parts (n : ), jewellery (n : ), batteries (n : ), "sharp" materials such as needles and pins (n : ), "large" amounts of food (n : ). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. / patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : hours, range min. - hours). chest and/or abdominal x-ray was performed as first-line investigation ( / objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in children. in / the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from . to years. methods: protocol n consisted of intravenous administration of diphtheria antitoxic serum, prednisolone ( mg/kg bw/day), plasmapheresis and supportive care. protocol n included the use of antitoxic serum against the background of high-dose dexasone ( - mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n consisted of children with malignant diphtheria, of them with severe malignant diphtheria (grade and ). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the children in the group died seven. the children of the second group were treated according to the protocol n . out of total of patients of this group. patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of children admitted to the trial survived, one child with malignant diphtheria of grade and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. - , l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a months period non-neonatal pediatric ards patients (age - months) in a "pre-ecmo"-situation (oi over h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia ( ), sepsis ( ), immunosuppression ( ), near drowning ( ), neurogenous ards ( ). a total of - mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. hrs. if there was no improvement of the oi by at least , further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of mg/kg in doses. patients with no benefit (oi remained unchanged or increased within the max. - hrs) were taken on ecmo. results: nine patients improved within hours after surfactant therapy: the oi decreased from a level of (mean, range - ) before our treatment to a level of (mean, range - ) thereafter. in patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within - days. the other patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient : in a boy with down's syndrome an avsd had been surgically corrected at month of age. at , yr of age a catheter examination revealed a pulmonary vascular resistance of % of systemic vascular resistance in room air and at an fin of . . prostacyclin ( . mcg/ml) was administered with a jet nebulizer at an fin of . . pvr declined to . systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion ( ng/kg rain) had to be stopped after minutes because of systemic arterial hypotension. patient : a month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing % oxygen via face mask pao was mmhg, arterial ph was . . systolic arterial pressure was mmhg, a rv-ra gradient of mmhg was measured by cw-doppler. while fio was maintained aerosolized prostacyclin was administered over minutes. rv-ra gradient was mmhg, systemic blood pressure mmhg, pao mmhg. two hours later nitric oxide ( ppm) was inhaled at an fio of ( , . rv-ra gradient declined from to mmhg, systemic systolic blood pressure remained stable at mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient the poor response to inhaled pgi compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years ( - ), infants weighing less than gr. admitted to our referral unit. thirty four of them ( %) survived, ( % of infants weighing - g and % of infants weighing - gr survived) for the years - - the survival of these infants was % and for the years - - , % (p< . ). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors ( ) to neonatal deaths ( ) shows: gestational age: . w ( ) to . w ( ) (s). birth weight: . g ( ) to . ( ) (s). apgar score: , ( ) to . ( ) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants . ",,( ) to % ( ) (s) use of surfactant: % ( ) to % ( ). bpd observed in % of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in % of the mothers. in conclusion: ) a great improvement in the survival rate observed in these infants the last years in our unit. ) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated healthy preterms less then weeks postnatal age (gestational age +_ . wks; x + sd, weight +_ g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair . . + twall . ). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress ( rain). ii) insulation increased by a cap ( min), iii) min after its removal, iiii) increased operant temperature by . + . ~ ( min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded ( ) or diagnosticated an upper pouch fistula ( ). in patients who suspected "h" fistula ( ) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible - french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients ( primary anastomosis, resections of tef, oesophagocoloplasty and died with gastrooesofagostomy). the late mortality . % ( ) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il- , il- ) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, children ( months- years) were admitted in our picu in with the following diagnosis: bacteriemia ( ) sepsis ( ) and septic shock ( ) according to jacob's r f criteria. measurements: il- , il- , tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, , , hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il- , il- and tne high levels of tnf and il- are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd ( %). in patients pd lasted less then one week and there were no complications; in patients it lasted - days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. patients ( %) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles ( %, %, ~ %, %) of the most important pulmonary parameters were determined non-parametrically in weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of - healthy newborns ageing between - days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised % scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. ; : - . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during year period. as the validation study started on july , data collection is still on going and validation analyses will be carried out on july . up to now centers recruited cases. at present, characteristics of the sample recruited are the following: most of the patients were male ( %); the mean age is years with % of patiens having less than days; more than half were medical cases ( %) admitted from emergency room or from hospital floor ( %); % cases were admitted with an organ failure while % to be intensively monitored. icu-mortality was l %. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from consecutive paediatdc icu admissions over a year pedod. a proportional weight (pwt) was calculated as a percentage from the th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors ( . kg and . kg medians p = ) a lower pwt ( % and % medians p = . " . the incidence of malnutdtion , in our icu population was %. the mortality of manoudshed patients was' significantly increased (p = . ), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from . to . ). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample . , az validation sample . ). the modified formula is: legit = - . + . *prism score - . *age + . *weight category, where the probability of mortality is exp(iog/t)/ + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to ppm inhaled no in patients( m, f, median age . months, range day to years) with signs of increased pulmonary vascular resistance, there were pre and postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a % reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders( / ) were characterised by left to right shunts or pulmonary venous hypertension( / ). patient# was weaned from ecmo with inhaled no. patient# , without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient# had a pulmonary hypertensive crisis which responded to no. all non-responders( / ) had evidence of anatomic obstruction to pulmonary blood flow (# , , )or a low pvr(# ) on subsequent cardiac catheterisation. in patient # , lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these years, the incidence of sdra was . % of the total of admissions. the most common etiology was meningococcic septic shock. since , there is a decrease of its incidence. (from % to %) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was , years ( % males, % females), total mortality by sdra was % and there is an increase up to % since mean time of stay of the dead was , days and , days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: cases of severe traumatism in children admitted to our icu in the period from january to june were reviewed. age of patient ranged from months to years, % were males. in our series, % of cases suffered traumatism due to a traffic collision and % had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in % of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: % of our patients suffered cranioencephalic traumadsm (ct); in % it was an isolated picture and in % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in % of cases. % of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was . %. in . % important sequelae remained. all of these patients presented tepas on admission equal or lower than . % of those with traumatises had slight sequelae. . % of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation % of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only % in spite of a ventilation of % of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m to nun h and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to ca/of the total pulmonary perfusion. after days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a year old girl. results: a total of children with ards were treated with bovine surfactant (alveofact| cases were evalable. the median age was . years (range weeks to , years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of clays (range - days) after first symptoms of ards witha median doseof mg/ kg (range - mg/kg). in patients doses of surfactant were applied. during the hour before therapy, the median pao / fio -ratio was - . within min. after application of exogenous surfactant the pao / fio -ratio increased to with successive decrease over a period of hours to . accordingly, an increase in pao and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. of treated patients survived ( of the , respectively). of the surfactant doses were applied in the surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of - hours. the effect often could repeatedly reproduced, in one case after applications. the increase in oxygenation often allowed the reduction of fio and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with patients detected antenatally and another diagnosed at months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a year period patients under y of age fullfilled diagnostic criteria; were detected prenatally ( - weeks) and one was diagnosed at age mo. the fetuses had intermitent svt during gestation. all of them had pjrt in the first month of life at rates between and bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in to days. one was in sinus rhytm until age y; he then showed persistent pjrt over % of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over % of the day (follow-up ran and y). the mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction %) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to ; rfca was performed at m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient ( to see) recovery of sinus rhytm. ff,s clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients ( %) died. these were meningitis, head injury, sub-arachnoid bleeds, status epileptieus, leukaemie, drowning, and multiple trauma. calculated from the a admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from - %. of the deaths, i were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p % & %) survived. the median length of stay was days (range - days). patlents( %) received ventilatn~ support and patienta( %) were transferred to specialist units ( neurosciences, liver, cardiac, bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths ( of ) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, m and y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was and bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of ~ there was a gradual decrease in heart rate in the following hours ( - bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in to hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september to december , a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n= ) were treated with et. in total newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was . ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of newborns, treated for culture-positive sepsis in the same icu during the first eight months in . students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: death of cases during the study period, compared to deaths among the controls (p< . ). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o. ) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for days. a significant increase of granulocytes was ever noted already after the second administration until hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, months old, submitted to a neurosurgical interventiol after days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after days an intrathecal administration of vancomyein m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january ' till april ' , all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied ( females, males)i out of which doctors, residents and nurses. it was considered sign!ficant as mild bo, a score between - and as severe, the score of bo >. . results: subjects ( %) resulted positive for bo, out of which were females ( %) and were males ( %). the subjects with mild bo were / : was a doctor, residents and nurses. the subjects with severe bo were / , out of which resident and nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses ( subjects), as well as residents ( subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of ~tc-dtpa from the lung. methods: three groups of white sealand rabbits (bw . kg) were anesthetized, tracheotomized and paralyzed. groups were ventilated for six hours at pressure regulated volume control, set to deliver ml/kg with a frequency of /rain, i/e ratio = : and peep = cm hzo using a modified servo ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either / or / [no (ppm) / fioz]. after six hours of ventilation in these groups and immediately after anesthesia in group (control), ~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for minutes, monoexponential curves were fitted to the data and the clearance half-time (t was calculated. the t~/ mean • sd of the different groups were: t~a (mean -sd) h"e,i witl~ arf : di.ff:erent kinds, aged .q-ore mon't.hes to [ gears o : (bodi weight .~rom ., to kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se... ~ .ards'- ; :~,;,,arf o~ ::entral genes:i s .- , ,~ :inc lud ing men ingeenceph it :is- ~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.." ). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba- 'i~ier was. about . tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to dayns.,~ < .-:in , "t -irl lo;and> davs'-in 'l~atierr~{s i'i"ai s:ltiol~ o ; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to - ml~ar, fi ~ecreased to , . lind less with a = /,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th f'f'v, teoph :i. : . l:i.r~ (is- .mg/kg/day), g lucecdr t icostei~oids ( .... :~;mg/kg/day), when r exceeded in , -.];, times normal va i tea the e aqes/,'!:l"oln ~j,, ite :i.~;::.!;, ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom ! . '.' i', to !..'; , - , mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i. e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl of: . health children (asa i, ii) facing single uncomplicated surgical elective procedures . various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones . unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods - ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure > % systemic pressure, critically depressed right, ventricular function or an oxygenation index > . assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in situations ( patients, age days- , years), ino was applied - h postoperatively. oxygenation was improved in situations from _+ to + mmhg pc ; pulmonary pressure was reduced in situations from -* % to _+ % of systemic pressure. in situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume + -* %, cardiac output + -* %). in situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n= ], multi-organ-failure [n= ]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under months of life belonging to asa group . they all had been anaesthetised in . in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in cases we tbund: lafingospasm during induction in anaesthesia ( , %), bronchospasm during induction in anaesthesia ( , %), impaired intubation ( , ~ postanaesthetic laringospasm ( , %), supposed aspiration ( , %),postanaesthetic inspiratory stridor ( , %), postinductional inngoedema ( , %), death after months in consequative of infection pneumonie ( , %), none of these disorders was correlated the prematurity, infants suffered of post anaesthetic apnea, of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb ) were investigated dudng mechanical ventilation over a pedod of hours. minipigs with hemorrhagic shock were intubated and ventilated for days in an animal intensive care unit (icu). after hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of hours of types of filters showed an increase in weight of + % and airway pressure. bactedal celonisation ccured in nearly all fillers ( of ) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: patients in respiratory failure, age range weeks to years in a total of patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from hours to days. indications for use ef the device were: ) to facilitate weaning from ippv ) prevent reintubation of patients following unsuccessful extubation, and ) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao :fio ratio after cnp and eov-nb (p < . , and p= . respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco both with eov-nb and cnp (p= . and p= . respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p< . ) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao :fio , the fall in paco and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: women with ~h-gestosis (i group) and somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was % in the ist group and % in the iind group; gaseous metabolism - % and %, respectively; central hemodynamics was ~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, ( males and females; mean age . • were enrolled in the study. all patients presented criteria for pneumonia with pao /fio ratio ~ and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = em h ; peep = emh ; trigger = -lemh ) continuously admires" tered ( ' before fob fio = . ; during fob, fio = and for ' alter fob, fio = . ). pao /fio ratio as well as saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis ( ), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: mechanically ventilated patients, male and female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia ( ), ards ( ) and septic shock ( ); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: consecutive patients with severe ards (murray-score > , ; pao / fit < mmhg; male, female, mean age years) were conventionally ventilated (pcv, peep - mbar, i:e=i:i, ppeak < mbar). if after hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every hours. beside ultimate survival, parameters investigated were aado , pao /fio , and venous admixture (qs/qt). results: during the first hours in prone position of patients showed a significant decrease in qs/qt ( . % vs. . %) and aado ( vs. mmhg), and an increase in pao /fio ( vs. mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than hours before first application of prone position. after an average of position changes ( to ) of patients could be weaned from the ventilator. patient could leave tile hospital. i the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co , and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the % of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < mbar, ppeak < mbar) pulmonary function does not improve within - hours prone position should be applied. when after - position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed % for h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged ,g • days in patients with ardsvs , _+ l, days in control . mean staylcuwas lg _+ ,g days in the ards group vs , • , days in control group postoperative mortality rate was % in ards patients vs , % in those without respiratory failure. -ards incidence in liver transplantation is low ( , % in our sene) but it causes high mortality ( %) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (< kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio of . , peep of cm h , imv of bpm, it of %, and tv of ml/kg), sheep weighing . ~ . ) kg had lung injury induced by instilling ml/kg of . n hc into the trachea. ten minutes after injury, sheep with pao < ton" were randomized to continue gas ventilation (control, n= ) or to institute page (n= ). page was instituted by instilling . l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, minutes after injury, minutes after injury, and then every minutes for hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age _+ y;fev~ . _+ . l) and ards patients (age _+ ; lis . _+ . ) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao , svo , hb and methb were measured (abl ,osm ). mean intratracheal concentrations of no and no were continuously monitored using a chemiluminescence analyzer (nox ) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no ppm, no ppm and basal . after a steady state of rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a % increment in pao . results: papm was similar in both groups and decreased significantly after no (ards, basal . _+ . mmhg, no . + . mmhg, p < . ) (copd, basal . _+ . mmhg, no- . _+ . nrmhg, p< . ). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio _+ mmhg) vs ards group (paojfio _+ mmhg)(p< . ). after no- , pao increased ( _+ mmhg to _+ mmhg, p< . ) and qs/qt decreased ( + % to _+ %, p< . ) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao /fio was not correlated with basal paoflfio . no responders were / ( %) in ards group and / ( %) in copd group (p< . ). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis / . objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest ; : - ) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co elimination in different clinical conditions. methods: we have studied adult patients ( controls, acute respiratory failure, severe ards and copd) mechalfically ventilated (servo and c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco /vt] =facoz, vco vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of % variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at levels of vt ( . , . and . l) and four levels of peep ( , , and cmh objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied groups of subjects. n: normal subjects scheduled for minor surgery; arf: critically ill patients with mild acute respiratory failure; ards: patients with early ards (< h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne ( . l) a peep level of , , and cmh was applied in all patients. we calculated ers (cmh /l), rmin, c~rrs (cmh /l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co slope beyond vae (sipco in contrast to synthetic surfactant natural suffactants (alveofact| are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe , innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days ( , ). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated ventilated patients with severe ards (entry criterias: murray score > , ) in the ct scann of the university hospital. after a stabilisation period of minutes in supine position a thoracic ct scan slice cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) degree of left lateral position, b) degree of right lateral position. each lateral position was held for minutes. at the end of each of these periods a thoracic ct scan slice cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) ( ). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: %; l: %) as well as a significant decrease of the noninflated compartment (s: %, l: %) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: %, l: %) whereas the noninflated compartment increased significantly (s: %, l: %). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who required no inhalation with a dose of ppm for at least days. entry criteria for no injaalafioa were murray score >i . aud pat/fie < nun hg with peep >~ em i~o for at least hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox , see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least % and was stopped when the change in pao /fio ~ was below % or when the patient presented a paojf > mm hg a~er minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao /fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was . +_ . years and mean lung injury score was . • . . mortality was % ( / ), metahemoglobinemia . • . %, and no concentrations zero. paojf~o always improved significantly al~er ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > . , and a pao /f~o < with peep ~ cm h for at least h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were men and women with a mean age of . years ( - ) and mortality was % ( / ). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to emh with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr ; : - ) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a ml v r with a respiratory rate of /min and i/e ratio : ; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who were treated with no ( ppm). the onta'y criteria for no inhalation were murray score >/ . and paojfo z < mm fig and peep >/ cm i~o for at least hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between and ml&g, with constant inspiratory flow, respiratory rate was - /rain, and i/e ratio between : to : . all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox , see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo above % was observed after the inhalation of ppm no (group r) . when the cha~age in paojfi z was below % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ %) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. patients (age: - years; males) with no serious complications al~er mitral or multiple valves replacements and with more than hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head # ; differential pressure transducer pm- -tc "statham" w amplifier "kistler ") and flow-volume recording of forced expiration (fleish head # ) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on ( nay) and ( day) days. airways specific conductance (sg aw) has been calculated as a mean of - consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at and % of vc on - f-v curves (mef .~ , mef ) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of patients ( • years) presenting with acute exacerbation of copd (fevi: • ml, paco : • , ph: . • . ) and nonmvasively ventilated (pressure support through a full-face mask) between april and may twenty-seven ( %) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while ( %) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score ( • vs • , p = . ), creatinine serum concentration ( • vs • gm/l, p = . ), blood urea nitrogen (bun : • vs mm/l, p = . ), age ( • vs • , p = . ) were higher and encephalopathy ( vs %, p = . ) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = , p = . ) older than years (or = , p = . ) and presenting with bun >_ mmyl (or = , p = . ) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco , pao , fev ) were unable to predict tile failure of niv. conclusion : copd patients older than years, presenting with acute exacerbation, encephalopathy and bun > ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december we studied pa_ timnts, males and females(mean age +/- ); of the se had emphysema,lo chronic bronchitis, dilatative car diomyopatia,with tracheostomy and emphysema.mean pac at admission in icu was +/- mmhg,while when weaningbegan, +/- .mean autopeep was cmh ( - ).all patients were ventilated in crpv as long as four hours to calculate st tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep cmh objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond hours after trauma), is more insidious. the clinical and radiographic picture has to clear within to hours. ( ) methods: all headtrauma patients admitted from january to december , in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao -fio ratio < . results: neurotrauma patients were admitted during . patients ( %) presented with severe head injury (gcs< ), patients ( . %) with moderate (gcs - ) and patients ( . %) with minor head injury (gcs - ). overall mortulity was . % early (within h. after trauma) and delayed onset respiratory incidents were distinguished, counting for ( . %), respectively patients ( . %), patients ( . %) had early and late respiratory complications. early respiratory insufficiency was caused in patients ( . %) by aspiration, in patients ( . %) by lung contusion, in patient ( . %) by fat embolism and in patients ( %) by npe. in the late onset group patients ( . %) presented with pneumonia, ( . %) with fat embolism and ( . %) with npe. the npe group, patients, presented as follows: patients ( . %) developed early npe, and ( . %) delayed onset npe. patients ( %) died within the first days after admission, showing high mortality. gcs was less than in patients ( . %), indicating severity of head injuries. conclusions: high incidence of arf with various etiology ( , ~ was found in this population. in about % of all admitted hcadtrauma patients ( , % of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of em h with demand-flow continuous positive airway pressure (cpap) cm h . after a minimum of minutes on the above sehiogs: gastric intramucosai pc'o , abg, and a p . were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for hours for any reason. results: twenty patients met criteria and were studied over one month period in october . six of the twenty patients ( %) failed weaning. the mean and standard deviation is outlined in failure . +/- . . +/- . . +/- . . +/- . comparison between roc areas shows phi and p . to each show a statistically significant difference from an area of . (p<o. ). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #' . results. of the newborns referred to us for ecmo-therapy developed barotrauma, of the required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates , d- giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in ards patients mechanically ventilated for - h (mean murray lung injury score [ ] . + . ). patients were randomized to receive either first no and then pgi (n= ), or vice versa (n= ), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi were compared to pro-and past-challenge values. no (mean dose . + . ppm) increased the mean oxygenation index (pao /fio ) from + to + mmi-ig (p < . ) and reduced the shunt-flow from . + . to . : . % (p < . ). aernselized pgi (mean dose . + . " ng/kg rain) augmented pao /fio from + to + mmhg (p < . ), and decreased shunt from . + . to . + . % (p < . ). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in patients, both no and pgi caused an increase in pao /fio by at least rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from . : . to . : . mmhg in response to no, and from . : . to . : . mmhg (p < . ) in response to pgi . cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co -removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since , patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf , pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: ) in mean, platelets dropped from /nl to /nl within h after onset of ela. ) tat levels were already elevated before ela start ( ug/]) and demonstrated an additional peak h after onset of ela (up to ug/[). ) in parallel, there was a transient peak of pai-i levels (from to au/ml} h after onset of ela. ) in mean, fibrinogen levels dropped from to mg/ ml within h after onset of ela. ) in mean, cl-inhibitor levels dropped significantly from % to % after onset of ela and reached the initial levels within h. ) after membrane change, there were significant changes for c -inhibitor, fibrin-d-dimers and tt. ) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep ( - - - - cm h ) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was - _+ mi (x _+ se) or a - . _+ . % error. increasing peep from to cm h induced a + ml change in eelv. a difference of + ml ( . % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was _+ ml/ min (fig.i) . validation of calibration showed a mean error of -+ . % after rain. the new rip had a higher drift when cold (cold + ml/ min vs warm _+ ml/ min), whereas the old rip was thermally stable (cold - + ml/ rain vs warm i + ml/ min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo ) values is higher than suspected. objectives: determine the incidence, the significance and the pro nosis value of those differences,if they exist,in severe head injured-patients. material and methods: severe head-injured patients, coma glasgow scale (gcs) , with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than hours, were studied. data from bilateral sjo were obtained simultaneously, every hours. discrepancies were considered significant, when differences in sjo were > %. no chan es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: men and women were studied, aged • yrs. at arrival at hospital, gcs were < in and ) in to. the incidence of high icp() mmhg) were sz at the entry. the mean therapy index level required to control lop was ~l all patients required vasopressor therapy to maintain upp over ds mmhg. in patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo . in the others , sj were intermittently controhed.the mean time of monitoring were d. • days. ten patients died within this period. a total of . blood samples were analized. at arrival, sjo discrepancies were found in patients, b %. at hours, the incidence were lower, / , . %. at th day, were h/ , z and at day , when the catheters were retired, ii[ , z showed discrepancies. the ct showed new injuries in g z of patients with differences > ~ in sd values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo values and sjo differences. conclusions: the incidence of discrepancies between sjo was higher than expected in severe head-injured patients. these situation could reflect disturbances between demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied healthy, non-atop• nonsmoking subjects ( m/ f, + yr), which were pre-treated with s ( ,ug) or placebo, with a randomized, double-blind, crossover, design, before paf ( ,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every min over a min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from to x /l), and pmn(from to _+ x /l), and increased aapo (from . _+ . to . + . mmhg, p<o. ) and rrs(from . . to . . cmh .s.i q ) (p < . each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r = . , p < . ). by contrast, after s, paf did not induce any change in white ceil count (from to + x /i), pmn count (from to x /i), aapo (from . to . + . mmhg), and rrs (from . . to . . cmh .s.i ). compared with placebo, after s there was a lower ~lln-pmn lung activity ( . . vs . . cts/mci/pixel, p<o. ), lower intrapulmonary pmn retention ( . . vs . . %, p=o, ), and a faster washout ( . . vs . . s ~, p= , ). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)> . - . has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied failed weaning trials in copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco > mmhg from baseline accompanied by a fall in ph< . ; a respiratory frequency (f) > /min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo pneumoeytes. sections of generation , and from bronchial tree were free of pathological changes. conclusion: alter h inhalation of p~ji no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation . objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = ) trauma patients with acute lung injury (ali), group if (n = ) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using . % marcain. the following parameters were analysed: pao , sao , paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao and sao , decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t pts. with ari (pao : , @ , kpa. paco : , i- , kpa, ph: ~: , , hco : , :~ , mmol/ , sao : , ~- , %) who were hospitally treated due to pneumonia( pts.),emboly of the pulmonary artery( pts.) and severe attack of bronchial asthma ( pts). among tham there were ( , %) males and ( , %) females, average age , ~: , years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during hours (sodium-na,potassium-k, chlorine-c , calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l o, the object of our investigation was a group of pts with massive pneumonias, males ( . %), females ( . %),mean age yrs.thirteen ( %) of them were smokers, ( %) nonsmokers. only pt ( . %) had pre-existing chronic respiratory disease, and ( . %) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in pts( . %),physicaly ~onchial breathing in i~s ( . %),white cell count onder x /l in pts( . %). radiographicly, bilateral massive homogeneous shadows were found in pts ( . %), onilateral in pts( . %),pleural effusion in pts ( . %). abnormal renal function was found in pts ( . %). sputum culture was positive in pts ( %): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in , , cases respectively. all patients had remarcable hypoxernia (pao range from , to , kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf intrapulmonary shunt) were measured at controlled flow. in dogs (treatment), data were collected at baseline, during long injury (obtained rain after intravenous administration of oa . ml/kg), and again after bos ( mg/kg intravenously). in dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < . vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar tension is measured by the application of the alveolar air equation in which the arterial pco is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of . . the purpose of this study was to estimate the effective alveolar tension (pao eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in normal subjects and patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po (pao (i)), measured from the alveolar air equation in which paco was substituted by the effective alveolar pco (paco eff) and with the alveolar po measured from the simplified alveolar air equation (pa ). this study is complemantary to previous work for the estimation of paco eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response and co analyzer. the method is a computerised calculation of the effective alveolar po quite similar to that of paco eff, obtained from the simultaneously recorded at the mouth expiratory flow, and co concentration versus time curves. results: the results showed a mean difference (pao eff-pa (i)) of - . kpa in normal subjects and - , in patients. the mean of the difference (pao eff-paq ) and (pad (i]-pao z) was much greater than . in all subjects. the limits of agreement for the difference (paozeff-pa (i))were - . to . kpa in normal subjects and - . to . in patients, while those for the differences (pao eff-pad ) and (pao (i)-pad ) were very large ( > - . to > . ) in all subjects. conclusions: the effective alveolar po is very close to the ideal one in normal subjects, tn patients pao eff may excessively deviate from pa (i) due to the observed significant difference between the alveolar/tidal volume ratio for o and that for co . the alveolar po measured from the simplified alveolar air equation (pao ) differed substantially from pao eff and pad (i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood -carrying capacity and increased affinity of hemoglobin for are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa >lo mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i . erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value • mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value . + . mu/ml in group i, . • mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i . we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, ( . <g-i < . ); and group iii comprises patients with severe ards (all score > . ). broncoalveolar lavage (bal) was obtained after infusion of normal saline at ~ to intubated patients and cooled immediately. cells were removed after mild centrifugation ( x g, min, oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of % acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than l]s. we have investigated patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was . l/s _+ . l/s. the maximal deliverable flow of l/s was obtained in of patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no ) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl ( ~g apob/ml) was incubated with cu + ( , ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from to ~m. oxidation of ldl was monitored continuously with conjugated diene formation ( nm) and hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to hour renal isehaemia following by hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (< . nmol/g tissue in sop versus . nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a -year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in patients ( %). at the hospital admission, pac was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years through , patients were admitted to hospital in acute asthma episode. there were ( , %) pts with complications; mean age of yrs; females ( %). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was yrs (range from months to yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from to days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during a total of patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco greater than , kpa and/or an altered state of consciousness. mean age was , -d: , yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was ~ /min and puts rate .d: bts/imn. arterial blood gases revealed a pao of , ~ , kpa, paco of , • kpa and ph of , -+- , . area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every hours the first day and every hours the second and third day. at the end of hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total patients, were finally included in the so group and in the po group.patients in so group and po group had comparable ages (p<. ).peep in both groups was comparable (=. ) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at h.(p<. ) and h. (p<. ).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at h. (p<. ) with respect to initial values.fifteen cases of ards ( . %) in the so group and twenty five cases ( . %) in the po group were identified. the time of onset of ards was _+ hours in the so group and + b hours in the po group.the final outcome was very similar th both groups : mortality of % in the so group versus % in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi ) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score > ) recived inhaled pgi at - ng.kg.min " using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after rain of pgi inhalation. results: short-terro p~i inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao ) increased from to mmhg in patient and from to in patient , the ratio pao to the fraction of inspired oxygen increased from to (patient ) and from to (patient ). venous admixture decreased from % to % and from % to % in patient and respectively. mean pulmonary artery pressure decreased slightly from to mmhg in patient and from to mmhg in patient . no effects on systemic haemodynamics were observed in any patient. conclusions: pgi inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated ards-patients (age yr ( - ) mean, range) during - . the lowest pao /fio -ratio was ( - ), the worst murray score . ( . - . ), icu-stay ( - ) days and hospital mortality %. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments ( - ). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) lowering cholesterol using iovastatin treating hypertension using nifedipine heart transplantation intensive care of ards-patients conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients ( ). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of . % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= ) to analyse the effect of different levels of peep ( , and omh ) on evlw during hpe; ) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in groups of dogs by inflating a foley catheter in left auricular to a pressure of - r~uhg. peep levels of , i or m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over kg) were randomly divided in three gmups immediately alter infusing via a central vein . ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n= ) : vt: - ml/kg; zeep. group :(n= ) : vt: - ml/kg; peep: cm h . group :(n= ) : vt: - ml/kg; peep: emil . (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi> kg/m ) we partitioned the total respiratory system mechanics into its lung ( ) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap ; : ) . at three different levels of peep ( , , cmh ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in - , selecting adult patients with arf defined as: intubation and pao /fio < mmhg plus an fio > . or peep> cm i . results. patients met the arf criteria: of them ( . %) underwent prone positioning (p+). prone position use began in the early phase of arf ( . • days from the beginning, range - , median ). out of p+ pts were treated with controlled ventilation (cppv or pcv), while were on assisted ventilation (simv+ps) and on spontaneous breathing (cpap). only pts were awake when turned prone, while pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght . • h, range . - h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least . mmhg increase in pao /fio : / patients ( . %.) were responders when first pruned. the pao /fio changes induced by prone position are reported in the figure. pao /fio increased when patients were pruned (*p< . ) and remained higher than baseline values when returning supine(*p< . ). paco remained unchanged. prone positioning was used at least twice in / ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis . +. ) submitted to mechanical ventilation randomly received inhaled no doses in increasing or decreasing sequence: . , , , , , and ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_ cmh ) and fit (. +. ). the changes in vt and fit due to the no ( ppm in n ) injection in the ventilator external circuit were compensated for. results . the dose of . ppm, ineffective on papm, significantly improved oxygenation. the increase of pat and the decrease of q'va/q' and papm were nearly maximal at - ppm. no deterioration of arterial oxygenation was observed at no doses as high as ppm. co exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [ ] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no ( - ppb) in hospital central-supplied medical air. [ ] we have realised nitric oxide measurements in ten healthy volunteers, ( smokers and non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat ry limb of the ventilator. unfiltered central supply medical air contained : - ppb of no and - ppb of no , whereas central supplied oxygen was no/no free. samples were taken after equilibration periods of minutes, with increasing fit levels of . , . and . for subsequent minutes periods; paired values were recorded every s. the mean no value was . ppb (sd . ) and n o significant differences were found for different fit levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [ ] and that, previously reported, differences between smokers and non-smokers are not always striking [ ] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either rag (n= ) or rag (n= ) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed , , and rain after the start of nebulization. results: compared with mg regimen, mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of ng of salbutamol is not more effective than rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of neutropenic patients (wbc < /mm and/or pmn < /ram ), admitted to the icu between and , were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients ( . %) had a h~ematologic malignancy, and ( . %) a solid tumor. fifty-nine of the patients died ( . %); the mortality rate did not differ between both groups ( . and % respectively, p = . ). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < - ) and cardiovascular (p < - ) failures, and the number of organ system failures (p < - ) are associated to the risk of death. the saps (p < - ) and saps ii scores (p < - ) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = - ); neither the features of the underlying malignancy (p > . ), nor the duration of neutropenia before admission in icu (p = . ), nor the severity scores figs ii: p = . ) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co removal. generally, this has been used for short periods of time (< h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used ( % of the patient's cardiac output), sufficient to achieve both co removal and oxygenation. satisfactory gas-exchanged was accomplished (pco = - mmhg) with nearly total lung rest for a prolonged period ( h). however, the long ecmo duration was associated with two severe complica-ti ns: ) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and ) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, : ; ) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt ), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects ( male, -+ yr) and copd patients ( male, -+ yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial - min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was < %. lung function tests were: in normals (mean-sd), fevl%pred = • fevl/fvc%= -+ % , and in copd patients, fev~%pred= __. and fevi/fvc%= --. %. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in , ~ os patients; inconsiderable improvement -in , ~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e , h and , ~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease ust afte~ the course o~ t~eatment ( , h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s months in , ~ os patients, till one yea~ in ~ ~). in , ~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in , ~ os patients a period os ~emission lasted s ha]s a yea~. , ~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: mechanically ventilated patients ( copd, ards, other pulmonary diseases) were studied in two phases: ) during the acute phase of respiratory failure; ) during recovery - days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with - cmh ) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase ) or for two hours when they could sustain sb indefinitely (phase ). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. . during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. . inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit pg/ml)in balf (lavage of x ml in the right middle lobe). results: lif was not detected in the balf of healthy control patients and in only one ( pg/ml) out of patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of out of patients with full-blown ards ( + , mean + sem, range - pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r: . , p= . ), albumin ( r: . , p= . ) and protein level (r: . , p= . ). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, ; : - ) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which % of a tcg~-labelled test meal was eliminated from the stomach (t / ). baseline data (do) were recorded after enteral nutrition reached to ml daily. scintigraphic measurements were repeated days after cisapride ( ml orally, q.i.d) had been added to this regimen (d ). patients were considered cisapride responders when gastric clearance improved by more than % from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be + min. in healthy volunteers (crit care med, ; : - ) . five patients responded to cisapride (t / : + rain vs. + min at do and d , respectively) and five did not (t / : + min vs. _+ rain at do and d , respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> ml) gastric residues, vomiting (> times/day and abdominal distension) which disappeared in of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove , czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval patients were enrolled in the study enteral feeding was begun within hours of injury in trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ l short bowel syndrome) and group b (intensive care, surgical patients). all patients likely to require pig for a period of days (group a) or days (group b) were studied. the pn regimens administered were the following : combination with g of mct/lct fat emulsion end , g of nitrogen, in liter end glucose requirements were met by imfizsion of l liter of glucose - % via a "y " connection. lipid thus provided % of the non introgen calories. total daily calorie intake was to ] kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf- , gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md , chiolero r md , pannatier a phd , berger l , cayeux c , voirol p , hurni m md . surgical icu, pharmacy, and cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, patients, aged _+ years (mean_+sd) were assigned to groups (anaesthesia: fentanyl gg/kg + midazolam): group (n= ): reference group, with normal hemodynamic status, easy recovery. group ('n= ): patients in low output syndrome, cardiac index < . i/m on day (d ) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol g, was given intragastrically on d + d : plasma levels measured (h.p.l.c), at administration (to), t - - - - - and rain. hemodynamic status assessed with pulmonary artery catheter. healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d in all patients (no difference between groups). on d , peak paracetamol level was significantly lower in group (low cardiac output): in group the area under the curve on d and d were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d in all patients, and reverted to normal between d and d in case of normal cardiac function, but not in case of low output syndrome. the decrease on d can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [ till november, there was carried out the randomized study of patients with general purulent peritonitis; among them persons constituted the control group and -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega- fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of . (std. dev. -+ . ). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg , hospal,uk, lactate concentration retool/l). date obtained hr before and - hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow ml/mlu, dialysate solution flow i /hr), and approximate daily lactate flux into the patient was calculated to be as high as mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels ( . + . axtd . + . ,respectively; p< . ,paired t-test). when patiente with increased basal lactete (~- ) were compared to paliente with normal basal values (n= ), no difference in laotete increase was fmmd (p= . , manova). patiente with severe liver dysfunction ( points in mop scomlg, n= ) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste ( or point in mof scoring, n=ll), rite values being . + . and . + . , respectively (p< . , student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p= . , manova). in pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t =o.ol; p--o. ) or oxygen consumption (reversed fie, k) (r -q).o ;p-- . ) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of polytraumafized patients, split in two groups of ten patients each, with mean ages of _+ an -+ years. due to their condition, all patients required p.n. for at least days. both groups were subjected to isocalorie and isonitrogenous solutions ( ci/kg/ day and . g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a % concentration and solution b %. blood samples determinations during days , , , after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < . ) and prealbumin (p < . ). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < . ). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p< . ) and prealbumin (p < . ) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < . ) when considering the interaction of both the type of diet and the time course of pn. conclusions: . solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. . no significant differences on plasma proteins were found between the two solutions having % or % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids g/i; glucose/xylito g/ g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids g/i; glucose g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on patients with severe brain injury. there was no difference in body mass index bmi (group a: . +/- . kg/m and group b: . +/- . kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started - hours after trauma or surgery with carbohydrates and aminoacids. fat was added h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for days in both groups. serum tipase steadily rose to lull in group a and . lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a -fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design groups (n= each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct- %), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct / - %) and group iii placebo (control), during h ( mg/kg/min each). we measured before, at the end of h infusion, and h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, -keto pgf~,, and ltb ). at the end of the fat emulsion, groups (i and il) to , • to , • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly ( . % and %) (p< . ). eicosanoids were increased at baseline compared to reference values (p< , ). a decrease (p<o, l in the arterio-venous difference of the vasodilatador prostanoid ( -keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of / in group i (n= ) , and a carbohydrate/fat ratio of / in group i] (n = ) during consecutive days. indirect calorimetry was set at baseline and hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day of enteral feeding. urine was collected during hours and was used to measure h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose -phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after h of nutrition patients in group i showed an increase in carbohydrate oxidation (from . % to . %, p= . ), and in group ii a decrease in protein oxidation (from % to %, p= . ). after days of enteral feeding a tendency to increase in glycogen concentration (group i, p= . ; and group ii,p= . ) was observed. while patients in group i showed a tendency (p= . ) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p = . ). in group i, the pao /fio ratio increased after days of treatment (from . to . ; p= . ), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in healthy humans (control) and in patients admitted to the intensive care unit (icu) at days , and . the apache ii of the patients studied was _+ . there was an increase of uer in the critically ill patients compared with controls (o. _+ . vs . _+ . ) (p< . ). in contrast, no changes in uer between days , and were observed ( . -+ . . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r= . ) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl /av). patients were divided for days of en according to the presence (group ) or absence (group ) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p< . for statistical significance. (vr%) group (vr%) tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: ) abdominal distension (abd), ) increase of gastdc residuals (igr), ) vomits (vom), ) diet regurgitation (reg), ) diarrhea (dia) and ) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t gr glucose/ h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only gr/ h glucose was provided, the mean values of vco output were _+_ and !-_ ml/min respectively. there was no difference in vco output between with low carbohydrates and high fat regimen and standard regimen, mean values were t . -+ and _+ respectively.the high calories regimen and tpn resulted in higher vco output, mean values _+ and • respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco output, in the contrary, high calories intake as well as tpn increased vco output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b raud j.j. m tabolic intensive care unit and nephrology department. lapeyronie hospital. montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january to january . patients (pts) (male: , female:lo; mean age: + y.o.) were admitted with rh defined as cpk> iu/ . etiologies were: traumatic and ischaemic , infectious , toxic , excess activity . factors studied were: simplified acute physiologic score (saps: . + . ), organ systemic failure (osf: . _-!- . ), diagnosis delay (d: +_ h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a hours period. arf was observed in pts. in non-arf and arf groups respectively, saps ( . _+ . vs . + . ), deshydratation ( vs ), sepsis ( vs ), phosphorus ( . + . vs . -+ . ), calcium ( . + . vs . _+ . ), ward score ( _+ . vs . + . ) were significantly different. however, no significance was observed in uar ( -+ vs -+ ) and ci ( _+ vs _+ ). patients required hemodialysis (hd) ( : sessions) and remained dialysis free. only osf ( . _+ . vs . -+ . ), ward score ( . _-/- . vs . _+ . ) and ci ( +_ vs -+ ) appeared significantly higher in pts requiring hd. pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over ) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x . . tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a hr infusion were investigated. methods: after approval by the local ethic committee healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using , -d glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor ( . pg/kg/min) and dob ( pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ ~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p< . , anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a / -adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet ( patients) or an isonitrogenous isocaloric parenteral support (tpn) ( patients) (total energy: kcal, nitrogen: . g, carbohydrates: g, fat: g, n/non protein calories: / ) at least for days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day in the tpn group and by day in the enteral group ( . % of the enterally fed patients and % of the tpn patients maintained in positive balance the day of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p= . ). thyroxine-binding prealbnmin rose significantly in both groups as well (p= . and . respectively). statistically significant rises in lymphocyte counts (p= . and . respectively), in levels of c (p= . and . ) respectively), iga (p= . ), igg (p= . and . respectively) and igm (p= . ) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group ( . %) and the tpn group ( %). by the end of the study the incidence of negative responsiveness was . % and . % respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in . % of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (> ml/ hours) fasting gastric aspirates. methods : antral ( sites separated each other from . cm), duodenal ( site) and jejunal ( site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder ( a recorder, hewlett-packard) then anal) ,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), ) presence of bursts (> min) of nonpropagated phasic pressure and ) presence of sustained (> min) uncnardinate pressure activity. patients with a volume of gastric aspirates of • (sd) [median ml/ hrs were investigated for - [median minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n= ) or migrated abnormally (retrograde propagation : n= ; retrograde and stationnary : n= ) in pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in pts and sustained uncoordinate pressure activity was found in pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n= }. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included patients with hypokalemia in whom rapid potassium replacement ( meq kci in h) was performed: patients received mg sulfate ( g in hours) and patients received a corresponding saline infusion. measurements were made at time , + , + and + hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time and h (p < . -students-newman-keuls). (table ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the " authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients . aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a months period and requiring artificial ventilation and enteral nutrition for a period of at least days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin ( rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. patients (male/female : / : mean age : . + . years; mean apacbell score : .t • . ) wore enrolled into the study.the procedure was performed within the dab,s following institution of mechanical ventilation. jejunal access was obtained in all patients without resort to enduscopy in , • . min.(total duration of the procedure). mean duration of fluoroscopy was . + . rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow % of normal values in or more of the above criteria were considered undernourished (und).statistical analysis was performed using % analysis.statistical significance was established at p<o.o . results: a total of patients( female, male) -with a mortality rate (~) of , % -were studied, aged -+ years and a median ]enght of stay of days. -nourished (nou) at ad and at dis - ; ~ %; -nou at ad and und at dis - ; ) , % ; -und at ad and und at dis - ; ~ . %; -und at ad and nou at dis - ; ~ , %. we observed a p< . when we compared groups and (p= . ), and groups and (p= . ). variation in nutritional status and longht of stay: -nou at ad and nou at dis = > median lenght of stay days; und at ad and und at dis = > median lengbt of stay days; nutritional status and age at admission: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) nutritional status and age at discharge: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) we observed a p<o. when we compared groups and (p=o. ) and groups and (p = . ) conclusion: such study permits to conclude that most of our patients ( . %), staying in icu for more than days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf- levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient baseline hlood samples for circulating gf-i were obtained over a hour period, prior to il!iection of p.g/kg of rhlgf- subcutaneously. a further l samples were taken fi'om each subject over the next hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age ( - ) years, apache ii score ( - ), and length of icu stay ( - ) days. baseiine circulating igf- levels were low ( < - ) ng/ml reaching a peak of only ( < - ) ng/ml. of the patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining patients tmax was at ( - ) hours, clearance was ( - ) ml/min, vd was . ( . - . ) l/kg, and tia . ( . - . ) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients ( . vs . l/kg), but clearance was greater ( vs ml/min), tmax earlier ( vs hours) and t~a shorter ( . vs i . homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january to march , patients underwent surgery for acute bacterial endocarditis: had native valve endocarditis and had prosthetic valve endocarditis. there were men and women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was + cc and in the aprotinin was :t: (p < , ). the mortality in icu was , %, the staphyloccocical endoearditis mortality was , % and the no-staphyloccocical endocarditis mortality was , % (p< , ). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality ( %). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: patients (ventilation period > days) were randomized and allocated to the sdd group (n= ) or the control group (n= ). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of rag polymi~ mg tobramycin and mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: smears were examined in the sdd group. mrsa strains were detected in samples ( . %) from patients, and in patients they were detected for a period of up to weeks. the positive smears were districted as follows: tracheal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal ( . %). severe mrsa-induced infections were observed in patients (infection rate . % of the colonized sdd patients). smears were examined in the control group. ivlrsa swains were r in samples ( . %) from patients, but only repeatedly over a period of up to days in patients. the po~tive snmars were distributed as follows: traclmal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal / ( . %). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip . g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: hospitalized patients have been documented in this multicenter trial during a year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > ~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip . g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n= ), followed by intraabdominal (n= ) and skin and soft tissue infections (n= ). % of the lrtis wvre nosocomial acquired and in % the treatment was conducted as monotherapy. in % the lrti was treated with taz/pip b.i.d, and in % t.i.d. pseudomonas spp. (n= ) and staph..aureus (n= ) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip . g b.i.d, and t.i.d, were % and % respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to . g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of cases of severe generalized tetanus (sgt), treated in our icu the last years. we review cases of sgt ( m, f), mean age . years. in eases the entry site of c.tetanus was a skin laceration, in case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first cases incubation period was short ( - days) and so was the period of onset ( - days). all patients needed mechanical ventilation (range - days), initally through an orotracheal tube,and later through a tracheostomy, performed • days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to weeks in the most severe cases. significant autonomic nervous system dysfunction was present in cases occurring - days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in cases. nosocomial infections occurred in eases, with sepsis and mof in one. average stay in the icu was - days. one patient died with severe septic complications and one was discharged with severe disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, out of examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi , intensive care unit, kb firule split spin~ideva ! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a -hour period.criteria for the suspected bacteremia were body temperature above c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in ( %) patients hemocultures was negative at both sites,whereas in the remaining ( %) they were positive.for twentyone ( ~ of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in ( . %) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than days in patients and for more than days in patients.from patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other had the catheter from - o days. we neither found significant differences in hemodynamic dates : objectives: , to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). . to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included consecutive ventilated patients ( male and female, _ + yr; apache ii score -+ ) over a period of months. et aspirated samples with and without saline were taken daily from day of intubation until pathogen~ were presented in counts of _> per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between to days. bacterial transfer between staff and patients was noted in % of patients until day of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was % with an overall mortality rate of %. acinetobacter anitratas (no ), staphylococcus aureus (no. ), klebsiella pna~moniae (no. ) and pscudomonas aeruginosa (no. ) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. . there is a close relationship between the microflora of personnel, patients and equipment. . bacteria transfer was noted both to and from patients. . strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to % of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from ~ to %. enteric gram-negative bacilli have been implicated in % to % of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for % to % of these pneumonias. importantly, epidemics of/ - actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than % of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and ;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from to ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i patients) or equal volumes of human albumin % (control group; patients). wig (sandoglobulin) was administered in a total dose of g/kg divided in a four time regimen on days , , and post-admission. three blood collections were performe& before the first dose (day ) and hours after the third and the fourth dose (days and respectively). complement, lgg fractions, the sba at ~ and at o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day ( +_ vs • ns and + vs + , ns), whereas they were significantly higher (p< ) in the v g group on days ( _+_ vs + , p< ) and ( _+ vs +i , p< . ). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas ( ~ c) at rain in ivig group vs control group were: - _+ vs - • ns for day , _+ vs - _+ p< for day and _+ vs - + p< for day . the mean (+sd) sbas ( ~ c) at rain presented a significant improvement over those of ~ c but for the control group remained negative a~d were respectively as following: -~ • vs - + , ns for day , +_ vs - _+ , p< . for day and _+ vs - _+ , p< . for day . the increase of temperature induced a -fold improvement of sba in iv g group and -fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p< ) in the ivig group than in the control ( vs , and vs , respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune , d- bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from the'overall resistance in intensive care units towards oftoxacin was %, whereas in other hospital wards the percentage of resistance was . %, in out-patients, however, only .$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid• in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of postoperative adults pts of mean age . + . years old, with a mean icu stay of . _+ . days, following an open heart surgery from july to april , pts ( %) remained in icu for more than days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole ( mg/day) ( patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of postoperative pts with prolonged jcu stay, pts ( . %) developed systemic candid• usually after the th postoperative day. they were males and females of mean age +_ . years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group ( min vs , and vs min). all these pts received inotropes per• (mean value= . ). during their icu stay, pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of days. the median icu and hospital stay was and days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: . a significant percentage of patients who remained in the postoperative icu for more than days developed systemic candidiasis. . all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. . fluconazole seems to be a very good alternative to amphotericin-b. . fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may to february . results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a -hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the th to th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis ( %), dry mouth ( %), dysfagia ( %), asthenia ( %), palpebral ptosis ( %), accomodation paralisis ( %) and urinary retention ( %). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an -month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high ( - %). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and month outcome. patients required mv; were immunocompromised (ic) including hiv. tb was pleuropulmonary in , disseminated in and meningeal in . results: mr was % in icu, % in hospital and % at month. / ( %) < . mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po /fio ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high ( % at month). need for mv increased mortality ( % vs %). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january tit march . in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total f pts were studied, males; mean age , _+ ( - ); apache ii score , + , ; glasgow coma scale (gcs) at admission , _+ , ; ( %) pts suffer from cronic pathology; ( %) pts diabetes mellitus (dm), ( , %) pts had had a previous cranial traumatism. in cases the source of infection was otic and also in ( %) episodes of pm there were bacteriemia. in out of ( %) pts that ct was performed no radiologic abnormalities were shown, of them presented cerebral oedema and pts a cerebral abscess. twenty-eight percent presented seixures, % hemiparesia, , % respiratory failure, , % shock, i % renal failure, , % multiple organ failure (mof). as for treatment refers , % pts recieved only penicillin, , % pts only tcg, , % pts tcg followed by penicillin and , % pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and , % vasoaetive drugs. the mean icu stay was , : days ( - ). twelve ( , %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the and hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at and hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of (} immunologycal parameters has been offered:t-calls total/cd +/;t-helper/cd +/;t-supressor/cd +/ th/ts ratio;b-cells/cd +/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd +mean= . %/ . %- . %/and cd +mean= . %/ % - . %/;inverted th/ts ratio ,mean=o. / . - , /;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population we prospectively studied pts, . % ( / -group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t and t type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in . %, tv in . %, av in % and pv in . ~ and was multiple in . %. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations ( % tr, pe %, mortality %). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type ,enterotoxin a and toxic shock syndrome toxin- .eight of patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from .futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from . these countermeasures for prevention of nosocomial infections after significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -- cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao : mmhg with fio : . , pco : mmhg, ph: . , hco : mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung / ). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was . mm , platelet: . ram , hematesrlt: %, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high ( / , negative<ill ) and iglm antibodies also very high (elisa, a= . , negative< . ) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period ( - ): group a, patients hiv+, and group b, patients hiv-. apache ii was identical in the groups (p=ns). group a required more often mechanical ventilation (p= ,o ), had a higher p(a-a)o (p= , ) and metabolic acidosis was more frequent (p= , ). regarding laboratorial parameters group a had a lower no. of linfocytes (p= , ), a higher ldh (p= , ) and a more marked hypoalbuminemia (p=o, ). mortality was higer in group a ( , %) than in group b ( , %), (p= , ). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p= , ). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o , metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, gr tid) plus netilmicin (netromycin, mg bid) (group b). a total of patients (pts) ( males and females, of mean age . + . years old) were included in the study over a period of one year; in group a and in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was . %; . % in group a and . % in group b (p=ns). pts ( . %) developed infection following cabg, pts ( . %) following valve replacement and pts ( . %) after other cardiac surgery. they were males ( . %) and females ( . %). endocarditis has occurred . % in group a and . % in group b. severe wound infection was recorded in . % in group a and in . % in group b. one case of sepsis ( . %) in group a and in group b ( . %). respiratory infection occurred in pts of group a ( . %) and in pts of group b ( . %). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in ( . %) in group a and ( . %) pts in group b. pts ( . %) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the patients with lpp admitted in our icu during the last years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . they represented . % of cap. seven patients were males and female, with mean age . + . years. tiss was . + . and apache ii . + . . all, but patient, were under mechanical yen tilation (mv) during a mean period of . • (min-l, max- ) days. two pneumonias occurred beyond the season, while patients had an epidemiological history. only patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in , renal failure in (due to rhabdomy~ lysis in ). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in patients. mortality rate was %. deceased patients had initially higher apache ii, (a-a) , and lower natriemia. comparing lpp with the other cap (n= ), both submitted to mv, mortality rate was similar ( , % versus . %). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > cfu cfu/ml. broncho-alv~lat lavage (bal) ~= c'fu/rnl or positive blood culture. were excluded for rapture of treatment ; were analysed (shift with oral antibiotic : ; prohibited antibiotics associations : ; resistant germ : ). clinical data : age , • , ; saps • , ; mac cabe i : , % -ii : , % -iii : , . , % of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in ( , %), copd ( , %), aspiration pneumonia ( , %). germs were isolated (psb , bal , blood culture ) : s. pneumoniac ( , %), h. influeazae ( , %), sttep~:occns ( , %), saar ns ( , %), enterobaetdrindr ( , %), mosexella catarrhalis ( , %), othem . / ( , %) were sensitive to freatment. the ltentment was mg/kg/d of ampiclllin and mg/kg/d of sulbactam in continuous iv adminisu'ation during at least days. clinical eff~ienev : success ( %), failures ( %) with superinfeetion , worsening or relapse , dead , side effects . there was no difference between etiologies : primiti~;e~ , %, copd , %, aspiration pneamoniae , %. the bacteriological effieieacy was evaluated only for patients with eradication ( , %), eradication but super~ection ( , %) : with pseadomoaas a&ogiuosa , eater~ac~ ; beeteriological failure ( , %). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > h were randomized to received topical application oga cl suspension ( %) qshrs, a cl gel ( %) q hrs or a placebo. in addition all vpts received a nasal and a op spray ( %) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n= n= n= n= / / / * / / / / * / / / / / / / / / i *p = , byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r enmmtion, * service des admissiens-urgeuces centre hospitalier g- ndral - saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a beds unit. the prospective study includes all the patients comn~ in from / / to / / . the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of nosoeomjal inthrtions: i abcess and bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed different periods: period ( / / to / / ) and period ( / / to / / ) for all .e peripheral catheters inserted in the i.c.u. period , % , % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo ) to measurements of the mechanical work of breathing (web) and p . . methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation ( - days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto , , , mbar far rain periods each. all patients had a peep between - mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display ( ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p . was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo and vco were measured using the pb metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p< . , compared to ips mbar. results: the values for breathing pattern, web, p . , vo and vco are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: episodes in consecutive patients (mean age: . years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, patients had nosocomial pneumonia, had urinary tract infection [ with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations ( - s cfu/ml) in occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over . mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p< . results: of the total admissions were fully evaluable. of them ( %) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for > h were compared: group = hme replaced every day, n= episodes of mv in patients; group = hme changed every h, n= episodes in patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~ cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every h. results: the groups were similar in terms of age, indication for and overall duration of mv ( +_ . vs +_ days, p= . ), and severity of illness (saps: --- . vs . +_ . , p= . ). the maximal values for peak airway pressure were identical in both groups ( . -+ . vs . • cmh , p= . ). obstruction of the tracheal tube was observed in only one instance in a group patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same ( / vs / , p= . ), as was duration of mv before the occurence of pneumonia ( • vs . +_ . , p= . ) and overall mortality rate ( vs , p= . ). conclusions: the clinical efficiency of this hme does not seem altered after days of use. indeed, replacing this hme every h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed patients (pts) admitted to our icu from july to december with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv ( cases), herpes zoster varicellae ( ), measle ( ), rabies ( ), unidentified ( ). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts ( / ) had an abnormal mri. among them, pts made a complete recovery, in comparison with / pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in cases (with poor outcome in one) and was improved in one. spect was found abnormal in / pts (among them, pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in % of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied pts, # ( , %), ( , %). hean age: , • years ( - ). mean stay in icu: , • days ( - ). they were divided in groups: a( pts) who did not suffer from pneumonia and b ( pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) times: on days o, and of the pneumonia for group b and respectively for g~oup a. conclusions: ) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts< years (p<o, ). ) the frequency of isolation of kp in icu pts during - was - %, but now ( - ) kp becomes more and more rare ( - %) and is isolated especially in elderly pts. ) its sensibility to antibiotics remains always the same. ) the prognosis of np in our study was independent from age (p<o, ), sex, presence of bacteremia (p< , ) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at and hours after the iv (bolus) or the et (nebulized) administration of mg of gm in seven criticcally ill patients ( with established renal failure). safety was defined as peak and trough plasma gm levels _< than and ijg/ml respectively and adequancy as gm levels in bronchial secretions _> , ijg/ml. results: gentamicin was administered by the et and iv routes in and separate sessions respectively. a total of samples were assayed, in bronchial secretions (bs) and in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route ( , + , vs , _+ , pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in % of patients after et administration, compared to % after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route ( , + , vs , • , pg/ml respectively, p _< . ). the et administration resulted in toxic bronchia~ gm levels in % of the specimens. % of these samples were from patients with renal failure, however toxic blood levels were reached in only % of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from groups of patients were tested. group i: patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of patients with a np without anaerobic bacteria, group ill: a control group of patients with dental stumps but without pulmonary infection, group iv: a control group of healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i ( • was statistically higher (p=o.o ) than in the control groups (respectively: + , _+ , _+ ). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t > . ~ c) in all adult patients admitted for >- hours in the icu during a two month period. these patients were studied for consecutive days. and werc classified in groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of patients studied, dec'eloped fever ( %). including (after complete evaluation) d, p and u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u ( . • versus . • and . -~ . ~ versus . - . , respectively p= . and p= . ). most common sources of infection in d were the lungs in patients ( %) and urina .ry tract in ( %). of these patients had positive blood cultures ( %). the overall mortality was % ( % in d, % in p and % in u. differences ns). antibiotics were given in % of d, % of p and % of u ( patients). in p there was a non significant lower mortality." in patients who received antibiotics ( / ( %) versus / ( %) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: patieet with a stay of more than days in inteaasive care were screened for candida infection. patients were treated with antifungal therapy due to either an increased antigen titre of -> : or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of : or higher. results: the median length of stay was (ranging from to ) days, the mean apache ii score on admission was (+_ . sd) points. of patients patients died ( . %). in the group treated with antifungnls ( patients) patients died ( . %). although of the patients only ( . %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher ( . % vs. . %, p < . [chi-square-test]). in patients an antigen concentration-> : was measured. seroconversion was found in patients. the most common fungus was candida albicans ( . %). furtberm re, candida glabrata was found in . %. most of the patients were treated with x mg fluconazole ( patients). in patients therapy was changed to amphotericin b/flucytosine. in patients therapy was started with amphotericine b and flucytosine. in patients a threefold decrease of candida antigen titre was found. patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied patients ( males and females, age + ) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at rpm for min revealed: i) more than leukocytes per optic field, ii) squamous epithelial cell less than percent and iii) one or more microorganisms per optic field on magnification. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap and patients did not. in cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was , -+ , , while in patients without vap was , + , . there was a significantly higher incidence of vap in patients who had i) coma (gcs < ) and ii) been receiving neuromuscular blockade (p< . ) . the sensitivity of the gram stain for vap diagnosis was %, the specificity , %, the positive predictive value %, and the negative predictive value , %. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october and february all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the psb performed for suspected mvp ( nosocomial and community-acquired pneumonia), yielded at least one micro-organism (positive psb : %). of positive psb demonstrated only aerobic bacteria and ( %) yielded with anaerobes. in out patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia ( / positive psb). strains of anaerobes were isolated. prevotella species represent out these strains ( %) the most frequent anaerobic species were prevotella oralis ( ) p. intermedia ( ) and p. buccae ( ). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied multi-trauma patients (mean age + years, apache ii . + ), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than and cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap, and patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)< (x = . , p< . ), the administration of neuromuscular blockade (x = . , p< . ), the duration of mechanical ventilation to be greater than days (x = . , p< . ), the flail chest (x = . , p< . ), the parenteral nutrition (x = . , p< . ), the ards (x = . , p< . ), the abbreviated injury scale (ais) of more than for thorax (:,: = . , p< . ), the pneumothorax (x = . , p< . ) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: . , p< . ), flail chest (f: . , p= . ), and gcs (< ) (f: . , p= . ). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (< ), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= , + , x~ + , x + , x , were x i-apache-ii score points, x -evidence of septic shock, x -endocrinopathy. the explanatory power of this quation was evidenced by roc of . , se (v - . introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in patients during the first week after trauma or the onset of sepsis. patients: group : (n = ) multiple trauma iss > , group : (n = ): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z- , pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. + . &-_ ( - ) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of granulocytopenie(wbc< , xl ) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in . % of patients before ss and in all patients after ss. only patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto ) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was . mt-m " , and le,~ and ie'i~ w~ . units in sic. we managed to maintain op of . - . ml.m " and ieto of . - . units in hst. patients from with mosf survived in sic and patients from survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and day survival were also recorded. biochemical data were compared with results from healthy subjects. results: xanthine oxidase activity was . + . units/i in patients (mean :t: sem) and . + . units/i in controls (p<o. , mann whitney u test), and was highest in the patients who survived (p< . ). malondialdehyde concentrations were elevated ( . • . prnol/i compared with . • . pmol/i in controls, p< . ). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco ) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p ) methods the pco electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography % of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on - % and general peripheral resistance increased on - %). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was - mg/kg during - days. as a result of such therapy was also the oliguric stage shortening (ac n ).in , % of the cases unsatisfactory datas of central hemodynamic,combined with high (more than cm h ) venous pressure,lung oedema.then nanipruss ( , - , mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton ( , - , mg/kg) and dopamine ( - mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during - days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; .ruptured aneurysm of the abdominal aorta (raaa) n = .elective vascular surgery (evs) n = .abdominal sepsis n - .trauma n = .others ( e.g. malignancy, obstetric emergencies) n- seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients % , group % , group % , group % , group % , group % . mortality increased with the number of failing organ systems; mortality for all patients with > failing organsysterns was % the only exception being the subgroup of trauma patients where mortality under these circumstances was o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( % ~ % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between and , and the function of , or organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol patients ( - years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: ) to increase the effect on microbic agents and the efficacy of treatment in combined infections; ) to lessen the possibility of the exciters'resistence to antibiotics; ) to prevent the development of superinfection: ) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was ( - ). when indicated, patients had blood volume measurements simultaneously based on the techniques of, i) dilution of ~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was ml ( - ml) compared with ml( - ml) with slcr labelled red cells (r= . ). regression analysis produced an intercept at ml. the slope of the regression line was . ( . - . , % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock ( - mmhg for rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of . mg/kg or . mg/kg at the end of resuscitation. results: infusion of . mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the h survival rate was significantly improved from . % in the control group to . % in the treatment group (p< . ). in contrast, the high dose of . mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest- ) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: patients have been hospitalized in our icu from january to may . , ( %) with mosf. among mosf pati~ts, ( %) have had hepatic dysfunction defined according to hebert (bilirubin ~ ttmop , chest ). thirty six of these patients acquired hepatic dysfunction after admission in the icu. these patients were compared with mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< . are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao /fio , arterial lactate, do were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to amag/~ factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f tm [fresenius] , ct tm [baxter] , and filtra tm [hospal]) were assessed. each filter was changed intentionally after a hours" use. together the data of filters were evaluated, each at three different times (immediately after its onset [ hi, after h, and after h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p< , ) showing the f tm membrane excelling filtra mand ct tm the more. the loss of inuline clearance ( mi/min/m ) after h, however, was insignificant for all filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after h (p< , ), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was years (y), patients ( % ) were < y, patients ( %) were >= y. the incidence of dialysed arf in our mixed intensive care departement is %/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively %, % and %. treatment: cavh (cd) or cvvh (cd) was used in patients ( %), hemedialysis (hd) was used in patients ( %). data: mean apache scores were the same for cd and hd ( for both groups), patients treated with continuous dialysis techniques had significantly (p<o, ) more need for ventilation ( % vs %), elevated bilirubin ( % vs %) and a higher vasopresser need ( % vs %@ than patients treated with hemedialysis. there was a trend towards more sepsis ( % vs %; p= . ) mad coaguiation disorders ( % vs %; p= . ) in cd. taere were sign/ficantly more younger patients (< y) treated with cd ( % vs %, p< .os). mean apache score was significantly lower in patiants< y than as patienta>= y ( vs ; p< . ). patients< y had significantly (i}< . ) more coagulation disorders ( % vs %) and elevated bilirabin ( % vs %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd ( % vs ~ p< . ). patiants>= y had a comparable sr vs patients< y ( ") */e vs %; p----a.s.). tha global survival rate (sr) was % ( patients). conclusions : diaiysed arf has a well known lowsurvival rate ( %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache score couldn't illustrate that. patient~ y with arf have the same survival rate as patients< y: although patients >=- y have a higher apache score they have less organ faille. the avacbe score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < . initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of . %. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo . sao , diuresis, pha, and hemoglobin were repeatedly registered during the first hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: measurements of co were obtained, ranging . objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo ) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do /vo > its co> relation to other parameters of do /vo disturbance, and with postoperative complications and clinical course. methods: patients ( male, female) undergoing cardiac surgical procedures were included ( myocardiai revascularizations, valvular substitutions, constrictive pericarditis). mean age was + years, mean weight _+ kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period ( ', ', ", ' and h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo ), diuresis and arterial ph (pha) were also recorded. results: phi values ranged . to . (mean . ( . ); the mean values of clinical evolution were: extubation time, _+ hr.; discharge from postoperative care unit, - hr.; and hospital total postoperative time, _+ . days. complications registered were: perioperative acute myocardial infarctions, cases of respiratory insufficiency, occlusion of coronary bypass, an ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > . ) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo , pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: ( ) gutidrrez g: lancet ; : - . ( ) landow i: acta anaesthesiol scand ; : - . the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p< . . according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb< and b: >sg/dl: i: ac< . and ii: > .smm. in either group blood transfusion results in zt significant increase in hb (a: . _+ . to . + . g/dl; b: .(~ . tt, . + . g/dl; i: . -+ . to . -+ . jdl; i : . -+ . to . + . g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: -+ to -+ ; b: + to + ; : -+ to -+ ; i : -+ to -+ ), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i -+ to -+ ; b: -+ to -+ ; i: -+ tu -+ ; : -+ to +_ ); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of > . -+ . g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in . % of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a -years-old worn an with a breast cancer, diagnosed in , initial staging t nim , treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of mg/m until august . the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of mg paclitaxel, given over hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to %. she died days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selectin (selam- ). sicam- , svcam- , selam- , and inflammatory cytokines were determined. methods: sicam- , svcam- , and selam- were determined by elisa. tnf-a, il- , and il- were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam- and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam- level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il- , but the level of il- . discussion and conclusion: the slcam- and svcam- levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il- also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam- , elam- , and vcam- , appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut- , reduced release of inflammatory cytokines (tnf, il-lg, il- ), activation of complement (c a, c a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut- on soluble adhesion molecules following cpb surgery. patients undergoing cpb surgery were divided into two groups, group a consisted of patients who received omg of fut- in priming solution, followed by a continuous infusion at mg/kg/hr during cpb in addition to initial heparin dose of mg/kg. group b, a control group, included patients who were injected with heparin only. the plasma slcam- , selam- , and svcam- concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam- and slcam- reached their peaks on hours after cpb and on pod respectively in both groups, but they remained lower in group a (selam-i: . + . vs. . • ng/ml, p< . , slcam-i: • vs. • ng/ml, p< . ), svcam- , in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut- reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, l- , il- , il- , s-cd- and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: - ml/h, filtration rate ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock ( ), the so called hepatorenal s~aldrome ( ) and a severe pancreatitis ( ). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha ( cases), scd ( cases), il- (l case) and il- ( cases), hemofiltrates contained no activities. only il- was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il- and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il- , il- could not be eliminated in cvvh with a filtration rate to ml/h. in contrast to findings of other investigators with a higher filtration rate (> ml/h), we found no significant concentrations of tnf-alpha and il in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (> ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~ ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed times using nafamostat mesilate as an anticoagulant in hours with l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~ -proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = consecutive pts treated with hd; n = consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = _+ ; mean number of organ system failure (osf) = . -+ ; mean blood pressure (mbp) = • mmhg (first day of application of hd). cvvhf-group : apachen score : + ; osf = -+ ; mbp = + mmhg (first day of application of cwhf discussion : during the first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) patients with mof were treated with cvvh in the period between january in to march in . the mean (• age of our patient population was , • years, being % male and % female the whole patient population was with mof iust at the moment the technique was accomplished; % was in mechanical ventilation, % needed vasopressor support and % required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was , ~: , (range - ) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine , st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta and dopaminergic receptor agonist. the specific beta adrenoceptor antagonist ici has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs ( - kg) divided into groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and pg/kg ici bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at - mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b antagonist blocked any protective effect of endogenous beta adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr chloride, a beta and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta adrenoceptor antagonist ici was infused to block any protective effect of dopexamine. nine anaesthetised pigs ( - kg) were randomised into groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition pg/kg ici bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at - mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. . _+ . , dopexamine . + . ", blocker . + . . data expressed as mean + sd. significance p< . . * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) ( patients) with bilirubin > . mg/dl and lung injury score < . , it) mof-ards ( patients) with respective values < . and > , iii) patients with head injury with respective values < and < , served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index > ml/min/m prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was . _+ . vs . _+ . (ns) and . _+ . respectively, of the paop was . +_ . vs +: (ns) and . + . respectively and of the cvp was .+. . vs . + . (ns) and . respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was . + . vs . + . ( < . ) and . _+ . respectively and lung injury score was . objectives: oxygen delivery (do ) and oxygen consumption (vo ) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo determination by either indirect calorimetry (vo m) or tick calculation (vo c). the purpose of this study was to investigate the changes in vo m and vo c following do increase. methods: the relatives of critically ill patients (mean age years, mean apache ii , mean mof-score ) gave their written informed consent to participate in this institutionally approved, prospective study. do was increased by fluid loading (hydroxyethylstarch %: mean volmne ml, mean duration of infusion min) and catecholamine support (dobutamine: mean dose , ~g/kg/min). changes in vo m and v c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor integrated in the ventilator (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in patients with peritonitis of different origins ( nonsurvivors and survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol ( +_ , nmol/ i), aldosterone ( , • , nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=- , , p < , between cortisol and pao ; r = , , p < , between cortisol and d (a-v) ; olso renin -cao r=- , , p < , , renin d ~,vl o r = , , p < , ). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b and p fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next hrs fzom + ml/ hrs to + ml/ hrs (ns). data expres,uxl as mean + sem. three patients ( %) became polyuric with urine output > ml/hr within days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was . + . days. serum potassium levels were well controlled. day or immediate pre-dialysis levels were . + . mmol/l compared with pre-lreatment . + . mmol/l overall mortality in this series was / ( %). duration of acute dialysis in survivors with renal recovery was . +_ . days. patients ( %) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do and vo calculations and temperature) and determined the respiratory mechanics (compliance and pao /fie relatinship) before starting the procedure, after minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler and minutes of zero fluid balance bemofiltration and after minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for minutes without filtered fluid production and after minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate ( mosm/l), was injected in the dose of .o ml/ kg into v. cephalica antebrachii after hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco and phi obtained simultaneously by gastric tonometry. material and method : in a patient ( y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : sets of data were obtained and are expressed in mean + sd. gastric pco z was , + compared to , + . mmhg for portal pco . phi was . +._ , vs . +._o, for portal ph. no correlation was found for these parameters. p (gast-a) c was . + mm hg vs + . mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = , ) [figure] but no correlation with p (gast-a) c . obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange ( ). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator dilution method, with indocyanine green and the stable isotope h as tracers, was used to measure evlw ( ). arterial bloodgases and endtidal co were registered. evlw data was compared to a group of renal healthy patients ( ). dcp n evlw, ml -pao , mmhg h~o +, nmol/l control group - -- l _+ "* -+ _+ crfgroup ii -+ ~ +- ns -+ "(" iii +- t _+ ns -+ t ** p < . dcp i from dcp , t p < . dcp li or i from dcp i, :~ p < . dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco ( . + . kpa) and etco ( . + . kpa) were unchanged while h o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao . the reduction in h + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao in bicarbonate dialysis. . prezant lung ; : - . . wallin j appl physio ; : - . a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value . , can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h -bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below . . results: among the patients, had a drop of phi below . during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi= . and the second . . the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the th day after operation and died on the th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other patients with a fall of phi had a lowest phi= . . . . . . . respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the th pest-operative day and a dehiscence of the surgical wound on the th day: the third one, operated on for a sigmoid carcinoma, had melena in h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these patients were discharged alive from the hospital. the other patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of hours duraticn. all l:atients nm.'-~ms_(~lly va~ ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e rain after the ~, ~ne at the middle, ~ne rain before lhe erd ard one rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re ( ve)emm to . ~ ~, ) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n to ~mg hg, ) i~ from to . mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o. ) of: i) s~o~ from to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: patients ( males and female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. patients were operated on eleetively, had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a month period ( / - / ), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to , deliveries occurring during that time period for a . % rate. the following procedural indications for icu transfer occured: insertions of endotracheal tubes, arterial catheterizations, temporary tracheostumy, and venous catheter. the following medical diagnoses were found: hypertensive/eclamptic episodes, hypotensive episodes, pulmonary embolism, mitral stenosis, and coagulation deficiency. there were four episodes of respiratory failure ( %). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for months in a multidisciplinary -bed icu. methods: data from consecutive patients admitted to the icu from / / to / / were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm , mpm and odin. data from days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = %) were evaluated from data collected during the day of admission (saps ii, rpm , odin) or after hrs (mpm ). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary -bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for consecutive patients admitted to the icu from / / to / / . data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied men and women, with a mean age of years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from clinical variables and laboratory items for patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i . thresholds were calculated with the youden's test (se+sp- ). results : patients were admitted in the intensive care unit (icu)and patients in medical units; patients died. the saps ii was higher in patients which died than the survivors ( , + , vs. , + , , p< , ; respectively), the best threshold was , se was de %, sp was %, the ac was / . thus for patients admitted in icu than in medical unit ( , : : , vs. , + , , p< , ; respectively); the best threshold was , se was %, the sp was %, the ac was %. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, hiv positive and hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure ( % versus %, p < . ) and septic shock ( % versus %, p , : ) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. , n= ) and january-march (period , n= ) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache scores. the icu and hospital mortalities decreased from . % to . % (p=ns) and . % and . % (p=ns) respectively in period . the mean icu and hospital stay was also decreased from . __+ . to . + . days (p=ns) and . __+ to . • . days (p=ns) respectively. the improved micu length of stay for survivors in period was statistically significant ( . • . days vs . • . days = . ). there was no significant differences in the vse of peritoneal dialysis ( . % vs . %) and mechanical ventilation ( . % vs . %). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from % in both to . % and . % respectively in period . conclusion: the duration of critical illness was reduced in period . hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, ald patients ( males; females; median age years, median apache ii score ; hospital mortality %)with severe complications ( [ %] with bleeding oesophageal varices, [ %] with hepatorenel failure, [ %] with respiratory failure, [ %] with septic shock, and [ %] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians and , p< . ), or more organ systems in failure in the first hours of icu admission was associated with a % ( ) mortality. mortality was also related to child's grading and the numbers of organs supported. of the patients who received more than one form of organ support only one survived. organs supported mortality a (n= ) % none / ; % b (n= ) . % one / ; % c (n= ) . % two or more / ; % the icu costs for the patients were only s but s ( %, patient days) was utilised by the patients who died, giving an effective cost per survivor (ecps) of s . although the cost of treating the patients with or more organ systems in failure on the day of admission was s ( % of the budget) the ecps was s . conclusion patients with ald who have or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which surgical patients were randomly assigned to either a control group (n = to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = ) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at / prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality days postoperatively. results: in the protocol group, there was a % reduction in mortality days postoperatively (p = . ), a significant reduction in the number of complications (p = . ) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a . % survival rate days postoperatively was s , , resulting in an average cost of s , per surviving protocol patient. the average cost of a control patient was s , to achieve a . % survival rate days postoperatively, resulting in an average cost of s , per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s , per patient together with a % reduction in mortality at days postoperatively. hence, the cost per surviving protocol patient was s . less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet ; : - ; madl et al. lancet ; : - ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: recordings of standard bilateral sensory evoked potentials were performed in patients with acute liver failure. findings were focused on cortical n peak -a stable negative deflection occuring • ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, patients were referred to emergency liver transplantation, and patients died. in all patients who recovered spontaneously, n peak was detectable between and ms. all patients who were referred to liver transplantation and of patients who died, developed a loss of a prior detectable n peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \ -test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows . . was used for statistical calculations. results: in-unit mortality: / patients ( . %) --oth . % > as . %> ni . % > ed . %; p = . . in-hospital mortality: / patients ( . %) --oth . % > ni . % > as . % > ed . %; p = , . mean survival time in days after discharge: as < ni < oth < ed ; p = . . conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january asd ]lay data from , patients over years of aqe who retained in the icu for mare than hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s . % and in-hospitul mortality . %. patients who died showed a higher spas score then survivors ( , ~ i ,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection ( ~ . ; % el, . to . ), pmo~othor~x related to mocasnical ventilation (@ . ; % cl, . to . ) and delay in the insertion of a fln~-quidod catheter (ii~ . ; % ic, i.i to . ). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~ , j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~ i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january and april , all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of intnmtsd patients amre stndied. self-extu~atien occurred in ( . %) patients and . % required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe ( . v_s . years, p = .~ ), ~verity of illness ( . ~ . spas score, p = . ), dia~isstia category ( s. % v_s . % of patients with res~iratury conditiono, p = , } and mean length of stay ( , ~ , days~ p = . ) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a . iir for mortality ( % ci, .i to . ) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber all patients who fulfilled criteria to be almittod to the ic who for waste~r reason retained in tile observation unit for more than hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of , patients who suffered no delay is admission to icii over a period of years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~ &ults: a total of patients remained in the observation nit for more than hours with a del w in igd admission of . _+ . hoers. assisted mechanical ventilation was requited in % of patients and only monitericatien in %. itsse patients were cspared with ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn % as compared with . % for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = . ; % ci, , to , ). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was for survivors and t for non-survivors (p < . ). the mortality ratio was . and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent ( ) of icu bed days were occupied by % of patients (non-sarvivors) which resulted in cancellation of cardiac sot#cat sessions in momhs. conclusions: this study concludes that apache t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a day period. ) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december through to april , patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score= , maximum day off scorn= , sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at . (two-sided), with n/group = [(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as to patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from % to % ( % relative risk reduction) where the n/group= . scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. . non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" . autonomy: in every particular medical attendance, the patient has ability to decide by himself. . justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. ( - ) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p = . ). the mean aiii score when considering all admissions was , ( - ) . the initial score between survivors and non survivors showed ststistical difference ( . vs . ) respectively (p < . ). univariate logistic regresion analysis demostrated a % increment in death probability for every points augmentation in the aiii score with a sensitlbity of . % and specificity of . %, the roc curve showed that the best cut off point for death prediction was points with a sensitivity of . % and specificity of . %. if a patient is classified as high risk (> ) the bayesian analysis showed a . probability of death and for one class(fed as low risk (< ) a death probability < %. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama , : ] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. , : - ] and then a step verbal scale (pqolv): best, good, fair, poor, worst. of the studied patients, at the first interview, were able to use both scales, but ( . %) understood only the verbal one. at the second interview, patients were not able to answer, used both scales and only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, pts ( . %) died in icu. they were males ( . %) and females ( . %). their mean age was (+ ) years and mean ef was . (+ . ). nineteen pts ( %) had low (< . ) preoperative ef. mortality was . % in the coronary artery bypass grafting (cabg) group (n= ) and . % in the valve replacement (vr) group (n= ). in the cabg +vr group, mortality was . % (n= ), and . % in the remaining pts (n= ). cardiogenic shock was the sole cause of death in pts ( %), septic shock in pts, whereas sepsis in combination with ards in pts, sepsis and stroke in two pts. in addition, pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p< . ) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: . although cardiogenic shock is the main cause of death ( %)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. . patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. . although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after and up to hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from / to / consecutive pts with ami were treated by primary ptca in the wuppertal heart center pts ( , %) were admitted to our hospital > hours and < hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was years ( - ). pts were male, four female. % had an anterior wall myocardial infarction, % suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in . %, multi-vessel-disease in . %. average time of onset of pain to recanalisation was min ( - ). angiography revealed timi-flow in . % of the pts, timi-flow i in . %, timi-flow ii in . %. average follow-up (fu) period was months ( - months). timi iii lv-ef ~ -day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality . % %/ % . % . % . % % early mortality occured in the two pts, who were in cardiogenic shock at admission no pt required emergency coronary artery bypass grafting.restenosis > % was seen in % of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about % (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of icu's representative of the italian icu system methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the patients recruited during one month period, a total of patients were included in this study. for the purpose of the comparison of the two scores, patients with less than years, or having cardiac surgery or staying in the icu less than hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$ ~: saps ii fits the data equally well compared to the older version (goodness-of-fit p= . and in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve . and . , respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p-- . ) and discrimination (receiver operating characteristics area= . ) were observed. c nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: ) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); ) assess the efficiency by means of a resource use index (rui); ) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. critical from ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was . and it ranged from . to . in the icus. the overall rui was and it ranged l~ont . to . . there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine ; : - ): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on random admissions to specialty icus with beds (range - ) between february i and december l, . post-operative solid organ transplant recipients were excluded. units included general medical, general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from . - . ; predicted risk of hospital death ranged from . - . %. standardized mortality ratios ranged from . to . with icus performing significantly better and performing worse than predicted (p< , ). los ratios and icu readmission rates ranged from . to . (ns) and . to . % respectively. patients predicted at low risk of requiring active icu treatment ranged from , to . % conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals ( - ; - ; - ; - ; > ) , we compared tcu mortality within age intervals (< ; - ; - ; - ; - ; > years - , - , - ) . in these groups mortality may be twice higher in the > years patients than in the _< years. mortality does not vary with age in low (physio a n = - ) and high (physio a n = > ) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear ( ), patients were treated at the unit, of them died, and ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only patients were evaluated on the fu. the, were classified into the follov ng three groups: acute medical, elective surge d and acute and emergency postoperative. the patients were seen at , and months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~ ous jobs. apache scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that / patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and % showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only % had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii i mpmp ii ), apach ii end gaprik were evaluated o~ patients with acute myocardial infurction admitted to intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii , saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii , and apache-saps were adapted and reduced to (mpm ii cor) end to variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to or variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included consecutive intensive therapy unit (itu) admissions with an itu stay> hrs in a month prospective study (exclusion criteria: burn injury and age < yrs). data were couectsd applying the criteria described by the developers [ , ] . the definition of coma (mpm ii) was modified and the best assessment within in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was yrs (ranse: - ) with a male:female ratio of . : . the actual hospital mortality was . %, mean predicted death rates were . % (mpmz ii) and . % (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p< . l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion %: apache ]/i . %, mpm ii . %). the area under the roc curve was . (ap iii) and . (mpm ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~ - , mpm b ;( - , lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -< % with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks > % ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of adult patients consecutively admitted to a medical-surgical itu over a period of months. patients with burn injury, age < yrs and itu stay < hrs were excluded. using a eomputerlsed database, we routinely recorded hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;( ) statistics and calibration curves [ ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the male and female patients was yrs (range: - yrs). of these patients, % were medical admissions, % were admired after emergency and % after elective surgery. the observed hospital mortality was . %, the overall mean predicted death rate was . %. mean predicted risks were siguifieanfiy greater for nonsurvivors ( . %o) than for survivors ( . %, p< . l, t-test). apache iii showed good calibration (z -~ , lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was . % (decision criterion: %). the area under the roc curve was . % confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected patients, in two months in summer in all over italy. regional data were echieved in a three years entlection ( -i ) in lombardia' icus from archidia group (arehivio diagnostieo), including patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr observed patients account for . % and % of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data ( . %) and extremely low mortality in regional data ( . %). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: . % and . %; .a.p.s. score: . and ; iength of stay: % and ). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively % vs. %, medical patients were % vs. % and traumatised were % vs. %. % of "observed patients" in national study and % in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than % did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients ( % vs. %); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during . the authors collected data of patients outcome and predictor variables. overall mortality rate was , %. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death ( %). average length of stay was . days ~. % patients died in the first ten hosiptal days and only % after days. age was directly correlated with death % of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose ( %) and natrium ( %) were in optimal physiological values. serum proteins ( %) and haemoglobin ( %) levels were inversely related to death. multivariate showed that alveolo-arterio difference in content was the most informative of all mortality predictors (mean value , mmhg in % patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. ; : - ) and the sepsis score of elebute and stoner (br. h surg. ; : - ) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past years for patients in our icu was calculated = % (prior probability). the outcome of patients who were admitted to our icu for > hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died ( . i . v~s . • . and . • v's . • . respectively p < . ). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at , and h while in the intervention group phi measurements were made hourly for h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure > mmhg, systolic arterial pressure > mmhg, urine output > . /ml/kg, haemoglobin > g/dl, blood glucose < mmol/ , arterial oxygen saturation > % and correction of uncompensated respiratory acidosis. if the phi was < . after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at then mcg/kg/h, titrated against phi. this additional therapy was continued until h after entry into the study. in each year patients were subdivided in two series with random selection, so that the st series contained abeat / and the nd / of the patients. the st series of all the years constituted the devdoping data set and the nd series the validation data set. with data of the st series ( patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die st, th, th and th day, calculating for each lime the apache ii score (for a total of records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january and february , anonymous questionnaires were sent to intensive care units, all over greece. results : questionnaires ( , %) were replied and returned back. of them , % were answered by male and , % by female. the doctors replied in the following rate : , % aged up to , % aged between and , % aged over . questions were answered and were divided into main topics, as following: . admission criteria: limited bed availability was the main cause for refusing admission in , % of icu's. , % evaluated each case's viability and only , % used some prognostic score system. , % of icu's accepted all cases and a significant percentage ( %) gave in to pressure coming from their colleagues ( , % female and , % male). . informing the patient/relatives: only , % was willing to tell the whole truth, while , % had given selective information.. in the case of iatrogenic incident, , % withheld it, because either they feared legal implications ( , %), or lost of trust ( , %). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of , %, while only , % found informed consent necessary for the proposed treatment procedure. . withdrawal of therapy/dnr orders/organ donation: , % were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in , % such a decis~n was unanimous, while the percentage of those carrying it out was , % ( , % female, , % male). in case of brain stem death , % ( , % female, , % male) withdrew any life support. , % would like therapy withdrawal to be legally established, while only , % would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only , %. , % considered organ donation to be a necessary proposal, while , % refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it ( , %), or because they doubted the procedures' objectivity ( , %). note: in greece, icu beds are less than % from the total number of hospital beds available. only a percentage of - % of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n= ) and mitral valve replacement (mvr) (n=t ). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at hour and at hours after operation. results: there were no significant hemodynamic changes during the first hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t trauma and sepsis patients randomly received either . mg/h (group i, n= ) or . mg/h (group , n= ) of enalaprilat i.v. or saline solution (control, n= ) as placebo for days. plasma levels of endothelin- (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion ( . mg/h: from . • to . • pg/ml; . mg/h: . • to . • whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p< . ) and decreased after . mg/h of enalaprilat. et remained almostunchanged in group , whereas et increased significantly in the control patients (from . • to .t• on the th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p< . ), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, min after the patient was placed in a trp and again min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo by monitoring partial concentration of o and co ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco without a similar trend in vo values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after min in trp. evaluation of vo from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after rain. since vo did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo ximetrix, abbott) allowing continuous svo monitoring was sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for hours and/or successful weaning (s-w). (j physiol ; ." - ) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and % co inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, year, performed three incremental exercise tests in a randomized order: ) inspiring air without vd (air control, ac); ) inspiring air with vd of ml (avd); ) inspiring % co ; % oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to % peak hr. results: ventilation (vi) was significantly greater (p< . ) during the avd and co tests than during the ac test at the same work rates. end-tidal co (petco ) and estimated arterial co (paco ) were significantly greater (p< . ) at w and w. oxygen saturation was significantly lower (p< . ) during the avd test than during the ac and % co exerdse. at matched hrequivalent to % peak hr, vi was significantly greater (p< . ) during the avd and % co tests than during the ac exerdse ( l, l, and /). conclusion: we conclude that the increase in xri and petco due to vd loading and % co inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu ( men and women; mean age . - . years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was . - . hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt ( , , and hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (< hours). table i list the variation of plasma eoneenlrations of tbars (mean -sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe time cuncentzatiens reveal a difference p<o. between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~). ) at , and hours fi-om de begin~g of'it. comparison ofthe , and hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in healthy subjects (group i) , patients with several risk factors of ischaemic cardiopathy (group if) and icu patients with possible aim ( were confirmed ami (group ma) and were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i . electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h , s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< . ) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i ( pts) -p:ithout d and group ii ( pts) -d ~as administered . mg/kg iv bolus hours prior to anesthesia and . mg/kg/h upon the end of operation and for at least hours post-operatively. iv was substituted by oval administration of x mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during minutes starting minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of o fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period ( hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), % survived, the remainder died of incomplete recovery. in vi with bvad, only % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in patients by comparing paopee, paoptmc, and paopnadir obtained within the first seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in patients (group a) but present in others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : +_ ; gr b : _+ cmh ). results : l. gr a : paopee ( _+ mmhg) differed (p < - ) from paopnadir ( • mmhg) and paoptmc ( _+ mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves ( " - , + , + mmhg respectively). . good correlatkm (r - (i. ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). . lung compliance (v t/bpah,) correlated well with i t in both groups (r - . ), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (> ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) hours after operation, e) hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline hours after operation. methods: patients with preoperative lvef< % undergoing coronary artery bypass grafting were studied. after surgery, a f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z- t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m ), intrathoracic bood volume (itbv,ml/m ), pulmonary blood volume (pbv,ml/m ) and extravascular lung water (evlw,ml/kg). with a f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while extraction ratio (o exr,%) and oxygen delivery (do ,ml/min/m ) was calculed. peak inspiratory pressure (pawp,cmh ) and mean airway pressure (mawp,cmh ) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after minutes (to) of volume controlled standard ratio ventilation (vc), and after minutes (ti) of stabilisation period of pcirv ( % inspiratory time, % pause). vt,ve and total peep were held constant in every mode of ventilation. +_ . " *'p < , versus to conclusions: these data show that pcirv : is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo . paw eight patients ( melee end females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so /co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o. ) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do and tissue oxygenation (pro ) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do and pro . objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight + kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio . ). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao ) and mixed venous (pvo ) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, pm microspheres (sephadex g coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to mmhg initially. rain after embolisation when haemodynamics were in a steady state (control ), inhaled no was administered. further measurements were taken min after ppm no (no ), min after ppm no (no ), and min after discontinuation of no administration (control ). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given rag of actij.yse as ~ hrs infusion, followed by heparin~single j. ihjaction snd iefusion ie doses - j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im . . hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao and sao in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed % improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation ( ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv ( ). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: normothermic who were stable (< % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit to insure both normal abg and a peak airway pressure < cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi. , ) with synchfjv runs interposed (hfjvz ). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > % ; n= and < %; n= ) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate h-bpap. trace mnou~ of ~fi-bpap ( gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of . pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g -a: %, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to • reflecting a • inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to :l: %, representing a -a: % inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as . x " sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi ) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from + to -+ dyn*s*cm (p< . ) and pulmonary artery pressure from . + . to + . mmhg (p < . ), cardiac output increased from . + . to . _+ . i/rain (p < . ), mixed venous oxygen saturation from . _+ . to . + . % (p < . ) and arterial oxygen saturation from . + . to . _+ . % (mean _+ sem of trials in patients). -month iloprost nebulisation in one patient ( gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selecfin (selam- ) levels in patients with acute myocardial infarction admitted within hours after onset. peripheral venous plasma-samples were collected at the time of admission, , , , , and hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into groups as follows: group ; killip's class (k) and without thrombolytie therapy, group ; k and with thrombolytic therapy and group ; k and . both plasma sicam- and svcam- concentrations in group and were elevated rapidly and significantly and maintained at a high level during the first days. plasma selam- level did not change in any of the groups. these results suggest that the adhesion molecules icam- and vcam- may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no no -(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were patients admitted within hours after onset. venous blood samples were collected at -hour intervals on the first day, -bour intervals on the nd day and -hour intervals on the rd day and th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about hours and - days after onset (basal level; . _+ . , first peak; . !-_ . , second peak; . + . ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of hours after onset were significantly correlated with maximal plasma creatine kinase level (r= . , p< . ). the levels of plasma nox in the early stage of myocardial infarction (from admission to the th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range - ) fullfilling the high risk criteria of shoemaker (colectomy , gastrectomy , pancreaticoduodenectomy , others ). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci > . l/min/m , pcwp > mmhg, hb > g/l, sat >. . objectives: evaluate the acute effects of , mg ipratropium bromide and , mg fenoterol (ibf) inhaled dose on pulmonary function in nonsmocers (nb:m) and smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and healthy subjects. methods: pulmonary function tests were performed < h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred puffs-ipratropium bromide ( , rag) and fenoterol ( , rag). for , h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in second(eevt) was , % (p< , t) improvement and , % (p< ,ob), forced expiratory flow betwen % and % of the forced vital capacity (fef . ) was , % (p< , ) and , % (p< , ) and maxamal voluntary ventilation (mw) was , % (p< , ) and , % (p<o, ) improvement. the mean changes in normal subjects were mild (fev increased by , %,fef - by , % and mvv by , %). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group (n= ) received both imas as grafts and group (n= ) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao /fio ratio was measured in all patients in the icu, at , , minutes on mechanical ventilation (mv), at , , minutes after extubation (ex) and on postoperative day (pod) . in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group had significantly longer bypass ( _+ vs + . p< , ) and aortic cross clamp time ( +- vs + , p= , ). pao /fi ratio was significantly higher in group only at minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group ( +_ vs _+ , p= , ), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group and one case of pulmonary infection in each group. after extubation m[n min min min* rain min pod ' _+ _+ + -+ - -+ _+ _+ _+ _+ -+ _+ _+ -+ mean_+sd, *p=o, o conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in patients without previous pulmonary disease undergoing elective cardiac surgery. there were males and females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were _. and • min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb- h after h after stemotomy closed chest cpb cpb estrs (cmh /l) . + . . - . " . _+ . * . - . * est cw " . _+ . . • . + . . -+ . est l " . -- . . - . " . -_ . " . _+ . " x + sd. * p < . : anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first hours and stabilizes hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied pts, % ( / ) developed dcm during the follow-up study. among this group % ( / pts) were in class i and ii and % ( / pts) in class iii and iv of the nyha classification. we divided this population in a acei+ ( pts) and acei-( pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t and t type cell population and its t /t ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography ( -de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new -de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of idcm pts, mean age _+ yrs, % male gender, using a combined -de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated -de software. computer processing and analysis of the tee images led to a -de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using -de derived indices. mean values of -de lvesv= -+ ml, lvedv= _+ ml, lvstv= _+ ml and lvef= -+ % were obtained. for all these -de parameters, good correlations were observed with -de lv measurements obtained through biplane tee analysis of the lv cavity (r>. ; p<. ) as well as regional analysis of sequential -de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new -de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of dcm patients, mean age _+ yrs, % male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ % pts; group slvd- % pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. -de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. + -_ . simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with (fl ) and (fl ) ml of dextl'an (rhemacrodex| in patients free of previous cardiopulmonary disease ( • yrs) with acf (ci < . l/rain/m ) due to angiographicalty proven mpe (miller score > ) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = . ) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep ( - - - cmh ) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in mechanically ventilated arf patients (lis = . ~- . sd). according to the response to peep , two groups of patients were defined: group a ( pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep , rvef decreased at peep (~ . %)~ rvesvi increased only at peep (+ . %) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep , 'rvesvi decreased only at peep (- . %), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the levels& peep were studied. rvedvi and ci were significantly correlated in out of:l patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in out of patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in patients of group b and in patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is . + . nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only • . nun hg is tolerated (p < . ). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at , and minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after minutes of reperfusion there was a highly significant increase (p < . ) in the mda values (mean = . /zmols/l). these returned to basal levels after and minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used "large white" pigs. mean weight was kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends patients studied as two groups of individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered rag/ hours of vitamin e (tocopherol acetate) hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a -year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of . u/hr for hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed ( . ui of streptokinase on days , , , ). fluid drainage was improved and cardiac output was also improved (day : . .min "i, day : . l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload ( , ) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of successive breaths of increasing magnitude ( , , , and ml/kg). a line of best fit is drawn between the minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh ). in mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of ml/kg of plasma expander. the initial mean downslope of the rsvt was -. + . mmhg/cmh . following volume loading the downslope decreased to -. + . (ns). at the same time, cardiac output (co) increased by . + . l/min (p<. ), end-diastolic area (determined by tee) increased from . + . to . + . cm (ns), and paop increased from + to + mmhg ( p < . ). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = . ) and the eda (r = . ). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion ~ . g/kg/ rain with a map --< mmhg. cardiovascular support was limited to na + dobutamine (db). c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t - ); and at the end of treatment (t - ) with c . conclusions: c does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to h. c is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope hzo in aorta (q v~ o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h ( . ml), h ( . ml) and icg ( mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and h in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= boluses) over group i (n= boluses) ( . <- . vs. . +- . , p= . ). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz ( ) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope h for the estimation of extravascular lung water (evlw hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg ( rag) and h ( . ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q n o, t o) ( ). data analysis: evlwg~av was reference for evlwzhzo calculated as q hzo times the difference in mean transit time between t nzo and rico (atm n). as reference for atzn o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for h was calculated as the sum of central blood volume and evlwg=v. boluses were administrated in a group (i) of anaesthetized pulmonary healthy sheep while q was altered. another boluses were administrated in a group (ii) of anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. results: for boluses h parameters were not significantly different from their respective reference parameter: at vao . +_ . s vs. atg~, . + . s, evlwzh o -+ ml vs. evlwg~,~ + ml. in group i the ratio between hzo parameters and respective reference parameters (n= ) were independent of qlco from . to . l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg ( mg) in water ( ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( men, women ) age from to were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in % ( pts), without high pvr-in % opts) and with left ventricular failure-in % ( pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed- ~-pat~e~ts ( males and woman), average , years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture ( ~ ~ ~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from . +__ . mm to . +_ . ram (p = . ) at ~ tilt angle, and from . + . mm to . + . mm (p = . ) at " tilt angle. the tidal volume increased from + to + rut a * tilt angle, and from + to + ml at * tilt angle (p = . ). two patients could not bear the horizontal position ( ' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion ( . + . mm to . + . ram). after an overnight respiratory assistance, pao increased from . +_. . to + . mmhg ( = . ), sao increased from . + . % to . +_. % (p = . ), and paco decreased from + . to . +_. mmhg (p = . ) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity and l/min) vs pressure triggering (-lcmh ) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized min. runs using a bird st ventilator at zero peep (zeep). trigger values for pressure (-lcmh ) and flow ( l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv ( . + . cmh: x sec) and a/c ( . + . ) with pressure triggering, as respect to psv ( . + . , p< . ) and a/c ( . + . , p< . ) with flow triggering ( l!m). no differences were observed between and l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being . + . cmh (psv flow trigger) vs . + . (psv pressure trigger) and . +_ . (a/c flow trigger) vs'f~ +l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung . we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in anaesthetized and paralyzed piglets we applied short periods ( s) of one-sided ventilation ( breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied -to-r and r-to-i cross-talk at ventilatory rates of , and bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during bpm the r-to-i crosstalk was _+ . % (mean +__ sd) of the tidal volume to the right lung and the -to-r crosstalk _ . % of the left tidal volume. both values increased at bpm to _ . % (p < . ) and _ . % (p < . ) respectively. the values at bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = ) or as a slow infusion (n = ) in infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every minutes with a lung function cart (sensormedics ) without interrupting ventilation. infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > %, maximum r > % of baseline value. retreatment was followed by an increase in r in all patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores > ) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l. and a peep level < cme=o} was not able to get either a p.ojf=o= ratio > or a s.o= > %. patients were sedated, paralyzed, and a ventilator (serve c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at . and peep removed. continuous gas flow ( • ml/kg] was humidified and jet delivered through a tube ( ram id, ml capacity, . ml/cm h=o compllancel ended in a nozzle ( . mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo ) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin . further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies - hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies - hz with the relative inspiratory time (ti) . - . . the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats ( - g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature ~ the arterial pressure was monitored. after traeheotomy the metal cannula ( mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = . kpa, ti = . . the dead space of ventilator including canula was . ml. the initial frequency was hz and rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : hz--> hz--> hz--> hz--> hz--> hz--~ hz--> hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco = + mm hg. the arterial po was always above mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt = . --+ . ml/kg for ft = hz, vt = . + . mukg for fz = hz, vt = . +_ . ml/kg forf = hz, vm = . + . ml/kg forf = hz andvmt= . + . mukg for fs = hz (presented as mean values _+ s.d., n = ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = . * f-e. . conclusions: the described system allowing ventilation in a wide frequency range - hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr - and gacr nr . s intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on ventilated patients (puritan bennett ac with metabolic monitor pb set to measure end tidal co ). all measurements were repeated with the patient stabilised at cm. cm and cm peep. inclusion criteria were: ) haemedynamic stab(l( .ty for hr; ) pulmonad" anon" flotation catheter in situ: ) volume control ventilation with plateau of . s: ) fio ~ > . to maintain pao~. > kpa with em peep: ) qs/ot > %; ) pao /fio ratio < . measured variab!es included: r minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co ; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p< . ), compliance (p<t). ) and o,'gcn deliver, index (do , p< . ) occurred with increasing peep (table ) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of men and women were studied (n= ). the mean distance of ett tip to carina was . cm. using the reference markings, cases ( . %) had the ett tip < cm from the carina and cases ( . %) > cm. one case resulted in an endobronchial intubation. the mean height of all patients were cm ( - ) for males and cm ( - ) for females. of the patients with ett tip < cm from carina, the mean height was cm and cm respectively. ~ onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases ( . %). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp- pulmonary monitor were recorded hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre . _+ . to post . _+ . , p < . ). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group included eight patients who were completely weaned from ventilator; group included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups ( . _+ . vs . + . in peepi; . _+ . vs . _+ . in cld, p < . ). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group ( % % in group vs . % in group ). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were survivors and deaths in this series (mortality %).using arterialpco values measured hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was %. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was %. arterial co accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po , pc ) and the ph (abg) was examined in stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for and ps plus intermitted mantatory ventilation in ii. [n each patient, speciments of abg were measured at min intervals during a - study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was . percent for po , . percent for pco and . percent for hco . the average sd for ph was . , the corresponding c for systolic bp, diastolic bp, cf, tv, rf were . , . , . , . , . percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in ; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, del, vo by improving the rheologic properties of blood and plasma, normalizes ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by . - . limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo ) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in critically ill patients treated with mechanical ventilation for at least h, who fulfilled at least of standard weaning criteria (vt> ml/kg; respiratory frecuency (f) < ; pimax > cm h ; pao /fio > ). baseline measurements: t, vt, p . , pimax, f/vt, p . *(f/vt), p . /pimax. study protocol: measurement of vo , vco (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after and minutes of spontaneous ventilation with cpap cm h . the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, were extubatad, and of them had to be reintubated before h, being considered also weaning failures. during cmv, vo /kg was . + . ml/kg/min, and . _+ . mlo- /kg/min after ' on cpap cm h (p < , ). of patients ( %) with standard criteria were extubated, while only of ( %) with criteria (p< , ). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa ( . - . ml/kg) in anesthetized, paralyzed, and intubated dogs (n= ) during volume controlled ventilation: rate= /min, peep= cmh , ti/ttot= . , fio = . , vt= ml/kg. animals were rotated during the oa infusion and the following minute stabilization period to assure uniform injury. in the supine position, peep was set - cmh above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of cmh : animals were ventilated in either the prone (n= ) or supine (n= ) position for four hours. pulmonary artery occlusion pressure was maintained constant ( - mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale - ). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at ( to ( cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two rain periods of chfo (sequences : ( and ) c/rain : group l, n = l: and c/rain : group , n = ). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa is not significantly modified during chfo at or c/rain. paco is slightly decreased at c/rain (p = .( ). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. ). there is no more effect of chfo for patieets who are at a flu higher than . (n = ). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < .ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = ): two levels of assist were studied; flow assist (fa) of cmh /i/sec and volume assist (va) of cmh /i (assist ), and fa of cmh /i/sec and va of cmh /i (assist ). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist ) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco of mmhg v t was increased significantly by . + . i and . + . i in protocol a with assist end , respectively, and by . _+ . i in protocol b with assist (p< . ). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ ( mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco ( mmhg), pdi and pmus at the end of neural inspiration decreased by . -+ . % and . + . %, respectively, from control values. neither change was significant (p> . ). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol ) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps ( - breaths each) from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p< . ); at i/min ttot, expressed as % of that at i/rain, was . +_ . % during sleep and . +_ . % during wakefulness. during wakefulness, at i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was % of that at i/min (p< . ). the corresponding value during sleep, was % (p> . ). in four sleeping subjects the increase in v~ was sustained for . - min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only - breaths. we conclude that ) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and ) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively asa i pediatrics patients ( to yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of mm. id and . m. long (children < kg), and a corrugated dar set of mm. id and . m. long (children > kg) were respectively used for ccl an cc values of . and . ml/cm h . a vtof mlg/kg and respiratory frequency was adjusted for an end-tidal co (etpco ) between mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was . % and reached as high as . %. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as . %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago- ) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically patients. the ventilatory setting is pressure support mode. the pressure level and fit were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: '(immediately before h/d), ', ', ', ', ', '. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp- ). the mean value of the breaths minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po and pco> however, a potential problem in mechanically ventilated patients is that the l mixing box (mb- l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb- l on the v c were compared with those produced by a new l mixing box (mb- l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb- l was substantially higher (vc= . *ppeak) than that provoked by the new mb- l (vc= . *ppeak). at a ppeak = cm h ~ the v c were ml (mb- l) and m{ (mb- l), respectively (p< . ). in a group of subjects ( m/ f, _+ years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb- l and mb- l fell on the identity line. it is concluded that the new mb- l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age ( - ) yrs . they all were connected to servo ventilators siemens c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo , swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with cm h cpap for at least one hour. we turned them to psv with inspiratory assistance (psv cm h ) and after rain we applied psv cm h , and after min psv cm h . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< . ) . respiratory rate (rr) decreased significantly (p< . ) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do ) decreased ns, oxygen consumption (vo ) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo and pvo values in a group of patients, following open heart surgery. methods: upon transfer to icu, patients ( males and females) of mean age _-+ years, were randomly assigned to receive (n= ), (n= ), or cm of peep (n= ). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of ml/kg. the fraction of inspired oxygen (fio ) was adjusted to keep a pao around mmhg. mixed venous po and svo were measured at min, and hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at hours post-extubation. differences at each study time were analysed by anova. results: mean svo and pvo values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo and pvo ) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from to ml, at rr from to bpm, while closely monitoring cpap airway pressure swings. at v v of and ml the rr was limited to bpm. for comparison we explored aps of a one l balloon cpap system, the cpap mode of the puritan bennett , and siemens ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one l balloon system was linear over a range from . to . l, with a cpl. of . l/em h .the cpl. of the l balloon ( . l/em h ) was linear between a volume of and . l. apf of the weighted balloon system was under em h at all v r (except at a v r of ml aps was . em h ), while the apf in the l balloon was up to em h . apf witli human volunteers with the two commercially available ventilators in the cpap mode was about cm h ; while under identical conditions apf in the l balloon system was . emhzo; and in the two l balloon system was below lcm h . conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co (etco ) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco -etco variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) ( + ml vs + ml, p< ) two individual behaviours were observed on tl in pl: four patients presented low etco: (range - mmhg)and normal pacoz (range - mmhg), while three patients had normal etco (range - mmhg) with high pac (range - mmhg). one hour before stopping ilv (t ), vt, etc and paco were the same in each lung. the pao /fio: ratio improved in all patients from the beginning ofllv cst of pl was + % of the normal lungs' cst on ti and improved to . + % ofnl's cst on t (p< . vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco and of the differential paco -etco gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med :$ , ) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from to % of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav %), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an month period. we compared assist/controlled mandatory ventilation (s-cmv, patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, patients) and biphasic positive airway pressure ventilation (bipap, patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation ( . h, p< . ) than patients treated with s-imv/-psv ( . h) and s-cmv ( . hi. with s-cmv . % of the patients required single or multiple doses of midazolam but only . % in the s-imv-/psv group and . % in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group ( . mg) than in the s-imv/psv group ( . mg, p< . ) and in the bipap group ( . mg, p< . ). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p< . ). after extubation the paco patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~ +pst, rs (bates) [ ] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h -h log t (hildebrandt) [ ] where h~ and h are parameters depending on lung volume, and initial time is s for analytical reasons. materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. was , -+ . , and using eq. was . + . . the values ofz~ (eq. ), however, decreased with increasing the tidal volume (vt) according to the following equation: "~ = . - . v t, similary, the values of h~ and h increased with increasing v t according to the following functions: h~ = . + v i and h = . + . v t. conclusions: the behaviour of "% of eq. suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range ( - ml/kg) in ventilated patients. infect this model predicts that "c is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r obtained by fitting an exponential for data of eq. , is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r wh t lsms ira tlmeand r given by:pv~c.~ = d~( -'e-~ )[ ] ere " ' p" tory " and "r are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c and r which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z + e~t.r= [ ] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~ by analyzing the time-course of ptr according to eq and next determining r according to eq. , using the expedmental values of p,i=~, ~ and ti, as well as "~ obtained with eq. . materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. was . . with v t of ml/kg, the mean values (+ sd) of ': and r of the subjects amounted to . • . s and . • . cmh i "~ s. with the traditional multi breath method the corresponding values were . + . s and . _+ . cmh i " s, respectively. with the t-test the difference between new and traditional "~ was statistically significant, between new and traditional r was not significant. conclusions: with the single breath method it is possible to compute ': and r . the mean values of r with v t of nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during - . a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. ) post tb respiratow failure ( %) ) neuromuscular diseases, ( %) } undiagnosed sas { %) ) cope) ( %) ( patients had a overlap syndrom). the programme consists of : ) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). ) group and individual respiratory therapy ) mobilization ) nutritional support ) educational classes for the members of the family. three from the patients passed away (during the year), are under nippv during night with or without supply, pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao /pao ) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients ( + yrs) suffering from ards (lung injury score > . ) for hours or less entered into the study. irv (volume controlled, decelerating flow, % inspiratory pause, lie = / ) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= / ). these two modes were applied for hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume ( . • . ml/kg), respiratory rate ( • "bpm) mad fit ( • %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after , , and hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a -year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * ( . rr of patients examined showed moderate to severe desynchronization. in of these patients a (re)activation of the hbr was found. the strength of hbr amounted to + %. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined patients after open-heart surgery and patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in of intubated patients, of which showed cs. in of these patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining patients. in of these patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k .w. the constant tube coefficients k~ and k are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k are changed rendering the p~,ch calculations inaccurate. therefore, k and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to %. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [ ] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [ ] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be inter- gil cano a, san pedro jm ~, sandar d, herntndez . , carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: ) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and ) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than mmhg or an absolute decrease in systolic blood pressure below to mhg within hour of intabatian. patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h ( ), cardiac arrest ( ) . there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < . ). high paco before lrasheal intubatian ( . - mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < . ) in patients who developed h. paw. ) thexe was a good relatienship between h and high arterial paco before traqueal intahatian and its fast "washing" with mechanical ventilation. ) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit ( ). application of ips has been shown to partly compensate this added work ( ). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd ( ). therefore, atc has been developed as a new form of assisted spontaneous breathing ( ), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls , dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls was connected to an artificial trachea (at, cm long, mm id). the at was intubated with three different tubes of . , . , . mm id and connected to an evita ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of and cm i and atc all with a peep of cm h . the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/ are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<. , **-p< , ns = non significant, f versus s neveltheless, in / patients, invasive ventilation was necessary in mean . _+ hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps & , clinical signs (rr, spo , heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : .a low value of pac on admission until the patients were intubated. . an increased level of cpk in relation with an acute myocardial infarction ( / cases in the failure group, vs / cases in the success group, x~(with continuity correction) : p<. ). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco (< mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators ( , ). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) ( , ). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls , driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls was connected to an artificial trachea (at), intubated with an . nun et]', end connected to one of four ventilators (servo c and servo , siemens,-elema, sweden; evita , driiges, liibeck, frg; pb ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips and mbar; trigger set at maximal sensitivity, peep always mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied paralyzed cppv ventilated ali patients (lung injury score = . • that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve c), first during cppv and then within the th psv hour. airway pressure and flow signals were recorded (cpi bicore) for subsequent analysis. an airway pressure plateau was defined as a flow tracing in which airway pressure was stable for at least . sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; ) during psv inspiratory plateaus are longer than the expiratory ones; ) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = patients). we selected ali patients (lis= . • in vcv mode who had available a chest ct scan performed within days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao > mmhg, irrespective of fie (peep < cmh ). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate < bpm, an increase in fie lower than . compared to vcv, a pace increase < % of vcv value and hemodynamic stability were maintained during the next hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with ventilated patients, after head injury and with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep ( - - cm h ). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: . + . for peep , . + . for peep and . + . l/rain for peep . simultaneously the bioimpedance device recorded a significant increase in cardiac output from . + . for peep to . + . l/mi for peep . (p < , ). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of patients who had been hypercapnic (paco > mmhg) for at least days (range to days) before the end of cv. the psv trial was started as soon as pao was > mmhg, irrespective of fie and with peep < cmh and the psv level had to be < (pplateau-peep) as measured during cv. pace , pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: ) . ) weaning is more difficult in pts with head injury(p<o, ) and iss> (p<o,ol), in elderly(p<o, ) and in pts who need fi > , (p<o, ) and peep>io cm h (p<o, ). ) pts with iss> need longer duration of mv (p<o,ol). ) the mortality rate is higher in pts with iss> (p<o,o ), with head injury (p<o,ol) and in elderly (p<o, ). ) paradoxally, compliance was found to be higher in pts> years than in pts< years (p<o, ). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in patients (individual analysis for each of the eight lungs) at peepe levels: peepe = cmh (zeep) and peepe amounting to %, % and % of the peep• at zeep ( . • cmh ). tidal volume was kept constant ( • . ml/kg). each lung section was divided into zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: pts with acute (a) (paco = • mmhg, pao /pao = . • and pts with chronic (c)(paco = • mmhg, pao /pao = . : . ) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh *s/min), which is an index of oxygen muscle consumption. measurements were collected, after rain of stabilization, at and cmh ps and at two different peep levels ( and cndd conclusions: during psv: ) breathing pattern and ptp are different between a and c; ) in both groups peep does not modify breathing pattern; ) peep reduces ptp in c but not in a; ) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p . and rr. changes in p . were remarkable, homogeneous and highly significant (p<. ), while changes in rr were less pronounced, inhomogeneous and less significant (p<. ). it seems therefore that p . is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. sets of measurements were obtained in each of ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to % of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=. .ctotlsf+l. , r =. ), and with cqs (cqs =. .ctotlsf + . , r =. ). the average difference between ctotlsf and cqs, yet, was much lower (+. + . ml/cmh ) than the one between ctotlsf and cdyn (+ . + . ml/cmh ). rtotlsf correlated much better with rmax (rmax=. *rtotlso+. , r =. ) than with rmin (rmin =. .rtotlsf +. . , r =. ). the average difference between rtotlsf and rmin was + . + . cmh / /s, while the one between rtotlsf and rmax was +. + . , confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. sets of measurements were obtained in mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all reference measurements, rcdyn (ve/v'epeak= . orcdyn-. , r =. ), rcstat (ve/v'epeak = . rcstat -. , r = . ), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period - we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao /fio < , despite maximal ventilator settings: peep > cm hz , fit > . . a total of patients matched these criteria, males and females with a median age of ( - ) years. seventeen suffered from severe trauma. chfjv was started following a median period of ( - ) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit . , pao /fio , peep cm h peak airway pressure (pap) cm h . chfjv consisted of high frequency jet ventilation with a frequency of to breaths/minute, driving pressure of . to . arm, and inspiration time of to percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l to breaths/minute and tidal volumes of to ml. results: following two days of chfjv of patients showed an improvement of ventilatory parameters; peep could be reduced to < cm h in patients, the pap was decreased with > cm h:o in patients, fio could be reduced to < . in patients and finally the median pao /fio ratio changed from to . during chfjv patients died, of respiratory failure and due to multiple organ failure, died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm ; ( ) :a ). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with groups to ventilate nonperfused, lavaged rat lungs with small vt ( to ml/kg) for hours and studied the effect on compliance and lung injury. group : peep>ping group : peep<pin f with an si, inflation to on h over sec, to recruit volume; group : peep<pinf without si; group : control group, lungs were inflated at peep<pin f, but not ventilated. results: in group and group static compliance did not change after ventilation (table) . in group static compliance fell significantly. group showed significant changes in the pv curve, which were less severe than in group . results from morphological examination are pending but preliminary results showed less lung injury in group , compared to group . conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy ( ) minimizes lung injury as well as, or better than using peep above pint" and ( ) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles eeg examinations with fourier spectral analysis' of patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co and blood gases during weaning. results: patients had invariable eeg activity or short -waves period (till one hour). the weaning of this patients was fast arid sucsessful. other patients have had a decreasing of a-activity, an appearence of -waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u , hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett ) but not others (siemens serve ). we studied the two types of systems in two groups of patients mechanically assisted with pressure support ventilation ( + cmh ). in the first group (pb ) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt ( + cmh .s/min - vs + , p< . ). by contrast no significant difference appeared in the second group (serve ), as predicted by the bench study despite marked interindividual differences ( + cmh .s/min - vs + , p = . ). we conclude that ) rigorously performed bench studies can predict in vivo effects, ) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs , lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: ( arf and ards) sedated paralysed patients were studied using a serve c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to - ml or a maximum paw of cm h (alfi). the quasistatic elastic pressure (pel,qs was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v from up to - ml were followed by a s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~ i~: in ards, mean cst was . + . and cqst . + . ml/cm h (us), lipst . + . and lipqst . + . cm h (us), uipst . + . and uipqst . + ~ cm h (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, ! ). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > colony forming units / ml. results: for the patients studied, the mean saps score (+ sd) was +_ , the initial pao /fio ratio was -&-_ , the duration of mechanical ventilation (mv) was + days. the mean delay before the onset of the first np was . + . days ( - ), and the mean pao /fio ratio was +- . respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in % of the patients studied. alteration of gas exchange was present in of the patients ( np) . a new pulmonary infiltrate was present in only np ( %). an increase of fever was noted in patients, an increase of leukocytosis > % in patients, an increase of volume and purulence of sputum in of the patients with np. the degree ofgazometric worsening (pao /fio before np minus pao /fio during np) during the first episode of np was + mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was in / patients, ( / ), ( / ) or ( / ). two patients only had a pulmonary colonization (ptc: < cfu / ml) before the first episode of np. the incidence of np is high ( %) during severe ards. the first episode occurs in average:at the th day, and is the cause of a severe hypoxemia (pao /fio ) . the onset of a np may contribute to the high mortality rate observed in our patients ( %). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of . • j/l, a mean value of inefflency, ~ of . ~= . and a mean value of resistance, ~, of . • cm h s/ . the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ---- . • . cm h s/l. during methacholine provocation~ ae rises more than five-fold up to . • j/l~ doubles to . • and t~ increases to a maximum of • cm h s/l, with armax : . • . cm h s/ . the variation in rk becomes more pronounced with higher doses of methacholine. methods: ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo , and aado decreased while paco increased and tda vo ume and co remained unchanged. on the contrary, in group v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo and aado increased with constant co, the pressure support level needed to achieve the target rate was much lower in group than in group ( , -+ . vs . _+ . ). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, - years)breathed spontaneously through an uncut ett of . mm id via a mouthpiece. the ett was connected with a prototype ventilator evita modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, ( ) asb ( mbarover mbar peep), ( ) atcin, ( ) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+ ), ,,equal" ( ) or ,,worse" (- ). inspiration and expiration were investigated separately by presenting mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within hours after cardiac arrest and repeated , , , , and hours later. results: we studied patients, females, mean age , + , years. the pi decreased s!gnificantly between th~ first and the last measurement from . _+ . to . + . (p = . ). cerebral oxygen extraction ratio decreased also from . + . to . + . (.p = . ). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels hours later, rasp. . pmol/ml (median; th . - th . ) and . pmol/ml (median; th . - th . ) (p = . ). eighteen and hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = . , p = . and r = . , p = . ). we.also found hours after the first measurement a significant correlation between pi and cgmp levels ( r = . , p = . ). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology ( , ). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged - years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m segment of the middle cerebral artery (mca). results: a total of phases of vf were investigated. duration of vf ranged from to seconds, with cbfvmc a (mean_+sd, cm sec - ) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: icu nurses were asked to inflate a testlung model times with a specific air volume ( ml, ,ml or ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated ml ( ml). the mean inflated volume for this group was ml. when the icu nurses had to inflate ml the mean absolute volume difference was ml with a mean inflated volume of ml. inflating ml produced an absolute volume difference of ml with an mean inflated volume of ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the ml and ml for all specific air volumes. % of the last inflations with feedback were significantly smaller than ml from the specific air volume (p < . ). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when ml and the smallest when inflating ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in domestic pigs. following rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for min. electrical defibrillation was then attempted and of animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of . ml/kg to . ml/kg (p< . ), and left ventricular stroke work index (lvswi) from . to . mmhg,ml/kg (p< . ). both svi and lvswi remained depressed for another hours. these decreases were associated with increases in heart rate from bpm to bpm (p< . ). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d- bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach - % depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from - min, mean min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after - min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report cases of prehospital cpr with primary asystolia terminated after - rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after - min and cpr efforts were immediately resumed, nevertheless, of the patients died at the scene, while could be hospitalized with stable circulation. one of them died hours after admission to the icu, the other survived for weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [ ]. we reported our results on such monitoring in [f.coaobbeb,r.fe enb~-kap~monorm~, ,n ,p. - ] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po ,pco (mm rg),so ,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po +pco ) in mm hg in ap blood. these data were derived from:i) subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [ j. in control group it is equal to , • mm hg. tests on sgp neither exclude nor substitute conventional (pc and pco ) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: ) using towel clips to reapproximate skin only, )i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, ) using loosely tied retention sutures for partial closure, ) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from to . all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died t days later of multiple organ failure. there were no complications related to the closure technique in any of the patients. conclusions; . selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from / through / , pts underwent open heart surgery at our hospital. of th~se, pts ( %) (age _+ yrs) had a cardiac arrest during the first hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) ( pts), valve replacement (vr) ( pts), cabg and vr ( pts) and aortic aneurysm ( pt). the preoperative ejection fraction was _+ % whereas bypass and aortic cross-clamp time were + and + rain, respectively. prior to arrest, they had a cardiac index of . _+ . l/min/m and were receiving . + inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation ( pts) and bradyarrythmia ( pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in pts. eighteen pts ( %) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t pts, %), tamponade ( pts, %), rupture of the proximal vein gra& anastomosis ( pt, %), graft occlusion ( pts, %); no cause was found in pts ( %). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (- % incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of doctors and nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between - . after the first screening we held a x hours training. doctors and nurses were trained for the endotracheal intubation (group it , t ) , doctors and nurses were trained to use the laryngeal mask (group lm , lm ) . all respondent were trained to use the bag-valve-mask device. day, month and month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. x hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and % responded the course "very useful". . % of doctors and . % of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies ( . %), penetrating abdominal wounds with injuries of mesenterial vessels ( . %), injuries of the liver ( . %), blunt abdominal trauma with lien ruption ( . %). in . % patients had the previous somatic pathology. blood loss volume was - ml, & the reihfuside blood volume was - ml, consisting - % of blood loss. it was needn't to fransuse donor blood in . % in further but - ml of contanined erythrocytes were frasfused for supporting of hb concentration on the g/l ( g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ functional indices of cardiorespiratory function were evaluated. results: pregnant women with ~h-gestosis were examined before and after i/v infusion of i ml of . % clophelin solution, . mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by %, of the heart rate by io. %, of the oardial output index by . %. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for patients. the mean iss value was . + . while the median value was . minor injuries were present in ( %) patients with iss less than , while ( %) patients with iss more than had severe injuries. increased mortality of the older patients was noted in the range - . all patients older than died while % of patients below yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp- ). results : fifteen patients were admitted in icu and in a medical unit. fifteen patients died. a group of patients had a lactatemia up to mmol.l" . in this group of patients, had acidocetosis, had asthma, had cerebral vascular ischemia, had neoplasia, had cardiogenic shock, was epileptic, had congestive heart failure, had acute respiratory failure, had septicaemia, had hyperosmolar status finally had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( . • vs. . + . , p<o.oool, respectively), the cut point of lactatemia was . mmoll" se was %, sp was % and the accuracy was %. on the other hand lactatemia was higher in patients admitted in icu than in medical unit ( . + . vs. . • p<o. , respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over months. setting: a -bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < x , neulropaenia < x , and lymphopaenia < . x ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days , , and . the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < . ) and hollow visceral intra-abdominal injury (p < . ). eight ( %) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < . ) and significant differences were found in absolute neutrophil counts (p < . ) and differential percentages (p < . ) on days and . no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that . % traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: . besides a physician specialized (vii --+ vii -~ viii gradus) in urgent medicine, it's necessary to introduce two ( ) medical technicians instead of one as is existing now ( ~ ). .the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). .the technicians have been specially educated for urgent cases (iv ~ v ~ vi). . such a new team structure should be more capable in professional sense. .we can see an importance of team-work in ects. . such teams should also be more economic in comparison with existing one. . we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis ( dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc , dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at - mg of cells per i kg of patients weight. dialysis was performed during - hrs. patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: . ml of hemosorbent and . ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of - ml/min, during - min. average volume of hemoperfusion made . . conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam ( . mg/kg),thiopentone ( mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone ( mg/ kg/die),mannitol ( .smg/kg) and diphenylydantoin ( mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in patients ( m., f., + y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is , - , mmol/l. although more than % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in % of patients ( ) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from st of january till st of december . results; in this four years period there were abdominal injures. there were blunt injures. among them in patients peritoneal lavage was applied. results were positive in patients and negative in patients. false positive finding were in patients and false negative in two patients. in patients with positive lavage there were more than , /mm red blood cells, more than , /mm white blood cells and the level of amylase was up to u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than , /mm red blood cells, less than /mm white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se en md, z.milo~evi md, *s.srdi md, k.sar ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the st of january till st of december , are presented. results: among operated patients there were females ( . %) and males ( . %). there were patients ( . %) with gastric bleeding and patients ( . %) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in patients ( . %), ulcer suture in patients ( . %), billroth i in patients ( . %) and billroth ii in patients ( . %). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in patients ( %), duodenotomy with ulcer suture was done in three patients ( . %) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj b ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in ( %) patients, while in ( %) it appeared unaffected. cpk-mb of % and more in comparison to total cpkwas . + , % in patients, and , + , % in pts (p< . ).c of . + . l/min in patients with cardiac contusion, measured hrs after admission at the latest, was significantly lower than in group of patients with co of . + . l/min (p< . ). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was % lower than in the blood inflowing. hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was % lower compared to the vb and did not change for hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent years patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. ( . %) underwent urgent surgery, of them ( . %) with cardiogenic shock and ( . %) with massive pulmonary embolism. patients ( . %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in cases ( . %), in ( . %) patients subsequent pulmonary embolectomy was performed. mortality rate was ( . %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and ( %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in % patients. pe mortality rate is repoted to be %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr ),free thyroxine(ft ),total tri-iodothyronine(tr ),free tri-iodothyronine (ff ) levels were measured within hr.of intensive care unit admission(first day) in multiple tratmmtized male patients(injury severity score higher than ). in fifth day the same hormone levels were repeated. tsh,et ,ft ,tr and ft levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt ,fr ,et and ft in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm ( ), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( , + , mmol& norm - , + , mmol/i ), whereas rats without glucagon had urea content , + , mmol/ ( p < , ). osmolarity of blood plasma in rats having glucagon injections was , + , mosm/kg h , without the drug - , + , mosm/kg h ( p < , ), ldh activity - , + . and , + , ( p < , ) mcmol cec- accordingly. lethality in the group of rats without glucagon was % /n= /, with glucagon - (n = ). in patients with hypovolemic shock in minutes after glucagon administration stroke volume increased to % (sv), miocardium contractility -to % (mc), peripheral resistance of vessels ( vr ) decreased to %. in - , hrs sv increased to %, mc-to %, pvr -decreased to %. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing, ), p. n, vasina (t) ,n.sebbota hph ( ). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k r-key, [ maine ( ), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i ~ mmol/l down to # mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal z, there of i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in % and morphologically in - % of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last years patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were patients with acute myocardial infarction treated before hospital by the cardiological groups. patients were treated with the early infusion of nitroglycerin and without. the results of treatment were: in the group with infusion: mortality in the first hours % the full disappear of pain % in the group without infusions mortality in the first hours % the full disappear of pain % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used mg prazosin tablets sub lingua and measured blood pressure ones during hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was . the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in % of patients. the general condition of patients became better in - min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: patients, aged _+ years (mean _+ sd), and burnt _+ % of body surface, were studied from day (d ) to d . serum samples were collected at do, serum and urine from d to d , and on dio, d , d . cutaneous exudates were extracted from the textiles surrounding the patients, collected over h periods from d to d , using bidistilled acidified water. mg supplements ( . to mmol/ h) were prescribed from d to dio according to the severity of hypomg (serum mg < . mmol/i). results: mg serum levels decreased below reference ranges in patients between d and d , and normalised thereafter during supplementation. urinary mg excretion was increased in patients, mean excretion being . _+ . mmol/ h until d . mean daily mg cutaneous losses, were mmol/ h, i.e. mmo; in days. large inter-& intra-patient variability: the daily exudative losses ranged from to mmol/ h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t / ,id ). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for seconds with ~ hot water to achieve a % tbs partial-thickness burn. blood samples were taken prior and after surgery as well as , minutes, , , and every further hours after thermal trauma. two control groups (each n= ) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n= ) received c -inhibitor concentrate at an initial dose of u/kg body weight either immediately or hours after thermal trauma (n- ), followed by three further applications (of reduced amounts) every hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin- (il- ) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of multiply injured ( ( ) ( ) ( ) il- (nis) ( ) ( ) ( ) il- (nin) ( ) ( ) ( ) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il- is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no ) and nitrates (no ). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included patients admitted to the intensive care unit of the burn center of brussels (age : - years; burned surfaee area - %), and control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age - years). the patients in both groups were fed enterally with to kcal/kg/day of a standard solution. in the burn group, daring the study period, patients were mechanically ventilated, required vasopressor (dopamine) therapy, and received antibiotics; patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no /no determinations (griess reaction) daily from day to day (n=ll) or to discharge (day , n= ; day , n= ) in the burn group, and once the control group. in the burn group, no /no levels were elevated at day ( _+ #moles/i), reached a maximal value on day ( - p.moles/l) and progressively decreased to day (fig). these values were higher than in the control group ( . :t: . #moles/l, all differences p< . ). however, no correlation was found between the plasma no /no levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no /no levels than those who did not ( + vs - #moles/i, ns). the present study indicates that burn noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no /no plasma levels, which are ao so consistent with an enhanced no pro- o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics ( ). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury ( ). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa , male, female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a gauge needle was inserted into the lumbar subarachnoid space at level l /l and connected to a trancducer (mx | medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco constant. after a resting period of minutes basline measurements were performed including csfp, cvp, map, hr, etco (cardiocap | datex). then the volunteers were given urapidil . mg/kg over a period of minute. minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p< . significant; values: mean_+sd, si = mmhg. - _ " _+ * _+ * - _+ " _+ csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map ( ). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of -min ischemia, reaching peak at . h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied patients with severe head injury (glasgow coma score < ) admitted to our icu between fobmaly and december . icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of patients were additionally icpc monitored by a fiber optic transducer (comma r~), and seven of patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = . (p < . ) with a regres~on line y = . + . x. four of ~hx lcpc -icpv patients had r > . when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = . (p < . ) with a regression line y = . + . x. corralalmu eoetfieiont was inwer than . in all seven patients. corrdation eoelfieients for levals of icpv > man hg, > mm hg and > tuna hg with icpe showed r = . , r = . and r = . respectively; and with icpe r = . , r = . and r = . . the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors ( , ). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam ( - mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam ( - mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, patients aged over years suffering from head injury, with glasgow coma scale (gcs) of or less, were studied retrospectively. twenty-six patients ( . %) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three ( . %) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs ( . vs. . ) and median age ( . vs. ). the incidence of brain pathology in the two groups was the following: epidural haematoma . % vs. . %, acute subdural! haematoma . % vs. . %, intracerebral haematoma . % vs. . %, subarachnoid haemorrhage . % vs. . %, diffuse haemorrhage . % vs. . %, contusion . % vs. . % and non-visible pathology (normal ct) . % vs. . %. unilateral pupilary dilatation was found to be . % in group a and , % in group b. the mortality during hospitalisation in the i.c.u. was almost the same: % iu group a and . % in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a month period ( - r the icp was monitored in p~,tients ( male, female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was . years ( - ). the glasgow coma scale at admission was . ( - ). in all cases the device was placed wfihln the first hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was . days ( - ) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c ntra!s by ct-scan. in case an intracranlai hemorrhage was detected and drained. the overall survival rate was . %. showed a complete resolutl n, in other . % psychological residuals like decreased mentatlon, in . % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and . % of the injured remained in a comatous status. in % of our cases the measured values correlated with clinical course and management. in cases ( . %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality . identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age . _+ . , injury severity score . + . ) admitted to icu were studied. incidence of arf was . % ( / ). arf was defined as persisteat plasma creatinine > mg/dl with or without oligoanuria . arf was defined as early when occurring within the first hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in patients while larf developed in patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis ( . % for both) were observed in larf group, when compared to earf ( % and % respectively). abdominal trauma was more frequent in earf group ( % vs %). the gs for earf and larf were respectively _+ . and _+ . while in the group who not developed arf (narf) the gs was . • conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher ( . versus . ) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc ( ) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied consequitive patients ( in group i) and ( in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was min. in group i we recorded r.o.s.c. (return of spontaneous circulation) , %, death %, continuation of cpr efforts , %. while in group ii, %, %, and , % respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: patients, aged _+ yrs (mean_+sd), with burns covering + % of body surface were studied from day (d ) to d post-injury, were randomised in groups (g): g -control receiving recommended te supplies + placebo; g -receiving in addition large supplements of cu, se and zn from d to d . enteral nutrition was started within hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d and d . infectious episodes and micro-organisms were monitored until d . results: the patients' characteristics were similar g & g . the total leukocyte counts were higher in g between d and d , due to increased neutrophils (significant from d to d ). total cd + and cdlg+ cells did not differ, whereas cd + (monocytes) were significantly increased at d . proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g with a mean of . _+ . infections during the first days versus . _+ . in the control group (p < . ). conclusions: the large te supplements for days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of % body surface area iiib-iv burn in rats. a burn eschar was excised on the rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the th day after burn infliction the activity of cathepsin d in injm'ed muscles is times lower under an adsorptive dressing than under a gauze bandage (control) (p< , ), the activity of trypsin-like proteases is . - . times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is - % and - %, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first hours icu data and icu admission had to occur within h of traumatic insult. over the last years, pts with severe head injury (gcs< ) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of . h after trauma, range between - h). in a total of pts (= . %), jugular bulb desaturatiens (< %) were noticed during this early h period. in pts (= %), jugular bulb saturations higher than % were observed, whereas pts (= . %) revealed no abnormal sjo data ( - %) during these first h. concerning the periods with too low jugular bulb saturations (n: ), we found the following correlation ; in pts (= . %) cerebral perfusion pressure (cpp) was below mmng, in pts (= . %) paco~ was below mmhg and finally in pts (= %) we found primary intracranial hypertension. for the high jugular saturations (n: ) we found a primary intracraniaf hypertension in f pts (= %), and a pace level above mmhg in pts (= %). in all patients we could restore jugular bulb saturation within normal range ( - %) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury ( ). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp> mmhg) and cerebral hyperaemia (sjo~> %) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of head injured pts received treatment for intracranial hypertension over the last months. six of them met the criteria set for the administration of indomethacine. in pts, no decrease in icp or in sjo was observed and both pts died due to therapy refractory intracranial hypertension. in the other pts, a significant fall in icp and in sjo was observed shortly after indomethacine administration. in pts we observed a catastrophic fall of sjo= even below %, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the pts, icp remained under control without further administration of indomethadne, but he died days later in multiple organ failure. the other pts, needed multiple indomethacine administrations (for pt even during consecutive days) to finally control icp. in all pts, icp was finally controlled, but only pt survived. both other pts died from systemic causes (multiple organ failure in pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in consecutive pts, suffering from severe head injury (gcs< ). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo information. pts revealing ich and sjo=values above %, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than %, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=> %). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed pts with ich, with an overall mortality of . % due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer pts, csf drainage alone was sufficient to control ice of the remaining pts, pts received mannitol and pts were hyperventilated as second approach. in the third line, pts were managed with barbiturates, with mannitol and pts with hyperventilation. finally, barbiturates were used as the final rescue in pts. these results reveal a less frequent use of mannitol as only pts received mannitol, compared to the pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier ( pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo -guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po , icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in piglets ( days old, , - kg) of either sex we produced a moderate brain injury ( , arm., msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and min. after brain trauma and after , and hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to mm hg being highly significant (before mean i mm hg, range - mm hg) could be observed: mean arterial blood pressure rose from appr. mm hg to ii mm hg for - min. in two animals this was followed by an ischemic period lasting min. accordingly icp values gradually returned to starting measures within hours; in the ischemic animals they remained at a level of about mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within hours. cerebral blood flow (ldf) fell from mean values being i before trauma to appr. zero and recovered to around . brain po started at mean values of mm hg (range - mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: ) suction of gastric contents from trachea during intubation, ) presense of a predisposing factor, e.g. coma. ) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale , because of cerebral injury, and patients who died within days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were and the non trauma patients . from the trauma patients, had cerebral injury and were polytreumatized without cerebral damage. from the non trauma patients, had malignant neoplasms, neurological diseases in terminal stage, old age, drug overdose, and several diseases. eighty seven from trauma patients ( %) and from non trauma patients ( %) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the groups (p> , ). the severity of complications was also proportional in the groups. eighteen deaths were recorded in the trauma patients (mortality %). only deaths correlated directly or indirectly with the aspiration ( %). in non trauma patients, deaths were recorded ( %). twelve deaths were recorded in patients with neoplasms, deaths in patients with neurological diseases, deaths in aged patients, death in drug overdose patients, and death in patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< , ). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p< , ). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in groups. the patients of group a ( patients) received fluconazole rag/day for days. and the patients of group ( patients) amfotericin-b rag/day for also days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from of the group a ( %), and from of the group b ( %), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p< , ). all the patients before treatment had positive for fungi blood cultures. after days of treatment, patients of group a and none of group b had positive cultures. eight patients (from who had positive cultures of bronchial secretions before treatment) of group a. and (from ) of group . had positive cuttures of bronchial secretions after days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p< , and p> , ): ten patients (from ) of group a and patients (from ) of group b had positive urine cultures, after days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p< , ). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in patients, chills and fever during infusion in patients, and hypokalemia in patients). three patients of group a and patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were . all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, in group b, in group c. and in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the groups as to number of complications ( twenty four patients had chest complications in group a, in group b, in group c, and in group d. p> , }. the duration of mechanical ventilation was not statistically different among the groups (the mean duration was , days in group a, , in group b, , in group c, and , in group d, p> , ). there was also no statistical difference in mortality among the groups (six patients died in group a. in group b, in group c, and in group d, p> , ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in trauma patients admitting between december , and july , in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in ( %) of survivors and in ( %) of non survivors .we noted low fncidence of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.( . vs . , . vs . meq/l, p< . )the duration of hyperlactemia also was higher in the patients with organ complications. ( . vs . days, p< . ). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.( . vs . , . vs . meq/l, p< . ) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj , kjdo and ceo in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and jo monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous hours, normal x ray chest and within the first hours from injury, were considered candidates. peep value where increased fro~ zero to cmh , in three different steps, each one with min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo , in the case of hemodynamic deterioration, during the study, ppc ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. patients were candidates, of them were exc;uded due to haemodynamic instability, k total of completed all steps and were e~amined, men and women, age ! . yrs. at hospite; arrival, glasgow were < in patients, and > in the rest . patients mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied patients( females), aged (mean-..+-sd) . _ . yrs, which were consecutively (from august to march ) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was . -+ . (range - ). the following data were evaluated: ) number of medical departments, where health care was provided until final admission to the icu, ) ambulance transportation conditions, ) catheters and tubes inserted before admission, ) vital signs upon admission ) information provided by referring physician (scored on a to scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), ) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu ( : . %, : . %, : . %, : %). thirty/ ( . %) patients were escorted by a physician. twenty-six/ ( . %) were transported on oxyge n, fio (mean__.sd): -+ %, pao : . -+ . mmhg. five of the remaining , for whom no oxygen was provided, had pao : . -+ mmhg. twelve/ ( . %) were intubated and ventilated during transportation. thirtyfour/ had a peripheral venous line, / had an arterial line, / had a nasogastdc tube, / had a urinary catheter. eleven/ were sedated and / were paralysed. three/ were on inotropes. vital signs upon admission were: arterial blood pressure, systolic . -+ mmhg, diastolic -+ mmhg, heart rate -+ bpm, temperature . -+ cc. patient information score was --. . . the actual state upon admission was found substantially different, as compared to the description of the referring physician, in / ( . %) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since . % of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from to years. dosing ranged from mglkg to mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from st june to july , all the calls for emergency care were analysed using a questionnaire of items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively % and %. the performance of the model is %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may to april , patients were admitted to our icu with bci ( m, f, age + ). a history of direct trauma was present in patients. admission gcs was in all patients, and was associated with hemiparesis in of them; the last became paretic hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan ( - days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table x in z test. res.cl~s: of patients were submitted to thrombolysts and died. the higher incidence of bracb, ar~lhmias (ii degree gg p t e and av block. i degree av block. avsb . <ii. sinus arrest) that requited the insertion of avsc . <cl temporar~ pace-maker was recorded in group a b vs c . ns in % of the cas ~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and , %nacl/ %dextran . methods:in polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q ) prevalid.replacement of the volume loss egan in institutions of general medicine,frequently by rl and dextran or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution dministered ~o the injured wer~ - ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the , %nacl/ %dextran @(groupii)( -sml/kg observed to the final surgical management,had statistically higher map(increase of % in relation to admission), cvp(average increase of , cmh~o),diuresis(averagely omi after min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q %,average increase c~ of cvp , cmh and average diuresis oml after omin). total quantity of the administered solutions was significantly smaller in the groupii( ml: ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl: ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of , %nacl/ %dextran lasted minutes. w.scherzer(l~,k.lechner( ),r.simon( ). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin !( )neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson, ) ,in general anaesthesia (bennet, ) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger, ) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter( ) ,basal stimulation (bienstein, ) , bobath-therapy( ) and facilitation of a physiological moving pattern(pnf)according to knott( ) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: patients (mean age: ) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n= ), acute congestive heart failure (n= ) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of rrd ice-cold glucose % non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of months. the tt is a eompetitve immunoassay which gives qualitative results within i minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study patients were enrolled, in of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of . and specificity of . . conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure > rorohg and diastolic blood pressure > nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below mmi-ig and diastolic blood pressure below mmi-ig within minutes after start of treatment. results: patients were included in our study, ( %) patients responded successfully to treatment. mean arterial pressure decreased in responders by . mmhg and in non-respenders by . mmhg (p< . ). responders and non-respenders differed signii'icantly concerning pra (p= . ), ace (p= . ) and ang ii (p= . ). . . the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r= . ; ang ii: r= . ). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july to june , files of patients consulting to the ed for smp have been retrospectively analyzed. results: patients, women and men, . + years old (range - ) have been admitted for episodes of smp ( % of all consultations) whose relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively . %, . % and , % of patients. the interval of time between the ingestion and emergency consultation was noted for % of smp ( + min, ranges - ). the involved products name was known in totality in % of cases with an average number by episode of . + drugs (ranges - ). the most often, ( %) or ( %) different products were interfered. the nonbarbiturate psychotropic drugs accounted for . % of the products (benzodiazepines %, antidepressants . %, neuroleptics %, carbamates . %, imidazopyridines . %, cyclqpyrrol nes . %). analgesics and nonsteroidal antiinflammatories represented . % of all drugs, anticonvulsants . %, cardiovascular drugs %, antiinfective agents . %, drugs against cough . %, muscle relaxants . % and antihistamines h . %. the benzodiaz pines were present in episodes, alone in episodes. in . % of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in . % of cases, activated charcoal in . % of cases, flumazenil in . % of cases, naloxone and acetylcysteine in . % of cases; orotracheal intubation was performed in patients. admission in hospital was effective for patients, in medical ward (n = ), psychiatry (n = ) or icu (n = ); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately . m is based on the principle of three identical bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate ( ). method: a plaque of electrodes, positioned on the left anterior ventricular wall of anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused ( . mg/kg/min iv) during rain; at t , dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least % of its value at to was demanded before the administration of b. results: dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively ( < . ) compared to values control fro)except qrs at t . the b ( + ml/kg; ranges . and . ml/kg) modified no studied dements outside of the ( }rs. to ti t t t t t a , + , , + , , + , , + , , + , , + , , +- ,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from (--full agreement) to (=disagreement). overall, "/. of the active emergency physicians participated in the survey. results: . % of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only % would not hesitate to go into action by dark. . % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only % would do it. . % of the doctors would rather stop flying. the most common reasons for ,uitting were wish of family and fear of an accident. . % conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. crash landings within less than years with deadcases and badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in patients (age - years). the indication for intubation was elective surgery in patients (asa i-ii) and emergency intubation in patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the emergency patients was dyspnea in , cardiac arrest in , trauma in, and unconsciousness due to drug overdose or seizures in patients. intubation was facilitated with medication in patients. results: of the elective patients, ( %) were successfully intubated. six patients ( %) needed two attempts before successful intubation. the duration of intubation exceeded seconds in patients ( %). of the emergency patients, ( %) were successfully intubated. six patients ( %) needed two attempts, and the duration of intubation was more than seconds in patients ( %). in % of all patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in patients ( %). oesophageal intubation occurred in patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < x e /i. in each treatment the same type (av- , fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between and s. the initial enoxaparin dose was rag every :th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels . and . mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin ( . + . h versus . + h, ns). the mean aptt-levcl during heparin treatment was s and during enoxaparin treatment s (ref. - s). the mean daily dose of heparin was nag, that of enoxaparin lg mg. the mean anti-xa activities were . u/mi and . u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load data obtained through direct measurement from the monitors and the lab, the program calculates parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for pts during aperiod of three .y~m , treama pts were . %), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g =- a p> . ; roc = , ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in dogs -~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made . + . ml/kg). the treatment was begun after .u+o. hrs of h. "sl", created on the basis of-sorblt and natrium lactate ( mosm/l) was injected into v. femofalls at the dose of io. ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to . mm hg and - . + . cm h (p .o ), while heart rate (hr)-increased to . + . per min (p<.o ). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by . % (p<.i) and . % (p~. ), while fibrin degradation products (fdp) enlarged by . % (p~ . ). after - min of treatment termination abp and cvp increased to . + . mmhg and . +o. cm h (p<.o ), and ~[r diminished to t . + . per min (p>. ). at the same time the indtces of pic and pf enlarged by . % and . % (p>.i), while fdp diminished by . % (p>.i). one of dogs survived. life duration of the other dogs was . + . hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo through opljcal fiber and the cerebral flux with tcd. methods: since january until june , we collected ht admitted to the icu, and of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ , with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< , sjo is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , ( m, f) , (m. age: + years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was > in all studied patients. tbe group a ( m, ") had no tremor and/or fever on admisskm, while em group b (tin, the above criteria were ix)sitive. bhx~d samplings were taken - hours after accident and - rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, healthy people were examined. _resu!_ts-il-lct, il-ii~, il- and tnf-tt levels were similar to control group levels in both groups a and b. i!,- and g-csf levels were found increased in both groups (p< jxjl), while il- levels were statistically significant comparing to group a. in con_tin_skin, during immediate post raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than ,. , cannot be detected,whereas .- was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep % burn, cornplicated by bloodshed the of blood grows at - times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to %, and % in hours. in hours after the trauma the-process is accompanied b ! , % lower inhibitory activity of blood, where as at the peak of the trauma it was , ~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and -fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at times; [ emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing - , kgr and at age of - months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and . ml (this volume assuring a normal incranial pressure after kaufman ) of autologous blood was injected into a depth of mm via a needle of . mm bore. the wound was closed and the animals were left to recover. nimodipine, of , mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into % formalin solution. tissue sections of ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was --. ~t while that of group b was + ~t (p< , ) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: pts with - % bsa second to third degree flame burns (respiratory tact injury excluded) to yrs of age were randomised to study (n= ) and control (n= ) groups. burn shock was treated with hypertonic saline -bicarbonate solutions ( mmol/l na +) ml/kg/%bsa for the first hours and ml/kg/%bsa for second day. analgesia in control group for the first hours was provided by regular hourly intramuscular administration of mg of morphine sulphate and mg of analgesic -antipyretic analgin with mg of diphenhydramine (dimedrol). from the rd day regular administration of morphine was finished. in the study group ixg of clonidine was added -hourly for hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, - ), sleeping time, spo , hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first days was . - . vs . - . in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only pt showed sign of aws. mean vas score was in . - . range for first postburn days. pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo values in study group were in - % range, among controls - %; vomiting was absent in. cionidine group vs cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs ( -+ kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter- polysulphone membrane) and remained stable for one hour (pao~= . • peco = -+ mmhg, ph= . -+ . , bp= -+ mmhg and pap= -+ mmhg). all was induced two hours after oleic acid infusion ( . ml/kgr) into the pulmonary artery (poo~= . _+ -p< . , paco~- . _+ -p< . , ph= . -+ . -p< . , bp= -+ -p=ns, and pap= _+ -p< . ). fio % for the next minutes did not significantly altered the b ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter ( l/min) while filtrate and gas were removed from the outlet port (bypass flow ml/min) b) through a thin intratracheal catheter positioned cm above the carina ( l/min). the fio given through the ventilator readjusted to %. results replacement fluids/filtrate during the next four hours were not exceed . l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.= . objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied randomised clinical groups of patients each containing patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the -st and on the -th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c and c fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the -st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the -st day, while on the -th and on the -th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, g. sofiiski str., sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a -year old male patient with extremely massive intraabdominal haemorrhage ( m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of ml of autologous blood using an original autotransfusion system developed by us ( pat. no / . . ) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first days after the trauma in patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over . results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the -nd and the -th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity ( + pg/ml compared to + . pg/ml in control) were found od day following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages h following trauma ( - % compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase ( %, h following trauma and % and % on days and after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days and following trauma, and initially but persistently high levels of plasma tnf alpha in da rats ( - fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, , iso , yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca - t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least wounded and sik people. in federal republic yugoslavia, it was proposed such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < % may be more dangerous than short-term levels of - %. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was / and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of % was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef %). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a year old male ingested mg doxepin (sinequanr), a tca and mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs / ), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of b.p.m., an arterial blood pressure of / mmh g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively gg/l (toxic level #g/l) and i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed / h later. nahco ( meq/ h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of patients treated" during a two year period ( - - to - - ) in our i.c.u who meeted the following criteria: ( ) coma score _< gcs within the first hours, ( ) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes - ), ( ) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ meq/l). survival was defined up to the th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = . ). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in [ ] the pre-existing equipment of the volunteers was completed with type a ambulances and special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, consecutive emergency interventions in the time period from novembre , to april , . the emergency physicians treated male ( %) and female ( %) patients, patients died before hospital admission and patients ( %) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period year earlier (november to april ) without attending physician the volunteer rescuers transferred all first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate ml/min in the recirculatory mode. its duration was - min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products - times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e tabli~mczr bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, yejin-l)a-l)ao, wuhan fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving ( . ~), the successful rate of saving tra ma ( .~), the suscessfut rate of saving shock ( .~) and the successful rate of cardioputmonary resuscitation ( . ~) daring the three years after t he network operated with these before ( . ~), ( ]. ~), ( . ~) and ( ft. ~), the successful rates after operating were remrk~iy higher ( p<i).o ). conctusions~ the above results showed estabiishmnt and mena emont of region at medical first-aid network had very si~nificont action during mergentty s avin the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med ; s. nouira. chest ) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among consecutive victims of se presenting to the emergency department, were managed using sav (group sav+). these patients were matched to envenomated patients who did not receive sav (group sav- chest injuries are the cause of death in % of trauma fatalities and a major contributing factor in an additional %. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january to december , all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao /fio < ) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: severe trauma patients were considered. of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais< ). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. patients with severe thoracic trauma (ais>= ) were admitted into the study. the mean iss was . ( - ). thirty-six patients required artificial ventilation for at least hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in cases pneumothorax was diagnosed an the initial cxr more patients had a pnx which was identified only on the ct. in cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, % ( / ) presented a pnx that required the insertion of a thoracic drainage. only one third ( / ) of the pneumothorax could be recognised on the initial cxr, while other were decompressed before performing the cxr. as many as % of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a % pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli , .~osid, m.zesevid, m.risovid, d.stepi , d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod: a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given - prcpofol bolus doses (o, ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion ~ ~ropafol. as sdflitianal were given fan-i~l (o, n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro o l.patients in gr~ o received i~ (an initial bolus dose of rg slowly intcavax~ rd mg as infusion over ~ rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj - rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, snd rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min -~a ~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than hour map < mmhg or use of vasoactive agents or blood lactates > mmol/ were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at . , , , hrs. an immunoradiometric assay was used to determine plasma concentrations of il (n< . ng/ml) and tnfc~ (n< pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n< . eu/ml)( endotoxine unit= pg). results : severe multiple trauma patients (age = _+ yrs, iss = -!-_ , saps = +'~, mean-+sd) were studied. they received + packed red cells during the first h. mean duration of collapsus before inclusion was . _+ . hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il level and saps was observed. in the early post-injury period phi, sao , svo , vo were significantly associated with ;il release (p< . at ho, h , h ). later a significant correlation existed between lactates and ii (h , h ). a peak of tnf was detected at and hrs. it was associated with low phi and low arterial ph of the early post-injury period (p< . iat ho, h , h ,h , h ) and with high lactate levels of later period (_>h ). only the late release of endotoxins (i{ ) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il in the early phase of trauma . i and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect brain wave monitor from january to june , . results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo ) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett ae ventilator with integrated metabolic monitor. inclusion criteria were; ) stable cardiorespirator s" status; ) systemic and pulmonary artery catheters already in situ; ) inspired oxygen < %. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, ug/kg, followed by an infusion of ug/kg/h to maintain the train-of-four ratio at . a further four sets of measured and calculated variables were obtained at min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo fick) , and measured oxygen consumption (vo gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than . and normal coronary circulation. for postoperation sedation propofol was administered in . mg/kg i.v. bolus, followed by a . mg/kgth perfusion. all data were registered before administering propofol and after minutes, the patients being hemodynamically stable and a rectal temperature of _+ . -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about years old, and the average period of aortic cross-clamp was . min. the adminstering of propofol caused a decrease in the coronary blood flow (- %), great curonary vein flow (- %), myocardial oxygen consumption (- %), regional myocardial oxygen constanption (- %), myocardial oxygen extraction (- %), regional myocardial ooxygen extraction (- %), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+ %) as well as in the great cardiac vein (+ %). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (- %), diastolic (- %) and mean (- %) arterial pressure, systemic vascular resistance (- %), and cardiac output (- %). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of . % "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than . and normal coronary anatomy, were selected. after the surgical operation, . "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of _+ . -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was -+ . years. during surgical operation the period of aortic cross-clamp was . _+ . rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (- %), coronary vascular resistance (- %), regional coronary vascular resistance (- %), regional myocardial oxygen consumption (- %), regional myocardial oxygen extraction (- %) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+ %) and in the great cardiac vein (+ %). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (- %), diastolic (- %), mean (- % ) arterial pressure, and systemic vascular resistances (- %) decreased, while cardiac output did not. discussion: the administering of . % "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: anaesthesias have been conducted in patients aged years ( females, males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of % insnffladon through mask. operations lasted from . - . h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao , t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics ( . mg/kg) and h -blockers ( . mg/kg) on the operational table was sumplemented by administration of . - . mg/kg of lidocaine, . . mkg/kg of clonidine, . - . mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: . - . mg/kg of droperidoi, .l- . mglkg of diazepam and analgesia: - mkg/kg of phentanyl, . -- . mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was - ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the -th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of - per rain. adequate external breathing: decrease (rr) by - per rain., with sao increase from % to - %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by -t % to the end of the operation with the initial moderate hyperglykhaemia of up to mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state ( - ) and analgesia lasted - b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt . /qtc . sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days and he received haloperidol, - mg/d (cumulative dose rag) for agitation and delirium. icu day : qt . /qtc . sec. icu day : for better control of delirium, trazodone " mg q hs was added. icu day : he developed frequent nonsustained ventdcular ectopy. icu day : qt . /qtc . sec, pha . , paco mm hg, pao mm hg, k . meq/l, mg . meq/l. later in icu day the patient had brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day : qt . /qtc . sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to mg/day. over past decade, cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental ( m), propofol ( p_g/ml), the solvent of propoful (intralipid), fentanyl ( ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm minutes, pmn minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp ( o - m) was measured in a two chamber well system for hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e ). % conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january to february , , data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of icus, of which enrolled at least one case. the total sample included patients. overall, . % of patients analyzed (t / ) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these patients in days in icu. although over drugs were reported to be used, pharmacological principles accounted alone for % of all prescriptions. opioids were actually used in % of prescriptions; propofol in % and benzodiazepine in . %. as regards the way of administration, intravenous administration was applied in % of cases and, followed by intramuscular in . %. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in % of patients and neuromuscular blockade agents (nmba) in %. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in , selected patients (pts) (> y, f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao > mmhg with flu < . ; d) stable ph> . with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (> .(i ml/kg/h); f) mediastinal bfeeding< ml/h for at least h; g) normothermia (core temp> ~ and no shivering). subsequent reintubation was for: ) rr> /min; ) spontancx)us ventilation for rain with paco > mmhg; ) pao < mmhg with fio > . ; ) ph> . ; ) heart rate>] bm; and/or ) non mental alertness; and ) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after h was considered: ) spontaneous breathing without any forrn of mechanical assistance; ) stability in haemodynamics; and ) elimination of fever threat. results: pts ( %) were extubated at h without complication; other pts ( %) at h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by h. only pts ( %) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation ( %= h and %= h), exempting pts with developed post-operative complications ( %=extubation< h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first - hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged - . group a ( patients) received baprenorphine hydrochloride (norphin) at dosages of #. • mg/kg. group b ( patients) received tramadel hydrochloride (t~aasl) st dosages of . z . mg/kg. before the procedure diazepam was administrated i.v. ( . ! . mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by . % on the onset of the procedure while sap and sv decreased by .%% and . %, respectively (p< . ). however, there were no reliable co chsnges. spoz ~educed by @. % (p< . ) and remained lower than the initial one after the procedure was oyez. when administrating tramsl min. after ste~ting the procedure sap and svr increased by ~ . % and . % respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o. ). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, , , ) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of up to microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by %. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate - rag/h) and antipyrine ( rag p.o.) were infused to ci with ( patients -group ) and without ( patientsgroup ) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group chypermetabolizer") as compared to group chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac ) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups ( pts a and pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl mg• kg < in ", pancuronium . mg x kg - and propofol mg • kg - in ". a blind investigator administered via another venous way at same speed ( ") saline in patients of group a and mg x kg - cac in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), ' after anesthesia induction (t ) and " after tracheal intubation (t ). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t . this trend was statistical significant (p < . ) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < . ) dramatically dropping at t , while in group b it showed a negligible decrease at t (p = ns) and value similar to baseline at t . svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was hours more than in control and made hours, though their average labor duration and in control was the same and made hours. % of the examined women experienced independant labor, as for the control group -only %. supplementary methods of anesthesia were required in % in control,that increased medicamentous depression of the fetus, but for the examined group -only in %. the examinees's newborns experienced favourable terminations, in control newborns were in state of light rate asphixia. analgetic effect was complete in control just in i %, and of examinees -in %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for % and in control only for %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of mg. than we were increased the dose to mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had -rhythm with slow and a-activity. an c~-activity index have been lower than %. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to %. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts ( - points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and min after each treatment. clonidine (i- ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of gg/mouse, tfl increased . -fold compared to time zero. l-arginine increased tfl at a high concentration (i gg/mouse). l-name suppressed tfl . -fold compared to time zero at a concentration of gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium ( , mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that , mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of female swine ( - kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n= ): induction with intravenous sodium thiopental in a dose of mg/kg, group b (n= ): propophol in a dose of mg/kg for induction'and mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with - it of cold ( oc) university of wisconsin solution for a period of hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c- , . mg/ml) was infused via portal vein and the liver was incubated at oc for rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (- oc). results: histology was completed in / experiments ( in group a and in group b). mononuclear infiltration (halothane type injury) was found in all specimens ( / ). focal zonal necrosis and acidophilic bodies were found in specimens from group a ( / and / respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b ( / ). smears of hepatocytes -beth formalin fixed and cryopreserved at - oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: patients were treated from jan. till dec. results: in patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic ( ml . ~ bupivacaine) when analgesia was first demanded; second group received ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature < ~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, ; i : - objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in during a period of months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of . % on the short stay unit and . % on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: ) there is no indication of a reduction in patient days with pressure sores (short stay unit: %, long stay unit: %); ) there is a reduction in severity of pressure sores (grade iv decreased from . % to . %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from . to . and the mean frequency of nursing patients on alternate sides decreased from . to . times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads ( - times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' x with the so called bookend method and are connected to a low vacuum suction system ( - kph) results: we studied the frequency of dressings changes on patients in the itu. in total they had days with an open abdomen. during this period there were dressing changes. this means one dressing change every days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo c: siemens), ventilation modes ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (- cm h ) was produced with a tv of ml and a frequency of /min in the ippv mode with trigger level on - cm h and a peep level of + cm h . the servo c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set ( cm h ), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( - cm h ) and the ippv mode the largest ( - cm h ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q , n = , items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by persons ( %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q , n = , items). at the issue, iger was created. results : q : responders ( %). law's misinformation : to %, disagreement for limitation of intensive cares : %, hope to improve collective decisions and creation of iger : % q : responders ( %) : satisfied by the training sessions : %, non satisfied : %, want to continue the project : %, refuse : %. iger was constitute by physicians, nurses, secretary, dietetic (n = ). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: ) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. ) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the st, nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in patients ( hmef and hme) h and h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, ( %) ventilator tubing yielded microorganisms in, at least, one sample as compared to ( %) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups ( % vs %, p:ns, for hmef and hme respectively). among the bacterial species isolated from ventilator tubing in the hmef group, ( %) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group ( / , %). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air ( % • vs % • .%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after h of mechanical ventilation in the hme group as compared to hme group ( . % • vs . % • %; p= . ). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in ~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in ~ end endocrine pancreatic tumors in %. in the intensive oere unit, these patients wore screened by noninwsive monitoring in ~ of cases: and invasive monitoring was applied in % of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of patient submitted to wchf during the last year. we used a total of filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x test to compare the results. results" from nurses of staff refused to flu the quetionary, and were not available. of the remaining, %were v~men and % men. the mean age were . % had an svable contract and ( eventual, the mean professional experience were of years and % worked in the unit since more than years. the % answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: ) to inform about patient mood. ) to inform about happenings from the last visit. ) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients ( male and female, aged + , and , , years respectively, ~+sym) being on therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without . during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa and sa were signif~canthy decreased in the case of oxygen discontinuation (p< , i). paco was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p< , ) . heart rate increased in all phases of the transport when administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s ~ment depression was progressively increasing and became very high at the end of transport in the case of therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao and sa , increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results: patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was , +- , . the duration of coma was from rain. to hour,average , +- ,sh.ln the group (the deads) the depth of come was , +- , .the artificial lung ventilation was used in all patients:in the group , +- , days,in the ~ , +- , days.apallish syndrome developed in cases,in patients diagnozed <<brain death~. the th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( group) and slow ( group) activity. the relative power of delta-and theta-ranges was about - per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the group decreased on , % and , z, in the -on , % and (; , %.general peripheral resistance (gpr) increased in the group on , %,in the -on (; , %.in the group brain blood flow was increased, in the -decreased on , n.in the group there was the normotonic type of reg-curve,in the -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on and n) and vascular ones (an increase of serotonin concentration on and %, hi stamine content on and %) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the group on %,in the - %.the level of middle molecules (mm) increased in the group on , %, in the -on , %,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was , . immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: . protective inhibition of brain and reduction of its energy requirements. . recovery of cell and vascular membrane fuoctions. .recovery of blood circulatiom .oxygenation of nervous tissue and drainage of brain disintegration products.in patientshbo carried out. .active methods of detoxication (homo-and enterosorption). .correction of cerebral metabolism. . dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. ( ). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co ( ), asystolia ( ), eclampsia ( ), acute renal failure ( ), control group ( ). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in monopolar channels. the estimation of eeg patterns by zhirmunskaya method ( ) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) , , , hz. we studied also the figures of absolute and relative power in such diapason : delta ( - hz), theta ( - hz), alpha ( - hz), betal( - hz), beta ( hz and more). results: all eeo changes were divided into following types: -organized ( , groups); iii -asynehronic ( group); iv -disorganized with prevalence of alpha-waves ( , , groups); v -disorganized with prevalence of delta-and theta activity ( , , ; , groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf- ,with effective area- cm,the volume of extracorporal contour- ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. ( ). post-operative cerebral neoplasm ( ), post-operative subdural hematoma ( ). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the minutes p st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) ( ) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: patients were included in this study: men and women. the mean age was years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was h. m. +/- h. m. hypertension was found in patients ( %), ischemic heart disease in patients ( %), hypercholesterolemia in patients ( %), hypertrigliceridemia in patients ( %), smoking in patients ( %), atrial fibrillation in patient ( %), history of epilepsy in patient ( %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in patients ( %), a single hypodense lesion in patients ( %). in patients electroencephalogram was normal ( %), in patients there were widespread nonspecific electrical changes ( %), in patients there were focal nonspecific eeg abnormalities ( %). conclusion: in our study tga was more common in women ( %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. march thirty-seven patients with intracranial aneurysms were operated on within hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n= ) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in ~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_. bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin ~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: ) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, )the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and ) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. ; : - ) . we wondered whether co vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: patients ( m/ f, age - yrs, glasgow - ) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco , , , and mmhg by increasing pico with mechanical ventilation kept constant. normal co vasoreactivity was defined as an increase in cbf of at least i ml/min. g per mmhg paco . results: patients had normal co vasoreactivity bilaterally, patients had altered co vasoreactivity at both sides, and patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the patients left cbf was in mean ! ml/min. g lower than right cbf (figure methods: following institutional approval piglets (body weight :tl . ) were anaesthetized by % fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs- abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at mmhg was achieved by appropriate balloon inflation for rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio =l on respirator (o). samples were obtained at time ( ), ' min at hypoperfusion ( ) arid at reperfijsion at ' ( ), ' ( ) and ' ( ). pao , pjo and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of ~tm hydrogen peroxide (h ). resul'~s: the mean pao was mmt-ig for group i and methods: we retrospectively reviewed all upper gi-endoscopies, performed in the period january -july in patients ( men and women) admitted at the icu's of our hospital. results: it concerned surgical, medical, eardiological and neurological patients with a mean age of . yrs (range: - ). in %, the endoscopy was performed at the icu and in % at the endoscopy department. in % of the cases, the endoscopy was primarily diagnostic, of which % was performed for localization of upper gi blood loss. in % the endoscopy was primarily thempentic, of which % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees ( %), duodenal ulcer ( %), oesophagitis ( %), gastric ulcer ( %), others ( %) and none ( %). as coincidental findings were noted: cesophagitis ( %), gastritis ( %), gastric deer ( %), duodenal ulcer ( %), duodenitis ( %), oesophageal ulcer ( %) and others ( %). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis ( ). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above mmoltl, measurement was repeated every hours for hours or until normaiisation (blood lactate level less than mmol/ ). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was , % and mortafity was , % in a group of patients with blood lactate level less than , mmol/l (n = ). frequency of complications ( , %) was significantly increased in a group of patients with blood lactate levels , - mmol/l (n = ), mortality was , %. mortality ( %) and frequency of complications ( %) were significantly increased in a group of patients with blood lactate levels above mmol/l (n = ). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo ) after major liver surgery, as shvo is considered an indirect parameter for splanchthc and hepatic blood flow. methods: consecutive patients were included in this study after liver resections for primary or secondary liver tumors. patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in out of attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein ( . _+ % rain vs. . + . rain; p < . ). the procedure requires a total of between and minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo showed a span from . % to . % with a mean of . % -+ . %. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without ( . % • . % vs. . % • . %; p < . ). (b) a negative correlation between shvo immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = - . ; p < . ). this correlation could also be seen for the first post-operative hours (r = - . ; p < . ). (c) a negative correlation between shvo and the difference between arterial and hepatic venous lactate levels was found (r = - . ; p < . ). conclusions: the routine measurement of shvo appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo findings cannot be given yet. methods: in cases after major liver resection, in which abnormally low readings of shvo suggested an impaired hepatic blood flow, pgi was applied at a dose rate of ng/kg/min. as shvo can be considered an indirect parameter for hepatic blood flow, the effect of pgi infusion on shvo was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo for all patients .was . % ( - - - ). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi lead to a sustained increase of shvo z to an average of . % ( . - , ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi infusion could be observed. both patients died and days respectively after operation in protracted liver failure. side effects of pgi included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to % as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h -receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/ could have hidden smaller differences between c and r as has been described in volunteers. between april , and april , , patients with severe acute pancreatitis were admitted to participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ ) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and were assigned to the control group. there were deaths in the control group ( %), compared with deaths ( %) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = . ). this difference was mainly caused by a reduction of late mortality (> weeks) due to significant reduction of gram-negative panreatic infection (p = . ). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < . ). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients ( %) and transient gramnegative pancreatic infection was seen in one ( %). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p- . active c provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline ( , - , % naci solution) in electrochemical set e~io- (russin,moscow). methods: . the nacio in concentration ragfl ( - ml/ h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis - /t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. . after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i mg/t in : ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca g/l), the concanlration of medium molecular oligopeptides (mm , ) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: . the intravenous administration of nac excels detoxicative effect of hemosortion by - % provides effictive presentation of protein components and blood cells and improves the transport function of albumin by %. . the return of exfused plasma after its purification ieco was - %. only the remaning - % of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of mos pts, m and f, mean age + . yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin days (mean . ). the mean peak level of ast, alt and ldh was iu/l (range to ), iu/l (range to ) and iu/l (range to ) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of . mg/dl (range . to . ), while altered coagulation paran-,ete's (pt> . times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt ( . %). renal impairment (cr> . mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure ( %) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range - pg/kg/min) and dopamine (range - pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired ( . %), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure ( %). it is concluded that: ) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. ) clinically significant coagulopathy is uncommon complication of ih. ) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, , and hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: pts, ~( , %), ( , %) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age: , • years. hean stay in icu: , • days. pts were operated, of them twice. hean value of ranson's scale: , • ( - ). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf - pts. b)early ards with typical xrf - pts( died), c)early arf with xrf(atelectasis,infiltrates)- pts( died). d)late ards with typical xrf- pts( died), e)pleural effusions in various combinations with the above patterns - pts. overall mortality rate: / = , %. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. )even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. )deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. )delay of surgical therapy is related to worse prognosis(p<o, ) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o, ). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of months all surgical patients on icu were registrated prospectively (n = ). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. , m!ll. of single patient informatioas. results: of patients didn't suffer from liver disease or have any operation on liver or bile tract. of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by patients. at a ,,cut-off" of rag% (total-bilirubine) the sensitivity was , , specivity , , positive predicive level , , negative predictive level , for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface . cm z, flow= itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight gr (min , max gr) were obtained and after a mean cold ischemia time of min were perfused fo} a mean of . h (min . , max h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of ( - ) mmhg at ~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t ) sampling: po , " + + " pco , ph, hco -, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from . at t o to . at t while mean output po fell from at t o to at t . mean output ast values increased from at t o to > at t while mean output alp values increased from . at t o to at t . mean output k + values increased from . at t o to > at t . histology revealed lesions of ischemic necrosis, more prominent after t . conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between and the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years organs were donated from potential donors i single, multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from ( ) ,to ( ) with a concomitant drastic reduction of donations from % in to % in ; amounting to a net unchanged number of donations over the last years ( = ; = ) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from % in to % in despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho ) provides online information about hepatic-splanchnic oxygen supply-demand ratio [ ]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [ ] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi , flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho ) in human liver transplantation. methods: with institutional approval and informed consent, consecutive patients, mean age - -_ years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of . gg/kg/min and pgi at ng/kg/min were initiated for a rain period. each sequence was followed by baseline therapy for rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p< , . results: ng at . gg/kg/min induced a decrease of mean arterial pressure (map) ( _ [baseline] vs. + mmhg) and pulmonary artery wedge pressure (pcwp) ( j: [baseline] vs. : mmhg). cardiac index (ci) ( - vs. + l/rain/m ), oxygen delivery index (do i) ( -+ vs. + mgnfin) and svho ( _~ vs. -l-_ %) were decreased (p< . ). pgi at ng/kg/min induced a reduction in map ( • nm~. _g) and pcwp ( + mmhg). ci ( _+ l/rain/m ), do i ( : ml/min) and svhoz ( + %) were increased (!o< . ). vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. pgi increased systemic oxygen delivery in parallel with svho , suggesting a corresponding improvement of hepatic-splanchnic okygenation. thus, if vasedilator therapy is indicated in th orient receiving liver grafting, pgi appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, until now donors were involved into this prospective study. every other donor was treated with mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group -steroid pretreatment goup (y~= ), and group -control group (n= ). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the - th, th and th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group had slightly lower ereatinin level ( -+ g.,non vs. -+ gmol/ ) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group . the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august and september , patients with transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n= ) included microbiologic evidence and more than secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n= ) referred as control groups. results: -year patient survival was . % ( / ): patients died due to serious infections, while died for other reasons. mean bilimbin, stnf-rii-, ifn- -, il- -, il- -, il- -, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: _+ pg/ml vs • pg/ml; ifn- : _+ pg/ml vs . -+ . pg/ml; il- : -+ pg/ml vs -+ pg/ml; il- : -+ pg/ml vs _+ pg/ml; il- : _+ pg/ml vs • pg/ml; laminin: -+ ng/ml vs -+ ng/ml; neopterin: _+ nmol/ vs _+ nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: -+ mg/l vs • mg/ . conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: patients with transplants were monitored for various cytokines on a daily basis between august and september . rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, patients ( . %) developed rejection; patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further patients required additional treatment with fk or okt (steroid-resistant rejection). patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn- , il-ib, il- r, il- , il- , il- , hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: . -+ . mg/dl vs . -+ . rag/all; tnf-rii: -+ pg/ml vs _+ pg/ml; il- : +- pg/ml vs -+ pg/ml; neopterin _+ nmol/ vs -+ nmol/ ; hyaluronic acid: _+ ~tg/l vs _+ ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: _+ mg/l vs _+ mg/ . ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il- , hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals ( beds) between december and may . exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms and days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that % is critical value for cpv loss and for cev it is %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june and april patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to days (mean . days) we reported of these patients as possible organ recipients to eurotransplant. all of these patients were transplanted within hours, ( %) of them even within hours. the principal aetiologies were hepatitis b ( ), hepatitis c ( ), nanb hepatitis ( ), mushroom poisoning (amanita phalloides ). after transplantation patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was %, patients died within months after transplantation due to various reasons. patients were not referred for liver transplantation. of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning ( ), paracetamol intoxication ( ) and fulminant hepatitis a ( ). patients suffering from fulminant hepatitis ( ) or intoxication ( ) were excluded from emergency liver transplantation for various contraindications. of these patients ( %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within hours. but the good survival rate in the transplantation group ( %) the % recovery rate in the group, where there was no transplant-indication in our opinion and the fatal outcome ( % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t ), increased reverse t (rt ) with normal levels of tetraiodothyronine ( " ) and tsh, this lacking in " " levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: .to study thyroidal pattern in brain death patients potential organ donors. .to avoid organ impairment by administration of t . .to study the hemodynamic and hormonal changes after the administration of t in these patients. material and methods:population: brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group ) and unstable (group ). group received a bolus of . p.gr/kg. and a perfusion at a dose of - . p.gr]h of t . hormonal assays: total t (tt ), total " (tt ), tsh. fxee t (ft ), free " (ft ) and rt were determine at the moment of clinical brain death ( hrs) and in group two these assays were repeted at hours , and . results: patients ( male) with a mean age of years (range to yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were patients in group ( , %) and patients in group ( , %). hormonal pattern: at the moment of brain death tt was normal in cases ( , %) and decreased in i ( , %); tt was normal in patients ( , %) and decreased in ( , %); ft was normal in cases (i , %), decreased in ( , %); fl' was normal in patients ( , %) , decreased in ( , %) .rt was normal in cases ( , %) and increased in cases ( , %). there were no statistically significant differences in hormonal pattern between the two groups. only t levels at hours , and were significant in group . in the cases with ft decreased, the tt was normal in ( %) and decreased in ( %), tt was decreased in ( , %) and normal in ( , %), tsh was decreased in i ( , %), normal in ( , %) and increased in i( , %) and ft decreased in ( , %) and normal in ( , %) and rt was normal in ( , %) and increased in ( , %). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t . conclusions: . the hormonal pattern most often find in brain death patients was: normal tt , decreased tt , normal tsh, decreased ft , normal fr and normal rt . . there were discrepancies in the values of ft and tt . there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace ) could be detected by mra. methods: following institutional approval and informed consent, mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace : , , mmhg; arterial blood gas probes; avl). mra was performed with a . tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight- d-angiography (tr: ms; te: ms; fl: deg; slab: mm) for vessel diameter detection and a flash- d-gradient-echo-sequence (tr: ms; te: ms; fl: dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace correlated with changing signal intensities, whereby under hyperventilation a decrease of , % (p . ) and under hypoventilation an increase of . % (p . ) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of - . % (p _< . ) and in peripheral diameter of - . % (p _< , ). a change from normo-to hypoventilation produced an increase in proximal diameter of + . % (p < . ) and of + . % (p -< . ) in peripheral diameter. conclusions: pace related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co -reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: ) bronchoscopic potency of airways and air bronchogram sign } resolution time of atelectasis and broncoscopic potency of airways. ) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were , men women , seventeen patients had atelectasis of whole lung, of upper lobe, and of lower lobe. ten patients had atelectasis in right and in left lung. eight from patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [ from patients with air bronchogram sign ( %) and from without air bronchogram sign ( %), had bronchoscopic potency of airways, p> . ], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency , days and in bronchoscopically closed bronchi , days, p> , ). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign , days, and without air bronchogram sign , days. p> ). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer ( . mhz) and a linear transducer ( . mhz) using acuson xp/ c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. ) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. ) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. ) . from march to april we did examinations by cs in icu patients ( male, female; age from - ). patients examinations pulmonary embolism pneumonia pleural effusion us-guided thoracic punctions were performed in patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an % concordance. results of the tee classification were : class : n = ( %) ; class : n = ( %) ; class : n = ( %) ; class : n = ( %) class : n = ( %). therapeutic implications of tee in class patients were : cardiac surgery in patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in patients with endocarditis and prescription of anticoagulant therapy in patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in patients admitted to the icu, an ultrasound-guided method (ultrasound group : patients) with an external landmark guided technique (control group : patients). all jugular vein accesses were performed by young interns with an experience of < procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in patients ( p.cent) in the control group (p < . ). average access time was longer in the control group ( • sec. vs • see. ; p = . ) and puncture of the carotid artery occurred in patients in each group (p = . ). patients ( p.cent) in the ultrasound group and patients ( p.cent) ia the control group (p < . ) were cannulated in rain. or less. the cannula was therefore unabie to be inserted within minutes in patients in the control group, with failure of eannulation in of these patients ( p.cent). failure was due to thrombosis (n = ), small calibre of the internal jugular vein (< ram) (n = ), abnormal vascular relations (n = ) or cervical irridation (n = ). among the primary failures of cannulation, an internal jugular vein catheter was able to be inserted in cases by an experienced physician on the side initially selected and with ultrasound guidance in cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining cases. jugular cannulation was obtained at the first attempt in p.cent in the control group and p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of - %. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from - %. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases ( male, i female, - years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in pts, a massive pe in pts. one patient (pt) had a cardiac arrest. in all cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in cases. all pts were treated with mg rt-pa, pts in a front-loaded regimen over minutes, pt over minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after to hours. all pts survived and were put on coumadine, pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein - cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a . mhz transducer (ultrasound depth - cm) fitted with a needle guide and a cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in patients catheters were introduced.in cases more than one ( - ) attempt was made and in patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > - cm), during the procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of days (range - ) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first patients cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following patients duplex scanning performed after removal of the catheter yielded another cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation - cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary -bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from consecutive patients admitted to the icu from / / to / / were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over years evaluate the necessary efforts and expenses to implement a cis in the routine of a -bed stcu. methods: in june a commercially available, unix-based cis was installed on a -bed surgical icu. the goal was a paperless documentation at the bedside. after more than years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, - trained health care professionals for configuration, system administrator. on a single icu ( - beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo ). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab ; : ). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo ), at ips (sipso ) and at mid jugular vein ( th cervical vertebra) (smj ) bilaterally. results: we found significant differences between right sjbo and both right sipso (p= . ) and right smjo ( p= , ) and between left sjbo and both left sipso (p= . ) and left smjo (p= . ) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : channel electroencephalograph, -channel roencephalograph, ad-converter ( analog inputs, bit resolution, k hz), ibm dx , software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal . ), expert programs for estimation objective and humoral patient status (written in clipper . ) and statistics. there were used following programme-language instruments : borland c++ . , nantucket clipper . , ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: . direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. . indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner % of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to centers and we received responses ( . %). there was at least one uncological (i.e. with > % of cancer patients) icu in (% % an -year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at /minute and a temperature of ~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was /mm ~ with neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv ( %) (n - %) and cardiac index ( , ) (n , - ). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n= , - pmo/l) went from , to , after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: patients were included in a prospective study from june to november . patients presented arf defined by creatinin serum leve(s greater than pmol/l and previous normal levels. patients were divided in three groups. group i (control) : patients with arf who did not receive substitutive techniques. group ih patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii : patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was . and mean length of tcu stay was . days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics ( ) cardiovascular drugs ( ), gastrointestinal system drugs ( ), enteral and parenteral nutrition ( ), respiratory system drugs ( ), sedative, analgesics and paralysing agents ( ), parenteral solutions with electrolytes, vitamins and trace elements ( ), anti-inflammatory agents ( ), protein substitutes and immunomodulation agents ( ), anticoagulative agents ( ). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was . drs ($ ). total tc/patient was . drs ($ . . ). ii) partitioning total tc per group of drugs reveals : ( ) %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %. t ) concerning antibiotics which consist the major cost component, group a and group b contributed by . % and . % to the total icu tc respectively. group b were administered to . % of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. -to determinate the variations of the rso during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso ) and rso were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam ( mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental ( mg/kg),fentanyl ( mcgr/kg) and atracnrium ( , mg/kg) have been used for induction of anesthesia. co te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane ( , - , %) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each minutes: blood pressure, cardiac frequency, pso , co te and rso . the rso cerebral variate in relation with: the anesthetic induction, blood ~ressure, co te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso cerebral was in relation with the :ross-clampping carotid ( minimal value: ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than anesthesia in chelyabinsk emergency hospital every year. to % patients of it emergency anesthesia is applied. more than patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than % of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of parameters simultaneously. results: pathients extremly needed peryoperative monitoring of hemodinamics. from these patients more % had stroke volume (sv) less than ml, n -co less than . /mim/m , % -ejection fraction (ef) less than n and % -puls bioimpedans microvessels (pbm) less than morn. patient had intensive care in special department. out of died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do ), oxygen consumption (vo ), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do ]. methods: patients ( males and females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo , do , ci, (a-v)do were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a -hour study period. a total number of measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p< . value was considered as significant. results: mean values were phi= . + . , do = + , vo = + , c. = . + . , (a-v)do = . + . . no correlation was found between phi and do , phi and vo , phi and c.i, phi and (a-v)do . on the contrary in patients phi remained below . for more than hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high ( %). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, berlingen) in combination with a standard gastroscope. measurements were performed on ventilated, traumatized patients (ages - years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of cm was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~ ) and standard p~ (ps st) consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in patients (mean age -* y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at ~ c using the abl radiometer for po , pco: and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the p~ at ph= . , pco:= mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps "in vivo" (i.e. the pso at the patient's value of ph, pco and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was . -* . houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo and oxygen extraction ratio (oer), r= . ,p= . . the improvement of ci ( . -* . to . + . l/min/m , p< . ) induced a significant increase in svo~ ( . -* . to . • . %, p<. ). a significant decrease in p ( . • . to . • . mmhg, p< . ) without any significant change in p~ st ( . • . to . • . mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: ( )personal data ( )patient's pathology ( ) laboratory and~ monitor lug data ( )drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. ( )reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month ( ) access to de questionnaired apache ii to load up new data. ( ) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: ( )detailed drugs interactions, including toxic elements ( )diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy ( ) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and ( )perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = days, los = - days, los = - da~, los = - days, los > day~. area under the carve (auc) was ud~ninted for each ro~. s~ing: imlimlcus. patents: of ~ pts comec~ively admired ~ a period of three yeet~ ( ) ( ) ( ) , a total of was i~leded in this study. pts without saps, p~ yolmger them yearn, p~ with los shorter ~ hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g : . , p > . ; auc = . i- . ) and in validation ~t (g.o.g g : . , p > . ; auc = . ..+ . ). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being . i- . in pts with los = days el . ~. ia tm with los > da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: .-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with items grouped in sub-scales: physiological functions ( items); functional capacity ( items) and subjective aspects ( items). qol is classified in levels: normality ( points); slight deterioration ( - points);moderate deterioration ( - points); significant deterioration (>i points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first hours as evaluated by tiss, and related factors. results: the sample was , patients, sge . ~ . years. severity by apache ii system was . • points. the tiss score was . • points, distributed as follows: i (<i points): %; ii (i - points): %; iii ( - points): %; iv (> points): %.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = . , p < . ), and a very weak but negative correlation between tiss and age (r = - . , p < . ), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v . for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of items classified in categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances ( . ), b) all generic names of medications available in our country for each substance, c) incompatibilities ( . cases) and d) interactions with other substances ( . cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on consexa~e ~ilically ill paliffas, subdivided in series following the diastases at the admission: medical pafiea~ ( s and ns), surgical patients ( s and ns), a~d poliwauntas ( s and ns). follow up was done at d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at fin~es (t): at fiae ~draission (t ), at x~ars from t (t ), at (f ), (y ), (t ), % (t ) and horus from t cf ). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < . w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t has a sensitivi .ly of ~ ,and a ~ecificity of . %: sv > . nffmin/n~, map> mmhg, pmap< nmalqg cvp<i nunhg will nmah& lvswi> g m/m , sxo > ~ do > mlhnin/m , o er< %. for lx~dical l~tienls at t the following criteric~a has a ser~tivi.ty of % and a ~zificity of . ~ cvp< . mn~g, sao > %, s,g) > ~ vo i< ml/nfin/m , o er< %, shunt< % survlvops' data of the series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n [ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh ~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure ) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied patients ventilated with a siemens c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of inflations ( , ,... ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves (( , c , c the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of paired act/p't-/" values was analysed in patients ( m., f., + y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of . u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within minutes. results ac-i -~ pi-f~ cact/~pi r = , ) cvc samples + + . v.femoralis samples " + + p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january through august , we studied filter surviva[ and hemorrhagic complications during filter periods in critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: filters ( %) were disconnected because of coagulation. mean survival of multiflow an filters was twofold shorter compared to survival of fh gambm filters. a total of hemorrhagic complications occurred of which three patients died at aptt values of respectively , and seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased % (relative risk . , %c . - . ) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased % (relative risk . , %ci . - . ) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between and seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure mmhg on admission and hours later were respectively . + . mmhg, . +_ . mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and hours later were respectively, pas; . _+ . , + . mmhg, ad; . _+ . , . _+ . ng/ml, nor; . _+ . , . + . ng/ml, pra; . _+ . , . _+ . ng/ml/hr. the ev values correlated significantly with ad (r= . , p= . , n= ), nor (r= . , p= . , n= ), pra (r= . , p= . , n= ). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p= . ) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf , , m , fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s- . the reaction is allowed to proceed at,. room temperature for minutes before discontinuation. the absorbance at nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied patients, who were admitted to our neurosurgical intensive care unit between january and march with head injury and no other associated pathology (age - yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) - hours after trauma (t ) and t hours later (t the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd( ).tbe n'mimum effect occurs after hours from the fast administration and is still present after hours. ~- patients, mean age --- yrs., weight --- kg were studied. ( . %) were male and ( . %) female. onderwent hip prosthesis ( previously plate and screw removal) hip revim'un ( stem, cop and stem + cop), tutal knee prosthesis, in the st anaesthesidogy depl from - to - - . as for antithromboembolic ptephylam, apart from hemodihitiun pts were with treated indobufen ndo), with heparin ealdum caeca) and with low mo!lecular weight hepam (lwr, ). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p= . l) in the padeats treated with indobufen ( . ) compared .'ith heca ( . %). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p= . l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro . ) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec, .a ( . -+ . days) and lmwh ( . +- a days) compared with indo(ll. _+ a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from - a + mmhg, which activates a peristaltic pump connected m drainage robes. the bt recovery display shows hourly bleeding in the first hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at - mmhg and then modified according to bleeding/minute, g bt recovery also has an alarm that sounds automatically if.' blood loss is more than ml/hour; air is in the circuit; the batteries are running low. materials and methods: pts were studied ( m and ~), aged . -+ .lyears, basal hemoglobin . -+ (range . - . )g/all, treated from st january, to mst december, in the st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision ( pts), cup revision (~ipts), stem revision ( pts), total knee revision ( pts). the average dumtion of the operations was -+ min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on pts. and integrated (epidural analgesia + light general) on the remaining t . anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in ( . %)pts., calalc heparin in ( . %)pts., low molecular weight heparin in ( . %)pts.; pt did not give a predepoalt of blood, gave unit, pts units, pts units, pts units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was -+ ml, average postop salvage was -+ mi the average intra+postop +- ml. average postop loss was -+ ml. the global incidence of postop complications was: h~natomata . %, dvt . %, pulmonary thromboembolism , , myocardiac ischemia . %, acute myocardic infarction . %, respiratory deflciecy . %, arrhythmia %, cystitis . % there were nn complications in . % of pts. postop bleeding over ml in under minutes (with bleeding alarm activation) occurred in pts ( . %). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p= . ) and with a significant decrease (p~ . ) in the pruthrombic activity detected about hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in % of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references )borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. patients with sah were studied. in of them sah was due to closed eraniocerebral injury and in the rema ining resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in patients with closed craniocerebral injury and in with vas. cular malformation despite the lack of clinical symptoms the tests in posttraumatic patients and in patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period ( - ) were reviewed. diagnosis of dic was based on the association of fibrinogen < g/ -platelets < / -fpd > ~tg/ml in the hours of the admission. results : patients -mean age + y -saps +_ -gestanional age _+ weeks -the two first conditions associated with d.i.c. were placental abruption ( %) and preeclampsia or eclampsia ( , %). bleeding episode was present in pts ( %) and surgical treatment has always been necessary. pts ( %) were given packed red ceils ( + u) and fresh frozen plasma ( + u). patients were given platelets packs. heparin was never administered. pts required mechanical ventilation and two patients hemodialysis. all the patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t h ~ % -f at t h , + , g/i). but platelets count remained low (plat. at t h + / ) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, patients with a dic score ( , japan) more than points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i- mg/kg/h for days or more) treated group and no foy group, each of patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, , and days after the admission. at-iii at units for days was administered if the at-iii at admission was less than %. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ %; group b, foy (+) and the at-iii < %" group c, foy (-) and the at-iii %; group d, foy (~) anffthe at-iii < %, each of patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day to were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were %, %, % and % in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: adults, mean age years and children, mean age months ( me.- yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and / of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, , . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction , to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group (control} dogs and group (no inhaled) dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of and n~ (f[q , ) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation ( ppm) in group was started rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan ) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and , , and rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < , . : no produced an increase in arterial oxigenation (pao /fio~ ratio) and reduced pap before pe induction in group . after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to % ) spills automatically into the circle, balancing o uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. - ( %) underwent conventional surgical therapeutics. " ( %) with resection of tracheal stenosis with end-to-end anastomosis(rts). i ( %) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. - ( , %) have received endoscopic laser ablation with or without calibration tubes. of them ( , %) are receiving continued endotracheal treatment until now. ( , %) have sp wihout continued treatment. -i ( , %) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas ( % oxxygen, % carbon dioxide, % nitrogen); gaseous no ( ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, , , , , , , , and minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at + , % (percentage of baseline value, mean -+ sd) until min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after minutes ( , + , % vs _+ , %(p< . ); min. , -+ , %vs , _+ , %(p< . ); min. , _+ , % ( p < . ). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at , +_ , % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to , _+ , %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp- , cd p) and glycoproteiu (gp , lysosomal protein, cd ) are expressed only after platelet activation and degranulation. activation was quantified in thrombin ( . u/ml) and adp ( . ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n= ), who had no drugs for at least days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i ( . mm) ( -morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd p (fitcconjugated), and cd (fitc). only cd a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was . + . %, and increased to . _+ . % after thrembin-activation, and to . + . % in adp-stimulated samples. addition of sin- attenuated p-selectin expression to . - - % in thrombin (p<. , two-tailed paired t-test), and . + . % (p<. ) in adpactivated platelets. basal gp expression was . _+ . % and increased to . + . % in thrombin, and to . _+ . % in adp-stimulated samples. with sin-l, gp expression decreased to _+ . % (p<. ) in thrombin, and . : . (p . ) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died days after exposure. the metal he had been working with was a % cadmium alleation. blood cadmilam concentration days after exposure was . mcg cd/g cr, and urine cadmium concentration was . mcg/l. on postmortem examination, tissue cadmium concentrations were: blood ng/ml, liver ng/g, kidney ng/g and lung ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : patients were studied. on admission, plc ranged from . to mmol/l, and bcc from . to gmol/l. mean systolic blood pressure was • mm hg, mean arterial ph . • . , mean anion gap was . + . mmol/l and mean pao . • . kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = . ), anion gap (p = . ) and bcc (p = . ) but not with heart rate, pao , paco and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> retool/ was a sensitive and specific indicator of a blood cyanide concentration > ~tmol/ . sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a -year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were mmol/l and gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r= . , p< . ). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r = . ) with a blood half-life of . h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r = . ) with a blood half-life of . h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a g dose of hoco ( g in case of cardiovascular collapse) was administered intravenously over min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were females and males. the mean blood cn concentration was • pmol/ . the mean blood carbon monoxide was . • . mmol/ . nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < ~mol/ ), there was no significant change in arterial blood pressure. in the cn-intoxicated patients (blood cn > gmol/ ) a significant increase in blood pressure was observed both immediately (p < . ) and hour later (p < . ) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ ] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a -beduniversity hospital from through . the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between + and * + + • the best correlation with severity grade was found with qrs-and qtc-duration (p= . ), the tca-dose (p= . ) and hf (p= . ); tqrs did not correlate. patients died ( . %). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative , -methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken tablets of "eve" (mdea, approx. g) and g of "smack" (heroin) by oral route approximately h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was ng/ml (lethal range - ng/ml). trace amounts of cocaine and substantial amounts of heroin ( ngtml; mean value in heroin-related deaths: ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients ( females, male) with documented aip were seen over a -year period in the university hospital. patient was in clinical remission and were with the acute attack of aip, among them with respiratory paralysis were required artificial lung ventilation and -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range - mkg per/ kgr creatinine) fill from mkg on admission . mkg, then on - day raise to mkg and then during treatment with normosong and prasmapheresis lowest level was . mgk. fatalities occured in two females during attacks with proforma cerebral involvement and patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in anaesthetized and ventilated (fio . ) minipigs and dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above mmhg. results: flow ( and ppa matched pvz data (mean-+sem) are shown in the table. [zo = hz impedance (z; {dyn.sec_em- }); zl = first harmonic z; zc = characteristic z; z phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < . between dog and minipig, and before v~. after embolisation respectively]. before case report: a -yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt= ml, rr= /mm, fio =l, the patient had ph= . , pao = mmhg and paco = . systemic bp was / mmhg and hr= b/min with low dose epinephrine ( . g/kg/min) a thrombolytic infusion (rtpa: mg/ h) through a peripheral vein was started tee imaging and clinical status hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from / mmhg to / mmhg, etco increased from to mmhg and sao improved from % to % three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also ~, properties. case report.an obese },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine . /die was used for prophylaxis of thrombotic disease, on day th at . a.m. she started to be hypoxic (sa %), tach ,tardic l l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to r mm/hg, the central venous pressure (cvp) raised [rom lb to mm/hg and the end tidal co was mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase x i(i u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp / mm/hg, cvs mm/hg, spo %) despite a cominnus infusion of dobutamine fag/kg/min and adrenaline . ~tg/kg/min. at this stage a bolus of aoradrenaline ,g was given followed by a cnntinous infusion of . !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was / mmhg, the cvp mm/hg, the sao % and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i pte patients (pts), males (average age , yrs) and .q females (average age , yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in , % of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain ( %), dyspnea ( %), hemoptysis ( %), cough ( %) with association of two or more symptoms in %. chest xrays findings were abnormal in % with diaphragmal elevation ( , ~ lung opaeilies ( , %), atelectasis ( , %), plemal effusion ( , %), main pulmonary brancah asimetry ( , ~ oligemia ( %), heart shadow changes ( , %) and pulmonary arteries "cut off' ( , %). the association of two or more abnormalities was found in , % while normal chest x-rot was found in ~ of the cases. hypoxemia with pao < , kpa was found in , % followed with hypocapnia and respiratory alealosis in , % in , % of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i , %, ecg changes-st q t type in "~ , %. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet : , ) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared patients arrived in our intensive care unit (i.c.u.) in. : (group a) with arrived in : (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of , +/- , (hb) and +/- (hct) in group a while in group b mean value are , -/- , (hb) and +/- (hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took ml of patients' blood and separated it within min with rotation frequensy of /rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made - procedures during - days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from + . g/l to _+ . g/ and from + . % to _+ . % respectively). the time of blood coagulation by lee-white has increased by - . times (up to - rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from . + . ml/min to . _+ . ml/min (p < . ); b) the regional brain blood flow from . _+ . ml/min to . + . ml/min (p < . ); c) the brain blood flow in internal carotid arteries from . _+ . ml/min to . + . ml/min (p < . ). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of consecutive patients, on verapamil, on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil ( mg over minutes) was followed by a rapid loading infusion ( . mg/min) for minutes and finally a maintenance infusion ( . rag/rain) for hours. results: a mean plasma level of verapamil of ng/ml was obtained only after more than hours. atrial fibrillation occurred in five out of patients who tolerated the verapamil infusion, and in out of patients on placebo (p = . ). verapamil infusion was not tolerated in patients because of hypotension or a heart rate of less than /min, within hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion ( + ) compared to placebo ( + ). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to . + . g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to . _+ . g/l, monofibrin soluble complex concentration to . -+ . g/l, blood plasmin level was enhanced to . + . mmol/ , plasminogen proactivator level was also enhanced to . + . ram, plateletes aggregation has decreased to %. after plasmopheresis aggregation was decreased in . times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in . times, and plasminogtnt activator level in . times. at the same time we have observed increase in total antifibrinalitic activity of blood in . times. activity of activators plasmine and plasminogene proactivators has decreased in . times and in the same time activity of activation inhibitors and antiplasmines has increased in times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc ) monitoring based on thermodilution technique in critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique ( ml of ice-cold saline solution) was used to compare cc with intermittent bolus cardiac output (ic ) measurements. the following subgroups were prospectively studied: i. heart rate (hr) > beats/min, . cardiac output > i/min . cardiac output < . i/min, . rectal temperature > . ~ and . pa catheter was inserted for more than days. results: a total of pairs of ic and cc measurements were obtained from the patients. bias (ico measurement minus cc measurement) of all measurements were . • i/min and the % confidence limits (mean difference• were - . / . i/min. also in the subgroups, cc measurement agreed closely with ico measurement (c > i/min: bias= . • i/min; co < . i/min: bias=- . • i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc measurement with ic measurement neither (> ~ bias= . • i/min). conclusions: monitoring of cc using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january , this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since , patients aged to years (mean years) underwent cas without ecc. the mortality in programmed surgery was %. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: units per patient, b) need of mechanical ~entilation: i , hours, c) postoperative bleeding: cc, d) days at icui , . we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments per patient (p< , ), b) need of mechanical ventilation: hours (p< , ), c) postoperative bleeding: cc (p< , ), d) days at icu: (p< , ), e) programmed surgery mortality: % (p< , ). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography ( and l days after beginning of the treatment), stress-echocardiography and stress ecg ( - -th day after the onset of mi). enalapril was included into the treatment of pts with mi (study group), with normal or increased blood pressure, from the -st day of the disease. the data were compared with pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in pts of the study group and of control group, the arrhythmia episodes during stress test -in and pts and episodes of silent nocturnal isehemia -in and pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh % stress test. in pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by %; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef< %). besides cesf, the ejection fraction was calculated before and after administration of mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) . • . ef (simpson) . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! ! [ gedv, itbv and pdr showed a significant decrease dufing the first - h after the operation, cfi and rvef si~canfly improved after k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at h. comparison of cold/explorer m~ements sb wed good correlations. discussion: concerning m ~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least . ~ (max rain apartl the patients were separated in two groups according to their cardiac function: patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below % and patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction ( er), the q er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied mechanically ventilated icu patients ( • years) in stable hemodynamic status (no change in vasoactive treatment for at least hours), separated in groups: group = deep sedation, cardiac dysfunction required dobutamine (n= )r group = deep sedation (barbiturates), unaltered cardiac function (h=lo), group = moderate sedation, altered cardiac function (h= ) and group = moderate sedation, unaltered cardiac function (n= ). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline ( x) and twice (q. min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of hypertensive surgical patients. patients were allotted to groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)< . , patients; eukinetic (ef> . ),i patients and hyperkinetic (ef> . ),i patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p< . ) while systolic arterial pressure (sap) (p< . ) and peripheral resistance (pr) (p< . ) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p< . t) stroke volume (sv) (p< . l) and co (p< . ) were increased while pr(p< . t), sap(p< . ) and diastolic arterial pressure(p< . ) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p< . ) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p< . i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : palients, post cardiac surgery, uremic ( men, women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a fr polyurethane catheter with end and multiple side holes over ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes( - ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht= % ,protein , gr/dl) in all cases. in first hours the mean volume of fluid removed was ml ( to ml). in that period echo showed no residual fluid. the catheter remained within the pericardium to days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by -d echo guided perieardiocentesis. a -fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this -d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after rain, min, and finally with one hour rate, within first hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate hbenzoyl-phe-ala-pro (bpap; p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi( -m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as . xf v. results: similar values of %m ( . + . vs . • and v ( . • vs . • were observed in both and one lung respectively. a~k~ decreased from • ml/min (both ltmgs) to :~ (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the % reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients ( females), with a mean age of + (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin hours postoperatively. square wave stimuli of . msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding . msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, . • msec for the left nerve and . • mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients ( . • msec) , whereas left pnct was normal in patients ( . • msec) and abnormal in patients (incidence . %). in patients the left phrenic nerve was inexcitable and in patient left pnct was prolonged ( . msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction ( • vs • p= . ). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper to cm of lima were preserved (p= . ). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo shifts. methods: we studied males ( - yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature - c we set pump flow at . i.m~ .min - . when map was higher than mmhg we calculated vo by using fick equation. then we infused sodium nitropruaside (sn) to control map at - mmhg for min and we calculated vq . without changing the sn infusion rate we set q at . i.m' .min " . ten min later we measured vo . we took vo changes into consideration if greater than %. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo response to sn infusion during low and high q we classified pts in four groups (table). i. vo increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo increases with sn but the addition of high q results in systemic shunt. iii. vo increase during high q proves that vasodilatation can turn flow insufficient. iv. vo does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > . l/min/m , _< paop < mm hg,and svri __< mmhg/ll/min/m decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: aaar patients and pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > . sec or cpk-mb > %. results are presented as means _ -. sd. opt was achieved in of ( %) and in of ( %) in the pvs and aaar group, respectively. events did nat differ between groups of ( , %) and of ( , %) in the pvs and aaar group, respectively (p>o. ). mort was of ( %) and of ( . %) in the pvs and aaar group, respectively (p > . ), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, of ( %) patients who developed events in the pvs group had a ci < . in contrast to of ( %)in the aaar group. the positive and negative predictive value were % and % in the pvs and % and % in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; . pvs patients who have net achieved opt are at extraordinary risk of perioperative events; . preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< . ; ratio >_ . may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade ( ). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in consecutive patients (pts) after uncomplicated cardiac surgery ( coronary artery bypass grafts; valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : . _+ . /minlm~; laotate: + rag/i; cvp range : - rnmhg; pewp range : - mmhg. mean cvp/pcwp ratio before extubation is . ( % confidence imerval : . - . ) and after extubation, . ( % confidence interval : . -. . ), (ns, paired t-test). in % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = - . ) and after extubation (r = - . ) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = . ) and after extubation (r = - . ), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< . ) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as within a large range of cvp ( - mmhg) and pcwp ( - mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : ( ) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from to minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period june to december , patients with chest paros were treated within a eriod varying from minutes to hours from onset of symtoms. ages ranged from to (average , ), oelng males and females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l m.. potentia ami patients were then admitted to the coronary unit, [)atients, under age with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was minutes ( . ), -he total median delay was minutes ~ -i h. min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to -i minutes and treatment delays to less than i minutes results. following rmplementatlon of this protocol in january , fts were glven, with an over all average delay of minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a % reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly c agents to patients with suspect of ami from the begin!ng of september until the end of february . in this time there are two different periods, during the first months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from to ) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to - ~ c. renal dysfunction was defined as a % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x when appropriate. data are expressed as mean +__sd. p value less than . was considered statistically significant. results: thirtytwo patients ( , %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p< , ). mortality rate was statistically different in patients with or without renal dysfunction(~, , % and %, respectively, p= , ). incidence of acute renal failure following renal dysfunction was , % ( patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature ( ). however renal dysfunction appeared significantly associated with a poor prognosis. reference: -settergren g, ohqvist g current opinion in anaesthesiology , : - r ; , tzelepis, g. , , late complications were observed in % of cannulations: local infection in (i, %), catheter displacement by the patient in cases ( , %), catheter displacement during nursing care in ( , %) and malfunction in cases ( , %). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, selected patients ( _+ years) undergoing peripheral vascular surgery (n= ) or carotid endarterectomy (n= ) were investigated. patients included had either documented cad (n= ) or two or more (n= ) dsk factors (age > years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia > mg/dl). -lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and h, h, h, and h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression > . mv and/or new t inversion). in addition results: of the patients investigated developed ecg-documented pmi, % occurdng in the immediate postoperative phase. troponin i levels > . ng/ml were found in of these patients thus, comparing a cardiac troponin i cut-off level of ng/ml with intermittent -lead ecg recordings, we found a sensitivity of % and a specificity of % methods: demographic, clinical and ecg data were analyzed. . % of patients were male; . % female. cad was the most common underlying cardiac disease ( . %) and . % underwent open heart surgery. % received proeainamide for supraventricular and % for ven~cular arrhythmias. % received a loading dose. maintenance was provided by iv route in . % and by po in . % ( . %sr end . % ir). . % of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was • years, for group b • years, and for group c • years. a history of previous myocardial infarction was present in pts of group a, in of group b and in of group c. three pts in group a, in group b and in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was days ( - ) for group a we used a continuous fixed intravenous a infusion at a dose of the sn was % in groups a and b, % in c, and sp % for group a, (fixed defects included) and % for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, pts -group b, and pts -group c), transient hypotension ( pt -group c), headache ( pts, group c), dyspnea ( pt -group a), while st depression was seen in pts of group b and in pts in group c. the rate of a infusion was decreased to /kgr/min in one group b pt due to development of chest pain s five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch bertschat, e betbes blanch, l del nogal saez e -meneza nolla, j. nolla-salas pilz~ u puig de la bellacasa e scarpa, n. van de wetering objectives: only % of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines ( ) able to induce pronounced conduction block ( ). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted g catheters in gbs-and cidp -patients under sterile conditions. some of them had not responded very well to established therapies. - ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed ( - ml) over one week. results: the gbs patients improved after days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after days (median). patients not at all treated before ( / ) responded better than patients that had been pretreated ( / ) with plasmaexchange or intravenous immunoglobulines. / cidp patients drew benefit from treatment, stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower ( / ) than those for plasmaexchange or intravenous immunoglobulines. references:( )wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve ( ) . ( ) clinical observations were made in patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok- apparatus, the exposure within minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in bed university hospital, covering . inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march th , were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score > , at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant ( th, ~ and th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from patients ( apache ii score: . ) admitted in the icu, ( . %) enter the study protocol. seven ( , %) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was . vs . . the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, / and mof, / . conclusions: . -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. . -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(> years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, patients were evaluated; ( %)had pancreatitis of unknown etiology . results:( )patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity( % vs %),mortality( % vs %),and longer hospital stays than p~tierf~ with pancreatitis of known cause.( )the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.( )curlously,no difference existed in associated medical conditions between patierts withknown and ur ~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for > days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were . % ie at ph . _+ . ( % limits) and between old & new catheters were . %, ie ph j _+ . ( % limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis . therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of < . according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n= ); ih analgesiajsedation plus pirenzepine mg/day (n= ); .[ih anatgcsia/sedalkm plus sncraltate x [ g/day (n= ); iv: analgesidsedatkm plus pirenzcpine mghlay plus sucralfate x e/day (n= ). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p < . . / patients ( . %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb ( . + . vs . -+ . ). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = . ) has been shown. the frequency of gisb among the groups is as follows: h . %; lh . %; llh . %; iv: . % (ch -~ = . ; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" = . ; p= . ) and reached an overall-value of . %.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a -h esophageal ph-metry was performed in patients in recumbent position at ~ who had a ngt for more than days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than ) (ger-t) more than %, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale= - ) was noted. two ph-metrles repeated on h and on days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than (tph< ). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: out of patients presented a ger-t> %. eleven out of group i group (n= ) i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise ( , and w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: . +/- . i/min, vtc: - +/- ml and frc: . +/- . liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol ; : - . objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co vasoreactivity in brain injury (stroke ; : - ), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: mechanically ventilated patients (age - yrs, glasgow - ) in coma due to acute brain injury were investigated during stepwise changes in paco ( , , , and mmhg) by increasing inspired pco . middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of % n through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco . objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at mm hg. . ringer's solution, . . % gelatine solution, . % hydroxyaethylstarch (mean m.w. . ). we evaluated the following parameters within intervals of min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in consecutive patients ( sepsis/sirs, acute heart failure, ards, transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z- system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f - , df = , p < . , (figure) ). aitbv was correlated to ac in a linear fashion (r = . , f = , df = , p < .o ). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in anaesthetized pigs using a centrifugal pump with a f arterial catheter and a f left atrial aspiration line. animals were supported for two hours at a mean flow of . liter ( ' rpm), a mean hematocrit of % and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in patients with ph of various causes: primary; post atrial septal defect closure (asd); chronic thromboembolic (te); anorexigen; iv drugs; collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me . we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to control humans (mean %met = . % _+ . % s.d.), the mean %met in ph patients was . % _+ %. the %met in ph patients correlated inversely with cardiac output (r= . ), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph ( + ml/min) as compared to controls ( _+ ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from % to less than %. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in patients with (raaa).a short acth stimulation test (synacthen test; ug - acth iv) was performed at hrs within hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > . umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the patients investigated died and survived. mean cort basal in nonsurvivors was significantly (p< .o ) higher than in survivors; . (range . - . ) vs . (range . - , ). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; . (range . - . ) vs . (range . - . ). while patients showed a cort basal < . , no cort stim < . was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; vs and vs . conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: pts. aged + years, admitted to our ccu from to with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters ( f, cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into groups. in group a we included pts with anterior ami, in group b, pts with inferior ami, and in group c, pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p< . was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were + mmhg and _+ mmhg respectively. despite the signifanf correlation (p< . ) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, pts ( %) presented cvp> mrnhg and of these ( %) had pwp_> mmhg. in group , the cvp was _+ mmhg and the pwp, _+ mmhg. significant correlation (p< . ) between the two variables also existed, however it was impossible to predict the pwp value. pts ( %) had cvp> mmhg but only of these ( %) had pwp> mmhg, similar was the relation between cvp and pwp in group c (p< . ). cvp averaged + mmhg, and pwp, _+ mmhg. pts ( %) had cvp> mmhg and from these ( %) presented pwp> mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as - ~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated pts (age + yeats nyha class: ii, hi, iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo max determination, a -mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was . ( - ) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every hours until somatic death. we we detected severe diffuse hypokinesia (ef< %) in patients and mild hypokinesia in others (ef - %). systolic function was strictly normal in only patients. corrected qt interval (qtc) in ecg was . _+ . msec (normal range - msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref- ; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc ( + . + . , p < . ) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from . + . to . + . (p < . ); redv was reduced from . -+ to . _+ . (p < . ); resv was reduced from • . to + . (p < . ). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group ( . + . |• . ) compared with the c"group ( • . ($ . • . ) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: elective cardiac surgery patients. interventions: dobutamine was infused at ug/kg/min for hours immediately after admission to the icu. hemodynamics were measured every minute periods until hours and again hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied patients . -+ t . years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< mmhg, urine output< ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the th post infarction day and months later. results: three patients refused iabp treatment and / survived on the th day. on the th day / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p < . ). on the th month / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p< . ). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from to ; females and males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n= ), valvular replacement (vr, n= ), combination of both (vr-cabg, n= ), or others (n= ) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x with continuity correction when appropriate. results: seventy hree patients ( . %) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p< . ). type of surgery, preoperative ef, and nyha classification are the first significant factors related to inotropic support (p< . ). most patients operated on for double-vr or vr=cabg required inotropic support ( and %, respectively). postoperative mortality was higher in patients receiving inotropic support ( , % vs , % 'overall mortality, p= . ). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every - seconds. cardiac output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa- sing do i to at least ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached mmhg. vasoactive or inotropic drugs were infused if the desired do was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics . materials: asa group ill-iv patients with a mean age of years (range - ) and a mean weight of kg (range - )) scheduled for major abdominal surgery were included. results: patients were excluded because do i could not be raised at all. mean do i was increased from ml/min/m (range - ) to ml/min/m (range - ). mean volume of preoperativdy infused colloid was ml (range - ). during surgery ml (range ) of colloid was infused. mean length of surgery was minutes (range - ). mean blood loss was ml (range ). expected mortality and morbidity rates ("possum") were % and %, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of % and %, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in patients (pts) with severe left heart faille (left veutrieul x filling pressure (lvfp) > mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in furosemide (f) + d butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n= ): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am ) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: consecutive patiens with ami, from october , to february , , admitted within hours alter onset of symptoms, undergone successfull systemic thrombolysis. of them were anterior (group a) and inferior (group b) . noradrenaline plasma levels at (na ), (na ) and (na ) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns . ns means -sem (normal limit for our laboratory: na < / pg/ml; ck < u/i ) conclusions: . the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. . the decrease in na is more evidence in amis with anterior location. . this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. . there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: patients scheduled for multiple cardiac valve replacement in were divided into two groups: group i ( patients) -without pp; group ii ( patients) -pp was performed preoperatively. the first pp was made ten days and the second - days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing ( - ~ and absence of dmso. this made it possible to preserve % functionally active platelets during days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the st p. o. d. was significantly greatest in the group i ( _+ ml) and all the patients required transfusion of the donor blood ( + ml) whereas the blood loss in group ii was +_ ml and olny patients required the donor blood. the number of platelets on the st p.o.d, was _+ . /l (group i) and + . /l (group ii), p < . .conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the patients of the swiss centers) and in the patients randomized in the isis- trail mortality rate in women was . % ( / ) compared to . % ( / ) in men; in switzerland: in-hospital mortality for women was . % ( / ), for men . % ( / ).the table shows the results of isis- in terms of odds ratios and their % confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. ( ) and bubble ( ) oxygenators a, ere used. anaesthesia was balanced and pts were extubated to hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia ( ), after induction of anaesthesia (i[), mins after cpb (iii) (iv) and (v) hrs afiter cpb, rains after extubation (vi), hrs after cpb (v[ ) and on the nd, rd, th, th and tb postoperative day (pd) (viii, x, x, xi, xi , respectively). analysis of data was performed by two-way analysis of variance, p < . being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the th pd. qs/qt reached maximum at rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over ubbie type oxygenators. the aim of this study was to evaluate the influence of x 'genator type on pulmonary shunt (as/at) after coronary surgery. methods: patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a (n = ) with a membrane o~genator and a (n = ) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), mins after extubation ( ), hrs alter cpb ( ) and on the nd, rd, th, th and th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < . being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at rains after extubation (ii) in a group. the difl'ereuce between the two groups was no more significant from hrs after cpb (iii) to the end of the investigated period. ! i * p < a. s betw~n ~o~ conclusions: membrane ox 'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation ( to hrs after cpb). the difference in qs/qt disappeared his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence ( to % ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent ( - % are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in patients on admission to the icu, using a nova electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs ). normal range of img++ was found between . - . mmot/l (healthy volunteers). results: for the entire population, we found a % prevalence ( / ) of hypomagnesemia (figure ) . among the surgical patients, the prevalence was highest after cardiac surgery ( %) and after thoracic surgery ( %) and was lowest after neurosurgery ( %). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure ( % for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i- %. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from / to / consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was % ( ). in all cases diagnosis was proven by a positive hipa. / patients died. in / hatt could be confirmed before severe thromboembolic complications occured. / patients developed a deep vein thrombosis (dvt), / dvt and pulmonary embolism (pe), / dvt, pe and arterial thrombosis (at) and / a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of pts. is %. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality ( / ). computed tom graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of patients with cardiogenic shock, as defined by an initial ct below . l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n= ), heart failure (n= ), valvulopathy (n= ) or recent cardiac surgery (n= ). after exclusion of patients with concurrently suspected/documented infection, this study included patients, of whom ( . %) survived. treatment of shock included dopamine (n= ), dobutamine (n= ), norepinephrine (n= ) and epinephrine (n= ). patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about - mm hg ayter single drug administration observed only in patients. ezap monotherapy accomplished during one week with mg daily dose. hypotensive effect observed in patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of mg. this new drug combination permits to lower ap in patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra( , %)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in % patients with ah and low pra. methods: in patients with cardiogenic shock due to ischemic heart disease (n= ), heart failure (n= ) and valvulopathy (n= ), hemod aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n= ), dopamine (n= ), norepinephrine (n=i ) and epinephrine (n= objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing - kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h it of heparinised r/l solution at ~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing - kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was ml. temperature was maintained at ~ and portal vein pressure at ( - ) mmhg. the flow was . - . ml/gr of graft liver mass per minute. observation period was hours (t ). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t = mmhg , t = mmhg), hr (t = , t = ), rap (t = mmhg , t = mmhg), pap (t = mmhg, t = mmhg), pcwp (t = mmhg, t = ~mhg), svr (t = dyn'sec/cm ' , t = dyn'seclcm~ pvr (t = dyn.sec/cm o, t = dyn.sec/cm ,'~), co (t = . t/min, t = . t/min), do (t = ml/min, t = . ml/min), vo (t = ml/min, t = ml/min), o er (t = . %, t = . % ), ph (to= . , t = . ), po (t = mmhg, t = mmhg), pco (t = mmhg, t = mmhg), pvo (t = mmhg, t = mmhg), svo (t = %, t = %), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. consumption, co production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: paced pts (mean age +- yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to . ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity %), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a -year-old woman presented a year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was mm hg, pulse beats/mir~ute, hematocrit . , seln~n protein /i, and se~um albumin q/l. an leg-kappa pa[apfotein was demostrated ( . g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, . units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in critically ill patients men and women ages from - who had sepsis -arf congestive heart failure postoperative multiple organ failure and polytrauma .this method was applied to these patients from to hours. % of the patients recovered completely their kidney function, % improved their kidney function and % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to groups: in group i, (n= ) airf was inducted by bilateral renal artery clamping for rain. group h (n= ) rats underwent a sham procedure. in each group, rats were dialyzed twice ( th and th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an ) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): . _+ . ; cupro: . _+ . ; hemo: . _+ . ; an : . _+ . ). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day (p< . ), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: patients who underwent first cadaver kidney transplantation in our unit between january and december in were involved. the recipients were divided into groups: group i." non functioning graft (n= ); group ii: delayed graft function (n= ), group ili: good graft function (n= ). the grouping criteria were: a/haemodialysis in the fii~t postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. ( . ) and croup ii. and iii. ( . and . , p< . ). intmo! emtive significant differences were found between the different groups in systolic blood pressure (group i. hgrmn, group ii. hgnnn, group iii. hgmm, p< . ), mean arterial pressure (group i. hgmm, vs. group ii. hgnun p< . , vs. group iii. hgmm p< . ), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. inin. vs. group ii. rain. p< . , vs. group i. rain. p< . !). the rejection rate was higher in the first days in the patients with non-functioning grafts (group i. % and group ii. % vs. group iii. %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord- -likfvwwj authors: jin, jian; okagu, ogadimma; yagoub, abu elgasim ahmed; udenigwe, chibuike c. title: effects of sonication on the in vitro digestibility and structural properties of buckwheat protein isolates date: - - journal: ultrason sonochem doi: . /j.ultsonch. . sha: doc_id: cord_uid: likfvwwj the present work investigated the effects of sonication at different amplitudes and durations on the in vitro digestibility of buckwheat protein isolates (bpis). the conformation, particle size and microstructures of the bpis were also studied to explicate the possible mechanisms of the sonication-induced changes. the results showed that sonication conditions of khz, pulsed on-time s, off-time s, amplitude of % and duration of min (sa t ) improved the digestibility of bpis from . % (control) to . %. the tertiary structure analysis showed that sonication exposed the hydrophobic core buried inside the protein molecules and broke the intramolecular crosslinks, based on the increase in the surface hydrophobicity and intrinsic fluorescence and the decrease in the disulphide content. the secondary structure analysis showed that sa t decreased the content of β-turn and β-sheet by . % and . %, respectively, and increased the content of anti-parallel β-sheet, random coil, and α-helix by . %, . %, and . %, respectively. the particle size of the control bpis ( . ± . nm) increased to . ± . nm in the sa t sonicated sample with a corresponding decrease in the polydispersity index from . ± . to . ± . . moreover, scanning electron microscopy indicated that sonication broke the macroparticles into smaller fragments and changed the surface state of the proteins. taken together, sonication has proven to be a promising approach for improving the digestibility of buckwheat proteins, which can be explored as a source of plant-based alternative protein for food applications. buckwheat is mostly grown in some european countries and china. it can be categorized into two types, common buckwheat (fagopyrum esculentum moench) and tartary buckwheat (fagopyrum tataricum l. gaertn), based on differences in the composition and biological features. the total protein content of buckwheat ranges from - % on a wet weight basis [ ] . buckwheat protein is rich in lysine, which is the first limiting amino acid in cereal proteins, such as corn and wheat proteins [ ] . the indispensable amino acid composition of buckwheat protein is comparable to the fao/who suggested requirements for children and adults. presently, studies on buckwheat proteins have focused on preparation of nutraceuticals with different beneficial effects on human health, such as cholesterol-lowering [ ] [ ] [ ] , antioxidative [ , ] , antihypertensive [ , ] , antimicrobial and antitumor activities [ ] [ ] [ ] . however, buckwheat proteins are underutilized in terms of human nutrition mostly because of their content of anti-nutritional factors, such as protease inhibitors, tannins, phytic acid and saponins, which decrease protein digestibility in the digestive tract [ ] . furthermore, the molecular structure of proteins also affects their digestibility [ ] ; for instance, corn protein contains numerous hydrophobic amino acid residues and α-helical domains that make it less digestible [ ] . according to an fao report, over million people globally were affected by hunger in and million people may be added to the total number of undernourished in the world in because of covid- pandemic [ ] . apart from lack of food, "hunger" encompasses food insecurity, reduced nutrient bioaccessibility and related health issues. meanwhile, the requirement for protein supply has increased due to the increasing world population and consumer preference for high-protein foods [ ] . recently, the awareness of the food industry and consumers about plant-based proteins has increased because of their lower carbon footprint compared to animal proteins. however, one of the drawbacks of plant-based proteins is their lower digestibility and bioavailability, which can be improved by suitable processing. therefore, there is a need to discover alternative proteins and to develop new methods for improving the digestibility and nutritional quality of the proteins. power ultrasound, also known as high-intensity ultrasound, with intensity ranging from to w·cm - and frequency of - khz, has extensively been used in the food industry. many studies reported that ultrasonic pretreatment of proteins before enzymolysis hydrolyzed the protein into smaller fragments and improved the reaction rate significantly [ ] [ ] [ ] . this occurred because acoustic cavitation generated drastic physical forces (micro jets, shear forces, shock waves, turbulence, etc.) and highly reactive free radicals [ , ] , which can attack the sidechains and backbone of protein molecules, leading to changes in their secondary and microstructures [ , ] . these changes result in the exposure of hydrolysis sites buried inside a protein, making them accessible to proteases. acoustic frequency, power intensity, temperature of the medium, and duration of treatment have been extensively studied as the major parameters of high-intensity ultrasound [ ] . however, the effect of the acoustic amplitude, square of which is directly proportional to the amount of energy applied to the sonication system [ ] , on the protein structure and digestibility has not been investigated. theoretically, when a higher acoustic amplitude is used at fixed frequency of an ultrasonic system, the cavitation bubbles can grow and collapse more easily within a few acoustic cycles, and these collapsed bubbles can generate numbers of nuclei bubbles that finally intensify the cavitation effect. therefore, this study was aimed at investigating the effects of sonication duration and acoustic amplitude on the in vitro digestibility of buckwheat protein isolates (bpis). in addition, the effects of sonication on the tertiary structures (surface hydrophobicity, intrinsic fluorescence, sulfhydryl and disulfide bond contents), secondary structure, particle size, zeta-potential and microstructure of bpis were studied to elucidate the structural mechanism underlying the effect of ultrasound on the digestibility of the proteins. buckwheat flour was obtained from bulk barn (ottawa, ontario, canada where m is the mass of the medium (g), c p is the heat capacity of the medium (j·g - ·°c - ), and dt/dt is the slope of the temperature vs. time plot. the dissipated acoustic power was expressed as watts per unit area of the emitting surface (w·cm - ), and the values are presented in table . the in vitro digestion of the control and sonicated bpis was conducted according to the cost consensus method [ ] of cacl ( . m) were added to the gastric digested mixture and the ph was adjusted to . using m naoh. the mixture was incubated at °c for another h with shaking at rpm. the in vitro digestion was terminated by boiling the mixtures for min, followed by centrifugation at g for min after cooling to room temperature. the in vitro digestibility (ivd) of the proteins was calculated using the following equation: where c is the concentration of peptides released (mg·ml - ) after deduction of a blank (buffer and digestive enzymes), which was determined using the lowry method [ ] , v is the volume of digestion (ml), n is the dilution factor, and m is the mass of the protein (mg). the surface hydrophobicity (h ) of the control and sonicated bpis was determined using ans as the fluorescence probe according to a previously reported method [ ] . the total sulfhydryl (tsh) content of bpis was determined using the same protocol as rsh determination, with the exception that the buffer contained m urea and g·l - sodium dodecyl sulfate. the rsh and tsh contents were calculated according to the following equation: where d is the dilution factor ( . ), c is the diluted protein concentration determined by the lowry method (mg·ml - ), Δabs is the difference in absorbance measured at nm with and without dtnb: the ss bond content of the control and sonicated bpis was determined according to the method developed by beveridge et al. [ ] with some modification. bpis ( mg the ss content was calculated using the following equation: ( ) where is the content of tsh and sh derived from the reduction of ss, which was ' sh c determined using eq. ( ), and is the total free thiol content which was calculated tsh c previously. the attenuated total reflectance fourier transform infrared spectra (atr/ft-ir) of the control and sonicated bpis were scanned at the wavenumber ranging from to cm - using a nicolet ftir (thermo fisher scientific, waltham, ma, usa) equipped with a 'itx smart orbit diamond' atr accessory. the surface of the crystal was cleaned with % ipa before the background was collected. after the background acquisition was completed, the sample was loaded onto the surface of the atr crystal and the pressing tip was pressed onto the sample to ensure good contact with the crystal surface. the spectra were collected using an omnic software (thermo fisher scientific cm - , β-turn: - cm - ) as previously reported [ , [ ] [ ] [ ] [ ] . the content of secondary structure was reported as relative peak area to the total area of amide i. the control and sonicated bpis ( mg) were dispersed in ml of milli-q water. then, the particle size and zeta-potential of the suspensions were determined with a nano zeta-sizer (malvern instruments ltd, uk) after equilibrating for s at °c. the morphologies of the control and sonicated bpis were observed with a jsm- f field emission scanning electron microscope (jeol, japan) at an acceleration voltage of . kv, after coating the sample with a layer of gold with a thickness of . nm using an em ace (leica, germany). all the data were reported as mean ± standard deviation (sd) of triplicate pretreatment experiments. statistical differences between the groups were analyzed by the one-way analysis of variance. significant differences between means were determined by tukey's test at p < . . all analyses were performed with the statistical software spss . (ibm corporation, ny, usa) and the graphs were plotted with origin pro . (origin lab corporation, ma, usa). the results of the in vitro digestibility (ivd) of bpis are shown in table acoustic amplitude is another parameter that influences the cavitation. generally, the intensity is proportional to the square of the amplitude of pressure waves [ ] . as shown in similarly, the increase in ultrasound power was reported to decrease the degree of hydrolysis of oat protein isolates [ ] . this observation may be because the smaller amplitude generated with lower intensity was dispersed in the protein solution and thus was not strong enough to disrupt the protein structure. however, the high intensity may have induced the formation of aggregates resulting in more compact protein particles that were less enzyme-digestible. bpis surface hydrophobicity, which correlates with the number of polar and non-polar groups on the surface of proteins, can reflect the change in molecular conformation of proteins. as shown in fig. (a) , higher amplitude and longer duration of sonication increased the surface hydrophobicity of bpis significantly (p < . ). this is possibly because acoustic amplitude is directly proportional to the amount of energy applied to the system. this increase in h resulted from unfolding of the bpis and exposure of hydrophobic amino acid residues buried in the interior of the protein molecules, due to the disruptive effects of the micro jets, shear forces, shock waves, and turbulence generated by ultrasound waves. the unfolding of a protein may expose its cleavage sites, thus facilitating interactions with the enzymes and increasing digestibility. similar findings have been reported on studying sonicated plant proteins [ , [ ] [ ] [ ] and animal proteins [ , ] , resulting in improved enzymatic hydrolysis of proteins. some hydrophobic amino acid residues such as tryptophan, tyrosine and phenylalanine can emit fluorescence when they are exposed outside or located at the surface of proteins. this indicator can be used to characterize changes in tertiary structures of proteins because these residues form the hydrophobic core of proteins where they interact by hydrophobic bonding. as shown in fig. (b) , sonication increased the fluorescence intensity of the bpis. at % amplitude, the fluorescence intensity decreased due to the increase in the ultrasound treatment duration, except for sa t . this result is because the exposed hydrophobic groups aggregated under the turbulence effect of sound waves. on the other hand, the fluorescence intensity of sonicated bpis increased with increase in ultrasound amplitude, which might be ascribed to protein unfolding resulting from the higher intensity. however, extremely high intensity may have led to refolding or aggregation of the proteins, as observed in the decreased fluorescence intensity of sa t . taken together, the increase in fluorescence intensity of bpis may be because sonication destroyed the hydrophobic interactions, hydrogen bonds and van der waals forces between the protein molecules [ ] . reactive sulfhydryl (rsh) groups, which are mostly located on the surface of protein molecules, can be involved in the formation of noncovalent bonds, such as hydrogen bonds. total sulfhydryl (tsh) is the sh group that exists in the free form rather than as ss bond, including tsh and sh groups buried inside the protein molecules. depending on the acoustic amplitude applied (fig. ) , some sonication intensities increased both rsh and tsh contents significantly (p < . ). compared to the control, sonication at sa t increased content of rsh from . to . μmol/g (by %), while sonication at sa t increased the content of tsh from to . μmol/g (by . %). however, sonication at sa t decreased the rsh content (p < . ) with no significant differences in tsh (p > . ). under the denaturation conditions, the content of tsh increased as a result of unfolding of the protein molecules. the increases in the content of rsh and tsh observed with the treatment may be ascribed to that sonication exposed the sh buried inside protein molecules and the ultrasound broke the disulfide bonds because of the physical and chemical effects of cavitation. the disulfide bond (ss) is one of the major forces that stabilize the structures of proteins. as shown in fig. [ ] . generally, the increase in sh content and the decrease in ss content is one of the mechanisms by which sonication improves the digestibility or enzymolysis of proteins [ , ] . peaks located in the amide i region of atr/ft-ir are widely used to determine the secondary structure of proteins. after deconvolution, extraction of the overlapping components (see supplementary information, fig. s ) and assignments of attribution, the secondary structure contents of bpis were determined. the β-sheet, random coil, α-helix, and β-turn contents of the native buckwheat protein isolate ( table ) were close to the values reported by choi and ma [ ] for globulin from common buckwheat, i.e., . % β-sheet, . % random coil, . % α-helix and . % β-turn. notably, the increase in the in vitro digestibility of bpis by sa t , sa t , sa t and sa t ( table ) might be because these treatments decreased the content of β-turn, which has a compact structure, compared to the control. moreover, these treatments, except sa t , increased the content of the anti-parallel β-sheet and, except sa t , increased the content of the β-sheet. however, only sa t decreased the content of the β-sheet and increased the content of the random coil substantially; this treatment also had the most pronounced effect on ivd. jin et al. [ ] attributed the improvement in enzymolysis of corn gluten meal to the increase in the β-sheet, but the study did not structurally differentiate the anti-parallel β-sheet from the β-sheet structure. in the present study, the total content of anti-parallel β-sheet and β-sheet of bpis treated by sa t ( . %) was close to that of the control ( . %). interestingly, all the secondary structure contents of the sa t -treated sample decreased and this might be attributed to the increase in the relative peak area of side-chain vibration ( cm - ) ( table s ). the random-coil, found in the control, was not detected in the sa t -treated sample, but the contents of other secondary structures increased compared to the control. this is likely because sonication made the protein molecules arrange more regularly or because of the shift of bands. the increase in content of the ordered structure might have occurred during the renaturation of the proteins. in general, sonication can increase the digestibility of proteins because of changes in the content of secondary structures resulting from cavitation-induced physical forces and free radicals [ ] . . . effects of sonication on particle size, polydispersity index and zeta potential of bpis the particle sizes of the control and sonicated bpis, which were presented as z-average values, and polydispersity index (pdi) are shown in table . the particle size of bpis was increased significantly by sonication, with the maximum -fold increase observed when the protein was sonicated at % amplitude for min (sa t ). some studies reported that ultrasound reduced the particle size of some animal and plant proteins [ , ] . o'sullivan et al. [ ] found that sonication of rice protein isolates by using a frequency of khz and power intensity of ~ w·cm - for min slightly increased the particle size by . %. jiang et al. [ ] also reported that sonication increased the particle size of black bean protein isolates owing to protein aggregation. the large increase in particle size of bpis might be due to the disruption of the protein microstructure by the ultrasound, leading to protein particle swelling after dispersion in water or induction of protein self-assembly due to hydrophobic interaction between the unfolded regions. pdi represents the disperse homogeneity of particles, with higher values indicating more heterogeneous particles in the system. as shown in table , the pdi of the control bpis indicated that the dispersed protein particles had a wide range of molecular weight distribution. sonication mostly decreased the pdi, suggesting that the dispersion of proteins occurred in a narrower molecular weight distribution, possibly ascribed to the formation of aggregates or to swelling of protein particles. similarly, stefanović et al. [ ] reported that an increase in the sonication duration from min to min, decreased the pdi and increased the particle size of egg white proteins; the reduced pdi was attributed to the random aggregation induced by excessive protein denaturation. jiang et al. [ ] indicated that the particle size of sonicated black bean protein isolates became larger because of the formation of soluble and unstable aggregates, and that the particle size distribution broadened due to the breakdown of the unstable aggregates. the surface of proteins shows a net charge resulting from the amino acid groups exposed to the exterior. typically, higher zeta potential means stronger electrostatic repulsion between protein molecules, and vice versa; thus, it plays a role in stabilizing the protein dispersion. in table , the control sample had the highest zeta potential magnitude, which was decreased significantly (p < . ) by sonication, except for sa t . moreover, longer sonication duration and higher amplitude resulted in the most pronounced decrease in zeta potential magnitude because of higher energy input into the system. the zeta potential values indicated that the sonication-induced structural changes ( table ) and protein aggregation, based on the increased particle size ( table ) , may have reduced the number of negatively charged residues on the protein surface. in contrast, jiang et al. [ ] reported that sonication increased the zeta potential of black bean protein isolates, resulting in the strengthening of the inter-particle electrostatic repulsion, thus disrupting protein aggregation and improving the dispersion stability of the proteins. the microstructures of the control and sonicated bpis are shown in fig. . the thin lamellar structure is the typical feature of bpis; however, some blocks were present in fig. (a)-(c). fig. (a) showed that the structure of the control bpi was compact. the sa t -treated sample revealed a loose structure having a larger size (fig. (b) ), which might be attributed to the destruction of the bonds between the protein subunits by the ultrasound waves, resulting in the extension of the distance between particles. in fig. (c), the sa t sample showed both blocks (marked with a circle) and multi-layer structure (marked with an arrow), which possibly resulted from the aggregation of the proteins. high intensity sonication can break the protein particles into smaller fragments, as observed in fig. (c)-(g) . the quantity and size of the fragments correlated positively with the input energy of ultrasound waves to the protein solutions. the formation of fragments might be a result of the destruction of the crosslinks between the polypeptide chains of proteins, including the ss bonds, hydrogen bond and van der waals interactions, due to effects of shear forces, micro streaming, shock waves and free radicals generated by the ultrasound radiation [ , ] . nonetheless, the fragmentation did not result in the reduction of particle size of the proteins ( table ) . therefore, we postulated that the proteins might absorb and hold water in their inner structures when dispersed in the aqueous solution, thus increasing the particle size. surface morphology, including roughness, has substantial influence on the bio-adsorption [ ] and mass transfer. as previously mentioned, the surface of the control bpi was tight ( fig. (a) ), which may have protected the proteins from adsorption of water, thus the protein particles had smaller sizes when dispersed in water ( table ) . sonicated samples (fig. (b)-(g) ), especially sa t ( fig. (f) ), presented a rough surface of proteins with many micropores, which can allow water to enter into the proteins, thus increasing the degree of swelling and leading to larger particle sizes. furthermore, the adsorption between the proteins and enzymes can be enhanced because of the larger contact areas of the sonicated bpis. longer sonication duration led to rougher protein surfaces because of the extension of cavitation. on the other hand, the surface of sa t sample ( fig. (g) ) appeared smoother than that of sa t ( fig. (f) ), which might be because the surface protruding edges were excised by the high intensity ultrasound. a previous report had demonstrated the flattened surface of glutelin extracted from the sonicated corn gluten meal [ ] . in general, the surface of the sa t -treated sample was substantially different from those of the other treated samples, which might be related to the lack of the random coil in the sa t sample ( table ) . our previous research on sonication of corn gluten meal indicated that sonication decreased the height and surface roughness of the proteins [ ] , suggesting differences that may originate from the nature of the materials. treatment of buckwheat protein by sonication improved the in vitro digestibility of the proteins significantly. specifically, sonication under khz, pulsed on-time s, off-time s, amplitude of % and duration of min (sa t ) improved the digestibility significantly by % compared to the control. the structural analysis showed that sonication changed the tertiary structure by increasing the surface hydrophobicity, intrinsic fluorescence intensity, and total sulfhydryl contents. the secondary structure analysis showed that sa t decreased the content of the β-turn and increased the content of the anti-parallel β-sheet, and the random coil. furthermore, sonication changed the particle size, dispersion characteristics, zeta-potential, and microstructures of the proteins. in conclusion, sonication can be further explored as an effective method to improving the digestibility of buckwheat proteins towards their utilization in food product development. sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min. different superscript letters on the bars represent significant differences (n = , p < . ). sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min. different superscript letters on the bars represent significant differences (n = , p < . ). ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: properties of protein concentrates and hydrolysates from amaranthus and buckwheat a buckwheat protein product suppresses gallstone formation and plasma cholesterol more strongly than soy protein isolate in hamsters preparation of tartary buckwheat protein product and its improving effect on cholesterol metabolism in rats and mice fed cholesterolenriched diet insoluble fraction of buckwheat (fagopyrum esculentum moench) protein possessing cholesterol-binding properties that reduce micelle cholesterol solubility and uptake by caco- cells fractionation and evaluation of radical scavenging peptides from in vitro digests of buckwheat protein physicochemical and antioxidant properties of buckwheat (fagopyrum esculentum moench) protein hydrolysates latent production of angiotensin i converting enzyme inhibitors from buckwheat protein purification and identification of angiotensin i-converting enzyme inhibitory peptide from buckwheat (fagopyrum esculentum moench) purification and characterization of the antitumor protein from chinese tartary buckwheat (fagopyrum tataricum gaertn.) water-soluble extracts a relatively stable antifungal peptide from buckwheat seeds with antiproliferative activity toward cancer cells biocontrol potential to inhibit fungal (botrytis cinerea) infection of cherry tomato influences of high hydrostatic pressure, microwave heating, and boiling on chemical compositions, antinutritional factors, fatty acids, in vitro protein digestibility, and microstructure of buckwheat protein substrate conformation and proteolysis effect of low-frequency ultrasonic-assisted enzymolysis on the physicochemical and antioxidant properties of corn protein hydrolysates the state of food security and nutrition in the world . transforming food systems for affordable healthy diets protein intake trends and conformity with the dietary reference intakes in the united states: analysis of the national health and nutrition examination survey enzymolysis reaction kinetics and thermodynamics of defatted wheat germ protein with ultrasonic pretreatment pretreatment of defatted wheat germ proteins (by-products of flour mill industry) using ultrasonic horn and bath reactors: effect on structure and preparation of ace-inhibitory peptides effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: kinetics and thermodynamics study applied sonochemistry: the uses of power ultrasound in chemistry and processing theoretical and experimental sonochemistry involving inorganic systems effects of multi-frequency power ultrasound on the enzymolysis and structural characteristics of corn gluten meal effects and mechanism of dual-frequency power ultrasound on the molecular weight distribution of corn gluten meal hydrolysates ultrasound technologies for food and bioprocessing physicochemical and functional properties of buckwheat protein product effect of ultrasound treatment on particle size and molecular weight of whey proteins a standardised static in vitro digestion method suitable for food-an international consensus the lowry method for protein quantitation comparison of protein surface hydrophobicity measured at various ph values using three different fluorescent probes an improved method to determine sh and-s-s-group content in soymilk protein determination of sh-and ss-groups in some food proteins using ellman's reagent conformational study of globulin from common buckwheat (fagopyrum esculentum moench) by fourier transform infrared spectroscopy and differential scanning calorimetry conformational study of globulin from rice (oryza sativa) seeds by fourier-transform infrared spectroscopy atr-ft/ir study on the interactions between gliadins and dextrin and their effects on protein secondary structure examination of the secondary structure of proteins by deconvolved ftir spectra ultrasonic treatment effect on enzymolysis kinetics and activities of ace-inhibitory peptides from oat-isolated protein effects of ultrasound on the structure and physical properties of black bean protein isolates effects and mechanism of ultrasound pretreatment on rapeseed protein enzymolysis effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein structural and functional changes in ultrasonicated bovine serum albumin solutions situ monitoring of the effect of ultrasound on the sulfhydryl groups and disulfide bonds of wheat gluten the effect of ultrasound treatment on the structural, physical and emulsifying properties of animal and vegetable proteins effect of the controlled high-intensity ultrasound on improving functionality and structural changes of egg white proteins, food and bioprocess technology sensitivity of surface roughness parameters to changes in the density of scanning points in multi-scale afm studies. application to a biomaterial surface sonication at % amplitude for min; sa t , sonication at % amplitude for min sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min sa t , sonication at % amplitude for min; sa t , sonication at % amplitude for min the authors declare that there are no conflicts of interest.  buckwheat protein isolates (bpis) were treated by varied sonication time and amplitude.  sonication increased the in vitro digestibility of bpis significantly compared to control.  sonication changed the molecular conformations (tertiary and secondary) of bpis.  sonication broke bpis into smaller fragments and induced surface modifications. key: cord- -g qe ml authors: di sotto, antonella; vitalone, annabella; di giacomo, silvia title: plant-derived nutraceuticals and immune system modulation: an evidence-based overview date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: g qe ml immunomodulators are agents able to affect the immune system, by boosting the immune defences to improve the body reaction against infectious or exogenous injuries, or suppressing the abnormal immune response occurring in immune disorders. moreover, immunoadjuvants can support immune system acting on nonimmune targets, thus improving the immune response. the modulation of inflammatory pathways and microbiome can also contribute to control the immune function. some plant-based nutraceuticals have been studied as possible immunomodulating agents due to their multiple and pleiotropic effects. being usually more tolerable than pharmacological treatments, their adjuvant contribution is approached as a desirable nutraceutical strategy. in the present review, the up to date knowledge about the immunomodulating properties of polysaccharides, fatty acids and labdane diterpenes have been analyzed, in order to give scientific basic and clinical evidence to support their practical use. since promising evidence in preclinical studies, limited and sometimes confusing results have been highlighted in clinical trials, likely due to low methodological quality and lacking standardization. more investigations of high quality and specificity are required to describe in depth the usefulness of these plant-derived nutraceuticals in the immune system modulation, for health promoting and disease preventing purposes. immunomodulators are defined as agents able to affect the immune response, which represents the set of reactions activated to protect the organism against infective agents, environmental injuries and illness; moreover, immune response can counteract the invasion of harmful native cells, such as precancerous and cancerous ones [ ] . immune response is mediated by a first line of defence, namely innate immunity (figure ), which is characterized by physical and biochemical barriers, alongside a non-specific cell-mediated immune response, including granulocytes, macrophages, natural killer cells and humoral elements, which cooperate to counteract pathogen infection and malignant transformation [ ] . moreover, an adaptive immunity is activated as a second defense line after a macrophage-mediated presentation of antigens to b lymphocytes, with the help of t lymphocytes; then, b cells can mediate the humoral immunity through the production of high-affinity antibodies and establish immunological memory [ ] . moreover, t lymphocytes can mediate cellular immunity after activation by cytokines released from helper t cells [ ] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [ ] [ ] [ ] [ ] [ ] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [ ] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [ ] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [ ] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. immunomodulating agents can affect immunity in a negative or positive manner, thus being categorized as suppressing or stimulant [ ] . particularly, immunosuppressors inhibit the activation of immune response or decrease the activity of its components, thus restoring normalcy. they are of interest in organ transplantations and in autoimmune disorders, wherein the immune system mistakenly activates an immune response against the own body tissues, leading to their destruction [ ] . for instance, vitamin d has been shown to counteract the aberrant immune responses of systemic lupus erythematosus, without compromising the physiological defences and to produce benefits in atopic dermatitis too [ , ] . conversely, immunostimulants boost the endogenous immune defences, thus allowing one to restore or maintain the body homeostasis [ ] . they can be usefully exploited as immunotherapeutic agents by individuals with immunocompromised conditions; however, they can represent suitable prophylactic strategies for healthy individuals or more susceptible subjects against viral infections [ ] . in support, during the current sars-cov- pandemic, the trained immunity by vaccines, which induce heterologous protection, have been proposed as a rational strategy to boost antiviral defences and reduce susceptibility to infection [ ] . a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [ ] [ ] [ ] [ ] [ ] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [ ] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [ ] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [ ] . furthermore, immunostimulants can act as adjuvant anticancer treatments, to counteract their immunosuppressive side-effects [ ] . particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [ ] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [ ] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors at higher doses [ ] . among polyphenols, resveratrol stimulated both cellular and humoral immunity in preclinical models, thus preventing pathogen replication and inflammation, and promoted antitumor immune response too [ ] . furthermore, cichoric acid from echinacea promoted phagocytic activity, both in vitro and in vivo [ ] . anti-inflammatory and immune-modulatory effects has been highlighted for curcumin too, although the poor bioavailability limits its clinical application [ ] . in the present review, up to date knowledge on the scientific basis for the immunomodulatory activity and clinical relevance of some emerging classes of plant-derived nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes, has been reported. a comprehensive search was made using pubmed and scopus electronic databases and selecting english as the preferred language, although no language limitations nor filters were applied. for more specific requirements, google scholar and clinicaltrials.gov were considered too. the following searching keywords and their combinations through the boolean logical operators were used: "herbal immunomodulators", "phytochemicals", "immune system", "nutraceuticals", "medicinal plants", "immunomodulation", "immune system boosters", "immunosuppressors", "immunoadjuvants", "gut microbiome", "natural occurrence", "chemical features", "preclinical studies", "clinical trials", "polysaccharides", "echinacea", "astragalus", "β-glucan", "fatty acids", "pufa", "oleic acid", "punicic acid", "γ-linolenic acid", "linoleic acid", "evening primrose oil", "borage oil", "flaxseed oils", "labdane diterpenes" and "andrographolide". this overview allows one to identify novel immune system modulators to be usefully exploited for health promoting and disease preventing purposes. polysaccharides are carbohydrate macromolecules containing at least monosaccharide units, joined by glycosidic linkages to form long-chain molecules, which can be both linear and highly branched. they are called homopolysaccharides when constituted of the same monosaccharide unit, while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [ ] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure ) [ ] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β → ), (β → ), and (β → ) or (α → ), (α → ), and (α → ), allow the production of linear and branched glucans. it seems that (β → )-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β → )-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α → ) (α → )-d-glucans, their structure activity relationship is less characterized [ ] . while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [ ] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure ) [ ] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β → ), (β → ), and (β → ) or (α → ), (α → ), and (α → ), allow the production of linear and branched glucans. it seems that (β → )-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β → )-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α → ) (α → )-d-glucans, their structure activity relationship is less characterized [ ] . mannans consist of a d-mannose backbone, linked mainly by β → bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [ ] . furthermore, (β → )-or (α → )-, (β → )-, and (β → )-or (α → )-dmannosidic bonds are reported [ ] . the (β → )-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [ ] [ ] [ ] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α- arabinofuranosyl and β-d-galactopyranosyl units linked via position at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β → )-and (β → )-d-galactopyranosyl units [ , ] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and mannans consist of a d-mannose backbone, linked mainly by β → bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [ ] . furthermore, (β → )-or (α → )-, (β → )-, and (β → )-or (α → )-d-mannosidic bonds are reported [ ] . the (β → )-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [ ] [ ] [ ] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α- -arabinofuranosyl and β-d-galactopyranosyl units linked via position at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β → )-and (β → )-d-galactopyranosyl units [ , ] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and molecular weight determine the structural diversity [ ] . moreover, flexible chain conformation and branched regions are the main ones responsible for the immunomodulatory properties [ , ] . galactans are polysaccharides rich in galactose and include, beside type i and ii arabinogalactans, carrageenans, chemically characterized by repeating disaccharide units of sulfated or unsulfated d-galactose, that are linked by (β → )-and (α → )-bonds. low molecular weight (< kda) and a high degree of sulfation have been reported as features that high influence their immunomodulatory properties [ , ] . fucoidans are heteropolysaccharides rich in l-fucopyranosyl sulfated units linked by (α → ), (α → ) or (α → ) bonds. other monosaccharides can be present, such as galactopyranosyl, mannopyranosyl, xylosepyranosyl and uronic acids [ ] . the naturally higher content of sulfate groups and the presence of acetyl groups are associated with a higher stimulatory activity [ ] . fructans are polysaccharides which constitute up to fructose units with a sucrolose terminal molecule. they are classified in inulin with a (β → )-d-fructofuranosyl, levan with a (β → )-d-fructofuranosyl, and mixed type, with both (β → )-and (β → )-linked d-fructofuranosyl moieties. a helical conformation has been associated with the modulatory activity on the immune system [ , ] . at last, xylans are polysaccharides containing predominantly a backbone of (β → )-dxylosepyranosyl units. other monomers attached to their backbone include α-dglucopyranosyl a units (glucuronoxylans) and α-l-arabinofuranosyl units (arabinoxylans). a correlation between their structure and activity has not been elucidated yet [ , ] . polysaccharides are naturally occuring in animal body fluids, cell walls, bacteria, yeast and fungi, extra cellular fluids, and in plant seeds, stems and leaves, which represent the focus of the present review. the main advantage of plant polysaccharides seems to be the low toxicity with respect to immunomodulatory bacterial polysaccharides and synthetic compounds [ ] . thus, they represent an ideal alternative for immune modulation. a variety of polysaccharides with immunomodulatory properties have been discovered in different species of plants (table ). among the most studied, there are type i and ii arabinogalactans from astragalus membranaceus (fisch.) bge., fructans from allium sativum l. [ ] , fucogalactoxyloglucan and type ii acidic arabinogalactan from echinacea purpurea l. (moench), ginsan and panaxanes from panax ginseng c.a. meyer, acemannan and aloeride from aloe vera l. [ ] , and glucomannan from amorphallus konjac koch [ ] . several studies have shown that polysaccharides from plants can modulate both innate and acquired intestinal immunity, by direct and indirect mechanisms. the former include the activation of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure ). of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure ). the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [ ] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [ , ] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca + concentration [ , ] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)- and the antiinflammatory cytokine il- in macrophage raw . , so showing a regulatory mechanism to maintain an equilibrium state [ ] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il- , tnf-α and interferon (ifn)-β production [ ] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor (tlr ), cd , complement receptor (cr ), scavenger receptor (sr), mannose receptor (mr), and dectin- . their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [ ] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il- and il- release induced by the synthetic lipopeptide p csk [ ] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il- /il- ratio and to reduce the release of ifn-γ, il- , il- , il- , il- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory proteins the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [ ] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [ , ] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca + concentration [ , ] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)- and the anti-inflammatory cytokine il- in macrophage raw . , so showing a regulatory mechanism to maintain an equilibrium state [ ] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il- , tnf-α and interferon (ifn)-β production [ ] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor (tlr ), cd , complement receptor (cr ), scavenger receptor (sr), mannose receptor (mr), and dectin- . their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [ ] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il- and il- release induced by the synthetic lipopeptide p csk [ ] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il- /il- ratio and to reduce the release of ifn-γ, il- , il- , il- , il- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory proteins (mip)- α, rantes and tnf-α by dendritic cells [ ] . polysaccharide enriched extracts of e. purpurea have been found to promote the phenotypic and functional maturation of dendritic cells by modulating c-jun n-terminal kinase (jnk), p mitogen-activated protein kinase (mapk) and nf-κb pathways [ ] . the activation of natural killer (nk) cells also contributes to the immunity modulation by polysaccharides. indeed, it has been shown that a. membranaceus polysaccharides can enhance the activity and killing effects of nk cells and promote their proliferation in rats with gastric cancer [ ] . moreover, they were able to increase cd -cd -cd + nks in peripheral blood lymphocytes [ ] . nks activation is probably due to the polysaccharides interaction with the killer cell lectin-like receptor k (klrk ) of nks [ ] . arabinoxylans extracted from wheat bran have been shown to inhibit the growth of transplantable tumors, and to promote the nk cell activity in s tumor-bearing mice [ ] . moreover, the mgn- rice bran arabinoxylan showed to enhance natural killer (nk) cell activity in aged c bl/ and c h mice upon its intraperitoneal injection [ ] . adaptive immunity is also modulated by plant polysaccharides. particularly, fan et al. have shown that polysaccharides from a. membranaceus significantly up-regulated the proliferation of b lymphocytes, probably through the interaction with immunoglobulin on the surface of b cells [ ] [ ] [ ] . a. membranaceus polysaccharides were also able to increase the number of cd +cd +cd + memory t helper (th) cells and cd +cd -cd + cytotoxic t cells [ ] . moreover, they also enhance the cd +/cd + t cell ratio [ ] . furthermore, arabinoxylan were found to increase the activation of tand b-cells and humoral and cell-mediated immunity in tumor bearing mice [ ] . at last, β-glucan microparticles enhanced t-cell activation and proliferation in vitro [ ] . their ability to affect the immune system by inducing th and/or th type immune response makes polysaccharides suitable adjuvants of the vaccine. among them, inulin, chitosan, glucans and mannans have been most extensively studied. particularly, the gamma and delta forms of inulin fructan have shown adjuvant activity against infectious pathogens by stimulating both th and th responses without inducing immunoglobulin e (ige) production [ ] . moreover, advax, a polysaccharide derived from delta inulin, has demonstrated to increase the immunization derived from influenza vaccine in mice. particularly, an induction of neutralizing antibody and memory b-cell against influenza, an increase in cd and cd t-cell proliferation, and enhanced levels of il- , ifn-γ, il- , il- were highlighted [ ] . advax also enhanced the immunogenicity of hepatitis b surface antigen (hbs) in mice and guinea pigs, by increasing both anti-hbs antibody titers and anti-hbs cd and cd t-cells. th , th and th responses were increased too [ ] . astragalus polysaccharides were also used as adjuvants of hepatitis b virus dna vaccine in a mice model, showing increased hbsag-specific antibody levels, higher activity of t cells, the production of il- , il- and ifn-γ by cd + t cells, and ifn-γ expression of cd + t cells. moreover, a stimulation of cytotoxic lymphocytes and dendritic cells maturation, and a reduction in the frequency of regulatory t cells were observed [ ] . mannans and fructooligosaccharide have also been shown to possess adjuvanticity [ , ] . furthermore, indirect effects are involved in the immunomodulatory properties of polysaccharides. in particular, dietary fibers (e.g., inulin, mannan, β-glucan, pectin) are metabolized by intestinal bacteria in the anaerobic environment of the cecum and colon, so generating scfa, such as acetate, propionate and butyrate [ ] . these molecules are able to cross the gut epithelium and interact with surface receptors on the immune cells, such as the g-protein coupled receptors (gprs) and [ ] . the activation of gprs by scfa modulates inflammatory signalling pathways, such as nf-κb, erk and p mapk [ , ] . moreover, it has been highlighted that scfa can reach t lymphocyte nucleus, so modulating several functions through a histone deacetylase (hdac) inhibition. recently, scfa have been reported able to induce t cells metabolic alterations by enhancing the mtor complex activity. particularly, after absorption into t cells, scfa can stimulate the activity of mtor complex, so increasing the conversion of pyruvate into acetyl-coa. moreover, the acetyl groups from scfa can be link to coa and enter the tricarboxylic acid cycle. the increased levels of citrate are exported from mitochondria into the cytoplasm, where the enzyme atp citrate lyase converts it into acetyl-coa, then used by histone acetyltransferases (hats) for histone acetylation and the regulation of cytokine gene expression [ ] . some clinical studies have been carried out on the potential immunomodulatory properties of polysaccharides, and a. membranaceus, e. purpurea and β-glucan have been most investigated. in a clinical trial on a. membranaceus by jiang et al. [ ] , twenthy-eight stable continuous ambulatory peritoneal dialysis patients were treated with peritoneal dialysis fluid containing astragalus ( ml/ l) for one week. an increase in the macrophage phagocytic capacity, no and tnf-α contents were observed in patients compared to those before the treatment [ ] . furthermore, ji et al. [ ] investigated the effect of astragalus pre-operative treatment of colorectal cancer patients (n = ) on immune function. results showed that astragalus pre-operative treatment promoted the nk cell activity in postoperative patients. in addition, the possible immunomodulatory activity of astragalus in patients with acute exacerbations of bronchial asthma (n = ) has been investigated [ ] . particularly, it was observed that the combination of conventional therapy with astragalus injection for days improved the effects of routine treatment, by enhancing t lymphocyte and nk-cells immune function. results of clinical trials on the immune system modulation by e. purpurea are controversial. particularly, a randomized blinded trial carried out on patients revealed that there was no significant difference in the incidence and severity of colds and respiratory infection between echinacea treatment ( weeks) and placebo groups. however, a small decrease of total lymphocyte counts was observed [ ] . another randomized, placebo controlled, double-blind clinical trial investigated the effect of different echinacea preparations, namely echinaforce ® (e. purpurea preparation from % herba and % radix), e. purpurea concentrate (same preparation at times higher concentration), special e. purpurea radix preparation (totally different from that of echinaforce ® ) on the reduction of the complaint index, defined by symptoms in healthy, adult volunteers who caught a common cold. the treatment continued until the enrolled patients felt healthy again, but not longer than days. the supplementation with echinaforce ® and its concentrated preparation showed to be significantly more effective than the special echinacea extract or placebo. moreover, all treatments were well tolerated [ ] . furthermore, prevention trials have been carried out, showing that echinacea products slightly reduce the risk of getting a cold in healthy individuals [ ] . however, the heterogeneity (e.g., different species and part used) of preparations used in the trials makes the conclusions on the potential immunomodulatory properties of echinacea difficult. clinical trials concerning the β-glucan immunomodulatory properties have also been carried out, although in some cases, yeast-derived-glucan were used. particularly, three randomized, double-blind, placebo-controlled studies have evaluated the effects of short-term β-glucan supplementation on children with chronic respiratory problems. after days' treatment, significant improvements in immunoglobulin, lysozyme, exhaled nitric oxide, and calprotectin production were found [ ] [ ] [ ] . furthermore, the combination of resveratrol plus carboxymethyl-β-glucan as a solution for aerosol has been tested in clinical trials. particularly, the ability of the combination to prevent or treat recurrent respiratory infections in children was studied [ , ] . in both cases, resveratrol plus carboxymethyl-β-glucan had a positive impact on children clinical conditions. indeed, nasal obstruction, rhinorrhea, sneezing, cough, fever, medication use, medical visits, and school absence were significantly reduced. moreover, resveratrol plus carboxymethyl-β-glucan have also been shown to relief nasal symptoms in children with allergic rhinitis, due to pollen allergy [ ] . at last, mannans should be mentioned. they have been reported to possess adjuvant-vaccine properties in clinical studies, probably mediated by its interaction with mannose receptors. particularly, it has been shown that oxidized mannan-mucin can be useful as an adjuvant in the breast cancer immunotherapy. indeed, a - years follow-up has highlighted that it decreases the cancer recurrence rate and prolongs recurrence time, without inducing toxicity or adverse reactions [ ] . fatty acids (fa) are a large group of lipids, characterized by a different number of carbons, arranged in a linear carbon chain skeleton of variable length with a terminal carboxylic group [ ] . based on the number of carbons in the chain, fatty acids can be classified as shortchain fatty acids (scfa; aliphatic tails up to a maximum of six carbons), medium-chain fatty acids (mcfa; aliphatic tails of - carbons), long-chain fatty acids (lcfa; aliphatic tails of to carbons) and very long-chain fatty acids (vlcfa; aliphatic tails of and more carbons) [ ] . among them, scfa, such as acetate, propionate, and butyrate, are produced by gut microbiota enzymes (i.e., propionate-coa transferase and propionaldehyde dehydratase) during the metabolism of carbohydrates and peptides containing branched-chain amino acids [ ] . bacteroidetes are reported to be mainly responsible for the production of acetate and propionate, while firmicutes are the primary contributors of butyrate; however, other bacteria such as lactobacillus and bifidobacterium spp. are involved too [ ] . based on the presence of different double bonds in this structure, fatty acids can be distinguished in saturated fatty acids (sfa), lacking double bonds in their carbon backbone, and unsaturated fa (ufa), which may contain one or more double bonds, thus leading to monounsaturated (mufa) and polyunsaturated fa (pufa) [ ] . sfa include palmitic acid (c : ), lauric acid (c : ), myristic acid (c : ), and stearic acid (c : ), whereas n- oleic acid (c : ) is an example of mufa. furthermore, pufa class includes fatty acids such as α-linolenic acid (ala; c : ), linoleic acid (la; c : ) and further long-chain metabolites [ ] . the number of carbon atoms and unsaturated bond position are used for the systematic nomenclature of fa. moreover, the greek letters omega (ω) and delta (∆) are included, to indicate how far a double bond is from the terminal methyl carbon and the presence and position of one or more double or triple bonds in the carbon backbone, respectively [ ] . a further "ω" or "n" classification designates the position of the first double bond in the skeleton from the end opposite to the carboxy group. accordingly, oleic acid is classified as a ω- (or n- ) fatty acid, while linoleic acid and α-linolenic acid are ω- (or n- ) and ω- (or n- ) fatty acids, as they contain the double bond nine, six and three carbons from the methyl end [ ] . nomenclature of the major representative fatty acids in the different fa classes is displayed in table . unsaturated fatty acids can be characterized on the basis of the cisor transorientation of the double bonds. usually, natural fatty acids carry a cisconfiguration, although some trans-fatty acids can also occur in foods as a consequence of the hydrogenation process, which can move double bonds from their naturally occurring position to a trans-configuration [ ] . trans-fatty acids are considered undesirable compounds in foods, as their intake is associated with an increased risk of cardiovascular and metabolic diseases [ ] . pufa can be further classified depending on the relative positions of the double bonds, as conjugated (double-bonded carbon atoms alternate with single bonds) and unconjugated (double bonds separated by one or more single bonds) [ ] . unconjugated pufa, especially ω- , ω- , and ω- series, are the most occurring in nature. the most common conjugated pufa are trienes, such as octadecatrienoic acids (e.g., punic acid, calendic acid). fatty acids within the series are biosynthetically related, being synthesized through enzymatic processes of desaturation, chain elongation, and chain shortening [ ] . particularly, the biosynthesis of ω- and ω- pufa starts from α-linolenic acid (ala or linolenate; , , - : ) and linoleic acid (la or linoleate; , - : ), respectively ( figure ). these precursors cannot be synthetized by mammals, which lack the ∆ and ∆ desaturases responsible for the convertion of : ω- fa to : ω- and : ω- pufa, and must be supplied by the diet, thus being considered as essential fatty acids. the initial rate-limiting step for the biosynthesis of ω and ω fatty acids is the insertion of a further double bond at the ∆ carbon into the carbon chain of ala and la, through the help of a ∆ desaturase enzyme: stearidonic acid (sa; , , , - : ) and γ-linolenic acid (gla; , , - : ) are formed, respectively. these compounds are converted to eicosatetraenoic acid (eta; , , , - : ) and dihomo γ-linolenic acid (dgla; , , - : ) by the elongase , being further converted to eicosapentaenoic acid (epa; , , , , - : ) and arachidonic acid (aa; , , , - : ) , by the addition of a double bond at the ∆ position, through a ∆ desaturase. further elongations convert epa and aa to docosapentaenoic acid ( , , , , - : ) and adrenic acid ( , , , - : ) ; then, a desaturation by ∆ desaturase generates docosahexaenoic acid (dha; , , , , , - : ) [ ] . both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [ ] . the byosynthetic pathways of ω and ω fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω and ω series is maintained. both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [ ] . the byosynthetic pathways of ω and ω fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω and ω series is maintained. figure . biosynthetic pathways of ω- and ω- fatty acids. fatty acids occur widely in nature, being identified in both animal tissue and plants. particularly, short-chain saturated acids are components of milk fats: in bovine milk, butanoic acid along with other scfa and mcfa have been reported [ ] . likewise, the mcfa lauric acid and myristic acid are the major components of the oils obtained from some lauraceae and myristiceae species [ ] . moreover, palmitic acid is the most representative sfa in vegetable oils, such as palm oil [ ] . fatty acids with immune modulating properties mainly belong to the long-chain classes; among them, punicic acid is a peculiar conjugated triene, found to be a unique component of pomegranate seed oil [ ] , while oleic acid is one of the most widely distributed fatty acids: it represents % to % of total fa in olive oil, although it does occur in high amounts in other oils, such as those from grape seeds, canola and sufflower [ , ] . moreover, ω and ω fatty acids have been highlighted in several natural sources, wherein both series co-occur (table ) , although in different amounts [ ] . table . major natural sources of long-chain monounsaturated (mufa) and polyunsaturated fatty acids (pufa) associated with immune system modulating activities and relative amounts. some vegetable oils, including rapseed, hemp seed, and sunflower oils, contain higher levels of la (essential ω pufa), while ala (essential ω pufa) is in lower proportion; a similar trend has also been reported for soybean, corn, and for dried black walnuts and brazilnuts; conversely, higher amounts of ala respect to la are reported in flaxseed oil and in the seeds of chia and perilla [ ] . likewise, green leafy vegetables seem to be an interesting source of ala [ ] . fish oils are also sources of both epa and dha (ω pufa), with lower amounts of dpa (ω pufa) [ ] . wild marine species showed to contain higher ω pufa levels compared to farmed ones, likely due to the feed composition [ ] . some vegetables can supply both the essential pufa and some derivative fatty acids. particularly, the oils obtained from the seeds of borago spp., echium spp., ranunculus spp. and oenothera biennis l. have been reported to contain high levels of both la and γ-linolenic acid (gla) [ , ] . fatty acids play energetic, metabolic, and structural functions, being the main component of phospholipids, triglycerides, diglycerides and monoglycerides. a separate category is represented by scfa, which act as metabolites of carbohydrates, produced by gut microbiota: their role in the modulation of immune function is described in section . . long-chain fatty acids have been found involved in immune modulation, being able to affect both innate and adaptive response. although specific profiles characterize each class of fatty acids, these effects are mainly ascribed to their ability to target the cell membrane, where they can be incorporated, thus changing membrane composition and fluidity and modulating membrane-protein interaction and signal transduction. furthermore, a role in the control of inflammation has been reported. epithelial growth factor receptors (a critical crossroad of multiple receptor pathways which is potentially implicated in the regulation of proliferation and possibly involved in atherogenesis) are considered possible targets for unsaturated fatty acids [ ] . mufa, especially oleic acid, have attracted great attention in the years as possible immunomodulating nutrients. preclinical studies demonstrated the ability of oleic acid to modulate the immune system, through affecting both innate and adaptive immunity response [ ] . indeed, it diminished nk cell activity [ ] and the expression of the leucocyte adhesion molecules, which have shown to be implicated in some pathophysiological conditions, such as rheumatoid arthritis [ ] . furthermore, it enhanced neutrophil aggregation and neutrophil-endothelial cell attachment, phagocytic and candidacidal capacities [ , ] . in regard to adaptive response, it inhibited the proliferation of immune cells, such as jurkat t cells and lymphocytes, likely through the regulation of the cell cycle, although the true mechanisms remain to be clarified [ ] . similar suppressive effects were also highlighted for its synthetic analogue minerval and confirmed in animal models [ , ] . furthermore, the treatment with oleic acid and minerval induced proapoptotic effects in jurkat (t lymphocyte) and raji (b lymphocyte) cells, likely due to mitochondrial depolarization and ros production [ ] [ ] [ ] . recently, oleate has been reported to be able to protect macrophages from palmitate-induced lipotoxicity; moreover, it has been associated with an increase in the regulatory phenotype of the myeloid msc- suppressor cells and suppression of activated t cells [ , ] . in the skin, oleic acid, along with other unsaturated fa, seems to be incorporated into the lipid moiety of staphylococcus aureus lpp, inducing an immune response against the pathogen [ ] . regarding conjugated pufa, punicic acid has been shown to improve the immune system development, stimulate the cd + and cd + lymphocyte-mediated immunity and increase the immune response against viruses [ ] . these immune boosting effects are due to nuclear peroxisome proliferator-activated receptor (ppar)γ-and δ-dependent mechanisms, as punicic acid is able to act as an agonist of these receptors; in support, the loss of pparγ in immune cells impaired its effects [ , ] . moreover, punicic acid inhibited the tnf-α-induced priming of ros production by inhibiting the ser -p phox phosphorylation and upstreaming kinase p mapk; likewise, it blocked the tnf-α-induced release of myeloperoxidase from neutrophils, and decreased neutrophil-activation and ros/mpo-mediated tissue damage in vivo [ ] . antinflammatory properties were found to be related to the activation of pparγ and the suppressed expression of inflammatory genes (encoding cytokines, chemokines, cyclooxygenase, no synthase, and metalloproteinases) [ ] . immunomodulatory properties of ω- and ω- pufa have been highlighted in different preclinical models and have been associated with their ability to modulate the inflammatory process [ ] . these fatty acids share common biosynthetic enzymes which mediate the production of different series of eicosanoids, starting from typical precursors, including dihomo-γ-linoleic acid (dgla), arachidonic acid (aa) and eicosapentaenoic acid (epa). among prostanoids, three types of prostaglandins (pg), including pg , pg and pg , can be obtained. pg is associated with beneficial effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg acts through a mixture of functions and is able to reduce the pg -mediated inflammation [ ] . starting from dgla, both anti-inflammatory pg and pro-inflammatory pg , through the conversion into arachidonic acid, can be produced (figure ) . effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg acts through a mixture of functions and is able to reduce the pg -mediated inflammation [ ] . starting from dgla, both anti-inflammatory pg and pro-inflammatory pg , through the conversion into arachidonic acid, can be produced (figure ) . this synthesis is controlled by the activity of Δ -desaturase and Δ -desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω- fatty acids are able to activate the conversion of dgla into pg , whereas low ω- intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [ ] . aa can also be released by the cell membranes through the action of phospholipase a during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while -lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a and leukotriene b are produced from aa, while tromboxane a and leukotriene b from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [ ] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [ ] . despite the antinflammatory role of ω- and ω- -based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω- with respect to ω- in the diet: a balance between ω- and ω- pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω- and ω- pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [ ] . dietary ω- pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [ ] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [ ] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [ ] . conversely, ω- pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [ ] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules;it also complexed with the anti-inflammatory this synthesis is controlled by the activity of ∆ -desaturase and ∆ -desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω- fatty acids are able to activate the conversion of dgla into pg , whereas low ω- intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [ ] . aa can also be released by the cell membranes through the action of phospholipase a during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while -lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a and leukotriene b are produced from aa, while tromboxane a and leukotriene b from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [ ] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [ ] . despite the antinflammatory role of ω- and ω- -based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω- with respect to ω- in the diet: a balance between ω- and ω- pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω- and ω- pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [ ] . dietary ω- pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [ ] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [ ] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [ ] . conversely, ω- pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [ ] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules; it also complexed with the anti-inflammatory molecule -antitrypsin, thus reducing lps-induced il- secretion in neutrophils [ ] . on the whole, preclinical evidence highlighted that these fatty acids could increase neutrophil function, thus promoting innate immunity. regarding adaptive immunity, ω- pufa have been reported able to improve the mitogen-mediated activation of immune cells and to promote the development of a th -type immune response [ ] . moreover, an increased production of associated anti-inflammatory cytokines like il- , in spite of a reduction of pro-inflammatory tnf-α, was found [ ] . similar effects were highlighted with both fish oil-enriched diets and the purified epa and dha [ ] . the beneficial influence of ω- pufa has been highlighted also on epithelial cells during inflammation, being able to restore impaired barrier function and reduce the production of pro-inflammatory mediators [ ] . moreover, a strictly interplay between omega- fatty acids, immunity and gut microbiota has been reported and seems to be an essential factor to maintain the intestinal wall integrity. these effects have been ascribed to the ability of ω- pufa to positively affect the microbiota composition and increase the production of anti-inflammatory compounds, like short-chain fatty acids [ ] . although a major interest over the years has been focused on marine sources of ω- -enriched oils or on pure compounds, some plant species have been studied for their immunomodulating and anti-inflammatory properties, likely ascribable to ω- and/or ω- pufa, although the major evidence has been highlighted for linum usitatissimun l., oenothera biennis l. and borago officinalis l. [ , , ] . the seed oil from l. usitatissimum, also known as flaxseed oil, has been reported to induce immunomodulating effects, likely through suppressing cell mediated immunity, without the involvement of humoral immunity. being a rich source of ala, its effects are mainly ascribed to this compound, although further studies suggested a possible contribution of bioactive phenolics [ ] . flaxseed oil was found to be effective in reducing skin inflammatory responses, although with a lower immunosuppressive power with respect to fish oil [ ] . moreover, it improved systemic and gut immunity, in a piglet model with intrauterine growth retardation: increased plasma concentration of immunoglobulin g, decreased cd +cd + t lymphocytes, and the downregulation of genes expression for proinflammatory factors have been reported [ ] . regarding o. biennis, the administration of the seed oil (namely, evening primrose oil) in animal models enhanced pge synthesis in peritoneal macrophages, decreased pge amounts in granulocytes, and suppressed the natural killer (nk) cell activity and lymphocyte proliferation; moreover, it decreased the serum levels of interferon γ (ifn-γ) and mcp- , while stimulating tnf-α [ ] [ ] [ ] [ ] [ ] . furthermore, anti-inflammatory effects have been found to be involved in the immunomodulation by evening primrose oil [ ] . these effects were ascribed to the content of gla, whose t-regulatory cell activity in autoimmune disease models was highlighted [ ] . however, a contribution of la to the antinflammatory effects seems to be likely; indeed, la can itself modulate inflammation as it is metabolized by lox to hydroxyoctadecadienoic acids (hodes) and oxo-hodes, characterized by antinflammatory properties [ ] . similarly, the seed oil from b. officinalis seeds produced immonomodulating and antinflammatory effects, likely through its gla content [ ] . a chemotactic migration of monocytes to necrotic site that differentiate into macrophages is associated with the administration of this product. moreover, it is known to reduce the levels of proinflammatory cytokines, such as tnf-α, and to promote pge generation; a reduced expression of inflammatory genes, especially those of macrophages involved in atherosclerosis, has been reported too [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . clinical studies mainly focused on the effects of fatty acid-enriched diet on inflammation, although specific immune-based pathological conditions associated with inflammation were assessed too. regarding mufa, few studies are available, and results differ from those in animal models. indeed, a mufa-rich diet (with highly refined olive oil for weeks) did not alter the immune function in healthy subjects; such effects could be due to the high amounts administered in animal models [ ] . conversely, clinical evidence about the immunomodulatory power of punicic acid in healthy or sick subjects is lacking [ ] . the ω- pufa series and the relative enriched fish oils have been mainly evaluated for their immunomodulating and antinflammatory effects in humans. although preclinical evidence highlighted their ability to influence both innate and adaptive immunity, the clinical relevance of these results remains to be clarified, due to lacking or inconclusive data [ ] . inadequacy of clinical results should be due to the different doses used in preclinical studies, wherein often high fatty acids levels were administered; moreover, other factors such as genetic and epigenetic heterogeneity of the recruited subjects, diet diversity, nutritional habits and microbiome can be considered as additional confounding factors [ ] . although limitations of clinical studies require further confirmation, ω- pufa intake produced significant clinical benefits and reduction of the symptoms in patients with autoimmune disorders, especially rheumatic diseases and systemic lupus erythematosus [ ] [ ] [ ] . in support, low levels of pufas have been found in the serum of patients with rheumatic diseases [ ] . conversely, inconsistent results are reported for multiple sclerosis, thus the possible usefulness of these fatty acids as supportive therapy requires more clinical trials [ ] . regarding ω- pufa, although they are associated with possible increased inflammatory conditions, being arachidonic acid a presursor of proinflammatory prostanoids, such a risk is not confirmed by clinical evidence [ ] . indeed, studies in healthy human adults highlighted that an increased intake of these fatty acids did not induce inflammation; conversely, epidemiological evidence reported reduced inflammatory conditions [ ] . the antinflammatory effects of la have been reported too [ ] . similarly, increased la intake was found to be not related to increased amounts of ara and proinflammatory factors; however, an inverse correlation with epa and dha was reported [ ] . this suggests that the interaction between ω-e and ω- series is regulated by complex mechanisms that requires further clarifications. major clinical studies have been performed using evening primrose oil (from the seeds of o. biennis), as a source of la and gla, in inflammatory diseases associated with immune system disorders, including atopic dermatitis, psoriasis, multiple sclerosis and rheumatoid arthritis. standardized oils for the content in la and ala (for instance, efamol is titred to contain % la and % gla) were usually used [ ] . the treatment with evening primrose oil ( and weeks) produced clinical improvements in patients with atopic dermatitis, as revealed by measuring the scoring atopic dermatitis [ ] . some beneficial effects were also reported in multiple schlerosis patients, although the few available studies limited the evidence in this disorder. conversely, evening primrose oil in combination with fish oil and vitamin e (efamol marine) failed to improve the symptoms of psoriasiac patients but produced antinflammatory effects [ ] . similarly, in association with ω- fatty acids, it did not induce improvements in patients with rheumatoid arthritis [ ] . a cochrane revision highlighted moderate evidence for oils containing gla (i.e., evening primrose, borage, or blackcurrant seed oil) to produce benefit in rheumatoid arthritis [ , ] . evening primrose oil along with borage oil were not effective to treat eczema too [ ] . highly variable results were also obtained for borage oil in the treatment of atopic dermatitis, although, in all the studies, a moderate efficacy degree was displayed [ ] . regarding flaxseed oils, some clinical trials higlighted a significant improvement of inflammatory parameters in subjects with cardiovascular diseases non-associated with the immune system [ ] . reported studies, although performed in pathological conditions associated with immune system disfunction, did not give a direct measure of the immunomodulatory effects of the treatments. furthermore, specific and high-quality studies are required for better characterizing the possible usefuleness of these pufa-enriched oils as anti-inflammatory and immunomodulating treatments. labdane compounds have a molecular formula c h with an average mass of . da ( figure ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [ ] . labdane compounds have a molecular formula c h with an average mass of . da ( figure ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [ ] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure ) (from andrographis paniculata (burm.f.) nees) [ ] , labda- ( ), -diene- , -dial (from curcuma amada roxb) [ ] , podoimbricatin c (a , -cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure ) (from andrographis paniculata (burm.f.) nees) [ ] , labda- ( ), -diene- , -dial (from curcuma amada roxb) [ ] , podoimbricatin c (a , -cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . figure . chemical structure of andrographolide. this has been obtained using chemspider ® chemical structure database. the body's defense responses can be improved through various properties induced by plants. some of them, referring to the plants in table , are shown below. for each plants and plant-derived nutraceutical, only properties potentially attributable to the labdane skeleton and useful to improve the immune system are reported. as both inflammation (biological response of body tissues to harmful stimuli) and oxidative stress (imbalance between reactive oxygen species and a biological system's ability to detoxify/repair the resulting damage of the reactive intermediates) are the main self-defend methods to eliminate pathogens and protect living bodies, plants with antinflammatory and/or radical scavenger properties are considered too [ ] . indeed, labdane diterpenoids have recently gained greater attention from the scientific point of view, due to a wide range of biological activities, including the anti-inflammatory modulation of immune cell functions [ ] . a. paniculata exhibited, in vitro and in vivo, various pharmacological activities, including antihyperglycemic, antiplatelet aggregation, anti-microbial, anti-inflammatory, anti-hiv, anti-cancer, anti-nociceptive activity, etc. it has also been used for autoimmune encephalomyelitis and, in indian and chinese medicine, for respiratory tract infections [ , ] . more recently, a. paniculata has been used to stimulate the immune system and treat myocardial ischemia [ ] . a. paniculata inhibited interleukin (il)- , tnf-α mrna, lps-induced expression, and suppressed levels of tnf-α, il- β, jnk, c-reactive protein, and nf-κb [ ] . many labdane diterpenoids compounds have been found to act on the latter. the activation of the nf-κb pathway leads to several physiological responses, including inflammatory or innate immune response [ ] . in vitro, andrographolide (the main phytoconstituent of a. paniculata) can inhibit inflammation, by regulating protein expression (cytokines, chemokines) and by reducing immune cell infiltration. andrographolide was shown to inhibit also oxidative stress by binding to adenosine a a receptor, by inducing nuclear factor (erthroid-derived )-like (nrf ) translocation, and by increasing the expression of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase- [ ] . these effects can contribute to the immunoregulatory activity of this plant-derived nutraceutical, as it can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and antibody productions [ ] . moreover, it was found to exert cytotoxic/anticancer effects on almost all types of tumour cell lines (human leukemia, renal tubular epithelial cells, breast cancer cells, etc.), mainly by cell cycle arrest, autophagy, cell death, anti-inflammatory and immune system mediated effects [ ] . other preclinical studies have highlighted pharmacological properties of labdane diterpenoidscontaining plants. data are limited, and consequently also their preclinical evidence. some examples are reported below. c. amada, also known as mango ginger, and its labdane-diterpenoids have shown antiflammatory, antibacterial, insecticidal, antifungal, antipyretic, antioxidant, anticancer, and antitubercolar properties in preclinical trials [ ] . d. imbricatus displayed cytotoxic and anti-neuroinflammatory activities, but it had no cytotoxic activity against human tumour cell lines [ ] . e. macrophyllus, brazilian plant, also known as "leather hat", is used as a methanolic (which contains mainly labane diterpenoids, steroids, alcaloids, etc.) or aqueous extract (rich in flavonoids) of the aerial parts, leaves in particular. in folk medicine, e. macrophyllus is used for various illnesses (respiratoy and urinary diseases, rheumatoid arthritis, atherosclerosis, etc.), as it has been shown to possess tissues protective activity and immunosuppressive effects (impaired secretion and function of b and/or t cells), on humoral or cellular immune responses and on autoimmune rheumatic diseases [ ] . in in vitro/vivo studies, the aqueous extract of e. macrophyllus exhibited strong antinflammatory activities by decreasing rats paw edema, inflammatory exudates, infiltrate tissues, no production, ltb release, and neutrophil migration [ ] . in preclinical studies, the methanolic extract of e. macrophyllus was not cytotoxic, genotoxic, mutagenic, and no acute toxicity (up to the maximum dose of mg/kg b.w.) has been observed in tested animals [ ] . however, the extrapolation of animal experiments to clinical practice must be done with caution [ ] . compounds from j. oblonga have shown anti-tumor effects, through moderate cytotoxicity against human tumor cell lines obtained from various human tissues, including: hepatocellular carcinoma (hepg ), breast cancer (mcf- ), and cervical carcinoma cancer (hela) [ ] . the berries from j. oblonga also have antimicrobial activity and anti-inflammatory effects. labdane diterpenoid (e.g., leonurine), extracted from l. japonicus, exhibited cytotoxicity and cell cicle arrest against cancer cell lines and presented immunomodulatory and antinflammatory activities (suppresses tnf-α, nf-κb, and down-regulated expression of inos, cox , and conseguently peg and no levels) [ ] . marrubium spp. (aschersonii, globosum, etc.) have multiple actions, including antimicrobial and anti-inflammatory activities. marrubasch a-f and marrubenol, isolated from the ethanolic extract of m. aschersonii, exhibited weak reduction in inos activity and, consequentely, no production [ ] . marrulibanoside, obtained from the aerial parts of m. globosum, inhibited catalytic activity of inos and cox- enzymes, and consequentely, the peg and no production. v. limonifolia, in preclinical trial, have shown a strong antiviral activity against coxsackievirus b , human rhinovirus b, and enterovirus (ev ). all of them could be responsible for various illnesses, ranging from common cold, hand, foot, and mouth diseases, to acute flaccid paralysis [ ] . the clinical efficacy of the medicinal plants, and plant-derived nutraceuticals discussed above are almost totally lacking. only for a. paniculata there are some evidence in humans. andrographis extract (various and not standardized), andrographolide, and its derivatives have been studied in the treatment of various disease (multiple sclerosis, infection disease, gastro-intestinal upsets, respiratory ailments, pain), and in the maintenance of immune function. in this last context, it seemed to improve the response to cough and sore throat, shortening the sick leave/time to resolution [ ] . the most interesting activity is the increase of cd + lymphocyte levels, in hiv-positive patients [ ] . the increase in these lymphocytes testifies an improvement in the state of the immune system. moreover, a chinese product containing andrographolide improved the efficacy of glucocorticoids and immunoglobulin in patients with severe hand, food, and mouth disease. however, andrographolide is considered a hazard, as it is irritating, and its injectable use is limited because it could induce allergic reactions (erythema, pruritus, etc.), which are sometimes life-threatening [ ] . preclinical data suggested that andrographolide could be responsible of pharmacokinetics interactions, as it induced cyp a [ ] . the european medicines agency (ema) reports a possibility of causing reproductive toxicity of andrographis extracts (decreases in sperm motility and counts) [ ] . on the other hand, no major adverse effects have been reported for a. paniculata; only minor side effects, mainly gastrointestinal, are known [ ] . notably, even if a. paniculata presents numerous pharmacological properties, andrographolide possess poor solubility (principally in dmso), which severely limits the possibility of achieving a therapeutic effect (if not properly formulated). its better absorption could be achieved by nano-formulations (e.g., nano-emulsion, nano-capsules). immunomodulation by plant-based nutraceuticals represents an interesting tool to be exploited for the treatment and preventing purposes of immune system disorders, due to their multiple bioactivities, well tolerability and good patient compliance. however, as often reported for several herbal medicinal products, some points require being underlined to improve the research in the field and provide solid evidence to support their rational use. according to previous stated critical issues [ , ] , herbal products under study must be characterized for the phytochemical composition, using validated analytical methodologies, and for the extraction procedures; moreover, the starting material should be fully defined in terms of origin (country and region), cultivation conditions, botanical identity and plant part. the content of specific compounds, used as analytical or active markers, should be determined too. these requirements are needed to ensure reproducible pharmacological/clinical activity and to compare different studies. indeed, using nonstandardized phytocomplexes increases variability of the biological response, thus limiting the reliability and validity of the studies. furthermore, to assess the pharmacological activity of specific compounds, purity (at least %) and identity should be characterized. indeed, when assessed as mixtures, the subtle interactions which can be established among phytochemicals make it difficult to understand whether the observed benefits are attributable to a specific class or to the whole phytocomplex. for instance, both fatty acids and polyphenols can be involved in the immunomodulating effects of pufa-enriched plant oils. moreover, as found for both polysaccharides and fatty acids, among the same class, different subclasses can co-occur, thus contributing to the whole effects. regarding preclinical studies, detailed methodologies, including information about specific extraction process, the choice of the tested concentrations and experimental procedures, vehicle effects, and comparison with standard effective compounds (positive controls) should be reported. in order to validate the "goodness" of the treatment, promising results in preclinical studies should be confirmed by clinical evidence of efficacy and lack of toxicological concerns for both the isolated compounds and the whole phytocomplex. at last, possible interactions with diet constituents or possible pharmacological treatments, as reported for andrographolide, which is a cyp a inducer, should be considered. as highlighted for a number of natural products, clinical evidence is a major challenge for plant-based immunomodulating nutraceuticals too, due to limited specific studies. moreover, methodological quality of the available trials was overall poor, the studies often being not blinded, protocol unavailable and lacking the standardization of tested products, thus making the claimed effect difficult to be reproduced. at last, standardized methodologies for systematic reviews and meta-analyses, such as the prisma guidelines [ ] , would allow a rational interpretation of the results and suggestions for future research. medicinal plants are rich sources of bioactive phytochemicals, characterized by multiple and often pleiotropic activities, which can be exploited both therapeutically and as nutraceutical strategies for preventive purposes. among plant-based nutraceuticals, immunomodulators have been highlighted to be of interest as boosters of the immune system, to counteract infectious or exogenous injuries, immunosuppressor, to control the abnormal immune response occurring during autoimmune diseases, or as adjuvants, which contribute by modulating nonimmune targets. in this review, we highlighted the scientific evidence about the immunomodulating properties of three emerging classes of nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes. some of them, especially polysaccharides and labdane diterpenes, act as immune system booster, while fatty acids (mufa and pufa) mainly act as immunosuppressor, although punicic acid (a conjugate pufa) exhibited immunostimulant properties. to date, these products are mainly used as well-tolerated food supplements, although the clinical evidence about their modulation of the immune functions is still limited. more investigations of better quality and specificity could strengthen the validity of using these plant-derived nutraceuticals in the immune system modulation. immune function and micronutrient requirements change over the life course key concepts in immunology drivers and regulators of humoral innate immune responses to infection and cancer developing phytocompounds from medicinal plants as immunomodulators immunomodulators in sle: clinical evidence and immunologic actions update on the role of systemic vitamin d in atopic dermatitis trained immunity: a tool for reducing susceptibility to and the severity of sars-cov- infection vaccine adjuvants: from to and beyond vaccine adjuvants: putting innate immunity to work the role of adjuvant immunomodulatory agents for treatment of severe influenza vaccine adjuvants as potential cancer immunotherapeutics immunotherapy of cancer. in innate immune regulation and cancer immunotherapy naturally occurring immunomodulators with antitumor activity: an insight on their mechanisms of action interaction between microbiota and immunity in health and disease microbiome and the immune system: from a healthy steady-state to allergy associated disruption changing scenario for promotion and development of ayurveda-way forward echinacea purpurea extract affects the immune system, global metabolome, and gut microbiome in wistar rats metabolome and gut microbiota variation with long-term intake of panax ginseng extracts on rats polysaccharides from the flowers of tea (camellia sinensis l.) modulate gut health and ameliorate cyclophosphamide-induced immunosuppression anti-inflammatory, and chemoprotective properties of acacia catechu heartwood extracts isolation, structure elucidation, antioxidative and immunomodulatory properties of two novel dihydrocoumarins from aloe vera extraction, purification, structural characteristics, biological activities and pharmacological applications of acemannan, a polysaccharide from aloe vera: a review characterization of aloeride, a new high-molecular-weight polysaccharide from aloe vera with potent immunostimulatory activity immunomodulatory activity of andrographolide on macrophage activation and specific antibody response artocarpus tonkinensis protects mice against collagen-induced arthritis and decreases th cell function a review of the pharmacological action of astragalus polysaccharide immunomodulatory activity of boswellic acids (pentacyclic triterpene acids) from boswellia serrata the immunomodulatory effect of green tea (camellia sinensis) leaves extract on immunocompromised wistar rats infected by candida albicans pharmacological review on centella asiatica: a potential herbal cure-all. ind immune modulation by curcumin: the role of interleukin- promotion of regulatory t cell induction by immunomodulatory herbal medicine licorice and its two constituents african potato (hypoxis hemerocallidea): a systematic review of its chemistry, pharmacology and ethno medicinal properties efficacy of ocimum basilicum for immunomodulatory activity in wistar albino rat ginseng: a dietary supplement as immune-modulator in various diseases potential of syzygium aromaticum (clove) leaf extract on immune proliferation response in balb/c mice infected with salmonella typhimurium tinospora species: an overview of their modulating effects on the immune system network ethnopharmacological evaluation of the immunomodulatory activity of withania somnifera withania somnifera (linn.) dunal: a review of chemical and pharmacological diversity some phytochemical, pharmacological and toxicological properties of ginger (zingiber officinale roscoe): a review of recent research immunomodulators inspired by use of quantitative flow cytometry to measure ex vivo immunostimulant activity of echinacea: the case for polysaccharides immunopotentiating significance of conventionally used plant adaptogens as modulators in biochemical and molecular signalling pathways in cell mediated processes th /th cytokine production by clove-treated mice azadirachta indica a. juss. (meliaceae). in handbook of medicinal plants herbal phytochemicals as immunomodulators the influence of dietary fatty acids on immune responses aristolochic acid in the etiology of renal cell carcinoma plant-derived immunomodulators: an insight on their preclinical evaluation and clinical trials health benefits and molecular mechanisms of resveratrol: a narrative review chicoric acid binds to two sites and decreases the activity of the yoph bacterial virulence factor a phase study to assess the immunomodulatory capacity of a lecithin-based delivery system of curcumin in endometrial cancer codex-aligned dietary fiber definitions help to bridge the 'fiber gap' structure-function relationships of immunostimulatory polysaccharides: a review mannans: an overview of properties and application in food products structural characterization of immunostimulating polysaccharide from cultured mycelia of cordyceps militaris immunostimulatory properties of coffee mannans characterization and immunostimulating activity of a water-soluble polysaccharide isolated from haematococcus lacustris in vivo anti influenza virus activity of an immunomodulatory acidic polysaccharide isolated from cordyceps militaris grown on germinated soybeans fractionation and characterization of biologically-active polysaccharides from artemisia tripartita polypotency of the immunomodulatory effect of pectins separation, structure characterization, conformation and immunomodulating effect of a hyperbranched heteroglycan from radix astragali structure elucidation and immunological activity of a novel pectic polysaccharide from the stems of avicennia marina carrageenan from solieria chordalis (gigartinales): structural analysis and immunological activities of the low molecular weight fractions immunomodulation effect of sulphated polysaccharide (porphyran) from porphyra vietnamensis natural glycans and glycoconjugates as immunomodulating agents fructooligosaccharides and fructans analysis in plants and food crops from radix ophiopogonis, stimulates the proliferation of cultured lymphocytes: structural and functional analyses studies on wheat bran arabinoxylan for its immunostimulatory and protective effects against avian coccidiosis comparison of the immunological activities of arabinoxylans from wheat bran with alkali and xylanase-aided extraction epiphanies of well-known and newly discovered macromolecular carbohydrates-a review botanical polysaccharides: macrophage immunomodulation and therapeutic potential peas in a pod isolation and structural characterization of an immunostimulating polysaccharide from fuzi, aconitum carmichaeli isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract mass spectrometry characterization of an aloe vera mannan presentin immunostimulatory activity sustainable food packaging materials based on future biorefinery products: xylans and mannans induction of secretory and tumoricidal activities in peritoneal macrophages activated by an acidic heteropolysaccharide (aragal) from the gum of anadenanthera colubrina (angico branco) linear mannan in the endosperm of schizolobium amazonicum immunological activities and structure of pectin from centella asiatica hydrolysis of isolated coffee mannan and coffee extract by mannanases of sclerotium rolfsii mannans as stabilizers of oil-in-water beverage emulsions structure identification of a new immunostimulating polysaccharide from the stems of dendrobium huoshanense macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of echinacea purpurea immunologically active polysaccharides of echinacea purpurea cell cultures polysaccharides isolated from acaí fruit induce innate immune responses nasal acai polysaccharides potentiate innate immunity to protect against pulmonary francisella tularensis and burkholderia pseudomallei infections immunomodulatory activity of dietary fiber: arabinoxylan and mixed-linked beta-glucan isolated from barley show modest activities in vitro characterization and immunostimulatory activity of an ( → )-α-d-glucan from the root of ipomoea batatas macrophage immunomodulatory activity of polysaccharides isolated from juniperus scopolorum studies on chemistry and immunomodulating mechanism of aglycoconjugate from lycium barbarum l structural characterization and immunostimulatory activity of a novel linear α-( → )-d-glucan isolated from panax ginseng c. a. meyer spruce-derived mannans-a potential raw material for hydrocolloids and novel advanced natural materials purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of prunus dulcis seeds physicochemical properties and structural characterization of a galactomannan from sophora alopecuroides l. seeds immunomodulatory activity of acidic polysaccharides isolated from tanacetum vulgare l immune stimulating properties of a novel polysaccharide from the medicinal plant tinospora cordifolia effect of fenugreek (trigonella foenum-graecum l.) galactomannan fractions on phagocytosis in rat macrophages and on proliferation and igm secretion in hb c cells advances in research on immunoregulation of macrophages by plant polysaccharides study on immunomodulatory effect of astragalus polysaccharide on mice peritoneal macrophage macrophage activation by polysaccharide isolated from astragalus membranaceus effects of dietary astragalus polysaccharides (aps) on growth performance, immunological parameters, digestive enzymes, and intestinal morphology of nile tilapia (oreochromis niloticus) astragalus membranaceus extract activates immune response in macrophages via heparanase molecular mechanisms of immunomodulatory activity by polysaccharide isolated from the peels of citrus unshiu dietary fiber pectin directly blocks toll-like receptor - and prevents doxorubicin-induced ileitis microbiota-dependent and -independent effects of dietary fibre on human health echinacea purpurea extracts promote murine dendritic cell maturation by activation of jnk, p mapk and nf-κb pathways extraction, characterization of astragalus polysaccharides and its immune modulating activities in rats with gastric cancer enhancement of astragalus polysaccharide on the immune responses in pigs inoculated with foot-and-mouth disease virus vaccine effect of radix astragali drug serum on nk cell activity and expression of klrk antitumor andimmunomodulatory activity of arabinoxylans: a major constituent of wheatbran enhancement of natural killer cell activity of agedmice by modified arabinoxylan rice bran (mgn- /biobran) a study on the immune receptors for polysaccharides from the roots of astragalus membranaceus, a chinese medicinal herb effects of astragalus polysaccharide liposome on lymphocyte proliferation in vitro and adjuvanticity in vivo mixing ratio optimization for functional complex extracts of rhodiola crenulata, panax quinquefolius, and astragalus membranaceus using mixture design and verification of immune functional efficacy in animal models supplementation with astragalus polysaccharides alters aeromonas-induced tissue-specific cellular immune response beta-glucans, history, and the present: immunomodulatory aspects and mechanisms of action vaccine adjuvants revisited advaxtm, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses a novel hepatitis b vaccine containing advaxtm, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing astragalus polysaccharides enhance immune responses of hbv dna vaccination via promoting the dendritic cell maturation and suppressing treg frequency in mice up to -year clinical follow-up of a pilot phase iii immunotherapy study in stage ii breast cancer patients using oxidized mannan-muc adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations short-chain fatty acids: bacterial messengers modulating the immunometabolism of t cells from dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites butyrate inhibits inflammatory responses through nfkappab inhibition: implications for crohn's disease shortchain fatty acids activate gpr and gpr on intestinal epithelial cells to promote inflammatory responses in mice addition of astragalus into peritoneal dialysate improve peritoneal macrophages function in capd patients comparison of perioperative immune function status in colorectal cancer patients and immune regulatory function of drugs influences of astragalus injection on clinical efficacy and cellular immune function in patients of bronchial asthma with acute exacerbation effect of oral administration of freshly pressed juice of echinacea purpurea on the number of various subpopulations of b-and t-lymphocytes in healthy volunteers: results of a double-blind, placebo-controlled cross-over study echinaforce and other echinacea fresh plant preparations in the treatment of the common cold. a randomized, placebo controlled, double-blind clinical trial echinacea for preventing and treating the common cold clinical trials of yeast-derived beta-( , ) glucan in children: effects on innate immunity beta-glucan affects mucosal immunity in children with chronic respiratory problems under physical stress: clinical trials placebo-driven clinical trials of yeast-derived beta-( - ) glucan in children with chronic respiratory problems resveratrol plus carboxymethyl-β-glucan in children with recurrent respiratory infections: a preliminary and real-life experience resveratrol plus carboxymethyl-β-glucan in infants with common cold: a randomized double-blind trial resveratrol plus carboxymethyl-β-glucan reduces nasal symptoms in children with pollen-induced allergic rhinitis update of the lipid maps comprehensive classification system for lipids fatty acids in foods and their health implications short-chain fatty acids, and herbal medicines mechanisms of action of trans fatty acids polyunsaturated fatty acid biosynthesis and metabolism in adult mammals comparison of bovine milk fat and vegetable fat for infant formula: implications for infant health laurie oil resources palm oil and palmitic acid: a review on cardiovascular effects and carcinogenicity health benefits of punicic acid: a review biological activities of phenolic compounds of extra virgin olive oil oleic acid and inhibition of glucosyltransferase omega- and omega- polyunsaturated fatty acids: dietary sources, metabolism, and significance-a review evaluation of oil content and fatty acid composition in the seed of grapevine varieties modification of fatty acid profiles of rapeseed (brassica napus l.) oil for using as food, industrial feed-stock and biodiesel bitter gourd (momordica charantia l.) seed oil as a naturally rich source of bioactive compounds for nutraceutical purposes flaxseed: its bioactive components and their cardiovascular benefits health effects of omega- , , fatty acids: perilla frutescens is a good example of plant oils fatty acid profiles and sn - fatty acid distribution of γ-linolenic acid-rich borago species the seed of industrial hemp (cannabis sativa l.): nutritional quality and potential functionality for human health and nutrition characterization of gamma-linolenic acid in ribes seed metabolite profiling of green, green/red, and red lettuce cultivars: variation in health beneficial compounds and antioxidant potential monounsaturated fatty acids and immune function effects of variations in the proportions of saturated, monounsaturated and polyunsaturated fatty acids in the rat diet on spleen lymphocyte functions oleic acid increases cell surface expression and activity of cd b on human neutrophils neutrophil migration inhibitory properties of polyunsaturated fatty acids. the role of fatty acid structure, metabolism, and possible second messenger systems minerval induces apoptosis in jurkat and other cancer cells comparative toxicity of oleic acid and linoleic acid on jurkat cells comparative toxicity of oleic acid and linoleic acid on raji cells oleate protects macrophages from palmitate-induced apoptosis through the downregulation of cd expression oleate but not stearate induces the regulatory phenotype of myeloid suppressor cells skin-specific unsaturated fatty acids boost the staphylococcus aureus innate immune response preventive and prophylactic mechanisms of action of pomegranate bioactive constituents modulation of peroxisome proliferator-activated receptor gamma(ppar γ) by conjugated fatty acid in obesity and inflammatory bowel disease punicic acid a conjugated linolenic acid inhibits tnfalpha-induced neutrophil hyperactivation and protects from experimental colon inflammation in rats role of omega- fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence polyunsaturated fatty acids and inflammation long-chain polyunsaturated fatty acids regulation of ppars, signaling: relationship to tissue development and aging dietary bioactive fatty acids as modulators of immune function: implications on human health impact of omega- fatty acids on the gut microbiota linseed essential oil-source of lipids as active ingredients for pharmaceuticals and nutraceuticals the chemical composition, botanical characteristic and biological activities of borago officinalis: a review. asian pac immunomodulatory activity of ether insoluble phenolic components of n-butanol fraction (epc-bf) of flaxseed in rat dietary fish oil and flaxseed oil suppress inflammation and immunity in cats flaxseed oil supplementation improves intestinal function and immunity, associated with altered intestinal microbiome and fatty acid profile in pigs with intrauterine growth retardation constituents in evening primrose oil with radical scavenging, cyclooxygenase, and neutrophil elastase inhibitory activities effects of different dietary oils on inflammatory mediator generation and fatty acid composition in rat neutrophils inhibition of natural killer cell activity by dietary lipids cytokine levels affected by gamma-linolenic acid the effects of fatty acids on lymphocyte functions fatty acids, the immune response, and autoimmunity: a question of n- essentiality and the balance between n- and n- hydroxyoctadecadienoic acids: oxidised derivatives of linoleic acid and their role in inflammation associated with metabolic syndrome and cancer botanical oils enriched in n- and n- fads products are equally effective in preventing atherosclerosis and fatty liver fish oil in recent onset rheumatoid arthritis: a randomised, double-blind controlled trial within algorithm-based drug use anti-inflammatory diet in rheumatoid arthritis (adira)-a randomized, controlled crossover trial indicating effects on disease activity omega- polyunsaturated fatty acids in critical illness: anti-inflammatory, proresolving, or both? the association between n- polyunsaturated fatty acid levels in erythrocytes and the risk of rheumatoid arthritis in korean women the role of diet in multiple sclerosis: mechanistic connections and current evidence omega- fatty acids and inflammation habitual dietary intake of n- and n- fatty acids in relation to inflammatory markers among us men and women effects of linoleic and gamma-linolenic acids (efamol evening primrose oil) on fatty acid-binding proteins of rat liver gamma-linolenic acid levels correlate with clinical efficacy of evening primrose oil in patients with atopic dermatitis a double-blind placebo controlled trial of efamol marine on skin and joint symptoms of psoriatic arthritis influence of different supplementation on platelet aggregation in patients with rheumatoid arthritis herbal therapy for treating rheumatoid arthritis oral evening primrose oil and borage oil for eczema borage oil in the treatment of atopic dermatitis lipid and blood pressure meta-analysis collaboration (lbpmc) group. a systematic review and meta-analysis of clinical trials investigating the effects of flaxseed supplementation on plasma c-reactive protein concentrations two rings in them all: the labdane-related diterpenoids overview of pharmacological activities of andrographis paniculata and its major compound andrographolide a bioactive labdane diterpenoid from curcuma amada and its semisynthetic analogues as antitubercular agents a new , -cyclo-labdane diterpenoid from the twigs of dacrycarpus imbricatus chapecoderins a-c, new labdane-derived diterpenoids from echinodorus macrophyllus labdane and abietane diterpenoids from juniperus oblonga and their cytotoxic activity phytochemistry and pharmacology of the genus leonurus: the herb to benefit the mothers and more labdane diterpenoids as potential anti-inflammatory agents labdane-type diterpenoids from vitex limonifolia and their antivirus activities andrographis paniculata (chuān xīn lián) for symptomatic relief of acute respiratory tract infections in adults and children: a systematic review and meta-analysis andrographolide, a diterpene lactone from andrographis paniculata and its therapeutic promises in cancer evaluation of the toxicokinetics and apoptotic potential of ethanol extract from echinodorus macrophyllus leaves in vivo flavonoid-enriched fraction from echinodorus macrophyllus aqueous extract exhibits high in-vitro and in-vivo anti-inflammatory activity immunosuppressive effects of echinodorus macrophyllus aqueous extract best practice in research-overcoming common challenges in phytopharmacological research a practical guide for transparent reporting of research on natural products in the british journal of pharmacology: reproducibility of natural product research preferred reporting items for systematic reviews and meta-analyses: the prisma statement this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license a.d.s. and s.d.g. fellowships were funded by grants from sapienza university (ateneo ) and regione lazio. the authors thank "enrico and enrica sovena" foundation (italy) for supporting the study. the authors declare no conflict of interest. key: cord- -apr oyqa authors: rosselló, jaume; becken, susanne; santana-gallego, maria title: the effects of natural disasters on international tourism: a global analysis date: - - journal: tour manag doi: . /j.tourman. . sha: doc_id: cord_uid: apr oyqa tourism is shaped by a wide range of factors and forces, including exogenous ones that have no direct link with the tourism sector. natural disasters and unexpected events are prime examples of such determining factors, as they have profound effects on individuals and society, and as a result have the potential to affect tourism flows considerably. several theoretical arguments exist why natural disasters and unexpected events could influence tourist destination choices. however, empirical research to confirm the nature and extent of impacts of disasters on tourism is lacking. to address this gap, this paper incorporates a dataset on natural and man-made disaster events into a model of international tourism flows to evaluate the effect of different types of disasters on international arrivals at the national level. findings provide evidence that the occurrence of different types of event change tourist flows to varying degrees. although in some cases a positive effect is estimated, in general the impacts are negative, resulting in reduced tourist arrivals following an event. understanding the relationship between disaster events and tourism is helpful for destination managers who make critical decisions in relation to recovery, reconstruction and marketing. earthquakes, tsunamis, floods, bush fires, hurricanes, droughts and heatwaves have always occurred. these events have formed part of the wider 'riskscape' that humans have learned to manage and live with. however, more recently the impacts of disasters have increased substantially, partly because of the exacerbating effects of climate change, but also due to the growing complexity of socio-ecological systems in a highly connected and globalized world (becken, mahon, rennie, & shakeela, ) . for instance, the year recorded a series of hurricanes (harvey, irma and maria) in the caribbean and a severe earthquake in mexico, amongst other events, and these resulted in the highest incurred losses ever recorded (us$ billion) (munich, ) . disasters constitute abrupt changes that shock the system in which tourism is embedded (shondell miller, ) . the nature and extent of impacts depend on the type of shock and the resilience of the affected system (oecd, ) . most disasters have profound impacts on individuals, organizations and communities, and consequently on tourism activities. the repercussions of a disaster are likely to affect tourism directly at a destination country, but indirect consequences for travel to and from the affected region are also conceivable (jin, qu & bap. ; ruan, quan & liu, ) . understanding, managing and responding to these risks, therefore, has to be an integral component of sustainable tourism management (shakeela & becken, ) . consequently, it is not surprising that the topic of risk management and disaster mitigation is attracting increasing attention in tourism research. an emerging body of literature has provided both theoretical and empirical insights into multiple aspects of disasters and tourism. research to date has largely focused on crisis management and disaster risk reduction (becken & hughey, ; faulkner, ; ritchie, ) . in particular, academics and practitioners have been interested in how sustainable development and marketing strategies should include plans to prepare, protect and rebuild a destination after a disaster, both in terms of physical assets and destination image (aljerf & choukaife, ; okuyama, ) . the perceptions of safety is an important aspect of destination image, and different types of risks and events have been studied in the context of visitor travel information seeking and decision making (sharifpour, walters, ritchie, & winter, ; trumbo et al., ; williams & bal� a� z, ) . re-establishing public perceptions of safety and attractiveness following a disaster is crucial to attract and reassure potential visitors to travel to the destination and, by doing so, assisting the affected area to regain functionality and economic recovery (wttc, ) . in addition to understanding visitor perceptions, it has been found that addressing risk perceptions and behaviours of relevant tourism stakeholder is critical for effective disaster response and recovery (kozak, crotts, & law, ; park & reisinger, ) . tourism is exposed and vulnerable to multiple types of hazards (becken, zammit, & hendrikx, ) , and disasters have the potential to deter visitors from travelling to affected destinations (bhati, upadhayaya, & sharma, ) . however, empirical research that confirms or quantifies the relationship between disasters and tourism activity is scant. existing studies have taken a case study approach (e.g. for chinese outbound tourism, see jin, qu, & bao, ) , but a global analysis is missing (ghaderi, mat som, & henderson, ; j� onsd� ottir, ; mazzocchi & montini, ; ruci� nska & lechowicz, ) . it is therefore timely to undertake a global study that uses a consistent approach to measuring the impact of disasters on international tourism movements. to increase the value of such a study for tourism managers, it needs to be designed in a way that includes a wide range of disaster types and magnitudes in the same model (ghimire, ) . consequently, the aim of this research is to explore the effect of various types of natural and man-made disasters on international tourism movements. to that end, this research integrates two different global datasets, namely one on disasters and another one on bilateral international tourist flows. a gravity model for international tourism flows is defined to quantify the effects of different disaster events on international tourist arrivals to the affected country. more precisely, we analyze the impact of droughts, earthquakes (ground movements and tsunamis), epidemics, cold and heat waves, floods, industrial accidents, landslides, wildfires, storms and volcanic activities. moreover, we use three different proxies to measure the impact of disasters; namely the number of deaths, affected people and economic costs. results will support the tourism sector and other key players (e.g. international insurance companies) in developing adequate responses to managing risk and recovery. to the best of our knowledge, the present research is the first attempt to undertake such an integrated analysis at a global scale. the rest of this article is organized as follows: the next section contains a literature review of the arguments behind the expected relationship between disaster events and tourism demand. the third section explains the methodology, data and the research design. the fourth section presents the empirical application. finally, a concluding discussion is presented that provides recommendations and an outlook on future research. the general perception might point towards an increase in the frequency of natural disasters over time, but this assumption needs to be verified. in fact, it has been suggested that, in some cases, the definition of disasters can become too fluid for statistical time series consideration (horlick-jones, fortune, & peters, ) . neumayer and barthel ( ) analyzed the economic damage from climate-related disasters and they found no significant upward trends in normalized data over the last years globally. however, the same study acknowledged that the frequency of weather-related natural disasters indicates an upward trend. other research suggests that the combination of climate change, industrialization and urbanization has accelerated the magnitude and occurrence of natural disasters around the world and the extent of the resulting damage park & reisinger, ) . population growth (often occurring in exposed areas such as coastal environments) is recognized as a key driver to explain why natural disasters affect more and more people (berke, ; wachinger, renn, begg, & kuhlicke, ) . aside from natural disasters, richardson ( ) notes that man-made disasters are becoming more severe because of the increasingly more powerful technology that is being used. perceptions of the frequency and extent of disasters are just as important as statistical facts. a key factor in this growing risk perception is the media (gierlach, belsher, & beutler, ) . for a the general public, who is exposed to mass media, it may appear that we live in an increasingly disaster prone world (faulkner, ) . the saying 'perceptions are reality' is nowhere more pertinent than in tourism, where potential visitors chose their destinations based on a mix of objective and subjective factors. destination (risk) perception has emerged as one of the critical factors in the decision-making process (becken, jin, chen, & gao, ) . disasters and other forms of crises (e.g. epidemics, conflict, pollution) can lead to a reduction in visitation to the affected area (bhati et al., ) . several examples in the literature provide empirical evidence of reductions in tourist arrivals following major events. for instance, mazzocchi and montini ( ) evaluated the impact on visitation to the umbria region in central italy, following a major earthquake in september . the data showed that arrivals fell drastically the first month after the main shock, with ongoing loss in tourism activity being recorded until june . a case study of a volcanic eruption at the eyjafjallaj€ okull glacier in iceland on th march showed that tourism numbers to iceland reduced by % until th april (j� onsd� ottir, ). huang and min ( ) analyzed the taiwan earthquake in september , using an integrated moving average model to explore the recovery process. their study revealed that the island's inbound arrivals had not yet fully recovered from the earthquake's devastation after months. kuo, chen, tseng, ju, and huang ( ) also used a time series model to investigate the impacts of infectious diseases, including avian flu and severe acute respiratory syndrome, on international tourist arrivals in asia. the empirical results indicated that the numbers of affected cases had a significant impact in the case of sars (see also mao, ding, & lee, ; mcaleer, huang, kuo, chen, & chang, ) , but for avian flu. man-made crises, such as the bp oil spill in the mexican gulf in , have also been found to reduce demand for travel to the affected area (ritchie, crotts, zehrer, & volsky, ) . often, declines in visitation spread to neighboring areas, even when they are not impacted by the event. a recent example has been the dramatic down turn in tourism in the caribbean region, following the devastating hurricane season in (wttc, ) . events within one country or a region can lead to notable structural breaks in international tourism arrivals, which was demonstrated by cr� o and martins ( a) in a recent study on various forms of crises in countries. there are several reasons why visitation to disaster areas declines in the immediate aftermath of an event. the most direct inhibitor relates to the damage inflicted by a disaster that prevents the affected areas from engaging in tourism activity. secondly, the decline in tourist arrivals is due to people's risk perceptions and avoidance of regions that are deemed unsafe (kozak et al., ; s€ onmez, apostolopoulos, & tarlow, ) . thirdly, and related to the second issue, is that potential travelers may feel uncomfortable or have ethical concerns about travelling to a disaster region. these underlying factors are discussed in more detail. in many cases, disasters pose significant physical constraints on the delivery of tourism services, thus severely limiting the supply side of tourism (shaw, saayman, & saayman, ) . depending on the type and extent of the disaster, critical infrastructure could be compromised or dysfunctional. prominent examples include airports and ports, land transport infrastructure, and electricity and telecommunication networks (ghobarah, saatcioglu, & nistor, ; parajuli & haynes, ) . in addition, core tourism assets could be damaged and not ready for business, such as accommodation establishments and key attractions. for instance, the earthquake in kathmandu, nepal, resulted in wide-spread destruction of unesco listed world heritage sites, and several trekking routes were deemed unsafe due to risks of rock fall and movements following further aftershocks or heavy rain events (becken, ) . even longer-term and insidious disasters, such as a drought, may impede the ability of a destination to cater for tourism. a recent example was the water shortage in cape town, south africa, that led to a reduction in tourism and a notable loss in income for local businesses. the decline was possibly influenced by requests to conserve waters, but also due to perceptions by visitors that the destination is not able to host tourists (wendell, ) . in addition to uncertainty around whether the destination is safe or tourism-ready, there are other psychological factors that influence tourists' decision making. frequently, media coverage of disasters conveys the resulting loss of life, human suffering, public and private property damage, and economic and social disruption. the ensuing negative publicity often characterizes the period after a disaster, lasting until full recovery is achieved and pre-disaster conditions resume (s€ onmez et al., ) . for instance, cohen ( ) points out that religious beliefs relating to the bodies of the tsunami victims trapped in sediment and rubble were behind a group of asian tourists deciding to abandon their plans to visit thailand after the tsunami. others may simply consider it inappropriate to visit a disaster zone. apart from religious or ethical concerns, some travelers do not wish to impede the recovery effort and place additional burden on the destination's resources and infrastructure (e.g. becken, ) . in some cases, the delayed recovery towards previously tourism figures is deliberate and led by the local tourism organizations. this was the case for the christchurch (new zealand) earthquake ( ), where extensive destruction of the city made tourism impossible, or at best would have led to unsatisfactory tourist experiences, leading christchurch canterbury marketing to de-market christchurch but promote surrounding regions instead (orchiston & higham, ) . optimal timing and stages of recovery were examined by okuyama ( ) for the case of avian flu in japan. whilst both theory and empirical evidence point to a decline in tourism following a disaster, several factors might promote travel to an affected area. providing information about hazards and their effects draws human attention and may even cause a level of fascination (e.g. the 'ring of fire', referring to tectonic activities around the edges of the pacific ocean). in this way, the number of tourists might be influenced by the coverage that media devote to natural disasters in other countries. media often use extreme natural phenomena as material for captivating stories, and travel bloggers, tourism campaigners and social media multiply the lure of these. an example of a disaster turning into a tourist attraction is the eyjafjallajokull volcano in iceland, with "the prospect of a new eruption bring[ing] a mix of trepidation and anticipation" (lawless, , p. ) . media coverage about a natural, or perhaps also man-made, phenomenon plays an informative role as a motivating factor to visit a region. ruci� nska and lechowicz ( ) argue that mass media and marketing are influential factors in the development of various forms of disaster-related tourism, as information on catastrophes popularizes the host location and the type of the phenomenon. such coverage could be both educational and simultaneously stimulate the interest of the audience. additionally, natural disasters and unexpected events can cause the arrival of people from other countries for humanitarian reasons but also for visiting friends and relatives who have been victims of those events. according to the statistical framework used by the united nations world tourism organization, these arrivals are captured as international tourists. finally, the decision to visit a disaster area for a range of motivations has been conceptualized as dark tourism (ruci� nska & lechowicz, ). this type of tourism involves travelling to places historically associated with death and tragedy (foley & lennon, ) . according to ruci� nska ( ), tourists might decide to travel to a region that has experienced a disaster because they want to feel emotions, risk, and the dynamics of natural hazards. overall, the present research hypothesizes a negative relationship between national disasters and inbound tourism; however, it also considers the motivating factors pointed out by ruci� nska ( ) that might lead to an increase in visitation after a disaster. the model developed in the following section will capture the cumulative impact of both effects. this research develops a gravity model for international tourism flows to quantify the effects of different types of natural and man-made disasters on tourist arrivals to the affected countries. gravity models are commonly used in the trade literature (anderson, ) , and increasingly in tourism research (fourie, rossell� o-nadal, & santana-gallego, ; khadaroo & seetanah, ; santeramo & morelli, ) . these models consider that international flows between two countries are directly proportional to their economic size, and inversely proportional to the distance between them. consequently, the level of bilateral tourism flows can be explained by a set of determining variables as in a demand equation. morley et al., have shown that gravity models to explain bilateral tourism can be derived from consumer choice theory. accordingly, the formulation of a gravity model can also be interpreted as a tourism demand equation. analytically: where, the dependent variable lntou ijt is the logarithm of tourist arrivals from country i, to destination country j, at year t; x d jt is a set of d destination-specific time-variant variables such as income level or population while x k ijt is a set of k country-pair time-variant determinants such as belonging to the same regional trade agreement. the variables of interests for this research are included in y l jt which is the set of l variables capturing the effect of l different disasters typologies (e.g. earthquake, tsunami, volcano, etc.) occurred in destination j during year t. this research uses three alternative proxies to measure the effect of disasters; namely number of deaths (d) in thousands, people affected (a) in millions and economic costs (c) in billions of us$. finally, β ; β k and β l are parameters to be estimated. due to the panel nature of data used in these kinds of models, and since our variables of interest are destination-country time variant, country pairs fixed effects λ ij and origin-year fixed effects λ it are also considered for estimation purposes. one of the consequences of this choice is that time-invariant country pair characteristics (such as distance or common borders) and time-variant origin country characteristics (such as income or population in the origin countries) are not explicitly included in the model. specific consideration is not necessary, because all these variables are captured by these fixed effects, as also suggested by balli, ghassan, and jeefri ( ), fourie et al. ( ) or giambona, dreassi, and magrini ( ) . this is a common practice in the development of gravity models in order to avoid omitted factor bias, and instead focus on the variables of interest for the particular research question. as dependent variable, lntou ijt , we consider the natural logarithm of international tourist arrivals from country i to country j in year t. this dataset originates from the compendium of tourism statistics compiled by the united nations world tourism organization (unwto, ) . this database contains tourism movements between countries for the period - , with missing data for some years and countries. in reference to the d variables determining tourism flows (x d jt ), and according to the considerations mentioned above about the no inclusion of time-invariant country pair characteristics and time-variant origin country characteristics, we consider the logarithm of the real gdp per capita (lngdppc jt ) as a proxy for the development level at each destination, and the logarithm of population (lnpop jt ) to control for the size of the destination country (lim, ; yap & saha, ) . both variables were taken from the world development indicators (wdi) elaborated by the world bank. third, we also consider an instability indicator that concerns safety and security of visitors when they travel to a destination. whilst there are different ways for evaluating safety and security at international level (see cr� o & martins, b; cr� o, martins, simões, & calisto, ; fourie et al., or santana-gallego, fourie, & rossell� o, in this case, and due to data coverage reasons, we use a proxy of the crime rate defined as the number of homicides per , inhabitants in the destination country (crime jt ). data also stem from the wdi. on the other hand, vector x k ijt includes a variable to control for the intensity of the economic relationship between a pair of countries, which is also time varying. the idea is to capture the presence of trade agreements between country pairs as an indicator of bilateral relationships that could boost tourism. this variable (rta ijt ) is a dummy variable for being a signatory to the same regional trade agreement and stems from the regional trade agreements information system compiled by the world trade organization. data for the occurrence and impact of disasters were retrieved from the centre for research on the epidemiology of disasters (cred), which makes data available through the emergency events database (em-dat). em-dat was created with the initial support of the world health organization (who) and the belgian government. the main objective of the database is to inform humanitarian action at national and international levels. the initiative aims to rationalize decision making for disaster preparedness, as well as provide an objective base for vulnerability assessment and priority setting. em-dat contains core data on the occurrence and effects of over , mass disasters in the world from to the present day. the database is compiled from various sources, including un agencies, non-governmental organizations, insurance companies, research institutes and press agencies. according to the objectives of this present research, the disaster types included in em-dat and considered in our analysis are presented in tables and . for the gravity equation estimation we are limited by the availability of the tourist database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . all other datasets provide data for this timeframe as well, leading to a database that covers a total number of events from the period of - . these are described using three types of impact metrics (table ) . more specifically, of all events, . % report information on the number of deaths, . % report the extent of affected people (beyond deaths) and . % state an estimated amount of damage measured in economic terms. with regards to people killed by different disaster types, table shows that ground movements emerge as the most fatal type of disaster, with a reported number of . deaths during the period - . tsunamis and storms accounted for almost , deaths in the same period. in terms of affected people, floods and droughts have the greatest impact, with about , and billion of people impacted upon, respectively. concerning the economic costs of disasters, storms rank first, with a total amount of , billion dollars of damage recorded in the database. storms make up % of total economic costs for the selected disasters in the em-dat during the period - . the distribution of disasters across different regions, indicates considerable variation both in terms of event type and resulting impacts. for instance, in the case of storms, although only . % take place in the americas, the impact in terms of deaths, affected people and costs is comparatively high ( . %, . % and . %, respectively) than in other regions. a similar result is obtained for earthquakes in the asia-pacific regions ( . % of events), with disproportionally high impacts in relation to the number of deaths ( . %), affected people ( . %) and costs ( . %). europe, with some exception, is characterized by a lower incidence of deaths and affected people, but a higher occurrence of costs. table shows the most important events for each type of disaster in the database. for example, it can be observed that the earthquake of haiti in january , which led to , deaths, was the worst event in terms of fatalities. the major storm (cyclone nargis) that occurred in myanmar during may resulted in , deaths, the second largest number in the records. in terms of affected people, the drought affecting india during and was the most significant event reported in the database ( million people impacted). disasters also cause substantial economic damage. the highest economic loss recorded was tropical hurricane katrina that made landfall in new orleans, usa. it caused a total amount of damage of $ billion. although the disaster database includes the exact day of the event, for the purpose of this analysis we are limited by the yearly nature of tourism data. following guidance from the previous literature another important issue to be considered is the multicollinearity that can arise between the different types of impacts related to the same specific disaster. thus, it is expected (and found) that the consequences of a certain disaster in terms of deaths will be correlated to other impacts measured in terms of affected people and economic costs. the increase in the variance of the coefficient estimates could drive them to be unstable and difficult to interpret. consequently, our first strategy is to consider the three impact metrics (i.e. deaths, affected people and cost) separately in different equations. additionally, for each of the three metrics, we evaluate the possibility to distribute the effects within and months. this results in specifications: three for each of the impacts, times the two evaluation periods ( and months). importantly, according to the theoretical argumentation, the relationship between disasters and arrivals is not unidirectional and necessarily negative, but an increase in tourist arrivals could be observed in certain circumstances. for this reason, a second research strategy considers the inclusion of all the variables in a general regression that is reduced using statistical testing strategies in order to get a specific regression encompassing every other parsimonious regression that is a table disaster typology and main descriptive magnitudes ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) notes: cold waves include severe winter conditions. epidemic episodes are not characterized by economic costs. valid restriction of the general regression perez, and ) . in other words, we integrate the three impact metrics into a single equation. through this strategy, it is possible to explore in detail if effects arise that counteract the initially expected negative relationship between disaster impacts and tourism flows. again, two impact timeframes are considered. the gravity model for bilateral tourism flows as defined in equation [ ] is estimated by using the correia ( ) procedure to estimate linear models with many levels of fixed effects. this procedure is a generalization of the panel-fixed effects estimator with both country-pair and origin-year fixed effects. database includes countries for the period - . table presents the results of estimating equation [ ] for the bilateral tourist arrivals (lntou ijt ) as dependent variable and including each disaster impact measure separately. as previously mentioned, because our variable of interest is destination-country time variant, country pairs fixed effects and origin-year fixed effects are included in the model to control for any type of determinant at origin or country-pair level. therefore, only time variant country-pair and destination-specific determinants are required. each column shows the estimate of different disaster consequences (d ¼ deaths, a ¼ affected people, and c ¼ economic costs) and the two alternatives for distributing the effects across the following and months. it is important to mention that we are interested in estimating the short-run effect of the natural disaster on inbound tourism. exploring how the tourism sector at the destination country recovers in the long-run is beyond the objective of the paper. in general, for all the estimates, control variables considered as de-terminants of tourism flows are statistically significant and with the expected sign. the coefficients for both lngdppc jt and lnpop jt are significantly positive and slightly higher that unity, implying that a % increase in the destination gdp per capita and population will lead to an increase higher than % on tourist arrivals to the country. the coefficient for the rta ijt , that controls for the existence of a trade agreement between country pairs during specific years, is also significant and positive. in this case, due to the binary nature of the explanatory variable used, the estimated coefficient (slightly higher that . ) implies that the existence of a trade agreement increases the number of tourists to a destination by more than %. finally, and as expected, the variable related to low levels of security and safety at the destination country (crime jt ) shows a negative effect, indicating that an increase in the number of homicides (per , inhabitants) reduces tourist arrivals. in reference to the different types of disasters, for events associated with tsunamis, floods and volcanoes, all the significant parameters are found to be negative, indicating that these three types of disasters constitute substantial negative motivators for prospective visitors. a more detailed examination of coefficients highlights that volcanic eruptions appear most deterring to international tourists. this circumstance could be related to the severity of the damage caused by volcanic eruptions, including potentially irreversible damage to infrastructure or the complete loss of a natural asset. for the occurrence of an eruption, and for every increase in the number of deaths (for every people), affected (in millions of people) and costs (in millions of us$), there will be a decrease in international tourists to the destination between . % and . %, . %- . % and . %- . %, respectively (according to whether the or months delay is considered). wildfires, earthquakes, industrial accidents, and storms present mixed effects on international tourist arrivals. for all types of disasters, and note: ***p < . , **p < . , *p < . . dyadic and origin-year fixed effects are included in the model but estimates are not reported. robust standard errors clustered by pairs. when economic costs are considered, a negative and significant relationship is found. in other words, the economic damage from these events, for example to infrastructure, is likely to reduce tourist arrivals. wildfires appear as the second most detrimental type of disaster when measured in economic damage, leading to an expected fall of . % of tourist arrivals for every million us$ cost associated with the disaster. interestingly and perhaps paradoxically, a significant positive relationship is evident between the number of people affected by wildfires and tourist arrivals. for every million affected people, an increase between . % and . % is expected. consequently, and considering the negative effect of economic damage mentioned above, the net effect of wildfires on tourism should consider the two different types of disaster impact measures. earthquakes show a similar negative impact compared with tsunamis (see above) in terms of the economic costs of the disaster, with falls around . % for every million us$ cost. however, the other impact metrics do not show a negative relationship. in terms of number of fatalities, there is even an increase in tourism for the number of deaths per people by . %. thus, the overall impact of an earthquake is a combination of decreases in response to economic damage and number of deaths. industrial accidents and storms show similar patterns in that there is a positive relationship between the number of fatalities and affected people, but a negative relationship between the economic impact of the disaster and tourism arrivals. for example, for storms there is a decrease in arrivals by . % for every million us$ cost but increases between . % and . % for every death/ people and between . %- . % and . - . % for every million people affected. droughts emerged as the only type of disaster that did not show a significantly negative relationship between disaster cost and tourism, but instead arrivals were significantly linked to the two other disaster impact metrics. more specifically, for every death/ people an increase higher than % is obtained, while for every million people affected a decrease of . % is estimated. it is perhaps not surprising that the relationship between disaster costs and tourism is not significant for drought. overall, it is less likely that drought conditions produce direct impacts on tourism-relevant infrastructures and supplies, as tourism businesses might absorb the extra costs of supplying water during water constrained times. there could be indirect costs, for example due to more expensive food supplies, but such effects do not seem to result in significant changes in visitation. epidemics, landslides, cold waves, and heat waves do not achieve significant results for any of the six regressions considered, and for this reason they were not considered in the final estimation presented in table . in the case of epidemics, and landslides we should note that these two variables have a strong structural component. for instance, epidemic episodes, such as cholera, dengue, and ebola, as well as land movements with consequences on people are recurrent in the same types of countries at different times, but rarely are these factors extended to other countries. in a similar way rossell� o, santana-gallego, and waqas ( ) evaluated the effects of dengue, ebola, malaria, and yellow fever on international tourism flows showing how these diseases have a strong structural component and are often recurrent in the same countries. the case of cold and heat waves is different. it should be noted how travel booking decisions (especially in international travel) are often taken months in advance, when no reliable weather predictions exist. although it is possible to cancel travel plans in case of extreme temperatures, tourist might assume the conditions are temporary and unlikely to impact their trip. in terms of longer lasting risk perceptions of a destination, heat or cold waves might not be seen as particularly threatening, and hence easily forgotten. visitors might expect that their tourism service provider is dealing with adverse conditions, for example by providing air conditioning or heating. instead, extreme temperatures are more likely to impact local people (e.g., farmers) leading to wider economic damage (but not attributed to tourism). regarding the distribution of the potential effects of each one of the disasters during the next months (columns from one to three) and during the next months (columns from four to six) no significant but only minor differences are found. additionally, different attempts to discriminate disaster by geographical regions did not yield significantly different conclusions. as mentioned earlier and in order to explore the bidirectional effects between disaster events and tourism flows, a second research strategy is implemented. based on two initial general regressions (one for each of the delay periods considered), including all the considered variables, a reduction is undertaken in order to get the specific regressions presented in table . regarding the distribution of the potential consequences of each type of disaster during the next months (columns one to three) and during the next months (columns four to six), in general, no significant differences are found. with the exception of floods and storms, the coefficients for the remaining disasters (in absolute terms) are higher for the month impact regressions than for the months ones, thus, indicating that effects are probably better captured by longer time lags. in contrast, the effects of floods and storms seem to have a shorter life span, since the month timeframe captures a higher impact. the analysis of the different disaster impacts reveals how, on the one hand, costs always present a negative relationship with international tourist arrivals. this confirms that the economic costs of a disaster are an important measure for tourism managers, probably because of the inherent damage to local infrastructure that is captured. on the other hand, the impact of some types of disaster evaluated in terms of deaths shows a positive relationship with tourist arrivals. this does not mean that the occurrence of these disasters will have a net positive effect on the arrival of tourists, since the negative effect of the associated costs must be taken into account when deriving an overall estimate of impact. as outlined earlier, the number of deaths could be related with the arrival of people for humanitarian reasons, or with a flow of people who travel to see (and support) friends and relatives affected by the event. this could present a significant effect in relative terms for those countries with a low base level of arrivals. the total effect also should note: ***p < . , **p < . , *p < . . dyadic and origin-year fixed effects are included in the model but estimates are not reported. robust standard errors clustered by pairs. consider the impacts of the number of affected people that for some disasters have a reducing effect (droughts, tsunamis and volcanoes), while for others there seems to be an increase in the number of tourists (industrial accidents, wildfires and storms). natural disasters and unexpected events have wide reaching effects on all spheres of life, including tourism. from a theoretical point of view, it has been assumed that a negative relationship between disasters and inbound tourism dominates (e.g. a cr� o & martins, a). however, because of some motivating factors identified in the literature, and due to the methodology and definition used by the unwto in collecting international tourist arrivals, an increase in visitation after a disaster seems also plausible. the number of inbound tourism arrivals directly impacts the performance of the national tourism industry, and ultimately the government, especially in countries where tourism is a major contributor to the national economy and fiscal revenue (massidda & mattana, ) . it is therefore of great importance for policymakers to improve their understanding of how disaster events affect visitor demand. this research highlights the need to consider different types of disasters and their varied consequences when assessing the consequences for tourism. the empirical research presented in this paper draws on two sets of data to explore in depth the relationship between international tourist arrivals and global disasters, measured through three different impact metrics (costs, deaths and affected people). the effects that these different disasters might have on inbound flows at a national level were investigated though a gravity model, estimated by panel data with destination-fixed effects and using yearly data. by doing so, spurious potential determinants related to the destination but not the disaster can be avoided. as a result, however, recurrent disasters affecting the same destination and those with a very short-run effect have not been captured. findings of this analysis provide evidence that the economic consequences of a disaster in a particular country generally affect international tourism arrivals negatively. this is likely due to damages to infrastructure, key attractions and a wider weakening of the economy in the host country. all of these reduce the destination ability to cater for tourism, undermine investment into tourism supply, and reduce destination attractiveness, at least in the short-term. at the same time, the analysis reveals that evaluating the tourism impacts of a disaster in terms of deaths and affected people is more ambiguous. our research found a dominance of positive effects in the case of deaths related to a disaster. thus, whilst disaster damage seems to prevent tourists to visit the affected destination, the number of fatalities and affected people seem to be less of a deterrent. tourists may not see a risk to their own safety. also, there could be an increase in tourism for some disasters due to the arrival of humanitarian 'tourists' and people visiting friends and relatives. whilst generally, this observation might be testimony to tourism resilience, and indeed reassuring for destination managers, there may be situations where continuous tourism demand after a disaster is hindering recovery works or impacting the well-being of residents. more research on 'optimum' recovery timeframes that take into account resident needs, would be useful (e.g. okuyama, ) . it is useful for decision makers to understand that not all disasters cause similar impacts. the comparison of different disaster types showed, for example, that volcanic eruptions typically cause the most significant and substantial negative impact on tourism. specifically, for every million people affected by an eruption a fall between . % and . % in the international tourism arrivals is expected, if a six-month period or a twelve -month period is considered, respectively. other disasters have smaller and shorter-term impacts (e.g. floods and storms). furthermore, floods and tsunamis are detrimental without nuance, although it is difficult to discerne whether the negative effect is due to the possible destruction or disablement of infrastructure or to the negative image of the destination generated by these types of event. when a destination is affected by a wildfire, an earthquake, an industrial accident, a storm or a drought, mixed effects may be expected. for example, when these types of disasters result in economic damage, a negative and significant relationship can be established, indicating that damage to infrastructure and built assets, and maybe business capability, is likely to reduce tourist arrivals. finally, this research revealed that some types of events are unlikely to have a major effect on arrivals, for example an unexpected epidemic, a landslide, a cold wave and a heat wave. it should be noted how these natural disasters are characterized by little or no impact on infrastructure and no long-term risk to tourists after the event has finished. natural disasters and unexpected events are traumatic experiences for the resident population and may cause lasting damage to destination infrastructures, which requires adequate and adaptive tourism management (hystad & keller, ) . strategies used to predict natural disasters and mitigate hazard risks in the first place need to be deployed to minimize the impacts. examples include the implementation of appropriate building codes, zoning regulations, and emergency training and preparedness for key stakeholders. new policies and practices may require additional resources, but investments into preparedness are likely to generate positive returns in the long term. in general, the empirical results in this paper confirm that disaster events are challenging news for tourism managers who need to deal with an unexpected fall in tourism demand. clearly, economic damage from an unexpected event leads to some reductions in tourist arrivals. in those cases, efforts by destination managers should focus on the recovery of necessary infrastructure and business capability. proactive planning, for example around business continuity, business support networks, and recovery assistance programs, could accelerate this effort (hystad & keller, ) . leadership may come from government agencies, destination management organizations, or businesses themselves. related research in new zealand revealed that leadership is "mainly provided by tourism stakeholders with a community-value orientation, and to a lesser extent by those who are mainly business-driven" (hughey & becken, , p. ) . in other words, response and recovery is often led by individuals who have a strong commitment to, and engagement with, the affected community. for some events, it is not necessarily the economic damage that is the most significant impact, but it could be the number of people affected or killed. for some disaster types, for example wildfires and storms, this research even established a positive impact. the positive relationship between number of tourists and affected people of fatalities by some disasters implies that these can attract visitors to the destination, a circumstance that should be taken into account by the managers of the destination. there are many different reasons why visitors might want to visit a destination that had been affected by a disaster (e.g. ruci� nska & lechowicz, on dark tourism), and understanding this non-orthodox typology of visitor types could be useful for destination managers. regardless, marketing activities have to be designed with great care to attract the right types of visitors at the right time, considering potentially ongoing limitations around tourism capacity (okuyama, ; orchiston & higham, ) . marketing campaigns implemented by businesses, local tourist destinations or national tourism bureaus should ideally align in their messaging and magnitude, implying a particular need for vertical integration following a disaster (hughey & becken, ) . this research has several limitations, including the availability, accuracy and granularity of data, which is outside the control of the research team. it could be argued that some impacts on tourism are significant, yet short-lived. given that the data used here is provided on an annual basis, short-term effects are likely to be missed or underestimated in this research. besides the limitations about the estimation method and the nature of the data of the unwto we have imposed a homogenization for each disaster. that means that a specific disaster in a developed country has the same effect than in an less developed one. in reality, this might not be the case. consequently, results obtained in this paper should be considered as average responses. future research should further explore this matter and investigate if differences among countries in reference to their level of development exist. our attempt to discriminate the different disaster by region did not obtain significant results. future research on the positive impacts of certain types of disaster consequences would also be beneficial in developing a potential tourist typology consisting of 'dark tourism' segments, humanitarian arrivals or other presently unidentified markets. jaume rossell� o-nadal as an expert in tourism demand modelling and the quantitative analysis of tourism has contributed with the knowledge of the specific literature of demand modelling and the identification of the gap in the literature. susanne becken as an expert in sustainable tourism and climatic change issues has contributed with the knowledge of the specific literature of natural hazards and potential effects on tourism and maria santana gallego as an expert within the fields of gravity models in a special way in the design of the methodology and in the exploitation of the results. he has also been responsible for the first model estimations. jaume rossell� o-nadal has a phd in business and economics, is full professor at the universitat de les illes balears (spain) and adjunct professor at griffith institute for tourism (australia). his research interests include tourism demand modelling and environmental issues with special interest in climatic change issues. jaume has led different research projects in spain and europe. dr susanne becken is a professor of sustainable tourism at griffith university, australia. susanne has led a large number of research programmes and consulting projects in asia pacific in the area of climate change, risk management and sustainable tourism. she contributes through various industry and government advisory roles and panels, and is on the editorial boards of nine tourism journals. professor in the department of applied economics, university of the balearic islands, spain. she teaches tourism economics and macroeconomics. her research interests include quantitative analysis of tourism and gravity models. sustainable development in damascus university: a survey of internal stakeholder views the gravity model towards understanding gcc outbound international tourism what can tourists do to help, not hinder, nepal's quake recovery? the conversation linking tourism into emergency management structures to enhance disaster risk reduction urban air pollution in china: risk perceptions and destination image the tourism disaster vulnerability framework: an application to tourism in small island destinations developing climate change maps for tourism: essential information or awareness raising? reducing natural hazard risks through state growth management national disaster management in the asean- : an analysis of tourism resilience tourism in science research. krak� ow-rzesz� ow: academy of physical education in krak� ow, university of information technology and management in rzesz� ow linear models with high-dimensional fixed effects: an efficient and feasible estimator structural breaks in international tourism demand: are they caused by crises or disasters? tourism management the importance of security for hostel price premiums: european empirical evidence effect of security on hostels' price premiums: a hedonic pricing approach towards a framework for tourism disaster management jfk and dark tourism: heart of darkness fatal attraction: how security threats hurt tourism when disaster strikes: the thai floods of and tourism industry response and resilience disaster management and post-quake impact on tourism in nepal the impact of the december earthquake and tsunami on structures and infrastructure. engineering structures tourism attractiveness in italy. some empirical evidence comparing origin-destination dom� estic tourism flows cross-cultural differences in risk perceptions of disasters data mining reconsidered: encompassing and the general-to-specific approach to specification search data mining reconsidered: encompassing and the general-to-specific approach to specification search measuring disaster trends part two: statistics and underlying processes earthquake devastation and recovery in tourism: the taiwan case value-engaged evaluation of a tourism-specific disaster management plan. research note. tourism management perspectives towards a destination tourism disaster management framework: long-term lessons from a forest fire disaster impact of crisis events on chinese outbound tourist flow: a framework for post-events growth líf-ogumhverfisvísindadeild, verkfraeði-ogn� attúruvísindasvið, h� ask� oli � islands the role of transport infrastructure in international tourism development: a gravity model approach the impact of the perception of risk on international travellers assessing impacts of sars and avian flu on international tourism demand to asia iceland eruptions bring tourism boom. stuff. available a survey of tourism demand modeling practice: issues and implications post-sars tourist arrival recovery patterns: an analysis based on a catastrophe theory a svecm analysis of the relationship between international tourism arrivals, gdp and trade in italy earthquake effects on tourism in central italy an econometric analysis of sars and avian flu on international tourist arrivals to gravity models for tourism demand: theory and use press release: natural catastrophe review: series of hurricanes makes year of highest insured losses ever normalizing economic loss from natural disasters: a global analysis guidelines for resilience systems analysis analysis of optimal timing of tourism demand recovery policies from natural disaster using the contingent behavior method knowledge management and tourism recovery (de)marketing: the christchurch earthquakes the earthquake impact on telecommunications infrastructure in nepal: a preliminary spatial assessment differences in the perceived influence of natural disasters and travel risk on international travel crisis management and the management strategy: time to &loop the loop tourism disaster planning and management: from response and recovery to reduction and readiness understanding the effects of a tourism crisis: the impact of the bp oil spill on regional lodging demand infectious disease risk and international tourism demand measuring tourism risk impacts on destination image natural disaster tourism as type of dark tourism natural hazard and disaster tourism the effect of safety and security issues on international tourism modelling tourism flows through gravity models: a quantile regression approach social amplification and attenuation of climate change risk in a vulnerable tourism destination investigating the role of prior knowledge in tourist decision making: a structural equation model of risk perceptions and information search identifying risks facing the south african tourism industry disaster tourism and disaster landscape attractions after hurricane katrina tourism in crisis: managing the effects of terrorism a cognitive-affective scale for hurricane risk perception the risk perception paradox-implications for governance and communication of natural hazards cape town's water crisis hitting tourism: officials tourism risk and uncertainty: theoretical reflections caribbean resilience and recovery: minimising the impact of the hurricane season on the caribbean's tourism sector do political instability, terrorism and corruption have deterring effects on tourism development even in the presence of unesco heritage? a cross-country panel estimate compendium of tourism statistics by the world tourism organization we acknowledge the agencia estatal de investigaci� on (aei) and the european regional development funds (erdf) for its support to the project<eco - -c - -r> (aei/erdf, eu) and unwto statistics department for kindly providing us with the tourist data for this study. we also wish to thank participants to the th international conference on tourism management & related issues. key: cord- - a uwjf authors: hughes, dyfrig a. title: acute chloroquine poisoning: a comprehensive experimental toxicology assessment of the role of diazepam date: - - journal: br j pharmacol doi: . /bph. sha: doc_id: cord_uid: a uwjf background and purpose: resurgence in the use of chloroquine as a potential treatment for covid‐ has seen recent cases of fatal toxicity due to unintentional overdoses. protocols for the management of poisoning recommend diazepam, although there are uncertainties in its pharmacology and efficacy in this context. the aim was to assess the effects of diazepam in experimental models of chloroquine cardiotoxicity. experimental approach: in vitro experiments involved cardiac tissues isolated from rats and incubated with chloroquine alone or in combination with diazepam. in vivo models of toxicity involved chloroquine administered intravenously to pentobarbitone‐anaesthetised rats and rabbits. randomised, controlled treatment studies in rats assessed diazepam, clonazepam and ro ‐ administered: (i) prior, (ii) during and (iii) after chloroquine and the effects of diazepam: (iv) at high dose, (v) in urethane‐anaesthetised rats and (vi) co‐administered with adrenaline. key results: chloroquine decreased the developed tension of left atria, prolonged the effective refractory period of atria, ventricular tissue and right papillary muscles, and caused dose‐dependent impairment of haemodynamic and electrocardiographic parameters. cardiac arrhythmias indicated impairment of atrioventricular conduction. studies (i), (ii) and (v) showed no differences between treatments and control. diazepam increased heart rate in study (iv) and as with clonazepam also prolonged the qtc interval in study (iii). combined administration of diazepam and adrenaline in study (vi) improved cardiac contractility but caused hypokalaemia. conclusion and implications: neither diazepam nor other ligands for benzodiazepine binding sites protect against or attenuate chloroquine cardiotoxicity. however, diazepam may augment the effects of positive inotropes in reducing chloroquine cardiotoxicity. linked articles: this article is part of a themed issue on the pharmacology of covid‐ . to view the other articles in this section visit http://onlinelibrary.wiley.com/doi/ . /bph.v . /issuetoc chloroquine and hydroxychloroquine are being repurposed as potential treatments for coronavirus disease (ferner & aronson, ) . the food and drug administration (fda, ) authorised their emergency use in the united states and clinical guidelines in belgium, china, france, india, iran, italy, south korea and the netherlands have made recommendations for their use in the prevention and treatment of covid- . case reports of cardiotoxicity and fatal poisoning relating to the use of chloroquine and hydroxychloroquine for covid- have emerged (agence régionale de santé, ; binding, ; busari & adebayo, ; simplicity, ; xuan, ) , as well as excess death with high doses in covid- clinical trials (borba et al., ) . the acute toxic effects of these drugs are well recognised (world health organization, ) and relate to their cardiotoxic effects of widening of the qrs complex, atrioventricular block, ventricular arrhythmias, negative inotropy, hypotension and severe hypokalaemia, which occur within - h of ingesting doses > g in adults. without intensive, supportive treatment, circulatory collapse and death can rapidly follow acute overdose. mortality due to acute toxicity is high, with of the cases reported in the literature between and (bondurand, n'dri, coffi, & saracino, , and a further from suicide attempts (weniger and world health organization, ) resulting in death. current recommendations for the management of acute toxicity include ensuring adequate ventilation, gastric lavage, administration of activated charcoal, adrenaline for its inotropic and vasoconstrictor effects, diazepam and correction of metabolic acidosis and hypokalaemia (jones, ) . the observation in of a patient who took g of chloroquine together with mg of diazepam and survived without symptoms of chloroquine toxicity (djelardje, ) , drew attention to the possible role of diazepam in chloroquine poisoning. subsequent case reports (jaeger, sauder, kopferschmitt, & flesch, ; meeran & jacobs, ; rajah, ) and a prospective non-randomised trial (riou, barriot, rimailho, & baud, ) , in which the odds of survival significantly favoured diazepam therapy, led to the recommendation of diazepam in the management of acute chloroquine toxicity. however, there remains controversy given some conflicting evidence of benefit (demaziere et al., ; clemessy et al., ) and limitations in study designs (yanturali, ) . experimental toxicity studies are also inconclusive. crouzette, vicaut, palombo, girre, and fournier ( ) demonstrated that an intraperitoneal injection of diazepam caused a significant decrease in the mortality of rats treated with chloroquine. riou, rimailho, galliot, bourdon, and huet ( ) observed an improvement in haemodynamics and a correction of the qrs interval prolongation when diazepam was administered to chloroquine-intoxicated pigs. gnassounou and advenier ( ) observed that clonazepam protected anaesthetised rats against chloroquine toxicity and that diazepam but not the translocator protein (tspo) agonist ( -chlorodiazepam) protected against the decrease in contractions, observed when guinea pig atria were exposed to chloroquine. in other studies, however, diazepam failed to improve the mechanical performance of rat cardiac papillary muscle exposed to chloroquine (riou, lecarpentier, barriot, & viars, ) and was ineffective in reversing chloroquine toxicity in anaesthetised rats (buckley, smith, dosen, & o'connell, ) . it would therefore appear that the effectiveness of diazepam in reversing chloroquine toxicity is equivocal and that the mechanism(s) by which diazepam may exert its effects remain unclear. due to the resurgence in the use of chloroquine and its structural analogue hydroxychloroquine for covid- , the aim of the present study was to investigate the potential cardioprotective effects of diazepam in experimental models of chloroquine toxicity. a series of experiments was conducted to assess the effects of chloroquine and diazepam alone and in combination on the contractility, refractoriness and beating rate of isolated rat cardiac tissues. all animal care and experimental procedures were performed in accordance with the uk animals (scientific procedures) act , approved by the institutional ethical review committee, and conducted under the authority of project licences held at the university of liverpool. animal studies are reported in compliance with the arrive guidelines (kilkenny et al., ; mcgrath, mclachlan, & zeller, ) and with the recommendations made by the british journal of pharmacology. • acute chloroquine poisoning manifests as cardiotoxicity and is often managed using diazepam. • diazepam does not attenuate the effects of chloroquine in isolated cardiac tissues nor in vivo. • inotropic support, which is essential for chloroquine poisoning, may be potentiated with diazepam. male wistar rats were bred in the departmental animal unit (the nuffield joint facilities) or in exceptional circumstances of supply shortage acquired from the biomedical services unit, faculty of medicine, or the department of veterinary pathology, university of liverpool. rats were kept under conditions of -h light/dark cycle at c with food (crm diets, sds, witham, essex) and water available ad libitum. the optimal weight range for experimental use was - g. rats were administered , iuÁkg − of sodium heparin by an intraperitoneal injection. after min, they were stunned by a blow to the head, exsanguinated and hearts were excised. isolated atria, ventricular strips (≤ mm in width) dissected longitudinally towards the apex of the heart and right papillary muscles were prepared and suspended in -ml organ baths, containing (in mm), nacl ; kci . ; mgs . ; kh po . ; nahco ; cacl . plus d-glucose . , gassed with % o , % co (boc medical gases, guildford) and maintained at c. each preparation was subjected to a resting diastolic tension of mn and stimulated with square wave pulses of -ms duration at a frequency of hz via a grass s or s stimulator (quincy, massachusetts). tissues were stimulated at twice threshold voltage ≤ v. right atria were allowed to equilibrate such that spontaneous, rhythmic beating occurred. in all cases, tissues were washed periodically throughout the stabilisation period. contractions were measured isometrically via dynamometer uf transducers (sensitivity range, mn) connected to lectromed preamplifiers (letchworth, hertfordshire). the beating rate of right atria was measured with a lectromed ratemeter preamplifier. these were housed within a mt p preamplifier unit, which relayed signals to a mt thermal pen recorder giving an output on heatsensitive paper. each channel was calibrated such that a full-scale deflection of mn could be observed. time to peak tension was measured from the onset of electrical stimulation to the peak of the contraction (penefsky, ) . the effective refractory periods (erps) of left atria, right papillary muscles and ventricular strips were measured using a modification of the extra stimuli method (reuter & heeg, ). a target of six samples of each cardiac tissue were assigned at random to one of four concentrations of chloroquine (calculated as the base; , , , or μm, dissolved in krebs solution) in the presence of the vehicle for diazepam ( % v/v propylene glycol). spontaneously beating right atria were exposed only to -μm chloroquine. the highest concentration of chloroquine ( μm) decreased the responsiveness of most tissues after approximately to min. as the threshold voltage for contraction gradually increased, tissues failed to respond to electrical stimuli. it was for this reason that μm was chosen for a subsequent experiment involving diazepam. in this second experiment, fresh tissues (target of six per group) were incubated with diazepam at concentrations of , or μm for min before the addition of -μm chloroquine. experimental models of toxicity were developed in spontaneously breathing rats, ventilated rats and ventilated rabbits, in which chloroquine was infused at different rates and measurements taken of haemodynamic and electrocardiographic parameters. studies were then conducted in which chloroquine-intoxicated rats were treated with combinations of diazepam, clonazepam, ro - , adrenaline or vehicle control. in developing a model of experimental toxicity, animals (six per group) were allocated at random to different doses of chloroquine diphosphate dissolved in . % w/v nacl. non-ventilated rats were randomised to intravenously infused doses (calculated as chloroquine base) of . , , or mgÁkg − Ámin − , ventilated rats , or mgÁkg − Ámin − and rabbits . , ,or mgÁkg − Ámin − for a maximum period of min or until death, after an initial period of stabilisation of at least min. six treatment randomised controlled trials were subsequently conducted to assess the efficacy of diazepam, clonazepam and ro - :-(i) prior, (ii) during and (iii) after chloroquine intoxication (table ) and the effects of diazepam:-(iv) in high dose, (v) in nonbarbiturate anaesthetised rats and (vi) co-administered with adrenaline. six rats were randomised to each treatment group within each of these studies. benzodiazepines (and vehicles) were administered as a slow intravenous bolus over min. diazepam was administered in a mgÁkg − intravenous bolus dose, based on and mgÁkg − in the study of high dose diazepam, which approximates to the dose recommendations for human cases of overdose (jones, ) when scaled allometrically (nair & jacob, ) . the doses of clonazepam ( . mgÁkg − ) and ro - ( . mgÁkg − ) were chosen to have the equivalent gabaergic and non-gabaergic activity respectively, to mgÁkg − of diazepam (wang, taniguchi, & spector, ) . these approximate to human equivalent doses of . and . mgÁkg − , respectively. the dose of adrenaline ( . μgÁkg − Ámin − ) was chosen t a b l e experimental protocols for randomised controlled trials (i) to (vi) conducted in anaesthetised rats to correspond to that which produced % increase in maximum rate of left ventricular pressure (lv + dp/dt max ) in anaesthetised rats (latini, zuanetti, conforti, schwartz, & lazzara, ) . however, this is appreciably lower (allometrically scaled human equivalent dose of . μgÁkg − Ámin − ) than infusion rates in human poisoning, which are titrated to maintain arterial pressure (median maximal rate of . μgÁkg − Ámin − ) (mégarbane et al., ) . male wistar rats (as above) and female new zealand white rabbits, which were either bred in the departmental animal unit or purchased from harlan interfauna (huntingdon, cambridgeshire), were used. rabbits were housed under ambient conditions of a -h light/dark cycle at c with food (r from sds, witham, essex) and given amprolium hci . % w/v and ethopabate . % w/v ( . ml per ml) drinking water for days as a prophylaxis against coccidiosis infection. for rabbits, the optimal weight range for experimental use was - kg. anaesthesia was induced in rats with sodium pentobarbitone of mgÁkg − intraperitoneally and, once venous access was established, maintained with intravenous boluses of mg as required. in the treatment study (v), urethane was prepared as a % w/v solution in isotonic saline and administered as an intraperitoneal dose of . gÁkg − . neuroleptanalgesia was induced in rabbits by an intramuscular injection of . mlÁkg − hypnorm ( . mgÁml − fentanyl citrate and mgÁml − fluanisone). surgical anaesthesia was achieved by administering sequential -mg boluses of sodium pentobarbitone into the marginal ear vein and then -mg boluses via a cannulated femoral vein, as required, upon commencement of ventilation. femoral veins were cannulated for venous access for drug administration. the right common carotid and a femoral artery were accessed for measurement of left ventricular pressure and recording of bp using a druck pdcr or a bell and howell type - - pressure transducer. a tracheotomy was performed to facilitate respiration, and a wide bore cannula secured in place. subcutaneous stainless-steel needle electrodes were inserted to each limb for the recording of the ecg. all animals were maintained at a rectal temperature of c. ventilation necessitated a thoracotomy at the fifth intercostal space as, in closed-chest rats, excessive contractions of the diaphragm and intercostal muscles were found to prevent effective respiration. a positive end-expiratory pressure was exerted and air ventilation provided at strokesÁmin − ( - . ml per stroke) using a harvard bioscience small animal respirator. rabbits were ventilated with air at strokesÁmin − ( - ml per stroke). a thoracotomy was not necessary in anaesthetised rabbits. blood gases were measured using a corning or ph/blood gas analyser. stroke volumes were adjusted for pre-drug po > mmhg and pco > mmhg. animals were excluded with pre-drug mean arterial bp < mmhg (in anaesthetised rats) or < mmhg (in ventilated rabbits), arterial po of < mmhg, arterial pco < or > mmhg or if arrhythmias occurred during the stabilisation phase of the experiment. in the randomised trials, rats were excluded if they died prior to the administration of chloroquine in trial (i) or the treament in trials (ii) to (vi). arterial bp, left ventricular pressure, and its first derivative (lv ± dp/dt max ) and contractility index lv + dp/dt max /p, left ventricular end-diastolic pressure, heart rate and ecg (lead ii) were measured and recorded using lectromed systems (letchworth, hertfordshire) or a grass d recorder (quincy, massachusetts) connected to a po-ne-mah digital data acquisition system (linton, diss, norfolk) and recorded at a sampling rate of , hz. blood samples for the determination of chloroquine concentration were obtained from trial (iv). approximately -μl arterial blood samples were drawn after , and min of chloroquine infusion from six rats for the analysis of whole blood chloroquine concentrations. four -μl aliquots were accurately pipetted on to a sheet of whatman grade blotting paper and protected from light exposure. standards were prepared by adding aliquots of chloroquine, giving final concentrations ranging from to μm, on to chloroquinefree blood spots. all samples were carefully cut from the surrounding chloroquine was detected using an isochrom lc spectra-physics pump equipped with a rheodyne injector, a spectra fluorescence detector, and a chromjet integrator. the excitation wavelength was nm, and a -nm emission filter was used (looareesuwan et al., ) . the column ( . m × . mm of internal diameter) was packed with spherisorb silica ( -μm particles; capital hplc) and eluted with an isocratic mobile phase consisting of acetonitrile:methanol:diethylamine ( : . : . ), flowing at . mlÁmin − . the limit of detection for chloroquine was . nm, and the precision of the method was . % at nm. in randomised trial (vi), arterial blood samples were analysed for blood gases, ph and electrolyte (k + , na + , and free ca + ) concentrations using statistical analyses were performed using arcus pro-stat version . . the data and statistical analysis comply with the recommendations of the british journal of pharmacology on experimental design and analysis in pharmacology (curtis et al., ) with the exception that the analysis was not blinded. tissues from rats ( ± g) were used. decreases in the developed tension of left atria were observed with chloroquine at the highest concentration of μm. the negative inotropic effect was time dependent, with maximal changes observed by min. chloroquine did not significantly alter the developed tension or time to peak tension of right ventricular strips or papillary muscles but significantly increased the time to peak tension in atria ( ± ms with chloroquine [ μm] compared to ± ms in the control group). chloroquine prolonged the erp of all tissues. in left atria, for instance, the prechloroquine erp was ± ms (in the μm group), which significantly increased to ± ms after -min exposure to chloroquine. diazepam alone was without effect on papillary muscles or ventricular tissue other than a small but significant increase from ± to ± ms in the time to peak tension of contracting right ventricular strips at μm. however, diazepam of μm evoked a positive inotropic response and prolonged the erp of left atria and had a significant negative chronotropic effect on right atria ( ± vs. ± beatsÁmin − ). diazepam in the concentration range of to μm did not appear to protect against the effects of -μm chloroquine (table ) . at the highest concentration, diazepam lengthened the erp and extended the time to peak tension in left atria and reduced rate of beating right atria. chloroquine caused dose-dependent negative inotropy in both species. reductions in lv + dp/dt max during the first to min of infusion seemed more pronounced than reductions in bp. for example, a % reduction in lv + dp/dt max occurred during the first min of infusion at mgÁkg − Ámin − compared with a % reduction in diastolic pressure for the same period in non-ventilated rats. cardiac lusitropy (lv − dp/dt max ) declined in a parallel manner to the negative inotropic response. increases in left ventricular end-diastolic pressure were observed with chloroquine in non-ventilated rats and ventilated rabbits. chloroquine caused a similar dose-dependent bradycardia over the time course of the experiment in both ventilated and nonventilated rats for the corresponding doses. in rabbits, however, heart rate declined abruptly by approximately half at time points corresponding to the onset of arrhythmias. in rats, increases in the pr intervals occurred with all doses of chloroquine and in proportion to the cumulative dose received. for example, the pr interval increased from ± to ± ms during the first min in ventilated rats receiving mgÁkg − Ámin − chloroquine, and from ± to ± ms during the first min at twice the infusion rate, with both groups receiving a total of mgÁkg − of chloroquine over these periods. chloroquine also caused a dose-dependent increase in the pr interval in rabbits. chloroquine broadened the qrs complex in all animals (figure ), although this was not as pronounced with the slower infusion rates as the changes in pr duration. in ventilated rats, for example, the qrs duration increased by % in the first min of chloroquine being infused at mgÁkg − Ámin − while a % increase in pr interval occurred over the same period. qt interval prolongations were observed with high infusion rates, but these were not as apparent when the qt was corrected for rate effects. a substantial increase in qtc occurred only with mgÁkg − Ámin − in ventilated rats. effects of chloroquine ( μm) in the presence of propylene glycol % v/v (control) or diazepam ( , and μm) on the developed tension, effective refractory period and time to peak tension of left atria, right ventricular strips, right papillary muscles and on the spontaneous beating rate of right atria in all rabbits, arrhythmias presented as mobitz type ii, seconddegree av block with a conduction ratio of : (two p waves for each qrs complex). the onset of arrhythmias was dose dependent and with higher degrees of block eventually occurring at the faster infusion rates. these largely degenerated to ventricular tachycardia and fibrillation. i efficacy of ligands for benzodiazepine binding sites (before infusion of chloroquine) twenty-seven rats entered in the study, but three died immediately following the administration of clonazepam and were excluded. there were no differences between pre-and min post-drug haemodynamics or ecg parameters or between randomised groups with the administration of diazepam, clonazepam, ro - , or vehicle. in the presence of these agents, chloroquine reduced bp, heart rate, contractility index and increased the pr, qrs and qtc intervals ( note: data are mean ± sem, of time-averaged measurements taken over min during administration of chloroquine in trial (i), and over min following administration of drug trestments in trials (ii) to (vi). a [+dp/dtmax/p] was measured in each experiment with the exception of experiment (v), which measured [+dp/dtmax]. *p < . versus control group. iv efficacy of high dose diazepam (during infusion of chloroquine) in contrast to trial (ii), an infusion of chloroquine for min did not cause significant changes in any of the haemodynamic or ecg parameters. chloroquine did not reduce mean bp and heart rate or increase the qtc interval significantly in those randomised to diazepam and did not increase the qrs interval in either group. following treatment, heart rate increased significantly in the diazepam group. there were no differences between treatment groups in other parameters and none developed arrhythmias (table ) . the whole blood chloroquine concentration in these rats was . ± . μm after min of infusion ( mgÁkg − Ámin − ), . ± . μm after min and . ± . μm after min. v efficacy of diazepam (during infusion of chloroquine) with a nonbarbiturate anaesthetic (urethane) over min of administration, chloroquine only significantly affected the pr and qrs intervals. there were no subsequent differences between groups, following administration of diazepam or vehicle control, in any of the measured parameters and none developed arrhythmias (table ) . a further randomised trial was initiated with chloroquine infused at a higher rate of mgÁkg − Ámin − in order to evaluate the effects of diazepam on more pronounced toxicity. however five of the first nine rats died and the study was terminated. vi efficacy of diazepam and adrenaline (during infusion of chloroquine) twenty-seven rats was included, but three died before the end of the experiment, one from each of the control, diazepam and diazepam + adrenaline groups. during the first min of infusion, chloroquine caused significant changes in all parameters, with the exception of the qrs and qtc intervals in the adrenaline group. the lack of an effect with diazepam ( mgÁkg − ) alone was consistent with trial (ii). the effects of adrenaline alone did not deviate significantly from the control group in any parameter other than the qrs interval, but this was not prolonged following chloroquine (figure ) . the combined administration of diazepam and adrenaline resulted in an improvement of cardiac contractility compared to the control and diazepam groups but not the adrenaline group ( ± vs. ± s − ). no significant differences were observed in the other parameters or incidence of arrhythmias (table ) . pre-chloroquine potassium concentrations were in the range expected for rats (burns & de lannoy, ) . chloroquine alone did not cause any significant changes in arterial po , pco or ph values over a period of -min infusion. the combined administration of diazepam and adrenaline, however reduced po when compared to t a b l e whole blood ph, gas, and electrolyte concentrations measured at baseline (pre-chloroquine), following min of chloroquine infusion (pre-drug treatment/vehicle), and min post-treatments pre-treatment values but not when compared to the other groups. chloroquine did not alter electrolyte concentrations; but pretreatment groups containing adrenaline were more hypokalaemic than the diazepam and control groups (table ). the results of the study indicate that chloroquine prolongs the erp of isolated rat atria. in vivo experiments also revealed that chloroquine induces cardiac arrhythmias and pathophysiologic changes in haemodynamic and electrocardiographic parameters. different protocols of diazepam administration did not result in significant improvement in cardiac function either in vitro or in vivo. however, the administration of diazepam and adrenaline in combination may be effective against chloroquine cardiotoxicity by improving cardiac contractility. the findings from the in vitro studies are congruent with previous experiments demonstrating the acute cardiotoxic effects of chloroquine (essien & ette, ; tona, ng, akera, & brody, ) . at the concentrations of chloroquine used, left atria were more sensitive to detrimental effects on mechanical performance than either ventricular or papillary tissue preparations. decreases in developed isometric tension, together with increases in times to peak tension, were observed, which are indicative of impaired atrial contractility. an increase in the time to peak tension by chloroquine reflects a prolongation of one or more phases of the cardiac excitation-contraction cycle and is consistent with the ability of chloroquine to block cardiac ion channels (essien & ette, ; rodríguez-menchaca et al., ; sánchez-chapula, salinas-stefanon, torres-jácome, benavides-haro, & navarro-polanco, ; tona et al., ) . ikhinmwin, sofola, and elebute ( ) demonstrated a negative inotropic response which was reversed in the presence of increased extracellular calcium aimed to promote calcium influx via unblocked l-type calcium channels. tona et al. ( ) demonstrated chloroquine to inhibit the treppe response in atrial guinea pig preparations, but without effect on postextrasystolic potentiation of contractile force, suggesting that chloroquine interferes with cellular calcium influx upon which the treppe response is dependent, but not the latter response which is dependent on intracellular calcium mobilisation. increases in the refractoriness of cardiac tissues are indicative of potassium and/or sodium ion channel blockade. using voltage-clamped cat ventricular myocytes, sánchez-chapula et al. ( ) observed that chloroquine blocked several inward and outward membrane currents. the order of potency ( - μm range) was inward rectifying potassium current > rapid delayed rectifying potassium current > sodium current > l-type calcium current. neither the transient outward potassium current nor the slow delayed rectifying potassium current was modified by chloroquine. salinas and cebada ( ) also demonstrated that chloroquine blocks the inward rectifying potassium current in dog cardiac myocytes but had no effects on either the transient outward or the delayed rectifier currents. rodríguez-menchaca et al. ( ) established that chloroquine blocks the inward rectifier k ir . channels, that underlie the cardiac inward rectifier potassium current, from the cytoplasmic surface. other quinolone antimalarials also have known actions in modulating cardiac electrical activity, including blockade of human ether-ago-go related gene (herg) potassium and l-type calcium channels (coker, batey, lightbown, díaz, & eisner, ; kim, lee, cha, kwon, & kim, ; michel, wegener, & nawrath, ) . diazepam had little effect on the function of myocardial tissue, other than at μm, where it increased the erp and peak developed tension in left atrial preparations and increased the times to peak tension in right ventricular strips. diazepam inhibits pde (collado et al., ) , suggesting a possible mechanism for cardioprotection, although this occurs at lower concentrations (ic of . μm) than required to elicit responses in the present investigation. the responses of cardiac tissues to chloroquine in the presence of diazepam were no different from vehicle controls, supporting previous observations that diazepam does not attenuate the cardiac effects of chloroquine via a direct action upon the heart (riou et al., ) . the in vivo experimental models of chloroquine toxicity indicated that impaired cardiac contractility was the primary event in the sequalae of toxicity. hypotension, bradycardia, changes in ecg intervals, arrhythmias and death followed in a similar manner as described previously (sofola, ) . however, cardiac arrhythmias may be less prevalent in cases of human chloroquine poisoning which occurs following oral ingestion (absorption half-life $ min) and where blood concentrations are predominantly governed by the distribution and redistribution processes from the various body compartments back to the intravascular space (mégarbane et al., ) . differences between species in cardiac electrophysiology may also explain varying arrhythmic manifestations. the provision of mechanical ventilation did not appear to influence the onset or the severity of these effects. significant changes in cardiovascular function occurred in the absence of changes in either arterial blood gas levels or ph, suggesting that toxic manifestations due to chloroquine are not secondary to hypoxia. chloroquine was about twice as potent in its toxic effects in rabbits than in rats (on a mgÁkg − basis), where whole blood concentrations were within the - μm range. this concentration range is comparable with the concentrations used in the in vitro experiments and observed in human toxicokinetic studies. clemessy et al. ( ) reported a mean whole blood chloroquine concentration of . μm (range . to μm) among patients admitted to intensive care. mégarbane et al. ( ) correlated mild cardiotoxicity with peak concentrations ≤ μm, moderate - μm and severe > μm. chloroquine is extensively distributed to extravascular tissues and some reversal of cardiotoxicity would be expected upon cessation of administration. the in vivo experimental models of chloroquine toxicity did not test this. however, in experiment (iii), the administration of chloroquine ( mgÁkg − Ámin − i.v.) ceased after min and recovery in cardiovascular parameters was observed in all treatment/vehicle groups. the series of randomised controlled trials was designed to assess whether modulation of the gaba a receptor or other effects of diazepam might account for previous reports of reduced toxicity with chloroquine. however, diazepam, whether administered prior, during or after the administration of chloroquine or at high dose, failed to attenuate chloroquine-induced cardiotoxicity in anaesthetised rats. these results are consistent with previous studies in spontaneously breathing rats anaesthetised with thiobutobarbitone (buckley et al., ) , but contrast with experiments performed in conscious rats (crouzette et al., ) and pentobarbitone-anesthetised, mechanically ventilated pigs . possible explanations for these discrepancies might include the choice of species, doses of chloroquine and diazepam, and anaesthesia. based on allometric scaling to human doses, and mgÁkg − of diazepam administered to rats correspond to . and . mgÁkg − in adults. effects were similar in the trial in which urethane was chosen as an anaesthetic for its lack of interaction with gaba a receptors. experiments aimed to differentiate any gaba mediated versus other effects of diazepam, used ro - , which has activity at the mitochondrial tspo benzodiazepine binding site distinct from the gaba a receptors in the cns and clonazepam, which rapidly crosses the blood-brain barrier and is a potent positive allosteric modulator of gaba a receptors, while having low affinity towards tspo. mitochondrial tspo is ubiquitously expressed in various tissues, including the heart with a putative role in regulating heart rate and contractility (surinkaew, chattipakorn, & chattipakorn, ) . as neither diazepam nor either of these agents protected against or attenuated chloroquine toxicity, it is unlikely that any cardiovascular effects in the context of chloroquine toxicity can be attributed to interaction with benzodiazepine binding sites. in view of the fact that the principal adverse effect of chloroquine is negative inotropy (sofola, ) and the absence of positive inotropic effects of diazepam under basal conditions (and negative inotropy under certain conditions [zeegers, van wilgenburg, & leeuwin, ]), the use of a positive inotrope seems essential for the improvement in the cardiac function following chloroquine toxicity. while neither diazepam nor adrenaline alone reversed any chloroquine-induced cardiovascular changes, the improvement in cardiac contractility observed with their combined administration may indicate a beneficial interaction. studies in rat ventricular tissues demonstrated that diazepam ( μm) augmented contractility due to isoprenaline (martinez, peñafiel, collado, & hernández, ) , noradrenaline (juan-fita, vargas, & hernández, ) and dopamine (juan-fita, vargas, & hernández, ) . these effects were not mimicked by gaba nor antagonised by the selective tspo inhibitor pk or flumazenil, an antagonist of the gaba a benzodiazepine binding site. rather, they were attributed to diazepam's ability to inhibit pde , the main isoenzyme responsible for the inotropic effect of β-adrenoceptor agonists in the rat myocardium. this offers a plausible mechanism for the observed effects in chloroquine-intoxicated rats. however, there are differences between species in the expression of pde , with a fivefold higher amount of non-pde activity in human hearts compared to rodents, and this will impact on the effect of enzyme inhibition (richter et al., ) . further mechanistic studies are warranted to assess the role of pde inhibition in this context. in conclusion, the results of this study do not offer compelling support for the use of diazepam in reducing chloroquine cardiotoxicity. ligands for benzodiazepine binding site, clonazepam, and ro - were similarly ineffective in the experimental models used. however, the results provide evidence that diazepam might enhance cardiac contractility when co-administered with adrenaline, although the lowering of whole blood potassium concentrations, consistent with agonism of β -adrenoceptors in skeletal muscle, might risk exacerbation of chloroquine-induced hypokalaemia (clemessy et al., ) and increased arrhythmogenicity. these new insights have important clinical and research implications in the current context of widespread publicity and use of chloroquine for covid- . chloroquine is widely used and available without prescription in many countries, including the united kingdom, presenting dangerous opportunities for unintentional overdose. for the management of patients with chloroquine poisoning, testing the efficacy of a positive inotrope with a greater selectivity for β adrenoceptors, such as dobutamine, would be desirable, as would a greater understanding of the non-cardiovascular roles for diazepam treatment given that chloroquine poisoning often causes convulsions, which can be intractable. press release-coronavirus: situation point in new aquitaine the concise guide to phar-macology / : ion channels an analysis of the time-relations of electrocardiograms coronavirus: man dies after ingesting chloroquine in attempt to ward off covid- . sky news effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial effects of catecholamines and diazepam in chloroquine poisoning in barbiturate anaesthetised rats compendium of normal blood values of laboratory animals with indication of variations. i. random-sexed populations of small animals nigeria records chloroquine poisoning after trump endorses it for coronavirus treatment therapeutic trial of diazepam versus placebo in acute chloroquine intoxications of moderate gravity hypokalaemia related to acute chloroquine ingestion effects of mefloquine on cardiac contractility and electrical activity in vivo, in isolated cardiac preparations, and in single ventricular myocytes functional and biochemical evidence for diazepam as a cyclic nucleotide phosphodiesterase type inhibitor experimental assessment of the protective activity of diazepam on the acute toxicity of chloroquine experimental design and analysis and their reporting ii: updated and simplified guidance for authors and peer reviewers effects of diazepam on mortality from acute chloroquine poisoning. annales françaises d'anesthèsie et de rèanimation intoxications volontaires par la chloroquine effects of chloroquine and didesethylchloroquine on rabbit myocardium and mitochondria chloroquine and hydroxychloroquine in covid- request for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of coronavirus disease les effets antagonistes du diazépam et du ro - dans l'intoxication aiguë par la chloroquine chez le rat et chez le cobaye sont-ils de nature centrale ou périphérique? the iuphar/bps guide to phar-macology in : updates and expansion to encompass the new guide to immunopharmacology the effects of calcium ions on the depression of cardiac contractility by chloroquine and quinine clinical features and management of poisoning due to antimalarial drugs critical care toxicology: diagnosis and management of the critically poisoned patient comparative actions of diazepam and other phosphodiesterase inhibitors on the effects of noradrenaline in rat myocardium diazepam enhances inotropic responses to dopamine in rat ventricular myocardium. anesthesia and analgesia animal research: reporting in vivo experiments: the arrive guidelines blockade of herg k(+) channel by antimalarial drug, primaquine demonstration of a different sensitivity to epinephrine in isolated and in vivo hearts cardiovascular toxicity and distribution kinetics of intravenous chloroquine diazepam potentiates the positive inotropic effect of isoprenaline in rat ventricle strips: role of cyclic amp transparency in research involving animals: the basel declaration and new principles for reporting research in bjp manuscripts chloroquine poisoning. rapidly fatal without treatment blood concentrations are better predictors of chioroquine poisoning severity than plasma concentrations: a prospective study with modeling of the concentration/effect relationships effects of quinine and quinidine on the transient outward and on the l-type ca + current in rat ventricular cardiomyocytes a simple practice guide for dose conversion between animals and human the determinants of contractility in the heart the use of diazepam in chloroquine poisoning action of antiarrhythmic and -receptor blocking drugs on functional refractory period and contractility of electrically stimulated guinea-pig atria conserved expression and functions of pde in rodent and human heart treatment of severe chloroquine poisoning diazepam does not improve the mechanical performance of rat cardiac papillary muscle exposed to chloroquine in vitro protective cardiovascular effects of diazepam in experimental acute chloroquine poisoning the molecular basis of chloroquine block of the inward rectifier kir . channel chloroquine selectively blocks the inward rectifying potassium channel in dog cardiac myocytes blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes -year-old girl near coimbatore dies after parents administer hydroxy-chloroquine drug as covid- preventive medicine the cardiovascular effect of chloroquine in anaesthetized dogs roles of mitochondrial benzodiazepine receptor in the heart depressant effects of chloroquine on the isolated guinea-pig heart structural requirements for the binding of benzodiazepines to their peripheral-type sites review of side effects and toxicity of chloroquine/by h. weniger. geneva: world health organization the cardiotoxicity of antimalarials. malaria policy advisory committee wuhan woman who was not infected with the new coronavirus, but bought and overdosed on prescription drugs, was sent to icu. the paper diazepam for treatment of massive chloroquine intoxication cardiac effects of benzodiazepine receptor agonists and antagonists in the isolated rat heart: a comparative study acute chloroquine poisoning: a comprehensive experimental toxicology assessment of the role of diazepam the author declares no conflicts of interest. this declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the bjp guidelines for design and analysis and animal experimentation, and as recommended by funding agencies, publishers, and other organisations engaged with supporting research. adrenaline diazepam diazepam + adrenaline key: cord- - fsl authors: wathes, d. claire; oguejiofor, chike f.; thomas, carole; cheng, zhangrui title: importance of viral disease in dairy cow fertility date: - - journal: engineering (beijing) doi: . /j.eng. . . sha: doc_id: cord_uid: fsl many viral diseases are endemic in cattle populations worldwide. the ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. there is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. these effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. this paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. acute infection with non-cytopathic bovine viral diarrhea virus (bvdv) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. bvdv infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. fertility may also be reduced by bvdv-induced immunosuppression, which increases the susceptibility to bacterial infections. bovine herpesvirus (bhv)- is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. previously infected animals subsequently show reduced fertility. although this may be associated with lung damage, ovarian lesions have also been reported. both bhv- and bhv- remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. while bhv- infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as escherichia coli and trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. both schmallenberg virus (sbv) and bluetongue virus (btv) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations. btv- also impairs the development of hatched blastocysts; furthermore, infection around the time of breeding with either virus appears to reduce conception rates. although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes. although viral disease remains a major cause of financial loss to the modern cattle industry, its potential impact on fertility is generally underestimated, and the main mechanisms of action are often unclear. factors including trade globalization, increases in herd size, and environmental change have contributed to the spread of existing pathogens and the introduction of disease into regions and animal populations that were previously free of it [ ] . poor fertility and udder health/milk quality remain the two major causes of concern among dairy producers [ ] . in terms of fertility, the ability of viruses to cause abortions and fetal malformations has probably received the most attention [ ] . the outcome is generally dependent on the stage of pregnancy during which the initial infection occurs. the effects of viral diseases on reproductive performance are, however, much more pervasive and can have many subtle effects through reductions in conception rates and increased risk of culling through failure to conceive in a timely fashion. excluding fertilization failure, approximately % of bovine embryos die in the first three weeks after service or insemination, with cows returning to estrus after - d. a further %- % of embryos are lost between days - of gestation [ ] . in comparison, abortion rates on cattle farms are usually quite low ( %- %) and have many potential etiologies that are often difficult to diagnose reliably [ ] . in addition to the loss of the fetus, an abortion does, however, often have adverse effects on the fate of the dam. depending on the stage of gestation when it occurs, the cow either may need to be rebred (thus increasing her calving interval) or may start the next lactation prematurely. in one study, for example, . % of holstein heifers aborted. this increased their risk of leaving the herd without completing a first lactation . times. one third of the animals which did not complete a first lactation either died or had to be culled within d of calving [ ] . the present short review focuses on five different viral infections showing a variety of mechanisms that can have an impact on dairy cow fertility. bovine viral diarrhea virus (bvdv) is discussed first and in the most detail, as its effects on fertility have been the most widely studied; thus, more is understood about its potential underlying mechanisms. bvdv is a flaviviridae pestivirus that is endemic in many countries worldwide, with a prevalence of %- % in individual cattle and %- % in cattle herds [ , ] . it comprises a single-stranded, positive-sense rna genome that is classified by sequence differences as type or (bvdv- or bvdv- ). there is also a third type, bvdv- (a hobi-like, atypical pestivirus). the virus exists as either non-cytopathogenic (ncp) or cytopathogenic (cp) biotypes, with the ncp biotype causing the majority of field losses [ ] . bvdv exhibits vertical transmission from mother to fetus, has a broad tissue tropism, and can infect the host either transiently or persistently [ ] . such rna viruses display significant genetic variation, facilitating the emergence of new species [ ] . mammalian cells normally produce type i interferons (ifns) in response to viral infection, which then trigger a cascade of antiviral pathways. bvdv causes immunosuppression through its ability to inhibit ifn production, thereby delaying the host's responses and enhancing the ability of the virus to complete its replication cycle [ , ] . bvdv infection generally occurs via the oronasal route, but direct transmission to the reproductive tract via semen or embryo transfer is also possible [ , ] . acutely infected animals usually eliminate the virus within - d, but transmissible virus can persist for much longer in some animals that have apparently recovered [ ] . in rare cases, bulls develop a persistent infection of the testes-an immune-privileged site. more commonly, bvdv is detectable by reverse transcription polymerase chain reaction (rt-pcr) in semen for some months after an initial acute infection, although the continued risk of viral transmission appears to be unlikely after nine weeks [ ] . fetal infection with ncp bvdv before the development of immune competence (i.e., prior to gestation day ) results in early embryonic death, later abortion, or the birth of an immunotolerant calf that is persistently infected (pi) [ ] . the pi calf can continuously shed virus from all secretions, and is therefore a major source of infection within a herd. the effects of acute bvdv infection vary extensively depending on both biotype and virulence, and this can lead to either avoidance or initiation of apoptotic and innate immune responses. ncp bvdv can dampen innate immune responses in several ways [ , ] . the virus is first detected by toll-like receptor (tlr)- or tlr- /tlr- located in intracellular compartments or by cytoplasmic pattern-recognition receptors (rig-i, ddx ), which detect single-stranded rna. the downstream signaling pathway from tlr- involves the ifn regulatory factor (irf)- and irf- , which usually upregulate the transcription of type i ifns. the bvdv protein n pro targets irf- toward proteasomal degradation, thus inhibiting downstream signaling and preventing the ifn rise [ ] . guanylate-binding protein (gbp ), an ifn-inducible gtpase, can also inhibit this pathway while leaving nf-jb signaling intact [ ] . in addition, the secreted bvdv structural protein e rns degrades viral rna through its extracellular function as a ribonuclease [ ] . many of the economic losses attributed to bvdv are due to suboptimal fertility, in addition to causing abortion and fetal deformity at later stages of gestation [ , ] . bvdv-induced immunosuppression increases susceptibility to other diseases, which may then also affect fertility. conception rates fell by up to % following experimental infections with bvdv either d before or d after insemination [ ] . the review by fray et al. [ ] cited many similar results that have been reported following ncp bvdv infection in the field, in spite of the occasional report to the contrary. since then, rüfenacht et al. [ ] measured fertility parameters in swiss dairy herds with a high prevalence of bvdv using individual seroconversion measurements to assess the time of likely exposure. infection during the first d of gestation did not influence non-return rates, but infection in mid-gestation was associated with an increased abortion rate from . % to . %. the timing of exposure is clearly critical, as rodning et al. [ ] reported that when pi animals were introduced to naïve heifers d prior to the start of breeding, they developed active immunity and there was no adverse effect on reproductive performance. newcomer et al. [ ] undertook a meta-analysis of studies to determine the potential benefits of vaccination against bvdv on three reproductive outcomes. vaccinated cows experienced a reduction in both abortion and fetal infection rates of nearly % and %, respectively, compared with unvaccinated cohorts, while the risk of becoming pregnant was smaller but nevertheless improved by about %. it is likely that a change of this magnitude would fail to reach significance in smaller studies due to a lack of statistical power. a variety of mechanisms have been suggested to account for such reductions in fertility via effects on the ovary, uterus, and early embryo. bvdv antigen was detectable in ovaries d after acute infection [ ] and in oocytes and follicular cells of pi heifers [ ] . animals infected with bvdv develop oophoritis [ ] and have impaired ovulation and ovarian steroidogenesis [ ] [ ] [ ] . when heifers were infected with acute ncp bvdv, follicular growth patterns were affected through the subsequent two estrous cycles, including reduced growth of dominant follicles [ ] . similarly, when heifers were infected d before a synchronized estrus, luteinizing hormone (lh) pulsatility was decreased, there was a delay from ovulation to the progesterone rise, and subsequent progesterone levels were lower [ , ] . these results align with studies showing that various types of stress can either delay or inhibit ovulation mechanisms [ , ] , while both heat stress and intramammary infection can reduce follicular steroidogenesis, disrupt follicular dominance, and reduce the pre-ovulatory lh surge [ ] . any acute infection occurring at this critical stage of the estrous cycle is likely to have a similar effect. the uterine endometrium is also recognized as a major site for bvdv infection [ , ] . bvdv was found in the uterus - d after infecting heifers with bvdv by either intravenous inoculation or by breeding to a pi bull [ , ] , while ncp bvdv was isolated from uterocervical mucus d after initial infection [ ] . bvdv antigen was also detected in macrophage-like cells of the endometrium in % of cows examined in a slaughterhouse survey [ ] . there is good evidence for two mechanisms by which the uterine presence of bvdv may have detrimental effects on fertility: first, by predisposing cows toward the development of endometritis; and second, by interference with the establishment of pregnancy. the bovine uterus is colonized with many bacterial species following calving in over % of cows [ ] . these bacteria should be cleared rapidly using mucosal defense systems and an innate immune response involving endometrial epithelial and stromal cells in addition to professional immune cells [ , ] . this early innate response is crucial to avoid the development of uterine disease; nevertheless, many dairy cows do develop metritis and/or endometritis (estimated at around % and % of all animals, respectively) [ ] . in cultured bovine endometrial cells, experimental infection with ncp bvdv inhibited a variety of immune pathways normally activated in response to a challenge with bacterial lipopolysaccharide (lps), including downregulation of many interferon-stimulated genes (isgs), which are an important part of uterine defense mechanisms [ , ] . infection with ncp bvdv was also able to switch endometrial prostaglandin production from prostaglandin (pg)f a to pge [ ] . pgf a is recognized as an immune enhancer, while pge acts as an immune suppressor and is luteotrophic [ , ] ; therefore, this switch may also reduce the endometrial immune response to bacteria and increase the likelihood of a cow developing a persistent corpus luteum [ ] , which is often found in association with uterine disease [ ] . maternal recognition of pregnancy in cows is achieved through the production of interferon tau (ifnt) by the trophectoderm of the elongating conceptus [ , ] , which inhibits the development of endometrial oxytocin receptors, thereby preventing luteolysis [ , ] . ifnt is a type i ifn that is structurally related to ifn-a and ifn-b but lacks viral responsive elements in its promoter and is therefore not upregulated by viral infection [ ] . ifnt does, however, bind to the same ifn-a/ifn-b receptor on the uterine endometrium. together with progesterone, ifnt programs the uterine endometrium to develop a receptive environment for implantation, including upregulation of many isgs [ , , ] . these are likely to have crucial roles in the establishment of pregnancy via modulation of uterine immunity, stromal remodeling, hyperplasia of the endometrial glands, and development of the uterine vasculature [ , ] . acute infection with ncp bvdv alone has been shown to have a limited influence on endometrial gene expression in vitro [ ] . however, infection did interfere with the isg regulatory irf-stat and stat pathways to inhibit ifnt-induced isg expression including isg , herc , usp (involved in protein modification via isgylation), ddx , ifih (cytosolic detection of viral rna) and ifit , mx , rsad , and samd (immune regulators with antiviral activity) [ ] . upregulation of the endometrial isgylation pathway is an important process in early pregnancy that is conserved across mammalian species [ ] . therefore, dysregulation of the antiviral ifn response by bvdv can undoubtedly interfere with ifnt signaling in the endometrium, suggesting another mechanism whereby infection in early gestation may reduce conception rates. there has been considerable research on the effects of bvdv on bovine embryos following concern that naïve cows might develop bvdv following embryo-transfer procedures. embryos produced using both in vivo and in vitro techniques have been infected with either ncp or cp virus at all stages from oocyte to hatched blastocyst. the affinity of bvdv for in vivo-derived embryos varied according to the strain of bvdv [ , ] . uterine inoculation with ncp bvdv- in the medium used for embryo transfer on day of a synchronized estrous cycle resulted in / heifers becoming pregnant d later, but these pregnancies had been lost within the following d [ ] . although bvdv replicated efficiently in cumulus cells surrounding bovine oocytes, this did not affect the development of the blastocysts subsequently produced by in vitro fertilization [ ] . similarly, when oocytes, zygotes, -cell embryos, morulae, and hatched blastocysts were infected with either ncp or cp virus, development was only adversely affected with cp bvdv and when the zona pellucida was not present [ ] . in a more recent study, cumulus-oocyte complexes were infected with bvdv- , bvdv- , or bvdv- at different doses [ ] . bvdv- had no effect on the embryos that did develop, and bvdv- infection actually increased cleavage rates but did not affect blastocyst rates. in both cases, however, the degenerate embryos tested positive. overall, the oocytes infected with bvdv- and bvdv- developed normally but carried the virus. bvdv- (hobi-like virus) reduced both cleavage and blastocyst rates, so would be expected to cause preimplantation embryo loss in vivo. bielanski et al. [ ] used semen from a pi bull on superovulated cows, collected day embryos, and transferred washed embryos to clean recipients. although bvdv was detected in the pre-transfer embryos, it did not infect the new host. from this work, it was concluded that the risk of transmission of bvdv to host cows via embryo transfer was minimal providing correct washing procedures were applied, as recommended by international embryo transfer society guidelines [ ] . this resulted in low copy numbers of virus, as measured by a sensitive quantitative polymerase chain reaction (qpcr) technique [ ] . in summary, acute ncp bvdv infection causes intracellular changes to ovarian and endometrial tissues through combined effects on pathways regulating immunity. these effects can reduce cow fertility by causing estrous cycle irregularities, early embryo mortality, and immunosuppression. infections during midgestation increase abortion rates or may give rise to the birth of pi calves. infectious bovine rhinotracheitis (ibr) is a highly contagious respiratory disease caused by bovine herpesvirus (bhv)- that is characterized by acute inflammation of the upper respiratory tract. bhv- is a virus of the family herpesviridae and subfamily alphaherpesvirinae. although some countries have achieved ibr eradication [ ] , the disease remains endemic in dairy herds in many parts of the world, including britain and ireland [ , ] . a recent metaanalysis found a pooled prevalence of bhv- of % in chinese cattle [ ] . it is a major contributing factor in calf pneumonia, which remains the most common cause of mortality and morbidity in dairy calves between and months of age [ ] . bhv- can also cause conjunctivitis, abortions, encephalitis, and generalized systemic infections [ , ] . after the first infection, the virus is never fully eliminated, remaining latent in nerve cells of the brain. from there, it can be reactivated in times of stress, mediated via increased glucocorticoids [ ] [ ] [ ] . bhv- is only one of a diverse range of pathogens that can contribute to bovine respiratory disease (brd) including several other viruses (i.e., bovine respiratory syncytial virus (brsv), parainfluenza iii virus (pi ), bvdv, and corona viruses), bacteria (e.g., mannheimia haemolytica, haemophilus somnus, pasteurella spp., and mycoplasma), and fungal genera (e.g., aspergillus) [ ] . numerous epidemiology studies in various countries around the world have determined that up to % of calves contract brd [ , ] . for the calves that survive, there is mounting evidence of longer term consequences of juvenile disease on adult performance [ , ] . brd-affected animals have reduced growth rates [ , ] , which in turn delay the age at first breeding and first calving. this is often associated with bronchopneumonic lesions and pleural adhesions [ ] . for example, first parity was delayed by a median of six months in heifers that had brd in the first three months of life [ ] . bach [ ] reported that calves experiencing four episodes of brd before first calving had . ± . greater odds of failing to complete their first lactation in comparison with healthy calves. another study found that calving intervals were increased by % in mature cows that had experienced severe brd as calves during their first three months [ , ] . in irish herds with a seasonal calving pattern that were identified as positive by a bulk tank bhv- enzyme linked immunosorbent assay (elisa), the three-week calving rate was significantly lower in multiparous cows in comparison with bhv- negative herds [ ] . two related epidemiology studies in ethiopia found significantly higher rates of uterine infection and retained fetal membranes in cows that were seropositive for bhv- [ , ] . a meta-analysis of over animals showed an overall decrease in abortion risk of % in pregnant cattle vaccinated against bhv- [ ] . a number of studies have investigated the effects of treating cattle with modified live ibr vaccine around the time of breeding. heifers inoculated at estrus [ ] , the day after [ ] , or on days or post-breeding [ ] developed mild oophoritis characterized by foci of necrosis, a few necrotic follicles, and mononuclear cell accumulation in the corpus luteum. heifers inoculated on days or post-breeding did not have lesions in the corpus luteum, but there were numerous necrotic follicles [ ] . such lesions were not found in ovaries from which bhv- was not isolated [ ] . vaccination at estrus was followed by a reduction in circulating progesterone [ , ] ; conception rates were also reduced [ , ] . although this review relates primarily to cows, there is evidence that young bulls exposed to bhv- at about six months of age had reduced sperm quality six months later [ ] . givens [ ] recently reviewed the effects of a number of viral diseases on bulls and the transmission risks of these diseases via semen. in summary, a high proportion of dairy calves experience brd, which is often associated with bhv- infection. this slows growth, leading to an increased age at first calving. fertility, risk of culling, and abortion rates are all subsequently increased. information on the direct effects on the reproductive tract is sparse, but there is some evidence that infection can have a direct effect on ovarian function. bhv- is a double-stranded dna virus that is highly prevalent in some dairy herds and has been associated with reduced fertility [ , ] . in common with other herpes viruses, it can remain latent in the host following an initial infection in several cell types including macrophages. this results in a persistent infection [ ] , which can be reactivated in vitro by glucocorticoids [ , ] . there is evidence from measuring seroconversion that it can also be reactivated in vivo during the periparturient period [ ] and in association with clinical metritis [ ] . like bvdv, bhv- can readily infect the uterus and has been associated with metritis and endometritis; however, its role in fertility is somewhat unclear, as it has often also been found in control cows that did not have uterine infection. in addition, tested cows were usually also positive for recognized bacterial pathogens including escherichia coli, trueperella pyogenes, streptococcus spp., and histophilus somni [ ] [ ] [ ] [ ] . nevertheless, there is evidence that bhv- can be associated with reduced fertility. a comparison between cows requiring one or two inseminations to conceive and those needing more than two inseminations found a higher prevalence of bhv- in the cows requiring more inseminations [ ] . klamminger et al. [ ] also recorded reduced risks of infected animals either being inseminated before d after calving or conceiving within d. unlike ncp bvdv, bhv- is cytopathic, and infection can kill endometrial epithelial and stromal cells [ , ] . accumulating evidence supports the view that bhv- can act as a co-factor with established uterine pathogens to promote the development of endometritis [ , , ] . replication of bhv- depends on immediate early gene (ie ) transactivation, and it has been shown that this promoter is upregulated by pge , tumor necrosis factor-a (tnf-a), escherichia coli, and lps, all of which are associated with bacterial infection of the endometrium [ , ] . bhv- in turn activates the interleukin (il)- gene promoter in endometrial cells [ , ] . this is a key chemokine that attracts granulocytes to the uterus. in a recent study, tebaldi et al. [ ] measured global gene transcription caused by the bhv- infection of cultured bovine endometrial stromal cells. in addition to il- , another main pathway that was activated involved the upregulation of matrix metalloproteinase (mmp)- . mmps are involved in the remodeling of the postpartum endometrium [ ] . they are also important in controlling the balance of immune responses. on the one hand, their proteolytic activity can promote immune cell migration and activate cytokines such as il- , il- , tnf-a, and defensins [ ] . on the other hand, over-activation of mmps has been associated with many immunopathological outcomes (reviewed in ref. [ ] ). in summary, the evidence to date suggests that bhv- infections are quite common in dairy cows. the virus on its own probably does not cause clinical uterine disease, but it can be reactivated from latency in the endometrium following calving and then act together with bacterial pathogens to increase the risk of uterine disease by disrupting innate immunity and impairing uterine repair mechanisms. schmallenberg virus (sbv) first emerged in europe in . phylogenetic analysis showed that it belongs to the simbu serogroup of the genus orthobunyavirus [ ] . sbv is transmitted by culicoides midges and affects both domestic and wild ruminants including sheep, goats, and cattle. the clinical signs of disease in adult cows are quite mild and include fever, a drop in milk yield, and diarrhea with peak viremia - d post-infection [ ] . sbv can both persist in and cross the placenta to replicate in the fetus itself [ ] . depending on the time of exposure, this may result in abortion or severe congenital malformations causing dystocia and the birth of non-viable calves [ , ] . a case control study on swiss dairy farms found that the abortion rate increased to . % in when the sbv infection started, in comparison with a rate of . % the year before [ ] . while these effects on the fetus are the most obvious sign of disease, there is also evidence for adverse effects on the establishment of pregnancy and/or early embryo development. similar to bvdv, it is possible that sbv infection during early pregnancy may disrupt ifnt production, thus compromising the survival of the conceptus. like bvdv, sbv uses a non-structural protein (in this case nss) that degrades cellular rna polymerase ii, resulting in the inhibition of type i ifn production and an increase in virulence [ ] . the impact of the epidemic on the productivity of dairy cattle in the netherlands and parts of germany was assessed at the herd level in a study by veldhuis et al. [ ] , who compared milk production, fertility, and mortality during the epidemic with those from an earlier reference period. in both countries, there was a small but demonstrable decline in fertility parameters during the epidemic, including a significant increase in the number of repeat inseminations required and a decrease of about % in the -day nonreturn rate (from . % to . %). a further analysis was undertaken based on the effects of sbv on swiss dairy cows [ ] . this was analyzed at the individual animal level and similarly found that the number of inseminations per cow was higher during the epidemic for cows showing clinical signs of infection in comparison with non-clinical animals from case and control herds. in this study, the non-return rate was not affected, although this may have been influenced by farms with affected animals stopping their services during the period of active infection. bluetongue virus (btv) is an important orbivirus virus infection of both domestic and wild ruminants. its geographical distribution is primarily dependent on the distribution of culicoides midges, which are the insect vectors [ ] . many serotypes of btv exist, including the btv- strain, which is currently circulating in europe [ ] . in addition to potentially causing high morbidity and mortality and reduced milk production, btv affects reproductive performance in dairy cows [ , ] . the virus can cross the placenta, and bovine fetuses infected before d of gestation develop fatal malformations of the central nervous system [ ] . later studies on btv- also found a higher incidence of congenital malformations in newborn calves [ ] . cows that were seropositive for btv in a californian study were significantly older at first calving [ ] . fetal mortality increased during an outbreak of btv- in belgium in [ ] . an early epidemiological study provided evidence for lower conception rates and longer calvingto-conception intervals in cattle [ ] . more recently, this was confirmed from data obtained after an outbreak of btv- in the netherlands [ ] . this study found that infected cows were five times more likely to return to service within d after their first artificial insemination (ai) and required . times more inseminations. using a different analytical approach, nusinovici et al. [ ] provided evidence that french cows infected with btv- experienced reduced fertility if they had been inseminated from four weeks before until five weeks after the date of disease detection within the herd. together, these studies provide good evidence that btv- infection prevents initial conception and/or has an adverse effect on early embryos. experimental infection of pre-implantation cattle embryos was only cytopathic in embryos with damaged zona pellucidae; there was no evidence of btv transmission to the early embryo in viremic donors [ ] . days - hatched blastocysts were, however, susceptible to btv- infection, showing growth arrest and increased apoptosis [ ] . there is again evidence that btv has the ability to inhibit ifn synthesis. in this case, viral ns protein is able to counteract the host's immune response by downregulating the expression of type i ifn and isgs [ ] . as discussed above for bvdv, this may potentially negate the signals normally associated with the maternal recognition of pregnancy. this literature review confirms that many common viral infections of cattle have adverse effects on dairy cow fertility. abortions and fetal abnormalities are easy to quantify, although in many cases the causal factor remains unknown. in contrast, reductions in conception rates are much more difficult to detect reliably. the effects are dependent on the exact stage of the reproductive cycle when the animal becomes infected, and are influenced by herd and season. some viruses can remain latent, and reactivation around calving is likely in association with the metabolic stress of early lactation. others have synergistic actions with other infectious agents, either directly or indirectly by promoting immunosuppression in the host. this may interrupt reproductive processes such as ovulation and implantation as well as predisposing the animals to bacterial infections of the reproductive tract. determination of significant effects on fertility rates in the field is dependent on having significant power in the study to detect potentially small changes. it is also complicated by our inability to capture reliable data on many other factors that influence fertility, such as the previous disease and vaccination history and the current metabolic status of individual cows. in vitro studies using primarily uterine endometrial cells and embryos have provided useful evidence on mechanisms of action. however, very few studies have made a thorough examination of the effects on the reproductive tract of viral infection in vivo. this is understandable, given the costs involved and the practicalities of maintaining infectious cows in containment facilities over a sufficient period of time. despite these limitations, the available data do strongly suggest that viral disease plays a key but currently under-recognized role in reducing cow fertility. given the importance of viral diseases in global cattle production, attempts to eradicate-or at least reduce-the prevalence of such diseases is vital. rigorous quarantine procedures can help prevent the spread of novel diseases between countries. national measures can incentivize farmers to increase their use of regular testing and vaccination. local regulatory organizations must remain vigilant to detect novel viral diseases or variant strains of existing viruses as rapidly as possible after their emergence. disease monitoring may also be facilitated by new technologies, such as a computational approach to pathogen discovery based on bioinformatic analysis of rna sequencing data from whole blood [ ] . such measures should pay dividends by improving conception rates and longevity within the dairy herd. potential applications for antiviral therapy and prophylaxis in bovine medicine setting priorities for non-regulatory animal health in ireland: results from an expert policy delphi study and a farmer priority identification survey common, emerging, vector-borne and infrequent abortogenic virus infections of cattle reproduction, events and management: mating management: fertility. in: reference module in food sciences infectious causes of embryonic and fetal mortality ruminant nutrition symposium: optimizing performance of the offspring: nourishing and managing the dam and postnatal calf for optimal lactation, reproduction, and immunity control of bovine viral diarrhea virus in ruminants herd-level prevalence of selected endemic infectious diseases of dairy cows in great britain bvdv genotypes and biotypes: practical implications for diagnosis and control pestiviruses: how to outmaneuver your hosts viral quasispecies establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type i interferon differential activation of interferon regulatory factors- and - by non-cytopathogenic and cytopathogenic bovine viral diarrhoea virus association of bovine embryos produced by in vitro fertilization with a noncytopathic strain of bovine viral diarrhea virus type ii epidemiology of prolonged testicular infections with bovine viral diarrhea virus infectivity of pestivirus following persistence of acute infection bovine viral diarrhoea: pathogenesis and diagnosis ubiquitination and proteasomal degradation of interferon regulatory factor- induced by npro from a cytopathic bovine viral diarrhea virus trim is a virus-and interferoninducible e ubiquitin ligase that restricts pestivirus infection pestiviral e rns blocks tlr- -dependent ifn synthesis by ll complexed rna a field investigation of the effects of bovine viral diarrhea virus infection around the time of insemination on the reproductive performance of cattle the effects of bovine viral diarrhoea virus on cattle reproduction in relation to disease control the effect of infection with bovine viral diarrhea virus on the fertility of swiss dairy cattle reproductive and economic impact following controlled introduction of cattle persistently infected with bovine viral diarrhea virus into a naive group of heifers efficacy of bovine viral diarrhea virus vaccination to prevent reproductive disease: a meta-analysis changes in ovarian follicles following acute infection with bovine viral diarrhea virus bovine viral diarrhoea virus: its effects on ovarian function in the cow the effect of bovine pestivirus infection on the superovulatory response of friesian heifers reproductive consequences of infection with bovine viral diarrhea virus detection of bovine viral diarrhea virus in the ovaries of cattle acutely infected with bovine viral diarrhea virus modulation of sex hormone secretion in cows by acute infection with bovine viral diarrhoea virus what is stress, and how does it affect reproduction? why is it getting more difficult to successfully artificially inseminate dairy cows? comparing the effects of heat stress and mastitis on ovarian function in lactating cows: basic and applied aspects embryos produced from fertilization with bovine viral diarrhea virus (bvdv)-infected semen and the risk of disease transmission to embryo transfer (et) recipients and offspring distribution of bovine viral diarrhea virus (bvdv) in the genital system tissues of cattle defining postpartum uterine disease in cattle defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle global transcriptomic profiling of bovine endometrial immune response in vitro. i. effect of lipopolysaccharide on innate immunity global transcriptomic profiling of bovine endometrial immune response in vitro. ii. effect of bovine viral diarrhea virus on the endometrial response to lipopolysaccharide acute bovine viral diarrhea virus infection inhibits expression of interferon s-stimulated genes in bovine endometrium bvdv alters uterine prostaglandin production during pregnancy recognition in cows steroidal regulation of uterine resistance to bacterial infection in livestock bacterial lipopolysaccharide induces an endocrine switch from prostaglandin f a to prostaglandin e in bovine endometrium effect of interferon-s on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin e risk factors for post partum ovarian dysfunction in high producing dairy cows in belgium: a field study sexual dimorphism in interferon-tau production by in vivo-derived bovine embryos interferon-s, a type interferon involved in maternal recognition of pregnancy the oxytocin receptor, luteolysis and the maintenance of pregnancy maternal-embryo interaction leading up to the initiation of implantation of pregnancy in cattle trophoblast interferons pregnancy recognition signaling mechanisms in ruminants and pigs conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant? the immunology of early pregnancy in farm animals isgylation: a conserved pathway in mammalian pregnancy different strains of noncytopathic bovine viral diarrhea virus (bvdv) vary in their affinity for in vivo-derived bovine embryos effects of oocytes exposure to bovine diarrhea viruses bvdv- , bvdv- and hobi-like virus on in vitro-produced bovine embryo development and viral infection intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo-derived bovine embryos noncytopathogenic and cytopathogenic bovine viral diarrhea-mucosal disease viruses do not affect in vitro embryonic development into the blastocyst stage replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona-free and zona-intact in vitro-produced bovine embryos and the effect on embryo quality bovine viral diarrhea virus (bvdv): epidemiologic concerns relative to semen and embryos bovine viral diarrhea virus (bvdv) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure pro and contra ibr-eradication genetic variability in the humoral immune response to bovine herpesvirus- infection in dairy cattle and genetic correlations with performance traits meta-analysis of prevalence of bovine herpes virus in cattle in the mainland of china disease management of dairy calves and heifers effect of primary and recurrent infections bovine rhinotracheitis virus infection on the bovine ovary characterization of dexamethasoneinduced reactivation of latent bovine herpesvirus a mutation in the latency-related gene of bovine herpesvirus disrupts the latency reactivation cycle in calves rates and risk factors for contagious disease and mortality in young dairy heifers analysis of pre-weaning feeding policies and other risk factors influencing growth rates in calves on commercial dairy farms the effect of early calfhood health status on survivorship and age at first calving the effect of group size on health and growth rate of swedish dairy calves housed in pens with automatic milk-feeders associations between several aspects of heifer development and dairy cow survivability to second lactation use of treatment records and lung lesion scoring to estimate the effect of respiratory disease on growth during early and late finishing periods in south african feedlot cattle effect of calfhood morbidity on age at first calving in new york holstein herds calving interval in dairy cows in relation to heifer rearing conditions in southwest sweden associations between housing, management, and morbidity during rearing and subsequent first-lactation milk production of dairy cows in southwest sweden associations between exposure to bovine herpesvirus (bohv- ) and milk production, reproductive performance, and mortality in irish dairy herds serological evidence of bovine herpesvirus- , bovine viral diarrhea virus and schmallenberg virus infections in relation to reproductive disorders in dairy cattle in ethiopia bovine herpesvirus- in three major milk sheds of ethiopia: serostatus and association with reproductive disorders in dairy cattle prevention of abortion in cattle following vaccination against bovine herpesvirus : a meta-analysis necrotic oophoritis in heifers vaccinated intravenously with infectious bovine rhinotracheitis virus vaccine during estrus ovarian lesions induced in heifers by intravenous inoculation with modified-live infectious bovine rhinotracheitis virus on the day after breeding experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy: effect on the bovine corpus luteum and conceptus the effects of vaccination on serum hormone concentrations and conception rates in synchronized naive beef heifers the effect of infectious bovine rhinotracheitis vaccine on reproductive efficiency in cattle vaccinated during estrus acute brsv infection in young ai bulls: effect on sperm quality review: risks of disease transmission through semen in cattle isolation and characterization of bovine herpesvirus (bohv- ) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome impact of bovine herpesvirus (bohv- ) on reproduction experimental inoculation of cattle with bovine herpesvirus- : evidence for a lymphoid-associated persistent infection experimental infection of bulls with a genital isolate of bovine herpesvirus- and reactivation of latent virus with dexamethasone a bovine macrophage cell line supports bovine herpesvirus- persistent infection virological and serological evidence of bovine herpesvirus type in cattle in northern ireland bohv- ) from a herd with a history of postpartum metritis endometritis in postparturient cattle associated with bovine herpesvirus- infection: cases bovine herpesvirus -associated postpartum metritis in a spanish dairy herd co-infection with bovine herpesvirus and histophilus somni significantly extends the service period in dairy cattle with purulent vaginal discharge uterine infection with bovine herpesvirus type in dairy cows the possible role of bovine herpesvirus type- infection in cow infertility bovine herpesvirus is tropic for bovine endometrial cells and modulates endocrine function bovine herpes virus type alters tnf-a and il- profiles and impairs the survival of bovine endometrial epithelial cells bacterial infection of endometrial stromal cells influences bovine herpesvirus immediate early gene activation: a new insight into bacterial and viral interaction for uterine disease bovine herpesvirus (bohv- ): general aspects of the biology and status in argentina bovine endometrial stromal cells support tumor necrosis factor alpha-induced bovine herpesvirus type enhanced replication the chemokine il is up-regulated in bovine endometrial stromal cells by the bohv- ie gene product, orf /rta: a step ahead toward a mechanism for bohv- induced endometritis virusmediated metalloproteinase induction revealed by transcriptome profiling of bovine herpesvirus -infected bovine endometrial stromal cells influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow transendothelial migration of lymphocytes across high endothelial venules into lymph nodes is affected by metalloproteinases a mutation 'hot spot' in the schmallenberg virus m segment schmallenberg virus experimental infection of sheep at and days of gestation with schmallenberg virus readily led to placental colonization without causing congenital malformations natural infection of pregnant cows with schmallenberg virus-a follow-up study the schmallenberg virus epidemic in europe- - a case-control study to estimate the effects of acute clinical infection with the schmallenberg virus on milk yield, fertility and veterinary costs in swiss dairy herds schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host schmallenberg virus epidemic: impact on milk production, reproductive performance and mortality in dairy cattle in the netherlands and kleve district association of clinical signs after acute schmallenberg virus infection with milk production and fertility in swiss dairy cows culicoides and the emergence of bluetongue virus in northern europe bluetongue in europe: vectors, epidemiology and climate change bovine infection with bluetongue virus with special emphasis on european serotype quantification and at-risk period of decreased fertility associated with exposure to bluetongue virus serotype in naïve dairy herds the impact of bluetongue virus on reproduction a cross-sectional study of bluetongue virus and mycoplasma bovis infections in dairy cattle: ii. the association between a positive antibody response and reproduction performance bluetongue in belgium: episode ii bluetongue virus serotype (btv- ) infection reduces fertility of dutch dairy cattle and is vertically transmitted to offspring bluetongue virus and embryo transfer in cattle susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype bluetongue virus ns protein is an interferon antagonist and a determinant of virus virulence unmapped reads from cattle rnaseq data: a source for missing and misassembled sequences in the reference assemblies and for detection of pathogens in the host the work in the authors' laboratory was funded by contributions from the royal veterinary college, the china scholarship commission, and the commonwealth scholarship commission. the authors thank professor joe brownlie and miss olivia anstaett for their generous provision of bvdv for use in in vitro experiments. rvc manuscript number is pps_ .compliance with ethics guidelines d. claire wathes, chike f. oguejiofor, carole thomas, and zhangrui cheng declare that they have no conflict of interest or financial conflicts to disclose. key: cord- -kt mg o authors: howell, amy b.; d'souza, doris h. title: the pomegranate: effects on bacteria and viruses that influence human health date: - - journal: evid based complement alternat med doi: . / / sha: doc_id: cord_uid: kt mg o pomegranates have been known for hundreds of years for their multiple health benefits, including antimicrobial activity. the recent surge in multidrug-resistant bacteria and the possibility of widespread global virus pandemics necessitate the need for additional preventative and therapeutic options to conventional drugs. research indicates that pomegranates and their extracts may serve as natural alternatives due to their potency against a wide range of bacterial and viral pathogens. nearly every part of the pomegranate plant has been tested for antimicrobial activities, including the fruit juice, peel, arils, flowers, and bark. many studies have utilized pomegranate peel with success. there are various phytochemical compounds in pomegranate that have demonstrated antimicrobial activity, but most of the studies have found that ellagic acid and larger hydrolyzable tannins, such as punicalagin, have the highest activities. in some cases the combination of the pomegranate constituents offers the most benefit. the positive clinical results on pomegranate and suppression of oral bacteria are intriguing and worthy of further study. much of the evidence for pomegranates' antibacterial and antiviral activities against foodborne pathogens and other infectious disease organisms comes from in vitro cell-based assays, necessitating further confirmation of in vivo efficacy through human clinical trials. pomegranates (punica granatum l.) have a long history of antibacterial use dating back to biblical times. egyptians used pomegranates to treat a number of different infections [ ] . it was utilized as a traditional remedy for thousands of years under the ayurvedic system of medicine, with extracts from the rind of the fruit and bark of the tree being effective against diarrhea and dysentery [ ] . over the years there have been many small studies undertaken in different areas of the world on the bactericidal effects of pomegranates on a number of highly pathogenic and drug-resistant strains. these studies normally determine bactericidal potency of different extracts of the pomegranate plant against a range of different bacteria, utilizing disc diffusion assays or minimum inhibitory concentration (mic). methanol extracts of the fruit, especially the peel, exhibit the broadest antibacterial activity [ ] [ ] [ ] [ ] [ ] [ ] ( table ) , which can vary depending on the pomegranate variety tested [ ] . methanol extracts of pomegranate are high in hydrolyzable tannins (punicalins and punicalagins), ellagic acid, a component of ellagitannins, and gallic acid, a component of gallotannins [ ] (figure ). mass spectrometry data shows that pomegranate contains oligomeric ellagitannin with a degree of polymerization of up to core glucose units [ ] . these molecules may be the most potent antibacterial compounds in pomegranate. however, other compounds also have activity and may contribute synergistically as mixtures to bring about the effects, including anthocyanins (pelargonidin- -galactose and cyanidin- -glucose) and flavonols (quercetin and myricetin) [ ] . bacteria. enteric bacteria can be either probiotic and exert beneficial effects on the gut microflora, or they can be pathogenic and cause life-threatening infections and disease. pomegranate has positive effects on both [ , ] , vibrio cholerae [ , ] , yersinia enterocolitica [ ] , shigella spp. [ , ] , and listeria monocytogenes (l. monocytogenes) [ , , ] . typhoid fever (causal agent, s. typhi) is a life-threatening enteric infection that can be transmitted by consuming food or drinking water contaminated with feces from an infected person. it is more common in less industrialized countries. extracts of pomegranate fruit pericarp were tested by agar well diffusion and found to be highly active when compared to a reference concentration-response curve for ampicillin [ ] . in another study which screened plants of importance in the ayurvedic system of medicine, strong antibacterial activity was exhibited by the methanol extracts of pomegranate [ ] . v. cholerae, the cause of cholera infections, is most commonly acquired from feces-infected drinking water. in one study examining bactericidal activity of plants used by peruvian people to combat cholera, pomegranate peel extract and tea infusions were effective [ ] . shigella spp. are an important cause of diarrhea and dysentery in the mexican population. in one study, ethanolic pomegranate extracts exhibited greater antibacterial activity than the antibiotic chloramphenicol, but lower activity than trimethoprim [ ] . shigella sonnei showed the highest susceptibility to the extracts. pomegranate extract was evaluated in several studies for the ability to decontaminate meat surfaces and maintain food quality. a pomegranate peel extract at g/ml was most effective at inhibiting antibiotic resistant strains of salmonella typhimurium (s. typhimurium) and staphylococcus aureus (s. aureus) on meat surfaces and improved sensory evaluations of quality [ ] . in another study, dipping raw chicken breasts in . % pomegranate fruit juice solution reduced protein oxidation, inhibited microbial growth, and increased sensory acceptability for up to days of refrigerated storage at ∘ c [ ] . dried pomegranate juice powder was heated to degrees c for , , , or minutes and added at % (wt/wt) to ground top round beef to determine if heat would alter the antilisterial activity of the powder [ ] . the meat was then cooked and inoculated with individual l. monocytogenes strains. samples of meat stored at ∘ c were taken at days , , , and and plated on media for evaluation of bacterial growth. all the heat-treated pomegranate juice powder treatments significantly inhibited growth of all five l. monocytogenes strains in refrigerated ground cooked beef by . to . log cfu/g at day , suggesting that heating does not impact the antilisterial activity of pomegranate. in another study, an % methanolic pomegranate peel extract resulted in a > log( ) reduction of l. monocytogenes in fish during storage at ∘ c [ ] . one study examined the effectiveness of pomegranate peel to inhibit growth of l. monocytogenes, evidence-based complementary and alternative medicine s. aureus, and salmonella enterica in cheese at room temperature (∼ ∘ c) [ ] . the pomegranate treatment increased the stability of cheese against lipid oxidation, improving shelf life. a pomegranate sour sauce had an antimicrobial effect when mixed with lettuce, spring onion, and parsley, either inoculated with s. aureus and e. coli o :h or containing the naturally existing bacterial flora [ ] . preservation and/or enhancement of probiotic bacteria in the gut is important for maintaining gastrointestinal health. a hydrolyzable tannin-rich pomegranate by-product (pomx) incubated with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins which enhanced the growth of bifidobacterium spp. and lactobacillus spp. [ ] . in another study, pomx significantly enhanced the growth of bifidobacterium breve and bifidobacterium infantis while inhibiting the growth of pathogenic clostridia and staphylooccus aureus [ ] . the effects of individual pomegranate ellagitannins were evident but less pronounced than the tannin mixture in the pomx formulation. pomegranate extract had a beneficial effect on rumen bacterial populations in lactating cows [ ] . the peel extract was fed at levels of , , or % on voluntary intake. the supplementation had a significant positive dose-dependent effect on the entire ruminal bacterial community, as determined by automated ribosomal intergenic spacer analysis. in cows fed at the % extract level, there were significant increases in digestibility of dry matter, crude protein, and neutral detergent fiber, as well as milk yields. healing. pomegranate skin preparations hold promise in increasing the rate of wound healing. pomegranate peel ( % methanolic extract) prepared as an ointment was applied to guinea pig wounds daily for days [ ] . the treatment significantly enhanced wound healing by increasing collagen, dna and protein synthesis as well as contraction rate and tensile strength. the extract exhibited significant antibacterial activity against wound bacteria, including strains of pseudomonas aeruginosa, s. aureus, e. coli, klebsiella pneumoniae (k. pneumoniae), salmonella anatum, s. typhimurium, and streptococcus pneumoniae. there were no toxic effects noted from use of the skin ointment. another study using the methanolic extract of pomegranate peels formulated into a % (wt/wt) water-soluble gel showed similar enhancements in wound healing in a wistar rat model compared to a commercial topical antibacterial product [ ] . the group treated with . % gel had a . % increase in contraction of the skin, a -fold increase in collagen content, and positive microscopic changes to the skin. participant's wounds were completely healed after days, compared to - days for the group receiving a blank control gel. the activity was postulated to be due to the high phenolic content ( %) in the peel extract. pomegranate flower extracts also hold promise for augmenting wound healing. a diethyl ether flower extract was applied to wounds in alloxan-induced diabetic rats at a dose of mg/kg/day [ ] . the extract-treated diabetic rats showed significant reduction in the wound area when compared with the control. another study in which flower extracts of pomegranate were applied to wounds resulted in decreased wound size compared to the control group and a significant increase in the rate of wound contraction and collagen turnover [ ] . bacteria. studies suggest a role for pomegranate extracts in reducing and preventing pathogenic dental bacteria and reducing the risk of plaque, gingivitis, and periodontal disease. many of these studies are human clinical trials. the effects of three different concentrations of a methanolic pomegranate peel extract at mg/ml, mg/ml, and mg/ml on growth of dental bacteria were compared using the disc diffusion method [ ] . all concentrations of the pomegranate extract had antibacterial activity against s. aureus and s. epidermidis. extract concentrations of mg/ml and mg/ml were effective against l. acidophilus, s. mutans, and s. salivarius. the extract did not inhibit actinomyces viscosus. in another similar in vitro study, ethanol and water extracts of pomegranate both had inhibitory effects against s. mutans and porphyromonas gingivalis (p. gingivalis) [ ] . a brazilian in vitro study investigated the antimicrobial effect of a pomegranate-based oral gel (made from an extract of dried peel combined with carbopol, water, and triethanolamine) against streptococcus sanguis, streptococcus mitis, and s. mutans [ ] . the mics required to inhibit adherence of the bacteria to glass were assessed using increasing and doubled concentrations of the diluted solution of the pomegranate gel at concentrations ranging from : to : . the mics of adherence of pomegranate gel against the bacteria were : for s. mutans and s. sanguis and : for s. mitis. these results suggest that pomegranate gel might be useful in the control of adherence of different bacteria in the oral cavity. in other in vitro studies, pomegranate extract also inhibited strains of periodontal bacteria, aggregatibacter actinomycetemcomitans, p. gingivalis, prevotella intermedia [ ] , klebsiella, e. coli, and proteus spp. [ ] . several clinical trials have explored the effectiveness of pomegranate extract rinses on reductions in oral plaque [ , ] . in one trial, the amount of plaque accumulation was measured at days and in thirty periodontally healthy volunteers who refrained from all mechanical oral hygiene measures for days and instead used either pomegranate extract, chlorhexidine, or a placebo rinse twice daily [ ] . at day , those volunteers using the pomegranate extract had significantly less plaque buildup ( < . ) than those using the placebo rinse. the pomegranate extract prevented as much plaque as the chlorhexidine rinse. these results on pomegranate's effect on plaque reduction are supported by another human trial in which pomegranate extract rinse was compared to chlorhexidine and placebo rinse [ ] . after hours without tooth brushing, plaque samples were taken from sixty healthy, younger patients between the ages of and who wore orthodontic appliances. dental plaque samples were plated on media for hours, and the number of colony forming units per milliliter (cfu/ml) showed that the pomegranate extract rinse was effective against dental plaque microorganisms, decreasing the cfu/ml by %, similar to chlorhexidine ( % inhibition), and significantly different from the control rinse ( % inhibition). authors speculated that the ellagitannin and punicalagin may be responsible for the antibacterial activity of the pomegranate extract rinse. gingivitis is an inflammation of the gums in response to bacterial plaque biofilms adhering to tooth surfaces. if left untreated, gingivitis may progress to periodontal disease and subsequent tooth loss. there is an incentive to use alternative plant-based preparations as an adjunctive to mechanical therapy in the prevention and treatment of gingivitis, due to the health risks imposed by long-term use of chemical and pharmaceutical preparations and the lack of available dental care in lesser-developed countries. results of a randomized clinical study of patients with chronic gingivitis showed that significant improvements were obtained in the group that used a pomegranate extract gel along with mechanical debridement for days when compared with patients using only control gel or mechanical debridement for the -day test period [ ] . another placebocontrolled human clinical trial of young adults examined salivary measures relevant to oral health and gingivitis after using a pomegranate extract mouth rinse three times per day for weeks or a placebo rinse [ ] . compared to the control group, those participants using the pomegranate rinse had reduced total protein associated with presence of plaqueforming bacteria, reduced activities related to cell injury, reduced levels of the sucrose-degrading enzyme alpha-glucosidase, and increased activity of the enzyme ceruloplasmin, which protects against oral oxidative stress. based on these results, the authors suggest the possibility of using pomegranate extracts in oral health products such as toothpaste and mouthwashes. a clinical study was undertaken to test pomegranate peel extract impregnated into biodegradable chips for use subgingivally as an adjunct to scaling and root planning for maintenance of periodontal disease [ ] . the pomegranate chips or placebo chips were implanted in patients with gum pocket depths of - mm. level of bacterial attachment, bleeding, and gingival and plaque indexes were initially measured and again at and months. after months, the pomegranate treatments resulted in decreased plaque and significant decreases in pocket depth and bacterial attachment compared to placebo. a marker of inflammation (il- beta) was also lower at and months compared to baseline. methicillinresistant staphylococcus aureus (mrsa) is any strain of s. aureus that has become resistant to beta-lactam antibiotics, including the penicillins and the cephalosporins. these mrsa strains are not necessarily more virulent than antibiotic-susceptible strains of s. aureus, but they are more dangerous because they do not respond to first-line antibiotics. mrsa can cause life-threatening infections in people with weakened immune systems, especially in lesser-developed countries where antibiotics are not readily available, and in hospitals and nursing homes. there is evidence from a number of in vitro experiments that pomegranate extracts moderately to strongly inhibit cultured mrsa strains [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in one study, ethanolic extracts of pomegranate were effective at inhibiting hospital isolates of mrsa at mics of . - . mg/ml [ ] . in another study, an extract of hightannin pomegranate polyphenols at and mg/ml was found to cause . - . log cfu/ml reduction of the two mrsa strains after hours at ∘ c, and to undetectable levels in most strains within hours [ ] . scanning electron microscopy of the bacteria showed that the pomegranate extract caused alterations in the bacterial cell walls after hours of treatment. multidrug-resistant acinetobacter baumannii (a. baumannii) is considered to be one of the most difficult bacterial infections to treat. a. baumannii survives for prolonged periods under a wide range of environmental conditions and causes serious infection outbreaks in hospitals. it is very difficult to control due to antibiotic resistance, so the need for alternative approaches is under investigation. pomegranate extract was tested as a resistance-modifying agent of the antibiotic novobiocin against a. baumannii using a growth inhibition assay [ ] . pomegranate extract at g/ml significantly enhanced the antibacterial activity of novobiocin at g/ml ( / × mic) against a. baumannii. helicobacter pylori (h. pylori) is the causal agent of stomach ulcers, which if left untreated can lead to stomach cancer. however, the bacteria are becoming resistant to the antibiotics used to treat ulcers, making this condition difficult to cure. disc diffusion was utilized to test the in vitro susceptibility of h. pylori isolated from patients with gastroduodenal complications to a pomegranate methanol extract [ ] . the pomegranate extract exhibited strong activity against h. pylori with a mean inhibition zone diameter of mm at g disc − . pomegranate peel extracts from nine iranian cultivars were further assayed against the h. pylori isolates. the results demonstrated that most all cultivars showed significant in vitro anti-h. pylori activity with the mean inhibition zone diameter ranging from to mm at g disc − . pomegranate enhanced the activity of antibiotics against mrsa in one study [ ] . synergistic activity between a methanolic pomegranate extract and the antibiotics chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin ranged from % to % against clinical isolates of mrsa. the bactericidal activity of the pomegranate extract ( . × mic) with ampicillin ( . × mic) was assessed and determined to be synergistic. this combination increased the lag time to bacterial growth by three hours over that of ampicillin alone and resulted in a . % reduction in mrsa. the mechanism evidence-based complementary and alternative medicine of action of the pomegranate extract was to either inhibit the mrsa nora efflux pump or to enhance the influx of the antibiotic. the antibiotic activity of ciprofloxacin was enhanced by a methanolic pomegranate peel extract against resistant strains of extended-spectrum beta-lactamase-producing e. coli, k. pneumoniae, and metallo-beta-lactamase-producing p. aeruginosa [ ] . synergy with ciprofloxacin was observed in of strains (fic of . - . for ciprofloxacin) possibly due to the bacterial efflux pump inhibitor activity of the pomegranate tannins. quorum sensing is an intercellular signaling mechanism used by bacteria to communicate as a colony about critical survival issues such as availability of nutrients, defense against other microorganisms, virulence, and biofilm formation. bacteria produce and detect signaling molecules important for pathogenic bacteria during infection of a host to coordinate their virulence in order to escape the immune response of the host and establish a successful infection. interference of quorum-sensing signals is a strategy for disease control. pomegranate inhibited quorum-sensing signals in two bacterial strains, interfering with purple pigment production and bacterial swarming motility in chromobacterium violaceum and p. aeruginosa, respectively [ , ] . the inhibition of these particular processes may be due to direct or indirect interference on quorum-sensing by pomegranate polyphenols or the interactive effect of different compounds present in the extract. limited studies have been conducted on the antiviral activities associated with pomegranate and its extracts. the fruit's antiviral effects have been reported against clinically relevant influenza virus, herpes virus, poxviruses, and human immunodeficiency (hiv- ) virus [ ] [ ] [ ] . the hydrolyzable tannins and anthocyanins are the main compounds associated with the beneficial effects of pomegranate consumption on other health effects "including antibacterial" [ ] , and may be responsible for the antiviral activity. in one study, among four flavonoid compounds associated with pomegranates (ellagic acid, caffeic acid, luteolin, and punicalagin), only punicalagin was shown to have inhibitory effects on influenza virus [ ] . natural antimicrobials from plant extracts have become increasingly popular for use as alternative antivirals [ ] [ ] [ ] [ ] . the increased research and need for such alternatives are based on the many advantages of natural plant antimicrobials. these include the absence of reported/ observed toxic effects at recommended doses along with additional benefits such as antioxidant, anticancer, antiinflammatory, and antimicrobial properties [ , , , , , , [ ] [ ] [ ] [ ] . it is possible that pomegranate juice and extracts could be potentially useful in inhibiting viruses transmitted via infected food products, bodily fluids, and so forth. recent studies indicate that viruses cause an estimated % of all foodborne illnesses ( . million), % of hospitalizations, and % of deaths in the united states alone [ ] . foodborne virus outbreaks are gaining immense attention due to their increased incidence and scope of illness. the epidemiologically significant foodborne viruses include human noroviruses, hepatitis a virus, rotaviruses, aichi virus, hepatitis e virus, astroviruses, adenoviruses, parvoviruses, and other human enteroviruses and small round structured viruses [ ] . among the foodborne viruses, human noroviruses are the main cause of viral gastroenteritis outbreaks worldwide [ , , ] . human noroviruses (hnovs) belong to the family of caliciviridae, being nonenveloped and round in shape with a diameter of to nm. there are currently five genogroups based on nucleic acid sequence analysis, of which primarily genogroup i and frequently genogroups ii and iv are associated with human norovirus outbreaks. earlier studies indicated that hnovs are estimated to be solely responsible for up to . million infections, , hospitalizations, and deaths per year in the us alone [ ] . as hnovs cannot be grown in cell culture, cultivable surrogates such as feline calicivirus (fcv-f ) [ ] , bacteriophage ms [ ] , and murine norovirus (mnv- ) [ ] are used in infectivity assays to study antiviral or inactivation effects, though the more recently cultivable tulane virus is also being researched [ ] . hnovs can be transmitted from person to person, through food, aerosols, water, and contact with fomites [ ] . they are reported to have a low infectious dose of to viral particles with symptoms including nausea, vomiting, diarrhea, abdominal pain, and low-grade fever. the infection is self-limiting in healthy individuals and lasts for up to hours. however, newly emergent strains (such as genogroup ii. ) have become highly virulent and life-threatening especially to the elderly and immunocompromised [ ] . currently, there are no known vaccines available to prevent norovirus infection or disease onset. in addition, there is no effective treatment option available besides rehydration therapy. the effectiveness of natural remedies such as pomegranate juice and extracts as alternatives for the treatment or prevention of foodborne norovirus infections needs to be explored more aggressively. recently, pomegranate juice and polyphenols were shown to have significant antiviral effects against foodborne viral surrogates, fcv-f , mnv- , and bacteriophage ms [ ] . these researchers showed that the juice could decrease low titers (∼ log pfu/ml) of fcv-f , mnv- , and ms by . , . , and . log pfu/ml, respectively, after hour at room temperature and high titers (∼ log pfu/ml) by . , . , and . log pfu/ml. the most potent effect was on fcv-f after treatment with , , or mg/ml of pomegranate polyphenol (extracted from fresh pomegranate fruit-pom obtained from pom wonderful). after hour at room temperature, low and high titers of fcv-f were completely undetectable. following -hour incubation with , , or mg/ml pomegranate polyphenol, low initial titers of mnv- were reduced by . , . , and . log pfu/ml and high initial titers by . , . , and . log pfu/ml, respectively. the titer reduction effect of pomegranate against mnv- is notable in that this surrogate is considered to be more appropriate than fcv-f by some researchers. bacteriophage ms at low initial titers was reduced by . , . , and . log pfu/ml and at high initial titers by . , . , and . log pfu/ml after incubation with , , or mg/ml of pomegranate polyphenol, respectively. overall effectiveness of pomegranate on reduction of virus titer rankings was fcv-f > mnv- > ms for the tested low-titer viruses ( log pfu/ml), while for the high-titer viruses ( log pfu/ml), the effectiveness rankings were fcv-f > ms > mnv- [ ] . pomegranate juice contains total phenolics of . mg/ml [ ] ; therefore, pomegranate polyphenols were tested at a similar concentration ( mg/ml) and exhibited consistently greater antiviral effects than that of pomegranate juice against all viruses at both high and low titers [ ] . this difference may be due to variability in composition and bioavailability of polyphenols in juice versus those extracted in pure form. in addition, the antiviral effects are not ph-dependent, as no differences in bioactivity were noted when juice ph was changed from . to . . given that mnv- is quite resistant to most treatment conditions, including ph and heat [ ] , but inhibited by pomegranate juice and its polyphenols, it is possible that additional research will reveal a role for pomegranate as a natural alternative for treating and/or preventing human norovirus infections. comparison of the effects of cranberry juice, grape juice, and orange juice on the infectivity of foodborne viral surrogates revealed that the titer reduction for fcv-f , mnv- , and ms followed the order of cranberry juice > pomegranate juice > grape juice > orange juice in general, with the exception that grape juice had a greater effect on high-titer fcv-f than cranberry juice [ ] . when the time-dependent effects of pomegranate juice and polyphenols at and mg/ml against foodborne viral surrogates were further studied, varied titer reduction rates of fcv-f , mnv- , and ms over hour at room temperature were obtained [ ] . there were no significant differences in titer reductions when comparing different brands of commercial pomegranate juice; however, titer reduction levels were affected by different storage times. for all three viruses, ≥ % of the total reduction was found to be achieved within minutes. interestingly, upon immediate mixing with pomegranate juice or or mg/ml pomegranate polyphenols, fcv-f was reduced by . , . , and . log pfu/ml, respectively, with further reductions of . , . , and . log pfu/ml within the next minutes. compared to fcv-f , bacteriophage ms and mnv- titers were not significantly reduced using this experimental regime. these results indicate an in vitro effect of pomegranate against human norovirus surrogates; however, further in vivo work is necessary to determine if clinically relevant therapeutic or preventive uses are viable. konowalchuk and speirs [ ] found that < % of log pfu poliovirus/ . ml survived after storage at ∘ c for hours in pomegranate juice, though the mechanism of action was unknown. poliovirus is transmitted through the fecal-oral route in a manner similar to other enteroviruses, being a nonenveloped rna virus. as preventive measures, poliovirus vaccines are available. in order to understand the mechanism of action, the host cell monolayers for the respective viruses were treated with pomegranate juice and polyphenols prior to or after infection, where reduced infectivity of fcv-f and mnv- was obtained [ ] . greater effects in titer reduction were observed when the treatment was performed prior to infection (corresponding to attachment/adsorption stage) than after infection (corresponding to replication stage), suggesting that pomegranate juice and its polyphenols may play a role in preventing virus binding to the host cell receptors by blocking the cell surface receptors or the virus surface ligands. it was postulated that further work using transmission electron microscopy may determine if these polyphenols cause structural damage to the virus or virus capsid. recently, a cranberry-pomegranate juice blend was shown to reduce the specific binding ability of human nov p particles to salivary human histoblood group antigens (hbgas) using an enzyme-linked immunosorbent assay (elisa), where the binding pattern is reported to correspond with the probability of infection [ ] . hbgas are complex carbohydrates present on red blood cell surfaces, the mucosal epithelium of respiratory, genitourinary, and digestive tracts and as free oligosaccharides in saliva, intestinal contents, milk, and blood. these researchers reported that cranberry juice at concentrations of and % and cranberrypomegranate at concentrations of % to % were found to reduce the binding of human nov strains specifically to certain types of human hbgas, in agreement with the study by su et al. [ ] that used infectivity plaque assays. li et al. [ ] also postulated that the interaction of plant polyphenolic compounds with the viral capsid protein may cause irreversible damage or reversible blocking of certain regions/ areas of the capsid protein. pomegranate extracts have also shown antiviral effects against influenza virus, hiv- and poxviruses [ , , ] . influenza virus continues to be a major cause of morbidity and mortality each year with , deaths reported yearly in the us, despite access to vaccines [ ] . however, frequent recombination events and viral evolution necessitate the change in vaccine composition requiring administration of new vaccines yearly. researchers have shown that pomegranate polyphenols were virucidal against influenza a virus, suppressed the replication of the virus in host cells, and inhibited agglutination of chicken red blood cells caused by the virus using real-time polymerase chain reaction, a plaque assay, and a median tissue culture infective dose % hemagglutination assay [ ] . they also showed that among four polyphenols (ellagic acid, caffeic acid, luteolin, and punicalagin), punicalagin was found to be the most effective anti-influenza sundararajan et al. [ ] also showed that the acidity of pomegranate juice and concentrated liquid pomegranate extract (pomxl) solutions contributed to rapid anti-influenza activity, whereas pomegranate polyphenol (pp) powder (pomxp) did not. a -minute treatment at room temperature with g/ml pp was shown to result in at least a log titer reduction of influenza viruses pr (h n ), x (h n ), and a reassortant h n virus derived from a human isolate. loss of hemagglutinating activity was reported to accompany the loss of influenza infectivity, with decreased antibody binding to viral surface molecules after treatments with pp. viral structural damage was also reported using electron microscopic analysis of pp-treated viral particles. however, they found that the antiviral activity was less against avian isolates of one coronavirus and reassortant h n influenza viruses. kotwal [ ] suggested that pomegranate juice can neutralize the infectivity of diverse enveloped viruses and a number of subtypes of a given enveloped virus, indicating potential for development as a treatment option that can be broadly effective against pandemic viruses like hiv, potentially pandemic viruses like influenza, and some carcinogenic viruses. it was shown that influenza a/hk/x (h n ), influenza a/vietnam/ / (h n ), and a reassortant x containing the ns gene segment of an h n isolate were inactivated when treated for minutes at ∘ c with pomegranate juice [ ] . in , the aids pandemic was reported to claim million lives that resulted in ∼ , new hiv- global infections daily [ ] . in the absence of vaccines, antiretroviral chemotherapeutics have been used to decrease hiv- symptoms mainly in developed countries. neurath et al. [ ] showed that hiv- entry inhibitors from pomegranate juice are adsorbed onto corn starch, block hiv- binding to cd and cxcr /ccr host cell receptors, and inhibit infection by primary virus clades a to g and group o. these researchers showed the potential of producing anti-hiv- microbicides from naturally safe food sources. several other mechanisms of antiviral activity have been proposed for nonpomegranate polyphenols, which could offer valuable insights for researchers studying the antiviral mechanisms related to pomegranate consumption. haslam [ ] suggested that plant polyphenols exert a direct action on the viral particles, inhibiting the adsorption of the virus to the host cell receptors. one study found that proanthocyanidin a- inhibited viral attachment and penetration and affected the late stages of herpes simplex virus type infection [ ] . liu et al. [ ] determined that the inhibitory effect of tea polyphenols is through multiple mechanisms of action, including inhibiting hiv- reverse transcriptase and protease activity, blocking gp -cd interaction by binding to cellular cd molecules, and destroying viral particles. further studies are needed to identify additional antiviral mechanisms of action associated with pomegranate and its constituents. there are a number of studies on pomegranate and their antimicrobial activities against bacteria and viruses, with mechanisms of actions including ph-independent bacterial and viral growth inhibition, effects on bacterial cell signaling, reductions in viral infectivity and binding to host cell receptors, and structural damage to viruses. however, many of the study results are based on in vitro and cell-based assays. therefore, the applicability of the results to human health is mainly focused on diseases and infections that occur topically, such as those in the oral cavity or on the skin surface. it is difficult to extrapolate in vivo effects on infection from in vitro results using unmetabolized pomegranate juice or compounds on microbes. in addition, it is difficult to compare results of studies that do not standardize the treatment extracts for the active components. standard procedures should be adopted by the pomegranate industry that utilize the same quantification methods. a method for determining ellagitannin levels using a novel pomegranate standard has been published, which could be helpful in addressing this issue [ , ] . the current studies do support potential benefit of pomegranate extracts in food preservation and decontamination. this application could be particularly useful in lesserdeveloped countries where food sanitation can easily be compromised. results of the studies on antibacterial benefits of pomegranate extracts against dental bacteria and infections hold promise because they are clinically-based. additional larger-scale trials should be conducted to confirm the benefits. pomegranate antimicrobial activities of pomegranate antibacterial activity of punica granatum pomegranate peel and fruit extracts: a review of potential anti-inflammatory and antiinfective effects studies on antibacterial and antifungal activity of pomegranate (punica granatum l.) antibacterial, antioxidant and tyrosinase-inhibition activities of pomegranate evidence-based complementary and alternative medicine fruit peel methanolic extract antimicrobial activity of pomegranate (punica granatum l.) fruit peels antibacterial activity directed isolation of compounds from punica granatum antimicrobial activity of six pomegranate (punica granatum l.) varieties and their relation to some of their pomological and phytonutrient characteristics antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from punica granatum l maldi-tof mass spectrometry of oligomeric food polyphenols effective medicinal plants against enterohaemorrhagic escherichia coli o :h medicinal plant extracts as anti-escherichia coli o :h agents and their effects on bacterial cell aggregation in vitro antibacterial activity of argentine folk medicinal plants against salmonella typhi antimicrobial evaluation of some medicinal plants for their anti-enteric potential against multidrug resistant salmonella typhi antibacterial activities of medicinal plants used for the treatment of diarrhoea in limpopo province, south africa the in vitro action of plants on vibrio cholerae antibacterial properties of some plants used in mexican traditional medicine for the treatment of gastrointestinal disorders potential application of spice and herb extracts as natural preservatives in cheese anti-listeria monocytogenes activity of heat-treated lyophilized pomegranate juice in media and in ground top round beef surface decontamination and quality enhancement in meat steaks using plant extracts as natural biopreservatives effect of dipping in pomegranate (punica granatum) fruit juice phenolic solution on the shelf life of chicken meat under refrigerated storage ( ∘ c) inhibitory effect of sour pomegranate sauces on some green vegetables and kisir the influence of pomegranate byproduct and punicalagins on selected groups of human intestinal microbiota the effect of pomegranate (punica granatum l.) byproducts and ellagitannins on the growth of human gut bacteria effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population hydroalcoholic extract based-ointment from punica granatum l. peels with enhanced in vivo healing potential on dermal wounds wound healing activity of malva sylvestris and punica granatum in alloxan-induced diabetic rats the wound healing activity of flower extracts of punica granatum and achillea kellalensis in wistar rats antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens minimum inhibitory concentration of adherence of punica granatum linn (pomegranate) gel against s. mutans, s. mitis and c. albicans ethnobotanical survey and antibacterial activity of plants used in the altiplane region of mexico for the treatment of oral cavity infections the antiplaque efficacy of pomegranate mouthrinse punica granatum (pomegranate) extract is active against dental plaque antibacterial properties of tropical plants from puerto rico activity of medicinal plant extracts against hospital isolates of methicillin-resistant staphylococcus aureus effect of certain bioactive plant extracts on clinical isolates of -lactamase producing methicillin resistant staphylococcus aureus in vitro activity of brazilian medicinal plants, naturally occurring naphthoquinones and their analogues, against methicillin-resistant staphylococcus aureus antibacterial effects of plant-derived extracts on methicillin-resistant staphylococcus aureus pomegranate extract inhibits staphylococcus aureus growth and subsequent enterotoxin production anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of s. aureus, mrsa and pvl positive ca-mssa thai ethnomedicinal plants as resistant modifying agents for combating acinetobacter baumannii infections in vitro antibacterial activity of some iranian medicinal plant extracts against helicobacter pylori study on wound healing activity of punica granatum peel efficacy of a herbal extract gel in the treatment of gingivitis: a clinical study pomegranate extract mouth rinsing effects on saliva measures relevant to gingivitis risk adjunctive periodontal treatment with centella asiatica and punica granatum extracts in supportive periodontal therapy synergic interaction between pomegranate extract and antibiotics against staphylococcus aureus pomegranate pericarp extract enhances the antibacterial activity of ciprofloxacin against extended-spectrum -lactamase (esbl) and metallo--lactamase (mbl) producing gram-negative bacilli screening of traditional chinese medicinal plants for quorum-sensing inhibitors activity screening of certain medicinal plants from india for their anti-quorum sensing activity pomegranate (punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir punica granatum (pomegranate) juice provides an hiv- entry inhibitor and candidate topical microbicide hbv and hcv) viruses and possible agents of bioterrorism (variola) by enveloped virus neutralizing compounds (evncs) pomegranate phenolics from the peels, arils, and flowers are antiatherogenic: studies in vivo in atherosclerotic apolipoprotein e-deficient (eo) mice and in vitro in cultured macrophages and upoproteins effects of pomegranate chemical constituents/intestinal microbial metabolites on cyp b in rv prostate cancer cells cranberry cocktail juice, cranberry concentrates, and proanthocyanidins reduce reovirus infectivity titers in african green monkey kidney epithelial cell cultures effect of cranberry juice constituents on haeamgglutination and infectivity of influenza virus time-dependent effects of pomegranate juice and pomegranate polyphenols on foodborne viral reduction cranberry juice constituents affect influenza virus adhesion and infectivity antiinflammatory effects of punica granatum linne in vitro and in vivo role of anthocyanins in the antioxidant ability of pomegranate antioxidant and antibacterial activities of punica granatum peel extracts phase ii study of pomegranate juice for men with rising prostate-specific antigen following surgery or radiation for prostate cancer foodborne illness acquired in the united states-major pathogens persistence of caliciviruses on environmental surfaces and their transfer to food reevaluation of epidemiological criteria for identifying outbreaks of acute gastroenteritis due to norovirus: united states norovirus illness is a global problem: emergence and spread of norovirus gii. variants food-related illness and death in the united states surrogate viruses for testing virucidal efficacy of chemical disinfectants survival of viruses on fresh produce, using ms as a surrogate for norovirus murine norovirus: a model system to study norovirus biology and pathogenesis new interventions against human norovirus: progress, opportunities, and challenges epidemiologic and molecular trends of "norwalk-like viruses" associated with outbreaks of gastroenteritis in the united states in vitro effects of pomegranate juice and pomegranate polyphenols on foodborne viral surrogates antiviral activity of fruit extracts effects of a variety of food extracts and juices on the specific binding ability of norovirus gii. p particles influenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins natural polyphenols (vegetable tannins) as drugs: possible modes of action in vitro anti-hsv- activity and mechanism of action of proanthocyanidin a- from vaccinium vitis-idaea theaflavin derivatives in black tea and catechin derivatives in green tea inhibit hiv- entry by targeting gp development of a novel pomegranate standard and new method for the quantitative measurement of pomegranate polyphenols pomegranate (punica granatum) supplements: authenticity, antioxidant and polyphenol composition the authors occasionally receive minimal research funds from pom wonderful, llc; however, they have no financial interest or gain from the sale or endorsement of pom products. key: cord- -mjjnkqv authors: jarach, natanel; dodiuk, hanna; kenig, samuel title: polymers in the medical antiviral front-line date: - - journal: polymers (basel) doi: . /polym sha: doc_id: cord_uid: mjjnkqv antiviral polymers are part of a major campaign led by the scientific community in recent years. facing this most demanding of campaigns, two main approaches have been undertaken by scientists. first, the classic approach involves the development of relatively small molecules having antiviral properties to serve as drugs. the other approach involves searching for polymers with antiviral properties to be used as prescription medications or viral spread prevention measures. this second approach took two distinct directions. the first, using polymers as antiviral drug-delivery systems, taking advantage of their biodegradable properties. the second, using polymers with antiviral properties for on-contact virus elimination, which will be the focus of this review. anti-viral polymers are obtained by either the addition of small antiviral molecules (such as metal ions) to obtain ion-containing polymers with antiviral properties or the use of polymers composed of an organic backbone and electrically charged moieties like polyanions, such as carboxylate containing polymers, or polycations such as quaternary ammonium containing polymers. other approaches include moieties hybridized by sulphates, carboxylic acids, or amines and/or combining repeating units with a similar chemical structure to common antiviral drugs. furthermore, elevated temperatures appear to increase the anti-viral effect of ions and other functional moieties. viruses are not living organisms per se, but small structures containing only a nucleic acid genome within a mostly protein based protecting membrane. unlike living organisms, viruses must penetrate a living host cells to reproduce and replicate [ ] . viruses are usually classified into seven major classes [ ] [ ] [ ] [ ] [ ] [ ] [ ] . class i includes viruses with double-stranded dna, like poxvirus, which uses asymmetric transcription to raise their mrna, much like "regular" cells, but depend on the hosting cell's polymerases to transcript their genome. class ii contains single-stranded dna viruses. the dna's polarity in these viruses is equal to their mrna's. this class contains the anelloviridae, circoviridae, parvoviridae, geminiviridae, microviridae, and more. class iii includes viruses with double-stranded rna. these viruses' mrna demonstrates an asymmetric transcription of the virus' genome. this group includes the reoviridae and birnaviridae viruses. class iv consists of viruses with single-stranded rna. these viruses' mrna is the sequence's base identical to their virion rna. these viruses are referred to as "positive-sense single-stranded rna viruses". this group contains togaviridae, astroviridae (causing diarrhea), caliciviridae, flaviviridae (including many different diseases such as hepatitis-c, bovine viral diarrhea virus, and more), picornaviridae (including the rhinoviruses, which causes the "common cold"), arteriviridae, and coronaviridae [including the severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome-related coronavirus (mers-cov), and severe acute as will be described later [ ] . ganciclovir and acyclovir are well known commercial antiviral drugs which, though not having any electrical charge, have a well-established effect on some virus polymerase. some other molecules with antiviral effects are -(n-ethyl-n-isopropyl)amiloride (eipa), verapamil, and diltiazem. some studies suggest their effect is a result of the ion-blocking effect. others suggested that the viral inhibition is based on interactions with virus proteins [ ] . other neutral antiviral molecules are ascorbic and dehydroascorbic acids [ ] , triclosan [ ] and the camphor imine derivatives. various studies conducted in recent years showed the antiviral potential of the latter, especially against influenza viruses [ ] [ ] [ ] . there are two main approaches to the use of polymers in the antiviral campaign. one includes the use of polymers with only a supportive role, using polymers for antiviral drug delivery. the second includes introducing the polymers in the anti-viral front-line using polymers containing ions and/or metal particles or polymers with antiviral moieties such as amines, ions moieties, carboxylic acid moieties, sulphates, phenols and more. although the first one has been in the focus of most relevant research, only a few examples will be demonstrated related this work, as the second approach is the focus of the current review. chapter : chapter : the enhancement of effective drug release has intensively occupied researchers in recent years. one route suggested is the use of drug-conjugated bio-degradable polymers as drug-delivery agents. by controlling the degradation rate, the drug release could also be controlled. based on this method, several antiviral-drug-conjugated polymers have been studied over the years. ahmad et al., for example, studied the uses of a novel chitosan/xanthan gum-based hydrogels as an acyclovir (a commercial antiviral drug) delivery. they found that this cross-linked polymer could be used for antiviral drug delivery, while its efficiency is depended on the drug to polymer ratio, ph, loading time, cross-linking density and other controlled parameters [ ] . other research groups have also studied antiviral drugs that release polymeric systems and mechanisms. based on their results, it was concluded that the degree of the antiviral effect (i.e., the drug release rate) was dependent on several controllable parameters such as the chemical structure of the polymer, the molecular weight, loading concentrations and the combination with the use of inherently antiviral-polymers [ ] [ ] [ ] . liu et al. also studied the effect of temperature on viruses [ ] . they found that tobacco plants gained better viral resistance at • c compared to at • c. haam et al. studied the efficiency of using poly(ethylene glycol)-block-poly(phenylalanine) as a drug delivery system, especially against the influenza a virus [ ] . they found that due to the better virus envelope penetrating ability of the polymer compared to the model drugs, the antiviral effect increased. chen et al. studied the use of poly-l-glutamine (pgn) conjugated with zanamivir and demonstrated that this system had an antiviral efficiency against early and late-stage influenza virus infection [ ] . antiviral drug delivery has also been studied using metal-organic frameworks (mofs), exploiting both their porous structure and their biodegradable properties. an example of this approach is the work done by gref et al. who studied the use of a mof obtained by reacting fe(iii) and trimesic acid as an anti-hiv azt-tp drug delivery system [ ] . other examples are the work of keskin and kızılel about the antiviral drug delivery uses of three different mofs developed by reacting zn o(co) and three different dicarboxylate linkers [ ] and the work novio et al. about the uses of iron-catechol-based mofs as antiviral drugs delivery systems [ ] . while polymer antibacterial and antimicrobial properties have been well studied over the years, only a few studies were conducted on the antiviral properties of polymers. several proposed approaches are discussed in this paper, as follows. first, polymerization of commercial antiviral drugs and/or polymers grafted with commercial drugs (pro)drugs. second, adding antiviral additives such as metal or ion-particles to achieve an antiviral effect. other approaches are amine-containing polymers and/or polycations, poly(carboxylic acid)s, polyanhydrides and/or polyanions, sulphate and sulphuric acid-containing polymers and hydroxyl-containing polymers, polyphenols, and/or some other non-ionic polymers. in contrast to their use in a supporting role, described above, when used in a front-line role, polymers do not go through any degradation process to accomplish their antiviral effects. "macromolecular (pro)drugs", polymerized commercial antiviral drugs or polymers grafted with commercial drugs as pendant groups, are straightforward examples for polymers with inherent antiviral activity. several studies have been conducted using this approach, such as zelikin and his group about ribavirin [ , ] . larson also studied the uses of (pro)drugs to overcome immunity against some commercial antiviral drugs. while this approach was found useful in some circumstances, it wasn't an efficient method for all drugs tested [ , ] , thus efficacy might be attributed to other mechanisms. bovin and associates, synthesized poly(n- -hydroxyethylacrylamide) ( sln-paa) by polymerization of the co-monomers neu acα - galβ - glcnacβ ( sln) and polyacrylamide as shown in figure [ ] . owing to the sln antiviral properties, the entire co-polymer gains antiviral effect against the influenza virus. an increase attraction between viruses and polymer enhanced the drugs' original antiviral effect. polymers , , x for peer review of sulphate and sulphuric acid-containing polymers and hydroxyl-containing polymers, polyphenols, and/or some other non-ionic polymers. in contrast to their use in a supporting role, described above, when used in a front-line role, polymers do not go through any degradation process to accomplish their antiviral effects. "macromolecular (pro)drugs", polymerized commercial antiviral drugs or polymers grafted with commercial drugs as pendant groups, are straightforward examples for polymers with inherent antiviral activity. several studies have been conducted using this approach, such as zelikin and his group about ribavirin [ , ] . larson also studied the uses of (pro)drugs to overcome immunity against some commercial antiviral drugs. while this approach was found useful in some circumstances, it wasn't an efficient method for all drugs tested [ , ] , thus efficacy might be attributed to other mechanisms. bovin and associates, synthesized poly(n- -hydroxyethylacrylamide) ( ′sln-paa) by polymerization of the co-monomers neu acα - galβ - glcnacβ ( ′sln) and polyacrylamide as shown in figure [ ] . owing to the ′sln antiviral properties, the entire co-polymer gains antiviral effect against the influenza virus. an increase attraction between viruses and polymer enhanced the drugs' original antiviral effect. based on a similar principle, carraher et al. studied the effect of two commercial antiviral drugsbased polymers. they found that cisplatin derivatives containing tilorone polymers have an antiviral effect on herpes simplex- , vaccinia, and varicella zoster and reovirus viruses and a tilorone derivative polymers have also an antiviral effect on both dna and rna viruses [ , ] . polymers (see figure ) were synthesized using tilorone or tilorone , (both commercially available) dissolved in distilled water, and later added to a stirred solution of potassium tetrachloroplatinate(ii) in distilled water at room temperature. they also found these two polymers require far lower concentrations to achieve the same antiviral effect, compared with k ptcl , acyclovir and/or cisplatin, as shown in figure . based on a similar principle, carraher et al. studied the effect of two commercial antiviral drugs-based polymers. they found that cisplatin derivatives containing tilorone polymers have an antiviral effect on herpes simplex- , vaccinia, and varicella zoster and reovirus viruses and a tilorone derivative polymers have also an antiviral effect on both dna and rna viruses [ , ] . polymers (see figure ) were synthesized using tilorone or tilorone , (both commercially available) dissolved in distilled water, and later added to a stirred solution of potassium tetrachloroplatinate(ii) in distilled water at room temperature. they also found these two polymers require far lower concentrations to achieve the same antiviral effect, compared with k ptcl , acyclovir and/or cisplatin, as shown in figure . the addition of metallic nanoparticles to achieve antiviral properties is one of the most studied approaches. an example is the study conducted by rashid et al. who examined the addition of silver nanoparticles to polyaniline. they report that this addition contributed to the polymer antimicrobial and antiviral properties [ ] . naka et al. registered a us patent ( / a ), based on a similar method, including the addition of metal ions (ag, cu, zn, al, mg, and ca) to textile to obtain an antiviral effect [ ] . imai et al. also explored the addition of copper ions to polymers to obtain an antiviral effect. they showed that when adding copper ions to zeolites (cuzeo), the cotton gained antiviral properties against avian influenza virus h [ ] . silver (ag) particles have an antiviral effect even in their metal form; yamamoto et al. showed that the addition of ag particles to a cotton textile granted it antiviral effect against influenza a and feline calicivirus [ ] . moreover, the addition of eagle's minimal essential medium decreased the efficiency of tag particles. elechiguerra et al. [ ] and lara et al. [ , ] also studied the addition of ag particles to polymers. they showed that small ag particles ( - nm) added to poly(n-vinyl- -pyrrolidone), interacted with hiv- ′s gp protein causing viral inhibition. shree et al. found that large ag particles (~ nm) interfere with respiratory syncytial virus cell attachment [ ] . rogers et al. showed that the addition of - nm ag particles to acacia gum (a natural polysaccharide) blocked monkeypox virus interactions with host cells [ ] . the same effect on tacaribe virus was reported by hussain et al. [ ] . ishihara et al. showed that the antiviral effect of ag-chitosan composites increase with the concentration of ag particles [ ] and balagna et al. showed that ag-silica composite nanoparticles coated masks via a sputtered coating method has an antiviral effect on sars-cov- virus [ ] . romero and his associates experimented with the addition of zinc acetate (zn(ac) ) to carrageenan-based gels. the combination possessed antiviral properties against herpes simplex virus (hsv- ) and simian immunodeficiency virus (sivs) both in vitro and in vivo (tested on mises) [ ] . the anti-viral properties of quaternary ammonium has been studied extensively in the literature [ ] [ ] [ ] . lino et al. explored the antimicrobial and antiviral effect of silica nanoparticles coated with didodecyldimethylammonium bromide (ddab) [ ] . two silica nanoparticles were tested -snp and snp , with nm diameter and nm diameter respectively, while the snp was used as a reference. as illustrated in figure , coating glass slides with snp followed by another layer of the addition of metallic nanoparticles to achieve antiviral properties is one of the most studied approaches. an example is the study conducted by rashid et al. who examined the addition of silver nanoparticles to polyaniline. they report that this addition contributed to the polymer antimicrobial and antiviral properties [ ] . naka et al. registered a us patent ( / a ), based on a similar method, including the addition of metal ions (ag, cu, zn, al, mg, and ca) to textile to obtain an antiviral effect [ ] . imai et al. also explored the addition of copper ions to polymers to obtain an antiviral effect. they showed that when adding copper ions to zeolites (cuzeo), the cotton gained antiviral properties against avian influenza virus h [ ] . silver (ag) particles have an antiviral effect even in their metal form; yamamoto et al. showed that the addition of ag particles to a cotton textile granted it antiviral effect against influenza a and feline calicivirus [ ] . moreover, the addition of eagle's minimal essential medium decreased the efficiency of tag particles. elechiguerra et al. [ ] and lara et al. [ , ] also studied the addition of ag particles to polymers. they showed that small ag particles ( - nm) added to poly(n-vinyl- -pyrrolidone), interacted with hiv- s gp protein causing viral inhibition. shree et al. found that large ag particles (~ nm) interfere with respiratory syncytial virus cell attachment [ ] . rogers et al. showed that the addition of - nm ag particles to acacia gum (a natural polysaccharide) blocked monkeypox virus interactions with host cells [ ] . the same effect on tacaribe virus was reported by hussain et al. [ ] . ishihara et al. showed that the antiviral effect of ag-chitosan composites increase with the concentration of ag particles [ ] and balagna et al. showed that ag-silica composite nanoparticles coated masks via a sputtered coating method has an antiviral effect on sars-cov- virus [ ] . romero and his associates experimented with the addition of zinc acetate (zn(ac) ) to carrageenan-based gels. the combination possessed antiviral properties against herpes simplex virus (hsv- ) and simian immunodeficiency virus (sivs) both in vitro and in vivo (tested on mises) [ ] . the anti-viral properties of quaternary ammonium has been studied extensively in the literature [ ] [ ] [ ] . lino et al. explored the antimicrobial and antiviral effect of silica nanoparticles coated with didodecyldimethylammonium bromide (ddab) [ ] . two silica nanoparticles were tested -snp and snp , with nm diameter and nm diameter respectively, while the snp was used as a reference. as illustrated in figure , coating glass slides with snp followed by another layer of based on their antiviral effect, some have suggested the use of metallic nanoparticles such as ag, mg, tio etc. as coating agents to achieve antiviral properties on polymeric surfaces and fabrics. for example, us patent number / a demonstrated tio , crystalline silver copper aluminum silicate, alumina and some more metallic-based coatings [ ] . the addition of tio was also demonstrated by thilagavathi and parthasarathi who also studied the addition of metal-based particles to textiles to obtain an antiviral effect aiming at the production of antiviral surgical gowns. it was found that tio particles in nonwoven polyester grants an antiviral effect eliminating hiv, hepatitis b, and hepatitis c viruses (only) under visible light [ ] . for further information about the uses of metal/metal oxide nanoparticles in polymeric matrices to obtain antiviral properties, see the review by hilal et al. [ ] . another method of antiviral coating was demonstrated by sahin et al. who studied the consequence of adding % sodium pentaborate pentahydrate, . % triclosan and % glucapon solution to cotton textile [ ] . they reported that after min in solution (and after drying it), the textile gained antimicrobial and antiviral properties. the antimicrobial effect was tested against five bacteria (such as escherichia coli, staphylococcus aureus, and salmonella enterica subsp), one yeast (candida albicans), two fungi (aspergillus niger and trichophyton mentagrophytes) and two viruses (adenoid strain of human adenovirus type and chat strain of human poliovirus type ). the use of organo-metal hybrid polymers was also suggested. one example is polymeric anhydride of magnesium and proteic ammonium phspholinoleates that demonstrated antiviral properties against hsv- , adenovirus and canine parvovirus, as shown by durán and nunes in their us patent number , , [ ] . organotin polymers have also been studied as antiviral polymers. frank and her group proposed several organotin polymers with antiviral effects against vaccinia virus and zika virus [ ] . those polymers, as shown in figure , were synthesized using organotin dihalides, camphoric acid and lamivudine. the mechanism for the antiviral effect of organotin polymers is described in the study of barot et al. during their study, they figured that organotin materials inhibited the cell proliferation and sister chromatid exchanges, which blocked the replication of the cells. since most viruses depend on the cell replication, these materials have inherent antiviral properties [ ] . some other organo-metalhybrid polymers that have been reported as antiviral polymers are bis(cyclopentadienyl)zirconium dichloride and diethylstilbestrol-based polymers [ ] . based on their antiviral effect, some have suggested the use of metallic nanoparticles such as ag, mg, tio etc. as coating agents to achieve antiviral properties on polymeric surfaces and fabrics. for example, us patent number / a demonstrated tio , crystalline silver copper aluminum silicate, alumina and some more metallic-based coatings [ ] . the addition of tio was also demonstrated by thilagavathi and parthasarathi who also studied the addition of metal-based particles to textiles to obtain an antiviral effect aiming at the production of antiviral surgical gowns. it was found that tio particles in nonwoven polyester grants an antiviral effect eliminating hiv, hepatitis b, and hepatitis c viruses (only) under visible light [ ] . for further information about the uses of metal/metal oxide nanoparticles in polymeric matrices to obtain antiviral properties, see the review by hilal et al. [ ] . another method of antiviral coating was demonstrated by sahin et al. who studied the consequence of adding % sodium pentaborate pentahydrate, . % triclosan and % glucapon solution to cotton textile [ ] . they reported that after min in solution (and after drying it), the textile gained antimicrobial and antiviral properties. the antimicrobial effect was tested against five bacteria (such as escherichia coli, staphylococcus aureus, and salmonella enterica subsp), one yeast (candida albicans), two fungi (aspergillus niger and trichophyton mentagrophytes) and two viruses (adenoid strain of human adenovirus type and chat strain of human poliovirus type ). the use of organo-metal hybrid polymers was also suggested. one example is polymeric anhydride of magnesium and proteic ammonium phspholinoleates that demonstrated antiviral properties against hsv- , adenovirus and canine parvovirus, as shown by durán and nunes in their us patent number , , [ ] . organotin polymers have also been studied as antiviral polymers. frank and her group proposed several organotin polymers with antiviral effects against vaccinia virus and zika virus [ ] . those polymers, as shown in figure , were synthesized using organotin dihalides, camphoric acid and lamivudine. the mechanism for the antiviral effect of organotin polymers is described in the study of barot et al. during their study, they figured that organotin materials inhibited the cell proliferation and sister chromatid exchanges, which blocked the replication of the cells. since most viruses depend on the cell replication, these materials have inherent antiviral properties [ ] . some other organo-metalhybrid polymers that have been reported as antiviral polymers are bis(cyclopentadienyl)zirconium dichloride and diethylstilbestrol-based polymers [ ] . polyethyleneimine (pei) is one of the most studied amine-containing polymers in the field of antimicrobial polymers. for example, vos et al. studied the antiviral effect of branched (pei) coating over polyether sulphone (pes) membrane [ ] . pei coating led to an increase of ≥ log -units (≥ . %) in ms bacteriophages hosting strain salmonella typhimurium wg reduction on the membrane surface. as shown in figure , the effect of pei has been examined on glass slides and by using bulk solution with . % pei. besides its inherent antiviral effect, yang et al. considered some modification of pei. they showed that owing to their effect on the ph and ability to block viral entry through electrostatic interactions, mannose derivatives containing pei had antiviral effects [ ] . polyethyleneimine (pei) is one of the most studied amine-containing polymers in the field of antimicrobial polymers. for example, vos et al. studied the antiviral effect of branched (pei) coating over polyether sulphone (pes) membrane [ ] . pei coating led to an increase of -units ( . %) in ms bacteriophages hosting strain salmonella typhimurium wg reduction on the membrane surface. as shown in figure , the effect of pei has been examined on glass slides and by using bulk solution with . % pei. besides its inherent antiviral effect, yang et al. considered some modification of pei. they showed that owing to their effect on the ph and ability to block viral entry through electrostatic interactions, mannose derivatives containing pei had antiviral effects [ ] . another study about modified peis was conducted by moeller et al. they examined several modified peis obtained by reacting pei and different cyclic carbonate derivatives [ ] . while their study focused on antimicrobial/antibacterial properties, generally, antimicrobial materials also exhibit antiviral properties against enveloped viruses. modified peis were also evaluated by alvarez-lorenzo et al. synthesizing modified pei by polymerization of aziridine and magnetite-silica core-shell particles. it was evident that these polymers inhibited bacteriophage ms , hsv- , enveloped viral hemorrhagic septicaemia viruses (vhsv), and nonenveloped infectious pancreatic necrosis virus (ipnv) [ ] . polymers , , x for peer review of oxide (cu o), sulphide (cu s), iodide (cui), and chloride (cucl) have high antiviral efficiency, compared to solid-state silver and cupric compounds [ ] . zinc is another example of a metal cation that has been well studied for its in vitro antiviral effect on the viral proteins (at low concentrations) and dna (at high concentrations) [ ] . read et al. in their review about the role of zinc in antiviral immunity, pointed out that the zinc concentrations that were needed to achieve the antiviral effect (mm [ ] ) are much higher than the physiological concentrations (μm), with some differences in the needed concentrations between different types of viruses [ ] . for example, van hemert and his associates demonstrated the inhibition effect of some zinc derivatives over the binding and elongation of coronaviruses' rdrp enzymes [ ] . they showed that a combination of - μm of pyrithione with - μm of zn(ac) has a major effect, with increasing effect at higher concentrations, over sars-cov and eav populations. moreover, they suggested that water-soluble zinc-ionophores might have a higher antiviral effect. hong et al. demonstrated the effect of - μm zncl on the rna polymerase of hepatitis c viruses [ ] . takagi et al. also demonstrated the effect of zncl , but with lower concentrations of - μm on the replication of hepatitis c viruses [ ] . further, - μm of znso has an antiviral effect, as shown by ahlenstiel et al. [ ] and by brendel et al. [ ] . some other zn salts that showed antiviral effects are pyrrolidine dithiocarbamate (pdtc), zinc gluconate (zn(glu) ) [ , ] , zinc lactate (zn(lac) ), zinc citrate (cizar), zinc picolinate (zn(pic) ), and zinc aspartate (zn(asp) ) [ ] as well as zinc ionophores pyrithione [ ] . also, it seems that the antiviral effect of zinc ions is temperaturedepended. while at lower temperatures ( - °c) zinc has no significant effect on the virus population, at elevated temperatures ( - °c) the antiviral effect is significantly increased [ ] . while only a few studies have been conducted on the effect of iron ions, it seems that these ions have some level of antiviral effect, as described in aagripanti et al. work [ ] . cobalt (iii) is also a metal ion with antiviral effects [ ] . some other metal-based anions showed antiviral properties like nickel [ ] , polyoxometalates, polyatomic ions containing three transitional metals such as titanium, vanadium, tungsten, molybdenum, etc. that form clusters with the surrounding oxygen (the transition metal oxyanions linked to each other with sharing oxygen atoms). those anions show antiviral properties by affecting larson studied modified pei composed of n,n-dodecylmethyl-pei that exhibited antiviral effect on hsv- and hsv- viruses (see also figure ) [ ] , influenza a virus [ ] and on poliovirus and rotavirus [ ] . other modified pei tested by larson was based on n,n-hexylmethyl-pei coated on polyethylene, a surface with antiviral effect eliminating poliovirus and rotavirus [ ] . another study about modified peis was conducted by moeller et al. they examined several modified peis obtained by reacting pei and different cyclic carbonate derivatives [ ] . while their study focused on antimicrobial/antibacterial properties, generally, antimicrobial materials also exhibit antiviral properties against enveloped viruses. modified peis were also evaluated by alvarez-lorenzo et al. synthesizing modified pei by polymerization of aziridine and magnetite-silica coreshell particles. it was evident that these polymers inhibited bacteriophage ms , hsv- , enveloped viral hemorrhagic septicaemia viruses (vhsv), and nonenveloped infectious pancreatic necrosis virus (ipnv) [ ] . larson studied modified pei composed of n,n-dodecylmethyl-pei that exhibited antiviral effect on hsv- and hsv- viruses (see also figure ) [ ] , influenza a virus [ ] and on poliovirus and rotavirus [ ] . other modified pei tested by larson was based on n,n-hexylmethyl-pei coated on polyethylene, a surface with antiviral effect eliminating poliovirus and rotavirus [ ] . chitosan has also been studied for its antiviral and antibacterial effects. as been described in chirkov review, chitosan (and chitin) can induce interferon synthesis, which leads to suppression of the virus replication by causing damage to the rna and/or mrna [ ] . the chitosan effect was tested chitosan has also been studied for its antiviral and antibacterial effects. as been described in chirkov review, chitosan (and chitin) can induce interferon synthesis, which leads to suppression of the virus replication by causing damage to the rna and/or mrna [ ] . the chitosan effect was tested on influenza a, influenza b, alfalfa mosaic virus, bean goldish mosaic virus, peanut stunt virus, tmv, tobacco necrosis virus and some other plant viruses. moreover, some chitosan sulphate derivatives also showed an antiviral effect on hiv- . another review about the antiviral effect of chitin polymers (i.e., chitin and chitosan) and their monomer (i.e., n,n-diacetylchitobiose dimer and n-acetylglucosamine) was conducted by rogers et al. deducing similar conclusions [ ] . lei et al. also studied the antiviral effects of chitosan and found that chitosan obtained from musca domestica l housefl's larvas has an antiviral effect, based on tests conducted on autographa californica multicapsid nucleopolyhedrovirus (acmnpv) and bombyx mori nuclear polyhydrosis virus (bmnpv) [ ] . wu et al. studied a combination of cytosinpeptidemycin and chitosan oligo-saccharide, found it has an antiviral effect on tms, most likely due to several mechanisms, such as a suppression effect of the viral rna, an effect on the virus's subcellular localization as well as punctate formation of tmv mp in some plants leaves [ ] . as opposed to the above, ishihara found chitosan to be ineffective against the influenza a virus [ ] . lembo et al. studied the effect of other amines-containing polymers like poly(amidoamine)s [ ] . six polymers labeled as isa , isa , agma , agma , and agma were studied and found to exhibit antiviral effects on hsv- , hsv- , human cytomegalovirus, human papillomavirus- , respiratory syncytial virus, human rhinovirus, and vesicular stomatitis virus. other amine-containing polymers were synthesized by xiao et al. by reacting polyhexamethylene guanidine hydrochloride and acrylamide using β-cyclodextrin (cd) with eight active and five active sites. they achieved star polymers with antiviral effect on non-enveloped adenovirus [ ] . pitha et al. researched the antiviral effect of amine-containing polymers. they showed that both poly( -vinyladenine) and poly(l-vinyluracil) exhibited better antiviral properties, compared to commercial antiviral drugs [ ] . as forementioned, quaternary ammonium moieties have been studied for their antiviral properties. duizer et al. investigated the antiviral effect of several hyperbranched quaternary ammonium containing polymers on influenza a (an envelope virus) and poliovirus sabin (a non-envelop virus) [ ] . they coated glass and plastic surfaces with the polymers, and then tested their effect on the respective viruses' populations. to obtain the hyperbranched quaternary ammonium polymers, they reacted in a polymer solution containing polyamine, k co , tert-amylalcohol and heptylbromide. while the envelope containing influenza a virus showed significant decay, the non-enveloped sabin showed almost no change. klibanov et al. also studied ammonium hybrid polymers for their antiviral properties on influenza viruses [ ] . polymers labeled a-c, a-c, , , and (see figure ) , exhibited an antiviral effect in correlation with remaining cationic polymers attached to the slide surfaces. it was postulated that the antiviral effect happened by contact between the viruses and the polymers. moreover, they postulated the polymers labeled as a-c and a-c possessed probably some other antiviral mechanisms. ammonium containing phenylene ethynylene based polymers and oligomers have also been studied as antiviral materials by whitten et al. [ ] . they tested several polymers and oligomers, as shown in figure , against two model non-enveloped viruses, ms and t bacteriophages. the first is an rna virus and the latter is a dna one. they discovered that these hybrid polymers caused a ammonium containing phenylene ethynylene based polymers and oligomers have also been studied as antiviral materials by whitten et al. [ ] . they tested several polymers and oligomers, as shown in figure , against two model non-enveloped viruses, ms and t bacteriophages. the first is an rna virus and the latter is a dna one. they discovered that these hybrid polymers caused a partial dissociation of the virus structure in the dark, while under visible light and/or uv, these materials led to photochemical damage to the viruses' capsid protein. ammonium containing phenylene ethynylene based polymers and oligomers have also been studied as antiviral materials by whitten et al. [ ] . they tested several polymers and oligomers, as shown in figure , against two model non-enveloped viruses, ms and t bacteriophages. the first is an rna virus and the latter is a dna one. they discovered that these hybrid polymers caused a partial dissociation of the virus structure in the dark, while under visible light and/or uv, these materials led to photochemical damage to the viruses' capsid protein. larson investigated the antiviral and antimicrobial effects of ammonium containing n,n-dodecylmethyl-polyurethane (synthesis as illustrated in figure ) [ ] . it was concluded that this polymer, used as a coating, has an antiviral effect against influenza a viruses (enveloped) but did not affect the non-enveloped poliovirus. larson investigated the antiviral and antimicrobial effects of ammonium containing n,ndodecylmethyl-polyurethane (synthesis as illustrated in figure ) [ ] . it was concluded that this polymer, used as a coating, has an antiviral effect against influenza a viruses (enveloped) but did not affect the non-enveloped poliovirus. zhao and associates studied the combined antiviral effect of phosphonium and ammonium, by synthesizing phosphonium-type cationic polyacrylamide as shown in figure [ ] . they showed that this copolymer has an antiviral effect against non-enveloped adenovirus. zhao and associates studied the combined antiviral effect of phosphonium and ammonium, by synthesizing phosphonium-type cationic polyacrylamide as shown in figure [ ] . they showed that this copolymer has an antiviral effect against non-enveloped adenovirus. oligomeric ammonium-silane based systems and their impact on herpesvirus has been studied by prusty et al. [ ] . their results are consistent with other works related to the antiviral effect of ammonium. shuto et al. patented the antiviral (and antimicrobial) properties of quaternary ammonium ion (us patent number , , b ), antiviral coatings containing acrylic melamine, quaternary ammonium, multi-valent aromatic carboxylic-acid, and phosphoric acid were included in the claims [ ] . it should be noted that the coating was tested against influenza-a viruses. quaternary pyridinium containing polymers have also been studied for their antiviral and antimicrobial properties. xiao and xue examined the antiviral effect of quaternary pyridinium containing co-polymers on several influenza viruses (a, pr , , ) , as demonstrated in figure [ ] . oligomeric ammonium-silane based systems and their impact on herpesvirus has been studied by prusty at el [ ] . their results are consistent with other works related to the antiviral effect of ammonium. shuto et al. patented the antiviral (and antimicrobial) properties of quaternary ammonium ion (us patent number , , b ), antiviral coatings containing acrylic melamine, quaternary ammonium, multi-valent aromatic carboxylic-acid, and phosphoric acid were included in the claims [ ] . it should be noted that the coating was tested against influenza-a viruses. quaternary pyridinium containing polymers have also been studied for their antiviral and antimicrobial properties. xiao and xue examined the antiviral effect of quaternary pyridinium containing co-polymers on several influenza viruses (a, pr , , ) , as demonstrated in figure [ ]. as shown by muñoz-fernández et al., caprolactam containing polymers also have antiviral properties. they synthesized poly(n-vinyl caprolactam)-nanogels with different levels of crosslinking agents and showed that a polymer with mg of vcl and % of bis crosslinking agent inhibited the replication of r -hiv- viruses in cells [ ] . it was concluded that the antiviral effect of this polymer is affected by its thermal properties, collapsing to nanoparticles at body temperatures. since copper ions are inherently antiviral, studies have been conducted on polymers conjugated with them. for example, in gabbay has registered a us patent consisting of adding copper as shown by muñoz-fernández et al., caprolactam containing polymers also have antiviral properties. they synthesized poly(n-vinyl caprolactam)-nanogels with different levels of cross-linking agents and showed that a polymer with mg of vcl and % of bis crosslinking agent inhibited the replication of r -hiv- viruses in cells [ ] . it was concluded that the antiviral effect of this polymer is affected by its thermal properties, collapsing to nanoparticles at body temperatures. since copper ions are inherently antiviral, studies have been conducted on polymers conjugated with them. for example, in gabbay has registered a us patent consisting of adding copper particles to some polymeric fibers, such as polyamide , fibers [ ] . moreover, a review on the uses of copper and silver particles has been conducted by sánchez et al. indicating that antiviral activity can be identified in chitosan with green seed extract, polyhydroxybutyrate (phb) with cinnamaldehyde (for more information about the antiviral effect of cinnamaldehyde, see [ ] ), poly(lactic acid) (pla) with silver ions, polyhydroxybutyrate valerate (phbv) with silver nanoparticles or with copper ions and more [ ] . finkelstein and merigan studied the antiviral effect of several commercial negatively charged carboxylate-based polymers, as illustrated in figure [ ] . based on their findings, it was concluded that a higher molecular weight with higher carboxylate free groups led to a higher antiviral effect, although in vivo experiments showed that the polymers were activated only when complexed with organic cations (arginine and poly l-ornithine (plo)). in addition, bounding the carboxylate groups by amidation reduced the antiviral effect to a minimum. they emphasized that, unlike their previous hypothesis, a higher antiviral effect was achieved using non-degradable polymers. finkelstein and merigan studied the antiviral effect of several commercial negatively charged carboxylate-based polymers, as illustrated in figure [ ] . based on their findings, it was concluded that a higher molecular weight with higher carboxylate free groups led to a higher antiviral effect, although in vivo experiments showed that the polymers were activated only when complexed with organic cations (arginine and poly l-ornithine (plo)). in addition, bounding the carboxylate groups by amidation reduced the antiviral effect to a minimum. they emphasized that, unlike their previous hypothesis, a higher antiviral effect was achieved using non-degradable polymers. some other polycarboxylates were also studied by loebenstein and stein [ ] . by assessing the antiviral effect of poly(ethylene-co-maleic anhydride), poly(acrylic acid), poly(methacrylic acid), poly(vinyl methyl ether-co-maleic anhydride), poly(vinyl methyl ether-co-maleic acid), poly(vinyl methyl ether-co-maleic anhydride-co-methyl ester), poly(styrene-co-maleic anhydride), poly(isobutylene-co-maleic anhydride) and poly( -olefin octadecene-co-maleic anhydride) they understood that these co-polymers had an antiviral effect on tmv only in vivo. regelson and feltz, who studied the antiviral effect of ethylene-maleic anhydride-based-polymers showed, like finkelstein and merigan, that those polymers had an antiviral effect also in vitro [ ] . based on the antiviral effect of the malic acid/anhydride derivatives, tsunekuni et al. registered a us patent number / a claiming several polymeric fibers containing an olefine-maleic acid copolymer, a styrene-maleic acid copolymer, a vinyl ester-maleic acid copolymer, a vinyl acetatemaleic acid copolymer and a vinyl chloride-maleic acid copolymer [ ] . styrene-alt-maleic acid copolymer was also found to inhibit r and x hiv- ′s infection by krebs et al. [ ] . for further information about the effect of maleic anhydride containing polymers on different viruses, see the manuscript by popescu et al. [ ] . using the alkyne-azide click chemistry, mata et al. studied the antiviral effect of carbosilanecontaining anionic dendrimers [ ] . they found that these negatively charged dendrimers displayed an antiviral effect against hiv- viruses and that phosphonate containing dendrimers did not show any antiviral effect. as a result of the similarity to carboxylate, carboxylic acids containing polymers have been suggested as antiviral polymers, as claimed by mandeville and neenan in their us patent number , , [ ] . humic acid (ha) based phenolic polymers are polyanion polymers exhibiting antiviral activity. helbig et al. studied these polymer systems, by reacting and analyzing the effect of different o-diphenolic compounds on the polymers' antiviral effect on hsv- viruses [ ] . their results indicate that increasing the number of carboxylic groups and the number of unsaturated moieties in the starting compounds led to an increase in the antiviral activity of the respective some other polycarboxylates were also studied by loebenstein and stein [ ] . by assessing the antiviral effect of poly(ethylene-co-maleic anhydride), poly(acrylic acid), poly(methacrylic acid), poly(vinyl methyl ether-co-maleic anhydride), poly(vinyl methyl ether-co-maleic acid), poly(vinyl methyl ether-co-maleic anhydride-co-methyl ester), poly(styrene-co-maleic anhydride), poly(isobutylene-co-maleic anhydride) and poly(α-olefin octadecene-co-maleic anhydride) they understood that these co-polymers had an antiviral effect on tmv only in vivo. regelson and feltz, who studied the antiviral effect of ethylene-maleic anhydride-based-polymers showed, like finkelstein and merigan, that those polymers had an antiviral effect also in vitro [ ] . based on the antiviral effect of the malic acid/anhydride derivatives, tsunekuni et al. registered a us patent number / a claiming several polymeric fibers containing an olefine-maleic acid copolymer, a styrene-maleic acid copolymer, a vinyl ester-maleic acid copolymer, a vinyl acetate-maleic acid copolymer and a vinyl chloride-maleic acid copolymer [ ] . styrene-alt-maleic acid copolymer was also found to inhibit r and x hiv- s infection by krebs et al. [ ] . for further information about the effect of maleic anhydride containing polymers on different viruses, see the manuscript by popescu et al. [ ] . using the alkyne-azide click chemistry, mata et al. studied the antiviral effect of carbosilanecontaining anionic dendrimers [ ] . they found that these negatively charged dendrimers displayed an antiviral effect against hiv- viruses and that phosphonate containing dendrimers did not show any antiviral effect. as a result of the similarity to carboxylate, carboxylic acids containing polymers have been suggested as antiviral polymers, as claimed by mandeville and neenan in their us patent number , , [ ] . humic acid (ha) based phenolic polymers are polyanion polymers exhibiting antiviral activity. helbig et al. studied these polymer systems, by reacting and analyzing the effect of different o-diphenolic compounds on the polymers' antiviral effect on hsv- viruses [ ] . their results indicate that increasing the number of carboxylic groups and the number of unsaturated moieties in the starting compounds led to an increase in the antiviral activity of the respective polymers. other acid-containing polymers reported as antiviral polymers are poly(lysine), poly(glutamic acid), and poly(acrylic acid) [ ] . sano has considered the effect of alginate on tmv [ ] , which is a natural anionic polymer, usually obtained from seaweeds [ ] . he indicated that alginate had an antiviral effect on tms, demonstrating greater effect with decreasing polymer chains' stiffness [ ] . as shown by mooney and lee, alginate also exhibits an antiviral effect on adenovirus [ ] . sulphate containing polymers have also been studied for their antiviral properties. görög et al. studied the effect of polyvinyl-alcohol-sulphate (pvas) and polyvinyl-alcohol-sulphate-co-acrylic acid (pavas) as inhibitors for hiv- , hiv- , herpesvirus, human cytomegalovirus, vesicular stomatitis virus, respiratory syncytial virus, sindbis virus, semliki forest virus, junin virus, tacaribe virus, and murine sarcoma virus [ ] . the two polymers were shown to have an antiviral effect over those viruses but do not affect non-envelop viruses such as reovirus and poliovirus. they also found that the greater the sulphonation degree and/or the greater the molecular weight of the polymers, the higher the antiviral effect became. hayashi et al. explored the antiviral effect of modified calcium spirulan, a sulphated polysaccharide. they established that several modified spirulan have an antiviral property. unlike other studies, non-toxic metal ions have a greater effect compared to copper and silver modified spirulan [ ] . patents have also been registered in the field of sulphate containing polymers, like the us patent of munson and tankersley who patented the use of sulphonated naphthalene formaldehyde condensates (snfc) as antiviral materials [ ] . the effect of sulphate on several viruses has also been analyzed by schelhaas at el. in their inquiry, they tested the antiviral effect of sulphated glycomimetic oligomers and polymers as described in figure and table [ ] . spontak et al. studied the antiviral and antimicrobial properties of poly[tert-butylstyrene-b-(ethylene-alt-propylene)-b-(styrene sulphonate)-b-(ethylene-alt-propylene)-b-tert-butylstyrene] (teset). as already mentioned, they found that the antiviral efficiency of the polymer increases with increasing the degree of sulphonation (in their study, they changed the degree of mol% of the mid-block) [ ] . polymers , , x for peer review of [ ] . polymers , , x for peer review of mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv- [ ] . neurath et al. also pointed the anti-hiv- effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [ ] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv- viruses [ ] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv- [ ] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv- and hsv- . they suggested the antiviral effect is due to blocking of virion adsorption to host cells [ ] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [ ] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv- [ ] . neurath et al. also pointed the anti-hiv- effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [ ] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv- viruses [ ] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv- [ ] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv- and hsv- . they suggested the antiviral effect is due to blocking of virion adsorption to host cells [ ] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [ ] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these [ ] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv- viruses [ ] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv- [ ] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv- and hsv- . they suggested the antiviral effect is due to blocking of virion adsorption to host cells [ ] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [ ] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv- [ ] . neurath et al. also pointed the anti-hiv- effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [ ] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv- viruses [ ] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv- [ ] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv- and hsv- . they suggested the antiviral effect is due to blocking of virion adsorption to host cells [ ] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [ ] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these mimura et al. suggested the use of sulphate modified cellulose and branched cellulose as antiviral polymers. they found that cellulose and branched cellulose with a high degree of sulphonation demonstrated an antiviral effect on hiv- [ ] . neurath et al. also pointed the anti-hiv- effect of sulphate modified cellulose as well as a similar effect of buffergel and aryl sulphonates [ ] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv- viruses [ ] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv- [ ] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv- and hsv- . they suggested the antiviral effect is due to blocking of virion adsorption to host cells [ ] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [ ] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these [ ] . sulphated derivatives of natural polymers as antiviral agents have also been demonstrated by matsuzaki et al. in their research, they studied the antiviral effect of sulphates of curdlan and its branched derivatives. by reacting piperidine n-sulphonic acid or so -iimethylformamide complex with curdlan, they obtained antiviral properties against hiv- viruses [ ] . hirsch et al. demonstrated an antiviral naphthalene-sulphonate containing polymer with inhibition ability of hiv- [ ] . the antiviral properties of sulphated polysaccharides have also been studied by dong et al. in their study, they found that two fucoidans obtained from brown algae sargassum henslowianum have an antiviral effect on hsv- and hsv- . they suggested the antiviral effect is due to blocking of virion adsorption to host cells [ ] . other antiviral sulphate-containing polymers and their effect on tmv have also been investigated by sano [ ] that showed that two types of polysaccharides, chondroitin sulphate types c and a, have some inhibition effect on tmv. in a continuing study, he found that the effect of these polymers on the virus might increase or decrease, depending on the molecular weight [ ] . furthermore, the antiviral effect might be attributed to the viral envelope surface protein decapsulation process caused by the polymers. combining both amine and sulphate moieties, sulphated cellobiose-polylysine dendrimers were studied by yoshida et al. [ ] . they found that a shorter distance between the terminal sulphate cellobiose units increases the anti-hiv- effect. sulphonic acid-containing polymers were studied by clercq et al. in the course of their study, they synthesized four polymers -poly( -styrene sulphonic acid) (pss), poly(anethole sulphonic acid) (pas), poly(vinyl sulphonic acid) (pvs) and poly( -acry-lamido- -methyl-l-propane sulphonic acid) (pamps). they concluded these four polymers exhibited antiviral effect on hiv- and hiv- in mt- cells, using concentrations which were not toxic to the hosting cells. they also reported these polymers inhibited the syncytium formation in co-cultures of molt- cells infected with hiv- and hiv- [ ] . pvs was also tested by shimonaski et al. with similar results [ ] . cardin et al. examined a sulphonic acid polymer named mdl , a biphenyl disulphonic acid urea copolymer [ ] . they found this polymer has an antiviral effect on hiv- viruses. sulphites containing polymers also show an antiviral effect, as was demonstrated by christopher et al. in their us patent number a [ ] . they demonstrated a change in the oxidation degree of the sulphite's ions using a catalyst, which led to novel antiviral polymers. several hybrid polymers containing anions moieties and sulphate moieties were demonstrated by orsi et al. [ ] . they revealed that dextran sulphate, scleroglucan, and lambda carrageenan have antiviral effects on hsv- which is greater than the effect of glyloid sulphate and locust bean gum. furthermore, these polymers influence hsv- , with similar effect like dextran sulphate, glyloid sulphate and lambda carrageenan which is higher than the effect of scleroglucan and glycogen sulphate . finally, they explained that those polymer efficacies were based on their ionic potential. polyphenols are another example of antiviral polymers. as was demonstrated by tempesta in his us patent number , , , proanthocyanidins-based polymers have antiviral properties against several viruses, including influenza a, b, and c, respiratory syncytial virus and herpes viruses [ ] . gilbert et al. also confirmed the antiviral effect of polyphenols. they showed an antiviral effect of sp- , a natural polyphenolic polymer, on respiratory syncytial and parainfluenza type viruses [ ] . due to the antiviral effect of polyphenols, lebrun et al. suggested the use of catechin polyphenol grafted non-woven cellulosic fabrics as bio-based cleaning wipes and filters [ ] . in their study, they used laccase enzymatic oxidation to obtain the catechin grafting over the cellulose-based fabrics. these treated fabrics showed antiviral effect ( -log after h) on t d bacteriophage virus of escherichia coli b. panarin et al. [ ] synthesized several co-polymers (as shown in figure ) by reacting -dimethylaminoethyl methacrylate and -diethylaminoethyl methacrylate with -deoxy- -methacryalamido-d-glucose and n-vinyl-n-methylacetamide with -dialkylaminoethyl methacrylate units. they established the antiviral properties of these polymers against influenza a virus subtype h n . panarin et al. [ ] synthesized several co-polymers (as shown in figure ) by reacting dimethylaminoethyl methacrylate and -diethylaminoethyl methacrylate with -deoxy- methacryalamido-d-glucose and n-vinyl-n-methylacetamide with -dialkylaminoethyl methacrylate units. they established the antiviral properties of these polymers against influenza a virus subtype h n . benzophenones were also studied in the field of antiviral polymers. sun et al. examined the antiviral effect of benzophenones particles (see figure ) on poly(vinyl alcohol-co-ethylene)-based membranes [ ] . they found that these particles performed under visible light (due to the formation of rnmh·) but could also affect viruses in the dark. however, only non-envelope double-stranded dna viruses were tested. this conclusion points to the potential of these articles as rechargeable antiviral materials. benzophenones were also studied in the field of antiviral polymers. sun et al. examined the antiviral effect of benzophenones particles (see figure ) on poly(vinyl alcohol-co-ethylene)-based membranes [ ] . they found that these particles performed under visible light (due to the formation of rnmh·) but could also affect viruses in the dark. however, only non-envelope double-stranded dna viruses were tested. this conclusion points to the potential of these articles as rechargeable antiviral materials. in addition, ′-fluorinated-aristeromycin analogs were proposed as antiviral materials, owing to their effect on the rna polymerase (rdrp) and the host cell s-adenosyl-l-homocysteine (sah) hydrolase. polymers based on this group have been discussed by jeong et al. as general antiviral polymers, however, some have only limited effect on few types of viruses. the most efficient of these polymers is illustrated in figure , as well as its antiviral test results [ ] . in addition, -fluorinated-aristeromycin analogs were proposed as antiviral materials, owing to their effect on the rna polymerase (rdrp) and the host cell s-adenosyl-l-homocysteine (sah) hydrolase. polymers based on this group have been discussed by jeong et al. as general antiviral polymers, however, some have only limited effect on few types of viruses. the most efficient of these polymers is illustrated in figure , as well as its antiviral test results [ ] . polymers , , x for peer review of tong and sankarakumar suggested the use of imprinted polymers. accordingly, a one-stage mini-emulsion polymerization, as illustrated in figure , could be used to entrap viruses instead of inhibiting their activity [ ] . notably, this method is virus-specific, depending on the imprinted polymer matching a specific virus. tong and sankarakumar suggested the use of imprinted polymers. accordingly, a one-stage mini-emulsion polymerization, as illustrated in figure , could be used to entrap viruses instead of inhibiting their activity [ ] . notably, this method is virus-specific, depending on the imprinted polymer matching a specific virus. polymers , , x for peer review of while most antiviral effects reported in literature stem from materials composition, some report on temperature-dependent antiviral effects. wout et al. studied short-time pasteurization effects on human milk. the process of heating the milk to °c for s is well effective against bovine viral diarrhea, hepatitis a and c viruses, hiv- and hiv- , porcine parvovirus, and pseudorabies viruses [ ] . while heating above °c may cause damage to most viruses, at lower temperatures, as was shown by rott and scholtissek [ ] and by goede et al. [ ] , the viral activity might increase when temperatures are increased. similar conclusions were reported by molla et al. while studying the effect of temperature on poliovirus formation and rna synthesis [ ] . yamaya et al., however, showed that, above °c, the influenza viruses' replication decrease [ ] , while reed showed that for panonychus-citri's nonoccluded virus, the biocidal temperature (above which the virus population is damaged) is °c [ ] . chan, et al. showed that for sars-cov, the biocidal temperature is < °c [ ] and harrison showed that rothamsted tobacco necrosis virus biocidal temperature is even lower than influenza's [ ] . therefore, it seems that biocidal temperature differs from virus to virus and that dna viruses tend to be more stable than rna viruses [ ] . along with the direct effect of temperatures on some viruses' populations, some researchers have studied the temperature-dependent antiviral effects of ions. for example, brendel et al. showed that the effect of zn ⁺ ions is temperature-dependent increased with increasing temperatures [ ] . sánchez et al. studied the antiviral effects of ag nanoparticles on norovirus [ ] . they tested this effect at two different temperatures, namely and °c, and found an increased antiviral effect at higher temperatures. kaufman et al., at their investigation about the thermodynamics of the antiviral effect of ions on the interaction between ns protein and single-stranded rna, also suggested that increasing the temperature may increase the antiviral effect [ ] . bisaillon et al. studied the kinetics of metal ions binding to hepatitis c's rna polymerase and suggested the reaction's kinetics is temperaturedependent; increasing the temperatures caused accelerated binding [ ] . surfactin is a surfactant used for antiviral and antimicrobial applications. pauli et al. showed that its antiviral effect on the non-enveloped hsv- , hsv- , suid herpes virus type (shv- ), vsv, siv, feline calicivirus (fcv), and murine encephalomyocarditis virus (emcv) increased with increasing temperatures [ ] . for shv- , the efficiency increased linearly with increasing temperature up to °c, above which the rate of inhibition was too fast to measure. hogle et al. showed that the efficiency of drugs that bind to poliovirus also increases with rising temperatures [ ] . while most antiviral effects reported in literature stem from materials composition, some report on temperature-dependent antiviral effects. wout et al. studied short-time pasteurization effects on human milk. the process of heating the milk to • c for s is well effective against bovine viral diarrhea, hepatitis a and c viruses, hiv- and hiv- , porcine parvovirus, and pseudorabies viruses [ ] . while heating above • c may cause damage to most viruses, at lower temperatures, as was shown by rott and scholtissek [ ] and by goede et al. [ ] , the viral activity might increase when temperatures are increased. similar conclusions were reported by molla et al. while studying the effect of temperature on poliovirus formation and rna synthesis [ ] . yamaya et al., however, showed that, above • c, the influenza viruses' replication decrease [ ] , while reed showed that for panonychus-citri's nonoccluded virus, the biocidal temperature (above which the virus population is damaged) is • c [ ] . chan, et al. showed that for sars-cov, the biocidal temperature is < • c [ ] and harrison showed that rothamsted tobacco necrosis virus biocidal temperature is even lower than influenza's [ ] . therefore, it seems that biocidal temperature differs from virus to virus and that dna viruses tend to be more stable than rna viruses [ ] . along with the direct effect of temperatures on some viruses' populations, some researchers have studied the temperature-dependent antiviral effects of ions. for example, brendel et al. showed that the effect of zn + ions is temperature-dependent increased with increasing temperatures [ ] . sánchez et al. studied the antiviral effects of ag nanoparticles on norovirus [ ] . they tested this effect at two different temperatures, namely and • c, and found an increased antiviral effect at higher temperatures. kaufman et al., at their investigation about the thermodynamics of the antiviral effect of ions on the interaction between ns protein and single-stranded rna, also suggested that increasing the temperature may increase the antiviral effect [ ] . bisaillon et al. studied the kinetics of metal ions binding to hepatitis c's rna polymerase and suggested the reaction's kinetics is temperature-dependent; increasing the temperatures caused accelerated binding [ ] . surfactin is a surfactant used for antiviral and antimicrobial applications. pauli et al. showed that its antiviral effect on the non-enveloped hsv- , hsv- , suid herpes virus type (shv- ), vsv, siv, feline calicivirus (fcv), and murine encephalomyocarditis virus (emcv) increased with increasing temperatures [ ] . for shv- , the efficiency increased linearly with increasing temperature up to • c, above which the rate of inhibition was too fast to measure. hogle et al. showed that the efficiency of drugs that bind to poliovirus also increases with rising temperatures [ ] . tannins are natural polyphenols, that have been studied for their antimicrobial and antiviral properties. mileva et al. showed that, much like other antiviral materials, tannins' antiviral effects are temperature-dependent, and are more efficient at • c than at room temperature [ ] . the antiviral battle has been the focus of numerous studies over the years. while originally focusing on small molecules based on antiviral drugs, in recent years, researchers started concentrating on hybrid and composite polymers as a promising approach to the global viral problem. the antiviral campaign is waged in two main approaches. the first employing polymers as drug delivery systems based on biodegradable polymers conjugated with antiviral drugs. in this approach, polymers are used in a supporting role, increasing efficacy and half-life of delivered drugs. the second exploiting hybrid and composite polymers. in this approach, polymers are incorporated with metal and/or metal-ions particles like zinc, silver, copper, zirconium, magnesium, tungsten, and more or otherwise hybridized antiviral polymers based on electrically-charged moieties, are employed, whether anionic or anionic, such as carboxylate and/or organic acids/anhydride, or cationic or cationic such as ammonium, phosphonium, and amines. some other approaches studied include sulphate containing polymers, phenol/hydroxyl-containing polymers and organometal polymers such as organotin-based polymers. moreover, some studies have suggested the polymerization of commercial antiviral drugs to achieve a more efficient treatment. lastly, it was shown that some antiviral effects are temperature dependent. most of the reported investigations pointed out that increased temperatures enhance the antiviral impact of ions and the hybrid polymers. thus, a combination of anti-viral polymers along with the ability to increase temperature may be an additional tool to increase antiviral properties. the authors declare no conflict of interest. expression of animal virus genomes polypeptide components of virions, top component and cores of reovirus type structure and classification of viruses the species problem in virology classification and identification of aquatic animal viruses the molecular mechanisms of copper and silver ion disinfection of bacteria and viruses early interactions of hepatitis a virus with cultured cells: viral elution and the effect of ph and calcium ions effect of ionic concentration on the infectivity of a virus of the citrus red mite, panonychus citri the significance of oxidation in chemical inactivation of poliovirus on the cytotoxicity of vitamin c and metal ions: a site-specific fenton mechanism roles of copper and superoxide anion radicals in the radiation-induced inactivation of t bacteriophage reversal of the silver inhibition of microorganisms by agar application of silver nanoparticles in viral inhibition: a new hope for antivirals pharmacology and toxicology of heavy metals: silver theory meets experiment: metal ion effects in hcv genomic rna kissing complex formation evidence for a non-catalytic ion-binding site in multiple rna-dependent rna polymerases effect of metal ion binding on the structural stability of the hepatitis c virus rna polymerase multivalent ion effects on electrostatic stability of virus-like nano-shells highly efficient antiviral and antibacterial activities of solid-state cuprous compounds the mechanism of the antiherpetic activity of zinc sulphate the role of zinc in antiviral immunity zn + inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture characterization of soluble hepatitis c virus rna-dependent rna polymerase expressed in escherichia coli zinc is a negative regulator of hepatitis c virus rna replication the antiviral role of zinc and metallothioneins in hepatitis c infection effect of treatment with zinc gluconate or zinc acetate on experimental and natural colds antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections virus inactivation by copper or iron ions alone and in the presence of peroxide cobalt complexes as antiviral and antibacterial agents broad spectrum anti-rna virus activities of titanium and vanadium substituted polyoxotungstates in vitro antimicrobial effects of virus block, which contains multiple polyoxometalate compounds, and hygienic effects of virus block-supplemented moist hand towels antibacterial/antiviral property and mechanism of dual-functional quaternized pyridinium-type copolymer. polymers (basel) studies of inhibitory effect of ammonium ions in several virus-tissue culture systems a common mode of antiviral action for ammonium ions and various amines antiviral effects of ferric ammonium citrate different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus enhanced antioxidant and antifungal activity of chitosan derivatives bearing -o-imidazole-based quaternary ammonium salts novel quaternary phosphonium-type cationic polyacrylamide and elucidation of dual-functional antibacterial/antiviral activity synthetic biologically active polymers chitosan/xanthan gum based hydrogels as potential carrier for an antiviral drug: fabrication, characterization, and safety evaluation quaternary ammonium-based biomedical materials: state-of-the-art, toxicological aspects and antimicrobial resistance from multifunctionalized poly(ethylene imine)s toward antimicrobial coatings inactivation of foot-and-mouth disease virus by commercially available disinfectants and cleaners synergistic antiviral effect of xanthates and ionic detergents mechanism of the antiviral activity of new aurintricarboxylic acid analogues nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis b and hepatitis d infection ion transport blockers inhibit human rhinovirus release antiviral effects of ascorbic and dehydroascorbic acids in vitro application of nanotechnology in textile engineering: an overview camphor-based symmetric diimines as inhibitors of influenza virus reproduction discovery of a new class of antiviral compounds: camphor imine derivatives broad range of inhibiting action of novel camphor-based compound with anti-hemagglutinin activity against influenza viruses in vitro and in vivo polymeric films as a promising carrier for bioadhesive drug delivery: development, characterization and optimization. saudi pharm a novel polymeric chlorhexidine delivery device for the treatment of periodontal disease release of antimicrobial and antiviral drugs from methacrylate copolymer system: effect of copolymer molecular weight and drug loading on drug release elevated ambient temperature differentially affects virus resistance in two tobacco species efficient antiviral co-delivery using polymersomes by controlling the surface density of cell-targeting groups for influenza a virus treatment polymer-attached zanamivir inhibits synergistically both early and late stages of influenza virus infection towards an improved anti-hiv activity of nrti via metal-organic frameworks nanoparticles biomedical applications of metal organic frameworks versatile iron-catechol-based nanoscale coordination polymers with antiretroviral ligand functionalization and their use as efficient carriers in hiv/aids therapy macromolecular (pro)drugs in antiviral research macromolecular prodrugs of ribavirin combat side effects and toxicity with no loss of activity of the drug antiviral polymeric drugs and surface coatings conjugating drug candidates to polymeric chains does not necessarily enhance anti-influenza activity polymer-bound sialyl-n-acetyllactosamine protects mice infected by influenza virus antiviral and anticancer activity of cisplatin derivatives of tilorone cisplatin derivatives as antiviral agents antibacterial activity of polyaniline coated silver nanoparticles synthesized from piper betle leaves extract anti-viral fiber, process for producing the fiber, and textile product comprising the fiber inactivation of high and low pathogenic avian influenza virus h subtypes by copper ions incorporated in zeolite-textile materials antiviral activity of silver nanoparticles immobilized onto textile fabrics synthesized by radiochemical process interaction of silver nanoparticles with hiv- pvp-coated silver nanoparticles block the transmission of cell-free and cell-associated hiv- in human cervical culture mode of antiviral action of silver nanoparticles against hiv- silver nanoparticles inhibit replication of respiratory syncytial virus a preliminary assessment of silver nanoparticle inhibition of monkeypox virus plaque formation interaction of silver nanoparticles with tacaribe virus antiviral activity of silver nanoparticle/chitosan composites against h n influenza a virus virucidal effect against coronavirus sars-cov- of a silver nanocluster/silica composite sputtered coating a modified zinc acetate gel, a potential nonantiretroviral microbicide, is safe and effective against simian-human immunodeficiency virus and herpes simplex virus infection in vivo nanoparticles and surfaces presenting antifungal, antibacterial and antiviral properties coating composition, resin composition and antiviral product development of tri-laminate antiviral surgical gown for liquid barrier protection polymeric membranes incorporated with metal/metal oxide nanoparticles: a comprehensive review developing novel antimicrobial and antiviral textile products polymeric anhydride of magnesium and proteic ammonium phospholinoleate with antiviral, antineoplastic and immunostimulant properties organotin polymers as antiviral agents including inhibition of zika and vaccinia viruses antiviral activity of metal-containing polymers-organotin and cisplatin-like polymers metal-containing and related polymers for biomedical applications virus reduction through microfiltration membranes modified with a cationic polymer for drinking water applications high selectivity, and resistance mitigation antiviral properties of polymeric aziridine-and biguanide-modified core-shell magnetic nanoparticles decreasing herpes simplex viral infectivity in solution by surface-immobilized and suspended n,n-dodecyl,methyl-polyethylenimine biocidal packaging for pharmaceuticals, foods, and other perishables hydrophobic polycationic coatings disinfect poliovirus and rotavirus solutions agricultural uses of chitin polymers antioxidant, antifungal and antiviral activities of chitosan from the larvae of housefly, musca domestica l. food chem novel combined biological antiviral agents cytosinpeptidemycin and chitosan oligosaccharide induced host resistance and changed movement protein subcellular localization of tobacco mosaic virus agmatine-containing poly(amidoamine)s as a novel class of antiviral macromolecules: structural properties and in vitro evaluation of infectivity inhibition tailor-made antimicrobial/antiviral star polymer via atrp of cyclodextrin and guanidine-based macromonomer uptake and fate of water-soluble, nondegradable polymers with antiviral activity in cells and animals polymeric coatings that inactivate both influenza virus and pathogenic bacteria cationic phenylene ethynylene polymers and oligomers exhibit efficient antiviral activity antiviral and antibacterial polyurethanes of various modalities anti-herpesviral effects of a novel broad range anti-microbial quaternary ammonium silane, k antimicrobial and antiviral polymeric materials inhibitory effect of cinnamaldehyde, derived from cinnamomi cortex, on the growth of influenza a/pr/ virus in vitro and in vivo polymers and biopolymers with antiviral activity: potential applications for improving food safety interferon-stimulating and in vivo antiviral effects of various synthetic anionic polymers induced interference by synthetic polyanions with the infection of tobacco mosaic virus ethylene maleic anhydride copolymers as viral inhibitors a styrene-alt -maleic acid copolymer is an effective inhibitor of r and x human immunodeficiency virus type infection biomedical applications of maleic anhydride copolymers synthesis of anionic carbosilane dendrimers via "click chemistry" and their antiviral properties against hiv antiviral polymers comprising acid functional groups and hydrophobic groups anti-herpes simplex virus type activity of humic acid-like polymers and their o-diphenolic starting compounds antiviral activity of alginate against infection by tobacco mosaic virus alginate: properties and biomedical applications sulphated polymers are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, respiratory syncytial virus, and toga-, arena-, and retroviruses effects of structural modification of calcium spirulan, a sulfated polysaccharide from spirulina platensis, on antiviral activity antiviral sulfonated naphthalene formaldehyde condensation polymers prophylactic antiviral activity of sulfated glycomimetic oligomers and polymers inherently self-sterilizing charged multiblock polymers that kill drug-resistant microbes in minutes synthesis, structure and antiviral activity of sulfates of curdlan and its branched derivatives anti-hiv- activity of anionic polymers: a comparative study of candidate microbicides naphthalene sulfonate polymers with cd -blocking and anti-human immunodeficiency virus type activities structural characterization and antiviral activity of two fucoidans from the brown algae sargassum henslowianum antiviral activity of polysaccharides against infection of tobacco mosaic virus antiviral activity of chondroitin sulfate against infection by tobacco mosaic virus elucidation of anti-hiv mechanism of sulfated cellobiose -polylysine dendrimers sulfonic acid polymers as a new class of human immunodeficiency virus inhibitors antiviral activity of an interferon-inducing synthetic polymer potent inhibition of human immunodeficiency virus by mdl , a novel sulphonic acid polymer inhibition of herpes simplex virus infection by negatively charged and neutral carbohydrate polymers tempesta proanthocyanidin polymers having antiviral activity and methods of obtaining same the antiviral activity of sp- , a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type viruses in cotton rats antiviral effects of polyphenols: development of bio-based cleaning wipes and filters synthesis and antibacterial and antiviral properties of silver nanocomposites based on water-soluble -dialkylaminoethyl methacrylate copolymers daylight-driven rechargeable antibacterial and antiviral nanofibrous membranes for bioprotective applications synthesis, and anti-rna virus activity of -fluorinated-aristeromycin analogues preventing viral infections with polymeric virus catchers: a novel nanotechnological approach to anti-viral therapy antimicrobial and antiviral effect of high-temperature short-time (htst) pasteurization apllied to human milk effect of temperature on the multiplication of an influenza virus effect of temperature on survival and growth of infectious pancreatic necrosis virus effects of temperature and lipophilic agents on poliovirus formation and rna synthesis in a cell-free system effects of high temperature on pandemic and seasonal human influenza viral replication and infection-induced damage in primary human tracheal epithelial cell cultures effects of temperature on virus-host relationships and on activity of the noninclusion virus of citrus red mites, panonychus citri the effects of temperature and relative humidity on the viability of the sars coronavirus studies on the effect of temperature on virus multiplication in inoculated leaves literature review of the effect of salinity on the larger marine algae antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems thermodynamic study of the effect of ions on the interaction between dengue virus ns helicase and single stranded mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis stabilization of poliovirus by capsid-binding antiviral drugs is due to entropic effects abstract tannins as antiviral agents this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -vmnc yg authors: minetti, giampaolo title: mevalonate pathway, selenoproteins, redox balance, immune system, covid- : reasoning about connections date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: vmnc yg it has been proposed that a degraded immune system is (one of) the condition(s) that predispose certain subjects to fatal consequences from infection by sars-cov- . it is unknown whether therapeutic regimens to which these patients may have been subjected to in the months/years preceding the infection could be immunocompromising. statins are among the most widely prescribed cholesterol-lowering drugs. as competitive inhibitors of hmg-coa-reductase, the key enzyme of the “mevalonate pathway” through which essential compounds, not only cholesterol, are synthesized, statins decrease the levels of cholesterol, and thus ldls, as an innate defense mechanism, with controversial results in decreasing mortality from cardiovascular disease. moreover, statins have pleiotropic, mostly deleterious effects on many cell types, including immune cells. in the attempt to decipher the enigma of sars-cov- infectivology, the hypothesis should be tested whether the population of subjects who succumbed to covid- may have developed a compromised immunity at sub-clinical levels and have become more susceptible to fatal consequences from sars-cov- infection due to statin therapy. it has been suggested that the presence of a degraded immune system is (one of) the condition(s) for the susceptibility of certain predisposed subjects, usually the elderly and those presenting with various co-morbidities, to infection by sars-cov- and its often fatal consequences. but a significant number of young and otherwise apparently healthy subjects has succumbed, and still is succumbing, to covid- . immunodepression has been linked to several possible environmental factors, such as pollution, electromagnetic fields, global warming, but also to drug induced side effects, such as in oncologic patients on chemotherapy. no attempts have been made, however, to carry out an anamnestic evaluation of the subjects, especially the younger ones, who have died from the consequences of covid- . in particular it is unknown whether a weakened immune system could be the result of possible adverse effects from therapeutic regimens to which these patients may have been subjected to in the months/years preceding the infection. several drugs have been considered as potential factors that predispose (anti-hypertensive, in particular angiotensin ii receptor blockers, mineralocorticoid-receptor antagonists, anti-diabetics) or protect (hydroxychloroquine, corticosteroids) from sars-cov- infection. however, to the best of our knowledge, no systematic studies have been carried out to evaluate the role played by one family of drugs that has been largely prescribed to the general population over the past couple of decades: the statins. a couple of studies have indeed been published that exclude a statistically significant correlation between covid- outcome and statin routine treatment of patients, but no data on compliance, or cholesterol levels were provided. , one of the articles has been retracted by the authors almost immediately. statins are cholesterol-lowering drugs that act as competitive inhibitors of the enzyme -hydroxy--methyl-glutaryl-coenzyme-a (hmg-coa)-reductase, the key enzyme of the "mevalonate pathway". starting from acetic acid as the building block, this pathway leads to the biosynthesis of isoprenic units, which are in turn assembled to produce such various and essential compounds for both plant and animal cells: dolichol, coenzyme q, isopentenyl for the modification of trnas, farnesyl and geranyl-geranyl for the prenylation of proteins, rubber, plant hormones, phytol chain of chlorophyll, carotenoids, vitamin a, vitamin e, vitamin k, vitamin d, bile acids, steroid hormones and cholesterol. since hmg-coa reductase is at the very root of the pathway, its inhibition will prevent the production of not only cholesterol, but also of all the essential compounds listed above. the theory enforcing cholesterol-lowering therapies holds that high levels of serum cholesterol, in the form of low density lipoproteins (ldl), are positively correlated with the prevalence of cardiovascular disease and mortality from myocardial infarction and stroke. however, metaanalyses of the outcome of several clinical trials have revealed that the correlation between cholesterol levels and mortality is either non-existing or inverse: the lower the cholesterol levels the higher the mortality from all causes, especially those related to infection. [ ] [ ] [ ] [ ] furthermore, the levels of ldl cholesterol are inversely associated with longevity in the elderly, and low serum cholesterol is even a negative prognostic factor in patients with advanced heart failure, independent from lipid-lowering therapy. ldl cholesterol as part of the innate immunity the role of lipoproteins as first line defense against microbial infection is well established. , prolonged infection with hepatitis b virus has been show to correlate with low blood cholesterol levels and cancer, and hypocholesterolemic men have significantly fewer circulating lymphocytes, total t cells and cd + cells compared with hypercholesterolemic subjects. in spite of this evidence, the prevailing theory has not been could not be overturned, and statins are still widely prescribed for the "treatment of blood cholesterol". indeed, although statins are ineffective in the secondary prevention of cardiovascular events, they appear to be beneficial in the primary prevention of mortality from myocardial infarction. however, the benefit is very modest, as one hundred subjects need to be treated to prevent one single infarction-related casualty. moreover, this efficacy appears to be related to one of the multiple pleiotropic effects of statins (see below), i.e. an anti-inflammatory effect, rather than a cholesterol-lowering action. a number of different cell types can be targeted by the adverse effects of statins, the best known being the skeletal muscle cell. severe rhabdomyolysis, leading to more than deaths, was reported in for subjects on treatment with "first generation" statins. all muscle cells, including the myocardium, are especially dependent on mitochondrial respiration for the production of atp. coenzyme q is an essential component of the electron transport chain in the inner mitochondrial membrane and it is synthesized in the mevalonate pathway. one of the most diffusely reported adverse effects of "new generation" statins is myopathy, which is linked to mitochondrial damage likely resulting from deficiency of coenzyme q. not surprisingly, coenzyme q supplementation has been found to ameliorate myopathy in subjects on statins. deleterious side-effects of statins on the central and peripheral nervous systems, including cognitive disorders have also been reported. because statins inhibit the biosynthesis of substances that are required for the post-translational modification of proteins (and the maturation of trnas, see below), virtually every cellular metabolic and signaling pathway may be affected. thus, statins exert a general inhibitory action on immune cells, probably by interfering with prenylation of g proteins: growth, proliferation, adhesiveness and chemotaxis are inhibited in monocytes. lymphoid cell function is suppressed by statins in vitro, although the mechanism has not been characterized. given their depressive action on immune cells, statins have been proposed as immunomodulators with milder effects than the conventional immunosuppressive drugs. it can be concluded that statin therapy may contribute to generalized immunosuppression. less investigated is the impact of statins on redox metabolism. an important contribution to the redox balance in cells and tissues comes from selenoproteins, which contain one or more selenocysteine (sec) residues in their sequence. the majority of seleoproteins are selenoenzymes, as the sec residue(s) are located in the catalytic site, with antioxidant and oxidative damage-repairing activities. the human selenoproteome comprises different selenoproteins. the best characterized are glutathione peroxidases, thioredoxin reductases, iodothyronine deiodinases, and selr, or methionine-r-sulfoxide reductase (msrb ) which reduces the l-methionine-r-sulfoxide and has an important role in repairing proteins that have lost function because of oxidation of lmethionine residues. of the less characterized selenoproteins, some are molecular chaperones in the endoplasmic reticulum, others (seln) are important for muscle function, as their mutation is associated with muscular disease. for a third class of all selenoproteins the role is still unknown. glutathione peroxidases are antioxidant enzymes in plasma and blood cells. msrb is highly expressed in neutrophils. all selenoproteins are synthesized through a mechanism of translational recoding of the stop codon tga (uga in the messenger) as a codon for sec insertion. key factor in this mechanism is the special trna sec , which must carry an isopentenyladenosine at position for being functional. the isopentenyl moiety required for this modification is produced in the mevalonate pathway, hence the link with a potential inhibitory effect of statins on selenoprotein expression. increased oxidative stress is one of the factors that explain statin-induced myopathy. this effect has been until now mainly linked to decreased levels of coenzyme q and associated increased production of reactive oxygen species in the mitochondrion. however, a contribution of selenoproteins downregulation to the generation of a statin-induced pro-oxidant state in muscle and other tissues has not been evaluated so far. pro-or anti-inflammatory effects of statins? as statins have been claimed to have anti-inflammatory effects, it may seem contradictory that they could contribute to the hyper-inflammatory state observed in covid- patients. however, statins interfere with the proper modification of essential g proteins in the signal transduction pathways that control innate immunity. inhibition of prenylation of ras-family g-proteins is associated with the generation of a hyper-inflammatory conditions with hyper-production of pro-inflammatory cytokines. interestingly, mevalonate-kinase-deficiency, a hereditary disorder associated with a defective mevalonate pathway, is typically characterized by an auto-inflammatory state of the patients. statins may also perturb acquired immunity by altering antigen presentation by antigen- it may be concluded that an intact redox balance is essential for white blood cells and that it would be worth investigating whether downregulation of selenoproteins may contribute to compromise leukocyte function in subjects on statin therapy. this study cannot be a retrospective analysis of pre-existing data, because, to the best of our knowledge, no quantification of selenoprotein activity has ever been carried out in vivo in subjects on statin treatment compared to normal subjects. a recent nutritional study reports on a beneficial effect of selenium supplementation on markers of muscle damage in subjects on statin therapy. selenoprotein expression levels were not changed, except for an increase in glutathione peroxidase activity, which correlated with a decrease in serum creatine kinase activity, in subject supplemented with selenium. however, the study lacked a control of normal subjects not on statins. therefore, until now it is not known how the modulation of the mevalonate pathway activity impacts on the levels and availability of the various intermediates produced in the pathway and in particular on selenoprotein activity. also lacking is the information whether hypercholesterolemia is associated with increased levels of mevalonate pathway intermediates. therefore, this hypothesis could only be tested within an "ad hoc" research project, possibly in a placebo controlled, double blind type of study. a second prediction of the hypothesis could be tested retrospectively, as discussed below. hmg-coa reductase as well as most enzymes for sterol biosynthesis are all integral membrane proteins in the endoplasmic reticulum of eukaryotic cells. all cells require cholesterol and actively synthesize it as an essential structural constituent of the plasma membrane. for the purpose of replicating, a virus heavily engages the host cell's molecular machinery for protein synthesis and vesicular trafficking and in this process it deranges the cell's metabolism. it is expected that any cellular reaction that takes place at the level of the endoplasmic reticulum membrane, like the mevalonate pathway, will be affected by the tremendous burden that an actively replicating virus poses on the intracellular membranous compartments. this view seems to be supported by a report revealing a sharp decrease in neutrophil, lymphocyte and cholesterol levels in covid- patients. thus, subjects with already low ldl levels and possibly weakened leukocyte function because of statin treatment would be even more susceptible to infection and its fatal consequences. this prediction of the hypothesis could be tested by retrospectively evaluating whether a correlation exists between the cholesterol levels of subjects infected by sars-cov- and the severity of the consequences suffered with the progression of the disease. the long list of deleterious effects that statins exert at all levels of biochemical cellular processes should be sufficient to outweigh the minor potential benefits that these drugs may show in preventing cardiovascular disease. the minimal benefit in reducing the absolute risk of myocardial infarction by one mere percent point may be linked to statins' an anti-inflammatory action, which, however, is associated with a plethora of adverse effects that include dysregulation of immune cells. therefore, the suggestion that statins should be used for the prevention of covid- is questionable. by decreasing the levels of ldls as an innate defense mechanism against pathogens and by unleashing the power of a drug that acts at multiple basic cellular levels with deleterious effects, we may have reared a population of subjects who have over time developed a compromised immune system. a new perspective ad alternative approaches are mostly wanted to address an issue of such general interest as a global infective pandemic. serious attempts to decipher the enigma of the sars-cov- infectivology must certainly include the analysis of factors that could compromise the immune system, among which statin therapy has to be included. hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis phenotypic characteristics and prognosis of inpatients with covid- and diabetes: the coronado study how statistical deception created the appearance that statins are safe and effective in primary and secondary prevention of cardiovascular disease lack of an association or an inverse association between low-density-lipoprotein cholesterol and mortality in the elderly: a systematic review ldl-c does not cause cardiovascular disease: a comprehensive review of the current literature low serum total cholesterol is associated with marked increase in mortality in advanced heart failure plasma lipoproteins are important components of the immune system infections may be causal in the pathogenesis of atherosclerosis prolonged infection with hepatitis b virus and association between low blood cholesterol concentration and liver cancer immune system differences in men with hypo-or hypercholesterolemia acc/aha guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the american college of cardiology/american heart association task force on practice guidelines bayer decides to withdraw cholesterol lowering drug effects of coenzyme q on statin-induced myopathy: an updated meta-analysis of randomized controlled trials antiinflammatory and immunomodulatory properties of statins suppression of lymphoid cell function in vitro by inhibition of -hydroxy- -methylglutaryl coenzyme a reductase by lovastatin immune modulatory effects of statins characterization of mammalian selenoproteomes mutations in sepn cause congenital muscular dystrophy with spinal rigidity and restrictive respiratory syndrome neutrophil granulocytes uniquely express, among human blood cells, high levels of methionine-sulfoxide-reductase enzymes inhibition of selenoprotein synthesis by selenocysteine trna[ser]sec lacking isopentenyladenosine selenoprotein synthesis and side-effects of statins oxidative stress as a possible mechanism of statin-induced myopathy control of the innate immune response by the mevalonate pathway simvastatin inhibits the mhc class ii pathway of antigen presentation by impairing ras superfamily gtpases association between creatine kinase activity, oxidative stress and selenoproteins mrna expression changes after brazil nut consumption of patients using statins low serum cholesterol level among patients with covid- infection in wenzhou statin therapy in covid- infection key: cord- - i gk et authors: bachmann, maría consuelo; bellalta, sofía; basoalto, roque; gómez-valenzuela, fernán; jalil, yorschua; lépez, macarena; matamoros, anibal; von bernhardi, rommy title: the challenge by multiple environmental and biological factors induce inflammation in aging: their role in the promotion of chronic disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i gk et the aging process is driven by multiple mechanisms that lead to changes in energy production, oxidative stress, homeostatic dysregulation and eventually to loss of functionality and increased disease susceptibility. most aged individuals develop chronic low-grade inflammation, which is an important risk factor for morbidity, physical and cognitive impairment, frailty, and death. at any age, chronic inflammatory diseases are major causes of morbimortality, affecting up to – % of the population of industrialized countries. several environmental factors can play an important role for modifying the inflammatory state. genetics accounts for only a small fraction of chronic-inflammatory diseases, whereas environmental factors appear to participate, either with a causative or a promotional role in % to % of patients. several of those changes depend on epigenetic changes that will further modify the individual response to additional stimuli. the interaction between inflammation and the environment offers important insights on aging and health. these conditions, often depending on the individual’s sex, appear to lead to decreased longevity and physical and cognitive decline. in addition to biological factors, the environment is also involved in the generation of psychological and social context leading to stress. poor psychological environments and other sources of stress also result in increased inflammation. however, the mechanisms underlying the role of environmental and psychosocial factors and nutrition on the regulation of inflammation, and how the response elicited for those factors interact among them, are poorly understood. whereas certain deleterious environmental factors result in the generation of oxidative stress driven by an increased production of reactive oxygen and nitrogen species, endoplasmic reticulum stress, and inflammation, other factors, including nutrition (polyunsaturated fatty acids) and behavioral factors (exercise) confer protection against inflammation, oxidative and endoplasmic reticulum stress, and thus ameliorate their deleterious effect. here, we discuss processes and mechanisms of inflammation associated with environmental factors and behavior, their links to sex and gender, and their overall impact on aging. the inflammatory response is different in men and women. adult females develop stronger innate and adaptive immune responses than males. these sex-related differences can determine the ability of immune cells to generate an effective inflammatory response, which translates into epidemiological differences on the prevalence of various pathologies, including allergies ( ), asthma ( , ), autoimmune diseases ( ), anaphylaxis ( ), neonatal sepsis ( ), and cancer ( ), among several pathologies. the immune response of women is polarized towards an increased production of th cells, t regulatory cells (treg), m macrophages, il , il , and gata- cytokines, and decreased th , th , tbet, and rorgt lymphocytes ( - ). on the contrary, men show an immune response that depends on th lymphocytes ( , ), high il production ( ) and low levels of reactive mast cells ( ) . men have also an increased response of microglia in the central nervous system (cns) and an increased presence of tnfa and prostaglandins in response to inflammatory stimuli ( ). differences in inflammatory response between men and women vary among specific tissues. in the cns inflammation, women show greater levels of b-cell (cd +, cd +, cd d hi b ) migration from the spleen to the site of injury than men, followed by an increase of macrophages/microglia (cd b+, cd ), which appears to generate a lower neuroinflammatory response in female compared with male mice ( ). in addition, women develop an increased immunoreactivity due to high numbers of ifn-producing dendritic cells ( , ). female mice tend to have m phenotype and activated eosinophils and mast cells show a higher reactivity than in male mice ( , ) . however, in response to an acute inflammatory stimulus, males produce higher amounts of inflammatory cytokines, cd a+ neutrophil and t cells infiltration of the injury site ( ). conversely, the inflammatory microenvironment in female mice is characterized by an increased production of antibodies ( , ) and a differential pattern migration of antibody-secreting cells ( ). the immune system responds differently in men and women not only because of the influence of sex hormones, but also differences in the patterns of autosomal methylation and x chromosome methylation, which determine distinctive profiles of gene expression ( , ). sex hormones exert antagonist effects on the immune system: both estradiol and testosterone have a suppressive effect on the immune response ( ). estrogen is the sex hormone with the greatest impact on the immune response, being described as one of the non-modifiable regulators of the immune system, due to its immunoregulatory and protective effects in many inflammatory models ( ). however, this is contradictory with the fact that women have a higher prevalence of autoimmune diseases than men, although estrogens should be a protective condition ( ). the sex-dependent difference in the immune response is time-, and estrogen dose-dependent ( ). variations on the estrogen concentration during the ovulatory cycle, puberty or menopause, can promote the development of immune-related diseases ( ). mice exposed to chronic estrogen-treatment generate hormone resistance, decreasing the clonal expansion of treg lymphocytes in autoimmune diseases ( , ) . estrogen regulates immune response primarily through aand b-estrogen receptors (era/b), mitogen-activated protein kinase (mapk) pathways, estrogen-dependent ′- ′-cyclic adenosine monophosphate (camp) response element-binding (creb), and modifications in the production of camp in immune cells ( ). in addition to estrogen receptors, the presence of il receptors influences the type of immune response; female macrophages express greater amounts of il receptors than males. il receptors favor the m phenotype when stimulated by estrogen. in agreement with that, estrogen induces an increased expression of il on naive cd + t cells ( , ) . for a better general view of estrogen´s mechanisms and effect on the innate immune system cells we recommend reviews that have extensively covered those topics ( - ). sex regulates gene expression in multiple human tissues, in fact, one third of the autosomal genes that are expressed in a sexbiased manner exhibit androgen or estrogen hormonal response elements ( , ). sex hormones play a strong role in sexually dimorphic gene networks ( ), inducing aberrant expression in immune response genes via differential methylation ccl cxcl il ( ) . there are changes in the methylation pattern of sex-dependent immune response genes during embryonic development, which are reinforced in puberty by the estrogenmediated induction of active forms of chromatins that are maintained during adulthood ( ) . immune response-related genes located in chromosomes and x are differently expressed in b lymphocytes depending on the sex of the individual ( ). among the differentially expressed genes that are relevant for the immune/inflammatory response, can be mentioned the toll-like signaling, cytokine receptors, jak-stat pathway and genes related to the activation of t-cell receptors ( ) . phenotypically, the different pattern of gene expression may explain the greater female t-cell expandable capacity when exposed to an antigen ( ) . female t cells present higher activation and division capacities than their male counterparts. however, male t cells can develop greater infiltration potential and a lower self-reactive phenotype than female ones ( , ) . these differences could be due to the high expression of peroxisome proliferator-activated receptors (ppars) ( ) , prostaglandins, and cyclooxygenase- (cox- ) in males ( ) . the influence of sex on the immune response is observed throughout life and is accentuated with aging. in the neonatal stage, women have a lower concentration of regulatory t lymphocytes than men ( ) . during childhood, men develop a more intense immune response and are more likely to develop infections by various pathogens compared with women ( , ) . with increasing age, the dynamics and proportion of lymphocytes and myeloid cells differ depending on the sex due to the differential expression of genes of the immune response in men and women ( ) . also, in aged individuals, epigenomic changes generate a more robust innate and pro-inflammatory response in men and an increased activity in the adaptive immune response in women ( , ) . in recent times, during the covid- pandemic, it has been observed that the infection by sars-cov- in older adults shows conspicuous differences; men have elevated plasma levels of il and il and a high amount of monocytes whereas women develop a robust activation of t lymphocytes ( ) . this differences in the immune response could explain the higher mortality of covid in men than in women ( , ) . to recapitulate, sex hormones and genetic expression patterns in men and women can generate distinct immune and inflammatory responses that determine singularities in the epidemiological distribution of immune diseases. research protocols in immune response and inflammation must be redefined to avoid results biased by sex. furthermore, research in women is urgently needed to define the efficacy for women of several therapies that were originally tested in men. the increase in noncommunicable diseases (ncds), such as obesity, hypertension and cancer as well as the low-grade chronic inflammation that characterizes most ncds ( ) can be affected by environmental factors that change the immune response. lifestyle factors like nutrition can modulate the immune system. it has been reported in mice that western diet-induced systemic inflammation and reprogramming of myeloid cell precursors is mediated by the activation of the nlrp inflammasome, which is a key sensor of the innate immune system for metabolic danger signals, such as uric acid and cholesterol ( ) . metabolic regulation appears to be very robust and long lasting, being reported that proper nutrition during pregnancy can reduce the risk for ncds in the offspring even at adult age ( , ) . the impact of the diet on the immune response and inflammation some diet types can result in metabolic and epigenetic changes that affect immune function ( ) , as reported in populations that consume a high-fat and low-fiber western diet, who show a prevalence of ncds higher than populations that consume a mediterranean diet or a diet based on bioactive compounds, like the hydroxytyrosol in olive oil ( ) ( ) ( ) . there is evidence supporting the anti-inflammatory activity of phenolic extracts from olive oil, such as their ability to reduce lipopolysaccharide (lps)-stimulated nitric oxide (no) production by the raw- . macrophage cell line. the hydroxytyrosol stearate and the hydroxytyrosol oleate decrease no production in a concentration-dependent manner ( ) . in addition, olive oil extracts increase total plasma glutathione concentration ( ) , increasing the antioxidative response of the individual. nordic diet has many similarities with the mediterranean diet, but its effects on low-grade chronic inflammation are less known. both diets include abundant fruits, vegetables, whole grain products, fish and vegetable oil, but restrict saturated fat and red and processed meats ( , ) . observational ( , ) and interventional ( , ) studies report an inverse association between the adherence to nordic diet and the concentration of high sensitivity c-reactive protein (hscrp). single intervention studies reported beneficial effects, reducing il receptor a (il ra) ( ) and cathepsin s ( ) , and downregulation of inflammatory mediators in the adipose tissue ( ) and peripheral blood mononuclear cells (pbmcs) ( ) . a key nutrient in fish are the n polyunsaturated fatty acids (pufas) ( ) . the greenland inuit population, which has a high dietary intake of n -pufas, have a lower incidence of myocardial infarction than the danish population ( ) . numerous studies associate the cardioprotective effects of n- pufas to their effect on immunomodulation ( ) ( ) ( ) , and control of inflammation, including neuroinflammation during aging ( ) . the mechanism of the anti-inflammatory effects of n -pufas n -pufas can regulate the transcription and expression of inflammatory mediators such as cytokines, chemokines and adhesion molecules in cardiomyocytes, fibroblasts, endothelial cells, and monocyte-macrophages ( ) ( ) ( ) ( ) . anti-inflammatory effect of eicosapentaenoic acid (epa), docosahexaenoic acid (dha) and their biologically active metabolites (d and e resolvinsmediators derived from omega- fatty acids, primarily epa and dha that block the production of proinflammatory mediators and regulate leukocyte trafficking to inflammatory sites) can be mediated through one of the mechanisms capable of reducing inflammation of raw- . cells and of primary intraperitoneal macrophages ( ) . one of the mechanisms is the activation of g-protein coupled receptors (gpr), ea. gpr inhibition of toll-like receptor (tlr )-mediated inflammatory response, which blocks nfkb activation. the other is mediated by nuclear receptors, particularly ppars-a/g. dha binds to ppars with high affinity resulting in the activation of anti-inflammatory cascades ( ) , which appears to be responsible for the beneficial health effects ( ) . the inhibition of nfkb-mediated pro-inflammatory activity ( ) is the common mechanism of immunomodulation by n -pufas, being dha more effective than epa in reducing lps-n -pufas induced inflammatory cytokine production by macrophages ( ) . n -pufas are incorporated into phospholipid bilayers and in human atherosclerotic plaques. their incorporation is associated with a reduction in the number of foam-and t cells, and a decrease in inflammation ( ) . the increased incorporation of n -pufas in membranes affects both the innate and adaptive immune responses, impairing the maturation of dendritic cells and the function of macrophages, as well as the polarization and activation of t and b cells ( ) ( ) ( ) . it is well known that n - pufas compete with n -pufas for being incorporated into cell membranes and for the active sites of cox- and lipoxygenase, resulting in the production of less potent pro-inflammatory or even anti-inflammatory mediators, such as the -series of prostaglandin and thromboxane ( ) . resolvins reduce also neutrophil-derived ros production, favoring neutrophil apoptosis and clearance by macrophages, and inhibit chemokine signaling ( ) . the partial agonist/antagonist activity of resolvin e (rve ) on the leukotriene b receptor on polymorphonuclear cells (pmns), inhibits nfkb activation, reduces release of pro-inflammatory cytokines and reduces infiltration by pmn ( ) . moreover, rve reduces tnfa and ifng presence in the aortic wall, decreases the levels of the inflammatory marker crp and reduces macrophage infiltration of the intima. thus, rve attenuates atherosclerosis and atherosclerotic plaque formation ( ) . aging is associated with the activation of inflammatory signaling pathways ( , ) , which can be targeted by specific nutrients with anti-inflammatory effects, such as n -pufas ( , ) . in the brain, the main n -pufa is dha, representing - % of total fatty acids ( ) . aging and neurological disorders are associated with decreased levels and turn-over rate of brain n -pufas ( ) ( ) ( ) ( ) . in aged mice, n -pufa supplementation and diets enriched in dha have been reported to revert age-induced spatial memory deficits and impairment on learning and memory ( ) ( ) ( ) . in older adults, a low consumption of n -pufas and decreased erythrocyte dha levels are associated with cognitive impairment ( , ) . dietary supplementation with dha is positively correlated with an improvement in declarative memory test performance, improved cognitive function ( , ) and a lower risk of developing neurological disorders ( ) . the probable mechanisms by which n -pufas mediate their effects in the resolution of age-related neuroinflammation are the increased synthesis of n -pufa-derived rvd and decreased n -pufaderived oxylipins, displaying an anti-inflammatory profile ( , ) . to recapitulate, the evidence indicates that n -pufas and their bioactive metabolites have immunomodulatory and antiinflammatory properties. potential cardioprotective lipid mediators, through multiple mechanisms, including changes in cell membranes composition, and modification of both cell signaling and gene expression, shift the pattern of lipid metabolites toward a more anti-inflammatory metabolite profile. dietary habits may be essential regulators of the inflammatory profile and promote healthy aging, reinforcing the recommendation of a n -pufa rich diet. the long term chronic psychological stress is increasing among the world's population ( ) . its circuit arises at high cortical centers through the limbic system to the hypothalamus, where corticotropin-releasing factor (crf) is produced, which is responsible for inducing the pituitary gland to liberate adrenocorticotropic hormone (acth) that signals the adrenal cortex to synthesize and secrete glucocorticoids (gcs) ( ) . stress also activates the sympathetic nervous system (sns), particularly the adrenal medulla, activating chromaffin cells to produce epinephrine (epi), a main stress hormone along with gcs. the latter plays a key regulation feature inhibiting the hypothalamic-pituitary-adrenal (hpa) axis through negative feedback at the pituitary gland, hypothalamus, and medial prefrontal cortex, reducing crf secretion [rewieved in ( ) ]. the interplay of social and environmental stressors induces inflammation through multiple biological mechanisms, including epigenetic factors ( ) . studies in rats show that the methylation patterns of genes involved in the stress response, such as the glucocorticoid receptor (nr c ) and crf, can be modified by psychosocial factors from early childhood ( ) . similarly, early life adversity induces acute and long-lasting epigenetic modifications in nr c genes, regulating hpa axis and cytokine production, reinforcing the importance of the activation inputs during critical periods of development ( , ) . acute short-term emotional stress, such as speaking in public, leads to a transient increase in circulating inflammatory biomarkers and natural killer (nk) cells by the sns catecholaminergic activity ( ) . on the contrary, chronic stress results in a reduction of cytotoxic nk activity, determining a poorer response to cytokines ( ) . therefore, stress appears to have short term beneficial immune effects, whereas chronic stress in the absence of immune challenge has the opposite effect ( , ) , activating constantly the hpa axis with the consequent persistent elevation of systemic gcs and reduction of nk cell responsiveness to cytokines ( ) , affecting the balance of the t helper cell type /type (th /th ) cytokine networks, predisposing to a wide range of diseases ( ) . the stress magnitude has been associated with il b mrna overexpression in peripheral pbmcs, providing a molecular mechanism by which psychological stress is translated into an immune system response ( , ) . when stress becomes chronic, such as in depression, there is a maintained overproduction of inflammatory cytokines, which have been associated with gcs resistance. immune cells become less sensitive to their anti-inflammatory effects because of their persistent secretion, leading to chronic low-grade inflammation ( , ) . activation of the innate and adaptive immune system by chronic mild stressors increases inflammatory cytokines gene expression, maturation and trafficking of dendritic cells (dc), increased macrophage number and t cells recruitment and activation. social stressors can induce an increase in inflammatory responses and a state of gcs resistance at different levels ( , ) . the acute repeated social defeat stress (rsds) and chronic restraint stress (crs) models induce an inflammatory response that results in neuroinflammation and depressive behavior ( ) . stress activates the hpa axis and the sympatho-adrenomedullar (sam) axis causing neuroinflammation by circulating cytokines that crossed the blood-brain barrier (bbb) at the circumventricular organs and by cytokine bbb transporters. an inflammatory response that promotes bbb permeability, allowing more inflammatory factors entering the brain, including crf, metalloproteinase- , il , and tnfa ( ) . additionally, microglia produce chemokines that attract monocytes into the brain ( ) . activation of sns and hpa axis through continuous psychological stress dysregulate cytokine production, and together with the stress hormones corticosteroids and catecholamines, can affect endothelial adhesion molecules, causing endothelial damage ( ) . corticosteroids could facilitate the infiltration of monocytes by increasing the expression of il and il receptors on endothelial cells. these monocytes and lymphocytes, after attaching to such sites, would commence the process of infiltration into the wall vessels, leading to foam cell formation and thrombotic events ( , ) . chronic unpredictable mild stress (cums) decreases body mass and impairs the metabolism of carbohydrates and lipids. a model for cums showed an increased liver and pancreas protein-lipid peroxidation and protein oxidation ( ) . high ros production in both organs could be a result of a response mechanism to stress at the cellular level. in the liver, protein oxidation can be due to the regulation of metabolic impairments by gcs and epi ( ). the antioxidant system of the liver is in general more efficient than the pancreas. however, it is insufficient to clear the reactive species increased as consequence of chronic stress, which could be due to alterations in the antioxidant enzymatic activity ( ) . altogether, stress appears to have short term beneficial effects on the immune function, whereas chronic stress ( , ) activates persistently the hpa, elevating systemic gcs, and impairing the cytokine balance. the overproduction of inflammatory cytokines lead to gcs resistance driven by immune cells that lose their sensitivity to gcs, leading to a state of chronic low-grade inflammation ( , ) . this gcs imbalance, shares common features with aging, mediating an enhanced neuroinflammatory priming ( ) . the presence of psychological stress potentiates the defective immune response observed in aging, which at the same time conditionate an exaggerated sickness response to immune challenges (such as chronic stress). thus, chronic stress contributes to the phenomenon of inflammaging, which promotes the development of several age-related pathologies, including atherosclerosis and diabetes among others [reviewed in ( ) ]. additionally, there is an impairment of the antioxidant defense system to manage ros production after chronic stress, resulting in the damage of various tissues ( ) . in addition, people exposed to chronic stress age rapidly, showing a faster telomere shortening in their cells ( ) ( ) ( ) . on the other hand, epigenetic changes acquired during critical developmental stages could shape chronic stress-response along the lifespan, either promoting or reducing pathological aging ( , ) . substance abuse, such as alcohol and drugs, are important triggers of chronic inflammatory processes ( , ) . the effects of alcohol on human health are complex and depend on multiple factors. however, many of those factors are associated with the generation of immunosuppression and increased morbimortality in heavy users. those effects, which have been previously reviewed by goral et al. ( ) will not be discussed in this review. here, we will describe the effect of cocaine and methamphetamine abuse. both drugs are potent psychostimulants that, when repeatedly consumed, significantly disrupt the functioning of the cns, and modify the regulation of the immune response, leading to a chronic neuroinflammatory state ( ) . in general, it is known that drug abuse, among other factors, increases nfkb transcription of multiple proinflammatory genes that spread across brain cell types further amplifying of nfkb transcription, as has been reviewed by crews et al. ( ) . cocaine (benzoylmethylecgonine according to the international common denomination) is a strong stimulant tropane alkaloid that acts by modulating the catecholaminergic neurotransmitter dopamine. studies of the striatum of mice after the administration of various drugs showed that h after administration of mg/kg cocaine, there is a significant increase in gene arrays for hypoxia-inducible factor (hif- ), transcription factors, and cytokine receptors (il r, tnfa). two hours after cocaine administration, there is an increased gene expression for various tnf receptors, inducible no synthase (inos) and adhesion molecules ( ) . in the nucleus accumbens of mice stimulated with cocaine, there is a significant increase in matrix metalloproteinase (mmp ), macrophage colony stimulating factor (mcsf) and major histocompatibility complex ii (mhc-ii) ( ) . the brain of human subjects consuming cocaine shows an increased density of macrophages and activated microglia ( ) . cocaine induces the activation of microglia through the endoplasmic reticulum stress and autophagy pathways ( ) . studies of human and rodent immune cell populations after cocaine administration show decreased numbers of t lymphocytes, modulation of nk activity and cytokine production ( ) . among brain glial cells, astrocytes are the most abundant, and perform critical functions, being involved in neurogenesis, promotion of neuronal survival, elimination of free radicals, and the production of no to maintain neuronal homeostasis ( ) . nevertheless, astrocytes can also be activated by toxic stimuli, leading to a new phenotype called "reactive astrocytes", similar with the changes observed after inflammatory activation. this phenomenon has been described in various neuropsychiatric disorders, such as alzheimer's and parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis ( ) . the reactivity of astrocytes to toxic stimuli, such as cocaine, infection or disease, potentiates the neuroinflammatory process ( ) . methamphetamine (desoxyephedrine; meth) is a synthetic adrenergic agonist with psychostimulatory effects, structurally related to the ephedrine alkaloid and adrenaline. studies on the effect of meth are limited. however, it has been determined that its abuse affects the immune response. animals exposed to both acute and chronic meth use show alkalization of normally acidic organelles in immune cells, inhibition of antigen presentation, and impairment of phagocytosis ( ) . meth also generates mitochondrial oxidative damage, dysfunction of t lymphocytes and decreased production of antibodies and cytokines ( ) . meth has effects in various tissues ( ) . in the lungs, the number of t lymphocytes decreases compared with that of untreated animals, indicating a reduction in circulating cd + cells, and levels of il and il increases. in the spleen, recruitment of pmn and the number of ly- g+ and f / + are increased, whereas cd + cells are significantly reduced. in addition, levels of tnfa, ifng, il , and il are higher than those of control mice. in the liver, there is an increase of t lymphocytes and macrophages, hepatocellular atrophy, and increased levels of ifng, tnfa, il b, - , - , - , and - in the group exposed to meth compared with control animals ( ) . in the cns, meth can induce the activation of calpains and caspases; the production of ros with the subsequent induction of oxidative stress, and the release of high amounts of glutamate, causing excitotoxicity ( ) . recently, raineri et al. reported that meth induces activation of astrocytes and microglia, increasing the levels of il and tnfa mrna and its receptor (tnfr ) in the mouse striatum and hippocampus ( , ) . medical advances have resulted in the increment of the average life expectancy in developed countries. the aging of the population is associated with an increase in the number of older people using drugs of abuse. from to , the number of cocaine users aged or older that required treatment for drug addiction in the us increased by % ( , ) . aging is associated with low-grade basal inflammation that can be compounded by substance use. as cocaine exposure is associated with elevated inflammation and altered immune functioning, the presence of cocaine use disorder might exacerbate inflammatory processes in aging adults ( ) . a recent report by soder et al, compared the levels of inflammation (through the neutrophil to lymphocyte ratio) in older adults with cocaine use disorder (cud) and in healthy older adults, finding that the group with cud had a significantly higher baseline level of inflammation ( ) . the use of illegal drugs such as cocaine or methamphetamine has not been shown to affect cognitive function in older adults at the clinical level. however, the evaluation of the cognitive function of young drug users reveals a decreased performance compared with healthy young people. in fact, the cognitive function of young drug users is similar to that of adults older than years of age ( , , ) . in summary, both cocaine and meth can directly impair the immune response, induce the activation of glial cells and stimulate the release of pro-inflammatory mediators in the cns. all those effects cause relevant changes in glial cell regulation and inflammatory activation, triggering chronic neuroinflammation and potentiating pathological aging. air pollution has become an important threat to public health. air pollutants consider a mixture of gases such as nitrogen oxides (nox), sulphur oxides (sox), tropospheric ozone (o ), volatile organic compounds (vocs), and particulate matter (pm) ( ) . pm can enter the respiratory tract leading to severe in situ damage as well as inducing additional systemic effects ( ) . the world health organization (who) suggests a maximum annual exposure of μg/m³ of pm . , however, the exposure of % of the world's population exceeds the proposed limit ( ) . exposure to air pollutants is associated with increased morbimortality associated with respiratory, cardiovascular, metabolic, neurological, carcinogenic and autoimmune diseases ( , ( ) ( ) ( ) . inflammation is the main pathophysiological mechanism induced by air pollutants. in terms of the molecular and cellular mechanism induced by pollutants, pm and sox can generate ros, inducing oxidative stress, together with mitochondrial dysfunction and the consequent energy deprivation ( ) ( ) ( ) . as a direct consequence, nfkb and mapk inflammatory pathways are activated, triggering an innate immune activation ( , ) . despite the attempts to resolve the inflammatory event, the outcome appears to be an imbalance in lymphocyte homeostasis and immune system dysregulation, with inhibition of th and treg lymphocytes ( ) . there is also an increase of th lymphocytes and recruitment of eosinophils, resulting in respiratory disorders such as asthma ( , , ) . in parallel, pm deactivates the nuclear factor erythroid pathway (nrf ), involved in antioxidant regulation and prevention of oxidative stress, a necessary process for the resolution of inflammation. therefore, to maintain oxidation-reduction reactions becomes impossible, becoming a breaking point towards increased ros production and the non-resolution of the inflammatory event ( ) . another mechanism of action of pollutants is the activation of the aryl hydrocarbon receptor (ahr) by toxic agents. the binding of pm to ahr increases circulating th and decreases treg lymphocytes. increase in th associates to the release of il , promoting an abrupt increase of th lymphocyte response. these changes promote the dysregulation of the immune response associated with the development of autoimmune processes ( ) . aberrant increases in th may result in increased inflammation, with consequences such as asthma and acute respiratory failure syndrome (ards), due to neutrophil infiltration and tissue damage ( ) . studies suggest the existence of a decline in treg levels and, therefore, an inability to suppress th , th and phagocyte responses ( , ) . in addition, exposure to pm has been associated with fibrotic events, where il increases synthesis and secretion of collagen in the lung parenchyma ( , ) . in addition, it has been described that pm also induces the expression of tgfb, directly promoting fibroblast differentiation, which could also induce collagen deposition followed by a lower antifibrotic process in the liver ( ) . pollutants may promote direct dna damage through oxidation of nitrogenous bases. hu and yu described in a paper different mechanisms and changes in mirna expression that comprise specific targets of dna methyltransferases, which can impair the methylation of tumor suppressor genes ( ) . furthermore, urban populations show increased levels of mitochondrial methylation genes due to pm exposure ( ) . there is evidence of the existence of methylation, acetylation and phosphorylation of histones h and h , markers found in genes involved in the activation of immune cells and cardiovascular diseases ( , ( ) ( ) ( ) ( ) . altogether, air pollutants can generate dna adducts promoting carcinogenesis and deteriorate telomerase activity, as reviewed by martens and nawrot ( ) , and contributing to continuous dna damage and premature aging ( , ) . in vivo studies suggest that the inflammatory activation is doseand time-dependent. mice exposed to pm show that both variables are determinant for the outcome. however, inflammatory effects and major genetic changes appear to be especially dependent on the exposure to high concentrations of pm. one possible explanation is that a prolonged exposure could induce an adaptive response of the inflammatory activation ( ) , which may be mediated by the inactivation of the nrf pathway, generating a loss of antioxidant capacity and deregulation of the immune system ( ) . the resolution appears to depend on the exposure context. acute exposure would result in high levels of ros and damage, whereas prolonged stimulation, even a low-grade one, generates a constant production of ros and chronic low-grade inflammation ( ) , consequent with the potentiation of disease risk and an epigenetic age acceleration ( ) , promoting pathological aging. direct causes of the deregulation of the inflammatory resolution process resulting from inhaled contaminants are still unknown, however, the burden of associated chronic diseases is expected to increase. it is mandatory to intensify environmental policies specifically in lower-middle-income countries in prevention of the development of inflammatory conditions and the subsequent chronic diseases. aging, characterized by a progressive loss of cellular functions, is an inevitable physiologic process inherent to all living beings ( ) . the number of older adults is increasing. during the next years, up to % of the world population will be older than years ( ) . this demographic change is accompanied by a higher incidence of ncds accumulated in the aging population ( ) . therefore, various strategies have been proposed to improve the health and quality of life of older adults ( ), along with recommendations for the development of public policies that support the fiscal expenditure resulting from ncds ( ) . one of the most studied events of aging is the impairment of the immune system, characterized by an aberrant-increased activation of the innate immunity ( , ) , and high levels of circulatory inflammatory mediators that establish an inflammatory environment, and a decrease of the adaptive immune response ( , ) and a decrease of the adaptive immune response ( ) due to this low-grade chronic inflammation ( , ) , which together would promote the inflammaging phenomenon ( ) . interestingly, it is proposed that age would not be the cause per se of these diseases associated with aging ( ) . thus, there is a deterioration of the immune system's response to external stimuli, which depends on the individual's history ( ) . also, several epigenetic mechanisms can modulate the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events ( ) . on the other hand, it is described that epigenetic clocks would be useful to analyze mechanisms associated with this environmental influence ( ) . finally, they would be capable of modulating the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events ( ). multiple age-dependent changes play important roles in the promotion of ncds, with increased oxidative stress standing out as one of the main mechanisms. over the last two decades, evidence has revealed that increased oxidative stress and inflammation are involved in various ncds such as alzheimer's disease ( ) , rheumatoid arthritis ( ) , cardiovascular diseases ( , ) , and cancer ( ), among others. also, recent studies propose that the activation of nfkb signaling pathways could be the main driver of these associations ( ) ( ) ( ) ( ) . interestingly, de almeida et al. showed different sources of low-grade chronic inflammation that promote cardiovascular disease ( ) . in the cns, high levels of ros lead to the activation of astrocytes and microglia, further increasing the overproduction of ros and proinflammatory cytokines that promote the development of neurodegenerative changes ( , , ) . in fact, several systemic biomarkers appear to be associated with neuroinflammation and the development of cns diseases associated with aging ( ) . these modifications trigger the phenotype of senescent or aged cells characterized as sasp ( , ) extensively studied in the context of the deleterious effects of aging. however, sasp is also essential for remodeling and promoting wound healing, which requires a strict control of the inflammatory response, thus avoiding the induction of cell aging phenotypes that contribute to the development of chronic inflammatory diseases ( ) . the immune imbalance in aging occurs due to various alterations in cellular behavior and phenotype, which cause functional deficiencies in immune cells ( ) . for example, this context induces polarization of macrophages towards an inflammatory phenotype characterized by strong activation of the inflammasome ( ) . thus, these events could induce il b and tnfa release, changes in the chemoattraction of neutrophils mediated by the reduction of the intercellular adhesion molecule (icam- ) expression, and the aberrant activation of the phosphoinositide lipid kinase- (pi k) ( ) . also, there is a decrease in the expression of pattern recognition receptors (prr), which leads to the activation of proinflammatory signaling promoting tissue damage ( , ) . finally, the reduced level of certain hormones due to the impaired hypothalamic function causes the loss of muscle mass and an increase in adipose tissue, further contributing to the release of inflammatory cytokines and changes in metabolism ( ) . despite the remarkable effort being made to understand the basis of the processes underlying the inflammatory imbalance during aging, it is not fully understood. in aging, there are cumulative epigenetic changes that promote low-grade inflammation ( , ) , including a decrease in the global genome methylation, with increased methylation in specific regions, as those with repressive histone marks of cd + and cd + t cells ( ) and bivalent chromatin domains ( ) and histone acetylation and methylation. however, the influence of genomic methylation during aging remains undetermined ( ) . several studies correlate the methylation of multiple sites on cpg islands with the increase of the low-grade inflammation marker, crp ( , , ) . nonetheless, stevenson et al. propose that the dna methylation could be better associated with the low-grade chronic inflammation than crp ( ) . in addition, the age-related mitochondrial dysfunction, with the resulting oxidative stress and decreased atp production ( ) , affect the expression and activity of dna methyltransferases, which are responsible for maintaining the methylation pattern of dna ( ) . the reduced methylation results in the demethylation of the tnfa promoter in leukocytes and macrophages ( ) and the adhesion of immune cells to the endothelium ( ) . also, many epigenetic events contribute to the differentiation of proinflammatory t cells, th ( ) , which can compromise immunocompetence, associated with repression of differentiation of immune cells, loss of treg function ( ) and the alteration of the hematopoietic stem cells differentiation ( ) . thus, epigenetic mechanisms appear to have a major role in the inflammatory imbalance, which are associated with the accumulation of damage in time that ultimately leads to the perpetuation of a constant inflammatory response. according to the who, % of the world population is sedentary, lacking the benefits of physical exercise ( ) . conditions such as sedentarism, unhealthy diet, overweight, obesity and aging induce chronic low-grade inflammation. physical exercise increases the anti-inflammatory potential and reduces the pro-inflammatory effect ( ) . this equilibrium is partly modulated through tlrs ( ) , which are fundamental for the recognition of prrs, including the damage-associated molecular patterns (damps) and the induction of an inflammatory response in the absence of pathogens. there is evidence that in young people, physical exercise decreases tlrs expression, co-stimulatory molecules cd / cd , and mhcii ( , ) in cd + monocytes. physical exercise also affects the adipose tissue. exercising reduces tlr mrna expression and tnfa production in adipocytes ( , ) in obese mice. chronic physical exercise decreases tnfa and tlr gene expression in the skeletal muscle ( ) . the evidence suggests that obesity-or cerebral ischemiainduced neuroinflammation, which are associated with the overexpression of tlr and tlr , may be reduced by physical exercise through the reduction of tlrs expression as well as their downstream signaling molecules (tnfa, il b, myd , traf , tak , and nfkb), together with the reduced microglial activation ( , ) . there is evidence that cigarette smoking induces inflammatory status [reviewed in ( ) ]. however, exercise training reduces smoke-induced inflammation. in that sense, training for min with endurance exercise for days in smoke-exposed mice demonstrated that therapeutic exercise training significantly reduces the expression of il b and tnfa mrna in rectus femoris ( ) . physical exercise has been used as a therapeutic tool in chronic pathological conditions. in that sense, obese older adults (body mass index ± kg/m ; ± years) undergoing an exercise program consisting in physical therapy, endurance, and resistance for min, days per week, show a reduced expression of tlr , il , and tnfa mrna in skeletal muscle ( ) . in older adults, -week physical exercise reduces the expression of tlr and tlr , as well as tlrs downstream mediators, such as myd , p , pp , trif, ikki/ikkϵ, irf , and pirf in pbmcs ( ) . similarly, dendritic cells from multiple sclerosis patients undergoing an exercise (endurance and resistance) program for weeks reduce tnfa and mmp secretion when stimulated with a tlr ligand (lps in combination with ifng, or a tlr ligand) ( ) , suggesting that long-term physical exercise decrease tlr responsiveness. on the other hand, high-intensity physical exercise in untrained individuals induces inflammation, resulting in the increased expression of tlr , ap , nfkb, and p in mice myocardium and in adipose tissue ( ) ( ) ( ) . physical exercise associated with eccentric contractions causes expression of tlr and nfkb in skeletal muscle and liver in rats ( , ) . furthermore, this phenomenon induces muscle damage, which can increase chemotaxis, attracting nk, cd + t cells, macrophages and neutrophils to the site of injury, promoting the production of cox , inos, monocyte chemotactic protein- (mcp- ), tnfa, il , and il b, in addition to the production of ros and the activation of nfkb ( , ) . in healthy young males, one session of intense endurance exercise ( h intense cycling immediately followed by h intense running), increases plasmatic concentrations of il and il , in addition to increased gene expression of proinflammatory il receptor (il r) and tlr signaling pathways. moreover, plasma myoglobin changes in correlation with neutrophil tlr gene expression (r= . ), suggesting that their transcriptional activity was particularly induced by damps ( ) . therefore, inflammation and muscle damage are mainly associated with the type and intensity of the exercise, with loads that exceed individual physical abilities. chronic physical exercise generates epigenetic modifications. the physical exercise associated with an energy expenditure > kilocalories per week, results in hypomethylation of the il gene and hypermethylation of the tnfa gene ( ) , with an inverse correlation between tnfa methylation and tnfa mrna expression ( ) . the methylation of the caspase recruitment domain (asc) of the apoptosis-associated specklike protein gene, the main regulator of inflammasome and promoter of the activation of il b and il , decreases with aging. however, older adults who maintain physical exercises regularly express higher levels of asc methylation than subjects not exercising, which would imply a decreased release of inflammatory cytokines ( ) ( ) ( ) ( ) . similarly, in review a -month walk training can induce hypermethylation of the nfkb- gene, suppressing inflammation through the inhibition of the nfkb pathway ( ) . as life expectancy increases, age-related diseases thrive. aging is a complex multifactorial process of molecular and cellular decline that renders individuals susceptible to disease and death. maintenance of cell integrity, cell metabolism and host-defense mechanisms are tightly regulated by the surrounding microenvironment. a growing body of evidence in different biological models has contributed towards identifying biological mechanisms that ward off structural and functional deterioration. these data offer us insights into healthy aging. molecular integrity of the genome, telomere length, epigenetic stability, and protein homeostasis are all features linked to more youthful stages (regardless of the age), associated with mitochondrial fitness, metabolic regulation, efficient intercellular communication, stem cell renewal, and regenerative capacity in tissues. a good understanding of the environmental and endogenous mechanisms that underlie agerelated normal and deleterious changes, and how these pathways interconnect, remains a major challenge for slowing pathological aging while extending older adults' healthy lifespan. the study of the environmental influence on the development of complex-chronic diseases shows that in addition to genetic predisposition, the pathogenesis is promoted by changes in metabolism and behavior, cellular environment, and epigenetic regulation patterns. the type of nutrient, or environmental cytokine milieu dramatically affects not only the homoeostasis of tissues but also of complete organs and even of the whole individual. thus, tissue stress, malfunction, and damage may induce inflammation alarm responses, which result either in resolution of tissue damage, restoration of normal cell function or development of chronic disease ( figure ). older adults often present inflammaging, characterized by increased levels of proinflammatory cytokines il , il , il , tnfa/crp ( ) . however, the cellular sources of these cytokines are partially unknown. the increased inflammatory cytokines have been proposed to be a driver of unsuccessful aging (increased morbidity, degenerative processes, or frailty) and shortened health-span. the inflammatory scenario is complex and occurs in response to various internal and environmental stimuli ( figure ) mediated mainly by a high level of proinflammatory cytokines. indeed, in healthy aging, increased production of the anti-inflammatory cytokines tgfb and il , can regulate the pro-inflammatory state ( , ) . research into the impact of environmental factors on inflammaging is at an early stage and the involved mechanisms are not completely understood. several hypotheses have been developed to explain the chronic inflammation: aging-related increase of stress ( ) and oxidative stress ( ) , dna damage in senescent cells [reviewed in ( ) ], and stem cell aging ( ) . the proposed mechanisms are likely interdependent, resulting in the generation of ros causing oxidative damage and amplification of the cytokines secretion, thus perpetuating a vicious circle of systemic inflammation where tissue injury and healing mechanisms proceed in parallel while damage slowly accumulates over the lifespan of the individuals. endocrine and metabolic alterations are linked to the shift towards a pro-inflammatory profile, which could explain some age-related pathologies, such as alzheimer's and parkinson's disease, osteoporosis, diabetes, cancer, and frailty ( , ) . regarding stress-induced immune modifications, new evidence suggests that cross talk signals between the cns, endocrine and immune system are required for optimal response to stress [discussed in ( ) ]. various stressors can affect the activity and regulation of immune cells via direct regulation by the autonomic and peptidergic system or through the release of neuroendocrine mediators. moreover, neuronal catecholamines modulate immune cell functions. these interactions are bidirectional, cytokines produced by immune cells, such as il , can modulate the production of corticotropin-releasing hormone (crh) by the hypothalamus. chronic diseases are favored by some modern living conditions, such as the intake of high-caloric foods and the low level of physical activity, or endogenous signals produced by the chronic stress of modern life. there are many challenges in conducting research on biosocial processes, which will define novel disease-trigger factors. tailor-made approaches will depend on genetics, epigenetics and a constellation of factors depending on the historical as well as the present exposure to the environment. although environmental factors also express themselves as epigenetic changes, the combinatorial effect of the multiple factors generates complex patterns of epigenetic regulation, and the concomitant exposure to environmental factors can further modify the individual response. all authors contributed equally on the conception of the work, the analysis of literature and preparing the content of the review. rbe drafted and organized the manuscript. all authors contributed to the article and approved the submitted version. endogenous and environmental factors can be mostly beneficial (in green) and deleterious (in red) or can have both beneficial and deleterious effects depending on the specific context. the interplay of lifespan endogenous and environmental factors regulates the aging phenotype depending on dna damage, epigenetic changes, and inflammation. these drivers can induce functional aging hallmarks: changes in endocrine and metabolic regulation, and defective immune regulation that will further determine the response of the individual. in yellow we show processes that can participate in both protection and damage. exposure to various alarm signals induce an acute inflammation that, when associated with deleterious environmental and biological factors, potentiates chronic inflammation, which can be further promoted by excess ros production and oxidative stress that results from mitochondrial dysfunction or nox activity, leading to inflammaging and eventually to age-related disease. on the contrary, in the presence of protective environmental and biological factors, the initial inflammatory activation will be resolved and lead to a healthy aging process. ros, reactive oxygen species. sarcopenic obesity and inflammation in the inchianti study physiological aging: links among adipose tissue dysfunction, diabetes, and frailty immunosenescence: the potential role of myeloid − derived suppressor cells ( mdsc ) in age − related immune deficiency the hallmarks of aging circulating c q complement/tnf-related protein (ctrp) , ctrp , ctrp and ctrp concentrations in type diabetes mellitus: in vivo regulation by glucose aging, inflammation and the environment both genetic and dietary factors underlie individual differences in dna damage levels and dna repair capacity pro ala polymorphism of the pparg gene interacts with a mediterranean diet to prevent telomere shortening in the predimed-navarra randomized trial micrornas linking inflamm-aging, cellular senescence and cancer metabolic control of longevity immunobiography and the heterogeneity of immune responses in the elderly: a focus on inflammaging and trained immunity epidemiology of parkinson disease mitochondrial dysfunction and longevity in animals: untangling the knot. sci cardiac oxidative stress in diabetes: mechanisms and therapeutic potential nadph oxidase and mitochondria are relevant sources of superoxide anion in the oxinflammatory response of macrophages exposed to airborne particulate matter evaluation of oxidative damage and nrf activation by combined pollution exposure in lung epithelial cells emerging role of air pollution in autoimmune diseases inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network child development and the physical environment psychological stress in childhood and susceptibility to the chronic diseases of aging: moving towards a model ofbehavioral and biological mechanisms sex steroidinduced dna methylation changes and inflammation response in prostate cancer integrative analysis of methylome and transcriptome in human blood identifies extensive sex-and immune cell-specific differentially methylated regions gender differences of b cell signature related to estrogen-induced ifi l/baff in systemic lupus erythematosus gender differences of b cell signature in healthy subjects underlie disparities in incidence and course of sle related to estrogen peroxisome proliferator-activated receptor (ppar)alpha expression in t cells mediates gender differences in development of t cell-mediated autoimmunity sex-specific t-cell regulation of angiotensin ii-dependent hypertension. hypertens (dallas tex peroxisome proliferator-activated receptor (ppar)alpha and -gamma regulate ifngamma and il- a production by human t cells in a sexspecific way sex differences in prostaglandin biosynthesis in neutrophils during acute inflammation sex disparity in cord blood foxp + cd t regulatory cells in infants exposed to malaria in utero sex differences in pediatric infectious diseases correlation between female sex, il b genotype, and the clinical severity of bronchiolitis in pediatric patients sex differences in the blood transcriptome identify robust changes in immune cell proportions with aging and influenza infection sexual-dimorphism in human immune system aging sex differences in older adults' immune responses to seasonal influenza vaccination sex differences in immune responses that underlie covid- disease outcomes gender differences in patients with covid- : focus on severity and mortality sex-specific clinical characteristics and prognosis of coronavirus disease- infection in wuhan, china: a retrospective study of severe patients noncommunicable diseases country profiles western diet triggers nlrp -dependent innate immune reprogramming the next innovation cycle in toxicogenomics: environmental epigenetics transgenerational effects of maternal diet on metabolic and reproductive ageing nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and dna methylation population differences in associations between c-reactive protein concentration and adiposity: comparison of young adults in the philippines and the united states diet and the epigenome effects of olive oil and its minor components on cardiovascular diseases, inflammation, and gut microbiota identification of hydroxytyrosyl oleate, a derivative of hydroxytyrosol with anti-inflammatory properties, in olive oil by-products olive phenolics increase glutathione levels in healthy volunteers effects of an isocaloric healthy nordic diet on insulin sensitivity, lipid profile and inflammation markers in metabolic syndrome -a randomized study (sysdiet) what is a healthy nordic diet? foods and nutrients in the nordiet study associations of the baltic sea diet with cardiometabolic risk factors-a meta-analysis of three finnish studies associations of the baltic sea diet with obesity-related markers of inflammation health effect of the new nordic diet in adults with increased waist circumference: a -mo randomized controlled trial a diet high in fatty fish, bilberries and wholegrain products improves markers of endothelial function and inflammation in individuals with impaired glucose metabolism in a randomised controlled trial: the sysdimet study influence of a prudent diet on circulating cathepsin s in humans healthy nordic diet downregulates the expression of genes involved in inflammation in subcutaneous adipose tissue in individuals with features of the metabolic syndrome healthy nordic diet modulates the expression of genes related to mitochondrial function and immune response in peripheral blood mononuclear cells from subjects with metabolic syndrome-a sysdiet sub-study eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? marine omega- fatty acids and inflammatory processes: effects, mechanisms and clinical relevance omega- fatty acids and inflammatory processes: from molecules to man cardioprotective mechanism of omega- polyunsaturated fatty acids neuro-immune dysfunction during brain aging: new insights in microglial cell regulation n- polyunsaturated fatty acids (pufa) modulate the expression of functionally associated molecules on human monocytes polyunsaturated fatty acids inhibit the antigenpresenting function of human monocytes effects of omega- -rich harp seal oil on the production of pro-inflammatory cytokines in mouse peritoneal macrophages dietary fish oil diminishes lymphocyte adhesion to macrophage and endothelial cell monolayers gpr is an omega- fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects human dendritic cell activities are modulated by the omega- fatty acid, docosahexaenoic acid, mainly through pparg:rxr heterodimers: comparison with other polyunsaturated fatty acids eicosapentaenoic acid prevents lps-induced tnf-a expression by preventing nf-kb activation docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human thp- macrophages more effectively than eicosapentaenoic acid eicosapentaenoic acid (epa) from highly concentrated n- fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque epa is associated with decreased plaque inflammation and increased stability docosahexaenoic acid prevents dendritic cell maturation, inhibits antigen-specific th /th differentiation and suppresses experimental autoimmune encephalomyelitis regulatory activity of polyunsaturated fatty acids in t-cell signaling n- pufa improves fatty acid composition, prevents palmitate-induced apoptosis, and differentially modifies b cell cytokine secretion in vitro and ex vivo effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of -lipoxygenase pathway products by ionophore-activated human neutrophils resolvin e and protectin d activate inflammation-resolution programmes resolvin e selectively interacts with leukotriene b receptor blt and chemr to regulate inflammation resolvin e (rve ) attenuates atherosclerotic plaque formation in diet and inflammation-induced atherogenesis human cns immune senescence and neurodegeneration microglial senescence: does the brain's immune system have an expiration date? ageassociated changes in the content and fatty acid composition of brain glycerophospholipids the aging human orbitofrontal cortex: decreasing polyunsaturated fatty acid composition and associated increases in lipogenic gene expression and stearoyl-coa desaturase activity modulation of brain pufa content in different experimental models of mice dha-enriched phospholipid diets modulate age-related alterations in rat hippocampus docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: a lipidomic analysis from inflammation to sickness and depression: when the immune system subjugates the brain fatty acid composition of brain phospholipids in aging and in alzheimer's disease chronic administration of docosahexaenoic acid improves the performance of radial arm maze task in aged rats docosahexaenoic acid-rich phospholipid supplementation: effect on behavior, learning ability, and retinal function in control and n- polyunsaturated fatty acid deficient old mice dha improves cognition and prevents dysfunction of entorhinal cortex neurons in xtg-ad mice association between mediterranean diet and late-life cognition red blood cell omega- fatty acid levels and markers of accelerated brain aging dietary intake of eicosapentaenoic and docosahexaenoic acids is linked to gray matter volume and cognitive function in elderly cognitive aging, childhood intelligence, and the use of food supplements: possible involvement of n- fatty acids elevated levels of proinflammatory oxylipins in older subjects are normalized by flaxseed consumption dietary n- long chain pufa supplementation promotes a pro-resolving oxylipin profile in the brain systematic review of the association between chronic social stress and telomere length: a life course perspective the developmental origins of chronic physical aggression: biological pathways triggered by early life adversity psychological stress, immune response, and atherosclerosis the epigenome at the crossroad between social factors, inflammation, and osteoporosis risk epigenetic programming by maternal behavior the role of dna methylation in stress-related psychiatric disorders selective mobilization of cytotoxic leukocytes by epinephrine immune dysregulation and chronic stress among older adults: a review chronic stress and age-related increases in the proinflammatory cytokine il- depression, mood, stress, and th /th immune balance in primary breast cancer patients undergoing classical massage therapy nuclear factor-kb is a critical mediator of stress-impaired neurogenesis and depressive behavior social signal transduction theory of depression inflammation as a psychophysiological biomarker in chronic psychosocial stress repeated social defeat activates dendritic cells and enhances toll-like receptor dependent cytokine secretion neuroinflammation caused by mental stress: the effect of chronic restraint stress and acute repeated social defeat stress in mice anger emotional stress influences vegf/vegfr and its induced pi k/akt/mtor signaling pathway chronic unpredictable mild stress generates oxidative stress and systemic inflammation in rats stress and aging act through common mechanisms to elicit neuroinflammatory priming the link between chronic stress and accelerated aging psychological wellbeing and healthy aging: focus on telomeres toward a deeper understanding of a triad of early aging tired telomeres: poor global sleep quality, perceived stress, and telomere length in immune cell subsets in obese men and women alcohol and epigenetic changes: summary of the alcohol and immunology research interest group (airig) meeting methamphetamine decreases cd t cell frequency and alters proinflammatory cytokine production in a model of drug abuse exposure-dependent effects of ethanol on the innate immune system cocaine induced inflammatory response in human neuronal progenitor cells induction of innate immune genes in brain create the neurobiology of addiction the dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum histone deacetylase epigenetically controls behavioral adaptations to chronic emotional stimuli decreased brain dopamine cell numbers in human cocaine users cocaine induces astrocytosis through er stress-mediated activation of autophagy immune system inflammation in cocaine dependent individuals: implications for medications development psychostimulant abuse and neuroinflammation: emerging evidence of their interconnection methamphetamine and its immune-modulating effects methamphetamine administration modifies leukocyte proliferation and cytokine production in murine tissues causes and consequences of methamphetamine and mdma toxicity modafinil abrogates methamphetamine-induced neuroinflammation and apoptotic effects in the mouse striatum methamphetamine-induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin trends in substance use admissions among older adults elevated neutrophil to lymphocyte ratio in older adults with cocaine use disorder as a marker of chronic inflammation age-and sex-dependent effects of methamphetamine on cognitive flexibility and -ht c receptor localization in the orbitofrontal cortex of sprague-dawley rats crack-cocaine dependence and aging: effects on working memory air pollution prevention and control policy in china air pollution and allergy: you are what you breathe air pollution associated epigenetic modifications: transgenerational inheritance and underlying molecular mechanisms biomarkers of the health outcomes associated with ambient particulate matter exposure the effects of exposure to air pollution on the development of uterine fibroids role of oxidative damage in toxicity of particulate mechanisms of heightened airway sensitivity and responses to inhaled so in asthmatics exposure scenario: another important factor determining the toxic effects of pm . and possible mechanisms involved in vitro toxicity of particulate matter (pm) collected at different sites in the netherlands is associated with pm composition, size fraction and oxidative potential -the raptes project exposure to ultrafine particulate matter induces nf-kb mediated epigenetic modifications diesel exhaust particle exposure in vitro impacts t lymphocyte phenotype and function acute nitrogen dioxide (no ) exposure enhances airway inflammation via modulating th /th differentiation and activating jak-stat pathway diesel exhausts particles: their role in increasing the incidence of asthma. reviewing the evidence of a causal link air pollution-derived pm . impairs mitochondrial function in healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells acute respiratory distress syndrome effects of pm . exposure on the notch signaling pathway and immune imbalance in chronic obstructive pulmonary disease the impact on tregulatory cell related immune responses in rural women exposed to polycyclic aromatic hydrocarbons (pahs) in household air pollution in gansu, china: a pilot investigation pm . induced pulmonary fibrosis in vivo and in vitro blocking il- a promotes the resolution of pulmonary inflammation and fibrosis via tgf-b -dependent and -independent mechanisms effects of sub-chronic exposure to atmospheric pm . on fibrosis, inflammation, endoplasmic reticulum stress and apoptosis in the livers of rats genetic and epigenetic alterations in normal and sensitive copd-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived effects of airborne pollutants on mitochondrial dna methylation prenatal particulate air pollution and dna methylation in newborns: an epigenomewide meta-analysis dose-and time-effect responses of dna methylation and histone h k acetylation changes induced by traffic-related air pollution air pollution and dna methylation: effects of exposure in humans epigenetic response profiles into environmental epigenotoxicant screening and health risk assessment: a critical review air pollution, particulate matter composition and methylation-based biologic age air pollution stress and the aging phenotype: the telomere connection the effect of exposure time and concentration of airborne pm . on lung injury in mice: a transcriptome analysis facing up to the global challenges of ageing coming of age: molecular drivers of aging and therapeutic opportunities find the latest version: review series introduction coming of age: molecular drivers of aging and therapeutic opportunities disability incidence and functional decline among older adults with major chronic diseases quality of life assessment instruments for adults: a systematic review of population-based studies comparative financing analysis and political economy of noncommunicable diseases the integration of inflammaging in age-related diseases scavenger receptor-a deficiency impairs immune response of microglia and astrocytes potentiating alzheimer's disease pathophysiology source of chronic inflammation in aging microglial cell dysregulation in brain aging and neurodegeneration aging and the immune system: an overview cellular senescence and alzheimer disease: the egg and the chicken scenario the epigenetics of inflammaging: the contribution of age-related heterochromatin loss and locus-specific remodelling and the modulation by environmental stimuli wandering along the epigenetic timeline myeloperoxidase as an active disease biomarker: recent biochemical and pathological perspectives the role of signaling pathways of inflammation and oxidative stress in development of senescence and aging phenotypes in cardiovascular disease oxidative stress and advanced lipoxidation and glycation end products (ales and ages) in aging and age-related diseases how the ageing microenvironment influences tumour progression unveiling the role of inflammation and oxidative stress on age-related cardiovascular diseases nf-kb-mediated neuroinflammation in parkinson's disease and potential therapeutic effect of polyphenols epigenetic upregulation of fkbp by aging and stress contributes to nf-kbdriven inflammation and cardiovascular risk nf-kb signaling in astrocytes modulates brain inflammation and neuronal injury following sequential exposure to manganese and mptp during development and aging neuroinflammation in frontotemporal dementia immunosenescence in aging: between immune cells depletion and cytokines up-regulation back to the future: epigenetic clock plasticity towards healthy aging immunity and inflammation: from jekyll to hyde update of inflammasome activation in microglia/macrophage in aging and aging-related disease aging of the immune system: focus on inflammation and vaccination inflammaging: a new immune-metabolic viewpoint for age-related diseases characterisation of an inflammation-related epigenetic score and its association with cognitive ability agerelated profiling of dna methylation in cd + t cells reveals changes in immune response and transcriptional regulator genes human aging-associated dna hypermethylation occurs preferentially at bivalent chromatin domains abnormal epigenetic regulation of immune system during aging do you remember mitochondria? mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondriallysosomal axis theory of aging agerelated loss of cpg methylation in the tumour necrosis factor promoter dysregulation of c-x-c motif ligand during aging and association with cognitive performance understanding intrinsic hematopoietic stem cell aging world health organization's global strategy on diet, physical activity and health: the process behind the scenes interleukin- responses from acute exercise in healthy subjects: a systematic review the physiological regulation of toll-like receptor expression and function in humans the influence of prolonged cycling on monocyte toll-like receptor and expression in healthy men exercise training inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from m to m macrophages in high-fat-diet-induced obese mice the anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and tlr mrna in rat skeletal muscle exercise therapy downregulates the overexpression of tlr , tlr , myd and nf-kb after cerebral ischemia in rats neuroprotective effects of endurance exercise against high-fat diet-induced hippocampal neuroinflammation ten hacken nht. acute effects of cigarette smoke on inflammation and oxidative stress: a review exercise training reverses inflammation and muscle wasting after tobacco smoke exposure exercise but not dietinduced weight loss decreases skeletal muscle inflammatory gene expression in frail obese elderly persons role of toll-like receptor and signaling pathways on the inflammatory response to resistance training in elderly subjects weeks of combined endurance and resistance training reduces innate markers of inflammation in a randomized controlled clinical trial in patients with multiple sclerosis efecto del ejercicio agudo sobre la expresioń del receptor tipo toll- y los mecanismos inflamatorios en corazoń de rata acute exercise activates myocardial nuclear factor kappa b exhaustive exercise increases inflammatory response via toll like receptor- and nf-kbp pathway in rat adipose tissue diclofenac pretreatment effects on the toll-like receptor /nuclear factor kappa b-mediated inflammatory response to eccentric exercise in rat liver diclofenac pretreatment modulates exercise-induced inflammation in skeletal muscle of rats through the tlr /nf-kb pathway exercise-induced increase in serum inferleukin- in humans is related to muscle damage honokiol protects rats against eccentric exercise-induced skeletal muscle damage by inhibiting nf-kb induced oxidative stress and inflammation transcriptome analysis of neutrophils after endurance exercise reveals novel signaling mechanisms in the immune response to physiological stress exercise and inflammation-related epigenetic modifications: focus on dna methylation impact of aerobic exercise and fatty acid supplementation on global and genespecific dna methylation role of physical exercise in the regulation of epigenetic mechanisms in inflammation, cancer, neurodegenerative diseases, and aging process epigenetic regulation on gene expression induced by physical exercise regulatory molecules involved in inflammasome formation with special reference to a key mediator protein exercise effects on methylation of asc gene nfkb gene as a novel candidate that epigenetically responds to interval walking training inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans role of tgf b signaling in the pathogenesis of alzheimer's disease age-dependent changes in the activation and regulation of microglia stress responses and innate immunity: aging as a contributory factor oxidative stress, inflamm-aging and immunosenescence dna damage response (ddr) and senescence: shuttled inflamma-mirnas on the stage of inflamm-aging emerging models and paradigms for stem cell ageing from inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation aging and parkinson's disease: inflammaging, neuroinflammation and biological remodeling as key factors in pathogenesis impact of stress on aged immune system compartments: overview from fundamental to clinical data the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © bachmann, bellalta, basoalto, goḿez-valenzuela, jalil, lépez, matamoros and von bernhardi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -i l cq h authors: law, betty yuen kwan; mok, simon wing fai; wu, an guo; lam, christopher wai kei; yu, margaret xin yi; wong, vincent kam wai title: new potential pharmacological functions of chinese herbal medicines via regulation of autophagy date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: i l cq h autophagy is a universal catabolic cellular process for quality control of cytoplasm and maintenance of cellular homeostasis upon nutrient deprivation and environmental stimulus. it involves the lysosomal degradation of cellular components such as misfolded proteins or damaged organelles. defects in autophagy are implicated in the pathogenesis of diseases including cancers, myopathy, neurodegenerations, infections and cardiovascular diseases. in the recent decade, traditional drugs with new clinical applications are not only commonly found in western medicines, but also highlighted in chinese herbal medicines (chm). for instance, pharmacological studies have revealed that active components or fractions from chaihu (radix bupleuri), hu zhang (rhizoma polygoni cuspidati), donglingcao (rabdosia rubesens), hou po (cortex magnoliae officinalis) and chuan xiong (rhizoma chuanxiong) modulate cancers, neurodegeneration and cardiovascular disease via autophagy. these findings shed light on the potential new applications and formulation of chm decoctions via regulation of autophagy. this article reviews the roles of autophagy in the pharmacological actions of chm and discusses their new potential clinical applications in various human diseases. autophagy is the catabolic process in which eukaryotic cells engulf and lysosomally digest intracellular contents. the process maintains metabolic balance and cellular quality by removing dysfunctional cytoplasmic constituents and recycling basic molecular building blocks. macroautophagy envelops and delivers the unfavoured intracellular contents to lytic compartment through the doubled-membraned autophagosome [ ] . autophagy-related proteins (atg) are the main underpinning mechanistic component of macroautophagy. about mammalian homologs of yeast atg have been discovered. these proteins are responsible for the nucleation and elongation of the isolation membrane through protein complex formation [ , ] . basal autophagy retains proper physiological functioning of cells through the control of cellular homeostasis, metabolic balance, and protein structural integrity. additionally, macroautophagy is known as type ii programmed cell death with its molecular regulation intricately interconnecting with the apoptotic mechanism [ ] [ ] [ ] [ ] . growing evidence also suggests involvement of macroautophagy in innate and adaptive immunity [ , ] . therefore hampered autophagy is closely associated with proteinopathies, metabolic and immunological disorders. in fact, macroautophagy failure is the molecular culprit of aberrant proteins accumulation in neurodegenerative diseases such as prion, alzheimer's and parkinson's [ ] [ ] [ ] . in metabolic diseases, altered macroautophagy is related to the mammalian tor performs its function by complexing with other proteins, and appears in two constitutively different forms which are the target of rapamycin complex and (mtorc and ) [ ] . canonically, mtorc masters the repression of autophagy responding to growth factor, hormone and amino acid signaling by direct interaction with the atg machinery and interference of autophagosome formation [ , ] . compared with mtorc , mtorc is better characterized which together with the tsc / (tuberous sclerosis complex / ) complex, an mtorc activities suppressor, underpinning the central molecular governance of mtor cascade. this pathway contributes mainly to the integration of growth factor and insulin signaling. the tsc / complexes collect the signals from cellular sensor such as insulin receptor at the plasma membrane. class i ptdlns k is then activated and phosphorylates phosphatidylinositol ( , )-bisphosphate (pip ) to phosphatidylinositol ( , , ) -bisphosphate (pip ). while class iii ptdins k product pi p is critical for autophagy, pip generated by class i ptdlns k are inhibitory. pip subsequently recruits and activates pkb [ ] . during cellular energy stress, the amp: atp ratio is increased because of intracellular atp depletion [ , ] triggering the upstream activator of ampk, liver kinase b (lkb kinase) [ ] . the activated ampk can interact directly with the regulatory-associated protein of mtor (raptor) subunit of mtorc inactivating the protein complex thereby upregulating autophagy [ ] . cytosolic calcium ion (ca + ) concentration is another positive stimulation of ampk-induced autophagy which is initiated by ca + /calmodulin-dependent kinase kinase β (camkkβ) and endoplasmic reticulum-localized bcl- [ ] . accumulation of misfolded proteins is the common feature of the different neurodegenerative diseases [ ] . the self-eating property of autophagy is cytoprotective which assists the clearance of the defective β-sheets enriched protein structure [ ] [ ] [ ] [ ] [ ] . clinically, the beclin level of ad's brain are found to be significantly depressed [ ] . transgenic mouse overexpressing beclin could improve parkinson's disease (pd) progression by reducing α-synuclein aggregation through the enhancement of autophagy [ ] . mutant huntingtin (htt) forms, the main source of neurotoxic activities of huntington disease (hd), are sensitive to beclin level as demonstrated by the increased accumulation of htt upon beclin deficiency [ ] . a mouse model of ad and in vitro studies showed that rapamycin effectively induced amyloid-β (aβ) clearance, and soothed the cognitive deficit [ ] . lithium inhibits inositol monophosphatase (impase) which reduces free inositol and ip levels promotes the removal of htt of hd and α-synuclein via autophagy [ , ] . repressing ip synthesis with the sodium salts of carbamazepine and valproate could induce similar therapeutic effects in the experimental models of hd [ ] [ ] [ ] . another mtor-independent autophagy inducer, trehalose, is also relevant to htt, α-synuclein and tau aggregations [ , ] . most tumor suppressors and oncogenes are actually cellular metabolism regulators responsible for metabolic pathways including aerobic glycolysis, glutaminolysis and one-carbon metabolism [ ] . the major cellular energy monitor ampk, and hence the downstream autophagy up-regulated by the kinase, are closely related to cancer progression in response to metabolic stresses. ampk activators such as -aminoimidazole- -carboxamide riboside (aicar) are strongly cytotoxic to the in vitro models of hepatic, gastric and prostate cancers [ ] [ ] [ ] . many other molecular messengers along the autophagy pathways are cancer-related. for example, the induction of the oncogenic mtorc attenuates autophagy and support cancer development [ ] [ ] [ ] [ ] . clinical trials with cytotoxic drugs rapamycin, temsirolimus and everolimus targeting mtorc to induce autophagy have been reported [ ] . therefore, cancer intervention with the use of autophagy inducers can trigger both autophagic cell death and physiological changes, such as cell cycle arrest, on cancer cells. this is of particular interest for most cancers and malignancies in regard to apoptotic resistance towards chemotherapies [ ] . the systemic balance of glucose and fatty acid is frequently lost in patients enduring metabolic syndromes such as obesity and glucose intolerance. it is evident that the corresponding complex regulatory networks are linked up by autophagy [ ] . chronic consumption of diets with high nutrient abnormally activates mtorc [ ] . the de novo hepatic lipogenesis can then be stimulated by mtorc and its substrate sterol regulatory element-binding protein c (srebp- c) during the obese state [ , ] , which further dampens the expressions of the atgs lc , beclin , atg and atg and thus the autophagic process [ ] . while activating autophagy with pharmacological inducers seems to be an ideal method for improving metabolic disorders, caloric restriction appears to be the best strategy for controlling these disorders [ ] . autophagy functions as an immune effector and directly delivers the intracellular microorganism or their components [ ] into the lysosome through a specific form of selective autophagy called xenophagy [ , ] . several medically important bacterial pathogens, such as mycobacterium tuberculosis (mtb), group a streptococcus, salmonella, shigella and listeria can be degraded by xenophagy. the sindbis virus, herpes simplex virus and the parasite toxoplasma gondii can also be eliminated by xenophagy [ , , ] . beyond the invading microbes, autophagy also targets the innate and adaptive immunity for preventing infections. autophagy negatively regulates il- β and il- expression through maintaining the quality of the intracellular milieu [ ] . the overexpression of il- β and il- of lipopolysaccharides (lps)-stimulated macrophages with the loss of atg l further clarified the role of autophagy in inflammatory immune responses [ ] . in vitro studies have verified the potential of some autophagic inducers in infection therapy. the hormonally active form of vitamin d , d , enhanced macrophage autophagy and prevented human immunodeficiency virus (hiv) replication [ ] . similarly, the small molecule pdk inhibitor ar cleared francisella tularensis [ ] and salmonella enterica serovar typhimurium [ ] . the therapeutic effects of the antibiotics cocktails containing isoniazid and pyrazinamide towards mtb-infected host cells also corresponded to their autophagy-inducing properties [ ] . autoimmunity is the aberrant activation of immune systems towards self-antigens due to central intolerance [ ] . autophagy is essential for loading the mhc ii compartment with intracellular antigens, a process involving the atg-lysosome interaction [ ] , implicating the importance of autophagy in the determination of cd + t lymphocytes receptor repertoire. also, the autoreactive thymocytes that have been programed in thymus can be eliminated by autophagy [ ] . besides, the autophagic activity is positively correlated with macrophagic expression of the proinflammatory cytokines tnf-α and il- which may also contribute to sle pathogenesis [ ] . glucocorticoids since autophagy dysregulation underlies a broad range of pathological conditions, the successful therapeutic outcomes of using autophagy modulators, along with the expanding discoveries regarding the molecular basis of autophagy, suggest the need of intensively investigating the pharmaceutical potential of compounds with autophagy-adjusting ability (table ) . while a number of synthetic autophagy regulators, such as tamoxifen, sorafenib and the water-soluble synthetic rapamycin temsirolimus, have been reported [ ] [ ] [ ] , natural autophagic compounds from chms are of interested because of their potential new therapeutic applications ( figure ). since autophagy dysregulation underlies a broad range of pathological conditions, the successful therapeutic outcomes of using autophagy modulators, along with the expanding discoveries regarding the molecular basis of autophagy, suggest the need of intensively investigating the pharmaceutical potential of compounds with autophagy-adjusting ability (table ) . while a number of synthetic autophagy regulators, such as tamoxifen, sorafenib and the water-soluble synthetic rapamycin temsirolimus, have been reported [ ] [ ] [ ] , natural autophagic compounds from chms are of interested because of their potential new therapeutic applications ( figure ). induction of autophagic cells death in glioblastoma, gastric adenocarcinoma [ ] ; inhibition of iav-induced autophagic cell death [ ] fructus piperis longi (bi bo) warms cold, expels cold, relieves pain piperlongumine promotion autophagic cell death of breast, kidney, prostate and lung cancer cells [ , ] blood regulating drugs rhizoma curcumae longae (jiang huang) regulates blood, moves blood, moves and regulates qi, descends the qi curcumin hinders α-synuclein accumulation in neural cells and suppression of the proliferation of glioma cells through induction of autophagy [ , ] radix salviae miltiorrhizae (dan shen) moves blood, breaks up blood stasis, cools heat, cools blood tanshinone iia induction of autophagic cell death of leukemia via activation of ampk/mtor, erk/mtor and p s k signaling [ ] ligusticum wallichii (chuan xiong) moves blood, moves and regulates qi, dispels wind ligustrazine induction of cytotoxic effects in hepatocellular carcinoma and protection of the kidney from neurotoxicity through autophagy [ , ] external using drugs venenum bufonis (chan su) opens the orifices, detoxifies, relieves pain bufalin induction of cell death in hepatoma cells and suppression of colon cancer cells proliferation through autophagy [ , ] gamboge (teng huang) detoxifies, disperses swelling, antiparasitic, alleviates itching gambogic acid amelioration of bladder cancer and induction of cytotoxic in leukemia cell through autophagy [ , ] spirit calming drugs radix polygalae (yuan zhi) anchors the yang, dislodges phlegm, opens the orifices onjisaponin b acceleration of the degradation of mutant α-synuclein and huntingtin in pc- cells through autophagy [ ] ganoderma lucidum (ling zhi) tonifies the heart and qi, calms and anchors the spirit ganoderic acid c reduction of accumulation of mutant huntingtins in pc- cells, induction of autophagic cell death in melanoma cells [ ] caulis polygoni multiflori (shou wu teng) calms and anchors the spirit, anchors the yang anthraquinones induction of autophagic cell death in c and u [ ] fructus schisandrae (wu wei zi) harmonizes and tonifies the yin and qi, secures the essence schisandra total lignin inhibition of d-galactose-induced brain tissue aging through autophagy [ ] semen ziziphi spinosae (suan zao ren) tonifies yin and blood, astringes and collects, anchors the yang jujuboside a, jujuboside b jujuboside b induces autophagic cell death in ags and hct human cancer cells and suppresses tumor growth [ ] succinum (ambrum) calms and anchors the spirit, sedates and cools the heart vitamin e succinate (ves) ves-induced autophagy participates in sgc- cell protection by inhibiting mtor axis phosphorylation [ ] . heat-clearing drugs are used to clear damp-heat, fire or heat in the blood and body fluids to maintain regular body temperature and normal hemostatis of body [ , ] . radix scutellariae (huang qin) has been shown to modulate inflammatory diseases like gastroenteritis and hepatitis, and is also effective in controlling tumorigeneses [ ] . the two main active components, baicalin and wogonin inhibit the release of proinflammatory mediators from different immunocellular components [ , ] . baicalin and wogonin might be effective for inducing cytotoxicity or inhibiting proliferation in various human hepatoma [ ] . the anti-cancer effects of radix scutellariae such as induction of cell death and cell cycle arrest could be mediated through autophagy [ ] . although the involvement of autophagy in the radix scutellariae-triggered anti-inflammatory property is still elusive, the progression of gastroenteritis and hepatitis are highly autophagic-related [ ] , suggesting the potential autophagic role of radix scutellariae in such diseases. all these observations suggest that a main part of the clinical functions of radix scutellariae as documented in chinese traditional medical references are manifested via autophagy. cortex phellodendri (huang bo) with berberine as its active ingredient after bark extraction has been traditionally prescribed for the treatment of pneumonia, tuberculosis, meningitis and liver cirrhosis [ , ] . cortex phellodendri is anti-inflammatory in nature, which helps to eliminate invading pathogens, ameliorates acetaldehyde-induced hepatic nf-κb activation during cirrhosis [ , ] , and inhibits glial proinflammatory inos (nitric oxide synthase) and tnf (tumor necrosis factor)-α activity [ ] . berberine modulates autophagic processes through the ampk/mtor signaling pathway [ , ] , therefore, the observed anti-inflammatory capability of cortex phellodendri was likely related to berberine-induced autophagy [ , ] . recently, the dietary supplement nexrutine ® which contains berberine, has been found to have therapeutic potential towards melanoma, multiple myeloma, prostate, pancreatic, breast and non-melanoma skin cancer [ ] [ ] [ ] [ ] [ ] . since berberine could induce both apoptosis and autophagy during tumorigenesis [ ] , autophagy may be responsible for the newly discovered anti-cancer properties of cortex phellodendri. rhizoma coptidis (huang lian), containing the active component berberine, has been traditionally used in alleviating inflammatory disorders, including type ii diabetes. pharmacological studies have reported the anti-inflammatory effects of rhizoma coptidis. for example, berberine was effective in repressing the adverse responses caused by atherosclerosis (as) [ ] . rhizoma coptidis was beneficial to the treatment of intestinal infections [ , ] , and gut-associated abnormalities such as irritable bowel syndrome [ ] . furthermore, berberine induced autophagy [ , ] and suppressed the pro-inflammatory phenotype of macrophages [ ] . in response to the anti-diabetic effect of rhizoma coptidis, autophagy was important for regulating the synthesis and secretion of insulin by pancreatic β-cells [ ] . recent studies have also reported the potential of rhizoma coptidis in neurodegeneration therapy [ , ] . with the protective role of autophagy in neurodegenative and inflammatory diseases, it is notable that the protective effect of rhizoma coptidis may be regulated through autophagy. radix sophorae flavescentis (ku shen) was used to alleviate toxicity, killed parasites and induced diuresis according to chinese medicinal theory [ ] . many traditional formulas contain radix sophorae flavescentis, for example, "xiaofeng san" was prescribed for treating cutaneous disorders [ ] , and "sanwu huangqin tang" has long been used for post-partum fevers arising from reproductive organ infections during childbirth [ ] . these therapeutic effects suggest the immunomodulatory and anti-inflammatory function of radix sophorae flavescentis may be attributed to the maintenance of systemic homeostasis by clearing off metabolic wastes through autophagy. the active component of radix sophorae flavescentis is matrine, which is pharmacologically related to the suppression of inflammation by inhibiting neutrophil infiltration and oxidative stress, as well as reducing the production of inflammatory mediators [ ] . also, matrine has been reported as t cell anergy inducer through regulating the expression of anergy-associated genes such as jumonji and cd [ ] . the autophagic effects of matrine have suggested radix sophorae flavescentis as a promising anti-cancer herb. for example, matrine was able to induce autophagic cell death against certain cancers [ , , ] , a pharmacological action which has not been reported in chinese medicinal documents. although no direct linkage between radix sophorae flavescentis-induced autophagy and its traditional immunity regulatory effects has been reported, owing to the significant role of autophagy in immunomodulation, the involvement of such a process cannot be neglected. herba rabdosiae (dong ling cao) has been extensively used in cancer therapy [ ] . this herbal drug was also effective in encountering inflammation, oxidative stress and pathogen invasion [ ] . oridonin, an active component of herba rabdosiae, attenuated neuroinflammation and associated oxidative stress by repressing the microglial production of nitric oxide and pro-inflammatory cytokines like il- b and il- [ ] . oridonin inhibited cancer growth by repressing the expression of proinflammatory mediators like il- and bone morphogenetic protein- (bmp- ) [ ] . therefore, the traditional anti-cancer property of herba rabdosiae is highly correlated to autophagy. radix isatidis (ban lan gen) is a popular herbal medicine, especially after the severe acute respiratory syndrome (sars) epidemic, for its clinical applications in upper respiratory tract infections, including the nose, throat, and sinuses [ ] . it is also prescribed for other viral infections like measles and hepatitis [ , ] , etc. the active components of radix isatidis are bis-benzylisoquinoline alkaloids-fangchinoline and tetrandrine. these active ingredients interact with invading pathogens and modulate the host responses. for example, fangchinoline could stop the replication of human immunodeficiency virus (hiv) type by disturbing the proteolytic process of viral gp [ ] ; tetrandrine suppressed hepatitis through the repression of nf-κb activation [ ] . in addition, fangchinoline activated autophagic cell death through the p /sestrin /amp pathway in hepatocellular carcinoma [ ], whereas tetrandrine inhibited leukemia cell proliferation and induced autophagy in vivo. to our knowledge, the use of radix isatidis in anti-cancer therapy has not been reported, therefore, these findings shed light to the development of the new usage of radix isatidis in oncology through induction of autophagy. however, the role of autophagy in mediating the traditional anti-inflammatory function of radic isatidis remains to be investigated. stephania japonica (qian jin teng) has been traditionally used for relieving fever, diarrhea, dyspepsia and urinary disease [ ] . cepharanthine and dauricine are the active components of the herb that have been commonly used for inflammatory and immunological disorders in chinese medicine [ , ] . animal and in vitro studies illustrated that cepharanthine suppressed cytokine synthesis, platelet aggregation, plasma membrane lipid peroxidation, and nuclear factor-κb (nf-κb) stimulation [ ] . the herb also facilitated the removal of free radicals and alleviated oxidative stress [ , ] . dauricine exhibited similar anti-inflammatory effects through repressing the expression of inflammatory indicators such as myeloperoxidase, il- bβ and tnf-α [ ] . recently, these two compounds have been postulated as novel anti-cancer drugs. for example, cepharanthine abolished the drug resistance property of cancer cells by modulating the activities of multidrug resistance factor abcc (mrp ) and atpase [ ] . dauricine had been reported to suppress cancer proliferation and invasion, and promote apoptosis through the nf-κb signaling pathway [ ] . although documentation associating autophagy with the immunomodulatory effects of cepharanthine, dauricine or stephania japonica is scarce, our laboratory discovered that both bioactive components induced autophagic cell death in apoptosis-resistant cancer cells [ ] . these findings suggest cepharanthine and dauricine repress cancer growth through autophagy induction, and stephania japonica may be a potential pharmaceutical agent for cancer. rhizoma polygoni cuspidati (hu zhang) has been used to relieve inflammation, coughing, fever, and provide diuretic, emmenagogue, emollient and stomachic actions [ , ] and against tumor activity [ ] . resveratrol, the active compound of rhizoma polygoni cuspidati, is a natural antibiotic [ ] which inhibits the growth of bacteria and fungi [ , ] . resveratrol exhibited its anti-inflammatory effect through inhibiting pro-inflammatory mediator synthesis [ ] . resveratrol also repressed tumorgenesis [ ] [ ] [ ] . increasing evidence has shown the autophagic role of resveratrol. for example, resveratrol attenuated the inflammatory phenotype of vascular endothelium through up-regulation of autophagy [ ] , and induced autophagic cell death in glioma and adenocarcinoma [ , ] . recently, resveratrol-induced cardioprotection was found to associate with mtorc -mediated autophagy and the up-regulation of antioxidant proteins [ , ] . therefore, autophagy could be the underlying molecular mechanism responsible for the protective effects of rhizoma polygoni cuspidati. herba scutellariae barbatae (ban zhi lian) has long been used for cancer therapy [ ] . it is also effective in treating inflammatory-related symptoms, for example, furunculosis, pyogenic infections, traumatic injury, edema, venomous snake bite, and jaundice [ ] . the active ingredient of the herb, pheophorbide, inhibited cytokines expression and monocyte activities, down-regulated macrophage expression of inos, and scavenged reactive oxygen species (ros) [ ] . in addition, pheophorbide was cytotoxic to hepatocellular carcinoma [ ] and breast adenocarcinoma [ ] . thus far, literatures correlating pheophorbide and autophagy were mostly about the photodynamic therapy of tumorigenesis involving the induction of autophagy and cancer cell death. in breast adenocarcinoma, pheophorbide induced autophagy through the erk signaling pathway [ ] . it also induced both apoptosis and autophagy via erk / and p in skin cancer [ ] . such autophagy-related therapeutic effect of pheophorbide has also been described in oral squamous carcinoma cells and hormone insensitive prostate cancer [ , ] . accordingly, the anti-cancer effect of herba scutellariae barbatae could be partly mediated by autophagy. nelumbo nucifera (lian hua) has been used for maintaining homeostasis, reducing anxiety, acting against bleeding, and repressing inflammation [ ] . neferine is one of the active components responsible for maintaining glucose and lipid balance during starvation [ ] . anti-inflammatory and anti-oxidative effects of neferine in neurodegenerative disease by suppressing nf-κb activation and lipid peroxidation have also been reported [ ] . protective effects of neferine towards inflammation and oxidative stress were observed in pulmonary fibrosis [ ] . emerging findings suggest that autophagy is one of the pharmaceutical targets of nelumbo nucifera. for example, neferine attenuated mutant huntingtin toxicity by inducing the ampk-mtor-dependent autophagic pathway [ ] . such findings correlated the psychopathological regulatory effects of neferine to autophagy. neferine induced autophagy via the ros mediated pathway in lung cancer [ ] , and was found effective in suppressing hepatocellular carcinoma [ ] . these findings confirmed the role of the compound in controlling inflammatory progression and its novel usage in tumorigenesis. therefore, triggering autophagy by nelumbo nucifera could be a new pharmaceutical strategy for encountering inflammatory, neurodegenerative and cancerous diseases. syzygium samarangense (lian wu) possessing the anti-free radical ability is indigenously used to manage inflammation-related conditions and removal of oxidative stress [ , ] . dimethyl cardamonin (dmc) is the active compound isolated from the leaves of syzygium samarangense contributing mainly to the anti-inflammatory and anti-oxidative properties. dmc protected the cells from ros damage by modulating the glutathione s-transferase and superoxide dismutase (sod) activities [ , ] . the compound also attenuated nf-κb activation and relieved cellular inflammatory phenotype with decreased serum level of proinflammatory cytokines [ , ] . recent studies have suggested autophagy induction by dmc was associated with proliferative arrest in colorectal carcinoma by stimulating the p /jnk-dependent signaling [ , ] . the therapeutic effects of dmc-induced autophagy have also been reported in cancers of the pancreas and prostate, myeloid leukemia and multiple myeloma [ ] . although the autophagic role of syzygium samarangense in anti-inflammation remains to be elucidated, the induction of autophagy is highly correlated to the syzygium samarangense-induced anti-cancer effects. trichosanthes kirilowii (cucumber) has been used as folk medicine for the treatment of inflammation and cancer [ ] . pharmacological research further proposed trichosanthes kirilowii as a potential remedy for suppressing hiv replication [ ] . cucurbitacin d, a major component of the herb, is an anti-inflammatory compound which inhibited pro-inflammatory mediator production via inos and nf-κb signaling [ , ] and cyclooxygenase- (cox- ) [ ] . cucurbitacin d was effective in the protection against hepatic and cardiovascular damages, the alleviation of diabetic condition, and the removal of invading microbes [ , ] . cucurbitacin d could be used for repressing tumorigenesis of colorectal carcinoma [ ] , breast adenocarcinoma [ ] , and leukemia [ ] . recent findings have demonstrated that structural analogues of cucurbitacin d, for example, cucurbitacins b, e and i, induced autophagy and are potentially beneficial to cancer intervention. cucurbitacin b triggered autophagy in breast cancer cells by manipulating the ros activities [ ] , and in melanoma cells via c-jun n-terminal kinase (jnk) activation [ ] . cucurbitacin e induced autophagy in cervical and breast cancer cells via activation of ampk signaling [ ] . similar therapeutic effects have also been reported in cucurbitacin i treatment of glioblastoma [ ] . all these observations point towards the likelihood of involvement of cucurbitacin d in autophagy-induced anti-tumor effects. mallotus philippensis (cu kang chai) has been used for alleviating inflammatory symptoms caused by bronchitis, rheumatism and infection. the herb is useful in eliminating parasite invasions such as tapeworm [ ] . rottlerin, one of the major active components of the herb, also manifested potent anti-inflammatory properties. rottlerin regulated inflammatory mediators including cox, protein kinase c δ, lipoxygenase, heme oxygenase and nf-κb [ ] . besides, it also suppressed the progression of malignant cancers by increasing the susceptibility of cancer cells towards apoptosis [ ] [ ] [ ] . in addition, the anti-tumor effects of rottlerin were associated with its autophagy activation property in certain cell types [ , , ] . rottlerin induced apoptosis and autophagic cell death in prostate [ ] and pancreatic cancers via the inhibition of pi k/akt/mtor signaling [ ] . rottlerin also triggered apoptosis and autophagic cell death in fibrosarcoma cells through a pi k/akt/mtor-independent pathway [ ]. these findings, together with the protective role of rottlerin in preventing the spreading of misfolded proteins including prion protein, amyloid aβ, and α-synuclein [ ], suggest that mallotus philippensis could be used as a novel therapeutic intervention for cancer-related and neurodegenerative disorders based on its autophagy-inducing ability. whether autophagy is involved in the traditional anti-inflammatory effects of rottlerin or mallotus philippensis is yet to be determined. rhizoma anemarrhenae (zhi mu) has been used for minor symptoms like cough, fever and constipation [ , ] . the herb was also effective in treating diabetes [ ] . one of the active components extracted from rhizoma anemarrhenae, timosaponin aiii, relieved inflammation and oxidative damages by regulating the cytosolic ca + concentration of endothelial cells [ ] , and neutrophilic superoxide generation stimulated by arachidonic acid [ ] . recent pharmacological studies demonstrated that the anti-tumor effect of timosaponin aiii was associated with autophagy. timosaponin aiii elicited autophagy and cytotoxicity in cervical cancer which was independent of apoptosis [ ] . it also repressed mtor activity, triggered er stress and autophagic cell death in breast cancer [ ] . as demonstrated in an insulin resistance rodent model, timosaponin aiii-induced autophagy may be responsible for its diabetes-ameliorating effect through activation of ampk [ ] . in neurodegeneration model, timosaponin aiii activated autophagy and facilitated the downstream sequestration of aggregation-prone ubiquitinated proteins [ ] . these findings implied the pharmaceutical potential of applying rhizoma anemarrhenae for the treatment of cancers and neurodegenerative diseases. these herbs are used when the zheng qi (normal body condition or upright qi) is weakened, for example, during recovery from illness, during childhood or in old age. the herbs have the ability to tonify (bu), nourish, supplement and strengthen human body [ , ] . radix glycyrrhizae (liquorice, gan cao) is traditionally used as adjuvant to modify the efficacy of other herbs in a single prescription of around % of chinese herbal formulas [ ] , which acted against inflammatory symptoms such as relieving cough, sore throat and phlegm production. it is important for maintaining a proper stomach function and is used for stomach ulcers. licochalcone a (la) and isoliquiritigenin (isl) are compounds extracted from radix glycyrrhizae. la exhibited anti-inflammatory effects which suppressed pro-inflammatory mediator expression [ , ] , and cleared cellular oxidative stress [ ] . isl demonstrated anti-oxidative [ ] and immunomodulatory effects [ ] . of note, emerging data suggest that the anti-cancer properties in both compounds are associated with autophagy. la induced autophagy via pi k/akt/mtor signaling which repressed cervical cancer growth [ ] . androgen-sensitive prostate adenocarcinoma and adenoid cystic carcinoma were sensitive to la-and isl-induced autophagic cell death mediated by mtor inhibition [ , ] . isl also suppressed breast cancer progression through autophagy induction [ ] . therefore, radix glycyrrhizae has the chemotherapeutic potential to function as an effective cancer therapeutic. further investigation is needed for clarifying the mediating role of autophagy in the traditional anti-inflammatory effects of the herb. radix dipsaci (xu duan) has been used for intervention in osteoporosis, strengthening of tendons and ligaments, and alleviating joint stiffness symptoms by promoting blood circulation in close proximity to the affected areas. the herb exhibits anti-inflammatory properties as reflected by its application in reducing abscesses, swellings, and sores [ ] . in addition, radix dipsaci helped to prevent abortion, which implies a regulatory role in the immune system [ ] . akebia saponin pa (as) is one of the bioactive components found in radix dipsaci, as induced autophagic and apoptotic cell death of gastric cancer cells through both the ampk/mtor and pi k/akt/mtor signaling and the downstream activation of p /jnk molecular pathway, which facilitated capase- -dependent apoptosis [ ] . this finding pointed towards the potential therapeutic role of radix dipsaci in cancers. also, the possibility that autophagy may participate in the immunomodulatory and anti-inflammatory functions of radix dipsaci should not be ignored. radix ginseng (ren shen) has been prescribed for maintaining bioenergetics balance as suggested by chinese herbalists for breathlessness, anorexia, hypodynamia, and diabetes [ ] . since, one of the main functions of autophagy is to retain energy homeostasis, it may be related to the traditional use of ginseng as mentioned. pharmacological studies revealed that the bioactive ginsenosides including rb , rg , rg , rh , re, and rd [ ] ameliorated inflammation, removed oxidative stress, stimulated immune system and regulated apoptosis. therefore, radix ginseng may cure more diseases than traditionally known. radix ginseng illustrated beneficial effects in neurodegenerations in part due to its anti-oxidative [ ] and anti-apoptotic properties [ ] . similar therapeutic uses of radix ginseng have also been reported in cardiovascular disease [ ] , and cancers [ ] . recent researches have focused on studying the autophagic mechanisms of radix ginseng. rb suppressed neurotoxicity through inhibiting autophagy by beclin- downregulation [ ] . the compound could also enhance cardiac muscle cell survival through autophagy [ ] . the minor rb -derived ginsenoside f [ ] and rg could regulate autophagy leading to the suppression of breast cancer stem cells [ ] and hepatocellular carcinoma [ ] , respectively, suggesting the role of autophagy in the potential new therapeutic action of ginseng. peschiera fuchsiaefolia (dao zhong sha ma cha) showed in vitro antimalarial activity against plasmodium falciparum [ ] . voacamine (voa), a bioactive alkaloid extracted from the herb [ , ] , has been reported to induce autophagic cell death of multidrug-resistant osteosarcoma, and inhibit the action of transporter p-glycoprotein (p-gp) [ ] . voa is the ligand of p-gp which expresses in kidney, gastrointestinal tract, brain, etc. [ ] . in fact, diabetes mellitus is associated with p-gp dysregulation [ ] . also, the blood brain barrier (bbb) p-gp was associated with abnormal protein aggregation in alzheimer's and parkinson's diseases, suggesting the potential use of the herb in neurodegenerative disorders [ ] . therefore, the well-known autophagic involvement in diabetes mellitus and neurodegenerations strongly advocated that peschiera fuchsiaefolia may act therapeutically as novel autophagy regulators under such pathological conditions. radix ophiopogonis (mai dong) has been used for treating inflammatory symptoms such as cough and phlegm production, and cardiovascular diseases [ ] . ophiopogonin (op)-b is one of the bioactive components, and was found to be an inducer of autophagy. in non-small cell lung cancer, op-b up-regulated autophagy of tumor cells through pi k/akt pathways, and induced apoptosis-independent cell death and silences [ ] . another active constituent, op-d exhibited anti-inflammatory effects through direct inhibition of mitochondrial ros synthesis [ ] . however, such an anti-inflammatory effect was not related to op-d-induced upregulation of autophagy, as the compound could in fact suppress autophagy per se [ ] . therefore, owing to the close relationship between inflammation progression and autophagy, together with the autophagy modulating role of op-b and op-d, it is predicted that the compounds may contribute to the anti-inflammatory activity of the herb by regulating autophagy. also, the findings of op-b in inducing autophagic cancer cell death suggest the alternative use of radix ophiopogonis in cancer therapy. these drugs help our body defenses against external stimulus, including invading pathogens, cold, heat, damp-wind or summer heat that may have a noxious effects on the human body, by maintaining a normal and healthy status of organ such as the stomach (wei qi) [ , ] . radix bupleuri (chai hu) has been used for counteracting different inflammatory conditions including pancreatitis, liver cirrhosis and fever, infections like malaria and common cold, modulating abnormal lipid metabolism and relieving depression [ ] . the bioactivities of the main components, the saikosaponins, are responsible for the mentioned clinical indications. for instance, saikosaponins modulated host immunity by manipulating pro-inflammatory mediator release and lymphocyte responses [ ] . saikosaponins also repressed viral replication [ ] and fever [ ] , reduced hepatotoxicity [ ] and acted as tranquilizers [ ] . direct evidence for the participation of autophagy in regulating such activities is however lacking. however, contemporary studies verified the anti-tumor effect of saikosaponins, in particular ssd (saikosaponin-d), through autophagy regulation. ssd was cytotoxic to different cancers, such as breast and cervical cancers by increasing autophagy-induced er stress via the camkkβ-ampk-mtor signaling [ , ] . in fact, formulated decoctions containing radix bupleuri have been prescribed for cancer therapy [ ] , which supports the idea that radix bupleuri is a novel autophagy enhancer exhibiting therapeutic effects towards cancers. rhizoma zingiberis recens (sheng jiang) has been used for centuries for the treatment of colds, arthritis, migraines, nausea and hypertension [ , ] . -gingerolis is the most abundant bioactive ingredient found in rhizoma zingiberis recens. it suppressed oxidative stress by inhibiting inos activity of activated macrophage [ ] . -gingerol affected the anti-inflammatory properties by regulating ca + , which may be related to the regulation of autophagy [ ] . the anti-mentic qualities of rhizoma zingiberis recens are also related to -gingerol through inhibition of the function of serotonin receptor [ ] . in addition, rhizoma zingiberis recens-mediated new anti-tumor functions via autophagy induction were further clarified. for instance, -gingerol induced cervical cancer cell death by upregulating caspase -mediated apoptosis, and autophagy partly via the repression of akt signaling [ ] . in pancreatic cancer, -gingerol induced cytotoxicity exclusively through autophagy by activating ampk-mtor signaling, which is a process independent of necroptosis and apoptosis [ ] . these herbs are responsible for treating painful obstruction (bi) syndromes due to wind (wind-bi syndrome), cold (cold-bi syndrome), dampness (dampness-bi syndrome) or heat (heat-bi syndrome), which are caused by poor qi or blood circulation [ , ] . radix tripterygii wilfordii (lei gong teng) has been used for the treatment of inflammation and overactive immune system, including rheumatoid arthritis, systemic lupus erythematosus, and dermatomyositis [ ] . celastrol is the bioactive component responsible for exhibiting the anti-inflammatory and anti-oxidative effects for alleviating autoimmune disorders such as chronic inflammation, neurodegenerative disease, and asthma [ ] . recent findings suggested the use of celastrol in cancer therapy as demonstrated by its inhibitory effects in different tumors [ ] . emerging data revealed that autophagy was one of the molecular machineries mediating these celastrol-induced therapeutic functions. by regulating the ros/jnk signaling pathway, celastrol triggered autophagy and apoptosis which further repressed the proliferation of osteosarcoma cells in both animal and cellular models [ ] . beside, autophagy induction by celastrol through the inhibition of pi k/akt/mtor signaling, have demonstrated therapeutic potential in inflammatory disorders such as crohn's disease, which implies that the traditional functions of radix tripterygii wilfordii are mediated through the regulation of autophagy [ ] . the compound also prevented neurodegeneration by inducing autophagy in affected neuronal cell death via the targeting jnk and pten-akt/mtor network [ ] . radix stephaniae tetrandrae (fang ji) is used for the treatment of edema, anti-hypertension and analgesic, and was usually used as decoction such as "fang ji huang qi tang" [ ] . the main bioactive constituents of the herb are fangchinoline and tetrandrine. both compounds are able to modulate cytokine expression and exhibit anti-inflammatory effects [ ] . fangchinoline reduced blood glucose levels, scavenged free radicals and reduced oxidative stress [ ] . tetrandrine exhibited anti-hypertensive action by disrupting ca + movement and no synthase activity [ ] . although the role of autophagy modulation in such alteration of glucose and ca + level remains elusive, both fangchinoline and tetrandrine have been reported to contribute their anti-tumor effect through autophagy. the proliferation and invasion of gastric cancer cells could be suppressed by fangchinoline mediated inhibition of pi k/akt signaling [ ] . the compound also triggered autophagic cell death by targeting the p /sestrin /ampk signaling in hepatocellular carcinoma [ ] . in leukemia cells, autophagy was induced by tetrandrine through the upregulation of notch and ros signaling [ ] . these autophagy-related anti-tumor activity stimulated by fangchinoline and tetrandrine encourage the further development of radix stephaniae tetrandrae as an effective drug for cancer therapy. radix plumbaginis zeylanicae (bai hua dan) has effects in relieving pain, activating blood circulation, and is used for menstrual disorders, detoxification, and elimination of intestinal worms. plumbagin, the bioactive component of the herb, is well known for its therapeutic safety and effectiveness [ ] . pharmacological studies demonstrated that plumbagin modulates the immune system and resolves inflammation [ , ] . the compound facilitates microbe clearance [ ] and modulates lipid metabolism [ ] . however, the role of autophagy in mediating these traditional functions of the herb remains to be elucidated. recently, the anti-tumor activities of plumbagin have been increasingly reported [ , ] . the anti-tumor properties of plumbagin were attributed to the autophagy induction ability of the compound. for instance, plumbagin induced autophagic cell death in breast cancer via the akt/mtor signaling pathway [ ] . the compound also triggered apoptosis and autophagic cell death in lung cancer and tongue squamous carcinoma cells through the mtor signaling pathway [ ] . these herbs are prescribed when there is an accumulation of dampness. this kind of disturbance in body fluid (water) metabolism is related to a dysfunction of the lung, spleen, kidney or bladder [ , ] . rhizoma alismatis (ze xie) is derived from the stem tuber of alisma orientale [ ] . the herb reduces the circulatory levels of cholesterol and blood sugar, and promotes urine production and perspiration, which helps to resolve symptoms like chronic nephritis and edema [ ] . alisol b is the active component of rhizoma alismatis which improved lipid metabolism by inhibiting the absorption and synthesis of cholesterol [ ] . alisol b -acetate, another important constituent of rhizoma alismatis, participates in inhibiting antibody-mediated allergic reactions, and lipopolysaccharide (lps)-induced inos and no production [ ] . up to now, it is still questionable if autophagy mediates the traditional functions of rhizoma alismatis or its bioactive constituents as mentioned. however, alisol b has been reported as a new autophagy inducer functioning through activation of camkk/ampk/mtor signaling, induction of apoptosis and triggering of cell death in breast cancer cells [ ] . the anti-tumor effects of alisol b -acetate have also been reported in hepatocellular carcinoma [ ] . therefore, rhizoma alismatis has high potential to be developed as an anti-cancer drug through regulation of autophagy. cortex magnoliae officinalis (hou po) helps remove chest stuffiness due to phlegm accumulation, and relieves distension, which suggest its regulatory role in the immune system [ ] . in consistence with the clinical indications of cortex magnoliae officinalis, the bioactive component, magnolol, alleviated acute pain and endothelial damage stimulated by inflammation [ ] . other pharmacological effects of the compound included the reduction of anxiety and irritability via the regulation of γ-aminobutyric acid (gaba) receptor expression [ ] . magnolol also possessed anti-fungal properties [ ] , and was applied for bone repair by regulating the activities of osteoclasts and osteoblasts [ ] . magnolol also interplayed with the autophagic process which was beneficial to cancer therapy, but seemed not to be related to the traditional immunomodulatory effects of the herb. it induced autophagic cell death of lung cancer by blocking the pi k/pten/akt pathway [ ] . ery , a compound derived from magnolol, activated autophagy and suppressed angiogenesis, causing apoptosis-independent cytotoxicity in prostate cancer cells [ ] . based on these observations, the effects of cortex magnoliae officinalis on autophagy appear to be a valuable research niche in the search of new cancer treatment modalities. these herbs are used to warm the interior organs, expel cold, tonify yang and rescue harmed yang qi and relieve pain. interior cold of the human body can be caused by exogenous cold, or kidney yang deficiency which finally results in spleen and heart yang deficiency [ , ] . fructus evodiae (wu zhu yu) was effective for the treatment of gastrointestinal and menstrual disorders, postpartum hemorrhage and headaches [ , ] . pharmacological studies of the bioactive component, evodiamine, showed that the compound is anti-inflammatory in nature by inhibiting cox- expression [ ] , and inducing blockage of preadipocytes differentiation [ ] . evodiamine also induced apoptosis and suppressed proliferation of cancer cells [ ] . in addition, evodiamine could induce autophagic cell death in glioblastoma by quenching calcium/jnk signaling and apoptosis [ ] . through the modulation of beclin- and bcl- expression, evodiamine induced autophagic cell death and apoptosis of gastric adenocarcinoma cells, respectively [ ] . in a drug screening test, evodiamine was found to inhibit autophagic cell death of infected cells upon viral inoculation of influenza a through ampk/tsc /mtor signaling [ ] . therefore, evodiamine regulated autophagy through a complex molecular network, further verification of such a circuit would help to discover and standardize the novel usage of evodiamine and fructus evodiae. further investigations correlating autophagy induced by evodiamine or fructus evodiae to their traditional use should also be undertaken. fructus piperis longi (bi bo) represses cough and fever, relieves allergic symptoms like asthma, helps cease pathogen invasions, reduces blood glucose level, induces coronary vasodilation and treats jaundice [ ] . the active components, piperlongumine and its derivatives, inhibit pro-inflammatory mediator synthesis [ ] . these compounds remove oxidative stress and prevent cardiac damage caused by ros [ ] . they also inhibit platelet aggregation and are used as crude drugs for promoting peripheral blood circulation [ ] . amongst the diverse pharmacological activities, the potential piperlongumine-induced cytotoxic effects towards the different cancer cells have aroused the most attention [ , ] . for example, autophagy induced by piperlongumine mediates the anti-tumor effects of the compound. piperlongumine attenuates akt/mtor signaling and promotes autophagic cell death of cancer cells originated from breast, kidney, prostate and lung [ , ] . piperlongumine induces autophagy by targeting the p signaling in osteosarcoma [ ] . apart from regulating tumorigenesis, the traditionally reported anti-inflammatory effect of the compound partly results from autophagy enhancement [ ] . these herbs are used to treat malfunctions in maintaining normal blood hemostasis, such as: ( ) mild forms of blood stagnation caused by slow blood flow which may lead to blood stasis; ( ) severe forms of stagnation due to congealment of phlegm, heat or cold, which lead to formation of solid masses and blood circulation stasis [ , ] . rhizoma curcumae longae (jiang huang) is a safe chm for alleviating intermittent fever and inflammation of the bronchi, kidney and gall bladder, counteracting infections such as leprosy and cold, and treating of edema, diarrhea and cancer [ , ] . curcumin, the main bioactive component of rhizoma curcumae longae, possesses a unique structure amongst other active constituents extracted from the herb. such characteristics conferred curcumin with pharmacological properties per se correlating with the clinical efficacy of rhizoma curcumae longae. the compound has been associated with the capability of preventing inflammation [ ] , tumor progression [ ] , and oxidative stress accumulation [ ] . of note, curcumin was pharmacologically beneficial to neurodegenerative disorders [ , ] . the compound was particularly suitable for neurodegeneration intervention since the compound could cross the bbb after oral administration, acting as an anti-inflammatory and antioxidant drug, and amyloid aggregation inhibitor [ , ] . in a parkinson's disease model, curcumin triggered autophagy in neural cells by suppressing the mtor/p s k signaling which hindered the downstream α-synuclein accumulation [ ] . in addition, curcumin-induced autophagy was correlated to its anti-cancer properties. by the up-regulation of erk / and akt/mtor/p s k signaling, curcumin induced autophagy and suppressed the proliferation of glioma cells [ ] , suggesting the possible autophagic role of curcumin in various disease models. radix salviae miltiorrhizae (dan shen) has been shown to prevent platelet aggregation and facilitate fibrinolysis [ ] . it is a traditional remedy for managing coronary heart disease [ ] . the herb was also used as ingredient in formulations for diabetes such as "tangzhiqing" [ ] , and was prescribed for hepatic and renal disorders [ ] . tanshinone iia is the main bioactive component abundantly found in radix salviae miltiorrhizae. it modulates inflammation and host immunity by acting on multiple targets depending on the cell types [ ] . for instance, tanshinone iia inhibited macrophagic nf-κb activation via the erk / , p and jnk pathways [ ] . in line with the clinical indications of radix salviae miltiorrhizae, tanshinone iia was cardioprotective through its action upon calcineurin/nfatc pathway [ ] . intriguingly, calcineurin regulated ampk-dependent autophagy of cardiomyocytes upon oxidative stress [ ] implying that the process may underlie the cardioprotective function of radix salviae miltiorrhizae. in terms of autophagy regulation, tanshinone iia has been reported to induce autophagic cell death of leukemia via activation of ampk/mtor and erk/mtor, as well as p s k signaling [ ] . such observations suggested new potential uses of radix salviae miltiorrhizae in the treatment of cancer via autophagy. rhizoma chuanxiong (chuan xiong) is well known for its efficacy in improving blood fluidity, coronary and systemic circulation [ ] . contemporary studies have demonstrated that the herb modulates the proliferation of vascular smooth muscle cells [ ] and prevents endothelial cell damage [ ] . ligustrazine (tetramethylpyrazine) is the active constituent which increases myocardial contractility and coronary circulation [ ] . in addition, this single molecule acts as an anti-oxidant to remove superoxide anion, hydroxyl and lipid peroxyl radical which induce oxidative damages in tissues [ ] . however, it is not known if autophagy is modulating the cardioprotective functions of the herb. ligustrazine exhibits neuroprotective and anti-inflammatory effects on brain disorders such as cerebral ischemia, through elevating nuclear factor e -related factor (nrf )/heme oxygenase- (ho- ) expression [ ] . recently, ligustrazine has been reported to induce autophagy which was associated with cytotoxic effects toward hepatocellular carcinoma [ ] . the ligustrazine-induced autophagic effect has also been demonstrated in protecting the kidney from neurotoxicity [ ] . therefore, the mechanisms underlying the rhizoma chuanxiong or ligustrazine-induced autophagic process are intricate. further investigations are needed to support the usage of the herb in pathological condition such as cancer and inflammation conditions. these kinds of compounds are applied to eliminate toxins, kill parasites, diminish swelling, relieve pain, expel pus and abscesses, improve wound healing, stop itching or bleeding [ , ] . venenum bufonis (chan su) is famous for its clinical application in resolving cardiovascular and inflammatory symptoms, including sore throat and tonsillitis, promoting urine production, and acting as an analgesic agent [ ] [ ] [ ] . in modern chinese medicine, venenum bufonis was used in liver cancer therapy [ ] . bufalin is one of the constituents of venenum bufonis exhibiting bioactivities related to the clinical indications described [ ] . recently, bufalin was found to regulate autophagy via cell type-dependent mechanisms to suppress tumorigenesis. in liver cancer, bufalin interacted with the atg , jnk, becn- and tnf signaling, and stimulated autophagic cell death of hepatoma cells like huh , hep b and ha t [ ] . on the other hand, bufalin stopped the proliferation of hepatocellular carcinoma by activating autophagy through the akt/mtor and ampk/mtor pathways respectively [ ] . the bufalin-induced autophagic cell death also effectively suppressed colon cancer cell proliferation via jnk activation [ ] , and the pten/akt pathways [ ] . through targeting ampk and the downstream p s k, bufalin modulated the apoptotic and autophagic activities of glioma cells [ ] . collectively, these findings support the traditional use of venenum bufonis in the treatment of cancerous diseases via autophagy regulation. garcinia hanburyi (teng huang) was used traditionally for treating inflammatory conditions and immunity dysregulation such as ulcerative gingivitis, skin infection, scald and burn, and chronic eczema [ ] . the herb was also applied to cease traumatic bleeding, attacking toxin and parasites [ ] . gambogic acid is the major active ingredient of garcinia hanburyi biologically alleviating pain, inflammation, and fever [ ] . recent investigations strongly suggest that gambogic acid is anti-tumor in nature by suppressing the proliferation of cancers of lung [ ] , liver [ ] , blood [ ] and stomach [ ] . further studies revealed that gambogic acid triggered autophagy and ameliorated bladder cancer by modulating the beclin- , p and nf-κb activities [ ] . by up-regulating the beclin- expression, gambogic acid induced cytotoxic in leukemia cells through the induction of autophagy and apoptosis [ ] . apparently, garcinia hanburyi and gambogic acid have the potential to be further developed as novel and effective anti-cancer therapeutic strategy. it is also important to verify if autophagy is mechanistically meditating the garcinia hanburyi-induced inflammatory and immunological regulations. these herbs can be used to treat mental syndromes related to heart (blood and yin) deficiency such as over-activity of the heart, nervousness, fright, restlessness, irritability, insomnia, palpitations or anxiety; or treat syndromes related to liver (yin and yang) deficiency such as dizziness and headaches. this group of drugs is also prescribed to pacify internal wind which can contribute to tremor, spasms, paraesthesias of the limbs, dizziness or difficulties in walking [ , ] . radix polygalae (rp) (yuan zhi) is commonly prescribed in many classical decoctions such as "kai xin san" [ ] , and "ding zhi wan" [ ] for the treatment of forgetfulness [ ] , anxiety [ ] , insomnia or depression [ ] . recent pharmacological studies have also reported the sedative-hypnotic [ ] , memory improving [ ] , cognitive recognition enhancing [ ] , antidepressant [ ] and neuroprotective effects [ ] of rp. rp was reported to inhibit the phosphatidylinositol -kinase (pi k)/akt or activate the n-methyl-d-aspartate (nmda) signaling pathways [ , ] . the active ingredients of rp such as onjisaponin b and other identified saponins, were proved to accelerate the clearance of neurodegenerative disease proteins such as huntingtin and α-synuclein, reduce aggregation and toxicity of mutant proteins through the induction of autophagy [ , ] . therefore, rp may play its traditional sedative effect through degradation of unwanted proteins or organelles by autophagy. ganoderma lucidum (ling zhi) is having efficacy in replenishing qi, stabilizing the nervous system and relieving cough and asthma, is commonly prescribed to treat insomnia, palpitation, cough and phlegm. it exerts tranquilizing effects through tonifying the heart, qi and blood. triterpenes are the major components of ganoderma lucidum. pharmacological studies have demonstrated that both ganoderma lucidum extract and ganoderic acid c could reduce accumulation of mutant huntingtins in pc- cells, alleviate neurotoxicity and behavioral deficits induced by -nitropropionic acid, and prevent or reverse memory loss resulting from sleep deprivation [ ] . additionally, it was also reported that ganoderma lucidum triterpene extract (glt) suppressed the proliferation of human colon cancer cells and inhibited tumor growth in a xenograft model, which were associated with the induction of autophagic cell death [ ] . furthermore, ganoderic acid activated autophagy, which facilitates immune recognition of cd + t cells; induced autophagic cell death and apoptosis of melanoma [ ] ganoderma lucidum triterpene extract induced autophagy which inhibited the development of colon cancer via p mapk signaling [ ] ; and suppressed gastric cancer cells through the repression of p [ ] . all these findings suggest that the traditional therapeutic role of ganoderma lucidum is in part related to autophagy regulation, which may also be responsible for the novel use of the herb in cancer treatment. caulis polygoni multiflori (shou wu teng) has been prescribed for nourishing blood, tranquilizing the mind and dispersing wind to treat insomnia, numbness of the skin and rheumatism in traditional chinese medicine. it was often combined with semen ziziphi spinosae (suan zao ren) and cortex albiziae (he huan pi) to treat insomnia, distraughtness and dizziness. modern pharmacological study has suggested an effect of caulis polygoni multiflori extract in protecting rats against ccl -induced hepatotoxicity through its antioxidant activities [ ] . the major active ingredients of shou wu teng, anthraquinones, possessed similar chemical structures but different bioactivities. for example, emodin, the most abundant anthraquinone in rhubarb, inhibits cellular proliferation and prevents metastasis of cancers through apoptosis. another major anthraquinone in rhubarb, rhein, inhibits the uptake of glucose and leads to cancer cell death caused by changes in membrane-associated functions [ ] . ananthraquinone-containing extract of shou wu teng possesses myocardial protective effects by maintaining antioxidant status under oxidative stress conditions [ ] . abnormal aggregation of tau protein is highly correlated with the pathogenesis of alzheimer's disease (ad), therefore, with the ability in mitigating aggregation and cytotoxicity of tau [ ] anthraquinones possesses high potential in modulating ad. together with the fact that ananthraquinones were able to induce autophagic cell death in cancer cells [ ] , shou wu teng may exert its traditional sedative function through the induction of autophagy, which was highly related to the modulation of neurodegenerative disease proteins, as well as cancers [ ] . fructus schisandrae (wu wei zi) has been prescribed to replenish qi, nourish the kidneys and tranquilize the mind to produce a sedative nephroprotective effect. besides, sc extract was co-administered with other medicine for reducing immunosuppressive drug (cyclosporine a)-induced side effects [ ] . schisandra total lignin (stl), the major active ingredient of fructus schisandrae, delayed mouse brain aging by attenuating apoptosis [ ] . in vivo experiments further demonstrated stl inhibited the d-galactose-induced brain tissue aging through regulating autophagy and inhibiting apoptosis in the mice. it has been reported that longevity-promoting regimens such as caloric restriction or inhibition of tor is associated with induction of autophagy [ ] . therefore, autophagy may be the mechanism responsible for the sedative and anti-ageing effect of fructus schisandrae. semen ziziphi spinosae (suan zao ren) is commonly prescribed as "suan zao ren tang" for sedation, nourishing the nerves, insomnia, palpitations, anxiety, dizziness, dry mouth and throat, red tongue, and clinical treatment of neurasthenia, heart neurosis and menopausal syndrome due to deficiencies of the heart and liver [ , ] . the active component of semen ziziphi spinosae, jujuboside b, was reported to inhibit platelet aggregation and target cardiovascular diseases associated with platelet hyperaggregation [ ] . furthermore, the anti-tumor activity of jujuboside b was reported to be associated with the induction of apoptosis and autophagy [ ] . another active neuroprotective component from semen ziziphi spinosae, jujuboside a, could mitigate learning and memory impairment in mice, by reducing the level of aβ , and inhibiting the activities of acetylcholinesterase (ache) and no in the hippocampus and cerebral cortex of mice [ ] . with its traditional effects in tranquilizing the mind and nourishing heart, blood and qi, current pharmacological studies have confirmed the neuroprotective and autophagic role of semen ziziphi spinosae. as autophagy was highly correlated to the maintenance of cellular homeostasis, which was important for normal function of brain, autophagy may be responsible for the pharmacological action and sedative effects of semen ziziphi spinosae. succinum (ambrum) has been prescribed for relieving convulsion, tranquilizing the mind, activating blood and removing stasis, inducing diuresis, treating irritability, epilepsy, algomenorrhea and amenorrhea in tcm [ ] . it was commonly prescribed with rhizoma acori graminei (shi chang pu) and radix polygalae (yuen zhi) in "hu po ding zhi wan" for treating palpitations, insomnia and forgetfulness [ ] . vitamin e succinate (ves), one of the active components of ambrum, was proved to induce autophagy via the inhibition of mtor [ ] . besides, ves worked as an anti-neoplastic agent through regulating apoptosis in cancer cells [ ] . as autophagy is also highly correlated with modulation of cancers, with the recently identified anti-cancer effect of jujuboside b and ves through induction of autophagy, both semen ziziphi spinosae and ambrum may possess new applications in anti-cancer therapy via its traditional tranquilizing effect, which was highly associated with the beneficial effect of autophagy. in addition, the chinese medicinal herbs including nelumbo nucifera, rhizoma acori graminei, radix salviae miltiorrhizae and radix ginseng also participate in tranquilization of the mind [ ] . nelumbo nucifera treats palpitations, insomnia and dreamful sleep [ ] in tcm. it is commonly prescribed as a formulation with radix polygalae, semen ziziphi spinosae and radix salviae miltiorrhizae. the active component of nelumbo nucifera, neferine, was identified as a novel autophagic enhancer which facilitates the degradation of mutant neurodegenerative disease proteins in vitro [ ] . with the beneficial effect of autophagy in maintaining normal homeostasis in cells, sedative tcms may play protective role in neurodegenerative diseases, through the removal of disease proteins by autophagy. preclinical or clinical models showed that pharmacological inhibition of autophagy can enhance the sensitivity of tumor cells towards multiple anti-cancer drugs. for example, inhibition of autophagy enhanced apoptosis induced by cetuximab [ ] or vorinostat [ ] . while cq enhanced the therapeutic efficacy of saracatinib [ ] in prostate cancer xenograft model, inhibition by -ma increased fluorouracil ( -fu) [ ] induced apoptosis with tumor regression in colon cancer xenografts. among these autophagy inhibitors, only cq or hcq were studied in humans as they cross the blood-brain barrier with hcq much preferred to human due to the less severe side effects [ ] . based on these preclinical data, phase i or ii trials were performed to evaluate the combinational use of autophagy inhibitors (hcq or cq) with various anti-cancer cytotoxic agents. however, there are limitations for their use in clinical practice due to the long half-life and high effective concentration of hcq. a phase i trial was performed to evaluate the combined use of hcq with temozolamide [ ] and radiation in glioblastoma patients. phase i or ii clinical trials evaluating the combination use of bortezomib and cq [ ] are ongoing in patients with recurrent carcinoma. a phase i trial of -deoxyglucose [ ] , an agent that blocks glucose metabolism, showed a reduction in autophagy, suggesting the role of autophagy in cancer therapy. similarly, the clinically used mood stabilizers lithium (valproate and carbamazepine) [ ] , induce mtor-independent autophagy and enhance the cellular degradation of aggregate-prone mutant huntingtin and α-synuclein. the hypertensive agent rilmenidine [ ] , a us food and drug administration-approved compound, showed protective effect in huntington's disease models and is now under further clinical trials. protein phosphatase a (pp a) agonists [ ] which favor induction of autophagy are currently under clinical trials for alzheimer's disease. the clinically used approved antidiabetic drug, metformin [ ] , attenuates disease development in some neurodegenerative diseases via ampk activation. -hydroxypropyl-β-cyclodextrin [ ] , effective in enhancing the clearance of lipids or autophagic substrate burden, can mitigate neurological deficits in an npc mouse model and is currently under early clinical trials for treating npc. another lipofuscinolytic agent, centrophenoxine [ ] , is an anti-aging antioxidant which possesses potential protective effect for late-stage dementia in small early clinical trials. however, factors such as blood-brain barrier penetration power of the compounds and cellular heterogeneity of brain tissue can also affect the clinical neuroprotective efficacy of autophagy drugs. for example, the different responses of neurons and glia to autophagy or drug must be considered in clinical trial evaluations [ ] . although the molecular mechanisms and functions of many autophagy modulators isolated from chm were intensively studied, we are still far away from translating these traditional herbs or compounds into clinical applications. knowledge concerning autophagy research is mainly related to non-selective autophagy describing the molecular responses upon starvation. however, increasing studies are evidencing the significance of selective autophagy which involves specific molecular mediators targeting particular kinds of unwanted intracellular materials [ , ] . therefore, it is important to clarify the potential discrepancies between selective and non-selective autophagy in terms of their response towards different therapeutic agents. on the other hand, the time of starting the autophagy modulatory treatment should also be considered. for example, early cancer development is likely to be prevented by autophagy induction, as cancer cells exploit nutrients generated by autophagy to survive under the stressful cellular environment at the later stages [ , ] . it is also important to take into account the cells type-specific property of autophagy which may otherwise minimize the efficacy of the applied herbs, and result in unfavourable side effects. the heterogeneous cell types as presented by hepatic lobules can well explain such a scenario. the liver consists mainly of parenchymal cells (hepatocytes) and around % of non-parenchymal cells such as hepatic stellate cells (hscs) [ ] . the functional consequences of autophagy induction on these cells vary under different diseased conditions. autophagy of hsc activated during the end stage of chronic liver disease and hepatic fibrosis could be alleviated by autophagy repression in vitro [ ] [ ] [ ] . in contrast, autophagy stimulation is beneficial upon most of the parenchymal cells-related hepatic malignancies. therefore special caution would need to be taken when applying the autophagic modulators in cancer therapy. an increasing number of chinese herbal medicines (chms) have been discovered as autophagy modulators. such autophagic-regulatory effects are therapeutically beneficial to a broad range of disorders which are consistent with their traditional usage. interestingly, many chm-induced autophagy findings described in this review also demonstrate novel applications in various pathological conditions. these potential therapeutic functions can be summarized into: ( ) anti-cancer; ( ) neuroprotective; ( ) cardiovascular-protective; and ( ) antiviral applications. for example, through the activation of autophagic cell death, tumorigeneses can be repressed by cortex phellodendri, radix sophorae lavescentis, radix isatidis, and stephania japonica which are not associated with cancer therapy according to chinese herbology. in fact, a plethora of chms having similar potential in preventing cancer progression have not been studied before. these herbs include nelumbo nucifera, syzygium samarangense, mallotus philippensis, rhizoma anemarrhenae, radix glehniae, radix ophiopogonis, radix glycyrrhizae, radix dipsaci, radix ginseng, radix codonopsis, rhizoma atractylodis macrocephalae, ganoderma lucidum, radix bupleuri, rhizoma zingiberis recens, radix tripterygii wilfordii, radix stephaniae tetrandrae, rhizoma alismatis, cortex magnoliae officinalis, fructus piperis longi, radix salviae miltiorrhizae, rhizoma chuanxiong, garcinia hanburyi, radix plumbaginis zeylanicae, and fructus evodiae. in contrast, fructus evodiae can inhibit autophagic cell death of influenza-infected cells which provides insight to the search of chms sharing such anti-viral properties. in the case of neurodegenerative disorders, nelumbo nucifera, mallotus philippensis, rhizoma anemarrhenae, radix ginseng, radix tripterygii wilfordii, and rhizoma curcumae longae can trigger previously unidentified autophagy-mediated neuronal survival. these groups of novel neuroprotective herbs may help remove misfolded protein aggregates via autophagy activation. also, rhizoma polygoni cuspidate can upregulate the cardiac myocytic autophagy to eliminate damaged proteins uncovering the innovative use of the herb in cardiovascular disorders. it should also be noted that a single chm usually contain more than one bioactive component. therefore, the downstream molecular networks modulated by these chms are complicated. precise investigations concerning how chms may influence the intricate autophagy machinery is needed, and will be a major challenge for further developing chm as practical autophagy regulators. when compared with western medicines, most of the reviewed autophagy modulators here are bioactive components constituting the chms, which have long been prescribed as decoctions or formulations in the chinese community with well-known pharmacological action, toxicity or side effects. therefore, clinical trials using these natural herbs may be more safe and reliable. also, the philosophy of chm emphasizes the comprehensive and persistent body balance, which tailors them to deal with the autophagy-related diseases, which are pathologically associated with the loss of overall cellular and physiological balance. in addition, the actions of chm are usually multi-targeting [ , ] , making them suitable medications for autophagy-related disorders which are usually polysymptomatic. therefore, detailed and systematic investigation concerning the interaction between chms and autophagy-related disorders in a comprehensive molecular approach is needed. positive findings in these areas could widen the scope of chm applications by suggesting novel intervention strategies, which have not been mentioned in the traditional chinese pharmacopeia. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: (suppl. s ), s -s . the machinery of macroautophagy characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins methods in mammalian autophagy research autophagic cell death: loch ness monster or endangered species? to die or not to die: that is the autophagic question self-eating and self-killing: crosstalk between autophagy and apoptosis autophagic cell death: the story of a misnomer autophagy and the immune system unveiling the roles of autophagy in innate and adaptive immunity role of galectin- in prion infections of the cns autophagy failure in alzheimer's disease and the role of defective lysosomal acidification the role of autophagy in parkinson's disease. cold spring harb atg deficiency exacerbates glucose intolerance in mice on high-fat diet the autophagy-related gene (atg ) is regulated by forkhead box o transcription factors and circadian rhythms and plays a critical role in hepatic autophagy and lipid metabolism defective regulation of adipose tissue autophagy in obesity extracellular nucleotides inhibit insulin receptor signaling, stimulate autophagy and control lipoprotein secretion macroautophagy in homeostasis of pancreatic β-cell autophagy in the pathogenesis of disease autophagy genes are essential for dauer development and life-span extension in c. elegans caloric restriction delays disease onset and mortality in rhesus monkeys genomic analyses reveal global functional alterations that promote tumor growth and novel tumor suppressor genes in natural killer-cell malignancies promotion of tumorigenesis by heterozygous disruption of the beclin autophagy gene reduced expression of lc b-ii and beclin in glioblastoma multiforme indicates a down-regulated autophagic capacity that relates to the progression of astrocytic tumors systemic treatment with the antidiabetic drug metformin selectively impairs p -deficient tumor cell growth saikosaponin-d, a novel serca inhibitor, induces autophagic cell death in apoptosis-defective cells natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells z) , , , -trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level autophagy in chronic obstructive pulmonary disease: homeostatic or pathogenic mechanism? autophagy in immunity: implications in etiology of autoimmune/autoinflammatory diseases autophagy in immunity and cell-autonomous defense against intracellular microbes hsc blockade by the therapeutic peptide p affects autophagic processes and endogenous mhcii presentation in murine lupus molecular therapies for systemic lupus erythematosus: clinical trials and future prospects fty increases cd expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death pharmacological regulators of autophagy and their link with modulators of lupus disease effects of fty in mrl-lpr/lpr mice: therapeutic potential in systemic lupus erythematosus autophagic effects of chaihu (dried roots of bupleurum chinense dc or bupleurum scorzoneraefolium wild) tetramethylpyrazine (tmp) exerts antitumor effects by inducing apoptosis and autophagy in hepatocellular carcinoma oridonin up-regulates expression of p and induces autophagy and apoptosis in human prostate cancer cells spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome curcumin inhibits autophagy and apoptosis in hypoxia/reoxygenation-induced myocytes honokiol-induced apoptosis and autophagy in glioblastoma multiforme cells attenuation of aβ - -induced parallel autophagic and apoptotic cell death by gypenoside xvii through the estrogen receptor-dependent activation of nrf /are pathways erk / -dependent phosphorylation of gα-interacting protein stimulates its gtpase accelerating activity and autophagy in human colon cancer cells a heterotrimeric g-protein controls autophagic sequestration in the human colon cancer cell line ht- amino acids interfere with the erk / -dependent control of macroautophagy by controlling the activation of raf- in human colon cancer ht- cells synthesis and function of -phosphorylated inositol lipids beclin-phosphatidylinositol -kinase complex functions at the trans-golgi network regulation of membrane traffic by phosphoinositide -kinases induction of autophagy and inhibition of tumorigenesis by beclin jnk -mediated phosphorylation of bcl- regulates starvation-induced autophagy dap-kinase-mediated phosphorylation on the bh domain of beclin promotes dissociation of beclin from bcl-xl and induction of autophagy eaten alive: a history of macroautophagy mtor and cancer: insights into a complex relationship molecules and their functions in autophagy role of ampk-mtor-ulk / in the regulation of autophagy: cross talk, shortcuts, and feedbacks mammalian autophagy: core molecular machinery and signaling regulation amp-activated protein kinase: the energy charge hypothesis revisited amp-activated/snf protein kinases: conserved guardians of cellular energy the energy sensing lkb -ampk pathway regulates p kip phosphorylation mediating the decision to enter autophagy or apoptosis ampk phosphorylation of raptor mediates a metabolic checkpoint control of macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and bcl- the roles of intracellular protein-degradation pathways in neurodegeneration examining the structure of the mature amyloid fibril structure of the cross-β spine of amyloid-like fibrils loss of autophagy in the central nervous system causes neurodegeneration in mice suppression of basal autophagy in neural cells causes neurodegenerative disease in mice molecular structure of amyloid fibrils: insights from solid-state nmr the autophagy-related protein beclin shows reduced expression in early alzheimer disease and regulates amyloid β accumulation in mice beclin gene transfer activates autophagy and ameliorates the neurodegenerative pathology in α-synuclein models of parkinson's and lewy body diseases regulation of intracellular accumulation of mutant huntingtin by beclin inhibition of mtor by rapamycin abolishes cognitive deficits and reduces amyloid-β levels in a mouse model of alzheimer's disease inositol and ip levels regulate autophagy: biology and therapeutic speculations lithium induces autophagy by inhibiting inositol monophosphatase small molecule enhancers of autophagy for neurodegenerative diseases novel targets for huntington's disease in an mtor-independent autophagy pathway stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy trehalose, a novel mtor-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and α-synuclein metabolic pathways promoting cancer cell survival and growth amp-activated protein kinase activators can inhibit the growth of prostate cancer cells by multiple mechanisms adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of amp-activated protein kinase sustained activation of amp-activated protein kinase induces c-jun n-terminal kinase activation and apoptosis in liver cells a mammalian protein targeted by g -arresting rapamycin-receptor complex target of rapamycin (tor): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression autophagy in tumor suppression and cancer therapy the hallmarks of cancer endoplasmic reticulum stress and the inflammatory basis of metabolic disease a branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance effects of isoenergetic overfeeding of either carbohydrate or fat in young men mtorc activates srebp- c and uncouples lipogenesis from gluconeogenesis defective hepatic autophagy in obesity promotes er stress and causes insulin resistance circadian rhythms and metabolic syndrome: from experimental genetics to human disease pkr-dependent autophagic degradation of herpes simplex virus type autophagy fights disease through cellular self-digestion protection against fatal sindbis virus encephalitis by beclin, a novel bcl- -interacting protein cd induces macrophage anti-toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial dna mediated by the nalp inflammasome loss of the autophagy protein atg l enhances endotoxin-induced il- β production vitamin d inhibits human immunodeficiency virus type and mycobacterium tuberculosis infection in macrophages through the induction of autophagy eradication of intracellular francisella tularensis in thp- human macrophages with a novel autophagy inducing agent host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action autophagy in immunity and inflammation autophagic compartments gain access to the mhc class ii compartments in thymic epithelium macroautophagy substrates are loaded onto mhc class ii of medullary thymic epithelial cells for central tolerance blockade of macrophage autophagy ameliorates activated lymphocytes-derived dna induced murine lupus possibly via inhibition of proinflammatory cytokine production ophiopogonin d attenuates doxorubicin-induced autophagic cell death by relieving mitochondrial damage in vitro and in vivo mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells akebia saponin pa induces autophagic and apoptotic cell death in ags human gastric cancer cells neuroprotective effect of ginsenoside rb on glutamate-induced neurotoxicity: with emphasis on autophagy ginsenoside re enhances the survival of h c cardiac muscle cells through regulation of autophagy the plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells anticancer effect of ginger extract against pancreatic cancer cells mainly through reactive oxygen species-mediated autotic cell death celastrol induces apoptosis and autophagy via the ros/jnk signaling pathway in human osteosarcoma cells: an in vitro and in vivo study celastrol prevents cadmium-induced neuronal cell death via targeting jnk and pten-akt/mtor network tetrandrine induces autophagy and differentiation by activating ros and notch signaling in leukemia cells plumbagin induces g -m arrest and autophagy by inhibiting the akt/mammalian target of rapamycin pathway in breast cancer cells alisol b, a novel inhibitor of the sarcoplasmic/endoplasmic reticulum ca + atpase pump, induces autophagy, endoplasmic reticulum stress, and apoptosis effect of magnolol on the function of osteoblastic mc t -e cells evodiamine: a novel anti-cancer alkaloid from evodia rutaecarpa a drug screening method based on the autophagy pathway and studies of the mechanism of evodiamine against influenza a virus piperlongumine promotes autophagy via inhibition of akt/mtor signalling and mediates cancer cell death piperlongumine induces apoptosis and autophagy in human lung cancer cells through inhibition of pi k/akt/mtor pathway curcumin ameliorates the neurodegenerative pathology in a t α-synuclein cell model of parkinson's disease through the downregulation of mtor/p s k signaling and the recovery of macroautophagy regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer. cell death dis tanshinone iia induces autophagic cell death via activation of ampk and erk and inhibition of mtor and p s k in kbm- leukemia cells tetramethylpyrazine (tmp) protects against sodium arsenite-induced nephrotoxicity by suppressing ros production, mitochondrial dysfunction, pro-inflammatory signaling pathways and programed cell death anticancer effects of bufalin on human hepatocellular carcinoma hepg cells: roles of apoptosis and autophagy bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and jnk activation. free radic gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (nf-κb, beclin- , p and nbr ) in human bladder cancer cells gambogic acid induces death of k cells through autophagy and apoptosis mechanisms onjisaponin b derived from radix polygalae enhances autophagy and accelerates the degradation of mutant α-synuclein and huntingtin in pc- cells method and composition for inducing autophagy progress in phytochemicals' induction of autophagic cell death in cancer cells and regulation of nuclear receptors. chin can autophagy promote longevity? nat protective macroautophagy is involved in vitamin e succinate effects on human gastric carcinoma cell line sgc- by inhibiting mtor axis phosphorylation literature research of chinese medicine recipes for the treatment of psoriasis vulgaris with blood-heat syndrome type characterization of flavonoids in the traditional chinese herbal medicine-huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry effects of wogonin, wogonoside, and , , , , -pentahydroxyflavone on chemical mediator production in peritoneal exduate cells and immunoglobulin e of rat mesenteric lymph node lymphocytes anti-inflammatory effects of scutellaria baicalensis extract via suppression of immune modulators and map kinase signaling molecules autophagy induced by baicalin involves downregulation of cd in smmc- cells in vitro autophagy: a primer for the gastroenterologist/hepatologist chinese traditional medicine chinese pharmaceutical science and technology publication co new protolimonoids from the fruits of phellodendron chinense acetaldehyde-induced interleukin- β and tumor necrosis factor-α production is inhibited by berberine through nuclear factor-κb signaling pathway in hepg cells extracts of bark from the traditional chinese herb phellodendron amurense inhibit contractility of the isolated rat prostate gland inhibition of gene expression and production of inos and tnf-α in lps-stimulated microglia by methanol extract of phellodendri cortex development of protein kinase activators: ampk as a target in metabolic disorders and cancer berberine attenuates autophagy in adipocytes by targeting becn phellodendron amurense bark extract prevents progression of prostate tumors in transgenic adenocarcinoma of mouse prostate: potential for prostate cancer management nexrutine ® inhibits tumorigenesis in mouse skin and induces apoptotic cell death in human squamous carcinoma a and human melanoma a cells regulation of cox- by cyclic amp response element binding protein in prostate cancer: potential role for nexrutine stat down regulates lc to inhibit autophagy and pancreatic cancer cell growth butanol fraction containing berberine or related compound from nexrutine inhibits nfκb signaling and induces apoptosis in prostate cancer cells berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor advance of studies on anti-atherosclerosis mechanism of berberine preventive effect of coptis chinensis and berberine on intestinal injury in rats challenged with lipopolysaccharides in vitro response of blastocystis hominis against traditional chinese medicine analgesic effect of coptis chinensis rhizomes (coptidis rhizoma) extract on rat model of irritable bowel syndrome berberine exerts neuroprotective actions against in vitro ischemia-induced neuronal cell damage in organotypic hippocampal slice cultures: involvement of b-cell lymphoma phosphorylation suppression berberine and total base from rhizoma coptis chinensis attenuate brain injury in an aluminum-induced rat model of neurodegenerative disease sophora flavescens ait.: traditional usage, phytochemistry and pharmacology of an important traditional chinese medicine the efficacy and safety of a chinese herbal product (xiao-feng-san) for the treatment of refractory atopic dermatitis: a randomized, double-blind, placebo-controlled trial simultaneous determination of baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the chinese herbal preparation of sanwu-huangqin-tang by ion-paired hplc antiinflammatory effects of matrine in lps-induced acute lung injury in mice matrine induces cell anergy in human jurkat t cells through modulation of mitogen-activated protein kinases and nuclear factor of activated t-cells signaling with concomitant up-regulation of anergy-associated genes expression autophagy is involved in anticancer effects of matrine on sgc- human gastric cancer cells antitumor effect of matrine in human hepatoma g cells by inducing apoptosis and autophagy reversal effects of rabdosia rubescens extract on multidrug resistance of mcf- /adr cells in vitro oridonin ameliorates neuropathological changes and behavioural deficits in a mouse model of cerebral amyloidosis study on the inhibitory mechanism of oridonin in pancreatic cancers bxpc- cells by dna microarray. zhejiang zhongyiyao daxue xuebao efficacy and safety of ban-lan-gen granules in the treatment of seasonal influenza: study protocol for a randomized controlled trial ten lectures on the use of medicinals from the personal experience of jiao shu-de (jiao clinical chinese medicine) chinese medicinal teas: simple, proven, folk formulas for common diseases & promoting health fangchinoline inhibits human immunodeficiency virus type replication by interfering with gp proteolytic processing tetrandrine protects mice from concanavalin a-induced hepatitis through inhibiting nf-κb activation pharmacognostical studies on leaves of stephania japonica var dauricine induces apoptosis, inhibits proliferation and invasion through inhibiting nf-κb signaling pathway in colon cancer cells therapeutic potential of the biscoclaurine alkaloid, cepharanthine, for a range of clinical conditions potent antiperoxidation activity of the bisbenzylisoquinoline alkaloid cepharanthine: the amine moiety is responsible for its ph-dependent radical scavenge activity direct radical scavenging by the bisbenzylisoquinoline alkaloid cepharanthine inhibitory effect of dauricine on inflammatory process following focal cerebral ischemia/reperfusion in rats modulation of the atpase and transport activities of broad-acting multidrug resistance factor abcc (mrp ) a dictionary of plants used by man medicinal plants of china resveratrol as a chemopreventive agent: a promising molecule for fighting cancer fungicidal effect of resveratrol on human infectious fungi resveratrol antibacterial activity against escherichia coli is mediated by z-ring formation inhibition via suppression of ftsz expression anti-inflammatory responses of resveratrol resveratrol isolated from polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in lewis lung carcinoma-bearing mice resveratrol interferes with akt activity and triggers apoptosis in human uterine cancer cells estrogen and resveratrol regulate rac and cdc signaling to the actin cytoskeleton of metastatic breast cancer cells resveratrol in cardiovascular health and disease cardioprotection by resveratrol: a novel mechanism via autophagy involving the mtorc pathway scutellaria barbata d don inhibits colorectal cancer growth via suppression of multiple signaling pathways research progress on anticancer effect of scuteiiaria barbata d.don and its mechanism anti-inflammatory activity of edible brown alga saccharina japonica and its constituents pheophorbide a and pheophytin a in lps-stimulated raw . macrophage cells pheophorbide a, a major antitumor component purified from scutellaria barbata, induces apoptosis in human hepatocellular carcinoma cells pheophorbide a-mediated photodynamic therapy induces autophagy and apoptosis via the activation of mapks in human skin cancer cells chinese drugs of plant origin: chemistry, pharmacology, and use in traditional and modern medicine neferine enhances insulin sensitivity in insulin resistant rats anti-amnesic activity of neferine with antioxidant and anti-inflammatory capacities, as well as inhibition of ches and bace neferine, a bisbenzylisoquinline alkaloid attenuates bleomycin-induced pulmonary fibrosis neferine isolated from nelumbo nucifera enhances anti-cancer activities in hep b cells: molecular mechanisms of cell cycle arrest, er stress induced apoptosis and anti-angiogenic response quantitative analysis of antiradical phenolic constituents from fourteen edible myrtaceae fruits antioxidant activities of some local bangladeshi fruits (artocarpus heterophyllus, annona squamosa, terminalia bellirica, syzygium samarangense, averrhoa carambola and olea europa). sheng wu gong cheng xue bao protective effects of , -dihydroxy- -methoxy- , -dimethylchalcone to pc cells against cytotoxicity induced by hydrogen peroxide hepatoprotective effects of , -dihydroxy- -methoxy- , -dimethylchalcone on ccl -induced acute liver injury in mice dimethyl cardamonin inhibits lipopolysaccharide-induced inflammatory factors through blocking nf-κb p activation blockade of nuclear factor-κb signaling pathway and anti-inflammatory activity of cardamomin, a chalcone analog from alpinia conchigera cardamonin induces autophagy and an antiproliferative effect through jnk activation in human colorectal carcinoma hct cells induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma hct and lovo cells cucurbitacins-a promising target for cancer therapy anti-hiv agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and g protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors inhibition of nitric oxide generation by , -dihydrocucurbitacin d in mouse peritoneal macrophages anti-inflammatory activity of two cucurbitacins isolated from cayaponia tayuya roots anticancer and antiinflammatory activities of cucurbitacins from cucurbitaandreana dihydrocucurbitacin b inhibits delayed type hypersensitivity reactions by suppressing lymphocyte proliferation cucurbitane triterpenoids from leucopaxillus gentianeus activated kras protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p and p trichosanthes kirilowii ethanol extract and cucurbitacin d inhibit cell growth and induce apoptosis through inhibition of stat activity in breast cancer cells apoptosis induction through proteasome inhibitory activity of cucurbitacin d in human t-cell leukemia cucurbitacin e induces autophagy via downregulating mtorc signaling and upregulating ampk activity cucurbitacin i induces protective autophagy in glioblastoma in vitro and in vivo a helicobacter pylori treatment strategies and options: a review rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase c rottlerin induces apoptosis via death receptor (dr ) upregulation through chop-dependent and pkc δ-independent mechanism in human malignant tumor cells pkcδ protects human breast tumor mcf- cells against tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis tissue transglutaminase inhibits autophagy in pancreatic cancer cells screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mtorc signaling application of timosaponin aiii in anemarrhena to preparation of antitumor drugs the genus anemarrhena bunge: a review on ethnopharmacology, phytochemistry and pharmacology rhizoma anemarrhenae extract ameliorates hyperglycemia and insulin resistance via activation of amp-activated protein kinase in diabetic rodents effect of timosaponin a-iii, from anemarrhenae asphodeloides bunge (liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension effect of steroidal saponins of anemarrhenae rhizoma on superoxide generation in human neutrophils timosaponin a-iii induces autophagy preceding mitochondria-mediated apoptosis in hela cancer cells timosaponin aiii is preferentially cytotoxic to tumor cells through inhibition of mtor and induction of er stress chinese medicinal herbs as source of antioxidant compounds-where tradition meets the future liquorice, a unique "guide drug" of traditional chinese medicine: a review of its role in drug interactions inhibition by licochalcone a, a novel flavonoid isolated from liquorice root, of il- β-induced pge production in human skin fibroblasts licochalcone a isolated from licorice suppresses lipopolysaccharide-stimulated inflammatory reactions in raw . cells and endotoxin shock in mice licochalcone a activates nrf in vitro and contributes to licorice extract-induced lowered cutaneous oxidative stress in vivo antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward ldl oxidation. free radic inhibitory effects of some natural products on the activation of hyaluronidase and their antiallergic actions licochalcone a induces autophagy through pi k/akt/mtor inactivation and autophagy suppression enhances licochalcone a-induced apoptosis of human cervical cancer cells licorice and licochalcone-a induce autophagy in lncap prostate cancer cells by suppression of bcl- expression and the mtor pathway microrna- regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin chinese materia medica: combinations and applications an illustrated chinese materia medica a comparison of the ancient use of ginseng in traditional chinese medicine with modern pharmacological experiments and clinical trials comparison of the pharmacological effects of panax ginseng and panax quinquefolium ginsenoside rg attenuates dopamine-induced apoptosis in pc cells by suppressing oxidative stress possible mechanisms of the protection of ginsenoside re against mptp-induced apoptosis in substantia nigra neurons of parkinson's disease mouse model cardiovascular diseases and panax ginseng: a review on molecular mechanisms and medical applications anti-cancer and potential chemopreventive actions of ginseng by activating nrf (nfe l ) anti-oxidative stress/anti-inflammatory pathways enzymatic biotransformation of ginsenoside rb and gypenoside xvii into ginsenosides rd and f by recombinant β-glucosidase from flavobacterium johnsoniae ginsenoside f induces apoptosis accompanied by protective autophagy in breast cancer stem cells (s)-ginsenoside rg is a novel inhibitor of autophagy and sensitizes hepatocellular carcinoma to doxorubicin antiplasmodial activity of the alkaloids of peschiera fuchsiaefolia elaborating the role of natural products-induced autophagy in cancer treatment: achievements and artifacts in the state of the art autophagy-mediated chemosensitizing effect of the plant alkaloid voacamine on multidrug resistant cells tissue distribution and chemical induction of multiple drug resistance genes in rats attenuated function and expression of p-glycoprotein at blood-brain barrier and increased brain distribution of phenobarbital in streptozotocin-induced diabetic mice blood-brain barrier p-glycoprotein function in neurodegenerative disease genetic distinction of radix adenophorae from its adulterants by the dna sequence of s-rrna spacer domains encyclopedia of medicinal plants in vivo and in vitro antiinflammatory activity of saikosaponins antiviral effects of saikosaponins on human coronavirus e in vitro antinociceptive and antipyretic properties of the pharmaceutical herbal preparation, radix bupleuri in rats discussion on the adverse drug reaction monitoring by community pharmacies evidence of nutriceutical effectiveness in the treatment of osteoarthritis zingiberis rhizoma: a comprehensive review on the ginger effect and efficacy profiles ]-gingerol inhibits nitric oxide synthesis in activated j . mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions gingerols: a novel class of vanilloid receptor (vr ) agonists anti- -hydroxytryptamine effect of galanolactone, diterpenoid isolated from ginger [ ]-gingerol induces caspase dependent apoptosis and autophagy in cancer cells: drug-dna interaction and expression of certain signal genes in hela cells immunosuppressant discovery from tripterygium wilfordii hook f: the novel triptolide analog ( r)- -hydroxytriptolide (lldt- ) comparative bioavailability study of two cefixime formulations administered orally in healthy male volunteers celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-jun n-terminal kinase and suppression of pi k/akt signaling pathways celastrol ameliorates experimental colitis in il- deficient mice via the up-regulation of autophagy anti-inflammatory effects of the partially purified extract of radix stephaniae tetrandrae: comparative studies of its active principles tetrandrine and fangchinoline on human polymorphonuclear leukocyte functions anti-inflammatory effects of fangchinoline and tetrandrine anti-hyperglycemic effect of fangchinoline isolated from stephania tetrandra radix in streptozotocin-diabetic mice haemodynamic effects of chronic octreotide and tetrandrine administration in portal hypertensive rats fangchinoline targets pi k and suppresses pi k/akt signaling pathway in sgc cells perspectives on medicinal properties of plumbagin and its analogs plumbagin, a vitamin k analogue, abrogates lipopolysaccharide-induced oxidative stress, inflammation and endotoxic shock via nf-κb suppression plumbagin suppresses dendritic cell functions and alleviates experimental autoimmune encephalomyelitis antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the nf-κb signaling pathway plumbagin ( -hydroxy- -methyl- , -naphthoquinone) suppresses nf-κb activation and nf-κb-regulated gene products through modulation of p and iκbα kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents plumbagin induces apoptotic and autophagic cell death through inhibition of the pi k/akt/mtor pathway in human non-small cell lung cancer cells ze xie diao xue zhi de yan jiu jin zhan chromatographic fingerprint analysis of herbal medicines alisol b acetate, a triterpene from alismatis rhizoma, induces bax nuclear translocation and apoptosis in human hormone-resistant prostate cancer pc- cells natural product agonists of peroxisome proliferator-activated receptor γ (pparγ): a review autophagy triggered by magnolol derivative negatively regulates angiogenesis magnolol, a major bioactive constituent of the bark of magnolia officinalis, exerts antiepileptic effects via the gaba/benzodiazepine receptor complex in mice antifungal activity of magnolol and honokiol magnolol-induced h cells death via autophagy but not apoptosis gastroprotective effect of fructus evodiae water extract on ethanol-induced gastric lesions in rats the protective effects of rutaecarpine on gastric mucosa injury in rats evodiamine abolishes constitutive and inducible nf-κb activation by inhibiting iκbα kinase activation, thereby suppressing nf-κb-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion evodiamine inhibits adipogenesis via the egfr-pkcα-erk signaling pathway cytotoxic effect of evodiamine in sgc- human gastric adenocarcinoma cells via simultaneous induction of apoptosis and autophagy inhibition of ccl ´i nduced liver fibrosis by piper longum linn anti-inflammatory and antiarthritic effects of piperine in human interleukin β-stimulated fibroblast-like synoviocytes and in rat arthritis models protective effect of piper longum l. on oxidative stress induced injury and cellular abnormality in adriamycin induced cardiotoxicity in rats piperlongumine, a constituent of piper longum l., inhibits rabbit platelet aggregation as a thromboxane a receptor antagonist piperlongumine selectively kills glioblastoma multiforme cells via reactive oxygen species accumulation dependent jnk and p activation antiproliferative effects of two amides, piperine and piplartine, from piper species piperlongumine induces autophagy by targeting p signaling development and mechanism investigation of a new piperlongumine derivative as a potent anti-inflammatory agent anticancer potential of curcumin: preclinical and clinical studies curcumin and cancer: an "old-age" disease with an "age-old" solution potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases curcumin and cancer cells: how many ways can curry kill tumor cells selectively? anti-tumour and antioxidant activity of natural curcuminoids the chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane dietary curcumin supplementation counteracts reduction in levels of molecules involved in energy homeostasis after brain trauma curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an alzheimer mouse model an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use long-term treatment with danshen-gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive protein kinase c-ε/mkatp pathway in rats regulation effects on abnormal glucose and lipid metabolism of tzq-f, a new kind of traditional chinese medicine the general situation and progress of the modern research of red sage root (radix salviae miltiorrhizae) anti-inflammatory and immunomodulatory mechanism of tanshinone iia for atherosclerosis tanshinone iia inhibits lps-induced nf-κb activation in raw . cells: possible involvement of the nik-ikk, erk / , p and jnk pathways tanshinone iia protects against cardiac hypertrophy via inhibiting calcineurin/nfatc pathway calcineurin suppresses ampk-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress advances of ligusticum chuanxiong inhibition of rat vascular smooth muscle cell proliferation by extract of ligusticum chuanxiong and angelica sinensis protective effect of ligusticum chuanxiong and angelica sinensis on endothelial cell damage induced by hydrogen peroxide ocular and cardiovascular pharmacology of tetramethylpyrazine isolated from ligusticum wallichii franch scavenging effects of tetramethylpyrazine on active oxygen free radicals tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats studies on cardiotonic steroids from the skin of japanese toad pharmacology and toxicology of toad venom bufotalin from venenum bufonis inhibits growth of multidrug resistant hepg cells through g /m cell cycle arrest and apoptosis cardiac glycosides are potent inhibitors of interferon-β gene expression bufalin induces g /m phase arrest and triggers autophagy via the tnf, jnk, becn- and atg pathway in human hepatoma cells bufalin inhibits hct colon cancer cells and its orthotopic xenograft tumor in mice model through genes related to apoptotic and pten/akt pathways bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress anti-inflammatory, analgesic and antipyretic activities of the extract of gamboge from garcinia hanburyi hook f general gambogic acids inhibited growth of human hepatoma smmc- cells in vitro and in nude mice gambogic acid, a novel ligand for transferrin receptor, potentiates tnf-induced apoptosis through modulation of the nuclear factor-κb signaling pathway gambogic acid-induced g /m phase cell-cycle arrest via disturbing cdk -mediated phosphorylation of cdc /p in human gastric carcinoma bgc- cells characterization of multiple constituents in kai-xin-san prescription and rat plasma after oral administration by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry a representative prescription for emotional disease, ding-zhi-xiao-wan restores -ht system deficit through interfering the synthesis and transshipment in chronic mild stress-induced depressive rats effects of tenuifolin extracted from radix polygalae on learning and memory: a behavioral and biochemical study on aged and amnesic mice anxiolytic and sedative-hypnotic activities of polygalasaponins from polygala tenuifolia in mice preclinical evidence of rapid-onset antidepressant-like effect in radix polygalae extract polygalasaponin xxxii from polygala tenuifolia root improves hippocampal-dependent learning and memory polygalasaponin f induces long-term potentiation in adult rat hippocampus via nmda receptor activation clionosterol and ethyl cholestan- -enol isolated from the rhizome of polygala tenuifolia inhibit phosphatidylinositol -kinase/akt pathway identification of novel autophagic radix polygalae fraction by cell membrane chromatography and uhplc-(q)tof-ms for degradation of neurodegenerative disease proteins triterpenes from ganoderma lucidum induce autophagy in colon cancer through the inhibition of p mitogen-activated kinase (p mapk) a possible cross-talk between autophagy and apoptosis in generating an immune response in melanoma methanolic extract of ganoderma lucidum induces autophagy of ags human gastric tumor cells antioxidant activities of polygonum multiflorum thunb., in vivo and in vitro anti-cancer properties of anthraquinones from rhubarb myocardial protective effect of an anthraquinone-containing extract of polygonum multiflorum ex vivo anthraquinones inhibit tau aggregation and dissolve alzheimer's paired helical filaments in vitro and in cells pharmacokinetic and nephroprotective benefits of using schisandra chinensis extracts in a cyclosporine a-based immune-suppressive regime schisandra total lignin attenuates apoptosis of endoplasmic reticulum pathway to delay mouse brain aging clinical efficacy of traditional chinese medicine, suan zao ren tang, for sleep disturbance during methadone maintenance: a randomized, double-blind, placebo-controlled trial suan zao ren tang as an original treatment for sleep difficulty in climacteric women: a prospective clinical observation zizyphus jujuba and its active component jujuboside b inhibit platelet aggregation antitumor activity of jujuboside b and the underlying mechanism via induction of apoptosis and autophagy jujuboside a, a neuroprotective agent from semen ziziphi spinosae ameliorates behavioral disorders of the dementia mouse model induced by aβ - identification and clinical application of amber analysis on characteristic of kaixin powder and its similar prescriptions vitamin e succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (apo ligand) in vivo analysis of rule and clinicance of mind-calming medicine purgative genome of the long-living sacred lotus the epidermal growth factor receptor antibody cetuximab induces autophagy in cancer cells by downregulating hif- α and bcl- and activating the beclin /hvps complex autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation autophagy blockade sensitizes prostate cancer cells towards src family kinase inhibitors inhibition of autophagy augments -fluorouracil chemotherapy in human colon cancer in vitro and in vivo model the role of autophagy in cancer: therapeutic implications pharmacokinetic analysis and pharmacodynamic evidence of autophagy inhibition in patients with newly diagnosed glioblastoma treated on a phase i trial of hydroxychloroquine in combination with adjuvant temozolomide and radination (abtc ) the antidepressants maprotiline and fluoxetine induce type ii autophagic cell death in drug-resistant burkitt's lymphoma targeting tumor metabolism with -deoxyglucose in patients with castrate-resistant prostate cancer and advanced malignancies autophagy modulation as a potential therapeutic target for diverse diseases pp a blockade inhibits autophagy and causes intraneuronal accumulation of ubiquitinated proteins amp-activated protein kinase: a potential player in alzheimer's disease chronic cyclodextrin treatment of murine niemann-pick c disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression gerontopsychological studies using nai ("nürnberger alters-inventar") on patients with organic psychosyndrome (dsm iii, category ) treated with centrophenoxine in a double blind, comparative, randomized clinical trial the role of autophagy in neurodegenerative disease selective autophagy mediated by autophagic adapter proteins atg -family interacting motif crucial for selective autophagy deconvoluting the context-dependent role for autophagy in cancer autophagy as a target for anticancer therapy cooperation of liver cells in health and disease liver fibrosis a role for autophagy during hepatic stellate cell activation effects of fufang biejia ruangan pills on hepatic fibrosis in vivo and in vitro effect of bupleuri radix extracts on the toxicity of -fluorouracil in hepg hepatoma cells and normal human lymphocytes key: cord- -fm ww authors: scanes, colin g.; butler, leasea d.; kidd, michael t. title: reproductive management of poultry date: - - journal: animal agriculture doi: . /b - - - - . - sha: doc_id: cord_uid: fm ww based on data from the un's food and agricultural organization, about million metric tons of poultry meat were produced globally in . in addition, about million metric tons of eggs were produced. one of the bases for this production is the reproductive efficiency of today's poultry. this, in turn, is due to their inherent reproductive physiology, intensive genetic selection and advances in husbandry/management. the system of reproduction in males in largely similar to that in mammals except that there is no descent of testes. in females, there are marked differences with there being a single ovary and oviduct; the latter being the name of the differentiated entire müllerian duct. moreover, females produce eggs with a yolky oocyte surrounded by albumen, membranes and shell. among the most successful reproductive management techniques are optimizing photoperiod, light intensity and nutrition. widespread employment of these has allowed maximizing production. laying hens can be re-cycled toward the end egg production. other aspects of reproductive management in poultry include the following: artificial insemination (almost exclusively employed in turkeys) and approaches to reduce broodiness together with cage free (colony), conventional, enriched and free-range systems. introduction poultry production has markedly increased globally over the last years. this is summarized in table . . the increase in rate of poultry production over years is over twice the rate of increase in human population. chicken production is divided into two distinct sectors: meat and eggs. there are disparate lines of birds derived from different strains with greatly different genetics. an analogous situation exists for ducks. production of poultry meat depends on three distinct reproductive phases: with pedigree flocks undergoing intensive genetic selection for improvement. these produce grandparent stock/great grandparent stock and, following multiplication, leads to broiler breeders (or their equivalent in other poultry species). in the usa, there were million broiler breeder pullets placed for broiler egg production (and hence broiler chick) in . broiler breeder production of fertile eggs. in the usa, there were . billion hatching eggs produced in . . incubation of the fertile eggs in hatcheries to produce broiler chicks, turkey poults, ducklings and goslings. in the usa, there were million broiler chicks placed per week in august (cf. million eggs set per week) and million turkey poults hatched in . production of eggs depends on three distinct reproductive phases: . primary breeding companies with pedigree flocks undergoing intensive genetic improvement. these produce replacement pullets. in the usa, there are million re-placement pullets per year. the average number of layers in the usa in was million. in the usa, the average hen produces eggs per year. . re-cycling hens toward the end of egg production cycle. embryonic development of the reproductive system in contrast to the situation in mammals, the sex chromosomes in male birds are zz (homozygous) compared to zw (heterozygous) in females. in males, the two testes are internal and accessory organs such as the prostate and seminal vesicles are absent. the testes develop due to gene dosing with increased expression of the z-linked transcription factor gene, doublesex and mab- -related transcription factor (dmrt ). , anti-m€ ullerian hormone (amh) is synthesized and secreted by the embryonic testis with greater expression in the embryonic testes than the ovaries. e amh directs the regression of the paired m€ ullerian ducts. e in females, only the left ovary and oviduct develop in all avian species and closely related dinosaurs; the latter based on fossil evidence from the early cretaceous period. the avian oviduct is derived from the embryonic m€ ullerian duct; the former term encompassing the entire reproductive tract from infundibulum to the cloaca. regression of the right oviduct is induced by amh. parenthetically, amh also plays an important role in development of tubules in the testes. , the embryonic female gonad expresses the rate-limiting enzyme for the production of estrogens, aromatase (cyp a ) but expression is not found in the embryonic male gonads. , in turn, the estrogens, such as estradiol, induce growth of the oviduct. the egg is comprised of the yolk, yolk membranes, egg white, shell membranes and finally the egg shell. each of these components are developed along specific regions of the female reproductive tract together with the ovary. the egg yolk is a mature ovum (oocyte) that is produced by the ovary. the maturation of the ovum involves multiple processes including deposition of yolk proteins/lipids. yolk protein/lipoproteins/phosphoproteins were assigned to three categories based on centrifugation of diluted yolk: • low-density fraction with a very high lipid composition • granules composed of heavy and light chain lipovitellins, phosvitin and a yolk glycoprotein. • soluble proteins. the soluble proteins consist of the following: • a livetins (serum albumen) • b livetins (serum a -globulin containing transport proteins) • g livetins (serum g -globulin predominantly immunoglobulin y). egg yolk livetins (a, b, and g-livetin) have recently been shown to exert anti-inflammatory properties. yolk precursors: yolk precursors are synthesized in the liver. two major yolk precursors are very-low-density lipoprotein (vldl) and vitellogenin. very-low-density lipoprotein (vldl) has the following characteristics: • globular micelle-like • non-polar core of triglycerides and cholesterol esters • coated with amphiphilic mix of phospholipid, free cholesterol (fc) and twp apolipoproteins. chicken vitellogenin has been purified from plasma of estrogen treated adult male chickens. it is a dimer with a molecular weight , . it is a dimer composed of two polypeptide monomers each with a molecular weight of about , . there are about e phosphate moieties per monomer vitellogenin and the lipid component is about %. hepatic expression of vitellogenin is induced by estrogens. yolk deposition: a specific receptor is responsible for transfer of vitellogenin and verylow-density lipoprotein (vldl) across the oocyte plasma membrane to fill the oocyte with yolk. , within the oocyte, vitellogenin is cleaved proteolytically to form the yolk proteins, heavy and light chain lipovitellin ( % lipid), phosvitin and a yolk glycoprotein. these are incorporated into yolk granules. deposition of g livetins is very high in small follicles < mg, but decreases during development of large follicles. for the last four days of development of the follicles, yolk is being deposited at . cm or greater per day. once the ovum (egg yolk) has matured, ovulation is stimulated by the pituitary hormone, luteinizing hormone (lh). an extensive explanation of hormonal control of female reproduction follows. if ovulation is successful the ovum is normally received into the infundibulum. the egg white or albumin of the egg is produced by the magnum of the oviduct. the magnum is the longest section of the oviduct where the ovum spends approximately h accumulating egg white proteins. among the constituents of egg white are the following proteins: • ovalbumen - % of egg white proteins • ovotransferrin (conalbumen) % (this chelates metal ions particularly iron) • ovomucoid - % • lysozyme - . % • ovomucin e % • avidin . %. antimicrobial peptides and proteins e are present in the egg white and include the following: • gallin or ovodefensin • b-defensin • cathelicidin • cystatin -a cysteine protease inhibitor • lysozyme-a bacteriolytic enzyme • ovoinhibitor. following albumin deposition and addition of water ("plumping fluid") to the developing egg, the eggshell membrane is added in the isthmus; this taking approximately h. the eggshell membranes are % protein contains proteins including collagens, ovoalbumin, bacteriolytic enzymes such as ovotransferrin and lysozyme together with clusterin peptides and ovodefensins/defensins such as gallin. , these are also glycosaminoglycans including galactosaminoglycan. , egg shell the formation of the egg shell in the uterus/ shell gland is the final yet it is of longest in duration taking approximately h. this is due to the extensive structure of the shell. the egg shell is % inorganic (calcium carbonate). , of the remaining % (the decalcified egg shell) is % protein with the matrix phosphoproteins including the following: ovocleidin- , ovocleidin- , ovocalyxin- and osteopontin. , the fully formed egg is retained in the shell gland just distal to the vagina of the oviduct until oviposition. unlike many mammals, the testes of poultry and other birds are in the abdominal cavity. , following sexually maturation, adult male birds produce semen containing large numbers of spermatozoa (see table . ). production of spermatozoa is critical to fertilization of the ovulated ovum and, hence, the production of broiler chicks (see broiler breeder reproduction section), layer pullet chicks and turkey poults (see artificial insemination section). spermatogenesis is a complex process that is tightly regulated by neuroendocrine and endocrine mechanisms (discussed later in hormonal control of reproduction). , spermatozoa are produced in the seminiferous tubules of the testes. , each mature spermatozoan consists of an acrosome containing the nucleus that holds the dna information, attached to a microtubular flagella enabling it to be propelled from the cloaca of the hen to the infundibulum of the oviduct. once spermatozoa enter the vagina of the oviduct, they are stored in the utero-vaginal junctions or the infundibular sperm storage tubules. similar number of utero-vaginal junctions can be found in commercial broiler breeders and turkeys. spermatozoa can retain their ability to fertilize the oocyte for several weeks, however fertility begins to decline post days after insemination and is markedly lower days after insemination. hormones are critically important to the optimal functioning of the gonads, the photoperiodic stimulation of reproduction, sexual and maternal behavior and induced molting. the major androgen produced by the testes is testosterone. interestingly, it was demonstrated by berthold in , in the first endocrine study, that a testicular factor was essential to both male behaviors such as crowing, mating and aggression in chickens and to the development of the secondary sexual characteristics such as the rooster's comb and wattle. pituitary gland and reproduction: the gonads are controlled by the anterior pituitary hormones, lh and follicle stimulating hormone (fsh). these gonadotropins play a critical role in the development and maintenance of the gonads. lh stimulates production of progesterone by granulosa cells from large follicles and testosterone by leydig cells of the testes. fsh increases proliferation of granulosa cells, expression of both steroidogenic acute regulator (star) and inhibin a genes in granulosa cells and release of progesterone with the effect progressively greater with tissue from larger follicles. , in addition, prolactin can exert an inhibitory effect on the chicken ovary. hypothalamic control of gonadotropin release: there are two gonadotropin releasing hormones (gnrhs) in the chicken (cgnrh-i and cgnrh-ii) and two receptors for gnrh (cgnrhr and cgnrhr ). gnrh-ii is much more potent than gnrh-i in hens in stimulating lh release by fold. however, gnrh-ii is not detected in the median eminence. , there is high expression of gnrhr in the pituitary gland. therefore, the releasing hormone for lh is chicken gnrh-i and the receptor is cgnrhr . chicken gonadotropin-inhibitory hormone (gnih) is a peptide with -amino-acids. while gnih inhibits both the synthesis and the release of gonadotropins in chickens, the physiological relevance of gnih still requires clarification. the ovary produces the following: • estrogens, primarily estradiol. estrogens induce the following: development of the oviduct, production of yolk precursors (vldl and vitellogenin) (see above) by the liver, production of egg white proteins by the oviduct and, with androgens, formation of medullary bone (a labile source of calcium). in addition, estrogens allow the expression of female behaviors and moderate the release of luteinizing hormone (lh). • progesterone. among its roles are stimulating production of a specific egg white protein (avidin) and stimulating the release of lh. • androgens, predominantly testosterone. androgens are essential to the development of medullary bone. , ovarian hormones and growth factors also play critical roles in follicular development. for instance, activin a increases expression of both fsh and lh receptors but decreases cell proliferation of granulosa cells. moreover, development of small follicles is suppressed by epidermal growth factor receptor ligands such as transforming growth factor a. in contrast, bone morphogenetic protein enhanced responsiveness to fsh. , testicular functioning is controlled in a similar manner to the ovary. lh stimulates the leydig cells to produce testosterone. the sertoli cells produce nutrients to the maturing spermatozoa and are under the control of fsh. testosterone is produced from the leydig cells. there is seasonality of egg production in chickens when held under a natural photoperiod in the temperate zone. egg production increases markedly after the winter solstice and declines beginning prior to the autumnal equinox. the physiological basis of this annual cycle is photoperiodic stimulation of reproduction by long daylengths; these inducing the development of functioning gonads. red light is detected by photo-pigments in the hypothalamus e with the most important photoreceptor influencing the hypothalamic release of gnrh-i being red opsin. the photoperiodic mechanism involves light coinciding temporarily with the light sensitive (photo-sensitive) phase of a circadian rhythm. this leads to release of gnrh-i, synthesis and secretion of lh and fsh and, hence, gonadal resurgence. chickens pullets are reared under short daylengths ( l: d or l: d). they are transferred to longer daylengths ( l: d) at breed specific physiological ages to stimulate gonadal development. in studies where pullets were transferred to daylengths of l: d or l: d, plasma concentrations of lh did not increase, but marked increases in plasma concentrations of lh daylengths were observed with daylengths l: d or greater. perhaps surprisingly, daylengths were interpreted differently depending on the previous photoperiod. transfer of pullets from photoperiods of either l: d or l: d to l: d were followed by, respectively increases and decreases in plasma concentrations of lh. thus, the same photoperiod can be interpreted as either photostimulatory or photoinhibitory. commercially, pullets are brought into lay by increasing both the photoperiod and the light intensity (table . ). there are breed and genetic line specific differences between ages at which photostimulation is performed (see table . ). egg production in broiler breeder hens is improved when photostimulation is delayed from to weeks old. there is also evidence that it may be advantageous to delay photostimulation of broiler breeders to weeks old. turkeys: turkeys come into lay with increasing daylengths. there are seasonal differences (see table . ). there is a rapid increase in plasma concentrations of both lh and fsh when males and females are exposed to long day-lengths. photorefractoriness is the loss of the ability to respond to the stimulatory effects of long photoperiods. photorefractoriness can be "broken" by re-exposure to short day-lengths. this is seen in turkeys with prolonged exposure to long day-lengths with the signs of photorefractoriness being decreased egg production and molting. , the decline in egg production during the production cycle of chickens might also be attributed to photorefractoriness. indeed, there is greater sensitivity of older hens to reduced daylength with an over % decrease in egg production in weeks old hens compared to a % decline in weeks old hens. moreover, plasma concentrations of lh were only decreased in the older hens. while, light intensities greater than lux are required for photoperiodic induction of egg production, considerably higher light intensities are employed in commercial poultry production (table . ). for instance, in broiler breeders, light intensity is increased from before photostimulation about lux in the pullet phase to > lux after photostimulation at or weeks of age. other effects of light intensity: light intensity has other effects. for instance, increasing light intensity in immature pullets is associated with increased plasma concentrations of fsh. moreover, the ability of a short pulse of light to photostimulate chickens is influenced by light intensity. in addition, the ratio of the light intensity during the subjective day to that during the subjective night is important in entraining the rhythm of oviposition. nutrition and reproductive management overview in poultry, nutrition is integrally linked to the hypothalamo-pituitary-gonadal axis. it has been known for years that egg production in hens stops quickly following fasting. the administration of mammalian or avian gonadotropin restores, albeit partially, egg production in starved hens ; this suggesting that underlying cause is the lack of pre-ovulatory lh surges. fasting is followed rapidly by decreases in plasma concentrations of lh, body weight together with precipitous declines in ovarian and oviductal weights. similarly, production of eggs and plasma concentrations of lh decrease quickly after reducing calcium or sodium in the diet of hens. , in young chickens, protein deficiency also has been demonstrated to rapidly cause atrophy of gonads, decrease circulating concentrations of lh and depress responsiveness to gnrh. the nrc nutrient requirements of poultry has been invaluable to researchers and important to the poultry industry recommending minimum levels of nutrients in the feed. these requirements are based on the published research prior to the development of the specific edition of nrc nutrient requirements of poultry. primary breeders publish age specific nutrient recommendations for each of their genetic lines. , e examples of such recommendations for energy, protein, lysine and calcium are summarized in table . . it is clear that the recommendations for calcium content in laying hen diets are very high due to the demands of eggshell formation. moreover, the levels of calcium in diets are higher as the production cycle proceeds presumably due to the increasing size of the egg. this is the case irrespective of whether the recommendations are for layer or broiler breeder hens (table . ). feedingprogramsare designed toachievetarget body weights throughout growth with markedly lower weights at weeks old (ad libitum fed . kg; restricted to achieve target weights . kg). these programs not only decrease the feed needs of the broiler breeder but also reduce mortality and increase egg production. broiler breeder pullets can be fed nutrient restricted diets by programs where birds are fed daily or skip-a-day or feeding four or five or six days per week. there were greater body weights and lower liver weights in week-old pullets fed daily than skip-a-day despite the birds despite their receiving the same amount of feed. there were also higher hepatic concentrations of both lipid and glycogen together with the expression of lipogenic enzymes in skip-a-day fed pullets. , mench considered that feed restrict of broiler breeder females may be associated with physiological stresses and increased incidence of abnormal behaviors. the severity of feed restriction needs to be progressively greater with generation exhibiting increased size/growth rates in broiler chickens. the strategy in feed restriction is to reduce caloric intake while maintaining amount of feed consumed. this goal is achieved by increasing the percentage of crude fiber in the diet. skip-a-day programs have been considered helpful in increasing uniformity within flocks and reducing abnormal behaviors. induced molting or re-cycling to increase egg production hens can be induced (or forced) to molting at the end of their laying cycle resulting in improved egg production at a lower cost than using replacement pullets (fig. . ). in the usa, . % of laying hens are molted (re-cycled) each month. this process can involve severe nutritional restriction including starvation and/or withholding water and/or reduction in photoperiod. , e alternate methods of induced molting include an extremely high zinc diet ( , ppm) followed by a conventional layer feed beginning at day and sodium/chloride-deficient diets. , broiler breeders are rarely molted, but under certain circumstances, molting may be performed. most broiler breeder molt programs are achieved by restricting feed consumption and supplementing water containing essential micronutrients allowing utilization of fat stores. this reduced fat stores such that hens achieve a more pullet-like body composition before being photostimulated again. in addition to feed restriction, to induce molting in broiler breeder hens, the daylength is decreased to l: d and light intensity is reduced. , production levels with molted broiler breeders are about % less than their previous laying cycle. , the lower production level appears to be due to there being fewer follicles after a forced molt compared to their initial lay cycle. the terms, forced or induced molt, are open to question as it presumes that molting (loss of feathers) causes rejuvenation of reproduction performance. molting occurs after resumption of normal feeding and is temporally shifted from ovarian recrudescence (see fig. . ). when feed is withdrawn for days and water withdraw for days, egg production had completely ceased by days (see fig. . ). molting occurred after the resumption of feeding and there were concomitant increases in circulating concentrations of t and corticosterone (see fig. . ). circulating concentrations of lh, estradiol (e ) and progesterone were lower in molting hens than in laying hens or fully recycled hens. the effects of an industry molting system on organ weights together with circulating concentrations of ions and corticosterone were evaluated. the approach was to combine salt and protein deprivation this approach is effective in stopping egg production and decreasing body weight and organ weights. it is accompanied by reductions in circulating concentrations of sodium and chloride (see table . ). the egg production cycle of about weeks of age to e weeks old can be extended with a molt between and weeks old. the physiological mechanism underlying induced molting included decreased release of gnrh from the median eminence and consequently lack of the pre-ovulatory lh surge. ovulation completely ceased with days of feed withdrawal. plasma concentrations of lh and progesterone were decreased with days of feed withdrawal. the gnrh content of the median eminence was similarly decreased but not until days of feed withdrawal. there are also decreases in the number of gonadotropes expressing lh. oviductal regression occurs due to lack of estrogens and is accompanied by increased expression of peptidases with, for example, expression of the peptidase, cathepsin l. it is questioned whether re-cycling/forced molting of hens is consistent with one of the "the five freedoms," namely: " . freedom from hunger and thirst" and the need for "ready access to fresh water and a diet to maintain full health and vigor." there is both evidence for and against the process being physiologically stressful. an indicator of stress, heterophil: lymphocyte ratio, was increased after days after feed withdrawal to a forced molt. similarly, induction of molting was accompanied by increased the percentage of heterophils (day and ) and of eosinophils in one study. however, the evidence of molt induction influencing plasma concentration of the stress hormone, corticosterone, is circumspect. no changes in plasma concentrations of corticosterone in force molted hens were reported (also see table . ). , in laying hens subjected to induced molting, plasma concentrations of corticosterone were higher on day in hens subjected to complete induced molting or re-cycling to increase egg production feed withdrawal than those in which molting was induced by a high zinc diet or in a wholegrain barley diet. there are also shifts in immune responses including cytokine expression. during starvation induction of molting, there is a reduced delayed type hypertensive response in the wattle. feed withdrawal for e days is associated with increased oviductal expression of the following: interleukin (il)- , il and interferon g. similarly, expression of cytokines in response to an endotoxin challenge increased in the uterus of molting compared to laying hens. other aspects of reproductive management in poultry artificial insemination (ai) is used very widely in turkey production but not in the production of layer pullets or broiler chicks. hens are inseminated weekly with diluted semen (for spermatozoa concentrations in turkey ejaculate see table . ). while the technique is labor intensive, it takes advantage of the presence of spermatozoa storage glands in the oviduct. these release spermatozoa after oviposition such that newly ovulated ovum can be fertilized prior to the addition of egg white, membranes and the she l. the ai techniques that were developed for poultry have been applied to the conservation of endangered wild birds when they fail to mate in captive conditions. ai can be employed alone or coupled with cryopreservation. it is advantageous to suppress broodiness (maternal behavior) and the consequent decrease in egg production in poultry. broodiness has been effectively genetically eliminated in commercial chickens irrespective of whether broilers or laying hens. approaches that could reduce the unfavorable effects of broodiness in turkeys include: genetics and active or passive immunization with antisera reducing available prolactin. administration of antisera to turkey prolactin increased egg production in turkeys. an alternate approach is active immunization. similarly, egg production was markedly increased in turkey hens actively immunized against vip (with vip being conjugated to keyhole limpet hemocyanin) due to impairment of prolactin release and consequently reduction in incubation behavior(s). , it was hoped that antisera could also enhance reproduction in males. young male chickens exhibited multiple changes after active immunization against both inhibin and vip. these included increases in semen volume and spermatozoa mobility together with spermatozoa concentrations. in contrast, this improvement in reproductive performance was not seen in old roosters actively immunized against both inhibin and vip. antibody approaches do not appear to be widely adopted due to expense of registration. cage free (colony), conventional, enriched and free-range systems both egg production and bird health are influenced by the systems employed to maintain (chicken) hens. egg production was greater in the hens in conventional caging compared to an aviary system. much lower (w %) mortalities were reported in hens in conventional caging compared to an aviary system. moreover, there was a greater incidence of keel deformities in hens in a cage free system compared to those in conventional cages. there are also differences in leg bone characteristics with hens under the cage free conditions having increased cortical cross-sectional area and cortical density of their humerus and tibia compared to those maintained in conventional cages. moreover, there was there was greater stiffness of both humerus and tibia and increased percentage ash in the humerus in hens in a cage free system. , . reproductive management of poultry however, in another study, there were little differences between tibia and humerus parameters between hens in cage-free or conventional cage systems. in laying hens at the end of the laying cycle, there were differences in bone characteristics depending on the environment under which the pullets were raised. production has been compared between lohmann brown layer hens in convention layer housing, enriched convention layer housing and free-range systems. perhaps unexpectedly, egg production was greater in hens in a freerange system (average production over cycle . %) compared to conventional layer housing ( . % with enrichment and . % without). in contrast, there was greater feed intake and higher feed conversion ratios in hens in a freerange system. in another study, egg production was greater in hens with an enriched environment compared to conventional caging. broiler breeders are prone to ovulation issues. examples include internal ovulation and double ovulation of a (double "yolked" eggs). the latter is due to pair of large yellow follicles in the follicular hierarchy. duplicate ovulation is considered to be a result of "over-stimulated" hens coming into production or pullets not ready for light stimulation or to over-feeding. , internal lay of the ovum is followed the constituents of ovum being absorbed, but can result in a substrate for bacterial growth like escherichia coli. uses of components of eggs yolk as source of antibodies a major soluble protein type in yolk are the g livetins or the immunoglobulins (discussed section above). yolk igy from chicken immunized against pathogens (rotaviruses, bovine coronavirus, enterotoxigenic e. coli and salmonella spp.) offers potential to replace antibiotics in livestock production. this may be particularly important with the restriction of antibiotic use and growing consumer resistance to their use. a meta-analysis of studies determine that hyperimmune yolk igy was effective against diarrhea in piglets. similarly, oral administration of igy was efficacious in poultry and calves. , other products of eggs egg white and its constituents have important functional properties. examples include the following. avidin is used in multiple biochemical tests. lysozyme (bacteriostatic and bactericidal) has been used as a preservative, for instance, in cheese making. egg shell membranes or egg shell membrane powder have been used as natural bandage for burns and injuries in traditional asian medicine. egg shell membrane powder and its carbohydrate constituents have been recently been demonstrated to exert anti-inflammatory effects , supporting their effectiveness. in the chick embryo, primordial germ cells migrate from the germinal crescent to the genital ridge ultimately becoming male or female gametes. the primordial germ cells carry the genetics to the next generation and, thus, if this can be manipulated we have a multi-generational method of producing transgenic animals. chicken primordial germ cells can be obtained from blood of chick embryos and maintained in culture. this has been used to both insert and/or "knock out" genes. transgenic hens can be used as bioreactors to produce pharmaceutical proteins in their egg white when oviduct specific promoters are used. this concept has been applied to the production of human erythropoietin and tumor necrosis factor receptors. usda national agricultural statistics service (nass) un food and agriculture organization e faostat usda national agricultural statistics service (nass) chicken and eggs summary the avian zlinked gene dmrt is required for male sex determination in the chicken the avian ovary and follicle development: some comparative and practical insights anti-m€ ullerian hormone is required for chicken embryonic urogenital system growth but not sexual differentiation overexpression of anti-m€ ullerian hormone disrupts gonadal sex differentiation, blocks sex hormone synthesis, and supports cell autonomous sex development in the chicken identification, expression, and regulation of anti-m€ ullerian hormone type-ii receptor in the embryonic chicken gonad ovarian follicles shed new light on dinosaur reproduction during the transition towards birds expression of p ( alpha) hydroxylase and p aromatase genes in the chicken gonad before and after sexual differentiation sites of estrogen receptor and aromatase expression in the chicken embryo precursorproduct relationship between chicken vitellogenin and the yolk proteins: the kda yolk plasma glycoprotein is derived from the c-terminal cysteine-rich domain of vitellogenin ii serum proteins and the livetins of hen's-egg yolk anti-inflammatory effects of egg yolk livetins (a, b, and g-livetin) fraction and its enzymatic hydrolysates in lipopolysaccharide-induced raw . macrophages relationships of plasma and very-low-density lipoprotein lipids and subfractions with abdominal fat in chickens vitellogenin synthesis in the avian liver. vitellogenin is the precursor of the egg yolk phosphoproteins estradiol-induced synthesis of vitellogenin. iii. the isolation and characterization of vitellogenin messenger rna from avian liver a single chicken oocyte plasma membrane protein mediates uptake of very-low-density lipoprotein and vitellogenin the receptor for yolk lipoprotein deposition in the chicken oocyte changes in specific sequestration of protein during transport into the developing oocyte of the chicken the macromolecular composition of hen's egg yolk at successive stages of maturation modeling ovarian follicle growth in commercial and heritage single comb white leghorn hens roles of body weight and feed intake in ovarian follicular dynamics in broiler breeders at the onset of lay and after a forced molt reproduction in the female the chicken egg white proteome gallin; an antimicrobial peptide member of a new avian defensin . reproductive management of poultry family, the ovodefensins, has been subject to recent gene duplication characterization of egg white antibacterial properties during the first half of incubation: a comparative study between embryonated and unfertilized eggs chemical composition of chicken eggshell and shell membranes chicken egg shell membrane associated proteins and peptides extraction of glycosaminoglycans from chicken eggshell galactosaminoglycan composition in chicken eggshell the eggshell: structure, composition and mineralization functional proteins and peptides of hen's egg origin reproduction in the male comparisons of sperm storage tubule distribution and number in strains of mature broiler breeders and in turkey hens before and after the onset of photostimulation analysis of sperm storage ability using duration of fertility in hens effects of mammalian lh, cyclic amp and phosphodiesterase inhibitors on steroidogenesis, lactate production, glucose uptake and utilization by avian granulosa cells genistein affects testosterone secretion by leydig cells in roosters (gallus gallus domesticus) follicle-stimulating hormone regulation of inhibin alpha-and beta (b)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells granulosa cell responsiveness to follicle stimulating hormone during early growth of hen ovarian follicles effects of nonglycosylated and glycosylated prolactin on basal and gonadotropinstimulated steroidogenesis in chicken ovarian follicles sex differences in the lh responses to chicken lhrh-i and -ii in the domestic fowl physiological roles of chicken lhrh-i and -ii in the control of gonadotrophin release in the domestic chicken distribution of luteinizing hormone-releasing hormones i and ii (lhrh-i and -ii) in the quail and chicken brain as demonstrated with antibodies directed against synthetic peptides the chicken type iii gnrh receptor homologue is predominantly expressed in the pituitary, and exhibits similar ligand selectivity to the type i receptor a novel avian hypothalamic peptide inhibiting gonadotropin release gonadotrophin inhibitory hormone depresses gonadotrophin and follicle-stimulating hormone subunit expression in the pituitary of the domestic chicken the role of hormones in the regulation of bone turnover and egg shell calcification effect of activin a and inhibin a on expression of the inhibin/activin beta-b-subunit and gonadotropin receptors in granulosa cells of the hen bone morphogenetic protein- enhances gonadotrophindependent progesterone and inhibin secretion and expression of mrna transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells photoperiodic control of reproduction in the domestic hen the role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens red light is necessary to activate the reproductive axis in chickens independently of the retina of the eye early photo-stimulation at the recommended body weight reduced broiler breeder performance effect of strain, feed allocation program, and age at photostimulation on reproductive development and carcass characteristics of broiler breeder hens the relationship of plasma thyroid hormone and prolactin concentrations to egg laying, incubation behavior, and molting by female turkeys exposed to a one-year natural daylength cycle critical day lengths for egg production and photorefractoriness in the domestic turkey effect of photoperiod on gonadotrophin concentrations in domestic fowl neuroendocrine control of reduced persistence of egg-laying in domestic hens: evidence for the development of photorefractoriness light intensity and age at first egg in pullets breeder management supplement: fast feather female: cobb light intensity can influence plasma fsh and age at sexual maturity in domestic pullets time of lay in hens exposed to intermittent lighting entrainment of oviposition in the fowl using bright and dim light cycles the induction of ovulation in starving pullets using mammalian and avian gonadotropins the effect of shortterm starvation on pituitary and plasma lh, plasma estradiol and progesterone, and on pituitary response to lh-rh in the laying hen (gallus domesticus) physiological changes in caged layers during a forced molt. . body temperature and selected blood constituents response of laying hens to low salt diet effects of gradation in protein-calorie restriction on the hypothalamo-pituitary-gonadal axis in the young domestic fowl nutrient requirements of poultry publication: history and need for an update w- commercial layer management guide ross nutrition specifications ross nutrition specifications effects of low dietary protein and different allocations of food during rearing and restricted feeding after peak rate of lay on egg production, fertility and hatchability in female broiler breeders an examination of the role of feeding regimens in regulating metabolism during the broiler breeder grower period. . hepatic lipid metabolism feed restriction significantly alters lipogenic gene expression in broiler breeder chickens broiler breeders: feed restriction and welfare. world's implications of changes to commercial broiler and broiler breeder body weight targets over the past years. world's the effect of dietary alterations during rearing on growth, productivity, and behavior in broiler breeder females the effects of alternative forced-molting methods on the performance and egg quality of commercial layers molting layers-alternative methods and their effectiveness review: feed withdrawal and non-feed withdrawal moult. world's recrudescence mechanisms and gene expression profile of the reproductive tracts from chickens during the molting period the potential use of dietary salt deficiency for the force resting of laying hens effectiveness of low sodium diets for recycling of egg production type hens changes in plasma thyroid hormones, luteinizing hormone (lh), estradiol, progesterone and corticosterone of laying hens during a forced molt morphological and physiological changes in various stages of non-feed withdrawal molt (ph.d. dissertation) median eminence and anterior pituitary degradation of luteinizing hormone releasing hormone in hens undergoing changes in luteinizing hormone secretion cell proliferation and apoptosis in the anterior pituitary of chicken during inhibition and resumption of laying expression and regulation of avian cathepsin l in the oviduct during molting farm animal welfare council. five freedoms the effects of long-term caging and molt of single comb white leghorn hens on heterophil to lymphocyte ratios, corticosterone and thyroid hormones physiological changes in caged layers during a forced molt. . leucocytes and packed cell volume effects of different forced molting methods on postmolt production, corticosterone level, and immune response to sheep red blood cells in laying hens effects of induced moulting on immune responses of hens cytokines in reproductive remodeling of molting white leghorn hens effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokines and the subsequent recruitment of immunocompetent cells in the oviduct of laying and molting hens success in artificial insemination: quality of semen and diagnostics employed implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species genetic loci inherited from hens lacking maternal behaviour both inhibit and paradoxically promote this behaviour prevention of the expression of incubation behaviour using passive immunisation against prolactin in turkey hens (meleagris gallopavo) increased egg production by active immunization against vasoactive intestinal peptide in the turkey (meleagris gallopavo) active immunization with vasoactive intestinal peptide in turkey hens the effect of active immunization against vasoactive intestinal peptide and inhibin on reproductive performance of young white leghorn roosters the effect of active immunization against vasoactive intestinal peptide (vip) and inhibin on reproductive performance of aging white leghorn roosters impact of commercial housing system and nutrition on egg quality parameters comparisons of bone properties and keel deformities between strains and housing systems in end-of-lay hens housing conditions alter properties of the tibia and humerus during the laying phase in lohmann white leghorn hens influence of age and housing systems on properties of tibia and humerus of lohmann white hens : bone properties of laying hens in commercial housing systems egg production and welfare of laying hens kept in different housing systems (conventional, enriched cage, and free range) production of recombinant tumor necrosis factor receptor/fc fusion protein by genetically manipulated chickens colibacillosis in caged layer hens: characteristics of the disease and the aetiological agent effect of chicken egg yolk antibodies (igy) against diarrhea in domesticated animals: a systematic review and meta-analysis passive immunization with hyperimmune egg-yolk igy as prophylaxis and therapy for poultry diseases-a review the extracellular matrix of eggshell displays anti-inflammatory activities through nf-kb in lps-triggered human immune cells processed eggshell membrane powder regulates cellular functions and increase mmp-activity important in early wound healing processes germline transmission of genetically modified primordial germ cells competitive bioreactor hens on the horizon production of recombinant human erythropoietin/fc fusion protein by genetically manipulated chickens performance and profitability of second-cycle laying hens as influenced by body weight and body weight reduction during molt sperm production and testicular development of broiler breeder males reared on shortened growth cycles biochemical characteristics and sperm production of turkey semen in relation to strain and age of the males management guidelines turkey breeders breeder management supplement: female: cobb reproductive management of poultry we are grateful to ann (ryn) laster mcdonald for her invaluable input. key: cord- - usqpy j authors: hassan, atef a.; mansour, mogda k.; el hamaky, ahmed m.; sayed el ahl, rasha m.; oraby, noha h. title: nanomaterials and nanocomposite applications in veterinary medicine date: - - journal: multifunctional hybrid nanomaterials for sustainable agri-food and ecosystems doi: . /b - - - - . - sha: doc_id: cord_uid: usqpy j nowadays, nanotechnology has made huge, significant advancements in biotechnology and biomedicine related to human and animal science, including increasing health safety, production, and the elevation of national income. there are various fields of nanomaterial applications in veterinary medicine such as efficient diagnostic and therapeutic tools, drug delivery, animal nutrition, breeding and reproduction, and valuable additives. additional benefits include the detection of pathogens, protein, biological molecules, antimicrobial agents, feeding additives, nutrient delivery, and reproductive aids. there are many nanomaterials and nanocomposites that can be used in nanomedicine such as metal nanoparticles, liposomes, carbon nanotubes, and quantum dots. in the near future, nanotechnology research will have the ability to produce novel tools for improving animal health and production. therefore, this chapter was undertaken to spotlight novel methods created by nanotechnology for application in the improvement of animal health and production. in addition, the toxicity of nanomaterials is fully discussed to avoid the suspected health hazards of toxicity for animal health safety. significant economic importance for the security of farmers (thornton, ) whereas the products of these animals such as milk, meat, hides, and leather are essential for human populations (patel et al., ) . nowadays, microbial infections cause several health hazards to humans and animals. despite progressive advances in diagnosis and therapeutic technologies, microbial spoilage still has a major economic impact on the world's food supplies and is considered the main source of outbreaks of human and animal diseases (taylor and rodwell, ) . in the ongoing race between the emergence of drug resistance and the development of novel antimicrobial agents, microbes appear to be the targets of several studies (hassan et al., a) . in addition, nanotechnology helps in the development of new diagnostic tools and treatments that improve the quality of life of veterinary animals. some studies employed nanomaterials in disease treatment such as african animal trypanosomiasis (kroubi et al., ) as well as foot and mouth disease in cattle (greenwood et al., ) and sheep (mohanty et al., ) . recently, several studies evaluated the use of nanobiosensors in the detection of estrus, hormone levels, and metabolite profiles (monerris et al., ; sagadevan and periasamy, ) . also, nanomaterial has the ability to preserve gonadal tissues, sperm, oocytes, and embryos (saragusty and arav, ) . meanwhile, quantum dots (qds) improve the understanding of mammalian spermatozoon and oocyte movements as well as their interactions in a different physiological setting of fertility, as detected by hill and li ( ) . furthermore, the nanocomposites can make products and applications for obtaining efficient results in treatments and diagnosis (gordon and sagman, ) . also, the composites of drug molecules with nanomaterials elevated the efficacy of drug solubility and passed through the blood supply to the targeted affected tissues . moreover, several nanocomposites can be used in various applications such as nanoshells to destroy cancer cells, alumino-silicate nanoparticles to reduce bleeding, carbon nanoparticles as sensors and for drug delivery, gold nanoparticles (au nps) for diagnosis, silver nanoparticles (ag nps) as antimicrobial agents, and iron oxide nanoparticles to improve mri imaging (chakravarthi and balaji, ; hassan et al., b) . additionally, other tools such as microfluidics, nanomaterial, and bioanalytical nanosensors have the potential to solve many challenges in the diagnosis and treatment of diseases (meena et al., ) . therefore, this chapter was undertaken to spotlight the recent advances in nanotechnology and their application in the field of veterinary medicine. also, this review includes a brief discussion of the use of nanomaterials and nanocomposites in human and animal science. moreover, practical applications and ways to overcome the suspected toxicity of nanomaterials in veterinary medicine are fully discussed. there have been rapid advances in the fields of nanomaterials and nanocomposite applications in biomedical science related to human and animal health. a brief illustration of the beneficial fields of nanomaterials and nanocomposite application in animal science is shown in fig. . . the metal nanomaterials constitute effective antimicrobial agents against common pathogenic microorganisms (gong et al., ; hassan et al., a, b; nabawy, ) . in addition, metal nanoparticles have greater useful advantages than largesized metal particles (stankic et al., ) . recently, abd-elsalam et al. ( ) reported that metal nanoparticles such as ag, silver oxide (ag o), titanium dioxide (tio ), silicon (si), copper oxide (cuo), zinc oxide (zno), au, calcium oxide (cao), and magnesium oxide (mgo) have potential antifungal activity, whereas most metals provide novel antimicrobial potential (taylor et al., ) . another study reported that the minimum inhibitory concentration (mic) of zinc nanoparticles (zn nps) was ( . - μg/ml) (hosseini et al., ) . however, hassan et al. ( a) showed the antimicrobial potential of zn nps against the dermatological infection of buffaloes through species such as trichophyton verucosum, dermatophilus congolensis, and staphyloccocus aureus. similarly, the authors detected the antimicrobial potential of zn nps against esherichia coli, s. aureas, candida albicans, and aspergillus flavus, which caused mastitis in cattle. regarding ag nps applications, they have several potential uses such as their antimicrobial, antiinflammatory, and anticancer properties (ge et al., ; hassan et al., a hassan et al., , . currently, ag nps showed antimicrobial effects against fungal causes of skin diseases in bovines (hassan et al., b) ; fungal and bacterial diarrhea and mastitis infection in buffaloes (hassan et al., a) ; and mastitis in goats (gurunathan et al., ; yuan et al., ) . however, refai et al. ( ) detected the antimicrobial effect of ag nps against s. aureus and c. albicans. meanwhile, the ag nps significantly reduced the viability of coliform bacteria in ilea contents of piglets (fondevilaa et al., ) . but in another study, no effects of enteric bacteria were observed in piglets administered ag nps (sawosz et al., ) . furthermore, hassan et al. ( b) detected the antifungal potential of the ag nps against c. albicans and trichophyton mentagrophytes. ag nps also showed the ability to inhibit the growth of fungal cells, particularly c. albicans and dermatophytes (lara et al., ) . kim et al. ( ) showed that the mic of ag nps against pathogenic candida spp. was mg/ml of ag nps had higher potential than crud silver. the antifungal potential of nanosilver against t. mentagrophytes and the candida species was detected by kim et al. ( ) . moreover, the application of nanosilver in the biostabilization of footwear materials ( % solution) inhibited the growth of some mold (falkiewicz-dulik and macura, ) . nanoparticles of iron oxide (fe o nps) exhibited strong antimicrobial activity sawai, ) . currently, fe o nps are known to have antifungal potential against the growth of mycotoxigenic a. flavus while also altering their ability to produce aflatoxin (ahmad et al., ; lopes et al., ; nabawy et al., ) . meanwhile, hassan et al. ( c) detected the antifungal effect of fe o nps against c. neoformance that was recovered from respiratory diseases in cattle. similarly, nabawy et al. ( ) and mouhamed et al. ( ) detected the antifungal potential of fe o nps against the mycotoxigenic aspergillus species that was isolated from feeds. in another study, hassan et al. ( b) yielded the efficient antimicrobial potential of fe o nps against trichophyton verrucosum, t. mentagrophytes, and the bacteria of dermatophilus sp., which is isolated from bovine skin infections. abd el-tawab et al. ( ) discovered that fe o nps have an more of an antifungal effect than fe o nps. nabawy et al. ( ) and mouhamed et al. ( ) detected that aflatoxin b (afb ) and ochratoxin production by respective fungal isolates was significantly diminished until complete inhibition by increasing the dose treatment with fe o nps. moreover, the antimicrobial action of metal nanoparticles was suggested as being due to disrupting and penetration of the cell membrane of microorganism, damage and rupture of the cell wall and leakage of cytoplasm contents (gajbhiye et al., ; hassan et al., hassan et al., , a . in another study, khandelwal et al. ( ) determined that ag nps were able to prevent the penetration of a ruminant virus into animal cells by the destructive action of nanomaterials on viral cells. in another study, the antibacterial effects of zn nps against gram-positive and gram-negative bacteria occurred due to the penetration of nanoparticles into the cell membrane of bacteria and led to cell death (arabi et al., ; auffan et al., ; rosi and mirkin, ) . furthermore, the antibacterial activity of zno-nps due to their interaction with bacterial cells caused microbial cell injury and could enter the cells ( jin et al., ; stoimenov et al., ; zhang et al., ) . currently, we evaluate the synthesis and characterization of some metal nanoparticles such as zn nps, fe o nps, ag nps, and selenium nps and their antimicrobial potential against the viability of microbial causes of cow mastitis, abortion, and diarrhea ( fig. . a-c). the viability and growth of bacterial cells (e. coli and s. aureas) and fungal spores (a. flavus and c. albicans) significantly decreased as the used concentration on metal nanoparticles increased. when these isolates were observed by scanning electron microscopy (sem), they showed complete destruction and death of the treated microbial cells (fig. . d) . recently, bai et al. ( ) detected that au nps decreased the cell viability of pathogenic bacteria in chicken. meanwhile, the antibacterial potential of au nps against the bacillus species and e. coli was detected by zhou et al. ( ) . recently, mohamed et al. ( ) detected the antibacterial activity of au nps against corynebacterium pseudotuberculosis, which caused chronic caseous lymphadenitis and resulted in a major economic loss in goats and sheep. on the other hand, regarding ( ) after treatment; c. albicans ( ) before and ( ) after treatment; e. coli o :h ( ) before and ( ) after treatment, and of s. aureus ( ) before and ( ) after treatment with metal nanoparticles. the single-walled carbon nanotubes (swcnts), it was detected that as the length decreased, the antibacterial activity increased . meanwhile, suspensions such as phosphate-buffered saline decreased swcnt toxicity and their interactions with bacterial cells, hence reducing the aggregation of cells ). however, graphene oxides (gos) exhibited stronger antimicrobial potential against food-borne bacteria . boor ( ) detected the antimicrobial potential of cnts against the activity of bacteria and fungi. in another study, the antimicrobial potential of swcnt was investigated against bacteria, which caused the failure of bacterial film formation and caused cell death (kang et al., ) . swcnts are also toxic to bacterial cells that have the ability to interrupt bacterial membranes . carbon nanotubes (cnts) showed selective antibacterial agents (fernandez-lopez et al., ) and different forms of graphene materials against listeria monocytogenes and salmonella species (ye et al., ) . however, swcnts have potential antiviral activity against reovirus (gurunathan et al., ) . similarly, ye et al. ( ) detected the antiviral activity of go against the pseudorabies virus dna and the porcine epidemic diarrhea virus. furthermore, other cnts (fullerenes) and their derivatives showed antiviral activity against hiv (friedman et al., ; sijbesma et al., ) and the vesicular stomatitis virus (kaesermann and kempf, ) . the antifungal potential of chitosan and cs nps against fusarium graminearum colonies was evaluated, may be used as a novel nano-biopesticides (kheiri et al., ) and a growth inhibitor for rhizopus sp. and aspergillus niger (chookhongkha et al., ) . cs nps also have antimicrobial activity against staphylococcus saprophyticus and e. coli (soutter, ) . in addition, nanosensors have antibacterial and antiviral potential, even if used in a very small amount, which improves the feedstock (kuswandi et al., ; vyas et al., ) . furthermore, nanoscience produces a novel nontoxic antimicrobial agent against various pathogens, including brucella, mycobacterium bovis, s. aureus, and rhodococcus equi (muktar et al., ) as well as fungi and bacteria that cause mastitis and aflatoxins in feeds (hassan et al., a in cattle. over the past decade, various mycotoxins were detected as the main contaminant for foods and feed products, infested with mycotoxigenic fungi which have the potential ability to produce one or more types of mycotoxins. mycotoxins are carcinogenic and make other adverse effects on human and animal health. hence, the degradation of mycotoxins can improve animal health and production. recently, it was reported that zno nps have antifungal potential against the growth of mycotoxigenic a. flavus while altering its ability for aflatoxin production (ahmad et al., ; hassan et al., a; lopes et al., ; nabawy, ) . meanwhile, hassan et al. ( a) demonstrated the ability of zinc nps to inhibit the growth of mycotoxigenic molds and respective mycotoxin production (aflatoxins, ochratoxins, fumonisin b ) at the concentrations of , , and μg/ml of zno nps, respectively. aflatoxigenic a. flavus strains that require higher concentration of metals nanoparticles ( μg/ ml) than nonaflatoxigenic strains ( μg/ml) to inhibit their growth in animal feeds and aflatoxins production (nabawy et al., ) . similarly, mouhamed et al. ( ) reported that zn nps inhibit the growth of ochratoxigenic mold in feeds. moreover, the inhibition of microbial growth by metal nanoparticles varies according to their particle size and dose levels of treatment (shawky et al., ; violeta et al., ) . the first step in the degradation of mycotoxins by metal nanomaterials, hassan et al. ( a hassan et al. ( , b, successfully eliminated the carcinogenic effects of aflatoxins on vital organs such as the liver and kidney of rabbits by the addition of zn nps in the feed. similarly, abd el-fatah et al. ( ) detected the ability of zn nps to eliminate the aflatoxicosis carcinogenic effects in rats. however, zno nps can cause toxicity to vital internal organs in sheep (allen et al., ) . similarly, the oral administration of zn nps in mice resulted in toxicity to their vital organs (wang et al., a) . nowadays, researchers detected that the treatment of trichothecenes-producing fusarium poae by zn nps and ag nps resulted in the inhibition of their growth viability as well as trichothecenes mycotoxin production while decreasing the density and quantity of biosynthetic genes as detected by rt-pcr. we also reported that the nanoemulsion of cinnamon and olive oils caused the complete inhibition of mycotoxigenic fusarium growth and mycotoxin production at concentrations of %- %. zia-jahromi ( , ) reported the ability of ag nps to degrade aflatoxin in broiler chickens feed, ag nps are significantly elevated body weight gain and feed consumption. the significant application of magnetic nanoparticles (mnps) in toxin degradation through dehydroxylation of the toxin (mishima et al., ) . moreover, gibson et al. ( ) modified nanodiamond substrates in carboxylation, hydrogenation, and hydroxylation for immobilization of the carcinogenic effects of mycotoxins, particularly afb and ochratoxin a (ota). currently, the antimicrobial potential of cnts against aflatoxigenic a. flavus and the prevention of aflatoxin production in feeds were investigated (hassan et al., ) . the obtained results showed that the viability of microbial cells was inhibited and complete prevention of aflatoxin production occurred at a concentration level of μg/ml of cnts. veterinary applications of metal nanoparticles have been used in various fields related to animal disease diagnosis and treatment as well as biological sensing. several metal nanomaterials such as zn nps have been significantly used in diagnostic and therapeutic activities in animal lymphomas, cutaneous cancers, transmissible venereal tumors, and equine sarcomas (borzacchiello and corteggio, ; carr et al., ; raguvaran et al., ; scott and miller, ) . recently, in dairy cattle, gurunathan et al. ( ) showed the antibacterial potency of ag nps against causes of endometritis (prevotella melaninogenica and arcanobacterum pyogenes). moreover, asgary et al. ( ) used ag nps as adjuvants of the rabies virus in mice and dogs and observed that no toxicity occurred. several studies applied au nps in the diagnosis and detection of some viral infections in chickens (nurulfiza et al., ) ; the foot and mouth disease virus (ding et al., ) ; and the bluetongue virus in cows (yin et al., ) and pigs (wang et al., ) . au nps were used in the detection and diagnosis of some bacterial diseases in chickens (moongkarndi et al., ) , mycoplasma suis in pigs (meng et al., ) , and bacterial toxins (zhu et al., ) . also, au nps were applied in serological diagnosis of cystic echinococcosis ( jahani et al., ) and e. coli o :h in feed and water in mastitis of cow (hassan et al., c) and canine parasitic infestation ( jiang et al., ) . other studies have detected that carbon-based nanomaterials (cbns) such as cnts, graphene (g), carbon fibers, and carbon nanoparticles have huge potential in biomedicine, nanoelectronics, and mechanical engineering (de-volder et al., ; le croy et al., ) . they can be used in disease diagnosis, gene therapy, and physiological treatments wang et al., ) . nowadays, the varieties of nanomaterials and nanosensors have a significant value in all aspects of animal science such as the diagnosis of tuberculosis in bovines and the resulting therapy (kuswandi et al., ; sekhon, ) . it is interesting to report here that zn nps can kill cancer cells without affecting on normal immune cells and hence can be used as anticancer agents and for cancer diagnostic devices in humans and veterinary animals ). on the other hand, a carbon nanoparticle suspension injection (cnsi) can act as a drug carrier, where it has the ability to adsorb drugs such as epirubicin and doxorubicin drugs (xie et al., ) , which are used in lymphatic chemotherapy (yang et al., ) . currently, cnsi has been applied for clinical treatments as part of an injection during oncological surgery (xie et al., a) . cnsi could be applied for lymphatic mapping as well as in parathyroid gland and lymph node tumor imaging, such as in gastric cancer zhu et al., ) and breast cancer (wu et al., ) . gu et al. ( ) and zhu et al. ( ) successfully investigated relevancy of cnsi in lymph node dissection in the diagnosis of thyroid carcinoma. furthermore, the cnsi might include in photothermal therapy, gene delivery, and serve as an immune adjuvant (liu et al., ; xie et al., a, b) . meanwhile, the qds polyethylene glycol (peg)-functionalized carbon nanoparticles were nontoxic to mice after intravenous exposure . moreover, cbns can be utilized in the imaging of organs such as lymph nodes as well as physiological treatments wang et al., ) . wang et al. ( b) evaluated silica nanoparticles (snps) in the imaging of tumor cells, where the primary or secondary antibodies immobilized onto the snps and attached them to cancer cells. similarly, he et al. ( ) detected that the in vitro adoption of snps in methylene blue dyes initiated fluorescent imaging of hela cells and can be injected directly into the tumors in mouse causing its necrosis after laser radiation treatment. snps can also be used for the photodynamic therapy of cancer by producing singlet oxygen through fluorescence energy (kim et al., ) and mnp employed in medical imaging (mishima et al., ) . recently in sheep, fe o nps were used for the diagnosis of tendon disease by fluorescent imaging (scharf et al., ) . the advance tools in nanotechnology developed new delivery systems and methods at the nanoscale level to produce chemical and biological reactions related to targeted sites and cells (tomanek and enbody, ) . in the early stage, the use of nanotechnology in the treatment of salmonella typhi infection in mice conjugated with ampicillin resulted in a decrease in the amount of ampicillin used. therefore, their residues in animal tissues were reduced, which reflected the safety of produced food (fattal et al., ) . significant applications of mnps include pathogen detection, protein purification, and drug delivery (mishima et al., ) . narducci ( ) injected the ag np-loaded rabies vaccine into dogs, checked them daily for days, the obtained data noted that no abnormal signs. recently, cs nps have been widely studied for the delivery of antibiotic drugs (cover et al., ; zaki and hafez, ) and anticancer drugs such as -fluorouracil, paclitaxel doxorubicin, letrozole, and saponin (rejinold et al., ; saboktakin et al., ) . several reports examined the application of cbns in animal disease diagnosis and drug delivery wang et al., ) , and carbon-based drug delivery systems for cancer treatment (reina et al., ) . furthermore, the use of nanotechnology in drug delivery reduces the amount of antibiotics administered for the treatment of diseased animals and hence reduces their residue in animal tissues. the novel tools of diagnosis and treatment of animal diseases by recent nanotechnology have potentiated progressive advances in animal production and reproduction. the feed supplements at the nanoscale of nanocopper and nanochromium are more bioavailable to animals and poultry, allowing more interaction to occur in the gut and better absorbance (gonzales-eguia et al., ; huang et al., ; sirirat et al., ; wang and xu, ; zha et al., ) . recently, several studies detected that the supplementation of znonps into poultry feeds to improve their growth performance, immune status, reproduction activity, and preventing microbial infections (partha et al., ) and piglets and poultry (lina et al., ; yang and sun, ) . however, the bulk of the zinc was added as a feed additive to pigs to treat diarrhea caused by enterotoxigenic e. coli (broom et al., ; case and carlson, ) . the dietary supplementation of zinc-methionine in feeds of mastitis cows caused an elevation in milk production (salama et al., ) . meantime, the addition of zn nps in cows that suffered from subclinical mastitis caused a reduction in the level of the somatic cell and improved milk production (rajendran et al., ) . however, the mastitis disease caused huge economic losses by reducing the milk yield. the most recovered organisms are s. aureus, e. coli, c. albicans, a. flavus and a. niger, and they can be successfully treated by zno nps (bajpa et al., ; hassan et al., ; jalal et al., ) . currently, bai et al. ( ) screened the antibacterial potential of zn nps against staphylococcus epidermidis, streptococcus agalactiae, klebsiella pneumoniae, and e. coli recovered from mastitis. on the other hand, in poultry, the addition of zno-nps to feeds of broiler chicks significantly improved the growth, production, and dress performance (lina et al., ; mishra et al., ) . within another function, it has the ability to potentiate economic importance in piglets via increasing their growth performance and feeding utility (yang and sun, ) . ag nps in broiler chickens caused antimicrobial and growth-promoting effects, but the level of plasma immunoglobulin (igg) was decreased (pineda et al., ) . furthermore, nanographene is monitored in the measurement of blood parameters and the milk of large and small ruminant animals mccomb et al., ; rahimi, ) . in addition, the oral administration of milk containing casein nanoparticles conjugated with vitamin d to humans or animals reached the stomach in which nanocasein hydrolyzed separated from vitamin d, resulting in the elevation of their bioavailability (haham et al., ) . several reports evaluated employed various nanoparticles to improve meat and egg quality. wang and xu ( ) added chromium nanoparticles ( μg/kg) in pig feed, which caused an increase in muscle mass and improved pork quality. similar results were detected in birds when chromium nanoparticles added to feed ( μg/kg) caused the decreased cholesterol content of muscles and raised the feed efficiency (zha et al., ) . huang et al. ( ) gave a nanocalcium compound to mice that resulted in its bioavailability and increased the density of bone minerals, which is higher than the use of calcium compounds on a large scale. the administration of cs nps loaded with chromium pigs feed resulted in an improvement in pork quality and the low-fat content of meat by decreasing the synthesis of fatty acids . regarding animal reproduction, nanotechnology develops nanobiosensors for detection of the reproductive and fertility status of animals for use in fertility control applications, the cryopreservation of gametes and embryos, and the detection of fertility hormones (saragusty and arav, ) . they added that nanobiosensor devices can help in the detection of diseases, pathogens, estrus, hormone levels, and metabolites as tools for reproductive management. similarly, the level of estrogen hormones in the blood of animals could be measured by implantation of nanotubes under the skin and hence the detection of estrus. this is due to the nanotubes binding with estradiol antibodies by fluorescence-producing signals that help in the control system of breeding (o'connell et al., ) . the state of animal breeding such as fertilization and the viability of sperm and eggs can be measured by nanofluid. in addition, metal nanoparticles are used for the cryopreservation of gonadal tissues, sperm, oocytes, and embryos to cause ultrafast cooling rates and homogeneous rewarming of the biological materials (tomanek and enbody, ). applications of nanomaterials are currently used for the meat and food industry, including the use of nanomaterials as carriers of food ingredients/additives that are placed directly into food or as a part of food packaging. in addition, this can improve the dispersing ability of fat-soluble additives in food products, enhance taste, and reduce the use of fat, salt, sugar and preservatives, preventing hypertension and cardiovascular disease in humans and animals. nowadays, several applications of metal nanomaterials in veterinary medicine used zn nps as a food preservative and feed additive. however, they are toxic to microorganisms and used as antimicrobial agents (hosseini et al., ) and their addition in cow feeds resulted in a significant increase in milk production (rajendran et al., ) . in addition, they can be used in catalysis, sensors, environmental remediation, and personal care products of humans and animals (raguvaran et al., ) . the nanocarriers enable nutritive substances to be resistant to protease enzymes and other desaturating compounds. also, it may be increasing its ability to transfer across the intestinal membrane into blood. in addition, nanocarriers controlled release and better dispersion of nutrients in aqueous systems to water-insoluble food ingredients (lee, ) . nanomaterials can produce novel tools for the detection of food-borne pathogens such as nanomaterials loaded with anti-s. aureus antibodies; gold and iron oxide nanoparticles yielded the rapid detection of s. aureus in milk (sung et al., ) . similarly, nanotechnology could be applied in the science of biomedicine, food systems, food system security, and disease treatment delivery (scott, ) . nowadays, the varieties of nanomaterials and nanosensors are of significant value in all aspects of animal science, and are used as additives to animal products and food safety (muktar et al., ) . in the near future, nanotechnology will potentiate in the production of "interactive" poultry meat that changes color, flavor, or nutrients (meena et al., ) . until now, the conjugated and functionalized nanoparticles have resulted in the formation of hybrid nanomaterials (nanocomposites) that have significant benefits to animal health and production. they can be used in drug delivery, mri imaging, the delivery of therapeutic agents such as anticancer drugs, and disease diagnosis, particularly tumor imaging. nowadays, several studies have employed nanomaterials to avoid the toxic doses of metal nanoparticles for animal health. hassan et al. ( a hassan et al. ( , reported that synergistic and combination therapy has the ability to overcome microbial resistance to traditional antibiotics, resulting in more efficient antimicrobial and antitoxin activity of metal nanoparticles for the treatment of human and animal diseases. however, mody et al. ( ) detected the availability of metal nanomaterial conjugation with drugs and other biomedical components of health importance. hassan et al. ( a) detected that the combination between ag nps and traditional antibiotics resulted in the requirement of lower concentrations from both to obtain the strong antimicrobial effects against c. albicans, a. flavus, salmonella, and s. aureas. in addition, the combined and synergistic therapy of low dose levels of antibiotics and ag nps enhanced their antibacterial and antifungal effects (gurunathan et al., (gurunathan et al., , . the conjugation of nanosilver together with fluconazole (antifungal) and florfenico (antibacterial) have more potential to inhibit the growth of microbial causes of disease in buffaloes rather than their single forms (hassan et al., a) . this combination resulted in a decrease in ag nps dosage to avoid toxicity in the animal. similarly, smekalova et al. ( ) screened the antibacterial activity in the combined therapy of ag nps with penicillin g against actinobacillus pleuropneumoniae. these activities are attributed to the potential of ag nps in killing and their destructive effects on microbial cells . in addition, the functionalized swcnts had an antiviral effect against reovirus (gurunathan et al., ) , hiv (friedman et al., ) , and vesicular stomatitis virus (kaesermann and kempf, ) . therefore, it is suggested that swcnts and multiwalled carbon nanotubes (mwcnts) could be functionalized and combined with bioactive molecules to be a benefit in diverse biological applications (endo et al., ) . the carbohydrate-functionalized cnts have antibacterial and antifungal potential against e. coli, c. albicans, a. flavus (elkin et al., ) , and bacillus anthracis (wang et al., c) . meanwhile, the functionalized cnts can influence the viability of cells by injection cnts with complex cells (kam et al., ) . also, cnts were employed as cancer biomarkers (thakare et al., ) and viruses therapy agent (patolsky et al., ) . cnts have the ability to detect particular dna sequences in cells (tu et al., ) . zhu et al. ( ) and determined the significant antibacterial effects of various carbon nanoparticles and nanocomposites against bacteria causing mastitis in cows. the well-functionalized cnts have more beneficial effects on biological cells than the nonfunctionalized cnts (khalid et al., ) . hence, the benefit activity of cnts is associated with the property of conjugation with other biological components (dumortier et al., ) . therefore, there is significant importance in functionalized swnts for clinical applications in human and animal health (fig. . ) . several studies illustrated that the antimicrobial potential of liposomal formulations against several pathogenic bacterial species (swenson et al., ) . the liposome-encapsulated gentamicin has antibacterial potential against causes of mastitis in bovines such as s. aureus (macleod and prescott, ) . however, liposomeencapsulating tobramycin has broad-spectrum antibacterial effects against different kinds of bacteria (sachetelli et al., ) . in a study by singla et al. ( ) , liposomeencapsulating phage showed strong antibacterial potential against multidrugresistant bacterial infections. moreover, the liposomal amb is an effective and less toxic antifungal than conventional agents in the treatment of mycotic respiratory infection in animals (lambros et al., ; leenders and de marie, ) as well as yeast infections in mice (khan et al., ) and dogs (krawiec et al., ) . in a recent study, al-qushawi et al. ( ) investigated the antimicrobial potentials of tilmicosin in poultry in its binding with the micelle's nanomaterials. similarly, the antiviral activity of liposomal-encapsulated ribavirin was used in animals, resulting in increasing the drug safety efficacy and preventing replication of the virus (kende et al., ; makabi-panzu et al., ) . also, it can be used as a carrier against different types of microorganisms, such as the influenza virus (boraschi and italiani, ) , hav, hiv (qiao et al., ) , and infectious plasmodium vivax (powles et al., ) . moreover, the conjugation of the hem-agglutinin-derived synthetic peptide with liposome induces antiviral protection against the lethal influenza virus (rhee et al., ) . recently, the antimicrobial potential of polymeric nanoparticles composed of chitosan was evaluated against various causes of diseases such as e. coli o : h (doavi et al., ) , pseudomonas aeruginosa (cui et al., ) , the influenza virus (oberoi et al., ) , hbv (lebre et al., ) , filariasis (malathi et al., ) , and dengue (hunsawong et al., ) . furthermore, the attached hybrid dendrimer-based nanomaterials with the biological molecules of the cell wall as proteins initiated the rapid destruction of bacterial cells (xiao et al., ) . the antimicrobial potential of dendrimer-based nanocomposites caused inhibition of the growth of several pathogenic bacterial species via their ability to penetrate microbial cell membranes and cytoplasm contents with the final death of cells (bai et al., ) . recently, meena et al. ( ) reported that lipophilic substances and protection from degradation are the significant advantages of nanoemulsions. essential oils obtained from mentha piperita and encapsulated in chitosan nanogels with cinnamon acid showed antifungal potential against a. flavus (beyki et al., ) . hassan et al. ( a, b) used forskolin emulsions as antifungals against mycotoxigenic fungi (hassan et al., a, b) . hassan and mansour ( ) and hassan et al. ( ) determined that nanoemulsions of rhamnus cathartica oil, molasses, and garlic extracts successfully eliminated the fungal contamination in tested feeds. moreover, the nanoemulsions of natural oils are characterized by their physical activity and avoid chemical action. in addition, the contact of oil nanodrops with the membranes of bacteria, fungi, or enveloped viruses caused the leakage of cell contents and the death of organisms (meena et al., ) . the administration of conjugated penicillin or methicillin (β-lactam drug) to polymeric polyacrylate nanoparticles may increase the antibacterial activities of drugs. this due to the nanoparticles improves the destroying action of β-lactamase in the animal body and used against penicillin and methicillin-resistant s. aureas (turos et al., ) . similarly, ghosh et al. ( ) evaluated the antibacterial potential of tetracycline loaded into polymeric cs nps against e. coli strains and the mic was μg/ml. the authors of this chapter evaluated the antifungal and antimycotoxin potentials of conjugated ag nps with olive oil against the mycotoxin-producing fusarium sp. they caused the inhibition of fungus growth and the prevention of trichothecene and zearalenone production. in addition to antiflatoxins and antishiglla toxins, the antimicrobial potentials for nanoemulsions of cinnamon oil and zn nps were evaluated against isolated a. flavus and e. coli from feeds and diarrheic buffaloes. the results showed the complete inhibition of microbial growth and that toxin production requires lower levels of the combined form ( μg/ml of zn nps and % of cinnamon oil) than the single use of zn nps ( μg/ml) and cinnamon oil ( %) in treatment. in other studies, the role of mnps in toxin decorporation was reported by leroux ( ) while the reduction and removal of toxins from contaminated materials was detected by wang et al. ( ) . similarly, mycotoxins such as aflatoxins and ota were completely detoxified by the addition of fe o nps to mycotoxicosis poultry feeds (mouhamed et al., ; nabawy et al., ) . it is interesting to report that the synergistic and combination therapy of the lower doses of zn nps and ozone fumigation were able to cause the complete detoxification of afb in poultry feed at lower nontoxic doses ( ppm of ozone + μg of zn nps/kg of yellow corn) . the authors have evaluated olive oil and cinnamon oil emulsions against the mycotoxigenic fusarium species and the production of trichothecene and zearalenone mycotoxins. these emulsions have antifungal and antimycotoxin potentials at concentrations of %- %. in recent years, there have been progressive advances in the diagnosis and therapy of animal diseases, particularly using nanocomposites to improve diagnostic tools. the main types of mnps are composed of iron oxide and have several applications in biomedicine through coating mnps by biofunctional molecules to increase their sensitivity for many biological applications. furthermore, biocompatibility, a large surface-to-volume ratio, and chemically reactive biomolecules provide a lot of chemically active sites for biomolecule conjugation, responsible for their functionalization and the encapsulation of other biological materials (gupta and gupta, ; shokrollahi, ; tartaj et al., ) . the mnp structures and coating schemes with many other beneficial materials are of significant health importance to humans and animals; they are illustrated in fig. . . in this direction, gu et al. ( ) used vancomycin-conjugated fept nanoparticles to detect vancomycin-resistant enterococci and other gram-positive bacteria at low concentrations by increasing the ability of vancomycin to penetrate the microbial cells after conjugation. moreover, kaittanis et al. ( ) detected the high potential of immunomagnetic nanosensors (superparamagnetic iron oxide nps) in the detection of mycobacterium avium paratuberculosis. in another study, e. coli o :h could be detected by dextran-coated iron oxide nanosensors or silica-coated iron oxide nanosensors (kaittanis et al., ) . furthermore, mnps will be useful for various biological applications in veterinary medicine and produce useful tools for disease diagnosis, mri imaging, drug delivery, and cancer therapy (michalet et al., ) . the recent exposure of murine models to fe o nps resulted in a significant decrease in inflammatory reactions such as footpad swelling and increased phagocytosis activity in the spleen (shen et al., ) . in addition, mnps offer a simple and versatile platform for separating six histidines ( xhis)-tagged protein and increasing the protein binding capacity (xu et al., a, b) . the production of heterodimers of two distinct nanospheres is a way to fabricate fe o -au heterodimers in a homogeneous organic solvent (yu et al., ) . in recent years, au nps have been characterized by their biocompatibility with human cells and the ability to conjugate with dna, rna, antibodies, and peptides (shah et al., ) , which has resulted in the initiation of novel nanodiagnostic tools (syed and bokhari, ) . some studies used au nps as biosensors in the synthesis of the immune-chromatographic strip for efficient diagnostic assays (halfpenny and wright, ; syed and bokhari, ) to detect antibodies against microbial antigens (cui et al., ) and bacterial and viral diseases (peng et al., ) . furthermore, the functionalized ag nps with some oils were used for treatment of dermatophytes diseases in animals (bansod et al., ) . the authors have determined that the conjugation of ag nps with olive oil resulted in significant and obvious antimicrobial effects against bacterial and fungal causes of mastitis in cows at concentrations of - μg/ml. however, parvongnukul and lumb ( ) detected the useful application of functionalized graphite in the treatment of bone disorders in ovine. in addition, functionalized nanographene has several advantages that help in clinical applications in the therapy of animal diseases (novoselov, ; sanchez et al., ) and can also be used in various biomedical applications (sun et al., a, b) . in another study, the polymers of nanomaterials can be prepared by adhesion of the natural biological compound as carbohydrates and protein molecules with a polymer that increases the efficacy of delivery of these compounds (meena et al., ) . in addition, several studies used natural polymeric nanoparticles in disease diagnosis such as inulin in anthrax (feinen et al., ) synthetic polymer nanoparticles were used in the treatment of several diseases (cho et al., ) . in addition, the synthetic polymeric nanoparticle drugs were used to detect several causes of animal diseases such as s. aureus (colonna et al., ; liu et al., ) , tuberculosis (lawlor et al., ; parlane et al., ) , brucella abortus, b. anthracis (zhao et al., a, b) , influenza (oberoi et al., ) , and plasmodium malariae (powles et al., ) infections. also, qds could be used to screen blood samples for certain proteins that may indicate certain diseases (mohanty et al., ) . qds were applied in microbial detection such as pathogenic mycobacteria with cdse qds conjugated with streptavidin (liandris et al., ) . hahn et al. ( ) used the brighter signal of streptavidin-conjugated cdse/zns (core/shell) qds for detection of the e. coli o :h strain. modified cs nps have the ability to decrease doses of used drugs in disease treatment, which can lower the costs associated with drug treatments (aruna et al., ; fang et al., ; wang et al., a) and also in antitumor treatments (ghadi et al., ) . moreover, chitosan nanocomposites could be used for tumor therapy (aruna et al., ; cao and zhou, ; fang et al., ) . other studies by lu et al. ( ) illustrated that the injection of a chitosan solution in articular cartilage increases the chondrocyte density and their genesis. the chitosan-based material can support chondrogenesis, activate produced new cartilage, and improve the long-term outcomes of cartilage repair in clinical settings (suh and matthew, ) . in addition, it can be used in gene therapy and have an adjuvant effect in vaccines in vivo (peniche and peniche, ; shi et al., ) . silica nanoparticles (snps) have been characterized by well-defined structures that enable them to be easily combined with other materials (burns et al., ) . in addition, their optical transparency allows excitation and emission light to pass through targeted cells (shirahata, ) . snps can also be conjugated with drug molecules and enter targeted cells in diseased animals, resulting from the effective accumulation of drugs inside cells and hence treatment (taylor-pashow et al., ; wang et al., b) . peled et al. ( ) diagnosed a respiratory mycobacterial infection in cattle using nanocomposite tools. nanomaterials have the ability to conjugate with biological elements such as viral and phage cells and are used for bacterial detection (billington et al., ) . recently, bai et al. ( ) reported that nanosensors can provide a diagnosis of subclinical bovine ketosis by using β-hydroxybutyrate (bhba). in addition, nanosensor tools are used in the measurement of different physical and chemical blood parameters (cui and mumper, ; hasuwa et al., ; rolfe, ) and the detection of the constituents of animal excretions such as sweat, breath, and temperature (neethirajan et al., ) . currently, various types of nanosensors can be utilized in the detection of cations, anions, organic compounds, and other toxic and microbial content in feed, food, and intelligent packaging (lu and bowles, ; neethirajan et al., ) . generally, it is suggested that the action potentials of nanosensors are due to two detection principles: catalytic sensing (using enzymes, cells, tissues/ organelles, and microorganisms) and affinity sensing (antibodies, nucleic acid, phages, bonding proteins, polymers, and synthetic proteins) as a diagnosis tools (akkoyun et al., ) . the first step in this direction, wang et al. ( b) used the nanocomposites of polyclonal antibodies and au nps for immunochromatographic strip detection of toxin contaminants in milk such as carcinogenic aflatoxin m . nanoshells are composed of a dielectric core material such as silica conjugated with a metallic material layer can be injected into animals with targeted agents that search and attach to cancer cell receptors (hirsch et al., ) . while qds have many advantages over organic fluorescent dyes, they are brighter and easier to visualize than organic dyes. hence, they can observe cell pathways and events inside the animal body as well as drug delivery to target tissues (chakravarthi and balaji, ) . qds also may be injected into the bloodstream of animals, and they may detect cells that are malfunctioning by illuminating the body with light and stimulating the qd to produce sufficient heat to kill the cancer cell (freitas, ) . the nanocomposites and nanomaterials have the advantages to enter into the cancer cells and agglomerated to form large clusters inside tumor cells (yigit et al., ) . this helps in drug delivery, imaging, and cancer therapy (fig. . ). schematics of different nanotechnology-based tools used for cancer therapy and imaging. liposomes are made up of lipid structures that can be made stealthy by pegylation and encapsulating different therapeutic agents; these are used as a potential nanocarrier for cancer therapy. nanocantilevers are array-like structures in which engineered tiny bars anchored at one end help in the detection of altered proteins present in certain types of cancers. quantum dots are fluorescent nanocrystals that can be conjugated to a ligand by coating a polymeric layer onto it; therapeutic agents are encapsulated and used for cancer therapy. new synthetic methods have been developed to design multifunctional nanoparticles, in which we can encapsulate both therapeutic and imaging agents in a single nanocarrier system that will conjugate with more than one ligand on the surface. thus, it will act as a novel multifunctional nanocarrier system with the capacity of targeted tumor imaging and the delivery of therapeutic agents. the most recent drug delivery in targeted tissues was achieved by nanocarrier composites with biological molecules such as fluorescent nanostructured glucoseand sucrose-derived nanoparticles, which help successfully deliver therapeutic agents to lung carcinoma (ajmal et al., ) . similarly, when injected, superparamagnetic nanoparticles made essentially from iron oxide target cancer cell receptors, enhance the location of cancer cells, and deliver the attached drug to kill the cancer cells. the encapsulation of qds with snps and coating with pegylated phospholipids and a paramagnetic lipid coating have been applied in animal science ( van-schooneveld et al., ) . moreover, qds also may be injected into the bloodstream of animals. platinum-containing nanoparticles (fept) without any surface coating may act as potential anticancer drugs yin et al., ) . magnetic nps encapsulated with silica could be used as carriers for anticancer drugs and fluorescence molecules (xu and sun, ) . in addition, the combination of fept@fe o with yolk-shell nanoparticles has high cytotoxicity and strong mr contrast enhancement (gao et al., b; peng and sun, ) . they added that it can encapsulate anticancer drugs. the functionalization of yolk-shell mnps can target a specific tissue for delivering therapeutic agents and monitoring the transformation of the tumor by mri. the mnps can combine with qds to exhibit magnetic and fluorescent properties (gao et al., a (gao et al., , a gu et al., ) and sequentially grow metallic nanocomponents (gu et al., b) . they added that exotic nanostructures such as yolk-shell nanoparticles are very stable because the biocompatible, compact fe o shells prevent the oxidative species from reaching the fept (gao et al., b (gao et al., , b . ag nps and fept nanocomposites remediate tumors and radiation therapy in cats (woods et al., ) . liposomes have the potential to provide several valuable tools in the diagnosis and therapy of tumors (hofheinz et al., ; johnston et al., ; sadozai and saeidi, ) . they also have the ability to functionalize and conjugate with biologically valuable materials for use in animals as anticancer drugs (alexis et al., ) . it is interesting to report that the intravenous administration of conjugated au nps with gum arabic (ga) to swine helped in the detection of tumor tissues by au np constructs (fent et al., ) . similarly, when conjugated, radioactive au nps with ga were injected intralesionally in canine animals suffering from prostate cancer. a successful entrance of au nps with drugs to the prostate tissues occurred, resulting in successful cancer therapy (axiak-bechtel et al., ) . au nps can be used for cancer detection and imaging in dogs and mice (chanda et al., ) . the variations in the thickness of the nanoshells help with their use in diagnostic assays and tumor detection (avaritt et al., ) . cnts without any additive were toxic to mice lungs (lam et al., ; shvedova et al., ) . the functionalization of cnts was nontoxic to animals in conjunction with other beneficial materials (schipper et al., ; wu et al., ) . hence, the nonfunctionalized, long mwcnts may be carcinogenic to mice (ding et al., ) . the drug is delivered for cancer therapy by liposomes characterized by the successful reach of drugs to the site of cancer cells that overcome the resistance of cancer for therapy (malam et al., ) . zamboni et al. ( ) also detected the efficacy of docetaxel encapsulated in the liposome bilayer for treatments of cancers in the mice prostate and pancreas as well as nonsmall-cell lung cancer. in a similar study, torchilin ( ) and sadozai and saeidi ( ) detected the ability of liposome nanocomposites to treat canine cancers in the spleen. meanwhile, kleiter et al. ( ) showed that liposome-encapsulated muramyl tripeptide showed antitumor potential and prolonged disease-free survival in canines. dendrimers are nanocomposites characterized by low cost, high potentiality in complexion or encapsulation with other beneficial biomaterials, and active destruction of the target cell membrane in cancer cells (baker et al., ; koda et al., ; sekowski et al., ) . in addition, these characteristics enable dendrimers to attach to drugs through a sphere on their surface and enter from the site of administration to targeted tissues via the blood vascular system, resulting in effective cancer therapy (al-jamal et al., ) . the polymers also adhered to albumin molecules, which potentiated their delivery and could be used in cancer detection and therapy (gradishar et al., ) . recently, meena et al. ( ) detected major benefits of cnts for animal health and production. they can be used as sensors, antimicrobial agents, anticancer agents, and for drug delivery. fernandez-lopez et al. ( ) and dilbaghi et al. ( b) illustrated that dendrimers can be used in the diagnosis and therapy of tumors in animals as well as the detection of their nature by their ability to penetrate tumor cells and change the biochemical content of cytoplasm, leading to cell death. various studies have shown that mesoporous silica nanoparticles (msnps) have potential applications (hom et al., ; slowing et al., slowing et al., , vivero-escoto et al., c) . msnps have a high surface area and volume, a stable mesostructure, a tunable pore diameter ( - nm), and a modifiable morphology (vivero-escoto et al., a, b) . recently, msnps were effectively used to protect conjugated molecules and agents ( juan et al., ) . they can be functionalized with nanostructures and employed for drug/gene delivery and sensing applications (igor et al., ) . the msnps were readily internalized by eukaryotic cells without detectable toxic effects (radu et al., ) . in addition, the surface functionalization of msnps was manipulated by the uptake efficiency of hela cancer cells (slowing et al., ) . in the meantime, the msnps were functionalized with folic acid to form folate receptors, which facilitated drug aggregation in target cells and hence gave a promising result in the regulation of human cancers in mice (lu et al., ) . today, there are different progressive advances in nanotechnology applications in the diagnosis and therapy of cancer that successfully target cancer cells without affecting normal, healthy cells (fig. . ) . . . . bioimaging (x-ray, fluorescent, magnetic resonance imaging (mri)) today, the right diagnosis of human and animal diseases depends on visions of the activities of body cells and organs by imaging technology, which has progressively advanced. the essential nanomaterial is the mnps that are used for various biological applications in veterinary medicine and produce useful tools for mri imaging (michalet et al., ) . the mnps can penetrate cell membranes and image the targeted tissues using magnetic resonance imaging (mri) (soenen et al., ) and produce fluorescence through light signal activation (ajmal et al., ; croissant et al., ) . cheon and lee ( ) reported that the conjugation of mnps and dyes will lead to efficient mri and optical imaging. furthermore, gu et al. ( a) and neuberger et al. ( ) revealed that porphyrin-modified fe o nanoparticles can act as a multifunctional nanomedicine that combines anticancer treatment and noninvasive mri imaging. similarly, gao et al. ( ) detected that the conjugation of fluorescent vancomycin with fluorescein-amine stains or fept@van causes the quick, sensitive, and low-cost detection of bacteria by using fluorescence microscopy; it also offers the ability to enhance contrast in mri. kolecka et al. ( ) incubated canine stem cells with superparamagnetic iron oxide nanoparticles for h. different approaches of nanotechnology such as gene therapy, photodynamic therapy, radio therapy, radiofrequency therapy, and cancer theragnostics are being applied for the treatment of cancer. these advanced technologies help target cancer cells only, without affecting normal cells. ultimately, this leads to the death of the cancer cells while the normal healthy cells survive. the nano-sized materials entered the cells by endocytosis and acted as contrasts for the detection of the activity of canine stem cells. long et al. ( ) also reported that the ultrasmall superparamagnetic nanoparticles could be used for mri imaging for temporal lobe epilepsy and the detection of any abnormalities. in addition, the fe o -cd-se heterodimer nanoparticles produced fluorescent mnps that allow their intracellular movements to be controlled using magnetic force and a fluorescent microscope (gao et al., a) . another study detected the reduced coated graphene oxide used in the treatment and diagnosis of diseases in humans and animals. it may employ as an opt-acoustic transmitter for producing high-pressure and highfrequency ultrasound (lee et al., ) . it is interesting to report that the hypernanocomposites of dendrimers can be used as alternative tools to the traditional dyes used as mri contrasts as well as in genes and drug delivery for therapy (kim and zimmerman, ; margerum et al., ; samad et al., ). nowadays, real-time sensitive imaging and sensing applications have been adopted with the development of qds, which are crystalline materials with a facet and lattice structure that are analogous to bulk semiconductor materials. several useful biomedical applications of qds in biology have highlighted its potential in nanobiotechnology (michalet et al., ) . they can be used as sensors and detecting tools for biomarkers, pathogens, and immune-labeling of cells and tissues (geszke-moritz and moritz, ; kaittanis et al., ) . most qds used for analytical applications are synthesized as core/shell structures (geszke-moritz and moritz, ). they have greater stability and resistance to photo-bleaching. they exhibit both fluorescence and magnetism, such as co@cd-se core-shell nanocomposites (kim et al., ) and fept-zn s nanosponges (gu et al., b) . similarly, the mnps can combine with qds to exhibit magnetic and fluorescent properties for imaging target cells and organs (gao et al., a (gao et al., , b, a . the modification of the qd surface gives rise to a variety of conjugation strategies with biomolecules such as vitamins, proteins, peptides, and antibodies, and initiates their entrance to targets in animal cells. similarly, maša ( ) illustrated that qds produced a signal of fluorescence (fluorophores) to detect different biological events and helped in enhancing sensitivity in analyzing different biological processes. he added that different fluorophores transfer energy from biological events into specific lights that can be detected. the most effective target detection is achieved by coupling biomolecules (such as enzymes, antibodies, small molecules, and oligonucleotides) into fluorescents (kaittanis et al., ) . furthermore, the combination of nano-sized particles and fluorescence has become a good alternative to detect biological events (h€ otzer et al., ; ruedas-rama et al., ) . giri et al. ( ) added that qd barcodes were conjugated with single-stranded oligonucleotides that can hybridize to a target sequence and conjugate to a specific dye, resulting in a genetic biomarker that is measured by its optical emissions by a fluorescence signal. moreover, qds have a unique property such as highly bright and extremely photo-stable with longer excited-state lifetimes than classical fluorescent dyes and chemical degradation (petryayeva et al., ) . fluorescent bioconjugated silica nps were also used in the encapsulation of fluorescent dye molecules, producing a highly amplified and reproducible signal (zhao et al., ) . moreover, the rapid detection of e. coli o :h was performed with a fluorescence microscope using snps encapsulated with dye (tuitemwong et al., ) . this produced effective detection rather than using dyes alone (he et al., ) . the use of doped snps with fluorescent dyes, qds, and metal nps did not cause any changes in encapsulated agents (tallury et al., ) . hence, snps have the advantage of robust materials while also being mechanically stable and transparent, enabling the stabilization and protection of encapsulated fluorophores. moreover, juan et al. ( ) quantified the biodistribution of snps doped in fluorescent dye and found that the main organs for accumulation were the liver and spleen, then passing to the stomach via fecal excretion. currently, mri is a recent imaging technology that is considered a noninvasive diagnostic tool with a high payload of molecules and molecular contrast agents (na and hyeon, ; villaraza et al., ) . in another study, msnps were used as an alternative to mri contrast agents and the produced signal loss in the liver was detected (taylor et al., ) . the liver, kidney, and spleen are the main organs of carryover of nanomaterial after intravenous administration (souris et al., ) . furthermore, msnps may applicable in biomedical imaging and develop new tools for clinical diagnosis and disease therapy (lai et al., ; lu et al., ; radu et al., ) . another study reported that the biodistributions and detection of disease pathogenesis by biomarkers for therapeutic treatments (cheon and lee, ) . the advances in nanotechnology enable us to develop delivery systems and tools at the nanoscale and to produce chemical and biological reactions related to targeted sites and cells (tomanek and enbody, ) . magnetic nanocomposites are useful for various biological applications in veterinary medicine and produce new tools for drug delivery and cancer therapy (michalet et al., ) . it is suggested that an efficient delivery system should have the capability to transport the desired guest molecules without any loss before reaching the targeted location (radin et al., ) . however, rejman et al. ( ) showed that msnps can be efficiently employed as carriers for intracellular drug delivery as well as cell tracers and cytoplasmic biosensors. in addition, liposomes have been successfully used for targeted drugs, imaging agents, vaccines, and gene delivery (bakker-woudenberg et al., ; hiszczy nska-sawicka et al., ) . mesosilica nanoparticles (msnps) can be encapsulated in drugs to increase the delivery to target organs (vallet-regi et al., ) such as cadmium sulfide (lai et al., ) , gold (liu et al., ; torney et al., ) , and iron oxide (giri et al., ; vivero-escoto et al., ) . dendrimers (radu et al., ) proteins (zhao et al., ) , and polymers have been developed radu et al., ) . several studies have detected the availability of fluorescent snps in gene therapy such as dna probes and carriers (lee et al., b; mintzer and simanek, ; wang et al., b; zhao et al., a, b) . they can also help in transferring genes in mice lungs using silica nanoparticles (ravi et al., ) . . nanomaterials and nanocomposite applications micelle nanoparticles are characterized by a hydrophobic core stabilized by a hydrophilic shell and used in transdermal therapeutics (lee et al., a) . the micelle nanoparticles are highly water soluble due to their hydrophilic shell; hence, they have low toxicity and are used for the drug delivery of therapeutic agents (koo et al., ) . scott-moncrieff et al. ( ) determined that insulin mixed with micelles was efficiently absorbed in dogs, resulting in successful insulin delivery and therapy. vail et al. ( ) currently uses the water-soluble micelle paclitaxel to efficiently treat tumors in dogs. the activity and safety of micelle paclitaxel are superior to lomustine by a high ability to enter the targeted cancer cells. several studies illustrated that polymer compositions, structures, and properties potentiated its use in a biomedical application such as drug delivery (moreno-vega et al., ; yang, ) . the advantages of using polymer-drug conjugates include their ability to overcome drug resistance and to elicit immune-stimulatory effects (ríhová et al., ; sirova et al., ) . moreover, some studies detected that skin administration is the better route for the administration of polymer-based nanoparticles such as hydrogels containing dexamethasone for the treatment of psoriasis (degim, ) . meanwhile, drug delivery can use polymers to coat mnps and to encapsulate drugs to form nanocapsules or micelles (gupta and gupta, ) , which may require complicated processes and result in modest efficiency. klajnert and bryszewska ( ) and stecko et al. ( ) formed a dendrimer-based nanocomposite with vaccine preparation, increased the efficacy of vaccine delivery, and potentiated its immunogenicity. furthermore, nanoemulsions of natural oils were successfully used in veterinary medicine as a drug delivery agent (kang et al., ; vandamme et al., ). . . . animal production, reproduction, nutrition, and breeding today, there is progressive exploration and refinement in nanotechnology, which plays a significant role in animal production and reproduction. some nanoparticles have been demonstrated to enhance fertility and protect spermatozoa through the functional groups they carry. recently, qds have been used to improve the detection of spermatozoon and oocyte movement and their interactions in a physiological setting. they potentiated the production of greater signal intensity than the old traditional method used in imaging gametes (druart et al., ; feugang et al., ; long et al., ) . feugang et al. ( ) and hasuwa et al. ( ) detected that the consumption of composed bioluminescence resonance energy transferconjugated quantum dot nanoparticles can be used in male pig gametes for the observation and imaging of fertilization events in deep gonadal tissues. the purification of semen is essential for successful artificial insemination in animals. recently, coated mnps with antibodies or lectins were used for the separation of damaged sperm from undamaged healthy sperm, while the conjugated antibodies with mnps directed directed to ubiquitin characteristic material for defective sperm (odhiambo et al., ; petruska et al., ) . in addition, the application of nanotechnology in sperm cryopreservation fixes the stability of sperm quality and prevents microbial pollution during storage (bryla and trzcinska, ) . antibiotic treatment may reduce the motility and activity of sperm; this problem will be solved by the use of nanoparticles that replace extender antibiotics (hargreaves et al., ) . also, mesoporous silica nanoparticles were loaded with dna and transferred the sperm in vitro without changes in its quality (barkalina et al., ) . pawar and kaul ( ) detected that the incubation of buffalo sperm with low levels ( μg/ml) of titanium led to successful fertilization. the administration of nanofunctionalized α-tocopherol dose for horses may increases the absorption and plasma concentration due to the oxidative status of racehorses become under intense training (rey et al., ) . the supplementation of ag nps singly or in combination with amino acids in chicken feed can improve their immune status (bhanja et al., ) . similarly, rey et al. ( ) demonstrated the potential of the administration of micellar nanoparticles conjugated with vitamin e to pigs in improving the health status of the animals. several studies have used nanobiosensors for animal reproduction and measurement of their fertility (monerris et al., ; sagadevan and periasamy, ) and detection of the viability of reproductive organ functions and fetus (saragusty and arav, ) . furthermore, engineered nanoparticles with fluorescent probes visualize the events during ovulation and pregnancy in reproductive tissues (feugang et al., ; hasuwa et al., ) . in addition, qds have the ability to detect the mammalian spermatozoon and oocyte movement, which significantly helps in animal production (hill and li, ) . moreover, the small size of the biosensors and the high surface-to-volume ratio enable them to act as signal reporters in biosensors and reduce the time of target cells to be detectable by spectrophotometer, fluorescence microscope, and luminometer (koedrith et al., ) . greenwood et al. ( ) suggested that the formation of reactive oxygen species (ros) and free radical production by nanomaterials causes the destruction of mitochondria, the denaturation of proteins, and the damage of dna. several studies have detected that ros production can be found in c fullerenes, swnts, and qds (dilbaghi et al., a) . the mechanism of action of nanoparticles is briefly illustrated in fig. . . the nanomaterial potentials such as anticancer, antimicrobial, and other activities against target cells resulted in the penetration and disruption of the cell membrane and cell death. particularly, when the microbial cells that were treated by metal nanoparticles were examined by a scanning electron microscope, cell membrane damage and adhered nanoparticles to the respiratory sequence of cytoplasm were observed, followed by the death of target cells (gajbhiye et al., ; hassan et al., b hassan et al., , hassan et al., , a nabawy, ) . another suggestion is the interaction between the constituents of target cells with the oxygen atom and metal ions of metal nanoparticles, which can cause damaged cell components and death (brayner et al., ; matei et al., ; moraru et al., ; violeta et al., ) . in spite of the progressive and valuable tools of biosensor applications in animal science, their toxicity risk to animals and the environment is the essential reason for limiting their use in human and animal science (donaldson et al., ; oberd€ orster and kuhlbusch, ) . in addition to nanotechnology applications in several aspects of biomedicine, there are potential toxicity hazards to the environment and users as well (baltic et al., ) . oxidative stress and inflammation are caused by affected tissue fibers or secondary mutations (aschberger et al., ) . in general, nanomaterial toxicity varies according to nano-sized particles, the health states of the humans and animals, the administered doses, and the time of exposure (aschberger et al., ; maynard, ; rim et al., ) . also, the efficient evaluation of nanoparticle toxicity risk must include their particle size and shape, crystalline form, functionalization, and purity (aschberger et al., ) . some nanoparticles are toxic to sperm; this is adversely reflected as it decreases its viability in vitro due to overdoses and long exposure to zinc oxide and titanium oxide nanoparticles (pawar and kaul, ) . the incubation of the sperm with the concentrations of - μg/ml of zn nps resulted in the death of sperm within a few minutes (barkhordari et al., ) . pawar and kaul ( ) detected that the incubation of buffalo sperm with μg/ml of titanium oxide nanoparticles reduced its viability. several studies have reported that metal nanoparticles may induce toxicity by their ability to easily access the skin, lungs, and brain, causing adverse effects in biological functions. the toxicity of zn np nanoparticles to animals and the environment must be briefly studied before they are used as a feed additive. therefore, the measurement of effective nontoxic doses of metal nanoparticles in laboratory animal models must be undertaken to study the suitability of their field application (abd el-fatah et al., ; asharani et al., ; hassan et al., ; shaw et al., ) . until now, there has been a lack of toxicological effects of nanoparticles on health (savolainen et al., ) . so, the toxicological aspects of nanomaterials require further continuous studies to be completely understood (fig. . ). regarding the route of exposure to nanomaterials, ingestion is the main exposure route for humans and animals (aschberger et al., ) . upon ingestion, the nanomaterials enter the gastrointestinal tract to the intestines and are eliminated rapidly through the liver and spleen (oberd€ orster et al., ) . the ingestion mainly results from the presence of nanoparticles in food due to direct contact of nanopackaging to food (baltic et al., ; bouwmeester et al., ) . the nanoparticles are distributed in the liver and spleen in circulation (baltic et al., ; silvestre et al., ) . inhalation and the skin are other routes of nanoparticle toxicity, which are detected mainly in the laboratory and in industry workers in nanomaterial production factories (aschberger et al., ) . another study described that inhalation and skin exposure to magnesium oxide nanoparticles allows them to enter the nerve cells and central nervous system (elder et al., ) . nurkiewicz et al. ( ) demonstrated that the inhalation of nano-sized titanium dioxide reached systemic circulation in rats. tinkle et al. ( ) and tsuji et al. ( ) detected the ability of nanomaterials to enter healthy human skin during constant flexing, causing many controversial effects (monteiro-riviere et al., ) . in other studies, the zn nps detected the availability of penetrated skin tissue (baltic et al., ; sharma et al., ) and have carcinogenic effects such as asbestos-caused fibrosis in the lungs (muller et al., ) . the inhalation of high doses of nano-tio also has the potential for lung carcinogenesis (aschberger et al., ) . furthermore, the entrance of nanoparticles via the bloodstream may affect blood vessel function and cause blood clot formation and other adverse effects on the cardiovascular system, as in the case of inhalation of ambient ultrafine particles (pekkanen et al., ) . similar microvascular dysfunction was observed in rats after inhalation exposure to low concentrations of nano-sized titanium dioxide (nurkiewicz et al., ) and platelet aggregation and vascular thrombosis after inhalation exposure to swcnts and mwcnts (radomski et al., ) . some studies reported that nanomaterials can penetrate the blood and are distributed in body organ cell tissues, and sometimes transmitted to the fetus via the blood supply (baltic et al., ; silvestre et al., ) . in the near future, more progressive advancement in nanobiomedical science and availability to improve animal health is needed. they will have the potential to solve many problems related to animal disease diagnosis, animal production, reproduction, and good hygienic practices during rearing and maintaining of food animals. the possible applications of the technology are almost incredible in relation to livestock. although much research is needed before nanotechnology applications are used in veterinary and animal sciences, in particular the toxicity risk. schematic mechanism of cytotoxic activity of nanoparticles (nps). until now, nanotechnology has offered significant advances in the development of novel technology in human and veterinary medicine as a synthesis of new materials and tools that help increase animal health and production. several applications of nanomaterials are used to produce strategies for disease diagnosis, drug delivery, animal nutrition, breeding and reproduction, additives to animal products, and finally food safety for human and animal health. in addition, they are used for tumor detection, production of tumor vaccines, tissue engineering, mri images, sensor development, and the detection of pathogens, proteins, and biological molecules. these applications resulted in the development of novel nanodrugs such as metal nanoparticles (particularly, zn nps, ag nps, au nps, and mnps), liposomes, polymeric nanoparticles, dendrimers, cs nps, qds, etc. the use of metallic nanoparticles in nanomedicine has the ability to improve imaging and therapeutic drug delivery for the treatment of cancer in humans and veterinary medicine. the nanomaterials and nanocomposites bind with biological fluids to help control drug release and hence are used in the synthesis of biological markers, imaging, drug delivery systems, and disease detection. in addition, the potential of nanoparticles in disease treatment and diagnosis, antimicrobial, anticancer and other activities of metals nanoparticles against target cells resulted from disruption of cell membrane, damage, rupture of cell wall and leakage in inter cellular components and finally cell death. the toxicity of nanomaterials is mainly due to oxidative stress. the exact mechanism of the formation and generation of ros requires more study to be completely understood. oxidative stress resulting from the use of nanomaterials can cause inflammation, fibrosis, genotoxicity, and cancers. in the future, there will be progressive advances in nanobiomedical science and use in improving animal health. it will have the potential to solve many problems related to animal health, animal production, reproduction, and good hygienic practices during rearing and maintaining of food animals. the possible applications of the technology are almost incredible in relation to livestock. however, special attention is required for the known toxicological aspects of nanomaterials prior to application in veterinary and animal sciences. because there is no brief knowledge about toxicology studies available, hence much research is urgently needed before nanotechnology applications can be used, in particular their toxicity risk. comparative study between the use of bulk and nanoparticles of zinc oxide in amelioration the toxic effects of aflatoxins in rats a comparative study on antifungal activity of fe o , and fe o nanoparticles nanobiotechnological strategies for molud and mycotoxin control extracellular biosynthesis of silver nanoparticles using the fungus fusarium oxysporum synthesis, characterization and in vitro evaluation of methotrexate conjugated fluorescent carbon nanoparticles as drug delivery system for human lung cancer targeting detection of sulphamethazine with an optical biosensor and anti-idiotypic antibodies nanoparticle technologies for cancer therapy tumor targeting of functionalized quantum dot-liposome hybrids by intravenous administration zinc toxicity in ruminants preparation and characterization of three tilmicosinloaded lipid nanoparticles: physicochemical properties and in-vitro antibacterial activities antimicrobial therapeutic determinants of outcomes from septic shock role of chitosan nanoparticles in cancer therpy analysis of currently available data for characterising the risk of engineered nanomaterials to the environment and human health; lessons learned from four case studies green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine toxicity of silver nanoparticles in zebrafish models towards a definition of inorganic nanoparticles from an environmental, health and safety perspective plasmon resonance shifts of au-coated au nanoshells: insight into multicomponent nanoparticle growth gum arabic-coated radioactive gold nanoparticles cause no short-term local or systemic toxicity in the clinically relevant canine model of prostate cancer theranostics aspects of various nanoparticles in veterinary medicine moderate intensity static magnetic field has bactericidal effect on e. coli and s. epidermidis on sintered hydroxyapatite the synthesis and testing of anti-cancer therapeutic nanodevices longcirculating sterically stabilized liposomes in the treatment of infections nanotechnology and its potential applications in meat industry in vitro effect of biogenic silver nanoparticles on sterilisation of tobacco leaf explants and for higher yield of protoplasts effects of mesoporous silica nanoparticles upon the function of mammalian sperm in vitro effect of zinc oxide nanoparticles on viability of human spermatozoa encapsulation of mentha piperita essential oils in chitosan-cinnamic acid nanogel with enhanced antimicrobial growth against aspergillus flvus in ovo administration of silver nanoparticles and/or amino acids influence metabolism and immune gene expression in chicken embryos prevention of bacterial foodborne disease using nano biotechnology comparison of two methods for blood lead analysis in cattle: graphite-furnace atomic absorption spectrometry and leadcare(r) ii system bacterial stress responses: what does not kill them can make them stronger from antigen delivery system to adjuvanticy: the board application of nanoparticles in vaccinology equine sarcoid: state of the art review of health safety aspects of nanotechnologies in food production toxicological impact studies based on escherichia coli bacteria in ultrafine zno nanoparticles colloidal medium effects of zinc oxide and enterococcus faecium sf dietary supplementation on the performance, intestinal microbiota and immune status of weaned piglets quality and fertilizing capacity of boar spermatozoa during liquid storage in extender supplemented with different antibiotics fluorescent core-shell silica nanoparticles: towards "lab on particle" architectures for nanobiotechnology progress in antitumor studies of chitosan. chin bovine papillomavirus dna in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western united states effect of feeding organic and inorganic sources of additional zinc on growth performance and zinc balance in nursery pigs applications of nanotechnology in veterinary medicine gold nanoparticle based x-ray contrast agent for tumor imaging in mice and dog: a potential nano-platform for computer tomography theranostics nanotechnology in nutraceuticals and functional foods synergistically integrated nanoparticles as multimodal probes for nanobiotechnology therapeutic nanoparticles for drug delivery in cancer effect of chitosan and chitosan nanoparticles on fungal growth and chilli seed quality sub-unit vaccine against s. aureus-mediated infections: set-up of nano-sized polymeric adjuvant synergetic effects of doxycyclineloaded chitosan nanoparticles for improving drug delivery and efficacy two-photontriggered drug delivery via fluorescent nanovalves chitosan-based nanoparticles for topical genetic immunization a simple and rapid immunochromatographic strip test for detecting antibody to porcine reproductive and respiratory syndrome virus d-mannose-modified chitosan microspheres enhance oprf-opri-mediated protection of mice against pseudomonas aeruginosa infection via induction of mucosal immunity combinatorial prospects of nano-targeted chemoimmunotherapy nanoparticle therapeutics: an emerging treatment modality for cancer new tools and approaches for predicting skin permeability carbon nanotubes: present and future commercial applications evaluation of tropicamide-loaded tamarind seed xyloglucan nano-aggregates for ophthalmic delivery nanoscale device for veterinary technology: trends and future prospective molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano-onions on human skin fibroblast a highly sensitive detection for foot-and-mouth disease virus by gold nanopariticle improved immuno-pcr chitosan-based intranasal vaccine against escherichia coli o :h in vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe gential tract functionalized carbon nanotubes are non-cytotoxic and preserve the functionality of primary immune cells nanomedicine(s) under the microscope human health risks of engineered nanomaterials: critical knowledge gaps in nanomaterials risk assessment immune-carbon nanotubes and recognition of pathogens potential applications of carbon nanotubes nanosilver as substance biostabilising footwear materials in the foot mycosis prophylaxis comparison of antitumor effects of chitosan nanoparticles from different sources in vitro impact of nanotechnology on drug delivery treatment of experimental salmonellosis in mice with ampiciline-bound nanoparticles advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant bio distribution of maltose and gum arabic hybrid gold nanoparticles after intravenous injection in juvenile swine antibacterial agents based on the cyclic d, l-alpha-peptide architecture application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa selfilluminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes silver nanoparticles as a potential antimicrobial additive for weaned pigs microbivores: artifcial mechanical phagocytes using digest and discharge protocol inhibition of the hiv- protease by fullerene derivatives: model building studies and experimental verification fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole antimicrobial activities of commercial nanoparticles against an environmental soil microbe, pseudomonas putida kt combining fluorescent probes and biofunctional magnetic nanoparticles for rapid detection of bacteria in human blood fluorescent magnetic nanocrystals by sequential addition of reagents in a one-pot reaction: a simple preparation for multifunctional nanostructures fept@cos yolkshell nanocrystals as a potent agent to kill hela cells multifunctional yolk-shell nanoparticles: a potential mri contrast and anticancer agent intracellular spatial control of fluorescent magnetic nanoparticles nanosilver particles in medical applications: synthesis, performance, and toxicity quantum dots as versatile probes in medical sciences: synthesis, modification and properties synthesis and optimization of chitosan nanoparticles: potential applications in nanomedicine and biomedical engineering nanosilver effects on growth parameters in experimental aflatoxicosis in broiler chickens effect of nanosilver on blood parameters in chickens having aflatoxicosis amelioration studies on optimization of low molecular weight chitosan nanoparticle preparation, characterization with potassium per sulfate and silver nitrate combined action with aid of drug delivery to tetracycline resistant bacteria immobilization of mycotoxins on modified nanodiamond substrates stimuli-responsive controlledrelease delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles preparation and antibacterial activity of fe o @ag nanoparticles effects of nanocopper on copper availability and nutrients digestibility, growth performance and serum traits of piglets nanomedicine taxonomy: briefing paper. canadian nanobusiness alliance phase iii trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer vaccination against foot-and-mouth disease virus using peptides conjugated to nano-beads using biofunctional magnetic nanoparticles to capture gram-negative bacteria at an ultra-low concentration facile one-pot synthesis of bifunctional heterodimers of nanoparticles: a conjugate of quantum dot and magnetic nanoparticles synthesis and cellular uptake of porphyrin decorated iron oxide nanoparticles-a potential candidate for bimodal anticancer therapy direct synthesis of a bimodal nanosponge based on potential role for carbon nanoparticles identification and preservation in situ of parathyroid glands during total thyroidectomy and central compartment node dissection synthesis and surface engineering of iron oxide nanoparticles for biomedical applications biosynthesis, purification and characterization of silver nanoparticles using escherichia coli green chemistry approach for the synthesis of biocompatible graphene enhanced antibacterial and antibiofilm activities of silver nanoparticles against gram-negative and gram-positive bacteria reduced graphene oxide-silver nanoparticle nanocomposite: a potential anticancer nanotherapy antibacterial efficacy of silver nanoparticles on endometritis caused by prevotella melaninogenica and arcanobacterum pyogenes in dairy cattle stability and bioavailability of vitamin d nanoencapsulated in casein micelles detection of single bacterial pathogens with semiconductor quantum dots nanoparticle detection of respiratory infection effects of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine on sperm function in vitro new trials of use of molasses and garlic extracts for competing mycotoxicosis study the availability of using buckthorn (rhamnus cathartica) plant extract in laboratory control of some bacterial and fungal diseases efficacy of use of forskolin plant extract in control of toxic effects of aflatoxicosis in food prevalence of yeast infections in small ruminants with particular references to their treatment by some natural herbal extracts effect of zinc oxide nanoparticles on the growth of mycotoxigenic mould biosynthesis of silver nanoparticles (ag-nps) (a model of metals) by candida albicans and its antifungal activity on some fungal pathogens (trichophyton mentagrophytes and candida albicans) using of molecular biology techniques for detection of c. neoformance in respiratory disorders in cow with references to its control by nanoparticles of iron oxide the possibility of using zinc oxide nanoparticles in controlling some fungal and bacterial strains isolated from buffaloes herbal biosynthesis of zinc nanoparticles and evaluation of their antifungal and antibacterial effect for buffaloes skin affections antimicrobial potential of iron oxide nanoparticles in control of some causes of microbial skin affection in cattle highly sensitive and rapid determination of escherichia coli o :h in minced beef and water using electrocatalytic gold nanoparticle tags the efficiency of using silver nanoparticles singly and in combination with traditional antimicrobial agents in control of some fungal and bacterial affection of buffaloes efficacy of zinc oxide nanoparticles and curcumin in amelioration the toxic effects in aflatoxicated rabbits evaluation of the efficacy of ozone fumigation and zinc oxide nanoparticles in control of aflatoxins contamination in cattle feeds chapter : toxic and benefit effects of carbon nanomaterials on human and animal health. in: carbon nanomaterials for agri-food and environmental applications transgenic mouse sperm that have green acrosome and red mitochondria allow visualization of sperm and their acrosome reaction in vivo ultra stable, highly fluorescent, and water-dispersed silicon-based nanospheres as cellular probes current and future prospects for nanotechnology in animal production nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance induction of immune responses in sheep by vaccination with liposome-entrapped dna complexes encoding toxoplasma gondii mic gene liposomal encapsulated anti-cancer drugs silica nanoparticles as a delivery system for nucleic acid based reagents advax™, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology antifungal effect of sodium dodecil sulfate and nano particle zno on growth inhibition of standard strain of candida albicans fluorescence in nanobiotechnology: sophisticated fluorophores for novel applications endostar-loaded peg-plga nanoparticles: in vitro and in vivo evaluation effects of nano calcium carbonate and nano calcium citrate on toxicity in icr mice and on bone mineral density in an ovariectomized mice model immunogenic properties of a bcg adjuvant chitosan nanoparticle-based dengue vaccine in human dendritic cells mesoporous silica nanoparticles for drug delivery and bios improved serodiagnosis of hydrated cyst disease using gold nanoparticle labeled antigen b in naturally infected sheep escherichia coli signal peptidase recognizes and cleaves the signal sequence of xylanase from a newly isolated bacillus subtilis strain r a novel dynamic flow immunochromatographic test (dfict) using gold nanoparticles for the serological detection of toxoplasma gondii infection in dogs and cats antimicrobial efficacy of zinc oxide quantum dots against listeria monocytogenes, salmonella enteritidis, and escherichia coli o :h characterization of the drug retention and pharmacokinetic properties of liposomal nanoparticles containing dihydrosphinomyelin inorganic-organic hybrid nanomaterials for therapeutic and diagnostic imaging applications photodynamic inactivation of enveloped viruses by buckminsterfullerene one-step, nanoparticle-mediated bacterial detection with magnetic relaxation rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation emerging nanotechnology-based strategies for the identification of microbial pathogenesis carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction controlled release of paclitaxel from microemulsion containing plga and evaluation of anti-tumor activity in vitro and in vivo single-walled carbon nanotubes exhibit strong antimicrobial activity microbial cytotoxicity of carbon based nanomaterials: implications for river water and wastewater effluent magnesium and iron nanoparticles production using microorganisms and various salts enhanced efficacy of liposome-encapsulated ribavirin against rift valley fever virus infection in mice. antimicrob. agents chemother toxicology of carbon nanotubes-a review incorporation of amphotericin b in tuftsin-bearing liposomes showed enhanced efficacy against systemic cryptococcosis in leucopenic mice silver nanoparticles impair peste des petits ruminant's virus replication application of chitosan and chitosan nanoparticles for the control of fusarium head blight of wheat fusarium graminearum in vitro and greenhouse applications of dendrimers in bio-organic chemistry synthesis and characterization of co/cdse core/shell nanocomposites: bifunctional magnetic-optical nanocrystals organically modified silica nanoparticles co-encapsulating photosensitizing drug and aggregationenhanced two-photon absorbing fluorescent dye aggregates for two photon photodynamic therapy antifungal effect of silver nanoparticles on dermatophytes multimodal drug delivery using gold nanoparticles dendrimers: properties and applications concomitant liposomal doxorubicin and daily palliative radiotherapy in advanced feline soft tissue sarcomas gene transfection into adherent cells using electroporation on a dendrimer-modified gold electrode recent trends in rapid environmental monitoring of pathogens and toxicants: potential of nanoparticle-based biosensor and applications behavior of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labeling for magnetic resonance imaging role of nanotechnology in targeted drug delivery and imaging: a concise review use of an amphotericin b lipid complex for treatment of blastomycosis in dogs development of a nanoparticulate formulation of diminazene to treat african trypanosomiasis biodistribution of a high dose of diamond, graphite, and graphene oxide nanoparticles after multiple intraperitoneal injections in rats chapter -nanosensors for the detection of food contaminants a mesoporous silica nanosphere-based carrier system with chemically removable cds nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules pulmonary toxicity of single wall carbon nanotubes in mice and days after intratracheal instillation disposition of aerosolized liposomal amphotericin b bacteriicidal effect of silver nanoparticles against multidrug-resistant treatment of mycobacterium tuberculosis-infected macrophages with poly (lactic-co-glycolic acid) microparticles drives nfκb and autophagy dependent bacillary killing functionalized carbon nanoparticles: syntheses and applications in optical bioimaging and energy conversion intranasal administration of novel chitosan nanoparticle/dna complexes induces antibody response to hepatitis b surface antigen in mice quality and safety aspects of meat products as affected by various physical manipulations of packaging materials synergistic anticancer effects achieved by co-delivery of trail and paclitaxel using cationic polymeric micelles synthesis of stable silicadye hybrid nanomaterial as dna carrier dual-color photoacoustic lymph node imaging using nanoformulated naphthalocyanines the use of lipid formulations of amphotericin b for systemic fungal infections injectable nanocarriers for biodetoxification nanomaterials in the application of tumor vaccines: advantages and disadvantages clinical study of harvesting lymph nodes with carbon nanoparticles in advanced gastric cancer: a prospective randomized trial porphyrin-based carbon dots for photodynamic therapy of hepatoma detection of mycobacteria based on functionalized quantum dots coupled with immunomagnetic separation effect of nano-zinc oxide on the production and dressing performance of broiler sharper and faster nano darts kill more bacteria: a study of antibacterial activity of individually dispersed pristine single-walled carbon nanotube ph-responsive nanogated ensemble based on gold-capped mesoporous silica through an acid-labile acetal linker understanding the toxicity of carbon nanotubes construction of poly (vinyl alcohol)/poly (lactide-glycolide acid)/vancomycin nanoparticles on titanium for enhancing the surface self-antibacterial activity and cytocompatibility genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein mri tracking of bone marrow mesenchymal stem cells labeled with ultra-small superparamagnetic iron oxide nanoparticles in a rat model of temporal lobe epilepsy sensory trial to assess the acceptability of zinc fortificants added to iron-fortified wheat products how will nanotechnology affect agricultural supply chains effects of chitosan on rat knee cartilages biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals the use of liposomally-entrapped gentamicin in the treatment of bovine staphylococcus aureus mastitis comparison of cellular accumulation, tissue distribution, and anti-hiv activity of free and liposomal t, t-dideoxycytidine liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens gadolinium(iii) do a macrocycles and polyethylene glycol coupled to dendrimers effect of molecular weight on physical and biological properties of macromolecular magnetic resonance imaging contrast agents nanotechnology in food safety and quality assessment: potentiality of nanoparticles in diagnosis of foodborne pathogens cobalt doped zno prepared by electrochemistry: chemistry, morphology, and magnetism nanotechnology: assessing the risks an investigation of blood selenium concentrations of goats in new york state applications of nanotechnology in veterinary therapeutics development of colloidal gold-based immunochromatographic assay for rapid detection of mycoplasma suis in porcine plasma quantum dots for live cells, in vivo imaging, and diagnostics nonviral vectors for gene delivery development of magnetic field control for magnetically targeted drug delivery system using a superconducting magnet colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery growth performance and serum biochemical parameters as affected by nano zinc supplementation in layer chicks introduction to metallic nanoparticles nanomedicine: prospective diagnostic and therapeutic potential polyvinylpyrrolidone-coated gold nanoparticles inhibit endothelial cell viability, proliferation, and erk / phosphorylation and reduce the magnitude of endothelial-independent dilator responses in isolated aortic vessels an overview of nanomedicine in veterinary science integrated electrochemical immunosensor with gold nanoparticles for the determination of progesterone nanotoxicology-characterization, dosing and health effects evaluation of an immunochromatographic assay for rapid detection of salmonella enterica serovars typhimurium and enteritidis nanotechnology: a new frontier in food science polymeric and ceramic nanoparticles in biomedical applications effect of metal nanoparticles on the growth of ochratoxigenic moulds and ochratoxin a production isolated from food and feed application of nanotechnology for animal health and production improvement: a review clastogenic and aneugenic effects of multi-wall carbon nanotubes in epithelial cells nanostructured t mri contrast agents effect of metal element nanoparticles in the growth of aflatoxogenic a. flavus and aflatoxin production in feed effect of metal nanoparticles in comparison with commercial antifungal feed additives on the growth of a. flavus and aflatoxin b production an introduction to nanotechnologies: what's in it for us? references national nanotechnology initiative recent advancement in biosensors technology for animal and livestock health management superparamagnetic nanoparticles for biomedical applications: possibilities and limitations of a new drug delivery system development and applications of mesoscopic hall microprobes nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction immunochromatographic gold-based test strip for rapid detection of infectious bursal disease virus antibodies band gap fluorescence from individual singlewalled carbon nanotubes in vivo effects: methodologies and biokinetics of inhaled nanomaterials translocation of inhaled ultrafine particles to the brain peg modified liposomes containing crx- adjuvant in combination with methylglycol chitosan enhance the murine sublingual immune response to influenza vaccination increased conception rates in beef cattle inseminated with nanopurified bull semen phosphatidylinositol di-mannoside and derivates modulate the immune response to and efficacy of a tuberculosis protein vaccine against mycobacterium bovis infection preparation and effects of nano mineral particle feeding in livestock: a review evaluation of polytetrafluoroethylene-graphite-coated total hip prostheses in goats superovulatory response to fsh and embryo recovery rate in pandharpuri buffaloes (bubalus bubalis) electrical detection of single viruses toxicity of titanium oxide nanoparticles causes functionality and dna damage in buffalo (bubalus bubalis) sperm in vitro nanocarriers as an emerging platform for cancer therapy particulate air pollution and risk of st-segment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the exposure and risk assessment for fine and ultrafine particles in ambient air (ultra) study detection of volatile organic compounds in cattle naturally infected with mycobacterium bovis synthesis and characterization of monodisperse hollow fe o nanoparticles development of an immunochromatographic strip for rapid detection of h subtype avian influenza viruses chitosan nanoparticles: a contribution to nanomedicine advax™, a novel microcrystalline polysaccharide particle engineered from delta inulin, provides robust adjuvant potency together with tolerability and safety antioxidant supplementation and purification of semen for improved artificial insemination in livestock species quantum dots in bioanalysis: a review of applications across various platforms for fluorescence spectroscopy and imaging effect of silver nanoparticles on growth performance, metabolism and microbial profile of broiler chickens the use of synthetic carriers in malaria vaccine design enhanced noninflammasome mediated immune responses by mannosylated zwitterionic-based cationic liposomes for hiv dna vaccines silica sol-gel for the controlled release of antibiotics. i. synthesis, characterization, and in vitro release nanoparticle-induced platelet aggregation and vascular thrombosis a polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent zinc oxide nanoparticles: opportunities and challenges in veterinary sciences lead and cadmium concentrations in goat, cow, sheep, and buffalo milks from different regions of iran enhancing the milk production and immunity in holstein friesian crossbred cow by supplementing novel nano zinc oxide cationic silica nanoparticles as gene carriers: synthesis, characterization and transfection efficiency in vitro and in vivo antimicrobial effect of biologically prepared silver nanoparticles (agnps) on two different obturator's soft linings in maxillectomy patients promises, facts and challenges for graphene in biomedical applications saponin-loaded chitosan nanoparticles and their cytotoxicity to cancer cell lines in vitro size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis short-and long-term effect of oral administration of micellized natural vitamin e (d-α-tocopherol) on oxidative status in racehorses under intense training lower oral doses of micellized α-tocopherol compared to α-tocopheryl acetate in feed modify fatty acid profiles and improve oxidative status in pigs immunization with a hemagglutinin-derived synthetic peptide formulated with a cpg-dna-liposome complex induced protection against lethal influenza virus infection in mice cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data oxidative dna damage from nanoparticle exposure and its application to workers' health: a literature review a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection micro-and nanosensors for medical and biological measurement nanostructures in biodiagnostics fluorescent nanoparticles for intracellular sensing: a review a novel hepatitis b vaccine containing advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing synthesis and characterization of biodegradable thiolated chitosan nanoparticles as targeted drug delivery system demonstration of a ion mechanism between a fluid bactericidal liposomal formulation and bacterial cells recent developments in liposome-based veterinary therapeutics recent trends in nanobiosensors and their applicationsa review nanotechnology: the future medicine effects of dietary supplements of zinc-methionine on milk production, udder health and zinc metabolism in dairy goats dendrimers: a class of polymers in the nanotechnology for the delivery of active pharmaceuticals biological interactions of graphenefamily nanomaterial: an interdisciplinary review current progress in oocyte and embryo cryopreservation by slow freezing and vitrification risk assessment of engineered nanomaterials and nanotechnologies-a review quantitative evaluation of antibacterial activities of metallic oxide powders (zno, mgo and cao) by conduct metric assay influence of hydrocolloidal silver nanoparticles on gastrointestinal microflora and morphology of enterocytes of quails superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury a pilot toxicology study of single-walled carbon nanotubes in a small sample of mice nanotechnology and animal health treatment with individualized homeopathic remedies unsuccessful enhancement of intestinal insulin absorption by bile salt-fatty acid mixed micelles in dogs drug-drug co-crystals dendrimers in biomedical sciences and nanotechnology gold nanoparticles: various methods of synthesis and antibacterial applications dna damaging potential of zinc oxide nanoparticles in human epidermal cells perturbational profiling of nanomaterial biologic activity evaluation of the antimicrobial effect of zinc oxide nanoparticles on listeria monocytogenes and candida albicans isolated from infected egyptian buffalo suffering from abortion iron oxide nanoparticles suppressed t helper cell-mediated immunity in a murine model of delayed-type hypersensitivity biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells silica nanocrystals: a controlled organic-inorganic interface and its implications of color-tuning and chemical design toward sophisticated architectures structure, synthetic methods, magnetic properties and biomedical applications of ferrofluids unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice synthesis of a fullerene derivative for the inhibition of hiv enzymes food packaging based on polymer nanomaterials encapsulation of bacteriophage in liposome accentuates its entry in to macrophage and shields it from neutralizing antibodies effect of different levels of nanoparticles chromium picolinate supplementation on performance, egg quality, mineral retention, and tissues minerals accumulation in layer chickens treatment with hpma copolymer-based doxorubicin conjugate containing human immunoglobulin induces long-lasting systemic anti-tumour immunity in mice effect of surface functionalization of mcm- -type mesoporous silica nanoparticles on the endocytosis by human cancer cells mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers mesoporous silica nanoparticles: structural design and applications enhanced antibacterial effect of antibiotics in combination with silver nanoparticles against animal pathogens intracellular nanoparticle coating stability determines nanoparticle diagnostics efficacy and cell functionality surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles chitosan nanoparticles properties and applications pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties synthesis of pyrrolizidine alkaloids via , -dipolar cycloaddition involving cyclic nitrones and unsaturated lactones metal oxide nanoparticles as bactericidal agents application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review magnetic nanoparticles in mr imaging and drug delivery nanographene oxide for cellular imaging and drug delivery novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of staphylococcus aureus in milk in vitro and in vivo antifungal activity of amphotericin b lipid complex: are phospholipases important? gold nanoparticle based microbial detection and identification nanobioimaging and sensing of infectious diseases the preparation of magnetic nanoparticles for applications in biomedicine outbreak of foetal infection with bovine pestivirus in a central queensland beef herd mesoporous silica nanospheres as highly efficient mri contrast agents reducing infections through nanotechnology and nanoparticles hybrid nanomaterials for biomedical applications carbon nanotubes in cancer theragnosis livestock production: recent trends, future prospects skin as a route of exposure and sensitisation in chronic beryllium disease science and application of nanotubes lipid-core micelles for targeted drug delivery mesoporous silica nanoparticles deliver dna and chemicals into plants research strategies for safety evaluation of nanomaterials, part iv: risk assessment of nanoparticles dna sequence motifs for structure specific recognition and separation of carbon nanotubes facile and sensitive epifluorescent silica nanoparticles for the rapid screening of ehec penicillin-bound polyacrylate nanoparticles: restoring the activity of β-lactam antibiotics against mrsa a randomized trial investigating the efficacy and safety of water-soluble micellar paclitaxel (paccal vet) for treatment of nonresectable grade or mast cell tumors in dogs a new property of mcm- : drug delivery system adjuvant effect of gantrez ® an nanoparticles during oral vaccination of piglets against f + enterotoxigenic escherichia coli improved biocompatibility and pharmacokinetics of silica nanoparticles by means of a lipid coating: a multimodality investigation macromolecules, dendrimers, and nanomaterials in magnetic resonance imaging: the interplay between size, function, and pharmacokinetics nanoparticles applications for improving the food safety and food processing photoinduced intracellular controlled release drug delivery in human cells by gold-capped mesoporous silica nanosphere tuning the cellular uptake and cytotoxicity properties of oligonucleotide intercalator-functionalized mesoporous silica nanoparticles with human cervical cancer cells hela mesoporous silica nanoparticles for intracellular controlled drug delivery mesoporous silica nanoparticles: synthesis and applications development of immunochromatographic strip test using fluorescent, micellar silica nanosensors for rapid detection of b. abortus antibodies in milk samples effect of chromium nanoparticle on growth performance, carcass characteristics, pork quality and tissue chromium in finishing pigs a biocompatible method of decorporation: bisphosphonate-modified magnetite nanoparticles to remove uranyl ions from blood acute toxicological impact of nano-and submicro-scaled zinc oxide powder on healthy adult mice bioconjugated silica nanoparticles: development and applications unique aggregation of anthrax (bacillus anthracis) spores by sugar coated single walled carbon nanotubes fluorescent dye encapsulated zno particles with cell-specific toxicity for potential use in biomedical applications recent advances of chitosan nanoparticles as drug carriers sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin m in milk effects of chromiumloaded chitosan nanoparticles on growth, blood metabolites, immune traits and tissue chromium in finishing pigs development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses effects of chromiumloaded chitosan nanoparticles on growth, carcass characteristics, pork quality, and lipid metabolism in finishing pigs host-guest carbon dots as high-performance fluorescence probes anti-microbial porcelain enamels nanocrystalline silver dressing and subatmospheric pressure therapy following neoadjuvant radiation therapy and surgical excision of a feline injection site sarcoma biocompatible carbon nanotubes generated by functionalization with glycodendrimers sentinel lymph node detection using carbon nanoparticles in patients with early breast cancer bioactive cell-like hybrids coassembled from (glyco) dendrimersomes with bacterial membranes drug-loaded carbon nanoparticle suspension injection, drug selection, releasing behavior, in vitro cytotoxicity and in vivo lymph node targeting bioaccumulation and toxicity of carbon nanoparticles suspension injection in intravenously exposed mice skeleton labeled c-carbon nanoparticles for the imaging and quantification in tumor drainage lymph nodes new forms of superparamagnetic nanoparticles for biomedical applications dopamine as a robust anchor to immobilize functional molecules on the iron oxide shell of magnetic nanoparticles nitrilotriacetic acid-modified magnetic nanoparticles as a general agent to bind histidine-tagged proteins physiochemical aspects of drug delivery and release from polymer-based colloids effects of nanometre zno on growth performance of early weaned piglets carbon dots as nontoxic and high performance fluorescence imaging agents antimicrobial activity of single walled carbon nanotubes length effect effects of gestational age and surface modification on materno-fetal transfer of nanoparticles in murine pregnancy antiviral activity of graphene oxide: how sharp edged structure and charge matter magnetic nanoparticles for cancer diagnosis and therapy formation of hollow nanocrystals through the nanoscale kirkendall effect nanoparticle-based bio-barcode assay for the detection of bluetongue virus dumbbell-like bifunctional au-fe o nanoparticles effects of silver nanoparticles on multiple drug-resistant strains of staphylococcus aureus and pseudomonas aeruginosa from mastitis-infected goats: an alternative approach for antimicrobial therapy enhanced antibacterial effect of ceftriaxone sodium-loaded chitosan nanoparticles against intracellular salmonella typhimurium tumor, tissue, and plasma pharmacokinetic studies and antitumor response studies of docetaxel in combination with -nitrocamptothecin in mice bearing skov- human ovarian xenografts efficacy of trivalent chromium on growth performance, carcass characteristics and tissue chromium in heat-stressed broiler chicks biofabrication of a novel biomolecule-assisted reduced graphene oxide: an excellent biocompatible nanomaterial investigation into the antibacterial behavior of suspensions of zno nanoparticles (zno nanofluids) degradable disulfide core-crosslinked micelles as a drug delivery system prepared from vinyl functionalized nucleosides via the raft process silver nanoparticles: synthesis, characterization, properties, applications, and therapeutic approaches bioconjugated silica nanoparticles for bioanalysis mesoporous silica nanoparticlebased double drug delivery system for glucose-responsive controlled release of insulin and cyclic amp advances in rapid detection methods for foodborne pathogens pegylated mesoporous silica as a redox-responsive drug delivery system for loading thiolcontaining drugs antibacterial activities of gold and silver nanoparticles against escherichia coli and bacillus calmette-gu erin recent developments in antibody-based assays for the detection of bacterial toxins carbon nanotube-based nanocarrier loaded with ribavirin against grass carp reovirus carbon nanoparticle-guided central lymph node dissection in clinically nodenegative patients with papillary thyroid carcinoma liposomes as immunological adjuvants cancer nanotechnology: application of nanotechnology in cancer therapy chapter : synthesis, characterization, and applications of carbon nanotubes functionalized with magnetic nanoparticles fungal nanotechnology: a new approach toward efficient biotechnology application ultrasensitive dna detection using highly fluorescent bioconjugated nanoparticles the authors of this chapter gratefully acknowledge dr. m.k. refai, professor of microbiology, cairo university, for his continuous assistance and advice in initiating the achievement of this work. acknowledgments key: cord- -woqb t authors: choukér, alexander; stahn, alexander c. title: covid- —the largest isolation study in history: the value of shared learnings from spaceflight analogs date: - - journal: npj microgravity doi: . /s - - - sha: doc_id: cord_uid: woqb t the world is currently experiencing the largest isolation experiment in history. in an attempt to slow down the spread of the covid- pandemic numerous countries across the world have been shutting down economies, education, and public life. governments have mandated strict regulations of quarantine and social distancing in an unprecedented manner. the effects of these measures on brain, behavior, neuro-humoral and immunological responses in humans are largely unknown. life science research for space exploration has a long history in using high-fidelity spaceflight analogs to better understand the effect of prolonged isolation and confinement on genes, molecules, cells, neural circuits, and physiological systems to behavior. we here propose to leverage the extensive experience and data from these studies and build a bridge between spaceflight research and clinical settings to foster transdisciplinary approaches to characterize the neurobehavioral effects on the immune system and vice versa. these approaches are expected to develop innovative and efficient health screening tools, diagnostic systems, and treatments to mitigate health risks associated with isolation and confinement on earth and during future exploratory spaceflight missions. throughout history infectious diseases have been one of the greatest threats to global public health. viruses have killed by far more humans than other disease, war or natural disaster. the outbreak of the novel coronavirus sars-cov- shows that the risk of infectious diseases can rapidly put our health systems to the test and turn the world economy, education, and public life upside down. research is just beginning to understand the full impact of the disease. there is increasing evidence that the current pandemic affects global health and well-being in many more ways than a respiratory disease, including the central nervous system and neurological diseases . the brain's susceptibility to sars-cov- could also be a potential risk factor for alzheimer's disease . in addition to the clinical manifestations associated with covid- , physical distancing policies to control the spread of the diseases have led to large-scale and unprecedented social isolation across the globe. the prevalence of social isolation and loneliness in response to the covid- pandemic is currently unclear, with estimates varying between and % . it is considered the greatest international biopsychosocial emergency the world has faced for a century . recent research reviewed the psychological implications of prolonged isolation and confinement associated with quarantine, reporting posttraumatic and acute stress symptoms, confusion, and anger in response to quarantaine . the interventions to mitigate the negative effects on mental health and well-being include befriending schemes, individual and group therapies, various shared activity programs, social prescription by healthcare providers, and diverse strategies using information and communication technologies, but their effectiveness and relevance to different age groups remains to be determined . reduced sensory stimulation and sensory monotony experienced in isolated, confined, and extreme (ice) environments are also expected to be a major risk during future spaceflight exploratory class missions . the "absence of environmental stimulation" is expected to account for sleep disruptions, impaired cognitive performance, negative affect, and interpersonal tension and conflict during exploratory spaceflight missions . space agencies and their human research programs have a long history of seeking to understand the effects of isolation and confinement on astronaut health and performance. consequently, developing strategies to mitigate the risks of adverse behavioral conditions and psychiatric disorders associated with human spaceflight are of critical importance . it may therefore not be surprising that the value of shared learnings from spaceflight during covid- has been emphasized in numerous news reports and social media channels, providing anecdotal evidence for the parallelism of isolation and confinement in extreme environments and in the current pandemic [ ] [ ] [ ] . here, we summarize the opportunities of spaceflight analogs to accelerate ( ) the understanding of the neurobehavioral and immunological consequences of social isolation during the covid- pandemic, and ( ) the development of innovative and efficient treatment strategies to mitigate adverse behavioral conditions. more than years of research in space has revealed how the unique physiological conditions and stresses of space affect almost every biological system in all kingdoms of life; from plants, to bacteria, to humans. man evolved in a world where newton's apple never fails to fall and at no stage did evolution prepare us for the eventuality that we may one day be exposed to life in zero gravity while confined in a hazardous environment. spaceflight affects the entire human body. the lack of gravity results in considerable bone loss much akin to osteoporosis and muscle wasting , cardiovascular deconditioning , renal stones and kidney dysfunction . however, besides these physiological effects of microgravity and multiple environmental toxicants in space, the psychological stressors are exhaustive and additive: life in a tin can, lack of privacy, separation from friends and family, high workload, operational and interpersonal distress, dietary restrictions, noise, circadian disorders and sleep loss . the development of adverse behavioral conditions and psychiatric disorders during future long-duration space missions (ldsm) are considered one of the most serious, but also least understood risks . isolation and confinement are significant contributors to these unmitigated risks, and can lead to mental health symptoms, impulse control deficits, workplace errors, and even increased mortality . the beginning of the study of isolation and confinement can be marked by polar expeditions, providing anecdotal evidence of the psychological and physiological challenges associated with prolonged isolation and confinement such as sleep disorders, mood disturbances, depression, anxiety, paranoia, and suicide . the european space agency (esa) early recognized the need to study the effects of isolation and confinement on mental wellbeing and performance to enable safe and successful spaceflight. since the s the european space agency (esa) has been consistently sponsoring studies in isolated, controlled and confined analogs (icc), starting with the -day campaign "isolation study for the european manned space infrastructure" (isemsi) campaign in bergen norway in , followed by the -day study "experimental campaign for the european manned space infrastructure (exemsi) carried out at the german aerospace center (dlr) in cologne, germany in . in esa cooperated with the institute of biomedical problems (ibmp) in moscow, russia, to further the understanding of the psychophysiological effects of isolation and confinement and extended the duration of the isolation period to days in the "human behaviour in extended spaceflight" (hubes) experiment, and up to days (two crews were isolated for days, and one crew was isolated for days, respectively) in the "simulation of the flight of the international crew on space station" (sfincss) experiment. these studies lay the groundwork for the longest isolation experiment in history, i.e., the mars study, which exposed six international crewmembers to days of isolation and confinement in ibmp's nek facility in . the success of the isolation studies at ibmp is now continued in close collaboration with nasa and esa in a series of isolation studies as part of the "scientific international research in unique terrestrial station" (sirius) project . in nasa started to simulate operations and test spacewalk techniques using a highly unique underwater habitat (nasa extreme environment mission operations, neemo) with mission durations of up to three weeks . since nasa has also been supporting prolonged isolation studies using an analog habitat hi-seas (hawai'i space exploration analog and simulation) on the island of hawaii . more recently, nasa has been using its human exploration research analog (hera) facility at johnson space center to investigate the effects of isolation and confinement for up to days . except for several russian studies using the nek complex [ ] [ ] [ ] , and chinese experiments in chinese celss (controlled ecological life support system) , laboratory-based isolation studies in isolated, controlled and confined analogs (icc) have often been rather short in duration (< days). submariners and antarctic stations can provide excellent opportunities to study the effects of prolonged isolation and confinement in real extreme environments (isolated, confined and extreme environments, ice). despite more than antarctic research stations, only very few qualify as high-fidelity analogs for space research. suitable stations are characterized by a small crew sizes (i.e., < ), extended mission duration of one year or longer, and include complex logistical operations with only limited or no rescue capabilities during the antarctic winter. the complications associated with isolations and confinement depend on a plethora of factors including individual issues such as personality, coping strategies, psychological support, crew dynamics, and mission duration. physical isolation may also not necessarily have any adverse effects per se. physical isolation and perceived social isolation, the subjective distressed feeling of being alone or separated can be related, but physical isolation is not a sufficient condition for loneliness . a common denominator of perceived social isolation is that it translates to physiological stress responses via the sympathetic nervous system and the hypothalamic-pituitary-adrenal (hpa) axis. loneliness has been shown to increase chronic sympathetic tone, oxidative stress, and hpa activation, and to decrease anti-inflammatory responses, and the expression of genes regulating glucocorticoid responses, ultimately leading to glucocorticoid resistance . two organ systems that are profoundly challenged by stress responses pertain to our host defense and our central control: the brain and the immune system. stress can impact nearly any brain region, but one brain structure that is of particular importance is the hippocampus. the hippocampus plays a pivotal role in episodic memory formation, mapping spatial relationships and performing navigational tasks. given the importance of visuospatial abilities during operations such as docking, landing, exploring and navigating in new environments and on planets with low gravity, it is imperative to understand the impact of spaceflight on spatial cognition and its neural basis. because of the high density of corticosteroid receptors in the hippocampus it is not surprising that this brain region is very vulnerable to increased stress levels . both short-and long-term social isolation has been shown to reduce hippocampal long-term potentiation in rodents , . the dentate gyrus (dg) of the hippocampus is likely to play a key role in defining the impact of stress on hippocampal functioning. we recently published data on neuroendocrine and brain changes in response to antarctic expeditions at the german neumayer iii station characterized by multiple stressors including environmental deprivation and prolonged physical and social isolation . our data demonstrated considerable decreases in dentate gyrus volume that were associated with changes in key neurotrophins such as brain-derived neurotrophic factor (bdnf). the reductions in dentate gyrus volume were also associated with lower cognitive performance in tests of spatial processing and the resolution of response conflicts test but there was no reduction in performance in other cognitive tests relying primarily on motor speed and attention, and manual dexterity. these data confirm the impact of environmental and social variation on hippocampal plasticity in humans . the distinct changes of the dentate gyrus to environmental deprivation in comparison to other hippocampal subfields is similar to findings from animal models, suggesting a possible link between hippocampal neurogenesis, stress-induced behavioral changes, and environmental deprivation [ ] [ ] [ ] . furthermore, whole-brain analysis using voxel-based morphometry revealed significant decreases in gray matter volume of the right dorsolateral prefrontal cortex (dlpfc), and left orbitofrontal cortex (ofc) after the -month expedition . the dlpfc and the ofc are pivotal for executive control such as response inhibition, working memory and cognitive flexibility , but also the generation of and regulation of emotion . projections between the hippocampus and orbitofrontal cortex (ofc), and to some extent also the dlpfc , can foster cross-structural communication, and might interact to influence behavior . data from the icc analog nek also suggest decreases in white matter integrity of the right temporoparietal junction (tpj) after prolonged isolation and confinement . whereas these data need to be interpreted cautiously because of their cross-sectional nature (reductions in white matter integrity were not reported within subjects over time), and a very small sample size, the results raise interesting questions about the effects of sensory deprivation on the brain during isolation and confinement that should be considered in future ice/icc experiments. the right tpj integrates multisensory information and has been suggested to play a critical role for reorienting of attention, i.e., being able to respond quickly to unexpected events in the surroundings, and social processes . we attribute these effects to both sensory deprivation as well as lack of diverse social interactions associated with the prolonged isolation and confinement. together, these data suggest that prolonged isolation and confinement can have considerable, differential effects on brain structures involved in various complex cognitive controls including learning and memory formation, spatial navigation, self-control, planning, problem-solving, and emotional control. notably, these data are characterized by considerable inter-individual differences in their response to social and environmental monotony. in addition, the recovery of these changes is currently unknown. the immune system could be a critical pathway in better understanding the specific neurobehavioral phenotypes. for instance, recent nasa funded research suggests that social isolation influences cytokine levels in the hippocampus . the immune system is among the largest human organs and consists of more than four trillion cells. it affects every organ and influences almost every disease state from infection, to cancer, to cardiovascular disease and bone homeostasis. most remarkable about the immune system is its adaptability to protect us rapidly and efficiently from the pathogenesis and progression of bacterial and viral infections. using an orchestra of innate and adaptive response, it regulates a critical and delicate balance between health and disease. any disruption of this equilibrium can lead to life threatening infections, autoimmune diseases, and cancer. understanding this balance is vital to ensuring adequate immunity and health, which in a case of imbalance can lead to immune aging, viral reactivation, and hypersensitivities/allergies . these gradual and inter-individual effects of psychological and environmental stressors are true for astronauts , overwintering crews and participants voluntarily isolated and confined for the sake of research , and also thereafter, when being re-exposed to everyday life again . at the same this applies as much to people on earth exposed to chronic emotional and physical stress-as it is happening right now around the world as a consequence of the covid- pandemic. the parallels between social distancing and isolation and confinement and spaceflight analogs offer opportunities for shared learnings between these settings. for example, shedding of dormant herpes virus in saliva might be a very helpful surrogate marker of immune dysfunction. this could be internationally set and include many cases as clustered in different areas. care points or pharmacies could enable data collections on a consensual basis. in addition, crowdsourcing could be supported by smart phone-based applications collecting self-reported data. an example is the epb (european polar board)-esa project 'choicee' for the monitoring immune deficiencies after isolation , and its potential applicability also in following the impact of infections with sars-cov- and the quarantine effects. a caveat of such epidemiological studies is often the lack of pre-pandemic reference data. cross-sectional data from health controls including pre-mission data from spaceflight and spaceflight analogs can serve as a comparison for evaluating the effects of the covid- pandemic. data collected before the pandemic could be particularly valuable for establishing such a reference. for instance, the combination of self-reported data, medical records, biospecimen and neuroimaging collected during the previous months prior to the covid- pandemic and medical records could be considered either as a reference for between-subject comparisons or extended by follow up data collections, to foster a pre/post comparison within subjects. in particular, investigating brain morphological changes in a longitudinal manner could provide valuable information about the effects of social isolation on the brain plasticity, and verify the data we previously reported in response to prolonged isolation and confinement in extreme environments . further, future isolation and confinement studies of healthy individuals without a history of covid- will remain a critical source for maximizing the synergies between spaceflight research and the clinical manifestations associated with social isolation. need for countermeasures against the detrimental effects of isolation and confinement to mitigate the adverse health effects associated with isolation and confinement, innovative countermeasures are critically needed. this need was recognized as early as the one of the first antarctic expeditions. the crew of the belgian expedition "belgica" led by adrien de gerlache de gomery became the first to endure an antarctic winter after being accidentally trapped among ice packs in the bellinghausen sea in . frederick cook's reports from that expedition can perhaps be considered as one of the observations of melancholy and depression observed during prolonged isolation and confinement. to counter these symptoms cook prescribed two actions, which may be considered as one of first exercise and light interventions to treat depression , . first, the "baking treatment" had crew members stay in front of the stove's fire for several hours each day . the radiant heat and light associated with the fire were expected to foster relaxation and restoration, and induce positive mood. second, cook requested the crew to take daily walks around the ship to engage in physical activity, which became known as the "madhouse promenade" . about years later, there is increasing scientific evidence about the role of exercise as a means of active release, enhancing brain structure and function, and improving cognitive performance , . acute and chronic exercise has also been shown to improve innate and adaptive immunity [ ] [ ] [ ] . recent research highlights physical activity is an important clinical target to sustain and improve mental health during the current pandemic . in addition to exercise, target-specific countermeasures will be needed to specifically address the risks associated with perceived social isolation and sensory deprivation. these could deliver similar restorative effects like the "baking treatment" during the belgica expedition. virtual reality (vr) technologies are likely to play an important role in meeting these requirements . they can foster a "virtual window", i.e., exploring virtual worlds and induce a high degree of a first-person immersive experience of "being there" , which has been suggested to be of central importance for eliciting restorative effects. the positive outcomes of exposure therapies in vr which include mood induction procedures , , stress reduction , treatment of ptsd , anxieties , and proenvironmentalist behavior with immersive vr altogether have demonstrated the usefulness of this medium in changing our cognitive and emotional mechanisms. currently, various projects sponsored by dlr, esa, nasa and trish have been investigating the potential of vr as a countermeasure to mitigate sensory deprivation and social isolation. for instance, the nasa sponsored project "hybrid training -a sensory stimulation countermeasure for long duration space exploration missions" combines physical exercise with an interactive virtual environment to enhance sensory augmentation and stimulate brain plasticity during prolonged isolation and confinement . the combination of vr technologies and physical exercise was also recently recognized as a. choukér and a.c. stahn a promising coping strategy to promote health and wellness during covid- . former astronaut jay c. buckey and his team developed the dartmouth path program, a series of self-help tools designed by to relieve stress, improve mood, and maintain relationships during prolonged isolation and confinement associated with spaceflight. ongoing research currently investigates whether the path program also help people cope with the emotional stresses brought on by covid- , . likewise, the nasa supported project ansible (a network of social interactions for bilateral life enhancement) delivered a multi-modal digital toolset that leverages virtual worlds to provide a space where humans and intelligent virtual agents can be companions, advisors, provided psychological support, and share experiences . it is intended to virtually connect with their family, friends, and the ground crew to provide a sense of social consistency and permanence. another digital resource to support astronauts during exploration class missions is the crew interactive mobile companion (cimon) project supported by dlr . cimon is a virtual voice-controlled interactive assistant that uses artificial intelligence to foster social interactions between humans and machines, and support astronauts during routine tasks. such approaches could also directly translate to immunological benefits. it has become clear that the immune system is not autonomous and is not solely affected by pathogens alone. instead how we feel, how we interact with others, the conditions in which we live all influence its function. this interaction between our social and emotional lives and the immune system take place via the central nervous system. stress-sensitive neural systems and cells together with humoral pathways coordinate immune cell maturation, responsiveness and their interactions . recently, an international team of esa, nasa and ibmp experts proposed a specific and personalized immune countermeasure prescription for prospective astronauts embarking on deep-space voyage . the combination of different physical, medical and behavioral interventions including therapeutics, nutrient-enriched diets, regular exercise, adequate rest, and stress-relief could be expected to go beyond supporting immunologic responses by affecting other organ systems including the central nervous system. in particular, it is possible that the full synergistic potential of the proposed measures could be further enhanced by combining it with some of the behavioral strategies outlined above. along these lines data collected before the pandemic could be particularly valuable for establishing such a reference. for instance, the combination of self-reported data, medical records, biospecimen and neuroimaging collected during the previous months prior to the covid- pandemic and medical records could be considered either as a reference for between-subject comparisons or extended by follow up data collections, to foster a pre/post comparison within subjects. in particular, investigating brain morphological changes in a longitudinal manner could provide valuable information about the effects of social isolation on the brain plasticity, and verify the data we previously reported in response to prolonged isolation and confinement in extreme environments . further, future isolation and confinement studies of healthy individuals without a history of covid- will remain a critical source for maximizing the synergies between spaceflight research and the clinical manifestations associated with social isolation. spaceflight-related research offers a variety of unique experimental approaches and designs that can foster basic research in life science and drive innovative medical and health-related applications (fig. ). for instance, bedrest studies which can serve as a model for the aging population, and provide highly standardized and controlled conditions to assess the efficacy of new interventions and treatments to mitigate musculoskeletal, cardiovascular, and neurobehavioral risks brought by a lack of physical activity and/or immobilization . likewise, given the specific requirements of spaceflight health monitoring technologies such as size, mass, ease of operation, and non-invasiveness, human spaceflight research programs can promote health technologies on earth. an example is the application of a heat flux-based approach to noninvasively determine core body temperature at the head . the technology was validated and tested in spaceflight analogs and on iss , . the system is now available for patient monitoring in the clinical setting because temperature monitoring is critical for treating infections, detecting complications, and reducing postoperative mortality rates . at the same emerging terrestrial biomedical research and technology development can accelerate the mitigation of risks associated with human spaceflight by providing promising new approaches, treatments, countermeasures or technologies that have practical application to spaceflight. the full scope of the synergies between terrestrial and spaceflight applications remains to be uncovered. recent reviews highlight the need to increase the awareness, training, and collaboration of the research community for revealing the true potential of applying these space technologies to global health settings . several space agencies have begun to address this need and developed initiatives to foster productive crossdisciplinary collaboration between universities, non-university institutions, companies, and governmental and international entities. for instance, the dlr initiatives innospace® masters and space health seek the development of innovative space technologies and services using expertise from various industries combined with existing technologies, services and applications care astronaut health and performance applications. the continuation and extension of these approaches is a key factor to help bridging the gap between experts in spaceflight life sciences, basic life science, and stakeholders in health technology innovation. this is particularly true for mitigating the risks of adverse behavioral conditions associated with isolation and confinement. along this line trish has announced an "industry program " grant call for industry and academic behavioral health experts to contribute towards the development of specific key areas of interest including novel anxiety and stress monitoring techniques, and unobtrusive health monitoring technologies. similar to the oecd council's "recommendation on responsible innovation in neurotechnology" the trish program seeks to provide guidance at each step of the innovation process so that the benefits are maximized and risks minimized. this includes providing companies and pre-companies with ( ) access to experts working in space health care and at nasa, ( ) rapid technology maturation and de-risking, ( ) preparation for transfer to commercial healthcare markets, and ( ) pathways to government sales in the emerging space market. "facts are the air of scientists. without them you can never fly" (linus pauling). that is why space agencies are taking research to the skies and are supporting innovative approaches to better understand how the human body copes with extreme conditions, and how this knowledge can be transferred to and benefit the people on earth. here we used the parallels of spaceflight analogs and the covid- pandemic to illustrate the value of shared learnings for gaining a better understanding of the neurobehavioral and immunological implications of social isolation and how to mitigate related adverse health conditions. since space research is addressing and increasingly influencing the traditional fields of medicine including neurobiology, clinical immunology, and public health of an aging society, this pandemic, the re-entry to normal life and its aftermath should strengthen current inter-and transdisciplinary collaborative research initiatives between space life sciences, basic biomedical science, clinical applications, and industrial stakeholders to mitigate the negative effects of physical and social isolation in health and disease. such collaborative research initiatives combined with historic data from spaceflight analogs could lead to a better understanding of the cause and effects of social distancing and quarantine on health and mental well-being. they can also provide the basis to develop innovative and efficient health screening tools, diagnostic systems, and personalized treatments to mitigate health risks associated with isolation and confinement. partnerships between space agencies, non-space funding bodies, academic institutions and companies will continue to play an important role to translate research from space to earth and scale it effectively. received: may ; accepted: september ; neurological manifestations of patients with covid- : potential routes of sars-cov- neuroinvasion from the periphery to the brain neurologic and immunologic complications of covid- : potential long-term risk factors for alzheimer's disease how the covid- pandemic is focusing attention on loneliness and social isolation research priorities for the covid- pandemic and beyond: a call to action for psychological science the psychological impact of quarantine and how to reduce it: rapid review of the evidence effects of reduced sensory stimulation and assessment of countermeasures for sensory stimulation augmentation psychological effects of polar expeditions space lessons for covid- isolation-and more covid- lockdown advice from astronauts and submariners confined in a small space due to covid- ? here's some tips from an astronaut -abc news muscle and bone-aging and space cardiovascular deconditioning in microgravity: some possible countermeasures men and women in space: bone loss and kidney stone risk after long-duration spaceflight the psychology of isolated and confined environments: understanding human behavior in antarctica the unengaged mind: defining boredom in terms of attention isolation study for european manned space infrastructures): a space psychology experiment exemsi: description of facilities, organization, crew selection, and operational aspects esa -the hubes experiment: a ground-based simulation of a -day manned spaceflight the mars project extreme environment mission operations multi-system adaptation to confinement during the -day controlled ecological life support system (celss) experiment the growing problem of loneliness social isolation stress modulation of hippocampal activity-spotlight on the dentate gyrus social isolation disrupts hippocampal neurogenesis in young non-human primates social isolation stress reduces hippocampal long-term potentiation: effect of animal strain and involvement of glucocorticoid receptors brain changes in response to long antarctic expeditions neural consequences of environmental enrichment social isolation delays the positive effects of running on adult neurogenesis environmental enrichment requires adult neurogenesis to facilitate the recovery from psychosocial stress neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and nmda receptor activation executive functions emotion and the prefrontal cortex: an integrative review nonhuman primate models of hippocampal development and dysfunction over the river, through the woods: cognitive maps in the hippocampus and orbitofrontal cortex changes of brain dti in healthy human subjects after days isolation and confinement on a simulated mission to mars the role of the right temporoparietal junction in attention and social interaction as revealed by ale meta-analysis overexpression of catalase in mitochondria mitigates the inflammatory effects of simulated microgravity and social isolation in mouse hippocampus stress challenges and immunity in space: from mechanisms to monitoring and preventive strategies stress related shift toward inflammaging in cosmonauts after long-duration space flight immune sensitization during year in the antarctic highaltitude concordia environment. allergy eur effects of isolation and confinement on humansimplications for manned space explorations influences of large sets of environmental exposures on immune responses in healthy adult men bold endeavors: lessons from polar and space exploration through the first antarctic night bridging animal and human models of exercise-induced brain plasticity acute aerobic exercise induces a preferential mobilisation of plasmacytoid dendritic cells into the peripheral blood in man a single exercise bout augments adenovirus-specific t-cell mobilization and function a systems biology approach to investigating the influence of exercise and fitness on the composition of leukocytes in peripheral blood physical activity in a pandemic: a new treatment target for psychological therapy being there: the subjective experience of presence positive mood induction procedures for virtual environments designed for elderly people virtual reality and stimulation of touch and smell for inducing relaxation: a randomized controlled trial restorative effects of virtual nature settings virtual reality exposure therapy for anxiety and related disorders: a meta-analysis of randomized controlled trials virtual reality exposure therapy in anxiety disorders: a systematic review of process-andoutcome studies virtual reality exposure therapy in anxiety disorders: a quantitative meta-analysis short-and long-term effects of embodied experiences in immersive virtual environments on environmental locus of control and behavior hybrid training-a sensory stimulation countermeasure for long duration space exploration missions virtual reality exercise as a coping strategy for health and wellness promotion in older adults during the covid- pandemic space medicine research program focuses on people struggling with confinement an interactive media program for managing psychosocial problems on longduration spaceflights maintaining psycho-social health on the way to mars and back cimon-the intelligent astronaut assistant-dlr portal specific immunologic countermeasure protocol for deep-space exploration missions bed rest and accelerated aging in relation to the musculoskeletal and cardiovascular systems and frailty biomarkers: a review the double sensor-a non-invasive device to continuously monitor core temperature in humans on earth and in space circadian rhythms in bed rest: monitoring core body temperature via heat-flux approach is superior to skin surface temperature increased core body temperature in astronauts during longduration space missions tcore tm temperature monitoring system safe, accurate, non-invasive applications of space technologies to global health: scoping review the work was supported by the german space agency (dlr) on behalf of the federal ministry of economics and technology/energy (bmwi) through grants wb , wb , wb , wb , wb , wb ; and the european space agency (esa, elips and scispace programs, topical team # / /nl/vj). we thank the alfred wegener institute, helmholtz centre for polar and marine research in bremerhaven, germany, and the french (ipev) and italian (pnra) polar institutes for continuous logistical and technical support in conducting research in icc. we also like to acknowledge the support from ibmp, dlr, esa and nasa for their initiatives and research support in studies at the nek and hera space analogs. we thank judith-irina buchheim, hanns-christian gunga and david f. dinges for the constructive discussions. the authors declare no competing interests.a. choukér and a.c. stahn correspondence and requests for materials should be addressed to a.c. or a.c.s.reprints and permission information is available at http://www.nature.com/ reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -h czy bh authors: koirala, prashamsa; jung, hyun ah; choi, jae sue title: recent advances in pharmacological research on ecklonia species: a review date: - - journal: arch pharm res doi: . /s - - - sha: doc_id: cord_uid: h czy bh the genus ecklonia (lessoniaceae, phaeophyceae), commonly called kelp (brown algae), is abundant on the coasts of japan and korea. during the past few decades, ecklonia species have received tremendous attention for their wide range of therapeutic properties and multiple health benefits, such as great nutritional value and being rich in vitamins, minerals, dietary fiber, proteins, and polysaccharides. several novel functional ingredients with diversified biological activities have been isolated and possess antimicrobial, antiviral, hepatoprotective, cardioprotective, anti-inflammatory, neuroprotective, anticarcinogenic, immunomodulatory, hypolipidemic, anti-diabetic, and antioxidant therapeutic properties. the present review discusses the phytochemical, pharmacological, therapeutic, nutritional, and health benefits of different species of genus ecklonia, as well as their use in the prevention of disease and maintenance of good health. seaweed is used as a vegetable and traditional medicine in east-asian countries such as japan, korea, and china. it is an important resource of the marine ecosystem, containing active metabolites with probable nutraceuticals that are rich sources of novel bioactive secondary metabolites and harbor diverse classes of health-promoting molecules, including essential dietary fiber, fatty acids, essential amino acids, and vitamins a, b, c, and e (kolb et al. ) . taxonomic classification divides seaweed into three major classes based more on their pigments and coloration than their genetics: rhodophyceae (red algae), phaeophyceae (brown algae), and chlorophyceae (green algae, also known as macroalgae) (rajasulochana et al. ). brown algae have been used for thousands of years, but only in modern times have they been recognized to contain bioactive substances such as polysaccharides, lipids, and polyphenols, with various pharmacological properties (kumar et al. ) . ecklonia is a genus of kelp (brown algae) belonging to the family lessoniaceae that has an abundance of eckoltype phlorotannins. there are nine species: ecklonia biruncinata, ecklonia brevipes, ecklonia cava (ec), ecklonia fastigiata, ecklonia kurome (ek), ecklonia maxima (em), ecklonia muratii, ecklonia radiate (er), and ecklonia stolonifera (es) (hornemann ; moon et al. ) . ec, an edible marine brown alga, is used as a food ingredient, animal feed, and fertilizer, as well as a raw material in the production of fucoidan and phlorotannin. ec is also used as an herbal remedy in the form of an extract called seanol, a polyphenolic extract, and ventol, a phlorotannin-rich natural agent with two major constituents, phlorotannins and sterols (kang et al. a) . es (turuarame), ek, and er are edible species traditionally eaten in japan and korea and are rich in phlorotannins and fatty acids. ecklonia species are known to exhibit antioxidant (heo et al. ) , anti-inflammatory , antibacterial , anti-diabetic (jung et al. ) , anticancer (kong et al. ), anti-photoaging (joe et al. ) , anti-hiv (artan et al. ) , anti-hypertensive (jung et al. ) , hepatoprotective (jung et al. a) , and anti-allergic activities (le et al. ). due to these numerous health benefits, they have been a focal point for researchers eager to elucidate their pharmacological potential. plenty of information regarding the pharmacological activities of terrestrial plants is available; however, such information is limited for marine species (shibata et al. ) . a handful of excellent studies are available regarding the pharmacological activities of ecklonia (wijesinghe and jeon ; thomas and kim ; li et al. ; . also, jiao et al. ( ) have reported the chemical structures and bioactivities of marine algae. fourteen years of research and nearly $ million of clinical studies demonstrate the importance of ecklonia species. ecklonia-derived polyphenols are unlike those found in land-based plants and are quite possibly the most powerful antioxidants found in nature, being - times more powerful than other polyphenols. the oxygen radical absorbance capacity (orac) score of such a polyphenol is more than . the water-soluble polyphenols found in land-based plants have a half-life of about min, and their antioxidant powers decrease very quickly (seanol ) . various phlorotannins, polysaccharides, enzymatic extracts, and organic extracts from ecklonia exhibit multifaceted beneficial effects when used in pharmaceuticals, nutraceuticals, cosmeceuticals, and functional foods. thus, this genus has been a target of special attention, and consumer-driven demand has led to the development of marine-derived medicines. our review summarizes the literature on the biological characterization and pharmacological bioactivity of various ecklonia species, focusing on recent developments in the therapeutic application of extracts and isolates. a shift in the balance between oxidants and antioxidants in favor of oxidants is called oxidative stress (table ) . it arises when the balance between the production of reactive oxygen species (ros) and antioxidant defenses changes. human cells have an inherited antioxidative defense system in the form of various enzymatic and non-enzymatic pathways for removing ros. elevated production of ros increases oxidative stress, leading to cellular dysfunction, and it can eventually contribute to many pathological conditions, including neurological disorders (agostinho et al. ) , diabetes (ceriello ) , cancer (perse ) , asthma , and dermal disease (trouba et al. ). an ethanolic extract of ec attenuated h o -induced comet tail formation and phospho-histone ch ax expression. furthermore, it enhanced the level of the phosphorylated form of nuclear factor erythroid -related factor (nrf ) and its nuclear translocation, which was associated with the induction of heme oxygenase- (ho- ) and nad'ph-quinone oxidoreductase- (nqo- ). thus, ec provided cytoprotective effects against oxidative stress in muscle cells via up-regulation of nrf -ho- and nqo- expression through the activation of the mitogen-activated protein kinases (mapk) pathway, which could be due to its phenolic content (choi ) . in another study, es and ek showed high total phenolic content (tpc) and high antioxidant activity, including , -diphenyl- -picryl-hydrazyl (dpph) radical and hydroxyl (•oh) radical scavenging, ferrous reducing power, and superoxide (•o -) radical scavenging activity. those results suggest that macroalgal beach-casts could be used as a new natural source for functional foods, cosmetics, medicines, and fertilizer instead of being processed into landfills or incinerated (kuda and ikemori ) . enzymatic extracts from er demonstrated more potent antioxidant ability in the ferric reducing ability of plasma (frap) and orac assays (tpc of . g) than conventional acidic extracts (tpc of . g), showing their potential for value-added nutritional products (charoensiddhi et al. ) . inflammation is a pathological condition that produces highly reactive species. nitric oxide (no), a small diffusible molecule responsible for vasodilatation, neurotransmission, and inflammation, is produced by organisms at a basal concentration. however, under stimulation by pathogens, no is generated in higher amounts by the inducible nitric oxide synthase (inos) in activated macrophages (moncada et al. ) . nonsteroidal anti-inflammatory drugs are commercially available medications for inflammatory pain, but their side effects have limited their use. anti-inflammatory drugs from natural resources have been sought due to the persistent deleterious side effects of commercial medications. ec dose-dependently suppressed inos and cyclooxygenase- (cox- ) protein expression and subsequently reduced no content in phorbol- myristate -acetate enzymatic extracts nf-jb lee et al. ( c) ( nmol/l) and a ( lmol/l) (pmaci)-stimulated human mast cell line- cells. no production decreased by . and . % with and lg/ml ec treatments, respectively. furthermore, ec dose-dependently inhibited both the mrna and protein expression of tumor necrosis factor (tnf)-a, interleukin (il)- b, and il- in pmacistimulated human mast cell line- cells without any cytotoxic effects. the inhibitory effects of ec on il- b ( . %) and tnf-a ( . %) production were greater than those on il- ( . %) at lg/ml. in addition, ec exerted anti-inflammatory action via the inhibition of the extracellular signal-regulated kinase (erk)/mapk signaling pathway, suggesting a potent and efficacious antiinflammatory agent against mast cell-mediated inflammatory diseases (kim d . the world health organization (who) defines diabetes as a chronic disease that occurs when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces, which leads to an increased concentration of glucose in the blood, i.e., hyperglycemia. type diabetes (previously known as insulin-dependent) is characterized by a lack of insulin production. type diabetes (formerly called non-insulindependent) is caused by the body's ineffective use of insulin, often from excess body weight and physical inactivity. the total number of people with diabetes is projected to rise from million in to million in (rathmann and giani ) . intensive insulin therapy carries a high risk of side effects, especially the occurrence of severe hypoglycemia; therefore, research into antihyperglycemic agents has focused on plants used in traditional medicine because they could provide better treatment than the currently used synthetic drugs (mccall ) . kimchi has been a popular side dish in korea since ancient times. baechu kimchi is a salt fermented cabbage widely consumed in traditional korean foods. a recent study revealed that kimchi with added ec extract showed high inhibition against a-glucosidase and a-amylase, with ic values of . and . mg/ml, respectively. both of those inhibitory activities of kimchi with added ecklonia extracts (ke) were higher than those of kimchi extract alone. the hypoglycemic effect of ke was higher than that of kimchi extract on starch loading. ke suppressed the postprandial blood glucose level in both streptozotocin (stz)-induced diabetic and normal mice, which indicated a delay in the absorption of dietary carbohydrates consumed . in another report, baechu kimchi with added ec extract protected human umbilical vein endothelial cells (huvecs) from damage induced by high glucose by restoring cell viability and reducing lipid peroxidation and intracellular ros in a dose-dependent manner. furthermore, it reduced the overexpression of inos, cox- , and nuclear factor-jb (nf-jb) proteins in huvecs, indicating its potential as a treatment against high glucose-induced oxidative stress . ek inhibited carbohydrate-hydrolyzing enzymes, decreased postprandial blood glucose levels, and improved glucose tolerance, decreasing both fasting blood glucose and insulin levels (xu et al. ) . ek effectively down-regulated blood glucose in both db/db mice and prediabetic c bl/ j mice, indicating the presence of the active compounds in the gametophytes. ek regulated metabolism by manipulating the balance among cytokines, including interferon-gamma (ifn-c) or leptin, resulting in the downregulation of blood glucose (dwiranti et al. ). the liver is involved in the metabolism of internal and external toxic agents. it has an astounding role in the performance, maintenance, and regulation of homeostasis in the body and is engaged in almost all biochemical pathways of growth, fight against diseases, nutrient supply, energy provision, and reproduction. it plays a vital role in the detoxification of xenobiotics and drugs (agrawal et al. ) . hepatic disease is a term that indicates damage to liver cells, tissues, structures, or function. because no drug currently available completely or effectively stimulates hepatic function, offers complete protection to the organ, or aids in regenerating hepatic cells, naturally derived hepatoprotective agents are needed to negate the factors that contribute to liver damage. a polyphenol-rich fraction of ec prepared in gijang prevented diabetes by regulating various metabolic processes, such as lipogenesis, lipolysis, inflammation, and the antioxidant defense system in livers and adipose tissues affected by nonalcoholic fatty liver disease in high fat diet (hfd)-fed mice. magnetic resonance imaging/magnetic resonance spectroscopy (mri/mrs) analysis showed that liver fat and liver volume in hfd-triggered obese mice decreased following gijang extract treatment. furthermore, the treatment reduced the mrna expression levels of inflammatory cytokines and hepatic lipogenesis-related genes and increased the mrna expression level of cholesterol a-hydroxylase , the key enzyme in bile acid synthesis. thus, gijang extract ameliorated hepatic steatosis by suppressing inflammation and improving lipid metabolism . apart from this, ec increased the activity of alcohol dehydrogenase, aldehyde dehydrogenase, and cyclic adenosine monophosphate (camp) concentration, suggesting that it plays a role in the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. ec inhibited cytochrome p e expression related to the production of ros (yamashita et al. ) . a study investigated the protective effect of es in alcoholic fatty liver and found that es treatment suppressed adipogenesis and increased the expression of fatty acid oxidation-related genes, e.g., peroxisome proliferator-activated receptor (ppar)-a and cpt- , but decreased the expression of sterol regulatory element-binding protein (srebp)- , a triglyceride (tg) synthesis-related gene, suggesting that es extract could be useful in preventing fatty acid oxidation and reducing lipogenesis in ethanol-induced fatty liver (bang et al. ) . neurodegenerative diseases are expected to surpass cancer as the second most common cause of death among the elderly by the s (lilienfeld and perl ) . alzheimer's disease (ad) is characterized by loss of memory and other cognitive functions and accounts for most of the deaths in the elderly. an increase in acetylcholinesterase (ache) level around b-amyloid plaques and neurofibrillary tangles is a common feature of ad neuropathology (lam et al. ). parkinson's disease (pd) is a multidimensional progressive disease with many motor and non-motor features, including cognitive dysfunction. adverse intra-and extracellular effects of toxic a-synuclein are believed to be central to the pathogenesis of pd and other nervous system disorders with lewy body pathology (goldman and postuma ; ingelsson ) . many categories of natural and synthetic neuroprotective agents have been reported. considering the devastating side effects of synthetic neuroprotective agents, there is growing interest in nutraceuticals or other herbal alternatives (pangestuti and kim ) . a study by kang et al. ( d) demonstrated that ec n-buoh extract regulated the expression and activity of csecretase and a-secretase, leading to a reduction in ab production by the stable cells and a reduction in the basal nuclear location of the psen responsible for chromosome mis-segregation in neurodegenerative disease. ec and ek together inhibited ache and bace by . ± . and . ± . %, respectively, reducing neuronal cell death and improving dementia, highlighting their synergistic potential. ek likewise showed the highest result of the , -azino-bis-( -ethylbenzothiazoline- -sulfonic acid) (abts) assay (ic = . ± . mg/ml), which the researchers attributed to its tpc (son et al. ) . kim et al. ( e) further highlighted the importance of ec for its potential analgesic effects in postoperative pain and neuropathic pain. ec extracts ( mg/ kg) significantly increased the mechanical withdrawal therapy value. the number of ultrasonic distress vocalizations in the treated group of rats was reduced at and h after plantar incision operation ( . %). ec also increased the paw withdrawal latency in hot-and coldplate tests in the plantar incision rats. after days of continuous treatment, ec ( mg/kg) alleviated the spared nerve injury-induced hypersensitivity response, which could revolutionize the use of natural resources for therapeutic treatment. matrix metalloproteinases (mmps), known as matrixins, are a large family of similar proteolytic enzymes involved in tissue remodeling associated with various physiological and pathological processes, such as morphogenesis, angiogenesis, tissue repair, arthritis, chronic heart failure, chronic obstructive pulmonary disease, chronic inflammation, and cancer metastasis. as a result, mmps are considered viable drug targets in the therapy of those diseases (dormán et al. ) . according to gelatin zymography results, extracts harvested from ec and ecklonia bicyclis showed higher inhibitory effects on mmp- and - activity than those from the other marine plants at concentrations of , , and lg/ml (bae et al. ) . the global epidemic of bacterial resistance to existing antibiotics such as b-lactams and quinolones necessitates the discovery of potent candidates from natural resources, both terrestrial and marine. the evolution of antibacterial biomolecules alongside bacteria over millions of years allow them to overcome strains such as methicillin-resistant staphylococcus aureus (mrsa) and fluoroquinoloneresistant pseudomonas (ramanan et al. ) . therefore, exploring and developing cheaper and more effective natural antimicrobial agents with better potential and fewer side effects than existing antibiotics, good bioavailability, and minimal toxicity is a public health priority (pérez et al. ) . a recent study showed that ec with ciprofloxacin evinced potent antibacterial activity against enterococcus faecalis, showing synergistic effects. etoac exhibited the strongest antibacterial activity, with a minimum inhibitory concentration (mic) value of lg/ml against e. faecalis strains. furthermore, the combination of ciprofloxacin and the etoac fraction resulted in a p fic min of . and p fic max range of . - , suggesting that the ciprofloxacin-etoac combination resulted in an antibacterial synergy effect against e. faecalis (kim et al. c) . ec led to the strongest growth effects on three lactic acid bacteria and fish pathogenic bacteria in a dose-dependent manner. secondary metabolites produced by ec significantly inhibited the growth of pathogen bacteria. in a further in vivo study, the co-treatment of ec and l. plantarum improved the growth and mortality of edwardsiella tardainfected zebrafish by regulating the expression of inflammatory molecules such as inos and cox- . altogether, ec played an important role as a potential prebiotic and protected against the infection caused by e. tarda injection in zebrafish . ec ( . %) improved the growth and body weight of olive flounder and decreased its mortality from e. tarda without changing its biochemical profile. the supplementation of . % ethanolic extract of ec also enhanced the innate immune response of the fish, as evidenced by a high respiratory burst and increased serum lysozyme and myeloperoxidase activity. thus, ec acted as a prebiotic by improving the innate immune response in fish infected with pathogenic bacteria (lee et al. c ). seanol, a seaweed extract rich in phlorotannins, stimulated mineralization with calcium phosphate, increased antibacterial activity, and increased compressive modulus. seanol and alkaline phosphatase (alp) interacted in a non-covalent manner. seanol exhibited antibacterial activity against mrsa with comparable cytotoxicity toward mg- osteoblast-like cells, suggesting its mineralizability and antibacterial activity (douglas et al. ) . venkatesan et al. ( ) reported the rapid biological synthesis of gold nanoparticles (au nps) using ec by reducing chloroauric acid. fourier transform infrared (ftir) spectroscopic analysis showed that au nps functionalized with biomolecules (a primary amine group, a hydroxyl group, and other stabilizing functional groups) showed good antimicrobial activity and biocompatibility with a human keratinocyte cell line. the results indicate that aunps might have promising applications in drug delivery, tissue engineering, and biosensor development. er extract prepared by using celluclast-assisted extraction induced significantly higher production of butyrate ( . lmol/ml) and promoted the growth of beneficial bacteria such as bifidobacterium and lactobacillus, improving gut health (charoensiddhi et al. ). ec and ek had the strongest inhibitory effects when tested using the agar disk diffusion method, with an mic of . mg/ ml and no cytotoxicity even at lg/ml, indicating their potential as therapeutic agents for acne vulgaris ). obesity is a leading preventable cause of death worldwide, with increasing rates in both developed and developing countries. as defined by who, it is a medical condition in which excess body fat has accumulated to the extent that it can have negative effects on health, and it is often comorbid with diseases such as type diabetes, hypertension, coronary disease, and cancer. in , who announced that obesity had reached epidemic proportions worldwide (kumar and rao ; caballero ) . commercial drugs, such as orlistat; lorcaserin; and a combination of phentermine, topiramate, and bariatric surgery, such as roux-en-y bypass or gastric banding, are available. however, concerns about perioperative mortality, surgical complications, and the frequent need for reoperation mean that those procedures tend to be reserved for morbid obesity (rodgers et al. ). thus, remedies from natural sources are vital, especially those from marine sources. the anti-adipogenic activity of ec was determined by measuring lipid accumulation in adipocytes. the n-buoh fraction particularly reduced lipid accumulation and glucose consumption; the adipogenic transcription factors pparc and srebp- c; and the adipogenic specific genes fatty acid binding protein (fabp)- , fabp- , fatty acid synthase (fas), lipoprotein lipase (lpl), hormone-sensitive lipase (hsl), and acyl-coa synthetase (acs ) . ec polyphenol extract regulated fat metabolism, inflammation, and the antioxidant defense system in hfd-induced obese mice. ec polyphenol extract supplementation reduced body weight gain, adipose tissue mass, plasma lipid profiles, hepatic fat deposition, insulin resistance, and the plasma leptin/adiponectin ratio derived from hfd-induced obesity. furthermore, ec polyphenol extract supplementation selectively ameliorated the hepatic protein levels associated with lipogenesis, inflammation, and the antioxidant defense system, as well as activation of ampk and sirtuin (sirt ) to inhibit obesity (eo et al. ) . there has been a worldwide increase in allergic diseases, including atopic dermatitis, asthma, allergic rhinitis, and food allergies, possibly because environmental factors are interacting with genetic factors to sensitize individuals (tanaka ) . numerous studies have been done in the search for anti-allergens, especially from marine resources. ec extract exhibited excellent inhibitory activity against crude histidine decarboxylase (hdc), reducing overall histamine production by . % and thereby enhancing the safety of mackerel muscle (kim et al. g) . another report presented a % inhibition of hdc at a concentration of mg/ml, reducing histamine poisoning by decreasing histamine production in mackerel (jung et al. b) . ec and ek together inhibited the degranulation of a rat basophilic leukemia cell line (rbl- h ), mitigating allergic symptoms and highlighting their synergistic potential (yoshioka et al. ) . phlorotannin-rich es inhibited enzymatic activity and degranulation in stimulated rbl- h cells in a dose-dependent manner. the meoh: chloroform ( : , v/v) (m/c) extract of es also inhibited enzyme activity and degranulation in stimulated rbl cells in a dose-dependent manner. the active compounds in the m/c extract might be phenolic compounds, such as phlorotannins, because the m/c extract became inactive when the phenolic compounds were removed (sugiura et al. ) . ionizing radiation produces deleterious effects, deterministic or stochastic, on living organisms, though it can have health benefits in the form of radiation therapy for the treatment of cancer or thyrotoxicosis. the benefits of ionizing radiation are compromised by the side effects that result from radiation-induced damage to normal tissue, and the synthetic agents used to combat those side effects, wr (amifostine), ok- , and ethiofos, have their own serious side effects, including decreased cellular function, nausea, hypotension, and death (baliga and rao ; park et al. ) . therefore, investigators have directed their attention toward plants and other natural products. enzymatic extracts of ec exhibited radioprotective properties, including the modulation of apoptosis via inhibition of the nf-jb signaling pathway ). ecklonia species and their constituents exhibit pronounced inhibitory effects against oxidative stress (table ) . ec phlorotannins, including phloroglucinol (pg), eckol, dieckol, eckstolonol, and triphlorethol-a (tpa), scavenged intracellular ros, inhibited lipid peroxidation, and suppressed , -azobis( -amidinopropane) dihydrochloride (aaph)-induced cell death in zebrafish embryos. these phlorotannins maintained the positive changes in morphological phenomena; pericardial edema, yolk sac edema, and growth retardation in zebrafish embryos exposed to aaph were not observed in the groups also exposed to phlorotannins, indicating that the phlorotannins possess prominent antioxidant activity against aaph-mediated toxicity (kang et al. a) . tpa further exhibited a protective effect against oxidative stress-induced dna-base damage, especially -oxoguanine ( -oxog), in v - cells. decreased level of -oxog induced by h o were confirmed by an increase in ogg mrna and ogg protein levels. tpa restored the expression of nuclear nrf , small maf protein, and the nrf -maf complex and also increased nrf binding to are sequences and the resulting ogg promoter activity. sequentially, it maintained the levels of the phosphorylated forms of akt kinase downstream of phosphatidylinositol -kinase (pi k) and erk, which are regulators of ogg , suggesting that ogg induction by tpa involves the pi k/akt and erk pathways . eckol from ec also attenuated the high intracellular ca ? levels stimulated by h o , decreased the augmented levels of mitochondrial ros, recovered h o -diminished atp level and succinate dehydrogenase activity, and induced manganese superoxide dismutase through phosphorylated ampk and forkhead box o a (foxo a), which showed a cytoprotective effect on chang liver cells ). fucoidan extracted from ec exhibited prominent effects on peroxyl radical scavenging activity and , -azobisdihydrochloride-induced oxidative stress in vero cells and reduced ros generation, lipid peroxidation, and cell death in a zebrafish model, proving its antioxidant capacities in vitro and in vivo despite being neither a polyphenol nor a flavonoid. although, it did not contain a benzene ring or conjugated structure, it exhibited antioxidant potential (kim et al. c ). in another study, , bieckol, -phloroeckol, dieckol, and phlorofucofuroeckol (pff-a) isolated from ec significantly inhibited high glucose-induced ros and cell death in zebrafish. dieckol significantly reduced heart rate, ros, no, lipid peroxidation generation, and cell death and also reduced the overexpression of inos and cox- , thereby preventing oxidative stress . es could be used as a natural antioxidant and cytoprotective agent. es inhibited ros even at a concentration of lg/ml, yielding five compounds (pff-a, dieckol, eckstolonol, pg, and eckol) that inhibited total ros, proving its use as a potent scavenger (kang et al. ) . three active compounds were isolated from es, pff-a, dieckol, and dioxinodehydroeckol (dhe), among which pff-a and dieckol significantly suppressed intracellular ros in lipopolysaccharide (lps)induced raw . cells, and dhe scavenged dpph radicals. pff-a also significantly inhibited the lps-induced production of no and prostaglandin e (pge ) through the down-regulation of inos and cox- protein expression ). dieckol and pff-a obtained from boiling water-and organic solvent extracts of ec and es showed almost -and -fold stronger antioxidant activity than the standard butylhydroxytoluene, and -and -fold greater activity than l-ascorbic acid in molar concentration, anti-inflammatory activity ecklonia cava dieckol decreased blood glucose level kang et al. ( b) insulin resistance lee and jeon ( ) akt up-regulation kim et al. ( b) phlorofucofuroeckol-a a-glucosidase and a-amylase fucodiphlorethol g uvb induced oxidative stress kim et al. ( f) respectively (chowdhury et al. ) . three known phlorotannins, eckol, pff-a, and dieckol, along with one new compound, eckstolonol, were obtained from es, and the new compound was found to be a potent radical scavenger through its elimination of dpph radicals (kang et al. b) . eckol suppressed the production of intracellular ros and increased glutathione peroxidase (gsh) level in hepg cells. it inhibited the production of ros in h o treated hepg cells in a dose-dependent manner, and the total relative level of lm eckol was estimated to be . ± . % compared to the non-treated group, making it a much stronger ros scavenger than n-acetylcysteine (nac). the intracellular gsh content in hepg cells was dose-dependently enhanced by eckol treatment, indicating higher antioxidant activity than nac. thus, eckol mediated the expression of ho- in hepg cells, which was regulated by nrf activation via the jun n-terminal kinases (jnk) and pi k/protein kinase b (akt) signaling pathways, suggesting that it is a natural antioxidant and cytoprotective agent (jun et al. (fig ) . ec extract and its major compound (dieckol) significantly increased the survival rate and attenuated liver and kidney damage in mice with a whole-body inflammatory condition by down-regulating pro-inflammatory factors (inos, cox- , tnf-a, il- , and hmgb- ) via a nik/tak / ikk/ijb/nf-jb pathway. additionally, ec increased nrf and ho- expression, reducing inflammation (yang et al. ) . dieckol ( and lm) inhibited the production of a macrophage-derived chemokine, c-c motif chemokine , induced by interferon-c ( ng/ml) in a dose-dependent manner and inhibited the nuclear translocation of signal transducers and activators of transcription (stat ). these results showed that dieckol produced anti-inflammatory effects via the down-regulation of stat activation . in addition, , -bieckol isolated from ec suppressed key inflammatory mediators such as no and pge in raw . macrophages. the inhibition of no occurred by suppressing lps-induced expression of inos at the mrna and protein levels in primary macrophages and raw . cells. likewise, , -bieckol reduced the production and mrna expression of the inflammatory cytokine il- , but not that of tnf-a, in raw . cells. furthermore, , -bieckol significantly reduced mortality in lps-induced septic mice, which indicates that the anti-inflammatory properties of , bieckol are associated with the suppression of no, pge , and il- via negative regulation of the nf-jb pathway and ros production in lps-stimulated raw . cells ). dieckol extracted from ec suppressed lpsinduced inos expression in mouse leukemic macrophage raw . cells, decreasing both lps-induced no production and inos promoter-driven transcriptional activity in a dose-dependent manner. furthermore, it prevented lps-mediated nf-jb activity. it also diminished lpsmediated p nuclear translocation or ijba phosphorylation dose-dependently and reduced lps-induced phosphorylation of mapks, especially p mapk. collectively, these findings suggest that dieckol acts as a negative regulator of lps-mediated inos induction by suppressing nf-jb activity, implying a mechanistic role for dieckol in the regulation of the inflammatory response . the in vivo anti-inflammatory effect of fucoidan from ec was studied using tail-cutting-induced recent advances in pharmacological research on ecklonia species: a review and lps-stimulated zebrafish models; it inhibited tail-cutting-induced and lps-stimulated ros and no generation and also showed a protective effect against the toxicity induced by lps exposure in zebrafish embryos ). pff-a isolated from es showed potential antiinflammatory properties in macrophages stimulated by lps. for this, lm of pff-a significantly inhibited inos and cox- mrna levels induced by lps stimulation. similarly, levels of pro-inflammatory cytokines such as il- b, il- , and tnf-a were significantly reduced. pff-a further inhibited the promoter activities of inflammatory mediators and transcriptional factors. thus, pff-a regulated inos and cox- expression through the nf-jbdependent transcriptional control associated with the inhibition of multiple signaling proteins, suggesting that pg derivatives could be potential treatments for inflammatory diseases . the etoac fraction of es, along with its isolated compounds -phloroeckol, , bieckol, pff-a, pff-b, and -b, inhibited the production of lps-induced no and pge and reduced the expression of inos and cox- in a dose-dependent manner (wei et al. ). most of the investigations on phlorotannins, particularly those derived from brown algae, indicate their promising anti-diabetic effects. for example, , -pg- , -bieckol isolated from ec improved postprandial hyperglycemia through a-glucosidase and a-amylase activity in stz-induced diabetic mice. it showed higher inhibitory activity than the positive control, acarbose (a-glucosidase ic of . lm; a-amylase ic of . lm) (lee et al. a ). in addition, , -bieckol purified from ec at concentrations of or lg/ml significantly inhibited high glucose-induced glucotoxicity and dose-dependently reduced the level of thiobarbituric acid reactive substances (tbars), generation of intracellular ros, and the level of no. furthermore, , -bieckol prevented the apoptosis of rat insulinoma cells under high-glucose conditions, attributed to increased expression of the anti-apoptotic protein bcl- and reduced expression of the pro-apoptotic protein bax, establishing ec as a potential nutraceutical candidate for protection against glucotoxicity (park et al. a ). , -bieckol from ec at concentrations of or lg/ml markedly suppressed high-glucose-induced cytotoxicity and dose-dependently decreased the increased levels of tbars, ros, and no caused by high glucose. in addition, it down-regulated the overexpression of inos, cox- , and nf-jb proteins in huvecs, indicating its therapeutic ability to treat diabetic endothelial dysfunction and related complications ). ec-derived dieckol noticeably decreased blood glucose level, serum insulin level, and body weight. furthermore, it reduced tbars and increased the activities of antioxidant enzymes, including superoxide dismutase (sod), catalase (cat), and gsh-px, in liver tissue. in addition, western blotting analysis revealed that dieckol increased the phosphorylation levels of ampk and akt observed in muscle tissues, suggesting that dieckol could be a therapeutic agent for type diabetes ). dieckol-rich extract from ec led to a significant decrease in postprandial glucose level, insulin, and c-peptide level after weeks without any adverse effects. in other words, ec supplementation significantly contributed to lowering postprandial hyperglycemia and reducing insulin resistance . in addition, dieckol improved blood glucose regulation, hepatic glucose metabolic regulation, and akt up-regulation in alloxan-induced hyperglycemic zebrafish ). pff-a isolated from ec showed prominent inhibitory effects against a-glucosidase and a-amylase activities, with ic values of . and . lm, respectively, which were higher than those of acarbose. moreover, the area under the curve was significantly lower after pff-a administration ( vs. mmol min/l) in diabetic mice, indicating its potent anti-diabetic activity (you et al. ) . the a-glucosidase inhibitory property of em and its isolated compounds pg, dibenzo [ , ] dioxine- , , , -tetraol, and eckol exceeded that of the positive control, suggesting their potency as oral anti-diabetic drugs or functional food ingredients, with a promising role in the formulation of medicines and nutritional supplements (rengasamy et al. ) . es exhibited inhibitory activity on glucose-mediated protein damage, advanced glycation end-products (age), and rat lens aldose reductase (rlar), hinting at potential antidiabetic activity. in spite of negligible activity against age, es phlorotannins, including eckol, dieckol, and -phloroeckol, possessed inhibitory activity on glycation, which indicates that pff-a could be used to prevent diabetic complications (jung et al. ) . likewise, fucosterol from es inhibited rlar, human recombinant aldose reductase, protein tyrosine phosphatase b (ptp b), and a-glucosidase activity (jung et al. a ). pff-a, dieckol, and -phloroeckol isolated from es were potent and non-competitive ptp b inhibitors, with ic values ranging from . to . lm, and a-glucosidase inhibitors, with ic values ranging from . to . lm. interestingly, pff-a and -phloroeckol were non-competitive, whereas dieckol exhibited competitive inhibition in a a-glucosidase assay. thus, isolated phlorotannins from both algae possessed marked ptp b and a-glucosidase inhibitory activity that could contribute to the development of therapeutic agents to control postprandial blood glucose level and prevent diabetic complications (moon et al. ) . published results clearly indicate the anti-diabetic potential of brown seaweed and its derived components, which could be used as nutraceuticals or functional foods to treat diabetes. pyrogallol-phloroglucinol- , '-bieckol fucoidan dieckol from ec exerted cytotoxicity in lx- , hsc-t , and hepg cells, with reduced fibrosis features (large, spread out, and flattened polygonal shapes) in lx- cells compared with the untreated control. in addition, it attenuated the expression of a-sma and tgf-b , increased the sub-g phase population, induced caspase- activation, and cleaved parp in hepatic stellate cells. thus, dieckol suppressed liver fibrosis via caspase activation, micro-rna-mediated jnk activation, and via nf-jb inhibition ). an in vitro study of dieckol showed the strongest protective effect and lowest cytotoxicity against ethanol-induced cell apoptosis in chang liver cells. western blot analysis revealed reduced cell apoptosis through the activation of b cell lymphoma-extra large (bcl-xl) and parp and down-regulation of bax and caspase- , providing evidence for this potential protective agent against ethanol-induced liver diseases. in an in vivo study in a zebrafish model, the dieckol-treated group scavenged intracellular ros and prevented lipid peroxidation and ethanol-induced cell death in embryos (kang et al. c cytochrome c from mitochondria to the cytosol in a dosedependent manner (lee et al. a ). pg isolated from es decreased the formation of lipid peroxide in acetaminophen ( mg/kg, i.p.)-induced rats. though the activities of cytochrome p- , aminopyrine n-demethylase, and aniline hydroxylase were unchanged, pg restored enzyme activity in the livers of pretreated-rats, suggesting that acetaminophen-induced hepatic lipid peroxidation could be reduced by enhancing the activity of glutathione s-transferase (gst) (park ) . all of these data suggest that ecklonia species could be potent hepatoprotective agents. dieckol isolated from ec suppressed the phosphorylation of erk in lps-stimulated bv- microglia ( lg/ml), attenuated akt phosphorylation, and increased the expression of gp phox , a catalytic component of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex responsible for microglial ros generation. furthermore, dieckol offered neuroprotection, as confirmed in an enhanced green fluorescent protein-transfected b neuroblastoma cell line (cui et al. ) . dieckol isolated from ec showed potent activity on rotenone-induced oxidative stress in sh-sy y cells, a human dopaminergic neuronal cell line. it reduced rotenone-induced cell death and retarded rotenone-induced a-synuclein aggregation in asynuclein-overexpressing sh-sy y cells, which prevented a-synuclein aggregation, adding to its role in the prevention of pd (cha et al. ) . pg ( , , -trihydroxybenzene) from ec attenuated the increase in ros accumulation induced by oligomeric ab - treatment in the ht- hippocampal cell line and ameliorated the reduction in dendritic spine density induced by ab - treatment in rat primary hippocampal neuron cultures. also, pg attenuated cognitive dysfunction in the hippocampal region, indicating its anti-ad effects (yang et al. b) . another report showed that pff-a from ec had particularly potent inhibitory activity (ic = . lm) for butyrylcholinesterase (bche), more than -fold greater than for ache. other polyphenols (pff-a, eckol, , -bieckol, , -bieckol, and dieckol) inhibited glycogen synthase kinase b, which is related to the formation of hyperphosphorylated tau and generation of ab. additionally, pff-a inhibited amyloid precursor protein biosynthesis and showed very strong bace inhibitory activity, with a submicromolar ic , making it an interesting potential drug candidate for ad ). an oligosaccharide sugar chain derived from ek inhibited the toxicity induced by the ab protein in both primary cortical cells and the sh-sy y cell line, inhibiting apoptosis and fibril formation and indicating its potency for ad (hu et al. ) . em exhibited potent activity against ache, as did its isolated compounds pg, dibenzo [ , ] dioxine- , , , -tetraol, and eckol, highlighting its potential as a functional food ingredient for the management of neurodegenerative disorders (kannan et al. ) . a phlorotannin preparation (pp) containing eckstolonol from es and ec exhibited a hypnotic effect by modulating the benzodiazepine site of the c-amino butyric acid receptor. pp ([ mg/kg) decreased sleep latency and increased non-rapid eye movement sleep (nrems). likewise, eckstolonol significantly decreased sleep latency ([ . mg/ kg) and increased the amount of nrems ( mg/kg). in addition, the hypnotic effects were completely abolished by pretreatment with flumazenil, suggesting that the phlorotannins could potentially be used as an herbal medicine for insomnia and offering a promising structure for the development of novel sedative-hypnotics . fucosterol and fucoxanthin from es showed noncompetitive and mixed-type inhibition against b-site amyloid precursor protein cleaving enzyme (bace ). furthermore, molecular docking simulation results demonstrated the effective binding of isolated compounds by the bace enzyme, suggesting that both compounds could be used beneficially in the treatment of ad and providing potential guidelines for the design of new bace inhibitors . isolated eckol and dieckol attenuated tnf-a induced expression of mmp- and basal expression, though the expression of timp- was not affected. however, they did reduce both nf-jb and ap- reporter gene activity, which strongly indicates their mmp inhibitory potential. one study demonstrated the inhibitory effect of eckol and dieckol isolated from es on mmp- expression in human dermal fibroblasts, suggesting the possibility of developing an agent to prevent and treat skin aging (joe et al. ) . obviously, marine sources outweigh terrestrial sources in abundance in the development of safe mmp nutraceuticals. anticoagulation occurs by inhibiting the key serine proteases thrombin and factor xa, facilitated by accelerating the activity of the major physiological serine protease inhibitor serpin-antithrombin iii. heparin, a highly sulfated polysaccharide present in mammalian tissues, is commercially used as a blood anticoagulant. it has antihemostasis, fibrinolytic potentiation, and anti-lipemic activity in addition to coagulation activity. though it is a primary anticoagulant, difficulty in isolating it and its hemorrhagic side effects limit its use and drive researchers to search for novel anticoagulants from natural resources free from cytotoxicity (shanmugam and mody ) . studies of the anticoagulant bioactivity of brown seaweeds suggest that they have more than one mechanism of action, including the direct and indirect inhibition of thrombin through the activation of thrombin inhibitors (e.g., antithrombin and heparin cofactor). interestingly, the algal fucans were found to have anticoagulant activity through a direct inhibition of thrombin, whereas the invertebrate fucans showed activity through an indirect inhibition of the enzyme, which required antithrombin and heparin cofactor ii, and that has driven the rapid discovery of anticoagulants from marine resources (jiao et al. ) . fucoidan isolated from ek significantly inhibited the generation of thrombin and factor xa in the intrinsic pathway. furthermore, it inhibited the formation of prothrombin-activating complex (i.e., prothrombinase); the ic of thrombin generation was one-tenth to one-seventh that of the activity of the thrombin in plasma, whereas the antithrombin activity of fucoidan was mediated by heparin cofactor ii in plasma. this further highlights the relationship between the molecular weight of sulfated polysaccharides and their anticoagulant activity such that the higher molecular weight fucans show greater anticoagulant activity than sulfates with a lower molecular weight (nishino et al. (nishino et al. , a . sulfated polysaccharides, mainly -linked and , -disubstituted fucopyranosyl residues isolated from ek, exhibited potent anticoagulant activity (nishino et al. b) . some potent and novel antiplasmin inhibitors such as pff-a and eckol isolated from ek inhibited the action of alpha -macroglobulin (ic = . lg/ml) and alpha -plasmin inhibitor (ic = . lg/ml), the main plasmin inhibitors in plasma (fukuyama et al. (fukuyama et al. , . thus, the development of antithrombotic algal polysaccharides would be advantageous because their use would avoid the potential for contamination with prions or viruses present in commercial heparins, which are obtained from pig and bovine intestines. moreover, with more specific activities or targets, algal sulfated polysaccharides could find applications complementary to heparin. dieckol from ec significantly blocked the cleavage of sars-cov cl(pro) in a cell-based assay without showing any toxic effects and exhibited a high association rate in the spr sensorgram, forming strong hydrogen bonds to the catalytic dyad (cys and his ) of the sars-cov cl(pro) ). both phlorotannins from ec and isolated epibiotic bacteria showed potent antibacterial activity in close affiliation with the genus bacillus (kanagasabhapathy et al. ) . another novel bacterial strain, designated ec t, was isolated from ec, and it showed potent antibacterial activity (kim et al. d) . another study suggested that the compounds eckol, dieckol, pff, and -phloroeckol exhibited potent antiviral activity, with ic ranging from . ± . to . ± . lm against porcine epidemic diarrhea. these compounds completely blocked the binding of viral spike protein to sialic acids at concentrations less than . lm by hemagglutination inhibition. pff and dieckol inhibited viral replication with ic values of . ± . and . ± . lm, respectively, in the post-treatment assay and exhibited stronger inhibition of viral rna and viral protein synthesis in later stages ( and h) than in early stages ( and h), suggesting their potential as natural therapeutic drugs against coronavirus infection ). dieckol and , -bieckol (hexamers) isolated from ek were tested against the food-borne pathogenic bacteria campylobacter jejuni with an mic of mg/l and . lmol/ml, respectively, which were effective against mrsa as determined by a broth microdilution method. the bactericidal effects of the phlorotannins were more pronounced than those of the catechins . one study showed that n-buoh fractions with isolated phlorotannins (tpa, eckol, and dieckol) increased glycerol secretion and reduced the regulation of adipogenic transcription factors, pparc, ccaat/enhancer-binding protein (c/ebpa), and tnfa. those phlorotannins also reduced the differentiation-dependent factor /srebp- c and downstream genes such as fabp- , fatty acid transport protein- , fas, leptin, and acs , whereas they increased the mrna expression of hormone-sensitive lipase while suppressing perilipin expression to treat obesity . dieckol from ec down-regulated the expression of pparc, c/ebpa, srebp- , and fabp- , showing anti-adipogenic effects on adipocyte differentiation through the activation and modulation of the ampk signaling pathway to improve obesity . enzyme-treated ec and its isolated compounds (eckol, dieckol and pff-a) exhibited potent adipogenic activity in t -l adipocytes. dieckol was found to be the major compound in the enzymatic extract, with a concentration of mg/g. in addition, lg/ml of ec extract inhibited glucose utilization and tg accumulation, as confirmed by oil red o staining. additionally, it decreased the expression of ccaat/(c/ebp)a, srebp- c, adipocyte-fabp, fas, and adiponectin. thus, ec prevented adipogenesis by affecting the activation of the c/ebpa signaling pathway and the resulting adipogenesis-related gene expression (kim and nam ) . in a similar report, dieckol from ec inhibited lipid accumulation via activation of ampka signaling and cell-cycle arrest in t -l cells and mouse and zebrafish models . seapolynol derived from ec inhibited triglyceride synthetic enzymes such as diacylglycerol acyltransferase , gpat , kruppellike factor (klf ), klf , c/ebpb, c/ebpd, and protein c-ets- , whereas it up-regulated klf , an anti-early adipogenic factor, preventing metabolic disorders . fucosterol was isolated from the strong anti-adipogenic ch cl -soluble fraction from es, with significant inhibition ( . %) of intracellular lipid accumulation at a non-toxic concentration. the anti-adipogenic activities of es, along with the isolated fucosterol, reduced lipid content in a concentration-dependent manner without showing any cytotoxicity. fucosterol treatment also yielded a decrease in the expression of the adipocyte markers pparc and c/ebpa in a concentration-dependent manner. taken together, these results suggest that fucosterol inhibits the expression of pparc and c/ebpa, resulting in a decrease in lipid accumulation in t -l pre-adipocytes, thereby indicating the potential of es and its bioactive component fucosterol as anti-obesity agents (jung et al. b ). similarly, fucosterol isolated from es down-regulated the insulin-triggered pi k/akt and erk pathways, which subsequently decreased the expression of adipogenic transcription factors, including pparc, c/ebpa, and srebp- . in addition, fucosterol enhanced sirt expression and decreased phospho-foxo expression, which resulted in the activation of foxo and revealed that fucosterol inhibited adipogenesis of t -l preadipocytes at concentrations of and lm through the modulation of the foxo signaling pathway (lee et al. b) . compound -b isolated from ek inhibited the differentiation of mouse embryonic fibroblasts and t -l cells into adipose cells by acting as the peptidyl prolyl cis/trans isomerase inhibitor responsible for the uptake of tg and the differentiation of fibroblasts into adipose cells in response to insulin stimulation without inducing cytotoxicity. this finding suggests that -b could be a lead drug candidate for obesity-related disorders (mori et al. ) . altogether, these results suggest that several ecklonia species possess potent activity that might be exploited in adjunct therapy for obesity. dieckol from ec inhibited mast cell activation and mast cell-mediated type i allergic reactions caused by igespecific antigen, mainly through the marked downstream signaling of fceri. a high dose of dieckol suppressed hypersensitive reactions, offering another target molecule for the prevention or treatment of mast cell-dependent allergic diseases (ahn et al. c) . six phlorotannins isolated from ek, pg, an unknown tetramer, eckol, pff-a, dieckol, and , bieckol, inhibited hyaluronidase at concentrations of , , [ , , , and lm, respectively. also, , , the strongest hyaluronidase inhibitor, acted as a competitive inhibitor with an inhibition constant (ki) of . lm . park et al. ( ) described the multi-faceted protection mechanisms of pg against oxidative stress caused by ionizing radiation in mice. pg inhibited apoptosis and strengthened hematopoiesis. it increased the viability of splenocytes without cytotoxicity and significantly enhanced the proliferation of splenocytes by limiting the increment of sub-g( ) dna contents via the inhibition of ros production in gy-irradiated splenocytes. in addition, pg significantly decreased dna damage and the number of apoptotic fragments in lymphocytes during oxidative stress and increased the counts of endogenous spleen colony forming units (cfus) compared with control mice exposed to ionizing radiation. a similar study confirmed that pg protected against small intestine damage caused by ionizing radiation, raising the apoptosis threshold of jejunal crypt cells. pg regenerated the intestinal crypts and down-regulated the expression level of proapoptotic molecules such as p , bax, and bak in the small intestine. pg further augmented antiapoptotic molecules such as bcl- and bcl-x(s/l) (ha et al. ) . cancer is the uncontrolled growth of cells that can invade and spread to distant sites of the body. cancer can have severe health consequences and is a leading cause of death. lung, prostate, colorectal, stomach, and liver cancer are the most common types of cancer in men, whereas breast, colorectal, lung, uterine cervix, and stomach cancer are the most common types among women. more than % of cancer deaths could be prevented by modifying or avoiding key risk factors. cancer results from a mutation in the chromosomal dna of a normal cell, which can be triggered by both external factors (tobacco, alcohol, chemicals, infectious agents, and radiation) and internal factors (hormones, immune conditions, inherited mutations, and mutations occurring in metabolism) (croce ; ferlay et al. ) . in korea, cancer accounts for one in four deaths ( . %), and more than , new cancer cases were diagnosed in . although antineoplastic drugs and chemotherapy are available, the deleterious effects of those medications have driven researchers to derive new drug candidates from natural products. dieckol isolated from ec prevented n-nitrosodiethylamine (ndea)-induced rat hepatocarcinogenesis. dieckol administered orally ( mg/kg body weight) for weeks with . % ndea through the drinking water reversed the activities of hepatic marker enzymes such as aspartate transaminase, alanine transaminase, alp, gamma glutamyl transferase, lactate dehydrogenase, a-fetoprotein, and total bilirubin and increased the elevation of cytochrome p . dieckol also decreased lipid peroxidative markers (tbars, lipid hydroperoxides, protein carbonyl content, and conjugated dienes) and decreased the antioxidant cascade viz enzymatic antioxidants (such as sod, cat, glutathione peroxidase, gst, glutathione reductase) and non-enzymatic antioxidants (such as reduced glutathione, vitamin c, and vitamin e). dieckol was more effective at mg/kg than at and mg/kg body weight and protected the liver from cancer (sadeeshkumar et al. ) . likewise, dieckol showed anti-breast cancer activity by regulating the expression of metastasis-related genes ). ec and its major phlorotannin (dieckol) increased the tumor growth-inhibitory effect of cisplatin and reduced cisplatin-induced nephrotoxicity and weight loss in skov -bearing mice. furthermore, they enhanced cisplatin-induced apoptosis by stimulating caspases in skov and a ovarian cancer cells via down-regulation of akt and nf-jb signaling. thus, ec suppressed cisplatin-induced ros production and cell death in normal hek kidney cells, and its major compound dieckol evidently enhanced the suppression of tumor growth by cisplatin with less weight loss and kidney damage in a mouse model . dieckol from ec exhibited cytotoxic effects on a and skov ovarian cancer cells, induced the apoptosis of skov cells, and suppressed tumor growth without any significant adverse effect in the skov -bearing mouse model. furthermore, it activated caspase- ,- , and - ; caused mitochondrial dysfunction; and suppressed the levels of anti-apoptotic proteins. thus, dieckol enhanced intracellular ros, and the antioxidant n-acetyl-l-cysteine (nac) noticeably reversed the caspase activation, cytochrome c release, bcl- downregulation, and apoptosis caused by dieckol through akt and p ). in another investigation, ahn et al. ( b) showed the anticancer effects of crude polysaccharides isolated from ec enzymatic extracts, using amyloglucosidase (amg), viscozyme, protamex, and alcalase enzymes against a colon cancer cell line. among the tested extracts, crude polysaccharides of protamex showed the highest inhibitory effect against the growth of ct- cells and dose-dependently increased the formation of apoptotic bodies and the percentage of sub-g dna content. furthermore, crude polysaccharides of protamex activated caspase and parp to regulate the expression of bax and bcl- and showed the highest inhibitory effect against the growth of ct- cells, attributed to their high fucose and sulfated group content, demonstrating anticancer effects on colon cancer cells via regulation of the bcl- /bax signal pathway. additionally, dieckol isolated from es reduced the number of viable cells and increased the number of apoptotic cells, increased the expression levels of cleaved caspases-( , , , and ) and cleaved poly(adp-ribose) polymerase, increased the permeability of mitochondrial membranes, and increased the release of cytochrome c from mitochondria into the cytosol with an apoptosis-inducing factor. thus, dieckol induced apoptosis via the activation of both death receptors and mitochondrial-dependent pathways in human hepatocellular carcinoma (hep b) cells (yoon et al. ). the skin is the human body's largest organ and is colonized by diverse microorganisms, most of which are harmless or even beneficial to their host. it acts as an anatomical barrier that is tough when intact and prevents the entry and colonization of many microbes (grice and segre ) . however, continuous exposure to ultraviolet (uv) light leads to various complications correlated with various pathological consequences, such as photo-damage of the skin, which is characterized by distinct alterations in the composition of the dermal extracellular matrix and results in wrinkles, laxity, coarseness, mottled pigmentation, and histological changes that include increased epidermal thickness and connective tissue alteration (rittie and fisher ) . therefore, varieties of anti-photoaging or photoprotective compounds from algae and other marine organisms have been isolated. dhe from ec exerted a preventive effect against uvbinduced apoptosis in human keratinocyte (hacat) cells, indicating its benefit as a repair agent for skin damage caused by uvb (ryu et al. ) . fucodiphlorethol g from ec reduced uvb radiation-induced loss of mitochondrial membrane potential, the generation of apoptotic cells, and active caspase- expression. thus, it prevented oxidative damage-mediated apoptosis induced by uvb irradiation (kim et al. f) . dhe from es inhibited cellular melanin content and the expression of melanogenesis-related proteins, including microphthalmia-associated transcription factor and tyrosinase and tyrosinase-related proteins trp- and trp- , stimulating the phosphorylation of akt in a dose-dependent manner without affecting the phosphorylation of erk (lee et al. b ). angiotensin-i-converting enzyme (ace) plays an important physiological role in the regulation of blood pressure by converting angiotensin i to angiotensin ii, a potent vasoconstrictor. some commercial drugs, such as captopril, ramipril, lisinopril, and enalapril, have unfortunate side effects like cough, taste disturbances, skin rashes or angioneurotic edema, so novel compounds such as phlorotannins have been derived from marine organisms as potential ace inhibitors . jung et al. ( ) reported that, among the seven enzymatically hydroxylated brown algal species, ec was a potent ace inhibitor with an ic value of . lg/ml. eckol, pff-a, and dieckol from es showed potent ace inhibiting activity with ic values of . ± . , . ± . , and . ± . lm, respectively, showing the importance of es. the human immunodeficiency virus (hiv) infects cells of the immune system by destroying or impairing their function, ultimately causing immune deficiency and opening the door to opportunistic infections, thereby causing acquired immune deficiency syndrome (aids). approximately . million people were living with hiv at the end of . as of mid- , . million people were receiving antiretroviral treatment worldwide. seven out of ten pregnant women living with hiv received anti-retroviral treatment (who ). more than approved commercial drugs are available for the treatment of aids. these drugs keep the disease under control; however, they are often associated with the emergence of cross-resistant hiv strains and various side-effects (sánchez and holguín ; haas et al. ). so, the need for potential nutraceuticals, especially from marine organisms, to fight this vicious disease is vital. , -dieckol from ec at noncytotoxic concentrations repressed hiv- activated syncytia formation, lytic effects, and viral p antigen production. furthermore, it selectively inhibited the activity of hiv- reverse transcriptase enzyme with % inhibition at lm. therefore, it showed high potential and could be considered as a drug candidate for the development of a new generation of therapeutic agents (karadeniz et al. ) . other pharmacological activities include immunomodulatory, aphrodisiac, anti-hair loss, hearing repair, urinary tract infection remedy, hair growth performance, cosmetic whitening, osteoarthritis, and bone-related conditions. one study suggested that sulfated polysaccharides from ec induced t and b cell responses via both the jnk and nf-kb pathways . ecklonia bicyclis together with the novel drug tradamixina improved male sexual function in elderly men, particularly libido, mild-moderate erectile dysfunction, ejaculation function, and sexual quality of life (sansalone et al. ). purified polyphenols from ec increased fibroblast survival in human dermal papilla cells, preventing hair loss . dieckol isolated from ec suppressed ea.hy cell proliferation induced by vascular endothelial growth factor, demonstrating its anti-proliferative and anti-migratory effects through a mapk molecular signaling cascade and suggesting its potential as an anti-angiogenic candidate . dhe from ec enhanced osteoblast differentiation, as evidenced by increased cell proliferation, alkaline phosphatase activity, and intracellular cell mineralization, along with enhanced levels of osteoblastogenesis markers at lm in mc t -e pre-osteoblasts. furthermore, dhe up-regulated phosphorylated erk and c-jnk, which were also stimulated by the bmp signaling pathway . a polyphenol-rich extract from ec showed potent radical-scavenging activity and decreased the abr threshold shifts, suggesting its potential as a preventive agent against temporary threshold shift . water-soluble sulfated fucans isolated from ec induced the degradation of ij-b and the phosphorylation of mapk in raw . cells, implying that they might stimulate raw . cells through the activation of the nf-jb and mapk pathways (cao et al. ) . the etoac fraction of ec resulted in elongation of the hair shaft in cultured human hair follicles and activated transition of the hair cycle from the telogen to the anagen phase in the dorsal skin of c bl/ mice. furthermore, it induced an increase in igf- expression in human dermal papilla cells (bak et al. ) . ec and its phlorotannins tpa, eckol, and dieckol attenuated the pathophysiological consequences of osteoarthritis and enhanced osteoblast differentiation, as indicated by increased alkaline phosphatase activity and raised levels of osteoblastogenesis, preventing osteoporosis . undoubtedly, ecklonia species and their active metabolites are potential candidates for drug development, as shown by their plethora of activities against various diseases. our review provides current information on the biological and pharmacological potential of this genus, which could be further used for the development of nutraceuticals. screening and study of the interactions between these algae and their constituents and human systems threatened by disease need to continue. therefore, we recommend rapid screening and isolation to develop novel functional ingredients from ecklonia species. the future of pharmaceuticals based on natural products seems promising. neuroinflammation, oxidative stress and the pathogenesis of alzheimer's disease review: hepatoprotective activity of spirulina species the jnk/nfjb pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from ecklonia cava dieckol, isolated from the edible brown algae ecklonia cava, induces apoptosis of ovarian cancer cells and inhibits tumor xenograft growth a sulfated polysaccharide of ecklonia cava inhibits the growth of colon cancer cells by inducing apoptosis dieckol, a phlorotannin of ecklonia cava, suppresses ige-mediated mast cell activation and passive cutaneous anaphylactic reaction dioxinodehydroeckol enhances the differentiation of osteoblasts by regulating the expression of phospho-smad / / anti-hiv- activity of phloroglucinol derivative, , -bieckol, from ecklonia cava evaluation of effective mmp inhibitors from eight different brown algae in human fibrosarcoma ht cells ecklonia cava promotes hair growth radioprotective potential of mint: a brief review protective effects of ecklonia stolonifera extract on ethanol-induced fatty liver in rats the global epidemic of obesity: an overview water soluble sulfated-fucans with immune-enhancing properties from ecklonia cava possible role of oxidative stress in the pathogenesis of hypertension dieckol, an edible seaweed polyphenol, retards rotenone-induced neurotoxicity and a-synuclein aggregation in human dopaminergic neuronal cells protective effect of a purified polyphenolic extract from ecklonia cava against noise-induced hearing loss: prevention of temporary threshold shift improved antioxidant activities of brown seaweed ecklonia radiata extracts prepared by microwave-assisted enzymatic extraction impact of extraction processes on prebiotic potential of the brown seaweed ecklonia radiata by in vitro human gut bacteria fermentation marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the gabaa receptor the cytoprotective effects of ethanol extract of ecklonia cava against oxidative stress are associated with upregulation of nrf -mediated ho- and nqo- expression through activation of the mapk pathway in vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, propionibacterium acnes suppression of nf-jb by dieckol extracted from ecklonia cava negatively regulates lps induction of inducible nitric oxide synthase gene dieckol, a major phlorotannin in ecklonia cava, suppresses lipid accumulation in the adipocytes of high-fat diet-fed zebrafish and mice: inhibition of early adipogenesis via cell-cycle arrest and ampka activation multifunctional activity of polyphenolic compounds associated with a potential for alzheimer's disease therapy from ecklonia cava comparison of ecklonia cava, ecklonia stolonifera and eisenia bicyclis for phlorotannin extraction oncogenes and cancer dieckol attenuates microglia-mediated neuronal cell death via erk, akt and nadph oxidase-mediated pathways enrichment of enzymatically mineralized gellan gum hydrogels with phlorotannin-rich ecklonia cava extract seanol Ò to endow antibacterial properties and promote mineralization effects of gametophytes of ecklonia kurome on the levels of glucose and triacylglycerol in db/db, prediabetic c bl/ j and ifn-c ko mice brown alga ecklonia cava polyphenol extract ameliorates hepatic lipogenesis, oxidative stress, and inflammation by activation of ampk and sirt in high-fat diet-induced obese mice cancer incidence and mortality worldwide: sources, methods and major patterns in globocan structure of an anti-plasmin inhibitor, eckol, isolated from the brown alga ecklonia kurome okamura and inhibitory activities of its derivatives on plasma plasmin inhibitors anti-plasmin inhibitor. vi. structure of phlorofucofuroeckol a, a novel phlorotannin with both dibenzo- , -dioxin and dibenzofuran elements, from ecklonia kurome okamura premotor and non-motor features of parkinson's disease the skin microbiome phloroglucinol protects small intestines of mice from ionizing radiation by regulating apoptosis-related molecules: a comparative immunohistochemical study pharmacogenetics of efavirenz and central nervous system side effects: an adult aids clinical trials group study antioxidative effect of proteolytic hydrolysates from ecklonia cava on radical scavenging using esr and h o -induced dna damage om fucus buccinalis lin acidic oligosaccharide sugar chain, a marine-derived acidic oligosaccharide, inhibits the cytotoxicity and aggregation of amyloid beta protein alpha-synuclein oligomers-neurotoxic molecules in parkinson's disease and other lewy body disorders seapolynol extracted from ecklonia cava inhibits adipocyte differentiation in vitro and decreases fat accumulation in vivo chemical structures and bioactivities of sulfated polysaccharides from marine algae the inhibitory effects of eckol and dieckol from ecklonia stolonifera on the expression of matrix metalloproteinase- in human dermal fibroblasts eckol enhances heme oxygenase- expression through activation of nrf /jnk pathway in hepg cells angiotensin-converting enzyme i inhibitory activity of phlorotannins from ecklonia stolonifera inhibitory activities of extracts from several kinds of seaweeds and phlorotannins from the brown alga ecklonia stolonifera on glucose-mediated protein damage and rat lens aldose reductase kinetics and molecular docking studies of an antidiabetic complication inhibitor fucosterol from edible brown algae eisenia bicyclis and ecklonia stolonifera inhibitory effects of histamine production in mackerel muscle by medicinal herbs and seaweed extracts protective effect of the edible brown alga ecklonia stolonifera on doxorubicininduced hepatotoxicity in primary rat hepatocytes anti-adipogenic activity of the edible brown alga ecklonia stolonifera and its constituent fucosterol in t -l adipocytes kinetics and molecular docking studies of fucosterol and fucoxanthin, bace inhibitors from brown algae undaria pinnatifida and ecklonia stolonifera antibacterial activities of marine epibiotic bacteria isolated from brown algae of japan antioxidant and anti-inflammatory activities of ventol, a phlorotannin-rich natural agent derived from ecklonia cava, and its effect on proteoglycan degradation in cartilage explant culture a new phlorotannin from the brown alga ecklonia stolonifera inhibitory phlorotannins from the edible brown alga ecklonia stolonifera on total reactive oxygen species (ros) generation protective effect of marine algae phlorotannins against aaph-induced oxidative stress in zebrafish embryo dieckol isolated from brown seaweed ecklonia cava attenuates type ii diabetes in db/db mouse model protective effect of dieckol isolated from ecklonia cava against ethanol caused damage in vitro and in zebrafish model phlorotannin-rich ecklonia cava reduces the production of betaamyloid by modulating alpha-and gamma-secretase expression and activity dieckol, a component of ecklonia cava, suppresses the production of mdc/ccl via down-regulating stat pathway in interferon-c stimulated hacat human keratinocytes acetylcholinesterase inhibitory activity of phlorotannins isolated from the brown alga, ecklonia maxima (osbeck) papenfuss anti-hiv- activity of phlorotannin derivative , -dieckol from korean brown alga ecklonia cava phlorotannins suppress adipogenesis in pre-adipocytes while enhancing osteoblastogenesis in pre-osteoblasts role of erk/mapk signaling pathway in antiinflammatory effects of ecklonia cava in activated human mast cell line- cells enzyme-treated ecklonia cava extract inhibits adipogenesis through the downregulation of c/ebpa in t -l adipocytes hepatoprotective constituents of the edible brown alga ecklonia stolonifera on tacrine-induced cytotoxicity in hepg cells isolation and identification of phlorotannins from ecklonia stolonifera with antioxidant and anti-inflammatory properties phlorofucofuroeckol a inhibits the lps-stimulated inos and cox- expressions in macrophages via inhibition of nf-jb, akt, and p mapk evaluation of inhibitory effect of phlorotannins from ecklonia cava on triglyceride accumulation in adipocyte triphlorethol-a from ecklonia cava upregulates the oxidant sensitive -oxoguanine dna glycosylase cytoprotective effect of eckol against oxidative stress-induced mitochondrial dysfunction: involvement of the foxo a/ampk pathway protective effect of fucoidan against aaph-induced oxidative stress in zebrafish model the edible brown seaweed ecklonia cava reduces hypersensitivity in postoperative and neuropathic pain models in rats fucodiphlorethol g purified from ecklonia cava suppresses ultraviolet b radiationinduced oxidative stress and cellular damage inhibitory effects of brown algae extracts on histamine production in mackerel muscle via inhibition of growth and histidine decarboxylase activity of morganella morganii protective effect of marine brown algal polyphenols against oxidative stressed zebrafish with high glucose first evidence that ecklonia cavaderived dieckol attenuates mcf- human breast carcinoma cell migration synergistic antibacterial activity of ecklonia cava extract against anti-biotic resistant enterococcus faecalis winogradskyella eckloniae sp. nov., a marine bacterium isolated from the brown alga ecklonia cava -bieckol suppresses inflammatory responses by downregulating nuclear factor-jb activation via akt, jnk, and p mapk in lps-stimulated microglial cells a marine algal polyphenol, dieckol, attenuates blood glucose levels by akt pathway in alloxan induced hyperglycemia zebrafish model dieckol, a phlorotannin isolated from a brown seaweed, ecklonia cava, inhibits adipogenesis through amp-activated protein kinase (ampk) activation in t -l preadipocytes evaluation of marine algae wakame (undaria pinnatifida) and kombu (laminaria digitata japonica) as food supplements induction of apoptosis by phloroglucinol derivative from ecklonia cava in mcf- human breast cancer cells edible brown alga ecklonia cava derived phlorotannin-induced anti-adipogenic activity in vitro minerals, polysaccharides and antioxidant properties of aqueous solutions obtained from macroalgal beachcasts in the noto peninsula periodontal disease and obesity seaweeds as a source of nutritionally beneficial compounds-a review in vitro antiviral activity of phlorotannins isolated from ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination anti-cholinesterases and memory improving effects of vietnamese xylia xylocarpa inhibitory effects of polyphenols isolated from marine alga ecklonia cava on histamine release efficacy and safety of a dieckol-rich extract (ag-dieckol) of brown algae, ecklonia cava, in prediabetic individuals: a double-blind, randomized, placebocontrolled clinical trial isolation and identification of phlorotannins from ecklonia stolonifera with antioxidant and hepatoprotective properties in tacrine-treated hepg cells dioxinodehydroeckol inhibits melanin synthesis through pi k/ akt signalling pathway in alpha-melanocyte-stimulating hormone-treated b f cells recent advances in pharmacological research on ecklonia species: a review anti-inflammatory effect of fucoidan extracted from ecklonia cava in zebrafish model alleviating effects of baechu kimchi added ecklonia cava on postprandial hyperglycemia in diabetic mice radio-protective effect of polysaccharides isolated from lactobacillus brevis-fermented ecklonia cava effect of baechu kimchi added ecklonia cava extracts on high glucose-induced oxidative stress in human umbilical vein endothelial cells microrna mediated upregulation of jnk and downregulation of nf-kb signaling are critically involved in dieckol induced antihepatic fibrosis a prebiotic role of ecklonia cava improves the mortality of edwardsiellatarda-infected zebrafish models via regulating the growth of lactic acid bacteria and pathogen bacteria a prebiotic effect of ecklonia cava on the growth and mortality of olive flounder infected with pathogenic bacteria a phlorotannin constituent of ecklonia cava alleviates postprandial hyperglycemia in diabetic mice fucosterol, isolated from ecklonia stolonifera, inhibits adipogenesis through modulation of foxo pathway in t -l adipocytes phlorotannins as bioactive agents from brown algae dieckol as a novel antiproliferative and anti-angiogenic agent and computational antiangiogenic activity evaluation projected neurodegenerative disease mortality in the united states caffeic acid phenethyl ester alleviates asthma by regulating the airway microenvironment via the ros-responsive mapk/akt pathway insulin therapy and hypoglycemia selecting australian marine macroalgae based on the fatty acid composition and anti-inflammatory activity nitric oxide: physiology, pathophysiology, and pharmacology protective effect of phlorotannin components phloroglucinol and eckol on radiation-induced intestinal injury in mice protein tyrosine phosphatase b and a-glucosidase inhibitory phlorotannins from edible brown algae, ecklonia stolonifera and eisenia bicyclis a high-throughput screen for inhibitors of the prolyl isomerase, pin , identifies a seaweed polyphenol that reduces adipose cell differentiation bactericidal activity of phlorotannins from the brown alga ecklonia kurome the influence of sulfate content and molecular weight of a fucan sulfate from the brown seaweed ecklonia kurome on its antithrombin activity an anticoagulant fucoidan from the brown seaweed ecklonia kurome inhibition of the generation of thrombin and factor xa by a fucoidan from the brown seaweed ecklonia kurome cancer statistics in korea: incidence, mortality, survival, and prevalence in neuroprotective effects of marine algae effects of phloroglucinol isolated from ecklonia stolonifera on the acetaminophen-metabolizing enzyme system in rat radioprotective properties of eckol against ionizing radiation in mice phloroglucinol (pg) purified from ecklonia cava attenuates radiation-induced apoptosis in blood lymphocytes and splenocytes dieckol, a sars-cov cl(pro) inhibitor, isolated from the edible brown algae ecklonia cava -bieckol isolated from ecklonia cava protects oxidative stress through inhibiting expression of ros and proinflammatory enzymes in high-glucose-induced human umbilical vein endothelial cells -bieckol protects insulinoma cells against high glucose-induced glucotoxicity by reducing oxidative stress and apoptosis polyphenol-rich fraction of ecklonia cava improves nonalcoholic fatty liver disease in high fat diet-fed mice antimicrobial action of compounds from marine seaweed oxidative stress in the pathogenesis of colorectal cancer: cause or consequence? antibacterial activity of the extracts of marine red and brown algae algaebacteria interactions: evolution, ecology and emerging applications global prevalence of diabetes: estimates for the year and projections for potential antiradical and alpha-glucosidase inhibitors from ecklonia maxima (osbeck) papenfuss uv-light-induced signal cascades and skin aging anti-obesity drugs: past, present and future dioxinodehydroeckol protects human keratinocyte cells from uvb-induced apoptosis modulated by related genes bax/bcl- and caspase pathway protective effects of dieckol on n-nitrosodiethylamine induced hepatocarcinogenesis in rats drug resistance in the hiv- -infected paediatric population worldwide: a systematic review alga ecklonia bicyclis, tribulus terrestris, and glucosamine oligosaccharide improve erectile function, sexual quality of life, and ejaculation function in patients with moderate mild-moderate erectile dysfunction: a prospective, randomized, placebo-controlled, single-blinded study jp renew distributors lombard street heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents inhibitory activity of brown algal phlorotannins against hyaluronidase antioxidant activities of phlorotannins isolated from japanese laminariaceae enhancement of human hair growth using ecklonia cava polyphenols in vitro screening for anti-dementia activities of seaweed extracts inhibitory effect of extracts from the brown alga, ecklonia stolonifera, on enzymes responsible for allergic reactions and degranulation in rbl- h cells flavonoids for allergic diseases: present evidence and future perspective potential pharmacological applications of polyphenolic derivatives from marine brown algae oxidative stress and its role in skin disease marine algae-mediated synthesis of gold nanoparticles using a novel ecklonia cava isolation and identification of anti-inflammatory compounds from ethyl acetate fraction of ecklonia stolonifera and their anti-inflammatory action prevent hiv, test and treat all-who support for country impact angiotensin-i-converting enzyme (ace) inhibitors from marine resources: prospects in the pharmaceutical industry phlorotannins from ecklonia cava (phaeophyceae): biological activities and potential health benefits exploiting biological activities of brown seaweed ecklonia cava for potential industrial applications: a review anti-diabetic effect of polyphenols from brown alga ecklonia kurome in genetically diabetic kk-ay mice ecklonia cava polyphenol has a protective effect against ethanolinduced liver injury in a cyclic amp-dependent manner -bieckol, isolated from edible brown algae, exerts its anti-inflammatory effects through inhibition of nf-jb signaling and ros production in lps-stimulated macrophages brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity phloroglucinol attenuates the cognitive deficits of the xfad mouse model of alzheimer's disease protective effect of brown alga phlorotannins against hyper-inflammatory responses in lipopolysaccharide-induced sepsis models dieckol, isolated from ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma hep b cells inhibitory effect of chlorophyll c from brown algae, sargassum horneri, on degranulation of rbl- h cells isolation and structural determination of two novel phlorotannins from the brown alga ecklonia kurome okamura, and their radical scavenging activities phlorofucofuroeckol a isolated from ecklonia cava alleviates postprandial hyperglycemia in diabetic mice acknowledgements this research was supported by the basic science research program through the national research foundation of korea (nrf), funded by the ministry of education ( r a a ). conflicts of interest the authors declare no conflicts of interest. key: cord- - vsynfgn authors: kostoff, ronald n.; briggs, michael b.; porter, alan l.; spandidos, demetrios a.; tsatsakis, aristidis title: covid- vaccine safety date: - - journal: int j mol med doi: . /ijmm. . sha: doc_id: cord_uid: vsynfgn in response to the sars-cov- outbreak, and the resulting covid- pandemic, a global competition to develop an anti-covid- vaccine has ensued. the targeted time frame for initial vaccine deployment is late . the present article examines whether short-term, mid-term, and long-term vaccine safety can be achieved under such an accelerated schedule, given the myriad vaccine-induced mechanisms that have demonstrated adverse effects based on previous clinical trials and laboratory research. it presents scientific evidence of potential pitfalls associated with eliminating critical phase ii and iii clinical trials, and concludes that there is no substitute currently available for long-term human clinical trials to ensure long-term human safety. the new outbreak of sars-cov- from december precipitated a world-wide crisis. globally, lockdowns of different severity levels were imposed ( ) . while the number of daily deaths attributable to covid- appears to have decreased substantially by june , the increasing numbers of 'cases' (positive test results for viral exposure) have raised some concerns regarding the ability of governments and decision-making authorities to reduce viral transmission and subsequent consequences ( ) ( ) ( ) . currently, at months following the outbreak, no specific treatment for severe forms of covid- has achieved consensus within the medical community, although several potential therapies appear to have produced more or less encouraging results ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the methods used to contain the spread of the virus have been the traditional social distancing, quarantine, use of disinfectant substances, and wearing of protective face masks ( ) ( ) ( ) . these measures have adverse consequences, both psychological and economic, and have resulted in substantial disagreement among the medical community and political decision-makers regarding their efficacy ( , , ) . in parallel with the imposed restrictions to prevent viral spread and the testing of (mainly) repurposed anti-viral treatments is the accelerated development of vaccines to prevent/restrict potential viral damage. questions have been raised as to whether an accelerated vaccine development can be accomplished safely, preventing potential adverse vaccine effects not only in the short-term, but also in the mid-and long-term (https://smartech.gatech.edu/handle/ / ). currently (mid-september, ), there is avid competition regarding the development and commercialization of a vaccine by early ( , ) . one candidate vaccine, sputnik- , was approved by the ministry of health of the russian federation on august , ( ) . these accelerated vaccine development efforts suggest that safety testing was performed in ≤ year, a time frame significantly shorter than that of - years typically associated with the commercialization of a vaccine ( ) . it is difficult to see how mid-and long-term safety testing for the proposed vaccine (or any vaccine or drug) can be performed credibly in such a compressed time frame (https://smartech.gatech.edu/handle/ / ) for reasons described below. the underlying models may be lacking. this could impact the credibility of any conclusions on safety or toxicity ( ) . thus, while these models may provide interesting insight, they cannot substitute for human trials, at least at this point in their development. animal experimentation. there are several examples where whole animal experiments have been poor predictors of human responses to environmental exposures or drugs. isotretinoin (acutane), for example, has been demonstrated to cause birth defects in rabbits, monkeys and humans, but not in mice or rats. as another example, corticosteroids are teratogenic in experimental animals, but not in humans. in addition, there is the well-known example of thalidomide, 'a teratogen in humans, but not in many experimental animal species' ( ) . why are some animal experiments poor predictors of human outcomes and responses? the studies may be designed poorly and may be inadequate methodologically; the studies may not be replicated or subject to meta-analyses; the metabolic pathways or drug metabolism of humans differ from those of the tested species or strains and 'disease manifestations in the animals are distinct from those encountered in humans' ( ) . laboratory animal experiments allow for the selection of test animals with short lifetimes, and can identify adverse health effects over the animals' lifetimes (the analog of long-term human effects), and perhaps one or two generations beyond. as previously stated ( ) , how well the response of the species selected for the experiments reflects the response of humans remains to be determined. additionally, laboratory animals are typically exposed to one toxic stressor (vaccines, in the present case), whereas human beings are exposed to myriad toxic stressors daily and over their lifetimes ( ) ( ) ( ) ( ) . these toxic stressor exposures may substantially alter the effects of a vaccine ( ) . to simulate the human real-life experience, a number of animal experiments would have to be performed to reflect the effects of various combinations of the thousands of toxic stressors (in conjunction with vaccines) to which human beings could be exposed (and other exposures that, by themselves are not toxic, but in combination are toxic) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . these experiments would require vast amounts of resources, particularly money and time. human clinical trials. human trials have at least two advantages over laboratory animal experiments. first, there are no concerns regarding species differences that occur when extrapolating from laboratory animal testing results to potential human impacts. second, humans are exposed to myriad toxic stressors before, during and after the trial period, providing results that mirror the real-life experience. in all cases, human trials will be most relevant if the characteristics of the trial population reflect those of the target/user population. the disadvantages of human clinical trials are as follows: i) the exposures to toxic stimuli are either not known, or, if they are known, have not been estimated accurately; and ii) the identification of the long-term effects requires long periods of time (https://smartech.gatech.edu/handle/ / ). how much time is required? in a previous study of vaccines and autoimmunity ( ), the authors concluded that 'latency periods can range from days to years for postinfection and postvaccination autoimmunity'. mid-term adverse effects of vaccines, such as central nervous system (cns) inflammatory demyelination ( ) and diabetes ( ) have been shown to emerge after approximately years. longer-term effects, such as cancer, alzheimer's disease, parkinson's disease, etc., have not been studied. in fact, vaccine inserts typically state that carcinogenic effects (and mutagenic and fertility effects) have not been studied ( ) [e.g., for the mmr vaccine it is stated that 'm-m-r ii has not been evaluated for carcinogenic or mutagenic potential, or potential to impair fertility ... animal reproduction studies have not been conducted with m-m-r ii'; and for the hpv vaccine it is stated that 'gardasil has not been evaluated for the potential to cause carcinogenicity, genotoxicity or impairment of male fertility' ( ) ]. several decades of close tracking would be required to identify such adverse effects. an overlooked issue associated with the vaccine discussions is potential transgenerational effects. transgenerational studies of adverse substance effects tend to be focused on environmental causes; however, there are some examples of such studies for drugs. a previous study on chemotherapy-induced late transgenerational effects ( ) has raised some concerns, both due to the scarcity of such studies in the literature and the transmission of adverse effects deep in the generational chain. due to the inadequate safety testing of several toxic stimuli in the past (including vaccines), it remains uncertain as to whether a number of diseases currently affecting humanity may be due in part to the actions of our predecessors passed on to us through transgenerational effects. it is uncertain as to whether any of the drugs, vaccines, foods or radiation exposures of our predecessors, which were not tested for transgenerational effects, are adversely affecting human life at present. of note, the question remains whether humanity is currently willing to pass on potential devastating diseases to future generations due to the present need for the speedy development of a vaccine, bypassing adequate long-term and transgenerational safety testing. there are also ethical issues of concern associated with accelerated vaccine development, particularly with the drastic reduction in time devoted to clinical trial phases ii and iii ( ) . the main target population for a vaccine is the most vulnerable demographically: the elderly with high comorbidities and dysfunctional immune systems. yet, the test demographic population being used for the initial clinical trials is the relatively young and healthy population (as discussed below). this leads to uncertainty regarding the efficacy of the trial, raising issues as to how the results from a young healthy population can be extrapolated to an elderly and vulnerable population. additionally, in myriad cultures, it is the elderly who sacrifice for the benefit of the young. this tradition is being inverted in the present accelerated testing regimen. for any new product, the decision to implement (whether for commercial or non-commercial purposes) typically involves a tradeoff between costs and benefits. in the ideal case, the projected benefits would far outweigh the projected costs. the potential costs and/or benefits may be known to high, modest, or low degrees of certainty. thus, a risk factor must be applied to the costs and benefits, reflecting the level of uncertainty about the projections. the vaccine costs in this discussion are the potential adverse health effects from a covid- vaccine, particularly for the mid-and long-term. for a vaccine with high levels of uncertainty as to the projected costs, a high risk factor is required. for the tradeoff to justify moving forward, a very high level of benefits would be required. the cost-benefit tradeoff for a covid- vaccine would be different for groups with different vulnerabilities to the disease. for simplicity, the target population for vaccination could be divided into groups: the highly vulnerable, and the remainder of the population. the demographic population most vulnerable to the more severe consequences of covid- tends to be the elderly with high comorbidities and others with compromised immune systems ( ) . it is a small fraction of the total population, although a somewhat greater fraction of the senior population. the remainder of the population, when infected with the sars-cov- virus, usually displays no symptoms or minimal symptoms. this demographic sub-division is similar to that for influenza and for the sars pandemic ( ) . the vaccine tradeoff analysis will differ for each of these two groups. for the most vulnerable, the main consideration is to survive the season. the mid-and long-term effects may be of lesser importance (although for the few younger members of this demographic population with highly compromised immune systems, the mid-and long-term adverse effects would not be negligible). for the least vulnerable (the vast majority of the population), the need for a vaccine is unclear, since the adverse effects of the virus appear to be minimal for most. this least vulnerable demographic population would have to bear the brunt of any potential mid-and long-term adverse health impacts that may result from a vaccine inadequately tested for these effects. thus, a vaccine that proved efficacious for the very short-term for all demographics may potentially be justifiable (albeit high-risk) for the most vulnerable demographic population. however, it is difficult to ascertain how such a vaccine could be justified for the remaining demographics. furthermore, the question remains of what are the present prospects for a vaccine efficacious even in the short-term. trial results for a highly-promoted covid- vaccine reported publicly have exhibited adverse effects of varying severity, where the test group was relatively young and very healthy ( , ) , unlike the highly vulnerable elderly target group with comorbidities. in other words, even short-term efficacy has not yet been demonstrated for the least vulnerable demographic population, much less the most vulnerable demographic population who would be the most justifiable target of the vaccine. in the present political environment, there is the potential that the majority of the population could be required to be vaccinated, even those demographics that were not vulnerable to the severe effects of covid- , and particularly those in the youngest demographic. the potential adverse consequences of such a mass inoculation with a vaccine not adequately tested for mid-and long-term adverse effects could be substantial. not applicable. no funding was received. not applicable. all authors substantially contributed to the conception, writing and revision of the work and approved the final content of the manuscript. not applicable. not applicable. das is the editor-in-chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. the other authors declare that they have no competing interests. a new threat from an old enemy: re-emergence of coronavirus (review) post-lockdown guidelines covid- in northern italy: an integrative overview of factors possibly influencing the sharp increase of the outbreak (review) the under-reported role of toxic substance exposures in the covid- pandemic editorial: nicotine and sars-cov- : covid- may be a disease of the nicotinic cholinergic system zinc and respiratory tract infections: perspectives for covid- (review) spandidos da, nikolouzakis tk, drakoulis n and tsatsakis a: comprehensive analysis of drugs to treat sars-cov- infection: mechanistic insights into current covid- therapies (review) spandidos da and tsatsakis a: a dissection of sars-cov with clinical implications (review) a perspective on emerging therapeutic interventions for covid- sars-cov- : repurposed drugs and novel therapeutic approaches -insights into chemical structure-biological activity and toxicological screening impact of self-imposed prevention measures and short-term government-imposed social distancing on mitigating and delaying a covid- epidemic: a modelling study nazarenko y: air filtration and sars-cov- protection and disinfection policies against sars-cov- (covid- ) effects of wearing n and surgical facemasks on heart rate, thermal stress and subjective sensations covid- : important potential side effects of wearing face masks that we should bear in mind fact sheet: explaining operation warp speed covid- vaccine tracker. regulatory affairs professionals society towards effective covid- vaccines: updates, perspectives and challenges (review) are we rushing too much? the role of toxic stimuli combinations in determining safe exposure limits covid- , an opportunity to reevaluate the correlation between long-term effects of anthropogenic pollutants on viral epidemic/pandemic events and prevalence human exposure to chemical mixtures: challenges for the integration of toxicology with epidemiology data in risk assessment simulating real-life exposures to uncover possible risks to human health: a proposed consensus for a novel methodological approach new challenges in risk assessment of chemicals when simulating real exposure scenarios; simultaneous multi-chemicals' low dose exposure a small jab -a big effect: nonspecific immunomodulation by vaccines setting safer exposure limits for toxic substance combinations behavioral impacts of a mixture of six pesticides on rats a mixture of routinely encountered xenobiotics induces both redox adaptations and perturbations in blood and tissues of rats after a long-term low-dose exposure regimen: the time and dose issue genotoxic, cytotoxic, and cytopathological effects in rats exposed for months to a mixture of chemicals in doses below noael levels the effect of chronic vitamin deficiency and long term very low dose exposure to pesticides mixture on neurological outcomes -a real-life risk simulation approach six months exposure to a real life mixture of chemicals' below individual noaels induced non monotonic sex-dependent biochemical and redox status changes in rats adverse and hormetic effects in rats exposed for months to low dose mixture of chemicals: rlrs part iii hormetic neurobehavioral effects of low dose toxic chemical mixtures in real-life risk simulation (rlrs) in rats vaccines and autoimmunity hepatitis b vaccine and the risk of cns inflammatory demyelination in childhood clustering of cases of insulin dependent diabetes (iddm) occurring three years after hemophilus influenza b (hib) immunization support causal relationship between immunization and iddm johns hopkins bloomberg school of public health: package inserts and manufacturers for some us licensed vaccines and immunoglobulins under-reporting of adverse events in the biomedical literature covid- vaccines: ethical framework concerning human challenge studies coronavirus disease: vs. the flu. johns hopkins medicine an mrna vaccine against sars-cov- -preliminary report key: cord- -oa twg z authors: pastorino, giulia; cornara, laura; soares, sónia; rodrigues, francisca; oliveira, m. beatriz p.p. title: liquorice (glycyrrhiza glabra): a phytochemical and pharmacological review date: - - journal: phytother res doi: . /ptr. sha: doc_id: cord_uid: oa twg z in the last years, consumers are paying much more attention to natural medicines and principles, mainly due to the general sense that natural compounds are safe. on the other hand, there is a growing demand by industry for plants used in traditional medicine that could be incorporated in foods, nutraceuticals, cosmetics, or even pharmaceuticals. glycyrrhiza glabra linn. belongs to the fabaceae family and has been recognized since ancient times for its ethnopharmacological values. this plant contains different phytocompounds, such as glycyrrhizin, β‐glycyrrhetinic acid, glabrin a and b, and isoflavones, that have demonstrated various pharmacological activities. pharmacological experiments have demonstrated that different extracts and pure compounds from this species exhibit a broad range of biological properties, including antibacterial, anti‐inflammatory, antiviral, antioxidant, and antidiabetic activities. a few toxicological studies have reported some concerns. this review addresses all those issues and focuses on the pharmacological activities reported for g. glabra. therefore, an updated, critical, and extensive overview on the current knowledge of g. glabra composition and biological activities is provided here in order to explore its therapeutic potential and future challenges to be utilized for the formulation of new products that will contribute to human well‐being. prepare a tea that is an excellent thirst quencher. the dried root has been described as a tooth cleanser (armanini et al., ) . actually, the most important industrial use of g. glabra is the production of food additives, such as flavours and sweetening agents (mukhopadhyay & panja, ) . in particular, the root is used as a flavouring agent for american-type tobacco, chewing gum, candies, baked goods, ice cream, and soft drinks (rizzato, scalabrin, radaelli, capodaglio, & piccolo, ) . in beers and fire extinguishers, the root extracts are used as foaming agents, whereas the root fibbers are used in insulation, wallboard, and boxboard materials, after removal of the medicinal and flavouring constituents. in the cosmetic field, g. glabra is described as a skin depigmentation agent and is being incorporated in topical products for that purpose. with regard to government approval, liquorice extract and glycyrrhizin have been allowed for use in foods by the united states food and drug administration, the council of europe, and the joint fao/who expert committee on food additives (fao, ) . indeed, the u.s. flavor and extract manufacturers association has recognized it as generally safe. to the best of our knowledge, a limited number of reviews have been published on this plant, particularly in what concerns to pharmacological aspects (asl & hosseinzadeh, ; fiore, eisenhut, ragazzi, zanchin, & armanini, ) . the objective of this review was to examine the bioactive compounds of g. glabra and the biological activities associated with these compounds. g. glabra is a typical herbaceous perennial, growing to m in height, presenting pinnate leaves with a length of to cm. the flowers are purple to pale whitish blue, being arranged in a hermaphrodite inflorescence, whereas the fruit is an oblong legume with to cm of length and containing several seeds. the genus glycyrrhiza (fabaceae) consists of about species, such as g. glabra, g. uralensis, g. inflata, g. aspera, g. korshinskyi, or g. eurycarpa. like the other plants of fabaceae, g. glabra is able to fix nitrogen, due to symbiosis with bacteria of the genus rhizobium, at the root level, being suitable for sandy and clay soils, though preferring humid soils. since the egyptian age, the therapeutic properties of g. glabra are well documented (fiore et al., ) . the roots are the most used parts whereas leaves are considered an agrochemical waste. however, in the last years, different authors studied the phytochemical composition of g. glabra leaves, demonstrating that certain compounds present in the roots are also identified in leaves, although in smaller quantities (hayashi & sudo, ; siracusa et al., ) . in the last years, the chemical constituents of liquorice have been extensively investigated by different authors siracusa et al., ) . nevertheless, few studies were carried out on the nutritional composition of g. glabra. nutritionally, liquorice is a source of proteins, amino acids, polysaccharides and simple sugars, mineral salts (such as calcium, phosphorus, sodium, potassium, iron, magnesium, silicon, selenium, manganese, zinc, and copper), pectins, resins, starches, sterols, and gums (q. . oestrogens, tannins, phytosterols (sitosterol and stigmasterol), coumarins, vitamins (b , b , b , b , e, and c), and glycosides have been reported (q. wang, qian, et al., ) . a large number of biological compounds have also been isolated, mostly triterpenes, saponins (responsible for the sweet taste), and flavonoids (rizzato et al., ; q. wang, qian, et al., ) . the liquorice saponins are present as glucuronides, whereas the aglycones are present as oleananes. the triterpene saponins are the major characteristic constituents of liquorice, being responsible for the sweet taste. the contents of these compounds may vary significantly due to geographic sources, harvesting, and processing, affecting the therapeutic effects of liquorice. the main constituent of roots is glycyrrhizin, a triterpenoid saponin that is almost times sweeter than sucrose, being the primary active ingredient (j. y. yu et al., ) . glycyrrhizin represents about % of the liquorice root dry weight, being a mixture of potassium, calcium, and magnesium salts of glycyrrhizic acid that varies between % and % (rizzato et al., ) . after oral administration, glycyrrhizin is metabolized to -glycyrrhetic acid omonoglucuronide and glycyrrhetic acid by intestinal bacteria (albermann, musshoff, hagemeier, & madea, ) . the yellow colour of liquorice is due to the flavonoid content. the flavonoids identified belong to different classes, including flavanones, flavones, flavanonols, chalcones, isoflavans, isoflavenes, isoflavones, and isoflavanones. the major flavonoids are glycosides of liquiritigenin ( ′, -dihydroxyflavanone) and isoliquiritigenin ( ′, , ′trihydroxychalcone), such as liquiritin, isoliquiritin, liquiritin apioside, and licuraside (rizzato et al., ) . five new flavonoids have been isolated from dried roots: glucoliquiritin apioside, shinflavanone, shinpterocarpin, prenyllicoflavone a, and -methoxyphaseolin. pinocembrin and licoflavanone were also isolated from the leaves (fukui, goto, & tabata, ) . glabridin is the principal isoflavone identified, ranging between . % and . % of roots' dry weight (simmler, pauli, & chen, liquorice is one of the oldest and most popular herbal medicines in the world. many of the liquorice historical uses are still practised the antioxidant activity of g. glabra is one of the major reasons for its uses. the phenolic content is probably responsible for the powerful antioxidant activity observed (rackova et al., ) . varsha and sonam ( ) attributed this activity to flavonoids, whereas singh et al. ( ) reported that mostly isoflavones, such as glabridin, hispaglabridin a, and -hydroxy- -o-methylglabridin, are the responsible compounds. biondi, rocco, and ruberto ( ) reported a huge antioxidant activity of the dihydrostilbene derivates present in g. glabra leaves. also, licochalcones b and d are present in g. glabra, showing a strong scavenging activity on dpph radical and the ability to inhibit the microsomal lipid peroxidation (biondi et al., ; v. sharma, katiyar, & agrawal, ) . these phenolic compounds are effective in the protection of biological systems against oxidative stress, being able to inhibit the onset of skin damages (haraguchi et al., ) . according to castangia et al. ( ) , the topical application of liquorice extract formulations may be of value in innovative dermal and cosmetic products as it counteracts oxidative stress damage, maintaining the skin homeostasis due to its high antioxidant content. table summarizes the most important studies of antioxidant activity. the anti-inflammatory activity of g. glabra and its use in the treatment of inflammatory diseases have been documented since ancient times (r. yang, yuan, ma, zhou, & liu, ) . shalaby, ibrahim, mahmoud, and mahmoud ( ) evaluated the anti-inflammatory activity of g. glabra in male rats after weeks of food intake. the authors observed a significant decrease in the total cholesterol and triglyceride levels as well as in the levels of serum liver enzymes. harwansh, patra, pareta, singh, and biswas ( ) reviewed the positive effects of g. glabra on the treatment of the upper respiratory tract and gastric system diseases. these pharmacological effects were due to an increase in the secretion of serotonin and prostaglandins in the stomach that led to a decrease of gastric inflammation (bahmani et al., ) . different authors described that the anti-inflammatory action is primary mediated by glycyrrhizin, which in vitro could inhibit factors responsible for inflammation as well as promote the healing of stomach and mouth ulcers (rackova et al., ; yin et al., ) . in fact, the anti-inflammatory effects of glycyrrhizin were described as similar to those of glucocorticoids and mineralocorticoids (kageyama, suzuki, & saruta, ) . furthermore, g. glabra is used in renal and liver complications on the basis of its strong antiinflammatory effects (y. xiao et al., ) . y. xiao et al. ( ) reported the inhibition of liver granuloma formation and the inflammatory cytokine production by glycyrrhizin, whereas x. r. wang, hao, and chu ( ) described the anti-inflammatory effects on endometriosis. moreover, liu et al. ( ) proved the anti-inflammatory activity of glabridin on raw cells. the antitussic and expectorant effects of liquorice have been reported by different authors, particularly its useful effects on the treatment of sore throat, cough, and bronchial catarrh (damle, ; fiore et al., (hasanein, ) glycyrrhiza glabra extract - mg/kg, days in vivo-oral administration to swiss young male albino mice production of antidepressant-like effect in mice in forced swim test and tail suspension test, probably by interaction with adrenergic and dopaminergic system (dhingra & sharma, ) g. glabra aqueous extract - mg/kg, days in vivo-oral administration to mice dose of mg/kg significantly improved learning and memory of mice (parle, dhingra, & kulkarni, ) sedative activity (cho et al., ) antidepressive activity g. glabra aqueous extract - mg/kg in vivo-forced swim test and tail suspension test applied to mice antidepressant-like effect of liquorice extract seems to be mediated by increase of brain norepinephrine and dopamine, but not by increase of serotonin (dhingra & sharma, ) oestrogenic activity female wistar rats stimulation of creatine kinase specific activity (tamir, eizenberg, somjen, izrael, & vaya, ) liquorice root extract μg/day, weeks in vivo-oral administration to female rats increase in creatine kinase activity (tamir et al., ) liquiritigenin - μg/ml in vitro-mcf- and t d cells induction of oestrogen responsive alkaline phosphatase activity in endometrial cancer cells, oestrogen responsive element luciferase in mcf- cells and tff mrna in t d cells (somjen et al., ) isoliquiritigenin - . mg/ml in vivo-intraperitoneal injection of female icr mice improvement of ivf rate (tung, shoyama, wada, & tanaka, ) skin effects glycyrrhizinic acid in vivo-double-blind clinical trial in human patients reduction of erythema, oedema, and itching scores (halder & richards, ) -in vitro-topical treatments in human patients during weeks lighten hand solar lentigines (nerya et al., ) glycyrrhetinic acid; glabridin - μm in vitro-human keratinocyte culture prevention of oxidative dna fragmentation and activation of apoptosis-associated proteins in human keratinocyte (grippaudo & di russo, ) (continues) in vitro-vero cells in vivo-ducks stimulation of immune and antiviral effect against dhv (soufy et al., ) . μg/ml (extract) in vitro-human foreskin cell line protection of host cells against ev infection (kuo, chang, wang, & chiang, ) in vitro-vero cells protection against coronavirus (cinatl et al., ) μg/ml (compound) in vitro-peripheral blood mononuclear cells inhibition of nonsyncytium-inducing variant of hiv replication (sasaki, takei, kobayashi, pollard, & suzuki, ) - , mg/day (compound) in vitro-human immunodeficiency virus type (hiv- ) p antigen inhibition of hiv- replication (hattori et al., ) mg in vivo-oral administration to humans (ldl isolation) (carmeli & fogelman, ) licochalcone - μg/ml dpph, superoxide anion, lipid peroxidation, red blood cells inhibition of the microsomal lipid peroxidation (haraguchi, ishikawa, mizutani, tamura, & kinoshita, ) hepatoprotective activity liquorice aqueous extract - mg/kg days in vivo-oral administration to wistar rats stimulation of the antioxidant enzymes and arrest of inflammatory cytokine production (huo, wang, liang, bao, & gu, ) g. glabra aqueous root extract g/day, months in vivo-humans alt and ast decrease (hajiaghamohammadi, ziaee, & samimi, ) , , mg/kg ). these effects are associated with the presence of glycyrrhizin that helps to expel congestion in the upper respiratory tract and accelerates tracheal mucus secretion (v. sharma et al., ) . likewise, liquiritin apioside, an active compound reported in the methanolic extract of liquorice, has the ability to inhibit capsaicin, a compound that induces cough (kamei, nakamura, ichiki, & kubo, ) . the effect on sore throat has been compared with that of carbenoxolone, a glycyrrhetinic acid derivative with a steroid-like structure, which stimulates gastric mucus secretion (damle, ) . the use of g. glabra extract as antiulcerative is widely known. for the gastrointestinal system, it is used in gastric and duodenal ulcers (bardhan, cumberland, dixon, & holdsworth, ) , whereas for the treatment of spasmodic pains of chronic gastritis, it is employed as an adjuvant (armanini et al., ) . the benefits of g. glabra in the treatment of duodenal and peptic ulcers have been reported since the s, and this traditional use is related to the presence of antiinflammatory saponins (krausse et al., ) . the major compound responsible for this activity is glycyrrhizin, which can raise the concentration of prostaglandins in the digestive tract, promoting stomach mucus secretion (jafarian & ghazvini, ) . in addition, liquorice prolongs the lifespan of stomach surface cells, demonstrating an antipepsin effect (ram, lachake, kaushik, & shreedhara, ) . furthermore, deglycyrrhizinated liquorice has shown some effects in the treatment of gastrointestinal ulcers, suggesting the presence of other active ingredients (zadeh, kor, & goftar, ) . indeed, carbenoxolone, a glycyrrhetinic acid analogue, is reported to inhibit two important enzymes for the metabolism of prostaglandin, -hydroxyprostaglandin dehydrogenase and Δ prostaglandin reductase, raising prostaglandin levels and leading to positive effects in clinical trials for gastric and duodenal ulcers (damle, ) . in fact, prostaglandins stimulate mucous secretion and cell proliferation leading to ulcer healing. nevertheless, the glycyrrhetic acid derivative carbenoxolone presents secondary effects such as the potential development of pseudo aldosteronism, which limits its use. in a clinical trial, bardhan et al. ( ) studied the effect of liquorice by oral administration in patients with gastric ulcer. the patients were randomly allocated to the treatment either with deglycyrrhizinated liquorice or with placebo. however, after weeks, no differences were observed between groups in the percentage of ulcer area reduction or clinical improvements (bardhan et al., ) . particular, glabridin, glabrol, glabrene, hispaglabridin a, hispaglabridin b, -methylglabridin, and -hydroxyglabrol, isolated from g. glabra, are responsible for this activity (l. . the mechanism behind this could be the decrease of bacterial gene expression, the inhibition of bacterial growth, and the reduction of bacterial toxin production (gupta et al., ; l. wang, yang, et al., ) . in , s.-j. ahn, song, mah, cho, and kook ( ) (fukai et al., a (fukai et al., , b l. wang, yang, et al., ) . on the other hand, the ability to inactivate methicillin-resistant s. aureus (mrsa) by decreasing the expression of saer and hla, the key virulence genes of mrsa, have also been reported by different authors (fukai et al., a (fukai et al., , b l. wang, yang, et al., ) . it is also suggested that licochalcone e could be used for chemical synthesis of novel anti-s. aureus compounds, reducing the production of α-toxin in both methicillin-sensitive s. aureus and mrsa (l. . besides, α-haemolysin is an important exotoxin in the pathogenesis of s. aureus infections (berube & bubeck wardenburg, ) . such infections are associated with a broad spectrum of diseases, ranging from endocarditis to minor skin infections, toxinoses, and lethal pneumonia. liquiritigenin, one of the most active compounds of liquorice, demonstrated the capacity to prevent human lung cells (a ) from α-haemolysin-mediated injuries, by decreasing αhaemolysin production (l. . similarly, glabrin and glycyrrhetinic acid have shown antibacterial activity against s. aureus . different authors reported the antibacterial action of g. glabra against mycobacterium tuberculosis (gupta et al., ) , demonstrating that glabridin is the responsible compound for this activity, instead of hispaglabridin b . the antitubercular phenolic compounds were previously identified as licoisoflavone and licochalcone a (chakotiya, tanwar, srivastava, narula, & sharma, ) . in a mice lung infection model, g. glabra was therapeutically active against multidrug-resistant strain of p. aeruginosa (chakotiya et al., ) , and the hydro-alcoholic extract led to a reduction of the microbial load in the blood, mainly due to the presence of stigmasterol, ergosterol, licochalcone, and glabridin (chakotiya et al., ) . the activity of g. glabra against helicobacter pylori has been also reported, as mentioned in the previous subsection (krausse et al., ) . according to krausse et al. ( ) , the compounds responsible for this activity are glabridin and glabrene. cao et al. ( ) also reported that β-glycyrrhetinic acid significantly attenuated the gastritis infection caused by h. pylori. asha et al. ( ) found that the flavonoid glabridin exhibits activity against h. pylori whereas glycyrrhizin did not present activity even at a concentration of μg/ml. these flavonoids also showed activity against h. pylori strains resistant to clarithromycin and amoxicillin (fukai et al., a (fukai et al., , b . the probable mechanism behind these action is the inhibition of the protein synthesis, dna gyrase, and dihydrofolate reductase (fukai et al., a (fukai et al., , b . moreover, the liquorice polysaccharides also present activity against porphyromonas gingivalis adhesion, which is of huge importance because no specific adhesion inhibitors have been described (chinsembu, ) . the antifungal activity of g. glabra is also detailed (sato, goto, nanjo, kawai, & murata, ) . sato et al. ( ) reported that the methanolic extract of liquorice presents fungicidal activity against arthrinium sacchari and chaetomium funicola, whereas glabridin was found to be the active compound responsible for the observed effects (sato et al., ) . in fact, isoflavonoids, such as glabridin, glabrol, and their derivatives, are responsible for the in vivo inhibition of mycobacterium smegmatis, shigella, salmonella, e. coli, s. mutans, and lactobacillus acidophilus (ajagannanavar et al., ) . recently, chandra and gunasekaran ( ) also proved the antifungal activity of crude methanolic extract of g. glabra against aspergillus niger. different authors reported that c. albicans is susceptible to liquorice extracts due to their richness in liquiritigenin, liquiritin, licochalcone a, and glabridin (chandra & gunasekaran, ; j. y. lee et al., ; singh et al., ) . nevertheless, according to karahan, avsar, ozyigit, and berber ( ) , the antimicrobial activity could be influenced by the environmental conditions that may affect the chemical compound contents and the biological activity. according to messier et al., licochalcone a and glabridin present a therapeutic potential against c. albicans oral infections, whereas glycyrrhizic acid had no effect (messier & grenier, ; moazeni et al., ) . fukui et al. ( ) isolated licoflavanone from the leaves of g. glabra, demonstrating its antimicrobial activity. indeed, chen et al. ( ) reported that licochalcone a is an antiparasitic compound with potential activity against human pathogenic protozoan leishmania species. the antiviral activity of g. glabra extracts against different viruses has been reported, including herpes simplex, varicella zoster, japanese encephalitis, influenza, and vesicular stomatitis virus (l. . different studies have demonstrated that two triterpenoids are responsible for the antiviral activity reported: glycyrrhizin and β-glycyrrhetinic acid (l. . these compounds have the ability to inhibit virus gene expression and replication, decreasing the adhesion force and stress and reducing hmgb binding to dna (l. . also, they can herpes simplex virus (hsv) is one of the most common viruses infecting humans and animals. during hsv infection, the cellular adhesion is increased, playing a key role in inflammatory response. w. huang et al. ( ) reported that the adhesion force and stress between the cerebral capillary vessel endothelial cells and the polymorphic nuclear leukocytes were amplified during hsv infection. glycyrrhizin stimulates the mouse defence system against hsv- infection (sekizawa, yanagi, & itoyama, ) . furthermore, glycyrrhizic acid was found to have a distinctive effect against kaposi sarcoma-associated herpes virus (kshv). it was proved that glycyrrhizic acid could terminate the latent infection of kshv when all current drugs are ineffective (damle, ) . also, glycyrrhizic acid down-regulates the expression of latency-associated nuclear antigen in b lymphocytes leading to natural cell death (apoptosis) of the kshv-infected cells (damle, ) . recently, the antiviral activity of glycyrrhizin against severe acute respiratory syndrome virus was evaluated (cinatl et al., ) . glycyrrhizin affects the cellular signalling pathways such as protein kinase c, casein kinase ii, and transcription factors, namely, activator protein and nuclear factor κb (cinatl et al., ) . furthermore, glycyrrhizin and its aglycone, β- the hepatoprotective activity of glycyrrhizin and β-glycyrrhetic acid by inhibition of free-radical generation and lipid peroxidation has been extensively reported (huo et al., ) . one of these studies indicated that the hydromethanolic root extract of g. glabra exhibits a significant protection against hepatotoxicity induced by carbon tetrachloride in the liver tissue of mice (v. sharma & agrawal, ) . the effects of liquorice on nonalcoholic fatty liver disease have also been investigated (hajiaghamohammadi et al., ) . according to rizzato et al. ( ) , glycyrrhizin and glycyrrhetinic acids prevent drug-induced liver injury and ensure the disruption of bile acid metabolism in humans. indeed glycyrrhetinic acid has been reported as anti-inflammatory and hepatoprotective compound (yin et al., ) , whereas glycyrrhizin, when compared with the placebo, induced a significant reduction in the serum aminotransferases and improved the liver histology (van rossum et al., ) . it has also been reported that the long-term use of glycyrrhizin prevents the development of hepatocellular carcinoma in chronic hepatitis c (van rossum et al., ) . in vitro studies have shown that glycyrrhizin modifies the intracellular transport and suppresses the hepatitis b virus surface antigen (sato et al., ) . in addition, it prevents the oxidative and hepatic damage caused by aflatoxins through increasing cyp a and glutathione-stransferase activity, contributing to the anticarcinogenic activity by metabolic deactivation of the hepatotoxin (y. . mahmoud, hussein, hozayen, and abd el-twab ( ) reported that the treatment with β-glycyrrhetinic acid significantly reduced the serum enzymes, bilirubin, and proinflammatory cytokines in the liver, decreasing the expression of p e . different studies suggest that the extract of g. glabra may be a potential supplemental source for different cancer treatments (c. s. lee, kim, lee, han, & lee, ; ohtsuki, oh-ishi, & nagata, ) . this activity is due to the β-glycyrrhetinic and glycyrrhizic acids that x. y. xiao et al., ) . the results indicated that licochalcone a inhibits gastric cancer cells growth in a dose-dependent way, by blocking cell cycle progression at the g /m transition, inducing apoptosis. in addition, licochalcone a induced apoptosis by its effects on the expression of parp, caspase- , bcl- , and bax (x. y. xiao et al., ) . kanazawa et al. ( ) and jung et al. ( ) showed that isoliquiritigenin inhibits the cell growth by g /m cell cycle arrest in breast and prostate tumour cells. different studies demonstrated that isoliquiritigenin suppresses pulmonary metastasis in mice (yamazaki et al., ) and human hepatoma cells (hsu, kuo, & lin, ) . apoptosis was primarily mediated through mitochondrial death cascade, as shown by loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase- . a possible explanation is that cell growth was arrested through up-regulation of p and p and down-regulation of cdk , cyclin e, and e f- while apoptosis was induced by increasing bax protein expression and activating caspase- (g. sharma et al., ) . glabridin exhibited antitumour properties in various human cancer cells . the results revealed that glabridin induced apoptosis in dose dependently in huh cells through caspase- , caspase- , and caspase- activation and parp cleavage (hsieh et al., ) . the effects of g. glabra on learning and memory were investigated in mice (dhingra & sharma, ; parle et al., ) . in , parle et al. ( ) administered the extract of g. glabra orally to mice during days at different concentrations ( - mg/kg). chakravarthi and avadhani ( ) and dhingra and sharma ( ) studied the effects of g. glabra root aqueous extract on the learning and memory of month-old male wistar albino mice at doses between and mg/kg, orally administered during six successive weeks. both studies demonstrated a significant improvement of learning and memory in mice, but the exact mechanism behind this action remains unknown (chakravarthi & avadhani, ; dhingra & sharma, ). these findings suggest a possible neuroprotective role of liquorice in the prevention of diseases such as alzheimer. the basis of alzheimer is the chronic inflammation of certain brain regions. thus, the antiinflammatory activity of liquorice might contribute to the observed memory-enhancing effects (yokota, nishio, kubota, & mizoguchi, ) . also, oxygen free radicals are implicated in the process of aging and could be responsible for the development of alzheimer's disease in elderly persons. the protective role of liquorice extract may be attributed to its antioxidant properties, resulting in reduced brain damage and improvement of neuronal function and memory. the combination of anti-inflammatory and antioxidant activities with neuroprotective role could lead to memory-enhancing effects. hasanein ( ) gamma-aminobutyric acid (gaba) is the major inhibitory neurotransmitter in the central nervous system, being gaba a receptors a target for anaesthetics and neuroleptic, anxiolytic, and anticonvulsant compounds . g. glabra acts as a modulator of gaba a receptors (hoffmann, beltrán, ziemba, hatt, & gisselmann, ) , being able to induce sedative and anxiolytic effects. glabridin was evaluated by examining gaba responses in acutely isolated dorsal raphe neurons of a rat . according to the authors, glabridin potentiated gaba-induced responses by positive modulation of gaba a receptors, exhibiting sedative and hypnotic effects . glabridin potentiation is not sensitive to flumazenil and uses a similar mechanism of the general anaesthetics involving the amino acids n and m , which are located in the second and third transmembrane domains on the β-subunit of gaba a receptors (hanrahan, chebib, & johnston, ) . also, glabridin could contribute to the hypnotic effect, as it is able to cross the blood-brain barrier . liquorice extract may have potential therapeutic value for the treatment of depressive disorders. recent studies have shown that liquorice extract produces significant antidepressant effects in mice during forced swim test (fst) and tail suspension test (tst; dhingra & sharma, ) . in the fst model, mice were forced to swim in a restricted space and induced to a characteristic behaviour of immobility. this situation reflects a state of depression. the tst model also induces a state of immobility that is claimed to reproduce a condition similar to human depression. both models are widely used to screen antidepressant drugs. the precise mechanisms by which liquorice extract produced this effect are not completely understood. however, it is suggested that the extract may interact with α -adrenoceptors and dopamine d receptors, increasing the levels of norepinephrine and dopamine in the mice brain (dhingra & sharma, ) . besides, p-cpa (a serotonin synthesis inhibitor) did not attenuate the antidepressant effect of liquorice extract, suggesting that this is not mediated by the serotonergic system. on the other hand, p-cpa reversed the antidepressant effect of fluoxetine, suggesting that fluoxetine acts in the serotonergic system. reserpine produces a significant depression by depleting biogenic amines in the brain of rodents. as liquorice extract reversed reserpine-induced depression, its antidepressant effect can be associated with the restoration of brain monoamines, such as norepinephrine and dopamine. since ancient times, the influences of liquorice on the action of cortisol, reduction of testosterone synthesis, and the influence on oestrogen activity are well known (armanini et al., ) . s. h. kim and park ( ) reported that isoflavones can influence sexual development and impair oestrous cycling and ovarian and hypothalamus and pituitary glands function (s. h. kim & park, ) . the oestrogenic effect of liquorice ethanolic extract could be explained by its agonist activity on mcf- breast cancer cells, being this action mediated by β-glycyrrhetinic acid (g. sharma et al., ) . glabridin is a common component of herbal remedies used for the treatment of menopausal symptoms, resulting in favourable outcomes similar to those of βoestradiol (su wei poh et al., ) . in concentrations between . and μg per animal, glabridin induces similar effects to the administration of oestradiol in a concentration of μg per animal. glabridin was found to be three to four times more active than ′-omethylglabridin and ′-o-methylglabridin (tamir et al., ) . moreover, according to tamir et al. ( ) , glabrene has a considerable oestrogenic activity. glabridin and glabrene are similar to βoestradiol in the stimulation of the specific activity of creatine kinase, although at higher concentrations, differing in the extension rate of action as well as in the interaction with other drugs. in human premenopausal bone cells, the response to β-oestradiol and glabridin (at a higher concentration) was higher than in postmenopausal cells, whereas glabrene (at a higher concentration) was more effective in postmenopausal cells (somjen et al., ) . isoliquiritigenin has a strong oestrogen-like activity, suggesting that this compound may be cyclized to liquiritigenin, which is an active flavonoid under physiological conditions (hajirahimkhan et al., ) . in vivo, the stimulatory effects of glabrene are similar to those of oestradiol (powers & setzer, ) . it is also interesting to observe that isoliquiritigenin and formononetin stimulate sperm during fertilization (tung et al., ) . this reveals that both phytoestrogens may be useful therapeutic agents for infertility treatments (tung, shoyama, wada, & tanaka, ) . zamansoltani, nassiri-asl, sarookhani, jahani-hashemi, and zangivand ( ) reported that the alcoholic extract of g. glabra has antiandrogenic effects probably by increasing the testosterone metabolism, down-regulating androgen receptors, or activating oestrogenic receptors. the main skin benefits reported for g. glabra are based on the antioxidant and anti-inflammatory activities as well as on the ultraviolet (uv) protection (halder & richards, ) . saeedi, morteza-semnani, and ghoreishi ( ) reported the use of liquorice mainly for skin eruptions, including dermatitis, eczema, pruritus, and cysts. in particular, the g. glabra flavonoids present depigmenting capabilities and tyrosinase inhibition effects (solano, briganti, picardo, & ghanem, ) . the presence of an α-keto group in flavonoids is responsible for this activity (y. j. kim & uyama, ; parvez et al., ) . castangia et al. ( ) have reported the skin protective effects of liquorice against damage from oxidative stress. according to the authors, liquorice extract can scavenge dpph free radicals with an inhibition of % and protect fibroblasts against oxidative stress (castangia et al., ) . nevertheless, when evaluated in the isolated form, glycyrrhizin showed a poor antioxidant activity, being not able to efficiently counteract the oxidative effect (castangia et al., ) . tyrosinase is essential for skin pigmentation due to its role in melanin biosynthesis (solano et al., ) . the use of tyrosinase inhibitors is important in the cosmetic and medicinal industries, due to their preventive effect on pigmentation disorders such as melasma, age spots, and sites of actinic damage (nerya et al., ) . alternatively, tyrosinase inhibitors may be targets for developing medicines to treat hypopigmentation-related problems, such as albinism and piebaldism (y. j. kim & uyama, ) . in particular, glabridin, glabrene, isoliquiritigenin, licochalcone a, and liquiritin have been reported as g. glabra compounds able to inhibit the tyrosinase activity (ebanks, wickett, & boissy, ; nerya et al., ) . recently, grippaudo and di russo ( ) described the effects of the topical application of glycyrrhetinic acid combined with fractional carbon dioxide laser for the benign treatment of hand hyperpigmentation during weeks. likewise, the treatment of human keratinocytes with βglycyrrhetinic acid and glabridin was documented to directly and indirectly prevent dna damage, avoiding the apoptosis activation caused by uv b radiation (veratti et al., ) . indeed, yokota et al. ( ) described that glabridin inhibits tyrosinase activity, melanogenesis, and skin inflammation. besides, glabrene acts as a tyrosinase inhibitor, preventing the formation of melanin in melanocytes, probably acting as skin-lightening agent. saeedi et al. ( ) liquorice has been traditionally used as a sweetener due to its taste, (tian, liu, zhen, & tong, ; tong, xie, rong, zhou, & meng, ) . according to bahmani et al. ( ) , liquorice can reduce diabetes symptoms, such as polydipsia and frequent urination, but cannot reduce blood glucose. takii et al. ( ) suggested that glycyrrhizin has an antidiabetic effect in noninsulin-dependent diabetes mice model, reducing the postprandial blood glucose rise. licochalcone a is the most promising one, inhibiting in vitro the growth of chloroquine-susceptible and chloroquine-resistant plasmodium falciparum strains (chen et al., ) . glabridin also showed in vitro activity against this parasite, probably by an induction of oxidative stress, mainly through the generation of reactive oxygen and nitrogen species that lead to apoptosis (cheema et al., ) . it is well known that myocardial ischaemia is one of the principal diseases in the western world. this disease occurs through occlusion or blockage of coronary arteries, resulting in myocardial cell death. however, the reperfusion produces the salvage of ischaemic tissue but also contributes to the myocardial cellular injury. the pretreatment with g. glabra significantly attenuates the ischaemic reperfusion, through an improvement of the heart antioxidant status, a positive modulation of the perturbed haemodynamic, and a recovery of left ventricular contractile function, along with histological salvage (di paola et al., ; ojha, golechha, kumari, bhatia, & arya, ) . in particular, glycyrrhizic acid induced protection against myocardial ischaemia in rats, probably due to its antioxidant potential (ojha et al., ) . similarly, nakagawa, kishida, arai, nishiyama, and mae ( ) demonstrated that g. glabra is safe for cardiomyocytes in a long-term administration. the extract of g. glabra has been used in the treatment of low bone mass, osteoporosis, fractures, bone defects, osteomalacia, osteogenesis imperfecta, bone disease, and periodontal diseases . rajesh ( ) described the inhibitory effect on bone reabsorption of g. glabra, whereas choi ( ) reported that glabridin is responsible for this activity. mitochondrial dysfunction, especially respiratory chain disruption, is responsible for aging-related bone diseases. hence, the target of glabridin is the reduction of mitochondrial dysfunction induced during aging and the prevention of osteoblast damage in osteoporotic patients (choi, ) . study on mice demonstrated that g. glabra, particularly glabridin, when integrated in a dietary supplement, could reduce the susceptibility of low-density lipoprotein (ldl) to oxidation and the atherosclerotic lesion area (fuhrman & aviram, ; grassi, desideri, & ferri, ) . these results could be related to the absorption and binding of glabridin to the ldl and a subsequent protection of the ldl from oxidation (fuhrman et al., ) . the methanolic extract of g. glabra rhizomes, at a dose of mg/kg, has antiarthritic activity in male rats by inhibition of leukocyte migration, autoantigen production, and exhibition of antiproteinase activity (choudhary, kumar, malhotra, & singh, ) . also, mishra, bstia, mishra, chowdary, and patra ( ) reported that a combined formulation of g. glabra and boswellia serrata ( : ) had a significant synergistic action on arthritis. shin et al. ( ) studied the antiallergic effects, namely, the antiscratching behaviour and the ige production inhibitory activity, of glycyrrhizin, β-glycyrrhetinic acid, isoliquiritin, and liquiritigenin in dermatitis and asthma (shin et al., ) . in particular, β- different adverse side effects were reported for high doses of g. glabra such as hypertension, hypokalaemia, or fluid retention (omar et al., ) . the exposure to high levels of glycyrrhizin can produce hypermineralocorticoid-like effects. glycyrrhetic acid and liquorice saponins can inhibit -β-hydroxysteroid dehydrogenase enzyme, leading to a cortisol-induced mineralocorticoid effect and a consequent tendency to the elevation of sodium and reduction of potassium levels (isbrucker & burdock, ) . for example, in , a -yearold woman was suspected to have suffered a lethal acute intoxication from eating liquorice over a period of several months (albermann et al., ) . albermann et al. associated the effects with the potential mineralocorticoid action of glycyrrhizin and its metabolite, glycyrrhetic acid, and quantified by liquid chromatography-tandem mass spectrometry these compounds in the blood. nevertheless, only traces of glycyrrhetic acid had been found in the blood and stomach content of the deceased woman, which means that the possibility of acute lethal glycyrrhetic acid intoxication could be eliminated (albermann et al., ) . based on in vivo assays and clinical evidence, the amount of liquorice ingested daily by patients with mineralocorticoid excess syndromes appears to vary over a wide range ( . - g/day ; isbrucker & burdock, ) . in addition to hypertension, patients may experi- thus, side effects remain an area of potential future study. this review not only details and explains the traditional use of this plant but also highlights its potential uses for other industries, such as cosmetic or pharmaceutical ones. more clinical trials should be performed to provide scientific basis for new uses. in conclusion, although evidence has grown in the past decade, there is still a need to conduct further robust double-blind randomized controlled trial about g. glabra. there is also an immense scope to explore different combinations of liquorice preparations in a wide range of disorders. anti-obesity effects of glabridin-rich supercritical carbon dioxide extract of licorice in highfat-fed obese mice determination of optimal concentration of deglycyrrhizinated licorice root extract for preventing dental caries using a bacterial model system effect of aqueous and alcoholic licorice (glycyrrhiza glabra) root extract against streptococcus mutans and lactobacillus acidophilus in comparison to chlorhexidine: an in vitro study determination of glycyrrhetic acid after consumption of liquorice and application to a fatality history of the endocrine effects of licorice in vitro anti-helicobacter pylori activity of a flavonoid rich extract of glycyrrhiza glabra and its probable mechanisms of action review of pharmacological effects of glycyrrhiza sp. and its bioactive compounds a review of the health effects and uses of drugs of plant licorice (glycyrrhiza glabra l.) in iran clinical trial of deglycyrrhizinised liquorice in gastric ulcer staphylococcus aureus αtoxin: nearly a century of intrigue new dihydrostilbene derivatives from the leaves of glycyrrhiza glabra and evaluation of their antioxidant activity the protective effects of β-glycyrrhetinic acid on helicobacter pylori-infected gastric mucosa in mongolian gerbils antioxidant effect of polyphenolic glabridin on ldl oxidation delivery of liquorice extract by liposomes and hyalurosomes to protect the skin against oxidative stress injuries alternative to antibiotics against pseudomonas aeruginosa: effects of glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in pseudomonas aeruginosa and its in vitro time-kill activity effect of aquo-alchoholic extract of glycyrrhiza glabra against pseudomonas aeruginosa in mice lung infection model beneficial effect of aqueous root extract of glycyrrhiza glabra on learning and memory using different behavioral models: an experimental study screening of the phytochemical, antimicrobial and antioxidant activity of glycyrrhiza glabra root extract glabridin induces oxidative stress mediated apoptosis like cell death of malaria parasite plasmodium falciparum licochalcone a, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of leishmania licochalcone a, a new antimalarial agent, inhibits in vitro growth of the human malaria parasite plasmodium falciparum and protects mice from p. yoelii infection plants and other natural products used in the management of oral infections and improvement of oral health hypnotic effects and binding studies for gabaa and -ht c receptors of traditional medicinal plants used in asia for insomnia glabridin protects osteoblastic mc t -e cells against antimycin a induced cytotoxicity medicinal plants with potential anti-arthritic activity chemical composition and antimicrobial activities of the essential oil from glycyrrhiza glabra leaves glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus glycyrrhiza glabra (liquorice)-a potent medicinal herb effects of glycyrrhizin in a mouse model of lung adenocarcinoma antidepressant-like activity of glycyrrhiza glabra l. in mouse models of immobility tests. progress in neuro-psychopharmacology and biological psychiatry protective effects of glycyrrhizin in a gut hypoxia (ischemia)-reoxygenation (reperfusion) model. intensive care medicine mechanisms regulating skin pigmentation: the rise and fall of complexion coloration evaluation of certain food additives a history of the therapeutic use of liquorice in europe flavonoids protect ldl from oxidation and attenuate atherosclerosis licorice extract and its major polyphenol glabridin protect low-density lipoprotein against lipid peroxidation: in vitro and ex vivo studies in humans and in atherosclerotic apolipoprotein e-deficient mice anti-helicobacter pylori flavonoids from licorice extract antimicrobial activity of licorice flavonoids against methicillin-resistant staphylococcus aureus two antimicrobial flavanones from the leaves of glycyrrhiza glabra flavonoids: antioxidants against atherosclerosis effects of topical application of β-resorcinol and glycyrrhetinic acid monotherapy and in combination with fractional co laser treatment for benign hand hyperpigmentation treatment antimicrobial potential of glycyrrhiza glabra roots the efficacy of licorice root extract in decreasing transaminase activities in non-alcoholic fatty liver disease: a randomized controlled clinical trial evaluation of estrogenic activity of licorice species in comparison with hops used in botanicals for menopausal symptoms topical agents used in the management of hyperpigmentation flavonoid modulation of gaba (a) receptors antioxidative and superoxide scavenging activities of retrochalcones in glycyrrhiza inflata pharmacological studies of glycyrrhiza glabra: a review chemopreventive effect of β-glycyrrhetinic acid via modulation of inflammatory markers and induction of apoptosis in human hepatoma cell line (hepg ) glabridin as a major active isoflavan from glycyrrhiza glabra (licorice) reverses learning and memory deficits in diabetic rats preliminary evidence for inhibitory effect of glycyrrhizin on hiv replication in patients with aids economic importance of licorice characterization of g. glabra and g. bucharica collected in tajikistan estimation of dietary intake of ochratoxin a from liquorice confectionery potentiating effect of glabridin from glycyrrhiza glabra on gaba a receptors glabridin induces apoptosis and autophagy through jnk / pathway in human hepatoma cells isoliquiritigenin induces apoptosis and cell cycle arrest through p -dependent pathway in hep g cells glycyrrhizin inhibits porcine epidemic diarrhea virus infection and attenuates the proinflammatory responses by inhibition of high mobility group box- protein inhibition of intercellular adhesion in herpes simplex virus infection by glycyrrhizin α-glycyrrhetinic acid induces apoptosis of hl- human leukemia cells through caspases-and mitochondria-dependent signaling pathways hepatoprotective and antioxidant effects of licorice extract against ccl( )-induced oxidative damage in rats risk and safety assessment on the consumption of licorice root (glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin in vitro susceptibility of helicobacter pylori to licorice extract glabridin inhibits cancer stem cell-like properties of human breast cancer cells: an epigenetic regulation of mir- a/smad signaling. molecular carcinogenesis potentiating effect of glabridin on gabaa receptor-mediated responses in dorsal raphe neurons isoliquiritigenin induces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells role of glucocorticoid in the development of glycyrrhizin-induced hypertension. clinical and experimental hypertension antitussive principles of glycyrrhizae radix, a main component of the kampo preparations bakumondo-to (mai-men-dong-tang) isoliquiritigenin inhibits the growth of prostate cancer antimicrobial and antioxidant activities of medicinal plant glycyrrhiza glabra var. glandulifera from different habitats β-glycyrrhetinic acid, the major bioactive component of glycyrrhizae radix, attenuates airway inflammation by modulating th cytokines, gata- , stat , and foxp transcription factors in an asthmatic mouse model effects of phytoestrogen on sexual development tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future intestinal absorption and biliary elimination of glycyrrhizic acid diethyl ester in rats. drug design in vitro anti-helicobacter pylori activity of extractum liquiritiae, glycyrrhizin and its metabolites biphasic calcium phosphate-casein bone graft fortified with cassia occidentalis for bone tissue engineering and regeneration water extract of glycyrrhiza uralensis inhibited enterovirus in a human foreskin fibroblast cell line β-glycyrrhetinic acid induces apoptotic cell death in siha cells and exhibits a synergistic effect against antibiotic anti-cancer drug toxicity liquiritigenin, a licorice flavonoid, helps mice resist disseminated candidiasis due to candida albicans by th immune response, whereas liquiritin, its glycoside form, does not synthesis and anticancer activities of glycyrrhetinic acid derivatives metabolomics analysis to evaluate the anti-inflammatory effects of polyphenols: glabridin reversed metabolism change caused by lps in raw . cells methotrexate hepatotoxicity is associated with oxidative stress, and down-regulation of ppargamma and nrf : protective effect of β-glycyrrhetinic acid antithrombotic effect of glycyrrhizin, a plantderived thrombin inhibitor effect of licorice compounds licochalcone a, glabridin and glycyrrhizic acid on growth and virulence properties of candida albicans glycyrrhizin exerts antioxidative effects in h n influenza a virus-infected cells and inhibits virus replication and proinflammatory gene expression antiarthritic activity of glycyrrhiza glabra, boswellia serrata and their synergistic activity in combined formulation studied in freund's adjuvant induced arthritic rats evaluation of immunomodulatory activity of glycyrhiza glabra l. roots in combination with zing glabridin triggers over-expression of mca and nuc genes in candida glabrata: is it an apoptosis inducer a novel process for extraction of natural sweetener from licorice (glycyrrhiza glabra) roots. separation and purification technology licorice flavonoids suppress abdominal fat accumulation and increase in blood glucose level in obese diabetic kk-a(y) mice glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots the stimulatory and inhibitory effects of glycyrrhizin and a glycyrrhetinic acid derivative on phosphorylation of lipocortin i by a-kinase in vitro glycyrrhiza glabra protects from myocardial ischemia-reperfusion injury by improving hemodynamic, biochemical, histopathological and ventricular function licorice abuse: time to send a warning message antibacterial effects of glycyrrhetinic acid and its derivatives on staphylococcus aureus licochalcone-a induces intrinsic and extrinsic apoptosis via erk / and p phosphorylation-mediated trail expression in head and neck squamous carcinoma fadu cells memory-strengthening activity of glycyrrhiza glabra in exteroceptive and interoceptive behavioral models naturally occurring tyrosinase inhibitors: mechanism and applications in skin health, cosmetics and agriculture industries the pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling a molecular docking study of phytochemical estrogen mimics from dietary herbal supplements mechanism of anti-inflammatory action of liquorice extract and glycyrrhizin protective activity of glycyrrhiza glabra linn. on carbon tetrachloride-induced peroxidative damage formulation and evaluation of floating tablets of liquorice extract a new exploration of licorice metabolome anti-hiv activity of indian medicinal plants the treatment of atopic dermatitis with licorice gel effect of glycyrrhizin, an active component of licorice roots, on hiv replication in cultures of peripheral blood mononuclear cells from hiv-seropositive patients therapeutic basis of glycyrrhizin on chronic hepatitis b antifungal activity of plant extracts against arthrinium sacchari and chaetomium funicola hair growth stimulating effect and phytochemical evaluation of hydro-alcoholic extract of glycyrrhiza glabra glycyrrhizin increases survival of mice with herpes simplex encephalitis some effects of glycyrrhiza glabra (liquorice) roots extract on male rats β-glycyrrhetinic acid induces apoptosis through modulation of akt/foxo a/bim pathway in human breast cancer mcf- cells in vivo antioxidant and hepatoprotective potential of glycyrrhiza glabra extract on carbon tetra chloride (ccl ) induced oxidative-stress mediated hepatotoxicity assessment of median lethal dose and anti-mutagenic effects of glycyrrhiza glabra root extract against chemically induced micronucleus formation in swiss albino mice glycyrrhiza glabra: chemistry and pharmacological activity in vitro and in vivo antiallergic effects of glycyrrhiza glabra and its components phytochemistry and biological properties of glabridin a polyphenolic flavonoid glabridin: oxidative stress response in multidrug-resistant staphylococcus aureus phytocomplexes from liquorice (glycyrrhiza glabra l.) leaves-chemical characterization and evaluation of their antioxidant, anti-genotoxic and anti-inflammatory activity hypopigmenting agents: an updated review on biological, chemical and clinical aspects estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts and prepubertal rat skeletal tissues antiviral and immune stimulant activities of glycyrrhizin against duck hepatitis virus estrogenicity of glabridin in ishikawa cells antidiabetic effect of glycyrrhizin in genetically diabetic kk-ay mice estrogen-like activity of glabrene and other constituents isolated from licorice root estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells successful treatment of latent autoimmune diabetes in adults with traditional chinese medicine: a case report detection of consensuses and treatment principles of diabetic nephropathy in traditional chinese medicine: a new approach improved in vitro fertilization ability of mouse sperm caused by the addition of licorice extract to the preincubation medium two activators of in vitro fertilization in mice from licorice review article: glycyrrhizin as a potential treatment for chronic hepatitis c pharmacokinetics of intravenous glycyrrhizin after single and multiple doses in patients with chronic hepatitis c infection phytochemical screening and determination of anti-bacterial and anti-oxidant potential of glycyrrhiza glabra root extracts β-glycyrrhetinic acid and glabridin prevent oxidative dna fragmentation in uvb-irradiated human keratinocyte cultures the antiviral and antimicrobial activities of licorice, a widely-used chinese herb metabolites identification of bioactive licorice compounds in rats β-glycyrrhetinic acid exhibits potent antitumor effects against colorectal cancer via inhibition of cell proliferation and migration liquorice, a unique "guide drug" of traditional chinese medicine: a review of its role in drug interactions glycyrrhizin inhibits lps-induced inflammatory mediator production in endometrial epithelial cells hypoglycemic effects of glabridin, a polyphenolic flavonoid from licorice, in an animal model of diabetes mellitus licochalcone a inhibits growth of gastric cancer cells by arresting cell cycle progression and inducing apoptosis β-glycyrrhetinic acid ameliorates acute propionibacterium acnes-induced liver injury through inhibition of macrophage inflammatory protein- α isoliquiritigenin suppresses pulmonary metastasis of mouse renal cell carcinoma the anti-inflammatory activity of licorice, a widely used chinese herb protective effects of hepatocyte-specific glycyrrhetic derivatives against carbon tetrachloride-induced liver damage in mice efficacy of intravenous glycyrrhizin in the early stage of acute onset autoimmune hepatitis glycyrrhetinic acid attenuates lipopolysaccharide-induced fulminant hepatic failure in d-galactosamine-sensitized mice by up-regulating expression of interleukin- receptor the inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation antiinflammatory activities of licorice extract and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in bv cells and mice liver licochalcone-e induces caspase-dependent death of human pharyngeal squamous carcinoma cells through the extrinsic and intrinsic apoptotic signaling pathways licorice (glycyrrhiza glabra linn) as a valuable medicinal plant antiandrogenic activities of glycyrrhiza glabra in male rats glycyrrhizin administration ameliorates coxsackievirus b -induced myocarditis in mice liquorice (glycyrrhiza glabra): a phytochemical and pharmacological review the authors declare that they have no conflict of interest. key: cord- - m lk authors: paterson, r. russell m. title: cordyceps – a traditional chinese medicine and another fungal therapeutic biofactory? date: - - journal: phytochemistry doi: . /j.phytochem. . . sha: doc_id: cord_uid: m lk abstract traditional chinese medicines (tcm) are growing in popularity. however, are they effective? cordyceps is not studied as systematically for bioactivity as another tcm, ganoderma. cordyceps is fascinating per se, especially because of the pathogenic lifestyle on lepidopteron insects. the combination of the fungus and dead insect has been used as a tcm for centuries. however, the natural fungus has been harvested to the extent that it is an endangered species. the effectiveness has been attributed to the chinese philosophical concept of yin and yang and can this be compatible with scientific philosophy? a vast literature exists, some of which is scientific, although others are popular myth, and even hype. cordyceps sinensis is the most explored species followed by cordyceps militaris. however, taxonomic concepts were confused until a recent revision, with undefined material being used that cannot be verified. holomorphism is relevant and contamination might account for some of the activity. the role of the insect has been ignored. some of the analytical methodologies are poor. data on the “old” compound cordycepin are still being published: ergosterol and related compounds are reported despite being universal to fungi. there is too much work on crude extracts rather than pure compounds with water and methanol solvents being over-represented in this respect (although methanol is an effective solvent). excessive speculation exists as to the curative properties. however, there are some excellent pharmacological data and relating to apoptosis. for example, some preparations are active against cancers or diabetes which should be fully investigated. polysaccharides and secondary metabolites are of particular interest. the use of genuine anamorphic forms in bioreactors is encouraged. too much about cordyceps is unsubstantiated. this literature is written to sell so-called medicines to potentially vulnerable people with serious diseases. on the other hand, there is convincing scientific information that indicates significant pharmacological properties which are worth assessing (see table ). the present large review undertakes this task and deals with papers that use the name cordyceps sometimes in its most general sense, especially when the revision of sung et al. ( ) is considered. there follows an extended introduction to the topic. the title relates to another in this journal concerning the fungal traditional chinese medicine (ftcm), ganoderma (paterson, ) . in that case, the fungus was indeed a biofactory in the sense that numerous compounds have been reported from the fungus. what is the situation with another ftcm, cordyceps? the immediate answer is that the state of the art is considerably less developed (see table for a list of secondary metabolites). there is a general impression that this fungus is being used in a modern context, before the benefits, and even what is being used, have been determined scientifically. cordyceps is one of a growing number of ftcm being considered as cures for mod- table medically related purported effects of various cordyceps taxa (or preparations) as described by various authors ern human diseases. many commercial products are available in the market (e.g. didanosine from cordyceps militaris). these nutraceuticals are considered to relieve the ''stress for humans of living in technologically developed societies" by stimulating basic and secondary responses of the immune system (lakhanpal and rana, ) . the fungus represents a genus of perithecial ascomycetes (phylum ascomycota) classified in the clavicipitaceae, a monophyletic group included in the order hypocreales. the genus contains over species and the anamorphs of most are unknown. paecilomyces is considered traditionally to host the anamorphs but this has been disputed. sung et al. ( ) should be consulted for an up-to-date revision (and see later). cordyceps are parasites of insects or fungi, often exhibiting a high degree of host specificity (fig. ) . however, the cordyceps species associated with lepidopteran hosts do not represent a monophyletic group. there is even a high degree of genetic variation within cordyceps sinensis which creates difficulties in verifying samples. a taxonomic review of the fungus is now available (see later section) a similar review is required for ganoderma (e.g. see paterson, . larval infection via meiotic and/or mitotic spores/conidia and multiplication within the insect is from yeast-like budding. however, the fungus grows through the insect by hyphae. the accumulation of the biomass eventually kills the host (and/or a toxin(s) may be involved). it would be interesting to determine the biochemical parameters that cause these changes but this is not reported in the literature. the fungus ruptures the host body following over wintering and forms the sexual perithecial stroma that are connected to the dead larva below ground which grow upward to emerge above the soil surface (fig. ) . the complete insect/fungus combination is used traditionally, but not exclusively, for medicinal purposes. the present reviewer has seen no reports of the insect per se being given as a treatment. c. sinensis (berk.) sacc., is one of the most famous traditional chinese medicines (tcm) and health foods. the fungus parasitises larvae of moths (lepidoptera), especially hepialus armoricanus (and thitarodes), and converts each larva into a sclerotium, from which the stroma and fruitbody grows. the complex (including the larva body) has been used as a health food and traditional medicine to ''invigorate the lung and nourish the kidney" in china for hundreds of years, and at least from the th century (dong and yao, ; kuo et al., ) . although what these preparations actually represented is impossible to determine given the difficulties in taxonomy of even modern times (burnett, ; korf, ; and see sung et al., ) . understandably, conservation and sustainable harvest are important issues. there is need for (a) research on biological screening, (b) a better understanding of the status in natural habitats, and (c) artificial cultivation of the fungus. cordyceps and products are available in ''western" countries as over-the-counter medicine/tonics which advertise them as chinese herbs with anti-aging, ''pro-sexual", anti-cancer and immune boosting effects, although with poor supporting scientific evidence. the believe is that c. sinensis (cs) has various beneficial effects on humans, including those of a psychological nature. the ftcm, is also called dong chong xia cao in chinese (=winter worm summer grass) (li et al., a) . primarily it is prescribed as a tonic for body strengthening after serious disease. more recently other treatments have been claimed such as for (a) respiratory, renal, liver, nervous system and cardiovascular diseases, and (b) tumours, aging, hyposexuality and hyperlipidemia (kuo et al., ; chen et al., ; wang and shiao, ) . it has been officially classified as a drug in the chinese pharmacopoeia since . furthermore, the outbreak of the severe acute respiratory syndrome (sars) in china in has increased use considerably. this would have been an excellent opportunity to have determined how effective it was. however, this does not appear to have been undertaken. the market demand for cs is growing sharply in many countries (dong and yao, ) . they would surely be hailed as medical breakthroughs if the efficacy of any of these treatments were confirmed. nevertheless, the identities of active components have not been determined (in all cases) (li et al., b) . research has shown that at least some of the traditional uses ''may" relate to pharmacological activities (zhu et al., a,b) (if not pharmacological activities then what?). herbs have been used throughout history to enhance physical performance, but scientific scrutiny with controlled clinical trials has only recently been used to study such effects (bucci, ) . the authors mention that cordyceps remain untested which is surprising given the interest in the fungus. the fungus is endemic to the alpine habitats of the tibetan plateau above m in south-western china, and there has been large-scale harvesting of the wild material from nepal and india more recently. it is agreed generally table example of the range of species and some of the low molecular-weight secondary metabolites from cordyceps secondary metabolites c. sinensis cyclic peptides, h -a c. militaris cyclic peptides, cordycepin, -membered macrolides, cepharosporolides c, e and f, pyridine- , -dicarboxylic acid and carboxymethyl- -( -hydroxybutyl) furan, dipicolinic acid c. pseudomilitaris bioxanthracenes c. brunnearubra cordyformamide c. sinclairii ( s, s, r)-(e)- -amino- , -dihydroxy- hydroxymethyl- -oxoeicos- -enoic acid c. cicadae ergosterol peroxide c. nipponica cordypyridones a and b c. ophioglossoides ophiocordin, glycoprotein containing nacetylgalactosamine c. heteropoda cicadapeptins i and ii, myriocin the number of compounds is small compared to that for ganoderma (paterson, ) . to have been over-harvested. furthermore, the price of natural products of cs is over us$ , kg À ( prices) for only ''average quality" (how this is determined is not clear) in the market and increasing (sharma, ) . so one can understand the pressures on supply. the socioeconomic implications of the ftcm are highly significant to the regions where it is harvested. the fungus has officially been classified as an endangered species by cites management authority of china and china customers and this scarcity is of considerable concern to all. consequently, living strains have been isolated from natural cs and cultivated in large quantity by bioreactor technology which is a promising method to meet the needs of human consumption and to reduce the pressure on natural resources of the species (dong and yao, ) . in vitro culture of the fungus has been employed increasingly and yang et al. ( ) state, ''it is generally accepted that its cultivated cs fungi possess the same functions as cs natural ''herbs" (sic)". some other issues that require addressing are that natural c. militaris is not readily available and is costly. thus, a growing number of socalled cordyceps products that derive from mycelial cultures of the asexual forms of these fungi have become commercially available (hamburger, ) . mycelia cultivation has resulted in establishing a number of cultures derived from the holomorphic cs. these are referred to by the anamorphic names paecilomyces hepiali and cephalosporium sinensis, although the anamorph of cs appears to be hirsutella sinensis (and see later). however, the situation is confused with some taxonomist using outdated names. to paraphrase buenz et al. ( buenz et al. ( , : while these strains undoubtedly support ecologically sustainable use of cs, the actual similarities between the wild fungus and the cultures are not clear. the consumption of complimentary medicine has increased dramatically, with over % of people in the united states of america reported as ''users". sales were us$ . billion in (buenz et al., ) . an important factor was the passage of the dietary supplement health and education act in in the usa which opened the market for tcm (cooper and chang, ) . one can appreciate how journals advocating these have increased concomitantly. why have they not been developed by big pharmaceutical companies and made available to the public in pure compound form? no doubt there could be many reasons why this has not happened (e.g. not enough profit, ''sticky" intellectual property rights issues (see paterson, ) , difficulty in mass production or synthesis, etc.)apart from the possibility that they simply may not be effective. buenz et al. mention that ''one of the most interesting supplements is the not yet well-characterized c. sinensis (berk.) sacc.". cs has attracted much research interest for anti-oxidant activity and there is considerable evidence of this from the fungus as a treatment of a wide range of diseases. however, unauthenticated material has been used in some cases. for example, a polysaccharide was isolated which can protect pc cells against hydrogen peroxide-induced neuronal cell toxicity, but the cordyceps mycelia used was from the wan fong pharmaceutical factory (zhejiang, china) and derived from ce. sinensis chen sp. nov. this is a nomenclaturally illegitimate fungal name, which raised doubts as to its relationship to cs. in fact, it was later proved to be a different species (dong and yao, ) and this is a specific example of a general problem in the field. an example of another problem is cordymax cs- , a commercial mycelial fermentation product that lowered fasting plasma levels of glucose and insulin, improved oral glucose tolerance and increased the glucose-insulin index, which measures insulin sensitivity, in rats (zhao et al., ) . however, the following statement is given on the web site of the product, ''these (health-related) statements have not been evaluated by the food and drug administration. this product is not intended to diagnose, treat, cure or prevent any disease". extracts from artificially cultivated fruit-bodies of cs from the xinhui xinhan artificial cordyceps factory (guangdong, china) could scavenge ros by inhibiting malondialdehyde formation by the peroxynitrite generator sin- . these results have been since referred to uncritically (e.g. buenz et al., buenz et al., , li et al., ) . however, the fungal material may have been unauthentic, because reports exist that cultivation of fruit-bodies of this fungus was not repeatable and that the manufacturer is actually selling c. militaris. furthermore, li et al. ( ) compared the anti-oxidant activities of natural cs and cultured cordyceps mycelia from different sources and were able to show the similar effects of the cultured mycelia to the natural products. however, the cultured material used was derived from a wide range of strains, and not from a valid cs. some of these were products from the chinese medicine factory of jiangxi and hebei boding pharmaceutical factory. in addition, the ftcm are boiled in water or soaked in alcohol to drink for medications/health foods. obviously, the various solvents and temperatures may have resulted in different compounds (dong and yao, ) . nevertheless, guo et al. ( ) state that the ongoing exploration of cs has shown that the species can produce ''many" bioactive compounds, and the medicinal benefits of cs have been demonstrated extensively. in addition, there is (unbelievably for scientific journals) the use of words such as ''yin" and ''yang" as a basis for undertaking scientific research on material activities. canney ( ) discusses kidney ''yang" and one needs to ask what this is from a scientific perspective. why include this word when it has no scientific currency? the fungus is also referred to as a herb and indeed the title of the piece asks, ''c. sinensis animal, vegetable or both?", whereas it is neither. various bioactive constituents from cordyceps species have been reported. these include cordycepin and other anti-bacterial and anti-tumour adenosine derivatives, ophicordin, an anti-fungal agent, and l-tryptophan. recent reports have indicated that cs contains polysaccharides exhibiting anti-oxidant activity and nucleosides that inhibit platelet aggregation (wu et al., ) . the bioactive compounds involved in the activities claimed include polysaccharides, modified nucleosides, and cyclosporin-like metabolites which are produced by this fungus and related species. the beneficial effects on (a) renal and hepatic function and (b) immunomodulation-related anti-tumour activities are most promising and deserve great attention. an increasing number of studies have used cultured mycelia in investigations. more mechanism-based, disease-oriented pharmacological studies are required to ensure clinical efficacy for particular diseases. however, pang et al. ( ) state revealingly that studies have demonstrated repeatedly that many natural products marketed as nutraceuticals or health food do not deliver the health benefit as claimed and are inconsistent from batch to batch. in the popular mind, cs first gained worldwide attention when it was revealed that several chinese runners who broke world records in had included this fungus in their diet as part of their training program. although scientist need to desist from quoting such reports as they are unsubstantiated and far-fetched. purported and unsubstantiated effects of the fungus include use as an aphrodisiac, analgesic, immune modulator, and free radical scavenger. a review of the literature uncovers the predictable collection of general papers concerning medical mushrooms some with a distinctly ''alternative" flavour. i have no concerns about being alternative but are they scientific? these overviews often are written in breathless, overblown and unscientific terms; others are well balanced. dasilva ( ) talks about ''novel mushroom-based healthcare products and therapeutics licensed for medical use can contribute to the good health status and feeling of the povertystricken strata of urban societies and populations. indeed, mushroom cultural practices and medicines are being widely accepted as the integral skeins in the fabric of the human society of tomorrow". a valid response to this situation of a wide range in the quality of papers would be to only review those papers that have a high impact factor (or any impact factor). temporarily putting the debate about the value of impact factors to one side, the reader may find it beneficial to concentrate only on those journals reviewed herein that do indeed have such ratings. of course, the disadvantage is that valuable information may be missed. it may be worth mentioning that, asian nations on the world stage have realised that biomedicine offers a unique chance to develop new industries and markets. universities in singapore, korea, hong kong and china are appearing concomitantly in world tables for the best and citations data suggest they are producing well-regarded papers (ince, ) . the following review concentrates on the biological activity of various preparations of cordyceps spp. (the name as used in the essentially non-taxonomic papers) because of the quantity of data and importance of medicinal claims, at the expense of taxonomic and quality control issues. the various pure compounds, extracts, whole fungus and other preparations as they relate to pharmacological activities are discussed next, as these divisions are considered to be most relevant to a biochemical/phytochemical perspectives of the topic. data from pure compounds are the most revealing in terms of determining effects of the fungus/insect. it is noted that these reports are scarce. some compounds from cordyceps (as defined here) are not particularly unusual. ng and wang ( ) review the chemical constituents and pharmacological properties. the chemical constituents include (a) cordycepin ( -deoxyadenosine) and its derivatives, (b) ergosterol, (c) polysaccharides, (d) a glycoprotein and (e) peptides containing a-aminoisobutyric acid. the activities ascribed to the fungus are anti-tumour, antimetastatic, immunomodulatory, anti-oxidant, anti-inflammatory, insecticidal, anti-microbial, hypolipidaemic, hypoglycaemic, anti-aging, neuroprotective and renoprotective effects: so a vast a range of properties from a narrow spread of compounds. polysaccharides account for the anti-inflammatory, antioxidant, anti-tumour, anti-metastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects. cordycepin contributes to the anti-tumour, insecticidal and anti-bacterial activity. ergosterol (a universal fungal compound) exhibits anti-tumour and immunomodulatory activity. finally, a dnase has been characterized. these are not particularly novel compounds and one wonders why there are so many reports of the effects of crude extracts rather than much more work on the effects of novel pure compounds. cordycepin (fig. ) , -deoxyadenosine, is a derivative of the nucleoside adenosine differing from the latter by the absence of oxygen in the position of its ribose entity. as such is may be quite common. initially, it was extracted from cordyceps; however, it is now produced synthetically. some enzymes do not discriminate between adenosine and so it can participate in certain reactions. for example, it can be incorporated into rna molecules causing premature termination of its synthesis. it is classified as an anticancer compound. cordycepin inhibited the growth of clostridium paraputrificum and clostridium perfringens, but had no effect on bifidobacterium spp. and lactobacillus spp. (ahn et al., ) . in addition, larvicidal activity against plutella xylostella after - days of treatment was observed . it is interesting that cordycepin, a compound originally isolated from c. militaris as much as years ago (cunningham et al., ) , is known to exert cytotoxic effects through nucleic acid methylation (kredich, ) , with possible implications for the pcr of these fungi (see paterson, , . if it is a truly useful compound it is surprising that it is not a well-known pharmaceutical by now. the presence of cordycepin in cs has been difficult to confirm, although it has been confirmed by nmr (chen and chu, ) . however, other groups have not been able to detect this compound . it is clearly important to confirm the presence of the compound in cs in terms of determining the active components of the fungus and ultimately for chemotaxonomic purposes. cho et al. ( ) state that cordycepin is isolated from c. militaris and is (claimed to be) an ingredient in tcm which is prescribed for various diseases, such as cancer and chronic inflammation (again note how vague this verbatim statement is). in this study, the novel effect of cordycepin inhibiting collagen-induced platelet aggregation was reported. the data suggests that the inhibitory effect of cordycepin might be associated with the down-regulation of [ca + ]i and the elevation of camp/cgmp production. this result has obvious significance for prevention of thrombus formation. finally, cordycepin inhibited the growth of b melanoma cells inoculated subcutaneously into right murine footpads (yoshikawa et al., ) . cordyheptapeptide a (fig. ) , a novel cycloheptapeptide, was isolated from a strain of cordyceps together with four known bioxanthracenes. there were only two previous reports on the isolation of cyclic peptides from this genus and these were from c. militaris and cs. the metabolite exhibited anti-malarial activity against plasmodium falciparum and cytotoxicity to vero cell lines. also, the anti-malarial and cytotoxic activities of the bioxanthracenes were reported (rukachaisirikul et al., ) . in an extensive and impressive report, compounds designated as es- s, were isolated from a verticillium strain and identified as bioxanthracenes (isaka et al., a) . these compounds were known to exhibit potent activity as n-methyl-d-aspartate (nmda) receptor antagonists. in addition, five novel es- analogues were isolated with nine known compounds from a cordyceps strain. a closely related strain provided cordyheptapeptide a, cordyheptapeptide b, and known es- s. the structures of the novel bioxanthracenes were -o-desmethyl analogues of the compounds described. furthermore, cordyheptapeptide b has an n-methyl-l-phenylalanine residue in place of the n-methyl-l-tyrosine. the isolation, structure elucidation, and anti-malarial activity of es- s and their analogues from the insect pathogenic fungus cordyceps pseudomilitaris (from a lepidoptera larva) were reported previously. cycloheptapeptide, cordyheptapeptide a, and some known es- s were isolated from a cordyceps strain from an elaterid larva. in a continuing search for bioactive compounds from insect pathogenic fungi it was noticed that culture extracts of six cordyceps strains, collected in the same location (from coleoptera larvae, at doi innthanon national park, chiang mai province, thailand), showed similar h nmr spectra. this suggested the presence of bioxanthracenes (es- s) and cordyheptapeptide a as major constituents. two of these strains were subjected to mass fermentation ( l) and chemical investigation. as a result, five new es- analogues and nine known compounds and cordyheptapeptide a were isolated from an undefined strain. cordyheptapeptide b, was isolated, together with other known compounds. some of these were tested for activity against p. falciparum and cytotoxicity to kb cells (oral human epidermoid carcinoma), bc cells (human breast cancer), nci-h cells (human small cell lung cancer), and noncancerous vero cells (african green monkey kidney fibroblasts). cordyheptapeptide a exhibited antimalarial activity, while cordyheptapeptide b was inactive and both cyclic peptides showed moderate cytotoxicity. furthermore, cordyformamide is a plausible biogenetic precursor of xanthocillin y, and was isolated from a culture broth of cordyceps brunnearubra bcc . cordyformamide was found to exhibit activity against p. falciparum, whereas it showed weak or no cytotoxicity (isaka et al., b) . production of the nonribosomal peptides cicapeptins i and ii (fig. ) were reported by krasnoff et al. ( ) which was the first report from fungi of consecutive hyp or pro residues in a nonribosomal linear peptide. the compounds exhibited anti-bacterial and anti-fungal activity. a novel immunosuppressant was isolated from the culture broth of isaria sinclairii, the anamorph of c. sinclairii, and characterized as ( s, s, r)-(e)- -amino- , -dihydroxy- -hydroxymethyl- -oxoeicos- -enoic acid, which was identical to anti-fungal substances, myriocin and thermozymocidin (fujita et al., ) . the suppressive activity was found to be equal to, or higher than cyclosporin a which is used clinically. the activities of the derivatives were also examined, indicating the following relationships between structure and activity -the: (a) lactone formation between the carboxy group at c- and the hydroxy group at c- , and the reduction of the carbonyl group at c- to the hydroxy group do not affect the suppressive activity; (b) hydrogenation of the double bond at c- resulted in activity; and (c) acetylation of the amino group and the thioketalization on the carbonyl group at c- ''drastically" reduced the suppressive activity. also, the compound suppressed the production of anti-bodies to sheep red blood cells and induction of cytotoxic lymphocyte t cells more strongly than cyclosporin a. obviously, this is an important lead compound and hence one of the more satisfactory papers. chen et al. ( ) isolated a pure compound (h -a) from cs and investigated whether autoimmune disease progression in mice was affected by administration of the metabolite. the authors are vague as to what the compound is and state that, ''it is a kind of ergosterol and looks like testosterone". the authors also provide a chemical structure which confirms that it is a common sterol and a systematic name could have been provided. their results demonstrated that mice treated daily exhibited a progressive reduction in anti-ds-dna production. in clinical presentation, the treated group had a reduction in lymphadenopathy, a delayed progression of proteinuria, and an improvement in kidney function. histological analysis of kidney tissue indicated that h -a inhibited mesangial proliferation that was evident in lupus nephritis. however, there was no change in immune complex deposition. h -a ''may be" useful for treating systemic lupus erythematosus in human patients. however, more work is required. h -a was claimed to be effective in the treatment of autoimmune disorders (yang et al., ) . results demonstrated inhibition of cell proliferation and promotion of apoptosis of activated human mesangial cells in vitro: the activities were not a result of cytotoxicity. in addition, h -a inhibited tyrosine phosphorylation of human mesangial proteins. these findings suggest that h -a modulated some (unspecific) subcellular signal-transduction pathways and changed the balance between proliferation and apop-tosis of mesangial cells in vitro and in vivo. the conclusions were that h -a may be effective in the management of autoimmune disorders, and the modulation of the signal-transduction proteins may represent a target for future pharmacologic interventions. more correctly, they probably do represent a target, and such vague statements should be avoided. in an older report, hi-a alleviated immunoglobulin a nephropathy (berger's disease) with histological and clinical improvement . hi-a inhibited the proliferation of human mesangial cells and promoted apoptosis by suppressing tyrosine phosphorylation of bcl- and bcl-xl (yang et al., ) and reduced antids-dna production and lymphadenopathy, delayed progression of proteinuria, improved kidney function and inhibited mesangial proliferation . moving on to more interesting compounds, cordypyridones a and b were detected from the uncommon species, c. nipponica. these are atropisomers, and demonstrated potent anti-malarial activity in vitro (isaka et al., b) . shin et al. ( ) state that, ''in an effort to evaluate the pharmacological effects, including the anti-aging effect" of the fruiting bodies of the cultivated paecilomyces japonica fungus, ''a new type" of cordyceps sp. was investigated. two pure compounds were isolated as active principles from low molecular-weight fractions, and a protein-bound polysaccharide that showed a marked increase in the liver enzyme activities, and a significant inhibition of lipid peroxidation was found. boros et al. ( ) reported that ophiocordin, an anti-fungal antibiotic from cordyceps ophioglossoides (kneifel et al., ) and balanol from verticillium balanoides are structurally identical. this may indicate a particularly close taxonomic relationship between the two taxa. the structure of ophiocordin was falsely assigned and balanol was the compound of interest. balanol was under development as an anti-cancer agent as it established to be a selective inhibitor of protein kinase c (see paterson, ) . it is more common for pure compounds to be tested in the fields of anti-bacterial, anti-fungal, anti-malarial and insecticidal activity which is to be recommended more generally. ophiocordin is an anti-fungal antibiotic isolated from submerged cultures of c. ophioglossoides. however, it is devoid of anti-bacterial activity (kneifel et al., ) . bioxanthracenes (see also previously) were isolated from c. pseudomilitaris (isaka et al., a; jaturapat et al., ) and appear to be anti-malarial. ten-membered macrolides, cepharosporolides c, e and f, cordycepin, pyridine- , -dicarboxylic acid and -carboxymethyl- -( hydroxybutyl) furan were reported from c. militaris by rukachaisirikul et al. ( ) . however, only cordycepin was anti-malarial. krasnoff et al. ( ) reported cicadapeptins i and ii (nonribosomal peptides containing aminoisobutyric acid), which were anti-bacterial and antifungal, and myriocin (anti-fungal) from c. heteropoda isolated from an australian cicada. finally, a glycoprotein containing n-acetylgalactosamine was isolated from c. ophioglossoides but activity data are not available (kawaguchi et al., ). an inhibitor of the prophenoloxidase activation was isolated from a culture filtrate of c. militaris and identified as dipicolinic acid (dpa). the production of dpa in a range of clavicipitaceae fungi was examined. entomogenous fungi that produce dpa were integrated into one group by a phylogenetic analysis based on s rdna. interestingly, it was suggested that the group acquired an ability to produce dpa during its evolution from plant pathogenic fungi to entomogenous fungi (watanabe et al., ) . in a useful comparison of crude extracts and pure compound, the anti-diabetic effect of various fractions of c. militaris, ccca (crude cordycepin containing adenosine), cmess (ethanol soluble supernatant), and cordycepin were evaluated in diabetic mice (yun et al., ) . cmess showed a potent inhibitory activity of . % in starchloaded mice: cmess reduced blood glucose level by . %. however, ccca, and cordycepin showed no difference. after days administrations of these drugs, cmess, and cordycepin dramatically reduced blood glucose level. ccca with a high concentration of cordycepin did not reduce blood glucose level. proliferation of t-lymphocyte was significantly decreased; while no production was increased more than two-fold in the cordycepin-administered group. the proliferation of macrophages and no production were significantly decreased in the cmess administered group. cmess and cordycepin may be (a) useful tools in the control of blood glucose level in diabetes and (b) promising new drugs as an anti-hyperglycemic agent without the defects of lowered immune responses and other side effects, the authors suggest. furthermore, cordycepin, -amino- -deoxyadenosine, homocitrullyl aminoadenosine, adenine, cordycepic acid and d-mannitol have been reported from cordyceps spp. (cunningham et al., ; chatterjee et al., ; kredich and guarino, ; guarino and kredich, ; kaczka et al., ; liu et al., ) . ergosterol peroxide isolated from c. cicadae inhibited phytohaemagglutinin-induced t cell proliferation, and arrested the progression of activated t cells from g to s phase of the cell cycle. early gene transcripts, in particular those of cyclin e, interferon, and interleukins were suppressed (kuo et al., ) . the glycosylated form of ergosterol peroxide from cs was more potent than the aglycone in inhibiting proliferation of tumour cells (bok et al., ) . however, ergosterol peroxide is widespread in fungi and cordyceps does not offer any particular advantage in its preparation. it is worth noting that eight different cordyceps species (cs. c. militaris, c. cicadae, c. ophioglossoides, c. heteropoda, c. pseudomilitaris, c. nipponica, c. sinclairii) are listed in the above paragraph indicating the extent of the possible diversity involved in the biology and activity (tables and ) . although whether they are distinct species is open to question. water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured c. militaris by hotwater extraction followed by ethanol precipitation. the polysaccharides were successively purified by chromatography giving three polysaccharide fractions. in the in vitro anti-oxidant assay, p - was found to possess hydroxyl radical scavenging activity. the polysaccharide is a heteropolysaccharide and is occasionally branched. the fundamental information obtained from this work is beneficial to the interpretation in the relationship of polysaccharide structure and its biological functions. this provides the ''experimental evidence and scientific explanation for the folkloric uses of c. militaris as a substitute for cs" (yu et al., a) . the effect of an exopolysaccharide fraction (epsf) from anamorphic strains of cs on the immunocyte activity of tumour-bearing mice was investigated. epsf significantly inhibited the h tumour growth, and elevated the activity of immunocytes. it enhanced the phagocytosis capacity of peritoneal macrophages and proliferation ability of spleen lymphocytes. epsf promoted (a) tnf-a expression of macrophages, (b) the cytotoxicity of spleen lymphocytes, and (c) tnf-a and ifn-c mrna expression of splenic lymphocytes . cs possesses anti-tumour, anti-oxidation and stimulation of the immune system activities . however, the identity of active component(s) has not been determined . towards this end, a polysaccharide was isolated from cultured cordyceps mycelia which had strong anti-oxidation activity, and which contained glucose, mannose and galactose. the pre-treatment of the isolated polysaccharide on cultured rat pheochromocytoma cells demonstrated strong protective effect against hydrogen peroxide (h o )-induced insult. treatment prior to h o exposure significantly elevated the survival of pc cells in culture. this was the first report that identified a polysaccharide from cordyceps, which protected against the free radical-induced neuronal cell toxicity. a water-soluble polysaccharide fraction, a poorly water-soluble polysaccharide, and a protein fraction stimulated steroidogenesis (huang et al., b) . interestingly, galactomannans isolated from the insect portion of c. cicadae demonstrate potent hypoglycaemic activity in mice (kiho et al., ) . in an investigation into a polysaccharide from cs mycelium hypocholesterolaemic and hypotriglyceridaemic activity in mice was exhibited (kiho et al., ) . chen et al. ( ) studied a polysaccharide fraction from cs as to its effect on the proliferation and differentiation of human leukaemia cells using an in vitro culture system. the conditioned medium had an activity that significantly inhibited proliferation. differentiated cells also possessed phagocytosis functions and supported superoxide production. antibody neutralization studies further revealed that the tumouricidal and differentiating effects of the compounds were mainly derived from the elevated cytokine concentrations. finally, galactosaminoglycan from c. ophioglossoides reacted with sera from patients with certain collagen diseases and its use as an index of serological activity is thus of diagnostic value (ikeda et al., ) . an aqueous extracted polysaccharide from cultured c. militaris demonstrated general anti-inflammatory activity (yu et al., a) as did ethanolic extracts of cultured fruiting bodies and mycelia of c. militaris applied topically in the croton oil-induced ear oedema test in mice. the fact that in vitro fruiting bodies were employed rather than in vivo is interesting as most papers report using fruiting bodies in vivo and/or in vitro biomass. however, the paper is flawed as the details of the cultivation of the fruiting bodies were not provided. antioxidant activity in the xanthine oxidase, haemolysis and lipid peroxidation assay systems was demonstrated by li et al. ( a) from water extracts, and a polysaccharide fraction, of cultured cs mycelia. interestingly, the fruiting body and the caterpillar parts of cs are claimed to be similar in chemical composition and hence anti-oxidant activity, because the fungus had presumable replaced the insect constituents with fungal . it would be interesting to determine (a) how this occurs in terms of insect substrate utilisation and optimisation of yields of bioactive fungal components, (b) when the preparation is at the correct stage for use as a medicinal treatment, and (c) if these data could be extrapolated to in vitro culture. an ambiguous statement is made by shin et al. ( ) , ''cordyceps is negative for its many biological activities and a tonic for restoring vital functions in traditional chinese medicine". it proceeds to state that p. japonica is a new type of cordyceps species, which is incomprehensible. it also mentions that p. japonica is (or produces) mushrooms. paecilomyces is considered to be an anamorph of cordyceps and so this appears to be incorrect and, what appear to be mushrooms, may be synnemata (compacted conidiophores). however, it is unwise to speculate what the material actually is as descriptions are vague. a protein-bound polysaccharide that inhibited lipid peroxidation and increased the activity of anti-oxidant was described from the fungus. finally, phaeochromocytoma cells were protected against h o -induced injury by a -kda polysaccharide from cs mycelium . yamada et al. ( ) reported that a water-insoluble extracellular glucan isolated from the culture filtrate of c. ophioglossoides suppressed potently the growth of sarcoma solid-type tumours. remarkably, a protein-bound polysaccharide fraction from c. ophioglossoides extended the life of mice bearing ehrlich carcinoma or a syngeneic tumour . also, ohmori et al. ( a,b) isolated a galactosaminoglycan that inhibited the proliferation of sarcoma cells and the growth of a syngeneic solid tumour in vivo: it exhibited cytotoxicity against cancer cells in vitro. chen et al. ( ) observed that medium from blood mononuclear cells stimulated with the polysaccharide fraction from cs inhibited the proliferation of human leukaemic cells, and induced approximately % to differentiate into mature monocytes/macrophages expressing non-specific esterase activity and certain surface antigens. the anti-proliferation and differentiating effects were demonstrated to be caused by an elevated production of cytokines, i.e. a tumour necrosis factor and an interferon. the exopolysaccharide fraction of cs inhibited metastasis of melanoma cells and down-regulated concomitantly the levels of bcl- protein into the lungs and the liver . the exopolysaccharide fraction of cultivated cordyceps stimulated peritoneal macrophages to take up neutral red and splenic lymphocytes to proliferate . crude and neutral polysaccharides of cs exerted hypoglycaemic activity in normal mice. however, the polysaccharide did not affect the circulating insulin level in normal mice (kiho et al., ) . the compound lowered the plasma glucose level in diabetic mice (kiho et al., ) . another unspecific polysaccharide from a hot-water extract of mycelia also lowered the plasma glucose level in normal, adrenaline-induced hyperglycaemic and diabetic mice (kiho et al., ) . some cordyceps-like strains have been isolated from the fruiting bodies of wild cs that have been reported to show the same properties as the natural product. however, care in interpretation is required as these could conceivably be contaminants (see later). an exopolysaccharide fraction was prepared from cultivated cs . the results showed that it enhanced significantly the neutral red uptake capacity of peritoneal macrophages and spleen lymphocyte proliferation in melanoma-bearing mice. the metastasis of b melanoma cells to lungs and livers was significantly inhibited. moreover, the levels of bcl- in the lungs and livers were decreased. the results suggest that the polysaccharide has an immunomodulatory function and anti-tumour activity. however, yang et al. ( ) state that although certain polysaccharides from cs are bioactive, the anti-tumour effect has not been confirmed. the authors investigated the effects of the exopolysaccharide fraction of cultivated cs fungus on c-myc, c-fos, and vascular endothelial growth factor (vegf) expression of tumour-bearing mice. the expression in the lungs and livers of treated mice were found to be significantly lower than those of untreated mice. the authors suggest that the fraction had inhibited tumour growth in the lungs and livers of mice, and that it is an adjuvant in cancer therapy. in addition, yu et al. ( a,b) isolated four polysaccharides from c. militaris, cps- was shown to possess a significant anti-inflammatory activity and suppressed the humoral immunity in mice but had no significant effects on cellular immunity and non-specific immunity. in a previous study using anti-oxidant activity-guided fractionation csp- from cultured cordyceps, mycelium was isolated. the hypoglycemic effect of csp- on mice and rats was demonstrated. csp- increased circulating insulin level in diabetic animals, which suggests that the compound(s) may stimulate pancreatic release of insulin and/ or reduce insulin metabolism. chen et al. ( ) undertook further work on the biological activity of the isolate: the polysaccharide from fungus and its anti-oxidant activity on h -tumour bearing mice was investigated. the h tumour growth was inhibited and sod activity of liver, brain and serum and gsh-px activity of liver and brain in tumour-bearing mice were enhanced. in general, beneficial effects were observed in the liver and brain of tumourbearing mice. finally, four exopolysaccharides with different molecular masses ranging from to kda were reported from c. militaris by kim et al. ( b,c) as part of yield optimisation studies. an extracellular polysaccharide extracted from the mycelia of cs with hot-water indicated that this d-glucan consisted of a backbone composed of ( ? )-b-d-glucosyl residues and carried a single ( ? )-b-linked d-glucosyl residue: sugar residues were linked with b-glycosidic bonds (wu et al., ) . a lectin from c. militaris exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human abo erythrocytes (jung et al., ) . however, the n-terminal amino acid sequence differed greatly from those of other lectins. it exhibited mitogenic activity against mouse splenocytes. the following section concerns solvent extraction of the fungi. in effect, this is often how the preparations will be consumed as a tcm. the significance of tests on extracts is much reduced compared to those of pure compounds. there is a great deal of data. in general, this type of work needs to be deemphasised in favour of that of pure compounds. reports on the metal chelating and reducing power from cs are not available in the scientific literature. therefore, there is a demand to obtain an overall measure of the anti-oxidant activity of extracts using reliable fungal material because of increasing interest in the relationship between anti-oxidants and diseases. the anti-oxidant activities from natural and cultured mycelia of cs were investigated in vitro. optimal effects were demonstrated on the inhibition of linoleic peroxidation. the results suggested that the cultured and natural mycelia have direct and potent activities and that the cultured mycelia could be used for the anti-oxidant activity which would tend to reduce the pressures on the natural fungus, which is, after all, an endangered species (dong and yao, ) . the anti-oxidant efficiency of c. militantis extract (cme) and cs extract (cse) in protecting lipid, protein, and lowdensity lipoprotein (ldl) against oxidative damage was reported (hui et al., ) . however, this study provoked a strong response from hamburger ( ) which was subsequently rebutted by one of the original authors (duh, ) . this is something of an unexpected bonus to a reviewer of the literature such as myself as another opinion is obtained. the questions are, what biological material is being worked with, and can other scientists obtain it to repeat the experiment? the current author has encountered this before (paterson, ) where commercial interests are involved. the hamburger response can be applied in a general sense to some of the other work cited in the present review. cme and cse showed weakly inhibitory effect on liposome oxidation. the inhibitory effect of cme on protein oxidation was inferior to that of cse. cme and cse showed inhibition of ldl oxidation. the contents of the bioactive ingredients cordycepin and adenosine in cme were higher than those of cse; however, cordycepin and adenosine showed no significant anti-oxidant activity. in addition, a polysaccharide present in cme and cse displayed anti-oxidant activity. the authors concluded that the protective effects of cme and cse against oxidative damage of biomolecules are a result of their free radical scavenging abilities. however, the experimental data and some of the conclusions need a critical comment (hamburger, ) . the author criticised the report on the bases of poor taxonomy, biochemistry and extrapolation of data to imply possible cures of diseases. in particular, the authors' claims of a potential treatment for human disease on the basis of in vitro data are called into question. the rebuttal by duh simply confirms that appropriate information about the strains was not provided; the comments on the inadequate analytical procedures are largely accepted. and there is no doubt that the conclusions could have been rewritten to indicate that the results were preliminary. hamburger (personal communication, ) stated that the rebuttal was evasive, an assessment with which i agree. kuo et al. ( ) describe the effects of cs against group a streptococcus infection in mice. the preparation protected by decreasing bacterial growth ''and dissemination", thereby increasing mouse survival rate. il- and ifn-gamma expression and macrophage phagocytic activity also increased. kuo et al. ( ) claim to demonstrate that cs increased phagocytosis in human monocytic cells and abrogated inhibition of phagocytosis by causing cytokine production. these two reports are sound and in good journals. however, the fungus used was from a company called simpson biotech and very few or no details are supplied about how the material was collected, identified, maintained, and grown. of course, this is unsatisfactory. shahed et al. ( ) refer somewhat unusually to cs as a ''black blade" fungus. their results showed that cs improved renal function and reduced the expression of inflammatory and apoptotic genes in rats. the authors make the very conditional statement that, cs extract may play a potential therapeutic role in renal transplantation"; on the other hand it may not play an actual role. after all, precision in what is written in such important areas of medicinal research is crucial. the authenticity of the fungal material can be questioned: it was obtained from a company from the united states of america and there is little indication about the standards of collecting, purity, identities and maintenance of the materials. basically, what level of accreditation applies to such organizations? a c. militaris inhibited the growth of human umbilical vein endothelial cells (huvec) and ht cells. it down-regulated, in dose-and time-dependent manners, bfgf gene expression in huvec cells and mmp- gene expression in ht cells. the growth of melanoma cells in mice was suppressed. in addition, anti-angiogenic activity was manifested (yoo et al., ) . it is gratifying that adequate details of the fungal material are provided in this chinese journal, which acts as a model for others generally. chiou et al. ( ) observed a hypotensive effect of cs in anaesthetized rats and a vaso-relaxant effect in isolated aorta. the fungus counteracted arrhythmia in rats and increased the dosage of ouabain required to produce arrhythmia in guinea-pigs. in addition, the heart rate in anaesthetized rats and the contractility of isolated papillary muscle or atria in guinea-pigs were decreased (mei et al., ) . cultured fruiting bodies of cs prevented deposition of cholesterol in the aorta of atherosclerotic mice by inhibiting free radical-mediated ldl oxidation in an investigation into hypolipidaemic activity (yamaguchi et al., a) . a hot-water extract of mycelia (a) lowered the total cholesterol concentration, (b) reduced the concentration of cholesterol carried by ldl and very-low-density lipoprotein, and (c) elevated the high density lipoprotein (hdl)-cholesterol concentration in the serum of mice fed a cholesterol enriched diet (koh et al., a) . water extracts of cs: increased survival time of mice inoculated with carcinoma cells or syngeneic fibrosarcoma cells (yoshida et al., ) ; inhibited spontaneously liver metastasis of carcinoma cells and melanoma in syngeneic mice and results suggested that the activity was not attributable to cordycepin (nakamura et al., a) ; prolonged the survival period of mice inoculated with b melanoma cells when coadministered with methotrexate (nakamura et al., ) and caused apoptosis of melanoma cells (nakamura et al., b ). an orally administered cs was considered to be ''quite" safe based on body weight gain, and liver/kidney weights of mice (nakamura et al., a,b) . the authors concluded the extract could inhibit aortic cholesterol deposition in atherosclerotic mice by scavenging free radicals in vivo. these extracts ''may have" beneficial effects on the process of atherogenesis and aging with few side effects (yamaguchi et al., a) . this is good news but surely more evidence is required. towards these ends, tsai et al. ( ) also demonstrated the hydroxyl radical scavenging activity of cs. whereas reduced lipid peroxidation in rats was demonstrated by shen and chen ( ) . antioxidant activity in the xanthine oxidase, haemolysis and lipid peroxidation assay systems was reported from water extracts, and a polysaccharide fraction, of cultured cs mycelia (li et al., a) . as mentioned previously, the fruiting body and the caterpillar parts of cs are claimed to be similar in chemical composition and hence anti-oxidant activity . it would be interesting to determine (a) how this occurs in terms of insect substrate utilisation by the fungus with a view to optimising yields of bioactive fungal components, (b) when the preparation is at the correct stage for use as a medicinal treatment, and (c) if these data could be extrapolated to in vitro culture. cho et al. ( ) and wang et al. ( ) also reported radical scavenging activity with wang et al. reporting activity against colorectal tumour cells. the influence of cs on the immunoactivity of macrophages was determined to probe the mechanism of its alleged tonic effect. the phagocytosis of macrophages were enhanced significantly (jia and lau, ) and ''maybe" the tonic effect of cs is accomplished by an enhancing effect on the immune system. some chemical fractions had insulin like and insulin release promoting activity and ''could be developed" as an anti-diabetic agent (young et al., ) . cordyceps possessed a strong anti-oxidation activity in all assays tested by li et al. ( a) . the cultured cordyceps mycelia had equally strong anti-oxidation activity compared to in vivo cordyceps. further, the anti-oxidation activities were increased  - in the partially purified polysaccharide fractions. in an intriguing report, liu et al. ( ) mentions that identification of an effective non-toxic biological radioactivity protector is a ''matter of some urgency". orally administered cs protected mice from bone marrow and intestinal injuries after total-body irradiation. the levels of free radical species within cells are suggested to be a likely mechanism for the purported effects. the biochemical mechanisms of anti-proliferative effects of c. militaris in human leukemia cells were investigated in a convincing study involving cancer treatment . it was found that they inhibited cell growth in a dose-dependent manner, which was associated with morphological change and apoptotic cell death such as formation of apoptotic bodies and dna fragmentation. furthermore, the treatment caused a dose-dependent inhibition of cyclooxygenase- (see paterson, ) and prostaglandin e accumulation. taken together, these results indicated that the anti-proliferative effects were associated with the induction of apoptotic cell death through regulation of several major growth regulatory gene products. the extracts ''may have therapeutic potential" in human leukemia treatment. in addition, corticosterone output by cultured rat adrenocortical cells was increased without increasing the intracellular camp level. the steroidogenic effect was abolished by the protein kinase c inhibitor calphostin c, indicating that its action may involve stimulation of protein kinase c (wang et al., ) . c. militaris reduced the fasting serum glucose level and enhances glucose utilisation in skeletal muscles in rats . the fungus demonstrated cytotoxic activities on the three kinds of human cancer cell lines, stomachic adenocarcinoma, colorectal adenocarcinoma, and hepatocellular carcinoma (lim et al., ) . cytotoxic activity-guided isolation and identification of active fractions afforded the well-known, cordycepin as an active component (see above). koh et al. ( b) reported that cs mycelia prolonged swimming endurance capacity and produced an anti-fatigue action in mice. cs reduced the hepatic content of malondialdehyde and the serum concentrations of transaminases and alkaline phosphatase in rats with hepatic fibrosis. treatment with the extracellular biopolymers resulted in a reduction in hepatic hydroxyproline content and normalization of morphological characteristics of the liver, indicating an anti-fibrotic action (nan et al., ) . finally, crystals of the fungus stimulated proliferation of erythroid progenitor cells in mouse bone marrow . in a very interesting report, the insect-body part (of the tcm) inhibited proliferation of enhanced human mononuclear cells (hmnc). any differences between the fungus and insect components are well worth further investigation. the production of interleukin- and interferon was stimulated by the aqueous methanolic extracts and inhibited by the methanolic extracts (weng et al., ) . treatment of patients with condyloma acuminata brought about an increase in interleukin- and a decrease in interleukin- , indicating a recovery in the balance of th /th cytokines. recurrence was also diminished (gao et al., ) . interestingly, the ergosterol esters concentrations were much higher in the (dead) caterpillar than the fruiting bodies (yuan et al., ) although ergosterol was similar. although why these compounds are of particular interest is ''mystifying" as they are universal in fungi. in a surprising paper, the following may be an example of the mystical nature of some of the reports (see paterson, ) . it represents a bizarre rational for undertaking the work and it is surprising that it was published from a scientific perspective. the rational for the work is that cs is a popular chinese tonifying herb, and was/is revered for being, what is referred to as, 'yin-nourishing' and 'yanginvigorating' in chinese medicine (siu et al., ) . in order to establish the pharmacological basis for the 'yinnourishing' and 'yang-invigorating' action of cordyceps, the effects of wild and cultured cordyceps on concanavalin a stimulated splenocytes, an in vitro bioassay for 'yinnourishment', and myocardial atp generation capacity, an ex vivo bioassay for 'yang-invigoration', were investigated in mice. the results indicated that wild and cultured cordyceps enhanced the con a-stimulated splenocyte proliferation in vitro and myocardial mitochondrial atp generation ex vivo in mice, with no significant difference in potency of action between the two types of cordyceps. while the immunopotentiating effect was associated with the increase in interleukin- production, the stimulation of myocardial atp generation was paralleled by an enhancement in mitochondrial electron transport. when compared with typical 'yin' and 'yang' tonifying chinese herbs, cordyceps was found to possess both 'yin-nourishing' and 'yang-invigorating' activities, with a lower potency in both modes of action. it is impossible to take reports such as these seriously from a scientific standpoint and is given considerable space here to indicate a general problem which may have motivated some of the published reports reviewed herein. this is without attempting to detract from the philosophical aspects of the concepts of yin and yang in terms of two mutually correlated opposites in a general sense. a comprehensive definition of the corresponding philosophy of science is inappropriate: it is enough to state that it is vitally important for science that the information about the surrounding world and the objects of study be as accurate and as reliable as possible. to continue, cs fruiting bodies inhibited various tumour cell lines (kuo et al., ) : two fractions were particularly potent. growth inhibitors other than cordycepin and polysaccharides may have been involved; two fractions significantly inhibited (a) the blastogenesis response, (b) nk cell activity and (c) il- production (kuo et al., ) . neither fraction was cytotoxic, and immunosuppressive ingredients were found to be intracellular. fruiting bodies inhibited human mesangial cells (hmc) activation by il- plus il- and liver toxicity or mutagenicity were not observed. the fraction was purified to obtain purified compound h -a (see above). the authors claim a novel treatment for human berger's disease ''in the future". a fraction (a) dose dependently suppressed bronchoalveolar lavage fluids (balf) cells proliferation, (b) reduced interleukin production in lps activated balf cell cultures and (c) affected interleukin mrnas in various significant manners (kuo et al., ) . the purported therapeutic activity may be related to modulation of cells functions in bronchial airways. furthermore, the molecular mechanism of cordyceps pruinosa pharmacological and biochemical actions of macrophages in inflammation has not been clearly elucidated . the authors suggest that an extract suppresses inflammation through suppression of nf-jb-dependent inflammatory gene expression, and hence may be beneficial for treatment of endotoxin shock or sepsis. shim et al. ( ) wrote that they attempted to develop ''a new type cordyceps". to obtain this, they investigated the effects of the fruiting bodies of the cultivated fungus of p. japonica grown on silkworm larvae on hyperglycemia in rats and mice and on immunological functions in mice. as mentioned previously, it is not at all clear whether paecilomyces (even) contains anamorphs of cordyceps. therefore, this report is a cause of some concern. immunostimulating activity and a significant anti-fatigue effect in mice were observed. kuo et al. ( ) reported unidentified substances which inhibited tumour cells, but which were not cordycepin or polysaccharides, in the methanolic extract of cs -this report requires further investigation. c. cicadae ascocarps enhanced hmnc proliferation (weng et al., ) . in contrast, the insect-body portion suppressed hmnc proliferation. this is a most interesting result in the current author's opinion because of the different effects of the two components. the action mechanisms of the fractions may involve the regulation of interleukin and interferon production in hmnc. overall, the results demonstrated that c. cicadae contained growth modulators for hmnc. unfortunately, the compounds responsible were not characterised. koh et al. ( ) demonstrated that a hot-water extract modulated interleukin- production by activation of macrophages and augmented the secretion of haematopoietic growth factors. whereas aqueous methanolic extracts of the ascocarp stimulated proliferation of phytohaemagglutinin-induced proliferation of hmnc. of course, such differences indicate that different compounds are involved or the same compounds are at different concentrations. c. ophioglossoides mycelia prevented cell death in neuronal cells and memory deficits in rats (jin et al., ) . two fractions from fruiting bodies inhibited (a) the blastogenesis response, (b) natural killer cell activity, (c) interleukin- production and (d) tumour necrosis factor production in phytohaemagglutinin-stimulated human mononuclear cells (kuo et al., ) . the levels of interferon, interleukin- and tumour necrosis factor produced by cultured rat kupffer cells were increased by the fungus (liu et al., a) . proliferation of cells in balf was inhibited which also reduced tumour necrosis factor (kuo et al., ) . in an effort to evaluate the pharmacological effects, including the anti-aging, of the fruiting bodies of the cultivated p. japonica fungus, a new type of cordyceps sp. was investigated (shin et al., ) . this statement by the authors is incomprehensible (see above). types of fungi are the typical specimens often held in culture collections. it is not proposed to discuss the described effects herein. hot-water extracts of cordyceps scarabaecola stromata exhibited potent intestinal immune system-modulating activity, while the methanol-soluble fraction manifested intermediate activity (yu et al., ) . xu et al. ( ) detected inhibition in melanoma colony formation in murine lungs by cs. the fruiting bodies of ''p. japonica" reduced tumour weight and volume and lengthened the life span of mice inoculated with sarcoma cells (shin et al., ) but what this fungus represents in unclear. water and ethanol extracts of cs possessed a potent anti-oxidant activity (yamaguchi et al., b) . anti-lipid peroxidation activities also were detected and accumulation of cholesteryl ester in macrophages was inhibited via suppression of ldl oxidation. hot-water extracts were particularly effective. ''p. japonica" exhibited immunostimulating activity. its ethanolic extract stimulated phagocyteosis and macrophage acid phosphatase activity (shin et al., (shin et al., , . c. militaris demonstrated general anti-inflammatory activity (yu et al., a) in mice. the fact that cultured fruiting bodies were employed is interesting rather than extraction from those from the wild and most papers seen for this review either use fruiting bodies from the wild and/ or in vitro biomass. however, it is disappointing that details of the cultivation of the fruiting bodies are not provided as this is required. nitric oxide production and inos gene expression in lps-stimulated raw . cells are suppressed by ethanolic preparations (won and park, ) . a unique insight into how the cultured fruiting bodies were produced in this report and should be referred to: this detail is required in future reports. cs (as supplied by the ''xinhui xinhan artificial cordyceps factory" (sic)) inhibited mda generation via hydroxyl radicals induced by the peroxynitrite generator sin- and macrophage accumulation of esterified cholesterol (yamaguchi et al., a,b) . the authors concluded that the cultured cs has anti-oxidant and anti-lipid peroxidation properties and inhibits accumulation of cholesteryl ester in macrophages via suppression of ldl oxidation. the authors conclude correctly that this cultured chinese medicine appears to merit further investigation as an anti-atherosclerotic. in the unusual use of this solvent to create an extract from cs mycelia, apoptosis in human pre-myelocytic leukaemia hl cells was induced. in addition, cell proliferation was inhibited . obviously, ethyl acetate may extract different compounds from the fungus compared to the more common water or methanol extracts and the constituents need to be determined. an alcoholic extract of cs inhibited abdominal aortic thrombus formation in rabbits by preventing platelet aggregation (zhao, ) . cs was extracted in pbs and dialyzed (chiou et al., ) the resulting macromolecule fraction was assayed in anesthetized rats for hypotensive effects and in isolated aorta for vasorelaxant effects. a constituent(s) in cs relaxed vascular beds directly. the in vivo and in vitro effects and its extracted fractions on the secretion of testosterone in mice were studied (hsu et al., a) . cs, watersoluble protein, and poorly water-soluble polysaccharide and protein significantly stimulated in vitro testosterone production in purified mouse leydig cells. the authors concluded that it is ''possible" that cs ''might" contribute to an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males, which is a large leap in conclusions. increase antigen expression was found in hepatoma cells and ''will" provide more effective host immune surveillance against tumour cells (chiu, ) from a cordyceps extract. in an interesting study, the beneficial effects of the ''traditional chinese medicine cs", on mice with hypoferric anaemia were evaluated by nmr spectroscopy (manabe et al., ) . the extract increased hepatic energy metabolism in anaemic mice and was concluded to be due to increased hepatic blood flow. zhang et al. ( ) concluded that ''their" extract ''is" effective in resisting the oxidative damage on liver mitochondria of diabetic mice. qiao and jian ( ) attempted to identify the signaling pathways for the induction of hl- cell apoptosis by cs mycelium extract (csme). csme induced nuclear fragmentation and dna degradation, two hallmark events of apoptosis, in the hl- cells within - h of treatment. concomitantly, several major events in the mitochondrial signal pathway occurred, including (a) the loss of mtp, (b) cytochrome c release into the cytoplasm, (c) the decrease in bcl- protein level, (d) the translocation of bax protein from cytoplasm into mitochondria, and (e) the activation of caspase- , - , and - . however, caspase- , the initiator caspase in the death receptor pathway, was not activated. these results suggest that csme induces apoptosis in hl- cell through the mitochondrial pathway rather than the death receptor pathway. treatment of d-galactose-induced-aged-mice with an unspecific cs extract resulted in (a) an improved learning ability and memory, (b) an increase in superoxide dismutase activity in erythrocytes, liver and brain, (c) an increase in catalase and glutathione peroxidase activity in blood, (d) reductions in malondialdehyde levels in brain and liver and (e) a reduction in monoamine oxidase activity in the brain . extracts enhanced the antibody response, restored the phagocytic activity of macrophages in tumour-bearing mice, and lengthened the survival period of the mice (yamaguchi et al., ). an extract down-regulated apoptotic genes in the rat kidney following ischaemia/reperfusion (shahed et al., ) . manabe et al. ( manabe et al. ( , found that a mycelial extract increased hepatic energy metabolism, as demonstrated by liver atp:pi value, in diet-induced hypoferric anaemic mice by increasing hepatic blood flow. the following section considers the use of the fungus as whole mycelium and/or fruit-bodies, although the prepara-tions may have to be prepared in water or ethanol in practise. cs down-regulated inflammation-related genes in the rat kidney following ischaemia/reperfusion (shahed et al., ) . a similar treatment improved lung function in guinea-pigs and airway inflammation in rats, suggesting a possible asthma treatment . c. pruinosa inhibited (a) gene expression of an interleukin tumour necrosis factor, (b) inducible nitric oxide synthase (inos) and cyclooxygenase- , and (c) nuclear transcription factor nf-jb activation in a lipopolysaccharide (lps)-stimulated mouse macrophage cell line: this indicated a role in the treatment of endotoxin shock or sepsis . anti-ds-dna production was inhibited and improved survival in mice indicated that cs may be beneficial to patients with systemic lupus erythematosus, an autoimmune disease with involvement of multiple organ systems . the fungus inhibited lymphadenectasis, reduced proteinuria and plasma anti-ds-dna antibody and improved renal function in mrl pr/ pr mice (fu and lin, ). an oral dose of - g daily for years prevented the recurrence of lupus nephritis and protected renal function in lupus nephritis patients (lu, ) . carcinogenesis in the murine forestomach was suppressed by cordyceps (lin, ) . results from liu et al. ( ) indicated that the levels of il- and inf, produced by cultured rat kupffer cells were increased from rats fed on cs. studies have demonstrated that polysaccharides extracted from these natural products have anti-hyperglycemic effects (lo et al., ) . these authors investigated the effects of intragastrically administered cordyceps sp. for alleviating fasting hyperglycemia in diabetic rats. animals had significantly increased serum levels of triglyceride, cholesterol and blood urea nitrogen, and significantly decreased body weight, serum albumin levels and weights of the thymus, lungs and gastrocnemius muscle compared to animals in the control group. in addition, blood glucose was significantly increased compared to the control group; these results suggest that enterally administered cordyceps sp. has potential anti-hyperglycemic ability. these findings reveal that the fungus ''may be used as a nutraceutical to alleviate hyperglycemia in diabetes". oral administration of cordyceps alleviates fasting hyperglycemia . recent evidence has shown that an extract has immunoregulatory activity. the objective was to investigate whether cordyceps has biological activity in regulating the lymphocyte subsets in diabetes. after weeks, body weight, thymus weight, thymocyte number, and the percentages of total t and t helper cells in the thymus were significantly lower in the treatment groups than in the controls. results demonstrate that stz-induced diabetic rats had significantly decreased numbers and subsets of t cells in the thymus. however, oral administration of cordyceps did not improve these changes. the results suggested that oral administration had no significant effect on lymphocyte subsets in stz-induced diabetic rats. hsu et al. ( b) demonstrated in a convincing study, that cs mycelium regulates mouse cell testosterone production and may suppress stimulated testosterone production via p scc enzyme activity. hsu et al. ( a) demonstrated that the fungus and fractions from it were capable of stimulating testosterone production. the steroidogenic activity was observed in vivo in male mice after days of treatment (huang et al., b) . chen et al. ( ) found that protein kinase a and protein kinase c pathways are acted upon to stimulate steroidogenesis in ma- mouse leydig tumour cells. inhibitors of protein kinase a, protein kinase c and phospholipase c and calmodulin antagonists (see paterson, ) reduce leydig cell steroidogenesis induced by the fungus. c. militaris inhibited (a) the growth and metastasis of lewis lung cancer cells, and (b) the growth of sarcoma s cells implanted in mice. in addition, the survival period of the mice was increased (liu et al., ) . liu et al. ( ) showed that p. sinensis inhibited lipid peroxidation but increases the amount of glutathione peroxidase and superoxide dismutase in mouse liver. again the name of the fungus raises similar questions as to those raised for p. japonica and consequently as to what material is being used and whether the experiments could be repeated. finally, cordyceps is included in a list of anti-aging tcm . cs stimulated mitochondrial electron transport and atp production (siu et al., ) . the effect of the fungus on hepatic fibrogenesis induced in rats was studied by liu and shen ( ) . it was found that it delayed cirrhotic development and improves liver function by inhibiting expression of transforming growth factor-and plateletderived growth factor and deposition of procollagen i and iii. zhou et al. ( ) presented evidence for the beneficial effects of cs on chronic hepatitis b. the fungus increased dna synthesis in primary cultured rat tubular epithelial cells (tian et al., ) . proximal tubular cells were protected from the toxic effects of gentamicin. the possible mechanisms include protection of sodium pump activity, reduction of lipid peroxidation and attenuation of lysosomal over-activity in tubular cells due to phagocytosis of gentamicin (zhen et al., ; li et al., ) . also, rat kidneys were protected from cyclosporin-induced nephrotoxicity and ameliorated glomerular and interstitial damage (zhao and li, ) . bao et al. ( ) reported that ''old" patients were protected from amikacin sulphate toxicity as demonstrated by decreases in urinary nephroaminoglycosidase and microglobulin. also, inhibition by c. militaris of ldl-induced proliferation of cultured human glomerular mesangial cells, which are involved in the development of glomerulosclerosis was observed (zhao-long et al., ) . treatment of mice with c. militaris lengthened the swimming time to exhaustion (jung et al., ). an unspecified cordyceps is one of the components of fuzheng huayu recipe, which is used to control the development of post-hepatic cirrhosis or to prevent complications from the disease (liu et al., b) . the macrophage-stimulating activity of natural fungus and cultured mycelia has been described by zhang ( ) . zhang and xia ( ) demonstrated immunosuppressant effects in the heterotropic heart allograft model in rats and determined that prolonged survival periods were possible. also, zhu and yu ( ) found prolonged mouse skin allograft survival time. the number of t helper cells was increased, as were lyt- /lyt- (t helper cells to t suppressor cells) in peripheral blood and spleen . a mitogenic action was demonstrated on splenic lymphocytes and interleukin- from spleen cells of rats with chronic renal failure was augmented (cheng, ) . in addition, natural killer cell activity was enhanced (xu et al., ) . treatment of patients with post-hepatic cirrhosis resulted in (a) enhancement of natural killer cell function, (b) increased number and improved ratio of cd þ and cd þ cells, and (c) reduction in iga and igg levels (zhu and liu, ) . treatment of chronic hepatitis b resulted in increased cd /cd ratios, and reductions in hyaluronic acid and procollagen type iii. the data indicate the usefulness of the fungus in adjusting the level of t lymphocyte subsets and treating hepatic fibrosis (gong et al., ) . liu et al. ( ) demonstrated increased peripheral natural killer cell activity from healthy subjects and leukaemia patients. improved renal function and augmented cellular immune function in chronic renal failure has also been observed (guan et al., ) . the fruiting body portion, but not the carcass portion, of cordyceps reduced weight loss, polydipsia and hyperglycaemia in diabetic rats (lo et al., ) . some other somewhat obscure papers are available on the effect of whole fungi (i.e. peizhong, ; chen, ; zhang, ; shao, ; liu and xu, ; zhang, ; chen, ; du, ; sun, ; li et al., ) . feng et al. ( ) reported the vasodilating effect of cultured mycelia of cs in an investigation of the cardiovascular system of dogs. thirty three cases of chronic hepatitis b patients treated with cultured cs mycelia have shown that the drug (a) improves liver function, (b) promotes negative transfer hbsag, (c) markedly helps to raise plasma albumin, (d) helps patients resist high gamma globulin and (e) adjusts body immunocompetence (zhou et al., ) . it is suggested that the fungus may be a medicine for chronic hepatitis b patients in adjusting protein metabolism and correcting inversion of albumin and globulin. since inflammation has been reported to be associated with chronic diseases inhibitory dietary factors such as the fungus may be beneficial in alleviating disease (hong and lin, ) . b-estradiol may directly influence the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. cs mycelium is ''believed" to enhance libido and fertility in both sexes. however, the mechanism of its effect in women has not been determined. this is surely a rather thin basis on which to undertake research. huang et al. ( a) concluded that treatment of granulosa-lutein cells with cs results in increased b-estradiol production due, in part, to increased star and aromatase expression. if these data are confirmed, cs may help in the development of treatment regimens to improve the success rate of in vitro fertilization, they state. zhu and liu ( ) found that cultivated cordyceps mycelia inhibited humoral immune hyperfunction and increase the serum complement level in patients with post-hepatic cirrhosis, and improved liver function. whole body insulin sensitivity in rats was increased (balon et al., ) . the fungus increased the basal plasma insulin level (zhang et al., ) and inhibited hepatic fibrogenesis in rats with ccl -induced liver fibrosis. previous studies by lo et al. ( ) demonstrated that the fruiting bodies of cs attenuated diabetes-induced weight loss, polydipsia, and hyperglycemia in rats. rats were orally administered, fruiting bodies, fermented mycelia, spent broth, or mycelia plus spent broth of the fungus. the results revealed that the mycelia and spent broth had anti-hyperglycemic activities similar to those of the fruiting bodies. the authors , bold claim that the fermented products could be developed as potential anti-diabetic agents or functional foods for persons with a high risk of diabetes mellitus needs further confirmation. yang et al. ( ) mention that mycelium can inhibit tumour growth and induce tumour cell apoptosis. however, the antitumour mechanisms are not fully understood. so, the molecular mechanism was determined. the authors conclude that cell apoptosis is induced by activating caspase- -dependent and caspase- -independent pathways and downregulating nf-jb protein expression. mycelia induced human granulosa-lutein cells to produce b-estradiol by upregulating expression of steroidogenic acute regulatory protein (star) and aromatase (huang et al., a) . also, steroidogenesis in mouse leydig tumour cells (huang et al., a) was stimulated without involvement of star (huang et al., ) . testosterone production was inhibited by human chorionic gonadotropin or dibutyryl cyclic amp. thus, its effect on the signal-transduction pathway for steroidogenesis may be after the production of cyclic amp. it is revealing to read the web sites of some of the commercial products: these are often written in unscientific terms. the best of them at least contain warnings that, ''these statements have not been evaluated by the (us) food and drug administration. this product is not intended to diagnose, treat, cure or prevent any disease". colson et al. ( ) undertook a revealing experiment to determine the effect on male cyclists' performance using a combination of cs and rhodiola rosea ''herbs" -a tradi-tional herbal medicine. importantly, and unusually, the study followed a double blind, randomized, placebo-treatment, pre-post test design. essentially no effect was observed. this is an example of why scientists need to circumspect about making unsubstantiated claims about the powers of these preparations even if spin does help project funding. ''in this regard, the maintenance of a balance of yin and yang -two opposing components involved in life activities as exemplified by the antagonistic action of the sympathetic and parasympathetic nervous systems-is essential in achieving a healthy condition" (leung et al., ) . statements such as these leaves the current reviewer bewildered. why these terms are used in a scientific publication does not appear to merit comment. previous studies have shown that long-term treatment with a ''yang-invigorating" chinese herbal formula (vi- ) could increase red cell cuznsuperoxide dismutase (sod) activity in male human subjects. yet the authors conclude, the beneficial effect of vi- treatment on mitochondrial functional ability and antioxidant capacity may have clinical implications in the prevention of age-related diseases. ka wai lee et al. ( ) investigated a commercial preparation of a cultivated strain of cs. they state that the immunomodulatory activities have been renowned for centuries. the report describes positively the immunomodulatory features: in vitro results demonstrated that the fungus induced the production of interleukin (il)- , il- , il- and tumour necrosis factor alpha, augmented surface expression of cd on lymphocytes, and elevated macrophage phagocytosis and monocyte production of h o . the authors state that, ''our results possibly provide the biochemical basis for future clinical trials". the current author emphasises that such over extrapolation of data needs to be discouraged. dai et al. ( ) found that cordymax cs- (a mycelial fermentation product of cs) ''improved the bioenergy status" in the mouse liver. these findings may explain why cordymax cs- is claimed to alleviate fatigue and improve physical endurance especially in aged subjects. reports of athletes' performance being improved may also be supported from these types of reports. cordymax cs- lowered fasting plasma levels of glucose and insulin, improved oral glucose tolerance and increased the glucose-insulin index, which measures insulin sensitivity, in rats (zhao et al., ) . an unspecified cordyceps is reported to be a component of fuzheng huayu recipe, which is used to control the development of post-hepatic cirrhosis or to prevent its complications (liu et al., b) . as part of the eternal search to find an excelsior of youth, many are interested in anti-aging activity but it is difficult to define what his means scientifically. are the authors stating that the aging process is slowed for example? do people live longer? in any case, this was published in the journal, chinese journal of integrated traditional and western medicine which is an admirable sentiment but difficult to do in practice. a prep-aration of cs referred to as jinshuibao capsule, increased the depressed superoxide dismutase activity and reduced elevated malondialdehyde (mda) level caused by aging when tested in senile patients. in addition, they enhanced the repair of damaged non human animal dna (zhang et al., ) . quite extraordinarily beneficial effects on senile patients are claimed and, of course, the constituents of the capsule may be difficult to verify. zhou and lin ( ) used ''jinshuibao", to restore cellular immune function and ''improve the quality of life" of patients with advanced cancer without affecting humoral immune function. obviously, quality of life is a subjective notion and is difficult to define. remarkably, a dried powder preparation of mycelia called bailing capsule (a) prevented rejection of renal transplants, (b) protected renal and hepatic function, (c) stimulated haematopoietic function, (d) improved hypoproteinaemia and hyperlipidaemia and (e) reduced the incidence of infections (sun et al., ) . however, such claims must surely carry considerable elements of doubt. in another case of a combined treatment with as many as six other components in addition to cs, the treatment was more effective at preventing acute renal failure than chronic in rats. obviously, it is difficult or impossible to assess the efficacy of the individual components (ngai et al., ) . inhibition of apoptosis is a novel area of clinical investigation with great promise and so merits a separate section in this review and will be of particular interest to pharmacologists. in one of the more critical papers, buenz et al. ( ) mention that there is a wide range of uses of cs in the literature, and those claiming altered apoptotic homeostasis are of the most intriguing. however, they emphasize problems created by the (a) difficulty of identifying the species of cordyceps and (b) many conflicting reports of pharmacological function. in response, the authors (a) outline what is known about the ability of cs to alter apoptotic homeostasis, (b) attempt to reconcile differences in function, and (c) identify the challenges and how to progress cs research. many disorders (e.g. stroke, myocardial infarction and hiv) incorporate apoptosis in their aetiology and pathogenesis. the ability to inhibit apoptosis has emerged as an important potential therapy (e.g. cancer). there are reports of cs extracts inhibiting and inducing apoptosis (shahed et al., ) ; this is not contradictory but allows a foundation to determine the molecular mechanism of activity. indeed, cordyceps may contain compounds that inhibit apoptosis; however, conflicting evidence has been obtained. the fungus can scavenge reactive oxygen species by inhibiting malondialdehyde formation by sin- , the peroxynitrite generator, which has been confirmed by xanthine oxidase, hemolysis, and lipid peroxidation assays. furthermore, an isolated extract of cs, h -a (this is reported as a pure compound (see above), inhibited induced apoptosis, by permeabilizing the cell membrane and upregulating nitric oxide synthase (trubiani et al., ) . on the other hand, extracts of cs did not inhibit hydrogen peroxide-induced apoptosis (buenz et al., ) , in a reactive oxygen species model (fauconneau et al., ) . alternatively, cs down-regulated apoptotic genes and modulated apoptosis in a rat kidney ischemia reperfusion model. reported anti-apoptotic effects of cs include in (a) the mouse (anti-cytotoxic activity, anti-oxidant activity, cell proliferation inhibition), (b) cell culture (anti-proliferation activity, cell proliferation inhibition, hemolysis inhibitory activity, lipid peroxide formation inhibition, radical scavenging effect), (c) human cells (proliferation inhibition, natural killer cell inhibition, tumour necrosis factor inhibition) and (d) the rat (gene expression inhibition). whereas the reported apoptotic effects of the fungus are in: (a) the mouse (anti-tumour activity, metastasis inhibition) and (b) cell culture (proliferation stimulation, cytotoxic activity). in reality these opposite effects are not incompatible. there are three prominent factors that may contribute to the apparent discrepancies (the first and third are fairly obvious). first, certain fungi contain different biologically active compounds. second, extracts may contain a prodrug; a metabolism step may be required to generate the biologically active form. third, different constituents may be assayed as there are multiple methods of extraction utilized in the literature. furthermore, decreases in fas, fas ligand and tumour necrosis factor expression and decreased caspase- activity have been demonstrated (shahed et al., ) . cs inhibited tnf-a expression (kuo et al., ) . however, when apoptosis was initiated via a fas agonist antibody (ch- ) (alderson et al., ) , aqueous and alcohol extracts of cs did not rescue cells induced by fas receptor ligation (buenz et al., ) . furthermore, cell cycle arrest and/or inhibition of proliferation yield cells resistant to apoptosis. papers concerning proliferation of leukemic u cells and glomerular mesangial cells inhibition (zhao-long et al., ; lin et al., ) may be explained by conferring a apoptotic resistance to cells: the alteration may involve p (fridman and lowe, ) or nf-jb (karin et al., ) . overall, aqueous, and organic extracts of cs have the ability to inhibit apoptosis. cells pre-incubated with cs extracts were equally sensitive to hydrogen peroxide and fas-mediated apoptosis. thus, the putative antioxidant and anti-apoptotic properties of cs were insufficient to rescue cells from apoptosis in vitro (buenz et al., ) . furthermore, cancer chemotherapeutics have involved the ability to induce apoptosis. hence the polysaccharide fraction (h -a) induced apoptosis by inhibiting (a) phosphorylation of bcl- and bcl-xl and (b) apoptosis induced by dimethyl sulfoxide (yang et al., ) . these data require to be confirmed by further work (buenz et al., ) . finally, direct cytotoxic activity may be a factor (nakamura et al., a,b; kuo et al., ; sato, ; buenz et al., ) . a factor which is not often considered is whether the activities of extracts and pure compounds are from the cordyceps of interest or from other contaminating fungi. the issues of anamorph/teleomorph associations are relevant here. for example, epicoccins a-d were isolated from cultures of a cordyceps-colonizing isolate of epicoccum nigrum (zhang et al., b) . gliocladinins a and b are of an isolate of gliocladium sp. that colonized cs (guo et al., ) . it is essential that such activity is differentiated from that obtained from the traditional medicine or cordyceps per se. in a series of papers, (a) paecilomyces militaris is shown to possess militarinones a, b, c, d and (b) farinosomes and a deoxymilitarinone substance were detected from paecilomyces farinosus (schmidt et al., (schmidt et al., , cheng et al., cheng et al., , . the authors do not make statements as to the holomophic connections to cordyceps. hamburger ( ) does make this link to c. militaris (teleomorph) from p. militaris (anamorph) apparently on the basis of the same pigment production. however, it is has been suggested that paecilomyces does not contain anamophic species of cordyceps . hence, comment on these reports is avoided by the current author because the relationships between paecilomyces and cordyceps remain ill-defined. another complicating facet is that the medicine may contain a proportion of the insect host and so what does this bring to the activity? this is an area of investigation which simply is not reported. indeed does the healthy insect have interesting activities, and/or at what stage does the activity occur in the infected insect? could the activity be greater at these stages? the accumulation of bioactive plant metabolites by insects is well documented (brown and trigo, ) and insects are used as medicines . these aspects are almost totally ignored in the case of the medicinal cordyceps. it appears as if entomologists have simply not been involved in the field. does the insect have to be dead before the system works or are more active components produced by the fungus as it is in the process of killing the insect? research into these factors could be fruitful. hence, it is worth reporting what is known about the differences between the insect and fungus parts: interestingly, galactomannans isolated from the insect portion of c. cicadae demonstrate potent hypoglycaemic activity in mice (kiho et al., ) . the fruiting body portion, but not the carcass portion, of cordyceps reduced weight loss, polydipsia and hyperglycaemia in diabetic rats (lo et al., ) . aqueous methanol extracts of c. cicadae ascocarps enhanced human mononuclear cells (hmnc) proliferation (weng et al., ) . in contrast, the methanol ( %) extracts of the c. cicadae insect-body portion suppressed hmnc proliferation. interestingly, the ergosterol esters concentrations were much higher in the (dead) caterpillar than the fruiting bodies (yuan et al., ) although ergosterol was similar. finally, the fruiting body and the caterpillar parts of cs are claimed to be similar in chemical composition and hence anti-oxidant activity because the fungus had presumable replaced the insect constituents with fungal . little scientific evidence exists to support the numerous herbs used to improve diabetes-related metabolic disorders (lo et al., ) . the dual organism medicine (i.e. fruiting body and carcass) has been proposed to have multiple medicinal activities. in one investigation, the effects of the fruiting body and carcass of the preparation on hyperglycemia were investigated. diabetic rats had significantly lower weight gain and higher blood glucose response in oral glucose tolerance test than the control rats; and these changes were significantly reduced by administrating the fruiting body of cordyceps. the results revealed that the fruiting body (not the carcass) of cordyceps attenuated the diabetes-induced weight loss, polydipsia and hyperglycemia, and these improvements suggest that fruiting body has a potential to be a functional food for diabetes. the water extracts from the fruiting body and ''worm" of natural cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had a chemical composition similar to the fruiting body . in addition, both the fruiting body and worm of cordyceps showed similar potency in their anti-oxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. these results suggest that the function of the worm is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by mycelia. aqueous methanol extracts of c. cicadae ascocarps enhanced human mononuclear cells hmnc proliferation (weng et al., ) . in contrast, the methanol ( %) extracts of the c. cicadae insect-body portion suppressed hmnc proliferation. this is a most interesting result in the current author's opinion. the following are references concerning the chemical constituents as determined by analytical profiles (guo, ; xiao, ; . yu et al. ( b) developed a method involving ''biospecific" extraction and hplc for potential immunological components in cs. the two active compounds were identified as guanosine and adenosine. li et al. ( b,c) found that cultured cs mycelia have a much higher content of nucleosides than natural cs. guo et al. ( ) described an hplc method for quantitative determination of adenosine and deoxyadenosine. li et al. ( ) determined adenosine in fermented products of cordyceps by reversed-phase hplc. ergosterol in cs can be determined by hplc li et al., ) , and, of course, ergosterol is present in all fungi. when there are many questions as to what are the active components, a section on optimisation may seem premature. however, kim and yun ( ) investigated the optimal culture conditions for the production of exopolysaccharides (eps) and cordycepin during submerged mycelial culture of c. militaris and cs. this was not done in the paper hamburger ( ) comments upon. fermentations were performed in flasks and in -l stirredtank bioreactors. the concentrations of mycelial biomass, eps and cordycepin achieved in submerged culture of c. militaris were higher than those of cs. as the authors claim, comparative studies between the two fungi are not available and the paper was the first report on the optimum medium composition for submerged culture of cs. cordyceps nutans pat. is another entomopathogenitic ascomycete belonging to the family clavicipitaceae which is parasitic on hemipteran insects (sasaki et al., ) . very few investigations have been made with this fungus. in this research, optimum temperature and ph for mycelial growth was determined. however, it appeared that growth remained low and commercial exploitation perhaps limited. however, it would be interesting to include this species in any future taxonomic treatments of the genus. optimisation of culture conditions for mycelial growth and production of polysaccharides and cordycepin were described by park et al. ( b park et al. ( , , xu et al. ( ) , kim et al. ( b,c) , xiao et al. ( ) , mao and zhong ( ) and hsieh et al. ( ) , although cordycepin is usually produced synthetically at present. two cases of lead poisoning were reported (wu et al., ) . these two patients took cordyceps herbal medicine for treatment of underlying diseases. loss of appetite and anemic signs of lead poisoning were manifested in one patient with a high blood lead level, while the other patient was asymptomatic. the lead content in the cordyceps powder was found to be as high as , ppm. after cessation of intake in the asymptomatic patient, and cessation of intake and treatment with chelating agents in the symptomatic patient, the blood lead levels returned to normal range. this report raises concerns about lead poisoning from unusual herbal medicine in general. this present review is not intended to be a taxonomic paper, but is essentially a review of the papers that employ the name cordyceps more or less loosely, to describe the fungus used in medicinally related experiments. however, in a most impressive paper, sung et al. ( ) state that cordyceps comprise over species in their modern phylogenetic classification of cordyceps and the clavicipitaceous fungi. cordyceps fr. host range is broad, ranging from orders of arthropods to the truffle-like genus elaphomyces, although most species are restricted to a single host species or closely related host species. the main objectives of the study was to reassess the (a) morphological traits used currently, (b) taxonomic utility of the anamorphic forms and ( ) classification in relation to phylogenetic relationships. it may be worth mentioning that pcrs may be subjected to inhibition, and nucleic acids may be affected by the medium in which the fungus was grown (see paterson, , . unfortunately, sung et al. ( ) do not provide details of how the fungi were grown for analysis and so it is difficult to conjecture. obviously, as discussed in this review, the fungus produces numerous bioactive compounds which may be inhibitors or mutagens and the effect of metabolite production on the dna preparations need to be determined or misleading results may be obtained. however, to continue, it is worth mentioning that c. militaris maintains its name in the study while c. sinensis is now classified as ophiocordyceps sinensis. the taxon was historically classified in the clavicipitaceae, based on cylindrical asci, thickened ascus apices and filiform ascospores, which often disarticulate into part-spores. the fungus was characterized by having a pathological ecology on elaphomyces and arthropods, with infrageneric classifications emphasizing ascospore morphology, host affiliation and arrangement of perithecia. the production of welldeveloped often stipitate stromata was typical. sung et al. re-classified on the basis of phylogenetic relationships between taxa by employing five to seven genetic loci. three clavicipitaceous clades were determined which rejected the monophyly of cordyceps and clavicipitaceae and most diagnostic characters used in cordyceps were not supported as being phylogenetically informative. however, the most consistent characters with the phylogeny were pigmentation, morphology and morphology of stromata. cordycipitaceae was validated based on c. militaris, the type of cordyceps, which included most cordyceps species that possess bright, fleshy stromata. the new family ophiocordycipitaceae was proposed. the majority of species here produce stromata that often possess aperithecial apices and are darkly pigmented, tough to pliant. elaphocordyceps was proposed for a subclade of the ophiocordycipitaceae, which includes species of cordyceps that parasitize (a) the fungal genus elaphomyces and (b) arthropods. the family clavicipitaceae included the core clade of grass symbionts, and the entomopathogenic genus hypoc-rella and relatives. the new genus metacordyceps is proposed for cordyceps species that are closely related to the grass symbionts in the clavicipitaceae s. s. metacordyceps includes teleomorphs linked to metarhizium and other closely related anamorphs. lists of accepted names for species in cordyceps, elaphocordyceps, metacordyceps and ophiocordyceps are provided. obviously, this work needs to be consulted in future work on the fungus for medicinal properties as does stadler et al. ( ) . too often authors have used highly conditional statements such as; the preparation ''may" have ''possible" activity against, for example, cancer, which are meaningless. it is claimed that modern studies are demonstrating anti-oxidant, vascular, immune, and anti-inflammatory effects (meletis and barker, ) and the mechanisms by which these mushrooms work are being elucidated. however, the journal in which the paper is published is dedicated to complimentary and alternative medicines. this implies it is not mainstream scientific and so scientists may not give it as much weight in comparison to, for example, phytochemistry or the journal of natural products. nevertheless, the former reports could be considered as providing leads for more scientific research. in general, problems include too many chemically uncharacterised crude extracts, inadequate taxonomic vigour, and over extrapolation of in vitro data to imply therapeutic value. this needs to be avoided as it falsely raises hopes for cures for serious diseases. furthermore, there are no biochemical data on how the insect are converted into fungal components, or if the insect host per se has pharmacological properties. poor (i.e. inadequate or obsolete) biochemical procedures and assays can also because problems (see hamburger, ) . the biological material being used often is not well characterised and there are few indications of specimens being available for other workers to repeat the described work for example (i.e. no voucher specimens are available), with some notable exceptions. a rather limited range of solvents have been employed to extract the components and, for example, chloroform/methanol is particularly efficacious for fungal compounds (e.g. paterson and bridge, ) . there is very little on the use of non-polar solvents. paterson et al. ( ) , paterson ( ) and paterson ( ) have advocated a method which may have utility in standardising bioactive fungi. in this scheme, a ''common, readily identifiable" morphological character is determined and then the fungus is analyzed for particular metabolites. in this case the steps may be: . produces stroma on lepidopteron insects. . produces detectable concentrations of, for example, cordycepin. the analysis for other compounds would be desirable and some potential marker compounds are provided in table . however, the phylogenic approach of sung et al. ( ) will set the standards for decades to come and will profoundly influence future publications in all fields relating to cordyceps. table summary of markers currently used for quality control of cordyceps and associated activities (after li et al., a) compound type pharmacological activities comments nucleosides anti-tumour activities; ca + antagonist; the release of various neurotransmitters presynaptically and anticonvulsant activity; stimulate axon growth in vitro and in the adult central nerve system nucleosides, especially adenosine, are usually used as markers, and the profiles can be applied for authentication of cordyceps polysaccharides anti-oxidation, immunopotentiation, anti-tumour, and hypoglycemic activity; anti-inflammatory activity and suppress the humoral immunity in mice represents the most biological properties of cordyceps, and less used as marker for quality control. thus, it should be developed ergosterol and its analogs cytotoxic activity, anti-viral activity, and anti-arrhythmia effect; suppress the activated human mesangial cells and alleviate immunoglobulin a nephropathy (berger's disease) ergosterol can be used as marker for quality control mannitol diuretic, anti-tussive and anti-free radical activities it sometimes is used as marker for quality control peptides anti-tumour and immunopotentiation activities a potential marker for quality control cordycepin: selective growth inhibitor derived from liquid culture of cordyceps militaris against clostridium spp regulation of apoptosis and t cell activation by fas-specific mab a fermentation product of cordyceps sinensis increases whole-body insulin sensitivity in rats amelioration of aminoglycoside nephrotoxicity by cordyceps sinensis in old patients antitumor sterols from the mycelia of cordyceps sinensis comparison of balanol from verticillium balanoides and ophiocordin from cordyceps ophioglossoides multi-level complexity in the use of plant allelochemicals by aposematic insects selected herbals human exercise performance the traditional chinese medicine cordyceps sinensis and its effects on apoptotic homeostasis cordyceps sinensis extracts do not prevent fas-receptor and hydrogen peroxide-induced t-cell apoptosis fungal populations and species cordyceps sinensis animal, vegetable or both? cordyceps sinesis (berkeley) saccardo: structure of cordycepic acid platelet hemopoiesis and ultrastructure observations in mice treated with natural cordyceps sinensis and its cultured mycelia the effect of natural cordyceps sinensis and its cultured mycelia on murine immuno-organs and function of the mononuclear macrophage system effects of cordyceps sinensis on murine t lymphocyte subsets the effects of chinese herbs on improving survival and inhibiting anti-ds dna antibody production in lupus mice morphological and genetic characterization of a cultivated cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in h tumor-bearing mice recent advances in studies on traditional chinese anti-aging material medica nmr and ir studies on the characterization of cordycepin and -deoxyadenosine. zhongguo kang sheng su za shi (chin cordyceps sinensis mycelium activates pka and pkc signal pathways to stimulate steroidogenesis in ma- mouse leydig tumor cells effect of cordyceps sinensis on the proliferation and differentiation of human leukemic u cells genetic diversity and taxonomic implication of cordyceps sinensis as revealed by rapd markers determination of the anamorph of cordyceps sinensis inferred from the analysis of the ribosomal dna internal transcribed spacers and . s rdna effect of cordyceps sinensis on cellular immunity in rats with chronic renal insufficiency farinosones a-c, neurotrophic alkaloidal metabolites from the entomogenous deuteromycete paecilomyces farinosus novel tetramic acids and pyridone alkaloids, militarinones b, c, and d, from the insect pathogenic fungus paecilomyces militaris protein constituent contributes to the hypotensive and vasorelaxant activities of cordyceps sinensis cordyceps sinensis increases the expression of major histocompatibility complex class ii antigens on human hepatoma cell line ha t/vgh cells cordycepin ( -deoxyadenosine) inhibits human platelet aggregation in a cyclic ampand cyclic gmp-dependent manner antioxidant and memory enhancing effects of purple sweet potato anthocyanin and cordyceps mushroom extract improvement of insulin resistance and insulin secretion by water extracts of cordyceps militaris, phellinus linteus, and paecilomyces tenuipes in % pancreatectomized rats cordyceps sinensis-and rhodiola roseabased supplementation in male cyclists and its effect on muscle tissue oxygen saturation asian herbals: opportunities for marketing traditional chinese medicines in the west cordycepin, a metabolic product isolated from cultures of cordyceps militaris (linn.) link cordymax cs- improves steady-state bioenergy status in mouse liver mushrooms in medicine and culture in vitro evaluation of antioxidant activities of aqueous extracts from natural and cultured mycelia of cordyceps sinensis antitumor activity of cordyceps sinensis and cultured cordyceps mycelia rebuttal on comparison of protective effects between cultured cordyceps militaris and natural cordyceps sinensis against oxidative damage induction of heat shock proteins (hsps) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death vasodilating effect of cultured cordyceps sinensis (berk) sacc. mycelia in anesthetized dogs control of apoptosis by p effect of cordyceps sinensis on inhibiting systemic lupus erythematosus in mrl pr/ pr mice a novel immunosuppressant, isp-i, of isaria sinclairii effect of cordyceps sinensis on the th / th cytokines in patients with condyloma acuminatum effects of cordyceps sinensis on t lymphocyte subsets and hepatofibrosis in patients with chronic hepatitis b effect of cordyceps sinensis on tlymphocyte subsets in chronic renal failure isolation and identification of -amino- -deoxyadenosine from cordyceps militaris determination of adenosine and -deoxyadenosine in cordyceps militaris (l.) link. by hplc bioactive pterphenyl derivatives from a cordyceps-colonizing isolate of gliocladium sp preliminary study of cordyceps barnesii -comparison of the chemical constituents of cordyceps barnesii and cordyceps sinensis comment on comparison of protective effects between cultured cordyceps militaris and natural cordyceps sinensis against oxidative damage evaluation of the anti-inflammation screening model of macrophages cell line by the proinflammatory mediators secretions medium optimization for polysaccharide production of cordyceps sinensis in vivo and in vitro stimulatory effects of cordyceps sinensis on testosterone production in mouse leydig cells regulatory mechanism of cordyceps sinensis mycelium on mouse leydig cell steroidogenesis effects of extracted cordyceps sinensis on steroidogenesis in ma- mouse leydig tumor cells upregulation of steroidogenic enzymes and ovarian b-estradiol in human granulosa-lutein cells by cordyceps sinensis mycelium effects of cordyceps sinensis on testosterone production in normal mouse leydig cells cordyceps sinensis and its fractions stimulate ma- mouse leydig tumor cell steroidogenesis in vivo stimulatory effect of cordyceps sinensis mycelium and its fractions on reproductive functions in male mouse comparison of protective effects between cultured cordyceps militaris and natural cordyceps sinensis against oxidative damage co-n reaction-a new serological activity index-on wegener's granulomatosis world university rankings. the times higher education supplement a xanthocillin-like alkaloid from the insect pathogenic fungus cordyceps brunnearubra bcc bioxanthracenes from the insect pathogenic fungus cordyceps pseudomilitaris bcc . ii. structure elucidation es- derivatives and cycloheptapeptides from cordycepssp. strains bcc and bcc structures of cordypyridones a-d, antimalarial n-hydroxy-and nmethoxy- -pyridones from the insect pathogenic fungus cordyceps nipponica bioxanthracenes from the insect pathogenic fungus cordyceps pseudomilitaris bcc . i. taxonomy, fermentation, isolation and antimalarial activity the immuno-enhancing effect of chinese herbal medicine cordyceps sinensis on macrophage j mycelial extract of cordyceps ophioglossoides prevents neuronal cell death and ameliorates beta-amyloid peptide-induced memory deficits in rats a mushroom lectin from ascomycete cordyceps militaris effect of medicinal plant extracts on forced swimming capacity in mice immunomodulatory activities of herbsnsenses tm cordyceps -in vitro and in vivo studies identification of cordycepin, a metabolite of cordyceps militaris, as -deoxyadenosine the ikk nf-kappa b system: a treasure trove for drug development occurrence of gal beta ( - ) galnac-ser/thr in the linkage region of polygalactosamine containing fungal glycoprotein from cordyceps ophioglossoides polysaccharides in fungi. xxxii. hypoglycemic activity and chemical properties of a polysaccharide from the cultural mycelium of cordyceps sinensis polysaccharides in fungi. xxv. biological activities of two galactomannans from the insect-body portion of chan hua (fungus: cordyceps cicadae) structural features and hypoglycemic activity of a polysaccharide (cs-f ) from the cultured mycelium of cordyceps sinensis polysaccharides in fungi. xxxvi. hypoglycemic activity of polysaccharide (cs-f ) from the cultural mycelium of cordyceps sinensis and its effect on glucose metabolism in mouse liver a comparative study on the production of exopolysaccharides between two entomopathogenic fungi cordyceps militaris and cordyceps sinensis in submerged mycelial cultures larvicidal activity against plutella xylostella of cordycepin from the fruiting body of cordyceps militaris methanol extract of cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing nf-jb activation optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by cordyceps militaris c production and characterization of exopolysaccharides from an enthomopathogenic fungus cordyceps militaris ng ophiocordin, an antifungal antibiotic of cordyceps ophioglossoides hypocholesterolemic effect of hot-water extract from mycelia of cordyceps sinensis antifatigue and antistress effect of the hot-water fraction from mycelia of cordyceps sinensis activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of cordyceps sinensis reinventing taxonomy: a curmudgeon's view of years of fungal taxonomy, the crises in biodiversity, and the pitfalls of the phylogenetic age cicadapeptins i and ii: new aib-containing peptides from the entomopathogenic fungus cordyceps heteropoda homocitrullylaminoadenosine, a nucleoside isolated from cordyceps militaris inhibition of nucleic acid methylation by cordycepin. in vivo synthesis of s- -deoxyadenosylmethionine by wil human lymphoblasts abrogation of streptococcal pyrogenic exotoxin b-mediated suppression of phagocytosis in u cells by cordyceps sinensis mycelium via production of cytokines cordyceps sinensis mycelium protects mice from group a streptococcal infection differentiation of cordyceps sinensis by a pcr-single-stranded conformation polymorphism-based method and characterization of the fermented products in taiwan growth inhibitors against tumor cells in cordyceps sinensis other than corydcepin and polysaccharides cordyceps sinensis as an immunomodulatory agent regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from cordyceps sinensis activation and proliferation signals in primary human t lymphocytes inhibited by ergosterol peroxide isolated from cordyceps cicadae medicinal and nutraceutical genetic resources of mushrooms a yanginvigorating chinese herbal formula enhances mitochondrial functional ability and antioxidant capacity in various tissues of male and female rats experimental study on effect of cordyceps sinensis in ameliorating aminoglycoside induced nephrotoxicity determination of nucleosides in natural cordyceps sinensis and cultured cordyceps mycelia by capillary electrophoresis anti-oxidation activity of different types of natural cordyceps sinensis and cultured cordyceps mycelia the contents and their change of nucleosides from natural cordyceps sinensis and cultured cordyceps mycelia simultaneous determination of ergosterol, nucleosides and their bases from natural and cultured cordyceps by pressurised liquid extraction and high-performance liquid chromatography the fruiting body and its caterpillar host of cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity quality control of cordyceps sinensis, a valued traditional chinese medicine hypoglycemic activity of polysaccharide, with antioxidation, isolated from cultured cordyceps mycelia a polysaccharide isolated from cordyceps sinensis, a traditional chinese medicine, protects pc cells against hydrogen peroxide-induced injury rp-hplc determination of adenosine in fermented cordyceps effect of cordyceps sinensis on erythropoiesis in mouse bone marrow determination of ergosterol in cordyceps sinensis and cordyceps black-bone chicken capsules by hplc antitumor activity of cordyceps militaris on human cancer cell line inhibition of activated human mesangial cell proliferation by the natural product of cordyceps sinensis (h - ): an implication for treatment of iga mesangial nephropathy inhibitory effect of cordyceps on carcinogenesis of the forestomach in mice oral administration of cordyceps does not affect lymphocyte subsets in stz-induced diabetic rats effects of fermented cordyceps powder on pulmonary function in sensitized guinea pigs and airway inflammation in sensitized rats effects of cordyceps sinensis (cs) on in vitro natural killer cells immuno-pharmacologic activity of cordyceps sinensis (berk) sacc anticarcinogenic effect and hormonal effect of cordyceps militaris link chemical constituents of cordyceps militaris (l.) link effect of fuzheng huayu recipe in treating posthepatitic cirrhosis influence of cordyceps sinensis (berk.) sacc. and rat serum containing same medicine on il- , ifn and tnf produced by rat kupffer cells protection against radiation-induced bone marrow and intestinal injuries by cordyceps sinensis, a chinese herbal medicine inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism anti-oxidation of paecilomyces sinensis (s. pnov.) intragastrically administered chinese herbal medicine cordyceps alleviates fasting hyperglycemia in diabetic rats anti-hyperglycemic activity of natural and fermented cordyceps sinensis in rats with diabetes induced by nicotinamide and streptozotocin the anti-hyperglycemic activity of the fruiting body of cordyceps in diabetic rats induced by nicotinamide and streptozotocin study on effect of cordyceps sinensis and artemisinin in preventing recurrence of lupus nephritis effects of the mycelial extract of cultured cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice effects of the mycelial extract of cultured cordyceps sinensis on in vivo hepatic energy metabolism in the mouse hyperproduction of cordycepin by twostage dissolved oxygen control in submerged cultivation of medicinal mushroom cordyceps militaris in bioreactors antiarrhythmic effects of cordyceps sinensis (berk.) sacc medicinal mushrooms: a selective overview combined effects of cordyceps sinensis and methotrexate on hematogenic lung metastasis in mice inhibitory effect of cordyceps sinensis on spontaneous liver metastasis of lewis lung carcinoma and b melanoma cells in syngeneic mice activation of in vivo kupffer cell function by oral administration of cordyceps sinensis in rats antifibrotic effect of extracellular biopolymer from submerged mycelial cultures of cordyceps militaris on liver fibrosis induced by bile duct ligation and scission in rats pharmacological actions of cordyceps, a prized folk medicine the nephroprotective effects of the herbal medicine preparation, wh +, on the chemicalinduced acute and chronic renal failure in rats isolation of galactosaminoglycan moiety (co-n) from protein-bound polysaccharide of cordyceps ophioglossoides and its effects against murine tumors the correlation between molecular weight and antitumor activity of galactosaminoglycan (co-n) from cordyceps ophioglossoides antitumor activity of protein-bound polysaccharide from cordyceps ophioglossoides in mice problems in the use of herbal and natural substances, with a specific example concerning the cardiovascular system growth inhibition of u leukemia cells by aqueous extract of cordyceps militaris through induction of apoptosis optimization of submerged culture conditions for the mycelial growth and exobiopolymer production by cordyceps militaris effect of agitation intensity on the exobiopolymer production and mycelial morphology in cordyceps militaris the coronamycin producer: a case of mistaken identity? ganoderma -a therapeutic fungal biofactory ganoderma disease of oil palm -a white rot perspective necessary for integrated control internal amplification controls have not been employed in diagnostic fungal pcr hence potential false negative results fungal enzyme inhibitors as pharmaceuticals, toxins, and scourge of pcr biochemical techniques for filamentous fungi solutions to penicillium taxonomy crucial to mycotoxin research and health inhibitory effect of cordyceps on carcinogenesis of the forestomach in mice cordyceps sinensis mycelium extract induces human premyelocytic leukemia cell apoptosis through mitochondrion pathway a cyclopeptide from the insect pathogenic fungus cordycepssp. bcc -membered macrolides from the insect pathogenic fungus cordyceps militaris bcc optimum temperature and ph for mycelial growth of cordyceps nutans pat. (ascomycetes) studies on anti-tumor activity of crude drugs. i. the effects of aqueous extracts of some crude drugs in shortterm screening test militarinone a, a neurotrophic pyridone alkaloid from paecilomyces militaris (+)-n-deoxymilitarinone a, a neuritogenic pyridone alkaloid from the insect pathogenic fungus paecilomyces farinosus down-regulation of apoptotic and inflammatory genes by cordyceps sinensis extract in rat kidney following ischemia/reperfusion treatment of hyperlipidemia with cultivated cordyceps -a double-blind, randomized placebo control trial trade of cordyceps sinensis from high altitudes of the indian himalaya: conservation and biotechnological priorities effect of cordyceps militaris on the damage of rats induced by n-hexane profiles of nucleosides and nitrogen bases in chinese medicinal fungus cordyceps sinensis and related species pharmacological activities of paecilomyces japonica, a new type cordyceps sp anti-tumour and immuno-stimulating activities of the fruiting bodies of paecilomyces japonica, a new type of cordyceps spp antioxidant and immunostimulating activities of the fruiting bodies of paecilomyces japonica, a new type of cordyceps sp pharmacological basis of 'yin-nourishing' and 'yang-invigorating' actions of cordyceps, a chinese tonifying herb chemotaxonomy of pochonia and other conidial fungi with verticillium like anamorphs clinical study on application of bailing capsule after renal transplantation cordyceps sinensis and cultured mycelia phylogenetic classification of cordyceps and the clavicipitaceous fungi effects of cordyceps sinensis, rhubarb and serum renotropin on tubular epithelial cell growth dmso modifies structural and functional properties of rpmi- cells by promoting programmed cell death rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radicalscavenging ability of aqueous extracts of food constituents free radical scavenging and apoptotic effects of cordyceps sinensis fractionated by supercritical carbon dioxide effects of a watersoluble extract of cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells pharmacological functions of chinese medicinal fungus cordyceps sinensis and related species an experimental study on anti-aging action of cordyceps extract entomogenous fungi that produce , -pyridine dicarboxylic acid (dipicolinic acid) immunomodulatory functions of extracts from the chinese medicinal fungus cordyceps cicadae anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of cordyceps militaris lead poisoning caused by contaminated cordyceps, a chinese herbal medicine: two case reports structural analysis of a neutral ( ? ),( ? )-b-d-glucan from the mycelia of cordyceps sinensis medicinal herbal extracts -renal friend or foe? part two: herbal extracts with potential renal benefits optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by cordyceps jiangxiensis jxpj studies on chemical constituents of cordyceps sinensis i application of statistically based experimental designs for the optimization of exopolysaccharide production by cordyceps militaris ng effects of cordyceps sinensis on natural killer activity and colony formation of b melanoma structure and antitumor activity of an alkalisoluble polysaccharide from cordyceps ophioglossoides augmentation of various immune reactivities of tumor-bearing hosts with an extract of cordyceps sinensis inhibitory effects of water extracts from fruiting bodies of cultured cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice antioxidant activity of the extracts from fruiting bodies of cultured cordyceps sinensis cordyceps sinensis mycelium induces ma- mouse leydig tumor cell apoptosis by activating the caspase- pathway and suppressing the nf-jb pathway effects of exopolysaccharide fraction (epsf) from a cultivated cordyceps sinensis fungus on c-myc, c-fos, and vegf expression in b melanoma-bearing mice efficacy of a pure compound h -a extracted from cordyceps sinensis on autoimmune disease of mrl lpr/lpr mice h -a extracted from cordyceps sinensis suppresses the proliferation of human mesangial cells and promotes apoptosis, probably by inhibiting the tyrosine phosphorylation of bcl- and bcl-xl effects of cordyceps militaris extract on angiogenesis and tumor growth antitumor activity of an extract of cordyceps sinensis (berk.) sacc. against murine tumor cell lines antitumour activity of cordycepin in mice hypoglycemic effect of cordyceps militaris pharmacological activities of stromata of cordyceps scarabaecola isolation and biological properties of polysaccharide cps- from cultured cordyceps militaris isolation, purification and identification of polysaccharides from cultured cordyceps militaris structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured cordyceps militaris. carbohydr macrophage biospecific extraction and high performance liquid chromatography for hypothesis of immunological active components in cordyceps sinensis simultaneous determination of free ergosterol and ergosteryl esters in cordyceps sinensis by hplc anti-diabetic effects of ccca, cmess, and cordycepin from cordyceps militaris and the immune responses in streptozotocin-induced diabetic mice prosecutable function of cordyceps sinensis extracts for hepatic mitochondrial oxidative injuries in diabetic mice induction of hl- apoptosis by ethyl acetate extract of cordyceps sinensis fungal mycelium activation of murine peritoneal macrophages by natural cordyceps sinensis and its cultured mycelia lewis lung cancer of mice treated with cordyceps sinensis and its artificial cultured mycelia effects of the exopolysaccharide fraction (epsf) from a cultivated cordyceps sinensis on immunocytes of h tumor bearing mice immunomodulatory and antitumour effects of an exopolysaccharide fraction from cultivated cordyceps sinensis (chinese caterpillar fungus) on tumourbearing mice dynamical influence of cordyceps sinensis on the activity of hepatic insulinase of experimental liver cirrhosis influence of cordyceps sinensis on pancreatic islet beta cells in rats with experimental liver fibrogenesis epicoccins a-d, pipolythiodioxopiperazines from a cordyceps-colonizing isolate of epicoccum nigrum clinical and experimental studies on elimination of oxygen free radical of jinshuibao capsule in treating senile deficiency syndrome and its deoxyribonucleic acid damage repairing effects cordyceps sinensis -i as an immunosuppressant in heterotopic heart allograft model in rats cordymax cs- improves glucose metabolism and increases insulin sensitivity in normal rats inhibitory effect of cordyceps sinensis and cordyceps militaris on human glomerular mesangial cell proliferation induced by native ldl investigation on distribution and habitat of blaps rynchopetera fairmaire (coleoptera: tenebrionidae) in yunnan cordyceps sinensis in protection of the kidney from cyclosporine a nephrotoxicity inhibitory effects of alcoholic extract of cordyceps sinensis on abdominal aortic thrombus formation in rabbits mechanisms and therapeutic effect of cordyceps sinensis (cs) on aminoglycoside induced acute renal failure (arf) in rats effect of jinshuibao capsule on the immunological function of patients with advanced cancer short-term curative effect of cultured cordyceps sinensis mycelia in chronic hepatitis b modulating effects of extractum semen persicae and cultivated cordyceps hyphae on immuno-dysfunction of inpatients with posthepatitic cirrhosis the scientific rediscovery of a precious ancient chinese herbal regimen: cordyceps sinensis part ii the scientific rediscovery of an ancient chinese herbal medicine: cordyceps sinensis part i immunosuppressive effect of cultured cordyceps sinensis on cellular immune response acknowledgements r.r.m. paterson is funded by grant sfrh/bpd/ / from fundac ßão para a ciência e a tecnologia, portugal. professor nelson lima, universidade do minho is gratefully appreciated for his unstinting support, as are other colleagues therein. l.e. gilbert, university of texas, austin was generous for allowing the use of his images of infected insects (fig. c-f ). marc stadler, university of bayreuth, germany is thanked for discussions. key: cord- -elbnzgx authors: mutua, victoria; gershwin, laurel j. title: a review of neutrophil extracellular traps (nets) in disease: potential anti-nets therapeutics date: - - journal: clin rev allergy immunol doi: . /s - - - sha: doc_id: cord_uid: elbnzgx activated neutrophils release neutrophil extracellular traps (nets) in response to a variety of stimuli. netosis is driven by protein-arginine deiminase type , with the release of intracellular granule components that function by capturing and destroying microbes, including viral, fungal, bacterial, and protozoal pathogens. the positive effects of pathogen control are countered by pro-inflammatory effects as demonstrated in a variety of diseases. components of nets are non-specific, and other than controlling microbes, they cause injury to surrounding tissue by themselves or by increasing the pro-inflammatory response. nets can play a role in enhancement of the inflammation seen in autoimmune diseases including psoriasis, rheumatoid arthritis, and systemic lupus erythematosis. in addition, autoinflammatory diseases such as gout have been associated with netosis. inhibition of nets may decrease the severity of many diseases improving survival. herein, we describe netosis in different diseases focusing on the detrimental effect of nets and outline possible therapeutics that can be used to mitigate netosis. there is a need for more studies and clinical trials on these and other compounds that could prevent or destroy nets, thereby decreasing damage to patients. neutrophil extracellular traps (nets) were discovered in [ ] and further detailed by brinkmann et al. who termed the process netosis [ ] [ ] [ ] . neutrophils are short-lived granulocytes that are the initial defense against invading pathogens. they achieve this through phagocytosis, degranulation, production of reactive oxygen species (ros), and production of chemokines and cytokines to recruit other immune cells maximizing the host's immune response [ ] [ ] [ ] . neutrophils enhance their antimicrobial properties by releasing nets, composed of extracellular chromatin decorated with histones and numerous granular proteins [ , ] and were identified as part of innate immune response which can either be beneficial or pathological [ , , ] . net formation starts with the activation of neutrophils through the recognition of stimuli and activation of nadph oxidase (nox) complex through protein kinase c (pkc)-raf/merk/erk [ ] [ ] [ ] which in turn activate myeloperoxidase (mpo), neutrophil elastase (ne), and protein-arginine deiminase type (pad ) [ , ] . pad catalyzes citrullination of histones and promotes chromatin decondensation [ ] [ ] [ ] , while the ros species promote netosis by inducing gradual separation and loss of the nuclear membrane with the release of chromatin outside the cell through membrane pores. cellular lysis with a final release of dna, citrullinated histones (cith ), and other intracellular granules form the extracellular traps [ ] . netosis is induced in response to stimuli promoting pathogen clearance by trapping, and either killing through microbial toxicity or immobilizing microbes facilitating phagocytosis by other neutrophils and phagocytes [ , , , ] . due to the non-specific effects of the released enzymatic proteins, nets may lead to uncontrolled inflammatory response causing tissue pathology. there is direct cell damage, recruitment of other pro-inflammatory cells and proteins, and formation of immune complexes that induce autoantibody production leading to tissue damage [ , ] . nets can capture metastatic tumors aggravating cancerous condition [ ] , and in diabetic cases, they lead to a delay in wound healing [ , ] . neutrophil can also form interactions with platelets mediated by p-selectin [ ] . this leads to induction of platelet-derived high-mobility group protein b (hmgb ) [ ] which stimulates nets [ , ] causing occlusion in the vasculature by promoting thrombosis and obstruction causing organ damage. although nets have been shown to promote inflammation, a study done by christine et al. shows that an accumulation of net aggregates can reduce inflammation in a mouse model of gout through the degeneration of cytokines and chemokines [ ] . this means that there is still much about nets we are not aware of, and thus the need for more studies to understand their specific mechanism and how to harness their benefits while limiting their negative effects. there are certain compounds that have been identified in some studies to either inhibit or disrupt netosis, but there is no available therapeutic that has been researched extensively or approved for human use. we propose that the limited therapeutics have been due to the different effects of nets in different disease conditions; thus, identifying a stand-alone compound might be a challenge. in this review, we briefly mention netosis in different diseases and try to reconcile different aspects of net biology highlighting possible compounds that can be considered therapeutic. new approaches in therapeutic design and efficacy testing will have to be developed to find a truly efficacious treatment. here is a brief discussion on the different researched effects of nets. nets have been shown to have positive effects in controlling bacterial infections. they possess antimicrobial properties with components including histones, cathepsin g, ne, mpo, lactoferrin, antimicrobial peptide-ll , pentraxin , gelatinase, proteinase , and peptidoglycan-binding proteins that are bactericidal [ , , ] . nets limit growth or kill bacterial as reviewed by vidal delgado-rizo et al. which include shigella flexneri, pseudomonas aeruginosa, escherichia coli, shigella sonnei, salmonella enteritidis, salmonella typhimurium, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus albus, staphylococcus aureus, and propionibacterium [ , ] . in viral infections including influenza, hiv, and respiratory syncytial virus, there is an excessive neutrophil recruitment [ , ] . these viruses stimulate netosis through tlr , and/or with the release of ros species and the nets trap, contain, and eliminate viruses [ ] [ ] [ ] or inhibit viral replication through the blockade of the pkc pathway. histones are also important for viral aggregation and neutralization leading to a significant decrease in viral replication [ , ] . fungi like aspergillus nidulans, candida albicans, aspergillus fumigatus, and cryptococcus spp. induce netosis through the recognition of β-glucan on hyphae by components of the extracellular matrix or activation of nox [ , , ] . nets have been shown to be important in trapping and clearing large pathogens in vivo, thus being critical for antifungal defense [ , , ] . in parasitic conditions including plasmodium falciparum and toxoplasma gondii, there is activation of platelets, monocytes, and neutrophils. net formation, which is dependent on mek-erk pathway, limits the dissemination of the parasites by trapping and killing them [ , ] . histones reduce the replication of the leishmania spp. [ ] and together with other nets-associated compounds, such as ne, mpo, and collagenase, were shown to kill these pathogens [ ] [ ] [ ] [ ] . most studies on nets have been done in mice and in vitro, but there is still a gap in knowledge on the exact mechanism of nets in vivo. this necessitates the need for more studies to clearly evaluate their effects in in vivo and in humans. the ability to detect nets may be used as a prognostic tool for patients with conditions presenting with a higher rate of net formation, facilitating clinicians to provide personalized treatment. for nets to be used as screening tools, there has to be studies to standardize and define normal from abnormal levels. this could involve measurement of net-associated products in the blood cfdna, cith , ne, and mpo. in colorectal and breast cancer patients, cfdna has been quantified in serum samples via a simple nucleic acid-staining assay [ ] [ ] [ ] [ ] . this can be used to classify the cancer; however, measuring circulating mpo/cfdna conjugates and cith may be more specific for net analysis than evaluation of cfdna alone [ ] . cith is highly specific to netosis making it a possible tool for understanding variances between net levels [ ] . thalin observed that high plasma content of cith was a significant indicator of short-term mortality in some cancer patients [ ] , and some observational studies inform on the significance of nets in progression of colorectal cancer [ ] . further human studies are needed to definitively quantify different levels of nets and associate them with poor cancer/disease outcomes. large amounts of circulating nets demonstrated in septic patients are associated with poor outcome and multiple organ failure [ , , ] . this could be due to increased netosis, apoptosis, and necrosis or decreased clearance of extruded products with studies suggesting that cfdna exacerbate inflammation by inducing tnf-α mrna [ , ] . histones also function as damage-associated molecular patterns and can induce organ damage by promoting pro-inflammatory cytokine release causing endothelial dysfunction by inducing cytotoxicity and increasing ros production [ , , ]. nets have been indicated in pathologic alterations in autoimmune and autoinflammatory diseases [ , ] . here, we discuss in brief a few of these diseases. psoriasis is a chronic immune-mediated disease characterized by demarcated erythematous plaques on the skin. some patients may also suffer from psoriatic arthritis with joint pains and deformities [ ] [ ] [ ] [ ] . studies show that neutrophils are recruited to psoriasis lesions where they cluster to form spongiform pustules and munro's microabscesses and produce pro-inflammatory cytokines including il- , il- , and il- s [ , ] . il- in keratinocytes increases the expression of ll , a cathelicidin-derived antimicrobial peptide, and defensins which mediate net formation in dermatological conditions [ , ] . these inflammatory compounds have been shown to promote netosis and pathology in the absence of infection [ ] in these patients. systemic lupus erythematosus (sle) is an autoimmune disease characterized by immune complexes and high levels of ifn-α with the activation of autoreactive b cells [ , ] . there is a possible production of autoantibodies against nucleic acids released by neutrophils undergoing netosis [ , ] with the generated immune complexes representing a source of self-antigens that enhance the autoimmune and inflammatory process. this in turn results in more injury and inflammation [ , ] . rheumatoid arthritis (ra) is a systemic autoimmune disease characterized by persistent synovial inflammation that leads to cartilage and bone injury in the joints [ ] . the synovial fluid at the synovial cavity of ra patients becomes infiltrated with neutrophils that readily form nets [ , ] . studies have demonstrated that circulating neutrophils of ra patients are more easily stimulated to netosis than those from healthy subjects [ , ] , and as in other autoimmune conditions, nets act as a source of extracellular autoantigens leading to excessive innate and adaptive immune responses in the joints and subsequent tissue injury [ , ] . type diabetes mellitus (t dm) is an autoimmune disease characterized by the destruction of β pancreatic cells leading to hyperglycemia [ ] . this causes production of autoantigens that are recognized by immune cells with production of autoantibodies [ , ] . t dm patients are at a risk of developing neutropenia, and neutrophils can be found within infiltrates in pancreatic islets where elevated tnf-α induces formation of nets [ ] . cytokines produced in this process lead to neutrophil recruitment to sites of inflammation, providing negative feedback and contributing to pathogenesis in autoimmune diabetes [ , ] . small vessel vasculitis (svv) is a systemic disease of unknown etiology where the patients exhibit blood vessel inflammation, with necrotizing inflammation in small blood vessels potentially leading to organ damage [ ] [ ] [ ] . these patients have been shown to have anti-neutrophil cytoplasmic antibodies (ancas) [ , ] . proteins released during netosis are the main cause of anca production by activating the complement system resulting in endothelial damage [ , ] . these studies have shown that α-pr and α-mpo ancas induce netosis during active disease perpetuating a feedback loop [ ] . gout is an autoinflammatory disease characterized by the deposition of monosodium urate (msu) crystals in the joints, stimulating immune responses by attracting leukocytes and inducing nets that promote inflammation [ , , [ ] [ ] [ ] . inflammatory bowel diseases (ibds) are disease affecting the gastrointestinal tract characterized by chronic uncontrolled inflammation. the two major forms of ibd include ulcerative colitis (uc) and crohn's disease (cd), which have different etiologies, pathogenesis, and diagnostic features with the differences not fully understood. cd clinically manifests as gastrointestinal disorders but is a systemic disease involving inflammation of the ileum and colon [ ] [ ] [ ] . net formation in cd has not been well-studied although studies indicate that ros production is enhanced, which could promote netosis [ , [ ] [ ] [ ] . uc is also characterized by inflammation of the gastrointestinal tract mostly restricted to the colon with nets observed in the colon accompanied by exacerbated inflammation [ ] . there are a few studies looking into the mechanism of nets in these conditions, but there are more studies needed to better inform on development of effective treatment options. metabolic diseases have been associated with chronic lowgrade inflammation with activation of the innate immune response and recruitment of mononuclear and polymorphonuclear leukocytes increasing cellular dysfunction [ , ] . this microenvironment favors netosis linking it to immune deregulation and hyperglycemia, oxidative stress, inflammation, and further complications of metabolic diseases. type diabetes is a chronic metabolic condition characterized by glucose level build-up in the bloodstream, hyperglycemia, and cells unresponsive to insulin. studies have shown that hyperglycemia predisposes neutrophils to release nets. net-related bioproducts (ne, mpo, and cfdna) are increased compared with non-diabetic subjects and also positively correlate with increased glycated hemoglobin (hba c) levels [ , ] . this suggests that the chronic proinflammatory conditions present during hyperglycemia promote netosis in both the type and type diabetes [ , ] ; thus, net formation is enhanced in hyperglycemic conditions independent of diabetes type and origin. obesity is a metabolic condition characterized by an excess of adipose tissue deposition, as a result of energy imbalance due to increased energy intake versus expenditure. obesity is frequently associated with other chronic complications including cardiovascular disease and diabetes [ , ] . studies have shown an association between obesity and chronic inflammation with enhanced neutrophil activity, increased superoxide radicals, and net formation [ , ] . moorthy et al. show that the neutrophils of mice fed with high-fat diet are more prone to spontaneous net formation, compared with neutrophils derived from mice fed with low-fat diet [ ] . the same is seen in mice fed with high-fat diet and infected with inlfuenza compared with the mice fed with low-fat diet [ ] . there is an increase in obesity globally, necessitating more studies into this condition and the link to the other metabolic conditions. this will inform on management of conditions caused or exacerbated by obesity and how nets play a role in this. although nets can be beneficial, the detrimental effect of nets can cause excessive tissue damage and pathology. there are studies evaluating the possible effects of certain compounds against nets as illustrated in table , and more studies need to be considered to mitigate the negative effects of netosis. acetylsalicylic acid (aspirin) is a non-steroidal drug with an antithrombotic and an anti-inflammatory effect used in the management of inflammatory symptoms. it functions through the irreversible acetylation of cyclooxygenase enzyme (cox), and suppresses prostaglandin generation [ , ] . it is used as an antiplatelet agent for prevention of arterial thromboses as it inhibits thromboxane a [ , ] . thromboxane a is a vasoconstrictor that activates new platelets increasing platelet aggregation, an important function during tissue injury, inflammation, and healing( [ ] ). platelets are the primary effector cells of hemostasis [ ] , but recent evidence indicates that they play a direct role in innate immunity by interacting with pathogens or recognize pathogen-associated molecular patterns (pamps) [ , ] . they facilitate innate immunity and activate netosis via platelet-neutrophil interaction [ , ] . platelet activation through tlr and tlr leads to the expression of pselectin which binds to neutrophil receptor (psgl- ) inducing netosis as demonstrated in mice [ , , ] . netosis is also mediated by the binding of αmβ (mac- ) on neutrophils to glycoprotein bα (gp bα) on platelets generating nets in liver and lungs during endotoxemia and in septic conditions [ , ] . in addition to lps, platelets can be activated by thrombin and arachidonic acid to form nets [ , ] . upon activation, platelets secrete soluble mediators including high-mobility group box- (hmbg- ) [ ], platelet factor (pf ), and ccl (rantes) that induce netosis via the neutrophil g protein coupled receptors [ ] . the interactions between platelets and neutrophils mediate netosis, and inhibition of this interaction using antiplatelet therapy has the potential to inhibit net formation [ , ] . in a study conducted on endotoxin-triggered acute lung injury, pretreatment of mice with aspirin showed a decreased intravascular net formation and reduced degree of lung injury [ , ] . in another study on effects of nets on transplantation in mice, they discovered that platelet activation was also inhibited by aspirin [ ] . lapponi et al. conducted another study where they treated neutrophils with a steroidal immunomodulatory drug (dexamethasone) or aspirin, and discovered that dexamethasone had no effect, while aspirin prevented net formation [ ] . they demonstrated that aspirin functions by inhibiting nf-κb, an inflammatory transcriptional regulator, that promotes netosis. these results show that aspirin could be a useful therapy in the management of pathologic netosis induced by platelets, but we have to keep in mind the side effects of aspirin. aspirin is a blood thinner and predisposes patients to stomach ulcers, so more studies are needed to find out which conditions could benefit from treatment with aspirin, without the excessive side effects. cyclosporine a is an immunosuppressant drug widely used in post-allogeneic organ transplant to reduce the activity of the patient's immune system, and therefore the risk of organ rejection [ , ] . it causes reversible inhibition of immunocompetent lymphocytes and has been used to manage fungal infections, rheumatoid arthritis, asthma, dermatologic drug, and immunosuppressive agent [ , ] . the mechanism of action of cyclosporine a involves binding to cytophilin, resulting in the downregulation of nfat (nuclear factor of activated t cells) transcription factor and inhibiting the calcineurin pathway subsequently inhibiting net formation [ , ] . gupta [ ] [ ] [ ] [ ] [ ] [ ] chloroquine/hydroxychloroquine mmps, timps antimalarial, inhibits cytokine production, interferes with the stimulatory effect of platelet aggregation, maintaining extracellular matrix homeostasis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diphenyleneiodonium chloride (dpi) nadph inhibits nadph oxidase ros production [ ] [ ] [ ] [ ] n-acetylcysteine (nac) inhibits ros production, prevents thrombus formation [ ] [ ] [ ] [ ] [ ] [ ] nucleases recombinant human dnase dna matrixes reduces neutrophil infiltration, cleaves dna matrixes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] staphylokinase plasminogen, alpha-defensins converting nets to deoxyadenosine mediating death of immune cells [ ] [ ] [ ] [ ] notable compounds probiotics pkc pathway dampening ros production [ ] [ ] [ ] [ ] vitamin d reduces endothelial damage, decreases cell apoptosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] pge prostagladin e , nfat nuclear factor of activated t cells, pad protein-arginine deiminase , ros reactive oxygen species, pkc protein kinase c, mmps matrix metallopeptidase, timps tissue inhibitors of metallopeptidase, mtorc mechanistic target of rapamycin complex , ampk- ′ amp-activated protein kinase. properties), and rapamycin (a cell anti-proliferative and immunosuppressive agent), on both extra and intracellular calcium pools and their modulation in netosis. their data indicated that a combination of ascomycin and cyclosporine a reduced netosis, but the same effect was not evident following treatment with rapamycin [ ] . this opens up the possibility to therapeutically suppress or modulate netosis using cyclosporine a or a combination therapy with ascomycin. there were no other studies we could find in this area, and this could be due to the fact that cyclosporine a and immunosuppressive drugs may impair normal host immune responses to microbes [ , ] , predisposing patients to frequent infections. therefore, there is a need for more studies into how these drugs could be formulated to manage netosis safely. chlor-amidine (cl-amidine) is a compound designed to irreversibly inhibit protein-arginine deiminase (pad) through covalent modification at the active site of the enzymes [ ] . as described above, pad is an enzyme involved in netosis; thus, inhibition of pad is a possible therapeutic target. avin et al. run a study to evaluate the effect of inhibition of pad in netosis using an antagomir- , a pleiotropic microrna important in the regulation of immune responses, demonstrating a decreased induction of pad mrna and subsequent reduced nets in response to pma challenge [ ] . in a mouse model of lupus, systemic treatment with the pad inhibitor (bb-cl-amidine) showed protection of the mice from developing net-mediated vascular damage, endothelial dysfunction, and kidney injury. the study indicated that pad inhibition markedly downregulates the expression of type i interferon-regulated genes and reduces proteinuria and immune complex deposition in the kidneys, while also protecting against skin disease [ ] . another study found that pad -deficient mice (both diabetic and nondiabetic) possess faster wound healing and re-epithelization processes than their wild-type counterparts. this effect was independent of wound infection suggesting that netosis could hinder wound healing by limiting keratinocyte migration and re-epithelization [ ] . it is therefore possible to target inhibition of pad to inhibit net formation; however, it is important to note that pad may have other important functions in immunity which may be impaired [ , ] . in one study, they reported mixed results of pharmacological pad inhibition using cl-amidine in human neutrophils, where netosis induced by smoking was blocked by inhibiting of pad , but netosis induced by cholesterol crystals was not blocked [ ] . with these results, developing a suitable targeted therapy for pad may be challenging thus the need for more carefully considered human studies on the function of pad before using its inhibition as a strategy for management of netosis. pge -prostaglandins (pgs) are members of the eicosanoid family synthesized from arachidonic acid via cox enzymes and produced by nearly all cells within the body. pge is the most abundant prostaglandin in the human body and has been shown to influence both inflammatory and in some cases anti-inflammatory effects [ , ] . shishikura et al. evaluated the effects of pge , agonists and antagonists of its receptors, and modulators of the camp-pka pathway on the formation of nets in vitro (in isolated neutrophils) and in vivo in a mouse model. they also discovered that pge inhibited pma-induced net formation in vitro through ep and ep . exogenous pge treatment limited netosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by camp-and protein kinase a-dependent manner demonstrating a physiologic inhibition of netosis. incubation with a cell-permeable camp analogue, dibutyryl camp, or various inhibitors of a campdegrading enzyme, rolipram (pde inhibitor), and butaprost, (ep receptor agonist) also suppressed net formation [ ] . interestingly, domingo et al. conducted a study in murine bone marrow transplant mice (bmt) where neutrophils overexpress cox- and overproduce pge , leading to defective intracellular bacterial killing. they wanted to determine whether netosis was defective after transplant and whether this was regulated by pge signaling. treatment of bmt neutrophils with rapamycin resulted in reduced net formation relative to control cells while the ep receptor antagonist (pf- ) or the ep antagonist (ae - ), gαscoupled receptors, restored net formation suggesting that blocking pge -ep or ep signaling pathway restores netosis [ ] . these findings will contribute to the development of novel treatments for netosis-related diseases although more studies need to be done to evaluate the effect of using pge as a therapeutic. although pge is beneficial in management of sle and other ifn-α-dependent, th driven diseases [ ] , it could pose a challenge in conditions like arthritis [ , ] associated with pain. pge is known to contribute to pain as part of the inflammatory response, thus the need for more studies to evaluate its effects in different diseases compared with its benefits. pa-dpeg is a peptide inhibitor of complement c (pic ) which mitigates peroxidase activity of mpo, hemoglobin, and myoglobin through a reversible process [ ] . defective complement action caused by dysregulation and acute and chronic tissue damage or transplants can lead to host cell attack contributing to inflammatory conditions [ , ] . this is more so in the kidney which has been shown to be particularly sensitive to complement-mediated damage [ , ] . it is known that complement effectors including c a and membrane attack complex (sc b- ) interact with and can stimulate human neutrophils to generate nets. subsequently, products of netosis can activate complements causing a destructive loop [ ] . therapeutic complement inhibition is successfully used in paroxysmal nocturnal hemoglobinuria showing a promise in its use in other clinical conditions [ , ] . an article by hair et al. demonstrated that pic showed dose-dependent antioxidant activity, acting via the single electron transport (set) and hydrogen atom transfer (hat) mechanisms interfering with oxidation of cysteine residues. they showed that pa-dpeg achieved complete inhibition with complement effector levels equivalent to background. pa-dpeg was also able to dose-dependently inhibit net formation by human neutrophils, stimulated by pma, mpo, or immune complex activated human sera [ ] . their results suggest that pa-dpeg inhibition of nets occurs by blocking the mpo pathway of net formation. this provides proof that peptides can potentially be developed to inhibit complement-induced netosis and be used to manage conditions worsened by nets, although since complements are important in immune response, this needs to be researched further. antibiotics have been used in the management of bacterial infections and also have immunomodulating effects by influencing the properties of numerous immune cells, including neutrophils [ , ] . bystrzycka et al. conducted a study to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of nets by neutrophils. their study indicated that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of nets, with azithromycin showing a concentration-dependent effect on the respiratory burst in bystrzycka et al.'s study [ ] . another article by manda-handzlik et al. looked into the effects of cefotaxime and gentamicin on nets and discovered that gentamicin inhibits net release by human neutrophils, while cefotaxime had no impact on this process [ ] . the information that antibiotics can modulate net release can be useful in the management of infectious diseases or patients suffering from net-related diseases. since different antibiotics have different effects, there is need for more studies on their mode of action. this will inform on a possible compound for use in the management of nets without interfering with their antimicrobial function. thrombomodulin is a protein cofactor expressed on endothelial cell surfaces that modifies the substrate specificity of thrombin by an allosteric mechanism [ ] . thrombinthrombomodulin complex activates protein c, initiating an essential anticoagulant pathway [ ] [ ] [ ] . thrombosis is the formation of a blood clot within a blood vessel caused by cytokines and other inflammatory mediators produced during an injury, obesity, and in some cases drugs, e.g., estrogen pills [ ] . large amounts of circulating cfdna, present in nets, can influence thrombus formation by impairing fibrinolysis creating a scaffold for the binding of red blood cells, platelets, fibrin, and coagulation factors [ ] . besides cfdna, other net components also exert procoagulant properties with extracellular histones inducing platelet activation, aggregation, and thrombin generation [ , , ] . immuno-thrombosis-induced coagulopathy may contribute to hypercoagulability which increases occurrence of multiple organ dysfunction and mortality [ ] [ ] [ ] . helms et al. did a study where they looked into the use of a recombinant human thrombomodulin (rhtm) and found out that it can limit procoagulant responses. rhtm was also shown to fully inhibit netosis in neutrophils cultured with platelets and in the presence of lps [ ] . there is not much research in effect of rhtm, but this provides a starting point for further research into its potential use to inhibit nets. activated protein c (apc) is a multifunctional serine protease produced in blood by vitamin k-activating protein c. apc has anticoagulant, cytoprotective, and anti-inflammatory activities [ , ] . protein c has been shown to be an important prognostic indicator in patients with sepsis. during sepsis, there is a reduction in the conversion of protein c to its active form due to the downregulation of thrombomodulin by inflammatory cytokines [ ] . the antithrombotic effects of activated protein c is mediated by its ability to inhibit the formation of clotting factors va and villa and disrupting extracellular histones [ , ] . healy et al. demonstrated that apc cleaves and detoxifies extracellular histones and prevents activated platelets from inducing netosis. the pretreatment of neutrophils with apc before inducing netosis inhibited platelet adhesion to nets. they also used antibodies against the neutrophil receptors endothelial protein c receptor (epcr), protease-activated receptor (par ), and macrophage- antigen (mac- ) which blocked apc inhibition of netosis [ ] . another study demonstrated that the blockade of protein c activation lead to exacerbated sublethal lps challenge to turn lethal, which was reversed by treatment with antibodies to histones [ , ] . these findings suggest that the anti-inflammatory function of apc may include inhibition of netosis. drotrecogin alfa is a recombinant human activated protein c produced by xigris approved for use in septic patients [ , , ] . studies should be done to evaluate if this and other similar compounds would be effective in minimizing netosis in sepsis and other conditions with minimal side effects. heparin is a medication and naturally occurring glycosaminoglycan used as an anticoagulant (blood thinner). specifically, it is used in the treatment of heart attacks and unstable angina, and also antagonizes the effects of histones [ , ] . high levels of circulating histones have been positively correlated with disease severity in many disease conditions as they activate nf-κb pathway inducing the secretion of cytokines that amplify inflammation leading to organ damage [ ] [ ] [ ] . heparin has been shown to significantly suppress histone-induced disease [ , ] . studies have been done to evaluate the effect of unfractionated heparin, low molecular weight heparin, e.g., parnaparin and non-anticoagulant heparin [ , , ] . these studies demonstrate that heparin was able to protect mice from organ damage and death by antagonizing circulating histones attenuating tissue damage. administration of heparin, especially the nonanticoagulant heparin, is a novel and promising approach that may be further developed to treat patients with high levels of circulating histones potentially inhibiting netosis without increasing the risk of bleeding. anti-high-mobility group box (hmgb ) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule [ ] [ ] [ ] . it plays a beneficial role in microbial eradication through its pro-inflammatory actions and modulation of neutrophil chemotaxis [ ] [ ] [ ] [ ] [ ] . platelets are the major source of hmgb within the thrombi and present it to neutrophils promoting netosis [ ] [ ] [ ] ] . vogel et al. determined that platelet-derived hmgb is a critical mediator of thrombosis from their study using generated transgenic mice with platelet-specific deletion of hmgb [ ]. these effects were mediated via tlr -and myd -dependent recruitment of platelet guanylyl cyclase (gc) toward the plasma membrane, followed by myd /gc complex formation and activation of the cgmp-dependent protein kinase i [ , ] . mice lacking hmgb in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/ hemorrhagic shock [ , ] . exposure of neutrophils to hmgb resulted in enhanced formation of nets in vitro through tlr -dependent processes contributing to inflammatory processes and tissue injury [ ] [ ] [ ] [ ] ] . studies show that the use of anti-hmgb antibodies may diminish net formation, as seen in a reduction of histone and free dna in the bal fluid of lps-treated mice that received neutralizing antibodies to hmgb [ , ] . however, decreased levels of cytokines in the lungs after administration of anti-hmgb antibodies to lps-treated mice may not necessarily be a direct result of diminished net formation but could reflect the effects of hmgb on other pro-inflammatory pathways [ , ] . this is a promising area necessitating more research into how anti-hmgb interrupts netosis, and possibly use it as a treatment option. c esterase inhibitor (c inh) is an acute phase protein found in blood and a serine protease inhibitor that targets the complement pathway, coagulation pathway (factor xiia), and the contact system protease kallikrein. it is an endogenous inhibitor of c protein in the complement system [ , , ] . c -inh concentrates are approved for use in the management of hereditary angioedema (hae), an autosomaldominant disease caused by c -inh deficiency due to a mutation in the c -inhibitor gene [ , ] . studies have been done to evaluate whether ci-inh may protect from lung injury in vivo possibly explaining the underlying mechanisms mediating protection. these studies demonstrated that application of c inh alleviates bleomycin-induced lung injury via direct interaction with extracellular histones [ , ] . in vitro, c inh was found to bind all histone types with the interaction being independent of its protease inhibitory activity, but dependent on its glycosylation status [ ] . in vivo, histone-c inh complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury [ ] . the reactivecenter-cleaved c inh attenuated pulmonary damage evoked by intravenous histones indicating that c inh administration may provide a new therapeutic option for disorders associated with histone release [ ] . wygrecka and his colleagues tested c inh for its ability to bind and neutralize histones and determined that c inh can bind purified histones in vitro reducing epithelial cell death by blocking histone interactions with cell surface proteins [ ] . in another study, there was evidence for active binding of the exogenous c inh to extracellular and citrullinated histones released during netosis suggesting an endogenous mechanism by which histones are potentially neutralized [ ] . this mechanism could be exploited for therapeutic management of excessive netosis in other conditions, but studies need to be done to evaluate the effectiveness of these and other compounds with similar effects. metformin, a widely prescribed blood glucose-normalizing antidiabetic drug, suppresses immune responses. it mainly achieves this through the induction of amp-activated protein kinase. ampk is an enzyme that plays a role in cellular energy homeostasis, by activating glucose and fatty acid uptake and oxidation when cellular energy is low. induction of ampk subsequently inhibits the mammalian target of rapamycin (mtorc ), a pathway that regulates mammalian metabolism and physiology, by inhibiting mitochondrial ros production [ ] . this results in its direct effect on the cellular functions of various pro-inflammatory immune cells. due to the ros inhibitory effect of metformin, studies are underway to evaluate it as a drug for regulating autoimmune diseases, treating chronic autoimmune diseases and gero-protection [ , ] . menegazzo et al. investigated the effect of metformin against netosis and discovered that compared with a placebo, it significantly reduced the concentrations of nets in vitro. they showed a reduction in elastase, proteinase- , histones, and cfdna, whereas glucose control with insulin exerted no significant effect [ ] . metformin was shown to prevent membrane translocation of pkc-βii and activation of nox in neutrophils altering pathologic changes in nuclear dynamics and dna release [ , ] . this resulted in a blunted netosis in response to pma and calcium influx. this provides information for a possible use of metformin on the pkc-nox pathway as an anti-netosis therapy. chloroquine/hydroxychloroquine is an antimalarial drug used to treat malaria and has effects on amoeba (a protozoa) and some viruses [ , ] . studies are currently underway to evaluate its effects on the novel corona- virus (covid ) that is currently causing a pandemic [ , ] . hydroxychloroquine (hdq) is a slightly less potent derivative of chloroquine that is used in the treatment of malaria and as an immunosuppressive drug for management autoimmune conditions including sle and ra [ , ] . it exerts its immunosuppressive effect through inhibition of cytokine production with modulation of co-stimulatory molecules and also inhibits leukocyte phagocytosis [ , ] . hdq interferes with the stimulatory effect of platelet aggregation even in the presence of a thrombin agonist [ ] . mmps are matrix metalloproteinases which are enzymes involved in extracellular matrix remodeling, and timps are counter regulatory tissue inhibitors of mmps [ , ] that have been extensively studied in sle [ , ] . research has shown that hdq modulated mmps-timps interaction assisting in maintaining homeostasis of the extracellular matrix [ , ] and may thus play a role in reducing nets. in another study that looked at tumor-derived extracellular vesicle (ev), transportable vesicles important in the exchange of biological molecules between cells and induce formation of nets [ , ] , hdq was shown to inhibit neutrophil uptake of tumor-derived evs, thus reducing netosis [ , [ ] [ ] [ ] . however, the precise mechanism of inhibiting the uptake is largely unknown. due to the associated complications of nets in autoimmune conditions and cancer metastasis, it is important for future research efforts to focus on further investigation of these drugs and other new specific targets for prevention or control of the detrimental effects of nets formation. diphenyleneiodonium chloride (dpi) is as a hypoglycemic agent able to block gluconeogenesis and respiration by inhibiting many enzymes; nadph oxidase, nitric oxide synthase, xanthine oxidase, nadph cytochrome p oxidoreductase and cholinesterase [ , ] . dpi works by binding the heme group of nadph oxidase, inhibiting of nadph oxidase and thus inhibits ros production [ ] . ostafin et al. evaluated the effect of dpi on ros production in the context of nets and discovered that addition of dpi to the sample led to a reduction of extracellular dna release with the strongest inhibition noticed after adding μm dpi. these findings confirmed that dpi is able to block net creation. however, the addition of dpi together with pma or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of net formation [ ] . these findings necessitate more studies to look into the mechanism of action of dpi under different conditions and in different diseases as a potential therapeutic for nets. n-acetylcysteine (nac), also known as acetylcysteine, is a medication used to treat acetaminophen overdose [ ] and to loosen thick mucus in individuals with cystic fibrosis or chronic obstructive pulmonary disease [ ] . it also functions as an antioxidant which helps mitigate symptoms for a variety of diseases exacerbated by ros species [ , ] . zawrotniak and his team evaluated the effect of nac, ketoprofen, and ethamsylate on netosis and observed a reduction of ros production in a dose-dependent manner. nac inhibited netosis, but in the presence of hydrogen peroxide, this neutrophil ability was restored indicating that nac influences net formation by modulating ros productivity [ ] . the administration of ethamsylate led to a significant reduction in net formation, but this effect was not restored by hydrogen peroxide suggesting an additional side effect of this drug. ketoprofen seemed to promote ros-independent net release, simultaneously inhibiting ros production [ ] . brianna et al. used an acute pulmonary thrombosis model in vivo where nac reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin [ ] . in vitro analysis of platelet activation revealed that nac reduced thrombin-induced platelet-leukocyte aggregate formation in mice model of mutated janus kinase , a common mutation found in patients with chronic hematologic malignancies (chm), and reduced net formation in primary human neutrophils from patients with chm as well as healthy controls [ ] . these results strongly suggest that the therapeutic strategies applied in many neutrophil-mediated diseases should take into account the net-associated effects and that studies should look at the effect of these compounds in other diseases. recombinant human dnase, marketed as pulmozyme (dornase alfa) by genentech, is a highly purified solution of recombinant human deoxyribonuclease i (rhdnase). this is an enzyme which selectively cleaves dna and has been used to hydrolyze the dna present in sputum/mucus of cystic fibrosis patients and reduces viscosity in the lungs promoting clearance of secretions [ ] . nucleases perform various functions like acquiring nucleotide nutrients, allowing or preventing uptake of foreign dna, controlling biofilm formation/dispersal/architecture, aiding some pathogens in invading host by tissue damage, degrading dna matrixes, and immunomodulating the host immune response [ ] [ ] [ ] . studies have demonstrated the destructive effect of dnase on dna-nucleoprotein, and immune complexes, providing a rational way to interfere with the disease processes in sle and lupus nephritis [ ] . numerous other studies have evaluated the effect of rhdnase on nets, with results showing a reduction of netosis with reduced neutrophil infiltration reducing the inflammatory response [ ] [ ] [ ] . albadawi et al. conducted a study where they observed reduced detection of extracellular traps in post-ischemic muscle but did not alter skeletal muscle fiber injury, levels of pro-inflammatory molecules, or atp level. rhdnase treatment enhanced postischemic hindlimb perfusion, decreased infiltrating inflammatory cells, and reduced the expression of thrombinantithrombin iii [ ] . in addition, dnase i decreases tumor volume in rats when injected intramuscularly or intraperitoneally in conjunction with other proteases (papain, trypsin, and chymotrypsin) [ ] ; however, it is not known whether these effects are due primarily to net inhibition, thus the need for more studies. findings from a different study, showed that early and concurrent treatment with dnase i and antibiotics resulted in improved survival, reduced bacteremia, and organ dysfunction in septic conditions [ ] ) suggesting a possible combination therapy to control netosis. additionally, dnase i injection may have off-target effects that need to be considered in its use for control of nets or they may fail to function as expected in vitro [ ] . staphylokinase is an exoprotein produced by staphylococcus aureus, which activates host plasminogen [ ] . it induces extracellular release of alpha-defensins from polymorphonuclear cells promoting a complex formation between alpha-defensins and staphylokinase. the effect of this interaction is an almost complete inhibition of the bactericidal effect of alpha-defensins [ ] . thammavongsa et al. reported that s. aureus escapes these defenses by converting nets to deoxyadenosine, which triggers the caspase- -mediated death of immune cells [ ] .thus, the pathogenesis of s. aureus infections has evolved to anticipate host defenses and to repurpose them for the destruction of the immune system [ , ] . secretory nucleases also provide means of survival to other bacteria like iron-reducing shewanella and such functions help them adapt and survive proficiently [ ] . other than their pro-pathogen roles in survival, nucleases can be used directly as therapeutics due to their biological functions and medical applications in diagnosis, immunoprophylaxis, and autoimmune therapy. in the future, these enzymes can impact human medicine positively by opening new avenues for therapeutics which have otherwise reached saturation due to multi-drug resistance. probiotics are live microorganisms promoted with claims that they provide health benefits when consumed, generally by improving or restoring the gut flora [ , ] . probiotics are considered generally safe for consumption but may cause unwanted side effects and bacteria-host interactions in rare cases. alterations in the gut microbiota, as well as the presence of local and systemic markers of inflammation, are strongly associated with the manifestation of a spectrum of intestinal disorders [ ] . linda et al. investigated the effects of a nonpathogenic, enteropathogenic, and probiotic bacteria on the dynamics of net formation using murine bone marrow-derived neutrophils and the neutrophil-differentiated human myeloid cell line dhl- . they demonstrate that the probiotic lactobacillus rhamnosus strain gg (lgg) inhibits both pma and s. aureus induced nets by inhibiting pkc pathway and dampening ros production disrupting netosis supporting its antioxidative capacity [ ] . given the presence of nets in inflamed intestine [ ] , it is possible that some of the beneficial effects of lgg are attributable to its action on local neutrophils. probiotics have been shown to protect against bacterial-induced cytotoxicity, but more studies need to be done to highlights the dynamic interaction between beneficial bacteria and neutrophils to inform on the usefulness of probiotics as gut-protective and immunomodulatory compounds. vitamin d is a group of fat-soluble secosteroids important for increasing intestinal absorption minerals including calcium, magnesium, and phosphate. vitamin d has other multiple biological effects including activating the innate immune system while dampening the adaptive immune systems [ ] [ ] [ ] [ ] . in humans, vitamin d (cholecalciferol) and vitamin d (ergocalciferol) are the most important [ ] . there are suggestions indicating the benefits of vitamins d on various conditions, but evidence is lacking on whether supplementation of vitamin d helps to reduce the risk of these diseases including asthma, tuberculosis, irritable bowel disease, depression, and other conditions [ , ] . in the case of netosis, handono et al. evaluated the effect of hypovitamin d on nets in sle patients [ ] . they demonstrated a significant decrease in early apoptosis with a moderate positive correlation between ne externalizations with early apoptosis. they concluded that vitamin d could reduce endothelial damage by decreasing netosis activity [ ] . this result may reveal the possibility of vitamin d as supplementary therapy for sle patients and other patients with hypo-vitamin d to prevent netosis and endothelial damage. investigation of the impact of one or more of the therapeutics discussed above in modification of net formation will vary depending upon the disease and drug of interest. where there are animal models of a target disease (e.g., rheumatoid arthritis or psoriasis) administration of the therapeutic drug can be done in a placebo-controlled study evaluating different doses. tools available include histological identification of nets and comparison of nets formed in placebo versus drugtreated animals manually or using available computer programs. one example from our own work involves using a bovine model of respiratory syncytial virus and examination of the role of ibuprofen, a cox-inhibitor which decreases proinflammatory prostaglandin production and thromboxane [ ] . our theory is that ibuprofen would reduce netosis by decreasing neutrophil-activating cytokines and platelet activation. in our study, lungs are harvested at necropsy, fixed and stained with antibodies against citrullinated histones and neutrophil elastase, to delineate the presence of neutrophil nets. another model is the use of neutrophils incubated in vitro with the drug to be tested and staining to determine if there is an effect on netosis under different drug doses. nets have been implicated in many disease processes, and although they have a positive effect by clearing pathogens, they are also destructive due to the release of enzymes and other proteins that cause tissue injury. control of nets is quickly becoming a target for therapeutics in the management of various disease, but it is clear to see that the different compounds that inhibit or clear nets may have other unwanted effects on the immune system. this makes it challenging to conclude that one compound works better that the other and thus the need for more research. there is a possibility that the management of nets may require using a combination therapy that incorporate conventional treatments such fluid therapy, antibiotics, antivirals, and net-targeted drugs. to potentially optimize treatment efficacy and outcome in clinical patients, it is important we run more studies to evaluate the mode of action of these compounds to pick the actual effective component of these drugs, while evaluating the effect in the overall immune system to ensure there are no other detrimental effects. rapid killing of human neutrophils by the potent activator phorbol -myristate -acetate (pma) accompanied by changes different from typical apoptosis or necrosis neutrophil extracellular traps: is immunity the second function of chromatin? beneficial suicide: why neutrophils die to make nets netting bacteria in sepsis social networking of human neutrophils within the immune system neutrophil function: from mechanisms to disease neutrophils in the activation and regulation of innate and adaptive immunity neutrophil extracellular traps kill bacteria neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps killing activity of neutrophils is mediated through activation of proteases by k+ flux neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus reactive oxidants and myeloperoxidase and their involvement in neutrophil extracellular traps mir- regulates pad -dependent formation of neutrophil extracellular traps inhibition of pad activity is sufficient to disrupt mouse and human net formation histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation neutrophil histone modification by peptidylarginine deiminase is critical for deep vein thrombosis in mice neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens how neutrophils kill microbes neutrophil extracellular traps (net) induced by different stimuli: a comparative proteomic analysis neutrophil extracellular traps profiles in patients with incident systemic lupus erythematosus and lupus nephritis cancer cells induce metastasis-supporting neutrophil extracellular dna traps nlrp activation induced by neutrophil extracellular traps sustains inflammatory response in the diabetic wound the influence of hyperglycemia on neutrophil extracellular trap formation and endothelial pathogenesis and therapy of psoriasis neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity regulating neutrophil apoptosis: new players enter the game mechanisms of b cell autoimmunity in sle systemic lupus erythematosus immunological pathogenesis and treatment of systemic lupus erythematosus genetic variations in a dub domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus review article neutrophil function in an inflammatory milieu of rheumatoid arthritis synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis proinflammatory mediators and neutrophils are increased in synovial fluid from heifers with acute ruminal acidosis nets are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis the role of neutrophil extracellular traps in rheumatic diseases nets spread ever wider in rheumatic diseases type diabetes: translating mechanistic observations into effective clinical outcomes neutrophil extracellular traps: the core player in vascular complications of diabetes mellitus small-vessel vasculitis the american college of rheumatology criteria for the classification of vasculitis: introduction a practical approach to the diagnosis, evaluation, and management of cutaneous smallvessel vasculitis abundant neutrophil extracellular traps in thrombus of patient with microscopic polyangiitis enhanced formation and disordered regulation of nets in myeloperoxidase-ancaassociated microscopic polyangiitis neutrophil extracellular trap formation is associated with il- β and autophagy-related signaling in gout neutrophil extracellular traps (nets) in autoimmune diseases: a comprehensive review chambers tj, morson bc ( ) the granuloma in crohn's disease gene and mirna regulatory networks during different stages of crohn's disease the epidemiologic and demographic characteristics of inflammatory bowel disease: an analysis of a computerized file of patients neutrophil activation and neutrophil extracellular trap formation in inflammatory bowel disease neutrophil extracellular traps in pediatric inflammatory bowel disease neutrophil extracellulartraps sustain inflammatory signals in ulcerative colitis. j crohn's colitis low-grade inflammation and the metabolic syndrome in children and adolescents low-grade inflammation and its relation to obesity and chronic degenerative diseases. rev médica del diabetes primes neutrophils to undergo netosis, which impairs wound healing the adipose tissue as a source of vasoactive factors adipocytes properties and crosstalk with immune system in obesity-related inflammation neutrophil extracellular traps and cardiovascular diseases: an update increased plasmatic nets by-products in patients in severe obesity in vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection effect of high-fat diet on the formation of pulmonary neutrophil extracellular traps during influenza pneumonia in balb/c mice role of thromboxane-dependent platelet activation in venous thrombosis: aspirin effects in mouse model platelets induce neutrophil extracellular traps in transfusion-related acute lung injury aspirin-triggered -epi-lipoxin a regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice activation of platelet function through g protein-coupled receptors aspirin-triggered resolvin d down-regulates inflammatory responses and protects against endotoxin-induced acute kidney injury aspirin, but nottirofiban displays protective effects in endotoxin induced lung injury neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation regulation of neutrophil extracellular trap formation by anti-inflammatory drugs mechanisms of action of cyclosporine cyclosporine: a review cyclosporine in veterinary dermatology molecular mechanisms of action of some immunosuppressive drugs efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine a. plos one: posttransplant infections in the tropical countries molecular targeting of protein arginine deiminases to suppress colitis and prevent colon cancer aa et alpeptidylarginine deiminase inhibition disrupts net formation and protects against kidney, skin and vascular disease in lupus-prone mrl/lpr mice netosis delays diabetic wound healing in mice and humans pad regulates proliferation of multipotent haematopoietic cells by controlling c-myc expression dysregulation of pad -mediated citrullination of nuclear gsk β activates tgf-β signaling and induces epithelialto-mesenchymal transition in breast cancer cells neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis prostaglandins and inflammation prostaglandins. in: encyclopedia of toxicology prostaglandin e inhibits neutrophil extracellular trap formation through production of cyclic amp inhibition of neutrophil extracellular trap formation after stem cell transplant by prostaglandin e prostaglandin e inhibits ifn-α secretion and th costimulation by human plasmacytoid dendritic cells via e-prostanoid and e-prostanoid receptor engagement complement and diseases: defective alternative pathway control results in kidney and eye diseases defective complement inhibitory function predisposes to renal disease netosis, complement, and coagulation: a triangular relationship the complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria paroxysmal nocturnal hemoglobinuria. a complement-mediated hemolytic anemia immunomodulatory properties of antibiotics antibiotics modulate the ability of neutrophils to release neutrophil extracellular traps azithromycin and chloramphenicol diminish neutrophil extracellular traps (nets) release microvascular thrombosis and multiple organ dysfunction syndrome immunoinflammatory activation in acute cardio-embolic strokes in comparison with other subtypes of ischaemic stroke primer on cerebrovascular diseases thrombomodulin favors leukocyte microvesicle fibrinolytic activity, reduces netosis and prevents septic shock-induced coagulopathy in rats the anticoagulant protein c pathway activated protein c the cytoprotective protein c pathway activated protein c efficacy and safety of recombinant human activated protein c for severe sepsis drotrecogin alfa (activated) treatment in severe sepsis from the global open-label trial enhance: further evidence for survival and safety and implications for early treatment histone variants in pluripotency and disease epigenetic protein families: a new frontier for drug discovery release and activity of histone in diseases heparins attenuated histone-mediated cytotoxicity in vitro and improved the survival in a rat model of histone-induced organ dysfunction nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis low molecular weight heparins-a new tool to disetangle from the nets low molecular weight heparins prevent the induction of autophagy of activated neutrophils and the formation of neutrophil extracellular traps hmgb : a novel protein that induced platelets active and aggregation via toll-like receptor- , nf-κb and cgmp dependent mechanisms circulating platelets as a source of the damage-associated molecular pattern hmgb in patients with systemic sclerosis disulfide hmgb derived from platelets coordinates venous thrombosis in mice high mobility group box protein interacts with multiple toll-like receptors high-mobility group box- in ischemia-reperfusion injury of the heart damageassociated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury high-mobility group box potentiates antineutrophil cytoplasmic antibody-inducing neutrophil extracellular traps formation deep vein thrombosis in mice is regulated by platelet hmgb through release of neutrophil-extracellular traps and dna intracellular hmgb inhibits inflammatory nucleosome release and limits acute pancreatitis in mice anti-high mobility group box- antibody therapy for traumatic brain injury anti-high mobility group box monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats c inhibitor, a multi-functional serine protease inhibitor hereditary angioedema with normal c inhibitor: clinical characteristics and treatment response with plasma-derived human c inhibitor concentrate (berinert®) in a french cohort hereditary angioedema with normal c inhibitor function: consensus of an international expert panel antihistone properties of c esterase inhibitor protect against lung injury bench-to-bedside review: the role of c -esterase inhibitor in sepsis and other critical illnesses c inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide the effect of c inhibitor on myocardial ischemia and reperfusion injury c -esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema neutralizing extracellular histones in acute respiratory distress syndrome the mechanisms of action of metformin encyclopedia of biomedical gerontology metformin improves healthspan and lifespan in mice the antidiabetic drug metformin blunts netosis in vitro and reduces circulating netosis biomarkers in vivo metformin prevents glucose-induced protein kinase c-β activation in human clinic rev allerg immunol umbilical vein endothelial cells through an antioxidant mechanism metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of pkc-nad(p)h oxidase pathway in human aortic endothelial cells in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine effects of chloroquine on viral infections: an old drug against today's diseases? of chloroquine and covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- antimalarials for treating rheumatoid arthritis regulation and involvement of matrix metalloproteinases in vascular diseases role of polymorphisms in mmp- and timp- as biomarkers for susceptibility to systemic lupus erythematosus patients enhanced neutrophil extracellular trap formation in acute pancreatitis contributes to disease severity and is reduced by chloroquine. front immunol: chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps the receptor for advanced glycation end products (rage) enhances autophagy and neutrophil extracellular traps in pancreatic cancer ) diphenyleneiodonium inhibits the cell redox metabolism and induces oxidative stress diphenyleneiodonium, an inhibitor of noxes and duoxes, is also an iodide-specific transporter hemin upregulates egr- expression in vascular smooth muscle cells via reactive oxygen species erk- / -elk- and nf-κb different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation a review of acetaminophen poisoning the role for nacetylcysteine in the management of copd the antioxidant action of n-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid n-acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why view of selected mucolytic, anti-inflammatory and cardiovascular drugs change the ability of neutrophils to form extracellular traps (nets) n-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm recombinant human dnase i reduces the viscosity of cystic fibrosis sputum nucleases of bacterial pathogens as virulence factors, therapeutic targets and diagnostic markers structure and function of nucleases in dna repair: shape, grip and blade of the dna scissors current and future delivery systems for engineered nucleases: zfn, talen and rgen the treatment of systemic lupus erythematosus (sle) in nzb/w f hybrid mice; studies with recombinant murine dnase and with dexamethasone deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma nets and cf lung disease: current status and future prospects neutrophil extracellular traps in the central nervous system hinder bacterial clearance during pneumococcal meningitis effect of dnase i treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice antitumor effects of systemic dnase i and proteases in an in vivo model recombinant human dnase i decreases biofilm and increases antimicrobial susceptibility in staphylococci neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death colonization and infection of the skin by s. aureus: immune system evasion and the response to cationic antimicrobial peptides staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study probiotic mechanisms of action the potential of probiotics: a review risk and safety of probiotics probiotic lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps toll-like receptor triggering of a vitamin d-mediated human antimicrobial response vitamin d and the immune system nonclassic actions of vitamin d an update on vitamin d and human immunity principles of nutrigenetics and nutrigenomics: fundamentals of individualized nutrition pp low-dose aspirin for preventing recurrent venous thromboembolism aspirin as an antiplatelet drug cyclooxygenase inhibitors and the antiplatelet effects of aspirin differential involvement of the p y and p y receptors in platelet procoagulant activity molecular basis of platelet granule secretion platelets and chemokines in atherosclerosis: partners in crime platelets: new bricks in the building of neutrophil extracellular traps human thrombinderived host defense peptides inhibit neutrophil recruitment and tissue injury in severe acute pancreatitis neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps mechanisms of pain in arthritis low-grade inflammation in symptomatic knee osteoarthritis: prognostic value of inflammatory plasma lipids and peripheral blood leukocyte biomarkers inhibition of immune complex complement activation and neutrophil extracellular trap formation by peptide inhibitor of complement c complement in human diseases: lessons from complement deficiencies the role of complement in physiology and pathology thrombomodulin and its role in inflammation structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex a systematic evaluation of laboratory testing for drug-induced immune thrombocytopenia neutrophil extracellular trapsthe dark side of neutrophils activated protein c: biased for translation activated protein c inhibits neutrophil extracellular trap formation in vitro and activation in vivo regulation of inflammatory responses by natural anticoagulants effectiveness and safety of drotrecogin alfa (activated) for severe sepsis: a meta-analysis and metaregression heparin. in: encyclopedia of toxicology the story of the discovery of heparin and warfarin high-mobility group box- in sterile inflammation high-mobility group box , oxidative stress, and disease the high mobility group box: the ultimate utility player of a cell high-mobility group box protein (hmgb ): nuclear weapon in the immune arsenal involvement of tolllike receptors and in cellular activation by high mobility group box protein late-acting cytokine hmgbi: mediatory functions and prospects for clinical application the role of high mobility group box in innate immunity high-mobility group box is involved in the initial events of early loss of transplanted islets in mice activated platelets present high mobility group box to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps inactivation of factor xii active fragment in normal plasma. predominant role of c -inhibitor contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma hydrochloroquine retinopathy: characteristic presentation with review of screening mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology chloroquine inhibits production of tnf-a, il- b and il- from lipopolysaccharidestimulated human monocytes/macrophages by different modes bourboulia d, stetler-stevenson wg ( ) matrix metalloproteinases (mmps) and tissue inhibitors of metalloproteinases (timps): positive and negative regulators in tumor cell adhesion association between serum matrix metalloproteinase-(mmp-) levels and systemic lupus erythematosus: a meta-analysis effect of chloroquine phosphate treatment on serum mmp- and timp- levels in patients with systemic lupus erythematosus chloroquine treatment influences proinflammatory cytokine levels in systemic lupus erythematosus patients role of tumor-derived extracellular vesicles in cancer progression and their clinical applications (review) tumor-derived exosomes induce the formation of neutrophil extracellular traps: implications for the establishment of cancer-associated thrombosis vitamin d and immune function vitamin d supplementation, -hydroxyvitamin d concentrations, and safety vitamin d prevents endothelial damage induced by increased neutrophil extracellular traps formation in patients with systemic lupus erythematosus an overview of clinical pharmacology of ibuprofen publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -tqc usry authors: godden, sandra title: colostrum management for dairy calves date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: tqc usry colostrum management is the single most important management factor in determining calf health and survival. unfortunately, a significant proportion of north american dairy calves suffer from failure of passive transfer of antibodies from colostrum, contributing to excessively high preweaning mortality rates and other short- and long-term losses associated with animal health, welfare, and productivity. a successful colostrum management program requires producers to consistently provide calves with a sufficient volume of clean, high-quality colostrum within the first few hours of life. this article reviews the process of colostrogenesis and discusses important components of colostrum. the key components of delivering and monitoring a successful colostrum management program are discussed. the placenta of the cow separates the maternal and fetal blood supplies, preventing in utero transmission of protective immunoglobulins (ig) [ ] . consequently, the calf is born agammaglobulinemic and so depends almost entirely on the absorption of maternal ig from colostrum after birth. the absorption of maternal ig across the small intestine during the first hours after birth, termed passive transfer, helps to protect the calf against common disease organisms until its own immature immune system becomes functional. calves are defined as having failure of passive transfer (fpt) if the calf serum igg concentration is less than mg/ml when sampled between and hours of age [ , ] . achieving early and adequate intake of highquality colostrum is widely recognized as the single most important management factor in determining health and survival of the neonatal calf ( fig. ) [ ] [ ] [ ] [ ] . in addition to reduced risk for preweaning morbidity and mortality, additional long-term benefits associated with successful passive transfer include reduced mortality in the postweaning period, improved rate of gain and feed efficiency, reduced age at first calving, improved first and second lactation milk production, and reduced tendency for culling during the first lactation [ ] [ ] [ ] [ ] . unfortunately, many producers continue to incur significant loss associated with fpt. in the united states mortality rates in preweaned dairy heifers are estimated to range between % and % [ , , ] . poor colostrum management is one of the key factors contributing to these excessive losses. in one study % of calves sampled between and hours of age had fpt (serum igg ! mg/ml) [ ] . it was estimated that approximately % of preweaning mortality events occurring in the first weeks of life were attributed to fpt [ ] . these studies point to the need for producers to adopt practices to improve colostrum management. this article reviews the process of colostrogenesis and discusses important components of colostrum. the key components of developing a successful colostrum management program are discussed. bovine colostrum consists of a mixture of lacteal secretions and constituents of blood serum, most notably ig and other serum proteins, which accumulate in the mammary gland during the prepartum dry period [ ] . this process begins several weeks before calving, under the influence of lactogenic hormones, including prolactin, and ceases abruptly at parturition. important constituents of colostrum include ig, maternal leukocytes, growth factors, hormones, cytokines, nonspecific antimicrobial factors, and nutrients. concentrations of many of these components are greatest in the first secretions harvested after calving (first milking colostrum), then decline steadily over the next six milkings (transition milk) to reach the lower concentrations routinely measured in saleable whole milk (table ) [ ] . igg, iga, and igm account for approximately % to %, %, and %, respectively, of the total ig in colostrum, with igg accounting for % to % of the total igg [ ] . although levels are highly variable among cows and studies, one study reported that mean colostral concentrations of igg, iga, and igm were mg/ml, . mg/ml, and . mg/ml, respectively [ ] . igg, and igg in particular, are transferred from the bloodstream across the mammary barrier into colostrum by a specific transport mechanism: receptors on the mammary alveolar epithelial cells capture igg from the extracellular fluid, and the molecule undergoes endocytosis, transport, and finally release into the luminal secretions [ ] . the alveolar epithelial cells cease expressing this receptor, most likely in response to increasing prolactin concentrations, at the onset of lactation [ ] . smaller amounts of iga and igm are largely derived from local synthesis by plasmacytes in the mammary gland [ ] . although not well understood, colostral transfer of ige also occurs and may be important in providing early protection against intestinal parasites [ ] . normal bovine colostrum contains greater than  cells/ml of immunologically active maternal leukocytes, including macrophages, t and b lymphocytes, and neutrophils [ , ] . at least a portion of colostral leukocytes are absorbed intact across the intestinal barrier [ ] . liebler-tenorio and colleagues [ ] reported that the preferential route of uptake of colostral leukocytes through the intestinal barrier is through the follicle-associated epithelium of peyer patches in the jejunum and ileum. reber and colleagues [ ] proposed that, after entering the neonatal circulation, maternal leukocytes traffic to neonatal nonlymphoid tissues and secondary lymphoid tissues, disappearing from the neonatal circulation by to hours after feeding colostrum. although their functional importance in calves is not routinely measured, early evidence suggests that colostral leukocytes enhance lymphocyte response to nonspecific mitogens, increase phagocytosis and bacterial killing ability, and stimulate humoral immune responses (igg formation) in the calf [ , [ ] [ ] [ ] . presumably these cells would not be viable in pasteurized colostrum or colostrum replacer products. the role and functional significance of colostral leukocytes remains areas of active research. other important components of colostrum include growth factors, hormones, cytokines, and nonspecific antimicrobial factors. bioactive components of colostrum with antimicrobial activity include lactoferrin, lysozyme, and lactoperoxidase [ ] [ ] [ ] . oligosaccharides in colostrum may provide protection against pathogens by acting as competitive inhibitors for the binding sites on the epithelial surfaces of the intestine [ ] . growth factors in bovine colostrum include transforming growth factor beta- (tgf-b ), growth hormone (gh), and insulin, but their function in colostrum is not fully understood (see table ) [ ] . colostral insulinlike growth factor-i (igf-i) may be a key regulator in the development of gastrointestinal tracts of bovine neonates, including stimulation of mucosal growth, brush-border enzymes, intestinal dna synthesis, increased villus size, and glucose uptake increased [ ] [ ] [ ] . trypsin inhibitor, a compound found in colostrum in concentrations nearly times greater than in milk, serves to protect igg and other proteins from proteolytic degradation in the intestine of the neonatal calf. although the immunologic importance of colostrum is frequently discussed, the nutritional significance of the first colostrum meal should not be overlooked. the total solids content (%) in first milking colostrum and whole milk in holstein cows has been reported to average . % and . %, respectively (see table ) [ ] . much of this increase in colostrum solids content is attributed to a more than fourfold increase in protein content of colostrum versus milk, this being because of significant increases in ig and casein content [ ] . the crude fat content of first milking holstein colostrum ( . %) is also significantly higher than for milk ( . %) [ ] . energy from fat and lactose in colostrum is critical for thermogenesis and body temperature regulation. certain vitamins and minerals, including calcium, magnesium, zinc, manganese, iron, cobalt, vitamin a, vitamin e, carotene, riboflavin, vitamin b , folic acid, choline, and selenium are also found in increased concentrations in bovine colostrum versus milk (see table ) [ , ] . to achieve successful passive transfer of igg, the calf must first consume a sufficient mass of ig in colostrum and then be able to successfully absorb a sufficient quantity of these molecules into its circulation. major factors affecting the mass of ig consumed by the calf include the quality and volume of colostrum fed. the major factor affecting the absorption of ig molecules into circulation is the quickness, after birth, with which the first colostrum feeding is provided. the remainder of this article reviews these and other important factors affecting passive transfer, management strategies for preventing bacterial contamination of colostrum, and the use of colostrum supplements and replacers, and provides recommendations for monitoring the colostrum management program. although it is recognized that colostrum contains a wide spectrum of important immune and nutritional components, because the relationship between ig concentrations and calf health is best understood, and because igg composes more than % of total ig in colostrum, the concentration of igg in colostrum has traditionally been considered the hallmark for evaluating colostrum quality. high-quality colostrum has an igg concentration greater than g/l [ ] . the igg concentration in colostrum can vary dramatically among cows. in one recent study, colostrum igg averaged g/l, but ranged from to g/l for individual holstein cows [ ] . some factors affecting colostrum quality, such as breed or age of the dam, may be out of the producer's ability to manipulate. several other important factors affecting colostrum quality, however, including preparturient vaccination, dry period length, and time to colostrum collection, can be managed by producers. this section reviews factors affecting colostrum quality and discusses cow-side testing of colostrum quality. comparative studies have reported that there can be a breed effect on colostrum quality [ , ] . in one study, igg concentration was greater in secretions from beef cows ( . g/l) than from dairy cows ( . g/l) [ ] . in another study, holstein cows produced colostrum with total ig content ( . %) that was numerically lower than for guernsey ( . %) and brown swiss ( . %) cows, and statistically lower than for ayrshire ( . %) and jersey ( . %) cows [ ] . breed differences could be attributed to genetic differences and/or dilutional effects. most, but not all, studies report a tendency for older cows to produce higher quality colostrum, presumably because of older animals have had a greater period of exposure to farm-specific pathogens [ ] [ ] [ ] [ ] . as one example, tyler and colleagues [ ] reported that the mean colostral igg concentration for holstein cows in their first, second, or third and greater lactations was , , and g/l, respectively. in the same study, however, there was reportedly no difference in igg concentration for guernsey cows in their first ( g/l), second ( g/l), and third and greater lactations ( g/l). producers should be discouraged from automatically discarding colostrum from first-calf heifers, because it may be of very good quality. studies generally have shown that ig content of colostrum is not affected by prepartum maternal nutrition [ ] . in a study feeding beef cows either % (co) or % (rs) of national research council (nrc) ( ) [ ] protein and energy requirements, maternal nutrition did not affect either colostrum igg concentration ( . versus . g/l for rs and co, respectively) or the calves' serum igg concentration at hours ( . versus . mg/ml for rs and co, respectively) [ ] . lacetera and colleagues [ ] reported that cows supplemented with injections of selenium and vitamin e in late pregnancy produced a greater volume of colostrum than unsupplemented cows, when all cows were fed a prepartum diet that was deficient in vitamin e and selenium. treatment had no impact on colostrum igg concentration, however. producers should feed dry cows and heifers nonlactating rations balanced according to nrc ( ) guidelines [ ] . some, but not all, studies have reported that exposure to high ambient temperatures during late pregnancy is associated with poorer colostrum composition, including lower mean concentrations of colostral igg and iga, and lower mean percentages of total protein, casein, lactalbumin, fat, and lactose [ , ] . these effects may be attributed to the negative effects of heat stress on dry matter intake resulting in nutritional restriction, reduced mammary blood flow resulting in impaired transfer of igg and nutrients from the blood stream to the udder, or impaired immune reactivity of mammary gland plasmacytes that produce iga [ ] . producers should adopt the similar heat-abatement strategies for prepartum cows and heifers as are routinely used for lactating animals. pritchett and colleagues [ ] observed that cows producing less than . kg of colostrum at first milking were more likely to produce high-quality (o g/l) colostrum than cows producing higher quantities of first milking colostrum (r . kg). this finding was presumed to be attributable to dilutional effects. however, more recent studies report that there is no predictable relationship between colostrum igg concentration and weight of colostrum produced at first milking [ , ] . persistent intramammary infection (imi) during the nonlactating period has not been associated with altered igg concentration. imi is associated with lower colostral volume produced, however [ ] . producers should not feed colostrum from cows with clinical mastitis. pooling of colostrum from multiple dams is generally discouraged because larger volumes of low-quality colostrum may dilute smaller volumes of higher-quality colostrum [ ] . furthermore, pooling raw colostrum may increase the number of calves potentially exposed to colostrum-borne pathogens. although not all studies have shown positive results, a body of research has established that vaccinating the pregnant cow or heifer during the final -to -week period preceding calving results in increased concentrations of protective colostral antibodies, and increased passive antibody titers in calves of vaccinated dams, for some common pathogens including pasteurella haemolytica, salmonella typhimurium, escherichia coli, rotavirus, and coronavirus [ ] [ ] [ ] [ ] [ ] . secretion of ig from the dam's circulation into the mammary gland begins approximately weeks before calving. in one observational study, dry period length (mean ¼ . ae days) was not associated with colostrum igg concentration [ ] . in a controlled study, rastani and colleagues [ ] also reported that colostrum quality was not different for cows with a -or -day dry period, respectively. cows with excessively short dry periods (! days) or no dry period produce colostrum with significantly lower igg concentrations [ , ] . furthermore, dry period length can affect the volume of colostrum produced: in a recent controlled field study cows with a short ( -day) dry period produced . kg less colostrum than did cows with a conventional ( -day) dry period [ ] . the concentration of ig in colostrum is highest immediately after calving, but begins to decrease over time if milking is delayed. in one study, delaying harvest of colostrum for hours, hours, or hours after calving resulted in a %, %, and % decrease in colostral igg concentration, respectively [ ] . to collect the highest quality colostrum, producers should aim to milk the cow within to hours after calving if possible, with a maximum delay of hours. empiric recommendations suggest rejecting colostrum that is visibly watery, bloody, or is from cows that leaked before calving [ ] . it is difficult to predict, based on such factors as dam parity, weight of colostrum produced at first milking, or visual consistency, which colostrum collected will be of high (o g/l igg) versus low quality [ ] . the colostrometer, a hydrometer instrument that estimates igg concentration by measuring colostrum specific gravity, is one rapid and inexpensive cow-side test that may be useful to differentiate high-from low-quality colostrum (specific gravity o . approximates igg concentration o g/l igg). factors such as content of fat and other solids, plus colostrum temperature, affect the hydrometer reading, however. pritchett and colleagues [ ] reported that the sensitivity and specificity of the instrument for detecting low-quality colostrum were . and . , respectively, meaning that the instrument would incorrectly classify two of every three low-quality colostrum samples as acceptable. pritchett and colleagues [ ] suggested that to avoid misclassification error, producers should alter the hydrometer cutoff points to , , or g/l if feeding either . , . , or . l of colostrum at first feeding, respectively. test specificity would be severely compromised by using higher cutpoints, however, resulting in an excessive portion of colostrums being misclassified as deficient [ ] . others have suggested that if a large enough volume (eg, . l) is fed at first feeding, then there may be limited value to using a hydrometer. despite its limitations, the hydrometer may still be useful to differentiate high-from low-quality colostrum used for first versus later feedings, respectively. an alternate tool for differentiating high-from low-quality colostrum may be a commercially available cow-side immunoassay kit (colostrum bovine igg quick test kit, midland bio-products, boone, iowa). chigerwe and colleagues [ ] recently reported that the sensitivity and specificity of this test kit to identify poor-quality colostrum (igg! g/l) were . and . , respectively. with this relatively low specificity, the immunoassay test would incorrectly classify one in every four high-quality colostrum samples as unacceptable. one additional limitation of the immunoassay is that it yields only a positive or negative result, but does not provide an estimate of the actual igg concentration. the immunoassay costs approximately $ (united states dollars [usd]) per sample and takes approximately minutes to run. to achieve successful passive transfer in an average -kg ( lb) holstein calf, experts calculate that producers should feed at least a minimum mass of g of igg in the first colostrum feeding [ ] . so what volume of colostrum should producers feed to meet or exceed this minimum dose? obviously the answer to this question depends on the igg concentration in the colostrum being fed. for example, if colostrum was known to contain g/l igg, then the producer would only need to feed . l ( qt) to achieve the minimum goal of ingesting more than g igg. if the colostrum contained only g/l of igg, however, then the producer would need to feed . l ( qt) to achieve the same ingested mass of igg. besser and colleagues [ ] noted that only % of colostrum samples tested would be of high enough quality to provide greater than g igg if calves were only fed . l. some % of colostrum samples tested would be of high enough quality to provide greater than g igg if calves were fed . l, however. because producers frequently do not know the concentration of igg in the colostrum being fed, it is currently recommended that calves be fed % to % of their body weight of colostrum at first feeding ( . l for a -kg calf). in one study mean serum igg at hours was significantly higher for calves fed l of high-quality colostrum at hours and a further l at hours ( . mg/ml igg) as compared with calves fed only l of high-quality colostrum at hours and a further l at hours ( . mg/ml) (fig. ) [ ] . another study reported that brown swiss calves fed . l (versus . l) of colostrum at first feeding experienced significantly higher rates of average daily gain and greater levels of milk production in both the first and second lactation [ ] . in national surveys, . %, . %, and . % of producers reported feeding or more quarts of colostrum within the first hours in , , and , respectively [ , , ] , indicating that increasing the volume of colostrum fed is still an area of opportunity for most dairy producers. the term ''open gut'' refers to the unique ability of the neonatal enterocyte to nonselectively absorb intact large molecules, such as ig, by pinocytosis [ ] . from there, ig molecules are transported across the cell and released into the lymphatics by exocytosis, after which they enter the circulatory system through the thoracic duct [ ] . in a process referred to as ''closure,'' the efficiency of colostral ig absorption through the intestinal epithelium of the calf decreases linearly with time from birth to completely close at approximately hours [ ] . feeding colostrum after the gut has closed still offers the benefit of local immunity in the gut lumen, but ig absorption into the circulation no longer occurs. the following section discusses factors affecting the efficiency of ig absorption, many of which are under management's control. the major factor affecting efficiency of ig absorption is age of the calf at feeding. the efficiency of ig transfer across the gut epithelium is optimal in the first hours postpartum, but after hours there is a progressive decline in the efficiency of ig absorption over time [ , ] . delaying the first colostrum feeding can only slightly postpone gut closure ( hours) [ ] . producers should aim to feed all calves within to hours after birth and by hours at a maximum. the method of feeding colostrum is worth considering because this can influence the time to first feeding, the volume consumed, and the efficiency of ig absorption. high rates of fpt have been reported in calves left to suckle the dam [ , ] . this finding may be attributable to failure of the calf to voluntarily consume a sufficient volume of colostrum and delays in suckling. edwards and broom [ ] reported that % of calves born to second parity and older cows had failed to suckle within hours after birth. by comparison, % of calves born to first-calf heifers had failed to suckle within hours after birth. these delays could be caused by numerous factors, including weak or injured cow or calf, mastitis or other illness in the cow, low pendulous udders or large teats, or poor mothering ability. it is for this reason that it is currently recommended that the calf be removed from the dam within to hours of birth, and that the calf then be hand-fed a known volume of colostrum using either a nipple bottle or esophageal feeder [ ] . in national surveys, . %, . %, and . % of calves were reportedly fed using a nipple bottle or esophageal tube in , , and , respectively [ , , ] , indicating that progressively fewer producers are relying on suckling the dam for colostrum delivery. producers may have a personal preference for using either a nipple bottle or esophageal feeder for the first colostrum feeding. although the esophageal feeder method is quicker, it is known that when fluid is given with an esophageal feeder, the esophageal groove reflex is not triggered, resulting in fluid being deposited into the forestomachs. this limitation is not significant, however, because outflow of colostrum from the forestomachs to the abomasum and small intestine occurs for the most part within hours [ ] . adams and colleagues [ ] reported that calves fed colostrum using a bottle had only slightly higher serum igg concentrations versus calves fed with an esophageal feeder, but that these differences were numerically small and statistically insignificant. it is generally accepted that either method of feeding achieves acceptable rates of passive transfer provided a sufficient volume of colostrum is consumed [ , ] . veterinarians should train interested producers on how to properly use and clean esophageal feeders. it has been reported that efficiency of ig absorption was improved when calves were housed with the dam [ ] . considering that acceptable levels of serum igg can be achieved without housing the calf with the dam, however, and given that the latter practice may increase the calf's risk for exposure to pathogens from the dam or her environment, it is currently recommended that the calf be removed from the dam within to hours of birth and then hand-fed a known volume of colostrum [ ] . decreased colostral ig absorption in the first hours has been reported in calves with postnatal respiratory acidosis, associated with prolonged parturition [ ] . although hypoxic calves may have delayed igg absorption initially, studies have reported that there is no difference in overall absorptive capacity between hypoxic and normoxic calves and that there is no difference in serum igg concentrations by the time of gut closure [ , ] . weaver and colleagues [ ] suggested that an increased rate of fpt seen in calves with metabolic or respiratory acidosis may be caused by a delay in the animal getting up to nurse, not by reduced absorptive capacity. absorption of ig may be impaired when newborn calves are exposed to extreme cold, possibly because of direct effects on intestinal absorption and transport and indirect effects on the calf's ability to stand and nurse [ ] . bacteria in colostrum may bind free ig in the gut lumen or directly block uptake and transport of ig molecules across intestinal epithelial cells, thus interfering with passive absorption of colostral ig [ ] [ ] [ ] . this effect was demonstrated in a recent controlled study wherein newborn calves were fed either . l of pasteurized ( c  min) colostrum or . l of raw colostrum, with the geometric mean total bacteria counts in the two colostrum treatment groups being cfu/ml or , cfu/ml, respectively [ ] . although the volume, timing, and quality of colostrum fed to the two feeding groups was not different, calves fed pasteurized colostrum had significantly higher mean serum igg levels at hours of age ( . mg/ml) versus calves fed raw colostrum ( . mg/ml). this improvement was attributed to reduced bacterial interference with igg absorption across the gut, resulting in higher efficiency of igg absorption in calves fed pasteurized colostrum ( %) versus calves fed raw colostrum ( %) [ ] . strategies for preventing or minimizing bacterial contamination of colostrum are discussed in the next section. although colostrum is an important source of nutrients and immune factors, it can also represent one of the earliest potential exposures of dairy calves to infectious agents including mycoplasma spp, mycobacterium avium subsp paratuberculosis, fecal coliforms, and salmonella spp [ ] [ ] [ ] . this exposure is a concern because pathogenic bacteria in colostrum could cause diseases such as diarrhea or septicemia. it is also a concern because bacteria in colostrum may interfere with absorption of ig [ ] [ ] [ ] . experts recommend that fresh colostrum fed to calves contain fewer than , cfu/ ml total bacteria count (tpc) and fewer than , cfu/ml total coliform count [ ] . unfortunately, average bacteria counts in colostrum fed on commercial dairies frequently far exceeds this cutpoint [ , ] . in one study of wisconsin dairy herds, % of samples tested exceeded the upper limit of , cfu/ml tpc [ ] . the following section describes management techniques for minimizing bacterial contamination of colostrum. preventing contamination during colostrum harvest, storage, and feeding procedures methods for reducing the risk for pathogen exposure to calves include avoiding feeding colostrum from known infected cows and avoiding pooling of raw colostrum. additionally, all producers should take steps to avoid contamination during colostrum harvest, storage, or feeding processes. in a study of colostrum harvesting and feeding practices on one dairy, total bacteria counts (tpc cfu/ml) were very low or nil in colostrum stripped directly from the gland (geometric mean udder tpc ¼ . cfu/ml). significant bacterial contamination occurred, however, during the process of milking the colostrum into the bucket (geometric mean bucket tpc ¼ , cfu/ ml) [ ] . these results emphasize the importance of minimizing colostrum contamination by properly prepping udders before harvesting colostrum, milking into a clean, sanitized bucket, and handling colostrum using clean, sanitized storage or feeding equipment. bacteria can multiply rapidly if colostrum or milk is stored at warm ambient temperatures [ ] . unless colostrum is to be fed right away, it should be frozen or refrigerated within hour after collection. it is generally accepted that colostrum may be frozen for up to year, provided multiple freeze-thaw cycles do not occur. when thawing frozen colostrum, producers should avoid overheating colostrum (avoid temperatures o c or f) or some denaturation of colostral ig can occur [ ] . options for producers who wish to store fresh colostrum include refrigeration with or without the use of preservatives such as potassium sorbate [ ] . igg in raw refrigerated colostrum is stable for at least week. average bacteria counts in raw refrigerated colostrum may reach unacceptably high concentrations (o , cfu/ml) after days of refrigeration, however. by comparison, average colostrum bacteria counts remained less than , cfu/ml for days of refrigeration when colostrum was preserved with potassium sorbate in a . % final solution [ ] . information on potassium sorbate sources and mixing directions can be found at http://www.atticacows.com/orgmain.asp?orgid¼ &storytypeid¼&sid¼&. an additional tool that may be useful to reduce bacterial contamination of colostrum is pasteurization. early studies tried to pasteurize colostrum using the same conventional methods and high temperatures as are typically used to pasteurize milk ( c [ f] for minutes or c [ f] for seconds). this process yielded unacceptable results, however, including thickening or congealing of colostrum and denaturation of approximately one third of colostral igg [ ] . despite these early setbacks, more recent research has determined that using a lower-temperature, longer-time approach ( c [ f] for minutes) to batch-pasteurize colostrum is sufficient to maintain igg activity and colostrum fluid characteristics, while eliminating or significantly reducing important pathogens including e. coli, salmonella enteritidis, mycoplasma bovis and mycobacterium avium subsp paratuberculosis [ , ] . in one recent on-farm controlled study, calves fed pasteurized colostrum ( c  minutes) experienced a significant reduction in colostrum bacterial exposure and significantly higher serum igg levels at hours of age versus calves fed . l of raw colostrum [ ] . if stored in a clean covered container, the shelf life of pasteurized refrigerated colostrum is at least to days [ ] . the potential short-and longterm health and economic benefits of feeding pasteurized colostrum have not yet been described. farms can occasionally experience periods in which an adequate supply of clean, high-quality, fresh or stored colostrum is not available to feed to all newborn calves. contributing to this problem, some producers may discard colostrum from cows that test positive for m avium subsp paratuberculosis, bovine leukosis virus, or m bovis mastitis. under such circumstances, using colostrum supplements (cs) or colostrum replacement (cr) products may offer producers a convenient way to improve levels of passive immunity in calves while reducing the risk for pathogen exposure through colostrum. powdered commercial cs or cr products contain bovine ig that is typically either lacteal-or plasma-derived. it is recommended that cs or cr products be mixed in water (according to label directions) and fed as a separate meal after any natural colostrum has been fed [ ] . there are important differences between the less expensive cs products ($ -$ per dose) and more expensive cr products ($ -$ per dose). colostrum supplement products typically contain less than g igg per dose, contain no nutrient pack, and are only intended to supplement (not replace) existing colostrum. if given alone, feeding cs products results in significantly lower serum ig and greater risk for fpt in calves as compared with feeding fresh colostrum [ ] . there is no added benefit of feeding cs products if already feeding to l of high-quality bovine colostrum [ , ] . by comparison, cr products contain a minimum of g igg per dose, provide a nutritional source of protein, energy, vitamins, and minerals, and are designed to completely replace (or feed in the absence of) maternal colostrum [ ] . results of cr studies have been mixed, with many products failing to routinely provide the necessary mg/ml igg in serum of calves fed cr [ , [ ] [ ] [ ] . in a controlled study of dairy herds in minnesota and wisconsin, swan and colleagues [ ] reported that commercial dairy calves fed a commercially available cr product (acquire, american protein corporation, inc., ames, iowa) had significantly lower serum igg concentrations ( . mg/ml igg) than calves fed maternal colostrum ( . mg/ ml igg). although a trend was present, the preweaning morbidity and mortality rates were not different for calves fed cr (morbidity ¼ . %; mortality ¼ . %) versus calves fed maternal colostrum (morbidity ¼ . %; mortality ¼ %). other studies have reported better rates of successful passive transfer (mean serum igg o . mg/ml), particularly when calves were fed two doses of cr product [ , ] . in one such study, the average -hour serum igg level for calves fed either one dose ( g igg) or two doses ( g igg) of a lacteal-derived cr, or . l of maternal colostrum, were . , . , and . mg/ml igg, respectively (land o' lakes colostrum replacement, land o' lakes inc., st. paul, minnesota) [ ] . feeding higher doses of cr products may increase the rate of successful passive transfer, but the cost-benefit of this practice has yet to be described. similarly, the effectiveness and cost-benefit of routinely using cr products in johne's or other infectious disease control programs has yet to be described. because of the highly variable performance among different products, veterinarians should review results of peer-reviewed controlled trials when selecting a cr product. veterinarians can help producers develop programs to routinely monitor colostrum management. possible laboratory-based test methods for directly measuring or estimating serum igg concentrations in calves include radial immunodiffusion (rid), turbidimetric immunoassay (tia), enzyme-linked immunosorbent assay (elisa), sodium sulfite turbidity test, zinc sulfate turbidity test, serum gamma glutamyltransferase (ggt) activity, and wholeblood glutaraldehyde coagulation test [ ] [ ] [ ] . in a recent review of these tests, weaver and colleagues [ ] raised concerns about unacceptably high levels of inaccurate results for the sodium sulfite turbidity test when using the % and % sodium sulfite test solutions, the zinc sulfate turbidity test if samples are exposed to co or are hemolyzed, ggt test results, and whole-blood glutaraldehyde coagulation test results. although rid, tia, or elisa would be acceptable tests for use in periodic outbreak investigations, the expense and inconvenience of routinely submitting serum samples to a veterinary diagnostic laboratory would generally discourage their adoption for ongoing monitoring programs. a lateral-flow immunoassay is one tool that could be used for on-farm testing (midland quick test kit -calf igg, midland bioproducts corp., boone, iowa). the manufacturer has reported the sensitivity, specificity, and overall accuracy of this assay to identify calves with serum igg less than . mg/ml as being . , . , and . , respectively [ ] . independent validation of this test is still required. one limitation of the immunoassay is that it yields only a positive or negative result, but does not provide an estimate of the actual serum igg concentration. the assay requires approximately minutes to complete and costs approximately $ . (usd) per sample. measurement of serum total solids (sts) by hand-held refractometer offers a convenient, simple, rapid, and inexpensive on-farm tool by which producers can monitor the colostrum feeding program. the refractometer instrument costs approximately $ (usd). in an early study of calves, sts had a good correlation with serum igg concentration as measured using rid (r ¼ . ) [ ] . calloway and colleagues [ ] , reported that sts concentration test endpoints of . and . g/dl yielded the most accurate results in estimating the adequacy of passive transfer as defined by serum igg . mg/ml or greater (sensitivity o . ; specificity o . ; proportion classified correctly o . ). in that study lower or higher test endpoints misclassified larger numbers of calves. because sts results do result in periodic misclassification of individual calves, the use of sts results as an individual animal diagnostic tool is discouraged. when results are interpreted at the group or herd level, however, sts results accurately reflect the proportion of calves that have fpt, thereby making it a useful on-farm tool for monitoring whether the colostrum management program is succeeding. it is recommended that serum samples be collected from a minimum of clinically normal (not scouring) calves between hours and days of age [ ] . wallace and colleagues [ ] reported that the results of sts refractometry from centrifuge-and noncentrifuge-harvested sources of serum were highly correlated (r ¼ . ), so producers can conduct this test onfarm without need of a centrifuge. mcguirk and collins [ ] suggest that a goal is for % or more of calves tested to meet or exceed a sts cutpoint of . g/dl. tyler suggests that % or more of calves tested should meet or exceed the more accurate sts cutpoint of . g/dl (j tyler, personal communication, ). if it is determined that a disproportionate number of calves have fpt, then the veterinarian and producer must investigate to identify and then correct the root causes of fpt within the colostrum management program. in addition to periodically sampling groups of calves to assess fpt, producers can also periodically submit frozen colostrum samples to a microbiology laboratory for culture. a goal is for a majority of samples submitted to have at total bacteria count of less than , cfu/ml and a total coliform count less than , cfu/ml [ ] . colostrum management is the single most important management factor in determining calf health and survival. unfortunately, a significant proportion of north american dairy calves suffer from failure of passive transfer, contributing to excessively high preweaning mortality. there is considerable opportunity for most dairy producers to improve their colostrum management practices, resulting in improved short-and long-term health and performance of the animal. a successful colostrum management program requires producers to consistently provide calves with a sufficient volume of clean, high-quality colostrum within the first few hours of life. colostrum replacers are useful tools if a sufficient quantity of clean, high-quality maternal colostrum is not available. ongoing monitoring helps producers to more quickly identify and correct problems within the colostrum management program. the development of the conceptus dairy : national dairy health evaluation project. dairy heifer morbidity, mortality, and health management focusing on preweaned heifers. ft. collins (co): usda-aphis veterinary services passive transfer of colostral immunoglobulins in calves national animal health monitoring system. national dairy heifer evaluation project. dairy herd management practices focusing on preweaned heifers. ft. collins (co): usda-aphis veterinary services in: the development, nutrition, and management of the young calf managing the production, storage and delivery of colostrum effects of passive immunity on growth and survival in the dairy heifer effects of passive immunity on subsequent production in dairy heifers factors associated with mortality to days of life in dairy heifers in the united states effects of colostrum ingestion on lactational performance part : reference of dairy health and management in the united states. ft. collins (co): usda-aphis veterinary services availability, storage, treatment, composition, and feeding value of surplus colostrum: a review immunoglobulin production and transport by the mammary gland immunological activities of milk effect of prolactin on in vitro expression of the bovine mammary immunoglobulin g receptor colostral transfer of bovine immunoglobulin e and dynamics of serum ige in calves cellular components of mammary secretions and neonatal immunity: a review intestinal absorption of maternal leucocytes by newborn lambs uptake of colostral leukocytes in the intestinal tract of newborn calves colostrum induced phenotypic and trafficking changes in maternal mononuclear cells in a peripheral blood leukocyte model for study of leukocyte transfer to the neonatal calf the influence of colostral leukocytes on the course of an experimental escherichia coli infection and serum antibodies in neonatal calves effects of the ingestion of whole colostrum or cell-free colostrum on the capacity of leukocytes in newborn calves to stimulate or respond in oneway mixed leukocyte cultures effect of maternal cells transferred with colostrum on cellular response to pathogen antigens in neonatal calves growth factors and antimicrobial factors of bovine colostrum effects of milk-derived bioactives: an overview immunoglobulins, growth factors and growth hormone in bovine colostrum and the effects of processing antioxidants in bovine colostrum effects of dietary insulin-like growth factor i on growth and insulin-like growth factor receptors in neonatal calf intestine peptide regulation of intestinal glucose absorption small intestinal morphology in eight-day-old calves fed colostrum for different durations or only milk replacer and treated with long-r -insulinlike growth factor i and growth hormone passive transfer of immunoglobulin g and preweaning health in holstein calves fed a commercial colostrum replacer regulation of colostrum formation in beef and dairy cows colostral immunoglobulin concentrations among breeds of dairy cattle factors associated with colostral specific gravity in dairy cows management and production factors influencing immunoglobulin g concentration in colostrum from holstein cows colostral immunoglobulin concentrations in holstein and guernsey cows effects of prepartum protein restriction in the beef cow on immunoglobulin content in blood and colostral whey and subsequent immunoglobulin absorption by the neonatal calf nutrient requirements of beef cattle influence of nutritional restriction during late gestation on production measures and passive immunity in beef cattle effects of selenium and vitamin e administration during a late stage of pregnancy on colostrum and milk production in dairy cows, and on passive immunity and growth of their offspring nutrient requirements of dairy cattle composition of colostrum from dairy heifers exposed to high air temperatures during late pregnancy and the early postpartum period use of mammary gland and colostral characteristics for prediction of colostral igg concentration and intramammary infection in holstein cows shortening the dry period from to days does not affect colostrum quality but decreases colostrum yield by holstein cows effects of mastitis on the volume and composition of colostrum produced by holstein cows passive protection of calves against experimental infection with salmonella typhimurium colostral and milk antibody titers in cows vaccinated with a modified live rotavirus-coronavirus vaccine a field trial to evaluate the efficacy of a combined rotavirus-coronavirus escherichia coli vaccine in dairy cattle immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes preparturient vaccination to enhance passive immunity to the capsular polysaccharide of pasteurella haemolytica a reducing dry period length to simplify feeding transition cows: milk production, energy balance and metabolic profiles metabolism and mammary secretion of serum protein in the cow effect of delayed colostrum collection on colostral igg concentration in dairy cows alliance on management and nutrition. a guide to colostrum and colostrum management for dairy calves evaluation of the hydrometer for testing immunoglobulin g concentrations in holstein colostrum evaluation of a cow-side immunoassay kit for assessing igg concentration in colostrum comparison of three methods of feeding colostrum to dairy calves effects of quality, quantity, and timing of colostrum feeding and addition of a dried colostrum supplement on immunoglobulin g absorption in holstein bull calves electron microsopic studies of the jejunal epithelium from neonatal pigs fed different diets the ultrastructure of neonatal calf intestine and absorption of heterologous proteins effect of colostral immunoglobulin g and immunoglobulin m concentrations on immunoglobulin absorption in calves intestinal transmission of macromolecules in newborn dairy calves of different ages at first feeding colostral immunoglobulin transfer in calves i. period of absorption effect of suckling followed by bottle feeding colostrum on immunoglobulin absorption and calf survival the period between birth and first suckling in dairy calves the failure of the oesophageal groove reflex, when fluids are given with an oesophageal feeder to newborn and young calves two methods for administering colostrum to newborn calves colostrum management in calves: effects of drenching vs. bottle feeding studies on dairy calves allowed to suckle their dams at fixed times postpartum decreased colostral immunoglobulin absorption in calves with postnatal respiratory acidosis hypoxia in neonatal calves: effect on intestinal transport of immunoglobulins effect of high arterial carbon dioxide tension on efficiency of immunoglobulin g absorption in calves effects of maternal nutritional restriction and cold stress on young calves: absorption of colostral immunoglobulins effect of orally administered duodenal fluid on serum proteins in neonatal calves influence of administered indigenous microorganisms on uptake of [iodine- ] gamma-globulin in vivo by intestinal segments of neonatal calves bacteria in colostrum: impact on calf health [abstract the effect of feeding heat-treated colostrum on passive transfer of cellular and humoral immune parameters in neonatal dairy calves survey of ontario bulk tank raw milk for foodborne pathogens isolation of mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis preventing bacterial contamination and proliferation during the harvest, storage and feeding of fresh bovine colostrum heat-treatment of bovine colostrum i: effects of temperature on viscosity and immunoglobulin g effect of on-farm commercial batch pasteurization of colostrum on colostrum and serum immunoglobulin concentrations in commercial dairy calves heat-treatment of bovine colostrum ii: effects of heating duration on pathogen viability and immunoglobulin g improving cleanliness and shelf-life of refrigerated colostrum using heat-treatment and chemical preservatives effects of a colostrum replacement product derived from serum on immunoglobulin g absorption by calves serum immunoglobulin concentrations after feeding maternal colostrum or maternal colostrum plus colostral supplement to dairy calves efficacy of a dried colostrum powder in the prevention of disease in neonatal holstein calves formulation of colostrum supplements, colostrums replacers and acquisition of passive immunity in neonatal calves effect of a why protein concentrate used as a colostrum substitute or supplement on calf immunity, weight gain, and health short communication: absorption of protein and immunoglobulin g in calves fed a colostrum replacer influence of pooled colostrum or colostrum replacement on igg and evaluation of animal plasma in milk replacer serum igg and total protein concentrations in dairy calves fed two colostrum replacement products development of an automated turbidimetric immunoassay for quantification of bovine serum immunoglobulin g evaluation of assays for failure of passive transfer in calves quantitation of bovine immunoglobulins: comparison of single radial immunodiffusion, zinc sulphate turbidity, serum electrophoresis, and refractometer methods evaluation of a lateral-flow immunoassay for use in monitoring passive transfer of immunoglobulins in calves an examination of the influence of husbandry on the plasma immunoglobulin level of the newborn calf, using a rapid refractometer test for assessing immunoglobulin content comparison of refractometers and test endpoints in the measurement of serum protein concentration to assess passive transfer status in calves a comparison of serum harvesting methods and type of refractometer for determining total solids to estimate failure of passive transfer in calves key: cord- -uj m t authors: hua, jinxi; zhang, yuanxun; de foy, benjamin; mei, xiaodong; shang, jing; feng, chuan title: competing pm . and no holiday effects in the beijing area vary locally due to differences in residential coal burning and traffic patterns date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: uj m t abstract the holiday effect is a useful tool to estimate the impact on air pollution due to changes in human activities. in this study, we assessed the variations in concentrations of fine particulate matter (pm . ) and nitrogen dioxide (no ) during the holidays in the heating season from to based on daily surface air quality monitoring measurements in beijing. a generalized additive model (gam) is used to analyze pollutant concentrations for sites by comprehensively accounting for annual, monthly, and weekly cycles as well as the nonlinear impacts of meteorological factors. a saturday effect was found in the downtown area, with about % decrease in pm . and % decrease in no relative to weekdays. on sundays, the pm . concentrations increased by about % whereas there were no clear changes for no . in contrast to the small effect of the weekend, there was a strong holiday effect throughout the region with average increases of about % in pm . and average reductions of about % in no concentrations. there was a clear geographical pattern in the strength of the holiday effect. in rural areas the increase in pm . is related to the proportion of coal and biomass consumption for household heating. in the suburban areas between the fifth ring road and sixth ring road there were larger reductions in no than downtown which might be due to decreased traffic as many people return to their hometown for the holidays. this study provides insights into the pattern of changes in air pollution due to human activities. by quantifying the changes, it also provides insights for improvements in air quality due to control policies implemented in beijing during the heating season. particles (pm . ) and nitrogen dioxide (no ), there have been conflicting results. (cui et al., ) found concentrations of thirteen elements in pm . were higher on weekends than on weekdays. (beirle et al., ; chen et al., ; liu et al., ) did not find a clear weekly pattern in the beijing area. these studies did not consider the effect of meteorology on pollutant concentrations. the regional transport plays an important role in the air pollution of beijing: a clear spatial pattern was observed with decreasing concentrations from south to north (li et al., ) . because meteorology has a strong impact on air pollution in the region, it must be taken into account to prevent biases in the estimates of human-related air pollution changes (sun et al., ; sun et al., ; wang et al., a; . in addition to meteorology, local emissions also affect air quality. the main anthropogenic sources of air pollution in beijing during the heating season are coal-burning for heating and vehicle emissions due to rapid urbanization xu et al., ) . a series of measures were implemented to improve air quality in the city, such as "action plan on prevention and control of air pollution" (zhang et al., ) , "joint prevention and control of atmospheric pollution" , "coal to electricity" and "coal to gas" projects (shuxue et al., ) . the actions have been found to effectively reduce the pm . and no concentrations (barrington-leigh et al., ; cheng et al., ; de foy et al., b; wang et al., b; zhang the holiday effect is similar to the weekend effect in that it is a measure of the difference in pollution levels between holidays and non-holidays (tan et al., ) . in general, it shows higher concentrations during non-holidays and lower concentrations during holidays. the holiday effect is due to changes in human activities which are influenced by lifestyle, urbanization, energy consumption structure, and traditional culture (forster and solomon, ; liu et al., ; seidel and birnbaum, ) . when there is no clear weekend effect, the holiday effect provides a useful tool to identify changes in air quality due to changes in human activities (chen et al., ) . (madhavi latha and highwood, ) found coarse particulate matter (pm ) concentrations are lower during the christmas holidays than non-holidays in the united kingdom, which are mainly due to reductions of local traffic emissions. (chen et al., ; tan et al., ) investigated the difference of six air pollutants in taipei between the spring festival and non-spring festival, finding significant reductions during the spring festival for nitrogen oxides (no x ), carbon monoxide (co), volatile organic compounds or non-methane hydrocarbon (nmhc), sulfur dioxide (so ), and pm , while o concentrations increased due to a reduction in the titration effect. (tan et al., ) reported a distinct spatial holiday effect associated with the degree of urbanization in taipei and found that the holiday effects of no x , co and nmhc become greater when the population, and hence the number of motor vehicles increases. was available from the beijing municipal environmental monitoring center (http://www. bjmemc.com.cn/). these sites are designed to reflect urban background conditions, regional transmission, traffic pollution, and urban air quality, covering most of the spatial range and multiple land use types (ji et al., ; sun et al., ) . for each site, daily averages were calculated from the valid data points when data was available for more than hours per day. the location of the sites and the number of observations as shown in table s . pm . and no were available for more than % of the days for most sites during the study period ( days in total). beijing includes administrative regions ( fig. ) , with a total population of . million in (beijing municipal bureau of statistics) and an area of , km (dong et al., ) . rapid urbanization caused a drastic expansion from core districts (dongcheng and xicheng). the area within the fifth ring road is considered the most densely populated area with around half of the inhabitants and only % of the surface area of beijing (xin , dong, et al., . due to relatively short commuting time and cheap housing, the area between the fifth ring road and the sixth ring road accounts for % of the population of beijing city (xin zhang, ). outside the sixth ring road, the population density is relatively sparse, and rural residents account for a large proportion, with approximately % of land area only % of the population (xin zhang, , dong, et al., . different population distribution and development patterns will lead to variations in air pollution due to variations in human activities. to explore the spatial variation of the holiday effect, the sites were clustered by geographical locations based on different population and development patterns (fig. ) . the sites within the fifth ring road were defined as downtown, the sites between the fifth ring road and the sixth ring road were defined as suburban, the sites outside the sixth j o u r n a l p r e -p r o o f journal pre-proof the holiday periods referred to new year"s day (january ), the spring festival (lunar new year), and the lantern festival based on the national legal holiday arrangements issued by the state council. the spring festival is the most important holiday involving family reunions, and it will bring travel peaks of people returning to their hometowns before the festival and then back to work afterwards. for the purposes of this study, the festival holiday period is defined as starting two days before the eve of the spring festival and finishing four days after the spring festival. this excludes the influence of the travel peaks which occur before and after that . the specific dates are listed in table s . overall, the holiday periods last around ten days each year. in this study, the non-parametric mann-whitney u test, which does not require the data to be normally distributed (chen et al., ) , was used to compare the pollutant concentrations and meteorological observations during the holidays and non-holidays, weekends and weekdays. the predictors in our gam model include time vectors to represent inter-annual, monthly, and weekday variations, as well as meteorological variables (boundary layer height, east-west wind component, south-north wind component, relative humidity, air temperature, dew point temperature, and surface pressure). the time factors were defined as linear terms, and the meteorological variables were defined as smooth terms. the equation is as follows: s (·) is the p-spline smoothing function that optimizes the fitting and controls the smoothness through a penalty term. s (blh) are the smoothers that characterize the non-linear influence of boundary layer height on the measurements. s (u, v) are interaction terms that denote the influence of horizontal ventilation due to zonal and meridional wind speeds. " " represent the meteorological variables selected in the candidate list based on their contribution to the model r . the candidate list included relative humidity, air temperature, dew point temperature, and pressure. to avoid the collinearity problem, the test process input only one variable to gam at a time. the one leading to the greatest increase in correlation coefficient was included. all meteorological variables were scaled linearly in order to approximate the normal distribution of zero mean to reduce the effects of extreme observations. after considering the impact of meteorological parameters, a net contribution to the time cycle can be obtained from the regression coefficient. in this way, temporal profiles are closer to changes caused by emissions than meteorological factors. the temporal indicators are input for each year, each month, and each day of the week, and therefore do not have a unique solution. as described j o u r n a l p r e -p r o o f journal pre-proof in (de foy, ), a weighting factor of one was used on the penalty term for the regression coefficients which solves the dummy variable trap problem while also forcing  to have the smallest possible values. the gam results can be interpreted using equation ( ): where p corresponds to the percentage change in the concentration during the time intervals relative to long-term averages. block-bootstrapping (de foy and schauer, ; requia et al., ) with seven-day chunks was used to estimate the uncertainty of the linear terms. the model was obtained times using a randomly resampled dataset each time. when resampling the dataset, the data points to be included were selected at random with replacement so that each dataset was of the same size as the original. the standard deviation of the temporal coefficients was obtained from the model simulations. the % confidence interval of the nonlinear terms in equation ( ) and % lower no concentrations ( table ) . the mean (± sd) pm . concentrations are ± μg/m and ± μg/m in holidays and non-holidays, respectively. the mean (± sd) no concentrations are ± μg/m and ± μg/m during holidays and non-holidays, respectively. daily means of pm . and no between the holidays and non-holidays, weekends and weekdays periods were found to be statistically different with p< . using the mann-whitney u test (table ) . meteorological variables were also statistically different, which means that it was important to consider these factors in the model in order to properly quantify the human-related air pollution changes. during the holidays, the mean blh is m lower than non-holidays and the air temperature is around ℃ lower. the main reason for the difference is that the holiday periods fall mainly within january and february which are colder than march. the same weather parameters from era and isd show a consistent bias: blh, d m, t m, rh, sp, and u are lower during the holidays and v is higher. the average standard deviations of pm . scaling factors are around . % and . % in non-holidays and holidays, respectively, and they are stable from site to site ( table s ). the relatively high uncertainty of holiday effects is due to the fact that there are fewer data points for the holiday periods than for the non-holidays. for no , the uncertainty of non-holidays is around . % and for holidays it is around . %. the uncertainty for pm . is higher than no mainly because pm . concentrations are affected by complex factors such as various emission sources, air mass transport, chemical transformation, day-to-day carry over, and complex interaction effect j o u r n a l p r e -p r o o f between synoptic conditions and pm . (wang, et al., , khuzestani, et al., . the performance of the model fit was assessed by calculating the regression coefficient (r ) and the root mean square error (rmse) using the bootstrap runs ( fig. s and fig. s ) . overall, the gam estimates of the holiday effects were found to be robust for each site with respect to the selection of the optimal set of meteorological variables. the list of variables selected as input to the gam for each site is shown in table s . daily average boundary layer height and isd winds were included in most cases, and the average relative humidity was the optimal choice at the majority of sites during the sensitivity tests. we will take the site yz as an example to discuss the impact of meteorology on pollutant concentrations. the site is located in the plain area and near a subway station, which means that its topographic features are similar to most of the other sites and it is strongly impacted by human activities. the increase in the mixing layer height led to the strong diffusion of pollutants (miao et al., ) , leading to approximately a % decrease in pm . with one standard deviation increase in the boundary layer height at yz (fig. s ) . high relative humidity promotes the hygroscopic growth of particulate matter (cheng et al., ) , with about a % increase in pm . with one standard deviation increase in the relative humidity. beijing is surrounded by mountains to the west, north, and northeast; and the southeast is a plain that slopes slowly towards the bohai sea. because of the topographical features, the air quality at most sites is influenced by southerly winds. frequent northerly winds during the heating periods also transport air pollution from downtown to j o u r n a l p r e -p r o o f journal pre-proof yz site which is a suburban site to the southeast. for no , an increase in one standard deviation of the boundary layer height is associated with a % decrease at yz (fig. s ) . higher relative humidity is associated with higher no concentrations. except for the influence from southerly winds, local emissions were found to have high contributions to no levels because the yz site is very busy as it is near a subway station. the % confidence interval range is very narrow for the meteorological parameters ( fig. s and for the day of the week profiles, the pm . scaling factors vary from site to site, but on average there is not much variation from monday to sunday (fig. , table figure s ) suggesting that the weekend effects are robust with respect to spatial variability. the weekend effect in pm . can be clearly seen even though pm . is influenced by carry over on the scale of to days. while this shows that the weekend effect is strong enough to be identified over other factors, future analysis would be required to estimate the impacts of carry over, for example using aerosol chemical speciation. the probability distribution of percentage changes by type of day shows that the differences between the days are statistically significant. as an example, the distributions of pm . factors at wsxg are clearly different for saturdays, sundays and holidays, and the distributions of no factors are clearly different for saturdays and holidays ( figure s ). this result updates the previous research on the weekend effect in beijing. (beirle et al., ; chen et al., ; liu et al., ) which showed that there was no obvious weekend effect for pm . or no in beijing, mainly because the previous study mostly did not consider the impact of meteorological parameters and of spatial patterns. this implies that the gam analysis provides valuable information with respect to temporal variation and meteorological influence, and that a network of widely distributed monitoring stations can provide more subtle information on the weekend effect. suggest that there are multiple factors involved in the change of human-activities, as will be discussed in section . . from downtown to suburban, to rural areas, there is a clear change in the holiday effects in the direction of a larger increase of pm . and a smaller decrease of no (fig. ) . for the most part, sites in close geographical proximity behave similarly, although there are a few outliers. dl, dgc, and tz exhibit different characteristics from the neighboring sites, probably because they are affected by local land use effects. for example, dgc is geographically close to pg, but dgc is surrounded by farmland and forest while pg is located in a residential area (fig. s ) , which suggests that the different holiday effects are due to local variations in land use type. dl was purposely designed to monitor urban background air quality surrounded by mountainous areas (chen et al., ; li et al., ) . these sites are excluded in the analysis that follows. for downtown, the changes are relatively consistent, with an increase in pm . concentrations ranging from . % to . % and a reduction in no ranging from . % to . % ( table ) . for most sites in the suburban region, the pm . changes are close to downtown sites with . ~ . % increases, and the changes in no are stronger than downtown with . ~ . % decreases. the effects of holidays in rural areas are clearly distinct. for the pm . holiday effect, the southwest and northeast rural areas demonstrate the largest increases, being on average . % higher than weekdays. the sites in the southeast and northwest areas show the lowest increases j o u r n a l p r e -p r o o f journal pre-proof with . % changes on average. this pattern is similar to the feature of pm . emissions from household heating in (cai et al., ) , who developed a village-based emission inventory of household combustion based on the investigation of all villages in beijing. for no , the sites in the southwest and northeast rural areas have the lowest decrease ( . % on average) while the sites in the southeast and northwest areas have the highest decrease ( . % on average). the pm . holiday effect consisted of increased concentrations ranging from % to % depending on the site. in contrast, the no holiday effect consisted of decreased concentrations ranging from % to % depending on the site. that pm . has a stronger holiday effect than no implies that there are greater differences in human activities for pm . emission sources. rural areas to the northeast of beijing experienced larger increases in pm . concentrations. these are mainly concentrated in miyun, huairou, and pinggu, which are districts in northeast beijing and are more heavily forested, and where coal and biomass burning is frequently used as a heating fuel (cai et al., ) . found that coal-fired boilers have been mostly eliminated from urban areas but remain in rural areas and especially in the districts of miyun, huairou, and pinggu in . we compared the strength of the holiday effect with household energy consumption by town in most districts of beijing in provided by (cai et al., ) . for each rural measurement site, we calculated the average coal and biomass consumption in neighboring districts. the strength of the holiday effect in pm . in rural areas was found to have a positive correlation with coal and biomass consumption (fig. ) . the northeast and southwest rural areas had a higher proportion of coal consumption for household heating than the northwest and southeast rural areas, and had a j o u r n a l p r e -p r o o f journal pre-proof correspondingly larger increase in pm . concentrations during the holidays. the spatial variation of the holiday effect suggests that indoor household heating activities could be a possible cause for the increases in pm . in rural areas. the results suggest that the promotion of the "coal to gas" project ( barrington-leigh et al., ; zhao et al., b) will play an important role in improving air quality in beijing in the next few years. household heating is also a significant source of no emissions (luo et al., ) , such that the no emitted by coal combustion probably offset the reduction of no due to reduced traffic. the area with more coal-fired activities have a smaller reduction in no than the areas with lower coal-fired activities. therefore, the net no holiday effect in rural areas is a result of two competing effects. for the area within the sixth ring road, although suburban areas and downtown show similar pm . increases, the suburban area exhibited an extra . % reduction in no relative to the downtown area. the difference is probably because the suburban area is home to a large number of commuters thanks to relatively cheap housing costs and short commute times, and consequently has a greater fraction of people traveling during the holidays (zhao et al., a) . changes in travel patterns are particularly strong during the spring festival when a large number of people return to their hometown leading to reduced transportation emissions during the spring festival itself. to distinguish between the effect of different holidays it will be necessary to have a longer time series of measurements. in this study, we used gam to estimate the holiday effects of pm . and no in beijing by concentrations increased by about % whereas there were no clear changes for no . although there is uncertainty due to meteorological factors such as temperature, wind speed and direction, and boundary layer height, the weekend variations are consistent at all downtown sites and robust with respect to temporal variability. the holiday effect was found to be much stronger than the weekend effects, and had opposite signs for pm . and no . there were increases in pm . ranging from % to % depending on the site. in contrast, no decreased from % to % depending on the site. furthermore, a clear spatial pattern was found in the strength of the holiday effect. in the rural areas, the strength of the pm . increases were associated with the extent of coal and biomass consumption for household heating in districts surrounding the measurement sites. it is worth noting that the promotion of renewable energy can therefore be expected to improve air quality in rural hotspots as well as in the greater beijing area. in addition, the suburban area where more people travelled during the holidays experienced greater reductions in no than the downtown area. the spatial variation in the holiday effect at different sites reflects two distinct ways that human activities impact air quality: increased residential heating tended to increase both pm . and no , whereas reduced traffic emissions leads to lower no . this study investigated the holiday effect in beijing, providing evidence for the influence of human activities on air quality on short time scales. studies of the holiday effects as well as other natural experiments such as the impacts of the olympic games (liu et al., ) , apec blue (gao et al., ; sun et al., ) , and new studies emerging on the impact of the covid- lockdown (bauwens et al., ; chauhan and singh, ; wang and su, ) will provide valuable information for the formulation of policies for both holiday and non-holiday periods. pollutant emissions from residential combustion and reduction strategies estimated via a village-based emission inventory in beijing decline in pm . concentrations over major cities around the world associated with covid- impacts of holiday characteristics and number of vacation days on "holiday effect diurnal, weekly and monthly spatial variations of air pollutants and air quality of beijing effects of meteorology and emission reduction measures on air pollution in beijing during heating seasons humidity plays an important role in the pm . pollution in beijing in situ continuous observation of hourly elements in pm . in urban beijing, china: occurrence levels, temporal variation, potential source regions and health risks city-level variations in nox emissions derived from hourly monitoring data in chicago changes in ozone photochemical regime in fresno, california from to deduced from changes in the weekend effect impacts of control strategies, the great recession and weekday variations on no columns above north american cities satellite no retrievals suggest china has exceeded its nox reduction goals from the twelfth five-year plan origin of high particle number concentrations reaching the st. louis, midwest supersite changes in speciated pm . california, due to nox concentrations in fresno, reductions and variations in diurnal emission profiles by day of week observations of a distinguishing the roles of meteorology, emission control measures, regional transport, and co-benefits of reduced aerosol feedbacks in characteristics and sources of aerosol pollution at a polluted rural site southwest in beijing spatial distribution differences in pm . concentration between heating and non-heating seasons in beijing spatial-temporal analysis on spring festival travel rush in china based on multisource big data diurnal, seasonal, and spatial variation of pm . in beijing temporal patterns in fine particulate matter time series in beijing: a calendar view source apportionment of fine-particle, water-soluble organic nitrogen and its association with the inflammatory potential of a statistical model to evaluate the effectiveness of pm . emissions control during the beijing olympic games source apportionment of black carbon during winter in beijing origins of aerosol nitrate in beijing during late winter through spring studies on particulate matter (pm ) and its precursors over urban environment of reading modeling the effect of weekday-weekend differences in motor vehicle emissions on photochemical air pollution in central california interaction between planetary boundary layer and pm . pollution in megacities in china: a review day-of-week patterns of particulate matter and its chemical components at selected sites in california in the united states: a trend analysis over the last years effects of independence day fireworks on atmospheric concentrations of fine particulate matter in the united states coal-to-electricity" project is ongoing in north china analysis of pm . pollution episodes in beijing from to : classification, interannual variations and associations with meteorological features the impact of relative humidity on aerosol composition and evolution processes during wintertime in beijing apec blue": secondary aerosol reductions from emission controls in beijing long-term real-time measurements of aerosol particle composition in beijing, china: seasonal variations, meteorological effects, and source analysis impact of urbanization on the air pollution "holiday effect" in taiwan air pollution "holiday effect" resulting from the chinese new year a joint prevention and control mechanism for air pollution in the beijing-tianjin-hebei region in china based on long-term and massive data mining of pollutant concentration a preliminary assessment of the impact of covid- on environment -a case study of china impacts of regional transport on black carbon in huairou effectiveness of temporary control measures for lowering pm . pollution in beijing and the implications relative impact of emissions controls and meteorology on air pollution mitigation associated with the asia-pacific economic results of investigation on population distribution of beijing's various ring roads. favorite big beijing nitrate dominates the chemical composition of pm . during haze event in beijing trends of multiple air pollutants emissions from residential coal combustion in beijing and its implication on improving air quality for control measures a study on the model for heating influence on pm . emission in beijing china air pollution and control action in beijing mass human migration and beijing"s urban heat island during the chinese new year holiday historic and future trends of vehicle emissions in beijing, - : a policy assessment for the most stringent vehicle emission control program in china long-term trends in no columns related to economic developments and air quality policies from to in china long commutes and transport inequity in china"s growing megacity: new evidence from beijing using mobile phone data effect of the "coal to gas" project on atmospheric nox during the heating period at a suburban site between beijing and tianjin pollution levels, composition characteristics and sources of atmospheric pm . in a rural area of the north china plain during winter human activities and urban air pollution in chinese mega city: an insight of ozone weekend effect in beijing village energy survey reveals missing rural raw coal in northern china: significance in science and policy this work was supported by the national natural science foundation of china (nsfc, no. j o u r n a l p r e -p r o o f key: cord- - i kv p authors: aucejo, esteban m.; french, jacob; araya, maria paola ugalde; zafar, basit title: the impact of covid- on student experiences and expectations: evidence from a survey date: - - journal: j public econ doi: . /j.jpubeco. . sha: doc_id: cord_uid: i kv p in order to understand the impact of the covid- pandemic on higher education, we surveyed approximately , students at one of the largest public institutions in the united states using an instrument designed to recover the causal impact of the pandemic on students’ current and expected outcomes. results show large negative effects across many dimensions. due to covid- : % of students have delayed graduation, % have lost a job, internship, or job offer, and % expect to earn less at age . moreover, these effects have been highly heterogeneous. one quarter of students increased their study time by more than hours per week due to covid- , while another quarter decreased their study time by more than hours per week. this heterogeneity often followed existing socioeconomic divides; lower-income students are % more likely than their higher-income peers to have delayed graduation due to covid- . finally, we show that the economic and health related shocks induced by covid- vary systematically by socioeconomic factors and constitute key mediators in explaining the large (and heterogeneous) effects of the pandemic. the disruptive effects of the covid- outbreak have impacted almost all sectors of our society. higher education is no exception. anecdotal evidence paints a bleak picture for both students and universities. according to the american council on education, enrollment is likely to drop by % in the fall of , while at the same time many institutions may have to confront demands for large tuition cuts if classes remain virtual. in a similar vein, students face an increasingly uncertain environment, where financial and health shocks (for example, lack of resources to complete their studies or fear of becoming seriously sick), along with the transition to online learning may have affected their academic performance, educational plans, current labor market participation, and expectations about future employment. this paper attempts to shed light on the impact of the covid- pandemic on college students. first, we describe and quantify the causal effects of the covid- outbreak on a wide set of students' outcomes/expectations. in particular, we analyze enrollment and graduation from college in the fall semester at more than twice the rate in previous years. historically, % of students who fail to re-enroll do not return to asu or another university after years (authors' calculations from asu first-time freshmen transcript data for the - spring semesters), suggesting that the pandemic may have a lasting impact on the educational achievement of current students. we also find that students report a decreased preference for online instruction as a result of their recent experiences. as expected, the covid- outbreak also had large negative effects on students' current labor market participation and expectations about post-college labor outcomes. working students suffered a % decrease in their wages and a % drop in weekly hours worked, on average. moreover, around % of students lost a job, internship, or a job offer, and % reported to have a family member that experienced a reduction in income. the pandemic also had a substantial impact on students' expectations about their labor market prospects post-college. for example, their perceived probability of finding a job before graduation decreased by almost %, and their expected earnings when years old (around years from the outbreak) declined by approximately . %. this last finding suggests that students expect the pandemic to have a long-lasting impact on their labor market prospects, which is qualitatively consistent with the literature on graduating during a recession. for instance, oreopoulos et al. ( ) and schwandt and von wachter ( ) find significant reductions in earnings and years after graduation, respectively, and kahn ( ) finds an even longer-lasting effect on wages. on the other hand, although we are measuring the probability of finding a job before graduating, not unemployment directly, our estimated quantitative effect on students' expectations of finding a job seems to be larger relative to the literature (kahn, ; altonji et al., ; and rothstein, ) . the data also show that while all subgroups of the population have experienced negative effects due to the outbreak, the size of the effects are heterogeneous. for example, compared to their more affluent peers, lower-income students are % more likely to delay graduation due to and are % more likely to report that covid- impacted their major choice. learning, the impact of covid- on honors students' academic outcomes is consistently smaller than the impact on non-honors students. finally, we evaluate the extent to which mitigating factors associated with more direct economic and health shocks from the pandemic (for example, a family member losing income due to covid- , or the expected probability of hospitalization if contracting can explain the heterogeneity in pandemic effects. we find that both types of shock (economic and health) are systematically correlated with students' covid- experiences. for example, the expected probability of delaying graduation due to covid- increases by approximately % if either a student's subjective probability of being late on a debt payment in the following days (a measure of financial fragility) or subjective probability of requiring hospitalization conditional on contracting covid- increases by one standard deviation. as expected, the magnitude of health and economic shocks are not homogeneous across the student population. the average of the principal component for the economic and health shocks is about . - . standard deviations higher for students from lower-income families. importantly, we find that the disparate economic and health impacts of covid- can explain % of the delayed graduation gap (as well as a substantial part of the gap for other outcomes) between lower-and higher-income students. this analysis should be viewed as descriptive in nature and not necessarily causal, since omitted factors that are correlated both with the shocks and the outcomes may be driving these relationships. to our knowledge, this is the first paper to shed light on the effects of covid- on college students' experiences. the treatment effects that we find are large in economic terms. whether students are overreacting in their response to the covid- shock is not clear. we do find that previous cumulative gpa is a strong predictor of expected semester gpa without covid- , suggesting that students' reported expectations are meaningful. however, we know a total of , respondents completed the survey. respondents were ineligible for the study (such as students enrolled in graduate degree programs or diploma programs) and were dropped from the sample. finally, responses in the st and th percentile of survey duration were further excluded, leading to a final sample size of , . the survey took minutes to complete, on average (median completion time was minutes). the first five columns of table show how our sample compares with the broader asu undergraduate population and the average undergraduate student at other large flagship universities (specifically, the largest public universities in each state). relative to the asu undergraduate population, our sample has a significantly higher proportion of first-generation students (that is, students with no parent with a college degree), and a smaller proportion of international students. the demographic composition of our sample compares reasonably well with that of students in flagship universities. our sample is also positively selected in terms of sat/act scores relative to these two populations. the sample may also differ from the student body at other large public schools in that % report living on campus, which is not always the norm at other large institutions and may play an important role in how disruptive the pandemic has been. the better performance on admission tests could be explained by the high proportion of honors students in our sample ( % compared to % in the asu population). the last four columns of table show how honors students compare with asu students and the average college student at a top- university. we see that they perform better than the average asu student (which is expected) and just slightly worse than the average college student at a top- university. the share of white honors students in our sample ( %) is higher than the proportion in the asu population and much higher than the proportion of white students in the top- universities. overall, we believe our sample of asu students is a reasonable representation of students at other large public schools, while the honors students may provide insight into the experiences of students at more elite institutions. though, it is important to acknowledge that elite institutions we next outline a simple analytic framework that guides the empirical analysis. let ( - ) i o covid be the potential outcome of individual i associated with covid- treatment. we are interested in the causal impact of covid- on student outcomes: where the first term on the right-hand side is student i 's outcome in the state of the world with covid- , and the second term being student i 's outcome in the state of the world without covid- . recovering the treatment effect at the individual level entails comparison of the individual's outcomes in two alternate states of the world. with standard data on realizations, a given individual is observed in only one state of the world (in our case, - = covid ). the alternate outcomes are counterfactual and unobserved. a large econometric and statistics literature studies how to identify these counterfactual outcomes and moments of the counterfactual outcomes (such as average treatment effects) from realized choice data (e.g., heckman and vytlacil, ; angrist and pischke, ; imbens and rubin, ) . instead, the approach we use in this paper is to directly ask individuals for their expected outcomes in both states of the world. from the collected data, we can then directly calculate the individual-level subjective treatment effect. as an example, consider beliefs about end-of-semester gpa. the survey asked students "what semester-level gpa do you expect to get at the end of this semester?" this is the first-term on the right-hand side of equation ( the approach we use in this paper follows a small and growing literature that uses supply. there is one minor distinction from these papers: while these papers elicit ex-ante treatment effects, in our case, we look at outcomes that have been observed (for example, withdrawing from a course during the semester) as well as those that will be observed in the future (such as age earnings). thus, some of our subjective treatment effects are ex-post in nature while others are ex-ante. the soundness of our approach depends on a key assumption that students have well-formed expectations for outcomes in both the realized state and the counterfactual state. since the outcomes we ask about are absolutely relevant and germane to students, they should have well-formed expectations for the realized state. in addition, given that the counterfactual state is the one that had been the status quo in prior semesters (and so students have had prior experiences in that state of the world), their ability to have expectations for outcomes in the counterfactual state should not be a controversial assumption. as evidence that students' expectations exhibit meaningful variation, appendix figure a shows that previous cumulative gpa is a strong predictor of expected semester gpa with covid- . we start with the analysis of the aggregate-level treatment effects, which are presented in table . the outcomes are organized in two groups, academic and labor market (see appendix for example, the average subjective treatment effect of covid- on semester-level gpa is a decline of . points. more than % of the students in our sample expect a decrease in their gpa due to the treatment (versus only % expecting an increase). additionally, on average, % of the participants delayed their graduation, % withdrew from a class during the spring semester, and % stated that their major choice was impacted by while almost no students report planning to drop out due to covid- , on average they expect to take a break from asu in the fall semester at nearly twice the historical rate (historically). admittedly, the decision to take a break during a pandemic may be different than in more normal times. however, a substantial increase in the share of students failing to continue their studies is concerning, as historically % of students who fail to re-enroll for a fall semester do not return to asu or another university within years. regarding the impact of the pandemic on major choice, students who report that covid- impacted their major choice were more likely to be in lower-paying majors before the pandemic; mean pre-covid major-specific annual earnings were $ , ($ , ) for students whose major choice was (not) impacted by impacted students were also . percentage points less likely to be in a science, technology, engineering, or math (stem) major before covid- . we are only able to observe pre-and post-covid major choices for the subset of students who had switched their major by the date of the survey. within this selected subsample of switchers, students chose to move into higher paying majors, with an average change in first-year earnings of $ , . these patterns are generally consistent with the finding that students tend to gravitate towards higher-paying majors when exposed to adverse economic conditions when in college (blom et al., ). table is that, on average, students are percentage points less likely to opt for online instruction if given the choice between online and in-person instruction due to their experience with online instruction during the pandemic. , however, there is a substantial amount of variation in terms of the direction of the effect: % ( %) of the participants are now more (less) likely to enroll in online classes. we explore this heterogeneity in more detail in the next section, but it seems that prior experience with online classes somewhat ameliorates the negative experience; the average treatment effect for students with prior experience in online classes is a . percentage points decrease in their likelihood of enrolling in online classes, versus a . percentage points decline for their counterparts (difference statistically significant at the . % level). this large variation in the treatment effects of covid- is apparent in several of the other outcomes, such as study hours, where the average treatment effect of covid- on weekly study hours is - . (that is, students spend . less hours studying per week due to . the interquartile range of the across-subject treatment effect demonstrates substantial variation, with the pandemic decreasing study time by hours at the th percentile and increasing study time by hours at the th. overall, these results suggest that covid- represents a substantial disruption to students' academic experiences, and is likely to have lasting impacts through changes in major/career and delayed graduation timelines. students' negative experiences with online teaching, perhaps due to the abruptness of the transition, also has implications for the willingness of students to take online classes in the future. turning to panel b in table , we see that students' current and expected labor market outcomes were substantially disrupted by covid- . as for the extensive margin of current employment, on average, % of the students lost the jobs they were working at prior to the pandemic ( % of the students were working prior to the pandemic), % of students had their internships or job offers rescinded, and % of the students reported that a close family member had lost their job or experienced an income reduction. the last statistic is in line with findings from other surveys of widespread economic disruption across the us. respondents experienced an there was no change in weekly earnings for % of the sample, which again reflects substantial variation in the effects of covid- across students. in terms of labor market expectations, on average, students foresee a percentage points decrease in the probability of finding a job by graduation, a reduction of % in their reservation wages, and a . % decrease in their expected earnings at age . the significant changes in reservation wages and expected earnings at age demonstrate that students expect the treatment effects of covid- to be long-lasting. qualitatively, this is broadly consistent with the literature on graduating during recession. oreopoulos et al. ( ) finds that graduating during a recession in which the unemployment rate increases % implies an initial loss in earnings of %, that decreases to . % within years and disappears after years for a sample of male college graduates in canada. similarly, schwandt and von wachter ( ) find a . % reduction in earnings years after graduation for a -percentage point increase in unemployment at graduation, and kahn ( ) finds an even longer-lasting effect on wages. a large literature has investigated the impact of graduating during recessions on unemployment rates. kahn ( ) we next explore demographic heterogeneity in the treatment effects of covid- . figure plots the average treatment effects across several relevant demographic divisions including gender, race, parental education, and parental income. honors college status and cohort are also included as interesting dimensions of heterogeneity in the covid- context. the figure shows the impacts for six of the more economically meaningful outcomes from table (additional outcomes can be found in figure a ). at least four patterns of note emerge from figure . first, compared to their classmates, students from disadvantaged backgrounds (lower-income students defined as those with below-median parental income, racial minorities, and first-generation students) experienced larger negative impacts for the academic outcomes, as shown in the first three panels of the figure. the trends are most striking for lower-income students, who are % more likely to delay graduation due to covid- than their more affluent classmates ( . increase in the proportion of those expecting to delay graduation versus . ), expect % larger negative effects on their semester gpa due to covid- , and are % more likely to report that covid- impacted their major choice (these differences are statistically significant at the % level). for some academic outcomes, covid- had similarly disproportionate effects on nonwhite and first-generation students, with nonwhite students being % more likely to report changing their major preference compared to their white peers and first-generation students being % more likely to delay their graduation than students with college-educated parents. thus, while on average covid- negatively impacted several measures of academic achievement for all subgroups, the effects are significantly more pronounced for socioeconomic groups which were predisposed towards worse academic outcomes pre-covid. the pandemic's widening of existing achievement gaps can be seen directly in students' expected semester gpa. without covid- , lower-income students the cutoff for median parental income in our sample is $ , based on analysis of asu administrative data including transcripts, we find that, relative to their counterparts, first-generation, lower-income, and non-white students drop out at higher rates, take longer to graduate, have lower gpas at graduation, and are more likely to switch majors when in college (see appendix table a ) j o u r n a l p r e -p r o o f second, panel (d) of figure shows that the switch to online learning was substantially harder for some demographic groups; for example, men are percentage points less likely to opt for an online version of a course as a result of covid- , while women do not have a statistically significant change in their online preferences. we also see that honors students revise their preferences by more than . times the amount of non-honors students. as we show later (in table ), these gaps persist after controlling for household income, major, and cohort, suggesting that the switch to online learning mid-semester may have been substantially more disruptive for males and honors students. while the effect of covid- on preferences for online learning looks similar for males and honors students, our survey evidence indicates that different mechanisms underpin these shifts. based on qualitative evidence, it appears that honors students had a negative reaction to the transition to online learning because they felt less challenged, while males were more likely to struggle with the learning methods available through the online platform. one speculative explanation for the gender difference is that consumption value of college amenities is higher for men (however, jacob et al. ( ), find little gender difference in willingness to pay for the amenities they consider). the third trend worth highlighting from figure is that honors students were better able to mitigate the negative effect of covid- on their academic outcomes (panels a, b, and c), despite appearing to be more disrupted by the move to online learning (panel d). honors students report being less than half as likely as non-honors students to delay graduation and change their major due to covid- . extrapolating from these patterns provides suggestive evidence that academic impacts for students attending elite schools-the group more comparable to these honors studentsare likely to have been small relative to the impacts for the average student at large public schools. finally, the last two panels of figure present the covid effect on two labor market expectations and show much less meaningful heterogeneity across demographic groups compared to the academic outcomes in previous panels. this suggests that, while students believe covid- will impact both their academic outcomes and future labor market outcomes, they do the difference is significant at % in both cases. honors students were as likely as non-honors students to say that classes got easier after they went online but, conditional on saying classes got easier, were % more likely to say "homework/test questions got easier." conversely, males were marginally more likely to say classes got harder after they went online ( % more likely, p= . ) and, conditional on this, were % more likely to say that "online material is not clear". the one notable exception to the lack of heterogeneity in panels (e) and (f) of figure are seniors, who on average revised their subjective probability of finding a job before graduation three times as much as other cohorts. figure a further breaks down the estimated covid- effects by expected year of graduation. perhaps unsurprisingly, the cohort expects much larger effects on immediate job market outcomes such as reservation wages and probability of finding a job before graduation. while average expected changes to job market outcomes are noisier for academically younger students, perhaps reflecting additional uncertainty about the longer-term impacts of covid- , they appear to anticipate meaningful changes to their future labor market prospects. conversely, younger students also expected larger disruptions to academic outcomes such as semester gpa and study time. this section presents mediation analysis on the drivers of the underlying heterogeneity in the treatment effects. the covid- pandemic serves as both an economic and a health shock. however, these shocks may have been quite heterogeneous across the various groups, and that could partly explain the heterogeneous treatment effects we documented in the previous section. we proxy for the financial and health shocks due to covid- by relying on a small but relevant set of covariates which capture more fundamental or first-order disruptions from the pandemic. financial shocks are characterized based on whether a student lost a job due to covid- , whether a student's family members lost income due to covid- , the change in a student's monthly earnings due to covid- , and the likelihood a student will fail to fully meet debt payments in the next days. to measure health shocks, we consider a student's belief about the likelihood that they will be hospitalized if they contract covid- , a student's belief about the likelihood that they will have contracted covid- by summer, and a student's subjective health table reports summary statistics of the different economic and health proxies by demographic group. given the results in figure , the remainder of the analysis will focus on three socioeconomic divisions: parental income, gender, and honors college status. our data indicate that lower-income students faced larger health and economic shocks as compared to their more affluent peers. in particular, they are almost percentage points more likely to expect to default on their debt payments compared to their higher-income counterparts. additionally, lower-income students are percentage points more likely to have had a close family member experience an income reduction due to covid- . regarding the health proxies, lower-income students rate their health as worse than higher-income students and perceive a higher probability of being hospitalized if they catch the virus. finally, the differences in economic and health shocks between lower and higher-income students, as summarized by the principle components of the selected proxy variables, are statistically significant. columns ( )- ( ) of table show that both economic and health shocks are larger for non-honors students. in fact, the average differences in the principal component scores for both the economic and health factors is larger for these two groups than for the income groups. likewise, the last three columns of the table show that women experienced larger covid- shocks due to economic and health factors. these differences are partly driven by the fact that, in our sample, females are more likely to report that they belong to a lower-income household than males ( % vs. %). in short, table makes clear that the impacts of covid- on the economic well-being and health of students have been quite heterogeneous, with lower-income and lower-ability students being more adversely affected. to investigate the role of economic and health shocks in explaining the heterogeneous eigenvalues indicate the presence of only one principal component for each of the shocks. table shows estimates of equation ( ) for four different outcomes (appendix table a shows the estimates for additional outcomes). for each outcome, five specifications are reported ranging from controlling for only demographic variables in the first specification to controlling for both economic and health factors in the fourth specification. finally, the last column includes only the principal component of each shock to provide insight about overall effects, given that certain shock proxies show high levels of correlation (see appendix table a for the correlations within each set of proxies). several important messages emerge from table . first, both shocks are (economically and statistically) significant correlates of the covid- effects on students' outcomes. in particular, f-tests show that the financial and health shock proxies are jointly significant across almost all specifications. this is also reflected in the statistical significance of the principal components. moreover, the fact that the effect of key proxy variables remains robust when we simultaneously control for both shocks demonstrates the robustness of our results. for example, we find that a percentage point increase in the probability of being late on debt payments is associated with an increase in the probability of delaying graduation and switching majors due to covid- of . and . percentage points, respectively. these effects are large given that they represent more than the only exception is the financial shock when explaining changes in the probability of taking classes online. journal pre-proof half of the overall covid- treatment effect for these variables. similarly, we find that an analogous increase in the probability of hospitalization if contracting covid- is associated with a and percentage points increase in the probability of delaying graduation and switching majors due to covid- . second, in terms of labor market expectations, we find that the change in the expected probability of finding a job before graduation strongly depends on having a family member that lost income (which is also correlated with the student himself losing a job). in particular, the size of this effect represents % of the overall covid- treatment effect. therefore, this finding suggests that students' labor market expectations are driven in large part by personal/family experiences. third, although the proxies play an important role in explaining the pandemic's impact on students, there is still a substantial amount of variation in covid- treatment effects left unexplained. across the four outcomes in table , the full set of proxies explain less than a quarter of the variation in outcomes across individuals. appendix figure a visualizes this variation by plotting the distribution of several continuous outcomes with and without controls. while the interquartile range noticeably shrinks after conditioning on the proxy variables, these plots highlight the large amount of variation in treatment effects remaining after conditioning on the proxies. finally, our results show that the financial and health shocks play an important role in explaining the heterogeneous effects of the covid- outbreak. in particular, columns ( ) and ( ) demonstrate that economic and health factors together can explain approximately % and % of the income gap in covid- 's effect on delayed graduation and changing major respectively. the gap between honors and non-honors students is likewise reduced by % and % for the same outcomes. taken together, these results imply that differences in the magnitude of covid- 's economic and health impact can explain a significant proportion of the demographic gaps in covid- 's effect on the decision to delay graduation, the decision to change major, and preferences for online learning. these results are important and suggest that focusing on the needs of students who experienced larger financial or health shocks from covid- may be an effective way to minimize the disparate disruptive effects and prevent covid- from exacerbating existing achievement gaps in higher education. journal pre-proof this paper provides the first systematic analysis of the effects of covid- on higher education. to study these effects, we surveyed , students at arizona state university, and present quantitative evidence showing the negative effects of the pandemic on students' outcomes and expectations. for example, we find that % of students have delayed graduation due to covid- . expanding upon these results, we show that the effects of the pandemic are highly heterogeneous, with lower-income students % more likely to delay graduation compared to their higher-income counterparts. we further show that the negative economic and health impacts of covid- have been significantly more pronounced for less advantaged groups, and that these differences can partially explain the underlying heterogeneity that we document. our results suggest that by focusing on addressing the economic and health burden imposed by covid- , as measured by a relatively narrow set of mitigating factors, policy makers may be able to prevent covid- from widening existing achievement gaps in higher education. notes: bars denote % confidence interval. notes: data in columns ( ), ( ) and ( ) *significant at %, ** %, *** %. a it refers to the mean of the first factor of a pca that uses the measures in the corresponding panel. b through scale where higher numbers mean better health. notes: p-value columns report the p-value of a difference in means test between the two columns indicated by the numbers in the heading. notes: data winsorized below % and above %. controls include cohort fixed effects, major fixed effects, and the economic/health proxies in table *significant at %, ** %, *** %. figure notes: table reports correlation matrix for indicated variables key: cord- -qy lccjq authors: mubagwa, kanigula title: chloroquine cardiac effects and toxicity.a short update. date: - - journal: int j antimicrob agents doi: . /j.ijantimicag. . sha: doc_id: cord_uid: qy lccjq there is currently an increased interest in using the antimalarials chloroquine and hydroxychloroquine for the treatment of other diseases, including cancer and viral infections such as covid- . however, risks of cardiotoxic effects tend to limit their use. the effects of these drugs on the electrical and mechanical activities of the heart, as well as on the remodeling of the cardiac tissue are presented, and the underlying molecular and cellular mechanisms discussed. the drugs can have proarrhythmic as well as antiarrhythmic actions, resulting from their inhibition of ion channels, including voltage-dependent na(+) and ca( +) channels, background and voltage-dependent k(+) channels, and pacemaker channels. the drugs also exert a vagolytic effect, due at least in part to a muscarinic receptor antagonist action. they also interfere with the normal autophagy flux, an effect which could aggravate ischemia/reperfusion injury or post-infarct remodeling. most of the toxic effects occur at high concentrations, following prolonged drug administration or in the context of drug associations. -the antimalarials chloroquine and hydroxychloroquine have been proposed for antiviral therapy, including for covid- . -their use is limited for fear of cardiotoxic effects. -the mechanisms underlying cardiac chloroquine effects include direct actions on ion channels and receptors, while others involve an inhibition of autophagy. -despite the effects on many ion channels, the drugs are not associated with prominent qt prolongation or life-threatening arrhythmias when used for a short duration and at low concentrations. -side effects are usually associated with long-term treatments or with very large doses. -the drugs are relatively safe, but care should be taken to avoid association with other drugs with known toxic effects on the myocardium. chloroquine (and its related drug hydroxychloroquine), introduced more than years ago and used traditionally to treat malaria or chronic inflammatory diseases, has re-emerged as possible therapeutic agent in viral diseases including covid- [ ] . renewed interest in this drug is not novel, as there have also been assessments of its potential effects as anti-cancer [ , ] , anti-arrhythmic [ ] or pulmonary vasodilator [ ] agent. whenever considering to (re)use a drug, one should weigh the benefits against the risks (mainly the side effects) associated with the drug use. the usefulness of chloroquine/hydroxychloroquine in the treatment of covid- is still being currently debated [ , ] . whereas on the one hand there is little doubt that chloroquine and hydroxychloroquine can display antiviral action in vitro [ , [ ] [ ] [ ] and in vivo [ ] , on the other hand controversy exists on whether this effect is of use in covid- patients. clinical studies are under way to answer the question concerning an anti-covid- protective action in humans [ ] . while waiting for a clear answer to this issue, it is necessary to reflect on risks of using the drug. the effects of chloroquine/hydroxychloroquine, especially those related to the immunomodulatory action have been reviewed very recently [ ] . among side effects of chloroquine/hydroxychloroquine, those involving the heart are frequently invoked as being life-threatening, and cardiotoxicity is a major concern for many other antimalarial drugs [ , ] . cardiotoxic effects have been presented as arguments against recent trials to repurpose these drugs for the treatment of covid- [ ] . in the present note, we describe the major known cardiac effects of the drugs, with the aim to determine which effects can be expected to occur at usual therapeutic concentrations and account for observed beneficial or side effects. in the presentation we do not make a distinction between the effects of chloroquine versus those of hydroxychloroquine, the underlying assumption being that, despite potential pharmacokinetic and potency differences, their pharmacological actions are essentially identical. we suggest ) that major side effects occur at high concentrations (e.g. following poisoning), after long-term treatment or in the context of drug associations, and ) that common effects after short-term treatment with low clinical doses are generally well tolerated. we also comment on the cellular or molecular mechanisms underlying the effects. chloroquine/hydroxychloroquine can be administered via parenteral or oral routes. acute toxic effects were recorded more frequently when the drugs were given via the parenteral (especially the intravenous) route [ ] . drug absorption is practically complete even when administered via the oral route, but the concentration profile reaches lower peak levels post enteral than post parenteral administration [ ] . the drugs accumulate into cells, due to ion trapping in more acidic environments, especially in lysosomes [ , ] , where the concentration can be many orders of magnitude higher than in the blood. the biological half-life is as long as - days [ , ] . hence there is possibility for progressive drug accumulation on repeated administration even of small doses, and the drug effects also develop following different time scales: from seconds or minutes, to hours or days, and to many months or years. there are many case reports of rapidly developing cardiovascular effects following intoxication by chloroquine/ hydroxychloroquine, ranging from bradycardia and hypotension to eventual cardiac arrest [ ] . as mentioned, fear or real risks of cardiotoxicity has limited the use of these drugs. cardiotoxic effects associated with chloroquine/hydroxychloroquine use in clinical conditions have been recently reviewed [ , ] . here we summarize major effects detected under either clinical or experimental conditions, and have grouped them into ) purely functional (electrophysiological and mechanical) effects, and ) those associated with structural changes. electrophysiological and mechanical effects effects on the cardiac rhythm and on the conduction and duration of the electrical activity electrocardiographic (ecg) changes associated with chloroquine/hydroxychloroquine treatment have been reported as proarrhythmic in some studies, or as antiarrhythmic in others. rhythm and electrophysiological effects of chloroquine/hydroxychloroquine include bradycardia as well as tachycardia, flattening of the t wave, prolongation of the qt interval, and various forms of conduction blocks. for example, sinus dysfunction with bradycardia [ ] and atrioventricular (av) or intraventricular conduction abnormalities [ ] were noted to be frequent in rheumatoid arthritis and lupus erythematosus patients treated with chloroquine/hydroxychloroquine. in a review of case reports till , conduction disturbances were the most prevalent among all side effects [ ] . however, although arrhythmias in such patients have been traditionally attributed to the drugs, a role of the underlying diseases (as direct cause or as favoring factor) cannot be excluded. indeed, in one study, ventricular conduction blocks observed in chloroquine/hydroxychloroquinetreated patients had a prevalence that was not different from the one in those not receiving these drugs [ ] . given the role played by the duration of ventricular depolarization (measured as qt interval on the ecg) in arrhythmogenesis, it has been of interest to examine whether chloroquine/hydroxychloroquine increase the qt duration. qt prolongations have been noted under clinical conditions with high chloroquine concentrations ( mg base.kg - .day - ; [ ] ) or in experimental conditions using parenteral administration [ ] . the qt prolongation is dominated by the effect on the qrs duration, and marginal dose-dependent prolongation is seen on the j-t interval [ ] , which measures more accurately the duration of depolarization. in most studies no or only marginal qt prolongation is induced by clinical doses (< mg.kg - .day - ) of chloroquine/hydroxychloroquine, especially using the oral route [ , ] , suggesting that chloroquine/hydroxychloroquine are in this respect safer than other quinoline-based antimalarial drugs (e.g. halofantrine [ ] ). clinically, low chloroquine doses ( - mg.kg - .day - ) were associated with significant qt prolongation when given over many months [ ] , suggesting a role of drug accumulation or of myocardial remodeling. in contrast to the pro-arrhythmic actions of chloroquine/ hydroxychloroquine, an antiarrhythmic action, known since the middle of the past century [ , ] , can be further demonstrated from experimental animal data [ ] and from clinical data of patients treated for chronic inflammation [ ] . recently, it could be shown that chloroquine ( - mg.day - ) can cause conversion to sinus rhythm in a patient with established atrial fibrillation [ ] . such an antiarrhythmic action is not unexpected given the drug action on voltage-dependent na + channels (see below). it should be noted that the "janus-faced" (pro-and anti-) arrhythmic action is also known for classic antiarrhythmics [ ] . the effect of chloroquine/ hydroxychloroquine on cardiac contraction remains unclear. early studies in amphibian hearts suggested that chloroquine exerts a positive inotropic effect, attributed to an indirect action due to the activation of brain sympathetic centers [ ] . similarly, a transient positive inotropic action was obtained in guinea-pig atria [ ] . it was suppressed by pretreatment with reserpine or propranolol, indicating that it was due to a release of catecholamines, which then acted on cardiomyocyte beta-adrenergic receptors [ ] . most subsequent studies in mammalian hearts either failed to show any inotropic action or demonstrated a negative action. no change of cardiac mechanical function assessed using echocardiography was observed in rats injected with mg.kg - .day - chloroquine for weeks [ ] . in contrast, negative inotropism was evident in atrial muscles, but only at concentrations above µm [ , ] . similarly, in guinea-pig cardiac muscle chloroquine decreased the contraction at - µm in isolated atria [ ] . in rat isolated left ventricular papillary muscles, chloroquine ( µm) was found either to have no effect [ ] or to decrease contractile force and the rates of shortening and relaxation [ ] . in langendorff-perfused guinea-pig ventricles chloroquine decreased the contraction at concentrations above µm [ ] , but this apparently higher potency in ventricular muscle could be an artifact due to a time-dependent rundown of the contraction in this preparation. in rat isolated perfused hearts, systolic function was decreased with chloroquine concentrations > µm and was completely suppressed at µm [ ] . this negative inotropic action was associated with a decrease in heart rate. unless heart rate is controlled it is difficult to accurately determine the inotropic effect since part of the decrease in contraction may be related to a positive force-frequency relation prevailing in the preparation. in addition, at high chloroquine concentrations loss of excitability and suppressed conduction (due to chloroquine effects on the upstroke of the action potential) might account for an apparent loss of contraction. thus, negative inotropic of chloroquine/hydroxychloroquine is typically obtained at high drug concentrations, above those reached by usual therapeutic regimens. the mechanism underlying the reduction of contraction could be related to the inhibition of i ca-l discussed below. it has been noted that the effects of chloroquine ( . mg. ml - , i.e. about µm) to reduce contractility in turtle hearts is similar to those of quinine, and that for both drugs, the effect is reversed by raising the external ca + concentration [ ] . this points to a role of decreased excitability and/or of decreased ca + influx, and makes less likely an effect on myofilament ca + sensitivity. however, in guinea-pig hearts [ ] , increasing external ca + was not able to fully reverse the chloroquine-induced contraction decrease. contractions obtained while inactivating na + channels with high external k + were as sensitive to chloroquine as those recorded in normal conditions, whereas post-rest potentiation of contraction was not affected by the drug. these data point to a role of diminished ca + influx in the negative inotropism of chloroquine. cardiovascular shock is common following chloroquine poisoning [ ] , but it is not clear whether the condition is mainly due to vascular or to cardiogenic mechanisms. since cardiogenic mechanisms can be primarily arrhythmic and not necessarily primarily inotropic, the contribution of decreased inotropy is unclear. it is of note that, beside studies of the mechanical dysfunction related to chloroquine/hydroxychloroquine cardiomyopathy, there are no studies that report on eventual acute inotropic effects in human patients or volunteers. a dose-dependent decrease of systolic pressure can also be demonstrated in experimental animals intoxicated with intravenous chloroquine [ ] . the hemodynamic failure was associated with decrease of maximum rates of pressure development or relaxation, bradycardia and qrs widening. this suggests the participation of an important non-vascular, cardiac component. chronic treatment (usually over many months or years) with chloroquine/ hydroxychloroquine can result in cardiomyopathy [ ] , characterized by wall thickening and microscopic structural changes. cardiomyopathy seems to occur more with chloroquine than with hydroxychloroquine, and involves at the microscopic level an enlargement and vacuolization of cardiac myocytes, with deposition of myeloid and curvilinear bodies. the cardiomyopathy is accompanied by functional changes, including av or bundle-branch conduction block [ ] as well as mechanical dysfunction, with diffusive hypokinesia and decreased ejection fraction [ ] . chloroquine cardiomyopathy can develop in the absence of skeletal myopathy or of retinal toxicity, although similar cellular mechanisms might be involved. in the context of short-term treatment, these effects are not expected. other structural changes occurring in the myocardium are not primarily due to chloroquine/hydroxychloroquine but are modified by these drugs. myocardial remodeling involves structural and functional changes triggered by many stress factors, including physiological conditions such as physical exercise, pregnancy or fetal body growth, as well as pathological conditions such as ischemia/reperfusion (infarction), pressure or volume overload, inflammation, metabolic factors (e.g., hyperglycemia and hyperlipidemia), reactive oxygen radicals [ ] . physiological remodeling is adaptive and lead to hypertrophy to increase function in athletes, pregnant women and growing fetus. in contrast, remodeling following pathological triggers is usually maladaptive, as it evolves with time towards heart failure and increased propensity to arrhythmias. characteristic structural changes during pathological remodeling include increase in cardiomyocyte size (causing hypertrophy or dilatation) and ultramicroscopic structure (e.g. loss of t-tubules; [ ] ), modifications of the extracellular matrix, proliferation of myofibroblasts with development of fibrosis. after myocardial infarction, concomitantly with the development of fibrosis and formation of a scar in the infarcted zone there are changes in the infarct border and in the remote non-infarcted myocardium. with time the heart undergoes chamber dilatation. functional changes in remodeled myocardium include modifications in the electrical properties (called electrical remodeling), as a result of altered expression of ion channels (e.g., kir k + channels, l-type ca + channels, connexins, ryanodine receptors, etc.), transporters (e.g., na + -ca + -and exchangers, sarcoplasmic reticulum atpases, etc.) and other proteins, which impact on the action potential and its conduction within the cardiac tissue, on the excitation-contraction coupling, and on the contractile properties. electrical remodeling is responsible for the increased propensity to arrhythmias. many weeks following myocardial infarction, there is progressive deterioration of myocardial function with increased left ventricular end-diastolic volume, diastolic dysfunction and decreased systolic function. many processes and cell types are implicated in the remodeling process [ , ] . inflammation, with the associated release of mediators, cytokines and chemokines by inflammatory cells, play an important role in the remodeling process, and an experimental model of inflammation-induced remodeling, resulting in dilated cardiomyopathy, is obtained in mice overexpressing tumor necrosis factor, tnf [ ] . the effect of chloroquine on remodeling does not necessarily involve a direct action on cardiomyocytes, and may involve primary effects on other cardiac cells such as fibroblasts. during post-infarct remodeling cardiac fibroblasts are activated and converted to myofibroblasts, which display a higher proliferative and secretory capacity. in the fibroblasts chloroquine significantly reduced the expression of proteins such alpha smooth muscle actinin (α-sma) and fibronectin. in myofibroblasts chloroquine reduced cell migration and contractility [ ] . chloroquine/hydroxychloroquine may also affect the electrical remodeling. as far as ion channel remodeling is concerned, reduced amplitude and slightly modified kinetics of voltage-dependent sodium current were evident in epicardial cells of the infarct border zone, and these changes were supposed to play a role in decreased conduction underlying reentrant arrhythmias [ ] . chloroquine ( µm), in addition to displaying its inhibitory action on the sodium current, was shown to completely suppress the camp-dependent kinase (pka)-mediated restauration of the ion current in the cells from the infarct border zone [ ] . remodeling in infarcted hearts can be reduced by calorie restriction or pharmacologic agents such as resveratrol or rapamycin, which probably act by promoting autophagy [ ] . chloroquine suppressed the anti-remodeling effects of food restriction and of resveratrol. this was shown in mice that developed ventricular dilation and dysfunction a few days/weeks after induction of myocardial infarction by left coronary artery ligation. restriction by - % of the food intake, started week after infarction and maintained for weeks, was accompanied by an improvement of the cardiac chamber size and of most contractile parameters [ ] . in addition the increase in cell size was also attenuated. treatment with chloroquine ( mg/kg - .day - , given by continuous subcutaneous injection) for weeks reversed the improvement afforded by food restriction. like the effect of food restriction, a two-week treatment with resveratrol significantly attenuated the ventricular dilation and improved cardiac function in mice that underwent left coronary artery ligation. this protective effect was absent in mice treated with chloroquine ( mg.kg - .day - ) simultaneously with resveratrol [ ] . as discussed below, these effects of chloroquine are apparently due to a suppression of observed increases in autophagy by food restriction or drugs in the cells. while chloroquine appears to have deleterious effect in the remodeling induced by primary cardiac lesions, the drug may have a protective action on remodeling of the right heart associated with pulmonary arterial hypertension. rats in which this condition was induced (by monocrotaline) and which were intraperitoneally injected with either chloroquine ( or mg.kg - .day - ) or hydroxychloroquine ( mg.kg - .day - ) for weeks displayed less right ventricular hypertrophy and improved function due to a decrease of pulmonary vascular resistance. in this case the remodeling in the pulmonary vasculature was suppressed by chloroquine/hydroxychloroquine [ ] . exploring the cellular and molecular mechanisms of chloroquine/hydroxychloroquine effects allows to understand possible synergy or antagonism with other drugs administered simultaneously or the interaction with changes already induced by pathological conditions. inactivation of voltage-dependent na + channels: local anesthetic effect chloroquine is structurally close to quinine and quinidine, which act on voltage-dependent na + (na v ) channels: they favor an inactivated state of the channels, therefore making them less available for opening upon depolarization (excitation). chloroquine exerts similar "local anesthetic" effects [ ] . given the involvement of na v channels in the genesis and transmission of the electrical impulse in nerves, chloroquine can suppress pain sensation as do classic local anesthetics, but the drug is not used for this effect because of its anticoagulant effect. na v channels are present in other excitable cells, including cardiac atrial contracting cells and all ventricular cell types, where they are responsible for the upstroke (phase ) of the action potential. chloroquine decreases all indices of na v channel contribution to the cardiac action potential: the maximal rate of depolarization during phase and the action potential amplitude [ ] , both of which determine the rate of conduction within the tissue. side effects concerning intraventricular conduction (qrs prolongation, bundle branch block) observed with the drugs [ , ] are likely explained on this basis. a persisting, late na + current is among determinants of the action potential duration and favors arrhythmogenesis [ ] . it contributes to intracellular na + overload and the subsequent na + -ca + exchange-mediated ca + overload during ischemia. suppression of this current by chloroquine [ ] may counterbalance the effect of blocking k + currents on the qt duration, and partly account for antiarrhythmic and anti-ischemic protective actions. decrease of the l-type ca + current chloroquine/hydroxychloroquine inhibit l-type ca + channels, since they decrease the amplitude of i ca-l in sino-atrial (sa) [ ] and in working myocardial cells [ ] . the effect in sa cells likely contributes to the slowing of the pacemaker activity. similar effects are expected in av nodal cells and can explain av conduction block. given the role of i ca-l in cardiac excitation-contraction coupling, negative inotropic action of the drugs [ , ] might also be explained on this basis, although most studies report a lack of significant inotropic effect at clinically useful concentrations. cardiac automatic activity is due to the presence of a spontaneous depolarization of nodal and conduction cells during the diastolic time. many processes contribute to this diastolic depolarization [ , ] , among them the opening of hyperpolarization-activated channels [ ] [ ] [ ] . these channels are called "funny channels" and the corresponding current "funny current, i f ", because of the apparently strange behavior in comparison with other channels, which usually open upon depolarization. i f amplitude (measured during hyperpolarizing steps under voltage-clamp) was decreased by hydroxychloroquine [ ] . there was no change in voltage-dependent activation, suggesting that the drug acts on the channel conductance, but the underlying molecular mechanism is unknown. this inhibitory action on i f was translated into a decrease of the rate of diastolic depolarization and of the beating rate of sa isolated cells or of atrial multicellular preparations [ , ] . based on its effect to decrease i f , hydroxychloroquine was proposed as a relatively safe bradycardic agent [ ] , and bradycardia was obtained with intravenous injections in anesthetized animals, without negative inotropic or hemodynamic action. therefore, the bradycardic effect was proposed to be of potential value in decreasing oxygen demand, of benefit in conditions such as angina. it should be noted that bradycardia occurred within a few (typically ) minutes using hydroxychloroquine in anesthetized animals, while tachycardia is observed for hours after a single dose of chloroquine in awake human volunteers (see below). it is likely that in humans the fast developing rate-slowing action on pacemaker tissue is followed and opposed by a slowly developing effect due to an interaction with the autonomic nervous system (see below). chloroquine also acts on various k + channels [ , [ ] [ ] [ ] [ ] [ ] [ ] . chloroquine blocks kir . inward-rectifier channels, largely responsible for the background conductance and i k current [ , ] that set the resting potential but also contribute to the repolarization and the duration of the action potential. the underlying mechanism involves an accession of chloroquine from the intracellular side and its binding near the intracellular mouth of the pore [ ] . there appears to be a dual action of chloroquine on kir . channels. in contrast to the acute decrease of channel conductance, an increase in the plasmalemmal expression of the channel protein has been reported in hek cells used as expression system [ ] . this effect developed over hours and was related to the inhibition of the lysosomal degradation of the channels. to our knowledge no study has tested this effect in cardiomyocytes, but such an increase in channel expression could have a corrective action against the decrease due to block, and partly contribute to limit qt prolongation. chloroquine/hydroxychloroquine also block the g-protein-activated inward rectifier k + (girk) channel present in cardiac supraventricular (atrial contracting and nodal) cells. the channel is also present in ventricular cells of some species but practically absent in human ventricular contracting cells. it is activated following acetylcholine (ach, the neurotransmitter of the parasympathetic system) binding on m muscarinic receptors, or adenosine binding on a receptors, hence the names i k-ach or i k-ado for the corresponding ion currents. the blocking action of chloroquine is also observed in hek cells expressing kir . and kir . channels, which contribute subunits forming girk channels [ , ] . the underlying mechanism is similar to that of kir . (i k ) channels described above: it involves the binding from the intracellular side to a site within the internal mouth of the channel. the ic of chloroquine for both channels was µm, a concentration similar to the plasma concentrations following oral doses of about mg.kg - . the blocking effect on i k , i k-ach or both should result in a lengthening of the action potential duration. as mentioned earlier no or only marginal qt prolongation is induced by low doses of chloroquine/hydroxychloroquine given via the oral route [ , ] . given that action potential shortening is associated with arrhythmogenesis, the action potential lengthening effect of chloroquine may be antiarrhythmic under pathological conditions which markedly shorten the action potential duration. such an antiarrhythmic effect is mostly obvious in case of short qt syndrome due to mutations of kir . channels [ ] , but it was also shown to occur in sheep hearts with sustained atrial fibrillation induced by chronic pacing [ ] . under the latter condition i k-ach is spontaneously activated in the absence of agonist binding on g-protein coupled receptors. chloroquine decreased the dominant fibrillation frequency and finally induced a conversion to sinus rhythm [ ] , an effect that was associated with the prolongation of the atrial action potential and could be reproduced by a toxin (tertiapin) known to suppress i k-ach . chloroquine/hydroxychloroquine also act on atp-sensitive k + channels. these channels monitor the cell metabolism and open following a decrease of intracellular atp and an increase of adp, as occur in conditions such as ischemia. in the heart the channels are formed by a pore unit made of kir . and a sulfonylurea-binding regulatory unit, sur a. chloroquine blocks the atp-sensitive current (i k-atp ) activated pharmacologically (by pinacidil) in ventricular cardiomyocytes [ ] and the corresponding channels units expressed in hek cells [ ] . the suppression involved two mechanisms: a pore block by chloroquine entering from the intracellular side and occluding ion movement within the channel, and an inhibition, given the amphiphilic character of chloroquine, of the channelactivating interaction between kir . and the membrane phospholipid phosphatidyl-inositol bisphosphate (pip ). i k-atp inhibition in cardiac myocytes occurred at micromolar concentrations (ic : . µm), indicating that chloroquine at therapeutic doses could also act on the heart through mechanisms involving atp-sensitive channels, e.g. to antagonize fibrillation following action potential shortening and inhomogeneity during ischemia. in sa node cells, purkinje fibers and ventricular myocytes, hydroxychloroquine was shown to decrease the amplitude of the rapid delayed rectifier i kr by % [ ] . the drug did not affect the slow component i ks . the effect on i kr is consistent with the inhibition of herg channels in expression systems (ic : . - . m) and could contribute to qt prolongation, especially at high concentrations [ , ] . channels carrying the transient outward current, i to , and other channels a transient outward current, i to , underlies phase- early repolarization in cardiac cells. it is a mixed current, made of a ca + -independent but voltage-dependent k + component, and a ca + -activated clcomponent [ , ] . chloroquine does not seem to have potent effect on i to [ ] . inhibitory effects occurred only at very high concentrations (k d : µm) [ ] ), which are not reached under therapeutic conditions. the effect on other (chloride-selective and non-selective sarcolemmal, sarcoplasmic reticulum, and mitochondrial) cardiac channels have not, to our knowledge, been investigated. in contrast to the slowing of the heart rate by chloroquine observed under experimental conditions as expected from the suppression of i f and i ca-l [ ] , the drug increases heart rate during more than hours after a single per os dose of . - mg.kg - in healthy human volunteers [ ] . this latter effect may be attributed to a dual effect on the autonomic nervous system. on the one hand there might be an activation of heart accelerating mechanisms resulting from a stimulation of the sympathetic system [ ] , but such a mechanism has not been demonstrated in humans. on the other hand there is inactivation of heart decelerating mechanisms resulting from an inhibition of the parasympathetic (or vagal) system. this latter effect has been more thoroughly investigated by the author. the heart rate increase was associated with a decrease of cardiovascular reflexes used as indices of the vagal influence on the heart. one of the most readily assessable parameters of the autonomic nervous control of the heart is the beat-to-beat variation of the heart cycle. chloroquine caused a narrowing of the average dispersion between heart cycle durations. other reflexes, such as the cardiac cycle increase during a valsalva maneuver or post deep inspiration, were also decreased, indicating that chloroquine exerts a vagolytic action on the heart. this effect is dose-dependent and its underlying mechanism seems to involve, at least in part, a direct atropine-like, antagonist action of chloroquine on muscarinic receptors. chloroquine antagonized the negative chronotropic effect of muscarinic receptor activation under experimental conditions using animal isolated hearts [ ] . chloroquine is known to bind on muscarinic receptors (k d : - µm [ ] ). its binding on cardiac muscarinic receptors could be demonstrated by its displacement of saturation binding curves of a radioactive label of these receptors [ ] . however, this interaction with receptors occurred at rather higher concentrations than those interfering with the negative chronotropic effect of muscarinic agonists. thus, despite the demonstrated interaction of chloroquine with muscarinic receptors, the mechanism of chloroquine effect on the heart rate appears to be complex. as discussed above, effects on channels, including a block of i k and i k-ach , which contribute to the regulation of the heart rate, could play a role and partly account for the increase of heart rate caused by chloroquine. besides the effects described above, which basically occur at the surface membrane, chloroquine also targets intracellular processes, mainly (but not exclusively) those involving the intracellular processing of proteins by autophagy. autophagy is an important homeostatic mechanism, which is responsible for the clearance of damaged intracellular protein complexes and organelles [ , ] , including in the heart. it plays an important role in the response to stress and in disease conditions such as ischemia/reperfusion [ , ] , hypertrophy [ ] , heart failure [ , ] , etc. drug-induced changes in autophagy may also underlie therapeutic or toxic effects. macro-autophagy involves the fusion of autophagosomes with lysosomes, to constitute autolysosomes, where cargo brought by the phagosomes is digested. chloroquine, which accumulates into lysosomes by ion trapping [ , ] and causes alkalization of the intralysosomal ph, inhibits the fusion between autophagosomes and lysosomes and the basic macro-autophagy flux [ ] . hearts pre-treated with chloroquine ( mg.kg - .day - , a dose within clinical range) for days show a decreased autophagy flux, with accumulation of autophagosomes. another type of autophagy, which involves chaperone-mediated transport of abnormal proteins into lysosomes, also appears to be inhibited by chloroquine [ ] . given the importance of ischemic diseases in cardiac pathology, it is of interest that ischemia/reperfusion is reported to modify autophagy, and that chloroquine has a potential to modulate the changes occurring under these conditions. an excessive decrease or increase of autophagy beyond its basal level may be expected to have harmful consequences. autophagy is upregulated during myocardial ischemia and reperfusion [ ] . whereas during the ischemia phase activation is adenosine monophosphate-activated kinase (ampk)-dependent, adaptive and protective, during reperfusion it is ampkindependent, excessive and it enhances myocardial death. protection during ischemia is likely due to the ability of autophagy to deliver metabolites and to clear damaged organelles such as mitochondria (mitophagy). excessive autophagy and cell death during reperfusion are attributed to the formation of reactive oxygen radicals. autophagy inhibition by chloroquine could limit the adaptive increase during ischemia and aggravate the condition. this is the case in isolated rats submitted to episodes of intermittent hypoxia for many weeks. in this experimental model set to mimic the condition of sleep apnea, inhibition of autophagy with chloroquine was associated with a development of myocardial dysfunction, while no dysfunction was present with intermittent hypoxia alone [ ] , suggesting that autophagy provided cardiac adaptive mechanisms to hypoxia. in contrast, attenuation of the excessive increase of autophagy during reperfusion could a priori be expected to confer protection. however, reperfusion by itself, despite upregulating autophagy, is associated with decreased clearance of autophagosomes [ ] . further suppression of autophagosome clearance by chloroquine can therefore aggravate this process. increased cell death caused by chloroquine during ischemia/reperfusion has been linked to a decrease of oxygen radical buffering action of mitochondria [ ] . chloroquine could be expected to be protective in other conditions that enhance autophagy flux, such as cardiac hypertrophy. the drug reversed the myocardial wall thickening caused by pressure overload in rats that underwent ascending aorta banding. it also appears to confer protection against ischemia/reperfusion in diabetic cardiomyopathy [ , ] . modification of autophagy is at least in part responsible for the effect of chloroquine/hydroxychloroquine on the cardiac remodeling induced by stress conditions such as myocardial ischemia/reperfusion. autophagy is decreased during post-infarction remodeling in rabbit heart [ ] . in the days following myocardial infarction in rats there was maintained cell death by controlled necrosis (necroptosis), including in the noninfarcted border zone. autophagy flux was increased during the first day but, despite maintained increase in autophagosome formation, with time autophagy flux was decreased at the level of autophagolysosome processing and there was an associated increase in heart dysfunction [ ] . the impaired autophagy appeared to be responsible for the necroptosis since the latter could be attenuated by genetic overexpression of beclin, an initiator of autophagy. in this model, chloroquine caused an aggravation of the structural and functional deterioration. as already mentioned, because of reduced autophagy during myocardial remodeling, various strategies, including food restriction and pharmacological agents, can be used to stimulate autophagy. a similar protection can also be demonstrated by targeting autophagy in fibroblasts using micro-rna [ ] . the cellular signaling involved in some of these strategies may be complex. for example, the effect of food restriction and resveratrol may implicate an increased ampk activity, and a downregulation of the mtor signaling [ ] . chloroquine seem to suppress the protective effect of these strategies by modifying the effects of these signaling pathways on autophagy. it is not known whether there are additional inhibitory effects at more proximal steps. the scope of effects due a modulation of autophagy may be larger than heretofore known. it is plausible that the inhibition of autophagy affects the expression of many proteins (e.g. of kir . channels mentioned earlier) and impact on function. chloroquine has been reported to enhance the loss of t-tubules observed in cultured ventricular cardiomyocytes from failing human and from healthy adult rat hearts cultured in the absence of glucocorticoids [ ] . chloroquine, by inhibiting lysosome function in cultured mouse neonatal cardiomyocytes, was shown to cause an accumulation of phospholamban [ ] , a negative regulator of the sarco-endoplasmic reticulum ca + atpase (serca; responsible for storing ca + needed for contraction in the lumen of the sarcoplasmic reticulum). it is not known to which extent this effect occurs in adult cardiomyocytes and impacts on contractility. reports of neurotoxic effects of chloroquine include the development of extrapyramidal symptoms (rolling of the eyeballs, involuntary movements, trismus) following a few hoursdays of treatment with low doses in children [ ] as well as in adults. these effects were reversible within - hours after stopping the treatment. behavior disturbances have also been described [ ] [ ] [ ] . whether they can be related to actions on ion channels and receptors, or to rapid consequences of the interference with autophagy is not clear. retinotoxicity, a noted frequent side effect of treatment with chloroquine/ hydroxychloroquine [ ] , and skeletal muscle myopathy, which is less frequent [ ] [ ] [ ] [ ] , are largely results of changes in autophagy. administration of chloroquine can cause a degeneration of skeletal muscle cells resulting in myopathy. chloroquine myopathy can be reproduced experimentally by high doses ( mg.kg - .day - ) of the drug over at least weeks. the degenerated muscle fibers contain numerous autophagic vacuoles [ ] , into which various proteins accumulate [ , ] . as mentioned earlier, a similar accumulation can occur in cardiomyocytes and is responsible for cardiotoxicity following long-term treatment [ , ] . such effects are unlikely to complicate treatments limited to a few days and involving low to moderate doses (≤ mg.kg - .day - ). chloroquine poisoning is known to induce hypokalemia [ ] . the degree of hypokalemia is largest the higher the dose ingested and the blood concentration. hypokalemia in patients is systematic at chloroquine concentrations of µm and above, but in a few cases it could be obtained at much lower concentrations [ ] . the condition can develop within two hours or less following large doses and its underlying mechanism is not very clear but increased renal loss has been excluded. it has been proposed to involve reduced cellular k + efflux due to channel block by chloroquine (described above). however, in the absence of an increased k + influx pathway, suppression of outflux is not a satisfactory explanation for the cellular uptake of k + that causes hypokalemia. since the cellular membrane potential of resting or active cells is positive to the k + equilibrium (nernst) potential, only outward k + movements can occur via channels. one should therefore explore the role of a non-channel influx pathway to account for the increased transport of k + into the cell. the effect of chloroquine on the na + -k + pump is unclear. chloroquine has been shown to inhibit the na + -k + atpase in vitro [ ] , but a transient stimulation could be obtained in vivo at low concentration [ ] . it is conceivable that the hypokalemia is triggered by conditions such as stress and exercise that stimulate the na + -k + atpase by enhancing the sympathetic tone, as is the case in hypokalemic periodic paralysis [ ] or by an eventual increase of insulin secretion. in addition, the vagolytic action of chloroquine (described above) could remove the known "accentuated antagonism" exerted by the parasympathetic system towards the sympathetic system. increased na + -k + pump activity could then result from increased -adrenergic receptor activation [ , ] . this or other possible mechanisms have not been examined, and the main basis for the hypokalemia thus remains unclear. low extracellular k + concentration, largely because of the decrease in conductance of inward rectifier channels and of the corresponding outward currents, is associated with a lengthening of cardiomyocyte action potential duration and of the qt interval, and is arrhythmogenic. indeed, ventricular tachyarrhythmias were the immediate cause of death in about a third of patients admitted for chloroquine poisoning [ ] . one should be careful when attributing any clinically observed qt lengthening to a direct effect of chloroquine, without excluding the eventual contribution of concurrent hypokalemia. the possible efficacy of chloroquine/hydroxychloroquine in the treatment of various diseases, including viral infections such as covid- [ , , , ] but also cancer [ , ] , has received increasing interest. beside the drug efficacy in patients, the issue concerning the safety is also important. evidently, if the side effects were to be such marked that they put the life of treated patients in danger, then, as long as beneficial effects have been confirmed, the drug use could be restricted to only very severe cases, when there is no other choice than to try everything that can potentially save the patients. such use can only be done with close monitoring of the patient. in contrast, if side effects are mild and tolerable, such that they do not jeopardize the patient's life, then one can or should consider using the drug for its proven or potential benefit. the side effects of chloroquine/hydroxychloroquine on the heart are mainly of two types: those involving changes in electrical conduction, heart rate and rhythm [ , , , , , , ] , and those involving structural changes associated with cardiomyopathy [ , ] or myocardial remodeling. conduction and rhythm disturbances can be seen under acute conditions before any structural change, but they can also be favored by the cardiomyopathy [ ] . on a time frame, the rhythm changes are acute and reversible upon drug withdrawal, whereas cardiomyopathy is induced after treatment for months and is largely irreversible. all effects are also dose-dependent, and cardiomyopathy [ ] and interference with remodeling can be experimentally induced over shorter periods (weeks) with high doses. the mechanisms underlying the conduction and rhythm changes mainly involve inhibitory actions on various ion channels. block of na + channels (local anesthetic action) is likely responsible for observed bundle branch and intraventricular block [ , , ] . block of k + channels will tend to lengthen the action potential duration, whereas the inhibition of na + and ca + channels with have opposite effects. that the drugs given alone at low or moderate concentrations and for a short duration produce only limited prolongation of the qt interval is likely due to this balanced contribution of opposite effects [ ] . heart rate changes also result from a balance between a bradycardic action of i f and i ca-l inhibition [ , ] and a tachycardic effect of i k and i k-ach inhibition [ , , ] , in addition to the suppression of the vagal tone on the heart [ , ] . the resultant action of chloroquine can be pro-or antiarrhythmic, depending on the underlying condition [ , , , , , , ] . the mechanism underlying chloroquine cardiomyopathy involves an inhibition of autophagy [ , , ] . even in the absence of cardiomyopathic changes, such an inhibition can aggravate cell damage under ischemia-reperfusion, which by itself causes a change in the autophagic flux and recruits additional cell death mechanisms [ ] [ ] [ ] [ ] [ ] ] . the trend to favor myocardial death may be opposed by protective mechanisms due to an inhibition of intracellular na + and ca + load and of arrhythmias resulting from the effects on ion channels or due to inhibition of apoptosis [ ] . it should be underlined that all cardiac effects of chloroquine/hydroxychloroquine could not be covered in this brief review. for example, metabolic effects due to phospholipase a inhibition have been proposed to mediate an anti-ischemic action of chloroquine [ ] . in addition, other effects may not yet be known, and the consequences of autophagy inhibition on the expression of various proteins could be more complex. finally the effect of the drugs and their mechanisms may depend on coexisting non cardiac (e.g. electrolyte) changes or cardiac diseases (e.g. the presence of myocarditis). the side effects of chloroquine/hydroxychloroquine are generally mild, but they can be exacerbated by drug associations. many macrolide antibiotics can cause a qt prolongation when used in isolation [ ] . if co-administered with chloroquine/hydroxychloroquine, the effects on the qt interval might add and become marked, even if the effect of every drug taken separately was mild. use of high doses and association with other potential qt-prolonging drugs are likely the cause of the marked qt prolongation and occurrence of arrhythmias that have recently led to interrupt clinical trials testing the benefit of chloroquine in the treatment of covid- [ ] . even when apparently moderate mean doses are reported to have been used in large registries of patient groups, there will be large variations among different patient groups and a significant number of them might have been exposed to high drug doses. in any case, drug associations and the role of complicating conditions such as hypokalemia that are themselves drug dose-dependent, should also be analyzed before concluding on the direct cardiotoxic effect of chloroquine/hydroxychloroquine. the antimalarial drugs chloroquine and hydroxychloroquine have been proposed for antiviral therapy, including for covid- . the drugs are relatively safe when used at low or moderate doses, with blood concentrations ≤ µm. at such doses they can display beneficial, e.g. antiarrhythmic, actions, and despite the many effects on k + channels, they do not seem to be associated with prominent qt prolongation acute life-threatening cardiac effects, including ventricular tachyarrhythmias, are obtained with high doses. given the long half-life of the drug, other side effects are usually associated with long-term treatments. the mechanisms underlying chloroquine effects include direct actions on ion channels and receptors, while others (especially a cardiomyopathy developing following long-term treatment) involve the inhibition of autophagy. care should therefore be taken to limit the dose and the duration of treatment with the drugs, as well as to avoid association with other drugs with known toxic effects on the myocardium. declarations funding: work of the author is supported by funding from vlir-uos in the frame of an institutional university cooperation between flemish universities and the université catholique de bukavu. new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? recycling antimalarial leads for cancer: antiproliferative properties of n-cinnamoyl chloroquine analogues current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a minireview evidence for cardiac safety and antiarrhythmic potential of chloroquine in systemic lupus erythematosus chloroquine is a potent pulmonary vasodilator that attenuates hypoxia-induced pulmonary hypertension chloroquine and hydroxychloroquine as available weapons to fight covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread a call for randomized controlled trials to test the efficacy of chloroquine and hydroxychloroquine as therapeutics against novel coronavirus disease (covid- ) mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology the arrhythmogenic cardiotoxicity of the quinoline and structurally related antimalarial drugs: a systematic review cardiotoxicity of antimalarial drugs effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial parenteral chloroquine for treating falciparum malaria the disposition of chloroquine in healthy nigerians after single intravenous and oral doses the contribution of lysosomal trapping in the uptake of desipramine and chloroquine by different tissues excessive lysosomal ion-trapping of hydroxychloroquine and azithromycin clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements pharmacokinetics of quinine, chloroquine and amodiaquine. clinical implications cardiac arrest after intravenous chloroquine injection cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature sinus node dysfunction in adult systemic lupus erythematosus flare: a case report hydroxychloroquineinduced cardiotoxicity in a -year-old woman with systemic lupus erythematosus and systolic dysfunction heart conduction disorders related to antimalarials toxicity: an analysis of electrocardiograms in patients treated with hydroxychloroquine for connective tissue diseases the pharmacokinetics and electrocardiographic effects of chloroquine in healthy subjects azithromycin/chloroquine combination does not increase cardiac instability despite an increase in monophasic action potential duration in the anesthetized guinea pig assessment of multi-ion channel block in a phase i randomized study design: results of the cipa phase i ecg biomarker validation study the effects of antimalarial drugs on ventricular repolarization chloroquine and qtc interval comparison of antifibrillatory potency of certain antimalarial drugs with quinidine and procaine amide chloroquine and hydroxychloroquine in the treatment of cardiac arrhythmias chloroquine terminates stretch-induced atrial fibrillation more effectively than flecainide in the sheep heart the antimalarial chloroquine reduces the burden of persistent atrial fibrillation antiarrhythmic drugs and cardiac ion channels: mechanisms of action increase of electrocardiogram voltage and contractile force of the frog's heart induced by chloroquine comparative effects of some antimalarial drugs on isolated cardiac muscle of the guinea pig highdose chloroquine is metabolically cardiotoxic by inducing lysosomes and mitochondria dysfunction in a rat model of pressure overload hypertrophy acute chloroquine poisoning: a comprehensive experimental toxicology assessment of the role of diazepam depressant effects of chloroquine on the isolated guinea-pig heart diazepam does not improve the mechanical performance of rat cardiac papillary muscle exposed to chloroquine in vitro revisiting the cardiotoxic effect of chloroquine the effects of calcium ions on the depression of cardiac contractility by chloroquine and quinine treatment of severe chloroquine poisoning chloroquine cardiomyopathy -a review of the literature chloroquine cardiomyopathy with conduction disorders molecular mechanisms of myocardial remodeling reduced synchrony of ca + release with loss of t-tubules-a comparison to ca + release in human failing cardiomyocytes molecular and cellular mechanisms involved in cardiac remodeling after acute myocardial infarction impaired protein quality control during left ventricular remodeling in mice with cardiac restricted overexpression of tumor necrosis factor inhibition of autophagy inhibits the conversion of cardiac fibroblasts to cardiac myofibroblasts alterations of na + currents in myocytes from epicardial border zone of the infarcted heart. a possible ionic mechanism for reduced excitability and postrepolarization refractoriness can pka activators rescue na + channel function in epicardial border zone cells that survive in the infarcted canine heart? the role of autophagy emerging in postinfarction cardiac remodelling restriction of food intake prevents postinfarction heart failure by enhancing autophagy in the surviving cardiomyocytes resveratrol reverses remodeling in hearts with large, old myocardial infarctions through enhanced autophagy-activating amp kinase pathway chloroquine prevents progression of experimental pulmonary hypertension via inhibition of autophagy and lysosomal bone morphogenetic protein type ii receptor degradation a new local anesthetic with anticoagulant properties, chloroquine (aralen) dihydrochloride blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes late sodium current: a mechanism for angina, heart failure, and arrhythmia drug potency on inhibiting late na + current is sensitive to gating modifier and current region where drug effects were measured hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current i f : novel electrophysiological insights and therapeutic potential pacemaking in cardiac tissue. from i k to a coupled-clock system a novel quantitative explanation for the autonomic modulation of cardiac pacemaker cell automaticity via a dynamic system of sarcolemmal and intracellular proteins pacemaker mechanisms in cardiac tissue a brief history of pacemaking the funny current has a major pacemaking role in the sinus node chloroquine blocks the kir . channels by an openpore blocking mechanism molecular mechanisms of chloroquine inhibition of heterologously expressed kir . /sur a channels structural bases for the different anti-fibrillatory effects of chloroquine and quinidine chloroquine blocks a mutant kir . channel responsible for short qt syndrome and normalizes repolarization properties in silico the molecular basis of chloroquine block of the inward rectifier kir . channel chloroquine blocks the background potassium current in guinea pig atrial myocytes lysosome mediated kir . breakdown directly influences inward rectifier current density structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule action potential clamp and chloroquine sensitivity of mutant kir . channels responsible for variant short qt syndrome specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets antimalarial drugs: qt prolongation and cardiac arrhythmias inhibition of herg k + currents by antimalarial drugs in stably transfected hek cells calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes two components of transient outward current in canine ventricular myocytes open channel block of the fast transient outward k + current by primaquine and chloroquine in rat left ventricular cardiomyocytes muscarinic antagonist action of clinical doses of chloroquine in healthy volunteers chloroquine interacts with muscarinic receptors and inhibits cholinergic effects in the heart binding and doseresponse studies with chick embryo cells the role of autophagy in the heart the role of autophagy in the heart is autophagy in response to ischemia and reperfusion protective or detrimental for the heart? the role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart distinct roles of autophagy in the heart during ischemia and reperfusion: roles of amp-activated protein kinase and beclin in mediating autophagy the altered expression of autophagy-related genes participates in heart failure: nrbp and calcoco are associated with left ventricular dysfunction parameters in human dilated cardiomyopathy autophagy-inflammasome interplay in heart failure: a systematic review on basics, pathways, and therapeutic perspectives chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice chloroquine and amodiaquine enhance ampk phosphorylation and improve mitochondrial fragmentation in diabetic tubulopathy progressive reduction in myocyte autophagy after myocardial infarction in rabbits: association with oxidative stress and left ventricular remodeling necroptosis mediated by impaired autophagy flux contributes to adverse ventricular remodeling after myocardial infarction microrna- repairs infarcted myocardium by accelerating cardiomyocyte autophagy glucocorticoids preserve the t-tubular system in ventricular cardiomyocytes by upregulation of autophagic flux metformin increases degradation of phospholamban via autophagy in cardiomyocytes extrapyramidal syndrome following chloroquine therapy toxic psychosis: a complication of antimalarial therapy lethality and behavioral side effects of chloroquine behavioral toxicity and equivocal suicide associated with chloroquine and its derivatives hydroxychloroquine retinopathyimplications of research advances for rheumatology care hydroxychloroquine-induced autophagic vacuolar myopathy with mitochondrial abnormalities experimental chloroquine myopathy immunohistochemical evidence for amyloid beta in rat soleus muscle in chloroquineinduced myopathy snake coiled fibres in rat soleus muscle in chloroquine induced myopathy share immunohistochemical characteristics with amyloid depositions in alzheimer's disease brain tissue hypokalaemia related to acute chloroquine ingestion the in vivo inhibition of transport enzyme activities by chloroquine in different organs of rat is reversible the interaction of chloroquine with transport atpase and acetylcholine esterase in microsomal membranes of rat in vitro and in vivo extracellular potassium homeostasis: insights from hypokalemic periodic paralysis regulation of the beta-stimulation of the na + -k + pump current in guinea-pig ventricular myocytes by a camp-dependent pka pathway effects of alpha-adrenergic stimulation on intracellular sodium activity and automaticity in canine purkinje fibers antimicrobials and qt prolongation hydroxychloroquine cardiotoxicity in systemic lupus erythematosus: a report of cases and review of the literature chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p independent hydroxychloroquine protects against cardiac ischaemia/reperfusion injury in vivo via enhancement of erk / phosphorylation effect of chloroquine in experimental myocardial ischaemia ethical approval: not required. key: cord- - sukdb authors: quade, bianca n.; parker, mark d.; occhipinti, rossana title: the therapeutic importance of acid-base balance date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: sukdb baking soda and vinegar have been used as home remedies for generations and today we are only a mouse-click away from claims that baking soda, lemon juice, and apple cider vinegar are miracles cures for everything from cancer to covid- . despite these specious claims, the therapeutic value of controlling acid-base balance is indisputable and is the basis of food and drug administration-approved treatments for constipation, epilepsy, metabolic acidosis, and peptic ulcers. in this narrative review, we present evidence in support of the current and potential therapeutic value of countering local and systemic acid-base imbalances, several of which do in fact involve the administration of baking soda (sodium bicarbonate). furthermore, we discuss the side effects of pharmaceuticals on acid-base balance as well as the influence of acid-base status on pharmacokinetic properties of drugs. our review considers all major organs systems as well as information relevant to several clinical specialties such as anesthesiology, infectious disease, oncology, dentistry, and surgery. the normal function of nearly all physiological processes in the body depends on maintenance of appropriate acid-base balance. the value of intracellular ph and interstitial ph strongly depends on the value of arterial blood ph, which ranges between . and . under normal physiological conditions. when ph deviates from its normal range, ph-dependent enzymes and membrane transport proteins may not work properly and metabolic pathways can be negatively affected. acidemia, which is defined as arterial ph lower than . , can cause a variety of disturbances including arterial vasodilation, insulin resistance, compromised immune function, and reduced neuronal excitability. alkalemia, which is defined as arterial ph greater than . , can also cause many disturbances including reduced myocardial blood flow and seizures. thus, it is imperative that the value of blood ph is tightly controlled. therapies for acid-base disturbances are not new. infusion of sodium carbonate (na co ) into cholera patients to compensate for loss of serum alkali in diarrhea was recorded in the s [ ] and the commercial production of sodium bicarbonate (nahco ) for use as an antacid (brioschi®) apparently dates back to the s. since then, decades of research advances have led to a broad appreciation of the importance of acid-base balance in health and disease. this research is now coming to fruition in the form of inspired and effective medical advances. at the same time, some in the alternative medical community have seized on anecdotes and the results of limited trials to generate ubiquitous clickbait headlines about the miraculous properties of household acids and bases such as baking soda and, in some cases, propagate conspiracy theories about suppression of this information. in the first major section of our review ( acid-base homeostasis) we discuss how the body controls the abundance and distribution of acids and bases in order to achieve acid base homeostasis. we describe the importance of the powerful co /hco buffer system, the vital functions of the lungs and kidneys in excreting excess acids and bases, the role of membrane transport proteins and carbonic anhydrases in the local redistribution of acids and bases, and the drugs that can be harnessed to control these processes. in the second major section of our review ( systemic acid-base disturbances) we discuss the causes and consequences of generic acidbase imbalance caused by disturbances in co and hco levels and how our knowledge of their etiology has informed therapeutic strategies. our third and fourth major sections ( applications by organ system and other applications by clinical specialty) bring together a wealth of information from in vitro, in vivo, and clinical studies that demonstrate the current and potential utility of acid-base-balance correcting therapies. for each organ system or clinical specialization, as appropriate, we provide the fundamental physiological aspects of normal acid-base balance, the pathological consequences of systemic and local acid-base disturbances, as well as considerations of corrective therapies based on restoring (or harnessing the agents of) acid-base balance. in our fifth and final major section ( ph-dependent aspects of pharmaceutical therapy) we discuss how acid-base chemistry can influence drug pharmacokinetic properties and how this phenomenon can be advantageous for optimizing therapeutic interventions. our review highlights an emerging and dynamic field of research that is in the process of translating numerous basic scientific findings into clinical therapies. these findings appear to touch on nearly all aspects of health. we note a wide array of therapeutic paradigms developed around the control of acid-base balance including numerous reports of the successful application of nahco , the so-called 'enemy of the pharmaceutical industry,' to the amelioration of disease signs in animal models and in limited clinical trials. although studies of the role of acid-base balance in health and disease have resulted in the generation of several fda-approved pharmaceuticals such as contraceptive gels and gastric-acid suppressors, systematic reviews of random trials of the clinical effectiveness of nahco itself tend to be circumspect in their conclusions. a note to the reader: we, the authors, are basic scientists and do not intend this review to serve as a diagnostic or therapeutic guide. in many cases, a lack of consistency among study methods and subject demographics makes it difficult to draw firm conclusions regarding outcomes. for these reasons it is also impossible to extrapolate findings of therapeutic effectiveness in animal models and limited trials into an assessment of general clinical utility. but, in as much as we are reporting potential, we have not discounted any positive outcomes. finally, we note that the scope of this narrative review is extremely broad and the literature is extensive. for this reason, we have often cited reviews instead of primary literature in order to simplify the document and provide a cue to further reading. we apologize in advance to any authors whose work we have omitted. the maintenance of blood ph in the face of a ~ - meq h + /day acid load imposed by diet and metabolism (net endogenous acid production: neap [ , ] ), requires robust homeostatic mechanisms. regulation of blood ph and, by extension, the entire extracellular fluid compartment depends on the interplay between (i) the urinary system, which controls the blood bicarbonate concentration ([hco − ]), and (ii) the neuro-respiratory systems, which control the partial pressure of co (pco , see figure ). the kidneys perform the tasks of generating hco -, depositing it into circulation, and recycling hco from filtered plasma back into circulation (see figure ). the lungs exhale co , with respiratory drive being controlled by chemosensitive neural circuitry [ ] . a third mechanism of defense, which does not exactly regulate ph but only tends to minimize its changes, is provided by the multitude of buffer systems present in the extracellular fluid compartment. among these, the most powerful is the co /hco − buffer system, the efficacy of which is conferred by the body behaving as an open system, with respect to co , from which co can escape [ ] . a feature of the co /hco − buffer system is that the first of the two-step reactions that describe the interconversion between co and hco − (co +h o ⇌ h co ⇌ hco − + h + ) is very slow unless catalyzed by a carbonic anhydrase (ca) enzyme. thus, efficient buffering requires the presence of a ca. the henderson-hasselbalch equation [ ] describes how [hco − ] and pco determine ph:   co [hco ] ph = p + log . pco k s four of the members of the monocarboxylate transporter family (mcts, slc a - [ ] ) are h + -coupled lactate transporters (mct - ). mct and mct , being expressed in tumors, are of main therapeutic interest. the directionality of their transport process is determined by kinetic, thermodynamic, and situational considerations; for example the widely expressed isoform mct typically mediates h + /lacimport, while mct , which is abundantly expressed in glycolytic (i.e., lactate-producing) cells such as cancer cells and astrocytes, mediates h + /lacexport [ ] . members of several other solute carrier families also cotransport h + with their substrates such as the h + -coupled oligopeptide transporters of the slc family (e.g., peptt [ ]) or the h + -coupled neurotransmitter transporters of the slc family (e.g., the excitatory amino acid transporter eaat [ ]). the mct inhibitor azd [ ] is currently in phase i clinical trial for its effectiveness in treating cancer (clinicaltrials.gov identifier: nct ). there are several membrane proteins that conduct h + but share no obvious commonality in protein sequence. one group act as h + -selective channels and include the voltage-gated h +channel hv (hvcn [ ] ) and the voltage-independent h + conductors slc a [ ] and otopetrin (otop [ ]). all permit the movement of h + down their transmembrane electrochemical gradient, but each differ in their regulation of gating. hv only opens when the gradient favors h + efflux, slc a favors h + influx (particularly at elevated phi [ ] , perhaps to defend phi), and otop also favors h + influx (particularly at low phe, perhaps consistent with its sensory role). in acidotic conditions, the transient receptor potential cation channel subfamily v member (trpv ) can also mediate a significant, but non-canonical, acid-loading h + conductance [ ] . we are unaware of any fda-approvals for inhibition of this class of proteins, with the exception of trpv agonists whose influence on h + conductance is untested. although there is no description of a hco --specific ion channel, several anion channels have significant hco permeability. the electrochemical gradient for hco typically favors hco -efflux. these channels include the cystic fibrosis transmembrane regulator (cftr [ ]), the ca +activated cl --channel anoctamin (ano [ ]), as well gaba-and glycine-activated cl -channels (gabr and glr families [ ] ). drugs such as ivacaftor (kalydeco®, increases cftr channel open probability), or cocktails that include ivacaftor and one or more of the cftr folding chaperone drugs elexacaftor/lumacaftor/tezacaftor (e.g., orkambi®, symdeko®, trikafta®) are indicated for the treatment of cystic fibrosis by the restoration of certain defective cftr channels. both ivacaftor and tezacaftor rescue the hco permeability of the Δ -cftr mutant. in fact the rescued mutant has a greater hco -:clpermeability ratio than wild-type cftr, which may be therapeutically valuable. the importance of cftr-mediated hco secretion is discussed in sections . the respiratory system, . the lower digestive system, and . the urinary system). five of the ten members of the slc family of proteins mediate some form of na + -coupled hco --transport [ ]. nbce and nbce (slc a and slc a ) are electrogenic na + / hco cotransporters that may either act as acid-extruders or acid-loaders, depending on the electrochemical gradient. for example, nbce mediates hco efflux in renal proximal tubule epithelia, but hco influx in pancreatic duct epithelia. the remaining three are all acid extruders. nbcn (slc a ) is an electroneutral na + -hco cotransporter, while ndcbe (slc a ) is an electroneutral na + -driven cl -/hco exchanger. nbcn /ncbe (slc a ) has been described as being capable of both actions. we are unaware of any fda-approvals for inhibition of this class of proteins, nor of any drug that is specific for this class of protein. we note however that the nonsteroidal anti-inflammatory drug tenidap, which failed clinical trials for the treatment of rheumatoid arthritis due to renal and hepatic toxicity, is an effective blocker of nbce and nbce [ , ]. equation ( ) and figure show that, provided that pco remains constant, (i) a fall in extracellular [hco − ] causes ph to decrease whereas (ii) a rise in extracellular [hco − ] causes blood ph to increase. these two cases describe states of metabolic acidosis (mac) and metabolic alkalosis (malk), respectively. equation ( ) also indicates that ph can return towards its normal physiological value by a decrease in extracellular pco (in case 'i') or an increase in extracellular pco (in case 'ii'). this compensatory normalization of the [hco -]:pco ratio to restore ph, describes the physiological response of the neuro-respiratory system to mac and malk. moreover, equation ( ) and figure show that, provided that [hco -] remains constant, (iii) a rise in extracellular pco causes blood ph to decrease whereas (iv) a decrease in extracellular pco causes blood ph to rise. these two cases describe states of respiratory acidosis (rac) and respiratory alkalosis (ralk), respectively. again, equation ( ) indicates that ph can return towards its normal value by an increase (case 'iii') or decrease (case 'iv') in extracellular [hco − ], describing the compensatory physiological response of the urinary system to rac and ralk. mac, malk, rac, and ralk are usually referred to as the four classic/simple acid-base disturbances. respiratory compensations usually occur quite rapidly, within an hour of the appearance of the metabolic disorders and are fully resolved within to hours. in contrast, metabolic responses to respiratory disorders occur more slowly and may take up to several days to fully resolve as they require remodeling of acid-base handling mechanisms in the urinary system. the most rapid response-and the first line of defense of our body-to an acid-base disorder is given by chemical buffering which usually occurs within minutes. in the following four sub-sections we will review the causes, consequences, and therapeutic paradigms for each of the four systemic acid-base disturbances. several of these considerations are also relevant to the resolution of local acid-base disturbances and will be revisited in sections and . for a more complete and clinical perspective on these disturbances in isolation, and in combination, we refer the reader to reference [ ] . metabolic acidosis is defined as acidemia due to a primary pathological deficit in [hco -] rather than a physiological, compensatory lowering of [hco -] in response to respiratory alkalosis [ ] . the body can counter acid shifts in plasma ph in the short term by increasing respiratory drive to lower co and, in the longer term, by increasing renal h + excretion/hco generation. however, if these compensatory systems are defective or overwhelmed, mac will result. for example: mac can result from diet, chronic kidney disease, and diabetes (diabetic ketoacidosis) or can follow acute myocardial infarction (lactic acidosis), mutations in renal acid-base transporters (renal tubular acidosis, see section . ), intoxication with compounds (e.g., aspirin), and diarrhea (loss of hco --rich secretions) [ ] [ ] [ ] [ ] [ ] . clinical manifestations vary depending on underlying cause, but generally include weakness, nausea, and flushed skin [ ] . as we shall see, chronic mac has severe consequences for long-term health. in cases where mac is secondary to another disturbance such as in diabetes or diarrhea, treatment of the underlying disorder is the ultimate goal. however, for short term remediation of mac, or for situations in which the primary defect is with acid-base homeostatic mechanisms, the typical course of action is 'alkali therapy' to address mac by normalizing plasma ph. this can be achieved via two mechanisms. the following paragraph describes therapies that increase base load while the final paragraph describes therapies that lower acid load. increasing base load. the simplest paradigm is administration of hco salts. a direct rise in plasma [hco -] can be achieved either intravenously or by peritoneal dialysis. an indirect rise in plasma [hco -] can be achieved by oral dosing; as the parietal cells of the stomach replace neutralized stomach acid, they also generate new hco -, which is absorbed into circulation [ ] . there are however a number of caveats associated with hco administration [ ] . one caveat is that the counter anion (usually na + or k + ) may contribute to fluid retention or k + imbalance. a second caveat is that the treatment has the potential to rapidly generate co . with oral administration this can manifest as bloating or even gastric rupture, whereas with intravenous administration, the co , if not effectively eliminated by the lungs, can enter cells causing a paradoxical intracellular acidification. a third caveat is that ph overshoot (i.e., overcompensation that creates its own ph disturbance) is possible if the dose is not well titrated. however, in practice, manifestation of the side effects associated with nahco administration is not a foregone conclusion [ , ] . alternative vehicles for intravenous alkali delivery such as na co and caco produce less co per neutralized h + and impose less of an osmotic stress [ ] . carbicarb is a mixture of nahco and na co that does not cause intracellular acidification [ , ] . citrate salts provide a gentler, indirect mean of raising hco as citrate is converted into hco in the liver. alternative buffers such as tham (tris-hydroxymethyl aminomethane aka tris-base aka tromethamine aka trometamol) bind h + without generating co and the protonated product is readily cleared by the kidneys [ ] . furthermore, because a certain proportion of tham is uncharged at physiological ph, it is cell permeable and can counter intracellular acidosis. other hco --replacing bases include lactate and acetate [ , ] . finally, potential side effects can be ameliorated by administering buffers at a lower dose as part of an intravenous cocktail of buffers. for example, tribonat is a mixture that includes nahco , na hpo , and sodium acetate [ , ] . an added bonus of that mixture is that the inclusion of phosphate counters the hypophosphatemia associated with mac. dietary acid load is associated with lower serum hco - [ , , ] and thus there is scope for dietary correction of mac by, for example, adherence to a very low protein [ ] or otherwise "alkaline" diet [ ] . ph imbalance in mac can also be redressed by increasing h + excretion. the thiazide diuretic hydrochlorothiazide increases h + secretion by the renal collecting duct and has been used as an adjunct therapy with nahco for mac [ ] . its role as a diuretic ought also to assist with excretion of the na + load associated with nahco treatment. veverimer is an orally dosed h + binding polymer that is in phase iii clinical trials at the time of writing for the treatment of mac in the context of chronic kidney disease (clinicaltrials.gov identifier: nct ). it binds h + in the stomach for eventual excretion in the feces [ , , ] . moreover, the raising of gastric ph by veverimer prompts parietal cells to deposit hco into circulation, mimicking the alkaline tide associated with feeding. another approach to counter mac, is to increase cellular h + consumption (and/or decrease lactic acid production) by metabolic means either by pyruvate administration or by stimulating pyruvate dehydrogenase using dichloroacetate (dca) [ , ] . malk is defined as alkalemia caused by a primary excess of hco -. malk may follow volume depletion or hyperaldosteronism (promotes renal h + secretion), vomiting (eliminate gastric acid, stimulating an alkaline tide), or the use of certain pharmaceuticals that mimic those responses (loop diuretics, antacids). clinical manifestations can include confusion and tetany [ ] . malk can also have a genetic cause. for example, liddle syndrome is associated with hyperactivity of the epithelial na + channel enac, the action of which promotes renal h + secretion [ ] . besides treatment of the underlying conditions, correction of malk has been achieved using the ca inhibitor acz, which by itself results in mac [ ] , by intravenous infusion of hcl [ ] , or (if malk follows loss of gastric acid) the use of h -receptor agonists to prevent alkaline tide [ ] (see section . . ). rac is defined as acidemia with a plasma pco > mmhg at rest and at sea level [ ] . it usually occurs when there is a disruption in the ventilatory system that causes a mismatch between the rate of co removal and the rate of co production, with consequent accumulation of co into the blood (i.e., co retention). this disruption can be caused by (i) inability of the lungs to remove the metabolically produced co (i.e., reduced ventilation), (ii) defects in co transport from tissue to lungs and (iii) overproduction of co . reduced ventilation can result from a depression of the respiratory center (e.g., due to sedative overdose or brain injury), airway obstruction (e.g., due to vomit aspiration or laryngospasm), neuromuscular disorders (e.g., due to guillain-barré syndrome) or restrictive defects of the chest (e.g., due to impaired functioning of the diaphragm) [ ] . defects of co transport that lead to hypercapnia are less common and usually the result of reduced pulmonary perfusion in response, for example, to cardiac arrest or pulmonary embolism. overproduction of co is rarely the sole cause of rac. in fact, under normal circumstances the body responds to increases in co production by appropriately increasing ventilation in order to remove the excess co and prevent hypercapnia. situations in which the lungs are unable to match the increased co production can occur in patients undergoing mechanical ventilation or with reduced respiratory reserve [ ] . in fact, for therapeutic reasons, individuals on mechanical ventilation are often deliberately maintained in a state of ''permissive hypercapnia'' (see section . ). as for metabolic disturbances, rac can be either acute or chronic. acute rac occurs when pco rises very rapidly and the kidneys are unable to adequately increase hco production to compensate in such a short amount of time. thus, only a very modest renal compensation occurs. on the contrary, during the longer timespan of chronic rac (such as with chronic obstructive pulmonary disease, copd), the kidneys are able to restore the acid-base balance by increasing acid excretion and hco production [ ] . treatment is usually directed towards reversing the underlying cause and also at restoring adequate alveolar ventilation, which can be accomplished by endotracheal intubation with mechanical ventilation or positive pressure ventilation [ ] . because the sum of pco and po must be constant in the alveolar gas of patients breathing room air, hypercapnia leads to hypoxemia, a condition that can have consequences far more dangerous than those caused by hypercapnia [ ] . consequently, management of acute respiratory acidosis is often also directed towards ensuring adequate oxygenation. administration of o must be performed carefully because it may lead to increased co retention, especially in patients with copd [ ] . correction of hypercapnia in chronic rac usually occurs slowly because rapid reduction of pco can lead to overshoot alkalosis due to the renal compensation that increases [hco -]. in the central nervous system (cns), rapid alkalinization of the cerebrospinal fluid (csf) can cause seizures and even coma [ ] . the use of alkali therapy in rac is controversial and indicated only in patients with acute hypercapnia and concurrent metabolic acidosis [ ] . administration of nahco is contraindicated because it may increase co production, reduce alveolar ventilation as well as cause a paradoxical acidosis in the cns. as noted above for mac, alterative alkali therapies such as carbicarb that do not generate as much co as nahco alone (see section . . ) may be preferable to correct ph in rac. in patients with copd, the ca inhibitor acz is sometimes used to stimulate respiration in order to improve oxygenation, reduce co retention and possibly remove the need for mechanical ventilation [ ] . however, because ca is ubiquitous, the inhibitory effect of acz may impact a variety of tissues and have potential negative consequences on patients with pulmonary diseases. for this reason the role of acz as a respiratory stimulant is controversial, especially in patients with severe copd with or without hypercapnia [ ] . finally, co can be de-gassed from blood using an extracorporeal co removal (ecco r) device or [ ] lowered by dialysis using a dialysate that has a low [hco -] [ ] . ralk refers to alkalemia with a plasma pco < mmhg at rest and sea level [ ] . it occurs when the ventilatory system does not work properly causing an increase in alveolar ventilation and/or reduced co production with consequent co depletion in the blood. hyperventilation can result from stimulation of the respiratory centers (e.g., due to drugs or disorders of the cns), hypoxemia or tissue hypoxia (e.g., due to high altitude), lung diseases (e.g., pneumonia). reduced co production can result from a decrease in the basal metabolic rate (e.g., due to hypothermia) or in physical activity (e.g., due to muscle paralysis). clinical manifestations can include rapid breathing and dizziness [ ] . although usually considered not life-threatening, severe ralk can have serious consequences on the brain, lungs and the heart. treatment is usually directed towards correcting the underlying disorders. hormone replacement therapy caused ralk in a study of postmenopausal women [ ] . abrupt correction of severe ralk should be avoided because of the risks of cerebral and pulmonary reperfusion injury. acz is used in the prevention and treatment of ralk associated with hyperventilation at high altitude (acute mountain sickness: ams) in part because it enhances hco excretion in the urine, providing a compensatory lower of ph [ ] . neuronal activity presents a substantial challenge to local acid-base balance. neurotransmitterfilled vesicles release h + into the synaptic cleft [ , ] (h + themselves may be considered to be neurotransmitters [ ] ) and are removed from the synaptic cleft by h + -coupled neurotransmitter transporters such as the excitatory amino acid transporter eaat . gaba-activated anion channels in neurons and astrocytes release hco - [ , ] , and the ca + /h + exchange activity of the plasma membrane ca + -atpase (pmca) in neurons causes a rise in extracellular ph as it restores intracellular ca + following an action potential [ ] . the acid load that results from intensive neuronal firing can result in a drop in phi that dampens neuronal activity: a mechanism that prevents excessive firing via effects upon ph-sensitive channels such as asic a and nmda receptors [ ] [ ] [ ] . conversely, alkalosis is associated with an increase in neuronal activity and seizures [ ] . neurons and astrocytes express numerous abts and cas to maintain ph homeostasis and their importance is highlighted by the effects of their disruption [ ] . for example, genetic disruptions in ae or nbcn are associated with epilepsy [ , ] , although the mechanism is not simply related to effects of neuronal phi on excitability as ae is an acidloader while nbcn is an acid extruder and may depend on whether the neurons in question are excitatory or inhibitory. several abts and cas are expressed in the choroid plexus epithelia where their action supports the secretion of cerebrospinal fluid (csf). genetic ablation of these transporters (e.g., nbcn , nbce ) in rodents is linked to reductions in ventricle fluid volume [ ] while pharmacological inhibition of cas results in reduction of intracranial pressure [ ] . however, it is unclear whether these changes are accompanied by a fall in ph of the csf. besides its role in determining ph, hco plays an important role in neuronal plasticity because the transmembrane gradients of cland hco determine the reversal potential of gabaactivated channels and consequently whether gabaergic signals are depolarizing and excitatory or hyperpolarizing and inhibitory [ ] . changes in these gradients are important in two ways. firstly, developmental changes in the gradient during central nervous system maturation promote the switch to inhibitory gaba signaling [ ] . secondly, activity-dependent changes in the gradient contribute to the pathophysiology of epilepsy by promoting a pathological switch to excitatory gaba signaling [ ] . neuronal cl − -hco − exchangers such as ae and ndcbe are likely to contribute to the status of these gradients [ ] . finally, mutations in endosomal nhe cause intellectual disability and are associated with defective synaptic remodeling [ ] . acidosis has a number of other consequences. for example in stroke, lactic acidosis is linked to ischemic damage [ ] . in protein-aggregating neurodegenerative diseases, acidic ph promotes the aggregation of alzheimer's amyloid proteins [ ] . a major genetic risk factor for alzheimer's disease is incidence of the apolipoprotein e allelic variant apoe , which causes the epigenetic downregulation of nhe [ ] . loss of nhe from endosomes causes aberrant acidification and defective clearance of amyloid deposits [ ] . brain acid-base status also has consequences for mental health (see mental health). the role of ph in the retina is considered in a later section (see the sensory systems). the link between ph and neuronal excitability is exploited in the anticonvulsant value of inhaled % co to induce hypercapnic acidosis [ ] . hypercapnia also has a neuroprotective role in stroke, by inhibiting caspase and other cytotoxic activities [ ] , and during reperfusion [ ] . ca inhibitors are used as adjunct therapies for epilepsy [ ] and have potential application for treatment of neuropathic pain [ ] , alzheimer's disease [ ] , and cognitive disorders [ ] . however, mac is a side effect of systemic ca inhibition [ ] . lowered seizure thresholds in some strains of abt-null mice suggest that abts may be potential targets for anticonvulsant therapy. however, the need for caution is shown by the observation that, at least in the case of nbcn -null mice, a reduced seizure-threshold does not mean reduced neuronal excitability [ ] . the role of abts and cas in csf secretion hints at the potential for targeting of these proteins to lower intracranial pressure in idiopathic intracranial hypertension (iih). the use of ca inhibitors in patients with iih produces some symptom relief, but the mechanism of action is uncertain [ ] . regarding therapies for neurodegenerative diseases, histone deacetylase inhibitors have shown potential to release nhe from its epigenetic restraints to restore amyloid protein processing in apoe mice [ ] . another strategy that has been proposed to have potential to reverse amyloid deposition in alzheimer's disease is the raising of brain ph [ ] . therapies that target the peripheral nervous system are discussed in the following section. sight. most ocular tissues express one or more abt or ca for the purpose of maintaining fluid and ph balance. perhaps the most therapeutically tractable tissue is the ciliary body that employs caii and a range of abts to secrete hco --containing aqueous humor into the anterior chamber [ , ] ( figure ). this fluid leaks into the corneal stroma to flush out metabolic wastes and is returned to the anterior chamber by corneal endothelial cells which express a similar array of abts including nbce , mct , and the h + channel slc a [ , ] . finally, the fluid is drained from the anterior chamber via the trabecular meshwork. individuals with mutations in nbce have band keratopathy, glaucoma, cataracts, and corneal edema linked to fluid/ph imbalance in the cornea, lens, and elsewhere [ ] . abts and hco are also important for retinal function [ ] [ ] [ ] , as suggested by the link between nbcn mutation and progressive rod-cone dystrophy [ ] , or retinal degeneration in mice with defective expression of nbce and mcts [ , ] . hearing loss is a symptom of several systemic diseases linked to defects in abts, including pendrin (pendred syndrome [ ] ), the h + /k + -atpase (distal renal tubular acidosis [ ] ), and slc a (harboyan syndrome [ ] ). all of these abts are expressed in the inner ear where they help to maintain inner ear fluid ph and endocochlear potential [ ] . although a human correlate has not yet been reported, progressive hearing loss is also a feature of nbcn null mice [ ] . disruption of the h + -channel otop and the anion exchanger pendrin in mice is associated with malformation of the caco crystals (otoconia) that are essential for maintenance of balance [ , ] . taste. in addition to its role in the inner ear, otop is required for sour taste sensation [ ]. pain sensation. it is generally recommended to keep the ph of injected formulation close to physiological ph to avoid injection-site pain, with the added note that the inclusion of certain buffers may increase pain (hence new citrate-free formulations of adalimubab aka humira®) [ ] . low phe exacerbates sensation of pain due to its effects on trpv channel activation in nociceptive neurons. furthermore, activation of these channels under acidotic conditions is associated with a drop in neuronal phi that is mediated in part by a trpv -mediated h + sight. ca inhibitors applied as eye drops have long been used to treat glaucoma by virtue of their ability to reduce the production of aqueous humor, although even their localized ophthalmic use has been documented to lead to the side-effect of systemic mac in some prone individuals [ , ] . corneal edema that results from the expression of mutant misfolded slc a may be amenable to correction by small molecule folding chaperones [ ] . nhe blockers are cytoprotective in a rat model of diabetic cataract formation and retinopathy [ ] . hypercapnia is protective against ischemia-reperfusion injury in the retina [ ] , as it is elsewhere in the central nervous system (see section . ). nahco solution is useful for softening and dispersing hardened ear wax [ ] . however, we are unaware of any therapies specifically targeted to restoring the acid-base chemistry necessary for correct generation of endolymph or ostoconia. on a related topic there is one side effect of ear drops that pertains to acid-base balance. the acetic acid in some ear drops used to treat outer ear infection can be ototoxic because acetic acid can move across the round window into the inner ear, resulting in a drop in endocochlear potential (perhaps by acid inhibition of the na + /k + -atpase) and endolymph and perilymph ph [ ] . we are unaware of any demonstrations of the usefulness of otop modulation in this area, but inhibitors of proteins that mediate bitter taste sensation have been used to mask bitter tastes, suggesting potential utility of otop block for masking sour tastes and increasing the palatability of sour-tasting medications [ , ] . pain sensation. adjuvant nahco raises the ph of an injectable lidocaine solution and lowers perception of pain associated with lidocaine injection in one study, but the mechanism of the effect is uncertain [ ] . see also sections . (anesthesiology). besides the increased respiratory drive to exhale co in response to rac [ ] and the bohr effect (see section . the circulatory system) the highest profile link between ph and respiration relates to the role of cftr. defects in cftr are devastating because the cland hco secretion that this channel normally mediates is a fundamental part of the mechanisms that drive fluid secretion in our bodies [ ] ( figure ). the majority of deaths associated with cf are caused by respiratory failure [ ] . in the lungs, secretions are required to provide a moist surface for gas exchange, to liquefy mucus, and to flush inhaled particles and pathogens out towards the throat (mucociliary clearance). besides the general importance of anion secretion, cftr-mediated hco secretion plays a further role in ph homeostasis in the airway surface liquid (asl); hco helps to unfold and hydrate mucus [ ] and, by defending airway ph, has been hypothesized to promote a healthy local immune response to airway bacteria [ , ] . hco secretion is modulated by epithelial h + secretion mediated by a host of acid-extruding transporters [ ] ( figure ). airway acidification is a feature of individuals with cystic fibrosis, as well as those with asthma and tuberculosis [ ] and is exacerbated by lactic acid production by airway pathogens and airway epithelia [ ] . the new personalized cf therapies have focused on stimulation of defective cftr to restore fluid secretion [ ] , but are targeted to individuals with specific cf genotypes and thus alternative general therapies are still required. strategies specifically focused on correcting asl ph include inhalation of nebulized bases such as nahco [ , ] and tham [ ] as well as block of airway h + secretion using h + /k + -atpase inhibitors [ ] . all these strategies result in improvements in asl ph and some also improve mucus viscosity and/or pathogen clearance. an in vitro study suggests that mct blockade could also be protective of asl ph in individuals with cf [ ] by reducing epithelial h + secretion. a newly described paracellular pathway for hco secretion by cf airway epithelia might also be amenable to therapeutic modulation [ ] . the new personalized cf therapies have focused on stimulation of defective cftr to restore fluid secretion [ ] (e.g. lumafactor/ivacaftor, see section . . ), but are targeted. heart. mac is associated with reduced cardiac contractility. this phenomenon is explained by diverse mechanistic elements such as the ph-dependence of the channels and transporters that regulate ca + handling in myocytes as well as the dampening effect of acidosis on the responsiveness of the contractile apparatus to ca + [ ] . whether the heart rate is lowered by acidosis is harder to predict because of the complex effects of acidosis upon the sympathoadrenal system [ ] . intracellular acidosis in myocardial infarction after a period of ischemia, is countered, during reperfusion, by the action of acid-extruders such as ncbts and nhes [ ] . however, the accompanying na + load can be sufficient to reverse the action of the na + / ca + exchanger, raising [ca + ]i and increasing susceptibility to ventricular arrhythmias [ , ] . paradoxically, the loss of nbce function can also result in ca + overload because compensatory acid-extrusion mediated by nbcn and nhe imposes double the na + load per hco equivalent; a mechanism proposed to promote hypertrophy of cardiomyocytes in spontaneously hypertensive rats [ ] . ca activity is also pro-hypertrophic [ ] . on the other hand, the action of the acidloading anion exchange ae is considered to be protective against hypertrophy [ ] . finally, nhe action in the mitochondria is proposed to contribute to mitochondrial damage in the diseased heart [ ] . vasculature. typically, acidosis causes arterial vessels to dilate resulting in a fall in peripheral resistance, while veins may constrict [ ] . it is perhaps then no surprise that numerous blood pressure traits are linked to polymorphisms in abt genes [ ] . at least at the level of the vascular response of arteries, nbcs and nhes are required for normal vascular smooth muscle contractility and sensitivity to vasodilators [ ] . however, blood pressure is a complex trait that is not determined by vascular response alone, so explanation of these linkages is not simple. another important aspect is that mac inhibits progression of vascular calcification [ ] . the bohr effect describes the influence of ph and pco upon the oxygen carrying capacity of hemoglobin. in systemic capillaries, metabolically produced co enters the red blood cells (rbcs) where it is hydrolyzed into hco and h + by the action of caii. the newly produced hco is then extruded by ae , causing a fall in rbc phi which, by the bohr effect, reduces the hb-o binding affinity, promoting o release from hb to tissue ( figure b ). the reverse process occurs in the pulmonary capillaries. here, as co leaves rbc phi rises thereby favoring o binding to hb ( figure b ). thus acidosis enhances o delivery into tissues, but diminishes o loading in the lungs [ ] . this relationship between ph and gas exchange is partly sensitized by the content of the hemoglobin-regulating molecule , -dpg (diphosphoglyceric acid) in rbcs, a parameter which itself is ph-dependent; , -dpg levels increase with chronic acidosis promoting o release [ ] . heart. exogenous expression of skeletal muscle caiii in mouse cardiomyocytes enhances defense of phi and preserves cardiac function during mac [ ] . nahco is used to counter lactic acidosis in cardiac arrest and during prolonged cardiopulmonary resuscitation, but aside from its value at normalizing pre-existing mac or hyperkalemia (acidosis promotes cellular k + release), compelling data that this treatment improves outcomes are lacking [ ] [ ] [ ] . nhe blockers have shown promise as cardioprotective agents in reperfusion injury [ ] and likely act by targeting both plasma membrane and mitochondrial nhe [ , ] . although the nhe blocker cariporide caused serious side-effects in clinical trials (see section . . ), alternative approaches are available. for example, a microrna that lowers nhe expression protects cardiomyocytes from apoptosis during prolonged endoplasmic reticulum stress [ ] . in addition, antibodies and drugs that block nbcs have also demonstrated cardioprotective properties in animal models of ischemia reperfusion injury [ , ] . just as blockade of acid-extruders is cardioprotective, so too is the stimulation of the acid loader ae . this has been achieved in cell models using the glycoside sasanqua saponin [ ] , an extract from a herb used in traditional chinese medicine. we are unaware of any reports of acid-base based therapies for blood pressure that directly target the vasculature, but a discussion of diuretics for lowering blood pressure in congestive heart failure is provided in the urinary system section. some alkali-containing therapies may enhance progression of vascular calcification [ ] while use of the ca blocker acz has therapeutic value in calcifying disease [ , ] , perhaps by lowering ph. one study has cautioned the use of nahco in congestive heart failure because, in the face of adaptively elevated , -dpg levels, a sudden rise in ph could result in a maladaptive increase in hb-o affinity and risk of myocardial ischemia [ ] . in skeletal muscles, the build-up of lactic acid during intense exercise correlates with muscle weakness and self-limiting fatigue. however, the contribution of lactic acidosis to those symptoms may not be as direct or major as once thought [ , ] . generalized acidosis may contribute to weakness via alterations in neuromuscular drive [ ] and/or a decreased driving force for lactate efflux [ ] . regardless, acidosis promotes degradation of muscle protein [ ] . a high estimated dietary acid load has been associated with frailty in elderly japanese women [ ] recovery from lactic acidosis is mediated by mcts, nbcs, and nhes [ ] , while caiii specifically has been shown to plays a role in defense from muscle fatigue [ ] . many studies suggest the utility of nahco for improving exercise performance. for example, induction of malk by ingestion of oral nahco solutions has been shown to improve exercise endurance [ ] and reduce perception of effort [ ] in limited trials. however, taking a broader view of the field, the results of trials that link ph and exercise performance are deemed inconclusive due to inconsistent methodology and subgroup effects [ , ] . it has also been suggested that any competitive benefits that could be gained from nahco administration, from an athletic viewpoint, may be outweighed by gastrointestinal side effects such as bloating [ ] . away from the arena, hco administration or a reduced dietary acid load could have value in maintaining muscle mass in older adults [ , ] . mineralized material is eroded by acids as is evident in the case of tooth enamel, which is subject to demineralization by dietary acids (see oral health). however acidosis also inhibits bone growth by inhibiting osteoblasts, stimulating the activity of bone-resorbing osteoclasts [ ] , and influencing hormonal axes [ , ] (see also section . about the effects of ph on the endocrine system). accordingly, serum [hco -] positively correlates with bone mineral density (bmd) [ ] and negatively correlates with levels of serum parathyroid hormone (which promotes bone resorption) [ ] . at a local level, the process of bone remodeling, as well as the hormonal mobilization of ca + and pi from bone, requires that osteoclasts secrete h + onto the bone surface. these cells express intracellular caii to generate h + and hco -, an apical v-type h + -atpase to secrete h + onto the bone surface, and basolateral ae to export hco and defend osteoclast phi from alkalosis during h + secretion ( figure ). mutations in the v-type atpase and caii disable bone resorption by osteoclasts and are associated with increased bone density and osteopetrosis in humans [ , ] . the acid-base regulating proteins of osteoclasts are amenable to pharmaceutical modulation and their blockade ought to be protective of osteoporosis. for example, ae may be a useful target for increasing bmd because bmd is elevated in ae -null mice and cattle (reviewed in ref. [ ] ). regarding caii, one study showed a fortuitous bone-sparing effect in post-menopausal women were chronic users of ca-inhibitors for glaucoma treatment, [ ] . another study showed a paradoxical, but therapeutically valuable, bmd-lowering effect of ca inhibition in three children with sclerosing bone dysplasias. in these children, osteoclasts are already defective so the predominant effect of ca-inhibition is induction of chronic mac which promotes bone resorption [ ] . the therapeutic utility of proton pump inhibitors (ppis) to treat osteoporosis is negated by their negative influence on intestinal ca + absorption [ ] . in fact, several studies link ppi use with fracture susceptibility and low bmd (reviewed in refs. [ , ] ). because of these side effects, ppis are used with caution in some groups who may take them as antacid therapy [ , ] (and see next section). the three major health-related aspects of acid-base in this system are the roles of salivary hco in defense of enamel (which are discussed in section . oral health), gastric acid secretion, and peptic ulceration with helicobacter pylori. gastric acid is required to activate digestive enzymes, stimulate downstream secretory processes, and to kill ingested pathogens. it is secreted by parietal cells using similar transport mechanisms employed by osteoclasts (described in the previous section). thus, the secretion of acid across the apical membrane is mediated by a h + /k + -atpase and is balanced by the extrusion of hco into the plasma via ae (the alkaline tide associated with feeding [ , ] , see figure ). stomach epithelia are protected from acid injury by a mucus lining. the pathogenic bacterium h.pylori is able to survive in gastric acid because it can take up urea from its environment, via a h + -gated urea channel, and convert it into nh to neutralize acid in its immediate environment [ ] . in the vicinity of the mucus layer, this action causes h.pylori to raise mucus ph, lowering its viscoelasticity, promoting bacterial infiltration, and ultimately resulting in inflammation, ulceration [ ] , and risk of gastric cancer [ ] . another condition, gastroesophageal reflux disease, is caused by reflux of gastric acid into the esophagus and can cause heartburn and, in severe cases, can lead to esophageal damage. salivary hco plays an important role in esophageal acid defense [ ] [ ] [ ] by neutralizing gastric acid [ ] . conversely, the action of nhe in esophageal epithelia may exacerbate the damage, perhaps by indirectly stimulating pro-apoptotic pathways [ ] . acid reflux symptoms can be relieved by neutralizing gastric acid with antacids, which at their simplest are just bicarbonate or carbonate salts (e.g., tums® is calcium carbonate). however, an early antacid regimen for peptic ulcers, based on administration of milk and caco and still observed in the modern age in self-medicating individuals, results in adverse outcomes: the so-called 'milk-alkali syndrome' characterized by malk and hypercalcemia [ , ] . an alternative approach to lowering gastric ph is to use ppis or h -receptor agonists which dampen the signaling pathways that stimulate h + secretion. h agonists, in addition, therapeutically lower the activity of esophageal nhe [ ] . some over-the-counter formulations combine these drugs with an antacid to lower the dose of each and minimize side effects of each such as bloating (from gastric co generation) and osteoporosis (from chronic inhibition of intestinal ca + reabsorption, see section . ). orally-dosed acid-chelators such as veverimer, also raise gastric acid ph [ ] but have not been tested as a therapy for heartburn. the achlorhydric phenotype of ae -null mice suggests that ae blockage may have potential as a therapeutic target [ ] . ppis in combination with antibiotics are used to treat h.pylori infections: it has been proposed that raising stomach ph permits faster bacterial growth, potentiating the effects of antibiotics that act on dividing bacteria [ ] . inhibitors of the urease and h + -gated urea transporter of h.pylori are potential therapeutic modalities that remain in development [ ] . the exocrine pancreas secretes a hco --rich fluid that is vital for neutralizing gastric juices passing into the duodenum. the alkaline ph of pancreatic juice holds digestive enzymes such as amylase and lipase in an inactive state until the secreted fluid is neutralized in the duodenal lumen by chyme, preventing damage to the pancreatic ducts. in cf, duodenal hyperacidity also holds pancreatic enzymes in an inactive state, but without the neutralization of chyme, they are not even active in the duodenum leading to malabsorption of nutrients such as lipids [ ] . all along the intestine, hco --containing fluid secretions are required to promote gastric motility. the loss of this fluid in feces represents a substantial acid load. consequently, cftr mutations result in intestinal blockage [ ] and secretory diarrhea can result in mac [ ] . balancing secretory processes, nhe and slc a promote fluid reabsorption ( figure ). accordingly, downregulation of intestinal nhe by the enterotoxigenic bacteria (e.coli and c.difficile) and inactivating genetic defects in nhe and slc a are all associated with hypersecretion and diarrhea [ ] [ ] [ ] [ ] . on the subject of gut microbiota, intestinal ph can both influence and be influenced by the composition of gut microbiome [ ] . in individuals with insufficient intestine to absorb nutrients (short bowel syndrome), unabsorbed carbohydrates promote the growth of lactic acid-producing bacteria, which can lead to d-lactic acidosis [ ] . finally, the absorption of many nutrients depends on the action of h + -coupled abts (e.g. the h + -coupled oligopeptide transporters of the slc family: [ ] ), which in turn require the presence of acid extruders such as nbce to maintain epithelial ph during nutrient absorption. indeed, nbce -null mice exhibit defective nutrient absorption, which contributes to their general failure to thrive [ ] . the ability of small molecule inhibitors of nhe (tenapanor) and slc a (the , dimethylcoumarin drug "drainh-a ") to reduce intestinal fluid absorption makes them valuable therapies for irritable bowel syndrome with constipation and for relief of constipation in cf [ , ] . the cftr corrector ivacaftor improves intestinal hco secretion and nutrient absorption in individuals with cf [ ] . the mode of action of the anti-constipation drug linaclotide (linzess®: a guanylate cyclase c receptor agonist) encompasses both paradigms by the cgmpmediated reduction of nhe [ ] and activation of cftr activities [ ] . nephron function. the kidneys are vital to whole body ph balance (see acid-base homeostasis). it is the kidneys that generate hco -, reabsorb hco from the glomerular filtrate to prevent its loss in urine, and excrete h + in the form of nh + or titratable acids such as phosphate [ ] ( figure ). thus, it is no surprise that defects in renal transport mechanisms result in mac. these acidifying diseases can be acquired or genetic. fanconi syndrome is a degeneration of the proximal tubule, while renal tubular acidosis (rta [ ] ) can result from mutations in acid-base transporters such as nbce (type ii proximal rta: prta), h + -atpase or ae (type i distal rta: drta), caii (type iii rta), or disruption of h + secretion due to hypoaldosteronism (type iv drta). chronic kidney disease (ckd) is also associated with mac [ ] , and mac itself promotes progression of ckd (see below). malk is a common finding in cf patients. cf-model mice are less capable of defending against hco loads than their wild-type counterparts, due to downregulation of the renal hco -secreting anion exchanger pendrin [ ] and presumably loss of direct cftr-mediated hco secretion. concurrently, acid-base status has profound influence on kidney function. the very mechanisms that allow the kidneys to increase acid excretion in response to acute increases in acid load (e.g., ammoniagenesis and the renal endocrine response to acidosis) can be maladaptive in chronic mac, leading to inflammation and fibrosis [ ] . it is perhaps then not coincidental that low serum [hco -] is linked to a higher risk of chronic kidney disease in both adults and children [ , ] . an additional set of renal pathologies follows the integration of acid/base and salt/water handling by the nephron. for example, states and conditions of increased sodium reabsorption by the proximal tubule (e.g., volume contraction) or collecting duct (e.g., hyperactivity of the epithelial na + channel enac in liddle's syndrome) result in malk (see metabolic alkalosis) while hyperkalemia can cause mac [ ] . stone formation. urinary ph can influence stone formation which can lead to inflammation and obstructive kidney injury. a high urinary ph can cause the formation of calcium oxalate or calcium phosphate crystals, while a low urinary ph promotes uric acid crystallization [ ] . urinary ph can be modified by uropathogenic bacteria. urease-expressing bacteria generate nh , which can substantially raise urinary ph, promoting deposition of struvite and apatite crystals [ ] . besides the consequences of stone formation in the urinary tract, these deposits can cause the encrustation and blockage of indwelling catheters [ ] . it is interesting to recall that the pathogenic action of another bacterium, h.pylori, in the stomach also depends on urease action (see section . ). nephron function. many studies point to the value of correcting mac for preserving the function of the failing kidney and slowing ckd progression [ ] [ ] [ ] . we outlined corrective strategies based around alkali therapy in section . . ), but there is an additional prophylactic value in emergency settings. nahco infusion is protective against the kidney damage that can result from traumatic rhabdomyolysis (due to a crush injury), preventing development of mac and tempering the renal toxicity of myoglobin [ , ] . another consideration related to therapies is that a number of drugs cause metabolic acid-base disturbances because they are nephrotoxic [ ] or incidentally interfere with the kidneys ability to excrete acid [ ] . for example, ca inhibitors such as acz that are used as diuretics, due to their ability to interfere with fluid reabsorption, also cause mac [ ] . on the other hand, loop diuretics use can cause malk [ ] . another example are penicillin antibiotics which, acting as significant non-reabsorbed anions in the collecting duct lumen, promote hypersecretion of k + and h + , resulting in hypokalemia and malk [ , ] . finally in this section, the ability of cf kidneys to secrete excess hco is restored by treatment with the cftr restoring drug cocktail lumacaftor/ivacaftor (orkambi®) [ ] . stone formation. both citrate and low ph discourage the formation of calcium precipitates [ ] . thus, ingesting lemon juice, which raises urinary citrate while lowering urinary ph, decreases the propensity to form kidney stones and catheter-blocking deposits [ ] . dietary supplementation with citrate salts is also effective for this purpose because, despite resulting in a rise in urinary ph, the accompanying rise in urinary citrate increases the ph of crystal nucleation to an even higher value [ , ] . urinary ph can also exert a meaningful influence on drug excretion as discussed later (see section . ). at this point in our review we have presented ample evidence that abts are necessary to sustain life, and now we will see that they are also necessary to create new life. in the male reproductive tract, h + secretion by clear cells in the tail of the epididymis is required to maintain an acidic luminal ph for storage of sperm [ ] . hco secretion along the length of epididymis is necessary to functionally activate sperm before ejaculation [ ] and prevent their inactivation by the acidic vaginal environment (discussed later). indeed low levels of hco are associated with lowered sperm motility [ ] . in the female reproductive tract, endometrial epithelial cells further secrete a hco --rich fluid that is necessary for sperm capacitation and fertilization [ ] . furthermore, the secretory phase of the uterine cycle is associated with a dramatic rise in the ph of the oviduct lumen, corresponding with a level of hco that is sufficient to promote thinning of mucus during ovulation to promote sperm mobility [ ] and to promote dispersal of the egg-surrounding corona cells to allow the sperm access for fertilization [ ] . ultimately hco is even a prerequisite for the acrosome reaction [ ] , by virtue of its ability to stimulate soluble adenylyl cyclase to produce camp and initiate requisite signaling cascades [ ] . finally, once fertilization has occurred, acidification of uterine fluid is a necessary prerequisite for embryo implantation [ ] . numerous abts are involved in these processes; for example, loss of ae , slc a , cftr, or nhe are all associated with infertility or reduced fertility in male mice [ , [ ] [ ] [ ] . the acidic ph of the vagina noted earlier is caused by the metabolic activity of lactobacilli and serves to defend against sexually transmitted disease pathogens. loss of the acidity in bacterial vaginosis is associated with increased susceptibility to std infection [ ] . with regard to ultimate reproductive success, maternal-fetal acid-base balance is an important determinant of perinatal outcomes [ ] . for example, obstructed labor has poor outcomes due to intermittent hypoxia and lactic acidosis [ ] . because vaginal acidity tends to dampen sperm motility, vaginal douching with nahco improves fertility [ , ] . conversely, vaginal acidification is contraceptive and prophylactic. the spermicidal properties of lemon juice have long been appreciated [ ] . phexxi™ (formerly known as acidform: [ ] ), is the most recent of a series of acidic contraceptive gels. phexxi™ is a vaginally-applied gel of lactic acid, citric acid, and potassium bitartrate that is indicated by the fda to prevent pregnancy [ ] . an alternate approach 'buffergel' utilizes an acidic polymer for the same purpose [ ] . by lowering vaginal ph these products also confer microbicidal benefits [ ] . finally, some abt-targeted drugs interfere with fertility: ca inhibitors, for example, prevent dispersal of corona cells [ ] and ppis inhibit sperm motility [ ] . finally, with regard to childbirth, peri-operative nahco infusion has been proposed as a possible measure to improve outcomes in obstructed labor [ ] . many metabolic reactions that consume or generate acids and bases can, in disease, result in mac. the liver makes important contributions to acid-base balance by consuming lactate (countering lactic acidosis), generating albumin (a weak acid: hypoalbuminemia is associated with malk) and producing keto acids (contributing to diabetic ketoacidosis) [ ] . furthermore, acidbase status impacts the activity of the enzymes that constitute several key metabolic pathways. for example, acidosis inhibits glycolysis [ ] and lipolysis [ ] but stimulates gluconeogenesis [ , ] . the ability of disturbed acid-base balance to interfere with glycemic control is further evidenced by the following observations: (i) acidosis and alkalosis both lower glucose-stimulated insulin release from pancreatic islets [ ] ; (ii) the hco content of plasma correlates with insulin solubility [ ] , (iii) acidosis is associated with decreased insulin sensitivity [ , ] . this latter observation is due in part to the ph-sensitivity of the interaction between insulin and its receptor: a bell-shaped ph-dependence that exhibits strongest binding at ph ~ . [ ] . in fact, insulin can also influence abt action. for example, insulin promotes renal nbce activity. in type diabetes the resulting pathological increase in renal fluid absorption caused by nbce upregulation is thought to contribute to hypertension [ ] . other hormones that influence acid-base balance include secretin (increases pancreatic hco secretion in response to duodenal acidity [ ] ), angiotensin ii and aldosterone (increases renal acid excretion in acidosis [ , ] ), and parathyroid hormone (increases renal acid excretion and excretion of urinary-buffer phosphate in acidosis: [ ] ). interestingly, licorice can cause malk because one of its constituent compounds (glycyrrhizic acid) indirectly causes overstimulation of the aldosterone receptor [ ] . hormones whose levels are pathologically altered in acidosis include aldosterone and endothelin (increased: [ , ] ) cortisone (increased: [ ] ), and igf- (decreased: [ ] ). metabolic reprogramming of cancer cells in the hypoxic and acidotic tumor environment is discussed further in a later section (see . oncology.) the influence and therapeutic relevance of ph upon endocrine and metabolic aspects of heart function, muscle mass, and bone growth have been discussed in earlier sections ( . , . , and . ). the link between dietary acid load and development of insulin sensitivity has made dietary control an appealing target for lowering the incidence of type diabetes, although overwhelming evidence of efficacy is currently lacking [ ] . because of the ph-dependence of insulin solubility, the dissolution time of administered insulin is slower in plasma from diabetics with dka than in otherwise normal plasma, suggesting at face value that combined insulin-bicarbonate therapy might be valuable [ ] . however, such treatment in practice may be of limited value and has been linked to development of cerebral edema in children [ ] . the influence of ph on drug solubility is discussed in more detail in section . . ph plays a role in all aspects of the immune response. first we will enumerate the subcellular effects: (i) the cytosolic alkalinization that promotes cytoskeletal rearrangement during neutrophil spreading (the morphological change that is important for capillary adhesion and extravasation) requires nhe action [ ] . (ii) inflammatory sites are usually acidic due to the metabolic activities of invading bacteria and neutrophils, an environment that promotes the production of proinflammatory cytokines [ ] (see also section . ). (iii) during bacterial killing, h + efflux into the phagosome is necessary for charge compensation during the respiratory burst that produces cytotoxic superoxide anions. this action is mediated by the voltage-gated h + channel hv [ ] . (iv) some of the generated superoxide is converted into cytotoxic hypochlorous acid (hocl) which is able to diffuse back into the neutrophil cytoplasm. thus the action of phagosomal hv , together with the action of nhe in the plasma membrane, defends the neutrophil cytoplasm from acidosis which would otherwise dampen nadph oxidase activity [ , , ] . secondly we can consider the effect of acid-base disturbance on the whole cell. although the ph sensitivity of individual immune cell types are well characterized in vitro (e.g., acidosis decreases leukocyte and neutrophil mobility [ ] , promotes complement activation [ ] , modulates expression of inflammatory mediators [ ] ), there are many subtleties to these effects. for example the type of acidosis, type of cell, activation state of the cell, and effects on phagocytic activity versus migration may all influence the effect of acid-base disturbance on the immune response mediated by a given cell type [ ] . thus, for example, acidosis enhances bacterial killing by neutrophils [ ] and leukocytes [ ] but not by macrophages [ ] . although it is complicated to tease out a set of concerted mechanisms, in general, systemic acidosis is associated with compromised immune function [ , ] . [ ] . the link between ph and disturbed immune response is demonstrated by the following example. single nucleotide polymorphisms in the acid-loading protein ae are linked to progression of primary biliary cholangitis. a mechanism is suggested by studies of ae -null mice in which loss of ae from cytotoxic t-cells causes a phi increase that promotes their proliferation, activation and survival, amplifying the autoimmune response against damaged liver cells. furthermore, type iv drta is linked to systemic lupus erythematosus, although the causal relationship is unclear [ , ] . several paradigms have been proposed to suppress a pathological immune response. oral nahco dosing in rats stimulates an anti-inflammatory response; this effect could be harnessed to prevent tissue damage in autoimmune diseases such as rheumatoid arthritis [ ] . one preliminary study in mice even suggests that oral nahco dosing could be useful to suppress peanut allergy [ ] . regarding the pharmacological aspect, mct inhibitors act as immunosuppressors by interfering with the disposal of lactate during t-cell activation [ ] . finally, the nhe blocker cariporide suppresses the systemic immune response to burn injury in rats, although the mechanism is unclear [ ] . on the other hand, knowledge of acid-base balance can be exploited to promote an immune response. it has been suggested that blocking ae to promote a stronger cytotoxic t-cell response could be useful for treatment of chronic infections [ ] . considerations about the role of ph in cancer immunotherapy are included in section . . neuronal activity is ph dependent (see section . ) and there is emerging evidence that agents of acid-base balance can influence behavior and progression of neuropsychiatric disorders. inhalation of co (despite its general dampening action on neuronal excitability, see section . . ) invokes anxiety and panic, and the influence of co is exacerbated in individuals with panic disorders [ ] . studies in mice indicate that lowering brain ph triggers the action of the acidsensing ion channel asic a in the regions of the brain responsible for stress, fear, and social behavior [ , ] . the acquisition of fear-related freezing behavior in mice is enhanced by asic a overexpression [ ] and dampened by asic a disruption [ ] . in humans, decreased brain ph is also associated with schizophrenia, bipolar disorder, and autism spectrum disorder [ ] . one study even suggests that a high dietary acid load correlates with incidence of emotional problems and hyperactivity in young children, but causality could not be established as could not be discounted that behavior influences dietary habits [ ] . in terms of the linkage between abts and neuropsychiatric disorders, slc a is a biomarker (both in terms of incidence of a specific single nucleotide polymorphism and in terms of reduced expression determined by microarray) of suicide ideation and completion, especially with bipolar disorder [ ] . although the mechanistic details of the linkage are unknown, the role of nbce in control of brain ph is likely to be relevant. perhaps also of tangential relevance, due to its impact upon systemic acid-base balance [ ] , is that estimated glomerular filtration rate in ckd, which typically correlates with ability to excrete acid, is inversely correlated to depressive symptoms and suicide ideation [ ] . the anxiety response to co inhalation is a useful clinical test to follow the effectiveness of treatments for panic disorders [ ] . the response itself can be quelled by acz [ ] but, as this is just a model of panic disorder, the use of ca blockers to treat actual panic disorders is unclear. the above-mentioned studies of asic a-null mice suggest that asic a inhibition could be therapeutic for panic disorder. regarding the link between slc a and suicide, it is unclear how modulating nbce activity might influence depression, although detection of the biomarker could help to identify high risk individuals and thereby inform therapeutic strategies [ ] . finally, it has been suggested that antipsychotic medications could contribute to lactic acidosis and be partly responsible for decreased ph in the brains of individuals with schizophrenia [ ] . as mentioned previously, plasma ph depends on adequate ventilation to exhaust co thus mechanical ventilation can induce respiratory acid-base disturbances. another important aspect for consideration in this section is that the bioavailability of anesthetic agents can be influenced by acid-base status. when using mechanical ventilation, two important considerations, related to acid-base balance, must be raised. the first consideration relates to low-flow anesthesia or closed-circuit rebreathing systems that return exhaled anesthetic gas mixtures. in these systems co must be removed from the recirculated air. for this purpose, co scrubbers are used to adsorb co . the archetypal scrubber is soda lime (a mixture of naoh and ca(oh) , which reacts with carbonic acid to form an insoluble caco precipitate), although a number of other technologies are available [ , ] (see also section . . ). the choice of technology can be important as many co scrubbers can have undesirable reactions with anesthetic gases, producing toxins such as carbon monoxide [ ] . the second consideration is for individuals in which low-tidal-volume ventilation is indicated, such as those with acute respiratory distress syndrome or copd. these individuals may be deliberately under-ventilated to prevent mechanical stress on the lungs. thus, patients are maintained in a state of compensated rac called "permissive hypercapnia". under some circumstances, such as in critically ill patients, this hypercapnic state may be protective due to its anti-inflammatory influence [ ] (see also the immune system). acid-base balance concerns are not limited to inhaled anesthetics. prolonged infusion with the intravenously administered anesthetic propofol can cause severe lactic acidosis [ ] . the influence of ph on the pharmacokinetics of drugs in general is discussed in section . perioperative interventions have the potential to disturb acid-base homeostasis with negative consequences for outcome. post-operative metabolic acidosis is a well described but complex phenomenon related to issues including lactate accumulation in poorly perfused tissues and hyperchloremic acidosis due to dilution/displacement of hco --containing plasma by infused saline [ ] [ ] [ ] [ ] . pre-operative acidosis has also been described and has been linked to stress and fasting [ ] . post-operative malk has also been described following general surgery and has been linked to the infusion of citrate-buffered plasma [ , ] . peri-operative malk is linked to the removal of stomach acid by nasogastric suction. poor outcomes have been associated with both post-operative mac [ ] and malk [ ] , although this is not a universal finding [ ] . on a related theme, the usefulness of stored blood for transfusion can be compromised by numerous storage lesions including low ph that follows anaerobic metabolism by stored cells [ , ] . finally, an acidic tissue environment appears to favor natural wound healing, yet alkaline ph favors the success of skin grafts [ ] . altering the chemistry of infused fluids is the obvious strategy to counter post-operative acid-base disturbances. with specific regard to post-operative mac, dichloroacetate treatment tempered the pathological rise in lactate following liver transplantation, although no effect on outcome was observed [ ] . other treatments for mac are discussed in section . . . treatment for malk are discussed in section . . . finally, a study in mice suggests that raising the ph of stored blood enhances red blood survival after transfusion [ ] . the ph-dependence of wound healing suggests that therapeutic maintenance of the ph of the wound or graft could aid healing [ ] . hco is a major buffer in stimulated saliva [ ] , defending oral ph against acidic foods and drinks and against those acids produced from sugars by acidophilic bacteria in the oral cavity. acid defense protects enamel from erosion and oral ph is also an important determinant of a healthy oral microbiome; low salivary flow and ph generally encourage the presence of pathogenic and cariogenic bacteria (e.g., [ , ] ). as a consequence, low salivary ph, [hco -], and/or buffer capacity are predictors of cavity formation [ ] [ ] [ ] . salivary ph is lowered in many groups of individuals such as patients undergoing chemotherapy for head/neck cancer [ ] , cocaine users [ ] , or tobacco smokers [ ] . low salivary ph is also described in individuals with diseases such as cf [ ] , sjögren's syndrome [ ] , and juvenile idiopathic arthritis [ ] . not all studies report a major impact on dental health in these cases as compensating factors may be in play [ ] . abts and cas play important roles in salivary secretion and well as in enamel formation [ , ] . for example, defective dentition is a feature of some individuals with nbce mutations [ ] and appears not to be a secondary consequence of acidemia [ ] . salivary ph is a useful biomarker for oral health [ ] and the usefulness of baking soda in oral hygiene was first suggested as long ago as [ ] . nahco delivery either as a mouth wash [ ] , mucoadhesive spray [ ] or sugar-free gum [ ] raises salivary ph and in some cases may lower colonization of acidophilic bacteria. in individuals undergoing chemotherapy for leukemia, use of a nahco mouthwash lowered susceptibility to mouth ulcers [ ] . in smokers the similar treatment reduced levels of the inflammatory biomarker il- β [ ] . nahco containing dentifrices have been shown to be effective at neutralizing plaque ph [ ] , enhancing plaque removal [ ] , and inhibiting formation of caries [ ] . ph-stabilizing resins used in restorative dentistry have also been suggested to be useful cariostatic by releasing oh - [ ] . finally, reducing dietary intake of sugars is also beneficial to oral ph because it limits the acidification that can be caused by acidophilic bacteria. hence sugar-free gum is effective at raising plaque ph [ ] . we have dealt with various aspects of acid-base-related medical microbiology in earlier sections (see sections on the respiratory system, the upper digestive system, the lower digestive system, the reproductive system, the immune system and oral health). in this section we will confine our considerations to viruses and parasites, using influenza and malaria as examples. . both of these mechanisms are necessary to support viral replication. in defending phi against the increased acid load, infected cells extrude h + , creating a concomitant acidification of phe at the cell surface [ ] . there are multiple acid-base-related aspects to the malarial lifecycle and its pathological impact. first of all, mosquitos are attracted by co and co sensitizes them to human odors [ ] . secondly, the erythrocytic phase of malaria infection requires that plasmodium invades red blood cells. one of the surface antigens that plasmodium exploits for host-cell recognition and invasion is the ae -glycophorin a complex. consequently, ae -null mice are immune to infection [ ] as are individuals with an ae defect that causes the abnormal red cell morphology (south east asian ovalocytosis, sao) [ ] . finally, malarial infection causes numerous metabolic disturbances, such as mac that, in part, follows the tissue hypoxia and hyperlactemia caused by blocked microvasculature. mac is a strong prognosticator of fatal outcome in infected individuals [ , ] . influenza. m h + -channel blockers have potential to target influenza strains that are resistant to currently available antiviral treatments [ ] . on the other hand, the use of ppis may paradoxically increase susceptibility to viral infection in the gastrointestinal tract by neutralizing the stomach acidity that typically destroys viral particles [ ] . lactic acidosis in malaria can be ameliorated by dichloroacetate treatment [ ] . malarial resistance in individuals with sao suggests that transfusions with sao blood may be a useful therapy for individuals infected with drug-resistant plasmodium [ ] . finally, the parasite's own abts may be a target for antimalarial action [ ] . the rapid proliferation of cancerous cells is associated with the warburg effect (also known as 'aerobic glycolysis'): a shift from aerobic to anaerobic metabolism even in the presence of oxygen [ , ] . in this state, cells increase their atp production by increasing their glucose uptake with the consequence of increased lactate and h + production. because of their high metabolic rates, such cells would tend to have a much lower phi than normal cells. however, cancer cells are able to maintain near-normal phi by upregulating acid-extruding abts such as nbce , nbcn , nhe , h + -atpase, and mct to facilitate the removal of h + , as well as extracellular cas (caix, caxii) to facilitate the removal of co [ ] [ ] [ ] [ ] [ ] . aquaporins (aqps)-which can serve as a conduit for transmembrane movements of co [ ] -also promote tumor growth and survival, but it is not clear that promotion of co removal is a major part of their pathological importance [ , , ] . as shown in cancer cell-lines [ ] , acidosis can also increase the drive on the tca cycle (promoting atp production for h + extrusion) and the pentose phosphate pathway (promoting nadph production to counter ros, promoting cell survival) [ ] . the combined action of these processes results in a drastic reduction in local phe. these changes allow cancer cells both to outcompete neighboring non-cancer cells and to mobilize and spread to other parts of the body. metastasis and tumor survival are further enhanced by acid-dependent remodeling of the extracellular matrix [ ] and suppression of anti-tumor immune responses [ ] (see also the immune system). it is noteworthy that a high level of net endogenous acid production was associated with higher mortality in breast-cancer recurrence in a cohort of early stage breast cancer survivors [ ] . moreover, acid-treatment of melanoma cells selects for more invasive phenotypes [ ] and the extent of upregulation of various abts and cas can be an adverse prognostic factor (e.g., [ ] [ ] [ ] ). the importance of acid-base balance in cancer has suggested that targeting tumor ph could be a valuable adjuvant therapy. the three main therapeutic modalities are (i) interfering with the ability of cancer cells to defend their phi, (ii) interfering with the ability of cancer cells to create an acidic extracellular environment, and (iii) bolstering the immune response in an acidic tumor microenvironment. interfering with phi defense. blocking the defense of phi by cancer cells can be achieved by inhibiting acid-extruding abts and/or cas [ , ] . the value of these approaches is demonstrated in diverse studies that report anything from delayed tumor-growth in nbcn -null mice [ ] to positive clinical outcomes with adjuvant use of proton pump inhibitors [ ] . however, reports from clinical trials are currently sparse. given the potential for side effects due to the physiological importance of these proteins in all organ systems, much attention has focused on inhibiting those proteins, such as caix, which are specifically upregulated in cancer cells (in response to hypoxia) and not highly expressed elsewhere [ ] . another approach, the combinatorial use of blockers, allows for a lower dose of each and thus fewer undesired effects on non-tumor cells. the use of a combination of five compounds, each of which targets a different abt/ca, revealed effectiveness at reducing intracellular brain tumor acidification in mice and the consequent activation of the pro-apoptotic marker caspase- in tumor cells, with little negative effect on non-tumor cells [ ] . interestingly, the potency of such approaches is enhanced by glucose loading, which increases the acid-load in these glycolytic tumor cells [ ] . finally, as different abts/cas are upregulated in different tumor types, generic approaches are also valuable to consider. for example, a number of anticancer drugs, such as salinomycin, that are not known to specifically target abts or cas, also promote cellular acidification [ ] . interfering with extracellular acidification. anecdotal evidence suggests that interfering with extracellular acidification might be achieved by dietary means [ ] . indeed, oral nahco supplementation can raise tumor phe and inhibit metastasis in mice [ ] , but the overall consequence is complex, with one recent study suggesting that such therapy may confoundingly promote tumor proliferation [ ] . however, the addition of adjuvant nahco to a chemotherapeutic agent that was fed directly into a hepatic tumor via catherization of tumorfeeding arteries (tila-tace: targeting intra-tumoral lactic acidosis with trans-arterial chemoembolization) was associated with markedly improved outcomes [ ] . in short, there is currently insufficient data to show that lowering dietary acid load improves outcomes, except by virtue of the association of such diets with general well-being [ ] and the role of alkalinization in enhancing the safety of certain chemotherapeutic drugs ( [ ] ). the acidic tumor microenvironment can also be exploited to promote local drug delivery as discussed in section . . bolstering antitumor immune response. the acidic tumor microenvironment promotes an antiinflammatory t-cell response (see section . . ), but the deletion of ae promotes a proinflammatory t-cell response (see section . . ). therefore inhibition of t-cell ae could be a valuable paradigm for enhancing a cytotoxic immune response [ ] . ph-dependent aspects of pharmaceutical therapy in this section, we will focus on the influence of ph upon pharmacokinetic properties of drugs. we will also consider how all of these ph-related properties can be harnessed to therapeutic advantage, particularly in diseases associated with acid-base imbalance. oral drug delivery is the most common and convenient method used to administer drugs whereby they can either directly access their targets or be absorbed into circulation to reach their intended targets. thus, oral drug delivery will serve as our paradigm for discussion. however, these concepts are also relevant to other, parenteral, methods of drug delivery such as inhalation of nebulized substances and injection into subcutaneous, intramuscular, or intravenous compartments. gastrointestinal (gi) ph is one of the major determinants of oral drug bioavailability as it affects various properties including solubility and dissolution rate, stability, and absorbability. ph varies drastically along the gi tract [ ] [ ] [ ] . starting from a value of ~ . in the stomach, it rises to ~ . in the duodenum, reaches ~ . in the terminal ileum, and falls again to ~ . in the colon [ ] . an additional consideration is that these values can vary with age, presence of food, diseases, and also by the co-administration of other drugs [ ] [ ] [ ] [ ] . finally, we note that the influence of ph on bioavailability could, in ph-disturbed states, result in underdose or overdose. most drugs are weak acids and bases. the relationship between the drug ionization constant (pka) and the ph of the environment to which the drug is exposed (hereafter referred to as the environmental ph) is a critical determinant of drug solubility and dissolution rate in aqueous compartments [ ] . this relationship determines the ratio of the concentration of the unionized (i.e., uncharged) form (u) to the concentration of the ionized (i.e., charged) form (i) of the drug and is described by the henderson-hasselbalch equation. now, according to equation ( ), . if ph<pka (i.e., ph−pka < ), the ionized form i (i.e., the acidic conjugate weak base bh + ) prevails; . if ph>pka (i.e., pka−ph > ) , the unionized form u (i.e., the basic neutral for b) prevails. thus, because the ionized form i is more water-soluble than the unionized form (due to better solvation between the ionized form i and the dipole of the water molecule), weakly acidic drugs have higher solubility at high ph whereas weakly basic drugs have higher solubility at low ph. this means that weakly acidic drugs tend to dissolve more easily in the intestine whereas weakly basic drugs tend to dissolve more easily in the stomach. because drug solubility is ph-dependent, the dissolution profile of the drug (i.e., the process by which a solid drug dissolves into solution) may also be ph-dependent [ , ] . drug solubility and dissolution may be enhanced by several techniques including (i) chemical derivatization to alter drug pka, (ii) administration of the drug in ionic form (as a salt) rather than as a free acid or base, or (iii) alteration of environmental ph by the adjuvant use of acids or bases to better match the drug's pka [ ] [ ] [ ] . in some cases, it may be desirable to lower the solubility and dissolution of parenterally-administered drugs in order to prolong their half-life. as we discuss in the next section, the influence of drug pka and environmental ph on drug ionization has important ramifications for drug absorption and distribution. besides solubility in aqueous solvent, the ionization state of a drug can influence its solubility in the lipid phase (e.g., drug pka can be modified to increase drug polarity and therefore reduce drug lipophilicity) and therefore affect its ability to permeate cell membranes [ , ] . assuming that passive diffusion (i.e., transmembrane concentration gradient is the driving force) is the mechanism by which a drug moves across a membrane and that only uncharged ions can freely diffuse across the membrane, we can turn again to the henderson-hasselbalch equation to describe the importance of drug pka and environmental ph to drug absorption and distribution between body compartments. we note that, according to point ) above, the henderson-hasselbalch equation predicts that weak acids tend to be absorbed in an acidic environment (e.g., in the stomach). similarly, according to point ) above, the henderson-hasselbalch equation predicts that weak bases tend to be absorbed in a basic environment (e.g., in the small intestine). in , shore and coworkers proposed the ph partition hypothesis (first described by jacobs in [ ] ) to describe the influence of pka and ph upon the gastric secretion of a variety of intravenously-administered weakly acidic and basic drugs [ ] . the results of their experiments could be explained with their theoretical model of drug absorption across a lipoid barrier (i.e., the gastric mucosa) that separates two compartments (i.e., the stomach lumen and the blood) with different ph and permeable only to the unionized form, which was assumed at equilibrium. they found that the extent to which a drug moves between compartments (the gi tract and blood) depends on the value of the drug pka and the environmental ph values of the two compartments such that (using equations ( ) and ( ) that is to say that weakly acidic drugs such as aspirin (pka= . ) can be effectively absorbed from the stomach. in the case of a weakly basic drug. in practice, the model has several limitations because the ph partition hypothesis ignores other substantial influences upon drug absorption. for example, the assumption of equilibrium is unrealistic in such a dynamic system. furthermore, even weakly acidic drugs can be substantially absorbed in the small intestine because of the large luminal surface area that it presents [ ] . finally, the ph-partition hypothesis does not consider the mechanism by which drugs can move across epithelial layers. today we know that both the transcellular and paracellular pathways play important roles in the absorption and elimination of both charged and uncharged drug forms [ ] . low-specificity membrane transporter proteins that contribute to transcellular drug transport around the body include organic anion transporters (oats), organic cation transporters (octs), some mcts, and members of the abc transporter superfamily, such as the p-glycoprotein transporters (p-gp) [ ] [ ] [ ] [ ] [ ] . in some cases, the transporters themselves may be ph-sensitive or coupled to the transport of acids and bases. the importance of such considerations is exemplified by lowered absorption of weakly basic drugs in individuals with an unusually high stomach ph such as those taking ppis or individuals with achlorhydria [ ] . for example, the dissolution and absorption rate of the weakly basic antifungal agent ketoconazole, which is soluble only at ph lower than , can be enhanced by coadministration of an acidic, carbonated beverage [ ] . a related example, albeit related to absorption of drugs by bacteria, is that the adjuvant use of nahco enhances the in vitro potency of antibiotics by interfering with the proton motive force that drives antibiotic efflux from bacteria [ ] . as we will see in section . , considerations of drug solubility and transepithelial movement also influence drug elimination in urine by the kidneys. when developing drugs for oral delivery it is important to account for the adverse acidic environment of the stomach, which can cause instability and rapid degradation of drugs before they reach the small intestine for absorption. this could happen because the polymers used for the tablet coating may be susceptible to ph. for this reason, carriers for oral drug delivery are tested for ph-sensitivity and endurance in acidic environment. acid-resistant polymers that only dissolve above certain ph values are sometimes used as enteric or gastro-resistant coatings [ ] [ ] [ ] [ ] . this is the case, for example, for oral delivery of insulin [ , ] . the ph partition hypothesis provides the theoretical framework for understanding how urinary ph influences renal drug excretion; acidification of urine favors elimination of weakly basic drugs (because their absorption is reduced) whereas alkalinization of urine favors elimination of weakly acidic drugs. for example, urine acidification via administration of ammonium chloride increases elimination of the weakly basic drug amphetamine [ ] , whereas urine alkalinization via intravenous administration of nahco enhances elimination of acidic drugs like salicylic acid (i.e., aspirin) and can be helpful in the management of drug poisoning like aspirin intoxication [ ] [ ] [ ] . as we considered earlier, partitioning is just one aspect of drug distribution. many oats and octs [ ] are expressed in nephron epithelia where their action is vital for delivering drugs from the peritubular capillaries into the nephron lumen for excretion in urine and for delivering diuretics (e.g., furosemide) to their therapeutic targets in the nephron lumen. the direct secretion of these drugs into the nephron lumen is necessary because many such drugs are substantially bound to albumin in circulation and are not effectively filtered into the nephron lumen at the glomerulus. . exploiting ph for targeted drug delivery acidity can be harnessed to target drug release to acidic environments such as the stomach or pathological acidotic microenvironments such as tumors. one example under development is a gastro-floating matrix tablet that contains adjuvant nahco with the drug, produces co upon reaction with gastric acid causing the dosage form to remain buoyant in the stomach for prolonged release [ ] . another example is the use of ph-sensitive vehicles such as micelles that could release cytotoxic chemotherapeutic agents only in the acidic tumor environment [ ] . similarly, as suggested by the disparity between the usefulness of an anticancer drug that was identified in an in vitro screening performed at neutral ph and its value in vivo in the acidic tumor microenvironment [ ] , it is possible that some drugs may be inactive in circulation and may not be activated until they reach an acidic environment. abts and cas play major roles in a variety of pathologies and provide an array of potential therapeutic targets, but many are currently lacking specific or safe drugs. the application of epigenetic modulators and other genetic tools to alter their expression is an underexplored area of research. it is interesting to note that, among the many treatment paradigms for diverse acid disturbances, nahco administration is a common thread. its low cost and ready availability have prompted its nickname "enemy of the pharmaceutical industry." however, despite the promise of numerous limited trials, robust evidence in favor of its broad effectiveness in many fields is currently lacking. this is perhaps due to a lack of appreciation of subgroup effects or a lack of standardization among trails. nonetheless, research into the therapeutic importance of balancing ph remains robust and promises the delivery of many more effective treatments in the coming years. figure . targeting carbonic anhydrase to treat glaucoma. glaucoma is retinal degeneration caused by increased intralocular pressure. eye drops containing ca-inhibitors such as acetazolamide (acz) target cas in the ciliary body and reduce the production of aqueous humor, lowering intraocular pressure. the ciliary body is a complex epithelial tissue comprised of two cell layers joined by gap junctions. a variety of abts and other transporters are required to move nacl, which is followed by water, from the interstitial fluid into the anterior chamber of the eye. figure . enhancing fluid secretion in the lungs. cftr promotes the movement of hco -containing fluid onto the airway surface to promote mucociliary clearance and lung health. drugs such as lumacaftor/ivacaftor rescue this function in some individuals with cf by helping misfolded cftr molecules to function normally. alternative ph-based strategies have been suggested as adjunct cf therapy, such as blockade of the many h + secreting abts. figure . the bohr effect. the action of ae and cas promote o release in systemic capillaries (panel a) and co release in pulmonary capillaries because of the ph-dependence of the affinity of hemoglobin for o (the bohr effect). figure . the role of abts and cas in bone remodeling. osteoclasts secrete acid onto the bone surface to resorb minerals during bone growth/remodeling and in response to hormonal requirements for release of mineralized ca + and phosphate. one report suggested an off-target bone-sparing effect of ca inhibitors, used to treat glaucoma, in a group of post-menopausal women. figure . targeting the stomach h + /k + -atpase to treat acid-reflux disease. proton pump inhibitors are widely used to reduce gastric acid secretion, as an alternative or adjunct strategy to neutralizing stomach acid with an antacid such as caco . figure . targeting intestinal abts to treat constipation. intestinal fluid absorption is promoted by the combined action of nhe and slc a , which perform the net uptake of nacl, and therefore water. inhibition of either, to reduce fluid absorption from the intestinal lumen is a useful therapy for irritable bowel syndrome with constipation. ivacaftor is similarly useful in cf by restoring intestinal fluid secretion. figure . the role of abts and cas in cancer. numerous acid-base handling proteins are upregulated in rapidly proliferating tumor calls to help them dispose of metabolic acids and create an acidic microenvironment that disadvantages non-tumor cells in their vicinity. as most of these abts and cas are gainfully expressed elsewhere in the body, therapies in development are focused on blocking those rarer targets that are preferentially expressed in the hypoxic tumor environment such as mct and caix. other approached include exploiting the acidic milieu of the tumor for the targeted delivery of chemotherapeutic drugs (see section . ). figure . the influence of ph on drug distribution. theoretical distribution of a hypothetical weakly acidic drug (pka = . , panel 'a') and a hypothetical weakly basic drug (pka = . , panel 'b') between two aqueous compartments with different ph (gi tract at ph = . and blood at ph = . ). assuming that only the unionized form u (ha in panel 'a' and b in panel 'b') can cross the membrane and that u is equilibrated across the plasma membrane, panel 'a' shows that a weakly acidic drug is more concentrated in the alkaline compartment. this result suggests that weakly acidic drugs tend to be absorbed from the more acidic compartment to the more basic compartment (blue arrows). panel 'b' shows that a weakly basic drug is more concentrated in the acidic compartment, indicating that weakly basic drugs are poorly absorbed in an acidic compartment (blue arrows). weakly basic drugs are in fact poorly absorbed from the stomach. [ the history of blood gases, acids and bases dietary acid, age, and serum bicarbonate levels among adults in the united states higher estimates of daily dietary net endogenous acid production (neap) in the elderly as compared to the young in a healthy, free-living elderly population of pakistan cellular mechanisms involved in co( ) and acid signaling in chemosensitive neurons mathematical modeling of acid-base physiology acid base physiology racial/ethnic-specific reference intervals for common laboratory tests: a comparison among asians, blacks, hispanics, and white centers for disease control and prevention chronic kidney disease surveillance team, race/ethnicity, dietary acid load, and risk of end-stage renal disease among us adults with chronic kidney disease nhanes iii: influence of race on gfr thresholds and detection of metabolic abnormalities ethnicity and young age influence the frequency of diabetic ketoacidosis at the onset of type diabetes admissions for diabetic ketoacidosis in ethnic minority groups in a city hospital differences in acidosisstimulated renal ammonia metabolism in the male and female kidney effect of age on blood acid-base composition in adult humans: role of age-related renal functional decline chapter -human carbonic anhydrases: catalytic properties role of carbonic anhydrases and inhibitors in acid-base physiology: insights from mathematical modeling statpearls, statpearls publishing, treasure island (fl), clinical physiology of acid-base and electrolyte disorders metabolic acidosis in ckd: core curriculum renal pathophysiology: the essentials renal tubular acidosis: developments in our understanding of the molecular basis drug-induced metabolic acidosis, f res metabolic acidosis: pathophysiology, diagnosis and management diet-induced low-grade metabolic acidosis and clinical outcomes: a review handbook of pathophysiology the alkaline tide phenomenon tribonat--a comprehensive summary of its properties bicarbonate therapy and intracellular acidosis short-and long-term effects of alkali therapy in chronic kidney disease: a systematic review treatment of acidified blood using reduced osmolarity mixed-base solutions brain ph effects of nahco and carbicarb in lactic acidosis carbicarb: an effective substitute for nahco for the treatment of acidosis the treatment of acidosis in acute lung injury with tris-hydroxymethyl aminomethane (tham) sodium acetate as a replacement for sodium bicarbonate in medical toxicology: a review lactate versus bicarbonate. a reconsideration of the therapy of metabolic acidosis estimated net endogenous acid production and serum bicarbonate in african americans with chronic kidney disease dietary acid load is associated with serum bicarbonate but not insulin sensitivity in chronic kidney disease very low-protein diet (vlpd) reduces metabolic acidosis in subjects with chronic kidney disease: the "nutritional light signal" of the renal acid load fruit and vegetable treatment of chronic kidney disease-related metabolic acidosis reduces cardiovascular risk better than sodium bicarbonate therapy of bicarbonate-losing renal tubular acidosis long-term safety and efficacy of veverimer in patients with metabolic acidosis in chronic kidney disease: a multicentre, randomised, blinded, placebo-controlled, -week extension veverimer: an advance in base therapy for metabolic acidosis pyruvate in the correction of intracellular acidosis: a metabolic basis as a novel superior buffer a controlled clinical trial of dichloroacetate for treatment of lactic acidosis in adults. the dichloroacetate-lactic acidosis study group pathophysiology of metabolic alkalosis: a new classification based on the centrality of stimulated collecting duct ion transport acetazolamide-mediated decrease in strong ion difference accounts for the correction of metabolic alkalosis in critically ill patients hydrochloric acid infusion for the treatment of metabolic alkalosis in surgical intensive care unit patients cimetidine in the management of metabolic alkalosis induced by nasogastric drainage respiratory acid-base disorders national kidney foundation primer on kidney diseases acid-base transport by the renal proximal tubule statpearls, statpearls publishing alkali therapy for respiratory acidosis: a medical controversy acetazolamide use in severe chronic obstructive pulmonary disease. pros and cons acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates co removal from blood a proof of concept study, demonstrating extracorporeal carbon dioxide removal using hemodialysis with a low bicarbonate dialysate hormone replacement therapy causes a respiratory alkalosis in normal postmenopausal women mechanisms of action of acetazolamide in the prophylaxis and treatment of acute mountain sickness minireview: ph and synaptic transmission protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala role of hco -ions in depolarizing gabaa receptor-mediated responses in pyramidal cells of rat hippocampus bicarbonate efflux via gabaa receptors depolarizes membrane potential and inhibits two-pore domain potassium channels of astrocytes in rat hippocampal slices rapid rise of extracellular ph evoked by neural activity is generated by the plasma membrane calcium atpase regulation and modulation of ph in the brain seizure termination by acidosis depends on asic a cull-candy, proton inhibition of n-methyl-d-aspartate receptors in cerebellar neurons brain alkalosis causes birth asphyxia seizures, suggesting therapeutic strategy emerging roles of na+/h+ exchangers in epilepsy and developmental brain disorders disruption of sodium bicarbonate transporter slc a in a patient with complex partial epilepsy and mental retardation association of the asp variant of the human anion exchanger gene with common subtypes of idiopathic generalized epilepsy na + dependent acid-base transporters in the choroid plexus; insights from slc and slc gene deletion studies acetazolamide lowers intracranial pressure and modulates the cerebrospinal fluid secretion pathway in healthy rats ionic mechanisms of neuronal excitation by inhibitory gabaa receptors the gaba excitatory/inhibitory developmental sequence: a personal journey chloride concentration in cultured hippocampal neurons increases during long-term exposure to ammonia through enhanced expression of an anion exchanger a christianson syndromelinked deletion mutation (Δ es ) in slc a impairs hippocampal neuronal plasticity cerebral acidosis in focal ischemia dramatic aggregation of alzheimer abeta by cu(ii) is induced by conditions representing physiological acidosis amyloid clearance defect in apoe astrocytes is reversed by epigenetic correction of endosomal ph % co is a potent, fast acting inhalation anticonvulsant the role of carbon dioxide in acute brain injury a novel neuroprotective strategy for ischemic stroke: transient mild acidosis treatment by co inhalation at reperfusion acetazolamide in the treatment of seizures a class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects a new kid on the block? carbonic anhydrases as possible new targets in alzheimer's disease carbonic anhydrase modulation of emotional memory. implications for the treatment of cognitive disorders type ii renal tubular acidosis secondary to topiramate: a review disruption of slc a augments neuronal excitability and modulates synaptic short-term plasticity preclinical update on regulation of intracranial pressure in relation to idiopathic intracranial hypertension, fluids and barriers of the cns studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium na+/h+ and ci-/hco -antiporters of bovine pigmented ciliary epithelial cells slc a and the pathophysiology of congenital hereditary endothelial dystrophy h(oh): a holiday perspective. focus on "mouse slc a expressed in xenopus oocytes is an ideally selective h + /oh -conductance pathway that is stimulated by rises in intracellular and extracellular ph functional roles of electrogenic sodium bicarbonate cotransporter nbce in ocular tissues retinal ph and acid regulation during metabolic acidosis sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in ambystoma tigrinum retina bicarbonate modulates photoreceptor guanylate cyclase (ros-gc) catalytic activity novel mutation in slc a gene causing autosomal recessive progressive rod-cone dystrophy severe neurologic impairment in mice with targeted disruption of the electrogenic sodium bicarbonate cotransporter nbce (slc a gene) role of monocarboxylate transporters in regulating metabolic homeostasis in the outer retina: insight gained from cell-specific bsg deletion genetics and phenomics of pendred syndrome novel atp v b and atp v a mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss congenital hereditary endothelial dystrophy with progressive sensorineural deafness (harboyan syndrome) genetic disorders of transporters/channels in the inner ear and their relation to the kidney early hearing loss upon disruption of slc a in c bl/ mice non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin calcium oxalate stone formation in the inner ear as a result of an slc a mutation subcutaneous injection of drugs: literature review of factors influencing pain sensation at the injection site loss of slc a /nhe impairs nociception in a mouse model of christianson syndrome metabolic acidosis and anaemia associated with dorzolamide in a patient with impaired renal function metabolic acidosis with ophthalmic dorzolamide in a neonate ophthalmic nonsteroidal anti-inflammatory drugs as a therapy for corneal dystrophies caused by slc a mutation na+/h+-exchanger- inhibition counteracts diabetic cataract formation and retinal oxidative-nitrative stress and apoptosis protective effects of hypercapnic acidosis on ischemia-reperfusion-induced retinal injury cerumenolytic efficacy of . % sodium bicarbonate versus docusate sodium: a randomized, controlled trial the preparation of acetic acid for use in otic drops and its effect on endocochlear potential and ph in inner ear fluid discovery and development of s and s as potent tas r antagonists modulation of bitter taste perception by a small molecule htas r antagonist effect of ph modification by bicarbonate on pain after subcutaneous lidocaine injection role of cftr in epithelial physiology pulmonary complications of cystic fibrosis a new role for bicarbonate in mucus formation extracellular ph and lung infections in cystic fibrosis airway acidification initiates host defense abnormalities in cystic fibrosis mice mechanisms of acid and base secretion by the airway epithelium airway ph homeostasis in asthma and other inflammatory lung diseases hyperglycaemia and pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter dependent lactate-h+ secretion cftr modulator theratyping: current status, gaps and future directions rheological properties of cystic fibrosis bronchial secretion and in vitro drug permeation study: the effect of sodium bicarbonate safety, tolerability, and effects of sodium bicarbonate inhalation in cystic fibrosis repurposing tromethamine as inhaled therapy to treat cf airway disease esomeprazole increases airway surface liquid ph in primary cystic fibrosis epithelial cells paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid ph effects of changes of ph on the contractile function of cardiac muscle the effects of acid-base disturbances on cardiovascular and pulmonary function relative contributions of na+/h+ exchange and na+/hco -cotransport to ischemic nai+ overload in isolated rat hearts impact of systemic acidosis on the development of malignant ventricular arrhythmias after reperfusion therapy for st-elevation myocardial infarction mechanisms underlying the emergence of post-acidosis arrhythmia at the tissue level: a theoretical study reduced sarcolemmal expression and function of the nbce isoform of the na + -hco₃ − cotransporter in hypertrophied cardiomyocytes of spontaneously hypertensive rats: role of the reninangiotensin system carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy loss of the ae anion exchanger in a hypertrophic cardiomyopathy model causes rapid decompensation and heart failure mitochondrial nhe : a newly identified target to prevent heart disease disturbed acid-base transport: an emerging cause of hypertension extracellular acidosis suppresses calcification of vascular smooth muscle cells by inhibiting calcium influx via ltype calcium channels regulatory mechanisms of hemoglobin oxygen affinity in acidosis and alkalosis transgenic expression of carbonic anhydrase iii in cardiac muscle demonstrates a mechanism to tolerate acidosis sodium bicarbonate on severe metabolic acidosis during prolonged cardiopulmonary resuscitation: a double-blind, randomized, placebo-controlled pilot study an evidence-based evaluation of the use of sodium bicarbonate during cardiopulmonary resuscitation sodium bicarbonate: basically useless therapy nhe- : still a viable therapeutic target cariporide (hoe ), a selective na+-h+ exchange inhibitor, inhibits the mitochondrial death pathway na+/h+ exchanger inhibitor cariporide attenuates the mitochondrial ca + overload and ptp opening inhibits endoplasmic reticulum stress-induced apoptosis by targeting na+/h+ exchanger- in the heart inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury antibodies against the cardiac sodium/bicarbonate co-transporter (nbce ) as pharmacological tools sasanquasaponin induces increase of cl-/hco -exchange of anion exchanger and promotes intracellular cl-efflux in hypoxia/reoxygenation cardiomyocytes bicarbonate therapy, phosphate binders, and risk for vascular calcification hyperphosphatemic familial tumoral calcinosis: response to acetazolamide and postulated mechanisms metabolic and hemodynamic consequences of sodium bicarbonate administration in patients with heart disease cellular mechanisms of muscle fatigue challenging the role of ph in skeletal muscle fatigue lactic acid and exercise performance : culprit or friend? k+ and lac-distribution in humans during and after high-intensity exercise: role in muscle fatigue attenuation? metabolic and clinical consequences of metabolic acidosis three-generation study of women on diets and health study group, higher dietary acid load is associated with a higher prevalence of frailty, particularly slowness/weakness and low physical activity, in elderly japanese women regulation of ph in human skeletal muscle: adaptations to physical activity carbonic anhydrase iii is expressed in mouse skeletal muscles independent of fiber type-specific myofilament protein isoforms and plays a role in fatigue resistance effects of sodium bicarbonate on high-intensity endurance performance in cyclists: a double-blind, randomized cross-over trial sodium bicarbonate supplementation does not improve elite women's team sport running or field hockey skill performance the impact of sodium bicarbonate on performance in response to exercise duration in athletes: a systematic review mechanistic insights into the efficacy of sodium bicarbonate supplementation to improve athletic performance effect of sodium bicarbonate on prolonged running performance: a randomized, double-blind, cross-over study potassium bicarbonate reduces urinary nitrogen excretion in postmenopausal women dietary patterns, skeletal muscle health, and sarcopenia in older adults the effects of acid on bone metabolic acidosis inhibits growth hormone secretion in rats: mechanism of growth retardation serum bicarbonate and bone mineral density in us adults acute metabolic acidosis enhances circulating parathyroid hormone, which contributes to the renal response against acidosis in the rat defects in tcirg subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis carbonic anhydrase ii deficiency in families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification mouse models of slc -linked disorders of hco -transporter dysfunction effect of chronic carbonic anhydrase inhibitor therapy on bone mineral density in white women can acetazolamide be used to treat diseases involving increased bone mineral density? chronic proton pump inihibitor therapy and calcium metabolism proton-pump inhibitor use and fracture risk: an updated systematic review and meta-analysis proton pump inhibitors therapy and risk of bone diseases: an update meta-analysis revisiting the parietal cell a h+-gated urea channel: the link between helicobacter pylori urease and gastric colonization helicobacter pylori moves through mucus by reducing mucin viscoelasticity helicobacter pylori and gastric cancer: factors that modulate disease risk salivary bicarbonate as a major factor in the prevention of upper esophageal mucosal injury in gastroesophageal reflux disease analysis of bicarbonate, phosphate and other anions in saliva by capillary electrophoresis with capacitively coupled contactless conductivity detection in diagnostics of gastroesophageal reflux disease role of saliva in esophageal defense: implications in patients with nonerosive reflux disease acid neutralizing capacity of human saliva physiology of na+/h+ exchangers (nhes) in the digestive system milk-alkali syndrome health-behavior induced disease: return of the milk-alkali syndrome regulation of na/h exchanger- in gastroesophageal reflux disease: possible interaction of histamine receptor mice with a targeted disruption of the ae cl -/hco -exchanger are achlorhydric the role of acid inhibition in helicobacter pylori eradication the development of urease inhibitors: what opportunities exist for better treatment of helicobacter pylori infection in children? the cystic fibrosis of exocrine pancreas intestinal obstruction syndromes in cystic fibrosis: meconium ileus, distal intestinal obstruction syndrome, and constipation acid-base disturbances in gastrointestinal disease update on slc a mutations in congenital chloride diarrhea differential regulation of na+/h+ exchange isoform activities by enteropathogenic e. coli in human intestinal epithelial cells inhibition and redistribution of nhe , the apical na+/h+ exchanger, by clostridium difficile toxin b reduced sodium/proton exchanger nhe activity causes congenital sodium diarrhea ph and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon d-lactic acidosis: an underrecognized complication of short bowel syndrome h+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine defective small intestinal anion secretion, dipeptide absorption, and intestinal failure in suckling nbce -deficient mice slc a inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation efficacy of tenapanor in treating patients with irritable bowel syndrome with constipation: a -week, placebo-controlled phase trial (t mpo- ) impact of cftr modulation on intestinal ph, motility, and clinical outcomes in patients with cystic fibrosis and the g d mutation linaclotide improves gastrointestinal transit in cystic fibrosis mice by inhibiting sodium/hydrogen exchanger linaclotide activates guanylate cyclase-c/cgmp/protein kinase-ii-dependent trafficking of cftr in the intestine medical physiology. a cellular and molecular approach renal tubular acidosis metabolic acidosis and subclinical metabolic acidosis in ckd downregulation of the cl-/hco -exchanger pendrin in kidneys of mice with cystic fibrosis: role in the pathogenesis of metabolic alkalosis mechanisms of metabolic acidosis-induced kidney injury in chronic kidney disease low serum bicarbonate and ckd progression in children serum bicarbonate levels and the progression of kidney disease: a cohort study mechanism of hyperkalemia-induced metabolic acidosis kidney stones: pathophysiology and medical management the primary cause of infection-induced urinary stones urinary catheter blockage depends on urine ph, calcium and rate of flow daily oral sodium bicarbonate preserves glomerular filtration rate by slowing its decline in early hypertensive nephropathy bicarbonate supplementation slows progression of ckd and improves nutritional status clinical evidence that treatment of metabolic acidosis slows the progression of chronic kidney disease a causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure drug-induced renal fanconi syndrome diuretics: a review metabolic alkalosis the behavior of carbenicillin as a nonreabsorbable anion hypokalaemia, metabolic alkalosis, and hypernatraemia due to "massive" sodium penicillin therapy impaired renal hco -excretion in cystic fibrosis citrate and renal calculi: an update lemonade therapy increases urinary citrate and urine volumes in patients with recurrent calcium oxalate stone formation long-term treatment of calcium nephrolithiasis with potassium citrate strategy to control catheter encrustation with citrated drinks: a randomized crossover study role of v-atpase-rich cells in acidification of the male reproductive tract the activating effects of bicarbonate on sperm motility and respiration at ejaculation lowered levels of bicarbonate in seminal plasma cause the poor sperm motility in human infertile patients critical role of cftr in uterine bicarbonate secretion and the fertilizing capacity of sperm a new role for bicarbonate secretion in cervico-uterine mucus release hydrogen ion and carbon dioxide content of the oviductal fluid of the rhesus monkey (macaca mulatta) bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction factors and pathways involved in capacitation: how are they regulated? acidification of uterine epithelium during embryo implantation in mice loss of the na+/h+ exchanger nhe causes male infertility in mice by disrupting acrosome formation disruption of the slc a -mediated anion transport is associated with male subfertility anion exchanger is essential for spermiogenesis in mice tests of buffergel for contraception and prevention of sexually transmitted diseases in animal models maternal and fetal acid-base chemistry: a major determinant of perinatal outcome effect of preoperative bicarbonate infusion on maternal and perinatal outcomes of obstructed labour in mbale regional referral hospital: a study protocol for a randomised controlled trial improvement of cervical mucus viscoelasticity and sperm penetration with sodium bicarbonate douching sodium bicarbonate douching for improvement of the postcoital test spermicidal effects of lemon juice and juices from other natural products, agriculture and natural resources acidform: a review of the evidence a novel vaginal ph regulator: results from the phase ampower contraception clinical trial bicarbonate ion; the corona cell dispersing factor of rabbit tubal fluid pantoprazole, a proton-pump inhibitor, impairs human sperm motility and capacitation in vitro acid-base disorders in liver disease effect of ph on the kinetics of frog muscle phosphofructokinase inhibition by acidosis of adenosine ', '-cyclic monophosphate accumulation and lipolysis in isolated rat fat cells bicarbonate in the treatment of metabolic acidosis: effects on hepatic intracellular ph, gluconeogenesis, and lactate disposal in rats renal gluconeogenesis in acidosis, alkalosis, and potassium deficiency: its possible role in regulation of renal ammonia production effect of extracellular ph on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets a physiological solvent for crystalline insulin insulin sensitivity and glucose homeostasis can be influenced by metabolic acid load dietary acid load, metabolic acidosis and insulin resistance -lessons from cross-sectional and overfeeding studies in humans the ph dependence of insulin binding. a quantitative study stimulatory effect of insulin on renal proximal tubule sodium transport is preserved in type diabetes with nephropathy molecular mechanism of pancreatic and salivary glands fluid and hco − secretion effect of mineralocorticoids on acid-base balance bartter or gitelman-how to differentiate? acid retention during kidney failure induces endothelin and aldosterone production which lead to progressive gfr decline, a situation ameliorated by alkali diet role of endothelin- in renal regulation of acidbase equilibrium in acidotic humans higher dietdependent renal acid load associates with higher glucocorticoid secretion and potentially bioactive free glucocorticoids in healthy children effect of chronic metabolic acidosis on the growth hormone/igf- endocrine axis: new cause of growth hormone insensitivity in humans in vivo bicarbonate deficiency and insulin dissolution combined insulin and bicarbonate therapy elicits cerebral edema in a juvenile mouse model of diabetic ketoacidosis intracellular ph regulation during spreading of human neutrophils extracellular acidosis is a novel danger signal alerting innate immunity via the nlrp inflammasome the intimate and controversial relationship between voltage-gated proton channels and the phagocyte nadph oxidase voltage-gated proton channels maintain ph in human neutrophils during phagocytosis na+/h+ exchange activity during phagocytosis in human neutrophils: role of fcgamma receptors and tyrosine kinases science review: extracellular acidosis and the immune response: clinical and physiologic implications the effect of ph and nucleophiles on complement activation by human proximal tubular epithelial cells unravelling the interplay between extracellular acidosis and immune cells acute asphyxia affects neutrophil number and function in the rat ph changes observed in the inflamed gingival crevice modulate human polymorphonuclear leukocyte activation in vitro acidosis differently modulates the inflammatory program in monocytes and macrophages the effects of extracellular ph on immune function anion exchanger is critical for cd (+) t cells to maintain phi homeostasis and modulate immune responses systemic lupus erythematosus associated with type renal tubular acidosis: a case report and review of the literature systemic lupus erythematosus with distal renal tubular acidosis presenting as hypokalemic paralysis with respiratory failure oral nahco activates a splenic anti-inflammatory pathway: evidence that cholinergic signals are transmitted via mesothelial cells sodium bicarbonate facilitates low-dose oral tolerance to peanut in mice monocarboxylate transporter mct is a target for immunosuppression inhibition of na+/h+ exchanger by cariporide alleviates burn-induced multiple organ injury a. van den hout, co vulnerability in panic disorder overshadowed by the amygdala: the bed nucleus of the stria terminalis emerges as key to psychiatric disorders acidsensing ion channel is localized in brain regions with high synaptic density and contributes to fear conditioning overexpression of acid-sensing ion channel a in transgenic mice increases acquired fear-related behavior decreased brain ph as a shared endophenotype of psychiatric disorders dietary acid load and mental health outcomes in children and adolescents: results from the giniplus and lisa birth cohort studies understanding and predicting suicidality using a combined genomic and clinical risk assessment approach metabolic acidosis of ckd: an update prevalence of depression and suicidal ideation increases proportionally with renal function decline, beginning from early stages of chronic kidney disease carbon dioxide test as an additional clinical measure of treatment response in panic disorder responses to hypercarbia induced by acetazolamide in panic disorder patients increased lactate levels and reduced ph in postmortem brains of schizophrenics: medication confounds possible alternatives to soda lime absorption of carbon dioxide interaction of inhalational anaesthetics with co absorbents hypercapnia and acidosis in sepsis: a double-edged sword? propofol infusion associated metabolic acidosis in patients undergoing neurosurgical anesthesia: a retrospective study perioperative metabolic acidosis: the bradford anaesthetic department acidosis study chasing the base deficit: hyperchloraemic acidosis following . % saline fluid resuscitation factors related to postoperative metabolic acidosis following major abdominal surgery cause of metabolic acidosis in prolonged surgery postoperative metabolic alkalosis following general surgery: its incidence and possible etiology citrate metabolism and its complications in non-massive blood transfusions: association with decompensated metabolic alkalosis+respiratory acidosis and serum electrolyte levels outcome of surgical patients who present acidosis postoperatively biochemical changes in stored donor units: implications on the efficacy of blood transfusion clinical impact of blood storage lesions influence of ph on wound-healing: a new perspective for wound-therapy? dichloroacetate stabilizes the intraoperative acid-base balance during liver transplantation ph modulation ameliorates the red blood cell storage lesion in a murine model of transfusion an analysis of the buffer systems in saliva change of saliva composition with radiotherapy effect of decreased salivation and ph on the adherence of klebsiella species to human buccal epithelial cells evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries salivary parameters and oral health status amongst adolescents in mexico salivary flow rate, ph, buffering capacity, total protein, oxidative stress and antioxidant capacity in children with and without dental caries salivary buffer capacity, ph, and stimulated flow rate of crack cocaine users effect of sodium bicarbonate mouth wash on salivary ph and interleukin- β levels among smokers salivary biomarkers and oral microbial load in relation to the dental status of adults with cystic fibrosis salivary changes and dental caries as potential oral markers of autoimmune salivary gland dysfunction in primary sjögren's syndrome unstimulated salivary flow, ph, proteins and oral health in patients with juvenile idiopathic arthritis new paradigms on the transport functions of maturation-stage ameloblasts a novel mutant na + /hco -cotransporter nbce in a case of compound-heterozygous inheritance of proximal renal tubular acidosis extrarenal signs of proximal renal tubular acidosis persist in nonacidemic nbce b/c-null mice salivary ph: a diagnostic biomarker baking soda dentifrices and oral health the effect of sodium bicarbonate oral rinse on salivary ph and oral microflora: a prospective cohort study salivary ph after a glucose rinse: effect of a new mucoadhesive spray (cariex) based on sodium bicarbonate and xylitol effect of chewing bicarbonate-containing sugar-free gum on the salivary ph: an in vivo study sodium bicarbonate solution versus chlorhexidine mouthwash in oral care of acute leukemia patients undergoing induction chemotherapy: a randomized controlled trial the effect of bicarbonate/fluoride dentifrices on human plaque ph enhancement of plaque removal efficacy by tooth brushing with baking soda dentifrices: results of five clinical studies bicarbonate-based powder and paste dentifrice effects on caries ph stabilizing properties of a posterior light cured resin composite: an in vivo study six months of daily high-dose xylitol in high-risk schoolchildren: a randomized clinical trial on plaque ph and salivary mutans streptococci the influence of virus infection on the extracellular ph of the host carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours merozoite surface protein recognition of host glycophorin a mediates malaria parasite invasion of red blood cells reduced risk of plasmodium vivax malaria in papua new guinean children with southeast asian ovalocytosis in two cohorts and a case-control study the pathophysiologic and prognostic significance of acidosis in severe adult malaria unidentified acids of strong prognostic significance in severe malaria put a cork in it: plugging the m viral ion channel to sink influenza proton pump inhibitors are risk factors for viral infections: even for covid- ? pharmacokinetics and pharmacodynamics of dichloroacetate in children with lactic acidosis due to severe malaria can exchange transfusions using red blood cells from donors with southeast asian ovalocytosis prevent or ameliorate cerebral malaria in patients with multi-drug resistant plasmodium falciparum? na(+) regulation in the malaria parasite plasmodium falciparum involves the cation atpase pfatp and is a target of the spiroindolone antimalarials the warburg effect: how does it benefit cancer cells? we need to talk about the warburg effect ph control mechanisms of tumor survival and growth regulation and roles of bicarbonate transporters in cancer role of ph regulatory proteins and dysregulation of ph in prostate cancer disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth carbonic anhydrases: role in ph control and cancer sharpey-schafer lecture: gas channels rapid co permeation across biological membranes: implications for co venting from tissue water transport proteins-aquaporins (aqps) in cancer biology acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress the acidic tumor microenvironment as a driver of cancer tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages increased acid-producing diet and past smoking intensity are associated with worse prognoses among breast cancer survivors: a prospective cohort study acid treatment of melanoma cells selects for invasive phenotypes the hypoxic response expression as a survival biomarkers in treatment-naive advanced breast cancer, asian pac increased expression of na+/h+ exchanger isoform predicts tumor aggressiveness and unfavorable prognosis in epithelial ovarian cancer monocarboxylate transporters in breast cancer and adipose tissue are novel biomarkers and potential therapeutic targets cellular acidification as a new approach to cancer treatment and to the understanding and therapeutics of neurodegenerative diseases disrupting na + , hco₃ − -cotransporter nbcn (slc a ) delays murine breast cancer development intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer development of a small molecule tubulysin b conjugate for treatment of carbonic anhydrase ix receptor expressing cancers brain tumor acidification using drugs simultaneously targeting multiple ph regulatory mechanisms glucose-dependent growth arrest of leukemia cells by mct inhibition: feeding warburg's sweet tooth and blocking acid export as an anticancer strategy manipulating ph in cancer treatment: alkalizing drugs and alkaline diet bicarbonate increases tumor ph and inhibits spontaneous metastases targeting the acidic tumor microenvironment: unexpected pro-neoplastic effects of oral nahco therapy in murine breast tissue a nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis pros and cons of dietary strategies popular among cancer patients irinotecan-induced neutropenia is reduced by oral alkalization drugs: analysis using retrospective chart reviews and the spontaneous reporting database martínez-climent, targeting the anion exchanger with specific peptides as a new therapeutic approach in b lymphoid neoplasms intestinal luminal ph in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs measurement of gastrointestinal ph profiles in normal ambulant human subjects advances in oral drug delivery for regional targeting in the gastrointestinal tract -influence of physiological, pathophysiological and pharmaceutical factors impact of gastrointestinal disease states on oral drug absorption -implications for formulation design -a pearrl review food, gastrointestinal ph, and models of oral drug absorption literature review of gastrointestinal physiology in the elderly prediction of ph-dependent drug-drug interactions for basic drugs using physiologically based biopharmaceutics modeling: industry case studies study of ph-dependent drugs solubility in water acidic and basic drugs in medicinal chemistry: a perspective drug solubility: importance and enhancement techniques, isrn pharm physicochemical properties, formulation, and drug delivery a quantitative assessment of herg liability as a function of lipophilicity some aspects of cell permeability to weak electrolytes the gastric secretion of drugs: a ph partition hypothesis surface area of the digestive tract -revisited tight junction modulation and its relationship to drug delivery polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics the human organic cation transporter oct mediates high affinity uptake of the anticancer drug daunorubicin the organic anion transporter (oat) family: a systems biology perspective the p-glycoprotein transport system and cardiovascular drugs drugs as p-glycoprotein substrates, inhibitors, and inducers impaired drug absorption due to high stomach ph: a review of strategies for mitigation of such effect to enable pharmaceutical product development bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force ionotropically cross-linked ph-sensitive ipn hydrogel matrices as potential carriers for intestine-specific oral delivery of protein drugs peppas, ph-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: effects of protein size, crosslinking density, and hydrogel degradation on protein delivery nanotechnology for protein delivery: overview and perspectives eudragit: a technology evaluation preparation of layer-by-layer thin films containing insulin and its ph-sensitive decomposition oral delivery of insulin using ph-responsive complexation gels fundaments of toxicology-approach to the poisoned patient the role of sodium bicarbonate in the management of some toxic ingestions acid-alkaline balance: role in chronic disease and detoxification., alternative therapies in health and medicine relationship between the urinary excretion mechanisms of drugs and their physicochemical properties gastro-floating matrices designed for simultaneous improvement of floating and drug release capabilities: an in vitro case study of matrices loaded with ciprofloxacin hydrochloride environmental ph-sensitive polymeric micelles for cancer diagnosis and targeted therapy a drug screening assay on cancer cells chronically adapted to acidosis key: cord- -y o ovf authors: guliyev, hasraddin title: determining the spatial effects of covid- using the spatial panel data model date: - - journal: spat stat doi: . /j.spasta. . sha: doc_id: cord_uid: y o ovf this study investigates the propagation power and effects of the coronavirus disease (covid- ) in light of published data. we examine the factors affecting covid- together with the spatial effects, and use spatial panel data models to determine the relationship among the variables including their spatial effects. using spatial panel models, we analyse the relationship between confirmed cases of covid- , deaths thereof, and recovered cases due to treatment. we accordingly determine and include the spatial effects in this examination after establishing the appropriate model for covid- . the most efficient and consistent model is interpreted with direct and indirect spatial effects. efforts directed toward interpreting the pathophysiology of covid- have led to the eu mobilising € , , into research that would "contribute to more efficient clinical management of patients infected with the virus, as well as public health preparedness and response" ("coronavirus: eu mobilises € million for research," , january ). further, us-based corporations such as co-diagnostics and the novacyt's molecular diagnostics division primerdesign have been developing covid- testing kits for use in the research setting ("primerdesign launches molecular test for new coronavirus," , january ). the uk government has also sanctioned £ , , to support the development of a covid- vaccine ("coronavirus: uk donates £ m to speed up vaccine," , february ). given the nature of the pandemic, covid- has been a subject of intense discussion since the beginning of . as the pandemic spreads exponentially, healthcare enterprises and non-profit organizations have already begun work to counter it. in this study, we investigate the propagation power and effects of covid- in light of published data. thus, the factors affecting covid- are examined together with spatial effects, and spatial panel data models are used to determine the relationship among the variables (factors) with spatial effects. using spatial panel models, we analyse the relationship between the rate of confirmed cases (r c ) of covid- , the rate of deaths (r d ), the rate of recovered cases (r r ) due to treatment, with spatial and temporal effects. we first estimate a standard linear panel data model devoid of spatial effects. this model can be used as a reference for the estimation results of spatial panel data models as well as to check the robustness of these estimation results (yang, chen, cao, li, & li, ) . the formulation of a standard linear regression model (slm) is as follows (lan, ; tatoglu, ) : where is the explained variable, denotes the individuals, and constitutes the regions ( = ). is the dimension of the time series, that is, from january to march . ′ is the × vector of observations of the explanatory variables and is the × vector of undetermined coefficients. is an individual effect that cannot be directly observed and quantified and is a disturbance term that varies with the individual and time. if is related to , the panel data model is a fixed effects model; otherwise, it is a random effects model (fotheringham & rogerson, ) . spatial panel data models include the spatial autoregression model (sar), spatial error model (sem), spatial autocorrelation model (sac), and spatial durbin model (sdm). these models consider the spatial effects based on the slm and they are estimated using the maximum likelihood principle. among them, the sar model considers the spatial spillover effect of the dependent variable. hence, its formula includes the spatial lag term of the dependent variable, which can be expressed as follows: where is the spatial lag of the dependent variable and = ∑ = is the contiguity based on the weighted rook matrix. is the spatial autoregression coefficient. if has statistical significance, it demonstrates the existence of a significant j o u r n a l p r e -p r o o f spatial dependence among the dependent variables. that is, a confirmed case in a region depends on the contiguous regions. the value of reflects the degree of the spatial dependence (gelfand, diggle, guttorp, & fuentes, ) . the sem discovers the effects of the omitted variables on the observation of the determined (dependent) variable in a provincial area, which contains a spatial error term. a spatial autocorrelation among residuals is thus practical and the sem can be formulated as follows: where λwε is the spatial error term, is the autoregressive factor, and is a random error term that is usually assumed to be independent and identically distributed (i.i.d.). we can confirm the existence of hidden independent variables with spatial autocorrelation if is statistically significant, which results in the trend of a noticeable spatial autocorrelation in the residuals. the sac model is a combination of the sar models and sems; it consists of the dependent variable spatial lag and a spatial error term, which can be expressed as follows: = + ′ + + = λwε + , ( ) in the above equation, corresponding to various research functions and needs, swm w and swm w (spatial weight matrix) can be the same or different (lesage, ; yang et al., ) . in this study, we used the same swm to estimate the model, that is, = = , and the residual terms are the same as those revealed above. the sdm includes the dependent variable spatial lags and explanatory variables. it uses the marginal effects of the explanatory variables from the nearby regions/state based on the sar model. the common specification for the sdm is as follows: = + ′ + δ + + ( ) where δ is the explanatory variables' spatial lag, is the × ( − ) constant independent variable matrix, and is the ( − ) × vector of the parameters that determine the marginal effects of the independent variables from nearby observations on , the dependent variable. (elhorst, ) , illustrates the relationships among the previously stated spatial panel models. first, we examine the slm estimated by ordinary least squares. we start with this model, as it is the simplest and most common. though it is a non-spatial effect model, it is frequently used as a diagnostic tool for model specification and is a benchmark for comparisons with spatial models. we also represent the sem, as the interpretation of the coefficients is similar to that of an slm. the sar is introduced in section . because of endogenous spatial dependence in this model, it is more challenging to interpret the coefficients. this section also examines the sac model (kelejian & prucha, ) , which is characteristically close to the sar model. in section , we present two regression models with spatial lags only in the independent variables-the spatially-lagged x model (slx) other than the spatial durbin error model, both of which include exogenous spatial dependence. in section , we consider the interpretation of coefficients for an sdm, which includes both exogenous and endogenous spatial dependencies, consequently complicating the interpretation more than for the preceding models (golgher & voss, ) . for the purpose of our objective, we include sampling data from january to march for the * regions in mainland china. the data are collected from the covid- situation reports ** by who. we analyse the relationship between the rate of confirmed cases (r c ) of covid- , the rate of deaths (r d ), and the rate of recovered cases (r r ), with spatial and temporal effects. the rates are calculated classifying each variable by the population in the province. population statistics for each province is collected from the national bureau of statistics of china *** . the statistics of the rate of confirmed deaths and recovered cases on march and average statistics are presented in table a . . table a. . shows that hubei had the highest concentration of the rate of confirmed cases ( . cases per , people), followed by guangdong ( . cases per , people) and henan ( . cases per , people). the least rate of confirmed cases was from ningxia, qinghai, and tibet. these data are up to march . in hubei, an average of . out of , people tested positive for covid- , . out of , patients recovered, and . out of , people lost their lives. for guangdong, these statistics are . , . , and . , respectively. before fitting spatial panel models, we require an swm matrix. an swm characterizes the spatial relationships among variables in a dataset (fotheringham & rogerson, ; zeren, ) . the in this research was × , row-standardized with zero diagonal factors and developed via the conceptualization of spatial relations of the polygon rook contiguity in stata . formally, this form is then transformed into a suitable format for stata . and is used in spatial panel regression. to manage the spatial autocorrelation effect of the dependent variable and correctly analyse the affecting factors and their spatial spillover effects, spatial panel data models can be used. compared with standard linear panel data models, spatial panel data models take on spatial effects, such as the spatial dependence and spillover effects. further, compared with the spatial model built on cross-sectional data, the spatial panel data model can grasp the individual heterogeneity of spatial units-that is, individual effects-and can escape missing variables and estimation errors more efficiently (elhorst, ) before estimating spatial panel data models, we need to test for cross-sectional dependence. the primary issue when confronted with spatially referenced data is to determine whether spatial dependence exists, that is, whether "nearby" cases are more correlated than distant ones. a flexible way of assessing whether dependence in the cross-section of a panel dataset is spatially related is the particularization of the pesaran ( ) test for general cross-sectional dependence (croissant & millo, ; tatoğlu, ) . table shows the cross-sectional dependence test reports; we can reject that the null hypothesis errors are i.i.d. this is not surprising given our hit map visual ( figure ) appraisal of confirmed covid- cases. consequently, we require spatial panel models. the estimation results for the slm and the six spatial panel data models are shown in table . the parameters of the spatial panel models are estimated using the quasi-maximum likelihood estimator derived by lee and yu ( ) and the p-value is calculated using the robust standard error. all of spatial panel data models include two-way effects: individual (cross) and temporal (time) effects. temporal effects for each spatial panel model are shown in table a. . firstly, we eliminated sem ( ), sdm ( ) and sdem ( ) models since there was spatial effect no statistically significant at % level. following, we had to choose from models such as sar ( ), sac ( ) and slx ( ). the estimated coefficient of the spatially lagged independent variables (lm r and lm d ) in the slx (spatially-lagged x) model was statistically significant at the % level. that is, the rate of confirmed covid- cases for provinces in china are spatially correlated. this further suggests that it is necessary to construct spatial panel data models rather than slms, which do not consider spatial effects, if our objective is to explore the influencing factors of the rate of confirmed cases and their spatial spillover effects. the pseudo-r ( . ), likelihood ratio-stat (lr-stat) ( ), and lagrange multiplier (lm) test of common spatial terms stat ( . ) for the slx are higher than sar ( ) and sac ( ) models. its value of the corrected akaike information criterion (aicc) (- . ), which is calculate for small samples, and bayesian information criterion (bic) (- . ) are also lower than the sar and sac models. the lm r , and lm d test statistics for the slx are significant at the % level, and, hence, spatial effects of explanatory variables (lm r , lm d ) are different from zero. hausman test statistics is . for slx; further, the fixed effects slx is more consistent in comparison with the random effects slx (prob< . ). consequently, the slx can be considered a betterfitting spatial panel regression model. therefore, we mainly interpret the influencing factors based on the estimation results of the slx in the following analysis. the average direct, indirect, and total effects of these explanatory variables are presented in table . the direct effect expresses the marginal effect of the change in the independent variable of one percent on the dependent variable of the same unit. the indirect effect is the marginal effect of the change in the independent variable in one percent on the dependent variable value of all neighbouring units. the total effect is the sum of both effects. the average direct effects of the rate of recovered cases and the rate of deaths are . (prob< . ) and - . (prob< . ), respectively, indicating that one-percent increase in the rate of deaths (example, in hubei) leads to % positive change in the rate of confirmed cases (in hubei) and a one-percent increase in the rate of recovered cases leads to . % negative change in the ratio of confirmed cases (in hubei), respectively. compared with the average direct effects and the estimated coefficients, the average indirect effects can more comprehensively reflect the actual effect of the influencing factors. the indirect effects of the rate of recovered cases are measured at . (prob< . ), indicating that a one-percent increase in the rate of deaths (in hubei) leads to . % positive change in the rate of confirmed cases (in neighbouring regions of hubei, namely, henan, anhui etc.). however, the indirect effect of the rate of recovered cases is not significant at the % level (prob> . ). table a . shows temporal effects of slx model. we consider that the rate of confirmed cases in the first days increased slightly and it was not statistically significant at % level. however, for the slx model, after february date, the rate of confirmed case increases had become statistically significant. the increase in the rate of confirmed cases since the beginning of march has become dramatic. so, we contemplate that the confirmed cases on march compared to january date increased by . cases in , people. built on the spatial panel data of regions in china from january to march , we investigated the influencing variables (the rate of deaths and recovered cases) and their spatial spillover effects of covid- . before we built and compared the spatial panel data models, we tested the cross-sectional dependence using the pesaran test. we thus found cross-sectional dependence between the units. among the panel data regression models estimated to capture spatial effects, the most efficient and consistent model was determined according to the maximum pseudo-r , lr-test, lm-test statistics, and minimum aicc and bic values. the results of the model comparison allowed us to select the slx from the predicted spatial panel data models for interpretation. in the slx model, the spatial effects of the dependent and independent variables were examined separately. specifically, the independent variables effects were split into the total, indirect (spatial spillover effects), and direct effects in order to improve the identification of the actual impacts and spatial interactions of the factor components on covid- . we thus draw the following conclusions: • as per the total effect, the rate of deaths has significant positive effects, while the rate of recovered cases has significant negative effects on covid- . • as per the direct effect, the rate of deaths has significant positive effects on covid- . that is, a one-percent increase in the rate of deaths leads to % the rate of confirmed positive changes. in addition, the recovered cases have significant negative effects on covid- . that is, a one-percent increases in the rate of recovered cases leads to . % confirmed negative changes. • as per the indirect effect, the rate of deaths has significant positive effects on covid- in the neighbouring region. that is, a one-percent increase in the rate of deaths leads to . % confirmed positive changes in the neighbouring regions. however, the rate of recovered cases did not have significant negative effects on covid- . • as a result of the temporal effect analysis, the rate of confirmed cases is increasing day by day. we compared the date of january with the date of march , the confirmed cases had increased nearly by . cases per , people, in other word, cases per , , people. some limitations need to be addressed while discussing the results of the present study. we cannot model the rate of deaths because of the presence of high proportion of zeros. in addition, we consider that the time period is short. future research can be examined with a big dataset. in general, this study had provided researchers with information about the effects of the spread of the covid- virus. therefore, the effects of the spread of the virus have been addressed both spatially and temporally, and efforts have been made to produce information that would be useful to all humanity. . ** . ** . ** . ** . ** . ** . ** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** / / . *** . *** . *** . *** . *** . *** . *** j o u r n a l p r e -p r o o f references coronavirus: eu mobilises € million for research coronavirus: uk donates £ m to speed up vaccine panel data econometrics with r applied spatial econometrics: raising the bar spatial panel data models the sage handbook of spatial analysis: sage handbook of spatial statistics how to interpret the coefficients of spatial models: spillovers, direct and indirect effects a generalized spatial two-stage least squares procedure for estimating a spatial autoregressive model with autoregressive disturbances analysis of cross-sectional data and panel data with application of stata estimation of spatial autoregressive panel data models with fixed effects an introduction to spatial econometrics outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle general diagnostic tests for cross section dependence in panels. primerdesign launches molecular test for new coronavirus İleri panel veri analizi stata uygulamalı clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan the spatial characteristics and influencing factors of modal accessibility gaps: a case study for guangzhou mekansal etkileşim analizi key: cord- - ju fcf authors: arthi, vellore; parman, john title: disease, downturns, and wellbeing: economic history and the long-run impacts of covid- date: - - journal: explor econ hist doi: . /j.eeh. . sha: doc_id: cord_uid: ju fcf how might covid- affect human capital and wellbeing in the long run? the covid- pandemic has already imposed a heavy human cost—taken together, this public health crisis and its attendant economic downturn appear poised to dwarf the scope, scale, and disruptiveness of most modern pandemics. what evidence we do have about other modern pandemics is largely limited to short-run impacts. consequently, recent experience can do little to help us anticipate and respond to covid- ’s potential long-run impact on individuals over decades and even generations. history, however, offers a solution. historical crises offer closer analogues to covid- in each of its key dimensions—as a global pandemic, as a global recession—and offer the runway necessary to study the life-course and intergenerational outcomes. in this paper, we review the evidence on the long-run effects on health, labor, and human capital of both historical pandemics (with a focus on the influenza pandemic) and historical recessions (with a focus on the great depression). we conclude by discussing how past crises can inform our approach to covid- —helping tell us what to look for, what to prepare for, and what data we ought to collect now. the health and economic toll of the covid- pandemic continues to expand throughout the globe, impacting countries both rich and poor. as it does so, the virus exposes the strengths and weaknesses of our healthcare systems, political institutions, media, and our economies themselves. much of the discussion to date has understandably focused on stemming the immediate costs of the covid- crisis: among them, mortality, business failures, job losses, and foreclosures. this pain is salient, and as such, very obviously demands an urgent response. however, there are potential outcomes of the current pandemic which, while perhaps less salient, also merit urgent attention: namely, long-run damage to human capital and wellbeing. it is to these particular long-run effects that we turn our attention in this paper. the potential for long-run harm to human capital arises from two main facts about the current pandemic. first, key features of covid- -among them its geographic reach, its relatively high ease of transmission, its comparatively low lethality, and its many emerging sequelae-have given rise to widespread and potentially lasting morbidity among its many survivors. second, the pandemic has sparked an unprecedentedly large downturn, which in its own right has the capacity to permanently scar trajectories of health and income, even for those who do not fall ill themselves. while the costs of these long-run effects may seem far away, they are latent today and could become massive down the line: burdening healthcare systems and government assistance programs, suppressing work capacity and human capital investment, and reducing economic prosperity more generally. luckily, the returns to avoiding these harms, or to acting swiftly to compensate for them before they have a chance to compound, tend to be much higher the sooner interventions can be made (see, e.g., heckman, ; almond & currie, ) . together, the potential for diffuse and latent adverse effects, and the cost-effectiveness of early remediation, suggest that in addition to any efforts to address the immediate pain of the pandemic, our eyes should also be on the future-and on actions we can take now to mitigate the long-run pain for affected cohorts, and therefore, the wider economy. but what, exactly, is the long-run human fallout of covid- likely to be-and who will bear the brunt of this crisis? to answer these questions, we need the sort of long-run view that only history can provide. an obvious starting point is to look to evidence from historical pandemics. despite potential differences in empirical settings and epidemiological characteristics, the sheer number and diversity of past pandemics means that covid- has many close historical analogues as a health crisis. for instance, while the current pandemic is frequently described as unprecedented, in many ways, its immediate effects on health are not altogether anomalous. with cases first appearing in december , sars-cov- , the pathogen behind covid- , spread throughout the world in a matter of weeks, with deadly consequences. by the end of april , worldwide cases had topped million, and fatalities exceeded , . as of this writing in late october , and with the pandemic still spreading, cases exceed . million, and fatalities have surpassed . million. for context, deaths from h n (swine flu) in - were smaller in magnitude, with estimates of over , deaths attributable to the virus (dawood et al., ) . while at million deaths, the hong kong flu of is comparable to covid- in its death toll to date, the asian flu of was substantially deadlier, killing million people. likewise, cholera, typhus, smallpox, measles, and tuberculosis all have had high death tolls, including during the th century. reaching even further back, the black death left a devasting imprint on the world, killing a third of europe's population. clearly, historical pandemics offer a rich evidence base that can help shed light on the range of possible long-run effects of covid- through morbidity and mortality. however, there is one crucial aspect of the current pandemic that sets it apart from all but the most catastrophic historical disease outbreaks : the presence of an acute public health crisis alongside massive and widespread economic disruptions. not just that-it is the fact that this health crisis has precipitated an economic one. to wit, efforts to stop the spread of the virus, alongside failures to contain it, have contributed to a dramatic slowdown of the global economy. consider, for instance, the economic dislocation experienced in the u.s., a country which quickly came to lead the world in both confirmed covid- cases and deaths. in march and april of , roughly percent of the united states' labor force filed unemployment claims. double-digit unemployment would continue through the summer. the dow jones fell by over percent. for contrast, during influenza pandemic, responsible for roughly , deaths in the u.s. (glezen, ; simonsen et al., ) , unemployment peaked at . percent, and the dow fell percent-certainly a recession, but nothing on the order of what we have already experienced during the covid- outbreak, just a few months in. the unprecedented scale of the covid- economic downturn relative to past pandemics is apparent in figure , which shows the evolution of u.s. gdp over time, with major epidemics highlighted. notes: annual gdp per capita data for through are taken from https://www.measuringworth.com. quarterly real gdp per capita data for and are taken from https://fred.stlouisfed.org/series/a rx q sbea and deflated to plague would continue to impact economies into the th century, with an outbreak in san francisco infecting individuals and killing (echenberg, ) . the san francisco outbreak presents interesting parallels to . despite health officials identifying the plague and urging action, california's governor, henry gage, denied there was an outbreak, partly out of a desire to prevent business losses from a quarantine. it took the intervention of federal authorities and a new governor to implement proper measures to stop the spread of the plague. california officials reacted quite differently to covid- , at least initially, swiftly imposing stay-at-home orders. a century later, the calculus of weighing an economic slowdown against the spread of disease has changed. despite a much larger population, and a much more widespread pandemic, covid- had claimed under lives in san francisco county as of late october , thanks in part to these relatively early and stringent interventions. for instance, the aids crisis and the black death may be some of the only other major pandemics where mass morbidity and mortality were accompanied by dramatic and widespread economic dislocation. dollars to match the historical data. code to generate the figure and the underlying data for it can be found at open-icpsr (https://doi.org/ . /e v ). clearly, this feature of the current pandemic calls for complementary evidence if we are to understand its potential for long-run harm: there is no suitable all-in-one historical analogue for covid- , and evidence from past pandemics alone is likely to understate the potential for damage to (or intervention in) health and welfare through income and labor-market channels. indeed, to continue with our u.s. example, the two-trillion-dollar coronavirus aid, recovery, and economic security act's closest comparison, is not to be found in past responses to health crises, but rather in the response to past macroeconomic crises-e.g., the american recovery and reinvestment act in the case of the great recession, and the new deal in the case of the great depression. federal outlays as a percentage of gdp rose from . percent at the start of the great depression to . percent by . the cares act alone is equal to roughly percent of gdp, and this does not account for additional relief that may be approved in the coming months. asset purchases in response to the great recession increased the federal reserve's balance sheet from $ . billion in to $ . trillion in . that balance sheet has gone from $ . trillion in february of to $ . trillion just four months later. by nearly any metric, covid- has generated both an economic crisis and a government response of historic scale. studying how individuals emerged from these primarily economic disasters, and what role government fiscal interventions played in their recovery, may therefore help us flesh out the incomplete perspective we would gain from studying past health shocks alone. turning to a combination of historical crises, then-past pandemics and recessions, both-allows us to consider events that in many ways more closely mirror current circumstances, and whose contextual differences can themselves be informative of our current situation first, and most crucially, these events have had time to fully unfold: the short-, medium-, and long-run consequences of these events can be directly observed. second, the diverse array of historical events, settings, and mechanisms provides a set of reasonable analogues for covid- , even as our understanding of covid- evolves. third, the economic history literature shows how much can be learned with clever analysis of even incomplete or imperfect data. missing and inaccurate health data is unfortunately directly relevant to assessing the spread of covid- , given, for instance, current issues with testing and coordination. thus, a historical perspective allows us to use rich data to look at not only the short-term effects of crises like covid- on health, labor, and human capital, but also the long-term and intergenerational impacts along these dimensions for both individuals and the wider economy. in so doing, it can offer us insight on the current crisis-telling us what to look for, what to prepare for, and what data we ought to collect now. put another way, understanding the lingering health and economic impacts of these past crises offers valuable insight for anticipating and responding to the potential long-term impacts of covid- . to examine how history can inform our view of the coronavirus pandemic and associated policy responses as they relate to long-run wellbeing, we begin in section ii by reviewing the features of covid- that will determine its potential health and economic impacts, and placing these features in historical context. then, in sections iii and iv, respectively, we narrow our focus to two of the closest analogues to the current pandemic-one, the influenza pandemic, which speaks to -direct‖ health-channel effects; and another, the great depression, which speaks to -indirect‖ effects through the labor market and wider economy. there, we review the economic literature on the short-and long-term effects on cohorts exposed to these massive shocks, and discuss how these short-run experiences can give rise to lasting, and sometimes hidden, damage. we conclude by discussing what economic historians and researchers of covid- can offer each other. before we can look to historical evidence on how covid- 's effects may unfold in the long run, it is useful to fix ideas about key features of the current crisis-its epidemiology, its demographics, and the policy responses to date. comparing these features to those seen in past pandemics offers a sense of which historical pandemics might serve as the most useful points of reference going forward. we draw here on the principles outlined by morens et al. ( ) to categorize pandemics. they point to eight characteristics common to most accepted definitions of a pandemic: ) wide geographic extension and ) disease movement, which speak to the disease's spatial reach; ) high attack rates and explosiveness, ) infectiousness, and ) contagiousness, which speak to how it spreads; ) severity, which speaks to its potential for population scarring and culling; and ) minimal population immunity and ) novelty, which speak to the scope for harm and the speed with which preventive and therapeutic responses can be marshalled. by all measures, covid- presents these hallmark features of a pandemic. understanding exactly how covid- reflects each dimension is essential for understanding the likely short-and long-run consequences of the pandemic. the widespread nature of pandemics makes their health and economic impacts particularly devastating: with effects felt everywhere, it becomes increasingly difficult to shift economic activity to, or medical resources from, unaffected areas. while the true extent and timing of covid- cases is yet to be determined, the evidence to date indicates that the global spread of the virus has been incredibly rapid. the earliest reported cases appeared in december in wuhan, china. that same month, the virus made it to france. by january , there were confirmed cases throughout asia, europe, north america, and africa, and by the end of the month, the number of cases worldwide reached , . in the months that followed, that number rose sharply-first to , in february, , in march, and over , , by the end of april. by may , only sovereign states had no confirmed cases, of which are island nations in oceania. covid- had become a truly global pandemic by the end of spring , and both cases and fatalities have continued to rise across the globe in the months that followed. this feature of covid- surely has much to do with the highly globalized nature of our modern economy. indeed, we see similar patterns in historical pandemics, reaching as far back as we have had extensive trade routes. nearly every country with reliable mortality statistics displayed excess deaths from the influenza pandemic . similarly, the plague pandemic originating in canton and hong kong in spread to ports across five continents (who, ) , and even the justinian plague of reached asia, africa, and europe. even in a historical era where countries were less tightly integrated than they are today, as of late october , there have been nearly million cases of covid- worldwide, and over million deaths-and, as winter dawns on the northern hemisphere, and as we enter a new and possibly more lethal wave of the pandemic in many parts of the world, these numbers seem poised to rise further. even though world economies are substantially more tightly integrated than in the past, even in the preindustrial era, alfani & murphy ( ) document that it was common for disease to be transmitted along trade routes or through inter-regional commercial contact. big trading centers in particular, such as amsterdam, london, and venice, frequently faced outbreaks of plague (see alfani ( ) , biraben ( ) , and curtis ( ) for the underlying studies). the only thing that truly spared an area from pandemics was isolation. with the increase over the last few centuries in both global connectedness and population density, the implications for our current crisis are clear. the speed with which a disease spreads directly impacts the difficulty of containing it. indeed, it is these transmissibility-related features that account for many of the public health measures seen in response to the current crisis-some, such as early international travel restrictions, which tried to contain a disease that in many countries was already being spread locally via community transmission; and some, such as stay-athome orders and mask-wearing, which have been more effective in slowing transmission once it was too late for a containment strategy to be tenable. high attack rates and explosiveness (multiple cases appearing in a short time span) make it hard to stay ahead of a disease. these characteristics are functions of a disease's infectiousness and contagiousness: its ability to spread from person to person. covid- is transmitted by respiratory droplets and aerosols produced when an infected person coughs or sneezes. this has led to covid- having a daunting rate of transmission, with early estimates of a basic reproductive rate of between and (sanche et al., ) . as a point of reference, these transmission numbers are akin to those seen for past sars, polio, mumps, yellow fever, and influenza outbreaks (see figure ). the economic history of these pandemics thus provides a guide for what we might expect from the covid health crisis. for contrast, the economic history of measles-which presents far higher transmission rates, with estimates of basic reproductive rates greater than (guerra et al., )-offers a sense of how much worse things could be. notes: case mortality rates are for untreated patients. for covid- , basic reproduction rates are taken from https://wwwnc.cdc.gov/eid/article/ / / - _article. all other reproduction and fatality rates are taken from https://docs.google.com/spreadsheets/d/ khcewy-d hxlwrft jjrq xf whqlmwyrxel wjxkw /edit#gid= (the data the opening up of regions to trade, or conquest, has generated a large literature on the role of disease in shaping economies. see in particular the literature on the columbian exchange (nunn & qian, ) . the basic reproductive rate is the number of expected cases directly generated by one case when all individuals in the population are susceptible to infection. like the disease's ease of transmission, the severity with which it manifests symptoms will also be a crucial determinant of both its consequences for individuals and the wider economy, and the nature and magnitude of the government response. for instance, a highly lethal pandemic may generate extensive and indiscriminate mortality; a less lethal pandemic may generate culling (selective mortality related to a specific health threshold); and an even less lethal pandemic may generate very little mortality, but substantial health scarring among survivors. if a disease is so mild that many of those who are infected remain asymptomatic, this can, in the absence of widespread testing, undermine efforts to slow transmission. likewise, rates of infection, in combination with severity considerations, will help determine whether governments intervene, or merely wait for the disease to -take its course‖ on the way to achieving herd immunity. in its april , covid- strategy update, the world health organization note that percent of those infected experience moderate disease, including pneumonia, and percent experience severe disease. they cite a crude clinical case fatality rate of over three percent that rises to percent or higher in individuals over the age of . as shown in figure , the crude mortality rate for covid- in its first months is similar to that of the influenza pandemic and of measles, but far lower than the deadlier recent outbreaks of mers, ebola, sars, and lower still than the truly devastating historical toll of smallpox, which had an average case fatality rate of percent (ellner, ) . this rich historical spectrum of pandemic severity, in turn, demonstrates that both mild and severe diseases impact the economy, albeit in very different ways. for instance, the eradication of hookworm in the u.s. south-a disease which is not fatal, but which primarily causes lethargy and anemia-improved returns to schooling, educational attainment, and incomes in areas with high prior infection rates, but did little to change to overall demographic, economic, or institutional structure (bleakley, ) . for contrast, the black death and other pre-modern outbreaks of plague, which had extraordinarily high death tolls, fundamentally reshaped the global economy through their effects on population size and demographic structure. in this context, covid- 's wide scale and relatively low lethality will surely have a bearing on the scope, magnitude, and timescale of damages. as we will see in later sections, it suggests that we might ultimately expect to see the greatest harm only in the long run, with widespread generational scarring arising from short-run morbidity and economic disruptions. the novelty of a pandemic virus contributes to its potential for destruction: it takes time to identify a new disease, understand key features of its epidemiology, develop treatments and vaccines, and achieve a degree of population immunity. in the meantime, everyone represents a potential victim. as a novel coronavirus, it is worth emphasizing that even if a patient survives covid- , they may still face substantial harm. for instance, emerging research suggests that some covid patients may experience persistent symptoms well beyond the normal recovery period, and that others-even some with relatively mild cases-may nevertheless suffer permanent respiratory, cardiovascular, and neurological damage. voigtländer & voth ( a , b argue that the increased wages due to labor shortages from plague mortality increased demand for urban products, driving a cycle of urbanization and additional disease that moved europe out of a malthusian trap into a modern world of permanently higher per capita incomes. dittmar & meisenzahl ( ) demonstrate that outbreaks of plague aided the spread of reformation laws and the expansion of public goods. for more on the economic history of plagues, see alfani & murphy ( ) . covid- struck a population with neither natural nor acquired immunity: wherever the virus spread, it had the potential to be devastating. with little immediate means of preventing, testing for, or treating it, some of the only short-run mitigation strategies available have been relatively brute-force ones such as lockdowns and border closures. consequently, economic shutdowns-resulting both from official government actions and from individuals taking actions to avoid exposure-have been widespread, leaving no major economies or populations spared. interestingly, because medical technology was limited for much of the past, and societies could only count on some degree of population immunity, even endemic (i.e., non-novel) diseases could have the sort of destructive potential we only typically see today in new disease variants such as the novel coronavirus. for instance, in a variety of past pandemics studied by economic historians, cases of an endemic disease would sporadically rise sharply, with substantial consequences for living standards and economic organization. indeed, a large literature considers the impact of such diseases on the growth trajectories of countries over the long run, often focusing on tropical diseases like malaria, yellow fever, dengue, and others. one strand of studies considers the direct impact of disease on human capital formation (see, for example, bleakley ( bleakley ( , on malaria). another strand focuses on the impact of these endemic diseases on institutional development, finding that disease environments inhospitable to colonial settlers drove them to rely on extractive institutions that were ultimately harmful to economic growth (acemoglu et al., ) . finally, scholars have considered the way that one society's acquired immunity to an endemic disease can devastate the economy of another society lacking that immunity (diamond, ; mcguire & coelho, ; tang, ) . together, this historical evidence gives us a picture of what our circumstances might look like today if we are unable to adequately ramp up our capacity for disease prevention and treatment, and are instead forced to rely on acquired immunity, the nature of which for covid- is still poorly understood. age has been at the forefront of discussions about the disparate impact of the current crisis, and shutdown efforts have been framed in part around protecting older individuals and other vulnerable populations, such as the immunocompromised, while societies work to expand medical capacity and develop a vaccine. in this respect, covid- is much like many infectious disease outbreaks in the past-though young people can both transmit the disease and become ill, it is the elderly and those with poor baseline health are at greatest risk. cdc estimates put the risk of hospitalization five times higher, and the risk of death from covid- times higher, for to year-olds compared to individuals in their twenties. likewise, despite claims in some quarters that covid- is -the great equalizer,‖ it is already becoming clear that socioeconomic status will be central to understanding the demographics of this crisis. one of the ways low-income populations will be affected is through differential exposure to pandemic risks. individuals who continue to do their jobs in person during the pandemic-including service-industry workers with extensive contact with customers, healthcare professionals, and other frontline workers-will that is, it is improved medical knowledge that has allowed us to escape from the sort of flare-ups of endemic disease so frequently observed in the past, and it is relative our lack of knowledge about covid- , as a new virus, that makes our current situation in some ways comparable to even past endemic disease outbreaks. economic historian joel mokyr expands on this idea regarding the evolution of knowledge regarding infectious disease outbreaks in a recent op-ed: https://www.cnn.com/ / / /opinions/struggle-between-people-and-microscopic-pathogens-mokyr/index.html. retrieved from https://www.cdc.gov/coronavirus/ -ncov/covid-data/investigations-discovery/hospitalizationdeath-by-age.html, august , . the mirror image of differential exposure is differential transmission risk. this is one reason why some have called for prioritizing the strategic testing of workers at highest risk of spreading the novel coronavirus to others, particularly asymptomatic ones. bear a disproportionate burden of the pandemic's health impacts. these workers are more likely to be in low-paying jobs, and are more likely to be women and minorities, than their counterparts with jobs allowing them to work from home. consider, for instance, meat and poultry workers in the u.s. the mean annual wage in the industry is only $ , (bls, ) . among laborers in the food manufacturing industry, percent are black and percent are hispanic (eeoc, ). three-quarters of full-time, year-round healthcare workers are female, with that share even higher among the lower paid nursing and health aide occupations, critical occupations with severe risk of exposure to this disproportionate exposure to virus for lower income groups, women, and minorities is exacerbated by differences in these groups' access to healthcare and the quality of that health care-factors that affect both vulnerability and resilience to pandemic disease. membership in more than one of these groups will tend to compound disadvantage even further. preliminary research suggests that black patients exhibiting covid- symptoms were six times less likely to get treatment or testing than white patients. this is not unique to covid- : similar patterns have been observed for other modern pandemics including the h n influenza outbreaks (quinn et al., ) . the outsized impact of pandemics on minority populations and people of lower socioeconomic status has historical precedent. the influenza pandemic hit the poor first and hardest (sydenstricker, ; mamelund, ) , a point we will return to in section iii. explanations for this relationship mirror modern ones: poorer populations lived in denser housing units under worse conditions, and had occupations that increased exposure to the virus. moreover, low incomes constrained their ability to avoid exposure and seek treatment. historical evidence shows that to escape a th century outbreak of yellow fever, wealthier residents often left the city-an option unavailable to low-income workers with tenuous job security. this is a pattern that we see as well during outbreaks of plague in earlier centuries, and is part of dittmar & meisenzahl's ( ) explanation for why the black death paved the way for institutional reform: the old elites simply left town (dinges, ; isenmann, ) . this sort of regional flight is unlikely to be a central dimension along which covid- has differential impacts across income levels-but it does raise important issues that set the current crisis somewhat apart https://www.bls.gov/oes/ /may/oes .htm https://www.eeoc.gov/statistics/employment/jobpatterns/eeo / /national-naics /table?naics= &state=&cbsa= figures are based on the u.s. census bureau's calculations using american community survey data (https://www.census.gov/library/stories/ / /your-health-care-in-womens-hands.html). https://www.nytimes.com/ / / /us/coronavirus-african-americans-bias.html the relationship between pandemic exposure and socioeconomic status has not always been constant. as alfani & murphy ( ) note, studies of the plague in europe find the black death to have been universally deadly. however, plagues of the fifteenth and sixteenth centuries exhibited the negative relationship between socioeconomic status and mortality found in more recent pandemics (see, for example, slack ( ) , alfani ( ) , and carmichael ( )). however, the final major plagues of the seventeenth century once again tended to have severe consequences across all classes. mamelund ( ) note that in norway, the impacts of the pandemic were most severe for transport, hotel and industry workers, paralleling the observations above about covid- . these factors also translated into worse outcomes for minorities. as the exception that proves the rule, black americans fared better than white americans during the pandemic (crosby, ; Økland & mamelund, ) . crosby ( ) argues that the black population had disproportionately high exposure to the early, less virulent summer wave of the pandemic due to their worse occupations and living conditions, conferring some degree of immunity to the more deadly later wave. a similar mechanism operated during historical yellow fever outbreaks, albeit resulting in advantages for native-born individuals over immigrants. individuals born in an area with endemic yellow fever and exposed at a young age often contracted a mild form of the disease, and then developed immunity. as a consequence, adult immigrants were far more likely to die in yellow fever outbreaks in the united states than either native-born whites or blacks (patterson, ) . saavedra ( ) exploits this pattern to demonstrate that earlylife yellow fever exposure negatively impacted adult occupational outcomes, with white males born to immigrant mothers during yellow fever pandemics less likely to become professionals than the sons of native-born mothers. from other historical pandemics: the spatial distribution of population within and across cities, the degree of interconnection between rural and urban areas, and the extent of urban health penalties. while cities are much healthier today than in the past, societies today are also much denser, more urbanized, and better connected-all factors that would tend to make modern pandemics both faster to spread and harder to control than in centuries prior. and indeed, while covid- has hit dense metropolitan areas particularly hard, as in the past, under the current crisis, rural communities have not been spared. this is in part because of the relative ease with which people circulate between communities with our modern transportation networks, but also because of the way that the nature of modern work tends to place individuals in close contact with each other, even in less densely populated areas. to wit, major rural clusters of covid- in the united states have been tied to large meat and poultry processing facilities, with workers at these facilities experiencing case rates an order of magnitude higher than the general u.s. population (dyal et al., ) . moreover, rural areas' demographic composition (often older and less affluent) and healthcare infrastructure (often sparser) can also contribute to their difficulties with pandemic disease. to respond effectively to pandemics in the moment, and to deal with their long-run fallout, will require an understanding of its distributional effects over time and space. we explore these central consideration in depth in section iii. evaluating the policy response to covid- and how it compares to historical pandemics requires recognizing that information on the disease and how to stop its spread has been limited to date, and is still evolving. this issue stems in part from covid- being a novel disease-developing treatments and vaccines takes time, and public health recommendations can change as knowledge advances. it also stems from incomplete and inaccurate data: limitations on covid- testing has often meant relying on mortality rates rather than case rates. incidentally, this is the same approach economic historians are often required to take. morbidity data are rare historically and, when available, may be unrepresentative and inaccurate. mortality data are both far more prevalent and reliable, even if it is morbidity that is typically more relevant to the economic impacts of a pandemic, particularly less lethal ones. for health officials today, the need to assess the spread of covid- through mortality data leads to the frustration of identifying the arrival of cases with a substantial lag. for the economic historian, this lag is irrelevant, but the issue remains that only those places experiencing excess mortality can be identified; diseases leading to widespread morbidity but little mortality may be equally important for the evolution of economies, but far more difficult to identify prior to modern medical records. again, this suggests that evidence from crises that have run their course can be informative of what to expect going forward. while some of the challenges in developing effective covid-control responses have stemmed from incomplete and rapidly evolving knowledge of the disease, they have also stemmed from issues of state capacity, political will, and ideology. for instance, policymakers, firms, and individuals have been hamstrung by not only limited testing and contact tracing capacity, but also by a failure at times to deploy these tools efficiently. constraints such as these are a product of both the limitations of medical technology, and broader issues of political leadership and coordination. the inability to identify and isolate individuals at risk of spreading the disease, in turn, has necessitated rather blunt policy tools, such as business closures and stay-at-home orders. in the u.s. in particular, these covid-control efforts have been aggressively decentralized, and have tended to prioritize both commerce and individual liberty-even where these might be at odds with each other, or lead to ineffective disease control. to wit, business owners and public officials have struggled to gain widespread compliance with (and have often declined to enforce) precisely the sorts of behaviors-e.g., mask-wearing, social distancing-that would allow for the safe reopening of businesses. indeed, it appears that the fear and uncertainty created by the failure to control the spread of disease, in turn, has contributed to prolonging economic pain (goolsbee & syverson, ) . challenges such as these, related to culture and institutions, are nothing new-in fact, they characterize the u.s.'s historical experience of managing epidemic disease. in his excellent the pox of liberty, troesken ( ) lays out how the very institutional features-among them a decentralized federal system, a focus on property rights and commerce, and protection of individual liberties-that led to the u.s.'s rapid economic development also often undermined its attempts to control past outbreaks of smallpox, typhoid, and yellow fever. the examples he provides have uncanny parallels to the u.s.'s approach so far to managing covid- . strategies to manage the spread of covid- have been varied, with many jurisdictions pursuing multiple complementary approaches, often including coordinated sourcing and distribution of protective equipment, reallocation of medical capacity, virus and antibody testing, contact tracing, frequent sanitizing of public facilities, social distancing, mask-wearing, managing congestion in public places by staggering timings and moving activities outdoors, limiting large gatherings, quarantining infected individuals, and minimizing the risk of disease exposure via closures of businesses and schools and broader stay-at-home orders. of these strategies, shutdowns and quarantines have been some of the most accessible, widely used, and hotly debated under covid- . a shutdown-centered approach such as this also has strong historical precedent. in fact, closures and quarantines were some of the only tools available to societies prior to the virology advances of the th and th centuries. though the shutdown of firms has been more comprehensive under covid- than in many past pandemics, the primary measures being taken now, such as quarantining sick individuals, restricting public gatherings, and closing schools, were all implemented during the pandemic (markel et al., ; hatchett et al., ) , albeit with a smaller scope and shorter duration. likewise, when england was combatting the other countries offer a contrast on one or more of these dimensions. for example, though some of these early gains have since dissipated, taking a more authoritarian and centralized approach, china and india had some initial success in containing the virus following swift and complete lockdowns, underscoring the potential importance of state capacity and centralization in pandemic control. likewise, south korea's response demonstrated the importance of relative novelty: due in part to experience with past respiratory pandemics, pre-existing public health infrastructure and greater public buy-in with mask-wearing allowed them to respond more quickly and effectively to the greater use of firm closures as a disease-control strategy today could be due, for instance, to improvements over time both in the safety net and in remote-work capabilities. this more widespread pause on non-essential activities may in turn lead to fairly different effects of covid- relative to past pandemics, e.g., in terms of patterns of disease transmission or total economic impact. while cities employed a diverse set of non-pharmaceutical interventions (npis) during the pandemic, only a small fraction of these interventions (namely, closures of public facilities, isolation policies, bans on public gatherings, and making influenza a notifiable disease) were widely practiced across localities (hatchett et al., ) . moreover, markel et al. ( ) document that npis were in place for only months or less in out of the cities in their sample; even the maximum duration in their sample ( days, in kansas city, missouri) still falls far short of the u.s.'s experience of covid- npis to date. indeed, barro ( ) suggests that the short average duration of npis during the pandemic led them to be relatively ineffective in curtailing mortality. the efficacy of npis depends on factors including the type of intervention, when it is first implemented, how long it is in place, and the strictness with which it is implemented. because of this, and although npis under covid- have been in place longer than during the pandemic, it is difficult to say at this stage whether this necessarily means that they will have been more effective in reducing morbidity and mortality-particularly because covid- npis in the u.s. have tended to be implemented somewhat late in the pandemic's course, have tended to be intermittent and noncomprehensive, and have tended to be leniently enforced, all factors which undermine efficacy and which may themselves be contributing to the need for longer npi duration. plague in the s, they quarantined ships from other countries, closed ale houses, and limited the number of lodgers allowed in a house, actions that would sound familiar to cruise ship passengers and restaurant owners during the covid- pandemic (bell, ) . indeed, it is striking-maybe even alarming-how little has changed about our best options for fighting pandemics, despite centuries of advances in medicine, public health, and living standards. this policy response, necessitated by factors including inadequate testing and broader uncertainty about key epidemiological parameters-even those as basic as precisely how and through whom the disease can be transmitted, and whether it is possible to become re-infected-makes the economic history of policy responses to pandemics particularly relevant for studying the current crisis. even when we contemplate a world where successful covid- vaccines are available, history sounds a note of caution: the same underlying issues that have made mask-wearing both incomplete and fraught in settings, like the u.s., with a strong institutional commitment to liberty and rugged individualism, could also be expected in the context of covid- vaccination. as troesken ( ) notes, anti-vaccinationism has a long history in the u.s., bolstered by the common failure to appreciate the extent of infectiousdisease externalities (the choice not to vaccinate can be individually rational, even if people understood externalities, which they largely do not), as well as by a belief in both minority rights (individuals cannot be forced to vaccinate) and federalism (individuals preferring not to vaccinate can sort into amenable jurisdictions). while troesken documents that mandatory vaccination was frequently enforced in the past via fines, or by denying access to schools or other public services, it is difficult based on the nature of the u.s. covid- response to date to imagine such enforcement mechanisms being implemented. instead, in heterogeneous, strongly pro-individual, pro-freedom societies, we may need to rely on a stylized fact that troesken demonstrates using data from th century smallpox epidemics in germany: vaccinations rates rise in pandemic years, because during pandemics, the risk of infection rises sharply, and the private costs of non-vaccination are clearly outweighed by the private benefits. for thinking about the direct effects of pandemics on the health and wellbeing of individuals in the shortand long-run, the influenza pandemic, or the -spanish flu,‖ provides a useful point of reference. covid- to date parallels the pandemic in several key ways, including its rate of transmission, global spread, and crude mortality rates. the spanish flu was one of the most acute and widespread natural disasters in modern history. taubenberger & morens ( ) estimate that during the pandemic, roughly million individuals, equivalent to roughly a third of the world's population at the time, were infected and symptomatic. case fatality rates, at over . percent, were at least times as high as in other influenza pandemics, making the virus especially lethal. all told, somewhere between and million individuals perished globally. the death toll in the u.s. alone exceeded that from all american combat deaths over the twentieth century (almond, ) . see for example markel et al. ( ) on the effectiveness of school closures in combatting the spread of the influenza pandemic, meyers and thomasson ( ) on the effects of polio-related school closures on educational attainment, and alfani & murphy ( ) on city-level quarantines during pre-modern plagues. cultural features like distrust of scientific expertise, and of institutions more generally, may also undermine vaccine compliance. troesken ( ) notes that the underlying legal theory behind vaccination non-compliance fines gained further credence after the supreme court upheld the affordable care act's insurance mandate in national federation of independent business v. sebelius, u.s. ( ). there are challenges to drawing lessons from the pandemic. much remains unknown about the origins and epidemiology of the virus and its economic impacts are confounded by the effect of world war i. the pandemic itself was sharp, sudden, and concentrated over the span of little more than a -month period. the virus, an h n strain similar to that which caused the swine flu outbreak, spread roughly simultaneously across europe, asia, and north america, in three distinct waves over the year beginning in spring . the first of these waves, appearing in march , was relatively mild. it was followed by a substantially more catastrophic one from september to november , and another in the early months of (taubenberger & morens, ) . in some parts of the world, particularly in east asia, a further major wave of pandemic influenza hit as late as (lin & liu, ; ogasawara, ) . this sort of timing and spacing was unprecedented among influenza pandemics, as was its distinctive mortality profile. where influenza death rates by age typically follow a u-shape, with high mortality rates among the very young and the very old (as is also the case with the sars-cov- , the virus behind covid- ), the strain followed a w-shape, with a sharp peak in mortality risk among young adults as well. indeed, almost half of all influenza-related deaths during the pandemic period accrued to those aged - (taubenberger & morens, ) . the age pattern associated with this strain of influenza was in fact so unusual that it has been exploited as a diagnostic tool in recent studies. for instance, while the influenza pandemic is typically thought to have emerged in full force in europe around the summer of , and in a milder form somewhere in the central u.s. in spring , detailed age-by-month mortality statistics allow olson et al. ( ) to uncover evidence that an early -herald‖ wave of pandemic influenza was actually present in new york city well beforehand, from february to april of . during this period, the age profile of excess influenza mortality had started to shift from the older ages typical of interpandemic seasons to the younger ages that characterize pandemic seasons. this underscores the value of accurate and disaggregated data in tracing the origins and spatiotemporal spread of pandemics, and the need to strengthen not only rapid-response public health infrastructure, but also that to support ongoing disease surveillance. turning to morbidity, those under the age of , and particularly, those aged - , had disproportionately high incidence of influenza-however, the latter group had a much lower death rate from influenza and pneumonia than other ages, further sharpening the middle peak in the morbidity-adjusted pandemic mortality curve (taubenberger & morens, ) . age, however, was not the only major factor that contributed to pandemic mortality risk, and a range of recent studies have emerged cataloging the often interrelated features of countries, cities, and individuals that led to disparities in the immediate mortality burden of the flu. on these mechanisms, the evidence is mixed-surely in part because of diverse empirical settings and disciplinary approaches-but certain patterns do emerge. first, baseline health status mattered: both pre-pandemic pneumonia, a bacterial condition with a strong biological interaction with the influenza virus, and infant mortality rates, a proxy for population health, contributed to higher pandemic flu mortality (acuna-soto et al., ; clay et al., ) . likewise, high levels of air pollution, an environmental factor that aggravates respiratory conditions and depresses baseline health, also raised pandemic mortality. for instance, clay et al. ( ) examine evidence from a panel of u.s. cities, and find that the air pollution generated by coal-fired electricity plants was a significant contributor to pandemic mortality, with effect sizes roughly half those associated with measures of population health and poverty. together, they estimate that these factors accounted for approximately half of all cross-city variation in pandemic mortality. in another study, they find that both infant and all-age mortality were impacted adversely by the presence of coal-burning plants, with poor air quality responsible for - percent of total pandemic mortality in high-and medium-pollution cities, a figure equivalent to some , - , excess deaths beyond those attributable to the pandemic alone (clay et al., ) . , second, population density and related concerns, such as housing quality and the number and composition of social interactions, were also important factors in pandemic mortality. in europe as in the u.s., the pandemic came to cities earlier, and was more devastating there, a phenomenon linked to urbanization and residential crowding (chowell et al., ; mamelund, ; murray et al., ) . transmission was localized, and influenza and pneumonia mortality exhibited significant and rather tight (e.g., - , m) spatiotemporal clustering (grantz et al., a,b; tuckel et al. ) , though proximity to high-risk population centers like wwi military bases appears to have had little effect (clay et al., ) . although urban centers were associated with higher pandemic mortality, the opposite population gradient prevailed when comparing among cities, or among rural areas: in both cases, smaller, less dense localities fared worse (acuna-soto et al., ; chowell et al., ) , suggestive perhaps of capacity constraints in the healthcare workforce and medical infrastructure. third, factors-such as illiteracy and foreign-born status-that might have prevented individuals from adopting public health recommendations were strong predictors of elevated mortality, often above and beyond their association with poverty. higher rates of illiteracy were linked to higher rates of influenza mortality during the pandemic, across both cities and neighborhoods (clay et al., ; grantz et al., a, b) . likewise, foreign-born status not only predicted higher pandemic mortality in hartford, connecticut, but the relationship between nativity and mortality persisted even after controlling for socioeconomic status, population density, and neighborhood ethnic composition, indicating perhaps a role for social factors, or language or cultural barriers to the adoption of relevant public health measures (tuckel et al., ) . crucially, the consequences of these barriers were not limited to the foreign-born: holding all else equal, native-born individuals living in areas with a higher share of foreign-born had higher mortality rates than their counterparts living alongside a lower share of foreign-born neighbors. this emphasizes the importance of neighborhood spillovers in infectious disease transmission-and, of course, demonstrates the interrelated nature of individual-and neighborhood-level mechanisms. it is possible that indoor pollution and seasonality also played a role in air quality-influenza interactions, both during and outside pandemic times. for instance, influenza is generally prevalent in the winter, a time when coal smoke from home heating also tended to peak in this era (barreca et al., ) . clay et al.'s ( ) observation that modern levels of pollution in parts of the developing world, including india and china, are on par with those in the early th century u.s., sounds an ominous note in light of the current crisis-though the circumstances today (e.g., improved medical technology, the higher baseline share of trafficrelated emissions, a fall in pollution due to widespread economic shutdowns) may be just different enough to ameliorate concerns over the lethal interaction between pollution and pandemic influenza. while pollution can lower baseline health by undermining the respiratory system, it is worth noting that pollution may also be associated with higher baseline health, insofar as it proxies economic activity. for instance, clay et al. ( ) find evidence of crucial tradeoffs between the income generated through industrial activity on the one hand, and the pollution generated on the other in the u.s. from the s to the s. in less developed localities, infant mortality followed a u-shaped pattern with respect to the expansion of coal capacity: first falling as rising incomes and cleaner residential energy sources buoyed infant health, and then rising as subsistence health needs were met and the concentration of pollution grew. the net health effects of a pandemic that dampens economic activity (and so reduces pollution), then, is therefore likely to be context-specific, depending on factors such as the level of baseline health and income, the extent of medical infrastructure, and the strength of social safety nets. troesken ( ) also points to individualism and liberty as cultural/institutional values that tend to lower individual-level compliance with public health recommendations. when considering these biological, demographic, and socioeconomic factors in quick succession, it is difficult not to see the overarching hand of income in all of these mechanisms-though, to be clear, several of these studies are careful to disentangle these factors from their association with income. in theory, income gradients in pandemic mortality could arise through a number of channels, including many of those hinted at above: e.g., the tendency of those with higher incomes to have better baseline health status, rendering them biologically less vulnerable and more resilient to infection; higher-quality and lower-density housing, reducing the chances of viral transmission; better public health knowledge, the human capital necessary for individuals to effectively assimilate this knowledge and to adopt life-saving recommendations, and timelier and more robust public health interventions, all slowing the spread of illness; better access to healthcare and medical infrastructure, improving the probability of survival conditional on infection; and a greater capacity for individuals to undertake avoidant, adaptive, and compensatory behaviors, both throughout and following the pandemic. crucially, these channels can operate at both individual and institutional (e.g., city or country) levels, with both richer people and localities-and certainly, the interaction of these-theoretically better equipped to weather the crisis. the fact that some of these channels are highly correlated, of course, can make it difficult to pinpoint the underlying mechanisms: higher-socioeconomic status (ses) individuals are likelier to be both healthier, protecting them from infection, and more educated, rendering them better able to adopt public health measures; cities tend to be richer in both income and infrastructure, but they are also more heterogeneous and densely populated than rural areas. nevertheless, the literature can still shed light on the role of income on net. while some studies explicitly looking at its role in pandemic severity have shown little relationship between pre- economic development and pandemic mortality (brainerd & siegler, ) , a great many indicate that poverty exacerbated mortality risk. for instance, murray et al. ( ) document tremendous (i.e., over thirty-fold) within-and cross-country variation in excess mortality due to the pandemic, with nearly half of this variation explained by baseline per capita income. taking a finer-grained look at these issues, grantz et al. ( a,b) explore the socioeconomic determinants of pandemic mortality and transmissibility using detailed data from chicago. among the associations they find between health and various poverty proxies are large, statistically significant, and negative associations between census tract-level homeownership rates and mortality. these findings are consistent with the lower baseline health of lower-ses neighborhoods, their poorer access to medical care, and their lower awareness and adoption of public health recommendations. shanks & brundage ( ) add that these factors may be proxying other features of low-ses populations, such as a higher risk of sequential infections (e.g., pandemic influenza followed by a secondary bacterial infection such as pneumonia), or the larger number and lower-ses composition of their social interactions. all of these could have contributed to higher cumulative pandemic mortality through faster and more widespread disease transmission, higher incidence of infection, or higher case fatality rates. these results suggest that rather than acting as a democratizing force, the pandemic further entrenched preexisting socioeconomic disparities. the clear implication of studies documenting the immediate health effects of the outbreak is that the damage from pandemics has, and remains likely to, fall disproportionately on disadvantaged communities. apart from its effects on health, however, the pandemic also had important consequences for population dynamics. one such effect pertains to temporal and cross-disease mortality spillovers resulting from pandemic-era mortality patterns. noymer ( ) shows that the influenza pandemic hastened the decline of tuberculosis in the u.s. through a harvesting mechanism. specifically, he suggests that independent competing risks may be responsible for this phenomenon, driven by substantial age overlap in the profile of prospective tuberculosis and (pandemic-type) influenza victims. this -passive selection‖ contrasts with -active selection‖ based on biological interactions between influenza and tuberculosis. this harvesting, in turn, had long-lived implications for sex differences in post-pandemic mortality rates: because tuberculosis morbidity disproportionately affects men, and because the influenza pandemic reduced the pool of those who might die of tuberculosis in the years following, the pandemic had the effect of eroding women's longevity advantage over men. we might expect similar outcomes in the context of covid- given that a large share of those dying have one or more co-morbidities, though the distinct age profile of pandemic deaths versus covid- deaths may complicate these dynamics. studying brazil, guimbeau et al. ( ) likewise find rather larger reductions in sex ratios at birth following the influenza pandemic, consistent with the greater vulnerability of male fetuses to adverse in utero shocks-a phenomenon often seen in the literature on famines and environmental disasters. such changes in the sex ratio, or in sex-specific survival, may well have had long-run implications for marriage and labor markets. another major area in which the pandemic affected demographic behavior relates to marriage and fertility. in some cases, this was largely a function of pandemic psychology. mamelund ( ) shows that a climate of fear and uncertainty in norway, alongside social distancing efforts and peculiarities of norwegian marriage laws (which imposed a one-year waiting period before widows could remarry), led to a drop in births in , as families deferred childbearing. higher rates of maternal mortality and miscarriage during the pandemic likely also contributed to a drop in birth rates. this pent-up demand for children (alongside -replacement‖ demand for children lost to the pandemic) was released after the crisis passed, resulting in a baby boom in . elsewhere, as was the case in nearby sweden, changes in fertility arose from the way that pandemic mortality affected markets for marriage and labor: boberg-fazlić et al. ( ) find evidence of a drop in fertility during the pandemic, followed by a short-lived rebound in post-pandemic fertility. the net effect in the long term, however, was to reduce fertility-due in part to persistent disruptions to marriage markets (particularly in rural areas and poorer cities); the adverse effects on income; as well as to behavioral changes induced by the pandemic, including a rise in female labor supply (and so, an increase in the opportunity cost of childrearing) in regions with high male pandemic mortality rates. perhaps most noteworthy, the short-run post-pandemic fertility increase was selective in nature: a child born during this boom was more likely born to mothers who were married or who were high-ses city-dwellers. this was largely driven by postponement fertility, and particularly, selective postponement. finally, pandemic-related mortality affected childbearing through its effect on survivors' incomes. donaldson and keniston ( ) show that the high pandemic death toll in some regions of india implied a substantial increase in per capita incomes, as survivors assumed the agricultural land of pandemic victims. in light of this rise in incomes, they find an increase in both the quantity and quality (given by literacy and height) of children born following the pandemic in india. phenomena such as these, which change the sex-and age-composition of the population-not to mention the average health status of successive cohorts-are likely to have long-lived effects on economic development, population health, and individual wellbeing. the lethality and peculiar age profile of the pandemic also give rise to long-run considerations. these may be especially relevant in light of covid- , where the vast majority of people who become sick ultimately survive. during the pandemic, young adults-including prime childbearing-age womenwere some of the likeliest to fall ill: in some parts of the u.s., roughly a third of all mothers (relative the about percent of the general population) became infected during the crisis (almond, ) . moreover, across settings, evidence of replacement fertility is rather more limited. note however that covid- appears to be less prevalent, and possibly less severe, among prime-aged people. consequently, it is possible that scarring through the health channel under covid- may end up being less severe, and/or less widespread, than that following the influenza pandemic. scarring through the income/labor-market channel, however (explored in more detail in section iv), could well be substantially worse following covid- than the pandemic, given the latter's relatively mild and short-lived effects on the economy. despite the very high mortality rates from this pandemic, most of those infected ultimately survived. this left considerable scope for maternal morbidity-and, through the impact of maternal stress and illness on intrauterine hormones, nutritional resources, and other factors-for insults to fetal health. in what is perhaps the seminal study in economics of the influenza pandemic's long-run effects on wellbeing, almond ( ) finds wide-ranging adverse effects on later-life human capital and labor market outcomes among u.s. cohorts exposed to the pandemic in utero. these include substantial reductions in high school completion rates, wages, and socioeconomic status, alongside large increases in the probability of living in poverty, the receipt of welfare payments, the likelihood of incarceration, andparticularly among men-the probability of physical disability. that these adverse outcomes exist in spite of a pandemic-induced increase in miscarriages, stillbirths, and infant mortality rates (see, e.g., guimbeau et al., ; mamelund, )-all culling forces which likely resulted in a pool of survivors if anything positively selected on health-is a testament to the catastrophic extent of post- scarring. almond's initial study has also since spawned a large and varied literature interrogating the long-run effects of the pandemic across a range of global settings. a first set of studies dig deeper into the u.s. case. one such study shows that birth cohorts (and in particular, those born in quarter of , who were in utero at the height of the pandemic), are percentage points (or percent) more likely to report fair or poor health than their counterparts born in surrounding years; see a statistically significant - percent increase in a range of functional limitations, including trouble hearing, speaking, lifting, and walking; and are also likelier to experience diabetes and stroke (almond & mazumdar; . others debate the possibility of pandemic-induced selection into fertility, which could confound estimates of the long-run health effects of early-life pandemic exposure. these studies ultimately conclude that the positive selection of wwi recruits, and the corresponding negative selection of pandemic-era fathers, does not substantially alter the conclusion that fetal exposure to the pandemic was a major and direct cause of these cohorts' later-life disadvantage (brown & thomas, ; beach et al., ) . a newer set of papers, focusing on non-western, and particularly, lower-income, settings, shows that the evidence on the pandemic's long-run penalties is robust across a range of empirical contexts, each with different levels of baseline income and health status, different institutional responses to the pandemic, and different degrees of involvement in wwi. for instance, as in the west, in taiwan there is evidence of permanent scarring: cohorts exposed to the pandemic in utero faced penalties with respect to educational attainment, heights, kidney disease, circulatory and respiratory issues, and diabetes (lin & liu, ) . in low-income settings with minimal public health intervention, even higher incomes only did so much to buffer these shocks: in a sample of high-ses children in japan, ogasawara ( ) finds that in utero exposure to the influenza pandemic reduced boys' and girls' heights by . cm and . cm, respectively-magnitudes which in other studies have been associated with substantial increases in the probability of type ii diabetes, osteoarthritis, and heart disease. the long-run results seen in japan, as in guimbeau et al. ( ) in brazil, are consistent with sex differences in resilience to adverse health shocks. now quite common and influential in economic research, the conceptual framework linking early-life conditions to later-life health and wellbeing is termed the -barker‖ or -fetal origins‖ hypothesis. this hypothesis holds that certain chronic conditions stem from deficits in the fetal environment (barker, ) . based on this initial literature in epidemiology and medicine, which focused on evidence from historical famines, a growing literature in economics has used these ideas to model the technology of human capital formation, and to identify sensitive and critical periods for the development of a range of outcomes contributing to labor market success and general wellbeing, including cognitive and non-cognitive skills, metabolism, and longevity (heckman, ; almond & currie, ) . meanwhile, swedish pandemic survivors saw reductions in life expectancy (helgertz & bengtsson, ) . the reduction in the health, human capital, and labor market prospects of cohorts exposed in utero also appears to have dampened their marriage market prospects in ways that continue to carry intergenerational consequences. while both men's and women's own educational attainment was lower among exposed cohorts, only exposed women appear to suffer a marriage market penalty: they marry earlier, to spouses with lower levels of education (fletcher, ) . these are factors generally understood to reduce household incomes, female control of household resources, and the budget share allocated to child-centric expenditure. as such, these effects could represent a mechanism-alongside, e.g., epigenetics, or the more direct role of parental education in facilitating children's access to quality healthcare and schooling-by which we see intergenerational persistence in the consequences of early-life exposure to the influenza pandemic of . indeed, moderate adverse effects on educational attainment, occupational prestige, and family socioeconomic status have been documented up to the third generation, i.e., the grandchildren of those exposed in utero (cook et al. ) . what action, if any, did households take to shield their children from these effects, or to help them recover? while surprisingly little has been written in the context of the pandemic on questions of individual-and household-level avoidance, adaptation, and remediation, parman ( ) is a noteworthy exception. drawing on linked microdata from the u.s., he finds evidence of reinforcing investments in response to the influenza pandemic: that is, families with a child in utero during the crisis shifted resources to the child's older siblings, leading the latter children to higher educational attainment. parman explicitly rules out changes in family size, birth spacing, or selectivity in any such changes, underscoring that the effects observed here are directly a function of parents reallocating limited resources away from affected children, and toward the child with a higher human capital endowment at birth. thus, household responses may have if anything compounded any early-life disadvantage associated with the shock. historical pandemics can help us think about potential long-run effects on wellbeing arising directly through the current pandemic's patterns of morbidity and mortality. but what about the impacts resulting from its disruption of daily economic life? one of the central features of the current coronavirus pandemic is the sudden, extreme, and widespread economic disruption it has caused. on this count, it has perhaps less in common with other recent pandemics. indeed, the immediate economic disruption caused by the pandemic pales in comparison to that caused by so, while this historical pandemic can some work has, however, addressed the broader policy responses (and lack thereof) to the pandemic in the u.s. for instance, hatchett et al. ( ) find that cities that simultaneously implemented multiple non-pharmaceutical interventions (consisting of, e.g., isolation of sick individuals, bans on public gatherings, mandatory notification of disease, and closure of public gathering places, staggered business hours, and no-crowding rules) early in the pandemic had peak mortality rates roughly half that of cities that did not implement such interventions, and substantially lesssteep epidemic curves. no single intervention was responsible for these gains; rather, it was the combination of multiple mutually reinforcing interventions that were effective. these findings are in line with markel et al. ( ) , who emphasize the importance of early and sustained non-pharmaceutical interventions during the pandemic. while many cities were successful in taking such a multi-pronged approach to pandemic management, on the whole the u.s. policy response to the pandemic was rather weak, undermined by a preoccupation with world war i-related efforts. until the covid- crisis, there had been relatively little work on the effects of the pandemic on economic activity, largely for lack of high-frequency, spatially disaggregated data on local economic conditions (see beach et al. (forthcoming) for an excellent overview of both the state of this literature and related empirical challenges). indeed, the precise magnitude and temporal reach of these economic effects are still being debated (see, e.g., basco et al. ( ) , barro et al. ( ) , correia et al. ( ) , lilley et al. ( ) , and velde ( )), and a challenge for many of these studies in identifying pandemic effects on the economy remains the confounding effect of world war give us insight into long-run effects on wellbeing through the health channel (-direct‖ effects), we must look elsewhere to think about the long-run consequences of pandemics through corresponding economic downturns (-indirect‖ effects). but where to look for a suitable comparison? in some ways, episodes such as the black death or the aids crisis in sub-saharan africa would seem to present closer analogues than the influenza pandemic, as health events with massive and lasting economic ramifications. the catastrophic loss of life under these pandemics fundamentally reshaped entire societies and economies, with, for instance, the resulting labor scarcity driving up the real wages of survivors, and, in some cases, precipitating other major demographic, economic, social, cultural, and institutional changes (young, ; alfani & murphy, ) . indeed, some point to the former plague as a major contributor to sustained rises in western european living standards even under a malthusian regime (voigtländer & voth, , a , and to the region's rapid economic development and eventual divergence from the rest of the world over the early modern period (clark, ) . notes: the insured unemployment rate is based on employees covered under unemployment insurance as reported to states by employers. covid- cases are relative to the entire state population. unemployment data were retrieved from https://oui.doleta.gov/unemploy/claims.asp. covid- data were retrieved from https://github.com/nytimes/covid- -data. the code and data needed to generate the figure are available at open-icpsr (https://doi.org/ . /e v ). in each of these pandemics, mass mortality led to rapid and dramatic changes in population density and age i. moreover, it is worth noting that the -focused studies that have emerged in the wake of covid- tend to conflate the economic effects of the pandemic that arise from within and outside the -direct health-shock‖ channel. to disentangle these channels and use a shock of comparable magnitude, we focus primarily on the great depression when examining the long-run human effects of economic dislocation. see alfani & murphy ( ) for an excellent and in-depth review of the literature on pre-industrial plagues, their long-run socioeconomic consequences, and parallels to modern pandemic control efforts. structure, which in turn affected factor prices and labor markets. thankfully, mortality rates under covid- are not on such a scale as to produce the sort of fallout seen with these events. instead, it appears it may be a combination of factors other than the virus's actual toll on morbidity and mortality that is the source of economic dislocation in this instance. indeed, as figure shows, the severity of the immediate health effects has not been a clear predictor of a locality's economic downturn. likewise, emerging evidence complicates the popular conception that pandemic-control measures themselves, such as stay-at-home orders, are primarily responsible for the downturn associated with covid- . for instance, while gupta et al. ( ) suggest that % of the decline in employment in the early months of the pandemic was driven by state and local social distancing policies, kahn et al. ( ) show that the labor market effects of covid- to date have been broader-based than is typically thought. all u.s. states exhibited a collapse in job vacancies in march , and a corresponding rise in unemployment insurance (ui) claims, irrespective of either the intensity of the virus's initial spread or the timing of stay-at-home orders. these phenomena were seen for the most part across both essential and non-essential sectors, directly-and indirectly-affected sectors, and across occupations with and without work-from-home capabilities. they conclude that -the current damage done to the economy is not solely caused by the stay-at-home orders; it is too large and pervasive.‖ exploring the drivers of the collapse in economic activity, goolsbee & syverson ( ) suggest that -individual choices were far more important [than government restrictions,] and seem tied to fears of infection.‖ these voluntary disease-avoidance strategies by individuals are likely connected to the lack of decisive and coordinated policy responses, and to broader uncertainty about this novel disease. it remains to be seen whether other plausible mechanisms may also have a role-e.g., global supply chains that allow covid-related firm slowdowns in one country or sector to propagate to others, or changes in firm production decisions under covid uncertainty. clearly, both the current crisis and our understanding of it are still rapidly evolving. what we do know, however, is that the downturn this pandemic has precipitated is substantially larger than in other modern pandemics, and unlike in some pre-modern plagues, is likely unrelated to either mortality-related changes in demography or to immediate reductions in labor supply or work capacity due to contemporaneous morbidity. as such, crises of primarily economic origin, such as historical recessions-and in particular, the great depression-may make the best analogues: while the coronavirus pandemic is a public health crisis, to be sure, it has manifested above all as a massive economic disruption, both in terms of magnitude and reach. accordingly, we might want to think about its health and human capital consequences through this -livelihoods‖ channel as well. indeed, it is these effects that are likely to be most relevant to our current situation. beginning with short-term effects, we can look to a large literature on business cycles and health. these studies indicate that the net effects of downturns on morbidity and mortality will likely be highly contextdependent. this is because health is multidimensional, there are many countervailing channels through which local economic conditions can affect wellbeing, and because the particulars of the empirical even while modern globalization has made disease transmission faster and harder to control, and even while increased efficiency in healthcare systems and global supply chains have complicated efforts to quickly ramp up treatment and control responses, other modern factors have made the current pandemic less dangerous to health than those that came before it-among them, improved medical technology, which has made it easier to manage secondary infections, and higher incomes, which have made human populations both less vulnerable and more resilient to infectious disease. this is certainly true at least in a distributional sense. while adverse effects will certainly be severe through direct morbidity/mortality channels, these will nevertheless be relatively concentrated. for contrast, adverse spillovers from these direct health effects, and from broader disease-control efforts, will be much more diffuse, even if less acute. consider, for instance, that unlike the health-channel scarring effects of pandemics discussed in section iii, the economy-channel shocks apply to everyone to one extent or another, not just those who survive infection. setting-e.g., the size, nature, and origin of the shock; the baseline level of population health; and the strength of social safety nets-will ultimately govern which of these effects dominate (arthi et al., ; cutler et al., ) . recessions have been shown to improve health, for instance, by freeing up time for health-promoting activities such as exercise, childcare, and breastfeeding (dehejia & lleras-muney, ; miller & urdinola, ; ruhm, ) ; by reducing the income available to sustain unhealthy behaviors such as alcohol, tobacco, and drug abuse (ruhm & black, ; ruhm, ) ; by reallocating high-skilled but displaced healthcare workers toward higher-risk populations (stevens et al., ) ; and by limiting individuals' exposure to environmental and work-related hazards, including traffic accidents, on-the-job injuries, and pollution (muller, ; chay & greenstone, ; miller et al., ). meanwhile, adverse income shocks can compromise access to basic needs such as nutrition, medical care, and housing (griffith et al., ; painter, ) ; and can cause psychological stress that in turn raises rates of self-harm and risky behaviors (eliason & storrie, ; sullivan & von wachter, ) . while in theory, the net effect of local economic shocks on health is ambiguous, in practice, the bulk of the evidence drawn from modern and rich-country settings suggests that on net, total mortality rates fall during recessions (arthi et al., ) . in addition to setting-specific features like higher baseline health and stronger safety nets, the fact that beneficial channels tend to dominate in these settings may be in part because this evidence comes principally from small fluctuations in local economic conditions: using cross-country evidence over two centuries, cutler et al. ( ) show that mild downturns lower mortality, while large ones raise it. the downturn caused by covid- would surely qualify as the latter. the evidence is much more mixed in developing-country and historical settings, where levels of baseline income and health are low, where safety nets are weak, and where cutting-edge medical technology is less accessible (see, e.g., baird et al. ( ) and ferreira & schady ( ) ). in such settings, even small losses in income can be devastating to health (costa, ; heckman, ) , and there is less scope for the sort of offsetting positive spillovers and behavioral changes seen in more modern and affluent settings. consequently, this evidence seems to more often indicate countercyclical mortality. for instance, arthi et al. ( a) show that even in the presence of adaptive migratory responses, the cotton famine, a major s downturn in britain's cotton textile-producing regions, substantially raised mortality in cotton regions, particularly amongst the elderly (who were more sensitive to income shocks), amongst cotton households (who faced unemployment and reduced hours), and amongst those working in non-tradeables the case of pollution in particular underscores how complex the interactions between health and the economy can be-all the more so during a respiratory pandemic that has precipitated an economic crisis. tied as pollution is to economic activity, a downturn that reduces pollution (and so reduces direct health hazards) also reduces income (and so raises indirect health hazards). moreover, it does so unevenly across space and demographic groups. add to this long-standing (i.e., baseline) distributional considerations around who is most exposed to environmental and pandemic hazards (see, e.g., chay & greenstone, ; currie et al., ) ; and who, conditional on exposure, is most sensitive to income shocks, environmental shocks, infectious disease shocks, or even all three simultaneously (see, e.g., hsiang et al. ; almond & currie, ) ; and a key question for assessing covid- 's effects through economy-environment interactions then becomes, from both an aggregate and distributional standpoint, whether and for whom the losses in health and human capital through the income channel are offset by the gains in health through actions taken to reduce the spread of influenza, the reduction of pollution, and the interaction of these factors. see arthi et al. ( ) for a much more detailed review. note as well that under covid- stay-at-home orders and supply-chain disruptions, the effects through many of these mechanisms are likely to be much more extreme, since the reduction in economic activity has been much more acute (in some cases, nearly absolute). this is the case even in rich countries, but especially in poor ones. in the latter, as discussed above, even smaller economic fluctuations can raise net mortality. consequently, we might expect developing countries to face the greatest tension between the desire to limit the direct health costs of covid- on the one hand, and the desire to limit those health costs arising from the corresponding economic contraction on the other. this is especially the case if pandemic-control measures are seen as helping the former objective while harming the latter, though it is worth noting that it is still unclear the extent to which pandemic-control measures are responsible for the contraction caused by (whose livelihoods depended on the success of the local cotton industry). diverse historical evidence such as this can help us think about how the effects of the covid- crisis might out play out differently in other economies, particularly in the long run-something we cannot get from modern data, and especially, from modern u.s. data, alone. likewise, turning to the great depression, a more recent and thus perhaps more comparable setting to today's, stuckler et al. ( ) find at best mixed evidence of a beneficial health effect of the downturn: while there was a small reduction in all-cause mortality during this crisis, only those reductions in heart disease (small) and traffic fatalities (rather larger) could plausibly be linked to contemporaneous local economic shocks; other recession-related causes of death identified in the literature, such as suicide, rose substantially. fishback et al. ( ) similarly find that had new deal relief spending not intervened, the great depression would have created a -demographic disaster,‖ depressing birth rates and elevating death rates relative to prior trends (particularly among infants, those perhaps most vulnerable to short-run income fluctuations). their results emphasize the importance of government responses to economic crises that in turn become health crises (and vice-versa): for instance, they note that while all-cause non-infant mortality rates were largely unaffected by relief spending, such income support nevertheless did help reduce rates of certain salient causes of death such as suicide, one of the few causes of adult mortality identified in stuckler et al. ( ) as seeing a marked increase during the great depression. while current debates around covid- are understandably focused on the immediate impact of pandemic-induced recession conditions, the economic history literature teaches us that we should be equally-perhaps even more-concerned about the long-run scarring effects arising from this economic dislocation. indeed, this channel may be especially relevant in more modern, high-income, and robustsafety net settings where most people survive an adverse shock, only to contend with the long-term and sometimes latent fallout. some of these scarring effects stem from the immediate impact on household incomes. depression-era resource deficits have been shown to affect cohorts that were in utero at the time well into adulthood, lowering their college completion rates and later-life incomes, and raising their rates of later-life poverty and disability-adverse effects that were only more pronounced in poorer areas, and areas that received less relief spending (arthi, ; fishback & thomasson, ) . meanwhile, other long-run penalties arise from disruptions to labor markets and human capital acquisition. a large contemporary literature studies the phenomenon of labor market scarring, or the idea that economic conditions at the time of labor market entry may have lasting effects on training decisions, occupational choice, career trajectories, and lifetime income. this evidence, much of it taken from college graduates around the recession, is mixed: some studies suggest that the impact of initial labor market conditions diminishes over the course of an individual's career-often within the first decade-while others find that some penalties associated with early-career shocks can be cumulative and permanent (see rothstein ( ) for an in-depth review; see also, kahn ( ) ). these effects are often heterogeneous by skill level, and may be driven by mismatch in initial job placement (faberman & mazumder , liu et al. , oyer , Şahin et al. , van den berge ), lower initial wages (which may be partially related to job mismatch; the stress of adverse shocks may also be transmitted intergenerationally through epigenetic channels. see, e.g., costa et al. ( ) . likewise, there is evidence that both pandemics and recessions-as traumatic and stressful events-can shape the attitudes and preferences of those exposed during formative years in ways that can have lasting political and economic consequences (see, e.g., campante et al. ( ) ; giuliano & spilimbergo ( ) ; malmendier & nagel ( ); and schoar & zuo ( ) ). while this literature focuses on adverse shocks at the time of labor market entry, note that compared to other recessions, long-run labor market scarring could even extend to a different and younger range of cohorts in the covid- case, because of widespread school closures. other covid- -related mechanisms, such as the loss of parental income, would tend to compound these effects further. oreopoulos et al. ( ) ), reduced working time (cockx & ghirelli ) , and delays in finding employment (genda et al. ) , among other factors. moreover, strategic responses to these shocks, such as migration (feigenbaum ) , temporary exit from the labor force (hershbein ) , and human capital acquisition (charles et al. , barr & turner , may themselves have implications for short-and long-run labor market prospects, as separate from those arising directly from the initial shock. these studies thus strongly suggest that downturns may have important -overhang‖ that may potentially -reduce prosperity for decades to come,‖ both for directly-affected cohorts and the wider economy (rothstein , p. ) . accordingly-and bearing in mind that under covid- , peak unemployment rates for younger workers have been nearly three times the national average -very-long-run and even intergenerational evidence on these issues can be especially valuable. recent work in economic history has looked to the great depression in order to offer precisely this sort of perspective. these studies show substantial and persistent penalties for all workers in severely-hit areas, but especially for new labor market entrants, who faced very different constraints and scope for adaptation than did incumbent workers. moulton ( ) , for instance, finds a substantial earnings penalty amongst less-educated american men just entering the labor market in . while there are large adverse effects for those born in severely-affected states, this age-at-downturn penalty disappears in lessaffected states. likewise, examining evidence on labor force transitions using large-scale linked microdata from the u.s., arthi et al. ( b) show that many younger workers during the depression accepted work that they otherwise might not have considered in better economic times-whether because of their now-dire need, the additional competition from older workers, or some combination of these factors. moreover, many young people seeking work were locked out of the labor market completely by their older counterparts, who now remained in the labor force (or even re-entered it) at higher rates. evidence on occupational transitions and socioeconomic mobility also suggest important career-stage gradients in scarring: younger workers were crowded out of the best local job opportunities by their older counterparts, with young workers in more rural areas pushed out of farming by older workers who retained these jobs at higher rates, and into general laborer and non-occupational positions; and those in more industrial areas being pushed into farming, the less desirable class of occupations in these areas. importantly, while both of these outcomes represent a short-run penalty for newer labor market entrants, the long-run implications for wellbeing may be very different, given the rapid urbanization and the incipient decline of the agricultural sector that was to come. indeed, by providing the impetus to leave agriculture (or by prompting higher rates of out-migration-younger labor market cohorts irrespective of sector were also likelier to have moved across state lines or into urban areas during the depression, perhaps in response to the dearth of local opportunities for inexperienced workers), the great depression may have had a small silver lining for young rural workers. however, at least in the short run, it served to hamper upward mobility-or even, to induce downward mobility. for instance, liu & fishback ( ) show that though concerns over skill depreciation and mismatch during spells of un-or underemployment animated depression-era policymakers, new deal programs largely failed to match workers to jobs that used their skills, often resulting in lower incomes and transitions into lower-skilled employment or unemployment-though at least some general human capital was maintained. meanwhile, feigenbaum ( ) finds that by , intergenerational mobility had fallen for men growing up in cities severely hit by the depression. migration-in particular, the superior destination choices of the sons of richer fathers-was an important mechanism behind these results, again emphasizing the capacity of large adverse shocks to exacerbate rather than level preexisting inequalities. the history of past pandemics and economic downturns provides sobering guidance for what we might expect from the current covid- crisis. there is a complicated relationship between health and economic productivity that will shape the immediate and latent effects of covid- in both obvious and subtle ways. given that these latent effects unfold over decades and even generations, economic history is uniquely capable of providing evidence on the potential long-term costs of the pandemic. experience from both historical pandemics and historical recessions can inform our view of the possible long-run effects of covid- , and how we might mitigate these costs. the experience of the influenza pandemic suggests that disease exposure can impact individuals throughout their lifetimes, both directly through poorer ongoing health, and indirectly through reduced investment in human capital. the costs were not limited to those individuals directly exposed; instead, they spilled over within households and across space, sectors and generations. moreover, while mortality is salient, and the saving of lives remains perhaps the primary objective during a pandemic, avoiding and compensating for morbidity is arguably as important a matter of policy concern, especially in the context of possible long-run effects. particularly in a pandemic where large shares of prime-aged people fall ill (as in the pandemic), or in pandemics where many are infected but ultimately survive (as in both the pandemic and covid- ), experiences of pandemic illness may have lasting effects over the life-course, either through the initial illness (which may, for instance, compromise fetal nutrition, reduce work capacity, or permanently damage health), or through its sequelae later in life. the great depression points to other long-term effects that are likely to emerge from the pandemic-related slowdown in economic activity: both being born or entering the labor market during the great depression led to economic penalties well into adulthood, and constraints on migration had adverse effects on individuals and firms. importantly, history shows us that these two types of harms are mutually reinforcing: damage to health tends to undermine labor market prospects in the long run, while damage to labor market prospects tends to undermine health in the long run. researchers and policymakers should therefore consider the potential for these long-run costs when weighing the short-term costs and benefits of pandemic control and fiscal intervention. history suggests potentially massive future costs for both the economy and the safety net arising from the dampened economic fortunes, chronic health issues, and foregone fertility of cohorts impacted by covid- . given that human capital investments are generally more productive the earlier they are implemented, this suggests that policy interventions undertaken now, such as cash relief, could be especially cost-effective, and their net long-run benefits tremendous. economic history also reveals that we cannot think of the health and economic impacts of covid- independently of one another. past pandemics indicate that regardless of the pathology of a disease, its impacts are often a function of economic conditions. while some pandemics spared no class, many disproportionately impacted individuals of lower socioeconomic status due to a variety of factors including their occupations, living conditions, and access to healthcare. these individuals are at greater risk of exposure, face greater harms conditional on exposure, and are less able to remediate these harms. we have already seen this taking place with covid- , and need to remain aware that the spread of the disease and the severity of its effects will be in part a function of the spatial distribution of residence, economic activity, and environmental harms. these disparate impacts of the virus itself will be compounded by the associated economic downturn. to the extent that the covid- economic downturn limits exposure to environmental and work-related hazards, or reduces spending on unhealthy behaviors, non-coronavirus related dimensions of health may actually improve. however, both the modern literature on developing countries and the u.s.'s experience during the great depression suggest that the severe economic downturn may compound health problems in areas with lower baseline incomes and weaker safety nets. identifying the channels through which income loss and general recession conditions impact health is necessary for properly interpreting any observed changes in population health levels during covid- , and for designing effective policies to safeguard health. successful implementation of these policies also requires a firm understanding of history-roadblocks to public health initiatives during past pandemics associated with institutional structures and individual attitudes offer cautionary tales for our current crisis. while economic history provides useful insights for the current pandemic, the way in which the pandemic is unfolding also provides a fresh perspective with which to revisit the past. we are witnessing the actions that individuals and families, workers and firms, citizens and public officials alike, take to guard against the pandemic, and the damage it has done to the economy. we are witnessing how these responses change as new information on covid- emerges. the current pandemic affords us unprecedently rich and disaggregated data that, even while still evolving, can give new insights into which groups might warrant additional study in past pandemics. all of these dimensions of covid- can help us reshape the roadmap for studying the economic history of pandemics. one of the most important ways the covid- experience can shape the direction of economic history may not be in seeking out the similarities but rather focusing on differences. while the rate of transmission and severity of the effects of covid- have historical analogues, many relevant features of the world are meaningfully different-among them, the global nature of production; flows of people, goods, and information; urbanization; baseline living standards; medical technology; public health infrastructure; and the role of government. these differences can help us understand both past and present pandemics better; moreover, they help us understand how and why things have changed. for example, the covid- shutdowns have been more far-reaching, and the corresponding economic downturn more damaging, than we might have predicted from previous pandemics. can these differences explain the far greater economic costs of covid- relative to similarly lethal pandemics of the th century? this suggests an important direction for future economic history work: identifying why the nature of the response to public health crises differed, and why the resulting economic consequences were often smaller historically. engaging in this work also allows us to grapple with challenging questions about tradeoffs between population health and economic activity. these tradeoffs are incredibly difficult to tackle head on in the face of an unfolding crisis; they force unfathomable but unavoidable choices on policymakers often working with limited information. by offering insight into not just the actions but also the short-and long-run outcomes of governments, firms, and individuals, economic history can guide us to better decisions in this and future crises. the colonial origins of comparative development: an empirical investigation infuenza and pneumonia mortality in large cities in the united states in years surrounding the pandemic plague in seventeenth-century europe and the decline of italy: an epidemiological hypothesis is the influenza pandemic over? long-term effects of in utero influenza exposure in the post- u human capital development before age five.‖ pages the influenza pandemic and subsequent health outcomes: an analysis of sipp data the dust was long in settling': human capital and the lasting impact of the american dust bowl . -recessions, mortality, and migration bias: evidence from the lancashire cotton famine estimating the recession-mortality relationship when migration matters.‖ nber working paper # labor market scarring in the very long run: evidence from large-scale longitudinal microdata -disease, downturns, and wellbeing: economic history and the long-run impacts of covid- aggregate income shocks and infant mortality in the developing world fetal and infant origins of adult disease: papers out of work and into school: labor market policies and college enrollment during the great recession smoke, and death‖ bituminous coal and american home heating.‖ nber working paper # non-pharmaceutical interventions and mortality in u.s. cities during the great influenza pandemic, - .‖ nber working paper # the coronavirus and the great influenza pandemic: lessons from the "spanish flu" for the coronavirus's potential effects on mortality and economic activity (no. w ) the redistributive effects of pandemics: evidence on the spanish flu.‖ universidad carlos iii de madrid working papers in economic history # - forthcoming. -the influenza pandemic and its lessons for covid- fetal shock or selection? the influenza pandemic and human capital development.‖ nber working paper # the great plague in london in . the great plague in london in les hommes et la peste en france et dans le pays européens et méditerranées disease and development: evidence from the american south disease and development: evidence from hookworm eradication in the american south malaria eradication in the americas: a retrospective analysis of childhood exposure disease and fertility: evidence from the influenza pandemic in sweden.‖ iza discussion paper # the economic effects of the influenza epidemic on the long term effects of the u.s. influenza pandemic the virus of fear: the political plague and the poor in renaissance florence housing booms and busts, labor market opportunities, and college attendance does air quality matter? evidence from the housing market the impact of air pollution on infant mortality: evidence from geographic variation in pollution shocks induced by a recession pandemic influenza and socioeconomic disparities: lessons from chicago.‖ proceedings of the national academy of sciences the - influenza pandemic in england and wales: spatial patterns in transmissibility and mortality impact.‖ proceedings. biological sciences a farewell to the alms: a brief economic history of the world what explains cross-city variation in mortality during the influenza pandemic? evidence from u pollution, infectious disease, and mortality: evidence from the spanish influenza pandemic canary in a coal mine: infant mortality, property values, and tradeoffs associated with mid- th century air pollution.‖ nber working paper # scars of recessions in a rigid labor market.‖ labour economics multigenerational effects of early-life health shocks.‖ demography pandemics depress the economy, public health interventions do not: evidence from the flu health and the economy in the united states from to the present intergenerational transmission of paternal trauma among us civil war ex-pows america's forgotten pandemic: the influenza of environmental health risks and housing values: evidence from , toxic plant openings and closings was plague an exclusively urban phenomenon? plague mortality in the seventeenth-century low countries economic conditions and mortality: evidence from years of data estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study guns, germs, and steel: the fates of human societies pest und staat: von der institutionengeschichte zur sozialen konstruktion. neue wege in der seuchengeschichte state capacity and public goods: institutional change, human capital, and growth in historic germany how positive was the positive check? investment and fertility in the aftermath of the influenza in india.‖ mimeo covid- among workers in meat and poultry processing facilities - states plague ports: the global urban impact of bubonic plague does job loss shorten life smallpox: gone but not forgotten is there a skills mismatch in the labor market?‖ chicago fed letter intergenerational mobility during the great depression aggregate economic shocks births, deaths, and new deal relief during the great depression hard times in the land of plenty: the effect on income and disability later in life for people born during the great depression the effects of in utero exposure to the influenza pandemic on family formation long-term effects of a recession at labor market entry in japan and the united states growing up in a recession emerging infections: pandemic influenza fear, lockdown, and diversion: comparing divers of pandemic economic decline .‖ nber working paper # disparities in influenza mortality and transmission related to sociodemographic factors within chicago in the pandemic of reply to shanks and brundage: many plausible mechanisms of pandemic mortality disparities.‖ proceedings of the national academy of sciences food expenditure and nutritional quality over the great recession the basic reproduction number (r ) of measles: a systematic review the brazilian bombshell? the long-term impact of the influenza pandemic the south american way.‖ nber working paper # effects of social distancing policy on labor market outcomes.‖ nber working paper # public health interventions and epidemic intensity during the influenza pandemic the economics, technology, and neuroscience of human capability formation.‖ proceedings of the national academy of sciences the long-lasting influenza: the impact of fetal stress during the influenza pandemic on socioeconomic attainment and health in sweden graduating high school in a recession: work, education, and home production the distribution of environmental damages die deutsche stadt im mittelalter - : stadtgestalt, recht, verfassung, stadtregiment the long-term labor market consequences of graduating from college in a bad economy labor demand in the time of covid- : evidence from vacancy postings and ui claims.‖ nber working paper # public health interventions and economic growth: revisiting the spanish flu evidence.‖ mimeo does in utero exposure to illness matter? the influenza epidemic in taiwan as a natural experiment good skills in bad times: cyclical skill mismatch and the long-term effects of graduating in a recession effects of new deal spending and the downturns of the s on private labor markets in / .‖ explorations in economic history depression babies: do macroeconomic experiences affect risk taking? can the spanish influenza pandemic of explain the baby boom of in neutral norway?‖ population pandemic morbidity: the first wave hits the poor, the second wave hits the rich a socially neutral disease? individual social class, household wealth and mortality from spanish influenza in two socially contrasting parishes in kristiania - nonpharmaceutical interventions implemented by u.s. cities during the - influenza pandemic parasites, pathogens, and progress: diseases and economic development can pandemics affect educational attainment? evidence from the polio epidemic of why are recessions good for your health? -cyclicality, mortality, and the value of time: the case of coffee price fluctuations and child survival in colombia what is a pandemic the great depression of income: historical estimates of the longer-run impact of entering the labor market during a recession business recession, alcohol consumption, drinking and driving laws: impact on oklahoma motor vehicle fatalities and fatal crashes estimation of potential global pandemic influenza mortality on the basis of vital registry data from the - pandemic: a quantitative analysis the influenza pandemic hastened the decline of tuberculosis in the united states: an age, period the columbian exchange: a history of disease, food, and ideas the long-run effects of pandemic influenza on the development of children from elite backgrounds: evidence from industrializing japan race and influenza pandemic in the united states: a review of the literature epidemiological evidence of an early wave of the influenza pandemic in new york city.‖ proceedings of the national academy of sciences the short-and long-term career effects of graduating in a recession the making of an investment banker: macroeconomic shocks, career choice, and lifetime income what happens to household formation in a recession?‖ th annual areuea conference paper childhood health and sibling outcomes: nurture forcing nature during the influenza pandemic.‖ explorations in economic history yellow fever epidemics and mortality in the united states racial disparities in exposure, susceptibility, and access to health care in the u.s. h n influenza pandemic the lost generation? scarring after the great recession are recessions good for your health? healthy living in hard times does drinking really decrease in bad times? early-life disease exposure and occupational status: the impact of yellow fever during the th century high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus . emerging infectious diseases shaped by booms and busts: how the economy impacts ceo careers and management styles,‖ the review of financial studies variable mortality during the influenza pandemic in chicago the impact of influenza epidemics on mortality: introducing a severity index the best of times, the worst of times: understanding pro-cyclical mortality banking crises and mortality during the great depression: evidence from u.s. urban populations, - job displacement and mortality: an analysis using administrative data the incidence of influenza among persons of different economic status during the epidemic of the engine and the reaper: industrialization and mortality in late nineteenth century japan influenza: the mother of all pandemics the pox of liberty: how the constitution left americans rich, free, and prone to infection the diffusion of the influenza pandemic of in hartford bad start, bad match? the early career effects of graduating in a recession for vocational and academic graduates what happened to the us economy during the influenza pandemic? a view through high-frequency data.‖ federal reserve bank of chicago working paper how the west ‗invented' fertility restriction malthusian dynamism and the rise of europe: make war, not love the three horsemen of riches: plague, war, and urbanization in early modern europe who report on global surveillance of epidemic-prone infectious diseases. world health organization. . covid- strategy update the gift of the dying: the tragedy of aids and the welfare of future african generations key: cord- -ifwjvvjj authors: beaver, annabelle; meagher, rebecca k.; von keyserlingk, marina a.g.; weary, daniel m. title: invited review: a systematic review of the effects of early separation on dairy cow and calf health date: - - journal: journal of dairy science doi: . /jds. - sha: doc_id: cord_uid: ifwjvvjj abstract concern from the public is growing regarding early cow-calf separation, yet proponents of this practice maintain that artificial rearing is critical for cow and calf health. early separation is assumed to reduce the risk of transfer of pathogens from dam to neonatal calf, but a wide range of health benefits associated with extended cow-calf contact has also been documented. the aim of this systematic review was to report and synthesize conclusions from the literature on dairy cow and calf health in conventional rearing versus cow-calf contact systems. peer-reviewed, published manuscripts, written in english, directly comparing dairy cow or calf health in artificial versus suckling systems, were eligible for inclusion. we conducted targeted searches using web of science to identify key literature on important health conditions. the resulting manuscripts underwent a -step appraisal process, and further manuscripts were sourced from reference lists. this process resulted in a final sample of articles that addressed cow and calf health. sufficient literature was available to assess mastitis in cows, and scours, cryptosporidiosis, johne's disease, pneumonia, immunity, and mortality in calves. the results for cryptosporidiosis, pneumonia, immunity, and mortality were mixed, with some differences between studies likely attributable to flawed comparisons between cohorts. overall, the articles addressing calf scours and mastitis pointed to beneficial or no effects of suckling. the studies addressing johne's disease did not find cow-calf contact to be a significant risk factor. in conclusion, the scientific peer-reviewed literature on cow and calf health provides no consistent evidence in support of early separation. on many commercial dairy farms, it is routine practice to separate the calf from the dam within h of calving (de passillé et al., ; stěhulová et al., ) . proponents of early separation consider it economically beneficial (due to an increase in saleable milk) and ethically preferable (as it is thought to preclude formation of a maternal bond that becomes progressively more difficult to break; flower and weary, ) . one oft-cited rationale for immediate cow-calf separation is the health benefit ostensibly afforded by artificial calf rearing. for example, faubert and litvinsky ( ) claim that this practice reduces the risk of cryptosporidium parvum infections. muskens et al. ( ) make a similar claim for johne's disease, and daugschies and najdrowski ( ) for eimeriosis. however, such literature does not always cite evidence to corroborate these assertions. the concern over disease transmission stems from the agammaglobulinemic state of the neonatal calf and its heightened susceptibility to disease during this time. artificial feeding of calves is thought to allow better control of colostral quality and quantity and thus improve transfer of maternal immunoglobulins to the calf. moreover, the dam's fecal coliform count increases by up to cfu during the periparturient period (pelan-mattocks et al., ) , leading to a concern that calves permitted to remain in the calving area are at an increased risk of exposure to pathogens (mcguirk, ) . despite these concerns, health benefits of prolonged contact have been documented for calves and cows, ranging from increased immunoglobulin absorption from colostrum (stott et al., ) , to decreased mortality rates for calves (alvarez et al., ) , to reduced risk of mastitis for cows (walsh, ) . thus, allowing the cow and calf to remain in contact presents a mosaic of purported health benefits and risks, for which there is a lack of consensus. the aim of the present review is to provide a critical and systematic evaluation of the scientific literature on the health implications of cow-calf contact versus artificial rearing; our compan-invited review: a systematic review of the effects of early separation on dairy cow and calf health ion paper (meagher et al., ) presents the results of a parallel review on the effects of cow-calf contact on measures of behavior, welfare, and productivity. a synthesis of conclusions from the literature is required to offer a measure of resolution to this debate. articles were eligible for inclusion if they were peerreviewed, written in english, complete (e.g., conference abstracts were excluded), available in full-text form, and contained a direct investigation of the effects of cow-calf contact or suckling on dairy cow or calf health. articles were removed if not based upon original data (e.g., review articles or literature-based mathematical models). any manuscript published after the completion of the literature search (may , ) was not included. exclusion and inclusion criteria for the systematic review were developed a priori and agreed upon by all co-authors. systematic searches were conducted using the web of science (wos) database, which allows for integration of boolean operators (i.e., and, or, not) to string together words or phrases, as well as wildcard truncations (denoted as *) to designate a range of possible word forms. the $ symbol was employed to account for alternate spellings (e.g., american versus british english). all searches contained the following fixed set: ("cow-calf" or "cow/calf" or "dam-calf" or "dam/ calf" or "dam rearing" or "reared by the dam" or "reared by cows "or "suckling system*" or "mother rearing" or "reared by the mother" or "contact of calves with adult*" or "leav* calves with dam*" or "stay* with the dam" or "remain* with the dam" or "kept with the dam") and (nurs* or suckl* or separation or contact or "risk factor*") and (calf or calves). seven specific searches were conducted, each containing targeted terms addressing the most relevant calf and cow health conditions: scours (scour* or diarr*), cryptosporidiosis (cryptosporidi*), johne's disease (johne's or paratuberculosis), pneumonia (pneumonia or respiratory), immunity (immunity or "passive transfer"), health and mortality (disease* or infection* or health or morbidity or mortality), and mastitis (mastitis or "intramammary infection*" or "udder health"). several additional search terms were tested but yielded no results meeting the predefined exclusion criteria; these terms included "e. coli," escherichia, salmonella, heidelberg, clostridium, campylobacter, "enteric bacteria," coccidi*, bvd, "bovine viral diarr*," rotavirus, coronavirus, neospor*, bluetongue, diphtheria, "mycoplasma bovis," "mycobacterium bovis," schmallenberg, sarcocystis, anaplasm*, cowdria, blv, "bovine leukemia virus," biv, "bovine immunodeficiency virus," bohv- , bhv- , "bovine herpes virus," piv- , piv , "parainfluenza virus," "mannheimia haemolytica," "pasteurella multocida," "histophilus somni," "arcanobacterium pyogenes," "retained placenta," "placental retention," "f$etal membrane," metritis, "uterine health," "uterine disease," "milk fever," hypocalc$emia, dd, "digital dermatitis," mange, and mites. the selection of these search terms was based upon expert opinion, in addition to several review articles on pathogenesis and health conditions in dairy cows and calves: mulligan et al., ; muktar et al., ; and francoz et al., . results from the unique searches were pooled, and duplicate results were excluded. articles were then selected based upon a -step screening and appraisal process: phase . conference proceedings and articles written in a language other than english were removed. the titles of the remaining articles were scanned to filter out irrelevant results (e.g., literature clearly pertaining to animals other than the dairy cow and calf). phase . abstracts were evaluated to identify and remove additional articles not relevant to the topic of dairy cow or calf health, disease, infection, or mortality (e.g., articles addressing dairy-herd economics). phase . reference lists were mined for additional relevant manuscripts. if full texts were not available online or in the university of british columbia's library system, they were requested via interlibrary loan, re-searchgate, or personal contacts. the reference lists of papers added at this stage were also considered as a source for further manuscripts phase . finally, review articles were removed, and full texts of the remaining papers were read in detail. articles containing experimental research were excluded if the experiment itself did not address the question of cow-calf contact (but rather reviewed literature on the issue as part of a larger discussion). mathematical models were excluded if parameters relating to cow-calf contact were sourced from other literature, or if insufficient information pertaining to real-world data collection was provided to permit recalculation of model parameters. the articles remaining at this stage were included in the systematic review, and in multiple sections if they described more than one relevant effect. to provide a comprehensive overview of the literature, no additional restrictions were placed upon publication year, study type, sample size, or overall quality; however, a quality assessment of the included manuscripts was conducted and is reported in the appendix. from each manuscript, where applicable, we have recorded authorship, publication year, country, breed of cattle under study, dam-calf contact type (e.g., restricted suckling or unrestricted contact), contact duration, sample size, the amount and type of milk provided to artificially reared calves, the author's conclusion, and the direction of this conclusion. inter-observer reliability for data extraction (for all categories except for authorship and publication year) was tested on a random subset of articles, with a result of % agreement. we also present diagnostic test information, relative risks (rr), odds ratios, hazard ratios, and the associated % confidence intervals (ci), where available. these statistics were provided in many of the articles addressing mastitis, johne's disease, and calf cryptosporidiosis. in an attempt to achieve consistency, we calculated the rr (or prevalence ratio in the case of cross-sectional studies) and ci for articles in these categories if not reported by the authors. this was only possible when sufficient information was provided to permit calculation. the following formulae were applied: where a, b, c, and d correspond to number of controls with the disease, number of disease-free controls, number of experimental animals with the disease, and number of disease-free experimental animals, respectively. where se lnrr a c a b a c ( )= + − + − + . results from the -step screening and appraisal process detailed in the materials and methods section are shown in figure . briefly, wos returned unique papers pertaining to cow-calf separation and health. the titles of these papers were scanned, and papers were subsequently excluded ( of these were written in another language, and concerned beef cattle or other species). following an assessment of the abstracts (n = ), an additional papers were excluded. at this stage, articles were sourced from the reference lists of the remaining papers, and from the reference lists of newly included papers. in the final exclusion stage, a further papers were removed because they were review articles (n = ), did not directly compare cow or calf health in relation to cow-calf contact (n = ), or were literature-based mathematical models (n = ). in total, articles (comprised of single-herd studies and multi-herd studies) were selected for inclusion; of these were included in of the specific sections below, and were included in sections. the final manuscripts included in this systematic review were authored between and . the studies originated from continents and countries, most commonly north america (n = : united states = ; canada = ; mexico = ) and europe (n = : central europe = ; scandinavia = ; western europe = ; southern europe = ). additionally, studies originated from south america, from east africa, from asia (middle east = ; south east asia = ), and from oceania (australia = ; new zealand = ). these statistics include only those studies that specified regional information. the single-herd studies included between and relevant groups with a total sample size of between and animals. the multi-herd studies included between and , herds with the total number of animals ranging from to , . scours and enteric pathogens. of the included articles, ( single herd and multi-herd studies) addressed dairy-calf scours of unspecified or multiple etiologies. nine additional articles specifically addressed calf cryptosporidiosis, predominantly infection with c. parvum. one of these studies represented a single-herd evaluation of prevalence, and the other were multi-herd trials (between and herds) that paired questionnaires on management practices with cross-sectional or repeated fecal samples from individual calves. results for scours are reported in table and results pertaining to cryptosporidium are reported in table . two additional articles addressed other enteric pathogens, namely campylobacter, giardia, eimeria, rotavirus, and coronavirus. as shown in table , several studies demonstrated benefits of cow-calf contact on scours (carias and vaccaro, ; nocek et al., ; rajala and castrén, ; weary and chua, ; boonbrahm et al., a; wagenaar and langhout, ) . conversely, studies (svensson et al., ; roth et al., ) reported a greater risk of scours in dam-reared animals. roth et al. ( ) hypothesized that the higher incidence in nurs-ing calves could be explained by the increased quantity of milk consumed by these animals, rather than by infectious causes. indeed, wagenaar and langhout ( ) reported that the type of scouring sometimes observed figure . flowchart depicting the manuscript screening and appraisal process. note that summing the studies included in the qualitative synthesis in each subsection yields studies, rather than . this discrepancy results from the inclusion of studies in specific sections and studies in specific sections. a deletion of the duplicates results in unique studies. listed for each study are country, breed of cattle, type of cow-calf contact, duration of this contact, how milk or colostrum (or both) was fed to calves in the control condition (i.e., in which calves were not allowed to suckle), the amount of milk provided in this condition, the total number of calves followed, the number of herds (and groups within a herd where relevant), and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction in suckling calves was characteristically distinct from that of bucket-fed counterparts, primarily because it did not lead to the animals dirtying their backsides. boonbrahm et al. ( a) found that mortality from scours and other conditions was higher in bucket-fed calves ( %) compared with dam-reared calves ( %), again suggesting a different etiology. it is now well known that high volumes of milk may contribute to looser manure in calves (see khan et al., ) , which need not reflect any infection. methods of evaluating scours in calves should therefore take into account differences in fecal consistency associated with level of milk intake. although the balance of the research seems to point to either neutral or positive effects of dam rearing on calf scours, results are mixed with respect to cryptosporidium (see table ). two of the included studies reported a protective effect of the dam's presence (kvac et al., ; duranti et al., ) , reported no difference (maldonado camargo et al., ; mohammed et al., ; delafosse et al., ; garro et al., ) , and the remaining (quigley et al., ; trotz-williams et al., , a indicated that dam-calf contact increases the risk of infection. this lack of consensus may stem from variation in study design and outcome measurements (e.g., herd versus sample-level prevalence), in breed (dairy versus mixed dairy-beef), or in the diagnostic methods chosen. despite the conflicting results, the collective burden of available odds ratios and risk ratios is skewed toward a protective effect of suckling. two papers addressed other specific enteric pathogens in relation to dam-calf contact. klein et al. ( ) found that herds leaving calves with the dam for > h had a . times greater odds of testing positive for campylobacter spp., and quigley et al. ( ) noted an increased risk of giardia in suckling calves, but no differences were found for eimeria, rotavirus, or coronavirus; further investigation of these and other pathogens in suckling systems is needed. johne's disease. of the articles included, directly addressed the relationship between the prevalence of mycobacterium avium ssp. paratuberculosis (map; the causal agent of johne's disease) and duration of cow-calf contact. all studies consisted of management questionnaires paired with serum (n = ) or milk (n = ) elisa testing, unspecified elisa testing (n = ), bulk-milk pcr (n = ), fecal culture (n = ), a combination of fecal culture and serum elisa (n = ), or clinical case reports (n = ). results are shown in table . a limiting factor in many of the cross-sectional studies presented in this review is the absence of a temporal relationship between johne's diagnosis and the imple- listed for each study are country, breed of cattle, study design, groups compared (in reference to length of cow-calf contact), total number of calves sampled, the number of herds included (and groups within herds where applicable), the type of herd, the diagnostic(s) used, and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction). hf signifies that breed was reported as holstein, friesian, or holstein-friesian. this designation includes country-specific variants such as danish holstein. pr (ci) = prevalence ratio ( % ci). mentation of management practices. however, none of the included articles were able to identify increased map prevalence among herds permitting cow-calf contact, even in preliminary univariable analyses (with the exception of pillars et al., ) . official johne's disease control programs frequently describe the risks associated with allowing the calf to suckle and remain within the maternity area, or directly recommend immediate separation of cow and calf following parturition; such programs include the three step calf rearing plan of australia (animal health australia, ), the voluntary bovine johne's disease control program of the united states (usda, ), and several regionspecific canadian programs [such as the atlantic veterinary college ( ) johne's disease initiative, which deems calf nursing to be a risky practice, even within test-negative herds]. given the lack of quantitative evidence on the value of immediate cow-calf separation, the persistence and regularity of this recommendation is surprising. a common theme that emerged in our systematic search of this topic was the presence of unsubstantiated claims regarding map prevalence and cow-calf separation. an example of the dissemination of such claims within the literature is shown in figure . the type of citation scheme demonstrates how a largely unsubstantiated claim (in this case, that calves should be immediately separated from the dam to prevent johne's disease) can achieve the status of "common knowledge" in the literature. the perpetuation of these assertions may lead to decisions such as that of norton et al. ( ) to include "duration of dam-calf contact" in multivariable risk assessment modeling due to "biological importance," despite the variable's lack of significance at the univariable screening stage. the hypothesis regarding biological relevance is not unfounded because calves under mo of age are most susceptible to map infection. mycobacterium avium ssp. paratuberculosis may be transmitted from an infected dam to calf in utero or through direct bacterial shedding into colostrum or milk; however, map infection occurs predominantly by means of a contaminated environment, via the fecal-oral route (lombard, ) . we may thus imagine that prompt calf removal could mitigate transmission. several mathematical models (e.g., collins and morgan, ; marcé et al., ) have demonstrated that removing the calf from the calving area, or minimizing the effective number of cow-calf contacts, reduces map prevalence, but such models are often parameterized based upon an assumption that transmission increases for calves contacting adult animals. yet, in some herds, cow-calf separation has supplanted control strategies for which concrete evidence exists to tie the respec-tive strategy to a reduction in map prevalence. for example, wells and wagner ( ) noted that herds in which cows and calves were separated at h (compared with > h) were more than times as likely to have had a previous johne's disease diagnosis. the authors posit that herd managers altered their management practices following the positive herd status report. in contrast, the practice of sourcing > % of animals from outside dairies was associated with a current johne's disease diagnosis in the herd, yet no evidence was provided to suggest that this practice underwent a similar modification (wells and wagner, ) . johnson-ifearulundu and kaneene ( ) reported that the process of cleaning maternity pens was linked to a -fold reduction in the odds of a positive herd-level map status. however, many commercial herds allow multiple animals in the calving area (e.g., % in the united states; usda, ) or permit sick cows to be housed in these areas ( %). of those herds that did use dedicated individual maternity pens, only to % (depending on herd size) cleaned the area after each calving (usda, ). there is evidence for a synergism of infection risk in the calving area, based upon the level of environmental cleanliness, udder hygiene, and presence of other lactating animals (beaver et al., ) . the evidence we have reviewed indicates that prompt calf removal should not be viewed as a substitute for proper hygiene and management in the maternity area. respiratory health. of the included articles, ( single-herd and multi-herd studies) addressed respiratory health in dairy calves. the results of these studies are shown in table . the majority of these papers did not investigate pneumonia as a primary outcome measure; all but one study (gulliksen et al., a) was included in other subsections of this review. five studies failed to find any association between respiratory risk and dam-calf contact or nursing (although of these studies were conducted using the same group of calves: lundborg et al., , and svensson et al., ) . the remaining studies presented opposing conclusions, with gulliksen et al. ( b) reporting a higher risk of pneumonia for calves kept > h with the dam and boonbrahm et al. ( a) noting a lower pneumonia incidence in suckling calves. further research is clearly required before meaningful conclusions can be reached, including studies for which the investigation of specific respiratory pathogens is a primary objective. immunity. of the included articles, described calf immunity or failure of passive transfer (fpt) in suckling compared with artificial systems. the majority (n = ) were single-herd trials, and were multi-herd studies (table ) . the cited studies present conflicting conclusions. for example, stott et al. ( ) and quigley et al. ( ) described a positive association between suckling and immunoglobulin absorption in neonatal calves, and selman et al. ( ) found that the presence of the dam led to improved passive transfer. in contrast, several articles concluded that suckling results in higher levels of fpt (nocek et al., ; besser et al., ; trotz-williams et al., b; beam et al., ). this discrepancy may, in part, be traced back to the source and quantity of colostrum offered to control groups. it is now well established that quantity, quality, and promptness of colostrum administration is pivotal in facilitating immunoglobulin absorption and thus lowering the rate of fpt (see godden, ) . the current recommendation stipulates that calves obtain to % of their bw in colostrum at first feeding, and additional benefits have been shown in calves receiving further doses of colostrum at h postpartum (godden, ) . in several of the older studies (stott et al., ; quigley et al., ) , the volume of colostrum fed to control groups would be considered insufficient by today's standards, and is likely not comparable to the amount a suckling calf would obtain from the dam. moreover, in stott et al. ( ) the colostrum provided to control calves was pooled; pooling has been reported to increase the bacterial burden, leading to inhibition of immunoglobulin absorption (stewart et al., ) . thus, differences in fpt between dam-reared and artificially raised calves may be attributable to colostrum allowance. other studies seeking to compare rates of passive transfer between the groups are often encumbered by different limitations. besser et al. ( ) collected data from separate herds, each with different colostrum management practices (tube feeding, bottle feeding, or suckling). because these herds likely differed in a variety of other dimensions, and successful immunoglobulin absorption is affected by numerous external factors (godden, ) , it is difficult to isolate the effect of suckling. besser et al. ( ) made some effort to measure colostrum quality in all groups, but large volumes were fed to the control animals, and the authors acknowledge that these would far exceed the expected intake of a suckling calf. in nocek et al. ( ) and beam et al. ( ) , calves fed high-quality and promptly administered colostrum were compared with an unmonitored suckling group, with unknown colostral quality and latency to feed. thus, the same concerns regarding insufficient colostrum allowance (stott et al., ; quigley et al., ) are replicated, but in this case, are transferred to the suckling group. several other studies have addressed the question cow-calf removal and fpt without drawing a direct comparison between artificial rearing and suckling systems. for example, mcaloon et al. ( ) found that increased time spent in the calving pen was associated with diminished immunoglobulin absorption, likely because many calves did not suckle in the first few hours after birth. arguably, these conclusions are attributable to latency until farmer intervention rather than time spent with the dam, per se. producers aim to provide prompt administration of high quality colostrum to separated calves and could provide similar interventions for the suckling calf. (goodger et al., ) represents an aggregate of the following management practices: ( ) whether colostrum was harvested from a clean udder (free of manure), ( ) whether the bottles used to store colostrum were clean, ( ) whether colostrum was pooled, and ( ) whether calves were permitted extended contact with the dam. map = mycobacterium avium ssp. paratuberculosis. listed for each study are country, breed of cattle, type of cow-calf contact, duration of this contact, how colostrum was fed to calves in the control condition (i.e., in which calves were not allowed to suckle), the amount of colostrum provided in this condition, the total number of calves followed, the number of herds (and groups within a herd), and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction). fpt = failure of passive transfer. it is important to recognize that a large proportion of dairy calves left with the dam fail to nurse within h after parturition ( % of calves born to dams of second or higher parity; edwards and broom, ) . calves may exhibit a higher latency to suckle if they have low vigor or if the dam has experienced a difficult calving (rajala and castrén, ) . thus, the farmer cannot rely upon nature alone but rather should supervise and intervene if necessary to promote adequate passive immune transfer to the calf. calves born to dams with low-hanging udders (ventorp and michanek, ) may also have more difficulty obtaining colostrum by suckling, suggesting that these calves in particular could benefit from active assistance with nursing or administration of colostrum by bottle. together these observations indicate that various types of farmer intervention, including careful observation and supplementary feeding, may be beneficial regardless of whether the calf is separated from the cow. given that bacterial contamination of colostrum can interfere with immunoglobulin absorption (see godden, ) , it is useful to compare bacterial counts in directly stripped colostrum (which a suckling calf would be expected to obtain) and corresponding counts in harvested and stored colostrum sources. one study (stewart et al., ) found that the process of harvesting colostrum into a bucket resulted in dramatically higher bacterial counts than in directly stripped colostrum. additionally, bacteria in colostrum can multiply precipitously when kept at ambient room temperature and may even reach concentrations exceeding , , cfu/ml after h of refrigeration (stewart et al., ) . bacterial growth in colostrum may add variation to how calves perform when fed by bucket or teat; artificial colostrum feeding is therefore likely to work best on farms able to mitigate this risk (e.g., through decontamination of storage containers). thus there may be challenges and benefits associated with both hand feeding and nursing colostrum from the dam. given the conflicting evidence presented, and the flawed comparisons between groups, the common recommendation to separate the cow and calf immediately after parturition to ensure successful immune transfer should not be considered to be evidence based. however, leaving the calf unsupervised with the dam cannot serve as a replacement for careful colostrum management. mortality. of the included articles, addressed the subject of general calf health and directly addressed calf mortality. six were single-herd trials and the remaining incorporated between and herds, with data obtained from questionnaires or surveys on management practices and calf mortality rates (table ). there appears to be little consensus regarding mortality in dam-reared versus conventionally raised calves. the reasons for this variation are likely similar to those previously described for the fpt outcomes. that is, conventionally reared calves may be at risk when study methodology results in inadequate colostrum uptake (as in quigley et al., ) . insufficient colostrum quantity has been definitively linked to fpt, which, in turn, has strong associations with mortality (see godden, ) . similarly, dam-reared calves may experience increased mortality rates when colostrum intake is unmonitored, unaided, or not quality-controlled. the confounding of dam rearing with low-input management may account for the conclusions from large-scale, multi-herd studies such as jenny et al. ( ) and wells et al. ( ) . indeed, jenny et al. ( ) stated that farmers leaving calves with the dam may wrongly assume these calves have obtained sufficient colostrum via suckling. in contrast, farmers separating the calf and dam must undertake the "mothering role" themselves and provide individualized attention. waltner-toews et al. ( ) noted an interesting phenomenon in calves provided assistance at suckling. in this study, calves that suckled colostrum naturally were less likely to require treatment for disease than were calves fed colostrum by bucket; however, calves requiring assistance to suckle also had higher odds of being treated for disease compared with calves suckling naturally. these findings could indicate that farmers were more concerned about the assisted suckling group and therefore were more likely to provide treatment, or that these animals were "weak calves to begin with" (p. ) and thus required more treatment. in any case, these findings do not imply that suckling assistance itself leads to increased disease rates. of the included articles, addressed mastitis in dairy cows. of these, were conducted using a single herd, wherein to groups were evaluated. the cohorts typically consisted of cow-calf pairs in either full or restricted-suckling systems in addition to variants of artificially reared controls; however, one of these articles (wagenaar et al., ) addressed the future udder health of heifer calves raised under contrasting management conditions. of the remaining multi-herd studies, one evaluated cohorts across separate herds, and the other employed a questionnaire on management paired with clinical mastitis assessments for herds. the assessments of udder health and mastitis rates were conducted using the california mastitis test ( ) ( ), electrical conductivity ( ), or unspecified methods ( ). table lists the included studies. consistent with the findings of johnsen et al. ( ) , the studies reviewed here demonstrate a beneficial effect of suckling systems in reducing the risk of mastitis in dairy cows. in addition to the advantages of suckling to remove residual milk from the udder, reduced mastitis rates in suckled cows could be attributed to lysozymes for bacterial inhibition present in calf saliva (mdegela et al., ) . no study in our systematic review demonstrated an increased risk of imi in suckled cows; however, one study did report higher rates of teat damage (thomas et al., ) . this damage was most pronounced in cows suckling calves twice daily for wk compared with machine milking. these authors recommend nursing for shorter durations to prevent the temporary teat damage associated with continuous, long-term suckling. walsh ( ) found that the greatest benefits in mastitis prevention afforded to nursing cows were in early and mid-lactation. together, these results suggest that producers interested in reducing the risk of mastitis should consider the use of suckling, particularly in early lactation (see also kälber and barth, ) . we systematically reviewed literature on the effect of suckling on calf health, with an emphasis on calf enteric health (including general scours, cryptosporidiosis, and johne's disease), respiratory health, immune status, and mortality. in sum, the evidence presented in this review does not support the recommendation of immediate separation to promote calf health. specifically, we found no consistent evidence of increased risk of pneumonia in calves reared with the cow; however, the available literature on this subject was limited and often did not address the subject of respiratory health as a primary research objective. with respect to enteric health, the majority of studies demonstrated that rearing the calf with the dam had no effect on scours, or was associated with a reduced risk. future research should aim to identify differences in etiology (e.g., infectious versus nutritional causes) between scours in dam-reared compared with artificially reared calves. the leading cause of mortality in dairy calves is diarrhea, for which a wide variety of enteric pathogens are responsible (cho and yoon, ) . with the exception of c. parvum (for which the evidence was mixed), common pathogenic causes of scours in the dairy calf have not been evaluated in detail in artificial compared with suckling systems (apart from the work of klein et al., , and quigley et al., ) . johne's disease is commonly considered an important risk associated with cow-calf contact, but we could find little evidence that rearing the calf with the dam increases map prevalence. contact with the dam after birth cannot be considered an appropriate surrogate variable for "contact with adult cow manure," unless aspects of cow hygiene, colostrum management, maternity-pen management, and overall cleanliness of the calving area are poorly governed. as several these variables have been strongly associated with map transmission risk (e.g., see ansari-lari et al., ; tiwari et al., ; donat et al., ) , initiatives to improve these measures should be prioritized over cowcalf separation. more targeted longitudinal research is required to assess whether immediate cow-calf separation provides any substantive benefit across herds with differing cow-level prevalence. for measures of calf immunity and mortality, no consistent pattern was observed, with studies split between those showing benefits versus risks associated with suckling. studies demonstrating benefits associated with suckling typically did not provide sufficient colostrum or milk to the artificially reared calves. similarly, articles detailing the risks of leaving the calf with the dam did not systematically monitor colostrum intake, quality, and suckling latency. interestingly, our systematic review uncovered only a single study addressing immunity and one addressing mortality authored within the last decade (beam et al., , and gulliksen et al., b, respectively) . in nocek et al. ( ) , several experimental groups were completely deprived of colostrum. given our current knowledge of the importance of colostrum quality, quantity, and timeliness of provision, further studies should assess the feasibility of keeping the calf with the dam and providing supplemental high-quality colostrum as soon as possible after parturition. the results of our systematic search into the effects of suckling systems on dairy cow health do not indicate any benefit to early separation. all of the studies included in this review demonstrated a reduced risk of mastitis in nursing dairy cattle or reported no difference. mastitis is one of the most prevalent disorders in dairy systems worldwide, and some evidence suggests that incidence is on the rise in north american herds (see usda, , versus usda, . as mastitis is also, arguably, the most economically significant challenge facing dairy producers (see seegers et al., ; jamali et al., ) , the favorable conclusions with regard to dam-calf suckling should not be taken lightly. our conclusions on mastitis are in agreement with other reviews on this topic (e.g., kamboj and kumar, ; kälber and barth, ; johnsen et al., ) ; the consensus among these authors is that some type of listed for each study are country, breed of cattle, type of cow-calf contact allowed, duration of this contact, how colostrum was fed to calves in the control condition (i.e., in which calves were not allowed to suckle), the amount of colostrum provided in this condition, the age range of calves under study, the total number of calves followed, the number of herds (and groups within a herd), and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction restricted suckling system has the potential to reduce mastitis on dairy farms. future research is necessary to determine the exact mechanisms responsible for reducing this risk (e.g., removal of residual milk by the calf, lysozymes present in calf saliva, and so on). surprisingly, there was an absence of literature addressing other common postpartum diseases and conditions such as metritis, ketosis, and retention of the fetal membrane. in their review, flower and weary ( ) address retained fetal membranes and cite the danish work of krohn et al. ( ) , which highlights a beneficial effect of suckling on lowering the risk of placental retention. flower and weary ( ) suggest that this evidence could partially explain the lower incidence of retained fetal membranes in beef compared with dairy cows (noakes, ) . another study demonstrated no effects of immediate calf separation on latency of placental expulsion or placentophagia (lidfors, ) . given the importance of postpartum diseases on the longevity and productivity of dairy cattle (mulligan and doherty, ) , this area of research requires further exploration. the evidence extracted from the included journal articles does not support a recommendation of early dairy cow-calf separation on the basis of calf or cow health. specifically, the body of literature on calf immunity, mortality, scours, and pneumonia does not indicate that early separation is advantageous. moreover, there is an absence of literature to suggest that immediate dam-calf separation confers benefits toward mitigating johne's disease. with respect to cow health, this review indicates that suckling is protective against mastitis. using material sourced from domains and of ro-bis (risk of bias in systematic reviews, whiting et al., ) , we have attempted to identify and address weaknesses in our methodology. we restricted our initial literature screening to a single search engine (wos), as different search engines employ distinct criteria and weightings to generate search results. in addition, the search terms themselves require alteration across different databases; certain search engines automatically account for similar terms whereas others do not. it therefore becomes difficult to integrate reference lists that have been generated based upon unique algorithms. we considered wos to be the best available resource for our search due to its status as a human-curated database with itemized, structured entries. additionally, wos only provides results from journals indexed by clarivate analytics and filters out non-peer-reviewed literature; however, articles from well-known, high-impact-factor journals may be overrepresented in our results. the use of a single search engine may be considered a weakness of the present review. thus, we have attempted to validate this approach by conducting targeted searches in pubmed to ensure no additional articles were discoverable. first, we ran the exact searches as listed in materials and methods in pubmed. results indicated that no exact match was found for the phrases "reared by the dam," "reared by cows," "reared by the mother," and "calf contact with adults." therefore, these phrases were removed to ensure that pubmed would not search within the quoted phrases. our final pubmed search included the following fixed set: ("cowcalf" or "cow/calf" or "dam-calf" or "dam/calf" or "dam rearing" or "suckling system" or "mother rearing") and (nurs* or suckl* or separation or contact or "risk factor*") and (calf or calves). in addition, the following targeted terms were included: (scour* or diarr*) or (cryptosporidi*) or ("johne's" or paratuberculosis) or (pneumonia or respiratory) or (immunity or "passive transfer") or (disease or diseases or diseased or infection or health or morbidity or mortality) or (mastitis or "intramammary infection*" or "udder health"). a total of articles were uncovered in these searches; of these were duplicates from our wos searches. of the unique articles, pertained to beef cattle or other species and were therefore excluded. the titles and abstracts of the remaining articles were scanned, and were considered to be irrelevant to the main research question. two full texts were reviewed and neither study made a direct comparison between cow or calf health in suckling or cow-calf contact systems and conventional rearing. pubmed searches were then conducted for all terms that yielded no results in wos (as listed in materials and methods). no additional relevant results were identified. the "topic" field of wos generates searches of the article title, abstract, and keywords, but not the body of the manuscript. papers assessing a variety of risk factors for a given disease may therefore have been overlooked, particularly if the risk factor of interest was not significantly associated with cow-calf contact. thus, it is conceivable that the results of the wos queries reported here demonstrate a bias away from the null. fortunately, this bias is expected to be present in both directions and is therefore unlikely to affect overall conclusions. to counter this undesired selectivity, we searched within reference lists to uncover additional papers. it must be acknowledged that the authors of these papers may be more inclined to cite papers in agreement with their own conclusions, but this type of selection bias is likely to arise in any systematic review using reference lists as additional resources. similarly, although we placed no restriction on publication year, we did not locate any manuscripts published in or , since very recent papers tend to have fewer citations (from even more recent manuscripts). in the initial scans of the reference lists, we only selected papers with titles that alluded to a potential comparison of cow or calf health in different rearing conditions. for example, a hypothetical paper titled "management decisions linked to heightened prevalence of cryptosporidium oocysts" would have been considered, whereas a paper titled "cryptosporidium parvum in cattle" would not have been investigated further. we may have missed papers that, in spite of a more general title, had in fact addressed the topic in question. this assessment is unavoidably qualitative. it is not possible to measure the number of primary literature articles that may have been overlooked due to our methodology; this highlights the importance of accurate and descriptive key words in manuscripts as emphasized by de boer et al. ( ) . we excluded conference proceedings (both papers and abstracts), as well as book chapters, as we could not be certain that these sources had been peer reviewed. we also excluded literature in languages other than english, as we were unable to critically assess the methods and evaluate the results. we are unable to determine to what extent these exclusions affected the conclusions of this review. to provide an inclusive overview of the topic in question, no exclusion criteria were developed concerning sample size, study quality, or journal quality. here we provide a quality assessment of the included journal articles on the basis of study design, thoroughness of reporting, and potential biases. in the field of human medicine, randomized controlled trials are typically considered the gold standard, and observational research is sometimes excluded from systematic reviews. the number of randomized controlled trials in agriculture and veterinary medicine is comparatively low (sargeant and o'connor, ) , so observational studies are often included out of necessity. with reference to study design, articles in our systematic review ( %) were experimental or quasi-experimental and ( %) were observational. randomization procedures for animals were described or referenced in studies ( % of the experimental studies), but only included some type of blinding (blinding was likely not feasible for some measures, given the obvious differences in management between groups with different levels of maternal contact). random assignment of animals to treatment groups was described in of the single-herd trials ( %), and random selection of herds (or a random selection of animals within herds) was described in ( %) of the multi-herd studies. in tables to , we have reported the authors' conclusions concerning their data, regardless of whether these conclusions were tested using inferential statistics. of the included papers, ( %) either did not confirm their conclusions using statistical testing or failed to report results. in these instances, the reported results often reflected secondary research objectives or descriptive information. as per the inclusion criteria, papers investigating other aspects of cow and calf contact or separation as a primary objective were not excluded, as long as health-related measures were also reported. a further studies ( %) reported conclusions based upon a . α-level threshold. these papers are indicated with footnotes in their respective tables. study limitations, potential biases, and sources of error were formally discussed in manuscripts ( %), although only paper included an assessment of inter-observer reliability. some level of missing data was reported in studies ( %), with explanation for the missing data provided in of these. seven of the single-herd studies ( %) did not contain complete information on the type and duration of cow-calf contact and feeding regimens for control groups. interestingly, ( %) of papers failed to provide information on country or region in which the study was conducted (although the authors' affiliations and the location of laboratories used for sample analysis most often suggested that these studies were conducted in the united states). an additional studies ( %) did not include any information pertaining to cattle breed (of these, were multi-herd trials). for manuscripts addressing johne's disease, cryptosporidiosis, or mastitis, ( %) adequately discussed the diagnostic test used, although only ( %) cited the sensitivity or specificity (or both) of the implemented diagnostic. of the articles in the immunity section that made reference to fpt, only ( %) provided a full definition for the condition and ( %) offered rationales or citations supporting the diagnostic cut points implemented. in the single-herd studies, there was a high variability in what constituted control groups in terms of the level of maternal contact. for example, in of these studies, control calves were given less than h of contact with the dam; in a further studies, control animals were provided with a minimum of d of unrestricted dam-calf contact. the feeding regimens were also different across studies. these discrepancies are likely to have affected conclusions, and thus it is difficult to make direct comparisons between articles. even in studies seemingly describing the same treatment (e.g., "restricted suckling"), the implementation of these treatments was variable. for example, in mejia et al. ( ) calves in the "restricted suckling" group were permitted suckling for min after milking (with one quarter left unmilked), twice daily for mo. in contrast, in fulkerson et al. ( ) , calves were reunited with the dam once per day (for an unspecified amount of time) for approximately mo. further, in several studies (e.g., fallon and harte, ; nocek et al., ) , dam-calf suckling is referenced, but the duration of contact between cows and calves in the control group is not described. several of the older studies had low sample sizes and are more accurately interpreted as case reports. at one extreme, rigby et al. ( ) presented conclusions regarding mastitis based upon a single cow nursing her calf; however, most of the other studies with small sample sizes were able to enroll at least animals. due to the variability in study type, methodology, use of controls, and the amount of information provided, it was not feasible to conduct a meta-analysis. we provided risk ratios, prevalence ratios, and odds ratios where possible, but the variation in study methodology prevented us from generating a meaningful estimate of the overall magnitude of the effects considered. instead, only the direction of association was highlighted, and our discussion is mainly based upon on the number of positive and negative associations. a weakness of this approach is that it weighs equally all studies, regardless of sample size, effect size, and any weaknesses in design and interpretation. it is important to consider that no temporal relationship between management factors (e.g., cow-calf separation) and outcomes (e.g., disease) has been examined in the included cross-sectional studies. thus, management decisions could plausibly have been implemented in response to the disease in question, rather than representing a risk factor for disease. effect on milk production and calf performance of milking crossbred european/zebu cattle in the absence or presence of the calf, and of rearing their calves artificially three step calf rearing plan risk factors for mycobacterium avium ssp. paratuberculosis in fars province (southern iran) dairy herds atlantic johne's disease initiative, risk assessment workbook relationship between frequent milking or suckling in early lactation and milk production of high producing dairy cows prevalence of failure of passive transfer of immunity in newborn heifer calves and associated management practices on us dairy operations comparative risk assessment for new cow-level mycobacterium avium ssp. paratuberculosis infections between dairy production types: organic, conventional, and conventional-grazing systems factor analysis of a johne's disease risk assessment questionnaire with evaluation of factor scores and a subset of original questions as predictors of observed clinical paratuberculosis comparison of three methods of feeding colostrum to dairy calves the influence of calf rearing methods and milking methods on performance traits of crossbred dairy cattle in thailand . calf performance the influence of calf rearing methods and milking methods on performance traits of crossbred dairy cattle in thailand - . milk yield and udder health rearing holstein friesian and brown swiss calves on nurse cows relationships between the presence of johne's disease and farm and management factors in dairy cattle in england prevalence, incidence and geographical distribution of johne's disease in cattle in england and the welsh borders an overview of calf diarrhea-infectious etiology, diagnosis, and intervention epidemiological model of paratuberculosis in dairy cattle herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in wisconsin risk associations to milk elisa result for paratuberculosis in dairy cows in northern portugal using a multilevel regression model path model of individual-calf risk factors for calfhood morbidity and mortality in new york holstein herds eimeriosis in cattle: current understanding invited review: systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows effects of twice-daily nursing on milk ejection and milk yield during nursing and milking in dairy cows cryptosporidium parvum infection and associated risk factors in dairy calves in western france management practices associated with mycobacterium avium ssp. paratuberculosis infection and the effects of the infection on dairy herds management of the calving pen is a crucial factor for paratuberculosis control in large dairy herds risk factors associated with cryptosporidium parvum infection in cattle the period between birth and first suckling in dairy calves methods of feeding milk to young calves natural transmission of cryptosporidium parvum between dams and calves on a dairy farm the effects of early separation on the dairy cow and calf respiratory pathogens in quebec dairy calves and their relationship with clinical status, lung consolidation, and average daily gain health and performance of holstein calves that suckled or were hand-fed colostrum and were fed one of three physical forms of starter effect of restricted suckling on milk yield, milk composition and udder health in cows and behaviour and weight gain in calves, in dual-purpose cattle in the tropics effect of suckling ('restricted suckling') on dairy cows' udder health and milk let-down and their calves' weight gain, feed intake and behaviour improvement in milk production of first calf heifers by multiple suckling prevalence and risk factors for shedding of cryptosporidium spp. oocysts in dairy calves of buenos aires province colostrum management for dairy calves effect of residual calf suckling on clinical and sub-clinical infections of mastitis in dual-purpose cows: epidemiological measurements epidemiologic study of on-farm management practices associated with prevalence of mycobacterium paratuberculosis infections in dairy cattle respiratory infections in norwegian dairy calves calf mortality in norwegian dairy herds calf mortality invited review: incidence, risk factors, and effects of clinical mastitis recurrence in dairy cows management factors associated with calf mortality in south carolina dairy herds is rearing calves with the dam a feasible option for dairy farms? current and future research managementrelated risk factors for m. paratuberculosis infection in michigan, usa, dairy herds practical implications of suckling systems for dairy calves in organic production systems-a review effect of weaning on performance and behaviour of calves and their dams in dairy cows-a review invited review: effects of milk ration on solid feed intake, weaning, and performance in dairy heifers prevalence and risk factors for shedding of thermophilic campylobacter in calves with and without diarrhea in austrian dairy herds comparison of the behaviour of newborn calves housed with the dam and in the calf-house long-term effect of colostrum feeding methods on behaviour in female dairy calves cow-calf relations. : the effect of vs. days suckling on behaviour, milk production and udder health of cows in different stabling age-related and housing-dependence of cryptosporidium infection of calves from dairy and beef herds in south bohemia behavioural effects of separating the dairy calf immediately or days post-partum influence of partial suckling of crossbred dairy cows on milk offtake and calf growth in the ethiopian highlands epidemiology and economics of paratuberculosis herd-level risk factors for infectious diseases in swedish dairy calves aged - days prevalence of and risk factors for shedding of cryptosporidium parvum in holstein friesian dairy calves in central mexico within-herd contact structure and transmission of mycobacterium avium subspecies paratuberculosis in a persistently infected dairy cattle herd restricted suckling of tropical dairy cows by their own calf or other cows' calves relationship between selected perinatal paratuberculosis management interventions and passive transfer of immunity in dairy calves disease management of dairy calves and heifers prevalence and determinants of mastitis and milk-borne zoonoses in smallholder dairy farming sector in kibaha and morogoro districts in eastern tanzania invited review: a systematic review of the effects of prolonged cow-calf contact on behavior, welfare, and productivity effects of restricted suckling versus artificial rearing on milk production, calf performance and reproductive efficiency of dual purpose mpwapwa cattle in a semi-arid climate effect of restricted suckling on milk yield, composition and flow, udder health, and postpartum anoestrus in grazing holstein cows maternal influence on defecation and urination in the newborn calf risk factors associated with cryptosporidium parvum infection in dairy cattle in southeastern new york state a review on major bacterial causes of calf diarrhea and its diagnostic method production diseases of the transition cow a herd health approach to dairy cow nutrition and production diseases of the transition cow herd management practices associated with paratuberculosis seroprevalence in dutch dairy herds effect of management practices on paratuberculosis prevalence in danish dairy herds fertility and obstetrics in cattle, edition . blackwell science influence of neonatal colostrum administration, immunoglobulin, and continued feeding of colostrum on calf gain, health, and serum protein a questionnaire-based cross-sectional study of clinical johne's disease on dairy farms in new zealand fecal shedding of coliform bacteria during the periparturient period in dairy cows management factors related to calf morbidity and mortality rates association between risk-assessment scores and individualcow johne's disease-test status over time on seven michigan, usa dairy herds effects of housing and colostrum feeding on the prevalence of selected infectious organisms in feces of jersey calves effects of housing and colostrum feeding on serum immunoglobulins, growth, and fecal scores of jersey calves serum immunoglobulin concentrations and health of dairy calves in two management systems from birth to weeks of age effect of compliance with recommended calf-rearing practices on control of bovine johne's disease rearing dairy calves by restricted suckling. vii. effect on mastitis development caused by staphylococcus aureus. can influence of artificial vs. mother-bonded rearing on sucking behaviour, health and weight gain in calves effects of restricted suckling versus artificial rearing on performance and fertility of bos taurus and bos indicus cows and calves in tanzania effects of restricted suckling versus artificial rearing on performance and fertility of crossbreed f (holstein friesian x local) cows and calves in vietnam issues of reporting in observational studies in veterinary medicine production effects related to mastitis and mastitis economics in dairy cattle herds absorption of immune lactoglobulin by newborn dairy calves. attempts to produce consistent immune lactoglobulin absorptions in newborn dairy calves using standardised methods of colostrum feeding and management response of dairy cows and calves to early separation: effect of calf age and visual and auditory contact after separation preventing bacterial contamination and proliferation during the harvest, storage, and feeding of fresh bovine colostrum colostral immunoglobulin transfer in calves. iv. effect of suckling morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases herd characteristics and management practices associated with seroprevalence of mycobacterium avium ssp. paratuberculosis infection in dairy herds influence of suckling by friesian cows on milk production and anoestrus risk factors associated with mycobacterium avium ssp. paratuberculosis seropositivity in canadian dairy cows and herds passive immunity in ontario dairy calves and investigation of its association with calf management practices association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario calf-level risk factors for neonatal diarrhea and shedding of cryptosporidium parvum in ontario dairy calves part iv: reference of dairy cattle health and management practices in the united states uniform program standards for the voluntary bovine johne's disease control program dairy cattle management practices in the united states national health monitoring system. dairy : health and management practices on u.s. dairy operations the importance of udder and teat conformation for teat seeking by the newborn calf effect of production system, alternative treatments and calf rearing system on udder health in organic dairy cows practical implications of increasing 'natural living' through suckling systems in organic dairy calf rearing milk secretion in machine-milked and suckled cows dairy calf management, morbidity and mortality in ontario holstein herds. iii. association of management with morbidity effects of early separation on the dairy cow and calf: . separation at h, day and days after birth herd-level risk factors for infection with mycobacterium paratuberculosis in us dairies and association between familiarity of the herd manager with the disease or prior diagnosis of the disease in that herd and use of preventive measures factors associated with mortality to days of life in dairy heifers in the united states robis: a new tool to assess risk of bias in systematic reviews was developed we are grateful to dairy australia (melbourne, australia) for soliciting this review and for providing support to rebecca meagher and annabelle beaver. we also thank angela yu and auguste de pennart (both students at the university of british columbia) for their assistance. key: cord- - acp m authors: lin, chih-yin; yao, chun-an title: potential role of nrf activators with dual antiviral and anti-inflammatory properties in the management of viral pneumonia date: - - journal: infect drug resist doi: . /idr.s sha: doc_id: cord_uid: acp m the outbreak of coronavirus disease (covid- ) pandemic has already caused a huge burden to the global healthcare system, with the death toll reached tens of thousands. although some antiviral agents were identified and used to inhibit viral replication, the management of cytokine storm is also a critical issue. in this article, we reviewed the literature on drug candidates for severe acute respiratory syndrome (sars-cov- ) and provided a brief overview of a class of drugs that exert antiviral and anti-inflammatory effects. these molecules mitigated inflammatory cytokine cascades induced by viral infections via nrf activating capacity and might have additional anti-fibrotic and anti-remodeling properties. besides, their effects on the regulation of scavenger receptors expression by macrophages may offer some benefits to the pulmonary antibacterial defense system after viral infection. the potential roles of these agents assessed on the basis of the pathophysiology of viral pneumonia and acute respiratory distress syndrome were also discussed. further research is needed to ascertain whether nrf activators are useful in the management of viral pneumonia. the coronavirus disease (covid- ) pandemic has already caused a tremendous burden on the healthcare system globally and poses a threat to all human beings. scientists and doctors have been desperately trying to find possible treatments since the outbreak of the disease. some potential treatment options, including nucleoside analogs, protease inhibitors, and interferon, were proposed and tested. besides the existing antiviral drug options, naturally occurring phytochemicals might also have a role in combating viral pneumonia. in an in vitro study of severe acute respiratory syndrome coronavirus (sars-cov- ), several compounds with antiviral activity, including diterpenes, sesquiterpenes, lupane-type triterpenes, lignoids, and curcumin were identified. interestingly, curcumin and some triterpenoids were proven to be nuclear factor erythroid -related factor (nrf ) activators, and the potential role of nrf activators in the management of respiratory viral infection has drawn some scientists' attention. , the nrf -antioxidant response element (are) pathway is known to maintain the redox balance in cells and reduces inflammation. these groups of plantderived chemicals and their analogs might provide a class of drugs that possesses both antiviral and anti-inflammatory properties and might help tackle the pathophysiological changes in viral pneumonia and acute respiratory distress syndrome (ards). among the nrf activators, curcumin is the most extensively studied and widely used product with established safety profile and biological effects. [ ] [ ] [ ] curcumin has been proven to have broad-spectrum antiviral properties against many rna viruses, including influenza a virus, respiratory syncytial virus (rsv), and norovirus. animal experiments, mostly using influenza a virus, showed decreased pulmonary viral titers, decreased production of cytokines (such as tnfα, il- β, and il- ) and matrix metalloproteinase- and (mmp- , ), decreased infiltration of inflammatory cells, decreased pulmonary histopathological injury score, and increased animal survival. [ ] [ ] [ ] [ ] interestingly, although curcumin did not affect the clearance of reovirus in a mouse model, it reduced collagen deposition in lung tissue and decreased the expression of myofibroblast phenotype. the observed anti-fibrotic and anti-remodeling effects might offer additional benefits if they translate into clinical practice because ct images of covid- pneumonia patients showed ground glass opacities with partial consolidation and were absorbed with formation of fibrotic stripes after improvement. whether the observed pulmonary fibrosis is a temporary phenomenon or may affect the functional recovery of patients remain to be studied. in another study, it was demonstrated that curcumin reduced the lipopolysaccharide (lps)-induced mucin ac secretion in a mouse model. moreover, it is also known that nrf , a key mediator that combats oxidative stress, can be upregulated by curcumin. nrf /are pathway targets more than genes, including genes which regulate oxidative stress (ho- , gclm, and gclc), and reduces inflammation by decreasing nf-κb and tgf-β. many studies have demonstrated the protective effect of nrf activators in hyperoxia-or lpsinduced ards models. besides their function in the lung tissue, nrf activators also have a renoprotective effect. whether nrf activating drugs are useful in acute kidney injury induced by ards remains to be further studied. although curcumin has been studied in various inflammatory and proliferative diseases, its effects on human respiratory tract infection have rarely been tested. in a clinical study, the effects of lactoferrin and curcumin in healthy children with recurrent respiratory infection were examined, and the results showed reduced infection and skewing of cd + t lymphocyte maturation. direct clinical evidence of curcumin in human viral pneumonia is still limited. besides, one of the major drawbacks of curcumin is its poor absorption and rapid metabolism. several formulation and conjugation strategies were used to increase the bioavailability of oral curcumin. different routes of administration, such as inhalation and intravenous routes, were also evaluated. , however, turmeric infusion by a naturopathic practitioner even caused mortality and was considered to be related to the presence of peg castor oil. therefore, pharmaceutical-grade preparation of intravenous curcumin should be used in medical institutions by qualified personnel. another group of molecules, namely terpenes, was also studied for its nrf activating effect. several semisynthetic and synthetic triterpenoids, including bardoxolone methyl and omaveloxolone, are currently undergoing clinical trials for kidney diseases. some evidence indicates that bardoxolone methyl suppresses rna viruses such as dengue virus and zika virus. although some experiments showed its positive effects in lps-induced acute lung injury mouse model, its effect on respiratory tract infection is still largely unknown. another nrf activator named sulforaphane was also studied for its antiviral capacity, and could suppress respiratory viruses, such as rsv and influenza virus. in a human study using live attenuated influenza virus on human subjects, sulforaphane was found to increase granzyme b production in natural killer (nk) cells after inoculation. lower granzyme b levels were related to the risk for influenza in institutionalized older adults, and serum granzyme b levels correlated with protection against influenza in older adults following vaccination. , besides, a study also showed that sulforaphane increased the expression of macrophage receptor with collagenous structure (marco) in alveolar macrophages, and thus, provided survival benefits in an animal model of postinfluenza bacterial pneumonia. however, although in vitro studies showed improved phagocytosis of bacteria by alveolar macrophages from patients with copd, an in vivo study of sulforaphane failed to induce nrf target gene expression in alveolar macrophages from the same disease population. therefore, the effects of sulforaphane observed in healthy subjects may not be accurately extrapolated to patients with copd. besides, another advantage of sulforaphane over curcumin is that it has good oral bioavailability. the overall benefits of curcumin and sulforaphane were demonstrated by their ability to increase survival rates in influenza and sepsis animal models. , , , [ ] [ ] [ ] however, in many experiments animals were pretreated with drugs or drugs were used on the same day of virus inoculation; the results may be different in clinical settings because patients may be at a more advanced disease stage. whether these agents are useful for chemoprevention or treatment remains to be elucidated. in vivo , in vivo: clp murine model of sepsis , in vivo: postinfluenza bacterial pneumonia mouse model according to a recent publication, azithromycin and hydroxychloroquine combination treatment was associated with sars-cov- viral load reduction and decreased the duration of virus carriage in a small group of patients. the efficacy of macrolides in respiratory viral infections and inflammatory diseases has been extensively researched, , and their anti-inflammatory profiles are similar to the aforementioned nrf activators. an in vitro study using human small airway epithelial cells revealed that clarithromycin decreased h o induced inflammation through upregulation of nrf expression. another study also found that azithromycin alleviated cigarette smoke extract induced inflammation in human airway epithelial cells by activating nrf . whether the observed effects of macrolides in viral infections are related to nrf activating property remains to be clarified. another group of antibiotics, including tetracycline, minocycline, and doxycycline, is a well-known class of antibiotics with anti-inflammatory features and was proposed as a potential repurposing candidate for the management of covid- diseases. although a study revealed that minocycline upregulated nrf in retrovirus infected astrocytes, another study showed that doxycycline inhibits malondialdehyde-acetaldehyde-induced activation of nrf in hek nrf /are cells. therefore, the impact of different drugs of the tetracycline group on the nrf pathway remains to be further clarified. the effects of nrf activators on the pathophysiology of viral pneumonia/ards based on evidence are summarized in table . in conclusion, the activation of nrf pathway by drugs has been researched for its antiviral and anti-oxidative mechanisms and can be the foundation for further clinical development. the nrf activators and their analogs might be tested for their potential antiviral efficacy and might become drug candidates, either alone or in combination with other antiviral agents, for further clinical trials in viral pneumonia. infection and drug resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. the journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. submit your manuscript here: https://www.dovepress.com/infection-and-drug-resistance-journal therapeutic options for the novel coronavirus ( -ncov) specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus role of nrf and its activators in respiratory diseases activators and inhibitors of nrf : a review of their potential for clinical development inhibition of enveloped viruses infectivity by curcumin a review on antibacterial, antiviral, and antifungal activity of curcumin anti-infective properties of the golden spice curcumin curcumin modulates the inflammatory response and inhibits subsequent fibrosis in a mouse model of viral-induced acute respiratory distress syndrome curcumin alleviates macrophage activation and lung inflammation induced by influenza virus infection through inhibiting the nf-κb signaling pathway. influenza other respi viruses inhibition of curcumin on influenza a virus infection and influenzal pneumonia via oxidative stress, tlr / , p / jnk mapk and nf-κb pathways curcumin ameliorates severe influenza pneumonia via attenuating lung injury and regulating macrophage cytokines production clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov- curcumin inhibits lipopolysaccharide-induced mucin ac hypersecretion and airway inflammation via nuclear factor erythroid -related factor role of nrf in protection against acute kidney injury immune modulation by lactoferrin and curcumin in children with recurrent respiratory infections bioavailable curcumin formulations: a review of pharmacokinetic studies in healthy volunteers inhalation treatment of idiopathic pulmonary fibrosis with curcumin large porous microparticles pharmacokinetics of liposomal curcumin (lipocurc tm ) infusion: effect of co-medication in cancer patients and comparison with healthy individuals death associated with intravenous turmeric (curcumin) preparation small molecule grp inhibitors block dengue and zika virus replication the protective effect of cddo-me on lipopolysaccharide-induced acute lung injury in mice antiviral activity of nrf in a murine model of respiratory syncytial virus disease nrf expression modifies influenza a entry and replication in nasal epithelial cells effect of broccoli sprouts and live attenuated influenza virus on peripheral blood natural killer cells: a randomized, double-blind study granzyme b: a marker of risk for influenza in institutionalized older adults granzyme b: correlates with protection and enhanced ctl response to influenza vaccination in older adults immunomodulators targeting marco expression improve resistance to postinfluenza bacterial pneumonia opsonic phagocytosis in chronic obstructive pulmonary disease is enhanced by nrf agonists lack of effect of oral sulforaphane administration on nrf expression in copd: a randomized, double-blind, placebo controlled trial sulforaphane: its "coming of age" as a clinically relevant nutraceutical in the prevention and treatment of chronic disease curcumin attenuates sepsis-induced acute organ dysfunction by preventing inflammation and enhancing the suppressive function of tregs sulforaphane reduces hmgb -mediated septic responses and improves survival rate in septic mice curcumin protects against sepsis-induced acute lung injury in rats hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial macrolide therapy in respiratory viral infections the immunomodulatory effects of macrolides a systematic review of the underlying mechanisms long-term treatment of clarithromycin at a low concentration improves hydrogen peroxide-induced oxidant/antioxidant imbalance in human small airway epithelial cells by increasing nrf mrna expression azithromycin induces sestrin expression through nrf signaling pathway in lung epithelial cells stimulated with cigarette smoke extract ( . ) doxycycline, a widely used antibiotic in dermatology with a possible anti-inflammatory action against il - in covid - outbreak attenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice novel antioxidant properties of doxycycline protective mechanism of sulforaphane in nrf and anti-lung injury in ards rabbits clarithromycin suppresses induction of monocyte chemoattractant protein- and matrix metalloproteinase- and improves pathological changes in the lungs and heart of mice infected with influenza a virus sulforaphane exerts anti-inflammatory effects against lipopolysaccharide-induced acute lung injury in mice through the nrf /are pathway intranasal curcumin protects against lps-induced airway remodeling by modulating toll-like receptor- (tlr- ) and matrixmetalloproteinase- (mmp- ) expression via affecting map kinases in mouse model regulation of neutrophil elastase-induced muc ac expression by nuclear factor erythroid- related factor in human airway epithelial cells erythromycin attenuates muc ac synthesis and secretion in cultured human tracheal cells infected with rv modulation of cd scavenger receptor expression by curcumin and vitamin e affects cellular uptake of lipids and bacteria ( . ) targeting nrf signaling improves bacterial clearance by alveolar macrophages in patients with copd and in a mouse model the preventive effect of macrolide antibiotics on postinfluenza bacterial pneumonia in copd model mice macrolide therapy is associated with reduced mortality in acute respiratory distress syndrome (ards) patients dovepress submit your manuscript | www.dovepress.com dovepress infection and drug resistance the authors report no conflicts of interest in this work. this work is not funded by research grants. key: cord- - cex gid authors: wu, guoyao title: important roles of dietary taurine, creatine, carnosine, anserine and -hydroxyproline in human nutrition and health date: - - journal: amino acids doi: . /s - - - sha: doc_id: cord_uid: cex gid taurine (a sulfur-containing β-amino acid), creatine (a metabolite of arginine, glycine and methionine), carnosine (a dipeptide; β-alanyl-l-histidine), and -hydroxyproline (an imino acid; also often referred to as an amino acid) were discovered in cattle, and the discovery of anserine (a methylated product of carnosine; β-alanyl- -methyl-l-histidine) also originated with cattle. these five nutrients are highly abundant in beef, and have important physiological roles in anti-oxidative and anti-inflammatory reactions, as well as neurological, muscular, retinal, immunological and cardiovascular function. of particular note, taurine, carnosine, anserine, and creatine are absent from plants, and hydroxyproline is negligible in many plant-source foods. consumption of g dry beef can fully meet daily physiological needs of the healthy -kg adult human for taurine and carnosine, and can also provide large amounts of creatine, anserine and -hydroxyproline to improve human nutrition and health, including metabolic, retinal, immunological, muscular, cartilage, neurological, and cardiovascular health. the present review provides the public with the much-needed knowledge of nutritionally and physiologically significant amino acids, dipeptides and creatine in animal-source foods (including beef). dietary taurine, creatine, carnosine, anserine and -hydroxyproline are beneficial for preventing and treating obesity, cardiovascular dysfunction, and ageing-related disorders, as well as inhibiting tumorigenesis, improving skin and bone health, ameliorating neurological abnormalities, and promoting well being in infants, children and adults. furthermore, these nutrients may promote the immunological defense of humans against infections by bacteria, fungi, parasites, and viruses (including coronavirus) through enhancing the metabolism and functions of monocytes, macrophages, and other cells of the immune system. red meat (including beef) is a functional food for optimizing human growth, development and health. the scientific conference entitled "frontiers in agricultural sustainability: studying the protein supply chain to improve dietary quality" hosted by new york academy of sciences highlighted growing controversies on meat consumption by humans in the u.s. (wu et al. ) . over the past decades, there have been growing concerns that consumption of red meat (e.g., beef) increases risks for obesity, type ii diabetes mellitus, cardiovascular disease, colon cancer and alzheimer's disease in humans (e.g., nelson et al. ; willett et al. ) . thus, beef consumption per capita in the u.s. has steadily declined from lb in to only lb in (usda ) . this has arisen, in part, from a lack of understanding of red meat as a nutritionally important source of functional amino acids (e.g., taurine and hydroxyproline), dipeptides (e.g., carnosine and anserine), and creatine (a metabolite of amino acids) with enormous physiological significance. for example, taurine is a nutritionally essential amino acid for children (particularly preterm infants) and a conditionally essential amino acid for adults (schaffer et al. ). in addition, dietary supplementation with -hydroxyproline improves anti-oxidative function and prevents colitis in the intestine (ji et al. ) . furthermore, carnosine and anserine are potent antioxidants (hipkiss and gaunitz ) , whereas creatine is both an antioxidant and a major component of energy metabolism in excitable tissues (brain and skeletal muscle) (wyss and kaddurah-daouk ) . of note, taurine, carnosine, anserine, and creatine are particularly abundant in beef skeletal muscle but are absent from plants (wu ) . also, -hydroxyproline is abundant in meat but negligible in many plant-source foods . for comparison, intramuscular concentrations of balenine (β-alanyl-l- -methylhistidine, an antioxidant and a buffering substance) are high in whale (~ mmol/kg wet weight), relatively low in swine (~ . to mmol/kg wet weight or - % of carnosine content), and very low in cattle, chickens and sheep (~ . to . mmol/kg wet weight; boldyrev et al. ; carnegie et al. ; harris and milne ) . this dipeptide is barely detectable in human and rat muscles (boldyrev et al. ) and, therefore, is not a focus of the present article. growing evidence shows that taurine, carnosine, anserine, creatine and -hydroxyproline play crucial roles in protecting mammalian cells from oxidative stress and injury (abplanalp et al. ; avgerinos et al. ; seidel et al. ; wu et al. ) . these results indicate that red meat provides not only high-quality protein for the growth of children but also functional amino acids, dipeptides and creatine for optimal human nutrition and health. however, the public is generally not aware of these beneficial nutrients in meat, including beef (e.g., kausar et al. ; uzhova and peñalvo ) . to provide accurate and complete information on animal-source foods, it is imperative to review pertinent articles regarding the benefits of dietary taurine, carnosine, anserine, creatine and -hydroxyproline on metabolic, retinal, muscle, immunological, and cardiovascular health as well as healthy ageing in humans and animal models. dietary sources of taurine, creatine, carnosine, anserine, and -hydroxyproline for humans taurine, creatine, carnosine, anserine, are chemically stable and soluble in water within a physiological range of ph values. they are abundantly present in animal-source foods (such as beef). however, their reported values are highly inconsistent in the literature and may differ by - fold . for example, the amounts of carnosine in g wet beef meat have been reported to be mg (szterk and roszko ) or g (clifford ) , and the amounts of anserine in g wet beef meat to be . mg (thornton et al. ) or mg (mateescu et al. ) . such large variations in nutrient composition may result, in part, from differences in either beef breeds or analytical techniques. to provide a much-needed database, we analyzed taurine, carnosine, anserine, creatine, and -hydroxyproline in cuts from three subprimals (chuck, round, and loin) of beef carcasses selected at three commercial packing plants in the united states (table ) . these nutrients are abundant in all beef cuts . in contrast, taurine, carnosine, anserine, and creatine are absent from all plants [e.g., corn grains, peanuts, pistachio nuts, potatoes, soybeans, sweet potatoes, wheat flour, and white rice ]. thus, vegetarians are at great risks for the deficiencies of taurine, carnosine, anserine, and creatine, particularly if they table the content of crude protein, amino acids, creatine, carnosine, anserine, and -hydroxyproline in beef and plant-source foods adapted from wu et al. ( ) , wu et al. ( ) , and hou et al. ( ) cp crude protein ( . × n%), oh-pro -hydroxyproline are active in physical exercise (rogerson ). in addition, most of plant-source foods contained little -hydroxyproline and little β-alanine (a precursor of carnosine in humans) (table ) . these data are useful for nutritionists and medical professionals to make quantitative recommendation for consumption of beef and plant-source foods by humans. biochemistry, physiology, and nutrition of taurine, creatine, carnosine, anserine, and -hydroxyproline in humans taurine ( -aminoethanesulfonic acid, a slightly acidic substance) is a sulfur-containing β-amino acid. it was originally isolated from the bile salts (taurocholate, also known as cholyltaurine) of the ox by austrian scientists f. tiedermann and l. gmelin in . taurine is widely distributed in the animal kingdom and to be present in relatively high concentrations in all mammalian and avian tissues, such as blood, intestine, liver, skeletal muscle, heart, brain, kidneys, and retina. a -kg person has ~ g taurine (huxtable ) . concentrations of taurine in mammalian and avian cells range from to mm, depending on species and cell type (wright et al. ). human skeletal muscle, heart, retina, and placenta contain - , - , - , and - mm taurine, respectively. because of its large mass [e.g., accounting for % of body weight (bw) in healthy, nonobese adults], skeletal muscle is the major site (about %) of taurine storage in the -kg adult. taurine is abundant in the milk of mammals (e.g., . mm in human, . mm in mouse, and . mm in cats) as a physiologically essential nutrient for infants and children (sturman ) . dietary taurine is taken up by the enterocyte across its apical membrane via na + -and cl − -dependent transporters, taut, gat and gat (lambert and hansen ; zhou et al. ) , as well as pat (a h + -coupled, ph-dependent but na + -and cl − -independent transporter), with taut being the major transporter under physiological conditions (anderson et al. ). taurine exits the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via specific transporters (fig. ). absorbed taurine is not degraded by the intestinal mucosa and, therefore, enters the portal circulation. taurine is transported in blood as a free amino acid for uptake by extra-intestinal tissues (wu ) . increasing dietary intake of taurine enhances its concentrations in plasma and tissues, such as skeletal muscle, brain and heart (schaffer et al. ; sirdah ) . taurine is synthesized from cysteine, a product of methionine catabolism, in the liver of many mammals (wu ) . in humans, the rate of taurine synthesis from methionine and cysteine is exceedingly low in comparison with rats (sturman ) , because the activity of hepatic cysteinesulfinate decarboxylase (a key enzyme in taurine synthesis) is about three orders of magnitude lower in the young and adult men than that in rats (sturman and hayes ) . compared with livestock (e.g., cattle, pigs, and sheep) and poultry (e.g., chickens and ducks), humans also have a very low ability to synthesize taurine at any stage of life. depending on the dietary intake of protein, nutritional status, and hepatic enzyme activity, a healthy adult may synthesize - mg taurine per day (jacobsen and smith ) . under stress or diseased conditions (e.g., heat stress, infection, obesity, diabetes, and cancer), taurine synthesis in the body may be impaired due to the suboptimal function of liver and the reduced availability of the amino acid precursors. infants and children do not sufficiently synthesize taurine despite adequate provision of its precursors in diets (geggel et al. ) . in addition, individuals who consume only plant-source foods but no animal products are at increased risk for taurine deficiency, because the precursors of taurine (methionine and cysteine) are present at low concentrations in most proteins of plant origin (e.g., corn, potato, rice, wheat, and vegetables) ). in animal cells, taurine undergoes limited catabolism by taurine:pyruvate transaminase or taurine:α-ketoglutarate transaminase, which catalyzes the transamination of taurine with pyruvate or α-ketoglutarate to form -sulfoacetaldehyde (also known as -oxoethanesulfonate, -hydroxyethanesulfonate, or isethionate) and l-alanine or l-glutamate (read and welty ) . these reactions also occur in aerobic and anaerobic bacteria (cook and denger ) . in addition, taurine dehydrogenase converts taurine into ammonia plus -sulfoacetaldehyde (an oxidation reaction) in aerobic bacteria in the presence of cytochrome c as the physiological electron acceptor (brüggemann et al. ) . furthermore, taurine is oxygenated by α-ketoglutarate-dependent taurine dioxygenase to generate sulfite and -aminoacetaldehyde in microorganisms (including e. coli eichhorn et al. ) . the initial fate of the degradation of the taurine's sulfur atom is sulfite in strictly anaerobic, facultatively anaerobic, or strictly aerobic bacteria (brüggemann et al. ). thus, taurine catabolism is initiated by transamination, oxidation, or oxygenation in a species and cell-specific manner. in humans, taurine is covalently conjugated with bile acids in the liver to form bile salts, which are then exported by the atp-dependent bile salt export pump out of the hepatocyte and stored in the gallbladder (hofmann ) . during feeding, bile salts are secreted from the gallbladder via the common bile duct into the duodenum, where they facilitate the digestion and absorption of dietary lipids. the bile salts are not absorbed by the proximal small intestine (duodenum, jejunum and proximal ileum) due to the lack of their apical transporters, and are resistant to deamidation by pancreatic and mucosal enzymes (including peptidases). instead, after the absorption of dietary lipids, bile salts enter the distal ileum, where a small fraction of them are hydrolyzed (and thus deconjugated) by microbial bile salt hydrolases to form bile acids and taurine (foley et al. ) . taurine, the remaining large proportion of bile salts, and bile acids are efficiently absorbed into the enterocyte of the distal ileum by specific transporters (figs. , ) . about % of the liver-derived bile salts and bile acids are absorbed primarily by the distal ileum into the portal circulation, and then taken up by hepatocytes of the liver (fig. ). this is known as the enterohepatic circulation for the ileal reabsorption of bile salts and bile acids. only ~ % of the liver-derived bile salts and bile acids enter the large intestine during each enterohepatic circulation. thus, it generally takes a prolonged period of time (e.g., months) to induce a taurine deficiency in humans and animals. it is now recognized that taurine plays major roles in human physiology and nutrition, including serving as: ( ) a nutrient to conjugate bile acids to form bile salts in the liver that facilitate intestinal absorption of dietary lipids (including lipid-soluble vitamins) and eliminate cholesterol in bile via the fecal route; ( ) a major antioxidant, antiinflammatory, and anti-apoptotic factor in the body; ( ) a physiological stabilizer of cell membranes; ( ) a regulator of modulation of ca + signaling, fluid homeostasis in cells, and retinal photoreceptor activity; ( ) a contributor fig. absorption of taurine, creatine, carnosine, anserine, and -hydroxyproline by the human small intestine and the transport of the nutrients in blood. dietary collagen is hydrolyzed by proteases, peptidases and prolidase to free amino acids as well as -hydroxyproline and its peptides. dietary taurine, creatine, carnosine, anserine, and -hydroxyproline are taken up by the enterocyte across its apical membrane via specific transports. inside the cell, taurine, creatinine and anserine are not degraded, some of the -hydroxyprolinecontaining peptides are hydrolyzed to -hydroxyproline and its peptides, some -hydroxyproline is oxidized to glycine, and carnosine undergoes limited catabolism. taurine, creatine, carnosine, anserine, and -hydroxyproline, as well as the products of carnosine hydrolysis (β-alanine and histidine) exit the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via specific transporters (wu ) . the absorbed nutrients are transported in blood in the free forms for uptake by extra-intestinal tissues via specific transporters. β-ala β-alanine, cat cationic amino acid transporter, cn carnosinase- (serum carnosinase), cn carnosinase- (tissue carnosinase), creat creatine transporter- , creat creatine transporter- , gat γ-aminobutyrate transporter, hypd -hydroxyproline-containing dipeptides, hypt -hydroxyproline-containing tripeptides, oh-pro -hydroxyproline, pat proton-(h + -coupled) and ph-dependent but na + -and cl − -independent transporter for taurine (low-affinity, high-capacity transporter), pept peptide transporter- , pept peptide transporter , pht / peptide/histidine transporters and , taut taurine transporters. note that the distribution of pht / in tissues is species-specific in that human skeletal muscle expresses pht but no pht , whereas mouse skeletal muscle expresses both pht and pht to osmoregulation; ( ) a key component of nerve and muscle conduction networks; ( ) a stimulator of neurological development; and ( ) an inhibitory neurotransmitter in the central nervous system (cns) (schaffer and kim ) . thus, taurine exerts beneficial effects on cardiovascular (including protection against ischemia-reperfusion injury, maintaining cell membrane structure, and reducing blood pressure), digestive, endocrine, immune, muscular, neurological, reproductive, and visual systems (ito et al. ; shimada et al. ; seidel et al. ) . for example, taurine protects cells and tissues (brain) from the toxicity of reactive oxygen species (ros), excess metals [e.g., nickel (xu et al. ) and manganese (ahmadi et al. ) ], and ammonia (jamshidzadeh et al. a ) by maintaining the integrity of plasma and organelle (especially mitochondrion) membranes of the cell. conversely, a deficiency of dietary taurine results in retinal degeneration in cats (hayes et al. ) and children (geggel et al. ) that can be corrected with taurine supplementation. furthermore, through the production of n-chlorotaurine by activated human granulocytes and monocytes, taurine contributes to the killing of pathogenic bacteria, fungi, parasites, and viruses (gottardi and nagl ) . since , taurine has been used for years as a dietary supplement to improve health in humans and animal models of metabolic syndrome (el idrissi ; hayes et al. ; ra et al. ) . for example, taurine supplementation can prevent diabetic rats from developing cardiomyopathy by inhibiting the expression of the angiotensin ii type- receptor (li et al. ) , modulating mitochondrial oxidative metabolism (militante et al. ) and electron transport activity (jong et al. ) , and reducing oxidative stress (schaffer et al. ). in addition, oral administration of fig. the transport of bile salts from the liver to the duodenum and the return of bile salt from the distal ileum to the liver via the enteralhepatic circulation in humans. conjugated bile acids are exported by the atp-dependent bile salt export pump out of the hepatocyte through its canalicular (apical) membrane into the canaliculus. the bile salts subsequently enter bile ducts, the common hepatic duct, and the gallbladder. during digestion, the bile salts are secreted from the gallbladder to the common bile duct and then the duodenum. in the distal ileum, a fraction of bile salts is hydrolyzed by microbial bile salt hydrolases to form bile acids and taurine or glycine. taurine, glycine and bile salts are efficiently taken up by the enterocytes of the distal ileum via specific transporters. the substances are transported in the blood for uptake by the hepatocyte via its sinusoidal basolateral membrane. during each enteral-hepatic cycle, about % of the liver-derived bile salts are reabsorbed to the liver. asbt apical sodium-dependent bile salt/acid transporter (in ileal enterocytes), ba bile acids (unconjugated), bsep bile salt export pump, cba conjugated bile acids, gly glycine, glyt glycine transporters, m multidrug resistance protein- , mbsl microbial bile salt hydrolases, ntcp na + -taurocholate cotransporting polypeptide, oatp organic anion transporting polypeptide family, ostα/β organic solute transporter subunit α/β, pd passive diffusion, tau taurine, taut taurine transporters taurine can ameliorate structural abnormalities of the pancreas (santos-silva et al. ) , while improving insulin production by pancreatic β-cells (nakatsuru et al. ) and whole-body insulin sensitivity in subjects with hyperglycemia (sarkar et al. ) . this role of taurine in diabetic patients are highly significant, because they have a % lower concentration of taurine in plasma than normal subjects (sak et al. ) and that the number of type- diabetic patients is increasing globally (willet et al. ) . taurine supplementation may also be needed for cancer patients maintained on total parenteral nutrition, because the concentration of taurine in their plasma is % lower than those in healthy subjects (gray et al. ) . of particular note, ingestion of . - g taurine per day for various days improved ( ) metabolic profiles in blood (including reductions in total cholesterol and low-density-lipoprotein cholesterol), and ( ) cardiovascular functions in healthy subjects, as well as in patients with overweight, diabetes, hypertension, or congestive heart failure (militante and lombardini ; xu et al. ). in addition, oral administration of g taurine/day for weeks resulted in clinically significant reductions in the frequency, duration, and intensity of muscle cramps in patients with chronic liver disease (vidot et al. ) . furthermore, long-term oral administration of taurine ( or g per day) for weeks can effectively reduce the recurrence of stroke-like episodes in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (melas), a rare genetic disorder caused by point mutations in the mitochondrial dna (ohsawa et al. ) . these beneficial effects of oral taurine are summarized in table . waldron et al. ( ) conducted a meta-analysis to evaluate the effects of oral ingestion of taurine at various doses on endurance performance in adult humans who consumed - g taurine/day in single doses for up to weeks. the authors found that the ingestion of taurine improved overall endurance performance in the subjects. this conclusion is consistent with the recent findings that taurine is a potential ergogenic aid for preventing muscle damage, attenuating muscle protein catabolism, decreasing oxidative stress, and improving performance in subjects with endurance exercise (de carvalho et al. ; page et al. ; paulucio et al. ; waldron et al. ). creatine (n-[aminoiminomethyl]-n-methyl glycine), "kreas" in greek meaning meat, was discovered by the french chemist michel e. chevreul in as a component of skeletal muscle in cattle. this neutral, water-soluble substance is abundant in skeletal muscle, heart, brain, and pancreas. a -kg adult has about g of total creatine (creatine phosphate plus free creatine), with about % of it being in skeletal muscle (casey and greenhaff ) . total creatine is about % more abundant in white (fast-twitch fibers, type ii) muscle than in red (slow-twitch fibers, type i) muscle (murphy et al. ) . in skeletal muscle and brain, creatine stores energy (primarily atp) as creatine phosphate through the action of creatine kinase. in a resting state, about two-third and one-third of total creatine exist as creatine phosphate and creatine, respectively (mcgilvery and murray ) . the irreversible loss of creatine as creatinine is . % of the whole-body creatine (brosnan and brosnan ) . this means that each day, . % of creatine is continuously recycled to store atp energy via creatine kinase. in human skeletal muscle, the concentration of creatine phosphate is about three to four times that of atp (gray et al. ; mcgilvery and murray ) . the energy of the γ-phosphate bond ( . kj/mol or . kcal/mol) in one mole of atp is transferred to one mole of creatine for storage in vivo (wu ) . in the small intestine of mammals including humans, the apical membrane of its enterocytes absorb dietary (luminal) creatine via na + /cl − -coupled (i.e., sodium-and chloridedependent) creatine transporter- (creat ) (santacruz and jacobs ) . all the dietary-free creatine ( %) is absorbed by the small intestine of humans into the portal circulation (deldicque et al. ) . ingested creatine phosphate, which is also abundant in meat, is hydrolyzed extracellularly by alkaline phosphatases (synthesized and released by enterocytes) into creatine and phosphate before intestinal absorption (fernley ) . absorbed creatine undergoes limited phosphorylation (only % of ingested creatine) in the intestinal mucosa, and nearly % of orally ingested creatine enters the portal circulation (jäger et al. ) . creatine is transported in blood as a free substance, and is rapidly taken up by extra-intestinal tissues and cells (e.g., liver, skeletal muscle, brain, kidneys, testes, and other tissues and cell types) via creat , and by testes and brain also via creat (bala et al. ) . creat protein shares % homology with creat . immunohistochemical analysis has shown that creat is mainly associated with the sarcolemmal membrane in all types of skeletal muscle (christie ; murphy et al. ) . creat is widespread in animal tissues, whereas creat is primarily present within the testes (snow and murphy ). creat is essential for normal brain and muscular function as mutations in the gene (slc a ) result in x-linked mental retardation, severe speech and language delay, epilepsy, autistic behavior, and muscular abnormalities (e.g., hypotonia) (bala et al. ; salomons et al. ) . increasing dietary intake of creatine adam et al. ( ) enhances its concentrations in plasma and tissues, such as skeletal muscle, brain and heart (derave et al. ; wyss and kaddurah-daouk ) . the absorption of creatine by the small intestine and its transport in the blood are illustrated in fig. . creatine is a metabolite of three amino acids (arginine, glycine and methionine) via the cooperation of multiple organs, primarily including the liver, pancreas and kidneys (wu ) . beef is an abundant source of arginine, glycine and methionine. in contrast, all plant-source foods contain low levels of glycine and methionine, and most of plant-source foods (except for soybean, peanuts and other nuts) also have low levels of arginine ). the pathway of creatine synthesis is initiated by arginine:glycine amidinotransferase, which transfers the guanidino group from arginine to glycine to form guanidinoacetate and ornithine. arginine:glycine amidinotransferase is expressed primarily in the renal tubules, pancreas, and to a much lesser extent in the liver and other organs. thus, the kidneys are the major site of guanidinoacetate formation in the body. the guanidinoacetate released by the kidneys is methylated by guanidinoacetate n-methyltransferase, which is located predominantly in the liver, pancreas, and, to a much lesser extent, in the kidneys to produce creatine (wu and morris ) . creatine synthesis is regulated primarily through: ( ) changes in expression of renal arginine: glycine amidinotransferase in both rats and humans; and ( ) the availability of substrates. dietary intake of creatine and circulating levels of growth hormone are major factors affecting de novo synthesis of creatine (wu and morris ) . activities and mrna levels for arginine:glycine amidinotransferase in rat kidney are greatly reduced by hypophysectomy or by feeding a diet containing creatine. neither creatine supplementation nor growth hormone influences the hepatic activity of guanidinoacetate n-methyltransferase in animals. thus, dietary supplementation with creatine helps to spare arginine, glycine and methionine for utilization via other vital metabolic pathways, such as the syntheses of protein, nitric oxide, and glutathione (wu ). this has important nutritional and physiological significance. a -kg healthy adult synthesizes . g creatine per day from . g arginine, . g glycine, and . g methionine (wu and morris ) , which represent %, % and %, respectively, of their daily dietary intakes. this amount of creatine is necessary to replace its daily irreversible loss ( . g/day) as creatinine from the subject through the excretion of urine. a greater loss of creatine from the body occurs in response to enhanced muscular activity (kreider et al. ; rogerson ) , and this amount of creatine should be replenished through enhanced endogenous synthesis there were no side effects for the ingestion of taurine, creatine, carnosine, anserine and hydroxyproline at the indicated dosages on all the studies chf congestive heart failure, hpt and dietary supplementation. for example, cycle ergometer exercise (approximately % of maximum o consumption for min) increases the loss of creatine (as indicated by urinary creatinine excretion) by % above pre-and postexercise values (calles-escandon et al. ) . there are new lines of evidence that endogenous synthesis of creatine is not sufficient for humans under many physiological (e.g., exercise, pregnancy and lactation) and pathological (e.g., tissue injury, ischemia, and diabetes) conditions. first, dietary creatine supplementation aids in increasing the power, strength, and mass of the skeletal muscle in athletes and bodybuilders, especially those who consume little meat (smith et al. ) . second, creatine supplementation is beneficial for ameliorating sarcopenia in elderly subjects that is characterized by reductions in the mass and function of skeletal muscle (candow et al. ) . third, patients with neurological and muscular disorders can respond well to creatine supplementation with improvements in health, as summarized previously. thus, adequate provision of dietary creatine may be necessary for maintaining homeostasis and optimal health in humans (brosnan and brosnan ) , particularly for vegan athletes who generally have low intake of creatine and its precursors (arginine, methionine and glycine) (rogerson ). creatine undergoes limited catabolism in mammalian cells (wu ) . however, creatine is reversibly converted by creatine kinase into creatine phosphate and is irreversibly cyclized into creatinine through spontaneous loss of one molecule of water, as indicated previously. like creatinine, creatine phosphate is also spontaneously converted into creatinine. the breakdown of creatine phosphate results in the losses of phosphate and one molecule of water. creatine kinase exists in the cytosolic and mitochondrial isoforms. this enzyme is not only most abundant in skeletal muscle and brain, but is also present in many tissues and cell types (keller and gordon ; trask and billadello ) . when healthy adults consume . g creatine monohydrate once, plasma creatine peaks [ µm; an -fold increase over the -min baseline value of about µm (jäger et al. )] in min (kreider et al. ) and its half-life is min (jäger et al. ). thus, creatine is rapidly cleared from plasma, and skeletal muscle is the major sink of creatine. creatinine is excreted in urine. the amount of urinary creatinine is proportional to (and, therefore, an indicator of) skeletal muscle mass in healthy subjects. creatine is essential for energy metabolism in the brain and skeletal muscle (wyss and kaddurah-daouk ) . in these two highly excitable tissues that can undergo rapid changes in their membrane potentials for the transport of electrical signals, creatine decreases intracellular calcium concentrations (e.g., by stimulating sarcoplasmic reticulum ca + -atpase in skeletal muscle) and extracellular glutamate concentrations (e.g., through uptake by synaptic vesicles in the brain), and prevents the opening of the mitochondrial permeability transition pore (bender et al. ; fortalezas et al. ) . creatine also plays an important role in antioxidative and anti-apoptotic reactions, scavenging free radicals, and protection against excitotoxicity in tissues (lawler et al. ) . for example, creatine can prevent mitochondrial disorders that are commonly associated with decreased atp production and increased ros production (rodriguez et al. ; wyss and kaddurah-daouk ) . by delaying the depletion of atp during oxygen deprivation and reducing the availability of ros (balestrino et al. ) , pretreatment with creatine can reduce the cardiac and neurological damages induced by ischemia or anoxia and that such treatment can also be useful even after the onset of stroke or myocardial infarction (balestrino et al. ; lensman et al. ; osbakken et al. ; perasso et al. ; prass et al. ; scheer et al. ; shen and goldberg ; whittingham and lipton ; wilken et al. ; zapara et al. ) . of note, creatine has been safely and beneficially administered to patients affected by age-related neurological diseases, including parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, long-term memory deficits, alzheimer's disease, and stroke (adhihetty and beal ; smith et al. ) , as well as patients with pathophysiological conditions, such as gyrate atrophy, post-stroke depression, congestive heart failure, chronic musculoskeletal pain disorders, atherosclerotic diseases, and cisplatin-induced renal damage (genc et al. ; hummer et al. ; persky and brazeau ; wyss and schulze ) . through increasing the availability of arginine for the generation of nitric oxide (a killer of pathogenic bacteria, fungi, parasites, and viruses), dietary supplementation with creatine plays an important role in protecting humans from infectious diseases (ren et al. ) . athletes and muscle builders are major users of supplemental creatine. this is primarily because ( ) both energy metabolism and production of oxidants are enhanced during exercise, and ( ) creatine participates in atp turnover in skeletal muscle and is a potent antioxidant. due to the very low rate of creatine loss ( . %) in the whole-body pool (creatine plus creatine phosphate), dietary supplementation with a low dose of creatine (e.g., g of creatine monohydrate per day for days in -kg men) results in a steady accumulation of creatine in skeletal muscle, and the saturation of creatine in the muscle ( mmol/kg dry matter) occurs by day (hickner et al. ; hultman et al. ) . for comparison, the content of creatine in the skeletal muscle of adult humans without creatine supplementation is mmol/kg dry matter of muscle (hultman et al. ) . thus, based on the current knowledge of creatine metabolism in humans, continuous supplementation with creatine is not necessary to achieve the saturation of creatine in skeletal muscle. in the case of discontinuous supplementation of creatine, such as disruption for short intervals (e.g., or days per week), a longer period of supplementation (e.g., by - days) should still be sufficient to achieve the saturation of creatine in skeletal muscle. thus, consumption of creatine monohydrate ( g/day) over a prolonged period of time can beneficially increase the concentration of creatine in skeletal muscle to the saturation level even if the supplement is not taken every single day. when an adult consumes daily g of dry beef that provides mg creatine ) as its sole dietary source, it will take about days to saturate creatine in skeletal muscle. because of increased losses of creatine during active muscular work as noted previously, oral administration of creatine is necessary for maintaining its homeostasis in exercising subjects and this nutritional strategy is beneficial for improving their performance. for example, dietary supplementation with a low dose of creatine (as g of creatine monohydrate per day) to adults can enhance the capacity of skeletal muscle to store energy by % [( - )/ = %] (hultman et al. ) . the rate of utilization of creatine phosphate by the skeletal muscle of adult humans during intensive exercise is mmol/l of muscle water/min (baker et al. ) . this is equivalent to mmol/whole-body skeletal muscle/ min ( × × % × . = ) for a -kg adult with l of muscle water. through creatine recycling via creatine kinase, ingestion of g of creatine monohydrate can help store kcal energy in the skeletal muscle of a -kg person for supporting a -min intensive exercise (i.e., mmol creatine phosphate/whole-body skeletal muscle/min × / = mmol = mmol × . kcal/mol × . × min = kcal). thus, a small amount of supplemental creatine makes a very significant contribution to energy storage and metabolism in the skeletal muscle of humans to enhance the power and duration of muscular work. in support of this view, dietary supplementation with creatine to young adults decreases oxidative dna damage and lipid peroxidation induced by a single bout of resistance exercise (rahimi ) . oral administration of creatine phosphate ( g/day on days - and g/day for days - ) offers a comparable ergogenic effect to that of equal amounts of creatine monohydrate (peeters et al. ) . in the international society of sports nutrition position stand (kreider et al. ) , multiple studies have been reviewed to indicate the following. first, there is consistent evidence that oral ingestion of g creatine monophosphate (equivalent to . g creatine) per day increases intramuscular creatine concentrations. this can be a biochemical basis for improvements in high-intensity exercise performance and training adaptations, as noted previously. second, creatine supplementation may enhance post-exercise recovery, injury prevention, thermoregulation, and rehabilitation, as well as concussion, spinal cord neuroprotection. third, creatine supplementation can improve muscular function and enhancing rehabilitation from injuries in subjects with neurodegenerative diseases (e.g., muscular dystrophy, parkinson's, huntington's disease), diabetes, osteoarthritis, fibromyalgia, aging, brain and heart ischemia, adolescent depression, and pregnancy. fourth, creatine supplementation can enhance anti-oxidative capacity and reduce oxidant-induced tissue injury. thus, creatine is one of the most popular and beneficial nutritional ergogenic aids for athletes (kreider et al. ). this conclusion continues to be supported by research findings over the past years. for example, wang et al. ( ) reported that creatine supplementation combined with exercise training for weeks improved maximal muscular strength and reduced exercise-associated muscle damage in adults. furthermore, oral consumption of creatine electrolyte improved overall and repeated short duration sprint cycling performance (crisafulli et al. ) , as well as anaerobic power and strength in athletes (hummer et al. ) . in addition to its ergogenic effect, creatine has also been used for humans to improve cognitive function (avgerinos et al. ) and reduce traumatic brain injury (balestrino et al. ; dolan et al. ) . furthermore, creatine supplementation ameliorates skeletal muscle dysfunction, enhances muscle functional capacity in patients with chronic obstructive pulmonary disease on long-term o therapy (de benedetto et al. ) , and supports the rehabilitation of tendon overuse injury in athletes (juhasz et al. ) . recently, there have been suggestions that creatine may delay or ameliorate sarcopenia in elderly subjects (candow et al. a, b) , reduce fat accumulation in the liver (da silva et al. ), improve muscle mass or function in cancer patients (fairman et al. ) , and maintain the mass and function of ageing bones (candow et al. a, b) . growing evidence over the past years shows that dietary supplementation with creatine may provide a safe and effective means in the therapeutic intervention of cancers. for example, creatine has an anti-tumor effect in vitro cell cultures and in vivo various transplanted human and rodent tumors (kristensen et al. ; lillie et al. ; miller et al. ). in addition, pal et al. ( ) reported that oral administration of creatine ( mg/kg bw per day) for consecutive days resulted in significant regression of tumor size in sarcoma- tumor-bearing mice and improved the overall survival of the animals. furthermore, creatine supplementation ( g/kg bw per day for days) to walker- tumor-bearing rats prevented skeletal muscle atrophy by attenuating systemic inflammation and protein degradation signaling, thereby enhancing their muscle mass and survival (cella et al. ) . similar beneficial effects of creatine supplementation have been obtained for humans with cancers (fairman et al. ). in , the russian biochemist w. gulewitsch discovered an abundant substance in the skeletal muscle of cattle and named this substance carnosine after "caro" or "carnis" (meaning meat in latin), which was identified in to be a dipeptide, β-alanyl-l-histidine. carnosine is characterized by three ionizable groups: the carboxylic group (pka . ), the amino group of the β-alanine residue (pka . ), and the imidazole ring in histidine (pka . ), with the pka of the whole molecule being . (tanokura et al. ) . thus, at physiological ph (e.g., ph . - . ), carnosine is present in the zwitterionic form and has a net positive charge. concentrations of carnosine in the skeletal muscle of humans without carnosine or β-alanine supplementation range from to mm (boldyrev et al. ) , or . - . mmol/kg dry weight of muscle. for example, the soleus and gastrocnemius muscles of adult males contain and mm carnosine, respectively, or . - . mmol/ kg dry weight of muscle (derave et al. ). these values are approximately , and times greater than those in the skeletal muscle of pigs, rats and mice, respectively (boldyrev et al. ) . note that bodybuilders can have carnosine concentrations as high as . mm or mmol/kg dry weight, with average values being mm or mmol/ kg dry weight (tallon et al. ) . the concentrations of carnosine in the olfactory bulb of the brain and the cardiac muscle are comparable to those in skeletal muscle, but those in other tissues (e.g., ~ . mm in kidneys and adipose tissue) are only . % to % of those in skeletal muscle. based on the report that the mean concentrations of carnosine in the skeletal muscles of women and men are . and . mmol/ kg dry weight, respectively (mannion et al. ) , it can be estimated that a -kg women and a -kg men have and g carnosine, respectively. in mammals (including humans), about % of carnosine is present in skeletal muscle (sale et al. ). carnosine is negligible or not detectable in the plasma of humans, but is present at low concentrations ( - µm) in the plasma of non-primate animals. carnosine is more abundant in the white muscle of humans than in their red muscle (hill et al. ). based on: ( ) the percentages of type i and type ii fibers in skeletal muscle of adult humans, which are % and %, respectively, in soleus muscle, and % and %, respectively, in gastrocnemius muscle (hill et al. ) , and ( ) the concentrations of carnosine in these two muscles (derave et al. ) , it can be calculated ( . x + . y = and . x + . y = ; x and y are carnosine concentration in type i and ii muscles, respectively) that type i and type ii muscle fibers contain . and . mm carnosine, respectively. this means that type ii muscle fiber has % more carnosine than type i muscle fiber. such information is important for developing an effective nutritional strategy to enhance muscular carnosine levels and improve muscular strength in athletes and elderly subjects. this is because of the following reasons. first, elite athletes can have up to % of type i or type ii muscle fiber, depending on the type of their sports, which is a determinant of their success at competition. for example, a sprinter with % of white muscle fibers will have a better performance than somebody with only % of white muscle fibers (komi and karlsson ) . second, age-related loss of muscle mass results from a decrease in the total number of both type i and type ii fibers and, but secondarily, from a preferential atrophy of type ii fibers (lexell et al. ; roos et al. ) . therefore, adequate availability of carnosine in skeletal muscle will be beneficial for healthy ageing. dietary carnosine is absorbed by the enterocytes of the small intestine across their apical membrane via peptide transporter- (pept ) (fig. ) . this is an h + -driven process. inside the enterocytes, a limited amount of carnosine is hydrolyzed by carnosinase- into β-alanine and histidine (sadikali et al. ) . the intracellular carnosine is exported by peptide/histidine transporters and (pht and pht ) out of the enterocyte across its basolateral membrane into the lamina propria of the small-intestinal mucosa. pept , pht and pht are members of the proton-coupled oligopeptide transporter family (pot-family or slc ), and have a broad specificity for di-and tri-peptides (including carnosine and its methylated analogs). the main difference between pept and phts is that the phts can transport l-histidine, in addition to di/tripeptides. within enterocytes, a small amount of carnosine is hydrolyzed by carnosinase- (also known as tissue carnosinase), and nearly all of the ingested carnosine enters the portal circulation (asatoor et al. ). in the blood of humans, carnosine is actively hydrolyzed by carnosinase- (also known as serum carnosinase; an enzyme that is synthesized and released from the liver) into β-alanine and histidine, which are then taken up by extra-intestinal cells via specific transporters for beta-amino acids and basic amino acids, respectively. carnosine in plasma, if any, is transported into extra-intestinal tissues and cells via pept (peptide transporter ) as well as pht and pht . pept , which has a broad specificity for di-and tri-peptides as does pept , is more widespread than pept in extra-intestinal tissues and cells. increasing dietary intake of carnosine enhances its concentrations in skeletal muscle, brain and heart (boldyrev et al. ) . ingestion of g synthetic carnosine alone by adult humans does not affect its concentrations in plasma due to high carnosinase- activity in plasma that rapidly hydrolyzes carnosine into β-alanine and histidine, but does increase the urinary output of carnosine (gardner et al. ) . urinary carnosine accounts for % of the ingested amount over a -h period and peaks at h after the consumption of carnosine. similarly, within min after the ingestion of . g carnosine by adult humans, the concentration of carnosine in serum does not change but the concentrations of histidine and β-alanine rapidly increase (asatoor et al. ) . this is consistent with the high activity of carnosinase- in the plasma of humans to hydrolyze carnosine. interestingly, consumption of beef by men and women can result in an increase in the concentration of carnosine in their plasma that peaks ( µm) at . h after intake and returns to the baseline value at . h after intake (park et al. ) . it is possible that some components in beef [anserine, amino acids (e.g., histidine and β-alanine), and copper] inhibit serum carnosinase (bellia et al. ; boldyrev et al. ) , so that the circulating levels of carnosine are substantially elevated. this can directly supply the dipeptide to extraintestinal tissues (e.g., heart and pancreas) and cells (e.g., red blood cells and immunocytes). in this regard, ingestion of beef as a whole food may be superior to oral administration of synthetic carnosine alone for humans. synthesis of carnosine from β-alanine and histidine is catalyzed by atp-dependent carnosine synthetase (a cytosolic enzyme) (wu ) , which is primarily present in skeletal muscle, heart, and certain brain regions (e.g., the olfactory bulb) (drozak et al. ; harding and o'fallon ) . the sources of β-alanine are diets and endogenous syntheses from the catabolism of aspartate (mainly in bacteria), malonic acid semialdehyde (transamination with glutamate), coenzyme a, pyrimidines, polyamines (wu ) . the sources of histidine are diets and the degradation of hemoglobin (which is very rich in histidine), actin, myosin, and other proteins in the body. based on the k d (fractional turnover rate) value of . / day for the loss of intramuscular carnosine at the physiological steady state (spelnikov and harris ) , the intramuscular concentrations of carnosine in women and men ( . and . mmol/kg dry weight, respectively), skeletal muscle mass ( % of bw), and its dry matter content ( %), it can be estimated that a -kg woman and a -kg man synthesizes and mg carnosine per day, respectively. the k m values of this enzyme for β-alanine and histidine are . - . mm (ng and marshall ) and . µm (horinishi et al. ) , respectively. the value of carnosine synthetase for β-alanine is much greater than or comparable to the intracellular concentration of β-alanine in the brain ( . mm) or skeletal muscle ( . mm), respectively (wu ) . in contrast, the value of carnosine synthetase for histidine is much lower than the intracellular concentration of histidine in the brain ( µm) and skeletal muscle ( µm), respectively (wu ) . as reported for nitric oxide synthase whose k m value for arginine is much greater in cells than that in assay tubes (wu and meininger ) , it is possible that the k m of carnosine synthetase for histidine in vivo is much greater than that (i.e., . µm) reported for the purified enzyme under in vitro assay conditions, due to complex protein-protein and enzyme-substrate interactions in vivo. availability of β-alanine primarily limits carnosine synthesis by carnosine synthetase in human skeletal muscle and the olfactory bulb, but adequate provision of histidine in diets is also critical for maximal production of carnosine because histidine is not synthesized de novo (hill et al. ; sale et al. ) . for example, supplementation with β-alanine to humans (e.g., - g/day) dose-dependently increases the concentrations of carnosine in skeletal muscle by - %, but dietary supplementation with . g histidine/ day for days has no effect on intramuscular carnosine concentrations in non-vegetarian adults (blancquaert et al. ; culbertson et al. ) . furthermore, dietary supplementation with . or . g β-alanine per day (as multiple doses of or mg) or with l-carnosine (isomolar to . g β-alanine) per day for weeks augmented intramuscular carnosine concentrations by %, % and % (harris et al. ) . these results reveal that histidine is not a limiting factor in carnosine synthesis in adults consuming adequate histidine from animal-source foods. thus, in nonvegetarian humans, consumption of an equal amount of β-alanine and carnosine effectively increases intramuscular carnosine concentrations to the same extent. it is unknown whether dietary intake of histidine limits carnosine synthesis in vegetarians with or without β-alanine supplementation. it is noteworthy that dietary supplementation with β-alanine ( g/day for days) to adults reduces the concentrations of histidine in their plasma and skeletal muscle by % and %, respectively, possibly due to reduced intestinal absorption of histidine and increased utilization of histidine for carnosine synthesis by skeletal muscle (blancquaert et al. ) . whether this reduction in histidine with β-alanine supplementation has a long-term adverse effect on human health is unknown, but it is prudent to ensure that dietary intake of histidine via supplementation or consumption of histidine-rich foods (e.g., meat) is sufficient. in contrast, oral administration of carnosine increases the concentration of not only β-alanine but also histidine in the plasma of humans (asatoor et al. ) , indicating an advantage of the consumption of synthetic carnosine or carnosine-rich foods (e.g., beef) over the consumption of β-alanine alone. within a mammalian species, the synthesis of carnosine is affected by multiple factors, including age, sex, muscle fiver type, muscular activity, and diet (harris et al. ) . for example, in a study with -to -year-old humans, baguet et al. ( ) found that the concentration of carnosine in skeletal muscle increased between and years of age but decreased thereafter with advanced ages. likewise, the content of carnosine in soleus muscle declines with age between -and -year-old subjects (everaert et al. ) . in adult humans, white muscle fibers contain - % more carnosine than red muscle fibers, and men have - % greater concentrations of carnosine in skeletal muscle than women (everaert et al. ; mannion et al. ) . for example, compared to age-matched women, men have , and % greater concentrations of carnosine in soleus, gastrocnemius and tibialis anterior muscles, respectively (everaert et al. ) . it is possible that androgens enhance carnosine synthesis in skeletal muscle, but the circulating levels of testosterone in healthy adult men do not appear to be related to their intramuscular carnosine concentrations (everaert et al. ). finally, long-term exercise (e.g., days per week for weeks for sprinters) increases intramuscular carnosine levels in male subjects by %, which is associated with a % increase in the mean power (e.g., during -s maximal cycle ergometer sprinting) was significantly increased following training (suzuki et al. ) ; similar results (a % increase in intramuscular carnosine levels) were obtained for resistance-trained bodybuilders (tallon et al. ) . finally, because plant-source foods contain much lower concentrations of β-alanine and histidine ) than animal products ) and because the formation of β-alanine in vegans is limited (harris et al. ) , the synthesis of carnosine in vegans is inadequate and its concentrations in soleus and gastrocnemius muscles are % and %, respectively, lower than those in omnivores who consume some meat that is an excellent source of both β-alanine and histidine (everaert et al. ; harris et al. ). carnosine is hydrolyzed by carnosinase- (a zn + -dependent cytosolic enzyme in tissues) and carnosinase- (a mn + -dependent extracellular enzyme in human blood), but not by proteases or dipeptidases, in humans and animals. expression of these two isoforms of carnosinase occurs in a tissue-specific manner. specifically, carnosinase- is expressed in the liver, brain, and kidneys but is absent from skeletal muscle and the small intestine (boldyrev et al. ; everaert et al. ). expression of carnosinase- in tissues other than the liver, brain, and kidneys is limited shortly after birth and gradually increases to adult levels during adolescence. accordingly, the plasma concentrations of carnosine are - µm in preterm infants, - µm in term infants, and absent in healthy adults (asatoor et al. ; valman et al. ) . in humans, carnosinase- that is present in serum at a very high activity is synthesized and secreted mainly by the liver. in contrast, the serum of healthy nonprimate mammals (except for the syrian golden hamster) and birds contains no carnosinase (boldyrev et al. ) . thus, carnosine is stable in the plasma of rats and farm animals but not humans (yeum et al. ). the concentrations of carnosine are - µm in the plasma of non-primate land animals. in contrast, carnosinase- is more widely expressed in mammalian tissues (including skeletal muscle and the small-intestine mucosa of some species), but is absent from the serum or cerebrospinal fluid of mammals or birds (bellia et al. ; boldyrev et al. ; everaert et al. ) . the gastric and colonic mucosae of mammals (including humans, pigs, cattle and rats) contain no carnosinase- or carnosinase- activity. as noted previously, in humans, dietary carnosine is absorbed intact by the small intestine into the portal circulation, but is rapidly hydrolyzed by serum carnosinase in plasma into β-alanine and histidine; both of the amino acids are reused for carnosine synthesis by skeletal muscle, heart, and the olfactory bulb of the brain (boldyrev et al. ; harris et al. ). in the human skeletal muscle (ph . ), which lacks carnosinase- , the degradation of carnosine by carnosinase- is very limited, because its optimal ph . is much greater than the intracellular ph and its v max is times lower than that of carnosinase activity in the human kidneys (lenney et al. ) . this explains the relatively high concentration of carnosine in mammalian skeletal muscle. the mean half-life of intramuscular carnosine in adult humans has been estimated to be days (a range of - days; spelnikov and harris ). this indicates that carnosine has a relatively high degree of biological stability in the body. consistent with this view, the turnover rate of carnosine in adult humans at the physiological steady state is . %/day (spelnikov and harris ) . excess carnosine, β-alanine and histidine are excreted from the body via the urine (sale et al. ). the healthy adult without ingesting carnosine excreted µmol carnosine in h (gardner et al. ) . in adult humans consuming g beef or chicken broth, urinary concentrations of carnosine within h after intake were -and -fold greater, respectively, than the values for no consumption of the meat (yeum et al. ) . carnosine has a functional imidazole ring, which can readily donate hydrogen to free radicals for their conversion into non-radical substances (kohen et al. ) . such an ability of the imidazole ring is enhanced by the β-alanine moiety in carnosine. the major physiological functions of carnosine include: ph-buffering, activation of muscle atpase to provide energy, metal-ion (copper, zinc and iron) chelation and homeostasis, antioxidant capacity (directly through scavenging ros and peroxyl radicals and indirectly through chelating metals), and protection against lipid peroxidation, protein oxidation, and the formation of advanced protein glycation (by inhibiting protein carbonylation and glycoxidation) and lipoxidation end products (by suppressing lipid peroxidation) (barca et al. ; boldyrev et al. ; nelson et al. ) . the pka value of the imidazole ring of carnosine ( . ) is closer to the intracellular ph in cells than the imidazole ring of free l-histidine ( . ) (wu ) . as a positively charged molecule, carnosine can neutralize atp (~ mm, a negatively charged molecule). thus, in skeletal muscle, carnosine ( - mm) would be a better buffering molecule than free histidine ( . mm). davey ( ) reported that carnosine, together with anserine, accounted for approximately % of the ph-buffering capability in the skeletal muscles of rabbits and pigeons. likewise, sewell et al. ( ) found that carnosine contributed about % of the buffering in type i muscle fibers and up to % in type iib fibers, and these results are consistent with the findings that type i muscle fibers have a lower glycolysis activity and accumulate less lactic acid than type iib muscle fibers. a similar buffering function of carnosine also applies to the olfactory bulb of the brain, which has only . mm histidine (wu ) . the β-alanine moiety of carnosine contributes to a reduction in its oxidation potential, and carnosine is oxidized at a lower oxidation potential than histidine to remove oxidants (kohen et al. ). thus, carnosine readily forms a charge-transfer complex with oxygen free radicals (e.g., superoxide radical, hydroxyl radicals, peroxyl radicals, and nitric oxide), non-radical ros (e.g., peroxynitrite, hypochlorous acid, singlet oxygen, and h o ), and deleterious aldehydes (e.g., malondialdehyde and formaldehyde) to confer anti-oxidative effect and protect cell membrane and intracellular organelles (e.g., mitochondria) from damage (kohen et al. ; pavlov et al. ) . besides the antiischemic effect of carnosine on the brain and heart, there is evidence that carnosine can maintain the integrity of the dna molecule, as indicated by studies with telomere (shao et al. ). the latter is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. specifically, carnosine can reduce telomere shortening rate possibly by protecting telomeres from damage, thereby contributing to the lifeextension effect of carnosine (shao et al. ). thus, carnosine is beneficial for healthy ageing. other physiological functions of carnosine include the regulation of sarcoplasmic reticulum ca + -release channels and sarcoplasmic reticulum ca + homeostasis in skeletal muscle, activation of its phosphorylase activity to promote glycogen breakdown (johnson et al. ) , inhibition of the angiotensin converting enzyme, enhancement of nitric oxide availability in endothelial cells, potentiation of cardiac and skeletal muscle contractilities, serving as a neurotransmitter or a neuromodulator, as well as the modulation of excitation-contraction coupling in skeletal muscle and the activity of the sympathetic nerve innervating the muscle (nagai et al. ; berezhnoy et al. ) . carnosine also plays a role in the inhibition of the growth and migration but induction of apoptosis of tumor cells, including human glioblastoma cells as well as colorectal and ovarian carcinoma cells (hipkiss and gaunitz ; hsieh et al. ; iovine et al. ) , as well as the suppression of the release of interleukin- by lipopolysaccharides plus interferon-γ-activated macrophages (caruso et al. ) . most recently, carnosine was reported to influence epigenetic regulation of gene expression in mammalian cells via increased histone acetylation (oppermann et al. ). compared with its constituent amino acids, the formation of carnosine can reduce intracellular osmolarity in the skeletal muscle and olfactory bulb, thereby protecting their integrity and function. in addition, being a positively charged dipeptide that is resistant to peptidases, carnosine does not readily exit cells and can be accumulated inside the cells at high concentrations within physiological ranges. through its intracellular actions, carnosine confers a vasorelaxing effect to reduce blood pressure (ririe et al. ) , stroke, and seizures (horning et al. ) , and carnosine is particularly beneficial for ameliorating ageing-related disorders such as cataract and neurological diseases (cararo et al. ; schön et al. ). thus, carnosine plays an important role in maintaining cell structure and function, as well as whole-body homeostasis and healthy aging, while reducing the risk of oxidative stress-related diseases, such as obesity, diabetes, hypertension, atherosclerosis, alzheimer's disease, stroke and seizures. much research has demonstrated the beneficial effects of dietary supplementation with carnosine on animal models of human diseases (derave et al. ). these effects include decreased plasma glucose and amelioration of diabetic complications (e.g., nephropathy, ocular damage, and retinopathy) in diabetic mice (lee et al. ; pfister et al. ); reduced lipid oxidation and augmented anti-oxidative capacities [e.g., restoring of blood glutathione and basal activities of antioxidant enzymes in aging rats (aydin et al. a,b; hipkiss and brownson ) ] and in pigs (ma et al. ); amelioration of acetaminophen-induced liver injury (yan et al. ), thioacetamide-or hyperammonemia-induced liver cirrhosis (aydin et al. a,b; jamshidzadeh et al. b) , and ethanol-induced chronic liver injury in rats (liu et al. ) . dietary supplementation with carnosine also results in decreases in malondialdehyde, oxidative stress (including ischemic oxidative stress and cerebral ischemia), and ethanol-induced protein carbonyls in brains (berezhnoy et al. ; fedorova et al. ); amelioration of neurological disorders, including autism spectrum disorder in children (chez et al. ; kawahara et al. ) ; protection against cardiovascular injury (abplanalp et al. ; artioli et al. ) and bleomycin-induced lung fibrosis in rats (cuzzocrea et al. ) ; cardiomyopathy in rats induced by chemotherapeutic agents that cause hydroxyl radical formation and lipid peroxidation (dursun et al. ) ; ros-induced renal damage in mice (fouad et al. ) ; and promotion of wound healing in rodents (ansurudeen et al. ) . through augmenting respiratory burst in neutrophils and their production of ros, as well as modulating the release of the virulent influenza virus from activated neutrophils, oral administration of carnosine and anserine reduces virus dissemination in humans (babizhayev and deyev ; babizhayev et al. ) . extensive animal experiments have laid a strong foundation for human clinical studies, which indicate beneficial effects of supplementation with carnosine ( . - g/day) for - months on subjects with chronic diseases (table ) . for example, oral administration of carnosine has been used to ameliorate syndromes in patients with gastric ulcers (sakae and yanagisawa ) , parkinson disease (boldyrev et al. ) , schizophrenia (chengappa et al. ) , autistic spectrum disorder (chez et al. ) , and ocular diseases (babizhayev et al. ) . carnosine supplementation has also been shown to attenuate elevated levels of glucose, triglycerides, advanced glycation end products, and tumor necrosis factor-α levels in patients with type- diabetes (houjeghani et al. ) and in overweight or obese pre-diabetic subjects (de courten et al. ; liu et al. ) , enhance cardiac output and improve the quality of life in patients with heart failure (cicero and colletti ) , as well as renal functional integrity and anti-oxidative capacity in pediatric patients with diabetic nephropathy (elbarbary et al. ); ameliorate insulin resistance (baye et al. ) , and increase leantissue mass in obese or overweight subjects (liu et al. ) . exercise is associated with increases in ca + influx into the myofibers of skeletal muscle (regulating myosin-actin cross bridging and action potential along the muscle fiber membrane) and in the production of ros and h + by skeletal muscle. excessive ros and h + must be removed rapidly, for example, by carnosine to sustain muscular activity and atp generation. therefore, dietary supplementation with β-alanine or carnosine improves muscular performance in humans (matthews et al. ). this notion is further substantiated by several lines of evidence. first, increases in carnosine concentrations in soleus (+ %) and gastrocnemius (+ %) through oral administration of β-alanine ( g/day) for weeks enhanced rowing performance in athletes (baguet et al. ) . similarly, supplementation with - . g β-alanine per day ( dosing of . or . g for each dosing) for weeks to non-vegetarian men (consuming . - . g β-alanine from histidine dipeptides in meat) augmented intramuscular carnosine concentrations by % ( % and % increase in type i and ii muscle fibers, respectively) and improved their high-intensity cycling performance (hill et al. ) . second, dietary supplementation with . g of a mix of anserine and carnosine ( : ) per day for weeks enhanced cognitive functioning and physical capacity (indicated by the senior fitness test) in the elderly (szcześniak et al. ) . third, oral administration of carnosine ( mg once daily) for months improved exercise performance (indicated by the -min walking test, peak o consumption, and peak exercise workload), as well as the quality of life in patients with chronic heart failure (lombardi et al. ) . after the discovery of carnosine in beef, scientists analyzed this substance in other animal species. in , n. tolkatschevskaya and d. ackermann independently identified a carnosine-like compound in goose skeletal muscle to be a dipeptide (methyl carnosine; β-alanyl- -methyl-l-histidine), which was named anserine after the taxonomic name for the goose. this peptide is characterized by three ionizable groups: the carboxylic group (pka . ), the amino group of the β-alanine residue (pka . ), and the imidazole ring in histidine (pka . ), with the pka of the whole molecule being . (bertinaria et al. ) . thus, at physiological ph (e.g., ph . to . ), anserine is present in the zwitterionic form and has a net positive charge. anserine is abundant in the skeletal muscles of birds, certain fish [e.g., salmon, tuna and trout (boldyrev et al. ) ], and beef , but is absent from human tissues (including skeletal muscle, heart and brain) (mannion et al. ). among the animal kingdom, anserine is the major histidine-containing dipeptide in the skeletal muscles of dog, cat, lion, rabbit, agouti, mouse, kangaroo, wallaby, opossum, cod, smelt, marlin, whiting, croaker, tuna, japanese char, salmon and trout (boldyrev et al. ) . intramuscular concentrations of anserine in these non-primate species range from mm for opossum to mm for marlin. high concentrations of anserine in the mm range are also present in the brains of the non-primate animals. in contrast, anserine is usually absent from the plasma of healthy adult humans without anserine intake (everaert et al. ) and is present at - µm in the plasma of non-primate animals depending on species (boldyrev et al. ) . intramuscular and cardiac concentrations of anserine are affected by a number of factors, including muscle fiber type, muscular contractility, and health status. for example, white muscle fibers contain much more anserine than red muscle fibers, as the concentrations of anserine and carnosine were . -and . -fold higher, respectively, in breast versus thigh muscle . in rat skeletal muscles (longissimus dorsi and quadriceps femoris), the concentrations of carnosine and anserine decrease by - % during senescence, and are - % lower in hypertensive animals than in normotensive ones (johnson and hammer ) . similarly, in rat cardiac muscle, the concentrations of total histidine dipeptides decline by % during senescence and, are % lower in hypertensive animals than in normotensive ones (johnson and hammer ) . in humans, dietary anserine is absorbed by the small intestine, transported in blood, and taken up by extra-intestinal tissues as described previously for dietary carnosine (fig. ) , except that the rates of catabolism of anserine to β-alanine and -methyl-histidine by serum carnosinase (carnosinase- ) in plasma and by carnosinae- in non-blood tissues are lower than those for carnosine (yeum et al. ) . a study with adult humans has shown that oral administration of g anserine/ kg bw without or with . g food increased the concentration of anserine in plasma, which peaked ( and µm, respectively) at min after consumption and returned to the baseline value at h after consumption (kubomura et al. ). in the absence of food intake, the oral administration of anserine increased the concentration of -methyl-histidine in the human plasma, which peaked ( µm) at min and declined thereafter to ~ µm at h after consumption. when anserine was consumed along with . g food, because the intestinal absorption of anserine was delayed, the oral administration of anserine increased the concentration of -methyl-histidine in the human plasma to the peak value of ~ µm) at min, which remained essentially unchanged at h after consumption. similar results were obtained for β-alanine (yeum et al. ) . in adult humans, the t / of orally administered anserine in plasma is . or . h, respectively, without or with food consumption (kubomura et al. ). thus, in humans, the circulating anserine is cleared rapidly as is carnosine. increasing dietary intake of anserine enhances its concentrations in skeletal muscle, brain and heart (boldyrev et al. ). humans and other primates do not synthesize anserine. however, non-primate animals can synthesize anserine from β-alanine and histidine at various rates, depending on species (boldyrev et al. ) . the atp-dependent pathways require anserine synthetase, as well as carnosine synthetase plus carnosine -methyltransferase (wu ) . the anserine synthetase pathway is a minor one, because -methylhistidine is limited in animal tissues. the carnosine n-methyltransferase is quantitatively important and physiologically active for anserine synthesis in skeletal muscle. because carnosine -methyltransferase and guanidinoacetate methyltransferase (the enzyme that converts guanidinoacetate into creatine) compete for s-adenosylmethionine (wu ) , there may be a close metabolic relationship between anserine and creatine syntheses in non-primate animals. like carnosine, the homeostasis of anserine in skeletal muscle is controlled by the availability of β-alanine or its degradation via transamination (blancquaert et al. ). besides using carnosine as a substrate, carnosinase also acts on anserine, but its enzymatic activity is lower for anserine than for carnosine (boldyrev et al. ; yeum et al. ) . thus, anserine is metabolized in humans and other animals as described previously for carnosine, except that serum carnosinase degrades anserine at a lower rate than carnosine and, therefore, oral administration of anserine or anserinecontaining food (e.g., beef) can transiently increase the concentration of anserine in the human plasma (everaert et al. ; yeum et al. et al. ) . excess anserine, β-alanine and -methyl-histidine are excreted from the body via the urine (sale et al. ) . oral administration of , and mg anserine/kg bw to young adults dose-dependently increased its urinary excretion, with peak concentrations of anserine in urine at min after intake (everaert et al. ) . even in subjects consuming mg anserine/kg bw, the peak concentration of anserine in the plasma was only µm, indicating extensive catabolism of this peptide by serum carnosinase. in adult humans consuming g beef or chicken broth, urinary concentrations of anserine within h after intake were -and -fold greater, respectively, than the values for no consumption of the meat (yeum et al. ) . anserine has physiological functions similar to those of carnosine, including h + buffering, antioxidation, modulation of muscle contractility (e.g., excitation and contraction through transmembrane potential maintenance and electromechanical coupling), and regulation of metabolism (boldyrev et al. ; everaert et al. ; kohen et al. ). however, as a methylated metabolite of carnosine, anserine has some biochemical properties that are different from those of carnosine. for example, in contrast to carnosine, anserine does not chelate copper and may not regulate nitric oxide availability in cells (boldyrev et al. ) . also, although anserine and carnosine exhibit an equal anti-oxidative activity (boldyrev et al. ) , anserine ( mm), but not carnosine ( mm), increased the protein and mrna levels of heat shock protein- in renal tubular cells treated with mm glucose or - µm hydrogen peroxide (peters et al. ) . furthermore, anserine, but not carnosine, inhibits carnosinase activity (derave et al. ). thus, anserine may potentiate the action of carnosine in the body. some studies have shown the beneficial effects of dietary supplementation with anserine on animal models of human diseases characterized by oxidative stress. for example, intraperitoneal administration of . or mg anserine to hyperglycemic rats ( - g of body weight) reduced hyperglycemia and plasma glucagon concentrations by suppressing sympathetic nerve activity . similarly, intravenous administration of anserine every days for days to -week-old diabetic db/db mice reduced blood glucose concentration by %, vascular permeability by one-third, and urinary proteinuria by % (peters et al. ). in addition, kaneko et al. ( ) reported that oral administration of anserine ( mg/mouse) to -month-old aβppswe/psen de mice (a model of alzheimer's disease) for weeks completely recovered the memory deficits, improved pericyte coverage on endothelial cells in the brain, and suppressed glial inflammatory reactions. these results indicate that anserine ameliorates neurovascular dysfunction and improves spatial memory in aged animals. interestingly, the effects of anserine on the renal function seem to be dependent on its doses via actions on the histaminergic nerve. for example, intravenous administration of µg anserine to urethane-anesthetized rats suppressed the renal sympathetic nerve activity, blood pressure, and heart rate, whereas intravenous administration of mg anserine had the opposite effects (tanida et al. ) . thus, because of its anti-oxidative and vasodilatory actions, dietary supplementation with anserine-rich chicken meat extracts ameliorated carbon tetrachloride-induced hepatic oxidative stress and injury in rats (peng and lin ) , while improving glucose homeostasis and preventing the development of hypertension in stroke-prone spontaneously hypertensive rats (matsumura et al. ) . besides animal studies, clinical investigations with humans have demonstrated beneficial effects of anserine on their metabolic, neurological, immunological, cardiovascular and renal functions. for example, oral administration of anserine ( or mg/ kg bw in ml of tap water) reduced blood glucose levels during an oral glucose tolerance test in humans . similarly, supplementing anserine plus carnosine ( g/day, : ratio, months) to healthy elderly subjects has been shown to preserve verbal episodic memory and brain perfusion (including blood flow in the prefrontal brain (ding et al. ; hisatsune et al. ; rokicki et al. ) , attenuate cognitive impairment (hisatsune et al. ; masuoka et al. ) , inhibit the production of inflammatory cytokines by peripheral blood mononuclear cells (katakura et al. ) , and enhance muscular strength and exercise endurance (hirohiko et al. ) . because avian muscle contains a large amount of anserine as noted previously, much evidence shows that consumption of a popular asian food known as chicken essence (chicken meat extract) by humans, particularly elderly subjects, has long been a nutritional means to improve human health and prevent chronic diseases. specifically, oral administration of anserine ( . - . g/day) to adults can effectively relieve stress and fatigue, ameliorate anxiety, promote post-partum lactation, improve physical capacity and exercise performance, reduce hyperglycemia and hypertension, enhance immunity, prevent ageing-associated neurological (e.g., cognitive and memory) dysfunction and inflammation, and accelerate wound healing szcześniak et al. ) . furthermore, oral administration of anserine can enhance the ability of humans to defend against infectious disease, as noted previously. collectively, these results indicate many health benefits of anserine supplementation on multiple systems in humans. tissue distribution of -hydroxyproline -hydroxyproline ( -hydroxypyrrolidine- -carboxylic acid; an imino acid; also often referred to as an amino acid) was originally produced from the acid hydrolysates of beef gelatin by e. fischer in . subsequently, -hydroxyproline was found to be an abundant constituent of collagen and elastin. studies in the s revealed that -hydroxyl-proline is derived from the post-translational hydroxylation of l-proline in proteins (primarily collagen). humans and animals also contain -hydroxyproline. in collagen and physiological fluid, the ratio of -hydroxyproline to -hydroxyproline is : , and these two imino acids exist in the trans form (wu et al. ) . the healthy -kg adult human contains . % protein (or . kg protein; wang et al. ) , including . kg collagens (meléndez-hevia et al. ). in humans and animals, collagen is the most abundant protein comprising % and % of body protein at birth and in adult life, respectively (wu et al. ) . collagen consists of . proline, . -hydroxyproline, . -hydroxyproline, and glycine residues per amino acid residues (or . g proline, . g -hydroxyproline, . g -hydroxyproline, and . g glycine residues per g collagen). thus, proline plus hydroxyproline accounts for approximately . % (g/g) of proteins in the body, with the ratio of proline to -hydroxyproline being . : (devlin ) . in the gly-x-y repeat of collagen, proline is in the x or y position but -hydroxyproline occurs only in the y position. -hydroxyproline is abundant in mammalian milk and plasma (mm range) in the peptide form [primarily glycyl-prolyl- -hydroxyproline (gly-pro-hyp)], and free -hydroxyproline is also present in these physiological fluids (µm range). for example, the concentrations of gly-pro-hyp in the plasma of -to -day-old pigs and sow's milk range from to mm and from to mm, respectively (wu et al. ) . for comparison, the concentrations of free -hydroxyproline in the plasma of mammals are to µm [e.g., - µm, adult humans (knight et al. ) and µm, -day-old pigs (wu et al. ) ], depending on diet, species, and age, as well as physiological (e.g., stress) and pathological (cancer) conditions. -hydroxyproline-containing proteins (primarily collagens) are hydrolyzed by proteases in the luminal fluids of the stomach (pepsin) and small intestine (trypsin, chymotrypsin, elastase, carboxypeptidases a and b, and aminopeptidase) to yield peptides and free amino acids (wu et al. ) . the oligopeptides are further hydrolyzed by peptidases to tripeptides, dipeptides, and amino acids. the mucosa of the small intestine secretes proline peptidase that specifically hydrolyzes proline-containing dipeptides (sjostrom et al. ) . some prolyl- -hydroxyproline (pro-hyp, a product of gly-pro-hyp degradation) is hydrolyzed to proline and -hydroxyproline by intestinal prolidase. this multifunctional enzyme possesses a unique ability to degrade imidodipeptides (x-pro or x-hyp) in which a proline or -hydroxyproline residue is located at the c-terminal end (lupi et al. ). thus, hydroxyproline-containing dipeptides and tripeptides in the lumen of the small intestine are transported intact into enterocytes (absorptive epithelial cells) by h + gradient-driven peptide transporters (fig. ) . in contrast, free proline and -hydroxyproline in the lumen are taken up into the cells primarily by the na + -dependent system imino transporter and the system nbb transporter (present on the brush border for the transport of neutral amino acids), as well as the na + -independent system l transporter (brandsch ) . inside the enterocyte, some of the -hydroxyprolinecontaining tri-and di-peptides undergo hydrolysis by peptidases and/or cytosolic prolidase to form free amino acids, including -hydroxyproline. the latter is metabolized locally to form glycine (wu et al. ) . the remaining di-and tripeptides exist the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via pht / (fig. ) . in contrast, proline and -hydroxyproline exit the enterocyte across its basolateral membrane into the lamina propria of the intestinal mucosa via specific transporters. some (about %) of the absorbed free proline and hydroxyproline are degraded by the intestinal mucosa and the remaining (about %) enters the portal circulation (li and wu ) . studies with rats have shown that free -hydroxyproline and -hydroxyproline-containing di-and tri-peptides in the portal vein account for approximately % and % of the total food-derived -hydroxyproline measured in the plasma (osawa et al. ) . note that gly-pro-hyp [a major small peptide from dietary collagen hydrolysates (yazaki et al. ) ] in the plasma was not determined in the published study (osawa et al. ) . these results indicate that small peptides from collagen hydrolysates are absorbed by the small intestine into the portal circulation predominantly as tri-and di-peptides. most of the diet-derived -hydroxyproline-containing di-and tri-peptides are not hydrolyzed by the small intestine during the first pass and, therefore, enter the portal circulation (shigemura et al. ) . the absolute bioavailability of collagen hydrolysates to the small intestine and extra-intestinal tissues in various amino acid and peptide forms is about - % in humans and animals, depending on species and age. in humans, ingestion of collagen hydrolysates increases the concentrations of at least hyp-containing peptides (pro-hyp, hyp-gly, ala-hyp, ile-hyp, leu-hyp, phe-hyp, glu-hyp, pro-hyp-gly, gly-pro-hyp, ala-hyp-gly, and ser-hyp-gly) in plasma (sato et al. ) . a majority of studies have shown that pro-hyp and hyp-gly, which are resistant to peptidases in the human plasma, are the most (about %) and second most abundant food-derived -hydroxyproline-containing peptides in the plasma of healthy adult humans, rats and mice after consuming collagen hydrolysates (osawa et al. ; sato et al. ; shigemura et al. ). the small peptides are cleared rapidly from the blood. the maximum concentration of food-derived -hydroxyproline-containing peptides in the plasma of individuals consuming collagen hydrolysates ( mg/kg bw) is µm (the mean value) at h after administration, with the maximum concentration of free -hydroxyproline in the plasma being µm (the mean value) at - h after administration (ohara et al. ). interestingly, gly-pro-hyp and pro-hyp are the most and second most abundant small -hydroxyproline-containing peptides in the plasma of mice, whereas ala-hyp, gly-pro-hyp and pro-hyp are the most, second most, and third most abundant food-derived -hydroxyproline-containing peptides in the plasma of adult men and women, at - h after the animals and human subjects consumed high amounts of tripeptide (gly-x-x)-containing collagen hydrolysates ( . - . g/kg bw for mice and . g/kg bw for humans) (yazaki et al. ) . thus, the abundance of -hydroxyproline-containing di-and tri-peptides in the plasma is affected by collagen hydrolysate preparations. proline, -hydroxyproline, and their small peptides are transported in blood in the free form. they are rapidly taken up by extra-intestinal tissues and cells [e.g., kidneys, skeletal muscle, skin, heart, and immunocytes (yazaki et al. ) ] via imino or peptide transporters (wu ) . inside these tissues and cells, the -hydroxyproline-containing di-and tri-peptides are hydrolyzed by peptidases and/or prolidase to yield their constituent imino acids ( -hydroxyproline or proline) and glycine. increasing dietary intake of -hydroxyproline in the free or peptide form enhances the concentrations of -hydroxyproline in plasma and tissues, such as the skeletal muscle, colon, joints, and skin (ji et al. ; yazaki et al. ). in fibroblasts, -hydroxyproline is formed from the posttranslational hydroxylation of proline residues in proteins, primarily collagen (li and wu ) , as noted previously. specifically, proline is incorporated into protein through the intracellular pathway of protein synthesis. thereafter, some proline residues are hydroxylated in the endoplasmic reticulum by prolyl -hydroxylase in the presence of oxygen, ascorbic acid, α-ketoglutarate, and fe + to form -hydroxyprolyl residues (gorres and raines ). hydrolysis of the protein containing hydroxylated proline residues releases -hydroxyproline as a free imino acid. other prolyl -hydroxylases, including hypoxia-inducible transcription factor α, can convert a limited number of proline residues to -hydroxyproline residues in non-collagen proteins (myllyharju and koivunen ). relatively little is known about -hydroxyproline catabolism in humans or animals. however, there is evidence that the kidneys of adult rats can rapidly take up extracellular -hydroxyproline and then convert it into glycine via -hydroxyproline oxidase (lowry et al. ) . other gibson et al. ( ) . adult humans consume . g protein/kg bw/ day b yu et al. ( ) . this value refers to the healthy adult consuming . g protein/day c meléndez-hevia et al. ( ) d the value is estimated from the plasma flux of glycine ( . g/day in the -kg healthy adult; ginson et al. ) and the net conversion of plasma glycine into serine (i.e., % of plasma glycine flux; butterworth et al. ) e starck et al. ( ) f the average value of . g/day for the healthy adult consuming . g protein/day (gibson et al. ) and . g/day for the healthy adult consuming . g protein/day (yu et al. ) g the value was calculated on the basis of the following: ( ) the rate of degradation of mature collagens in the extracellular matrix is equal to the rate of net synthesis of collagen (i.e., the rate of collagen secreted from fibroblasts to the extracellular matrix; . g/day) in the healthy adult human (meléndez-hevia et al. ); ( ) the content of -hydroxyproline in collagen is . g/ g collagen; wu et al. ); and ( ) % of collagen-derived -hydroxyproline is catabolized to form glycine in the healthy adult human (knight et al. extra-intestinal tissues (e.g., liver, skeletal muscle, and skin) also synthesize glycine from -hydroxyproline (hu et al. ) . the half-lives of free hydroxyproline ( to min) and its di-and tri-peptides ( - min) in blood are relatively short and vary with the species and age of mammals. for example, we found the half-lives of free -hydroxyproline in the blood of -and -day-old pigs are . ± . and . ± . min (mean ± sem, n = ), respectively, as determined by intravenous administration of -hydroxyproline ( mg/kg bw) and the single exponential model of its pharmacokinetics (wu et al. ). the half-lives of gly-pro-hyp and pro-hyp in the blood of adult humans are estimated to be about and min, respectively, but are about min in mice (yazaki et al. ) . similarly, based on the work of kusubata et al. ( ) , the half-life of pro-hyp in the blood of mice is estimated to be about min. the metabolic pathway for glycine synthesis from -hydroxyproline, with glyoxylate as the most immediate intermediate (wu et al. ) . interestingly, the estimate rate of whole-body glycine synthesis in the -kg healthy human adult ( . g/day) is comparable to the measured rate of . g/day (gibson et al. ) and . g/day (yu et al. ) in the healthy human adult consuming . and . g protein/day, respectively. a small fraction of the glyoxylate is converted into oxalate and glycolate in the liver (knight et al. ) . in adult humans, about % of the collagenderived -hydroxyproline may be converted into glycine, and the remaining % oxidized to oxalate and glycolate (knight et al. ) . the amounts of -hydroxyproline, oxalate and glycolate excreted daily in the urine of adult humans are - , - , and - mg, respectively. we estimated that milk-borne and collagen-derived -hydroxyproline contribute to . % and . %, respectively, of glycine needed by the young pig, whereas milk-borne glycine and non-hydroxyproline amino acids contribute to . % and . %, respectively, of glycine needed by the neonate (wu et al. ) . in adult humans, -hydroxyproline may contribute to about % of total glycine requirement (table ) . -hydroxyproline oxidase is the rate-controlling enzyme in the conversion of -hydroxyproline into glycine (wu et al. ) and is induced by cortisol (phang et al. ) . thus, the circulating levels of hydroxylproline are reduced in animals with fatigue (caused by deprivation of rest and sleep; a physical stress condition) or oxidative stress (kenéz et al. ; kume et al. ) . as a key component of collagen, proline and -hydroxyproline permit the sharp twisting of the collagen helix. this allows for establishing and maintaining the rigid structure of the collagen molecule in connective tissues, particularly skin, tendon, cartilage, bone, blood vessels, and the basement membrane (e.g., the intestinal lamina propria; a thin, fibrous, extracellular matrix of tissue that separates an epithelium from its underlying tissue), as well as protecting other tissues in the body (phang et al. ). in addition, the presence of -hydroxyproline in the gly-x-y collagen peptides reduces chemotaxis and blocks apoptosis in neutrophils, while the hydroxylation of two proline residues in hypoxia-inducible factor-α to form -hydroxyproline under normoxic oxygen conditions triggers the proteasomal degradation of the protein to regulate its abundance (wu et al. ) . multiple tissues in humans and many animals synthesize glycine from -hydroxyproline, as noted previously. this metabolic pathway is not dependent on folate (whose provision in diets is quantitatively low relative to glycine requirement) and, therefore allows for the provision of glycine from the inter-organ metabolism of amino acids (e.g., arginine, glutamine, glutamate, ornithine and proline) other than the tetrahydrofolate-dependent hydroxymethyl transferation of serine. the synthesis of glycine from -hydroxyproline plays an important role in maintaining the homeostasis of glycine [an amino acid with enormous nutritional and physiological importance, including heme formation and anti-oxidative reactions (wu ) ], as typical diets can meet at most % and % of daily glycine needs in milk-fed neonates (hou et al. ) and adult humans (table ). in addition, glycine is a major limiting factor for the syntheses of glutathione (the most abundant low-molecular-weight antioxidant in cells) (mccarty et al. ) , as well as bile salts, purines, heme and creatine in the body (wu ) . growing evidence shows that -hydroxyproline can scavenge oxidants (phang et al. ) , suppress nf-kb activation (ji et al. ) , regulate the intracellular redox state, and stimulate the expression of anti-oxidative enzymes in cells (wu et al. ) . furthermore, -hydroxyproline inhibits the production of hydroxyl radical via the fenton reaction possibly through: (a) formation of coordinate bonds with iron, (b) sequestration of fe + , and (c) interactions with intermediates of the reaction via hydrophobic hydration (milić et al. ) . in vitro anti-oxidative effects (e.g., scavenging of radicals and inhibition of lipid peroxidation) have also been demonstrated for the enzymatic hydrolysates of porcine collagen (ao and li ) . similarly, -hydroxyprolinecontaining collagen hydrolysates reduce inflammation and promote collage synthesis in dermal fibroblasts (offengenden et al. ) , while enhancing bone density and strength (watanabe-kamiyama et al. ) . finally, as an activator of the apoptotic cascade, oxidation of -hydroxyproline by -hydroxyproline oxidase to ros can inhibit the growth of cancer cells and promote their death (cooper et al. ) , thereby inhibiting tumorigenesis. furthermore, through the action of ros to kill pathogenic bacteria, fungi, parasites, and viruses (vatansever et al. ) , -hydroxyproline can enhance the ability of humans against infectious diseases. thus, in humans and animals, the generation of -hydroxyproline is enhanced in response to oxidative stress as an adaptation mechanism for defense and survival (wu et al. ) . research on the health benefits of -hydroxyproine has focused on its role in improving joint, skin and bone health (moskowitz ; zhang et al. a ) and in preventing gut inflammation in animal models. for example, pro-hyp stimulates the growth and migration of fibroblasts in the mouse skin (shigemura et al. ) and, therefore, would have important implication for maintaining dermal hydration and accelerating wound healing (li and wu ) . furthermore, pro-hyp inhibits the differentiation of chondrocytes into mineralized cells to maintain their normal physiological activity, while increasing glycosamino-glycan content in the extracellular matrix (nakatani et al. ). consistent with this view, dietary supplementation with . % pro-hyp reduced the breakdown of articular cartilage and ameliorates osteoarthritis in c bl/ j mice (nakatani et al. ). likewise, dietary supplementation with -hydroxyprolinecontaining collagen peptides ( . g/kg bw per day) to mice attenuated uv-b-induced decreases in skin hydration and soluble type i collagen, hyperplasia of the epidermis, and skin damage (tanaka et al. ). furthermore, oral administration of the collagen hydrolysates enhanced bone density (wu et al. ) and femur fracture healing in rats (tsuruoka et al. ) , as well as cutaneous wound healing in diabetic rats (zhang et al. b ) and injured nondiabetic rats (zhang et al. c) . there is growing interest in the role of -hydroxyproline in intestinal health. ji et al. ( ) reported that oral administration of -hydroxyproine ( % in drinking water) attenuated dextran sulfate sodium (dss)-induced colitis (characterized by the infiltration of mononuclear cells and colonic mucosal damage) in mice through inhibiting the nf-κb signaling and oxidative stress. likewise, intragastric administration of a -hydroxyproline-containing dipeptide (pro-hyp) to dss-challenged mice at the dose of mg/kg bw per day ( mmol or mg -hydroyproline/kg bw per day) for days mitigated dss-induced colitis (zhu et al. ) . similarly, intraperitoneal administration of a synthetic -hydroxyproline-containing polypeptide [(gly-pro-hyp) ] ameliorated clinical symptoms and histopathological colonic changes in mice with acute dss colitis (heimesaat et al. ) . because colitis increases the risk for colon cancer (the third most common type of cancer diagnosed in the us) (healy et al. ) , dietary supplementation with -hydroxyproline may help to prevent and treat this disease. future studies are warranted to test this novel and important hypothesis. clinical studies with humans have shown tremendous benefits of dietary supplementation with -hydroxyprolinecontaining peptides on skin, joint and bone health. for example, in a randomized double-blind, placebo-controlled trial involving - year-old women, oral administration of . or . g of -hydroxyproline-containing collagen peptide once daily for weeks enhanced skin elasticity (proksch et al. ). in addition, daily consumption of collagen hydrolysates enhances the moisture content in the epidermis of women during winter (matsumoto et al. ) and ameliorates joint pain in subjects with knee osteoarthritis (deal and moskowitz ) . likewise, dietary supplementation with collagen hydrolysates ( g/day) to - year-old women for weeks increased collagen density in the dermis, while decreasing the fragmentation of the dermal collagen network, with the effects occurring at week and persisting through week (asserin et al. ) . furthermore, daily oral administration of g of a collagen-hydrolysate food product (containing . or g pro-hyp and hyp-gly per kg product) by - year-old women for weeks resulted in dose-dependent improvements in facial skin conditions, such as skin moisture, elasticity, wrinkles, and roughness (inoue et al. ) . of particular interest, -month daily oral administration of g collagen hydrolysates (containing -hydroxyproline) augmented bone mineral density in postmenopausal women (könig et al. ) , suggesting their role in preventing osteoporosis. similarly, dietary supplementation with -hydroxyproline-containing collagen hydrolysates ( g/day) in combination with calcitonin for weeks has shown positive effects on mitigating osteoporosis in postmenopausal women (adam et al. ) . the beneficial effects of collagen peptides on humans can result from not only -hydroxyproline but also proline and glycine. the latter two amino acids are also abundant in beef ). humans and animals have physiological requirements for taurine, creatine and carnosine (wu ) . at present, dietary requirements of taurine, creatine, carnosine, anserine, and -hydroxyproline have not been established for humans or animals. the institute of medicine (iom ) recommended the dietary requirements of infants, children and adults for the so-called nutritionally essential indispensable) amino acids that are not synthesized by their tissues, but did not recommend dietary requirements for the so-called nutritionally nonessential dispensable) amino acids (e.g., arginine and taurine) that are synthesized de novo by their tissues. this does not necessarily mean that humans have no dietary requirements for taurine, creatine or carnosine. historically, growth or nitrogen balance was the sole criterion to define whether or not an amino acid or a nitrogenous substance was nutritionally essential for animals (hou et al. ) . similarly, the criterion for assessing nutritional essentiality of amino acids for humans was a short-term ( -day) nitrogen balance in healthy young adults (rose ) . whether an amino acid was classified as nutritionally essential or nonessential is only the matter of definition, and should not reflect the physiological needs of animals or humans for the nutrient wu , ) . it must be recognized that short-term nitrogen balance studies are not always sensitive to identify the essentiality of all amino acids for the organisms. for example, nitrogen balance was maintained in normal adult humans fed histidine-free diets for days (rose ) , but there are no metabolic pathways for histidine synthesis in mammalian cells (wu ) . a negative nitrogen balance did not occur in healthy adults during a short period (e.g., days) of consuming a histidine-free diet because the hydrolysis of histidine-rich hemoglobin in blood and of carnosine in skeletal muscle provides histidine for protein synthesis in tissues at the expense of health. furthermore, it has been known over years that feeding an arginine-deficient diet to adult men for days did not result in a negative nitrogen balance, but decreased sperm counts by ~ % and increased the percentage of non-motile sperm ~ fold (holt and albanese ) . this striking observation underlines a crucial role of arginine in spermatogenesis and argues that functional needs should be a criterion for the classification of arginine and any other amino acid as an essential or nonessential nutrient in diets . of note, the institute of medicine ( ) stated that dietary arginine was not required by healthy adults because arginine is synthesized via the hepatic urea cycle. however, there is no net synthesis of arginine in the mammalian liver via the hepatic urea cycle (wu and morris ) . this demonstrates the importance of ensuring that recommendations for dietary amino acid requirements be based on up-to-date knowledge of new developments in the field of amino acid metabolism. taurine, creatine, and carnosine must be provided in diets for individuals who do not synthesize these nutrients due to inborn errors of metabolism (wu ) . by the criterion of maintaining the integrity of tissues (e.g., eyes, heart and skeletal muscle), taurine is clearly a nutritionally essential amino acid for humans without inborn errors of metabolism, particularly the young, as noted previously. based on functional needs, creatine can be classified as a conditionally essential nutrient for humans, particularly athlete vegans who may not have adequate intakes of arginine, glycine and methionine. because glycine intake from typical diets meets < % of physiological needs and the endogenous synthesis of glycine may be insufficient for the optimal requirements of healthy adults (table ) , dietary supplementation is expected to beneficial for their optimal health, particularly under stress conditions and during ageing (wu ) . likewise, while a lack of carnosine or anserine in diets is not fatal, consumption of these two dipeptides can improve human health, especially neurological, immunological, retinal, cardiac and muscular functions. furthermore, ingestion of -hydroxyproline (an antioxidant and a precursor of glycine) may protect the gastrointestine from oxidative stress and inflammation, thereby possibly reducing risk for stomach, colon and breast cancers as well as possibly other types of cancer. physiological requirement of a healthy -kg adult for taurine may be mg/day or . mg/kg bw per day based on its urinary excretion of mg/day (laidlaw et al. ). because of their growth and development, infants and children likely have greater physiological requirements for taurine per kg bw than do the human adults. likewise, based on the urinary excretion of creatinine, a -kg healthy adult needs . g creatine/day to meet physiological needs. dietary provision of creatine can spare the use of amino table estimated physiological requirements of the -kg adult human for taurine, creatine, carnosine, anserine, and -hydroxyproline for optimal health "-" denotes no basal physiological requirement a % of the daily glycine intake by the healthy adult consuming . g protein/day (gibson et al. ) nutrient physiological requirement provision of nutrient from -g dry weight of beef (mg) beef ( acids for protein synthesis and other important pathways such as the production of polyamines and nitric oxide to improve the function of multiple systems. thus, dietary intake of creatine is beneficial for metabolic health. based on the whole-body turnover rate of . %/day for carnosine and the total amount of carnosine ( . g) in a -kg man (spelnikov and harris ) , it can be estimated that the physiological need for this dipeptide by a -kg subject is mg/day. anserine is not a physiological constituent in humans without consuming this dipeptide and, therefore, humans do not appear to have a basal physiological need for it. however, oral administration anserine in the amount of mg/day can enhance whole-body insulin sensitivity in subjects with hyperglycemia ). thus, dietary intake of anserine can improve human health. finally, because -hydroxyproline is the post-translational product of protein-bound proline, humans have no dietary requirements for -hydroxyproline. however, dietary -hydroxyproline can augment intestinal anti-oxidative capacity and endogenous glycine synthesis. based on studies with mice receiving intragastric administration of mg prolyl-hydroxyproline/kg bw per day ( mmol or mg -hydroyproline/kg bw per day) (zhu et al. ) and the mouse to human conversion factor of . (fda ), we suggest that dietary intake of . mg -hydroxyproline/kg bw per day for adult humans may be beneficial for improving intestinal health and preventing colitis. beef is a rich source of high-quality protein as well as highly bioavailable vitamin b , zinc, and iron (wu et al. ) . notably, compared with an isocaloric plant foodbased snack composed of corn, beans and greens, daily supplementation with a -g beef ( . g protein)-based snack ( kcal) to plant-source diets containing little or no animal-source protein (mean = . g animal-source protein/day) for months prevents nutritional growth stunting, while enhancing skeletal muscle mass and improving immune function in -to -year-old children (grillenberger et al. ; neumann et al. ). an important concept emerging from this review is that beef is an abundant source of taurine, creatine, carnosine, anserine, and -hydroxyproline as physiologically important nutrients for infants, children, and adults to maintain their health and prevent chronic diseases. for example, g of dried beef can provide . mg taurine, which can meet % of daily taurine requirement of the -kg adult (table ) . similarly, g of dried beef can provide mg carnosine, which can meet % of daily carnosine requirement of the -kg adult (table ) . likewise, meat provides a large amount of creatine, and the consumption of beef can substantially contribute to physiological needs of humans for this nutrient (table ). finally, beef contains abundant anserine and -hydroxyproline to improve the health and well-being of the young, adult, and ageing populations. thus, beef is a functional food for humans. there are concerns that consumption of red meat increases risks for chronic diseases or metabolic disorders in humans, including obesity, type ii diabetes mellitus, cardiovascular disease (the number one killer in the developed nations), ageing-related dysfunction and atrophy of organs (particularly, the brain and skeletal muscle), and cancers (willet et al. ). however, there is no direct evidence to support this view (johnston et al. ; leroy and cofnas ) . compelling evidence shows that taurine, creatine, carnosine, anserine, and -hydroxyproline, which are all abundant in red meat (e.g., beef, lamb and pork), play an important role in inhibiting oxidative stress (a common trigger of chronic diseases) and inflammation, ameliorating tissue (e.g., brain, heart, skeletal muscle, kidney, liver, gut, eye, and connective tissue) injury, improving metabolic profiles and the health of multiple systems, and enhancing immunity in animals and humans (fig. ) . these nutrients may help to kill pathogenic bacteria, fungi, parasites, and viruses (including coronavirus) through enhancing the metabolism and functions of monocytes, macrophages and other cells of the immune system. of note, taurine, creatine, carnosine, anserine can inhibit tumorigenesis and promote the apoptosis of tumor (e.g., colon cancer) cells, whereas -hydroxyproline prevents colitis and thus possibly reduces risk for colon cancers. thus, red meat is a functional food for optimizing human growth, development and health. as part of a healthy diet, regular consumption of red meat may actually prevent oxidative stress, thereby reducing risks for chronic diseases in humans. future studies with animal models and human subjects are warranted to test this hypothesis. fig. major functions of dietary taurine, creatine, carnosine, anserine and -hydroxyproline on improving the health of multiple systems in humans. these beneficial effects of the nutrients are summarized on the basis of available evidence in the current literatrure. some of the effects are tissue-and nutrient-specific. however, because all the systems of the body are integrated, the health of one system can affect that of other systems dietary taurine, creatine, carnosine, anserine, and -hydroxyproline (which are all abundant in beef) play an important role in inhibiting oxidative stress (a common trigger of chronic diseases) and inflammation, ameliorating tissue (e.g., brain, heart, skeletal muscle, kidney, liver, and gut) injury, and improving metabolic profiles in animals and humans. such a comprehensive update, along with recent advances in amino acid nutrition and physiology, will be highly informative to educate the public and policy makers about animal-source foods (e.g., beef) for human consumption. the health and ergogenic effects of dietary taurine, creatine, carnosine, anserine, and -hydroxyproline are expected to reverse the drastic decline in consumption of red meat (e.g., beef) in the u.s. due to an inadequate understanding of animal-source foods to provide functional amino acids, peptides, and creatine. finally, new knowledge presented herein can be used to guide future research priorities involving meat (including beef) in improving human nutrition, health and well-being. carnosine supplementation mitigates the deleterious effects of particulate matter exposure in mice postmenopausal osteoporosis. treatment with calcitonin and a diet rich in collagen proteins creatine and its potential therapeutic value for targeting cellular energy impairment in neurodegenerative diseases taurine prevents mitochondrial membrane permeabilization and swelling upon interaction with manganese taurine uptake across the human intestinal brush-border membrane is via two transporters: h + -coupled pat (slc a ) and na + -and cl − -dependent taut (slc a ) carnosine enhances diabetic wound healing in the db/db mouse model of type diabetes amino acid composition and antioxidant activities of hydrolysates and peptide fractions from porcine collagen carnosine in health and disease intestinal absorption of carnosine and its constituent amino acids in man the effect of oral collagen peptide supplementation on skin moisture and the dermal collagen network effects of creatine supplementation on cognitive function of healthy individuals effect of carnosine against thioacetamide-induced liver cirrhosis in rat the effect of carnosine treatment on prooxidant-antioxidant balance in liver, heart and brain tissues of male aged rats management of the virulent influenza virus infection by oral formulation of nonhydrolized carnosine and isopeptide of carnosine attenuating proinflammatory cytokine-induced nitric oxide production n-acetylcarnosine, a natural histidine-containing dipeptide, as a potent ophthalmic drug in treatment of human cataracts l-carnosine modulates respiratory burst and reactive oxygen species production in neutrophil biochemistry and function: may oral dosage form of nonhydrolized dipeptide l-carnosine complement anti-infective antiinfluenza flu treatment, prevention and self-care as an alternative to the conventional vaccination? important role of muscle carnosine in rowing performance the influence of sex, age and heritability on human skeletal muscle carnosine content exogenous creatine delays anoxic depolarization and protects from hypoxic damage: doseeffect relationship role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage potential of creatine or phosphocreatine supplementation in cerebrovascular disease and in ischemic heart disease interaction among skeletal muscle metabolic energy systems during intense exercise slc transporters: structure, function, regulation, disease association and therapeutics differences in muscle histidine-containing dipeptides in broilers carnosine modulates the sp -slc a /ctr copper-sensing system and influences copper homeostasis in murine cns-derived cells carnosine supplementation reduces plasma soluble transferrin receptor in healthy overweight or obese individuals ) carnosinases, their substrates and diseases creatine supplementation lowers brain glutamate levels in huntington's disease carnosine as an effective neuroprotector in brain pathology and potential neuromodulator in normal conditions synthesis, physicochemical characterization and biological activities of new carnosine derivatives stable in human serum as potential neuroprotective agents effects of histidine and β-alanine supplementation on human muscle carnosine storage carnosine increases efficiency of dopa therapy of parkinson's disease: a pilot study physiology and pathophysiology of carnosine creatine: endogenous metabolite, dietary, and therapeutic supplement enzymes and genes of taurine and isethionate dissimilation in paracoccus denitrificans the absorption of glycine and its conversion to serine in patients with sprue influence of exercise on urea, creatinine, and -methylhistidine excretion in normal human subjects creatine supplementation and aging musculoskeletal health variables influencing the effectiveness of creatine supplementation as a therapeutic intervention for sarcopenia effectiveness of creatine supplementation on aging muscle and bone: focus on falls prevention and inflammation carnosine and related peptides: therapeutic potential in age-related disorders ophidine (β-alanyl-l- -methylhistidine, 'balenine') and other histidine dipeptides in pig muscles and tinned hams carnosine modulates nitric oxide in stimulated murine raw . macrophages does dietary creatine supplementation play a role in skeletal muscle metabolism and performance? creatine supplementation in walker- tumor-bearing rats prevents skeletal muscle atrophy by attenuating systemic inflammation and protein degradation signaling a preliminary, randomized, double-blind, placebocontrolled trial of l-carnosine to improve cognition in schizophrenia double-blind, placebo-controlled study of l-carnosine supplementation in children with autistic spectrum disorders functional insights into the creatine transporter nutraceuticals and dietary supplements to improve quality of life and outcomes in heart failure patients the effect of cold storage on the carnosine content of muscle metabolism of taurine in microorganisms: a primer in molecular biodiversity? a novel function for hydroxyproline oxidase in apoptosis through generation of reactive oxygen species creatine-electrolyte supplementation improves repeated sprint cycling performance: a double blind randomized control study effects of betaalanine on muscle carnosine and exercise performance: a review of the current literature protective effect of orally administered carnosine on bleomycin-induced lung injury dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet the significance of carnosine and anserine in striated skeletal muscle supplementation with qter ® and creatine improves functional performance in copd patients on long term oxygen therapy taurine: a potential ergogenic aid for preventing muscle damage and protein catabolism and decreasing oxidative stress produced by endurance exercise effects of carnosine supplementation on glucose metabolism: pilot clinical trial nutraceuticals as therapeutic agents in osteoarthritis: the role of glucosamine, chondroitin sulfate, and collagen hydrolysate kinetics of creatine ingested as a food ingredient textbook of biochemistry with clinical correlations plasma guanidino compounds are altered by oral creatine supplementation in healthy humans beta-alanine supplementation augments muscle carnosine content and attenuates fatigue during repeated isokinetic contraction bouts in trained sprinters an update on carnosine and anserine research anserine/carnosine supplementation preserves blood flow in the prefrontal brain of elderly people carrying apoe e beyond muscle: the effects of creatine supplementation on brain creatine, cognitive processing and traumatic brain injury molecular identification of carnosine synthase as atp-grasp domain-containing protein (atpgd ) protection against adriamycininduced cardiomyopathy by carnosine in rats: role of endogenous antioxidants characterization of alpha-ketoglutarate-dependent taurine dioxygenase from escherichia coli taurine regulation of neuroendocrine function the effect of weeks carnosine supplementation on renal functional integrity and oxidative stress in pediatric patients with diabetic nephropathy: a randomized placebo-controlled trial vegetarianism, female gender and increasing age, but not cndp genotype, are associated with reduced muscle carnosine levels in humans gene expression of carnosine-related enzymes and transporters in skeletal muscle development and validation of a sensitive lc-ms/ms assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers the potential therapeutic effects of creatine supplementation on body composition and muscle function in cancer oxidative status in different areas of the cerebral cortex of wistar rats during focal ischemia and its modulation with carnosine mammalian alkaline phosphatases enzymes bile salt hydrolases: gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract creatine protects against cytosolic calcium dysregulation, mitochondrial depolarization and increase of reactive oxygen species production in rotenone-induced cell death of cerebellar granule neurons protective effect of carnosine against cisplatin-induced nephrotoxicity in mice intestinal absorption of the intact peptide carnosine in man, and comparison with intestinal permeability to lactulose nutritional requirement for taurine in patients receiving long-term, parenteral nutrition effect of free creatine therapy on cisplatin-induced renal damage endogenous glycine and tyrosine production is maintained in adults consuming a marginal-protein diet n-chlorotaurine, a natural antiseptic with outstanding tolerability taurine-supplemented total parenteral nutrition and taurine status of malnourished cancer patients skeletal muscle atp turnover and single fibre atp and pcr content during intense exercise at different muscle temperatures in humans s) food supplements have a positive impact on weight gain and the addition of animal source foods increases lean body mass of kenyan school children the subcellular distribution of carnosine, carnosine synthetase, and carnosinase in mouse olfactory tissues the identification of the n tau-methyl histidine-containing dipeptide, balenine, in muscle extracts from various mammals and the chicken the absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis determinants of muscle carnosine content retinal degeneration associated with taurine deficiency in the cat screening high-risk populations for colon and rectal cancers the synthetic hydroxyproline-containing collagen analogue (gly-pro-hyp) ameliorates acute dss colitis effect of days of creatine ingestion on muscle metabolism and performance of a simulated cycling road race influence of beta-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity a possible new role for the antiageing peptide carnosine inhibition of tumour cell growth by carnosine: some possible mechanisms efects of carnosine and anserine supplementation on relatively high intensity endurance effect of anserine/ carnosine supplementation on verbal episodic memory in elderly people the continuing importance of bile acids in liver and intestinal disease observations on amino acid deficiencies in man purification and characterization of carnosine synthetase from mouse olfactory bulbs endogenous mechanisms of neuroprotection: role of zinc, copper, and carnosine nutritionally nonessential amino acids: a misnomer in nutritional sciences nutritionally essential amino acids dietary essentiality of "nutritionally nonessential amino acids" for animals and humans composition of polyamines and amino acids in plant-source foods for human consumption l-carnosine supplementation attenuated fasting glucose, triglycerides, advanced glycation end products, and tumor necrosis factor-α levels in patients with type diabetes: a double-blind placebo-controlled randomized clinical trial carnosine suppresses human colorectal cell migration and intravasation by regulating emt and mmp expression the hydroxyproline-glycine pathway for glycine synthesis in neonatal pigs muscle creatine loading in men creatine electrolyte supplement improves anaerobic power and strength: a randomized double-blind control study physiological actions of taurine ingestion of bioactive collagen hydrolysates enhance facial skin moisture and elasticity and reduce facial ageing signs in a randomised double-blind placebocontrolled clinical study l-carnosine dipeptide overcomes acquired resistance to -fluorouracil in ht human colon cancer cells via downregulation of hif -alpha and induction of apoptosis the effect of taurine on chronic heart failure: actions of taurine against catecholamine and angiotensin ii biochemistry and physiology of taurine and taurine derivatives comparison of new forms of creatine in raising plasma creatine levels analysis of the efficacy, safety, and regulatory status of novel forms of creatine taurine treatment preserves brain and liver mitochondrial function in a rat model of fulminant hepatic failure and hyperammonemia carnosine ameliorates liver fibrosis and hyperammonemia in cirrhotic rats histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles regulation of muscle phosphorylase activity by carnosine and anserine unprocessed red meat and processed meat consumption: dietary guideline recommendations from the nutrirecs consortium hydroxyproline attenuates dextran sulfate sodium-induced colitis in mice: involvement of the nf-κb signaling and oxidative stress creatine supplementation supports the rehabilitation of adolescent fin swimmers in tendon overuse injury cases mechanisms underlying the antioxidant activity of taurine: prevention of mitochondrial oxidant production anserine (beta-alanyl- -methyl-l-histidine) improves neurovascular-unit dysfunction and spatial memory in aged aβppswe/psen de alzheimer'smodel mice anserine/carnosine supplementation suppresses the expression of the inflammatory chemokine ccl in peripheral blood mononuclear cells from elderly people a review on functional ingredients in red meat products zinc, carnosine, and neurodegenerative diseases discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells lower plasma trans- -hydroxyproline and methionine sulfoxide levels are associated with insulin dysregulation in horses hydroxyproline ingestion and urinary oxalate and glycolate excretion antioxidant activity of carnosine, homocarnosine, and anserine present in muscle and brain skeletal muscle fibre types, enzyme activities and physical performance in young males and females specific collagen peptides improve bone mineral density and bone markers in postmenopausal women international society of sports nutrition position stand: safety and efficacy of creatine supplementation in exercise, sport, and medicine creatine and cyclocreatine treatment of human colon adenocarcinoma xenografts: p and h magnetic resonance spectroscopic studies intestinal absorption and blood clearance of l-histidine-related compounds after ingestion of anserine in humans and comparison to anserinecontaining diets effect of anserine ingestion on hyperglycemia and the autonomic nerves in rats and humans potential biomarkers of fatigue identified by plasma metabolome analysis in rats detection of endogenous and food-derived collagen dipeptide prolylhydroxyproline (pro-hyp) in allergic contact dermatitis-affected mouse ear plasma and urine taurine levels in vegans regulation of taurine transport systems by protein kinase ck in mammalian cells direct antioxidant properties of creatine improved functional recovery of ischemic rat hearts due to singlet oxygen scavengers histidine and carnosine histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation characterization of human tissue carnosinase intracerebroventricular administration of creatine protects against damage by global cerebral ischemia in rat should dietary guidelines recommend low red meat intake? what is the cause of ageing atrophy? total number, size, and proportion of different fiber types studied in whole vastus lateralis muscle from -to -year-old men roles of dietary glycine, proline and hydroxyproline in collagen synthesis and animal growth taurine may prevent diabetic rats from developing cardiomyopathy also by downregulating angiotensin ii type receptor expression bioactivities of chicken essence cyclocreatine ( -carboxymethyl- -iminoimidazolidine) inhibits growth of a broad spectrum of cancer cells derived from solid tumours beneficial effects of histidine and carnosine on ethanol-induced chronic liver injury a dietary supplement containing cinnamon, chromium and carnosine decreases fasting plasma glucose and increases lean mass in overweight or obese pre-diabetic subjects: a randomized, placebo-controlled trial effects of oral administration oforodispersible levo-carnosine on quality of life and exercise performance in patientswith chronic heart failure hydroxyproline metabolism by the rat kidney: distribution of renal enzymes of hydroxyproline catabolism and renal conversion of hydroxyproline to glycine and serine human prolidase and prolidase deficiency: an overview on the characterization of the enzyme involved in proline recycling and on the effects of its mutations dietary supplementation with carnosine improves antioxidant capacity and meat quality of finishing pigs carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans effects of anserine/ carnosine supplementation on mild cognitive impairment with apoe genetic parameters for carnitine, creatine, creatinine, carnosine, and anserine concentration in longissimus muscle and their association with palatability traits in angus cattle clinical effect of fish type i collagen hydrolysate on skin properties preventive effect of a chicken extract on the development of hypertension in stroke-prone spontaneously hypertensive rats the physiological roles of carnosine and β-aalanine in exercising human skeletal muscle dietary glycine is rate-limiting for glutathione synthesis and may have broad potential for health protection calculated equilibria of phosphocreatine and adenosine phosphates during utilization of high energy phosphate by muscle a weak link in metabolism: the metabolic capacity for glycine biosynthesis does not satisfy the need for collagen synthesis the relationship of physicochemical properties to the antioxidative activity of free amino acids in fenton system treatment of hypertension with oral taurine: experimental and clinical studies the role of taurine in the pathogenesis of the cardiomyopathy of insulin-dependent diabetes mellitus inhibition of rate of tumour growth by creatine and cyclocreatine role of collagen hydrolysate in bone and joint disease creatine transporter protein content, localization, and gene expression in rat skeletal muscle hypoxia-inducible factor prolyl -hydroxylases: common and specific roles topical application of l-carnosine to skeletal muscle excites the sympathetic nerve innervating the contralateral skeletal muscle in rats chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo taurine improves glucose tolerance in stz-induced insulindeficient diabetic mice alignment of healthy dietary patterns and environmental sustainability: a systematic review biochemical characterization of the catecholaldehyde reactivity of l-carnosine and its therapeutic potential in human myocardium meat supplementation improves growth, cognitive, and behavioral outcomes in kenyan children regional and subcellular distribution of homocarnosine-carnosine synthetase in the central nervous system of rats chicken collagen hydrolysates differentially mediate anti-inflammatory activity and type i collagen synthesis on human dermal fibroblasts comparison of quantity and structures of hydroxyproline-containing peptides in human blood after oral ingestion of gelatin hydrolysates from different sources taurine supplementation for prevention of stroke-like episodes in melas: a multicentre, open-label, -week phase iii trial carnosine influences transcription via epigenetic regulation as demonstrated by enhanced histone acetylation of the pyruvate dehydrogenase kinase promoter in glioblastoma cells absorption and metabolism of orally administered collagen hydrolysates evaluated by the vascularly perfused rat intestine and liver in situ creatine and cyclocreatine effects on ischemic myocardium: p nuclear magnetic resonance evaluation of intact heart acute taurine supplementation enhances thermoregulation and endurance cycling performance in the heat creatine supplementation with methylglyoxal: a potent therapy for cancer in experimental models quantitation of carnosine in human plasma after dietary consumption of beef taurine supplementation improves economy of movement in the cycle test independently of the detrimental effects of ethanol the mechanism of interaction of carnosine with superoxide radicals in water solutions effect of oral creatine monohydrate and creatine phosphate supplementation on maximal strength indices, body composition, and blood pressure effects of chicken extract on antioxidative status and liver protection under oxidative stress therapeutic use of creatine in brain or heart ischemia: available data and future perspectives clinical pharmacology of the dietary supplement creatine monohydrate protective actions of anserine under diabetic conditions oral carnosine supplementation prevents vascular damage in experimental diabetic retinopathy proline metabolism and microenvironmental stress improved reperfusion and neuroprotection by creatine in a mouse model of stroke oral supplementation of specific collagen peptides has beneficial effects on human skin physiology creatine supplementation decreases oxidative dna damage and lipid peroxidation induced by a single bout of resistance exercise effects of taurine supplementation on vascular endothelial function at rest and after resistance exercise synthesis of taurine and isethionic acid by dog heart slices roles of arginine in cellmediated and humoral immunity vasodilatory actions of the dietary peptide carnosine beneficial effects of creatine, coq , and lipoic acid in mitochondrial disorders vegan diets: practical advice for athletes and exercisers daily carnosine and anserine supplementation alters verbal episodic memory and resting state network connectivity in healthy elderly adults age-related changes in motor unit function the amino acid requirements of adult man carnosinase activity of human gastrointestinal mucosa the relationship between plasma taurine levels and diabetic complications in patients with type diabetes mellitus oral treatment of pressure ulcers with polaprezinc (zinc l-carnosine complex): -week open-label trial effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance x-linked creatine-transporter gene (slc a ) defect: a new creatine deficiency syndrome structural correlates of the creatine transporter function regulation: the undiscovered country taurine supplementation ameliorates glucose homeostasis, prevents insulin and glucagon hypersecretion, and controls β, α, and δ-cell masses in genetic obese mice prophylactic role of taurine and its derivatives against diabetes mellitus and its related complications generation of bioactive prolylhydroxyproline (pro-hyp) by oral administration of collagen hydrolysate and degradation of endogenous collagen effects and mechanisms of taurine as a therapeutic agent role of antioxidant activity of taurine in diabetes creatine and creatine pyruvate reduce hypoxia-induced effects on phrenic nerve activity in the juvenile mouse respiratory system the potential of carnosine in brain-related disorders: a comprehensive review of current evidence taurine: a regulator of cellular redox-homeostasis and skeletal muscle function estimation of the carnosine content of different fibre types in the middle gluteal muscle of the thoroughbred horse ) l-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts creatine pretreatment protects cortical axons from energy depletion in vitro effect of prolylhydroxyproline (pro-hyp), a food-derived collagen peptide in human blood, on growth of fibroblasts from mouse skin identification of a novel food-derived collagen peptide, hydroxyprolyl-glycine, in human peripheral blood by pre-column derivatisation with phenyl isothiocyanate dose-dependentchanges in the levels of free and peptide forms of hydroxyproline in human plasma after collagen hydrolysateingestion role of ros production and turnover in the antioxidant activity of taurine protective and therapeutic effectiveness of taurine in diabetes mellitus: a rationale for antioxidant supplementation purification and specificity of pig intestinal prolidase a review of creatine supplementation in age-related diseases: more than a supplement for athletes a kinetic model of carnosine synthesis in human skeletal muscle endogenous amino acid losses from the gastrointestinal tract of the adult human-a auantitative model the biology of taurine in nutrition and development taurine in development the effect of sprint training on skeletal muscle carnosine in humans simultaneous determination of free amino acids, l-carnosinem purine, pyrimidine, and nucleosides in meat by liquid chromatography/single quadrupole mass spectrometry anserine and carnosine supplementation in the elderly: effects on cognitive functioning and physical capacity the carnosine content of vastus lateralis is elevated in resistancetrained bodybuilders effects of collagen peptide ingestion on uv-b-induced skin damage effects of anserine on the renalsympathetic nerve activity and blood pressure in urethaneanesthetized rats h nuclear magnetic resonance studies of histidine containing di and tripeptides. estimation of the effects of charged groups on the pka value of the imidiazole ring effects of dietary potato by-product and rumen-protected histidine on growth, carcass characteristics and quality attributes of beef tissue-specific distribution and developmental regulation of m and b creatine kinase mrnas promotion by collagen tripeptide of type i collagen gene expression in human osteoblastic cells and fracture healing of rat femur economic research service. livestock, dairy, and poultry outlook mediterranean diet and cardio-metabolic health: what is the role of meat? protein intake and plasma amino acids of infants of low birth weight antimicrobial strategies centered around reactive oxygen species -bactericidal antibiotics, photodynamic therapy and beyond randomised clinical trial: oral taurine supplementation versus placebo reduces muscle cramps in patients with chronic liver disease the effects of an oral taurine dose and supplementation period on endurance exercise performance in humans: a meta-analysis oral taurine improves critical power and severe-intensity exercise tolerance total body protein: a new cellular level mass and distribution prediction model effects of -week creatine supplementation combined with complex training on muscle damage and sport performance absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats cerebral synaptic transmission during anoxia is protected by creatine food in the anthropocene: the eat-lancet commission on healthy diets from sustainable food systems creatine protects the central respiratory network of mammals under anoxic conditions taurine: biological update amino acids: metabolism, functions, and nutrition amino acids: biochemistry and nutrition arginine nutrition and cardiovascular function arginine metabolism: nitric oxide and beyond arginine nutrition in development, health and disease assessment of effectiveness of oral administration of collagen peptide on bone metabolism in growing and mature rats s) pharmacokinetics and safety of arginine supplementation in animals proline and hydroxyproline metabolism: implications for animal and human nutrition dietary requirements of "nutritionally nonessential amino acids" by animals and humans production and supply of highquality food protein for human consumption: sustainability, challenges and innovations composition of free and peptide-bound amino acids in beef chuck, loin, and round cuts metabolism, nutrition and redox signaling of hydroxyproline significance, challenges and strategies of animal production creatine and creatinine metabolism health implications of creatine: can oral creatine supplementation protect against neurological and atherosclerotic disease? the potential health benefits of taurine in cardiovascular disease the neuroprotective effects of taurine against nickel by reducing oxidative stress and maintaining mitochondrial function in cortical neurons protective effects from carnosine and histidine on acetaminophen-induced liver injury oral ingestion of collagen hydrolysate leads to the transportation of highly concentrated gly-pro-hyp and its hydrolyzed form of pro-hyp into the bloodstream and skin profiling histidine dipeptides in plasma and urine after ingesting beef, chicken or chicken broth in humans quantitative aspects of glycine and alanine nitrogen metabolism in postabsorptive young men seasonal differences and protection by creatine or arginine pretreatment in ischemia of mammalian and molluscan neurons in vitro carnosine pretreatment protects against hypoxia-ischemia brain damage in the neonatal rat model oral administration of skin gelatin isolated from chum salmon (oncorhynchus keta) enhances wound healing in diabetic rats oral administration of marine collagen peptides from chum salmon skin enhances cutaneous wound healing and angiogenesis in rats deletion of the γ-aminobutyric acid transporter (gat and slc a ) gene in mice leads to changes in liver and brain taurine contents gelatin versus its two major degradation products, prolyl-hydroxyproline and glycine, as supportive therapy in experimental colitis in mice ethical statement this review article does not require either human consent or the approval of animal use.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -fe u oi authors: nirk, eliise laura; reggiori, fulvio; mauthe, mario title: hydroxychloroquine in rheumatic autoimmune disorders and beyond date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: fe u oi initially used as antimalarial drugs, hydroxychloroquine (hcq) and, to a lesser extent, chloroquine (cq) are currently being used to treat several diseases. due to its cost‐effectiveness, safety and efficacy, hcq is especially used in rheumatic autoimmune disorders (rads), such as systemic lupus erythematosus, primary sjögren's syndrome and rheumatoid arthritis. despite this widespread use in the clinic, hcq molecular modes of action are still not completely understood. by influencing several cellular pathways through different mechanisms, cq and hcq inhibit multiple endolysosomal functions, including autophagy, as well as endosomal toll‐like receptor activation and calcium signalling. these effects alter several aspects of the immune system with the synergistic consequence of reducing pro‐inflammatory cytokine production and release, one of the most marked symptoms of rads. here, we review the current knowledge on the molecular modes of action of these drugs and the circumstances under which they trigger side effects. this is of particular importance as the therapeutic use of hcq is expanding beyond the treatment of malaria and rads. antimalarial drugs have a long history, starting around years ago when quinine, a substance in the bark of the cinchona tree, was first used to fight plasmodium falciparum infections (woodward & doering, ; haładyj et al, ) . cq was the first potent and massproducible drug against malaria and was synthesized as an analogue of quinine (shanks, ) . despite its remarkable antimalarial efficiency, cq was deemed too toxic due to its side effects such as gastrointestinal and skin complications, retinopathy, cardiotoxicity or myopathy (kalia & dutz, ; haładyj et al, ) . the discovery of hcq mitigated this issue, and hcq is now regularly used in clinics under the brand name plaquenil (furst, ; aviña-zubieta et al, ; al-bari, ; haładyj et al, ) . already during the second world war, the positive effects of these two antimalarial drugs on rads were observed. soldiers taking cq and hcq as prophylaxis reported improvement of rashes and inflammatory arthritis. today, cq and particularly hcq are commonly used to treat rheumatic and dermatological diseases, and are further being tested in clinical trials as potential drug candidates for covid- , several types of cancer, diabetes type i and ii, multiple sclerosis, recurrent miscarriages and myocardial infarction (al-bari, ; clinicaltrials.gov) . rads, such as systemic lupus erythematosus (sle) (ruiz-irastorza et al, ; willis et al, ; , rheumatoid arthritis (ra) (khraishi & singh, ) and primary sjögren's syndrome (pss) (oxholm et al, ; rihl et al, ; kumar & clark, ; demarchi et al, ) , are caused by a malfunctioning immune system that targets healthy tissues (smith & germolec, ) such as joints (kumar & clark, ) . cqs and hcqs therapeutic role in rads is linked to its anti-inflammatory and immunomodulatory effects (plantone & koudriavtseva, ) . these effects are achieved through the modulation of the autoimmune response by (i) impairing functions of the endolysosomal system through its lysosomotropic effects (ziegler & unanue, ; kaufmann & krise, ; yoon et al, ) , (ii) decreasing the levels of circulating pro-inflammatory cytokines (sperber et al, ; van den borne et al, ) , (iii) inhibiting t-cell proliferation (landewe et al, ; costedoat-chalumeau et al, ) , (iv) blocking tolllike receptors (tlrs) (kyburz et al, ) and (v) autophagy inhibition (an et al, c) . however, numerous questions remain regarding both the mechanism of action of cq and hcq in rads and the side effects caused by this compound. in this review, we report on hcq and cq modes of action at the molecular and cellular levels in the context of rads. additionally, we discuss the relevance of these drugs in the treatment of cancer and infectious diseases. finally, we summarize the side effects reported in patients taking hcq for rads and discuss how some of those can be explained by the current knowledge on cq and hcq. mobilization from the endoplasmic reticulum (er). they might further modulate other cellular and organismal processes, e.g. golgi trafficking (mauthe et al, ) , but the underlying mechanisms remain to be identified. inhibition of lysosomal activity and autophagy cq and hcq are weak bases that easily cross cell membranes and accumulate in acidic subcellular compartments such as lysosomes and endosomes, where they remain trapped in a protonated state (ohkuma & poole, ) . this leads to a ph increase in lysosomes from to , causing inhibition of acidic proteases and other enzymes within the endolysosomal compartments ( fig a) (ohkuma & poole, ; poole & ohkuma, ; ziegler & unanue, ; haładyj et al, ) . as a result, antigen processing and subsequent presentation by mhc-ii complex on the cell surface of both macrophages and lymphoid dendritic cells are impaired (guidos et al, ; chesnut & grey, ; fox, ) , dampening the adaptive immune response (fig ) (fox, ) . cq and hcq also increase ph levels within the golgi stacks. this causes functional alterations of this organelle that possibly contribute to the cellular effects of these two drugs, e.g. by impairing transforming growth factor beta (tgf-b) activity (perkett et al, ; rivinoja et al, ; mauthe et al, ) . the ability to block lysosomal degradation also makes cq and hcq potent macroautophagy inhibitors ( fig a) . macroautophagy, hereafter called autophagy, is a conserved intracellular degradation pathway that is required to maintain cellular homeostasis by recycling damaged or unwanted cytoplasmic proteins, complexes and organelles (eskelinen & saftig, ). autophagy plays a role in many physiological processes, and its misregulation is linked to pathologies such as cancer, neurodegeneration and inflammatory diseases (mizushima et al, ; levine et al, ; dikic & elazar, ; levine & kroemer, ) . during autophagy, cytoplasmic cargoes are sequestered by double-membrane vesicles called autophagosomes, which fuse with lysosomes to generate autolysosomes (eskelinen & saftig, ) . fusion with lysosomes and activity of the lysosomal enzymes are required to break down the autophagosomal cargoes and recycle the resulting metabolites. impairment of both autophagosome-lysosome fusion and lysosomal degradative activity blocks autophagy (klionsky et al, ) . although cq and hcq decrease the acidity of lysosomes (seglen et al, ; poole & ohkuma, ; mizushima et al, ) , the glossary antigen-presenting cells (apc) cells that process proteins derived from pathogens or from dying/ dead cells, into peptides that get presented on their surface, thereby activating t cells and initiating an immune response. autophagy an intracellular process that delivers unwanted cytoplasmic material into lysosome for degradation. b cells a type of lymphocytes (white blood cells) that plays a crucial role in the adaptive immune response by producing antigen-specific antibodies. calcium (ca + ) is the most abundant mineral in the human body and is vital for a multitude of cellular and physiological function. it is also an important second messenger in numerous signal transduction pathways. chloroquine (cq)/hydroxychloroquine (hcq) originally developed to fight malaria, these drugs are used to treat rheumatic autoimmune diseases and are currently tested in clinical trials as therapies for other conditions. cytokines small secreted proteins that mediate communication and modulate interactions between cells, including immune cells. endosomes intracellular organelles that mainly function as a sorting and recycling hub for endocytosed and biosynthetic components, on their route to lysosomes. immune system a network consisting of a variety of different cell types that defend the body against infections and other potentially harmful anomalies, and which, when misregulated, contributes or causes the development of an inflammatory disease. lysosome intracellular organelles containing a large battery of digestive enzymes that degrade extracellular and cytoplasmic material delivered to their interior by endocytosis and autophagy, respectively. a membrane-bound multi-subunit enzymatic complex at either the plasma or endosomal membrane, which participates in a variety of cellular functions, ranging from cellular signalling and gene expression to host defence mechanisms. primary sjögren's syndrome an autoimmune disease that belongs to the group of rheumatic autoimmune diseases, which affect saliva-producing glands leading to symptoms such as dry mouth and dry eyes. retinopathy condition characterized by a damaged retina, which causes vision impairment, and is a documented adverse effect that can occur when taking hcq and cq. rheumatic autoimmune diseases a group of conditions characterized by a dysregulated immune system, which primarily affect the muscles, joints, connective tissue and bones. systemic lupus erythematosus an autoimmune disease that belongs to the group of rheumatic autoimmune diseases, which is the most common form of lupus and is associated with symptoms such as severe fatigue, joint pain and joint swelling. t cells a type of lymphocytes (white blood cells) that is a key component of the adaptive immune system and that orchestrates other cell types in response to antigens. toll-like receptors (tlr) transmembrane proteins that recognize specific molecules at either the plasma membrane or endosomes, and subsequently initiate signalling pathways that are crucial for the innate immune response. primary inhibitory effect of these drugs on autophagy is blocking the fusion of autophagosomes and lysosomes, which is at least in part mediated by the dysregulation of the recruitment of specific snare proteins onto autophagosomes (mauthe et al, ) . this block results in an accumulation of autophagosomes in the cytoplasm (mauthe et al, ) , which can contribute to an enhanced autophagosome-mediated signalling output (martinez-lopez et al, ; barrow-mcgee et al, ) and even compromise tumour cell viability (button et al, ) . although hcq and cq have been extensively described as autophagy inhibitors, there is emerging evidence that these drugs induce a non-canonical form of endocytosis (florey et al, ; jacquin et al, ) . activation of tlrs, especially in macrophages, monocytes and t helper cells, but also in neutrophils and endothelial cells, induces the production and secretion of pro-inflammatory cytokines, a hallmark of rads (beutler & cerami, (a) cq and hcq are weak bases that accumulate inside acidic subcellular compartments, e.g. endosomes and lysosomes. they remain trapped in a protonated state, causing an increase of ph and thereby inhibiting the functions of these cellular compartments. impairment of the autophagosome-lysosome fusion leads to autophagy inhibition. (b) cq and hcq alter endosomal tlr activation by increasing endosomal ph, by blocking the interaction between nucleic acids and endosomal tlrs (trl , tlr and tlr ) and by preventing translocation of tlr to endosomes. hcq also blocks the correct assembly of the nox complex by preventing the translocation of the nox subunit gp phox onto endosomes and consequently the formation of an active nox . (c) cq and hcq impair the release of ca + from the er, resulting in inhibition of ca + -dependent signalling pathways. hcq further inhibits the replenishing of intracellular ca + stores from the extracellular space. kim & moudgil, ). hence, inhibition of endosomal tlrs by hcq or cq is a powerful therapy approach for these diseases (lafyatis et al, ) . tlr , activated by dna in immune cells, can thus be inhibited by hcq and cq (yi et al, ; ahmad-nejad et al, ) . tlr , activated by guanosine analogues, can also be inhibited by cq, but to a lesser extent than tlr (lee et al, ) , indicating different inhibitory mechanisms. tlr is mainly activated by poly(i-c), but also by debris originating from necrotic synovial fluid cells in ra patients, and both modes of activation are hampered by hcq and cq (brentano et al, ; jolly et al, ; imaizumi et al, ) . in general, inhibition of tlr , tlr and tlr by hcq and cq has been attributed to their ability to impair endosomal acidification (macfarlane & manzel, ; lafyatis et al, ; schrezenmeier & dörner, ) , as activation of endosomal tlrs and subsequent downstream signalling only takes place within acidified compartments ( fig b) (blasius & beutler, ) . beside endosomal acidification, kuznik and colleagues discovered a second mechanism by which cq impairs tlr signalling. they showed that cq could inhibit endosomal tlr signalling after stimulation with nucleic acids at concentration too low to influence the endosomal ph. under those conditions, cq blocks endosomal tlr activation by directly interacting with tlr ligands, such as nucleic acids, which changes the nucleic acid secondary structure and prevents their binding to endosomal tlrs (macfarlane & manzel, ; ku znik et al, ) . this notion is further supported by the observation that hcq specifically blocks activation of dendritic cells and macrophages by dna but not by lps, although lps also stimulates these cells via a signalling cascade emanating from endosomes (häcker et al, ) . a third mechanism that interferes with inflammatory cytokine production is the ability to disrupt gmp-amp synthase (cgas) signalling . cgas is a crucial component of the cgas-stimulator of interferon gamma (ifn) genes (sting) signalling cascade that is required for the ifn type i response in immune cells (sun et al, ) , making it an important player in activation of pro-inflammatory response in autoimmune diseases (gao et al, ; kato et al, ) . cgas is also upregulated in a portion of sle patients (an et al, a,b) , and interestingly, hcq and cq can inhibit cgas binding to its ligands, e.g. dna, in vitro and in a t-cell line . importantly, inhibition of cgas activation results in reduced ifnb expression ( fig c) . inhibition of nadph oxidase nox is a protein complex involved in numerous pro-inflammatory signalling cascades, such as tumour necrosis factor alpha (tnfa)and interleukin (il)- b-induced cascades. activation of endosomal nox, which leads to the generation of reactive oxygen species (ros), requires the endocytic internalization and delivery to endosomes of cell surface ligand-receptor complexes (müller-calleja ) . hcq blocks the nox-mediated signalling cascades triggered by tnfa and il- b in monocytes by blocking translocation of gp phox, the catalytic subunit of nox, from the cytosol onto endosomal membranes without changing the endosomal ph (müller-calleja et al, ) . this inhibition prevents the correct assembly and activation of nox, hindering the downstream cellular events and the production of the pro-inflammatory cytokines tnfa and il- . hcq also prevents the redistribution of tlr from the er to endosomes, which is necessary to mediate the inflammatory response (müller-calleja et al, ) ( fig b) . inhibition of ca + signalling ca + mobilization from both the er and extracellular space into the cytoplasm and subsequent ca + -dependent signalling is an important mechanism to activate cells of the immune system, such as t and b cells (feske, ) . high cytoplasmic levels of ca + act as a second messenger for the activation of signalling pathways and transcription factors that regulate the expression and secretion of cytokines and other immune regulatory factors (izquierdo et al, ) . ca + release from the er can be impaired by hcq (goldman et al, ; xu et al, ; , leading to the inhibition of intracellular signals. in particular, t-cell and b-cell receptormediated intracellular ca + mobilization from both intracellular stores and the extracellular milieu is inhibited by hcq in a dosedependent manner (goldman et al, ) . this impairment of ca + mobilization is at least partially caused by the reduction of the ca + stored intracellularly and the inability to replenish these intracellular stores with extracellular ca + (goldman et al, ) . this further enhances its negative impact on the ca + -dependent signalling pathways ( fig c) (feske, ) . the precise mechanism of hcq-induced reduction of internal ca + mobilization remains unknown. however, it has been shown that hcq does not reduce the availability of inositol , , -trisphosphate, but rather the binding to its intracellular receptors that promotes ca + release (misra et al, ) . autoimmunity is characterized by an overreaction of the immune system (smith & germolec, ) , which is linked to both innate and adaptive immunity (mescher, ) . the innate immune system is responsible for the initial recognition of pathogens, which is mostly carried out by antigen-presenting cells (apcs), e.g. dendritic cells, and eventually triggers the activation of the adaptive immune system (mescher, ) . in particular, when apcs get directly activated through exposure to pathogen-associated molecular patterns, they initiate both cell-and antibody-mediated immune responses, which are mediated by the t and b cells, respectively (christmas, ) . the cell-mediated response is executed by t cells that get activated by apcs through antigen presentation at their surface via mhc molecules. in contrast, b cells are activated through t helper (th) cells and cytokines that are secreted by apcs (mescher, ) . activated b cells produce and secrete additional pro-inflammatory cytokines and antibodies to further stimulate the immune reaction (mescher, ) . hcq and cq negatively regulate many aspects of these innate and adaptive immune responses by reducing inflammation, and ultimately the severity of autoimmune diseases (fig ) . through the inhibition of endosomal tlr signalling, hcq and cq treatment decreases the levels of pro-inflammatory cytokines produced by peripheral mononuclear cells in the blood, including ifnc (van den borne et al, ), tnfa (picot et al, ; van den borne et al, ; jang et al, ) , il- (picot et al, ; sperber et al, ; jang et al, ) , il- (sperber et al, ; van den borne et al, ; jang et al, ) and il- (landewe et al, ) . the reduction of tlr signalling-mediated activation of immune cells by both drugs consequently decreases the aberrant immune response and diminishes inflammation symptoms observed in rheumatic patients (da silva et al, ) . in addition to directly inhibiting endosomal tlr signalling, cq and hcq can interfere with the intracellular signals that lead to both the release of phorbol esterinduced arachidonic acid and the block of pro-inflammatory cytokines secretion (e.g. tnfa and il- ) in mouse macrophages (bondeson & sundler, ) . in particular, activation of phospholipase a by phorbol esters, but not by ca + , is inhibited by hcq and cq, which blocks the synthesis of arachidonic acid. furthermore, these compounds negatively impact the generation of zymosan-induced formation of inositol phosphates, a product of phospholipase c activity (matsuzawa & hostetler, ) , suggesting that they have an inhibitory effect on this enzyme as well (bondeson & sundler, ) . hcq also inhibits ca + -activated k + channels in macrophages, and consequently k + efflux, which could result in impaired inflammasome activation and pro-inflammatory cytokine release (eugenia schroeder et al, ) . high levels of pro-inflammatory cytokines are a central characteristic of the ra pathogenesis (mcinnes & schett, ; blasius & beutler, ; pollard et al, ; schinnerling et al, ; muskardin & niewold, ) . in particular, stimulatory cytokines (i.e. il- , il- , il- , il- , il- , il- and type i and ii ifn for t cells, and b-cell activating factor (baff) for b cells) activate t and b cells, which in turn produce pro-inflammatory cytokines and autoantibodies, respectively. pro-inflammatory cytokines contribute to ra pathogenesis by promoting autoimmunity, maintaining chronic inflammatory synovitis and stimulating the destruction of joint tissues. they also play a role in the maturation and activation of osteoclasts, the cells responsible for breaking down bone tissue (mcinnes & schett, ) . excessive production of baff, a cytokine essential for b-cell physiology, alters the immune tolerance by contributing to the maturation and survival of self-reactive b cells, the major source for autoantibodies contributing to joint inflammation (mahdy et al, ) . reduction of the high baff levels in the serum from ra patients by hcq (mahdy et al, ) improves symptoms of rads, both in animal models and in clinical trials (sun et al, ) . cytokines like baff, tnfa, ifna and ifnc are also major contributors to sle severity, by promoting b-cell survival and autoantibody production, and contributing to organ inflammation (rönnblom & elkon, ) . thus, the modulation of their levels represents a potential therapeutic avenue (rönnblom & elkon, ) . this is supported by a cohort study showing that treatment of sle patients with hcq results in a decrease of type i ifn levels and concomitant reduction of disease severity (willis et al, ) . hcq can also directly affect the production of autoantibodies by b cells through tlr inhibition. particularly, hcq interferes with the differentiation of memory b cells into antibody-producing plasmablasts, a subset of b cells, by inhibiting tlr activation (torigoe et al, ) . although the pathogenesis of pss is not fully understood yet, activation of exocrine gland epithelium cells is thought to lead to the release of pro-inflammatory cytokines such as ifna and ifnb (both type i ifn), il- and baff, and chemokines (retamozo et al, ) . these factors stimulate further activation of apcs, but also of t and b cells, which promotes inflammation and autoimmunity (retamozo et al, ) . only a few studies investigated hcq administration in pss patients. nonetheless, pss patients treated with hcq have a significant lower baff levels in the serum, and an improvement in saliva production (mumcu et al, ) , indicating that this drug might be a promising therapy for pss as well. through t-cell receptors (tcrs) on their surface, t cells recognize antigens that are presented by apcs and get activated (goldman et al, ) . this results in both their proliferation and the release of various cytokines, including il- and tnfa (sperber et al, ) . one important step in the signalling cascade downstream of tcrs is the increase of intracellular ca + levels, which is released from internal ca + storages such as the er. as previously mentioned, hcq can impair the release of ca + from the er, which consequently inhibits t-cell activation (goldman et al, ; xu et al, ; schmidt et al, ) . hcq also negatively influences the expression and activity of cd on t cells, which is needed for b-cell activation dewitte et al, ) . cd expression is controlled by the nuclear factor of activated t cells (nfat), a transcription factor that relies on ca + release from the er . by impairing this event, hcq inhibits nfat nuclear translocation, resulting in decreased gene expression of cd . altogether, these studies show that blocking ca + release from the er by hcq leads to a multilevel inhibition of t-and b-cell activation, thereby hindering the immune response (fig ) . alterations in autophagic activity play an important role in the pathophysiology of t-and b-cell-mediated autoimmunity (weindel et al, ; van loosdregt et al, ; alessandri et al, ; mocholi et al, ; zhang et al, ) . in this context, autophagy is required to maintain cellular homeostasis in t cells (an et al, c) and autophagy deficiency impairs mhc class ii presentation and contributes to the generation of autoreactive t cells by thymic epithelial cells (levine et al, ) . moreover, plasma cells require autophagy to sustain immunoglobulin production and b-cell development (wu & adamopoulos, ). an imbalance within the t-cell populations, more specifically an increase in the number of th cells and a decrease in that of t reg cells, has been linked to pathogenesis of autoimmune diseases (yang et al, a; jadidi-niaragh & mirshafiey, ; Á lvarez-rodríguez et al, ) , including sle (an et al, c; Á lvarez-rodríguez et al, ) . this imbalance leads to an increased secretion of pro-inflammatory cytokines such as il- and il- , and a reduction of the levels of circulating factors like tgf-b, which suppresses inflammation and autoimmunity (an et al, c; geng et al, ) . this latter effect can be dampened with hcq and cq, as those drugs rebalance the th /t reg ratio ( geng et al, ) . mechanistically, this could be caused by an alteration of autophagy, as an induction of this process is observed in sle patients (an et al, c) . thus, an and colleagues thought to suppress hyperactivated autophagy by administrating hcq to lupus mlr/pr mice, an animal model for sle. in addition to lowering autophagic activity in this model, hcq rebalanced th and t reg cell numbers, which led to a decrease in pro-inflammatory cytokine levels (fig ) and a concomitant augmentation of antiinflammatory cytokines, resulting in the suppression of the autoimmune response (an et al, c) impact of nox inhibition on the immune system nox inhibition by hcq impairs the production of pro-inflammatory cytokines and the correct distribution of tlr , thereby dampening the immune response (müller-calleja et al, ) . this inhibition also positively affects nitric oxide (no) bioavailability (gómez-guzmán et al, ) . no is involved in a multitude of physiologic functions, including the regulation of blood vessel tone and vasodilation, and is rapidly inactivated by ros (nagy et al, ) . in sle patients, no bioavailability is severely lowered by high ros levels, particularly o À , resulting in endothelial dysfunction (griendling & alexander, ; landmesser & harrison, ; gómez-guzmán et al, ) . by blocking nox, the major producer of o À in the vascular wall, hcq treatment reduces ros levels and helps to prevent endothelial dysfunction in a mouse model for sle (gómez-guzmán et al, ) . in agreement with this concept, nox inhibition by hcq reduces thrombus formation, which is a well-known clinical manifestation in sle, in a venous thrombus mouse model (mü ller-calleja et al, ; miranda et al, ) (fig ) . thus, at the cellular level, hcq and cq inhibit antigen presentation, nox signalling, b-and t-cell activation, and rebalance t reg / th cell ratio. these multifaceted effects on different immune cells synergistically result in a decreased production and release of proinflammatory cytokines, a common hallmark of rads (fig ) . hcq is administered orally in tablet form as hydroxychloroquine sulphate . it is absorbed in the gastrointestinal tract (mclachlan et al, ) before being widely distributed throughout the body to muscles, liver, spleen, lungs, kidneys, pituitary and adrenal glands, and tissues that contain melanin (haładyj et al, ) . daily dosage of hcq ranges from to mg for rads, from to mg for dermatological disorders (ben-zvi et al, ), from to , mg in cancers (chude & amaravadi, ) and from to mg for various infectious diseases. its half-life in the body ranges between and days (mclachlan et al, ) , and - % of hcq is protein-bound (furst, ) , resulting in - % unbound, pharmacologically active drug (rang et al, ) . the majority of hcq is excreted through the kidneys, while the rest is metabolized by the liver or excreted through faeces (furst et al, ; haładyj et al, ) . contraindications for taking hcq are a history of retinopathy or visual field changes, hypersensitivity to -aminoquinoline compounds and long-term therapies in children (https://www.fda.gov/). hcq is, however, considered safe during pregnancy (kaplan et al, ; haładyj et al, ) . hcq ameliorates classical rad symptoms, such as skin problems and joint pain, predominantly by decreasing the inflammation reaction in patients (fig ) . in sle, hcq is given to patients as either a single or a combinatorial therapy together with steroids and immunosuppressive drugs, to improve patients' life expectancy by reducing lupus flares and accrual of organ damage (ponticelli & moroni, similarly, hcq treatment produces significant clinical improvement and functional capacity in ra patients (smolen et al, ; haładyj et al, ) . in ra, prevention of cartilage degradation, which causes joint destruction, is an important aspect of the therapeutic approach (kumar & clark, ) . cartilage degradation is mostly caused by pro-inflammatory cytokines, such as il- , il- and tnfa, and their production can be repressed by hcq treatment (picot et al, ; sperber et al, ; van den borne et al, ; jang et al, ; mcinnes & schett, ; da silva et al, ) . in vitro experiments have also established that cq inhibits proteoglycan turnover (fulkerson et al, ; ackerman et al, ; schug & kalbhen, ; rainsford et al, ) , and early autoradiographic studies following tritium-labelled hcq have revealed that this drug accumulates in the cartilage of mice (cecchi & porzio, ) . these findings and its water-soluble properties led to the proposition that hcq accumulates in the cartilage by binding acidic proteoglycans and protecting them from degradation by proteolytic enzymes (rainsford et al, ) . although an early study pointed out that cq and hcq can indeed inhibit cartilage breakdown, slowing down the disease progression and preventing further joint damage in ra patients (julkunen et al, ) , more recent investigations could not confirm a positive effects on joint damage (sanders, ; smolen et al, ; haładyj et al, ) . the therapeutic benefits of hcq administration on pss classical symptoms, e.g. sicca symptoms, remain controversial; some studies documented beneficial effects (tishler et al, ; rihl et al, ; yavuz et al, ; mumcu et al, ) , while others reported none (gottenberg et al, ; yoon et al, ; wang et al, ) . hcq treatment, however, ameliorates extraglandular symptoms (fox et al, ; demarchi et al, ) , and according to the sjögren's syndrome foundation's clinical practice guidelines (https://www.sjo grens.org/), disease-modifying anti-rheumatic drugs are recommended to treat musculoskeletal pain, with hcq being the therapeutic approach of choice (carsons et al, ) . hcq also reduces immunological alterations of pss, such as decreased levels of immunoglobulins, erythrocyte sedimentation rate, serology and il- production (tishler et al, ; yavuz et al, ; mumcu et al, ) . furthermore, in a retrospective analysis, hcq administration to pss patients significantly improved saliva production (rihl et al, ) . this improvement was more pronounced in patients who were positive for autoantibodies against anti-a-fodrin, an intracellular filamentous cytoskeleton protein. while the cause for this difference remains unknown, a possible explanation is that hcq could improve saliva production by decreasing elevated levels of cholinesterase, an enzyme that counteracts saliva production (dawson et al, ) . anti-viral effects the anti-viral function of hcq and cq has mainly been linked to their ability to increase the ph of the endosomal system and the trans-golgi network (tgn) (savarino et al, ) . thus, these drugs are able to inhibit cell entry of numerous viruses, as a low endosomal ph is required for the fusion of endocytosed virions with the limiting membrane of endosomes. in this context, cq and hcq decrease replication of viruses such as dengue virus (denv ), chikungunya virus, hepatitis a and c virus, influenza a virus, zika virus, severe acute respiratory syndrome coronavirus (sars-cov) and borna disease virus in cellular models (bishop, ; gonzalez-dunia et al, ; keyaerts et al, ; vincent et al, ; blanchard et al, ; de clercq, ; eng et al, ; di trani et al, ; sourisseau et al, ; khan et al, ; ashfaq et al, ; boonyasuppayakorn et al, ; farias et al, ; delvecchio et al, ; shiryaev et al, ) . for some viral structural proteins, a maturation step involving post-translational modification and/or processing in the tgn is crucial for their function and ultimately for the assembly of infectious viral particles, e.g. glycosylation of hiv gp (tsai et al, ; savarino et al, ) or cleavage of the denv prm protein (randolph et al, ) . glycosylation in the tgn is also required for the correct assembly of ace , the entry receptor for sars-cov (vincent et al, ) . thus, hcq and cq contribute to inhibit viral infections by neutralizing the ph of intracellular organelles, interfering with important processes required for viral life cycle. although hcq and cq have shown beneficial therapeutic effects in animal models for denv , hepatitis c virus, avian influenza a virus, zika virus and sars-cov infections, clinical trials have so far failed to conclusively prove their anti-viral potential in humans (rodrigo et al, ; fragkou et al, ; mckee et al, ) . this might be due to the fact that drug concentrations required to deacidify intracellular compartments cannot easily be reached in humans (al-bari, ) . therefore, neither hcq nor cq is currently recommended as anti-viral drugs (rodrigo et al, ) . during the sars-cov- pandemic in , the need to find an effective medication has brought major attention to hcq and cq due to their ability to both inhibit viral infections and dampen the massive cytokine response that is observed in sars-cov- -infected patients (badgujar et al, ; ibáñ ez et al, ; moore & june, ) . the effectiveness of hcq and cq against sars-cov- , however, has so far not been proven in humans, and the results at the time that this review was completed were still controversial (boulware et al, ; fragkou et al, ) . ª the authors embo molecular medicine : e | anti-cancer therapy cq and hcq are being increasingly used in clinical trials to treat cancer (https://clinicaltrials.gov/). because high doses are required to achieve anti-tumoural effects in monotherapies, they are often used in combination with radiotherapy and/or other chemotherapeutical drugs (plantone & koudriavtseva, ). we briefly discuss here possible mechanisms of action for hcq and cq in cancer. for a more detailed discussion on this topic, more specific reviews are available (manic et al, ; pascolo, ; levy et al, ; shi et al, ; verbaanderd et al, ) . elevated autophagic activity is crucial for tumour cell survival and growth as it supplies the high demand of nutrients within a developed tumour (amaravadi et al, ) . this is especially relevant for autophagy-dependent cancers that rely on this pathway when faced with metabolic stress. consequently, hcq or cq treatment has been successful in regressing the growth of some of those cancers in preclinical studies (e.g. with ras pathway mutations (guo et al, ; lock et al, ) , such as specific pancreatic cancers (mancias & kimmelman, ; yang et al, b; sousa et al, ) , or braf-driven tumours (levy et al, ; strohecker et al, ; xie et al, ) . the effectiveness of hcq and cq in cancer therapy is, however, controversial. in animal models, hcq dosages are often mg/kg/day or higher, which is too high to be administered in humans (pascolo, ) , and with lower dosages, autophagy is not sufficiently inhibited to achieve tumour regression (pascolo, ) . moreover, some cancer cells (e.g. derived from breast tumours or melanomas or kras-driven cancer cell lines) have shown cq-mediated cell growth inhibition that was independent of autophagy (maycotte et al, ; maes et al, ; eng et al, ) . various cancer cells express high levels of tlr , e.g. breast and prostate cancer cells (merrell et al, ; verbaanderd et al, ) , which is linked to cancer invasiveness in vitro and associated with poor prognosis (väisänen et al, ; verbaanderd et al, ) . tlr -mediated nf-jb signalling is required for cancer cell migration and proliferation in gastric cancer cell models, which is inhibited by cq (zhang et al, ) . the exact molecular mechanism of tlr signalling inhibition in cancer cells remains unknown. another mechanism by which hcq affects cancer growth is by modulating the immune system. tumour-associated macrophages (tams), which are phenotypically described as m macrophages, play a role in promoting tumour growth and immune escape, angiogenesis and metastasis (mantovani et al, ; li et al, ) . in contrast, tumour killing macrophages (m macrophages) have an opposite effect and are activated by cytokines such as ifnc, which are released from t cells (de palma & lewis, ; ostuni et al, ) . interestingly, in a melanoma-bearing mouse model, intraperitoneal injection of mg/kg cq effectively inhibited melanoma growth in a t-cell-dependent manner, and prolonged animal survival (chen et al, ) . mechanistically, cq can switch tams into m macrophages by raising lysosomal ph, and thereby mobilizing lysosomal ca + through upregulation of the lysosomal ca + channel mucolipin . the release of lysosomal ca + then activates the p and nf-jb pathways, but also the transcription factor eb, resulting in an enhanced anti-tumour t-cell response (chen et al, ) . by stimulating the t-cell-mediated immune response and simultaneously decreasing immune inhibitory cells, including tams and t regs , and cytokines such as tgf-b and il- , cq treatment reduced breast cancer growth and prolonged mice survival in a breast xenograft model . another important aspect of anti-cancer immunity is the activation of immune cells by sensing danger signals (e.g. hmgb ). danger signals are subsequently recognized by receptors, such as tlr on dendritic cells (apetoh et al, ) . one function of tlr is to preserve engulfed tumour antigens from enhanced degradation, and thereby favour antigen presentation. the loss of in rads, hcq treatment predominantly alleviates the symptoms (purple boxes) by inhibiting the production and release of pro-inflammatory cytokines. as a consequence, hcq diminishes skin conditions. there are also indications that hcq both decreases cartilage degradation and consequently reduces joint and muscle pain, and helps to restore saliva production. usage of hcq can cause side effects (orange boxes); the most common are gastrointestinal disturbances, skin discoloration, cutaneous eruptions and elevated muscle enzymes, whereas retinopathy, cardiac myopathy and myotoxicity are rare, but severe. of embo molecular medicine : e | ª the authors antigen presentation capacity in tlr -deficient dendritic cells can be restored by cq, possibly by raising lysosomal ph, which contributed to tumour size reduction in a tlr À/À thymoma mouse model (apetoh et al, ) . along these lines, cq reduced breast cancer growth in mice after irradiation by enhancing apoptotic and immunogenic tumour cell death (ratikan et al, ) . the enhanced immune response was attributed to a decreased degradation of tumour antigens in dendritic cells, resulting in an increased antigen presentation (ratikan et al, ) . hcq and cq can also inhibit cxcl /cxcr signalling, which is involved in chemotaxis and adhesion of tumour cells and of growth factors secretion that are key for cancer progression (sun et al, ; kim et al, ; verbaanderd et al, ) . moreover, hcq and cq interfere with the activation of growth-promoting pathways in cancer stem cells, thereby suppressing the regrowth of tumours (li et al, ; balic et al, ; choi et al, ) . multiple reports further describe the mechanisms by which cq triggers cell death in tumour cells. cq induces apoptosis of cancer cells by either stimulating the mitochondrial apoptotic pathway (du jiang et al, ) or activating the p -dependent transcription of pro-apoptotic genes (zhou et al, ; loehberg et al, loehberg et al, , maclean et al, ; kim et al, ; bieging et al, ) . additionally, several studies have suggested that cq intercalates into dna and disturbs chromatin topology (o'brien et al, ; sternglanz et al, ; field et al, ; yin et al, ) , which could lead to an impairment in dna repair mechanisms, and in turn cause dna damage and enhance cell death (michael & williams, ; liang et al, ; weyerhäuser et al, ) . besides directly targeting tumour cells, cq also affects tumour angiogenesis by altering endothelial cell functionality. cq administration leads to notch accumulation in endothelial cell endosomes, stimulating the downstream signalling that leads to tumour vessel normalization, and resulting in reduced tumour invasion and metastasis (maes et al, ) . therefore, cq also improves the delivery and efficacy of other chemotherapeutics (maes et al, ) . hcq and cq thus show potential in inhibiting tumour growth and modulating tumour immune response through various mechanisms. it is, however, important to reiterate that the doses used to achieve relevant effects in cancer therapies are often substantially higher than the doses used to treat rads. moreover, when treating cancer or viral infections, one has to keep in mind that hcq and cq also have immune suppressive functions that could negatively influence its beneficial effect for the patients. side effects of hcq treatment are rare, but nonetheless exist, and can be very serious, especially during prolonged administration (haładyj et al, ) . in table ev , we provide a comprehensive overview of the known side effects caused by hcq in rads and their prevalence. overall, the most common side effects in rad patients taking hcq or cq are gastrointestinal disturbances, skin discolorations, cutaneous eruptions and elevated muscle enzymes. although rare, retinopathy, neuromuscular and cardiac toxicities (fig ) are the most serious and life-threatening side effects potentially triggered by hcq (plantone & koudriavtseva, ) . prolonged administration of hcq or cq can cause retinopathy and loss of retinal function that, when ignored, can result in permanent vision loss (jorge et al, ) . the primary site of toxicity in the retina is the photoreceptor layer, with secondary degeneration occurring later in retinal pigment epithelium (rpe) cells (de sisternes et al, ; yusuf et al, ) . some studies offer a potential explanation for this severe side effect. by inhibiting the lysosomal degradation capacity and possibly endocytosis in rpe cells, hcq and cq are preventing the degradation of old and spent outer segments of photoreceptors in the rpe, a process that is required to maintain its function and preserve vision (kevany & palczewski, ; yusuf et al, ) . furthermore, hcq entrapment in the rpe might lead to an accumulation of lipofuscin, which is associated with photoreceptor function impairment and consequent vision loss (kevany & palczewski, ; yusuf et al, ) . it has been speculated that, due to this entrapment, retinopathy still continues in some cases after cessation of hcq treatment (michaelides et al, ) . accumulation of cq in the pigmented ocular tissue, which comprises rpe cells, the iris, the choroid and the ciliary body, and eventually in the retina, was also observed in rhesus monkeys when cq was administered for months (rosenthal et al, ) . this caused an initial damage to the photoreceptors and the ganglion cells, followed by a disruption of both the rpe and choroid, which ultimately led to visual impairments and retinopathy (rosenthal et al, ) . high levels of hcq inhibit the function of the organic anion transporting polypeptide a (oatp a ), a plasma membrane importer expressed in many tissues, including rpe cells (xu et al, ) . in particular, oatp a transports all-trans-retinol (atrol), a retinol precursor essential for the classic visual cycle (chan et al, ) , into rpe cells. by blocking this transporter, hcq causes an extracellular accumulation of atrol and disrupts the classic visual cycle (xu et al, ) . cardiac side effects and myotoxicity hcq can cause acute and chronic cardiac adverse effects (chatre et al, ) . acute adverse effects are linked to a very high dose of hcq, which provokes a block of na + and ca + channels. this inhibition can lead to membrane-stabilization effects in cardiac muscle cells, which in turn causes conduction disturbances with atrioventricular block and qrs interval widening (white, ) . chronic adverse effects are connected to long-term treatment with a high cumulative dose of hcq (chatre et al, ) . as described above, hcq treatment impairs the degradative activity of lysosomes, which leads to an accumulation of material such as glycogen and phospholipids in their interior (chatre et al, ) . in myocytes, this causes a vascularization of the cytoplasm and myofibrillar disorganization, which contributes to the development of cardiac myopathy and myocardial fibrosis (yogasundaram et al, ) . this phenomenon can also be seen in the fabry and danon lysosomal storage diseases, which have similar phenotypes (roos et al, ; d'souza et al, ; chatre et al, ) . moreover, hcq-mediated accumulation of autophagosomes in muscles and peripheral nerves can lead to myotoxicity or myotoxicity combined with peripheral nerve dysfunction (shukla et al, hcq is nowadays widely used for the treatment of rads and has shown great success in improving the quality of life of many patients. over the years, research on the molecular and cellular mode of action of hcq (and cq) revealed that this compound modulates molecular processes and cellular responses in multiple ways. at least four mechanisms of action that, directly or indirectly, influence the immune system by synergistically dampening pro-inflammatory responses, have been described. although lysosomal inhibition and autophagy impairment are the most studied, hcq also influences other important immune regulatory pathways by inhibiting specific steps, such as activation of endosomal tlr-, cgas and nox signalling and ca + mobilization for the er. the beneficial therapeutic effect of hcq in rads probably lies in its multifaceted properties, which also makes it a promising candidate in other medical fields, such as oncology (onorati et al, ) and microbiology (savarino et al, ; cortegiani et al, ; yao et al, ) . generally, hcq is considered a safe drug with low prevalence of side effects. these side effects nevertheless exist and can impact the life of a patient tremendously. among them, the most severe, i.e. retinopathy and cardiomyopathy, is linked to the induced lysosomal activity inhibition. this suggests that the unwanted negative effects of hcq could be due to its lysosomotropic properties. in this context, it has been reported that the effect of hcq on endosomal and lysosomal ph at therapeutic concentrations is negligible (ku znik et al, ) and that the ph changes observed in vitro might not reflect the in vivo reality. therefore, a higher dose of hcq (or a higher cumulative dose) could lead to a ph increase in the compartments of the endolysosomal system and thus cause more side effects (latasiewicz et al, ; jorge et al, ) . the well-documented list of side effects caused by hcq during the treatment of rads should be considered when using hcq to treat other pathologies such as cancer (onorati et al, ) , neurodegenerative disorders (hedya et al, ) , metabolic diseases (pasquier, ) and microbial infections (savarino et al, ) , especially since treatment of some pathologies requires high hcq doses (leung et al, ) . while the search for a unifying mechanism of action for hcq is tempting, current knowledge shows that this small molecule has more than a single target. as a result, future research should aim at identifying potential additional cellular and organismal pathways specifically modulated by hcq. the mechanisms by which hcq causes side effects could also provide important information. increasing our understanding of hcq mode of action would improve patient outcome by promoting therapeutic benefits while reducing side effects. (i) investigate whether all hcq modes of action described with in vitro experiments are relevant in patients, and whether one of these mechanisms is predominantly causing the observed side effects. (ii) determine whether hcq has other molecular effects than the ones described, which could help to better understand hcq treatment outcomes in patients. (iii) chemically improve hcq to make it more effective and less toxic, and thereby render it more suitable for the treatment of other diseases (e.g. specific cancers). (iv) understand how the anti-inflammatory role of hcq influences the anti-viral and anti-tumorigenic action of this drug in patients, and whether this could explain the observed discrepancies between the in vitro and in vivo results. lupus erythematosus but not primary antiphospholipid syndrome recent insights into the function of autophagy in cancer cutting edge: antimalarial drugs inhibit ifn-b production through blockade of cyclic gmp-amp synthase-dna interaction expression of cyclic gmp-amp synthase in patients with systemic lupus erythematosus antimalarial drugs as immune modulators: new mechanisms for old drugs chloroquine autophagic inhibition rebalances th /treg-mediated immunity and ameliorates systemic lupus erythematosus inhibition of cyclic gmp-amp synthase using a novel antimalarial drug derivative in trex -deficient mice toll-like receptor -dependent contribution of the immune system to anticancer chemotherapy and radiotherapy lysosomotropic agents as hcv entry inhibitors long term effectiveness of antimalarial drugs in rheumatic diseases hydroxychloroquine for covid- : a review and a debate based on available clinical trials/case studies chloroquine targets pancreatic cancer stem cells via inhibition of cxcr and hedgehog signaling beta -integrin-c-met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes hydroxychloroquine: from malaria to autoimmunity the biology of cachectin/tnf -a primary mediator of the host response unravelling mechanisms of p -mediated tumour suppression examination of potential inhibitors of hepatitis a virus uncoating hepatitis c virus entry depends on clathrin-mediated endocytosis intracellular toll-like receptors antimalarial drugs inhibit phospholipase a activation and induction of interleukin b and tumor necrosis factor a in macrophages: implications for their mode of action in rheumatoid arthritis amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- rna released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll-like receptor accumulation of autophagosomes confers cytotoxicity sjögren's foundation clinical practice guidelines affinité de l-hydroxychloroquine pour les tissues articulaires human organic anion transporting polypeptide a (oatp a ) mediates cellular uptake of all-trans-retinol in human retinal pigmented epithelial cells accidental hydroxychloroquine overdose resulting in neurotoxic vestibulopathy cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward m phenotype antigen presenting cells and mechanisms of antigen presentation chloroquine eliminates cancer stem cells through deregulation of jak and dnmt toll-like receptors: sensors that detect infection targeting autophagy in cancer: update on clinical trials and novel inhibitors a systematic review on the efficacy and safety of chloroquine for the treatment of covid- hydroxychloroquine: a multifaceted treatment in lupus hydroxychloroquine therapy in patients with primary sjögren's syndrome may improve salivary gland hypofunction by inhibition of glandular cholinesterase potential antivirals and antiviral strategies against sars coronavirus infections macrophage regulation of tumor responses to anticancer therapies localization of damage in progressive hydroxychloroquine retinopathy on and off the drug: inner versus outer retina, parafovea versus peripheral fovea chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models catalan pellet a et al ( ) primary sjögren's syndrome: extraglandular manifestations and hydroxychloroquine therapy cd induces interleukin- secretion by kidney tubular epithelial cells under hypoxic conditions: inhibition by chloroquine different ph requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza a viruses mechanism and medical implications of mammalian autophagy danon disease clinical features, evaluation, and management antitumor and antimetastatic activities of chloroquine diphosphate in a murine model of breast cancer in vitro inhibition of human influenza a virus replication by chloroquine macroautophagy is dispensable for growth of kras mutant tumors and chloroquine efficacy autophagy: a lysosomal degradation pathway with a central role in health and disease pro-inflammatory ca++-activated k+ channels are inhibited by hydroxychloroquine antiviral activity of chloroquine against dengue virus type replication in aotus monkeys anti-tnfa therapy of rheumatoid arthritis: what have we learned? calcium signalling in lymphocyte activation and disease inhibition of precursor incorporation into nucleic acids of mammalian tissues by antimalarial aminoquinolines v-atpase and osmotic imbalances activate endolysosomal lc lipidation mechanism of action of hydroxychloroquine as an antirheumatic drug treatment of primary sjögren's syndrome with hydroxychloroquine: a retrospective, open-label study in vitro hexosamine depletion of intact articular cartilage by e-prostaglandins: prevention by chloroquine pharmacokinetics of hydroxychloroquine and chloroquine during treatment of rheumatic diseases dose-loading with hydroxychloroquine improves the rate of response in early, active rheumatoid arthritis: a randomized, double-blind six-week trial with eighteen-week extension activation of cyclic gmp-amp synthase by self-dna causes autoimmune diseases reduced let- f in bone marrow-derived mesenchymal stem cells triggers treg/th imbalance in patients with systemic lupus erythematosus hydroxychloroquine inhibits calcium signals in t cells: a new mechanism to explain its immunomodulatory properties chronic hydroxychloroquine improves endothelial dysfunction and protects kidney in a mouse model of systemic lupus erythematosus mechanism of borna disease virus entry into cells effects of hydroxychloroquine on symptomatic improvement in primary sjögren syndrome: the joquer randomized clinical trial oxidative stress and cardiovascular disease a comparison of the stimulatory activities of lymphoid dendritic cells and macrophages in t proliferative responses to various antigens activated ras requires autophagy to maintain oxidative metabolism and tumorigenesis cpg-dna-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation antimalarials -are they effective and safe in rheumatic diseases hydroxychloroquine antiparkinsonian potential: nurr modulation versus autophagy inhibition hydroxychloroquine and chloroquine in covid- : should they be used as standard therapy? chloroquine attenuates tlr /ifn-b signaling in cultured normal human mesangial cells: a possible protective effect against renal damage in lupus nephritis ataxia telangiectasiamutated and p are potential mediators of chloroquine-induced resistance to mammary carcinogenesis akt and p are potential mediators of reduced mammary tumor growth by chloroquine and the mtor inhibitor rad increased autophagy in cd + t cells of rheumatoid arthritis patients results in tcell hyperactivation and apoptosis resistance the role of t cell interleukin- in conducting destructive arthritis: lessons from animal models antagonism of immunostimulatory cpgoligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds targeting lysosomal degradation induces p -dependent cell death and prevents cancer in mouse models of lymphomagenesis tumor vessel normalization by chloroquine independent of autophagy therapeutic potential of hydroxychloroquine on serum b-cell activating factor belonging to the tumor necrosis factor family (baff) in rheumatoid arthritis patients targeting autophagy addiction in cancer chloroquine and hydroxychloroquine for cancer therapy tumourassociated macrophages as treatment targets in oncology autophagy proteins regulate erk phosphorylation inhibition of lysosomal phospholipase a and phospholipase c by chloroquine and , -bis(diethylaminoethoxy)a, bdiethyldiphenylethane chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy cytokines in the pathogenesis of rheumatoid arthritis candidate drugs against sars-cov- and covid- bioavailability of hydroxychloroquine tablets in patients with rheumatoid arthritis toll-like receptor agonists promote cellular invasion by increasing matrix metalloproteinase activity junqueira's basic histology text & atlas choloroquine inhibition of repair of dna damage induced in mammalian cells by methyl methanesulfonate retinal toxicity associated with hydroxychloroquine and chloroquine: risk factors, screening, and progression despite cessation of therapy hydroxychloroquine reverses the prothrombotic state in a mouse model of antiphospholipid syndrome: role of reduced inflammation and endothelial dysfunction chloroquine, quinine and quinidine inhibit calcium release from macrophage intracellular stores by blocking inositol , , -trisphosphate binding to its receptor autophagy fights disease through cellular self-digestion methods in mammalian autophagy research autophagy is a tolerance-avoidance mechanism that modulates tcr-mediated signaling and cell metabolism to prevent induction of t cell anergy cytokine release syndrome in severe covid- hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase salivary and serum b-cell activating factor (baff) levels after hydroxychloroquine treatment in primary sjögren's syndrome type i interferon in rheumatic diseases central role of nitric oxide in the pathogenesis of rheumatoid arthritis and sysemic lupus erythematosus cardiac safety of off-label covid- drug therapy: a review and proposed monitoring protocol reactions of quinine, chloroquine, and quinacrine with dna and their effects on the dna and rna polymerase reactions fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents macrophages and cancer: from mechanisms to therapeutic implications rational drug therapy recommendations for the treatment of patients with sjogren's syndrome chloroquine modulates inflammatory autoimmune responses through nurr in autoimmune diseases time to use a dose of chloroquine as an adjuvant to anticancer chemotherapies autophagy inhibitors review: hydroxychloroquine and chloroquine for treatment of sars-cov- (covid- ) chloroquine normalizes aberrant transforming growth factor beta activity in cystic fibrosis bronchial epithelial cells chloroquine inhibits tumor necrosis factor production by human macrophages in vitro current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review interferon-c and systemic autoimmunity hydroxychloroquine in systemic lupus erythematosus (sle) effect of weak bases on the intralysosomal ph in mouse peritoneal macrophages therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases acidotropic amines inhibit proteolytic processing of flavivirus prm protein rang and dale's pharmacology chloroquine engages the immune system to eradicate irradiated breast tumors in mice cytokines as therapeutic targets in primary sjögren syndrome treatment of sicca symptoms with hydroxychloroquine in patients with sjögren's syndrome elevated golgi ph impairs terminal n-glycosylation by inducing mislocalization of golgi glycosyltransferases clinical evidence for repurposing chloroquine and hydroxychloroquine as antiviral agents: a systematic review cytokines as therapeutic targets in sle chloroquine cardiotoxicity: clinicopathologic features in three patients and comparison with three patients with fabry disease chloroquine retinopathy in the rhesus monkey clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review a review of controlled clinical trials examining the effects of antimalarial compounds and gold compounds on radiographic progression in rheumatoid arthritis effects of chloroquine on viral infections: an old drug against today's diseases? anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors the role of interleukin- signalling and its therapeutic blockage in skewing the t cell balance in rheumatoid arthritis chloroquine inhibits human cd + t-cell activation by ap- signaling modulation mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology influence of chloroquine and other substances on the collagenolytic activity in human osteoarthritic cartilage in vitro inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin historical review: problematic malaria prophylaxis with quinine research progress of hydroxychloroquine and autophagy inhibitors on cancer repurposing of the antimalaria drug chloroquine for zika virus treatment and prophylaxis pearls & oy-sters: hydroxychloroquine-induced toxic myopathy mimics pompe disease: critical role of genetic test hydroxychloroquine decreases th -related cytokines in systemic lupus erythematosus and rheumatoid arthritis patients introduction to immunology and autoimmunity eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: update characterization of reemerging chikungunya virus pancreatic stellate cells support tumour metabolism through autophagic alanine secretion selective regulation of cytokine secretion by hydroxychloroquine: inhibition of ª il- -a) and il- in human monocytes and t cells nuclear magnetic resonance studies of the interaction of chloroquine diphosphate with adenosine -phosphate and other nucleotides autophagy sustains mitochondrial glutamine metabolism and growth of brafv e -driven lung tumors baff-targeting therapy, a promising strategy for treating autoimmune diseases cxcl / cxcr / cxcr chemokine axis and cancer progression cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway hydroxychloroquine treatment for primary sjoegren's syndrome: its effect on salivary and serum inflammatory markers hydroxychloroquine efficiently suppresses inflammatory responses of human class-switched memory b cells via toll-like receptor inhibition inhibition of human immunodeficiency virus infectivity by chloroquine expression of toll-like receptor- is associated with poor progression-free survival in prostate cancer chloroquine and hydroxychloroquine equally affect tumor necrosis factor-a, interleukin , and interferon-c production by peripheral blood mononuclear cells repurposing drugs in oncology (redo) -chloroquine and hydroxychloroquine as anti-cancer agents chloroquine is a potent inhibitor of sars coronavirus infection and spread is hydroxychloroquine effective in treating primary sjogren's syndrome: a systematic review and metaanalysis b cell autophagy mediates tlr -dependent autoimmunity and inflammation re-purposing chloroquine for glioblastoma: potential merits and confounding variables cardiotoxicity of antimalarial drugs effect of hydroxychloroquine treatment on pro-inflammatory cytokines and disease activity in sle patients: data from lumina (lxxv), a multiethnic us cohort the total synthesis of quinine hydroxychloroquine inhibits cd expression in cd + t lymphocytes of systemic lupus erythematosus through nfat, but not stat , signaling atg overcomes senescence and promotes growth of brafv e-driven melanoma chloroquine inhibits ca signaling in murine cd + thymocytes chloroquine and hydroxychloroquine are novel inhibitors of human organic anion transporting polypeptide a recovery of the immune balance between th and regulatory t cells as a treatment for systemic lupus erythematosus pancreatic cancers require autophagy for tumor growth hydroxychloroquine inhibits the differentiation of th cells in systemic lupus erythematosus in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine improves dry eye symptoms of patients with primary sjogren's syndrome cpg motifs in bacterial dna activate leukocytes through the ph-dependent generation of reactive oxygen species kinetics of dna binding with chloroquine phosphate using capacitive sensing method hydroxychloroquine-induced cardiomyopathy: case report, pathophysiology, diagnosis, and treatment induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured arpe- cells effect of hydroxychloroquine treatment on dry eyes in subjects with primary sjögren's syndrome: a double-blind randomized control study chloroquine inhibits mgc gastric cancer cell migration via the toll-like receptor /nuclear factor kappa b signaling pathway chloroquine (cq) exerts anti-breast cancer through modulating microenvironment and inducing apoptosis autophagy in regulatory t cells: a double-edged sword in disease settings control of mammary tumor cell growth in vitro by novel cell differentiation and apoptosis agents decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to t cells license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited authors apologize in advance to those authors whose contributions have been omitted due to lack of space and felt that their work was one of the highlights. the authors declare that they have no conflict of interest. ackerman nr, jubb sn, marlowe sl ( ) effects of various antiinflammatory and anti-rheumatic agents on the synthesis, secretion, and key: cord- -l i rn authors: kim, hong-bumm; park, jung-ho; lee, seul ki; jang, soocheong (shawn) title: do expectations of future wealth increase outbound tourism? evidence from korea date: - - journal: tour manag doi: . /j.tourman. . . sha: doc_id: cord_uid: l i rn as international tourism is generally considered a luxury good, models to date have shared an understanding that demand is dependent on discretionary income. however, consumption theories predict that a shift in demand can be induced without changes in actual earnings when expectations for future income are adjusted. this presumes demand for international tourism can be influenced by “wealth effects” from real estate and financial assets. this study tested for the wealth effect on korean outbound travelers during the years between and . korea is a unique place to examine in that korean households possess housing assets and financial assets that are traded actively in markets. the results of this study favored the possibility of a significant wealth effect from housing on outbound travel demand, but not from financial assets. this may be explained by data sensitivity and the relative importance of financial assets in the korean people's wealth portfolios. implications and suggestions for future research are provided along with the findings of the study. literature on international tourism demand often shares an explicit assumption that demand is a function of real discretionary income. although it is unrealistic to think that expenditures on overseas travel could precede day-to-day necessities, the assumption in the literature directly follows a syllogism based on two fundamental premises. first, it is widely acknowledged that international tourism is considered a luxury good rather than a normal good (lim, ) , in that tourists must commit significant expenditures on costly goods such as airfare and hotels (bakkal & scaperlanda, ) . second, since it is a luxury good, demand should be dependent on the discretionary income of consumers (crouch, ) , which is the proportion of real earnings after normal expenses (i.e. food, living, medical) are deducted. accordingly, the concept that international tourism has a higher sensitivity to income than a normal good (income elasticity higher than unity) has been thoroughly tested by academics (lanza, temple, & urga, ; smeral, ) . these studies commonly found that the income elasticity of international tourism is greater than unity. concurrent with evidence that international tourism is indeed a luxury good, a number of studies have advocated using real discretionary or disposable income in tourism demand models whenever it is available rather than crude income per capita variants, such as gross domestic product or gross national income (durbarry and sinclair, ; lim, ; song, romilly, & liu, ) . however, while real disposable income may be the best income variable for determining international tourism demand, it is not sufficient proof that demand for international tourism is solely dependent on residual income after living expenses are deducted from total earnings. despite the high significance and compelling results (crouch, ) of said income variables, it is still possible that other income-related factors play a role in determining international tourism demand. certainly, the absolute availability of cash flows is likely to place a strict upper limit on tourism demand. if an individual or household does not have the wealth to purchase luxury goods, without doubt there will be less consumption. yet, we cannot safely extend this logic to suggest that demand will consistently lead to the consumption of tourism goods or travel by using a constant proportion of discretionary income. meanwhile, a stream of studies in the consumption literature has argued that consumer spending behaviors cannot be explained by current earnings alone, if at all. when consumer spending is not guided by extremes of myopia and liquidity constraints (shea, a) , the distribution of wealth consumption across various stages of a consumer's life depends not only on current earnings but also on their beliefs regarding their future earnings and permanent income. although movements in real discretionary income will undoubtedly increase international tourism demand, demand may also shift based on adjustments in adaptive expectations for future earnings or total wealth regardless of whether the potential gain is actually realized. in this light, the two popularly cited assets that guide expectations on future earnings of aggregate demand are housing and financial assets. the way in which changes in the value of these two prominent asset classes impact consumption is popularly known as the 'wealth effect.' the "wealth effect" has been examined numerous times since the seminal work done by ando and modigliani ( ) and has been empirically validated to some extent (case, quigley, & shiller, ) . nevertheless, there have been mixed results (attanasio, blow, hamilton, & leicester, ) , as well as ongoing discussions and efforts by researchers to scrutinize the relationship between consumption and unrealized wealth. modigliani ( ) suggested that, holding labor income fixed, a wealth gain of a dollar increases consumer spending by five cents roughly. carroll ( ) affirmed the wealth effect of both housing and wealth on consumption, with housing (non-financial) wealth having a greater influence on consumption. case et al. ( ) concluded that there was a significant increase in consumption explained by gains in housing and financial wealth in the us. however, contradicting results have also been reported. elliott ( ) concluded that only financial assets affect consumption, while both skinner ( ) and levin ( ) argued against the wealth effect from housing. lettau and ludvigson ( ) contended that most changes in asset values are "transitory and unrelated" to aggregate consumption. differences were also found across countries. case et al. ( ) found a significant housing wealth effect for a panel of developed countries, but an insignificant financial wealth effect. likewise, campbell and cocco ( ) observed a relatively large housing wealth effect using micro data in the uk. the persistent debates and discussion stem from a few inevitable challenges in verifying this relationship at the aggregate level, as the wealth of consumers may respond differently to a directional movement of asset prices. for example, an increase in housing prices may lead to enhanced expectations of future wealth (or in some cases an actual gain should the house be sold) for home owners. at the same time, housing costs such as rent would generally increase along with housing prices. consequently, an increase in housing prices would imply a net negative wealth effect for those who do not own their homes. this part of demand may decrease consumption based on unfavorable expectations regarding future income and costs. another example can be drawn from ogawa, kitasaka, yamaoka, and iwata ( ) , who found that the liquidity of unrealized wealth is also an important determinant of consumption. even if the asset appreciates the owners may not view it as a capital gain due to illiquidity of the asset, a lack of information on reference prices, or consumption needs such as continued residence. furthermore, even when asset appreciation is perceived as a net gain in wealth, the influence of the wealth effect on the propensity to consume may differ for individuals and households of various wealth levels. based on a michigan survey, starr-mccluer ( ) reported that only those who own a considerable amount of equity investments were significantly affected by movements in capital asset prices. this is intuitive as the amount of investment is likely to determine the potential income or return, and therefore govern expectations for future income. also, poterba ( ) posited that "wealth elasticities" may differ by product type, arguing that household consumption affected by wealth gains from the stock market mainly consisted of luxury goods. with the exception of households in significant need, there is certainly no need to consume more inferior or normal goods under the expectation that future income will increase. in effect, testing the wealth effect on aggregate consumption is complicated and difficult due to the sizable number of criteria to be met. in order to evenly distribute the wealth effect of price gains across individuals or households, various income classes must hold approximately equal proportions of non-financial and financial assets. a significant absolute amount of wealth should also be invested in these assets, even by low income households. further, the asset's value should remain relatively stable over time and be traded constantly while price information needs to be consistently disseminated. in order to ensure that owners actively respond to the wealth effect by changing consumption patterns, the effect on luxury goods should be tested rather than normal or inferior goods. although participation in international tourism is likely to be regarded as a luxury good for the most part, it is difficult to presume that on average those individuals and households who engage in international tourism have considerable investments in similar assets that are consistently traded in the market. to this end, the current study proposes that korean outbound travelers constitute a unique sample for testing the wealth effect on international tourism demand. the korean residential market is dominated by a unique housing type referred to as 'apartments,' which are analogous to condominiums in the us (ham & lee, ) . consistent price increases have led korean apartments to become an actively traded asset in the wealth portfolios of middle-class households. information on prices and rents are readily available (hwang, quigley, & son, ) . according to the national statistics office of korea, in the wealth of korean households was deeply concentrated in real estate ( . %) and financial assets ( . %), while other assets accounted for only . percent. in essence, the wealth of the korean people is concentrated in just two asset types, which are broadly held and actively traded with accurate and immediate dissemination of price information. therefore, the current study purports to investigate the wealth effect of housing and financial assets on korean outbound travel demand. it is crucial to examine this causal relationship in order to improve our understanding of deterministic tourism demand models (song & li, ) . although the wealth effect may not be universal or present in every economy, in cases where tourism demand is significantly affected by not only current earnings but also expectations on future earnings, estimation of tourism consumption behavior based on contemporaneous income may be erroneous. in turn, misunderstanding consumption behaviors implies ineffective and misguided pricing and marketing strategies for tourism products and targeting of demand segments. moreover, by identifying another driver of tourism efficiency in demand forecasting and modeling may be improved. implications and suggestions are presented along with the findings of the study. the housing market in korea is dominated by multi-family residential buildings referred to as 'apartments,' (kim, yang, yeo, & kim, ) . nearly percent of all new housing in the country between and were apartments, while as of they accounted for more than half of the housing stock in korea (ham & lee, ) . apartments are highly standardized in terms of amenities, floor plans, and building materials. the housing market shares similar expectations when information on an apartment's location, size, and year built are provided. homogeneous expectations combined with low transaction costs have led to easy trading and rental of apartments among individuals. as a result, the korean apartment market is deeper and more active than most other housing markets (hwang et al., ) . this high volume of active trading and ease of comparability facilitate the construction and dissemination of reliable reference prices. such information is usually offered free of cost and flows instantaneously as brokers are located in most neighborhoods and provide daily information on price movements. thus, individuals have a very accurate estimate of housing prices. an active market, consistent and stable price inflation, a lack of permanent rental accommodations in korea, and a high desire to own real estate (park, ) have all contributed to making apartments an important consumption asset. apartments are also considered a crucial investment in the wealth portfolios of the korean people. moreover, the proportion of real estate in wealth portfolios for various income groups in korea is consistent. according to the korea statistical office, in the highest income group (top percent) reported that . % of their wealth was in real estate whereas the bottom % reported . % in real estate assets. given the unique characteristic of korean apartments being actively traded investment assets, the efficiency of the korean housing market, and the broad ownership of apartments throughout various income classes, the appreciation of apartments is likely to be perceived as permanent wealth gains by the korean people. consequently, outbound tourism, a luxury good, may be consumed to a greater extent by aggregate demand when expectations of permanent increases in wealth are pervasive. thus, we developed the following hypothesis: hypothesis . appreciation of apartments will increase demand for outbound travel in korea. meanwhile, a similar effect can be anticipated from the korean stock market. although much smaller than the equity markets of developed countries, the korean stock exchange is fairly sizable in terms of market capitalization and is the th largest stock exchange in the world (kim, baek, noh, & kim, ) . the equity market involves frequent trading and is as liquid as the stock markets of many developed countries, such as the us, england, germany, and japan. the korea statistical office reported in that financial assets are consistently held by different income groups; the proportion of household wealth invested in the financial market is practically the same across society, with . % for the highest income group and . % for the lowest income group. the high volatility of the korean stock market has been noted (fratzscher & oh, ) and investments in the equity market by korean households are considerably lower than in the housing market. it is possible, however, that increases in financial wealth can also stimulate the consumption of outbound tourism if stock price increases are perceived as permanent wealth gains and consumption is adjusted according to the adaptive expectations of future earnings. therefore, the second hypothesis was developed based on the wealth effect from the stock market: hypothesis . appreciation of stocks will increase demand for outbound travel in korea. in order to examine the wealth effect from apartments and the stock market on korean outbound tourism demand, quarterly timeseries data from the first quarter of through the fourth quarter of (n ¼ ) was obtained from the following sources: number of outbound tourists from the korea tourism organization (kto), average household incomes and apartment sales price index from the bank of korea, real effective exchange rates from the bank for international settlements, composite korean stock price index from korea stock exchange, and singapore kerosene-type jet fuel spot price from the u.s. energy information administration. average household income and apartment sales price index were initially obtained as monthly data and were aggregated into quarterly observations for consistency. the singapore kerosene-type jet fuel spot price is the mean of platts singapore (mops), used as the jet fuel price index by most airlines in asia. the sample period starts in , which is the first year the korean government relaxed restrictions on international travel. fig. illustrates the trends of data series. summary statistics and pearson correlations of the data are provided in tables and , respectively. all the variables tend to be highly correlated, as they are collectively subject to greater movements of the economy and common causality. there are two noteworthy observations to be made based on the data in the figure and tables. price appreciation, and, therefore, cumulative returns on apartments, were greater than stocks during the sample period, while at the same time the movements were less volatile. in effect, it is possible that the appreciation of apartments is more likely to be perceived as a permanent gain in wealth than stock appreciation. it should also to be noted that incomes and the prices of apartments and stocks are highly and significantly correlated. when the wealth effect is significant, omitting asset variables and including only income as an explanatory variable is likely to cause a misspecification bias, which would result in overestimation of the income elasticity of demand. a log-linear specification, or elasticity model, tests for the wealth effects from apartment and stock market appreciation on outbound travel demand in south korea: lnðtourist t Þ¼aþb lnðincome t Þþb lnðex t Þþb lnðkospi t Þ þb lnðapt t Þþb lnðfuel t Þþb w x q w where tourist is the number of outbound travelers departing from south korea, income is the average household income index, ex is the real effective exchange rate that accounts for changes in relative purchasing power parity in both prices and currency valuation, kospi is the index value of the korean stock market, apt is the aggregate sales price index of apartments in korea, fuel is jet fuel price proxying for airfare, sq w are the quarter dummies, the afc dummy ( if :q e :q , elsewhere) for the asian financial crisis, / dummy for the september th attack ( if :q , elsewhere), and the sars dummy ( if : q , elsewhere) are dummy variables taking on the value of unity upon the occurrence of a macro event and zero if not, and t is the time subscript denoting the pertaining quarter. following a popular approach, the number of outbound tourist departures was used to measure tourism demand. income and purchasing power parities were expected to be significant in the model (song & li, ) . the hypotheses regarding the wealth effect from financial and housing assets were tested by coefficients on kospi and apt,b andb , respectively. following wang's ( ) approach, jet fuel price was used as a proxy for airfare while binary variables controlled for the effects of macro socioeconomic events on tourism demand, namely the asian financial crisis, the september th attack, and spread of the severe acute respiratory syndrome (sars). the use of a log-linear model allows easy interpretation of the results as the elasticity of demand where the units of data for the current study are difficult to interpret (wang, ), while the log-linear form is also widely advocated for its robustness against heteroscedasticity (wang, ) . before estimation, heteroscedasticity and multicollinearity were examined using the breuschepagan test and the variation inflation factor (vif). the breuschepagan test did not reject the null at pvalue of . , supporting the homoscedasticity assumption. in addition, no variable had a vif exceeding the potential problematic level of . we inferred that much of the collinearity expected from table is alleviated after the logarithmic transformation (buongiorno, ; grime & smith, ) . also, in order to address the concerns of endogeneity from omitted-variable bias we identified the factors that influence housing prices in asia as: construction costs, household income, household formation, housing supply, stock prices, and monetary supply (chen & patel, ) . among these factors, those that may have an effect on tourism demand (income and stock prices) entered the regression equation as explanatory variables as ln(income t ) and ln(kospi t ), relieving concerns of endogeneity. as autocorrelation of the dependent variables is expected, even using quarterly data, many studies employ a lagged dependent variable as an explanatory variable (witt & witt, ) . although including the lagged dependent variable does not create a bias in the coefficients, it is expected to underestimate the variance of the estimator in the presence of positive autocorrelation, thereby overstating the significance of the coefficients (wooldridge, ) . in order to conduct an efficient estimation and obtain reliable results, the current study implemented two different estimation techniques: praisewinsten and ordinary least squares (ols) with neweyewest standard errors. estimators and statistics from the praisewinsten procedure are asymptotically efficient and valid for autoregressive models of the first-order, while the neweyewest procedure provides a robust inference of serial correlations of a higher-order (traub & jayne, ) . more specifically, if the model to be estimated includes a first-order autoregressive disturbance term, the praisewinsten method transforms the error vector that yields serially uncorrelated classical disturbances (baltagi, song, jung, & koh, ) . in the presence of higher-order lags, the new-eyewest method allows for the robust inference of standard errors on the coefficients (ferson, sarkissian, & simin, ) and is standard in the economics literature (greene, ) . for the praisewinsten estimation, a cochraneeorcutt transformation was done on ( ) after estimation of the autocorrelation parameter, r. for data quarters t and t- this can be written as: where x w are all independent variables on the right-hand side of ( ). by expressing the first-order autocorrelation in the error term as r, the error term can be expressed as a markov scheme of an autoregressive and random component: where y t is serially independent random disturbances. substituting ( ) into ( ), the equation can be rewritten as an equivalent system: an estimation of ( ) using the generalized least squares (gls) method yields coefficients and a variance structure unaffected by autocorrelation parameter r. results of estimation by both praisewinsten and neweyewest procedures are displayed in table . the results are similar in general, representing the reliability of the model, although there are some differences in significance and magnitude of the coefficients for variables ex, apt and dummy variables, as noted. the since the neweyewest procedure is only expected to outperform the praisewinsten method in the presence of higher-order autocorrelation, inferences were subsequently made based on the results from the praisewinsten estimation method. the coefficients of income and real effective exchange rates were the greatest in size and highly significant, consistent with the results of preceding studies (lim, ) . a one percent increase in average household income caused . percent increase approximately, in the number of quarterly outbound travelers. the effect from purchasing parity changes was relatively smaller at . percent. as hypothesized, the income elasticity greater than unity confirmed that outbound travel is considered as a luxury good by the korean people. accordingly, the consumption of international travel increased at a rate greater than that of income. the coefficient of apt was significant and stable across both models. a one percent increase in apartment prices in korea caused an approximately . percent increase in the number of outbound travelers per quarter. although the magnitude of the effect on aggregate demand was less than one third of that from income, the results nevertheless serve as evidence of the effect of adjusted expectations of wealth on consumption patterns. korean demand for outbound tourism seems to not only rely on actual income, but also on expected future earnings based on housing price gains during the sample period. as a result, hypothesis was supported and the current study found that housing assets had a significant wealth effect on korean demand for outbound tourism. on the other hand, hypothesis was rejected in both models with a low probability. a number of potential factors may have contributed to this finding. one possibility is that the high volatility of stock prices might prevent owners from viewing the appreciation as a permanent gain. the insignificant results could also be attributed to inefficient estimation; quarterly data may have insufficient sensitivity as stock prices are highly volatile in nature, as seen in fig. . yet another possibility is that the wealth effect from financial assets could be insignificant at the aggregate level. this may be due to a comparably low allocation of relative wealth in the stock market, which accounts for less than % of total household wealth, whereas housing assets account for more than %. the smaller amount of total investments in equity stocks, which resulted in a trivial absolute increase in expected wealth, may also account for the result (cho, ) . among the quarter dummies, the third quarter, which includes the korean summer vacation season, was the most sizable and significant. the dummy variables for macro events, except for the september dummy, were significant. the asian financial crisis, the net effect of depressed income and purchasing parity, decreased outbound travel demand by roughly percent. severe acute respiratory syndrome (sars) decreased outbound travel demand by approximately percent. a possible explanation for the insignificant coefficient of the / dummy variable is the effect of substitution to other destinations. although trips to the us from korea significantly declined immediately after the / attack, total outbound demand may have been sustained by substituting other nearby destinations, such as southeast asia or oceania, that had a relatively lower perceived risk of terrorism (lim, ) . as observed, the effect of unrealized wealth, the net of income and purchasing power parity changes, seems to influence the outbound travel of the korean people. although the effect is rather small compared to the two most important variables that govern international travel, we nevertheless found a significant causal relationship between expectations of wealth and the decision to travel internationally. consequently, distinguishing wealth and income in travel demand may be an important task based on analytic results. even though the wealth effect may only be conditionally present for certain economies and asset types, korean economy tourism demand can be partly explained by appreciation in housing assets. for example, tourist surveys often utilize the income levels of tourists to explain tourism demand or expenditure (jang, bai, hong, & o'leary, ; jang, ismail, & ham, ) . however, in order to produce more efficient estimates of income and/or price effects on tourism goods, changes in net assets and the relative location of a person in the society's wealth distribution curve can be taken into account. income and wealth variables are likely to be correlated with one another since they are both subject to the greater movements of the national economy. this suggests that efficiency of conventional tourism demand models can be enhanced and their accuracy improved by utilizing important wealth variables. ando and modigliani's ( ) life cycle hypotheses (lch) predicts that a shift in demand can be induced without an actual change in income, but instead based solely on a change in expectations for future income. based on lch, the current study hypothesized that consumption of international tourism is also subject to changes in unrealized, expected wealth tied to the prices of housing and financial assets. since examining the relationship at the aggregate level requires liquidity, active trading, an efficient market, and broad ownership of assets throughout demand segments, the korean housing and financial markets, which fulfill these conditions, were used to investigate the recursive relationship with outbound travel demand. estimating the constant elasticity model using time-series data with praisewinsten and neweyewest procedures, it was found that the appreciation of apartments, the dominant type of housing in korea, significantly increased outbound travel demand from to . conversely, the appreciation of stocks was found to have no effect on outbound travel demand. rather than naively interpreting these results as proof that the financial wealth effect is invalid, it should be noted that low data sensitivity, the relatively lower importance of financial assets in household wealth portfolios, and high price volatility may have led to the insignificant empirical results for financial assets. by identifying unrealized wealth or adaptive expectations based on future income, as a potential determinant of tourism demand the current study makes a significant contribution to the contemporary tourism literature. the deterministic system of travel demand will be better understood by taking the consumption behavior and wealth portfolios of tourism demand into consideration. wealth effects may appear insignificant and vary depending on the characteristics of the demand, product, and asset, and caution should be employed when drawing on the effect of unrealized wealth. however, the possibility of its presence should be considered to achieve better results in the analysis of tourism demand. managerial implications for the industry are straightforward. when the wealth effect is significant, forecasting systems can make use of new asset variables to improve efficiency and predictability. developing better tourism forecasting models requires identifying new variables and integrating quantitative and qualitative forecasting approaches (song & li, ) . in this regard, micro and qualitative data on demand characteristics, including demand elasticities, destination preferences, and the propensity to travel, may enter the econometric equations to improve model fit and forecasting accuracy. such efforts are not new and have been gaining attention in relatively new demand models (stepchenkova & eales, ) . although the current study operationalizes two assets suitable for the korean economy e housing and stocks e demand models for other groups, countries, or economies will benefit from testing and utilizing other potentially relevant asset variables, such as cultural beliefs (guiso, sapienza, & zingales, ) or hopes regarding future economic conditions (canina & carvell, ) . consequently, organizations promoting tourism, such as destination marketing organizations and conventions and visitors bureaus, can better allocate limited resources and coordinate marketing and sales efforts in order to respond to expected demand fluctuations from changes in the consumption behaviors of potential visitors that do not result from realized income. for example, instead of relying on crude forecasts based solely on gross domestic product, consumer price index, and travel costs, analysts may use market-specific data on destination preferences of sending countries regarding respective receiving countries and income elasticity to those destinations. this approach may be further augmented by connecting spending behaviors with cultural beliefs and expectations regarding future economic conditions. despite its relevance, this study has several limitations in terms of generalizing and extending the results to other settings. the korean economy and travel demand are a unique case among the numerous countries constituting the global economy and the demand for international tourism. as such, there is no guarantee that the wealth effect is universal or common across a number of economies, as it is jointly dependent on the nature of the economy, the asset type in question, and characteristics of the demand. it should also be noted that incorporating the effect of wealth in demand modeling or marketing strategies when it is insignificant could also sacrifice efficiency and reliability. furthermore, inflation and volatility of housing and stock prices differ greatly case by case, while adaptive expectations based on future income resulting from price changes are also likely to be inconsistent or distinctive on the part of the consumers. the initial wealth of sending countries and destination price levels are expected to play a significant role as well. for example, in an extreme situation the wealth-elasticity of high income demand for low cost tourism destinations may be negative, as wealthier travelers will shift toward more prestigious and costly alternatives. although the current study utilized korean data to overcome the limitations originating from the 'aggregation economy,' the theory of wealth effect on consumption has been supported and empirically validated to a significant extent, especially by studies using micro data (poterba, ) . accordingly, the effect might be universal, despite the limited testability with macroeconomic data. indeed, it would not be easy to find a similar developed economy where a considerable mass of wealth is concentrated in one or two asset classes. failing to satisfy these two conditions e namely the concentration of wealth into a few assets and the liquidity of these assets e creates noise in attempts to find the significant effects of wealth on consumption. as shown, the current study could not find a significant wealth effect from financial assets, even though almost % of total household wealth in korea is concentrated in them. a potential explanation for the results could be that financial assets in the korean economy may not generate a sufficient wealth effect for the people, presumably due to the volatility of stock prices. myopia, liquidity and credit constraints (rosenzweig & wolpin, ; shea, b; zeldes, ) , the main factors that also govern spending, vary greatly even for people of comparable net wealth. thus, the direction of the wealth effect may also differ for social and income classes. some may readjust their total wealth higher when housing price soars. others may face reductions in future discretionary spending. in this regard, collecting refined income and tourism spending data, segmented by income and wealth level, would be invaluable in examining the marginal sensitivity of the wealth effect and provide a base of comparison across cultures and societies. as such, further research efforts on this topic should focus on the behavior and response of travel demand to economic cycles. in this regard, refining estimation techniques and examining psychological and cultural differences are viable options to pursue. considering other potentially influential factors on demand such as liquidity, savings, credit constraints, and myopic behavior are also warranted. although the controversy around the effect of wealth on consumption is unlikely to be resolved conclusively (poterba, ) in the near future, untangling the complex dynamics among income, wealth, and consumption will be central in the future enhancement of forecasting techniques and models for travel demand. the 'life cycle' hypothesis of saving: aggregate implications and tests booms and busts: consumption, house prices and expectations characteristics of u.s. demand for european tourism: a translog approach testing for serial correlation, spatial autocorrelation and random effects using panel data long-term forecasting of major forest products consumption in developed and developing economies how do house prices affect consumption? evidence from micro data lodging demand for urban hotels in major metropolitan markets in: paper prepared for the academic consultants meeting of the board of governors of the federal reserve system comparing wealth effects: the stock market versus the housing market house price dynamics and granger causality evidence of a stock market wealth effect using household level data demand elasticities in international marketing: a metaanalytical application to tourism market shares analysis: the case of french tourism demand wealth and wealth proxies in a permanent income model spurious regressions in financial economics? why are korean markets so volatile econometric analysis mortgage allocation in salford and manchester does culture affect economic outcomes a quantitative analysis of the apartment unit types in south korea the dividend pricing model: new evidence from the korean housing market understanding travel expenditure patterns: a study of japanese pleasure travelers to the united states by income level heavy spenders, medium spenders, and light spenders of japanese outbound pleasure travelers the role of stochastic volatility and return jumps: reproducing volatility and higher moments in the kospi returns dynamics development of a housing performance evaluation model for multi-family residential buildings in korea the implications of tourism specialization in the long run: an econometric analysis for oecd economies understanding trend and cycle in asset values: reevaluating the wealth effect on consumption are assets fungible? testing the behavioral theory of life-cycle savings review of international tourism demand models the major determinants of korean outbound travel to australia monetary policy and consumption an empirical re-evaluation of wealth effect in japanese household behavior are consumers forward looking? evidence from fiscal experiments stock market wealth and consumption credit market constraints, consumption smoothing, and the accumulation of durable production assets in low-income countries: investments in bullocks in india union contracts and the life cycle-permanent income hypothesis myopia, liquidity constraints, and aggregate consumption: a simple test housing wealth and aggregate saving tourism demand modeling and forecasting e a review of recent research an empirical study of outbound tourism demand in the uk stock market wealth and consumer spending destination image as quantified media messages: the effect of news on tourism demand the effects of market reform on maize marketing margins in south africa: an empirical study. msu international development working paper number joint test for functional forms and heteroscedasticity with property sales and valuation data the impact of crisis events and macroeconomic activity on taiwan's international inbound tourism demand. tourism management forecasting tourism demand: a review of empirical research econometric analysis of cross section and panel data consumption and liquidity constraints: an empirical investigation this study was supported by sejong university research fund. key: cord- -ccauw y authors: yang, fude; dong, xiaoxv; yin, xingbin; wang, wenping; you, longtai; ni, jian title: radix bupleuri: a review of traditional uses, botany, phytochemistry, pharmacology, and toxicology date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: ccauw y radix bupleuri (chaihu) has been used as a traditional medicine for more than years in china, japan, korea, and other asian countries. phytochemical studies demonstrated that this plant contains essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols. crude extracts and pure compounds isolated from radix bupleuri exhibited various biological activities, such as anti-inflammatory, anticancer, antipyretic, antimicrobial, antiviral, hepatoprotective, neuroprotective, and immunomodulatory effects. however, radix bupleuri could also lead to hepatotoxicity, particularly in high doses and with long-term use. pharmacokinetic studies have demonstrated that the major bioactive compounds (saikosaponins a, b( ), c, and d) were absorbed rapidly in rats after oral administration of the extract of radix bupleuri. this review aims to comprehensively summarize the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of radix bupleuri reported to date with an emphasis on its biological properties and mechanisms of action. radix bupleuri, also called "chaihu" in chinese, is derived from the dried roots of bupleurum chinense dc. and bupleurum scorzonerifolium willd. [ ] . as a traditional herbal medicine, radix bupleuri has been used widely for the treatments of influenza, fever, inflammation, malaria, menstrual disorders, and hepatitis in china, japan, korea, and other asian countries [ , ] . according to ancient chinese medical literatures, radix bupleuri is capable of regulating the exterior and interior metabolisms, dispersing evil heat from the superficies, soothing the liver, and promoting yang and qi (representing "life energy" or "life force" in tcm theories). in recent decades, investigations of radix bupleuri have focused on its biological activities, including its anti-inflammatory [ , ] , anticancer [ , ] , antipyretic [ ] , antimicrobial [ ] , antiviral [ ] , hepatoprotective [ ] , and immunomodulatory effects [ ] . in addition, radix bupleuri also exhibited significant effects on membrane fluidity [ ] . these studies have resulted in the isolation of essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols from this plant [ ] . among them, triterpenoid saponins are known to be the major bioactive compounds [ , ] . saikosaponins a and d are commonly used as chemical standards for quality evaluation of radix bupleuri in the current chinese pharmacopoeia and recent publications. however, an increasing number of recently published studies have reported adverse effects of radix bupleuri. the purpose of this review is to provide updated, comprehensive information on the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of radix bupleuri based on scientific literatures in the past few decades. this study will facilitate exploring the therapeutic potential of this plant and evaluate future research opportunities. radix bupleuri, which is characterized by a wide spectrum of biological and pharmacological effects, has been used as a famous traditional chinese medicinal herb with a history of medical use in china. according to tcm theory, radix bupleuri is thought to regulate the exterior and interior curing rhinitis and nasosinusitis bioactive compounds owing to their antifungal and antiinflammatory activities [ , ] . in one study, the essential oils in radix bupleuri were extracted by steam distillation and solvent extraction and then analyzed by gc/ms; peaks were identified. among these peaks, the major volatile compounds were -methylbutanal ( . %), pentanal ( . %), hexanal ( . %), furan- -carbaldehyde ( . %), and heptanal ( . %) [ ] . however, in another study, the results showed that e- -heptanal, furan, -pentyl, and e- -nonenal were some of the main compounds of the oil [ ] . triterpenoid saponins are the main active components of radix bupleuri, which exhibit a broad spectrum of biological and pharmacological effects, including analgesic, immunomodulatory, hepatoprotective, immunomodulatory, anti-inflammatory, antitumor, and antiviral activities [ , [ ] [ ] [ ] . currently, approximately saponins have been isolated from radix bupleuri ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among them, saikosaponins a, c, and d are the major bioactive constituents found in radix bupleuri; however a variety of minor saikosaponins have also been isolated [ ] . the cytotoxic and antiproliferative effects of saikosaponins a and d have been attracting much interest in recent years [ ] . additionally, more information about the intimate relationship between the structural characterization of saikosaponins and their cytotoxic evaluations is very necessary. radix bupleuri have been identified, including ( z, z, e)pentadecatriene- , -diyne- -ol ( ), ( z, e, e)-pentadecatriene- , -diyne- -ol ( ), ( z, z, e)-heptadecatriene- , -diyne- -ol ( ), and bupleurynol ( ) ( figure ) [ , ] . radix bupleuri exerts a great variety of pharmacological activities due to its complexbioactive compounds. an overview of the pharmacological studies on radix bupleuriis presented in detail in the following sections. widely used for the treatment of several types of chronic inflammatory diseases. the crude polysaccharides ( mg/kg) isolated from the roots of bupleurum chinense dc. significantly attenuated lung injury by inhibiting the level of myeloperoxidase (mpo), tumor necrosis factor-(tnf-), and serum nitric oxide (no) [ ] . chun et al. reported that saikosaponins from radix bupleuri exhibited anti-inflammatory activity on inflammatory processes including inhibition of inflammatory exudation, capillary permeability, inflammatory mediators release, migration of white cells, connective tissue hyperplasia, and a variety of allergic inflammation [ ] . ma et al. was the first to show that saikosaponins exerted anti-inflammatory activity on paw edema mainly via regulating the nicotinate and nicotinamide metabolism and arachidonic acid metabolism [ ] . zhu et al. found that saikosaponin a (ssa) exhibited an inhibitory effect on proinflammatory cytokines in lps-stimulated macrophages. the mechanism of these actions involved the regulation of mapk and nf-b signals pathways [ ] . in another study, ssa dose-dependently inhibited the production of ros, tnf-, il- , cox- , and inos in lps-stimulated human umbilical endothelial cells (huvecs) by upregulating of the lxr -abca signaling pathway [ ] . moreover, lee et al. showed that saikosaponin c (ssc) was also shown to inhibited lps-induced apoptosis in huvecs via inhibition of caspase- activation and caspase- -mediated-fak degradation [ ] . zhao et al. showed that ssa also suppressed tnf-and il- concentrations in the intestines of septic rats through the inhibition of the nucleotide-binding oligomerization domain (nod )/nf-b signaling pathway [ ] . saikosaponin d (ssd) has been reported to inhibit pge production and intracellular free ca + concentration ([ca + ]i) in a concentration-dependent manner with an ic value of m in c rat glioma cells [ ] . in addition, several studies showed that a wide range of radix bupleuri preparations also exhibited antiinflammatory effects in in vitro and in vivo model. li et al. showed that saireito and its active components (ssd) could suppress the proliferation of mesangial cells and expansion of the mesangial matrix in the rat glomerulonephritis model [ ] . in experimental chronic pancreatitis rats model, "chai-hu-shu-gan powder" exerted anti-inflammatory and antifibrotic effects by inhibiting the expression of nuclear factor-b (nf-b) and tnf-mrna in the pancreas [ ] . furthermore, it also reduced the abnormally high plasma level of cholecystokinin, improved the gastric movement, and avoided nausea and flatulence [ ] . in another experiment, a chinese herbal formula called "rcm- " (containing flos magnoliae, radix bupleuri, radix glycyrrhizae, radix angelicae sinensis, etc.) inhibited the no production and inos protein expression in lps-stimulated rat aorta and raw . macrophages [ ] . radix bupleuri also possessed anticancer/antitumor effect. the acetone extract of bupleurum scorzonerifolium could inhibit the proliferation of a human lung cancer cells in a dose-dependent manner via causing cell cycle arrest in the g /m phase, increasing microtubule stabilization, suppressing telomerase activity, activating erk / and caspase- / in a cells [ ] [ ] [ ] . saponins isolated from radix bupleuri also exhibited significantly anti-proliferative activity in human non-small cell lung cancer a cells through fas-dependent apoptotic pathway [ ] . su et al. found that the water extracts of radix bupleuri could enhance -fluorouracil-induced cytotoxicity in hepg hepatoma cells through cell arrest at the late g /early s phase, while protecting normal blood lymphocytes [ ] . ssd showed very potent activity against the hepg cell line with an ic value of . mg/ml. the mechanism of cytotoxicity was attributed to the induction of apoptosis through activation of caspase- and caspase- , which subsequently resulted in poly-adp-ribose polymerase (parp) cleavage [ ] . sarcoplasmic/endoplasmic reticulum ca + atpase (serca), leading to the increase of intracellular calcium ion levels and activating the ca + /calmodulin-dependent kinase kinase--(camkk -) amp-activated protein kinase-(ampk-) mammalian target of rapamycin (mtor) signaling cascade, endoplasmic reticulum (er) stress, and unfolded protein responses (upr) [ ] . several chinese medicine preparations containing radix bupleuri also have been traditionally used in the treatment of tumors and cancer. the water extracts of "long dan xie gan wan" exerted a significant growth inhibitory effect in hl and ht cancer cell lines, indicating that this formulation may possess some chemotherapeutic potential [ ] . treatment with "xiao-chai-hu decoction" exhibited a significantly lower incidence of hepatocellular carcinoma and reductions in cancer pain and tumor size. the underlying mechanism of the antitumor activities is based on stimulation of the reticuloendothelial system (res) and is closely related of tnf production [ ] [ ] [ ] . wen et al. reported that the acetone extract of radix bupleuri possessed a significant antivirus effect on acute respiratory tract infections with h n virus infection and suppressed influenza a virus-induced rantes secretion in h n -infected a cells at a concentration of and g/ml, suggesting that radix bupleuri might be beneficial for the treatment of chronic inflammatory conditions followed by viral infection [ ] . ssc has been reported to show effective anti-hbv activity through inhibiting dna expression of hbsag, hbeag, and hbv [ ] . treatment with "xiao-chai-hu decoction" ( g/ml, days; g/ml, days) could inhibit the production of hbv ( < . ) and the expression of hbeag. moreover, crude saponins of bupleurum chinense dc. could inhibit the replication of hbv ( < . ) [ ] . similarly, in another study, yin et al. showed that ssd isolated from the meoh extract of bupleurum chinense dc. exhibited significant bioactivity in inhibiting dna replication of hbv [ ] . the antiviral activity of saikosaponins (a, b , c, and d) and their mode of actin were examined. the results showed that all saikosaponins exerted antiviral activity on human coronavirus- e at concentrations of . - m, and the strongest activity was observed for saikosaponin b with an ic of . m. this mechanism might involve interference in the early stage of viral replication, such as absorption and penetration of the virus [ ] . the water extract of radix bupleuri was reported to exert its antipyretic effect on dry yeastinduced high fever rats. the mechanism is related to the adjustment of synthesis and exudation of cyclic adenosine monophosphate (camp) and arginine vasopressin (avp) [ ] . a novel in situ gel system for nasal delivery of the essential oil from radix bupleuri was prepared. the results suggested that radix bupleuri in situ gel can be more effective than the solution in the treatment of fever [ ] . a similar study showed that the essential oil extracted from the herb exhibited dose-dependent antipyretic capacity on both fevered rabbits and rats [ ] . the ethanol extract of bupleurum chinense dc. exerted a remarkable bacteriostatic effect on gram-negative microorganism helicobacter pylori. the bioactive minimum inhibitory concentration (mic) value was mm [ ] . saikosaponins isolated from radix bupleuri have been reported to exhibit antibacterial activity, particularly against pseudomonas aeruginosa and listeria monocytogenes. the protective effect was attributed to the immunomodulatory action on macrophages [ ] . "chaihu injection" has also been tested for possible antimicrobial activity in vitro. the results demonstrated that mild inhibition of staphylococcus aureus was observed but no effects were observed against staphylococcus albus, neisseria gonorrhoeae, diplococcus pneumoniae, haemolytic streptococcus, or pseudomonas aeruginosa [ ] . the liver protective effects against ccl induced liver injury were investigated after treatment of mice with raw and vinegar-baked radix bupleuri ( g/kg/day) for days. the results showed that both raw and processed radix bupleuri showed liver protective effects against ccl induced liver injury, and the vinegar-baked radix bupleuri exerted better effects than that of raw radix bupleuri [ ] . pretreated with saikosaponins, especially ssa or ssd, showed remarkable inhibition of d-galactosamineinduced hepatic injury through decreasing the activity of glucose- -phosphatase and nadph-cytochrome c reductase and increasing -nucleotidase activity [ ] . similarly, bupleurosides iii, vi, ix, and xiii and saikosaponin b isolated from bupleurum scorzonerifolium willd. were also found to exhibit protective effect on the d-galactosamineinduced cytotoxicity in primary cultured rat hepatocytes [ ] . further studies also demonstrated that the protective effects of saikosaponins isolated from bupleurum chinense dc. could prevent hepatocyte injury through regulating intracellular calcium levels [ ] . in a rat model with ccl induced acute hepatic injury, the hepatic enzyme levels (got, gpt, and alp) and the lipid peroxidation in the liver were significantly reduced by the administration of ssd [ ] . additionally, ssd significantly reduced collagen i deposition and alanine aminotransferase level on liver fibrosis rats and decreased the concentration of transforming growth factor (tgf- ). moreover, ssd was able to alleviate hepatocyte injury from oxidative stress. the effect of ssd on liver fibrosis may be related to its ability to reduce lipid peroxidation [ ] . the effects of radix bupleuri on spontaneous lymphatic vessel activity. the results indicated that radix bupleuri significantly increased the amplitude of spontaneous activity of lymphatic vessels in a concentration-dependent manner, and the mechanisms of this effect seem to be independent of endothelial function [ ] . eugenin ( ) and saikochrome a ( ) isolated from the meoh extracts from bupleurum scorzonerifolium possessed immunosuppressive effect on human peripheral blood t cells via inhibiting cd -costimulated activation [ ] . ssd ( mg, intraperitoneally) significantly activated peritoneal macrophages in terms of enhancement of phagocytic activity, increased level of cellular lysosomal enzyme, and suppressed the response of plaque-forming cells to heterologous erythrocytes by stimulating t and b cells in a dose-dependent manner [ ] . moreover, ssd modulated lymphocyte activity through suppressing the t cell response and increasing the b cell response to different mitogens and the interleukin-(il-) /il- production through a receptor-bypassed pathway [ , , ] . in another experiment, wong et al. found that ssd was shown to inhibit okt /cd -costimulated human t cell proliferation and pma, pma/ionomycin, and con a-induced mouse t cell activation in vitro. the underlying mechanisms involved downregulation of nf-kb signaling by suppression of ikk and akt activities [ ] . autophagy is a complex process in cells, which occurs through the formation of doublemembrane vesicles (autophagosomes), which are engulfed by cytoplasmic molecules. then, the autophagosome fuses with the lysosomes, leading to degradation of long-lived proteins, aggregated proteins, and damaged organelles [ ] [ ] [ ] . moreover, autophagy might be triggered by hypoxia, nutritional deprivation, radiation, chemical drugs, and other stimulants [ ] . autophagy contributes to the pathogenesis of diverse diseases, such as neuronal degeneration, inflammatory bowel disease, aging, and cancer [ , ] . in the previous study, law et al. demonstrated that the protective pharmacological effects of radix bupleuri might be attributed to its autophagy induction. the autophagic effect of radix bupleuri played an important role in relieving liver disease-related symptoms through anti-inflammatory, organ-protective, and aggregate removal functions. furthermore, the anticancer effects of radix bupleuri could be attributed to its autophagy induction. radix bupleuri has been found to be an effective treatment against depression by regulating metabolite, hormone, and neurotransmitter levels via autophagy-mediated lipid metabolism [ ] . the effect of the ethanol extract from radix bupleuri on cytochrome isoform activities using a six-drug cocktail approach was evaluated; the results demonstrated that radix bupleuri had strong induction activity on the cyp e , cyp d , and cyp a , which may lead to potential plant drug-drug interactions [ ] . radix bupleuri was shown to be the inhibitor ofglucuronidase. the inhibition rate of radix bupleuri extracts rb (high molecular weight polysaccharides), rb (ethanol soluble/water insoluble component), rb (extracted by nbutanol, soluble in water), and rb (low molecular weight water soluble parts) on the activity of -glucuronidase was found to be . %, . %, . %, and . %, respectively [ ] . in pentylenetetrazol (ptz) induced epilepsy rats model, ssa isolated from radix bupleuri significantly reduced seizure severity and duration while it markedly elevated seizure latency and downregulated the cytokines expression of p-mtor, p- s k, l- , and tnf-through inhibiting mtor signaling pathway [ ] . he et al. demonstrated that ssa obviously reduced lipoprotein uptake to block foam cell formation and the expression of lox- and cd , boosted cholesterol efflux, and the expression of abca and ppar through inhibiting pi k/akt/nf-b/nlrp signaling pathway [ ] . in another experiment, ssc exerted a potent effect on inducing human umbilical vein endothelial cells (huvecs) viability and growth. furthermore, ssc also induced endothelial cells migration and capillary tube formation. the underlying mechanisms might be related to the gene expression or activation of matrix metalloproteinase- (mmp- ), vascular endothelial growth factor (vegf), and the p /p mitogenactivated protein kinase (mapk, erk) [ ] . in addition, ssc was shown to exhibit inhibitory activities against alzheimer's disease (ad) via suppressing the secretion of a peptides and abnormal tau hyperphosphorylation-mediated microtubule depolymerization. moreover, ssc suppressed a peptideinduced brain endothelial apoptosis, indicating that ssc might be a novel therapeutic tool for treating human ad and other neurodegenerative diseases [ ] . it was shown by liu et al. for the first time that four polyacetylenes ( -) from radix bupleuri potently exhibited an antidepressant activity by inhibiting the reuptake of serotonin, norepinephrine, and dopamine. the mechanism might be mediated by increasing the level of monoamines, particularly -ht and ne [ ] . zhu et al. suggested that ssa and ssd exhibited the anthelmintic activity against dactylogyrus spp. infecting goldfish. the effective concentration (ec ) values for ssa and ssd were . and . mg − , respectively [ ] . bupleuri possesses a wide spectrum of pharmacological effects, including anti-inflammatory effect, anticancer effect, antiviral effect, antipyretic effect, antibacterial effect, hepatoprotective effect, and immunomodulatory effect (table ). based on these pharmacological effects, we can conclude that the extracts and the compounds from this plant can prevent or treat certain diseases, such as cancer, fever, malaria, hepatitis, and ad. however, there is not enough systemic data of these chemical compounds and their pharmacological effects. thus, in the future, the pharmacological effects and the possible molecular mechanisms of the pharmacological activities of radix bupleuri must be urgently explored on our modern understanding of these diseases' pathophysiologies. radix bupleuri has been used for thousands of years as an important traditional herb in china. however, the toxic effects of radix bupleuri in clinical applications have been gradually reported. several studies have found that the liver is the main organ affected by toxicity, particularly in longterm use. major symptoms of liver injury induced by radix bupleuri included transaminase lifts, hepatitis, and jaundice. however, liver functions can return to normal levels after a specific period [ ] . radix bupleuri has been reported to exhibit acute hepatitis and acute hepatic necrosis. the mean total daily dose was . ± . g, which was more than the chinese pharmacopoeia recommended range of to g [ ] . moreover, radix bupleuri had been implicated in multiple cases of acute hepatitis both as an ingredient alone and within a particular formulation "xiao-chai-hu-tang" (also known as syo-saiko-to in japanese) [ ] . lee et al. demonstrated that two chinese herbal products containing radix bupleuri might increase their risks of liver injury in hbv-infected patients. however, further mechanistic research on the hepatotoxicity of radix bupleuri in the presence of hbv infection is warranted [ ] . in addition, the essential oil of radix bupleuri induced acute hepatotoxicity with asynchronous state, higher heart rate, and fast breathing [ ] . the total saponins isolated from radix bupleuri could also cause evidently liver damage in dose-dependent manner manifested as hepatocyte organic lesion and liver function changes, as well as hepatocyte death [ ] . a selective and sensitive lc-ms/ms method was developed and validated for simultaneous determination of ssa, b , c, and d in rat plasma afteroral administration of the ethanolwater ( : , v/v) extract of radix bupleuri for the first time. the results demonstrated that ssa, c, and d were absorbed rapidly with max less than min [ ] . in another pharmacokinetics experiment of rats, liu et al. was the first to develop an uplc-pda-ms method to determine the pharmacokinetics of four polyacetylenes after i.g. administration of % ethanol extract of radix bupleuri. the results showed that compounds and were not detected in rat serum, whereas compounds and exerted a fast distribution phase followed by a relatively slow elimination phase ( / , - h) [ ] . in traditional chinese medicine, radix bupleuri has long been used regulate the exterior and interior metabolisms, disperse evil heat from superficies, sooth the liver, and promote yang and qi. it has been widely used to treat various diseases in china, japan, korea, and other asian countries for many centuries. a total of compounds including essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols have been isolated and identified from radix bupleuri . pharmacological studies have revealed that radix bupleuri possesses a variety of biological effects, including anti-inflammatory, anticancer, antiviral, antipyretic, antibacterial, antiobesity, immunomodulatory, hepatoprotective, neuroprotective, and autophagic effects . however, there are some aspects that need to be further investigated. radix bupleuri is an ingredient of many patent medicines or prescriptions. although modern experiments have confirmed that this drug alone exhibits multiple pharmacological activities, it is important to investigate the molecular mechanisms of radix bupleuri combined with other herbs based on traditional uses. furthermore, the pharmacological effects of only a few of the ingredients, such as saikosaponins, flavonoids, and the essential oils, have been investigated. some polyacetylenes, lignans, and sterols have not been sufficiently researched in terms of their pharmacological effects. radix bupleuri shows both hepatoprotection and in vitro [ ] antipyretic effect dry yeast-induced high fever rats adjusts the synthesis and exudation of camp and avp in vivo [ ] turpentine-induced fever rabbits decreases body temperature the essential oil in vivo [ ] turpentine-induced fever rabbits and rats the essential oil in vivo [ ] antibacterial effect in vivo [ ] hepatotoxicity, which appears to be contradictory. this phenomenon is similar to that of polygonum multiflorum thunb. [ ] . based on the literature, the main reasons are likely the administration dosage and delivery time. high doses and long-term drug delivery are more likely to result in liver toxicity, whereas low doses and short-term drug delivery might result in liver protection. therefore, this issue needs further study. in conclusion, this review summarized the traditional uses, botany, phytochemistry, pharmacology, and toxicology of radix bupleuri. moreover, it has provided a new foundation for further research on its mechanism of action and the development of better therapeutic agents employing radix bupleuri in the future. it is anticipated that the comprehensive and detailed research on toxicity, pharmacodynamics, pharmacokinetics, and molecular mechanism are necessary to be explored to develop its bioactive compounds as effective drugs. the authors have declared that there are no conflicts of interest regarding the publication of this paper. fude yang and xiaoxv dong contributed equally to this work and share first authorship. editorial committee of chinese pharmacopoeia, chinese pharmacopoeia pharmacology and applications of chinese genus bupleurum: a review of its phytochemistry, pharmacology and modes of action in vivo and in vitro antiinflammatory activity of saikosaponins anti-inflammation effects and potential mechanism of saikosaponins by regulating nicotinate and nicotinamide metabolism and arachidonic acid metabolism effect of bupleuri radix extracts on the toxicity of -fluorouracil in hepg hepatoma cells and normal human lymphocytes antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines antinociceptive and antipyretic properties of the pharmaceutical herbal preparation, radix bupleuri in rats development and evaluation of a herbal formulation with antipathogenic activities and probiotics stimulatory effects in vitro anti-influenza a h n effect of extract of bupleuri radix scorzonerosides a, b and c, novel triterpene oligoglycosides with hepatoprotective effect from chinese bupleuri radix, the roots of bupleurum scorzonerifolium willd beneficial effect of bupleurum polysaccharides on autoimmune disease induced by campylobacter jejuni in balb/c mice the effects of saikosaponins on biological membranes bupleurum species: scientific evaluation and clinical applications evaluation of traditional chinese herbal medicine: chaihu (bupleuri radix) by both high-performance liquid chromatographic and highperformance thin-layer chromatographic fingerprint and chemometric analysis fast determination of saikosaponins in bupleurum by rapid resolution liquid chromatography with evaporative light scattering detection construction of a full-length enriched cdna library and analysis of ests from roots of bupleurum chinense dc identification of crude drugs from chinese medicinal plants of the genus bupleurum using ribosomal dna its sequences acute hepatitis induced by chinese hepatoprotective herb, xiao-chai-hu-tang medicinal plants of the world: an illustrated scientific guide to important medicinal plants and their uses studies on the antidepression effect of xiaoyao powder in mic identification and quantification of the major volatile constituents in antidepressant active fraction of xiaoyaosan by gas chromatography-mass spectrometry metabonomic study on chronic unpredictable mild stress and intervention effects of xiaoyaosan in rats using gas chromatography coupled with mass spectrometry editorial board of flora of china, flora of china determination of species of medical bupleunum the coloured atlas of the medicinal plants from genus bupleurum in china evaluation of root quality of bupleurum species by tlc scanner and the liver protective effects of "xiao-chai-hu-tang high performance liquid chromatographic assay of saikosaponins from radix bupleuri in china analysis on saikosaponin of sixteen bupleurum chinense from shanxi a highperformance liquid chromatographic method for saikosaponin a quantification in rat plasma simultaneous hplc-elsd determination of saikosaponins a evaporative light scattering detection: trends in its analytical uses determination of saikosaponin derivatives in radix bupleuri and in pharmaceuticals of the chinese multiherb remedy xiaochaihu-tang using liquid chromatographic tandem mass spectrometry ms/ms analyses of saikosaponins-a and -c as markers of bupleuri radix samples interaction of the main components from the traditional chinese drug pair chaihu-shaoyao based on rat intestinal absorption determination of saikosaponins a, c, and d in bupleurum chinese dc from different areas by capillary zone electrophoresis in vivo antifungal activity of the essential oil of bupleurum gibraltarium against plasmopara halstedii in sunflower pharmacological activity of the essential oil of bupleurum gibraltaricum: anti-inflammatory activity and effects on isolated rat uteri analysis of volatile compounds in radix bupleuri injection by gc-ms-ms analysis of the essential oil from radix bupleuri using capillary gas chromatography the effects of saikosaponin on macrophage functions and lymphocyte proliferation inactivation of measles virus and herpes simplex virus by saikosaponin d saikosaponin-d, a novel serca inhibitor, induces autophagic cell death in apoptosisdefective cells separation and determination of saponins of bupleuri radix by droplet counter current chromatography (dcc) new hepatoprotective saponins, bupleurosides chemical evaluation of bupleurum species collected in yunnan, china cytotoxic triterpenoid glycosides (saikosaponins) from the roots of bupleurum chinense studies on chemical constituents of bupleurum genus. part ii. isolation of triterpenoid glycosides (saikosaponins) from bupleurum kunmingense and their chemical structures identification and determination of the saikosaponins in radix bupleuri by accelerated solvent extraction combined with rapid-resolution lc-ms isolation, characterization, and nuclear magnetic resonance spectra of new saponins from the roots of bupleurum falcatum l saikosaponin v- from roots of bupleurum chinense dc novel cleavage of the glycosidic bond of saponins in alcoholic alkali metal solution containing a trace of water species discrimination of radix bupleuri through the simultaneous determination of ten saikosaponins by high performance liquid chromatography with evaporative light scattering detection biomed research international ionization mass spectrometry new derivatives of saikosaponins cytotoxic activity and inhibitory effect on nitric oxide production of triterpene saponins from the roots of physospermum verticillatum (waldst & kit) (apiaceae) a qualitative, and quantitative determination and pharmacokinetic study of four polyacetylenes from radix bupleuri by uplc-pda-ms qualitative and quantitative determination of polyacetylenes in different bupleurum species by high performance liquid chromatography with diode array detector and mass spectrometry the chemical constituents from the roots of bupleurum chinense dc a new chromone glycoside from bupleurum chinense flavonoids from the roots of bupleurum chinense dc new isoflavonoside from bupleurum scorzonerifolium immunosuppressive flavones and lignans from bupleurum scorzonerifolium studies on the constituents of umbelliferae plants. xviii. minor constituents of bupleuri radix: occurrence of saikogenins, polyhydroxysterols, a trihydroxy c fatty acid, a lignan and a new chromone the chemical constituents from the roots of bupleurum chinense analysis of the fatty acid from bupleurum chinense dc in china by gc-ms and gc-fid bupleurum chinense dc polysaccharides attenuates lipopolysaccharide-induced acute lung injury in mice anti-inflammatory effect of corymbocoumarin from seseli gummiferum subsp. corymbosum through suppression of nf-b signaling pathway and induction of ho- expression in lps-stimulated raw . cells saikosaponin a mediates the inflammatory response by inhibiting the mapk and nf-b pathways in lps-stimulated raw . cells saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in human umbilical vein endothelial cells via preventing tlr translocation into lipid rafts saikosaponin c inhibits lipopolysaccharide-induced apoptosis by suppressing caspase- activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells saikosaponin a protects against experimental sepsis via inhibition of nod -mediated nf-b activation dual effect of saikogenin d: in vitro inhibition of prostaglandin e production and elevation of intracellular free ca + concentration in c rat glioma cells suppressive effects of saireito on monoclonal antibody - - -induced glomerulonephritis: analysis of effective components therapeutic effects and mechanisms of chaihushugan decoction in experimental chronic pancreatitis rats effect of chaihu shugan pulvis on dysfunction of pancreatic exocrine secretion in patients with chronic pancreatitis inhibition of inducible nitric oxide production and inos protein expression in lipopolysaccharide-stimulated rat aorta and raw . macrophages by ethanol extract of a chinese herbal medicine formula (rcm- ) for allergic rhinitis acetone extract of bupleurum scorzonerifolium inhibits proliferation of a human lung cancer cells via inducing apoptosis and suppressing telomerase activity requirement for erk activation in acetone extract identified from bupleurrum scorzonerifolium induced a tumor cell apoptosis and keratin phosphorylation anti-proliferative activity of bupleurum scrozonerifolium in a human lung cancer cells in vitro and in vivo the antiproliferative activity of saponin-enriched fraction from bupleurum kaoi is through fas-dependent apoptotic pathway in human non-small cell lung cancer a cells cytotoxicity and anti-hepatitis b virus activities of saikosaponins from bupleurum species the role of saikosaponin d in regulating hif- /cox- signal transduction pathway in human hepatocellular carcinoma cells an in vitro based investigation of the cytotoxic effect of water extracts of the chinese herbal remedy ld on cancer cells prospective study of chemoprevention of hepatocellular carcinoma with sho-saiko-to (tj- clinical observation of the treatment of primary liver cancer by using xiao-chai-hu decoction antitumor activities and tumor necrosis factor producibility of traditional chinese medicines and crude drugs sho-saiko-to (xiao-chai-hu-tang) and crude saikosaponins inhibit hepatitis b virus in a stable hbvproducing cell line saikosaponins from bupleurum chinense and inhibition of hbv dna replication activity antiviral effects of saikosaponins on human coronavirus e in vitro experimental study on material basis, efficacy and mechanism of antipyretic effect of bupleuri radix a novel nasal delivery system of a chinese traditional medicine, radix bupleuri, based on the concept of ion-activated in situ gel preparation of bupleurum nasal spray and evaluation on its safety and efficacy in vitro anti-helicobacter pylori action of chinese herbal medicines used to treat ulcer diseases protective effect of saikosaponin a, saikosaponin d and saikogenin d against pseudomonas aeruginosa infection in mice the pharmacological activity of chinese "chaihu"-the root of bupleurum species chemical and biological comparison of raw and vinegar-baked radix bupleuri pharmacological actions of saikosaponins isolated from bupleurum falcatum. i. effects of saikosaponings on liver function new hepatoprotective saponins, bupleurosides effects of the extracts from bupleurum chinese dc on intracelluar free calcium concentration and vincristine accumulation in human hepatoma bel- cells protective effect of saikosaponin-d isolated from bupleurum falcatum l. on ccl -induced liver injury in the rat saikosaponin-d attenuates the development of liver fibrosis by preventing hepatocyte injury effects of the chinese herbal medicines bupleuri radix, ginseng radix, and zingiberis rhizoma on lymphatic vessel activity in rats activation of murine peritoneal macrophages by saikosaponin a, saikosaponin d and saikogenin d cell type-oriented differential modulatory actions of saikosaponin-d on growth responses and dna fragmentation of lymphocytes triggered by receptor-mediated and receptor-bypassed pathways characterization of the immunoregulatory action of saikosaponin-d mechanistic study of saikosaponin-d (ssd) on suppression of murine t lymphocyte activation autophagy: a potential target for thyroid cancer therapy (review) autophagy: process and function autophagy modulation as a potential therapeutic target for diverse diseases the role of autophagy in cancer: therapeutic implications the dual role of autophagy in cancer role of autophagy in aging autophagic effects of chaihu (dried roots of bupleurum chinense dc or bupleurum scorzoneraefolium wild) assessment of the effects of radix bupleuri and vinegarbaked radix bupleuri on cytochrome activity by a six-drug cocktail approach effect of saikosaponins and extracts of vinegar-baked bupleuri radix on the activity of -glucuronidase saikosaponin a functions as anti-epileptic effect in pentylenetetrazol induced rats through inhibiting mtor signaling pathway saikosaponin-a attenuates oxidized ldl uptake and prompts cholesterol efflux in thp- cells saikosaponin c induces endothelial cells growth, migration and capillary tube formation a potential therapeutic effect of saikosaponin c as a novel dual-target anti-alzheimer agent anthelmintic activity of saikosaponins a and d from radix bupleuri against dactylogyrus spp. infecting goldfish analysis of cases of druginduced liver disease drug-induced liver injury associated with complementary and alternative medicine: a review of adverse event reports in an asian community from liver injuries induced by herbal medicine, syo-saiko-to (xiaochai-hu-tang) risk of liver injury associated with chinese herbal products containing radix bupleuri in , patients with hepatitis b virus infection acute toxicity of volatile oil from bupleurum chinense in rats and mice dose-time-toxicity" relationship study on hepatotoxicity caused by multiple dose of total bupleurum saponin crude extracts to rats analysis of saikosaponins in rat plasma by anionic adducts-based liquid chromatography tandem mass spectrometry method traditional usages, botany, phytochemistry, pharmacology and toxicology of polygonum multiflorum thunb.: a review this work was financially supported by the collaborative innovation construction plan of beijing university of chinese medicine (no. -xtcx- ). key: cord- - y jy c authors: hetland, geir; johnson, egil; bernardshaw, soosaipillai v.; grinde, bjørn title: can medicinal mushrooms have prophylactic or therapeutic effect against covid‐ and its pneumonic superinfection and complicating inflammation? date: - - journal: scand j immunol doi: . /sji. sha: doc_id: cord_uid: y jy c medicinal mushrooms have documented effects against different diseases, including infections and inflammatory disorders. the related basidiomycota agaricus blazei murill (abm), hericium erinaceus (he), and grifola frondosa (gf) have been shown to exert antimicrobial activity against viral agents, gram‐positive and gram‐negative bacteria, and parasites in vitro and in vivo. since the mechanism is immunomodulatory and not antibiotical, the mushrooms should be active against multi‐drug resistant microbes as well. moreover, since these basidiomycota also have anti‐inflammatory properties, they may be suited for treatment of the severe lung inflammation that often follows covid‐ infection. an abm‐based mushroom extract (andosan™), also containing he and gf, has been shown to significantly reduce bacteraemia and increase survival in mice with pneumococcal sepsis, and to improve symptoms and quality of life in ibd patients via an anti‐inflammatory effect. hence, such mushroom extracts could have prophylactic or therapeutic effect against the pneumonic superinfection and severe lung inflammation that often complicates covid‐ infection. here, we review antimicrobial and anti‐inflammatory properties of abm, he and gf mushrooms, which could be used for the battle against covid‐ . effects of abm on infection, inflammation and tumour have been reviewed previously in sji, including that of the abm-based mycelium extract, andosan™, which also contains he ( %) and gf ( %). it has been used in three placebo-controlled randomized clinical trials as supplement to regular treatment for inflammatory bowel disease (ibd); ulcerative colitis (uc) ( patients) and crohn's disease (cd) ( patients), [ ] [ ] [ ] multiple myeloma (mm) ( patients) and pollen allergy and asthma ( blood donors) without adverse effects. it reduced proinflammatory cytokines and improved symptoms and quality of life in ibd patients, [ ] [ ] [ ] reduced allergy and asthma symptoms, specific ige and basophil sensitivity in allergics, and increased il- receptor antagonist (il- ra), il- , t regulatory cells, dendritic cells (dcs) and expression of ig, killer ig receptors (kirs) and hla genes in mm patients. moreover, since the beginning of this millennium there have been quite a few other reports on antimicrobial ( table , ) and anti-inflammatory (table ) effects of medicinal mushrooms such as abm, he and gf. the outbreak of a novel coronavirus (sars-cov- ) (covid- )-induced disease in china that causes serious respiratory illness, was declared a pandemic by the world health organization on march . most of those who got sick from the infection, developed lymphopenia and pneumonia and in severe cases also high levels of proinflammatory cytokines. this is similar to the 'cytokine storm' observed in sars, , which gives rise to viraemia and inflammatory lung injury and may be followed by multi-organ failure and death. in some cases, this pathogenesis will be enhanced by secondary bacterial superinfection in the lung as well as development of septicaemia. immune dysregulation may play an important role in many viral diseases such as respiratory syncytial virus infection, where a disease-enhancing inflammation similar to the covid- situation is dependent on the immune response in the airways mucosa. in viral infections, treg and th cells have a complex interaction in which tregs may inhibit immune activation and subsequent disease progression and maintain immune homeostasis, while th cells will induce immune activation and propagate the inflammation. as the covid- pathogenesis is unknown, similar to that of sars, there are no approved drugs for the disease and vaccines are yet to be developed. corticosteroids that otherwise are used for treatment of acute respiratory distress syndrome and severe lung injury, strongly inhibit antiviral immunity and may be counter-indicated. an inhibition of a proximal immune response event such as activation of ifn-related pattern recognition receptors (prrs) that are activated by pathogen-(pamps) and danger-associated molecular patterns (damps) at the mucosa, would seem unwise because of its general host defence regulatory function. therefore, targets should be limited to proinflammatory and th cytokines, such as oxygen radicals, tnfα, il- , il- , il- , il- and il- production. these, except for the il- that has not been examined, are the very same effector arms that are counteracted by andosan™ treatment. [ ] [ ] [ ] [ ] the aim of this article was to review possible effects that the much used and related medicinal mushrooms abm, he and gf might have against covid- infection and its complications. selection criteria were inclusion of pubmed/ medline indexed articles on antimicrobial and anti-inflammatory effects with these mushrooms. abm has been shown to counteract the cytopathic effect induced by western equine encephalitis (wee) virus on vero cells in vitro, and in a plaque reduction assay with poliovirus to reduce the number of plaques suggestively by acting in the initial stage of viral replication (table ) . in patients with chronic hepatitis b virus (hbv) and c virus (hcv) infection, abm extracts have been found to normalize liver function, and to slightly decrease hcv plasma load. also antiviral effect of gf alone or combined with ifnα has been demonstrated against hbv in hepg cells, in which hbv dna was inhibited. there are several reports regarding mushroom treatment of herpes virus (hsv- ) and (hvs- ): a protein isolated from gf inhibited hsv- replication in vitro and reduced severity of the viral infection upon topical administration in a mouse model. further, abm polysaccharides inhibited hsv- infection in hep- cell cultures. , another abm mycelium polysaccharide given orally to mice, reduced ocular, cutaneous and vaginal (hsv- ) infections by inhibition of virus attachment, entry and cellto-cell spreading as shown by plaque reduction assay. suggestively, this occurred through interference with early events of viral penetration. yet, another gf polysaccharide was shown to block replication of enterovirus (ev- ) -the major agent for foot, hand and mouth disease -suppress viral protein expression and exhibits apoptotic activity in vitro. with respect to influenza, one report found that abm metabolites had direct antiviral effect against influenza virus among others in vitro, and another reported inhibition by abm extract against h n influenza virus in a plaque formation test after the viral invasion of host cells. also, antiviral effects have been proved for he: it was effective against intestinal damage of muscovy duck reovirus in ducklings, in which injured mucosal immunity was restored. moreover, he is reported to counteract dengue virus infection in vitro as shown by inhibition of attachment and penetration in plaque reduction assays and reduction in viral gene expression. there are several publications regarding antibacterial and antiparasitic properties of abm (table ) . we reported years ago that the abm-based extract, andosan™, when given orally day before or at time of intraperitoneal (i.p.) inoculation of bacteria, significantly reduced bacteraemia and increased the animals' survival in two lethal bacterial sepsis models in mice. , one model was with gram-positive pneumococci (streptococcus pneumonia serotype b) that tend to give pneumonia as superinfection in elderly covid- infected patients. the other sepsis model was with a suspension of air-exposed mouse fecalia, predominantly containing gram-negative bacteria and their toxins, which are feared culprits for sepsis development with its life-threatening complications from organ failure. in the pneumococcal infection model, also effects of other japanese abm extracts were studied in a blinded fashion after administration orally h before i.p. bacterial challenge. however, only andosan™ gave a statistically significant (p < . ) decrease in bacteraemia (> log, day ) and increase in survival rate; % ( / ) survival day in andosan™ group and none day in saline controls ( figure ). in fact, whereas % or % of the mice survived when given andosan™ by gavage hours before or at time of i.p. injection of the bacteria, respectively, only % of the saline gavage controls survived the days of experimentation in follow-up experiments. in the more lethal faecal sepsis model, % of mice given andosan™ hours prior to i.p. bacterial challenge survived during the days experiment in contrast to the saline controls that were all dead on day . when testing natural products against synthesis of the bacteriocin mutacin by streptococcus mutans in dental plaque biofilm, it was found to be inhibited by erinacine c from he. quorum sensing plays an important role for virulence, biofilm formation and survival of many pathogenic bacteria, including gram-negative pseudomonas aeruginosa. interestingly, an abm extract has been shown to have antiquorum sensing effect as demonstrated by reduction of virulence factors of p aeruginosa and its biofilm forming capability, which may be used as weapon against such pathogens. with regard to parasitic infections, abm has been shown to counteract murine visceral leishmaniasis by induction of a th immune response, and also to improve the consequence of cerebral malaria, by reduction of plasmodium berghei fluorescence labelled red blood cells in blood of mice, following their i.p. instillation. a gf furanone is found to inhibit opportunistic pseudomonas sp. pathogens in a plaque formation test. there are two reports on inhibitory effect of he extract against helicobacter pylori in vitro. , he also affected microbiota, which resulted in improved colonic health. johnson et al reported that andosan™ had predominantly anti-inflammatory effect in vivo, as demonstrated by systemic reduction in proinflammatory cytokines and antioxidant effect in peripheral leucocytes (table ) . another abm extract given orally to rats with carcinogen-induced lung damage, attenuated the pulmonary inflammation and ensuing gross pulmonary consolidation. as mentioned, abm did improve cerebral inflammation from malaria. moreover, he mycelium and he-derived erinacine a protected against neural cell death induced by brain ischaemia in a rat model by inhibiting inos and map kinase, proinflammatory cytokines tnfα, il- β and il- , and promoting nerve growth properties. in a randomized clinical study (rct), multiple myeloma patients undergoing high-dose chemotherapy and given add-on placebo-controlled treatment with the abmbased andosan™ for months, were found to have the following immunomodulatory effects : reduced il- ra levels in plasma and increased t regulatory cells indicating anti-inflammatory effect, and increased plasmacytoid dc in blood and increased expression of genes in bone marrow aspirate at end of study vs before for kirs and mhc antigens, the latter being important for antigen presentation. in another placebo-controlled rct with ibd patients, , therkelsen et al showed that andosan™ given orally for weeks reduced symptoms and increased quality of life especially of the patients with ulcerative colitis, by an anti-inflammatory mechanism. in a rat ibd model, also he extract and isolated polysaccharide were shown to improve ibd-induced colonic mucosa damage by reducing mpo activity. in colonic mucosa, also nf Κ b and tnfα expression was decreased and t cells were activated and growth of beneficial gut bacteria was promoted. additionally, a he polysaccharide was shown to attenuate colitis in mice by reversing gut dysbiosis from potentially proinflammatory microbes, for example corynebacterium and staphylococcus, to potentially anti-inflammatory microbes, for example bacteroides and bifidobacterium. the mechanism was downregulation of oxidative stress and inflammatory signalling pathways and maintenance of the intestinal barrier by blocking phosphorylation of nf Κ b, and protein kinases mapk and act in mice with induced colitis. moreover, abm dry feed has been shown to prevent non-alcoholic steato-hepatitis (nash) in a mouse model by preventing oxidative stress. similarly, a gf polysaccharide was shown to ameliorate lipid metabolic disorders in rats by beneficial regulation of microbiota. interestingly and in line with this, it was found that erinacine a-enriched he mycelia given orally to aged mice, increased their longevity by induction of endogenous antioxidant enzymes. immunomodulating β-glucans constitute the main part of the cell wall in fungi, including abm, he and gf mushrooms. such polysaccharides have been found to have anticancer and anti-infection effects when given i.p. in mouse models. [ ] [ ] [ ] previously, we have also shown that yeast β-glucan given orally can protect against systemic s. pneumoniae infection in mice. moreover, owing to abm-induced humoral and cellular responses, they are capable of adjuvating positive effects of hepatitis b virus and foot-and-mouth disease dna vaccines in mice. , abm, he and gf share pamps and damps with other highly poisonous and health-threatening fungi and macrofungi. accordingly, this must be the reason for the observed strong and rapid engagement of innate immunity and subsequent skewing of adaptive immunity from th towards th responses in the host when encountering edible and harmless mushrooms such as abm, he and gf. , , , prr of the innate immune system such as tlr , dectin- and cr , [ ] [ ] [ ] recognize immediately pamp such as β-glucans from the main cell wall in mushrooms and fungi. tlrs and nucleotide-binding oligomerization domain (nod)-like receptors are the two main prr, which interact, for example in aspergillus fumigatus infection. furthermore, β-glucan f i g u r e bacteraemia (a) (no. of cfu after plating of ul blood drawn daily from the animals' lateral femoral vein) and survival (b) of mice ( per group) challenged i.p with streptococcus pneumoniae serotype b h after oral administration (gavage) of different abm extracts. extract a is andosan™, which was the only extract that showed significant differences (p < . ) compared with saline control. control was phosphate-buffered saline (pbs) (modified from ref. [ ] with permission from scand j immunol for republication) activation of dendritic cells to il- β production occurs subsequent to nod inflammasome activation. candida albicans exists as a benign, commensal member of microbiota on mucosal surfaces in most humans, where it triggers numerous innate responses, but overgrowth can give localized mucosal or systemic infection. interestingly, c. albicans hyphae of the mycelium evoke prr activation by their damps and stimulate production of antimicrobial peptides by epithelial and innate immune cells, , whereof defensins are the largest group. other properties than just their β-glucans probably are important triggers of defensins and other antimicrobial peptides. in fact, it was a surprise when it recently was revealed that the β-glucan content of andosan™ was very low, probably due to its source being mycelia and not fruiting body. nevertheless, this mixed mushroom extract did stimulate tlr in monocytic cells. such abm stimulation is shown to promote differentiation of m to m cells and induce a potent antitumour effect. abm also stimulates the expression of nkg d/ncr cell surface receptors on nk cells. since port of entry for most non-vector-borne viruses is the mucosa, which also is the body surface exposed to the mushroom extracts upon intake, in vitro virus studies referred to (see table ) are quite similar to the in vivo situation. hence, abm and gf may very well have similar antiviral effect in vivo against polio virus and ev- , respectively, as demonstrated in vitro. , enteroviruses such as polio and ev- infect by the faecal-oral route and target gastrointestinal epithelium where they are detected and trigger innate immunity signalling, which they yet are masters in evading. this deregulation of inflammatory responses that results in a cytokine storm may play a critical role in pathogenesis of ev- pulmonary oedema and that of covid- infection as well. [ ] [ ] [ ] hence, one may assume that abm and gf also could counteract the covid- inflammatory lung injury. regarding hsv- and hsv- infections the host fails in initiating an effective early innate antiviral response and dc function, which should be targets for prophylactic strategies for preventing infections with these viruses. this may be the very same mechanism(s) as for the reported antiherpetic action of a gf protein and a abm mycelial polysaccharide in vivo. , moreover, the amelioration by abm of the wee virus-induced cytopathic effect demonstrated in vitro may reflect the antiviral effect against flaviviruses such as dengue virus also shown for he. in muscovy duck reovirus infection, there was a net loss of beneficial bacteria that produce short chain fatty acids (scfa) and compensatory proliferation of pathogenic bacteria (gram-negative enterobacteriaceae). this disruption of intestinal microbiota then results in severe pathology of intestinal mucosa and acute diarrhoea. the injured mucosa and its immunity could be restored by he, which was effective against this viral disease in ducklings. furthermore, oral intake in mice of andosan™ is shown to promote growth of bacteroides, potentially anti-inflammatory microbes, and production of scfa (commun. prof. t. ogita, shinshu univ, nagano, japan). this is also supported by our previous finding in the gram-negative faecal sepsis mouse model of significantly increased survival after oral treatment with andosan™. moreover, the antiviral effect of abm on influenza virus , is interesting because influenza corona viruses can give similar lung problems as covid- . although there was only insignificant reduction of hcv load in a few patients with chronic hcv infection who ingested andosan™ for a week, there was an increased expression in peripheral mononuclear leucocytes of genes related to g protein-r signalling, cell cycling and transcriptional regulation. g protein-coupled receptors for chemotaxins such as il- chemokine, leukotriene b, the complement activation product c a and bacteria-derived formyl peptides are associated with inflammation and microbial defence. since complement is involved in the pathogenesis of several of the diseases, due to self-attack or contribution to the overall pathology, [ ] [ ] [ ] which the mushrooms contained in andosan™ have proposed or documented health effects against, the mechanism of action of andosan™ may very well be linked to complement activity. examples of diseases with beneficial effect of these mushrooms are as follows: alzheimer's disease, ibd, , bacterial infections, , malaria, and allergy and asthma. this is supported by abm's ability to activate the alternative pathway of complement. also, there is new evidence for intracellular complementthe complosome-that is thought to be involved in immune cell regulation and metabolism in t cells and monocytes. this may further explain the involvement of complement in pathogenesis of such quite different diseases. the existence of intracellular complement was detected more than years ago by the first and second authors of this paper who showed that mononuclear phagocytes could produce all components for a functional complement system. , | abm, he, gf and covid- in the current situation with a seemingly non-curable pandemic at hand and where candidate drugs and vaccines just are in the testing stage, one must look at alternative prophylactic and therapeutic principles. one candidate is immune prophylaxis and/ or therapy by use of immunomodulatory mushrooms. the agaricomycota among the bacidiomycetes mushrooms, abm, he and gf, are wellknown medicinal mushrooms that have been used worldwide for a range of diseases in traditional medicine. in fact, many of those applications have been confirmed in preclinical and clinical studies. focus has especially been on antitumour effects where cytotoxicity and apoptotic mechanism have been revealed. however, in addition to an anti-inflammatory property, the mushrooms have also been found to induce enhanced th cellular immune response, as demonstrated by increase in ifnγ, il- and il- cytokines. , , cells participating in the th response are activated nk cells and cytotoxic th cells and γ/δ t cells, which besides tumour attack, also destroy virus-infected cells. moreover, γ/δ t cells play an important role in bridging the gap between innate and adaptive immunity, for example by being able to present antigen to conventional t cells, and they are predominantly localized in mucosa and epithelial sites, which are entry points for viruses. type iii interferons (ifn-λ) are thought to be especially important in antiviral immunity. , hence, the induction of the th response by medicinal mushrooms could be tested as a novel modality for prophylactic and/or therapeutic measures against covid- infection, as well as against its hazardous bacterial superinfection. in fact, bacterial infection, and especially with s. pneumonia, is found in % of the admitted elderly covid patients and in % of the dead. besides elderly patients, also those with complicating underlying diseases such as chronic obstructive lung disease, cardiac diseases, diabetes and other chronic diseases with systemic affection are at risk. in pneumococcal sepsis in mice caused by the strain s. pneumonia serotype b, initially isolated from a patient, the disease could be counteracted by andosan™ both when given orally either before or simultaneously with i.p. challenge with the pneumococci. hence, the extract seems to have both a prophylactic and a therapeutic effect against pneumococcal disease. moreover, since the effect of andosan™ was not antibiotical but immunomodulatory, this mushroom extract should be as effective against antibiotic-resistant bacteria as against antibiotic-sensitive bacteria. this aspect is especially interesting in context with the grave covid- situation that has been experienced in northern italy and spain, where pneumonia superinfection with multi-resistant bacteria may be an additive cause of death. also, a more effective riddance of a bacterial superinfection would dampen the immune response and the ensuing inflammation that otherwise may complicate the covid- disease. non-digestible carbohydrates with prebiotic effect, such as β-glucan polysaccharides from medicinal mushrooms, stimulate growth of gut microbes that are favourable to the host's health, and spur on the production of scfa, which energize anaerobic gut microbes, suppress pathogens (eg salmonella sp.) and improve host immunity. , in this context, the increased production observed of scfa by microbiota would probably stabilize colonocytes by being their main nutritional substrate, especially β-oh-butyrate ( %), generally as well as in ibd. such a trophical effect per se would normalize and equalize the physiological reaction to the body as such, which would benefit both healthy individuals prophylactically and patients therapeutically in the struggle against pathological agents, for example viral attacks such as from besides in the bacterial sepsis models, , the mushroom mixed product andosan™ has also given positive results both in murine models for allergy and colorectal cancer, where an increased t helper cell (th) immune response was found in both models in addition to a proinflammatory response (increased il- β, mcp- , tnfα) in the latter. the proinflammatory response in mice is probably due to uptake of β-glucans from the murine gut as opposed to the anti-inflammatory effect in humans where β-glucan is hardly taken up or to a lower degree, but may stimulate peyer's patches in the gut-associated lymphoid tissue (galt). therefore, other absorbable less defined low molecular weight substances (eg flavonoids) with anti-inflammatory and/or antioxidant activity probably contribute to this effect. in addition, in a placebo-controlled rct in individuals with pollen allergy and asthma, andosan™ supplementation before the pollen season resulted in decreased symptoms, medication, specific plasma ige levels and basophil sensitivity, owing to a skewing of th /th cells towards the th phenotype. the th response is besides its induction of antitumoral and anti-allergic activity, also driving an antiviral immune response as discussed. in conclusion, from the literature it seems possible that the related medicinal basidiomycetes mushrooms, abm, he and gf would have merit as prophylactic or therapeutic add-on remedies in covid- infection, especially as countermeasures against a pneumococcal superinfection, even when caused by multi-resistant bacteria, as well as for the immune overreaction and damaging inflammation that occurs with covid- attack. geir hetland is a cofounder and shareholder of immunopharma, oslo, norway. the other authors declare no commercial or financial conflict of interest. immunopharma had no other role than providing andosan™ free of charge for the studies referred to. geir hetland wrote the manuscript, and egil johnson, soosaipillai bernardshaw and bjørn grinde revised the manuscript. all authors have previously been much involved in several of the articles that this review is built upon. https://orcid.org/ - - - bioactivities and health benefits of mushrooms mainly from china therapeutic effects of substances occurring in higher basidiomycetes mushrooms: a modern perspective anti-inflammatory and anti-allergic effect of agaricus blazei extract in bone marrow-derived mast cells suppression of the inflammatory response by triterpenes isolated from the mushroom ganoderma lucidum anti-inflammatory activity of methanolic extracts from edible mushrooms in lps activated raw . macrophages pharmacological and pharmacokinetic studies with agaricoglycerides, extracted from grifola frondosa, in animal models of pain and inflammation antiinflammation and antioxidant effect of cordymin, a peptide purified from the medicinal mushroom cordyceps sinensis, in middle cerebral artery occlusion-induced focal cerebral ischemia in rats effects of the medicinal mushroom agaricus blazei murill on immunity, infection and cancer effect of a medicinal agaricus blazei murill-based mushroom extract, andosan™, on symptoms, fatigue and quality of life in patients with ulcerative colitis in a randomized single-blinded placebo controlled study effect of the medicinal agaricus blazei murill-based mushroom extract, andosantm, on symptoms, fatigue and quality of life in patients with crohn's disease in a randomized single-blinded placebo controlled study cytokine levels after consumption of a medicinal agaricus blazei murill-based mushroom extract, andosan™, in patients with crohn's disease and ulcerative colitis in a randomized single-blinded placebo-controlled study immunomodulatory effects of the agaricus blazei murill-based mushroom extract andosantm in patients with multiple myeloma undergoing high dose chemotherapy and autologous stem cell transplantation. a randomized, double blinded clinical study agaricus blazei-based mushroom extract supplementation to birch allergic blood donors: a randomized clinical trial clinical features of patients infected with novel coronavirus in wuhan lung pathology of fatal severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome protective and harmful immunity to rsv infection regulatory t cells and t helper cells in viral infection candida innate immunity at the mucosa coronavirus infections and immune responses effect of an extract based on the medicinal mushroom agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo an extract of the medicinal mushroom agaricus blazei murill can protect against allergy effect of an extract based on the medicinal mushroom agaricus blazei murill on expression of cytokines and calprotectin in patients with ulcerative colitis and crohn's disease effect of andosan™ on expression of adhesion molecules and production of reactive oxygen species in human monocytes and granulocytes in vivo inhibition by agaricus blazei murill fractions of cytopathic effect induced by western equine encephalitis (wee) virus on vero cells in vitro linhares antiviral activity of aqueous and ethanol extracts and of an isolated polysaccharide from agaricus brasiliensis against poliovirus type the mushroom agaricus blazei murill extract normalizes liver function in patients with chronic hepatitis b effects on gene expression and viral load of a medicinal extract from agaricus blazei in patients with chronic hepatitis c infection inhibition of hepatitis b virus by d-fraction from grifola frondosa: synergistic effect of combination with interferon-alpha in hepg . . isolation, identification and function of a novel anti-hsv- protein from grifola frondosa antiviral properties of polysaccharides from agaricus brasiliensis in the replication of bovine herpesvirus antiherpetic activity of an agaricus brasiliensis polysaccharide, its sulfated derivative and fractions in vivo anti-herpes simplex activity of a sulfated derivative of agaricus brasiliensis mycelial polysaccharide structural characterization and antiviral activity of a novel heteropolysaccharide isolated from grifola frondosa against enterovirus antiviral properties of basidiomycetes metabolites in vitro anti-influenza virus activity of agaricus brasiliensis ka hericium erinaceus polysaccharide facilitates restoration of injured intestinal mucosal immunity in muscovy duck reovirus-infected muscovy ducklings anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype : an in-vitro study an extract of the mushroom agaricus blazei murill administered orally protects against systemic streptococcus pneumoniae infection in mice an extract of the mushroom agaricus blazei murill protects against lethal septicemia in a mouse model of fecal peritonitis coronavirus disease in elderly patients: characteristics and prognostic factors based on -week follow-up the role of gut microbiota in sepsis screening for inhibitors of mutacin synthesis in streptococcus mutans using fluorescent reporter strains agaricus blazei hot water extract shows anti quorum sensing activity in the nosocomial human pathogen pseudomonas aeruginosa prophylactic or therapeutic administration of agaricus blazei murill is effective in treatment of murine visceral leishmaniasis evaluation of adjuvant activity of fractions derived from agaricus blazei, when in association with the recombinant lihyp protein, to protect against visceral leishmaniasis effect of mushroom agaricus blazei on immune response and development of experimental cerebral malaria extraction, identification and antimicrobial activity of a new furanone, grifolaone a, from grifola frondosa anti-helicobacter pylori activity of bioactive components isolated from hericium erinaceus vitro and in vivo inhibition of helicobacter pylori by ethanolic extracts of lion's mane medicinal mushroom, hericium erinaceus (agaricomycetes) isolation, purification and physicochemical properties of polysaccharide from fruiting body of hericium erinaceus and its effect on colonic health of mice royal sun medicinal mushroom agaricus brasiliensis (higher basidiomycetes) and the attenuation of pulmonary inflammation induced by -(methylnitrosamino)- -( -pyridyl)- -butanone (nnk) protective effects of hericium erinaceus mycelium and its isolated erinacine a against ischemia-injury-induced neuronal cell death via the inhibition of inos/p mapk and nitrotyrosine extracts from hericium erinaceus relieve inflammatory bowel disease by regulating immunity and gut microbiota polysaccharide of hericium erinaceus attenuates colitis in c bl/ mice via regulation of oxidative stress, inflammation-related signaling pathways and modulating the composition of the gut microbiota agaricus brasiliensis ka may prevent diet-induced nash through its antioxidant, anti-inflammatory, and anti-fibrotic activities in the liver grifola frondosa polysaccharides ameliorate lipid metabolic disorders and gut microbiota dysbiosis in high-fat diet fed rats erinacine a-enriched hericium erinaceus mycelia promotes longevity in drosophila melanogaster and aged mice antitumor beta glucan from the cultured fruit body of agaricus blazei host-mediated antitumor effect of grifolan nmf- n, a polysaccharide obtained from grifola frondosa glucan-induced enhancement of host resistance to selected infectious diseases protective effect of betaglucan against systemic streptococcus pneumoniae infection in mice coimmunization of agaricus blazei murill extract with hepatitis b virus core protein through dna vaccine enhances cellular and humoral immune responses extract from agaricus blazei murill can enhance immune responses elicited by dna vaccine against foot-andmouth disease amelioration of skewed th /th balance in tumor-bearing and asthma-induced mice by oral administration of agaricus blazei extracts interactions of toll-like receptors with fungi soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type (cd b/cd ) generates a primed state of the receptor capable of mediating cytotoxicity of ic b-opsonized target cells pattern recognition receptors and inflammation the crosstalk between tlr and nod in aspergillus fumigatus keratitis syk-dependent glycolytic reprogramming in dendritic cells regulates il- β production to β-glucan ligands in a tlr-independent manner hbd- : a novel beta-defensin from human plasma expression of beta-defensin and mrna by human monocytes, macrophages and dendritic cells defensins: transcriptional regulation and function beyond antimicrobial activity the polar high molecular weight fraction of the agaricus blazei murill extract, andosan™, reduces the activity of the tumor-associated protease, legumain, in raw . cells the medicinal and antitumor mushroom agaricus blazeii murill activates nf-kappab via tlr in monocytic cells and induces expression of cell surface markers and production of cytokines in human monocyte-derived dendritic cells (mddc) in vitro immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on nk cell-mediated cytotoxicity polysaccharide agaricus blazei murill stimulates myeloid derived suppressor cell differentiation from m to m type, which mediates inhibition of tumour immune-evasion via the toll-like receptor pathway enteroviruses: a gut-wrenching game of entry, detection, and evasion cytokine immunopathogenesis of enterovirus brain stem encephalitis herpes simplex virus evasion of early host antiviral responses muscovy duck reovirus infection disrupts the composition of intestinal microbiota in muscovy ducklings viral exploitation and subversion of the immune system through chemokine mimicry a review of human diseases caused or exacerbated by aberrant complement activation complement in the pathogenesis of alzheimer's disease the intestinal complement system in inflammatory bowel disease: shaping intestinal barrier function the cyanthin diterpenoid and sesterterpene constituents of hericium erinaceus mycelium ameliorates alzheimer's disease-related pathologies in app/ ps transgenic mice activation f the alternative complement pathway by agaricus blazei murill intracellular complement − the complosome − in immune cell regulation formation of the functional alternative pathway of complement by human monocytes in vitro as demonstrated by phagocytosis of agarose beads mononuclear phagocytes have the potential to synthesize the complete functional complement system the agaricus blazei-based mushroom extract, andosan™, protects against intestinal tumorigenesis in the a/j min/+ mouse phenotypic and functional plasticity of gamma-delta (γδ) t cells in inflammation and tolerance the impact of the interferon-lambda family on the innate and adaptive immune response to viral infections type iii interferons in viral infection and antiviral immunity prebiotic effects: metabolic and health benefits the prebiotic potential of brewers' spent grain on livestock's health: a review effects of orally administered yeast-derived beta-glucans: a review immunomodulation of fungal β-glucan in host defense signaling by dectin- can medicinal mushrooms have prophylactic or therapeutic effect against covid- and its pneumonic superinfection and complicating inflammation? key: cord- -ix un c authors: teixeira, maria c.; carbone, claudia; sousa, maria c.; espina, marta; garcia, maria l.; sanchez-lopez, elena; souto, eliana b. title: nanomedicines for the delivery of antimicrobial peptides (amps) date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: ix un c microbial infections are still among the major public health concerns since several yeasts and fungi, and other pathogenic microorganisms, are responsible for continuous growth of infections and drug resistance against bacteria. antimicrobial resistance rate is fostering the need to develop new strategies against drug-resistant superbugs. antimicrobial peptides (amps) are small peptide-based molecules of – amino acids in length, with potent and broad-spectrum antimicrobial properties. they are part of the innate immune system, which can represent a minimal risk of resistance development. these characteristics contribute to the description of these molecules as promising new molecules in the development of new antimicrobial drugs. however, efforts in developing new medicines have not resulted in any decrease of drug resistance yet. thus, a technological approach on improving existing drugs is gaining special interest. nanomedicine provides easy access to innovative carriers, which ultimately enable the design and development of targeted delivery systems of the most efficient drugs with increased efficacy and reduced toxicity. based on performance, successful experiments, and considerable market prospects, nanotechnology will undoubtedly lead a breakthrough in biomedical field also for infectious diseases, as there are several nanotechnological approaches that exhibit important roles in restoring antibiotic activity against resistant bacteria. the discovery of new antimicrobial molecules in the early s was a landmark in the field of pharmacology allowing the reduction of morbidity and mortality from infectious diseases, which were among the main causes of death worldwide [ ] . however, the widespread and indiscriminate use of nanomaterials , , of burrer et al. have used several strains of murine coronavirus in cell culture and in vivo in mouse models for the assessment of the antiviral properties of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (p-pmos) [ ] . the authors have reported the targeting effect of p-pmos against various target sites in the viral genome in cell culture and protected mice against virus-induced tissue damage. ykeda et al. have used an antimicrobial peptide isolated from the skin of xenopus tropicalis (pxt- ), and its modified peptide (modify-pxt- ) to produce self-assembled discoidal nanoparticles composed of amphiphilic alpha-helical scaffold proteins or peptides organised in a lipid bilayer [ ] . both the peptides pxt- , having hydrophobic and hydrophilic faces, behaved like general surfactants and can be used as carriers. bacteriocins, while very active at low concentrations, usually have low in vivo stability, being susceptible to degradation by proteolytic enzymes [ ] . another major limitation on the use of these peptides is the difficulty encountered in their large-scale production, compromising altogether their clinical application. an approach to overcome their limited stability in vivo is their loading into nanoparticles. nanomedicine is currently a well-established approach intimately related to the design and development of nanomaterials with unique therapeutic and diagnostic properties [ ] . nanotechnologies have also shown great potential in almost every aspect of the management of microbial infection with more than ten nanoparticles (nps)-based products marketed for bacterial diagnosis, antibiotic delivery and medical devices in . with unique physicochemical characteristics, nanomaterials are sensitive and selective in the detection of bacterial signalling and may also exbibit intrinsic antimicrobial properties. furthermore, nanomaterials can be used for antimicrobial drug delivery, and the incorporation of antimicrobial nanomaterials in medical devices and implants can prevent microbial adhesion and infection [ , ] . all these facts are instrumental against antimicrobial resistance by compromising bacterial mechanisms of resistance [ ] . focusing on nanomaterials-based drug delivery systems, these offer an improved strategy to increase the therapeutic index, by decreasing the dosage and frequency of administration. besides, nanomaterials promote intracellular drug delivery, mitigating the development of drug-resistant bacteria and also allowing targeted organ accumulation by functionalized surface modifications, thus limiting systemic side effects and immunosuppression [ ] . despite these promising outcomes, the main challenge of establishing clinical use is related to the evaluation of interactions of nano-antibiotics with cells, tissues and organs achieving information about their possible toxic effects, together with the production on a large scale [ , , ] . several types of nanomaterials have shown potential in the pharmaceutical field. they have also been studied as potential drug carriers with applications in the delivery of amps, promising antimicrobial molecules which, due to their nature and physicochemical characteristics, have limited bioavailability. the aim of this work is to revise the state-of-the-art on the approach that combines the advantages of the design of new drug delivery systems for the improvement of antimicrobial bioavailability, taking into account the recent developments in nanomaterials for antimicrobial peptide delivery. amps are small natural oligopeptides that have recently showed a potential activity against antibiotics resistance mechanisms, due to their ability in lysing bacterial membranes, thus providing broad-spectrum effects, targeting microorganisms from viruses to parasites. the main interesting property of amps is their ability to selectively disrupt bacterial membranes without affecting mammalian cells, thus being safe. in addition, amps are referred to in the literature as host-defence peptides with anti-inflammatory and anticancer activities, because they are synthesized molecules that take action on the defence mechanisms against biological threats of the living organism of origin [ ] . the discovery of amps dates back to the first half of the th century when in , dubos extracted an antimicrobial agent from a soil bacillus strain, proven to be effective in mice pneumococci infection. this extract was then fractioned allowing the identification of gramicidin. despite some systemic toxicity, gramicidin has shown to be effective in the topical treatment of wounds and ulcers. the first animal-originated amp to be reported is defensin, isolated from rabbit leukocytes in [ ] . nowadays, more than amps have been described and current molecular developments can be consulted in a series of databases available on the web (table ) , including natural identified molecules, as well as peptidomimetic molecules and analogues that are pharmacologically designed thanks to the use of bioinformatics [ ] . current food and drug administration (fda)-approved amps with well-established use include bacitracin, colistin and polymyxin b (although only for topical administration) [ ] . despite the lack of consensus on the influence of peptide sequence in biologic activity of amps, some common characteristics seem to be important and fairly related to their antimicrobial property. the main one is primarily charge, with % of amps being cationic, and secondly, hydrophobicity or amphipathicity, influencing solubility profiles and consequently bioavailability [ ] . associated with structural characteristics, these are also the main physicochemical properties that should also be taken into account in the design of new synthetic amps [ ] . compared to conventional antibiotics, amps demonstrate significant advantages such as potency and board spectrum of activity, as well as an additional activity to modulate the immune system responses and low resistance rates. they also show some limitations that impair their safe therapeutic use, such as sensitivity to proteolysis, influencing stability and undefined toxicological data for systemic use [ ] . in addition, their cationic and amphiphilic nature lead to high binding to serum proteins after parenteral administration, with consequent rapid elimination from bloodstream circulation and accumulation in the reticuloendothelial system, thus resulting in toxic effects and reduced activity [ ] . to overcome these limitations, some strategies have been developed, maximizing the proven therapeutic potential of amps. there are numerous methods for obtaining amp delivery systems. amps could be immobilized onto a variety of materials or onto a variety of surfaces and still retain their antibacterial activity. also, amps can be targeted through loading them in nanoparticulate systems with selective delivery capacities, including polymers, liposomes, hydrogels, nanospheres, nano-capsules and carbon nanotubes [ ] . as practical examples on of these of strategies, the studies of mishra et al. were focused on the development of an amp-coated surface, specifically immobilizing lassioglossin-iii onto silicone-based catheters and its activity against urinary tract infections [ ] . at the same time, water et al. developed the possibility of encapsulating plectasin, a cationic amp, into polymeric nps and evaluated their efficacy on s. aureus strains [ ] . bacteria show resistance to antibiotics drugs through a variety of mechanisms. moreover, the development of even new mechanisms of resistance has resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (mdr) bacterial strains [ , ] . however, when bacteria are drug-resistant it does not mean that they stop responding to an antibiotic, but that occurs only at higher concentrations [ , ] . of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial agent, in particular antibiotics, and proliferate and spread under the selective pressure of use of that drug. one approach to address this challenge is to design analogues of drugs [ , ] that are already in clinical use and that have activity against resistant organisms. however, bacteria are constantly succeeding to develop a resistant mechanism to new antibiotic drugs as well as to their analogues [ , ] . nanotechnology offers opportunities to re-explore the biological properties of already known antimicrobial compounds such as antibiotics by manipulating their size to modify their effect. the combination of amps with nanomaterials is not new. out of the available , papers indexed in the web of science, a total of , combining amps with nanoparticles or nanomaterials have been published over the last years in a range of disciplines ( figure ). nanomaterials , , x for peer review of water et al. developed the possibility of encapsulating plectasin, a cationic amp, into polymeric nps and evaluated their efficacy on s. aureus strains [ ] . bacteria show resistance to antibiotics drugs through a variety of mechanisms. moreover, the development of even new mechanisms of resistance has resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (mdr) bacterial strains [ , ] . however, when bacteria are drug-resistant it does not mean that they stop responding to an antibiotic, but that occurs only at higher concentrations [ , ] . of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial agent, in particular antibiotics, and proliferate and spread under the selective pressure of use of that drug. one approach to address this challenge is to design analogues of drugs [ , ] that are already in clinical use and that have activity against resistant organisms. however, bacteria are constantly succeeding to develop a resistant mechanism to new antibiotic drugs as well as to their analogues [ , ] . nanotechnology offers opportunities to re-explore the biological properties of already known antimicrobial compounds such as antibiotics by manipulating their size to modify their effect. the combination of amps with nanomaterials is not new. out of the available , papers indexed in the web of science, a total of , combining amps with nanoparticles or nanomaterials have been published over the last years in a range of disciplines ( figure ). evidence collected in the review work of huh and kwon [ ] , pelgrift and friedman [ ] , brooks and brooks [ ] , diab et al. [ ] and shimanovich and gedanken [ ] clarifies microbial drug resistance mechanisms and how nanotechnology may be considered a tool against this issue. development of drug resistance occurs in (at least) three steps: (i) acquisition by microbes of resistance genes; (ii) expression of those resistance genes; (iii) selection for microbes expressing those resistance genes. first, bacteria acquire resistance to single and multiple drugs through horizontal gene transfer by transformation, conjugation and transduction [ ] . bacteria can also acquire resistance genes by spontaneous mutation of existing genes [ ] . mdr is acquired when a bacterial cell already containing one type of drug resistance gene acquires another type of drug resistance gene [ , ] . second, in response to exposure to an antimicrobial drug, microbes express the resistance gene [ ] . third, resistance becomes widespread when there is selection for microbes that express resistance genes against the antimicrobial drug. this selective pressure in favour of resistance occurs whenever evidence collected in the review work of huh and kwon [ ] , pelgrift and friedman [ ] , brooks and brooks [ ] , diab et al. [ ] and shimanovich and gedanken [ ] clarifies microbial drug resistance mechanisms and how nanotechnology may be considered a tool against this issue. development of drug resistance occurs in (at least) three steps: (i) acquisition by microbes of resistance genes; (ii) expression of those resistance genes; (iii) selection for microbes expressing those resistance genes. first, bacteria acquire resistance to single and multiple drugs through horizontal gene transfer by transformation, conjugation and transduction [ ] . bacteria can also acquire resistance genes by spontaneous mutation of existing genes [ ] . mdr is acquired when a bacterial cell already containing one type of drug resistance gene acquires another type of drug resistance gene [ , ] . second, in response to exposure to an antimicrobial drug, microbes express the resistance gene [ ] . third, resistance becomes widespread when there is selection for microbes that express resistance genes against the antimicrobial drug. this selective pressure in favour of resistance occurs whenever microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug nanomaterials , , of followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials , , x for peer review of microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: i) controllable and relatively uniform distribution in the target tissue; ii) improved solubility; iii) sustained and controlled release; iv) improved patientcompliance; v) minimized side effects; and vi) enhanced cellular internalization [ , ] . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: (i) controllable and relatively uniform distribution in the target tissue; (ii) improved solubility; (iii) sustained and controlled release; (iv) improved patient-compliance; (v) minimized side effects; and (vi) enhanced cellular internalization [ , ] . nanomaterials , , x for peer review of microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: i) controllable and relatively uniform distribution in the target tissue; ii) improved solubility; iii) sustained and controlled release; iv) improved patientcompliance; v) minimized side effects; and vi) enhanced cellular internalization [ , ] . current advances in the use of inorganic nanoparticles for amp delivery involve essentially the development of silver nanoparticles (agnps) and gold nanoparticles (aunps), as well as silicon derivates nano-systems [ ] . the studies revised in this work are summarized in table . the development of polymeric nanoparticles for amp delivery may offer an excellent technological strategy to improve drug bioavailability and safety, avoiding drug chemical and enzymatic degradation, preventing aggregation, enhancing controlled release. chitosan (cs) nps (csnps) are particularly interesting as the broad spectrum of antibacterial activity of cs is well known and documented, offering the possibility of synergistic effects with antimicrobial molecules. moreover, due to its biocompatibility properties, cs nanostructures have been extensively studied for drug delivery, and that is no different for amp delivery. in recent years, an increasing number of papers reporting on a new generation of antimicrobial metallic nps has been published [ ] . consequently, many of the information on the application of nanotechnology in the infectious disease field regards the use of silver (ag) and gold (au) nps [ , , ] . recently, derivatives of other metals have been studied for antimicrobial applications, and the antibacterial effects of zero-valent bismuth nps and uncoated au, nickel (ni) and silicon (si) nps were reported [ , ] . despite the demonstrated intrinsic antimicrobial properties, dispersed metallic nps tend to aggregate and separate in solution, resulting in a decrease in their antimicrobial efficiency. with the aim of improving antibacterial properties, functionalization of nps has been attempted with surfactants, polymers or antibiotics resulting in more stable, less aggregated nps suspension and innovative, synergistic antibacterial agents. for instance, silver nps stabilized by polymers(polyvinylpyrrolidone) and surfactants (sds and tween ) exhibit enhanced antibacterial activities [ ] . nps can also act as drug-carriers able to pass through cell membranes [ , ] . widely used antibiotics such as ciprofloxacin may benefit from the association with nps, and conjugation may result in an antibacterial effect also against micro-organisms resistant to the same molecule in the naturally occurring form [ ] . when the antimicrobial agents are covalently linked to or contained within, nps, a higher drug concentration is attained in the area of interest, resulting in better efficacy at comparable doses and in slower release over time that may be exploited for preventing bacterial colonization [ , ] . moreover, specific biological sites can be attacked after modification of nps with target molecules [ , ] . as the nps themselves may have antibacterial properties, the combination of nps and loaded drugs exert a synergistic action [ ] . current advances in the use of inorganic nanostructures for amp delivery involve essentially the development of ag and aunps, as well as silicon derivate nano-systems. the studies revised in this work are summarized in table . research on gold nps is increasing thanks to their many advantages, such as their ease of synthesis and conjugation to biomolecules, their capability to maintain their own structure when in circulation and their improved effectiveness against bacteria, thus demonstrating their high potential in the field of nanomedicine. even if at the beginning, the research in this field was focused on the possibility to exploit gold nps in combination to laser radiation, thus significantly reducing bacteria viability due to cell lysis and mechanical disruption [ ] . currently, two recent studies have been reported exploring the use of au nanostructures for amp delivery. photoluminescent au nanodots (aunds) were prepared by chen et al. [ ] . these aunds were functionalized with hybridized ligands, an antimicrobial peptide (surfactin; sft), and -dodecanethiol (dt), on aunps. ultrasmall sft/dt-au nds (size ≈ . nm) were achieved and exhibited highly efficient antimicrobial activity. the photoluminescence properties and stability as well as the antimicrobial activity of sft/dt-au nds were also studied, and it was shown that these characteristics are highly dependent on the density of sft on au nds. relative to sft, sft/dt-aunds exhibited greater antimicrobial activity, not only to non-multidrug-resistant bacteria but also to the multidrug-resistant bacteria. the minimal inhibitory concentration values of sft/dt-aunds were much lower (> -fold) than that of sft. the authors considered that the antimicrobial activity of sft/dt-aunds was mainly achieved by the synergistic effect of sft and dt-aunds on the disruption of the bacterial membrane. in vitro cytotoxicity and hemolysis, analyses were also performed and had revealed superior biocompatibility of sft/dt-aunds than that of sft. moreover, in vivo methicillin-resistant s. aureus infected wound healing studies in rats showed faster healing, better epithelialization. this study suggested that the sft/dt-aunds system may be a promising antimicrobial candidate for preclinical applications in treating wounds and skin infections [ ] . rai et al. also reported a one-step methodology to generate amp-conjugated (aunps) [ ] . the amp-conjugated aunps prepared showed controlled size ( nm) and low polydispersity and allowed the inclusion of high concentration of amps. further, these systems demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble amp, as well as low cytotoxicity against human cells [ ] . interestingly, akrami et al. showed the possibility to exploit gold nps as a novel anticancer platform [ ] . results of this research confirm the improvement of cell internalization of gold nps, with higher cytotoxicity and cellular uptake for smaller nps compared to larger nanospheres and nanorods, suggesting that anticancer effects of the selected peptides were modulated by the size and shape of the gold nanoparticles [ ] . geilich et al. developed novel delivery systems by combining silver nps and ampicillin so to achieve a synergistic dose-dependent effect on bacterial cells [ ] . the obtained polymersomes were safe on human fibroblasts and more effective in inhibiting bacterial cells with a silver-to-ampicillin ratio of one to . , respectively. recent advances in amp delivery by ag nanostructures, pazos et al. [ ] reported on supramolecular assemblies of novel peptide amphiphiles (pas) containing aldehyde functionality in order to reduce ag ions and subsequently nucleate ag metal nps in water. this proposed system spontaneously generates monodisperse ag particles at regular distances along the length of the filamentous organic assemblies. the metal−organic hybrid structures exhibited antimicrobial activity and significantly less toxicity toward eukaryotic cells. metallized organic nanofibers of the type described offer the possibility to create other structures. for instance, hydrogels, that can be potentially applied in wound dressing development [ ] . also addressing the wound infection problem, salouti et al. investigated the synergistic antibacterial effect of plant peptide mbp- and agnps on infected wounds caused by s. aureus [ ] . the mic and mbc of mbp- and agnps both on their own and in combination form were determined against s. aureus via macrodilution and microdilution methods. the mic and mbc of mbp- were found to be . and . mg/ml, respectively. mic and mbc of agnps were determined to be . and . mg/l, respectively. mic and mbc of the agnps and mbp- combination were found to be . mg/ml, . mg/l; and . mg/ml, . mg/l, respectively. the synergistic antibacterial effect of ag nps and mbp- was investigated on infected wounds caused by s. aureus in a mouse model, and the infected wound healed properly after the combined use of mbp- and agnps [ ] . it is worth to note recent findings by chaudhari et al. concerning the conjugation of silver-coated carbon nanotubes with the antimicrobial peptide tp . herein, authors underline the occurrence of an additive effect of silver-coated nanotubes and tp regarding antibacterial activity. the innovative nano-system was found to be safe to murine macrophages and hep cells at the mic concentrations [ ] . as delivery systems for amps, silicon and silicon derivates nanostructures have also been investigated recently. membrane interactions are critical for the successful use of mesoporous sinps. in order to elucidate these, braun et al. have studied the effects of np charge and porosity on amp loading and release, as well as consequences of this for membrane interactions and antimicrobial effects [ ] . anionic mesoporous sinps were found to incorporate considerable amounts of the camp ll- , whereas loading was found to be much lower for non-porous or positively charged sinps. the results also demonstrated that due to preferential pore localization, anionic mesoporous particles, but not the other particles, protects ll- from degradation by infection-related proteases. for anionic sinps, membrane disruption is mediated almost exclusively by peptide release. in contrast, non-porous sinps built up a resilient ll- surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. for positively charged mesoporous sinps, ll- incorporation promoted the membrane binding and disruption displayed by the particles in the absence of peptide, but also caused toxicity against human erythrocytes. thus, the use of mesoporous sinps as amp delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nps [ ] . the properties of amps adsorbed on inorganic or organic surfaces are of interest for their potential applications in intracellular drug delivery. in the work of syryamina et al., continuous-wave (cw) electron paramagnetic resonance (epr) and pulsed electron-electron double resonance (peldor) techniques were applied to study the adsorption of the amps trichogin ga iv and ampullosporin a on monodisperse colloidal silica nanospheres (sins) of nm diameter [ ] . the results obtained by cw epr supported the view that the adsorbed peptides form close-packed clusters. peldor data show that both trichogin and ampullosporin adsorbed on the silica surface possess a more disordered conformation as compared to that in solution. for ampullosporin, disordering is much more pronounced than for trichogin. after desorption, the peptides restored their conformations; upon adsorption, the peptides in some cases may lose partly their biradical character [ ] . these results may be of interest as the antimicrobial activity is often related to peptide conformation. nano-clays or layered silicates are an interesting nanostructure that has been used for remediation of environmental contaminants, delivery of drugs and various active molecules, and to enhance polymer mechanical and barrier properties in packaging films. they typically present a stacked arrangement of silicate layers with a nanometric thickness [ ] . meira et al. studied three different nano-clays (bentonite, octadecylamine-modified montmorillonite, and halloysite) as potential carriers for the amps nisin and pediocin, known bacteriocins, the first referred above as having application as a food preservative. higher adsorption at room temperature of nisin and pediocin was obtained on bentonite. the antimicrobial activity of the resultant bacteriocin-nano-clay systems was analysed using skimmed milk agar as food simulant, and the largest inhibition zones were observed against gram-positive bacteria for halloysite samples. bacteriocins were intercalated into the interlayer space of montmorillonites as deduced from the increase of the basal spacing measured by x-ray diffraction (xrd) assay. these results indicate that nano-clays, especially halloysite, are suitable nano-carriers for nisin and pediocin adsorption, and the results may be considered interesting for the food industry [ ] . different biodegradable polymeric nano-systems have been explored as carriers for antimicrobial agents that exhibit a high bactericidal activity. the efficacy of this strategy is well proven, highlighting polymeric nano-systems can effectively improve the cellular penetration, intracellular retention and specific subcellular distribution of antimicrobial agents, and even evade intracellular inactivation of antimicrobial agents [ ] . controlled drug release using biocompatible and biodegradable polymers further emerged in the s [ ] . after the first polymer-based delivery of macromolecules using poly[ethylene vinyl acetate] polymer was demonstrated in [ , ] . antimicrobial drug delivery using polymeric nps offers several advantages: (i) structural stability in biological fluids and under harsh and various conditions for preparation; (ii) precisely tuneable properties, such as size, zeta-potentials, and drug release profiles, by manipulating polymer lengths, surfactants, and organic solvents used for np preparation [ ] , and (iii) facile and versatile surface functionalization for conjugating drugs and targeting ligands [ ] . concerning antimicrobial nanoparticulate delivery systems, two major types of polymers have been explored: linear polymers (e.g., polyalkyl acrylates and polymethyl methacrylate) and amphiphilic block copolymers. the majority of polymeric nps prepared with linear polymers are nano-capsules or solid nanospheres [ ] . in polymeric nano-capsules, a polymeric membrane that controls the release rate surrounds the drugs that are solubilized in aqueous or oily solvents. in solid nanospheres, drugs are homogeneously distributed in the polymeric matrices of variable porosities [ , ] . amphiphilic block copolymers form self-assemble micellar nps with the drug being encapsulated in the hydrophobic core and surrounded by a hydrophilic shield. this shied allows the core to be protected from degradation [ ] . several biodegradable polymers, including poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(lactide-co-glycolide) (plga), poly(caprolactone) (pcl), and poly(cyanoacrylate) (pca), have been used as the hydrophobic core of the amphiphilic copolymers, whereas peg has been the most commonly used hydrophilic segment [ , [ ] [ ] [ ] [ ] . targeting ligands can also be conjugated on the termini of peg for targeted and selective delivery [ , ] . polymeric nps have been explored to deliver various antimicrobial agents and greatly enhanced therapeutic efficacy in treating many types of infectious diseases has been reported. for instance, encapsulated ampicillin in polymeric nps was effective for s. typhimurium infection treatment [ ] and intracellular l. monocytogenes infection in mouse peritoneal macrophages [ ] . the use of polymeric nps can overcome the limited oral administration of unstable or inadequately absorbed drugs, [ ] and, in addition, pegylation of nps, can increase drug half-life in serum, and improve mucoadhesive capabilities by reducing phagocytosis [ ] thus, among nanoparticle platforms, polymer nps may be the most suitable system that can be used for antimicrobial drug delivery. biodegradable polymers and bioorganic polymers are also promising materials in the delivery of peptide-based drugs due to their compatibility, degradation behaviour, and nontoxic nature of administration [ ] . the development of polymeric therapeutic nanostructures for amp delivery may offer an excellent technological strategy to improve drug bioavailability and safety. cs-based nps (csnps) are particularly interesting as the broad spectrum of antibacterial activity of cs is well known and documented, offering the possibility of synergistic effects with antimicrobial molecules. moreover, due to its biocompatibility properties, cs nanostructures have been extensively studied for drug delivery, and that is no different for amp delivery. in fact, the majority of the studies performed so far involves cs nanostructures and was already revised in previous work [ ] from which we reproduce table . research has been covering, the technological development of new carrier systems and their full characterization, and the evaluation of their efficacy as drug delivery improvers. in addition to cs-based nanostructures, recent studies on other polymeric nanostructures for amp delivery have been developed (table ). cs-based nanoparticles vancomycin cs particles were prepared by ionic gelation and freeze-drying or spray-drying as recovery methods. antibacterial activity against s. aureus. cerchiara et al. [ ] cs-based nanoparticles lysozyme as model development of a nanoparticle model with commercially available cs loaded with lysozyme as antimicrobial protein drug model. piras et al. [ ] cs and poly-gammaglutamic acid composites ll- the results indicated that both ll- and no were co-loaded successfully in micro particles, and the composite particles could sustain ll- and no release at physiological ph, in vitro. cryptdin- preparation of cs tripolyphosphate (cs-tpp) nps by ionotropic gelation. the formulation was then characterized on the basis of particle size, zeta potential and polydispersity, and antimicrobial in vivo assays against salmonella enterica were performed. cs-based nanoparticles temporin b cs-nps were prepared based on the ionotropic gelation between cs and sodium tripolyphosphate. the nano-carrier evidenced a sustained antibacterial action against various strains of s. epidermidis. nanogel composites -prepation of agnps embedded in a biocompatible nanogel comprising degradable, natural polymers. in this study, hybrid nanogels were prepared with varying polymer content and their potential by determining their antibacterial properties against e. coliand s. aureus strains. coll ferrer et al. [ ] glycol-cs nanogels -study of the biocompatibility of a glycol cs nanogel by evaluation of effects on metabolic activity, cell cycles blood compatibility. overall, the results demonstrated the safety of the use of the gc nanogel as drug delivery system. nanofibers and films cs thin films hlf - immobilization performed onto cs thin films as a model for an implant coating due to its reported osteogenic and antibacterial properties. cs thin films were produced by spin-coating on au surfaces. activity against methicillin-resistant s. aureus (mrsa). nanofibers defensin- , dermaseptin ll- magainin alternate deposition of polycation (cs) and polyanion over cotton gauzes. antimicrobial assays were performed with two strains: s. aureus and k. pneumonia. food packaging systems study of the efficiency as antimicrobial carriers of hydroxypropyl methylcellulose [ ] , chitosan (cs), sodium caseinate (sc) and polylactic acid [ ] films, in the release rates of fluorescently labeled nisin z, evaluating their potential as food packaging polymers. imran et al. [ ] nanomaterials , , of plla-l -pllain situ gel ap- potential application of amps in wound healing, by developing a biodegradable poly (l-lactic acid)-pluronic l -poly (l-lactic acid) (plla-l -plla) in situ gel-forming system li et al. [ ] food preservation systems nisin study of a safe suitable antimicrobial system to be used in food industry. influence of pectin degree of acetylation on np properties. krivorotova et al. [ ] chitosan/pga nanoparticles nisin influence of chitosan coating on colloidal stability, loading capacity and encapsulation efficiency wu et al. [ ] d'angelo et al. designed and developed a system of nano-embedded microparticles (nem) for sustained delivery of cationic amps (camps) [ ] in the lung, studying its effect on p. aeruginosa, a known lung infection pathogen. to this purpose, plga nps containing a model camp, colistin (col), were produced by the emulsion/solvent diffusion technique, and then spray-dried in different carriers (lactose or mannitol), thus producing nem. the most promising nem formulations were selected from bulk and flow properties, distribution of nps in the carrier and aerosolization performance upon delivery through a breath-actuated dry powder inhaler. col-loaded nem were found to kill p. aeruginosa biofilms and to display a prolonged efficacy compared to the free col. [ ] . another camp, plectasin, was encapsulated into plga nps using the double emulsion solvent evaporation method, in the work of water et al. [ ] the plectasin-loaded nps displayed a high encapsulation efficiency ( - %) and mediated release of the peptide over h. the antimicrobial efficacy was investigated using bronchial epithelial calu- cell monolayers infected with s. aureus, and encapsulated plectasin displayed improved efficacy as compared to non-encapsulated plectasin. the author also assessed the subcellular localization of the prepared nps in different relevant cell lines: calu- epithelial cells, thp- macrophages and a epithelial cells. here the results have shown good patterns of penetration on calu- epithelial cell lines, as well as in thp- macrophages [ ] . hydrogels and nanogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. the biological performance of these systems, particularly those used as wound dressing; can be complemented with antimicrobial activity capable of preventing colonization of the wound site by opportunistic bacterial pathogens [ , , ] . these types of structures have also been studied recently for amp delivery. continuing their study of the antimicrobial activity of multi-domain camps (md-camps) in solution, jiang et al. investigated the same effect of self-assembled -d hydrogels supramolecular nanostructures and its rheological properties [ ] . among the studied md-camps solutions, the bactericidal activity of peptide hydrogels was found to be improved. the improved antimicrobial activity of the self-assembled peptide hydrogels was found to be related to the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. thus, the structure-property-activity relationship developed through this study may provide important knowledge for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications [ ] . the water et al. group also designed novel nanogel-based novicidin delivery system. the peptide novicidin was self-assembled with a nano-ctenyl succinic anhydride-modified analogue of hyaluronic acid, and this formulation was optimized using a microfluidics-based quality-by-design approach. the encapsulation efficiency of novicidin ( - %) and the zeta potential (− to − mv) of the nanogels could be tailored by changing the preparation process parameters, with a maximum peptide loading of ± %. the nanogels exhibited good colloidal stability under different ionic strength conditions and allowed complete release of the peptide over days. furthermore, self-assembly of novicidin with hyaluronic acid into nanogels significantly improved the safety profile at least five-fold and six-fold when tested in huvecs and nih t cells, respectively, while showing no loss of antimicrobial activity against e. coli and s. aureus [ ] . li et al. explored the potential application of amps in wound healing, by developing a biodegradable poly(l-lactic acid)-pluronic l -poly(l-lactic acid) (plla-l -plla) in situ gel-forming system [ ] . an injectable formulation composed of human amps (ap- ) loaded nps, and thermosensitive hydrogel was prepared. ap- peptides were enclosed with biocompatible nps (ap- -nps) with high drug loading and encapsulation efficiency. ap- -nps were further encapsulated in a thermosensitive hydrogel (ap- -nps-h) to facilitate its application in cutaneous wound repair. as a result, ap- -nps-h released ap- in an extended period and exhibited quite low cytotoxicity and high anti-oxidant activity in vitro. the in vivo wound healing assay using a full-thickness dermal defect model of sd rats indicated that ap- -nps-h could significantly promote wound healing. at day after an operation, the treated group showed nearly complete wound closure of , ± , % [ ] . other studies of nisin nanoencapsulation were performed, with the purpose of protection to ensure the stability of this amp during food processing and storage period. nisin-loaded pectin nps (nlp-nps) were prepared and analysed by krivirotova et al. by a simple complexation method [ ] . three types of pectin biopolymer were tested and it was found that the methoxylation degree of pectin influenced nisin loading efficiency and particle size. for the complex formation, both electrostatic and hydrophobic interactions were important. nlp-nps exhibited antimicrobial activity that was dependent on the type of biopolymer. overall, the results indicated that nlp-nps might be a suitable antimicrobial system to be used in the food industry [ ] . nisin nanoencapsulation in self-assembly chitosan-and poly-glutamic acid, was recently reported by wu et al. [ ] . herein, the authors showed that the use of a coating layer of chitosan improved colloidal stability, loading capacity and encapsulation efficiency. furthermore, chitosan layer composite nanoparticles were more effective compared to uncoated nps and nisin in inhibiting the growth of escherichia coli and listeria monocytogenes. solid lipid nanoparticles (sln) and nanostructured lipid carriers (nlc) stand for nanoparticles composed of lipid materials that melt at temperatures above • c [ ] . their main purpose is to modify the release profile of the payload attributed to their solid core. while sln are composed of solid lipids only, nlc matrix is a blend of solid and liquid lipids (which also melts above the body temperature) to improve the solubility of lipophilic compounds. both sln and nlc are biodegradable and non-toxic [ ] [ ] [ ] , and their versatility of is also attributed to their capacity to load a range of chemically different bioactives, including peptides and proteins [ ] [ ] [ ] [ ] , and be modified on their surface [ , ] . fumakia et al. have developed sln for the simultaneous delivery of an endogenous host defence peptide (ll ) with antimicrobial activity and an elastase inhibitor serpin a (a ) for the treatment of wound infections [ ] . the authors reported that sln could modify the release profile of simultaneous delivery of both bioactives, accelerate wound healing in bj fibroblast cells and keratinocytes and promote antibacterial activity against staphylococcus aureus and escherichia coli in comparison to ll or a alone. ll loaded into nlc have also been proposed by garcia-orue for the topical treatment of chronic wounds [ ] . ll -loaded nlc did not affect cell viability tested against human foreskin fibroblasts while the in vitro bioactivity assay showed that the peptide remained active after the loading into lipid nanoparticles as the system reversed the macrophages activation as happened with the ll in solution. ll -loaded nlc were also active against escherichia coli. the in vivo testing in mice also demonstrated the systems' capacity to improve healing by promoting wound closure, reepithelization grade and restoration of the inflammatory process. lewies et al. demonstrated that biodegradable nlcs enhance the antibacterial activity of antimicrobial peptides [ ] . the authors have studied the effect of nisin z when loaded into nanostructured lipid carriers on staphylococcus aureus, staphylococcus epidermidis and escherichia coli. nisin z exhibited additive interactions with numerous conventional antibiotics, in particular, with novobiocin. the presence of edta improved the antimicrobial activity of free nisin z towards escherichia coli significantly. nisin z-loaded nlcs were effective against gram-positive species at physiological ph, also showing an increased efficacy when adding edta. nisin z-loaded nlcs were shown potential to enhance the antibacterial activity of nisin z towards gram-positive bacteria commonly found in skin infections. other types of nanomaterials, such as dendrimers and carbon nanodots, have also been successfully proposed for the delivery of amps. due to their ease of synthesis and low manufacturing costs, antimicrobial polymers including dendrimers have been exploited to mimic the antibacterial mechanism host defence peptides, by compromising bacterial cell membranes [ ] . gide et al. developed nanomaterials-lipidated amphiphilic dendrimers which displayed potent and selective antimicrobial activity against both gram-positive and gram-negative bacteria, including multidrug-resistant strains [ ] . these dendrimers are also shown to inhibit bacterial biofilms effectively. carbon nanodots or carbon quantum dots were originally discovered due to their bright fluorescence emissions as those found in conventional semiconductor quantum dots, and can also be used for drug delivery [ ] . carbon dots modified with vancomycin were proposed for the treatment of gram-positive bacterial infections [ ] . zhong et al. synthesized surface-modified carbon dots with vancomycin and successfully tested them against bacillus subtilis, listeria monocytogenes, salmonella, pseudomonas aeruginosa and escherichia coli. the modified carbon dots showed affinity to the tested gram-positive bacteria owing to the ligand-receptor interactions between vancomycin and the cell walls. pramanik et al. synthesis red/blue fluorescent carbon dot-attached magnetic nanoparticles for selective separation and identification of superbugs from infected blood samples [ ] . the nanoparticles were capable of isolating methicillin-resistant staphylococcus aureus (mrsa) and salmonella dt superbug from whole blood samples, followed by accurate identification via multicolor fluorescence imaging. the authors also described the design of antimicrobial peptide-conjugated multicolor fluorescent magneto-carbon dots for separation, identification and disinfection of multidrug resistance superbugs from infected blood. amps are antimicrobial compounds recognized as among the most promising drug candidates against infections. they exhibit distinct mechanisms of action and may show additional biological activities, e.g., as signalling molecules and biomarkers, and even as tumoricidal agents. amps, however, show low stability and bioavailability. the formulation of amps into nanomaterials seems to offer a very appealing and effective manner to improve these limitations, and several systems have been designed and studied for this purpose (e.g., inorganic, polymeric and lipid nanoparticles, carbon dots and dendrimers). however, concerns on how to regulate the distribution of nanomaterials in the body or specific organs are also raised. nano-drugs are foreign substances to the body and may produce inflammation. therefore, safety data for long-term therapy or repeated dosage are needed to circumvent the potential risk. biodegradable nanomaterials have been proposed to reduce the risk of toxicological events both in vitro and in vivo. to date, few studies have investigated the toxicological and environmental effects of direct and indirect exposure to nanomaterials, and no clear guidelines exist to quantify these effects. therefore, there is an urgent need for developing guidelines, which can assure the safe use of nanomaterials. moreover, more powerful ex vivo models or animal models are needed to assess the safety issues and to comply with government regulations. how to extend the shelf life of nanodrugs is also a problem due to their agglomeration also being a problem. the production methods for nanostructures should also be improved, and scalable studies for industrial production are also of great importance in order to promote cost effectiveness of these new formulations. the cost and production of nanomaterials on a large scale is one of the hurdles in effective implementation of these products. hence, the scientific community should also pay attention to developing affordable methodologies so that nanotechnology can reach patients. in conclusion, it seems that, although the promising research results in this area are rising, it is also urgent to start directing efforts in making these new drug formulations a reality as therapeutic agents. nanoantibiotics": a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era therapeutic strategies to combat antibiotic resistance nanotechnology as a therapeutic tool to combat microbial resistance potent antimicrobial peptides and the fight against multi drug resistant species: resistance is futile? antibiotics antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and nanosystems as vehicles for the delivery of antimicrobial peptides (amps) antiviral effects of antisense morpholino oligomers in murine coronavirus infection models lipid nanodisc formation using pxt- peptide isolated from amphibian (xenopus tropicalis) skin, and its altered form, modify-pxt- nanomedicines for antimicrobial interventions silver nanoparticles-composing alginate/gelatin hydrogel improves wound healing in vivo development of chitosan/silver sulfadiazine/zeolite composite films for wound dressing nanomedicine in the management of microbial infection-overview and perspectives nanopharmaceutics: part ii-production scales and clinically compliant production methods nanopharmaceutics: part i-clinical trials legislation and good manufacturing practices (gmp) of nanotherapeutics in the eu delivery systems for antimicrobial peptides antimicrobial peptides antimicrobial peptides: an alternative for innovative medicines? antimicrobial peptides: possible anti-infective agents therapeutic peptides: new arsenal against drug resistant pathogens effects of pegylation on membrane and lipopolysaccharide interactions of host defense peptides nanomaterials site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens nanoparticle-mediated delivery of the antimicrobial peptide plectasin against staphylococcus aureus in infected epithelial cells the rise of the enterococcus: beyond vancomycin resistance the future of antibiotics and resistance selection of resistant bacteria at very low antibiotic concentrations a self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics sampling the antibiotic resistome bacteria on the rampage resistance to antibiotics mediated by target alterations tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. microbiol insights in nanoparticle-bacterium interactions: new frontiers to bypass bacterial resistance to antibiotics nanotechnology solutions to restore antibiotic activity antibacterial properties of nanoparticles effects of salt stress on the antimicrobial drug resistance and protein profile of staphylococcus aureus antibiotic resistance: an overview of mechanisms and a paradigm shift nanotechnology based anti-infectives to fight microbial intrusions metal-based nanoparticles as antimicrobial agents: an overview self-assembly of antimicrobial peptides on gold nanodots: against multidrug-resistant bacteria and wound-healing application one-step synthesis of high-density peptide-conjugated gold nanoparticles with antimicrobial efficacy in a systemic infection model haririan, i. tuning the anticancer activity of a novel pro-apoptotic peptide using gold nanoparticle platforms silver nanoparticle-embedded polymersome nanocarriers for the treatment of antibiotic-resistant infections nucleation and growth of ordered arrays of silver nanoparticles on peptide nanofibers: hybrid nanostructures with antimicrobial properties nanomaterials synergistic antibacterial activity of plant peptide mbp- and silver nanoparticles combination on healing of infected wound due to staphylococcus aureus a novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides peptides on the surface: spin-label epr and peldor study of adsorption of the antimicrobial peptides trichogin ga iv and ampullosporin a on the silica nanoparticles adsorption of nisin and pediocin on nanoclays design and characterization of chitosan/zeolite composite films-effect of zeolite type and zeolite dose on the film properties zerovalent bismuth nanoparticles inhibit streptococcus mutans growth and formation of biofilm photogeneration of reactive oxygen species on uncoated silver, gold, nickel, and silicon nanoparticles and their antibacterial effects aggregation kinetics of citrate and polyvinylpyrrolidone coated silver nanoparticles in monovalent and divalent electrolyte solutions oligonucleotide-modified gold nanoparticles for intracellular gene regulation the effects of size, shape, and surface functional group of gold nanostructures on their adsorption and internalization by cells investigations of the antibacterial properties of ciprofloxacin@sio in vivo supramolecular templating enhances the activity of multivalent ligands: a potential therapeutic against the escherichia coli o ab toxins gold nanocages covered by smart polymers for controlled release with near-infrared light selective binding of mannose-encapsulated gold nanoparticles to type pili in escherichia coli role of size scale of zno nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells silver-nanoparticle-embedded antimicrobial paints based on vegetable oil photothermal killing of staphylococcus aureus using antibody-targeted gold nanoparticles antimicrobial nanostructures in food packaging biodegradable nanoparticles for intracellular delivery of antimicrobial agents biodegradable polymeric nanoparticles as drug delivery devices polymers for the sustained release of proteins and other macromolecules controlled release of microquantities of macromolecules development of nanoparticles for antimicrobial drug delivery nanotechnology applied to the treatment of malaria new old challenges in tuberculosis: potentially effective nanotechnologies in drug delivery poly(alkylcyanoacrylates) as biodegradable materials for biomedical applications drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles innovative hybrid vs polymeric nanocapsules: the influence of the cationic lipid coating on the " s eco-friendly aqueous core surface-modified nanocapsules polymeric nanoparticles augment the ocular hypotensive effect of melatonin in rabbits revisiting the role of sucrose in plga-peg nanocarrier for potential intranasal delivery formulation of functionalized plga-peg nanoparticles for in vivo targeted drug delivery presenting vancomycin on nanoparticles to enhance antimicrobial activities improved oral therapeutic potential of nanoencapsulated cryptdin formulation against salmonella infection effect of nanoparticle-bound ampicillin on the survival of listeria-monocytogenes in mouse peritoneal-macrophages preparation, characterization and in vitro antimicrobial activity of ampicillin-loaded polyethylcyanoacrylate nanoparticles amoxicillin-loaded polyethylcyanoacrylate nanoparticles: influence of peg coating on the particle size, drug release rate and phagocytic uptake chitosan and its derivatives for drug delivery perspective chapter -delivery of antimicrobials by chitosan-composed therapeutic nanostructures a -ficai hollow chitosan/alginate nanocapsules for bioactive compound delivery chitosan based micro-and nanoparticles for colon-targeted delivery of vancomycin prepared by alternative processing methods preparation, physical-chemical and biological characterization of chitosan nanoparticles loaded with lysozyme chitosan microparticles ionically cross-linked with poly(gamma-glutamic acid) as antimicrobial peptides and nitric oxide delivery systems chitosan nanoparticles loaded with the antimicrobial peptide temporin b exert a long-term antibacterial activity in vitro against clinical isolates of staphylococcus epidermidis designing nanogel carriers for antibacterial applications biocompatibility of a self-assembled glycol chitosan nanogel incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applications controlled release of nisin from hpmc, sodium caseinate, poly-lactic acid and chitosan for active packaging applications overcoming barriers in pseudomonas aeruginosa lung infections: engineered nanoparticles for local delivery of a cationic antimicrobial peptide self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity hyaluronic acid-based nano gels produced by microfluidics-facilitated self-assembly improves the safety profile of the cationic host defense peptide novicidin in situ gel-forming ap- peptide delivery system for cutaneous wound healing nisin-loaded pectin nanoparticles for food preservation. food hydrocoll formation, characterization and release kinetics of chitosan/gamma-pga encapsulated nisin nanoparticles the chemistry and engineering of polymeric hydrogel adhesives for wound closure: a tutorial antimicrobial polymeric hydrogels chapter -solid lipid nanoparticle formulations pharmacokinetic and biopharmaceutical aspects in drug delivery preclinical safety of solid lipid nanoparticles and nanostructured lipid carriers: current evidence from in vitro and in vivo evaluation comet assay reveals no genotoxicity risk of cationic solid lipid nanoparticles nanotoxicology applied to solid lipid nanoparticles and nanostructured lipid carriers-a systematic review of in vitro data lipid nanoparticles (sln ® , nlc ® ) for cutaneous drug delivery:structure, protection and skin effects solid lipid nanoparticles as a drug delivery system for peptides and proteins oral bioavailability of cyclosporine: solid lipid nanoparticles (sln) versus drug nanocrystals cyclosporine-loaded solid lipid nanoparticles (sln): drug-lipid physicochemical interactions and characterization of drug incorporation perillaldehyde , -epoxide loaded sln-tailored mab: production, physicochemical characterization and in vitro cytotoxicity profile in mcf- cell lines surface-tailored anti-her /neu-solid lipid nanoparticles for site-specific targeting mcf- and bt- breast cancer cells nanoparticles encapsulated with ll and serpin a promotes wound healing and synergistically enhances antibacterial activity ll loaded nanostructured lipid carriers (nlc): a new strategy for the topical treatment of chronic wounds interactions of the antimicrobial peptide nisin z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity nano-sized lipidated dendrimers as potent and broad-spectrum antibacterial agents employing carbon dots modified with vancomycin for assaying gram-positive bacteria like staphylococcus aureus magnetic multifunctional carbon dots for selective separation, identification, and eradication of drug-resistant superbugs this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -t r xqj authors: grundler, franziska; mesnage, robin; goutzourelas, nikolaos; tekos, fotios; makri, sotiria; brack, michel; kouretas, demetrios; wilhelmi de toledo, françoise title: interplay between oxidative damage, the redox status, and metabolic biomarkers during long-term fasting date: - - journal: food chem toxicol doi: . /j.fct. . sha: doc_id: cord_uid: t r xqj obesity and its related metabolic disorders, as well as infectious diseases like covid- , are important health risks nowadays. we recently documented that long-term fasting improves metabolic health and enhanced the total antioxidant capacity. the present study investigated the influence of a -day fasting on markers of the redox status in subjects. reducing power, abts radical scavenging capacity, and hydroxyl radical scavenging capacity increased significantly, and indicated an increase of circulating antioxidant levels. no differences were detected in superoxide scavenging capacity, protein carbonyls, and superoxide dismutase when measured at baseline and after days of fasting. these findings were concomitant to a decrease in blood glucose, insulin, hba c, total cholesterol, ldl and triglycerides as well as an increase in total cholesterol/hdl ratio. in addition, the well-being index as well as the subjective energy levels increased, documenting a good tolerability. we documented an interplay between redox and metabolic parameters, as lipid peroxidation baseline levels (tbars) affected the ability of long-term fasting to normalize lipid levels. a machine learning model showed that a combination of antioxidant parameters measured at baseline predicted the efficiency of the fasting regimen to decrease ldl levels. in conclusion, we demonstrated that long-term fasting enhanced the endogenous production of antioxidant molecules, that act protectively against free radicals, and in parallel improved the metabolic health status. our results suggest that the outcome of long-term fasting strategies could be depending on the baseline values of the antioxidative and metabolic status of subjects. achieve these objectives could be long-term fasting, that is defined as voluntary food abstinence from days to several weeks (wilhelmi de toledo et al., b). the study of various forms of fasting on metabolic health has been gaining ground in recent years with more and more studies showing beneficial effects such as inactivating of the mechanistic target of rapamycin (mtor) signaling pathways as well as several others resulting in metabolic normalization, enhanced autophagy and apoptosis followed by cell regeneration, increased brain-derived neurotrophic factor (bdnf) levels in brain leading to enhanced cognition, mood as well as increased neuronal plasticity and regeneration. fasting also enhanced the transcription of cytoprotective enzymes, mitochondrial biogenesis all of these effects leading to restoration and conservation of functional integrity of cells and tissues (de even the occurrence of heart attacks and stroke (knott et al., ) . earlier studies also suggest that this can be applicable to understand redox systems as a recent study successfully developed a neural network to predict oxidative damage using measurements of patients. the bdnf, which has its production stimulated by ketosis during fasting (mattson et al., ) , is known to control the nuclear translocation of the transcription factor nrf to j o u r n a l p r e -p r o o f activate antioxidant defenses (bouvier et al., ) . furthermore, the mild oxidative stress caused by the reduction in adenosine triphosphate (atp)/adenosine monophosphate (amp) ratio has a similar effect to activate transcription of cytoprotective enzymes sulfiredoxin , thioredoxin reductase , heme oxygenase- , as well as several others conjugation and elimination enzymes (burton et al., ) . this article is a continuation of our previous study on the effect of a -day fasting on indicators of redox status of humans which showed that while the total antioxidant capacity (tac) was enhanced, tbars, an important indicator of lipid peroxidation, were reduced (wilhelmi de toledo et al., a). the aim of the present article was to analyze additional redox parameters and to correlate the redox status with markers of glucose and lipid status, the improvement of which leads to a better metabolic health and a reduced risk of metabolic diseases. it was also evaluated if the baseline levels in markers of the redox status can predict the success of the fasting regimen using a machine learning approach. the study participants were recruited out of a total of subjects who were admitted to the buchinger wilhelmi clinic (bwc) and fulfilled the following criteria: subjects were aged between and years and underwent at least to maximum ( ± days) days of fasting. two blood samplings at the beginning and at the end of the experiment were conducted. one week prior as well as during the fasting period the intake of micronutrient supplements was advised to be stopped, except for magnesium supplementation. clinical experience showed that magnesium intake during the fasting course seems to protect against muscle cramps that possibly could be provoked by the recommendation to drink sufficient liquids during fasting. subjects were excluded when they had a predefined contraindication to fasting as described in the guidelines of fasting therapy (wilhelmi de toledo et al., ) like cachexia, anorexia nervosa, advanced kidney, liver or cerebrovascular insufficiency, dementia or other chronic psychiatric diseases, as well as pregnant or lactating women. furthermore, subjects who could not follow the study procedure due to an inability to speak german, english or french, or subjects that were participating in another study, were also excluded. altogether, subjects did not meet the j o u r n a l p r e -p r o o f inclusion criteria, and subjects declined to participate. one subject terminated the study earlier as defined in the protocol due to low hemoglobin and sodium levels. a kcal diet of either rice and vegetables or fruits. to initiate the fasting period, a laxative ( - g na so in ml water) was administered. during fasting, an enema was applied every other day to remove intestinal remnants and desquamated mucosal cells. a calorie intake of ~ kcal/day was ensured by the daily intake of ml freshly squeezed organic juice at midday, and ml of vegetable soup in the evening, as well as g honey per day. the subjects were advised to drink daily at least - l of water or non-caloric herbal teas. a stepwise reintroduction of food with an ovo-lacto-vegetarian organic diet from to kcal/day followed the fasting period. was done at the ± fasting day (time-point # , post). subjects' height was measured with seca (seca, hamburg, germany) and the waist circumference was assessed with a measuring tape, placed halfway between the lowest rib and the iliac crest. body weight was measured daily (seca / , seca, hamburg, germany) between : am and : am by trained nurses, while subjects were lightly dressed. additionally, blood pressure and heart rate were measured on the non-dominant arm after a rest, while subjects were seated (boso carat professional; bosch +sohn gmbh u. co. kg). subjects self-reported their energy level on a numeric rating scale from (weak) to (powerful) before and after fasting. furthermore, the well-being index (who- ) was self- assed by answering five statements scored from (at no time) to (all of the time). after building the sum of the five scores and multiplying it with the who- was given as a percentage between % and % (bech, ) . possible adverse events were documented in a report form by the medical staff. germany) (in . n hcl) for the sample, or μl of . n hcl for the blank, were added to the pellet. the samples were incubated in the dark at room temperature for h with intermittent vortexing every min and were centrifuged at , x g for min at ˚c. the supernatant was discarded and ml of % tca was added, vortexed and centrifuged at , x g for min at ˚c. the supernatant was discarded and ml of ethanol-ethyl acetate ( : v/v) was added, vortexed and centrifuged at , x g for min at ˚c. this washing step was repeated twice. the supernatant was discarded and ml of m urea (ph = . ) was added, vortexed and incubated at ˚c for min. the samples were centrifuged at , x g for min at ˚c and the absorbance was read at nm. the calculation of the protein carbonyls concentration was based on the molar extinction coefficient of dnph. total plasma protein was assayed using the bradford protein assay. the superoxide anion radical-scavenging ability of plasma was measured using a slightly modified protocol of ak and gülçin, (ak and gülçin, ) . in this method, superoxide anion in the reducing power assay, a plasma sample was dissolved in phosphate buffer ( . m, ph = . ) at different concentrations. an aliquot ( μl) of the sample solution was added to μl of % potassium ferricyanide and incubated at ° c for min. the samples were cooled on ice for min. then, μl of % tca was added and the samples were centrifuged ( g, min, and ° c). subsequently, μl of dh o and μl of . % ferric chloride were added to the supernatant and the samples were incubated at rt for min. the absorbance was monitored at nm (yen and duh, ) . incubated at ° c for min. then, the samples were cooled on ice for min, centrifuged ( g, min, and ° c), and the absorbance was monitored at nm. in each experiment, the sample without h o was considered as blank and the sample without the free radical-scavenging activity of the samples was also determined by abts radical cation (abts•+) decolorization assay as previously described by cano et al. (cano et al., ) , with some modifications. in brief, abts•+ radical was produced by mixing mm abts with μm h o and µm horseradish peroxidase (hrp) enzyme in mm pbs (ph = . ). immediately, following the addition of the hrp enzyme, the contents were vigorously mixed, incubated at room temperature in the dark and the reaction was monitored at nm until stable absorbance was obtained. subsequently, µl of plasma were added in the reaction mixture and the decrease in absorbance at nm was measured. in each experiment, the tested sample alone containing mm abts and µm h o in mm pbs (ph = . ) was used as a blank, while the formed abts•+ radical solution alone with µl h o was used as a control. the determination of sod activity in rbcl was based on the method of nitroblue tetrazolium salt (nbt) as described in the study by oberley and spitz (oberley and spitz, ) . more specifically, this assay included a negative control which was prepared by mixing µl of the aim of this study was to understand the interplay between markers of the redox status and the metabolic changes observed during a long-term fasting in individuals. for this purpose, markers of the redox status were evaluated (figure ). this included markers of oxidative stress/damage (tbars, carbonyls), as well as markers of the antioxidant capacity (gsh, tac, superoxide scavenging capacity, reducing power, hydroxyl radical scavenging capacity, abts•+ radical scavenging capacity, as well as gpx, sod and gr activities). the mean age of the study population was years and % of the participants were female ( the heatmap displays the correlations between the redox parameters (rows) and the metabolic parameters (columns). dendrograms shows the relationships between the different parameters evaluated using the hierarchical clustering of euclidean distance. the colour scale shows the coefficient of correlations. statistical significance was tested (* p< . ; ** p< . ; *** p< . ). ), and show that these metabolic improvements are reproducible. in addition, well- being improvements were confirmed with new methods (i.e. energy levels, figure h ; who- index, figure i ). next, the changes in biomarkers of antioxidant systems caused by long-term fasting were studied ( figure j to u). gender differences were limited to tbars levels which were higher at baselines in males. gender did not have a substantial effect on the effects of fasting on redox parameters. glutathione levels ( figure j ) and its regulation through gr ( figure m ) and gpx activities ( figure n ) were unchanged. however, the antioxidant capacity reflected by the tac ( figure l ) and the reducing power ( figure u ) were increased. this ultimately increased ros scavenging potential as showed by an increase hydroxyl radical scavenging activity ( figure r ) and an increased abts scavenging ( figure s ). this improvement of the antioxidant capacity was concomitant to a decrease in markers of oxidative stress/damage tbars ( figure t ). the model was validated on an independent test set consisting of patients ( % of the data), with a statistically significant correlation between the predicted ldl decrease and the actual ldl decrease (p= . ). it was also evaluated if metabolic parameters at baseline (described in figure ) could predict the changes in lipid peroxidation during fasting but failed to establish a model with good predictive abilities. it was demonstrated that long-term fasting can increase the endogenous production of a number of antioxidant molecules that act protectively against free radicals. these findings are in contrast to the preconceived idea that the antioxidant reserves would decrease during fasting due to a lack of absorbed micronutrients with known antioxidative roles (e.g. vitamin e and b, zinc, selenium) after the cessation of food intake. when exogenous antioxidants are missing, endogenous antioxidants are sufficient to maintain homeostasis. this includes uric acid and bilirubin, two important endogenous antioxidants (ames et al., ; sedlak et al., ). although bilirubin was not measured in this study, we measured the increase in uric acid and its association with tac, that was discussed in detail in the previous article besides showing that long-term fasting improves the redox status and metabolic health indicators, an interplay between these parameters was described. the strongest correlation observed was between uric acid levels and the tac, which was expected, since much of the tac is due to the circulating uric acid (janaszewska and bartosz, ) . however, the most important biological correlation was observed between tbars and lipid metabolism. in general, tbars decreased significantly after long-term fasting, as did total cholesterol, ldl and triglyceride levels, with a parallel decrease in waist circumference and bmi. higher lipid peroxidation levels at baseline, reduced the ability of long-term fasting to normalize our machine learning algorithm confirmed that the levels of tbars is a reliable predictor of how well a person will respond to long-term fasting. this model was trained and evaluated number of studies are showing that response to diet is personal and that these differences can affect disease susceptibility (zeevi et al., ) . this can be due to differences in lifestyle, genetics, or even gut microbiome composition (berry et al., ) . we suggest in this study that the response to fasting can also be individual and showed that it can depend on the baseline antioxidative status. factors driving these differences would have to be evaluated in further studies including measurement of gut microbiome composition, as it was repeatedly showed to be an important factor driving personal susceptibility to disease (tierney et al., ) . in addition, it was also shown that the gut microbiome dramatically changed during fasting, and were correlated to changes in energy metabolism ( since lipids are used as energy substrates during fasting, it is reasonable to assume that the decrease in lipid levels, could partly lead to decreased lipid peroxidation. we documented in an unpublished study that the more atherogenic, small dense ldl particles diminished significantly after fasting days. since they are the most oxidizable, it seems logical that the lipid peroxidation levels decrease (chaudhary et al., ) . furthermore, hdl is the greatest antagonist of lipid oxidation and it was documented that total cholesterol/hdl increased significantly during long-term fasting (brites et al., ) . the results of our study conclude that fasting improves oxidative stress indicators by increasing the antioxidant capacity of the blood plasma through the increase in tac, reducing power, abts radical scavenging capacity and hydroxyl radical scavenging capacity. at the same time, fasting reduces lipid peroxidation and improves various metabolic indicators, especially lipids. furthermore, total cholesterol/hdl improved significantly. although the prognostic ability of the redox status to predict metabolic changes caused by long-term fasting will have to be confirmed on a larger cohort, the remarkable performance of this model considering the relatively small number of patients suggest that the antioxidant status is a crucial determinant of the normalization of lipid levels during long-term fasting. altogether, our results show that the effects of long-term fasting on lipid metabolism are influenced by the redox status, and that these effects can be forecasted based on the levels of redox parameters before fasting through the use of machine learning approaches. we recommend that long-term fasting strategies should be personalized and adjusted to the metabolic and antioxidative baseline status of the subjects. mechanisms and consequences of lipid peroxidation in biological systems ] catalase in vitro antioxidant and radical scavenging properties of curcumin uric acid provides an antioxidant defense in humans against oxidant-and radical-caused aging and cancer: a hypothesis prediction of gestational diabetes based on nationwide electronic health records hyperbilirubinemia of fasting measuring the dimension of psychological general well-being by the who- . quality of life newsletter the ferric reducing ability of plasma (frap) as a measure of "antioxidant power": the frap assay human postprandial responses to food and potential for precision nutrition quizalofop- p-ethyl induces adipogenesis in t -l adipocytes variations in the prevalence of obesity among european countries, and a consideration of possible causes nrf -dependent persistent oxidative stress results in stress-induced vulnerability to depression antioxidative activity of high-density lipoprotein (hdl): mechanistic insights into potential clinical benefit fasting supervision and lifestyle care in the tradition of natural hygiene vulkan a method to measure antioxidant activity in organic media: application to lipophilic vitamins low-density lipoprotein : a novel predictor of coronary artery disease severity hydroxyl radical-scavenging effects of spices and scavengers from brown mustard (brassica nigra) food additives such as sodium sulphite, sodium benzoate and curcumin inhibit leptin release in lipopolysaccharide-treated murine adipocytes in vitro effects of intermittent fasting on health, aging, and disease a machine-learning approach to the prediction of oxidative stress in chronic inflammatory disease nrf plays a critical role in mitigating lipid peroxidation and ferroptosis elevated plasma levels of bilirubin in patients with neurotrauma reflect its pathophysiological role in free radical scavenging effects of periodic fasting on fatty liver index-a prospective observational study dietary reference values for nutrients summary report sex-specific metabolic responses to hours of fasting during the active phase in young mice relations between metabolic syndrome, oxidative stress and inflammation and cardiovascular disease assay of total antioxidant capacity: comparison of four methods as applied to human blood plasma alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma effect of corticosteroid therapy on serum and csf malondialdehyde and antioxidant proteins in multiple sclerosis longitudinal associations of in utero and early life near-roadway air pollution with trajectories of childhood body mass index the prognostic significance of quantitative myocardial perfusion: an artificial intelligence-based approach using perfusion mapping analysis of the intricate effects of polyunsaturated fatty acids and polyphenols on inflammatory pathways in health and disease. food and chemical toxicology chronic inflammation in the context of everyday life: dietary changes as mitigating factors intermittent metabolic switching, neuroplasticity and brain health changes in human gut microbiota composition are linked to the energy metabolic switch during d of buchinger fasting the effect of fasting on total serum bilirubin concentrations mangsbo s. ( ) reactive oxygen species as an initiator of toxic innate immune responses in retort to sars-cov- in an ageing population, consider n-acetylcysteine as early therapeutic intervention the nrf -antioxidant response element signaling pathway and its activation by oxidative stress ] assay of superoxide dismutase activity in tumor tissue effects of change in body image and psychological well-being during behavioral obesity treatment: associations with weight loss and maintenance thiol redox state (trs) and oxidative stress in the mouse hippocampus after pentylenetetrazol-induced epileptic seizure obesity-a risk factor for increased covid- prevalence, severity and lethality oxidative stress and metabolic disorders: pathogenesis and therapeutic strategies exercise and psychologic well-being in the community bilirubin and glutathione have complementary antioxidant and cytoprotective roles. proceedings of the national academy of sciences obesity and impaired metabolic health in patients with covid- alternate day fasting improves physiological and molecular markers of aging in healthy, non-obese humans determining how best to support overweight adults to adhere to lifestyle change: protocol for the swift study the predictive power of the microbiome exceeds that of genome-wide association studies in the discrimination of complex human disease spectrophotometric assays for measuring redox biomarkers in blood fasting therapy-an expert panel update of the consensus guidelines safety, health improvement and well-being during a to -day fasting period in an observational study including subjects ( a) influence of long-term fasting on unravelling the health effects of fasting: a long road from obesity treatment to healthy life span increase and improved cognition intermittent fasting with or without exercise prevents weight gain and improves lipids in diet-induced obese mice exercise elicit sexual-dimorphic and tissue-specific adaptations in diet-induced obese mice increasing oxidative stress with progressive hyperlipidemia in human: relation between malondialdehyde and atherogenic index scavenging effect of methanolic extracts of peanut hulls on free-radical and active-oxygen species personalized nutrition by prediction of glycemic responses metabolomics analysis reveals the association between lipid abnormalities and oxidative stress, inflammation, fibrosis and nrf dysfunction in aristolochic acid-induced nephropathy • long-term fasting increases the antioxidant capacity and decreases oxidative damage • it improves the metabolic health status • high tbars levels at baseline limit the ldl reduction during long-term fasting • the antioxidant status is related with the lipid lowering effect of long-term fasting ☐ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work ☒the authors declare the following financial interests/personal relationships which may be considered as potential competing interests the funders had no role in the design of the study; in the collection, analyses, or interpretation of data key: cord- - flw xk authors: kreiner, jenny; pang, edwin; lenon, george binh; yang, angela wei hong title: saposhnikoviae divaricata: a phytochemical, pharmacological, and pharmacokinetic review date: - - journal: chinese journal of natural medicines doi: . /s - ( ) - sha: doc_id: cord_uid: flw xk abstract saposhnikoviae divaricata (turcz.) schischk (sd) is a traditional chinese herb commonly used to treat clinical conditions such as rheumatism and allergic rhinitis. this review article evaluates a collection of works on in vitro and biochemical studies of sd. the discourse on the diverse class of chromones and coumarins in sd offers an insight to the pharmacological effects of these bioactive constituents as anti-inflammatory, analgesic, immunoregulatory, antioxidative, and anti-proliferative agents. it is highlighted that there is a structural relationship between the constituents and bioactive activities, which in effect provides a valid reasoning and reaffirm the use of sd in the treatment of the pathologies in chinese medicine. saposhnikoviae divaricata (turcz.) schischk (sd), an umbelliferous genus, is widely found in the northern and northeastern territories of china and cultivated in many other areas such as anhui, shanxi, and gansu provinces. it is known as "fang feng" in china, "bou-hu" in japan and "bangpung" in korea. the chinese terminology of this herb literally translates "to avert wind", and the dried roots of this plant is generally applied to treat pathogenic conditions of wind-damp-cold in chinese medicine (cm). sd is an important component in traditional chinese medicine clinical practice and commonly used for the treatment of rheumatism, arthralgia, general aches, headaches, stroke, fever, cold, and allergic rhinitis. the chinese pharmacopoeia commission [ ] ranked sd as a top-grade herb, and it is described to possess pungent, sweet and slight warm properties. sd has a wide spectrum of use in the cm. its applications are recorded and well established in the cm classical text the shen nong's materia medica (shen nong ben cao jing) which is the oldest pharmaceutical monograph in cm, dating back to qin-han dynasty. in the shen nong's materia medica, sd is graded as a premium-grade herb cited for its multiple effects: immunoprotective capability in warding off cold (also otherwise known as dispelling wind-cold and relieving exterior pathogens in cm), relief of edema-induced pain (expelling dampness from the acupuncture meridians) and anti-spasmogenic activity (expelling wind and relieving convulsion). similarly, in the collected works of materia medica (ben cao hui yan), authored by ni zhu-mo in the ming dynasty, a herbal monograph outlined the multiple use of sd for the treatment of arthritis instilled with dampness, joints pain, tetanus, atrophy and flaccidity of muscles, headaches, cold, fevers, cough, adiaphoresis, nasal congestion, pharyngeal dryness syndrome, cerebrovascular accident, early onset of small pox, and anxiety in children. cm clinicians today still use sd for the same pathologies cited. in response to the pressing need to align cm for an evidence-based approach, there is an emergence of scientific research on the bioactive constituents and the therapeutic effects of sd. therefore, the objective of this review article is to provide a systematic review of phytochemical, pharmacological, and pharmacokinetic perspectives of the experimental studies on sd. the following databases were searched from their respective receptions up to january : pubmed; embase; amed; cinahl; cochrane library; medline; sciencedirect; scopus; web of science; china network knowledge infrastructure; cqvip; and wanfang data. the keywords used for the literature search included: fang feng and its english, botanical and pharmaceutical names. the papers identified from the search were screened according to the criteria laid out in the australian regulatory guidelines for complementary medicines and therapeutic goods orders for quantitative and qualitative analysis of herbal medicine by the australian therapeutic goods administration (tga). tga adopts the british pharmacopoeia and therapeutic goods orders as the official guidelines for testing and acceptance criteria for herbal substances, herbal preparations, and herbal medicinal products, inclusive of traditional herbal medicinal products [ ] . tga also recognizes other international pharmacopoeias for guidelines on assessing and testing botanicals in complementary medicine. the selection criteria included process controls of the herbal substances, reporting reference standards such as authentication of reference materials and profile chromatograms, and analytical procedures and validation data. papers in english or chinese language are considered for this review. scientific rigors were called upon to determine the chemical markers of herbs through the use of strict parameters in testing, quantitative and qualitative measures of the bioactive components, such as fingerprint spectrum, correlations differentiation, and stability evaluation, reference standards, and toxicological assessments. according to the tga, reporting referencing standards includes naming the origin of the plant materials and its authentication [ ] . a plant specimen voucher number (psvn) is an upmost requirement for referencing standards in botanical testing [ ] . it enables traceability of the plant material, allows access to the storage of crude plant for researchers who seek to cross verify data, further scientific investigations or commercial purposes, should there be discrepancies in the drug products produced from the plant source. essentially, a lack of psvn essentially thwarts scientific research from the onset and may cause legal repercussions if used for commercial purposes. attempts to compare bioactive yield outcomes of these studies was unfeasible in this review, owing to differences in extraction weights and yield units used in the papers such as relative standard deviation, microliters or milligrams per liters. the scientific process of reporting chemical profiling by spectroscopic and/or chromatographic fingerprint should include calibration curves that depict the peak area ratio and the percentage of relative abundance of the bioactive compounds yielded from the plant material in study. a total papers were identified through the literature search. among them, in vitro studies, phytochemistry studies, and phytochemistry with in vitro studies were considered for screening after non-saposhnikoviae divaricata and non-phytochemistry related papers were excluded. phytochemistry papers were further screened according to the selection criteria and papers were excluded due to non-psvn (n = ), different reporting standards of measure standard (n = ), standard procedures of reporting chromatographic profile not followed (n = ) and study on soil element of sd (n = ). as a result, sd phytochemical studies only [ ] [ ] [ ] [ ] adhered to the scientific rigors that ensure the integrity in validation and robustness in quantitative outcomes. among phytochemistry papers with in vitro studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , [ , [ ] [ ] [ ] [ ] were selected based on their relevance, but the phytochemistry component did not adhere to the selection criteria. a total of in vitro studies only were included in this review [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . fig. illustrates the study selection process. crude plant of sd contains chromones, coumarins, polyacetylenes, and acid esters. a summary of these bioactive compounds of sd reported in the included studies are shown in table . the crude dried roots of sd contain chromones and coumarins, which are both heterocyclic compounds, derived from the flavonoids. chromone is a class of oxygen-containing heterocyclic compound with a benzo annelated γ-pyrone ring, belonging to a subclass of the flavonoids known as flavone [ ] ; while coumarin, a benzopyrone, structurally consists of a benzene ring fused to a pyrone ring. a total of chromones are identified in the included studies. these chromones are discovered to exhibit strong pharmacological activities in attenuating inflammatory [ ] , scavenging free radical [ ] , and inhibiting pain [ ] in in vitro studies. aside from the chromones, a total of coumarins are identified in sd from the included studies. new discoveries of coumarins in sd have also emerged over recent years. three new coumarin compounds are discovered to have effects against porcine epidemic diarrhea virus (pedv): divaricoumarin a, divaricoumarin b, and divaricoumarin, with molecular formulae being c [ ] (fig. ) . among the sd coumarins tested, praeruptorin b is found to have the strongest effect against pedv in vero cells. in vitro results reveal praeruptorin b is able to inhibit viral replication at the stage of protein syntheses. although pedv is essentially a viral infection in pigs, this coronavirus stems from the same family of the human coronaviruses, such as severe acute respiratory syndrome and middle east respiratory syndrome [ ] . praeruptorin b, a coumarin from sd, could potentially offer a novel anti-viral drug research for the intractable human coronavirus diseases. in addition, phenylpropanoid fatty acid ester, defined chemically as (±)- -hydroxy- -( -hydroxy- -methoxyphenyl)- -methoxypropyl nervonic acid ester and divaricatol are discovered to inhibit nitric oxide (no) in lipopolysaccharide (lps)-induced mouse polyacetylene panaxynol h- c nmr/cc/tlc [ ] macrophages raw . [ ] . it is noteworthy that the diversity of these sd bioactive compounds could offer pharmacological scaffold for developing new agents with anti-inflammatory, antipyretic, anti-viral, anti-oxidant, and analgesic effects. the analgesic effects of sd are mainly due to c, gh (chromones) and anomalin (coumarin), while other chromones such as gv, gc, and mv are found to exert no significant analgesic effects. hamaudol with its algycone portion and ledebouriellol and divaricatol (both pyranochromones) increased pain threshold, by imparting an opioidergic effect experienced in mice when administered with a high dose of gh [ ] . significant writhing inhibition was observed, but no significant hypothermic effect was detected on the pyrexic mice even at a higher dose of mg·kg − , and writhing inhibitory effect was easily reversed by naloxone injection [ ] . evidence suggested that the sd analgesic effects were acting on an opioid receptor in the cns and its pathway, not on the pns pain-associated inflammation generally produced on the peripheral sensory nerve. these analgesic effects were also detected in a pain threshold tail writhing test with intramuscular injection of gc and gv in murine models at doses of , and mg·kg − . pain deterrent at higher doses were prolonged with a considerable reduction of spasmodic pain were observed in the models [ ] . anomalin is particularly effective against hyperalgesia and allodynia, exerting strong anti-nociceptive effects after hour at mg·kg − , inhibiting carrageenan-induced hyperalgesia [ ] . the anti-nociceptive anti-inflammatory effects gc, c, mv, and anomalin appear to exert considerable inhibition in major inflammatory pathways, namely nuclear factor (nf)-κb, mitogen activated protein kinases (mapks), camp response element-binding protein (creb), and nitric oxide production. anti-inflammatory effects of sd chromones were revealed in arthritic rats, including significant decline in the arthritis score for pain and swelling, with a reduction of the inflammatory cytokines levels, although suppression of the tumor necrosis factor (tnf) α and il- β was only observed in rats treated with higher dose of sd [ ] . a key indicator in inflammation, prostaglandin (pge ), was also decreased in both sera and joints. treatment of sd suppressed the activation of nf-κb binding to its dna sequences. deactivation of nf-κb binding was facilitated by the suppressions of signal-transduction inflammation induced by tnfα-mediated phosphorylation of erk, jnk, and p , which is amplified in both collagen-induced arthritic rats and human fibroblast-like synoviocytes ( . , . , and . fold, respectively) [ ] . the inhibition of mapks phosphorylated -erk, jnk and p was due to the action of gc. among all the mapk subtypes, p-jnk was discovered to be most effectively suppressed. the same gc inhibitory effects on erk / , jnk and p were detected in lps-induced inflammation in severe acute lung injury and no cytotoxic effect with the % lethal dose (ld ) of sd-gc from . to mg·l − [ ] . interestingly, although gc is an inhibitor of no and inos production [ ] , both c and mv have stronger effects than gc [ ] . sd chromones and anomalin are highly effective against acute and chronic inflammation. obvious anti-pyretic effects in acute carrageenan paw swelling of rats are associated with sd chromones [ ] as well as anomalin [ ] , and similar inhibition of chronic complete freund's adjuvant (cfa)-induced edema, with lesions being significantly reduced [ ] . both urine hplc-ms analysis and titre levels in cfa models indicated that gc and gv were mainly responsible for the anti-inflammatory effects in sd. gc, in particular, counteracted the lps-induced inflammation in severe pulmonary inflammatory disorder, manifested in acute respiratory distress syndrome [ , ] . in vivo and in vitro results have demonstrated gc attenuated lps-induced cytokine levels; tnf-α, il- β and il- were far lower in the gc group than that in the control group [ ] . anomalin-mediated inhibitions on inos, cox- , tnf-α and il deactivated the transcriptional onset of nf-κb in lps-induced raw . cells [ ] . cytoplasmic expression of iκbα was drastically reduced, along with subunits of nf-κb, p , p and c-rel being translocated; these were marked indications of anomalin obliterating nf-κb-dna binding activities. although anomalin was able to interrupt protein synthesis of iκbα and phosphorylation, there was no inhibitory effect on the protein initiating factor, eig α phosphorylation. anomalin is effective against hyperalgesia and allodynia associated inflammation. elevated mark-erk and p signaling and nuclei translocation are precursors to activation of the creb pathway [ ] . anomalin, besides its ability to deactivate the nf-κb pathway, is also effective in deactivating creb pathway, which is generally associated with pro-nociceptive mediators that induce prolonged pain in chronic inflammation. mark-erk and p- expressions are exponentially reduced, abolishing the creb-dna binding activity [ ] . similarly, gv, gc, c and gh are matrix metalloproteases (mmp) inhibitors [ ] . mmp and mmp- are responsible for clearing collagens fragments generated by collagenases in tissues repair and remodeling. enhancement of both mmp- and mmp- activities induced by the il secretion are inherent biomarkers of cancer [ ] . in another study, gv, gc, c and gh were shown to have concentra-tion-dependent inhibitory effects in in vitro mmp assay, with ic values being . , . , . , and . μmol·l − , respectively [ ] and gv being the strongest inhibitor of mmp- . when analyzed among other herbs studied in a herbal formula, although sd was discovered to inhibit mmp- , mmp- , and mmp- activities, it also promoted tnf α-induced -il- secretion, which indirectly promoted angiogenesis [ ] . four cancer cell lines, i.e., human mammary adenocarcinoma mcf and mda-mb- , leukemia k , and myelomonocytic hl cells, and murine raw . macrophage/monocyte cells were tested in vitro with camptothecin (cam), paclitaxel (ptx), and in combination with sd [ ] . among them, hdl was most susceptible to sd ethanol extract ( g/ ml) while mcf the least. the estimated % inhibitory concentration (ic ) were at / , / , / , and / dilutions for k , hl , mcf and mda-mb- cells, respectively [ ] . synergistic effects with the combined treatment of chemotherapeutic agents, cam or ptx, on the four cancer cells have been demonstrated; sd extracts effectively reduced the ic values of cam in k and hl cells and that of ptx in mcf and mda-mb- cells (table ) , compared with cells treated with chemotherapeutic agents alone. co-administration of sd with a lower concentration of chemotherapeutic agents could effectively achieve the same anti-proliferative effects as compared to a high cytotoxic concentration of cam or ptx. however, this antagonistic effect on cell proliferation was highly dependent on the concentrations of sd extracts and the chemotherapeutic agents. panaxynol, an active component of sd root was discovered to suppress cell proliferation in k , raji, wish, hela, calu- and vero cells by . % ± . %, . % ± . %, . % ± . %, . % ± . %, . % ± . % and . % ± . %, respectively, at μmol·l − [ ] . complete inhibitory effects were observed in vitro at μmol·l − with evidence of cell cycle arrest from g /g phases to s and g /m phases. remarkably, this inhibitory effect of panaxynol on the tumor cells proliferation and the cell arrest cycle could possibly suggest an impairment of cyclin e mrna level. laser densitometry analysis confirmed a reduction of the ratio of cyclin e mrna to β-actin mrna in vitro [ ] . the immunoregulatory effects of sd polysaccharides were tested using spleen proliferation index and spleen index as well as the macrophage and its phagocytic rate, showing marked difference in spleen proliferation index [ ] , but no significant difference in the spleen index. obvious increases in the phagocytic rate and macrophage index in mice coincided with the increased doses (table ) . lymphocyte subsets ratio for cd +cd increased from . % ± . % ( mg·kg − dose) to . % ± . % ( mg·kg − ), while cd +cd + was significantly high at . % ± . % ( mg·kg − ) but decreased by . % ± . % ( mg·kg − ). significant differences were detected in subsets cd +cd + ratio from . % ± . % ( mg·kg − ) to . % ± . % ( mg·kg − ), as well as cd + from . % ± . % ( mg·kg − ) to . % ± . % ( mg·kg − ) [ ] . the bioavailability of drugs is largely influenced by solubility and human intestinal permeability [ ] . a study evaluated sd with microsomal testosterone β-hydroxylation as an index marker for cyp a activity in response to sd furanocoumarins in human liver [ ] . samples of sd tested had no significant cyp a inhibition. compounds anomalin, -methoxy- -hydroxy psoralen, decursin and decursinol angelate, c and gh were found to be well-absorbed using the cacao- monolayer model at concentration of μmol·l − [ ] . these sd compounds were deemed to be well absorbed, with '-o-angeloylhamaudol and gh being moderate absorbent compounds. increased linear kinetic curves depicted influx measurement across cacao- monolayer indicated a passive diffusion mechanism for sd compounds, except for gh and '-o-angeloylhamaudol. both compounds possessed a comparably smaller permeability magnitude in the first-pass metabolism. all the bioactive constituents of sd were easily absorbed in the first-pass metabolism and did not seem to manifest any interference with cyp a . table effects of saposhnikoviae divaricata polysaccharides on phagocytic rate and macrophage index in mice [ ] blank control sd polysaccharides mg·kg − an interesting mechanism in the absorption and elimination of two sd main chromones gc and c has been discovered. the conversion of gc to c was marked by the presence of higher concentration in the blood after absorption. this was indicative that gc is essentially a prodrug of c. gc exhibited low bioavailability, with evidence in its rapid elimination in the concentration-time curve, with t / being . ± . h after oral administration [ ] . a delayed double peak occurred in mass spectrometry after the administration of the sd extract at . and . h with an increased area-under-curve. both absorption and elimination of cimifugin were longer as a combined sd extract than a single administration of c monomer solution alone [ ] . the collection of works reviewed herein illustrates the diverse bioactive properties of sd associated with active pharmacological actions in vitro. clinical applications of sd demonstrated in these experimental studies underscored the potency of the bioactive compounds of sd and its pharmacological effects. modern day diseases challenge medical researchers to look beyond the synthetic drugs and consider the bioactive compounds in natural products. sd chromones could provide analgesic, antinociceptive and anti-inflammatory effects in neuropathic pain management. its capability to downregulate the mediators and proinflammatory proteins in the process of signal transduction cascade, in particular, nf-κb and mapks inflammatory pathways. both pathways are constituted in rheumatoid arthritis (ra) manifesting pathological symptoms such as pannus formation, synovitis and cartilage damage, and bone erosion [ ] . proinflammatory cytokines such as tnf-α and il- β, chemokines, bacterial and viral products, uv radiation and free radicals could activate inhibitory protein iκb, inducing the phosphorylation, ubiquitination and degradation of iκb subunits by the iκb kinase (ikk) complex [ ] . although in vitro evidence has demonstrated that sd chromones deactivate proinflammatory cytokines tnf-α and il- β in ra via a disruption to mapks subtypes erk, jnk and p phosphorylation, thus effectively interrupting the canonical pathway of nf-κb, the molecular mechanism by which the sd chromones deactivate erk, jnk, and p has yet to be elucidated. the course of inflammation is often accompanied by tissue remodeling and repair, which involve mmps, which are a group of zinc dependent endopeptidases, involved in the degradation and removal of extracellular matrix molecules from tissues. these tissues are responsible for the regeneration of the extracellular matrix of skin manifested in wound healing, tissue repair, and remodeling in response to injury such as myocardial injury, atheroma, arthritis, cancer, and chronic tissue ulcer [ ] . one hallmark of cancer proliferation is the result of il- signaling which promotes angiogenesis and metastasis of malignant tumors. anti-cancer drug designs for mmp inhibitors are aimed at several targets, namely chelating zinc ion at mmp's active sites, inhibiting mmp's enzymatic activity as well as synthesis, and the latest utilizing bisphosphates and d-tryptophan derivatives to inhibit gelatinases [ ] . findings on sd capability to inhibit mmp- and mmp- , along with the discovery that sd indirectly promoted angiogenesis via production of il- were conflicting [ ] , had casted questions as to whether effects of sd could be diminished or adulterated when used in a combined herb formula. nonetheless, merits of gv in sd reflected its high affinity to enzyme mmp- and sd chromones' inhibitory effects on il- expressions in the signaling cascade that could potentially halt the tumorigenesis process and curtail mmp- activities vigorously. mmp- has a distinct role in tumor angiogenesis because it regulates vascular endothelial growth factor (vegf), the most potent inducer of tumor angiogenesis and a major therapeutic target [ ] . these chromones could potentially be used as a therapeutic intervention in targeting mmp- and vegf downstream effectors and targets, although further study on the mechanisms responsible for the inhibitory effects of sd chromones on mmp- and mmp- is still needed. immunomodulation of proinflammatory state in innate and adaptive immunity can easily be reversed into a stabilized anti-inflammatory mode. sd polysaccharides could be used as an immunomodulation agent to treat autoimmune diseases commonly characterized with chronic proinflammatory. plant-derived polysaccharides are posited to exert an immunomodulatory effect by the binding to specific receptors on immune cells in the gut-associated lymphoid tissues and activate intracellular signals [ ] . the t cell receptors in t cell activation indicate the 'status' of the adaptive immunity. low ratios of cd + and cd + are essentially associated with the immunosenescence of the immune system and is also a distinct feature of viral infection such as human immunodeficiency virus, and tuberculosis infections [ ] . lymphocyte subsets cd +cd + and cd + cd + mediate immune response in b cells differentiation and macrophage activation. a recovery of these phenotype biomarkers elicited an immune reconstitution in immunodeficiencies could be facilitated by the sd polysaccharides. the basis of this biologic activity of polysaccharides could possibly lie in the binding of its monosaccharide composition of rhamnose, mannose, glucose, and galactose; promoting sialylated changes on glycoproteins or otherwise reverting to a normalized anti-inflammatory mode in igg, while an absence of it would promote pro-inflammatory effector function [ ] . the potential role of sd polysaccharides could offer promising outcome for chronic persistent infections, in particular, with the elucidation of sd polysaccharides based on chemical-structural relationship to the receptors. this would provide a rational basis for clinical research to develop the novel drugs for autoimmune diseases. the concept of co-administration with complementary medicine such as herbal formulae is not a novel practice in cm, although it needs to be substantiated with scientific findings. co-administration with sd extracts and chemotherapeutics exhibited no cytotoxicity or side effects in vivo. coumarins and its derivatives such as , hydroxy coumarins and psoralen are known to possess anti-oxidant properties against human cancer cell lines [ ] [ ] [ ] ; it appears that the number of hydroxyl groups on the coumarin correlates with the role as a suppressor of reactive oxygenated species [ ] . structurally, natural products with phenolic groups with at least two polar groups in benzene ring at positions , or , are postulated to possess apoptotic properties while presence of hydroxyl groups at positions , , , and of aromatic nucleus exhibit anti-inflammatory properties of hydroxycoumarins [ ] . as part of the umbelliferae family, sd contains many natural coumarins and some of the sd pyranocoumarins substitution pattern indicated for praeruptorin f, cis- '-isovaleryl- '-acetylkhellactone, praeruptorin b, cis- ', '-disenecioylkhellactone, cis- '-isovaleryl- '-senecioylkhellactone, (−)-cis-khellactone were at c- /c- '/c- '/ /c- , with divaricoumarins at c and c [ ] . moreover, strong synergistic interactions were also featured when pyranocoumarins were combined with common anti-tumor drugs such as vincristine, doxorubicin, and ptx [ ] . these structural properties of sd engender anti-neoplastic attributes that could pose as an anti-cancer drug or an adjuvant in mainstream therapeutics with lowered cytotoxicity for chemotherapeutics. in view of this recommendation, future clinical research needs to pave the way to understand the mechanism of combining natural products as mainstream medicine. the bioavailability of drugs and herbs alike, is subjected to absorption, distribution, metabolism, excretion and toxicity (admet). metabolism of drugs is influenced by cytochrome p enzymes, such as cyp a and cyp d . drug interaction with cyp enzymes could pose as potential inhibitor or an inducer of the drug with actions of decreasing or nullifying or even amplifying the enzyme activity. no drug to drug interference was present when sd was co-administered with chemotherapeutic agents cam and ptx. passive diffusion in absorption and elimination was associated with almost all of the sd compounds. particularly, sd chromone c manifested lingering absorption and elimination in auc [ ] , a striking characteristic which highlighted naturally derived c from sd, engendered a slow onset of bioactivity and prolonged pharmacological effects. this hallmark is synonymous to properties of chinese herbal medicine. in summary, the compounds of the crude plant sd have exhibited a wide diversity of pharmacological effects. sd pharmacological efficacies are rooted in its diverse chro-mones, coumarins and derivatives. these biochemical structures of sd could provide a strong scaffold for potential drug development for the treatment of various diseases. it should be noted that this review is by no means exhaustive. further clinical trials would cast further insights into the mechanisms of the chemical constituents of sd and warrant its use as a drug for various diseases. pharmacopoeia of people's republic of china therapeutic goods administration. australian regulatory guidelines for complementary medicines standard operating procedure for the collection and preparation of voucher plant specimens for use in the nutraceutical industry anti-inflammatory constituents from the roots of saposhnikovia divaricata characterization of compounds from the roots of saposhnikovia divaricata by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry isolated perfused lung extraction and hplc-esi-ms n analysis for predicting bioactive components of saposhnikoviae radix application of response surface methodology to optimise ultrasonic-assisted extraction of four chromones in radix saposhnikoviae analgesic components of saposhnikovia root saposhnikovia divaricata a sensitive liquid chromatography-mass spectrometry method for simultaneous determination of two active chromones from saposhnikovia root in rat plasma and urine three new coumarins from saposhnikovia divaricata and their porcine epidemic diarrhea virus (pedv) inhibitory activity ultrafiltration lc-esi-msn screening of mmp- inhibitors from selected chinese medicinal herbs smilax glabra roxb., smilax china l. and saposhnikovia divaricata (turcz.) schischk as potential functional food ingredients inhibitory potential of herbal medicines on human cytochrome p -mediated comparative pharmacokinetics of prim-o-glucosylcimifugin and cimifugin by liquid chromatography-mass spectrometry after oral administration of radix saposhnikoviae extract, cimifugin monomer solution and prim-o-glucosylcimifugin monomer solution to rats biotransformation of prim-o-glucosylcimifugin by human intestinal flora and its inhibition on no production and dpph free radical study of therapeutic basis and pharmacokinetics of saposhnikoviae divaricata prime-o-glucosylcimifugin attenuates lipopolysaccharide-induced acute lung injury in mice suppression of lps-induced inflammatory and nf-κb responses by anomalin in raw . macrophages effects of volatie oil of schizonepeta tenuifolia briq herb and saposhnikovia divaricata schischke root on proinflammatory cytokine expression and regulation mechanism underlying anti-hyperalgesic and anti-allodynic properties of anomalin in both acute and chronic inflammatory pain models in mice through inhibition of nf-κb, mapks and creb signalling cascades anti-proliferative and antioxidant activities of saposhnikovia divaricata the suppressive effects of saposhnikovia divaricata (fangfeng) chromone extract on rheumatoid arthritis via inhibition of nuclear factor-κb and mitogen activated proteinkinases activation on collagen-induced arthritis model the effect of chinese herbal medicines on tnf-α induced matrix metalloproteinase- , - activities and interleukin- secretion a tumor cell growth inhibitor from saposhnikovae divaricata reactions of macrophage and lymphocyte subsets in normal mice to radix saposhnikoviae polysaccharide intestinal permeability of the constituents from the roots of saposhnikovia divaricata in the human caco- cell monolayer model a pharmacodynamic research on chromone glucosides of fangfeng increases in the activated forms of erk / , p mapk, and creb are correlated with the expression of at-level mechanical allodynia following spinal cord injury coumarins: old compounds with novel promising therapeutic perspectives the effect of cytochrome p metabolism on drug response, interactions, and adverse effects mechanisms of disease: the pathogenesis of rheumatoid arthritis iκb kinases: key regulators of the nf-κb pathway new facets of matrix metalloproteinases mmp- and mmp- as cell surface transducers: outside-in signaling and relationship to tumor progression matrix metalloproteinases: regulators of the tumor microenvironment an open-label dosing study to evaluate the safety and effects of a dietary plantderived polysaccharide supplement on the n-glycosylation status of serum glycoproteins in healthy subjects cd +cd −cd − (double negative) t cells: saviours or villains of the immune response in vitro cytotoxic potential and mechanism of action of selected coumarins, using human renal cell lines coumarin-based benzopyranone derivatives induced apoptosis in human lung (a ) cancer cells a study of the role of cell cycle events mediating the action of coumarin derivatives in human malignant melanoma cells studies on coumarins and coumarin-related compounds to determine their therapeutic role in the treatment of cancer antioxidant effects of coumarins include direct radical scavenging, metal chelation and inhibition of ros-producing enzymes recent advances and therapeutic journey of coumarins: current status and perspectives saposhnikoviae divaricata: a phytochemical, pharmacological and pharmacokinetic review key: cord- - ffnejwj authors: kwon, seungwon; lee, wonhaeng; jin, chul; jang, insoo; jung, woo-sang; moon, sang-kwan; cho, ki-ho title: could herbal medicine (soshihotang) be a new treatment option for covid- ?: a narrative review date: - - journal: integr med res doi: . /j.imr. . sha: doc_id: cord_uid: ffnejwj background: while the world struggles under the coronavirus disease (covid- ) pandemic, a variety of antiviral agents and symptomatic treatments are being administered to patients and urgent clinical trials are underway. under these circumstances, it is important to explore various possibilities for the treatment of covid- including herbal medicines. among various herbal medicines, soshihotang (ssht,xiao chai hu tang in chinese) has been prescribed to treat various viral diseases and is used in combination with other herbal medicines depending on the patient’s symptoms. methods: for conducting the present review, we searched electronic databases focusing on the antiviral effect of ssht in experimental and clinical study until april . the search keywords included ssht, constituents of ssht, and antiviral effect. we also searched for materials related to topic directly from websites and published books. based on these search results, we summarized the results of the included materials in the form of a narrative review. results: in a number of recent clinical studies, treatment with ssht improved the infection status of the respiratory and hepatobiliary systems, and experimental studies demonstrated the antiviral effect of ssht and its components. furthermore, ssht are being used in china—where covid- outbreak first took place—and offer a new option to treat covid- . conclusion: based on the present evidences, it is believed that ssht is likely to be a new therapeutic option for covid- . conducting further studies might provide improved understanding regarding the use of ssht in treating covid- . on march , , the world health organization declared that the outbreak of coronavirus disease (covid- ) is a global pandemic. the decision came as a result of , covid- infections and , deaths worldwide. currently, there are no established treatments other than supportive therapies that treat the symptoms of covid- . however, as covid- is a viral infectious disease caused by sars-cov- , some antiviral agents have been used for treatment. antiviral agents such as lopinavir-ritonavir, chloroquine (including hydroxychloroquine), remdesivir, umifenovir, and ribavirin are being used, and clinical trials are ongoing. however, clinical trials of lopinavir-ritonavir (a combination of the hiv protease inhibitors) in patients admitted with severe covid- did not show a significant effect. in addition, ribavirin is known to have specific adverse effects such as psychiatric problems, making it unsuitable as a first-choice medication. clinical trials involving remdesivir and chloroquine are still in progress. in addition to treatment with antiviral agents, interferons, steroids, intravenous immunoglobulin, antibiotics, and convalescent blood plasma are used depending on the patient's condition. there is no conclusive evidence that these antiviral agents and other treatments are effective against covid- . consequently, there is a need to search for candidate drugs other than conventional therapies. in this situation, china, the first country to experience covid- , has used traditional chinese herbal medicine (tchm) for the prevention and treatment of covid- based on the experience of using tchms during the past epidemic of severe acute respiratory syndrome (sars). , in the chinese guidelines for the prevention, diagnosis, and treatment of novel coronavirus-induced pneumonia ( th edition), qingfei paidu tang (qpt) has been suggested as a herbal medicine that can be used regardless of the stage of the disease, along with prescriptions for herbal medicines that are stage-specific. based on this guideline, herbal medicine treatment has been used in more than % of confirmed patients with covid- in j o u r n a l p r e -p r o o f a region. in a recent study aimed at evaluation of the combined effect of herbal medicine and conventional treatments, the rate of symptom loss at discharge, the rate of chest computed tomography (ct) image improvement, and the rate of clinical cure were significantly improved and the rate of disease exacerbation was significantly decreased compared with those after conventional treatments only. in addition, clinical trials of various oral medications and injections based on herbal medicine are underway in china. in this short narrative review, we will discuss the current clinical use and antiviral effects of soshihotang (ssht, xiao chai hu tang in chinese, shosaikoto in japanese), which has been used in the past mainly for infectious diseases with chronic progression. in addition, we suggest the possibility of utilizing ssht as a new therapeutic option for covid- . for conducting the present review, we searched electronic databases (pubmed, scopus, recens, zizyphi fructus, glycyrrhizae radix et rhizoma, and antiviral effect. there was no language restriction in the searching process. in addition to searching for electronic databases, we also searched for materials related to topic directly from websites and published books. based on the search results, we summarized the following items in the form of a narrative review; i) indications of ssht in classical medicine and in symptoms of covid- , ii) utilization status and clinical evidence of ssht in infectious diseases, especially viral diseases, iii) antiviral effect of ssht in experimental studies, iv) potential role of ssht in repurposing j o u r n a l p r e -p r o o f the treatment of covid- , v) ssht variants that can be used according to various clinical symptoms, and vi) cautions when using ssht. through the electronic databases search, we found articles evaluating the clinical usefulness of ssht for infectious diseases, especially viral diseases (n= ), [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and experimental studies (n= ) for antiviral effects of ssht and its constituents. in addition to these, we wrote this review using articles found through direct searches on websites and published books. ssht is a traditional herbal medicine prescription that consists of a total of seven herbs (bupleuri radix, scutellariae radix, pinelliae tuber, ginseng radix, zingiberis rhizoma recens, zizyphi fructus, and glycyrrhizae radix et rhizoma) ( table ). ssht appeared first in shanghan zabing lun, a classic of traditional east asian medicine that is thought to have been published before ad . one part of shanghan zabing lun is shanghan lun, translated as "treatise on cold-induced diseases"-a medical classic that contains traditional medical treatments for infectious diseases with fever. in shanghan lun infectious diseases are categorized as "greater yang," "lesser yang," "yang brightness," "greater yin," "lesser yin," or "reverting yin" according to the stage of disease progression, and the text describes the indications and the prescriptions that could be used at each stage. ssht has been classified as a representative prescription of "lesser yang," but it is suggested as a prescription that can be used at almost any stage of the disease. ssht appears in several verses of shanghan lun, and the representative indications are as follows: -after suffering from zhong feng syndrome with cold damage for five or six days, there is alternating chills and fever, discomfort and fullness in the chest and hypochondriac region, taciturnity with no desire to eat, a sick and discomforting feeling in the stomach and easily vomiting with any of the following possible symptoms and signs; vexation in the chest and absence of vomiting, thirst, abdominal pain, fullness and hardening below the hypochondriac region, palpitation in the epigastrium and abnormal urination, absence of thirst and slight fever on the body or cough. ssht should be prescribed. -in yang brightness disease, when there is tidal fever, loose stool, normal urination, and fullness in the chest and costal/hypochondriac region that has not abated, ssht might be prescribed. -in zhong feng syndrome of yang brightness disease, there are wiry, floating and big pulses, shortness of breath, fullness in the abdomen, pain in the epigastric region and costal/hypochondriac region, a sense of qi blockage under prolonged pressure, a dry nose, the absence of sweating somnolence, yellow coloration all over the body and the eyes, difficult urination, tidal fever, hiccup at times, and swelling in front of and behind the ears. the condition is slightly better after needling, but the exterior syndrome remains unchanged. if the disease has lasted for more than ten days and the pulse is still floating, ssht might be prescribed. considering the indications above, ssht should be prescribed when there are symptoms in fever and chest discomfort along with the symptoms of digestive tract (loss of appetite, nausea, vomiting, diarrhea, or constipation, etc.) after initial onset or within days of the occurrence of infectious diseases. based on the indications and recommendations in the shanghan lun, ssht has been used for various infectious diseases, especially respiratory, gastrointestinal, and liver diseases with or without fever. the typical clinical symptoms of covid- are lower respiratory symptoms (including dry cough), high fever, and difficulty breathing. in a chinese study of , diagnosed covid- cases, approximately % revealed severe dyspnea (oxygen saturation of % or less, lung infiltration %), and % showed fatal symptoms such as respiratory failure, organ j o u r n a l p r e -p r o o f failure, dysfunction, and septic shock. there have also been reports of accompanying gastrointestinal symptoms such as nausea, vomiting, and abdominal discomfort (although the frequency is low) after the initial respiratory symptoms. in another investigation in wuhan, hubei, the most commonly reported clinical manifestations were fever ( . %), cough ( . %), fatigue ( . %), and gastrointestinal symptoms ( . %). in addition, there have been some cases that have shown only gastrointestinal symptoms from the onset. the first covid- case in the united states is the representative example. this patient was hospitalized with nausea and vomiting on the second day of onset and was confirmed as a covid- case after the symptom of loose stool was observed on the second day of hospitalization. the clinical appearance of covid- , which is accompanied by fever, respiratory and gastrointestinal symptoms at a relatively early stage, is similar to the indications of ssht described in the medical classic, shanghan lun. ssht has been used to treat infectious diseases caused by various bacteria and, particularly, viruses. with respiratory system infections, in particular, there have been reports of ssht used to treat viral infections such as influenza or the common cold. in a study comparing the effects of oseltamivir in patients with influenza a, ssht treatment had an antipyretic effect equivalent to that of oseltamivir. the time required for the antipyretic effect was evaluated according to the treatment: oseltamivir (n = participants), mahwangtang (mht, ma huang tang in chinese, maoto in japanese; n = ), or ssht (n = ). the oseltamivir group required . ± . days for antipyretic effect, and both the mht and ssht groups showed antipyretic effect after . ± . days, with no significant difference between the two groups. ssht has j o u r n a l p r e -p r o o f also been reported to be a treatment for the common cold with more than five days progress. ssht (n = ) or placebo (n = ) was administered to patients ( to years old) with colds more than days after the onset of illness, accompanied by oral discomfort, anorexia, and tiredness. within one week ( days) after treatment, ssht group showed better results than placebo in the overall improvement (total effective rate: . % vs . %, ssht vs placebo). the ssht group showed significant improvement in the symptoms of sore throat and tiredness in to days compared with the placebo group. after the final ssht administration, appetite, arthralgia, and muscle pain were significantly improved compared with placebo administration. this result suggests that ssht might be an effective treatment of the common cold with delayed progress. in a report of a non-viral infection, pulmonary tuberculosis, ssht has also been used in the treatment of idiopathic interstitial pneumonia. tanaka et al. divided idiopathic interstitial pneumonia patients into a control group (n = ) without additional medication administration and a treatment group (n = ) administered ssht and compared the treatment results after more than months. improvement was shown in . % ( / ) of the patients in the treatment group and slight improvement ( . %, of patients) was shown in the n treatment group. j o u r n a l p r e -p r o o f ssht has attracted much more attention for its antiviral effect in chronic hepatitis b and c. although the shanghan lun did not intend for ssht to be used in liver disease, ssht has been used for this purpose based on indicative phrases in the text such as "yellow coloration all over the body and the eyes" and "discomfort and fullness in the chest and hypochondriac region". in cases of hepatitis c, it was confirmed that fever, alopecia, and leukocyte levels were significantly improved following the combination therapy of ssht and interferon compared with those following interferon monotherapy. improvements in the levels of aspartate transaminase (ast), alanine transferase (alt), and knodell's histology activity index, in % ( / patients) were evident when patients were administered ssht for months. in another study, ssht was administered for three years, and fibrosis markers were regularly followed (n = ). as a result, alt levels were reduced in both chronic active hepatitis and chronic persistent hepatitis (cph) patients. in addition, in cph, procollagen iii peptide (piiip) was normalized in % of cases and s collagen in % of cases, which suggests a liver fibrosis suppressive effect of ssht in chronic hepatitis c. there are an increasing number of reports of the use of ssht in the treatment of chronic hepatitis b. in most of these studies, patients are given combination therapy of ssht with interferon-based chemotherapy. although there was a slight difference in the results of each study, the adjunctive or single administration treatment using ssht showed a tendency to improve liver function, partial hbeag negative, and reduced viral load in patients with hepatitis b compared with chemotherapy alone or placebo. [ ] [ ] [ ] [ ] these results suggest that ssht has an antiviral effect against the chronic hepatitis virus. j o u r n a l p r e -p r o o f as described above, ssht has been applied to the treatment of viral diseases of the respiratory system and hepatobiliary system. the clinical results of ssht treatment can be explained by the antiviral effects of ssht itself and the effects of each constituent herb. ssht has been shown to inhibit coxsackievirus b infection by inducing the expression of type i interferons , and has been reported to inhibit antigen production through chemical promoters against epstein-barr virus. it has also been reported that ssht could prevent hepatitis c virus (hcv) disease progression by promoting interleukin- and - production. experimental research has shown that each constituent herb in ssht has antiviral effects. in particular, saikosaponin, a major component of bupleuri radix, has been reported to have antiviral activity against hcov- e , a coronavirus similar to covid- . in addition, saikosaponin a inhibits influenza a replication. it has been found that ginsenoside rb , a major component of ginseng, can remove cytoprotective macrophages found in human immunodeficiency virus type (hiv- ) infection by inhibiting the akt pathway. the most extensively studied antiviral herb is scutellariae radix. the major component of scutellariae radix, baicalin, is known to have antiviral activity against influenza a, , hiv- , , respiratory syncytial virus (rsv), dengue, chikungunya, duck hepatitis virus (dhv), , enterovirus, and hbv. another component of scutellariae radix, wogonin, also has an antiviral effect against the influenza virus and hbv. lastly, glycyrrhizae radix et rhizoma and its components have been reported to have antiviral activity against hcv, coxsackievirus b , dhv, influenza, rotavirus, rsv, and herpes simplex virus , (table in china, which saw the first manifestation of covid- and experienced local epidemics, treatment for the disease included tchms in combination with conventional therapies. , in the chinese guidelines for the prevention, diagnosis, and treatment of novel coronavirus-induced pneumonia ( th edition), qpt has been suggested as a general treatment regimen for the results of a network pharmacology analysis of the effect of ssht on covid- also suggested that ssht may be effective for early covid- prevention and treatment. the authors investigated components of ssht that show anti-sars-cov- activity (baicalein, beta-carotene, coptisine, formononetin, glycyrrhizic acid, kaempferol, moupinamide, quercetin, saikosaponin a, saikosaponin b , saikosaponin d, and -methoxy- -methyl isoflavone) could be effective for pneumonia treatment and immunoregulation by acting on a total of key targets (il- , nos , esr , etc.). based on these results, it was assumed that ssht could have effects such as inhibition of sars-cov activity, blocking of sars-cov invasion pathway, and suppression of cytokine storm expression. this phenomenon demonstrates multi-target and multi-directional activity, which is an advantage of herbal medicine composed of various herbs (table ) . furthermore, it is necessary to pay special attention to the immunomodulatory effect of ssht. in an experimental study using a cyclophosphamide-induced immunosuppression animal model, ssht was shown to exert an immunomodulatory effect that increases lymphocytes only in the immunosuppressive model (and is invalid in the normal animal model). in an observational study of patients with covid- , early lymphocyte degradation was observed to be a characteristic observation in the patients, suggesting that early lymphocyte degradation would have diagnostic value. considering these serologic abnormalities associated with early covid- and the immunomodulatory effect of ssht, it is possible that the use of ssht in the early to mid-term stages of the disease could be helpful (table ) . most minor liver injuries will recover without special treatment, but severe liver injuries will result in significant disease burden. as mentioned earlier, ssht has been utilized in patients with hbv or hcv infection and has shown significant antiviral and hepato-protective effects. [ ] [ ] [ ] [ ] [ ] [ ] [ ] these clinical and pharmacological evidences of ssht will be expected to play a role in the management of liver injury in patients with covid- (table ) . traditionally, ssht has been used in combination with various prescriptions depending on the symptoms accompanying various infectious diseases. an example is given in table . as we discussed earlier, ssht (as part of qpt) has been utilized in the covid- epidemic as a basic prescription that can be used at various stages. , this history of ssht use suggests that each prescription for each patient can be modified according to symptoms and that the antiviral action of ssht can also form the basis of prescription. ssht should be used with caution. the korean ministry of food and drug safety permits the use of ssht, except in the following contraindications (table ) : patients receiving interferon; patients with liver cirrhosis and liver cancer (use of ssht can lead to interstitial pneumonia, which can further cause serious consequences such as death); patients with platelet counts of less than , /mm owing to liver dysfunction. it is known that administering ssht in the above situations may cause interstitial pneumonia; therefore, use of ssht in these situations should be avoided. in particular, because of the risk of interstitial pneumonia, using ssht requires caution with combination with interferon. according to the covid- treatment guideline of korean association of internal medicine, use of type i interferon as monotherapy is not recommended, but use of combined therapy is recommended. therefore, when considering the use of ssht in clinical practice, it is necessary to confirm whether interferon is used or not. moreover, there have also been case reports of pulmonary edema occurring after ssht was used for liver dysfunction of unknown etiology. , therefore, it is necessary to pay attention to the use of ssht when there is a liver dysfunction of unknown cause. ssht has been used for infectious diseases for perhaps the last , years, and its efficacy in infectious diseases has been scientifically established. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in particular, ssht has been used in various viral diseases based on its antiviral effect and-if contraindications are dully considered-it can be regarded as a relatively safe medication given its long history of use. in addition, considering the indications of ssht, and the scope of its previous usage, we think that ssht could be prescribed for covid- patients with persistent fever, respiratory symptoms such as cough or sputum, and liver injury caused by conventional therapies. based on the evidence so far, it is believed that ssht is likely to be a repurposing medication for covid- . conducting further studies might provide improved understanding regarding the use of ssht in treating covid- . the authors declare that there is no conflict of interest regarding the publication of this paper. not applicable. data will be made available upon request. all data used for this study are from previous studies which are included in references. the authors declare that no funding was received in relation to this study. ) patients with liver cirrhosis and liver cancer (interstitial pneumonia can occur, which can lead to serious consequences such as death) ) patients with platelet counts less than , /mm because of liver dysfunction j o u r n a l p r e -p r o o f table . antiviral effect of soshihotang as observed in experimental studies tonsillitis and peritonsillitis. these conditions accompanied by sore throat: ssht; soshihotang * the indications are based on drug approvals from korea and japan. sars-cov- and covid- : the most important research questions discovering drugs to treat coronavirus disease (covid- ) a trial of lopinavir-ritonavir in adults hospitalized with severe covid- managing psychiatric side effects of antiviral therapy in chronic hepatitis c can chinese medicine be used for prevention of corona virus disease (covid- )? a review of historical classics, research evidence and current prevention programs covid- : an update on the epidemiological, clinical, preventive and therapeutic evidence and guidelines of integrative chinese-western medicine for the management guidelines for the prevention, diagnosis, and treatment of novel coronavirus-induced pneumonia, the th traditional chinese medicine applied in over half of confirmed patients in coronavirus-hit hubei: official application of integrative medicine protocols in the treatment of coronavirus disease traditional chinese medicine in the treatment of patients infected with -new coronavirus (sars-cov- ): a review and perspective efficacy of coadministration of maoto and shosaikoto, a japanese traditional herbal medicine (kampo medicine), for the treatment of influenza a infection, in comparison to oseltamivir placebo contrast comparison test between double blind test groups of tj- tsumura sho-saiko-to for the cold combination effect of kampo medicine as adjuvant therapy for pulmonary tuberculosis effects of sho-saiko-to on idiopathic interstitial pneumonia: comparison with the control group combination therapy of interferon and sho-saiko-to in chronic hepatitis c. abstracts of the a single arm phase ii study of a far-eastern traditional herbal formulation (sho-sai-ko-to or xiao-chai-hu-tang) in chronic hepatitis c patients examination of various fibrosis markers during long-term administration of sho-saiko-to in chronic hepatitis c effect of sho-saiko-to(xiao-chai-hu-tang) on hbeag clearance in children with chronic hepatitis b virus infection and with sustained liver disease clinical effects of sho-saiko-to on chronic hepatitis b study on the efficacy of sho-saikoto for chronic hepatitis b in hbeag positive children therapeutic effect of ifn-β (feron) and shosaikoto combination therapy on chronic active hepatitis b antiviral natural products and herbal medicines xiao chai hu tang inhibits cvb virus infection of ccfs- cells through the induction of type i interferon expression inhibitory effects of kampo medicine on epstein-barr virus antigen induction by tumor promoter effects of the japanese herbal medicine "sho-saiko-to" (tj- ) on interleukin- production in patients with hcv-positive liver cirrhosis saikosaponin a inhibits influenza a virus replication and lung immunopathology ginsenoside rb eliminates hiv- (d )-transduced cytoprotective human macrophages by inhibiting the akt pathway in vivo effect of quantified flavonoids-enriched extract of scutellaria baicalensis root on acute lung injury induced by influenza a virus the inhibitory effect of sodium baicalin on oseltamivir-resistant influenza a virus via reduction of neuraminidase activity flavonoid baicalin inhibits hiv- infection at the level of viral entry baicalin, an inhibitor of hiv- production in vitro baicalin from scutellaria baicalensis blocks respiratory syncytial virus (rsv) infection and reduces inflammatory cell infiltration and lung injury in mice. sci rep baicalin, a metabolite of baicalein with antiviral activity against dengue virus. sci rep deciphering the potential of baicalin as an antiviral agent for chikungunya virus infection assessment of the effect of baicalin on duck virus hepatitis inhibition mechanisms of baicalin and its phospholipid complex against dhav- replication the antiviral effect of baicalin on enterovirus in vitro baicalin benefits the anti-hbv therapy via inhibiting hbv viral rnas wogonin, a flavonoid isolated from scutellaria baicalensis, has anti-viral activities against influenza infection via modulation of ampk pathways anti-hepatitis b virus activity of wogonin in vitro and in vivo anti-hepatitis c virus compounds obtained from glycyrrhiza uralensis and other glycyrrhiza species the antiviral and antimicrobial activities of licorice, a widelyused chinese herb integrated drug information system treatise on cold-induced and miscellaneous diseases). beijing digital museum of tcm a study on the transmutation among six-channels in shanghanlun a study on the complex efficacy of soshihotang characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan clinical characteristics of patients infected with sars-cov- in wuhan first case of novel coronavirus in the united states expression of animal virus genomes clinical observation of qingfeipaidu decoction in the treatment of novel coronavirus pneumonia. pharmacology and clinics of chinese materia medica mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus study on the effect of kampo treatment for infectious diarrhea. recent progress of kampo medicine in traditional chinese medicine master professor zhou zhongying's interpretation for "traditional chinese medicine differentiation and treatment of new coronavirus pneumonia tcm differentiation and treatment of new coronavirus pneumonia in jiangsu province network pharmacological analysis and mechanism prediction of xiaochaihu decoction in treatment of covid- with syndrome of pathogenic heat lingering in lung and obstructive cardinalate immunoregulatory activity of bupleuri decoction on the expression of costimulatory molecules on the lymphocytes in immunosuppressed mice expression of lymphocyte subsets in peripheral blood of patients with novel coronavirus pneumonia and its clinical significance liver injury in covid- : management and challenges covid- treatment guideline of korean association of internal medicine a non-cardiogenic type of pulmonary edema after administration of chinese herbal medicine (shosaikoto)--a case report pulmonary edema associated with the chinese medicine shosaikoto group v: influenza group of virus are determined based on baltimore classification group i: viruses possessing double-stranded dna group ii: viruses possessing single-stranded dna group iii: viruses possessing double-stranded rna genomes group iv: viruses possessing positive-sense single-stranded rna genomes group v: viruses possessing negative-sense single-stranded rna genomes group vi: single-stranded rna viruses that replicate through a dna intermediate group vii: viruses possessing double-stranded dna genomes and replicating using reverse transcriptase key: cord- -hn pmcto authors: li, yao; yao, jiaying; han, chunyan; yang, jiaxin; chaudhry, maria tabassum; wang, shengnan; liu, hongnan; yin, yulong title: quercetin, inflammation and immunity date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: hn pmcto in vitro and some animal models have shown that quercetin, a polyphenol derived from plants, has a wide range of biological actions including anti-carcinogenic, anti-inflammatory and antiviral activities; as well as attenuating lipid peroxidation, platelet aggregation and capillary permeability. this review focuses on the physicochemical properties, dietary sources, absorption, bioavailability and metabolism of quercetin, especially main effects of quercetin on inflammation and immune function. according to the results obtained both in vitro and in vivo, good perspectives have been opened for quercetin. nevertheless, further studies are needed to better characterize the mechanisms of action underlying the beneficial effects of quercetin on inflammation and immunity. quercetin, a flavonoid found in fruits and vegetables, has unique biological properties that may improve mental/physical performance and reduce infection risk [ ] . these properties form the basis for potential benefits to overall health and disease resistance, including anti-carcinogenic, anti-inflammatory, antiviral, antioxidant, and psychostimulant activities, as well as the ability to inhibit lipid peroxidation, platelet aggregation and capillary permeability, and to stimulate mitochondrial biogenesis [ ] . therefore, there is a pressing need for well-designed clinical trials to evaluate this novel dietary supplement further. this article reviews effects of quercetin on inflammation and immunity in mental and physical performance and health. quercetin is categorized as a flavonol, one of the six subclasses of flavonoid compounds. the name has been used since , and is derived from quercetum (oak forest), after quercus. it is a naturally occurring polar auxin transport inhibitor [ ] . the international union of pure and applied chemistry (iupac) nomenclature for quercetin is , , , , -pentahydroxyflvanone (or its synonym , , , , -pentahydroxy- -phenylchromen- -one). this means that quercetin has an oh group attached at positions , , , , and . common forms of quercetin were shown in figure . quercetin (c h o ) is an aglycone, lacking an attached sugar. it is a brilliant citron yellow needle crystal and entirely insoluble in cold water, poorly soluble in hot water, but quite soluble in alcohol and lipids. a quercetin glycoside is formed by attaching a glycosyl group (a sugar such as glucose, rhamnose, or rutinose) as a replacement for one of the oh groups (commonly at position ). the attached glycosyl group can change the solubility, absorption, and in vivo effects. as a general rule of thumb, the presence of a glycosyl group (quercetin glycoside) results in increased water solubility compared to quercetin aglycone [ , ] . a quercetin glycoside is unique by the attached glycosyl group. generally, the term quercetin should be used to describe the aglycone only; however, the name is occasionally used to refer to quercetin-type molecules, including its glycosides in research and the supplement industry. quercetin-type flavonols (primarily as quercetin glycosides), the most abundant of the flavonoid molecules, are widely distributed in plants. they are found in a variety of foods including apples, berries, brassica vegetables, capers, grapes, onions, shallots, tea, and tomatoes, as well as many seeds, nuts, flowers, barks, and leaves. quercetin is also found in medicinal botanicals, including ginkgo biloba, hypericum perforatum, and sambucus canadensis [ ] [ ] [ ] . in red onions, higher concentrations of quercetin occur in the outermost rings and in the part closest to the root, the latter being the part of the plant with the highest concentration [ ] . one study found that organically grown tomatoes had % more quercetin than chemically grown fruit [ ] . quercetin is present in various kinds of honey from different plant sources [ ] . food-based sources of quercetin include vegetables, fruits, berries, nuts, beverages and other products of plant origin [ ] . in the determined food, the highest concentration is mg/ g of edible portion in capers (raw), the lowest concentration is mg/ g of edible portion in black or green tea (camellia sinensis) [ ] . dietary intake of quercetin was different in several countries. the estimated flavonoid intake ranges from to mg/day (quercetin accounts for %), mostly depending on the consumption of fruits and vegetables and the intake of tea [ ] . in the suihua area of northern china, quercetin intake was reported to be . mg/day, where the main food sources of flavonol was apples ( . %), quercetin (c h o ) is an aglycone, lacking an attached sugar. it is a brilliant citron yellow needle crystal and entirely insoluble in cold water, poorly soluble in hot water, but quite soluble in alcohol and lipids. a quercetin glycoside is formed by attaching a glycosyl group (a sugar such as glucose, rhamnose, or rutinose) as a replacement for one of the oh groups (commonly at position ). the attached glycosyl group can change the solubility, absorption, and in vivo effects. as a general rule of thumb, the presence of a glycosyl group (quercetin glycoside) results in increased water solubility compared to quercetin aglycone [ , ] . a quercetin glycoside is unique by the attached glycosyl group. generally, the term quercetin should be used to describe the aglycone only; however, the name is occasionally used to refer to quercetin-type molecules, including its glycosides in research and the supplement industry. quercetin-type flavonols (primarily as quercetin glycosides), the most abundant of the flavonoid molecules, are widely distributed in plants. they are found in a variety of foods including apples, berries, brassica vegetables, capers, grapes, onions, shallots, tea, and tomatoes, as well as many seeds, nuts, flowers, barks, and leaves. quercetin is also found in medicinal botanicals, including ginkgo biloba, hypericum perforatum, and sambucus canadensis [ ] [ ] [ ] . in red onions, higher concentrations of quercetin occur in the outermost rings and in the part closest to the root, the latter being the part of the plant with the highest concentration [ ] . one study found that organically grown tomatoes had % more quercetin than chemically grown fruit [ ] . quercetin is present in various kinds of honey from different plant sources [ ] . food-based sources of quercetin include vegetables, fruits, berries, nuts, beverages and other products of plant origin [ ] . in the determined food, the highest concentration is mg/ g of edible portion in capers (raw), the lowest concentration is mg/ g of edible portion in black or green tea (camellia sinensis) [ ] . dietary intake of quercetin was different in several countries. the estimated flavonoid intake ranges from to mg/day (quercetin accounts for %), mostly depending on the consumption of fruits and vegetables and the intake of tea [ ] . in the suihua area of northern china, quercetin intake was reported to be . mg/day, where the main food sources of flavonol was apples ( . %), followed by potatoes ( . %), lettuce ( . %) and oranges ( . %) [ ] , whereas the average quercetin intake was . mg/day, with apple ( . %), potato ( . %), celery ( . %), eggplant ( . %), and actinidia ( . %) being the main food sources of quercetin in harbin, china [ ] . the most recent study showed that quercetin intake is about mg/day for chinese healthy young males. in the usa, flavonol intake is about mg/day for u.s. adults, while quercetin represents three-quarters of this amount. the mean quercetin intake was approximately . to . mg per day. onions, tea, and apples contained high amounts of quercetin [ ] . in japan, the average and median quercetin intakes were . and . mg/day, respectively; the quercetin intake by men was lower than that by women; and the quercetin intakes showed a low correlation with age in both men and women. the estimated quercetin intake was similar during summer and winter. quercetin was mainly ingested from onions and green tea, both in summer and in winter. vegetables, such as asparagus, green pepper, tomatoes, and red leaf lettuce, were good sources of quercetin in summer [ ] . in australia, black and green teas were the dominant sources of quercetin. other sources included onion, broccoli, apple, grape, and beans [ ] . analysis of the -h recall data indicated an average adult intake of total flavonoids (> years) of mg/day. apple was the most important source of quercetin until age - years, after which onion became an increasingly important prominent source [ ] . in spain, the average daily intake of quercetin is . mg/day, which is significantly higher than that in the united states ( . mg/day), based on sources like tea, citrus fruits and juice, beers and ales, wines, melon, apples, onions, berries and bananas [ ] . the first investigation on the pharmacokinetics of quercetin in humans suggested very poor oral bioavailability after a single oral dose (~ %). the estimated absorption of quercetin glucoside, the naturally occurring form of quercetin, ranges from % to % in healthy individuals receiving mg. the relatively low bioavailability of quercetin may be attributed to its low absorption, extensive metabolism and/or rapid elimination. quercetin glycosides might be differently absorbed based on the type of sugar attached [ ] . available evidence indicates that quercetin glucosides (like those found predominantly in onion or shallot flesh) are far better absorbed than its rutinosides (the major quercetin glycoside in tea). the glucosides are efficiently hydrolyzed in the small intestine by beta-glucosidases to the aglycone form, much of which is then absorbed [ ] . quercetin glucuronic acid and its sulfuric acid derivatives were more easily absorbed than quercetin [ ] . thereafter, its absorption is affected by differences in its glycosylation, the food matrix from which it is consumed, and the co-administration of dietary components such as fiber and fat [ ] . thus different sugar types and sugar group conjugation sites will result in absorption variation. quercetin and derivatives are stable in gastric acid; however, there were no reports whether they can be absorbed in stomach. studies suggest that quercetin is absorbed in the upper segment of small intestinal [ , ] . among quercetin's derivatives, conjugated forms of its glycosides are better absorbed than quercetin. purified quercetin glucosides are capable of interacting with the sodium dependent glucose transport receptors in the mucosal epithelium and may therefore be absorbed by the small intestine in vivo [ ] . after absorption, quercetin becomes metabolized in various organs including the small intestine, colon, liver and kidney. metabolites formed in the small intestine and liver by biotransformation enzymes include the methylated, sulfo-substituted and glucuronidated forms [ , ] . a study regarding the tissue distribution in rats and pigs has shown that the highest accumulation of quercetin and its metabolites are found in (rat) lung and (pig) liver and kidney [ ] . quercetin and derivatives are transformed into various metabolites (phenolic acid) by enteric bacteria and enzymes in intestinal mucosal epithelial cells. these metabolites are absorbed, transformed or excreted later. moreover, bacteria ring fission of the aglycon occurs in both the small intestine and colon, resulting in the breakdown of the backbone structure of quercetin and the subsequent formation of smaller phenolics [ ] . quercetin metabolites analyzed in plasma and liver samples have shown that the concentrations of its derivatives in the liver were lower than those in plasma, and the hepatic metabolites were intensively methylated ( %- %) [ ] . limited studies suggest that quercetin was methylated, vulcanized and glucuronidated in liver [ ] . continuous intake of diet containing quercetin accumulated in blood and significantly increased quercetin concentration in plasma, which was significantly correlated to its dietary content [ ] . quercetin is present in a conjugated form in human blood whose major form is glycoside [ ] . while isorhamnetin and glucoside acid-sulfated derivatives of quercetin account for . % of its metabolites, other metabolites include its glucuronoside and methylated form [ ] . boulton also found that quercetin conjugated plasma protein (albumin account for . %), thus decreased its bioavailability in cells [ ] . the limited research suggests that quercetin and its metabolites tend to accumulate in the organs involved in its metabolism and excretion, and that perhaps mitochondria might be an area of quercetin concentration within cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] . kidney is a major organ of excretion. quercetin concentration in urine increased with the increasing dose and time after intake of fruit juice was ingested in human [ ] , perhaps benzoic acid derivatives are common metabolite of quercetin [ ] . human subjects can absorb significant amounts of quercetin from food or supplements, and elimination is quite slow, with a reported half-life ranging from to h [ ] . the average terminal half-life of quercetin is . h [ ] . the total recovery of c-quercetin in urine, feces and exhaled air is highly variable, depending on the individual [ ] . a high amount of absorbed quercetin is extensively metabolized and eventually eliminated by the lungs [ ] . additional literature suggests that isoquercetin (glycosylated quercetin) is more completely absorbed than quercetin in the aglycone form, and that the simultaneous ingestion of quercetin with vitamin c, folate and additional flavonoids improves bioavailability [ , ] . all of these studies indicate that quercetin glucosides is absorbed in the upper segment of small intestinal, then is methylated, sulfo-substituted and glucuronidated by biotransformation enzymes in the small intestine and liver, and is finally excreted by kidney in urine. quercetin was reported as a long lasting anti-inflammatory substance that possesses strong anti-inflammatory capacities [ , ] . it possesses anti-inflammatory potential that can be expressed on different cell types, both in animal and human models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is known to possess both mast cell stabilizing and gastrointestinal cytoprotective activity [ ] . it can also play a modulating, biphasic and regulatory action on inflammation and immunity [ ] . additionally, quercetin has an immunosuppressive effect on dendritic cells function [ ] . several studies in vitro using different cell lines have shown that quercetin inhibits lipopolysaccharide (lps)-induced tumor necrosis factor α (tnf-α) production in macrophages [ ] and lps-induced il- production in lung a cells [ ] . moreover, in glial cells it was even shown that quercetin can inhibit lps-induced mrna levels of tnf-α and interleukin (il)- α, this effect of quercetin resulted in a diminished apoptotic neuronal cell death induced by microglial activation [ ] . quercetin inhibits production of inflammation-producing enzymes (cyclooxygenase (cox) and lipoxygenase (lox)) [ , ] . it limits lps-induced inflammation via inhibition of src-and syk-mediated phosphatidylinositol- -kinase (pi k)-(p ) tyrosine phosphorylation and subsequent toll like receptor (tlr )/myd /pi k complex formation that limits activation of downstream signaling pathways in raw . cells [ ] . it can also inhibit fcεri-mediated release of pro-inflammatory cytokines, tryptase and histamine from human umbilical cord blood-derived cultured mast cells (hcbmcs); this inhibition appears to involve in inhibition of calcium influx, as well as phospho-protein kinase c (pkc) [ ] . the study of quercetin against h o -induced inflammation showed the protective effects of quercetin against inflammation in human umbilical vein endothelial cells (huvecs) and indicated that the effect was mediated via the downregulation of vascular cell adhesion molecule (vcam- ) and cd expression [ ] . quercetin significantly induces the gene expression as well as the production of th- derived interferon-γ (ifn-γ) and down-regulates th- derived interleukin (il- ) by normal peripheral blood mononuclear cells (pbmc). furthermore, quercetin treatment increased the phenotypic expression of ifn-γ cells and decreased il- positive cells by flow cytometry analysis, which corroborate with protein secretion and gene expression studies. these results suggest that the beneficial immuno-stimulatory effects of quercetin may be mediated through the induction of th- derived cytokine, ifn-γ, and inhibition of th- derived cytokine, il- [ ] . quercetin is able to inhibit matrix metalloproteinases, which are normally inhibited by plasminogen activator inhibitor (pai- ) in human dermal fibroblasts [ ] . il- -stimulated il- production from human mast cells is regulated by biochemical pathways distinct from ige-induced degranulation, and quercetin can block both il- secretion and two key signal transduction steps involved [ ] . quercetin is known to possess both mast cell stabilizing and gastrointestinal cytoprotective activity. a study demonstrates that quercetin has a direct regulatory effect on basic functional properties of immune cells which may be mediated by the extracellular regulated kinase (erk ) mitogen-activated protein (map) kinase signal pathway in human mitogen-activated pbmc and purified t lymphocytes [ ] . the property proves inhibitory to a huge panoply of molecular targets in the micromolar concentration range, either by down-regulating or suppressing many inflammatory pathways and functions. quercetin has shown a biphasic behavior in basophils at nanomolar doses and hence its action on cells involved in allergic inflammation. quercetin affects immunity and inflammation by acting mainly on leukocytes and targeting many intracellular signaling kinases and phosphatases, enzymes and membrane proteins are often crucial for a cellular specific function. however, the wide group of intracellular targets and the elevated number of natural compounds potentially effective as anti-inflammatory therapeutic agents, asks for further insights and evidence to comprehend the role of these substances in animal cell biology [ ] . in vitro treatment of activated t cells with quercetin blocks il- -induced tyrosine phosphorylation of jak , tyk , stat , and stat , resulting in a decrease in il- -induced t cell proliferation and th differentiation [ ] . taken as in vitro together, the possible pathway of quercetin on inflammation and immune function is as follows (figure ) . the main action of quercetin on inflammation and immune function in vitro is summarized in the table . the main action of quercetin on inflammation and immune function in vitro is summarized in the table . - mmol/l human umbilical cord blood-derived cultured mast cells (hcbmcs) inhibition of il- -induced il- secretion, p and pkc-theta phosphorylation [ ] ě mmol/l or ď mmol/l inhibition of dc activation; dc apoptosis; downregulation of the cytokines and chemokines, disturbance of immunoregulation; attenuation of lps-induced dc maturation and limitation of immunostimulatory activity; downregulate of endocytosis and impairment of ag loading; suppression of dc migration and disconnection of the induction of adaptive immune responses [ ] . . in vivo quercetin exerts inflammation and immune modulating activity in several murine models of autoimmunity. in vivo, animal experiments also support an anti-inflammatory effect. quercetin ameliorates the inflammatory response induced by carrageenan [ ] and a high-fat diet [ ] . quercetin reduced visceral adipose tissue tnf-α and nitric oxide production and downregulated nitric oxide synthase (nos) expression in obese zucker rats [ ] . in chronic rat adjuvant induced arthritis, quercetin decreased clinical signs of arthritis compared to untreated controls [ ] . in rats, post-trauma administration of quercetin improves recovery of motor function after acute traumatic spinal cord injury. intraperitoneal (ip) doses of - micromoles quercetin/kg body weight resulted in half or more of the animals walking, although with deficit [ ] . this ability to promote recovery from spinal cord injury appears to be highly dependent on the dose and frequency of dosing. in this study a lower ip dose was ineffective. in another study, compared to an untreated control group of animals (none of which recovered motor function sufficient to walk), quercetin administration twice daily for three or days resulted in about percent of the animals recovering sufficient motor function to walk. however, when quercetin was injected three times daily, none of the nine animals recovered the ability to walk [ ] . study has shown that quercetin exerted protective effect against irradiation-induced inflammation in mice through increasing cytokine secretion [ ] . quercetin possesses activity against isoproterenol-induced myocardial oxidative injury and immunity function impairment, and that the mechanism of pharmacological action was related at least in part to the antioxidant activity of quercetin [ ] . quercetin decreased histological signs of acute inflammation in the treated animals in a dose-dependent manner via suppressing leucocyte recruitment, decreasing chemokine levels and levels of the lipid peroxidation end-product malondialdehyde, and increasing antioxidant enzyme activity in experimental rat model [ ] . quercetin ameliorated experimental allergic encephalomyelitis (eae) by blocking il- signaling and th differentiation [ ] and experimental autoimmune myocarditis (eam) in dark agouti rats by interfering with production of pro-inflammatory (tnf-α and il- ) and/or anti-inflammatory (il- ) cytokines [ ] . quercetin most likely universally suppresses the accumulation and activation of immune cells, including anti-inflammatory cells, whereas it specifically increased gene expression associated with mitochondrial oxidative phosphorylation in western diet-induced obese mice. suppression of oxidative stress and nf-κb activity likely contributed to the prevention of the accumulation and activation of immune cells and resulting chronic inflammation of epididymal adipose tissue in western diet-induced obese mice [ ] . diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduces cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes in healthy seniors and hypercholesterolemic subjects [ ] . in a randomized, double-blinded, placebo-controlled trial, subjects took or mg/day quercetin or a placebo for weeks. for the group as a whole, quercetin supplementation had no significant influence on rates of upper respiratory tract infections (urti) compared to placebo. in a subgroup of subjects age or older who self-rated themselves as physically fit, mg/day quercetin resulted in a statistically significant reduction in total sick days and symptom severity associated with urti [ ] . female subjects were supplemented with or mg/day quercetin or placebo for weeks. while quercetin supplementation significantly increased plasma quercetin levels, it had no influence on measure of immune function [ ] . quercetin ( mg/day) did not alter exercise-induced changes in several measures of immune function following three days of intense exercise in trained athletes, but it significantly reduced urti incidence ( of subjects in active versus of in placebo group) during the two-week post-exercise period [ ] . a similar lack of effect on strenuous exercise-induced immune system perturbation was found in subjects who took mg/day of quercetin for three weeks before, during, and continuing for two weeks after the -km western states endurance run. in this study, however, there were no differences in the post-race illness rates between quercetin and placebo groups [ ] . there are several studies in humans investigating the correlation of quercetin and its immunomodulatory effects. quercetin does indeed reduce illness after intensive exercise. again, under double-blind conditions, nieman et al. showed that a supplement of mg of quercetin alone three weeks before, during and two weeks after a three-day period of h of cycling in the winter resulted in a markedly lower incidence of urti in well-trained subjects in the two weeks after the intensified training, but had no effect on exercise-induced immune dysfunction, inflammation and oxidative stress [ ] . the literature is supportive of the anti-pathogenic capacities of quercetin when it is cultured with target cells and a broad spectrum of pathogens including urti-related rhinoviruses, adenoviruses and coronaviruses. the impact of the co-ingestion of two or more flavonoids increases their bioavailability and the outcomes on immunity. nieman et al. determined the influence of two weeks of mg/day quercetin compared with placebo supplementation on exercise performance and skeletal muscle mitochondrial biogenesis in untrained, young adult males. it resulted in significantly reduced post-exercise measures for both inflammation and oxidative stress, with a chronic augmentation of granulocyte oxidative burst activity [ ] . when taken together, quercetin showed a successful reduction in the illness rates of exercise-stressed athletes as well as a chronic augmentation of their innate immune function. most in vitro research suggests that quercetin possesses anti-inflammation and immunological improvement. however, the results from a double-blinded, placebo-controlled, randomized trial indicated that quercetin supplementation at and mg/day for weeks significantly increased plasma quercetin levels but had no influence on measures of innate immune function or inflammation in community-dwelling adult females [ ] . the main action of quercetin on inflammation and immune function in vivo is summarized in the table . increase of activity of endogenous antioxidant enzymes and inhibition of free radical generation [ ] or µg sjl/j mice blockage of interleukin- signaling and th differentiation [ ] or mg/kg (oral administration) dark agouti rat interference of pro-inflammatory (tnf-α and il- ) and/or anti-inflammatory (il- ) cytokines production [ ] human and mg/person elderly human subject inhibition of proteasome (nitric oxide, c-reactive protein, γ-glutamyltransferase) activity [ ] and mg/day human subject reduction of upper respiratory tract infection and total sick days; improvement in -min treadmill time trial performance no effect [ ] mg/day human in treadmill no effect [ ] and mg/day human subject no effect on innate immune function or inflammation, illness rates no effect [ ] mg/day human cyclist no effect [ ] mg/day human runner no effect [ ] mg/day human cyclist no effect [ ] these results suggest that quercetin exhibited anti-inflammation and immune-enhancement in vitro (cells) and in vivo (animals), however, studies in human did not totally support these results from cells and animals. the effect, in which quercetin acts as an immune booster in humans, needs to be further verified for future broad application. as a widespread flavonoid, quercetin is a safe and dietary supplement based on its broad range of biological effects in animal. the results of these effects are not consistent, however, and the outcomes need to be carefully evaluated, as they are dependent on the type of subject and their level of health. taken together, we know definitively that a quercetin glycoside is much more efficient than other forms of quercetin. in the majority of the literature, we find references to the benefits of prolonged supplementation with quercetin. the future challenge is to investigate optimal benefits of quercetin, especially to the recommendation for the protracted intake. for example, a carbohydrate drink may have a better effect than pure quercetin preparation. the research in this area continues to determine the proper outcomes, dosing regimen and adjuvants that may amplify any perceived bioactive effects of quercetin in vivo. effects of the dietary flavonoid quercetin upon performance and health beneficial effects of quercetin on obesity and diabetes induction of zygotic polyembryos in wheat: influence of auxin polar transport dietary flavonoids: bioavailability, metabolic effects, and safety the sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man content of the flavonols quercetin, myricetin, and kaempferol in edible berries bioavailability and bioefficacy of polyphenols in humans. ii. review of intervention studies quercetin from shallots (allium cepa l. var. aggregatum) is more bioavailable than its glucosides genetic analysis of quercetin in onion (allium cepa l.) lady raider ten-year comparison of the influence of organic and conventional crop management practices on the content of flavonoids in tomatoes analysis of flavonoids in honey by hplc coupled with coulometric electrode array detection and electrospray ionization mass spectrometry biologically active substances of plant origin. flavonols and flavones: prevalence, dietary sources and consumption usda database for the flavonoid content of selected foods estimated dietary flavonoid intake and major food sources of u.s. adults using an ffq to assess intakes of dietary flavonols and flavones among female adolescents in the suihua area of northern china. public health nutr dietary flavonol and flavone intakes and their major food sources in chinese adults flavonol and flavone intakes in us health professionals estimated daily intake and seasonal food sources of quercetin in japan dietary flavonoid sources in australian adults estimation of dietary sources and flavonoid intake in a spanish adult population (epic-spain) interactions affecting the bioavailability of dietary polyphenols in vivo bioavailability and metabolism of the flavonol quercetin in the pig. free radic dietary fat increases quercetin bioavailability in overweight adults part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal l: umen quercetin metabolites in plasma of rats fed diets containing rutin or quercetin absorption, metabolism and bioavailability of flavonoids. in flavonoids in health and disease conjugation position of quercetin glucuronides and effect on biological activity. free radic tissue distribution of quercetin in rats and pigs metabolism of quercetin by human intestinal bacteria and its relation to some biological activities comparison of the bioavailability of quercetin and catechin in rats. free radic in vitro glucuronidation of kaempferol and quercetin by human ugt- a microsomes bioavailability of various polyphenols from a diet containing moderate amounts of berries absorption and excretion of conjugated flavonols, including quercetin- -o-beta-glucoside and isorhamnetin- -o-beta-glucoside by human volunteers after the consumption of onions plasma metabolites of quercetin and their antioxidant properties extensive binding of the bioflavonoid quercetin to human plasma proteins effect of fruit juice intake on urinary quercetin excretion and biomarkers of antioxidative status pharmacokinetics and bioavailability of the flavonol quercetin in humans polyphenols and prevention of cardiovascular diseases evaluation of quercetin as a countermeasure to exercise-induced physiological stress quercetin pharmacokinetics in humans carbon dioxide is the major metabolite of quercetin in humans a critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties naturally occurring anti-inflammatory agents basic, i. immunomodulatory and antimetastatic action of propolis and related polyphenolic compounds quercetin inhibits lps-induced nitric oxide and tumor necrosis factor-alpha production in murine macrophages dietary flavones and flavonols are inhibitor of poly (adp-ribose) polymerase- in pulmonary epithelial cells resveratrol and quercetin, two natural polyphenols, reduce apoptotic neuronal cell death induced by neuroinflammation effects of naturally-occurring flavonoids and bioflavonoids on epidermal cyclooxygenase and lipoxygenase from guinea-pigs protective effect of quercetin against arsenite-induced cox- expression by targeting pi k in rat liver epithelial cells quercetin disrupts tyrosine-phosphorylated phosphatidylinositol -kinase and myeloid differentiation factor- association, and inhibits mapk/ap- and ikk/nf-κb-induced inflammatory mediators production in raw . cells flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase c theta phosphorylation in human mast cells protective effects of quercetin and taraxasterol against h o -induced human umbilical vein endothelial cell injury in vitro the role of quercetin, flavonols and flavones in modulating inflammatory cell function role of mast cells in gastrointestinal mucosal defense immunosuppressive effect of quercetin on dendritic cell activiation and function the flavonoid, quercetin, differentially regulates th- (ifng) and th- (il ) cytokine gene expression by normal peripheral blood mononuclear cells inhibition of mammalian collagenase, matrix metalloproteinase- , by naturally-occurring flavonoids regulation of il- -induced selective il- release from human mast cells and inhibition by quercetin quercetin, a flavonoid phytoestrogen, ameliorates experimental allergic encephalomyelitis by blocking il- signaling through jak-stat pathway in t lymphocyte inhibitory effect of quercetin on carrageenan-induced inflammation in rats quercetin transiently increases energy expenditure but persistently decreases circulating markers of inflammation in c bl/ j mice fed a high-fat diet quercetin ameliorates metabolic syndrome and improves the inflammatory status in obese zucker rats therapeutic and preventive properties of quercetin in experimental arthritis correlate with decreased macrophage inflammatory mediators quercetin promotes functional recovery following acute spinal cord injury quercetin in an animal model of spinal cord compression injury: correlation of treatment duration with recovery of motor function effect of quercetin on impaired immune function in mice exposed to irradiation evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats protective effect of quercetin against oxidative stress and brain edema in an experimental rat model of subarachnoid hemorrhage quercetin ameliorates experimental autoimmune myocarditis in rats quercetin suppresses immune cell accumulation and improves mitochondrial gene expression in adipose tissue of diet-induced obese mice suppression of nitric oxide production and cardiovascular risk factors in healthy seniors and hypercholesterolemic subjects by a combination of polyphenols and vitamins quercetin supplementation and upper respiratory tract infection: a randomized community clinical trial a -week supplementation with quercetin does not affect natural killer cell activity, granulocyte oxidative burst activity or granulocyte phagocytosis in female human subjects quercetin reduces illness but not immune perturbations after intensive exercise post- -km race illness rates and decreases in granulocyte respiratory burst and salivary iga output are not countered by quercetin ingestion effects of quercetin and egcg on mitochondrial biogenesis and immunity quercetin's influence on exercise performance and muscle mitochondrial biogenesis the authors declare no conflict of interest. key: cord- -q fa m authors: misra, durga prasanna; gasparyan, armen yuri; zimba, olena title: benefits and adverse effects of hydroxychloroquine, methotrexate and colchicine: searching for repurposable drug candidates date: - - journal: rheumatol int doi: . /s - - - sha: doc_id: cord_uid: q fa m repurposing of antirheumatic drugs has garnered global attention. the aim of this article is to overview available evidence on the use of widely used antirheumatic drugs hydroxychloroquine, methotrexate and colchicine for additional indications. hydroxychloroquine has endothelial stabilizing and anti-thrombotic effects. its use has been explored as an adjunctive therapy in refractory thrombosis in antiphospholipid syndrome. it may also prevent recurrent pregnancy losses in the absence of antiphospholipid antibodies. hydroxychloroquine favourably modulates atherogenic lipid and glycaemic profiles. methotrexate has been tried for modulation of cardiovascular events in non-rheumatic clinical conditions, although a large clinical trial failed to demonstrate a benefit. colchicine has been shown to successfully reduce the risk of recurrent cardiovascular events in a large multicentric trial. potential antifibrotic effects of colchicine require further exploration. hydroxychloroquine, methotrexate and colchicine are also being tried at different stages of the ongoing coronavirus disease (covid- ) pandemic for prophylaxis and treatment. while the use of these agents is being diversified, their adverse effects should be timely diagnosed and prevented. hydroxychloroquine can cause retinopathy and rarely cardiac and auditory toxicity, retinopathy being dose and time dependent. methotrexate can cause transaminitis, cytopenias and renal failure, particularly in acute overdoses. colchicine can rarely cause myopathies, cardiomyopathy, cytopenias and transaminitis. strong evidence is warranted to keep balance between benefits of repurposing these old antirheumatic drugs and risk of their adverse effects. rheumatologists are at the forefront of research and practice of using various anti-inflammatory and immunomodulatory drugs. their decades-long clinical experience can be valuable at the time of drug repurposing and attempting to use widely tested antirheumatic drugs for newer indications [ ] . numerous immunomodulatory drugs have been employed to induce remission of autoimmune disease and prevent adverse effects of concomitant high-dose corticosteroid and other synthetic disease-modifying anti-rheumatic drug (dmard) therapies, particularly in elderly patients and those with comorbidities [ ] . although precise mechanisms of action of most old antirheumatic drugs remain unknown, these are exemplified for their relative safety and utility, allowing corticosteroid dose tapering and long-term disease control. a scopus search conducted on th august of the number of publications mentioning these drugs to this day revealed an ongoing interest in these drugs in the published literature. overall, , documents are retrievable for methotrexate, , for colchicine, and , for hydroxychloroquine (fig. ) . the repurposing of traditionally used dmards has garnered significant attention recently with the advent of the coronavirus disease pandemic [ ] . this opinion article aims to sensitize readers towards the potential utility of these drugs in cardiovascular disease and fibrosis (both of which have been reported as sequelae of [ ] [ ] [ ] as well as serve as a reminder of their adverse effect profile. such potential adverse effects need to be kept in mind by specialists who do not routinely use these drugs when they are explored for other indications. in this article we overview some of the effects of hydroxychloroquine, methotrexate and colchicine and reflect on their positive and negative sides to guide repurposing of drug therapies. the choice of articles for review reflects the authors' understanding and viewpoints regarding the potential therapeutic roles of these drugs. hydroxychloroquine (hcq), a relatively safe derivative of chloroquine, is effectively used for the treatment of systemic lupus erythematosus and other inflammatory rheumatic diseases. it acts by accumulating in lysosomes, where its basic ph modifies the normally acidic milieu. this interferes with the loading and presentation of antigens by class ii major histocompatibility complex (mhc) protein. also, it interferes with the activation of toll-like receptors (tlr) by deoxyribonucleic acid (dna) and ribonucleic acid (rna) moieties [ ] . the use of hcq reduces the risk of thrombosis and diminishes vascular inflammation and endothelial dysfunction in a murine model of antiphospholipid syndrome (aps) [ ] . in patients with antiphospholipid antibodies (apl) fig. annual publication activity in scopus as of august , with the keywords "methotrexate", "colchicine" and "hydroxychloroquine" for articles published up to . graph might appear to dip for some entries as the year is still ongoing. the graph seems to suggest that there is still ongoing interest in these older disease-modifying antirheumatic drugs treated with hcq, levels of soluble tissue factor reduced after three months of hcq therapy. however, other markers of thrombogenic potential, such as annexin activity and anti-domain immunoglobulin g activity, and complement activation did not differ [ ] . in patients with systemic lupus erythematosus (sle) from italy ( positive for apl) followed up over a median of years, there was a markedly reduced risk of vascular events in those treated with hcq for five years (hazard ratio [hr] . , % confidence interval [ci] . - . ) [ ] . another retrospective study analysed insurance databases in taiwan ( patients with sle on hcq during first year compared with a group of sle patients not on hcq during the same period) [ ] . over a mean period of . years, hcq therapy was associated with a small but non-significant reduction in the risk of vascular events (hr . , % ci . - . ). the data was limited due to the lack of data about apl for these patients [ ] . a multicentric randomized controlled trial (rct) reported patients with apl positivity in the absence of any systemic autoimmune disease who received either hcq or placebo. over a mean of . years of follow-up, neither group developed thrombotic events, resulting in premature termination of the trial and a subsequent inability to draw meaningful conclusions [ ] . another recent open-label study in greece with primary aps treated with standard anticoagulation regimen (with or without antiplatelets) randomized patients to receive either hcq in addition to standard care or standard care only. over mean . years follow-up, those on hcq had a lower risk of incident thrombosis, which did not remain significant (hr . , % ci . - . ) after adjusting for confounders. possibly, a larger sample size might have attained results with statistical significance [ ] . the european league against rheumatism (eular) recommends hcq therapy (based on expert opinion and anecdotal evidence) for patients with aps and recurrent pregnancy loss despite optimal doses of anticoagulation and antiplatelet agents during pregnancy [ ] . various ongoing clinical trials are evaluating the role of add-on hcq in aps. the hibiscus multicentre multinational trial is evaluating the effect of hcq ( mg daily for the duration of pregnancy) compared to placebo, in addition to standard therapy (i.e. preventative dose of low molecular weight heparin with aspirin), on live births in women with primary aps. a related study, the hibiscus-t trial, is evaluating the preventative role for recurrent thrombosis in patients with thrombotic aps treated with hcq or placebo in addition to oral anticoagulation with vitamin k antagonists for months [ ] . the hypatia trial is another multicentric trial spanning multiple european nations assessing the effect of hcq in a placebo-controlled rct involving patients with either aps or persistent apl positivity. this trial intends to assess the impact of hcq, started before pregnancy and continued for months, on pregnancy morbidity associated with apl, i.e. a composite of early pregnancy losses, pre-term delivery (< weeks) or placental insufficiency [ ] . another french multicentric trial, the hydrosapl trial, is also assessing the effect of hcq on pregnancy outcomes in primary aps [ ] . apart from aps, hcq is also being explored for its potential utility in patients with recurrent pregnancy loss. in this french rct (the bbq trial), patients with recurrent pregnancy loss shall be treated with hcq mg daily or placebo starting pre-conception until the th week of pregnancy, with the objective of assessing whether treatment improves pregnancy outcomes in such patients [ ] . other cardioprotective effects of hcq, apart from reduction of thrombotic events, have also been proposed. in a model of induced myocardial infarction in rats, the administration of hcq was associated with reduced apoptosis in the infarcted myocardial tissue after weeks [ ] . in patients with rheumatoid arthritis (ra) and sjogren's syndrome, the use of hcq has been associated with a reduced risk of developing diabetes mellitus (table ) [ ] [ ] [ ] [ ] . in animal models of insulin resistance induced by a high fat diet, the addition of hcq was associated with better survival of the insulin-producing islet of langerhans, as well as decreased release of inflammatory adipokines and markers of endothelial stress. thus, a beneficial effect of hcq on mechanisms driving insulin resistance has been postulated [ ] . in a small proof-of-concept study of patients with type diabetes mellitus (n = ) inadequately controlled with a combination of metformin and sulfonylurea, the addition of hcq mg daily was compared with pioglitazone mg daily. although pioglitazone was more effective in reducing levels of glycated haemoglobin and achieving glycaemic control, nearly two-thirds of the patients on hcq could attain less than . % level of glycated haemoglobin [ ] . another small clinical trial of patients with pre-diabetes to receive hcq (n = ) or placebo (n = ) demonstrated improvements in glycaemic control over months hcq therapy [ ] . these studies suggest the potential for hcq as an add-on agent for diabetes mellitus. however, until the utility of hcq is proven for the indications of diabetes mellitus, dyslipidemia or cardiovascular disease, its indiscriminate use for these indications should be avoided [ ] . considering the evidences for anti-platelet and anti-thrombotic actions of hcq, as well as favourable lipid profile in patients treated with hcq, it is possible that this drug might have an adjuvant role in the prevention of incident or recurrent cardiovascular events. this might need exploration in future clinical trials [ ] . methotrexate is the first-line dmard for the management of inflammatory arthritides such as rheumatoid arthritis and psoriatic arthritis. the drug's anti-folate mechanism of action is historically implicated in the treatment of neoplastic (lymphoblastic) diseases. the immunosuppressive action of low-dose methotrexate therapy (up to mg/week) primarily relies on the inhibition of the enzyme -aminoimidazole- -carboxamide ribonucleoside (aicar) transformylase (atic), resulting in higher levels of aicar that inhibits adenosine monophosphate deaminase and adenosine deaminase. the latter results in higher extracellular levels of adenine, further converted to adenosine, which acts via adenosine receptors to exert antiinflammatory actions. further, inhibition of the enzyme dihydrofolate reductase (dhfr) also results in uncoupling of nitric oxide (no) synthetase, by increasing levels of dihydrobiopterin and tetrahydrobiopterin, resulting in greater release of no as opposed to reactive oxygen species. methotrexate also exerts an effect to decrease downstream inflammatory signalling operating via nuclear factor kappa b (nfκb). while supplementation with folic acid antagonizes the anti-folate action of methotrexate (responsible for most of its side effects), it does little to inhibit other anti-inflammatory pathways [ ] . recently accumulated evidence suggests a favourable modulation of cardiovascular risk in patients with rheumatic diseases treated with methotrexate [ , ] . a pilot study evaluated the safety of methotrexate therapy ( mg/week) in patients with coronary artery disease undergoing percutaneous coronary intervention (pci), started weeks prior to the procedure and continued for weeks after the procedure. no major safety signals were observed during this period [ ] . a rct, the tethys trial, evaluated the role of methotrexate in patients with st segment elevation myocardial infarction (stemi). methotrexate administered at a dose of . mg/ kg, repeated h after pci in patients, was compared to patients receiving placebo. this trial aimed to analyse area under the curve for serial measurements of creatine kinase and troponin i over the first h after stemi as a surrogate for area of infarct. no significant differences in the area of infarct measured either using creatine kinase or troponin i could be demonstrated between the two groups. a matter of concern was that the left ventricular ejection fraction (lvef) was significantly lesser in patients treated with methotrexate compared to those receiving placebo, three months after completion of the treatment [ ] . another large multicentric rct, the cirt trial, evaluated the role of methotrexate in the secondary prevention of cardiovascular events. in this trial, patients who had a prior myocardial infarction or had triple vessel coronary artery disease on a background of diabetes mellitus or metabolic syndrome were randomized to receive methotrexate - mg/week ( patients) or placebo ( patients). the primary endpoint was a composite of first occurrence of a major adverse cardiovascular event (non-fatal myocardial infarction, stroke, or death due to cardiovascular cause), later modified to include hospitalization for unstable angina resulting in a revascularization procedure. the results of this trial did not favour the use of methotrexate, with a small but insignificant reduction in the risk of developing the primary end point with methotrexate (hr . , % ci . - . ). apart from a higher incidence of non-basal cell carcinoma in the group receiving methotrexate, no other significant safety signals were observed [ ] . these studies do not seem to suggest a role for methotrexate in the prevention of cardiovascular events. an ongoing study (nct ) is evaluating the targeting of left ventricular remodelling following stemi with a nano-emulsion containing methotrexate. it remains to be seen whether such a strategy is useful to favourably modulate cardiovascular events. colchicine has been used for the management of gout, familial mediterranean fever (fmf) and behcet's disease for decades [ ] . it acts predominantly on neutrophils, monocytes and macrophages, decreasing their chemotaxis and release of various inflammatory cytokines. it interferes with the activation of the nlrp- inflammasome complex, thereby reducing the secretion of downstream cytokines such as il- ß and il- [ , ] . knocking out the expression of nlrp- has been shown to reduce atherosclerosis as well as dampen the severity of myocardial injury following myocardial infarction in animal models [ ] . in a murine model, the administration of colchicine improved survival and residual left ventricular function following myocardial infarction [ ] . in a model of atherosclerosis induced by high fat diet in rabbits, the administration of colchicine reduced the uptake of -fluodeoxyglucose in atherosclerotic plaques in the abdominal aorta. moreover, in those rabbits with an increased cholesterol level in the peripheral blood, the burden of atherosclerotic plaques was significantly lesser along with greater circulating high-density lipoprotein (hdl) cholesterol [ ] . in platelet-rich plasma from healthy volunteers, in-vitro treatment with colchicine significantly reduced platelet aggregation [ ] . refractoriness to colchicine when used in the longer term in disease states such as fmf has been associated with oxidative stress [ ] . several clinical studies have also assessed the modulation of cardiovascular risk by colchicine. in a parallelgroup observational study, patients with coronary artery disease (cad) were treated with standard care and compared with another patients also treated with colchicine . mg daily in addition to standard care. multivariable analysis revealed a significant effect of colchicine on the reduction of the volume of atherosclerotic plaques [ ] . in a double-blind placebo-controlled trial of patients with cad short-term colchicine therapy significantly reduced systemic inflammation and improved endothelial function [ ] . in another study, patients with acute coronary syndromes (acs) planned for pci were randomized to treatment with add-on colchicine (n = ) or just standard treatment (n = ); those on colchicine had lower levels of chemokines, suggesting a reduction in local inflammatory activity at the site of pathology [ ] . in the low dose colchicine for myocardial infarction (lodoco-mi) study, patients with acute myocardial infarction were randomized to receive colchicine or placebo in addition to standard care, and c-reactive protein (crp) levels were compared at month. there was no significant difference in the proportions of patients attaining crp levels lesser than mg/l amongst patients treated with colchicine or placebo [ ] . the ongoing lodoco trial is evaluating clinical cardiovascular outcomes in , patients with stable cad treated with add-on colchicine or placebo [ ] . the colin trial, evaluated treatment with add-on colchicine (n = ) compared to placebo (n = ) in patients with stemi. neither the primary outcome of difference in peak serum crp during hospital admission between the two groups, nor secondary outcomes for differences in other biochemical, clinical or imaging outcomes (assessed by echocardiography or magnetic resonance imaging) at month were successfully met [ ] . a large multicentric trial evaluated the role of add-on colchicine (n = ) compared to placebo (n = ) in patients following myocardial infarction. the trial assessed the composite end-point of cardiovascular death, successful resuscitation after cardiac arrest, stroke, recurrent myocardial infarction, or angina requiring an intervention for revascularization. followed up for a median . months, treatment with colchicine was associated with a reduced risk of the composite outcome compared to placebo (hr . , % ci . - . ). gastrointestinal side-effects were similar, however, a greater incidence of pneumonia was observed in patients receiving colchicine ( . % vs . % on placebo) [ ] . a recent systematic review also confirmed an atheroprotective effect of colchicine, with a significant reduction in future cerebrovascular ischemic events with colchicine (odds ratio . , % ci . - . ) as observed in six clinical trials [ ] . colchicine may also exert anti-fibrotic effects. clinical trials have demonstrated the potential role of colchicine in reducing the occurrence of atrial fibrillation following catheter ablation and cardiac surgeries [ ] . it has been postulated that the modulation of atrial fibrosis by colchicine is mediated by its effects on interleukin- and renin-related pathways [ ] . in a trial of patients undergoing coronary artery bypass grafting, perioperative treatment with colchicine was associated with reduction in post-surgical constrictive pericarditis [ ] . in a rat model of hypertension induced by nephrectomy, treatment with colchicine ameliorated both glomerular and interstitial fibrosis in the remaining kidney [ ] . in another animal model of renal injury induced by ureteric obstruction in rats, treatment with colchicine reduced both cortical fibrosis and tubulointerstitial injury [ ] . anecdotal reports also exist of the regression of retroperitoneal fibrosis in patients treated with colchicine [ ] . in ten patients with keloids of the ear, treatment with colchicine for a month prior to keloid excision (along with pressure therapy and intralesional corticosteroid therapy in some) was associated with lack of recurrence of the keloids, although long-term follow-up data were unavailable for a majority of patients [ ] . a potential role of colchicine in retarding the progression of oral submucous fibrosis has also been proposed [ ] . treatment with topical colchicine was also effective in reducing induced spinal epidural fibrosis in an experimental model in rats [ ] . in a clinical trial involving patients with liver cirrhosis of different aetiologies, patients were treated with standard treatment alone and another with add-on colchicine. followed up over a mean of . years, those treated with colchicine had a greater proportion of survival. biochemical tests revealed that colchicine therapy was associated with lower serum levels of pro-collagen iii peptide, a marker of fibrosis [ ] . another report described the combination of colchicine with ursodeoxycholic acid (udca) in patients with primary biliary cirrhosis who did not respond to udca alone. after a followup of years, two-thirds of patients continued to survive when treated with this combination treatment regimen [ ] . it is possible that the anti-fibrotic effects of colchicine are mediated by its inhibitory actions on cytotoxic t lymphocytes as well as stimulation of endogenous mechanisms to regress fibrosis such as collagenases [ ] . the potential antifibrotic effects of colchicine merit systematic evaluation by clinical trials in patients with systemic fibrosing diseases such as systemic sclerosis, or major organ fibrosis such as interstitial lung diseases. the covid- pandemic has swept the world. based on preliminary evidence, abnormal activation of the immune system and cytokine storm appear to be associated with disease severity. in this context, commonly used antirheumatic drugs are being explored in covid- [ ] . while hydroxychloroquine has been recommended for prophylaxis of high-risk contacts of covid- , there is little evidence at present to support this [ ] . occasional reports have even described patients on long term hydroxychloroquine therapy for rheumatic diseases who eventually developed covid- [ ] . overall, the level of enthusiasm regarding the use of hydroxychloroquine in covid- has dampened due to mixed, often negative preliminary results of trials [ ] . it has been hypothesized that the timing (early initiation) of hydroxychloroquine might play a role in protection against severe covid- , and the results of trials on chemoprophylaxis of covid- with hydroxychloroquine might help answer this question [ ] . in this context, it is necessary to mention that hydroxychloroquine (and its analogue, chloroquine) have been previously tried in other viral infections such as zika virus and ebola virus infections. while in-vitro efficacy had been observed, this did not translate into clinical efficacy, possibly due to the fact that the drugs were unable to attain a concentration in the endosome necessary for their anti-viral properties to take effect [ , ] . anecdotal reports have suggested reduced severity of covid- in patients on colchicine for other indications [ ] . another retrospective review of records of patients from an european centre compared patients treated with standard of care (varying combinations of hydroxychloroquine, lopinavir/ritonavir and corticosteroids) with patients treated with colchicine at . - mg/day in addition to standard of care. treatment with colchicine was associated with % reduction in hazard of death (hazard ratio . , % confidence intervals . - . ) [ ] . however, in another recent study, six out of seven children with paediatric autoinflammatory syndromes who developed covid- were on colchicine [ ] . ongoing clinical trials identified on a search on clinicaltrials.gov on august are summarized in table . the results of these trials shall help better understand the role of antirheumatic drugs in covid- in the coming times. it is increasingly being understood that recovery from covid- is prolonged. nearly one-half of patients with covid- from italy had persistence of symptoms, including fatigue and joint pains, after months of onset of clinical disease [ ] . neuro-cognitive disorders are also being described in covid- survivors, at least in part driven by the endothelial injury due to the infection [ ] . a significant proportion of patients with pneumonitis due to covid- are likely to develop residual pulmonary fibrosis [ , ] . covid- causes endothelial injury, and this might be responsible for long-term cardiovascular consequences of the disease, including a predisposition towards future cardiovascular events [ , ] . therefore, age-old rheumatic drugs should be explored in such select recovered populations. particularly, it can be hypothesized that the anti-fibrotic effects of colchicine might help in post-covid- fibrosis, including lung fibrosis. table summarizes side effects of the examined drugs. hcq entails the risk of toxicities in the long term [ ] . ocular toxicity occurs in the form of retinal damage. up to in patients might develop retinopathy after five years of using hydroxychloroquine, and this is often silent in the earlier stages [ ] . in the later stages, macular vision may be affected, resulting in maculopathy and vision loss. fundoscopy and perimetry are insensitive for picking up earlier changes of hcq retinopathy. recent recommendations suggest limiting the dose of hcq to mg/kg/day, since higher doses increase the risk of retinopathy after years [ ] . before initiation of hcq, baseline fundus evaluation with the macula assessment is recommended. if this is abnormal, more sensitive techniques such as spectral domain optical coherence tomography may be employed and repeated annually [ ] . other adverse effects hcq-related skin pigmentation due to sun exposure, myopathy, and conduction blocks [ ] . cardiac toxicity may be potentiated by other drugs resulting in prolongation of the qt interval such as azithromycin [ ] . rare instances of sensorineural hearing loss have also been noted [ ] . in the context of the ongoing covid- pandemic, potential cardiac toxicity of hcq has received widespread attention. however, speaking from personal experience as well as the published literature in rheumatic diseases [ ] , cardiac toxicity with hcq (including qt prolongation) is very rare. pharmacological interaction with azithromycin can potentially prolong qt interval when these two drugs are taken together, as has been tried in the management of covid- . hence, due caution and pre-treatment with follow-up ecgs to seek any developing cardiac arrhythmias might be suggested if and when hcq is combined with azithromycin or any other drug that can cause qt prolongation [ ] . the adverse effect profile of methotrexate is mainly due to its antifolate actions [ ] . methotrexate toxicity can result in mucositis with oral ulcers, crusting of lips and diarrhoea, skin rashes, pancytopenia, transaminitis, and acute kidney injury [ ] . the risk of toxicities is greater in the presence of renal failure. in a series of patients with rheumatoid arthritis with concomitant renal impairment treated with methotrexate, nearly % developed features of methotrexate toxicity such as leucopenia, transaminitis and renal failure. interestingly, a protective effect towards methotrexate toxicity in the presence of renal failure was observed in those individuals also taking hydroxychloroquine [ ] . acute methotrexate toxicity requires discontinuation of the drug and administration of folinic acid. varying dose regimens of folinic acid are used, although often mg hourly for doses is administered. supportive treatment in the form of granulocyte colony stimulating factor, blood component transfusions, treatment of coexistent infections and nutritional care are also essential. untreated methotrexate toxicity is associated with significant risk of mortality [ , ] . rarely, methotrexate may result in acute interstitial pneumonitis [ ] . colchicine therapy can result in diarrhoea and other gastrointestinal adverse effects. long-term colchicine therapy is rarely associated with myopathy, polyneuropathy, transaminitis, and rhabdomyolysis [ ] . the risk of toxicity is increased in the presence of renal failure. also, coadministration with drugs inhibiting the enzyme cytochrome table adverse effect profile a risk of retinopathy generally occurs after years of use, and is related to cumulative doses. those with renal failure or on concomitant tamoxifen therapy are at greater risk b per se, cardiac toxicity is very rare when hydroxychloroquine is used in the context of rheumatic diseases c generally seen with methotrexate toxicity, such as in overdose (daily rather than weekly administration), or with concomitant underlying renal impairment p oxidase a (cyp a , such as clarithromycin) or the drug efflux protein p-glycoprotein portends a greater risk of adverse effects [ ] . occasional reports exist regarding the use of intravenous colchicine for prolonged periods for difficult to treat conditions such as familial mediterranean fever. despite intravenous use for longer term, little toxicity was observed in most patients, other than gastrointestinal side effects [ , ] . drug withdrawal is recommended if such toxicities develop [ , ] . rarely, these drugs can have cardiovascular side effects also, as holds true for most anti-rheumatic drugs: methotrexate has been associated with pericardial and myocardial injury, colchicine rarely causes myocardial injury, and hcq therapy occasionally results in conduction system abnormalities [ ] . clinicians should be aware for the risk of cardiac adverse events when these drugs are considered for repurposing and combined therapies in different patient cohorts. keeping in mind the diverse impacts on different body systems seen with these drugs, it is imperative to generate high-quality evidence for the use of hydroxychloroquine, methotrexate and colchicine for other indications. they are already being tried in patients with cardiovascular disease for secondary prevention. clinical trials might also attempt to decipher the potential utility of hydroxychloroquine, methotrexate or colchicine for primary prevention of cardiovascular events in a high risk population, such as those with metabolic syndrome and in individuals with inflammatory arthritides which predispose to greater cardiovascular risk. furthermore, the potential anti-fibrotic effects of colchicine need to be systematically studied in local fibrosing diseases (such as idiopathic interstitial lung diseases) or systemic fibrosing diseases (such as systemic sclerosis) by suitably powered clinical trials. although recent promising developments in vaccines for covid- hold promise for community level prevention, these are still preliminary and it is likely that millions of individuals shall be infected before such vaccines come into regular clinical use [ ] . therefore, the role of drugs with anti-fibrotic potential such as colchicine in preventing or managing long-term sequelae of covid- should also be explored. repurposing of old antirheumatic drugs is being attempted for numerous indications today. hcq, methotrexate and colchicine are primarily used for favourable modulation of cardiovascular risk. clinicians must be aware of the propensity of these drugs to cause side effects, such as cytopenias with methotrexate, retinopathy with hcq, and gastrointestinal adverse effects with colchicine. in view of the risk of adverse events, it is essential to carefully consider the risk-benefit ratio before prescribing these drugs for newer, unlicensed indications, taking particular care to identify high-risk populations such as those with underlying renal impairment and avoid drug interactions. the role of these drugs in covid- shall be clarified in ongoing trials. it may also require evaluation whether these drugs have a role in managing sequelae of covid- . drug repurposing to improve treatment of rheumatic autoimmune inflammatory diseases reflections on 'older' drugs: learning new lessons in rheumatology rheumatologists' perspective on coronavirus disease (covid- ) and potential therapeutic targets covid- pathways for brain and heart injury in comorbidity patients: a role of medical imaging and artificial intelligence-based covid severity classification: a review for the gemelli against covid-post-acute care study group ( ) persistent symptoms in patients after acute covid- long term respiratory complications of covid- mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology hydroxychloroquine reverses the prothrombotic state in a mouse model of antiphospholipid syndrome: role of reduced inflammation and endothelial dysfunction the effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies longterm hydroxychloroquine therapy and low-dose aspirin may have an additive effectiveness in the primary prevention of cardiovascular events in patients with systemic lupus erythematosus effect of long-term hydroxychloroquine on vascular events in patients with systemic lupus erythematosus: a database prospective cohort study hydroxychloroquine in the primary thrombosis prophylaxis of antiphospholipid antibody positive patients without systemic autoimmune disease the effect of hydroxychloroquine on thrombosis prevention and antiphospholipid antibody levels in primary antiphospholipid syndrome: a pilot open label randomized prospective study eular recommendations for the management of antiphospholipid syndrome in adults hibiscus: hydroxychloroquine for the secondary prevention of thrombotic and obstetrical events in primary antiphospholipid syndrome hydroxychloroquine to improve pregnancy outcome in women with antiphospholipid antibodies (hypatia) protocol: a multinational randomized controlled trial of hydroxychloroquine versus placebo in addition to standard treatment in pregnant women with antiphospholipid syndrome or antibodies hydroxychloroquine to obtain pregnancy without adverse obstetrical events in primary antiphospholipid syndrome: french phase ii multicenter randomized trial, hydrosapl hydroxychloroquine for prevention of recurrent miscarriage: study protocol for a multicentre randomised placebo-controlled trial bbq study effects of low-dose hydroxychloroquine on expression of phosphorylated akt and p proteins and cardiomyocyte apoptosis in peri-infarct myocardium in rats risk of diabetes mellitus associated with disease-modifying antirheumatic drugs and statins in rheumatoid arthritis association between use of disease-modifying antirheumatic drugs and diabetes in patients with ankylosing spondylitis, rheumatoid arthritis, or psoriasis/psoriatic arthritis: a nationwide, population-based cohort study of , patients immunosuppressive treatment and the risk of diabetes in rheumatoid arthritis hydroxychloroquine was associated with reduced risk of newonset diabetes mellitus in patients with sjogren syndrome favorable outcomes of hydroxychloroquine in insulin resistance may be accomplished by adjustment of the endothelial dysfunction as well as the skewed balance of adipokines randomized controlled trial comparing hydroxychloroquine with pioglitazone as third-line agents in type diabetic patients failing metformin plus a sulfonylurea: a pilot study the effect of hydroxychloroquine on glucose control and insulin resistance in the prediabetes condition the marketing of unproven drugs for diabetes and dyslipidaemia in india hydroxychloroquine, a promising choice for coronary artery disease? methotrexate and its mechanisms of action in inflammatory arthritis cardiac involvement in primary systemic vasculitis and potential drug therapies to reduce cardiovascular risk cardiovascular effects of methotrexate in rheumatoid arthritis revisited percutaneous coronary intervention: safety of methotrexate and its possible benefits on restenosis after bare-metal stent deployment methotrexate therapy in st-segment elevation myocardial infarctions: a randomized double-blind, placebocontrolled trial (tethys trial) low-dose methotrexate for the prevention of atherosclerotic events colchicine as an anti-inflammatory and cardioprotective agent colchicine revisited the nlrp inflammasome and the emerging role of colchicine to inhibit atherosclerosis-associated inflammation colchicine improves survival, left ventricular remodeling, and chronic cardiac function after acute myocardial infarction effects of colchicine on atherosclerotic plaque stabilization: a multimodality imaging study in an animal model colchicine reduces platelet aggregation by modulating cytoskeleton rearrangement via inhibition of cofilin and lim domain kinase can the thiol/disulfide imbalance be a predictor of colchicine resistance in familial mediterranean fever? colchicine therapy and plaque stabilization in patients with acute coronary syndrome: a ct coronary angiography study effect of shortterm colchicine treatment on endothelial function in patients with coronary artery disease colchicine as a novel therapy for suppressing chemokine production in patients with an acute coronary syndrome: a pilot study the low dose colchicine after myocardial infarction (lodoco-mi) study: a pilot randomized placebo controlled trial of colchicine following acute myocardial infarction the effect of low-dose colchicine in patients with stable coronary artery disease: the lodoco trial rationale, design, and baseline characteristics colin trial: value of colchicine in the treatment of patients with acute myocardial infarction and inflammatory response efficacy and safety of low-dose colchicine after myocardial infarction role of colchicine in stroke prevention: an updated meta-analysis the role of colchicine in treating postoperative and post-catheter ablation atrial fibrillation comparative transcriptome analysis to elucidate the therapeutic mechanism of colchicine against atrial fibrillation the effect of colchicine on the echocardiographic constrictive physiology after coronary artery bypass graft surgery colchicine attenuates renal injury in a model of hypertensive chronic kidney disease effects of colchicine on renal fibrosis and apoptosis in obstructed kidneys effectiveness of colchicine therapy in cases of retroperitoneal fibrosis associated with autoinflammatory diseases use of colchicine to prevent recurrence of ear keloids. a new approach efficacy of oral colchicine with intralesional hyaluronidase or triamcinolone acetonide in the grade ii oral submucous fibrosis altinors n ( ) topical use of colchicine to prevent spinal epidural fibrosis in rats colchicine reduces procollagen iii and increases pseudocholinesterase in chronic liver disease colchicine or methotrexate, with ursodiol, are effective after years in a subset of patients with primary biliary cirrhosis colchicine to decrease inflammation and fibrosis in patients with metabolic dysregulation a systematic review of the prophylactic role of chloroquine and hydroxychloroquine in coronavirus disease- (covid- ) a case of breakthrough covid- during hydroxychloroquine maintenance swinging the pendulum: lessons learned from public discourse concerning hydroxychloroquine and covid- usefulness of hydroxychloroquine for covid- : does answer lie in timing to start? chloroquine could be used for the treatment of filoviral infections and other viral infections that emerge or emerged from viruses requiring an acidic ph for infectivity hydroxychloroquine inhibits zika virus ns b-ns protease colchicine as a possible therapeutic option in covid- infection association between treatment with colchicine and improved survival in a single-centre cohort of adult hospitalised patients with covid- pneumonia and acute respiratory distress syndrome management of childhood-onset autoinflammatory diseases during the covid- pandemic immediate and long-term consequences of covid- infections for the development of neurological disease long-term pulmonary consequences of coronavirus disease (covid- ): what we know and what to expect thrombosis in coronavirus disease (covid- ) through the prism of virchow's triad adverse neuropsychiatric effects of antimalarial drugs frequency and clinical characteristics of hydroxychloroquine retinopathy in korean patients with rheumatologic diseases recommendations on screening for chloroquine and hydroxychloroquine retinopathy ( revision) safety management in treatment with antimalarials in rheumatology. interdisciplinary recommendations on the basis of a systematic literature review antimicrobials and qt prolongation hydroxychloroquine-induced auditory toxicity when there is a pandemic there is no time to waste: should we have hydroxychloroquine in our armoury against covid- infected patients? covid- and hydroxychloroquine: is the wonder drug failing? preventing and managing toxicities of high-dose methotrexate methotrexate-related toxicity in patients with rheumatoid arthritis and renal dysfunction methotrexateinduced pancytopenia: a case series of patients drug-induced interstitial lung disease update on colchicine colchicine intoxication in familial mediterranean fever patients using clarithromycin for the treatment of helicobacter pylori: a series of six patients safety and efficacy of intravenous colchicine in children with familial mediterranean fever efficacy and safety of long-term treatment with intravenous colchicine for familial mediterranean fever (fmf) refractory to oral colchicine colchicine poisoning: the dark side of an ancient drug adverse cardiovascular effects of antirheumatic drugs: implications for clinical practice and research safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -czcs y w authors: zhao, yang; aarnink, andrÉ j. a.; de jong, mart c. m.; groot koerkamp, peter w. g. title: airborne microorganisms from livestock production systems and their relation to dust date: - - journal: crit rev environ sci technol doi: . / . . sha: doc_id: cord_uid: czcs y w large amounts of airborne microorganisms are emitted from livestock production. these emitted microorganisms may associate with dust, and are suspected to pose a risk of airborne infection to humans in vicinity and to animals on other farms. however, the extent to which airborne transmission may play a role in the epidemic, and how dust acts as a carrier of microorganisms in the transmission processes is unknown. the authors present the current knowledge of the entire process of airborne transmission of microorganisms—from suspension and transportation until deposition and infection—and their relation to dust. the sampling and the mitigation techniques of airborne microorganisms and dust in livestock production systems are introduced as well. pathogenic microorganisms may occur in high concentrations in the air inside the livestock houses. along with ventilation, they can be emitted to the ambient environment and pose airborne infection risk to healthy animals on other farms and to humans living in the vicinity . y. zhao et al. the extent to which pathogenic microorganisms are transmitted to nearby recipients and cause disease spreading through the airborne route remains largely unclear. actually, airborne transmission only has been considered as a possible route in historical disease outbreaks in livestock production when the outbreaks could not be attributed to other known routes such as direct contact transmission or fecal-oral transmission (elbers et al., ; gloster et al., ) . attempts to link pathogen transmission (between farms) to prevalent wind directions-an apparent epidemiological proof of airborne transmission-have not been always successful, and the transmission is assumed to only be favored at picky atmospheric conditions mikkelsen et al., ) . all these facts induce doubts on the importance of the role of airborne transmission in disease outbreaks; however, no one can conclusively exclude this transmission route due to its potentially extensive and intensive impacts if truly involved in epidemics. given the lack of knowledge on airborne transmission it is important to stimulate relevant research in order to better understand what role it can play. here we define airborne transmission in livestock production as "an entire transmission process that involves pathogenic microorganisms releasing from the infected animal's excrement or secretion to air, transporting air, inhaled by a healthy animal, and eventually infecting the recipient." the airborne transmission of certain pathogenic microorganisms from animal to animal has been demonstrated in lab-scale experiments in which healthy animals separated physically but not aerially from infected animals became infected (berthelot-herault et al., ; brockmeier and lager, ) . furthermore, some microorganisms collected kilometers away from the source farm were found to be capable of infecting healthy animals intramuscularly or intratracheally (otake et al., ) . however, there is still uncertainty, because of the incomplete knowledge about the entire process of airborne transmission of microorganisms, from generation and transportation through inhalation and finally to infection (stark, ) . dust probably plays a role as the carrier of the microorganisms in the air. in , the importance of the relationship between airborne microorganisms and dust from livestock production systems was reviewed by muller and wieser ( ) . the authors separately described the indoor properties (source, concentration, and constitute) of airborne microorganisms and dust, and the dispersion in ambient air outdoors. since then, much research has been done on specific processes involved in the transmission of the airborne microorganisms and dust. however, we lack an integrated overview of and insight into all the processes involved in the airborne transmission of microorganisms in association with dust. the objective of this article is to review current knowledge on airborne microorganisms from production systems for typical livestock species (swine, poultry and cattle), and their relation to dust. specifically, in section , we identify the sources, species, size distributions, and concentrations of identifying the source of microorganisms and dust in livestock production systems helps to elucidate how airborne transmission is generated, and ultimately can help to develop and implement strategies that prevent such transmission from beginning (bull et al., ; cambra-lopez, ) . sources of dust in livestock production systems have been identified and assessed qualitatively and quantitatively (aarnink et al., ; donham and gustafson, ) . it is generally accepted that all dust sources are also sources of airborne microorganisms because these source materials somehow contain certain microbial species that may be generated together with dust. however, the source identification of airborne microorganisms has not yet been extensively investigated, and it is thought to be more complicated than the source identification of dust in at least two ways. the first way is associated with the complexity of microbial species in a source. a source material always contains a microbial flora composed of many different microbial species. the second way is associated with the dynamic viability of microorganisms in the generation process (milne et al., ) ; microorganisms may either decay or multiply in the source material. thus, the source identification for microorganisms should also be dynamic. animals shed microorganisms mainly by means of fecal excretion, which may contain large amount of microorganisms (letellier et al., ; pell, ) . consider two common zoonotic bacterial species, salmonella and escherichia, both have been found in feces: salmonella at a concentration of - log cfu g − feces (gray and fedorka-cray, ; himathongkham et al., ) and e. coli at - log cfu g − feces (mcgee et al., ; omisakin et al., ) . feces are also an important pathway for virus shedding from infected animals (fouchier et al., ) . a list of viral species that may be excreted by cattle was proposed by pell ( ) , including infectious bovine rhinotracheitis and foot-and-mouth disease (fmd) virus. many other viruses have been recovered from feces of other animal species, such as avian influenza a virus (webster et al., ) and newcastle disease virus (spradbrow et al., ) in poultry, and swine fever virus (van oirschot, ) , hepatitis e virus (de deus et al., ) , and porcine reproductive and respiratory syndrome virus (prrsv) (yoon et al., ) in pigs. the microorganisms in feces can become airborne when dried fecal particles are disturbed by air flow or animal activity. some studies have managed to identify feces as the source of airborne microorganisms. a study by duan et al. ( ) found the airborne e. coli strains inside and downwind from the pig houses were closely associated with those isolated from pig feces. water content binds particles in feces and prevents their suspension, so, the microorganisms in dry feces that have low water content become airborne more easily than microorganisms in fresh feces. the water content of fresh feces (or manure, when urine is not separated) is the range of - % (derikx et al., ) , depending on the animal species. under typical livestock housing environmental conditions, it may take hours or days to dry the feces to a water content less than %-the water content of airborne dust in livestock production systems (aarnink et al., ; zhao et al., ) . this means the microorganisms must undergo a latent period between the moment they are excreted in the feces and the moment they become airborne. during inhalation and exhalation the surface of the mucus in the respiratory tract is destabilized through an interplay between surface tension and viscous forces (edwards et al., ) . this can result in mucus's microorganisms to become airborne and expelled from the body via breathing, coughing, and sneezing. this source of airborne microorganisms is widely accepted in the human model of disease transmission, and pathogenic microorganisms have been frequently recovered from the exhaled aerosols (fabian et al., ; weber and stilianakis, ) . only a few studies have been carried out to directly detect the microorganisms in exhaled air of animals. by sampling exhaled air in masks placed over the heads of infected pigs, cho et al. ( ) recovered prrsv and hermann et al. ( ) recovered mycoplasma hyopneumoniae and bordetella bronchiseptica. in contrast, hermann et al. ( ) failed in recovering prrsv, porcine circovirus , swine influenza virus, and porcine respiratory coronavirus in the exhaled air of infected pigs, although they were found in the oral and nasal swabs. similarly, zhao et al. ( ) could not recover infectious brusal disease virus in the exhaled air of infected broilers. these results indicate that some microorganisms in animal respiratory tracts might not readily become suspended in the air or be expelled out of the body, which implies that this is perhaps not an major source of airborne microorganisms as compared to animal feces (zhao et al., ) . however, the reason the microorganisms may not be detected could be because the quantities of exhaled microorganisms were below the detection limit of the sampling devices. therefore, animal respiratory tracts can only be excluded as a source of airborne microorganisms until it has been incontrovertibly established that every single microorganism is detectable. organic materials such as feed may serve as carriers for variety of microorganisms. the microorganisms may originate from the soil and are transferred to standing crops by wind, rain, mechanical agitation, or insects (maciorowski et al., ) . both nonpathogenic and pathogenic microorganisms have been recovered from feed; analysis has revealed concentrations of gram-negative bacteria in feed as high as log cfu g − (hofacre et al., ) . these microorganisms can be disseminated together with feed particles during feeding (andersson et al., ; chang et al., ) ; the extent of dissemination depends greatly on how the feed is given (e.g., dry vs. wet feed delivery and powder vs. pellet feed delivery; pearson and sharples, ) . litter is a mixture of bedding materials (e.g., wood shavings, chopped straw, sawdust, and rice hulls) animal feces, dander, and feed (torok et al., ) . the provision of litter in livestock production systems may improve animal welfare by increasing the incidence of natural behaviors (appleby and hughes, ) , which, however, may result in more microorganisms being present in the air than in housing systems without litter (madelin and wathes, ; vucemilo et al., ) . the microorganisms arrive in litter during the harvesting and processing of the bedding material, and through animal excretion and secretion. the concentrations of aerobic bacteria in poultry litter range from to log cfu g − (lu et al., ; martin et al., ) . most of the bacteria in the poultry litter are gram-positive. gramnegative bacteria and mold account for a small fraction of the total microbial count, but due to the high concentration of the total microorganisms, their numbers can still be high in some cases (martin et al., ) . surprisingly, some pathogenic bacteria that are commonly recovered from animal feces (e.g., e. coli, salmonella, and campylobacter) are not always detectable in litter (lu et al., ) . the reason could be the less favorable microenvironment for microbial survival in the feces-bedding mixture than in feces alone. microorganisms, including globicatella sulfidofaciens, corynebacterium ammoniagenes, corynebacterium urealyticum, clostridium aminovalericum, arthrobacter sp., and denitrobacter permanens, which may be involved in degradation of wood and cycling of nitrogen and sulfur have been identified in poultry litter (lu et al., ) . other possible sources of airborne microorganisms in livestock houses include animal skin (baird-parker, ; gailiunas and cottral, ; kloos et al., ) and animal products (e.g., broken eggs, spoiled milk (de reu et al., ; donaldson et al., ; doyle, ; doyle and roman, ) , farm workers and visitors (newell and fearnley, ; nishiguchi et al., ) , and ambient air martin et al., ) . sources of airborne dust include feed, animal skin and feather debris, feces, litter, microorganisms, pollen, and insect parts (aarnink et al., ; donham et al., ) . the contribution of these sources to airborne dust varies, depending on the animal species and the housing system. heber et al. ( a) reported that the main source of airborne dust in pig houses was feed, which is consistent with the findings of donham et al. ( ) and aarnink et al. ( ) . muller and wieser ( ) found that - % of the airborne dust in floor layer systems with litter originated from the bedding materials in litter, while - % of the airborne dust in layer systems with battery system originated from feedstuff (table ). in floor systems with wood shavings as litter for three-week old broilers, aarnink et al. ( ) found that airborne dust mainly (> %) originated from down feathers and urine components. the contribution of feed to the airborne dust largely depends on its composition and how it has been processed (pearson and sharples, m; e.g., crumbles or pellets). the contribution of feces is probably related to the housing system (e.g., with or without litter [straw bedding vs. liquid manure]). table lists the main sources of dust and gives an estimation of their contributions. a large fraction of the airborne microorganisms in livestock production systems are bacteria, of which the most dominant are gram-positive bacteria, accounting for approximately % of the bacterial flora (zucker et al., ) . the most common species of these gram-positive bacteria are staphylococcus, streptococcus, and enterococci (clark et al., ; hartung, ; matkovic et al., ) . the gram-negative bacteria account for only a small fraction of airborne bacteria (zucker et al., ) . bakutis et al. ( ) reported that in terms of the total bacterial count, the proportion of gramnegative bacteria was approximately % in cattle houses, . % in pig houses, and . % in poultry houses. zucker et al. ( ) found that the airborne gram-negative bacteria in pig and cattle houses were aerobic and include enterobacteriaceae, pseudomonadaceae, and neisseriaceae; no culturable obligate anaerobic gram-negative bacteria were isolated. possible reasons for the smaller proportion of airborne gram-negative bacteria in livestock production systems are that their excretion by animals is less than their counterparts and these bacteria are more vulnerable to environmental stress such as oxidation, radiation, and dehydration, probably because of their thinner cell walls (pal et al., ; theunissen et al., ) . the proportion of fungi, molds, and yeasts in the airborne microbial flora in animal houses is low (hartung, ; lee et al., ) . the most frequently reported fungi in poultry, pig, and dairy houses are aspergillus sp., alternaria sp., cladosporium sp., penicillium sp., fusarium sp., scopulariopsis sp., and yeast (chang et al., ; cormier et al., ; martin et al., ; matkovic et al., ; vittal and rasool, ; wilson et al., ) . the size of an airborne particle determines its transportation, sedimentation, and resuspension, as well as its deposition in the respiratory tracts of recipients. investigations of the size distribution of microorganisms and dust in livestock production systems may provide a useful overview of their quantitative importance, indicate the health risk for human and animals, and facilitate the establishment and evaluation of control techniques. according to interests by different scientific sectors, sizes of airborne particles are differently categorized. for concerns in occupational health, particle sizes are categorized into three categories: inhalable (< μm), thoracic (< μm), and respirable (< μm or μm; cambra-lopez et al., ; curtis et al., ; madelin and wathes, ; zhang, ) . in environmental science, recent research is increasingly focusing on dust with aerodynamic diameter smaller than μm (pm ) and . μm (pm . ). the size distribution of airborne dust has been expressed either in mass or in counts. zhao et al. ( a) found that in three pig houses about - % of the airborne bacteria were in the nonrespirable range ( figure ) . a similar result was reported by curtis et al. ( ) : nonrespirable bacteria accounted for approximately - % of the airborne bacteria in pig houses. the size distribution of microorganisms in poultry houses depends on the type of housing system. in broiler rooms with wood-shaving litter, most of the bacteria were nonrespirable in the full life cycle of broilers (eight weeks); a similar distribution pattern was found in broiler rooms with raised netting floor only after the birds were older than six weeks. when the birds were two to five weeks old, the proportions of airborne respirable and nonrespirable bacteria were similar (madelin and wathes, ) . heber et al. ( a) reported that nonrespirable particles (> μm) in pig houses accounted for more than % in mass, but less than % in terms of count. expressed as percentage of total dust, lai et al. ( ) found the mass of pm was - % in pig houses, - % in poultry houses, and % in cattle houses. in all three types of house, pm count was more than % of the total dust. the equivalent figures for pm . mass were - % in pig houses, - % in poultry houses ( ), and % in cattle houses. for pm counts the figures were - % in pig houses, - % in poultry houses and % in cattle houses. the difference in the mass and numeric size distribution is caused by the fact that small dust particles contribute little to mass. that more microorganisms and less dust particles are found in the nonrespirable range indicates that a nonrespirable dust particle is more likely to be loaded with microorganisms than a respirable one. this is a reasonable hypothesis, because the larger a particle is, the greater the chance it may contain microorganisms. nowadays, the size distribution of airborne microorganisms is normally determined with the andersen stage impactor (andersen, ; zhao et al., a) . this sampler actually gives the counts of particles (in seven different size ranges) that contain microorganisms. in some cases, for instance a biosecurity assessment for occupational health, it is more important to quantify the individual microorganism in the collected particles rather than the microbial-containing particles themselves, because the former will give a better idea of the risk of infection. for this purpose, predetachment of microorganisms from dust particles or visually microbiological counting techniques (yamaguchi et al., ) may be required. concentrations of airborne microorganisms and dust in livestock production systems have been investigated in previous studies (kim et al., ; radon et al., ; zhao et al., a) . due to the huge spatial and temporal variations in microorganism and dust concentrations and the difference in sampling techniques used, it is difficult to compare the data between studies. so far, the studies by seedorf et al. ( ) and takai et al. ( ) still provide the most representative concentration data on microorganisms and dust in livestock production systems, and sampling methods were detailed in the publications by phillips et al. ( ) and wathes et al. ( ) . these data are summarized in table , together with the pm and pm . concentration measured by lai et al. ( ) . for microorganisms, the highest concentrations of airborne bacteria and fungi were found in broiler houses. the concentrations found in layer, pig and cattle houses were lower than those in broiler houses, but still much higher than those in ambient air (wang et al., ) . for dust, the highest dust concentrations were found in poultry houses, and the lowest dust concentrations were in cattle houses. the concentrations of airborne microorganisms and dust in animal houses are affected by animal, housing system and management. in this section, these factors are discussed separately, but one should realize that these factors always interactively affect the concentration because they are intercorrelated. for instance, animal activity is associated with animal age, weight, and light schedule, and ventilation rate is affected by outdoor and set-point temperature, humidity, and animal species and age. the animal factor can be further detailed into subfactors such as age, weight, activity, and stocking density. the concentrations of airborne microorganisms and dust generally increase concomitantly with animal age and weight predicala et al., ; yoder and van wicklen, ) . however, an inverse relationship has also been found. madelin and wathes ( ) found a decrease of microorganisms and dust concentrations in the late fattening period of broilers. a similar result was reported by saleh et al. ( ) . the decrease in concentration of microorganisms and dust is probably because the older broilers occupied all the floor space, which limited their activity. in general, higher concentrations of bacteria, fungi, and dust are measured when the animals are more active, as can be inferred from the finding that their concentrations were higher in day time than at night takai et al., ) . image and infrared technology allows animal activity to be automatically detected (gloster et al., ; pedersen and pedersen, ) . using an infrared detector, haeussermann et al. ( ) demonstrated that the indoor concentrations of pm were associated with pig activity. a similar study by heber et al. ( ) showed that both total dust and pm were correlated with the pig activity. however, gloster et al. ( ) failed to establish the correlation between the concentration of airborne fmd virus and pig activity quantified by taking sequential pictures. the reason is not clear, but the authors explained that the virus production appears to be more closely associated with other factors (e.g., physiological symptoms; gloster et al., ) . hardly any other information is available on the relation between quantified animal activity and concentrations of microorganisms. compared to a cage system, an aviary system contained higher concentrations of microorganisms (de reu et al., ) and dust (appleby and hughes, ) . this is because in an aviary system, laying hens have more scopes for moving horizontally and vertically and perform dust bathing behavior in the litter. housing systems with bedded floors caused more air quality problems, although such housing systems are generally thought to be more beneficial for animal welfare (kim et al., ; madelin and wathes, ; quarles et al., ) . the type of bedding material also affects the concentration of microorganisms in the air. for instance, in broiler houses, straw bedding released less bacteria in the air than wood shavings did (banhazi et al., a) . the authors argued that this was probably because wood shavings provided a better microenvironment for bacteria viability and multiplication. comparisons of dust concentrations between natural and mechanical ventilation systems showed that with mechanical ventilation, less respirable (phillips, ) and total dust was found in pig houses (chiba et al., ) and there was less total dust in turkey houses (janni and redig, ) . by contrast, concentrations of total bacteria and fungi were lower in naturally ventilated pig houses without bedding materials (deep-pit manure system with slats, and manure removal system by scraper) than in mechanically ventilated houses. the contradictory results found for the effect of type of ventilation (natural or mechanical) on microorganisms and dust are not fully understood, but it seems likely that the situations (including the management) of the ventilation systems vary between the different studies, making the data less comparable. feed management may play an important role in dust concentration in livestock production systems. previous studies have shown that dust concentrations were reduced by giving pelleted feed rather than powdered feed, wet feed rather than dry feed, and coated feed rather than uncoated feed (clark and mcquitty, ; pearson and sharples, ; zeitler et al., ) . the effect of feeding management on airborne microorganism concentration has not been extensively studied. maintaining good hygiene in livestock production systems may help to improve the air quality with respect to microorganisms and dust. for instance, cleaning (e.g., removing litter, scrubbing surfaces, and disinfecting the house) between two production circles may reduce both airborne microorganisms and dust (banhazi et al., a) . investigations of the relation between hygiene and air quality have found they are not always positively correlated. duchaine et al. ( ) reported that a housing system that appeared cleaner contained more airborne bacteria than one that appeared dirtier. the probable explanation is that houses with more settled dust on the surfaces are more readily ranked as dirtier, but dust accumulated on surfaces is not an appropriate indicator of the concentration of bacteria in the air. ventilation management (e.g., adjusting the rate at which indoor air is exchanged with outdoor air by mechanical ventilation systems) is to control the temperature and other aerial variables such as humidity and gas concentrations inside livestock houses. previous studies have shown that lower concentrations of microorganisms and dust can be achieved by increasing the ventilation rate in livestock production systems (duchaine et al., ; hinz and linke, ; kim et al., b) . however, a nonsignificant correlation between these variables has also been reported in livestock production systems (banhazi et al., b; seedorf et al., ) . the probable reason for these contradictory findings is that ventilation affects the concentration of microorganisms and dust in two ways: by exhausting airborne microorganisms and dust to outdoors through air exchange, thereby reducing their indoor concentrations, and by producing airflow turbulence above surfaces, agitating the particles and causing them to suspend in the air, thus compromising the removal effect. smaller airborne particles seem to be more effectively removed from livestock houses by ventilation than bigger particles (kuehn, ) because they are readily transported in the air streams. a high relative humidity in the air reduced the dust concentration in livestock production systems (guarino et al., ) . in humid environments, the dust particles bind to the surface and are not easily suspended, and those in the air aggregate and to settle faster (heber et al., b; takai et al., ) . in contrast, humidity seems not to affect the concentrations of total and gram-negative bacteria (attwood et al., ; banhazi et al., b; nicks et al., ) . a high humidity favors survival and/or multiplication of dehydration-sensitive microbial species in the sources (de rezende et al., ) , which might trade off the physical settlement of airborne microorganisms. experimental validation of this hypothesis is of importance, because it may strengthen the understanding of the interaction between airborne microorganism concentration and air humidity. this will help to formulate effective air humidification strategies for reducing both, dust and microorganisms, instead of achieving one by compromising the other. this information will also be valuable for understanding the bioenvironment in livestock houses where air humidification (e.g., water spray and evaporation cooling pad) is used to ameliorate animal heat stress in summer time (brown-brandl et al., ; tao and xin, ) . indoor temperature management is well regulated for the poultry and pig industries, to optimize productivity. al homidan et al. ( ) reported that the total dust in broiler rooms increased when the temperature was set • c above the recommended level. the correlation between dust concentration and temperature reverses from positive to negative when the temperature is extremely high, apparently because animal activity decreases at high temperature and thus fewer particles from surfaces are disturbed and suspended (donkoh, ; guarino et al., ; wylie et al., ) . as well, a high temperature triggers a series of events that may affect the dust concentrations in livestock production systems, such as increasing the ventilation rate and activating wet cooling systems (simmons and lott, ) . although some researchers (banhazi et al., a) have suggested there is a relation between the concentration of microorganisms and temperature, little information is available so far. the decay of microorganisms and dust is a parameter that cannot be ignored in empirical and theoretical models of airborne transmission (lighthart and frisch, ; yu et al., ) ; it may help when assessing health impacts from exposure. the term "decay" encompasses both physical and biological means. physical decay is the physical elimination of a particle from the air by means of a series of processes such as gravitational sedimentation, impaction and electrostatic precipitation; biological decay is the loss of biological activity of an airborne microorganism owing to loss of enzyme activities or denaturing of membrane phospholipids, proteins or nucleic acids (cox, ) . for biological decay, the situation may be more complicated in some cases when the microorganisms go into a viable but nonculturable (mckay, ; oliver, ; trevors, ) or dormancy state (locey, ) . it is clear that physical decay applies to the elimination of dust particles, whereas both physical and biological decay apply to airborne microorganisms. mechanisms affecting physical decay of particles are listed in table . in reality, these mechanisms may have collective effect on a particle, but the dominant mechanism varies mainly with particle size (e.g., decay of larger particles is prone to drive by gravimetric sedimentation whereas decay of smaller particles is mainly determined by diffusion (abadie et al., ; he et al., ) . physical decay can be quantified by the rate of deposition (deposition rate loss coefficient or deposition velocity; deshpande et al., ) . lai ( ) reviewed the deposition rate for particles ranging from . to μm and found it had a u-shaped pattern. the lowest deposition rate was found for particles ranging from . to μm because particles in this range were less affected by either sedimentation or diffusion than the mean transport of a particle by the mean motion of the atmosphere, and occurs when the spatial gradient is nonzero and the particle is transported along the mean wind (baldocchi et al., ) brownian diffusion the process of mass transfer of particles brought about by a random molecular motion (brownian motion) and associated with a concentration gradient (vaithiyalingam et al., ) thermophoresis the motion of a particle under the influence of a temperature gradient (langer and holcombe, ) gravitational sedimentation the separation of dispersed particles from gaseous phase under action of gravity (wunsch, ) impaction the deposition of particles due to their momentum causing them to deviate from airflow streamlines and impacting at bifurcations (katz et al., ) electrostatic precipitation the use of an electrostatic field for precipitating or removing charged particles from a gas flow in which the particles are carried (shen and pereira, ) [a] some definitions in this table that were originally for gases or molecules in nonaerial environments have been modified to make them suitable for describing the associating mechanisms of microorganisms and dust in the air. their counterparts do. besides particle size, deposition rate is affected by other factors such as room furnishing and air speed. thatcher et al. ( ) found increasing surface area (provide more furniture) and air speed (by means of elevating ventilation rate) increased the deposition rate of particles ( . - μm) in an experimental room. the biological decay of airborne microorganisms has been expressed in different ways (e.g., decay rate [or death rate], survival, or half-life). the decay rate is the decrease in concentration of viable microorganisms over time. a proportionality constant (k) indicates the extent of decay rate, and is shown in equation , where c o is the initial concentration of airborne microorganisms, c t is the concentration of microorganisms at time t after initial (phillips et al., ) . the survival represents the percentage of viable microorganisms left at a certain moment vis-à-vis the initial microbial count (wu, ). the half-life, t / , is the time taken for the concentration of viable microorganisms in the air to decrease by half (see equation ). previous studies showed that the biological decay of airborne microorganisms was species-dependent and was determined by many external factors, such as humidity, oxygen concentration, temperature, ozone concentration, radiation (uv, γ -ray, x-ray), air ions, and air pollutants (co, so , and no x ; benbough, ; lighthart, ) . for long distance airborne transmission between farms, microorganisms may be exposed to unfavorable environmental conditions that induce death or dormancy of microorganisms (lennon and jones, ; locey, ; wu et al., ) . in this section we present an introduction of several environmental factors and their working mechanisms on biological decay of microorganisms. the effect of humidity on the biological decay of airborne microorganisms has been investigated since the s. in the early studies, the measure most used for humidity was relative humidity (rh; the ratio of the actual water vapor pressure of the air to the water vapor pressure of saturated air at a certain temperature). the results of these studies showed that different microorganisms were prone to decay either at low rh (lighthart, ) , at median rh (wright et al., b) , or at higher rh (songer, ; theunissen et al., ) . more recently, a few studies have used absolute humidity (ah; the actual water content of the air) as another measure of humidity. for instance, shaman and kohn ( ) reported that the survival of airborne influenza virus was more significantly constrained by ah than by rh. the authors argued that rh is a meaningful physical quantity and for certain organisms may affect biological response; however, the ah can be of greater biological significance for many organisms. some studies reported significant effects of other measures of humidity, such as evaporation potential (ep; the difference between actual water vapor content in the air and the water vapor content in saturated air at the same temperature), on microbial survival (zhao et al., ) . although a bunch of studies have been carried out, it is not yet fully understood how humidity influences microbial decay. temperature profoundly affects the biological decay of airborne microorganisms. in general, the higher the ambient temperature is, the faster the microorganisms decay. for instance, the decay rate of flavobacterium sp. is . log min − at - to • c, but increases to . log min − at to • c (ehrlich et al., a) . a faster decay at higher temperature has also been reported for other microbial species, such as e. coli, s. marcescens (ehrlich et al., b) , and newcastle disease virus (kournikakis et al., ) . prescott et al. ( ) have stated that high temperature may damage microorganisms either by denaturing the enzymes, transport carriers, and other proteins, or by melting and disintegrating the lipid bilayer, or both. the ambient environment is full of various types of radiation, including uv ( - nm) and visible light ( - nm), which may inactivate the microorganisms. the wavelength of uv between nm and nm has the highest energy, but this type of uv is blocked by normal dioxygen in air and cannot reach the ground. the rest of uv radiation, from low to high energy, is categorized as uv-a ( - nm), uv-b ( - nm), and uv-c ( - nm), of which uv-c is considered to be the most germicidal. the uv-c wavelength between - nm can be effectively absorbed by microbial genetic material, and is the uv wavelength most lethal to microorganisms (keyser et al., ) . the germicidal effect of uv-c light on bacteria and viruses is primarily due to the formation of pyrimidine (thymine and cytosine) dimers that inhibit the replication and function of genetic material (giese and darby, ; prescott et al., ) . bacteria decay more readily under uv radiation than rna viruses (harris et al., ; hijnen et al., ) . the probable reason for this is that the thymine of bacterial dna is more vulnerable to dimerization induced by uv than the uracil of viral rna. the mechanism whereby uv-a and uv-b (also called near-uv) inactivates microorganisms is suspected to be the breaks in strands of genetic materials that are induced by the uv itself and toxic tryptophan photoproducts (prescott et al., ) . visible light may also damage microorganisms. microbial pigments become excited when they absorb light energy, and are transferred to o , generating singlet oxygen ( o ), which is a highly reactive oxidant (valduga et al., ) . as a self-protecting mechanism, some microorganisms may possess carotenoids that can absorb the excitation energy and reduce the formation of singlet oxygen, thus preventing cells from being damaged by light radiation (mccambridge and mcmeekin, ) . oxygen may accept electrons forming other toxic derivatives such as superoxide radical, hydrogen peroxide, and hydroxyl radical, which may easily destroy the cellular constituents. many aerobic microorganisms contain enzymes such as superoxide dismutase and catalase, which protect the cell against oxidation by the derivatives; all the strictly anaerobic microorganisms lack these enzymes (prescott et al., ) . the effect of oxygen level on decay of airborne serratia marcescens uk and e. coli b was studied by cox ( ) , who reported that oxygen was toxic for these microorganisms only when rh was lower than %. the toxicity increased with oxygen concentrations up to %; higher concentrations produced no additional toxicity. this finding was generally in agreement with other similar studies (benbough, ; hess, ) . viruses seem to be less sensitive to oxygen than bacteria. the decay of airborne viruses that included semliki forest virus, langat virus, t coliphage, poliovirus, and encephalomyocarditis virus was no different whether they were aerosolized in the air or in nitrogen (benbough, ; de jong et al., ). bacteria exposed to ozone (o ) may be inactivated due to damage to the cell surface (giese and christensen, ; scott and lesher, ) and destruction of the intracellular enzymes, protein and genetic material (barron, ; ingram and haines, ; kim et al., ) . ozone may damage the viral nucleic acids of viruses and alter the polypeptide chains of the viral protein coat kim et al., ; roy et al., ) . investigations of the ozone effect on decay of airborne microorganisms showed that fungi seemed more resistant to ozone than bacteria; gram-positive bacteria were more resistant than gram-negative ones (heindel et al., ; kowalski et al., ) . ozone alone can be toxic to airborne microorganisms; however, its toxicity is enhanced when ozone reacts with compounds in the ambient air, known as open air factors (oaf). studies have shown that mixture of ozone with olefins druett and packman, ) and ozone with negative air ions (fan et al., ) are more toxic to airborne microorganisms than ozone alone. the dust particles to which microorganisms adhere may protect them from biological decay. when microorganisms are carried by the dust, they may suffer less radiation and exposure to toxic gas, and less fluctuation in micro-climate (milling et al., ) . it has been found that individual bacteria are effectively inactivated by ozone; however, when these bacteria were covered with a coating of organic matter, as a bio aerosols, ozone in permissible concentration had no effect (elford and van den ende, ) . as well as providing physical protection, the composition of dust particles might give bio-chemical support to microorganisms. in order to metabolize, microorganisms require carbon, oxygen, nitrogen, phosphorus, sulfur, and other elements (maus et al., ) . compounds containing these elements are abundant in airborne dust from livestock production systems (aarnink et al., ; muller and wieser, ) . an interesting hypothesis is that microbial decay in particles differs, depending on the composition of the particles. actually, in some laboratory experiments, it appears that decay of microorganisms differs as they are aerosolized from liquid suspensions with different chemical compounds. (benbough, ; hess, ) . there have been extensive studies on the biological decay of microorganisms at different temperature and rh levels, using aerosol experiments. in these experiments, microorganisms were aerosolized in airspace that was sampled at certain intervals. the biological decay was indicated by the amounts of collected microorganisms at different sampling moments. the results of selected studies are summarized in tables and . the selection of references follows the procedures and screening criteria as below: first, all references related to biological decay of any species of airborne microorganisms were searched in literature databases (web of science, scopus, google scholar); second, only references in which biological decay was investigated by excluding confounding effect of physical decay (e.g., using inert tracers or labeled microorganisms) were selected; third, due to the fact that some microorganisms showed a biphase biological decay (with a fast initial decay rate in the first few seconds or minutes after aerosolization, followed by a slow secondary decay) and the airborne transmission concerned is long distance and time (brankston et al., ; cox, ; songer, ; webb, ) , only the secondary biological decay is presented in the tables. the tables show the highest biological decay at the least favorable temperature/rh, and the lowest biological decay at the most favorable temperature/rh. the values of biological decay have been presented in three ways: decay rate, survival and half-life. by reviewing the results in tables and , it proofs that the biological decay largely varies between the microbial species and is profoundly affected by temperature and rh. most of the previous studies have used wet aerosolization in which microbial suspensions were aerosolized. wet aerosolization does mimic the fate of microorganisms expelled from animal respiratory tracts in wet aerosols. however, after they have been generated, the large wet aerosols containing microorganisms settle on surfaces and the small ones shrink into dry nuclei; both processes are very fast, taking only seconds to minutes (kincaid and longley, ; sun and ji, ; wells, ) . therefore, the microorganisms in wet aerosols can only be transported over short distances and in brackets is the time span (in minutes) between which survival rate corresponds to. h = highest biological decay of airborne microorganisms (i.e., worst survival and shortest half-life time); l = lowest biological decay of airborne microorganisms (i.e., optimal survival and longest half-life time). [b] calculated by dividing the amount of virus collected in the last air sample ( min) by that in the first air sample ( min). [c] in brackets is the time span (in minutes) between which survival rate corresponds to. induce infections in limited areas (brankston et al., ) . for microorganisms released from dry sources such as feces and litter, which may be more closely associated with long distance transmission, dry aerosolization is recommended. dry aerosolization may give a picture of the biological decay of microorganisms that differs from the decay in wet aerosolization because the microorganisms may suffer either dehydration stress at low ambient rh or rehydration at high ambient rh (cox, ). according to heyder et al. ( ) , the probability of deposition will be different for each particle even if all the particles in the air inhaled in one breath are identical, because the inhaled air with particles penetrates the respiratory tract to different depths where it remains for different periods of time, and because of the stochastic nature of particle transport. therefore, particle deposition (in the respiratory tract) refers to the "mean probability" of an inspired particle being collected on airway surfaces. particle deposition in the respiratory tract depends on particle characteristics (e.g., size, shape, density) and breathing pattern (e.g., nasal/oral breath, respiratory flow rate, cycle period), and is commonly expressed as a function of particle size. particle deposition in the human respiratory tract has been well documented (brown et al., ; james et al., ; lippmann et al., ) . in principle, the deposition of particles is governed by the mechanisms of diffusion for particles < . μm, or by diffusion and sedimentation for . - μm particles, or by sedimentation and impaction for particles > μm (heyder, ) . on the basis of previous experimental studies, heyder et al. ( ) developed a semiempirical deposition model for particles ranging from . μm to μm. the model-simulated deposition pattern for slow inspiration over a long period for both oral and nasal breathing is shown in figure . the deposition has been shown for three regions of the respiratory tract-extra-thoracic, bronchial, and alveolar-based on how far down the tract particles may be deposited. in addition, total deposition is given (the sum of the deposition in the three regions). it can be seen that the total deposition is least for particles of . - μm, and that the deposition increases as the size of small particles (< . μm) decreases, and the size of the larger particles (> μm) increases. particles larger than μm are mainly deposited in the extrathoracic region, and cannot penetrate the alveolar region during slow and fast oral and nasal inspiration. the deposition in the human respiratory tract cannot be extrapolated to livestock because of the discrepancy in morphology of human and animal respiratory systems (corbanie et al., ) . particle deposition in guinea pig head, trachea, and lungs was investigated by harper and morton ( ) . they found an increasing regional deposition in guinea pig head for larger particles: . % of the μm particles were deposited in the guinea pig head region, compared with . % of the μm particles. the reverse was true for lung deposition: . % of μm particles and . % of μm were deposited in this region. the particle size most deposited in the trachea was . μm. a model of particle deposition in the guinea pig respiratory tract was established by schreider and hutchens ( ) , who found that % of unit density particles of μm or more could be deposited in the nasopharyngeal-tracheobronchial region (figure ). the lowest deposition was % for a particle size of . μm. about % of particles ranging from . to μm could be deposited in the pulmonary region. however, this model was compromised by several assumptions that were made by the authors (e.g., laminar airflow in the respiratory tract, equal expansion of all lobes and alveoli, and complete mixing of the particles in the alveoli. hayter and besch ( ) investigated the regional deposition of five particle sizes ( . , . , . , . , and . to μm) in chicken. the particles deposited in the head and anterior trachea were in the . - μm size range, those deposited in the lung and posterior air sacs were . μm size, and those deposited in the caudal regions of the birds were in the size classes . and . μm. those of size . μm were deposited mainly figure . deposition of unit density spherical particles in the respiratory tract of guinea pig at a tidal volume of . cm and a respiratory rate of breaths min − . np-tb: nasopharyngeal-tracheobronchial region. p = pulmonary region. adapted from schreider and hutchens ( ). in upper airways. these early data of particle deposition in chicken airways are suspected to be compromised by the use of anesthetized chickens, because anesthesia alters the animals' breathing pattern. corbanie et al. ( ) investigated the deposition of particles in a wider range ( , , , , and μm) in unanaesthetized chickens of three ages. unlike the definition of deposition that was proposed by heyder et al. ( ) , corbanie et al. ( ) defined deposition as the percentage of particles deposited in a particular region of the respiratory tract among those in the entire tract. they found that particles larger than μm were too large to be deposited in the lungs and air sacs in -and -week-old chickens, as low percentages of particles were recovered in these regions (figure ). for -day-old chicks, however, the particle deposition in lungs and air sacs was independent of particle size and even particles of μm were deposited in the lower airway, possibly due to the chicks having a different breathing pattern than older chickens. the deposition pattern of monodispersed particles ( . μm) in calf airways was studied by jones et al. ( ) . they found those particles were preferentially deposited in the trachea and major bronchi. inhalation of pathogens may result in infection of recipients. infection is likely to fit a single-hit model, which means that one pathogenic microorganism may trigger an infection in a recipient. from this point of view, the infective dose (id; or occurrence of infection) is therefore related to the probability that a recipient becomes infected after taking in a certain dose of pathogenic microorganisms, following a poisson distribution. the id of pathogenic microorganisms to human and animals has generally been expressed in two ways. in one way, id is expressed as the number of infected recipients out of a population after a dose of microorganisms has been administered. the other way is to determine the microbial concentrations required to infect % of a population (id ). table lists the id of several pathogenic microorganisms. the id of the same microorganism varies, depending on the recipient animal species. for instance, a lower id of fmd virus is needed to infect sheep and cattle than to infect a pigs (alexandersen and donaldson, ; donaldson et al., ; gibson and donaldson, ) . it can be also seen that a certain dose of a microorganism is not always capable of infecting all recipients, probably because of a difference in the resistance of individual recipients (due to e.g., age, breed; roy, ) . furthermore, the route by which the microorganisms are administered may also be responsible for variation in id (cafruny and hovinen, ; zimmerman et al., ) . in previous studies, the administration routes were either nasally or orally, or via aerosols, and these reflect different deposition situations and regions for microorganisms in the respiratory tract. nasal administration simulates infection because larger microbial particles are deposited in the upper airways, the oral ministration may simulate oral breathing, and the aerosol administration simulates infection because smaller microbial particles are deposited in deeper airways. it was reported that the microorganisms had preferential sites for multiplication and infection because of the complex vulnerability of regions in respiratory tracts (cafruny and hovinen, ; druett et al., ; druett et al., ) . this being so, infection occurs more readily when the microorganisms are administered to the more vulnerable region. baskerville ( ) summarized the preferred infection regions of some microorganisms to animals by categorizing nose, pharynx, and tonsils as the upper respiratory tract, and the trachea, bronchi and bronchioles, and alveoli as the lower respiratory tract (table ) . sampling protocols for dust in ambient air have been legislated by the u.s. environmental protection agency ( ) and the european committee for standardization (european commission, . nowadays, the official sampling protocols focus increasingly on small particles (e.g., pm and pm . ) because these have the potential to be suspended for longer time, transported for longer distance, and deposited in the lower respiratory tract, thus are more hazardous to human and animal health. to ensure unbiased sampling, these protocols specify many details, such as sampling duration, type of sampler, and sample handling. the protocols for ambient air may not be directly applicable for dust sampling in livestock production systems where the dust concentrations are much higher than those in ambient air. in addition, to date there is no gold standard for sampling airborne microorganisms. given that the sampling of microorganisms and dust in livestock production systems is increasingly being performed for assessing the biosecurity of air environments and for evaluating mitigation techniques, the sampling protocol must be well designed in order to assure reliable data. subsequently, sampling strategies and samplers for airborne microorganisms and dust are highlighted, taking into consideration their sampling in livestock production systems. isokinetic sampling is the ideal sampling method, because it has been devised to sample the true numbers of particles in the air. such sampling can be achieved if the sampler inlet is in alignment with and facing the direction of air flow and if the air velocity within the sampler is the same as the ambient air velocity (zhang, ) . however, true isokinetic sampling is impossible in practice due to variations in the surrounding air flow pattern (air direction and velocity) and the limitations of some samplers (liu and pui, ) . as a concession, the current legislation aims to reduce the sampling bias in nonisokinetic samplings by stipulating the range of conditions under which the samplings may be performed. the sampling location should be chosen bearing in mind the research purpose. when human health is of concern, sampling should be carried out near the human breathing zone. one option is to fit a portable sampler on a worker's body at a height of - cm above floor level and within a radius of cm around the mouth ouellette et al., ) . there are difficulties in doing the same with animals, therefore stationary samplers in their breathing zones are recommended. the recommended level of the breathing zone is - cm above floor level for pigs, - cm for poultry, and shoulder height for cattle (kim et al., a; topisirovic, ; zhao et al., d) . for growing animals these figures should be adjusted according to the animal height at a certain age. when emissions of microorganisms and dust are of interest, the best sampling location is in or near the air outlet. care should be taken not to place the samplers at a location where the air speed is too high, because the efficiency of the sampler (the ratio of the aerial pollutant concentration calculated from the air samples to the true concentration in the air) may drift far from % (grinshpun et al., ; hofschreuder et al., ) . for ambient air, the daily and annual thresholds for pm have been set at and μg m − , respectively (european commission, ) ; the annual threshold for pm . has been set at μg m − (european commission, ). to assess the dust concentration, the sampling period is generally hr. the sampling duration for ambient air may also be applied when sampling dust in animal houses to obtain daily mean concentrations. for studies collecting information on dust fluctuations during the day, continuous samplings for short periods should be performed. this kind of sampling can be achieved by interval sampling or sampling with real-time optical samplers. due to the lack of a standard, the sampling duration for airborne microorganisms in different studies varies, but is normally less than hr. the duration is determined by taking account of the estimated concentrations of microorganisms and the characteristics of the sampler in use. the sampling duration can be set at a short period (a few minutes) when the total microorganism is abundant in livestock houses. when the microorganism of interest is sporadically present, the period should be set long enough to collect enough microorganisms for further quantification analysis. some samplers have not been designed to be used for long sampling duration. for instance, severe evaporation of liquid medium in all glass impinger (agi- ) occurs when the sampling period is long and this may affect its sampling efficiency. the recommended maximum duration of the sampling period for the agi- is min. impactors may easily become overloaded when samples are taken in livestock houses (thorne et al., ) , therefore, the sampling duration is limited to minutes or even to seconds. filtration method would not encounter the problems (evaporation of sampling liquid and overloading) of impingers and impactors; however, long sampling duration by filtration may not benefit in microorganism collection because of the biological decay of microorganisms owing to dehydration (griffin et al., ; wang et al., ) . in order to collect detectable amounts of microorganisms and to get representative samples over a longer period, some high-volume and durable samplers that can be continuously operated over hours have been developed and applied, including omni- and high-volume impinger (griffin et al., ; kesavan and schepers, ) . other aspects in sampling and detection strategies include practical and economic considerations. the portability of the instrument and its ancillaries (e.g., weight and dimensions) are factors that should be considered. a highquality pump is required for dust filtration sampling, and it should be able to provide a constant airflow when ambient temperature changes or when pressure differences increases because of dust accumulation on the filter. the culture-dependent techniques for microorganism numeration are time consuming and particularly expensive; culture-independent techniques such as quantitative pcr provide possibilities for rapid and accurate analysis which is critical for biological emergency (postollec et al., ; ståhl et al., ) , but its incapability to distinguish between viable and dead microorganisms needs to be considered (yáñez et al., ). current samplers for airborne microorganisms are generally based on one of three main principles (i.e., impaction, impingement, or filtration; table ). these different sampling principles have their advantages and disadvantages. samplers using the impaction principle (e.g., andersen six stage impactor) can be used to distinguish the microorganisms according to their sizes (andersen, ) . in addition, the bacteria impacted on agar plates may be directly incubated for viable counts. however, its susceptibility to overloading limits its sampling in livestock houses to short periods, which may result in nonrepresentative samples. the problem of overloading can be overcome by using samplers with the impingement principle, because the liquid samples may be decimally diluted and analyzed after sampling. a disadvantage of this sampler is that it may not be able to sample for a long time due to evaporation of the collection liquid (lin et al., ) . filtration is a user-friendly method in a practical situation, but not suitable for sampling microorganisms that are vulnerable to dehydration stress. a sampler with known efficiency is a prerequisite for a reliable evaluation of the microbial concentration. the efficiency of a bioaerosol sampler includes physical and biological efficiency. the physical efficiency describes how well nonviable airborne particles are aspirated by the device's inlet and transported to the collection medium, referred to as inlet sampling efficiency, and how well the bioaerosol sampler retains these particles in its medium, referred to as collection efficiency (griffiths and stewart, ; nevalainen et al., ) . for particles in the range from to μm and an airflow rate between and cm s − , the inlet sampling efficiency of the andersen six stage impactor is - % when the opening faces in direction of the air flow and - % when it is oriented perpendicularly to the horizontal aerosol flow (grinshpun et al., ) . the inlet efficiency of the agi- for particles of μm is close to %, but it is reduced to - % for μm particles and to - % for μm particles (grinshpun et al., ) . when the % collection efficiency of the andersen six stage impactor and agi- was investigated it was found that the andersen six stage impactor has % collection efficiency for . - . μm particles at the first stage and . - . μm particles for the last stage (andersen, ; nevalainen et al., ) . the % collection efficiency of agi- was for particles of . μm (nevalainen et al., ) . filters vary in their physical efficiency. some are highly effective. by measuring the particle concentration upstream and downstream of filters with an optical particle counter, polytetrafluoroethylene (ptfe) filters and gelatin filters were confirmed to collect more than % of particles, even down to nm (burton et al., ) . if the particles are living organisms, they may be inactivated during sampling due to impaction stress (stewart et al., ) , impingement stress , and/or dehydration stress (li et al., ) . therefore, in order to indicate how well a bioaerosol sampler maintains the microbial viability and prevents cell damage during sampling, the concept of biological efficiency has been introduced (griffiths and stewart, ) . in some studies, the efficiency has been evaluated by comparing samplers side by side in an environment with unknown microbial concentration (engelhart et al., ; henningson et al., ; thorne et al., ) . this method easily ranks the performance of different samplers; however, it neither reveals whether the amount of microorganisms collected in the samples accurately represents the microorganism content of the air nor distinguishes between the physical and biological efficiency. other studies separately investigated the physical and biological efficiency of bioaerosol samplers in aerosol experiments, in which a known amount of microorganisms together with an indicator (either labeled microorganisms, or inert tracer compound) were nebulized in an isolator. the physical efficiency can be determined by comparing the amount of tracer collected by a bioaerosol sampler with that collected by the reference sampler (a sampler that has a high physical efficiency); the biological efficiency is subsequently indicated by the change in the ratio of microorganisms/indicator. using this method, zhao et al. ( b; c) reported the efficiency of the andersen six stage impactor, agi- , omni- , and gelatin filter in sampling enterococcus faecalis, e. coli, campylobacter jejuni, mycoplasma synoviae, and gumboro vaccine virus. european reference samplers use the filtration principle to collect dust from the air (european commission, ; european commission, ) ; while in us, a list of reference and equivalent samplers, including tapered element oscillating microbalance (teom), filter, and optical sampler, have been proposed by the u.s. environmental protction agency ( ). for sampling dust in certain size fractions (e.g., pm and pm . ), a preseparator for separating the coarse dust from the target dust particle sizes has to be installed in front of the filter/sensor/dust collector. sampling systems with pre-separators using the impaction principle have been legislated as reference methods for measuring dust in ambient air in the united states (u.s. environmental protction agency, ) and european countries (european commission, . these systems show steep collection curves for sampling fine dust in ambient air where dust load is low (kenny et al., ) . however, the preseparator with impaction principle may become overloaded when sampling in dusty livestock production systems, thereby resulting in overestimated concentrations of fine dust (zhao et al., ) . in contrast, a preseparator with cyclone principle has been found to be less vulnerable to overloading in dusty environments than preseparators based on impaction (zhao et al., ) . optical dust samplers are now commercially available. these dust samplers can monitor the real-time dust concentrations, and no further process is needed after sampling (unlike the gravimetric method, in which filters must be weighed). moreover, some optical samplers may separately record the concentrations of dust in different size ranges. however, the optical samplers have limitations in humid environments, because it cannot distinguish between droplets and dry dust. another limitation of using optical samplers lies to the difficulty in converting count to mass concentrations without knowing the true density of the particles in concern. . mitigation techniques for airborne microorganisms and dust many techniques have been applied in practice to reduce the concentrations and emissions of airborne microorganisms and dust in the livestock industry. they vary in their utility, but can be grouped into two main principles. the first principle, to control particles at source, includes techniques such as feed coating and oil spraying, which has been stated to be "the most effective means" of controlling airborne particles in the space (pearson and sharples, ) . the second principle is air purification. ionization (electrostatic) and air scrubbers are examples. the fatty substances often added to feed to increase metabolizable energy content may reduce the airborne microorganisms and dust in animal houses (pearson and sharples, ) . gore et al. ( ) reported that concentrations of airborne bacteria were reduced by %, and settled dust by - %, when % soybean oil was added to the pig diet. this result is consistent with the study by welford et al. ( ) , who found a % reduction of inhalable dust with % oil addition to the feed. other substances, such as tallow, lecithin and lignin have also been used as effective feed additives for the purpose of particle control in animal houses (dawson, ; pearson and sharples, ) . spraying techniques reduce the particle concentrations mainly by coating the surfaces, thereby, preventing particles from being suspended or resuspended from their sources (takai, ) . kim et al. ( ) reported that spraying ml m − of tap water, salt water, treated manure, microbial additive, soybean oil, artificial spice, and essential oil may all reduce particles in pig houses. these authors found an average reduction of % for airborne bacteria, % for fungi, and % for total dust. the substance for spraying found to be the most effective additive for reducing dust was soybean oil. in broiler rooms, aarnink et al. ( ) reported that pm reduction increased linearly from to % when daily rapeseed oil application rates increased from to ml m − . the pm . reduction was not related to application rate and was about %. in aviary systems for layers, a % reduction for pm and % reduction for pm . were achieved by daily spraying with ml m − . although a high oil application rate achieves high pm reduction in broiler rooms, it may adversely affect animal health . when spraying ml m − daily, there was a tendency for statistically higher footpad lesion in broilers. aarnink et al. ( ) recommended limiting oil application to ml m − . the ionization (also referred to as electrostatic) technique produces negative ions in the air, which causes airborne particles to become negatively charged. the negatively charged particles are attracted to earthed or positively charged surfaces. previous studies have shown promising reduction effects on dust in animal houses using ionization techniques, total dust was reduced by - % in poultry houses (lyngtveit and eduard, ; mitchell et al., ; mitchell et al., ; richardson et al., ) , and by - % in pig houses (rosentrater, ; tanaka and zhang, ) . cambra-lopez et al. ( ) reported that the technique produced average reductions of % for pm and % for pm . . the disparity is probably caused by the different charging mechanisms to particles. the small particles (< . μm) are charged by the thermal charging mechanism, which is proportional to the diameter; large particles (> . μm) are charged by field charging mechanism, which is proportional to the square of the diameter (bundy, ) . ionization has the potential to prevent airborne transmission of microorganisms holt et al., ) , however, no reduction of airborne bacteria, fungi and mold was found in broiler houses by this technique ). knowledge of ionization on reduction of airborne microorganisms in animal houses is still limited and needs to be augmented. end-of-pipe techniques, such as dedusters, air filters, and scrubbers have been installed at air outlets of animal houses to minimize emissions of air pollutants. zhang et al. ( ) found a deduster removed % total dust from the air exhausted from a pig house. because dedusters are based on the centrifuge principle, they are more effective at removing larger particles than smaller ones. the deduster studied by zhang et al. ( ) had a removal efficiency of % for particles larger than μm, % for μm particles, and % for μm particles. the low removal efficiency for small particles was assumed to be particle re-entrainment due to high air turbulence. acid and biological air scrubbers were originally developed to reduce ammonia and odor emissions, and they also appear to be effective in reducing particle emissions. an acid scrubber that uses sulfuric acid may achieve a % reduction of total bacteria . in a lab-scale experiment, aarnink et al. ( a) found that e. faecalis and gumboro virus could be reduced by % when per-acetic acid was used as the circulation solution in a scrubber. the biological scrubbers are not consistent in reducing microorganisms (seedorf and hartung, ) , probably because the microorganisms for digesting odorous compounds can also be emitted to the ambient air. the removal of total dust by biological scrubbers was found to be - % (seedorf and hartung, ) . combined techniques have been applied in practice: for instance, oil spraying combined with feed coating (takai and pedersen, ) and multistage air scrubbers . these techniques are more consistent and effective in reducing emissions of airborne microorganisms and dust, as well as other gaseous pollutants (ogink and bosma, ; zhao et al., ) . a disadvantage of combined scrubbing techniques is the relatively high energy use and the complexity for use on practical farms. therefore, further research is needed to develop energy-saving and simple-to-operate combined scrubbing techniques. • the most important source of airborne microorganisms is the animals themselves by means of excretion. the sources of dust include excrement, litter, feed, skin, and feathers. • airborne microorganisms in animal houses are mostly bacteria, with grampositive bacteria predominating; fungi account for only a small proportion of microorganisms. • concentrations of both microorganisms and dust are high in animal houses. they are affected by animal type, housing system, management, and environmental factors. because these factors play an interrelated role on the concentrations of microorganisms and dust, integrated research on the effects is required. • the microorganisms transmitted in the air suffer physical and biological decay. the physical decay largely depends on their size, and the biological decay is mainly determined by environmental factors, such as humidity, temperature, radiation, and toxic gases. in airborne transmission, microorganisms may be carried by dust particles that may protect microorganisms from biological decay. knowledge of the role of dust in the transportation of microorganisms is still lacking, and needs to be expanded. • microorganisms are deposited on different regions in the respiratory tract, mainly depending on their size. they have different preferred infection regions in the respiratory tract. the amount of microorganisms needed to induce an infection varies with microorganism species and animal species. • reference methods for microorganism and dust sampling in animal houses need to be legislated. these methods should be resistant to highly microbial and dusty environments, and the efficiency of the sampling devices needs to be investigated. • different techniques have been applied to reduce airborne microorganisms and dust in and from animal houses. combining several abatement techniques may achieve higher and more consistent reduction. energysaving and simple-to-operate combined techniques are of interest for further development. scrubber capabilities to remove airborne microorganisms and other aerial pollutants from exhaust air of animal houses options for dust reduction from poultry houses dust sources in animal houses dust reduction in broiler houses by spraying rapeseed oil indoor particle pollution: effect of wall textures on particle deposition aerosol stability of infectious and potentially infectious reovirus particles effect of temperature, litter and light intensity on ammonia and dust production and broiler performance further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs human influenza resulting from aerosol inhalation new sampler for the collection, sizing, and enumeration of viable airborne particles dust borne bacteria in animal sheds, schools and children's day care centres welfare of laying hens in cages and alternative systems: environmental, physical and behavioural aspects a study of the relationship between airborne contaminants and environmental factors in dutch swine confinement buildings the occurrence and enumeration, according to a new classification, of micrococci and staphylococci in bacon and on human and pig skin analyses of airborne contamination with bacteria, endotoxins and dust in livestock barns and poultry houses measuring biosphereatmosphere exchanges of biologically related gases with micrometeorological methods identification of the risk factors for high airborne particle concentrations in broiler buildings using statistical modelling identification of risk factors for sub-optimal housing conditions in australian piggeries: part . airborne pollutants comparison of the aerosol stabilities of foot-and-mouth disease virus suspended in cell culture fluid or natural fluids the role of free radicals and oxygen in reactions produced by ionizing radiations mechanisms of infection in the respiratory tract death mechanisms in airborne escherichia coli some factors affecting the survival of airborne viruses airborne microorganisms and dust in livestock houses effect of simulated solar radiation and sodium fluorescein on the recovery of venezuelan equine encephalomyelitis virus from aerosols experimental airborne transmission of streptococcus suis capsular type in pigs escherichia coli o :h infection of calves: infectious dose and direct contact transmission transmission of influenza a in human beings experimental airborne transmission of porcine reproductive and respiratory syndrome virus and bordetella bronchiseptica airborne survival of rift valley fever virus. army medical research institute of infectious diseases ultrafine particle deposition and clearance in the healthy and obstructed lung water spray cooling during handling of feedlot cattle sources of campylobacter spp. colonizing housed broiler flocks during rearing rate of dust decay as affected by relative humidity, ionization and air movement physical collection efficiency of filter materials for bacteria and viruses the relationship between route of infection and minimum infectious dose: studies with lactate dehydrogenaseelevating virus control of particulate matter emissions from poultry and pig houses airborne particulate matter from livestock production systems: a review of an air pollution problem ionization for reducing particulate matter emissions from poultry houses exposure of workers to airborne microorganisms in open-air swine houses effect of dietary fat on pig performance and dust levels in modified open-front and environmentally regulated confinement buildings the impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs air quality in farrowing barns airborne bacteria, endotoxin and fungi in dust in poultry and swine confinement buildings deposition of differently sized airborne microspheres in the respiratory tract of chickens airborne microbial contents in two types of swine confinement buildings in quebec transmission and infectious dose of escherichia coli o :h in swine aerosol survival of escherichia coli b disseminated from the dry state aerosol survival of pasteurella tularensis disseminated from the wet and dry states airborne bacteria and viruses aerosol survival of pasteurella tularensis and the influence of relative humidity experimental infection of calves and adult cattle with escherichia coli o :h relative and qualitative aspects of aerial bacteria and dust in swine houses minimizing dust in livestock buildings: possible alternatives to mechanical separation detection of hepatitis e virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions airborne microorganisms and dust in livestock houses factors in the inactivation of encephalomyocarditis virus in aerosols mechanisms of inactivation of bacteriophage phix and its dna in aerosols by ozone and ozonized cyclohexene survival of aerosolized bacteriophage x in air containing ozone-olefin mixtures bacterial eggshell contamination in conventional cages, furnished cages and aviary housing systems for laying hens bacterial contamination of table eggs and the influence of housing systems effect of dry litter and airflow in reducing salmonella and escherichia coli populations in the broiler production environment differences in aerosol survival between pathogenic and non-pathogenic strains of legionella pneumophila serogroup effect of ph on the behaviour of volatile compounds in organic manures during dry-matter determination modeling of the penetration of ambient pm . to indoor residential microenvironment the influence of relative humidity on the aerosol stability of different strains of foot-and-mouth disease virus suspended in saliva infection of cattle by airborne foot-and-mouth disease virus: minimal doses with o and sat strains quantitative data on airborne foot-and-mouth disease virus: its production, carriage and deposition human occupational hazards from swine confinement characterization of dusts collected from swine confinement buildings ambient temperature: a factor affecting performance and physiological response of broiler chickens association of campylobacter jejuni with laying hens and eggs prevalence and survival of campylobacter jejuni in unpasteurized milk studies on respiratory infection. i. the influence of particle size on respiratory infection with anthrax spores the germicidal properties of ozone-olefin mixtures studies on respiratory infection. ii. the influence of aerosol particle size on infection of the guinea-pig with pasteurella pestis airborne stability of tailless bacterial viruses s- and ms- influence of building maintenance, environmental factors, and seasons on airborne contaminants of swine confinement buildings inhaling to mitigate exhaled bioaerosols effects of atmospheric humidity and temperature on the survival of airborne flavobacterium relationship between atmospheric temperature and survival of airborne bacteria aerosol stability of bovine parainfluenza type virus factors associated with the introduction of classical swine fever virus into pig herds in the central area of the / epidemic in the netherlands. the veterinary record an investigation of the merits of ozone as an aerial disinfectant air sampling of aspergillus fumigatus and other thermotolerant fungi: comparative performance of the sartorius md airport and the merck mas- portable bioaerosol sampler airborne microorganisms and dust in livestock houses air quality: determination of the pm fraction of suspended particulate matter: reference method and field test procedure to demonstrate reference equivalence of measurement methods. european committee for standardization, brussels. belgium european commission ambient air quality-standard gravimetric measurement method for the determination of the pm . mass fraction of suspended particulate matter influenza virus in human exhaled breath: an observational study interaction of ozone and negative air ions to control microorganisms animal influenza virus surveillance presence and persistence of foot-and-mouth disease virus in bovine skin application of negative air ionization for reducing experimental airborne transmission of salmonella enteritidis to chicks exposure of sheep to natural aerosols of foot-and-mouth disease virus. research in veterinary science effects of ozone on organisms sensitivity of microorganisms to different wavelengths of uv light: implications on modeling of medium pressure uv systems airborne transmission of footand-mouth disease virus from burnside farm, heddonon-the-wall, northumberland, during the epidemic in the united kingdom footand-mouth disease: quantification and size distribution of airborne particles emitted by healthy and infected pigs the effects of soybean oil on nursery air quality and performance of weanling pigs minimal infective dose of rotavirus survival and infectivity of salmonella choleraesuis in swine feces observations on the use of membrane filtration and liquid impingement to collect airborne microorganisms in various atmospheric environments performance of bioaerosol samplers used by the uk biotechnology industry inlet characteristics of bioaerosol samplers dust concentration and mortality distribution in an enclosed laying house particulate emissions from deep-bedded growing-finishing pigs some factors affecting airborne survival of pseudomonas fluorescens indoors airborne micro-organisms: survival tests with four viruses the respiratory retention of bacterial aerosols: experiments with radioactive spores ultraviolet inactivation of selected bacteria and viruses with photoreactivation of the bacteria emissions of airborne substances from stalls of domestic animals enhanced recovery of airborne t coliphage and pasteurella pestis bacteriophage by means of a presampling humidification technique safety and efficacy of the neuraminidase inhibitor gg in experimental human influenza airborne-particle deposition in the respiratory tract of chickens particle deposition rates in residential houses qualityassured measurements of animal building emissions : particulate matter concentrations size distribution and identification of aerial dust particles in swine finishing buildings influence of environmental factors on concentrations and inorganic content of aerial dust in swine finishing buildings effect of ozone on airborne microorganisms a comparative study of apparatus for sampling airborne microorganisms detection of respiratory pathogens in air samples from acutely infected pigs effect of temperature and relative humidity on the stability of infectious porcine reproductive and respiratory syndrome virus in aerosols effects of oxygen on aerosolized serratia marcescens deposition of inhaled particles in the human respiratory tract and consequences for regional targeting in respiratory drug delivery. the proceedings of the deposition of particles in the human respiratory tract in the size range , - μm inactivation credit of uv radiation for viruses, bacteria and protozoan (oo) cysts in water: a review survival of escherichia coli o :h and salmonella typhimurium in cow manure and cow manure slurry a comprehensive experimental study of aerial pollutants in and emissions from livestock buildings. part : results characterization of antibiotic-resistant bacteria in rendered animal products measurement protocol for determining fine dust emission factors of animal housing systems effect of induced molting on the susceptibility of white leghorn hens to a salmonella enteritidis infection use of negative air ionization for reducing airborne levels of salmonella enterica serovar enteritidis in a room containing infected caged layers survival characteristics of airborne human coronavirus e studies on the survival of aerosolized bovine rotavirus (uk) and a murine rotavirus comparison of the airborne survival of calf rotavirus and poliovirus type (sabin) aerosolized as a mixture effect of relative humidity, atmospheric temperature, and suspending medium on the airborne survival of human rotavirus inhibition of bacterial growth by pure ozone in the presence of nutrients the respiratory tract deposition model proposed by the icrp task group factors affecting the occurrence of aspergillosis in turkeys deposition and clearance of radiolabelled monodisperse polystyrene spheres in the calf lung effect of relative humidity on the airborne survival of rhinovirus- factors affecting the deposition of aerosolized insulin development of a sharp-cut cyclone for ambient aerosol monitoring applications characteristics and sampling efficiencies of omni aerosol samplers. edgewood chemical biological center application of ozone for enhancing the microbiological safety and quality of foods: a review farmer and pig exposure to aerial contaminants in a pig confinement building effect of spraying biological additives for reduction of dust and bioaerosol in a confinement swine house assessment of airborne bacteria and fungi in pig buildings in korea effect of ventilation rate on gradient of aerial contaminants in the confinement pig building a water droplet evaporation and temperature model preliminary studies on the characterization and distribution of staphylococcus and micrococcus species on animal skin effects of temperature and relative humidity on the survival of newcastle disease virus aerosols in the rotating drum. defence research and development canada bactericidal effects of high airborne ozone concentrations on escherichia coli and staphylococcus aureus predicting air flow patterns and particle contamination in clean rooms particle deposition indoors: a review size distribution of airborne particles in animal houses thermophoretic collection and analysis of submicrometer ag particles emitted from a graphite tube-type electrothermal vaporizer aerosol stability and respiratory infectivity of japanese b encephalitis virus personal exposure to airborne dust and microorganisms in agricultural environments microbial seed banks: the ecological and evolutionary implications of dormancy distribution of salmonella in swine herds in québec evaluation of microbial samplers for bacterial microorganisms survival of airborne bacteria in a high urban concentration of carbon monoxide estimation of viable airborne microbes downwind from a point source effect of sampling time on the collection efficiency of all-glass impingers deposition, retention, and clearance of inhaled particles aerosol sampling inlets and inhalable particles synthesizing traditional biogeography with microbial ecology: the importance of dormancy dose determination for acute salmonella infection in pigs evaluation of broiler litter with reference to the microbial composition as assessed by using s rrna and functional gene markers reduction of dust exposure by negative air ionisation effects on poultry and livestock of feed contamination with bacteria and fungi air hygiene in a broiler house: comparison of deep litter with raised netting floors microbiological survey of georgia poultry litter bacterial and fungal flora of dust deposits in a pig building qualitative structure of airborne bacteria and fungi in dairy barn and nearby environment survival of bacterial and mold spores in air filter media assessment of aerosol mixtures of different viruses effect of solar radiation and predacious microorganisms on survival of fecal and other bacteria experimental human salmonellosis: iii. pathogenicity of strains of salmonella newport, salmonella derby, and salmonella bareilly obtained from spray-dried whole egg the survival of escherichia coli o :h in slurry from cattle fed different diets viable but non-culturable forms of potentially pathogenic bacteria in water investigation of airborne foot-and-mouth disease virus transmission during lowwind conditions in the early phase of the uk epidemic survival of bacteria on wood and plastic particles: dependence on wood species and environmental conditions survival of anaerobic fungi in feces, in saliva, and in pure culture reducing dust in a caged layer room: an electrostatic space charge system application of an electrostatic space charge system for dust, ammonia, and pathogen reduction in a broiler breeder house the effect of relative humidity and temperature on the survival of bovine rotavirus in aerosol dust and microbial emissions from animal production performance of bioaerosol samplers: collection characteristics and sampler design considerations sources of campylobacter colonization in broiler chickens temperature, air humidity and air pollution levels in farrowing or weaner pig houses risk factors for the introduction of avian influenza virus into commercial layer chicken farms during the outbreaks caused by a low-pathogenic h n virus in japan in multi-phase air scrubbers for the combined abatement of ammonia, odor and particulate matter emissions recent findings on the viable but nonculturable state in pathogenic bacteria concentration and prevalence of escherichia coli o in cattle feces at slaughter long distance airborne transport of infectious prrsv and mycoplasma hyopneumoniae from a swine population infected with multiple viral variants a portable environmental monitoring system to assess barn worker indoor air exposure photocatalytic inactivation of gram-positive and gram-negative bacteria using fluorescent light airborne dust concentrations in livestock buildings and the effect of feed animal activity measured by infrared detectors manure and microbes: public and animal health problem effect of air ions on bacterial aerosols dust levels in mechanically versus naturally ventilated hog barns. asae paper no. - the development of robust methods for measuring concentrations and emission rates of gaseous and particulate air pollutants in livestock buildings recent advances in quantitative pcr (qpcr) applications in food microbiology dust and bioaerosol concentrations in two swine-finishing buildings in kansas microbiology bacterial contamination in poultry houses and its relationship to egg hatchability stability of st. louis encephalitis virus in the airborne state air contaminants in different european farming environments inactivation of respiratory syncytial virus in aerosol effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust performance of an electrostatic dust collection system in swine facilities mechanism of enteroviral inactivation by ozone factors affecting susceptibility of calves to disease experimental campylobacter diarrhea in chickens influence of animal age and season on bio-aerosol concentrations in a broiler house effect of relative humidity on the airborne survival of rotavirus sa survival of pseudorabies virus in aerosol particle deposition in the guinea pig respiratory tract effect of ozone on survival and permeability of escherichia coli reduction efficiencies of a biofilter and a bioscrubber for bioaerosols from two different piggeries concentrations and emissions of airborne endotoxins and microorganisms in livestock buildings in northern europe absolute humidity modulates influenza survival, transmission, and seasonality handbook of environmental engineering bacterial aerosol samplers. ii. development and evaluation of the shipe sampler evaporative cooling performance resulting from changes in water temperature influence of relative humidity on the survival of some airborne viruses serological response of chickens to oral vaccination with newcastle disease virus the use of quantitative pcr for identification and quantification of brachyspira pilosicoli, lawsonia intracellularis and escherichia coli fimbrial types f and f in pig feces the role of infectious aerosols in disease transmission pigs effect of environmental factors on aerosol-induced lassa virus infection recovery of airborne streptococcal l-forms at various relative humidities effect of impact stress on microbial recovery on an agar surface viability of hospital staphylococci in air transport of droplet expelled by coughing in ventilated rooms factors influencing dust reduction efficiency of spraying of oil-water mixtures in pig buildings a comparison study of different dust control methods in pig buildings concentrations and emissions of airborne dust in livestock buildings in northern europe dust settling efficiency and electrostatic effect of a negative ionization system surface wetting and its optimization to cool broiler chickens effects of room furnishings and air speed on particle deposition rates indoors influence of temperature and relative humidity on the survival of chlamydia pneumoniae in aerosols airborne microorganisms and dust in livestock houses survival of toxigenic pasteurella multocida in aerosols and aqueous liquids comparison of bioaerosol sampling methods in barns housing swine. applied and environmental microbiology influence of different ventilation systems on inhalable and respirable dust particles concentrations distribution in weaning and finishing fattening pig houses influence of different litter materials on cecal microbiota colonization in broiler chickens viable but non-culturable (vbnc) bacteria: gene expression in planktonic and biofilm cells bacterial aerosol samplers. i. development and evaluation of the all-glass impinger bacterial aerosol samplers. iii. comparison of biological and physical effects in liquid impinger samplers ambient air monitoring reference and equivalent methods ambient air monitoring reference and equivalent methods national ambient air quality standards for particulate matter; final rule preparation and characterization of a customized cellulose acetate butyrate dispersion for controlled drug delivery effect of extracellularly generated singlet oxygen on gram-positive and gram-negative bacteria experimental production of congenital persistent swine fever infections: i. clinical, pathological and virological observations enumeration of airborne molds in some indoor environments of madras city (india) by cultural and non-cultural volumetric samplers the effect of animal age on air pollutant concentration in a broiler house bioaerosols as contributors to poor air quality in taichung city effect of sampling time and air humidity on the bioefficiency of filter samplers for bioaerosol collection human rotavirus studies in volunteers: determination of infectious dose and serological response to infection the survival of escherichia coli in an aerosol at air temperatures of and c and a range of humidities emissions of aerial pollutants in livestock buildings in northern europe: overview of a multinational project factors affecting the viability of air-borne bacteria. i. bacteria aerosolized from distilled water inactivation of influenza a viruses in the environment and modes of transmission: a critical review intestinal influenza: replication and characterization of influenza viruses in ducks pig building dustiness as affected by canola oil in the feed on air-borne infection: study ii. droplets and droplet nuclei airborne microbial flora in a cattle feedlot reaction of airborne rhizobium meliloti to some environmental factors role of relative humidity in the survival of airborne mycoplasma pneumoniae survival of airborne mycoplasma as affected by relative humidity antibacterial and static dissipating composites of poly (butylene adipate-co-terephthalate) and multi-walled carbon nanotubes meteorological factors and ambient bacterial levels in a subtropical urban environment oscillating sedimentation of spheres in viscoplastic fluids. rheologica acta airborne microorganisms and dust in livestock houses effects of ambient temperature and restricted feeding on the growth of feathers in growing turkeys global dispersion of bacterial cells on asian dust quantification of viable legionella pneumophila cells using propidium monoazide combined with quantitative pcr respirable aerosol generation by broiler chicken persistent and contact infection in nursery pigs experimentally infected with porcine reproductive and respiratory syndrome (prrs) virus effect of challenge dose and route on porcine reproductive and respiratory syndrome virus (prrsv) infection in young swine evidence of airborne transmission of the severe acute respiratory syndrome virus effect of the type of feed (meal, pellets or fluid) and season on the concentration and particle size of dust particles in pig houses an aerodynamic deduster to reduce dust and gas emissions from ventilated livestock facilities viral shedding and emission of airborne infectious bursal disease virus from a broiler room effectiveness of multi-stage scrubbers in reducing emissions of air pollutants from pig houses effect of temperature, relative humidity, absolute humidity and evaporation potential on survival of airborne gumboro vaccine virus investigation of the efficiencies of bioaerosol samplers for collecting aerosolized bacteria using a fluorescent tracer. i: effects of non-sampling processes on bacterial culturability investigation of the efficiencies of bioaerosol samplers for collecting aerosolized bacteria using a fluorescent tracer. ii: sampling efficiency, and half-life time detection of airborne campylobacter with three bioaerosol samplers for alarming bacteria transmission in broilers removal efficiency of dust and bacteria by multi-stageair scrubbers in a pig house evaluation of an impaction and a cyclone pre-separator for sampling high pm and pm . concentrations in livestock houses influence of dose and route on transmission of encephalomyocarditis virus to swine airborne gram-negative bacterial flora in animal houses key: cord- -gsg akaz authors: wu, mengjie; yang, chunping; du, cheng; liu, hongyu title: microplastics in waters and soils: occurrence, analytical methods and ecotoxicological effects date: - - journal: ecotoxicol environ saf doi: . /j.ecoenv. . sha: doc_id: cord_uid: gsg akaz microplastics (mps) are ubiquitous in the environment and more abundant in the marine environment. consequently, increasing focus has been put on mps in oceans and seas, while little importance has been attached to their presence in freshwaters and soils. therefore, this paper aimed to provide a comprehensive review of the occurrence, analysis and ecotoxicology of mps. the abundance and distribution of mps in several typical freshwater systems of china were summarized. it suggested that the surface water of poyang lake contained the highest concentration of items/l mps among all the freshwater systems, and the content of mps in sediments were higher than that of the surface water. net-based zooplankton sampling methods are the most frequently utilized sampling methods for mps, and density separation, elutriation and digestion are three major pretreatment methods. fourier transform infrared spectroscopy, raman spectroscopy and pyrolysis-gas chromatography coupled to mass spectrometry are often used to identify the polymer types of mps. besides, mps might damage the digestive tract of various organisms and negatively inhibit their growth, feeding and reproduction. the ways of human exposure to mps are by ingestion, inhalation and dermal exposure, digestive and respiratory system might be adversely influenced. however, potential health risks of mps to humans are remained insufficiently researched. overall, by showing the presence of mps in freshwaters and soils as well as possible ecotoxicological effects on the environment and humans, this paper provided a framework for future research in this field. the increasing production of plastics in recent years has led to severe plastic pollution in the environment. surveys show that the annual plastic production in has exceeded million tons (plasticseurope, ) , and it is estimated that the global annual production of plastics may be up to billion tones by the year of (cincinelli et al., ) . besides, the current outbreak of covid- pandemic has resulted in a dramatic increase in personal protective equipment (e.g. gloves and masks) in which plastics and rubbers are two of the major components. it is reported that the accumulated medical waste in china from january to march in was about kilotons (klemes et al., ) . this will lead to a severer plastic pollution in the environment. microplastics (mps) that are plastics less than mm in length are even more harmful than larger plastic items, and microplastic pollution (mp) has become an emerging environmental issue in the last two decades (laskar and kumar, ; wright et al., a) .studies have shown that . to . million tonnes of plastic waste entered the oceans and seas of countries in and this number would increase by % by (jahnke et al., ; jambeck et al., ; kühn et al., ; thompson et al., ; zarfl et al., ) ; however, mps have also been detected in freshwater systems as well as terrestrial environment. investigations of mps in freshwaters and soils are far from adequate, as knowledge on mps in freshwater and terrestrial environment is insufficient (rillig, ) . widely-recognized problems facing organisms are entanglement, suffocation and ingestion that are caused by mps (lo and chan, ; wu et al., ; yang et al., yang et al., , , and many studies have shown that mps might pose threat to marine zooplanktons, fish, mussels and algae (besseling et al., ; desforges et al., ; galloway et al., ; lusher et al., ; qin, ) . apart from mps itself, manifold plastic additives are another hidden danger. for example, nonylphenol as an endocrine disrupting chemical is frequently added in polyvinylchloride (pvc) and high-density polyethylene (pe) to achieve high thermostability of plastics (lam et al., ) . moreover, due to the small sizes and difficulty in degradation under natural condition, chances are that mps can be ingested or respired by a wide range of organisms and therefore will cause harm to them (wright et al., b) . worse still, mps have a high affinity for other pollutants. for example, mps may adsorb heavy metals and hydrophobic organic chemicals on their surface and act as a vector for transporting these pollutants in the environment (eerkes-medrano et al., ; fries et al., ; koelmans et al., ; lee et al., lee et al., , lin et al., ; mizukawa et al., ; tang et al., ; yang et al., ; zhou et al., ) . despite that information on the detection, identification and quantification of mps can be found in a number of papers (claessens et al., ; lebreton et al., ; martin et al., ; reddy et al., ; xiong et al., ; zobkov and esiukova, ) , comprehensive studies on mps which involve in their occurrence, distribution, sampling, pretreatment, characterization in freshwaters and sediments, as well as ecological influences on aquatic and soil organisms and humans are still needed. therefore, the review aimed to: ( ) present the occurrence and abundance of mp in some typical freshwater systems in china; ( ) summarize frequently used sampling, pretreatment and characterization methods for mps in waters and sediments; ( ) elucidate ecological impacts of mps on aquatic organisms, soil biota and human health; ( ) point out knowledge gaps in current studies of mps and make corresponding recommendations for further research. a literature review was conducted on database of web of science and sciencedirect for peer-reviewed publication from to february . a combination of keywords was applied as the criteria such as "microplastics" or "microplastic" and "freshwater" or "water", "microplastics" or "microplastic" and "soil", "microplastics" or "microplastic" and "effects", and these keywords were retrieved in any topic, title or text. a total of papers were identified as candidate publications, and they were classified into several subtopics, namely, occurrence and distribution in major freshwater systems of china, sampling and pretreatment, identification, and ecotoxicology. vast numbers of mps are accumulated in oceans every year; however, they have also been widely detected in freshwater systems, which has sparked high-profile campaigns recently (xu et al., ) . it was reported that the annual plastics production in china was about . million tons in , which accounted for . % of the whole plastics production all over the world (plasticseurope, ) . moreover, mismanagement of waste plastics is a common phenomenon in china. for instance, the mismanagement rate of plastic wastes of china has reached . % in , which made china on the top among the listed countries ranked by percentage of total mismanaged plastic wastes (jambeck et al., ) . therefore, some freshwater systems of china were chosen as the typical example to analyze the occurrence and abundance of mps in freshwaters. according to a couple of studies (di and wang, ; eerkes-medrano et al., ; kukulka et al., ; wang et al., b) , factors such as human activity, climatic and hydrological conditions could have a direct effect on the abundance and distribution of mps in freshwater environment. di and wang ( ) suggested that the concentration and distribution of mps in freshwater systems were mainly influenced by human, agricultural, fisheries and industrial activities. for example, the study of yin et al. ( ) suggested that the abundance of microplastics in the urban area sediment of dongting lake is lower than that of the rural area and the reason behind this phenomenon was that more environmental protection measures were taken by urban areas than rural areas. table summarized the abundance of mps in surface water and sediment of several typical freshwater systems in china. as shown in table , among those major freshwater systems in china, pearl lake in zhanjiang which is one of the developed industrial cities in guangdong province was detected with the highest mps concentration of items/m . mps were also found in lakes of rural areas such as tibet, though the concentration of which was lower than those dense populated areas. however, how mps were moved to rural areas remained ambiguous. thus, more studies should be conducted to investigate the transportation and distribution of mps in freshwater environment from a broad point of view. in addition, it was noticeable that fiber was the most dominant polymer component in all listed references as presented in table . nowadays, synthetic textile fibers are widely applied, napper and thompson ( ) pointed out that domestic sewage derived from washing machines comprised high concentrations of fibers, which might be an important source of microfiber. besides, fisheries activities could produce plenty of ageing fishing gears including fishing nets, thus it should be another source of microfiber yuan et al., ) . from table , it could also be seen that pe, pp (polypropylene) and ps (polystyrene) were the most commonly detected plastic types, as the three kinds of polymers are frequently used in fishing tools, packaging and decorations (wang et al., a) . concerning the particle size of mps detected in these freshwater systems, most of them were less than mm. it was also reported that small-sized mps were found more abundant in many investigations (eriksen et al., ) . however, the mps concentrations in lakes of tibet had larger particle size than that of other areas. this was probably owing to a fast dissipation of small plastic items by high ultraviolet radiation intensity and big temperature difference in tibet . because sediments were the final destination of high-density mps, the concentration of mps in sediments were higher than that of the surface water as seen in table . throughout the literature referred to, it could be found that different units were used to describe the concentration of mps, which made it inconvenient to compare the abundance of mps among different locations. for example, beside common unit of "items/m " and "items/l , less used unit such as "g/l" and "fibers/ ml" were also seen in some researches (baztan et al., ; browne et al., ) . therefore, using unified units to measure the abundance of mps would help the comparison and analysis of data. to date, various methods have been employed to sample and detect mps in waters; however, standardized methods have not been proposed (besley et al., ; hanvey et al., ; miller et al., ) . one of the commonly used tools for sampling mps from waters is net-based zooplankton sampling methods (lattin et al., ) . in order to make the samples more representative, large volumes of water should be collected. nets with different mesh sizes are used to estimate the concentrations of mps in open water bodies. three kinds of zooplankton nets including manta trawl, neuston and bongo net are wildly used. manta trawl net and neuston net are suitable for sampling mps that float on the surface of the water bodies (cincinelli et al., ; collignon et al., ; mauro et al., ) . neuston net can be used under strong wind and wave condition while manta net is suitable for calm water (frias et al., ; lima et al., ) . common sizes of neuston and manta trawl nets range from μm to μm, and the mesh size around μm is the most frequently used one (hidalgo-ruz et al., ) . advantages of both nets are that large volumes of water can be sampled in a rather short time (ruiz-orej� on et al., ), but this may underestimate the actual concentrations of mps in seawater, for mps with particle sizes smaller than μm that are more toxicologically significant will be readily escaped (doyle et al., ) . because small-mesh-size nets are easily blocked, nets whose mesh size are less than μm are rarely studied. bongo net is usually used for collecting water-column samples, and it can sample on sea surface as well as at mid-ocean depths. bongo net is a reliable single unit tow platform for sampling in shallow water (wang et al., a) . one of the unique features of bongo net is its opening and closing mechanism that allows discrete known-depth sampling (mauro et al., ) . the most widely used mesh sizes of bongo net are also about μm (c� ozar et al., ) . for instance, mauro et al. ( ) deployed a bongo net with a diameter of cm fitted with μm mesh size in an oblique tow from a depth of m to collect mps samples from northern gulf of mexico; in the experiments of lattin et al. ( ) , paired cm diameter bongo nets with a length of m and the mesh size of μm were used to sample mps in the southern california shore. detailed information about neuston, manta and bongo net is presented in table . as sediments in coastal beaches or lakefront are more convenient to be sampled, samples are usually taken from these places when studying mps in sediments. commonly used tools for collecting mps on beaches are forceps, sieves, grab samplers, box corers and spade corers (tsang et al., ; vianello et al., ) . because of the properties of mps and various environmental factors, the distribution of mps in sediments is not even. therefore, when sampling mps from sediments, the selecting of tide line, sampling depth and sampling volumes matters (bergmann et al., ) . in general, the main sampling areas are in the high tide line where more mps may be accumulated (claessens et al., ) . the sampling depths in sediments are the top cm, and a sampling area of cm is often chosen turner and holmes, ) . before characterizations being carried out, isolation and extraction processes are needed because organic matters (oms), algae and other impurities are mixed with mps samples (zhao et al., ) . the separation of mps samples involves in two major categories: physical methods such as density separation, filtration and sieving, and biochemical methods including acidic/alkaline digestion, oxidization and enzymatic degradation (qiu et al., ) . the density of mps varies depending on their polymer types and manufacturing processes, and the specific density values usually range from . to . g/cm (hidalgo-ruz et al., ) . density separation is based on the differences in the density of mps. as the typical density of sediments is about . g/cm which is heavier than most mps, the lighter items can be separated from the heavier ones by mixing with saturated salt solutions and shaking for a certain period of time (shim et al., ) . after that, less dense mps will float on the surface of the brine solutions while denser sediments will sink at the bottom. different brine solutions are applied to separating different mps, and the density of brine solutions that are used for density separation often should be higher than . g/cm . saturated sodium chloride (nacl, density of . g/cm ) solution is one of the most commonly used flotation agents owing to its cheap price, high availability and environmental benignity (nuelle et al., ) . however, saturated nacl solutions are not suitable for separating mps whose density is heavier than . g/cm such as pvc and pet (polyethylene terephthalate) and the recovery rates of which mauro et al. ( ) are less than % (quinn et al., ) . in addition, sediment samples need to be washed for three times when using nacl as separation solutions. other frequently used brine solutions are zinc chloride (zncl ), zinc bromide (znbr ), and sodium iodide (nai) (hidalgo-ruz et al., ; nuelle et al., ) . quinn et al. ( ) showed that nai and znbr were effective to separate mps with particle sizes of - μm, and both of them had good recovery rates (> %), and it only required a single wash of sediments. mps that can be separated by nai are similar to environmental samples, while znbr solution is more suitable for separating samples of single type (rocha-santos and duarte, ). moreover, znbr is harmful to the environment and may lead to a secondary pollution while using nai is environmentally safe (prata et al., a) . therefore, nai is a recommended brine solution for the separation of mps from sediments. however, nai should not be used with cellulose filters, as it can react with them. sodium polytungstate (na o w ) solution is also used for density separation, but the recovery rate test of which is scarce and its price is relatively high. more specific information about the density values of different polymers and commonly used brine solutions are provided in table . although shaking time and settling time of density separation is often about s to h, min to h, respectively, it actually requires less time in both process (hidalgo-ruz et al., ) . as shown in table , it took - min for mixing mps samples and brine solutions and - min for separation of mps in most study. in addition, besides salt solution separation, hydrocyclonic separation technology is also applied in mps separation (yuan et al., ) . elutriation is another separation method for extracting mps samples by injecting some fluid such as water at the bottom of a column so that the buoyant mps can be isolated from the settling oms and sediments (kedzierski et al., ) . filtration and sieving are also employed to separate mps from water and sediment samples that are obtained from the density separation process (prata et al., a) . the plastic particles will be filtered from the supernatant by a filter which is usually assisted by a vacuum (ng and obbard, ) . in order to sort out larger particles before the filtration step, water samples can firstly pass through a sieve whose mesh size ranges from . to . mm (andrady, ) . another category for processing mps samples with the purpose of removing oms is digestion. there are four major types of digestion methods namely acidic, alkaline, enzymatic and oxidative digestion. for acidic digestion, nitric acid (hno ), hydrofluoric acid (hf) and hydrochloric acid (hcl) are most frequently used. for example, davidson and dudas ( ) used - % of hno to separate mps from clam tissues; naidoo et al. ( ) found that % of hno could fasten the digestion process when heating; dubaish and liebezeit ( ) utilized hf to digest oms. although hcl is reported to have relatively low digestion efficiency, studies using hcl to extract mps can also be found (cole et al., ; desforges et al., ; karami et al., ) . many types of plastics such as nylon and pet can be easily degraded by acid at high acid concentration and high processing temperature, and acidic digestion may also underestimate the effects of mps on the environment (qiu et al., ) . thus, acidic digestion should be used with caution. the effects of acid on the integrity of mps have also been reported. for example, prata et al. ( a) summarized that when using hno as acid digestion reagent, it may cause loss of some types of polymers such as ps and pet. moreover, as heating is often required to assist the digestion process, polymers that have low resistance to acid may be more easily degraded at high temperature. alkaline digestion which serves as an alternative to acidic digestion has also been widely used in the separation of mps. among those available literature, sodium hydroxide (naoh) and potassium hydroxide (koh) are two commonly used alkaline solutions (maes et al., ) . cole et al. ( ) found that mol/l of naoh solution was the optimal alkaline concentration among the tested ones. as for koh, a concentration of % is often used when extracting mps (dehaut et al., ; foekema et al., ) . alkaline digestion has high digestion efficiency of oms and good recovery rate of mps; however, it may damage the structure of plastics and causing discoloration to pe, pvc and nylon (cole et al., ) . enzymatic digestion is a promising digestion method for the removal of oms since it is less hazardous to both the environment and plastic structures (courtene-jones et al., ) . proteinase k, tripsin, collagenase and papain are used for digesting oms; a sequential use of enzymes (protease, cellulase, chitinase) has achieved high removal efficiency of oms (l€ oder et al., ) . enzymatic digestion usually has no effect on the integrity of mps and obtains high recovery rates (cole et al., ) . however, despite the eco-friendly property of enzymatic digestion, the utilization of enzymes in the separation of mps is limited by the high prices, long digestion time and small application scales (crichton et al., ) . but as the industrial enzymes including industrial proteases, lipases and corolase are relatively affordable, they can be used to efficiently digest soft tissues and have good prospect in application (catarino et al., ) . oxidative digestion may digest oms more efficiently than acidic/ alkaline digestion. for example, nuelle et al. ( ) showed that % of hydrogen peroxide (h o ) was the ideal solution to digest oms and it achieved higher digestion efficiency than % of hcl and , , and % of naoh. different concentrations of h o are often employed as oxidant to remove oms. zhao et al. ( ) used % of h o to digest labile background organic matter in marine snow samples; shim et al. nuelle et al. ( ) also reported that incubation using % of h o at room temperature for days could only remove % of biogenic material. therefore, the efficiency of oxidative digestion by h o varies with its concentration and is related to the incubation temperature. a combined use of h o oxidization and enzymatic digestion has been shown in the study of karlsson et al. ( ) . nevertheless, degradation and color change of mps and the generation of foam after being treated by h o seem unavoidable according to avio et al. ( b) . fenton's reagent is an advanced oxidant as well as a potential alternative to h o . the u.s. national oceanic and atmospheric administration marine debris program has made a recommendation that % of h o is used in the presence of an iron (fe(ii)) catalyst for extracting mps from water and sediment samples (li et al., ; masura et al., ; he et al., ) . when comparing with h o , the fenton digestion can effectively destroy organic components and inorganic compounds that cannot be easily digested by h o (tagg et al., ) . besides, it takes shorter time to finish the whole reaction and makes lower demand on reaction temperature, but the ph of the fenton's reagent must be adjusted (to . - . ) to optimize the reaction (hermosilla et al., ) . the diverse components, shapes, sizes and sources of mps make the characterization of mps an important but arduous work. in addition, the characterization of mps is particularly vital as it allows obtaining more information about the sources and chemical composition of them. a comparison among different characterization methods is presented in table , where the requirement for samples as well as advantages and disadvantages of each method was shown. direct observation of mps by visual inspection is adopted in most previous studies (lares et al., ; wagner et al., ) . microscopes including ordinary optical microscope, stereoscope, fluorescent microscope and scanning electron microscope can be used to record the classification and abundance of large mps with distinctive colors or morphologies (politikos et al., ; vermeiren et al., ; wang et al., c) . however, visual inspection is a subjective method for the identification of mps because different observation results may be obtained by different observers, and a considerable amount of time is needed to conduct this work (prata et al., a) . furthermore, it is rather difficult to distinct those mps from other organic or inorganic matters whose particle sizes are similar to mps (fries et al., ) . thus, spectroscopic approaches such as fourier transform infrared spectroscopy (ftir), raman spectroscopy and pyrolysis-gas chromatography coupled to mass spectrometry (pyr-gc-ms) serve as more accurate methods to identify the polymer types of mps. owing to the attributes of cost, efficiency and reliability, these methods are widely used and highly recommended by many researches (cauwenberghe and janssen, ; cho et al., ; dümichen et al., dümichen et al., , li et al., ; ruiz-orej� on et al., ; thushari et al., ) . the polymer types of mps can be identified by comparing the ftir spectra with known spectra. ftir can identify mps with a minimum particle size of μm while raman spectroscopy allows the characterization of mps whose particle size is smaller than μm . multiple ftir techniques have been applied to analyzing mps. for instance, ftir microspectroscopy (μ-ftir) can detect smaller mps ( - μm) and provide high-resolution map (harrison et al., ; l€ oder et al., ; rocha-santos and duarte, ) ; attenuated total reflectance-ftir (atr-ftir) is capable of identifying thick or opaque mps (nor and obbard, ; shim et al., ; prata et al., a) . stimulated raman scattering (srs) is suitable to characterize mps on low raman background filter membrane when no pretreatment processes is conducted (zada et al., ) , but the cost of srs is relatively high. as raman spectroscopy (including micro-raman spectroscopy (μ-raman)) can identify extremely small-sized mps, it is always used table basic information of frequently-used characterization instruments of mps (atr-ftir: attenuated total reflectance-ftir; μ-ftir: together with ftir spectroscopy (ghosal et al., ; karami et al., ) . however, the accuracy of raman spectroscopy would be affected by colored matters, additives and other pollutants that are attached to the mps (lenz et al., ) . pyr-gc-ms has also been applied to analyzing the chemical composition and structural characteristics of higher molecular weight polymers by detecting their thermal degradation products (nuelle et al., ) . fries et al. ( ) used sequential pyr-gc-ms to simultaneously identify polymer types of mps and analyze the content of organic plastic additives. this method could provide a more complete overview of the sample and could more accurately evaluate the actual chemical property of the sample (tianniam et al., ) . nevertheless, wrong results may be obtained by this method for different polymers may produce similar thermal degradation products (nuelle et al., ) . besides, pyr-gc-ms is destructive to samples and cannot provide any information about the sizes, shapes and concentrations of mps (fabbri et al., ) . another deficiency of this method is that it can only analyze a very small number of simple and homogenous samples ( . mg) per run (dümichen et al., ) . comparing with pyr-gc-ms, thermo-extraction and desorption coupled to gas chromatography-mass spectroscopy (ted-gc-ms) has been used to measure relatively large numbers of sample masses ( mg), and it is able to analyze complex and heterogeneous samples (dümichen et al., ) . apart from that, pretreatment of standard mps particles is not required when employing ted-gc-ms to characterize mps (elert et al., ) . liquid chromatography is a less frequently used characterization method of mps, and it is usually applied with the assist of uv detection. elert et al. ( ) used size exclusion chromatography analyze ps and pet, and they found that changes in the molecular mass could be observed. based on the different solubility of mps, polymers can be dissolved by appropriate eluents. the advantage of this method is that larger numbers of samples can be analyzed and thus it can improve the representativeness of the characterization, but it is also destructive to samples and only shows the chemical composition of the samples. different molecular weight distribution can be measured by high performance liquid chromatography (hplc) coupled with size exclusion systems. as shown in the studies of hintersteiner et al. ( ) , hplc-uv and hplc-orbitrap-ms have high recoveries of selected polymers, although this method cannot determine the particle size of the samples and can only analyze certain types of polymers such as ps and pe. therefore, more investigations are required to verify this method for mps identification. several less frequently used methods for the characterization of mps have been reported in some studies. for instance, ceccarini et al. ( ) used gel permeation chromatography to determine the molecular weight and molecular weight distribution of the polymeric fractions; turner ( ) applied a portable x-ray fluorescence spectrometer to analyzing the elemental compositions of polymers; shim et al. ( ) has reported a simple staining method by using nile red to stain highly hydrophobic polymers, and which could differentiate fragmented polypropylene particles from sand particles. mps have complicated chemical characterizations and are potential carriers for other contaminants such as heavy metals and persistent organic pollutants. moreover, mps may also undergo complex natural processes and dramatic morphological change in the environment (fu et al., ) . therefore, it is urgent to develop new analytical technologies or combine existed methods to obtain more reliable characterization data of mps. the aforementioned section showed the abundance of mps in several typical freshwater systems in china, which proved that mps had wide distribution in the environment. as a result, mps may pose potential threat to the environment even humans; however, little is known about their actual effects on organisms and human health. it is noticeable that increasing attention has been paid to the ecotoxicology of mps, and escalating studies have investigated possible adverse effects of mps on different organisms in recent years. toxicological experiments usually used known polymer types of mps to study the according impacts. therefore, it is necessary to study the ecotoxicological effects of mps after polymer types and physic-chemical properties of mps were analyzed. nowadays, research focus of the impact of mps on aquatic organisms is on marine organisms. it was reported that plankton, bivalves, fish and mammals could be affected by the presence of mps (besseling et al., ; cauwenberghe and janssen, ; desforges et al., ; foekema et al., ) . evidence has shown that mps exist in manifold variety of organisms, which suggests the transport of mps along food chain and food web (besseling et al., ) . besides, as ingested mps mainly accumulate in the gastrointestinal tract, it could be deduced that one of the major risks that mps might cause to organisms is physical and chemical damage to their digestive process (davidson and dudas, ; wright et al., b) . negative effects of mps on different aquatic organisms such as growth inhibition and low reproduction rate were found in some studies. effects of mps on three classifications including plankton, invertebrate and vertebrate were summarized in table . from table , it could be seen that ps is the most frequently used material to investigate the toxicity of mps on plankton. another characteristic of the effects of mps on plankton is that small-sized mps are more easily to exert detrimental influence. one of the possible reasons might be that extremely smallsized mps could act as the substitute of nutrients that plankton needed, resulting in a sustained loss of energy inputs, and consequently leading to death of organisms (botterell et al., ) . impacts of mps on phytoplankton were seen on microalgae. some studies have suggested that the growth rate, chlorophyll content and photosynthesis activity of microalgae were negatively affected by the presence of mps (prata et al., a; xu et al., ) . moreover, according to the investigation of prata et al. ( b) , the disturbing effects of mps could decrease available nutrients that microalgae needed by functioning as substrates, which revealed a potential assimilation metabolism of mps by microalgae. as surface waters contain relatively high abundance of mps than deep water, zooplankton which predominately feeds in this area is more easily to encounter and take in them (c� ozar et al., ) . known effects of mps on zooplankton include but not limited to inhibition of growth and feeding, reproduction and life span (botterell et al., ) . for example, cole et al. ( ) demonstrated that mps could influence the feeding of copepod, and the ingestion rates of copepod centropages typicus were testified to have a significant dose-response relationship with mps concentration and total algal ingestion rates. lee et al. ( ) showed that the fecundity of copepod tigriopus japonicas was decreased by exposing to different concentrations of ps microbead. lo and chan ( ) found that gastropod crepidula onyx could ingest ps microbead, which led to slow growth. ecotoxicologic experiments that investigate the effects of mps on invertebrate often utilize mytilus galloprovincialis as a bioindicator, by which the quality of marine environments can be evaluated and monitored (capillo et al., ) . as invertebrate usually have no specific enzymes to digest synthetic polymers, and thus once mps are ingested by these organisms, they will not be digested or absorbed (andrady, ) . however, the ingestion of mps might cause a series of problems to creatures. some investigations showed that the main adverse effect of mps is cellular damage resulting from oxidative stress (avio et al., a; pagano et al., ; sureda et al., ) . these studies demonstrated that mps could reduce the energy input and further damage the immune functions, and continuous accumulation of mps could also exert chronic effects on the life span of invertebrates. the ecotoxicological effects of mps on vertebrate are represented by the influences that mps could have on fishes, for fishes are typical vertebrates in aquatic environments. as shown in some studies, major impacts of mps on fishes include intestinal damage, oxidative damage and reduction of predatory activity (ferreira et al., ; proki� c et al., ) . because fishes are important food for humans, chances are that individual fitness and population fitness would be decreased when mps-polluted fishes are consumed by human. another nonnegligible fact is that mps could change the bioavailability and ways of adsorbing metal contaminants, which indicate that other pollutants such as heavy metals could co-exist with mps, and might cause certain co-effect to aquatic organisms (guzzetti et al., ) . mammals such as whales were also reported to have mps ingestion and bioaccumulation. fossi et al. ( ) showed that organochlorine compounds were found in fin whales suggesting that mps might have potential effects on them. nevertheless, the metabolism mechanisms and effect mechanisms of mps on aquatic organisms are still remained to be explored. the investigation of ng et al. ( ) has showed that % of global plastic wastes is accumulated in terrestrial environment. nevertheless, compared with the aquatic environments, the studies of mps in soils are relatively insufficient. mps can change the structure and function of soils and can also affect microbial diversity (rillig, ) . whether soils are health or not determines food quality as well as food safety, which will ultimately threaten human health (murugan et al., ) . on the other hand, complex terrestrial environment is also able to impact the ecotoxicology of mps, and interaction between mps and soils may lead to unpredictable effects on the environmental behavior of other contaminants, which could result in more complicated ecotoxicological impact on soil (guo et al., ) . the effects of of mps on terrestrial plants are scarcely reported, and only limited plants are investigated. the study of jassby et al. ( ) showed that mps might be taken in by agricultural plants such as wheat and then further entered into food chain. the experiments of qi et al. ( ) demonstrated that the presence of mps had negative impacts on wheat growth tissue elemental composition at both vegetative and reproductive stages. furthermore, leaf and root traits together with total biomass of allium fistulosum were reported to be affected by three kinds of mps, namely, microfiber, microbead and micro-fragment . this study found that all three kinds of mps would significantly change the biomass, tissue elemental composition (e.g. water content and leaf nitrogen content), root traits (e.g. root length, diameter, density) and root symbioses . moreover, several potential positive and negative mechanisms that mps might work on plants were proposed by rillig et al. ( ) , and their study showed that the effects of mps would vary in terms of plant species. however, these mechanistic pathways should be tested in the near future. mps could exert direct and indirect influence on soil animals. mps may adhere to the surface of some soil animals so that their movement will be hindered . if mps are mistakenly ingested by soil organism, reduce in carbon biomass ingestion and further energy depletion would happen (cole et al., ) . earthworms are the most commonly researched targets when studying the toxic effects of mps on soil animals. wang et al. ( a) found that mps at low concentrations (< % (w/w)) had no significant impacts on growth and death rates of earthworms (eisenia fetida); whereas the growth of oligochaeta lumbricidae was significantly inhibited when the concentrations of mps were higher than % (huerta lwanga et al., ) . in addition to earthworms, other small soil invertebrates including springtails, nematodes and snails could also ingest mps. for instance, mps could decrease the gut microbial community, reproduction and avoidance behaviors of springtails ju et al., ) ; mps could disrupt energy metabolism, lower down the locomotor behavior and decrease the body length of nematodes (caenorhabditis elegans) lei et al., ) ; mps inhibited food intake and excretion of snails (achatina fulica) and affected oxidative stress (song et al., ) . the indirect effects of mps mainly refer to their adsorption and combination of other pollutants such as heavy metals and organic contaminants, which will aggravates soil pollution and amplify the risks posed to soil organisms and humans (hüffer et al., ; wang et al., a) . results from hodson et al. ( ) suggested that the bioavailability of zinc increased by adsorbed onto mps, and earthworms ingesting zinc-adsorbed mps accumulated more zinc in their guts. on the contrary, high concentration of mps in soils might reduce the accumulation of polycyclic aromatic hydrocarbons and polychlorobiphenyls as reported by wang et al. ( b) . therefore, the adsorption and desorption mechanisms of mps to other environmental pollutants need to be further studied. microorganisms as the primary decomposers are one of the good indicators of the soil health condition, but current studies of the effects of mps on soil microorganisms are limited. soil properties are closely related to soil microbial activity. it was held that the transport of mps in soils might change soil microbial community . changes in soil aggregation by mps could affect microbial evolution, and mps could provide a novel ecological habitat for soil microorganisms wang et al., b) . on the other hand, mps could also change soil porosity and soil moisture, which would affect the relative distribution of anaerobic and aerobic microorganisms in soils (rubol et al., ) . changes in soil pores may result in loss of microhabitat and extinction of indigenous microorganisms (veresoglou et al., ) . bacteria, fungi and soil enzymes are three parameters to assess the oliveira et al. ( ) impacts of mps on soil microbiota. for example, some bacteria (vibrionaceaeor and pseudoalteromonadaceae) were detected in large amounts on the surface of microplastic debris, while they were seldom found in natural soil surroundings (tender et al., ) . bl€ asing and amelung ( ) reported that fungus zalerion maritimum could biodegrade pe and promote their growth. however, mps were also found accumulated in fungi such as yeasts and filamentous fungi (chae and an, ) . as for soil enzymes, both positive and negative effects of mps were showed. liu et al. ( ) demonstrated that enzymatic activity was enhanced by the addition of mps, benefiting the input of c, n and p into soils. whereas adding small polyacrylic and pe particles would have negative effects on the enzymatic activity of fluorescein diacetate hydrolase, as shown in the study of de souza machado et al. ( ) . to sum up, how the presence of mps in soil affected microorganisms still remained unknown, and more efforts should be made to address issues in this field. mps are ubiquitous in the environment, which makes it unavoidable for humans to be exposed. nevertheless, currently there are inadequate studies on health risks that mps may cause to human. similar to other pollutants, humans contact mps mainly via three main pathways, namely ingestion, inhalation and dermal exposure. correspondingly, the effects of mps on human health are embodied in these aspects, but dermal contact was rarely reported to have significant influence on human health., as only particle with sizes < nm could transverse dermal barrier (revel et al., ) . overall, mps exposure to humans may result in particle toxicity, with oxidative stress, inflammatory lesions and increased uptake or translocation, and as the immune system cannot remove mps, it may cause chronic inflammation and increase risk of neoplasia (prata et al., ) . ingestion is regarded as the major way that humans expose to mps . as mps were detected in some food such as fish and table salt, the consumption of these stuffs might lead to possible ingestion of mps by humans karami et al., ) . mps could enter into the gastrointestinal system once mps-polluted foodstuff are taken up by human, specialized m-cells in the intestine might absorb small microplastic particles, and consequently covering intestine lymphoid tissues, causing inflammatory response and changing gut microbe composition and metabolism (ensign et al., ; salim et al., ) . powell et al. ( ) suggested there was a possibility that insoluble mps might penetrate intestinal mucus to get solubility increased. impacts including gene expression, inhibited cell viability, induced pro-inflammatory responses and morphological changes were recorded in the research of forte et al. ( ) , when mps were internalized by human gastric adenocarcinoma cells. although food and drinking water are widely contaminated by mps making mps ingestion unavoidable by humans, how mps negatively affect human health is little known for relative studies are limited. because mps can release into air from various sources such as synthetic textiles and car tires, humans might also inhale mps in the environment. dris et al. ( ) showed that mps concentrations in outdoor air ranging from . to . particles/m . moreover, the study of prata ( ) estimated that average mps concentration inhaled by individuals was - mps/d. respiratory symptoms such as dyspnea and interstitial inflammatory responses might be induced when considerable amount of mps were inhaled by susceptible individuals, especially those who exposed to microplastic environments (industrial workers) for a long time (prata, ) . a couple of studies showed that interstitial lung disease occurred in synthetic textile, flock, vinyl chloride and pvc industries (atis et al., ; pimentel et al., ; xu et al., ) . studnicka et al. ( ) reported that vinyl chloride and pvc industries workers who exposed for years had diseases like pneumoconiosis, narrowing of bronchial segments, foreign body granulomas, and fibrotic changes of the alveolar wall. in addition, cancer risk may be increased when chronically exposing to synthetic fiber dust, though more confirmed evidences were not founded (mastrangelo et al., ) . studies of mps adverse effects on human health are rather limited. nonetheless, as the accumulation of mps along tropic chain, the potential health risks that mps may cause to humans should not be ignored. moreover, measurement of human exposure to mps should be more precisely conducted by using diagnostic tools including mps identification and toxicity determination. further studies focusing on human exposure to mps are urgently needed to fully understand the risks of mps to humans. this work presented a comprehensive review on the status quo of abundance, distribution, sampling and analyses of mps in both waters and soils. moreover, potential ecological risks that mps may pose to the environments were also elucidated. four key conclusions were made as follows: ( ) freshwater environments in cities with dense population and large agricultural, fishery and industrial scales contained higher mps concentrations, but mps were also detected in rural freshwater systems. mps with particle size less than mm were most abundant and microfiber was a common polymer shape in surface waters and sediments of several typical freshwater systems in china. besides, among all the detected mps samples, polymer types of pe and pp were the most common two. ( ) zooplankton nets were the major tools for sampling mps from waters, and forceps, sieves, grab samplers, box corers and spade corers were often used to collect mps from sediments. however, deficiencies including limited sampling volumes and nonuniform sampling methods have hindered quantification of mps in the environment. ( ) pretreatment methods including density separation, filtration, acidic/alkaline digestion, oxidization and enzymatic digestion could efficiently remove impurities from mps samples. advanced characterization technologies such as ftir, raman and pyr-gc-ms were frequently employed to analyze the chemical composition and other properties of mps. ( ) mps have been reported to have ecological impacts on aquatic organisms, soil biota and human health. inhibition in growth and decrease in assimilation efficiency were seen in both aquatic and terrestrial organisms. digestive and respiratory damages to humans might be caused by the ingestion and inhalation of mps. nevertheless, metabolic mechanisms of mps in organisms and humans were generally lacking. although the studies of mps have been springing up in the last decade, research gaps still existed and should be filled in the future. suggestions were proposed on the grounds of the current knowledge of mps: ( ) current studies provided limited perspectives and information about mps in freshwaters and soils due to that the research of mps is first focused on marine environment. the study of mps including their occurrence, fate and toxicity in freshwater and terrestrial environment are still insufficient. in addition, freshwater as the source of drinking water and soil as the matrix of cereals and vegetables, both of them are closely related to human health. thus, more studies should be conducted to better understand how mps distribute and influence freshwater and soil health. ( ) different sampling and analytical methods will lead to different results of mps concentration and chemical properties. it is known that mps can act as carriers of other contaminants including heavy metals and persistent organic pollutants. furthermore, mps may undergo complicated environmental processes such as solar radiation, mechanical abrasion and biodegradation, which will hamper the characterization and quantification of mps. as a result, it is necessary to establish a standard and reliable procedure to sample, separate, purify and identify mps. noval characterization tools or a proper combination use of existed technologies are needed to analyze mps in complex environmental matrix. ( ) the detrimental effects of mps on soil biota and humans are inadequately researched compared with aquatic organisms, so that future investigations should concentration on the two aspects. besides, the mechanism that mps interact with different organisms in nature is remained to be revealed. realistic mps samples in the environment are rather complicated, and thus complex composition of mps and actual exposure conditions should be considered when carrying on ecotoxicological experiments in laboratory. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. microplastics in the marine environment the respiratory effects of occupational polypropylene flock exposure pollutants bioavailability and toxicological risk from microplastics to marine mussels experimental development of a new protocol for extraction and characterization of microplastics in fish tissues: first observations in commercial species from adriatic sea marine microplastic debris: an emerging issue for food security, food safety and human health protected areas in the atlantic facing the hazards of micro-plastic pollution: first diagnosis of three islands in the canary current high quantities of microplastic in arctic deep-sea sediments from the hausgarten observatory a standardized method for sampling and extraction methods for quantifying microplastics in beach sand microplastic in a macro filter feeder: humpback whale megaptera novaeangliae fate of nano-and microplastic in freshwater systems: a modeling study nanoplastic affects growth of s. obliquus and reproduction of d. magna physical adsorption of charges plastic nanoparticles affect algal photosynthesis microplastics affect assimilation efficiency in the freshwater amphipod gammarus fossarum plastics in soil: analytical methods and possible sources bioavailability and effects of microplastics on marine zooplankton: a review accumulation of microplastic on shorelines worldwide: sources and sinks assessment of electrolytes and metals profile of the faro lake (capo peloro lagoon, sicily, italy) and its impact on mytilus galloprovincialis development and optimization of a standard method for extraction of microplastics in mussels by enzyme digestion of soft tissues microplastics in bivalves cultured for human consumption the hidden microplastics: new insights and figures from the thorough separation and characterization of microplastics and of their degradation byproducts in coastal sediments current research trends on plastic pollution and ecological impacts on the soil ecosystem: a review abundance and characteristics of microplastics in market bivalves from south korea a potpourri of microplastics in the sea surface and water column of the mediterranean sea microplastic in the surface waters of the ross sea (antarctica): occurrence, distribution and characterization by ftir occurrence and distribution of microplastics in marine sediments along the belgian coast microplastic ingestion by zooplankton isolation of microplastics in biota-rich seawater samples and marine organisms neustonic microplastic and zooplankton in the north western mediterranean sea optimisation of enzymatic digestion and validation of specimen preservation methods for the analysis of ingested microplastics plastic debris in the open ocean a novel, density-independent and ftir-compatible approach for the rapid extraction of microplastics from aquatic sediments microplastic ingestion by wild and cultured manila clams (venerupis philippinarum ) from baynes sound, british columbia microplastics can change soil properties and affect plant performance impacts of microplastics on the soil biophysical environment microplastics in seafood: benchmark protocol for their extraction and characterization occurrence and spatial distribution of microplastics in sediments from norderney widespread distribution of microplastics in subsurface seawater in the ne pacific ocean ingestion of microplastics by zooplankton in the northeast pacific ocean microplastics in surface waters and sediments of the three gorges reservoir microplastics in surface waters and sediments of the wei river, in the northwest of china plastic particles in coastal pelagic ecosystems of the northeast pacific ocean a first overview of textile fibers, including microplastics, in indoor and outdoor environments suspended microplastics and black carbon particles in the jade system assessment of a new method for the analysis of decomposition gases of polymers by a combining thermogravimetric solid-phase extraction and thermal desorption gas chromatography mass spectrometry fast identification of microplastics in complex environmental samples by a thermal degradation method microplastics in freshwater systems: a review of the emerging threats, identification of knowledge gaps and prioritisation of research needs comparison of different methods for microplastic detection: what can we learn from them, and why asking the right question before measurements matters? oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers microplastic pollution in the surface waters of the laurentian great lakes analysis of poly (vinyl chloride) and other polymers in sediments and suspended matter of a coastal lagoon by pyrolysis-gas chromatography-mass spectrometry effects of multistressors on juveniles of the marine fish pomatoschistus microps: gold nanoparticles, microplastics and temperature plastic in north sea fish polystyrene nanoparticles internalization in human gastric adenocarcinoma cells fin whales and microplastics: the mediterranean sea and the sea of cortez scenarios evidence of microplastics in samples of zooplankton from portuguese coastal waters identification of polymer types and additives in marine microplastic particles using pyrolysis-gc/ms and scanning electron microscopy separation, characterization and identification of microplastics and nanoplastics in the environment interactions of microplastic debris throughout the marine ecosystem molecular identification of polymers and anthropogenic particles extracted from oceanic water and fish stomach -a raman micro-spectroscopy study source, migration and toxicology of microplastics in soil microplastic in marine organism: environmental and toxicological effects a review of analytical techniques for quantifying microplastics in sediments the applicability of reflectance micro-fourier-transform infrared spectroscopy for the detection of synthetic microplastics in marine sediments treatment of organic wastewater containing high concentration of sulfate by crystallization-fenton-sbr optimization of conventional fenton and ultraviolet-assisted oxidation processes for the treatment of reverse osmosis retentate from a paper mill microplastics in the marine environment: a review of the methods used for identification and quantification characterization and quantitation of polyolefin microplastics in personal-care products using hightemperature gel-permeation chromatography plastic bag derived-microplastics as a vector for metal exposure in terrestrial invertebrates microplastics in the terrestrial ecosystem: implications for lumbricus terrestris (oligochaeta, lumbricidae) polyethylene microplastics influence the transport of organic contaminants in soil reducing uncertainty and confronting ignorance about the possible impacts of weathering plastic in the marine environment marine pollution. plastic waste inputs from land into the ocean delivery, uptake, fate, and transport of engineered nanoparticles in plants: a critical review and data analysis effects of polyethylene microplastics on the gut microbial community, reproduction and avoidance behaviors of the soil springtail, folsomia candida a high-performance protocol for extraction of microplastics in fish the presence of microplastics in commercial salts from different countries comparison between manta trawl and in situ pump filtration methods, and guidance for visual identification of microplastics in surface waters screening for microplastics in sediment, water, marine invertebrates and fish: method development and microplastic accumulation efficient microplastics extraction from sand. a cost effective methodology based on sodium iodide recycling uptake of nanopolystyrene particles induces distinct metabolic profiles and toxic effects in caenorhabditis elegans soil microplastics inhibit the movement of springtail species minimising the present and future plastic waste, energy and environmental footprints related to covid- microplastic as a vector for chemicals in the aquatic environment: critical review and model-supported reinterpretation of empirical studies marine microplastic: preparation of relevant test materials for laboratory assessment of ecosystem impacts the effect of wind mixing on the vertical distribution of buoyant plastic debris a comprehensive analysis of plastics and microplastic legislation worldwide occurrence, identification and removal of microplastic particles and fibers in conventional activated sludge process and advanced mbr technology plastics and microplastics: a threat to environment a comparison of neustonic plastic and zooplankton at different depths near the southern california shore river plastic emissions to the world's oceans estimating microplastic-bound intake of hydrophobic organic chemicals by fish using measured desorption rates to artificial gut fluid sorption capacity of plastic debris for hydrophobic organic chemicals size-dependent effects of micropolystyrene particles in the marine copepod tigriopus japonicus polystyrene (nano)microplastics cause size-dependent neurotoxicity, oxidative damage and other adverse effects in caenorhabditis elegans a critical assessment of visual identification of marine microplastic using raman spectroscopy for analysis improvement focus topics on microplastics in soil: analytical methods, occurrence, transport, and ecological risks microplastics in commercial bivalves from china removal of triazophos pesticide from wastewater with fenton reagent distribution patterns of microplastics within the plankton of a tropical estuary occurrence and distribution of microplastics in an urban river: a case study in the pearl river along guangzhou city preparation of size-controlled silver phosphate catalysts and their enhanced photocatalysis performance via synergetic effect with mwcnts and pani response of soil dissolved organic matter to microplastic addition in chinese loess soil negative effects of microplastic exposure on growth and development of crepidulaonyx focal plane array detector-based micro-fourier-transform infrared imaging for the analysis of microplastics in environmental samples interactions between polystyrene microplastics and marine phytoplankton lead to species-specific hetero-aggregation microplastic and macroplastic ingestion by a deep diving, oceanic cetacean: the true's beaked whale mesoplodon mirus a rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with nile red occurrence of microplastics in raw and treated drinking water epidemiologic evidence of cancer risk in textile industry workers: a review and update methods for the analysis of microplastics in beach samples. laboratory methods for the analysis of microplastics in the marine environment: recommendations for quantifying synthetic particles in waters and sediments. noaa technical memorandum abundant plankton-sized microplastic particles in shelf waters of the northern gulf of mexico recovering microplastics from marine samples: a review of current practices monitoring of a wide range of organic micropollutants on the portuguese coast using plastic resin pellets belowground carbon allocation by trees, understory vegetation and soil type alter microbial community composition and nutrient cycling in tropical eucalyptus plantations are nitric acid (hno ) digestions efficient in isolating microplastics from juvenile fish? release of synthetic microplastic plastic fibres from domestic washing machines: effects of fabric type and washing conditions an overview of microplastic and nanoplastic pollution in agroecosystems prevalence of microplastics in singapore's coastal marine environment microplastics in singapore's coastal mangrove ecosystems a new analytical approach for monitoring microplastics in marine sediments single and combined effects of microplastics and pyrene on juveniles ( þ group) of the common goby pomatoschistus microps (teleostei, gobiidae) effects of microplastics and mercury in the freshwater bivalve corbicula fluminea (müller, ): filtration rate, biochemical biomarkers and mercury bioconcentration evaluation of functionality and biological responses of mytilus galloprovincialis after exposure to quaternium- (methenamine -chloroallylochloride) respiratory disease caused by synthetic fibers: a new occupational disease an analysis of european latest plastics production, demand and waste data modeling the fate and distribution of floating litter particles in the aegean sea (e. mediterranean) dietary microparticles and their impact on olerance and immune responsiveness of the gastrointestinal tract airborne microplastics: consequences to human health? environmental exposure to microplastics: an overview on possible human health effects methods for sampling and detection of microplastics in water and sediment: a critical review effects of microplastics on microalgae populations: a critical review ecotoxicological effects of microplastics: examination of biomarkers, current state and future perspectives macro-and micro-plastics in soil-plant system: effects of plastic mulch film residues on wheat (triticum aestivum) growth assessing environmental risks through fuzzy parameterized probabilistic analysis extraction, enumeration and identification methods for monitoring microplastics in the environment validation of density separation for the rapid recovery of microplastics from sediment description of the small plastics fragments in marine sediments along the alang-sosiya ship-breaking yard micro(nano)plastics: a threat to human health? microplastic in terrestrial ecosystems and the soil? evolutionary implications of microplastics for soil biota microplastic effects on plants a critical overview of the analytical approaches to the occurrence, the fate and the behavior of microplastics in the environment improving cost-efficiency for mps density separation by zinc chloride reuse modeling soil moisture and oxygen effects on soil biogeochemical cycles including dissimilatory nitrate reduction to ammonium (dnra) floating plastic debris in the central and western mediterranean sea air pollution effects on the gut microbiota microplastic and charred microplastic in the faafu atoll identification methods in microplastic analysis: a review identification and quantification of microplastics using nile red staining uptake and adverse effects of polyethylene terephthalate microplastics fibers on terrestrial snails (achatina fulica) after soil exposure pneumoconiosis and systemic sclerosis following years of exposure to polyvinyl chloride dust microplastics in taihu lake acute exposure to sunscreen containing titanium induces an adaptive response and oxidative stress in mytillus galloprovincialis fenton's reagent for the rapid and efficient isolation of microplastics from wastewater sequential vertical flow trickling filter and horizontal flow reactor for treatment of decentralized domestic wastewater with sodium dodecyl benzene sulfonate bacterial community profiling of plastic litter in the belgian part of the north sea lost at sea: where is all the plastic? effects of microplastics on sessile invertebrates in the eastern coast of thailand: an approach to coastal zone conservation pyrolysis gc-ms-based metabolite fingerprinting for quality evaluation of commercial angelica acutiloba roots microplastic pollution in the marine waters and sediments of hong kong in situ elemental characterisation of marine microplastics by portable xrf adsorption of trace metals by microplastic pellets in fresh water extinction risk of soil biota microplastic identification and quantification from organic rich sediments: a validated laboratory protocol microplastic particles in sediments of lagoon of venice, italy: first observations on occurrence, spatial patterns and identification novel method for the extraction and identification of microplastics in ocean trawl and fish gut matrices negligible effects of microplastics on animal fitness and hoc bioaccumulation in earthworm eisenia fetida in soil microplastics as contaminants in the soil environment: a mini-review microplastics in the surface sediments from the beijiang river littoral zone: composition, abundance, surface textures and interaction with heavy metals microplastics pollution in inland freshwaters of china: a case study inurban surface waters of wuhan microplastics in surface waters of dongting lake and hong lake sem/eds and optical microscopy analyses of microplastics in ocean trawl and fish guts microplastic ingestion decreases energy reserves in marine worms the physical impacts of microplastics on marine organisms: a review insights into atrazine degradation by persulfate activation using composite of nanoscale zerovalent iron and graphene: performances and mechanisms occurrence and fate of microplastic debris in middle and lower reaches of the yangtze river -from inland to the sea pulmonary toxicity of polyvinyl chloride particles after a single intratracheal instillation in rats. time course and comparison with silica microplastics in aquatic environments: occurrence, accumulation, and biological effects simultaneous removal of multicomponent vocs in biofilters microplastic pollution in table salts from correlations of nonlinear sorption of organic solutes with soil/sediment physicochemical properties comparison of the abundance of microplastics between rural and urban areas: a case study from east dongting lake accumulation of polystyrene microplastics in juvenile eriocheir sinensis and oxidative stress effects in the liver study on the hydrocyclonic separation of waste plastics with different density microplastic abundance, distribution and composition in water, sediments, and wild fish from poyang lake fast microplastics identification with stimulated raman scattering microscopy microplastics in oceans microplastic pollution of lakeshore sediments from remote lakes in tibet plateau a simple method for the extraction and identification of light density microplastics from soil an approach for extraction, characterization and quantitation of microplastic in natural marine snow using raman microscopy effect of zinc ions on nutrient removal and growth of lemna aequinoctialis from anaerobically digested swine wastewater microplastics in a marine environment: review of methods for sampling, processing, and analyzing microplastics in water, bottom sediments, and coastal deposits key: cord- -a ni s e authors: jackson, ronald s. title: wine, food, and health date: - - journal: wine science doi: . /b - - - - . -x sha: doc_id: cord_uid: a ni s e wine has historically been associated with religious rights, used as a salubrious beverage, employed as a medication as well as a medicinal solvent, and consumed as a food accompaniment. it is the last use that is most intimately associated in the minds of most modern consumers. despite this, there is little flavor commonality on which pairing could be based. the first section of the chapter examines this feature and wine's primary role as a palate cleanser and food condiment. the synergistic role of food and wine in suppressing each other's least pleasant attributes is also explained. the final section deals with the latest evidence relating to the many beneficial health effects of moderate wine consumption, shortfalls in the data, headache induction, dental erosion, and conditions under which wine intake is contraindicated. wine and its association with food have generated an incredible cornucopia of literature. amazingly, few of the views and assumptions have been subjected to scientific scrutiny. only a few research papers deal with the topic. this may relate to their subject disciplines (enology and food science) being in separate departments. also, the funding is different. that for enology and viticulture comes largely from government or industry grants, often based on levies on vineyards and wineries. funding in the food sciences comes largely from commercial firms, much of it done internally by food companies, and such research remains proprietary. funding from disparate industries provides little incentive for collaboration on interdisciplinary projects, such as food and wine combination. much of what is written is by those with an arts rather than a science background. sommeliers, for whom the combination of wine and food is their field, are more practitioners than researcher/academicians. thus, the topic has experienced languor, devoid of scientific rigor. only recently have scientists ventured into a subject normally the prerogative of the epicurean. scientists, being inherently doubters, demand experimental verification. the latter is difficult as designing adequate controls is next to impossible. in addition, the laboratory conditions, required for experimentation, make the relevance of finding to "real life" situations dubious. with food and wine combinations, the social context is often more important to perception than actual sensory clues. finally, social pressures often lead to acceptance of supposed norms. what follows is a brief discussion of the issue of food and wine combination and what can be said with a degree of confidence. in addition, it is hoped that it will provide "food for thought" to those seriously interested in wine. the traditional view is that wine is in some fundamental way designed to be consumed with food. however, when one searches for evidence, it seems to be a mythda view so oft repeated as to be taken as gospeld an example of social contagion of collective memory. wine appears to have been the standard food beverage in the greek or roman worlds. in the near east and egypt, wine was almost the exclusive preserve of the secular and religious oligarchy. in contrast, beer was the beverage of the laboring classes. wine is mentioned in the bible but recommended primarily in relation to religious ceremonies and weddings. it is only in regions where wild grapes grew indigenously that vines came to provide the drink for the masses. its acidity, alcohol, and phenolic contents enhanced its antimicrobial property, making it considerably safer to drink than water. wine's mildly antiseptic properties became increasingly important as population numbers grew, hygienic conditions deteriorated, and water supplies became more polluted. although production was seasonal (unlike beer based on dry grains), wine's comparative resistance to spoilage (isolated from air) permitted wine to become the preferred beverage. in addition, wine's higher alcohol content could temporarily numb the afflictions of a peasant life. although safe and nutritious (calorie rich), there is nothing in its attributes that inherently makes wine ideal as a food beverage. it is actually better suited for one of its ancient secondary roles, a solvent in preparing extracts from medical herbs. the sharp acidity of many ancient wines, often extended with aged seawater and vinegar (especially for slaves), can hardly be considered an ideal food accompaniment, at least from a modern perspective. the resinous flavor donated by storage in standard amphoras (made watertight with an inner coating of pitch) would not have facilitated detecting any subtle fragrances they might have had. the quality of the ordinary wine available to the average roman is probably best left to the imagination. finer wines were available for the patrician classes. these were probably stored in amphoras with a vitreous inner surface, without the need for a pitch lining. the most famous seem to have been or became highly concentrated with age, being almost syrup-like, usually diluted before being consumed. several roman poets eulogized the wonders of particular vintages. even modern wines, with their predominant acidic, bitterish, and astringent character have little to suggest food compatibility. these characteristics are simply the natural consequence of grape chemistry, not conscious intent. admittedly, consuming wine with food does mollify its less pleasant aspects, unless one develops an appreciation for sour beverages with a bitter/astringent aspect. some humans seem adept at coming to appreciate, even crave, perceptions initially repulsive to painful. black coffee, capsicum peppers, durian, and limburger cheese are classic examples. in some instances, the iron content of the wine can induce a metallic sensation, by catalyzing lipid oxidation. this may be masked in combination with food. however, this appears to be unrelated to the fishy aftertaste associated with some white wines taken with seafood (tamura et al., ) . in most "compatible" combinations, both the cheese and wine appear betterdnot by directly enhancing their respective qualities but by mutually suppressing their less pleasing attributes (nygren et al., (nygren et al., , a (nygren et al., , b . other studies have extended these findings, lending support to the view that red wines pair better with cheeses, due to wine tannins appearing more silky (bastian et al., ) . the fatty acid content of the cheese may also contribute to reduced bitterness (homma et al., ) , possibly by coating taste receptors. lipoproteins, typically found in foods, can react with taste receptors, suppressing the perception of bitterness (katsuragi et al., ) . however, the bitter-suppressing aspect of cheeses may also relate to its salt content (frijters and schifferstein, ; breslin and beauchamp, ; keast et al., ) or the presence of glutamate and adenosine monophosphate (keast and breslin, ) . the fruity aspect of wine may also be enhanced when sampled with cheese (galmarini et al., ) . although most cheese/wine associations seem mutually beneficial, some are not, for example, sweet wines and cheese (bastian et al., ) . that compatibility originates from the negation of unpleasant sensations may not be what wine pundits and sommeliers profess, but it seems closer to the truth. this phenomenon may also apply to many supposedly food and wine "marriages." salt's suppression of bitterness (nakamura et al., ) might explain the seemingly ancient habit of adding seawater to wine, e.g., oinos thalassikos (younger, , p. ) . pliny (historia naturalis, . ) also records that salt enhanced the perceived smoothness of wines. columella (de res rustica . ) recommends the addition of salt, apparently to avoid moldy tastes. he also notes salt addition in a recipe for preparing "greek" wine. salt water was even used until recently in preparing new barrels to receive wine (nègre and françot, ) . salt is well known as a flavor enhancer. this may involve disrupting weak, nonvolatile complexes between matrix and aromatic compounds, promoting their liberation and retronasal detection (linscott and lim, ) . in addition, sodium ion hydration may decrease "free water," changing solution polarity. although salt increases aromatic volatility, saltiness is by itself appreciated (bolhuis et al., ) . when one searches for affinities among the attributes of food and wine, one comes up empty-handed. in contrast, there is extensive incongruity. table wines possess gustatory attributes predominantly characterized by sourness, bitterness, astringency, and burning sensations. although pronounced sour tastes are inherently unpleasant, wine's acidity is of value when used as a marinadedpromoting acid-induced hydrolysis of food proteins. wine phenolics can also act as antioxidants, reducing the toxicity of heterocyclic amines (viegas et al., ) and acrylamide (qi et al., ) generated during frying (viegas et al., ) . phenolics are also antimicrobial (nisiotou et al., ) . by comparison, sourness is a rare attribute in most world cuisines (see moskowitz et al., for a marked exception). acids typically are added only as a component in some condiments or flavorants, notably vinegar, lemon juice, or tamarind. they can enhance the flavor of otherwise bland foods. the bitterness and astringency of most red wines also find no equivalent in meat and fish. the protein content of food reacts with both wine acids and phenolics, limiting their activation of taste and touch receptors. in comparison with wines, solid foods are characterized salty, savory (glutamate), sweet, and sebaceous (fatty acids) sensations. sour, bitter, astringent, and hot spicy attributes are (or have been) less common in western cooking and then usually in condiments. the inherent, aversive reactions to such sapid sensations probably arose as a protective response to avoid or limit the consumption of potentially toxic (or rotten) foods. conversely, bitter/astringent/toxic compounds were probably selected during plant evolution to discourage their consumption, with the principal exception of ripe fruit. during domestication, crop variants with reduced aversive and enhanced pleasant-tasting constituents have been propagated. thus, lettuce and other vegetables became less bitter; apples, cherries, and other fruits sweeter and less sour or astringent, citrus fruit less acidic, and legumes less flatulent. food preparation, notably cooking, further facilitated the inactivation or removal of potential food toxins and antimetabolites. examples include fungal toxins, potato alkaloids, and casava cyanogenic glycosides. cooking meat also facilitates digestion (promoting collagen and protein fiber breakdown) and enhances flavor. disappointingly, some cooking processes generate their own toxins, notably roasting and searing. examples are acrylamide (a maillard by-product) and a variety of toxic, pyrolytic, smoke by-products. fermentation is another ancient technique that helped destroy antimetabolites. an example is the action of rhizopus oligosporus degrading soybean flatulence compounds during tempeh production. lactobacillus can also destroy soy saponins. fermentation also has the potential to break down difficult-to-digest oligosaccharides as well as help preserve perishable foods. the aromatic aspects of food and wine equally show little similarity, on which supposed compatibility could be based. wine aromas are most frequently described in terms of fresh fruit, jam, or flowers. none of these is characteristic of the main components of a meal and would be considered odd if present. the hints of "apple" in chardonnay wine may be compatible with chicken, the "pepper" of a shiraz pair with pepper steak, and the "walnut" of some sherries combine with nutcontaining salads (without vinaigrette). however, does the box wood or cat urine of sauvignon blanc, the rose of riesling, and the black currant of cabernet sauvignon really match with any main course? in addition, does the vanilla/coconut of oak or the leather aspect of aged red wines have any inherent compatibility with food? the supposed spiciness of gewü rztraminer wines is given as a reason for its combination with spicy asian foods. however, the wine has more litchi aroma than spiciness, making the stated logic dubious. it is only its intense aroma that may permit its presence to remain noticeable. personally, the best aspect of wine and food association is not their complementary natures but their contrasts. each cleanses the palate between alternate samplings, allowing fresh perceptions of each. thus, wine permits swift shifts in savory sensitivity. without comparable tastes or flavors, where is the supposed inherent rapport between food and wine? is its only justification the reduction of undesirable aspects of the partnership and as a palate cleanser? certainly, wine's ability to partially rinse the palate between food samples is important. it offers both gustatory, olfactory, and trigeminal receptors time to reestablish their native receptive state, countering adaptation and loss of sensory appeal. thus, food appreciation is enhanced by being sampled afresh. wine can, unromantically, be viewed as a savory mouthwash. in addition, volatility of food and wine flavorants is influenced by the dynamically changing concentrations of ethanol, phenols, carbohydrates, etc. supplied with the wine. conversely, food dilutes, masks, and eliminates most wine flavors. the result being that the next sample can be appreciated to its full, adaptation having been avoided. the other aspect of potential compatibility arises from their dissimilar attributes. they act in concert, in a manner similar to condiments, providing flavor accents to enhance and maintain flavor interest throughout the meal. the result can be the creation of a stimulating holistic experience. the typical starchy elements in a meal (rice, potatoes, pasta, bread) supplement the synergy, helping to cleanse the palate and generate a feeling of satiation. however, statements like "wine cuts through fat" are unsupported. if this view has any validity, the partial fat solubility of ethanol may reduce the oily mouth-feel of fats as well as limit the activation of fatty acid taste receptors (running et al., ) , but this is no more than speculation. the acids in wine might also have a similar effect. in addition, wine tannins may denature the g protein receptors responsible for detecting fatty acids in the oral epithelium, giving the impression of less fattiness. conversely, fatty acids can reduce the perception of sourness, astringency (peyrot des gachons et al., ) , and bitterness (mattes, ) , possibly improving mouth-feel. wine phenolics can either reduce (jung et al., ) or enhance (mitropoulou et al., ; villamor et al., ) volatility, depending on the phenolic and aromatic involved. carbohydrates also have the ability to bind aromatics, reducing volatility and significantly modifying wine flavor and its retronasal attributes (voilley and lubbers, ; villamor et al., ) . thus, both synergies of flavor as well as suppression could be involved in "ideal" pairings. saliva-induced changes in flavor chemistry is another compounding issue little investigated. whether such potential reactions are sufficiently marked and rapid to have significance, within the time frame of food consumption, is currently terra incognita. in all the standard discussions of wine and food combinations, the obvious is rarely if ever mentioned. it limits the rate and maximum amount of alcohol reaching in the blood ( fig. . ) . correspondingly, it reduces breath-alcohol content (sadler and fox, ) and diminishes alcohol's performance impairment (millar et al., ) . in addition, by reducing alcohol uptake, its tendency to promote overeating is represseddby limiting activation of hypothalamic agrp neuron activity (cains et al., ) . many aspects of food preference appear to develop in utero, based on what the mother ate during pregnancy (mennella et al., ) and infancy. thus, early exposure can play a significant role in developing personal preferences. subsequently, peer pressure and cultural influences may combine to modify these early dispositions. habituation may not be imbued with the effusive and social appeal associated with the image of predestined food and wine marriages but far better fits the facts than any supposedly heaven-inspired pairings. thus, there is little wonder why there has been scant inclination to investigate lovingly held shibboleths about food and wine associations. however, a dose of reality does not have to destroy long-held views. knowing that michelangelo's sistine chapel consists of no more than brush strokes of pigment on plaster, perceived by photoreceptors in the eye, converted to synaptic impulses reconstructed piecemeal into a perception at the back of the brain, need not ruin art appreciation. ideally, it should enhance the wonder it instills. scientific understanding augments adds new layers of appreciation. if supplemental knowledge were not considered to augment appreciation, why would connoisseurs be so concerned with vintage dates, wine geography, cultivars, or details of vineyard sites and wine producers? with knowledge, it really is the more the merrier. admittedly, scientific realism injected at the wrong time may be a despoiler. for special events, psychological appeal can be more significant than sensory reality. as in other aspects of life, science can occasionally be set aside to permit spontaneity and anticipated pleasure presides. the intrigue of magic is being fooled so effectively. under most circumstances, though, some basic rules of pairing should be kept in mind, to avoid glaring mistakes. in most fine cuisine, flavor balance, combined with suitable complexity, is central. nonetheless, the vagueness of this concept is evidenced by the almost infinite variety of combinations seen in cookbooks, food magazines, and culinary shows. accepted norms also vary markedly among cultures (rozin, (rozin, , . thus, it should not be surprising that almost any wine can pair with almost any meal. there are limits, however, for example a dry gewü rztraminer with dessert or a riesling auslese with bouillabaisse. to almost everyone, these would not be considered "marriages made in heaven." cabernet sauvignon and dark chocolate is another clash but seeming appreciated by some connoisseurs. except where there are clear flavor or intensity disparities, notably sweet/ acid or sweet/bitter-astringent, almost any combination will be found pleasing to some and acceptable to most. of the generalities oft quoted, the "white with white, red with red" dictum bears logic, within the context of balanced flavor intensity. nonetheless, it is often the food preparation mode (e.g., poached, fried, baked, broiled, barbequed) or the condiments added (e.g., chilies, curry, relish, olive oil, tomato sauce, garlic, herbs) that often have the greatest influence on flavor intensity, the basic character of the meal, and correspondingly a compatible wine. in most instances, premium-quality table wines are best sampled prior to the meal and premium-quality dessert wines after the meal. because of their aromatic complexity, detection of these attributes is compromised by combination with food or dessert. alternatively, the food or dessert should be designed to be a foil for the wine and be mild in character. in contrast, the more markedly acidic or bitter/astringent the wine, the more effectively these features will be mollified by association with food. might not this be the most justifiable reason for the pairing most wines with food (other than reduced alcohol uptake)? in addition, a lack in the wine's aromatic interest can be camouflaged by flavors from the food. where little attention is likely to be paid to the wine, inexpensive, nondescript red or white wines are both financial and logical choices. if some food and wine pairings are ill-conceived, are there seraphic duets? clearly some do pair better than others, but transcendental experiences? published accounts of such paradisaical experiences may be no more than figments of a fertile imagination, created to sell wine, newspapers, or magazines. admittedly there is an incredible range in human sensory sensitivityd those having higher than average acuity tending to prefer milder flavored foods, and those with below average acuity tending to prefer more intensely flavored foods. but these are no more than tendencies, with experience and social pressures capable of inducing significant shifts in preference, if not perception. psychological influences and a desire to be influenced can distort perception, creating impressions that are "real," only because of the conditions under which the experience occurred. the more unexpected and astounding the sensation, the stronger the memory trace created. the more the mind is studied, the more we come to realize how the brain can distort perception, based on past experiences. they generate mental models of reality, against which sensations are judged, interpreted, and potentially modified. thus, it is wise to doubt perceptions and attempt to separate experience-based memory patterns from actuality, unless it is a selective choice to allow the mind to potentially deceive us. in addition to the sensory pleasure wine can supply when taken with a meal, it also has health benefits. among other direct benefits noted below, dining with wine can reduce the absorption of oxidized lipids and thereby limit their cardiovascular damage (natella et al., ) . the contrasting social and antisocial effects of alcohol consumption must have become evident shortly after the discovery of winemakingdthe negatives being noted early in the old testament (genesis . ) and vividly described by pliny (historia naturalis, . ). time has only expanded our understanding of this dr. jekyllemr. hyde relationship. it is clear that excessive alcohol consumption, both acute and chronic, can have devastating effects on physical and mental well-being. abusive ethanol consumption can cause cirrhosis of the liver, increase the likelihood of hypertension and stroke, favor the development of breast and digestive tract cancers, induce fetal alcohol syndrome, among others. many of these effects seem to arise from excessive alcohol intake activating of free-radical release and associated immune perturbations (meagher et al., ) . other, more severe, negative consequences may arise indirectly, via the accumulation of acetaldehyde, a major breakdown product of ethanol metabolism (lachenmeier et al., ) . because the problems associated with alcoholism (abrams et al., ; schmitz and gray, ) and its eventual, irreversible, chemical modifications in the brain (nestler and malenka, ; heinz, ) have been extensively reported, they need not be elaborated here. on the other hand, it is becoming equally clear that moderate wine consumption (approximately ml/day) can potentially have health benefits. this is considerably less than the two bottles per day that brillat-savarin ( ) considered a healthy man could consume and live long; or the amounts considered appropriate in times past (younger, , p. ) . mark twain, in characteristic style, crystalized moderation in his view of the temperance movement: "temperate temperance is best" (mark twain's notebook, ) multiple epidemiological studies suggest that daily, moderate, alcohol consumption (thun et al., ; doll et al., ) , and notably wine (grønbaek et al., ; renaud et al., ) , is associated with a reduction in allcause mortality. this is expressed in the now famous jshaped (hormesis) curve ( fig. . ), with earlier mortality being associated with both excess alcohol intake and abstinence. this is particularly evident in the reduced incidence of cardiovascular disease in moderate alcohol consumers. in addition, it reduces the likelihood of type diabetes, combats hypertension, and is correlated with reduced frequency of certain cancers (boffetta and garfinkel, ) . although encouraging to those who enjoy wine, the sharp rise in death risk at much above moderate consumption is of concern. presumably, this relationship would apply equally to morbidity figures, were such data available. however, morbidity unlike mortality is qualitative rather than quantitative and thus its measurement fraught with difficulty. additional dangers associated with anything more than moderate consumption, especially alone, comes from alcohol's progressive enhancing of the memory circuitry involving addictive cravings. these epidemiological correlations are supported by in vivo studies that provide potential molecular explanations for these associations. the principal elements missing, in confirming a causal relationship, involves detailed information on the dynamics of absorption, metabolism, and elimination of the proposed active ingredients. faced with a chemical and beverage that can be not only salubrious but also addictive, the fluctuations in society's attitude toward alcohol are not surprising (musto, ; pittman, ; vallee, ) . thankfully for those in the wine industry, wine drinkers appear less likely to become heavy drinkers (jensen et al., ) or to illustrate those alcohol-related problems that have given alcohol a bad reputation (smart and walsh, ) . in addition, wine has a more positive social image than other alcohol-containing beverages (klein and pittman, ; unwin, ) . the major caveat is the derogatory epithet, wino, ascribed to some unfortunate members of society. the use of wine as a medicine or carrier for herbal extracts has an extensive history. it goes back at least to pharaonic egypt (lucia, ; soleas et al., ) . ancient greek and roman society used wine extensively as a solvent for medicinal infusions. this practice continued largely unabated until the beginning of the twentieth century. the excessive abuse of distilled alcoholic beverages, combined with religious and political conservatism, created a backlash against all beverages containing alcohol, notably in north america. alcohol was viewed as an agent of corruption to be annihilated. following the failure of prohibition, humans themselves, not alcohol, came to be viewed as the source of iniquity. alcoholism is now viewed as a developmental, multistage, chronic dependence, possessing a complex etiology (nurnberger and bierut, ) , with both genetic and environmental aspects. in this regard, it is similar to other addictions (ersche et al., ) . thus, the social climate is changing and the relationship between wine (as opposed to alcohol) and health is again being reassessed and investigated seriously. it is unlikely that doctors will soon be prescribing wine for its health benefits. too often, people have difficulty recognizing the limits of rational use and differ markedly in their metabolism (gross et al., ) . in addition, detrimental influences rapidly counter any benefits at more than light to moderate consumption (often viewed as< mg ethanol/day) (rehm et al., ) . erring on the side of restraint seems judicious, without excessively assuaging pleasure, especially if combined with food. even dietary flavonoid supplements (one of the benefits of wine consumption) can be detrimental, if taken in excess (skibola and smith, ) . wine can be wonderful in moderation but is no panacea. alcohol is the primary by-product of fermentation in many organisms. ethanol is also an energy source for an even larger number of species. thus, it is not surprising that enzymes involved in ethanol metabolism are found in most life forms. in humans, ethanol enters the bloodstream either via the consumption of beverages containing alcohol, and/or from ethanol synthesized by members of the intestinal flora. when the concentration of alcohol is low, most of it is metabolized in the liver before it enters the systemic blood supply. most of the blood coming from the digestive tract passes through the liver before being dispersed to the rest of the body. the liver metabolizes about % of the blood alcohol content, at about ml/h. the rest tends to be lost via the breath or secreted in the urine and other bodily fluids. the rate of alcohol loss is relatively constant over time, with w % reduction within h ( fig. . ). the liver possesses two ethanol metabolizing pathways. the primary, constitutive mechanism involves the oxidation of ethanol to acetaldehyde, via cytoplasmic alcohol dehydrogenases (adhs). of the seven known adh genes (crabb et al., ) , three function in the liver. the others act in the gastric epithelium and other tissues. subsequent metabolism converts acetaldehyde to acetic acid. this occurs principally under the action of mitochondrial acetaldehyde dehydrogenase (aldh ). the cytoplasmic acetaldehyde dehydrogenase (aldh ) is less active. acetic acid is subsequently secreted into the blood or directly converted to acetyl coa. from this point, metabolism may flow along any standard biochemical pathway (see fig. . ). alcohol metabolizing enzymes frequently occur in allelic forms (isozymes). their relative occurrence also tends to vary among ethnic groups. some isozymes possess distinct physiological attributes. for example, adh b* codes for a subunit that oxidizes ethanol slowly, whereas adh b* encodes a highly active subunit of the dimeric enzyme (about times more efficient) (thomasson et al., ) . correspondingly, those individuals who are homo-or heterozygous for the adh b* subunit, eliminate alcohol from the blood more rapidly. rapid alcohol oxidation may donate a degree of protection against alcoholism, by quickly converting ethanol to acetaldehyde. however, if combined with slow-acting alleles for acetaldehyde dehydrogenase (aldh ) (crabb et al., ) , the accumulation of toxic acetaldehyde is enhanced, predisposing the bearer to cancers of the oropharynx and esophagus. those also possessing malfunctional glyoxalase and methylglyoxalase repair enzymes are those most susceptible to such damage (see dingler and patel, ) . a second, ethanol-degradation hepatic pathway becomes activated when blood-alcohol levels become elevated. it involves a microsomal cytochrome, p e (cyp e ). it oxidizes ethanol to acetaldehyde, using molecular oxygen rather than nad þ . regrettably, the microsomal pathway generates free oxygen radicals (meagher et al., ) dmolecules (or ions) with one or more unpaired electrons. these highly reactive oxidants, or reactive oxygen species (ros), can be generated long after alcohol intake ceases. another variant of cyp e occurs in the gastric mucosa, seemingly being more active in men. although most ros are inactivated by glutathione, superoxide dismutase, and catalase, long-term exposure to their presence may induce the slow, progressive accumulation of irreparable cellular damage. metabolizing ethanol to acetate (acetic acid) has the advantage that tissue cells can regulate its transport. this is not the situation with ethanol, which can diffuse freely across cell membranes. transport control is a central tenet in proper cellular function. the ability of ethanol to displace water and its unregulated passage across cell membranes explains much of alcohol's toxicity. in addition, its oxidation to acetaldehyde tends to be more rapid than acetaldehyde's oxidation to acetate. thus, acetaldehyde may accumulate in the blood and other bodily fluids. this is often viewed as an important contributor to the toxicity associated with excessive alcohol consumption (lachenmeier et al., ) . differentiating between these direct and indirect toxic effects of excessive ethanol intake has proven difficult. one of the first physiologic effects of alcohol consumption is a suppression of cognitive brain function. this is most noticeable in enhanced sociabilitydby blocking social inhibitions regulated by higher brain functions. for others, it quickly induces drowsiness (stone, ) . this probably explains why taking a small amount ( e ml) of wine before sleeping often helps those suffering from insomnia (kastenbaum, ) . this amount often provides the benefits of sleep induction, without causing subsequent agitation and sleep apnead often associated with greater alcohol consumption. the effect on sleep may arise from alcohol's modulating the action of inhibitory g-aminobutyric acid (gaba) receptors, while suppressing the action of excitatory glutamate receptors. gaba and glutamate are estimated to be involved in about % of the neurocircuitry of the brain. the level of melatonin in wine is well below those prescribed as an insomnia medication (rodriguez-naranjo et al., ) . another effect on brain function results from a reduction in the secretion of vasopressin. as a consequence, urine production increases, producing the frequently reported diuretic effect associated with alcohol consumption. less well known is how alcohol acts as a crucial regulator of the hypothalamicepituitaryeadrenal axis, modulating the release of hormones such as adrenocorticotropic hormone and corticosterone (haddad, ) . although alcohol has a general depressive action on brain function, the levels of some brain modulators show transitory increases. examples are serotonin and histamine. the latter may activate a cascade of reactions leading to headache production. another of the multiple influences of alcohol is the conversion of hepatic glycogen to sugar. this results in a short-lived increase in plasma glucose content. this, in turn, can cause glucose loss in the urine as well as an increase in insulin release by the pancreas. both result in a drop in blood sugar content. if sufficiently marked, hypoglycemia results. this apparently causes the temporary weakness occasionally associated with alcohol consumption, especially excess intake. in addition to direct effects, the accumulation of acetaldehyde, as a by-product of ethanol metabolism, may have several undesirable consequences. at low rates of alcohol intake, acetaldehyde metabolism is sufficiently rapid to limit its accumulation and liberation from the liver. at higher concentrations, acetaldehyde production rapidly consumes the liver's glutathione reservesda central cellular antioxidant. this coincides with activation of the microsomal ethanol oxidation pathway that generates toxic free-oxygen radicals. in the absence of sufficient glutathione, free-oxygen radicals can accumulate, disrupting mitochondrial function. elsewhere in the body, acetaldehyde can bind with proteins and cellular constituents, forming stable complexes (niemela and parkkila, ) . these can lead to the production of immunogenic determinants, which can stimulate antibody production against acetaldehyde adducts (romanazzi et al., ) . this may induce some of the chronic tissue damage associated with alcohol abuse (niemela and israel, ) . the binding of acetaldehyde to the plasma membrane of red blood cells is known to increase rigidity. by limiting their ability to pass through the narrowest capillaries, oxygen supply to tissue cells may be restricted. this could participate in suppressed brain function. it is estimated that the brain consumes up to % of the blood's oxygen supply but constitutes only about . % of body mass. in addition, acetaldehyde can disrupt dna repair mechanisms. fortified wines (notably sherries) can be a significant source of acetaldehyde (lachenmeier and sohnius, ) . although ethanol and acetaldehyde can produce severe, progressive, and long-term damage to various organs, and incite alcohol dependence, these consequences are minimal to undetectable when alcohol consumption is moderate and taken with meals (serianni et al., ) . as the sections below demonstrate, moderate, daily, wine consumption can have health benefits for the majority of people. wine's major nutritional value comes from its rapidly metabolized, ethanolic, caloric content. alcohol does not need to be digested, prior to being absorbed through the intestinal wall. in rural viticultural areas, wine historically provided a significant source of metabolic energy for the adult population. the caloric value of ethanol ( . kcal/g) is nearly twice that of carbohydrates ( . kcal/g). thus, it constituted a valuable caloric source. it is estimated that alcohol may supply about % of the energy in the average american diet (halsted, ) .wine was also a potable beverage and helped disinfect water to which it was addeddsome bacterial inactivation occurs within seconds (vaz et al., ) . wine contains small quantities of several vitamins, notably several b vitamins, such as b (thiamine), b (riboflavin), and b (cobalamin). however, wine is virtually devoid of vitamins a, c, d, and k. in excess, ethanol can impair vitamin uptake. wine contains various minerals in readily available forms, especially potassium and iron (in the ferrous state). nevertheless, excessive alcohol consumption can disturb the uptake of calcium, magnesium, selenium, and zinc and increase the excretion of zinc via the kidneys. the low sodium/high potassium content of wine makes it one of the more effective sources of potassium for individuals on diuretics. although wine contains soluble dietary fiber, especially red wines (díaz-rubio and saura-calixto, ) , it is insufficient to contribute significantly to the daily recommended fiber content in the human diet. wine has several direct and indirect effects on food digestion. its phenolic (hyde and pangborn, ) and alcohol (martin and pangborn, ) contents activate the release of saliva. in addition, wine promotes the release of gastrin as well as gastric juices. the principal constituent activating the release of gastric juices in red wines is apparently succinic acid, whereas in white wines it is malic acid (liszt et al., ) . they do not activate gastrin release, however. the substance(s) involved in stimulating gastrin secretion are unknown. wine also significantly delays gastric emptying, both on an empty stomach (franke et al., ) or when consumed with food (benini et al., ) . the latter favors digestion by extending the duration of acid hydrolysis. delayed gastric emptying may be a consequence of wine phenolics activating stanniocalcin- cells in the stomach. these possess the same tas r system as bitter-sensitive receptors in the mouth (see finger and kinnamon, ). on stimulus, they release cholecystokinin, a peptide hormone that reduces gut mobility. in addition, wine slows plasma glucose uptake, independent of any insulin response (benini et al., ) . furthermore, at the levels found in most table wines, ethanol activates bile release. wine acids and aromatics also have the same effects. in contrast, the high alcohol contents (e.g., distilled spirits) can suppress digestive juice flow, the release of bile, and induce stomach spasms. wine also aids digestion indirectly by inactivating gastrointestinal pathogens. despite the general beneficial effects of moderate amounts of alcohol on digestion, the phenolic content of red wine may counter some of these influences. for example, tannins and phenolic acids can interfere with the action of certain digestive enzymes, notably a-amylase, lipase and trypsin (rohn et al., ; gu et al., ) . digestion may be further slowed by phenolics polymerizing with food proteins. these effects may be mollified by the presence of ionic carbohydrates found in food (gonçalves et al., ) as well as by salivary proteins. both monomers and proanthocyanidins bind with basic, proline-rich, and histatin proteins in the saliva. their bonding is reversible, depending on equilibrium conditions. they can become irreversible, though, in the presence of metal ions, upon oxidation, or with ph changes (luck et al., ) . normally, these insoluble saliva/tannin complexes remain stable in the stomach and upper alimentary tract (lu and bennick, ) . thus, the inactivation of digestive enzymes or the disruption of mineral uptake by tannins may be limited. degradation of tannin-protein polymers and their moieties subsequently occurs in the colon. in contrast, some pepsin-activated protein breakdown is activated by monomeric phenolics, notably quercetin, resveratrol, catechin, and epigallocatechin gallate (tagliazucchi et al., ) . clearly the action of wine phenolics is complex and much more needs to be known. not only are the effects potentially different in the stomach from that in the small and large intestines, but also the chemical composition of wine phenolics changes during passage through the digestive tract. the wine's phenolic content can also decrease iron and copper absorption in the intestinal tract (cook et al., ) . although nutritionally undesirable, limiting iron bioavailability may reduce the formation of toxic lipid hydroperoxides during digestion. the antioxidant effect of polyphenolics also applies to peroxide generation in the stomach kanner and lapidot ( ); fig. . . the activation of gastric juice release not only aids food digestion but also inactivates enzymes involved in ulceration. even more significant may be the antibiotic action of wine constituents against helicobacter pylori (fugelsang and muller, ) . h. pylori is often considered the primary causal agent of stomach ulceration. thus, moderate wine consumption may have a prophylactic effect in limiting ulcer initiation (brenner et al., ) . the bacterium has also been implicated in gastritis, vitamin b malabsorption, and gastric adenocarcinoma. however, chronic secretion of gastric juice can produce irritation and may provoke ulceration, heartburn, and favor the development of adenocarcinomas in the lower esophagus. wine may further aid human sustenance by increasing nutrient uptake. congeners in wine combine with metallic ions, vitamins, and fatty acids, facilitating their transport across the intestinal wall. consuming wine with food slows the rate of alcohol uptake in the blood ( fig. . ) . in the absence of food, w % of the alcohol is absorbed through the intestinal wall. although the absolute proportion absorbed via the intestines increases when wine is jointly consumed with food, uptake is dispersed over a much longer period. this results primarily by food retarding gastric emptying. consequently, alcohol transfer into the intestines is delayed. this gives the liver more time to metabolize the alcohol, lowering the maximal blood-alcohol level reached. however, taking sparkling wine on an empty stomach can increase short-term alcohol uptake by about % (ridout et al., ) . because the same wine, with its carbon dioxide removed, did not have the same influence, it is suspected that carbon dioxide was the active ingredient (ridout et al., ) . it has occasionally been proposed that carbon dioxide relaxes the pyloric sphincter, allowing earlier transfer of fluids from the stomach into the duodenum and thereby its absorption into the blood. the rate of alcohol metabolism differs considerably among individuals, with rates commonly varying between and mg/kg/h. a person's hormonal and nutritional state also affects their ethanol metabolic rate. gender is also an influencing factor (kaltenback et al., ) . the tendency of women to have a higher body fat content (into which ethanol does not infiltrate), results in their being more rapidly influenced by similar amounts of alcohol (kalant, ) . additional benefits that may accrue from wine consumption are derived from metabolic by-products of proanthocyanin degradation in the colon. they assist protecting the colonic mucosa from the toxic effects of p-cresol production (generated from l-tyrosine) (wong et al., ) . furthermore, wine phenolics may prevent or delay intestinal diseases associated with inflammation and oxidative stress (e.g., inflammatory bowel disease) (biasi et al., ) . finally, wine can have a beneficial cultural/psychologic effect on food intake and digestion. the association of wine with refined eating promotes slower food consumption, potentially permitting biofeedback mechanisms to regulate food intake. in addition, wine consumption can promote a more relaxed lifestyle, something increasingly valuable in our overly compulsive society. whether this explains the reported improved appetite of many elderly and anorectic patients, when wine is taken with the meal, is unknown. wine taken with a meal can enhance the pleasures derived from both but not necessarily those suffering gastroesophageal reflux (acid reflux). this is apparently correlated more with the consumption of white than red wines (pehl et al., ) . most investigations on the health benefits of moderate wine consumption have involved population (epidemiologic) and tissue-culture studies. however, to be confident in their interpretation, intermediate stages needs to be known. this involves details on the uptake and degradation in the intestinal tract, metabolism in the liver, transport, binding, and modification in the blood and lymph, elimination by the kidneys, and cellular uptake and metabolism. although absorption via the intestinal tract is a priori requirement for most activity, it alone does not imply bioavailability at the cellular level. however, this does not apply to influences in the oral cavity and digestive tract. in the mouth and upper intestinal tract, a wine's phenolic constituents remain largely unmodified, except for binding with proteins. in contrast, considerable degradation occurs in the colon. here, large phenolics tend to be metabolized, depending on an individual's colonic flora. for example, hydrolyzable tannins are converted to more easily absorbed, antiinflammatory/anticarcinogenic urolithins (tomás-barberán et al., ) . if absorbed, most phenolics are quickly metabolized in the liver, conjugated with various moieties (e.g., methyl or sulfur groups) by plasma enzymes, and/or eliminated via the kidneys. amounts found in the plasma are often % of that consumed, although this may increase with repeated daily exposure. the proportion of wine-derived flavonoids is estimated at about mg/day/person in the united states (chun et al., ) . this compares with about mg/day/person from all sources. this value would increase to about mg flavan- -ols and mg procyanidin dimers, based on ml of red wine per day (forester and waterhouse, ). that volume is near the upper limit of what is typically considered moderate wine consumption. in the mouth, mid-sized flavonoid polymers often bind to salivary proteins, forming stable complexes (de freitas and mateus, ; pizarro and lissi, ) , slightly delaying their transport to the stomach. passage through the stomach does not modify the majority of wine phenolics. among wine flavonoids, anthocyanins appear to be those that most quickly traverse the stomach and pass into the blood (passamonti et al., ) . flavonoid glucoside uptake is facilitated by gastric glucose transporters (oliveira et al., ) , whereas aglycone uptake occurs by passive diffusion. flavonoids are also effectively translocated across the small intestine lining (talavéra et al., ) . phenolic acids, such as caffeic acid (simonetti et al., ) and resveratrol (soleas et al., ) also readily pass into the plasma via the intestinal tract. in contrast, flavonoid polymers tend to remain in the intestine, until degraded to phenolic acids and aldehydes by the colonic flora (aura, , fig. . ) . also metabolized in the colon are any anthocyanins or catechins monomers that have not already been absorbed and/or microbially degraded. these may enhance the growth of beneficial bacteria (e.g., bifidobacterium and lactobacillus spp.) or their attachment to the intestinal wall (bustos et al., ) . depending on the compound, variable amounts may be absorbed into the blood (ward et al., ) . studies on the bioavailability of phenolics, once in the bloodstream, are still preliminary (williamson and manach, ) . although many simple flavonoids are quickly absorbed into the plasma, most appear to be rapidly conjugated (methylated or sulfated), bound to proteins, transformed to glucuronides, or otherwise modified (williams et al., ; forester and waterhouse, ; xiao and högger, ) . hydroxycinnamic acids are also rapidly absorbed and metabolized into glucuronide and sulfate conjugates (nardini et al., ). this both reduces their toxicity (potential carcinogenicity) as well as facilitating their excretion by the kidneys. however, the latter reduces their potential beneficial effects. in contrast, anthocyanin seems to be efficiently absorbed via the stomach wall, and their metabolites appear to remain in the plasma for several days (kalt et al., ) . short-term studies primarily detect the uptake of phenolic metabolites, whereas long-term studies detect more parental constituents (sandoval-ramírez et al., ) . small amounts of cinnamic acid-tartrate esters are also found in the plasma. these transformations could significantly affect their antioxidant and other attributes as well as their ability to move into tissue cells and their surrounding fluids. most phenolic metabolites retain one or more hydroxyl groups and thus may still possess antioxidant properties. nevertheless, there is growing evidence that phenolic metabolites act primarily as signaling molecules, notably in oxygen-stress-related pathways (williams et al., ) . consequently, smaller amounts are needed than for direct antioxidant reactions. this might explain the discrepancy between the low levels of free phenolics in the plasma and their apparent effects. an example may be the increased activity and the presence of phenolics in the plasma permits their likely uptake into most body tissues. this generality does not necessarily apply to the brain. except where there are specific transport proteins, most compounds above a molecular weight of da are excluded from the brain by the bloodebrain barrier. the barrier consists of tight connections between the endothelial lining of cerebral capillaries. it prevents the diffusion of most molecules from the blood into the cerebrospinal fluid. however, with anthocyanins (passamonti et al., ) and simple flavonols (youdim et al., ) , access to the brain apparently can occur within minutes of consumption. initial animal studies suggest uptake levels in the brain occur at about % that of other tissues (wu et al., ) . although fascinating, consumers are more interested (if at all) in what wines provide the maximal health benefits and under what conditions. regrettably, data on these vital concerns are lacking. grape cultivar, maturity, wine production, maturation and aging conditions all influence the amounts and types of phenolics present and thus their activity. the prophylactic action of wine against gastrointestinal diseases has been known for millennia, long before their microbial origins were ever suspected. in spite of this, the mechanism(s) by which this occurs remain poorly understood. the antimicrobial effect of alcohol was discovered in the late s. nevertheless, alcohol is not particularly antimicrobial, certainly at the concentrations found in wine (its sterilant action is optimal at about %). the antimicrobial action of wine is closely related to that of crushed grapes (Ö ncü l and karabiyikli, ). thus, the antibiotic action of wine likely relates more to its phenolic and acidic (vaz et al., ) contents, although wine's alcohol content undoubtedly augments their effectiveness. anthocyanins, which are weakly toxic to viruses, protozoans, and bacteria, become more so as a consequence of fermentation. other phenolic compounds in wine are bacteriostatic and fungistatic. for example, p-coumaric acid is particularly active against grampositive bacteria (e.g., staphylococcus and streptococcus), whereas compounds, such as quercetin, inhibit pathogenic gram-negative bacteria (e.g., escherichia, shigella, proteus, and vibrio) (vaquero et al., ) . phenolics may also be inhibitory to intestinal pathogens such as clostridium difficile, c. perfringens, and bacteroides (lee et al., ) . despite wine being more effective than mildly antimicrobial agents, such as bismuth salicylate (weisse et al., ) , full action may take several hours (møretrø and daeschel, ; dolara et al., ) . although most studies have involved bacteria grown on culture plates, wine has also been shown to be antimicrobial under simulated gastrointestinal conditions (vaz et al., ) . an indirect effect, limiting intestinal problems, is illustrated by the action of the colon flora on anthocyanin structure. it favors the growth of bifidobacterium and lactobacillus-enterococcus spp. (hidalgo et al., ) . these have been associated with a healthy gut microflora (hord, ) . there is also considerable variation in the effects of different flavanols and procyanidins, both promoting and inhibiting the adhesion of probiotic lactobacilli to the intestinal wall, depending on their metabolic modification during passage through the intestinal tract (bustos et al., ) . in addition, viniferin (a resveratrol derivative) can inhibit biofilm formation by pathogenic pseudomonas aeruginosa and escherichia coli (cho et al., ) . red wine can suppress biofilm formation by oral pathogens (muñ oz- . in most instances, the mechanism by which phenolics have their action is unknown. however, in the case of quercetin, the effect may be partially attributed to its inhibition of dna gyrase, whereas with epigallocatechin, disruption of cell membrane function appears central to its antibiotic action. alternative methods of action may involve suppression of cell adherence and colony formation on the gut lining (selma et al., ; truchado et al., ) . adherence is often a prerequisite for the cascade of events leading to disease development. low ph and the presence of various organic acids appear to accentuate the antimicrobial action of both wine phenolics and ethanol. organic acids may themselves be antimicrobial, as is the case with bacillus cereus (vaz et al., ) . wine is also active against several viruses including the herpes simplex virus, poliovirus, hepatitis a virus as well as rhinoviruses and coronaviruses. the effect on the latter two groups appears reflected in the reduced incidence of the common cold in moderate alcohol consumers (cohen et al., ) , particularly those drinking red wines (takkouche et al., ) . if you have to gargle, port is certainly one of the more pleasant. the antioxidant action of wine phenolics not only appears to play an important role in limiting low-density lipoprotein (ldl) peroxidation (maxwell et al., ; rice-evans et al., ) but also the action of lipoxygenases and enzymes generating ros. phenolics can also directly scavenge (quench) these radicals (e.g., superoxide and hydroxyl radicals), contributing to the action of cellular antioxidants. the oxidized flavonoid byproducts are much more stable (nonreactive) and tend to be quickly metabolized or eliminated by the kidneys. a flavonoid's quenching ability is largely dependent on the location and number of its oh groups as well as its glycosylation, sulfation, methylation, and acylation status (plaza et al., ) . in addition, phenolics can chelate iron and copper, limiting their involvement in radical formation (morel et al., ; rice-evans et al., ) or access to bacterial pathogens. phenolic can also limit the influx of calcium ions associated with oxidative stress (ishige et al., ). an antioxidant relatively unique to wine is resveratrol. it is a stilbene phenolic produced in response to plant stresses. it has greater antioxidant activity than common dietary antioxidants, such as vitamin e and ascorbic acid (frankel et al., ) . there is also direct evidence that resveratrol can enter the blood system at levels sufficient to suppress cyclooxygenase and lipoxygenase pathways. these are involved in the synthesis of proinflammatory mediators (bertelli, ) . in addition, resveratrol can activate proteins involved in nerve cell differentiation, synaptic plasticity, and neuronal survival (tredici et al., ) . supplemental protection may result from ethanol activating the cellular biosynthesis of hydroxytyrosol (a dopamine metabolite) (de la torre et al., ) . hydroxytyrosol is an important antioxidant and antiinflammatory agent. the most clearly established benefit of moderate alcohol consumption, notably wine, relates to a nearly %e % reduction in death rate due to cardiovascular disease (klatsky et al., (klatsky et al., , renaud and de lorgeril, , fig. . ) . alcohol consumption is also correlated with a decrease in the likelihood of intermittent claudication (pain or cramping in the calf of the leg). claudication is a common indicator of peripheral arterial disease. recent studies have confirmed that incidental factors, such as gender, race, lifestyle, educational level, etc. do not affect these results (see mukamal et al., ) . studies have also demonstrated that daily consumption of alcohol significantly reduces the incidence of other cardiovascular diseases, such as hypertension (keil et al., ) , heart attack (gaziano et al., ) , stroke (truelsen et al., ; hillbom, ) , and peripheral arterial disease (camargo et al., ) . those who consume wine moderately live, on average, . e . years longer than teetotalers and considerably longer than heavy drinkers. the prime area of contention is the degree to which these benefits accrue from the effects of ethanol vs. phenolic and/or other constituents (rimm et al., ) . atherosclerosis is the principal cause of most cardiovascular disease (libby, ) . it apparently results from chronic injury to the arteries ( fig. . ) . although associated with several independent factors, most damage is correlated with lipid oxidationda subgroup of cholesterol-apoproteins complexes (ldls). because of the hydrophobic nature of cholesterol and triglycerides, their transport in the plasma requires a special structure. as illustrated in fig. . , lipoprotein complexes consist of an outer membrane of phospholipids, within which apoproteins and free cholesterol occur. they enclose a hydrophobic core possessing numerous triglycerides and cholesteryl esters. metabolism of the enclosed lipids is regulated by the apoproteins in the outer membrane. normally, ldls supply cholesterol for cellular membrane repair and steroid synthesis. however, in high concentrations, they may accumulate in the artery wall. if they remain there for an extended period, their lipid content tends to become oxidized. in an oxidized state, lipids are cytotoxic and indirectly irritate the artery wall. as a consequence, special adhesion proteins attach to the artery wall. monocytes and helper t-cells of the immune system bond to these proteins. in addition, affected endothelial cells may secrete compounds, such as endothelin- . endothelin- activates monocyte and t-cell migration into the artery wall. procyanidins, principally found in red wines, are particularly effective in suppressing the production of endothelin- (corder et al., ) . anthocyanin metabolites are also effective modulators of endothelial function (edwards et al., ) . in the layer just underneath the endothelial lining (intima), accumulated monocytes mature into macrophages. both macrophages and t-cells may release a range of cytokines that further activate the immune system, involving localized inflammation. activated macrophages tend to engulf oxidized ldls. however, as the ldls are not degraded, their progressive accumulation gives the macrophage the appearance of being full of bubbles (termed foam cells). they are the first clear evidence of localized arterial swelling (plaques). occasionally plaques bulge into the vessel. more frequently, they initially enlarge outward into the surrounding tissue. action of immune cells in the plaque also induces migration of smooth muscle cells from the artery wall into the intima. here they proliferate and produce collagen, forming a fibrous cap over the plaque. additional ldls slowly collect, provoking further rounds of inflammation and plaque enlargement. these accretions may develop their own vasculature, becoming fibrous and inelastic. as the plaques enlarge, they may produce irregular protrusions into and block the artery lumen. even without restricting blood flow, plaques set the stage for platelet aggregation, clot formation (thrombus) and the blockage that can precipitate a heart attack or stroke. in the later phases of plaque formation, unknown factors enhance inflammatory changes in the plaque. these disrupt the integrity of the cap. for example, collagenases secreted by macrophages inhibit collagen synthesis by smooth muscle cells. sudden rupture of a plaque permits blood infiltration into the plaque. because plaques contain potent blood clotting factors, thrombus development is almost instantaneous. it is currently thought that plaque rupture is the principal factor inducting thrombus formation and precipitating a heart attack, stroke, or other cardiovascular trauma. atherosclerosis appears to be at least partially reversible, if risk factors such as smoking, high blood pressure, high dietary sources of cholesterol, and possibly infection by pathogens such as chlamydia pneumoniae and cytomegalovirus are eliminated. part of the reversal process involves the action of high-density lipoproteins (hdls). of the two principal forms, ethanol augments the presence of hdl , whereas exercise increases the level of hdl . the effect of ethanol on hdl concentration appears independent of beverage type (van der gaag et al., ) . either form of hdl favors the removal of cholesterol from the arteries, transferring it to the liver for metabolism. hdls also appear to interfere with ldl oxidation. because the hdl/ldl ratio affects the degree and rate of cholesterol turnover, the slower the rate, the greater the likelihood of oxidation (walzem et al., ) and eventual plaque formation. the beneficial effect of moderate alcohol consumption on the hdl/ldl ratio is now relatively clearly established. less well understood is its effect in lowering the concentration of c-reactive protein (crp) (levitan et al., ) . crp is an indicator of inflammation. its level usually rises in correlation with the risk of atherosclerosis. moderate alcohol consumption also reduces the incidence of another risk factor for cardiovascular diseased type diabetes. chronically high values of circulatory glucose, associated with type diabetes, appear to generate high plasma triglyceride and ldl levels. however, the benefits of wine's alcohol content on glucose and insulin metabolism appear not to occur if intake is not coincident with meal consumption (augustin et al., ) . phytoestrogens, such as resveratrol, have a similar effect in reducing triglyceride and ldl contents in the circulatory system (see bisson et al., ) . another of alcohol's beneficial influences involves disruption of events leading to clot formation. platelets are less "sticky" in the presence of alcohol and thus less likely to aggregate, limiting clot formation. alcohol also increases the level of prostacyclin (interferes with clotting) and raises the level of plasminogen activator (a clot-dissolving enzyme). clots, adhering or becoming stuck to the roughened surfaces of narrowed atherosclerotic vessels, may block blood flow. the oxygen deficiency and cell death that result are central to the damage caused by a heart attack or stroke. thus, it is not surprising that inhibitors of platelet aggregation reduce the frequency of these cardiovascular crises and their sequelae. it is the rationale for recommending the daily consumption of acetylsalicylic acid (an inhibitor of platelet aggregation). ethanol (renaud and ruf, ) as well as wine phenolics, such as resveratrol and anthocyanins, have similar effects (fig. . ). an additional example of the beneficial effects of limited alcohol intake is the relation between alcohol dehydrogenase (adh) genotype and the incidence of myocardial infarction. individuals homozygous for adh c* (slow metabolizers of ethanol) are significantly less likely to have a heart attack than heterozygous individuals and even less likely than homozygous individuals for adh c* (fast metabolizers of ethanol) (hines et al., ) . individually, many phenolics, such as resveratrol, catechin, epicatechin, and quercetin appear to have inhibitory effects on platelet aggregation (keli et al., ) . in an in vitro study, though, monomeric or low-molecular-weight flavonoids and hydroxycinnamic acids enhanced platelet aggregation and ldl oxidation, with only large polymers being inhibitory (shanmuganayagam et al., ) . in another investigation, the combined effect of several phenolics was superior to single compounds (wallerath et al., ) . the action partially results from the enhanced synthesis and release of nitric oxide by endothelial cells. this has been found to occur at resveratrol concentrations associated with moderate wine consumption (gresele et al., ) . chlorogenic acid also appears to activate nitric oxide production (mubarak et al., ) . nitric oxide induces vasodilation (by relaxing vascular smooth muscle), reduces blood pressure, and limits platelet adhesion to blood vessel endothelia. indicative of the complexities of such interactions is the observation that flavonoids may also inactivate nitric oxide (verhagen et al., ) . in addition, nitric oxide, notably as peroxynitrite, oxidizes ldls. clearly, much more still needs to be known before a clear picture emerges. in addition to affecting platelet aggregation, wine phenolics can bind directly with ldls (limiting their oxidation), indirectly reduce macrophage-mediated oxidation and preserve the action of paraoxonase (further protecting ldls from oxidation) (aviram and fuhrman, ) . furthermore, red wine phenolics directly or indirectly limit the migration of smooth muscle cells into the intima of artery walls. these influences probably explain some of the added benefits of wine vs. other alcoholic beverages in reducing the incidence and severity of cardiovascular disease. although flavonoids tend to suppress inflammation, conflicting observations put the clinical significance of their antiinflammatory action to atherosclerosis in question. whether this might also apply to the antiinflammatory effects of wine phytoprostanes (degradation products of linolenic acid) is unknown. red wines usually have been credited with superior health-related benefits than white wines, especially relative to cardiovascular disease. this presumably results from their higher flavonoid content (tian et al., ) . this view is supported by studies where white wine has shown the same effects as red wine, when supplemented with grape polyphenolics (fuhrman et al., ) . nevertheless, prolonged skin contact, or choice of particular cultivars, can enhance the presence of phenolic acids in white wine. common phenolics in white wine, such as caffeic and coumaric acids as well as flavonols such as quercetin, are well-known potent antioxidants. the low sodium content of wine is an incidental benefit. it may permit wine consumption by those on a low-sodium diet, for example those with high blood pressure or heart attack victims. the high potassium to sodium ratio of wine ( : ) is also advantageous. as noted, many of the beneficial influences of alcohol and wine consumption show a j-shaped curve ( fig. . ). this also applies to its effect on age-related macular degeneration (obisesan, ; fraser-bell et al., ) . the disease expresses itself as a progressive degeneration of the central region of the retina (macula), leading to blurred or distorted vision. it results as a consequence of local atherosclerosis that deprives the retina of oxygen and nutrients. it is the leading cause of blindness in adults over the age of . a similar relationship has been found for cataract development. in both conditions wine antioxidants are suspected to be the active protective agent. in this regard, quercetin appears to be more protective (against light-induced lipid peroxidation) than either anthocyanin-or phenolic acid-rich constituents (liu et al., ) . however, wines high in ethanol content may undo these benefits, by promoting pro-oxidant action. alzheimer's, a devastating neurodegenerative disease, afflicts more than million people. not surprisingly, researchers have investigated whether wine consumption affects the incidence of this and other neurodegenerative diseases (barnham et al., ) . flavonoids not only activate key respiratory enzymes in mitochondria (schmitt-schillig et al., ) , but also decrease the production of reactive oxygen species, by stimulating the production of catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase . a pattern appears to apply here, as with most health-related benefits of wine and alcohol consumptiondmoderate intake bing beneficial, whereas high consumption or abstinence is deleterious. alzheimer's disease has been correlated with the accumulation of extracellular amyloid b-peptide (plaque) and the formation of intracellular neurofibrillary tangles containing tau-protein. many in vitro studies have shown that antioxidant compounds, such as vitamin e, protect neurons from b-amyloid accumulation. tannins also inhibit the formation of and destabilize preexisting b-amyloid fibrils (ono et al., ; guéroux et al., ) , whereas resveratrol promotes the degradation of amyloid b-peptides (marambaud et al., ) . wine consumption is also linked to a reduction in the incidence of alzheimer's disease (truelsen et al., ; letenneur, ; luchsinger et al., ) . even mild cognitive impairment and the progression of idiopathic dementia may be reduced with moderate alcohol consumption (solfrizzi et al., ) . grape juice has also been found to be effective in this regard (krikorian et al., ) . like other health benefits, these finding may not, in and by themselves, justify wine consumption, but they are encouraging to those who choose wine as part of their preferred lifestyle. age-related bone mass loss affects both sexes but is more frequent in postmenopausal women. many risk factors including dietary influences and hormonal supplements, can affect its progress and severity. of these factors, moderate alcohol consumption has been found to favor bone retention (ganry et al., ; ilich et al., ) . tucker et al. ( ) found data consistent with higher benefits from wine than other alcoholcontaining beverages. the source of these benefits may be a combination of enhanced calcium uptake, associated with alcohol consumption (ilich et al., ) , the phytoestrogen effects of phenolics, such as resveratrol and kaempferol, or other unsuspected influences. a number of drugs used in treating arthritis tend to irritate the stomach lining. this side-effect may be counteracted by the mildly acidic, dilute alcohol content of table wines. other beneficial effects connected with moderate wine consumption may accrue from its mildly diuretic and muscle relaxant properties. the diuretic action of wine can help reduce water retention and minimize joint swelling. wine can also directly reduce muscle spasms and the stiffness associated with . wine, food, and health arthritis. the antiinflammatory influences of wine phenolics, notably resveratrol (yang et al., ) , may also play a role in diminishing the suffering associated with arthritis and other diseases associated with chronic inflammation (schueller et al., ) . wine consumption has been shown to attenuate insulin-resistance in type diabetes (dixon et al., ; napoli et al., ) . this may result from wine phenolics quenching oxygen radicals, thought to be pivotal in the damage associated with the disease. type diabetes appears to result when body cells fail to respond properly to the presence of insulin. the incidence of metabolic syndrome is also lower in wine drinkers (rosell et al., ) . these effects may be due to one or more of the following: the influences of alcohol on metabolism; the antidiabetic properties of the element vanadium (for which wine is a significant source) (brichard and henquin, ; teissèdre et al., ) ; the hypoglycemic and hypolipidemic effects of phenolics such as resveratrol (su et al., ) ; and/or through some effect on endothelial nitric oxidase synthase (leighton et al., ). it appears there may be considerable specificity. for example, in a comparison between malvidin-and delphinidin- -o-glucosides, only the predominant anthocyanin in grapes (malvidin) seems to have a significant hypoglycemic effect (lida et al., ) . relative to diabetes mellitus (type diabetes), moderate consumption of dry wine was found to present no adverse effects on sugar control (gin et al., ; bell, ) . although wine does contain residual sugars, the most common, fructose, is poorly transported across the gastrointestinal tract. most of what is absorbed is rapidly removed from the blood by the liver, where it is metabolized into glycerol and often stored as fat. it does not stimulate pancreatic insulin release. red wine (and a diet rich in antioxidants) appear to slow the progression of kidney damage (necropathy), occasionally associated with diabetes (zhu et al., ) . in an epidemiological study, knudsen et al. ( ) found a strong link between alcohol consumption and a reduced prevalence of goiter and solitary thyroid nodules. the origin of this apparent protective effect is unknown. drinking water has long been associated with reducing the development of kidney stones. increased urine production is thought to limit calcium oxalate crystallization. what is new is the observation that wine consumption further reduces the production of these painful and dangerous inclusions (curhan, ) . moderate wine consumption has been correlated with reduced incidence in some cancers (see bianchini and vainio, ) (e.g., the kidney) but increased risk for others (notably the throat and gastrointestinal tract) (ebeler and weber, ; parry et al., ) . this potential varies markedly, primarily based on the amounts habitually consumed. for other cancers, moderate consumption appears to be neither protective nor a risk factor (e.g., prostate cancer) (chao et al., ) . conversely, excessive consumption of alcoholic beverages increases the risk of a range of cancers, notably those of the oropharynx, larynx, esophagus, liver, colon, rectum, and breast (connor, ) . because ethanol is not directly carcinogenic, negative associations with alcohol consumption presumably relate to carcinogens potentially present in wines (e.g., ethyl carbamate). thankfully, its carcinogenicity is reduced at the alcohol concentration typical of table wines. more significant, though, maybe acetaldehyde, a common denominator in gastrointestinal cancers (salaspuro, ) . depending on the type of wine consumed, short-term but high concentrations of acetaldehyde may occur in the saliva and gastric juice (lachenmeier and sohnius, ) . acetaldehyde may also accumulate due to the action of alcohol dehydrogenase in the digestive tract, microbial alcohol metabolism (notably the colon), and malfunction of cellular acetaldehyde dehydrogenase. certain wine phenolics can be protective, whereas others mutagenic, especially at high concentrations. for example, quercetin can induce mutations in laboratory tissue cultures but is a potent anticarcinogen in whole-animal studies (fazal et al., ). this apparent anomaly may result from differences in the concentrations of quercetin used, and/or the low levels of metal ions and free oxygen found in the body (vs. tissue culture). in addition, quercetin, along with several other phenolics that are potential carcinogens, lose this attribute when present as a glycoside. most phenolics in the plasma occur in some conjugated state, not as free phenolics. in addition, phenolics may detoxify the small quantities of nitrites commonly found in food. however, in the presence of high nitrite concentrations (a preservative found in smoked and pickled foods), nitrites are converted into diazophenols (weisburger, ) . these appear to favor the development of oral and stomach cancers. potential health issues several phenolics can limit or prevent cancer development through a diversity of effects, such as dna repair, carcinogen detoxification, enhanced apoptosis (programmed cell death), disrupted cell division (hou, ; aggarwal et al., ) , or enhanced immunostimulation (tong et al., ) . for example, resveratrol induces the redistribution of the fas receptor. it is a cellular attachment site for tnf (tumor necrosis factor). its action is part of a sequence that can lead to cancer cell apoptosis (delmas et al., ) . resveratrol is an inhibitor of angiogenesisdthe production of new vasculature essential for most tumor growth. other effects of resveratrol include inhibition of cyclooxygenase- (subbaramaiah et al., ) and cytochrome p a (chun et al., ) . cyclooxygenase- is thought to be involved in carcinogenesis, whereas p a is an important hydroxylase. it can convert several environmental toxicants and procarcinogens into active carcinogens. flavones and flavonols strongly restrict the action of common dietary carcinogens, notably heterocyclic amines (kanazawa et al., ) . it is estimated that these amines, produced during cooking, are consumed at a rate of approximately . e mg per day (wakabayashi et al., ) . antitumor activity is also associated with acutissimin, a flavono-ellagitannin found in oakmatured red wine. the antiallergic and antiinflammatory properties of flavonoid phenolics probably also contribute to the anticancer aspects of these flavonoids (see middleton, ) . the major exception to the general benefit of moderate wine consumption may be breast cancer (viel et al., ) . the connection is more evident in those with the adh c* (fast metabolizers of ethanol to acetaldehyde) (terry et al., ) . however, findings from the long-duration framingham study indicate no relationship between moderate alcohol consumption and the incidence of breast cancer (zhang et al., ) . ethanol, although not itself a carcinogen, can enhance the transforming effect of some carcinogens. another example of a negative effect of wine consumption, at least in excess of moderate intake, is to increase the incidence of mouth and throat cancers (barra et al., ) . alcoholic beverages may also induce a diversity of allergic and allergy-like reactions. in sensitive individuals, these may express as rhinitis, itching, facial swelling, headache, cough, or asthma. the primary culprit inducing bronchial constriction, at least in some asthmatics, is sulfur dioxide (dahl et al., ) . wine containing an abnormally high sulfur dioxide concentration ( ppm sulfite) induce a rapid drop in forced expiratory volume (vally and thompson, ) , recovery taking about e min. the same individuals did not respond to wine containing , , or ppm sulfite. fig. . illustrates the range of sulfur dioxide contents potentially found in californian wine. thus, the sulfite levels normally found in wine seem not to be a major factor in wine-induced asthmatic responses (vally et al., ) . why sensitive asthmatics episodically react to wines with low so content may be related to changes in their asthma control. surprisingly, red wines appear to provoke more asthma problems than white wines, even though red wines typically have lower sulfur dioxide contents than white wines. californian wines (mg/l). reprinted from peterson, g.f., kirrane, m., hill, n., agapito, a., . a comprehensive survey of the total sulfur dioxide concentrations of american wines. am. j. enol. vitic. , e , permission conveyed through copyright clearance center. . wine, food, and health the rapidity of the reaction to sulfite suggests some malfunction in the amount of glutathione in lung tissue or the activity of glutathione s-transferase in reducing sulfite to glutathione s-sulfonate. normally, sulfite is rapidly converted to sulfate by sulfite oxidase in the blood. however, low levels of this enzyme could permit sulfite to persist, provoking a heightened response in hypersensitive individuals. at greater risk are individuals afflicted with a rare, autosomal, genetic disease, caused by a deficiency in sulfite oxidase (shih et al., ; crawhall, ) . affected individuals must live on a very restricted diet, low in sulfur-containing proteins. it is estimated that the synthesis of sulfite, associated with normal food metabolism, generates approximately . g sulfite/day. the sulfites in wine contribute only marginally to this amount but may be temporally significant. because of the gravity of sulfite oxidase deficiency, most affected individuals do not reach adulthood. another allergy-like reaction provokes rapid facial and neck flushing (cutaneous erythema). it develops shortly after alcohol consumption. other symptoms often include peripheral vasodilation, elevated heart rate, nausea, abdominal discomfort, and bronchoconstriction. the syndrome is associated with a malfunctional form of mitochondrial acetaldehyde dehydrogenase (aldh * ) (enomoto et al., ; eriksson et al., ) and is particularly pronounced in the homozygous state. aldh is the principal enzyme oxidizing acetaldehyde to acetic acid. it is estimated that up to % of eastern asians possess at least one malfunctional aldh allele and express some degree of allergy-like reaction to alcohol consumption. it has been suggested that the aldh mutant, frequently found in eastern asians, may reflect an evolutionary adaptation to the endemic occurrence of hepatitis b (lin and cheng, ) . the mutation could have induced alcohol aversion, thereby avoiding synergism between alcohol and hepatitis b-induced liver damage. elevated levels of acetaldehyde appears to activate the release of histamine from mast cells. it subsequently induces vasodilation and an associated influx of blood (flushing). the connection between acetaldehyde and histamine is supported by the action of antihistamines in reducing the reaction, if taken in advance of an alcohol challenge (miller et al., ). an alternative proposal is that this flushing reaction results from a direct, cutaneous, alcohol-induced vasodilation. the phenomenon tends to be suppressed by acetylsalicylic acid (aspirin), if taken in advance (truitt et al., ) . the unpleasant side-effects of acetaldehyde accumulation is used in treating alcoholism. it involves taking disulfiram (a potent inhibitor of aldh) prior to alcohol consumption. a facial flushing, concomitant with alcohol consumption but devoid of other symptoms, is occasionally experienced by caucasians. whether this is related to an aldh malfunction is unclear. the histamine content of wine has frequently been thought to contribute to several allergy-like reactions. however, wine is typically low in histamine content. fig. . illustrates the range found in some wines. thus, it seems unlikely that a wine's histamine content is a major inducer of allergy-like reactions. this is supported by a double-blind study of people, selfreportedly wine intolerant. reactions to two pinot noir wines, differing in histamine content ( . vs. . mg/ l) were not significantly different (kanny et al., ) . other common foods are considerably higher in histamine content, for example cheeses. this does not necessarily exonerate biogenic amines from being somehow involved. people vary in diamine oxidase activity (a histamine inactivator) (wantke et al., ) and how alcohol (via acetaldehyde) suppresses its action . alcohol also can enhance the permeability of the intestinal lining to histamine. in addition, acetaldehyde accumulation can activate histamine release (harada et al., ) . this may explain the benefit antihistamines have in diminishing the rhinitis occasionally associated with wine consumption (andersson et al., ) . in addition, antihistamines counteract the broncho constriction in individuals showing histamine intolerance. idiopathic allergic and other immune hypersensitive responses to wine are difficult to predict or diagnose. reactions may include the induction of headaches, nausea, vomiting, general malaise, or a combination of these. in a few instances, ige-related anaphylaxis reactions have been reported to grape pr proteins (endochitinase and thaumatin) (pastorello et al., ) . the effects may involve urticaria/angioedema (red patches or wheals on the skin/swelling) and occasionally shock. residual amounts of fining agents, such as egg whites, have also been implicated in some allergic reactions (marinkovich, ) . in a double-blind, placebocontrolled trial, wines fined with egg white, isinglass, or nongrape derived tannins presented "an extremely low risk of anaphylaxis" to egg-, fish-, or peanutallergic consumers (rolland et al., ) . in an elisa analysis, only egg white and lysozyme could be detected in wine samples (weber et al., ) . nevertheless, with more than compounds potentially occurring in wine, it is not surprising that some individuals may occasionally show some form of adverse reaction to some wines. in addition to physiological reactions to wine constituents, there is a wide range of equally important psychological responses (rozin and tuorila, ) , both positive and negative. traumatic memories, associated with the first exposure to, or excessive consumption of, a particular beverage can create an association that lasts a lifetime. other people have come to associate certain products with social groups, lifestyles, or behaviors. such attitudes can make the beverage either unacceptable or desirable as the case may be. in the s, there were many reports linking gout with wine consumption, notably port. gout is caused by the localized accumulation of uric acid crystals in the synovium of joints. their presence stimulates the synthesis and release of humoral and cellular inflammatory mediators (choi et al., ) . gout is also associated with reduced excretion of uric acid in the kidneys. mutations in the gene that encodes urease, the enzyme that metabolizes uric acid to allantoin (a soluble by-product), is often implicated in gout. dietary predisposing factors for gout include red meat, seafood, and beer. this is presumably because purines, the principal source of uric acid, are found in higher concentrations in these products than many other foods or beverages. alcohol consumption may occasionally aggravate gout by increasing lactic acid synthesis. it, in turn, favors uric acid reabsorption by the kidneys. despite this, wine consumption appears not be associated with an increased risk for gout. in contrast, it seems to favor reduced serum urate levels (choi and curhan, ) . medical historians suspect the nineteenth century gouteport association was connected with leadinduced kidney damage (yu, ; emsley, emsley, / . samples of port from the nineteenth century show high lead contents. lead contamination probably came from the stills used in preparing the wine spirits added in port production. in addition, the former use of pewter and lead-glazed drinking cups and prolonged storage of port in lead crystal decanters or stemware could have further augmented lead content (falcone, ; guadagnino et al., ) . people occasionally avoid wine because it induces headaches. regrettably, the wine/headache connection is still poorly understood. however, effective differentiation between wine-induced headaches may be pivotal to discovering their causes and possible solutions. one of the most severe headache syndromes, potentially associated with wine consumption, is the migraine. migraines appear to be induced, but inconsistently, by a wide range of environmental stimuli, often in tandem. an association between it and red wine consumption has been noted since roman times. the dilation of cerebral blood vessels, partially associated with histamine release, appears to be a common element in many headache syndromes. migraines may be one of them, although current thought suggests a neurological rather than a vascular origin. in addition, a double-blind study seemingly has exonerated histamine in most redwine-induced migraine headaches (masyczek and ough, ) . in addition, migraine attacks are more often associated with consuming spirits and sparkling wines, both lower in histamine content that table wines or beer (nicolodi and sicuteri, ) . however, the former are often taken alone, leading to more rapid and higher spikes in blood alcohol content. alcohol may be directly involved in migraine induction through vasodilationdby activating meningeal vessel-associated trigeminal neurons (nicoletti et al., ) . alcohol's potential to reduce cerebral glucose metabolism (volkow et al., ) could also be a contributing factor. other potential disruptive aspects on brain function may relate to the slow rate of alcohol metabolism by cerebral adh. thus, more alcohol may be metabolized by cytochrome p e, a process that generates acetaldehyde as well as ros. the more frequent association of red wines with several headache sequelae may be due to their higher phenolic content. on average, red wines contain about mg/l phenolics, vs. mg/l for white wines. some phenolics can suppress the action of platelet phenol sulfotransferase (pst) (jones et al., ; yeh and yen, ) , several isozymic forms (m and p) of which detoxify biogenic amines and phenolics via sulfation. low levels of platelet-bound pst-p have been correlated with migraine susceptibility (alam et al., ) . the accumulation of small phenolics (those readily absorbed) in the blood could prolong the action of potent hormones and nerve transmitters (e.g., histamine, serotonin, dopamine, adrenalin, and noradrenaline). small phenolics can also promote platelet aggregation and blood vessel dilation. the associated increase in intercranial pressure may participate in a migraine attack (pattichis et al., ) . abnormal and cyclical patterns in platelet sensitivity to -ht release in migraine-prone individuals (jones et al., ; peatfield et al., ) may explain the inconsistent association of wine consumption with migraine induction. in the treatment of cluster-headaches, small doses of lithium have been suggested as preventive (steiner et al., ) . because some red wines have a higher than average lithium content, the possibility exists that they might limit the development of, rather than induce, this headache syndrome. another recognized headache syndrome is called the red wine headache (kaufman, ) . it may develop within minutes of consuming red wine, often being dose-related. the headache reaches its peak within w h, tends to fade but returns roughly h later. the headache seems related to the release of type e prostaglandins, important chemicals involved in dilating blood vessels. their release can be activated by phenolics (padilla et al., ) as well as alcohol (parantainen, ) . prostaglandins may also activate pain receptors around blood vessels (wienecke et al., ). this association may explain why prostaglandin synthesis inhibitors (e.g., acetylsalicylic acid, acetaminophen, or ibuprofen) may limit the development of some winerelated headaches (if taken about h before consumption) (kaufman, ) . a separate wine-related headache has been dubbed the red head (goldberg, ) . it develops within an hour of waking, after drinking no more than two glasses of red wine the previous evening. the headache, associated with nausea, is particularly severe when reclining. although the headache is somewhat relieved by standing, it itself exacerbates the nausea. the headache usually lasts a few hours before dissipating. a similar phenomenon has been reported with some white wines, or mixtures of white wine, taken alone or with coffee or chocolates. its chemical cause is unknown (kaufman, ) . because tannins are poorly absorbed in the upper digestive tract, in contrast to monomeric phenolics, the latter are likely the primary phenolic headache activants. this may explain why aged red wines (in which most phenolics occur as large polymers) tend to be less associated with headaches than their younger counterparts. large tannin polymers remain largely unmodified until reaching the colon, where bacteria degrade them (déprez et al., ) . because this can take up to days, they presumably are not (or not recognized to be) involved in wine-induced headaches. phenolic absorbed into the blood are primarily detoxified by being methylated or sulfated but may also become more "toxic" (to o-quinones). the latter can retard the breakdown of dopamine and restrict access to m-opioid (painkilling) receptors, exacerbating the pain associated with cerebral blood vessel dilation. nonetheless, some phenolics (e.g., resveratrol) limit, rather than augment, headache development. it inhibits the expression of cyclooxygenases, involved in the synthesis of prostaglandins (jang and pezzuto, ) . although red wines are generally associated with headache production, white wines are occasionally associated with their production (relja et al., ) . their characteristics and etiology are even less well understood than those evoked by red wines. in some individuals, this situation may be associated with a sensitivity to sulfites but atypically. one of the most recognized alcohol-related headache phenomena is associated with binge drinkingdthe hangover (veisalgia) (wiese et al., ) . although not consistently associated with a headache, it is frequently part of the sequelae. hangovers are characterized by tremulousness, palpitations, tachycardia, sweating, loss of appetite, anxiety, nausea, and possibly vomiting and amnesia . when accompanied with a headache, it possesses symptoms resembling a migraine. the headache may be global but frequently concentrated anteriorly, associated with heavy, pulsesynchronous throbbing. it usually starts a few (w h) after the cessation of drinking, when blood alcohol level is declining and other hangover symptoms have already developed (sjaastad and bakketeig, ; verster et al., ) . duration is seldom more than h. despite its all-too-frequent occurrence, the causal mechanism(s) remains unclear. most data suggest that alcohol-induced cerebral inflammation is the primary cause . this may operate directly via tissue dehydration and electrolyte imbalance (due to vasopressin enhanced urination) or indirectly via the toxic effects of acetaldehyde (quertemont et al., ) . ethanol can also promote hepatic glycogen breakdown, glucose release, loss via the kidneys, and induction of hypoglycemia. in addition, activation of the liver's microsomal ethanol oxidation pathway releases ros, causing cellular damage and multiple metabolic disruptions. that the severity of a hangover may be reduced by prostaglandin synthesis inhibitors suggests that they may also play a role in hangover sequelae (kaivola et al., ) . as the old spanish proverb noted: wine hath drowned more men than the sea because glutathione inactivates free radicals, taking an amino acid supplement (n-acetyl-cysteine) has been suggested as a partial remedy. it is rich in cysteine, an amino acid that forms the core of glutathione. in addition, glutathione facilitates the conversion of acetaldehyde to acetic acid. disruption of membrane function and cerebral neurotransmitter action by acetaldehyde is presumably the rationale for commercial products, such as hangover helper and rebound. they are designed to counter the effects of acetaldehyde. congeners (such as fusel alcohols and methanol) could exacerbate the effects of ethanol and acetaldehyde. however, because their content in wine is low, they are unlikely to be involved in wine-induced hangovers. some purported remedies, such as artichoke extract, have not stood up to rigorous clinical testing (pittler et al., ) , but others, such as an opuntia fiscus-indica (prickly pear) extract, apparently reduced the severity of some hangover symptoms (wiese et al., ) . that hangovers have been associated with deregulation of cytokine pathways (kim et al., ) , may explain the reported value of pyritinol (a vitamin b derivative) as a treatment (khan et al., ) . mineral deficiencies have also been correlated with hangovers (min et al., ) . combined with restraint, taking wine with a meal is probably the best means by which to avoid a hangover. food delays the movement of alcohol into the intestinal tract, thereby slowing alcohol uptake ( fig. . ) and correlating uptake closer to the liver's ability to metabolize ethanol. in addition, delayed transfer to the intestinal tract slows phenolic uptake (and other potential provocateurs). wine tasting is not normally considered a hazardous occupation. however, recent studies show that dental erosion is an occupational hazard (mok et al., ; mandel, ; mulic et al., ) . damage results from the frequent and extended exposure to wine acids, correspondingly, white wines are generally more corrosive than reds (willershausen et al., ) . saliva is diluted and washed away, resulting in the oral ph falling to that of the wine (obreque-slier et al., ) . this causes calcium to dissolve out of tooth enamel, softening and making it susceptible to erosion by masticatory forces and tooth brushing. exposure times as short as min can be harmful (lupi-pegurier et al., ) . demineralization commences at about ph . . dental erosion is unlikely to be a significant problem for the typical consumer who takes wine with meals. food and salivary secretion limit, if not prevent, tooth enamel demineralization. after many years, professional wine tasters may experience tooth disfiguration, affecting both tooth shape and size. cupping, a depression in the enamel, exposing dentine at the tip of molar cusps, is a frequent clinical sign. erosion can also contribute to severe root abrasion at the gum line. the good news is that not all tasters are equally at risk (mulic et al., ) . protection is partially achieved by rinsing the mouth with an alkaline mouthwash after tasting, application of a fluoride gel (such as apf) and refraining from tooth brushing for at least h after tasting. the delay permits minerals in the saliva to rebind with enamel. for more protective protocols see ranjitkar et al. ( ) . the use of remineralizing agents, such as tooth mousse, also helps prevent dental erosion (piekarz et al., ) . in contrast to this risk factor, consuming red wine may have some direct oral benefits. proanthocyanidins can limit the adherence and biofilm-forming activity of caries-inducing streptococcus mutans (daglia et al., ) . gibbons ( ) provides a fascinating insight into the association of this bacterium with changes in human diet which resulted from a switch from a hunter-gather to an agriculture lifestyle. mark twain also made pronouncements about dental health, which might be equally applied to wine: "i always take it (scotch whiskey) at night as a preventive of toothache. i have never had the toothache; and what is more, i never intend to have it". from europe and elsewhere. fetal alcohol syndrome refers to a set of phenomena including suppressed growth, mild mental retardation, and subtle facial abnormalities (wattendorf and muenke, ) . it was first described in and appeared most markedly in the children of alcoholic mothers. they tended also to be heavy smokers, users of illicit drugs, consumers of large amounts of coffee, had poor nutrition, or a combination of these (scholten, ; whitten, ) . although associated with alcohol uptake, the accumulation of acetaldehyde may be the principal toxicant. even more subtle effects have now been associated with alcohol consumption, generating the fetal alcohol spectrum disorders. because the consequences may be lifelong, it is generally recommended that pregnant women, or those desirous of becoming pregnant, refrain from alcohol consumption. although abstinence may be unnecessary (kesmodel et al., ) , erring on the side of caution can supply desired peace-of-mind. this also applies to breast feedingd alcohol in breast milk could be detrimental to infant development. the presence of toxins in wine is seldom mentioned, outside academic circles, presumably because of their minimal presence. the only mycotoxin for which there may be regular analysis is ochratoxin a (o'brien and dietrich, ; varga and kozakiewicz, ) , produced by several black aspergilli (notably aspergillus carbonarius) (somma et al., ) . preliminary data suggests that most ochratoxin a is eliminated (destroyed/precipitated) during and after fermentation/maturation (fernandes et al., ) . other potential mycotoxins that could occur in wine include isofumigaclavine, festuclavine, and roquefortine, all produced by penicillium spp. (moller et al., ) , aflatoxins (el khoury et al., ) from aspergillus flavus, fumonisins from aspergillus niger (mogensen et al., ) , and trichothecenes by trichothecium roseum (schwenk et al., ) . because these fungi are secondary saprophytes, they typically occur only on rotted grapes (thankfully, unlikely on noble-rotted grapes). although the exclusion of all diseased grapes is essentially impossible, their inclusion is limited as much as feasibly possible. pesticide residues are other potential toxins. their levels are usually below those known to be toxic, partially due to regulations limiting their use, precipitation or metabolism during winemaking and degradation during maturation. in addition, most importing countries possess regulations on permissible levels and systems to check for compliance. achieving a zero concentration is probably impossible, if only because of our increasing technical ability to detect their presence at increasingly infinitesimal levels. methanol is present but in amounts insufficient to have any known negative consequences. the same also appears to be true for diacetyl and other potentially toxic compounds. ethyl carbamate, a carcinogen, is no longer likely to occur, since its origin during wine production can be effectively avoided. it may initially be disconcerting to think of trace amounts of toxins in wine, but this situation applies to all food, water, and air. xenobiotics are an inescapable aspect of life, both modern and ancient. their universal presence in the natural environment presumably provided the selective pressures that favored the evolution of organs of detoxification (e.g., the liver and kidneys) and the presence of multiple detoxifying enzyme systems. thankfully, our bodies inactivate most xenobiotics rapidly and effectively, without our conscious knowledge. in addition, governmental agencies set regulations and assess compliance to limit most toxicants to well below known safe limits. as long as exposure to toxicants is kept to a bare minimum, consumers can basically forget they exist. the most important wine contraindication relates to those with a past history of alcohol abuse. for the majority of adults (except pregnant women), moderate wine consumption appears to have significant health benefits. nevertheless, there are several situations in which wine consumption, even in moderate amounts, can complicate or diminish the effectiveness of disease treatment. the acidic nature of wine can aggravate inflammation and slow the healing of ulcers in the mouth, throat, stomach, and intestinal tract. other constituents in wine may also be detrimental in this regard. thus, all beverages containing alcohol are usually contraindicated in cases of gastritis, gastric cancer, and bleeding in the upper digestive tract. nevertheless, the prophylactic action of red wine against helicobacter pylori and the suppression of histamine production by the gastric mucosa (masquelier, ) may require a reconsideration of the old prohibition in mild cases. in the presence of pancreatitis, alcohol is absolutely contraindicated. wine, along with other alcoholic beverages, may provoke gastroesophageal (acid) reflux in individuals prone to this syndrome. with liver disease, the consumption of wine is normally contraindicated. the presence of alcohol puts additional stress on an already weakened vital organ. chronic alcohol abuse can lead to cirrhosis of the liver. in acute kidney infection, wine should be avoided. the consumption of alcohol increases the burden on an organ essential to eliminating toxic metabolic wastes. with prostatitis or genitourinary infections, the consumption of alcohol can complicate matters. the diuretic action of wine may increase the frequency of urination, or conversely it may induce highly painful urinary retention. in epilepsy, the consumption of even moderate amounts of wine may increase the frequency of seizures. consumption should be strictly limited in most situations of hypertension, hemorrhagic stroke, or atrial fibrillation. patients, about to undergo surgery, are advised to avoid all alcoholic beverages well before surgery. this avoids increasing any tendency to enhance intra-and postoperative bleeding (wolfort et al., ) , due to alcohol's reduction of platelet clotting. the consumption of alcohol is also ill advised when eating certain mushrooms. the most well-known example is the antabuse reaction associated if alcohol is consumed with coprinus atramentarius (inky cap). another mushroom generating the same response is boletus luridus (budmiger and kocher, ) . the antabuse reaction derives its name from the trade name of disulfiram, a medication used in the treatment of alcoholism. it functions as an inhibitor of acetaldehyde dehydrogenase. even small amounts of alcohol consumed while taking disulfiram can generate very unnerving reactions (e.g., flushing, sweating, weakness, vertigo, blurred vision, difficulty breathing, nausea, chest pain, palpitation, and tachycardia). in severe cases, the reaction can provoke acute congestive heart failure, convulsion, and death. similar symptoms may develop in sensitive individuals when alcohol beverages are consumed while taking certain medications (e.g., cephalosporins, griseofulvin, chloramphenicol, sulfonylurea, metronidazole). in addition to the reactions noted above, consumption of alcohol while taking certain medications can generate dangerous conditions. most of the literature comes from studies on alcoholics or binge drinkers. this limits the potential applicability of the data to conditions of moderate consumption and when taken with food. nevertheless, even small amounts of alcohol may cause loss of muscle control in people taking tricyclic antidepressants. in addition, red wines can reduce the effectiveness of mao (monoamine oxidase) inhibitors, used in controlling hypertension. long-term acetaminophen use can enhance alcohol-induced kidney damage. other contraindications involve the intensification of the effects of barbiturates and narcotics. in combination with certain antidiabetic agents, such as tolbutamide and chlorpropamide, alcohol can cause dizziness, hot flushes, and nausea. mild reactions may occur with a wide range of other medications, such as sulfanilamide, isoniazid, and aminopyrine. additional details may be found in adams ( ) , fraser ( ) , and weathermon and crabb ( ) . in conclusion, mark twain crystalizes what so often seems to be the relationship between food, wine, and health: the only way to keep your health is to eat what you don't want, drink what you don't like, and do what you'd druther not." mark twain in following the equator. interactions between alcohol and other drugs role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies platelet sulphotransferase activity, plasma sulfate levels, and sulphation capacity in patients with migraine and tension headache effects of loratadine in red wineinduced symptoms and signs of rhinitis alcohol consumption and acute myocardial infarction: a benefit of alcohol consumed with meals? epidemiology microbial metabolism of dietary phenolic compounds in the colon wine flavonoids protect against ldl oxidation and atherosclerosis the relative antioxidant potencies of some polyphenols in grapes and wines neurodegenerative disease and oxidative stress type of alcoholic beverage and cancer of the oral cavity, pharynx and oesophagus in an italian area with high wine consumption waiter, is there histamine in my wine? comparisons between australian consumers' and industry experts' perceptions of ideal wine and cheese combinations understanding consumer preferences for shiraz wine and cheddar cheese pairings alcohol and the niddm patient effect of red wine, minor constituents, and alcohol on the gastric emptying and the metabolic effects of a solid digestible meal modulatory effect of resveratrol, a natural phytoalexin, on endothelial adhesion molecules and intracellular signal transduction wine and resveratrol: mechanisms of cancer prevention? wine consumption and intestinal redox homeostasis the role of moderate ethanol consumption in health and human nutrition effects of salt and fat combinations on taste preference and perception alcohol drinking and mortality among men enrolled in an american cancer society prospective study relation of smoking, alcohol and coffee consumption to active helicobacterium pylori infection: cross sectional study suppression of bitterness by sodium: variation among bitter taste stimuli salt enhances flavour by suppressing bitterness the role of vanadium in the management of diabetes boletus luridus and alcohol. case report (in german) effect of flavan- -ols on the adhesion of potential probiotic lactobacilli in intestinal cells agrp neuron activity is required for alcohol-induced overeating prospective study of moderate alcohol consumption and risk of peripheral arterial disease in us male physicians red wine consumption and risk of prostrate cancer: the california men's health study inhibition of pseudomonas aeruginosa and escherichia coli :h biofilm formation by plant metabolite ε-viniferin beer, liquor, and wine consumption and serum uric acid level: the third national health and nutrition examination survey pathogenesis of gout estimated dietary flavonoid intake and major food sources of us adults resveratrol is a selective human cytochrome p a inhibitor smoking, alcohol consumption, and susceptibility to the common cold alcohol consumption as a cause of cancer the effect of red and white wines on nonheme-iron absorption in humans endothelin- synthesis reduced by red wine overview of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology a review of the clinical presentation and laboratory findings of two uncommon hereditary disorders of sulfur amino acid metabolism, b-mercaptolactate cysteine disulfideuria and sulfite oxidase deficiency epidemiology of stone disease isolation of red wine components with anti-adhesion and anti-biofilm activity against streptococcus mutans red wine asthma: a controlled study resveratrol-induced apoptosis is associated with fas redistribution in the rafts and the formation of a deathinducing signaling complex in colon cancer cells polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids dietary fiber in wine of sizzling steaks and dna repair alcohol consumption in the severely obese: relationship with the metabolic syndrome inhibitory activity of diluted wine on bacterial growth: the secret of water purification in antiquity mortality in relation to alcohol consumption: a prospective study among male british doctors wine and cancer phenolic metabolites of anthocyanins modulate mechanisms of endothelial function fungal contamination and aflatoxin b and ochratoxin a in lebanese winedgrapes and musts when the empire struck lead genotyping of the aldehyde dehydrogenase (aldh ) gene using the polymerase chain reaction: evidence for single point mutation in the aldh gene for aldh -deficiency functional relevance of human adh polymorphism abnormal brain structure implicated in stimulant drug addiction migration of lead into alcoholic beverages during storage in lead crystal decanters strand scission in dna by quercetin and cu(ii): identification references of free radical intermediates and biological consequences of scission changes in ochratoxin a concentration during winemaking changes in antioxidant endogenous enzymes (activity and gene expression levels) after repeated red wine intake metabolites are key to understanding health effects of wine polyphenolics effects of ethanol and some alcoholic beverages on gastric emptying in humans inhibition of human ldl oxidation by resveratrol pharmacokinetic interactions between alcohol and other drugs smoking, alcohol intake, estrogen use, and age related macular degeneration in latinos: the los angeles latino eye study antibacterial, antiviral, and antifungal properties of wines and winery products in relation to their flavonoid content perceptual interactions in mixtures containing bitter tasting substances the in vitro effect of red wine on helicobacterium pylori white wine with red wine-like properties: increased extraction of grape skin polyphenols improves the antioxidant capacity of the derived white wine use of multi-intake temporal dominance of sensations (tds) to evaluate the influence of cheese on wine perception effect of alcohol intake on bone mineral density in elderly women: the epidos study. epidemiologie de l'osteoporose type of alcoholic beverage and risk of myocardial infarction how sweet it is: genes show how bacteria colonized human teeth short-term effect of red wine (consumed during meals) on insulin requirement of glucose tolerance in diabetic patients resveratrol, at concentrations attainable with moderate wine consumption, stimulates human platelet nitric oxide production in vitro bioconversion of polyphenols from black tea and red wine/grape juice by human intestinal microbiota displays strong interindividual variability type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer inhibition of key digestive enzymes by cocoa extracts and procyanidins co-operative study on the release of lead from crystalware how wine polyphenols can fight alzheimer disease progression: towards a molecular explanation alcoholism and neuro-immuneeendocrine interactions: physiochemical aspects alcohol: effects of consumption on diet and nutritional status aldehyde dehydrogenase deficiency as cause of facial flushing reaction to alcohol in japanese metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth oxidants, antioxidants, alcohol and stroke genetic variation in alcohol dehydrogenase and the beneficial effect of moderate alcohol consumption on myocardial infarction identification of bitterness-masking compounds from cheese eukaryotic-microbiota crosstalk: potential mechanisms for health benefits of prebiotics and probiotics potential mechanism of cancer chemoprevention by anthocyanins parotid salivation in response to tasting wine to drink or not to drink: how are alcohol, caffeine and past smoking related to bone mineral density in elderly women? flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. free radic resveratrol blocks eicosanoid production and chemically-induced cellular transformation: implications for cancer chemoprevention wine and headache alcoholic beverage preference and risk of becoming a heavy drinker reduced platelet phenolsulphotransferase activity towards dopamine and -hydroxytryptamine in migraine platelet aggregation in migraine patients during the headache-free interval study of interactions between food phenolics and aromatic flavors using one-and two-dimensional hangover headache and prostaglandins: prophylactic treatment with tolfenamic acid effects of food and body composition on blood alcohol curves anthocyanin metabolites are abundant and persistent in human urine après consommation des vins de champagne, cinétique du devenir de l'éthanol antimutigenicity of flavones and flavonols to heterocyclic amines by specific and strong inhibition of the cytochrome p a family the stomach as a bioreactor: dietary lipid peroxidation in the gastric fluid and the effects of plant derived antioxidants. free radic no correlation between wine intolerance and histamine content of wine wine and the elderly person proceedings of wine selective inhibition of bitter taste of various drugs by lipoprotein the red wine headache: a pilot study of a specific syndrome the red wine headache and prostaglandin synthetase inhibitors: a blind controlled study suppression of bitterness by sodium salts an overview of binary taste-taste interactions alcohol, blood pressure and hypertension dietary flavonoids, antioxidant vitamins and the incidence of stroke: the zutphen study the effect of different alcohol drinking patters in early to mid pregnancy on the child's intelligence, attention, and executive function alcohol-induced hangover. a double-blind comparison of pyritinol and placebo in preventing hangover symptoms effects of alcohol hangover on cytokine production in healthy subjects alcohol consumption before myocardial infarction: results from the kaiser-permanente epidemiologic study of myocardial infarction wine, liquor, beer, and mortality drinker prototypes in american society alcohol consumption is associated with reduced prevalence of goitre and solitary thyroid nodules concord grape juice supplementation and neurocognitive function in human aging the relationship of alcohol consumption to arteriosclerotic heart disease the role of acetaldehyde outside ethanol metabolism in the carcinogenicity of alcoholic beverages: evidence from a large chemical study carcinogenicity of acetaldehyde in alcoholic beverages: risk assessment outside ethanol metabolism effect of tea phenolics and their aromatic fecal bacterial metabolites on intestinal microbiota a central role of e-nos in the protective effect of wine against metabolic syndrome risk of dementia and alcohol and wine consumption: a review of recent results association between consumption of beer, wine, and liquor and plasma concentration of high sensitivity creactive protein in women aged to years complementary approaches to gauge the bioavailability and distribution of ingested berry polyphenolics why can't chinese han drink alcohol? hepatis b virus infection and the evolution of acetaldehyde dehydrogenase deficiency retronasal odor enhancement by salty and umami tastes identification of organic acids in wine that stimulate mechanisms of gastric acid secretion visible light-induced lipid peroxidation of unsaturated fatty acids in the retina and the inhibitory effects of blueberry polyphenols interaction of tannin with human salivary proline-rich proteins alcohol intake and risk of dementia a history of wine as therapy. lippincott, philadelphia williamson, . polyphenols, astringency and proline-rich proteins in vitro action of bordeaux red wine on the microhardness of human dental enamel dental erosion due to wine consumption resveratrol promotes clearance of alzheimer's disease amyloid-beta peptides allergic symptoms from fining agents used in winemaking human parotid secretion in response to ethyl alcohol neuroprotective properties of spanish red wine and its isolated polyphenols on astrocytes azione portettrice del vino sull'ulcera gastrica the red wine reaction syndrome effects of linoleic acid on sweet, sour, salty, and bitter taste thresholds and intensity ratings of adults wine antioxidants and their impact on antioxidant activity in vivo red wine and antioxidant activity in serum alcohol-induced generation of lipid peroxidation products in humans prenatal and postnatal flavor learning by human infants effect of plant flavonoids on immune and inflammatory cell function reduction of alcoholinduced performance impairment by prior ingestion of food antihistamine blockade of alcoholinduced flushing in orientals the application of minerals in managing alcohol hangover: a preliminary review aroma release of a model wine solution as influenced by the presence of non-volatile components. effect of commercial tannin extracts, polysaccharides and artificial saliva widespread occurrence of the mycotoxin fumonisin b in wine dental erosion: in vitro model of a wine assessor's erosion toxin-producing species of penicillium and the development of mycotoxins in must and homemade wine role of flavonoids and iron chelation in antioxidant action acute effects of chlorogenic acid on nitric oxide status, endothelial function, and blood pressure in healthy volunteers: a randomized trial alcohol consumption and risk for coronary heart disease in men with healthy lifestyles red wine and oenological extracts display antimicrobial effects in an oral bacteria biofilm model wine is bactericidal to foodborne pathogens the effect of various substances on the suppression of the bitterness of quinine-human gustatory sensation, binding and taste sensor studies red wine prevents the postrandial increase in plasma cholesterol oxidation products: a pilot study manuel practique de vinification et de conservation des vins the addicted brain ethanol causes neurogenic vasodilation by trpv activation and cgrp release in the trigeminovascular system of the guinea pig wine and migraine: compatibility or incompatibility? hemoglobinacetaldehyde adducts in human alcohol abusers alcoholic macrocytosis e is there a role for acetaldehyde and adducts? effect of wine-based marinades on the behavior of salmonella typhimurium and background flora in beef fillets seeking the connections: alcoholism and our genes perceived flavour changes in white wine after tasting blue mould cheese effects of tasting technique e sequential tasting vs. mixed tasting e on perception of dry white wine and blue mould cheese perceived flavour changes in blue mould cheese after tasting white wine wine: protective in macular degeneration wine ph prevails over buffering capacity of human saliva experimental and theoretical data on the mechanism by which red wine anthocyanins are transported through a human mkn- gastir cell model effects of grape seed-derived polyphenols on amyloid b-protein self-assembly and cytotoxicity ochratoxin a: the continuing enigma relationship between vasodilation capacity and phenolic content of spanish wines alcohol consumption and noncommunicable diseases: epidemiology and policy implications the stomach as a site for anthocyanins absorption from food fast access of some grape pigments to the brain identification of grape and wine allergens as an endochitinase , a lipid-transfer protein, and a thaumatin -hydroxytryptamine release from platelets by different red wines: implications for migraine prostacyclin, tyramine and red wine different effects of white and red wine on lower esophageal sphincter pressure and gastroesophageal reflux a comprehensive survey of the total sulfur dioxide concentrations of american wines an in vitro assessemt of the ole of tooth mousse in preventing wine erosion effectiveness of artichoke extract in preventing alcohol-induced hangovers: a randomized controlled trial crosscultural aspects of drinking, alcohol abuse and alcoholism red wine antioxidants. evaluation of their hydrophobicity and binding extent to salivary proteins substituent effects on in vitro antioxidizing properties, stability, and solubility of flavonoids epicatechin adducting with -hydroxymethylfurfural as an inhibitoury mechanism against acrylamide formation in maillard reactions is ethanol a pro-drug? acetaldehyde contribution to brain ethanol effects the relation between different dimensions of alcohol consumption and burden of disease: an overview wine alcohol, platelets and the french paradox for coronary heart disease alcohol and mortality from all causes effects of alcohol on platelet functions structure-antioxidant activity relationships of flavonoids and phenolic acids. free radic the effects of carbon dioxide in champagne on psychometric performance and blood-alcohol concentration review of moderate alcohol consumption and reduced risk of coronary heart disease: is the effect due to beer, wine, or spirits? melatonin: a new bioactive compound in wine inhibitory effects of plant phenols on the activity of selected enzymes potential food allergens in wine: doubleblind, placebocontrolled trial and basophil activation analysis immune response to acetaldehyde-human serum albumin adduct among healthy subjects related to alcohol intake low prevalence of the metabolic syndrome in wine drinkers e is it the alcohol beverage or the lifestyle? the structure of cuisine the use of characteristic flavourings in human culinary practice simultaneous and temporal contextual influences on food acceptance oleogustus: the unique taste of fat intra-individual and inter-individual variation in breath alcohol pharmacokinetics: the effect of food on absorption acetaldehyde as a common denominator and cumulative carcinogen in digestive tract cancers anthocyanin tissue bioavailability in animals: possible implication for human health. a systematic review flavonoids and the aging brain alcoholism: the health and social consequences of alcohol use moderate drinking in pregnancy proceedings of wine, health and society. a symposium. grt books sulfated and glucuronated trans-resveratrol metabolites regulate chemokines and sirtuin- expression in u- macrophages untersuchungen zur bedeutung toxischer stoffwechselprodukte des pilzes trichothecium roseum link ex fr. fü r den weinbau resveratrol and some glucosyl, glucosylacyl, and glucuronide derivitives reduce eschericia coli o :h , salmonella typhimurium, and listeria monocytogenes scott a adhesion to colonic epithelial cell lines sulfite oxidase deficiency plasma levels of caffeic acid and antioxidant status after red wine intake hangover headache: various manifestations and proposal for criteria. vågå study of headache epidemiology potential health impacts of excessive flavonoid intake. free radic wine as a biological fluid: history, production, and role in disease prevention ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection alcohol consumption, mild cognitive impairment, and progression to dementia diversity of black aspergilli and mycotoxin risks in grape, wine and dried fruits doubleblind placebo-controlled trial of lithium in episodic cluster headache sleep and low doses of alcohol resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin induced diabetic rats effect of some phenolic compounds and beverages on pepsin activity during simulated gastric digestion intake of wine, beer, and spirits and the risk of clinical common cold anthocyanins are efficiently absorbed from the small intestine in rats iron is an essential cause of fishy aftertaste formation in wine and seafood pairing contribution to wine in vanadium dietary intake: geographical origin has a significant impact on wine vanadium levels adh genotype, alcohol intake, and breast cancer risk adh gene polymorphism are determinants of alcohol pharmacokinetics alcohol consumption and mortality among middleaged and elderly us adults red and white wines inhibit cholesterol oxidation induced by free radicals ellagic acid metabolism by human gut microbiota: consistent observation of three urolithin phenotypes in internention trials, independent of food source, age, and health status immunomodulatory and antitumor activities of grape seed proanthocyanidins is dopamine behind the health benefits of red wine? inhibition of quorum sensing (qs) in yersinia enterocolitica by an orange extract rich in glycosylated flavonones intake of beer, wine, and spirits and risk of stroke: the copenhagen city heart study amount and type of alcohol and risk of dementia: the copenhagen city heart study aspirin attenuation of alcohol-induced flushing and intoxication in oriental and occidental subjects effects of beer, wine, and liquor intakes on bone mineral density in older men and women alcohol consumption stimulates early steps in reverse cholesterol transport alcohol in the western world role of sulfite additives in wine induced asthma: single dose and cumulative dose studies changes in bronchial hyperresponsiveness following high-and low-sulphite wine challenges in wine-sensitive asthmatic patients antibacterial effect of phenolic compounds from different wines ochratoxin a in grapes and grapederived products the antimicrobial effect of wine on bacillus ceresus in simulated gastro-intestinal conditions nitric oxide radical scavenging by wines the alcohol hangover research group consensus statement on best practice in alcohol hangover research inhibitory effect of antioxidant-rich marinades on the formation of heterocyclic aromatic amines in pan-fried beef alcoholic calories, red wine consumption and breast cancer among premenopausal women effect of ethanol, tannin and fructose on the headspace concentration and potential sensory significance of odorants in a model wine flavorematrix interactions in wine low doses of alcohol substantially decrease glucose metabolism in the human brain a blend of polyphenolic compounds explains the stimulatory effect of red wine on human endothelial no synthase atherosclerotic cardiovascular disease and antioxidants older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease histamine in wine e bronchoconstriction after a double-blind placebocontrolled red wine provocation test supplementation with grape seed polyphenols results in increased urinary excretion of -hydroxyphenylpropionic acid, an important metabolite of proanthocyanidins in humans fetal alcohol spectrum disorders alcohol and medication interactions investigation of the allergenic potential of wines fined with various proteinogenic fining agents by elisa nutritional approach to cancer prevention with emphasis on vitamins, antioxidants, and carotenoids wine as a digestive aid: comparative antimicrobial effects of bismuth salicylate and red and white wine wine in context: nutrition, physiology, policy prostaglandin e (pge ) induces headache in healthy subjects effect of opuntia fiscus indica on symptoms of the alcohol hangover the alcohol hangover prolonged in vitro exposure to white wines enhances the erosive damage on human permanent teeth compared with red wines flavonoids: antioxidants or signaling molecules? free radic bioavailability and bioefficacy of polyphenols in humans. ii. review of intervention studies alcohol and preoperative management deleterious effect of p-cresol on human colonic epithelial cells prevented by proanthocyanincontaining polyphenol extracts from fruits and proanthocyanin bacterial metabolites pharmacokinetics and blood-brain barrier penetration of (þ)-catechin and (e)-epicatechin in rats by microdialysis sampling coupled to high-performance liquid chromatography with chemiluminescence detection stability of dietary polyphenols under the cell culture conditions: avoiding erroneous conclusions resveratrol alleviates rheumatoid arthritis via reducing ros and inflammation, inhibiting mapk signaling pathways, and suppressing angiogenesis effects of phenolic acids on human phenolsulfotransferase in relation to their antioxidant activity flavonoid permeability across an in situ model of the bloodbrain barrier. free radic gods, men, and wine. the wine and food society ltd lead nephropathy and gout alcohol consumption and risk of breast cancer: the framingham study revisited association of alcohol intake with risk of diabetic retinopathy: a meta-analysis of observational studies measuring sensory perception in relation to consumer behavior on food and cooking e the science and lore of the kitchen molecular gastronomy: exploring the science of flavor flavor in food potential mechanisms by which polyphenol-rich grapes prevent obesity-mediated inflammation and metabolic diseases antibacterial, antiviral, and antifungal properties of wines and winery products in relation to their flavonoid content anthocyanins: from sources and bioavailability to cardiovascular-health benefits and molecular mechanisms of action wine and headache the effects of grape and red wine polyphenols on gut microbiotada systematic review wine consumption and renal diseases: new perspectives resveratrol as an anti-cancer agent: a review moderate alcohol consumption and the immune system. a review resveratrol and health e a comprehensive review of human clinical trials bioavailability of wine-derived phenolic compounds in humans: a review allergic and asthmatic reactions to alcoholic drinks effect of flavonoids on learning, memory and neurocognitive performance: relevance and potential implications for alzheimer's disease pathophysiology the alcohol hangover intracellular polyphenols: how little we know key: cord- -uceukiie authors: jones, arwel wyn; davison, glen title: chapter exercise, immunity, and illness date: - - journal: muscle and exercise physiology doi: . /b - - - - . - sha: doc_id: cord_uid: uceukiie abstract it is generally accepted that moderate amounts of exercise improve immune system functions and hence reduce the risk of infection whereas athletes engaged in regular prolonged and/or intensive training have a higher than “normal” incidence of minor infections, especially of the upper respiratory tract (urt, e.g., common cold and influenza). this is likely related to regular acute (and possibly chronic) periods of exercise-induced changes in immune function. urt infections can compromise performance directly if suffered shortly before or during competition or indirectly if suffered at other times via effects on training and/or physiological adaptations. this chapter covers the effects of exercise (acute and chronic), both positive and negative, on immune function and consequent infection risk, and considers the current state-of-the-art for monitoring and assessing this in athletes. it is generally accepted that moderate amounts of exercise improve immune system functions and hence reduce the risk of infection. however, there is strong evidence that athletes engaged in regular prolonged and/or intensive training have a higher than "normal" incidence of minor infections, especially of the upper respiratory tract (urt, e.g., common cold and influenza) (gleeson and walsh, ) . this is particularly apparent in endurance athletes such as cyclists, runners, swimmers, and triathletes, but any athletes with a high training load and/or suboptimal recovery may be at increased risk. such infections can compromise training and/or competition performance . upper respiratory tract infections (urti) are among the most frequent presentations to general practitioners (hasham and hall, ) . these do not usually require hospital admission, but such illnesses have a significant economic and social impact-e.g., absence from work, healthcare costs, increased morbidity, reduced feelings of well-being, health and quality of life, and reduced social interaction. broadly, moderate amounts of exercise are associated with enhanced immunity and resistance to such infections whereas high amounts or prolonged and/or vigorous training may increase the risk. indeed, urti are also suggested to be the most common type of infection in the athletic population (roberts, ; cannon, ; peters, ; gleeson and walsh, ) . in fact they have shown to be a highly prevalent medical condition in athletes at clinics in both the summer and winter olympic games (e.g., robinson and milne; engebretsen et al., engebretsen et al., , . observational and experimental studies have investigated the proposed greater resistance to pathogens with moderately active lifestyles. animal investigations have demonstrated that brief bouts of moderate physical activity ( À min treadmill running) compared to inactivity prior to or immediately following inoculation with pathogens leads to decreased mortality and morbidity from infection (davis et al., ; lowder et al., ) . early exercise training studies of older and obese humans also demonstrated that À weeks of moderate exercise [ min walking at %À % of maximal oxygen uptake ( _ vo max )] resulted in lower incidence or duration of selfreported urti compared to sedentary individuals (nieman et al., b (nieman et al., , . these effects have been supported by several longitudinal studies of the wider general population (ages À years) where maintenance of a moderately active lifestyle leads to lower self-reported or laboratory confirmed uri/urti episodes (kostka et al., ; matthews et al., ; kohut and senchina, ; ciloglu, ; kostka and praczko ; kostka et al., ; nieman, ) ; (spence et al., ; barrett et al., ) . although patterns vary between sports, a review by walsh et al. ( b) suggested that athletes tend to report urti either during the high-intensity and tapering period prior to competition (e.g., swimming, team sports) or in the period following competition (e.g., long distance running). it has long been hypothesized that a j-shaped relationship exists between exercise workload and susceptibility to urti (nieman, ) . this model suggests that an individual involved in regular moderate exercise is less likely to contract urti compared to a sedentary individual but prolonged high-intensity exercise or periods of strenuous exercise training are associated with an above-average risk of urti (see fig. . ). indeed, the j-shaped model was initially based on findings of increased self-reporting of urti in the À week period following participation in competitive endurance races (e.g., nieman et al., a) . further support for an adverse effect of prolonged/ strenuous exercise on susceptibility to urti has come from animal studies (davis et al., ; gross et al., ; folsom et al., ; lowder et al., ; murphy et al., ) . prolonged exercise (treadmill running for . h) has been shown to increase morbidity and mortality of mice inoculated with respiratory viruses (e.g., herpes simplex type virus, influenza) prior to (davis et al., ; murphy et al., ) or following (lowder et al., ) this type of exertion compared to resting and/or moderately exercised mice. in addition to acute exertion, equine studies have also demonstrated that intensified periods ( À days) of exercise training prior to or following inoculation with influenza leads to greater severity of infection in vaccinated (folsom et al., ) and nonvaccinated horses (gross et al., ) . generalizing these results to the human in vivo environment is questionable (albers et al., (albers et al., , bermon et al., ) and such approaches (i.e., pathogen challenge) with human volunteers do have considerable ethical constraints (hope and mcmillan, ) . although not yet investigated in an exercise context, pathogen challenge studies in humans have demonstrated lower resistance to urti due to other life stressors (e.g., psychological stress, sleep disturbance) (cohen et al., (cohen et al., , prather et al., ) . in addition to the potential exposure to these stressors in a training and competition environment (coutts et al., ; hausswirth et al., ) , the relevance of these findings to athletes is emphasized by the impact of transient modulation in host resistance to urti by a stressor. exercise immunology research (epidemiological and experimental) has grown substantially over the last few decades to investigate the relationship between exercise and urti in humans (shepard, ) . in contrast to animal research, human studies (attempting to discern the effects of prolonged exercise/intense training on urti) have mainly involved monitoring athletes following heavy exertion (i.e., relied on natural exposure to pathogens) but only a limited number of these have verified that symptoms are due to infectious agents (pathogens) (spence et al., ; schwellnus et al., ; hanstock et al., ) . this has raised concerns regarding the validity of urti episodes (i.e., self-reported) in athletes that occur in and around competition or heavy periods of training (bermon, ; walsh et al., b) . discrepancies between physician and laboratory diagnosed urti has also highlighted the limitations with evaluation of urti episodes (cox et al., ) . upon presentation of symptoms at a sports medicine clinic, the study of cox et al. ( ) observed that only % of cases were found to be indicative of infection with laboratory methods (e.g., identified pathogen) while % were diagnosed as urti by physicians. in a surveillance study of a range of athletes (recreational and elite) and sedentary controls, spence et al. ( ) demonstrated that the first two days of symptoms with infectious or noninfectious (see later) cases were similar but duration and severity of symptoms on subsequent days were greater with infectious cases. on the other hand, the common symptom scoring methods applied in self-report questionnaire studies usually require symptoms to be present for two or more days in order to be counted as an "episode" (e.g., fricker et al., ; gleeson et al., ) , which may offer some protection against such limitations. it is also worthy of note that a subsequent uk-based study observed an % agreement rate between self-report and laboratory-confirmed pathogen detection (hanstock et al., ) . the difference to earlier studies could be due to time of year, location, or perhaps the sensitivity of detection methods has improved since the earlier studies (e.g., spence et al., ) . nevertheless, for clarity the term upper respiratory tract symptoms or upper respiratory illness (uri) are generally accepted in this area unless infection has been clinically confirmed (when urti can be used). although there were numerous early anecdotal reports and retrospective survey data to support the proposed jshaped relationship (simon, ; shepard et al., ; nieman, ) , such observations alone cannot validate the influence of exercise on uri. however, further support to the j-shaped model (i.e., heavy exercise workload) was provided by a number of prospective and retrospective studies which suggested that marathon or ultramarathon runners suffer from an increased risk of uri (e.g., À weeks following competitions) (nieman et al., a; peters et al., ) . the study of peters and bateman ( ) was a seminal study in highlighting this by randomly recruiting a sample of runners who competed in the two oceans marathon in cape town. in the days following the km event, % of runners reported uri compared to % of age-matched controls that did not participate in the marathon but shared living space with runners (i.e., to control for exposure to pathogens and other environmental factors). nieman et al ( a) went on to show that, compared to equally experienced runners who did not compete, there was a sixfold increase of uri in runners during the seven days following the los angeles marathon. taking into account other factors influencing risk of uri (age, stress levels, and illness at home), the likelihood of uri was doubled in those who ran . km compared to those who ran , km as part of their weekly training programmes leading up to the event. heath et al. ( ) also highlighted running mileage as a significant risk factor for incidence of uri in a cohort of runners followed for a period of months. more recently, matthews et al. ( ) have also suggested that runners with higher training loads tend to be more prone to uri and that endurance athletes in particular suffer from longer episodes of uri than their recreational counterparts. however, such findings have not been demonstrated consistently as shorter observational studies have failed to observe any associations of uri with differences in training mileage, intensity, and load . despite much interest, there remains more uncertainties than evidence based facts regarding the notion that high volumes of training are associated with an increase in the incidence of uri (walsh et al., b) . one suggestion is that such inconsistent findings may be related to whether participants within studies are considered "elite" or "highly trained." malm ( ) suggests that a prerequisite to achieving elite athlete status is an immune system which can withstand the strenuous nature of training and competition as susceptibility to infections is incompatible with elite performance. for this reason, malm ( ) proposed an s-shaped rather than a j-shaped curve to include elite training which is associated with a lower risk of infection compared to high exercise workload (see fig. . ). however, it is highly likely that elite/ professions athletes will have considerable support (financial, medical, sports science, and nutrition, etc.) and their support team may implement preventive and treatment strategies to reduce the risk and limit the effects of uri and this may also contribute to better management of other stressors. it could be, therefore, that rather than being naturally able to withstand strenuous training and infections, it is simply that they are better supported (compared to counterparts who lack such support mechanisms) to reduce controllable risk factors. nieman ( ) suggested most athletes may not report uri or suffer from an increased risk if they avoid periods of overreaching or overtraining (nieman et al., ) . it seems that an increase in uri may only be attributed to participation in acute prolonged exertion (e.g., marathon, ultra-marathon) or more importantly when athletes are exposed to a greater strain of training through exceeding individual training thresholds coupled with inadequate recovery or other life stressors (e.g., sleep disturbance) (foster, ; pyne and gleeson, ; hausswirth et al., ) . in other words, training load or volume alone does not give full information on the level of stress that an athlete (and their immune system) is under. indeed, the way training in distributed or periodized is of key important also. in support of this, svendsen et al. ( ) have shown that rapid changes in training load (i.e., increasing too quickly) are better predictors of uri risk than total load alone, which goes some way to explaining the above mentioned discrepancies where only total load or volumes were considered. in line with the general population, when pathogen identification has been attempted, bacteria are rare causes of urti in athletes, with viruses being responsible in most cases (in particular rhinovirus, adenovirus, and parainfluenza) (roberts, ; mäkelä et al., ) . urti of viral origin last approximately À days but clearance of the particular virus from the system may take longer (winther et al., ; heikkinen and järvinen, ) . the route of entry into the body by most viruses is the respiratory tract where symptoms reflect the perturbations in function of infected cells and the attempts of the immune system to contain the infection (roberts, ) . as the nature of symptoms of some (i.e., infectious) uri may be similar to noninfectious inflammatory factors and/or presentation of allergic conditions, identification of antibody titers, and isolation of specific pathogens from body fluids of athletes have been recommended to clarify causes of symptoms and provide the most appropriate treatment or management strategies (gleeson, ; cox et al., ) . it was generally believed that upper respiratory symptoms in athletes were due to an infective cause, however, it is only more recently that other causes ("noninfectious hypothesis") have been proposed during training and competition (bermon, ; schwellnus et al., ) . robson-ansley et al. ( ) reported that a higher incidence of uri in runners following a marathon ( %) compared to nonrunners ( %) was significantly associated with positive responses in the allergy questionnaire for athletes. as the prevalence of inhalant allergy may be as common as %À % of highly trained athletes, it may partly provide an explanation to incidence of uri (langdeau et al., ; lumme et al., ; schwellnus et al., ) . environmental influences have been considered relevant to certain groups of competitive athletes, in particular swimmers who are exposed to chlorine derivatives from swimming pool disinfectants while inhaling large amounts of air above the water surface (piacentini et al., ; bougault et al., ). in addition to inhaled irritants, the combination of high ventilation rate in heavy training and surrounding cold, dry air is another possible source of noninfectious, nonallergenic inflammatory stimuli to uri in some athletes (e.g., runners, cyclists) (bermon, ; cox et al., ) . there is also potential for direct damage in epithelial tissue of the urt and/or fibers of the contracting skeletal muscle following strenuous exercise to contribute to the noninfectious, local, and systemic inflammatory origin of uri (peters, ) . schwellnus et al. ( ) reported that the use of an antiinflammatory agent reduced uri in participants following an ultra-marathon event, but as this nasal, buccopharyngeal spray also contained antimicrobial properties it does not provide any conclusive evidence to support a purely noninfectious inflammatory cause for uri. in contrast, administration of an antiinflammatory throat spray in the period leading up to and following a half-marathon event had no influence on the incidence of uri in runners but did reduce the severity of recorded symptoms (cox et al., ) . although conflicting evidence here may purely reflect site-specific differences between agents, together these studies do provide evidence that noninfectious inflammation may not be the underlying cause of all uri, but may interact with infectious causes to potentiate the symptoms that occur as a result of responses to pathogen challenge. in their surveillance study, spence et al. ( ) demonstrated that the distribution of uri closely followed the j-shaped curve in terms of training status (i.e., both the control and elite athlete group suffered from greater days of illness than their recreational counterparts), but only % of reported illnesses were confirmed by identification of common respiratory pathogens. it is worthy to note that only specific pathogens were tested in spence et al. ( ) , thus uri were possibly caused by known pathogens not tested for, unknown pathogens and/or new strains of viruses which were yet to be identified (bermon, ) . despite the low number of identified pathogens highlighted with laboratory evaluation of uri in some studies (i.e., spence et al., ; cox et al., ) , it must be emphasized that this does not rule out infectious causes for these cases as such diagnostics procedures do have inherent limitations in identifying causative agents from an evolving diverse pool of pathogens (e.g., b common cold viruses) (heikkinen and järvinen, ; eccles, ) . furthermore, a more recent (albeit smaller) study based in the united kingdom during the winter months (i.e., typical uri season) observed that a much higher proportion ( %) of reported illnesses were confirmed by identification of urti-causing pathogens (hanstock et al., ) . in this study, recreational-level athletes completed the study, which included a -week monitoring period during which subjects reported uri using the daily jackson common cold questionnaire. of these subjects, urti-causing pathogens were detected in (all were positive for rhinovirus and one was concurrently positive for coronavirus). not all cases of uri can present as a typical response to a primary viral infection of initial upper respiratory symptoms followed by local and systemic inflammation (e.g., fever, aches) (gleeson et al., ) . occasionally symptoms may be minor and/or short-lasting ( À days) resulting in training being unaffected or be a reflection of persistent fatigue and recurrent infections as a result of chronic heavy exertion (reid et al., ) . in these cases it has been suggested that, in addition to exercise-induced inflammation, uri following exercise may be related to reactivation of latent viruses within the upper airways rather than the incidence of primary infections in the recovery period (gleeson et al., ) or prior urt infections that have not been fully eliminated even after symptoms have subsided. ekblom et al. ( ) found in a group of recreational runners that prerace uri was significantly associated with uri incidence following the marathon. this was supported by a longitudinal observational field study, where it was suggested that reactivation of prerace viruses and exercise-induced inflammatory responses were the primary causes of elevated uri incidence prior to and following an . km marathon . the % of runners, who recorded incidence of uri during the À days following the race, also recorded symptoms in the time leading up to the race. these findings may support the aforementioned animal studies whereby participation in prolonged exercise may worsen symptom severity induced by existing infection (malm, ) . it has also been suggested that the reactivation of latent viruses (e.g., epsteinÀbarr virus, ebv) could be implicated in the etiology of urt infections (gleeson et al., ) . ebv is a herpes virus that typically infects b %À % of the world's adult population (gleeson et al., ; staras et al., ; bate et al., ) . after initial primary infection, these viruses lay dormant within the cells of the immune system and immunocompetent individuals are generally asymptomatic (kano and shiohara, ; crawford, ) . however, under significant physical and/or psychological stress the immune system's ability to control these infections (keeping latent) may be lost and such latent viruses may become reactivated (tingate et al., ; glaser et al., ; mehta et al., b; stowe et al., ) . in an exercising population, ebv has received attention due to its ability to replicate continuously or intermittently from the oropharynx (faulkner et al., ; nadal et al., ) . gleeson et al. ( ) found a significant relationship between previous ebv infection and uri in elite swimmers: while all seronegative swimmers remained unaffected (no reported uri) during a day period of intensive training out of the seropositive swimmers had ebv dna detected in saliva during the study with of these going on to develop uri which appeared À days following first detection of ebv dna. it was postulated that ebv was implicated in the symptoms. however, cox et al. ( ) treated a group of endurance runners with a herpes-virus-specific antiviral treatment and although this was able to significantly reduce ebv expression, this had no effect on uri. it is possible, therefore, that ebv is not directly involved/the cause of uri per se, but rather an in vivo indication of immunodepression/compromised immunity and, therefore, increased susceptibility to other uri-causing pathogens. indeed, for the reactivation of ebv to occur, there must be a disturbance within certain parameters of the immune system which usually keep the virus tightly-regulated and latent. this reflection of immune perturbations within the host (mehta et al., a) occur in line with the most researched hypothesis ("open window") behind an increased risk of primary infections in athletes who participate in prolonged exercise. this theory is still it its infancy however and requires further study, although it does seem to be supported by studies showing athletes to have greater levels of detectable ebv dna compared to controls (hoffmann et al., ) . in a clinical investigation of elite athletes suffering recurrent episodes of uri, ebv viral shedding was detected in % of the cohort (reid et al., ) . yamauchi et al. ( ) also found in an intensive training period with rugby players that salivary expression of ebv dna was . times greater in participants with uri compared to those without uri. the "open window" hypothesis suggests that prolonged/heavy exercise causes depression of the immune system which leaves the body less resistant to viruses and bacteria and thus increases the risk of subclinical and clinical infection for between and h (pedersen and ullum, ; nieman, ) , which reflects that the increased risk of uri with heavy exertion was due to a decrease in immunosurveillance (and vice versa, the decreased risk with moderate exercise was due to an increase in immunosurveillance). the immunological response to acute exercise is deemed a subset of stress immunology (hoffman-goetz and , where responses have been likened to those caused by infection, sepsis, burns, or trauma (pedersen and hoffman-goetz, ) . although exercise does share some similarities to the hormonal and immunological responses of these clinical physical stressors, there are also important distinct differences in the magnitude and temporal responses (shepard, (shepard, , . strenuous exercise induces an ordered sequence of modest changes in pro-inflammatory signaling followed predominantly by antiinflammatory responses which down-regulates immune function whereas some of the clinical stressors listed above (e.g., sepsis) trigger an excessive and overwhelming elevation in systemic pro-inflammatory responses (shepard, ) . exercise immunologists have controlled and adapted the duration and intensity of the stress model of exercise to gain a deeper understanding into how alterations in immune function following acute exercise and training may lead to changes in susceptibility to pathogens. the immune system has evolved to protect the human body from pathogens (viruses, bacteria, and parasites). it encompasses the ability to maintain homeostasis even when exposed to a wide range of foreign and self molecules (i.e., antigens). the components of the nonspecific innate system and the specific acquired system overlap to ensure that a state of immunity against infection is established. one of the main mechanisms responsible for changes in host defense with moderate activity seems to be a greater immunosurveillance associated with moderate activity. regular bouts of moderate intensity activity generally induce transient improvements in the immune system . there are various factors which mediate these relationships, one of which is the fitness status of participants, although many studies assessing the immune response to moderate exercise have focused on interventions for previously sedentary individuals. many components of the immune system are temporarily reduced (exercise-induced immunodepression) after strenuous and/or prolonged bouts of exercise (gleeson and walsh, ) . this may persist for as little as a few hours or a long as a few days (depending on the nature of the exercise). in particular, if subsequent bouts are commenced too soon, before the immune system has fully recovered, then a progressive accumulation of immunodepression may ensue. periods of depressed immunity, whether small acute periods or more chronic periods, are termed "open windows" (as discussed earlier) and this is a likely mechanism explaining increased susceptible. however, it is important to point out that this does not necessarily mean that changes in isolated immune markers alone can predict illness risk. there is considerable redundancy in the immune system, so it is important to note that changes in isolated in vitro and ex vivo markers may not give a good representation of the ability of the whole immune system to mount an effective response. even if it was possible to measure every component of the immune system (in vitro) concurrently, it would still be virtually impossible to calculate how such results could be combined to predict the whole integrated (in vivo) immune response. for this reason in vivo measures or markers that represent the ability of the whole immune system to mount a coordinated, integrated response are the most useful and clinically relevant markers (see albers et al., , and albers et al., for detailed reviews). it remains important to study such in vitro and ex vivo markers nonetheless as they provide mechanistic information and insight on the effects of exercise on immunity, but for most of these measures researchers (and practitioners) must exercise caution in their interpretation of such data, which should be used to supplement more clinically relevant markers and provide additional mechanistic insight. the number of circulating immune cells (leukocytes) is profoundly influenced by acute exercise with reports from over a century ago highlighting the exercise-induced mobilization following the boston marathon (larrabbe, ) . circulating leukocytes consist of the granulocytes (neutrophils, eosinophils, and basophils; %À % of total), monocytes ( %À %), dendritic cells (less than %), and the lymphocytes ( %À %) which can be divided into innate [natural killer (nk) cells] and acquired (t helper, t cytotoxic, and b cells) cells. it is now clear that leukocytosis, which is an increase in the total number of circulating leukocytes (mainly neutrophils and lymphocytes) occurs (up to %) during and immediately post exercise (simpson, ) . the observed changes are dependent on the exercise intensity and duration (gleeson, ) with prolonged endurance exercise (. . h) causing a greater leukocytosis (three-to fourfold increase) than brief ( À min) high-intensity exercise (robson et al., b) . at rest, it is estimated that an equal amount of leukocytes are circulating within the blood and located within marginated pools, adhered to blood vessel walls of the circulatory system (athens et al., ; berkow and dodson, ) . foster et al. ( ) suggested the increased cardiac output during exercise and the subsequent shear stress (increased blood flow) within blood vessels induces leukocytes to enter circulation in a process known as demargination. however, it is anticipated that marginal pools within the liver, lung, spleen and other vital organs (e.g., bone marrow, intestines) also contribute to the large leukocytosis following exercise as pools in the lung alone possess lymphocytes which are present in times larger amounts than the circulatory pool (hogg and doerschuck, ; simpson, ) . activation of the sympathetic nervous system (elevated concentrations of plasma catecholamines) and the hypotha-lamicÀpituitaryÀadrenal (hpa) axis (cortisol release) during prolonged exercise also play an integral role in exercise-induced leukocytosis (nieman, ; atanackovic et al., ; anane et al., ). the immediate leukocytosis, particularly neutrophilia (increased neutrophil count) upon onset of exercise is suggested to be due to the haemodynamic and catecholamine induced demargination of vascular and pulmonary pools but this is later followed by a cortisol-induced release of neutrophils from the bone marrow otherwise known as delayed leukocytosis (allsop et al., ) . this effect is seen simultaneously with the immediate leukocytosis during prolonged exercise of . h as cortisol level is sufficiently elevated and its action is prolonged, explaining the previously mentioned greater leukocytosis than following shorter, higher intensity exercise (robson et al., b) . neutrophils released from the bone marrow following stimulation by cortisol are suggested to include a greater proportion of immature cells (e.g., band cells) compared to neutrophils that demarginate from the endothelial walls upon onset of exercise which have similar maturity levels to those already in circulation (hetherington and quie, ; mccarthy et al., ) . although effects on dendritic cells remain unclear, the bone marrow is also a source of maturation for both monocytes and b cells where monocytes also increase proportionately with exercise duration and b cells are associated with limited redistribution (pedersen et al., ; shek et al., ; nieman et al., ; lancaster et al., a; okutsu et al., ) . nk and t cells (mostly cytotoxic) also increase proportionately with exercise intensity and duration unlike neutrophils where increases are predominantly influenced by duration (mccarthy and dale, ; gabriel et al., ; shek et al., ; campbell et al., ) . even though lymphocytes are present at numerous sites within the body, evidence suggests that mobilization of these cells during exercise mainly occurs from secondary lymphoid organs, for example, spleen, intestinal peyer's patches (pp) rather than primary lymphoid organs such as the thymus or bone marrow (simpson et al., (simpson et al., , campbell et al., ). the spleen is considered to be an abundant source of the lymphocytes deployed during exercise (baum et al., ; nielsen et al., ) , with shear stress and catecholamines being important release mechanisms (kappel et al., ; benschop et al., ; shepard, ; timmons and cieslak, ; dimitrov et al., ) . there is a clear consensus developing that exercise triggers a redistribution of t cells with a longer history of antigen exposure rather than naive t cells (campbell et al., ; simpson, ) . following the lymphocytosis during and upon completion of prolonged exercise, the high concentration of adrenaline and cortisol often cause lymphocytopenia (lymphocyte count below resting levels) (nieman et al., ; nieman, ) during the recovery. this biphasic response (lymphocytosis during exercise and lymphocytopenia during recovery) has been found with various protocols requiring heavy/prolonged exertion or exhaustive exercise (e.g., fry et al., ; shek et al., ) , with lymphocyte count up to % below resting levels reported post exercise (simpson, ) . lymphocytopenia is due to the selective extravasation of lymphocyte subsets, nk and t cytotoxic cells, from blood to the surrounding tissues (gabriel et al., ; simpson et al., ; kruger et al., ) . the return of total leukocyte count to resting levels generally begins immediately post exercise with diminishing activation of the sympathetic nervous system and hpa axis, but in the case of very intense exercise, leukocyte (primarily neutrophils) count may continue to increase as described above (mccarthy and dale, ; allsop et al., ) . given these leukocyte perturbations, an increase in neutrophil:lymphocyte ratio is suggested to be an indicator of the overall magnitude of the stress response induced by exercise . the immune system consists of many physical barriers (e.g., skin, mucus, cilia) which act to prevent entry of pathogens into the human body. if such attempts fail, infectious agents will be detected by receptors present on the cellular components of the innate immune system such as the granulocytes (mostly neutrophils, but include basophils and eosinophils), nk lymphocytes, monocytes (which mature into macrophages within tissue), and dendritic cells. the recognition of foreign material is one way in which the innate and acquired immune systems differ from one another. unlike acquired immunity, the innate system does not exhibit memory of previous encounters with antigens (i.e., foreign molecule), therefore a similar response takes place during any future exposure. present on the cell surface of innate cells are pattern recognition receptors (prr) that distinguish self from nonself material by recognizing molecules on microbes which have been conserved through evolution due to their essential function i.e., bacterial dna, lipopolysaccharides (lps), and other bacterial cell wall components (kimbrell and beutler, ; beutler and rietschel, ) . the majority of these structures comprise of the toll-like receptors (tlr) which are crucial components of the antigen presentation cells (apc) (discussed later in this section) of innate immunity (monocytes/macrophages). neutrophils also known as polymorphonuclear (pmn) cells (due to their multilobed nucleus) are the most abundant circulating leukocyte population of the immune system. these leukocytes are considered specialised short-lived cells with deficits in numbers and/or function of this subset being associated with increased risk of potentially fatal bacterial infections (smith et al., ; boxer, ; viscoli et al., ) . once released from the bone marrow, neutrophils migrate out of the circulation within À h to marginated and tissue pools where they reside for a further À days (smith, ; summers et al., ) . neutrophils respond to chemotactic stimuli (e.g., formylated peptides such as formyl methionyl leucyl phenylalanine, fmlp) from fragments of invading microorganisms and tissue damage at sites of infection and inflammation respectively where they engage in numerous intrinsic conformation adaptations during and following their migration (chemotaxis) (smith and pyne, ) . similar to other phagocytes (e.g., apc, discussed later in this section), the detection of pathogens or tissue damage through prr (e.g., fpr , a fmlp receptor) on the neutrophil surface leads to internalization (phagocytosis) and holding of these fragments within the cytoplasm (via a membrane bound vesicle known as phagosome) thereby activating a series of effector functions in the cell (nathan, ; borregaard, ) . the mechanistic detail of engulfment by neutrophils may depend on the involvement of the specific ligand (i.e., antigen) attached to the receptors or whether fragments have been opsonised by soluble immune factors, but the process will ultimately involve internalization into a phagosome (amulic et al., ) . soluble components which act as opsonins (promote attachment of antigen to phagocyte) include acute phase proteins, antibodies (see section . . ) and complement proteins (e.g., c ) (thiel et al., ; gordon, ; brekke et al., ) . it is worthy to note that elevations in circulating concentrations of acute phase proteins (c-reactive protein, crp) in response (i.e., acute phase response) to perturbations in homeostasis (e.g., trauma, tissue damage) are considered one example of why the strenuous nature of prolonged exercise is likened to medical conditions (e.g., sepsis, burns) (kushner and rzewnicki, ; pedersen and hoffman-goetz, ; fallon, ) . neutrophils are also known as granulocytes due to the characteristic release (degranulation) of primary azurophil [defensins, elastase, myeloperoxidase (mpo)] and secondary specific (lactoferrin, lysozyme) granule contents into the cytoplasm to fuse with the phagosome or the plasma membrane to create an antimicrobial milieu inside and outside of the cell. these granules do show varying readiness to mobilize in response to inflammatory signaling, with the azurophilic subset being the most difficult to mobilize (amulic et al., ) . the degradation of particles within the phagosome or extracellular space of infected or damaged tissue is aided further by the process of neutrophil oxidative burst involving the formation of reactive oxygen species (ros) through the nadph oxidase system (weiss, ; babior, ) . depending on the stimulus (see section . . . for relevant stimuli of this thesis), the nadph oxidase system may assemble on the membrane of the phagosome and/or the cell where the main ros produced are the superoxide anions which react to form other intermediates including hydrogen peroxide, hypochlorous acid, hydroxyl radical, and singlet oxygen (peake, ) . it has been suggested that components of azurophilic (e.g., mpo) and specific (e.g., flavocytochrome b ) granules may regulate the activity of the nadph oxidase (tal et al., ; amulic et al., ) , highlighting that intracellular signaling cascades triggered by attachments of ligands to surface receptors, may lead to coordinated degranulation and oxidative burst responses to invading pathogens (daniels et al., ; pyne, ) . it has also been demonstrated that neutrophils also possess antimicrobial capacity independent of phagocytosis known as neutrophil extracellular traps (nets) (mantovani et al., ) . although mechanisms of nets are not completely understood, it has been established that classical effector functions of neutrophils (degranulation and oxidative burst) play important roles in mediating the response (fuchs et al., ; patel et al., ; metzler et al., ; amulic et al., ) . nets are suggested to form upon an active form of cell death which results in the release of a network of nuclear filaments (dna and histone) into the extracellular space from the degrading neutrophil (brinkmann et al., ; fuchs et al., ) . on the basis of the exercise-induced changes in neutrophil counts it is not surprising that exercise also affects functional responses, but due to the variation in study design the early evidence was conflicting (peake, ) . nevertheless, it can be argued that when responses are controlled for on a per cell basis the effects of exercise duration (i.e., prolonged exercise) are clearer. neutrophils may present on a continuum of state of activation from dormant to primed through to being fully activated (smith, ) . during exercise, there is a release of agents into the circulation which may prime (e.g., induce assembly of nadph on membrane) or desensitize (internalization of receptors) the capacity of neutrophils for enhanced responsiveness to later stimulation or inhibit such functional responses respectively (pyne, ; peake, ; amulic et al., ) . the number of neutrophils engaging in phagocytic activity is increased following prolonged exercise but the phagocytic capacity of each neutrophil is decreased within the circulation and the nasal cavity (gabriel et al., ; müns, ; blannin et al. a; nieman et al., ; chinda et al., ) . albers et al. ( ) suggested that neutrophil phagocytosis may have low suitability as a marker of the immunomodulatory effects of exercise and assessing the killing capacity (degranulation and oxidative burst) may be more sensitive to reflect a susceptibility to infection. simultaneous with neutrophilia following prolonged exercise there is an increase in unstimulated degranulation and oxidative burst responses (e.g., measured by total plasma elastase, spontaneous ros production) (blannin et al., b; suzuki et al., ; bishop et al., ) . these findings provide support that prolonged exercise induces neutrophil activation (possibly due to muscle damage) that may result in the cells entering a "refractory period" in the recovery from prolonged exercise whereby there is a transient inability to respond to subsequent stimulation (e.g., in vitro) (peake, ) . numerous studies have found significant decreases in neutrophil degranulation and/or oxidative burst responses to in vitro stimulation by bacterial peptides (fmlp and lps) and/or synthetic stimuli (phorbol- -myristate- acetate, pma) during the recovery from prolonged ( . . h) exercise (chinda et al., ; suzuki et al., ; davison and gleeson, laing et al., ; davison and diment, ) . this decline in killing capacity has not been coupled to changes in cell surface receptors, suggesting that the modulatory effects of exercise on in vitro stimulants occur downstream in intracellular signal transduction pathways of neutrophils such as phosphorylation cascades or secondary messengers (e.g., cyclic adenosine monophosphate, calcium) (mooren et al., ; peake, ) . the magnitude of the effects of prolonged exercise on neutrophil effector functions will depend on the balance of immunosuppressive (e.g., "refractory period") and immunostimulating factors (e.g., priming agents) which largely depend on the extent of neutrophilia (peake, ) . as previously mentioned, prolonged exercise (i.e., elevations in stress hormones) induces a mobilization of immature neutrophils into the circulation which have been shown to exhibit reduced nadph oxidase activity and granular content (hetherington and quie, ; berkow and dodson, ) . the immuodepressive effects of cortisol and the subsequent release of subpopulations from the bone marrow was supported by robson et al. ( b) who found that the degree of exercise-induced decrease in stimulated degranulation per neutrophil and neutrophilia was greater following prolonged exercise (b h) compared to short, intense exercise (b min). although considered to be a major factor, activation of the hpa axis alone cannot wholly account for changes in the neutrophil function (laing et al., ) , as some of the noted inhibitory effects of cortisol on receptor mediated responses (e.g., fmlp) would not explain decreased responses to stimulants that activate neutrophils independent of surface receptors (e.g., pma) (o'flaherty et al., ; tomchek et al., ; peake, ) . thus other mechanisms have also been suggested to be involved in neutrophil dysfunction, where elevations in catecholamines, cyclic adenosine monophosphate, complement proteins (c a), direct cellular oxidative damage and growth hormone may occur with the inflammatory response to prolonged exercise and have been shown to interfere with calcium signaling or other intermediates of intracellular pathways (henson et al., ; hack et al., ; suzuki et al., ; thibault et al., ; tintinger et al., ; robson et al., ; laing et al., ) . the susceptibility of phagocytes to modulation by strenuous exertion are also demonstrated by the decreased expression of tlr on the cell surface of monocytes immediately and up to h following prolonged exercise (lancaster et al., b; oliveria and gleeson, ) which are not explained by exercise-induced changes in monocyte numbers alone (simpson et al., ) . the phagocytic function of monocytes has been shown to increase following prolonged exercise which may be related to the increased pro-inflammatory phenotype observed with exercise in this leukocyte (steppich et al., ; hong and mills, ) . once monocytes reach the tissues they will form mature macrophages, therefore the biological significance of changes in monocytes is unclear as it may not reflect exercise-induced changes in immunosurveillance within tissue (i.e., at sites of inflammation or infection) (simpson et al., ; walsh et al., b) . these macrophages along with other phagocytes (dendritic cells) are present in majority of body tissues where unlike the direct killing capacity of neutrophils, macrophages and dendritic cells mostly act as professional apc (beutler, ; iwazaki and medzhitov, ) . investigations within exercise stress models thus far have been limited to animal studies which are difficult to generalize to the human response, but do suggest dysfunction in the capacity of these cells to present antigens following prolonged exercise (davis et al., ; woods et al., ; ceddia and woods, ; ceddia et al., ; woods et al., ; murphy et al., ; liao et al., ; chiang et al., ) . nk cells are large granular cells that can sense structures of high-molecular weight glycoproteins expressed on virus-infected cells via prr on their cell surface. they form up to % of the lymphocytes within the body and are vital in defense against viral infection. however, compared to other peripheral lymphocytes (b and t cells, see section . . ), prior sensitisation is not required (cerwenka and lanier, ) . thus upon activation nk cells trigger apoptosis or lysis of a virusinfected cell by releasing granule contents such as the pore forming proteins perforin and cytolysin. the initial investigations into nk cell activity (nkca) showed that exercise-induced responses were largely mediated by the duration and intensity of the bout (gannon et al., ) . the nkca was demonstrated to mirror the increases in nk cells following moderate or exhaustive exercise (gannon et al., ; woods et al., ) . however, when the exercise bout is intense and prolonged, ncka on a per cell basis has been shown to be reduced for several hours (kappel et al., ; nieman et al., ; mcfarlin et al., ) . binding of microorganisms to prr (e.g., tlr) of innate immune cells not only triggers activation of innate parameters but also induces the generation of protein messengers (e.g., cytokines) and other signaling components to stimulate acquired immunity (takeda and akira, ) . however, compared to innate immunity, the acquired immune response to a pathogen is delayed due to a lag period for recognition by the vast range of antigen receptors within b and t lymphocyte populations, clonal selection/expansion of the pertinent lymphocytes and clonal elimination for tolerance of self to occur (kimbrell and beutler, ; medzhitov, ) . t and b cells form %À % and %À % of the circulating pool of lymphocytes respectively, where the t cell population mature in the thymus gland and the b cells in the bone marrow. t lymphocytes are subdivided further into cytotoxic (tc, cd ), helper (th, cd ), and regulatory (treg) where the treg are formed from a naive cd cell and modulate immune responses compared to the primary involvement in removal of pathogens by tc and th. the induction of a primary immune response to pathogens involves presentation of antigens to the cell surface receptor of t lymphocytes (t cell receptor) by dendritic cells (lanzavecchia and sallusto, ; mellman and steinman, ) , while all other apc (e.g., monocytes, macrophages) are involved in the initiation of secondary immune responses via memory t cells which have encountered the antigen previously (gallucci and matzinger, ) . t cells are able to recognize antigens via major histocompatibilty complex (mhc) class i and ii molecules on the cell surface of an apc (banchereau and steinman, ) . the number of antigens encountered by an individual will determine the proportion of naive (unactivated) or memory (activated) t cells circulating within the body. although structurally similar to the t cell receptor, the surface receptor of the b lymphocyte is a membraneanchored immunoglobulin (ig) (also termed antibody). upon interaction of antigen and receptor, the b cell undergoes clonal expansion to form both short-lived plasma effector cells and long-lived memory cells or internalize the bound antigen and act as an apc to t cells (lebien and tedder, ) . the short-lived plasma cells secrete igs into the blood to aid destruction of the pathogen while the memory cells with their greater affinity to the antigens can remain in the circulation throughout life to rapidly differentiate into plasma effector cells if the same antigen is encountered again (walsh et al., b) . the circulating igs fall into five major classes, igm, igg, iga, igd, and ige where each of these classes can be divided further into subclasses. this diverse repertoire of igs possess functional regions capable of binding to a vast range of antigen sites (epitopes) to form immune (antigenÀantibody) complexes that neutralize the toxicity of certain antigens or common ig regions which can activate phagocyte ingestion and soluble innate factors (e.g., complement) . following a lag period for accumulation of igs within the blood, the igm class predominates during any primary response to an antigen whereas igg predominates under normal resting conditions as well as in the rapid ig response to secondary antigen exposure (mckune et al., ) . therefore the acquired immune system consists of a cellular (i.e., t cells) and humoral component (i.e., ig). determining which component of the acquired immune system predominates, are the subpopulations of th cells (th and th ) and the cytokine profile they produce and release (walsh et al., b) . cytokines act as protein messengers between one immune cell and other, where the cell receiving the signal may proliferate, secrete additional cytokines, migrate to the area of origin of the signal, differentiate into another type of cell or die (undergo apoptosis) (curfs et al., ) . the main cytokine group responsible for leukocyte communication is the interleukin (il) family but other major cytokine groups are the colony stimulating factors (e.g., granulocyte macrophage colony stimulating factor, granulocyte-colony stimulating factor), tumor necrosis factors, and interferons (ifn) where their main roles are stimulation of cell growth, tumor cytoxicity, and inhibition of viral replication respectively (curfs et al., ) . the stimulation of th cells and production of cytokines such as il- , and ifn γ promotes cell-mediated immunity (tc responses) for defense against intracellular pathogens while activation of th cells (il- , il- , il- , and il- ) coordinates humoral immunity (b cells) and release of igs for defense against extracellular pathogens (seder, ) . it has been suggested that intense/prolonged exercise can modulate this balance by decreasing the proportion of th cells in circulation whereas th cells remain unaffected (steensberg et al., ; lancaster et al., lancaster et al., , a . this shift in immunity has been proposed to provide an explanation for potential increases in the susceptibility to uri following prolonged exercise given the importance of type responses towards viral infection (steensberg et al., ; fabbri et al., ) . such effects of exercise are suggested to be mediated via the suppression of type cytokine production by elevations in stress in hormones (adrenaline and cortisol) and the release of cytokines from contracting skeletal muscle (primarily il- ) that favor production of type cytokines (gleeson, ) . supporting evidence for cytokine modulations following intense exercise has been provided by decreases in il- and ifn and no changes in il- shown with in vitro mitogen-stimulated isolated cells and whole blood culture (tvede et al., ; moyna et al., ; smits et al., ; starkie et al., ) . these changes in cytokine production overlap with influences on other stages of t cell activation where decreases in mitogeninduced t cell proliferation have been observed following intense/prolonged exercise (fry et al., ; nieman et al., nieman et al., , henson et al., ; bishop et al., ) . despite these observations, the evidence suggesting that t cell activation is down-regulated by prolonged exercise is confounded by numerous methodological limitations (walsh et al., b) . t cell proliferation assays generally use a fixed amount of total lymphocytes or whole blood so changes following exercise may only reflect changes in the proportion of lymphocyte subsets due to the greater increase in nk cells post exercise which do not respond to mitogen (green and rowbottom, ) . depletion of nk cells from cell culture has been found to remove the significance of changes in mitogeninduced proliferation following exercise (green et al., ) . nieman et al. ( ) found that proliferative responses were substantially different based on adjustments for t cell populations, but there was still a significant fall in proliferation following intense exercise compared to preexercise. nevertheless, the validity and sensitivity of mitogen-induced culture to assess t cell function have been questioned due to their nonspecific effects (activating other cell types, e.g., b cells) and lack of ability to detect subtle changes in individual t cell populations (bishop et al., ) . demonstrated that ex vivo migration of cd and cd cells to a human-rhinovirus infected bronchial (lung) epithelial cell line was decreased following a prolonged exercise bout ( h of running). it is also difficult to extrapolate such observations on isolated cells from a circulating pool that is considerably lower than total lymphocyte mass within the complex in vivo environments at the skin, mucosa, and lymph nodes (gleeson, ) . although investigations on whole blood maintains the proximity of leukocytes and the extracellular milieu of leukocytes compared to leukocyte isolation, the use of in vivo measures to assess response to antigenic challenge may be more clinically relevant (albers et al., ; albers et al., ; walsh et al., b; bermon et al., ) . both cell-mediated and adaptive parameters contribute to the largest component of the immune system, mucosal immunity (total surface area of m ) (brandtzaeg et al., ) . the importance in host defense against pathogens becomes apparent when recognizing that the mucosal surfaces of the upper and lower respiratory tract account for b %À % of total immune protection by the body and the small intestine along with the colon are responsible for % of all igs produced (kudsk, ) . increases in illness and morbidity have been attributed to impairment in mucosal immunity (daele and zicot, ) , highlighting the importance that immune competence at mucosal surfaces has in the health and well-being of athletes (west et al., ) . although not functioning independently of the systemic immune system, mucosal immunity is also considered a distinct entity due to its autonomously regulated, localized defense mechanisms (toy and mayer, ) . the gut-associated lymphoid tissue, urogenital tracts, lacrimal glands, lactating mammary glands, and respiratory tracts which include the bronchus-associated lymphoid tissue (balt), salivary glands and nasalassociated lymphoid tissue are all mucosal surfaces which fall under the network of immune structures known as the common mucosal immune system (cmis) (gleeson and pyne, ) . the immunological protection provided by this network may be via organized tissue with wellformed follicles (muscosa-associated lymphoid tissue) such as pp of the small intestine or as a diffuse accumulation of leukocytes (lymphocytes, plasma cells, and phagocytes) as found in the lung and the lamina propria (connective tissue) of the small intestine (kyd and cripps, ) . these immune structures are pivotal for the monolayered epithelial layer of mucosal surfaces which is continually exposed to a wide array of antigens or allergens including pathogenic bacteria or viruses, gut microflora and ingested food (johansen et al., ) . indeed, the mucosae are considered to be the first line of defense as they are the sites where most pathogens enter the body (macpherson et al., ) . the cmis are differentiated into inductive and effector sites, where the induction sites (primarily pp) involve the sensitization of immune response following antigen presentation while the effector sites consist of interconnected distal sites (respiratory tract, lamina propria) where the array of activated b cells and plasma cells home and migrate to provide local protection (kyd and cripps, ; kudsk, ) . at least % of the body's plasma cells (activated b cells) reside in the mucosal effector tissues, with local production of igs representing a major immunological barrier at all mucosal surfaces and iga being the predominant antibody (brandtzaeg et al., ; . within the bloodstream, iga under most circumstances is found as a monomeric peptide (yel, ) . however, in all mucosal secretions iga exists as a dimeric protein covalently linked by a j chain containing another peptide termed the secretory component (gleeson and pyne, ) . the secretory component is the cleaved segment of the polymeric ig receptor (pigr) that is produced by the mucosal and glandular epithelial cells and expressed on the basolateral membrane (teeuw et al., ; ). the proteolytic cleavage of pigr occurs following its binding and induction of the active transport (exocytosis) of dimeric iga through epithelial cells on to the mucosal surface (strugnell and wijburg, ) . the remaining secretory component wrapped around the j chain-linked dimeric iga forms secretory iga (siga) which is considered to be resistant to proteases secreted at mucosal sites (e.g., intestinal mucosa) (underdown and dorrington, ; lindh, ; johansen et al., ; strugnell and wijburg, ) . it is this local production of siga that forms the major effector function of mucosal immunity . only in the neonate or situations of iga deficiency does igm represent a significant defense at mucosal surfaces with igg also being found in low quantities at mucosae (brandtzaeg et al., ; gleeson and pyne, ) . iga can be further divided into subclasses, where iga is the most abundant in the distal gastrointestinal tract ( %), whereas iga predominates in the salivary glands ( %À %) and nasal lymphoid tissue ( . %) (gleeson, ) . protection of mucosal surfaces via siga occurs through multiple mechanisms. one such mechanism known as immune exclusion involves the binding of antigens to regulate the commensal microorganisms (microbiota) and prevent the attachment and invasion of mucosal surfaces by pathogens (strugnell and wijburg, ; sutherland and fagarsan, ) . other mechanisms include the binding of antigens which have already crossed the mucosal barrier and actively transporting them back across the epithelial layer into the lumen or intracellular neutralisation of viruses when bound to pigr within the mucosal epithelia (lamm, ) . the presence of siga (formed by b cells adjacent to salivary ducts and glands) in saliva has tended to be the mucosal immune marker of choice due to the ease of collection (korsrud and brandtzaeg, ; gleeson, ; sari-sarraf et al., ; ). most of the salivary fluid itself is formed by three pairs of major salivary glands (parotid, submandibular, sublingual) but production is supplemented by a vast amount of small submucosal glands that lie on and around the tissue (e.g., palate, tongue) within the oral cavity (proctor and carpenter, ) . although saliva drains from the acini (cluster of cells) of each of these glands into the mouth via striated and excretory ducts, the nature of the secretion differs whereby a serous (watery) fluid is produced by the parotid, mucous fluid by the submandibular and sero-mucous mixture by the sublingual (aps and martens, ) . in unstimulated saliva secretion, the proportion of the fluid provided by parotid, submandibular, sublingual and the remaining submucosal glands are suggested to be %, %, %, and % respectively on average (dawes, ) . in addition to the aforementioned transcytosis of siga into the mucosal secretion, saliva benefits from the import of several antimicrobial peptides (amps) that contribute to the first line of defense by providing innate defences compared to the specific nature of iga (bals, ) . amps are categorized as being small cationic peptides (, amino acids) that represent inducible, constituent factors of mucosal secretions (west et al., ) . although numerous amps require some form of enzymatic modification prior to their functional configuration, they act in synergy with other components of the innate immune system to prevent and aid clearance of infections (wakabayashi et al., ; bowdish et al., ; de smet and contreras, ; ibrahim et al., ; radek and gallo, ) . several amps can modulate other immune processes such as leukocyte cytokine secretion, chemotaxis and remodeling of injured epithelia (ganz, ; bowdish et al., ; tjabringa et al., ) . the most abundant amps in the secretions of the urt are lysozyme and lactoferrin (singh et al., ) . lysozyme is a small protein ( kda) released into saliva by neutrophils, macrophages, and the submucosal glands that possesses bactericidal capacity through hydrolyzing the polysaccharide of bacterial cell walls (jolles and jolles, ; travis et al., ; bosch et al., ; west et al., ; fabian et al., ) . the antimicrobial properties of the smaller molecule lactoferrin ( kda) from neutrophils and submucosal glands are due to its ability to bind free iron, depriving bacteria of this nutrient that is essential for growth and multiplication (legrand et al., ; bowdish et al., ; ward et al., ) . although not an inhibitor of the main causative agent of urti (rhinovirus), lactoferrin is considered to be effective against other common respiratory viruses [adenovirus, respiratory syncytial virus (west et al., ) ]. there is a wide variety of other amps, most of which have been grouped into three main families; cathelicidins (e.g., ll ), defensins (α and β subfamilies), and histatins (bals, ) . these are primarily released into the oral cavity by the epithelial cells, salivary glands, and/or neutrophils (de smet and contreras, ; fabian et al., ) . the amps work synergistically in low concentrations to destabilize cell walls of microorganisms and provide a broad spectrum of activity against gram-positive and gram-negative bacteria (bals, ) . other salivary proteins that contribute an important line of defense via the inhibition of adherence and growth of specific bacteria (e.g., streptococcus) at the oral mucosa include α-amylase (scannapieco et al., ) . in addition to influx of invading microorganisms from the external environment, the human mouth has a constant microbial presence that needs to be regulated (bender et al., ) . surfaces of the oral cavity are bathed with saliva, whereby the fluid is recognized to provide a "fingerprint" of the vast range of the resident microorganisms (li and gleeson, ; boutaga et al., ; fabian et al., ; dewhirst et al., ) . reduced salivary flow rate can directly impact on the oral microbiome by inducing a shift towards colonization via pathogenic microorganisms (meurman, ) . indeed, the entire surface of the respiratory tract is a source of commensal microorganisms which, similar to exogenous antigens, possess the ability to become pathogenic in the host (watson et al., ; bosch et al., ) . amps not only play a crucial role in the protection against foreign pathogens, but also act as a synergic arsenal of molecules that regulate the response to commensal bacteria (boman, ; davison et al., ; jones et al., ) . therefore the importance of amps is twofold; preventing disruption of the epithelial layer by acting as a critical first line of defense (biofilm) against pathogens, and maintaining homeostasis of the commensal community that act to outcompete invading microorganisms (blaser and falkow, ; murphy et al., ; . the overlapping nature of these defenses is highlighted when reduction in expressions of amps in in vitro models lead to changes in bacterial colonization (bals et al., ; liu and modlin, ) . subsequently, perturbations in the balance of the microbiota on the mucosal surface can lead to overgrowth and further amplification of microbes which may have direct effects locally (e.g., oral infection, urti) or have indirect effects through predisposing to respiratory illness with the many established interactions between microorganisms (bacteriaÀbacteria, viralÀbacteria) (slots and genco, ; blaser and falkow, ; murphy et al., ; meurman, ; bosch et al., ) . such debilitating effects are likely to occur if the imbalance in microbiota occurred due to a lack of immune competence (e.g., decreased amps) (bosch et al., ) . the resistance of the microbiota within the urt to the immune perturbations associated with prolonged exercise is currently unclear. the most popular parameter of mucosal immunity that has been investigated in an acute exercise setting has been salivary siga due to the notion that individuals who suffer from iga deficiency contract urti regularly (gleeson and pyne, ) . physiological changes (e.g., nervous stimulation, dehydration) during exercise can influence the secretion of saliva and its protein components (walsh et al., ; ). the salivary glands are innervated by both the parasympathetic and sympathetic nervous system, with changes in stimulation of either of these having an influence on the volume, viscosity, protein and mucin concentration (aps and martens, ) . parasympathetic nervous stimulation via vasodilation of the salivary glands is believed to trigger a high volume of watery saliva, low in protein concentration ). on the other hand, sympathetic stimulation produces salivary secretions which are low in volume but high in protein that is primarily due to enhanced active transport of proteins from salivary cells (proctor and carpenter, ) . thus similar to the other discussed immune parameters, salivary siga concentration is susceptible to effects (sympathetic and parasympathetic responses) of exercise intensity and duration (walsh et al., b) . the following discussion will consider those studies which have used the most reproducible collection method, unstimulated whole saliva collection (also termed passive drool), due to the potential for stimulated saliva flow (e.g., chewing) and other collection methods (e.g., swabs) to preferentially induce secretion from certain glands and/ or influence saliva composition once secreted (navazesh and christensen, ; navazesh, ; proctor and carpenter, ; harmon et al., ; beltzer et al., ; granger et al., ; allgrove et al., ) . although there is inconsistency in study design within the literature to draw a definitive conclusion, walsh et al. ( b) suggested that siga concentration in saliva generally decreases (e.g., tomasi, et al., ; nieman et al., nieman et al., , palmer et al., ) or remains unchanged (e.g., mackinnon and hooper, ; sari-sarraf et al., ) following prolonged exercise ($ . h at %À % maximum oxygen uptake, _ vo max ). it seems that the combination of high-intensity and exercise of a long duration has the most significant impact (i.e., depressive) on salivary siga concentration (mackinnon, ; nieman et al., ) . the discrepancies in the literature are also believed to be partly due to the way in which salivary siga concentration is expressed relative to the exerciseinduced changes in physiological responses (walsh et al., ) . in an attempt to account for such changes, salivary siga concentration has been expressed as a secretion rate or relative to total salivary protein/albumin/osmolality (gleeson, ) , but this makes the comparison between studies difficult ). the secretion rate or expression relative to saliva osmolality are preferred over measures of siga as ratio to total protein (blannin et al., ) . one reason for this is that other salivary proteins (e.g., amylase) are known to increase with exercise without a change in siga (walsh et al., ) . furthermore, expression as a secretion rate may better reflect the amount of available siga on the mucosal surface (mackinnon et al., ) . in contrast, it has been argued that salivary siga concentration is of greater significance as secretion rate may only provide an explanation to how salivary flow rate has changed . having said that, salivary siga when expressed as a secretion rate has been found to be the best predictor of uri incidence in athletes following a km race or during intense training and competitive phases (fahlman and engels, ) . additionally, given that the majority of saliva is water, expression relative to secretion rate also accounts for the concentrating effect of other salivary components following any dehydration (bishop et al., ; oliver et al., ) . as saliva osmolality reflects the inorganic electrolyte concentration (rather than protein content) and hence falls in proportion with decreases in flow rate, siga:osmolality provides an alternative method (blannin et al., ; . although iga is the dominant ig in mucosal secretions, some have also attempted to identify changes in salivary igg and igm concentrations following exercise. limited findings suggest igg remains unchanged but igm parallels decreases in salivary siga, highlighting the potential effects that acute strenuous exercise has on salivary immune parameters (gleeson and pyne, ; ). another aspect of mucosal immunity which has received little attention to date is the responses of amps to exercise (walsh et al., b) . despite the interest of the dentistry field in the role of amps in oral health and infection, (putsep et al., ; tanida et al., ; tao et al., ; dale et al., ) , the relationship of amps with exercise-induced immune dysfunction is yet to be explored conclusively (davison et al., ) . to date, the few investigations that have been conducted suggest in accordance with other immunological measures, responses of amps may be dependent on the intensity and duration of the exercise bout (hoffman-goetz and . a h cycling bout at b % _ vo max resulted in a significant decrease in salivary lysozyme (slys) concentration, slys secretion rate and slys:osmolality which recovered after h of rest (davison and diment, ) . in contrast, expressions of other amps (ll and defensins: human neutrophil peptide À ) following similar stressors ( . h at b % _ vo max ) have been shown to significantly increased immediately post exercise. in the recovery period ( À . h post) following a km mountain trail race (mean running time b h) there was a significant decrease in salivary lactoferrin (slac) concentration and nonsignificant decreases in slac and slys secretion rate (gillum et al., ) . the mechanism behind the effects of prolonged exercise on salivary parameters (amps, siga) remains unclear (walsh et al., b) . the flow rate of saliva is considered to be the major source of variation in concentration of mucosal parameters (gleeson and pyne, ) . in general, saliva flow rate decreases in response to a prolonged exercise bout (walsh et al., ; bishop et al., ) . decreases have been attributed to a withdrawal of parasympathetic stimulation rather than sympatheticinduced vasoconstriction of salivary glands . parasympathetic withdrawal associated with sensations of a dry mouth in response to other acute stressors (e.g., psychological) supports such proposals as does the lack of effect on saliva flow rate with interventions aimed to increase sympathetic stimulation (bosch et al., ; bishop et al., ) . a crucial determinant of siga release into saliva is the presence of pigr to permit transport across the epithelial layer (bosch et al., ) . evidence from animal studies suggests that increased mobilization of pigr occurs only above a certain threshold of increased sympathetic stimulation (proctor et al., ) . this may explain why a brief bout of high-intensity exercise leads to increases in salivary siga (e.g., davison, ) . however, this does not explain the decrease found with prolonged exercise bouts ). it has been speculated that this nervous stimulation over a longer period (i.e., prolonged exercise) may deplete the available iga (proctor et al., ; allgrove et al., ) or there may be a further threshold or interaction with duration where pigr mobilization is down-regulated (walsh et al., b) . as brief bouts of maximal effort exercise result in increased slac and slys, it is reasonable to suggest that mobilization of amps during exercise is also influenced by sympathetic stimulation in relation to certain thresholds (allgrove et al., ; west et al., ; usui et al., ) . however, the reductions in slys following prolonged exercise may rather be related to an increased stimulation of the hpa axis and hence increases in salivary cortisol concentration, which have been associated (note, not cause, and effect) with reduced concentration of slys (perera et al., ; west et al., ) . additionally, it is worthy to note that the source of slac and slys (phagocytes, epithelial cells) is different to siga. therefore it is possible that increases in amps during high-intensity exercise may be as a result of the effects of hyperventilation (e.g., drying of the mucosal surfaces) given that some of these changes are lost or reduced when expressed relative to osmolality or as a secretion rate (davison, ) . additionally, the exercise-induced damage to the epithelial layer could induce an inflammatory response whereby epithelial cells increase release of amps and other sources of amps are recruited (e.g., neutrophils) (west et al., ) . the relative contribution of amps from each of these sources to saliva levels at both rest and following exercise is unclear. however, it is reasonable to suggest that an airway inflammatory response partly accounts for the increases in amps identified immediately following prolonged exercise (davison et al., ) . certain amps (α defensins) have been suggested to make up % of the protein found in neutrophil azurophilic granules (radek and gallo, ) . müns ( ) demonstrated a significant (twofold) increase in the neutrophil count of mucosal secretions (nasal lavage fluid) following a prolonged exercise bout. although neutrophils continually migrate into saliva from the circulation via gingival crevices (lukac et al., ; bender et al., ) , the extent of neutrophila that occurs in the circulation following exercise may lead to the presence of neutrophils and their contents in saliva (e.g., amps) being substantially increased (davison et al., ) . increased levels of amps (e.g., α defensins) have been observed in other body fluids (e.g., plasma) following a circulating neutrophila (shiomi et al., ) . the variation in the responses of different amps to prolonged exercise (davison et al., ; davison and diment, ) , therefore, may be due to the changes in the maturity of circulating neutrophils (as expressions of amps can vary throughout the maturation of the neutrophil in the bone marrow) or decreases in some amps may merely reflect neutrophils undergoing a refractory period post exercise (cowland et al., ; borregard and cowland, ; gullberg et al., ; nagaoka et al., ; sorensen et al., ; nagaoka et al., nagaoka et al., , peake, ) . based on the evidence presented to date regarding acute prolonged exercise, it may be expected that the highly trained athletes demonstrate lower immune function at rest compared to nonexercising controls. however, numerous cross-sectional studies have attempted to discern such differences and found that when measures of immune function are gathered in a "resting state" (at least h following the previous bout) there seems to be very little difference between athletes and controls (gleeson, ) . a review of early investigations in the area (nieman, ) suggested even when significant immune perturbations had been observed in athletes participating in strenuous exercise, investigators had limited success in identifying such measures that influence alteration in rates of uri (nieman et al., b; mackinnon et al., ; nieman et al., nieman et al., , . this actually adds further support to the open window theory, whereby these periods of depressed immunity are transient. however, for athletes who train many times per week, they will experience multiple open windows and the total time of increased susceptibility will be greater, meaning a higher overall risk of contracting an illness. despite the large inter-individual variation in concentration of salivary siga, (walsh et al., b) some of the early work (salivary siga responses to training) reported lower concentrations in endurance athletes compared to sedentary counterparts (tomasi et al., ) . in contrast, most of the subsequent investigations support evidence of other parameters by showing salivary siga to be broadly similar in the two populations (gleeson and pyne, ; ). conversely, findings in elite rowers had %À % lower slac concentration than controls at the start and mid-way through a month training period (west et al., ) , suggesting investigations of the mucosal immune compartment in a "resting state" between these populations requires further attention. inverse relationships between salivary siga concentration/secretion and uri incidence in some studies of athletes undergoing periods of intensive training (i.e., athletes not necessarily in "resting state") (fahlman and engels, ; neville et al., ) further supports the need for longitudinal research on mucosal immunity with large athletic populations (nieman, ) . the common observations of this mucosal parameter within intensive training studies represents a narrow range that have established a link between an immune measure and uri in athletes (gleeson, ; walsh et al., b) . given the complexity of the immune system, it is unlikely that salivary siga alone would explain uri risk for all athletes, thus other factors in combination with the immune measure need to be determined (nieman and bishop, ) . investigators have begun attempts to address this by examining differences between illness prone and healthy athletes with early evidence suggesting that the blood obtained from the illness prone athletes has greater ex vivo production of antiinflammatory cytokines (e.g., il- and il- ) in response to multiantigen challenge, indicative of a downregulation of cell-mediated immunity gleeson and walsh ; gleeson and bishop, ) . multifactorial mechanisms beyond salivary siga alone in uri incidence become more apparent when athletes intensify their training over a short period of time (gleeson, ) . indeed, À weeks of intensified training has been shown to induce marked reductions in neutrophil and monocyte function, lymphocyte proliferation and the circulating number of t cells (verde et al., ; robson et al., a; lancaster et al., lancaster et al., , . in addition to intensive experimental protocols, longitudinal monitoring over a competitive seasons showed that team sports (rugby) and endurance athletes (cyclists) are sensitive to the intensive training periods with decreases in t cell counts, slys, neutrophil oxidative burst, and il- production (baj et al., ; cunniffe et al., ) . indeed, rapid changes (increases) in training load/stimulus may be the key risk factor (more so than total training load) for uri occurrence in athletes (e.g., svendsen et al., ) . these findings suggest that there is a cumulative effect of repeated bouts of strenuous exercise due to an inadequate recovery time for the immune system (papacosta and gleeson, ) . furthermore, it may be that responses to acute prolonged exercise are more relevant than resting immunity in athletes where the immediate recovery period represents the "open window" when athletes are most vulnerable to infection (nieman et al., b (nieman et al., , pedersen and bruunsgaard, ; abbasi et al., ) , since the chronic effects represent the accumulation of each acute response. despite the lack of clear difference in measures at rest, as mentioned earlier, as athletes are exposed to more frequent acute bouts ("open windows") of immunodepression, the overall risk may be considered greater than nonexercising controls and each acute response dictates the size and duration of each individual "open window." the presence of any other risk factors (e.g., dietary deficiency, sleep disturbance, psychological stress) will have additive effects on the transient immune responses and increase the risk of illness following prolonged exercise as seen in many studies (gleeson, ) . although underlying causes of uri remain uncertain, decrements in performance as a result of uri have been reported (reid et al., ; pyne et al., ) . the monitoring of the risk status of an athlete is paramount in order to determine the appropriate preventive or therapeutic interventions required during stages of strenuous training/ competition. regular moderate activity in both sedentary and active populations is considered to promote an antiinflammatory environment. this is an important underlying mechanism in the protection against chronic inflammatory conditions gained from physical activity in older populations (e.g., cardiovascular disease, type diabetes, obesity) (walsh et al., a) . despite moderate activity induced decreases in resting concentrations of inflammatory mediators (e.g., acute phase reactants, crp) (mcfarlin et al., ) , evidence of an influence of regular moderate activity on resting leukocyte function remains equivocal however. it is likely that additional mechanisms are also responsible for the benefits of physical activity on overall immunity and infection risk. moderate activity (cycling) of h duration has been shown to increase the capacity of blood neutrophils to respond to both receptor independent and dependent in vitro stimulation (smith et al., ) . this exercise-induced priming of neutrophil microbiocidal capacity has been suggested to be due to an increased presence of immunostimulating factors in the circulation and a greater proportion of responsive cells. dhabar ( ) suggests that the immunopotentiation from moderate exercise is due to the bidirectional effect of stress hormones on immunity where subtle elevations are beneficial whereas significant and sustained elevations (as seen with prolonged and/or intensive exertion) are detrimental to the host. it is these short-lived changes in cell-mediated immunity that occur during and shortly after each acute moderate exercise bout itself that are proposed to contribute to the lower risk of urti (nieman et al., ) . to further investigate the effect of moderate activity on cell-mediated immunity, animal and human experimental studies have monitored the production of cytokines by t lymphocytes. it is well established that intracellular pathogens trigger a (type ) cell-mediated immune response resulting in the differentiation of naive cd and cd t cells into t helper and t cytotoxic type lymphocytes (th /tc ) characterized by a phenotype of interferon (ifn)-γ and il- production (seder, ) . in contrast, extracellular pathogens, induce a humoral (type ) immune response resulting in the differentiation of naive cd and cd t cells into th / tc lymphocytes characterized by a phenotype of il- , il- , il- , and il- production (seder, ) . type and type phenotypes are mutually inhibitory, whereby up-regulation or downregulation of either leads to an imbalance in immune responses (zhao et al., ) . for example, it has been suggested that moderate activity can induce heightened type responses and, given the importance of type responses towards viral infection (steensberg et al., ; fabbri et al., ) , contribute to decreased urti risk. baum et al. ( ) showed that an acute bout of moderate cycling caused increases in ifn-γ production by peripheral blood lymphocytes of young adults where concentrations remained elevated compared to a control group h post exercise. longitudinal studies ( . À years) of moderate activity (cycling or walking for min each day) have to date only been investigated in the older population where an age-associated decline in type lymphocytes and cytokines have been reversed (ogawa et al., ; shimizu et al., ) . although a type response is critical for immmunosurveillance and early viral clearance, a sustained or excessive shift towards this phenotype has been implicated in tissue damage within lung pathology (van reeth, ) . martin et al. ( ) have hypothesized, albeit from animal models, that moderate activity also potentiates immunoregulatory mechanisms to skew towards a th phenotype and antiinflammatory environment during responses to infection. further research is warranted to determine the role of type /type balance and cell-derived cytokines towards infection risk. longitudinal interventions (b weeks) also suggest that aerobic activities of a moderate intensity mediate the resting levels of other immune parameters in the circulatory and mucosal (saliva) compartments. this may include increasing ("restoring") plasma ig or preventing further age-related decline in salivary concentrations (secretory iga, siga) in the elderly or increasing levels (e.g., siga secretion rate) in the younger adult population klentrou et al., ; akimoto et al., ; martins et al., ; sloan et al, ) . siga of the mucosal immune system expressed as a saliva secretion rate has previously been found to be the best predictor of urti incidence (risk) during exposure to intense, prolonged exertion . as a reduction in incidence of urti symptoms during weeks of moderate activity has been associated with an increase in siga (klentrou et al., ) , it is likely that this parameter plays an important role in the differential doseÀresponse relationship of physical activity and infection risk. the underlying mechanisms of these changes are unclear but evidence from animal studies suggest that moderate activity may mediate an up-regulation of siga production by stimulating the expression of cytokines involved in the synthesis of the ig (drago-serano et al., ) . based on the available evidence, regular aerobic moderate activity should be promoted as a preventive strategy against urti due to a combination of short-term acute changes after each bout and a summation effect over a longer period, with the exact temporal pattern being dependent on the investigated immune parameter. resistance exercise has been studied less comprehensively in the context of its effects on immunity. the majority or research in this area has focused on the postexercise changes in leukocyte counts (freidenrick and volek, ) . following an acute bout of resistance exercise, nk cells, monocytes and neutrophils increase in the circulation whereby the number of some of these subsets (monocytes, neutrophils) remain elevated for up to h post exercise (ramel et al., ) . the duration and magnitude of the postexercise leukocytosis is diminished in the older population as a consequence of age-related decline in leukocytes (freidenrick and volek, ) . differentiation of monocytes into macrophages and their infiltration into muscle tissue with neutrophils is essential for the regeneration and repair of muscle tissue after resistance exercise induced damage (arnold et al., ; tidball et al., ) . this leukocyte redistribution has been shown to be dependent on individual components of the resistance exercise (e.g., rest length, training load) (mayhew et al., ; carlson et al., ) . the extent of this damage depends largely on the eccentric component of the exercise bout (sorichtere et al., ) . the infiltration of immune cells and release of chemokines and other inflammatory mediators associated with this muscle damage creates a localized pro-inflammatory environment (edwards et al., ) . although focus of investigations with resistance exercise has not been on changes in infection risk per se, this heightened inflammatory environment has been proposed to be an effective adjuvant that may enhance responses to vaccination as discussed in the vaccination section below. as discussed earlier, in vitro and ex vivo markers can help understanding of how the immune system responds to exercise but the most valuable (clinically relevant) measures are those which assess the whole integrated immune response (albers et al., (albers et al., , . in the exercise immunology literature, such measures include challenge-type tests [such as delayed type hypersensitivity (dth) or contact hypersensitivity (chs) tests], response to vaccination, and possibly the reactivation of latent viruses (as discussed earlier). dth is a complex local immunological reaction to an intracutaneous antigen (following previous encounter/sensitisation) that is primarily t-cell-mediated but involves interaction of various cell types and chemical mediators of the immune system to induce a cell-mediated immune response (albers et al., ) . although the antigen presentation to t cells within a dth reaction is similar to the response to intracellular pathogens (kaufmann, ) , it can be considered an amplified reflection of the stages that occur with a "conventional" antigen which allows for the cell-mediated immune response to be noninvasively quantified (kobayashi et al., ) . the dth reaction is characterized by an infiltration of predominantly peripheral blood mononuclear cells [t cells (antigen-specific) and monocytes/macrophages] which release cytokines and other inflammatory mediators that present as eczematous plaques at the site (i.e., skin) (bruunsgaard et al., ; schwarz, ; kaplan et al., ; malaijan and belsito, ) . such epidermal indurations (edema and erythema) that peak in the À h following antigen reexposure can then provide the means by which cell-mediated immune response (the outcome) can be measured (albers et al., ) . bruunsgaard et al. ( ) showed that triathletes who completed prolonged exercise prior to intradermal injections of seven previously encountered antigens had significantly reduced skin responses compared to resting controls, indicating immunodepression. this method, however, is limited to the recall (elicitation) of preexisting immunological memory and does not provide any evidence on the effects of prolonged exercise on the primary immune (induction) response to a novel antigen (harper-smith et al., ) . contact sensitisation which involves topical skin exposure to novel synthetic chemicals provides an experimental model where both the elicitation and induction of t-cell-mediated responses can be measured (palmer and friedmann, ; albers et al., ; friedmann, ) . harper -smith et al. ( ) found that participating in prolonged exercise ( h of running) compared with seated rest prior to cutaneous sensitisation with a novel antigen (diphenylcyclopropenone, dpcp) reduced the recall (elicitation) of the immune response (skin erythema and edema) to that same antigen four weeks later. when participants completed the same prolonged exercise immediately prior to the elicitation phase (recall at four weeks), there was also a reduction in skin responses to dpcp compared to resting controls. however, the magnitude of reduction in response to dpcp at this phase (elicitation, %) was considerably lower than the reduction observed when prolonged exercise was completed prior to dpcp sensitisation (induction, %). this suggests that the primary immune response to antigens (apc and induction of t cell specific memory) is more susceptible to the perturbations induced by prolonged exercise than responses to recall (previously exposed) antigens, which may have relevance regarding the susceptibility of athletes to new/ novel compared to nonnovel pathogens. a number of follow-up studies using the chs model have since demonstrated that this is a controllable, reproducible, sensitive, and valid in vivo marker of exercise-induced immunodepression (diment et al., ; davison et al., ; jones et al., ) . the study by diment et al. ( ) also demonstrated no effect of the same exercise ( h of treadmill running) on the skin's response to the irritant croton oil, which provided strong evidence that the observed decrease in in vivo immunity assessed by this chs method of immune induction to dpcp is an antigen-specific, t-cell-mediated, response. simultaneously to the aforementioned assessment of cell-mediated immunity, bruunsgaard et al. ( ) also vaccinated all groups with previously encountered antigens that would stimulate b cell function. there were no differences between the prolonged exercise and control group in the ability of b cells to generate antibody responses to these recall antigens in the days following vaccination. this may provide further supporting evidence to proposals that primary immune responses are more susceptible to the effects of exercise or it may suggest that perturbations are more pronounced in the immediate period following prolonged exercise ( À h, i.e., proposed open window). the response of circulating igs to prolonged exercise has not been extensively researched with the studies that have investigated the predominant igs in blood (a, g, m) reporting conflicted findings (decreases, increases, and no change) peters et al., ; walsh et al., b; bishop, ) . this may not be surprising given the small proportions of b cells ( %À %) in the circulating lymphocyte pool and little redistribution of this subset following exercise. for the investigation of activated b cells and ig responses to exercise, studies have focused primarily on other immune compartments which may have greater relevance to urti (i.e., mucosa). the role of moderate exercise on in vivo immune markers has received relatively little attention in the literature. the majority of relevant research has studied the response to vaccination. however, most of these studies were designed to assess the utility of exercise as a vaccine adjuvant (e.g., in individuals who are less responsive to vaccination such as the elderly or those with certain chronic diseases) rather than exploring the effects of exercise on in vivo immunity per se. such studies can provide some insight into the benefits of exercise to immunity but the extent to which this can be translated to other populations requires much further study. long-term physical activity in such populations (pre and post vaccination) has been shown to improve antibody responses and immunogenicity to influenza, pneumococcus and meningococus vaccination (kohut et al., keylock et al., ; yang et al., ; grant et al., ; woods et al., ; bachi et al, ) , which support the beneficial effects of moderate exercise on in vivo immunity. however, there are a number of potential confounding factors (such as social interaction and support, psychological factors etc, from the exercise interventions) that may contribute to the benefit but are difficult to control in such studies. the acute effects of various types of exercise has also been investigated in a number of studies (edwards et al., (edwards et al., , campbell et al., ; edwards et al., edwards et al., , long et al., ; ranadive et al., ) . generally, these studies have been conducted with younger rather than older subjects and in most of these cases no benefits are observed. this could be because younger populations generally mount a strong (optimal) response to vaccination anyway, leaving little scope for benefit. as such, in this context, response to vaccination may not be the best model of in vivo immune response since it does not completely mimic a real infective challenge (i.e., as the virus is usually not live, or is attenuated). nevertheless, more work is needed in this area and other in vivo markers are desirable. athletes and individuals involved in heavy training programmes and/or prolonged bouts of exercise appear to have an increased risk of contracting uri. this is likely related to regular acute (and possibly chronic) periods of exercise-induced immunodepression (open windows). regular moderate exercise, on the other hand, appears to have the opposite effect and reduces infection risk. although in vitro and ex vivo measures and markers can contribute mechanistic information to the understanding of these relationships, they should not be considered as a substitute for clinically relevant/in vivo immune markers as most in vitro/ex vivo markers do not predict infection risk well. this highlights the importance of studying clinically relevant outcomes and the complex nature of the immune system. hence, future research in this area requires more "clinically" relevant outcomes, and in vivo assessment of immune function. changes in spontaneous and lps-induced ex vivo cytokine production and mrna expression in male and female athletes following prolonged exhaustive exercise effect of months of exercise training on salivary secretory iga levels in elderly subjects monitoring immune modulation by nutrition in the general population: identifying and substantiating effects on human health markers to measure immunomodulation in human nutrition intervention studies effects of exercise intensity on salivary antimicrobial proteins and markers of stress in active men stimulating whole saliva affects the response of antimicrobial proteins to exercise. scand intrasplenic blood cell kinetics in man before and after brief maximal exercise neutrophil function: from mechanisms to disease mobilization of gammadelta t lymphocytes in response to psychological stress, exercise, and beta-agonist infusion review: the physiology of saliva and transfer of drugs into saliva inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis acute psychological stress alerts the adaptive immune response: stress-induced mobilization of effector t cells leukokinetic studies. iv. the total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects immunological status of competitive cyclists before and after the training season epithelial antimicrobial peptides in host defense against infection augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide dendritic cells and the control of immunity meditation or exercise for preventing acute respiratory infection: a randomized controlled trial cytomegalovirus seroprevalence in the united states: the national health and nutrition examination surveys the role of the spleen in the leucocytosis of exercise: consequences for physiology and pathophysiology moderate and exhaustive endurance exercise influences the interferon-gamma levels in whole-blood culture supernatants salivary flow and alphaamylase: collection technique, duration, and oral fluid type novel rinse assay for the quantification of oral neutrophils and the monitoring of chronic periodontal disease beta -adrenergic stimulation causes detachment of natural killer cells from cultured endothelium purification and functional evaluation of mature neutrophils from human bone marrow functional analysis of the marginating pool of human polymorphonuclear leukocytes airway inflammation and upper respiratory tract infection in athletes: is there a link? consensus statement immunonutrition and exercise innate immunity: an overview innate immune sensing and its roots: the story of endotoxin acute and chronic effects of exercise on markers of mucosal immunity carbohydrate and fluid intake affect the saliva flow rate and iga response to cycling effect of prolonged exercise and carbohydrate on total neutrophil elastase content lymphocyte responses to influenza and tetanus toxoid in vitro following intensive exercise and carbohydrate ingestion on consecutive days human t lymphocyte migration towards the supernatants of human rhinovirus infected airway epithelial cells: influence of exercise and carbohydrate intake effects of submaximal cycling and long-term endurance training on neutrophil phagocytic activity in middle aged men the effect of exercising to exhaustion at different intensities on saliva immunoglobulin a, protein and electrolyte secretion what are the consequences of the disappearing human microbiota? peptide antibiotics and their role in innate immunity neutrophils, from marrow to microbes granules of the human neutrophilic polymorphonuclear leukocyte stress and secretory immunity viral and bacterial interactions in the upper respiratory tract the respiratory health of swimmers comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction regional specialization in the mucosal immune system: what happens in the microcompartments? the role of complement c opsonization, c a receptor, and cd in e. coli-induced up-regulation of granulocyte and monocyte cd b/cd cr , phagocytosis, and oxidative burst in human whole blood neutrophil extracellular traps kill bacteria in vivo cell-mediated immunity and vaccination response following prolonged, intense exercise acute exercise mobilises cd t lymphocytes exhibiting an effector-memory phenotype the effects of vaccine timing on the efficacy of an acute eccentric exercise intervention on the immune response to an influenza vaccine in young adults exercise and resistance to infection carbohydrate supplementation and immune responses after acute exhaustive resistance exercise exercise suppresses macrophage antigen presentation intracellular mechanisms responsible for exercise-induced suppression of macrophage antigen presentation natural killer cells, viruses and cancer modulation of dendritic cells by endurance training a competitive marathon race decreases neutrophil functions in athletes the effect of exercise on salivary iga levels and the incidence of upper respiratory tract infections in postmenopausal women psychological stress and susceptibility to the common cold sleep habits and susceptibility to the common cold monitoring changes in performance, physiology, biochemistry, and psychology during overreaching and recovery in triathletes hcap- , a cathelin/pro-bactenecin-like protein of human neutrophil specific granules valtrex therapy for epsteinÀbarr virus reactivation and upper respiratory symptoms in elite runners clinical and laboratory evaluation of upper respiratory symptoms in elite athletes respiratory symptoms and inflammatory responses to difflam throat-spray intervention in half-marathon runners: a randomised controlled trial biology and disease associations of epsteinÀbarr virus mucosal immunity and illness incidence in elite rugby union players across a season a primer on cytokines: sources, receptors, effects, and inducers humoral immunodeficiency in recurrent upper respiratory tract infections. some basic, clinical and therapeutic features oral antimicrobial peptides and biological control of caries priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin b results from the recruitment of previously non-responsive cells exercise, alveolar macrophage function, and susceptibility to respiratory infection innate immune responses to a single session of sprint interval training influence of acute vitamin c and/or carbohydrate ingestion on hormonal, cytokine, and immune responses to prolonged exercise the effect of weeks vitamin c supplementation on immunoendocrine responses to . h cycling exercise in man the effects of acute vitamin c supplementation on cortisol, interleukin- , and neutrophil responses to prolonged cycling exercise bovine colostrum supplementation attenuates the decrease of salivary lysozyme and enhances the recovery of neutrophil function after prolonged exercise antioxidant supplementation and immunoendocrine responses to prolonged exercise salivary antimicrobial peptides ll- and alpha-defensins hnp - , antimicrobial and iga responses to prolonged exercise carbohydrate supplementation does not blunt the prolonged exercise-induced reduction of in vivo immunity salivary flow patterns and the health of hard and soft oral tissues human antimicrobial peptides: defensins, cathelicidins and histatins the human oral microbiome stress-induced augmentation of immune functionthe role of stress hormones, leukocyte trafficking, and cytokines exercise intensity and duration effects on selective mobilization of cytotoxic leukocytes by epinephrine moderate exercise enhances expression of siga in mouse ileum understanding the symptoms of the common cold and influenza acute stress exposure prior to influenza vaccination enhances antibody response in women eccentric exercise as an adjuvant to influenza vaccination in humans meningococcal a vaccination response is enhanced by acute stress in men exercise intensity does not influence the efficacy of eccentric exercise as a behavioural adjuvant to vaccination acute exercise enhancement of pneumococcal vaccination response: a randomised controlled trial of weaker and stronger immune response infectious episodes before and after a marathon race sports injuries and illnesses during the winter olympic games sports injuries and illnesses during the london summer olympic games t lymphocytes salivary genomics, transcriptomics and proteomics: the emerging concept of the oral ecosystem and their use in the early diagnosis of cancer and other diseases salivary defense proteins: their network and role in innate and acquired oral immunity the acute phase response and exercise: the ultramarathon as prototype exercise the ins and outs of ebv infection exercise alters the immune response to equine influenza virus and increases susceptibility to infection monitoring training in athletes with reference to overtraining syndrome leukocytosis of exercise: role of cardiac output and catecholamines influence of training loads on patterns of illness in elite distance runners the relationships between exposure dose and response in induction and elicitation of contact hypersensitivity in humans cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities novel cell death program leads to neutrophil extracellular traps circulating leucocyte and lymphocyte subpopulations before and after intensive endurance exercise to exhaustion suppressed pma-induced oxidative burst and unimpaired phagocytosis of circulating granulocytes one week after a long endurance exercise danger signals: sos to the immune system natural killer cells: modulation by intensity and duration of exercise defensins: antimicrobial peptides of innate immunity salivary antimicrobial protein response to prolonged running the differential impact of training stress and final examination stress on herpesvirus latency at the united states military academy at west point mucosal immunity and respiratory illness in elite athletes immune system adaptation in elite athletes immune function in sport and exercise special feature for the olympics: effects of exercise on the immune system: exercise effects on mucosal immunity uri in athletes: are mucosal immunity and cytokine responses key risk factors? epsteinÀbarr virus reactivation and upper-respiratory illness in elite swimmers daily probiotic's lactobacillus casei shirota reduction of infection incidence in athletes respiratory infection risk in athletes: association with antigen-stimulated il- production and salivary iga secretion. scand the bases expert statement on exercise, immunity, and infection pattern recognition receptors: doubling up for the innate immune response focus on methodology: salivary bioscience and research on adolescence: an integrated perspective cardiovascular exercise intervention improves the primary antibody response to keyhole limpet hemocyanin klh in previously sedentary older adults exercise-induced changes to in vitro t-lymphocyte mitogen responses using cfse exercise and tlymphocyte function: a comparison of proliferation in pbmc and nk cell-depleted pbmc culture effect of moderate exercise on the severity of clinical signs associated with influenza virus infection in horses biosynthesis, processing and sorting of neutrophil proteins: insight into neutrophil granule development pmn cell counts and phagocytic activity of highly trained athletes depend on training period tear fluid siga as a noninvasive biomarker of mucosal immunity and common cold risk measuring salivary cortisol in studies of child development: watch out-what goes in may not come out of saliva collection devices exercise-induced stress inhibits both the induction and elicitation phases of in vivo t-cell-mediated immune responses in humans respiratory syncytial virus in healthy adults: the cost of a cold influence of vitamin d status on respiratory infection incidence and immune function during months of winter training in endurance sport athletes exercise and the incidence of upper respiratory tract infections intracellular control of human neutrophil secretion. i. c a-induced stimulus-specific desensitization and the effects of cytochalasin carbohydrate supplementation and the lymphocyte proliferative response to long endurance running human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool: function and granule protein content exercise and the immune system: a model of the stress response? elevated epsteinÀbarr virus loads and lower antibody titers in competitive athletes leukocyte traffic in the lung effects of an exercise challenge on mobilization and surface marker expression of monocyte subsets in individuals with normal vs. elevated blood pressure challenge studies of human volunteers: ethical issues processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach toll-like receptor control of the adaptive immune responses role of j chain in secretory immunoglobulin formation the j chain is essential for polymeric ig receptor-mediated epithelial transport of iga whats new in lysozyme research-always a model system, today as yesterday effects of bovine colostrum supplementation on upper respiratory illness in active males the effects of bovine colostrum supplementation on in vivo immunity following prolonged exercise: a randomised controlled trial current understanding of cytomegalovirus infection in immunocompetent individuals early immune events in the induction of allergic contact dermatitis evidence that the effect of physical exercise on nk cell activity is mediated by epinephrine immunity to intracellular microbial pathogens higher antibody, but not cell-mediated, responses to vaccination in high physically fit elderly the evolution and genetics of innate immunity effect of moderate exercise on salivary immunoglobulin a and infection risk in humans immunopathogenesis of delayed-type hypersensitivity reversing age-associated immunosenescence via exercise exercise and psychosocial factors modulate immunity to influenza vaccine in elderly individuals moderate exercise improves antibody response to influenza immunization in older adults quantitative immunohistochemistry of immunoglobulin-and j-chain-producing cells in human parotid and submandibular salivary glands interrelationship between physical activity, symptomatology of upper respiratory tract infections, and depression in elderly people the symptomatology of upper respiratory tract infections and exercise in elderly people physical activity and upper respiratory tract infections exercise-induced redistribution of t lymphocytes is regulated by adrenergic mechanisms current aspects of mucosal immunology and its influence by nutrition the acute phase response: general aspects. bailliere's nontypeable haemophilus influenzae: challenges in developing a vaccine human blood neutrophil responses to prolonged exercise with and without a thermal clamp the iga mucosal immune system effect of acute exhaustive exercise and a -day intensified period of intensified training on immune function in cyclists effects of acute exhaustive exercise and chronic exercise training on type and type t lymphocytes effect of prolonged exercise and carbohydrate ingestion on type and type t lymphocyte distribution and intracellular cytokine production in humans the physiological regulation of toll-like receptor expression and function in humans airway hyperresponsiveness in elite athletes regulation of t cell immunity by dendritic cells leucocytosis after violent exercise b lymphocytes: how they develop and function lactoferrin and host defence: an overview of its immuno-modulating and antiinflammatory properties the effects of carbohydrate supplementation during the second of two prolonged cycling bouts on immunoendocrine responses effect of a periodized exercise training and active recovery program on antitumor activity and development of dendritic cells increased risistance of immunoglobulin a dimers to proteolytic degradation after binding of secretory component human macrophage host defense against mycobacterium tuberculosis vaccination response following aerobic exercise: can a brisk walk enhance antibody response to pneumococcal and influenza vaccinations? moderate exercise protects mice from death due to influenza virus phagocytic functions of salivary neutrophils in oral mucous membrane diseases airway inflammation, bronchial hyperresponsiveness and asthma in elite ice hockey players immunoglobulin, antibody, and exercise mucosal secretory immune system responses to exercise of varying intensity and during overtraining temporal relationship between exercise-induced decreases in salivary iga concentration and subsequent appearance of upper respiratory illness in elite athletes homeland security: iga immunity at the frontiers of the body viruses and bacteria in the etiology of the common cold cutaneous delayed-type hypersensitivity in patients with atopic dermatitis susceptibility to infections in elite athletes: the s-curve. scand neutrophils in the activation and regulation of innate and adaptive immunity exercise and respiratory tract viral infections effects of aerobic conditioning on salivary iga and plasma iga, igg and igm in older men and women moderate to vigorous physical activity and risk of upper-respiratory tract infection a self-reported questionnaire for quantifying illness symptoms in elite athletes rest-interval length affects leukocyte levels during heavy resistance exercise the leucocytosis of exercise. a review and model brief exercise induces an immediate and a delayed leucocytosis carbohydrate intake during endurance exercise increases natural killer cell responsiveness to il- physical activity status, but not age, influences inflammatory biomarkers and toll-like receptor influence of ultra-endurance exercise on immunoglobulin isotypes and subclasses toll-like receptors and innate immunity epsteinÀbarr virus reactivation associated with diminished cellmediated immunity in antarctic expeditioners reactivation and shedding of cytomegalovirus in astronauts during spaceflight dendritic cells: specialized and regulated antigen processing machines myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity functional foods/ingredients and oral mucosal diseases decoupling of intracellular calcium signaling in granulocytes after exhaustive exercise lymphocyte function and cytokine production during incremental exercise in active and sedentary males and females effect of long-distance running on polymorphonuclear neutrophil phagocytic function of the upper airways effects of moderate exercise and oat beta-glucan on lung tumor metastases and macrophage antitumor cytotoxicity exercise stress increases susceptibility to influenza infection isolation of cdna encoding guinea pig neutrophil cationic antibacterial polypeptide of kda cap and evaluation of cap mrna expression during neutrophil maturation evaluation of the expression of human cap gene during neutrophil maturation in the bone marrow synergistic actions of antibacterial neutrophil defensins and cathelicidins neutrophils and immunity: challenges and opportunities methods for collecting saliva a comparison of whole mouth resting and stimulated salivary measurement procedures the effects of acute moderate exercise on lymphocyte function and serum immunoglobulin levels salivary iga as a risk factor for upper respiratory infections in elite professional athletes splenectomy impairs lymphocytosis during maximal exercise exercise immunology: nutritional countermeasures clinical implications of exercise immunology the effects of acute and chronic exercise on immunoglobulins nutritional strategies to counter stress to the immune system in athletes, with special reference to football the effects of moderate exercise training on natural killer cells and acute upper respiratory tract infections effects of high-vs moderate-intensity exercise on natural killer cell activity lymphocyte proliferative response to . hours of running immune response to exercise training and/or energy restriction in obese women change in salivary iga following a competitive marathon race immune and oxidative changes during and following the western states endurance run immune response to a -minute walk tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene b , and plateletactivating factor. dissociation from priming in human polymorphonuclear neutrophils abitual exercise did not affect the balance of type and type cytokines in elderly people the effects of acute exercise-induced cortisol on ccr expression on human monocytes the influence of prolonged cycling on monocyte toll-like receptor and expression in healthy men salivary immunologyoglobulin a response at rest and after exercise following a h period of fluid and/or energy restriction ultraviolet radiation causes less immunosuppression in patients with polymorphic light eruption than in controls influence of vitamin c supplementation on oxidative and salivary iga changes following an ultramarathon effects of intensified training and taper on immune function nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation exercise-induced alterations in neutrophil degranulation and respiratory burst activity: possible mechanisms of action nk cell response to physical activity: possible mechanisms of action how physical exercise influences the establishment of infections exercise and the immune system: regulation, integration, and adaptation indomethacin in vitro and in vivo abolishes post-exercise suppression of natural killer cell activity in peripheral blood salivary lysozyme: a noninvasive marker for the study of the effects of stress on natural immunity exercise, immunology and upper respiratory tract infections postrace upper respiratory tract 'infections' in ultramarathoners-infection, allergy or inflammation? s. afr ultramarathon running and upper respiratory tract infections. an epidemiological survey vitamin c supplementation reduces the incidence of postrace symptoms of upper-respiratory-tract infection in ultramarathon runners upper respiratory tract infection symptoms in ultramarathon runners not related to immunoglobulin status airway inflammation in elite swimmers behaviorally assessed sleep and susceptibility to the common cold chewing stimulates secretion of human salivary secretory immunoglobulin a regulation of salivary gland function by autonomic nerves salivary secretion of immunoglobulin a by submandibular glands in response to autonomimetic infusions in anaesthetised rats deficiency of antibacterial peptides in patients with morbus kostmann: an observation study regulation of neutrophil function during exercise effects of intensive exercise training on immunity in athletes characterising the individual performance responses to mild illness in international swimmers antimicrobial peptides: natural effectors of the innate immune system acute impact of submaximal resistance exercise on immunological and hormonal parameters in young men effect of acute aerobic exercise on vaccine efficacy in older adults clinical investigation of athletes with persistent fatigue and/or recurrent infections viral illnesses and sports performance medicine at the sydney olympic games: the new zealand health team the effect of an acute period of intense interval training on human neutrophil function and plasma glutamine in endurancetrained male runners effects of exercise intensity, duration and recovery on in vitro neutrophil function in male athletes antioxidant supplementation enhances neutrophil oxidative burst in trained runners following prolonged exercise prevalence of allergy and upper respiratory tract symptoms in runners of the london marathon salivary alpha-amylase: role in dental plaque and caries formation fusafungine reduces symptoms of upper respiratory tract infections urti in runners after a km race acquisition of lymphokine-producing phenotype by cd t cells strenuous exercise and immunological changes: a multiple-timepoint analysis of leukocyte subsets, cd /cd ratio, immunoglobulin production and nk cell response sepsis and mechanisms of inflammatory response: is exercise a good model? cytokine responses to physical activity, with particular reference to il- : sources, actions, and clinical implications adhesion molecules, catecholamines and leucocyte redistribution during and following exercise development of the discipline of exercise immunology personal health benefits of masters athletics competition effect of moderate exercise training on t-helper cell subpopulations in elderly people establishment of radioimmunoassay for human neutrophil peptides and their increases in plasma and neutrophil in infection exercise and infection ageing, persistent viral infections, and immunosenescence: can exercise "make space the effects of exercise on blood leukocyte numbers the effects of intensive, moderate and downhill treadmill running on human blood lymphocytes expressing the adhesion/activation molecules cd icam- , cd beta integrin and cd high-intensity exercise elicits the mobilization of senescent t lymphocytes into the peripheral blood compartment in human subjects senescent t-lymphocytes are mobilised into the peripheral blood compartment in young and older humans after exhaustive exercise toll-like receptor expression on classic and pro-inflammatory blood monocytes after acute exercise in humans synergistic and additive killing by antimicrobial factors found in human airway surface liquid effects of exercise on s-iga and urs in postmenopausal women black-pigmented bacteroides species, capnocytophaga species, and actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction neutrophils, host defense, and inflammation: a double-edged sword exercise, training, and neutrophil function moderate exercise triggers both priming and activation of neutrophil subpopulations cytokine release and its modulation by dexamethasone in whole blood following exercise the human antibacterial cathelicidin, hcap- , is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils effects of unaccustomed and accustomed exercise on the immune response in runners incidence, etiology, and symptomatology of upper respiratory illness in elite athletes seroprevalence of cytomegalovirus infection in the united states effect of adrenergic blockade on lymphocyte cytokine production at rest and during exercise strenuous exercise decreases the percentage of type t cells in the circulation selective mobilization of cd cd monocytes by exercise elevated stress hormone levels relate to epsteinÀbarr virus reactivation in astronauts the role of secretory antibodies in infection immunity neutrophil kinetics in health and disease iga synthesis: a form of functional immune adaptation extending beyond gut endurance exercise causes interaction among stress hormones, cytokines, neutrophil dynamics, and muscle damage impact of a competitive marathon race on systemic cytokine and neutrophil responses training-related and competition-related risk factors for respiratory tract and gastrointestinal infections in elite cross-country skiers toll-like receptors in innate immunity cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of nadph oxidase activation decreased excretion of antimicrobial proteins and peptides in saliva of patients with oral candidiasis salivary antimicrobial peptide expression and dental caries experience in children neuroendocrine regulation of salivary iga synthesis and secretion: implications for oral health adenosine receptor occupancy suppresses chemoattractantinduced phospholipase d activity by diminishing membrane recruitment of small gtpases the concentration of the c-type lectin, mannan-binding protein, in human plasma increases during an acute phase response regulatory interactions between muscle and the immune system during muscle regeneration human natural killer cell subsets and acute exercise: a brief review antarctic isolation: immune and viral studies the antiinflammatory interactions of epinephrine with human neutrophils in vitro are achieved by cyclic amp-mediated accelerated resequestration of cytosolic calcium immune parameters in athletes before and after strenuous exercise role of corticosterone in modulation of eicosanoid biosynthesis and antiinflammatory activity by -lipoxygenase -lo and cyclooxygenase co inhibitors antimicrobial peptides and proteins in the innate defense of the airway surface the effect of light, moderate and severe bicycle exercise on lymphocyte subsets, natural and lymphokine activated killer cells, lymphocyte proliferative response and interleukin production studies on the structural and conformational basis for the relative resistance of serum and secretory immunoglobulin a to proteolysis changes in salivary antimicrobial peptides, immunoglobulin a and cortisol after prolonged strenuous exercise cytokines in the pathogenesis of influenza potential markers of heavy training in highly trained distance runners infections in patients with febrile neutropenia: epidemiology, microbiology, and risk stratification lactoferricin derived from milk protein lactoferrin the effects of high-intensity intermittent exercise on saliva iga, total protein and alpha-amylase salivary iga response to prolonged exercise in a cold environment in trained cyclists saliva flow rate, total protein concentration and osmolality as potential markers of whole body hydration status during progressive acute dehydration in humans position statement. part two: maintaining immune health position statement. part one: immune function and exercise multifunctional roles of lactoferrin: a critical overview upper respiratory tract bacterial carriage in aboriginal and non-aboriginal children in a semi-arid area of western australia tissue destruction by neutrophils antimicrobial peptides and proteins, exercise and innate mucosal immunity the effect of exercise on innate mucosal immunity sites of rhinovirus recovery after point inoculation of the upper airway effects of exercise on the macrophage mhc ii response to inflammation effects of maximal exercise on natural killer nk cell cytotoxicity and responsiveness to interferon-alpha in the young and old exercise and cellular innate immune function special feature for the olympics: effects of exercise on the immune system: exerciseinduced modulation of macrophage function cardiovascular exercise training extends influenza vaccine seroprotection in sedentary older adults: the immune function intervention trial virus activation and immune function during intense training in rugby football players effects of a taiji and qigong intervention on the antibody response to influenza vaccine in older adults effects of moderate and high intensity exercise on t /t balance key: cord- -r i d v authors: dimitrova-shumkovska, jasmina; krstanoski, ljupcho; veenman, leo title: potential beneficial actions of fucoidan in brain and liver injury, disease, and intoxication—potential implication of sirtuins date: - - journal: mar drugs doi: . /md sha: doc_id: cord_uid: r i d v increased interest in natural antioxidants has brought to light the fucoidans (sulfated polysaccharides present in brown marine algae) as highly valued nutrients as well as effective and safe therapeutics against several diseases. based on their satisfactory in vitro antioxidant potency, researchers have identified this molecule as an efficient remedy for neuropathological as well as metabolic disorders. some of this therapeutic activity is accomplished by upregulation of cytoprotective molecular pathways capable of restoring the enzymatic antioxidant activity and normal mitochondrial functions. sirtuin- has been discovered as a key player for achieving the neuroprotective role of fucoidan by managing these pathways, whose ultimate goal is retrieving the entirety of the antioxidant response and preventing apoptosis of neurons, thereby averting neurodegeneration and brain injuries. another pathway whereby fucoidan exerts neuroprotective capabilities is by interactions with p-selectin on endothelial cells, thereby preventing macrophages from entering the brain proper. furthermore, beneficial influences of fucoidan have been established in hepatocytes after xenobiotic induced liver injury by decreasing transaminase leakage and autophagy as well as obtaining optimal levels of intracellular fiber, which ultimately prevents fibrosis. the hepatoprotective role of this marine polysaccharide also includes a sirtuin, namely sirtuin- overexpression, which alleviates obesity and insulin resistance through suppression of hyperglycemia, reducing inflammation and stimulation of enzymatic antioxidant response. while fucoidan is very effective in animal models for brain injury and neuronal degeneration, in general, it is accepted that fucoidan shows somewhat limited potency in liver. thus far, it has been used in large doses for treatment of acute liver injuries. thus, it appears that further optimization of fucoidan derivatives may establish enhanced versatility for treatments of various disorders, in addition to brain injury and disease. the increasing prevalence of multiple chronic diseases has triggered massive exploration of novel pharmaceuticals for their proper management [ , ] . however, therapies are often accompanied by side effects. given that the impaired homeostasis of reactive oxygen species (ros) is often the source for these illnesses, extensive efforts are being made to produce synthetic antioxidants that are beneficial and very encouragingly, fucoidan has shown satisfactory results concerning in vitro scavenging and reducing and antioxidant potentials [ ] . since the areal of distribution of the brown algae f. vesiculosus, u. pinnatifida, or l. japonica is restricted to various separated regions distributed worldwide, efforts are being made to uncover additional natural sources (algae species) suitable for exploitation, including extraction of satisfactory amounts of biologically active polysaccharides and polyphenols from the species in question [ ] . a search is on for "optimal fucoidan sources," with the idea to expand the present pool of commercially available supplements, and add potent prophylactics [ ] . differences in the antioxidant potency, and sulfate or polyphenol content, of the various fucoidan extracts can depend on the type and/or quality of algal species used, the different extraction methods, methods of fractionation of extracts, and purity and yield of the polysaccharides attained. sulfate content vs. molecular weight and uronic acid percentage of the isolated polysaccharides also contribute to alteration in their antioxidant profiles [ , ] . these reported observations emphasize the importance for the analysis of the in vitro antioxidant profiles of commercially available seaweeds and comparing them with fucoidan supplements. in general, it is accepted that the seaweed fucoidan is a modest . diphenyl- -picrylhydrazyl (dpph) inhibitor, when compared to radical scavenging rates of ascorbic acid or other synthetic antioxidants [ ] . studies have indicated that a high sulfate content does not necessarily invoke high dpph quenching and established the relevance of the monosaccharide distribution as a contributing factor [ , ] . high inhibitory concentrations (ic ) of fucoidan supplements manufactured by marinova (supafuco), together with crude fucoidan extracted from purple laver (porphyra sp.) and nori (p. tennera) (ranging from . ± . mg/ml for supafuco and . ± . and . ± . mg/ml for purple laver and nori; summarized in table ), also endorsed these claims (own unpublished data). high polyphenol and chelating properties of purple laver significantly contribute to the dpph neutralization rates (own unpublished data). fortunately, according to the food and drug administration (fda), fucoidan has been recognized as safe ingredient with no side effects; the treatment should be able to intensify its primary antioxidant response in vivo by application of large doses of it [ ] . there is consensus that fucoidan also possesses nitric oxide (no) scavenging capabilities, but little information is available. for example, it was reported that fucoidan from sigma aldrich exhibits high no scavenging potential [ ] . in another study, fucoidan isolated from s. polycystum, showed complete neutralization of no free radical at concentration of mg/ml [ ] . a recent study by us, comparing the in vitro antioxidant profiles of isolated fucoidan from commercially obtained algae, versus fucoidan supplements, acknowledged that commercial dietary supplements were relatively potent in inhibiting difenylpicrylhydrazine (dpph) radicals and no radical scavengers (own unpublished data). in particular, pills containing fucoidan manufactured by marinova ltd. showed approximately two times and times lower ic values than the algae of purple laver and wakame, respectively (own unpublished data, summarized in table ). furthermore, while lower than the supplements from marinova, daiso fucoidan supplements still present higher antioxidant activity than extracted polysaharides, independent of low sulfate content. in parallel, isolated fucoidans from the algae shown significantly higher radical scavenging activity for superoxide radicals (ic values of . ± . for wakame and . ± . mg/ml, reached for nori algae, summarized in table ), which demonstrated that the isolated fucoidan from commercially procured algae are relative efficient scavengers of moderately potent oxidants (own unpublished data). nonetheless, it was found that fucoidan supplement from marinova ltd. showed the highest scavenging rates for this super oxide radical (ic value . ± . ), with approximately two times lower ic value than both of the abovementioned algal species (wakame and nori). interestingly, investigations of qu et al. confirmed higher superoxide scavenging activity of crude fucoidans obtained from l. japonica and e. maxima than those from ascorbic acid used as standard [ ] . the effectiveness of marinova fucoidan may simply relate to its high fucoidan content ( %) ( table ) . thus, it appears that anti-oxidant activities of fucoidan and their derivatives can be further optimized. the established positive correlation between the superoxide (o − ) scavenging activity and the sulfate content of the polysaccharides of all the investigated specimens (table , own unpublished data) , corroborated other studies [ , ] . high fucoidan contents of commercial supplements followed by wakame, could be crucial for their high (o − ) scavenging rates (own unpublished data). adequate hydroxyl radical (oh − ) scavenging abilities were also published regarding marine algae polysaccharides, appointing that multiple factors such as the reducing power, sulfate or the polysaccharide content could contribute to the oh − free radical inhibition [ , ] . taken together, the in vitro antioxidant potentials encouraged pharmacologic studies about fucoidan's prophylactic properties. in the light of the positive, curative effects of fucoidans on various form of brain disorders it is becoming interesting to gain further insights in the behavior of fucoidans in the organism. relevant for clinical application, few studies have been done to determine the efficiency of uptake of fucoidan via the digestive system. it can be assumed that the complexity of the fucoidan structure also affects its permeability and absorption rates through the gastrointestinal tract, mostly due to the limited enzymatic potentials of the human organism. in accordance with this hypothesis, earlier studies considered the sulfated polysaccharide molecule with high molecular weight and rich content of dietary fiber as "indigestible" given the partial in vitro degradation of the chemical constituents of different brown algae such as laminarans to monosaccharide units, and complete resistance to digestion of sulfated fucans and alginates by human fecal bacteria [ ] . on the other hand, elisa quantification of fucoidan levels in blood circulation, as a more reliable method for determining the digestibility of this molecule, showed that after a -day dosing of galactofucan capsules from u. pinnatifida to human individuals, only . % persisted in plasma [ ] . high urinary levels of fucoidan and low permeability coefficient concerning its transport across caco- cells reported by newer studies also support the findings about low uptake of fucoidan by the gastrointestinal system [ , ] . with regards to the mechanism of absorption of fucoidan, a study in suggested the involvement of the round mononuclear cells of the jejunum given the higher concentration of fucoidan observed by immunochistochemistry methods in rats fed standard chow containing % fucoidan for one or two weeks. increased accumulation of fucoidan in the sinusoidal non-parenchymal cells together with the kupffer cells also indicated these as crucial players in the internalization of fucoidan in the liver [ ] . n-butyl-n-( -hydroxybutyl) nitrosamine (bbn) was found to enhance fucoidan uptake in the small intestine and liver [ ] . since its intracellular transport hardly can be completed by simple diffusion, it is highly possible that transporters are involved in its cellular influx. researchers predict that the sodium glucose transporter (sglt ) and/or the glucose transporter (glut ) might be involved in fucoidan uptake over the blood-brain barrier (bbb), given their affinity to polysaccharides and phenols with sugar substitutes [ , , ] . the latest research has confirmed that fucoidan enters in caco- cells through clathrin-mediated endocytosis because its influx is modified by chemical inhibitors of this process [ ] . to stipulate very briefly, fucoidan appears to interact with selectin on endothelial cells, preventing leukocytes from entering the brain from the blood vessels via the blood brain barrier (bbb), i.e., closing the bbb. this of course reduces inflammatory responses inside the brain tissue proper. intracellularly, fucoidan interacts with sirtuin (sirt ) in brain cells, which in turn modulates mitochondria activity and cell nuclear gene expression, which, for example, reduces oxidative stress, ros generation, and mitochondrial apoptosis (via mitochondrial activity modulation), and inflammatory responses, regeneration, angiogenesis, and wound healing (via cell nuclear gene expression modulation). this will be discussed in the following sections in more detail below. fucoidan binds to p-selectin with high affinity and exerts antagonism of selective function [ ] . p-selectin is found on the cell surface of endothelial cells of the bbb. it binds to glycoprotein on the cell surface of leukocytes; p-selectin is involved in rolling and arresting leukocytes on the endothelium prior to leukocyte migration into the extravascular space [ ] [ ] [ ] [ ] . thus, the permeability of the bbb for leukocytes can be affected by p-selectin; for example, enhanced levels of p-selectin leads to a higher bbb mar. drugs , , of permeability, while decreased levels of p-selectin leads to lower permeability of the bbb. thus, briefly, by binding to p-selectin on endothelial cells of the bbb, fucoidan can inhibit the entry of leukocytes into the brain proper, and thereby, reduce the inflammatory response [ ] . another major pathway for fucoidan to ameliorate brain damage due to injury and disease appears to be its interaction with sirt ( figure ). sirt is a protein that in humans is encoded by the sirt gene (sirtuin (silent mating type information regulation homolog) (s. cerevisiae)) [ ] . sirt is a member of the mammalian sirtuin family of proteins. sirt exhibits nad+ dependent deacetylase activity. the human sirtuins present an interesting range of molecular functions and have emerged as important proteins in aging, stress resistance, and metabolic regulation. in addition to protein deacetylation, studies have shown that the human sirtuins may also function as intracellular regulatory proteins including mono adp ribosyltransferase activity. inflammatory response [ ] . another major pathway for fucoidan to ameliorate brain damage due to injury and disease appears to be its interaction with sirt ( figure ). sirt is a protein that in humans is encoded by the sirt gene (sirtuin (silent mating type information regulation homolog) (s. cerevisiae)) [ ] . sirt is a member of the mammalian sirtuin family of proteins. sirt exhibits nad+ dependent deacetylase activity. the human sirtuins present an interesting range of molecular functions and have emerged as important proteins in aging, stress resistance, and metabolic regulation. in addition to protein deacetylation, studies have shown that the human sirtuins may also function as intracellular regulatory proteins including mono adp ribosyltransferase activity. endogenous sirt is a soluble protein located in the mitochondrial matrix ( figure ) [ ] . overexpression of sirt in cultured cells increases respiration and decreases the production of ros. interestingly, there is a strong association between sirt alleles and longevity in males. in addition to controlling metabolism at the transcriptional level, sirtuins also directly control the activity of metabolic enzymes. the presence of the sirtuin deacetylase sirt in the mitochondrial matrix suggests the existence of lysine acetylated mitochondrial proteins. indeed, sirt deacetylates and activates the mammalian mitochondrial acetyl-coa synthetase (acecs ). furthermore, sirt and acecs are found complexed with one another, suggesting a critical role for control of acecs activity by sirt [ ] . mechanism of action of fucoidan in traumatic brain injury (tbi). fucoidan alleviates brain injury through upregulation of sirtuin, which decreases reactive oxygen species (ros) overproduction by inhibiting the mptp ( -methyl- -phenyl- , , , -tetrahydropyridine) opening, and restores normal mitochondrial function via stimulation of atp synthesis, and attenuates mitochondria-initiated apoptosis by decreasing leakage of cytochrome c from the mitochondria into the cytosol. additionally, fucoidan stimulates expression of foxo a and nrf- -are genes, thus increasing glutathione (gsh) production and mn-sod and cat activity. in addition to its reported mitochondrial function, some researchers have proposed a very small pool of active sirt exists in the cell nucleus ( figure ). this pool is reported to consist of the figure . mechanism of action of fucoidan in traumatic brain injury (tbi). fucoidan alleviates brain injury through upregulation of sirtuin, which decreases reactive oxygen species (ros) overproduction by inhibiting the mitochondrial permeability transition pore (mptp) opening, and restores normal mitochondrial function via stimulation of atp synthesis, and attenuates mitochondria-initiated apoptosis by decreasing leakage of cytochrome c from the mitochondria into the cytosol. additionally, fucoidan stimulates expression of foxo a and nrf- -are genes, thus increasing glutathione (gsh) production and mn-sod and cat activity. endogenous sirt is a soluble protein located in the mitochondrial matrix ( figure ) [ ] . overexpression of sirt in cultured cells increases respiration and decreases the production of ros. interestingly, there is a strong association between sirt alleles and longevity in males. in addition to controlling metabolism at the transcriptional level, sirtuins also directly control the activity of metabolic enzymes. the presence of the sirtuin deacetylase sirt in the mitochondrial matrix suggests the existence of lysine acetylated mitochondrial proteins. indeed, sirt deacetylates and activates the mammalian mitochondrial acetyl-coa synthetase (acecs ). furthermore, sirt and acecs are found complexed with one another, suggesting a critical role for control of acecs activity by sirt [ ] . in addition to its reported mitochondrial function, some researchers have proposed a very small pool of active sirt exists in the cell nucleus ( figure ). this pool is reported to consist of the long form of sirt and has been suggested to have histone deacetylase activity [ ] . the observation that sirt has cell nuclear activity came from a report that sirt protected cardiomyocytes from stress mediated cell death and that this effect was due to deacetylation of a nuclear factor, ku- [ ] . presently, not many brain research studies have linked fucoidan with sirt , thus, the question of potential fucoidan-sirt interactions is worthwhile to address. the most clear-cut fucoidan-sirt interaction is established in a traumatic brain injury (tbi) model, see section , here below. as said, with regard to brain damage, to date there is only one study of brain injury and neurodegeneration directly targeting the link between fucoidan and sirt . depending on its severity, tbi can lead to death and/or disabilities. outcomes of tbi are mainly characterized by disturbances in the normal physiology and structure of the brain caused by extrinsic mechanical insults due to assault or other accidents [ , ] . mechanical forces can inflict direct neuronal and astrocytic death, axonal degeneration, and vascular damage, also known as primary injuries [ ] . subsequently, the death and damage of neurons and activation of astrocytes are accompanied by additional systemic and intracranial complications, typically involving microglial activation, cytokine release, oxidative stress, and inflammation, which eventually lead towards apoptosis and necrosis [ ] . this heterogenic and diverse nature of human tbi, together with the limited success of bringing adequate treatment to the patients, further emphasizes the need for broadening the research for efficient yet safe therapeutic treatments for this condition [ ] . earlier reports about successful implementation of fucoidan treatment for cardiac dysfunction or renal ischemia reperfusion injury, disorders with similar molecular response as tbi, encouraged the exploitation of this polysaccharide component as efficient therapy [ , ] . regarding tbi itself, wang et al. conducted a thorough study concerning the effects of this novel therapeutic as prevention or treatment for brain injuries and discovered that low molecular weight fucoidan (lwmf) at doses of and mg/kg significantly reduced both cortical and hippocampal lesion volume [ ] . importantly for clinical considerations, lmwf was effective even when administered up to h after tbi. given prior to tbi, fucoidan prevented contusion injuries and tissue loss in the cortex and hippocampus, which was associated with positive outcomes of behavioral tests [ ] . this protection was associated with reduced neuronal apoptosis, as evidenced by tunel staining. moreover, administration of fucoidan significantly reduced oxidative stress as confirmed by the decreased levels of mda, -hydroxynonenal ( -hne), protein carbonyls levels, and ros levels, as well as reversed glutathione peroxidase (gpx), catalase (cat), and sod activity accompanied by restoration of mitochondrial cytochrome c levels [ ] (for illustration, see figure ). finally, and importantly to understand better the workings of fucoidan, the authors reported significantly elevated levels of sirt after tbi ( figure ). the expression of sirt was detected by rt-pcr and western blot. sirt , as mentioned above, is a protein involved in the activation of enzymatic ros quenching mechanisms. fucoidan treatment of tbi further enhanced sirt levels, which might be part of one of the regulatory pathways that triggered the restoration of gpx, sod, and cat levels [ ] . application of intracerebroventricular injection of small interfering rna (sirna) to induce knockdown of sirt partially prevented the therapeutic effects of lmwf. another study presented similar results after administration of commercial fucoxanthine in in vitro and in vivo tbi models, further revealing that these protective effects are only enabled through nrf -induced activation of the antioxidant response element (are) [ ] . in summary, it appears that fucoidan achieves its neuroprotective role via "triple impact." namely, rather than depending solely on its own chelating and radical scavenging properties, fucoidan further protects neuronal integrity by stimulating important genetic/molecular pathways capable of retrieving the entirety of the cellular antioxidant mechanisms. furthermore, as mentioned, by interacting with p-selectin, fucoidan prevents the entry of leukocytes from the blood stream into the brain tissue proper ( figure ). mar. drugs , , x of figure . effects of fucoidan on brain disease. fucoidan reduces inflammatory response in brain diseases by inhibiting microglial activation, thus resulting in significantly decreased neuronal and astrocyte degeneration due to diminishing production of pro-apoptotic agents and improving antioxidant responses of the cell. furthermore, fucoidan prevents leukocyte adhesion to the brain by blocking p-selectin. neurodegeneration is commonly defined as progressive atrophy and loss of function of neurons reflected in neurodegenerative diseases such as ad or pd [ ] . in particular, neurodegenerative disorders are characterized by a progressive decline of motor and/or cognitive functions caused by the selective degeneration and loss of neurons within the cns [ ] . while substantial progress has been made in revealing the cellular and molecular pathways causing these conditions, which mostly involve dysregulation of genetic expression indubitably accompanied by synthesis of truncated protein triggering neuroinflammatory response [ ] , they still pose a significant threat for human health in general, mainly because of their severe and aggressive symptomatology, and a pathophysiology that is still a diagnostic challenge [ ] . for treatment, or rather, for the alleviation of symptoms, ad patients mostly rely on cholinesterase inhibitors, while pd is mainly treated with dopamine precursors; however, to this date, no decisive progress achieved ad, pd, and other neurodegenerative disease, including neurodegeneration following brain injury [ ] . giving hope, however, favorable results in treatment of neurodegeneration have been reported with fucoidans, fucoxanthins, and other bioactive compounds from various algal sources, associated with characteristics such as: (i) scavenging potentials, which can avert neuronal damage given the linkage of this condition to neuronal ros imbalance caused by mitochondrial dysfunction [ ] ; (ii) reported acetylcholinesterase (ache) or butyrilcholinesterase (bche) inhibitory activity of polyphenol rich extracts [ , ] ; (iii) reported beta secretase (bace- ) inhibitory activities of crude extracts and polysaccharides, which may preclude amyloid beta (aβ) accumulation [ , ] . (see also figure ) newer reports about the molecular pathways involved in fucoidan neuroprotective effects on dopaminergic nerve precursor cells (mn d) treated with -methyl- -phenyl pyridine (mpp + ) suggest its involvement in increasing superoxide dismutase (sod) activity and reduced glutathione (gsh) concentration and decreasing the apoptosis levels by downregulation of bax expression [ ] . a study by zhang et al. demonstrated that acute high dose co-treatment with fucoidan isolated from l. japonica significantly reduced rotenone-induced loss of substantia nigra pars compacta and striatal neurons [ ] . this resulted in a significant improvement of the animals' behavior and alleviation of pd symptoms by increasing mitochondrial respiratory function together with the figure . effects of fucoidan on brain disease. fucoidan reduces inflammatory response in brain diseases by inhibiting microglial activation, thus resulting in significantly decreased neuronal and astrocyte degeneration due to diminishing production of pro-apoptotic agents and improving antioxidant responses of the cell. furthermore, fucoidan prevents leukocyte adhesion to the brain by blocking p-selectin. neurodegeneration is commonly defined as progressive atrophy and loss of function of neurons reflected in neurodegenerative diseases such as ad or pd [ ] . in particular, neurodegenerative disorders are characterized by a progressive decline of motor and/or cognitive functions caused by the selective degeneration and loss of neurons within the cns [ ] . while substantial progress has been made in revealing the cellular and molecular pathways causing these conditions, which mostly involve dysregulation of genetic expression indubitably accompanied by synthesis of truncated protein triggering neuroinflammatory response [ ] , they still pose a significant threat for human health in general, mainly because of their severe and aggressive symptomatology, and a pathophysiology that is still a diagnostic challenge [ ] . for treatment, or rather, for the alleviation of symptoms, ad patients mostly rely on cholinesterase inhibitors, while pd is mainly treated with dopamine precursors; however, to this date, no decisive progress achieved ad, pd, and other neurodegenerative disease, including neurodegeneration following brain injury [ ] . giving hope, however, favorable results in treatment of neurodegeneration have been reported with fucoidans, fucoxanthins, and other bioactive compounds from various algal sources, associated with characteristics such as: (i) scavenging potentials, which can avert neuronal damage given the linkage of this condition to neuronal ros imbalance caused by mitochondrial dysfunction [ ] ; (ii) reported acetylcholinesterase (ache) or butyrilcholinesterase (bche) inhibitory activity of polyphenol rich extracts [ , ] ; (iii) reported beta secretase (bace- ) inhibitory activities of crude extracts and polysaccharides, which may preclude amyloid beta (aβ) accumulation [ , ] . (see also figure ) newer reports about the molecular pathways involved in fucoidan neuroprotective effects on dopaminergic nerve precursor cells (mn d) treated with -methyl- -phenyl pyridine (mpp + ) suggest its involvement in increasing superoxide dismutase (sod) activity and reduced glutathione (gsh) concentration and decreasing the apoptosis levels by downregulation of bax expression [ ] . a study by zhang et al. demonstrated that acute high dose co-treatment with fucoidan isolated from l. japonica significantly reduced rotenone-induced loss of substantia nigra pars compacta and striatal neurons [ ] . this resulted in a significant improvement of the animals' behavior and alleviation of pd symptoms by increasing mitochondrial respiratory function together with the inhibition of malondialdehyde (mda), -hydroxy- -deoxyguanosine ( -ohdg), and -nitrotyrosine ( -nt) formation in rat ventral midbrain [ ] . these authors also reported that fucoidan treatment resulted in restoring the normal expression of peroxisome proliferator-activated receptor gamma coactivator -alpha (pgc- α) and nuclear transcription factor (nrf- ), thereby rescuing the mitochondrial functioning [ ] . a study by park et al. confirmed that pretreatment with fucoidan extracted from e. cava also showed antioxidant effects and protection of mitochondrial health in mice with cognitive dysfunction caused by trimethyltin injection [ ] . it was also reported that fucoidan has a beneficial influence on inhibiting bax expression and cytochrome c release, in accordance with previously reported in vitro findings [ ] . (see also figure ) finally, these same authors found significant decreases in aβ formation and phosphorylation of tau protein suggesting that fucoidan could be an efficacious agent for prevention for neurodegenerative disorders [ ] . fucoidan, neurodegeneration, and sirtuin as mentioned above, fucoidan may exert its beneficial effects via sirt (as seen with tbi), and it appears that sirt commonly presents a component of neurodegenerative diseases, as for example discussed by meng et al. [ ] . the most common neurodegenerative diseases are ad, pd, and huntington disease (hd). in the wake of several recent failures of ad therapies targeting beta-amyloid in plaques, growing evidence has suggested that infection with the herpes simplex virus (e.g., (hsv)- ) may play a role in ad. it is known that hsv- induces formation of beta-amyloid, and abnormally phosphorylated, ad-like tau (p-tau), which are the characteristic abnormal molecules of ad brains [ ] . furthermore, it has been found that sirt may be a relevant therapeutic target in als. this suggests that sirt may present a target for treatment of various neurodegenerative disorders [ ] . neurons have high energy demands, and dysregulation of mitochondrial quality and function is an important cause of neuronal degeneration (see also figure ). meng et al. discussed that the mitochondrial deacetylase sirt has been found to have a large effect on mitochondrial function [ ] . recent studies have also shown that sirt has a role in mitochondrial quality control, including the refolding or degradation of misfolded/unfolded proteins, mitochondrial dynamics, mitophagy, and mitochondrial biogenesis, all of which are part and parcel of neurodegenerative diseases. thus, the finding that fucoidan appears to interact with sirt , as seen with neurodegeneration due to tbi, may be very relevant to recapitulate a bit, sirtuins are highly conserved nad+ dependent class iii histone deacetylases and catalyze deacetylation and adp ribosylation of a number of non-histone proteins [ ] . in the recent past, clusters of protein substrates for sirt were identified in mitochondria and are now considered to be in association with protection from stress induced mitochondrial integrity and energy metabolism ( figure ). in this way, sirt may be protective regarding the pathogenesis of almost all neurodegenerative diseases. some recent findings demonstrated that sirt overexpression could prevent neuronal derangements in certain in vivo and in vitro models of aging and neurodegenerative brain disorders, including ad, pd, hd, tbi, stroke, etc. similarly, loss of sirt has been found to accelerate neurodegeneration in the brain challenged with excitotoxicity, which may explain the increase of sirt levels found in the wang's et al. study [ ] of tbi and its treatment with fucoidan, described above. fucoidan treatment of meningitis in rats reduced all inflammatory changes, while fucoidan treatment of animals without meningitis increased blood white cell count [ ] . this study also validated that selectins are involved in the early phase of pneumococcal meningitis and, possibly, are a target for adjunctive therapy with fucoidan [ ] . in a rabbit meningitis model based on intracisternal injection of live streptococcus pneumoniae, inhibition of leukocyte rolling by i.v. application of the polysaccharide fucoidan prevented the enhanced leukocyte extravasation into the subarachnoid space (sas) and also attenuated the leakage of plasma proteins over the bbb [ ] . thus, fucoidan's ability to block leukocyte rolling via binding to p-selectin presents the potential to reduce leukocyte-dependent cns damage in bacterial meningitis. in addition, in c glioma cells it was found that fucoidan can suppress tnf-α and ifn-È-induced no production and inos expression, another way whereby fucoidan can attenuate inflammatory responses (see figure ). (furthermore, fucoidan can inhibit: ( ) tnf-α and ifn-È-induced ap- , irf- , jak/stat activation; ( ) p mitogen-activated protein kinase (mapk) activation; and ( ) induced scavenger receptor b (sr-b ) expression [ ] . in support, in vitro experiments with primary microglia indicated that the excessive production of tnf-α and ros in lps-induced primary microglia was significantly inhibited by fucoidan administration [ ] . finally, in animal studies, it was found in lps treated rats that fucoidan significantly improved the behavioral functioning, prevented the loss of dopaminergic neurons, and inhibited the deleterious activation of microglia in the substantia nigra pars compacta [ ] . these studies indicate the anti-inflammatory properties of fucoidan at cellular as well as systemic levels. creutzfeldt-jakob disease is a serious and lethal brain damaging condition. it has been demonstrated that sulfated glycans such as fucoidan and pentosan polysulfate, as well as amyloidophilic compounds such styrylbenzoazole derivatives, and phenylhydrazine derivatives present efficacies in prion-infected animals [ ] . wozniak et al. moved on to investigate the antiviral activity of sulfated fucans from five brown algae (scytothamnus australis, marginariella boryana, papenfussiella lutea, splachnidium rugosum, and undaria pinnatifida) in relation to the hsv -induced formation of beta-amyloid, and ad-like tau [ ] . four sulfated fucan extracts each prevented the accumulation of hsv -induced beta-amyloid and ad-like tau in hsv -infected vero cells [ ] . thus, knowledge regarding fucoidan as an antiviral agent, beyond anti-inflammatory effects, may be relevant for brain disorders. as the burden of viral infections is increasing, culminating with highly contagious new corona viruses displaying heterogeneous structures, complicating the design of targeted therapy, it is valuable to consider novel and safe antiviral agents [ , ] . in light of these notions, we assume that together with its antiviral and anti-inflammatory properties, fucoidan may deliver protective effects against many illnesses, including neurodegenerative and hepatic disorders, as reported and reviewed here and elsewhere. these effects of polysaccharides are mostly achieved by stimulating the production of viral antibodies and upregulation of interleukins (particularly il- and il- ), hence increasing the activation of macrophages and natural killer (nk) cells and promoting phagocytosis. in support of the above, it is assumed that the sulfate groups in fucoidan molecule may also contribute to the antiviral activity by acting as polyanions, and inhibiting cell surface interactions of positively charged viral domains with the host cells, thus preventing their penetration and/or adsorption. boosting the humoral immune response by increased immunoglobulin synthesis has also been acknowledged as one of the antiviral effects of polysaccharides [ ] . early in vitro reports suggested that algal polysaccharides successfully inhibited the herpes simplex virus (hsv and hsv ), human cytomegalovirus, and bovine viral diarrhea virus [ , ] , which may also have implications for brain diseases, including alzheimer [ ] . a study conducted by hidari et al. placed the dengue virus type (den ), in the list of successfully inhibited viral species by fucoidan extracted from c. okamurans in the bhk- cell line, additionally defining glucuronic acid and the sulfated fucose contents as key elements involved in antiviral activity of this macromolecule [ ] . these authors also established the significance of arginine- as a vital region in the envelope glycoprotein (epg), which enables the interactions of glucuronic acid with the virus, while its possible substitution or "mispositioning" may decrease the antiviral effects of fucoidan as shown for den- and den- viruses [ ] . conversion of the complex structure of polysaccharides into monosaccharides could also diminish their virucidal activity [ ] . from recent published results, sun et al. verified these findings by establishing the inhibitory activities of two low molecular fractions of fucoidan isolated from l. japonica against influenza a, adenovirus, and parainfluenza virus type (hpiv ) in hep- , hela, and mdck cells [ ] . these authors also reported that acute intraperitoneal treatment with these fucoidan fractions prolonged the average survival time and increased the viability of lung, thymus, and spleen cells [ ] . similar research conducted by wang et al. revealed the inhibition of neuraminidase and epidermal growth factor cellular pathway (egfr) of fucoidan as crucial molecular mechanisms for preventing the penetration of influenza h n virus into host cells [ ] . another study showed the inhibitory properties of crude fucoidans obtained from the brown algae d. bartayesiana and t. decurrens of the human immunodeficiency virus (hiv) in infected peripheral blood mononuclear cells (pbmcs) at the extremely low inhibitory concentrations of . µg/ml and µg/ml, respectively, thus exhibiting significantly higher antiviral effects than ribavirin [ , ] . prokofjeva et al. also acknowledged fucoidans as potent anti-hiv agents irrespective of their degree of sulfation and carbohydrate structure [ ] . since fucoidan has been proven quite successful in inhibiting single stranded rna (ssrna) respiratory viruses such as influenza a and hpiv , in addition to virucidal activity against den (positive ssrna virus), we are also led to believe that it could be influential in treating coronaviruses induce diseases such as covid- , given covid- 's similarities with the genetic material and symptomatology of the virus species mentioned here [ ] . the variety of the cellular mechanisms by which this polysaccharide achieves its antiviral effects probably contributes to its potency. fucoidan was shown to suppress increased oxidative stress in bovine brain microvessel endothelial cells (bbmecs) in culture after exposure to diesel exhaust particles (deps) [ ] . in addition, permeability of bbmecs induced by dep exposure was decreased by fucoidan treatment. this study provides evidence that fucoidan might protect the cns against toxic effects of dep exposure [ ] . in cultured cortical neurons from one-day old wistar rats, fucoidan suppressed nmda induced ca + responses by % [ ] . however, the ca + responses of hippocampal neurons induced by glutamate, acpd, or adrenaline showed only slight decreases following fucoidan treatment. nonetheless, in cortical as well as hippocampal neurons, fucoidan treatment significantly decreased mrna expression of the nmda-nr receptor and the primer pair for l-type ca + channels, namely, pr /pr . in this way, fucoidan may counteract excitotoxicity, at least in the cerebral cortex [ ] . previous work showed that the glycosaminoglycan (gag) dextran sulfate ( kda) altered the binding and channel properties of alpha-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa)-type glutamate receptors [ ] . dextran sulfate was more potent in inhibiting high-affinity ampa binding to solubilized receptors (ec ( ) of nm) than fucoidan, another gag (ec( ) of nm). additionally, dextran sulfate ( nm), produced a three-to four-fold increase in open channel probability and a three-fold increase in mean burst duration of channel activity elicited by nm ampa. fucoidan produced similar effects, but at a concentration several times higher than that of dextran sulfate [ ] . these findings suggest that gag components of proteoglycans can interact with and alter the binding affinity of ampa receptors and modulate their functional properties. thus, in addition to nmda receptors, fucoidan can also prevent excitotoxity by altering the affinity of ampa receptors. we hope that these fucoidan effects can be optimized, for example, by producing more efficacious fucoidan derivatives. as mentioned above, fucoidan can counteract ad-like adverse effects of hsv infection, such as the formation of beta-amyloid and abnormal p-tau. other studies showed with rat behavioral tests that fucoidan can ameliorate aβ ( - )-induced learning and memory impairments [ ] . furthermore, fucoidan reversed the decreased activity of choline acetyl transferase (chat), superoxide dismutase (sod), glutathione peroxidase (gsh-px), and acetylcholine (ach) content, as well as the inhibitory effects on malondialdehyde (mda) synthesis in hippocampal tissue of aβ-injected rats [ ] . moreover, these effects were accompanied by an increase of bcl- /bax ratio and a decrease of caspase- activity [ ] . thus, apparently, by regulating the cholinergic system, reducing oxidative stress, and inhibiting apoptosis, fucoidan can ameliorate aβ-induced ad. park et al. investigated the sea weed ecklonia cava (e. cava) for the effects of fucoidan extract on cognitive function [ ] . they applied y-maze, passive avoidance, and a morris water maze to a trimethyltin (tmt)-induced cognitive dysfunction model. this demonstrated that the fucoidan extract promoted learning and memory improvements. in mouse brain tissue taken after such behavioral tests, fucoidan extract was shown to provide inhibitory effects on lipid peroxidation and improvement of cholinergic system activity. mitochondrial activity was improved as seen from associated mitochondrial ros content and mitochondrial membrane potential (mmp, ∆Ψm) levels, and was also detected by mitochondria-mediated protein (bax, cytochrome c) analysis for apoptosis induction. it appeared that the fucoidan-rich substances from e. cava could improve cognitive functions by downregulating amyloid-β production and tau hyperphosphorylation [ ] . jhamandas et al. showed that fucoidan reduced cell death rates otherwise induced by a beta ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) or a beta ( - ) to rat cholinergic basal forebrain cultures [ ] . in this study, it was also found that fucoidan attenuated a beta-induced downregulation of phosphorylated protein kinase c. furthermore, a beta ( - )-induced generation of ros was blocked by prior exposure of the cultures to fucoidan. regarding apoptosis, a beta activation of caspases and is blocked by pretreatment of cultures with fucoidan [ ] . caspases and are well known components of the signaling pathways of apoptotic cell death induction. these results show that fucoidan has neuroprotective effects against a beta-induced neurotoxicity in basal forebrain neuronal cultures, which may have implications for ad and other neurodegenerative diseases as fucoidan can affect sirt function, it is interesting to know what sirt may do in ad. lee et al. have discussed mitochondrial dysfunction in connection with the pathogenesis of ad [ ] . in particular, sirt mrna and protein levels are significantly decreased in ad cerebral cortex, and ac-p k is significantly increased in ad mitochondria. in this context, sirt prevented p -induced mitochondrial dysfunction and neuronal damage in a deacetylase activity-dependent manner. notably, mitochondrial targeted p (mito-p ), directly reduced mitochondria dna-encoded nd and nd gene expression, resulting in increased ros and reduced mitochondrial oxygen consumption. interestingly, nd and nd gene expressions are significantly decreased in patients with ad and increased p occupancy in mitochondrial dna in ad. lee et al. further found that sirt overexpression restored the expression of nd and nd and improved mitochondrial oxygen consumption by repressing mito-p activity [ ] . these results indicate that sirt dysfunction leads to p -mediated mitochondrial and neuronal damage in ad. therapeutic modulation of sirt activity may ameliorate mitochondrial pathology and neurodegeneration in ad. in the light that fucoidan can modulate sirt activity, it emphasizes that fucoidan may present an agent to ameliorate ad. the effects of fucoidan has been studied intensively on several pd models, for example: (i) in cell culture of dopaminergic nerve cells; (ii) mptp ( -methyl- -phenyl- , , , -tetrahydropyridine) application to mice; and (iii) application of -hydroxydopamine and rotenone application to rats, as given in some detail below. in a study by liang et al. on a cultured dopaminergic nerve precursor cell line (mn d), cell viability decreased by % within h of µm mpp+ application [ ] . -methyl- -phenylpyridinium (mpp+), the toxic bioactivation product of mptp, is a toxic compound that via the dopamine transporter is brought into neurons, in which it initiates neuronal death by inhibiting complex i of the mitochondria. pretreatment with µm fucoidan in this paradigm reversed the reduction of sod and gsh, as well as the decreased cell viability and induced apoptotic cell death, otherwise brought about within h by mpp+. furthermore, preceding this fucoidan reduced cellular expression of lc -ii and catd within h and suppressed the induction of bax protein. thus, liang et al. suggested that fucoidan may have a positive, curative effect regarding pd [ ] . regarding animals, luo et al. applied mptp to c /bl mice [ ] . when fucoidan was administered prior to mptp, behavioral deficits were reduced, and levels of striatal dopamine and its metabolites were enhanced, cell death was reduced, and a marked increase in tyrosine hydroxylase expression relative to mice treated with mptp alone was also observed. furthermore, as in the liang et al. study [ ] , it was found that fucoidan inhibited mptp-induced lipid peroxidation and reduction of antioxidant enzyme activity. in addition, pre-treatment with fucoidan significantly protected against mpp(+)-induced damage in mn d cells [ ] . in a -hydroxydopamine ( -ohda) rat model of pd, chronic fucoidan administration mitigated the motor dysfunction otherwise induced by -ohda [ ] . similarly, fucoidan reduced the loss of da neurons in the snc and da fibers in the striatum in -ohda-lesioned rats. moreover, fucoidan inhibited the -ohda-stimulating expression of nox in both tyrosine hydroxylase (th)-positive neurons as well as non-th-positive neurons, and prevented nox -sensitive oxidative stress and cell damage in snc neurons. finally, fucoidan also effectively inhibited nigral microglial activation [ ] . in a rotenone-induced pd rat model, it was found that chronic treatment with fucoidan significantly reversed the loss of nigral dopaminergic neurons, striatal dopaminergic fibers, and reduction of striatal dopamine levels [ ] . fucoidan also alleviated rotenone-induced behavioral deficits. interestingly, in the substantia nigra of these pd rats, the reduced mitochondrial respiratory function, detected by the mitochondrial oxygen consumption and the expression of peroxisome proliferator-activated receptor gamma coactivator -alpha (pgc- α) and nuclear transcription factor (nrf ), was markedly reversed by fucoidan [ ] . furthermore, oxidative products induced by rotenone were significantly reduced by fucoidan. these results thus also suggest some functional pathways modulated by fucoidan in relation to the neurodegenerative disease of pd. taken together, these studies indicate that fucoidan attenuates pd characteristics induced in various animal and cell culture models for pd. thus, fucoidan provides a promising venue for treatment of pd by modulating its various underlying molecular biological mechanisms. sirtuin and parkinson disease (pd) it may be that as in tbi, and also suggested for ad, fucoidan exerts its ameliorating effects regarding pd via interactions with sirt . interestingly, while it was shown that sirt null mice do not exhibit motor and non-moto deficits compared with wild-type controls, sirt deficiency dramatically exacerbated the degeneration of nigrostriatal dopaminergic neurons in -methyl- -phenyl- , , , -tetrahydropyridine (mptp)-induced pd mice [ ] . sirt null mice exposed to mptp also exhibited decreased sod , a specific mitochondrial antioxidant enzyme, and reduced glutathione peroxidase expression compared with wild-type controls [ ] . taken together, these findings strongly support that sirt has a possible role in mptp-induced neurodegeneration via preserving free radical scavenging capacity in mitochondria. thus, it would be worthwhile to study fucoidan-sirt interactions in models for pd. it indeed is very tempting to assume that fucoidan also interacts with sirt in pd. stroke is one of the leading causes of death. growing evidence indicates that ketone bodies have beneficial effects in treating stroke, but their underlying mechanism remains unclear [ ] . the potential of fucoidan to ameliorate stroke injury in the brain has been of interest for more than two decades now. because intracerebral hemorrhage (as induced by injection of bacterial collagenase into the caudate nucleus) is associated with more inflammation than seen with ischemic stroke, this stroke model attracted early interest for fucoidan treatment testing, as fucoidan can counteract inflammatory responses [ ] . fucoidan-treated rats exhibited evidence of impaired blood clotting and hemodilution, had larger hematomas, and tended to have less inflammation in the vicinity of the hematoma after three days. interestingly, fucoidan-treated rats showed significantly more rapid improvement of motor function in the first week following hemorrhage and better memory retention in the passive avoidance test, as compared to untreated controls [ ] . this early study from stated that investigations of more specific anti-inflammatory agents and hemodiluting agents would be warranted in intracerebral hemorrhage [ ] . as it was also understood that leukocyte-endothelial adhesion is a key step to initiate post-ischemic reperfusion injury in many organs, this potential contribution to stroke, including fucoidan interference, was also studied. uhm et al. found that the expressions of p-selectin mrna and protein were increased in the ipsilateral hemisphere with a peak at h after hypoxia-ischemia in immature brain [ ] . such temporal profiles of p-selectin expression followed by hypoxia-ischemia are consistent with a role in the subsequent brain injury. because fucoidan is known to inhibit p/l-selectin mediated leukocyte adhesion, it was examined whether the treatment of fucoidan attenuates hypoxia-ischemia-induced neural damages [ ] . indeed, fucoidan presented a substantial neuroprotective effect, including significant inhibition of the leukocyte adhesion, as revealed by myeloperoxidase activity. these results suggest that anti-adhesion strategy as can be provided by fucoidan may be an effective therapeutic application for perinatal hypoxic-ischemic encephalopathy [ ] . the following study presented further interest in fucoidan as an anti-inflammatory agent. fucoidan treatment inhibited the expressions of some brain cytokine or chemokine mrna, such as il- , tnf-α, and inos in the brain of the rats treated only with lps [ ] . moreover, fucoidan treatment dramatically decreased the infarct size in accelerated cerebral ischemic injury induced by lps treatment. in addition, the immunoreactivity of myleoperoxidase (mpo), a marker for quantifying neutrophil accumulation, was distinctively decreased in the ischemic brain of the fucoidan-treated rat. in brief, the results of kang et al. [ ] indicated that fucoidan provides a neuroprotective effect on lps accelerated cerebral ischemic injury through inhibiting the expression of some cytokine/chemokine and neutrophil recruitments [ ] . intracerebral hemorrhage (ich) is the most fatal stroke subtype, with no effective therapies [ ] . additionally, fucoidan did not have effects on brain water content, neurological deficits, and hemoglobin content after ich. the authors suggested that this may be so because crude fucoidan was used in this study, and high-molecular-weight fucoidans (hmwf) are reported to have less therapeutic potential than lmwf's [ ] . the effects of fucoidan on cerebral ischemia-reperfusion injury (iri) including the inflammatory and other underlying mechanisms were further explored in sprague-dawley (sd) rats [ ] . the results showed that administration of fucoidan significantly reduced the neurological deficits and infarct volume in a dose-dependent manner. additionally, fucoidan significantly decreased the levels of: (i) inflammation-associated cytokines (interleukin (il)- β, il- , myeloperoxidase (mpo), and tumor necrosis factor (tnf)-α); (ii) oxidative stress-related proteins malondialdehyde (mda) and sod; (iii) apoptosis (in particular, apoptosis-related proteins (p- , bax, and b-cell lymphoma (bcl)- )); and (iv) the mapk pathway mitogen-activated protein kinase (mapk) pathway (in particular, phosphorylation-extracellular signal regulated kinase (p-erk), p-c-jun n-terminal kinase (jnk), and p-p ). thus, fucoidan's protective role in cerebral iri includes anti-inflammatory effects, anti-apoptotic effects, anti-oxidative stress affects, and potentially gene expression regulation [ ] . studies directed at better determining the stroke-fucoidan interaction in various brain cell types, at systemic, in situ levels, showed that pretreatment with fucoidan confers neuroprotection against transient global cerebral ischemic injury in the gerbil hippocampal ca area via reducing of glial cell activation and oxidative stress [ ] . in some detail, the neuroprotective effect of fucoidan against transient global cerebral ischemia (tgci) included inhibition of activation of astrocytes and microglia in the ischemic ca area. furthermore, it significantly reduced otherwise increased -hydroxy- -noneal and superoxide anion radical production in the ischemic ca area with subsequent increased expressions of sod and sod in the ca pyramidal neurons before and after tgci [ ] . additionally, in obese gerbils, fucoidan treatment attenuated acceleration and exacerbation of tgci-induced neuronal death in the ca - hippocampal areas, and levels of oxidative stress indicators (dihydroethidium, -hydroxyguanine, and -hydroxy- -nonenal) were significantly reduced, while levels of antioxidant enzymes (sod and sod ) were significantly increased in pre-and post-ischemic phases [ ] . these findings indicate that pretreatment with fucoidan can relieve the acceleration and exacerbation of ischemic brain injury in an obese state via the attenuation of obesity-induced severe oxidative damage, and related factors. as mentioned at a few occasions above, sirts are a family of nad+ dependent histone deacetylase (hdac) proteins implicated in aging, cell cycle regulation, and metabolism. these proteins are involved in the epigenetic modification of neuromodulatory proteins after stroke via acetylation/deacetylation. the specific role of sirt , a mitochondrial sirtuin, in post-stroke injury has been relatively unexplored. nonetheless, verma et al. [ ] showed that sirt knockout (ko) mice show significant neuroprotection at days after ischemia/reperfusion (i/r) or stroke injury. the deacetylation activity of sirt , measured as the amount of reduced acetylated lysine, was increased after stroke [ ] . in male sirt ko mice and wild-type littermates (wt), stroke-induced increases in liver kinase (lkb ) activity were also appeared reduced in ko mice at days after stroke. yin et al. investigated whether mitochondrial sirt could mediate the neuroprotective effects of ketone bodies after ischemic stroke. the ketone treatment did enhance mitochondrial function, reduced oxidative stress, and probably in this way reduced infarct volume. this was associated with improved neurologic function after ischemia, including the neurologic score, the performance in rotarod, and in open field tests. they further presented that ketones' effects were achieved by upregulating sirt and its downstream substrates forehead box o a (foxo a) and superoxide dismutase (sod ) in the penumbra region. this appeared likely, since knocking down sirt in vitro diminished ketones' beneficial effects [ ] . it also indicates that upregulation of sirt after stroke is beneficial for amelioration of brain damage caused by stroke. these results provide us a foundation to develop novel therapeutics targeting this sirt -foxo a-sod pathway. of course, in the framework of this review, we would like to suggest the potential of fucoidan, based on its known modes of action and its efficaciousness. on further investigation, verma et al. found that the levels of sirt , another important member of the sirtuin family, were increased in the brains of sirt ko mice after stroke [ ] . to determine the translational relevance of these findings, they tested the effects of pharmacological inhibition of sirt . they found no benefit of sirt inhibition despite clear evidence of deacetylation. overall, it was concluded that sirt ko mice show neuroprotection by a compensatory rise in sirt rather than the loss of sirt after stroke [ ] . further analysis will determine the importance of using both pharmacological and genetic methods in pre-clinical stroke studies to better understand molecular biological pathways [ ] . additionally, it is important to further investigate the interaction between sirt and sirt . more studies approached the question of sirt involvement in brain damage due to stroke. the role of sirt in microglial cell migration and invading macrophages in ischemic stroke was studied. the middle cerebral artery occlusion (mcao) animal model of focal ischemia was used [ ] . lentivirus-packaged sirt overexpression was applied, and also knock down in microglial n cells in culture, to investigate the underlying mechanism driving microglial cell migration. more microglial cells appeared in the ischemic lesion side after mcao. the levels of sirt were increased in macrophages invading after ischemia. cx cr levels were increased with sirt overexpression. furthermore, sirt promoted microglial n cells migration by upregulating cx cr in both normal and glucose deprived culture media [ ] . these effects were g protein-dependent. thus, sirt promotes microglia migration by upregulating cx cr . this appears counter intuitive, finding that sirt promotes microglia migration, while the consensus appears to be that sirt acts anti-inflammatory. however, as mentioned, it would be nice also to further investigate interaction between sirt and sirt . after transient middle cerebral artery occlusion (tmcao) in adult male sirt ko and wild-type (wt) mice, it was found that the level of sirt in infarct region is decreased after ischemic stroke [ ] . in addition, it was found that sirt ko mice showed worse neurobehavioral outcome compared with wt mice, accompanied by decreased neurogenesis and angiogenesis, as shown by the reduction in number of dcx+/brdu+ cells, neun+/brdu+ cells, and cd +/brdu+ cells in the perifocal region during the recovery phase after ischemic stroke [ ] . furthermore, sirt deficiency reduced the activation of vascular endothelial growth factor (vegf), akt, and extracellular signal-regulated kinases (erk) signaling pathways [ ] . these results indicate that sirt is beneficial to neurovascular and functional recovery following chronic ischemic stroke. as a concluding remark, as tbi and stroke are brain injuries, it could be very fruitful to investigate fucoidan-sirtuin interactions in stroke models, and see whether similarities with tbi models will be seen. regarding the previously mentioned conclusions about antioxidant, anti-inflammatory, and anti-cell death impact of fucoidan, it can be deduced that this polysaccharide could have a significant therapeutic effect against systemic and whole organism disorders and inflammations rather than just impairments of tissues. it could also have a significant impact on regenerating the antioxidant potentials of more dynamic cells with significantly higher levels of ros production such as hepatocytes [ ] . we will discuss fucoidan-liver interactions here. an additional point, not to be dwelt on, may be the effect of liver damage on brain health, as for example with hepatic encephalopathy, or maybe also atherosclerosis associated with impaired liver functions. importantly, restricting ourselves to the liver, hepatocytes are easily prone to structural alterations, cellular deterioration, and damage, given their rates of metabolic activity; thus they are ideal models for confirming the protective effects of fucoidan [ ] . indeed, fucoidan has shown cytoprotective effects against the hepatotoxicity of several xenobiotics, such as acetaminophen (apap) [ ] or carbon tetrachloride (ccl ) [ ] (figure ). li et al. also confirmed the effects of fucoidan, extracted from f. vesiculosus, against liver fibrosis induced by acute ccl treatment (figure ) or bile duct ligation (bdl) through decreasing serum transaminase activity and hydroxyproline concentration; however, relative large doses of fucoidan were needed to restore normal levels of serum transaminase activity and hydroxyproline concentration [ ] (see also figure ). moreover, they discovered that fucoidan significantly reduced synthesis of collagen type and alpha smooth muscle actin (α-sma) proteins, which are typically upregulated in hepatocyte injury stimulating liver fibrosis by transformation of hepatic stellate cells (hscs) to myofibroblasts [ ] . furthermore, the authors proved the inhibitory effect of fucoidan on transforming growth factor beta (tgf-β)/smad molecular pathways, by the decreased expression of beclin- , a transcription factor activated by this pathway which influences autophagosome occurrence, which was considerably increased in the ccl or bdl injured hepatocytes ( figure ) [ , ] . a recent study confirmed these findings with fucoidan extracted from s. fluitans on wistar rats [ ] . thus, it can be concluded that fucoidan exerts its hepatoprotective effects through the alteration of pathways directly included in modification of liver microenvironment. effects against apap liver injury mostly through activating the nrf -are molecular pathway in addition to suppressing cytochrome member cyp e , responsible for metabolizing acetaminophen to its toxic product n-acetyl-p-benzoquinoneimine (napqi) and triggering gsh depletion [ , ] (see also figure ). however, large doses of fucoidan were able to increase the antioxidant response of liver cells and suppress serum alt, ast, and ldh levels only at the early stages of injury (up to h after of apap injection) [ ] (see also figure ). figure . effects of fucoidan on liver injury. damaging agents at the top left side, apap and carbon tetrachloride (ccl ), and protective fucoidan at the top right side. fucoidan averts liver fibrosis by inhibiting hscs production through optimal synthesis of collagen and alpha smooth muscle actin and prevents tissue damage by reducing transaminase release and restoring antioxidant potentials of cells. it decreases cyp e activity, which reduces levels of toxic metabolites and inhibits tgfβ/smad pathway, thereby hindering the occurrence of autophagosomes. fucoidan also stimulates expression of sirtuin- in the liver, which activates ampk and alleviates insulin resistance. reminiscent of neurons, fucoidan also stimulates expression of sirtuin molecules (sirt ) in hepatocytes, mostly involved in regulation of glucose and lipid metabolism in the liver, thus being one of the crucial factors included in the pathophysiology of the metabolic syndrome (mets) and figure . effects of fucoidan on liver injury. damaging agents at the top left side, apap and carbon tetrachloride (ccl ), and protective fucoidan at the top right side. fucoidan averts liver fibrosis by inhibiting hscs production through optimal synthesis of collagen and alpha smooth muscle actin and prevents tissue damage by reducing transaminase release and restoring antioxidant potentials of cells. it decreases cyp e activity, which reduces levels of toxic metabolites and inhibits tgf-β/smad pathway, thereby hindering the occurrence of autophagosomes. fucoidan also stimulates expression of sirtuin- in the liver, which activates ampk and alleviates insulin resistance. the contribution of sulfate content for the beneficial effects of this algal polysaccharide on hepatic injury has also been studied. namely, liu et al. reported more significantly decreased levels of serum lactate dehydrogenase (ldh) levels in mice with acute ccl treatment after co-treatment with more sulphated fractions of fucoidan ( . % and . %) isolated from k. crassifolia [ ] (see also figure ). however, the authors did not find major differences in the inhibition of lipid peroxidation and gsh restoration in animals treated with different doses of fucoidan fractions [ ] . similar results were also found for diminishing ast and mda levels during treatment with high sulphated fraction of fucoidan ( . %) from s. japonica, supported by histopathological analyses, which revealed a complete inhibition of liver necrosis by this fraction of fucoidan, with a mega dose applied [ ] . findings about the dependence of sulphate content on the biological potency of algal polysaccharides are in corroboration with those published by wang et al., who reported that low sulphate fraction ( %) of fucoidan from c. costata did not achieve suppression of alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels [ ] . recent investigations about fucoidan hepatoprotective effects against drug induced liver injury also provided promising results. a study by abdel-daim et al. has shown that acute treatment of rats with high quantity of fucoidan efficiently restores transaminase levels, creatine kinase, and acetylcholinesterase activity, as well as cholesterol and creatinine serum concentrations, after diazinon-induced hepatic injury, together with the dose dependent decrease of inflammatory biomarkers, such as tnf-α and il- [ ] . similarly, neurons, hepatocytes, renal cells, and cardiomyocytes also exhibited improvement of the enzymatic antioxidants via the restoration of cellular cat, sod, and gpx activities and a significant decrease in lipid peroxidation and nitric oxide levels ( figure ) [ ] . the same results were obtained using mice as animal models for microcystin-lr injury [ ] . discoveries of wang et al. suggested that fucoidan exerts its protective effects against apap liver injury mostly through activating the nrf -are molecular pathway in addition to suppressing cytochrome member cyp e , responsible for metabolizing acetaminophen to its toxic product n-acetyl-p-benzoquinoneimine (napqi) and triggering gsh depletion [ , ] (see also figure ). however, large doses of fucoidan were able to increase the antioxidant response of liver cells and suppress serum alt, ast, and ldh levels only at the early stages of injury (up to h after of apap injection) [ ] (see also figure ). reminiscent of neurons, fucoidan also stimulates expression of sirtuin molecules (sirt ) in hepatocytes, mostly involved in regulation of glucose and lipid metabolism in the liver, thus being one of the crucial factors included in the pathophysiology of the metabolic syndrome (mets) and insulin resistance [ ] . considering the severity of these illnesses and the increasing trend of obesity worldwide, it is of great importance to discover safe therapeutics that would decrease these alarming rates [ ] . in this context, a study by zheng et al. reported significant reduction of plasma and liver cholesterol and triglycerides in db/db mice after sub-chronic treatment with low molecular fucoidan isolated from l. japonica, but failed to acknowledge reduction in fasting glucose levels [ ] . hepatoprotective effects were confirmed by inhibition of transaminase release, but only at the highest dose applied. as expected, the anti-inflammatory and antioxidant effects of fucoidan in vivo were underlined by the significant decreases in the cytokines and ros markers, which was confirmed by the reduced expression of nf-κb in the fucoidan treated mice [ ] . most importantly, the authors reported significantly elevated levels of sirt and ' adenosine monophosphate-activated protein kinase (ampk), whose activation is typically associated with increased glucose uptake, fatty acid oxidation, and glycolysis, which certainly explains these antilipotoxic effects of fucoidan ( figure ) [ ] . treatment with f. vesiculosus fucoidan on palmitate induced insulin resistant hepg cells also resulted in increased expression of ampk, additionally confirmed by their increased glucose consumption and decreased lipid profile [ ] . furthermore, sub-chronic administration of fucoidan isolated from the same species also affected low-density lipoprotein cholesterol (ldl-c) and high-density lipoprotein cholesterol (ldl-c). in particular, this fucoidan application also resulted in reduction of ldl-c and elevation of hdl-c levels in high fat diet fed mice, thus showing similar results with metformin in most of the analyzed parameters both in vivo and in vitro (except for hdl-c), including body weight decreases, which additionally promotes fucoidan as an efficient remedy for metabolic disorders, considering the ongoing controversies of the effects of metformin [ , ] . another study dealt with aspects of homeostasis, including glucose metabolism, influenced by fucoidans [ ] . briefly, the effects of low-molecular-weight fucoidan (lmwf) in terms of antihyperglycemic, antihyperlipidemic, and hepatoprotective activities, were investigated in a mouse model of type ii diabetes. blood sugar levels and increased serum adiponectin levels, were lowered by lmwf intake; thus they are relatively effective at improving hepatic glucose metabolism [ ] . from the findings about hypolipidemic effect of fucoidans, the possible suppression of atherosclerosis by this molecule can be derived from complex pathology of atherosclerosis, which combines dyslipidemia, inflammation, and atherothrombosis, mostly affected by increased levels of plasma ldl and its oxidative transformations as a trigger for occlusion of peripheral arteries [ ] . in parallel, the explained hepatoprotective features of fucoidan also go in favor of this hypothesis considering the impacts of impaired hepatic functioning on lipid homeostasis, regulation of metabolic pathways and lipoprotein uptake [ ] . the revealed contribution of ros in the development of atherosclerotic plaques, as confirmed by increased lipid peroxidation, glutathione depletion, and plasma and tissue protein carbonylation levels, also endorsed investigations of fucoidan as suitable therapeutic for this disease [ ] [ ] [ ] . a study by park et al. reported the significant decreases in plasma lipid profiles in fucoidan-treated mice with chemically-induced hyperlipidemia, which were comparable with statin effects [ ] . furthermore, it was concluded that this marine derived polysaccharide influences significant reduction in the development of aortic lesions in chronic atherosclerosis model and reduced expression of lipogenic enzymes fatty acid synthase (fas) and acetyl coa carboxylase (acc) in hepg cells [ ] . this study also confirmed the significant changes in the expression of srebp- and the ldl receptor, which makes them the probable molecular targets for hypolipidemic effects of fucoidan [ ] . these findings were confirmed on apolipoprotein e deficient mice (apoe −/− ) in the study of yin et al., using fucoidan isolated from a. nodosum [ ] . beside the amelioration of the lipid profiles and transaminase activity in the high fat diet treated mice, the sub-chronic treatment with fucoidan favored cellular cholesterol efflux by upregulation of atp-binding cassette transporters (abca and abcg ) and suppressed srebp and peroxisome proliferator-activated receptor gamma (pparγ), thereby inhibiting fatty degeneration in the liver. however, the treatment did not influence changes in ldl-r expression in apoe −/− , in contrast with the effects of fucoidan isolated from f. vesiculosus, thus suggesting the disparity of the effects of polysaccharides with different structures as well as the chemical composition [ ] . to summarize, fucoidan averts hepatic injury and necrosis by: (i) inhibiting the profibrotic pathways in the extracellular matrix that promote hscs production; (ii) modulating the cytochrome p enzyme activity and influencing the expression of nrf- transcription factor, which stimulates the antioxidant response of hepatocytes; (iii) increasing the expression of sirt and other molecules involved in regulation of lipoprotein metabolism. while fucoidan research is still in its early stages, even though it exhibits versatile molecular response in liver cells, thus far, research only proved that fucoidan can alleviates acute injury at relatively large doses. thus, it would be worthwhile to go the road of optimizing fucoidan effects by enhancing the efficaciousness of its derivatives. furthermore, possibly with its protective functions in the liver, fucoidan can suppress atherosclerosis, which of course would also present a beneficial factor regarding reduction of the incidence of stroke and explain some of its healing effects. another question arises: which type of fucoidan in general would be relatively advantageous to use-high molecular weight fucoidan (hmwf) or low molecular weight fucoidan (lmwf). the bioactivity of fucoidan is primarily dependent on its molecular weight and sulfate content. in general, it is accepted that the therapeutic potentials of hmwf are limited due to its lower cellular uptake and bioavailability, as reported by several studies [ , ] . these limitations derive mainly from the difficulties of hmwf to cross lipid bilayers, thereby suggesting superior effects of lmwf regarding their anticancer and antioxidant activity, simply deriving from their relatively efficacious membrane permeability [ , ] . in one study, the structure/function relationships of fucoidans from ascophyllum nodosum regarding their pro-angiogenic effect and cellular uptake in human endothelial cells were investigated [ ] . it was evidenced that lmwf have relatively high pro-angiogenic and pro-migratory potential. this may be interesting knowledge for the potential application lmwf to vascular repair in ischemia. in contrast, hmwf seems to have greater immunostimulatory effects than lmwf in the spleen, as indicated by the increased activation of natural killer nk cells in addition to the higher levels of interleukins and tnf-α [ ] . in accordance with the latter, a study of liu et al. revealed that the hmwf exhibited more significant neuroprotective effect than the lmwf in sh-sy y cells, thereby suggesting that the amount of sulfate is an important factor for improving therapeutic properties of fucoidan [ ] . this study also uncovered the advantageous efficient blockage of hmwf, which further reveals the complexity of its steric configuration providing more binding sites to the complementary factors [ ] . in addition to distinctions in molecular weight also differences in presence of sulfated groups may contribute to the beneficial effects of fucoidans. overall, a positive correlation exists between the sulfate content and antioxidant capability, which generally implies increased therapeutic impact of more sulfated fucoidans against diseases whose etiologies include oxidative damage [ ] . more specifically, highly sulfated fucoidans have shown significant attenuation of lipid accumulation and antitumor activity [ , ] . in one review focusing on bone tissue regeneration, it was concluded that sulfated polysaccharides, including fucoidans, have exceptional properties in terms of hydrogel-forming ability, scaffold formation, mimicking the extracellular matrix, alkaline phosphatase activity, biomineralization ability, and stem cell differentiation [ ] . in a recent review by us, we discussed the potential involvement of a mitochondrial protein (the kda translocator protein; tspo ) in brain disorders [ ] . it is acknowledged that tspo can serve to maintain homeostasis at organism, tissue, and cellular levels, including curative effects. thus, in general, it is interesting to further investigate the potential of targeting mitochondria for curative effects on various aspects of brain disorders, including liver issues. as discussed throughout this review (indicated in figures and ) , fucoidans via their interactions with sirtuins and , can affect mitochondrial functions. this includes homeostasis and metabolism, which are essential components for maintaining brain and liver health, including curative responses to injury and disease. in this respect, a study of nogueiras et al. [ ] recognized that by deacetylating a variety of proteins that induce catabolic processes, sirt and sirt coordinately increase cellular energy stores and ultimately maintain cellular energy homeostasis. it is also known that effects in the pathways controlled by sirtuins and can result in various metabolic disorders [ ] . thus, our study suggests that studying the interactions of fucoidans with sirtuins can elicit multiple metabolic benefits regarding various forms of brain disorders and liver injuries. fucoidan presents beneficial effects in brain and liver damage, due to injury and disease. an interesting consideration is that it is possible to modify fucoidan derivatives to modulate fucoidan effects. additionally, it appears that fucoidan can interact with sirtuins; in the brain (sirt ), this appears to be associated with mitochondrial function and modulation cell nuclear gene expression. in the liver (sirt ), this appears to be associated with the regulation of glucose and lipid metabolism. finally, in the brain, in particular the bbb, fucoidan interacts with p-selectin, thereby blocking macrophages from crossing and thus attenuating the inflammatory response in the brain proper. in this context, to emphasize here, it is becoming more and more recognized that prion, viral, and bacterial infections can induce neurodegeneration, as for example observed with alzheimer disease (ad) [ ] . thus, fucoidans' anti-prion, anti-viral, and anti-bacterial functions may become relevant in this respect. we believe that since fucoidans have demonstrable curative effects on various brain disorders (and also other diseases not discussed in this review) it would be worthwhile to deepen research of the various effects of fucoidans at molecular and cellular levels and the whole organism in general. leading avoidable cause of premature deaths worldwide: case for obesity evolution of inflammatory diseases free radicals, antioxidants and functional foods: impact on human health antioxidant treatment and alcoholism role of marine macroalgae in plant protection & improvement for sustainable agriculture technology. beni-suef univ phenolic composition, antioxidant activity, anticholinesterase potential and modulatory effects of aqueous extracts of some seaweeds on β-amyloid aggregation and disaggregation isolation and antioxidant capacity of fucoidan from selected malaysian seaweeds. food hydrocoll evaluation of sea mustard (undaria pinnatifida) sporophylls from south korea as fucoidan source and its corresponding antioxidant activities. fish effects of molecular weight and hydrolysis conditions on anticancer activity of fucoidans from sporophyll of undaria pinnatifida fucoidan extracted from undaria pinnatifida: source for nutraceuticals/functional foods sulfated polysaccharides from brown seaweeds saccharina japonica and undaria pinnatifida: isolation, structural characteristics, and antitumor activity therapeutic effects of fucoidan: a review on recent studies the effect of fucoidan on cellular oxidative stress and the catd-bax signaling axis in mn d cells damaged by -methyl- -phenypyridinium fucoidan protects dopaminergic neurons by enhancing the mitochondrial function in a rotenone-induced rat model of parkinson's disease protective effect of fucoidan from fucus vesiculosus on liver fibrosis via the tgf-beta /smad pathway-mediated inhibition of extracellular matrix and autophagy fucoidan alleviates acetaminophen-induced hepatotoxicity via oxidative stress inhibition and nrf translocation fucoidan extracts ameliorate acute colitis fucoidan prevents the progression of osteoarthritis in rats antioxidant activities of enzymatic extracts from brown seaweeds phycochemical constituents and biological activities of fucus spp protective activity of fucoidan and alginic acid against free radical-induced oxidative stress under in vitro and cellular system antioxidant activity of sulfated polysaccharide fractions extracted from undaria pinnitafida in vitro structure characterization and antioxidant activity of fucoidan isolated from undaria pinnatifida grown in new zealand structural characteristics and antioxidant activities of fucoidans from five brown seaweeds fucoidan from new zealand undaria pinnatifida: monthly variations and determination of antioxidant activities possibilities of fucoidan utilization in the development of pharmaceutical dosage forms prabhu, n.m. investigation of antioxidant and anticancer potential of fucoidan from sargassum polycystum isolation and characterization of fucoidans from five brown algae and evaluation of their antioxidant activity antioxidant fucoidans obtained from tropical seaweed protect pre-osteoblastic cells from hydrogen peroxide-induced damage in vitro fermentation by human faecal bacteria of total and purified dietary fibres from brown seaweeds a quantitative method to detect fucoidan in human plasma using a novel antibody intestinal absorption of fucoidan extracted from the brown seaweed, cladosiphon okamuranus detection of fucoidan in urine after oral intake of traditional japanese seaweed dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases brown seaweed egregia menziesii's cytotoxic activity against brain cancer cell lines study on absorption mechanism and tissue distribution of fucoidan therapies from fucoidan; multifunctional marine polymers reduced blood brain barrier breakdown in p-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke elevated levels of soluble p-selectin in mice alter blood-brain barrier function, exacerbate stroke, and promote atherosclerosis inhibition of leukocyte entry into the brain by the selectin blocker fucoidin decreases interleukin- (il- ) levels but increases il- levels in cerebrospinal fluid during experimental pneumococcal meningitis in rabbits the human silent information regulator (sir) homologue hsirt is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase sirt , a human sir homologue, is an nad-dependent deacetylase localized to mitochondria sirt is a nuclear nad+-dependent histone deacetylase that translocates to the mitochondria upon cellular stress sirt is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of ku salvianolic acid b attenuates brain damage and inflammation after traumatic brain injury in mice the epidemiology of traumatic brain injury pathophysiology of traumatic brain injury kda translocator protein in mitochondria-related pathology: the case of traumatic brain injury animal models of traumatic brain injury: a review low molecular weight fucoidan against renal ischemia-reperfusion injury via inhibition of the mapk signaling pathway low molecular weight fucoidan alleviates cardiac dysfunction in diabetic goto-kakizaki rats by reducing oxidative stress and cardiomyocyte apoptosis low-molecular-weight fucoidan attenuates mitochondrial dysfunction and improves neurological outcome after traumatic brain injury in aged mice: involvement of sirt fucoxanthin provides neuroprotection in models of traumatic brain injury via the nrf -are and nrf -autophagy pathways pathology of neurodegenerative diseases sirt regulation of mitochondrial quality control in neurodegenerative diseases neuroinflammation as a common feature of neurodegenerative disorders a battle you would never choose to fight": the management of neurodegenerative diseases as a societal challenge the treatment strategies for neurodegenerative diseases by integrative medicine an updated review of parkinson's disease genetics and clinicopathological correlations anti-alzheimers and anti-inflammatory activities of a glycoprotein purified from the edible brown alga undaria pinnatifida advances in algal drug research with emphasis on enzyme inhibitors in vitro screening for anti-dementia activities of seaweed extracts fucoidan-rich substances from ecklonia cava improve trimethyltin-induced cognitive dysfunction via downregulation of amyloid β production/tau hyperphosphorylation anti-hsv activity of brown algal polysaccharides and possible relevance to the treatment of alzheimer's disease sirt and mitochondrial metabolism in neurodegenerative diseases mitochondrial sirt and neurodegenerative brain disorders fucoidin, a polysaccharide inhibiting leukocyte rolling, attenuates inflammatory responses in experimental pneumococcal meningitis in rats the polysaccharide fucoidin inhibits the antibiotic-induced inflammatory cascade in experimental pneumococcal meningitis suppression of inos expression by fucoidan is mediated by regulation of p mapk, jak/stat, ap- and irf- , and depends on up-regulation of scavenger receptor b expression in tnf-alpha-and ifn-gamma-stimulated c glioma cells fucoidan protects against lipopolysaccharide-induced rat neuronal damage and inhibits the production of proinflammatory mediators in primary microglia doh-ura, k. amyloidophilic compounds for prion diseases evaluation and treatment coronavirus (covid- ). in statpearls biological activities of fucoidan and the factors mediating its therapeutic effects: a review of recent studies interactions of bovine viral diarrhoea virus glycoprotein e(rns) with cell surface glycosaminoglycans novel antiviral fucoidan from sporophyll of undaria pinnatifida (mekabu) structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga in vitro anti-hiv- activity of the bioactive compound extracted and purified from two different marine macroalgae (seaweeds) (dictyota bartayesiana studies on antiviral and immuno-regulation activity of low molecular weight fucoidan from laminaria japonica inhibition of influenza a virus infection by fucoidan targeting viral neuraminidase and cellular egfr pathway in vitro characterization of the antiviral activity of fucoidan from cladosiphon okamuranus against newcastle disease virus fucoidans as potential inhibitors of hiv- . mar. drugs characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention fucoidan extracted from hijiki protects brain microvessel endothelial cells against diesel exhaust particle exposure-induced disruption inhibitory effects of fucoidan on nmda receptors and l-type ca( +) channels regulating the ca( +) responses in rat neurons modulatory effects of dextran sulfate and fucoidan on binding and channel properties of ampa receptors isolated from rat brain fucoidan, a sulfated polysaccharide from brown algae, improves cognitive impairment induced by infusion of abeta peptide in rats fucoidan inhibits cellular and neurotoxic effects of beta-amyloid (a beta) in rat cholinergic basal forebrain neurons sirt deregulation is linked to mitochondrial dysfunction in alzheimer's disease fucoidan protects against dopaminergic neuron death in vivo and in vitro therapeutic effects of fucoidan in -hydroxydopamine-lesioned rat model of parkinson's disease: role of nadph oxidase- sirt attenuates mptp-induced nigrostriatal degeneration via enhancing mitochondrial antioxidant capacity sirtuin mediates neuroprotection of ketones against ischemic stroke effect of fucoidan treatment on collagenase-induced intracerebral hemorrhage in rats effective treatment with fucoidin for perinatal hypoxic-ischemic encephalopathy in rats neuroprotective effect of fucoidin on lipopolysaccharide accelerated cerebral ischemic injury through inhibition of cytokine expression and neutrophil infiltration fucoidan from fucus vesiculosus fails to improve outcomes following intracerebral hemorrhage in mice protective role of fucoidan in cerebral ischemia-reperfusion injury through inhibition of mapk signaling pathway pretreated fucoidan confers neuroprotection against transient global cerebral ischemic injury in the gerbil hippocampal ca area via reducing of glial cell activation and oxidative stress antioxidant properties of fucoidan alleviate acceleration and exacerbation of hippocampal neuronal death following transient global cerebral ischemia in high-fat diet-induced obese gerbils evaluation of the neuroprotective effect of sirt in experimental stroke sirtuin promotes microglia migration by upregulating cx cr sirt deficiency impairs neurovascular recovery in ischemic stroke specific metabolic rates of major organs and tissues across adulthood: evaluation by mechanistic model of resting energy expenditure protective effect of fucoidan against acetaminophen-induced liver injury the effects of fucoidan extracts on ccl -induced liver injury hepatic stellate cells and liver fibrosis low-molecular-weight fibroblast growth factor attenuates hepatic fibrosis by epigenetic downregulation of delta-like hepatoprotective effect of a fucoidan extract from sargassum fluitans borgesen against ccl -induced toxicity in rats studies on the hepatoprotective effect of fucoidans from brown algae kjellmaniella crassifolia the positive effects of fucoidans extracted from the brown seaweed saccharina japonica on protection against ccl -induced liver injury structural characterisation of algae costaria costata fucoidan and its effects on ccl( )-induced liver injury fucoidan protects against subacute diazinon-induced oxidative damage in cardiac, hepatic, and renal tissues fucoidan alleviates microcystin-lr-induced hepatic, renal, and cardiac oxidative stress and inflammatory injuries in mice licochalcone a upregulates nrf antioxidant pathway and thereby alleviates acetaminophen-induced hepatotoxicity ampk functions as an adenylate charge-regulated protein kinase the epidemiology of obesity: a big picture low molecular weight fucoidan attenuates liver injury via sirt /ampk/pgc alpha axis in db/db mice anti-metabolic syndrome effects of fucoidan from fucus vesiculosus via reactive oxygen species-mediated regulation of jnk, akt, and ampk signaling years of metformin use: a glance at the past and a look to the future effects of low-molecular-weight fucoidan and high stability fucoxanthin on glucose homeostasis, lipid metabolism, and liver function in a mouse model of type ii diabetes liver lipid metabolism the kda translocator protein as a potential participant in atherosclerosis chronic high fat, high cholesterol supplementation decreases kda translocator protein binding capacity in association with increased oxidative stress in rat liver and aorta the kda translocator protein and atherosclerosis in mice lacking apolipoprotein e. lipoprotein metabolism fucoidan improves serum lipid levels and atherosclerosis through hepatic srebp- -mediated regulation the fucoidan from the brown seaweed ascophyllum nodosum ameliorates atherosclerosis in apolipoprotein e-deficient mice effects of middle molecular weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells antithrombotic activity of oral administered low molecular weight fucoidan from laminaria japonica identification of a pro-angiogenic potential and cellular uptake mechanism of a lmw highly sulfated fraction of fucoidan from ascophyllum nodosum characterization and immunomodulatory effects of high molecular weight fucoidan fraction from the sporophyll of undaria pinnatifida in cyclophosphamide-induced immunosuppressed mice the effect of different substitute groups and molecular weights of fucoidan on neuroprotective and anticomplement activity antibacterial and antioxidant capacities and attenuation of lipid accumulation in t -l adipocytes by low-molecular-weight fucoidans prepared from compressional-puffing-pretreated sargassum crassifolium oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities sulfated polysaccharides from macroalgae for bone tissue regeneration diagnostic and therapeutic potential of tspo studies regarding neurodegenerative diseases, psychiatric disorders, alcohol use disorders, traumatic brain injury, and stroke: an update sirtuin and sirtuin : physiological modulators of metabolism role of viruses, prions and mirna in neurodegenerative disorders and dementia key: cord- - gogju n authors: swire-thompson, briony; degutis, joseph; lazer, david title: searching for the backfire effect: measurement and design considerations date: - - journal: j appl res mem cogn doi: . /j.jarmac. . . sha: doc_id: cord_uid: gogju n one of the most concerning notions for science communicators, fact-checkers, and advocates of truth, is the backfire effect; this is when a correction leads to an individual increasing their belief in the very misconception the correction is aiming to rectify. there is currently a debate in the literature as to whether backfire effects exist at all, as recent studies have failed to find the phenomenon, even under theoretically favorable conditions. in this review, we summarize the current state of the worldview and familiarity backfire effect literatures. we subsequently examine barriers to measuring the backfire phenomenon, discuss approaches to improving measurement and design, and conclude with recommendations for fact-checkers. we suggest that backfire effects are not a robust empirical phenomenon, and more reliable measures, powerful designs, and stronger links between experimental design and theory could greatly help move the field ahead. one of the most concerning notions for science communicators, fact-checkers, and advocates of truth is the backfire effect. a backfire effect occurs when an evidence-based correction is presented to an individual and they report believing even more in the very misconception the correction is aiming to rectify (lewandowsky, ecker, seifert, schwarz, & cook, ) . this phenomenon has extremely important practical applications for fact-checking, social media, and all corrective communication efforts. however, there is currently a debate in the literature as to whether backfire effects exist at all, as recent studies have failed to find them, even under theoretically favorable conditions (e.g., . in this article, we discuss the current state of the worldview and familiarity backfire effect literatures, examine barriers to measuring the correction of misinformation, and conclude with recommendations for fact-checkers and communicators. there are numerous barriers to changing inaccurate beliefs after corrections have been presented. the continued influence effect is where individuals still use inaccurate information in their reasoning and memory after a credible correction has been presented (johnson & seifert, ; lewandowsky et al., ) . there is also belief regression, where individuals initially update their belief after being exposed to the correction, but this belief change is not sustained over time (berinsky, ; kowalski & taylor, ; . in contrast to the backfire effect, these barriers are where people at least still update their beliefs in the intended direction promoted by the correction. the term backfire effect only pertains to cases where a correction inadvertently increases misinformation belief relative to a precorrection or no-correction baseline. it has also been referred to as the boomerang effect (hart & nisbet, ) or backlash (guess & coppock, ) . two backfire effects have gained popularity in the literature: the worldview backfire effect and the familiarity backfire effect. these both result in increased belief after a correction yet are thought to have different psychological mechanisms. the worldview backfire effect is said to occur when people are motivated to defend their worldview because a correction challenges their belief system . it is more likely to occur with items that are important to the individual, such as politicized "hot-button" issues or information that the individual believes in strongly (flynn, nyhan, & reifler, ; lewandowsky et al., ) . in contrast to the mechanisms of the worldview backfire effect, the familiarity backfire effect is presumed to occur when misinformation is repeated within the correction (schwarz, sanna, skurnik, & yoon, ) . for example, if one were to try to correct a misconception and stated that "eating apricot seeds does not cure cancer," the correction repeats both "apricot seeds" and "curing cancer," thus making the original misinformation more familiar. this increased familiarity is problematic because people are more likely to assume that familiar information is true-a phenomenon called the illusory truth effect (begg, anas, & farinacci, ) . in other words, this boost in familiarity when correcting misinformation is thought to be sufficient to increase the acceptance of the misinformation as true, even though it is paired with a retraction. the logic behind the worldview backfire effect stems from the motivated reasoning literature, where one's ideology and values influence how information is processed (kunda, ; wells, reedy, gastil, & lee, ) , and information that counters preexisting beliefs is evaluated more critically than belief-congruent information (taber & lodge, ) . a possible reason for the backfire effect is that people generate counter-arguments consistent with their pre-existing views to contradict the new information or correction (nyhan & reifler, ) . the landmark paper regarding the worldview backfire effect is nyhan and reifler ( ) . their first experiment corrected the misconception that weapons of mass destruction were found in iraq during the invasion. liberal individuals, whose worldview aligned with the correction, were able to successfully update their belief, whereas conservatives increased their belief in the misconception. although nyhan and reifler's second experiment failed to replicate the backfire effect for this item with the conservative group as a whole, they did find the phenomenon in a subset of conservative respondents who rated iraq as the most important problem facing the country at that point in time. the authors suggested that backfire effects may only occur when a belief is strong, and the issue is currently connected with an individual's political identity. this suggestion aligns well with subsequent research demonstrating that worldview backfire effects have almost exclusively been found in either political or attitudinal subgroups, rather than communi- there are several studies that suggest that people misremember false information to be true more often than they misremember true information to be false (peter & koch, ; skurnik, yoon, park, & schwarz, ) . although this asymmetry could indeed stem from a familiarity process (see , this does not meet the criteria of a backfire effect. see appendix a for details regarding articles that are frequently cited in support of backfire effects but do not meet backfire criteria. ties as a whole. one major problem is that beyond the scientific literature, the media and online science blogs have often overgeneralized backfire effects found in subgroups to the population as a whole and to all corrective information (e.g., science, ) . there have subsequently been worldview backfire effects reported in a variety of subgroups with misinformation regarding vaccines (in respondents with least favorable vaccine attitudes, nyhan, reifler, richey, & freed, ; in respondents with high levels of concern about vaccine side effects, nyhan & reifler, ) , climate change (in republican participants, hart & nisbet, ; in republicans with high political interest, zhou, ) , the existence of death panels (in politically knowledgeable palin supporters, nyhan, reifler, & ubel, ) , and with a fictitious scenario detailing that right-wing politicians generally misappropriate public funds more than left-wing politicians (in right-wing attentive participants, ecker & ang, ), see appendix b. in addition to observing backfire effects in widely varying subgroups, a further complication is that the dependent variable has also varied substantially between studies. these dependent variables roughly fall into three categories: belief in or agreement with a claim (e.g., nyhan & reifler, ) , behavioral intentions (e.g., nyhan and reifler, ) , or use of misinformation when answering inference questions (e.g., ecker & ang, ) . regardless of the dependent variable used, failures to find or replicate previously observed backfire effects have been widespread (e.g., garrett, nisbet, & lynch, ; nyhan, porter, reifler, & wood, ; schmid & betsch, ; swire, berinsky, lewandowsky, & ecker, ; swire-thompson, ecker, lewandowsky, & berinsky, ; weeks, ; weeks & garrett, ) , even when using identical items that previously elicited the phenomenon. for example, haglin ( ) used identical methods and vaccine-related items to those from nyhan and reifler ( ) and failed to find any evidence of a backfire effect. the largest failure to replicate to-date was by wood and porter ( ) , conducting five experiments with over , participants. the items were specifically chosen to be important ideological issues that would be theoretically conducive to a worldview backfire effect. the authors found that out of issues corrected, no items triggered a backfire effect. much of the literature has interpreted these failures to replicate to indicate that either (a) the backfire effect is difficult to elicit on the larger group level, (b) it is extremely item-, situation-, or individual-specific, or (c) the phenomenon does not exist at all. see appendix b for details regarding which studies found a worldview backfire effect, which did not, and the dependent variable(s) used in each. in contrast to the ideological mechanisms behind the worldview backfire effect, familiarity backfire effects are often presumed to occur due to the correction increasing the misinformation's processing fluency. in other words, the correction of "apricot seeds do not cure cancer" increases the ease in which "apricot seeds" and "cancer" are retrieved and processed (schwarz, newman, & leach, ) . however, the specific mechanisms of how repetition could lead to an increase in perceived truth are currently under debate (see unkelbach, koch, silva, & garcia-marques, , for a review). furthermore, the familiarity backfire effect has often been conflated with the more well-established illusory truth effect. the former refers to increasing belief due to information repetition within a correction and has little to no empirical support, whereas the latter refers to increasing belief due to information repetition in the absence of a correction and is a robust empirical phenomenon (fazio, brashier, payne, & marsh, ) . the original notion of the familiarity backfire effect stems from an unpublished manuscript , as cited in schwarz et al., ) where participants who viewed a flyer with "myth vs. fact" information regarding the flu vaccine reported less favorable attitudes toward vaccination than those who did not view the flyer. although this paper is highly cited (e.g., berinsky, ; cook, bedford, & mandia, ; gemberling & cramer, ; lilienfeld, marshall, todd, & shane, ; peter & koch, ; pluviano, watt, ragazzini, & della sala, ; , it is difficult to evaluate given that it remains unpublished. there have been failures to directly replicate this study (cameron et al., ) , and the phenomenon has not been elicited under theoretically conducive circumstances, including a three-week delay between corrections being presented and belief being measured . furthermore, since worldview backfire effects have been demonstrated using vaccine stimuli (e.g., nyhan et al., ) , it is unclear whether the skurnik et al. ( ) backfire effect was due to worldview or familiarity mechanisms. this potential misattribution also applies to pluviano, watt, and della sala ( ) , pluviano et al. ( ) , and berinsky ( ) , where the backfire effects were reportedly due to familiarity mechanisms yet could have been due to worldview since the experiments exclusively used politicized information. see appendix c for details regarding which studies found a familiarity backfire effect, which did not, and the dependent variable(s) used in each study. there have also been recent findings that do not align with the familiarity backfire notion. for instance, simply tagging misinformation as false-with no further explanation as to why it is false-has shown to substantially reduce belief, both relative to a pre-correction within-subject baseline and in comparison to a control group who did not receive a correction at all (ecker, o'reilly, reid, & chang, ) . furthermore, if the familiarity backfire effect were genuine, then a practical recommendation would be to avoid repeating the misconception when presenting the correction. however, a recent meta-analysis of studies found that there was no significant difference in belief updating when comparing whether or not the initial misconception was repeated within the correction (walter & tukachinsky, ) . several recent studies not included in this meta-analysis found that repeating the misconception immediately prior to the correction facilitated belief updating (carnahan & garrett, ; ecker, hogan, & lewandowsky, ) , and that explicitly repeating misinformation prior to the correction is more effective than only implying it (rich & zaragoza, ) . although these findings collectively oppose the familiarity backfire notion, they align well with theoretical accounts that the co-activation of the misconception and corrective information facilitates knowledge revision (kendeou & o'brien, ) . it is possible that pairing the misconception and correction increases the likelihood that people notice discrepancies between the two, facilitating the integration of new information into their existing mental model (elsey & kindt, ; kendeou, butterfuss, van boekel, & o'brien, ) . finally, the illusory truth effect and familiarity backfire effect are thought to rely on the same mechanisms, and evidence suggests that the illusory truth effect can be eliminated when veracity is made salient to participants. brashier, eliseev, and marsh ( ) found that when participants were simply asked to rate statements for accuracy, the illusory truth effect was wiped out. in other words, if participants knew that the item was false, the illusory truth effect was not elicited if participants were instructed to focus on accuracy both immediately and after a two-day period (also see rapp, hinze, kohlhepp, & ryskin, ) . in sum, although repeated exposure to misinformation alone certainly increases belief, the weight of evidence suggests that this rarely, if ever, occurs when the misinformation is paired with a clear and salient correction. it remains theoretically possible that there are circumstances where the familiarity boost of the misconception outweighs the corrective element (for example, when attention is divided, troyer & craik, ) , but this has not been observed empirically. future research can more specifically investigate how familiarity boosts that increase belief and corrections that decrease belief interact. for instance, pennycook, cannon, and rand ( ) found that the increase in belief due to a single prior exposure of fake news was approximately equivalent to the reduction of belief when the fake news was accompanied by a "disputed by third-party fact-checkers" tag. the above review suggests that backfire effects are not a robust empirical phenomenon and it could be the case that they represent an artifact of measurement error. misinformation is still a relatively new field and more reliable measures and more powerful designs are needed to move the field ahead and determine the fate of backfire effects. here we suggest some experimental and theoretical steps that could improve the quality of the evidence. in particular, we suggest that future studies should carefully consider measurement reliability, when possible use more powerful designs with greater internal validity, be aware of sampling and subgroup issues, and take care in linking and chan ( , study ), nyhan and reifler ( ) , cobb, nyhan and reifler, ( , study ), huang ( , study and ), thorson ( , study ; measures with particular theories. the recommendations below could also be applicable to misinformation studies in general, rather than studies that specifically examine backfire effects. reliability is defined as the consistency of a measure, that is, the degree to which a test or other measurement instrument is free of random error, yielding the same results across multiple applications to the same sample (vandenbos, ) . although other areas of psychology have been highly focused on measuring the reliability of their assessments (e.g., individual differences, neuropsychology, attitude research ), this has largely not been the case with misinformation science. a common methodological weakness in this area is the reliance on a single item to measure a belief or agreement. single items are noisy and often have poor reliability (jacoby, ; peter, ) , and under these conditions statistical significance may convey very little information (loken & gelman, ) . given that % of backfire effects found in our review of the worldview and familiarity literatures are found with single item measures, we must consider that poor item reliability could be contributing to this phenomenon. see appendices b and c for details regarding the number of items in the measures of each study. indeed, we found that the proportion of backfire effects observed with single item measures ( %) was significantly greater than those found in multiple item measures ( %; z = . , p = . ). quantifying item-level reliabilities could greatly aid in interpretation, given that a backfire effect observed on a more reliable item would have greater meaning than if found on an unreliable item. perhaps the simplest approach to measure reliability for a single item is to include a test-retest group where participants rate their beliefs and then re-rate them after an interval has passed. this approach can be done in a control group or during a pre-baseline period in a waitlist design, if effects are expected to be extremely sample-specific. although multi-item measures are typically more reliable than single item measures, there are occasions where single items can be sufficiently reliable (sarstedt & wilczynski, ) . it is typically recommended that single-item test-retest reliability should be ≥. (nunnally, ; wanous & hudy, ) . unfortunately, because so few studies in the field of misinformation have reported any measure of reliability, it is hard to know which, if any, of the items have sufficient reliability to adequately measure backfire effects. implementing multi-item measures could both produce more reliable measures and inspire confidence that we are measuring generalizable constructs (e.g., whether items are perceived to be important or "hot-button" issues) rather than item-specific effects. one noteworthy study by horne, powell, hummel, and holyoak ( ) incorporated a -item scale (reliability: α = . ) to measure vaccine attitude changes, which correlated well with whether parents have ever refused a vaccination (r = − . ). notably, these data were subsequently reanalyzed by another group and interpreted at the individual item level because they thought the items represented separate constructs (betsch, korn, & holtmann, ) . this example not only shows that a multiitem measure can be highly reliable, but also demonstrates the challenges of creating a widely accepted multi-item measure and the current bias in the field toward analyzing individual items. in light of the replication crisis in psychology and beyond (open science collaboration, ) , all fields in the behavioral sciences have begun to focus more on measurement reliability, and a greater consideration of this issue in misinformation research could substantially improve the interpretation and replicability of our findings, particularly with regards to backfire effects. a related issue in measuring the reliability of beliefs is that some beliefs may be stronger and more well-formulated than others. less formulated beliefs may themselves be highly variable independent of measurement error. one approach to addressing this is to use several items to measure participants' within-belief consistency (e.g., higher consistency would indicate more formulated beliefs) as well as explicitly asking participants to rate how well-formed they perceive their beliefs to be. a final measurement issue that could influence backfire effects is that unreliable measures have more random error and are more susceptible to regression to the mean, where extreme values at baseline testing become less extreme at follow-up testing (bland, ) . a regression-to-the-mean effect may be particularly problematic for individuals or subgroups in pre-post design studies who report low pre-correction belief, given that the effect could increase post-correction belief. thus, this phenomenon could potentially result in spurious backfire effects. in figure we plot simulated data to illustrate this point. panel a shows test-retest data for an item with poor reliability (pearson's r = . ) whereas panel b shows test-retest data for an item with good reliability (pearson's r = . ). note that at retest, data points at the extremes in the unreliable measure move more toward the mean from the line of equality (line where time = time ) compared to the reliable measure. panels c and d shift these data points down . points as if a correction has been elicited. the gray area represents the "backfire zone" where post-correction belief is higher than pre-correction belief. participants with low pre-correction belief are more likely to be found in the backfire zone when the item is unreliable (panel c) than when it is reliable (panel d). though this is an oversimplified example, it shows how poor reliability and regression to the mean can give rise to spurious backfire effects in individuals and subgroups. it should be noted that effects of regression to the mean can be substantially mitigated by limiting exploratory subgroup analyses as well as including a well-matched test-retest or placebo group for comparison. in terms of design, studies of the backfire effect have varied widely, with most examining between-groups differences of correction versus no correction groups (using -point scales, garrett et al., ; nyhan & reifler, ; -point scales, wood & porter, ; zhou, ; percentages of participants accepting, rejecting, or unsure about the misinformation, berinsky, ; or counting the mean number of references to corrected misinformation after reading a fictitious scenario, ecker & ang, ) . in these studies, participants are typically randomly assigned to treatment or control, and participants' beliefs are only measured at one time point, with the experimental group being assessed after the correction. in addition to these post-only with control studies, a handful of studies have used within-subject pre versus post correction differences (using -point belief scales, aird, ecker, swire, berinsky, & lewandowsky, ; swire-thompson et al., ) , though nearly all have lacked test-retest control groups (for an exception, see horne et al., ) . other studies have used idiosyncratic approaches such as performing qualitative interviews (prasad et al., ). post-test only with control designs have an advantage in that they may be more practically feasible, often only requiring a single testing session. another advantage of this design is that researchers are able to test belief without a further familiarity boost, which is potentially important for studies attempting to examine the familiarity backfire effect. post-test only with control designs are also thought to limit carryover effects associated with pre-post designs, although it is questionable whether carryover effects are a concern in misinformation studies. if carryover effects were problematic, participants in pre-post studies would provide post-correction responses that are similar to their initial response, and the belief change observed in these designs would be significantly smaller than post-test only with control designs. however, effect sizes of belief change in pre-post studies are similar in magnitude to post-test only with control designs, suggesting that the impact of carryover effects is likely minimal. in fact, found larger decreases in belief in false claims if the manipulation was within-subjects rather than between subjects. furthermore, effect sizes for belief change in pre versus post-correction studies are typically large, especially in conditions where there is no delay between pre and posttest, where one would expect carryover effects to be most pronounced prasad developed a technique called "challenge interviews" where interviewers presented participants with substantive challenges to their political opinions. they themselves do not claim their findings were a backfire effect in their paper but are frequently cited in support of the worldview backfire phenomenon. they found that the most popular strategy was "attitude bolstering", bringing facts that support one's position to mind without directly refuting the contradictory information. belief change was not measured. simulated data of participants on a misinformation item. panel a illustrates test-retest data for an item with poor reliability (r = . ) and panel b for an item with acceptable reliability (r = . ). the dotted lines in panel a and b represent the line of equality, on which all data points would lie if the measurements at time and time were identical. note greater regression-to-the-mean effects in the unreliable data than the reliable data, indicated by the arrows. panels c and d shift these data points down . points as if a correction has been elicited. the gray area represents the "backfire zone." panel c shows pre/post data demonstrating that subjects reporting low pre-correction beliefs may be more likely to result in spurious backfire effects if the measures are unreliable. an important issue with designs such as the post-test only with control, is that even with randomization the groups may not be adequately matched at baseline (morris, ) . this issue may be particularly problematic with smaller sample sizes. the prevalence of this problem is hard to assess because many studies fail to report important demographic and political characteristics and rarely compare these variables between experimental and control groups. it is easy to imagine small group differences in demographics and political polarization producing the appearance of backfire effects. pre-post designs are a viable alternative to post-test only designs and are not as affected by sampling issues. however, pre-post designs without a control group (e.g., could also potentially suffer from problems related to repeated testing effects such as regression to the mean (vickers & altman, ) , which could drive backfire effects, particularly at the subgroup level (see figure ) . a more powerful design that can overcome many of these issues is a pre-post control group design, where participants are randomly assigned to intervention or control conditions, and participants are measured both before and after the intervention or control is administered (schulz, altman, & moher, ) . this design is common in clinical intervention studies, and compared to post-test only between-subject designs, it offers a boost in statistical power. further, because the experimental manipulation is within-subjects, the internal validity of this design does not solely depend on random assignment (charness, gneezy, & kuhn, ) . one important question for this design with regards to the backfire effect is what the best control condition would be. though a test-retest control is a starting point, a more sophisticated approach would be to employ one or multiple placebo conditions (e.g., where participants are given information related to the misinformation that does not correct nor confirm it). the only study that we are aware of that has used this design in the context of backfire effects is horne et al. ( ) . they compared vaccine attitudes in adults before and after random assignment to either (a) autism correction, (b) disease risk information, or (c) reading an unrelated scientific vignette (control condition). notably, they did not find backfire effects at the group level. when exploring subgroups, those with the least favorable attitudes toward vaccines were the most receptive to change. however, those with the most favorable attitudes to vaccines did show a backfire, which the authors interpreted as regression to the mean. though this type of design may be more participantand resource-demanding than post-only with control or pre-post designs, it could help provide a more powerful evaluation of the possible presence of backfire effects. we finally turn to demand characteristics and whether participants' expectations for how they are meant to behave facilitate backfire effects (orne, ) . demand characteristics generally lead participants to be a "good subject," encouraging them to behave in a manner that confirms the hypothesis of the experimenter (nichols & maner, ) . if the participant does receive cues that the experiment is about the efficacy of belief updating, they are likely to further reduce their belief after viewing a correction, rather than report increasing their belief. the only study, to our knowledge, that explicitly asked subjects about the purpose of the experiment in a debriefing questionnaire was conducted by ecker, lewandowsky, and tang ( ) , and they found that virtually all participants did indeed correctly assume that the experiment was about memory updating. it is nonetheless important that future studies quantify the extent to which demand characteristics influence misinformation experiments in general. should future investigations deem them problematic, one method to reduce demand characteristics is to blind participants to the goals of the study and, for in-lab studies, blind experimenters (see orne, ). another step forward for backfire studies is to be more aware of sampling and subgroup issues because the subgroups in which backfire effects have been found vary substantially. as we previously noted, the internal validity of between-groups post-test only designs can be seriously undercut by demographic differences between the groups, and more thorough between-groups demographic comparisons in these studies is essential. further, though some studies that test for backfire effects have used previously defined subgroups (e.g., ecker & ang, ; haglin, ; , some of the subgroup analyses reported may have been post hoc. post hoc subgroup analyses have been harshly criticized in the clinical literature (wang, lagakos, ware, hunter, & drazen, ) because it is often unclear how many are performed and whether they are motivated by inspection of the data. thus, in future studies, subgroup analyses derived from data inspection should be explicitly identified as exploratory (as done by nyhan & reifler, ) , and subgroup analyses should be pre-specified, or better yet, pre-registered. regarding the worldview backfire effect, fact-checkers can rest assured that it is extremely unlikely that, at the broader group level, their fact-checks will lead to increased belief in the misinformation. meta-analyses have clearly shown that corrections are generally effective and backfire effects are not the norm (e.g., chan, jones, hall jamieson, & albarracín, ; walter & murphy, ) . furthermore, given that research has yet to systematically show backfire effects in the same subgroups, practitioners should not avoid giving corrections to any specific subgroups of people. fact-checkers can therefore focus on other known issues such as getting the fact-checks to the individuals who are most likely to be misinformed. regarding the familiarity backfire effect, avoiding the repetition of the original misconception within the correction appears to be unnecessary and could even hinder corrective efforts kendeou & o'brien, ) . we therefore instead suggest designing the correction first and foremost with clarity and ease of interpretation in mind. although the familiarity backfire effect lacks evidence, we must be aware that the illusory truth effect in the absence of corrections or veracity judgments is extremely robust. therefore, when designing a correction, the misinformation should always be clearly and saliently paired with the corrective element, and needless repetitions of the misconception should still be avoided. for instance, given that many individuals do not read further than headlines (gabielkov, ramachandran, chaintreau, & legout, ) , the misconception should not be described in the headline alone with the correction in smaller print in the text below (ecker, lewandowsky, chang, & pillai, ; . adding the corrective element within the headline itself, even if it is simply a salient "myth" tag associated with the misconception, can be considered good practice. although improvements in both experimental measures and designs are important, oberauer and lewandowsky ( ) highlight that another cause of poor replicability is weak logical links between theories and empirical tests. future research could more explicitly manipulate key factors presumed to influence belief updating, whether it be fluency, perceived item importance, strength of belief, complexity of the item wording, order of corrective elements, internal counter-arguing, source of the message, or participants' communicating disagreement with the correction. focusing on theoretically meaningful factors could help to better isolate the potential mechanisms behind backfire effects or the continued influence effect in general. further-more, it would be beneficial to be aware of other competing factors to avoid confounds. for example, when investigating the effects of familiarity, one could avoid exclusively using issues presumed to elicit worldview backfire effects (e.g., vaccines, skurnik et al., ) . additionally, given that responses to corrections are likely heterogeneous, it would be beneficial to use a wide variety of issues in experiments that vary on theoretically meaningful criteria to dissociate when backfire effects occur and when they do not. future research should also empirically investigate common recommendations that stem from the familiarity backfire effect notion which have yet to be thoroughly examined. for example, it is unclear whether belief updating is fostered by presenting a "truth sandwich" to participants, stating the truth twice with the falsehood between (sullivan, ) . preliminary findings suggest that a "bottom-loaded" correction, which first states the misconception followed by two factual statements, could be more effective than the truth sandwich (anderson, horton, & rapp, ) , although further research is required prior to firm recommendations being made. finally, there are additional occasions where corrections could be counter-productive that require empirical investigation. for instance, correcting facts in public political debate might not always be advisable, because it involves the acceptance of someone else's framing, allowing the person who promulgated the original falsehood to set the agenda (lakoff, ; lewandowsky, ecker, & cook, ) . furthermore, broadcasting a correction where few people believe in the misconception could be a legitimate concern, since the correction may spread the misinformation to new audiences (kwan, ; schwarz et al., ) . for example, if the bbc widely publicized a correction to a misconception that its readership never believed to begin with, it will not reap the benefits of belief reduction, and those who do not trust this source may question its conclusion. the next crucial step is to examine such instances with real-world scenarios on social media or fact-checking websites. in today's fast-paced information society it is extremely important to understand the efficacy of corrections, the exact circumstances under which they have no impact, or even backfire. the current state of the literature calls into question the notion of the backfire effect and more rigorous studies are needed to determine if there are circumstances when these effects reliably occur. indeed, given the current coronavirus pandemic and the rampant misinformation that has accompanied it, understanding the parameters of misinformation correction is particularly crucial. in sum, the current review suggests that backfire effects are not a robust empirical phenomenon, and more reliable measures, powerful designs, and stronger links between experimental design and theory could greatly help move the field ahead. bst conceived of the idea and wrote the majority of the manuscript. jd contributed sections to the manuscript, particu-larly the measurement and design considerations. bst, jd, and dl edited the manuscript. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.. this project was funded by a hewlett packard foundation grant. all authors acknowledge that the material presented in this manuscript has not been previously published and is not under consideration by any other journal. we thank nicholas miklaucic for proofreading and verifying the information in appendices a, b, and c. article type of backfire reason for exclusion skurnik et al. ( ) familiarity this study did not meet the criteria of a backfire effect since post-correction belief is not compared to a pre-correction or no-correction baseline. they considered a backfire to be misremembering more false items as true than true items to be false. for a critique, see . weaver, garcia, schwarz, & miller ( ) familiarity this study is regarding the illusory truth effect, but since it does not provide corrections to participants it cannot comment on the backfire effect. prasad et al. this study consisted of qualitative interviews and belief change was not measured. peter and koch ( ) familiarity this study did not meet the criteria of a backfire effect since post-correction belief is not compared to a pre-correction or no-correction baseline. they considered the backfire to be misremembering more false items as true than true items as false. holman and lay note. dependent variables and studies that report backfire effects are highlighted in gray. a in addition to intent to vaccinate, skurnik et al. ( ) also considered the misremembering of more myths thought to be true than facts thought to be false to stem from famili arity mechanisms. we have excluded this element from the table because they do not meet the criteria of a backfire effect since it is not in comparison to a pre-correction or no-correction baseline. b berinsky ( )'s second experiment found the trend th at repetition of the misinformation prior to the correction made respondents less likely to reject it than if the misinformation was not repeated. however, we exclude this study since there is no pre-correction or no-correction baseline. c we exclude the first experiment from pennycook et al. ( ) it was investigating the illusory truth effect. since it did not provide corrections to participants it cannot comment on the backfire effect. does truth matter to voters? the effects of correcting political misinformation in an australian sample hungry for the truth: evaluating the utility of dissociation of processes in belief: source recollection, statement familiarity, and the illusion of truth rumors and health care reform: experiments in political misinformation don't try to convert the antivaccinators, instead target the fence-sitters the sage encyclopedia of communication research methods an initial accuracy focus prevents illusory truth patient knowledge and recall of health information following exposure to "facts and myths" message format variations processing style and responsiveness to corrective information debunking: a meta-analysis of the psychological efficacy of messages countering misinformation experimental methods: between-subject and within-subject design beliefs don't always persevere: how political figures are punished when positive information about them is discredited raising climate literacy through addressing misinformation: case studies in agnotologybased learning the debunking handbook political attitudes and the processing of misinformation corrections do people keep believing because they want to? preexisting attitudes and the continued influence of misinformation reminders and repetition of misinformation: helping or hindering its retraction? the effects of subtle misinformation in news headlines do people keep believing because they want to? preexisting attitudes and the continued influence of misinformation correcting false information in memory: manipulating the strength of misinformation encoding and its retraction explicit warnings reduce but do not eliminate the continued influence of misinformation the effectiveness of short-format refutational fact-checks techniques of attitude scale construction tackling maladaptive memories through reconsolidation: from neural to clinical science knowledge does not protect against illusory truth the ab scales: an operational definition of belief and attitude the nature and origins of misperceptions: understanding false and unsupported beliefs about politics social clicks: what and who gets read on twitter? proceedings of the acm sigmetrics international conference on measurement and modeling of computer science undermining the corrective effects of media-based political fact checking? the role of contextual cues and naïve theory: undermining corrective effects expert testimony on sensitive myth-ridden topics: ethics and recommendations for psychological professionals does counter-attitudinal information cause backlash? results from three large survey experiments the limitations of the backfire effect boomerang effects in science communication how motivated reasoning and identity cues amplify opinion polarization about climate mitigation policies they see dead people (voting): correcting misperceptions about voter fraud in the us presidential election countering antivaccination attitudes a war of (mis) information: the political effects of rumors and rumor rebuttals in an authoritarian country consumer research: how valid and useful are all our consumer behavior research findings? a state of the art review sources of the continued influence effect: when discredited information in memory affects later inferences integrating relational reasoning and knowledge revision during reading processing inaccurate information: theoretical and applied perspectives from cognitive science and the educational sciences reducing students' misconceptions with refutational teaching: for long-term retention, comprehension matters the case for motivated reasoning responsible reporting in an age of information disorder moral politics: how liberals and conservatives think misinformation and its correction: continued influence and successful debiasing beyond misinformation: understanding and coping with the "post-truth" era a method of constructing an attitude scale. scaling: a sourcebook for behavioral scientists the persistence of fad interventions in the face of negative scientific evidence: facilitated communication for autism as a case example measurement error and the replication crisis estimating effect sizes from pretest-posttestcontrol group designs the good-subject effect: investigating participant demand characteristics estimating the reproducibility of psychological science taking fact-checks literally but not seriously? the effects of journalistic fact-checking on factual beliefs and candidate favorability when corrections fail: the persistence of political misperceptions effective messages in vaccine promotion: a randomized trial does correcting myths about the flu vaccine work? an experimental evaluation of the effects of corrective information the hazards of correcting myths about health care reform addressing the theory crisis in psychology the nature of hypnosis: artifact and essence demand characteristics and the concept of quasicontrols prior exposure increases perceived accuracy of fake news reliability: a review of psychometric basics and recent marketing practices when debunking scientific myths fails (and when it does not). the backfire effect in the context of journalistic coverage and immediate judgments as prevention strategy there must be a reason": osama, saddam, and inferred justification misinformation lingers in memory: failure of three pro-vaccination strategies parents' beliefs in misinformation about vaccines are strengthened by provaccine campaigns reducing reliance on inaccurate information the continued influence of implied and explicitly stated misinformation in news reports more for less? a comparison of single-item and multi-item measures effective strategies for rebutting science denialism in public discussions consort statement: updated guidelines for reporting parallel group randomised trials metacognitive experiences and the intricacies of setting people straight: implications for debiasing and public information campaigns making the truth stick and the myths fade: lessons from cognitive psychology what is the backfire effect education about flu can reduce intentions to get a vaccination processing political misinformation: comprehending the trump phenomenon the role of familiarity in correcting inaccurate information they might be a liar but they're my liar: source evaluation and the prevalence of misinformation how warnings about false claims become recommendations instead of trump's propaganda, how about a nice 'truth sandwich'? the washington post motivated skepticism in the evaluation of political beliefs belief echoes: the persistent effects of correction misinformation belief echoes: the persistent effects of corrected misinformation identity and epistemic emotions during knowledge revision: a potential account for the backfire effect the effect of divided attention on memory for items and their context truth by repetition: explanations and implications apa dictionary of psychology analysing controlled trials with baseline and follow up measurements how to unring the bell: a metaanalytic approach to correction of misinformation a meta-analytic examination of the continued influence of misinformation in the face of correction: how powerful is it, why does it happen, and how to stop it statistics in medicine-reporting of subgroup analyses in clinical trials single-item reliability: a replication and extension inferring the popularity of an opinion from its familiarity: a repetitive voice can sound like a chorus emotions, partisanship, and misperceptions: how anger and anxiety moderate the effect of partisan bias on susceptibility to political misinformation electoral consequences of political rumors: motivated reasoning, candidate rumors, and vote choice during the information distortion and voting choices: the origins and effects of factual beliefs in initiative elections the elusive backfire effect: mass attitudes' steadfast factual adherence boomerangs versus javelins: how polarization constrains communication on climate change note. dependent variables and studies that report backfire effects are highlighted in gray. we also highly recommend viewing guess and coppock ( ) , although the findings are omitted from this table because they presented counter-attitudinal information rather than corrective information to participants. a trevors et al. ( ) found that self-concept negatively predicted attitudes after reading refutation text more than expository text, which the authors concluded as evidence of a backfire effect. we exclude the attitude dependent variable from this table because there was no between-subject control group and the pre-post attitude tests could not be compared. key: cord- -qw pf r authors: greaves, peter title: vii digestive system date: - - journal: histopathology of preclinical toxicity studies doi: . /b - - / - sha: doc_id: cord_uid: qw pf r publisher summary this chapter deals with the digestive system. the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity, and derangement of saliva production may lead to loss of integrity of the oral mucosa. drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. inflammation of the oral cavity may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis), and the peridontal tissues (peridontitis). therapeutic agents can induce inflammatory lesions in the tongue. moreover, a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species. the oral mucosa can be damaged by excessive local trauma from foreign materials, hard fragments in food and damaged teeth may produce ulceration of the mucosa with subsequent infection. however, the oral mucosa may show manifestations of local or systemic disease or derangement produced by therapeutic agents. excessive contact by therapeutic agents such as aspirin, potassium supplements and corticosteroids have been reported to produce local ulceration in the mouth (zentler-monro and northfield, ) . the increased use of mouthwashes over the last years has resulted in a number of reported adverse effects to the buccal mucosa in people (gargari and kabani, ) . systemic disorders produced by anticoagulants or chemotherapeutic drugs may also be evident by bleeding or ulceration in the oral cavity (goepp, ) . buccal ulceration is also described as part of a generalised hypersensitivity reaction to drugs (zentler-monro and northfield, ) . the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity and derangement of saliva production may lead to loss of integrity of the oral mucosa. drugs that effect motor co-ordination can give rise to drooling and disruption of cricopharyngeal co-ordination (wyllie et al., ) . drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. indeed, many alterations in the oral mucosa are those that are more readily detected by careful clinical and macroscopic observation rather than exhaustive histopathological examination of the buccal mucosa in laboratory animals -provided the basic toxicity profile of a novel agent is adequately assessed in the usual preclinical studies. oral irritation studies are used in the testing of products for use in the oral cavity, mainly for surgical, dental and hygiene purposes but also therapeutic agents administered by the sublingual route. this route may be selected for substances that are broken down in the stomach or show a rapid first pass effect. as it is technically not feasible to perform full preclinical toxicity studies by the sublingual route, conventional oral or parenteral routes are preferred for systemic toxicity studies on such compounds. the choice of the best route will to a large extent be dictated by pharmacokinetic considerations. however, it is necessary to assess local irritancy potential to oral mucosa using a laboratory animal model. test species for oral irritation studies are usually rats, hamsters (cheek pouch), guinea pigs, dogs or primates using gross and histopathological assessment. a similar scheme to that employed in the histological assessment of skin irritancy is appropriate. inflammation of the oral cavity (stomatitis) may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis) and the peridontal tissues (peridontitis). although inflammatory lesions are found sporadically in untreated laboratory rodents, dogs and primates, stomatitis can be induced by systemic administration of high doses of therapeutic agents. anticancer and antimitotic agents are particularly liable to induce stomatitis. a notable example is bleomycin that is capable of producing stomatitis as part of its general effect on squamous cells (thompson et al., ) . in humans, the adverse effects on therapeutic ionising radiation on the salivary glands may also give rise to inflammatory changes in the oral cavity (fox, ) diuretics and other agents, which are capable of producing severe electrolyte disturbances and uraemia at excessive doses, can also produce stomatitis when then are administered in high doses to laboratory animals (garthoff et al., ) . these lesions may be analogous to the well-described association of ulcerative stomatitis and uraemia in man and laboratory animals (boyd, ; barker and van dreumel, ) . the dog appears very sensitive to the ulcerogenic effects of uraemia in the oral cavity, although as there is a poor correlation between actual levels of blood urea and stomatitis, other biochemical factors are undoubtedly involved. compounds, which effect pigmentation of the skin, can produce similar changes in pigmented oral mucosa. a number of drugs including chlorpromazine, quinacrine, chloroquine, amodiaquine and pyrimethamine cause pigmentation of the oral mucosa in man notably over the hard palate. chloroquine and pyrimethamine have also been shown to significantly increase numbers of active melano-cytes within the palatal mucosa of pigmented da rats when treated orally for weeks (savage et al., ) . melanocytes in treated rats were shown to be enlarged and packed with melanin pigment and to possess extensive arborisation of cell processes between squamous cells. an experimental inhibitor of platelet aggregation, which produced pigment loss in the dark hair of long-evans rats and the skin of beagle dogs, also induced pallor of the normally pigmented oral mucous membranes in dogs (gracon et al., ; walsh and gough, ) . apart from loss of pigment, the histology of the mucous membranes and skin was normal. the tongue is conveniently sectioned for histological study, although reliance is often placed on careful visual inspection, because the usefulness of systematic histological examination of the tongue in routine preclinical safety studies has not been clearly established. a few lesions occur which are fairly specific to the tongue. amyloid may become deposited in the muscular and connective tissue of the tongue in amyloid-prone species, particularly mice (dunn, ) . mice, especially dba and dba/ ncrj strains, are liable to develop calcification in the lingual muscle spontaneously, even at a young age (imaoka et al., ) . calcified lesions are seen in the longitudinal muscle under the dorsolateral epithelium and the central part of the tongue, which, when severe, are associated with inflammation, granulation tissue, polypoid change, hyperplasia of the overlying squamous epithelium and ulceration. the histogenesis of this lesion is uncertain. in the dba/ ncrj mice, mineralisation of the tongue is associated with myocardial and aortic mineralisation (doi et al., ) . therapeutic agents can induce inflammatory lesions in the tongue. an example is provided by the investigational anticancer immunotoxin, zd , a mouse monoclonal antibody (c ) against colorectal carcinoma antigen conjugated to recombinant ricin a-chain. when administered to wistar-derived rats, this agent produced myocyte necrosis and inflammation specifically located below the ventral subepithelial surface of the tongue (westwood et al., ) . as the changes were different to the low grade myositis seen elsewhere in treated animals, these authors speculated that the changes in the tongue may have been related to the particular receptor profile of this area targeted by the monoclonal antibody. in common with other changes induced in the digestive tract of rats and cynomolgus monkeys by the administration of recombinant human epidermal growth factor, the tongue showed squamous epithelial hyperplasia characterised by a uniform increase in the thickness of the squamous epithelium in both species (breider et al., ; reindel et al., ) . at high doses, the squamous epithelium of the tongue of the primates was twice the thickness of control mucosa associated with elongation of rete pegs. teeth are usually only inspected by naked eye in conventional toxicity studies and this is appropriate for the assessment of a mature dentition. however, there has been an increasing awareness of dental lesions in toxicity studies, particularly as the teeth are visualised when the maxilla is examined histologically in inhalation studies. study of the rodent dentition in inhalation studies has shown that spontaneous lesions of the dentition are quite common. in one laboratory, malformations (dental dysplasia) of the maxillary incisors were observed in % of female and % of male cd- mice and . % female and . % sprague-dawley rats in and month inhalation studies respectively (losco, ) . the rat incisor and its pathology has been the subject of an excellent review (kuijpers et al., ) . unlike in humans, the rodent incisor continues to grow and differentiate throughout life and is renewed every - days. located at the centre of the tooth is the vascular pulp. this is surrounded by proliferating ondotoblasts which form predentin which when calcified becomes dentin. surrounding ameloblasts when induced by the presence of dentin produce the overlying enamel layer. it is these active cellular layers, which can be modified or damaged by xenobiotics, vitamin deficiencies, calcium, phosphate or magnesium deficiency, parathyroidectomy, hypophysectomy, hyperparathyroidism, adrenal insufficiency and fluorosis (kuijpers et al., ) . although in humans the mature dentition is no longer growing, in children the dentition is in a growth phase that starts in utero and lasts into the second decade. as increasing numbers of children survive malignant disease, damage to the mature dentition can occur as a result of cytotoxic therapy during childhood. clinical study of the teeth of children treated for malignancy have shown increased incidence of enamel hypoplasia and missing teeth (welbury et al., ) . histological examination of teeth from children treated with vincristine or combination chemotherapy for malignant disease has demonstrated prominent incremental lines in dentine correlating with the number of times the intravenous cytotoxic agents were administered (macleod et al., ) . it has also been shown that vincristine, a drug which interferes with the assembly of microtubules and reduces secretory activity in a number of cells including osteoblasts and chondroblasts, also effects dentine formation in the rat incisor (stene and koppang, ) . two weeks following a single intravenous dose of vincristine to young adult rats, a faint incremental line in the dentine was observed, probably a reflection of a direct effect of the drug on the dentinogenic tissue at the time of injection. at higher doses, focal niche-like or punched out defects in dentine were observed, expression of more severe injury to highly sensitive dentinogenic populations at the time of injection (stene and koppang, ). the precise mechanism of damage is not fully understood although decreased secretion of dentine matrix by odontoblasts has been demonstrated. calcification appears unaltered (stene and koppang, ) . administration of the alkylating anticancer agent cyclophosphamide or a sin-gle exposure to ionising radiation, produces localised niche-like or punched out defects in the rat incisor, rather than the more diffuse changes induced by vincristine. this presumably reflects more localised injury to a sensitive subpopulation of dentinogenic cells (koppang, ; vahlsing et al., ) . anticonvulsant drugs also produce changes in the dentition of man and experimental animals. in humans, reported alterations include tooth root resorption, small teeth, delayed shedding of deciduous teeth and retarded eruption of permanent teeth, features similar to those found in hypoparathyroid or pseudohypoparathyroid conditions (robinson et al., ) . tooth root alterations were also reported in a study in which young male wistar rats were treated with diphenylhyantoin for month. treated rats showed evidence of molar root resorption lacunae that penetrated the cementum and involved the dentine. the lacunae contained a dense infiltrate of cells contiguous with similar cells in the surrounding periodontal ligament. robinson and harvey ( ) showed that the changes were similar to those occurring in parathyroidectomized rats but not those in rats made hypocalcaemic with a calcium deficient diet. they suggested that the changes induced by diphenylhydantoin in rats were similar to those in pseudohypoparathyroidism in which resistance of tooth roots to resorption is reduced. discoloration of teeth and bone is a well-described side effect of tetracycline administration and it has also been reported in patients treated with the semisynthetic derivative, minocycline (cale et al., ) . interestingly the ameloblastic epithelium of the enamel forming tissues of growing incisors in wistar rats treated with high doses of human recombinant epidermal growth factor showed hyperplasia characterised by pseudostratification, increased nuclear-cytoplasmic ratio and increased cytoplasmic eosinophilia (breider et al., ) . this finding is consistent with the presence of epidermal growth factor receptors in the cells of the enamel organ (martineau et al., ) . periodontitis is a common and important disease in man and animals although overt cases are not usually seen in toxicity studies. however, periodontitis of a degree sufficient to disrupt chronic rat toxicity and carcinogenicity studies has been reported. robinson ( ) described periodontitis in alpk/ap rats in which there were erosive granulomatous cavities adjacent to molar teeth with fistulas opening into the nasal cavity. these changes were associated with penetrating food fibres in the gingival sulcus and it was suggested that the presence of long pointed food fibres in the powdered diet was the main reason for occurrence of periodontitis. peridontitis in rodents also results from the effects of dental pathology such as fractures, malformation or malposition of incisors (losco, ) . drug-induced overgrowth of the gingival tissues is a well-described phenomenon in both humans and laboratory animals including dogs, cats, and rats. in man, these changes have been associated with diphenylhydantoin (phenytoin) (beghi et al., ) nifedipine, calcium channel blockers (ledermann et al., ) , cyclosporin a (barthold, ) and valproic acid (syrjamen and syrjamen, ) . cyclosporin a, diphenylhydantoin and calcium channel blockers have been associated with similar changes in laboratory animals (do'nascimento et al., ; latimer et al., ; waner et al., ) . in most instances there is swelling of the gingiva by firm nodular overgrowths around the teeth. histologically, these overgrowths are characterised by marked acanthosis of the squamous epithelium overlying connective tissue that is infiltrated by large numbers of chronic inflammatory cells. fibrovascular proliferation may be marked. in patients treated with cyclosporin, myxomatous degeneration is described in association with dense infiltration of plasma cells and lymphocytes (barthold, ) . secondary acute inflammation in association with food debris and hair shafts is described in dogs treated with oxodipine (waner et al., ) . the forces behind these changes are unclear. studies of changed induced by nifedipine and hydantoin have shown increases in extracellular ground substance and increased numbers of fibroblasts containing sulphated acid mucopolysaccharides (kantor and hassel, ; lucas et al., ) . these drugs may alter fibroblastic proliferative and synthetic activity, possibly by selection of a subpopulation of fibroblasts (hassel et al., ) . it has also been suggested that an underlying mechanism in phenytoin-induced gingival hyperplasia involves the decrease in salivary iga that develops in some patients (beghi et al., ) . study of cyclosporin a-induced changes have suggested that impairment of t lymphocyte function may permit overgrowth of oral bacteria and bacterial products which may influence fibroblast function (barthold, ) . a spontaneous form of gingival hyperplasia has been described in non-human primates (macaca mulata). this is characterised by an enlargement of the marginal and alveolar gingiva by connective tissue consisting of relatively poorly cellular bundles of collagen fibres. the lesions show little inflammatory alterations and the overlying squamous epithelium shows mild hyperkeratosis only (schiødt et al., ) . this pathology is similar to hereditary gingival fibromatosis in humans. sessile or pedunculated squamous papillomas and infiltrating squamous carcinomas are occasionally found in the oral cavity of most laboratory animals including rodents (odashima, ; emminger and mohr, ; leiniger and jokinen, ; takahashi and okamiya, ; mohr, ) , rabbits (sundberg et al., ; sundberg and everitt, ) , and beagle dogs (watrach et al., ) . the microscopic structure of these neoplasms in rodents resembles those occurring in squamous epithelium in other sites. although a number of agents induce squamous neoplasms in the oral cavity, spontaneous squamous carcinomas are generally uncommon spontaneous lesions in laboratory animals. however, some strains of rodent may develop squamous neoplasms more commonly. for instance, in life time studies ad libitum fed brown-norway rats, % of males and % females developed oral squamous cell carcinomas although only % and % in food-restricted animals respectively (thurman et al., ) . it was suggested that certain pedigrees possessed a genetic predisposition to these neoplasms. papillomas occurring in rabbits and dogs are of note because they can occur in quite young animals, apparently as a result of infection with viruses of the papilloma group. viral inclusions may be seen in histological sections. the implications of papilloma viruses in laboratory species are that the progression of virally induced papillomas to malignant squamous carcinomas can be potentiated by nonviral factors including application of xenobiotics (howley et al., ) . in rabbits, the prevalence of oral papillomas varies considerably but they are quite common in some laboratory strains. they are overlooked because of their small size and a distribution limited to the ventral surface of the tongue (sundberg et al., ) . microscopically, they are typical squamous papillomas composed of irregular acanthotic squamous epithelium and a fibrovascular stalk of variable size. squamous cells at the margins of papillomas at the junction with normal mucosa, often show large, oval nuclei, marginated chromatin and central, basophilic, intranuclear inclusions, which electron microscopic examination shows to contain viral particles. oral papillomas in dogs develop as multiple growths, regressing spontaneously after a few months. they are also caused by a virus of the papilloma group, which possesses a high degree of specificity for the mucosa of the oral cavity and adjacent skin (watrach et al., ) . histologically, they are composed of proliferative masses of epithelial cells, keratinised on the surface and resting on an irregular connective tissue stroma or pedicule. large vesicular cells with basophilic intranuclear inclusions are also found in the granular cell layer, identifiable as virus arrays by electron microscopy (cheville and olson, ) . malignant change has been described in these canine lesions and this can occur in young beagle dogs (watrach et al., ) . although many types of papilloma viruses have been identified in both man and animals (pfister, ) , common antigenic determinants exist between viruses in different species. this immunological cross-reactivity can be exploited in the immunocytochemical localisation of papilloma viruses in epithelial lesions of many animal species. papilloma virus antigen has been demonstrated in oral papilloma of dogs and rabbits using antisera to bovine papilloma virus type i (sundberg et al., ) . cells positive for virus and viral inclusions are located in the upper layers of the epithelium, especially within cells of the granular layer. spontaneously developing odontogenic tumours are rare in rodents but they have been induced in laboratory animals given carcinogens such as nitrosoureas or exposed to ionising radiation (gössner and luz, ) . a range of tumours originating from dental tissues with epithelial, mesenchymal or mixed appearances has been reported in rodents (kuijpers et al., ) . the classification of odontogenic tumours is complex and confusing. they range from benign anomalies and cystic structures through to malignant neoplasms. the ameloblastoma comprises cords, nests, anastomosing strands or islands of ondontogenic epithelial cells within a fibrous stroma. the tumour cells resemble ameloblasts with the cords of spindle shaped cells similar to the stellate epithelium bounded by a peripheral layer of cuboidal or columnar cells resembling the inner enamel epithelium. other tumours of the odontogenic epithelium show induction of the mesenchymal elements or develop a complete sequence of odontogenic epithelium, odontogenic mesenchyme and dental hard tissues including dentine, enamel and cementum. in the rat these have been classified as odontoma characterised by the presence of all dental hard tissues and odontogenic fibroma composed of undifferentiated or primitive mesenchymal cells of developing dental tissue (mohr, ) . odontogenic tumours developing in fischer rats treated with aflatoxin were located in the upper jaw associated with the incisor teeth and were composed of proliferating fibroblast-like cells within which ovoid calcified bodies resembling cementum were seen (cullen et al., ) . occasional inclusions of solid epithelial nests were also seen. no metastatic deposits were found although the neoplasms were locally aggressive. in addition, squamous tumours and neoplasms of mesenchymal origin typical of other organs, bones and soft tissues are found in this region. although salivary glands may not represent vital organs in the same sense as the kidneys or heart, severe derangement of their secretions can alter both the quality and quantity of saliva. depending on the particular glands and cells affected, dry mouth, mucositis, and dental caries may develop . the severe oral complications of irreversible salivary damage and dysfunction, which can occur patients with head and neck cancer as a consequence of local irradiation, may have a significant impact on the efficacy of therapy, quality of life and survival (fox, ) . a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. these mucins usually contain more than % carbohydrate in the form of neutral and acidic oligosaccharide chains, o-glycosidically linked to threonine or serine. mucins possess several roles including mechanical flushing of the oral cavity, protection and lubrication of soft and hard tissues, modulation of oral microbial flora, buffering activity, regulation of calcium/phosphate equilibrium, digestion and extracellular post translation processing of molecules present in saliva . the heterogeneity of salivary glycoproteins suggests that they act as a defence against pathogenic microorganisms by competing with microbial binding sites of similar structure on the surface of cells lining the digestive tract (schulte, ) . minor salivary glands may also play an important part in the local immunosurveillance of the oral cavity for their ducts are anatomically closely associated with lymphoid tissue (nair and schroeder, ; nair et al., ) . salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species (junqueira et al., ) . the phylogenetic association of the salivary glands with the thyroid gland is evident functionally because salivary glands are capable of concentrating iodide in their secretions, although this is not under control of thyroid stimulating hormone (ingbar, ) . it has been shown that thyroxine accelerates the differentiation of the granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse (chabot et al., ) . the structure of the salivary glands differs among laboratory species, between different glands in the same species and between sexes. it is usually considered that there are three major salivary glands, the parotid, the sublingual and the submandibular (submaxillary) glands. minor salivary glands are scattered in other locations throughout the mouth and oropharynx. in dogs and other carnivores, the zygomatic (infra-orbital) gland, located just below the zygomatic arch and the buccal (molar) gland are also often referred to as major salivary glands. microscopically, salivary glands are composed of secretory glands or 'endpieces' attached to a connecting system of intralobular and extralobular (secretory) ducts. secretory endpieces may be acinar or tubulo-acinar in nature. the secretory cells have been subdivided into serous, mucous, seromucous and special serous types. controversy remains about the precise nature of the secretory cells found in the various salivary glands of different species and this makes critical interspecies comparisons difficult (see detailed discussion of this problem by pinkstaff, ). the duct system is less complex. this comprises an intercalated duct which leads from the secretory endpiece into a striated (secretory or intralobular) duct, so termed because their lining cells are striated by delicate eosinophilic cytoplasmic rods. the striated ducts converge into interlobular ducts and a main excretory duct system. in rats, mice and hamsters, an overall similarity in gross and microscopic anatomy of the various salivary glands exists although there are histochemical differences (munhoz, ; glucksmann and cherry, ; dawe, ; pinkstaff, ; emmiger and mohr, ) . moreover, it has been demonstrated that salivary glands in rodents as well as a number of other species show morphological and histochemical sexual dimorphism (pinkstaff, ) . the sublingual gland in rats, mice and hamsters is composed principally of mucous acini, with indistinct serous demilunes. acini open into fairly long intercalated ducts lined by flat or cuboidal cells devoid of granules. the parotid gland is composed of serous-type secretory cells containing zymogen granules and prominent hyperchromatic basal cytoplasmic poles. the submandibular gland is anatomically the most complex salivary gland in rodents. secretory endpieces are composed of small or moderately sized cells with foamy cytoplasm and basophilic basal poles. the most striking feature is the presence of an additional duct segment interposed between the intercalated and striated ducts. this segment is lined by cylindrical epithelium with basal nuclei and eosinophilic cytoplasm containing secretory granules. this duct segment is termed the granular duct or granular convoluted tubule. these granular cells are of special interest because they contain a large number of heterologous biologically active peptides including nerve growth factor, epidermal growth factor, renin, and kallikrinins (barka, ; mori et al., ). the precise physiological role of many of these peptides in salivary gland remains uncertain. epidermal growth factor was originally isolated from the mouse salivary gland. it initiates premature eyelid opening and incisor eruption when injected into the neonatal mouse (cohen, ) . study of the mouse submandibular gland has shown that both epidermal growth factor and nerve growth factor are released into saliva following the administration of phenylephrine, sympathomimetic amine acting mainly on αreceptors and isoprenaline (isoproterenol), a β-adrenergic agent (murphy et al., ) . immunohistochemical study also demonstrates that epidermal growth factor becomes depleted in mouse salivary tissue following administration of phenylephrine and similar agents (tsukitani and mori, ) . phenylephrine has been shown to cause marked secretory activity accompanied by loss of granules from granular cells, as well as loss of immune reactive carbonic anhydrase, an enzyme which participates both in membrane transport of bicarbonate ions into saliva and glandular secretion (noda, ) . morphological studies have shown that both acinar and granular tubular cells participate in this response to adrenergic agents (murphy et al., ) . this is in contrast to the effects of pilocarpine, a cholinergic agent, which elicits the secretion of saliva deficient in serous proteins with little or none of the growth factors, as its effects are more limited to acinar cells. glycoprotein secretion of rodent salivary glands has stimulated histochemical study using both conventional mucin histochemical techniques and labelled lectins which possess affinity for specific sugars or sugar sequences (tables and , pages and ). studies of rat, mouse and hamster salivary glands using batteries of labelled lectins have shown a greater heterogeneity of oligosaccharides in salivary glands than seen by classical histochemical techniques. there are considerable species differences and variations between murine strains and sexes of the same strain as well as heterogeneity among morphologically similar cells within one gland (schulte and spicer, , ; schulte, ) . the results of histochemical studies are in excellent agreement with studies using biochemical methods but suggest a significant influence of genetic and hormonal factors on the synthesis of salivary glycoproteins. less attention has been paid to the structure and cytochemistry of the dog salivary glands. there appears to be little variation between the structure of salivary tissues between beagles and other strains although variation with age has been reported (reifel and travill, ; nagoyo and tandler, ) . munhoz ( ) has described the histochemical features of the dog parotid gland. the dog parotid is of seromucinous type secreting both acidic and neutral mucosubstances, in contrast to the more neutral mucosubstances secreted by rodent glands. the salivary glands of non-human primates are similar to those in man. they possess parotid glands of serous or seromucous type, submandibular glands with both serous and mucous acini and sublingual glands of mainly mucous type. the salivary glands of the non-human primate react to adverse stimuli such as ionising radiation in a similar manner to human salivary tissue . focal chronic inflammation of the salivary glands occurs sporadically in untreated rats, mice, hamsters, dogs or primates employed in toxicology although severity and prevalence is variable. sialoadenitis as a result of a corona virus, the sialodacryoadenitis virus, is a well-known and fairly ubiquitous condition in rats, first described by innes and stanton ( ) . the condition is characterised histologically by oedema and congestion of submandibular and parotid salivary glands as well as extra-orbital lachrymal and harderian glands. it is accompanied by inflammation of variable severity and chronicity in both glandular and connective tissue as well as degeneration and necrosis of duct epithelium (fig. ) . the regenerative hyperplasia of the duct epithelium may be quite intense about a week after infection but all changes regress after about weeks and glands are essentially normal after or weeks (carthew and slinger, ; percy and wojcinski, ) . there may be a delay in the appearance of inflammatory cells and the onset of repair in rats immunosuppressed with cyclophosphamide (hanna et al., ) . depletion of salivary gland epidermal growth factor also occurs during the infection (percy et al., ) . suppurative infections in the neck region of the rat such as those produced by klebsiella aerogenes also cause acute and chronic inflammation of salivary glands with fibrosis and glandular proliferation of salivary tissue (arseculeratne et al., ) . sialadenitis occurs spontaneously in autoimmune-prone strains of mice such as the nzb/nzw and sl/ni strains and it has been reported in ageing female, but not male bdf mice (hayashi et al., ) . the non-obese diabetic mouse known for its spontaneous insulin-dependent diabetes mellitus also develops immune mediated damage to submandibular glands (fujino-kurihara et al., ; törnwall et al., ) . in ageing bdf females the submandibular gland was shown to be involved by a destructive inflammatory process characterised by an intense infiltration by small and medium sized lymphocytes, associated with mild inflammation in other organs such as the parotid and sublingual glands, pancreas and kidney. immunocytochemistry showed that most of the lymphocytes were t cells (thy- . and lyt- positive) of the helper/inducer subset (l t or cd positive) and less than % were of suppresser/cytotoxic (lyt- or cd fig. . section from salivary tissue from a sprague-dawley rat during an infection with the sialodacryoadenitis virus showing intense ductular inflammation. (he, × .) positive) type (see haemopoietic and lymphatic systems, chapter iii). circulating anti-salivary duct antibody of igg was also detected in afflicted mice. it was suggested that helper/inducer t cells played a key role in the production of this change, unlike induced autoimmune sialoadenitis in which cytoxic t-cell subsets may directly destroy glandular tissue. it has been suggested that this process in ageing females is related to the decline in the number of splenic lyt- cells in mice with advancing age (hayashi et al., ) . these cells are believe to be the most susceptible to ageing (see haemopoietic and lymphatic systems, chapter iii) in the non-obese diabetic strain of mouse derived from jcl-icr mice, a periductal chronic inflammatory infiltrate is found in the submandibular gland at about the same time that immune-mediated insulitis is most marked. this suggests that there is an extension of the autoimmune process to salivary tissue (fujino-kurihara et al., ) . it is probable that helper/inducer cd t cells are essential components of this infiltrate and a number of cytokines and their receptors such as ip- (interferon-γ inducible protein ) and rantes (regulated upon activation normal t cell expressed and secreted) may have an important role (törnwall et al., ) . an autoimmune type of sialoadenitis can also be experimentally induced certain strains of mice. crj:cd mice, thymectomized at days, a time point at which lyt- positive cells (cd suppresser t lymphocytes) can be maximally reduced, followed by immunisation at and days after birth with homogenates of salivary gland and complete freund's adjuvent, develop a distinctive sialoadenitis in the submandibular and to some extent the parotid glands (hayashi et al., ) . this sialoadenitis is characterised by degenerative changes in salivary glandular tissue associated with an extensive and intense infiltrate of small and medium sized lymphocytes. these cells appear shortly after immunisation but increase in number with time. immunocytochemical study has shown that many of these cells are reactive to antisera to thy- . and lyt- (cd ) features of suppresser/cytotoxic t lymphocytes. later appearing cells demonstrate features of plasmacytoid lymphocytes and contain immunoglobulin of mainly igg class (hayashi et al., ) . these authors therefore suggested the sialoadenitis appeared as both a result of cytotoxic/suppresser t-cell activity and an antibody-dependent cell-mediated cytotoxicity. in the hamster salivary glands, interstitial infiltrates of lymphocytes and plasma cell are quite common and may become more marked with advancing age (mcmartin, ) . whereas necrosis of the parotid gland of uncertain aetiology sometimes occurs in the dog, mild focal chronic inflammation is quite a common incidental finding in canine salivary glands and has been reported in about % of normal beagle dogs (kelly et al., ) . although the inflammation in salivary tissue which results from ionising radiation is only indirectly relevant to drug safety evaluation, it is of interest in view of the notable species differences in sensitivity to this form of insult. serous acinar cells in man and rhesus monkey appear least resistant to the effects of ionising radiation, where damage is characterised by widespread degranulation and degeneration of acini, infiltration by polymorphonuclear cells followed by lymphocytes, plasma cells and subsequent atrophy and fibrosis . these changes contrast with the lesser effects of ionisation radiation on the rodent salivary glands in which there is little or no acute inflammatory response. lymphoid bodies are sharply circumscribed collections of lymphoid cells generally located between the parotid and sublingual glands close to a cervical lymph node in mice. they are apparently normal aggregates of lymphoid tissue. like many other glandular organs, the size of the secretory tissue of the salivary gland is responsive to functional demand and is subject to age-related changes. in man, the gland parenchyma frequently becomes atrophic and replaced by connective tissue or fat with advancing age, possibly partly related to vascular changes (waterhouse et al., ; scott, ) . in ageing rats, the extent and height of granular ducts and their content of mature secretory granules has also been shown to decrease with age (sashima, ) . dietary factors influence salivary gland size. decreased food consumption or protein starvation can reduce the weight of salivary glands in rats. there is shrinking of mucous and serous glands and loss of zymogen granules associated with decreased rna but unchanged dna content, attributable to the reduced requirements for protein synthesis , mcbride et al., . as salivary gland function is responsive to adrenergic stimulation, it is not surprising that atrophy occurs following adrenergic blockade. the weights of the submandibular gland in mice were shown to decrease following administration of the β-adrenergic blocking agent, propranolol (smith and butler, ) . this was associated with a reduction in stainable neutral mucins and a decrease in the thickness of the acinar cells making the gland lumens appear larger than normal. the cytotoxic agent, alloxan, known primarily for its specific effect on pancreatic b cells, has also been shown to produce weight loss of the rat submandibular gland, associated with lipid inclusions in the acinar cells, capillary basement membrane thickening and reduced salivary flow (reuterving et al., ) . it is probable that alloxan exerts a cytotoxic effect on the acinar cells of the rat submandibular gland (sagström et al., ) . methotrexate, a folic acid antagonist, has also been reported to cause vacuolization of acinar and ductular cells with reduction of secretory granules in rat salivary glands (mcbride et al., ) . ligation of the main excretory ducts has frequently been used as an ex-perimental model for study of salivary gland atrophy as well as the regeneration that follows removal of the ligature. there is marked atrophy of all cell types but most markedly the acinar cells through apoptosis. although overt necrosis has been reported following ligation of the excretory duct, it appears that this may have been the result of constriction of the vasculature for acinar cells are relatively intolerant to a decrease in oxygen and nutrient (denny et al., ) . a number of therapeutic agents increase salivary gland size in man, although the scarcity of biopsy data precludes a critical assessment of the precise mechanism in many cases. drugs reported to produce salivary gland enlargement in man include iodide-containing radiological contrast media, isoprenaline and anti-inflammatory agents phenylbutazone and oxyphenbutazone. enlargement may also occur after endotracheal anaesthesia and upper gastrointestinal tract endoscopy in man (riddell, ) . some of these agents and procedures may produce spasm of large salivary ducts and retention of secretions. several pharmacological agents, particularly sympathomimetic amines, have been shown to produce increases in salivary gland size in rodents following repeated dosing (brenner and stanton, ) . there is an intimate relationship of sympathomimetic amines with the control of the secretory process in salivary tissue. whereas a single injection of isoprenaline (isoprotorenol) in the range of - mg/kg induces discharge of preformed secretory granules followed by gradual re-synthesis and reconstitution, repeated injections produces an increase in the size of salivary glands (simson et al., ) . histologically, the enlarged glands are composed of secretory cells of increased size that contain increased amounts of secretory substances in the cytoplasm (simson et al., ) . although these histological features are principally those of diffuse cellular hypertrophy, the increase in dna content and radioactive thymidine uptake described in the salivary tissue following repeated administration of isoprenaline suggests that a degree of hyperplasia also occurs (barka et al., ) . these effects do not depend on the integrity of the autonomic nerves because they occur after ablation of the autonomic ganglia (barka et al., ) . they appear to be mediated by an effect on adrenergic β-receptors. the effects can be blocked by propranalol, a β-receptor antagonist but not by phenoxybenzamine, an α-receptor antagonist (brenner and stanton, ) . as theophylline and caffeine also elicit salivary gland enlargement in rats, a role for cyclic ', -adenosine monophosphate (camp) in salivary gland enlargement has been postulated (brenner and stanton, ) . detailed study of hypertrophy, protein synthesis, and intracellular camp activity in the salivary glands of rats treated for days with isoprenaline (isoproterenol), a series of β-adrenergic receptor agonists and the phosphodiesterase inhibitors, theophylline and caffeine, showed that similar effects occurred with all agents although differences in the degree of hypertrophy, the nature of pro-tein and glycoprotein synthesis and golgi membrane enzyme activity were recorded (wells and humphreys-beher, ) . the parotoid gland showed the most pronounced hypertrophy followed by the submandibular gland but the sublingual gland appeared to be unaffected by treatment. the degree and nature of the changes induced by the various β /β receptor agonists suggested that most of these effects were mediated through β receptors which are present in greatest numbers on the parotid and salivary cells. it was suggested that the effects of β-adrenergic agonists on salivary gland are produced by a receptor-mediated stimulation of adenylate cyclase activity causing an increase in levels of intracellular camp. however, other factors may be important for wells and humphreys-beher ( ) also showed that although isoproterenol and caffeine increased salivary cell camp to comparable levels, the hypertrophy was greater with isoproterenol. cardioactive phosphodiesterase inhibitors were shown to produce submaxillary hypertrophy in rat subacute toxicity studies (rogers et al., ; jayasekara et al., ; smith et al., ) . parotid and submaxillary glands were those most affected by the inotropic phosphodiesterase inhibitor ici , (westwood et al., ) . as the agents produced their positive inotropic action via selective inhibition of the cardiac phosphodiesterase subfraction iii specifically requiring camp as its substrate, it was suggested that the salivary gland hypertrophy was a result of phosphodiesterase inhibition (smith et al., ) . other classes of drugs can also produce salivary gland enlargement in rats in repeated dose studies. doxylamine, a representative of the widely used ethanolamine group of antihistamines, has been reported to produce marked cytomegaly in the fischer rat parotid gland. enlarged cells were characterised by a basophilic and coarsely granular or vacuolated cytoplasm (jackson and blackwell, ) . the b c f mouse did not develop these changes after a similar treatment schedule. in view of the presence of considerable amount of epidermal growth factor in salivary glands, it is of interest to note the effects of its administration to laboratory animals. salivary gland weights were increased in rats and cynomolgus monkeys infused with high doses of recombinant human epidermal growth factor (breider et al., ; reindel et al., ) . however histological features seen are primarily those of ductular epithelial hyperplasia (see below under hyperplasia). epithelial cells characterised by abundant granular eosinophilic cytoplasm as a result of the accumulation of mitochondria are often referred to as oncocytes, a term used by hamperl ( ) to describe similar cells in hürthle tumours of the thyroid gland. they may be found in various focal nodular and neoplastic states of the salivary glands in both man and laboratory animals. the precise significance of these cells is uncertain. the mitochondria usually appear unremarkable except for lack of dense granules and it has been suggested that the mito-chondrial changes represent an adaptive phenomenon or compensatory hyperplasia (ghadially, ) . in human salivary tissue their prevalence seems to increase with advancing age and they can be associated with hyperplastic lesions or neoplasms such as oxyphil adenomas and adenolymphomas. eosinophilic cells also occur in the salivary glands of certain strains of aged rats (bogart, ) and in mice with experimentally induced autoallergic sialoadenitis (takeda et al., ) . in the study of takeda et al. ( ) the eosinophilic cells appeared to arise predominantly in the secretory (glandular) ducts of the submandibular glands, although eosinophilic cells can apparently develop from either duct or acinar cells. well-defined, unencapsulated foci of enlarged acinar cells occur spontaneously in the salivary glands, particularly the parotid of rats, mice (chiu and chen, ) , and hamsters although their reported incidence varies between laboratory. the enlarged cells possess greatly expanded cytoplasmic volume that retains a vesicular, vacuolated or foamy appearance or possesses a pale eosinophilic granular texture. the basal parts of the cells usually stain intensely blue in haematoxylin and eosin stained sections and contain large, dense, irregular hyperchromatic or pyknotic nuclei showing little evidence of mitotic activity. although there has been little ultrastructural study of these foci, the cytoplasmic alterations appear to be distinct from those of so-called oncocytes that characterised by granular eosinophilic cytoplasm packed with mitochondria. the biological nature of these foci is uncertain. the lack of any prominent mitotic activity, cell proliferation or expansive growth suggests that they are most aptly regarded as hypertrophic lesions (chiu and chen, ). although they increase in prevalence with increasing age in certain strains of rat, there is no evidence to suggest that they represent pre-neoplastic lesions or possess any relationship with development of neoplasia in salivary tissue (dawe, ) . hyperplasia and squamous metaplasia of the salivary ducts are common features of many inflammatory and reactive conditions in the salivary glands of rodents, dogs, non-human primates and man and can be associated with the presence of stones and calculi with the duct system. squamous metaplasia and regenerative change in the ducts occurs in rats afflicted with sialodacryoadenitis (carthew and slinger, ) . it is also described specifically located in the ducts of the sublingual glands in the wistar rat in the absence of obvious sialodacryoadenitis or evidence of any specific disease. similar regenerative hyperplastic duct changes are also seen in necrotic and inflammatory conditions in the dog salivary gland . detailed morphological examination with immunocytochemical study of epi-dermal cytokeratins of the rat salivary gland after arterial ligation has shown that the acinar units can also undergo squamous metaplasia (dardick et al., ) . it appears that the acinar-intercalated duct complexes can rapidly reprogram to produce epidermal cytokeratin filaments in ischaemic or inflammatory states. hyperplasia of the ductular epithelium appears to be the principle result of the administration of epidermal growth factor to rats and cynomolgus monkeys. in rats histological features were primarily of ductular epithelial hyperplasia without evidence of significant acinar hyperplasia (breider et al., ; reindel et al., ) . in primates the changes were most striking in the interlobular and large intralobular ducts where the epithelium showed multilayered and papilliform projections. however, mitotic activity was evident throughout the duct epithelia and acinar cells showed hypertrophy with depletion secretory granules and the presence of large vesicular nuclei . focal duct and acinar hyperplasia, showing minimal compression of the surrounding parenchyma and distinct from focal hypertrophy is also described in the classification of rat salivary lesions (mohr, ) . primary neoplasms of salivary glands are uncommon in the usual strain of rats and mice employed in carcinogenicity bioassays (haseman et al., ) . acinar and tubular adenomas and adenocarcinomas as well as squamous carcinomas are reported in rats (mohr, ) , mice (frith and heath, ) and hamsters (takahashi and okamiya, ) . some carcinomas showing squamous or glandular differentiation may be observed infiltrating the salivary gland that originate in other local structures of the head and neck region. occasionally, salivary gland neoplasms show adenomyomatous differentiation. mixed glandular and lymphoid tissue patterns resembling wartin's tumour in man are also sometimes seen. neoplasms of soft tissues also develop in and around the major salivary gland in rodents (see integumentary system chapter i). in humans the oesophagus is not considered a common site for drug-induced injury although some studies have suggested that medication-induced changes are more prevalent than previously supposed (bonavina et al., ) . severe damage can occur following prolonged contact between mucosa and ingested tablets or capsules which results in local high concentrations of potentially irritant substances (bott and mccallum, ; brors, ; kikendall, ; levine, ) . damage as a result of local contact may be more common in elderly subjects as the amplitude of oesophageal contractions decrease with age and capsules more liable to lodge in the lumen of the oesophagus (bonavina et al., ) . however patients of all ages may be affected. women have been injured more frequently than men probably because of the greater likelihood of their being treated with potentially injurious drugs (kikendall, ) . the shape and surface coating of tablets may influence their tendency to adhere to the mucosa and lodge in the oesophagus (marvola et al., ) . a wide variety of drugs have been implicated. in the united states the majority of cases appear to be caused by ingestion of tetracycline or doxacycline (levine, ) . some of the causative agents such as potassium chloride, aspirin and other non-steroidal anti-inflammatory drugs are also implicated in ulceration lower in the gastrointestinal tract. over recent years the bisphosphonate, alendronate has been one of the most commonly reported causes of adverse effects in the oesophagus with severe injury being reported. although injury is linked to ingestion without water or failing to remain upright after swallowing the medication, alendronate is particularly caustic (kikendall, ) . oesophagitis due to candida albicans is a well-described complication of antibiotic therapy. administration of immunosuppressive drugs may predispose to viral infections in the oesophagus. a number of agents affecting neuromuscular co-ordination may also predispose to gastro-oesophageal regurgitation and reflux oesophagitis (bott and mccallum, ) . in laboratory rodents spontaneous lesions of the oesophagus are occasionally seen. oesophageal impaction has been described in untreated srl:bhe rats. this is characterised by massive dilatation of the oesophagus with food or bedding (ruben et al., ) . histologically, the muscle fibres in the wall of the oesophagus show varying degrees of degeneration including swelling or shrinking of fibres, myofibrillar fragmentation, cytoplasmic vacuolation and mineralisation. so-called megaoesophagus, characterised by enlargement of the oesophagus, degeneration of muscle fibres and ganglion cells in the myenteric plexus has also been described in certain strains of rats and mice (harkness and ferguson, ; randelia and lalitha, ) . its cause is unknown. a commonly occurring lesion reported in fischer rats is oesophageal hyperkeratosis, which occurs at all ages . in the study by maeda et al. ( ) , it occurred more commonly in rats fed a protein-restricted, calorie unrestricted diet than in rats fed ad libitum with normal diet. it was suggested that the particular high prevalence of oesophageal hyperkeratosis observed in all groups in this particular study was related to acidification of drinking water . another pathological findings in rodents is perforation of the oesophagus as a result of a gavage accident. under these circumstances there is a variable inflammatory and purulent exudate localised around the perforation or spread within the pleural or occasionally the pericardial cavities. the oesophagus and surrounding tissues need careful examination by the pathologist for it is not always clear from clinical findings that oesophageal damage has occurred. spontaneous oesophageal lesions are uncommon in laboratory beagles, even though emesis and vomiting are frequent responses of this species following dosing in toxicity studies. local oesophageal irritancy potential of drugs has been assessed in a number of animal models, notably the cat and pig (carlborg and densert, ; olovson et al., ) . in these models, the test drugs are placed in the upper oesophagus using endoscopic techniques for periods of several hours to allow dissolution of the preparation. subsequently, the animals are followed for - days and histopathological assessment performed on the oesophagus. the degree of inflammation, erosion of mucosa or deep ulceration is recorded in a semiquantitative manner. the degree of ulcerogenic activity of drugs in these models seems to correlate with reported ulcerogenic activity in the human oesophagus (carlborg et al., ) . systemic administration of drugs with radiomimetic or antimitotic activity can cause hypoplastic changes in the oesophageal mucosa as well as the remaining gastrointestinal tract mucosa (tucker et al., ) . conversely, hyperplasia with increased keratinization has been reported in the oesophagus of the rat following chronic high dose administration of alcohol (mascrès et al., ) . acanthosis with hyperkeratosis and parakeratosis has been reported in the oesophagus but not stomach of rats treated for up to months with mesuprine hydrochloride, a β-adrenergic receptor stimulator (nelson et al., ) . as part of its effects on the gastrointestinal tract, the oesophagus in rats and primates has been reported to develop uniform hyperplasia of the squamous epithelium following infusion of recombinant epidermal growth factor (breider et al., ; reindel et al., ; vinter-jensen, ) . in the rat, mouse and hamster the forestomach occupies about two-thirds of the proximal stomach area and is lined by cornified stratified squamous epithelium. the limiting ridge is a distinct elevated mucosal fold at the junction between the forestomach and the mucosa of the glandular part of the stomach. as humans lack a forestomach, the relevance of changes produced by drugs and chemicals in the rodent forestomach is disputed. studies in rats in which the forestomach has been removed have suggested that the forestomach acts as a storage organ releasing relatively undigested food into glandular stomach in response to energy demand (gärtner and pfaff, ) . hence, the forestomach mucosa may be exposed to xenobiotics mixed in undigested food for far longer periods than elsewhere in the gastrointestinal tract. the interpretation of forestomach changes should take into account physiological factors, residence time and exposure differences to drugs between the rodent forestomach and human oesophagus. however the squamous mucosa lining the oesophagus in species without a forestomach may react to xenobiotics in a similar way to the forestomach mucosa of rodents if equivalent exposure levels are attained. inflammation and ulceration of the forestomach mucosa are some of the commonest spontaneous gastrointestinal lesions in laboratory rats, mice and hamsters. the prevalence of these gastric lesions varies between species, strains of laboratory rodents as well as between different laboratories. the precise causes of forestomach ulceration remain unclear although a variety of factors have been associated with its development including advanced age, infection, parasitism, diet, feeding regimens and stress. in rats, conflict-induced ulceration occurs in the forestomach and there is an age-related susceptibility, older rats developing more ulcers than younger rats (sawrey and sawrey, ) . in rats and mice dying of spontaneous disease, ulceration of the forestomach is also quite frequently observed. protein restriction or starvation has also been shown to produce forestomach ulceration in rats . ulceration of the forestomach in rodent toxicology studies may be incidental, particularly if the lesions are few and show no clear relationship to dose. if limited to high dose groups, ulceration may be a result of non-specific toxicity and stress-related. however, administered chemicals may have direct local effects of sufficient severity to cause focal damage to the forestomach mucosa. histological features of ulcers and inflammatory lesions of the forestomach are similar in rats, mice and hamsters. in mild cases, a scattering of acute inflammatory cells is seen in the intact squamous mucosa. ulcers can be single or multiple and are characterised by loss of squamous epithelium with a variable accumulation of neutrophils, mononuclear cells, cellular debris, fibrin and hair fragments in the ulcer crater. the inflammatory process may extend deeply into the stomach wall and be associated with intramural inflammation, oedema, endarteritis and fibrosis. haemosiderin pigment is also found in the ulcer margins. profuse haemorrhage may follow erosion of large blood vessels and complete perforation of the stomach wall with peritoneal involvement also occurs (greaves and faccini, ) . in long-standing cases of ulceration, hyperplasia of the adjoining squamous epithelium occurs, characterised by irregular acanthosis and down-growths of squamous epithelium into the submucosa . xenobiotics may produce inflammatory changes in the forestomach mucosa following initial dosing but subsequently, repair occurs even though treatment continues. an example of this phenomenon is illustrated by butylated hydroxyanisole. after week of administration of this agent in a % mixture in diet to rats, a vesicular inflammatory reaction characterised histologically by the presence of subepithelial vesicles containing inflammatory cells and exudate was seen (altmann et al., ) . after further treatment, only hyperplasia of the squamous epithelium was evident, presumably as an adaptive response to the effects of the continued insult. hyperkeratosis associated with hyperplasia of the squamous epithelium is seen sporadically in untreated aged rodents. these changes may be localised to the margins of chronic forestomach ulcers or they can be associated with diffuse inflammation of the mucosa. occasionally, the forestomach mucosa of untreated, aged rodents exhibits hyperkeratosis with hyperplasia without inflammation (fig. ). such changes may be diffuse or focal, but they are often localised to the zone adjoining the glandular stomach mucosa. there may be evidence of basal cell proliferation and downgrowth of the epithelium into the underlying stroma. dietary factors also influence the thickness of the forestomach mucosa. vitamin a deficiency, known to produce squamous metaplasia in glandular tissues may produce forestomach hyperplasia and hyperkeratosis in rats. when spf fischer rats were maintained in a vitamin a deficient state for over months, hyperplasia with hyperkeratosis, not unlike that produced by known carcinogens was reported (klein-szanto et al., ) . administration of a wide range of both industrial chemicals, therapeutic agents including both genotoxic and non-genotoxic carcinogens produces hyperkeratosis and hyperplasia of the forestomach epithelium which may be followed by preneoplastic lesions and squamous carcinoma (see below). histologically, the changes are characterised by hyperkeratosis, parakeratosis with varying degrees of acanthosis and papillomatosis (greaves and faccini, ) . the changes can be florid and it may be difficult to make a clear distinction between severe hyperplasia and neoplasia. nevertheless, it has been shown that the florid hyperplasia of the forestomach epithelium without evidence of cellular atypia can be completely reversible following the withdrawal of an inciting stimulus, ethyl acrylate (ghanayem et al., ) . hence, a critical feature may be the presence of cellular atypia in view of its association with agents with potent (genotoxic) carcinogenic activity. neoplasms arising in the forestomach of rodents are usually squamous carcinomas although basaloid features are also seen (fukushima and leiniger and jokinen, ; mohr, ; tatematsu, ) . squamous carcinomas, as at other sites, show variable differentiation being composed of proliferating squamous epithelium with moderate to marked cellular atypia, pleomorphism and mitotic activity with clear evidence of invasion into the muscularis. although they are relatively uncommon spontaneous lesions in aged rodents, they can be induced in rodents by administration of nitroso compounds (tatematsu ) as well as a range of non-genotoxic agents (see below). some authors report basal cell carcinoma when basaloid features are pronounced (tatematsu, ) . a wide range of agents is capable of producing squamous hyperplasia of the rodent forestomach and a number of these also induce squamous carcinomas. in kroes and wester reviewed over genotoxic and non-genotoxic compounds that were reported to produce hyperplasia and carcinoma in the forestomach of rats, mice or hamsters. a well-studied example is butylated hydroxyanisole (bha) an important food antioxidant (reviewed by whysner and williams, ) . structurally related phenols and acids produce similar changes (rodrigues et al., ) . ethyl acrylate, used in the production of materials for dental and medical devices is also capable of inducing marked squamous hyperplasia, papillomas and carcinomas after long-term treatment of f rats and b c f mice (ntp, ; ghanayem et al., ) . sk&f , an experimental histamine h receptor antagonist produced atypical forestomach hyperplasia in rats following administration by gavage for year by a mechanism which appeared unrelated to the inhibition of the h receptor (betton and salmon, ) . other therapeutic agents associated with squamous hyperplasia and neoplasia include the -hydroxy- -methylglutaryl coenzyme a (hmg-coa) reductase inhibitors (kloss et al., ; bueld et al., ; akiba et al., ; physicians' desk reference, ) and aristolochic acid (göggelmann et al., ; schmeiser et al., ) . some cytoprotective prostaglandins appear capable of inducing hyperkeratosis and hyperplasia without neoplasia presumably through a mechanism related to their pharmacological activity (levin, ) . this occurs in rats treated with misprostol, a synthetic prostaglandin e methyl ester analogue with gastric anti-secretory and anti-ulcer activity (kotsonis et al., ) , cl , , a synthetic analogue of prostaglandin e type (kramer et al., ) and , -dimethyl prostaglandin e (reinhart et al., ) . even the extensively used antibiotic, ampicillin has been associated inflammation, ulceration with acanthosis and hyperkeratosis in mice but not rats treated for years (national toxicology program technical report, ) . sodium saccharin is also reported to produce hyperplasia without neoplasia of the forestomach in f rats (hibino et al., ) . among its wide range of pharmacological effects on the gastrointestinal tract, the forestomach has also responds to recombinant epidermal growth factor when infused into rats (breider et al., ; vinter-jensen, ) . histological examination showed hyperkeratosis and hyperplasia of the squamous epithelium. the large body of studies performed with butylated hydroxyanisole illustrates the various factors that can influence the development of treatment-induced hyperplasia and neoplasia of the rodent forestomach and subsequent assessment of human risk. butylated hydroxyanisole possesses little or no mutagenic activity in vitro but when administered to rats for years as a % mixture in the diet, it produced squamous hyperplasia, squamous papillomas and squamous carcinomas of the forestomach. at . % in the diet butylated hydroxyanisole induced only hyperplasia . it also produces proliferative lesions in the forestomach of both mouse and hamster (ito et al., ) . studies in which butylated hydroxyanisole was fed in the diet to rats for shorter periods have shown that squamous epithelial hyperplasia occurs after only week of treatment preferentially over the lesser curvature, the site at which carcinomas developed in the -year studies (altmann et al., ) . after weeks' treatment, mucosal hyperplasia characterised by pronounced hyperkeratosis, parakeratosis and acanthosis most pronounced over the lesser curvature, was present in rats given % butylated hydroxyanisole in diet but not in rats given . , . and . % mixtures . abundant mitoses were found in the basal cells layers and tritiated-thymidine labelling confirmed that the changes were accompanied by a high rate of cell proliferation. following cessation of administration of butylated hydroxyanisole after weeks, the tritiatedthymidine labelling index rapidly reverted to control levels within about week although hyperplasia took longer to regress. nearly complete regression of the hyperplasia occurred after about weeks of normal diet . the distribution of squamous hyperplasia induced in the rodent stomach by butylated hydroxyanisole is influenced by the mode of administration. whereas following feeding of rats with butylated hydroxyanisole mixed in the diet lesions tended to be located near the limiting ridge, altmann et al. ( ) showed that gavage of butylated hydroxyanisole in corn oil produced similar changes at the apex of the forestomach. it was suggested that this difference was due to incomplete mixing of butylated hydroxyanisole in the stomach lumen when given by gavage and prolonged contact of the gavage mixture with the upper segment of the forestomach (altmann et al., ) . more recently, it was shown that fischer , shr, lewis and sprague-dawley rats differ in their response to the hyperplastic and carcinogenic effects of % butylated hydroxyanisole in pelleted diet. the most sensitive was the shr strain followed by the f rats and the differences correlated with the cytotoxic effects of butylated hydroxyanisole in the different strains (tamano et al., ) . it was suggested that the presence of vascular damage in the stomachs of the shr rats might have contributed to the response to cytotoxicity and subsequent carcinogenicity. residence time of administered compounds in the forestomach may influence the development of lesions. although it has been demonstrated that butylated hydroxyanisole does not produce hyperplasia in the oesophagus of animals without a forestomach, have shown that high-doses given to primates are capable of producing an increase in mitotic activity in the lower end of the oesophagus similar to that occurring at equivalent exposure levels in the rat. the implication is that these interspecies differences may simply be a question of differences in exposure of the squamous mucosa to compound. this underlines the fact that mechanisms of action and exposure levels of xenobiotics attained in the gastrointestinal tract of rodent and non-rodent species as well as of man need to be carefully assessed when hyperplastic changes are induced in the forestomach mucosa of rodents. such information can be helpful in facilitating regulatory decisions in this area (moch, ) . on balance, the evidence suggests that the tumour development by butylated hydroxyanisole in rodents represents an epigenetic phenomenon related to largely reversible cytotoxicity and increased cell proliferation (whysner and williams, ) . in view of the low levels of exposure to butylated hydroxyanisole that occurs with the usual use of this agent, carcinogenic hazard for the human stomach is therefore probably very small. similar phenomena have also been reported in studies of phenols and acids that are structurally related to butylated hydroxyanisole (rodrigues et al., ) . these agents include n-butyl and n-propyl- -hydroxybenzoic acid esters, propionic acid and -methoxyphenol. however, these studies suggested that certain areas of the forestomach epithelium react differently to structurally related chemicals, possibly due to the variable levels of activating enzymes within different zones of the forestomach epithelium. co-administration of acetylsalicylic acid was shown to abrogate some of these effects, suggesting that prostaglandin synthetase may be involved in the hyperplastic response (rodrigues et al., ) . a number of hmg-coa reductase inhibitors with different chemical structures including marketed products such as lovastatin, simvastatin and fluvastatin are also associated with the development of squamous hyperplasia of the rodent forestomach. the hyperplasia is time and dose dependent and may be associated with oedema and some inflammation of the submucosa. some, but not all of these agents are also capable of producing squamous neoplasia of the forestomach mucosa of rats, or mice or both after long-term treatment (kloss et al., ; bueld et al., ; akiba et al., ; physicians' desk reference, ) . the mechanism of action remains unclear although the degree of hyperplasia seems related to pharmacological potency. their carcinogenic potential in rodent bioassays does not seem to relate to the degree of hyperplasia in shortterm studies. moreover, the development of hyperplasia depends on local high concentrations of drug because when administered by non-oral routes, hyperplasia does not occur (kloss et al., ) . as most of these drugs are non-mutagenic, these findings are presumably also epigenetic in origin and possess relatively little risk for humans when given in the usual therapeutic doses. a contrasting example is provided by aristolochic acid, a nitrophenanthrene derivative of the ancient medicinal plant aristolochia clematis which was used as an anti-inflammatory component in a number of medicinal preparations in germany until (göggelmann et al., ; schmeiser et al., ) . aristolochic acid is a direct acting mutagen in salmonella typhimurium. when fed to rats at doses of . and mg/kg/day, aristolochic acid produced severe papillomatosis of the entire forestomach within a period of months. this was characterised histologically by the presence of branched squamous papillomas up to mm high with focal dysplastic features. invasive squamous carcinomas with metastases were found subsequently, or months later without further treatment (mengs et al., ) . even at a low dose of . mg/kg/day papillomas and squamous carcinomas developed months after a -month period of treatment. quite clearly the complexity of the hyperplastic response of the rodent stomach to xenobiotics, the association of hyperplasia induced by non-mutagenic compounds with the development of forestomach carcinomas, and the similarity of response in the forestomach to that of the oesophagus, dictates the need for a careful analysis of hyperplasia induced by novel drugs in the forestomach. the prelude to this assessment is careful histopathological characterisation of the changes. unlike the mouth and oesophagus through which tablets, capsules, gavage fluids and drug/diet mixtures pass relatively rapidly, the human stomach mucosa remains in contact with high local concentrations of administered compounds for much longer periods of time. administration of compounds in liquid or solid form, particle size, fasting and feeding all affect the gastric motility pattern. in the fasted state there is a cyclical pattern of motility consisting of three main phases. the first is a quiescent phase, followed by a phase of irregular contractions that increase in amplitude and frequency to reach a maximum in a third phase. feeding results in the replacement of this cyclic pattern by regular tonic contractions that move food towards the antrum and mix it with gastric secretions. these patterns have been well studied in both dog and man and appear to be qualitatively similar in the two species (sarna, ) . these motility patterns may have an impact on the length of time drugs remain in contact with stomach mucosa. for instance, the residence time of large non-disintegrating capsules or tablets administered in the fasting state is more dependent on the frequency of powerful phase iii contraction than if drugs are given as fluids or mixed with diet. for dosage forms released in the stomach, gastric residence time will influence drug supply to the main absorptive surfaces in the small intestine, which in turn may affect drug absorption (dressman, ) . gastric acid is also important in making ingested salts soluble. although the presence of food in the stomach is a stimulus of acid production, the ph in the forestomach of rats is highest in full stomachs and lowest when empty, presumably as a consequence of the buffering action of food (ward and coates, ) . the glandular stomach is conveniently divided into the fundus characterised by mucosal folds or rugae and the smoother antrum, which opens into the pylorus and duodenum. in species devoid of a forestomach, the proximal stomach mucosa or cardia is also lined by glandular mucosa. the glandular mucosa is covered by surface epithelium of regular columnar cells that extends downwards to form small gastric pits or foveolae. the gastric glands are simple tubular structures usually considered to comprise three segments. the base is the deepest part, the neck the mid-region, and the most superficial is the isthmus, continuous with the gastric pit. the upper part of the gastric gland contains mucous neck cells. small cuboidal chief or zymogenic cells, which secrete pepsinogen and stain blue or purple in haematoxylin and eosin sections, are located in deeper parts of the gland. the eosinophilic-staining parietal (oxyntic) cells, which produce hydrochloric acid, are distributed more randomly throughout the gastric glands. parietal cells can also be visualised by immunocytochemical staining with antibodies directed at h + k + -atpase (canfield et al., ) . the gastric glands situated near the limiting ridge in rodents, show a modified structure. in species not endowed with a forestomach, the mucosa near the cardia is composed of simplified branched glands lined by columnar epithelium. the antral mucosa is covered by a surface epithelium with gastric pits similar to that of the fundus but mucous secreting columnar glands line the glands. the stomach mucosa is richly endowed with endocrine cells, not all of which have been well characterised. enterochromaffin cells are quite numerous in the basal parts of the gastric glands of the fundus, particularly in the rat . they are generally argyrophilic, staining with silver staining techniques such as that of grimelius (grimelius, ; grimelius and willander, ) that utilise exogenous reducing agents. these cells contain histamine and histamine-related enzymes such as histidine decarboxylase in the rat and other species . endocrine cells which are argentaffin in type stain with silver stains such as that of masson ( ) because of the presence of endogenous reducing substances including -hydroxytryptamine and catechol-amines are also reported in the mucosa of the fundus of some species including man but apparently not in the rat . enterochromaffin cells are characterised ultrastructurally by the presence of numerous rounded or oval, vesicular, electron-lucent granules frequently containing a small eccentric electron dense core. gastric enterochromaffin cells can also be stained by immunocytochemical techniques using antisera to histamine and histidine decarboxylase as well as to non-specific enolase and chromogranin a betton et al., ) . immunocytochemical study of the rat fundus using a battery of antisera to a variety of gastrointestinal peptides has shown somatostatin containing (d) cells and glucagon staining cells but no cells with gastrin (g cells) or serotonin reactivity (bishop et al., ) . gastrin or g cells possess apical processes reaching the stomach lumen believed to be important in stimulation of gastrin release as a result in increases in antral lumen ph or the presence of amino acids or peptides . glucagon and serotonin containing endocrine cells have also been located in the rat antral mucosa (bishop et al., ) . increased gastric acid secretion is initiated by activation of central vagal efferent pathways but acid secretion is maintained by both neural and endocrine reflexes activated by the presence of food in the stomach. gastrin secreted from the g cells of antrum is the main stimulant of acid secretion. somatostatin is secreted from antral d cells when the luminal ph falls to below . to act by a paracrine mechanism to suppress g cell function thus forming a negative feedback loop (dockray, ) . the two main endocrine cell types from the body mucosa integrate neurohumoral stimuli rather than respond to luminal chemicals. although gastrin is capable of stimulating parietal cells directly, it has an even greater effect through stimulation of enterochromaffin cells to release histamine, a potent paracrine stimulator of parietal cells (hinkle and samuelson, ) . gastrin stimulates release of histamine from enterochromaffin cells of the body mucosa, which increases acid secretion through activation of parietal cell histamine-h receptors. both parietal and enterochromaffin cells are inhibited by somatostatin released from the d cells of the body mucosa in response to a variety of neurohumoral stimuli such as noradrenaline, vasoactive intestinal peptide, calcitonin gene-related peptide, and cholecystokinin. it should also be noted that gastrin has a role in kinetics and differentiation of both parietal and enterochromaffin cells (dockray, ) . gastrin acts at the gastrin/cholecystokin-b receptor that is expressed by gastric epithelial cells and by neurones in the central nervous system (kopin et al., ; wank, ) . the gastrin receptor is simply the cholecystokinin-b receptor located in the stomach. the other cholecystokinin receptor, cholecystokin-a has high affinity for cholecystokinin. stimulation of this receptor in the stomach mediates secretion of pepsin from gastric chief cells and release of somatostatin from d cells resulting in inhibition of acid secretion. in the central nervous system cholecystokinin and its receptors contribute to the regulation of satiety, anxiety, analgesia and dopamine-related behaviour (wank, ) . finally, it is worth recording that both progesterone and oestrogen receptors have been identified in both normal and pathological gastric tissues of humans (wu et al., ) . generative cells in the gastric mucosa as shown by uptake of tritiated thymidine for dna synthesis are distributed principally in the isthmus (inokuchi et al., ) . tracing of cells using thymidine labelling have shown that most of the cells in the generative zone migrate in a successive manner to the mucosal surface to form columnar epithelium. the life span of surface epithelium in the stomach of rats, mice and hamsters has been calculated to be about - days. studies of cell cycle and dna synthesis time in the proliferative zones in the stomach of rat, hamster and man have suggested that the generative cells in the isthmus undergo mitoses at about -hour intervals in rodents and -hour intervals in man (inokuchi et al., ) . although this process of migration from the proliferating cell zone of the isthmus renews surface epithelial cells rapidly, cell migration to the lower parts of the gastric glands is much slower and more complex. detailed studies have show that undifferentiated cells in the region of the isthmus represent a common source for surface mucous cells and mucous neck cells (karam and leblond, ) . electron microscopic and ultrastructural cytochemistry has suggested note: saccharide binding specifications are much more complex than the inhibition by simpler sugars outlined above suggests. see review by nicholson ( ) . *source: nicholson, ; goldstein and hayes, ; schulte and spicer, ; giannasca et al., . that chief cells in the adult rat stomach develop from undifferentiated stem cells in the isthmus (suzuki et al., ) . graft experiments in mice have also suggested that immature cells of the isthmus differentiate into chief cells as well as parietal cells (matsuyama and suzuki, ) . studies in transgenic mice have shown that mature parietal cells influence the fate of other gastric epithelial cells because targeted degeneration of parietal cells is associated with loss of chief cells suggesting interactions between these cell populations in determining their differentiation (li et al., ; canfield et al., ) . gastrin is also an important regulator of parietal cell and enterochromaffin differentiation and number (dockray, ; montgomery et al., ) . labelling experiments in the hamster stomach have shown that both chief and parietal cells possess a similar but quite long life span of about days (hattori, ; hattori and fujita, ) . it has been suggested that the relative distribution of chief and parietal cells in the gastric gland represents an expression of their different migration patterns downwards from the proliferative zones in the isthmus. this type of migration pattern in which cells are able to overtake each other has been termed a 'stochastic flow system' (inokuchi et al., ) . the origin and kinetics of endocrine cells of the stomach has also been the subject of debate but the available morphological, cytochemical and kinetic evidence suggests that the majority of these cells develop from the same stem cells as the other non-endocrine cells of the gastric mucosa, although self replication also occurs (matsuyama and suzuki, ; inokuchi et al., ; solcia et al., ) . much of our knowledge about mucins produced by the epithelial cells lining the gastrointestinal tract has been obtained using histochemical techniques and these approaches continue to be helpful in the understanding of spontaneous and drug-induced gastrointestinal disease (sheahan and jarvis, ; filipe, ; tsiftsis et al., ; jass and roberton, ) . for these reasons, mucin histochemical techniques represent useful tools for the characterisation and elucidation of experimentally or drug-induced changes in the glandular mucosa of gastrointestinal tract. techniques commonly employed are presented in table . the physiochemical properties of gastrointestinal mucins are dependent on their glycoprotein constituents. these glycoproteins are high molecular weight compounds with large numbers of sugar chains attached to a polypeptide backbone by o-glycosidic linkages between n-acetylgalactosamine and serine or threonine (berger et al., ) . the principle monosaccharides present are fucose, galactose, n-acetylgalactosamine, n-acetylglucosamine and sialic acid. traces of mannose may be present and ester sulphate residues are common (filipe, ) . due to this extensive glycosylation, mucins have a filamentous conformation, which is often negatively charged. this is believed to be important in forming a protective barrier to the cell. however, this property is a two-edged sword because when opposing cells have specific receptors for mucins, adhesion may become the predominant factor (van klinken et al., ) . although mucins are important in the gastrointestinal tract, it should be remembered that other products secreted by goblet cells might be important in mucosal defence. it has been recently recognised that trefoil proteins, a family of small proteins secreted by goblet cells and present on the mucosal cell surface, can also protect against a variety of deleterious agents, including bacteria, toxins and drugs (podolsky, ) . there are considerable regional variations in glycoprotein constituents in the gastrointestinal tract and these differences are probably related to physiological and functional factors. furthermore, synthesis and secretion of glycoproteins alter with changes in cell differentiation. alterations also occur in mucins in various inflammatory and neoplastic disease states as well as following administration of certain drugs and chemicals (ishihara et al., ) . terminal sugars or sugar sequences can be demonstrated histochemically by the use of labelled lectins, mostly plant proteins which combine non-enzymatically with particular sugar molecules, see table (goldstein and hayes, ; debray et al., ; rudiger, ) . magenta: all mucosubstances containing hexoses schiff d-pas (pearse, ) and deoxyhexoses with vicinal glycol groups. some non-sulphated acid mucosubstances. neutral mucosubstances. periodate-borohydride/ magenta: pas activity following periodate borosaponification/pas, hydride/potassium hydroxide indicates pb/koh/pas presence of o-acylated sialic acids. (reid et al., ; periodate borohydride reduces periodate culling et al., ; generated aldehydes. potassium hydroxide removes o-acylesters from potential vicinoldiols and sialic residues linked glycosidically to a potential vicinoldiol. alcian blue ph . basophilia: weakly sulphated mucins. carboxyl (pearse, ) groups of sialomucins alcian blue ph . basophilia: sulphated mucins (lev & spicer, ) alcian blue ph . -magenta: neutral mucins periodic acid schiff, ab/pas basophilia: acid mucins (mowry & morard, ) purple-blue: neutral and periodate reactive acid mucins high iron-diamine, brown-black: sulphated mucins hid (spicer, ) unstained: sialomucins high iron-diamine-alcian brown-black: sulphated mucins blue ph . , hid/ab basophilia: non-sulphated acid mucins (sialomucins) (spicer, ) source: adapted from filipe, . when gastrointestinal mucins were studied in several species using histochemical techniques under uniform conditions, species differences were most obvious in the stomach and duodenum (sheahan and jarvis, ) . neutral mucins generally predominate in the stomach, contrasting with acid mucins in the small intestine, and sulphated mucins in the colon. in the stomach neutral mucins staining purple with the pas/alcian blue stain, predominate in the surface and foveolar mucosa, whereas mucous neck cells and antral glands contain acidic mucins that stain blue with pas/alcian blue procedure. sulphated mucins, as shown by the high iron diamine technique (hid) are also found in the deep glandular mucosa of the antrum in rat, mouse and man (filipe, ; jass, ; greaves and boiziau, ) . extremely heterologous staining patterns are seen in the gastric mucosa with labelled lectins, each lectin staining quite different cell populations. there are considerable interspecies differences in staining patterns with the same lectins (kuhlmann et al., ; suganuma et al., ) . the so-called paradoxical concanavalin a stain, in which conjugated concanavalin a is used to label mucins before and after periodate oxidation, has also been used to classify the alterations in mucins in proliferative and neoplastic conditions of the rat stomach mucosa (kobayasi et al., ; tatematsu ). although gastric erosions and ulcers in the glandular mucosa occur quite commonly in laboratory animals in toxicity studies, it is often difficult to determine whether such lesions in treated animals indicate a real ulcerogenic risk for the test compound. there is little that is histologically specific to drug-induced ulceration of the gastric glandular mucosa. mucosal haemorrhage, depletion of mucin, erosions and ulcers with or without inflammation may all be found. erosions represent mucosal breaks superficial to the muscularis mucosa. ulcers are lesions that extend through the muscularis mucosa. whilst the histopathological features of gastric erosions and ulcers are themselves relatively non-specific, it is important to look for any associated pathology in the stomach such as mucus depletion, epithelial hyperplasia or dysplasia, intestinal metaplasia and vascular lesions (see below). in humans, biopsy data suggests that drug-induced ulceration is characteristically devoid of an inflammatory component, but the most usual histological appearances are those of underlying gastric pathology (riddell, ) . formation of gastric and duodenal ulcers is dependent on the presence of both acid and peptic activity in gastric juice because acid without pepsin appears to have little digestive power. important predisposing factors in human patients with peptic ulceration include helicobacter pylori (campylobacter pylori) infection of the antrum, cigarette smoking and ingestion of non-steroidal anti-inflammatory drugs (soll, ) . helicobacter pylori is believed to infect over half the human population and its presence in the gastric mucosa is associated with chronic atrophic gastritis and peptic ulceration (cover and blaser, ) . it is a microaerophilic, gram-negative organism that possesses potent urease activity crucial for its survival at acidic ph. genome sequence analysis has shown that helicobacter pylori has well developed sequences for motility, scavenging iron and dna restriction and modification systems used by bacteria to degrade foreign dna. the link between helicobacter pylori infection and peptic ulceration is related to increases in gastrin release, perhaps through bacterial products or cytokines released from activated lymphocytes (richter-dahlfors et al., ) . although helicobacter pylori can infect other species, apart from non-human primates, the usual laboratory animal models do not appear to develop the inflammatory disease seen in humans (nedrud, ) . erosions and ulcers also develop following stress, reflux of intestinal contents and bile, changes in acid secretion and hypoxia, all of which may develop under the conditions occurring in high-dose toxicity studies. the requirement to give the test compound in high doses may also dictate the need to administer exceedingly high concentrations of test agent. this may produce damaging high local concentrations on the mucosa not relevant to therapeutic doses used in clinical practice. it has been demonstrated that hyperosmolar solutions of quite innocuous substances such as glucose can cause haemorrhage, erosions and ulcers of the rat gastric mucosa (puurunen et al., ) . the well-known association of gastric erosions and haemorrhage with uraemia may also be manifest following administration of high doses of drugs such as diuretics which severely derange fluid and electrolyte balance (garthoff et al., ) . stress ulceration may be linked to temporary ischaemia of the mucosa (dubois, ) . synergism between the ulcerogenic action of drugs and stress is a well-described phenomenon (rainsford, ; beattie, ) . protein depletion and starvation is also capable of inducing gastric ulceration in rats . although gastric pathology represents the largest cause of morbidity and mortality in man following therapy with non-steroidal inflammatory agents (fowler, ) , the reasons for this are probably multifactorial. the acidic properties of some of these drugs may cause direct local damage of gastric epithelial cells, demonstrable by the fact that appropriate formulation can reduce gastric toxicity of these agents in man (brors, ) . anti-inflammatory agents are capable of decreasing synthesis of glycoproteins and this may adversely influence protective mucus production of gastric mucosa (azuumi et al., ; ishihara et al., ) . it has also been suggested that non-steroidal anti-inflammatory agents cause cellular damage to the gastric mucosa by back-diffusion of gastric acid into mucosal tissues (davenport, ) or by causing damage to the gastric capillary bed with subsequent mucosal infarction (robins, ) . the theory that has gained widespread acceptance is that the ulcerogenic potential of non-steroidal anti-inflammatory drugs is related to their pharma-cological activity. vane ( ) proposed that the ulcerogenic potential of these agents was largely a result of their ability to inhibit prostaglandin synthetase, thereby reducing the protective effects of prostaglandins. pharmacokinetic factors may also be important. lipid solubility in the low ph environment of the stomach may influence local penetration into the mucosa (mccormack and brune, ) . moreover, it has been proposed that certain anti-inflammatory agents may possess lesser ulcerogenic potential in man because their inhibition of prostaglandin production is more limited to sites of inflammation, sparing gastric mucosa (whittle et al., ; whittle and vane, ) . it has also been demonstrated that factors altering the enterohepatic circulation of drugs can influence the expression of gastric damage (overvold et al., ) . comparative studies of the ulcerogenic activity of indomethacin in beagle dogs and domestic pigs has suggested that the dog may be an excessively sensitive species as a result of extensive enterohepatic circulation of indomethacin in this species (hanhijarvi et al., ) . prediction of ulcerogenic potential for man based on data from animal models is clouded by the lack of good comparative data on the relative ulcerogenic potential of non-steroidal anti-inflammatory agents in man because of extensive differences in side effect reporting (fowler, ) . moreover proper comparisons in man require not only equivalent therapeutic doses but also comparable dosage forms (brors, ) . in laboratory animals, a variety of different patterns of drug-induced gastric damage have been described. the study by shriver et al. ( ) in which a wide variety of different anti-inflammatory drugs were administered to fasted sprague-dawley rats under identical conditions, suggested the drugs could be divided into three groups based on their profiles of gastrointestinal toxicity. immunological agents such as azathiaprine, cyclophosphamide, methotrexate and d-penicillamine produced gastric mucosal haemorrhage whereas aspirin and related agents produced gastric mucosal haemorrhage and ulcers. the powerful nonsteroidal anti-inflammatory drugs indomethacin and phenybutazone produced gastric mucosal erosion and ulcers as well as small intestinal damage. comparative single oral dose studies of several different non-steroidal antiinflammatory agents at three different dose levels by suwa et al. ( ) in the rat using histology and measurement of faecal blood loss with cr-labelled blood cells have also shown that different patterns of ulceration can be produced by different agents when administered under identical conditions. single oral doses of some non-steroidal anti-inflammatory drugs including aspirin produced widespread superficial damage and desquamation of gastric epithelium with little or no inflammation at hours following dosing which completely healed weeks later. this damage was associated with transient faecal blood loss. by contrast, indomethacin and ibuprofen produced both gastric damage and circumscribed, penetrating ulcers along the mesenteric border of the jejunum and ileum. furthermore, ulcers were still present after weeks and were associated with prolonged or biphasic blood loss (see small intestine). in addition feeding conditions can influence the distribution of erosions and ulcers in laboratory animals. in fasted rats, erosions due to indomethacin treatment are found in the body of the stomach whilst in conventionally fed rats they are most prominent in the small intestine. detailed studies by satoh et al. ( ) showed that rats fed for hour after a hour-fast and given a single dose of indomethacin within hours of re-feeding developed erosions and ulcers in the antrum primarily along the lesser curvature. indomethacin given to fasted rats produced erosions in the body mucosa. a further factor that needs to be kept in mind is that chronic administration of ulcerogenic compounds may produce quite different pathological appearances to those found following single dose administration. administration of aspirin to rats for weeks has been shown to stimulate epithelial proliferation of the gastric body but not antral mucosa, possibly by an effect on cyclic adenosine ', ' monophosphate (cyclic amp) or though increasing the rate of epithelial exfoliation (eastwood and quimby, ) . such a response may be the basis for increased resistance of the gastric mucosa to the chronic affects of these agents. it also may explain the tendency for ulcers to occur in the antrum following chronic administration of aspirin-like drugs as the proliferative response and presumably the adaptive potential appears less in this part of the gastric mucosa. both interspecies variations and strain differences have been reported in the response to ulcerogenic compounds. rainsford et al. ( ) showed that extravasation of red blood cells and greater vascular damage was observed in rats treated with aspirin or benoxprofen than in pigs given similar doses. sprague-dawley rats appear less susceptible to the ulcerogenic effects of cold-restraint stress than wistar rats (goldenberg, ) . diuretics and some angiotensin converting enzyme (ace) inhibitors and angiotensin ii antagonists have been associated with the development of gastric erosions and ulceration when administered in high doses to laboratory animals ( fig. ) (imai et al., ; garthoff et al., ) . however, these effects appear related to the severe electrolyte disturbances produced by excessive doses of these drugs. this is perhaps analogous to the well-known association of gastrointestinal tract erosion and haemorrhage with uraemia. dogs appear to have a particular predisposition to this effect where it may be associated with deposition of basophilic ground substance and mineral in connective tissues and blood vessels in the mucosa (barker and van dreumel, ) . although inflammatory conditions due to microorganisms are generally uncommon in the stomach, gastritis is reported in laboratory rhesus monkeys in association with the presence of helicobacter organisms (reed and berridge, ) . as in the analogous condition in man, the stomach of affected animals shows an infiltration of the central mucosa by small lymphocytes and plasma cells, associated with reactive or atrophic changes in the mucosa and the presence of small curved bacteria in glands, visualised best with the warthin-starry stain. infiltration of the stomach by lymphocytes in rats treated with human recombinant interleukin- without ulceration was reported as part of a multisystem involvement induced by this agent (anderson and hayes, ). decrease in gastric mucus secretion may accompany both spontaneous inflammatory conditions and drug-induced lesions in the stomach of man and experimental animals. mucus depletion is characterised histologically by the presence of an intact epithelial layer in which cells show loss of the normal clear cytoplasm replete with mucous substances by more basophilic cells that contain little or no mucin. qualitative changes in mucus composition can also accompany mucus depletion. gastric epithelium in man may show decreases in sulphated mucosubstances following stress, high alcohol consumption or after aspirin administration (filipe, ) . similar changes occur in laboratory animals subjected to ulcerogenic regimens. stress ulceration in the rat is accompanied by decreased sulphation of gastric glycoproteins, presumably an expression of the changes in gastric cellular activity accompanying stress (lambert et al., ) . administration of aspirin and other anti-inflammatory agents including adrenocortical steroids to laboratory animals also reduces the content of sulphomucins in the gastric mucosa, probably by reducing their synthesis (denko, ; gerard, ; ishihara et al., ) . rather surprisingly, administration of histamine h -receptor antagonists and fig. . section from the glandular stomach from a rat treated with ah high dose of an angiotensin ii antagonist that shows superficial degeneration and ulceration (erosion) of the mucosa. (he, × .) proton pump inhibitors associated with reduction of gastric acid output and increases in gastrin secretion have also been associated with alterations in gastric mucus. administration of omeprazole or famotidine to rats for weeks was shown to inhibit prostaglandins pge as well as the synthesis of both total and sulphated glycoprotein synthesis along with histochemical evidence of reduction in pas staining of the surface mucus (yoshimura et al., ) . although the mechanism for this change is unclear, the reduction in mucus, particularly sulphated mucus that is believed to be particularly resistant to peptic digestion, may have implications for mucosal defence. intestinal metaplasia of the stomach is characterised by the presence of differentiated epithelium, which resembles small intestine on the basis of light microscopic and ultrastructural morphology, mucin patterns and enzyme histochemistry (morson, ; planteydt and willighagen, ; lev, ; goldman and ming, ; . it develops in man in gastric mucosa altered by chronic atrophic gastritis and its significance is due to the fact that a link exists between intestinal metaplasia and gastric cancer. although intestinal metaplasia is found much less commonly in laboratory animals, it has also been reported to occur in association with gastric cancer induced by polychlorinated biphenyls (ward, ) . in view of this association with gastric cancer, it has been suggested that intestinal metaplasia represents a pre-neoplastic lesion. however, over recent years prospective clinical studies and experimental data have suggested that it is an epiphenomenon, coexisting with, but unrelated to the development of cancer. in man, several forms of intestinal metaplasia have been described. these variants fall into two main groups, an incomplete type and a complete form (teglbjaerg and nielson, ; jass and filipe, ; jass, ) . complete intestinal metaplasia is characterised by the presence of goblet cells, paneth cells and absorptive cells with brush borders and variably developed intestinal villi. incomplete forms are more heterogeneous characterised by goblet and mucous columnar cells but no absorptive cells and variable patterns of mucin. the routine alcian blue: ph . , periodic acid-schiff stain (ab/pas) ( table ) distinguishes between the intestinal acid mucins (blue) from the neutral mucins of gastric type. however, variable sialomucin and sulphomucin staining patterns are seen in intestinal metaplasia in man with the high iron-diamine/alcian blue stain (hid/ab) (jass, ) . the incomplete form of intestinal metaplasia, showing marked sulphomucin secretion, has been found more commonly in association with gastric cancer in man (jass and filipe, ; jass, ; wells et al., ) . however, prospective studies have tended to indicate that intestinal metaplasia with sulphomucin secretion may be an age-related form of chronic atrophic gastritis and not a premalignant lesion (ectors and dixon, ) . it has been suggested that intestinal metaplasia represents an adaptive response to long-standing chronic inflammation and reduced acid secretion. it may also represent an adaptive defensive response to long-standing helicobacter pylori infection because intestinal mucosa is more resistant to these organisms (steer, ; ectors and dixon, ) . intestinal metaplasia has been found in association with gastric cancer in laboratory animals. fischer rats treated with the polychlorinated biphenyl, aroclor , mixed in the diet for years developed foci of intestinal metaplasia in the stomach epithelium in association with gastric adenocarcinomas (ward, ) . these lesions were characterised by abundant mucin-containing cells and alkaline phosphatase activity typical of the small intestine (morgan et al., ; ward, ) . similar, but more diffuse intestinal metaplasia was reported in the stomach of primates treated with polychlorinated biphenyls, although unassociated with gastric neoplasia (allen, ; mcconnell et al., ) . intestinal metaplasia is also found in the stomach of laboratory animals treated with powerful genotoxic gastric carcinogens. although tsiftsis et al. ( ) showed hyperplasia and foci of atypical changes (dysplasia) but little or no intestinal metaplasia in rats following administration of n-methyl-n´-nitro-n-nitroguanidine, tatematsu et al. ( ) were able to show intestinal metaplasia in rats treated with the same agent. however, intestinal metaplasia can be induced in rodents by a variety of different procedures that are not associated with the development of gastric cancer. intestinal metaplasia can be induced in the glandular stomach of rodents by fractionated, localised, ionising radiation (watanabe, ; watanabe et al., ) , injection of xenogenic stomach antigens as well as propantheline bromide and the non-carcinogen, iodoacetamine shirai et al., ) . the characteristics of intestinal metaplasia in laboratory rodents are similar to those seen in man with early increases in intestinal enzyme activity (alkaline phosphatase, lactase, trehalase, sucrose and maltase), development of goblet cells containing neutral, sialo-, or sulphomucins, and intestinal crypts with or without paneth cells. both the fundus and antrum can show changes although as in man, males appear more prone to develop intestinal metaplasia than females . based on these experimental findings, have also proposed that intestinal metaplasia is not a precancerous condition but an adaptive response to a chronic elevation in ph in gastric secretion due to the early loss of parietal cell mass brought about by these various procedures. on balance therefore, the evidence to date suggests that although intestinal metaplasia is associated with cancer and may consequently be considered a helpful morphological feature in the evaluation of human gastric biopsies, the finding of isolated intestinal metaplasia in safety studies does not indicate a preneoplastic state. finally, a form of metaplasia in which hepatocytes have been found has been reported as rare incidental findings in the glandular stomach of mice sacrificed at the end of -year carcinogenicity bioassays (leiniger et al., ) . histologically, focal accumulation of well-differentiated hepatocytes were found in the submucosa and lamina propria adjacent to the limiting ridge with dilated adjacent gastric glands showing epithelial hyperplasia and mineralisation with herniation into the submusosa. whilst these foci were not believed to be related to treatment with xenobiotics, it is not clear whether represented metaplasia or congenital ectopia. the gastric glandular epithelium is predisposed to the deposition of calcium possibly as it is a site at which marked ion exchange normally takes place. focal aggregates or concretions of densely blue-staining mineral are fairly commonly observed in haematoxylin-stained sections from the stomachs of aged rats where they are associated with cystic dilatation of the gastric glands (greaves and faccini, ) . mice and hamsters occasionally show similar changes. small concretions are also observed in gastric glands in the beagle dog. these appear to represent aggregates of calcium around mucoid material. gastric mineralisation may become marked in rodents and dogs when there is disturbance of mineral metabolism, particularly in association with renal pathology. this has been well described in rats with severe renal disease (glomerulosclerosis) and parathyroid hyperplasia (snell, ) . a similar phenomenon has been described in the stomach of dogs in uraemic states (cheville, ) . identical changes result from the administration of drugs that induce prolonged azotemia or electrolyte disturbances. these changes are characterised by diffuse deposition of mineral in the intestinal tissue of the mucosa of the gastric body but not cardia, antrum or pylorus. mineral deposits develop around basement membranes surrounding epithelium and blood vessels. the lamina propria becomes expanded by oedema and fibroplasia of the interstitium also develops. the gastric glands themselves become distorted with swelling and degeneration of parietal cells and atrophy of chief cells. erosion of the glandular epithelium with haemorrhage occurs presumably as a result of the ischaemia caused by diffuse vascular injury and altered parietal cell function. focal atrophy of the gastric glandular mucosa is a sporadic occurrence in laboratory rodents, usually as a result of previous focal gastric inflammation, ulceration, mineralisation or vascular occlusion. these changes, characterised histologically by focal fibrosis of the mucosa, gastric glandular dilatation and atrophy variably accompanied by polymorphonuclear cells and mast cells are common in certain strains of rats when years or more in age (anver et al., ) . whereas diffuse mucosal atrophy occurs following severe inflammatory insult, diffuse atrophy of the stomach glandular mucosa without inflammation can be a result of surgically or drug-induced reduction in trophic factors necessary for the maintenance of normal gastric morphology and function. this is observed in man and experimental animals following antrectomy because this removes the peptide-producing cells of the antrum (gjurldsen et al., ; neilsen et al., ) . in the rat, antrectomy is accompanied by hypogastrinaemia, reduced weight and height of the oxyntic mucosa and a reduced number of argyrophil cells (håkanson et al., . this is in contrast to procedures such as antral exclusion that lead to hypergastrinemia and increased thickness of the oxyntic mucosa. mice with genetic deletion of the gastrin gene also show reduction in the thickness of the gastric mucosa. whilst all cell types are present, there is a most pronounced decrease in the numbers of parietal cells as well as enterochromaffin cells associated with an increase in surface mucous cells. these changes are linked to a profound decrease in acid secretion, which becomes unresponsive to histaminergic, cholinergic and gastrinergic stimulation (hinkle and samuelson, ) . analogous atrophic changes have been reported following pharmacological removal of trophic stimuli. for instance, administration of the cholecystokinin-b/gastrin receptor antagonist, ci- to cynomolgus monkeys for periods of up to weeks was associated with an initial phase of multifocal degeneration of gastric glands primarily in the fundus followed by diffuse reduction in the thickness of the glandular mucosa with little or no qualitative changes to the cell populations (dethloff et al., ) . although bilateral vagotomy produces profound functional changes in the stomach, notably reduction of gastric acid secretion, morphological changes in the fundal mucosa are not marked either in experimental animals or in man (crean et al., ; aase and roland, ) . studies in the rat have shown that diffuse atrophy of the gastric glands characterised by a decrease in the number and size of parietal, chief and mucous cells occurs transiently following truncal vagotomy but histological features return to normal by about month after surgery (nakamura, ) . by contrast, unilateral vagotomy in the rat leads to marked and persistent atrophy of the oxyntic zone on the denervated side. this is characterised histologically by reduced height of the mucosa and reduced numbers and staining intensity of argyrophil cells (hakanson et al., ) . håkanson and his colleagues argued that this unilateral atrophy was due to the removal of the trophic action of the vagus. the lack of lasting atrophy after bilateral but not unilateral vagotomy was explained by the subsequent rise in gastrin that occurs after bilateral vagotomy as a result of lack of acid feedback inhibition of gastrin release (håkanson et al., ) . removal or reduction in extra-gastric trophic factors or hormones may also reduce the thickness of the gastric mucosa. this is has been demonstrated in the rat by hypophysectomy which causes a reduction in thickness of oxyntic and antral mucosa, compared with pair-fed controls. although there was little or no change in peptic:parietal cell ratios, a significant decrease in cell volume and secretory activity of gastric glandular cells were demonstrated which suggested a widespread disturbance of synthesis and secretory mechanisms (bastie et al., ) . atrophic changes in the chief cells were observed in rats treated for months with high doses of omeprazole, an inhibitor of acid secretion. the findings were considered to represent disuse atrophy secondary to the inhibition of acid secretion (hansson et al., ) . another inhibitor of gastric acid secretion, the tricyclic agent pirenzepin, also produced atrophy of the fundic mucosa of rats following months but not month of treatment (lehy et al., ) . the atrophy was characterised by reduction in parietal cell numbers associated with lower numbers of gastrin-containing cells in the antrum, features unlike those following prolonged treatment with histamine h -receptor antagonists. an increase in the thickness of the gastric mucosa can be the result of hypertrophy or hyperplasia of the mucosal cells and this occurs both spontaneously or following administration of drugs and chemicals. in view of the different cell populations in the gastric mucosa and the variety of morphological alterations that occur, it is difficult to make a clear distinction between hypertrophy and hyperplasia without morphometric techniques. morphometric techniques have shown that hypertrophy of some mucosal cells can coexist with hyperplasia of other gastric cell populations. a distinction also needs to be made between diffuse or uniform hyperplasia involving one or more of the cell populations from the hyperplasia associated with proliferative or adenomatous overgrowth. adenomatous hyperplasia also needs to be evaluated for atypical cytological features (dysplasia), which are linked to development of gastric carcinoma (see below). cells of gastric glandular mucosa undergo increases in size or number in response to the effects of gastrointestinal trophic hormones or their synthetic analogues. similar changes also follow administration of compounds that inhibit gastric acid secretion or modify other trophic hormones or growth factors. when gastrin or its synthetic analogue, pentagastrin is administered subcutaneously to rats and mice for several weeks, there is both an increase in the number and size of parietal cells without concomitant increase in zymogenic chief cells (willems and lehy, ; crean et al., ; balas et al., ) . in addition, diffuse hyperplasia of enterochromaffin cells also occurs. by contrast, cholecystokinin, a trophic peptide found in the duodenum and sharing the same c-terminal tetrapeptide sequence as gastrin, increases in the number of chief cells but not parietal cells when administered to mice under similar conditions . drugs which inhibit or neutralise gastric acid secretion such as histamine h antagonists, proton pump inhibitors and antacids also induce hypertrophy or hyperplasia of the parietal cell population (witzel et al., ; crean et al., ; mazzacca et al., ; kaduk and hauser, ; betton et al., ; white et al., ) . these agents are associated with a rise in serum gastrin levels, probably as a result of loss of feedback inhibition of low antral ph on gastrin-producing g cells (witzel et al., ) . not all histamine h antagonists produce identical effects. other cytological changes have been reported with famotidine, another h -receptor antagonist. this agent produced a dose-related increase in the prevalence and degree of eosinophilic granularity in chief cells of the stomach in toxicity studies in rats but not dogs (burek et al., ) . electron microscopy showed an increase in electron density of zymogen granules and it was argued that these effects were the result of secondary inhibition of pepsin secretion or turnover due to inhibition of acid secretion. cytoprotective agents of prostaglandin type produce different forms of diffuse gastric hyperplasia. rats treated with , -dimethyl prostaglandin e hourly for weeks, not only developed forestomach alterations (see above) but also thickening of both the body and antral mucosa. in the body mucosa, these changes were the result of a proportional increase in the total mass of surface and foveolar mucous cells, mucous neck cells, chief cells, parietal and endocrine cells as well as connective tissue. this was largely as a result of increase in cell number, although parietal cells also increased in size (reinhart et al., ) . unlike treatment with gastrin and gastrin analogues there was an increase in number of surface and foveolar mucous cells associated with increase in mucus content. misprostal, a synthetic prostaglandin e methyl ester analogue also produced diffuse glandular hyperplasia, characterised by lengthening of gastric pits and increased mucous secretion in the preclinical safety studies in dogs and rats (kotsonis et al., ) . this glandular hyperplasia not only affected the body but also the antral mucosal. studies with tritiated thymidine showed that the labelling index was reduced in rats treated with misprostal, suggesting hyperplasia following administration of prostanoids is a result of an increase in cell survival and decrease in cell shedding rather than an increase in cell proliferation (fich et al., ) . levin ( ) has reviewed the effects of prostaglandins of the e series on the gastrointestinal tract of dogs and rodents. a dose-related diffuse hyperplasia of the gastric glandular mucosa has been reported in both rats and cynomolgus monkeys given human recombinant epidermal growth factor. the gastric mucosa was thickened and there was a increase in the number of undifferentiated cells particularly in the neck region and upper part of the gastric glands (breider et al., ; reindel et al., ) . mitotic figures were also numerous in the upper reaches of the mucosa. the lower parts of the gastric glands were generally less affected. the large increase in the number of undifferentiated cells may have a functional effect on gastric acidity and function (vinter-jensen, ) . administration of recombinant growth hormone has also been reported to induce thickening of the gastric glandular mucosa in dog toxicity studies along with typical growth hormone-induced changes in other organs, body weight increases and insulin-like growth factor (prahalada et al., ) . the pyloric and fundic mucosa showed histological evidence of hyperplasia of the mucous neck cells. thickening of the gastric glandular mucosa as a result of an irregular proliferation and cystic dilatation of gastric glands associated with inflammation characterises a number of non-neoplastic conditions in the stomach of man and laboratory animals. cystic change with chronic inflammation and foveolar hyperplasia is observed in biopsies taken from the edge of chronic gastric ulcers in man (franzin and novelli, ) . ménétrier's disease ('polydadenomes en nappes'), a rare disease found primarily in middle-aged men is also characterised by enlarged gastric folds, foveolar hyperplasia and gastric glandular cystic dilation (berenson et al., ; wilkerson et al., ) . although its pathogenesis remains elusive, increased expression of transforming growth factor α (tgfα) and the epidermal growth factor receptor has been described (demsey et al., ) . as tgfα is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin, and transgenic mice that overexpress tgfα in gastric mucosa develop a similar condition (see below), it was suggested by demsey et al. ( ) that tgfα might have an important role in this condition. similar changes have been observed in animals in association with infestation of the gastrointestinal tract by parasites (jubb and kennedy, ; cook et al., ) . laboratory rodents may develop a similar pattern of changes spontaneously with advancing age, although the cause of this change remains uncertain. the distinction between adenomatous hyperplasia and adenoma is not clear cut. nevertheless, adenomas are usually defined as localised or focal proliferative lesions with well-ordered glandular patterns with a clear boundary with the surrounding normal mucosa. they are usually exophytic or polypoid in nature but adenomas with localised downward growth are described (mohr, ) . proliferation of the gastric glandular mucosa has been well characterised in the laboratory mouse because certain strains have a particular tendency to develop this condition spontaneously with advancing age (stewart and andervont, ; rowlatt et al., ) . hyperplasia also occurs spontaneously in conventional laboratory strains employed in carcinogenicity bioassays. its prevalence can be influenced by environmental factors such as housing (chvédoff et al., ) , food restriction (rehm et al., ) and the administration of xenobiotics (poynter et al., ; betton et al., ) . similar gastric changes have also been reported to occur in mice thymectomised shortly after birth (suzuki et al., ) . histologically, these changes in mice are characterised by hyperplasia of the foveolar and neck regions of the body mucosa (fig. ) . in advanced cases this is accompanied by elongated, tortuous, or dilated glands lined by simple columnar or cuboidal epithelium, devoid of parietal or chief cells. the abnormal cells show only mild cellular pleomorphism and mitotic activity. the abnormal glands dis-place normal glandular tissue and may penetrate through the muscularis mucosa to reach the muscularis externa and serosa. step sections demonstrate continuity between these glandular elements and a total absence of metastatic spread in the adjacent tissues and lymph nodes. the lamina propria also shows increased amounts of smooth muscle and collagen accompanied by variable numbers of lymphocytes and other chronic inflammatory cells. oedema may be observed and blood vessels are often dilated. the antral mucosa remains relatively unaffected. histochemistry has shown variable mucin secretion of the altered glands. some glands are devoid of mucin, others show an increase in sulphomucin as revealed by the high-iron diamine technique (greaves and boiziau, ) . it has been suggested that these features are similar to ménétrier's disease in man and might have a similar pathogenesis (dempsey et al., ; takagi et al., ) . the aetiology of the spontaneous condition in the mouse is uncertain. the occurrence of similar lesions in thymectomized mice has given rise to the suggestion that autoimmune damage to the gastric mucosa may be responsible (kojima et al., ) . the presence of circulating anti-parietal antibodies and the decrease in the number of parietal cells in thymectomized mice suggested that autoimmune damage can occur to parietal cells with compensatory chronic stimulation and proliferation of the generative zones (suzuki et al., ) . however, based on findings in female han nmri mice, rehm et al. ( ) showed that this proliferative condition can develop in mice in the absence of antiparietal antibodies. they demonstrated that this change is associated with an increase in the number of antral gastrin cells, raising the possibility of a hormone or paracrine mechanism. a similar proliferative form of gastropathy has been reported in mice which overexpression transforming growth factor α (tgfα), a potent mitogen and member of the epidermal growth factor family of peptides. tgfα acts by binding to and activating the tyrosine kinase of the epidermal growth factor receptor. transgenic mice overexpressing tgfα develop severe adenomatous and cystic hyperplasia of the gastric glandular mucosa starting from about months of age along with loss of mature parietal cell numbers and a diminution in gastric acid production (takagi et al., ) . the degree of change was to some extent dependent on the genetic background on which the transgene operated. an increased prevalence of similar changes have been reported in cd- mice treated with the novel histamine h -receptor antagonist sk&f for months (betton et al., ; . although treated mice developed hyperplasia of gastric neuroendocrine cells similar to that observed in rodents treated with other antisecretory agents, they also showed an increase in the severity of glandular hyperplasia. like the spontaneous condition, these changes were characterised by thickening of the mucosa by hyperplasia of the foveolar and neck regions, and downward proliferation of glandular elements into gastric glands (betton et al., ) . poynter et al. ( ) have also reported similar glandular hyperplasia in the mouse stomach associated with histamine h -blockade with the agent ioxtidine. these findings were similarly associated with hyperplasia of neuroendocrine cells. adenomatous polyps have also been reported in the pyloric antrum of c bl/ j mice treated for weeks with the synthetic progestin, cyproterone acetate (tucker et al., ) . these were single, pedunculated and well-differentiated lesions showing little evidence of dysplasia. the mechanism for the induction of these polyps is unclear although they may have been hormonally mediated as progesterone receptors have been identified in gastric tissue (wu et al., ) . although usually less prevalent and less exuberant than in mice, the aged rat also develops proliferative gastric glandular changes spontaneously. these changes are characterised by hyperplasia of the foveolar and mucin-secreting cells of the body mucosa, development of cystic glands lined by simple mucous or flattened cells, accompanied by chronic inflammatory cells, prominent blood vessels and smooth muscle in the lamina propria (greaves and faccini, ) . the antrum remains relatively unaffected. proliferative alterations can be induced by administration variety of xenobiotics as well as following surgical procedures that induce chronic reflux of normal intestinal contents. for instance, hyperplasia of the gastric mucosa, notably over the lesser curvature has been described in rats following the so-called bilroth ii gastrectomy that allows reflux of intestinal and biliary secretions into the stomach (kobayashi et al., ) . a proliferative condition of the gastric mucosa has been shown to develop following long-term treatment of rats with an ulcerogenic regimen of aspirin. female sprague-dawley rats given mg/kg of aspirin in % methylcellulose once daily orally by gavage for months followed for periods of up to months without treatment, developed focal proliferative changes at the sites of healed ulcers, mainly in the mucosa of antrum or antral-body junction (st john et al., ) . these lesions were characterised by the presence of proliferating gastric glands lined by columnar, cuboidal or flattened epithelial cells in the mucosa, which also extended through the muscularis mucosa. mucus content of these glands was variable but when present was principally acidic in type, as shown by staining with alcian blue at ph . . the lesions were accompanied by increased collagen in the lamina propria, endarteritis and an infiltration of lymphocytes, plasma cells and mast cells. the lesions were not associated with the development of carcinoma following months' observation and it is probable that they were the result of the chronic damage and repair induced by aspirin treatment. hyperplasia of the gastric glandular mucosa also occurs in rats following the administration of powerful genotoxic carcinogens, although characteristically in association with of atypical histological changes and ultimately carcinoma. these changes have been best characterised in sequential studies with the rat using the carcinogen n-methyl-n´-nitro-n-nitrosoguanidine at doses low enough to avoid overt gastric ulceration and regenerative hyperplasia. it was shown that hyperplasia developing under these conditions occurs diffusely both in the body and antral mucosa. furthermore, the changes occurred earlier in the antrum than in the body and were focal or polypoid in character (tsiftsis et al., ) . involvement of the antrum in this way is quite unlike the spontaneous hyperplasia of the rat gastric mucosa. histologically, this form of hyperplasia is characterised by lengthening of the foveolae and neck regions both in the antrum and body. hyperplastic pits or foveolae show increased secretion of sialomucins and sulphomucins with a concomitant loss of neutral mucins. polychlorinated biphenyls such as arochlor , which produce intestinal metaplasia and adenocarcinoma in the stomach of rats, also induce proliferative alterations characterised by proliferative cystic lesions in the mucosa associated with inflammation and fibrosis (morgan et al., ) . in common with lesions induced by genotoxic agents, these changes are found primarily in the antrum and pyloric regions, zones of predilection for the development of gastric carcinoma in man and experimental animals. it is important to distinguish between the various hyperplastic and adenomatous conditions found in the gastric glandular mucosa in laboratory animals that are not associated with neoplasia from those which precede the development of carcinoma. this distinction is complicated by the fact that proliferative changes associated with the development of cancer both in man and laboratory animals possess features in common with lesions not associated with neoplasia. however, a key distinctive feature is the degree of epithelial dysplasia. dysplasia is considered to be the lesion common to gastric conditions in man such as atrophic gastritis and gastric polyps that have been linked with a significantly increased risk of gastric cancer. although the term dysplasia may be less widely employed in experimental pathology, similar dysplastic changes to those occurring in man have been characterised in laboratory animals in which precancerous gastric lesions have been studied (tsiftsis et al., ) . it therefore represents a unifying concept in the assessment of proliferative changes in the gastric glandular mucosa of laboratory animals. as defined by an international group concerned with the diagnosis of preneoplastic conditions in the stomach of man, the principle features of dysplasia are (i) cellular atypia; (ii) abnormal differentiation; and (iii) disorganised mucosal architecture (morson et al., ; nagayo, ) . cellular atypia is characterised by nuclear pleomorphism, hyperchromasia and stratification of nuclei, increased nuclear-cytoplasmic ratio and loss of cellular and nuclear polarity. abnormal differentiation is shown by reduction or alteration in the normal secretory products of the mucosa. disorganised mucosal architecture is shown by irregularity of crypt structure, back-to-back glands, budding and branching of crypts and intraluminal and surface papillary growths. it is important to assess gastric mucosa very carefully for the features of dysplasia when hyperplastic gastric changes are found in treated animals. in the rat gastric cancer model employing the carcinogen n-methyl-n´-nitro-n-nitrosoguanidine, dysplastic changes were shown to start in the proliferating neck region of hyperplastic zones (tsiftsis et al., ) . these changes were characterised histologically by irregular growth patterns of glandular cells showing reduced mucin secretion, numerous mitoses and enlarged pleomorphic nuclei. these atypical glands were observed to extend downwards, eventually replacing normal gastric glands and ultimately penetrating the muscularis mucosa forming infiltrating adenocarcinomas of variable differentiation. the antrum developed these changes earlier than the body mucosa (tsiftsis et al., ) . these considerations were important in the safety evaluation of the histamine h receptor antagonist, tiotidine (ici , ) a guanidino-thiazole derivative that also produced proliferative gastric lesions in the stomach of rats in a month carcinogenicity study (streett et al., ; . these changes were found mainly in the pyloric region and were characterised histologically by superficial erosions and irregular pyloric glands lined by cells with basophilic cytoplasm and enlarged hyperchromatic nuclei. some atypical glands penetrated the muscularis mucosae. dysplastic lesions situated primarily in the pyloric region were also associated with the development of invasive carcinoma in some rats. extensive histological sectioning of the stomach in rats treated with tiotidine for only months also revealed evidence of early proliferative changes (streett et al., ) . therefore, these lesions produced by tiotidine possessed more in common with those induced by powerful carcinogens such as n-methyl-n´-nitro-n-nitrosoguanidine than the benign, species-specific proliferative change of little or no relevance for human safety. interestingly, mice treated for months with tiotidine were devoid of dysplastic changes in the gastric mucosa (streett et al., ) . one of the most remarkable examples of drug-induced gastric alterations in rodent bioassays is the hyperplasia of enterochromaffin cells and development of carcinoid-like neoplasms in the stomach of rats treated with omeprazole (havu, ) . omeprazole is a substituted benzimidazole which inhibits gastric acid secretion by blocking the enzyme h + , k + -atpase, the proton pump of the parietal cells, in a specific and dose-dependent manner (fellenius et al., ) . although in rats there is a increase in number of gastric argyophilic cells with increasing age, rats treated with omeprazole for weeks showed a marked, dose-related and diffuse increase of argyophilic, non-argentaffin cells in the basal half of the oxyntic fundal mucosa (havu, ) . these changes were more marked in female than in male rats but were not observed in the bioassay in which cd- mice were treated with similar doses of omeprazole for weeks. these diffuse changes in the rat stomach were associated with focal hyperplasia of argyrophilic cells. these focal lesions were also associated with a doserelated increase in larger focal nodular lesions of argyrophilic cells, some of which were undoubtedly locally infiltrating carcinoid tumours. these nodular argyrophil lesions posed the usual problems of differential diagnosis of endocrine hyperplasia and neoplasia (see endocrine system, chapter xiii) and distinction of hyperplasia from neoplasia was uncertain. histologically, nodular lesions were composed of multifocal anastomosing solid or pseudoacinar cords of proliferating, regular cells with uniform nuclei and moderately abundant fine granular pale cytoplasm. these nodules showed little or no cellular pleomorphism or mitotic activity but clear evidence of submucosal infiltration without involvement of the muscularis externa was observed in some cases. the overall light microscopic features were similar to those of gastrointestinal carcinoid tumours reported in man. the incidence of gastric carcinoids was reported to be as high as % in females in the high dose group but only a few cases observed in similarly treated males (ekman et al., ; havu, ) . electron microscopy of the altered argyophil cells confirmed the presence of electron-lucent, vesicular granules, frequently with small irregular dense cores characteristic of enterochromaffin cells of the stomach. immunocytochemical study showed that these cells contained histidine decarboxylase which is found normally in gastric enterochromaffin cells which produce and store histamine . other findings reported in rats treated with omeprazole have been a proportional increase in the number and size of non-endocrine cells of the fundus (blom, ) , an increase in the number and immunostaining properties of the antral gastrin-containing g cells and hypergastrinaemia (bishop et al., ; creutzfeldt et al., ) . all functional and morphological changes following treatment for days were fully reversible after days' drug withdrawal (creutzfeldt et al., ) . as a result of these treatment-related increases of these normally rare gastric carcinoids in the rat bioassay with omeprazole, clinical trials with this agent were suspended until it was agreed that the endocrine alterations were a result of prolonged drug-induced achlorhydria. it was postulated that omeprazole causes a prolonged inhibition of acid secretion in the rat, which causes activation, and subsequently hyperplasia of antral gastrin cells and marked hypergastrinaemia. hypergastrinaemia in turn stimulates enterochromaffin cells of the fundus, which in time results in enterochromaffin hyperplasia . this argument is supported by the fact that similar morphological findings are reported in chronic atrophic gastritis and other achlorhydric sites in man (solchia et al., ; müller et al., ) and that antrectomy in the rat prevents the appearance of enterochromaffin hyperplasia following treatment with omeprazole . although mild dose-related gastric argyrophil cell hyperplasia was noted in dogs treated with omeprazole for year, neoplasms of the stomach were not observed during this time period. why mice neither developed neither argyrophil hyperplasia nor gastric carcinoids with a similar treatment regimen is not clear, as the mechanism of action of omeprazole is similar in rat, dog and mouse. however, as the duration of action of omeprazole is shorter in the mouse, it was postulated that sustained inhibition of gastric acid secretion over hours is necessary to activate increased gastrin secretion from antral cells (havu, ) . it has also been suggested that the mouse possesses fewer gastric enterochromaffin cells than the rat and shows a much lower serum gastrin response to omeprazole treatment (ekman et al., ) . duration of action or potency may also be the explanation for the lack of reports of carcinoid neoplasms in rats following inhibition of gastric acid secretion by the histamine h -receptor blockers cimetidine and ranitidine. neither of these drugs completely inhibits gastric acid secretion in the rat for hours (leslie and walker, ; larsson et al., ) , although mild gastric neuroendocrine hyperplasia has been recently described in cimetidine-treated rats (hirth et al., ) . however, the long-acting h -receptor antagonist sk&f produced gastric carcinoid neoplasms when administered at a high dose ( mg/kg) to rats for years (figs. and ) (betton et al., ; . although this dose level of sk&f did not entirely suppress gastric acid secretion and control gastric ph over hours, plasma gastrin levels remained elevated at - times control values over this period. in a -month oral carcinogenicity study in cd- mice at the same dose level ( mg/kg), a diffuse neuroendocrine cell hyperplasia and multifocal glandular hyperplasia or neoplasia was also observed (betton et al., ) . similarly, loxitidine, a potent, non-competitive, insurmountable histamine h -antagonist produced hyperplasia of neuroendocrine cells and carcinoid tumours in the gastric fundus of both rats and mice after years' treatment in diet and drinking water, respectively (poynter et al., (poynter et al., , . other histamine antagonists bl- and ici have been reported to produce neuroendocrine neoplasms in the stomach of rats and rats and mice, respectively (hirth et al., ; streett et al., ) . wormsley ( ) reviewed the considerations in the risk-benefit analysis of agents intended for long-term administration for peptic disease. drugs of other classes also cause hyperplasia of gastrin-containing cells. immunocytochemical study using antigastrin antibody revealed increased gastrin cell numbers in the antral mucosa of dogs given high doses of adrenocorticosteroids for weeks and these changes were accompanied by enhanced serum and tissue gastrin levels (delaney et al., ) . these results suggest that adrenocorticosteroids have a trophic effect on gastrin-containing cells. in human patients hypergastrinaemia is also produced by pharmacologically induced hypochlorhydria although this is usually only slight and hyperplasia of enterochromaffin cells has not been observed (soll, ) . a complicating factor in drug safety evaluation is the association of gastric cancer in both man and laboratory animals with n-nitroso compounds. some of the most effective stomach carcinogens in laboratory animals have proved to be nnitroso compounds particularly since sugimura and fujimura ( ) induced gastric adenocarcinomas in rats with n-methyl-n´-nitro-n-nitrosoguanidine dissolved in drinking water. furthermore, epidemiological evidence associating nnitroso compounds with human cancer is also fairly strong for the stomach (corea et al., ; pocock, ) . the formation of n-nitroso compounds is theoretically possible with a variety of compounds that contain amino groups. it has been suggested that the formation of nitrosamines occurs in vivo under the acidic conditions in the stomach following dietary ingestion of nitrite, nitrates and secondary amines (mirvish, (mirvish, , . low levels of preformed nitrosamines are also present in some commercial pelleted diets for laboratory animals, principally derived from fishmeal (edwards et al., ) . calculations based on dietary intake and nitrosatability of precursors and carcinogenicity of derivatives have suggested that the risk that arises from endogeneous nitrosation is highly variable but highest from ureas and aromatic amines (shephard et al., ) . a number of drugs in widespread clinical use have been shown to produce nnitroso products in acidic aqueous media, although the extent to which this occurs in actual therapeutic use is unclear (gillatt et al., ) . some clinical evidence suggests that nitrosation of therapeutic agents can occur in clinical practice. for instance piperazine, a cyclic secondary amine, widely used as an antihelmintic drug, has been shown to form small quantities of n-mononitrosopiperazine in the human stomach as measured by gas chromatography-thermal energy analysis (bellander et al., ) . however, n-mononitrosopiperazine has not been shown to be carcinogenic in rodents (love et al., ) . n,n´-dinitrosopiperazine, carcinogenic to the upper gastrointestinal tract in rodents (lijinsky and taylor, ) , was not detected in man after administration of piperazine under the same circumstances (bellander et al., ) . the possibility of nitrosation is not usually taken into account in the testing of carcinogenic potential of novel drugs as bioassays are usually only performed with parent compound. however, concerns about nitrosation have arisen in subsequent clinical practice. an example of this was the proposal that a few gastric cancers found in patients whilst being treated with the histamine h receptor antagonist cimetidine, were the result of treatment (elder et al., ; reed et al., ; hawker et al., ) . it now seems likely that all those observed cancers associated with cimetidine were incidental (penston and wormsley, ) . however, at that time concerns were increased by the theoretical possibility that cimetidine has the potential be nitrosated in vivo (elder et al., ) . a further factor was the concept that the treatment-induced gastric secretory inhibition with subsequent bacterial colonisation of the stomach rendered the conditions conducive to the generation of n-nitroso compounds from normal dietary constituents (reed et al., ; penston and wormsley, ) . these concerns appear to be unfounded. long-term surveillance studies with cimetidine have shown no causal link between its clinical usage and gastric malignancy (colin-jones et al., ; langman, ) . in addition, carcinogenicity bioassays performed with cimetidine, cimetidine plus nitrite and nitrosocimetidine have not shown any tumorigenic effect in the gastric mucosa (anderson et al., ) . a -year study in dogs in which multiple gastric biopsies were taken at intervals of approximately months have also shown no indication of gastric hyperplasia, dysplasia, intestinal metaplasia or neoplastic change . although complacency is certainly not warranted with respect to the nitrosation of therapeutic agents in vivo, the risks of development of gastric malignancy from such drugs when administered on a short-term basis are probably very small (world health organisation, ) . even for gastric antisecretory agents which may be administered for longer periods of time, the balanced view would also permit development of novel agents provided they are not obviously mutagenic or carcinogenic in the usual preclinical studies and are not particularly liable to undergo rapid nitrosation. most carcinomas of the glandular mucosa are adenocarcinomas, whether induced by the potent genotoxic carcinogens or therapeutic agents. these tend to develop in the antral region in rodents (streett et al., ; szentirmay and sugar, ) . they range from those with well differentiated tubular or papillary features to poorly differentiated forms with trabecular, mucoid or signet ring features (tatematsu, ) . squamous metaplasia within adenocarcinoma can also be observed. stroma may be abundant with pronounced chronic inflammatory infiltration and hyalinisation. metaplastic cartilage and bone has also been described (szentirmay and sugar, ; mohr, ) . gastric adenocarcinomas induced in dogs by n-methyl-n´-nitro-n-nitrosoguamide show similar histological features although their reported distribution in the stomach appears more variable (fujita et al., ) . histological criteria for the diagnosis of invasive adenocarcinoma in experimental animals may vary between individual pathologists. some retain the old criteria of stewart and co-workers ( ) who defined invasive cancer as a neoplastic growth reaching the serosa. it is now considered more appropriate to apply criteria of use in human diagnostic pathology (see mohr, ) . unequivocal invasion of the submucosa is sufficient evidence of an invasive and therefore malignant process (greaves and faccini, ) . the small intestine is of major importance in drug safety evaluation for it represents the primary site of drug absorption. in view of its length and the presence of villi, it possesses an enormous surface area of specialised absorptive epithelium. furthermore, ingested substances have an extended residence time in this part of the gastrointestinal tract. the canine model has been one of the most popular for the study of drug absorption because the dimensions of the canine gastrointestinal tract permit administration of dosage forms intended for clinical use in humans. for this reason, factors, which influence drug absorption, have been better studied in dog and man than many other species. however, data from dog and man not only suggest that diverse factors influence drug absorption from the small intestine but that there are considerable species differences. residence time is of particular importance for drugs, which are incompletely absorbed because differences in mucosal contact time can be expected to result in differences in the fraction absorbed. dressman ( ) has shown using the heidelberg capsule technique that small intestine transit time in dogs is varies from between and over minutes whereas in man equivalent times are between and minutes. these results suggest that absorption of poorly absorbable drugs is likely to be quantitatively less although more variable in dogs than in man. however, these differences do not explain why some poorly lipophilic drugs such as chlorothiazine, acyclovir and phosphalinic acid are more extensively absorbed in dogs than in man. intestinal ph is consistently higher in dogs than in man so that drugs with half maximal absorption ph in the range ph - may also be expected to be absorbed at different rates in man and dog (dressman, ) . physiological and anatomical differences in the small intestine of other test species particularly rodents and humans are also likely to have an impact on drug absorption although many of these factors are still poorly understood. in addition to the small intestine acting as an absorptive surface, it is becoming increasingly obvious that it plays an important part in the metabolism of drugs (breckenridge, ) . although monoxygenase activity is relatively low in the gut compared with the liver, conjugation mechanisms are efficient and activity of udp-glucuronosyltransferase and glutathione-s-transferase are as high or even higher than in the liver (hänninen et al., ) . in addition, the gastrointestinal microflora not only possesses metabolic capacity itself but also can influence the turnover rate of mucosal cells and subsequent exfoliation and release of enzymes into the lumen (hänninen et al., ) . gastrointestinal metabolising activity is important because that the mucosa is exposed to high concentrations of xenobiotics in toxicity studies, and this can influence their overall bioavailability (chhabra and eastin, ) . studies in untreated rats have shown that the concentration of total cytochrome p in small intestinal microsomes is only about % of that found in liver microsomes (bonkovsky et al., ) . however, it exists in at least two forms and as in the liver, its activity can be induced by xenobiotics. it has been shown that in the rat, the concentration of cytochrome p and drug metabolising enzyme activity increases in intestinal epithelial cells as they move from crypt to villous tips and they are found in greater concentration in the proximal two-thirds of the small intestine than in the distal third (hoensch et al., ; bonkovsky et al., ) . bonkovsky et al. ( ) also showed that the phenobarbital-inducible form of cytochrome p represents less than % of total p in the small bowel, but as in the liver, phenobarbital treatment can increase this form to about % of total cytochrome p in small intestine cells. furthermore, it has been shown that drug metabolising activity in the tips of the villi in the duodenum is greater in rats fed a conventional diet than a semisynthetic diet and that the activity depends critically on the absorption of iron from the intestine (hoensch et al., ) . glutathione is also present throughout the entire mucosa, although in rats, cells at the tips of the villi contain less than cells located more basally, whereas related enzymes γ-glutamyl transpeptidase and glutathione-s-transferase show highest activity in the villous tip region (ogasawara et al., ) . the fact that these enzyme activities are highest in the duodenum and lowest in the terminal ileum suggests that detoxification systems for exogenous compounds are greater in the proximal small intestine. the small intestinal mucosa is constructed not only to act as an absorptive surface but also as a barrier to potentially pathogenic substances and microorganisms. although the main cell population of the epithelium is composed of absorptive cells, other major epithelial cell types, the mucous (goblet) cells, paneth cells and endocrine cells have important protective functions. in addition, specialised epithelial cells, the microfold (membranous or m) cells are located in the epithelium over peyer's patches. these cells form part of the other important protective system of the intestine, the gut associated lymphoid tissue (galt) or mucosal associated lymphoid tissue (malt). the mucosal lining is in a constant state of renewal. enteric epithelium possesses the fastest rate of turnover of any tissue exceeded only by a few rapidly growing neoplasms . in normal circumstances, the constant turnover of small bowel mucosa is maintained by equilibrium between cell production in the crypts and cell loss at the tips of the villi. there are intrinsic controls within the mucosa itself. exogeneous substances, intraluminal secretions, mechanical and neural factors as well as alterations in blood flow all possess potential to influence mucosal cell kinetics . all main epithelial cell types are believed to arise from undifferentiation columnar cells at the crypt base (cheng and leblond, ) , although mucous cells may also arise by proliferation of partly differentiated mucous cells in the crypts. as cell division is limited to crypts, the cell population in the crypts have high activities of enzymes such as thymidine kinase that are involved in nucleic acid synthesis (imondi et al., ) . the complete cell cycle lasts about - hours in rodents and at least hours in man. enteric epithelium is completely replaced within - days in mice and rats and within - days in man . after two or more divisions in the crypt cells migrate to the villus, lose ability to incorporate thymidine and differentiate into mature cells equipped with enzymes associated with nutrient absorption (imondi et al., ) . cell migration in the rat is completed more rapidly in the ileum than in the jejunum principally as a result of the lower villous height in the ileal mucosa (altman and enesco, ). migration terminates by loss of cells from the tip of the villi. surrounding the crypt is a sheath of fibroblastic cells. these cells also undergo synchronous division and migration with the epithelial cells, maintaining the intimate relationship between the epithelium and supporting tissues (parker et al., ) . mature absorptive cells are important in the active and passive transport of nutrients as well as in the endocytosis of macromolecules. they are characterised by the presence of a striated or brush border which is seen in haematoxylin and eosin-stained sections as a refractile bi-laminar band. the inner, wider lamina corresponds to the microvillous region that is associated with the presence of neutral mucosubstances in most species. the outer, thinner band corresponds to the glycocalyx, which is composed principally of acidic mucosubstances (sheahan and jarvis, ) . this outer band of the brush border shows histochemical staining predominantly for sulphomucins in most species including mouse, hamster, dog and rhesus monkey, although in the duodenum of the rats and in the entire human small bowel sialomucins predominate in this layer. electron microscopy of the absorptive cells shows that the surface of absorptive cells is covered by tightly packed and well developed microvilli approximately µm long and . µm wide. these are considered the first site of entry of food substances into the cell. the plasma membrane of microvilli is associated with fine filamentous projections, which probably represent the polysaccharide chains of the glycocalyx (bennett, ) . as the glycocalyx is composed of a network of polysaccharides, it has been suggested that it may behave like an ionexchange resin, be able to bind certain lectin-like molecules or trap substances in its matrix so providing a site for efficient intraluminal digestion (bennett, ; goldberg et al., ; king et al., ). the plasma membrane shows a trilamina structure at ultrastructural level. freeze fracture replicas from the microvilli which cleave this membrane through the plane of apposed non-polar groups of the lipid bilayer, demonstrate smooth complementary surfaces studded with small particles. these particles represent integral globular proteins of the plasma membrane. some of these intramembranous particles, mostly those of nm diameter show irregular outlines with a central pit and are believed to represent gap junctions or transport channels (yamamoto, ) . a particularly important aspect of the absorptive cell membrane is its high concentration of disaccharidases such as sucrase, maltase and lactase, related to the absorption of sugars. alkaline phosphatase activity is also abundant on the surface of absorptive cells, although its precise role here uncertain (owen and bhalla, ) . immunocytochemical demonstration of alkaline phosphatase provides a useful tool to examine the effects of xenobiotics on intrinsic membrane glycoproteins in the small intestine (hasegawa et al., ) . enterokinase, the glycoprotein enzyme, which initiates the activation of pancreatic zymogens by converting trypsinogen to trypsin, is also present in the brush border and glycocalyx of the small intestinal epithelium, both in man and animals. immunocytochemical studies have demonstrated that in man this enzyme is located in the duodenum and proximal jejunum but not ileum, colon and stomach (hermon-taylor et al., ) . these enzymes constitute integral structural proteins of the cell membrane with active sites protruding from the cell surface. they are synthesised by the absorptive cells (blok et al., ) . the lateral surfaces of absorptive cells are in direct contact with neighbouring cells and firmly attached to each other by terminal bars or junctional complexes. a terminal bar comprises an apically situated tight junction or zonula accludens, a central zone, the zonula adherens, below which is situated a desmosome or macula adherens. the junctional complexes are relatively impermeable to macromolecules. studies with labelled tracers in the rat jejunum have shown that horseradish peroxidase (molecular weight , , diameter nm) and ferri-tin (molecular weight , , diameter nm) do not penetrate junctional complexes (yamamoto, ) . below the junctional complexes, the cell membranes interdigitate and the intercellular space widens towards the cell base, a feature that may be important in the movement of electrolytes and water across the intestinal epithelium (rhodin, ) . the cytoplasm of absorptive cells contains smooth and granular endoplasmic reticulum, free ribosomes and mitochondria. the golgi apparatus is located above the nucleus. the apical part of the cytoplasm is devoid of organelles except for a tight meshwork of filaments called the terminal web. filaments of actin within the microvilli are linked to the terminal web and this is believed to be important in the movement of microvilli (moosker and tilney, ; moosker et al., ) . goblet cells are much fewer in number than absorptive cells in the small intestine but they increase in number from the duodenum to the lower ileum. they are important in the production of mucus, which remains on the surface of the mucosa as a viscous layer and acts as the first line of defence against intestinal pathogens. goblet cells are characterised by the presence of abundant mucous droplets formed by the golgi complex and which accumulate in the apical part of the cell cytoplasm. histochemical study shows that neutral mucosubstances are present in the goblet cells found in crypts and on the villi in the entire small bowel mucosa of most species including man but there is an interspecies variation in the population of sialo-and sulphomucins (sheahan and jarvis, ) . in the mouse, sulphomucins predominate but among rats considerable individual variation in the proportion of sialo-and sulphomucins is reported. in the hamster, sulphomucins are more prominent in the proximal and sialomucins in the distal small bowel. in the dog, both sulphomucins and sialomucins are found with predominance of one or other in individual animals. staining for acidic mucins is less intense in the goblet cells of the small intestine in man compared with non-human primates but sialomucins are predominant in both species. a few goblet cells in the distal ileum in man also contain sulphated mucins (sheahan and jarvis, ) . paneth cells remain located near the crypt base throughout the small intestine (sandow and whitehead, ) . they are found in rodents and man but typically not in carnivores such as dog and cat (rhodin, ; . they are characterised by the presence of numerous eosinophilic cytoplasmic secretory granules between about . and µm diameter that contain various enzymes and mucosubstances. particular care is needed in fixation and staining for optimal demonstration of paneth cells for they rapidly degranulate after death and granules are destroyed by acetic acid fixation. formalin and mercuric fixatives appear appropriate methods and they permit staining with methylene blue, lendrum's phloxine-tartrazine and masson's trichrome (lewin, ) . the apical parts of paneth cells show glucose- -phosphatase, carbonic anhydrase and monoamine oxidase activity and they have been shown to contain lysozyme and immunoglobulins, particularly iga (speece, ; riecken and pearse, ; ghoos and vantrappen, ; . staining for lysozyme appears to be the most practical immunocytochemical stain for the detection of paneth cells in formalin fixed paraffin wax embedded tissue sections. however, other immunocytochemical reagents have been employed in human and rodent tissues. these include antibodies to the cd antigen (ariza et al., ) and to a rat paneth cell zinc binding protein (sawada et al., ) as well as the use of pokeweed lectin for murine paneth cells (evans et al., ) . the paneth cell granules not only contain lysozyme but also a range of antimicrobial peptides such as secretory phospholipid a , α-defensins, also called cryptins. these are believed not only to possess antimicrobial activity but also important in the regulation of cell volume, chemotaxis, mitogenesis and inhibition of natural killer cell activity (ouellette, ) . it has been shown that these substances are released from paneth cells when germ free rats are dosed with the intestinal flora from specific pathogen free rats (satoh and vollrath, ; . however, it has been shown that paneth cells develop under germ free conditions and do not require luminal bacteria or dietary material for their development (ouellette, ) . endocrine cells are also scattered throughout the small intestinal mucosa. they are of both argentaffin and argyrophil types and are situated predominately in crypts. immunocytochemical study shows that they contain a variety of different peptides although gastrin, secretion and serotonin-containing cells have been those most extensively studied (inokuchi et al., ) . in addition to the barrier formed by mucus and epithelial cells, lymphocytes, plasma cells, macrophages, dendritic cells and mast cells also form part of the protective function of the small intestine. some lymphocytes are located within the epithelium mostly, above the basal lamina but below epithelial nuclei (pabst, ) . these lymphocytes are termed intraepithelial lymphocytes and are predominantly of t-suppresser/cytotoxic type in man and laboratory animals (selby, ; martin et al., ; pabst, ) . most lymphocytes in the lamina propria are also t cells but t-helper (cd positive) cells outnumber the t-suppresser/cytotoxic or cd positive phenotype (hirata et al., ; pabst, ; bruder et al., ) . intraepithelial lymphocytes are also mainly t cells (bruder et al., ) . many plasma cells present in the lamina propria produce iga, the major immunoglobulin of mucosal secretions (michalek et al., ) . iga represents another important component of the mucosal barrier between the gastrointestinal mucosa and intraluminal antigens. the main function of iga is to effect immune exclusion by intimate co-operation with non-specific defence mechanisms (brandtzaeg et al., ) . plasma cells in the lamina propria produce dimeric iga with two dimeric molecules joined by a joint (j) piece. a secretory component (sc), a glycoprotein expressed on the basolateral surface of epithelial cells acts as a receptor for dimeric iga and as a transport system for iga to the gut lumen where monomeric iga is secreted (brandtzaeg et al., ) . morphometric analysis of iga-containing immunocytes in the rat ileal mucosa using immunocytochemical staining has shown that the number of these cells varies with alterations in the microbiological status of intestinal contents (rodning et al., ) . a significant reduction in iga-containing lymphocytes and plasma cells was observed following microbial reduction associated with gnotobiosis, probably reflecting decreased microbial antigenic stimulation. experimental studies using labelled mesenteric lymphocytes also suggests that local microenvironments are important in the distribution of there cells in the intestinal wall (mcdermott et al., ) . peyer's patches are the most prominent aggregates of lymphoid tissue in the gastrointestinal tract and constitute important sites at which antigens from the gut lumen encounter immune competent cells which are responsible for the initiation of immune responses. peyer's patches are located on the ante-mesenteric wall of the small bowel and consist principally of collections of lymphoid follicles. in man, peyer's patches are more common in the ileum (cornes, ) but in mice they are more uniformly distributed (owen and neumanic, ) . in rats they are also more numerous in the distal than in the proximal small intestine and the number of follicles in patches usually varies from to but sometimes many more may be seen in any particular section (martin et al., ) . peyer's patches vary between rat strains. a comparative study showed that in fischer rats they are smaller than those in wistar rats (bruder et al., ) . particular care in selection and orientation of tissue blocks is therefore essential for any form of quantitative or semiquantitative assessment of peyer's patches. peyer's patches are more than simple aggregates of lymphoid follicles. they consist of lymphoid follicles surrounded by a corona of small lymphocytes principally of b-cell type. the interfollicular area contains post-capillary venules with specialised cobblestone type epithelium (yamaguchi and schoefl, ) and many t lymphocytes. beneath the epithelium, over the bulging follicles (dome area), mixtures of t and b lymphocytes, plasma cells and macrophages can be seen (pabst, ) . immunohistochemical study in rats demonstrates the presence of w / -positive t (cd ) cells in the interfollicular area. in the rat, many cells with macrophage morphology also stain with the w / (cd ) monoclonal antibody in the interfollicular zone (bland and warren, ; martin et al., ) . immunocytochemical study of the peyer's patches in the mouse has also shown considerable heterogeneity of staining patterns, particularly under the dome epithelium (ermak and owen, ) . similar patterns have been observed following immunohistochemical study of peyer's patches in man (spencer et al., ) . the epithelium overlying the peyer's patch follicles (dome area) contains specialised epithelial cells, called microfold, membranous or simply m cells. these cells have been identified in many species including rats, mice, hamsters, dogs, monkeys and man (owen and bhalla, ; wolf and bye, ) . these cells differ functionally from other enterocytes by their ability to transport large molecules such as ferritin and horseradish peroxidase and particulate matter from the lumen to the underlying lymphoid tissue (owen, ; jeurissen et al., ) . they have also been shown to be the site of penetration of reoviruses into the epithelium and they can transport vibrio cholerae and other organisms (wolf and bye, ; smith et al., ) . m cells therefore form weak points in the intestinal wall which transport intact antigen and macromolecules to the follicles where they can be processed and be transported to lymph nodes with consequent iga immune responses. this contrasts with the uptake of soluble antigens, which can be taken up by ordinary epithelial cells and transported in the circulation of the villi to be ultimately trapped in the spleen possibly to evoke an igm/ igg response (jeurissen et al., ) . understanding of these cellular and molecular characteristics is critical for the design of mucosal vaccines for pathogens that exploit this pathway (neutra, ) . no simple, specific histological markers for m cells have been identified for use in routine histological sections although many specialist techniques can be applied (reviewed by gebert et al., ) . this has hampered their study because they are still best identified on the basis of their ultrastructural characteristics or ability to capture and transport macromolecules (hamzaoui and pringault, ) . the m cell shares tight junctions and desmosomes with adjacent epithelial cells but it has fewer and shorter microvilli than absorptive cells. there is lack of a well organised terminal web. vesicles are abundant in the apical cytoplasm but lysosomes are reduced in number. attenuated cytoplasmic processes may be seen embracing lymphocytes (owen and nemanic, ; wolf and bye, ) . a cardinal feature is the presence of a intraepithelial pocket on the basal membrane which acts as an internal docking site for lymphocytes, such that they are filled by b and cd positive lymphocytes, macrophages and dendritic cells that move between underlying follicles and the epithelium (ermak et al., ; farstad et al., ) . with care, these attenuated microvilli can be seen at light microscopy in semithin plastic sections (wolf and bye, ) . cytochemical analysis has also demonstrated that they can be distinguished at light microscopic level by markedly reduced alkaline phosphatase activity on their terminal surface, in contrast to the dense reaction product produced by other enterocytes (owen and bhalla, ) . as the glycoproteins on the surface of m cells are different to those on surrounding cells, they can be selectively stained by labelled lectins, although these patterns are different in different species. for instance, mouse m cells are labelled by the fucose-specific probe ulex europaeus and to some extent anguilla anguilla (giannasca et al., ) . mucosal mast cells also appear to be involved in the immunological defence of the gastrointestinal tract. they respond by proliferation, migration and discharge of granules during nematode infestations (miller, ) . it has been shown in the rat that mucosal mast cells of the gut differ in several ways from connective tissue mast cells. these differences result in poor preservation of mast cells of the gut if the usual metachromatic staining techniques employed for the demonstration of mast cells in tissue sections (wingren and enerbäck, ) . histochemical study suggests that mucosal mast cells differ from connective tissue mast cells by a lower degree of sulphation of glycosaminoglycans and different spatial relationships of protein and glycosaminoglycans in their granules. these cross link following formalin fixation in a way, which is sufficient to prohibit cationic dye binding. wingren and enerbäck ( ) showed that these staining difficulties can be surmounted in tissues fixed in formaldehyde by staining in toluidine blue for prolonged periods of time ( - days), a procedure which allows adequate penetration of the toluidine blue molecule. optimal histopathological study of the small intestine is complicated by its length and mucosal fragility. it is important to avoid vigorous washing procedures or any form of excessive manipulation of the unfixed bowel, as artefact caused by washing may confound interpretation of changes induced by xenobiotics (roe, ) . combination of artefact due to washing, autolysis and the presence of neutrophils can produce a histological appearance that mimics in vivo damage. although careful visual inspection of the intestine and sampling of appropriate segments for histological examination is usually sufficient for routine examination, various forms of 'swiss roll' techniques are helpful for more complete study. rolling the unfixed, opened rodent intestine around a wooden stick prior to freezing or fixation is one proposed method (moolenbeck and ruitenberg, ) , although this method risks undue manipulation of the unfixed tissue. another more versatile technique applicable to rodent, large animal and human intestine can be performed after fixation. the unfixed opened bowel is pinned flat on a cork or board and fixed in a bath of formal saline. after fixation, the full thickness of rodent intestine can be rolled, transfixed by a pin and embedded in paraffin wax. likewise the mucosa of the intestine of large animal species or humans can be rolled after fixation by separating it from the muscularis externa (filipe and branfoot, ) . inflammation and ulceration of the mucosa occurs as a result of stress, infection with bacteria, viruses, and infestation by parasites or as a direct result of the effects of xenobiotics or ionising radiation. antimitotic or radiomimetic agents as well as ionising radiation are liable to adversely affect the rapidly dividing cells of the small intestine with resulting breakdown of the mucosal barrier. the ulcerogenic activity of non-steroidal anti-inflammatory drugs may also be expressed in the small bowel mucosa. different agents also act in synergy to enhance damage to the small bowel mucosa. an important example is the effect of drugs that depress the immune system and permit the development of pathological infections by microorganisms of the opportunistic type in the small intestine. the histological features of the inflammatory process in the small intestine are not usually specific for a particular agent. it is important to search for evi-dence of microbiological organisms and viral inclusions, which can indicate the cause of intestinal inflammation and ulceration. associated features in non-ulcerated mucosa such as morphology of the villi, accumulation of abnormal cells or foreign substances and changes in lymphoid cells or blood vessels are also important in the assessment of these changes. a number of organisms including those, which are normal residents of the gastrointestinal tract, can cause inflammatory changes in the intestinal mucosa of laboratory animals. with the notable exception of non-human primates, inflammatory bowel disease caused by microbiological organisms is not usually evident or of concern in most toxicity or carcinogenicity studies. however, when animals are treated with antibiotics, immunosuppressive agents or other drugs, which alter the normal intestinal flora, conditions may favour the proliferation of potentially pathogenic organisms in sufficient quantities to cause overt damage to the mucosa. certain bacterial flora may also act synergistically with intestinal protozoans to produce pathological changes (boorman et al., ) . in non-human primate colonies, gastrointestinal disease remains one of the most important causes of death . in contrast to other laboratory species, histological evidence of intestinal infectious disease is relatively common and may confound the interpretation of gastrointestinal alterations occurring in toxicity studies. although the majority of potentially pathogenic organisms affect the primate colon, a number of bacteria, protozoa and metazoa occur in the small intestine. a detailed review of the protozoa and metazoa occurring in the primate gastrointestinal tract has been published (toft, ) . owen ( ) has reviewed the parasites of laboratory animals including those of the gastrointestinal tract and the principle reference for identification of metazoa in tissue sections remains that of chitwood and lichtenfels ( ) . organisms which can cause inflammatory disease in the small bowel but which are primarily agents that produce inflammatory disease in large bowel are reviewed under 'colon' (see below). bacillus piliformis is the agent responsible for tyzzer's disease produces intestinal inflammation and ulcers in rats, mice and hamsters. susceptibility of different species and strains to experimental infection with bacillus piliformis is variable. for instance, c bl, balb mice and f rats appear more resistant to infection than outbred syrian hamsters (waggie et al., ) . lesions of variable severity usually occur in the ileum but may also extend into the caecum and colon. severe infections are characterised histologically by ulceration of the mucosa, oedema and acute inflammation of the submucosa and muscle coats. muscle may also show focal necrosis. non-ulcerated mucosa is typically infiltrated by polymorphonuclear cells and crypt abscesses form. there is blunting and fusion of villi and reactive hyperplasia and mucin depletion of the overlying epithelium . mucosal lymphoid tissue may also show reactive changes or hyperplasia. filamentous bundles of bacillus piliformis can usually be found in the cytoplasm of both epithelial cells and smooth muscle cells at the edges of necrotic zones. methylene blue, giemsa or silver impregnation techniques such as warthin-starry or levaditi stains are the best stains for the demonstration of these organisms although with care they can be visualised in haematoxylin and eosin stained sections (weisbroth, ) . they are gram negative and pas positive. intestinal infections due to salmonella species are relatively common in the mouse but also occur in the hamster and rat . salmonella typhimurium and salmonella enteritidis are regarded as the organisms typical of murine salmonellosis (weisbroth, ) . lesions occur in the ileum and may extend into the jejunum and caecum. they are characterised by the presence of ulcers covered by fibrinous exudate and associated with diffuse infiltration of the adjacent mucosa by macrophages, neutrophils and lymphocytes. intact crypt epithelium shows mucin loss and reactive proliferative changes. a characteristic feature is the presence of poorly defined granulomatous lesions composed of macrophages mainly in associated lymphoid tissue or peyer's patches. clostridia species, especially clostridia difficile which cause a pseudomembranous colitis in man and laboratory animals (especially hamsters) may also produce inflammation and ulceration in the terminal ileum with histological features similar to those found in the colon (see following). proliferative ileitis (transmissible ileal hyperplasia) is a striking lesion of hamsters affecting the distal segment of the ileum that is associated with intracellular invasion of the intestine mucosa epithelium by bacteria. the organism has not been cultivated, but has been suggested that it is a campylobacter (jacoby, ) although other organisms have been identified in this condition (fox et al., ; peace et al., ) . although it is characterised by hyperplasia of the ileal mucosa in its early stages, an inflammatory phase intervenes in which there is focal necrosis and haemorrhage of the mucosa, crypt abscesses and infiltration of the lamina propria by acute inflammatory cells and macrophages. the histological features of the associated hyperplasia are characteristic. the mucosa is covered by immature, mucin-depleted pseudostratified hyperchromatic epithelium with mitoses extending to the tips of villi and densely basophilic intracytoplasmic inclusions (jacoby, ) . helicobacter jejuni (campylobacter jejuni) is a common cause of diarrhoea in man and may be the causative agent in small intestinal inflammation in laboratory dogs and primates. campylobacter species may be more prevalent in beagle dogs and primates than commonly appreciated. it is important to recognise that dogs colonised with these agents may be susceptible to stress-induced, acute onset gastroenteritis (fox et al., ; reed and berridge, ) . in man this form of bacterial disease is characterised histologically by mucin-depletion, flat-tening and reactive changes in the small bowel epithelium, crypt abscesses, oedema and infiltration of the mucosa by neutrophils, lymphocytes and plasma cells. similar histological findings have been reported in dogs infected with this organism (prescott and monroe, ) . the organisms are gram-negative curved, slender rods, which can be visualised in tissue sections with the warthin-starry stain, a recognised technique for spiral bacteria. the carbol fuchsin technique of gimenez ( ) , first used for the identification of ricketsiae in yolk sac culture and a cresyl fast violet technique are also useful methods for the identification of campylobacter species in paraffin sections (burnett et al., ; mcmullen et al., ) . another campylobacter-like organism, helicobacter pylori (campylobacter pylori) has been identified in human patients with gastritis, gastric and duodenal ulcers. it is an aetiological or predisposing factor in these forms of gastrointestinal disease (marshall and warren, ; rouvroy et al., ; richter-dahlfors et al., ) . spironucleus muris (hexamitis muris) is also a cause of inflammation in the small bowel of rats, mice and hamsters. during overt infestation, organisms are seen extracellularly in crypts and intervillous spaces associated with blunting of intestinal villi, epithelial degeneration and mucin-depletion, reactive epithelial hyperplasia, oedema and leucocyte infiltration (boorman et al., ; wagner et al., ) . the morphological expression of damage is accompanied by decreased levels of disaccharidases such as maltase, sucrase and lactase, which may represent a direct effect of the trophozoites on the brush border enzymes (gillon et al., ) . trophozoites are characteristically elongated symmetrical flagellates approximately - µm wide, - µm long. giardia species represent marginally pathogenic flagellates, which are found in the upper gastrointestinal tract. they are opportunistic agents that can become important in both animals and man with depressed immune function. studies in mice infected experimentally with giardia muris have shown that an early response is an increased infiltration of the epithelium by lymphocytes, predominantly t cells (gillon et al., ) . this has led to the suggestion that the response to infection by these parasite is primarily a cell-mediated immune reaction similar to experimental graft versus host reaction in the small intestine of mice (mowat and furguson, ; gillon et al., ) . depression of the immune response by treatment with corticosteroids has been shown not only to increase parasite numbers in murine giardiasis but also cause recrudescence of occult infections (nair et al., ) . it has also been suggested that decreased gastric acidity can predispose to giardiasis in man (nalin et al., ) . giardia muris (lamblia muris) is sometimes found in the small intestine of rat and mouse but it is common in the hamster (fig. ) . trophozoites appear in histological sections as crescent-shaped structures on the brush border of the intestinal mucosa or in the adjacent lumen. mucosal lesions may be totally ab-sent or there may be blunting of villi, reactive epithelial hyperplasia. a typical feature is increased infiltration of the epithelium and lamina propria by mononuclear cells (boorman et al., ) . another important finding is that lactase, sucrase and maltase levels have been shown to decrease in the small intestine in mice infested with giardia muris (gillon et al., ) . giardia lamblia may colonise the small intestine of non-human primates and of man and produce similar morphological appearances to those found with infestations in rodents by giardia muris. other flagellates such as tritrichomonas muris are also be found in the small intestine of mice, rats and hamsters. the coccidian protozoan parasite cryptosporidium represents a striking example of the close relationship between some human and animal diseases. this organism was first recognised in the gastric glands of mice by tyzzer in and has since been confirmed as a cause of diarrhoea in animals and as a human pathogen. it causes mild diarrhoea in normal subjects especially children and young adults but it can produce severe intestinal disease in immunocompromised individuals (casemore et al., ) . histological examination of the small intestine of laboratory animals infested by cryptosporidium reveals the presence of organisms attached to the mucosal surface, often associated, as in man, with other parasites or infections. they are rounded, weakly basophilic structures - µm diameter in haematoxylin and eosin stained sections but are strongly basophilic following romanowsky staining. transmission electron microscopy reveals the detailed internal structure of cryptosporidium attached to the microvillous surface of the epithelial cells. the various stages in the life cycle have been visualised by light and electron microscopy. infection starts with ingestion of an oocyst containing four sporozoites, which are probably released by the action of digestive enzymes. these attach themselves to the intestinal mucosa and undertake their life cycle attached to the epithelial cells (casemore et al., ) . these organisms have been demonstrated in laboratory species including mice, hamsters, rabbits, dogs and nonhuman primates (cockrell et al., ; rehg et al., ; toft, ; fukushima and helman, ; davis and jenkins, ) . hymenolepis nana (dwarf tapeworm) and hymenolepis diminuta (rat tapeworn) are described in the intestine of rats, mice, hamsters, non-human primates and man (hsu, ) . a variety of other metazoan patients are found in the small intestine of non-human primates, see review by toft ( ) . certain viruses produce inflammatory small bowel changes in mice. mouse hepatitis virus (lethal intestinal virus of infant mice) can cause mucosal epithelial necrosis and inflammation with characteristic compensatory epithelial hyperplasia and the formation of epithelial syncytia . murine rotavirus (epidemic diarrhoea of infant mice) produces swollen enterocytes of small and large bowel with fine cytoplasmic vesiculation with little or no inflammation but dilated lymphatics and vascular congestion. cytoplasmic acidophilic inclusions, - µm diameter, are characteristic findings . electron microscopic examination shows cytoplasmic vesicles arising from the rough endoplasmic reticulum, which contain virus particles and electron dense granular material . a mouse adenovirus may also produce large basophilic intranuclear inclusions in the epithelial cells of the small intestine and caecum. k virus infection produces lesions in the jejunal and ileal mucosa of mice. histological features are characterised by mild polymorphonuclear infiltration, with ballooning of occasional endothelial cells within intestinal villi. intranuclear inclusions can be demonstrated in these endothelial cells by light microscopy using appropriate fixation (greenlee, ) . a variety of viruses have been isolated from the gastrointestinal tract of nonhuman primates including viruses of man (kalter, ) . however, they appear to be relatively infrequent causes of gastrointestinal disease and when disease is caused by these viruses it usually affects other organs. not only can non-steroidal anti-inflammatory agents such as indomethacin and phenylbutazone produce gastric ulceration but also penetrating ulcers of the small bowel of laboratory animals (shriver et al., ) . imaging studies with indium-labelled leukocytes in man have also suggested that subclinical intestinal inflammation is associated with long-term therapy with non-steroidal anti-inflammatory drugs (bjarnason et al., ) . it has been postulated that indomethacin-induced intestinal ulcers in rats and dogs are produced by a prostaglandin-independent mechanism, different from the manner in which gastric ulceration is induced (tabata and okabe, ; whittle, ) . conversely, satoh et al. ( ) have suggested that similar mechanisms are responsible for both gastric and intestinal ulceration. they showed that indomethacin-induced gastric ulceration developed in the body mucosa in fasted rats but in the antrum and small intestine in rats given indomethacin minutes after a -hour period of refeeding following a -hour fast. there was good temporal correlation between the development of intestinal ulcers and inhibition of prostaglandin synthesis (satoh et al., ) . the ulcers in the small intestine were morphologically similar to those occurring in the stomach and they were distributed mainly in the mucosa on the mesenteric aspect of the bowel wall. single-dose studies with indomethacin and ibuprofen in rats conducted by suwa et al. ( ) have demonstrated differences between pathology of induced gastric and intestinal damage. gastric damage was superficial, occurred within hours and was fully repaired weeks after dosing. ulcers in the jejunum and ileum reached a maximum area at - hours after dosing, occurred on the mesenteric border, penetrated through the muscularis mucosa and were accompanied by inflammation and oedema. ulcers were still present weeks later. rainsford ( ) has shown that potent intestinal ulcerogens such as indomethacin inhibit the incorporation of radioactive s sulphate into glycoproteins of the upper intestinal mucosa as well as the stomach of rats. this may decrease the capacity of the mucus in the intestine to act as a buffer for hydrogen ions. indomethacin given orally to dogs in doses of . mg/kg/day for - days was also shown to produce intestinal ulceration. these ulcers were deep, punchedout lesions, many of which were lying over peyer's patches (stewart et al., ) . some ulcers involved the whole circumference of the small intestine wall. histologically, the ulcers were associated with an intense inflammatory response principally of mononuclear cells, which infiltrated the bowel wall to the serosa particularly adjacent to peyer's patches. it was suggested that this distribution of ulcers was a result of an exaggerated immune response to normal intestinal antigens. these antigens may have been produced by inhibition of suppresser cells in peyer's patches, following depression of prostaglandin synthetase by indomethacin (stewart et al., ) . special dye techniques, scanning and transmission electron microscopy have also shown that non-steroidal anti-inflammatory drugs also produce adverse effects on the small intestine mucosa without overt pathological changes being evident by light microscopy (brodie et al., ; djaldetti and fishman, ) . following administration of aspirin to mice for weeks, shortening and erosion of microvilli and increased numbers of goblet cells were only demonstrated in the duodenum and jejunum by scanning and transmission electron microscopy (djaldetti and fishman, ) . morphometric studies of the intestinal mucosa of indomethacin-treated mice have also shown widespread alterations to columnar cells, goblet cells and paneth cells suggesting generalised effects on mitotic activity and crypt loss (ettarh and carr, ) . such submicroscopic findings support the idea that non-steroidal anti-inflammatory agents may induce damage to the small intestine more commonly than supposed. although non-steroidal, anti-inflammatory drugs are the best-known drugs with adverse effects on gastrointestinal mucosa, small intestinal inflammation and ulceration can also produced by other agents through different mechanisms. anticancer drugs and other therapeutic agents, which affect cell proliferation, depress the bone marrow or the immune system can also produce intestinal mucosal necrosis, haemorrhage, inflammation and opportunistic gastrointestinal overgrowth when administered to dogs or rodents in high doses (fig. ) bregman et al., ) . lymphoid infiltrates without tissue fig. . section of small intestine from a wistar rat treated with a -day course of an antiproliferative anticancer drug. the mucosa shows mucus-depletion, loss of villous architecture as well as a reparative and inflammatory response. (he, × .) damage were also reported in the small intestine of rats treated with human recombinant interleukin- (anderson and hayes, ) . agents of particular toxicological interest are cysteamine, propionitrile and their structural analogues as well as -methyl- -phenyl- , , , -tetrahydropyridine (mptp) which are capable of producing ulcers of chronic type in the duodenum of rats and mice (szabo and cho, ) . these compounds vary in their ulcerogenic capacity but they are all able to produce ulcers of chronic type with crater formation, granulation tissue and reactive changes in adjacent mucosa in the anterior and posterior wall of the proximal segment of the duodenum of rodents. although these different agents influence gastric acid secretion in different ways, structure-activity relationships suggest that they produce duodenal dysmotility, decrease bicarbonate production and reduce its delivery from the distal to proximal duodenum. these factors decrease the neutralisation of gastric acid in the first part of the duodenum and this may contribute to the development of ulceration (szabo and cho, ) . furthermore, these effects can be attenuated or prevented by dopamine agonists or their precursors whereas dopamine antagonists can potentiate their effects. this suggests that the central or peripheral dopamine-mediated actions of these agents may be involved in the pathogenesis of duodenal ulceration (szabo, ; . using appropriate fixation and staining procedures, fine granular lipid droplets can be normally visualised in the apical parts of epithelial cells covering the upper third of small intestinal villi. administration of drugs and chemicals may produce an excessive accumulation of lipid through specific effects on lipid metabolism or as part of general cellular toxicity. in the preclinical toxicity studies with , -di-tert-butylamino- -acetyl- -methylpyridine (sa h - ), an inhibitor of glucose transport intended for use as an anti-obesity drug, lipid accumulation occurred in the lamina propria of the small intestinal villi of sprague-dawley rats and guinea pigs but not in dogs or primates (visscher et al., ) . after administration of sa h - to rats, there was progressive accumulation of lipid droplets in the epithelial cells over the tips of the duodenal villi demonstrable by osmium tetroxide staining. ultrastructural examination revealed uniform electron-lucent droplets within profiles of the smooth endoplasmic reticulum and golgi apparatus. lipid droplets increased with time, accumulated and coalesced to form large droplets in the lamina propria. larger droplets were phagocytosed by macrophages in the lamina propria but there was no evidence of epithelial damage or necrosis. changes were most pronounced in the duodenum but were also noted to a lesser extent in jejunum and ileum but not in colon or stomach. sequential studies using electron microscopy showed that lipid rapidly accumulated within several hours in the profiles of smooth endoplasmic reticulum and golgi apparatus of the epithelial cells and formed droplets or chylomicra in the intercellular space. the absence of any other subcellular changes or evidence of derangement of protein synthesis suggested that sa h - altered the pathways of lipid resynthesis or transport. this was consistent with the distribution of the lipid in the upper third of the jejunal villous epithelium, a zone most active in lipid absorption, resynthesis and transport (dobbins, ) . it was suggested fatty change might have taken place because of alterations in the sugar moiety of chylomicra brought about by interference with glucose transport (visscher et al., ) . lipid droplets which stained with oil-red-o in formalin-fixed frozen sections and showed uniform electron density characteristic of neutral lipid were also observed in the epithelial cells and macrophages in the lamina propria of jejunum and duodenum and mesenteric lymph nodes in rats given a synthetic '-dodecyl glutaramide ester of erythromycin (gray et al., ) . unlike the erythromycin base, rats poorly tolerated this ester. it appeared that the ester was absorbed unhydrolyzed and converted to chylomicron-like droplets, which then accumulated in the macrophages of the lamina propria and local mesenteric lymph nodes, without overt damage to epithelial cells. accumulation of lipid in epithelial cells of intestinal villi has been observed in rats following administration of puromycin (friedman and cardell, ) and ethionine (hyams et al., ) , agents which have inhibitory effects on protein synthesis. detailed morphological study of the intestinal epithelial cell in rats treated with puromycin have shown that there is concomitant accumulation of lipid with a decrease in the quantity of rough endoplasmic reticulum and golgi membranes (friedman and cardell, ) . these changes were in keeping with the concept that lipid accumulates as a result of inhibition of the synthesis of membrane components of the golgi by the rough endoplasmic reticulum which are important for the transport of lipid. in addition to lipid droplets forming as a result of altered lipid metabolism, they may form in the epithelial cells of the intestinal mucosal as a result of a direct toxic effect of the ingested drugs on the small intestinal mucosa. in such instances atrophy of villi and degenerative changes in the epithelial cells may also be observed (see following). the small intestinal mucosa is also one of the many sites at which drug-induced accumulation of polar lipids form laminated structures (myeloid bodies) or crystalloid structures within lysosomes. this form of lipid storage disorder is produced by diverse amphiphilic cationic drugs in both man and laboratory animals probably as a result of drug interaction with polar lipids rendering them difficult to digest (lüllmann-rauch, ) . species differences in susceptibility and tissue distribution of phospholipid are probably not only related to physiochemical characteristics of the inducing drugs which influences their ability to permeate selective biomembranes and react with different lipids, but also to tissue concentrations of drugs achieved and the ability of organs to metabolise parent drug to less amphiphilic products. in general terms, this form of disorder is characterised by membrane-bound, acid phosphatase-positive cytoplasmic inclusions, which on ultra-structural study are seen as lamellated or crystalloid structures in lysosomes. these appearances are characteristically reversible on cessation of treatment with the inciting agent. an example of this phenomenon occurring in the small intestine is provided by the iodinated amphiphilic drug, amiodarone, which has been used clinically in europe for the past years in the treatment of angina and more recently in the control of supraventricular cardiac arrhythmias. its adverse effects in man are believed to be the result of accumulation of drug in lysosomes particularly in liver, skin and eye (d'amico et al., ; shepherd et al., ) . when high doses of amiodarone were administered orally to rats and beagle dogs, multilamellated lysosomal inclusion bodies accumulated first in the jejunal mucosa and mesenteric lymph nodes before becoming widely distributed in other organs particularly in the lungs (mazué et al., ) . in both rats and dogs the small intestinal lesions were characterised by the presence of foamy macrophages with pale finely vacuolated cytoplasm and condensed eccentric nuclei within the lamina propria of the jejunal villi (mazué et al., ) . mesenteric lymph nodes were also involved early after the onset of treatment. in the dog, jejunal villi were somewhat flattened and widened or showed a variable degree of villous atrophy, most marked in the proximal and middle jejunum (vic et al., ) . electron microscopy confirmed the presence of lamellated lysosomal bodies distending macrophages. the early accumulation of foam cells in the jejunal macrophages was probably a reflection of the disposition of drug following oral absorption. although similar lipidosis was seen in many organs following intravenous administration in dogs, more lipidosis was seen in the jejunum after oral dosing. moreover, there were species differences in sensitivity to these changes, baboons being relatively insensitive compared to dogs. fischer rats were very sensitive to these changes compared to sprague-dawley rats and wistar rats were almost completely resistant to lipidosis induced by amiodarone under similar conditions (mazué et al., ) . similar cytological changes have been reported in cells of some organs in patients treated with amiodarone (d'amico et al., ; shepherd et al., ) . villous shortening or stunting results when the proliferative activity of the crypt epithelium is reduced or under circumstances in which crypt cell proliferation is insufficient to compensate for increased cell loss as a result of mucosal cell damage. decreased cell proliferation can be seen segments, which are surgically bypassed, or following decreased food intake, parenteral nutrition, hypophysectomy or thyroidectomy bastie et al., ) . as adrenergic factors are important in the control of small intestinal epithelial cell division, drugs that alter α or β adrenoreceptor activity may influence the proliferative capacity of the epithelium. in mice, increased α or β receptor stimulation by appropriate agonists (e.g. phenylephrine) diminishes proliferation of crypt cells but proliferation is increased by stimulation of α receptor activity (kennedy et al., ) . yohimbine, a α -antagonist also reduces cell proliferation in the same animal model. some of the effects of these agents may be mediated by changes in splanchnic blood flow . the detailed morphological study of the small intestinal mucosa in the rat following hypophysectomy by bastie et al. ( ) has shown a reduction in the height of the small intestinal villi associated with reduction in mitoses in the crypt epithelium. the number of goblet cells was shown to fall particularly in the jejunum and the number of paneth cells increase in the ileum. ultrastructural examination showed decreased height of the microvilli of absorptive cells and a lower number of their intracytoplasmic organelles and ribosomes. there were also significant decreases in brush border enzyme activities of alkaline phosphatase, aminopeptidase, maltase and lactase reported about week following hypophysectomy. substances which reduce mitotic activity and therefore lower regenerative capacity of the intestinal epithelium also produce shortening or stunting of small intestinal villi and eventually flattening of the mucosa. a wide variety of anticancer agents and antiviral drugs with radiomimetic properties interfere with cell division in the crypts thereby reducing the number of epithelial cells produced. histologically, the effects of such agents are characterised by blunting, shortening or complete atrophy of villi. mitotic activity is reduced in the crypts and the crypts become dilated and lined by flattened cells. the overlying epithelium loses its normal regular arrangement and cells show pleomorphic nuclei with irregular chromatin patterns. increased numbers of inflammatory cells may infiltrate the lamina propria and epithelium. ulceration, haemorrhage and secondary infection of the gut wall ensue if there is overwhelming cell damage. comparison of the gastrointestinal toxicity expressed by antimitotic anticancer drugs of different classes in rodents, dogs, non-human primates and man have suggested that there is a higher degree of correspondence between effects in man and dog than between man and other species (owens, ; schein et al., ) . in studies with the antiviral agent acyclovir, a radiomimetic effect was noted in the gastrointestinal tract of dogs at high doses but not rodents (tucker et al., ) . another example is the villous atrophy described in rats following treatment with an antibacterial agent ici , . this was believed to arise as a result of both interference with cell division and a direct effect on the surface epithelial cells (murgatroyd, ) . the effects of ici , were characterised by atrophy of villi with dilatation of crypts and atypical features in the crypt epithelium suggestive of an effect on mitotic activity. in addition, vacuolated lipid-laden epithelial cells were observed over the tips of villi accompanied by reductions in the numbers of goblet cells and reduced activity of acid and alkaline phosphatase, esterase, adenosine triphosphatase, glucose- -phosphatase and succinic dehydrogenase, compatible with a direct adverse effect on superficial mature epithelial cells. a variety of factors stimulate cell proliferation in the small intestinal epithelium. these include enterectomy, increased feeding and stimulation of autonomic nerves. administration of neurotransmitters, thyroxine, growth hormone, corticosteroids, testosterone, gastrin, glucagon and recombinant epidermal growth factor may also stimulate epithelial cell proliferation breider et al., ; reindel et al., ; vinter-jensen, ) . in rats hypothalamic damage, hyperthyroidism, tube feeding, diabetes mellitus and insulin injections have been shown to produce intestinal hyperplasia (mackay et al., ; levin and smyth, ; jarvis and levin, ; forrester, ) . most causes of greater cell production lead to increased villous height and mucosal hyperplasia, although intense crypt cell proliferation as a compensatory regenerative response can be associated with villous atrophy (see previous). the compensatory response to the surgically resected or bypassed intestine has been the focus of the most detailed studies of increased cell renewal in the small intestine. partial resection in both rats and man is accompanied by increased villous height and crypt length (hanson et al., ) . this is primarily the result of hyperplasia for it has been shown that the numbers of cells per unit length of villus remains unchanged (hanson et al., ) but there is an overall increase in the cell population of villus and crypt (hanson and osborne, ) . dna/rna ratios also remain largely unaltered . no gross changes in villous shape have been reported after resection and the total number of crypts remains constant. although increased intestinal uptake of substances from the bowel lumen occurs in hypertrophied segments per unit length of bowel, disaccharide and dipeptidase activities are normal or even decreased after resection suggesting a comparative immaturity of cells in the residual mucosa. functional adaptation therefore is achieved by a larger number of cells, the individual absorptive capacity of which is not increased . increased numbers of specific goblet cell populations are also seen in hyperfunctional states. following jejuno-ileal bypass operations in rats, increased numbers of pas-positive goblet cells develop in the villi and crypts of the hyperfunctional segments of the duodenum, jejunum and ileum (olubuyide et al., ) . mucin histochemistry using the high-iron diamine and alcian blue techniques have shown that the goblet cells in the hyperfunctional segments contain increased sialomucins in the villi and crypts of the jejunum and ileum but not in the duodenum and increased sulphomucins in the distal ileal segment. sialomucin production may reflect relative cellular immaturity of the more rapidly proliferating cells under these circumstances. however, as sialic acid conveys more viscoelastic properties to mucin, it has been suggested that the goblet cells change following intestinal bypass fulfils a protective function against the increased flow of gastrointestinal contents (olubuyide et al., ) . a number of nutritional factors, particularly dietary fibre, can influence the proliferative characteristics of the small bowel mucosa. carefully controlled studies in rats given different forms of dietary fibre have shown that the prolif-erative characteristics of the small intestine can be modified by both the quantity and the quality of the fibre. one study has shown a decrease in the length of villi, crypt cell hyperplasia and shorter transit times in rats fed pectin-supplemented diet but an increase in mucosal growth without alteration in relative differences in crypt and villous length with guar supplementation compared with rats fed fibre-free diet (jacobs, ) . another rat study has shown that pectin feeding leads to increased mucosal area and height associated with an increase muscle mass (stark et al., ) . these different effects may be the result of differences in solubility, gel formation, water holding capacity, effect on transit time and ion exchange activity or bile acid adsorption of the different fibres. interactions between dietary constituents are complex. for instance, in rats the effects of % dietary cholestyramine, a non-absorbable ion exchange resin, on small intestinal histomorphology have be shown to depend on interaction of dietary factors (burkhardt et al., ) . administration of an inhibitor of cholesterol biosynthesis, α-cholest- -( )en- β-ol- -one, to rats for up to days was also shown to produce enlargement of the small intestine in a way which was morphologically similar to the changes found following intestinal bypass (smith et al., ) . the enlargement was most marked in the proximal segment of the small intestine and progressively diminished towards the ileocaecal junction, sparing the stomach, caecum and colon. histological examination and morphometric analysis revealed an increase in smooth muscle mass, lengthening of the villi as well as an increase in the depth and cellular proliferation in the crypts of lieberkuhn without evidence of cell damage or fatty change. like the changes following jejunal bypass procedures, there was also an increase in acid mucosubstances in the goblet cell population overlying the villous mucosa (smith et al., ) . the mechanism for this change in the rat was not clear, particularly as intestinal hyperplasia was not seen in baboons treated with this -ketosterol for long periods. however, it was suggested that it was an adaptive response, possibly related to inhibition of cholesterol metabolism and cholesterol absorption from the diet, particularly as the laboratory diet employed in the rat study was particularly low in cholesterol. local and systemic changes in hormones and various transmitter substances also influence the number of cells in the small intestinal epithelium. morphometric studies of the small intestinal mucosa in mice following gastrin administration have shown increases in villous area associated with decreases in microvillous area, increased number of goblet cells and paneth cells . studies in which rats were treated with the prolactin-inhibitor, ergocryptine, have shown that the total number of mucous cells and the number staining with alcian blue at ph . increase in the ileal crypts, possibly as a result of increased synthesis of sulphated mucosubstances (gona, ) . in this context, it is of interest that chronic treatment with the rauwolfia neuroleptic, reserpine, causes an increase in the sulphation of goblet cell mucin in the small intestine as demonstrated by alcian blue staining at ph . and the high iron diamine technique without changes in the goblet cell numbers (park et al., ) . agents which affect activity of the sympathetic nervous system can also alter epithelial cell proliferation in the small (and large) intestine. treatment of rats with adrenaline, isoprenaline, phenylephrine, phentolamine and yohimbine all result in decreased mitotic activity of jejunal and colonic crypt cells (tutton and helme, ; kennedy et al., ) . by contrast, administration of metaraminol, clonidine, propranolol, prazosin and labetolol as well as simultaneous injection of propranolol and adrenaline all resulted in an increased rate of crypt cell proliferation (kennedy et al., ) . these results suggest that agents that stimulate α adrenergic receptor activity and those that are α -antagonists and β-adrenergic receptor antagonists increase proliferative activity in the rodent intestinal mucosa. caffeine is also reported to produce an increase in thickness of the intestinal mucosa when administered in high doses to rats (lachance, ) . this raises the possibility that intestinal mucosal hyperplasia can be produced by phosphodiesterase inhibition and resultant increases in intracellular camp in a similar way to the hypertrophy induced in salivary tissue. this is supported by recent findings in rats treated for periods of up to months with the inotropic vasodilator, ici , , a phosphodiesterase inhibitor intended for treatment of congestive cardiac failure. administration of high doses not only produced salivary gland hypertrophy but also marked thickening of the small and large intestinal mucosa. this was characterised histologically by an increase in villous length and deepening of intestinal glands, with a relatively unchanged number of epithelial cells per unit length of gland or villus (westwood et al., ) . although prostaglandin e analogues produce most of their effects in the stomach, increased thickness of the small intestine characterised by longer villi, deeper crypts and increase in cell size have been reported in rats treated with these agents (levin, ) . focal hyperplasia, focal avillous hyperplasia, focal atypical hyperplasia, duodenal plaque, polypoid hyperplasia, polyp -mouse irregular, atypical single or multiple foci of glandular hyperplasia may be found in the small intestinal mucosa of several strains of aged, untreated mice. the lesions are usually located in the first part of the duodenum where they form discrete, raised plaques composed of elongated, irregular or branched glands which replace the normal villous structure of the mucosa (rowlatt and chesterman, ) . the glands are lined by hyperchromatic columnar cells, which show marked pseudostratification and proliferative activity. paneth cells and mucinsecreting goblet cells may also be prominent. some glands are cystic and the stroma is fibrous and infiltrated by chronic inflammatory cells. the lesions become pedunculated or polypoid in appearance and show a fibrovascular core that is infiltrated by inflammatory cells. they resemble adenomatous polyps described in man. the cause of these changes in the mouse small intestine is unknown but their prevalence can be altered by dietary fibre and panthothenic acid deficiency as well as by administration of drugs and chemicals (hare and stewart, ; seronde, ; ito et al., ) . in their study of dba mice, hare and stewart ( ) considered that the lesions were not genuine neoplasms since they were composed of a mixture of cell types, which normally populate the mucosa. furthermore, they suggested that the presence of an inflammatory component in the stroma and the fact that the prevalence of these lesions was increased in mice fed a high roughage diet were consistent with the concept that they represent an inflammatory adenomatoid hyperplasia. seronde ( seronde ( , reported these lesions in mice fed purified diets, particularly when deficient in panthothenic acid. panthothenic acid deficiency was also associated with inflammation and deep penetrating chronic ulcers of the duodenum in affected mice, compatible with an inflammatory aetiology of the lesions. an increase in the prevalence of these duodenal changes was described in cd- mice treated with the synthetic prostaglandin e analogue, misoprostol for months . these authors suggested that the findings posed no real concerns for the safety of patients treated with misoprostol on the grounds that the mouse was unique in this aspect of the response to misoprostol because the mouse had a particular liability to develop such changes in the small intestine. the proliferative lesions were found in a few control cd- mice in the same study. in addition, it was also argued that the lesions were neither neoplastic nor preneoplastic in nature . they were not seen in rats treated with misoprostol for years . lesions characterised by such intense proliferative activity may be difficult to distinguish from neoplastic lesion. indeed chronic administration of hydrogen peroxide to c bl/ j mice in drinking water was not only shown to potentiate the development of a similar type of duodenal hyperplasia but also to produce frankly invasive adenocarcinomas (ito et al., ) . anatomically the large intestine is broadly similar in man and laboratory animals but there are significant functional differences. the rat colon is probably one of the best studied of the laboratory animal species because the rat is widely used for experimental work on colon carcinogenesis . as the canine model is popular for oral dosage-form testing, differences in colonic physiology between dog and man may be better understood than between man and many other species (dressman, ) . in man, as well as in the non-human primates, the large intestine can be divided anatomically into caecum, appendix, ascending colon, transverse colon, sigmoid colon rectum and anal canal. like the small bowel, the colon comprises mucosa, submucosa, muscularis mucosa and serosa. mucosal plicae are only found in the rectum although plicae semilumaris, formed by folds of the entire thickness of the bowel wall, are found in the colon. the large intestine of the dog resembles that of man more than that of most other domestic species. it is a simplified tubular structure only slightly larger in diameter than the small intestine. the colon of the dog is divided anatomically into ascending, transverse and descending parts, but there is no well-defined sigmoid segment. the caecum in dog is a small diverticulum, similar to that found in other carnivorous species and it communicates directly with the colon. the colon of the rat and mouse is shaped like an inverted v that can be divided into ascending and descending segments. there is no clearly defined transverse colon. a characteristic feature in both rat and mouse is the presence of a curved kidney-shaped caecum. its size is intermediate between the large and anatomically complex caecum of herbivores such as the rabbit and the small caecum of carnivorous species. this probably reflects the omnivorous nature and flexibility of the rat and mouse in their dietary habits, particularly their ability to breakdown cellulose (rerat, ) . the caecum of the rat and mouse is a blind pouch from which the colon and ileum exit in close proximity and in which antiperistaltic movements occur. this structure and the presence of bacteria undoubtedly contribute to its ability to function as a fermentation organ in which breakdown of substances can occur in a reasonably controlled milieu (snipes, ) . in rat and mouse, the caecum is the site of absorption of many substances including calcium, magnesium, water and electrolytes vitamin k and fatty acids (snipes, ) . caecectomy has been shown to decrease digestion of carbohydrates and protein and increase loss of faecal water in these species (ambuhl et al., ) . the activity of intestinal microflora in the metabolism of both endogenous and exogenous substances has been demonstrated in the rodent caecum rowland, ) . the usual stock diets for rodents contain abundant plant fibre which provides bulk and fermentable carbohydrate for the microbial population in the caecum. rats fed stock diets have been shown to possess high levels of reductive and hydrolytic enzyme activity (e.g. azoreductase, nitroreductase, nitrate reductase, β-glucosidase and β-glucuronidase) in their caecal contents compared with rats fed purified fibre-free diets . intestines of germ free animals have thinner lamina propria, lower cell turnover, enlarged caecum, altered metabolism of cholesterol, bilirubin and bile salts and larger amounts of mucin in faeces compared with animals possessing normal gastrointestinal microflora (midtvedt, ) . species differences in microflora are also reported. comparative studies with human and rat intestinal microflora have suggested that each population of organisms possesses a degree of autonomous self-regulation and capable of responding quite differently to dietary changes (mallett et al., ) . comparative studies have shown large differences in the numbers of facultative anaerobic gram-negative bacteria in the gastro-intestinal tract of mice from three, major specific pathogen free units in australian laboratories. it was shown that these differences could influence the immune system, susceptibility to infection and experimental results (o'rourke et al., ) . the colon and caecum in man and laboratory animals are lined by a fairly uniform mucosa devoid of villi. columnar cells of two main types cover the surface epithelium. these are absorptive and mucous cells similar to those found in the small intestine. intestinal glands or crypts of lieberkuhn extend downwards from the surface generally as simple, unbranched tubules lined principally by mucous cells with smaller populations of absorptive, endocrine and undifferentiated cells. the mucosa in man and laboratory animals is not entirely flat but shows a slightly corrugated or uneven pattern, which varies with the particular site within the colon. in histological sections of the colon in man, this corrugated pattern is seen as an anthemion-like structure of crypts reminiscent of a greek architectural feature (filipe and branfoot, ) . this is also seen in larger laboratory animal species. in rats and mice the crypts of the caecal mucosa are wider near the lumen than in the crypt base and crypts may be branched, features which may be related to the absorptive function of this zone (snipes, ) . the proliferative zone in the large bowel is found in the lower part of the gland and mitotic figures are normally limited to this zone. as in the small bowel, multipotent, undifferentiated stem cells situated in the gland base give rise to the principle cell types which migrate to the cell surface with subsequent differentiation and alteration of their enzyme activities and morphological features (chang and leblond, ). in studies with mouse aggregation chimaeras in which mosaic cell populations of the intestinal epithelium were localised immunocytochemically, it was demonstrated that the entire epithelium of each adult gland descended from a single progenitor cell (ponder et al., ) . the single progenitor may itself give rise to several stem cells responsible for the cell renewal in the complete crypt. absorptive cells are found most commonly in the surface epithelium but also to a lesser extent in the glandular epithelium. they are morphologically similar to those in the small intestine each possessing apical plasma membranes with uniform microvilli and a well-formed glycocalyx. microvilli of absorptive cells have been shown by electron microscopic study to become longer and denser with increasing distance distally in the gastro-intestinal tract. thus, they are longer and more dense in the ileum than in the caecum and least dense and shortest in the colon, perhaps reflecting their relative absorptive capacity (snipes, ) . this is reflected at light microscopic level by the less conspicuous brush border in the large intestine compared with that in the small intestine. there are species and regional differences in the glycoconjugates found in the brush border of the large intestine, although they generally contain predominantly acidic mucosubstances. in the mouse and rat, sialomucins with some neutral mucins are found. in hamsters, dogs, non-human primates and man both sulphomucins and sialomucins may be seen in the brush border (sheahan and jervis, ) . the glycocalyx is important in the protective function of the colonic mucosa for its disruption by agents such as salicylates has been shown to increase absorption of xenobiotics from the rat rectal mucosa (sithigorngul et al., ) . although there has been some debate about the precise nature of the mucous cell populations based on structural studies in mice and rats (chang, ; , for practical purposes two principle types of mucous cell can be defined. one of these is the typical goblet cell with cytoplasmic mucous droplets forming a goblet shape, which is found both in glandular and surface epithelium. the other type, the so-called vacuolated cell or mucous cell, is found only in the crypts (chang and leblond, ; thomopoulos et al., ) . these vacuolated or mucous cells show empty-appearing vacuoles in the cytoplasm which rather than being empty contain abundant sialomucins of a type different from those in goblet cells (spicer, ; wetzel et al., ) . detailed structural studies and cytochemistry using lectin probes have suggested that these vacuolated cells are able to differentiate into absorptive cells with the cellular apparatus to produce the cell surface glycoconjugates of the glycocalyx (thomopoulos et al., ) . sulphomucins, as demonstrated by the high-iron diamine technique (table ) , generally predominate in the distal colonic segment. in both rat and mouse, goblet cells of the proximal colonic mucosa contain largely sialomucins in the lower parts of the crypts with sulphomucins predominating in the upper parts of the crypt. the distal colon contains largely sulphomucins. the only difference between rat and mouse appears to be the fact that the mouse caecum contains almost exclusively sulphomucins but sulphomucins and sialomucins are found in the rat caecal mucosa. in hamsters, the entire colon contains predominantly sulphomucins. neutral mucins and sulphomucins predominate throughout the dog colon with occasional goblet cells containing sialomucin. in non-human primates, neutral mucins, sialomucins and sulphomucins are seen throughout the colon with sialomucins generally more prominent in the proximal colon and sulphomucins in the distal segment. in man, neutral mucins are found mostly in the caecum. in the caecum and ascending colon, sulphomucins are found in the upper crypts and sialomucins in the crypt base. the converse occurs in the distal colon where sulphomucins predominate in the lower two-thirds of the glands and sialomucins in the upper third of the glands and in the surface epithelium. lectin-labelling (table ) shows even greater heterogeneity of mucins in the colonic mucosal cell population, probably reflecting differentiation patterns and changes in glycosyltransferase activity as cells migrate upwards (freeman et al., ; thomopoulos et al., ) . it has been suggested by jass and roberton ( ) that the two principle changes in pathological processes in the human colonic mucins are loss of oacylation substituents at sialic acid c and c , , and increased sialylation, neither being specific for neoplasia. the colon, like many other tissues also possess drug metabolising activity, although less than in the liver. it has been shown that the activity of cytochromes p involved in hydroxylation of benzo[a]pyrene in microsomes prepared from the colons of sprague-dawley rats retain their activity and responsiveness to inducers better than those in the liver with advancing age (sun and strobel, ) . the lamina propria of the large bowel is arranged in a similar way to that of the small bowel. by virtue of the presence of lymphocytes, plasma cells, macrophages and dendritic cells as well as scattered small lymphoid aggregates or patches, it forms an integral and important part in the mucosal immune defence system. most of the lymphocytes in the lamina propria of the human colonic mucosa, like that of the ileum, have been shown to be t cells with helper t cells out numbering the t-suppresser phenotype (hirata et al., ; pabst, ) . this contrasts with intra-epithelial lymphocytes of the human colonic mucosa which are also t cells but more than % of which possess characteristics of the suppresser/cytotoxic phenotype and only - % being helper t cells. this distribution of lymphocyte subsets is seen immunocytochemically in the rat colon using the monoclonal antibodies w / , w / and mrc ox (see haemopoietic and lymphatic systems, chapter iii). the pan t-cell marker w / shows the presence of t lymphocytes in the lamina propria and most of these are labelled by w / demonstrating their cd helper phenotype (bland and warren, ) . mrc ox demonstrates that few lymphocytes in the lamina propria are of suppresser/cytotoxic (cd ) type, which contrasts, with the high proportion of mrc ox positive lymphocytes in the colonic epithelium (bland and warren, ) . the monoclonal antibodies mrc ox and mrc ox , specific for the rat ia antigen, also label numerous cells with macrophage and dendritic cell morphology in the large bowel of the rat (bland and warren, ; martin et al., ) . mature, small b lymphocytes are relatively uncommon in the colonic lamina propria of man and laboratory animals. however, the lamina propria contains large numbers of plasma cells, which are mainly of iga type, followed by smaller numbers of igm an igg subtypes (pabst, ) . a feature of the colonic mucosa is the presence of lymphoid aggregates also called lymphoid nodules, patches, lymphoid-glandular complexes or microbursa. these are similar to peyer's patches of the small intestine as they are composed principally of lymphoid cells of the b-cell series arranged in follicles with germinal centres with interfollicular and perifollicular zones composed of t cells (pabst, ) . they are distributed along the entire length of the colonic mucosa although they are generally smaller than peyer's patches. in sprague-dawley rats, lymphoid aggregates are usually about mm diameter except in the distal colon where they attain sizes of up to mm in maximum diameter (martin et al., ) . unlike peyer's patches which are characteristically not associated with crypts or villi, the colonic lymphoid aggregates frequently contain irregular atypical mucosal glands which may enter deeply in the lymphoid tissue and penetrate below the muscularis mucosa both in man and laboratory animals scott, ; martin et al., ) . in some strains of rat, cells in these glands express the ia antigen, unlike the other parts of the colonic epithelium (martin et al., ) . these glandular structures, which are intimately associated with lymphoid tissue, may be important in the immune protection of the colonic mucosa, perhaps by acting as a special local receptor for antigens . it has been proposed that these glandular structures represent sites of predilection for the spread of inflammatory disease to the submucosa by allowing microorganisms to pass through the muscularis mucosa (scott, ) . it has also been suggested that they constitute physical weak points in the bowel wall and may play a part in the pathogenesis of diverticular disease of the colon in man . colonic carcinomas induced by dimethylhydrazine in the rat also appear to develop more commonly in the lymphoid aggregates than in other zones (martin et al., ) . m cells have been described over the lymphoid aggregates in the caecum of the mouse (owen and nemanic, ) , see small intestine. although microorganisms are important causes of inflammatory disease in the large intestine of man and animals, among laboratory animals they are usually only significant problems in non-human primates and hamsters. in the strains of rats and mice and in beagle dogs commonly employed in drug safety evaluation, spontaneous disease of the colon as a result of infectious agents is uncommon. nevertheless, treatment with some therapeutic agents may alter the normal bacterial flora to permit overgrowth of pathogenic organisms or disturb the normal balance between antigens in the lumen or control mechanism to evoke inflammation. inflammation induced by organisms may also confound the histological assessment of drug-induced changes in the colon. ulceration and inflammation of the colon as a direct result of administration of potential therapeutic agents is reported in humans although less commonly that in the small intestine. it has been suggested from studies of the effects of anticancer compounds on neoplastic colonic cells that intestinal cells may possess inherent protective properties in the form of an accelerated efflux pump which can serve to protect them from potentially damaging agents (klohs and steinkampf, ) . ulceration and inflammation can be induced by the local application of drugs and vehicles to the rectal mucosa. assessment of these effects in an appropriate animal model is important in the safety evaluation of preparations designed for use in man as rectal suppositories. although inflammatory conditions of the large bowel may possess morphological features typical for some inducing agents, inflammation of the large intestinal mucosa is usually characterised by non-specific histological features. in early or mild inflammation, the surface and glandular mucosa remains intact but shows mucin depletion. this is characterised by reduction in the mucus in goblet cells and increased cytoplasmic basophilia. scattered neutrophils may be seen in the epithelium and adjacent lamina propria. in more severe cases, crypts become filled or distended with acute inflammatory cells (crypt abscesses). the lamina propria is variably hyperaemic and congested and contains increased numbers of mononuclear cells. severe changes are characterised by attenuation or frank erosion of the epithelium and the formation of penetrating ulcers filled with fibrinous exudate and surrounded by intense inflammation, granulation tissue and eventually fibrosis. residual glands may be dilated and lined by flattened epithelium or show reactive changes and mitotic activity. regenerative hyperplasia, which can become florid in chronic ulcerative conditions, is characterised by lengthening, irregularity and cystic dilatation of glands which are often lined by hyperplastic epithelial cells and goblet cells distended with mucin. where ulcerative damage has destroyed glands and supporting stroma, regeneration of glands may not occur in the normal regular fashion and branching of crypts may be evident. clostridium difficile may cause inflammatory changes in the colon of laboratory animals, particularly hamsters, and this may extend into the distal ileum. as in man this form of colitis, often referred to as pseudomembranous colitis, is usually associated with antibiotic therapy. in man it was originally associated with lincomycin and clindamycin therapy but other antibiotics have been implicated. it has been shown that both in man and the hamster experimental model that the enteritis is the result of the toxin produced by clostridium difficile (bartlett et al., ; milligan and kelly, ) . in man this condition is histologically characterised by the presence of plaques or pseudomembranes on the colonic mucosal surface. the pseudomembrane is composed of mucus, fibrin, blood cells, inflammatory cells and cell debris, which has an appearance of streaming from the underlying mucosa. the mucosa may be partly necrotic or mucosal glands are dilated and lined by flattened or hyperplastic cells. the ileal mucosa may show similar changes (milligan and kelly, ) . similar features are observed in the antibiotic-treated hamster although the pseudomembrane is less prominent and it may be distributed more proximally with involvement of the terminal ileum (rehg, ) . in the hamster, the condition is characterised histologically by erosion of the colonic epithelium and the variable presence of a pseudomembranous plaque of mucin and cell debris. intact but affected mucosa is thickened with reactive changes accompanied by mucin loss in the epithelium and infiltration of a hyperaemic and oedematous lamina propria and submucosa by polymorphonuclear cells (rehg, ) . although most instances of this form of clostridia colitis in the hamster have been associated with antibacterial therapy, it has also been reported in untreated hamsters (rehg and lu, ) and those treated with antineoplastic drugs (cudmore et al., ) . similar changes have been reported in antibiotic-treated guinea pigs and rabbits (rehg and lu, ; rehg and pakes, ) . guinea pigs are particularly sensitive to antibiotics especially those active against gram-positive organisms (young et al., ) . as in man, these drugs are believed to alter the intestinal flora, permitting overgrowth of clostridium difficile as well as gram-negative organisms, resulting in a severe and frequently fatal enterocolitis. a study of the disposition of ampicillin administered parenterally to guinea pigs showed that this drug was rapidly eliminated from the systemic circulation and excreted in urine and bile, possibly favouring this effect on flora in the colon (young et al., ) . citrobacter freundii, a gram-negative, short, plump rod and member of the family of enterobacteriaceae, is the causative agent of naturally occurring transmissible colonic hyperplasia of mice. this agent usually produces a mild or even asymptomatic enteritis in susceptible mouse populations, although it is a cause of rectal prolapse in mice (ediger et al., ) . marked strain differences have been noted in mice infected with this organism. nih swiss mice show the most severe histological changes and c bl/ j mice appear the least affected (barthold et al., ) . rats and hamsters seem to be unaffected by citrobacter freundii (barthold et al., ) . microscopic changes are found primarily in the descending colon, although proximal segments of the colon and the caecum may also be affected. an important morphological feature is epithelial hyperplasia, which occurs maximally - weeks after experimental inoculation with citrobacter freundii (barthold et al., ) . the colonic glands are elongated and lined by cells that show mucin depletion or loss of goblet cells, considerable immaturity and mitotic activity. the surface epithelium may be covered with numerous coccobacilli, which can be visualised in routine haematoxylin, and eosin stained sections. crypt abscesses, inflammatory cells in the lamina propria, mucosal erosions and ulceration are also features (barthold et al., ; . in regressing lesions there is a rebound increase in goblet cells, which are often distended with mucin. the colonic glands may be branched or irregular (barthold et al., ) . most laboratory animals are naturally resistant to shigella infections but this is not the case for most non-human primates (takeuchi, ) . in infections with shigella, the colon shows a superficial acute inflammatory reaction comprising oedema, congestion, haemorrhage and infiltration by acute inflammatory cells. the surface epithelium shows mucin loss and formation of microulcers where total destruction of the epithelium has occurred. ulcers can extend into the lamina propria but in general terms the inflammatory process remains relatively superficial (takeuchi, ) . organisms are also located predominantly in the superficial epithelium. another bacterial infection of the gastrointestinal tract, which affects the colon in primates, is that produced by non-tuberculous mycobacteria . large intestinal lesions are characterised by massive accumulation of epitheloid macrophages in the lamina propria, which may extend into the submucosa and muscular layers and along lymphatics to involve mesenteric lymph nodes. small intestinal lesions may also occur, characterised by the presence of similar large macrophages in the lamina propria of villus tips. superficial ulcers may occur in severely affected segments of intestine . acid-fast bacteria are typically found within macrophages. other organs, including spleen, liver, bone marrow and lungs, may also be involved by focal accumulations of bacteria-laden macrophages or occasionally discrete granulomas with multinucleated giant cells. numerous protozoa and metazoa have been described as inhabitants of the caecum and colon of the non-human primate (toft, ) . far fewer are observed in the usual laboratory rodents and beagle dogs. amoebiasis caused by entamoeba histolytica is a widespread disease among non-human primates. it is characterised histologically by the presence of necrotizing ulcers, which reach the muscularis mucosa to form typical flank-shaped ulcers containing or surrounded by trophozoites. extensive haemorrhage may be seen as well as an inflammatory infiltrate composed of neutrophils and mononuclear cells (toft, ) . the ciliate, balantidium coli, can also cause an ulcerative process in the colon of primates, characterised by ulcers which extend down to the muscularis mucosa accompanied by lymphocytic infiltrate and balantidium coli trophozoites of up to µm in greatest diameter (toft, ) . a variety of metazoan parasites can be observed in the primate colon and usually can be reasonably well identified in tissue sections (see review by chitwood and lichtenfels, ) . the nematode of species strongyloides is an important parasite, which may be observed in the intestinal mucosa of primates. oxyurids commonly known as pinworms are essentially innocuous parasites seen in man, non-human primates and rodents. enterobius vermicularis is found in the large intestine and appendix of man and non-human primates, syphacia muris and syphacia obvelata in rodents. oesophagostomum species (nodular worms) are especially common nematode parasites of non-human primates forming characteristic nodules up to mm diameter most frequently on the serosal surface of the large intestine and caecum and adjoining mesentery as well as in other sites in the peritoneal cavity. histologically, the nodules are composed of parasite cell debris surrounded by fibrous tissue and a variable mantle of chronic inflammatory cells and occasional foreign-body giant cells. they are frequently found in close proximity to small arteries and arterioles in the submucosa and subserosa of the colon and may be associated with a local granulomatous arteritis (lumb et al., ) . the inflammatory process may spread to surrounding or draining tissues, particularly if nodules rupture. mild periportal hepatic chronic inflammation is sometimes associated with the presence of this parasite in the mesentery, which may confound interpretation of drug-induced hepatic changes in the non-human primate. although the stomach and to a certain extent the small intestine remain the primary sites of predilection for the ulcerogenic action of non-steroidal anti-inflammatory, the colonic mucosa may become involved under certain conditions. less common complications of non-steroidal anti-inflammatory drugs and potassium chloride therapy in humans are colonic strictures. it appears that nonsteroidal anti-inflammatory drugs produce local inflammation followed by focal scarring of the submucosa with constriction and formation of a mucosal diaphragm whereas potassium causes segmental full thickness scarring and constriction (fellows et al., ; haque et al., ; van velzen et al., ; wolfe et al., ) . another form of induced colon damage has been reported in children with cystic fibrosis, the majority of who take high strength pancreatic-enzyme supplements to control malabsorption (smyth et al., ; fitzsimmons et al., ) . this condition has distinctive pathological features. there is involvement a long segment of ascending colon by a fusiform stenosis primarily as a result of submucosal thickening by deposition of mature collagen. the mucosa appears relatively spared but shows some ulceration and reparative changes (van velzen et al., ) . although it has been suggested that the changes may have been linked to the methylacrylate copolymer used for enteric coating of the high-strength preparations, a case-control study showed a strong relation between high daily doses of the enzyme supplements, accentuated by more recent availability of high dose forms (fitzsimmons et al., ) . in view of their usage for over years, preclinical data on this material is scarce. colonic damage can be induced experimentally by administration of therapeutic agents. dogs administered . mg/kg indomethecin orally each day for periods of up to days developed not only gastric and small intestinal ulceration but also scattered haemorrhagic erosions in the colon and rectum. histologically, these lesions were characterised by loss of superficial epithelial cells, mucus-depletion of glandular epithelium, crypt abscesses, frequently with acute inflammation in adjacent lymphoid aggregates in the submucosa (stewart et al., ) . an example of chemically induced colitis of relevance to safety assessment of therapeutic agents is that induced by degraded carrageenans or synthetic sulphated dextrans. carrageenans are a heterogeneous group of sulphated polysaccharides composed mainly of long chains of d-galactose subunits (d-galactan) derived from red seaweed species which are widely used as food emulsifiers, stabilisers, thickeners and gelling agents (ishioka et al., ) . when carrageenans are degraded by acid hydrolysis into smaller molecular weight fragments of about , - , and administered orally in high doses (e.g. % of diet) to rats, mice, guinea pigs, rabbits and rhesus monkeys, colitis results (sharratt et al., ; marcus and watt, ; benitz et al., ; fath et al., ; kitano et al., ) . similarly, colitis has been induced in rats following administration of a % dietary admixture of dextran sulphate sodium, a sulphated polymer of glucose (a d-glucose) of molecular weight of , (hirono et al., ) and a very high molecular weight d-glucan, amylopectin sulphate (ishioka et al., ) . although histological features of this form of induced colitis vary between study, species and strain, the colitis is generally characterised mucosal ulceration mainly in the caecum but also in the distal ileum, distal colon and rectum. there is mucus-depletion with variable acute inflammatory infiltrate of the in-tact epithelium, crypt abscesses, inflammatory infiltrate of the lamina propria with oedema, hyperaemia and even vascular thrombosis in the submucosa (hirono et al., ; fath et al., ) . increased proliferative activity of the mucosa is confirmed by an increase in the tritiated thymidine index compared with controls (fath et al., ) . in the caecum of rats, ulcers are linear but often circulating the entire circumference of the intestinal wall with subsequent scarring and stricture formation (oohashi et al., ) . ulcerating lesions in the rectum and at the anal margin are associated with squamous metaplasia. both the squamous metaplasia and the regenerative hyperplasia of the columnar epithelium have been shown to progress even after cessation of treatment (oohashi et al., ) . foamy macrophages containing metachromatic material, presumably polysaccharide, are also seen in the lamina propria, submucosa, regional lymph nodes, liver and spleen (hirono et al., ; oohashi et al., ) . the cause of this colitis is unclear. low dose levels, which may be expected to mimic human exposure, do not produce colitis. dextrans, carrageenans and other polysaccharides of molecular weights outside the range , - , tend not to incite colitis. an exception to this is the agent amylopectin sulphate, which has a far higher molecular weight. however, amylopectin is composed of polysaccharide chains, which can be degraded by amylase, and therefore smaller molecular weight fragments may be formed in vivo (ishioka et al., ) . it has been suggested that colonic disease produced by these agents is in some way linked to induced changes in intestinal microflora (marcus and watt, ) although the evidence for this is conflicting (ishioka et al., ) . a recent study in guinea pigs and rats using permeability markers of different molecular weights has suggested that degraded carrageenans enhance intestinal permeability in the absence of overt ulceration (delahunty et al., ) . it was therefore proposed that carrageenan-induced colitis could be the result of increased intestinal permeability to antigenic or inflammatory substances normally resident in the large intestine. moreover, long-term administration of high doses of these agents to rats leads to the development of colorectal cancer despite their being devoid of any mutagenic potential (see below). the only obvious features, which are common to a number of these non-genotoxic agents, is chronic inflammation and increased proliferative activity. the rectal administration of therapeutic agents and surfactants may also induce similar ulcerative and inflammatory changes. chemical colitis resembling pseudomembranous colitis has been reported in man as a result of chemical cleaning agents accidentally induced by endoscopic examination (jonas et al., ) . cellular degeneration, with loss of mitotic activity and mucin depletion can also occur in the colon following treatment with antimitotic drugs. lymphoid infiltrates without tissue damage were reported in the large bowel of rats treated with human recombinant interleukin- (anderson & hayes ) . melanosis coli is a well-described phenomenon in man associated with chronic ingestion of anthraquinone purgatives. it is considered to be due to the excessive accumulation of lipofuscin-like pigment in the macrophages of the colonic lamina propria (schrodt, ; ghadially and parry, ; steer and colin-jones, ) . this pigment probably originates from organelles of epithelial cells or macrophages, which are damaged by treatment. similar morphological changes have been induced in laboratory animals (guinea pigs) by treatment with anthraquinones (walker et al., ) . as a result of these animal studies, walker et al. ( ) suggested that the primary process is a treatment-induced increase in apoptotic bodies in the surface colonic epithelium that are phagocytosed by intraepithelial macrophages and transported to the lamina propria. lipofuscin and iron pigment is occasionally observed in the lamina propria of untreated rodents, notably hamsters, presumably a result of ageing, previous inflammatory processes and haemorrhage. as in other glandular epithelial tissues, hyperplasia may be focal or diffuse with or without atypical cellular features. the term used for hyperplasia with atypical features is atypical hyperplasia in the iarc classification (mohr, ) although others use the term dysplasia. like small intestine, cell proliferation in the large intestinal mucosa can be stimulated by a variety of different factors although these functional adaptive responses have been less well studied. physical stimulation by distension or increased dietary bulk is sufficient to initiate hyperplasia including thickening of the muscle coats (dowling et al., ; stragand and hagemann, ) . one of the most clearly documented forms of compensatory hyperplasia is that which occurs as a response to surgical resection or bypass of a segment of the colon. following resection of a segment of colon in rats, barkla and tutton ( ) showed that the remaining proximal segment of the right side of the colon showed an increase in the thickness of the mucosa and the muscularis externa as well as enlargement of lymphoid aggregates. histologically, the mucosa of the right side of the colon was uniformly thickened showing accentuated folds, elongated mucosal glands with increased height of the surface columnar cells. the changes were most marked up to days following surgery but were less pronounced after days. there was also a significant increase in the mitotic index in the proximal segment at days although at days and later the mitotic index had returned to normal. the distal, down-stream segment showed little or no morphological change but rather a long-lived increase in mitotic activity. it was suggested that these differences were related to the different embryological origin of the segments (barkla and tutton, ) . a similar form of uniform colonic hyperplasia affecting principally the caecal and right-sided colonic mucosa also occurs in rats following oral administration of sulphated dextrans of molecular weight of approximately , (figs and ). oral administration of a wide range of compounds such as raw and chemically modified starches, various dietary fibres, caramels, sugar alcohols (lactitol, sorbitol, mannitol, xylilol), lactose, a synthetic polydextrose, polyethylene glycol and magnesium sulphate to rats or hamsters has also been linked to an increase caecal size and colonic mucosal hyperplasia (leegwater et al., ; roe and bär, ; newberne et al., ; stark et al., ) . the characteristic histological appearance of the caecum following administration of these agents is lengthening of the mucosal glands which are lined by epithelium composed of increased numbers of enlarged epithelial cells (i.e. hypertrophy and hyperplasia) showing increased proliferative activity and more rapid incorporation of tritiated thymidine (newberne et al., ) . in addition, mucosal and submucosal oedema has been reported in association with the administration of lactose and increased mucosal lymphoid aggregates following lactose or xylitol feeding (newberne et al., ) . changes in the colon due to fibre are complex. morphometric analysis has shown that changes to the mucosa depend on the fibre type (stark et al., ) . there may also be an interaction between dietary fibre content and colonic microflora that influences mucosal growth, although the mechanism is unclear (whiteley et al., ) . hypertrophy of the muscularis external is also reported in rats fed high fibre diets (stark et al., ) . as the increase in caecal size and mucosal hypertrophy appears generally related to the osmotic activity of the caecal contents in rodents treated with these agents, it has been postulated that the changes represent a physiological adaptation to increased osmotic forces, irrespective of the contributing compounds (leegwater et al., ) . treatment of rodents with antibiotics also causes caecal enlargement or dilatation without significant histopathological changes, probably as a result of changes in caecal microflora. it has been suggested that the enlargement relates to accumulation of urea as a result of inhibition of bacterial ureases (juhr and ladeburg, ) . however, histochemical studies of the intestinal mucosa of rats treated with neomycin have also shown treatment-related reductions in activities of nad tetrazolium reductase, succinate dehydrogenase, esterase, alkaline and acid phosphatase in the distal ileum, suggesting that some antibiotics also posses the potential to directly influence absorption and metabolic functions of mucosal cells (van leeuwen et al., ) . long-term administration of , -dimethyl prostaglandin e to rats also produced thickening of the proximal colonic mucosa, although this was less fig. . similar area of colonic mucosa to that seen in fig. at the same magnification but from a rat treated with % dextran (molecular weight , ) in the diet for weeks. this shows diffuse hyperplasia of the mucosa with elongation of colonic glands that are lined by relatively normal epithelial cells with abundant mucin and prominent vesicular nuclei. (he, × .) marked than in the stomach and duodenum (reinhart et al., ) and similar changes have been reported in rats treated with other prostaglandin e analogues (levin, ) . as in the small intestine, administration of epidermal growth factor to rats and cynomolgus monkeys induces hyperplasia of the colonic mucosa characterised histologically by hyperplasia and increased mitotic activity of crypt cells and reduction in goblet cell numbers with an increase in crypt depth and a slight increase in the numbers of infiltrating neutrophils (breider et al., ; reindel et al., ) . in common with other epithelial surfaces, atypical hyperplasia is associated with the development of colonic cancer in both man and laboratory animals. the early alterations observed in rats treated with colonic carcinogens are similar to those found in the immediate vicinity of human colorectal carcinomas. the changes are characterised by lengthening, dilatation and branching of glands. the epithelium lining these glands shows mucous cell hyperplasia (goblet cell hyperplasia, see fig. ), goblet cells containing predominantly sialomucin instead of the normal sulphomucin (filipe and branfoot, ; filipe, ; olubuyide et al., ) . despite mucin alterations, activities of glucose- -phosphatase, glucose- -phosphate dehydrogenase and gly- fig. . section from the colon of an aged hamster from a colony that developed intestinal inflammation and neoplasia spontaneously. this shows focal mucous cell hyperplasia characterised by enlargement and lengthening of the colonic glands with lining cells replete with mucins. (he, × .) ceraldehyde- -phosphate dehydrogenase were shown to be normal in this epithelium in rats treated with , -dimethylhydrazine (mayer et al., ) . in man, this form of hyperplastic mucosa associated with cancer, has been termed 'transitional mucosa' (filipe and branfoot, ) . as lesions become more atypical, these dilated, branched glands become more complex and lined by epithelium that shows increasing pseudostratification and vesicular cell nuclei. for example in rats treated with the carcinogens azoxymethane or , -dimethylhydrazine, crypts show diminution of mucus secretion, increased cytoplasmic basophilia, prominent, rounded or enlarged nuclei which show variable degrees of pseudostratification and which eventually develop into frankly invasive glands . in contrast to goblet cell hyperplasia, these atypical zones show increased activity of glucose- -phosphate, glucose- -phosphate dehydrogenase and glyceraldehyde- -phosphate dehydrogenase (mayer et al., ) . described similar alterations in rats treated with azoxymethane and the non-genotoxic agent dextran sulphate. these authors also demonstrated that these atypical foci could be identified by low power microscopy as aberrant crypt foci by translumination of the whole mounts of the fixed mucosa stained with methylene blue. note: some compounds may induce qualitative and quantitative changes in mucin content in the colonic mucosa without marked morphological alterations. an example of this phenomenon was described in rats treated with reserpine for days. the colonic mucosa showed an increase in sulphomucin (high-iron diamine positive) containing goblet cells in the surface epithelium (park et al., ) . adenomas and adenocarcinomas of the small and large intestine are infrequent spontaneous neoplasms in laboratory animals compared with man where colorectal carcinoma is one of the most prevalent neoplasms in the western world. adenomas and adenocarcinomas probably occur spontaneously in older dogs more than in any other animal species and as in man these are located most frequently in the distal colon and rectum (lingeman and garner, ) . in nonhuman primates glandular neoplasms of the intestine occur with increasing age in the ileum and in the colon with a predilection for the zones near the ileocaecal valve (depaoli and mcclure, ) . in rats and mice, spontaneous intestinal neoplasms are uncommon although adenocarcinomas are occasionally observed in the ileum or colon in mice and rats used in chronic toxicity studies and carcinogenicity bioassays (burn et al., ; wells, ; maeda et al., ; greaves and faccini, ; zwicker et al., ) . most of these arise in the small intestine and appear to originate in the distal part of the small intestine, caecum and right side of the colon. they may produce metastases, mostly to liver and lungs. in a review of spontaneous adeno-carcinomas developing over a -year period in wistar rats, vandenberghe et al. ( ) identified adenocarcinomas, all in ascending colon. in of these cases there appeared to be an intimate relationship with campylobacter-like organisms together with diverticulae and chronic inflammation. these authors suggested that the organisms and the associated inflammation were involved in the pathogenesis of these cancers. in view of the importance of colon cancer in humans, a number of new genetic mouse models predisposed to colon cancer have been developed over recent years (heyer et al., ) . some hamster colonies, liable to develop inflammatory bowel disease (see above), also have a high incidence of small and large intestinal polyps, adenomas and adenocarcinomas (fortner, ; van hoosier et al., ; personal observations) . poorly differentiated carcinomas may infiltrate local lymph nodes and it may be difficult to locate the primary neoplasm. polyps are predominantly adenomatous in nature although inflammatory or regenerative polyps are observed (van hoosier et al., ) . adenocarcinomas are induced experimentally in the rodent intestine by the carcinogens , -dimethylhydrazine and azoxymethane. the histogenesis of these induced carcinomas has been extensively studied and it is generally accepted that they resemble human colorectal cancer (ward, ; freeman, ) . however, there are differences between reported studies. some have shown that these experimental carcinomas arise from pre-existing adenomas consistent with the 'adenoma-carcinoma sequence' theory (ward, ; ward et al., ) . others suggest that they arise 'de novo' from altered mucosa as microinvasive carcinomas (sunter et al., ; maskens and dujardin-loits, ; rubio et al., ) . these differences may be partly the result of different dosage schedules. rubio et al. ( ) have shown that a single dose of , -dimethyhydrazine produces non-polypoid, micro-invasive carcinomas, particularly in the mucosa overlying lymphoid aggregates, whereas in their earlier studies using multiple doses in the same strain of rat, an adenoma-carcinoma sequence was more evident. in addition, there are undoubtedly species and strain differences in the response to these agents. teague et al. ( ) demonstrated clear differences in the distribution and both macroscopic and histological types of adenomas and adenocarcinomas between three different inbred strains of rat given a similar dosage regimen of , -dimethylhydrazine. in general, many of these carcinomas develop in the distal colonic segments similar to the distribution of human colorectal cancer, although tumours also develop in the proximal colon and in the ileum in rats treated with this agent (ward, ; teague et al., ) . neoplasms occurring in the rat colon following administration of high doses of degraded carrageenans and sulphated dextran also commonly occur in the distal colon and rectum and are commonly polypoid adenomas and adenocarcinomas (hirono et al., ; oohashi et al., ; ishioka et al., ) . however, in these models adenomas and adenocarcinomas also occur in the caecum and proximal colon and squamous carcinomas are sometimes seen in association with squamous metaplasia at the colorectal junction (oohashi et al., ) . the pathogenesis of neoplasms induced by carrageenans and dextrans remains unexplained. although they are biologically active compounds, they are non-mutagenic in the usual short-term tests (ishioka et al., ) . it has been suggested that carrageenans act as tumour promoters as they potentiate the appearance of carcinomas in rats treated with standard intestinal carcinogens hirono et al., ) . conversely it has been proposed that these agents are tumour initiators based on the development of carcinomas in rats treated with degraded carrageenans for only months (oohashi et al., ) . however, despite only a short period of treatment, inflammation, regenerative changes and squamous metaplasia persisted throughout a period of months after treatment was withdrawn before development of cancer in these rats. the only consistent association of carrageenans with development of carcinoma in rats is that of chronic inflammation and increased cell proliferation. although dose levels needed to produce inflammation are far higher than any exposure likely to be achieved in man, interpretation of this inflammation-cancer sequence in rats remains a challenge in safety assessment for similar xenobiotics. this situation is interest in view of the unquestionable risk of carcinogenesis in ulcerative colitis in man (riddell et al., ) . a similar range of neoplasms can be defined histologically in both human and experimental pathology. it is appropriate, to use the same classification for all species including man. lingeman and garner ( ) who reviewed a range of tumours from domestic and laboratory animals were able to employ the classification for human gastrointestinal neoplasms. a similar approach has been used in the iarc classification of rat intestinal tumours (mohr, ) . this classification can be summarised as follows: these represent localised, sessile or polypoid neoplasms composed of proliferating tubular glands, which show varying degrees of nuclear hyperchromatism, pseudostratification and cellular pleomorphism. a useful scheme for grading the carcinogenic potential of hyperplastic mucosa and adenomatous polyps in man based on the degree of epithelial pseudostratification has been proposed by kozuka ( ) . although experimental neoplasms may not always show the full spectrum of these changes reported in man, this grading provides a useful baseline concept for the assessment of these non-invasive proliferative lesions. with increased nuclear pseudostratification and atypical branching of the glandular structures of these polyps becomes more prominent. if neoplastic cells or glands are seen in the stroma of the stalk or base the diagnosis of carcinoma is made. villous adenoma is a form of adenoma in which the epithelial proliferation takes the form of elongated villi with a sparse fibrovascular stroma. they can be graded in a similar way to other adenomas. these are glandular neoplasms of variable differentiation, sometimes originating in adenomatous polyps or villous adenomas but which show infiltration of the intestinal wall, i.e. beyond the boundary of the muscularis mucosa. squamous carcinomas also occur in the anorectal zone but are similar to those which occur in squamous epithelium elsewhere. similarly, mesenchymal neoplasms also are found in the small and large intestinal wall (see integumentary system, chapter i). light and electron microscopical studies of parietal cells before and one year after proximal vagotomy in duodenal ulcer patients six-month repeated oral toxicity study of nk- in rats response of the non-human primate to polychlorinated biphenyl exposure cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats induction of early lesions in the forestomach of rats by -tert-butyl- -hydroxyamisole (bha) effects of caecetomy in the young adult female rat on digestibility of food offered and libitum and in restricted amounts toxicity of human recombinant interleukin- in rats. pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement effects of cimetidine, cimetidine plus nitrite, and nitrosocimetidine on tumors in mice following transplancental chronic lifetime exposure age-associated lesions in barrier-reared male sprague-dawley rats: a comparison between hap: (sd) and crl:cobs[r] cd[r] (sd) stocks expression of cd in normal and metaplastic paneth cells of the digestive tract an epizootic of klebsiella aerogenes infection in laboratory rats correlation of quantitative changes of gastric mucosal glycoproteins with aspirin-induced gastric damage in rats long-term comparative effect cholecystokinin and gastrin on mouse stomach, antrum, intestine, and exocrine pancreas the effect of -hydro-xydopamine on rat salivary glands and on their response to isoproterenol biologically active peptides in submandibular glands the alimentary system proliferative and morphologic changes in rat colon following bypass surgery cyclosporin and gingival overgrowth the etiology of transmissible murine colonic hyperplasia dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia transmissible murine colonic hyperplasia mouse hepatitis virus infection, intestine, mouse murine rotavirus infection, intestine, mouse adenovirus infection, intestine, mouse clindamycinassociated colitis due to a toxin-producing species of clostridium in hamsters antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia histological variations jejunal and ileal mucosa on days and after hypophysectomy in rat: morphometric analysis in light and electron microscopy comparative study of histological and kinetic variations of the digestive mucosa and pancreatic parenchyma after hypophysectomy in the rat effect of drugs on rats exposed to cold-restraint stress adverse effects of anticonvulsant drugs: a critical review formation of n-mono-nitrosopiperazine in the stomach and its secretion in the urine after oral intake of piperazine intestinal effects of carrageenans in the rhesus monkey (macaca mulatta) the cell surface: components and configurations ménétrier's disease. serial morphological, secretory, and serological observations structure, biosynthesis and functions of glycoprotein glycans. experientia pathology of the forestomach in rats treated for year with a new histamine h -receptor antagonist, sk&f trihydrochloride fundic mucosal ecl cell hyperplasia and carcinoids in rodents following chronic administration of the histamine h -receptor antagonist sk&f and other antisecretory agents gastric ecl-cell hyperplasia and carinoids in rodents following chronic administration of the h antagonist sk&f and oxmetidine and omeprazole gastric regulatory peptides in rats with reduced acid secretion non-steroidal anti-inflammation in humans immunohistologic analysis of the t-cell and macrophage infiltrate in , -dimethylhydrazine-induced colon tumors in the rat turnover of brush-border glycoproteins in human intestinal absorptive cells: do lysosomes have regulatory function? alterations in gastric mucosal morphology induced by long-term treatment with omeprazole in rats the effect of aging on the rat submandibular gland. an ultrastructural, cytochemical and biochemical study drug-induced esophageal strictures cytochrome p of small intestinal epithelial cells. immunocytochemical characterization of the increase in cytochrome p caused by phenobarbital synergistic role of intestinal flagellates and normal intestinal bacteria in a post-weaning mortality of mice medication-induced oesophageal injury. survey of the literature resistance to starvation in albino rats fed from weaning on diets containing from to % of protein as casein diseases of the kidney the human gastrointestinal secretory immune system in health and disease clinical aspects: an overview single-dose and multiple-dose intravenous toxicity studies of bmy- in rats cellular hyperplasia in rats following continuous intravenous infusion of recombinant human epidermal growth factor adrenergic mechanisms responsible for submandibular salivary glandular hypertrophy in the rat aspirin: intestinal damage in rats gastrointestinal mucosal lesions: a drug formulation problem intestinal t lymphocytes of different rats strains in immunotoxicity effects of propionic acid and pravastatin on hmg-coa reductase activity in relation to forestomach lesions in the rat famotidine: summary of preclinical safety assessment effects of cholestyramine and diet on small intestinal histomorphology in rats spontaneous carcinoma of the colon of the rat cresyl fast violet staining method for campylobacter-like organisms pigmentation of the jawbone and teeth secondary to minocycline hydrochloride therapy genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa esophageal lesions caused by orally administered drugs. an experimental study in the cat tetracycline induced esophageal ulcers. a clinical and experimental study diagnosis of silodacryoadenitis virus infection of rats in a virulent enzootic outbreak cryptosporidium species a 'new' human pathogen thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse. a fine structural immunocytochemical study renewal of the epithelium in the descending colon of the mouse. i. presence of three cell populations: vaculated-columnar, mucous and argentaffin two types of mucous cells in the colon crypt origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. iii entero-endocrine cells cytology of the canine oral papilloma uremic gastropathy in the dog intestinal absorption and metabolism of xenobiotics in laboratory animals parasitological review. identification of parasitic metazoa in tissue section spontaneous basophilic hypertrophic foci of the parotid glands in rats and mice effects of housing conditions on food intake, body weight and spontaneous lesions in mice. a review of the literature and results of an -month study cryptosporidiosis in the intestines of rhesus monkeys (macaca mulatta) isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal post marketing surveillance of the safety of cimetidine: mortality during second, third, and fourth years of follow-up hyperplastic gastropathy in the rat due to taenia taeniaeformis infection: parabiotic transfer and hypergastrinaemia. gastroenterology number, size and distribution of peyer's patches in the human small intestine a model for gastric cancer epidemiology helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention the effects of vagotomy on the gastric mucosa of the rat the effect of prolonged administration of large doses of cimetidine on the gastric mucosa of rats effect of short-and long-term feeding of omeprazole on rat gastric endocrine cells clostridial enterocolitis produced by antineoplastic agents in hamsters and humans odontogenic tumours in fischer rats the histo-chemical demonstration of o-acylated sialic acid in gastrointestinal mucins: their association with the potassium hydroxide-periodic acid-schiff effect a new histochemical method for the identification and visualization of both side chain acylated and non-acylated sialic acids amiodarone keratopathy, drug-induced lipid storage disease salivary gland components involved in the formation of squamous metaplasia gastric mucosal injury by fatty and acetylsalicylic acids cryptosporidosis and proliferative ileitis in a hamster specificity of twelve lectins towards oligosaccharides and glycopeptides related to n-glycosylproteins intestinal permeability changes in rodents. a possible mechanism for degraded carageenan-induced colitis adrenal corticosteroids cause gastrin cell hyperplasia possible role of transforming growth factor alpha in the pathogenesis of menetrier's disease: supporting evidence from humans and transgenic mice the effect of hydrocortisone and cortisone on fixation of s in the stomach the effect of phenylbutazone and its derivatives, oxyphenbutazone and sulfinpyrazole, on s sulfate incorporation in cartilage and stomach salivary glands: a paradigm for diversity of gland development gastrointestinal neoplasms in non-human primates: a review and report of new cases gastric gland degeneration induced in monkeys by the cck-b/gastrin receptor antagonist ci- the effect of aspirin on small intestinal mucosa morphologic aspects of lipid absorption gastric and gastric epithelial physiology two-year evaluation of misprostol for carcinogenicity in cd sprague-dawley rats distribution and incidences of calcified lesions in dba/ ncrj and balb/canncrj mice interaction of phenytoin and inflammation induces gingival overgrowth in rats the intestinal response to high bulk feeding in the rat comparison of canine and human gastrointestinal physiology stress ulceration-clinical relevance of animal and human studies pathology of laboratory rats and mice effect of chronic aspirin ingestion on epithelial proliferation in rat fundus, antrum and duodenum the prognostic value of sulphomucin positive intestinal metaplasia in the the development of gastric cancer colitis in mice with high incidence of rectal prolapse volatile nitrosamine contamination of laboratory animal diets toxicological studies on omeprazole possible role of cimetidine and its nitrostated products in human stomach cancer cimetidine and gastric cancer tumours of the oral cavity, check pouch, salivary glands, oesophagus, stomach and intestines differential distribution of lymphocytes and accessory cells in mouse peyer's patches phenotypically distinct subpopulations of t cells in domes and m-cell pockets of rabbit gut-associated lymphoid tissues morphometric analysis of the small intestinal epithelium in the indomethacin-treated mouse expression of pokeweed lectin binding in murine intestinal paneth cells heterogeneity of m-cell-associated b and t cells in human payer's patches degraded carrageenan-induced colitis in cf mice. a clinical, histo-pathological and kinetic analysis substituted benzimidazoles inhibit acid secretion by blocking (h ++ k + ) atpase nonsteroidal anti-inflammatory drug induced jejunal and colonic diaphragm disease: a report of two cases effect of chronic misoprostol ingestion on rat gastric morphology and turnover abnormal patterns of mucous secretion in apparently normal mucosa of large intestine with carcinoma mucous secretion in rat colonic mucosa during carcinogenesis induced by dimethylhydrazine. a morphological and histo-chemical study mucins in the human gastrointestinal epithelium: a review. invest high-dose pancreatic-enzyme supplements and fibrosing colonopathy in children with cystic fibrosis the number of villi in rat's jejunum and ileum: effect of normal growth, partial enterectomy and tube feeding spontaneous tumors including gastrointestinal neoplasms and malignant melanoma, in syrian hamster aspirin, paracetamol and non-steroidal anti-inflammatory drugs. a comparative review of side effects campylobacter jejuni/coli in commercially reared beagles. prevalance and serotypes antigen specificity and morphological characteristics of chlamydia trachomatis, strain sfpd, isolated from hamsters with proliferative ileitis acquired salivary dysfunction. drugs and radiation gastritis cystica profunda application of lectins for detection of goblet cell glycoconjugate differences in proximal and distal colon of the rat lectin histochemistry of , -dimethylhydrazine-induced rat colon neoplasia effects of puromycin on the structure of rat intestinal epithelial cells during fat absorption morphological aspects on pancreatic islets of non-obese diabetic (nod) mice carcinoma and related lesions in dog stomach induced by oral administration of n-methyl-n´-nitro-n-nitrosoguanidine cryptosporidiosis in a pup with distemper. vet.pathol squamous cell carcinoma, forestomach, rat adverse effects of mouthwash use. a review. oral surg.oral med.oral pathol tyzzer's disease, intestine, mouse, rat, hamster salmonellosis, intestine, mouse, rat, hamster adequate substitution with electrolytes in toxicological testing of 'loop' diuretics in the dog the forestomach in rats and mice, a food store without bacterial protein digestion m cells in peyer's patches of the intestine histochemie de la muqueuse gastrique fundique du chien traité par des drogues ulcérigène an electron-microscope and histo-chemical study of melanoisis coli mitochondria. in: ultrastructural pathology of the cell and matrix sustainability of forestomach hyperplasia treated with ethyl acrylate for weeks and regression after cessation of dosing the cytochemical localization of lysozyme in paneth cell granules regional differences in glycoconjugates of intestinal m cells in mice: potential targets for mucosa vaccines susceptibilities of drug to nitrosation under simulated gastric conditions features of small intestinal pathology (epithelial cell kinetics, intra-epithelial lymphocytes, disaccharidases) in a primary giardia muris infection staining rickettsiae in yolk sac cultures alterations of gastric mucosa following a graded partial gastrectomy tumours of the salivary glands the oral cavity aristolochic acid is a direct mutagen in s. typhimurim studies of the binding of trypsin and chymotrypsin by human intestinal mucosa study of cold plus restraint stress gastric lesions in spontaneously hypertensive, wistar and sprague-dawley rats mucins in normal and neoplastic gastrointestinal epithelium the lectins: carbohydrate-binding proteins of plants and animals prolactin and ergocryptine effects mucus glycoproteins of the rat ileum tumours of the jaws hypopigmentary changes with a platelet aggregation inhibitor (abstract no. ). fed.proc drug induced enteropathy characterized by lipid in macrophages altered patterns of mucin secretion in gastric hyperplasia in mice digestive system. in: rat histopathology. a glossary for use in toxicity and carcinogenicity studies k virus infection, intestinem mouse a silver nitrate stain for alpha- cells in human pancratic islets silver stains in the study of endocrine cells of the gut and pancreas effects of antrectomy or porta-aval shunting on the histamine-storing endocrine-like cells in oxyntic mucosa of rat stomach. a fluorescence histochemical, electron microscopic and chemical study the vagus exerts trophic control of the stomach in the rat activation and hyperplasia of gastrin and enterochromaffin-like cells in the stomach gastrin and the trophic control of gastric mucosa onkocytes and so-called hürthle cell tumor interaction of microorganisms, epithelium, and lymphoid cells of the mucosa-associated lymphoid tissue dog and swine as models for testing indomethacin-induced gastrointestinal irritation sialodacryoadenitis in the rat: effects of immunosuppression on the course of the disease role of gut in zenobiotic metabolism epithelial cell kinetics in the small intestine of the rat days after resection of percent of the ileum and jejunum compensation by the residual intestine after intestinal resection in the rat proceedings of the lst international symposium on omeprazole a cecal diaphragm associated with the use of nonsteroidal anti-inflammatory drugs chronic gastritis of the glandular stomach, adenomatous polyps of the duodenum, and calcareous pericarditis in strain dba mice idiopathic megaoesophagus in rat immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment spontaneous neoplasm incidences in fischer rats and b c f mice in two-year carcinogenicity studies: a national toxicology program update diphenyldydantoin (dilantin) gingival hyperplasia: drug-induced abnormality of connective tissue on cell proliferation and differentiation of the fundic mucosa of the golden hamster. fractographic study combines microscopy and h-thymidine autoradiography tritiated thymidine autoradiographic study on cellular migration in the gastric gland of the golden hamster enterochromaffin-like cell carcinoids of gastric mucosa in rats after life long inhibition of gastric secretion gastric cancer after cimetidine in a patient with two negative pre-treatment biopsies induction of experimental allergi sialadenitis in mice spontaneous development of auto-immune sialodenitis in aging bdf mice i mmunofluorescent localization of enterokinase in human small intestine mouse models for colorectal cancer morphologic changes in the urinary bladder and stomach after long-term administration of sodium saccharin in f rats lessons from genetically engineered animal models iii. lessons learned from gastrin gene deletion in mice immunohistological characterization of intra-epithelial and lamina propria lymphocytes in control ileum and colon and inflammatory bowel disease induction of intestinal tumors in rats by dextran sulphate sodium gastric enterochromaffin-like hyperplasia and neoplasia in the rat: an indirect effect of the histamine h -receptor antagonist bl- oxidative metabolism of foreign compounds in rats small intestine: cellular localization and dependence on dietary iron clinicopathological studies of gastrointestinal disease in macaques non-tuberculous myobacterial disease in rhesus monkeys on human papillomaviruses the laboratory rat. biology, diseases inhibition of intestinal protein synthesis and lipid transport by ethionine experimental toxicity studies with captopril, an inhibitor of angiotesin -converting enzymes . one month studies of chronic toxicity of captopril in rats development of spontaneous tongue calcification and polypoid lesions in dba/ ncrj mice changes in enzyme levels accompanying differentiation of intestinal epithelial cells textbook of endocrinology acute disease of the submaxillary and harderian glands (sialodacryoadenitis) of rats with cytomegaly and no inclusion bodies cellular kinetics of gastrointestinal mucosa, with special reference of gut endocrinecells changes of gastric mucus glycoproteins with aspirin administration in rats induction of colorectal tumours in rats by sulpated polysaccharides induction of duodenal tumors in mice by oral administration of hydrogen peroxide carcinogencity of butylated hydroxyanisole in f rats modifying effects anti-oxidants on chemical carcinogenesis a week feeding study of butylated hydroxyanisole: the subsequent regression of the induced lesions in male fischer rat forestomach epithelium an -day study of butylated ydroxyanisole in the cynomolgus monkey subchronic studies of doxylamine in fischer rats effects of dietary fiber on mucosal growth and cell proliferation in the small intestine of the rat: a comparison of oat, bran, pectin, and guar with total fiber deprevation transmissible ileal hyperplasia, hamster anatomic adaption of the alimentary tract of the rat to the hyperphagia of chronic alloxan-diabetes a variant of intestinal metaplasia associated with gastric carcinoma: a histochemical study role of intestinal metaplasia in the histogenesis of gastric carcinoma colorectal mucin histochemistry in health and disease: a critical review uptake of particulate and soluble antigens in the small intestines of the rat chemical colitis due to endoscopic cleaning solutions: a mimic of pseudomembranous colitis pathology of domestic animals intestinal accumulation of urea in germ-free animals: a factor in caecal enlargement digestive enzymes in the parotid and submandibular glands of mammals morphologishe veränderungen der magenmukosa von ratten nach chronischer antazidagabe enteric viruses of non human primates increased accumulation of sulfated glycoaminoglycans in cultures of human fibroblasts from phenytoin-induced gingival overgrowth new insights into the stem cells and the precursors of gastric epithelium colonic lymphoid-glandular complex (microbursa): nature and morphology lymphoid tissue and lymphoid-glandular complexes of the colon: relation to diverticulosis histology of salivary gland infarction in the dog adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse pill esophagitis immunogold localization of ingested kidney bean (phaseolus vugaris) lectins in epithelial cells of the rat small intestine epithelial dysplasia of the rabbit colon induced by degraded carrageenan hyperkeratinization and hyperplasia of the forestomach epithelium in vitamin a deficient rats intrinsic resistance of colon tumors to anthrapyrazoles and antracyclines may be linked with a detoxification mechanism of intestinal cells (abstract no. ) studies on the effects of -hydroxy- -methylglutaryl coenzyme a reductase inhibitors on the rodent forestomach reversibility of adenomatous hyperplasia in the gastric stump after diversion of bile reflux in rats experimental production of possible autoimmune gastritis followed by macrocytic anemia in athymic mice expression cloning and characterization of the canine parietal cell gastrin receptor histomorphologic investigations on the effect of cyclophosphamide on dentinogenesis of the rat incisor. scand preclinical toxicology profile of misoprostol premalignancy of the mucosal polyp in the large intestine: i. histologic gradation of the polyp on the basis of epithelial pseudostratification and glandular branching morphologic changes in the gastric mucosa of rats and dogs treated with an analog of prostaglandin e forestomach carcinogens: possible mechanisms of action lectin-peroxidase conjugates in histopathology of gastrointestinal mucosa the rat incisor in toxicologic pathology the pharmacology and toxicology of caffeine incorporation of radiosulfate in the gastric mucosa of the rat subjected to restraint antisecretory drugs and gastric cancer plasma gastrin and gastric enterochromaffin-like cell activation and proliferation. studies with omeprazole and ranitidine in intact and antrectomized rats effects of cyclosporin a administration in cats gingival hyperplasia associated with nifedipine therapy the aetiology of caecal enlargement in the rat effect de l'administration prolongée d'un antisécrétoire gastrique, le pirenzepin, sur les populations cellulaires de l'estomac de rat hepatocytes in the mouse stomach tumours of the oral cavity, pharynx, oesophagus and stomach a toxicological profile of cimetidine specific staining of sulphate groups with alcian blue at low ph the mucin histochemistry of normal and neoplastic gastric mucosa the effect of the thyroid gland on intestinal absorption of hexoses structural changes of the gastrointestinal mucosa induced by prostaglandins structural aspects of salivary glycoproteins drug-induced disorders of the esophagus histochemical observations on paneth cells diphtheria toxin-mediated ablation of parietal cells in the stomach of transgenic mice carcinogencity of methylated dinitro-sopiperazines in rats comparative study of intestinal adenocarcinoma of animals and man dental dysplasia in rats and mice chronic oral administration of -nitrosopiperazine at high doses to mrc rats nifedipine-induced gingival hyperplasia: a histochemical and ultrastructural study drug-induced lysosomal storage disorders oesophagostomiasis in feral monkeys (macaca mulatta) hyperalimentation in normal animals produced by protamine insulin effects of cytotoxic chemotherapy on dental development nutritional influences on aging of fischer rats: ii the use of rats associated with human faecal flora as a model for studying the effects of diet on the human gut microflora colonic ulceration in young rats fed degraded carrageenan unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration gut-associated lymphoid tissue and , -demethylhydrazine intestinal tumors in the rat: a histological and immunoenzymatic study leucocyte and bone marrow effects of a thiomorpholine quninazosin antihypertensive agent localization of epidermal growth factor receptors in cells of the enamel organ of the rat incisor effect of dosage form and formulation factors on the adherence of drugs to the esophagus morphologic changes of esophageal mucosa in the rat after chronic alcohol ingestion experimental adenomas and carcinomas of the large intestine behave as distinct entities: most carcinomas arise de novo in flat mucosa la glande endocrine de l'intestin chez l'homme differentiation of immature mucous cells into parietal, argryophil, and chief cells in stomach grafs sequential histochemical and morphometric studies on preneoplastic and neoplastic lesions induced in rat colon by , -dimethylhydrazine recovery from amiodarone-induced lipidosis in laboratory animals: a toxicological study parietal cell hyperplasia indued by long-term administration of antacids to rats methotrexate-induced changes in rat parotid and submandibular gland function a spontaneous outbreak of polychlorinated biphenyl (pcb) toxicity in rhesus monkeys (macaca mulatta): toxicopathology classical absorption theory and the development of gastric mucosal damage associated with non-steroidal anti-inflammatory drugs mesenteric lymphoblast localization throughout the murine small intestine: temporal analysis relating intestinal length and lymphoblast division morphologic lesions in ageing syrian hamsters histological identification of campylobacter using gimenez technique in gastric antral mucosal the carcinogenic action of aristolochic acid in rats rat immunoglobulins in serum and secretions: comparison of iga and igg in serum, colostrum, milk and saliva of protein malnourished and normal rats influence of ofloxacin on the faecal flora the structure, origin and function of mucosal mast cells. a brief review pseudomembranous colitis in a leukaemia unit: a report of five fatal cases formulation of n-nitroso compounds: chemistry kinetics, and in vivo occurrence the etiology of gastric cancer: intragastric nitro-samide formation and other theories forestomach lesions induced by butylated hydroxyanisole and ethylene dibromide: a scientific and regulatory perspective international classification of rodent tumours. part , the rat the 'swiss roll'. a simple technique for histological studies of the rodent intestine organization of an actin filament-membrane attachment in the microvilli of intestinal epithelial cells charaterization and localization of myosin in the brush border of intestinal epithelial cells development of the human gastrointestinal tract: twenty years of progress aroclor -induced intestinal metaplasia and adenocarcinoma in the glandular stomach of f rats immuno-histochemical localization of epidermal growth factor in rodent submandibular glands intestinal metaplasia of the gastric mucosa precancerous conditions and epithelial dysplasia in the stomach hypersensitivity reactions in the small intestine. . pathogenesis of the graft-versus-host reaction in the small intestinal mucosa of the mouse the distribution of acid mucopoly-saccharides in normal kidneys as shown by the alcian blue feulgan (ab-f) and alcian blue-periodic acid-schiff (ab-pas) stains gastric endocrine cell hyperplasia and carcinoid tumors in atrophic gastritis type a histochemical classification of acini and ducts of parotid glands from artiodactyles, carnivores and rodents a morphological and histrochemical study of a drug-induced enteropathy in the alderley park rat the mouse submandibular gland: an exocrine organ for growth factors dysplasia of the gastric mucosa and its relation to the precancerous state ultrastructure of dog parotid gland corticosteroid treatment increases parasite numbers in murine giardiasis duct-associated lymphoid tissue (dalt) of minor salivary glands and mucosal immunity minor salivary gland duct-associated lymphoid tissues (dalt) in monkeys, changes with age quantitative light and electron microscopic studies on the effect of vagotomy on parietal cells in rats toxicology and carcinogenesis studies of ampicillin trihydrate in f /n rats and b c f mice animal models for gastric helicobacter immunology and vaccine studies fundal gastritis after billroth-ii type resection in patients with duodenal ulcer mesovarial leiomyomas in rats in a chronic toxicity study of musuprine hydrochloride current concepts in mucosal immunity v. role of m cells in transepithelial transport of antigens and pathogens to the mucosal immune system the influence of food additives and related materials on lower bowel structure and function the interactions of lectins with animal cell surfaces immunohistochemical localization of carbonic anhydrase in submandibular salivary glands of mice and hamsters treated with phenylephrine, testosterone or duct ligation tumours of the oral cavity, pharynx, oesophagus and stomach distribution of glutathione and its related enzymes in small intestinal mucosa of rats the ulcerogenic effect on the oesophagus of three β-adrenoceptor antagonists, investigated in a new porcine oesophagus test model goblet cell hyperplasia is a feature of the adaptive response to jejunoileal bypass in rats a study on carcinogenesis induced by degraded carrageenan arising from squamous metaplasia of the rat coloretum differences in thegastro-intestinal mirobiota of specific pathogen free mice: an often unknown variable in biomedical research paneth cells and innate immunity in the crypt microenvironment. gastroenterology mucosal immunity and inflammation iv. paneth cell antimicrobial peptides and the biology of the mucosal barrier entero-hepatic cycling in rats plays a major role in fatal nsaid intestinal ulcerogenicity sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of peyer's patches in the normal unobstructed mouse intestine: an ultrastructural study antigen processing structures of the mammalian intestinal tract: an sem study of lymphoepithelial organs cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse peyer's patch m cells predicting anticancer drug effects in man from laboratory animal studies the anatomical basis for the immune function of the gut morphological and histochemical changes in intestinal mucosa in the reserpine-treated rat model of cystic fibrosis the pericryptal fibroblast sheath. iv. replication, migration and differentiation of the subepithelial fibroblasts of the crypts and villus of the rabbit jejunum comparative analysis of the s rna gene sequence of the putative agent of proliferative ileitis of hamsters histochemistry: theoretical and applied h -receptor antagonists and gastric cancer diagnostic exercise: inter-mandibular swelling in rats depletion of salivary gland epidermal growth factor by silodacryoadenitis virus infection in the wistar rat biology and biochemistry of papillomaviruses lescol (fluvastatin sodium) the cytology of salivary glands salivary gland sexual dimorphism: a brief review enzyme histochemistry of the human stomach with special reference to intestinal metaplasia nitrates and gastric cancer mucosal immunity and inflammation v. innate mechanisms of mucosal defense and repair: the best offense is the best defense derivation of mouse intestinal rypts from single progenitor cells twenty-one month evaluation of misoprostol for carcinogenicity in cd- mice association of long lasting unsurmountable histamine h blockade and gastric carcinoid tumours in the rat changes in the gastric mucosa of the mouse associated with long lasting unsurmountable histamine h blockade pharmacological and toxicological effects of chronic porcine growth hormone administration in dogs campylobacter jejuni enteritis in man and domestic animals is ethanol-induced damage of the gastric muosa a hyperosmotic effect? comparative studies on the effects of ethanol, some other hypperosmotic solutions and acetyl-salicylic acid on rat gastric mucosa synergistic interaction between aspirin, or other non-steroidal antiinflammatory drugs, and stress which produces severe gastric mucosal damage in rats and pigs the effects of aspirin and other non-steroid anti-inflammatory/analgesic drugs on gastrointestinal mucus glycoprotein biosynthesis in vivo: relationship to ulcerogenic actions electron microscopic observations comparing the gastric mucosal damage induced in rats and pigs by benoxaprofen and aspirin, reflecting their differing actions as prostaglandin-synthesis-inhibitors megaoesophagus in icrc mice compylobacter-like organisms in the gastric mucosa of rhesus monkeys gastric cancer in patients who have taken cimetidine effect of cimetidine on gastric juice n-nitrosamine concentration cryptosporidium cuniculus in the rabbit clostridium difficile colitis in a rabbit following antibiotic therapy for pasteurellosis clostridium difficile antitoxin neutralization of cecal toxin(s) from guinea pigs with penicillin-associated colitis clostridium difficile typhlitis in hamsters not associated with antibiotic therapy clostridial enteropathies, hamster spontaneous non-neoplastic gastric lesions in female han: nmri mice, and influence of food restriction throughout life saponification-induced increase in the periodic-acid-schiff reaction in the gastrointestinal tract. mechanism and distribution of reactive substance structure and carbohydrate histo-chemistry of postnatal canine salivary glands recombinant human epidermal growth factor - -induced structural changes in the digestive tract of cynomolgus monkeys (macaca fascicularis) influence of long-term , -dimethyl prostaglandin e treatment on the rat gastrointestinal mucosa digestion and absorption of carbohydrate and nitrogeneous matter in hindgut of the omnivorous non-reminant animal salivary glands in longterm alloxan-diabetic rats. a quantitative light and electron-microscopic study digestive system: intestines. in: histology. a text and atlas helicobacter pylori-infected human antral primary cell cultures: effect on gastrin cell function the gastrointestinal tract dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications histochemical study on the paneth cell in the rat ultrastructural observations on the pathogenesis of aspirin-induced gastric erosions dietary related periodontitis and oro-nasal fistulation in rats abnormal skeletal and dental growth in epileptic children tooth root resorption induced in rats by diphenylhydantoin and parathyroidecotomy light microscopic morphometric analysis of rat ileal mucosa. i. component quantitation if iga-containing immunocytes short-term effects of various phenols and acids on the fischer male forestomach epithelium enzootic and epizootic adrenal medullary proliferative diseases of rats: influence of dietary factors which affect calcium absorption campylobacter pylori, gastritis, and peptic ulcer disease in central africa the effect of diet on the mammalian gut flora and its metabolic activities interactions of the gut microflora and the host in toxicology naturally occurring tumors and other lesions of the digestive tract in untreated c bl mice pathology of tumours in laboratory animals. tumours of the mouse esophageal impaction in the bhe rats minimal invasive carcinoma of the colon in rats early effects of alloxan on rat submandibular gland cyclic motor activity; migrating motor complex age-related changes of rat submandibular gland: a morphometric and ultrastructural study indomethacin produces gastric antral ulcers in the refed rat quantitative electron microscopic observations on paneth cells of germ-free and ex-germ-free wistar rats immunohistochemical observations of immunoglobin a in the paneth cells of germ-free and formerly-germ-free rats pigmentary changes in the rat oral mucosa following antimalaria therapy monoclonal antibodies to a zinc-binding protein of rat paneth cells age, weight and social effects on ulceration in rats the evaluation of anticancer drugs in dogs and monkeys for the prediction of qualitative toxicities in man gingival fibromatosis, macaca mulatta identification and mutagenicity of metabolites of aristolochic acid formed by rat liver melanosis coli: a study with the electron microscope light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. i. mouse light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates genetic and sex-related differences in the structure of subnmandibular glycoconjugates mucosal microhernias in the nonhuman primate colon: their role in the pathogenesis of colonic diseases quantitative age changes in the histological structure of human submandibular salivary glands immunohistological characterization of intra-epithelial lymphocytes of the human gastrointestinal tract chronic duodenal ulcers in pantothenate deficient mice focal avillous hyperplasia of the mouse duodenum colon epithelium. i. light microscopic, histochemical, and ultrastructural features of normal colon epithelium of male fischer rats colon epithelium. ii. in vivo studies of colon carcinogenesis. light microscopic, histrochemical, and ultrastructural studies of histogenesis of azoxymethane-induced colon carcinomas in fischer rats two types of mucous cells in the colon crypt carrageenan ulceration as a model for human ulcerative colitis. lancet, ii comparative histochemistry of gastro-intestinal mucosubstances assessment of risk of formulation of carcinogenic n-nitroso compounds from dietary precursors in the stomach granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study marked epithelial hyperplasia of the rat glandular stomach induced by long-term administration of iodoacetamide a profile of the gastrointestinal toxicity of drugs used to treat inflammatory diseases morphology and cyto-chemistry of rat salivary gland acinar secretory granules and their alteration by isoproterenol. i. parotid gland effects of sodium salicylate on epithelial cells of the rectal mucosa of the rat: a light and electron microscopic study the effects of long-term propranolol on the salivary glands and intestinal mucosa of the mouse effects of cardiotonic phosphodiesterase inhibitors on rat salivary gland. presented at the th annual acvp meeting inhibitors of sterol synthesis. morphological studies in rats after dietary administration, administration of α-cholest- ( )-en- β-ol- -one, a potent hypocholesterolemic compound m cell numbers increase after transfer of spf mice to a normal animal house environment strictures of ascending colon in cystic fibrosis and high-strength pancreatic enzymes pathology of laboratory rats and mice anatomy of the cecum of the laboratory mouse and rat gastric carcinoids and related endocrine growths pathogenesis of peptic ulcer and implications for therapy histochemical distribution of lysozyme activity in organs of normal mice and radiation chimeras human peyer's patches: an immunohistochemical study diamine methods for differentiating mucosubstances histochemically metabolic and morphometric changes in small and large intestine in rats fed high-fiber diets melanosis coli: studies of the toxic effects of irritant purgatives surface morphology of the gastroduodenal mucosa in duodenal ulceration the effect of vincristine on dentino-genesis in the rat incisor. scand autoradiographic investigation of dentine production in rats incisosrs after vincristine administration. scand acute and late radiation injury in rhesus monkey parotid glands. evidence of interphase death unique radiosensitivity of serous cells in rhesus monkey submandibular glands pathologic observations on the adenomatous lesions of the stomach in mice of strain i carcinoma of the glandular stomach of rats ingesting n,n´ ulcerative enterocolitis in dogs induced by drugs aspirininduced glandular dysplasia of the stomach. histologic and histochemical studies in rats effect of lumenal contents on colonic cell replacement pathologic findings in the stomach of rats treated with the h -receptor antagonist tiotidine morphologic stomach findings in rats and mice treated with the h -receptor antagonists, ici and ici lectin-peroxidase reactivity in rat gastric mucosa tumour production in glandular stomach of rat by nmethyl-n´nitro-n-nitrosoguanidine ageing affects the drug metabolism systems of rat liver, kidney, colon and lung in a differential fashion oral papillomatosis in new zealand white rabbits diagnostic exercise: lingual growths in rabbits immunoperoxidase localization of papillomaviruses in hyperplastic and neoplastic epithelial lesions of animals hypergastrinemia after blockade of acid secretion in the rat. trophic effects pathological features of the colonic tumors induced in rats by the administration of , -dimethylhydrazine comparative studies on the gastrointestinal lesions caused by serveral non-steroidal anti-inflammatory agents in the rats cells intermediate between mucous neck cells and chief cells in rat stomach fine structure of giant hypertrophic gastritis developed in thymectomized mice hyperplastic gingivitis in a child receiving sodium valproate treatment dopamine disorderin dodenal ulceration biology of disease. pathogenesis of duodenal ulcer disease from cysteamine to mptp: structure-activity studies with duodenal ulcerogens digestive system. monographs on pathology of laboratory animals role of salivary mucins in the protection of the oral cavity effects of , -dimethyl prostaglandin e -methyl ester on aspirin-and indomethacin-induced gastrointestinal lesions in dogs hypertrophic gastropathy resembling menetrier's disease in transgenic mice overexpressing transforming growth factor α in the stomach tumours of the oral cavity, buccal pouch, oesophagus, forestomach and salivary glands nodular hyperplasia of oncocytes in mouse submandibular glands early colonic lesions in experimental shigella infections in rhesus monkeys: revisited variation in susceptibility to the induction of forestomach tumours by butylated hydroxyanisole among rats of different strains pathology of neoplasia and preneoplasia in rodents independent induction of intestinal metaplasia and gastric cancer in rats treated with n-methyl-n´-nitro-n-nitrosoguanidine the response of three inbred strains of rat to the carcinogen , -dimethylhydrazine small intestinal type' and 'colonic type' intestinal metaplasia of the human stomach and their relationship to the histogenetic types of gastric adenocarcinoma light and electron microscopic cytochemistry of glycoconjugates in the recto-sigmoid colonic epithelim of the mouse and rat preclinical toxicologic evaluation of bleomycin (nsc ), a new anti-tumor antibiotic oral squamous cell carcinoma in ad libitum-fed and food restricted brown-norway rats the pathoparasitology of the alimentary tract and pancreas of non-human primates: a review t cell attractant chemokine expression initiates lacrimal gland destruction in nonobese diabetic mice altered patterns of mucin secretion in the precancerous lesions induced in the glandular part of the rat stomach by the carcinogen n-methyl-n´nitro-n-nitrosogaunidine immunohistochemistry and radioimmunoassay of egf in submanidbular glands of mice treated with secretogogues carcinogenicity of cyproterone acetate preclinical toxicology studies with acylovir: acute and sub-chronic tests the influence of adrenoreceptor activity on crypt cell proliferation in rat jejunum a sporozoan found in the peptic glands of the common mouse cyclophosphamide-induced abnormalities in the incisors of the rat spontaneous adencarcinoma of the ascending colon in wistar rats: the intracytoplasmic presence of a campylobacter-like bacterium inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs spontaneous hyperplastic and metaplastic duct epithelium in the sublingual salivary glands of wistar rats spontaneous tuors of the syrian hamster: observations in a closed breeding colony and a review of the literature mucin gene structure and expression: protection vs. adhesion morphological effects of high dose neomycin sulphate on the small and large intestine comparative and experimental pathology of fibrosing colonopathy intestinal pathology in the dog induced by sublethal doses of amiodarone pharmacological effects of epidermal growth factor (egf) with focus on the urinary and gastrointestinal tracts chemically induced lipidosis of the small intestinal villi in the rat lesions of experimentally induced tyzzer's disease in syrian hamsters, guinea pigs, mice and rats hexamitis in laboratory mice, hamsters, and rats melanosis coli: a consequence of anthraquinone-induced apoptosis of colonic epithelial cells safety evaluation of cimetidine: report at the termination of a seven-year study in dogs hypopigmentation in dogs treated with an inhibitor of platelet aggregation gingival hyperplasia in dogs induced by oxodipine, a calcium channel blocking agent cholecystokinin receptors gastrointestinal ph measurement in rats: influence of microbial flora, diet and fasting morphogenesis of chemically induced neoplasms of the colon and small intestine in rats natural history of intestinal neoplasms induced in rats by a single injection of methyl (acetoxymethyl) nitrosamine proliferative lesions of the glandular stomach and liver in f rats fed diets containing aroclor experimentally induced intestinal metaplasia in wistar rats by x-ray irradition. gastroenterolgy effect of dietary undegraded carrageenan on colon carcinogenesis in f treated with azoxymethane or methyl-nitrosourea induction of intestinal metaplasia in the rat gastric mucosa by local x-irradiation the effect of sex difference on induction of intestinal metaplasia in rats distribution of the histaminergic neuron system in the central nervous system of rats; a fluorescent immunohistochemical analysis with histidine decarboylase as a marker replacement of functional parenchymal cells by fat and connective tissue in human submandibular salivary glands. an age related change canine papilloma: progression of oral papilloma to carcinoma bacterial and mycotic disease dental health of survivors of malignant disease analysis of protein synthesis in rat salivary glands after chronic treatment with β-receptor agonists and phosphodiesterase inhibitors mucinous carcinoma of the ileum in the rat mucin histochemistry of gastric intestinal metaplasia long-term effects of an inotropic phosphodiesterase inhibitor (ici , ) on rat salivary gland, hardarian gland and intestinal mucosa the synovial membrane, liver, and tongue: target organs for a ricin a-chain immunotoxicin (zd ) ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains quantitation of glandular gastric changes in rats given a proton pump inhibitor for months with emphasis on sampling scheme selection aberrant crypt foci in the colonic mucosa of rats treated with genotoxic and nongenotoxic colon carcinogen the interactions of diet and colonic microflora regulating colonic mucosal growth selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis, and the gastro-intestinal damage iinduced by indomethacin in the rat a biochemical basis for the gastro-intestinal toxicity of non-steroid antirheumatoid drugs butylated hydroxianisole mechanistic data and risk assessment: conditional species-specific cytotoxicity, enhanced cell proliferation, and tumor promotion menetrier's disease presenting as iron deficiency anaemia radioautographic and quantitative studies on parietal and peptic cell kinetics in the mouse: a selective effect of gastrin on parietal cell proliferation intestinal adaptation. structural, functionaland cytokinetic changes instestinal adaptation. mechanisms of control mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties with special reference to protein blocking and solubility of the granular glycosaminoglycan effect of mixtures of dietary fibres on the enzyme activity of the rat caecal microflora effect of prolonged metiamide medication on the fundic mucosa the membraneous epithelial (m) cell and the mucosal immune system medical progress: gastrointestinal toxicity of nonsteroidal antiinflammatory drugs nitrosatable drugs: an assessment of the risks assessing the safety of drugs for the long-term treatment of peptic ulcers the mechanism of mitrazepam-induced drooling and aspiration blood vessels of the peyer's patch in the mouse. iii high endothelial venules ultrastructural basis of intestinal absorption the effects of omeprazole and famotidine on mucin and pge release in the rat stomach forestomach ulcers in crj:b c (c bl/ ncrj × c h/hencrj) f mice forestomach ulcers in crj:b c (c bl/ ncrj × c h/hencrj) f mice an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs drug-induced gastro-intestinal disease naturally occurring intestinal neoplasms in aged crl:cd ® br rats key: cord- -rprrkr authors: ahmad, md faruque; ahmad, fakhruddin ali; ashraf, syed amir; saad, hisham h; wahab, shadma; khan, mohammed idreesh; ali, maksood; mohan, syam; hakeem, khalid rehman; athar, md tanwir title: an updated knowledge of black seed (nigella sativa linn): review of phytochemical constituents and pharmacological properties date: - - journal: j herb med doi: . /j.hermed. . sha: doc_id: cord_uid: rprrkr n. sativa (n. sativa) has been used since ancient times, when a scientific concept about the use of medicinal plants for the treatment of human illnesses and alleviation of their sufferings was yet to be developed. it has a strong religious significance as it is mentioned in the religious books of islam and christianity. in addition to its historical and religious significance, it is also mentioned in ancient medicine. it is widely used in traditional systems of medicine for a number of diseases including asthma, fever, bronchitis, cough, chest congestion, dizziness, paralysis, chronic headache, back pain and inflammation. the importance of this plant led the scientific community to carry out extensive phytochemical and biological investigations on n. sativa. pharmacological studies on n. sativa have confirmed its antidiabetic, antitussive, anticancer, antioxidant, hepatoprotective, neuro-protective, gastroprotective, immunomodulator, analgesic, antimicrobial, anti-inflammatory, spasmolytic, and bronchodilator activity. the present review is an effort to explore the reported chemical composition and pharmacological activity of this plant. it will help as a reference for scientists, researchers, and other health professionals who are working with this plant and who need up to date knowledge about it. n. sativa belongs to family ranunculaceae and it is possibly one of the most significant medicinal plants in history. it is mentioned in different historical and religious text books. in southern asia, the seed of this plant is popularly known as 'kalonji' in the middle east most common name is 'habbat us sauda' and popular name of this plant in english is 'black cumin' (gilani et al., ; tavakkoli et al., ) . the seed of this plant is used in ancient medicine for a number of ailments including back pain, asthma, fever, bronchitis, cough, chest congestion, dizziness, paralysis, chronic headache, inflammation, infertility, and other gastrointestinal disorders like dyspepsia, flatulence, diarrhea, and dysentery (durmuskahya and ozturk, ; gholamnezhad et al., ; nasir et al., ; shah, ) . additionally, n. sativa seed oil is used for the remedy of an abscess, nasal ulcer, swollen joint, orchitis and eczema. n. sativa oil is also used in combination with honey for asthmatic problems, bronchospasms and chest congestion (gholamnezhad et al., ; nasir et al., ) . in ancient literature, n. sativa is also attributed as an analgesic, liver tonic, diuretic, appetite stimulant, analgesic and digestive (gilani et al., ; rajsekhar and kuldeep, ; tavakkoli et al., ; ziaee et al., ) . the literature was searched using pubmed and google scholar search engines. papers from between and were included. the search was made using keywords such as n. sativa the n. sativa plant is a green colour with finely divided linear leaves. the flower is pale blue and white in colour with - petals. the fruits are found in the form of inflated capsules. the capsules are further divided into - united follicles. each follicle contains numerous black seeds which are oval in shape and black measuring about mm in diameter ( figure ) (gholamnezhad et al., ; randhawa et al., ) . it is a plant of the family ranunculaceae. this family is also known as buttercup or crowfoot. it is comprised of over , known species of flowering plants in genera, which is distributed all over the world. the largest genera are ranunculus. it comprises species that also includes n. sativa. the seeds of the plants are known as 'black cumin' in english, in arab countries, they are known as 'habba al-sauda' or 'habba al-barakah'. the popular urdu name of this plant is 'kalonji'. it is known as 'siyah danch' and cork out in persian and turkish respectively. the plant is also known as love-in-a-mist, habatul barakah, sonez, krishana, jiraka and sidadanah (sultan et al., ) . n. sativa is native to africa and south west asia. it is also grown in india, bangladesh, turkey, pakistan, sri lanka, syria and other mediterranean regions. high quality seeds are known to come from egypt as egypt has the most suitable environment for growing this plant (naz, ). j o u r n a l p r e -p r o o f n. sativa has a long religious and historical background and is mentioned in various religious literature. it is mentioned in the old testament of bible and is found in the book of isaiah where it is mentioned as "ketzah" a spice for bread and cake that can be used in many ways (naz, ) . it is mentioned as melanthoin and gith in old literature (rahmani and aly, ) . it is also mentioned in the chinese and indian traditional medicine. it has been used to treat various disease from thousands of years ago and known as a vital drug in indian medicine (sharma et al., ) . the n. sativa has been discussed in the traditional arab and islamic medicine (taim) with the name "habb-e-sauda". it is known as prophetic medicine as use of this seed has been mentioned by prophet (saw). it has been mentioned in the book of bukhari that "n. sativa is healing of all the diseases (bukhari )". the first report of the chemical components has been documented in the s. it was reported by greenish et al that it is composed of oils, proteins, carbohydrates and fibres (greenish, ) . there have been many studies to find out the chemical nature of n. sativa and it was found that the medicinal value of n. sativa is mainly due to the presence of its quinone constituent which is also known as thymoquinone (tq) (sahak et al., ) . tq is the main constituent of the volatile oil and has a variety of pharmacological properties such as hepatoprotective (hassanein et al., ; laskar et al., ; saheb et al., ) , anti-inflammatory (abd-elbaset et al., ; j o u r n a l p r e -p r o o f shaarani et al., ) , antibacterial (goel and mishra, ) , antioxidant (erol et al., ) , fungicidal (almshawit and macreadie, ), nephroprotective (kotb et al., ) and anticancer (almoyad, ; majdalawieh et al., ; shaarani et al., ) . there is also literature showing evidence for the molecular mechanism of this molecule (gholamnezhad et al., ) . other components found in the n. sativa includes p-cymene, carvacrol, thymohydroquinone (thq), dihydrothymoquinone (dhtq), -thujene, thymol, t-anethole, -pinene, -pinene, andterpinene (sahak et al., ) . the important constituents of the n. sativa have been summarized in table and the chemical structure has been presented in figure . n. sativa has been reported to have a variety of pharmacological activities, which are categorized as follows. . . antimicrobial activity . . . antibacterial effect thymohydroquinone obtained from the volatile oil of n. sativa has a high significant effect against gram-positive microorganisms, including staphylococcus aureus. diethyl-ether extract of n. sativa was investigated to possess the concentration-dependent inhibitory effect on gram-positive bacteria s. aureus and gram-negative bacteria pseudomonas aeruginosa and escherichia coli (aljabre et al., ) . n. sativa showed an additive effect with various drugs such as doxycycline, chloramphenicol, erythromycin, nalidixic acid and lincomycin (hanafy and hatem, ) . in the management of neonates with staphylococcal pustular skin infections, n. sativa extract demonstrated almost similar results to topical antibiotic mupirocin (rafati et al., ) . n. sativa exhibited promising outcomes against many multi-drug-resistant gram positive and gram negative j o u r n a l p r e -p r o o f bacteria including resistant s. aureus and p. aeruginosa (mashhadian and rakhshandeh, ; morsi, ; salman et al., ) . honey, together with n. sativa possesses synergistic antibacterial effects in treatment of p. aeruginosa infection. chlorhexidine gluconate is a germicidal mouthwash that is used in bacterial infection. it was shown that n. sativa oil extract exhibits better results than chlorhexidine gluconate when treating the infection of s. mutans and other most common dental pathogens (al attas et al., ) . it was reported that n. sativa increases t helper cells (t ) and suppressor t cells (t ) as well as increases natural killer (nk) cell activity in healthy volunteers (aljabre et al., ) . in addition to improvement in immunity, n. sativa extract also shows some inhibitory effects on the human immune deficiency virus protease (khan, ) . in a study n. sativa oil capsules of mg were given three times a day to hepatitis c virus-infected patients for three months. overall there was considerable improvement in oxidative stress, a reduction in viral load and significant improvements were reported in albumin, total protein, platelet and rbc levels. the improvement in rbc count help to decrease the level of membrane lipid peroxide and reduce the chance of hemolysis (barakat et al., ; forouzanfar et al., ) . due to the excessive use of antifungal drugs, resistance is a widespread problem. plant based medicines have attracted researchers as potential alternative treatments for fungal infections (alsaidy, ; doudi et al., ) . antifungal activity is chiefly due to tq of n. sativa. tq, thymohydroquinone and thymol confirmed antifungal activity against dermatophytes, moulds and yeasts (taha et al., ) . the ether extract of n. sativa inhibits the growth of candida yeast (khan et al., ) . further, antifungal effects of n. sativa extracts were also seen against c. albicans j o u r n a l p r e -p r o o f (moghim et al., ) . it was reported that tq inhibits in vitro aspergillus niger and fusarium solani activity similar to the antifungal drug amphotericin-b (al-qurashi et al., ; randhawa et al., ) . moderate antifungal effects were found by tq in three main groups of dermatophytes trichophyton, epidermophyton and microsporum (abd-el-kader et al., ) . n. sativa ether extracts also exhibit antifungal results but produce a more favorable action in higher concentrations. extracts inhibit - % growth of the most dermatophytes in range of mg/ml, while the minimum inhibitory concentration of tq against different dermatophytes ranged from . to . mg/ml (aljabre et al., ) . cardiovascular diseases (cvds) that include coronary heart disease, cerebrovascular disease, heart failure, hypertension and peripheral vascular diseases are leading cause of death worldwide and it was estimated that by , almost . million people are likely to die from cvd (ahmad et al., b; ibrahim et al., ) . numerous cardio protective effects of n. sativa have been observed. n. sativa counteracts several risks of cardiovascular diseases by its versatile pharmacological action due to its antioxidant (leong et al., ) , diuretic (zaoui et al., ) , calcium channel blocking (boskabady et al., ) and cardiac depressant properties (el-taher et al., ; el tahir and ageel, ; el tahir et al., ) . it has been reported in a study that n. sativa oil exhibits positive results against hypertension through a reduction in systolic blood pressure (sbp), lactate dehydrogenase (ldh), asymmetric dimethylarginine, plasma creatine kinase (ck), attenuation of oxidative injury (decrease in malondialdehyde), increase in tissue na + k + atpase activity and plasma nitric oxide level (tasar et al., ) . it has been reported in a randomised controlled trial j o u r n a l p r e -p r o o f of patients that the consumption of n. sativa seed for months can lower the bp in the mildly hypertensive patients (dehkordi and kamkhah, ; fallah huseini et al., ) . globally, atherosclerosis is the most common reason for morbidity and mortality. it is a multifactorial disease with many different threats (joshi et al., ) . hyperlipidemia, low-density lipoproteins (ldl), clotting factors and inflammation all collectively contribute to the development of an atherosclerotic plaque (d'souza et al., ) . it has been illustrated that the volatile oils of n. sativa and its active constituent tq have valuable results for hyperglycaemia and hyperlipidema (ali and blunden, ; asgary et al., ) . tq has been reported to improve hyperlipidemia and protect against the development of atherosclerosis. endothelial dysfunction is a pathological state that contributes to the pathogenesis of a number of cardiovascular diseases, diabetes mellitus, obesity, dyslipidemia, atherosclerosis and ageing. these disorders are linked with overproduction of reactive oxygen species in the arterial vessel wall (idris-khodja and schini-kerth, ; vanhoutte et al., ). it has been found that inhibition of oxidative stress and normalization of the angiotensin system by tq improved endothelial function (idris-khodja and schini-kerth, ). n. sativa oil increases gastric levels of mucin and glutathione, and decreases gastric mucosal histamine content. therefore it can play a significant role in treating gastric ulcers induced by indomethacin and ethanol (el-dakhakhny et al., ; rifat-uz-zaman and khan, ) . the pepsinnogen proenzyme of pepsin is released from stomach cells and mixes with the gastric juice hydrochloric acid, and is converted to pepsin (kanter et al., ; tanaka et al., ) . tq j o u r n a l p r e -p r o o f stimulates pepsinogen to activate pepsin in gastric juice and exhibits protective action against gastric ulcers (kanter et al., ) . anti-inflammatory result of tq has also been reported in the treatment of gastric injury. tq reduces neutrophil invasion via decreasing myeloperoxidase which acts as a marker of acute inflammation. the scavenging of free radicals and antioxidant activities of tq play a significant role in the effect the plant has on gastrointestinal disorders (magdy et al., ) . the bioactive constituents affecting the gastric physiology are documented in table . in a comparative study between n. sativa seeds and a triple therapy using clarithromycin, amoxicillin, and omeprazole, it was reported n. sativa was clinically significant against helicobacter pylori (h. pylori). in an experiment of adult patients with positive h. pylori infection, h. pylori eradication was observed respectively . %, . %, . % and . % with triple therapy, gm ns + mg omeprazole, gm ns + mg omeprazole and gm ns + mg omeprazole. eradication rate with gm ns was significant in reference to g and gm (salem et al., ) . the brain is susceptible to oxidative stress injury due to its high rate of oxidative metabolic action, reactive oxygen species metabolites production and a relatively high content of polyunsaturated fatty acids with low antioxidant capacity, non-replicating character of its neuronal cells and low repair mechanism activity (al-majed et al., ; evans, ) . tq the major component of n. sativa volatile is observed to have potent antioxidant properties (darakhshan et al., ) . tq defends the body against induced oxidative injury caused by free radical generating mediators such as carbon tetrachloride-induced hepatotoxicity (nagi et al., ) , nephropathy produced by j o u r n a l p r e -p r o o f cisplatin (badary et al., ), doxorubicin-induced cardiotoxicity (nagi and mansour, ) , ischemia-reperfusion evoked gastric mucosal injury and allergic encephalomyelitis (mohamed et al., ; mohamed et al., ) . n. sativa encourages modulation properties on memory mutilation, averts hippocampal pyramidal cell loss and increases memory consolidation stored information and decreases neuronal cell death of hippocampal ca region (sayeed et al., ) . flumazenil is a particular antagonist of gabaa-bzd receptor complex (brogden and goa, ; file and pellow, ) . it is used to find out the role of bzd receptors involvement in the anticonvulsant properties induced by tq. it was reported that ptz diminishes the gabaergic tone by the inhibition of bzd site of the gaba receptors (rehavi et al., ) . it was observed from seizure induced by ptz with flumazenil that tq support preventive action of gabaergic system possibly via a competitive agonist action in gaba receptors of bzd site (macdonald and barker, ) . cancer has become a rising public health concern across the world (ahmad, ). thymoquinone of n. sativa exhibits promising anti-carcinogenic, anti-neoplastic, anti-mutagenic and antiproliferative activities against various tumor cells (khan et al., ; shoieb et al., ) . additionally it also acts as chemopreventive agent and is used with the combination of the therapeutic agents to reduce toxic effects of treatment (khader et al., ) . thymoquinone is the major constituent of n. sativa oil extract that induces apoptosis and inhibits proliferation in pancreatic ductal adenocarcinoma (pda) cells (chehl et al., ) . tq induces proapoptotic modes and anti-inflammatory actions and acts as a novel inhibitor of pro-j o u r n a l p r e -p r o o f inflammatory pathways. the high molecular weight glycoprotein mucin is abnormally expressed in cancer of the pancreas and contributes to the regulation of proliferation, differentiation and metastasis of pancreatic cancer cells. tq exhibits a down-regulatory effect on mucin in pancreatic cancer cells (torres et al., ) . hepatocellular carcinoma is one of the widespread malignant diseases, and globally the number of cases has rapidly increased over the past decades. the cytotoxic action of n. sativa seed was exhibited on human hepatoma hepg cell lines following -hr incubation with different concentrations of the n. sativa extract (thabrew et al., ) . it was found that oral administration of tq is useful in rising the actions of glutathione transferase and quinine reductase and acts as a potential prophylactic source against toxicity in hepatic cancer and chemical carcinogenesis (nagi and almakki, ). tq has been shown to be useful for the management of hormone refractory and hormone-sensitive prostate cancer (yi et al., ) . tq prevents prostate tumour growth with almost no chemotoxic side effects. it has also been reported that endothelial cells were more susceptible to thymoquinone-induced cell proliferation, cell apoptosis and cell migration inhibition in contrast to pc cancer cells. tq inhibited growth of vascular endothelial factor-induced extracellular signalregulated kinase activation. diabetes mellitus (dm) is a chronic metabolic disorder that affects . % population of the world. improper management of this disease leads to secondary diseases that include retinopathy, cataract, neuropathy and cardiac problems . it has been reported that j o u r n a l p r e -p r o o f the treatment of diabetes type patients with n. sativa seed dose of gm/day for the duration of months decreases postprandial glucose levels, fasting blood glucose levels, insulin resistance, as well as decreases glycosylated haemoglobin (bamosa et al., ) . it has also been shown that n. sativa seed considerably enhances high-density lipoprotein concentration (hdl-c), decreases serum total cholesterol (tc), low-density lipoprotein, cholesterol (ldl-c) and triglyceride (tg) levels (bamosa et al., ) . n. sativa active antioxidant constituents and tq enhance secretion of insulin via improving mitochondria energy metabolism as well as improving insulin receptors intracellular pathways (mansi, ) . among all mechanisms, antioxidant mechanisms are an important way to control the hyperglycemic stage of diabetic patients. n. sativa enhances the antioxidant enzymes and leads to oxidative stress reduction, and consequently it facilitates pancreatic beta-cells regeneration (abdelmeguid et al., ; houcher et al., ; kanter, ; sultan et al., ) . furthermore, it increases the numbers of islets cells, declines the resistance of insulin and enhances insulin secretion (bamosa et al., ; mansi, ; rchid et al., ; salama, ) . n. sativa and its tq decrease gluconeogenesis, expression of gluconeogenic enzymes like glucose- phosphatase and fructose , -bisphosphatase and hepatic glucose production, consequentially exhibit significant effect in controlling sugar levels particularly in diabetic patients. (abdelmeguid et al., ; houcher et al., ) . it has been revealed that n. sativa extracts and essential oils possess strong antioxidant activity (ashraf et al., ; sultan et al., ) . the antioxidant effect of tq has been found in different diseases, including diabetes, asthma, carcinogenesis and encephalomyelitis. tq preserves the activity of a variety of antioxidant enzymes such as glutathione peroxidase, glutathione-s-j o u r n a l p r e -p r o o f transferase and catalase and also acts as free radical and superoxide scavenger. it has been reported that tq acts as a nephroprotective and decreases ssat and cyp a gene expression via antioxidant mechanisms (awad et al., ; mansour et al., ) . it reacts with gsh, nadh and nadph and forms glutathionyl-dihydro-thymoquinone, offering evidence for potent free radical scavengers (khalife and lupidi, ) . influential chemo preventive action of tq has been shown against mc-induced fibrosarcoma tumours due to the antioxidant activity and its interference with dna synthesis (badary and gamal, ) . treatment with n. sativa extract prevented liver damage induced by lipid peroxidation (meral et al., ) . the favourable safety profile of herbal medicines is one of the reasons people often favour herbal medicines. it has been reported that a mixture of n. sativa with honey exhibits protection against methylnitrosourea-induced oxidative stress and carcinogenesis (ahmad, (ahmad, , ahmad et al., a; ahmad et al., ; mabrouk et al., ) . antioxidant properties of n. sativa ethanol extract exhibit protection from diabetes by improving antioxidant enzyme glutathione peroxidase and decreasing blood glucose levels, lipids levels (kaleem et al., ) . n. sativa containing flavonoids enhance gastric mucus and strengthen mucosal immune defense by scavenging superoxide and hydroxyl free radicals (badary et al., ) . inhibition of lipid peroxidation non-enzymatically has been revealed by tq and n. sativa oil (houghton et al., ) . formation of lipid peroxide and lactate dehydrogenase are suppressed by n. sativa oil even in low concentrations and increase the availability of superoxide dismutase and glutathione (gsh) simultaneously falling lipid peroxidation and free radical generation (houghton et al., ; mansour, ) . dyslipidemia is a wide term covering diverse lipoprotein and lipid disorders. it exhibits a significant role in the provocation of cardiovascular ailments. the research was carried out to assess the therapeutic result of a combination of black seed with garlic in the treatment of dyslipidemia. patients that were treated with garlic, black seed and simvastatin for weeks show considerable differences between cholesterol, triglyceride, non-hdl, and ldl levels (hamed ahmad alobaidi, ). various preparations of n. sativa include oil, extract and powder show a significant role in the modification of plasma lipids and act as a remedy in various disorders like cardio metabolic diseases, obesity, fatty liver, diabetes and metabolic syndrome (table s supplementary data). tq has been reported for its curative potential in dyslipidemia. a number of scientific trials also demonstrated in people with dyslipidemia with n. sativa. n. sativa seeds supplementations have a positive effect in the management of hypercholesterolemia and hyperlipidemia and particularly in patients suffering from diabetes (razavi and hosseinzadeh, ) . obesity is the physiological consequence of a complex genetic interaction, and psychosocial and environmental factors (rodríguez et al., ) . restriction of dietary calories is used as a conventional therapy while various functional foods individually or in combination can play an important role to overcome this problem. a clinical trial of black seed powder was conducted for adult male patients with central obesity for the duration of three months with a dose of . gm daily. significantly positive effects were observed in waist circumference, waist and hip ratio, body weight and also systolic blood pressure, while the reduction in serum testosterone, fasting blood sugar, triglyceride, diastolic blood pressure, uric acid, sgpt and sgot levels were reduced appreciably. (mathur et al., ; najmi et al., ; vanamala et al., ) . the immunomodulatory effects are one of the most valuable properties of n. sativa. active constituents of n. sativa augment the immunomodulatory properties through t cells and nk cells (el-kadi and kandil, ) . significant effects were shown with treatment of n. sativa oil in most of the participating subjects by a % increase in cd and cd t cell ratios and improving the function of nk cells (haq et al., ) . n. sativa oil has a strong potentiating effect on the cellular immunity mediated by t cells, whilst suppressor activity on immunity mediated by b cells has been reported by other constituents. n. sativa stimulatory properties on cellular immunity are linked to the nature of the immune response (salem, ) . the effects of n. sativa and tq on the cellular and humoral immunity have been compared and documented in table . in vitro results of black seed soluble fractions on human peripheral blood mononuclear cells response to various mitogens were observed. it was found that major stimulatory results were not exhibited by components on peripheral blood mononuclear cells response to t cell mitogens phytohemagglutinin while components showed a stimulatory outcome on the peripheral blood mononuclear cells response to pooled allogeneic cells. moreover, n. sativa fractionated proteins illustrated stimulatory activity in lymphocyte cultures (haq et al., ; salem, ) . oxidative stress is one of the most important causes of t cell-mediated autoimmune disorders of the central nervous system and is important for the progression of experimental allergic encephalomyelitis (eae) (majdalawieh and fayyad, ; mohamed et al., ) . this can enhance severity upon astrocyte proliferation and infiltration of inflammatory cells. tq expresses its valuable effects against allergic encephalomyelitis (chakrabarty et al., ) . it has been observed that tq treatment significantly raises glutathione levels in red blood cells and acts as a detoxifying agent due to of its defensive activity against oxidative stress linked with reactive j o u r n a l p r e -p r o o f oxygen species (ros), as well as leading to inhibition of the infiltration of mononuclear cells in the brain and spinal cord (majdalawieh and fayyad, ; pompella et al., ) . traditional use of n. sativa seeds and its active constituents have a significant value on the inflammatory disorders mediated through histamine. nigellone in comparatively small concentrations exhibits excellent results to prevent the release of histamine encouraged by the secretagogues. the mechanism is expected to be through preventing calcium uptake and efflux stimulation, which can lead to a reduction in the concentration of calcium intracellularly. furthermore, n. sativa crude extract shows beneficial calcium channel blocker properties against asthma, high blood pressure and diarrhea have been observed (boskabady et al., b; chakravarty, ) . nigellone suppressed various symptoms of bronchial asthma when it was given orally to asthma patients. it has been used as a traditional medicine for different age groups without any notable toxicity. it has been proved in a clinical study that n. sativa exhibits significant activity to control allergic disorders through reducing the eosinophil count, ige and endogenous cortisol levels in plasma and subsequently confirms it as an effective remedy for bronchial asthma, atopic eczema and allergic rhinitis (el-dakhakhny, ) . several studies have shown that n. sativa and its essential oil have anthelmintic action against earthworms, tapeworms, nematodes and cestodes (agarwal et al., ; akhtar and riffat, ) . n. sativa induces oxidative stress against mature worms which was revealed by declining activities of an antioxidant enzymes, glutathione peroxidase and superoxide dismutase glutathione as well as hexokinase and glucose- -phosphate dehydrogenase. this plays a key role in the control of the overgrowth of helminthes (mohamed et al., ; salem, ) . it has been reported in an in vitro study of n. sativa seeds against s. mansoni, cercariae, miracidia and adult worms that they exhibit strong actions against the entire phases of the parasite as well as an inhibitory result on eggs. black seeds reduce the action of glutathione peroxidase, glutathione reductase, superoxide dismutase and glucose metabolism enzymes, hexokinase and glucose- -phosphate dehydrogenase (g pd) and consequently exhibit oxidative stress to worms (forouzanfar et al., ; mohamed et al., ) . praziquantel is used as an anthelmintic medicine. it averts recently hatched insect larvae development in the body. n. sativa oil exhibits an additive effect in the treatment of schistosomiasis when it is given with praziquantel. a randomized double-blind placebo-controlled clinical trial was conducted whereby it was reported that ml of n. sativa oil improved sperm count and motility, semen ph, semen volume, and its round cells after months treatment of infertile men (kolahdooz et al., ) . it has been reported that mg/kg b.w of n. sativa seeds taken for days increased the number of spermatocytes, total sperm count and motility, weight of the reproductive organs and the number of mature leydig cells (mohammad et al., ) . n. sativa oil also demonstrated the ability to repair testicular degeneration, increase lipid peroxidation and abnormal sperms against sodium valproate testicular toxins. in relation to oxidative stress, tq has exhibited some defensive roles such as superoxide anion scavengers, direct cytoprotective effects and androgen activities. consequentially this leads to protect sperm and semen against testicular toxins. (hala, ; mahdavi et al., ) . n. sativa produces significant anti-nociceptive & anti-inflammatory properties. n. sativa oil and tq exhibit antinociceptive properties through supraspinal opioid activation. it has recommended that n. sativa inhibits the generation of eicosanoids in lipid peroxidation and leukocytes. they inhibit -lipoxygenase ( -lox) and cyclooxygenase (cox) pathways of arachidonic acid metabolism (pise and padwal, ) . in the progression of inflammatory diseases, inflammatory factors such as eicosanoids and ros play a significant role. the anti-inflammatory properties were widely searched in two major inflammatory disorders that include allergic encephalomyelitis and ulcerative colitis (majdalawieh and fayyad, ; nieto et al., ) . it has been found that tq through anti-inflammatory and antioxidant properties significantly improve allergic encephalomyelitis and ulcerative colitis (choudhary et al., ; koch et al., ; mahdavi et al., ) . topical n. sativa oil was reported to have a good curative effect on the healing of chemically induced oral ulcers. oral ulcers were treated with topical applications of n. sativa twice a day for days. the results illustrated an important healing process with n. sativa treatment and a noticeable anti-inflammatory action (muñoz-corcuera et al., ; porter and leao, ) . it has been reported in various studies that tq demonstrates a key role in various pathogenic conditions of kidneys. tq attenuates oxidative stress and inflammation by providing protection from various toxic agents (benhelima et al., ) . it reduces gentamicin-induced nephrotoxicity indexes and degenerative changes. tq supplementation prevents acute renal failure induced by gentamicin by recovering mitochondrial function and enhancing the production of atp. tq during ifosfamide treatment improves the severity of ifosfamide-induced renal damage, phosphaturia, glucosuria, high serum creatinine level and urea and stabilise clearance rate of creatinine. pyelonepheritis induced oxidative damage of the kidneys can be protected by tq administration. also the cisplatin antitumor action is potentiated by tq as well as it protects against nephrotoxicity induced by cisplatin (badary et al., ; darakhshan et al., ; el daly, ) . it has also been reported that n. sativa shows a significant nephroprotective activity on paracetamol-induced nephrotoxicity (canayakin et al., ) . it has been reported that the number of inflammatory joints and the extent of morning stiffness are improved by tq. in a placebo-controlled study of females with rheumatoid arthritis treated with n. sativa, tq showed protective results against rheumatoid arthritis, and a decrease in arthritis scoring (disease activity score (das- ) and bone resorption were reported (hadi et al., ) . published reports clearly suggest that n. sativa is an important medicinal plant in j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f inhibition of histamine release from mast cells by nigellone anti-inflammatory effects of the nigella sativa seed extract, thymoquinone, in pancreatic cancer cells novel antioxidants zolimid and aeol ameliorate colitis in rats efficacy study of the bioactive fraction (f- ) of acorus calamus in hyperlipidemia thymoquinone and its therapeutic potentials antihypertensive effect of nigella sativa seed extract in patients with mild hypertension comparing the antifungal effects of five essential oils plants eucalyptus, cinnamon, wormwood, sagebrush and iranian rose damascena on three standard strains of candida albicans in vitro ethnobotanical survey of medicinal plants used for the treatment of diabetes in manisa studies on the egyptian nigella sativa l. part iv: some pharmacological properties of the seed's active principle in comparison to its dihydro compound and its polymer effects of nigella sativa oil on gastric secretion and ethanol induced ulcer in rats effect of nigella sativa (the black seed) on immunity dethymoquinonated nigella sativa volatile oil and its major components alpha-pinene and p-cymene in rats protective effect of cysteine and vitamin e, crocus sativus and nigella sativa extracts on cisplatin-induced toxicity in rats effect of the volatile oil of (nigella sativa) on the arterial blood pressure and heart rate of the guinea-pig the cardiovascular actions of the volatile oil of the black seed (nigella sativa) in rats: elucidation of the mechanism of action comparison of combined antioxidants and thymoquinone in the prevention of testis ischemia-reperfusion injury free radicals in brain metabolism and pathology blood pressure lowering effect of nigella sativa l. seed oil in healthy volunteers: a randomized, double-blind, placebo-controlled clinical trial intrinsic actions of the benzodiazepine receptor antagonist ro - black cumin (nigella sativa) and its constituent (thymoquinone): a review on antimicrobial effects preclinical and clinical effects of nigella sativa and its constituent, thymoquinone: a review a review of medicinal uses and pharmacological activities of nigella sativa thymoquinone inhibits biofilm formation and has selective antibacterial activity due to ros generation contribution to the chemistry of nigella sativa effects of nigella sativa oil extract on inflammatory cytokine response and oxidative stress status in patients with rheumatoid arthritis: a randomized, double-blind, placebo-controlled clinical trial black cumin seed essential oil, as a potent analgesic and antiinflammatory drug protective effect of nigella sativa, linseed and celery oils against testicular toxicity induced by sodium valproate in male rats effect of nigella sativa and allium sativum coadminstered with simvastatin in dyslipidemia patients: a prospective, randomized, double-blind trial studies on the antimicrobial activity of nigella sativa seed (black cumin) nigella sativa: effect on human lymphocytes and polymorphonuclear leukocyte phagocytic activity immunomodulatory effect of nigella sativa proteins fractionated by ion exchange chromatography prophylactic effects of thymoquinone against carbon tetrachloride-induced hepatic damage in sprague-dawley rats effects of black seed (nigella sativa) on metabolic parameters in diabetes mellitus: a systematic review effects of methanolic extract and commercial oil of nigella sativa l. on blood glucose and antioxidant capacity in alloxan-induced diabetic rats fixed oil of nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation a randomised controlled trial on hypolipidemic effects of nigella sativa seeds powder in menopausal women thymoquinone improves aging-related endothelial dysfunction in the rat mesenteric artery effects of black seeds (nigella sativa) on male infertility: a systematic review immunomodulatory and anti-inflammatory action of nigella sativa and thymoquinone: a comprehensive review anti-cancer properties and mechanisms of action of thymoquinone, the major active ingredient of nigella sativa effects of oral administration of water extract of nigella sativa on serum concentrations of protective effects of thymoquinone and desferrioxamine against hepatotoxicity of carbon tetrachloride in mice effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and dt-diaphorase in different tissues of mice: a possible mechanism of action antibacterial and antifungal effects of nigella sativa extracts against s. aureus, p. aeruginosa and c. albicans antidiabetic properties of a spice plant nigella sativa effect of nigella sativa on glucose concentration, lipid peroxidation, anti-oxidant defence system and liver damage in experimentallyinduced diabetic rabbits antifungal effects of zataria multifora and nigella sativa extracts against candida albicans thymoquinone inhibits the activation of nf-kappab in the brain and spinal cord of experimental autoimmune encephalomyelitis improvement of experimental allergic encephalomyelitis (eae) by thymoquinone; an oxidative stress inhibitor sativa seeds against schistosoma mansoni different stages. memórias do instituto oswaldo cruz effects of black seeds (nigella sativa) on spermatogenesis and fertility of male albino rats antimicrobial effect of crude extracts of nigella sativa on multiple antibioticsresistant bacteria oral ulcers: clinical aspects. a tool for dermatologists. part ii. chronic ulcers thymoquinone supplementation induces quinone reductase and glutathione transferase in mice liver: possible role in protection against chemical carcinogenesis and toxicity protective effect of thymoquinone against doxorubicininduced cardiotoxicity in rats: a possible mechanism of protection effect of nigella sativa oil on various clinical and biochemical parameters of insulin resistance syndrome therapeutic uses of shoneez (nigella sativa linn mentioned in unani system of medicine-a review nigella sativa: the miraculous herb experimental ulcerative colitis impairs antioxidant defense system in rat intestine evaluation of anti-inflammatory activity of nigella sativa: an experimental study the changing faces of glutathione, a cellular protagonist review article: oral ulcers and its relevance to systemic disorders anti-microbial effect of nigella sativa seed extract against staphylococcal skin infection nigella sativa and its active constituents thymoquinone shows pivotal role in the diseases prevention and treatment pharmacognosy and pharmacology of nigella sativa-a review thymoquinone, an active principle of nigella sativa, inhibited fusarium solani a review of the effects of nigella sativa l. and its constituent, thymoquinone, in metabolic syndrome nigella sativa seed extracts enhance glucose-induced insulin release from rat-isolated langerhans islets effects of tetrazole derivatives on [ h] diazepam binding in vitro: correlation with convulsant potency gastroprotective and anti-secretory effect of nigella sativa seed and its extracts in indomethacin-treated rats effects of two energy-restricted diets containing different fruit amounts on body weight loss and macronutrient oxidation the role of nigella sativa and its active constituents in learning and memory hepatoprotective effect of nigella sativa seed in sterptozotocine induced diabetic albino rats: histological observations hypoglycemic effect of lipoic acid, carnitine and nigella sativa in diabetic rat model comparative study of nigella sativa and triple therapy in eradication of helicobacter pylori in patients with non-ulcer dyspepsia immunomodulatory and therapeutic properties of the nigella sativa l. seed antimicrobial activity of nigella sativa linn. seed oilagainst multi-drug resistant bacteria from clinical isolates the effect of nigella sativa linn. seed on memory, attention and cognition in healthy human volunteers the influence of pluronic f and f nanocarrier on physicochemical properties, in vitro release, and antiproliferative activity of thymoquinone drug the general principles of avicenna's canon of medicine central council for research in ayurveda & siddha, deptt. of ism & h, min. of health & family welfare, government of india in vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone supplementation of nigella sativa fixed and essential oil mediates potassium bromate induced oxidative stress and multiple organ toxicity nigella sativa fixed and essential oil supplementation modulates hyperglycemia and allied complications in streptozotocin-induced diabetes mellitus antifungal effect of thymol, thymoquinone and thymohydroquinone against yeasts, dermatophytes and non-dermatophyte molds isolated from skin and nails fungal infections the role of nitric oxide in the gastric acid secretion induced by ischemia-reperfusion in the pylorus-ligated rat protective effects of nigella sativa against hypertension-induced oxidative stress and cardiovascular dysfunction in rats black seed (nigella sativa) and its constituent thymoquinone as an antidote or a protective agent against natural or chemical toxicities (suppl. ) a review on therapeutic potential of nigella sativa (kalonji) seeds cytotoxic effects of a decoction of nigella sativa, hemidesmus indicus and smilax glabra on human hepatoma hepg cells effects of thymoquinone in the expression of mucin in pancreatic cancer cells: implications for the development of novel cancer therapies mitigation of obesity-promoted diseases by nigella sativa and thymoquinone endothelial dysfunction and vascular disease thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing akt and extracellular signal-regulated kinase signaling pathways diuretic and hypotensive effects of nigella sativa in the spontaneously hypertensive rat review of pharmacological and toxicological effects of nigella sativa and its active constituents j o u r n a l p r e -p r o o f key: cord- -qwdjb vk authors: jukic, igor; calleja-gonzález, julio; cos, francesc; cuzzolin, francesco; olmo, jesús; terrados, nicolas; njaradi, nenad; sassi, roberto; requena, bernardo; milanovic, luka; krakan, ivan; chatzichristos, kostas; alcaraz, pedro e. title: strategies and solutions for team sports athletes in isolation due to covid- date: - - journal: sports (basel) doi: . /sports sha: doc_id: cord_uid: qwdjb vk in december of , there was an outbreak of a severe acute respiratory syndrome caused by the coronavirus (sars-cov- or covid- ) in china. the virus rapidly spread into the whole world causing an unprecedented pandemic and forcing governments to impose a global quarantine, entering an extreme unknown situation. the organizational consequences of quarantine/isolation are: absence of organized training and competition, lack of communication among athletes and coaches, inability to move freely, lack of adequate sunlight exposure, inappropriate training conditions. based on the current scientific, we strongly recommend encouraging the athlete to reset their mindset to understand quarantine as an opportunity for development, organizing appropriate guidance, educating and encourage athletes to apply appropriate preventive behavior and hygiene measures to promote immunity and ensuring good living isolation conditions. the athlete’s living space should be equipped with cardio and resistance training equipment (portable bicycle or rowing ergometer). some forms of body mass resistance circuit-based training could promote aerobic adaptation. sports skills training should be organized based on the athlete’s needs. personalized conditioning training should be carried out with emphasis on neuromuscular performance. athletes should also be educated about nutrition (vitamin d and proteins) and hydration. strategies should be developed to control body composition. mental fatigue should be anticipated and mental controlled. adequate methods of recovery should be provided. daily monitoring should be established. this is an ideal situation in which to rethink personal life, understanding the situation, that can be promoted in these difficult times that affect practically the whole world. in december of , there was an outbreak of a severe acute respiratory syndrome caused by the coronavirus (sars-cov- or covid- ) in wuhan, hubei province, china. the virus rapidly spread across the country and then into the whole world [ ] , causing an unprecedented pandemic [ ], forcing governments to impose an almost global quarantine. at the beginning of (january-march), the whole world, including the world of sports, entered an extreme and unknown situation [ ], where, gradually, all sports competitions were postponed and any organized training or practice was banned [ ] . the health of the athletes, coaches and spectators became a priority. the major local and international competitions, such as the european football championship and the olympic games in tokyo, were postponed for a year. this unusual global crisis has caused a major organizational, financial and social disruption to athletes, coaches, clubs and sports federations. all teams have allowed their athletes to return home, where they are in mandatory home isolation following government guidelines. isolation, of course, does not allow athletes to follow their usual training and competition schedule. regardless of duration, isolation could have a significant impact on the physical and mental state of an athlete. however, to the best of our knowledge, no previous evidence on this particular topic has been published. the organizational consequences of quarantine/isolation are: absence of organized training and competition, lack of adequate communication between athletes and coaches, inability to move freely, lack of adequate sunlight exposure, inappropriate training conditions [ , ] . staying in quarantine can have negative effects, not only on most physiological systems, but also in the players' lives. for example, isolation at home can lead to poor and inappropriate nutrition, poor quality of sleep, addictions, loneliness, just to name a few negative lifestyle changes. the physiological adverse effects of isolation include an increase in body fat content and a decrease in muscle mass, impaired immunity, loss of mental sharpness and toughness, insomnia and depression [ , ] . all of these consequences can have both a short-and long-term negative effect on the athletes' physical fitness and competitive performance. although it is difficult to predict the duration of the global covid- crisis at this time, it is possible to predict the loss of training-induced adaptation [ ] [ ] [ ] [ ] [ ] . therefore, first, it is extremely important to identify these effects and to understand the mechanisms and effects on all physiological systems, as well as their impact on athletic performance. second, but no less important, it's important to provide practical recommendations to coaches and athletes to reduce the unwanted consequences of the forced quarantine. the principle of training reversibility states that stop or markedly reduce training induces a partial or complete reversal of the previous developed adaptations, thus compromising athletic performance [ ] . the reversibility principle is also known as detraining. the concept of detraining refers to the total or partial loss of the training-induced adaptation achieved through training [ ] . although athletes experience transition periods throughout their sports careers, usually coinciding with the end of their competition period, illness, injury, or other factors, the loss of physical activity is not comparable to the restriction that the current "stay at home" confinement represents. however, detraining is one of the biggest negative consequences of the forced quarantine. detraining affects different physiological systems (e.g., neuromuscular, cardiovascular, respiratory or muscle-skeletal) and their corresponding physical capacities (e.g., strength and power, endurance, speed or flexibility). although some investigations have concluded that neural changes are long-lasting and did not affect the elements of h-reflex pathways [ ] , there is strong evidence to think the opposite. for example, it was reported that neuromuscular performance was impaired in top-level male kayakers after weeks of either reduced training or complete training cessation [ ] . a recent systematic review [ ] revealed that the concurrent (ct) training-induced gains may be compromised with a short-term detraining period ( - weeks), leading to a return to baseline values. the authors also explained that a -week period of training cessation after ct with different resistances or aerobic training loads compromised training-induced gains in young men. they concluded that, despite scarce evidence, it seemed that regardless of the intensity of the previous endurance and resistance training during ct, only - weeks of training cessation can cause a significant and marked loss of performance. to date, the most used ct method is resistance circuit-based training (rcbt) [ ] . rcbt is an effective training method for the concurrent development of maximum oxygen consumption (vo max ) and one repetition maximum ( -rm) bench press in healthy adults, independent of participant and load characteristics, as shown in the authors' review and meta-analysis [ ] . therefore, some forms of home-based rcbt could easily be performed with simple equipment at home and promote both neuromuscular and metabolic adaptations, thus minimizing neuromuscular detraining effects [ ] . reductions in maximal and submaximal exercise performance occur within weeks after the cessation of training. these losses in aerobic performance decline cardiovascular function and muscle metabolic potential. specifically, significant reductions in vo max have been described within to weeks of detraining [ ] . the detraining effects were mainly: ( ) an initial rapid decline in vo max ; ( ) decrease in blood volume; ( ) changes in cardiac hypertrophy; ( ) decrease in the total hemoglobin content; ( ) decreased skeletal muscle capillarization; and ( ) disruption of temperature regulation. when absence of training continued beyond to weeks [ ] , the detraining effects became more severe. this results in: ( ) further declines in vo max ; ( ) reductions in maximal arterial-venous (mixed) oxygen difference; ( ) changes in maximal oxygen delivery, which may result from decreases in total hemoglobin content and/or maximal muscle blood flow and vascular conductance; ( ) declines in skeletal muscle oxidative enzyme activity; and ( ) reductions in submaximal exercise performance, which may be related to changes in the mean transit time of blood flow through the active muscle and/or the thermoregulatory response (i.e., degree of thermal strain) to exercise. therefore, athletes must incorporate some type of endurance exercise their daily routine to try and reverse some of the aforementioned effects of detraining. flexibility is the ability to move a joint through its optimal range of motion. the ability to move a joint without restriction or pain depends on the condition of different structures, such as bone, muscle, and connective tissue. it also depends on the muscle's ability to produce an adequate amount of force [ ] . decreases in flexibility have been reported after weeks of detraining [ ] . given that the current isolation period could be longer than a month, it is recommended to incorporate exercises to maintain and improve flexibility. for example, neurodynamic treatments [ ] or tai chi, ioga or thai chi may be a useful therapy for vestibular rehabilitation, improving dynamic balance control and flexibility [ ] . short-term detraining may specifically affect eccentric strength and the size of the type ii (ft: fast twitch) muscle fibers [ ] . it has been suggested that performing eccentric muscle actions during training is essential to promote greater and longer-lasting neural adaptations to training [ ] and that speed-strength is better maintained during periods of reduced training if previously the focus of training was on power development [ ] . loturco et al. [ ] concluded that it may be important for coaches to include plyometric training, even in detraining periods, in order to avoid possible impairments in the stretch-shortening function [ ] . this simple advice could help in maintaining/improving all the neuromuscular indices relevant to athletes' performance and could constitute the basis of an ideal detraining strategy in sports like track and field [ ] . reduced or complete absence of strength training can cause loss of muscle mass. muscle atrophy results from an imbalance between protein degradation and synthesis in favor of the former [ ] . when inactivity exceeds weeks, there is a transition of ft fibers into type i (st: slow twitch), especially in sports, characterized by explosive actions, with the ft being more vulnerable to periods of inactivity than the st type [ ] . although when training periods do not exceed two weeks, the changes in the distribution of muscle fibers are not noticeable in long distance runners or in strength and power athletes [ ] , after the first days, there is a decrease in the transverse fibrillar area of approximately . % per day [ ] . this decrease in muscle size translates to a % and % reduction in strength and team sports athletes, after a period of inactivity ranging from to weeks. a decrease in ft fiber content has been observed in footballers and weightlifters [ ] and a decrease in the ability to apply force to the water in swimmers [ ] . similarly, some fibrillar conversion of fta fibers to ftx fibers has been observed in long-distance runners and cyclists [ ] . periods of prolonged inactivity negatively affect the anti-gravitational muscle groups and the posterior extensor muscle chain [ ] . in general, inactivity affects different muscles and muscle chains depending on whether they are tonic or phasic, causing muscle shortening and/or hypertonia or laxity and/or hypotonia depending on the muscle type (figure ). these imbalances can be the onset or worsening of pathologies such as groin pain [ ] . other authors hypothesized that inactivity also caused a decrease in collagen synthesis in the human tendon, with progressive decreases in collagen synthesis being recorded between and days of complete inactivity [ ] . periods of prolonged inactivity negatively affect the anti-gravitational muscle groups and the posterior extensor muscle chain [ ] . in general, inactivity affects different muscles and muscle chains depending on whether they are tonic or phasic, causing muscle shortening and/or hypertonia or laxity and/or hypotonia depending on the muscle type (figure ). these imbalances can be the onset or worsening of pathologies such as groin pain [ ] . other authors hypothesized that inactivity also caused a decrease in collagen synthesis in the human tendon, with progressive decreases in collagen synthesis being recorded between and days of complete inactivity [ ] . fundamental characteristics of the tonic and phasic muscles, as well as the main physiological adaptations to pathology or inactivity. characteristics and habitual response of the tonic and phasic muscles (cos and cos, ) . reducation in activity results in a reduced energy expenditure, which consequently requires a reduction in energy intake to prevent unwanted body fat gains. in terms of an absolute amount of protein per day, when increasing protein to . g/kg, body mass reduces muscle loss during periods of reduce caloric intake [ ] . thus, athletes may benefit from increasing their protein intake to counter the immobilization-induced anabolic resistance, as well as to attenuate the associated losses in muscle mass [ ] . it is accepted that when reducing energy intake, macronutrients should not be cut evenly, as maintaining a high-protein intake will be essential to attenuate loss in lean muscle mass. for instance, leucine consumption, which is a key and critical amino acid for stimulating the cell signaling pathways that control muscle protein synthesis, should be emphasized in the protein sources consumed [ ] . a major consideration when training athletes in home isolation is compliance, especially regarding the intensity and volume of exercise. it is difficult to monitor and ensure that the load that athletes use at home is appropriate to maintain physical fitness and performance at the required level. for recreational and ordinary people in isolation, maintaining an acceptable level of physical fitness is possible with moderate exercise [ ] , but high-level athletes need precise exercise prescription. maintaining a high level of physical and mental fitness requires relatively high loads of submaximal and maximal intensity exercise [ ] . because of the lack of appropriate space (e.g., a football field) and the subsequent inability to perform sport-specific and/or high-intensity exercises, such as sprints, athletes returning to sport after the quarantine must be aware of an increased chance of injury. reducation in activity results in a reduced energy expenditure, which consequently requires a reduction in energy intake to prevent unwanted body fat gains. in terms of an absolute amount of protein per day, when increasing protein to . g/kg, body mass reduces muscle loss during periods of reduce caloric intake [ ] . thus, athletes may benefit from increasing their protein intake to counter the immobilization-induced anabolic resistance, as well as to attenuate the associated losses in muscle mass [ ] . it is accepted that when reducing energy intake, macronutrients should not be cut evenly, as maintaining a high-protein intake will be essential to attenuate loss in lean muscle mass. for instance, leucine consumption, which is a key and critical amino acid for stimulating the cell signaling pathways that control muscle protein synthesis, should be emphasized in the protein sources consumed [ ] . a major consideration when training athletes in home isolation is compliance, especially regarding the intensity and volume of exercise. it is difficult to monitor and ensure that the load that athletes use at home is appropriate to maintain physical fitness and performance at the required level. for recreational and ordinary people in isolation, maintaining an acceptable level of physical fitness is possible with moderate exercise [ ] , but high-level athletes need precise exercise prescription. maintaining a high level of physical and mental fitness requires relatively high loads of submaximal and maximal intensity exercise [ ] . because of the lack of appropriate space (e.g., a football field) and the subsequent inability to perform sport-specific and/or high-intensity exercises, such as sprints, athletes returning to sport after the quarantine must be aware of an increased chance of injury. therefore, sport governing bodies must offer appropriate time to the athletes and teams to prepare for high-level competition. lack of competition poses an additional problem to teams and athletes, because it is through competitions that athletes can best maintain their physical fitness and sport form. competing activities in many sports with a congested competition schedule [ ] is also a key factor which is an important developmental stimulus [ ] . in addition, preparatory, control and official competitions are an important tool to establish and maintain optimal performance [ ] . consequently, the absence of competition has a negative impact on athlete's performance and peak sports form. in spite of everything, some positive effects of isolation should also be kept in mind. in such conditions, the athlete can fully recover from all stresses, injuries and previously accumulated loads (overreaching and overtraining). for example, in team sports there are very few situations in the regular annual calendar in which a player can have a prolonged period of complete recovery from specific training and competition demands. only off-season/transition periods can offer some opportunity for rest [ ] . isolation and the absence of intensive specific training and competition enable both complete cellular recovery and the avoidance of common daily mental stress. this is also an opportunity to implement developmental programs of certain physical abilities for which an athlete in team sports does not have enough time under the regular periodization regimen [ ] . off-season/transition periods like this exceptional situation are also a rare opportunity to have enough time for extensive injury prevention and individual athletic development work. that work prepares athletes for a rushed pre-re-season, including high-intensity work in wide spaces, and good performance with low injury risk, when the competitions resume. a very similar situation occurs after an athlete suffers a serious injury. those athletes who use rehabilitation as an opportunity for athletic development generally return to competition in a better shape for the rest of the season, which consequently positively affects their future career [ ] . in other words, this isolation is an opportunity for both a complete physiological and mental reset as well as for the athlete's integral development. all the previously mentioned training and recovery programs should be strictly personalized [ ] . based on the current scientific and practical evidence, we strongly recommend the following points: -encourage, provoke and motivate the athlete to reset their mindset and use this break as an opportunity for personal development [ ] ; -organize appropriate guidance and support to athletes by experts (sports coach, strength and conditioning coach, nutritionist, doctor, psychologist) by using technology (video call, e-mail, telephone, text messages); -educate and encourage athletes to apply appropriate preventive behavior and hygiene measures to promote immunity and protect their own health and the health of the people in their immediate environment [ ] ; -ensure good living conditions in isolation (space, equipment, food, telecommunications). if possible, the athlete's living space should be equipped with cardio equipment (treadmill, bicycles, rowing ergometer, etc.), resistance training equipment (dumbbells, elastic bands, abdominal wheels, medicine balls, etc.) and other equipment for frequent use (mats, foam rollers, self-massagers, etc.). if not, some forms of body mass resistance circuit-based training could promote (or maintain) neuromuscular and aerobic adaptations [ ] ; -organize alternative sports skills training (kinesthetic ball training in a small space, visualization, virtual reality technical aids, video analysis, theoretical training) based on the athlete's deficits and needs; -organize personalized strength and conditioning training at home with available space and material resources that are tailored to the athlete's individual characteristics and current needs [ ] . focus on neuromuscular plyometrics (i.e., vertical and horizontal jumping) and eccentric training (i.e., elastic bands), to maintain some key adaptations related to the stretch-shortening cycle, strength and power performance. adaptations of the stabilizer muscles as an indispensable element and facilitator of the efficient sensorimotor action of any act is also extremely important [ , ] ; -educate the athlete about nutrition, supplementation (especially vitamin d, zinc and proteins) and hydration in isolation conditions, and about strategies to control body mass and body composition [ , , , ] . it is important to consume food to fight off viral infections, thus advising against lower carbohydrate/intermittent fasting approaches is likely important [ ] ; -organize mental fatigue monitoring and mental training (mental self-help techniques and/or the support of a psychologist by telecommunication) [ ] ; -provide adequate methods of recovery (supplementation, sleep, breathing and meditation exercises, self-massage, myofascial relaxation, stretching, low back heat, etc.) [ ] ; -use forms of self-assessment (heart rate monitoring, hearth rate variability, hearth rate recovery, orthostatic test, simple movement functional tests, simple vo max tests, etc.) that an athlete can use on a daily basis and share data with a strength and conditioning coach [ ] ; -establish daily monitoring of the athlete's health, wellness, physical fitness, recovery [ ] and workload by using technology (phone, applications, e-mail, text message) [ ] ; -even though many athletes are not currently injured, the time off is similar to the time off after an injury [ ] ; -finally, muscle memory is important to educate athletes, given that any losses are rapidly regained. this should quell some anxiety [ ] . to conclude, an athlete's life in isolation due to a covid- crisis and imposed quarantine should have another, positive meaning. this is an ideal situation to rethink and reorganize one's personal life and value system. humility, gratitude, understanding of the global situation, empathy for other people, family values, attitude towards knowledge, spirituality, and helping the needy are just some of the values that can be promoted and truly lived in difficult times that affect practically the whole world. as athletes are often role models, in this challenging time they should take responsibility and promote "good values". thus, they can contribute to building better societies and better people and promote better human behavior. covid- ): a perspective from china football cannot restart soon during the covid- emergency. a critical perspective from the italian experience and a call for action coronavirus disease (covid- ): the need to maintain regular physical activity while taking precautions vitamin d and the athlete: current perspectives and new challenges covid- epidemic: exercise or not to exercise; that is the question! asian in endurance training: science and practice effect of training cessation on muscular performance: a meta-analysis. scand detraining in young soccer players muscular characteristics of detraining in humans effects of short-term in-season break detraining on repeated sprint ability and intermittent endurance according to initial performance of soccer player peak performance: training and nutritional strategies for sport neural drive preservation after detraining following neuromuscular electrical stimulation training physiological effects of tapering and detraining in world-class kayakers concurrent training and detraining: brief review on the effect of exercise intensities effectiveness of resistance circuit-based training for maximum oxygen uptake and upper-body one-repetition maximum improvements: a systematic review and meta-analysis acute effects of two different resistance circuit training protocols on performance and perceived exertion in semiprofessional basketball players effect of reduced training on muscular strength and endurance in competitive swimmers seasonal variation in physiological fitness of a semiprofessional soccer team effects of neurodynamic treatment on hamstrings flexibility: a systematic review and meta-analysis impact of tai chi exercise on balance disorders: a systematic review the effects of detraining on power athletes effects of detraining following short term resistance training on eccentric and concentric muscle strength maintaining the training effect during work stoppage effects of detraining on neuromuscular performance in a selected group of elite women pole-vaulters: a case study mechanisms responsible for disuse muscle atrophy: potential role of protein provision and exercise as countermeasures mechanisms for fiber-type specificity of skeletal muscle atrophy crosstalk proposal: the dominant mechanism causing disuse muscle atrophy is decreased protein synthesis adaptive changes in work capacity, skeletal muscle capillarization and enzyme levels during training and detraining atrophy and hypertrophy of skeletal muscles: structural and functional aspects interpretación de las alteraciones del sistema músculo esquelético. beneficios del trabajo excéntrico y concéntrico. efectos de la inactividad y de la inmovilización en el músculo the temporal responses of protein synthesis, gene expression and cell signaling in human quadriceps muscle and patellar tendon to disuse increased protein intake reduces lean body mass loss during weight loss in athletes case study: muscle atrophy and hypertrophy in a premier league soccer player during rehabilitation from acl injury current concepts and unresolved questions in dietary protein requirements and supplements in adults. front. nutr. , , detraining and tapering effects on hormonal responses and strength performance discrepancy between exercise performance, body composition, and sex steroid response after a six-week detraining period in professional soccer players physical activity during a prolonged congested period in a top-class european football team training in team sports: structured training in the fcb an integrated, multifactorial approach to periodization for optimal performance in individual and team sports the transition period in soccer: a window of opportunity sport preparation system in team sports: synergy of evidence, practical experience and artistic expression a grounded theory of sport injury-related growth strength and conditioning in top level team sports: an individual discipline combined effects of fatigue and decision making on female lower limb landing postures: central and peripheral contributions to acl injury risk integrative neuromuscular training in youth athletes. part ii: strategies to prevent injuries and improve performance issn exercise & sports nutrition review update: research & recommendations zinc for the common cold opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation the application of mental fatigue research to elite team sport performance: new perspectives brief ideas about evidence-based recovery in team sports the development of a personalized training framework: implementation of emerging technologies for performance evidence-based post-exercise recovery strategies in rugby: a narrative review monitoring the athlete training response: subjective self-reported measures trump commonly used objective measures: a systematic review nutritional support for exercise-induced injuries muscle memory and a new cellular model for muscle atrophy and hypertrophy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no funding sources. the authors declare no conflict of interest. key: cord- -w spqqt authors: huan, yuchen; kong, qing; mou, haijin; yi, huaxi title: antimicrobial peptides: classification, design, application and research progress in multiple fields date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w spqqt antimicrobial peptides (amps) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. amps have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. the emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of amps, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. this review introduces the progress of research on amps comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. the research progress on antivirus peptides, especially anti-coronavirus (covid- ) peptides, has been introduced given the covid- pandemic worldwide in . alexander fleming discovered lysozyme in , and this discovery marked the birth of modern innate immunity. since then, antibiotics and antimicrobial peptides (amps) have been discovered. a total of , amps have been reported in the antimicrobial peptide database (apd ) updated on august , . different types of amps have the following commonalities: their number of amino acid residues is between and (average: . ), and almost all amps are cationic (average net charge: . ). however, several anionic amps also exist, and they have several acidic amino acids like aspartic acid and glutamic acid (malkoski et al., ; schittek et al., ; lai et al., ) . the anti-microbial resistance of microorganisms is becoming increasingly serious with the abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing countries. research from kenya has detected substantial amounts of antibiotic residues in edible meat (ayukekbong et al., ) . the prevalence of vancomycin-resistant enterococcus (vre) and methicillin-resistant staphylococcus aureus (mrsa) is increasing in clinical medicine, so the countermeasures are urgently needed to address these bacterial infections. however, from the http://aps.unmc.edu/ap/ perspective of pharmaceutical companies, the development of new antibiotic drugs results in low profit. thus, replacing antibiotics has become a consideration in the pharmaceutical, agricultural, animal husbandry, and food industries. research on amps is continuously developing and considerable amounts of data on amps have been stored in amp databases. however, the mechanism of amps remains incompletely understood, and further work needs to be performed to determine the relationship between different physicochemical properties to obtain low-cost and highly safe amps with remarkable antimicrobial effects and the specificity and a high capacity for synergies of amps should also be further developed (lazzaro et al., ) . the diversity of natural amps causes difficulty in their classification. amps are classified based on ( ) source, ( ) activity, ( ) structural characteristics, and ( ) amino acid-rich species (figure ). the sources of amps can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in apd . the amps found in oceans have also attracted widespread attention. mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. cathelicidins and defensins are the main families of amps. defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds (reddy et al., ) . human host defense peptides (hdps) can protect human from microbial infections but show different expressions in every stage of human growth. for example, cathelicidin ll- , a famous amp derived from the human body, is usually detected in the skin of newborn infants, whereas human betadefensin (hbd- ) is often expressed in the elderly instead of the young (gschwandtner et al., ) . hdps can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. besides, amps in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breastfeeding infants (field, ) . what's interesting is that casein (peptide derived from β-casein - aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums . dairy is an important source of amps, which are generated through milk enzymatic hydrolysis. several amps have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin b (lfcinb) (sibel akalın, ) . furthermore, whether the amps derived from dairy products can be used for dairy preservation is also an interesting subject to develop. in addition to antimicrobial activity, hdps, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing (wang, ) . antimicrobial peptides from amphibians play an important role in the protection of amphibians from the pathogens that have induced the global amphibian population decline (rollins-smith, ). frogs are the main source of amphibian amps and the most famous amp from frogs is magainin; the skin secretions of frogs from genera xenopus, silurana, hymenochirus, and pseudhymenochirus under the pipidae family are rich in amps (conlon and mechkarska, ) . furthermore, cancrin, which has an amino acid sequence of gsaqpykqlhkvvnwdpyg, has been reported as the first amp from the sea amphibian rana cancrivora (lu et al., ) . this marks a broader source of amps of amphibians. figure | classification of antimicrobial peptides. (semreen et al., ) . myticusin-beta is an immune-related amp of mytilus coruscus and a promising alternative to antibiotics (oh et al., ) . moreover, ge , known as pardaxin, is a marine amp and the ge -based vaccine has shown the ability to enhance antitumor immunity in mice (huang et al., ) . the activity of amps can be divided into categories according to the statistics of the adp database. these categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (hiv), and anti-tumor peptides (figure ). antibacterial peptides account for a large part of amps and have a broad inhibitory effect on common pathogenic bacteria, such as vre, acinetobacter baumannii, and mrsa in clinical medicine and s. aureus, listeria monocytogenes, e. coli in food and salmonella, vibrio parahaemolyticus in aquatic products. many natural and synthetic amps like nisin, cecropins and defensins have shown good inhibition activity to gram-positive bacteria and gram-negative bacteria. in recent research, amps p (yirkirrffkklkkilkk-nh ) and p (syerkinrhfktlkknlkkk-nh ), which are designed based on aristicluthys nobilia interferon-i, inhibit mrsa and show a low cytotoxicity . antifungal peptides are a subclass of amps that address fungal infections with enhanced drug resistance. many afps have shown excellent anti-fungal activities against common pathogenic fungi, such as aspergillus and candida albicans in clinical medicine, yeast, filamentous fungi (e.g., aspergillus flavus), mold in food and agriculture. except for brevinin, ranatuerin, cecropins, many synthetic peptides also show good antifungal activity. for example, aurh , derived from aurein . , can effectively treat c. albicans infection, which has a lethal rate up to % (madanchi et al., ) . aflatoxin, which is a carcinogen produced by a. flavus, is harmful to the human body. many afps can inhibit the growth of a. flavus. for example, an afp with a sequence of fpshtgmsvppp can inhibit the growth of a. flavus md . a total of antifungal peptides isolated from lactobacillus plantarum te and their mixture can reduce a. flavus spore formation in fresh maize seeds (muhialdin et al., ) . moreover, two chemically synthesized radish amps show a good inhibitory effect against different yeast species, such as zygosaccharomyces bailii and zygosaccharomyces rouxii (shwaiki et al., ) . viruses cause serious harm to human life and huge economic losses to the animal husbandry. the covid- , which is the recent outbreak, has caused great loss of lives and properties. furthermore, foot-and-mouth disease virus, avian influenza virus (aiv), and hiv are long-term threats to human life. so, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. antiviral peptides show a strong killing effect on viruses mainly by ( ) inhibiting virus attachment and virus cell membrane fusion, ( ) destroying the virus envelope, or ( ) inhibiting virus replication (jung et al., ) (shown in figure ). a recent report has shown that amp epi- mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus . moreover, infectious bronchitis virus (ibv) is the pathogen of infectious bronchitis and the inoculation of swine intestinal amp (siamp)-ibv mixed solution remarkably reduced the mortality of chicken embryos compared with the ibv infection group, showing the good inhibitory activity of siamp on ibv (sun et al., ) . anti-hiv peptides are a subclass of anti-viral peptides. the most important examples of these peptides include defensins (including αand β-defensins, which have different mechanisms), ll- , gramicidin d, caerin , maximin , magainin , dermaseptin-s , dermaseptin-s , siamycin-i, siamycin-ii, and rp (madanchi et al., ) and antiviral peptide fuzeon tm (enfuvirtide) has been commercialized as an anti-hiv drug (ashkenazi et al., ) . due to the global spread of the covid- (figure a ), the antiviral peptides against the coronavirus will be discussed in more detail. coronaviruses (covs) belong to the family coronaviridae; they are enveloped viruses with a positive-sense single-stranded rna genome and have a helical symmetry (franks and galvin, ) . covs, including severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (mustafa et al., ) , and the recent outbreak of covid- have caused serious threats to human life and property. covs can cause lifethreatening respiratory diseases and the viral particle is formed by spike glycoprotein (s), the envelope (e), the membrane (m), and the nucleocapsid (n) (vilas boas et al., ) . it should be noted that their infectivity requires viral spike (s) protein. fusion inhibitor peptides combine with the s protein to interfere with its folding and prevent infection. besides, the s domain of the sars-cov s protein contains heptad repeat hr and hr sequences. peptide hr (hr : sltqinttlldltyemlslqqvvkalnesyidlkel) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of s protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (fds) (du et al., ; park and gallagher, ) . according to recent research, the lipopeptide ek c , derived from ek (sldqinvtfldleyemkkleeaikkleesyidlkel), is the most effective fusion inhibitor against covid- s proteinmediated membrane fusion . homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against mers-cov (marimuthu et al., ) . two amps from the non-structural protein nsp of sars-cov, k , and k , can inhibit sars-cov replication (ke et al., ) . furthermore, rhesus thetadefensin (rtd- ) treated animals have a marked reduction in mortality in the presence of sars-cov while the peptide alone shows airway inflammation and the one possible mechanism of action for rtd- is immunomodulatory (wohlford-lenane et al., ) . in general, amps against coronavirus can be roughly classified as i) peptides derived from hr , hr and rbd subunits of the spike protein, ii) peptides derived from other amps, iii) peptides derived from non-structural protein (mustafa et al., ) . furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between covid- and ace (angiotensin-converting enzyme ) to inhibit covid- entrance in cells ( figure b ) (souza et al., ) . finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. this reminds us that nasal drug delivery (ndd) is a potential therapy for amps as anti-coronavirus drugs. besides, the antiviral database avpdb includes numerous antiviral peptides. parasitic protozoa can cause diseases in human and animals through a variety of routes, including animal-to-person or person-to-person contact, water, soil, and food (chalmers et al., ) . and with the increase in parasite drug resistance, the need for new treatments has increased. antiparasitic peptides show their killing effect on parasites which cause diseases such as malaria and leishmaniasis (mangoni et al., ; rhaiem and houimel, ) and amps like cathelicidin, temporins-shd show high inhibition activity against parasites (abbassi et al., ) . in recent research, epi- , a marine synthetic amp, can remarkably inhibit trichomonas vaginalis by destroying its membrane (neshani et al., ) . the peptide jellein derived from bee royal jelly which has introduced above and -amino acid amp kdel (lysine, aspartic acid, glutamic acid, and leucine) has shown a significant effect on the leishmania parasite (cao et al., ; zahedifard et al., ) . however, it should be noted that their mechanisms are not the same. cyanobacterial peptides differ from higher-eukaryote amps because their antiparasitic action depends on specific protein targets. thus, these target parasites can be distinguished accurately even though they belong to the same family or genus (rivas and rojas, ) . the acps show anticancer mechanisms by ( ) recruiting immune cells (such as dendritic cells) to kill tumor cells, ( ) inducing the necrosis or apoptosis of cancer cells, ( ) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and ( ) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells (wu d. et al., ; ma et al., ) . tritrpticin and its analogs induce considerable toxicity toward jurkat cells in vitro, whereas indolicidin and puroindoline a can also act as acps (arias et al., ) . it should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of acps and they can constrain and influence each other. thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin- , and interleukin- β) and some of them also inhibit inflammatory signals like nf-κb, mapk, and jak-stat pathways (meram and wu, ; gao et al., ) . anti-diabetic peptides play their function by modulating the g proteincoupled receptor kinase (grk / ) or activating glucagonlike peptide- (glp- ), glucagon receptors (marya et al., ; graham et al., ) . however, it is not accurate to classify these types of peptides as amps and bioactive peptides may be more convincing. proline is a typical non-polar amino acid. pramps behave differently from other amps, that is, they enter bacterial cytoplasm by the inner membrane transporter sbma instead of killing bacteria through membrane destruction (mattiuzzo et al., ) . once in the cytoplasm, pramps target ribosomes and block the binding of aminoacyl-trna to peptidyltransferase center or trap decoding release factors on the ribosome during the termination of translation to interfere with protein synthesis (seefeldt et al., ) . for instance, tur a, which is an orthologous amp of bovine pramp bac discovered from tursiops truncatus, interferes with the transition from the initial phase to the extension phase of protein synthesis by binding to ribosomes. in addition, different pramps lack a high sequence similarity but have short motifs containing repeating proline and arginine (arg) residues (e.g., -ppxr-in bac and -prpxin bac ) (mardirossian et al., (mardirossian et al., , . although pramps mainly kill gram-positive bacteria, ppr-amp , a proline-rich amp identified from crab (scylla paramamosain), exhibits antimicrobial activity against gram-positive and gram-negative bacteria (imjongjirak et al., ) . besides, pieces of research have shown that pramps have immunostimulation activity (li w. et al., ) . tryptophan (trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane's abundant anionic component. and it seems that trp residues play the role of natural aromatic activators of arg-rich amps by ion-pair-π interactions (walrant et al., ) , thereby promoting enhanced peptide-membrane interactions (chan et al., ) . in addition to indolicidin and triptrpticin which both are famous amps that rich in arg and trp residues. octa (rrwwrwwr) is also a typical trp-and arg-rich amp that inhibits gram-negative e. coli and pseudomonas aeruginosa and gram-positive s. aureus. and short trp-and arg-rich amps designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria (strøm et al., ; bacalum et al., ) . histidine is a common basic amino acid, and histidine-rich amps show good membrane permeation activity. hv is a histidinerich amp designed based on rr(xh) xdpgx(yh) rr-nh (where x represents i, w, v, and f). this peptide increases the permeability of bacterial cell membranes to cause cell membrane rupture and death. in addition, hv inhibits bacterial movement in a concentration-dependent manner and shows a strong anti-inflammatory effect by inhibiting the production of tumor necrosis factor α (tnf-α) ). an amp designed based on octa has shown good therapeutic potential by replacing its arg residues with histidine (bacalum et al., ) . furthermore, l h , which is designed based on the linear cationic amphiphilic peptide magainin, also shows good antibacterial activity and cell penetration properties by inserting four histidine sequences in leucine and alanine (lointier et al., ) . the r group of glycine is generally classified as a non-polar amino acid in biology. glycine-rich amps, such as attacins and diptericins, widely exist in nature (lee et al., ; kwon et al., ) . these peptides contain % to % glycine residues, which have an important effect on the tertiary structure of the peptide chain. a glycine-rich amp derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional amps (d'este et al., ) . furthermore, the glycine-rich central-symmetrical gg is an ideal commercial drug candidate against clinical gramnegative bacteria . antimicrobial peptides can be divided into four categories based on their structures including linear α-helical peptides, β-sheet peptides, linear extension structure, and both α-helix and β-sheet peptides ( figure ) (lei et al., ) . moreover, progressively cyclic peptides and amps with more complex topologies (including lasso peptides and thioether bridged structures) are reported (koehbach and craik, ) . the membrane-targeting mechanisms of amps can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models (figure ). the toroidal pore model is also known as the wormhole model. in this model, amps vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of - nm (matsuzaki et al., (matsuzaki et al., , . the typical examples of this model are magainin , lacticin q, and arenicin. furthermore, cationic peptides, including tc , tc , and bp , compromise the membrane barrier by creating fluid domains (omardien et al., ) . antimicrobial peptides aggregate with each other, penetrate the bilayer of the cell membrane in the form of multimers, and form channels that result in the cytoplasmic outflow. in severe cases, amps can induce cell membrane collapse and lead to cell death (lohner and prossnigg, ). for instance, alamethicin performs its pore-forming activity by using this model. besides, hairpin amp protegrin- can form stable octameric β-barrels and tetrameric arcs (half barrels) in implicit and explicit membranes by simulations (lipkin and lazaridis, ) . antimicrobial peptides are arranged parallel to the cell membrane. their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. amps will cover the membrane surface that similar to a carpet and destroy the cell membrane in a 'detergent'-like manner (oren and shai, ) . however, this pore-forming mechanism requires a certain concentration threshold and the required concentration of amps is high. human cathelicidin ll- exhibits its activity through this mechanism, and amps with β-sheet structure also play a role in this model (shenkarev et al., ; corrêa et al., ) . polarized light-attenuated total reflection fourier transform infrared spectroscopy (atr-ftir) was used to study the effect of amp cecropin p on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen's cell membrane to destabilize and eventually destroy the cell membrane . membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in figure ) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. the main lipids in cell membranes include glycerophospholipids (gpls), lysolipids, sphingolipids, and sterols. phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (pc), phosphatidylinositol (pi), pe, and phosphatidic acid (pa) are the main gpls in fungal cell membranes (ejsing et al., ; singh and prasad, ; li et al., ) . furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher pc content. meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol (faruck et al., ) . many amps take advantage of differences in membrane components to exert their effects. antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (cpps) which typically comprise - amino acids and can translocate across the cell membrane. cpps could be categorized according to physicochemical properties into three classes: cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. anti-biofilm peptides target the biofilms by different mechanisms including ( ) degradation of signals within biofilms; ( ) permeabilize within cytoplasmic membrane/eps; ( ) modulating eps production etc. and then can address chronic multi-resistant bacterial infections (pletzer et al., ; ribeiro et al., ; guidotti et al., ; derakhshankhah and jafari, ; rajput and kumar, ) . for instance, saap- , synthesized based on ll- , showed activity to prevent biofilm formation by s. aureus and a. baumannii (crunkhorn, ) . the way of amps entering cells is direct penetration or endocytosis. after entering the cytoplasm, amps will identify and act on the target. depending on the target, amps can be divided into the following categories. antimicrobial peptides affect transcription, translation, and assembly into functional peptides through molecular chaperone folding by interfering with related enzymes and effector molecules. for example, bac - targets ribosomes to inhibit protein translation (mardirossian et al., ) , whereas tur a inhibits protein synthesis in e. coli and thermus thermophilus by inhibiting the transition from the initial phase to the extension phase. however, the differences between tur a and bac also lead to various ways of binding to ribosomes and interacting with the ribosomal peptide exit tunnel (mardirossian et al., ) . but some amps' have different targets. for instance, genome-wide transcription shows that the amp dm can affect many important intracellular pathways of protein biosynthesis . chaperones are key proteins for correctly folding and assembling newly synthesized proteins and make them have stereoisomerism, which makes amps have cell selectivity and can prevent cytotoxicity. according to a previous review: both pyrhocoricin and drosocin can prevent dnak from refolding misfolded proteins by inducing a permanent closure of the dnak peptide-binding cavity (kragol et al., ; le et al., ; wrońska and boguś, ) . antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. indolicidin, a c-terminal-amidated cationic trprich amp with amino acids, specifically targets the abasic site of dna to crosslink single-or double-stranded dna and it can also inhibit dna topoisomerase i (subbalakshmi and sitaram, ) . tfp (tissue factor pathway inhibitor) - tc , which is an amp from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades dna and rna (he et al., ) . many amps can inhibit various metabolic activities by inhibiting protease activity. for example, histatin has a strong inhibitory effect on the proteases secreted by the host and bacteria. amps enap- and indolicidin inhibit microbial serine proteases, elastase, and chymotrypsin (le et al., ) . cathelicidin-bf is a peptide isolated from the venom of bungarus fasciatus, it can effectively inhibit thrombin-induced platelet aggregation and further block protease-activated receptor (shu et al., ) . antimicrobial peptides inhibit cell division by inhibiting dna replication and dna damage response (sos response), blocking the cell cycle or causing the failure of chromosome separation (lutkenhaus, ) . for instance, app (glaraltrllrqltrqltra), which is an amp with amino acid residues, can efficiently kill c. albicans because of its cell-penetrating efficiency, strong dna-binding affinity, and ability to induce s-phase arrest in intracellular environment . mciz, which has amino acid residues, is an effective inhibitor of bacterial cell division, z-ring formation, and localization (cruz et al., ) . moreover, it has been reported that several afps have damaging effects on the organelles of fungi. for example, histintin can interact with mitochondria, causing the production of ros, and inducing cell death (helmerhorst et al., ) . in addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (lps), lipid a and mannoproteins, are potential targets for amps. specifically, gram-positive and gram-negative bacteria are classified based on their bacterial cell wall structure. gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of lps. lps has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., ca + and mg + ) to form an electrostatic network (nikaido, ) . this electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of gram-negative bacteria. the main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of , -β-n-acetylglucosamine) and the mutations in the relevant genes of the lps pathway and phospholipid trafficking provide resistance to the amps (cabib, ; spohn et al., ) . mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). these proteins differ from human cell membrane proteins and are potential targets of afps (rautenbach et al., ) . furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of amps and these theories could support the design of amps with low cytotoxicity. antimicrobial peptides have good application prospects. however, amps have the following problems. ( ) amps damage the cell membrane of eukaryotes and cause hemolytic side effects; ( ) rising production costs and technical problems limit their manufacture; ( ) their stability is limited at certain ph; ( ) amps have reduced activity under the presence of iron and certain serum; ( ) amps are easily hydrolyzed by proteases. therefore, the ideal amp should meet the following characteristics: (i) high antimicrobial activity; (ii) low toxicity to mammalian membranes; (iii) high protease and environment stability; (iv) low serum binding capacity and (v) ease of access and low cost production . therefore, designing amps to achieve the desired effect has attracted increasing attention. the rational design of antibacterial peptides should focus on the following five aspects: chain length, secondary structure, net charge, hydrophobicity, and amphiphilicity and these have been mentioned in many studies and this review will focus more on several specific methods of antimicrobial peptide design. site-directed mutation refers to the redesign of natural antimicrobial peptides by adding, deleting or replacing one, or several amino acid residues (torres et al., ) . the de novo design of peptides attaches importance to the design of amphiphilic amps (guha et al., ) . for example, gala is a well-known de novo-designed amp. amphipathic α-helical peptide gala is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + (goormaghtigh et al., ) . the repeated sequence (xxyy)n, where x and x are hydrophobic amino acids, y and y are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical amps and successfully designs broadspectrum α-helical amps. sequences (lkkl) and (wkkw) . have the highest selectivity (khara et al., ) . moreover, l l k m w model peptides are also de novo-designed peptides. amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. among the model peptides, l k w has good anti-mrsa activity (lee et al., ) . sequence templates can be obtained by comparing a large number of structurally homologous fragments of natural amps (such as hdps) and extracting conservative patterns based on the type of residue (such as charged, polar, hydrophobic, etc.) (zelezetsky and tossi, ) . based on the modification, the parameters, such as helix formation tendency, cationic, amphiphilicity and overall hydrophobicity, can be systematically changed. for instance, cecropin, magainin, protegrin, and lactoferrin have all been used as amp templates (fjell et al., ) . peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to amps during the self-assembly of peptides. for example, kld- (kld) is a self-assembling peptide with amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. the addition of arg residues in kld shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures (tripathi et al., ) . dimer structure can also be used to enhance the antimicrobial activity of amps and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation (malekkhaiat häffner and malmsten, ). various chemical modifications of amps, including residue phosphorylation, the addition of d-amino acids or unnatural amino acids (homoarginine), cyclization, halogenation, acetylation, and peptidomimetics, have been used to improve the stability of peptides against proteases. given that the enzyme is stereospecific, the incorporation of unnatural d-amino acids into the amp sequence can reverse the stereochemistry and prevent protease degradation (zhong et al., ) . the so-called peptidomimetics, whose main elements mimic the structure of peptides, are usually produced by modifications, such as chain extension or heteroatom incorporation of existing peptides (patch and barron, ) . ornine, which is an unnatural residue with a positive charge and has a high resistance to protease activity, is also used in non-chemical modification. replacing trp residues with family residues, such as β-dihydrophenylalanine, can stabilize secondary structures and improve antibacterial properties (maurya et al., ) . halogenation is highly related to the activity, specificity, and stability of amps. in the latest report, halogen is introduced into jelleine-i which is a short peptide isolated from the royal jelly of honeybees (apis mellifera) by replacing phenylalanine with a halogenated phenylalanine analog, increasing the antibacterial activity in vitro and anti-biofilm activity. in addition, the proteolytic stability of jelleine- is increased by - times by halogenation (jia et al., ) . the halogenated peptidomimetic α,α-disubstituted β-amino amides are also promising bacteriostatic drugs that have inhibitory effects on more than multi-resistant clinical isolates of gram-positive and gram-negative bacteria (paulsen et al., ) . halogenation is also related to the specificity of amps. the o-fluorine substitution in phenylalanine residues maintains the activity of temporin l on e. coli but leads to the loss of activity on s. aureus and p. aeruginosa (setty et al., ) . three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural amps. the synthesis of disulfide bonds often complicates the development of synthetic peptides. the circularisation of the main chain of arenicin- molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity (orlov et al., ) . the hdps tachyplesins i, ii, and iii and their cyclic analogs cti, ctii, and ctiii, respectively, have similar structures and activities and can resist bacterial and cancer cells. the cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities (vernen et al., ) . capping refers to the addition of specific motifs or modifications, such as amidation at the c-terminus and acetylation at the n-terminus, rendering amps with more natural peptide characteristics. post-translational modifications play an important role in the function of amps and are the most commonly used in peptide design. the c-terminal rana box (consisting of a c-terminal cyclic heptapeptide with a conservative disulfide bond) and amide group are important c-terminal capping methods. for example, the c-terminal amide group of maximin h can enhance antibacterial efficacy without increasing lytic ability (dennison et al., ) . the n-terminal lipidated analog c vg krkp shows enhanced antibacterial activity against various gram-negative bacteria. the functions of n-terminal lipidation include (i) increasing lps neutralization, (ii) increasing stability to proteases and peptidases, and (iii) reducing cytotoxicity (datta et al., ) . furthermore, hydrophobic end labeling is a commonly used method to increase the activity of antimicrobial peptides. acyl lipid peptides have a linear or cyclic structure in which one or more hydrocarbon tails are connected to the n-terminus of a short oligopeptide (chu-kung et al., ) . lipopeptides have covalently attached hydrophobic moieties, such as sterols or fatty acids. aromatic amino acid terminal labeling is also the main hydrophobic terminal labeling method. tryptophan (w) and phenylalanine (f) are the commonly used aromatic amino acids. their large and polarisable residues have an affinity for the interface, and the w/f tag is also sensitive to the differences between ergosterol and cholesterol and can prevent self-assembly. this condition results in low aggregation numbers and high critical aggregation concentrations (schmidtchen et al., ) . peptide conjugation has been the goal of most research in recent years to produce active and stable amps with high selectivity. different side chains or amp fragments can be used aside from the repetition of the same amino acid motifs. for example, conjugating fatty acids with a length of - carbon atoms to the th or th side chain of the d-amino acids of ano-d , improves antibacterial selectivity and anti-biofilm activity. in addition, the new peptide exhibits high stability against trypsin, serum, salt, and different ph environments (zhong et al., ) . the conjugation of different amps can also be performed. for example, the hybrid peptide (pa -gnu ) constructed by the addition of pa to gnu has a high activity and specificity to p. aeruginosa . smamps include a broad family of molecular entities based on the structure and function of amps. however, their backbones are not entirely based on α-amino acids, including β-amino acid oligomers, arylamide oligomers, and phenylene ethynylenes (michael henderson and lee, ) . for instance, smamp , which is a potential drug for intravenous treatment, causes no drug resistance and has a strong inhibitory effect on mrsa and vancomycin-resistant enterococcus faecium (tew et al., ) . peptoids are peptide isomers, in which the side chain is bonded to the main chain nitrogen instead of α-carbon or poly-nsubstituted glycine in which the side chain is connected to amide nitrogen instead of the α-carbon on the main chain (andreev et al., ) . for example, the cationic peptide sa (iowagolfolfo-nh ) and its poly-n-substituted glycine homolog spo (ninonwnangnonlnfnonlnfno-nh ) inhibit the planktonic and biofilm formation of a. baumannii strains, which are susceptible to multi-drug resistance (sharma et al., ) . motifs with specific functions have been reported increasingly. these motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. this motif includes two tripeptide structures, including gly-gly-his or val-ile-his, which are added at the end of the peptide chain. atcun-containing amps in the presence of hydrogen peroxide and ascorbic acid combine with cu + to induce the valence of copper ions between + and + oxidation states and form an atcun-cu (ii) complex, generating ros by fenton-like reactions. extracellular polymeric substances (eps) are important for biofilms and can enhance the resistance of cells to antibacterial agents (flemming, ) . atcun-amps have been used to degrade environmental dna, which is one of the major components of eps. several related practical applications have been reported. for example, the biological activity against carbapenem-resistant enterobacteriaceae is increased by adding this motif to the n-terminus of an alpha-helical amp (such as cm ). besides, the cu-atcun derivative of ov- containing a c-terminal ggc sequence showed high levels of membrane permeation and lipid peroxidation. the concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions (alexander et al., ; agbale et al., ) . rana box: rana box is a heptapeptide motif (cglxglc) from the nigrocin family. rana box consists of two cysteine residues that are separated by four or five other residues on the side and can form a cyclic disulfide bond. rana box peptide has shown structural analogies with polymyxin (colistin), and the primary structure of the rana box motif is important in determining bacteriostatic activity (kozić et al., ) . the deletion of the 'rana box' motif will cause the amp antibacterial effect to disappear, but replacing the natural 'rana box' sequence of amps with amidated phenylalanine can expand its efficacy against antibiotic-resistant microorganisms, including mrsa and p. aeruginosa, and reduce cytotoxicity. this phenomenon also shows that the effect of the motif on amps needs to be determined based on the specific situation and is not completely beneficial (bao et al., ) . the lps binding motif (g-wkrkrf-g) can produce a broad spectrum of antibacterial activity when introduced into the c-terminus of temporin- ta and temporin- tb (close isoforms of temporin) (mohanram and bhattacharjya, ) . antifungal peptides have a conserved gxc(x − ) c γ-core motif (residues - , gkcykkdnic; d-isomer) at its n-terminus, which is a cation part of the ring. this conserved motif interferes with the integrity of the plasma membrane of the cell (yount and yeaman, ) . conserved γ-core motifs are directly involved in protein-membrane interactions and strongly contribute to membrane binding (utesch et al., ) . if replace d-phe -pro sequence in peptide chain with d-phe- -abz turn motif ( -abz is an abbreviation of -aminobenzoic acid d-amino acid) in amp tyrc a, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. unlike the traditional β-turn motif, the d-phe- -abz motif can be used as a tool for β-hairpin libraries. the hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the d-phe- -abz motif. moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broadspectrum antibacterial activity and low hemolysis rate (cameron et al., ; cameron et al., ) . the ngr motif is composed of asn-gly-arg, and amps with this structure have strong cytotoxicity ( table ). the data indicate that the new amps containing ngr may bind to cd + or αvβ + tumor cells by binding to cd or αvβ , respectively, to exert anti-tumor activity, especially on cd + tumor cells . the central gxxxg motif can induce strong self-assembly and have been already used in the design of amps (brosig and langosch, ; krauson et al., ) . bovine lactoferrin b is an amp composed of amino acid residues and has antibacterial, antifungal, and antiparasitic activities. the multivalent molecules lfcinb ( - ) and lfcinb ( - ) contain the lfcinb ( - ) motif (rrwqwr) and show inhibition activity against e. coli, p. aeruginosa, and s. aureus. chimeric peptide chimera containing two motifs, namely, the rrwqwr of lfcinb ( - ) and the rllr of bfii computer design includes simple statistical modeling, structureactivity relationships study (abdel monaim et al., ) , neural networks (müller et al., ) , deep learning (veltri et al., ) , word embedding (hamid and friedberg, ) and machine learning. for example, a machine learning method by matlab is proposed based on the concept of scoring the contribution of each amino acid's antibacterial activity (wu x. et al., ) . the genetic algorithm was used to design the amphiphilic α-helical peptide guavalin , which has an uncommon amino acid composition (three tyrosine and three glutamine residues) and interestingly causes membrane hyperpolarization, which is a different mechanism from those of other amps (porto et al., ) . two research methods have been developed based on the research background of quantitative structure-activity relationships: prediction method based on amp therapeutic index and the identification of novel potential amps from the expressed sequence tag database based on the principles of the highly conserved signal peptide subclasses related to amps (juretić et al., ) . in this way, a variety of amp variants can be obtained. if combined with high-throughput screening, it can effectively obtain the desired amp. for instance, some new amps are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity (krauson et al., ) . cations, such as na + and mg + , may affect amp activity . however, the different valences of metal ions have varied effects on amps. for example, divalent cations show stronger antagonism to bacteria than monovalent cations with thanatin and s-thanatin, which are insect amps (wu et al., ) . in the presence of nacl, the signal response during the association phase remarkably decreased in single-cycle and multi-cycle kinetic experiments, resulting in a decreased association rate. this occurrence may be caused by the shielding effect of nacl between the cationic peptide and the zwitterionic membrane. another possible reason is that na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (sabapathy et al., ) . the reduced activity of synthetic peptide [rllr] under high salt concentration is possibly caused by the destruction of its α-helix structure. table shows that several amps, including histatin, myxinidin, and hepcidin, contain atcun motifs (amino terminal copper and nickel with xxh sequence). iron is the most abundant metal ion in human saliva, but the combination with this metal ion results in the loss of the α-helix of histatin and greatly reduces its antifungal activity (puri et al., ) . however, the coordination of copper (ii) and nickel (ii) ions can induce the formation of ros, which is essential for bactericidal activity (jeżowska-bojczuk and stokowa-sołtys, ). anionic amps have a large number of negatively charged aspartic and glutamic acid residues (lakshmaiah narayana and chen, ) . they require zinc as a functional cofactor and the zinc complex shows stronger antibacterial activity (jiang et al., ) . several of these amps use metal ions to form cationic salt bridges with the negatively charged components of the microbial membrane to penetrate the membrane. anionic amps may attach to ribosomes or inhibit ribonuclease activity when in the cytoplasm (jeżowska-bojczuk and stokowa-sołtys, ). metal ions also affect the self-assembly of peptides. these ions can recognize specific amino acids, such as lysine and glutamic acid, and may form salt bridges between peptide molecules to induce peptide self-assembly. for example, zn + can stabilize the aggregation of peptides on the cell membrane, which results in the enhanced antibacterial effect of dcd- l in the presence of zn + (tian et al., ) . ph many amps are stable and retain their antimicrobial activity in a wide ph range. amps have enhanced activity at low ph because of their basic properties. this condition is related to the protonation of histidine at acidic ph, which promotes electrostatic interactions with anionic surfaces, including lps and the anions of phospholipids, and subsequently enhances antibacterial properties. the effect of ph on the antibacterial activity of amps varies. for example, thanatin's activity at neutral ph is slightly higher than that under acidic conditions. by contrast, the activity of xylan on e. coli, listeria, and c. albicans is remarkably higher at ph . than at ph . (holdbrook et al., ) . the inactivation of the histidinecontaining amp c g-his under low ph conditions involves ph-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to ph and lipid composition, may be affected by binding and conformation. peptides can also enhance bacterial membrane permeability at low ph (hitchner et al., ) . thrombin-derived c-terminal peptides (tcps) will also change the mode of cd (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from ph . to ph . (holdbrook et al., ) . a dimer (e.g., p- ) can be created to provide amps with resistance to a higher ph range. the sensitivity of this ph-sensitive amp can be used to achieve a certain targeting effect in practical applications. in addition, charge interaction is one of the most important factors in peptide self-assembly. ph affects the charge state of amino acid and substituent functional groups. therefore, adjusting the ph is the most common method for controlling peptide assembly and disassembly (tian et al., ) . proteases have a strong destructive effect on amps. for instance, ll- , which has the strongest inhibitory effect on chlamydial infection, is inhibited by the protease chlamydial protease-like activity factor (cpaf) secreted by chlamydia (tang et al., ) . studies have been focused on the design of amp carriers to solve this problem (lewies et al., ; nordström et al., ) . the presence of chitosan-silica solid support of kr- peptide can protect it be hydrolyzed by α-trypsin, and the degree of protection is increased by % compared with the free kr- (diosa et al., ) . however, several enzymes, such as protease , esterase and phosphatase , cut the blocking group of the peptide and trigger the self-assembly of the peptide, which positively affects amps (tian et al., ) . antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases (de la fuente-núñez et al., ) . for example, α-defensins hnp- , hnp- , and hnp- showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of amps (guaní-guerra et al., ) . likewise, amps secreted by the paneth cells in the mammalian gut are important to shape the gut microbiota (bevins and salzman, ) . the application of amps in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. but there are only three amps that have been approved by fda including gramicidin, daptomycin, and colistin. dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. dental caries is a prevalent oral disease and some acidogenic bacteria like streptococcus sp. are the main cariesassociated pathogens (izadi et al., ) . several amps have good application potential. for instance, peptide zxr- (fkiggfikklwrslla) has shown potent activities against pathogenic bacteria of dental caries, streptococcus mutans, streptococcus sobrinus, and porphyromonas gingivalis and peptide pac- (clinical trial identifier: nct ) that has been sold over the counter in taiwan for treating oral candidiasis (chen l. et al., ) . in surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life (thapa et al., ) . several amps have shown the therapeutic potential of these diseases. for example, amp pxl shows pronounced efficacy as an anti-infective agent in burn wounds in mice and amp d a has been in the third phase of clinical trials for treating burn wound infections (björn et al., ) . in ophthalmology: human eyes are prone to be infected by several organisms including bacteria and fungi in which s. aureus, streptococcus pneumoniae, p. aeruginosa, aspergillus spp., and c. albicans are the most relevant pathogens (silva et al., ) . although amps such as lactoferricin b, protegrin- exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. with the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology (khan and lee, ) . additional methods need to be performed for the application of amps as drugs in medicine. the main strategies include ( ) constructing precursors to reduce cytotoxicity and improve protease stability, ( ) using amps in combination with existing antibacterial agents, ( ) inducing the correct expression of amps with appropriate drugs and using engineering probiotics as vectors to express amps. for example, in the field of wound repair, different formulation strategies, such as loading amps in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver amps to the wound (borro et al., ; thapa et al., ) . in recent research, the sponges developed from modified starch and hs-peg-sh are covalently immobilized with amp showed effective antibacterial activity (yang et al., ) . more technical means, including pheromone-labeled amps, local environment-triggered amps (enzyme precursor drug release system, ph-activated amps, etc.), have been developed to improve the targeting mechanism of amps. furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of amps (magana et al., ) . hybrid peptides have also been used to build targeting peptides. for example, pa , which is a p. aeruginosa-targeting peptide, was combined with gnu (a broad-spectrum amp) to construct a hybrid peptide (pa -gnu ) that targets oprf protein and has good bactericidal activity and specificity . furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a -membered cyclic peptide consists of amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic (durand et al., ; lampejo, ) . although they are antibiotics, they have provided broader ideas for the design of amps. food preservatives have potential harm to the human body. therefore, natural preservatives are being advocated by more people. amps have a good inhibitory effect on common bacteria and fungi in food, and many amps are resistant to acids, alkalis, and high temperatures are easily hydrolyzed by proteases in the human body. thus, amps are a promising alternative to preservatives. nisin is a bacteriocin produced by l. lactis subspecies. lactic acid bacteria have been widely used as food preservatives. nisin is categorized as generally recognized as safe (gras) by the us food and drug administration (fda) and is used as a food preservative in other countries (khan and oh, ) . however, only nisin and polylysine are currently approved by the fda as food additives (santos et al., ) . pedocin pa- , a bacteriocin consisting of amino acids produced by a diplococcus, is also used as a food preservative and is sold on the market under the trade name alta . pedocin pa- is used as a food additive to inhibit the growth of l. monocytogenes, which can cause meat deterioration (settanni and corsetti, ) . enterocin as- is an amp used to preserve cider, fruit and vegetable juices, and enterocin ccm is used to preserve soy milk (rai et al., ; santos et al., ) . encapsulating bacteriocins into liposomes is a new method used to overcome the problems of amps in food applications (such as proteolytic degradation or interaction with food ingredients) (da silva malheiros et al., ) . moreover, active packaging by adding amps is a novel packaging method that has great potential in the food industry. for instance, ε-poly-l-lysine is used in conjunction with starch biofilms to show good inhibitory effects on aspergillus parasiticus (aflatoxin producer) and penicillium expansum and nisin have the potential to be dairy preservative because it is a highly surface-active molecule (luz et al., ) . the european union banned the use of animal growth promoters in animal feed in . thus, a new antibacterial strategy is needed. many amps are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance (liu et al., ; bao et al., ) , immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics cote et al., ) . for example, siamp has a good effect on the treatment of ibv in chicken (sun et al., ) . by adding swine gut intestinal antimicrobial peptides (sgamp), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions (hu et al., ) . frog caerin . , european sea bass dicentracin and nk-lysine peptides (nklps) have good inhibitory effects on nodavirus, septicaemia haemorrhagic virus, infectious pancreatic necrosis virus and spring viremia carp virus, which are devastating to fish farming (león et al., ) . the amp in soybean meal fermented by b. subtilis e effectively inhibits v. parahaemolyticus and vibrio alginolyticus and enhances the resistance level of litopenaeus vannamei against v. parahaemolyticus when added to feeds (cheng et al., ) . for agriculture, the plant pathogenic infection of bacteria and fungi causes the loss of economy, for instance, aspergillus flavus infection of corn and peanuts, citrus green mold caused by penicillium digitatum, gray mold disease caused by botrytis cinerea on strawberries and geotrichum citriaurantii infection of citrus fruit all cause great harm to the growth and post-harvest of agricultural products (liu et al., ; liu et al., ) . several afps have shown prospect to control these problems. however, the practical application of antimicrobial peptides in the transportation and preservation of agricultural products is still lacking, because the use of antimicrobial peptides will greatly increase the cost in the transportation of fruits and vegetables (application examples of amps in these four fields are shown in table ). antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. several restrictive factors hinder the application of amps. the interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective amps. computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of amps. how to further understand the correlation between amps and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. on this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of amps under complex physiological conditions. several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing amps to solve the problem of protease hydrolysis. most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. in addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. furthermore, studying the amp expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more amps in nature are discovered. further research is needed on the reported amps to solve the problem on structure-function relationship. as a branch of peptide drugs, amps need to progress with the advancement of medical science against the background of the current low success rate of the clinical application of amps. more attention can be focused on food, agriculture, and animal husbandry. qk and yh: conceptualization, methodology, writing -original draft preparation, and writing -review and editing. all authors contributed to writing and reviewing the manuscript. antibacterial and leishmanicidal activities of temporin-shd, a -residue long membrane-damaging peptide teixobactin as a scaffold for unlimited new antimicrobial peptides: sar study antimicrobial and antibiofilm activities of helical antimicrobial peptide sequences incorporating metal-binding motifs amino terminal copper and nickel binding motif derivatives of ovispirin- display increased antimicrobial activity via lipid oxidation hydrophobic interactions modulate antimicrobial peptoid selectivity towards anionic lipid membranes selective anticancer activity of synthetic peptides derived from the host defence peptide tritrpticin multifaceted action of fuzeon as virus-cell membrane fusion inhibitor the threat of antimicrobial resistance in developing countries: causes and control strategies modulating short tryptophan-and arginine-rich peptides activity by substitution with histidine effects of pig antibacterial peptides on growth performance and intestine mucosal immune of broiler chickens modification targeting the "rana box" motif of a novel nigrocin peptide from hylarana latouchii enhances and broadens its potency against multiple bacteria paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis efficacy and safety profile of the novel antimicrobial peptide pxl in a mouse model of infected burn wounds microgels and hydrogels as delivery systems for antimicrobial peptides the dimerization motif of the glycophorin a transmembrane segment in membranes: importance of glycine residues two novel techniques for determination of polysaccharide crosslinks show that crh p and crh p attach chitin to both β( - )-and β( - )glucan in the saccharomyces cerevisiae cell wall tyrocidine a analogues bearing the planar d-phe- -abz turn motif: how conformation impacts bioactivity acyclic peptides incorporating the d-phe- -abz turn motif: investigations on antimicrobial activity and propensity to adopt β-hairpin conformations yeast-based synthetic biology platform for antimicrobial peptide production in vitro leishmanicidal activity of antimicrobial peptide kdel against leishmania tarentolae parasite detection in food: current status and future needs for validation tryptophan-and arginine-rich antimicrobial peptides: structures and mechanisms of action a novel antimicrobial peptide against dental-caries-associated bacteria as-cath - , novel cathelicidins with potent antimicrobial and immunomodulatory properties from alligator sinensis, play pivotal roles in host antimicrobial immune responses isolation and characterization of antimicrobial peptides derived from bacillus subtilis e -fermented soybean meal and its use for preventing vibrio infection in shrimp aquaculture chain length dependence of antimicrobial peptide-fatty acid conjugate activity host-defense peptides with therapeutic potential from skin secretions of frogs from the family pipidae fundamentals on the molecular mechanism of action of antimicrobial peptides combinations of early generation antibiotics and antimicrobial peptides are effective against a broad spectrum of bacterial biothreat agents synthetic peptides eradicate resistant infections photochemically-generated silver chloride nanoparticles stabilized by a peptide inhibitor of cell division and its antimicrobial properties food applications of liposome-encapsulated antimicrobial peptides designing potent antimicrobial peptides by disulphide linked dimerization and n-terminal lipidation to increase antimicrobial activity and membrane perturbation: structural insights into lipopolysaccharide binding antimicrobial peptides: role in human disease and potential as immunotherapies the role of c-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin h cell penetrating peptides: a concise review with emphasis on biomedical applications antimicrobial and host cell-directed activities of gly/ser-rich peptides from salmonid cathelicidins formation mechanisms of chitosan-silica hybrid materials and its performance as solid support for kr- peptide adsorption: impact on kr- antimicrobial activity and proteolytic stability bioactivity and bactericidal mechanism of histidine-rich β-hairpin peptide against gramnegative bacteria the spike protein of sars-cov-a target for vaccine and therapeutic development antibiotic discovery: history, methods and perspectives beneficial role of insect-derived bioactive components against inflammation and its associated complications (colitis and arthritis) and cancer global analysis of the yeast lipidome by quantitative shotgun mass spectrometry an overview of antifungal peptides derived from insect the immunological components of human milk and their effect on immune development in infants designing antimicrobial peptides: form follows function eps-then and now coronavirus, " in viruses and the lung purification and characterization of new anti-adrenocorticotropin rabbit neutrophil peptides (defensins) sturgeon proteinderived peptides exert anti-inflammatory effects in lps-stimulated raw . macrophages via the mapk pathway secondary structure and orientation of the amphipathic peptide gala in lipid structures: an infrared-spectroscopic approach glucagon from the phylogenetically ancient paddlefish provides a template for the design of a long-acting peptide with effective anti-diabetic and antiobesity activities fetal human keratinocytes produce large amounts of antimicrobial peptides: involvement of histone-methylation processes antimicrobial peptides: general overview and clinical implications in human health and disease mechanistic landscape of membrane-permeabilizing peptides cell-penetrating peptides: from basic research to clinics identifying antimicrobial peptides using word embedding with deep recurrent neural networks a tfpi- peptide that induces degradation of bacterial nucleic acids, and inhibits bacterial and viral infection in half-smooth tongue sole, cynoglossus semilaevis the human salivary peptide histatin exerts its antifungal activity through the formation of reactive oxygen species purification and primary structure of pediocin pa- produced by pediococcus acidilactici pac- . . archiv activity and characterization of a ph-sensitive antimicrobial peptide influence of ph on the activity of thrombin-derived antimicrobial peptides effects of antimicrobial peptides on growth performance and small intestinal function in broilers under chronic heat stress grouper (epinephelus coioides) antimicrobial peptide epinecidin- exhibits antiviral activity against foot-andmouth disease virus in vitro a cancer vaccine based on the marine antimicrobial peptide pardaxin (ge ) for control of bladder-associated tumors acquired resistance to the rice blast in transgenic rice accumulating the antimicrobial peptide thanatin characterization and antimicrobial evaluation of sppr-amp , a proline-rich antimicrobial peptide from the mud crab scylla paramamosain oral antimicrobial peptides and new therapeutic strategies for plaque-mediated diseases peptides having antimicrobial activity and their complexes with transition metal ions the effect of halogenation on the antimicrobial activity, antibiofilm activity, cytotoxicity and proteolytic stability of the antimicrobial peptide jelleine-i preparation and identification of peptides and their zinc complexes with antimicrobial activities from silver carp (hypophthalmichthys molitrix) protein hydrolysates envelopedeforming antiviral peptide derived from influenza virus m protein knowledge-based computational methods for identifying or designing novel, non-homologous antimicrobial peptides short peptides derived from the interaction domain of sars coronavirus nonstructural protein nsp can suppress the '-o-methyltransferase activity of nsp /nsp complex integration of nisin into nanoparticles for application in foods recent progress and strategies to develop antimicrobial contact lenses and lens cases for different types of microbial keratitis disruption of drug-resistant biofilms using de novo designed short α-helical antimicrobial peptides with idealized facial amphiphilicity development of a novel hybrid antimicrobial peptide for targeted killing of pseudomonas aeruginosa the vast structural diversity of antimicrobial peptides predicting the minimal inhibitory concentration for antimicrobial peptides with rana-box domain the antibacterial peptide pyrrhocoricin inhibits the atpase actions of dnak and prevents chaperone-assisted protein folding conformational fine-tuning of pore-forming peptide potency and selectivity gain-of-function analogues of the pore-forming peptide melittin selected by orthogonal high-throughput screening comparative analysis of two attacin genes from hyphantria cunea the human anionic antimicrobial peptide dermcidin induces proteolytic defence mechanisms in staphylococci antimicrobial peptides: possible anti-infective agents dalbavancin and telavancin in the treatment of infective endocarditis: a literature review antimicrobial peptides: application informed by evolution transcriptome analysis of streptococcus pneumoniae treated with the designed antimicrobial peptides intracellular targeting mechanisms by antimicrobial peptides diptericinlike protein: an immune response gene regulated by the anti-bacterial gene induction pathway in drosophila de novo generation of short antimicrobial peptides with simple amino acid composition the antimicrobial peptides and their potential clinical applications exploring small cationic peptides of different origin as potential antimicrobial agents in aquaculture interactions of the antimicrobial peptide nisin z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity two optimized antimicrobial peptides with therapeutic potential for clinical antibiotic-resistant staphylococcus aureus membrane active antimicrobial peptides: translating mechanistic insights to design mechanism of antifungal activity of antimicrobial peptide app, a cell-penetrating peptide derivative, against candida albicans: intracellular dna binding and cell cycle arrest membrane interactions of proline-rich antimicrobial peptide, chex -arg , multimers effects of the peptide h-ooww-nh and its derived lipopeptide c -ooww-nh on controlling of citrus postharvest green mold implicit membrane investigation of the stability of antimicrobial peptide β-barrels and arcs control of sour rot in citrus fruit by three insect antimicrobial peptides effects of rabbit sacculus rotundus antimicrobial peptides on the intestinal mucosal immunity in chickens effect of an antifungal peptide from oyster enzymatic hydrolysates for control of gray mold (botrytis cinerea) on harvested strawberries biological activity and structural aspects of pgla interaction with membrane mimetic systems membrane pore-formation correlates with the hydrophilic angle of histidine-rich amphipathic peptides with multiple biological activities the first antimicrobial peptide from sea amphibian regulation of cell division in e. coli antimicrobial packaging based on ε-polylysine bioactive film for the control of mycotoxigenic fungi in vitro and in bread nucleation and growth of pores in , -dimyristoyl-sn-glycero- -phosphocholine (dmpc) / cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin p in vitro and md simulation study to explore physicochemical parameters for antibacterial peptide to become potent anticancer peptide antimicrobial peptides of the vaginal innate immunity and their role in the fight against sexually transmitted diseases the value of antimicrobial peptides in the age of resistance influence of self-assembly on the performance of antimicrobial peptides kappacin, a novel antibacterial peptide from bovine milk temporins, small antimicrobial peptides with leishmanicidal activity the host antimicrobial peptide bac - binds to bacterial ribosomal proteins and inhibits protein synthesis the dolphin proline-rich antimicrobial peptide tur a inhibits protein synthesis by targeting the bacterial ribosome proline-rich peptides with improved antimicrobial activity against e. coli, k. pneumoniae, and a. baumannii insilico alpha-helical structural recognition of temporin antimicrobial peptides and its interactions with middle east respiratory syndromecoronavirus structural characterization of lacticin , a two-peptide lantibiotic with synergistic activity anti-diabetic potential of peptides: future prospects as therapeutic agents translocation of a channel-forming antimicrobial peptide, magainin , across lipid bilayers by forming a pore an antimicrobial peptide, magainin , induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation role of the escherichia coli sbma in the antimicrobial activity of proline-rich peptides mechanism of action of novel synthetic dodecapeptides against candida albicans anti-inflammatory effects of egg yolk livetins (α, β, and γ-livetin) fraction and its enzymatic hydrolysates in lipopolysaccharideinduced raw . macrophages promising antimicrobial agents designed from natural peptide templates lollipop'-shaped helical structure of a hybrid antimicrobial peptide of temporin b-lipopolysaccharide binding motif and mapping cationic residues in antibacterial activity antifungal activity determination for the peptides generated by lactobacillus plantarum te against aspergillus flavus in maize seeds recurrent neural network model for constructive peptide design current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review epinecidin- , a highly potent marine antimicrobial peptide with anticancer and immunomodulatory activities molecular basis of bacterial outer membrane permeability revisited. microbiol degradable dendritic nanogels as carriers for antimicrobial peptides myticusin-beta, antimicrobial peptide from the marine bivalve bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer mode of action of linear amphipathic α-helical antimicrobial peptides ponericins, new antibacterial and insecticidal peptides from the venom of the ant pachycondyla goeldii redesigning arenicin- , an antimicrobial peptide from the marine polychaeta arenicola marina, by strand rearrangement or branching, substitution of specific residues, and backbone linearization or cyclization expression systems for heterologous production of antimicrobial peptides lipidation increases antiviral activities of coronavirus fusion-inhibiting peptides mimicry of bioactive peptides via nonnatural, sequence-specific peptidomimetic oligomers antimicrobial activity of amphipathic α,α-disubstituted β-amino amide derivatives against esbl -carba producing multi-resistant bacteria; effect of halogenation, lipophilicity and cationic character use of click chemistry for obtaining an antimicrobial chimeric peptide containing the lfcinb and buforin ii minimal antimicrobial motifs anti-biofilm peptides as a new weapon in antimicrobial warfare in silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design iron binding modulates candidacidal properties of salivary histatin antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food anti-biofilm peptides: a new class of quorum quenchers and their prospective therapeutic applications antifungal peptides: to be or not to be membrane active antimicrobial peptides: premises and promises targeting leishmania major parasite with peptides derived from a combinatorial phage display library new frontiers for anti-biofilm drug development cyanobacterial peptides as a tour de force in the chemical space of antiparasitic agents the role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines revisiting the interaction of melittin with phospholipid bilayers: the effects of concentration and ionic strength nisin and other antimicrobial peptides: production, mechanisms of action, and application in active food packaging dermcidin: a novel human antibiotic peptide secreted by sweat glands effect of hydrophobic modifications in antimicrobial peptides the proline-rich antimicrobial peptide onc inhibits translation by blocking and destabilizing the initiation complex primary structures of six antimicrobial peptides of rabbit peritoneal neutrophils recent updates of marine antimicrobial peptides application of bacteriocins in vegetable food biopreservation modulating the antimicrobial activity of temporin l through introduction of fluorinated phenylalanine nkl- : a novel antimicrobial peptide derived from zebrafish nk-lysin that inhibits bacterial growth and enhances resistance against vibrio parahaemolyticus infection in yesso scallop cationic antimicrobial peptide and its poly-n-substituted glycine congener: antibacterial and antibiofilm potential against a. baumannii the unique antimicrobial peptide repertoire of stick insects molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers antimicrobial peptide cathelicidin-bf inhibits platelet aggregation by blocking protease-activated receptor . intern anti-yeast activity and characterisation of synthetic radish peptides rs-afp and rs-afp against food spoilage yeast dairy-derived antimicrobial peptides: action mechanisms, pharmaceutical uses and production proposals the importance of antimicrobial peptides and their potential for therapeutic use in ophthalmology comparative lipidomics of azole sensitive and resistant clinical isolates of candida albicans reveals unexpected diversity in molecular lipid imprints a molecular docking study revealed that synthetic peptides induced conformational changes in the structure of sars-cov- spike glycoprotein, disrupting the interaction with human ace receptor integrated evolutionary analysis reveals antimicrobial peptides with limited resistance antimicrobial activity of short arginine-and tryptophan-rich peptides mechanism of antimicrobial action of indolicidin swine intestine antimicrobial peptides inhibit infectious bronchitis virus infectivity in chick embryos chlamydiasecreted protease cpaf degrades host antimicrobial peptides antimicrobial peptides from different plant sources: isolation, characterisation, and purification antimicrobial activity of an abiotic host defense peptide mimic topical antimicrobial peptide formulations for wound healing: current developments and future prospects role of peptide selfassembly in antimicrobial peptides peptide design principles for antimicrobial applications variants of self-assembling peptide, kld- that show both rapid fracture healing and antimicrobial properties a computational modeling approach predicts interaction of the antifungal protein afp from aspergillus giganteus with fungal membranes via its γ-core motif synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the rwqwr motif against gram-positive and gram-negative strains deep learning improves antimicrobial peptide recognition characterization of tachyplesin peptides and their cyclized analogues to improve antimicrobial and anticancer properties antiviral peptides as promising therapeutic drugs evolutionary plasticity of insect immunity ionpair-π interactions favor cell penetration of arginine/tryptophanrich cell-penetrating peptides human antimicrobial peptides and proteins high specific selectivity and membrane-active mechanism of the synthetic centrosymmetric α-helical peptides with gly-gly pairs antimicrobial peptides: promising alternatives in the post feeding antibiotic era control of green and blue mold and sour rot in citrus fruits by the cationic antimicrobial peptide paf rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease heat shock proteins (hsp , , , and ) in galleria mellonella (lepidoptera) hemolymph are affected by infection with conidiobolus coronatus (entomophthorales) peptide-based cancer therapy: opportunity and challenge effects of cations and ph on antimicrobial activity of thanatin and s-thanatin against escherichia coli atcc and b. subtilis atcc in vitro and in vivo activities of antimicrobial peptides developed using an amino acidbased activity prediction method inhibition of sars-cov- (previously -ncov) infection by a highly potent pancoronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion fabricating antimicrobial peptide-immobilized starch sponges for hemorrhage control and antibacterial treatment multidimensional signatures in antimicrobial peptides secretory production of antimicrobial peptides in escherichia coli using the catalytic domain of a cellulase as fusion partner comparative study of different forms of jellein antimicrobial peptide on leishmania parasite identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect sphodromantis viridis alpha-helical antimicrobial peptides-using a sequence template to guide structure-activity relationship studies antimicrobial activity and mechanism of the human milk-sourced peptide casein an antimicrobial peptide containing ngr motif has potent antitumor activity against cd + and cd -tumor cells antimicrobial peptides conjugated with fatty acids on the side chain of d-amino acid promises antimicrobial potency against multidrug-resistant bacteria isolation and structure of corticostatin peptides from rabbit fetal and adult lung characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity we thank the national key r&d program of china ( yfd ). key: cord- - ks bopm authors: nejatifard, marzieh; asefi, sohrab; jamali, raika; hamblin, michael r.; fekrazad, reza title: probable positive effects of the photobiomodulation as an adjunctive treatment in covid- : a systematic review date: - - journal: cytokine doi: . /j.cyto. . sha: doc_id: cord_uid: ks bopm background covid- , as a newly-emerged viral infection has now spread all over the world after originating in wuhan, china. pneumonia is the hallmark of the disease, with dyspnea in half of the patients and acute respiratory distress syndrome (ards) in up to one –third of the cases. pulmonary edema, neutrophilic infiltration, and inflammatory cytokine release are the pathologic signs of this disease. the anti-inflammatory effect of the photobiomodulation (pbm) has been confirmed in many previous studies. therefore, this review study was conducted to evaluate the direct effect of pbm on the acute lung inflammation or ards and also accelerating the regeneration of the damaged tissues. the indirect effects of pbm on modulation of the immune system, increasing the blood flow and oxygenation in other tissues were also considered. methodology the databases of pubmed, cochrane library, and google scholar were searched to find the relevant studies. keywords included the pbm and related terms, lung inflammation, and covid- -related signs. studies were categorized with respect to the target tissue, laser parameters, and their results. results seventeen related papers were included in this review. all of them were in animal models. they showed that the pbm could significantly decrease the pulmonary edema, neutrophil influx, and generation of pro-inflammatory cytokines (tumor necrosis factor-α (tnf-α), interleukin beta (il- β), interleukin (il- ), intracellular adhesion molecule (icam), reactive oxygen species (ros), isoform of nitric oxide synthase (inos), and macrophage inflammatory protein (mip- )). conclusion our findings revealed that the pbm could be helpful in reducing the lung inflammation and promoting the regeneration of the damaged tissue. pbm can increase the oxygenation indirectly in order to rehabilitate the affected organs. thus, the infra-red lasers or light-emitting diodes (leds) are recommended in this regard. coronaviridae is a family of the enveloped rna viruses, which can infect the humans or other mammals. the two beta corona viruses known as sars cov and mers cov are among some of the well-known viruses within this family. in the recent decades, two epidemics of the acute respiratory syndrome (ars) including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) have occurred. mild to severe pneumonia was the main feature of these syndromes. beginning in december , a novel type of coronavirus was emerged in wuhan, china that caused a disease known as corona virus disease (covid- ) ( ) . high transmissibility is the hallmark of this virus. the rapid worldwide distribution of the covid- led to the declaration of a pandemic by the world health organization (who). epidemiologic data have shown that the median age of the patients is between - years old ( ) . high fever, myalgia, dry cough ,and dyspnea are the most common clinical symptoms of the disease ( ) . pneumonia is the hallmark of this infection. it can be distinguished from other probable types of pneumonia by the "crazy paving" lesions observed in the chest ct scans. they are the bilateral ground glass opacities in the parenchyma of the lungs ( ) . diarrhea, sputum production, headache, haemoptysis, rhinorrhea, sneezing and sore throat are other common symptoms of this disease ( , ) . clinical data have suggested that the pneumonia could cause dyspnea in roughly half of the patients (liu, ) . one -third of the patients may develop the acute respiratory distress syndrome (ards) and require admission to an intensive care unit (icu). invasive mechanical ventilation is needed in and % of the patients who are candidates for the extracorporeal membrane oxygenation (ecmo). older or smoker patients are more likely to require the invasive life support ( ) . lower blood oxygenation and tissue hypoxia can be expected following the damage to the respiratory system. concomitant tissue hypoxia can lead to a subsequent damage to the heart or central nervous system, considering their sensitivity to the blood oxygen supply. the paraclinical findings include the hypoalbuminemia, leukopenia, lymphopenia, and low platelet counts. increased serum level of c-reactive protein, aminotransferase ( ) ,and lactate dehydrogenase (ldh) can be seen in these patients. the number of cluster of differentiation (cd ) cells may be reduced during the course of the disease ( ) . levels of some inflammatory cytokines, such as il- b, il- , interferon-gamma (ifnγ), interferon-gamma-inducible protein- (ip ) and monocyte chemotactic protein- (mcp ) increase in the serum. these cytokines increase the number and function of t helper type (th ) lymphocytes. meanwhile, activation of t helper type (th ) lymphocytes following the release of interleukin (il- ) and interleukin (il- ) could also be expected. in the patients with severe disease admitted to the icu, high levels of granulocyte colony-stimulating factor (gcsf), ip , mcp , macrophage inflammatory protein a (mip a) ,and tnfα have been commonly found ( ) . in a recent study, it was suggested to consider a "cytokine storm" in the covid- . cytokine storm results in an influx of a lot of immune cells into the infection site, damage of the vascular barrier or capillaries, and may also lead to the multi-organ failure. an increase was observed in the level of many cytokines in the covid- disease including il- β, interleukin (il- ), interleukin (il- ), interleukin (il- ), il- , fibroblast growth factor (fgf), g-csf, granulocyte macrophage colony-stimulating factor (gm-csf), ifn-γ, ip- , mcp- , mip- a, macrophage inflammatory protein b (mip -b), platelet-derived growth factor (pdgf), tnf-α, and vascular endothelial growth factor (vegf). il- , tnf-α, and il- are the most important pro-inflammatory cytokines. it was found that the il- has a pivotal role in the covid- disease especially it is associated with higher mortality. ( ) although, the clinical features of the covid- are less severe than the sars or mers, it is more easily transmitted. the mortality rate of the covid- is equal to - %, while it is equal to and % in the sars and mers, respectively. presently, there is no gold standard treatment protocol available for the covid- . supportive protocols are recommended most often. there is a considerable controversy about the beneficial effects of the antiviral medications and corticosteroids, although dexamethasone has been shown to be effective in reducing the deaths recently. the efficacy of the lopinavir plus ritonavir ( ) has also been reported in treating the mers. in the meantime, remedesivir ( , ) has been indicated to have a good antiviral effect in treatment of the sars/mers. since, the corticosteroids can alleviate the inflammation and subsequent injury, they are also recommended for the severe cases in which standard supportive procedures were not adequate. however, there is no evidence stating that the steroids can reduce the mortality rate in ards in general ( ) . corticosteroids can prolong the virus clearance ( ) and might cause immune suppression with concomitant susceptibility to the secondary infections ( , ) . hyperglycemia, heart attack, hypertension, and gastrointestinal bleeding are some important side effects of the corticosteroids. ( ) . it should also be mentioned that nearly half of the covid- patients have underlying systemic diseases like diabetes, hypertension, and cardiovascular diseases. therefore, corticosteroid prescription might worsen the patient's underlying systemic disease. another point is the existence of genetic polymorphisms, which can influence the patients҆ response to the corticosteroids. this issue might explain why some patients with ards may experience harmful outcomes after steroid administration ( ) . the pandemic of covid- underlines the importance of supportive protocols, which can reduce the inflammation in the severe cases. as previously mentioned, comprehensive evaluation of the patients҆ medical history is necessary to catalogue the underlying systemic diseases and the used medications. the situation is challenging in emergencies where there are many infected patients along with a shortage of medical services as observed in the covid- pandemic. at the moment, there is no definitive treatment, so the therapeutic protocols should be confined to supportive treatments in order to alleviate the inflammation. photobiomodulation could be a promising novel treatment approach. in this non-invasive method, light-emitting diodes or low-level lasers are used to irradiate on the tissue in order to activate the cellular photo-acceptors. irradiation is absorbed by the internal photo-acceptors like porphyrins, cytochrome c oxidase, and light -sensitive ion channels. cytochrome c oxidase is unit iv of the mitochondrial respiratory chain, absorbing the red and near infrared wavelengths. this leads to higher electron transport, increased mitochondrial membrane potential and increased production of the adenosine triphosphate (atp). light-sensitive ion channels absorb the photons, which increases the concentration of the intracellular calcium (ca +) ions. these processes activate several signaling pathways via reactive oxygen species (ros), cyclic adenosine monophosphate (camp), nitric oxide (no) ,and ca + ( , ) (figure ). these pathways influence the cellular processes like proliferation and differentiation, and can also influence other processes like inflammation as histamine release, prostaglandin production, or cyclo-oxygenase expression ( ) ( ) ( ) ( ) . in this way, pbm can be helpful in accelerating the regeneration processes like wound healing and reducing the inflammation ( ) . pbm has mainly local effects and can be irradiated just to a target organ without any side effects on the distant areas. considering the anti-inflammatory and biostimulatory effects of the pbm, it seems to be a reasonable approach to be applied in controlling the covid- symptoms especially in the cases with ards. therefore, this study was conducted to evaluate the direct effect of the pbm on the acute lung inflammation or ards and accelerating the regeneration of the damaged tissue. moreover, the indirect effects of the pbm on modulation of the immune system or blood flow oxygenation within the tissues were also investigated. our search was categorized based on the common covid- symptoms including respiratory problems and also paraclinical changes influencing the blood cells or cytokines. the review strategy was constructed according to the preferred reporting items for systematic reviews and meta-analyses (prisma) checklist. the databases of medline, pubmed, cochrane library, and google scholar were comprehensively searched to find the relevant studies. initially, there were no time or language limitations, in order to avoid missing any relevant papers. in the next step, the non-english papers were excluded from the study. the included papers were evaluated for the effect of light therapy, pbm, or low -level laser therapy on the lung inflammation, ards, lymphocytes, neutrophils, and lung parenchyma. papers were categorized with respect to the type of light source, wavelength, target tissue/ organ, light source parameters, and pbm results. references in all of the included papers were also reviewed. totally, available papers were retrieved from the pubmed database. twelve papers were excluded due to non-english language and three papers were duplicates. there were available papers on the cochrane library and none of them were related. search in the google scholar database yielded papers. fig. shows the final included papers in the form of a diagram. there were some related studies evaluated the effect of the pbm on the chronic respiratory inflammation (such as chronic obstructive pulmonary disease (copd) and asthma). table shows the related papers included in this study. all the studies confirmed that the pbm can reduce the lung inflammation, neutrophil recruitment, and pro-inflammatory cytokine production. during the clinical course of the covid- disease, dyspnea can start as early as days after the infection. coughing is a major sign, indicating an inflammatory reaction in the respiratory system. sputum production is another common sign of the disease. ground glass opacities can be found in the chest radiographs and there are more of them in the severe cases. ards may occur after days and it can be observed in one-third of the patients. it is a life-threatening condition, which might require invasive mechanical ventilation in % of the patients and extracorporeal membrane oxygenation (ecmo) in % of the patients. elderly patients and smokers are more likely to require the invasive life support ( ) . although, there is no definititive treatment protocol for the covid- disease yet, supportive care is the mainstay for management of the covid- infection. chloroquine, kaletra ( ), remedesivir ( , ) , tocilizumab, favipiravir ( ) ,and a combination of lopinavir and ritonavir ( ) are some of the treatment options in this regard, but no definite effect has been observed on the patients҆ survival after treatment with these medications ( ). corticosteroids are the most common anti-inflammatory option. inhibiting the edema formation, leukocyte extravasation, fibrin deposition, capillary dilation, and phagocytosis are some of positive effects of the corticosteroids. reduction of inflammation can also be induced by triggering the apoptosis of the eosinophils and lymphocytes ( , ) . it should be considered that prescription of the corticosteroids should be confined to the icu-admitted cases with ards ( ). they may lead to higher susceptibility to the secondary infections ( , ) and delay the viral clearance ( ). chan et al., suggested that the corticosteroids should only be used in the severe ards cases where standard supportive measures are insufficient. corticosteroids should not be prescribed when there is a remaining infection ( ) . also, genetic polymorphisms play a pivotal role in the patient's response to the corticosteroids. a wide range of adverse reactions make the use of corticosteroids questionable in the ards induced by the covid- . other potential side effects of the corticosteroids may affect other organs especially the liver and kidneys as major organs for metabolism. other side effects include the hyperglycemia, polyneuropathy in the patients staying in the icu for a long time, higher risk of necrotizing myopathy, risk of secondary infections, delayed wound healing, suppression of calcium absorption leading to the osteoporosis and avascular necrosis of the hip, and gastrointestinal ulcers ( ) . also, the corticosteroids may have negative interactions with other medications taken by the patients. this situation is more noticeable in the covid- patients where nearly half of them are older and usually have underlying systemic diseases like diabetes, hypertension, cardiovascular diseases, etc., requiring the additional medications or have compromised immune systems. moreover, covid- sharply lowers the blood oxygenation, which can directly damage the pulmonary tissues or indirectly damage the other organs like the heart or brain by inducing the hypoxia. our experience in the recent covid- pandemic suggests that the clinicians should consider a comprehensive evaluation of the patient's medical condition, medications, systemic diseases, genetic polymorphisms that may affect the drug reactions. these factors make it complex for the clinicians to manage an emergency like the covid- , involving large numbers of the infected people along with insufficient medical services. as mentioned above, there is not yet any definite treatment for the covid- therefore, every possible intervention ,which may help to reduce the inflammation and restore the respiratory system or other impaired tissues could be tried for managing the covid- progression. this could be achieved by direct rehabilitation of the damaged tissue, or indirectly by increasing the oxygenation and blood flow. so, reducing the inflammation and aiding the tissue regeneration are the major goals of the clinicians. it is important for the clinicians to choose the treatment modalities with the least drug interactions or side effects. photobiomodulation is an innovative approach in this regard. it is a non-invasive approach in which the leds or low -level lasers are used to produce the red or near-infrared (nir) light absorbed by the cellular photo-acceptors. this light absorption produces the ros , such as singlet oxygen, hydrogen peroxide (h o ), and superoxide ( - ). these ros affect many cellular processes ,such as proliferation, differentiation ( , , ( ) ( ) ( ) , adenosine triphosphate (atp) formation ( , ) and also can reduce the inflammtion ( ). so, pbm is a helpful approach when the cellular function is impaired especially by the hypoxia ( ). previous studies have shown the advantages of the pbm including anti-inflammatory effects and acceleration of the wound healing ( ) . these features operate alongside the general effects of the pbm for balancing the metabolic, analgesic, and immunomodulatory conditions. one advantage of this approach is that it is applied locally without any systemic side effects on the other organs ( ). based on the aforementioned advantages of the pbm and the current lack of established treatments for covid- disease, it seems that the pbm could be helpful in controlling the covid- disease as an alternative or adjunctive treatment, particularly in the severe cases with ards. table shows the related papers used the pbm for treatment of lung inflammation or ards. the majority of studies have used the red diode lasers ( and nm) with an energy density within the range of - . j/cm and power density within the range of . - mw/cm . three papers ( , , ) have used the infra-red lasers ( and nm) with an energy density within the range of - j/cm and there was not sufficient information about the power density except in one paper ( . mw/cm ( )). all the papers have shown the anti-inflammatory effects of the pbm including reducing the lung edema, cytokines in the bronchoalveolar lavage (bal) fluid, neutrophil influx, myeloperoxidase (mpo) activity, and damage to the endothelial cytoskeleton. the mechanisms of action of the pbm involve many factors related to the inflammation such as: -activating the interferons (ifns) having a pivotal role in the defense against viruses and the modulation of the immune system. huang et al., ( ) found a higher level of ifn-γ in the covid- patients. ifn-γ has antiviral and anti-tumor activity, and also increases the t lymphocytes and natural killer cells. it can regulate the immune system reactions. ifnα and ifn-β stimulate the natural killer cells and macrophages, they can also stimulate the lymphocytes and macrophages in order to improve the antiviral or anti-tumor activity ( - ). -pbm can also activate the phagocytes engulfing and removing the microorganisms (like bacteria or viruses) as well as apoptotic cells ( , ). -pbm can increase the micro and macro-circulation in order to increase the tissue or organ resistance against the external harmful factors ( ). -it also can increase the oxygen saturation of the tissues, which in turn increases the cell metabolism and capacity for proliferation or regeneration of the damaged tissue ( ). based on table , there are several cytokines influenced by the pbm. according to the literature, pbm can reduce the expression of tnf-α mrna and its production levels ( , , , , , ( ) ( ) ( ) ( ) ( ) . neutrophil adhesion and activation can be influenced by the tnf-α and also il- generation can be stimulated by the tnf-α. moreover, tnf-α can promote the coagulation and edema in the acute lung inflammation ( , ). (il- β) il- β, as the main inflammatory cytokine contributes to the initiation of inflammation. neutrophils are the main source of this cytokine and it increases the survival rate of the neutrophils in a reciprocal manner. severe cases of ards with poor prognosis show higher levels of il- β. many studies have confirmed its effects in the acute lung inflammation. it has been found that the pbm can reduce the production of this cytokine ( , , , ) . mafra de lima et al., ( ) revealed that the il- β expression and concentration were not reduced by the energy dose of j/cm , but on the other hand, they were reduced by higher doses ( , and . j/cm ). therefore, it is necessary to provide a sufficient energy dose to the target tissue, especially the lung parenchyma. laser irradiation will be attenuated by the passage through overlying tissue, such as intercostal muscles and skin. -interleukin- (il- ) pbm can reduce the il- levels during the acute lung inflammation or ards ( - ). this cytokine prolongs the duration of inflammation ( , ) and is related to the poor prognosis in ards ( , - ). studies have shown that the low -level laser can increase the il- generation. il- , as an anti-inflammatory cytokine can modulate the production of other inflammatory cytokine or reduce the tissue injury. it has been suggested that there is a balance between the tnf and il- production ( ) . so, the tnf production can be reduced by promoting the il_ formation by the pbm. this protective effect has been demonstrated in several studies ( , , , ). it has been found that the pbm increases the il- production. in contrast, two studies ( , ) have shown that the low -level laser does not influence the il- production. neutrophil recruitment and their chemotaxis from the bloodstream to the lung parenchyma is one of the hallmarks of the acute lung inflammation or ards . there are some cytokineinduced neutrophil chemoattractants (cincs) present on the neutrophils and endothelial cells. cd integrin is present on the polymorphonuclear leukocytes (pmns). however, icam- is a related adhesion molecule on the endothelial cells. it has been shown that the pbm reduces the icam- expression ( , , ) . it has been suggested that the inhibitory effect of the pbm on icam- expression may be related to the suppression of tnf-α and il- β production. this may be another mechanism by which the pbm reduces the lung inflammation. there are two approaches to deliver the light used in the pbm. this involves a direct irradiation of the target organs ( ). in the covid- disease, the lungs can be irradiated through the chest skin or through the back area when the patient is in the prone position. studies on the transcranial irradiation have demonstrated that the light can penetrate through the scalp and skull to reach the brain ( ). as mentioned above, red and nir wavelengths have been used in this regard. pbm should irradiate through the interchondral space so that, the laser clusters are applied over the entire surface of the thorax. infrared wavelengths (e.g., nm) are suggested for the pbm, because less laser irradiation will be absorbed by the overlying tissues. infrared lasers have lower tissue absorption compared to the red lasers. high-intensity laser therapy (hilt) may be helpful to achieve deeper penetration of the laser light to the underlying tissues, especially lung tissues. hilt has been shown to reduce the inflammation by a non-invasive approach ( ). the lack of any long-term toxicity, gene mutations, and damage to other organs are among the potential advantages of this approach. there has not been any bacteremia observed after irradiation, which is beneficial for the immune-suppressed patients. this approach increases the oxygenation of the red blood cells, and influences the biomarkers in order to indirectly decrease the inflammation or regenerate the damaged tissues. this approach can be carried out intravenously, transmucosally, or transcutaneously over the superficial arteries. blood vessels in the easily accessible areas like the nasal mucosa, under the tongue, behind the knee, or on the wrist can be irradiated with the laser or leds. oxygenation can be increased by the green lasers however; viruses could be cleared by the blue laser irradiation. red lasers improve the atp production. ( , ) therefore, using visible lasers is recommended in this approach with an adjusted dosage. two studies have supported the use of this approach ( , ). they have shown that the laser parameters (wavelength, energy density, etc.) have a critical role in the therapeutic effects ( ). it has been shown that the pbm could reduce the ros production in the neutrophils in peripheral blood ( ), and pbm could regulate the immune system by increasing the lymphoctes and neutral killer cells involved in the defense against the viruses ( ). there was just one clinical study which successfully treated a female patient with ards following a secondary viral infection ( ) . clinicians used the pbm ( nm and mw) in order to reduce the lung inflammation. laser was delivered to the skin between the ribs. this study was a casereport, so it was excluded from our study. other immunomodulatory effects of the pbm may be expected by irradiating the lymphoid tissues like the thymus, spleen, bone marrow ,or the lymphatic system. despite the lack of available studies on the pbm effects on the covid- , but it is assumed that the pbm may be a helpful adjunctive treatment in management of the covid- disease. pbm can reduce the lung edema, neutrophil influx and promote the regeneration of the lung tissue and better oxygenation for all the related organs. infra-red lasers are recommended because of their higher ability for penetration into the lung tissue. dosimetry of . - . j/cm energy density is suitable for the red lasers and an amount of . - . j/cm is a suitable dose for the infra-red lasers. continous mode irradiation at different points of the respiratory system may be helpful in management of the covid- pneumonia. pbm may be used as a preventive approach in the high -risk patients who could receive pre-treatment pbm while being still at a relatively mild stage of the disease. also, pbm may be considered as a therapeutic approach in the hospitalized patients before their condition worsens sufficiently to require the icu admission. therefore, randomized clinical trials should be carried out on the pbm effects for the covid- disease, and indeed some have already been started in various parts of the world. clinical features of patients infected with novel coronavirus in wuhan, china. the lancet infection. covid- , sars and mers: are they closely related? clinical microbiology and infection clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury the covid- cytokine storm; what we know so far treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β b (miracle trial): study protocol for a randomized controlled trial broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov use of glucocorticoids in the critical care setting: science and clinical evidence immune regulation by glucocorticoids the laser therapy handbook. grangesberg, sweden: prima books ab proposed mechanisms of photobiomodulation or low-level light therapy effect of photobiomodulation on mesenchymal stem cells biological responses of stem cells to photobiomodulation therapy effect of lllt ga-al-as ( nm) on lps-induced inflammation of the airway and lung in the rat low-level laser therapy induces dose-dependent reduction of tnfα levels in acute inflammation low level laser therapy (lllt) decreases pulmonary microvascular leakage, neutrophil influx and il- β levels in airway and lung from rat subjected to lps-induced inflammation low level laser therapy (lllt): attenuation of cholinergic hyperreactivity neutrophil-derived il- β is sufficient for abscess formation in immunity against staphylococcus aureus in mice infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an exvivo perfused swine model anti-inflammatory mechanisms of apolipoprotein ai mimetic peptide in acute respiratory distress syndrome secondary to sepsis impact of human interleukin- on vector-induced inflammation and early graft function in rat lung transplantation repeated transcranial low-level laser therapy for traumatic brain injury in mice: biphasic dose response and long-term treatment outcome the beneficial effects of high-intensity laser therapy and co-interventions on musculoskeletal pain management: a systematic review comparative in vitro study: examining nm laser and nm ultraviolet interaction with blood acute respiratory distress syndrome successfully treated with low level laser therapy this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord- -pxnl wgc authors: liang, yu-lei title: thoughts on the effects of moxa smoke in the epidemic prevention: 关于艾烟防疫作用的思考 date: - - journal: world j acupunct moxibustion doi: . /j.wjam. . . sha: doc_id: cord_uid: pxnl wgc through summarizing and analyzing the modern mechanism researches and controversial questions of moxibustion fumigation in the epidemic prevention, the thoughts on tackling the critical points are proposed in the paper, such as the recognition of moxa smoke in traditional chinese medicine (tcm), the mechanism of moxa smoke in air disinfection and the characteristics of clinical application of moxa smoke so as to provide the references to the prevention and control of covid- in tcm. in the chapter of sùwân ( 《素问》 plain questions), it is recorded that "where the epidemic disease arrives, it is easy to infect each other, no matter young or old, with similar symptoms". it is the first record of the epidemic disease, describing its characteristics. it is recorded in wēnbìng tiáobiàn (《温病条辨》systematic differentiation of warm diseases) that for the patient with plagues, the epidemic pathogens are related in etiology, mostly combined with turbid and toxic pathogens. wēnrè lùn (《温热论》treatise on warm-heat diseases) points that the treating principle of this disease should be eliminating pathogens with aromatic herbal medications and in association with detoxification. the ancient scholars believe that Àiyâ (艾叶 folium artemisiae argyi) is pungent and warm in nature and aromatic in flavor, acting on strengthening the body, preventing disease and removing disorders by fumigation. hence, moxibustion by fumigation is the common method in the epidemic prevention. atractylodis) is applied for the epidemic prevention. the literature research indicates that the essential oil produced by burning folium artemisiae argyi is mainly composed of eucalyptol, borneol, -terpene alcohol, etc., acting on sterilization, anti-inflammation, anti-virus, suppressing cough, relieving asthma and strengthening immunity [ ] . clinical trial proves that moxa smoke fumigation achieves the same or even better effect as compared with the room air disinfection and ultraviolet disinfection and this method is not influenced by temperature and humidity [ , ] . the author was intended to propose some approaches to moxibustion in the battle against corona virus disease (covid- ) by analyzing the mechanism of moxa fumigation in the epidemic prevention and the related controversial issues. it is anticipated to provide the references to the prevention and control of covid- in traditional chinese medicine (tcm). there are very few reports on the mechanism researches of moxa fumigation for the epidemic prevention. generally, two aspects are involved. one is related to the block of transmission route and another is to the improvement of body immunity. in chapter of sù wân (《素问》plain questions), it is explained that the epidemic toxin invades the body through the respiratory tract. when the virus carrier sneezes and coughs, the tiny droplets containing the pathogenic microorganisms are ejected from the mouth and nose, forming droplet particle bacteria, hosting in the aerosol and floating in the air, which forms a chain of infection. it can be seen that blocking aerosol transmission route is of great significance for the prevention of the epidemic. the researches show that moxa smoke achieves its prevention role by killing pathogenic bacteria and forming micromembrane barrier. rui-hong li, et al. [ ] burnt moxa sticks, once every two days, h each time in the ward during the flu season. it is found that moxa fumigation effectively kills virus and reduces the incidence of influenza to be %. the disinfection effect is better than that of dynamic air disinfection machine. hongmei zhao, et al. [ ] had adopted the same method to sterile baby ward, m and it also indicates that moxa fumigation presents a certain of inactivation effect on hepatitis b virus hbaag and hbeag. xiao-rong hu et al. [ ] fumigated the hematology ward with g moxa stick for min. it is found that moxa fumigation has significant sterilization effect on common pathogenic bacteria such as pseudomonas aeruginosa, staphylococcus aureus and candida albicans. xiao-ping zhan, et al. [ ] used moxa stick fumigation for disinfection in the intensive care unit (icu), about . g/m and the elimination rate of natural bacteria in the air is %. ya-qin tang, et al. [ ] provided moxa stick fumigation in the ward, about . g/m , for hour each time and the average qualified rate of disinfection in a year is . %, meeting the requirements of hospital air disinfection. cai-ping cai [ ] observed the disinfection effect of moxibustion among different doses. the maternal-neonatal unit of hospital ward was disinfected by . g/m , g/m and g/m respectively. it is discovered that the antibacterial effect of each dose group is % in h after disinfection but it is no effect in h after disinfection in the . g/m group and in h after disinfection in the g/m and g/m groups. hence, it is believed that the persistent time of disinfection with fumigation of folium artemisiae argyi is within days. some scholars think [ ] that the fumigation of folium artemisiae argyi not only inhibits or kills bacteria and viruses in the air, but also forms a micromembrane barrier in the mouth and nose to prevent the invasion of influenza viruses. improving body immunity is significant in preventing from epidemic infection. the researches show that moxibustion regulates the concentrations of immune factors such as interleukin, tumor necrosis factor, interferon and immunoglobulin and improves the degree of erythrocyte aggregation, which affects the body immune system from different aspects [ ] . ping liu, et al. [ ] found that the long-term intervention of moxa smoke down-regulates the proportion of cd + cd + treg in cd + t cell in the peripheral blood of the rats. hong cai, et al. [ ] found that a certain concentration of moxa smoke condensate may improve the activity and phagocytic function of macrophages in the pulmonary alveolus of rats and chang huang, et al. [ ] discovered that moxa smoke could increase wbc count and thymus index in serum of mice with leukopenia caused by cyclophosphamide and reduce spleen index, thus effectively protect immune organs. some researches [ , ] indicate that moxa smoke has a significant positive effect on serum icam , vcam , mcp , tnf-а, hs-crp and vwf in apoe-/-mice, as well as suppresses the inflammatory responses. it has been discovered in our previous researches [ , ] that moxibustion increases the levels of immunoglobulin and cre in serum of fatigue rats, reduces the levels of ldh and ck and enhances the body adaptability. in clinic, the research by bin yang, et al. [ ] indicates that moxibustion increases cd + and the ratio of cd + to cd + of serum t cell subsets, improves patient's immunity, promotes lesion absorption and enhances clinical effect in the patents with tuberculosis. hong li, et al. [ ] found that moxibustion improves the immune function of red blood cells and relieves the dysfunction of t cell subsets in athletes and improves their immunity. in the research by zhen-wei liu, et al. [ ] , it is discovered that moxibustion is assisted in the antiretroviral therapy for human immunodeficiency virus (hiv). it improves the immune indicators, such as cd + /cd + , il- and il- , significantly reduces the incidence of adverse reactions and improves the quality of life in the patients. some studies think that moxibustion plays an irreplaceable role in the treatment of diseases [ ] , but its safety remains controversial. some studies suggest that a certain concentration of moxa smoke is relatively safe. bai-xiao zhao's research team [ , ] evaluated the accumulated toxic reaction of particulate matter in moxa smoke and its influence on serum free radicals, and concluded that the toxic reaction of moxa smoke in low and medium concentration groups was not significant and its harm to human body was not obvious. yu -hai huang et al. [ ] proved the safety of moxa smoke because moxa smoke could not cause abnormal changes in physiological indicators such as blood pressure, respiratory rate, heart rate, electrocardiogram and blood oxygen saturation in healthy people. however, some clinical investigations found that the incidence of chronic pharyngitis among the physicians of the department of acupuncture and moxibustion in moxibustion smoke environment was . %, and . % of the physicians had pharyngeal discomfort or cough after moxibustion therapy [ ] . ran jin et al. [ ] conducted qualitative analysis on the combustion products of folium artemisiae argyi and found that a few of its components had certain toxicity at a high concentration. li han [ ] calculated that the limit of human safety concentration was . mg/m according to animal experiments. most researchers believe that harmful substances can be controlled within a safe range by constructing a good exhaust ventilation system, but the attention should still be paid to the air quality problems caused by moxa smoke [ ] . in modern researches, the role of essential oil in folium artemisiae argyi has been briefly classified as the function of moxa smoke [ ] , resulting in unclear effect and application and inducing some safety controversy of toxic side reactions. to face up to the therapeutic effects of moxa smoke, we should primarily improve the understanding of moxa smoke in traditional chinese medicine (tcm), identify its yin or yang property and classify its application range of cold, heat, deficiency or excess syndrome so as to lay the theoretical foundation of tcm for the modern study of moxa smoke as well as its promotion. the composition of moxa smoke is complex and most of the current researches focus on the composition or effect of its ingredients. there is still a gap in the study of what substances or material groups play a role in moxa smoke. the author believes that the researches should emphasize on this aspect to make clear what substances or material groups play the roles of disinfection and sterilization, those of barriers as well as of aerosol deposition, etc. in addition, the ancient literature records that some aromatic herbs, such as rhizoma atractylodis, are fumigated for the epidemic prevention and control. it is wonder whether its combustion may be the same materials (groups) or with a similar structure. hence, it is necessary to specify the harmful threshold dose and non-harmful threshold dose to research and develop the related products, ensure the accurate target of function as well as determine the curative effect. eventually, the significance of tcm is displayed the epidemic prevention. besides the effects aforementioned, it is discovered by xiao-nan meng, et al. [ , ] that moxa smoke plays its anti-fatigue effect in the responses of nervous system and the regulation of free radical metabolism through nerve-endocrine-immune system. jia yang, et al. [ ] believe that moxa smoke may prevent from atherosclerosis by observing the impacts of moxa smoke on blood lipid metabolism and microcirculation. it is found by dan li, et al. [ ] that moxa smoke extract may regulate the expressions of neuronal apoptotic proteins, bcl- and active-caspase- and antagonize apoptosis to delay the progress of neurodegenerative diseases such as parkinson's disease and alzheimer's disease. current researches are extensive in content, but not deep in scientific exploration, which limits the clinical guidance of tcm. the author summed up the contents mentioned above according to zangfu functions of tcm and has discovered that the heart and lung functions are involved in all of the researches, focusing on the relationship of qi and blood. it is believed that the research on moxa smoke should be on the base of tcm theories, take the lung as the core and highlight the clinical characteristics of tcm. moxibustion therapy is a very important part in the treasure of tcm, characterized by simple in operation and low in expenditure. it is valuable for this therapy to be promoted in the aspect of prevention from virus transmission, especially in primary-care hospital, ordinary families, as well as public. it indicates the great potential space of research and development. therefore, it is required to further strengthen the collaboration of multi-disciplinary research staffs, such as medicine, engineering and experimental science and promote the modern research of moxibustion and product development so that the diversified clinical demands can be met constantly. qualitative analysis on components of moxa combustion products by solid-phase microextraction-gas chromatography-mass spectrography modern research progress of the safety effects of moxibustion smoke comparison of air disinfection effect between moxa stick fumigation and ultraviolet irradiation study on air disinfection of moxa stick fumigation in the outpatient department of the primary hospitals clinical observation on the prevention of influenza by air disinfection with moxa stick fumigation study on effect of inactivation to hbsag by using of moxa fumigation in baby-friendly ward hematology ward disinfection by moxa stick fumigation comparison of efficacy of three kinds of chinese herbal medication fumigation in disinfection of air in icu observation on the effect of moxa sticks fumigation on air disinfection in hospital rooms observation on the disinfection effect of artemisia argyi leaf in the maternal-neonatal unit of hospital ward characteristics and thoughts of epidemic virus prevention of moxa smoke acupuncture and moxibustion and immunity; the actuality and future effects of long-term intervention of moxa smoke on tlymphocytes subets and cd + cd + tregin peripheral blood of wistar rats effects of moxibustion and moxa smoke on mice with leukopenia caused by chemotherapy influences of moxa-smoke condensate on activity and phagocytosis of alveolar macrophages (nr ) in rats influence of moxibustion and moxa smoke on proinflammatory factors in apoe knock-out mice effects of moxibustion and moxa smoke on tnf-α, hs-crp, vwf in serum of atherosclerosis mice study on the effect of moxibustion at "shénquè" ( 神阙 cv ) on the immune system of rats taking long-term exhaustive exercise difference in the anti-fatigue effect of moxibustion at different acupoints in one-off exhausted rats influence of moxibustion apparatus as adjuvant treatment for pulmonary tuberculosis and patient's immune function impacts of moxibustion on erythrocyte immune function and t-lymphocyte subsets in athletes moxibustion combined with highly active antiretroviral therapy for cd + and γ chain cytokines of hiv infected patients mechanism of moxa-smoke in mooxibustion and its safety experiment study on systemic anaphylaxis reaction in guinea pigs under the average clinical concentration of moxa smoke effects of moxa smoke with different concentrations on experession of sod and mda in lung and serum of rats the effect of moxa smoke on blood pressure, respiratory rate, heart rate, ecg and oxygen saturation of healthy adults clinical effect of moxibustion smoke on chronic laryngitis in acupuncture practitioners qualitative analysis on components of moxa combustion products by solid-phase microextraction-gas chromatography-mass spectrography experiment research of toxicology of moxa smoke influence of moxa smoke on indoor air quality and strategies for its control research progress on pharmacological activities and development utilization of artemisia argyi levl. et vant effects of folium artemisiae argyi burning products on monoamine neurotransmitters contents in brain of senescence-accelerated mouse p effect of moxa products after burning on sod， mda and gsh-px in the brain of senescence accelerated mice effect of long-term intervention of moxa smoke with different concentrations on vwf, ox-ldl in rats blood serum effects of moxa smoke extract on neuronal apoptosis and its mechanism key: cord- - p hv authors: gambardella, jessica; khondkar, wafiq; morelli, marco bruno; wang, xujun; santulli, gaetano; trimarco, valentina title: arginine and endothelial function date: - - journal: biomedicines doi: . /biomedicines sha: doc_id: cord_uid: p hv arginine (l-arginine), is an amino acid involved in a number of biological processes, including the biosynthesis of proteins, host immune response, urea cycle, and nitric oxide production. in this systematic review, we focus on the functional role of arginine in the regulation of endothelial function and vascular tone. both clinical and preclinical studies are examined, analyzing the effects of arginine supplementation in hypertension, ischemic heart disease, aging, peripheral artery disease, and diabetes mellitus. l-arginine, hereinafter referred to as arginine, is a semi-essential or conditionally essential amino acid, since it can be synthetized by healthy individuals but not by preterm infants [ ] . from a chemical point of view, arginine is a -amino- -guanidinopentanoic acid ( figure ). its name derives from the greek word ἄργυρoς (silver), indicating the color of arginine nitrate crystals. arginine is involved in a number of biological processes, it is the substrate for a series of reactions leading to the synthesis of other amino acids, and it is a substrate for two enzymes, namely nitric oxide (no) synthase (nos) and arginase, which are fundamental for the generation of no and urea, respectively. arginine is known to act as a substrate for no production by endothelial cells, thus regulating vascular tone and, overall, cardiovascular homeostasis [ ] . no is synthesized from arginine by the enzyme nos in a reaction that involves the transfer of electrons from nicotinamide adenine dinucleotide phosphate (nadph)-via the flavin adenine dinucleotide (fad) and flavin mononucleotide (fmn) in the c-terminal reductase domain [ , ] -to the heme in the n-terminal oxygenase domain, where the substrate arginine is oxidized to citrulline and no [ , ] , as shown in figure . arginine is also implicated in t-cell proliferation and host immune responses, as well as in creatine and collagen synthesis [ ] [ ] [ ] [ ] [ ] . there are three isoforms of nos, two of which-endothelial (enos) [ , ] and neuronal (nnos) [ ] [ ] [ ] -are constitutively expressed, while the third one, inducible nos (inos) [ ] [ ] [ ] , is expressed in response to cytokines and is related to the inflammatory response [ , ] . no generation in normal conditions, nos catalyzes the transformation of arginine, o , and nadph-derived electrons to no and citrulline ( figure ). however, in the presence of pathologic conditions like atherosclerosis and diabetes, the nos function is altered, and the enzyme catalyzes the reduction of o to superoxide (o − ), a phenomenon that is generally referred to as "nos uncoupling" [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and has been linked to a limited bioavailability of tetrahydrobiopterin (bh , also known as sapropterin) [ ] [ ] [ ] [ ] [ ] [ ] . indeed, the donation of an electron by bh to produce a transient bh •+ radical is required for the oxidation of arginine to citrulline and the associated formation of a ferrous iron-no complex at the nos heme catalytic center [ ] [ ] [ ] [ ] . bh is synthesized from guanosine triphosphate (gtp) by gtp cyclohydrolase i (gtpch) and recycled from , -dihydrobiopterin (bh ) by dihydrofolate reductase (figure ). of note, nos is inhibited by arginine analogs that are substituted at the guanidino nitrogen atom, like ng-monomethyl-arginine or ng-nitro-arginine [ - ]. as mentioned above, in the urea cycle arginine is converted by arginase, a manganese metalloenzyme, in ornithine and urea; this cycle is crucial not only for allowing urea excretion, but also for producing bicarbonate, which is critical for maintaining acid/base homeostasis [ - ]. arginase exists in two distinct isoforms, arginase i and ii, that share~ % sequence homology; arginase i is a cytosolic enzyme mainly localized in the liver, whereas arginase ii is a mitochondrial enzyme with a wide distribution and is expressed in the kidney, prostate, gastrointestinal tract, and the vasculature [ - ]. the enzyme arginase is a key modulator of no production by competing for arginine: in other words, no generation is dependent on the relative expression and activities of arginase and nos. more specifically, increased arginase activity may lead to a decreased bioavailability of arginine for nos, thereby diminishing no production. this mechanism has emerged as an essential factor underlying impaired endothelial functions [ , ] . specifically, an increased arginase activity has been associated with endothelial dysfunction in a number of experimental models of hypertension, atherosclerosis, diabetes, and aging . indeed, endothelial dysfunction is a leading cause of several pathological conditions affecting the cardiovascular system, including hypertension, atherosclerosis, diabetes, and atherothrombosis [ , . moreover, in april , we were the first group to show that the systemic manifestations observed in coronavirus disease (covid- ), caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), could be explained by endothelial dysfunction [ ] . indeed, alterations in endothelial function have been linked to hypertension, diabetes, thromboembolism, and kidney failure, all featured, to different extents, in covid- patients [ ] [ ] [ ] . other investigators have later confirmed our view [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . on these grounds, based on the positive effects of arginine on endothelial function, we can also speculate that arginine supplementation could be helpful, while not being harmful, for contrasting endothelial dysfunction in covid- patients. an increasing interest in the potential therapeutic effects of arginine supplementation, especially in cardiovascular disorders, has recently emerged. an impaired no synthesis is considered a main feature of a dysfunctional endothelium [ , [ ] [ ] [ ] ; however, several studies suggest that arginine supplementation in healthy subjects does not lead to a significant increase in no production [ , [ ] [ ] [ ] [ ] . for instance, the daily administration of arginine for week did not affect the serum concentration of the major determinants of cardiovascular risk, including dyslipidemia, glucose intolerance, smoking, hypercholesterolemia, and aging, have a direct impact on the endothelium [ ] [ ] [ ] . exposing the vasculature to these conditions induces endothelial dysfunction and alterations as an early phenomenon, able to evolve and contribute to the progression towards clinically relevant disorders like hypertension, atherosclerosis, and diabetes mellitus. hence, the endothelium plays a key role in cardiovascular physiology and pathophysiology [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . fervent research has been conducted in recent years in order to understand the underlying mechanisms and identify therapeutic strategies to prevent or counteract endothelial dysfunction. the ability of the endothelium to regulate vascular homeostasis is largely dependent on no production, making endothelial vasodilator failure the main sign of endothelial dysfunction and a hot point to be targeted. the impaired endothelial no availability in perturbed vasculature can be attributable to a diminished synthesis of no or, indirectly, to an increased ros production, which inactivates the no source [ , ] . in addition to counteracting oxidative stress, the stimulation of no synthesis represents an alternative and a potentially effective approach [ , ] , for instance, by providing further substrates to no synthase. theoretically, arginine supplementation meets these needs, and thus, it has been tested in many cardiovascular disorders as a potential therapeutic strategy [ ] . however, human studies on arginine supplementation have often been a source of debate. indeed, in healthy subjects as well as in patients suffering from cardiovascular disorders, levels of plasma arginine range from~ to~ µmol/l [ , [ ] [ ] [ ] , significantly higher than the enos k m of . µmol/l [ ] . endocrine mechanisms may also contribute to vasodilation induced by arginine. indeed, arginine stimulates the release of both insulin [ ] [ ] [ ] and glucagon [ ] from pancreatic islets of langerhans. interestingly, an intravenous infusion of arginine has been shown to induce vasodilation and insulin release in healthy humans, but when insulin secretion was blocked by octreotide co-infusion, no vasodilation occurred, whereas vasodilation was restored by insulin co-administration [ ] . since high intravenous doses of arginine ( g) have also been shown to induce growth hormones (ghs), and secretion [ ] , the vasodilation induced by arginine could also be mediated by ghs via a signaling pathway that includes insulin-like growth factor- [ , ] . substantial data indicate that endothelial dysfunction is highly prevalent in elderly individuals [ , ] . endothelial dysfunction has also been implicated in age-associated declines in cognitive function, physical function, as well as in the pathogenesis of stroke, erectile dysfunction, and renal dysfunction. clinical trials testing the effects of arginine in aging-induced endothelial dysfunction have yielded controversial results. an acute intravenous infusion of arginine ( g/min for min) had no effect on endothelial-dependent vasodilation in healthy older individuals [ ] . similarly, the intravenous infusion of arginine induced a significant increase in the renal plasma flow, glomerular filtration rate, natriuresis, and kaliuresis, in young but not in aged hypertensives [ ] . another study conducted in healthy postmenopausal women taking g of arginine per day for month confirmed that plasma arginine increased without a concomitant significant change in flow-mediated dilation [ ] . on the contrary, in a prospective, double-blind, randomized crossover trial in healthy, old participants (age . ± . years), chronic arginine supplementation ( g/day for weeks) markedly increased their plasma levels of arginine ( . ± . vs. . ± . mm) and significantly improved endothelial-dependent vasodilation [ ] . the majority of studies in animal models supports a beneficial effect of arginine supplementation in hypertension, especially in the presence of salt-sensitive hypertension. for instance, both oral [ ] [ ] [ ] and intraperitoneal [ , ] arginine administration in dahl salt-sensitive (dss) rats was shown to prevent the increase in blood pressure induced by a high salt diet. however, arginine was not effective in dss pretreated with high salt for three weeks [ ] , suggesting that arginine is able to prevent and counteract hypertension when it is in the early stages, but probably not when some changes and pathological remodeling have already occurred. the outcome of arginine supplementation could also depend on the method of administration. for instance, renal medullary interstitial infusion of arginine prevents the increase in blood pressure in high salt-treated rats, while the intravenous dose necessary to obtain a similar increase in plasma arginine does not affect blood pressure [ ] . a rat model of type diabetes mellitus shows an important reduction in blood pressure after weeks of oral arginine treatment [ ] ; oral arginine administration prevents fructose-induced hypertension [ ] . oral arginine administration does not correct hypertension in spontaneously hypertensive rats, although markedly reduces renal damage [ ] . although the beneficial effect of arginine supplementation in hypertension appears to be largely attributable to its impact on no synthesis, arginine has also been shown to have antioxidant properties, thus affecting the activity of redox-sensitive proteins and lowering blood pressure [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, supplementation with g/day arginine for two months increases the serum total antioxidant capacity in obese patients with prediabetes [ ] ; of note, in vitro experiments performed in endothelial cells have revealed that arginine reduces superoxide release and the cell-mediated breakdown of no [ ] . in the clinical scenario, the oral administration of arginine acutely improves endothelium-dependent, flow-mediated dilatation of the brachial artery in patients with essential hypertension [ ] ; however, the long-term effects of arginine were not investigated in this study [ ] . in a japanese population, the acute intravenous infusion of arginine ( mg/kg for min) is able to decrease arterial pressure of both salt-sensitive and salt-insensitive patients [ ] . in a similar study, conducted on african-americans, the same amount of arginine administration reduces blood pressure with a greater effect in the salt-sensitive population [ ] . interestingly, in hypertensive patients in which the control of blood pressure with angiotensin converting enzyme (ace)-inhibitors and diuretics for three months was unsuccessful, the addition of oral arginine ( g/day) was effective in reducing both systolic and diastolic blood pressure levels [ ] . unfortunately, many of the findings on the effects of arginine supplementation in hypertension derive from small clinical studies and, despite the promising efficacy, further investigations are needed, especially large, randomized, and controlled trials. the ability to modulate the renin-angiotensin-aldosterone system (raas) is another mechanism by which arginine can regulate blood pressure: specifically, arginine inhibits ace activity, reducing angiotensin ii production and its effects on vascular tone [ ] . alongside the preservation of endothelial-dependent vasodilation, the enhanced bioavailability of no reduces the activation of pro-inflammatory genes and the expression of endothelial adhesion molecules [ ] . these events strongly regulate the development and the fate of atherosclerosis [ ] [ ] [ ] . for these reasons, it is not surprising that arginine has a powerful effect on atherogenesis and its evolution. in particular, preclinical investigations have shown that chronic arginine administration in ldl-receptor ko mice significantly reduces the extension of atherosclerotic plaques [ ] . similarly, arginine supplementation in humans reverses the increased monocyte-endothelial adhesion, mirrored by a normalization of platelet aggregation [ ] . these effects make arginine a promising drug for disorders like coronary artery disease (cad), heart failure, and peripheral artery disease (pad). in , two important studies investigating the effects of arginine in cad were published [ , ] . in a placebo-controlled study, adams and collaborators showed that oral administration of arginine ( g/day for days) significantly improved the vasodilatory response of the brachial artery in premature cad [ ] . a double-blind placebo-controlled study conducted on patients with stable angina pectoris revealed that the administration of arginine was able to improve their exercise capacity in just days [ ] . the following year, a clinical study confirmed the beneficial effects of long-term arginine supplementation ( g for months), showing significantly enhanced vascular responses to acetylcholine in patients with coronary atherosclerosis [ ] . preclinical studies were consistent with these findings. for instance, oral administration of arginine reduced the intimal hyperplasia in balloon-injured carotid arteries in spontaneously hypertensive rats [ ] . this first encouraging evidence prompted further investigations about arginine's effects on cad. again, arginine treatment for weeks preserved endothelial function in cad patients, markedly reducing ldl oxidation [ ] . another study highlighted the method of administration as a major determinant of the efficacy of high dose arginine supplementation: intra-arterial infusion, but not oral administration, was able to improve endothelial-dependent vasodilation in patients with stable angina pectoris [ ] . the therapeutic potential of arginine has been also investigated in heart failure [ ] [ ] [ ] [ ] [ ] and ischemia-reperfusion injury [ ] [ ] [ ] , often yielding controversial results. endothelium-dependent vasodilation in response to acetylcholine and ischemic vasodilation during reactive hyperemia is attenuated in the forearm of patients with heart failure [ ] . in a seminal paper, hirooka and collaborators demonstrated that the intra-arterial infusion of arginine was effective in reversing the blunted endothelium-dependent vasodilation observed in heart failure [ ] . moreover, oral arginine supplementation ( g twice a day for weeks) enhanced endurance exercise tolerance in heart failure patients, an important determinant of daily-life activity in patients with chronic stable heart failure [ ] . in line with these results, a clinical study carried out in patients with class ii/iii heart failure (new york heart association, nyha) established that improved endothelial function following exercise training is associated with increased arginine transport [ ] . however, another investigation in patients with nyha class iii/iv heart failure demonstrated that responses to acetylcholine and sodium nitroprusside determined using forearm plethysmography were not affected by arginine ( g/day every day for days), although the actual levels of arginine in the blood were not measured [ ] . exogenous arginine ( g three times a day for months) administered to patients after an acute myocardial infarction did not improve vascular stiffness measurements or ejection fractions; this clinical trial had to be interrupted due to excess mortality in the treated patients [ ] . the improvement in peripheral circulation is critical in patients with pad, as in severe cases the extensive damage of leg tissues can result in gangrene and amputation [ ] [ ] [ ] . intravenous arginine administration to pad patients is able to increase the calf blood flow and walking distance [ ] . similarly, an acute intravenous arginine infusion ( g in min) improves no production and blood flow of the femoral artery in pad patients [ ] . the oral consumption of arginine for weeks is able to increase the pain-free walking distance, improving the quality of life of patients with hypercholesterolemia [ ] . nevertheless, if the short-term arginine administration seems to be effective in treating pad, the results on long-term administration are less consistent. a randomized clinical trial testing the long-term ( months) effects of arginine supplementation was conducted on subjects. despite an increase in plasma levels of arginine, the study revealed no significant effect of arginine treatment on no-dependent vasodilation, as well as on the relative functional phenotype of pad patients [ ] . given the fundamental pathogenic role of endothelial dysfunction in diabetes and its complications [ , ] , the therapeutic use of arginine supplementation has been tested. in addition to the direct impact of arginine on endothelial vasodilator capacity, a crosstalk with the insulin pathway has been suggested [ , ] . in particular, as mentioned above, arginine can induce the release of insulin from pancreatic beta cells [ ] [ ] [ ] . on the other hand, insulin is able to reduce adma concentrations [ ] and to stimulate the secretion of arginine [ , ] . the stimulation of insulin receptors induces no release, producing an insulin-dependent vasodilation [ ] [ ] [ ] [ ] [ ] . of note, such a protective effect of insulin on arginine mobility and endothelial no production is compromised in diabetes [ ] . henceforth, diabetic patients could be an optimal target population for arginine supplementation. preclinical studies corroborate this theory: in diabetic rats, the oral administration of arginine reverses endothelial dysfunction [ ] , restoring endothelium-dependent relaxation and decreasing oxidative stress [ ] . arginine administration in tap water (free base, mg/kg/day) for months has been shown to reduce both cardiac [ ] and renal [ ] fibrosis in db/db mice, by the interaction of arginine with reactive carbonyl residues of glycosylation adducts of collagen, thereby inhibiting glucose-mediated abnormal cross-linking of collagenous structures. these results were later confirmed in a clinical setting, showing that g of arginine free base administered orally as two daily doses of g each reduced the lipid peroxidation product malondialdehyde in diabetic patients [ ] . clinical studies confirmed the reduction in blood pressure, platelet aggregation, and hemodynamic function in diabetic patients treated with intravenous arginine [ ] . while in healthy subjects arginine treatment does not seem to affect insulin receptor sensitivity or density [ ] , in conditions of insulin resistance, arginine improves insulin sensitivity; indeed, the intravenous injection of arginine in obese or type diabetic patients stimulates insulin responsiveness, restoring insulin-dependent vasodilation [ , ] . similarly, the oral administration of arginine improves hepatic and peripheral insulin sensitivity in a cgmp dependent fashion [ ] . a prospective, crossover clinical trial conducted in mildly hypertensive type diabetic patients revealed a significant decrease in blood pressure in response to arginine, occurring two hours after the oral administration; the effect of lowering blood pressure was associated with increased plasma levels of citrulline, whereas no significant changes in insulin levels were detected, suggesting that the observed phenotype was dependent on arginine-induced no synthesis [ ] . overall, the mentioned studies substantiate the use of arginine in the diabetic population, at least as a prophylactic treatment able to prevent cardiovascular complications of diabetes. one potential limitation for the use of arginine is the risk of reaction with precursors of advanced glycosylated products [ ] , which are particularly abundant in diabetes. since the addition of methylglyoxal (abundant in diabetic patients [ ] ) to arginine has been shown in vitro to produce potent superoxide radicals in a dose-dependent manner [ ] , arginine supplementation has been suggested to be combined with antioxidants. a double-blind study on diabetic patients verified this assumption evaluating the combination of n-acetylcysteine and arginine oral treatments: the combined treatment was able to reduce systolic and diastolic blood pressure, total cholesterol, c-reactive proteins, vascular adhesion molecules, and improved the intima-media thickness during endothelial post-ischemic vasodilation [ ] . this last evidence indicates that the combination of arginine with an antioxidant agent should be potentially effective and well-tolerated. overall, data available in the literature support and encourage the use of arginine supplementation in cardiovascular disorders, especially in preventing the evolution of hypertension and atherosclerosis. one limitation of using arginine supplementation remains the selection of the optimal target population. in this sense, we believe that adma levels could be very useful in selecting the target population, and patients with increased adma/arginine ratios are probably the most suitable population, in which arginine supplementation can actually be effective. another limitation about arginine use concerns its dose. indeed, available studies suggest a number of different doses, sometimes effective, sometimes not. for instance, the acute oral administration of arginine ( g/day) has been shown to be not successful in inducing an effective no production [ ] . instead, chronic administration of oral arginine (e.g., vials containing arginine salts-free . g/ ml), has been shown to favor the utilization of arginine for no synthesis [ ] , and we have data showing that oral arginine ( g/day of bioarginina ® , farmaceutici damor, vials/day) improves endothelial function in hypertensive patients via the regulation of non-coding rnas (gambardella et al., personal communication) . large, prospective randomized clinical trials are needed to better define the target population for arginine supplementation, alongside with correct dosage definitions. to date, a dose of~ g/day of arginine (e.g., bioarginina ® , vials/day) seems to be effective in favoring the utilization of arginine for no synthesis, without toxic effects. arginine de novo and nitric oxide production in disease states no formation by a catalytically self-sufficient bacterial nitric oxide synthase from sorangium cellulosum recruitment of governing elements for electron transfer in the nitric oxide synthase family the principles, development and application of microelectrodes for the in vivo determination of nitric oxide enzymatic function of nitric oxide synthases arginine stimulates wound healing and immune function in elderly human beings arginine enhances wound healing and lymphocyte immune responses in humans physiological cyclic stretch directs l-arginine transport and metabolism to collagen synthesis in vascular smooth muscle creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo increase in fasting vascular endothelial function after short-term oral l-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center endothelium-derived relaxing factor release on activation of nmda receptors suggests role as intercellular messenger in the brain reaction of neuronal nitric-oxide synthase with oxygen at low temperature. evidence for reductive activation of the oxy-ferrous complex by tetrahydrobiopterin regulation of neuronal nitric oxide synthase in rat adrenal medulla endothelial nos and the blockade of ltp by nos inhibitors in mice lacking neuronal nos functions of arginase isoforms in macrophage inflammatory responses: impact on cardiovascular diseases and metabolic disorders cardiovascular effects of arginase inhibition in spontaneously hypertensive rats with fully developed hypertension inhibition of p mapk reduces adipose tissue inflammation in aging mediated by arginase-ii aging modulates the influence of arginase on endothelial dysfunction in obesity aging alters the production of inos, arginase and cytokines in murine macrophages mechanisms of endothelial dysfunction induced by aging: role of arginase i involvement of increased arginase activity in impaired cavernous relaxation with aging in the rabbit arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels huangqiguizhiwuwu decoction prevents vascular dysfunction in diabetes via inhibition of endothelial arginase effect of experimental diabetes on tissue arginase levels red blood cells in type diabetes impair cardiac post-ischemic recovery through an arginase-dependent modulation of nitric oxide synthase and reactive oxygen species erythrocytes from patients with type diabetes induce endothelial dysfunction via arginase, i serum exosomes mediate delivery of arginase as a novel mechanism for endothelial dysfunction in diabetes mechanisms of diabetes-induced endothelial cell senescence: role of arginase arginase inhibition improves microvascular endothelial function in patients with type diabetes mellitus prevention of diabetes-induced arginase activation and vascular dysfunction by rho kinase (rock) knockout the role of arginase i in diabetes-induced retinal vascular dysfunction in mouse and rat models of diabetes arginase inhibition improves endothelial function in patients with coronary artery disease and type diabetes mellitus hematopoietic arginase deficiency results in decreased leukocytosis and increased foam cell formation but does not affect atherosclerosis identification of macrophage arginase i as a new candidate gene of atherosclerosis resistance endothelial cell dysfunction and the pathobiology of atherosclerosis nishigaki, i. the vascular endothelium and human diseases endogenous nitric oxide synthase inhibitors, arterial hemodynamics, and subclinical vascular disease: the prevencion study covid- as a cardiovascular disease: the potential role of chronic endothelial dysfunction revisiting pharmacology of oxidative stress and endothelial dysfunction in cardiovascular disease: evidence for redox-based therapies. free radic association of transient endothelial dysfunction induced by mental stress with major adverse cardiovascular events in men and women with coronary artery disease significance and mechanistic relevance of sirt -mediated endothelial dysfunction in cardiovascular disease progression new therapeutic implications of endothelial nitric oxide synthase (enos) function/dysfunction in cardiovascular disease endothelial dysfunction, increased arterial stiffness, and cardiovascular risk prediction in patients with coronary artery disease: fmd-j (flow-mediated dilation japan) study a effects of the mean amplitude of glycemic excursions and vascular endothelial dysfunction on cardiovascular events in nondiabetic patients with coronary artery disease abo group, endothelial dysfunction and cardiovascular disease: multiple connections, multiple implications endothelial dysfunction and racial disparities in mortality and adverse cardiovascular disease outcomes increased levels of anti-heat-shock protein (anti-hsp ) indicate endothelial dysfunction, atherosclerosis and cardiovascular diseases in patients with mixed connective tissue disease endothelial dysfunction and cardiovascular disease in early-stage chronic kidney disease: cause or association? endothelial dysfunction as a target for prevention of cardiovascular disease significance of a multiple biomarkers strategy including endothelial dysfunction to improve risk stratification for cardiovascular events in patients at high risk for coronary heart disease risk prediction in cardiovascular disease: the prognostic significance of endothelial dysfunction eurodiab prospective complications study group. levels of soluble receptor for age are cross-sectionally associated with cardiovascular disease in type diabetes, and this association is partially mediated by endothelial and renal dysfunction and by low-grade inflammation: the eurodiab prospective complications study the editor's roundtable: endothelial dysfunction in cardiovascular disease endothelial dysfunction and reduced antioxidant protection in an animal model of the developmental origins of cardiovascular disease caveolae and endothelial dysfunction: filling the caves in cardiovascular disease estrogen protection, oxidized ldl, endothelial dysfunction and vasorelaxation in cardiovascular disease: new insights into a complex issue markers for endothelial dysfunction, but not markers for oxidative stress correlate with classical risk factors and the severity of coronary artery disease. (a subgroup analysis from the ludwigshafen risk and cardiovascular health study) endothelial dysfunction and cardiovascular disease: the role of predictive adaptive responses endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease: additive value of flow-mediated dilation to ankle-brachial pressure index predictive value of noninvasively determined endothelial dysfunction for long-term cardiovascular events in patients with peripheral vascular disease endothelial dysfunction and cardiovascular disease: the promise of blocking the renin-angiotensin system is covid- an endothelial disease? clinical and basic evidence hypertension, thrombosis, kidney failure, and diabetes: is covid- an endothelial disease? a comprehensive evaluation of clinical and basic evidence coronavirus infections-more than just the common cold the continuing -ncov epidemic threat of novel coronaviruses to global health-the latest novel coronavirus outbreak in wuhan sars-cov- endothelial infection causes covid- chilblains: histopathological, immunohistochemical and ultraestructural study of paediatric cases covid- neurologic complication with cns vasculitis-like pattern endotheliitis and endothelial dysfunction in patients with covid- : its role in thrombosis and adverse outcomes the vascular endothelium: the cornerstone of organ dysfunction in severe sars-cov- infection biomedicines diffuse alveolar damage (dad) from coronavirus disease infection is morphologically indistinguishable from other causes of dad intracerebral haemorrhage and covid- : clinical characteristics from a case series dysfunctional coagulation in covid- : from cell to bedside pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- endothelial cell infection and endotheliitis in covid- covid- and kawasaki disease: novel virus and novel case nitric oxide and endothelial dysfunction the histone deacetylase inhibitor tubacin mitigates endothelial dysfunction by up-regulating the expression of endothelial nitric oxide synthase the evaluation of flow-mediated vasodilation in the brachial artery correlates with endothelial dysfunction evaluated by nitric oxide synthase metabolites in marfan syndrome patients biochemical responses of healthy subjects during dietary supplementation with l-arginine acute l-arginine supplementation does not increase nitric oxide production in healthy subjects acute effects of l-arginine supplementation on oxygen consumption kinetics and muscle oxyhemoglobin and deoxyhemoglobin during treadmill running in male adults no effect of short-term arginine supplementation on nitric oxide production, metabolism and performance in intermittent exercise in athletes pharmacokinetic and pharmacodynamic properties of oral l-citrulline and l-arginine: impact on nitric oxide metabolism isolation of nitric oxide synthetase, a calmodulin-requiring enzyme regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs arginine as a potential ergogenic aid in healthy subjects l-arginine supplementation improves exercise capacity after a heart transplant nutritional ergogenic aids and exercise performance a combination of oral l-citrulline and l-arginine improved -min full-power cycling test performance in male collegiate soccer players: a randomized crossover trial l-arginine supplementation does not improve muscle function during recovery from resistance exercise to give or not to give? lessons from the arginine paradox insulin vasodilatation and the "arginine paradox insulin and the arginine paradox endogenous dimethylarginine as an inhibitor of nitric oxide synthesis evaluation of endothelial (dys)function, left ventricular structure and function in patients with chronic kidney disease adma): a promising biomarker for cardiovascular disease? adma as a possible marker of endothelial damage. a study in young asymptomatic patients with cerebral small vessel disease transport of asymmetric dimethylarginine (adma) by cationic amino acid transporter (cat ), organic cation transporter (oct ) and multidrug and toxin extrusion protein (mate ) interference of l-arginine analogues with l-arginine transport mediated by the y+ carrier hcat- b arginase- deficiency regulates arginine concentrations and nos -mediated no production during endotoxemia asymmetric dimethylarginine (adma): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia asymmetrical dimethylarginine (adma) in critically ill patients: high plasma adma concentration is an independent risk factor of icu mortality asymmetrical dimethylarginine is associated with renal and cardiovascular outcomes and all-cause mortality in renal transplant recipients asymmetric dimethylarginine predicts cardiovascular events in patients with type diabetes plasma asymmetric dimethylarginine (adma) concentration is independently associated with carotid intima-media thickness and plasma soluble vascular cell adhesion molecule- (svcam- ) concentration in patients with mild-to-moderate renal failure plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study plasma concentrations of asymmetric dimethylarginine are increased in patients with type diabetes mellitus plasma levels of asymmetric dimethylarginine (adma) are related to intima-media thickness of the carotid artery: an epidemiological study risk of acute coronary events and serum concentration of asymmetrical dimethylarginine reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetric dimethylarginine in men with essential hypertension association of the endogenous nitric oxide synthase inhibitor adma with carotid artery intimal media thickness in the framingham heart study offspring cohort association of circulating levels of asymmetric dimethylarginine (adma) with carotid intima-media thickness: evidence from participants piper sarmentosum promotes endothelial nitric oxide production by reducing asymmetric dimethylarginine in tumor necrosis factor-alpha-induced human umbilical vein endothelial cells asymmetric dimethylarginine (adma) and symmetric dimethylarginine (sdma) concentrations in patients with obesity and the risk of obstructive sleep apnea (osa) asymmetric dimethylarginine limits the efficacy of simvastatin activating endothelial nitric oxide synthase plasma arginine/adma ratio as a sensitive risk marker for atherosclerosis: shimane cohre study asymmetric dimethylarginine as an independent risk marker for mortality in ambulatory patients with peripheral arterial disease asymmetric dimethylarginine predicts survival in the elderly evaluation of asymmetric dimethylarginine and homocysteine in microangiopathy-related cerebral damage a caveolar complex between the cationic amino acid transporter and endothelial nitric-oxide synthase may explain the "arginine paradox role of lipotoxicity in endothelial dysfunction cigarette smoking and cardiovascular disease: pathophysiology and implications for treatment cardiac biomarkers in clinical practice most, p. cardiomyocytes, endothelial cells and cardiac fibroblasts: s a 's triple action in cardiovascular pathophysiology hemodynamic shear stress and the endothelium in cardiovascular pathophysiology micrornas distinctively regulate vascular smooth muscle and endothelial cells: functional implications in angiogenesis, atherosclerosis, and in-stent restenosis relationships between endothelial, inflammatory and angiogenesis markers in rheumatoid arthritis: implications for cardiovascular pathophysiology endothelial alpha -adrenoceptors regulate neo-angiogenesis endothelium-derived hyperpolarizing factor in vascular physiology and cardiovascular disease camk gene deletion induces hypertension diabetes is a vascular disease: the role of endothelial dysfunction in pathophysiology of cardiovascular disease in diabetes endothelial cells are able to synthesize and release catecholamines both in vitro and in vivo endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology endothelial beta adrenergic signaling to akt: role of gi and src endothelial cell injury in cardiovascular surgery: the pathophysiology of vasomotor dysfunction ischemic neoangiogenesis enhanced by beta -adrenergic receptor overexpression: a novel role for the endothelial adrenergic system endothelial function and oxidative stress l-arginine is an effective medication for prevention of endothelial dysfunction, a predictor of anthracycline cardiotoxicity in patients with acute leukemia novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis arginase inhibition restores peroxynitrite-induced endothelial dysfunction via l-arginine-dependent endothelial nitric oxide synthase phosphorylation arginine nutrition and cardiovascular function clinical consequences of urea cycle enzyme deficiencies and potential links to arginine and nitric oxide metabolism reference intervals for plasma l-arginine and the l-arginine:asymmetric dimethylarginine ratio in the framingham offspring cohort correlation between plasma levels of arginine and citrulline in preterm and full-term neonates: therapeutical implications synthesis and hyperpolarisation of enos substrates for quantification of no production by ( )h nmr spectroscopy insulin secretion from pancreatic b cells caused by l-arginine-derived nitrogen oxides biomedicines effect of amino acids and proteins on insulin secretion in man stimulus-secretion coupling of arginine-induced insulin release: comparison between the cationic amino acid and its methyl ester inhibition of pancreatic glucagon responses to arginine by somatostatin in normal man and in insulin-dependent diabetics the vascular effects of l-arginine in humans. the role of endogenous insulin plasma growth hormone after arginine infusion. clinical experiences ng-monomethyl-l-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to igf-i: possible role of nitric oxide in muscle protein synthesis l-arginine stimulates no-dependent vasodilation in healthy humans-effect of somatostatin pretreatment aging and endothelial function in normotensive subjects and patients with essential hypertension non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis impaired flow-mediated dilation with age is not explained by l-arginine bioavailability or endothelial asymmetric dimethylarginine protein expression aging abolishes the renal response to l-arginine infusion in essential hypertension effects of oral l-arginine on endothelium-dependent vasodilation and markers of inflammation in healthy postmenopausal women oral l-arginine improves endothelial function in healthy individuals older than years hypertensive nephrosclerosis in the dahl/rapp rat. initial sites of injury and effect of dietary l-arginine supplementation l-arginine improves endothelial function in renal artery of hypertensive dahl rats l-arginine reverses p phox and gp phox expression induced by high salt in dahl rats l-arginine administration normalizes pressure natriuresis in hypertensive dahl rats l-arginine abrogates salt-sensitive hypertension in dahl/rapp rats renal medullary interstitial infusion of l-arginine prevents hypertension in dahl salt-sensitive rats reversal effects of l-arginine treatment on blood pressure and vascular responsiveness of streptozotocin-diabetic rats effects of l-arginine on blood pressure and metabolic changes in fructose-hypertensive rats l-arginine reverses severe nephrosclerosis in aged spontaneously hypertensive rats l-arginine supplementation improves antioxidant defenses through l-arginine/nitric oxide pathways in exercised rats protective effects of l-arginine on pulmonary oxidative stress and antioxidant defenses during exhaustive exercise in rats potential ergogenic effects of l-arginine against oxidative and inflammatory stress induced by acute exercise in aging rats beneficial effects of antioxidants and l-arginine on oxidation-sensitive gene expression and endothelial no synthase activity at sites of disturbed shear stress antioxidant activity and free radical scavenging capacity of l-arginine and nahs: a comparative in vitro study the alpha-amino group of l-arginine mediates its antioxidant effect the effects of l-arginine, alone and combined with vitamin c, on mineral status in relation to its antidiabetic, anti-inflammatory, and antioxidant properties in male rats on a high-fat diet protective effects of dietary arginine supplementation against oxidative stress in weaned piglets effects of l-arginine supplementation on antioxidant status and body composition in obese patients with pre-diabetes: a randomized controlled clinical trial vascular effects of l-arginine: anything beyond a substrate for the no-synthase? oral l-arginine improves endothelial dysfunction in patients with essential hypertension renal response to l-arginine in salt-sensitive patients with essential hypertension effect of l-arginine on systemic and renal haemodynamics in salt-sensitive patients with essential hypertension study of supplemental oral l-arginine in hypertensives treated with enalapril + hydrochlorothiazide intravenous administration of l-arginine inhibits angiotensin-converting enzyme in humans nitric oxide decreases cytokine-induced endothelial activation. nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines the transcription factor erg inhibits vascular inflammation by repressing nf-kappab activation and proinflammatory gene expression in endothelial cells integrating diet and inflammation to calculate cardiovascular risk adhesion molecules and atherogenesis the effects of l-arginine on atherosclerosis and heart disease l-arginine reduces human monocyte adhesion to vascular endothelium and endothelial expression of cell adhesion molecules oral l-arginine improves endothelium-dependent dilatation and reduces monocyte adhesion to endothelial cells in young men with coronary artery disease effect of supplemental oral l-arginine on exercise capacity in patients with stable angina pectoris long-term l-arginine supplementation improves small-vessel coronary endothelial function in humans oral administration of l-arginine reduces intimal hyperplasia in balloon-injured rat carotid arteries l-arginine improves endothelial function and reduces ldl oxidation in patients with stable coronary artery disease endothelium-dependent vasodilation is independent of the plasma l-arginine/adma ratio in men with stable angina: lack of effect of oral l-arginine on endothelial function, oxidative stress and exercise performance l-arginine administration prevents reperfusion-induced cardiomyocyte hypercontracture and reduces infarct size in the pig randomized, double-blind, placebo-controlled study of supplemental oral l-arginine in patients with heart failure l-arginine supplementation prolongs exercise capacity in congestive heart failure association of l-arginine supplementation with markers of endothelial function in patients with cardiovascular or metabolic disorders: a systematic review and meta-analysis correction of endothelial dysfunction in chronic heart failure: additional effects of exercise training and oral l-arginine supplementation the role of l-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat l-arginine treatment in ischemia/reperfusion injury direct detrimental effects of l-arginine upon ischemia-reperfusion injury to myocardium endothelium-dependent vasodilation is attenuated in patients with heart failure effects of l-arginine on impaired acetylcholine-induced and ischemic vasodilation of the forearm in patients with heart failure chronic l-arginine supplementation enhances endurance exercise tolerance in heart failure patients augmentation of endothelial function following exercise training is associated with increased l-arginine transport in human heart failure dietary supplementation with l-arginine fails to restore endothelial function in forearm resistance arteries of patients with severe heart failure l-arginine therapy in acute myocardial infarction: the vascular interaction with age in myocardial infarction (vintage mi) randomized clinical trial update on peripheral artery disease: epidemiology and evidence-based facts serum metabolic signatures of chronic limb-threatening ischemia in patients with peripheral artery disease american heart association council on peripheral vascular disease; et al. perfusion assessment in critical limb ischemia: principles for understanding and the development of evidence and evaluation of devices: a scientific statement from the american heart association biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease cardiovascular effects of l-arginine nutritional therapy for peripheral arterial disease: a double-blind, placebo-controlled, randomized trial of heartbar l-arginine supplementation in peripheral arterial disease: no benefit and possible harm endothelial dysfunction in diabetes: the role of reparatory mechanisms endothelial dysfunction-a major mediator of diabetic vascular disease effect of -month l-arginine supplementation on insulin resistance and tumor necrosis factor activity in patients with visceral obesity seljeflot, i. decreased levels of asymmetric dimethylarginine during acute hyperinsulinemia activation of l-arginine transport (system y+) and nitric oxide synthase by elevated glucose and insulin in human endothelial cells regulation of amino acid and glucose transporters in endothelial and smooth muscle cells insulin enhances endothelial alpha -adrenergic vasorelaxation by a pertussis toxin mechanism roles for insulin receptor, pi -kinase, and akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells cooperation between insulin and leptin in the modulation of vascular tone nitric oxide release accounts for insulin's vascular effects in humans akt participates in endothelial dysfunction in hypertension cardiovascular actions of insulin short-term oral administration of l-arginine reverses defective endothelium-dependent relaxation and cgmp generation in diabetes l-arginine reduces heart collagen accumulation in the diabetic db/db mouse the effect of substance l on glucose-mediated cross-links of collagen in the diabetic db/db mouse l-arginine reduces lipid peroxidation in patients with diabetes mellitus. free radic review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction arginine does not influence insulin binding on circulating monocytes effects of low-dose l-arginine on insulin-mediated vasodilatation and insulin sensitivity long-term oral l-arginine administration improves peripheral and hepatic insulin sensitivity in type diabetic patients oral arginine reduces systemic blood pressure in type diabetes: its potential role in nitric oxide generation inhibition of l-arginine metabolizing enzymes by l-arginine-derived advanced glycation end products methylglyoxal impairs insulin signalling and insulin action on glucose-induced insulin secretion in the pancreatic beta cell line ins- e methylglyoxal interaction with superoxide dismutase . redox biol. , , long-term n-acetylcysteine and l-arginine administration reduces endothelial activation and systolic blood pressure in hypertensive patients with type diabetes a slow-compared with a fast-release form of oral arginine increases its utilization for nitric oxide synthesis in overweight adults with cardiometabolic risk factors in a randomized controlled study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -by b gy authors: nishinari, katsuyoshi; fang, yapeng title: molar mass effect in food and health date: - - journal: food hydrocoll doi: . /j.foodhyd. . sha: doc_id: cord_uid: by b gy it is demanded to supply foods with good quality for all the humans. with the advent of aging society, palatable and healthy foods are required to improve the quality of life and reduce the burden of finance for medical expenditure. food hydrocolloids can contribute to this demand by versatile functions such as thickening, gelling, stabilising, and emulsifying, controlling texture and flavour release in food processing. molar mass effects on viscosity and diffusion in liquid foods, and on mechanical and other physical properties of solid and semi-solid foods and films are overviewed. in these functions, the molar mass is one of the key factors, and therefore, the effects of molar mass on various health problems related to noncommunicable diseases or symptoms such as cancer, hyperlipidemia, hyperglycemia, constipation, high blood pressure, knee pain, osteoporosis, cystic fibrosis and dysphagia are described. understanding these problems only from the viewpoint of molar mass is limited since other structural characteristics, conformation, branching, blockiness in copolymers such as pectin and alginate, degree of substitution as well as the position of the substituents are sometimes the determining factor rather than the molar mass. nevertheless, comparison of different behaviours and functions in different polymers from the viewpoint of molar mass is expected to be useful to find a common characteristics, which may be helpful to understand the mechanism in other problems. . molar mass effect on the diffusion . molar mass effect on mechanical properties of gels and solids . minimum chain length for helix formation to contribute to the universal demand for secure and sustainable food supply, food colloids science and technology can do many things. it can create palatable foods from underutilized resources, and can reduce the food loss. it is reported that there are many people who die from the insufficient supply of foods and malnutrition while food was wasted by improper distribution in some regions as pointed out by the world health organization (who). international union of food science and technology (iufost) aims to supply sufficient foods helping secure the world's food supply and eliminate world hunger. while the life expectancy in japan is the longest in the world, it is demanded to extend the healthy life expectancy, which is defined as the period in which the person can enjoy life without any limitation in day-to-day activities resulting from health problems, because the increase of the life-style related diseases make some people ill, which lowers quality of life and causes a significant financial burden. palatable, healthy, and sustainable foods are demanded. from the nutritional standpoint it is possible to have a completely adequate diet in the form of fluid foods that require no mastication. however, few people are content to live on such a diet. modern food and pharmaceutical companies sell manna-like foods, which proclaim to be palatable and healthy. but, people don't eat them every day. bourne ( ) raises the great number of dentists in the developed countries which supports his argument. even if ready-made convenient foods are palatable and good for health, most people like to eat something different every time when the situation allows except in a serious calamity such as the earthquake and typhoon/hurricane. palatable and healthy foods are even more demanded in such a situation as in the age of covid- because disadvantaged persons, such as refugees and patients, are under the harsh stresses. unchangeable products are required in the other industry, food should be fragile in a sense that it should be chewable and digested by enzymes to be processed in oral and gastrointestinal organs. it is well known that not only the molar mass but also the structural characteristics such as linkage mode of glucose in polysaccharides make the performance diversity even for similar molar mass compounds. functional proteins have their specific molar mass because their role in the life phenomenon is designed naturally. therefore, it seems to be absurd to dare to write an overview on the effects of molar mass in food quality and health related properties. the authors tried to find some common characteristics or rules governing the mechanism which could be applied to other materials or phenomena within food and health problems. steak consisting of minced meat with granular protein. size and shape of this granular soy protein can be controlled by changing the extruder outlet, composition of raw materials, running condition of extrusion (isobe & noguchi, ) . not only the fibrous texture but also juiciness is an important texture characteristics to produce an authentic meat mouthfeel (puolanne, ; warner, ). low molar mass and high molar mass emulsifiers act differently on the surface of oil droplets. while low molar mass emulsifiers may adsorb faster on the surface of oil droplets because their diffusion coefficient is larger than that of high molar mass ones, high molar mass emulsifiers may show a steric stabilisation effect (dickinson, ). the steric repulsion is dependent not only on molar mass of the polymer forming layers surrounding droplets but also on the conformation and branching (dickinson, ). all the hydrocolloid scientists know the importance of molar mass, for example, to control the viscosity of fluid foods, high molar mass polysaccharides can increase the viscosity at a very low dose (concentration) if the conformation is not completely random coil. random coil polysaccharides such as pullulan and dextran are not effective to increase the viscosity. the solution viscosity of low molar mass saccharide is much lower than that of higher molar mass saccharide, i.e. polysaccharide. though there have been many papers studying food processing or health related problems by changing the dose (concentration) of different polysaccharides without controlling the molar mass, it is evident that it is not possible to compare the concentration dependence or the different effects of these polysaccharides when the molar mass of each polysaccharide is different. in the industrial application of these thickening and gelling polysaccharides, the solubility and the hydration rate and extent are important, which are related to the molar mass and other structural characteristics. in electrolytic copolymers such as pectins and alginates or chitosans, the degree of blockiness, type of cations/anions, the position of cations/anions, degree of substitution (methoxylation/acetylation) are also determining factors of the function. in cellulose derivatives, the degree of substitution and its position and length are determining factor of the function. although the conformation or the stiffness of polysaccharide chain plays important roles in their performance, it is worthwhile to pay attention to the molar mass effect. in the present paper, the terms molar mass, molecular mass, and molecular weight represented in da (dalton) or g/mol are used interchangeably. since it is generally time/energy consuming to obtain food polymers with narrow molar mass distribution, most published papers used polydisperse polymers. nevertheless, high molar mass and low molar mass have been shown to affect the function /property differently as will be discussed in the present review. the molar mass of some polysaccharides have not been determined with high precision because their structures are heterogeneous and difficult to purify to be subjected to molar mass determination. since fibrils of globular protein appeared, the concept of globular protein that forms a gel only at high concentrations changed drastically. it was reported that fibrils of β-lactoglobulin can form for the smooth movement of joints of knee or any other part conferring the lubrication as well as shock absorption. hyaluronan (also called hyaluronic acid, ha) is a main ingredient, also occurring in connective, epithelial, and neural tissues e.g., in eyes, umbilical cord, skin, blood vessels, heart valves (laurent, & fraser, ) . many elderly persons suffer from knee joint pain from osteoarthritis (oa), and the traditional therapy was an injection of hyaluronic acid (balazs & denlinger, ) . since hyaluronan is hydrolyzed by endogenous hyaluronidase and cannot function for a long time, cross-linked hyaluronan was introduced, which can keep longer (balazs et al., ) . ha a copolymer consisting of glucuronic acid and n-acetyl-glucosamine, and occurring naturally, but is also produced by microbial fermentation. its molar mass is reported to j o u r n a l p r e -p r o o f specific problem, the degradation and reconstruction of molecules are governed by specific enzymes and thus different problems cannot be compared directly each other, but methods of study are sometimes similar thus a study on one problem can learn from another study, and finally the total aspects should be overviewed to solve each specific problem. it is the authors' hope that each specialists share their own area with other areas to collaborate to find a better solution. the viscosity of the liquid foods is in most cases determined by food macromolecules and the molar mass is the determining factor together with the structural characteristics, conformation, molecular shape, stiffness, and degree of branching. some problems of the mixture of high molar mass and low molar mass polymers, which have an important practical significance in medical problems such as hyaluronate related with knee pain, and alginate related with cystic fibrosis, and also the interaction between polysaccharides and proteins in food industry are also discussed. there have not been many studies on the effect of molar mass distribution by mixing monodisperse polymers because it is time/energy consuming to get well fractionated polymers with wide molar mass range. the elasticity of solid or semi-solid, gel-like foods is also dependent on molar mass and also the structure of junction zones, the network density and the elastically active network chains, which are also dependent on molar mass. electric charges, ionic strength and ph are also important factors, and therefore the effect of molar mass is not isolated from these factors. nevertheless, it is expected that extraction of common features in various phenomena which are strongly influenced by the molar mass of polymers which play an important role is useful. from this overview, unresolved problem may find a clue from the other problems. in food and health are overviewed in the present paper. there are many methods to obtain or analyse polysaccharides and proteins with different molar masses (table ) . each methods have advantages and disadvantages. for example, oligomers of sulphated polysaccharides have been attracted much attention because of their biological activities, and depolymerized by ultrasonication, gamma-irradiation, acid or enzymatic hydrolysis. sulphate groups tend to be removed by acid hydrolysis, while the structure of the repeating units was retained by gamma-irradiation in fucoidan (choi & kim, ) . in another report, the sulphate content of fucoidan slightly increased by ultrasonic degradation (guo, ye, sun, wu, wu, hu, et al., ). in ultrasonic degradation of xanthan, removal of pyruvate groups from native xanthan increased the thermal stability keeping the helical conformation, and lowered the sensitivity of molecular conformation to the salt concentration, and thus led to lower degradation efficiency (li & feke, ) . some methods of molar mass change, decrease or increase, applied to polysaccharides and proteins in food and health industries are shown in table . the intrinsic viscosity (also called limiting viscosity number) is often determined by an ubbelohde type capillary viscometer. the relative viscosity is defined as the ratio of the viscosity of the solution to that of the solvent η rel = η/η s , and the specific viscosity (relative viscosity increment) η sp = (η -η s )/η s = η rel - per unit concentration is extrapolated to zero concentration to obtain the intrinsic viscosity [η]= lim (c→ ) η sp /c. the intrinsic viscosity has a unit of the inverse of the concentration, and represents the volume the polymer occupies in the solution. therefore where k and a depend on solvent quality, a < / for poor solvent, a = / for θ solvent, a > / for good solvent (doi, ; tanaka, ) . the mhs exponent a is reported as . for amylose (in . molar kcl), dextran (water), and . for stiff chains such as locust bean gum, . ~ . for anionic polysaccharides such as carboxymethyl cellulose, alginate, κ-carrageenan, xanthan, pectin (walstra, ). solution properties of pullulan was extensively studied (kawahara, ohta, to zero shear rate is used to understand the concentration dependence systematically. fig. the critical coil overlap concentration c* is approximately given by /[η], and at this concentration the zero shear viscosity was found η sp ~ . the slope above the critical concentration was found about . for most random coil polymers including the data for polystyrene in toluene, but this slope was approximately . for guar gum and locust bean gum for which the critical concentration was found a little bit lower than for the other random coil polymers. the deviation of the behavior for galactomannans (guar gum and locust bean gum) may be due to specific attractive interactions between side groups on the polymer chains or the stiffness of polymer chains. it should be reminded that a typical behaviour shown in fig. is limited to the viscosity at very low shear rates. as typical shear rate dependence of the viscosity for the solutions of water soluble polymers, data for cellulose derivatives and cereal β-glucans are shown in fig. a and b. while all the glucose residues are linked byβ-( → ) in cellulose which is insoluble in water, in cereal β -glucans about one third of the linkages between the glucose residues are β-( → ) linkages in addition to β-( → ) linkages, and more soluble in water. cellulose derivatives are soluble and have been studied extensively. for cellulose derivative compounds with different molar masses but approximately the same degree of substitution, the steady shear viscosity of % solutions as a function of shear rate is shown in fig. a . a similar shear rate dependence of the steady shear viscosity for beta glucans from oat, barley, wheat flour and wheat bran is shown in fig. b . the following features are noted: ) the viscosity at lower shear rates increases with increasing molar mass; ) the viscosity decreases with increasing shear rate, which is called shear thinning; ) the viscosity of the solution of the lowest molar mass does not depend so much on the shear rate, and shows approximately a constant value at lower shear rates. this is called a newtonian plateau. because of the limited sensitivity of the rheometer, the viscosity at very low shear rates cannot often be measured. in this case, the viscosity of the solution of the lowest molar mass was not measured below the shear rate of s - (fig. a) or . s - (fig. b ) or s - (fig. c ). it should be noted that some published papers erroneously showed a steep rise of the viscosity at lower shear rate with decreasing shear rate, which were probably caused by neglecting the low sensitivity limit of the sensor. diffusion is a ubiquitous phenomenon of motion of molecules or particles in any phase (gas, liquid, or solid) caused by the concentration gradient. this is caused not by an external force but where k is the boltzmann constant ( . × − j k − ), t is the temperature (k), r h is the hydrodynamic radius of a spherical particle (m), η the viscosity of the surrounding medium (pa.s). this equation is valid only for an infinitely dilute solution, and for a particle which is larger than the solvent molecule. the hydrodynamic radius r h is related with the radius of gyration r g = km ν (flory equation, ν is called flory exponent) by ρ = r g / r h.. for linear, flexible chains in the theta solvent ρ = . , while in the limit of large excluded-volume effects (v = . ) a value of ρ = . is obtained. polydispersity increases this value by % (i.e. ρ = . ) for a most probable (schulz-flory) distribution with a polydispersity index pi, the ratio of weight average molecular weight m w and the number average molecular weight m n, pi = m w /m n = (burchard, ). denaturing solutions increased tended to approach . , similar to the exponent expected for a random coil polymer in a good solvent as mentioned above. this difference in the ν value can be used to distinguish between folded, disordered and denatured proteins (evans, ). dudás & bodor ( ) acquired diffusion coefficients of globular proteins and intrinsically disordered proteins (idps). they reported ν = . for native folded globular proteins, and ν= . for intrinsically disordered proteins (fig. ) . the value close to . indicated that the . fig pullulan has been studied extensively as a water soluble, model random coil polysaccharide. the diffusion coefficient of pullulan as a function of molar mass is shown in fig. a where ξ h is hydrodynamic correlation length, should be used (de gennes, ) . diffusion coefficient of pullulan was found to decrease with increasing concentration for lower mw ( . kda - . kda) observed by ultracentrifuge and for higher mw ( . kda- kda) observed by pcs (nishinari et al., ) . diffusion coefficient of α s -casein as a function of concentration is shown in fig. a (kusova, sitnitsky, idiyatullin, bakirova, & zuev, ). this dependence can be generalised by scaling as shown in fig. b (nesmelova, melnikova, ranjan, & skirda, ). and increased with further cooling, as shown in fig. (b) . the decrease of solute gellan concentration at t i( ) might cause a decrease in the local viscosity because solubilized random coil gellan molecules were incorporated into helices and their aggregates. the increase of d gel at t i( ) was more pronounced than expected from the decrease of local viscosity. therefore, it is considered that the mw distribution of the gellan remaining as a random coil as a solute among the network of aggregates was shifted to lower mw values because higher molar mass gellan chains were preferentially incorporated to junction zones than lower mw chains. this is consistent with the observation that shorter chains preferentially eluded out from gellan gels when gels were immersed in solvents (hossain & nishinari, the diffusion coefficient of pullulan decreased with decreasing temperature, and it was concluded that pullulan chains were not involved directly in the aggregation of gellan, and remained dissolved during the gelling process, probably due to the high solubility of pullulan in water. the diffusion of pullulan is thought to be restricted by the hydrodynamic interaction with the solute gellan as well as the network of aggregates. the restriction became smaller with decreasing solute gellan concentration and increasing network pore size, both of which result from the thickening of the aggregates. since probe molecules (pullulan) in the solution have no intermolecular interaction with the host polymer (gellan) except the hydrodynamic interaction, the diffusion is expressed as follows (cukier, ; de gennes, ; matsukawa & ando, ) : where d inhost and d inpure are d of the probe molecule in the host polymer solution and that in pure dilute solution, respectively, and ξ is the hydrodynamic mesh size which represents the hydrodynamic mesh size made of the solute gellan and the network of aggregates. values of ξ are shown in the right vertical axis in fig. d . the change in the microscopic surroundings of pullulan during the cooling process is shown schematically in fig. . when a physical polymer gel is immersed in a solvent, molecular chains which are not connected to the junction zones were found to release out from the gel to the solvent (djabourov et al., ). it has been observed that the immersion of potassium type deacylated gellan gels in water or electrolyte solution induces chain release, and this release is more noticeable for shorter chains. ultimately the gel becomes eroded and then disintegrates, and the rate of collapse depends on polymer concentration, original molecular mass and the initial salt content of the gels and the solvent. salt diffusion from the gels into the solution is faster than chain release; chains which lose condensed or bound ions cannot retain a helical conformation, and so they diffuse out into the immersion in pure water. they estimated the diffusion coefficient ( . ~ . ) × - m s - , which was slightly larger than diffusion coefficient ranging from . × - m s - to . × - m s - at ℃ in mm nacl for molar mass range from . × to . × reported by takahashi et al. ( ) . this is reasonable since molecular chains released out from the gel network were shorter than un-released chains de silva, poole-warren, martens, & in het panhuis ( ) studied the chain release of also deacylated but ca + cross-linked gellan gels at ℃ in a phosphate buffer saline (pbs). these authors also found the chain release as mass loss up to days. since the cd ellipticity was proportional to the gellan concentration, they estimated the diffusion coefficient of gellan from the cd data d = . × - m s - , which was two orders of magnitude smaller than the reported value of deacylated gellan at ℃ (takahashi et al., ). they ascribed this difference to the retardation of the mobility of gellan molecules in gel network. molar mass is a key for the gel formation. when the material changes from sol state to gel state, the molecules are connected each other spanning the whole space in the vessel, and this is called the percolation at which the molar mass is thought to diverge to infinity (de gennes, ; nishinari, ; tanaka, ; tokita, ) . gelling polysaccharides and proteins have been used widely for controlling the food texture. mechanical strength of gels can be indexed by the elastic modulus and/or fracture stress/strain. although the oral processing is a dynamic process, that is time dependent, only the fracture stress has been used to characterize the mechanical j o u r n a l p r e -p r o o f properties quite often neglecting the intermediate stress/ strain of the compression/shearing in the mastication. for example, in fishery industry in japan a so-called "gel rigidity", which was defined as the ratio of the force to deformation at break using a spherical probe of mm diameter, has been used widely. though it may have some merit, it does not distinguish two concave and convex curves connecting the origin and the break point in the force-deformation plot. we hope that these distinction will be taken into account in the near future. since the average number of residues per helical turn to form α-helix has been established as . , and atoms are involved in the ring formation by hydrogen bond, α-helix is also called . -helix. the average number of residues per helical turn for other protein helices is also determined as . for amylose gelation is known to be a triggering in the early stage of retrogradation of starch, but only a few studies have been published on the gelation kinetics using amylose samples with different chain lengths (clark, gidley, richardson, & ross-murphy, ). fig. a shows the gelation process of % solutions of amylose with different chain lengths. storage shear modulus g' increased fast and then reached a plateau value earlier in lower molar mass samples. the plateau value increased with increasing molar mass within the dp range from to however the highest molar mass dp still continued to increase even after min (clark et al., ) . the concentration dependence of plateau modulus of amylose gels and β-glucan gels is shown in fig. . while the plateau storage modulus of amylose gels in the concentration range from to %, the modulus is higher for higher molar mass than for lower molar mass (fig. a) , the modulus of β-glucan gels in the concentration range from to %, the modulus is higher for lower molar mass than for lower molar mass (fig. b ), in amylose gelation, the plateau value increased with increasing molar mass as shown in fig. a , while in β-glucan gelation, the plateau value seemed to decrease with increasing molar mass as shown in fig. b , though for slow gelling samples with higher molar mass did not reach the plateau within h. the apparent inconsistency between fig. a and fig. b , that the apparent plateau modulus increases with increasing mw in the former (amylose) while it is smaller for higher mw in the latter (β-glucan), originates from the non-equilibrium nature of these apparent plateau moduli. as shown in fig. b , the gelation proceeds much slower in β-glucan than in amylose, it is impossible to compare directly with fig. a and fig. b . this reminds us the slow gelation rate of gelatin solutions. the storage modulus of . % (w/w) gelatin/water increased faster at lower temperatures but it has not reached an equilibrium plateau but continued to increase even after h (te nijenhuis, ) . it is also necessary to take into account the molar mass range and in addition molecular conformation, flexible or stiff, when the modulus of different gelling polymers are compared as a function of molar mass. the effect of minor component of intermediate dp higher than and a small amount of protein in β-glucan seems to be difficult to be quantified accurately, and is a future problem. it should be noted that there is a critical molar mass and a critical concentration below which no gelation occurs. for amylose this critical molar mass was in between dp= and dp= , and below dp= only precipitates were observed. the critical concentration for gelation of amylose was found to depend on the molar mass (gidley & bulpin, ). since it is difficult to prepare samples with different molecular weight and with a narrow molecular weight distribution, there have not been so many studies on the molecular weight dependence of elastic modulus of gels. saunders &ward ( ) studied rheological properties of gelatin gels with different molecular weights indexed by intrinsic viscosity, and showed that the elastic modulus increased with increasing molecular weight up to a certain value and then levelled off whilst the breaking stress continued to increase with increasing molecular weight (saunders & ward, shear modulus of alginate gels as a function of weight average degree of polymerization was reported to show two regions; the initial steep ascending region and then levelled off and show a long horizontal region. smidsrød ( ) showed this tendency for both alginate gels and κ-carrageenan gels. he suggested that the difference of the plateau values, independent of the dp, is due to the difference in the number or the strength of the junctions in alginate and κ-carrageenan. twenty years later, the same group draget, skjåk braaek & smidsrød ( ) reported the molar mass dependence of apparent young's modulus e app and storage shear modulus g' of gels of alginate. in the range of molar mass from kda to kda, they observed that both e app and g' increased with increasing molar mass and did not show the molar mass independency as shown in fig. . it should be mentioned that they noticed that elastic modulus increased with increasing guluronic acid residues as reported previously from the same group (smidsrød & haug, ) . this comparison is difficult because it is difficult to obtain the series of samples with the same molar mass and only different in the guluronic acid residue content. quite a similar behavior was reported for κ-carrageenan gels with different molecular weights (rochas, rinaudo, & landry, ). these tendencies can be summarised as shown in fig. . the elastic modulus increases steeply with increasing molar mass, and then it levels off above a certain molar mass. the temperature dependence of the elastic modulus has been attracting much attention. whether the elasticity of agarose is entropic or energetic has been debated for a decade just after the nd world war in japan (nishinari, a). nishinari, watase, & ogino ( ) tried to separate the entropic term and energetic term from the observed temperature dependence of young's modulus e′ for gels of agarose with different molar masses (indexed by the intrinsic viscosity) and with the same sulphate content and the , anhydro-l-galactose content, both of which are important factors influencing the helix and gel formation (nishinari & fang, ). the young's modulus e′ increased with increasing molar mass and temperature for higher molar mass fractions (fig. a) . the tendency observed for the increase of e′ with increasing molar mass is consistent with the above fig. for gels of gelatin, κ-carrageenan, and alginate. the elastic modulus was determined as dynamic young's modulus e′ by observing longitudinal vibrations of a cylindrically moulded gel, which is free from slippage. while e′ decreased monotonically for a gel of agarose with a low molar mass (f ), e′ of other gels with higher molar masses (f , f , f , f ) increased gradually from ° c up to a certain temperature t max , and then decreased (nishinari et al, ) . this temperature t max shifted to higher temperatures with increasing molar mass and concentration (fig. a) . it was found that the entropic part decreased while the energetic part increased with increasing temperature. the increase of e′ with increasing temperature was explained by a reel-chain model (nishinari, koide, & ogino, ) . stress relaxation of agarose gels with different molar masses were observed, and the relaxation spectra were obtained using time℃ temperature superposition (watase & nishinari, ) . it was shown that box type spectra extended to longer relaxation times with increasing molar mass at a constant concentration, and that both breaking stress and breaking strain increased with increasing molar mass (fig. b) . more recently, moritaka. yamanaka, kobayashi, ishihara, & nishinari ( ) examined the size distribution of the masticated fragments of agarose gels, and found that the size distribution depended on molar mass as well as a mouthful size. as expected, the size of masticated fragments decreased with decreasing molar mass of agarose, which forms more brittle gels broken at lower fracture strains. high-sensitivity isothermal titration calorimetry (itc), ca + -selective potentiometry, and relative viscometry. the results revealed three distinct and successive steps in the binding of calcium to alginate with increased concentration of ca ions. they were assigned to (i) interaction of ca + with a single guluronate unit forming monocomplexes; (ii) propagation and formation of egg-box dimers via pairing of these monocomplexes; and (iii) lateral association of the egg-box dimers, generating multimers. the boundaries between these steps were found critical, and they were closely correlated with the ca/guluronate stoichiometry expected for egg-box dimers and multimers with / helical chains. in this multi-step binding of ca to alginate, the dimerization process was found to be highly critical, only occurring when the stoichiometry of egg-box dimers is met, that is, ca/ g = . , one calcium ion per four guluronate residues. the formation of egg-box dimers and their subsequent association are thermodynamically equivalent processes and j o u r n a l p r e -p r o o f can be fitted by a model of independent binding sites. in addition, the step (iii) showed different association modes depending on the molar mass of alginate. while the relative viscosity η r continues to increase in (iii) indicating that lateral association of egg-box dimers for lower molar mass alginate occurs between different dimers, η r decreases in (iii) for higher molar mass alginate indicating the association makes the volume of aggregates smaller. fig. a shows the gelation profiles of mg/ml alg with and without addition of mg/ml oligoguluronate, guluronate block (gb) at various concentrations of ca-edta. it is well known that alginate forms an inhomogeneous gel by a rapid ionic reaction, and the slow release of calcium from calcium carbonate or ca-edta in the presence of slow acidifier like glucono-delta-lactone is used to make a homogeneous alginate gel (draget, Ǿstgaard, & smidsrød, ; thu, smidsrød, & skjåk braaek, ) . gelation kinetics and equilibrium gel properties of alginate aqueous solutions induced by in-situ release of ca ions from ca-edta during d-glucono-delta-lactone (gdl) hydrolysis were observed and the modulatory effects of gb were analysed quantitatively. it is apparent that when ca-edta < . mm the systems without mg/ml gb gelled faster than those with the addition of mg/ml gb and also attained a higher value of saturated equilibrium storage modulus. it manifests an inhibitory effect exerted by the addition of gb. on the contrary, when ca-edta > . mm, the systems with mg/ml gb tended to end up with a higher saturated equilibrium storage modulus than those without mg/ml gb. moreover, the gelation kinetics seems not to be altered significantly by the addition of gb. the effect of guluronate block gb depends on the ratio of calcium ions to guluronate unit, r = ca/g. the addition of gb shows an inhibitory effect in the range of . < r (ca/g) < . , and promotive effect in the range of r > . based on static and dynamic viscoelastic measurements. mixed egg-box dimers between alg and gb were ruled out because of cooperativity requirement of dimerisation. the promotive effect in the higher ca concentration regime was assigned to the role of gb dimers participating in and enhancing the lateral aggregation of alg dimers. in conclusion, short chain guluronate block is found to inhibit the gelation of alginate at low concentration of ca + ions while it enhances the gelation at higher ca + concentration. inhibitory effect is attributed to the binding of calcium ions to shorter guluronate chains. those findings may be useful in food processing and also have some therapeutical significance in the rheology of sputum as discussed in the following section. significantly different from the case of alginate where no gelation could be induced at all. in the range of . < r < . , the addition of gb was found to inhibit the gelation of lmp, whereas it had a negligible effect on the gelation of alginate as long as a fixed r was considered. in the range of r > . , gb was found to promote the gelation of lmp again, which is similar to the case of alginate as described above (fig. b) . polysaccharides are frequently used to reduce the fat content, and to improve the texture and water holding capacity (whc) of protein gels. yao, zhou, chen, ma, li, & chen ( ) recently examined the effect of the addition of sodium alginate (sa) with three different molar masses on the whc of chicken breast myosin gels. they found that whc increased simultaneously with turbidity and thermal stability, accompanied with the decrease in surface hydrophobicity with increasing molar mass of added sa. they noticed that the addition of sodium alginate shifted the thermal transition temperature to higher temperatures detected by dsc with the higher contribution by a higher molar mass sa, which is consistent with the same stabilisation by the addition of flaxseed gum to salt-soluble meat protein, but is contradictory with the reported thermal destabilisation of myofibrillar, sarcoplasmic and connective tissue protein by the addition of sa. they observed that inhomogeneity of cavities formed in sa-protein gel network was enhanced by the higher molar mass sa, which resulted in the increase in whc because they thought that large cavities could store more water. some polysaccharides such as locust bean gum and cereal β-glucan as well as poly(vinyl alcohol) (pva) form cryogels after freezing and thawing cycles while most polysaccharides such as agarose, carrageenans donot but only framework structure remains after freezing because water force-extension curves for cryogels of pva with different dps (curve ( )), (curve ( )), (curve ( )), and (curve ( )). however, the storage shear modulus g′ of oat β -glucan cryogels showed opposite tendency as illustrated in fig. c . the finding that g′ increased with increasing concentration of βglucan is a common phenomenon. however, the g′ values of cryogels decreased with increasing molecular size. lazaridou & biliaderis ( ) had found a similar trend in their earlier study for cereal β-glucan gels formed at room temperature and it had been attributed to the higher mobility of shorter chains (lazaridou et al., (lazaridou et al., , . their interpretation was as follows: the molecular size has a significant impact on the viscosity of polymer solutions, thus influencing the diffusion rates of the interacting polysaccharide chains. however, it is difficult to understand this explanation because the stress-strain curves shown in fig. a , the young's modulus estimated from the initial slope of the curve is the largest for the gel from the higher molar mass oat glucan kda and the smallest for the gel from the lowest molar mass oat glucan kda. unfortunately, gels from kda were so weak to keep the shape to do the compression measurements. the inconsistency between the young's modulus from the slope of stress-strain curves in fig. a and the storage shear modulus shown in fig. c should be studied further. heat set gel formation also depends on molar mass. the gelation process of solutions of methyl cellulose (mc) with different molar masses and with approximately the same methoxy content ( wt%) was observed by measuring g' and g" at a constant temperature ℃. the gelation time at which g' began to increase above the base line was shorter in a higher molar mass mc solution. this rheological tendency is in accordance with dsc observation in which an endothermic peak appeared on heating accompanying the gelation. the longer chains have a greater tendency to form junction zones which are induced mainly by hydrophobic interaction (nishinari, hofmann, moritaka, kohyama, & nishinari, ) . whether the gel formation of mc is caused by spinodal decomposition or by the nucleation and growth is still a matter of hot debate (mcallister, schmidt, dorfman, lodge, & bates, ). more detailed studies on the gelation of mc changing molar masses at a constant degree of substitution, preferably using regioselective substitution, are expected in the future. the gelation of kgm occurs through the alkali-induced removal of acetyl groups which confer the solubility for this polysaccharide (nishinari, b). kgm forms a thermally stable gel by deacetylation upon addition of alkaline coagulant and the gelation of kgm is promoted by heating, in contrast to many other cold-set thermo-reversible gels. the increase in the concentration or molecular weight of kgm shortened the gelation time and increased the rate constant of gelation, and the resulted gels show higher modulus (yoshimura & nishinari, ; zhang, yoshimura, nishinari, williams, foster, & norton, ) . this is consistent with the general tendency observed in gelatin, carrageenan, alginate and agarose gels as described above. the effect of the degree of acetylation (da) on the gelation of kgm was studied (huang et al., ; gao et al., ) . to understand the effect of molar mass on the gelation, it is necessary to obtain kgm sample with a constant degree of acetylation (da) with different molar masses, but this has not yet been done because of the difficulty of sample preparation. the same difficulty is encountered in the study of the interaction of kgm with other polysaccharides such as κ-carrageenan, agarose, starch or proteins such as gelatin and myofibrillar proteins although these mixtures have been used in food products without understanding the detailed mechanism. the more detailed knowledge will improve further the products. crystallinity of solid fat is generally much higher than high molar mass polysaccharides and proteins, and the linear elastic range is much narrower than in semi-solid foods mainly composed of polysaccharides and proteins. spreadability of semi-crystalline solid fat such as butter and margarine is an important functionality during consumption, and this characteristics is different from high molar mass polysaccharide and protein food gels. as is shown in fig. , the gel strength was found to increase with mass fraction of ec in a power law fashion, and also to increase with increasing molar mass of ec. from the cryo-sem observation of partially de-oiled gels, it was found that the internal structure consisted of oil droplets entrapped within a network of interconnected strands or bundles of ec, which formed a scaffolding to support the gel (zetzl, gravelle, kurylowicz, dutcher, barbut, & marangoni, ). rennet-induced gelation of casein micelles has been studied extensively because it is the basis of cheese production. niki, kohyama, sano, & nishinari ( ) prepared the casein micelles with different sizes by differential centrifugation. since the κ-casein, the substrate of rennet enzyme chymosin, exists mainly on the surface of casein micelles, the smaller micelles are richer in κ-casein content. therefore, glycomacropeptide is liberated faster in smaller casein micelles as shown in fig. a . the saturated value of the storage shear modulus was largest in the gel formed by the smallest casein micelles (fig. b) . then, the smallest casein micelles can form a gel faster and can form a stronger gel. in this case, the gelation is dominated not only the size of the casein micelles but the difference of the distribution of κ-casein in the casein micelles should also be taken into consideration. gluten network determines the dough rheology, and thus governs the quality of bread. it has been generally accepted that glutenin contributes to the elasticity and gliadin is for the viscosity, and this is mainly ascribed to the high molar mass of the glutenin and the lower molar mass of of mixing (arrival time) lmw and hmwglutenins were found to form aggregates, which were then distorted to form a uniform network with gliadin when mixed until the dough reached peak time. the development of this gluten network formed by the assembly of the three gluten subunits was thought to be related to the significant increase of dough strength. further mixing after peak led to departure showing less dense structure characterized by the increase in mean lacunarity. recent trend to exploit the possibility to use biowaste or to add more values to plant proteins is becoming more and more active (petrusan, rawel, & huschek, ) . felix, perez-puyana, romero, & guerrero ( ) examined enzymatic hydrolysates of pea protein isolate expecting the improvement of functional properties. they found that although the solubility increased by hydrolysis, the gelling ability was reduced. it should be noticed that the lipid content and the surface hydrophobicity decreased by enzymatic hydrolysis, and therefore, the decrease in gelling ability was the result of all these interactions. gelling ability of plant proteins was reported to decrease by enzymatic hydrolysis, but a limited enzymatic hydrolysis makes the buried peptide groups exposed on the surface, and the ionizable groups liberated thus increasing the protein-protein interaction and leading to strengthen the gelling ability (wouters, rombouts, fierens, brijs, & delcour,. ). since transglutaminase (tgase) can make new covalent bonds, ε-(γ-glu)-lys crosslinks, it has been used widely to increase the gelling ability of many proteins such as soy proteins, sunflower protein, canola proteins (wouters et al, ). here, the molar mass decrease by hydrolysis and then its increase by tgase are the main governing principle of the decrease and increase in the gelling ability. to control the mechanical properties of solid foods, it may be useful to remind the fundamentals of glass transition (bhandari & roos, ). glass means the amorphous solid not only the glass used for windows or cups. on lowering the temperature, the solid behaviour changes from rubber-like to solid-like at the glass transition temperature t g . this transition is different from a simple liquid to a simple solid (crystal) transition, and was studied extensively for amorphous polymers. the solid-like amorphous glass is in a non-equilibrium super cooled liquid state, and different from an equilibrium crystalline state. large scale main chain motion is allowed and the amorphous solid is ductile/plastic above t g , while the large scale motion is frozen and the solid becomes brittle below t g . this can be detected as a drastic decrease of elastic modulus from ca pa to pa on raising the temperature. in the first-order transitions such as melting and boiling, there is a discontinuity in the volume-temperature (v-t) or enthalpy-temperature (h-t) plot, while in the glass transition, these changes are gradual. therefore, the first order temperature derivative above the glass transition temperature t g , the amorphous polymer behaves rubber-like and shows a larger strain at break, but below t g , the polymer showed a brittle fracture. the glass transition temperature of synthetic polymers has been studied extensively, and a fox-flory equation t g = t g -k/m, where t g is the t g of the polymer when the molar mass m approaches infinity, and k is a constant (nunes et al., ) . unfortunately, this equation was not found to fit well to experiments for polysaccharides and proteins, though not so many papers have been published. since fractural behaviour of solid state is crucial in the application of plastics, effects of molar mass was studied using polystyrene with different molar masses from . kda to kda in the early days (onogi, matsumoto, & kamei, ) as shown in fig. . the elongation test of film ( cm length and cm width) was performed at the elongation rate from . to . cm/s and at a temperature range from to ℃. they found that stress σ f and strain γ f at fracture as a function of elongation rate dγ f /dt could be superposed by shifting horizontally as had been done for linear viscoelastic studies (ferry, ) . the shift factor was found to be similar to that used in wlf equation. the resulted master curves of σ f or strain γ f as a function of dγ f /dt was divided into four regions, i. brittle to ductile transition, ii. ductile, iv. viscous fluid, and iii. between ii and iii. the molar mass dependence was more clearly observed at regions iii and iv. in other words, mechanical properties are less sensitive to molar mass in the region i and ii. at regions iii and iv in master curves log σ f or γ f vs log s t dγ f /dt were obtained by superposition with a suitable shift factor s t (fig. ). higher mw film showed a higher fracture stress or fracture strain at the same elongation rate where the elongation rate is slow. in food science/technology, it should be reminded that water plays an important role of plasticiser (bhandari & roos, ). amorphous solid polymers can be made more pliable by lowering its t g , and this can be achieved by incorporating quantities of high-boiling, low-molar-mass compounds in the material. these are called plasticisers and must be compatible which is similar to non-degraded starch. however, the reported values donot agree exactly with ca ℃ (roos & karel, ). the effectiveness of the concept of the glass transition in low temperature preservation of food was shown in many foods, e.g. in frozen starch products. below the glass transition temperature amylose and amylopectin chains mobility is reduced, preventing the molecular association leading to the restructuring (retrogradation) (zaritzky, ) . maltodextrins are used widely as drying aids in spray drying of, for example, skim milk. it is desirable to prevent powders sticking and it is effective to raise the t g so that the surface of powders might become less sticky; above the glass transition temperature the powders are in glassy state and less sticky. bakery products formulations taking into account the glass transition were recently reviewed (wang & zhou, ) . since polysaccharides and proteins have hydrophilic residues such as hydroxyl groups or amide groups, the glass transition is influenced by these intermolecular interactions. main-chain motion is restricted owing to these intermolecular interactions and the glass transition temperature is higher than that of the hydrophilic polymer in the completely dry state (hatakeyama & quinn, ) . in most dried proteins and polysaccharides no glass transition or melting is observed until decomposition of the main chain occurs because intramolecular and intermolecular hydrogen bonds stabilise the high order structure of these polymers. glycerol, sorbitol, or dimethylsulphoxide were used to plasticise the amylose/starch films to study the glass transition at since gelatin has been widely used in food industry although its usage in photographic film has been reduced, understanding the interaction of water and gelatin remains important. dsc heating curves for gelatin gels of different concentrations from . to . wt%, quenched from room temperature to - ℃ using liquid nitrogen, are shown in fig l a. a step-like change in baseline observed at around - ℃ was attributed to glass transition. the glass transition temperature t g is shown by an arrow in fig a. t g shifted to higher temperatures with decreasing gelatin concentration beyond %. it is reported that various kinds of hydrogels form glassy state by quenching from a sol state to - ℃, and a part of water molecules associated closely with matrix polymers solidify into an amorphous state (hatakeyama & hatakeyama, ). by heating, amorphous ice associated with matrix become mobile. the free molecular motion commences in gelatin gels with amorphous ice, which is detected as t g in heating dsc curves. a broad exothermic deviation is observed in samples containing a small amount of water (fig. a, curves a-c), which was attributed to the cold crystallization. water molecules solidified in an amorphous state are mobilized at t g on heating, and molecular motion is enhanced by successive heating and reorganised as unstable ice just before melting. this is confirmed by the fact that the exotherm disappeared if the sample is annealed at a temperature lower than melting. this suggests that cold crystallization can be observed for thermally unstable samples prepared by quenching (hatakeyama & hatakeyama, ). the phase transition temperatures of gel are shown as a function of water content w c in fig. b. the water content w c is defined as w c (g/g) = w w /w s (g/g), where w w is the weight of water in the system, and w s is that of completely dried sample. at water content range higher than w c = . (a water content between curves a and b in fig. a ), movement of gelatin molecules is restricted by the presence of ice, and on this account, t g shifted to higher temperatures with increasing water content. it was difficult to obtain a complete dissolution for gelatin solutions above % (molar mass of gelatin was da and the isoelectric point was . ). however, t g water, they also might have found the shift of t g to higher temperatures with increasing water content above a certain w c . it is well known that t g of a polymer shifts to lower temperatures by the addition of a plasticiser (cowie & arrighi, ). t g shifted to lower temperatures with decreasing gelatin concentration, i.e. with increasing water content below w c = . , since water molecules play a role of plasticiser. it is also well known that t g shifts to higher temperatures in the presence of a large amount of water, because in such a case the whole system cannot be converted to glassy state, but some ice crystals are formed, and they inhibit the molecular motion of polymer chains (hatakeyama & hatakeyama, ). the glass-transition temperature t g of gelatin gels as a function of gelatin concentration, therefore, showed a minimum around gelatin concentration % (w c = . ) (fig. b) . this phenomenon which shows the minimum of t g as a function of water content is widely observed in many polysaccharides-water systems. the relation between w c and the temperature t g,min at which t g becomes minimum is shown in fig. c for gellan (closed circle),xanthan gum, cellulose sulphate, alginic acid, carboxymethylcellulose, hyaluronic acid (hatakeyama, nakamura, takahashi & hatakeyama, ). the glass-transition temperature t g and the melting temperature t m of % gelatin gels containing sugars (deoxyribose, ribose, glucose, sucrose, raffinose, maltotriose, maltotetraose, α-cyclodextrin, maltohexaose) or polyols (ethylene glycol, propylene glycol, glycerin) with different concentrations as a function of molecular weight of sugars or polyols is shown in fig. d . t g shifted to higher temperatures with increasing molecular weight of sugars or polyols added, and it ranged from to k. t m also shifted to higher temperatures with increasing molecular weight of sugars or polyols added, but it ranged from to k, and the shift was not so pronounced in comparison to that for t g . it is reasonable to see that t g was lowered by adding higher sugar or polyol. however, the efficiency as a plasticizer of each sugar or polyol to lower the glass transition temperature is reduced with increasing molar mass of sugar because their glass transition temperature is higher than that of water (- ℃ = k), and their unfrozen water content per unit mass is also increasing with molar mass (levine & slade, ) . in addition, the edible films made from polysaccharides, proteins, lipids, or the combination, are used to protect foods from contamination of harmful microorganism and oxidation, and also used as are water vapor permeability (wvp) and oxygen permeability (op). these parameters are known to be influenced by molar mass, plasticizer, film production methods including casting, extruding, temperature control, and drying rate. there have been not so many papers reporting the effect of molar mass. in contrast, many papers report the effects of plasticisers, methods of production of films without controlling the molar mass. chitosan films are widely used in food and pharmaceutical industry. chitosan (ch) is a cationic polysaccharide composed of randomly distributed ( , )-linked -amino- -deoxy-β-d-glucose units, and is obtained by deacetylation of chitin (rinaudo, ) . as expected, ts of chitosan films prepared from chitosan solutions with different solvents, acetic acid, citric acid, lactic acid, and malic acid, all increased with increasing molar mass, while eb also showed the same tendency except the film prepared using citric acid. the eb of films prepared from citric acid was found that wvp was not influenced significantly by the molar mass of chitosan. they also found that op of films prepared with malic acid was the lowest, followed by acetic, lactic, and citric more recently, films of ch with three different molar masses ( kda, kda and kda) and with approximately the same degree of deacetylation . %) were prepared with and without glycerol as a plasticiser to see the effect of molar mass (liu, yuan, duan, li, hu, liu et al., ). as expected ts and eb increased with increasing mw in all the films with different glycerol contents. wvp and op decreased with increasing mw. they further applied these ch films for packaging fresh strawberries. they found the weight loss was reduced and the colour was maintained by ch films, and in addition by virtue of antibacterial properties of ch, thus it was useful to extend the shelf life. they found that the film with high mw ( kda), high ch content and % glycerol/chitosan (w/w) showed the best performance among types of films with different ch and glycerol contents the effect of molar mass of chitosan on the film properties of chitosan (ch) -corn starch (cs) composite ( : mixing ratio) was studied using low, medium and high molecular weight (lmw, with hmm ch is approximately the same to that of hmw cs alone (fig a) , values of wvp for the cs/ch blend film with lmw ch and mmw ch were significantly lower than those for lmw ch and mmw ch alone. this was interpreted as follows: the developed interactions between ch and cs, mainly hydrogen bonding type, reduced the hydrophilic groups availability of chitosan matrices, leading to a reduction in water vapor transmission rate. more recently, effects of molar mass on blend films of galactomannan and κ-carrageenan were studied using partially hydrolysed galactomannans (rodriguez-canto, cerqueira, chel-guerrero, pastrana, & aguilar-vega, ). from a widely accepted view that the film strength may decrease with decreasing molar mass, it seems to be of no use to lower the molar mass, however, the molar mass reduction leads to lower the viscosity of the solution, which is advantageous for fluid transportation by pumping to lower the energy cost and also for spray drying coating (garcia, ( : weight % mixing ratio). while ts was increased by mixing drgm hydrolysate with κ-carrageenan and the composite with lwwh shows the lowest ts, this composite showed the lowest wvp (fig. b) , which was attributed to a more compact molecular arrangement with physical bonding interactions, such as higher hydrogen bonding, in the structure of the films as a consequence of galactomannan shorter chain length (rodriguez-canto et al, ). this is the opposite tendency as described above for cc/ch film where the longer chain ch was believed to make the structure more compact. the finding that ts of the composite of κ-carrageenan with hmwh was lower than that of κ-carrageenan with mmwh was interpreted that the molar mass above mwwh was higher than the limiting value of the molar mass above which no increase of mechanical strength was observed. as was widely known, the mechanical strength of solid polymers is not increased with increasing molar mass above a certain limiting value of molar mass while eb decreased with increasing molar mass of peg. it was also found that the higher mw peg (> ) showed higher wvp, and the authors concluded that lower mw peg were more effective to decrease the moisture transfer between the food and the surrounding atmosphere. as described above, some contradictions among previous papers about the molar mass effects on the film properties, ts, eb, and wvp, have been found, and these should be clarified to develop further application of films. more systematic study should be done for polysaccharides and proteins to get more basic knowledge to develop further the utilization of polysaccharide and protein films. film thickness, plasticizers, deformation rate, production method, all these affect the film characteristics. not only trial and error approach but also more systematic study to make films are expected in future. interaction of polysaccharide and protein has been studied by many research groups because of dsc curves of κ-car during cooling at different mixing ratio r= β-lg/κ-car were not influenced by the addition of β-lg at higher ph than the isoelectric point (iep) of β-lg (ph = . ) where no electrostatic complexation occurs. on the other hand, at lower ph= . , the dsc exothermic peak on cooling accompanying the conformational and gelling transition of κ-car was suppressed with increasing mixing ratio r= β-lg/κ-car. it was suggested that β-lg/κ-car forms soluble complexes on cooling at low r, but insoluble complexes are formed at high r. relative extent of conformational transition of κcar φ (r) was defined as a ratio of dsc enthalpy change ∆h (r) for the mixture with mixing ratio r to that of κ-car without addition of β-lg ∆h( ) : φ (r)=∆h(r) /∆h( ). the extent φ (r) as a function of β-lg/κ-car mixing ratio r at different phs decreased more steeply at lower ph below φ = . (fig. b ). this decrease of φ (r) as a function of r was analyzed more quantitatively by using mcghee-von hippel ( ) theory which was used to understand the binding of protein to dna. the number of binding sites consecutively occupied by one protein molecule to κcar and the binding constant were found to increase with decreasing ph. in addition to dsc results, the number of binding sites consecutively occupied by one protein molecule to κ- car and the binding constant determined from itc measurement also showed a good agreement with the results from dsc. this inhibitive effects of β-lg on the helix formation and gelation of κ-car were further studied by using β-lg hydrolysates with different molar masses. it was found and gelation temperature shifted to higher temperatures on cooling by the addition of glucose or mannose. on heating the gellan gum gels containing glucose or mannose after cooling, the storage modulus began to decrease accompanying the gel to sol transition at a certain temperature, and this temperature shifted to higher temperatures with increasing concentration of added glucose or mannose. as shown in the previous section . . effect of molar mass of β-lactoglobulin hydrolysates on the gelation of κ-carrageenan, lysine and s (lower molar mass (< da) hydrolysate of β-lg) enhanced the gelation of κ-carrageenan but β-lg and its higher molar mass (> da) hydrolysate inhibited the gelation of κ-carrageenan. since these two gel-promotion or gel-inhibition phenomena show a common effect, there may be a boundary molar mass for kgm and its hydrolysate above which the inhibition by adding kgm and below which the promotion by adding kgm hydrolysate occurs. although the promotion of gelation and the increase in the elastic modulus of resulted gels by the addition of a monosaccharide and disaccharide for agarose, κ-carrageenan, or gellan is well documented (nishinari and fang, ), the mechanism whether it is due to the increase in the effective concentration of polymers by the hydration of a monosaccharide and disaccharide or it is due to the direct binding of residue in the polymer with the hydroxyl group in a monosaccharide and disaccharide is still a matter of debate (see stenner, matubayasi, & shimizu, ). since starch is an important ingredient of many cereals, tubers, pulses and other crops and an important energy source, and used to control texture of many processed foods, its gelatinization amylose. these nsps increased the effective concentration of starch and rva peak viscosity, but never led to gel formation judging from small deformation oscillatory measurements, g' < g" and tan δ was increased. these nsps accelerated the short term retrogradation but retarded the long term retrogradation. since the interaction between nsp and starch depends on the molar mass of nsp, amylose/amylopectin ratio and the concentration of starch, it is necessary to compare the effects of nsp with different molar masses using different starches with different amylose/amylopectin ratios to understand better the mechanism. funami, kataoka, omoto, goto, asai, & nishinari ( b), using guar gums with eight different molar masses (g/mol) (g = . × , g = . × , g = . × , g = . × , g = . × , g = . × , g = . × , g = . × ), and three maize starch samples with different amylose contents %, % and % prepared by mixing high amylose maize ( . % amylose), normal maize ( . % amylose) and waxy maize ( . % amylose), studied the effect of molar mass employing the same methods as mentioned above. for the % and normal maize, the addition of . % guar gum increased the rva peak viscosity and setback values (with some exceptions), and this increase was found more pronounced with increasing molar mass of guar gum. the onset of the viscosity increase on heating was shifted to lower temperatures with increasing molar mass of guar gum which was thought to interact with amylose, and the increasing of the rva peak viscosity was attributed to the interaction between guar and amylopectin. it was shown that the short term retrogradation was retarded by the addition of . % guar gum to % maize starch, which was suggested by the increase of the loss tangent as a function of molar mass, and this retarding effect was shown to be correlated with the decreasing leached amylose. this leached amylose in starch-guar system was found to decrease with rva peak viscosity values (funami, kataoka, omoto, goto, asai, & nishinari ( c). for the longer term retrogradation, higher molar mass guar g and g were effective to retard the retrogradation in higher amylose % starch ( % amylose) than in low amylose starch ( % amylose), which indicated that high molar mass guar suppress effectively the gelation of amylose until days. for a higher starch concentration ( % maize starch), the rate constant k of retrogradation kinetic the rate constant) was found to decrease with increasing molar mass of the added guar gum ( . %) for higher molar mass than . × (g ), however, the lower molar mass guar gum, g and g were found to promote the retrogradation ( fig a) . syneresis for % normal starch was quantified with and without guar ( . %) (fig. b ). for the control, syneresis was estimated to be . ± . and . ± . % after storage at ℃ for and after freeze-thawing, and reported that syneresis was increased with increasing cycle of freeze-thawing and that pectin reduced the syneresis. however, in this experiment the lower mw pectin was more effective to reduce the syneresis, which is opposite in the result for maize-guar system shown in fig. b . whether this contradiction is due to the difference in the syneresis observed in the starch stored at ℃ and that subjected to freeze-thawing is not clear, and should be studied further. corn fibre gum (cfg) has been attracting much attention because of its adhesive, thickening, properties of rice flour mixed with native β-glucan and with partially hydrolysed β-glucan, and found that the rva peak viscosity decreased by adding β-glucan, and the extent of the decrease was more pronounced by the addition of hydrolysed β-glucan. dangi, yadav, & yadav ( b) reported that the rva peak viscosity for barley starch decreased by adding lower mw pectin (pectin hydrolysate) but the peak viscosity was higher when native pectin (high mw) was added. dangi, yadav, & yadav ( a) reported that the rva peak viscosity for pearl millet starch increased by adding even lower mw guar (guar hydrolysate), and the peak viscosity was higher when high mw guar was added. this was also reported for wheat starch-arabinoxylan system (hou, zhao, tian, zhou, yang, gu, et al., ). the rva peak viscosity in the gelatinization of wheat starch during heating was found to be suppressed by the presence of arabinoxylan (ax), and this effect was more pronounced for lower molar mass ax. it was interpreted by the insertion of lower molar mass ax in the molecular chain of amylose which limit the formation of double helices. this interpretation was based on the lowering of the enthalpy of amylose -lipid complex formation and melting on cooling and heating dsc, and this decrease was more pronounced in the presence of lower molar mass ax. although the amylose -lipid complex formation has been studied extensively by x-ray diffraction, nmr, and other methods, the insertion of short polysaccharide chains have not been studied, and should be studied further. as for the rva peak viscosity, non-starch polysaccharides inhibit the gelatinization and decreased the value, but opposite tendency was also found. whether the increase in the peak viscosity by the addition of non-starch polysaccharide is due to its own high viscosity or not should be clarified the so-called setback values of rva were not consistent among literatures probably because of its structure break down nature in the measurement as pointed out in chantaro, pongsawatmanit & nishinari ( ). in addition to molar mas effect, the structural complexity of starch, the chain length distribution of amylose and amylopectin, degree of branching, and the mixing ratio and concentration effect also influence the gelatinisation and retrogradation. it is not easy to examine all these aspects in one laboratory, and the collaborative research is required. gluten network determines the dough rheology, and thus governs the quality of bread. it has been generally accepted that glutenin contributes to the elasticity and gliadin is for the viscosity, kda) and f (mw= kda). they found that the added weax reduced the heat-induced polymerisation of gluten, and this reduction rate was more pronounced for the lower molar mass fraction f . since the loaf volume was found to decrease with increasing heat-induced polymerisation of gluten, it was suggested that the lower molar mass fraction f was more suitable for the production of steamed bread. however, they also stated that excessive inhibition of gluten polymerisation resulted in the weakened gas retention capacity and thus the loaf volume could not be increased. in addition, the two fractions are different not only in molar mass but also in ferulic acid content, and monosaccharide (especially arabinose and mannose), more detailed study is required to understand the mechanism. when two polymer solutions are mixed, phase separations due to thermodynamic incompatibility or synergistic interaction due to weak secondary molecular forces occur. phase fractionation for higher molar mass ga (em ) with mw of . × g/mol and a polydispersity index (m w /m n ) of . than for lower molar mass std (standard gum arabic) with . × g/mol and . . in the phase separation induced fractionation of gum arabic /hyaluronan mixed systems at a fixed ha concentration of . %, the agp content was found to increase from % to % when the em concentration was decreased from % to . %. the agp content of standard gum arabic also increased from % to % when the concentration of std was decreased from % to %. the extent of increase in agp content was much more significant for em /ha system than std/ha system. this is in line with the previous findings that the fraction of higher molecular weight tends to segregate while the lower molecular weight fraction has relatively biopolymer are also used for stabilising dispersed particles or emulsion droplets by forming a steric stabilising layer, and higher molar mass biopolymers are more effective to make a thicker selenium is an essential micronutrient although it is toxic in large doses. its deficiency is believed to cause some diseases such as kashin-beck disease, and thus selenium nanoparticles (senps) were prepared by the stabilisation of gum arabic (kong, yang, zhang, fang, nishinari, & phillips, ). senps are not stable without any dispersant and show aggregation. chitosan was found effective to stabilise the senps (son, chen, zhao, sun, che & leng, ), which will be discussed later. ga was found effective to stabilise senps, and senps (particle size of . nm) were found to be stabilised in gum arabic aqueous solutions for ca. days (fig. b) . the alkali-hydrolysed ga (ahga) was also prepared and its efficiency in stabilising senps was found to be less effective than ga. it was concluded that the branched structure of ga as well as molar mass were important for the stabilisation. it was also found that hydroxyl radical scavenging ability and , -diphenyl- -picrylhydrazyl (dpph) scavenging ability of ga-senps were found higher than those of ahga-senps. agp thus the emulsification ability of asy was enhanced. asy modified in this way contained a two-fold agp content and . times higher average molar mass, and asy-stabilised emulsions were found resistant against severe conditions, such as high saline or low ph conditions (fig. c) . more recently, in the study of the stability of emulsions stabilised by conjugates prepared by maillard reaction between whey protein hydrolysates and linear dextrin with three dps ( , , ), the highest stability was found for the emulsion prepared with the linear dextrin with the highest dp, which conferred the highest steric hindrance ( chain to the hydrophilic sodium alginate chain, and prepared ugi-alg with three different molar masses ( kda, kda and kda). it was found that this amphiphilic ugi-alg formed self-assembled micelles, and the critical aggregation concentration was lowest for the highest molar mass ugi-alg, and this micelle with higher molar mass ugi-alg formed a more compact and stable micelle. it was also found that emulsions of soybean oil/water and styrene/water were stabilised better by higher molar mass than lower molar mass ugi-alg, which were proposed to be used as a drug delivery based on a model experiment of curcumin release. it is expected that the study on the safety of this kind of hydrophobically modified polysaccharides in food use will be clarified in the near future maltodextrin is often used with a small molecule emulsifier to stabilise the emulsion. anti-tumour activity of β-( → )glucans such as schizophyllan, lentinan, grifolan, and others have been studied extensively. it is well known that schizophyllan (spg) takes a triple helix conformation in water, and random coil in dmso or alkaline solvent (norisuye, ) , and it has been used as anti-tumour agent. tumour inhibition ratio was evaluated by the inhibiting effect of and below that molar mass spg was found random coil, and examined the molecular weight dependence of the tumour inhibition ratio using sarcoma ascites for the random-coil and the triple helix species of spg. they concluded that schizophyllan has anti-tumour activity against sarcoma only when it has a molecular weight higher than x and a triple helical structure in aqueous solution (fig. ) . zhang, li, xu, & zeng ( ) also reported that the triple helix conformation is necessary for lentinan, also a β-( → ) glucan having β-( → ) branching structure as spg, to show anti-tumour activity, and thus lower molar mass lentinan is not effective. they found that the highest anti-tumour activity for uspg which has a lower mw than non-degraded spg and uspg (fig. ) , which seems to be contradictory to the results shown in decreased below the molar mass of ca , and the lower anti-tumour activity found for uspg than for uspg (fig. ) is in accordance with the previous finding shown in fig. . degraded spg inhibited the production of no, but the lower mass spg was more effective. however, the criteria used to estimate the anti-tumour activity are different in research groups unfortunately, and thus the direct comparison is not possible. this should be further clarified. saccharomyces cerevisiae had anti-tumour effects in s tumour cells, and the effect was found dose dependent but the molar mass of β -glucan was not changed in their experiments. to inhibit the hemolysis was found more pronounced for high mw β -glucan (dotted line) than for low mw one (broken line). all these evidences suggest that the immune response is in part non-specific, determined by and low mw oat β-glucans were found beneficial, but high mw oat β -glucan was proved to be more effective in decreasing stress oxidation in animals with lps induced enteritis. low mw oat β -glucan was found more effective in alleviating parameters in animals without administered a similar difference between high and low mw barley β -glucans in the antioxidant activity the molar mass was found in the order of oat ( kda), wheat ( kda) and barley ( kda). the reduction of total cholesterol, triglyceride and malondialdehyde as well as the increase of sod in the liver by the administration of β -glucans were found more conspicuous with increasing mw. although the polydispersity in the mw distribution was different in three cereals, the difference in the effectiveness was believed due to mainly the difference in mw. since it has been demonstrated that molar mass, conformation, chemical modification, and solubility of β -glucans significantly affect their anti -cancer, immune -modulating, and anti - inflammatory properties, further studies are necessary to understand the mechanism using better balazs ( ) reported mechanical spectra for synovial fluids taken for a healthy young adult, and a healthy elderly person and an osteoarthritis (oa) patient. a cross-over frequency at which the storage modulus and loss modulus coincide was found to shift to higher frequencies with increasing age, and no such crossover was found for a synovial fluid from an old person. similar but more complete data were collected from normal samples and oa samples by kawata, okamoto, endo, & tsukamoto ( ). kawata et al ( ) showed that the mechanical spectra of an oa patient becomes closer to that of a normal healthy person by adding high molar mass ha (fig. c) . since it was reported that molar mass of ha decreases or the concentration of ha decreases with aging, it is an important therapeutic problem how the rheology changes when short chains (sha) coexist with long chains ha. if g′ and g of ha are decreased by adding sha, is it better to remove sha? welsh, rees, morris, & madden ( ) reported that the addition of disaccharide units drastically changed the mechanical spectra of ha from an entanglement solution behavior in which g′ and g〞show a crossover at ca - rad/s to a dilute solution behavior in which g′ was smaller than g〞at all the angular frequency range from . rad/s to rad /s and both moduli are strongly frequency dependent ( fig. a(a) ). however, fujii, kawata, kobayashi, okamoto, & nishinari ( ) found a slight increase of both moduli on adding disaccharide units (fig. a(b) ). they prepared ha short chains with various chain lengths not includes a chain-length effect, a sugar effect, and a salt effect. therefore, effects of the addition of sha were explained in terms of the superposition of these three distinct effects. a shift factor "a" which depends on these three effects was developed to explain these additive effects quantitatively. bursitis is known to be a common knee pain symptom which is induced by excessive synovial fluids. effects of injection of high or low molar mass ha to the joint synovial fluid of oa patients were studied, and it was found that higher mw ha decreased some specific protein such as mammary gland branching and kda ha fragments strongly stimulate branching, but the activity of ha decreases with increasing molecular weight and kda ha strongly inhibited this morphogenetic process. ha was used as wound healing therapy. here again, the physiological function is found to depend strongly on molar mass, and high molar mass ha and low molar mass ha were reported to function in opposite directions: while high molar mass ha has already been used in would healing based on its anti-inflammatory and immunosuppressive properties, low molar mass ha was reported to be a potent proinflammatory molecule. oligo ha, - disaccharide units, were shown to stimulate endothelial proliferation at a lower concentration than native higher molar mass ha. in addition, the lesion area was found completely recovered when the endothelial layer was exposed to oligo ha for h after injury, while the wound remained half recovered pretreated with native ha at the same condition (gao, pseudomonas aeruginosa produces extracellular alginate which interacts with tracheobronchial mucin. thick and sticky mucus is accumulated. lung disease results from clogging of the airways due to mucus build-up, decreased mucociliary clearance, and resulting inflammation. this is one of main signs and symptoms of cystic fibrosis. in the pulmonary clearance mechanism, tracheobronchial mucus traps inhaled particles and bacteria, which should be carried away by mucociliary transport. however, pseudomonas aeruginosa, an opportunistic human pathogen cannot be easily removed by this mechanism. mucin is known to consist of heavily glycosylated regions and sparingly glycosylated region, and these units are linked by intra-and inter-molecular disulfide bonds (fuongfuchat, jamieson, sputum sample from a cystic fibrosis patient after pre-shear ( s at s - ) decreased by the addition of sodium chloride, but much more decreased by guluronate block, gb (degree of polymerisation, dp ) and by the addition of gb (dp ) decreased to / of the control sputum without addition of nacl or gb (fig. ). fig immunogenic response was also reported for high molar mass alginate, which was lost by jaas grew on all gos up to dp , k grew only on gos up to dp , and s grew only on go . since they found the different growth rates for each strains, they expect that further study on the molar mass effect will be interesting. ulvan is a sulphated polysaccharide, extracted from green algae ulva and entermorpha, mainly composed of rhamnose, xylose, glucose, glucuronic acid, iduronic acid, and sulphate, with increased, which is more beneficial, in comparison with diet for control rats. however, the tc and ldl were rather increased in comparison with control and non-degraded ulvan although the difference was not significant. since the main difference between non-degraded ulvan and degraded fractions u , and u was the molar mass because the sulphate and uronic content were not so different, the above mentioned physiological difference found in rats fed with the non-degraded ulvan and degraded u and u samples might be attributed to the molar mass difference. however, it is difficult to find a conclusive mechanism from these data because lower molar mass u increased hdl but increased also tc, tg and ldl than in u and non-degraded ulvan fed rats. antioxidant activity of carrageenans was found to increase by gamma-irradiated degradation into oligosaccharides (abad, relleve, racadio, aranilla, & de la rosa, ). among κ-, ι-, λ-carrageenan samples, κ-carrageenan showed the highest dpph radical scavenging activity, and this activity was increased with increasing concentration of carrageenan, and with decreasing molar mass. dpph scavenging activity of carrageenans was found lower than that of ascorbic acid. oligosaccharides prepared by four methods, using free radical depolymerisation, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis. as in a previous report (abad et al, ) , they also recognized the importance of the reducing sugar content which increased simultaneously with decreasing molar mass in addition to the sulphate content. low molecular weight carrageenans were reported to exert anti-tumour effects by promoting the immune system in mice inoculated with s tumour cell suspension (fedorov, ermakova, zvyagintseva, & stonik, ). however, as will be discussed below, some negative function such as inducing colitis has been also reported. the induction of colitis in c bl/ j mice. they found some differences in these three carrageenans, for example, κ-carrageenan seemed to be a promoter for the growth of helicobacteraceae, while ιand λ-carrageenan appeared to be inhibitors for the growth of this bacterium. they also found that non-fermentable κand ι-carrageenans inhibited the growth of this bacterium desulfovibrio, probably due to the antibacterial activity of κand ι-carrageenans, while λ-carrageenan promoted the growth of this bacterium. most importantly, the authors found all κ-, ι-, λ-carrageenans significantly decreased the populations of akkermansia muciniphila j o u r n a l p r e -p r o o f which is a potent anti-inflammatory commensal bacterium in the gut. thus, they concluded that this decrease of anti-inflammatory bacterium caused by the intake of carrageenans led to induce the colitis. this is surely an interesting point as the authors stated. although the pathological analyses were done in detail, unfortunately the detailed information on the molar mass and the sulphate content, which might have some influences on the pathology, were not described. pretreatments of most of polymeric carrageenans at - µg/ml were found significantly to increase the tnf-α level, implying the co-inflammatory effects with lps. the co-inflammatory effectiveness of pure carrageenans at µg/ml was notable for λ-carrageenan, followed by ι-carrageenan, and insignificantly for κcarrageenan. sulphate content is κcar < ιcar < λcar. this suggested a non-negligible role of sulphate content. oligo ι-carrageenan and κ-carrageenan were found to decrease tnf-α significantly, and were expected to be anti-inflammatory agents. the authors found that anti-inflammatory effects of carrageenan oligosaccharides in their study were comparable to those found for chitin oligosaccharides at - µg/ml ai et al. found the highest activity in f - k , and the reason for the lower activity of the lowest mw f < k was attributed to the removal of sulphate groups during hydrolysis because they also believed that sulphate played an important role as in previous reports. they also examined the conformation of fucoidan hydrolysates based on the relation between the radius of gyration and the weight average molecular weight obtained from the hpsec-malls measurement. they reported that the highest mw fraction f > k was compact spherical conformation and the anionic sulphate groups available to bind proteins on the cell surface may be hidden inside the chains while f - k fraction took more loose and entangled conformation allowing sulphate groups in the chain are available to bind the proteins. it was unfortunate that the sulphate content of f - k fraction was higher (ca . %) than the other two fractions f > k and f < k , which makes the speculation difficult; which of the two, sulphate content or the conformation, was more influential to the activity. in addition, the monosaccharide composition was reported to be slightly different. it is indeed not so easy to obtain clear-cut sample fractions. they also pointed out that the affinity of heparin to anti-thrombin is so high that in vivo, i.e. in the presence of anti-thrombin, all other activities are considerably weakened due to its binding to anti-thrombin. then they concluded that their in vitro data were in line with animal experiments showing that the anti-inflammatory activity of fucoidan is superior to that of heparin (pomin, ) . bioactive characteristics of a polysaccharide ascophyllan extracted from a brown seaweed ascophyllum nodosum have been studied extensively. it has similar but distinct structure of experimental concentration ( - µg/ml) were found, and rather both these saccharides were shown to promote significantly hsf cell proliferation. moreover, the promotion of lmwas-l was more significant than that of native ascophyllan. poa was found to decrease with the lapse of time after , and h after treatment by ascophyllan and lmwas-l, and the decrease was more pronounced when treated by lmwas-l than by native ascophyllan (fig. ) . although the detailed mechanism for the difference of effectiveness between a high and low mw polysaccharides was not elucidated, the authors interpreted as follows: since the molecular size of lmwas-l is much lower than that of native ascophyllan, the molar concentration of lmwas-l should be much higher than those of native ascophyllan under the same mass concentration conditions. therefore, the higher molar concentration of lmwas-l than ascophyllan can effectively and sufficiently bind with some receptors on the cell-surface of hsf cells, which promotes the migration of hsf cells. fig. the authors further examined the antibacterial activity using a gram-positive bacteria lmwas-l were found to inhibit the growth of these bacteria, and the effect was found more concentration up to µg/ml but hp with highest sulphate content decreased the activity above that concentrations, which was attributed to negative impact of hp on cell viability at higher concentrations (song et al, ) . this negative effect of hp was in good agreement with a previous study (peschel et al., ) . it was believed that higher sulphate contents as well as konjac glucomannan has been traditionally used in asian countries, and many papers on its texture modifying function, physiologically beneficial function such as prebiotics, lowering assay and superoxide radical assay showed that the scavenging activity of these mucilages were increased with increasing concentration and with decreasing molar mass. anti-mutagenic activities showed the same order as in antioxidant activities although authors were not sure for the mechanism of these beneficial functions. although body weights of mice did not decrease significantly by the administration of nps, the tumour volume and weight were reduced significantly (fig. ) . therefore, the authors concluded that cs-se nps have promising anti-tumour activity with less side effects, and the higher molar mass cs ( kda) was more effective. since it was clarified that the highest mw chitosan among several mw chitosans (< kda, kda, kda, kda, and kda) showed the highest performance, it is expected in the future to be clarified whether higher molar mass chitosan (> kda) or a chitosan with mw in between kda and kda shows a stronger bioactivity or not. sporotrichosis is a zoonotic mycosis, caused by species belonging to the s. schenckii complex, digestion is the breakdown process of food substances to small molecules which can be absorbed. digestion begins from the oral processing, and then digestive organs which secrete digestive enzymes. carbohydrates are decomposed into monosaccharides, proteins are into amino acids and fats are into fatty acids and glycerol, monoacylglycerol. food digestion process consists of physical process and chemical process. physical process is the breakdown foods from the larger size food to smaller size, increasing the surface area thus more susceptible to digestive enzymes. chemical process is the catalytic reaction which breakdowns food fragments into molecular length scales and further into structural elements, monosaccharides, amino acids and fatty acids and glycerol, which are ready to be absorbed at intestines. food processing and cooking improve the palatability and facilitate the enzymatic reaction. gelatinized starch, denatured proteins, and emulsified lipids are more susceptible for enzymatic reaction. while in vivo test is required finally to understand the digestion and absorption of foods, in vitro test has many advantages, rapid, less expensive, reproducible, easy sampling, free from the individual differences, and therefore is suitable for screening better candidates from many samples, and in addition it is possible to compare the results obtained in different laboratories. thus, the international protocol for the digestion study was proposed (minekus, alminger, alvito, ballance, dietary fibre has been studied more than years for its beneficial effects on health, but its definition has been a matter of debate. after codex alimentarius issued the definition of dietary fibre in : dietary fibre is carbohydrate polymers that are not digested in the small intestine of humans and categorised into the following three materials ) edible carbohydrate polymers naturally occurring in the food; ) carbohydrate polymers, which have been obtained from food raw material by physical, enzymatic, or chemical means; and ) synthetic carbohydrate polymers (jones, ). in the early stage of the discussion, oligosaccharides with degree of polymerisation dp < were excluded, but now these are also included if these are not hydrolysed in the small intestine and fermented in the large intestine (jones, ). the importance of glycemic index (gi ) as a valid and reproducible method of classifying carbohydrate foods was confirmed and there was consensus that diets low in gi and glycemic load in contrast, the rate of amylolytic hydrolysis of maize starch were not so much influenced by granular sizes of maize starch; larger granules (> µm) compared with smaller granules (< µm) were hydrolysed almost at the same rate. this similarity in hydrolysis extent was explained by the structural difference in large and small granules: there are surface pores and channels in maize starch granules that can dramatically increase the available surface area for amylase catalysed hydrolysis while there are no such pores, channels and cavity in potato starch (dhital et al., ). the same research group compared the starch hydrolysis of barley and sorghum, and found that the hydrolysis of barley was faster than sorghum for the similar grain fragment sizes. it was interpreted that more coexisting protein bodies/matrices in sorghum hinder the amylase accessibility toward the starch granules. the hydrolysis extent was found negatively correlated with particle size, as milling to smaller fragments can break down the cell wall and protein matrices increasing the accessibility of amylases towards the starch granules (dhital et al., ). as was described for the rennet gelation of casein micelles with different micelle sizes ( . molar mass effect in protein gelation), the gelation was governed by not simply the micelles sizes but was influenced by the microstructural difference. it is now widely recognised that postprandial increase of gi is lower in the slowly digestible concluded that starch samples with higher amounts of amylose short-medium chains and relatively shorter amylose medium chains showed slower digestion, which they attributed to the denser small cells forming the gel matrix, limiting its susceptibility to digestion enzymes (fig. ). since amylopectin is digested faster, and was not found to decrease in the digestion rate. pectin. this increase was found more pronounced in the presence of pectin and its effect was more conspicuous for higher molar mass pectin, which was consistent with the relative crystallinity estimated by x-ray diffraction; starch in the presence of higher mw pectin showed a higher relative crystallinity. however, the relative crystallinity was found lower in the starch with pectins than in starch alone. although it is believed that the higher crystalline starch was believed to be slowly or hardly digested than less crystalline starch, this was found not to be the case in this study, and should be studied further. β-glucan viscoelastic gels containing low mw β-glucan lg (▲) was not found effective to reduce the blood glucose rise. it is clear that the higher mw is effective to lower the blood glucose, and it is lost when it is in a gel state. to see what is the key factor of the dietary fibre which lowers glycemic index, two solutions of xyloglucan with high and low molar masses but with approximately the same steady shear viscosity at a lower shear rate . s - were ingested by nine healthy male subjects. the blood was taken every min up to min after the ingestion of the mixed solution of glucose and xyloglucan. the rise of blood glucose was blunted by ingesting both solutions of xyloglucan with high and low molar masses, and the effect of the solution of higher molar mass xyloglucan was slightly higher (fig. b) , which was in line with the previous report that the lowering effect was mainly governed by the product of the peak value of molar mass and the concentration of the ) asserting that the high viscosity is the determining factor because the polymer solution viscosity is higher when the molar mass is high at the fixed concentration as shown in fig. in although more detailed studies are required to get a clearer insight on the mechanism of the blood-glucose-lowering-effect of xyloglucan, the results exclude an explanation that attributes this lowering effect to the binding of glucose with dietary fibre xyloglucan which are expelled out from the body without being absorbed. if this explanation is valid, the xyloglucan b sample would have shown a greater effect, which was not the case. in the intervention study on health benefit of dietary fibres, baseline diet, mode of feeding (e.g., whether fed alone or with meals, as a single bolus in a liquid or added to foods), the total dose, characteristics of the subjects, and other important study conditions should be taken into account (jones, ). although an intervention study such as glucose intake is useful to assess the acute glycemic effects, long-term (multimonth) data from well-controlled intervention clinical studies are necessary to establish a clinically meaningful health benefit for improved glycemic control although the molar mass seems to be the key factor to determine the activities, slight difference in monosaccharide composition or linkage patterns may inhibit the comparison or not is expected to be clarified. the authors submitted another paper before these two reports bbp- ( mw= kda), and noticed that primary structure was retained. a slight increase of reducing sugar with decreasing mw may have some effect, which also was noticed in the degradation performed to improve the bioactivity in other polysaccharides. fig. shows the ic (the concentration of the polysaccharide required to inhibit % of the α-glucosidase activity) of bbp, bbp- , bbp- , bbp- , together with acarbose, an α-glucosidase inhibitor, widely used for diabetes treatment. digestion of lipids dispersed as oil droplets in foods has been studied actively and as wood believed that the high viscosity of β-glucan in the gut is the governing factor to lower the cholesterol (tosh, ). his group obtained β-glucan from various cereals, oat, barley, wheat flour and wheat bran. ldl cholesterol was significantly lowered in the subjects who were given high molar mass β-glucan cereals, and low molar massβ-glucan was not effective. they concluded that intake of g of oat β-glucan/d with a high molar mass ( . × g/mol) or a medium molar mass ( . × g/mol) lowered ldl cholesterol, but a lower molar mass ( . × g/mol) β-glucan was less effective (wolever, tosh, gibbs, & brand-miller, ). they recognized that the viscosity is a very important criterion for lowering the ldl cholesterol as was asserted (p = . ), and concluded that log(mw × c) was a significant determinant of ldl cholesterol (p = . ) by analysis of covariance (fig. a) . they also noticed that treatment effects were not significantly influenced by age, sex, study centre, or baseline ldl cholesterol. the ineffectiveness of low molar mass β-glucan to lower ldl cholesterol was confirmed later (hu, sheng, li, liu, zheng & chen, ) effects of molar mass of β-glucan added to canola oil emulsion on the lipolysis were recently studied (zhai, gunness & gidley, ). as has been reported, the lipolysis of emulsion foods is influenced by mixing methods and emulsifier types. non-food emulsifier triton x- was used because the initial droplet shape using this emulsifier was more uniform than that using wpi or wt% rapeseed oil, four of which were heated at ℃ to gel. they found that the proteolysis in gastric phase (ph ) was faster in a liquid food at the initial stage but at later ( min), more proteins were digested, and in addition, they found the similar tendency also in the intestinal phase. this was explained by the higher susceptibility of heat-denatured whey protein than native whey protein. they found it was consistent with in vivo study in which a higher protein content was found in the caecum for the liquid-food-fed-rats than for the solid-food-fed-rats. the degree of lipid hydrolysis in the intestinal phase (ph ) is shown for four different food models, liquid fine coarse emulsion with a modified o/w interface (gcei) in fig. . . wt% guar gum solution stirred at room temperature for h, then heated at °c for h. sample (g ), . wt% guar gum solution stirred at room temperature for h, then heated at °c for h and sterilized at °c for min. sample (x ), . wt% xanthan solution stirred at room temperature for h, then heated at °c for h and sterilized at °c for min. all these solutions contain x-ray contrast medium which was affirmed to be non-toxic for lung. the order of the magnitude of the viscosity at higher shear rates and that at lower shear rates is opposite: η (x ) > η (g ) > η (g ) at lower shear rate but η (x ) < η (g ) < η (g ) at higher shear rates. thirty-two observations were analyzed, and it was found that the incidence of aspiration was for x , for g and for g . the experimental observation that a higher molar mass guar g showed more shear thinning than a lower molar mass g is consistent with widely observed behaviour for water-soluble polymer solutions (see fig. ). thus, there is a general tendency that the viscosity at lower shear rate seems to be more important rather than that at s - or higher shear rate. the probability of the aspiration seems to decrease with increasing the viscosity at lower shear rate (table ). since the number of observation is not sufficiently high, this supposition should be confirmed with larger number of data in the future. it should be mentioned here that when the concentration of guar gum was higher to increase the viscosity at lower shear rates, panellists found the difficulty to swallow. therefore, solutions of the guar gum with lower molar mass which is less shear thinning than xanthan solutions seem to be not suitable as a thickening agent to lower the risk of aspiration. on the other hand, shear thinning solutions were generally evaluated easy to swallow. extensional rheology has been studied in dysphagia because the bolus is believed to be subjected to extensional flow when bolus is squeezed between the tongue and the palate and also in the subsequent pharyngeal phase in swallowing process. in addition, both bolus elasticity and its cohesiveness of bolus were found to be important for dysphagia treatment, and the extensional the rate of aspiration / (▲x ) < / (■g ) < / (•g ) table the relation between the viscosity at lower shear rates and the rate of aspiration (nishinari et al., ) palatability and the subsequent reduction of liquid intake, leading to dehydration and malnutrition, the optimum thickening level should be identified. at the same time, apart from its viscosity, the bolus cohesiveness is another key factor which influences the aspiration. the cohesiveness is shown to be highly correlated with the extensibility which can be characterized by the break-up it is generally known that oligosaccharides with higher degree of polymerization (dp) and polysaccharides donot give sweet taste, but it is also empirically known that when starch is masticated for a long time in the mouth, it gives some sweetness sensation as mentioned earlier. to understand this problem more quantitatively, it is necessary to notice that carbohydrates have test in which subjects are asked to discriminate samples including blank stimuli containing tasteless methylcellulose to balance the viscosity, they found that subjects discriminated s and s but not s . they noticed that the detectability of the average dp was higher than that of the average dp at both % and % concentrations. they concluded that humans can perceive the taste of α-glucan with dp up to but below , and the upper limit is still not known (lapis, penner & lim, ; ). they further examined the discriminability of maltooligosaccharides, dp - , dp - , dp - together with glucose (dp ), maltose (dp ) and maltotriose (dp ) in the presence and absence of sweetness inhibitor lactisole (fig. ) . all six stimuli were perceived, and the discriminability (d′) among all six stimuli was not significantly different in the absence of lactisole, but in the presence of lactisole it was found that glucose (dp ), maltose (dp ) and maltotriose (dp ) were not discriminated because their sweetness was suppressed, while maltooligosaccharides, dp - , dp - , dp - were detected indicating that these maltooligosaccharides were discriminated. these results led these authors to think that there are unidentified receptor which is different from those already discovered recently by zuker's group. who ( ) recommends to reduce intake of monosaccharides and disaccharides added to foods and beverages, and sugars naturally present in honey, syrups and fruit juices (di monaco, miele, cabisidan & cavella, ). . since oligosaccharides have been attracting much attention by virtue of their prebiotic function, their sweetness intensity has been studied (ruiz-acceituno, hernandez-hernandez, kolida, moreno & methven, ) using ten commercial and four novel prebiotics ( -galacosyl-kojibiose, lacturosucrose, lactosyl-oligofructosides and raffinosyl-oligofructosides). based on the sensory evaluation and principal component analysis, ruiz-aceitunoo et al. ( ) concluded that chain length was the most important determining factor for sweetness intensity of these carbohydrates rather than the types of linkage or the presence of ketose groups. disaccharides (lactose, lactulose, kojibiose, leucrose, maltulose, turanose and others) showed higher sweetness (relative sweetness . - . to sucrose sweetness ), than trisaccharides ( -galactosyl-kojibiose and lacturosucrose) ( . - . ), which in turn exhibited higher sweetness than mixtures of oligosaccharides having dp above ( . - . ). it is generally believed that the perceived sweetness intensity is decreased with increasing concentration of coexisting thickening agents (morris, ) . experimental results of morris showed that this decrease of sweetness intensity did not depend on the kind of thickening agent such as guar gum, alginate, carboxymethyl cellulose but only on the viscosity (fig. ) . nottingham group using guar and dextran with different molar masses examined the saltiness intensity as a function of the zero shear viscosity and polymer concentration (fig. ) they found that saltiness intensity decreased with increasing viscosity at lower shear rate, which was consistent with results on sweetness intensity by morris ( ) shown in fig. . the difference in saltiness intensity was recognized only when the zero shear viscosities are very different. they also found that the saltiness intensity increased with increasing polymer concentration at a constant zero shear viscosity. this observation became possible because they used dextran which is not commonly used as a thickener in food industry because dextran is not so effective as a thickener in comparison with guar, locustbean gum, and xyloglucan. the reason why dextran is not effective as a thickener may be attributed to its chain flexibility caused by alpha , linkage than other polysaccharides. therefore, it was necessary to add a large amount of dextran with lower molar mass to make a solution with the same zero shear viscosity of solutions of guar or high molar mass dextran. the saltiness increasing effect of the addition of a low molar mass dextran may be attributed to one of the following reasons: the effective concentration of salt increases because water is hydrated with dextran, or the direct interaction of salt with some part of glucose residues of dextran. it is difficult to conclude which one or the other mechanism is responsible for this effect at present. as for solid foods, morris ( ) found a good correlation between the intensity of the flavour release and the fracture strain; he found that the sweetness intensity and strawberry flavour intensity decreased with increasing fracture strain. it was understood as the decrease in the surface area of the fragments from which taste and aroma compounds were released. clark ( ) showed that the overall flavour intensity decreased linearly with increasing hardness for dessert gels. however, a deformable/cohesive gel consisting of % low acyl gellan, xanthan, and locust bean gum did not show a good flavour release than other gels having the same hardness because the flavour remained trapped in the gel. although gelatin gel is deformable/cohesive, it showed a better flavour release in the mouth than other gels having the same hardness because it melted at body temperature. therefore, both interpretations of morris ( ) and clark ( ) are not contradictory but complementary. as described in section . elastic modulus and fracture stress of gels as a function of molar mass, lower molar mass gelling agents form less deformable and brittle gels, these will form gels with higher flavour release. in addition to the fracture strain, the flavour release is known to be enhanced by syneresis (nishinari & fang, ) . the relation between water and aroma holding capacity and the molar mass of the food matrix should be further studied. to develop further a better method for aroma retention or release, it is necessary to understand the flavour characteristics and carrier (polysaccharides, proteins and lipids) characteristics, sample preparation before encapsulation (e.g. initial emulsion formation before spray drying), method of encapsulation (e.g spray drying emulsification, extrusion), and operating condition of encapsulation (operating conditions of spray drying) (saifullah, shishir, ferdowsi, rahman, & vuong, ). since molar mass plays an important role in flavour characteristic and carrier, these two factors are discussed in this section. the general tendency of the retention has been found is in the order of acids < aldehydes < esters ≤ ketones ≤ alcohols, indicating that acids tend to most easily to release. therefore, it can be to understand systematically the aroma retention or release, it is logical to examine the influence of molar mass, chemical groups, polarity and volatility of aroma compounds retained in various carriers, glucose, maltose, maltodextrin, starch, thickening or gelling polysaccharides, proteins, and lipids. a general tendency that higher molar mass aroma compounds are better however, the retention of ethyl butyrate was higher than that of isoamyl butyrate in maltose and maltodextrin (de . ), which was an exception to the above mentioned general rule. zhao, su, & sun, ). it seems that more release (less retention) was found in the following order; alcohols ( -pentanol, -hexanol, -octen- -ol, -octenol) > aldehydes (pentanal, hexanal, heptanal, octanal) > ketones ( -pentnon, -hexanone, -heptanon, -octanone), and longer chain (higher molar mass ) compounds seem to be more retained probably because of stronger hydrophobic interactions. the hydrophobicity is represented by partition coefficient. the water-air partition coefficient (logpwa) representing the volatility (from water) of aroma compounds and an decreases with decreasing molar mass and moisture content. the mobility of aroma compounds encapsulated by these polymers is higher in rubbery state than in glassy state. therefore, to prevent the loss of aroma compounds during storage, wall/carrier materials should be kept in glassy state. it is generally found that the concentration of oxidation products of encapsulated flavours during storage is lower for low molecular weight matrices, in particular when produced by spray drying. this is related to the matrix free volume, which decreases with the decreasing with dp= ) to cd (cd with dp= ) were believed to be different from cd (α-cd) to cd (γ-cd) while still providing sufficient rigidity to form stable complexes. thus, cd to cd were thought to act as chiral selectors (sonnendecker et al., ) . therefore, the flexibility is other factors such as binding of aroma compounds with carriers, taste-odour interaction should also be taken into account to understand whole aspects of aroma release (gupta et al., as has been widely reported, texture, taste and aroma interact with each other before and during eating. for example, when isoamylacetate (iaa) which is a key compound of banana and sucrose solution are injected to the mouth, most subjects perceived banana flavour, but when the sucrose solution is replaced by water, most subjects donot perceive the banana flavour. this is understood by the fact that most humans have never eaten banana from which sugars are removed, and they always perceive a banana flavour key compound in the presence of sugars (hort & hollowood, ). it is well known that gel formation of food biopolymers is strongly influenced by the molar mass, ionic strength, ph, temperature and other environmental conditions. some typical examples were discussed. however, needless to say, it is important to study the effect of detailed chemical structure, the degree of substitution and the position of the substituent in cellulose derivatives, kgm, pectin, alginate, galactomannans, native gellans, and other gelling polysaccharides and proteins. although it is possible to prepare the rigorously controlled cellulose derivatives by regio-selective substitution, it is difficult to get a large amount of sample at present, which makes the collaborative works very difficult. as for the polyelectrolytes such as ionic polysaccharides, gellans, carrageenans, pectins, alginates, chitins, and others, effects of the type of cations or anions present are sometimes more important than a small difference in molar mass. the large scale preparation methods to study polysaccharides with narrow molar mass distribution are not well established in addition to the above-mentioned requirement of chemical purity. collaborative research using a common sample is expected to be useful. molecular level study such as rearrangements of biopolymers during structure formation has been reported and this may be distinguished from syneresis (this is also a kind of rearrangement) causing slippage in rheological measurements. although pickering emulsion is widely studied, the mechanism is not yet clarified. it is necessary to study the emulsion activity and stability using the solid particles with different lengths and flexibilities and lipophilicities. again, a common sample distributed in the collaborative work will be useful. it is necessary to combine the fundamentals and applications in different disciplines. collaborative research work done in ifogest using a common protocol of in vitro digestion study and in the japanese research group using a common gellan sample proved to be fruitful, and such collaborative research is expected to flourish in many food hydrocolloids related areas. the research was supported by the grants from the national natural science while it decreases when it is degrade or digested fig. renneting. casein micelle solution ( %) in artificial milk serum. the enzymatic reaction was performed at °c (niki et al., ) . for the control (without guar); . × - (funami et al., c). fig. b syneresis for % normal maize starch/ . % guar system after storage at ℃ for either or days as a function of molecular weight of guar gum. dotted line in the figure represents the syneresis for the control ( % normal maize starch without added guar) after storage for days; . %, whereas solid line stands for the syneresis for the control after storage for days; . %. presence (dark bars) of the sweetness inhibitor lactisole. subjects performed discrimination tests. the proportion of correct responses (right y-axis) was used to obtain the discriminability (d′). the mm) with mm thick was clamped between metal plates having mm diameter hole in the center, and were subjected to compression using mm diameter spherical metal probe at mm/min (watase & nishinari, ). and investigative medicine effect of degree of acetylation on gelation of konjac glucomannan butyrate improves insulin sensitivity and increases energy expenditure in mice antifungal activity of different molecular weight chitosans against planktonic cells and biofilm of sporothrix brasiliensis edible films and coatings. fundamentals and applications nitric oxide releasing polyamide dendrimer with anti-inflammatory activity aggregation of amylose in aqueous systems: the effect of chain length on phase behavior and aggregation kinetics physico-chemistry of ( , )-β-glucans chemistry, biochemistry, and biology of - beta glucans and functional categorisation of dietary fibre in foods: beyond 'soluble' vs 'insoluble' distribution of short to medium amylose a study of self-diffusion of molecules in polymer gel by pulsed-gradient spin-echo h nmr hyaluronan: more than just a wrinkle filler thermodynamics of aqueous methylcellulose solutions theoretical aspects of dna-protein interactions : cooperative and non-cooperative binding of large ligands to a one-dimensional homogeneous lattice clarifying the confusion between poligeenan, degraded carrageenan, and carrageenan: a review of the chemistry, nomenclature, and in vivo toxicology by the oral route evidence-based approach to fiber supplements and clinically meaningful health benefits, part : what to look for and how to recommend an effective fiber therapy evidence-based approach to resolving enduring effects of the gel size before ingestion and agarose molecular weight on the textural properties of a gel bolus rheological and organoleptic properties of food hydrocolloids food hydrocolloids structures, properties and functions gelation of gellan -a review transglutaminase and its use for food processing insight into the stabilization mechanism of emulsions stabilized by maillard conjugates: protein hydrolysates-dextrin with different degree of polymerization characteristics of different molecular weight chitosan films affected by the type of organic solvents antihyperlipidemic effects of different molecular weight sulfated polysaccharides from ulva pertusa (chlorophyta) modulation of osteogenic activity of bmp- by cellulose and chitosan derivatives protein-rich vegetal sources and trends in human nutrition: a review improvement of canola protein gelation properties through enzymatic modification with transglutaminase. lwt -food science and technology sulfated glycans in inflammation conformations and interactions of pectins. ii. influences of residue sequence on chain association in calcium pectate gels effects of γ-irradiation on molar mass and properties of konjac mannan developments in our understanding of water-holding capacity in meat κ-carrageenan from marine red algae, kappaphycus alvarezii-a functional food to prevent colon carcinogenesis compound formation and glassy solidification in the system gelatin-water chitin and chitosan: properties and applications effects of sucrose, guar gum, and carboxymethylcellulose on the release of volatile flavor compounds under dynamic conditions role of the molecular weight on the mechanical properties of kappa carrageenan gels spectroscopic characterisation and conformation of oligo kappa carrageenans delonix regia galactomannan-based edible films: effect of molecular weight and k-carrageenan on physicochemical properties delonix regia galactomannan hydrolysates: rheological behavior and physicochemical characterization chemoenzymatic synthesis of ultralow and low-molecular weight heparins. bba -proteins and proteomics non-equilibrium states and glass transitions in bakery products non-equilibrium states and glass transitions in foods processing effects and product-specific implications the eating quality of meat -iv water-holding capacity and juiciness medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides role of extractive components of scallop in its characteristic taste development (taste-active components of scallop part ii effect of sodium alginate with three molecular weight forms on the water holding capacity of chicken breast myosin gel the influence of non-ionic surfactant on lipid digestion of gum arabic stabilized oil-in-water emulsion dynamic viscoelastic study on the gelation of konjac glucomannan with different molecular weights molecular characteristics of partially hydrolyzed fucoidans from sporophyll of undaria pinnatifida and their in vitro anticancer activity a low-molecular-weight ascophyllan prepared from ascophyllum nodosum: optimization, analysis and biological activities the adsorption of alpha-amylase on barley proteins affects the in vitro digestion of starch in barley flour chemical and physical deterioration of frozen foods chemical deterioration and physical instability of food multicomponent biopolymer gels, fig. back extrusion force for oleogels (soybean oil) as a function of mass fraction of ec (Φ ec ) with different molar masses, . ± . kda (•) casein micelle solution ( %) in artificial milk serum. the enzymatic reaction was performed at °c (niki g" sat (closed circles) and loss tan δ (open squares). the experiments were carried out at °c. casein micelle solution: % (niki et al l ( kda), and l ( kda) at °c, where s t represents a shift factor a dsc heating curves for gelatin gels of different concentrations: (a) . %; (b) . %; (c) . %; (d) . %; (e) . % (f) . %; gels were quenched from the room temperature to - ℃ by liquid nitrogen, and then heated at ℃/min relationship between phase-transition temperatures and water content w c (g/g) for gelatin water system. t g : glass-transition temperature, t pm : pre-melt crystallization, t cc : cold crystallization temperature, t m : melting temperature c relation between the lowest t g value (t g,min ) and the w c where the lowest t g was observed for various kinds of water-polysaccharide systems from light to dark gray from left to right: control (evaporation without starch), maize, potato, and pea. , acetaldehyde (mw= da); , dimethyl sulfide (mw= da); , diacetyl (mw= da); , allyl isocyanate (mw= da) error bars indicate ±sd. different letters denote significant difference (p< . ) between starches competitive inhibition in fig. a (a) and fig. b (a) and enhancement of entanglement or association in fig. a (b) and fig. b(b) . solid curves in fig. a represent g′ and g〞of ha while broken lines stand for g′ and g〞of ha to which disaccharide ( fig. a(a) ) or disaccharide ( fig. a(b) ) units were added (fujii et al., ) . ha short chains inhibit the entanglement or association of longer ha molecular chain in fig. b (a) (competitive inhibition) while they can rather contribute to the entanglement or association in fig. b (b) . there is no conflict of interest. key: cord- -ly fop d authors: faustini, annunziata; davoli, marina title: attributable risk to assess the health impact of air pollution: advances, controversies, state of the art and future needs date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: ly fop d despite the increased attention given to the health impact assessment of air pollution and to the strategies to control it in both scientific literature and concrete interventions, the results of the implementations, especially those involving traffic, have not always been satisfactory and there is still disagreement about the most appropriate interventions and the methods to assess their effectiveness. this state-of-the-art article reviews the recent interpretation of the concepts that concern the impact assessment, and compares old and new measurements of attributable risk and attributable fraction. it also summarizes the ongoing discussion about the designs and methods for assessing the air pollution impact with particular attention to improvements due to spatio-temporal analysis and other new approaches, such as studying short term effects in cohorts, and the still discussed methods of predicting the values of attributable risk (ar). finally, the study presents the more recent analytic perspectives and the methods for directly assessing the effects of not yet implemented interventions on air quality and health, in accordance with the suggestion in the strategic plan − from the health effect institute. the impact of exposure on public health is assessed by measuring its contribution to the total disease incidence or mortality [ ] . the attributable risk and the attributable fraction are the most important measurements of this impact. the attributable risk (ar) is the rate (proportion) of a health outcome (disease or death) in exposed individuals, which can be attributed to the exposure [ ] . ar assesses how much greater in absolute terms the frequency of an outcome is among the exposed compared with the non-exposed [ ] . it is measured as the difference in the rates of an outcome among unexposed individuals (iu) from the rates among those who have been exposed (ie), according to the formula: ar = ie − iu. this estimate is based on the assumption that all other possible causes of the studied outcome have equal effects in exposed and non-exposed individuals. the concept of ar was first proposed by levin in [ ] in the context of studies on cancer, which were mainly cohort studies where people were assembled on exposure and followed over a defined period of time. in the light of the discussion about the attributable measures by greenland and robins in [ ] , steenland and armstrong in [ ] , rothman [ ] and greenland in [ ] we underline that this ar is very similar to a difference of incidence proportions or average risks, since it uses as denominators the exposed and not-exposed individuals and not the time-persons. the attributable fraction (af) is the proportion of all cases (or overall incidence) that can be attributed to a specific exposure in a population since it combines relative risk and prevalence of exposure. it is measured as ar divided by the incidence risk in the exposed, according to the formula: af = ((ie − iu)/ie). thus giving an estimate of the proportion of cases that would not have occurred if exposure had been totally absent [ , ] . the no-exposure hypothesis is usually defined as counterfactual-i.e., far from the experience-in a causal approach framework, since it postulates that the same population is followed in an identical situation where only the exposure level changes to the reference value of [ ] . this estimate as well as ar is based on the assumptions that ( ) all other possible causes of the studied outcomes have equal effects in the exposed and nonexposed groups, ( ) the studied association is causal. the af, like the ar, uses proportions or risk fractions, but divides the difference estimated in the ar by the proportion of cases among the exposed, thus giving an indication of the magnitude of excess risk from exposure as a proportion of total incidence, unlike ar. to extend this definition to the general population, ar should be theoretically estimated from a life-time follow-up of exposed and non-exposed cohorts in the studied population [ ] . in general, using ar, af and ef similarly in those exposed and in the population as a whole, is not acceptable as it has been pointed out by rothman [ ] and greenland [ ] , essentially because populations are not closed cohorts and causality requires many assumptions relative to biological evidence. attributable fraction has been renamed excess fraction (ef) by greenland and robins [ ] , to resolve the ambiguity introduced by the term "attributable" whose meaning might be either a case in which an exposure played an etiological role (etiological fraction) or a case that would not have occurred, had the exposure not occurred (excess fraction). the difference is important since all excess cases are etiological cases, but not all etiological cases contribute to the fraction attributable to a specific exposure. the authors then proposed three different meanings of attributable fraction: excess fraction, etiologic fraction and incidence-density fraction, that uses the rate per person-years per year. these definitions should be chosen and estimated according to the questions of the research and to their relevance to public health, as discussed below. if the essential question is "whether" the outcome occurs by time t due to an exposure, and if then the public health objective is to control the dangerous exposure overall, then the excess fraction is an adequate estimate of an attributable fraction. if the essential question is "when" the exposure occurs, and the public health objective is identifying the specific age and condition when preventing or reducing exposure, the excess fraction could adequately represent the af, on the condition that hypotheses about periods or conditions of vulnerability are made explicit. if the essential question is "whether the exposure caused the outcome", attributable fraction (af) by itself is not an appropriate estimate, because according to the traditional definition, the af measures only the excess cases that could in total be much fewer than the etiological cases, because they are related only to the studied exposure. we would like to point out here that excess fraction could be an etiological fraction (ef) and therefore, the proportion by which the incidence rate of the outcome in the population would be reduced on condition that the exposure was eliminated; in the above first and second cases, it is also required that ( ) a causal (etiological) relationship between the studied exposure and the effect was assessed, and ( ) the many biological assumptions about a causal relationship were respected. ar has been used in the literature to mean various concepts and measures. these other measures include the population attributable risk (par), the population excess rate (per), and the rate difference (rd), but none of them can be considered entirely equivalent to af. their definitions follow according to the last dictionary, [ ] to lead to a better understanding in the literature, and to review briefly the possible pitfalls connected to the concept of causality [ , ] . the population attributable risk (par) helps determine which exposures are most important in a specific community and is calculated as the incidence of a disease in the total population, minus the incidence in the group of those unexposed to a specific risk factor (it − iu), thus giving the risk attributable to that risk factor in the population. par is often used instead of af. it is measured as: par = ((it − iu)/(it × ), where "it" is the incidence rate for the total population, iu is the incidence rate among the unexposed, or par = (((pe(ie − iu)/(pt × it) × ), where pe is the number of people exposed, ie is the incidence rate among the exposed, iu is the incidence rate among the unesposed, pt is the number of people in the population, and it is the incidence rate in the population. the population excess rate (per) is very similar to par; it measures the disease associated with exposure to a putative cause in the population, but it is calculated merely as the difference between the rates of disease in the total population and in the nonexposed. per = pt − pu. the rate difference is used as a measurement that is equivalent to the population excess rate, but it is once again calculated as absolute difference between incidence rate in an exposed population group and a non exposed population group, according to the formula: rd = ie − iu. to complete this brief review of the concepts related to the definition of attributable risk we would also like to consider here the definitions of health impact assessment and accountability. the definition of health impact assessment (hia), as published in the gothenburg consensus paper by the who regional office for europe in , is "a combination of procedures or methods by which a policy, program or project may be judged as to the effects it may have on the health of a population [ ] ." impact assessment is an important step in public health epidemiology, and can be seen as related to risk characterization, the last step in risk assessment, as defined by epa [ ] , together with hazard assessment, dose-response assessment and exposure assessment. risk characterization ( ) reports the epidemiological results, including estimates of population exposed with harmful effects, ( ) makes explicit assumptions and uncertainties, and ( ) provides indications for public-health interventions to reduce exposure. the fields in which impact assessment has been applied include studies about the environment where people live, either natural [ ] or man-made (through urban planning or house building [ ] ), their health care systems [ ] , their medical treatments [ ] , but also the clinical prediction models [ ] and the impact of reducing exposure through controlling sources and passing legislation to reduce exposure [ ] . on the other hand, it has long been recognized that health and its determinants are strongly influenced by policies, programs, and projects outside of the health care sector. thus we have the obvious interface between the epidemiological results for the hia and the more complex policy impact assessments (pias), which define formally evidence-based procedures to assess the economic, social, and environmental effects of public policy [ ] . these procedures are not analyzed here, nor are the possible interactions with various levels of pias. we would like just to touch upon the complexity of pias which require many different kinds of expertise. the pia procedures have been incorporated into policy making in the oecd countries and the european commission [ ] . we would instead like to verify here how much of the prediction made many years ago by irvapicciottohas in fact come to pass. she wrote at the time: "a serious appraisal of current methods and practice suggests that much can be done to improve scientific rigor in risk assessment [ ] . accountability which has been termed "evaluating the extent to which air quality regulations improve public health" is part of a broad effort to assess the performance of environmental regulatory policies. the methods for accountability research have been promoted by the health effect institute [ ] . one of the strengths of this approach consists in analyzing the compliance to the most important steps of the chain of effectiveness to add or change regulatory actions. in the case of air pollution, the crucial steps involve emissions, ambient air quality, exposure/dose, human-health response. the health effects of air pollution have been well known to the scientific community since the early th century, when serious epidemics of mortality and respiratory diseases were linked to concurrent extraordinary peaks of air pollution in natural experimental or extraordinary scenarios such as london fog [ ] ; fog in the meuse valley [ ] ; still mill in utah valley [ ] . toward the end of the th century, researchers started to focus their attention on the possible adverse health effects induced by "common" levels of outdoor air pollutants [ , ] . clinicians have also acknowledged the causal role of air pollution on human health since , when the american thoracic society (ats) first defined the concept of an "adverse respiratory effect of air pollution" (ats, ) as "medically significant physiologic or pathologic changes" [ ] . all these scientific contributions provided a fundamental support for the definition of standardized legislation on air quality in the united states (u.s. epa, ) and in europe (european council, ; european parliament and council, ). today's research is exploring the possible effects of air pollution below the current limits of exposure both for short-term exposure-going from aphena in europe and the usa [ ] up to the recent almost worldwide studies [ , ] -and for long-term exposure (e.g., in the netherlands [ ] in europe [ ] , in the usa [ , ] and in china [ ] ). in the late s, two important multicity studies-one in europe (aphea, ) and another in the usa (nmmaps, )-were started with the aim of providing answers to the unresolved uncertainties which had emerged from previous studies about the short-term effects of air pollution which had been carried out mainly in single cities, apart from the milestone air pollution andmortality in six us cities [ ] . we summarize below the essential points of these studies since they are the basis of many subsequent methodological developments right up to today. both aphea and nmmaps were multicity studies which benefited from the following advantages over single-city studies: they involve larger populations and can recruit a larger number of cases, thus increasing the power of association; . they can observe the same relationship under different circumstances, such as seasons, and in different populations thus making causality a more plausible interpretation of any possible association, providing that heterogeneity has been controlled for or assessed; . even a "weak" effect of air pollution on health will constitute an important public health problem, since a unique characteristic of air pollution exposure is its ubiquity for large populations. the most important problems with these studies were recognized by the researchers themselves, i.e., ( ) not having studied other factors potentially responsible for the effects, including air pollutants other than particulate matter (pm), ( ) not having individual measurements of exposure, which could prefigure exposure measurement errors, ( ) having measured mortality that was premature only by a few days, which is an effect of limited public-health impact, ( ) having used different methods to study this association in different cities. to respond to the above problems: . the researchers thereafter proposed a "measurement error model for time-series studies of air pollution and mortality" which was a combination of the berkson model; and the bayesian hierarchical generalized additive model (gam). the berkson model [ ] deals with the relationship between ambient concentration and personal exposure by comparing the two measurements to estimate the error between them. although the berkson error-unlike the misclassification-causes little or no bias in the association, the difference between the average personal exposure and the true ambient level has been identified as an important source of bias in log-linear models [ ] . the bayesian hierarchical generalized additive model was the tool for modeling variability across the studies of the relationship between personal and ambient exposure concentrations. two different approaches were available: a hierarchical multivariate regression with missing predictors for either continuous or categorical data [ ] . . they included gaseous pollutants in subsequent studies on air pollution effects. this innovation was supported by using a hierarchical model to assess exposure-health outcome association which made it possible to estimate the independent effects of multiple pollutants in the presence of measurement error [ ] . they adopted a longer scale of mortality linked to short-term exposure which overcame the mortality displacement [ ] , and helped to introduce the long-term studies. . they took into account the temporal structure of the exposure-response relationship which led to including effects of up to days after exposure peak or harvesting. the distributed lag models played the major role in assessing the new exposure-response relationship of short-term effects [ , ] . later, the study by gasparrini and leone [ ] extended the definition of attributable risk within the framework of distributed lag models. . they revised the frailty hypothesis-which restricted mortality to the frail people-by showing that larger effects occurred in frail people only at shorter time scales [ ] . in parallel, a rich discussion developed about which was the better design, to assess the effects of long-term exposure between time-series and cohort study. the idea that cohort design might have been more appropriate for assessing effects related to long-term exposure was introduced in nmmaps in [ ] . the discussion began with kunzli's observations [ ] about the double action of air pollution on both the long-term underlying diseases and the short term risk of death: the author identified four different combinations: (a) air pollution increases the risks of both underlying diseases, leading to frailty, and of short-term mortality among the frail; (b) air pollution increases the risk of chronic diseases but is unrelated to the timing of death; (c) air pollution is unrelated to chronic diseases but short term exposure increases mortality among the frail; (d) neither underlying chronic disease nor death is related to air pollution exposure. the author concluded that the cohort design was more powerful for studying long-term exposure, as opposed to the time-series approach, because the later captures deaths in categories (a) and (c) only, whereas the cohort design assesses cases in categories a, b and c. hence the impact assessment of air pollution on mortality should be based on cohort studies. this position has been discussed claiming that "lifetime lost due to short air pollution peaks, may not be sufficiently captured in cohort studies because long-term mean concentrations are insensitive to such hidden peaks" [ ] . although no available design could fully assess the contribution of air pollution to life experience, cohort studies might be more powerful for case attribution, if long-term exposure was used, since it also reflects past peak exposures. the spatio-temporal extension of short-term analysis [ ] as well as the studies of the short term effects in cohorts [ , ] contributed important developments in assessing the effects of this sort of cumulative exposure to both short-term and long term exposures: • the first approach overcame the limited capacity of the time-series so as to take account of spatial variation [ ] and the introduction of cox proportional hazards models in spatio-temporal analysis introduced the possibility of estimating results for different geographic levels, such as cities and states; • the second approach included in the short-term assessment the effect due to the sensitivity of some population subgroups, which is itself due to long-term exposure [ , ] . the current situation seems to promise further interesting developments in the methods of dealing with spatial-temporal analysis of short-term health effects. two recent studies show the potential of using the time-series approach in studying numerous areas [ , ] , strengthened by the concentration-response function whose important role has been recognized as a metric for impact assessment in addition to the cause-effect metric [ ] [ ] [ ] (see also below page , the role of dose-response curve). in the above-cited recent multi-area papers [ , ] , the contribution of short-term exposure in increasing mortality emerged as independent of long-term exposure, and a no-threshold linear relationship has been confirmed for the pm-mortality function, with continuous increases starting even at concentrations below those of the current guidelines. it is also worth noting that the authors used the excess fraction to estimate the risk attributable to short-term exposure, thus highlighting the opportunity of regulatory interventions [ , ] . daily increases of µg of pm have been associated, within days, with an additional . % ( % cis, . − . ) of daily mortality, . % ( % ci, . − . ) of cardio-vascular mortality and . % ( % ci, . − . ) of respiratory mortality. the studies of long-term (l-t) effects are characterized by prolonged exposures to air pollution, measured by annual average and by health effects in the exposed population. health effects are usually measured as all-cause and cause-specific mortality, and as diseases diagnosed in health services (hospitals, emergency rooms, medical offices). health effects are also estimated as decreased survival which is measured in terms of either life expectancy or years of life lost (yll). all of these measurements are appropriate for estimating attributable risks or excess fractions, both which are important metrics of risk assessment. the long-term approach led to important methodological improvements after the first studies on mortality [ , [ ] [ ] [ ] , that facilitated estimating ars: . an increasingly accurate assessment of exposure (although not yet at the individual level) ranging from continuous monitoring of pm and pm . components [ ] to models for spatial analysis [ ] up to the current, almost worldwide coverage [ ] ; . a more comprehensive picture of the health effects of air pollution, which include cancer [ , ] , metabolic diseases [ ] , maternal and birth outcomes [ , ] , developmental effects [ ] , cognitive impairment [ ] and central nervous system (cns) diseases [ ] . all these additional diseases have a progression that is consistent with cumulative exposure and progressive mechanisms of damage, such as chronic or degenerative diseases; . an updated definition of "adverse health effects of air pollution" that includes asymptomatic signs of health deterioration, such as biological effects, altered biomarkers and reduced functions [ ] the extension of exposure-lag-response models to allow for the health effects due to the protracted exposures to environmental factors [ ] . the most frequently used approaches for assessing l-t effects were cohort studies and the studies that use functions to link exposure to health effects [ ] . cohort studies had been focused on specific sources that were both noxious to human health and susceptible to reduction, such as traffic density [ ] [ ] [ ] , industrial emissions [ ] and home wood combustion [ ] . or they used cohorts created for different reasons but appropriate for representing population and assuring long prospective follow-ups to assess exposure changes after interventions [ , , [ ] [ ] [ ] . in addition, they also used populations that were resident in defined areas thanks to the availability of wide-ranging administrative and environmental data [ ] [ ] [ ] [ ] [ ] . studies using functions to link long-term exposure to health effects were promoted by the global burden of disease (gbd) for estimating the burden of diseases attributable to ambient pm . in areas with extremely high concentrations of air pollution, but no direct epidemiological evidence [ ] , such as india [ ] . the integrated response function that these studies implemented uses satellite data to assess air pollution and makes deaths data and mortality rates comparable through the use of a standardized method [ ] . this makes it possible to compare attributable risks across countries, where, briefly, the risk function to link exposure to health effects is estimated in each country for different exposures (ambient air pollution, second-hand smoke, active smoking and household solid cooking fuel), and the population attributable fractions (pafs) for each of them are calculated by using worldwide ambient pm . concentrations [ ] . both short-term and long-term studies of air pollution effects contribute to health impact assessment, since both of them give estimates of damage due to increasing noxious exposures, by using population attributable fractions or excess fractions, as the metric of effects. an additional metric called "impact fraction" has also been proposed [ ] for assessing "the potential efficacy, effectiveness, adequacy, and efficiency of planned intervention strategies", in studies that aim to estimate the health gains achievable by potential reductions of air pollutants levels [ ] [ ] [ ] . this approach could contribute increasingly to supporting the political decision to cut back air pollution, but the methods used in estimating the potential benefits should be further enhanced, so that the hei indicates it as the first pointer for accountability in the strategic plan − for understanding the health effects of air pollution, nd mar, : − [ ] . the theoretical concept of these potential effect measures is that they are a special case of attributable fraction that compare the effect of an observed exposure with that of a counterfactual case in which exposure is completely absent. as a matter of fact, different potential conditions of exposure are usually chosen [ ] (possibly on the basis of the knowledge about the available technologies). hence, a current choice of new limits to which air pollution concentrations should be decreased cannot rely on a counterfactual case that would posit zeroing air pollution, nor on a safe tested level, as we know that no threshold exists in the dose-response curve of air-pollution and health effects. finally, the definition of an "acceptable" damage not based on scientific evidences should involve larger social, economic and-political institutions. these studies have been and could be still useful also in revising the methods used to predict the possible beneficial effects of interventions, along the same lines as what the hei did by supporting, in the s, intervention studies on air pollution to test whether the proposed methods were able ( ) to assess the population exposure, ( ) to identify an effective dose of exposure ( ) to identify the best health outcomes (defined as those associated with several pollutants and detectable shortly after exposure change), ( ) to identify the best populations as those which make it possible to assess differences between areas, and to study the effects related to intervention [ ] . other studies help in choosing priorities for the intervention studies on the basis of the observed risk assessments for different exposures (behavioral, environmental, occupational), like those provided by the gbd studies [ ] , or from different sources (industrial, residential, vehicular and multiple sources) with respect to their effects on mortality, morbidity and on changes in pollutant concentrations [ ] . promoting an intervention to reduce air pollution with the purpose of reducing the health effects, requires evidence of a causal relationship between exposure to that pollution and those effects. it also requires estimates of excess fractions for the exposure so as to be able to evaluate to what extent the interventions have met expectations. a no-threshold linear concentration-response (c-r) curve has been assumed to exist by researchers on the basis of a linear relationship of the observed intermediate values in most of the initial studies on air pollution effects. likewise, a further assumption was that a linear relationship of not measured values there could be on both the left curve, i.e., at the lowest levels of pollutants, and the right one, at the highest levels. the assumptions about the lowest levels of pollutants were discussed from the point of view of public health, since the shape of the c-r curve is an important parameter for predicting the benefits of reducing high-level exposure [ , ] . just a bit of evidence of a threshold was found, whereas the two methods of assessing regression linearity (penalized splines and model averaging) were equivalent. the possibility of predicting c-r curves at the highest levels at which to intervene first of all so as to reduce damages, has been discussed most recently by cox [ ] . the author questions the possibility that the observed c-r relationship might be used to predict the effects of an intervention at the highest levels of pm . , because this would imply that the magnitude or the direction of the observed curve was similar to those of the predicted curve after intervention. this assumption could not be necessarily true, since the observed values are fixed, whereas the predicted values are based on changing pollutant levels. additionally, pope, even though he recognized that both the burden of disease attributable to pm . and the benefits of reducing pollution depend upon the observed c-r function, found that pollution abatement may yield greater benefits in relatively clean areas than in highly polluted ones. hence, he concluded that the shape of the c-r function should be further explored to better understand this relationship for more effective abatement of air pollutant effects in the future [ ] . new types of evidence were proposed for estimating air pollutant regulatory strategies on the basis of direct experiments, rather than deriving predictions from observed situations [ ] . the most important feature of this approach-that justifies the use of the term 'causal inference'-is that different areas are analyzed to allow for the assumption that the intervention to reduce exposure in certain areas is assessed at random, as in a hypothetical randomized experiment. thus the researchers "anchored" the exposure assessment to the estimates of the causal consequences of well-defined interventions, and could conclude that the observed health effects, observed after the intervention, had diminished due to a given reduction in concentrations. the new methods were applied by zigler and colleagues to two case studies: • the first one, the "causal" health impact on mortality-among medicare beneficiaries-in areas which exceeded the pm limits, was compared with the mortality rates among medicare beneficiaries in areas where pm limits were not reached. the members of the first group were assumed to be randomly assigned to "treatment", after testing their comparability with the second group (controls) for potential confounders by using the propensity-score method. in other words, this causal approach allows the consideration of mortality rates among controls as the mortality that might have occurred among cases had their area's exposure been below the pm limit. the second case study examined the extent to which sulfur dioxide (so ) affects emissions of so , nitrogen oxides (no(x)), and carbon dioxide (co ). the authors tested a range of scrubber technologies to reduce multiple gaseous pollutants (so , no and co ) in emissions and outdoor pm . concentrations in areas where plants were either equipped with scrubbers or were not so equipped. the causal estimates were supported by applying principal stratification and causal mediation methods to assess the exposure, while a bayesian nonparametric method was used to evaluate whether the effect on pm . was really mediated by reducing gas emissions. scrubber systems (chemical or gas scrubbers) control air pollution by removing particulate matter and/or gases from industrial exhaust streams. propensity scores [ ] , principal stratification [ ] , causal mediation analysis [ ] , spatial hierarchical models [ ] , and bayesian estimation [ ] are the methods that support and allow us to evaluate a causal relationship [ ] . by grounding accountability research in a potential-outcome framework and applying these methods to national data sets, the authors provide additional evidence of the health effects of long-term, large-scale air quality regulations. the experiences briefly presented above remind us of the difficulties of this discussion. if, from a scientific point of view, it is always possible and appropriate to improve on the available knowledge, as was done in developing the first studies on air pollution effects (see above pp. - ), from a public heath point of view, taking a decision on the basis of the contemporaneously available knowledge (as it was done at the end of the th century in both europe and the usa) would be at times desirable or even necessary. epidemiologists have expressed doubts about policy indecision in the face of clear scientific evidences [ , ] . on the other hand, the problems of uncertainty [ ] , verification of hypothesis [ , ] and heterogeneity still affect important studies. uncertainty is due to errors in estimating mean values in a population, especially when parts of it are commuters; uncertainty may have multiple explanatory factors and is currently quantified by montecarlo simulation, a statistical technique requiring many assumptions. interventions implemented to reduce traffic have not achieved the expected results, but the verification of this hypothesis could rely on many factors not considered in carrying out the interventions such as the spatial and temporal variability of a similar "preventive" intervention. heterogeneity, the first recognized in the multicity studies, has been partly explained by the different composition of particulate matter in different areas [ ] , or controlled by using hierarchical bayesian models to combine the city-specific estimates [ ] but the discussion about the assumptions of these models is still open [ , ] . we can but wish for increasingly well-defined research questions, more accountability studies and further improvements in research methods so as to provide more solid answers to the scientific questions and the policy indecision. meanwhile we would like to go back here to what pope and dockery wrote in about the effects of fine particulate air pollution [ ] . "despite important gaps in scientific knowledge and continued reasons for some skepticism, a comprehensive evaluation of the research findings provides persuasive evidence that exposure to fine particulate air pollution has adverse effects on cardiopulmonary health". the accountability studies, as we saw before, arose as an attempt to "evaluate the extent to which air quality regulations improve public health to assess the performance of all environmental regulatory policies" [ ] . we return now to this first mission of accountability, to reconsider its strengths and weaknesses. three fundamental steps were identified in evaluating these regulatory policies, in logical sequence they are as follows: ( ) reducing emissions, ( ) improving ambient air quality, ( ) reducing adverse health effects; we could summarize these steps as the impact of air quality improvement on public health. a few of the most important accountability studies which applied these criteria are reported as follows: . the ban of coal sales in dublin was followed by an important decrease in black smoke concentrations ( %); natural mortality decreased by . %, respiratory mortality by . % and cardiovascular mortality by . % [ ] . reducing the sulfur content in fuel in hong kong was followed by a substantial reduction in seasonal deaths during the first months, followed by a peak death rate in the subsequent cool-season. it seemed that the intervention led to a significant decline in the annual trend of deaths from all causes ( . %; p = . ), respiratory ( . %; p = . ) and cardiovascular ( . %; p = . ) diseases, but not from other causes. the average gain in life expectancy attendant upon the lower pollutant concentration was to days [ ] . some measures to reduce traffic were implemented in more than one situation. the results in these cases are conclusive as regards neither exposures nor health effects. among the initial studies, those carried out in atlanta during the olympic games have produced contradictory results. the first [ ] found promising evidence, as the decreased led to a prolonged reduction in ozone levels and lower rates of childhood asthma events. the other study [ ] instead found that meteorological conditions contributed significantly to the reduction in ozone, emergency department visits did not decrease, and the strategy adopted for the occasion would not be sustainable beyond that exceptional event. in contrast, both the studies carried out in london to assess the impact of the congestion charging scheme (ccs) did not find conclusive results: the first, appearing five years after the introduction of the ccs, showed a modest benefit in air pollution levels and life expectancy. the explanation given was that the greater reductions in air pollution in deprived areas have had only a small impact in counteracting the socioeconomic inequalities in exposure and mortality rates [ ] . the second study, published eight years after the introduction of the ccs, deals with the oxidative potential (op) of pm as a parameter of traffic exposure. it shows a remarkable variation of op between roadside and urban background locations, which was attributed to varying pm components. this result is consistent with the increased vehicle use throughout london in recent years and a decreased number of vehicles entering the ccs [ ] . another well-conducted study evaluates, in several dutch cities, the air quality and health effects of local traffic policies including low emission zones; however, apart from one urban street in the hague where traffic flow and air pollution were drastically reduced, the study did not find that "reductions in air pollution related to abatement policies lead to actual improvements in respiratory function" [ ] . the experience that the world is currently facing thanks to covid- reduces mobility-worldwide and simultaneously-as regards both long distance and local urban traffic, thus offering the framework for a "natural" experiment which the health effect institute also suggested to study [ ] more specifically about the health effects of reducing traffic and long-distance transports. what has emerged until now are few points which, however, support the hypothesis of a natural experiment, such as the reduction of particulate matter in the atmosphere during the pandemic [ ] , the association of air pollutants (pm, no and o ) with an increased risk of covid- infection [ ] and a possible role in transmission of severe covid- infections by air pollutants [ ] . some people would like causal accountability studies to support the more effective political interventions and thus replace traditional assessment which would be based on expensive studies about the exposure-response relationship in estimating the burden of diseases [ ] . but the authors themselves of the causal accountability studies have concluded that their methods are "an important addition to the toolkit and should continue to be further explored, but cannot wholly substitute for accountability assessments that rely on evidence from other scientific methods, including more traditional epidemiology analyses." in general, the impact of the interventions on public health should be maintained both to verify the full achievement of the results and to identify the potential necessary corrections, since the process of accountability involves so many scientific, administrative and political institutions. in addition, it is possible that many interventions together, or in sequence, are needed to reduce pollutants or that they require more time to exert effects on total population health and so on. this is well evidenced in a not very recent paper which anticipated the importance of longer term, wide-ranging actions or events such as the complex changes associated with the reunification of germany [ ] . this approach is also evocative of experiments that respect the natural times for changes. both the kind of accountability that assesses the regulations already in place, and the causal accountability approach, which promises stronger support to the future air quality scenarios, might be necessary for a long time. modern epidemiology a dictionary of epidemiology essentials of medical statistics the occurrence of lung cancer in man conceptual problems in the definition and interpretation of attributable fractions an overview of methods for calculating the burden of disease due to specific risk factors concepts and pitfalls in measuring and interpreting attributable fractions, prevented fractions, and causation probabilities world health organisation. health impact assessment: main concepts and suggested approach health risk assessment, basic information global estimates of mortality associated with long-term exposure to outdoor fine particulate matter health impact assessment on urban development projects in france: finding pathways to fit practice to context. glob. health promot environmental impacts of the u.s. health care system and effects on public health the impact of isoniazid resistance on the treatment outcomes of smear positive re-treatment tuberculosis patients in the state of andhra pradesh risk prediction models: ii. external validation, model updating, and impact assessment particulate air pollution and mortality in the united states: did the risks change from to ? policy assessment: the state of the art public governance and territorial development directorate regulatory policy committee promoting inclusive growth through better regulation; oecd regulatory policy working papers epidemiology and quantitative risk assessment: a bridge from science to policy assessing the health impact of air quality regulations: concepts and methods for accountability mortality from fog in london mortality and air pollution: associations persist with continued advances in research methodology respiratory disease associated with community air pollution and a steel mill an association between air pollution and mortality in six particulate air pollution as a predictor of mortality in a what constitutes an adverse health effect of air pollution? acute effects of ambient particulate matter on mortality in europe and north america: results from the aphena study short term association between ozone and mortality: global two stage time series study in locations in countries ambient particulate air pollution and daily mortality in cities effects of long-term exposure to traffic-related air pollution on respiratory and cardiovascular mortality in the netherlands: the nlcs-air study long term exposure to ambient air pollution and incidence of acute coronary events: prospective cohort study and meta-analysis in european cohorts from the escape project air pollution and mortality in the medicare population particulate matter air pollution and national and county life expectancy loss in the usa: a spatiotemporal analysis ambient particulate matter concentrations and hospital admissions in of china's largest cities commentary: a structural approach to berkson's fallacy and a guide to a history of opinions about it biases in estimating the effect of cumulative exposure in log-linear models when estimated exposure levels are assigned a measurement error model for time-series studies of air pollution and mortality estimating the independent effects of multiple pollutants in the presence of measurement error: an application of a measurement-error-resistant technique harvesting and long term exposure effects in the relation between air pollution and mortality the distributed lag between air pollution and daily deaths the temporal pattern of mortality responses to air pollution: a multicity assessment of mortality displacement attributable risk from distributed lag models testing the harvesting hypothesis by time-domain regression analysis. i: baseline analysis the national morbidity, mortality, and air pollution study. part ii: morbidity and mortality from air pollution in the united states assessment of deaths attributable to air pollution: should we use risk estimates based on time series or on cohort studies? assessment of deaths attributable to air pollution: should we use risk estimates based on time series or on cohort studies? extended follow-up and spatial analysis of the american cancer society study linking particulate air pollution and mortality a comparison of short-term and long-term air pollution exposure associations with mortality in two cohorts in scotland estimating acute air pollution health effects from cohort study data estimating the exposure-response relationships between particulate matter and mortality within the aphea multicity project the effect of dose and timing of dose on the association between airborne particles and survival. environ. health perspect impact of the hong kong legislation for restriction on sulfur content in fuel health effects of fine particulate air pollution: lines that connect estimation of long-term average exposure to outdoor air pollution for a cohort study on mortality lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution development of a continuous monitoring system for pm and components of pm . use of satellite observations for long-term exposure assessment of global concentrations of fine particulate matter particulate matter air pollution components and risk for lung cancer air pollution: another cause of lung cancer ambient air pollution and early manifestation of type diabetes association of stillbirth with ambient air pollution in a california cohort study the association between air pollution and preterm birth and low birth weight in guangdong, china neurodevelopmental deceleration by urban fine particles from different emission sources: a longitudinal observational study living close to heavy traffic roads, air pollution, and dementia air pollution: mechanisms of neuroinflammation and cns disease influence of exposure to coarse, fine and ultrafine urban particulate matter and their biological constituents on neural biomarkers in a randomized controlled crossover study biomarkers of the health outcomes associated with ambient particulate matter exposure a joint ers/ats policy statement: what constitutes an adverse health effect of air pollution? an analytical framework modeling exposure-lag-response associations with distributed lag non-linear models constituents and related air quality variables as predictors of survival in a cohort of u.s. military veterans long-term exposure to traffic-related air pollution and cardiovascular mortality effects of long-term exposure to air pollution on natural-cause mortality: an analysis of european cohorts within the multicentre escape project a population-based birth cohort study of the association between childhood-onset asthma and exposure to industrial air pollutant emissions impact of wood combustion for secondary heating and recreational purposes on particulate air pollution in a suburb in finland mortality in the medicare population and chronic exposure to fine particulate air pollution in urban centers long-term exposure to air pollution and cardiorespiratory disease in the california teachers study cohort long term exposure to ambient fine particulate matter and incidence of stroke: prospective cohort study from the china-par project long-term exposure to ambient air pollution and mortality due to cardiovascular disease and cerebrovascular disease in shenyang relation between concentration of air pollution and cause-specific mortality: four-year exposures to nitrogen dioxide and particulate matter pollutants in neighborhoods in oslo, norway a cohort study of traffic-related air pollution and mortality multicity study of air pollution and mortality in latin america (the escala study) long-term exposure to urban air pollution and mortality in a cohort of more than a million adults in rome cardiovascular disease and fine particulate matter applying integrated exposure-response functions to pm . pollution in india global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study an integrated risk function for estimating the global burden of disease attributable to ambient fine particulate matter exposure a method for using epidemiologic data to estimate the potential impact of an intervention on the health status of a target population towards a fuller assessment of benefits to children's health of reducing air pollution and mitigating climate change due to fossil fuel combustion estimated public health impacts of changes in concentrations of fine particle air pollution in canada air pollution interventions and their impact on public health strategic plan for understanding the health effects of air pollution. - . fist draft commuting-adjusted short-term health impact assessment of airborne fine particles with uncertainty quantification via monte carlo simulation evaluating the effectiveness of air quality interventions global, regional, and national comparative risk assessment of behavioural, environmental and occupational, and metabolic risks or clusters of risks for countries and territories, - : a systematic analysis for the global burden of disease study interventions to reduce ambient air pollution and their effects on health: an abridged cochrane systematic review do causal concentration-response functions exist? a critical review of associational and causal relations between fine particulate matter and mortality health benefits of air pollution abatement policy: role of the shape of the concentration-response function causal inference methods for estimating long-term health effects of air quality regulations estimating causal associations of fine particles with daily deaths in boston: table principal stratification-uses and limitations mediation analysis: a practitioner's guide spatial variability of the effect of air pollution on term birth weight: evaluating influential factors using bayesian hierarchical models fine particulate air pollution and daily mortality. a nationwide analysis in chinese cities do we really need another time-series study of the pm . -mortality association? assessment of the health impacts of particulate matter characteristics ambient ozone pollution and daily mortality: a nationwide study in chinese cities using spatio-temporal lagged association pattern to unravel the acute effect of air pollution on mortality tracking national and regional spatial-temporal mortality risk associated with no concentrations in canada: a bayesian hierarchical two-level model effect of air-pollution control on death rates in dublin, ireland: an intervention study cardiorespiratory and all-cause mortality after restrictions on sulphur content of fuel in hong kong: an intervention study impact of changes in transportation and commuting behaviors during the summer olympic games in atlanta on air quality and childhood asthma impact of improved air quality during the summer olympic games in atlanta on multiple cardiovascular and respiratory outcomes air pollution and mortality benefits of the london congestion charge: spatial and socioeconomic inequalities the impact of the congestion charging scheme on air quality in london. part . analysis of the oxidative potential of particulate matter respiratory effects of a reduction in outdoor air pollution concentrations accountability studies of air pollution and health effects: lessons learned and recommendations for future natural experiment opportunities effect of lockdown amid covid- pandemic on air quality of the megacity delhi association between short-term exposure to air pollution and covid- infection: evidence from china effects of air pollutants on the transmission and severity of respiratory viral infections the influence of improved air quality on mortality risks in erfurt this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: i thank karen christenfeld for the accurate revision of the english text. neither financial nor other relationships might lead to a conflict of interests.note: manuscript has neither been published previously nor is under consideration for publication in another journal. key: cord- - zx o p authors: rico, timóteo matthies; dos santos machado, karina; fernandes, vanessa pellegrini; madruga, samanta winck; santin, mateus madail; petrarca, cristiane rios; dumith, samuel carvalho title: use of text messaging (sms) for the management of side effects in cancer patients undergoing chemotherapy treatment: a randomized controlled trial date: - - journal: j med syst doi: . /s - - -x sha: doc_id: cord_uid: zx o p cancer patients are often not sufficiently oriented to manage side effects at home. sending text messages with self-care guidelines aimed managing side effects is the main objective of this randomized controlled trial. patients who started outpatient chemotherapy treatment between march and december at a hospital in southern brazil were invited to participate in this study and were allocated to the intervention or control group (ratio : ). each patient in the intervention group received a daily sms (short message service) with some guidance on management or prevention of side effects. all text messages were sent to the intervention group patients in an automated and tailored way by our app called chemotherapp. side effects experienced by patients were verified using the european organization for research and treatment of cancer quality of life questionnaire core- (eortc qlq-c ). results showed intervention group patients experienced fewer side effects compared to the control group in cycle (p < . ), in general. in addition, intervention group experienced less nausea in relation to the control group, in the cycle and cycle (p < . ). this study indicate text messaging may be a tool for supporting side effect management in patients receiving chemotherapy. this study was enrolled in clinicaltrials.gov with the identification number nct . this research was performed in accordance with the declaration of helsinki. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. chemotherapy is one of the main options in the treatment of cancer, and it is used to eradicate neoplastic cells through the administering of drugs, and can be applied in combination with radiotherapy or surgery [ , ] . available antineoplastic drugs do not specifically act on tumor cells, which often leads to the elimination of healthy cells [ ] . consequently, it can cause toxicity to organs, inducing side effects such as fever, nausea, vomiting, fatigue, diarrhea, mucositis, pain, among others [ , ] . these adverse effects can compromise quality of life, increase financial costs, diminish adherence to treatment and cause medical complications [ , ] . in addition, the financial costs of managing side effects of chemotherapy and the demand for better health care are increasing, potentially compromising cancer treatment [ ] . thus, there is an incentive for the development of cost-effective methods to prevent or ameliorate undesirable side effects [ ] . most people receive cancer treatment on an outpatient basis and manage side effects of the disease and treatment while at home [ ] . thus, developing patients' self-care skills is critically important to ensure safe and high quality care at home [ ] . the mobile phone is cited as the most widely adopted electronic supplementary material the online version of this article (https://doi.org/ . /s - - -x) contains supplementary material, which is available to authorized users. technology on the planet [ , ] . the ubiquity and capabilities of this type of mobile telecommunication technology has spawned a rapidly growing line of research and practice in the health care field, called mhealth (mobile health) [ ] . mhealth can be broadly defined as the usage of information technology and communication applied to the health field through mobile technology, assisting patients, populations and health professionals in improving health care. mhealth has the potential to transform the face of health service delivery across the globe [ ] , leading to the adequacy of health care and patients' quality of life, and ultimately the improvement of health care services. thus, mhealth can play an important role in health care procedures in the treatment of cancer, tackling the management of side effects, and empowering patients and care providers in regard to self-care [ ] . in this covid- era, mhealth interventions are an even more attractive strategy, to minimize patient's exposure [ , ] . one of the most used features in mhealth is text messaging (short message service -sms), since this technology is one of the most utilized functions in mobile phones, representing one of the most widely employed communication methods in the world, and an inexpensive intervention modality [ ] . review studies concluded that sms text messaging can serve as a tool to support patient self-management [ , ] . also, sms text message interventions are capable of producing positive change in the form of preventive health behaviors [ ] . the majority of patients desired to receive as much information as possible about chemotherapy-related side effects [ ] . the degree of satisfaction that cancer patients express about the information they receive has been associated with positive health outcomes, specifically regarding quality of life, performance status, side effects, and psychological well-being [ ] . however, cancer patients may not receive adequate support to manage side effects at home [ ] . some studies report that patients received little information from their health team about the management of treatment-related side effects [ , ] . several approaches based on text messaging (sms)method for communication have been used in cancer treatment, both in preventive measures [ ] [ ] [ ] [ ] [ ] [ ] and in clinical care situations [ ] [ ] [ ] [ ] . specifically in chemotherapy, there are different intervention methods that employ sms to monitor side effects as nausea, vomiting, and other symptoms, as well as in relation to patient adherence to their treatment. few published articles focus on the usage of sms messaging to manage the side effects of chemotherapy in combating cancer. we can highlight the following studies, conducted in singapore from november to january , and in united states from november and september , respectively [ , ] . both studies proved the feasibility of the usage of this technology to monitor side effects provoked by chemotherapy considering not randomized controlled trials. in addition, these studies found that text messaging does not interfere in the daily activities of patients. however, these studies did not focus on the prevention of side effects. in our previous study, we showed the acceptance and perception of cancer patients undergoing chemotherapy on an outpatient basis receiving daily text messages about prevention and managing side effects, as well as emotional support [ ] . some patients reported, through text messages, they felt more confident in treatment, felt supported and encouraged, therefore facilitating self-care. other patients reported they received new information about self-care, and that the messages helped them to take better care of themselves. thus, all patients participating in that study reported receiving text messages helped them better cope with treatment because they received useful self-care information about treatment through text messages. in addition, the adherent patients reported they would recommend these text messages to other patients initiating chemotherapy for cancer [ ] . from our previous results [ ] , we improved the present study expanding it to a considerable number of cancer patients, in order to verify other outcomes. to our knowledge, the present study is the first clinical trial employing text messaging (sms) with preventative advice and side effect management tips sent to cancer patients in outpatient chemotherapy. the primary objective of this study was to determine if the text messages received by the patients lead to a reduction in side effects brought on by chemotherapy. in addition, we verified clinical feasibility of sending sms text messages with self-care content to oncological patients. study design and management (ufpel). this he/ufpel has full capability to execute projects related to new actions and policies directed to sus (unified health system), since it is a reference in the area of oncology and it is supported % by sus, as well as characterized as a sentinel hospital. in addition, he/ufpel serves patients from municipalities. adult patients ( to years of age) diagnosed with cancer, who started the first outpatient chemotherapy treatment scheme between march and december of in he/ ufpel, were invited to participate in the study. participants needed to have their own cell phone, be literate and be able to speak and read in portuguese. all participants also needed to sign the free and informed consent form. participants were randomized to one of two trial arms (intervention and control) in a : ratio, one by one, as they started chemotherapy treatment. the participants assigned to the intervention group received a sms text message on their own cell phones on a daily basis, free of charge. in addition, patients in the intervention group were not required to respond to these text messages. all participants received standard care, which included instructions and information provided by oncologists, nurses, nutritionist, psychologist, pharmacists, among others. these text messages have content about the prevention of side effects as well as emotional support. the messages were sent automatically on a daily basis to each patient in the intervention group utilizing the application chemotherapp, developed by our group according to the methodology presented by rico et al. [ ] . the text messages containing advice were drafted in simple, clear, and objective language, in order to facilitate understanding with the patients. these messages were based on guidelines from the national cancer institute [ ] , as well as international cancer manuals [ ] and were prepared by a multidisciplinary team, with the support of the medical and nursing team of the oncology service of the he/ufpel. all of the messages were drafted and sent in brazilian portuguese. the messages are listed in the supplementary file . in order that each patient in the intervention group receive advice on dealing with the most common side effects, the messages were divided by themes. thus, the configuration of each message in chemotherapp, beyond registering the message itself, was also configured according to the theme covered. the messages were drafted and divided in nine themes: physical activity, hydration, emotional support, nausea/vomiting, constipation/diarrhea, hygiene, eating, immunity, and changes in the skin and sense of taste. for each of these themes, various pieces of advice were drafted and sent. in many cases the messages dealt with more than one side effect. the algorithm of chemotherapp for the automatic sending of text messages is timed with the treatment period of each patient [ ] , and does not send messages with the same theme for at least consecutive days. in addition, the algorithm does not repeat messages for a period of at least days. thus, the process of receiving the text messages is dynamic because each day every patient received a tailored message with a different topic. the patients in the control group received only standard care, and answered the questionnaires periodically. the first interview with each study patient was performed at the time of their first chemotherapy session. in this interview, the patients answered the socio-demographic questionnaire (age, sex, schooling, and marital status), as well as signed the free and informed consent term of the research. in addition, the ecog scale (eastern cooperative oncology group) was applied aiming to qualify the patients' well-being and capacity for self-care. the type of cancer of each patient was verified directly in the medical records of the he/ufpel oncology service. the european organization for research and treatment of cancer quality of life questionnaire core- (eortc qlq-c ) [ ] was employed as the base to verify the presence of side effects. in this questionnaire, patients from both groups responded if they experienced a side effect. the presence of side effects were verified: changes in the skin, changes in the sense of taste, fatigue, fever, diarrhea, pain, lack of appetite, shortness of breath, indigestion, mouth lesions, weakness, constipation, nausea, and vomiting. on the first day of the cycle , cycle , and cycle cycle, patients from both groups were interviewed using the questionnaire to measure the side effects experienced in the previous cycle. or rather, in the beginning of the cycle , the side effects experienced in the cycle were verified, in the beginning of the cycle the side effects experienced in the cycle , and finally, in the beginning of the cycle the side effects experienced in the cycle . in addition, in the beginning of the cycle the patients of the intervention group were asked if they read the received messages on a daily basis, and if the received text messages were considered helpful in their treatment. in addition, these patients were asked whether or not they had followed the guidelines received through text messages (followed all guidelines, followed almost all guidelines, followed half guidelines, followed a few guidelines). besides, these patients were asked about the level of satisfaction of the text messages received (very satisfied, satisfied, neither satisfied nor dissatisfied, dissatisfied, very dissatisfied). all interviews were conducted face-to-face with consenting patients while they were receiving the chemotherapy infusion. all the questionnaires were read to the patients, which has been shown to reveal more detailed and accurate information than written surveys, in order to reduce possible misclassification bias [ ] related to the low levels of education of some patients in brazil. all interviews were conducted by main researcher. we used the exact fisher test and the chi-square test to compare groups (intervention and control) regarding side effects. to compare the number of symptoms for each group along the cycles we used the wilcoxon paired test. the significance level was set as . for two-tailed tests. intentionto-treat analysis was applied to all cycles. data analysis were performed in march using stata software, version . . in total, patients participated in this study, in each group (fig. ) . the losses of participants ( patients in the intervention group, and patients in the control group) were due to death, abandonment of treatment, patient hospitalization, or treatment interruption. in addition, some patients ended their treatment in less than four cycles. almost % ( patients) of the patients were not included in the study for being illiterate, and thus, they were excluded from randomization. no patient refused to participate in the study. in the cycle , patients of the intervention group and patients of the control group were included in intention-totreat analysis. in the cycle , patients of the intervention group and patients of the control group were included in intention-to-treat analysis. in the cycle , patients of the intervention group and patients of the control group were included in intention-to-treat analysis. the most prevalent types of cancer in the participants were breast cancer, colon cancer and lung cancer. the majority of the participants were older than and were married. also, it was observed in patients of both groups that a large majority had never finished elementary school. most patients were between the scale of - on the ecog, which implies good capacity for self-care and completion of daily tasks. both groups were similar in the studied characteristics (table ) . each patient in the intervention group received, on average, sms text messages from the first day of treatment to the beginning of the cycle . all text messages were sent automatically via chemotherapp in the morning. all patients in the intervention group attested to have read daily the received text messages. besides, these patients have considered text messages received were helpful to cope with treatment. regarding the satisfaction in receiving the text messages, patients ( . %) reported being very satisfied, and patients ( . %) reported being satisfied. regarding the followed guidelines from text messages received, patients ( . %) reported to have followed all text messages, patients ( . %) reported to have followed almost all text messages, and ( . %) patient reported to have followed half of them. it can be observed on table intervention group experienced less multiple side effects in the cycle , compared to the control group. however, this result was in the limit of statistical significance (p = . ). in the cycle and cycle , both groups were equivalent with regard to multiple experienced side effects. more than % of the patients in the control group experienced multiple side effects in the first three cycles of treatment, on average. however, in the intervention group, the rate of multiple side effects throughout the first three cycles was closer to % on average (p = . ). in the cycle , cycle and cycle the patients from the intervention group had, on average, . , . , and . side effects, respectively. on the other hand, the patients from the control group in the cycle , cycle and cycle had, on average, . , . , and . side effects, respectively. the side effects most observed in the first three cycles of treatment in the control group were nausea ( %), fatigue ( %), and changes in the sense of taste ( %). in the intervention group, the most common side effects were fatigue ( %), weakness ( %) and nausea ( %) (fig. ) . table shows how many patients experienced (or not) each of the side effects analyzed, by group and cycle. two (nausea and indigestion) of the side effects measured showed statistical significance between both groups (table ). there was significantly higher reports of nausea in the control group in the cycle (p = . ) and in the cycle (p = . ), compared to the intervention group. whilst not reaching statistical significance, there is a trend for intervention group experience less nausea in the cycle compared to the control group (p = . ). besides, there was significantly higher reports of indigestion in the control group in the cycle (p = . ), compared to the intervention group. the intervention group showed far less average of side effects in the cycle , compared with the control group (p = . ). in the cycle , the intervention group also showed less average of side effects when compared with the control group, but in the limit of statistical significance (p = . ). in the cycle , there was no significant difference in side effects between both groups (p = . ). there was a change in the mean of the total sum of experienced side effects in intervention group. it was observed intervention group had an increase total sum of experienced side effects from cycle to cycle , from cycle to cycle , and from cycle to cycle . on the other hand, the total sum of the experienced side effects remained stable in the control group over the three cycles analyzed, from cycle to cycle , from cycle to cycle , and from cycle to cycle (table ). this intervention sent one text message a day from the st day of chemotherapy to the beginning of the fourth cycle for each patient of the intervention group, with general and accessible guidelines on self-care for cancer patients. in addition, this intervention did not focus on the prevention and management of a specific side effect, but informed the patients regarding the relief of the most experienced side effects. however, more self-care behaviors text messages were used for nausea than for the other side effects experienced by subjects. thus, the results of this clinical trial are directly related to the elaborate text messages, and to the algorithm of sending the sms by chemotherapp. the prevention and management of chemotherapy-induced nausea and vomiting (cinv) is a priority in the oncology setting [ ] . incidence of vomiting has been substantially reduced, but efforts to control nausea have been less successful, with nausea continuing to effect upward of % of patients [ ] . nausea was the symptom most frequently encountered in the patients of the control group in our study. seventy-three per cent of the patients in the control group, on average, reported some degree of nausea throughout the first three cycles, whereas % of the patients of the intervention group experienced some degree of nausea in the first three cycles on average. this clinical trial significantly reduced the symptoms of nausea in the patients that were subject to intervention, in the first and second cycle. the messages sent to the patients of this study focused on preventing nausea, taking into account that this was the side effect most feared by the patients. this intervention reduced side effects in cycle compared to the control group, on average. however, the mean of the experienced side effects remained stable in the control group over the three cycles analyzed, but in the intervention group it increased. the increase of total sum of experienced side effects in intervention group from cycle to cycle could be because the intervention group showed an immediate improvement, reducing the side effects in cycle in comparison to the control group. although the total sum of experienced side effects in intervention group from cycle to cycle has increased, on average the side effects of intervention group was lower in cycle and cycle compared to the control group, however, this average was statistical significance only in cycle . no strategy for managing the side effects used by control patients was effective, because there was no prevention of any side effects and no decrease in average of side effects in the three cycles analyzed. the intensity of side effects vary among patients receiving the same antineoplastic drugs [ ] . thus, the side effects may be more diverse and the reactions of patients more variable and individualistic [ ] . besides, many side effects increase and became more severe over time, including changes in taste, nausea, and vomiting [ , ] . spichiger et al. showed a significant increase in number of symptoms experienced from . at the start of chemotherapy to . by cycle [ ] . another study also found a gradual increase in experienced side effects from the first until fifth cycle [ ] . in addition, some side effects are difficult to prevent and to control through self-care behaviors, including taste changes, nausea, vomiting, weakness, and fatigue [ , ] . hence, management and prevention of side effects using self-care behaviors may not always be effective in all cycles of chemotherapy. we have decided to use the sms focusing one-way communication, which might restrict the potential of this technique. a review study identified two-way (sending text messages and receiving replies) text messaging is better than one- way text messaging (sending text messages only) to improve medication adherence [ ] . another study suggested interventions that provided and combined information, monitoring, feedback, and self-management could be more promising than interventions that only provided information to the patients [ ] . it is possible that the intervention patients did not feel as engaged with text messages in cycle and cycle as in cycle , since % of the intervention patients did not followed all guidelines received through the text messages, but, they followed almost all these guidelines. thus, the increase of total sum of experienced side effects in the intervention group, and the general improvement only in cycle , may be partly attributed to the one-way communication that may have affected patient engagement. side effects can frequently be prevented and/or managed with supportive medications when they are recognized early [ ] . however, in this research we have decided to elaborate the text messages only with non-pharmacological orientations. it is possible that because we did not use pharmacological orientation in this intervention, made it difficult to reduce the side effects in all cycles, which it were only reduced in the first cycle. still, study participants received pharmacological and non-pharmacological guidance from the treatment team. related works using mhealth applied to the management of side effects guided patients only after they indicated which symptom they were experiencing [ , ] . in addition, these studies instructed patients with pharmacological and non-pharmacological orientations. rather than wait for side effects to develop, intervention might be used to prepare patients for treatment and the expected challenges to quality of life [ ] . besides, the goal of symptoms management may not always be to prevent symptoms from occurring, but rather reduce impact on daily life [ ] . in our study, we instructed patients regarding prevention and management of side effects in antecipatory mode, sending daily guidelines on self-care even if patients were not experiencing a particular symptom. sending daily pieces of advice via text messaging helped patients to learn more about self-care, leading them to take better care of themselves. according to d'haese et al., providing information in a stepwise fashion may be more beneficial than provide information all at once [ ] . according to schofield and chambers [ ] , a new intervention must also be acceptable to both patients and clinicians. this present intervention found great acceptance with the participating patients, as well as all the health professionals involved (doctors, nurses, nutritionists, psychologists, social workers and other professionals). all of the participants in the intervention group were very grateful for all the text messages received, and they reported that the text messages helped to better confront their treatment. in addition, even though they were not requested to respond to the text messages, many patients expressed gratitude for the advice they received. all intervention patients who started cycle were satisfied or very satisfied about the received text messages. thus, this intervention showed clinical feasibility. the results of this study indicate the use of text messaging may be a tool for supporting side effect management in patients receiving chemotherapy. patients require preparation before beginning chemotherapy, which is commonly a very stressful time [ ] . incorporating text messages into prechemotherapy education may be useful. for example, changes in the taste could be more easily prevented if, since prechemotherapy, patients already received guidelines for prevention of this symptom based on behavior changes [ ] , since changes in the taste was the most increased experienced side effect from cycle to cycle in the intervention group. the side effect evaluation questionnaire was not carried out in the residence of each patient when they experienced side effects. side effects tend to be under-reported both qualitatively and quantitatively if they are reported after the fact [ ] . nevertheless, if this occurred, it occurred with both groups. possible additional factors could have influenced in the effect of the intervention. the type or quantity of additional information that the patients from both groups could have received from family members, friends, and the media (newspapers, television, and internet) could have interfered in this study. likewise, even in outpatient treatment, the information that patients could have received from the treatment team could have interfered in the intervention. we did not control or evaluate this kind of information. however, the demographic characteristics and patient illness of both groups were similar and, thus, the results should not contain significant estimation errors. text messaging technology in cell phones can lead to the so-called hawthorne effect [ ] , which provokes a change in behavior, in the case of the receipt of the messages, for the simple fact of the patient feeling cared for and appreciated. it is also believed that this effect is established by the patient's perception of their health care, which most certainly helps their self-care. another limitation is possible contamination between both groups. some side effects experienced by the patients showed themselves to be almost statistically significant in comparing both groups. nevertheless, other associations could not be found due to lack of statistical power. despite text message-based communication presenting numerous advantages, it is not also without its limitations. a potential limitation to the use of sms text-message-based interventions is the possible marginalization of certain populations, such as those that are illiterate. reduced visual acuity could be a potential limiting factor, making reading text messages difficult. in addition, unfamiliarity with sms text messaging technology is another limitation, particularly among senior citizens. in this study, patients with cognitive impairments and above years old were excluded. this mhealth intervention proved to be an acceptable and feasible means to educate and support cancer patients undergoing chemotherapy treatment. the results of this study demonstrate that text messaging based intervention has the potential to manage side effects. results of this clinical trial suggests that this intervention is associated with better chemotherapy-induced nausea related. further studies with larger sample sizes are required to confirm these results and to further explore other benefits from text messaging in oncology setting. this randomized controlled trial covered a broad spectrum of cancer patient characteristics. thus, this intervention can be easily adapted and applied in other hospitals and clinics that carry out chemotherapy in treating cancer, benefiting the general oncological population. in conclusion, the results of this clinical trial could be used to guide the development and testing of new interventions. in addition, the present intervention can be expanded and applied in other health areas, whether it be via text messages with advice on the management of certain diseases, or even text messages about prevention of health-risk factors. future studies may adapt this intervention to prevent a specific side effect, or even a symptom cluster. in addition, a possible adaptation of this intervention would be related to the algorithm of sending the text messages. increasing frequency of sending text message could show other results. besides, future studies could a d a p t t h i s i n t e r v e n t i o n e x p l o r i n g t w o -w a y communication. side effects of chemotherapy among cancer patients in a malaysian general hospital: experiences, perceptions and informational needs from clinical pharmacists doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems symptoms and treatment burden associated with cancer treatment: results from a cross-sectional national survey in the us the social construction of cancer chemotherapy toxicity: the case of taiwan the economic burden of toxicities associated with cancer treatment: review of the literature and analysis of nausea managing symptoms during cancer treatments: evaluating the implementation of evidence-informed remote support protocols the patient remote intervention and symptom management system (prisms)-a telehealth-mediated intervention enabling real-time monitoring of chemotherapy side-effects in patients with haematological malignancies: study protocol for a randomised controlled trial world in : ict facts and figures : ict facts and figures the performance of mhealth in cancer supportive care: a research agenda mhealth: new horizons for health through mobile technologies rapid scaling up of telehealth treatment for tobacco-dependent cancer patients during the covid- outbreak self-removing passive drain to facilitate postoperative care via telehealth during the covid- pandemic text messaging as a tool for behavior change in disease prevention and management mobile text messaging for health: a systematic review of reviews mobile phone messaging telemedicine for facilitating self management of long-term illnesses preventive health behavior change text message interventions: a meta-analysis information needs of early-stage prostate cancer patients: a comparison of nine countries effects of an interactive mhealth innovation for early detection of patient-reported symptom distress with focus on participatory care: protocol for a study based on prospective, randomised, controlled trials in patients with prostate and breast cancer understanding the concept of chemotherapy-related nausea: the patient experience the healthytexts study: a randomized controlled trial to improve skin cancer prevention behaviors among young people comparative effectiveness of a multifaceted intervention to improve adherence to annual colorectal cancer screening in community health centers: a randomized clinical trial effect of short message service as a reminder on breast self-examination in breast cancer patients: a randomized controlled trial can skin cancer prevention and early detection be improved via mobile phone text messaging? a randomised, attention control trial text-message reminders increase uptake of routine breast screening appointments: a randomised controlled trial in a hard-toreach population text-message reminders in colorectal cancer screening (triccs): a randomised controlled trial feasibility and acceptance of a pharmacist-run tele-oncology service for chemotherapy-induced nausea and vomiting in ambulatory cancer patients a health belief messaging framework and a randomized controlled trial of an sms-based intervention for cancer patient outcomes bidirectional text messaging to monitor endocrine therapy adherence and patient-reported outcomes in breast cancer text messaging (sms) helping cancer care in patients undergoing chemotherapy treatment: a pilot study instituto nacional do câncer reproducibility of the brazilian portuguese version of the european organization for research and treatment of cancer core quality of life questionnaire used in conjunction with its lung cancer-specific module the validity of self-reported smoking: a review and metaanalysis the effect of a standardized ginger extract on chemotherapy-induced nausea-related quality of life in patients undergoing moderately or highly emetogenic chemotherapy: a double blind, randomized, placebo controlled trial nausea, vomiting and quality of life of patients with cancer undergoing antineoplastic treatment: an evaluation by pharmacists update on anti-emetics for chemotherapy-induced emesis discordance between physicians' estimations and breast cancer patients' self-assessment of side-effects of chemotherapy: an issue for quality of care the effect of education in managing side effects in women receiving chemotherapy for treatment of breast cancer effects of web-based interventions on cancer patients' symptoms: review of randomized trials prevalence of symptoms, with a focus on fatigue, and changes of symptoms over three months in outpatients receiving cancer chemotherapy new insights into potential prevention and management options for chemotherapy-induced peripheral neuropathy one-way versus two-way text messaging on improving medication adherence: meta-analysis of randomized trials evaluation of a mobile phone-based, advanced symptom management system (asyms©) in the management of chemotherapy-related toxicity efficacy and costs of two forms of stress management training for cancer patients undergoing chemotherapy the effect of timing of the provision of information on anxiety and satisfaction of cancer patients receiving radiotherapy effective, clinically feasible and sustainable: key design features of psycho-educational and supportive care interventions to promote individualised selfmanagement in cancer care impact of a novel nurse-led prechemotherapy education intervention (chemoed) on patient distress, symptom burden, and treatment-related information and support needs: results from a randomised, controlled trial self-care strategies to cope with taste changes after chemotherapy immediate versus delayed self-reporting of symptoms and side effects during chemotherapy: does timing matter? the hawthorne effect: a randomised, controlled trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this research was a two-arm, randomized controlled trial (rct), not blinded. the research is registered in plataforma brasil and received the approval of the research ethics committee of the faculty of medicine of the federal university of pelotas with the certificate of presentation for ethical appreciation (caae) , , . . . . also, this study was enrolled in clinicaltrials.gov with the identification number nct . this research was performed in accordance with the declaration of helsinki.acknowledgments the authors thank all the patients who participated in the study and all the professionals of the oncology service of the school hospital of the federal university of pelotas. also, this study was enrolled in clinicaltrials.gov with the identification number nct . this research was performed in accordance with the declaration of helsinki. this manuscript does not contain individual information. participants were referred to generically as "patients," "participants," etc. authors have no conflict of interest to declare. key: cord- -okw la b authors: nan title: chapter health effects date: - - journal: interface science and technology doi: . /s - ( ) - sha: doc_id: cord_uid: okw la b abstract there are beneficial as well as harmful aerosols. according to their nature, harmful particles can be classified into three categories: chemically toxic, infectious, and radioactive. in general, there is a relationship between the response and the dose received. a biochemically active particle may contain only a small amount of active agents. in this respect, an inhaled particle simply acts as a carrier that facilitates delivery of deleterious or beneficial components to specific surface areas of lung airways. in view of the tortuous narrow passageways and sharp turns they have to go through, aerosol particles are an effective carrier. as an indication of their effectiveness, about one half of all -m particles inhaled through the mouth deposit in the alveolar region. there are beneficial as well as harmful aerosols. according to their nature, harmful particles can be classified into three categories: chemically toxic, infectious, and radioactive. in general, there is a relationship between the response and the dose received. a biochemically active particle may contain only a small amount of active agents. in this respect, an inhaled particle simply acts as a carrier that facilitates delivery of deleterious or beneficial components to specific surface areas of lung airways. in view of the tortuous narrow passageways and sharp turns they have to go through, aerosol particles are an effective carrier. as an indication of their effectiveness, about one half of all -~tm particles inhaled through the mouth deposit in the alveolar region. in addition to respiratory disease, some types of deposited particles can cause systemic diseases following their dissolution and absorption. the period between first exposure to an agent and the appearance of symptoms varies depending on the characteristics of the agent and disease. some symptoms appear within hours from an acute exposure, whereas others may have latency periods as long as years. synergistic or antagonistic effects play an important role. synergism represents the interaction of two or more agents that produces an adverse effect greater than the sum of the effects resulting from exposure to each agent separately. antagonism is an interaction between two or more agents that results in a reduction in adverse effect of each agent. host factors have a strong influence on biological responses to harmful particles. the main host factors include state of health, genetic trait, immunological status, and psychological state such as anxiety and stress. inhalation of particulate toxicants can lead to various types of responses including fibrotic, systemic, irritant, allergic, mutagenic, teratogenic, and carcinogenic responses. injury of tissues may result from the inhaled compound or its metabolic products. effects may differ considerably between direct exposure of epithelial cells to toxicants and exposure to the toxicants after phagocytosis. toxicity also depends on particle size and physicochemical properties of the agent. concentration of the agent and duration of exposure are two important factors influencing response. acute and chronic exposure may result in different adverse effects. pneumoconioses, characterized by scars due to increase in interstitial fibrous tissue, are diseases resulting from retention of certain types of particles in the alveolar region. different types of dusts may produce different patterns of fibrotic lesions. for example, crystalline free silica found in mines, foundries, blasting operations, and glass manufacturing can give rise to a nodular type of fibrosis (silicosis). on the other hand, asbestos fibers can cause a diffuse fibrosis (asbestosis) if they exceed ~tm in length and . ~tm in width (lippmann, ) . coal workers' pneumoconiosis has a complicated form. most pneumoconioses result in shortness of breath and chronic cough, but pneumoconioses from certain types of dust, such as iron oxide, may have no evidence of functional impairment. systemic response arises from chemical toxicants that affect certain organs or tissues. target sites include respiratory tract, gastrointestinal tract, hematopoietic system, liver, kidney, bones, endocrine system, and nervous system. herbicides, pesticides and heavy elements such as mercury, lead, cadmium, arsenic, molybdenum, and selenium are known systemic toxicants. an example of systemic response is metal fume fever, an acute flu-like illness with symptoms of throat irritation and dry cough. the symptoms appear within hours following heavy exposure to fumes of metal oxides. irritant response generally results from inhalation of particulate sulfates, pesticides, and droplets of sulfuric acid or other strong acids. they cause inflammation in affected regions such as rhinitis, pharyngitis, laryngitis, bronchitis, pulmonary edema, and pneumonia. allergic response involves sensitization by initial exposure and reaction to subsequent exposure. such response can lead to bronchial asthma. examples of allergens include nickel, cobalt, arsenic, chromium, organic dusts, herbicides, and insecticides. inhalation is a route of entry for airborne mutagens (agents that produce changes in dna), teratogens (agents that cause developmental malformations), and carcinogens. among known or suspected chemical mutagens are ddt, sodium arsenate, cadmium sulfate, and some lead salts. examples of known or suspected chemical teratogens include dioxin, organic mercury, cadmium sulfate, and sodium arsenate. there is a broad variety of chemical carcinogens including cigarette smoke, soot, asbestos, arsenic, chromates, and certain petroleum products. changes in lung tissues can begin when an individual is exposed to a cancer-causing substance. a few abnormal cells may appear in the lining of bronchi. if exposure to the carcinogen continues, more abnormal cells can appear and lead to formation of a tumor. in addition to asbestosis, asbestos fibers can cause bronchial cancer and mesothelioma. the latency period is years or longer. to have chronic toxicity, inhaled asbestos fibers must exceed the following critical length limits" ~tm for mesothelioma and ~tm for lung cancer. in addition, fiber width must be less than . ~tm to induce mesothelioma and larger than this limit to cause lung cancer (lippmann, ) . cigarette smoke, containing particles and vapors, has been identified to be the cause of a plethora of diseases. in addition to cancers of the lungs, larynx, esophagus, bladder, cervix, kidney, pancreas, and stomach, smoking can give rise to emphysema, chronic bronchitis, pneumonia, chronic heart and cardiovascular diseases, abdominal aortic aneurysms, acute myeloid leukemia, cataracts, and gum disease. central bronchial airways are the most common sites for lung cancer in cigarette smokers (schlesinger and lippmann, ) . the diseases resulting from exposure to ambient aerosols include pulmonary emphysema, bronchitis, and, perhaps, lung cancer. asthmatics are particularly susceptible to the adverse effects of aerosol acidity in ambient air. emphysema is a condition of over-inflation of structures in the alveolar region. the over-inflation arises from a breakdown of alveolar walls. early symptoms of emphysema include shortness of breath and cough. the primary cause of emphysema is cigarette smoking. there is also an inherited form of emphysema due to deficiency of a protein called alpha -antitrypsin. bronchitis is an inflammation of the lining of bronchial airways. when bronchial airways are inflamed, less air is able to flow to and from the alveolar region and a heavy mucus or phlegm is coughed up. cigarette smoking is the number one cause of chronic bronchitis. dusts and fumes in ambient air and workplaces are other common causes of this disease. higher rates of chronic bronchitis are found among workers exposed to particles of high concentrations, such as coal miners, grain handlers, and metal molders. emphysema and chronic bronchitis in combination comprise chronic obstructive pulmonary disease (copd). a copd patient has difficulty breathing because ( ) the airways lose their elasticity and therefore cannot keep open properly; ( ) some alveolar walls are destroyed; ( ) airway walls are inflamed; and ( ) cells in airways make more mucus than usual, which tends to clog the airways. ambient aerosol is a mixture containing numerous chemical species of natural and anthropogenic origins. these particles are either directly emitted into the atmosphere from many different types of sources or formed in air by gas-to-particle conversion processes. harmful components identified in highly polluted air include acid sulfate species, heavy metals, reactive organic compounds, and peroxides. at sufficiently high doses these components can produce local respiratory diseases or systemic disorders. however, none of the harmful components mentioned above exists in ambient particles at sufficiently high concentration levels to cause a specific disease. ambient particles are harmful when their concentration exceeds a certain limit; epidemiological studies have indicated a strong association of increases in human morbidity and mortality rates with increased concentrations of ambient particles in a certain size fraction (wilson and spengler, ; vedal, ) . complexity in chemical characteristics of ambient particles has led to considerable difficulty in identifying the components responsible for adverse health effects. thus, it remains largely unclear which specific components are responsible. concentration alone cannot explain the causal relationship. according to the threshold limit values recommended by american conference of governmental industrial hygienists, a worker can be exposed to nuisance or inert dust at the concentration of mg/m in the respirable fraction without measurable injury. questions have been raised whether ambient particles are a carrier of shortlived, difficult-to-quantify, but harmful compounds. friedlander and yeh ( ) have provided supporting evidence for the involvement of peroxides in the adverse health effects of particulate pollutants. reactive oxygen species (ros) in particles also have been shown to play an important role. highly reactive oxygen-containing species, such as hydroxyl radical and hydrogen peroxide, are collectively described as ros. in ambient air, photochemical reactions involving reactive organic gases can produce ros, which are distributed in the gas and particulate phases. combustion aerosols, such as vehicular exhaust, also contain relatively high concentrations of ros. hung and wang ( ) have reported that the concentrations of particulate ros in ambient air are strongly associated with photochemical activities. there is a good correlation between the concentrations of ambient ozone and ros in submicron particles, especially in the ultrafine fraction that are freshly produced by either photochemical reactions in ambient air or combustion processes in vehicular engines. aerosol particles can serve as an effective carrier for ambient peroxides and reactive oxygen species to reach the alveolar region. in the absence of particles, inhaled peroxides and ros mainly deposit in tracheobronchial airways because of their high solubility and diffusivity. when these reactive species are adsorbed on particle surfaces, they can easily reach the alveolar region and thereby lead to an adverse effect greater than in tracheobronchial airways. because of their small sizes and surface characteristics, ultrafine particles appear to be more toxic than larger particles. respiratory effects are shown to be associated with the number of ultrafine particles (peters et al., ) . a recent study indicates that ultrafine particulate pollutants are capable of inducing oxidative stress and mitochondrial damage (li et al., ) . pulmonary effects of ultrafine particles have been reviewed by oberd rster ( ) . studies on rodents indicate that ultrafine particles administered to the lungs cause a greater inflammatory response than larger particles of the same total particle mass do. surface chemistry appears to be an important contributing factor of their high toxicity. furthermore, it appears that deposited ultrafine particles largely escape capture by alveolar macrophage and therefore have higher probabilities to enter the pulmonary interstitium. these results lend support to the hypothesis that ultrafine particles are causally involved in adverse responses seen in susceptible groups of the population. the need for further studies on ultrafine particles has been stressed in a couple of recent reports (national research council, ; friedlander and pui, ) . inhalation is an important route of entry for bacterial and viral pathogens. single bacterial particles can remain airborne for a long time and therefore have high probabilities to be transmitted through air. viruses in droplets generated by sneezing and coughing of a patient can survive for hours and gain access to another host while they are airborne. droplet size plays an important role in determining the atmospheric spread of infectious diseases. sneezing and coughing can produce droplets of various sizes. the distance to which a droplet can be transported depends on the rate at which the droplet evaporates. large droplets settle out of air quickly and, even if they are inhaled, may not reach the pulmonary region. for many pulmonary diseases, the susceptible part is the alveolar region of the respiratory tract. however, smaller droplets can evaporate rapidly to less than ~m in diameter. it is mainly these residual particles, named droplet nuclei (wells, ) , that can spread the diseases. tuberculosis and bacterial pneumonia are two common respiratory diseases caused by bacteria. symptoms of tuberculosis include feeling tired, loss of appetite, fever, coughing up blood, and night sweats. pneumonia is a serious infection of the lungs. when the lungs are infected, the alveoli fill with pus and other liquid, thereby greatly reducing the ability to transfer oxygen to the bloodstream. legionnaires' disease is a form of bacterial pneumonia. it has acquired its name, because the first known outbreak occurred in the bellevue stratford hotel in philadelphia where a convention of the pennsylvania department of the american legion was held. in that outbreak, over people contracted this previously unknown type of pneumonia as a result of exposure to mist from comaminated water that was used to cool the air in the hotel's air conditioning system. the disease can also be contracted by inhaling mist from water sources such as whirlpool baths and showers that are contaminated with legionella bacteria. anthrax is a disease caused by bacillus anthracis, a spore-forming bacterium. its symptoms are similar to a common cold initially, but progress to severe breathing problems and shock in a few hours to a few days. to contract the pulmonary form of anthrax, it requires inhalation of to thousand spores into the alveolar region of the lungs. droplets that deposit in the nose are not likely to cause the disease. examples of diseases due to viruses include influenza, common cold, viral pneumonia, and severe acute respiratory syndrome (sars). when a person is infected by influenza virus, the tissues of respiratory tract become swollen and inflamed. prominent symptoms of flu are high fever, muscle aches, severe headache, dry cough, and sore throat. patients with common colds have milder fever, stuffy nose, sneezing, coughing, and also sore throat. flu and common colds mostly spread through transfer of virus-containing droplets by inhalation or hand-to-hand contact. sars is a respiratory disease due to a type of coronavirus. first reported in asia in february , its symptoms include high fever (temperature higher than . ~ shortness of breath, headache, coughing, diarrhea, and malaise. most sars patients develop pneumonia. according to the world health organization, a total of , people worldwide were infected during the outbreak. of these, died. a probable transmission route of the sars virus is the spread of droplets produced by the cough or sneeze of a patient. infection can occur if these droplets deposit on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. the most common radioactive particles in ambient air are radon daughters. after release from soil, radon gas continues to decay and generate a series of radioactive products. these radon daughters easily attach to ambient particles and become a source of radiation exposure for the general population. occupational exposure to radioactive particles mainly occurs in uranium, tin, and hematite mines. the primary health effect from inhalation of radioactive particles is cancer. damage caused by ionizing radiation at the cellular or molecular level can give rise to uncontrolled growth of cells. this is a stochastic effect re:~ulting from long-term, low-level exposure to radiation. increased doses lead to increase in probability of occurrence. all regions of the respiratory tract are susceptible, but the frequency of incidence differs from region to region. the types of radiogenic tumors and target tissues have been reviewed by icrp ( ) . soluble radionuclides in deposited particles can be absorbed into bloodstreams. depending on their metabolic behavior, these radionuclides can be retained in various organs for different periods of time. a broad variety of pharmaceutical agents can be effectively delivered through inhalation to treat respiratory and systemic diseases. examples of aerosolized agents for treating respiratory disorders include bronchodilators and mucolytics. antibacterial and antiviral agents have been used in aerosol form for treating pulmonary infections. for aerosol therapy of systemic illnesses, various degrees of success have been achieved with hormones, vaccines, antibiotics, and cardiovascular drugs. among the agents tested for systemic medication, aerosolized insulin and heparin appear to be very promising. two proven classes of aerosolized drugs for treatment of asthma and chronic obstructive pulmonary disease are -adrenergic agonists and glucocorticoids. inhalation of -adrenergic agonists can relax bronchial smooth muscle, stimulate skeletal muscle, and inhibit release of inflammatory mediators. short-acting -adrenergic agonists can provide bronchodilation within minutes of inhalation. glucocorticoids are useful for treatment of inflammation. anticholinergics are among other common agents for treatment of asthma and chronic obstructive pulmonary disease. as bronchodilators, anticholinergics agent have a slower onset but a comparable duration of action as -adrenergic agonists. aerosolized formulations of corticosteroids have become commonly used agents in treating respiratory diseases such as asthma, cystic fibrosis, and obstructive lung disorders. these formulations are able to influence existing inflammation. mucolytics such as n-acetylcysteine can give rise to rapid liquefaction of viscid mucus. when administered in aerosol form, they can help maintain adequate mucus viscosity in patients with chronic obstructive airway disease. . a review of the sars epidemic in indicates that, in some countries, a majority of cases were caused by nosocomial infection. discuss possible transmission routes of the sars coronavirus in hospitals. . a potential terrorist weapon is the release of plant and animal pathogens in aerosol form. these pathogens can have devastating effects on agriculture if they are released at points where they can spread out rapidly. what strategies can be used to protect agriculture from such terrorist attacks? the submicron atmospheric aerosol as a carrier of reactive chemical species: case of peroxides emerging issues in nanoparticle aerosol science and technology (nast) experimental determination of reactive oxygen species in taipei aerosols human respiratory tract model for radiological protection ultrafine particulate pollutants induce oxidative stress and mitochondrial damage effects of fiber characteristics on lung deposition, retention, and disease research priorities for airborne particulate matter. i. immediate priorities and a long-range research portfolio pulmonary effects of inhaled ultrafine particles respiratory effects are associated with the number of ultrafine particles selective particle deposition and bronchogenic carcinoma ambient particles and health: lines that divide on air-borne infection; ii. droplets and droplet nuclei particles in our air: concentrations and health effects key: cord- -agwdmtug authors: shankar, venkatesh; kushwaha, tarun title: omnichannel marketing: are cross-channel effects symmetric? date: - - journal: nan doi: . /j.ijresmar. . . sha: doc_id: cord_uid: agwdmtug the rapid growth in omnichannel (e.g., web, call center, sales agent, store) shopping and the need to effectively allocate resources across channels are prompting managers and researchers to better understand cross-channel effects, that is, the effects of marketing efforts in one distribution channel on shopping outcomes in other channels. we develop a broad set of hypotheses about cross-channel effects based on channel richness and influence roles (informative, persuasive). to test the hypotheses, we model the effects (own and cross) of channel marketing efforts on shopping outcomes in different channels through a simultaneous equation system. we estimate these models using data from the auto insurance industry that comprises the exclusive agent, the independent agent, the web, and the call center channels. our results offer novel insights. they show that cross-channel effects and elasticities are significant and asymmetric. while the effect of marketing efforts in a channel on shopping outcomes in a dissimilar (with a different primary influence role) channel is positive (e.g., exclusive agent, the web, and the call center channels are complementary), the magnitudes of the cross-channel effects are asymmetric. similarly, while the effect of marketing efforts in a channel on shopping outcomes in a similar (with the same primary influence role) channel is negative (e.g., independent agent and exclusive agent channels are substitutional), they are also asymmetric. exclusive agent efforts have a greater negative effect on the outcomes of independent agent efforts than vice versa. based on the results, we develop a channel influence vs. influenceability analysis tool for managers to better plan their channel efforts. we also illustrate a resource allocation model that shows substantial incremental profits from the reallocation of marketing efforts based on our model with cross-channel effects relative to a model without cross-channel effects. omnichannel marketing-the practice of simultaneously offering shoppers information, products, services, and support through two or more synchronized distribution channels in a seamless manner-is continuing to grow at a phenomenal rate (verhoef, kannan, and inman, ) . an overwhelming majority of consumers shop in more than one channel (sopadjieva, dholakia, and benjamin, ) , and these consumers are more profitable than others (montaguti, neslin, and valentini, ) . different distribution channels include the web, call center, and direct sales force (neslin and shankar, ) . omnichannel marketing offers organizations greater opportunities to interact with customers through different channels and efficiently use all the channels (kauferle and reinartz, ; kumar and venkatesan, ; montoya-weiss, voss, and grewal, ) . omnichannel marketing typically goes beyond multichannel marketing to include coordination of the distribution channels that offer a seamless customer experience. as channels evolve, cross-channel effects are becoming a significant imperative for omnichannel marketers (mark et al., ; watson, storm, palmatier, and ganesan, ) . cross-channel effects refer to the effects of marketing efforts in one distribution channel or format on shopping outcomes in other channels (gauri, ; talukdar and gauri, ; the dma, ) . the rapid growth in e-commerce and multichannel shopping is significantly altering shopping behavior (nielsen, ) and prompting managers and researchers to better understand cross-channel effects, to effectively allocate resources across distribution channels, managers need to better understand the effects of efforts in different channels on shopping outcomes (e.g., inquiries, quotes, orders, purchases) in other channels. cross-channel effects are important in several industries with multiple distribution channels. for example, in the auto insurance industry, state farm, like other leading brands, uses the for expositional use, we use the terms, shopper, consumer, and customer interchangeably throughout the paper. complementary, substitutional, or insignificant in nature. two channels are complementary (substitutional) if efforts in one channel positively (negatively) influence purchases in another channel. in addition, some cross-channel effects can be high or low. a better understanding of the nature, direction, and magnitude of cross-channel effects is critical for researchers and managers from theoretical and practical standpoints, respectively. to deeply understand cross-channel effects, many firms do not have data on each customer's use of different channels. although more data on online customer journey are available through advanced google and adobe analytics, much data on offline customers' activities are still private and proprietary. furthermore, privacy regulations, such as general data protection regulation (gdpr) and california consumer privacy act, make it harder to track every customer's data. thus, many firms cannot always fully track each customer's exact journey (e.g., whether she first visited the web, called the firm, or contacted an agent). however, firms typically have access to channel level data on variables such as channel efforts and aggregate shopping outcomes in each channel. therefore, an important question is: how can managers use channel level aggregate data to estimate and infer cross-channel effects? we address the research questions by first developing a broad set of hypotheses about crosschannel effects. we then formulate a model of cross-channel effects. we estimate the model and the cross-channel effects using channel level aggregate data from the auto insurance industry. finally, we discuss the results, derive useful theoretical and managerial implications, and illustrate a resource allocation exercise based on our model of cross-channel effects. our results offer novel insights, showing cross-channel effects and elasticities are significant and asymmetric. while the effect of marketing efforts in a channel on shopping outcomes in a dissimilar (with a different primary influence role and richness) channel is positive (e.g., j o u r n a l p r e -p r o o f journal pre-proof exclusive agent, the web, and the call center channels are complementary), the directions and extent of cross-channel effects are asymmetric. similarly, while the effect of marketing efforts in a channel on outcomes in a similar (with the same primary influence role) channel is negative (e.g., independent and exclusive agents are substitutional), it can be asymmetric. exclusive agent efforts have a greater negative effect on independent agents' outcomes than vice versa. our research makes important contributions to the omnichannel management literature. first, it offers an approach for researchers and practitioners to develop and estimate a cross-channel effects model using only channel level aggregate data that are more commonly available. second, it develops broad hypotheses about cross-channel effects based on channel richness and influence roles (informative vs. persuasive), leading to complementary and substitutional crosseffects. third, it produces several interesting substantive insights regarding asymmetric crosschannel effects. finally, it offers a useful managerial tool of influence vs. influenceability analysis and a resource allocation model to better allocate resources across channels. the web channel has significantly reshaped shopping behavior in all the channels (kannan and li, ; peterson et al., ; verhoef et al., ) . studies on the addition of web to existing channels show little or no cannibalization between the channels. biyalogorsky and naik ( ) show that online activities do not significantly cannibalize offline sales for compact discs. pozzi research on the introduction of physical stores on sales in existing channels offers mixed results. avery et al. ( ) find that when an online retailer adds a physical store, it temporarily cannibalizes the catalog channel, but not the web, and over the long-run, enhances sales in both the catalog and the web channels. similarly, pauwels and neslin ( ) show that the introduction of physical stores of a retail firm dampens catalog sales without affecting web sales, resulting in a net increase in shopping frequency and revenues across channels. wang and goldfarb ( ) report that the effect of a new physical store is associated with a decline (spike) in online sales in places where the retailer has a strong (weak) presence. bell, gallino, and moreno ( ) find that eyewear showroom openings increased online and overall sales. fisher, gallino, and xu ( ) report that the opening of a distribution center to reduce delivery time enhanced online sales and spilled over to offline sales. some studies suggest that interactions among channels depend on product type. analyzing data on electronic and physical channels for products of certain quality (new cars) and uncertain quality (used cars), overby and jap ( ) find that more transactions involving low (high) quality uncertainty occur in the electronic (physical) channels, suggesting little cross-channel effect for the same product type. brynjolfsson et al. ( ) show that the internet competes fiercely with brick-and-mortar channel for mainstream products, but the effects could be complementary across the internet, catalog, and store channels for niche products. a few studies analyze the effects of shopping in a channel on shopping in other channels. based on survey responses in six product categories, verhoef et al. ( ) find significant cross-j o u r n a l p r e -p r o o f journal pre-proof channel effects within and across shopping tasks such as search. teerling ( ) also concludes that the role of informational websites on cross-channel shopping behavior may be significant. while these studies examine the effects of shopping across channels, our focus is on the effects of the firm's efforts in each channel on outcomes in the other channels. a related research stream focuses on the drivers and consequences of channel choice, switching, use, and offerings, without explicitly addressing cross-channel effects. a firm's channel efforts and customers' channel experience play significant roles in determining customers' channel selection (ansari et al., ) . venkatesan et al. ( ) study the timing of adoption of a new channel by a customer and find differences between the drivers of adoption of second and third channels. the results from these studies suggest that customers' channel choice, channel switching behavior, and price effects are fairly complex and depend on factors such as product category and customer shopping traits. our research extends these studies by examining the effects of the firm's marketing efforts in each channel on the outcomes in the other channels. another less directly related research stream is on communication channels that play the informative influence role, but our work differs from this stream in important ways. de haan et al. ( ) , dinner et al. ( ) , li and kannan ( ) , and naik and peters ( ) analyze the effects of efforts in communication or media channels in the earlier stages of the purchase funnel (e.g., search advertising, display/banner advertising) on outcomes in the later stages of the purchase funnel. similarly, wiesel et al. ( ) examine the effects of communication or media efforts (e.g., email, adwords, fax, flyer). dinner et al. ( ) estimate the effects of advertising spending in traditional, online display, and online search communication channels on sales in j o u r n a l p r e -p r o o f journal pre-proof online and offline channels. mark et al. ( ) find significant effects of catalogs on web and store purchases. in contrast, we examine the effects of each distribution channel's (e.g., exclusive agent, web, call center) efforts on shopping outcomes in other distribution channels in advertising's presence. thus, our research complements this research stream. these articles focus on the impact of the presence or addition of one channel on the existing channel(s), the drivers of channel choice, or communication channels. however, most firms have multiple distribution channels and seek to understand the effects of marketing efforts in one distribution channel on the outcomes in other channels. moreover, prior studies do not study cross-channel effects by controlling for the effect of advertising that is influential in many industries such as the insurance industry (guitart and hevet, ) . furthermore, these studies are mostly about retailers, precluding generalization to service providers, who outnumber retailers. finally, prior studies do not focus on a managerial cross-channel effort guidance tool. our study focuses on the cross-channel effects of marketing efforts in each channel on outcomes in other distribution channels after controlling for advertising effects in the same framework. our empirical application is in the services context and offers a new managerial cross-channel influence-influenceability tool (a channel management tool) and resource allocation insights. table presents a review of selected relevant studies in cross-channel effects, including our research. some research relating to cross-channel effects focuses on firm outcomes when a new purchase channel is added to existing purchase channels. some studies are about channel choice, while others are on communication channels, which may also play informative roles similar to distribution channels. some studies (e.g., ansari et al. ) do include email messages. we recognize that research on the 'news' product category has examined cross-effects across print and online formats (e.g., chen, ho, and smith ; kanuri, mantrala, and thorsen ) . these formats are similar in spirit to channels but fundamentally differ from channels in that shoppers consume the product or service in these channels. entails convincing prospects through a rational appeal, an emotional appeal, or both. these roles add to the richness of each channel. but each channel has a primary influence role that can help us understand and predict the effects of marketing efforts in that channel on shopping outcomes in the other channels. relative to informativeness, persuasiveness differs more across channels, making it the key differentiator among channels in their impact on shopping outcomes. among the channels, the catalog channel and web channel primarily serve to inform shoppers. these leaner channels primarily allow for presentation of factual information. the web is richer than the catalog in that it allows retargeting, so it is more persuasive. mobile app, smart speaker, and kiosk are more informative as well as persuasive than the web and the catalog. the call center is richer than the web, catalog, mobile app, smart speaker, kiosk channels. it can provide information but only upon queries from the shopper, making its informative role specific. at the same time, it is interactive, so it can also serve to persuade shoppers. thus, the call center typically offers a balance of information and persuasion or moderate richness, so we call it a balanced channel. independent stores and independent agents are richer than the balanced channel. exclusive stores, exclusive agents, value added resellers, and manufacturers' representatives are also richer than the call center channel. these channels interact with shoppers more than do the other channels. during the interactions, these channels can use rational and emotional appeals to persuade prospects. thus, these channels' primary influence role is persuasion. their role goes beyond the call center's persuasive role because these channels allow face-to-face and long interactions with the shopper. a graph depicting the roles, leanness, and richness of the channels relative to one another appears in figure . (avery et al., ; kushwaha and shankar, ) . for example, the web, a primarily informative channel, offers instantaneous access to product information, the ability to search, sort, and compare products, and a low pressure sales environment (balasubramanian et al., ) . the electronic channel offers the benefits of convenience and efficacy of information acquisition (choudhury and karahanna, ) . however, it is still lean in that it does not allow physical inspection, demonstration, trial, instantaneous acquisition, or instant gratification. in contrast, the call center channel, with a mix of information and persuasion, is richer, offering personal interactions, authentic answers to questions, and ubiquitous access. at the same time, it does not permit visual inspection and product and price comparisons. exclusive sales agents represent a richer channel and are primarily persuasive. they are highly knowledgeable advocates and credible stewards of the firm's products. however, prospective customers may not regard the information obtained from exclusive agents to be entirely objective and may be unable to gather accurate information about competitor brands (zweifel and ghermi, ) . independent sales agents, who constitute another rich and primarily persuasive channel, offer brand variety and are more unbiased than exclusive agents about the focal firm. however, prospective customers may not expect to get the most accurate information about all the competing brands and may feel that they would be unable to obtain a better deal for a brand than they could get from an exclusive agent for that brand (zweifel and ghermi, ). in addition, customers may perceive both exclusive and independent agents as pushy. from the firm's viewpoint, these distribution channels are direct or indirect based on the nature of contact and interaction with customers (sa vinhas and anderson, ). the web and the call center are direct channels, allowing the firm to directly interact with customers. in j o u r n a l p r e -p r o o f contrast, the exclusive agent and the independent agent channels are indirect channels as the firm relies on these intermediaries to communicate with the customers. this classification loosely maps with the categorization of informative vs. persuasive (narayanan et al., ) . firms use direct channels mainly to exercise an informative role and to some extent a persuasive role. in contrast, firms typically utilize indirect channels to implement a persuasive role. another way to classify the channels is as owned versus independent, consistent with dual distribution--a hybrid of vertically integrated and market governance channels (e.g., srinivasan, ) . viewed from this perspective, all the channels in our channel except the independent agent channel are owned channels. the main difference between these two types of channels lies in the extent of the firm's control over marketing efforts. firms can exercise greater control in their owned channels than in the independent channels. this classification corresponds with our categorization of informative versus persuasive channels in the following way. firms can use their owned channels to perform either an informative role, a persuasive role, or both. but firms typically utilize independent channels to persuade customers. the classification of channels in terms of their primary influence role as informative or persuasive is consistent with channel or information richness theory, which is quite fundamental, requiring fewer assumptions. therefore, we adopt this classification to develop the hypotheses. to understand the effects of marketing efforts in one channel on shopping outcomes in other channels, we analyze the relative richness and the primary influence roles of the channels. in cases where the channels are dissimilar in richness and if the primary influence roles of two channels are complementary, the benefits of one channel spill over to another channel, producing complementary cross-channel effects. in cases where the richness and primary influence roles of the channels are similar, the limitations of one channel extend to other channels, creating j o u r n a l p r e -p r o o f substitutional cross-channel effects. still in other cases, efforts in any one channel may have only stand-alone effects and not affect outcomes in other channels. by combining their richness and primary influence roles, some channels may complement one another. for example, efforts in the lean, primarily informative web channel can enhance outcomes in other channels such as the call center that erves both informative and persuasive roles. as discussed earlier, the addition of the web does not cannibalize sales in other channels (biyalogorsky and naik, ) as it acts an al informative channel for shoppers who may prefer to shop in rich, persuasive channels. furthermore, trusted websites can direct prospects to make purchases from a more persuasive channel like the call center (teerling, ) . similarly, call center agents can provide a high level of information and personal service to prospective customers, combining both informative and persuasive roles, empowering shoppers to complete shopping task from a primarily informative channel such as the web or the catalog. a call center agent can offer a richer human touch to a prospective customer that could increase the likelihood of buying from a primarily informative channel. by personally answering customers' questions, call center agents can enhance customer trust to effect favorable outcomes in informative channels. shoppers can make decisions in an informative channel based on the intent developed from their interactions with call center agents, consistent with the theory of reasoned action (fishbein, ) . after a shopper receives persuasive communication from the balanced channel, she evaluates the arguments over time. if the shopper is convinced, she could minimize the time and effort expended in making her decision by directly going to the lean, primarily informative channel (the web) and practice self-service. if she goes back to the j o u r n a l p r e -p r o o f balanced channel, she would have to wait for an agent to connect and go through social protocols before being able to make her decision. these arguments lead to the following hypotheses. a lean and primarily informative channel such as the web or the catalog can combine with a rich and primarily persuasive channel such as a brick-and-mortar store or a sales agent to positively influence shopping outcomes in each other's channel. marketing efforts in a primarily informative channel can direct shoppers to a primarily persuasive channel to complete their task. the phenomenon of research shopping in which a shopper searches for product information on the web and buys the product at the store is well documented (verhoef et al., ) . indeed, efforts in an online (primarily informative) channel complements outcomes in the direct sales force (primarily persuasive) channel in b b markets (lawrence et al., ) . thus, we expect the effect of marketing efforts in a primarily informative channels like the web to positively influence outcomes in primarily persuasive channels such as exclusive and independent agents. a rich and primarily persuasive channel such as the store or the exclusive agent channel can also facilitate positive shopping outcomes in a lean and primarily informative channel. through extra efforts to accept and handle product returns, stores can assure customers to purchase more on the web (mahar et al., ) . furthermore, in banking, complementary marketing efforts in more persuasive channels such as branches facilitate customer adoption of the web channel (campbell and frei, ) . indeed, customers who adopt the outlet store channel increase their purchases on channels such as the web and the catalog (soysal and krishnamurthi, ) . similarly, exclusive agents act as strong advocates for the company's brand. although these agents expend their efforts to consummate shopping outcomes in their own channel, their overall j o u r n a l p r e -p r o o f goal is to maximize the probability of prospects buying from the company even if it is in a primarily informative channel. the role of the exclusive and independent agents is similar to that of the stores, branches, and outlet stores in that both these channels perform a persuasive role focused on the company's offerings. this reasoning leads to the next hypotheses. h a : marketing efforts in a primarily informative channel has a positive effect on the shopping outcome in a primarily persuasive channel. h b : marketing efforts in a primarily persuasive channel has a positive effect on the shopping outcome in a primarily informative channel. we expect channels with a balanced mix of information and persuasion such as the call center to have complementary cross-channel effects on outcomes in richer, primarily persuasive channels. shoppers touched by call center agents may be well informed about the focal firm's products. if the call center agent provides a high level of service, shoppers may even be inclined toward the firm's products. such shoppers can consummate their shopping tasks in a richer, primarily persuasive channel like exclusive agent or independent agent. by the same token, efforts in a richer primarily persuasive channel such as the exclusive agent or the independent agent should also facilitate outcomes in a leaner primarily balanced channel. exclusive agents and independent agents can move shoppers toward a strong purchase intent for the focal brand. such shoppers may call the call center and resolve their final questions in an interactive manner and complete their shopping task, consistent with the theory of reasoned action (fishbein, ) . thus, the efforts of primarily persuasive channels can complement outcomes in balanced channels. these arguments lead to the following hypotheses. in contrast, a rich, primarily informative channel when combined with another rich, primarily informative channel may not enhance the effect on the shopping outcomes in the other channel. shoppers may perceive the marketing efforts in each informative channel as substitutes for obtaining similar information. similarly, a rich, primarily persuasive channel when combined with another rich, primarily persuasive channel may not have a positive effect on the shopping outcomes in the other channel. in fact, two persuasive channels may be perceived as perfect substitutes by shoppers and may compete with each other for shopper attention. channels that are primarily lean, informative (rich, persuasive) may act as substitutes for other primarily informative (persuasive) channels. prior research offers some evidence for substitution (e.g., gong et al., ) . customer adoption of the primarily informative web channel in banking is associated with substitution from other primarily informative channels such as self-service channels (automated teller machines, voice response systems) (campbell and frei, ) . even in cases where a primarily persuasive channel opens in a location containing another primarily persuasive channel, the cross-channel effect may be substitutional. indeed, the growth in web channel sales has come at the expense of the catalog channel, another primarily informative channel (avery et al., ; pauwels and neslin, ) . the channels in these cases are similar in their benefits and limitations, so they serve as substitutes. the same logic may extend to the effects of marketing efforts in rich and primarily persuasive channels on outcomes in channels with the same primary influence role. efforts in similar channels can lead to customer perceiving the channels as substitutes because the net benefits in those channels may be similar. consequently, efforts in one channel may help outcomes in that channel but hurt outcomes in similar channels. although exclusive and independent agents serve as two primarily persuasive channels in many industries, each channel j o u r n a l p r e -p r o o f may independently promote face-to-face human persuasion (shrum et al., ) . consequently, customers perceive these channels as similar in net benefits and treat them as substitutes. these channels typically compete with each other to get a share of the customers' business. for example, in the auto insurance industry, the compensation of agents is commission based and both exclusive agents and independent agents do not have the incentive to refer a customer to other agents. therefore, efforts by exclusive agents may hinder shopping outcomes through independent agents and vice versa. the competition between similar channels could also lead to potential conflict (sa vinhas and anderson, ) . thus, we predict that these two channels may be more substitutional than complementary. therefore, we propose the following hypothesis. h : marketing efforts in a channel have a negative effect on the shopping outcome in a similar (with the same primary influence role) channel. a complete list of all the predicted cross-channel effects appears in table , which shows the directional effects of efforts in each channel on outcomes in other channels. we expect all the complementary (primarily informative, primarily persuasive, balanced) cross-channel effects, except those between the exclusive agent and independent channels, to be positive. because two similar (primarily informative or primarily persuasive) channels often compete for the same business, the efforts of one of these channels will likely negatively influence outcomes in the other channel. the extent to which efforts in each channel will influence outcomes in the other channels is an empirical question we will address in the subsequent sections. < table about here > to test our hypotheses, we analyze data from the automobile insurance industry. a representation of how cross-channel effects occur in this industry appears in figure . in this context, consumers can ask for an auto insurance brand quote from one of four channels, the exclusive agent, the independent agent, the web, and the call center channels. a consumer can get information from one channel but obtain a quote or buy a policy from another channel. thus, the informative and persuasive roles of the distribution channels are important in the channel outcomes. exclusive agents sell only the focal firm's products. in contrast, independent agents sell products from multiple competitors. in each channel, the key outcome variable is the number of quotes, a big milestone and a forerunner of policies. unlike in other product categories, a prospect in the auto insurance category needs to provide detailed and confidential personal information to elicit a quote. therefore, eliciting a quote is a strong signal of purchase intent, and quotes correlate highly with purchases. quotes and policies are equivalent to sales leads and sales, respectively in most industries. marketing efforts in each channel impact the outcomes in the same channel as well as in the other channels. advertising also affects shopping outcomes. all the marketing channel and advertising efforts are mainly directed at increasing quotes. < figure about here > we collect data on quotes, policies, and channel efforts for the focal brand, one of the largest brands in the industry, for weeks. because channel effects occur in the presence of advertising, we need to control advertising effects. therefore, we gathered weekly advertising data for the focal brand from adtracker during the same time frame. the advantages of our dataset are that it offers rich industry-specific information and captures the shopping outcomes in each channel. the operationalization of the variables in the data appears in table . indeed, the correlation between quotes and policies is rather high in our data ( . ). we are unable to disclose the real brand names and the years to preserve confidentiality. j o u r n a l p r e -p r o o f < table about here > table provides summary statistics for the key variables in our model. these summary statistics suggest that the exclusive agent channel is the leading channel for the focal brand, accounting for a majority of the quotes generated, followed by the web, the call center, and the independent agent channel in that order. the focal brand also has many more exclusive agents than independent agents. this situation is common for the leading brands in the industry. the purpose of this research is to rigorously investigate the cross-channel effects through our models. < table about here > table presents the correlation matrix for the key variables in our data. an analysis of the correlations and collinearity diagnostics suggests that the variance inflation factors do not exceed , suggesting that multicollinearity is not an issue in our data. < table about here > to capture the cross-channel effects, we model the number of quotes generated in each channel, that is, quotes by exclusive agents (eaq), quotes by independent agents (iaq), quotes from the web (wq), and quotes from the call center (ccq) using the following system of equations ( ) we do not model policies for cross-channel effects because qualification of prospects, the stage before policies are issued, could not be fully performed by the web and call center channels, so the number of policies issues through the web was negligible and through the call center was limited. furthermore, the number quotes is highly correlated with the number of policies in the channels in which they were issued ( . ). j o u r n a l p r e -p r o o f where t is week, ea and ia are the number of exclusive agents and independent agents, respectively; web is the number of unique visitors on the website; and cc is the number of call center agents. ad is the number of advertising impressions. busd is the number of business days in the week, ucar is the number of used cars registered in the country, and , , and high correlation between web content and traffic to a website (kalhor and nikravanshalmani, ) . therefore, we use web as the measure of the firm's efforts in the web channel. to control for state dependence, we include a one period lagged dependent variable in each equation. we also include lagged quotes from each of the other channels. we log transform the dependent, channel effort, and advertising variables, so the coefficients serve as elasticities. while the focal brand's efforts and outcomes are temporally separated, given the high degree of autocorrelation observed in efforts, we need to account for the potential endogeneity of these efforts in equations ( ) are endogenous. we use one time period lags for these variables to rule out possible reverse causality. we also use appropriate instruments for each endogenous variable. for each endogenous variable, we select instruments based on both relevance and exclusion restriction. the number of exclusive agents depends on the wage rate for brokers. if the wage rate is high (low), a firm will hire a fewer (greater) number of agents, satisfying the relevance condition. but the brokers' wage rate is not directly related to the outcome variables. thus, the exclusion restriction is met. therefore, we the wage rate for brokers is a good instrument. similarly, the number of independent agents depends on the independent service agents' wage rate. as in the case of exclusive agents, the firm will hire fewer (greater) independent agents if this wage rate is higher (lower), meeting the relevance criterion. however, the wage rate of the independent service agents is not directly correlated with the outcome variables. thus, the exclusion restriction is satisfied and the wage rate of independent service agents is a good instrument for the number of independent agents. we collected wage rate information from the bureau of labor statistics (https://www.bls.gov/oes/tables.htm). data on suitable instruments for web are generally difficult to obtain. good instrument candidates such as the number of unique visitors to competitors' website are hard to collect, in particular, at the weekly level. the wage rate of web developers determines the number and quality of web developers to design and run a website. although web developers' wage rate is correlated with the number of developers (satisfying the relevance condition), it is uncorrelated with the outcome variables. therefore, the exclusion restriction is satisfied and wage rate of software professionals is a good instrument for the number web development employees. the wage rate of telemarketers determines the number of telemarketers who would be hired in a call center. although telemarketers' wage rate is correlated with the number of call center employees (maintaining the relevance condition), it is uncorrelated with the outcome variables. therefore, the exclusion restriction is satisfied and wage rate of telemarketers is a good instrument for the number of call center employees. because competing brands vie for the same customers, the focal brand's advertising will be correlated with the past advertising of its closest competitor brands. thus, the instrument relevance criterion is met. however, past competitor brand advertising is not directly related with the error terms in equations - (low correlation of . to . in our data), satisfying the exclusion restriction. thus, for the focal brand's advertising efforts, we use the past advertising impressions of its three closest competitors as good instruments. to ensure that the results are consistent for different instruments, we also use another set of instruments, the average of the past four weeks of wage rates in a subsequent robustness check. j o u r n a l p r e -p r o o f we estimate equations ( - ) as a simultaneous equation system with correlated errors. because the values of the outcome variables are large numbers and are log-transformed, they are ratioscaled and normally distributed. in equations ( - ) , the system of recursive equations, the random error components are likely to be correlated, so to increase the efficiency of the system, we jointly estimate the system and model the correlated errors through a multivariate normal distribution as follows: where the equation-specific random errors that are correlated and ∑ is the unconstrained variance-covariance matrix. therefore, we estimate the simultaneous equation systems using a seemingly unrelated regression (sur) procedure, consistent with mullahy ( ) . a single equation estimation would produce consistent estimates, but accounting for covariance in the error structure across equations produces more efficient estimates. we test the validity and strength of our instrumental variables. the results of the first stage regressions and the fit statistics appear in table < table about here > based on the results from the first stage regression and the relevant sargan-hansen test, we can conclude that the instruments are valid. we also test the strength of our instrumental variables through the staiger and stock ( ) and stock and yugo ( ) tests. the f-statistic in all cases for single instruments is greater than , consistent with staiger and stock ( ) . the f-statistic for the multiple instrument case (focal brand ad) is greater than . , in line with stock and yogo ( ) . therefore, we conclude that the instruments are sufficiently strong. to check how well the instruments control for endogeneity, we examine the correlations between the instruments of marketing efforts and residuals from eq ( ) through eq ( ). the correlation between and wage rate of brokers is . (p= . ); and wage rate of independent service agents is . (p= . ); and wage rate for web developers is . (p= . ); and and wage rate for telemarketers is . (p= . ). these low and insignificant correlations suggest that these instruments control for endogeneity well. the results of the cross-channel appear in table . we discuss the results in the order of the similarly, the number of independent agents has a significant but smaller negative effect on exclusive agent quotes (- . ; p < . ). taken together, the results reveal that the effects of efforts in the exclusive agent and independent agent channels on each other are asymmetric. thus, in general, efforts in a rich, primarily informative channel, a primarily persuasive channel, and a balanced channel reinforce one another and have complimentary effects on outcomes in one another's channel. however, there are some interesting asymmetries between the channels in these effects. while the effect of efforts in a primarily informative channel on shopping outcomes in a balanced channel is positive, the effect of efforts in a balanced channel on shopping outcomes in a primarily informative channel is insignificant. a possible explanation follows. a shopper typically encounters a leaner, informative channel earlier in the shopping journey. as a result, marketing efforts in such a channel will have positive effects on outcomes in richer, persuasive channels such as the call center and exclusive agent. in contrast, once a shopper moves to a balanced channel such as the call center, efforts in that channel will unlikely move the shopper back to a primarily informative channel for a desired shopping outcome. efforts in the rich, primarily persuasive, independent agent channel have some positive and significant effects on shopping outcomes in the other channels, but efforts in the other channels do not have a positive outcome in the independent agent channel. this asymmetry can be explained as follows. unlike the independent agent channel, the web, call center, and exclusive agent channels are firm-controlled channels. the efforts in these channels directly benefits the firm. in contrast, because independent agents deal with multiple brands, not all of their efforts are directed toward the focal brand. as a result, the complementary effect of marketing efforts in a dissimilar channel (primarily informative or balanced) is typically weak or insignificant. however, the substitutional effect of a similar channel (another primarily persuasive channel) on the independent agent channel is strong given the focal firm's stakes in its own exclusive agent channel. given the dedicated sales efforts of exclusive agents, the efforts of independent agents do not make much dent in the number of quotes issued through the exclusive agents. we now discuss the other effects in the models. the effects of efforts in a channel on outcomes within the same channel are positive and significant; exclusive agents (p < . ), independent agents (p < . ), website (p < . ), and call center (p < . ). the magnitudes of own channel and cross-channel elasticities reveal several surprising findings. table shows the significant own-and cross-channel elasticities and within channel advertising elasticities. the confidence intervals for these elasticities, obtained by bootstrapping given data points, appear in web appendix table a . with regard to own channel elasticities, we < table about here > to ensure that the results are robust, we performed several alternative analyses. first, we use cost shifters and hausman style instruments to account for the endogeneity of cross-channel and advertising efforts, respectively. a potential concern with contemporaneous cost shifters like wage rates is that managers may be able to make their effort decisions based only on past values of cost shifters, rendering lagged values of such variables to be more appropriate instruments. as a robustness check, we reestimate our models with averages lagged wage rates as instruments. the results (web appendix table a ) are consistent with those of our main model. second, we focus on quotes for our cross-channel model because quote is a key metric that the industry uses analyze at the channel level and because only a limited number of policies were issued in the web and call center channels. nevertheless, we did a robustness check of the model with policies as the dependent variable primarily for exclusive and independent agents. the results (web appendix table a ) are substantively similar to those of our proposed model. third, to assess the value of estimating cross-channel effects in the presence of advertising, we estimated two alternative models, one without advertising and one with no channel efforts. the results appear in web appendix tables a and a . the fits of these models are inferior to those of the proposed models. furthermore, the results differ from those of the proposed full models. thus, these results underscore the importance of including both channel effects and advertising in the model to accurately estimate cross-channel effects on shopping outcomes. web efforts facilitate outcomes from exclusive agents ( . ) and the call center ( . ). similarly, call center efforts enhance quotes from exclusive agents ( . ) as well as from the web ( . ). we note that var models are typically data intensive and their estimates and impulse response functions may not fully converge to stable values when the number of observations is small. furthermore, we are testing theory-driven hypotheses. for these reasons, we retain the results from the proposed set of equations that we developed to test the hypotheses. < figure about here > to summarize, our results largely support our hypotheses of complementary channel effects among a primarily persuasive channel (exclusive agent), a primarily informative channel (the web), and a balanced channel (call center). however, cross-channel complementarity is asymmetric in two ways. the first asymmetry is in the direction. in the case of the web and the omnichannel management is a burgeoning area for research and practice. our research on crosschannel effects has critical implications for research and practice. the test of hypotheses offer new insights on asymmetric cross-channel effects. our results could lead to a broader theory of channel effects. in such a theory, channel efforts make significant our research also offers scope for the development of a theory about the relative magnitudes of channel effects on shopping outcomes. we find that within each channel, advertising elasticities are lower than those of channel elasticities, including cross-channel elasticities. this result suggests that within a distribution channel such as the independent agent channel and the exclusive agent channel, the role of the communication vehicle such as advertising may be muted. these findings provide a fertile ground for the development of a more nuanced theory on j o u r n a l p r e -p r o o f the roles of the communication and distribution channels and the interplay between the two channel types in effecting purchases, extending dinner et al. ( ) and li and kannan ( ) . the surprising finding that a rich, primarily persuasive independent agent channel positively affects quotes in a lean, primarily informative web channel without a reciprocating effect poses intriguing theoretical possibilities. when independent agents inform shoppers about alternative brands, some shoppers may visit their websites and indicate their interest in those brands. some of these shoppers may view the brands' websites as more accurate and perhaps unbiased sources of a brand's information than that provided by independent agents, who might be perceived as promoting slanted information on brands better aligned with their compensation. this opens up the role of channel credibility in additionally explaining asymmetric cross-channel effects. the results offer valuable implications for managerial practice. first, managers should use a rich, primarily persuasive channel such as the exclusive agent, a lean, primarily informative channel such as the web, and a balanced channel such as the call center in a coordinated way. such cross-channel integration is critical to firm's growth (cao and li, ) . because exclusive agents have the highest own elasticity as well as high cross-elasticity on the call center, firms should allocate more efforts to exclusive agents. moreover, the web has the second highest own elasticity and high levels of cross-channel elasticities on exclusive agents and the call center. therefore, enhancing website capability to offer better information, navigation, visibility, and direct fulfillment is critical. furthermore, because the call center has high own elasticity and high cross-elasticity on exclusive agents, investing in the call center is also advantageous. thus, allocating more resources to dissimilar channels with varying richness--such as the exclusive agent, the web, and the call center--reinforces these channels in a synergistic manner. the j o u r n a l p r e -p r o o f findings have broad implications in other contexts. consumer packaged goods and durables firms should invest in own stores, own websites and call centers to provide a seamless omnichannel experience to the customer and benefit from positive cross-channel effects. second, the result that the rich, primarily persuasive channels, the exclusive and the independent agent channels are substitutional and asymmetric offers practical guidelines. in general, firms may not desire too much competition between exclusive and independent agents lest that leads to price erosion and service variance across the agents. however, because marketing efforts in at least one of these two channels have a negative effect on the other, firms will have to act creatively. to boost overall impact, managers may want to minimize cannibalization between these two agencies by redefining their roles and incentives. they could use independent agents more for reaching new shoppers or for new product launches, and exclusive agents more to develop deep relationships and repeat purchases for core products. because exclusive agents have a stronger negative effect on independent agent quotes than vice versa, managers could reexamine the nature and magnitude of incentives to align these channels together. again, we can extend this result broadly to other contexts and suggest that firms should distribute their products judiciously between exclusive outlets and independent stores and align margins or commission levels for these channels based on the cross-channel elasticities. third, by understanding this nuanced effect of advertising relative to channel effects, capitalize on cross-channel effects, within each channel, managers should assign fewer resources to advertising than to channel efforts. finally, from the cross-channel elasticities, managers can derive greater actionable insights by using an important tool, namely, a graph of influence and influenceability of the different channels. managers can compute influence and influenceability as follows. j o u r n a l p r e -p r o o f where ince c is influence of channel c, c is the total number of channels, e ci is the elasticity of efforts in channel c on outcome in channel i, and inty c is the influenceability of channel c, and e ci is the elasticity of efforts in channel i on outcome in channel c. the influence and influenceability graph for our data appears in figure . the web has the highest total influence on the other channels. in contrast, the exclusive agent is influenced most by the other channels but has the least positive influence on the other channels. the call center channel is in the middle with regard to both influence and influenceability. finally, the independent agent channel has a negative overall influence on the other channels and is also influenced lowest by other channels. < figure about here > managers can use the results from this graph together with own channel elasticities to prioritize the relative importance of each channel for marketing efforts. managers can see that the exclusive agent channel is most valuable because it has the highest own channel elasticity as well as the highest influenceability. they may find the web channel to be important because it has the second highest own channel elasticity and the highest influence on the other channels. managers can also appreciate the call center's value with moderate levels of own elasticity, influence and influenceability. contrary to the efforts expended in it, the independent agent channel has the least own elasticity and cross-channel influence and influenceability. therefore, managers should deemphasize this channel. by determining the relative importance of each channel through this graph, managers can better coordinate the levels of efforts in each channel. j o u r n a l p r e -p r o o f we illustrate the managerial usefulness of our model through a resource allocation exercise. we recognize that closed form optimal marketing efforts based on the estimated model is tedious to derive. therefore, we use numerical analysis to compute the optimal levels of marketing efforts using the estimated own-and cross-elasticities, the levels of other variables, and the unit costs of efforts in each channel. the results appear in table . they show the allocation percentages across the channels at different levels of marketing spending or budget relative to the actual marketing expenditures. these budget levels are akin to different spending scenarios proposed by dinner et al. ( ) . the table also reveals the profit differentials for different marketing spending levels relative to the actual marketing spending. to highlight the value of our crosschannel effects model, the table displays resource allocation outputs based on two models: model without cross-channel effects and with cross-channel effects (see web appendix table a ). < table about here > the profit levels for resource allocation based on the model with cross-channel effects are consistently higher than those for the model without cross-channel effects for all marketing budget levels. although the profit from optimization based on a model without cross-channel effects is greater than the firm's actual profit ( . % more), the profit from optimization based on our model with cross-channel effects is much greater ( . % more). the incremental profits from our model is highest ( . % more) when the marketing budget expands by %. the optimal reallocation of marketing budget across the channels reveals interesting insights. the firm's actual channel allocation mix was . % for exclusive agents, . % for independent agents, . % for the web, and . % for call center. a reallocation based on optimization through a model without cross-channel effects produces an optimal channel mix of j o u r n a l p r e -p r o o f . % for exclusive agents, . % for independent agents, . % for the web, and . % the call center. these numbers suggest a significant shift in resource allocation from the independent agent, the web, and the call center channels toward exclusive agents mainly because the exclusive agents have the highest own elasticity. however, a reallocation based on our proposed model with cross-channel effects suggests a channel mix of for . % for exclusive agents, . % for independent agents, . % for the web, and . % for call center. these results suggest that the firm needs to move substantially away from exclusive and independent agents toward the web and call center. these results are consistent with the influence-influenceability matrix. the cross-channel effects of a primarily informative channel and a balanced channel on primarily persuasive channels explain the shift in allocation. without considering these crosschannel effects, the firm overspent on primarily persuasive exclusive and independent agents. these findings based on cross-channel effects are consistent with the general trend in channel allocation. across industries, there is a growing shift in allocation of resources toward leaner and more efficient channels such as the web channel. banks have moved more toward the web and mobile channels. so have most retailers, in particular, post covid- . service organizations are reallocating toward leaner and balanced channels. theoretically, the allocation findings suggest a trade-off between maximizing the high own elasticities of rich, persuasive channels and leveraging the high cross-channel elasticities of lean, informative and balanced channels. our research has limitations that future research could address. first, our models do not include competitor channel efforts because data on competitor brands were unavailable. with competitor data, a more complete model of own and competitor channel efforts could be developed. second, channel roles may vary across categories and stages in customer journey. future research could examine cross-channel effects for categories in which the catalog channel and industrial channels are relevant, extending kauferle and reinartz ( ) . with disaggregate data, future work could explore differential channel roles in customer journey and customer relationship management across channels, furthering verhoef et al. ( ) . third, with data on prices, promotions and customer satisfaction, a more comprehensive model of shopping outcomes can be developed. online and offline prices can differ (ancarani and shankar, ) and may result in differential cross-channel effects. furthermore, offline satisfaction can affect online purchases for services (montoya-weiss et al. ). fourth, our study could be extended to examine cross-channel integration (cao and li, ) . fifth, our model is at the channel level as data at the consumer level are difficult to obtain. with shopper level data, we could analyze the mechanisms behind cross-channel effects. finally, cross-channel effects could be studied in conjunction with cross-buying as the drivers and consequences could be somewhat similar (kumar, george, and pancras, ) . in conclusion, we sought answers to important questions relating to the growing phenomenon of cross-channel effects. our research offers fresh insights. it shows that cross-channel effects are significant and asymmetric. while channels with dissimilar primary influence roles (e.g., the exclusive agent, the web and the call center channels) are complementary, the direction and magnitude of the cross-channel effects are asymmetric. likewise, while channels with similar primary influence roles (e.g., the independent agent and exclusive agent channels) are substitutional or competitive, their cross-channel effects are also asymmetric. within each channel, own channel elasticity is greater than advertising elasticity. the findings offer important implications for theory and practice with regard to allocation of channel efforts across channels. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f we use hausman style instruments, i.e., advertising decisions made by competing brands as instruments for the focal brand's advertising. these advertising spending decisions may be directly influenced by common advertising cost shock but not demand shock. the sargan-hansen test for over-identifying restriction is insignificant (p > . ), suggesting the instruments are valid. note: * p < . ; ** p < . ; *** p < . . for each instrument, f-statistic is well above , satisfying staiger and stock ( ) , stock and yogo ( ) tests. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f price levels and price dispersion within and across multiple retailer types: further evidence and extension customer channel migration adding bricks to clicks: predicting the patterns of cross-channel elasticities over time consumers in a multi-channel environment: product utility, process utility, and channel choice offline showrooms in omnichannel retail: demand and operational benefits clicks and mortar: the effect of online activities on offline sales battle of retail channels: how product selection and geography drive cross-channel competition cross-channel effects of promotions: an empirical analysis of multi-channel grocery sector the impact of cross-channel integration on retailers' sales growth cost structure, customer profitability, and retention implications of selfservice distribution channels: evidence from customer behavior in an online banking channel the impact of ebook distribution on print sales: analysis of a natural experiment the relative advantage of electronic channels: a multidimensional view organizational information requirements, media richness and structural design the effectiveness of different forms of online advertising for purchase conversion in a multiple-channel attribution framework what makes commercial web pages popular? an empirical investigation of web page effectiveness driving online and offline sales: the crosschannel effects of traditional, online display, and paid search advertising theory of reasoned action: some applications and implications in beliefs, attitudes, intentions, and behavior: an introduction to theory and research the value of rapid delivery in omnichannel retailing benchmarking retail productivity considering retail pricing and format strategy substitution or promotion? the impact of price discounts on cross-channel sales of digital movies the impact of contextual television ads on online conversions: an application in the insurance industry the economic aspects of advertising correlation between content and traffic of universities' websites digital marketing: a framework, review and research agenda optimizing a menu of multiformat subscription plans for ad-supported media platforms distributing through multiple channels in industrial wholesaling: how many and how much cross buying in retailing: drivers and consequences who are multichannel shoppers and how do they perform? correlates of multichannel shopping behavior are multichannel customers really more valuable? the moderating role of product category characteristics multichannel strategies for managing the profitability of business-to-business customers attributing conversions in a multichannel online marketing environment: an empirical model and a field experiment the own-wage elasticity of labor demand: a metaregression analysis multichannel retailing: a review and research agenda optimizing marketer costs and consumer benefits across "clicks" and "bricks catalogue as a tool for reinforcing habits: empirical evidence from a multichannel retailer can marketing campaigns induce multichannel buying and more profitable customers? a field experiment determinants of online channel and overall satisfaction with a relational, multichannel service provider instrumental-variable estimation of count data models: applications to models of cigarette smoking behavior a hierarchical marketing communications model of online and offline media synergies temporal differences in the role of marketing communication in new product categories information and consumer behavior advertising as information key issues in multichannel customer management: current knowledge and future directions its time to think omnichannel shopper, not just omnichannel electronic and physical market channels: a multiyear investigation in a market for products of uncertain quality modern (multiple) time series models: the dynamic system building with bricks and mortar: the revenue impact of opening physical stores in a multichannel environment exploring the implications of internet for consumer marketing communication and persuasion: central and peripheral routes to attitude change the effect of internet distribution on brick-and-mortar sales opportunities and challenges in multichannel marketing: an introduction to the special issue how potential conflict drives channel structure: concurrent (direct and indirect) channels persuasion in the marketplace: how theories of persuasion apply to marketing and advertising, in the persuasion handbook a study of , shoppers shows that omnichannel retailing works how does adoption of the online channel impact customers' spending in retail stores: conflict or synergy? instrumental variables regression with weak instruments testing for weak instruments in linear iv regression the economics of information home internet access and usage in the usa: trends in the socioeconomic digital divide multichannel marketing report. the direct marketing association determining the cross-channel effects of informational websites, doctoral dissertation dual distribution and intangible firm value: franchising in restaurant chains multichannel shopping: causes and consequences multichannel customer management: the research shopper phenomenon from multi-channel retailing to omni-channel retailing: introduction to the special issue on multi-channel retailing crm in data-rich multichannel retailing environments: a review and future research directions can offline stores drive online sales? the evolution of marketing channels: trends and future research directions marketing's profit impact: quantifying online and offline funnel progression exclusive vs. independent agencies: a comparison of performance. the geneva papers on risk & insurance theory in-sample and out-of-sample predictive model validation for quotes in each channel key: cord- - klf qr authors: saponaro, federica; saba, alessandro; zucchi, riccardo title: an update on vitamin d metabolism date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: klf qr vitamin d is a steroid hormone classically involved in the calcium metabolism and bone homeostasis. recently, new and interesting aspects of vitamin d metabolism has been elucidated, namely the special role of the skin, the metabolic control of liver hydroxylase cyp r , the specificity of α-hydroxylase in different tissues and cell types and the genomic, non-genomic and epigenomic effects of vitamin d receptor, which will be addressed in the present review. moreover, in the last decades, several extraskeletal effects which can be attributed to vitamin d have been shown. these beneficial effects will be here summarized, focusing on the immune system and cardiovascular system. vitamin d is a steroid hormone which exerts a crucial role in the maintenance of bone and calcium homeostasis. the discovery dates back to one hundred years ago, but vitamin d has become a hot topic in endocrinology research only in the last decades, and it has recently emerged as a burning issue due to the covid- pandemic, because of the alleged correlation between hypovitaminosis d and high risk of chronic pulmonary diseases and mortality [ ] . it is now clear that vitamin d displays a complex multistep metabolism and acts as a hormone on many extra-skeletal targets [ ] . the aim of this review is to focus on some new, intriguing, and still incompletely clarified aspects of vitamin d metabolism, such as novel concepts in enzyme regulation, new pleiotropic effects of vitamin d receptor (vdr) activation, and epigenetic effects. vitamin d exists in two forms: vitamin d , which is the most important source in animals and is produced in the skin; and vitamin d which differs from d for a methyl group in c and a double bond in c -c and is produced by plants [ ] . in the skin, vitamin d is produced from -dehydrocholesterol ( dhc), an intermediate in cholesterol synthesis. exposure to ultraviolet b (uvb) light, in the range of - nm, determines an electrocyclic rearrangement of the ring in the c -c position, yielding pre-vitamin d (pred ). once pred is formed, thermal isomerization to vitamin d (vitd ) occurs, with the shift of a hydrogen from c to c [ ] (figure ). this reaction is reversible and pred and vitd both coexist. from an evolutionary point of view, the observation that vitd production is strictly dependent on uvb sheds light on the ancient origin of the hormone, at least . billion of years ago, when algae began to produce cholesterol [ ] . this process has probably developed as a scavenger mechanism to protect from uvb radiation, which is absorbed and dissipated in the rearrangement of double bonds [ ] . as a matter of fact, the synthesis of vitd depends on the concentration of dhc, which in turn depends on dehydrocholesterol reductase (dhcr ) activity. this enzyme catalyzes the reversible reduction of dhc into cholesterol. this is part of the biochemical pathway first described this reaction is reversible and pred and vitd both coexist. from an evolutionary point of view, the observation that vitd production is strictly dependent on uvb sheds light on the ancient origin of the hormone, at least . billion of years ago, when algae began to produce cholesterol [ ] . this process has probably developed as a scavenger mechanism to protect from uvb radiation, which is absorbed and dissipated in the rearrangement of double bonds [ ] . as a matter of fact, the synthesis of vitd depends on the concentration of dhc, which in turn depends on dehydrocholesterol reductase (dhcr ) activity. this enzyme catalyzes the reversible reduction of dhc into cholesterol. this is part of the biochemical pathway first described by kandutsch and russel in (alternative to the bloch pathway), in which six isoprene units from acetyl-coa are converted in a cyclized isoprenoid hydrocarbon (lanosterol) and subsequently through oxidative/reductive steps into zymosterol, zymostenol, dhc, and finally cholesterol ( figure ) [ ] . it was only in that the kandutsch/russel pathway was completely elucidated and found to have a high activity in the skin, providing the substrate for vitd production [ , ] . in the last fifty years, the discovery of a rare syndrome called smith-lemli-opitz syndrome (slos, omim # ) which is caused by mutations in the dhcr gene provided interesting information [ , ] . slos has an incidence of : , and it is clinically characterized by morphogenic and congenital aberrations, with cognitive retardation and altered behavior [ ] . currently different mutations of the dhcr gene have been described, that cause enzyme inactivation and accumulation of dhc [ , ] . the most frequent mutations are: a null mutation ivs - g>c and a nonsense mutation w x; the others being missense mutations [ ] . slos is more common in countries with low sun exposure and this observation has been interpreted as an heterozygous advantage for mutation carriers to avoid vitamin d (we will refer to vitd if not differently specified) deficiency [ ] . vitamin in the last fifty years, the discovery of a rare syndrome called smith-lemli-opitz syndrome (slos, omim # ) which is caused by mutations in the dhcr gene provided interesting information [ , ] . slos has an incidence of : , and it is clinically characterized by morphogenic and congenital aberrations, with cognitive retardation and altered behavior [ ] . currently different mutations of the dhcr gene have been described, that cause enzyme inactivation and accumulation of dhc [ , ] . the most frequent mutations are: a null mutation ivs - g>c and a nonsense mutation w x; the others being missense mutations [ ] . slos is more common in countries with low sun exposure and this observation has been interpreted as an heterozygous advantage for mutation carriers to avoid vitamin d (we will refer to vitd if not differently specified) deficiency [ ] . vitamin d levels have been measured in few slos patients. in rossi et al. did not find any difference in vitamin d levels in patients with slos, compared to healthy matched controls [ ] , but this finding might be attributed to the photosensitivity of slos patients, leading to reduced exposure to the sunlight. on the other hand, movassaghi et al. evaluated pediatric patients with slos and found significantly higher levels of hydroxyvitamin d ( ohd), the marker of vitamin d status, across all seasons ( . ± . ng/ml vs. . ± . ng/ml, p < . ), without signs of vitamin d intoxication (normal serum calcium) [ ] . moreover, the genetic locus dhcr /nadsyn was found to be a determinant of vitamin d status in two contemporary mendelian studies or large-scale genome-wide association studies (gwas) [ , ] , but the hypothesis that some dhcr polymorphisms are correlated with vitamin d status is still controversial and has not been confirmed by some studies [ ] . biochemical regulation of dhcr seems to be a crucial aspect in vitamin d production, since reduced activity of this enzyme can redirect the pathway from cholesterol to vitamin d biosynthesis. indeed, at the transcriptional level both vitamin d or cholesterol can reduce dhcr expression [ ] . at the post-translational level, enzyme phosphorylation seems to be important: prabhu et al. showed that inhibition of amp-activated protein kinase and protein kinase a significantly reduced dhcr activity, enhancing vitamin d synthesis and reducing cholesterol production [ ] . in summary, dhcr enzyme is the first line of regulation of vitamin d biosynthesis in the skin, even if the actual production is also modulated by other factors including genetic polymorphisms, age, geographical location and latitude, exposure behavior and cultural conducts, uvb dose, clothing and body surface area (bsa) exposed [ ] . concerning regarding sunscreen photoprotection and vitamin d status have recently demonstrated to be inconsistent by an international panel of experts who reviewed the literature and concluded that vitamin d production is not affected by sunscreen use [ ] . the skin has long been known as the major source of vitamin d. moreover, keratinocytes of epidermis and hair follicles express the hydroxylases needed to produce the active hormone , dihydroxy vitamin d [ , (oh) d] (see section ), and vdr has been shown to be present on keratinocytes [ ] . as a matter of facts, vitamin d produces autocrine and paracrine effects in the skin [ ] . in keratinocytes vitamin d has been shown to control differentiation, proliferation, barrier activity and immune response [ ] . in selective epidermis vdr knockout animals, predisposition to cancer and impaired wound healing has been observed [ ] . moreover, vitamin d deficiency has been related to skin inflammatory diseases and vitamin d analogues have been found to be effective in psoriasis, a proliferative inflammatory skin disease [ ] . it is well established that vitamin d requires two subsequent hydroxylation steps to become the active hormone , (oh) d ( , -dihydroxy vitamin d, calcitriol). the first hydroxylation, in the c position, occurs mainly, but not exclusively, in the liver, with a non-regulated and substrate dependent mechanism, as originally reported. as a matter of fact, several enzymes display -hydroxylase activity and among them cyp r has been found to play the major role in the liver and testis. cyp r is located in the microsomal p fraction of hepatocytes, as reported quite recently by cheng et al. [ ] . there are few studies on catalytic properties of the enzyme: in a yeast system, shinkyo et al. demonstrated that cyp r can hydroxylate either vitd or vitd , with higher affinity for the first compared to the latter [ ] . subsequently, this observation has been confirmed in escherichia coli and the crystallographic structure of the enzyme has been determined. notably, the pocket for vitamin d entrance faces the hydrophobic membrane domain [ , ] . the human cyp r gene is located on chr. p . ( . kb) and contains highly conserved exons, codifying for a -amino acid protein. in cyp r knockout mice, ohd levels have been found to be decreased by about %, the other % being ensured by other -hydroxylase enzymes [ ] . in humans, five cyp r mutations have been identified in patients with different phenotypes, including rickets and low ohd levels [ ] [ ] [ ] [ ] . moreover, more than single-nucleotide polymorphisms (snps) of cyp r are known and could explain the population variability in ohd concentration observed in some genome-wide association studies [ , ] . particularly, a recent meta-analysis suggested that the rs polymorphism has a role in the genetically determined vitamin d deficiency [ ] . despite the previous hypothesis that cyp r is a non-regulated substrate dependent enzyme, recent evidences challenged this dogma, suggesting that the enzyme expression is modulated by age and metabolic environment. ohd levels decrease and are less responsive to supplementation in older patients. roizen et al. attributed this finding to a reduction in cyp r activity in aging, since cyp r mrna and protein content in hepatic tissue of male mice progressively decreased from to and weeks (one-way anova, p = . ). moreover, the ohd /vitd ratio was positively correlated with cyp r mrna and consistently declined with age [ ] . the metabolic layout also affects cyp r expression. it is known that ohd levels are significantly reduced in patients with obesity and type diabetes. the current hypothesis is that vitamin d could be sequestered in adipose tissue or diluted in the high surface of obese people. however, a reduction in cyp r activity has been proposed as an alternative explanation. cyp r activity was diminished in an animal model of high fat diet (hfd) obesity, since cyp r mrna was significantly lower ( %) compared to lean mice, whereas other -hydroxylase enzymes were not altered by hfd. cyp r protein expression and enzyme activity was reduced by % in obese mice liver homogenates compared to controls [ ] . other investigators observed that h-fasting strongly reduced cyp r mrna and the effect was even higher after h ( % and % respectively), both in mice or rat models [ ] . protein expression and enzymatic activity were reduced accordingly. in different models of diabetes (hfd induced type diabetes and streptozocin induced type diabetes) a similar suppression of liver cyp r activity was observed [ ] . at least two potential signaling pathways have been involved in cyp r modulation: the peroxisome proliferator-activated receptor γ coactivator -α/estrogen related receptor α (pgc α/errα) and the glucocorticoid receptor (gr) axis. the pgc α/errα pathway is physiologically activated during fasting and it is pathologically induced in diabetes [ , ] : overexpression of this signaling strongly decreased cyp r hydroxylate activity [ ] . however, other mechanisms are likely to exist, since suppression of cyp r by starving was observed also in pgc α knockout mice. aatsinki et al. showed that pharmacological inhibition of gr prevented cyp r induction by fasting, suggesting a role for this pathway in hydroxylase regulation [ ] . these findings suggest a complex crosstalk between vitamin d and several metabolic pathways, so that ohd levels undergo a refined control, and do not simply mirror vitamin d intake, as usually assumed. in addition, further hydroxylase activities have been found in the liver. they include cyp a , which is located in the mitochondria and has a major role in cholic acids formation [ ] , and cyp a , which has a compensatory c hydroxylase activity [ ] . besides the well-known role of vitamin d in calcium and bone metabolism, in the last ten years additional effects have been described, with special regard to the immune system. from an evolutionary point of view, specific investigations and genome-wide association studies demonstrated that the ancient and initial role of vitamin d was likely the regulation of genes involved in energy metabolism [ ] . during vertebrate evolution, skeletal and immune systems evolved quite simultaneously and vitamin d was a central driver of the osteo-immune bidirectional interactions [ ] . as a matter of fact, the primary reason for these extra-skeletal effects of vitamin d is the ability of different tissues to produce the active hormone, i.e., , (oh) d, locally, thanks to the enzyme α-hydroxylase. despite the existence of several -hydroxylase enzymes, cyp b has been demonstrated to be the only α-hydroxylase in human, and different tissues isoforms exist [ ] . it is noteworthy that whereas ohd is easily detectable in blood and urine (in the order of ng/ml), the concentrations of , (oh) d are much lower (order of pg/ml) and are largely regulated peripherally with autocrine and paracrine mechanisms, which escape systemic endocrine control and detection. in , renal cyp b was identified and the kidney was thought to be the only source of , (oh) d [ ] . the renal form of cyp b is regulated by at least three hormones, with a crucial role in calcium-bone metabolism ( figure ): parathyroid hormone stimulates the hydroxylation, whereas fgf and , (oh) d itself inhibit it, in response to calcium and phosphate concentrations [ , ] . calcitonin has also been shown to stimulate renal cyp b and leptin to inhibit, probably via fgf ( figure ) [ ] . beyond classical renal cyp b modulation, the novelty in the field is represented by a completely different regulation of cyp b in the other tissues, particularly in the immune system. in the s, it was observed that the administration of , (oh) d to blood myeloid cells induced their maturation into white cells [ ] . the contemporary report of hypercalcemia and high levels of , (oh) d in an anephric patient with sarcoidosis, suggested that c hydroxylation could occur outside the kidney [ ] . in , adams et al. observed , (oh) d production from macrophages in sarcoidosis patients [ ] . it is now known that macrophages are involved in the pathophysiology of many inflammatory and/or autoimmune diseases (sarcoidosis, tuberculosis, chron's disease, foreign body granulomata, cryptococcosis and others), and that they are able to produce , (oh) d at high levels by their own cyp b [ ] . differently from renal cyp b , the macrophage isoform is not controlled by pth. , (oh) d formation depends only on substrate availability and is not limited by product accumulation, which was interpreted as absence of catabolic enzymes control [ , ] . this is likely the reason why in sarcoidosis , (oh) d production is persistent and eventually leads to systemic hypercalcemia [ ] . the regulation of cyp b in macrophages and monocyte has been elucidated and it is under the control of cytokines and inflammation. macrophages' cyp b is stimulated by interferon-γ (infγ), tumor necrosis factor α (tnfα), interleukin (il) , and , but not by pth. moreover, dexamethasone inhibits cyp b [ ] . in addition to macrophages, also dendritic cells (dc), th lymphocytes and b lymphocytes express cyp b , but only when they are activated. in these cells , (oh) d functions as a αhydroxylase inhibitor, thus controlling their activation and proliferation. as described further in details in paragraph , , (oh) d exerts many autocrine and paracrine functions on immune system cells, ensuring a feedback control on immune cells themselves [ ] . beyond classical renal cyp b modulation, the novelty in the field is represented by a completely different regulation of cyp b in the other tissues, particularly in the immune system. in the ′s, it was observed that the administration of , (oh) d to blood myeloid cells induced their maturation into white cells [ ] . the contemporary report of hypercalcemia and high levels of , (oh) d in an anephric patient with sarcoidosis, suggested that c hydroxylation could occur outside the kidney [ ] . in , adams et al. observed , (oh) d production from macrophages in sarcoidosis patients [ ] . it is now known that macrophages are involved in the pathophysiology of many inflammatory and/or autoimmune diseases (sarcoidosis, tuberculosis, chron's disease, foreign body granulomata, cryptococcosis and others), and that they are able to produce , (oh) d at high levels by their own cyp b [ ] . differently from renal cyp b , the macrophage isoform is not controlled by pth. , (oh) d formation depends only on substrate availability and is not limited by product accumulation, which was interpreted as absence of catabolic enzymes control [ , ] . this is likely the reason why in sarcoidosis , (oh) d production is persistent and eventually leads to systemic hypercalcemia [ ] . the regulation of cyp b in macrophages and monocyte has been elucidated and it is under the control of cytokines and inflammation. macrophages' cyp b is stimulated by interferon-γ (infγ), tumor necrosis factor α (tnfα), interleukin (il) , and , but not by pth. moreover, dexamethasone inhibits cyp b [ ] . in addition to macrophages, also dendritic cells (dc), th lymphocytes and b lymphocytes express cyp b , but only when they are activated. in these cells , (oh) d functions as a αhydroxylase inhibitor, thus controlling their activation and proliferation. as described further in details in paragraph , , (oh) d exerts many local production of , (oh) d by cyp b for autocrine/paracrine purpose has been described in many other tissues, including epithelial tissues, placenta, bone, endocrine glands (parathyroid, pancreatic islets, thyroid, adrenal medulla, gonads), brain, liver and endothelia [ ] . in the majority of cases, experimental data suggest that the regulation of local cyp b escapes the classical patters of the renal isoform and is due to local tissue-specific stimuli [ ] . in figure the main regulators of extrarenal cyp b isoforms are summarized. more than metabolites of vitamin d have been described in the last decades and some of them display a certain interest because of their biological activity. the best-known catabolic enzyme is cyp a , belonging to mitochondrial p fraction and encoded by the cyp a gene on chr. q . [ ] . cyp a can hydroxylase both ohd and , (oh) d, producing r, (oh) d and , , (oh) d, respectively. the same enzyme further catalyzes the hydroxylation of these products in multiple steps, yielding a series of -and -hydroxylated derivatives. the final products are the inactive calcitroic acid or , -lactone excreted with bile or urine (figure ). cyp a is up-regulated by calcitriol and fgf and is inhibited by pth and hypocalcemia. cyp a has been detected in many tissues expressing vdr, and it plays a crucial role in the local modulation of vitamin d activity [ ] . pathogenic variants of cyp a have been described and they are responsible for idiopathic infantile hypercalcemia (iih, omim ), a rare disorder due to impaired vitamin d catabolism and subsequent hypercalcemia. in particular, biallelic variants (in homozygosis or heterozygosis) have a severe phenotype with hypercalcemia that may occasionally lead to death in infant age [ ] . in addition to cyp a , other minor metabolic pathways have been described and still need further evaluation. recently, the c- epimerization pathway has been identified, which leads to the production of several c- epimer metabolites, in which the hydroxyl group on c has the alpha rather than the beta orientation in the space. epimeric metabolites have been shown to be highly expressed particularly in neonates and young children, but the physiological role of this redundant pathway needs to be elucidated [ ] . the presence of so many metabolites is stimulating the development of novel and more accurate analytical techniques. since , when high performance liquid chromatography techniques have been introduced, they have been continuously improved and today the most recent lc-ms-ms assay is referred as the "gold standard" method [ ] [ ] [ ] [ ] . this is due to the high sensitivity, reproducibility and accuracy, this latter also being influenced by the capability to discriminate ohd and ohd , as well their epimeric forms. moreover, it offers the possibility to measure different vitamin d metabolites at the same moment [ ] . right now, five intermediates have been measured with standardized techniques, namely: vitamin d, ohd, α, (oh) d, r, (oh) d and c -epi (oh)d and procedures are listed in the joint committee for traceability in laboratory medicine (jctlm) database [ ] [ ] [ ] [ ] . in the use of lc-ms-ms for vitamin d metabolites measurement was advised by the nutritional health and nutrition examination survey [ ] in usa and from the uk food standard agency (fsa) in their national diet and nutrition survey [ ] . the hope is that with lc-ms-ms further insight in the complexity of vitamin d metabolites will be achieved [ , ] . transport of vitamin d metabolites is accounted for % by vitamin d binding protein (dbp) with high affinity and % by albumin with low affinity [ ] . dbp, initially known as gc-globulin, is a multitasking protein which is very conserved in vertebrates' evolution. the human gene for dbp is located on chr. , close to other genes for albumin family proteins and encodes for a amino acid single chain protein [ ] . all metabolites of vitamin d can be bound by the same binding site of dbp, even if ohd and , (oh) d have the highest affinity [ ] . free ohd represents . % and , (oh) d . % of the total amount of the metabolites and have been classically interpreted as the only active hormone to enter cells (free hormone hypothesis [ ] ). however, at least in some organs like kidney, the free hormone hypothesis has been recently revised. indeed, it has been shown that the large transmembrane protein megalin is present on the apical side of the proximal tubule cells and acts as a receptor for the complex vitamin d-dbp, together with cubulin and disabled- proteins [ ] . accordingly to this hypothesis, knockout mice for lrp (encoding for megalin) show severe osteomalacia and poor survival, demonstrating the pivotal role of dbp binding capacity in the kidney [ ] . on the other hand, the role of this mechanism in the other tissues is still debated: megalin is expressed in several tissues in which vitamin d exerts extraskeletal functions, but megalin-mediated uptake of dbp has not been completely elucidated. summarizing recent evidences, dbp functions as a large pool reservoir of circulating ohd, which prevents for vitamin d deficiency when supply is low. moreover, dbp also functions as a regulator for vitamin d access to cells in kidney and most likely in the other peripheral tissues [ ] . the human vitamin d receptor gene (vdr) is located on chr. and contains nine exons. in the last twenty years vdr cdnas were obtained and cloned from several species (human, mice, rats, chicken, frog, quail), revealing a great homology among species and many conserved regions [ , ] . vdr is a polypeptide of , da formed by a single amino acid chain. it is almost ubiquitous in the body since it is expressed in at least thirty tissues, involved in bone metabolism (intestine, bone, cartilage, kidney) or in other extra-skeletal functions (heart, immune system, adipose tissues and many others) [ , ] . vdr belongs to the nuclear receptor superfamily along with the receptors of other steroid hormones. these receptors share the ability to bind their ligands at nanomolar concentrations in a specific conserved ligand binding domain (lbd), with a pocket of - a [ , ] . when vdr binds to , (oh) d, it can reach the nucleus and forms a heterodimer with retinoid x receptor (rxr), able to interact with gene response elements. this interaction is crucial for assembling the transcriptional machinery at the promoters of , (oh) d targets genes [ ] . the crystallographic structure of the receptor bound to its major ligand , (oh) d was resolved by rochel et al. [ ] . the ligand binding domain (lbd) is formed by αhelices (h - ) packed in three layered α helical sandwich and three stranded β sheets. when the ligand binds, h is able to shift and deeply closes the ligand into the pocket binding site [ ] . the dna binding domain is formed by two zinc fingers, where four cysteines residues maintain zinc in a tetrahedral configuration [ ] . more than % of the entire genome from zebrafish to human is under direct or indirect vdr control, so that more than , genes have been identified as putative targets for vdr, controlling many pivotal mechanisms such as metabolism, cells adhesion, tissue differentiation, development and angiogenesis [ ] . some of the major signaling pathways activated by vdr are summarized in table . table . major signalling pathways activated by vdr. as a matter of fact, vitamin d has recently become a hot topic in nutrigenomics that is the discipline studying the environmental factors able to affect the transcriptome and the epigenome. the latter is a novel and interesting field in vitamin d research. chromatin is the structure in which genomic dna, nucleosome-forming histone proteins and non-histone proteins are packed in the nucleus, and it represents the scaffold of the entire human heritable information [ ] . chromatin exists in at least two different forms: less dense and transcription-available euchromatin and compact, functionally repressed heterochromatin. these different conformations are largely related to post-translational changes of chromatin proteins. epigenomic studies all the modifications (such as histone methylation or acetylation) which occur in chromatin in the absence of genomic changes [ ] . these epigenetic alterations can be very stable and heritable or unstable and transient and are catalyzed by the so-called chromatin modifier enzymes. a few hundred genes for chromatin modifier enzymes have been described, which as carlberg brilliantly wrote: "add (write), interpret (read) or remove (erase) post translational histone modifications" [ ] . vdr acts as a transcription factor and is able to modulate genes encoding for chromatin modifier enzymes, thus modulating the human epigenome. one example is kdm b/jmjd , a histone h lysine demethylase, which plays a crucial role in development. it has been shown to be induced by , (oh) d/vdr and in turn to modulate vitamin d metabolism. pereira et al. showed that , (oh) d/vdr induced jmjd rna in human colon cancer cells, suggesting a role for , (oh) d in colon cancer epigenomic events [ ] . another level of epigenomic control by vdr is the direct interaction between vdr and chromatin proteins. furthermore, vdr has been shown to interact with co-activators, such as those of the ncoa family, or co-repressors, such as ncor proteins, which respectively lead to local chromatin opening or closing [ ] . the enrolment of co-regulators is also important in the tissue specific and cell-specific regulation of differentiation. an example is the skin were the change of different , (oh) d/vdr genes controlling the process of differentiation of the keratinocytes is regulated by the subsequent recruitment of different co-regulators of the mediator complex family (med) in the early stage and steroid receptor co-activator (src ) in the later stages [ ] . vdr seems to interact with more than nuclear proteins, and deeply affects chromatin remodeling [ ] . vdr has also been detected in a different subcellular location, namely as a transmembrane receptor which appears to be activated by vitamin d analogues with a different configuration ( s-cis), if compared to those that activate the nuclear vdr ( s-trans). transmembrane vdr is one of the receptors by which vitamin d exerts non genomic, rapid effects on its target cells and tissues. some non-genomic effects of vitamin d, which occur rapidly over minutes or hours, include rapid intestinal absorption of calcium (transcaltachia), secretion of insulin by pancreatic cells, opening of voltage-gated ca + and cl-channels in osteoblasts, and the rapid migration of endothelial cells [ ] . the classical role of , (oh) d in calcium/bone metabolism, namely the regulation of intestinal calcium absorption, renal calcium reabsorption and mobilization of calcium and phosphate from bone, has been known for decades and is beyond the aims of the present review. on the other hand, in the last decade a mean of papers per year have been published on vitamin d, pushed by new findings concerning extra-skeletal effects of this hormone [ ] . in this section we will summarize part of these discoveries and the effects of , (oh) d on several districts, particularly the immune system and the cardiovascular system ( figure ) . actions of vitamin d on skin have been summarized in the first paragraph. methylation or acetylation) which occur in chromatin in the absence of genomic changes [ ] . these epigenetic alterations can be very stable and heritable or unstable and transient and are catalyzed by the so-called chromatin modifier enzymes. a few hundred genes for chromatin modifier enzymes have been described, which as carlberg brilliantly wrote: "add (write), interpret (read) or remove (erase) post translational histone modifications" [ ] . vdr acts as a transcription factor and is able to modulate genes encoding for chromatin modifier enzymes, thus modulating the human epigenome. one example is kdm b/jmjd , a histone h lysine demethylase, which plays a crucial role in development. it has been shown to be induced by , (oh) d/vdr and in turn to modulate vitamin d metabolism. pereira et al. showed that , (oh) d/vdr induced jmjd rna in human colon cancer cells, suggesting a role for , (oh) d in colon cancer epigenomic events [ ] . another level of epigenomic control by vdr is the direct interaction between vdr and chromatin proteins. furthermore, vdr has been shown to interact with co-activators, such as those of the ncoa family, or co-repressors, such as ncor proteins, which respectively lead to local chromatin opening or closing [ ] . the enrolment of co-regulators is also important in the tissue specific and cell-specific regulation of differentiation. an example is the skin were the change of different , (oh) d/vdr genes controlling the process of differentiation of the keratinocytes is regulated by the subsequent recruitment of different co-regulators of the mediator complex family (med) in the early stage and steroid receptor co-activator (src ) in the later stages [ ] . vdr seems to interact with more than nuclear proteins, and deeply affects chromatin remodeling [ ] . vdr has also been detected in a different subcellular location, namely as a transmembrane receptor which appears to be activated by vitamin d analogues with a different configuration ( scis), if compared to those that activate the nuclear vdr ( s-trans). transmembrane vdr is one of the receptors by which vitamin d exerts non genomic, rapid effects on its target cells and tissues. some non-genomic effects of vitamin d, which occur rapidly over minutes or hours, include rapid intestinal absorption of calcium (transcaltachia), secretion of insulin by pancreatic cells, opening of voltagegated ca + and cl-channels in osteoblasts, and the rapid migration of endothelial cells [ ] . the classical role of , (oh) d in calcium/bone metabolism, namely the regulation of intestinal calcium absorption, renal calcium reabsorption and mobilization of calcium and phosphate from bone, has been known for decades and is beyond the aims of the present review. on the other hand, in the last decade a mean of papers per year have been published on vitamin d, pushed by new findings concerning extra-skeletal effects of this hormone [ ] . in this section we will summarize part of these discoveries and the effects of , (oh) d on several districts, particularly the immune system and the cardiovascular system ( figure ) . actions of vitamin d on skin have been summarized in the first paragraph. immune system: vitamin d seems to influence both the innate and the acquired immune system with complex effects, which are still not completely elucidated. we have already outlined that α-hydroxylation produces the active hormone within different cells of immune system, where it exerts autocrine and paracrine effects. a very recent study on vitamin d target genes evaluated and compared all the available transcriptome-wide datasets from human monocytes treated with , (oh) d in vitro [ ] . vdr target genes with a potential pivotal role in the immune response were identified and classified in three groups. group included camp, cd , fn , and trem genes, which have a low basal expression but are highly inducible after , (oh) d/vdr activation and encode proteins involved in the immediate response to infection. in general, effectors of the lps/tlr signaling pathways seem to be coded by these genes. group includes lilrb , lrrc , mapk , sema b, thbd, and themis genes, which are required in the general response to infection. group includes acvrl , cd , cebpb, ninj , srgn, genes involved in long-term autoimmunity mechanisms, which do not require ligand binding to vdr and are likely involved in epigenomic regulation [ ] . mathieu et al. recently reported a comprehensive revision of vitamin d effects on the immune system [ ] . both monocytes and macrophages express vdr, the latter at higher levels than the former. , (oh) d has been shown to stimulate differentiation and proliferation of monocytes, whereas on activated macrophages the overall effect leads to reduced inflammatory response. indeed, , (oh) d stimulates the production of il- (anti-inflammatory) and decreases the release of pro-inflammatory effectors such as il- β, il- , tumor necrosis factor-α (tnfα), receptor activator of nuclear factor kappa-b ligand (rankl), and cyclo-oxygenase- (cox- ) [ ] . the signaling pathways proposed to mediate the anti-inflammatory effect include: (i) upregulation of mapk and mkp and inhibition of lps/p , (ii) modulation of thioesterase superfamily member , with subsequent cox inhibition, (iii) direct antimicrobial effect by cathelicidin antimicrobial peptide (camp) induction, (iv) anti-oxidative effect due to increased glutathione reductase (gr) with drop of reactive oxygen species [ , ] . on the other hand, vitamin d inhibits the acquired immune system, mainly reducing the expression of mhc class ii and co-signaling molecules on antigen presenting cells, decreasing the activity of th and th cells, and up-regulating regulatory t cells. the final result is to promote the regulatory and protective phenotype of t cells [ ] . from a clinical perspective, vitamin d deficiency is strongly associated with increased risk of infections, dysregulation of the immune system and autoimmune diseases [ ] . a specific issue that recently gained a great relevance is the relationship between hypovitaminosis d and pulmonary infections. vitamin d concentration is inversely related to the risk of multiple pulmonary injuries such as pneumonia, community acquired pneumonia, ards, sepsis, heart failure and mortality from pulmonary infections [ ] [ ] [ ] [ ] [ ] . a recent large metanalysis on more than , subjects demonstrated that vitamin d supplementation had a protective role in acute respiratory infections, in adults [ ] . due to these findings, an important role for vitamin d has very recently been suggested in the treatment or prevention of covid- . in march , covid- spread as a pandemic emergence due to the new β coronavirus severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] . in the absence of specific treatments, public health measures are required to characterize risk factors and prevent the infection or the progression of the disease. among the factors that might contribute to the development of severe covid- , vitamin d status was proposed as a credible candidate [ , ] , even if the evidence is still preliminary. at present, the following observations suggest a possible role of vitamin d in reducing sars-cov- risk: (i) the seasonal flare of covid- , which coincides with the nadir of vitamin d levels, (ii) the previously mentioned association between hypovitaminosis d and pulmonary infections, (iii) the anti-inflammatory role of vitamin d which could be of benefit against the so called "cytokine storm", which seems to be a pathophysiological pivotal player in sars-cov morbidity and mortality [ ] . several short reports have been published and are still emerging at the moment of this writing, encouraging to analyze the relationship between vitamin d and covid- [ , , ] . in the setting of autoimmune diseases, there is an interesting association between low levels of vitamin d and increased risk of developing multiple sclerosis (ms). some prospective studies have demonstrated that increasing levels of ohd significantly reduce the risk of ms, among caucasian people [ ] ; moreover, cholecalciferol supplementation associated with interferon β b significantly reduced the activity of the disease as evaluated by mri, compared to interferon β b alone [ ] . cardiovascular system: in the early s, robert scragg proposed the hypothesis that the increase in cardiovascular diseases usually observed in winter might be a result of low ohd levels, due to the reduced sunlight exposure [ ] . this idea turned on a great interest in the potential cardiovascular benefits of vitamin d, leading to several publications over the last ten years. however, the physiological role of vitamin d in the cardiovascular system is still unclear. vdr is expressed in rat and human heart tissue and has a potential role as a modulator of cardiac hypertrophy and failure. this hypothesis is based on the concept that altered intracellular handling of ionized calcium is related to the impaired contractility of the myocardium in heart failure (hf), since , (oh) d is directly involved in calcium-dependent cellular processes, including synthesis of calcium-binding protein, activation of adenylate cyclase, rapid activation of voltage-dependent calcium channels, and modulation of sarcoplasmic reticulum calcium uptake and release [ ] . another possible mechanism is the putative role of , (oh) d as a negative regulator of the renin-angiotensin system (ras). in both normotensive and hypertensive subjects, , (oh) d serum levels are inversely associated with pra (plasma renin activity), suggesting a potential role of vitamin d in hypertension via renin regulation. recent evidence showed that nuclear hormone receptors, including vdr, liver x receptor (lxr) and peroxisome proliferators-activated receptor (ppar), regulate renin gene transcription via specific elements in the renin promoter [ ] . strong support for the involvement of vitamin d in the pathogenesis of cardiovascular diseases comes from vdr knockout mice (vdr −/− ). these mice develop typical signs of hf, including activation of the renin-angiotensin-aldosterone system, cardiac hypertrophy, high blood pressure, and increased levels of atrial natriuretic peptide. furthermore, the development of hypertension in vdr −/− mice can be corrected with the administration of ace inhibitors, only as long as vitamin d levels are sufficient [ ] [ ] [ ] . additional effects of , (oh) d concern the vasculature; indeed, vitamin d can modulate the growth of smooth muscle and endothelial cells and can induce the activation of vasodilatory and antithrombotic genes [ ] . moreover, vitamin d seems to suppress inflammation and to reduce update of oxidized ldl, giving potential vascular benefits. vdr −/− mice show hypercoagulability and atherosclerosis [ , ] . from a clinical point a view, low serum ohd levels have been associated with increased risk of cardiovascular diseases, including hypertension, coronary artery disease, ischemic heart disease, hf, stroke, and type diabetes [ ] [ ] [ ] [ ] [ ] [ ] . convincing data has been produced particularly on the association between hypovitaminosis d and hf, which still is a major public health disease with poor prognosis. a large cohort of patients with hf has been evaluated by gotsman et al. who described the variations of serum ohd during the year and the effect of vitamin d deficiency and supplementation on mortality. in their population, vitamin d deficiency strongly predicted low survival rate and vitamin d supplementation succeeded in reducing mortality [ ] . our group recently reported results from an italian cohort of patients with hf in whom low serum levels of ohd were inversely correlated with a validated score of hf mortality, namely the metabolic exercise cardiac kidney index or mecki score [ ] . in a subgroup of these patients, cardiovascular outcomes have been also reported: patients with hf had mean serum levels of ohd statistically lower than healthy subjects ( . ± . nmol/l vs. . ± . nmol/l, p < . ) and a higher prevalence of vitamin d insufficiency (serum ohd < nmol/l or ng/ml) ( . % vs. . %, p < . ), associated with higher mortality risk [ ] . several meta-analyses on retrospective studies [ , [ ] [ ] [ ] [ ] [ ] [ ] confirmed a consistent association between a low vitamin d status and cardiovascular endpoints (myocardial infarction, hypertension, hf) and/or mortality. on the other hand, randomized controlled trials (rcts) and mendelian randomization studies so far have not succeeded in proving a benefit of vitamin d supplementation. however, it is likely that the latter investigations are affected by some methodological limitations (no clear and homogeneous dosage of vitamin d, no measurement of basal ohd levels, design of the study targeted on skeletal outcome) therefore it is still unclear if vitamin d has a causative role in cardiovascular diseases or is rather a marker of poor health in chronic disease. adipose tissue and glucose/lipid metabolism: recent studies showed an association between low levels of vitamin d and almost all aspects of the metabolic syndrome, namely type diabetes mellitus (t dm), impaired fasting glucose, hypertension, dyslipidemia, obesity, and insulin resistance. therefore, several investigations focused on the role of vitamin d in adipose tissue biology. some studies have shown a negative correlation between vitamin d and leptin or resistin, as well as and an inverse correlation with adiponectin. [ , ] old preclinical studies have demonstrated that a normal activity of pancreatic β cells required adequate levels of vitamin d and functioning vdr [ ] , while more recent studies on vitamin d deficient mice showed impaired glucose-stimulated insulin secretion in pancreatic islets [ ] . a recent review by mathieu supported role of vitamin d in diabetes. indeed, vdr is present in all tissues involved in t and t dm, either in pancreatic islets and immune cells, adipose tissue, liver and muscle. in all these tissues and organs, the machinery responsible for the local production of , (oh) d and its metabolites is also present. moreover, low levels of vitamin d are associated with increased risk of t and t dm and in several animal models vitamin d supplementation improved islets function and insulin sensitivity. however, convincing randomized prospective studies in humans are still lacking and necessary. muscle: it has been ascertained that vitamin d deficiency is responsible for low muscle strength, balance disorders and an increased risk of falls. in a model of vdr null mice, a specific phenotype due to immature muscle-specific genes developed and cardiomyocyte-specific vdr knockout induced cardiac hypertrophy and failure [ , ] . observational studies have confirmed as association between low vitamin d levels and muscle weakness in children and in the elderly, while supplementation improved muscle function as well as energy recovery after exercise [ , ] . cancer: vdr has been shown to be expressed by cancer cell lines and it is hypothesized to play a role in the pathogenesis and progression of cancer. cyp b is also expressed at high level in many cancer cells and tissues and , (oh) d has been proved to have an anti-proliferative effect firstly on myeloma and melanoma cells and subsequently on other cancerous cell lines [ ] . on the other hand, cyp a is overexpressed in cancer and several vitamin d analogues and/or cyp a inhibitors have been tested in preclinical studies [ ] . animal models with vdr knockout do not spontaneously develop neoplasia, however, they have been demonstrated to be more susceptible to develop malignancies under stimulus [ ] and in several studies on different animal models vitamin d analogues slowed cancer progression and the development of metastases [ ] . an interesting example is that of melanomas: , (oh) d and analogues have been clearly showed a beneficial effect as inhibitors of proliferation, plating efficiency and anchorage-independent growth of melanomas cells in vitro experiments and in vivo animal models and have been proposed as potential adjuvant therapies, especially if topically delivered [ , ] . clinical data regarding vitamin d and cancer are more controversial: low vitamin d levels have been associated with a higher risk of cancer, but rcts have not been revealed any significant benefit from vitamin d supplementation. however, international multicenter rcts are still ongoing, specifically designed for the extra-skeletal effects of vitamin d, and they will hopefully shed light on the controversies [ ] . vitamin d has been clearly recognized to be a molecule with several endocrine, paracrine and autocrine effects on multiple tissues and organs, beyond skeletal homeostasis maintenance. research is still very active in this field, with the aim of clarifying many aspects of the complexity of vitamin d and its metabolites. new concepts have emerged in the last years, namely the special role of the skin, the metabolic control of liver hydroxylase cyp r , the specificity of α-hydroxylase in different tissues and cell types and the genomic, non-genomic and epigenomic effects of vdr. many issues need further investigation and many questions are still waiting for answers, which will hopefully become available in the near future. funding: this research received no external funding. the authors declare no conflict of interest. consensus statement from nd international conference on controversies in vitamin d skeletal and extraskeletal actions of vitamin d: current evidence and outstanding questions vitamin d in plants: a review of occurrence, analysis, and biosynthesis. front catalyzed thermal isomerization between previtamin d and vitamin d via β-cyclodextrin complexation vitamin d and evolution: pharmacologic implications sunlight and vitamin d: a global perspective for health enzymatic reduction of the delta bond of -dehydrocholesterol flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways dhcr : a vital enzyme switch between cholesterol and vitamin d production birthday of a syndrome: years anniversary of smith-lemli-opitz syndrome smith-lemli-opitz syndrome determination of the allelic frequency in smith-lemli-opitz syndrome by analysis of massively parallel sequencing data sets age and origin of major smith-lemli-opitz syndrome (slos) mutations in european populations malformation syndromes caused by disorders of cholesterol synthesis vitamin d status in patients affected by smith-lemli-opitz syndrome vitamin d levels in smith-lemli-opitz syndrome genome-wide association study of circulating vitamin d levels common genetic determinants of vitamin d insufficiency: a genome-wide association study cholesterol-mediated degradation of -dehydrocholesterol reductase switches the balance from cholesterol to vitamin d synthesis phosphorylation regulates activity of -dehydrocholesterol reductase (dhcr ), a terminal enzyme of cholesterol synthesis sunscreen photoprotection and vitamin d status target cells for , -dihydroxyvitamin d in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid new aspects of vitamin d metabolism and action-addressing the skin as source and target the biological activities of vitamin d and its receptor in relation to calcium and bone homeostasis, cancer, immune and cardiovascular systems, skin biology, and oral health disruption of vitamin d and calcium signaling in keratinocytes predisposes to skin cancer in vitamin d: fourth edition de-orphanization of cytochrome p r : a microsomal vitamin d -hydroxylase structural analysis of cyp r in complex with vitamin d behavior of human cytochromes p on lipid membranes properties of purified cyp r in a reconstituted membrane environment and its -hydroxylation of -hydroxyvitamin d the angiotensin-converting enzyme /angiotensin ( - )/mas axis protects against lung fibroblast migration and lung fibrosis by inhibiting the nox -derived ros-mediated rhoa/rho kinase pathway genetic evidence that the human cyp r enzyme is a key vitamin d -hydroxylase cyp r mutations impair generation of -hydroxyvitamin d and cause an atypical form of vitamin d deficiency mutation of the cyp r vitamin d -hydroxylase in a saudi arabian family with severe vitamin d deficiency vitamin d-dependent rickets type b ( -hydroxylase deficiency): a rare condition or a misdiagnosed condition? effects of cyp r gene variants on vitamin d levels and status: a systematic review and meta-analysis decreased serum -hydroxyvitamin d in aging male mice is associated with reduced hepatic cyp r abundance obesity decreases hepatic -hydroxylase activity causing low serum -hydroxyvitamin d fasting-induced transcription factors repress vitamin d bioactivation, a mechanism for vitamin d deficiency in diabetes control of hepatic gluconeogenesis through the transcriptional coaotivator pgc- pgc- α, glucose metabolism and type diabetes mellitus cerebrotendinous xanthomatosis: clinical course, genotypes and metabolic backgrounds cyp a mutation causes vitamin d-dependent rickets type evolving concepts in bone-immune interactions in health and disease cloning of human -hydroxyvitamin d- α-hydroxylase and mutations causing vitamin d-dependent rickets type unique biosynthesis by kidney of a biologically active vitamin d metabolite genetic diseases of vitamin d metabolizing enzymes cytochrome p -mediated metabolism of vitamin d effects of leptin on the skeleton differentiation of mouse myeloid leukemia cells induced by α, -dihydroxyvitamin d hypercalcemia in an anephric patient with sarcoidosis: evidence for extrarenal generation of , -dihydroxyvitamin d metabolism of -hydroxyvitamin d by cultured pulmonary alveolar macrophages in sarcoidosis physiologic and pathophysiologic roles of extra renal cyp b : case report and review regulation of renal and extrarenal α-hydroxylase vitamin d metabolism revised: fall of dogmas the measurement of vitamin d metabolites: part i-metabolism of vitamin d and the measurement of -hydroxyvitamin d. hormones hereditary hypercalcemia caused by a homozygous pathogenic variant in the cyp a gene: a case report and review of the literature epimers of vitamin d: a review progress of liquid chromatography-mass spectrometry in measurement of vitamin d metabolites and analogues measuring -hydroxyvitamin d in a clinical environment: challenges and needs -hydroxyvitamin d assays: the quest for accuracy analysis of vitamin d metabolic markers by mass spectrometry: current techniques, limitations of the "gold standard" method, and anticipated future directions mass spectrometric profiling of vitamin d metabolites beyond -hydroxyvitamin d candidate reference measurement procedures for serum -hydroxyvitamin d and -hydroxyvitamin d by using isotope-dilution liquid chromatography-tandem mass spectrometry development of a candidate reference measurement procedure for the determination of -hydroxyvitamin d and -hydroxyvitamin d in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry a candidate reference measurement procedure for quantifying serum concentrations of -hydroxyvitamin d and -hydroxyvitamin d using isotope-dilution liquid chromatography-tandem mass spectrometry standardizing vitamin d assays: the way forward nhanes monitoring of serum -hydroxyvitamin d: a roundtable summary uk food standards agency workshop consensus report: the choice of method for measuring -hydroxyvitamin d to estimate vitamin d status for the uk national diet and nutrition survey vitamin d metabolite profiling using liquid chromatography-tandem mass spectrometry (lc-ms/ms) simultaneous profiling of vitamin d metabolites in serum by supercritical fluid chromatography-tandem mass spectrometry vitamin d binding protein: a historic overview the multifunctional properties and characteristics of vitamin d-binding protein vitamin d is much less effective than vitamin d in humans the free hormone hypothesis: a physiologically based mathematical model megalin-mediated endocytosis of vitamin d binding protein correlates with -hydroxycholecalciferol actions in human mammary cells vitamin d and dbp: the free hormone hypothesis revisited relationship of structure and function of dna-binding domain in vitamin d receptor regulation of gene expression the nuclear vitamin d receptor: biological and molecular regulatory properties revealed vitamin d: not just the bone. evidence for beneficial pleiotropic extraskeletal effects vitamin d receptor signaling and its therapeutic implications: genome-wide structural view . can vitamin d receptor : novel ligands and structural insights epigenome-wide effects of vitamin d and their impact on the transcriptome of human monocytes involve ctcf structural aspects of vitamin d endocrinology regulation of the vitamin d receptor gene by environment, genetics and epigenetics epigenetics, cellular memory and gene regulation nutrigenomics of vitamin d kdm b/jmjd histone demethylase is induced by vitamin d and modulates its effects in colon cancer cells molecular endocrinology of vitamin d on the epigenome level structural considerations of vitamin d signaling minireview: vitamin d receptor: new assignments for an already busy receptor extraskeletal actions of vitamin d key vitamin d target genes with functions in the immune system vitamin d's effect on immune function modulation of inflammatory and immune responses by vitamin d vitamin d action: lessons from vdr and cyp b null mice vitamin d supplementation for the prevention of acute respiratory tract infection: a randomized, double-blinded trial among young finnish men vitamin d status has a linear association with seasonal infections and lung function in british adults serum -hydroxyvitamin d and the incidence of acute viral respiratory tract infections in healthy adults cord blood vitamin d deficiency is associated with respiratory syncytial virus bronchiolitis the association between vitamin d deficiency and community-acquired pneumonia vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data a pneumonia outbreak associated with a new coronavirus of probable bat origin letter: covid- , and vitamin d covid- and vitamin d-is there a link and an opportunity for intervention? evidence that vitamin d supplementation could reduce risk of influenza and covid- infections and deaths letter: covid- and vitamin d-authors' reply serum -hydroxyvitamin d levels and risk of multiple sclerosis a randomised, double blind, placebo controlled trial with vitamin d as an add on treatment to interferon β- b in patients with multiple sclerosis seasonality of cardiovascular disease mortality and the possible protective effect of ultra-violet radiation vitamin d regulation of the renin-angiotensin system heart extracellular matrix gene expression profile in the vitamin d receptor knockout mice , -dihydroxyvitamin d is a negative endocrine regulator of the renin-angiotensin system effects of vitamin d analogs on gene expression profiling in human coronary artery smooth muscle cells disruption of nuclear vitamin d receptor gene causes enhanced thrombogenicity in mice vitamin d and cardiovascular disease prevention circulating levels of hydroxy-vitamin d and risk of cardiovascular disease: a meta-analysis of prospective studies optimal vitamin d supplementation levels for cardiovascular disease protection vitamin d deficiency and risk for cardiovascular disease vitamin d, parathyroid hormone, and cardiovascular events among older adults hypovitaminosis d: a novel risk factor for coronary heart disease in type diabetes? endocrine vitamin d status and cardiovascular outcome vitamin d deficiency is a predictor of reduced survival in patients with heart failure; vitamin d supplementation improves outcome hypovitaminosis d in patients with heart failure: effects on functional capacity and patients' survival vitamin d measurement and effect on outcome in a cohort of patients with heart failure -hydroxyvitamin d levels and risk of ischemic heart disease, myocardial infarction, and early death: population-based study and meta-analyses of and studies vitamin d status and ill health: a systematic review vitamin d and risk of death from vascular and non-vascular causes in the whitehall study and meta-analyses of deaths lower vitamin d status is associated with an increased risk of ischemic stroke: a systematic review and meta-analysis serum -hydroxyvitamin d and the risk of cardiovascular disease: dose-response meta-analysis of prospective studies - vitamin d and mortality: individual participant data meta-analysis of standardized -hydroxyvitamin d in individuals from a european consortium vitamin d and dysfunctional adipose tissue in obesity serum -hydroxyvitamin d levels are inversely associated with systemic inflammation in severe obese subjects the role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro vitamin d and diabetes: where do we stand? is the vitamin d receptor found in muscle? endocrinology cardiomyocyte-specific deletion of the vitamin d receptor gene results in cardiac hypertrophy calcium intake and hip fracture risk in men and women: a meta-analysis of prospective cohort studies and randomized controlled trials relevance of vitamin d in muscle health , -dihydroxyvitamin d and malignant melanoma: the presence of receptors and inhibition of cell growth in culture vitamin d analogues: potential use in cancer treatment function of the vitamin d endocrine system in mammary gland and breast cancer the role of vitamin d in reducing cancer risk and progression on the role of classical and novel forms of vitamin d in melanoma progression and management vitamin d signaling and melanoma: role of vitamin d and its receptors in melanoma progression and management this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -why t ld authors: carneiro, lara; afonso, josé; ramirez-campillo, rodrigo; murawska-ciałowciz, eugenia; marques, adilson; clemente, filipe manuel title: the effects of exclusively resistance training-based supervised programs in people with depression: a systematic review and meta-analysis of randomized controlled trials date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: why t ld the purpose of this study was to systematically review the effects of supervised resistance training (rt) programs in people diagnosed with depression or depressive symptoms. the following databases were used to search and retrieve the articles: cochrane library, ebsco, pedro, pubmed, scopus and web of science. the search was conducted in late june . search protocol required the title to contain the words depression or depressive or dysthymia. furthermore, the title, abstract or keywords had to contain the words or expressions: “randomized controlled trial”; and “strength training” or “resistance training” or “resisted training” or “weight training”. the screening provided results. after the removal of duplicates, records remained. further screening of titles and abstracts resulted in the elimination of papers. therefore, records were eligible for further scrutiny. of the records, nine were excluded, and the final sample consisted of four articles. results were highly heterogeneous, with half of the studies showing positive effects of resistance training and half showing no effects. in two of the four combinations, the meta-analysis revealed significant benefits of rt in improving depressive symptoms (p ≤ . ). however, considering significant differences with moderate (effect size = . ) and small (es = . ) effects, the heterogeneity was above %, thus suggesting a substantial level. to draw meaningful conclusions, future well-designed randomized controlled trials (rcts) are needed that focus on understudied rt as a treatment for depression. data from has revealed that depression is the leading cause of mental health-related disease burden globally, affecting an estimated million people worldwide [ ] . the who declared the sars-cov- (covid- ) outbreak a global pandemic in march , which has affected the lives of , people globally [ ] , and may potentially generate an increase in depressive states as a result of psychosocial stressors like life disruption, fear of illness, or fear of negative economic effects [ , ] . therefore, it is important to understand how to approach depression and depressive symptoms in order to better prepare for an expected increase of people affected by it. the conventional treatment to treat depression is antidepressant medication. besides pharmacotherapy, clinicians recommend cognitive behavior therapy and mindfulness-based therapy [ ] , as well as physical exercise [ ] . thus, treatment guidelines for mental illnesses from leading international organizations now recommend the integration of physical activity-based interventions as part of routine psychiatric care [ ] . exercise promises to be an efficacious treatment for people with depression. indeed, several systematic reviews and meta-analyses have positively assessed the effects of exercise on depression [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, persons with depression are at an increased risk of sedentary behavior [ ] , and exercise contributes to physical health in addition to mental health [ ] . specifically, evidence-based recommendations for the prescription of exercise for patients with major depressive disorders (mdds) propose interventions of - sessions of supervised aerobic and/or aerobic and resistance training exercise of - min duration with moderate intensity per week [ ] , although it has been suggested that the volume of training may be more relevant than frequency [ ] . to achieve optimal outcomes and decrease dropouts, the evidence also indicates that physiotherapists and qualified exercise professionals should lead and supervise physical exercise programs [ ] . even home-based programs, which may occur due to a plethora of reasons (e.g., distance, difficulties with transportation, costs), could benefit from being supervised through the utilization of new technological tools [ ] . this could become more relevant if future scenarios similar to the covid- situation are to be repeated and, therefore, force people to stay at home for prolonged periods of time. the benefits of aerobic exercise (ae) for depression have been well documented; namely, it has been recognized that it promotes neurophysiological effects of which results may be similar to those observed after antidepressant drug treatments [ ] . a meta-analysis of seven randomized controlled trials [ ] showed that there was also good evidence that aerobic exercise over roughly weeks improved cardiorespiratory fitness in people with depression. however, the effects of resistance training (rt) are less investigated, and the literature regarding rt as a stand-alone therapeutic intervention for mdd is limited [ ] . earlier studies that focused on rt for people with depression reported positive effects. however, these must be interpreted with caution due to the heterogeneity of the interventions and poor methodology by reporting several confounding variables that could interfere and introduce the potential for bias. as an example, kim et al. [ ] aimed to investigate the effects of weeks of the rt program on depression in older women and their neurotransmitters. however, the rt intervention was composed of a min warm-up consisting of walking and dynamic stretching, and then a min cool-down with static stretching. therefore, min of the program, % of the session in part i, . % in part ii and . % to % in part iii, were devoted to activities that were not resistance training, and this may have contaminated the data. to date, only one meta-analysis examined the efficacy of rt on depressive symptoms. gordon and colleagues [ ] analyzed papers meeting their inclusion criteria. their review concluded that rt significantly reduced depressive symptoms in adults regardless of health status, the total prescribed volume of rt, or significant improvements in strength. however, in the present systematic review and meta-analysis, which differs from the previous, more strict inclusion and exclusion criteria were defined. namely, we excluded other comorbidities (e.g., parkinson's, alzheimer's, cancer and dementia) and only supervised exclusively resistance training-based interventions, with minimal warm-up activities outside the scope of the main exercise mode. therefore, this systematic review and meta-analysis allows for a more comprehensive assessment of the potential benefits of rt for depression pathology. hence, this systematic review with meta-analysis (srma) addresses one question: how effective is a stand-alone supervised intervention with rt at improving depression? this systematic review and meta-analysis followed the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines and the cochrane collaboration guidelines for the evaluation of risk of bias in randomized studies. the protocol was published in the international platform of registered systematic review and meta-analysis protocols with the number inplasy and doi . /inplasy . . . articles were eligible if they were published or in press in a peer-reviewed journal, with full text in the english language. no limitations were placed regarding publication date, and articles in press were considered. the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines were adopted [ ] . p.i.c.o.s. was established as follows: (i) participants were humans explicitly diagnosed with any form of depression according to established criteria (e.g., the american psychiatric association's [ ] diagnostic and statistical manual of mental disorders-dsm- ® or previous versions, or the world health organization's [ ] international classification of diseases-icd- or previous versions) or those with depressive symptoms above clinical threshold (cutoff values) determined by a validated screening measure (e.g., beck depression inventory-bdi or bdi-ii-by beck et al. [ ] , the hamilton rating scale for depression-ham-d-by hamilton [ ] , the geriatric depression scale-gds-of yesavage [ ] ), but without other major disease (e.g., parkinson's, alzheimer's, cancer, dementia); (ii) only supervised exclusively resistance training-based interventions were considered, with minimal warm-up activities outside the scope of the main exercise mode; comparators were control groups not performing any training protocol and/or supervised contrast groups also performing an alternative exercise program (i.e., yoga, stretching, aerobic exercise); outcomes were any effects on performance, health and quality of life; study design was limited to randomized controlled trials (rcts). the following databases were used to search and retrieve the articles: cochrane library, ebsco, pedro, pubmed, scopus and web of science. the search was conducted in late june . search protocol required the title to contain the words depression or depressive or dysthymia. furthermore, the title, abstract or keywords had to contain the words or expressions: (i) "randomized controlled trial"; and (ii) "strength training" or "resistance training" or "resisted training" or "weight training". no limitations were established for publication date, and in press articles were considered. for cochrane library, only trials were considered. for ebsco, the title and abstract had to be searched separately, and, therefore, we chose to search (i) "randomized controlled trial"; and (ii) "strength training" or "resistance training" or "resisted training" or "weight training" without any limitations in the field. for pubmed, combined search only afforded the selection of title/abstract, not keywords. the same was valid for pedro, besides only affording one field at a time. therefore, multiple searches were needed in pedro. in addition, the criterion of "clinical trial" was selected, therefore automatically excluding practice guidelines and systematic reviews. in web of science, the combination of title, abstract and keywords was termed "topic". the initial screening provided results. after the removal of duplicates, records remained. screening of titles and abstracts resulted in the elimination of papers. this was due to the following reasons: (i) non-scientific production (e.g., protocol registrations without actual research; book chapters, letters, replies); (ii) reviews (e.g., narrative or systematic reviews, meta-analyses); (iii) abstract-only records; (iv) original research where the exercise intervention did not apply resistance training; (v) original research where there was no group exclusively using resistance training; (vi) participants with other major health problems (e.g., cancer, parkinson's disease, dementia, end-stage renal disease); (vii) single group experiments; and (viii) papers unrelated to our topic. therefore, records were considered eligible for further scrutiny, and all were written in the english language. of the records eligible for full-text analysis, nine were excluded. the paper by kim, o'sullivan and shin [ ] was excluded because the warm-up consisted of min of walking and dynamic stretching, and the training session was followed by a min warm-down consisting of static stretching. therefore, min of the program (representing % of the training in part i of the program, . % in part ii and . % to % in part iii) was devoted to activities that were not resistance training, and this may have contaminated the results. similarly, the paper by pereira et al. [ ] included a min warm-up consisting of walking and stretching, thereby meaning that % of the training session duration was not composed of resistance training. in a similar vein, sims et al. [ ] stated that there were warm-up and warm-downs besides resistance training, but there was no reporting of the duration and type of activities performed in these two stages. the paper of teychenne et al. [ ] was excluded because the strength and conditioning group had a mixture of resistance training and aerobic training. the papers of ansai and rebelatto [ ] , chin et al. [ ] , kekäläinen et al. [ ] , lecheminant et al. [ ] , and levinger et al. [ ] were excluded because there was no diagnostic of depression and the average values of the utilized scales for evaluation of depression or depressive symptoms did not reach the cut-off value indicative of depressive symptoms. the final sample consisted of four articles: krogh et al. [ ] , moraes et al. [ ] , sims et al. [ ] and singh et al. [ ] . the process is synthesized in figure . two of the four articles included had more than one main outcome, resulting in the need to perform more than one meta-analysis ( figure ). ja and fmc conducted the initial search and the screening and exclusion process independently. lc later reviewed the entire process. after this stage, the entire process was compared step by step, and disagreements were discussed with all the authors of this manuscript until consensus was achieved. study characteristics: (i) sample size and general characteristics (e.g., age, sex/gender, physical activity habits); (ii) duration and characteristics of the intervention; (iii) adherence rates to the training protocol. primary outcomes: mean change in depressive symptoms in the exercise group assessed by any validated scale, from baseline to post-intervention, in comparison with the mean change of the control and/or comparison groups. if an author reported the results of two outcome measures meeting our criteria (i.e., mean change/pre and post-test change in depressive symptoms according to two different measures), we used the primary outcome chosen by the author. if this was not clear, we used the ham-d or the bdi to increase homogeneity in our results. these outcome measures were also prioritized since they were commonly used in the exercise and depression literature [ ] . secondary outcomes: (i) physical (e.g., performance tests, body composition, perceived exertion); (ii) psychosocial (e.g., body image and appearance, reporting of positive or negative feelings, self-esteem, cognitive evaluations, memory and concentration tasks). the revised cochrane risk-of-bias tool for randomized trials (rob ) was applied to evaluate the individual studies, considering its five dimensions: bias arising from the randomization process, bias due to deviations from intended interventions, bias due to missing outcome data, bias in the measurement of the outcome and bias in the selection of the reported results. ja and fmc completed the risk-of-bias evaluation independently. after completion of the first coding, the figures were compared, and all disagreements were discussed with all authors of the manuscript and reanalyzed until consensus was achieved. the analysis and interpretation of results in this srma were only conducted if at least three study groups provided baseline and follow-up data for the same measure [ ] . means and standard deviations for a measure of pre-post rt interventions were converted to hedges' g effect size (es). the inverse-variance random-effects model for meta-analyses was used because it allocated a proportionate weight to trials based on the size of their standard errors [ ] and enabled analysis while accounting for heterogeneity across studies [ ] . the ess were presented alongside % confidence intervals (cis) and interpreted using the following thresholds [ ] : < . , trivial; . - . , small; > . - . , moderate; > . - . , large; > . - . , very large; > . , extremely large. the analyses were carried out using the comprehensive meta-analysis program (version ; biostat, englewood, nj, usa). to estimate the degree of heterogeneity between the included studies, the percentage of total variation across the studies due to heterogeneity was used to calculate the i statistic [ ] . low, moderate and high levels of heterogeneity corresponded to i values of < %, - % and > %, respectively [ ] . finally, the extended egger's test [ ] was used to assess the risk of bias across the studies. in case of bias, a sensitivity analysis was conducted. as described in the methods, the initial search provided articles, of which remained after the removal of duplicates. screening delivered articles eligible for full-text analysis, after which nine papers were excluded with reasons that were previously explained. four articles were included in the qualitative synthesis and meta-analysis. table presents the general data items for the individual studies. the article by krogh, saltin, gluud and nordentoft [ ] was an rct with patients diagnosed with unipolar depression according to the icd th revision. the sample of patients ( . % of which were women) was by far the largest among the included articles, potentially affording greater confidence in terms of the generalizability of the results. furthermore, it was the longest trial, with weeks of intervention. three randomized groups of patients each were formed: (i) rt; (ii) aerobic training; and (iii) relaxation, which had characteristics that approximated it to a true control group. primary outcomes were assessed with the hamilton scale, and there were no changes after the -week intervention. there were also no changes in secondary outcomes, such as quality of life and cognitive abilities. however, the resistance training group improved in repetition maximum ( rm) testing, while the aerobic group improved in maximal oxygen uptake. most importantly, there were reduced percentages of days absent from work in the rt group. the lack of broader and better results may, however, be due to very poor adherence rates to the training programs, with the top value of . % being attributed to the rt group. table synthesizes the details concerning the rt protocols, table synthesizes the parallel group protocols, while table synthesizes the results for primary outcomes. exercise did not change primary outcomes, but rt reduced absences to work. rt group improved in rm chest press, while at group improved in maximal oxygen uptake. no effect on cognitive abilities. moraes et al. [ ] randomized trial with three exercise groups as adjunct treatments to pharmacotherapy (antidepressants and anxiolytics) for persons diagnosed with major depressive disorder (mdd) according to dsm-iv, not engaged in physical exercise outside of the treatment setting. patients were over years old and sedentary for more than months. exclusion criteria: psychiatric comorbidities, score > points in ham-d, score < on the mini-mental state examination, cerebrovascular infarction, neurodegenerative disease, severe cardiovascular disease, illiteracy, poor mobility, balance disorders, and severe deficits in visual and/or auditory function. a -week intervention. resistance training (n = ). aerobic training (n = ). low intensity exercise control (n = ). all patients had a minimum of % attendance rate. none. rt and at groups showed significant reductions in depressive symptoms in both scales compared to controls, therefore improving upon the efficacy of pharmacological treatment only. authors report the rt group had lower depression scores after the intervention, but not at the -month follow-up. however, the rt group already had much lower depression scores at baseline. rt group improved significantly in strength, but ultimately there were no significant changes in ces-d from pre-to post or at follow-up. singh et al. [ ] randomized controlled trial with adults ( women and men, > years old) with major or minor depression or dysthymia, determined through dsm-iv, and who also had gds score ≥ . exclusion criteria: dementia, folstein mini-mental state examination score ≤ , medical contraindications for exercise, bipolar disorder, active psychosis, perceived suicidal tendencies, currently seeing a psychiatrist, prescribed antidepressant drugs in the previous months, or participating in any exercise training more than twice a week.an -week intervention. high intensity rt (n = ). eighteen completed the study. low intensity rt (n = ). seventeen completed the study. controls (n = ). nineteen completed the study. there were six drop-outs. of those who completed the study, adherence rates were > %. ham-d . gds. physical outcomes: rm chest press, upright row, shoulder press, leg press, knee extension and knee flexion. moraes, silveira, oliveira, matta mello portugal, araújo, vasques, bergland, santos, engedal, coutinho, schuch, laks and deslandes [ ] performed a -week, three-armed randomized intervention with patients diagnosed with mdd according to dsm- , and all taking medication, with the three groups (i) rt; (ii) aerobic training; and (iii) low-intensity exercise control. the small sample, especially the small number of patients per group, advises caution when generalizing the results. the adherence to the intervention was solid, with all patients engaging in ≥ % of the training sessions. after the intervention, both exercise groups outperformed the controls, showing significant reductions in both the hamilton scale and the beck depression inventory, suggesting that exercise improves the efficacy of exclusively pharmacological interventions. no secondary outcomes were assessed. in their investigation, sims, galea, taylor, dodd, jespersen, joubert and joubert [ ] investigated stroke survivor patients (stroke > months before the investigation) diagnosed with depressive symptoms, but that were otherwise healthy at the beginning of the intervention, including in cardiovascular terms (see exclusion criteria in table ). the -week program compared an rt group with a non-exercise control group. adherence was solid, with an average of % attendance rate to training sessions. the authors reported lower depression scores in the rt group after the -week intervention, but not after the -month follow-up. this should be interpreted as a normal detraining effect; after all, the patients exercised for weeks, but later went through a detraining process lasting weeks. however, even the short-term comparisons after the -week intervention should be taken with a grain of salt, as the differences in the centre for epidemiologic studies for depression scale (ces-d) were already significant in baseline testing, with the exercise group presenting lower scores even before the intervention had started. there were no changes in secondary outcomes, such as quality of life, social support, self-esteem and other psychosocial assessments. unsurprisingly, the rt group improved in rm strength testing. finally, singh, stavrinos, scarbek, galambos, liber and singh [ ] selected adults with some form of depression or depressive symptoms and randomized them into a high-intensity rt group, a low-intensity rt group and a control group. adherence rates to the -week program were excellent, with over % average attendance rate to the training sessions. primary outcomes were assessed using the hamilton scale and gds, and showed improvements in depressive symptoms for both experimental groups. interestingly, this was the only study analyzing a dose-response relationship, and the improvements observed in the high-intensity group were much superior to those registered in the low-intensity group. however, the differences between the two experimental groups may have been exaggerated due to an excessive dissimilarity in exercise intensities. firstly, one group worked with % rm, while the other was limited to % rm, which was already a very significant difference. most importantly, though, while the % rm group was regularly re-tested to keep the loads adjusted at %, the % rm group kept the initial loads. therefore, with adaptation to training, it is possible that by the later weeks, the low-intensity group was actually working with - % rm. additionally, this study showed that strength gains were associated with a decrease in depressive symptoms. the high-intensity group also experienced greater increases in quality of life. risk of bias was assessed using cochrane's rob (see table ). risk of bias arising from the randomization process was low for the articles by krogh, saltin, gluud and nordentoft [ ] and singh, stavrinos, scarbek, galambos, liber and singh [ ] , but there were concerns with the other two papers [ , ] . risk of bias due to deviations from intended interventions (effect of assignment to intervention) was low for all articles. risk of bias in the effect of adhering to intervention was high in the paper of krogh, saltin, gluud and nordentoft [ ] , but low for the remaining articles. all articles had a low risk of bias due to missing outcome data. risk of bias in measurement of the outcome was low for the paper of krogh, saltin, gluud and nordentoft [ ] , but there were some concerns with the other three papers. finally, risk of bias was uniformly low for the selection of the reported results. out of six dimensions, the article by krogh, saltin, gluud and nordentoft [ ] had a high risk of bias in one dimension and low risk of bias in the remaining dimensions. the article by singh, stavrinos, scarbek, galambos, liber and singh [ ] presented some concerns in one dimension, but low risk for the others. finally, the articles by moraes, silveira, oliveira, matta mello portugal, araújo, vasques, bergland, santos, engedal, coutinho, schuch, laks and deslandes [ ] and sims, galea, taylor, dodd, jespersen, joubert and joubert [ ] raised some concerns in two dimensions, but had a low risk of bias in the others. two of the articles [ , ] had two primary outcomes, which required different combinations resulting in four separate meta-analyses (figures - ) . the relaxation group in the article by krogh, saltin, gluud and nordentoft [ ] was considered a control group for practical purposes. first, the exercises were relaxation-based and with extremely low intensity, and therefore did not fit into recognizable categories of training programs. furthermore, adherence rates to the sessions was < %, meaning that this experimental group behaved very similarly to a true control group. the following results represent the possible combinations for detecting the effects of rt in primary outcomes related to depressive symptoms. in the first combination, four randomized-controlled studies provided data for depressive symptoms, involving five experimental and four control groups (pooled n = ). there was no significant effect of resistance training on depressive symptoms (es = . ; % ci = − . to . ; p = . ; i = . %; egger's test p = . ; figure ). the relative weight of each study in the analysis ranged from . % to . %. in the second combination, four randomized-controlled studies provided data for depressive symptoms involving five experimental and four control groups (pooled n = ). there was a significant effect of resistance training on depressive symptoms (es = . ; % ci = . to . ; p = . ; i = . %; egger's test p = . ; figure ). the relative weight of each study in the analysis ranged from . % to . %. in the third combination, four randomized-controlled studies provided data for depressive symptoms, involving five experimental and four control groups (pooled n = ). there was no significant effect of resistance training on depressive symptoms (es = . ; % ci = − . to . ; p = . ; i = . %; egger's test p = . ; figure ). the relative weight of each study in the analysis ranged from . % to . %. in the fourth combination, four randomized-controlled studies provided data for depressive symptoms, involving five experimental and four control groups (pooled n = ). there was a significant effect of resistance training on depressive symptoms (es = . ; % ci = . to . ; p = . ; i = . %; egger's test p = . ; figure ). the relative weight of each study in the analysis ranged from . % to . %. this meta-analysis is, to the best of our knowledge, the first to examine rcts aimed at measuring the efficacy of exclusively rt supervised programs in people having depression. although multimodal interventions are usually advised, understanding the role of each exercise modality is paramount to understanding the effects and necessity of such a training component. this is highly relevant health-wise, but also to assess whether time should be invested in a given training modality, or if better investments would be applied elsewhere. therefore, the purpose of this study was to systematically review the effects of supervised resistance training programs in people diagnosed with depression or depressive symptoms. upon retrieval of papers ( after the removal of duplicates), only four papers fulfilled the inclusion criteria, meaning the effects of resistance training on depressive symptoms is still not widely studied, against our expectations. the risk of bias was assessed with cochrane's rob , with only a few selected concerns having arisen, but overall the four analyzed studies had a low risk of bias, and so we believe the reported results are trustworthy. primary outcomes focused on depressive symptoms. here, results were highly heterogeneous, with half of the studies showing positive effects of resistance training and half showing no effects. in two of the four combinations (figures - ) , the meta-analysis revealed significant benefits of rt in improving depressive symptoms (p < . ). although considering significant differences with moderate (es = . ) and small (es = . ) effects, shown respectively in figures and , the heterogeneity was above %, thus suggesting a substantial level [ ] . in fact, in combinations and ( figures and ) , the experiment of sims, galea, taylor, dodd, jespersen, joubert and joubert [ ] was favorable to the control group, and the experiment of moraes, silveira, oliveira, matta mello portugal, araújo, vasques, bergland, santos, engedal, coutinho, schuch, laks and deslandes [ ] largely benefited the rt group. however, no experimental group experienced a worsening of symptoms as a result of resistance training. the reason different combinations lead to different results appears to be a factor of analysis. it is important to note that several rating scales were used to assess the severity of depression in research and clinical settings. these measures were categorized as clinician-rated, such as the ham-d, montgomery Åsberg depression rating scale (madrs) [ ] or quick inventory of depression symptomatology clinician rating [ ] , and self-reported scales, such as bdi and its revised version bdi-ii, and patient health questionnaire- [ ] . in this study, as a primary outcome, ham-d ( -item version), bdi, ces-d and gds were used. nevertheless, other studies utilized different assessment measures for their different interests. choosing appropriate outcome measures is a fundamental component of any assessment. therefore, selecting outcome measures could be considered as a compromise between factors. for example, depression measures should be selected based on the patient population. indeed, depression occurs in children, adolescents, adults and the elderly. as a matter of example, gds was specifically designed to screen and measure depression in geriatric patients. it contains forced-choice "yes" or "no" questions, a format that is helpful for individuals with cognitive dysfunction [ ] . although various scales for rating depression severity have been developed to date, the ham-d ( -item version) is the most regularly used clinician-rated scale in research and clinical settings. originally published by max hamilton in , the first version of the ham-d, the ham-d , comprised items [ ] . hamilton recommended, nonetheless, to use only the first items of the ham-d since the last four symptoms (i.e., diurnal variation, depersonalization, derealization, paranoid and obsessional/compulsive symptoms) were either not considered part of the illness, or they were relatively infrequent or not considered features related to depression severity [ ] . on the other hand, the bdi is one of the most widely used self-rating scales. thus, both the ham-d and the bdi/bdi-ii are frequently adopted as the primary outcome to assess depression severity in this scope [ ] . in this line, it should be important to know what a given total score or a change score from baseline on one scale means in relation to the other scale. this notion is supported by a review carried out by furukawa et al. [ ] , these results can help clinicians interpret the ham-d or bdi scores of their patients in a more versatile way and also help clinicians and researchers assess such scores reported in the literature when scores on only one of these scales are provided. these dates were obtained from the rcts of psychological and pharmacological treatments for major depressive disorders. however, despite being extensively used, the clinician-administered ham-d [ ] has been identified to present various psychometric problems, including lack of unidimensionality and poor ability to detect changes among persons with mild to moderate depressive symptoms [ ] . these problems may be particularly relevant to studies investigating the antidepressant effects of exercise because exercise is a treatment that is particularly recommended for individuals with mild to moderate symptom severity. consequently, the use of the ham-d scale may not accurately reflect the magnitude of the antidepressant effect of exercise [ ] . another key point to address is related to a clear definition in the rcts, which is the scale, aimed at measuring the main outcome. therefore, it is crucial to provide clear information about it. frequent omissions of key details, namely on the primary outcome and the scale used to measure the main outcome, may impair interpretability, replicability and synthesis of rcts that could interfere with decision-making. in the first meta-analysis [ ] , which examined the efficacy of rt on depressive symptoms, there was wide variability in the scales used to measure depression, including beck depression inventory another important topic is how to better frame the primary outcomes. it is very important to analyze adherence rates (i.e., compliance with training sessions). these were excellent in three of the four papers, but were very low in the study of krogh, saltin, gluud and nordentoft [ ] . therefore, despite being the longest study and having the greatest sample size, the very low adherence rates may have compromised the results of the interventions. dropouts from exercise treatment represent a huge barrier that impedes individual benefit from an intervention, and appear to be a particular problem in persons with depression [ ] . in this line, stubbs, vancampfort, rosenbaum, ward, richards, soundy, veronese, solmi and schuch [ ] conducted the first systematic review and meta-analysis which explored the incidence and predictors of dropout rates among adults with depression participating in exercise rcts. these authors found that in mdd patients, higher baseline depressive symptoms predicted an increased dropout. some strategies may serve as facilitators to reduce the impact of dropouts in those with mdd, namely sessions supervised by physiotherapists and exercise physiologists. moreover, schuch, vancampfort, richards, rosenbaum, ward and stubbs [ ] identified that, in people with depression who have higher social support, the likelihood of having symptom improvements in response to exercising increases. additionally, incorporating a motivational component into exercise interventions for depression may be needed to decrease dropouts. vancampfort et al. [ ] found that autonomous motivation (i.e., acting out of choice and pleasure) was the key to adopting and maintaining physical activity behavior in patients with severe mental disorders defined as schizophrenia, bipolar disorder or mdd. additionally, understanding that exercise is not a "one size fits all" intervention leading to immediate results is a key step to achieving progress [ ] . at a certain point, [ ] state that the american college of sports medicine's guidelines suggest three weekly sessions, and lament the fact that their study could only provide two weekly sessions. however, as became apparent, even those two sessions had very low compliance rates. the classic study of dunn, trivedi, kampert, clark and chambliss [ ] assessed the dose-response relationship between frequency of activity (three or five times per week) and the amount of exercise based on energy expenditure ( or . kcal/kg/week). the results demonstrated that the most important factor for decreasing depressive symptoms in people with mild to moderate depression seems to be the amount/dose of activity instead of frequency. with these populations, a home-based supervised protocol could help. indeed, home-based exercise programs may be an effective method to overcome barriers to exercise and increase exercise adherence. as a matter of example, blumenthal, babyak, doraiswamy, watkins, hoffman, barbour, herman, craighead, brosse, waugh, hinderliter and sherwood [ ] randomized individuals into four groups for weeks of either home-based aerobic exercise, supervised group exercise, taking a placebo pill or taking sertraline. the authors concluded that individuals receiving active treatments tended to achieve higher remission rates than the placebo group: supervised exercise = %; home-based exercise = %; medication = %; placebo = % (p = . ). however, further research with larger sample sizes is needed to confirm adherence and efficacy for improving depressive symptoms. in any case, home-based exercise may be a good alternative to practicing physical activity, in particular for vulnerable people. even in the other three studies, which had very good adherence to the programs, no study had more than two weekly training sessions. this may be a manifestly low frequency, perhaps insufficient to promote more expressive gains. it should also be highlighted that the study of singh, stavrinos, scarbek, galambos, liber and singh [ ] presented the greater improvements in primary outcomes, despite being the shortest trial of only weeks. the adherence rate of > % presence in each training session can potentially explain this success. therefore, training programs should be designed in a motivating manner to ensure high adherence rates, especially if only two weekly training sessions are performed. motivation is a crucial predictor of success and a critical factor in supporting sustained exercise. concerning secondary outcomes, only one study failed to assess physical outcomes [ ] . this is unfortunate because the other three studies showed that even when depressive symptoms do not ameliorate, there are still benefits in terms of strength and cardiovascular response, which is positive in itself. several reviews and studies have shown that people with severe mental illness, including people with mdd, have an excess mortality, being two or three times as high as those within the general population, and this excess mortality is mainly due to physical disease [ ] . in this sense, and as an example, the rate of type ii diabetes mellitus in individuals with mdd is around %, representing an increase of around % in comparison to the rate achieved for people without mdd [ , ] . a meta-analysis [ ] revealed that individuals with depression could achieve clinically relevant improvements in cardiorespiratory fitness levels in response to exercise interventions. furthermore, previous studies have found that exercise can improve physical and psychological domains of quality of life in individuals with severe mental illness [ ] , and specifically in people with depression [ ] . thus, exercise may act in these two directions, not only by improving symptoms of depression but also by improving the cardiovascular health and wellbeing of this vulnerable population. additionally, none of the four articles reported the rest times between exercises, although this a highly important and well-recognized variable to consider when prescribing rt. indeed, when prescribed appropriately with other key prescriptive variables (i.e., volume and intensity), the amount of rest between sets could influence the efficiency, safety and effectiveness of an rt intervention [ ] . even if rest times were self-regulated in the studies, that should have been reported. relatively to psychosocial variables, effects on cognitive abilities and quality of life were scarce and heterogeneous. moreover, only one study tested for a dose-response effect [ ] , with the high-intensity group having experienced not only the greatest improvements in depressive symptoms, but also in the quality of life. this raises the necessity of designing training programs that avoid excessively low intensities. however, despite the evidence which supports a dose-response relationship, people with depression typically have low cardiorespiratory fitness, so it seems to be idealistic to start with high volumes or intensities of exercise. literature within this scope has shown that intensity is not critically significant for symptom management, and that exercise of even a light intensity can lead to short-term improvements in mood in this specific population [ ] . for example, people with severe mental illness may face additional challenges towards exercise such as inexperience with intense physical efforts, associated fatigue and discomfort, increased risk of physical injuries, limited availability of physical activity facilities and specialized equipment, and costs associated with access to facilities or training [ ] . moreover, other barriers may include psychiatric medication side effects (e.g., sedation, fatigue, weight gain), lack of motivation and low self-confidence [ ] . lastly, recommendations for the prescription of exercise for patients with mdd should not be discarded. small, incremental improvements can be obtained through real-life interventions aimed at improving the health of people with severe mental illness [ , ] . the existence of only four articles fulfilling inclusion criteria is daunting and reveals that this field has potentially not been adequately investigated. moreover, the considerable heterogeneity of the studies (i.e., sample size and characteristics, experimental protocols, evaluated parameters, and so on) makes comparisons difficult and conclusions tentative. also, only one study evaluated a dose-response effect [ ] . furthermore, it should be underlined that the four studies referred to samples from brazil, denmark and australia. therefore, out of the entire american continent, only south america is represented (and only by one country), while europe also has a single representative. no data on the topic is available from asia and africa. this is troublesome, as the prevalence of depression may vary depending on a country's human development index [ ] . patel et al. [ ] carried out a systematic review and meta-analysis, which included studies on developed countries. approximately two-thirds of all studies and five out of six longitudinal studies showed a statistically significant positive relationship between income inequality and the risk of depression. moreover, because societal context is paramount for understanding access, motivation and engagement with structured physical activity [ , ] , cultural specificities may be important in understanding why, how and how often people have access to and engage with supervised exercise programs [ , ] . in the case of depression, country of origin, ethnicity and cultural differences are suspected of playing a major role in the patients' symptoms and on the behaviors of their family aggregates [ ] . in summary, the majority of interventions in this specific scope have been focused on aerobic exercise, and it is perhaps time to change the paradigm and invest in more research to assess the effects of rt in treating depression. notwithstanding, to achieve this aim, it is essential to be rigorous and first assess the efficacy of exclusively rt supervised programs; otherwise, it may lead to misinterpretations. additionally, it is crucial for those involved with the prescription of rt, namely exercise physiologists, to acquire an understanding of the program variables (e.g., loading and volume, exercise selection and order, rest periods, frequency) and the importance of their application [ ] . overall, to draw meaningful conclusions, future well-designed rcts are needed that focus on understudied rt as a treatment for depression. addressing the burden of mental, neurological, and substance use disorders: key messages from disease control priorities the outbreak of coronavirus disease (covid- )-an emerging global health threat anxiety and depression among general population in china at the peak of the covid- epidemic the psychological impact of the covid- epidemic on college students in china mental health strategies to combat the psychological impact of covid- beyond paranoia and panic is the comparison between exercise and pharmacologic treatment of depression in the clinical practice guideline of the american college of physicians evidence-based? epa guidance on physical activity as a treatment for severe mental illness: a meta-review of the evidence and position statement from the european psychiatric association (epa), supported by the international organization of physical therapists in mental health (ioptmh) effects of yoga on depressive symptoms in people with mental disorders: a systematic review and meta-analysis exercise for depression effects of exercise on depression and anxiety in persons living with hiv: a meta-analysis comparison among aerobic exercise and other types of interventions to treat depression: a systematic review exercise parameters in the treatment of clinical depression: a systematic review of randomized controlled trials physical exercise and clinically depressed patients: a systematic review and meta-analysis exercise as a treatment for depression: a meta-analysis adjusting for publication bias physical activity and sedentary behavior in people with major depressive disorder: a systematic review and meta-analysis exercise treatment for depression: efficacy and dose response dropout from exercise randomized controlled trials among people with depression: a meta-analysis and meta regression cost-effectiveness of supervised versus unsupervised rehabilitation for rotator-cuff repair: systematic review and meta-analysis exercise and pharmacotherapy in the treatment of major depressive disorder exercise improves cardiorespiratory fitness in people with depression: a meta-analysis of randomized control trials can weeks strength training reduce feelings of depression and increase neurotransmitter in elderly females? association of efficacy of resistance exercise training with depressive symptoms: meta-analysis and meta-regression analysis of randomized clinical trials preferred reporting items for systematic reviews and meta-analyses: the prisma statement diagnostic and statistical manual of mental disorders (dsm- ® ) who. international classification of diseases for mortality and morbidity statistics an inventory for measuring depression rating depressive patients geriatric depression scale effects of physical exercise on plasma levels of brain-derived neurotrophic factor and depressive symptoms in elderly women-a randomized clinical trial exploring the feasibility of a community-based strength training program for older people with depressive symptoms and its impact on depressive symptoms general strength and conditioning versus motor control with manual therapy for improving depressive symptoms in chronic low back pain: a randomised feasibility trial effect of two physical exercise protocols on cognition and depressive symptoms in oldest-old people: a randomized controlled trial effects of resistance and all-round, functional training on quality of life, vitality and depression of older adults living in long-term care facilities: a 'randomized effects of a -month resistance training intervention on quality of life, sense of coherence, and depressive symptoms in older adults: randomized controlled trial. qual effect of resistance training on body composition, self-efficacy, depression, and activity in postpartum women resistance training improves depressive symptoms in individuals at high risk for type diabetes the demo trial: a randomized, parallel-group, observer-blinded clinical trial of strength versus aerobic versus relaxation training for patients with mild to moderate depression is strength training as effective as aerobic training for depression in older adults? a randomized controlled trial assessing the feasibility of a strength-training program to enhance the physical and mental health of chronic post stroke patients with depression a randomized controlled trial of high versus low intensity weight training versus general practitioner care for clinical depression in older adults is muscular fitness associated with future health benefits in children and adolescents? a systematic review and meta-analysis of longitudinal studies analysing data and undertaking meta-analyses the cochrane collaboration a re-analysis of the cochrane library data: the dangers of unobserved heterogeneity in meta-analyses progressive statistics for studies in sports medicine and exercise science measuring inconsistency in meta-analyses bias in meta-analysis detected by a simple, graphical test a new depression scale designed to be sensitive to change qids-sr): a psychometric evaluation in patients with chronic major depression the phq- : validity of a brief depression severity measure development of a rating scale for primary depressive illness translating the bdi and bdi-ii into the hamd and vice versa with equipercentile linking randomized clinical trials underestimate the efficacy of antidepressants in less severe depression a critical review of exercise as a treatment for clinically depressed adults: time to get pragmatic dropout from individual psychotherapy for major depression: a meta-analysis of randomized clinical trials autonomous motivation is associated with the maintenance stage of behaviour change in people with affective disorders integrating physical activity as medicine in the care of people with severe mental illness physical illness in patients with severe mental disorders. i. prevalence, impact of medications and disparities in health care metabolic syndrome and metabolic abnormalities in patients with major depressive disorder: a meta-analysis of prevalences and moderating variables diabetes mellitus in people with schizophrenia, bipolar disorder and major depressive disorder: a systematic review and large scale meta-analysis. world psychiatry off physical activity interventions for people with mental illness: a systematic review and meta-analysis exercise improves physical and psychological quality of life in people with depression: a meta-analysis including the evaluation of control group response rest interval between sets in strength training chapter -exercise for the prevention and treatment of depression. in exercise-based interventions for mental illness exercise for adults with depressive symptoms: beyond the weight loss paradigm why moving more should be promoted for severe mental illness prevalence of depression in the community from countries between and income inequality and depression: a systematic review and meta-analysis of the association and a scoping review of mechanisms. world psychiatry off neighborhood social cohesion as a mediator of neighborhood conditions on mothers' engagement in physical activity: results from the geographic research on wellbeing study longitudinal examination of social and environmental influences on motivation for physical activity structural and cultural factors influencing physical activity in switzerland socio-cultural determinants of physical activity across the life course: a 'determinants of diet and physical activity' (dedipac) umbrella systematic literature review cultural differences in the development and characteristics of depression designing resistance training programmes to enhance muscular fitness: a review of the acute programme variables this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord- - vpehvf authors: overbeck, silke; rink, lothar; haase, hajo title: modulating the immune response by oral zinc supplementation: a single approach for multiple diseases date: - - journal: arch immunol ther exp (warsz) doi: . /s - - - sha: doc_id: cord_uid: vpehvf zinc is required for multiple cellular tasks, and especially the immune system depends on a sufficient availability of this essential trace element. during the last decades, many studies attempted to affect the outcome of various diseases by zinc supplementation. these efforts either aimed at supporting immunity by zinc administration or at correcting a loss of zinc secondary to the disease to restore the zinc-dependent functions of the immune system. this review aims to summarize the respective findings and to discuss possible molecular mechanisms by which zinc could influence viral, bacterial, and parasitic infections, autoimmune diseases, and the response to vaccination. zinc supplementation in diseases such as diarrhea, chronic hepatitis c, shigellosis, leprosy, tuberculosis, pneumonia, acute lower respiratory infection, and leishmaniasis seems beneficial. in contrast, the results for the common cold and malaria are still not conclusive, and zinc was ineffective in most vaccination and rheumatoid arthritis studies. for aids and type diabetes, zinc supplementation may even be a risk factor for increased mortality or deterioration of the glucose metabolism, respectively. in these cases, zinc supplementation should be used with care and limited to clearly zinc-deficient individuals. in , the severe consequences of zinc deficiency in humans were first described by prasad et al. [ ] . since then, large parts of the molecular basis for the essentiality of zinc have been identified. it was shown that it is a component of more than enzymes from all six classes [ ] , where it acts as a catalytic, cocatalytic, structural, or regulatory ion. zinc-dependent biological functions include dna replication [ ] , rna transcription [ , , ] , signal transduction [ ] , enzymatic catalysis [ ] , redox regulation [ ] , cell proliferation [ , , , ] , cell differentiation [ ] , and apoptosis [ , ] . in light of these observations it is easy to understand that zinc ions are crucial for multiple aspects of the immune system, including the normal development, differentiation, and function of cells belonging to both innate and acquired immunity [ , ] . among the immune cells that are affected by zinc deficiency, t lymphocytes seem to have the highest susceptibility [ ] and are influenced on several levels ( fig. ) . zinc deficiency reduces the number of peripheral and thymic t cells, their proliferation in response to phytohemagglutinin, and the functions of t helper (th) and cytotoxic t cells, but also acts indirectly by reducing the levels of active serum thymulin. on the molecular level, zinc stimulates the autophosphorylation of the protein tyrosine kinase lck by non-covalent interaction with the cytoplasmic tails of cd and cd , leading to t cell activation [ , , ] . as one result, the delayed-type hypersensitivity reaction is usually reduced in zinc-deficient individuals. however, other cells are also affected, leading to reduced antibody production and compromised function of cells of the innate immune system, such as natural killer cell activity, cytokine production by monocytes, and the chemotaxis and oxidative burst of neutrophil granulocytes [ , ] . another important aspect is the interaction between inflammation and zinc. pro-inflammatory cytokines have a direct influence on zinc homeostasis. it has been shown that il- induces the expression of the zinc trans-porter zrt-and irt-like protein (zip) , thereby increasing zinc uptake into hepatocytes [ ] . il- also upregulates the zinc binding protein metallothionein (mt) and increases cellular zinc in hepatocytes [ ] . experiments with mt knockout mice confirmed that during endotoxin-induced inflammation, mt is required for zinc sequestration in the liver, leading to a significant reduction in plasma zinc levels [ ] . hence, during the acute-phase response, hypozincemia is caused by zip-mediated translocation of zinc into the liver and sequestration bound to mt (fig. ) . conversely, zinc affects several aspects of monocyte signal transduction and the secretion of pro-inflammatory cytokines by these cells [ ] , and zinc supplementation has been shown to reduce the production of tumor necrosis factor (tnf)-α and interleukin (il)- β in healthy human subjects [ ] . the clinical manifestations associated with zinc deficiency comprise growth retardation, thymic atrophy, hypogonadism, infertility, dermatitis, delayed wound healing, alopecia, poor pregnancy outcomes, teratology, anorexia, diarrhea, and increased susceptibility to infectious diseases caused by bacterial, viral, and fungal pathogens [ , , ] . those symptoms are hallmarks of the autosomal recessive inheritable disease acrodermatitis enteropathica. it is characterized by low serum zinc levels [ ] . these result from reduced enteral zinc absorption, which is based on a mutation in the slc a gene that encodes the intestinal zinc import protein hzip [ , , ] . successful treatment of all symptoms was achieved by oral zinc supplementation [ , ] , which is still the standard therapy for acrodermatitis enteropathica. in addition to the impressive effects on acrodermatitis enteropathica, which demonstrate the physiological importance of zinc, many studies have been performed investigating the effects of zinc supplementation on other diseases. these studies often gave highly contradictory results. resolving these discrepancies is difficult. a major problem is the different amounts of elemental zinc that were administered, which sometimes differed by more than one order of magnitude. matters are complicated even further by the fact that the metal content varies significantly between different zinc salts, and several zinc supplements even exist in different salt forms (table ) . if this form is not specified, the amount of elemental zinc that has been administered cannot be calculated reliably. furthermore, even if the same supplement is used in various studies, the amount of calculated elemental zinc diverges between different research groups. other studies do not report the zinc status of the patients, and zinc-deficient subjects will react differently to zinc treatment than zinc-sufficient ones. even when data regarding the patients zinc status are provided, the parameters measured in most studies are total serum or plasma zinc. the significance of these values is questionable. while they are well suited to detect severe forms of zinc deficiency, they are not adequate parameters to indicate marginal zinc deficiency [ , ] . another important difference between studies can result from the differential bioavailability of the zinc supplements [ ] . salts with organic anions such as acetate, methionine, or histidine generally have a higher bioavailability than zinc sulfate, while zinc oxide is of lower availability. furthermore, zinc uptake does not only vary between different salt forms, but also depends on the source and manufacturing process of the supplements, particularly with regard to zno [ ] (table ) . inflammation and zinc homeostasis. pro-inflammatory cytokines induce the production of zip and metallothionein (mt). this leads to enhanced zinc transport via zip into liver cells, where it is stored bound to mt. as a consequence of this zinc translocation from plasma into the liver, a significant decrease in plasma zinc occurs during inflammation. on the other hand, zinc affects pro-inflammatory cytokine production by monocytes (m). hence plasma zinc levels can regulate cytokine secretion. solid lines show a direct release or translocation of a substance, while dashed lines indicate a modulation of expression. concerning bioavailability, one has also to consider the nature of the diet consumed by the subjects. the uptake of zinc is more efficient with an intake of animal protein than with a dietary intake predominantly consisting of cereal protein. the absorption of zinc is negatively influenced by zinc-chelating phytates and phosphates as well as by augmented levels of other bivalent cations, such as cu, mg, ca, ni, cd, and fe. on the other hand, it is increased by high amounts of proteins and single amino acids, in particular histidine and methionine [ , ] . in turn, high-dose zinc supplementation can also interfere with the uptake of other nutrients. this has been documented for iron [ ] , and in particular for copper. impaired copper uptake by excessive zinc supplementation induces severe copper deficiency, which can lead to anemia and neutropenia, abrogating the potential bene-ficial effects of zinc therapy [ , , ] . finally, gastric acidity enhances zinc absorption, especially for zinc oxide. this can be a particular problem for the elderly. they have a high incidence of hypo-or achlorhydria [ ] and show a general tendency for lower zinc levels, which leads to an impairment of immune function [ , ] . this review aims to summarize current knowledge about both the beneficial and adverse effects of zinc supplementation on the immune system and to discuss the possible molecular mechanisms by which these effects could be mediated. table specifies different zinc supplementation studies dealing with diseases induced by viral pathogens. the common cold is a syndrome caused by a multitude of different viruses, many of them belonging to the rhino-and coronaviruses. for this disease, the use of zinc has been extensively investigated. the individual studies will not be discussed in detail here because they have already been summarized and compared elsewhere [ , , ] . in these studies, which used . - . mg elemental zinc per single dose, the results are as inconsistent as the treatment conditions. in a review by hulisz [ ] it was concluded that zinc can be effective in reducing the duration of the common cold when administered within h after the onset of symptoms. a recent study in children indicates that zinc may also have a prophylactic effect and the administration of zinc results in a lower mean number of colds [ ] . however, two metaanalyses did not confirm an effectiveness of zinc- -lozenges in reducing the symptoms of the common cold after seven days [ , ] . many potential mechanisms have been suggested that could explain a potential beneficial effect of zinc. these include effects of zinc on viral replication and infection of cells on the one hand and the immune system, in particular cytokine production and modulation of the activity of immune cells, on the other. zinc inhibits the formation of viral capsid proteins and the rhinovirus c protease, thus preventing the replication of rhinoviruses in vitro. however, this remains to be demonstrated in vivo [ , , , ] . alternatively, zinc could interact with the binding of the rhinovirus to the intercellular adhesion molecule- (icam- ), an event that is required for invasion of cells of the nasal epithelium. it was hypothesized that the positively charged zinc ions can bind to the negatively charged regions at the carboxyl termini of rhinovirus coat proteins, thereby preventing the binding to icam- [ , ] . in addition, zinc may protect or stabilize the cell membrane [ ] , which could also contribute to an inhibition of the entry of the virus into the cell. in a similar manner to viral binding of icam- , zinc might also interfere with the binding of leukocyte function-associated antigen- to icam- , thus suppressing inflammation [ ] . z -zinc, p -placebo, c -control. a values are given as the amount of supplement unless indicated otherwise. the elemental zinc content is given as provided in the respective publication and may not always correspond to table . among the immunomodulatory effects of zinc that could counteract viral infections is its influence on the synthesis of cytokines. in vitro, zinc induces the production of antiviral interferon (ifn)-α as well as ifn-γ [ , ] and it can potentiate the antiviral action of ifn-α, but not of ifn-γ [ ] . besides this, clearance of viral infections requires cytotoxic t lymphocytes, which are highly dependent on zinc, as discussed above. other potential mechanisms by which zinc could act against the common cold include an inhibitory effect of zinc on human prostaglandin metabolism [ ] , which may account for the ability of zinc to reduce symptoms of the common cold. finally, a zinc-induced alteration of the capillary epithelium might inhibit transcapillary movement of plasma proteins and reduce local edema, inflammation, exudation, and mucus secretion [ ] . so far it is not known which of these explanations are relevant for the proposed effect of zinc treatment in vivo. the lack of statistical confirmation of effectiveness and the plethora of possible mechanisms illustrate the need for in-depth research, which may allow using zinc with higher efficiency. infection with the human immunodeficiency virus (hiv) results in the acquired immune deficiency syndrome (aids), a disease where zinc application was tested as a supporting therapeutic intervention [ ] . given the importance of zinc for the development and function of t cells [ , ] , this seems to be a sensible approach. the initial study found an increase in hla-dr positive cells, a stimulation of lymphocyte transformation by phytohemagglutinin and concanavalin a, and augmented phagocytosis by polymorphonuclear neutrophils [ ] . a report by mocchegiani et al. [ ] described even more promising beneficial effects of zinc, including an increase in the number of th cells and a reduced frequency of opportunistic infections with pneumocystis jiroveci (formerly p. carinii) and candida. to antagonize a loss in th cells, zinc could either stimulate t-lymphocyte production or enhance their survival. a reason for the former effect may be an action of zinc through thymulin on the maturation of t cells. thymulin is a zinc-dependent nonapeptide hormone [ ] that regulates the differentiation of immature t cells in the thymus [ ] and the function of mature t cells in the periphery [ , ] . on the other hand, an antiapoptotic action of zinc ions [ ] at both the peripheral and thymic level could result in an increase in the number of th cells. it is known that zinc inhibits caspases- , - , and - [ , , , ] . moreover, zinc can increase the bcl- /bax ratio, thus enhancing the cells' resistance to apoptosis [ ] . unfortunately, the results of the supplementation trials are not consistent. in contrast to the observations of mocchegiani et al. [ ] , there was no alteration of the cd /cd ratio in the initial study [ ] and several recent papers were unable to find effects of oral zinc on hiv- viral load, immune response to tuberculosis, lymphocyte subsets, cd + , cd + , and cd + cell counts, or antibody response to a pneumococcal conjugate vaccine [ , , , , ] . in addition, when fawzi et al. [ ] investigated the effects of zinc supplementation on pregnant hiv-positive women, zinc had no effect on pregnancy outcome, but the authors reported lower increases in hemoglobin, red blood cell count, and packed cell volume after the women had given birth. to make matters worse, two studies by tang et al. [ , ] indicated an increased risk for the progression to aids and a lower survival after zinc intake by hiv-positive individuals. one explanation for these contradictory results may be a different zinc status of the patients. while moderate zinc supplementation to zinc-deficient subjects can advance their immune responses, it may have harmful effects when given to zinc-sufficient ones. zinc deficiency is frequent in patients with aids without treatment; however, anti-retroviral therapy has been shown to counteract the zinc deficiency [ ] . hence, zinc supplementation should be seen as a potential hazard to these patients and be strictly limited to individuals with documented zinc deficiency. finally, several studies have investigated the effect of zinc supplementation on hepatitis c, which is induced by an infection with the hepatitis c virus (hcv). after zinc treatment, decreases in the incidence of gastrointestinal disturbances, body weight loss, and mild anemia were found in patients with chronic hepatitis c [ ] . in addition, zinc given in combination with ifn-α was more effective against chronic hepatitis c than a therapy with ifn-α alone [ ] . in addition to the effects of zinc on immune function and antiviral defense discussed above, its role as an antioxidant may be important in hepatitis. oxidative stress is a major contributor to cellular damage during viral hepatitis [ ] . in mice it was shown that zinc enhances the expression of mt in liver tissues [ ] , which can function as a free radical scavenger [ , ] and may prevent oxidative damage to liver tissue [ ] . several in vitro studies indicate that zinc may be able to inhibit viral replication of hcv [ ] , but also herpes simplex virus [ ] and rhinovirus [ ] , all at concentrations of µm zinc ions in the culture medium. for hiv, a concentration of µg/ml (~ . mm) was effective [ ] . all these amounts seem to be relatively high. hence, the in vivo relevance of an inhibition of viral replication as a mechanism for antiviral actions of zinc in humans remains to be demonstrated. different zinc supplementation studies dealing with diseases caused by bacterial pathogens are listed in table . diarrhea can be either of viral or bacterial origin, but because there seem to be no obvious differences in the effects of zinc treatment, we will discuss all forms together in this paragraph. diarrhea is a target for successful zinc treatment. this has been extensively studied, and the results have already been summarized in detail, showing that zinc can reduce the duration, sever-ity, and incidence of diarrhea [ , ] . two pooled analyses were conducted by the zinc investigators' collaborative study group assessing the use of zinc for the prevention or treatment of diarrhea in children in developing countries [ , ] . the combined results of seven trials of continuous zinc supplementation confirmed that zinc significantly reduces the incidence and prevalence of diarrhea [ ] . when zinc was used for the treatment of acute and persistent diarrhea, the probability of continuation was reduced and the rate of treatment failure or death was diminished by %. these results caused the authors to conclude a substantial benefit of zinc supplementation for the treatment of both acute and persistent diarrhea in children [ ] . however, a recent report points out that this may not be the case for infants younger than six months of age [ ] . diarrhea leads to increased intestinal loss and malnutrition with micronutrients, including zinc. this loss can be corrected by oral zinc supplementation, which may improve the absorption of water and electrolytes by the intestine [ , , ] , lead to a faster regeneration of the gut epithelium [ ] , and increase the levels of enterocyte brush-border enzymes [ , ] . finally, the loss of zinc may negatively affect immune function, which can be antagonized by zinc supplementation to improve the clearance of bacterial pathogens from the intestine [ ] . when zinc supplementation was examined in patients with shigellosis, which is induced by different species of shigella, several studies report improvements. these include increased intestinal mucosal permeability, alkaline phosphatase activity and better nitrogen absorption [ ] , but also augmented lymphocyte proliferation in response to phytohemagglutinin and increased antigen-specific igg titers [ ] . in addition, augmented serum antibody titers together with an increase in cd + cells (b cells) and cd + /cd + cells (plasma cells) were observed [ ] . all these effects indicate that the effect of zinc is likely mediated by a modulation of immune function. leprosy is induced by infection with the pathogen mycobacterium leprae. leprosy patients with borderline tuberculoid leprosy, borderline lepromatous leprosy, and lepromatous leprosy have reduced serum zinc levels [ ] . hence, several zinc supplementation studies have been conducted. all of them reported different, but beneficial, effects. one study found a reduction in the required dose of clofazimine, a withdrawal of primarily essential steroids, and an improved toleration of dapsone after zinc treatment. furthermore, they observed a reduced incidence and severity of erythema nodosum leprosum, a gradual decrease in the size of granuloma, and a gradual increase in the number of lymphocytes [ ] . another study reported a decreased incidence of erythema, edema, and infiltration as well as a reduced bacterial index of granuloma. in addition, there was a regrowth of eyebrows and an increase in neovascularization and endothelial cell proliferation [ ] . those beneficial effects of zinc treatment were confirmed by two other studies [ , ] , which detected decreased size of skin nodules, improved delayed hypersensitivity reactions, a disappearance of erythema and a regrowth of eyebrows [ ] as well as improvements regarding frequency, duration, and severity of erythema nodosum leprosum reactions, and a reduction in steroid requirements [ ] . another form of mycobacterial infection is tuberculosis, which is caused by the pathogen mycobacterium tuberculosis and associated with lower serum zinc levels [ ] . here, one study reported an increase in plasma retinol concentration, earlier sputum conversion, and resolution of x-ray lesion areas in response to zinc supplementation [ ] . effective clearance of mycobacterial infections requires a th -mediated activation of infected macrophages by ifn-γ [ ] . studies in mice showed that zinc may improve an imbalance in t cell subpopulations, which reflects a disturbed helper/suppressor cell ratio. here, zinc acts by inducing t cell activation or alteration of lymphokine production, which in turn may activate macrophages to promote bacterial clearance [ ] . zinc can induce the production of ifn-γ in human peripheral blood mononuclear cells [ ] . furthermore, zinc deficiency leads to a th shift, which is mainly characterized by a reduction of il- and ifn-γ [ , , ] . it was also reported that zinc promotes a th immune response by augmenting the gene expression of il- and ifn-γ [ ] . zinc supplementation has also been investigated against acute lower respiratory infection. two studies reported decreased episodes of infection [ ] and increased recovery rates from illness and fever after zinc therapy [ ] , whereby the effect in the latter study was only significant in boys. lower respiratory infection may be caused by different bacterial or viral pathogens, the nature of which was not investigated in these studies. hence it can be concluded that zinc treatment reduces the symptoms, but an effect of zinc on the immune response against the underlying pathogens cannot be concluded from these data. a pooled analysis of four trials in which continuous supplementation was investigated confirmed that zinc is efficient for the prevention of pneumonia. here, zinc supplementation reduced the incidence of pneumonia in children in developing countries by % [ ] . pneumonia is a major factor of childhood mortality; it accounts for approximately % of childhood deaths in developing countries [ ] , making zinc supplementation a promising approach for a significant reduction in childhood mortality. in addition, a recent study indicates that zinc may also be helpful for the elderly. serum zinc concentrations were negatively associated with the incidence of pneumonia in nursing home residents, indicating that zinc supplementation may be a measure to prevent pneumonia in the elderly [ ] . low gastric mucosal zinc concentrations in heliobacter pylori-infected patients were correlated with the severity of inflammation, measured as infiltration by polymorphonuclear cells into the gastric mucosa [ ] . treatment with polaprezinc (zinc l-carnosine), which is used as an anti-ulcer drug in japan, led to an improved cure rate when administered together with antimicrobial triple therapy [ ] . this indicates that zinc could also be effective as an adjunct therapy for the treatment and eradication of h. pylori infection. various zinc supplementation studies dealing with diseases caused by parasites are listed in table . one of these is acute cutaneous leishmaniasis, induced by different forms of leishmania. patients with cutaneous, mucosal, and visceral leishmaniasis display lower plasma zinc levels [ ] . as a result of zinc therapy, a dose--dependent decrease in erythemas and size of induration and an increased cure rate were found [ ] . a direct anti-leishmanial effect was shown for zinc which could be demonstrated in vitro by zinc-induced inhibition of several enzymes from leishmania [ , ] . however, the lowest concentration investigated in the assays was µm, and many inhibitions were only observed at even higher concentrations. therefore, the physiological relevance of these observations may be limited. another area of zinc application is malaria. during acute malaria, plasma zinc levels are reduced and inversely correlate to c-reactive protein, indicating a decrease of zinc as a consequence of the acute-phase response [ ] . there are some studies dealing with the usefulness of zinc supplementation against this disease, with contradictory results. two papers reported beneficial effects, including a reduced incidence in p. falciparum-mediated febrile episodes [ ] and a trend toward fewer malaria episodes [ ] . however, the lastmentioned study was statistically not significant and found no zinc-mediated effect on diarrhea and respiratory infection [ ] . two other studies also found no effect of zinc on the incidence of malaria, but demonstrated that zinc supplementation decreased morbidity from diarrhea [ , ] . in addition to these trials, which focused on the prevention of malaria by zinc supplementation, the therapeutic value of zinc as an adjunct to standard chemotherapy for the treatment of acute malaria has also been investigated in a large multicenter study in ecuador and four african countries [ ] . here, no effect of zinc was found on any of the parameters that were investigated. with regard to a potential mechanism, it seems remarkable that zinc protects against morbidity mediated by p. falciparum, but not by p. vivax. this selectivity indicates that zinc may act on a specific pathogenic process, for example the sequestration of mature parasite-infected erythrocytes in the microvasculature, which is associated only with p. falciparum [ ] . table summarizes zinc supplementation studies in patients who suffer from one of two autoimmune diseases, namely rheumatoid arthritis (ra) and type i or insulin-dependent diabetes mellitus (iddm). when the effect of zinc supplementation on ra patients was investigated, one study detected positive changes after zinc therapy regarding joint swelling, morning stiffness, and walking time [ ] . however, two other studies found no antirheumatic activity of zinc [ , ] . conversely, it was shown that zinc supplementation modulated ex vivo phagocytosis and oxidative burst in phagocytes from ra patients [ , ] . at present, these contradicting data do not allow concluding an effectiveness of zinc for treating ra. patients with ra show reduced serum zinc levels [ , ] , which may be due to a malabsorption of zinc [ ] . a way in which zinc deficiency could affect the pathogenesis of ra is its influence on pro-inflammatory cytokine secretion. in patients with ra, the serum zinc level correlates negatively with levels of tnf-α and il- β as well as parameters for inflammation such as acute-phase proteins and erythrocyte sedimentation. this corresponds to a cellular in vitro model for zinc deficiency in which the levels of pro-inflammatory cytokines such as tnf-α, il- β, and il- were increased [ ] , and to other observations demonstrating that monocytic production of pro-inflammatory cytokines is inhibited by zinc ions [ , ] . iddm is typically accompanied by a loss of zinc due to increased urinary excretion, resulting in a decrease in total body zinc. this secondary zinc deficiency might contribute to diabetic complications [ ] . on the one hand, zinc is necessary for insulin maturation and storage as a solid hexamer bound with two zinc ions per hexamer in insulin secreting pancreatic β cells [ , , ] . on the other hand, zinc deficiency comprises the potential to hinder immune function, interacting with pro-inflammatory cytokine production as discussed above. both effects suggest a potential benefit for zinc as a supporting therapeutic intervention in diabetic patients. besides its action on the immune system, zinc supplementation may have an additional effect on patients with iddm or ra. zinc deficiency is known to induce oxidative stress [ ] , and in both diseases, reactive oxygen species contribute to the pathogenesis [ , ] . the influence of zinc on redox metabolism is well established [ , ] and several mechanisms have been suggested by which zinc can act as an antioxidant. because zinc itself is not redox active in biological systems, its antioxidant function is indirect, for example through the expression of mt [ , ] . moreover, zinc can bind to thiolate groups and protect them from oxidation by lowering their susceptibility to oxidation [ ] . other trace metals can also play a role in ra. excess iron aggravates inflamma-tion [ ] and zinc has been shown to counteract transition metal-mediated oxidation by interfering with the haber weiss cycle [ ] . finally, zinc is important for antioxidant enzymes. it was suggested that zinc may exert an effect on lipid peroxidation by protecting the active site of the selenium-glutathione peroxidase, which is important for detoxification of reactive oxygen species, against the binding of toxic ligands with subsequent inactivation of the enzyme [ ] . in addition, the zinccontaining metalloenzyme cu/zn superoxide dismutase, which catalyzes the degradation of superoxide to hydrogen peroxide, is effective against iddm [ ] and ra [ ] in vivo. how much does the zinc deficiency observed in both types of patients contribute to oxidative damage? although this has not been directly investigated so far, the improvement in parameters for oxidative stress in diabetic patients indicates that the antioxidant effect of zinc is relevant for disease progression in vivo. one study found a positive effect on oxidative stress, measured by an increase in selenium-glutathione peroxidase activity, and a decrease in plasma thiobarbituric acid reactive substances, which are an indicator for lipid peroxidation [ ] . on the other hand, two other studies detected an increase in the glycosylated form of hemoglobin, hba c, indicating a further deterioration of metabolic control [ , ] . taken together, it seems that zinc supplementation can be helpful against oxidative stress, but its effect on glucose metabolism may limit its usefulness in diabetic patients. antibody production during both the first and an immunological memory response is disturbed by zinc deficiency [ , , ] , suggesting that zinc supplementation could improve vaccination results. experiments in mice showed that antibody production in response to t cell-dependent antigens is more sensitive to zinc deficiency than in response to t cell-independent antigens [ ] and zinc deficiency impairs th cell function [ ] . the various effects of zinc supplementation on different forms of vaccination are listed in table . although both the elderly and hemodialysis patients have a high risk for being zinc deficient, there was no influence of zinc on influenza vaccination in either group [ , , ] . conversely, there is one study from which a relationship between zinc status and vaccination response can be concluded. in this report, a correlation between failure to respond to diphtheria vaccination by elderly chronic hemodialysis patients and low serum zinc level was found [ ] . other studies analyzed the effect of zinc on cholera vaccination and had contradictory results. on the one hand, an increase in vibriocidal antibody titers after zinc therapy could be found [ , ] , while on the other, a suppression of antibody formation against cholera toxin was detected [ , ] . so far, only one study has reported an entirely beneficial effect of zinc on vaccination. in contrast to all other studies, the patients started with zinc treatment one month prior to tetanus vaccination, but stopped taking zinc during vaccination. following this treatment the patients showed an increase in the anti-tetanus toxin igg titer and also in the number of circulating t lymphocytes and an improved delayed type hypersensitivity reaction toward several different antigens [ ] . potentially, zinc is required to restore normal th cell function, but because it has a direct inhibitory effect on t lymphocytes [ ] , supplementation during vaccination may hinder efficient vaccination response. it may be a promising approach to investigate the time-and concentration-dependent effect of zinc on vaccination in order to define an optimal treatment protocol. it is well established that zinc status is an essential aspect of an intact immune system. however, in the studies discussed above it could not always be distinguished if zinc acts solely as an immune-modulator or to what extent other functions, for example its antioxidant properties, contribute to an in vivo effect of zinc supple- z -zinc, p -placebo, c -control. a values are given as the amount of supplement unless indicated otherwise. the elemental zinc content is given as provided in the respective publication and may not always correspond to table . fig. . interaction between zinc homeostasis and disease. while many diseases affect zinc homeostasis, the latter can modulate several components of the immune system, but also general metabolic processes such as the production of reactive oxygen species (ros). these can lead to complications such as secondary infections and cellular damage, but also contribute to the initial disease. mentation. due to the clear effects of zinc deficiency and supplementation on numerous immune parameters, especially pro-inflammatory cytokines and t lymphocytes, it can be safely assumed that its effect on the immune system contributes significantly to the results observed in supplementation trials for different diseases. in most cases it is not known to what extent zinc deficiency is causal for a disease or if it occurs secondary to the disease and only contributes to its severity or the occurrence of complications or secondary infections (fig. ) . in any event, zinc supplementation can be effective in correcting both states. therapeutic zinc supplementation in diseases such as acute lower respiratory infection, chronic hepatitis c, diarrhea, shigellosis, leprosy, tuberculosis, and acute cutaneous leishmaniasis is beneficial. unfortunately, these observations cannot be generalized. the results for the common cold and malaria are still not conclusive, and zinc was ineffective in many vaccination trials as well as most ra studies. it is unclear if this can be overcome by changes in dosage or duration. in aids and type diabetes, zinc supplementation may even be a risk factor for increased mortality or deterioration of the glucose metabolism, respectively. in these cases, zinc supplementation should be avoided, or at least limited to clearly zinc--deficient individuals. future work should aim at two aspects. first, by paying more attention to zinc dosage and the patient's zinc status before and during the supplementation, it should be possible to administer the optimal amount of zinc and thereby significantly improve the therapeutic effects. here the standard parameter, i.e. total serum or plasma zinc, does not adequately reflect the patient's zinc status, but the development of new methods such as the use of fluorescent probes for the measurement of labile intracellular zinc [ ] may lead to improvement. secondly, elucidating the molecular mechanisms by which zinc acts will help to provide a successful treatment, in particular when zinc is given in combination with other substances. here an important first step will be evaluating which in vitro observations are relevant. given that many effects can only be seen when supra-physiological concentrations of zinc are used, as in the different cases of the inhibition of viral replication, the likelihood for an in vivo relevance is questionable. there is still great potential for improving the use of zinc as a therapeutic agent and successful application for the modulation of the immune response. rheumatoid arthritis and metal compounds -perspectives on the role of oxygen radical detoxification the assessment of zinc status: a personal view enteric protein loss and intestinal permeability changes in children during acute shigellosis and after recovery: effect of zinc supplementation supplementation with zinc, but not vitamin a, improves seroconversion to vibriocidal antibody in children given an oral cholera vaccine the mechanism behind the antileishmanial effect of zinc sulphate the mechanism behind the antileishmanial effect of zinc sulphate. i. an in-vitro study zinc coordination sphere in biochemical zinc sites zinc modulates mrna levels of cytokines a trial of zinc supplementation in young rural gambian children changes in cytokine production and t cell subpopulations in experimentally induced zinc-deficient humans zinc potentiates the antiviral action of human ifn-alpha tenfold a critical physiological role of zinc in the structure and function of biomembranes functions of zinc in signaling, proliferation and differentiation of mammalian cells prevention of diarrhea and pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized controlled trials. zinc investigators' collaborative group therapeutic effects of oral zinc in acute and persistent diarrhea in children in developing countries: pooled analysis of randomized controlled trials safety and efficacy of zinc supplementation for children with hiv- infection in south africa: a randomised double--blind placebo-controlled trial nutrient regulation of cell cycle progression the physiological role of zinc as an antioxidant. free radic zinc supplementation reconstitutes the production of interferon-α by leukocytes from elderly persons dysregulation between th and th t cell subpopulations in the elderly zinc, insulin and diabetes changes in serum selenium, copper, zinc levels and cu/zn ratio in patients with pulmonary tuberculosis during therapy zinc deficiency and supplementation in hiv/aids interleukin regulates secretion of zinc-thymulin by human thymic epithelial cells and its action on t--lymphocyte proliferation and nuclear protein kinase c hyperzincuria in individuals with insulin-dependent diabetes mellitus: concurrent zinc status and the effect of high-dose zinc supplementation a zinc dependent epitope of the molecule of thymulin, a thymic hormone effect of zinc and vitamin a supplementation on antibody responses to a pneumococcal conjugate vaccine in hiv-positive injection drug users: a randomized trial interference in the development of a secondary immune response in mice by zinc deprivation: persistence of effects effects of zinc supplementation in patients with type diabetes the role of assembly in insulin's biosynthesis zinc and the gene beneficial effects of oral zinc supplementation on the immune response of old people plasma zinc concentrations are depressed during the acute phase response in children with falciparum malaria transcriptional regulation of the mouse metallothionein-i gene by heavy metals bioavailability of zinc in several sources of zinc oxide, zinc sulfate, and zinc metal effect of oral zinc supplementation on the cell mediated immunity in lepromatous leprosy role of zinc in insulin biosynthesis: some possible zinc-insulin interactions in the pancreatic b-cell zinc in developmental biology: the role of metal dependent transcription regulation lipid peroxidation in insulin-dependent diabetic patients with early retina degenerative lesions: effects of an oral zinc supplementation trial of zinc supplements in relation to pregnancy outcomes, hematologic indicators, and t cell counts among hiv- -infected women in tanzania zinc and the risk for infectious disease an essential role for interferon gamma in resistance to mycobacterium tuberculosis infection regeneration of t-cell helper function in zinc-deficient adult mice interrelationships between zinc and immune function zinc deficiency and the immune function reprogramming of the immune system during zinc deficiency zinc suppresses apoptosis of u cells induced by hydrogen peroxide through an increase of bcl- /bax ratio treatment of acrodermatitis enteropathica with zinc sulphate effect of severe zinc deficiency on activity of intestinal disaccharidases and -hydroxy- -methy-glutaryl coenzyme a reductase in the rat in vitro activity of zinc salts against human rhinoviruses serum zinc/copper ratio in subtypes of leprosy and effect of oral zinc therapy on reactional states transport of electrolytes, water, and glucose in zinc deficiency kinetic aspects of compartmental storage and secretion of insulin and zinc a randomised controlled trial of oral zinc on the immune response to tuberculosis in hiv-infected patients zinc as a second messenger of mitogenic induction. effects on diadenosine tetraphosphate (ap a) and dna synthesis flow cytometric measurement of labile zinc in peripheral blood mononuclear cells correlation between zinc status and immune function in the elderly signal transduction in monocytes: the role of zinc ions imbalance between pro-oxidant and pro-antioxidant functions of zinc in disease inhibition of hiv- infection by zinc group metal compounds effect of intragastric ph on the absorption of oral zinc acetate and zinc oxide in young healthy volunteers zinc aspartate in vivo and in vitro modulation of reactive oxygen species production by human neutrophils and monocytes zinc in the treatment of acute diarrhea: current status and assessment efficacy of zinc against common cold viruses: an overview zinc-altered immune function zinc and the common cold: a meta-analysis revisited a meta--analysis of zinc salts lozenges and the common cold intestinal and systemic immune responses to an oral cholera toxoid b subunit whole-cell vaccine administered during zinc supplementation a double-blind, placebo-controlled study of vitamin a and zinc supplementation in persons with tuberculosis in indonesia: effects on clinical response and nutritional status polaprezinc, a mucosal protective agent, in combination with lansoprazole, amoxycillin and clarithromycin increases the cure rate of helicobacter pylori infection copper and zinc inhibit the metabolism of prostaglandin by the human uterus the effect of zinc supplementation on the treatment of chronic hepatitis c patients with interferon and ribavirin inhibition by zinc of rhinovirus protein cleavage: interaction of zinc with capsid polypeptides zinc ions inhibit replication of rhinoviruses the impaired immune response to diphtheria vaccination in elderly chronic hemodialysis patients is related to zinc deficiency transgenic copper/zinc superoxide dismutase modulates susceptibility to type diabetes the mechanism of the antiherpetic activity of zinc sulphate the prophylactic and therapeutic effectiveness of zinc sulphate on common cold in children identification of slc a , a gene involved in acrodermatitis enteropathica interleukin- regulates the zinc transporter zip in liver and contributes to the hypozincemia of the acute--phase response absorption of zinc in acrodermatifis enteropathica dietary factors influencing zinc absorption the role of zinc in growth and cell proliferation oral zinc therapy in recurrent erythema nodosum leprosum: a clinical study randomized, double-blind, placebo-controlled clinical trial of the efficacy of treatment with zinc or vitamin a in infants and young children with severe acute lower respiratory infection metallothionein and tissue damage zinc coordination environments in proteins as redox sensors and signal transducers zinc requirements and the risks and benefits of zinc supplementation oral zinc in recurrent erythema nodosum leprosum reaction oral zinc as an adjunct to dapsone in lepromatous leprosy zinc sulphate in rheumatoid arthritis characterization of caspase processing and activation in hl- cell cytosol under cell-free conditions. nucleotide requirement and inhibitor profile serum zinc and pneumonia in nursing home elderly bioverfügbarkeit von zink-präparaten benefit of oral zinc supplementation as an adjunct to zidovudine (azt) therapy against opportunistic infections in aids experimental zinc deficiency: effects on cellular responses and the affinity of humoral antibody acrodermatitis enteropathica: a lethal inherited human zinc-deficiency disorder effect of zinc supplementation on malaria and other causes of morbidity in west african children: randomised double blind placebo controlled trial zinc metabolism in rheumatoid arthritis: plasma and urinary zinc and relationship to disease activity zinc therapy in acrodermatitis enteropathica trace metal composition of synovial fluid and blood serum of patients with rheumatoid arthritis how does zinc modify the common cold? clinical observations and implications regarding mechanism of action zinc-induced suppression of inflammation in the respiratory tract, caused by infection with human rhinovirus and other irritants free radicals and diabetes new insights about iron bioavailability inhibition by zinc effects of short term isolated zinc deficiency on intestinal growth and activities of brush border enzymes in weaning rats a novel form of host defense: membrane protection by ca + and zn + effect of zinc on leukocyte sodium transport in vivo effects of zinc supplementation on the phagocytic functions of polymorphonuclears in patients with inflammatory rheumatic diseases phosphorylation of the lymphoid cell kinase p lck is stimulated by micromolar concentrations of zn + zinc is a potent inhibitor of the apoptotic protease, caspase- . a novel target for zinc in the inhibition of apoptosis inhibition of myoblast differentiation by lack of zinc endotoxin-induced inflammation does not cause hepatic zinc accumulation in mice lacking metallothionein gene expression anaemia and low serum-copper during zinc therapy the antioxidant properties of zinc discovery and importance of zinc in human nutrition effects of zinc deficiency on th and th cytokine shifts antioxidant effect of zinc in humans zinc deficiency affects cell cycle and deoxythymidine kinase gene expression in hut- cells hypocupremia induced by zinc therapy in adults zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypogonadism effect of zinc or zinc plus arginine supplementation on antibody titre and lymphocyte subsets after influenza vaccination in elderly subjects: a randomized controlled trial suppressive effect of zinc on antibody response to cholera toxin in children given the killed, b subunit-whole cell, oral cholera vaccine effects of zinc supplementation as adjunct therapy on the systemic immune responses in shigellosis effect of zinc supplementation on immune and inflammatory responses in pediatric patients with shigellosis lack of beneficial effect of zinc sulphate in rheumatoid arthritis zinc supplementation does not enhance antibody formation to influenza virus vaccine in the elderly zinc and iron supplementation and malaria, diarrhea, and respiratory infections in children in the peruvian amazon extracellular and immunological actions of zinc zinc homeostasis and immunity absorption of zinc sulfate, methionine, and polyascorbate in the presence and absence of a plant based rural mexican diet influence of highly active antiretroviral therapy on micronutrient profiles in hiv-infected patients thymulin modulates cytokine release by peripheral blood mononuclear cells: a comparison between healthy volunteers and patients with systemic lupus erythematodes zinc induces thymulin secretion from human thymic epithelial cells in vitro and augments splenocyte and thymocyte responses in vivo induction of interferon-γ in human leukocyte cultures stimulated by zn + zinc status and immune system relationship zinc as an essential micronutrient: a review excessive oral zinc supplementation oxygen free radicals and metallothionein. free radic zinc supplementation reduces the incidence of acute lower respiratory infections in infants and preschool children: a double-blind, controlled trial bioavailability of zinc from zinc-histidine complexes. i. comparison with zinc sulfate in healthy men interleukin regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures low concentrations of zinc in gastric mucosa are associated with increased severity of helicobacter pylori-induced inflammation the influence of zinc supplementation on morbidity due to plasmodium falciparum: a randomized trial in preschool children in papua new guinea zinc and immune function: the biological basis of altered resistance to infection oral zinc sulphate in the treatment of acute cutaneous leishmaniasis oral zinc sulphate in rheumatoid arthritis oxidative stress in viral hepatitis and aids biochemical characteristics of caspases- the influence of zinc on apoptosis zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis c dietary micronutrient intake and risk of progression to acquired immunodeficiency syndrome (aids) in human immunodeficiency virus type (hiv- )-infected homosexual men effects of micronutrient intake on survival in human immunodeficiency virus type infection the role of zinc in caspase activation and apoptotic cell death effects of zinc supplementation on the immune system and on antibody response to multivalent influenza vaccine in hemodialysis patients interaction of the unique n-terminal region of tyrosine kinase p lck with cytoplasmic domains of cd and cd is mediated by cysteine motifs the treatment of rhinovirus infections: progress and potential effect of iron, tin and copper on zinc absorption in humans the biochemical basis of zinc physiology zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study zinc supplementation to hiv- -infected pregnant women: effects on maternal anthropometry, viral load, and early mother-to-child transmission zinc-dependent suppression of tnf-alpha production is mediated by protein kinase a-induced inhibition of raf- , ikappab kinase beta, and nf-kappab zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses tnf-alpha and il- beta production in monocytes by elevation of guanosine ', '-cyclic monophosphate zinc during and in convalescence from diarrhea has no demonstrable effect on subsequent morbidity and anthropometric status among infants < mo of age a novel member of a zinc transporter family is defective in acrodermatitis enteropathica zinc bioavailability in feed-grade sources of zinc bioavailability of zinc from inorganic and organic sources for pigs fed corn-soybean meal diets the immunobiology of zinc zinc inhibits il- dependent t cell stimulation the temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis zinc in dna replication and transcription zinc is a negative regulator of hepatitis c virus rna replication childhood pneumonia -progress and challenges effect of zinc on the immune status of zinc-depleted aids related complex patients effect of zinc on the treatment of plasmodium falciparum malaria in children: a randomized controlled trial serum zinc and copper in active rheumatoid arthritis: correlation with interleukin beta and tumour necrosis factor alpha key: cord- -gwbi nlt authors: Álvarez-castro, josé m. title: gene–environment interaction in the era of precision medicine – filling the potholes rather than starting to build a new road date: - - journal: front genet doi: . /fgene. . sha: doc_id: cord_uid: gwbi nlt gene–environment interaction is a key part of evolutionary biology, animal, and plant breeding, and a number of health sciences, like epidemiology and precision medicine. however, bottlenecks in models of gene–environment interaction have recently been made manifest, particularly in the field of medicine and, consequently, specific improvements have been explicitly requested—namely, an implementation of gene–environment interaction satisfactorily disentangled from gene–environment correlation. the present paper meets those demands by providing mathematical developments that implement classical models of genetic effects and bring them up to date with the prospects current available data bestow. these developments are shown to overcome the limitations of previous proposals through the analysis of illustrative examples on disease susceptibility, with special attention paid to precision medicine. indeed, a number of misconceptions about the application of models of genetic/environmental effects to precision medicine are here identified and clarified. the theory here provided is argued to strengthen, in particular, the methodology required for high-precision characterization of strain virulence in the study of the covid- pandemic. scientific progress is often accompanied with expectations beyond objective appraisal. on the one hand, quantitative trait locus experiments became prominent years ago and substantial resources were soon after expectantly invested for elucidating genetic architectures of traits of economic importance (see e.g., rifkin, ; Álvarez-castro, ) . in turn, the latest decade witnessed major efforts to aid livestock production and plant breeding to undergo a swift switch toward genomic prediction (see e.g., gondro et al., ) . on the other hand, although initially developed for model species, genetic mapping of human traits became possible at the beginning of the current century by means of the international hapmap project (international_hapmap_consortium., ) and genome-wide association studies (gwas; see e.g., gondro et al., ) but-in line with the fate of quantitative trait locus experiments-its potential for dissecting the genetic basis of diseases is openly questioned nowadays (see e.g., teperino, a) . the aforementioned advances in genetics methodologies have enabled increasingly accurate medical predictions, particularly in regards to treatment efficiencies and prevention strategies for different (groups of) individuals, an approach that has been coined as precision medicine. in this context, the first half of the title above, "gene-environment interaction in the era of precision medicine, " has been stolen from a recent paper in which bottlenecks of classical models of genetic effects and their use in genetic mapping are discussed (li et al., ) . this is so because the present paper takes the baton of the aforementioned one by reviewing whether it is realistic and worth it to try and further amend classical models of genetic effects or whether it proves more sensible (or even necessary) to undertake alternative theoretical strategies instead. in order to further feed into that debate, the present paper dissects the advantages and limitations of the current theory of gene-environment interaction stemming from the classical models of genetic effects and provides a new mathematical implementation that overcomes their historical limitations. next, the advantages of the theory here provided are shown through built-in cases on disease susceptibility, which also serve to further illustrate the application of this theory to make predictions in the aid of precision medicine. then, in the discussion, all the above is argued to endorse both the general flexibility of the classical models of genetic effects to serve as a basis for further implementations and, particularly, the theory here provided to enable a demanded leap in the application of gene-environment interaction for medical purposes, like the achievement of a detailed understanding of important facts of the current covid- pandemic. firstly, the basic conceptual definition of gene-environment interaction is here discussed. the genetic and environmental components act independently as long as environmental changes cause the same effects on phenotypes for all genotypes. if, for instance, genotype g displays phenotypes and under environments e and e , respectively (i.e., the environmental change causes an increase of two phenotypic units to this genotype), and genotype g displays a phenotype of under environment e , then it is said that there is gene-environment interaction whenever genotype g displays a phenotype different from under environment e . in the aforementioned paper, li et al. ( ) echo the message that techniques using conventional genetic models do not often provide insightful enough results and that, in particular, they have so far provided no clear-cut evidence on whether disease etiologies are due to rare alleles with strong effects or to common alleles with weak effects. more to the point, li et al. ( ) have carried out a simulation by means of which certain genetic models are shown not to be able to capture the complexity of realistic underlying factors of a disease-particularly, involving epistatic effects (gene interactions, i.e., departures from the sum of the marginal contributions of the effects of the genes involved). further on, li et al. ( ) provide a probabilistic approach based on a bayesian framework to hierarchically model geneenvironment interaction, leading to a population-dependent index, c, called the genetic coefficient of the disease (at a population)-"a large c indicates large distinguishability of case genomes from control genomes." then they illustrate the performance of the proposed methodology using a built-up example in which the disease susceptibility is by default very low ( . ) and it significantly increases due to either environmental (exposure) or genetic (risk allele) factors or both, to . , . , and . , respectively. that case is hereafter referred to as the risk and exposure (rae) case (see table ). with an exposure frequency of . and a frequency of the risk allele of . , li et al. ( ) report the genetic coefficient of the disease of the rae case to be c = . . about half a dozen years earlier, ma et al. ( ) provided a model of gene-environment interaction based on the natural and orthogonal interactions (noia) model of genetic effects (Álvarez-castro and carlborg, ) , stemming from the classical models. in these models, the parameter α can be used to reflect the "difference between the additive expectations of case genomes and control genomes, " thus providing an alternative measure for the genetic coefficient of the disease, c from the work of li et al. ( ) . assuming hardy-weinberg proportions at the risk allele locus and an equal risk of heterozygotes and homozygotes for the risk allele (since it is not explicitly specified otherwise in that paper), the model from ma et al. ( ) can be used to compute a difference between the additive expectations of case genomes and control genomes of α = . (or, to be more precise, α g = . , using the specific notation from ma et al. ( ) ). the departure between this value and the genetic coefficient of the disease, c = . , from the work of li et al. ( ) , could be due to the choices necessary in relation with dominance and the hardy-weinberg proportions. along with the aforementioned statistical formulation of genetic effects, both noia (Álvarez-castro and carlborg, ) and the extension of it to gene-environment interaction by ma et al. ( ) entail a so-called functional formulation. whereas the statistical formulation is population-referenced and thus its parameters reflect properties of populations, the functional formulation is individual-referenced and thus its table | phenotypes (disease susceptibility) of the four individual classes (risk allele carriers and non-carriers under exposed and non-exposed environments) for the two cases considered in the text-the case taken from li et al. ( ) , here called the risk and exposure (rae) case and the genetic risk to exposure (rte) case. complete dominance of the risk allele is assumed so that homozygotes for the risk allele and heterozygotes are equally susceptible to the disease. parameters reflect plane effects of substitutions from a reference class (a genotype at an environment) to the others. applying that functional formulation from the default (non-exposed and non-risk) individual reference ( . ), the additive, dominance, environment, additive-by-environment, and dominance-byenvironment effects reflecting the aforementioned substitutions are . , . , , , . , and . , respectively (see table ). those values show that, although the rae case by li et al. ( ) entails both genetic and environmental effects, it can hardly be considered a gene-environment interaction case as intended, since the gene-environment interaction effects are extremely small relative to both the genetic and the environmental marginal contributions-the interaction effects actually lie about two orders of magnitude below the marginal effects. hitherto, it has been shown that relatively recent implementations of the classical models not only enable the analysis of the rae case built up by li et al. ( ) to illustrate their theoretical proposals but are also adequate to easily and precisely quantify basic properties of that case itself, which have apparently been missed by those authors. more generally speaking, theoretical developments stemming from the classical models are not always fairly acknowledged. to this regard, it is worth noting that both noia (Álvarez-castro and carlborg, ) and the extension of it to gene-environment interaction by ma et al. ( ) can properly deal with departures not only from complete dominance but also from hardy-weinberg proportions, which were assumed above only due to the absence of any explicit specifications of departures from those features. nevertheless, the general warning li et al. ( ) post on the use of genetic models still holds-the current state-ofthe-art implementations of classical models of genetic effects, whether unfairly acknowledged or not, still leave room for further improvement. indeed, the original noia proposal fails to properly account for nonrandom associations of marginal genotypic frequencies (i.e., assumes linkage equilibrium between/among the loci involved) and ma et al. ( ) inherit that limitation in regards to nonrandom associations between aei and dei stand for additive-environment interaction and dominanceenvironment interaction, respectively. for each of the two cases, this table shows both functional (i.e., individual-referenced) effects from the reference of the default (non-risk and non-exposed class, with a disease susceptibility of . ) and statistical (i.e., population-referenced) effects from the reference of the average phenotype. the reference population of the statistical effects has a frequency of exposure of . , a frequency of the risk allele of . , hardy-weinberg proportions, and random associations of (i.e., absence of correlation between) genotypes and environments. by virtue of the latest, the results here reported may be equally obtained using the former noia setting and arnoia (ultimately from expression ), as explained in the text. the marginal frequencies of genotypes and environments (i.e., gene-environment correlation). thus, those association-pending models shall hereafter be referred to as the former noia setting. incidentally, it is imperative to overcome that limitation both because correlations between/among marginal frequencies may occur in populations and because they are in any case likely to achieve significant levels in the actual samples used in real data analyses. opportunely, it is hereafter shown that the gaps of the former noia setting for gene-environment interaction can be bridged. indeed, new mathematical developments for studying gene-environment interaction are provided below, in which gene-environment correlation is properly implemented. since the resulting theoretical proposal bridges the aforementioned associations-pending gap, it shall be referred to as arnoia (associations-resolved noia). a biallelic locus a (with alleles a and a ) and two environmental instances (e and e ) of an environmental variable e are initially considered. this setting leads to six possible classes-combinations of genotypes and environments-and thus to six phenotypic expectations (e.g., six values of disease susceptibility). those values are gathered in the column-vector of genotypic values, g = (g ijk ), where the subscripts indicate genotype a j a k at environment e i . the genotypic values can be expressed in terms of genetic effects by means of regression model in which the explanatory variables are the mean phenotype µ, the environmental effect, e = υ = (e , e ) t (where t stands for the transpose operation), the genetic additive effect, α = υ = (α , α ) t , the dominance effect, δ = υ = (δ , δ , δ ) t , and the additive-by-environment effect, αe = υ = (αe , αe , αe , αe ) t , and the residual term is the dominance-by-environment effect, δe = η = (δe ijk ). let (m) be a column vector of length m with all its scalars equal to , i (n) an identity matrix of dimension n, n = t , and ⊗ the kronecker product. then, the design matrices in expression ( ) can be expressed as: regression ( ) with design matrices ( ) is meant to be solved sequentially, as follows. let the population frequencies be p ijk and let p be the diagonal matrix of those frequencies, p = diag(p ijk ). then, the mean phenotype is µ = p ijk g ijk , the mean-corrected vector of genotypic values is η = g - ( ) µ, and the expressions for the remaining explanatory variables and the residual term of regression ( ) come from computing, υ l =h l η l− and η l = m l η l− , l = to , (expression ) whereh l = n t l pn l − n t l p and m l = i ( ) -n lhl . with this, a theory of population-referenced (i.e., statistical, orthogonal) genetic/environmental effects that properly accounts for both gene-environment interaction and gene-environment correlation is provided, which shall hereafter be referred to as a correlationwise orthogonal interactions (coia) model. in order to fully integrate coia within the aforementioned noia framework (Álvarez-castro and carlborg, ) , regression ( ) has to be expressed in the form of a standardized statistical formulation. such a formulation is where e = (µ, α, δ, e, αe, δe) t is the vector of genetic/environmental effects and the genetic/environmentaleffects design matrix, s, is computed via its inverse, s − , whose rows can be obtained using expressions ( - ) where: the first one is (p ijk ), the set of coefficients of µ = p ijk g ijk , the second, third, fourth and fifth ones are, analogously, the sets of coefficients of g ijkl in α = (α -α ), δ = δ -((δ + δ )/ ), e = e -e and αe = (αe -αe -αe + αe ), respectively, and the sixth one from expression ( ) it is easy to perceive how critical building fine-tuned genetic/environmental-effects design matrices becomes in order to perfectly grease the machinery of the noia model, (here upgrading it in particular to an arnoia level, i.e., resolving the implementation of any kind of associations by means of coia). indeed, expression ( ) is a compact way of representing how a genotype-to-phenotype map (essentially, a g vector) can be translated into its evolutionary properties. as noted by Álvarez-castro and carlborg ( ), the statistical, orthogonal genetic (and, here, also environmental) effects reflecting evolutionary properties of two populations, " " and " , " can easily be transformed into each other by equating the genotypic values in expression ( ), i.e., simply as e = (s ) − s e . and in what regards the individual-referenced (i.e., functional, natural) side of noia, this expression holds when one of the vectors of genetic/environmental effects (or both) and its corresponding matrix do not reflect allele substitutions made from the reference of a population, but of an individual genotype/environment instead. as pointed out above, functional (natural) genetic/environmental effects design matrices for gene-environment interactions imply a biallelic locus and two environments have been provided by ma et al. ( ) . using previous extensions of classical models of genetic effects (Álvarez-castro and yang, ; alvarez-castro and crujeiras, ), the coia regression framework for gene-environment interaction developed above and its implementation into an arnoia model can be extended to several, possibly multiallelic, loci with arbitrary epistasis and arbitrary departures from linkage equilibrium and simultaneously to several environmental variables with multiple environmental instances, with nonrandom associations (i.e., correlations) of environmental variables and of genotypes and environments. the details of such extensions are, though, beyond the scope of this paper. the advantage arnoia confers over the shoulders it stands on-the ones of the former noia setting (Álvarez-castro and carlborg, ) and, particularly, of its implementation with gene-environment interaction (ma et al., )-is discussed hereafter. as a baseline, the population-referenced genetic/environmental effects of the rae case in the absence of gene-environment correlation are shown in table and they can be equally computed using either of the two methods. the whole range of possible correlations between the risk allele and environmental exposure is inspected in figure a . the thick vertical line marks the point of random association (i.e., no correlation) where all values provided by the former noia setting by ma et al. ( ) are correct and meet the ones provided by arnoia (i.e., as mentioned right above, the values in table ). then, the model from ma et al. ( ) still provides, within the whole range of correlations, those same values shown in table to fit to the random association scenario (gray horizontal lines) whereas arnoia (black lines) shows instead how the genetic/environmental effects actually change with negative (to the left of zero) and positive (to the right) riskexposure correlations. roughly, the effects decrease and increase with negative and positive correlations, respectively, although a slight decrease of the additive effect toward the maximum positive correlations and slightly more capricious behavior of the dominance effect for intermediate positive correlations can also be noticed. in view of figure a , it could seem that settling for the relatively simpler formulae of the former noia setting (not accounting for nonrandom associations of genes and environments) by ma et al. ( ) would not come with a high cost. indeed, values that are correct for circumstances known beforehand (precisely, nonrandom associations) are retrieved regardless the nonrandom associations involved. however, that is but a mirage for such a constraint shall, on the one hand, severely hamper the flexibility of the model for making predictions (as illustrated in the following section) and, on the other hand, make the models less efficient in disclosing genetic architectures (as explained below). in what follows, a case of actual gene-environment interaction is considered. it is a case of genetic risk to environmental exposure (thus referred to hereafter as rte), where the risk allele increases disease susceptibility only when combined with exposure, hence actually interacting with the environment. thus described, the interaction behaves as a switch-the environmental effect shall either be switched on (when carrying the risk allele) or turned off (otherwise), as shown in table . table shows that the functional additive and dominance effects (i.e., the marginal genetic effects) of the rte case from the reference of the individual default class (no genetic risk and no exposure) are zero, which is in accordance with the genetic risk being turned off in the absence of exposure. in table it is also illustrated that large gene-interaction effects frontiers in genetics | www.frontiersin.org figure | genetic and environmental effects of disease susceptibility influenced by a risk allele and environmental exposure, for the whole range of possible correlations (including negative and positive associations) of the risk allele and environmental exposure. the risk allele frequency is . , with genotypic frequencies under hardy-weinberg equilibrium, and the environmental exposure frequency is . . the rae case (see table ) is shown in panel (a), where the former noia setting and arnoia, shown with gray and black lines, respectively, are compared. the thick black solid vertical line marks the case of random association (i.e., no correlation) between risk and exposure. the genetic effects obtained with arnoia for the rte case (see table ) are shown in panel (b). the marginal genetic and environmental effects are shown with the same black lines as in panel (a) and gray lines are used here for the interaction effects. frontiers in genetics | www.frontiersin.org actually have a noticeable influence on the lower level effects, since marginal genetic effects become not nil under a different genetic/environmental background (i.e., when expressing the effects from a different reference) and also the environmental effect is significantly modified. in the rae case, only the additive effects change noticeably under different references, which is an effect of dominance interaction under backgrounds with differential presence of the alleles. for a broader scope, figure b shows all the genetic/environmental effects of the system as obtained using arnoia, for the whole range of possible gene-environment correlations. marginal effects are displayed as in figure a and gene-environment interaction effects are shown in gray. the marginal genetic effects of the rte case are small in the absence of gene-environment correlation. indeed, this case entails a visual example of a warning issued above since it illustrates that marginal effects approach zero as an occasional outcome (of a particular set of population frequencies), making it tricky to spot them in a mapping experiment. the trouble vanishes though as long as the (larger) gene-environment interaction effects are inspected (despite the apparent absence of marginal genetic effects) and disclosed. note also that although the marginal genetic effects get closer to zero under certain negative correlations (toward the far-left end of the graph), the additiveby-environment interaction effect increases accordingly. thus, in any case, eventually out-of-reach marginal effects may be unveiled by diligently fishing interaction effects. overall, for properly detecting marginal (genetic and environmental) and interaction (gene-gene and geneenvironment) effects (and, therefore, identify their corresponding loci and environmental variables) in mapping experiments it is essential that the genetic models entail not only any interactions between/among the effects themselves but also any departures from equilibrium genotype/environment frequencies, as figure shows arnoia to accomplish. moreover, it is hereafter illustrated that the advantages of arnoia over the former noia setting are also crucial for using detected genetic and environment underlying factors of traits in the formulation of predictions, particularly in the context of precision medicine. figure a shows the genetic coefficient of the disease-as defined by li et al. ( ) -for the rae case, which, as mentioned above, in the context of the developments stemming from the classical models of genetic effects is given by the parameter α. on top of the variables already considered in figure a , figure a has one dimension added for enabling predictions in the context of a hypothetical decrease of the environmental exposure. the black solid line in figure a marks random association and shows that the genetic coefficient of the disease is simply not affected by decreasing the exposure frequency in the population. this is as expected under the lack of interplay between gene and environment (i.e., no interaction and no correlation). indeed, although the trait is subject to both genetic and environmental influence, as long as there is no (or very little) interplay between them, the genetic parameter remains virtually constant in the face of variations in the environmental exposure. however, as already shown above in relation with figure (where the additive genetic effect, α, was shown instead of the genetic coefficient of the disease, α), such an interplay may come not only by means of gene-environment interaction but also through gene-environment correlation. thus, whereas the genetic coefficient of the disease remains constant in figure a against diminishing exposure in the absence of significant geneenvironment interaction, it is in point of fact affected by risk-exposure correlations. in particular, negative associations between the risk allele and exposure causes the genetic coefficient of the disease to decrease, as the surface to the left of the black line shows. conversely, positive associations make it increase, to the right of the black line, although this occurs up to a maximum followed by a slight decrease. this is, naturally, the same kind of behavior the additive effect, α, displays in figure a . note also that the range of possible risk-exposure correlations (shown by the light gray area at the bottom of the figure) narrows down as the exposure frequency approaches zero, which explains the tip of the surface at the end of the black line. in figure b , the rte case of figure b is resumed and further extended in a way analogous to figure a from figure a . as figure b shows, for the rte case the genetic coefficient of the disease decreases for decreasing values of exposure under random associations of risk and environment (decreasing black line). that coefficient also decreases for decreasing (increasingly negative) associations between the risk allele and environmental exposure, as the left tip of the surface shows. in plain language, the figure shows that the problem of increased disease susceptibility of the carriers of the risk allele may be reduced (and eventually removed) either by reducing exposure for the whole population or by restricting the access to the exposed environment only for the risk population, or even through any intermediate alternative (any reduction of the exposure in the population biased toward the carriers of risk alleles). optimal management would then depend just upon the reluctance of the average individual to avoid the exposed environment (or even the actual feasibility of bringing the whole population out of it) and the cost of tests to detect the risk allele, which would enable personalized warnings. overall, the rae and rte cases considered in figure deal with rather singular instances (virtually absent and switchtype, respectively) of gene-environment interaction, for which some predictions would be feasible even without mathematical modeling. the results obtained using arnoia not only reassuringly agree with the conceptually attainable predictions but also further illustrate how to precisely quantify any desired genetic/environmental parameter. such an advantage can hereafter be applied to more complex real cases of interest undergoing less intuitive behaviors. interactions are known to encrypt the map where a pursued genetic architecture could be spotted. this is known to occur because interactions of any kind (from just dominance to geneenvironment interaction) may make lower level effects (like environmental effects or genetic additive effects) vanish under a certain genetic/environmental composition of a population or of an experimental sample (see e.g., Álvarez-castro, ) . this fact, which has been further illustrated in figure a , is unfortunately not always properly taken into account. in relation with this, the commendable review by malosetti et al. ( ) on models of gene-environment interaction in the context of plant breeding reasonably recommend to adhere to a strategy where effects are inspected sequentially-as they also are in expressions ( - ) above-, but it oversteps the mark when more specifically proposing a conditional sequential procedure, by claiming that "dominance effects should be tested conditioned on the additive effects present in the model." indeed, since interactions make effects on the phenotype able to cancel out in average at the group of individuals under study and to thus be missed in mapping experiments, unveiling interactions actually becomes doubly imperative rather than something to be subject to the condition of first having found their (possibly masked) lowerorder effects. thus, in order for the theoretical genetic/environment models and the estimation strategies used in mapping methods to become accurate enough to address the difficulty of dealing with possibly masked effects, it is necessary in the first place to opportunely implement such models with interaction effects, as thoroughly recalled by li et al. ( ) , particularly in regards to precision medicine. but interactions are deceitfully puzzling and thus trying to properly map complex genetic architectures makes it crucial to also improve the flexibility of the models to accurately fit to the frequencies of the genotypes in the population/sample under study. indeed, zan et al. ( ) have recently shown to what extent departures from linkage equilibrium frequencies may condition the models to strikingly distort the genetic architecture of a trait, particularly in regards to genetic interactions. to this regard, it has become particularly opportune that an elusive implementation of models of genetic interactions, even claimed to be beyond reach, has recently been attained, enabling genetic interaction (epistasis) and genotype frequencies correlation (linkage disequilibrium) to be disentangled (alvarez-castro and crujeiras, ). the coia regression framework and the arnoia model developed above attain an analogous goal in what regards a joint implementation of gene-environment interaction and geneenvironment correlation, which is particularly timely for aiding precision medicine, as further discussed below. affordable data is an ever-changing variable and it is thus sensible to assume that, likewise, theoretical models required in data analyses shall need to keep on being worked out every now and then. in this context, it is as essential to make the best possible use of the models available at a particular time-spot as it is to point out in which precise way they are at that time imposing limitations on the analyses. in what has been recently understood as the, at least relative, "failure of gwas" (teperino, b) , gene-environment interaction has been pointed out as a key factor. indeed, the importance of gene-environment interaction in human health has been stressed in relation with a broad spectrum of disorders ranging from obesity, cardiometabolic diseases, and other metabolic disorders, through to cancer, autoimmune diseases, and mental disorders (e.g., karl and arnold, ; lopizzo et al., ; flouris et al., ; cust, ; smith, ; teperino, a) . all in all, the lack of a finetuned theory of gene-environment interaction has, thus, been imposing serious limitations in this field. more precisely, developments implementing the effects of both gene-environment interaction and gene-environment correlation in a properly disentangled manner have explicitly been demanded within the field of precision medicine. indeed, in the context of mental health, assary et al. ( ) have recently advocated that "[i]dentifying which form of gene-environment interplay contributes to a particular disorder or behavior is absolutely crucial in order to select suitable intervention efforts" because theoretical developments that enable a joint analysis of both phenomena are needed, in particular for "ensuring that the outcomes of one do not bias the effects of the other." the present paper meets that demand and it does so within a theoretical framework capable of simultaneously addressing many other genetic facts of relevance. indeed, the arnoia model here provided illustrates the possibilities of mathematical developments stemming from the classical models of genetic effects in regards to their potencial to be continuousy improved and thus address eventual demands to come. in other words, since the machinery here provided has proven useful to fill in inconvenient potholes of the classical road network, it should be deemed to be applicable to increasingly complex challenges in the future. for instance, the need to consider gene-gene-environment interaction (e.g., zan and carlborg, ) and/or geneenvironment-environment interaction (e.g., keers and pluess, ) has already arisen and it is to this regard worth highlighting here that the advantages of arnoia can also be applied to address such complexities (and actually gene-environment interaction/correlation, multiple alleles, dominance, epistasis and departures from hardy-weinberg equilibrium and from linkage equilibrium, simultaneously) by merging the mathematical developments provided above with previous theory (Álvarez-castro and yang, ; alvarez-castro and crujeiras, ). the previous is, however, not to say that alternative roadslike li et al. ( ) -should never be built. it looks sensible in any case to assume that a new road will consume significant resources before providing benefits comparable to the already existing ones, especially in regards to the wealth of experience amassed in the use of them. therefore, it would be reasonable to first thoroughly inspect the possibilities of the existing roads to be fixed and as well to guarantee the added value the new road is intended to bring. on top of that, it would also make perfect sense to assume that the new road would only provide its best service when adequately connected with the previous road network. whenever developed along these lines, alternative perspectives in genetic modeling could aim to open doors to novel analyses and/or double check the already existing ones and thus enrich the application of mathematical models of genetic and environmental effects in precision medicine. as a final remark, it would be regrettable in the context of the current covid- pandemic not to explicitly point out that arnoia improves the methodology that can in particular be applied to dissect the behavior, and thus help to eventually overtake, such a global threat. epidemiology relies on a thorough study of interactions (see e.g., dewan, ) and the particularly strong link between epidemiology and gene-environment interaction has already been underscored in relation to the covid- pandemic (rodriguez-morales et al., ) . in the cases analyzed above, arnoia has been applied to disease susceptibility and from those instances it becomes easy to perceive that it is equally applicable to other traits of interest in epidemiology, including, for instance, mortality caused by a disease. generally, the dynamics of a pandemic shall depend upon how the different strains of the infectious agent affect different (groups of) individuals, with different (proportions of) genotypes and under different environmental conditions. it becomes particularly useful to notice in this regard that although virulence variability is underlain by mutations (and thus conceptually related to genetics), arnoia may naturally integrate the presence of different strains (with differential virulence) simply as an (additional) environmental variable, since that is how they are perceived from the perspective of the susceptible individualsthe genetic component of the model. bearing that in mind, it is easier to perceive why it is crucial, for the study of covid- , that arnoia considers together (but disentangled) geneenvironment interaction and gene-environment correlation. indeed, the various geographical regions affected by the disease do not only undergo different proportions of virus strains (environmental component of the model) but also different genetic backgrounds of the susceptible individuals (genetic component), thus setting a human genotype-strain (gene-environment) correlation scenario in which human genotype-strain (gene-environment) interaction needs to be properly understood. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. the author confirms being the sole contributor of this work and has approved it for publication. current applications of models of genetic effects with interactions across the genome genetic architecture a unified model for functional and statistical epistasis and its application in quantitative trait loci analysis orthogonal decomposition of the genetic variance for epistatic traits under linkage disequilibrium-applications to the analysis of bateson-dobzhansky-muller incompatibilities and sign epistasis multiallelic models of genetic effects and variance decomposition in non-equilibrium populations the role of gene-environment interaction in mental health and susceptibility to the development of plychiatric disorders, " in beyond our genes. pathophysiology of gene and environment interaction and epigenetic inheritance gene-environment interactions and melanoma risk gene-gene and gene-environment interactions role of ucp gene variants in interethnic differences in the development of cardio-metabolic diseases genome-wide association studies and genomic prediction the international hapmap project shizophrenia: a consequence of gene-environment interactions? switzerland: frontiers media sa childhood quality influences genetic sensitivity to environmental influences across adulthood: a life-course gene x environment interaction study gene-environment interaction in the era of precision medicine gene-environment interaction in major depression: focus on experience-dependent biological systems natural and orthogonal interaction framework for modeling gene-environment interactions with application to lung cancer the statistical analysis of multi-environment data: modeling genotype-by-environment interaction and its genetic basis quantitative trait loci (qtl) genomic epidemiology and its importance in the study of the covid- pandemic gene environment interactions. nature and nurture in the twenty-first century beyond our genes, pathophysiology of gene and environment interaction and epigenetic inheritance preface, " in beyond our genes. pathophysiology of gene and environment interaction and epigenetic inheritance dynamic genetic architecture of yeast response to environmental perturbation shed light on origin of cryptic genetic variation on the relationship between high-order linkage disequilibrium and epistasis. g the author acknowledges insightful comments by dr. ania pino-querido and by two reviewers, who improved the final form of this paper. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © Álvarez-castro. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -mwgccbfo authors: casado-aranda, luis-alberto; sánchez-fernández, juan; viedma-del-jesús, maría i. title: analysis of the scientific production of the effect of covid- on the environment: a bibliometric study date: - - journal: environ res doi: . /j.envres. . sha: doc_id: cord_uid: mwgccbfo the fight against covid- since january has become the top priority of more than countries. in order to offer solutions to eradicate this global pandemic, the scientific community has published hundreds of articles covering a wide range of areas of knowledge. with the aim of synthesizing these publications, academics are resorting to bibliometric analyses from the perspectives of the disciplines such as biology, medicine, socioeconomics and tourism. yet no bibliometric analysis has explored the diffuse and little-known growth of covid- scientific publications in the field of environmental studies. the current study is the first of this type to fill this research gap. it has resorted to scimat software to evaluate the main topics, authors and journals of publications on the subject of covid- combined with environmental studies spanning the period between december and september . the search yielded a collection of articles published in scientific journals indexed on by web of science and scopus databases. these publications can be broken down into six main themes: (i) a sharp reduction in air pollution and an improvement of the level of water pollution; (ii) the relationship of wind speed (positive), ultraviolet radiation (positive) and humidity (negative) with the rate of infections; (iii) the effect of the pandemic on the food supply chain and waste habits; (iv) wastewater monitoring offers a great potential as an early warning sign of covid- transmission; (v) artificial intelligence and smart devices can be of great use in monitoring citizen mobilization; and (vi) the lessons gleaned from the pandemic that help define actions to mitigate climate change. the results of the current study therefore offer an agenda for future research and constitute a starting point for academics in the field of environmental studies to evaluate the effects of covid- . the struggle initiated in january to combat the severe acute respiratory syndrome coronavirus (sars-cov- or covid- ) has become the top priority for more than countries. the pandemic is putting a massive strain on, among others, health care personnel, law enforcement agencies, public administrations and information and communication professionals. in order to offer solutions to this global health problem, the scientific community is also facing one of the most important challenges in recent times. it is responding by publishing hundreds of articles every day in a variety of fields, from medicine and epidemiology to psychology and the environment. many also appear in the field of economics due to the severe financial consequences of the virus. the ongoing scientific contributions confirm that the academic response to covid- is both massive and multifaceted (bonilla-aldana et al., ; felice and polimeni, ; nowakowska et al., ) . a revealing fact in this regard is that since the pandemic's outset, the number of scientific papers on covid- have doubled every days (torres-salinas, ) . this sheer volume of the scientific output in response to the pandemic renders it difficult even when resorting to accurate keywords to pinpoint information from the databases such as pubmed, web of science and scopus. in order to facilitate the search for scientific information related to covid- , academics are following two different alternative paths. the first, applied by academics from various branches, consists of carrying out comprehensive reviews and bibliometric analyses so as to synthesize and simplify the results. papers between december and april . bonilla-aldana et al. ( ) and radanliev, de roure and walton ( ) proceeded further by evaluating the scientific literature on coronavirus types and potential vaccine treatments. verma and gustafsson ( ) in the sphere of socioeconomics conducted a bibliometric study of covid- literature in the domains of business and management so as to identify current areas of research and propose future lines of research. sigala ( ) , in the field of tourism, likewise undertook a critical review on the impacts and implications of covid- so as to offer solutions to reset the industry. at the same time, efforts are being conducted to build covid- -oriented databases of scientific papers to continually update the scientific contributions to the subject. examples are the databases of the world health organization ( ) and the center for disease control and prevention ( ). in this period of pandemic, the audiovisual and written media bear the responsibility (now more than ever) to disseminate true and quality information, and prevent the proliferation of hoaxes and fake news in the face of an uncertain future. nevertheless, the spread of fake news and misunderstandings is occurring, more than ever, and at a very rapid pace. this is due to the growth of accessibility to internet, the popularity of social media and the nature of how it works (i.e., post sharing) combined with the appetite of the media to increase their visibility via click-baiting techniques and sensational headlines. in their effort to avoid misconceptions, information and communication professionals have turned to scientific texts on virology, epidemiology, law, economics or psychology to attempt to transfer the scientific results through their newsletters in a simple, informative and accessible manner. journalists from the newspaper el país (de benito, ) , for example, reported on the findings of crucial scientific research with the aim to explain the drugs available to treat covid- . the new york times (debgupta, ) , likewise based on scientific data, offered a guide on how to attend to supermarket food purchases. the world health j o u r n a l p r e -p r o o f organization ( ) also reported on how to cope with the stress related to covid- . this reporting on the effect of covid- at pharmacological, nutritional and psychological levels contrasts with the little data advanced by the international media as to the consequences of covid- on the environment. exceptions are reports by the bbc (henriques, ) that offered scientific details from other pandemics to answer the query "will covid- have a lasting impact on the environment?" the online newspaper eldiario.es (garcía-charton, ) likewise delved into the environmental consequences of new methods of consumption, migration and mobility subsequent to the crisis. it is also noteworthy that different media reported opposite data as to covid- 's biomedical-environmental origin and ecological consequences (anon., ; o'callaghan, ) . due to the low quality of the current information regarding the main environmental consequences of covid- and the diffuse and little-known growth of reports on the subject, a descriptive and visual quantification of scientific research on the virus and its effect on the environment would be of assistance to information professionals and environmental academics to gain an objective perspective of the evolution, current scope and main results to include in their reporting and research. this task would lend a hand to information professionals to disseminate the effects of covid- on the environment consistently and clearly and depict the encroaching phenomenon of the coming months. although certain bibliometric analyses on covid- exist in a general level (e.g., bonilla-aldana et al., ; chahrour, ; felice and polimeni, ; hossain, ; nowakowska et al., ; torres-salinas, ) , no study has explored the evolution of publications related to covid- or identified its main thematic axes and environmental consequences in the specific area of the environment. prior environmental research resorting to bibliometric analyses have examined the characteristics and implications of the patterns in shale gas literature between and j o u r n a l p r e -p r o o f (wang and li, ) or have attempted to identify a research profile on the natural gas acquired from these types of deposits (wang and lin, ) . closer to the current covid- topic are studies delving into the effects of temperature and relative humidity on the viability of previous types of coronavirus (casanova et al., ; chan et al., ) . other studies have focused on the survivability of earlier strains of coronavirus in water and wastewater (gundy et al., ; ye et al., ) . recent environmental literature is placing great weight on the antecedents and consequences of covid- on the environment and its interaction with human activities (e.g., sims and kasprzyk-hordern, ; wang and su, ; xie and zhu, ) . the intention of the current study is to offer a first straightforward report on the evolution of publications combining the effect of covid- on the environment since the outset of the pandemic, as well as to identify the main lines of research that are surging as a result of the crisis and establish a research agenda for environmental scholars. the study specifically pursues five objectives: (i) quantification of the volume of production of covid- -related scientific articles in the field of environment studies including peer-reviewed publications indexed in the main databases (i.e. web of science and scopus) spanning december to september . environmental studies is a multidisciplinary field which systematically delves into human interaction with the environment. it specifically draws together principles from the physical sciences, commerce/economics, and humanities and social sciences to address complex contemporary environmental issues (soulé and press, ) . (ii) identification of the main authors and scientific journals publishing research on covid- and the environment. this study is not only a first in the evolution of covid- research from the specific perspective of environmental studies, but will be of extreme utility for information professionals to disseminate objective and quality coverage on the effects of the virus on the environment. furthermore, the results will offer insight into the authors, research journals and themes worth considering in future environmental research. the databases web of science core collection and scopus served for this study. the information gleaned from them was from a consultation carried out on september . the query equation for scopus was the following: all (( " -ncov" or "covid- " or "covid- " or "sars-cov- " or the initial search yielded over , papers in english, of which were duplicates (i.e., indexed in both databases) and thus excluded from further analysis. the corpus was then narrowed down to articles within the scope of environmental studies so as to eliminate contributions linked to covid- treatments, or others related to the implications of the virus on public health. this manual screening process led to a collection of documents published between december and september . the data analysis was carried out with scimat software (cobo et al., ) , a technique serving to examine the social, intellectual and conceptual framework of a specific field. based on the two files extracted from consulting the scopus and web of science databases, the scimat software: (i) sorted and ranked all the documents by year of publication, number of citations, and titles of journals; and (ii) carried out a co-word analysis aiming to identify emerging themes related to covid- stemming from the realm of environmental research (cobo et al., ) . thus the co-word analysis applied text-mining techniques to the titles, abstracts, and keywords leading to the design, based on fundamental bibliometric indicators such as the number of publications, of a strategic diagram illustrating the main themes of interest related to the issue of environment in covid- research. to accurately detect the main topics garnered from the co-word analysis, the authors specifically followed the steps outlined in the study by cobo et al. ( ) consisting of isolating the author and journal keywords after an initial manual elimination of the documents not directly linked to the environment. the second step was to collect the relevant information from the raw data of these documents. as noted by cobo et al. ( ) , this information is gathered by analyzing the co-occurrence of keyword frequency. individually, this frequency j o u r n a l p r e -p r o o f of two keywords is extracted from the corpus by computing the number of documents in which the two keywords appear together. the third step is to calculate the similarities between the items collected in the second step. the similarities are calculated from keywords co-occurrence frequency. following the recommendation of cobo et al. ( ) , an equivalence index is the most appropriate means to normalize the frequency of co-occurrence. the fourth step was to determine the clusters that serve to identify subgroups of linked keywords that signal topics of interest. likewise, following the method suggested by cobo et al. ( ) , the simple center algorithm (with the values and representing the minimum and maximum size of the network) was applied to detect the themes of relevance. finally, the values of the "number of citations" and "number of documents" served to measure the quality of the strategic diagram. the results of the bibliometric analysis applying the web of science and scopus databases yielded a corpus of articles published in peer-reviewed scientific journals since the outset of the pandemic (december ) combining the subject of covid- and the environment. % were identified through scopus. the remaining % were through both scopus and web of science. of note is that % indexed through the web of science finds are of open access, while . % of this type were identified through scopus. the recent interest of the effects triggered by the international covid- health crisis (table ). the authors with the greatest number of publications the effects of covid- on the environment are primarily chinese and american universities and research institutes (table ) . the two-dimensional graph generated by scimat software (figure and ( ) lessons learnt from covid- applicable to climate action. one of the motor themes of environmental study literature linked to the covid- pandemic is the role of the virus in air and water quality. a number of studies have explored the reduction of air pollutants (e.g., nitrogen dioxide, no ; carbon dioxide, co ; particulate matters, pm) and the decrease in fossil fuel emissions stemming from the daily lockdown. another thematic area that stands out among the main analyses of covid- and the environment is the effect of meteorological factors (i.e., temperature, humidity, wind speed and ultraviolet radiation) on the rate of covid- infection. xie and zhu ( ) other research has focused on the relationship between wind, ultraviolet radiation and covid- rates. rendana ( ) specifically concluded that low wind speed correlates with a rise in covid- cases (r = - . ). the study also reveals that low temperatures and the number of sunshine hours match with a higher number of covid- cases. Şahin ( ) environmental studies are now paying particular attention to the effects of covid- on wildlife and ecosystem conservation. certain initial enquiries, such as that by yuan et al. environmental specialists have more recently attempted to evaluate the effects of covid- on food supply chain efficiency and security, i.e., the availability and delivery of food and its accessibility. this is an important issue in environmental research as after the covid- crisis rose to the top of policy agendas, several decision makers warned of the problem of food supply and spikes of food prices (keulertz et al., ) . deaton and deaton ( ), for instance, in their appraisal of canada's food security and agricultural systems during the pandemic, noted that despite surges in demand and supply chain disruptions, there was no broad, rapid hike in food prices suggesting an adequate short-term supply of food. kerr ( ) environmental study academics also have explored how the monitoring of environmental settings can serve to prevent and predict such outbreaks and, consequently, improve public health. sims and kasprzyk-hordern ( ) their results suggest the great potential of wastewater monitoring to offer early warning signs on the extension of covid- circulation in a community, especially among those marked by mild or no symptoms. similar findings were reported by la rosa et al. ( ) in italy, kumar et al. ( ) in india, and mlejnkova et al. ( ) in the czech republic. in order to implement strategies to prevent the spread of covid- , environmental researchers have also evaluated aerosol transmission. morawska and cao ( ) , for example, recommend that the authorities take into account the airborne spread of covid- in their regulations to prevent transmission in indoor spaces. tang et al. ( ) review the evidence of aerosol transmission and concluded its plausibility and scored, based on the weight of the combined evidence, at out of . j o u r n a l p r e -p r o o f applying the concept of smart cities to tourism is known as smart tourism, that is, the creation of innovative spaces and improve services founded on a state-of-the-art technological infrastructure taking advantage of the surge of information and communication technologies. its objective is to guarantee a sustainable development of a territory, accessible to all, and facilitate the interaction and integration of the visitor with the environment. in the framework of the current pandemic, tourism companies and institutions are increasingly boosting these intelligent environments as a way to monitor and guarantee tourist security. jamal and budke ( ), for example, explain the importance of smart destinations and offer directions for tourism research and practice subsequent to the pandemic. specifically, the authors state that there is a need of a greater responsibility among residents and tourists to seek correct scientific facts about the virus and take sensible precautions, as well as exercise care to those suffering its adverse impact. they also advise of the need of a greater global coordination and attention to vulnerable destinations and an increase in the use of smart devices. chang et al. ( ) likewise advance the need of development of a more sustainable tourism as a way to balance tourism, travel, and the hospitality industry. among other measures, the authors highlight the urgency of personal protection equipment during travel, the need to implement comprehensive and frequent monitoring to control diseases and pandemics, as well as the necessity to impose updated rules to monitor medical facilities and highly trained on-board healthcare workers. gallego and font ( ) also advanced a method of the early detection of the reactivation of tourist markets to help mitigate the effects of the covid- crisis resorting to analyzing skyscanner data as to flight searches between november and december . their results reveal how big data can offer timely information crucial to identify highly volatile situations and that destination firms must improve their big data analytical competence. environmental studies are also evaluating the effects of artificial intelligence (a field of science and technology investigating the combination of algorithms designed to create machines emulating human capabilities) on how to control covid- . ghazaly et al. ( ) , for example, conclude that artificial intelligence can be applied to deploy intelligent diagnostic and treatment devices. in addition, they surmise that it can serve for teleworking, distance education and intelligent production to ensure a minimal disruption of daily life. along a same vein, nadikattuan et al. ( ) propose an innovative localization method to track through sensors the position of individuals in an outdoor environment. specifically, the authors suggest a novel smart device resorting to artificial intelligence to maintain social distancing as well as to detect covid- symptoms. similarly, simsek and kantarci ( ) recommend an artificial intelligence-driven strategy for mobile assessment agents during epidemics/pandemics. by means of simulations of real street map mobile crowd-sensing simulator, the authors signal "… that on the th day following the first confirmed case in the j o u r n a l p r e -p r o o f city under the risk of community spread, ai-enabled mobilization of assessment centers can reduce the unassessed population size down to one fourth of the unassessed population under the case when assessment agents are randomly deployed over the entire city" (simsek and kantarci, , p. ). main thematic networks serving to evaluate the interaction between covid- and smart cities a last research theme among the documents identified by the searches of the databases focuses on the future tasks that we, as a society, and environmental researchers, must develop to mitigate climate change. howarth et al. ( ) , for example, posit that covid- has raised our awareness of how vulnerable we are in the face of climate change. according to these authors, mitigating climate change requires a more carefully planned, inclusive, less disruptive and greater sustained response through deliberative engagement mechanisms aiming to build a social mandate for post-covid climate action. in an effort to clarify the measures of sustainability that we must assume in the future help mitigate climate change, meles et al. ( ) examined the impact of the covid- pandemic on the eu co emissions target. the authors claim that although existing climate policy measures in the wake of the pandemic will reduce emissions more than % by , this will not be enough to meet the guidelines of the paris agreement. hence a need for more strict and sustainable measures. klenert et al. ( ) summarized the main lessons of covid- applicable to reduce climate change: (i) delayed action increases mitigation costs; therefore, institutions and long-term incentives should be drawn up; (ii) there is a need to involve citizens in the reasons of climate change, thereby policies should be more appealing; (iii) actions in the future should address distributive concerns as well as measures of mitigation; (iv) individuals, governments and organizations must work together to encourage multilevel collaboration, tying in large emitters; and (iv) citizens must be inoculated against misinformation. environmental researchers have likewise highlighted that covid- should serve as a starting point to promote social sustainable habits. kleinschroth and kowarik ( ) resorted to google trends to estimate changes in online searches for basic activities typically carried out in urban green areas (e.g., walking) before and after the onset of the pandemic. their results indicate that searches for outdoor walks and parks during the pandemic increased exponentially. the authors therefore suggest that the crisis underscores the value of preserving and further developing urban green infrastructure. along the same line, mukanjari and sterner ( ) propose that efforts to revitalize the economy after covid- should resort to green and renewable resources as more polluting means and materials (e.g., airports, fossil fuel, carbon) experienced the largest decreases in stock value during the crisis. furthermore, goffman ( ) states that the best way to simultaneously alleviate the rapidly moving pandemic crisis and the slower moving environmental crisis is through glocalization, that is, a world in which people live far more local lives than in recent decades but foster a greater global awareness through a connective world. the author adds that these measures j o u r n a l p r e -p r o o f should be accompanied by a reduction of air and automobile travel and an increase in local production and smart growth. the global spread of the severe acute respiratory coronavirus syndrome and the response to the pandemic by national authorities is unprecedented in its speed and scope. the current crisis has likewise placed information professionals in the centre of an information pandemic (torres-salinas, ) as they are required to turn to scientific texts as sources to offer the public simple, instructive and accessible information. moreover, academics are turning to bibliometric surveys to facilitate navigating through the scientific databases to synthesize and simplify access to the findings of research on covid- . despite the fact that covid- bibliometric studies have been carried out in the fields of biology and medicine (bonilla-aldana et al. ), socioeconomics (verma and gustafsson, ) and tourism (sigala, ) , no study of this ilk has explored the growing number of covid- - this also suggests that populations suffering from poorer air quality suffer more covid- infections, hospital admissions and deaths. water pollution improved during the covid- lockdown as evidenced by decreases in spm and metal concentrations in lakes and rivers. (ii) covid- and meteorological factors. environmental studies indicate significant correlations between wind speed, air pressure, humidity, ultraviolet radiation and covid- rates. despite certain findings suggesting an opposite effect between temperature and virus cases, little research corroborates such a link. (iii) effects of covid- on wildlife and agricultural conservation. environmental study specialists exploring the changes provoked of covid- on food supply chains and changes in waste habits during the pandemic offer arguments as to its impact on the conservation of biodiversity. (iv) covid- and epidemiology. environmental researchers claim that monitoring wastewater has a great potential to offer early warning signs on the degree of distribution of covid- in a community, especially among individuals bearing mild symptoms or no symptoms at all. (v) covid- and smart cities. environmental and urban researchers coincide that the use of artificial intelligence, sig and smart devices can serve in monitoring the mobilization of citizens in urban and tourism destinations, and thus play a vital role in preventing an advance of the pandemic. propagation. hence prospective studies are now in a good position to evaluate this relationship in different tropical zones. thirdly, future studies exploring social changes in energy consumption, real environmental purchases, waste behavior and leisure activities is required to advance in the understanding of the effects of pandemics on social daily routines. fourthly, although studies have identified airborne transmission of covid- , no analysis to date has specifically enquired into what extent meteorological factors or urban contexts affect this type of transmission. lastly, more environmental studies are required to assess the manner in which smart devices and monitoring platforms can assist prevention and avoidance of covid- propagation. finally, the current study only isolated documents indexed in the web of science and scopus and has not identified all published materials. this study likewise did not take into account preprints, early versions of scientific articles, which also represent a rapid means of dissemination of information (torres-salinas, ). however, despite the fact that preprints enable a rapid dissemination of findings, commentaries and critical reviews through open access platforms, we contend that they increase the risk of spreading false information as they are posted devoid of independent quality control. considering the daily coverage that covid- receives from the international media, erroneous conclusions could be quickly replicated beyond the scientific sources leading to misinformation. finally, the current study did not account for the different methods applied by environmental researchers. future research should discuss these diverse methods applied to each of the above-mentioned thematic networks. hence the findings of this article offer information professionals with a true and objective framework of the main current scientific research combining covid- and the environment. journalists as well as information and documentation professionals, by incorporating these findings into their news stories and narratives, would bolster and guarantee their credibility in a time marked by an unprecedented information explosion. a bibliometric analysis. travel medicine and infectious disease a spatio-temporal analysis for exploring the effect of temperature on covid- early evolution in spain impact of covid- event on the air quality in iran effects of air temperature and relative humidity on coronavirus survival on surfaces covid- research articles downloadable database a bibliometric analysis of covid- research activity: a call for increased output exposure to nitrogen dioxide (no ) from vehicular emission could increase the covid- pandemic fatality in india: a perspective the effects of temperature and relative humidity on the viability of the sars coronavirus a charter for sustainable tourism after covid- smart korea: governance for smart justice during a global pandemic an approach for detecting, quantifying, and visualizing the evolution of a research field: a practical application to the fuzzy sets theory field scimat: a new science mapping analysis software tool air pollution exposure and covid- in dutch municipalities. environmental & resource economics, - lockdown for covid- in milan: what are the effects on air quality? the science of the total environment air pollution and covid- : the role of particulate matter in the spread and increase of covid- 's morbidity and mortality can atmospheric pollution be considered a co-factor in extremely high level of sars-cov- lethality in northern italy? environmental pollution la agencia del medicamento ha aprobado en un mes ensayos de fármacos para el covid- . el país, de abril will the coronavirus threaten our food? cleaning the river ganga: impact of lockdown on water quality and future implications on river rejuvenation strategies coronavirus disease (covid- ): a machine learning bibliometric analysis changes in air passenger demand as a result of the covid- crisis: using big data to inform tourism policy el escenario post-covid- , una ventana de oportunidades. eldiario.es, de abril novel coronavirus forecasting model using nonlinear autoregressive artificial neural network the wake of covid- , is glocalization our sustainability future? sustainability: science, practice and policy agriculture, transportation, and the covid- crisis survival of coronaviruses in water and wastewater air pollution aggravating covid- lethality? exploration in asian cities using statistical models will covid- have a lasting impact on the environment? bbc current status of global research on novel coronavirus disease (covid- ): a bibliometric analysis and knowledge mapping. ssrn, april nd building a social mandate for climate action the nexus between covid- , temperature and exchange rate in wuhan city: new findings from partial and multiple wavelet coherence smart tourism destinations: ecosystems for tourism destination competitiveness covid- virus outbreak lockdown: what impacts on household food wastage? environment covid : lets act now: the urgent need for upscaling agroecology in uganda ( ) air pollution improvement and mortality rate during covid- pandemic in india: global intersectional study the impact of covid- on water and food systems: flattening the much bigger curve ahead covid- crisis demonstrates the urgent need for urban greenspaces five lessons from covid- for advancing climate change mitigation first proof of the capability of wastewater surveillance for covid- in india through detection of genetic material of sars-cov- first detection of sars-cov- in untreated wastewaters in italy potential impacts of covid- on canadian farmland markets bat origin of a new human coronavirus: there and back again containing the spread of coronavirus disease (covid- ): meteorological factors and control strategies conserving africa's wildlife and wildlands through the covid- crisis and beyond effects of temperature variation and humidity on the death of covid- in wuhan, china. science of the total environment air quality variation in wuhan, daegu, and tokyo during the explosive outbreak of covid- and its health effects can a paper-based device trace environmental science & technology covid- and eu climate targets: can we now go further? environmental & resource economics preliminary study of sars-cov- occurrence in wastewater in the czech republic airborne transmission of sars-cov- : the world should face the reality charting a "green path" for recovery from covid- novel economical social distancing smart device for covid- (ssrn scholarly paper id ) preventing bat-born viral outbreaks in future using ecological interventions when science goes viral: the research response during three months of the covid- outbreak salud planetaria y covid- : la degradación ambiental como el origen de la pandemia actual. instituto de salud global, de abril assessing nitrogen dioxide (no ) levels as a contributing factor to coronavirus (covid- ) fatality. science of the total environment mental health and covid- impact of covid- lockdown on pm , so and no concentrations in salé city (morocco) investigating a serious challenge in the sustainable development process: analysis of confirmed cases of covid- (new type of coronavirus) through a binary classification using artificial intelligence and regression analysis temperature significantly changes covid- transmission in (sub)tropical cities of brazil data mining and analysis of scientific research data records on covid- mortality, immunity, and vaccine development-in the first wave of the covid- pandemic (ssrn scholarly paper id ) smart sanitation-biosensors as a public health tool in sanitation infrastructure covid- impact on fruit and vegetable markets covid- and the canadian cattle/beef sector: some preliminary analysis impact of weather on covid- pandemic in turkey covid- challenges to pakistan: is gis analysis useful to draw solutions? science of the total environment imprints of pandemic lockdown on subsurface water quality in the coastal industrial city of tuticorin, south india: a revival perspective increased ozone levels during the covid- lockdown: analysis for the city of rio de janeiro, brazil. the science of the total environment tourism and covid- : impacts and implications for advancing and resetting industry and research future perspectives of wastewater-based epidemiology: monitoring infectious disease spread and resistance to the community level artificial intelligence-empowered mobilization of assessments in covid- -like pandemics: a case study for early flattening of the curve converting home spaces into food gardens at the time of covid- quarantine: all the benefits of plants in this difficult and unprecedented period what is environmental studies? -day lockdown in india dramatically reduced air pollution indices in lucknow air quality development during the covid- pandemic over a medium-sized urban area in thailand sunlight ultraviolet radiation dose is negatively correlated with the percent positive of sars-cov- and four other common human coronaviruses in the u.s. science of the total environment aerosol transmission of sars-cov- ? evidence, prevention and control changes in air quality during the lockdown in barcelona (spain) one month into the sars-cov- epidemic ritmo de crecimiento diario de la producción científica sobre covid- análisis en bases de datos y repositorios en acceso abierto investigating the emerging covid- research trends in the field of business and management: a bibliometric analysis approach a preliminary assessment of the impact of covid- on environment -a case study of china natural gas from shale formation: a research profile research status of shale gas: a review covid- . global literature on coronavirus disease effects of temperature and humidity on the daily new cases and new deaths of covid- in countries association between ambient temperature and covid- infection in cities from china the -ncov epidemic control strategies and future challenges of building healthy smart cities survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater regulating wildlife conservation and food safety to prevent human exposure to novel virus. ecosystem health and sustainability covid- and surface water quality: improved lake water quality during the lockdown. the science of the total environment drastic improvements in air quality in ecuador during the covid- outbreak air quality changes in new york city during the covid- pandemic nox emission reduction and recovery during covid- in east china this study was supported by an excellence project awarded by the junta de andalusia [ref:b-sej- -ugr ] and by a grant from the fundación ramón areces [rareces - ]. adams, m. d. ( ). air pollution in ontario, canada during the covid- state of emergency. total environment, , . anónimo ( ). trump reaviva la teoría de que el virus surgió en un laboratorio chino. la razón, key: cord- -n zqc gm authors: bzdok, danilo; dunbar, robin i.m. title: the neurobiology of social distance date: - - journal: trends cogn sci doi: . /j.tics. . . sha: doc_id: cord_uid: n zqc gm abstract never before have we experienced social isolation on such a massive scale as we have in response to covid- . yet we know that the social environment has a dramatic impact on our sense of life satisfaction and well-being. in times of distress, crisis, or disaster, human resilience depends on the richness and strength of social connections, as well as active engagement in groups and communities. over recent years, evidence emerging from various disciplines has made it abundantly clear: loneliness may be the most potent threat to survival and longevity. here, we highlight the benefits of social bonds, choreographies of bond creation and maintenance, as well as the neurocognitive basis of social isolation and its deep consequences for mental and physical health. j o u r n a l p r e -p r o o f are conventionally most concerned about all had much less impact on survival rates. key factors included obesity, diet, alcohol consumption, how much exercise was taken, the drug treatments prescribed, and local air pollution. these authors conducted a follow-up analysis of studies of longevity in older people, which followed ~ . million people over an average of ~ years [ ] : social isolation, living alone and feeling lonely increased the chances of dying by about %, even after accounting for age, sex and health status. many other studies have shown that social isolation (though not self-reported feelings of loneliness) was a significant predictor of the risk of death. for example, a longitudinal analysis of ~ , british men and women in their fifties [ ] found that being socially isolated increases the risk that you will die in the next decade by about %. quantitative analysis of nearly ~ , married couples in the american medicare database revealed that, for men, the death of their spouse increased their own chances of dying in the immediate future by %. the death of the husband in turn increased the wife's risk of dying by % [ ] . similar effects on morbidity rates have been found with respect to social support. a series of elegant prospective studies using data from the framingham heart study [ , ] found that the chances of becoming happy, depressed or obese were all strongly mirrored by similar changes in the closest friend. there was a smaller significant effect due to the behaviour of the friends' friend. even a just detectable effect was present due to the friend of a friend's friend, but nothing beyond. this contagion phenomenon was especially strong if the friendship was reciprocal (i.e., both individuals listed each other as a friend). if the friendship was not mutual, the social contagion effect was negligible. the investigators also documented a strong effect of "geographical contagion". if you have a happy friend who lives within a mile radius, you are % more likely to become happy. and you are % more likely to be happy if your next-door neighbour is happy. people who belong to more groups are less likely to experience bouts of depression. such findings emerged from the uk longitudinal study of ageing (elsa) that repeatedly profiled around ~ , people from the age of onwards. previous research showed [ ] that depressed people reduced their risk of depression at a later time point by almost a quarter if they joined a social group such as a sports club, church, political party, hobby group or charity. indeed, joining three groups individuals were more immersed in their local community and trusted their neighbours more [ ] [ ] [ ] . the causal directionality was difficult to pin down in these cases because of the cross-sectional nature of the data. nevertheless, path analysis provided some indication that intensity of social exchange was the candidate driver. the impetus to access social capital in the wider community [ ] extends beyond humans. there is now a wealth of evidence from long-term field studies of wild baboons that socially wellconnected females experience less harassment by other monkeys [ , ] , have lower levels of cortisol stress hormones [ , ] , faster wound healing [ ] , produce more offspring and live longer [ ] [ ] [ ] [ ] . such ramifications of social capital appear to hold up across a diversity of species, including chimpanzees [ ] , macaques [ ] [ ] [ ] , feral horses [ , ] and dolphins [ ] . a key underlying reason for these effects, at least in humans, is likely that loneliness directly impairs the immune system, making you less resistant to diseases and infections. research found [ ] that freshmen students who reported feeling lonely had a reduced immune system response when they were given a flu vaccine compared to students who felt socially well engaged. moreover, those students with only - close friends had significantly poorer responses than those with - friends. these two effects seemed to interact with each other: having many friends (a large social group of nineteen or twenty friends) seems to buffer against a weakened immune response. yet, feeling lonely and having few friends results in a particularly poor immune defence. other investigators [ ] used data from the framingham heart study to show that people with fewer contacts in their social network had elevated serum fibrinogen concentrations. in contrast, people enjoying many social contacts had low fibrinogen levels. fibrinogen plays an important role in blood clotting when a blood vessel has been ruptured, as well as facilitating wound healing and tissue repair more generally: high concentrations thus signal poor health. endorphins constitute a core component of the psychoendocrine mechanism underpinning friendship (see box ). other research found [ ] that social bonds stimulate the release of the body's natural killer cells, one of the white blood cells of the innate immune system whose core function is to destroy harmful bacteria and viruses. people who are more socially integrated have better adjusted biomarkers for physiological function, as indexed by lower systolic blood pressure, lower body mass index, and lower levels of creactive protein -the latter being another molecular response to inflammation. this insight was evident in each of four age groups (adolescents, young adults, middle age and old age) based on data from four large longitudinal american health databases [ ] . the investigators found that, in j o u r n a l p r e -p r o o f adolescence, lack of social engagement had as big an effect on risk of inflammation as lack of physical activity. in old age, lack of friends had a bigger effect on risk of hypertension than the usually cited clinical causes like diabetes. even more worrying, the effects of social relationships on these physiological measures of good health during adolescence and young adulthood can persist into old age. in a longitudinal study of males, for example, research found [ ] that the more socially integrated a child was at six years of age, the lower their blood pressure and body mass index (a measure of fatness) two decades later in their early thirties. this result held up when they controlled for race, body mass index in childhood, parental socioeconomic status, childhood health and extraversion. social isolation may well have pervasive effects on brain connectivity. if rats are socially isolated when young (a condition that would give rise to feelings of loneliness in humans), neural function and plasticity are altered [ ] [ ] [ ] [ ] . in particular, episodes of social isolation can irretrievably alter the function of the prefrontal cortex (the part of the brain that is central to managing our social relationships [see below]), as well as its axon myelinisation (the laying down of the fatty sheaths around neurons that enable them to transmit signals faster and more efficiently) [ ] . while short periods of loneliness in humans rarely have any long-term adverse outcomes, persistent loneliness escalates the risk of alzheimer's disease and depression [ , ] . loneliness also leads to poor sleeping habits, with adverse psychological and physiological consequences [ ] . the fact that friends can have such dramatic effects on our health and well-being may lead us to suppose that the more friends we have, the better. however, the number of friends and family relationships we can manage at any given time is limited by cognitive constraints to ~ [ , ] . there is, however, considerable individual variation, with social network sizes ranging between approximately - . a number of fairly conventional factors are responsible for this variation: age (younger people typically have larger social networks than older people [ ] ), sex (females usually have larger social networks than males [ , ] ; though this does vary with age [ ] ), personality (extraverts have larger social networks than introverts [ ] ; women who score high on the neuroticism personality dimension have fewer acquaintances than those who score lower [ ] ). friendships, however, require the investment of considerable time to create and maintain. the emotional quality of a friendship depends directly on the time invested in a given social link [ ] [ ] [ ] . one prospective study estimated that it takes around hours of face-to-face contact over a three-month period to turn a stranger into a good friend [ ] . conversely, the emotional quality of a j o u r n a l p r e -p r o o f relationship will decline rapidly ( figure ) if contact rates drop below those appropriate to the relationship quality [ ] . time resources, however, are naturally limited: we devote only around % of our day to direct social interaction (excluding business-related interactions), equivalent to about . hours per day [ ] . given that our relationships are not all of equal value to us (friends serve a variety of different functions for us [ , ] ), we allocate our valuable time across our social network in such a way as to maximise the different benefits that friends of different quality provide [ ] . this dynamic results in a specific social fingerprint that is unique to each of us [ ] . nonetheless, there are some broadly consistent patterns: a % share of our time is devoted to our five closest friends and family, and a further % to the ten next closest individuals. in other words, % of the . hours a day we spend in social interaction are devoted to just people. social partners in the outermost layers of the social network each receive just secs of our time a day on average. this gives rise to a very distinctive layering to our social networks, with layers that have a characteristic fractal pattern: the innermost layers of closest friends is very small (typically people) but intense, the outermost ( ~ ) very large but more casual [ , ] . it is that inner circle of five closest friends and family that seems to matter most in terms of the buffering of both loneliness and disease. geographical distance also imposes strong constraints on the organization of friendship. the ' -min rule' provides an empirical reminder that people are less willing to visit friends and family who live more than mins away -no matter whether that involves travel on foot, by bicycle or by car [ ] . cutting across this effect is the influence of genetic relatedness: the kinship premium (i.e., the strong mutual benefits that kinship typically affords) incentivizes us to travel an extra mile to maintain contact with family than we are with friends [ ] . while the role of close contacts, like friends, is pivotal, other regular contacts can also contribute to one's social capital. previous authors [ ] famously claimed that weak -as opposed to strong, or close -ties provide important sources of external information. analyses of information flow in social networks suggest that sources outside the closest friendships offer few benefits [ ] . other benefits of interaction with more loose social ties can, of course, include heightened subjective well-being and sense of belonging to the local community [ ] . however, as is often the case in such studies, it is crucial to precisely define the meaning of weak versus strong ties, since all weak ties belonged to the same community (a student class). regular interaction with different people at the periphery of social networks can give rise to heightened perceived social and emotional fulfillment in ways that act as psychological buffers [ ] , although this might depend on personality or social style [ ] . social-affective processes in the presence of others take a different form than during the others' physical absence. already in a nursery, if a baby starts crying, other nearby babies hear the distress signal and typically also start crying by mere emotional contagion. in addition to utterances and prosody, humans tend to align their communication towards each other by imitating vocabulary, grammar, mimics and gestures. for instance, humans tend to unconsciously synchronize their facial expressions even with people who are directing gaze at somebody else [ ] . such subliminal motor and emotional resonance is typically found to be intrinsically rewarding [ ] . on the positive side, contagion processes can uplift an individual's happiness through people within the close neighborhood, but also miles apart [ ] . on the negative side, loneliness also spreads rapidly through an individual's social interaction partners, thus affecting even friends of friends of friends [ , ] . reading others' faces -impossible during a conventional phone call -may be an evolutionarily conserved means for exchanging pivotal information, which coevolved with the corresponding decoding machinery in brain and behavior responses (see next section). faces offer a plethora of social information about an individuals' sex, age, ethnicity, emotional expression and potentially their intentions and mental state (all of which influence the strength of the bond between two individuals [ ] ). throughout development, learning and maturing critically hinge on joint attention of two individuals on the same object [ , ] . such mentalizing and eye gaze processes have been repeatedly linked to the higher associative and the striatal reward circuitry [ , [ ] [ ] [ ] . some authors even argue that the importance of such facets of interpersonal exchange may explain why humans developed wide and white sclera in the eyes -more easily visible than in most animals [ ] . what may lead to greater vulnerability to predators for some species (by making the individual and her intentions more visible and exploitable) may have boosted learning and cooperation in human primates [ ] . such evolutionary adaptations facilitate how humans automatically represent the (visual) perspective of nearby others. making statements about objects channels [ ] . compared to actual interpersonal encounters, a surprising number of psychological constants exist in how humans entertain and juggle with social relationships in digital environments. for example, the upper bound of ~ contacts (cf. above), as well as the structure of these networks, appears to hold across both the real world and a variety of virtual online contexts [ , , , ] , suggesting that group size in today's society is still orchestrated by the same principles as when we were hunter-gatherers. indeed, several neuroimaging studies [e.g., , ] broadly confirm that our online social networks correlate with the volumes of the same core brain regions that resonate with the size of our offline networks [ , ] . these constancies suggest that lively virtual social interaction may similarly entrain faculties like memory and concept generation. conversely, paucity of social interaction and loneliness may have deleterious effects on the cognitive and memory systems. it is conceivable that enhancement or decline of cognitive and neural reserve may be mediated by analogous pathways potentially involving dendritic arborization in the hippocampal and prefrontal regions [ ] . the need for personalized interactions may already be reflected in the way that stock market traders sometimes add coded numbers to money transfers (e.g., , , instead of , , shares) as a potential replacement for the recognition of somebody's unique facial identity rather than remaining anonymous [ , ] . this attractor for a full range of face-to-face cues during social interactions may explain why emojis have become so popular: they replace the important emotional signals in the absence of the ostensive facial cues that we use for the interpretation of utterances in the face-toface environment. these considerations raise the important question how the brain implements toggling between real-world social interactions and virtual or imagined social interaction in the absence of physical contact [ ] . the right temporoparietal junction was proposed as a key switching relay between two antagonistic classes of neurocognitive processes: those more anchored in one's current external sensory environment and more stimulus-independent ones relying on internally generated information [ ] . this idea was later substantiated by a multi-modal neuroimaging study in , humans [ ] : the right and left temporoparietal junction explained most variation in functional coupling changes between all major brain networks. hence, these two association cortex regions may help mediate shifts of focus from the person in front of you to a person you are texting with on the phone, who is out of sight or touch. taken together, evidence of digital communication suggests that this new medium does not in fact change the general pattern of our social interactions or the numbers of people we contact [ , , , ] . the sizes of the layers in our social networks are unchanged by using digital media or virtual communication. also, the frequencies with which we contact certain people in each social j o u r n a l p r e -p r o o f layer are strikingly similar in the online and offline worlds. some digital vehicles, however, lack the communicative richness of real face-to-face interactions: when asked to rate their satisfaction with interactions with their five closest friends each day, participants rated face-to-face and skype interactions as equally satisfying and both as significantly more satisfying than interactions with the same individual by phone, text messaging, sms messaging, email or text-based social media such as facebook [ ] . human and non-human primates live in groups mainly to minimize external ecological threats, including predators, raiding by neighbors, and environmental risk. advanced forms of cooperation are rare in non-primate species [ , ] and probably emerged in non-human primates several million years ago. today, the average humans spends up to % of waking hours in the presence of others [ , ] . investing cognitive resources in keeping track of friends, family and colleagues is highly demanding -more costly than contemplating the physical facts [ , ] . not only time limits (cf. above) but also neurocognitive limits [e.g., ] effectively constrain how close one can be to how many individuals. but how is regular social stimulation reflected in neurobiology? in monkeys [ , ] and in humans [ , , , ] , various indices of sociality and measures of social network size are robustly associated with specific regions of the neocortex. these same regions are responsible for processing social information such as predicting others' intentions [ , ] . at least some of these brain-behavior associations may be cross-culturally consistent in humans, as evidenced by a structural neuroimaging study in the usa and china [ ] . whole-brain analyses have repeatedly highlighted a relationship between the ventromedial prefrontal cortex and measures of social network complexity and social competence [ , , , [ ] [ ] [ ] [ ] . the ventromedial prefrontal cortex and striatal nucleus accumbens have been found to play a key role in both social reward behaviors and the amount of social stimulation in humans [ ] and other mammals [e.g., , ] . functional neuroimaging has shown that these neural correlates are also implicated in tracking others' popularity status in real-world social networks [ ] . similarly, positron emission tomography has shown that, in humans, the density of mu-receptors for betaendorphin, especially in the ventromedial prefrontal cortex, correlates with social attachment style, for which endorphins are more important than other neuropeptides [ ] . other evidence, such as in a functional neuroimaging study on maintenance and manipulation of social working memory [ ] , has also related the dorsomedial prefrontal cortex to social network properties. there are similar correlations for social cognitive skills like mentalizing that are crucial to maintaining functional social relationships [ ] [ ] [ ] . analyses of social richness and brain morphology in humans tend to identify a neural network involving the prefrontal cortex with several parts of the so-called default mode network as being crucial for managing social networks (e.g., noonan et al., ) . this major brain network of the higher association cortex has probably recently expanded in primate evolution [ ] . its constituent regions are often thought to support several of the most sophisticated neurocognitive processes [ , ] . in monkeys, there is evidence that experimental manipulation of social group size results in adaptations in the volume of frontal brain regions, the posterior superior temporal sulcus or temporo-parietal junction, as well as the amygdala and other parts of the limbic system [ , ] . in humans, there is evidence for structural coupling between social network size measured by number of online friends and parts of the default mode network, including the hippocampus [ ] . from a clinical perspective, functional connectivity alterations in the default mode network have been demonstrated as a consequence of feelings of loneliness in younger adults [ ] . moreover, the default mode network is especially subject to vulnerability in normal cognitive aging [ ] , and is among the main brain circuits to be impacted by neuropathology in alzheimer's disease [ , ] . complementing higher associative parts of the human social brain [ ] , amygdala volume is larger in individuals with more extensive social networks in humans [ , ] . amygdalar functional connectivity was also reported to increase with canonical brain networks implicated in face perception and approach-avoidance behaviour [ ] . indeed, previous authors reported [ ] that a patient with complete bilateral amygdala lesions lacked a sense of appropriate personal space vis-àvis other people (figure ). this patient exhibited no discomfort when at close distances from another person, even to the point of touching the other's nose -despite the fact that their conceptual understanding of people's private physical space was intact. in contrast, healthy individuals typically show amygdala activation in response to close personal proximity. in a similar vein, the grey-matter volume of the amygdala correlated negatively with social phobia [ ] . the amygdala may hence be required to trigger the strong emotional reactions normally associated with personal space violations, thus regulating interpersonal distance in humans. such reports on the social brain often seemed to be in conflict about whether they highlight the prefrontal cortex or the amygdala of the limbic system. this apparent discrepancy was reconciled in a recent population neuroimaging study [ ] : social traits such as daily exchange with family, friends, and work colleagues were associated with brain morphology in ~ , uk biobank participants. particularly prominent findings were reported in the limbic system, where volumes varied consistently with various indicators of social isolation. less socially stimulated participants showed volume effects in various parts of the social brain including the ventromedial prefrontal j o u r n a l p r e -p r o o f cortex and the amygdala, in addition to the nucleus accumbens of the reward circuitry. volume effects in these regions were reported for several markers of brittle social integration, such as living in a socially "emptier" household, knowing fewer individuals with whom to regularly share experiences and concerns, feeling unsatisfied with one's friendship circles, as well as having grown up without brothers or sisters and being unhappy with one's family situation [ ] . this analysis also demonstrated wide-ranging sex differentiation in how traits of social isolation are linked to brain morphology. these findings underscore evidence from animals for a sex specific co-evolutionary relationship between the primate brain and social complexity [social brain hypothesis: , ]. the perspective of brain network integration in loneliness was investigated in a seminal neuroimaging study of intrinsic functional connectivity in ~ , humans [ ] . careful analysis showed that feelings of loneliness especially affect the neural communication strength between the limbic system and the default mode network as well as the communication strength inside of the default mode network. as a particularly discriminatory pattern for loneliness, impoverished functional modularity was found for the default mode network and its interacting brain networks. in contrast, a positive sense of one's meaning in life was linked to strengthened functional differentiation of the canonical network ensemble. the collective evidence led the investigators [ ] to argue that the default mode network and its coupling partners represents a neural signature reflecting one's own purpose in life versus social disconnection to others. according to unicef estimates, ~ million children worldwide live deprived of parents who could provide comfort and support. ~ million of these children grow up in institutions without the socioemotional context of a regular family. in one of the earliest randomized clinical trials of its kind, orphans raised in institutions were systematically compared to orphans who were later welcomed into a foster home [ ] . abandoned children were randomly assigned either to remain under the care of the institution or to transition to the care of foster-parents. their cognitive trajectories were monitored over several years. those children who remained in the institution showed significantly lower development indices and lower iqs [of around : ] than the adopted orphans. being deprived of social bonds with caregivers also led to a pernicious reduction in grey-and white-matter tissue and lower fiber tract integrity as evidenced by brain mri [ ] . institutional rearing was also shown to exacerbate the decay of the telomeres in cell nuclei [ , ] . these protection caps normally prevent chromosome deterioration, which acts like a cellular sand clock of aging. their shortening has major consequences for various biological pathways and health outcomes. the younger the children were when adopted by a foster family, the better the cognitive performance later [ ] . impoverished cognitive domains include memory and executive function: for orphans who transitioned to a foster home, some cognitive facets remained below-average throughout later life (e.g., short-term visual memory and attention allocation). other cognitive dimensions (e.g., visual-spatial memory and spatial working memory) caught up with a normal trajectory at age [ ] . such unique evidence underlines the fact that lack of socioemotional context in early life severely impedes brain development and maturation of the cognitive repertoire, which can be partially mitigated by developing social bonds to non-genetic parents (see box ) . early psychosocial deprivation also shows inter-generational effects, which are probably mediated through maternal and epigenetic effects [ ] . social isolation in childhood leads to molecular annotations of the genetic strand (such as methylation or phosphorylation of the histones that provide the structure for dna strands) that are passed on to influence how children cope with stress and in turn how they raise their own children. for instance, in rats, socioemotional experience as a pup has an impact on how the rat's own pups later deal with stress and high anxiety levels [ ] . epigenetic regulation of gene transcription is involved in how maternal care promotes the rat pup's brain development and cognitive maturation. more licking and grooming by the mother increases protein expression of the grm gene in the pup's hippocampus. this up-regulated gene transcription leads to greater availability of glutamate receptor proteins in hippocampal cells for inter-neuronal signaling [ ] . in humans, a longitudinal neuroimaging study indeed showed that social support from the mother promotes volume growth trajectories in the hippocampus, and predicts socioemotional development and emotion regulation in early adolescence [ ] . in young rhesus monkeys, loss of social contact to the mother leads to behavioral aberrations that last right into adulthood. such social isolation was shown to entail down-regulated dendritic growth in the prefrontal cortex and reduction in gene expression in the amygdala [ ] . social adversity undergone by children with institutional upbringing led to disturbed functional connectivity between the prefrontal cortex and the amygdala [ ] . such perturbed brain maturation through social deprivation may be mediated by glucocorticoids, which are known to be inhibited by maternal care in primates [ ] . hence, maternal care is a critical enrichment of the social environment that promotes maturation, expression of growth hormones, and synaptogenesis in various brain circuits. in contrast, social neglect leads to disturbed social attachment, as well as increased aggression and hyperactivity, often potentially lifelong [ , ] . how vulnerable an individual is to parental deprivation is subject to complex nature-nurture interactions that are strongly conditioned on personality and overall genetic endowment [ , ] . rats separated early from their mothers were impaired in adult life in emotion regulation and arousal management [ ] . early socioemotional isolation of rat pups had impact on whether these rats later showed healthy responses to stress by mounting adequate cortisol levels [ ] . hormones of the hypothalamic-pituitary-adrenocortical (hpa) axis are an important endocrine mechanism of stress neurobiology that plays a key role in social isolation. in baboon monkeys, infant survival is jeopardized for mothers who are more socially isolated and not well integrated in the local communities including ties to sisters, adult daughters, and other mothers [ ] . monkey mothers with a thinner social network are less likely to have infants which themselves have high fitness [ ] . female baboon monkeys with a larger close social circle of grooming partners have healthy cortisol levels and typically deal better with stressful situations [ , , ] . when one of these strong social bonds is disrupted, such as when a close member of the social group is killed by predators, cortisol titres rise in the blood. such monkeys then tend to seek out new connections to "repair" the lost link in their social network [ ] . a lower-than-usual cortisol level in the morning is indicative of extended stress periods in adults [ ] . the same diurnal cortisol dynamic is frequently observed in disturbed child-caregiver relationships [ ] . in rhesus monkeys, a low hormone response has been observed after repeated separations from the mother. the same observation has been reported for children who were moved between several caregivers. an intact child-caregiver relationship probably provides a stress reserve to adrenoreceptor responses so that children get over stressful episodes quicker [ , ] . after undergoing adversity in early childhood, such as emotional or physical neglect, maltreatment, or maternal separation, enhancement of the child-caregiver relationship can mitigate the effect of previous hits to the hpa system. early disturbance in important social relationships is linked to dysfunctional cortisol hemostasis in adult life [ ] . in some neglected children, ensuing problems and behavioral disruptions can even be exacerbated in adult life [ ] . abnormal blood cortisol levels can potentially be prevented, mitigated or restored by family-based therapy and other interventions [ ] . nonetheless, dysregulated diurnal cortisol levels are further linked to various mental disorders including major depression, substance abuse, and post-dramatic stress disorder [ ] , in addition to stress-induced impact on the immune, cardiovascular, and metabolic systems [ , ] . further insight into the neurobiology of social isolation has also been derived from rigorous experiments with adult primates (see also box ). in one study, monkeys were separated from others to live alone for . years [ ] . subsequently, monkeys were re-integrated into social groups of four monkeys housed together. repeated positron emission tomography (pet) scanning revealed increased levels of d receptors in the basal ganglia, which includes key nodes of the reward j o u r n a l p r e -p r o o f circuitry (see above), after being socially housed. this neurochemical adaptation in the monkeys' brain circuitry was apparent after as few as months of social rehabilitation [ ] . these authors also reported several differences in respect of social integration and social rank: monkeys of higher rank were groomed more by others. in contrast, subordinate monkeys spent more time by themselves. as a consequence at the behavioral level, the lower-rank monkeys were also significantly more willing to self-administer cocaine, which may also relate to heightened drug abuse in lonely humans [ ] . such molecular imaging evidence shows that changing from social deprivation to an environment with constant social stimulation causes neural remodeling in the dopaminergic neurotransmitter pathways in non-human primates, which may be clinically relevant for substance abuse disorders in humans. we are social creatures. social interplay and cooperation have fuelled the rapid ascent of human culture and civilization. yet, social species struggle when forced to live in isolation. the expansion of loneliness has accelerated in the past decade. as one consequence, the united kingdom has launched the 'campaign to end loneliness' -a network of over national, regional and local organizations to create the right conditions for reducing loneliness in later life. such efforts speak to the growing public recognition and political will to confront this evolving societal challenge. these prospects should encourage us to search for means to mitigate possible negative backlash. we offer some suggestions in box . additional insight into stress-responsive brain systems is imperative to tailor clinical decision making and therapeutic interventions to single individuals. there is also a dire need for additional longitudinal research on the hpa axis and the cortisol response to psychological stressors. we are grateful to guillaume dumas and tobias kalenscher for valuable comments on a previous version of the manuscript. db was supported by the healthy brains healthy lives initiative (canada first research excellence fund), and by the cifar artificial intelligence chairs program (canada institute for advanced research). primates service their relationships through social grooming. grooming triggers the endorphin system in the brain through a very specific neural system: the afferent ct fibres [ ] . these axon bundles have receptors at the base of most hair follicles, have the unusual properties of being unmyelinated (and hence very slow, especially compared to the pain receptors in the skin), with no return motor loop (unlike pain and other proprioceptive neurons), respond to a very specific stimulus (light slow stroking at ~ . cm per sec) and directly trigger the endorphin reward system [ ] . although humans no longer have the full fur covering that encourages social grooming, we still have the receptors and instead use physical contact in the form of touching, stroking, caressing, and hugging as a means for strengthening social ties in our more intimate relationships [ , ] . physical touch is intimate, and hence limited mainly to close family and friends ( figure ). to bond our wider range of relationships as well as our more intimate ones, humans exploit a number of behaviours that turn out to trigger the endorphin system. these joint activities include laughing [ , ] , singing [ , ] , dancing [ , ] , feasting [ ] and emotional storytelling [ ] . an important feature of all these behaviours is that behavioural synchrony seems to ramp up the level of endorphin release [ , ] . in baby primates, close social interaction is not only beneficial, but critical for maturation and resilience. experiments in baby monkeys showed that upbringing in social isolation during the first years causes a variety of social deficits. when separated early from their mothers, baby monkeys showed strong symptoms of social withdrawal: self-hurting behaviour like biting, stereotypical and repetitive motor behaviour, excessive avoidance behavior towards others as well as poor social and maternal skills as adults. when separated later from their mothers, baby monkeys tended to indiscriminately approach unknown monkeys without fear [cf. ] . reports of human children in some crowded russian and rumanian orphanages painted a strikingly similar picture: socially and emotionally abandoned children showed either forward-j o u r n a l p r e -p r o o f backward rocking tics and social escape or overly strong attachment style, analogous to neglected baby monkeys [cf. ] . these cases invigorated the then-contested claim that mother-child bonds are indispensable for normal development, and that foster-care parents can compensate many of these needs [ , , , ] . disruption of social interplay during critical development impacts negatively on cognitive, verbal, social and motor performance, and predisposes to mental health issues. in other words, early neglect remains measurable in brain and behaviour in later life. the socioemotional dialogue between caregiver and baby is mediated in several important ways. mothers speak to their offspring in "baby talk", which potentially evolved only recently in humans [ ] . accompanied by direct face-to-face exchanges, these communication bouts with characteristic vocabulary and prosody promote infant development milestones. the interpersonal stimulation grabs the baby's attention, she gains weight faster, modulates her emotional state, and enhances various health outcomes. mother-infant communication is also delivered through direct skin-to-skin contact [ ] . postnatal touching bolsters mother-infant bonding, alleviates anxiety, and provides intrinsic pleasure through endorphin release [ ] [ ] [ ] . throughout life and quite independent of geography, primate societies are orchestrated by the creation, curation, and cultivation of social bonds though purposeful social closeness. among the many consequences of loneliness on body and mind, the scarcity of social contact encourages drug compensation behaviour, such as alcoholism, possibly via non-social rewards triggering dopaminergic neurotransmitter pathways [ ] . at the genetic level, loneliness was shown to entail under-expression of anti-inflammatory genes involved in glucocorticoid response and over-expression of genes related to pro-inflammatory immune responses [ ] . fortunately for future clinical intervention, loneliness may be a modifiable determinant in healthy aging [ ] . as people grow older, the social network typically becomes smaller -naturally diminishing the cognitive stimulation through frequent and intense social interaction on a daily basis, thus potentially reducing the neural reserve. over the last century, the average human lifespan in developed nations has increased by nearly three decades. on the other hand, older people were also reported to show a decline in the capacity to take other people's point of view, as demonstrated in three separate mentalizing tasks [ ] . these authors showed that social cognition j o u r n a l p r e -p r o o f deficits were related to decreased neural activity responses in the medial prefrontal default mode network [ ] . this capacity is likely to be particularly important when introspecting other people's minds who are not physically present -where social cues like facial expression, mimics, and gestures are missing. both limited social stimulation and weakening social reflection capacities relate to the sense of loneliness in complicated and important ways [ ] . once lonely, bias for negative information processing of cues from others hinders social rehabilitation in a downward cycle [ , ] . many recent studies have corroborated the corpus of empirical evidence that the feelings of loneliness escalate the risk of certain neurological diseases and especially alzheimer's disease in later life [ ] . social isolation at massive scale risks creating cohorts of individuals who are socially dysfunctional. it may therefore be important to identify ways of mitigating the worst of the effects so as to alleviate the consequences. the following possible countermeasures may be worth exploring:  one promising intervention would involve creating opportunities where mutual social support relationships (friendships) could develop naturally. you cannot, however, force people to become friends: both parties need to be willing to devote resources to each other in a context where available time budget for social engagement is limited [ , ] and there are competing friendship interests [ ] . however, by providing more opportunities for people to meet in congenial environments, new friendships may blossom.  social neuroscientists [ ] undertook a longitudinal intervention study on matched adults who underwent regular training sessions. several months of cognitive training improved empathy for others' affective state or perspective-taking of others' mental state, which resulted in structural remodeling in brain regions belonging to the social brain network, including the frontoinsular network and the default mode network. daily affective training resulted in thickening of the right anterior and mid-insula, with correspondingly enhanced compassion ratings. different training regimes correlated with different brain regions. further research is urgently needed to explore therapeutic interventions using training of social capacities in socially deprived humans. j o u r n a l p r e -p r o o f  one important lesson is that joining clubs can have important benefits in reducing both a sense of loneliness and psychological or psychiatric conditions [ ] . one obvious solution is to encourage vulnerable individuals to join social groups and communities that suit their interests and abilities. establishing a wide range of such clubs is likely to be much cheaper than paying for carehomes and prisons.  singing is known to have a dramatic, immediate effect on creating a sense of social engagement and elevating psychological well-being [the "ice-breaker effect" : ] . vulnerable individuals could be encouraged to join choirs and community singing groups. encouragement and funding may need to be invested in establishing a network of choirs.  use of video-embedded digital communication is likely to gain in importance. this is especially true where family and friendship groups can meet in the same virtual space. the visual component of the interpersonal encounter appears to play a key role in creating a more satisfying experience of digital social media [ ] . emotional closeness at the start of the study is set at for both groups. redrawn from [ ] . regions. in , people from several countries, this study investigated the permissibility of social touch [ ] . the authors showed that human social touch is particularly dependent on the nature of the relationship. the topography of accepted social touching depends on many factors, including a) emotional relationship, b) type of interpersonal bond including kinship, c) sex, and d) power dynamics. close acquaintances and family members are touched for more different reasons. culture influence, measured in five countries, was small. female, rather than opposite-sex, touch was evaluated as more pleasant, and it was consequently allowed on larger bodily areas. reproduced from [ ] .  what further refinements of online digital media might improve people's function in creating and maintain friendships, especially for the housebound? it is insufficiently known which types of modern medium best mimic which neurocognitive facets of real social interaction.  which neurobiological mechanisms explain how the default mode network and its connections to subordinate brain systems support higher social capacities, and their decline in social deprivation? this associative brain network needs to be more completely understood; especially regarding the congruencies and idiosyncrasies between healthy aging trajectories, the experience of social isolation, and vulnerability to neurodegenerative pathologies. in terms of progress towards causal understanding, putting a premium on longitudinal studies holds out unprecedented promise.  across the entire lifespan, to what extent does reduced social stimulation or too few social contacts lead to loss in general capacities of the cognitive repertoire? how much do people struggling with cognitive load have issues maintaining many active social relationships? or both? progress in this chicken-and-egg problem will shed light on the aetiopathology of the loneliness, and usher towards new intervention strategies. the phenotype of loneliness evolutionary mechanisms for loneliness perceived social isolation and cognition the growing problem of loneliness loneliness and attention to social threat in young adults: findings from an eye tracker study the cultural context of loneliness: risk factors in active duty soldiers it is all about who you know: social capital and health in lowincome communities counting on kin: social networks, social support, and child health status impact of social connections on risk of heart disease, cancer, and all-cause mortality among elderly americans: findings from the second longitudinal study of aging (lsoa ii) associations of social networks with cancer mortality: a meta-analysis social and emotional support and its implication for health the effect of social relationships on survival in elderly residents of a southern european community: a cohort study social networks and health social isolation, social activity and loneliness as survival indicators in old age; a nationwide survey with a -year follow-up social relationships and mortality risk: a meta-analytic review loneliness and social isolation as risk factors for mortality: a meta-analytic review social isolation, loneliness, and all-cause mortality in older men and women the effect of widowhood on mortality by the causes of death of both spouses dynamic spread of happiness in a large social network: longitudinal analysis over years in the framingham heart study connected: the surprising power of our social networks and how they shape our lives social group memberships protect against future depression, alleviate depression symptoms and prevent depression relapse breaking bread: the functions of social eating social structure as a strategy to mitigate the costs of group living: a comparison of gelada and guereza monkeys religiosity and religious attendance as factors in wellbeing and social engagement. religion focused grooming networks and stress alleviation in wild female baboons social stressors and coping mechanisms in wild female baboons (papio hamadryas ursinus) social affiliation matters: both same-sex and opposite-sex relationships predict survival in wild female baboons social bonds of female baboons enhance infant survival strong and consistent social bonds enhance the longevity of female baboons the benefits of social capital: close social bonds among female baboons enhance offspring survival network connections, dyadic bonds and fitness in wild female baboons social support reduces stress hormone levels in wild chimpanzees across stressful events and everyday affiliations family network size and survival across the lifespan of female macaques decoupling social status and status certainty effects on health in macaques: a network approach responses to social and environmental stress are attenuated by strong male bonds in wild macaques sociality increases juvenile survival after a catastrophic event in the feral horse (equus caballus) social bonds between unrelated females increase reproductive success in feral horses social and genetic interactions drive fitness variation in a free-living dolphin population loneliness, social network size, and immune response to influenza vaccination in college freshmen social connectedness is associated with fibrinogen level in a human social network opiate antagonist prevents μ-and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth social relationships and physiological determinants of longevity across the human life span friends with health benefits: the long-term benefits of early peer social integration for blood pressure and obesity in midlife postweaning social isolation enhances morphological changes in the neonatal ventral hippocampal lesion rat model of psychosis abnormalities of presynaptic protein cdcrel- in striatum of rats reared in social isolation: relevance to neural connectivity in schizophrenia region-specific impairments in parvalbumin interneurons in social isolation-reared mice a critical period for social experience-dependent oligodendrocyte maturation and myelination feelings of loneliness, but not social isolation, predict dementia onset: results from the amsterdam study of the elderly (amstel) loneliness and risk of alzheimer disease loneliness is associated with sleep fragmentation in a communal society online social network size is reflected in human brain structure social brain volume is associated with in-degree social network size among older adults emotional arousal when watching drama increases pain threshold and social bonding social networks, support cliques, and kinship sex differences in social focus across the life cycle in humans extraverts have larger social network layers: but do not feel emotionally closer to individuals at any layer individual differences and personal social network size and structure social relationships and the emergence of social networks the anatomy of friendship how many hours does it take to make a friend? managing relationship decay: network, gender, and contextual effects the social brain hypothesis closeness, loneliness, support: core ties and significant ties in personal communities different strokes from different folks: community ties and social support hamilton's rule predicts anticipated social support in humans persistence of social signatures in human communication social network size in humans calling dunbar's numbers did distance matter before the internet? going that extra mile: individuals travel further to maintain face-to-face contact with highly related kin than with less related kin the strength of weak ties structure and function in human and primate social networks: implications for diffusion, network stability and health social interactions and well-being: the surprising power of weak ties limited communication capacity unveils strategies for human interaction what's in a smile? neural correlates of facial embodiment during social interaction fairness and cooperation are rewarding: evidence from social cognitive neuroscience alone in the crowd: the structure and spread of loneliness in a large social network attraction and close relationships a second-person approach to other minds joint attention, shared goals, and social bonding the computation of social behavior reward value of attractiveness and gaze ale meta-analysis on facial judgments of trustworthiness and attractiveness the eyes have it: the neuroethology, function and evolution of social gaze unique morphology of the human eye seeing it their way: evidence for rapid and involuntary computation of what other people see effects of duration and laughter on subjective happiness within different modes of communication: happiness and mode of communication technology-mediated communication in familial relationships: moderated-mediation models of isolation and loneliness human social networks the structure of online social networks mirrors those in the offline world the social network-network: size is predicted by brain structure and function in the amygdala and paralimbic regions ventromedial prefrontal volume predicts understanding of others and social network size the modular neuroarchitecture of social judgments on faces the social brain: allowing humans to boldly go where no other species has been characterization of the temporo-parietal junction by combining datadriven parcellation, complementary connectivity analyses, and functional decoding subspecialization within default mode nodes use of social network sites and instant messaging does not lead to increased offline social network size, or to emotionally closer relationships with offline network members coevolution of neocortical size, group size and language in humans games people play-toward an enactive view of cooperation in social neuroscience a survey method for characterizing daily life experience: the day reconstruction method gossip, reputation, and social adaptation higher order intentionality tasks are cognitively more demanding mental time travel into the past and the future in healthy aged adults: an fmri study social working memory predicts social network size in humans social network size affects neural circuits in macaques baboons (papio anubis) living in larger social groups have bigger brains amygdala volume and social network size in humans orbital prefrontal cortex volume predicts social network size: an imaging study of individual differences in humans ventromedial prefrontal volume predicts understanding of others and social network size orbital prefrontal cortex volume correlates with social cognitive competence gray matter volume of the anterior insular cortex and social networking segregation of the human medial prefrontal cortex in social cognition the brain structural disposition to social interaction the structural and functional brain networks that support human social networks orbital prefrontal cortex volume correlates with social cognitive competence neural mechanisms tracking popularity in real-world social networks adult attachment style is associated with cerebral μ-opioid receptor availability in humans: opioids and attachment building blocks of social cognition: mirror, mentalize, share? social cognition and the brain: a meta-analysis are there theory of mind regions in the brain? a review of the neuroimaging literature surface-based and probabilistic atlases of primate cerebral cortex dark control: the default mode network as a reinforcement learning agent. hum brain mapp the brain's default network: anatomy, function, and relevance to disease loneliness and meaning in life are reflected in the intrinsic network architecture of the brain social-cognitive deficits in normal aging cortical hubs revealed by intrinsic functional connectivity: mapping, assessment of stability, and relation to alzheimer's disease structural covariance of the default network in healthy and pathological aging computing the social brain connectome across systems and states personal space regulation by the human amygdala reduced amygdalar and hippocampal size in adults with generalized social phobia social brains: sex differentiation in human brain anatomy. science advances neocortex evolution in primates: the 'social brain' is for females primate brain evolution: genetic and functional considerations cognitive recovery in socially deprived young children: the bucharest early intervention project telomere length and early severe social deprivation: linking early adversity and cellular aging accelerated telomere shortening: tracking the lasting impact of early institutional care at the cellular level long-term effects of institutional rearing, foster care, and brain activity on memory and executive functioning nongenomic transmission across generations of maternal behavior and stress responses in the rat behavior of adult rats is modified by the experiences their mothers had as infants variations in postnatal maternal care and the epigenetic regulation of metabotropic glutamate receptor expression and hippocampal function in the rat preschool is a sensitive period for the influence of maternal support on the trajectory of hippocampal development amygdala gene expression correlates of social behavior in monkeys experiencing maternal separation early developmental emergence of human amygdala-prefrontal connectivity after maternal deprivation psychobiological mechanisms underlying the social buffering of the hypothalamic-pituitary-adrenocortical axis: a review of animal models and human studies across development maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations early adversity and critical periods: neurodevelopmental consequences of violating the expectable environment beyond diathesis stress: differential susceptibility to environmental influences biological sensitivity to context: ii. empirical explorations of an evolutionary-developmental theory critical periods for the effects of infantile experience on adult learning maternal and environmental influences on the adrenocortical response to stress in weanling rats social relationships among adult female baboons (papio cynocephalus) i. variation in the strength of social bonds sociophysiology of relationships in squirrel monkeys. i. formation of female dyads behavioural and hormonal responses to predation in female chacma baboons ( <i>papio hamadryas ursinus</i> ) stress and disorders of the stress system the potential role of hypocortisolism in the pathophysiology of stressrelated bodily disorders maternal behavior predicts infant cortisol recovery from a mild everyday stressor dampening of adrenocortical responses during infancy: normative changes and individual differences disentangling psychobiological mechanisms underlying internalizing and externalizing behaviors in youth: longitudinal and concurrent associations with cortisol effects of a therapeutic intervention for foster preschoolers on diurnal cortisol activity importance of studying the contributions of early adverse experience to neurobiological findings in depression the influence of social hierarchy on primate health social dominance in monkeys: dopamine d receptors and cocaine selfadministration loneliness and alcohol abuse: a review of evidences of an interplay the neurophysiology of unmyelinated tactile afferents social touch modulates endogenous μ-opioid system activity in humans topography of social touching depends on emotional bonds between humans cross-cultural similarity in relationship-specific social touching sharing the joke: the size of natural laughter groups social laughter triggers endogenous opioid release in humans the ice-breaker effect: singing mediates fast social bonding singing and social bonding: changes in connectivity and pain threshold as a function of group size naltrexone blocks endorphins released when dancing in synchrony synchrony and exertion during dance independently raise pain threshold and encourage social bonding emotional arousal when watching drama increases pain threshold and social bonding rowers' high: behavioural synchrony is correlated with elevated pain thresholds social recovery by isolation-reared monkeys research network on early experience and brain development early adverse care romania's abandoned children function of infant-directed speech stroking modulates noxious-evoked brain activity in human infants the communicative functions of touch in humans, nonhuman primates, and rats: a review and synthesis of the infants autonomic cardio-respiratory responses to nurturing stroking touch delivered by the mother or the father social regulation of gene expression in human leukocytes examining the visual processing patterns of lonely adults time as a limited resource: communication strategy in mobile phone networks cognitive resource allocation determines the organization of personal networks structural plasticity of the social brain: differential change after socioaffective and cognitive mental training key: cord- - qenzjiu authors: shorter, john r.; maurizio, paul l.; bell, timothy a.; shaw, ginger d.; miller, darla r.; gooch, terry j.; spence, jason s.; mcmillan, leonard; valdar, william; pardo-manuel de villena, fernando title: a diallel of the mouse collaborative cross founders reveals strong strain-specific maternal effects on litter size date: - - journal: g (bethesda) doi: . /g . . sha: doc_id: cord_uid: qenzjiu reproductive success in the eight founder strains of the collaborative cross (cc) was measured using a diallel-mating scheme. over a -month period we generated , litters, and provided , weaned pups for use in different published experiments. we identified factors that affect the average litter size in a cross by estimating the overall contribution of parent-of-origin, heterosis, inbred, and epistatic effects using a bayesian zero-truncated overdispersed poisson mixed model. the phenotypic variance of litter size has a substantial contribution ( %) from unexplained and environmental sources, but no detectable effect of seasonality. most of the explained variance was due to additive effects ( . %) and parental sex (maternal vs. paternal strain; . %), with epistasis accounting for . %. within the parental effects, the effect of the dam’s strain explained more than the sire’s strain ( . % vs. . %), and the dam’s strain effects account for . % of total variation explained. dams from strains c bl/ j and nod/shiltj increased the expected litter size by a mean of . and . pups, whereas dams from strains wsb/eij, pwk/phj, and cast/eij reduced expected litter size by a mean of . , . , and . pups. finally, there was no strong evidence for strain-specific effects on sex ratio distortion. overall, these results demonstrate that strains vary substantially in their reproductive ability depending on their genetic background, and that litter size is largely determined by dam’s strain rather than sire’s strain effects, as expected. this analysis adds to our understanding of factors that influence litter size in mammals, and also helps to explain breeding successes and failures in the extinct lines and surviving cc strains. size in wild populations. a controlled laboratory environment is therefore necessary to measure genetic effects on litter size (jinks and broadhurst ; roberts ; bandy and eisen ; hoornbeek ; peripato et al. ; gutiérrez et al. ; varona and sorensen ) . the collaborative cross (cc) and its eight founder strains are an important resource for studying complex traits, for establishing mouse models for human disease, and for understanding the mouse diversity outbred (do), which originated from the cc (ferris et al. ; chesler ; rogala et al. ; rasmussen et al. ; gralinski et al. gralinski et al. , schoenrock et al. ; maurizio et al. ) . the cc and its founder strains are also useful for studying reproductive ability due to their established record of breeding successes and failures. for example, litter size in the early cc lines shows a steady decline over the first six generations of inbreeding (philip et al. ). in addition, nearly % of all cc lines have become extinct, primarily due to subspecific genomic incompatibilities (shorter et al. ) . although standard reproductive phenotypes have been measured, mostly in male cc founders (odet et al. ) , the genetic and non-genetic control over cc breeding success has never been thoroughly characterized, and is likely to contain vital information that may be used to improve cc breeding in the future. given the growing popularity of the cc as a community resource, we investigated reproductive ability across the eight founders of the cc to better understand the genetic and non-genetic factors that affect their fertility and breeding. using an · diallel design, we measured weaned litter sizes from , litters arising from crosses across four years of breeding. adapting a recently developed statistical model of diallel effects (lenarcic et al. ) , we quantified both the genetic contributions that shape litter size and the contributions of several environmental factors. our results provide a detailed account of breeding patterns across and between the eight founders. this experiment also informs us about genetic combinations that are highly or marginally productive in the cc, helping us to better understand cc fertility problems and line extinction. the mouse inbred strains used in these experiments are the eight founder strains of the collaborative cross (cc) (collaborative cross consortium ) . the founders of the cc include five classical strains, a/j (aj), c bl/ j (b ), s /svimj ( s ), nod/shiltj (nod), nzo/ hlltj (nzo), and three wild-derived strains, cast/eij (cast), pwk/ phj (pwk), and wsb/eij (wsb). mice originated from a colony maintained by gary churchill at the jackson laboratory, and were transferred to the fpmv lab at the university of north carolina (unc) in . the original colony also produced most of the g breeders that populated the inbred funnels at ornl, tau and geniad (srivastava et al. ) . all mice described here were reared by the fpmv lab at unc. mice were bred at the unc hillsborough vivarium from - and bred at the unc genetic medicine building (gmb) vivarium from - . a total of , litters resulting from crosses between , individual dams and , individual sires were born from all eight inbred crosses and of reciprocal f hybrid crosses, excluding hybrids between nzo·cast and nzo·pwk, which are known unproductive crosses (chesler et al. ) . the directions of all crosses are described as female by male (i.e., dam.strain · sire.strain), unless otherwise noted. litter size and sex were determined at weaning by visual inspection. animals were kept on a -hour, -hour light/dark schedule with lights turned on at : am; temperature was maintained at °- °with relative humidity between - %. mice were housed in standard · -cm ventilated polysulfone cages with standard laboratory grade bed-o-cob bedding. water and purina prolab rmh were available ad libitum. mouse chow was supplemented with fenbendazole (feb ) two weeks before and two weeks after transportation to the gmb facility to eliminate possible pinworms. selamectin treatment was dropped onto the coats of mice before transfer to remove mites from the cages. these treated cages were not opened until after their arrival at unc gmb. all animal rearing and breeding was conducted in strict compliance with the guide for the care and use of laboratory animals (institute of laboratory animals resources, national research council , https://www.ncbi.nlm. nih.gov/books/nbk /). the institutional animal care and use committee of the university of north carolina approved all animal use and research described here. testing environmental interactions significant environmental interactions were determined using anova, using jmp software (jmp, version . sas institute inc., cary, nc, - . tested effects included a season effect (average weaned litter size in each month over every year), non-seasonal factors using a yearby-month effect (average weaned litter size for all months across all years), and a litter order effect (average weaned litter size in each subsequent dam litter). we performed the anova on litter size counts from the eight inbred matings only, because of their robust sample sizes throughout the four years of breeding. to estimate the overall contributions of heritable factors affecting litter size in our population, we adapted a previously published linear mixed model, bayesdiallel (lenarcic et al. ) , which performs this estimation for continuous phenotypes, to the setting of a discrete count-based phenotype. to do this we reimplemented the bayesdiallel gibbs sampler as a generalized linear mixed model (glmm) using the r software package mcmcglmm (hadfield ) . let y i be the number of pups born to litter i, where y i is a positive integer (zeros are not observed). for any categorical variable x, let the notation x½i indicate the value of x relevant to i: in particular, let h½i denote litter i's batch h ¼ ; . . . ; ; let r½i denote its parity order r ¼ ; . . . ; ; and let ðj; kÞ½i denote its parentage, defined by maternal strain j and paternal strain k, where ðj; kÞ f ; . . . ; g . the effect of parental strains on y i was modeled using an overdispersed zero-truncated poisson (ztp) regression (data scales of the model in brackets): where ztpoisðl i Þ denotes a poisson distribution with an expected value e½y i ¼ li e l i but is conditional on having observed that y i ¼ (appendix a), g is the link function gðxÞ ¼ logðxÞ, with inverse g ðxÞ ¼ e x , that relates l i to a latent scale ℓ i , and h i is a linear predictor on that latent scale with an error term e i $ nð ; s Þ providing overdispersion. the linear predictor h i is composed of the following: an intercept m; a litter (parity) order effect, modeled as the combination of a fixed effect slope, ra, and a random effect with an independent level for each litter order, i.e., order r $ nðra; t order Þ, where a is a fixed effect and t order is the variance of the random deviations around ra; a batch effect, batch h $ nð ; t batch Þ; and a linear predictor, d t ðj;kÞ b, modeling the effect of the parental strain combination ðj; kÞ. the contribution of parental strains, d t ðj;kÞ b, is equivalent to the 'fullu' (full, unsexed, 'babmvw') model described in (lenarcic et al. ) , namely, where subscripted variables a; m; b; v; w model the effects of specific strains or strain-pairs, b inbred models an overall effect, i fag is an indicator and equal to if a is true and otherwise, s fag is a sign variable equal to if a is true and otherwise. in more detail, the a ("additive") class represents strain-specific dosage effects, with a ; . . . ; a modeled as a j $ n stz ð ; t a Þ, where n stz is a normal distribution subject to the sum-to-zero constraint p j a j ¼ [using the approach of crowley et al. ( ) , appendix a]. the m ("parental sex") class represents parent-of-origin effects, modeled as m j $ n stz ð ; t m Þ, where a positive value of m j implies that strain j increases litter size more when inherited through the maternal line, with the difference between maternal and paternal being m j (since the design matrix entry for m j is coded þ for dam and for sire). the effect of being inbred is composed of an overall (fixed) effect b inbred and strainspecific (random) effects b j $ n stz ð ; t b Þ. epistatic effects are modeled as strain-pair specific deviations, with "symmetric" effects v jk $ n stz ð ; t v Þ representing the overall deviation from the rest of the model induced by the (unordered) strain combination j with k, and "asymmetric" effects w jk $ n stz ð ; t w Þ modeling a further deviation induced by differences in parent-of-origin. to provide directional parent-of-origin effects for maternal and paternal strain we defined, by reparameterization of the additive and parental sex effect, the following two additional types of effect: for example, dam b ¼ a b þ m b is the b -specific dam (maternal strain) effect and sire b ¼ a b m b is the b -specific sire (paternal strain) effect. posterior samples of these quantities were obtained as a post-processing step by reparameterizing posterior samples of a j and m j . obtaining these as a post-processing step, rather than explicit modification of equation , preserves the original bayesdiallel model and therefore has no effect on the effect estimates (or variance projection estimates) for the other diallel categories. (parameter definitions summarized in table s .) the decomposition of diallel effects in equation and its dam vs. sire reformulation have parallels in earlier diallel literature. a reduced version of the decomposition composed of additive (a) and symmetric epistasis (v) estimate, respectively, the generalized combining ability (gca) and specific combining ability (sca) described by sprague and tatum ( ) ; including also the (reciprocal) asymmetric epistasis (w) term recapitulates griffing's method , model (griffing ). the inbred penalty (b inbred , b) is comparable to dominance measures defined in, for example, hayman ( ) . the parental sex effects (m) are akin to the maternal effects of topham ( ) and the "extranuclear" effects in the "bio" model of cockerham and weir ( ) and zhu and weir ( ) , and the bio model's reparameterization to indirectly estimate dam and sire effects has been described in, for example, lynch and walsh ( ) . the estimation of dam and sire effects directly, as explicit model parameters for the diallel, was proposed by robinson ( , ) . the main differences between these earlier analyses and ours are the simultaneous inclusion of all parameter groups, the use of a bayesian random effects framework to allow all these parameters to be fitted, and the extension to modeling a non-gaussian response. priors were chosen to be minimally informative. for fixed effects (m, a, and b inbred ) we used nð ; · Þ. for variances of random effects (s , t a , t m , t b , t v , t w , t order , t batch ) we used an inverse gamma distribution with scale and shape both equal to . . posterior effect estimates are presented as posterior mean (and median), and the % highest posterior density (hpd) interval. in order to stably estimate the contribution of each variance class to the overall phenotype, we used the diallel variance projection [varp; crowley et al. ( ) ]. this is a heritability-like measure that partitions the overall phenotypic variance of an idealized future diallel experiment into additive, parent-of-origin (parental sex), inbred (dominance), epistatic, and other random/fixed effects categories in the diallel. rather than being based on estimated variance parameters (e.g., t a ; t b ; . . .), which are typically ill-informed by the data and thus both uncertain and sensitive to priors, the varp uses the estimated effects themselves, both fixed (e.g., b inbred ) and random (i.e., the best linear unbiased predictors, or blups, such as a ; a ; . . .), since these are well-informed, precise and regularized by shrinkage. the varp calculation involves ratios of sums-of-squares in similar fashion to r but for an idealized diallel that is both complete and balanced. as an r -like measure, the varp is reportable for both fixed and random effects, and includes confidence intervals arising from posterior uncertainty in those effects' values. varps were calculated both from the ztp model described above, and, for comparison, from the standard gaussian-outcome bayesdiallel model (using the bayesdiallel software) (appendix a). the standard bayesdiallel model was applied to our data after litter size was subject to a variance-stabilizing transformation, the square root, this corresponding to the linear mixed model approximation to the ztp, diallel analysis of sex ratio to model diallel effects on sex ratio we recast the bayesdiallel model as a binomial glmm. letting y i be the number of males out of a total of n i pups for litter i, we model where p i is the expected proportion of males predicted for litter i, the link function is gðxÞ ¼ logitðxÞ ¼ logðxÞ=ð xÞ, with inverse link g ðxÞ ¼ logit ðxÞ ¼ expitðxÞ ¼ e x =ð þ e x Þ, and ℓ i , which represents p i on the latent scale, is modeled using the bayesdiallel hierarchy as in equation . additional details about our statistical modeling approaches are provided in appendix a (litter size) and appendix b (sex ratio). file s contains all breeding data used for the analysis in this study. file s contains the scripts and software used for the analysis in this study. litter size is affected by housing facility but not season we bred litters in an · inbred diallel of the cc founder strains, and generated all eight inbreds and of possible reciprocal f hybrids (figure , figure s -s ). the eight inbred strains, displayed across the diagonal, were mated at higher frequencies both for maintenance of inbred strains and propagation of the diallel. for all genetic inbred and hybrid crosses, we recorded the following information: mated pairs, wean dates, litter size at weaning, including total and sex-specific counts (file s ). this diallel cross was originally designed and maintained for the generation of f mice for several experimental projects (koturbash et al. ; aylor et al. ; mathes et al. ; kelada et al. ; didion et al. ; collaborative cross consortium ; calaway et al. ; crowley et al. ; phillippi et al. ; odet et al. ; crowley et al. ; morgan et al. ; percival et al. ; shorter et al. ; oreper et al. ; maurizio et al. ) . as a result, certain reproductive measurements such as time between litters and maximum number of offspring per cross were necessarily biased by experimental breeding requirements. average weaned litter size, however, is a reproductive trait that should be well-estimated independently of these factors. we measured litter size and report the mean number of weaned pups per litter for the viable crosses in the diallel (figure ) . a wide distribution of litter sizes was observed, ranging from an average of . weaned pups for wsb·wsb crosses, to an average of . weaned pups for nod·pwk crosses, with an overall mean of . weaned pups per litter. examining average litter sizes of the inbred strains across all years, we found neglible evidence of consistent seasonal effects (f ; ¼ : p = . ) ( figure s ) but positive evidence of non-seasonal patterns: a modeled 'year.month' covariate significantly affected litter size (f ; ¼ : p , . ; see also figure s ). the non-seasonal effect could be driven by the relocation of mice between vivariums, which occurred in february . the housing facility may have an impact on litter size as well. to test this, we measured average litter size differences between the two housing facilities and found that two founder strains, s and wsb, had significantly larger average litter sizes at the hillsborough facility than at unc gmb with a difference of . to . weaned pups per litter (p , . ) for s , and . to . weaned pups per litter (p = . ) for wsb. the six other founder strains did not significantly differ in their average litter sizes between the two facilities, but tended to have smaller weaned litters at unc gmb (see discussion). for average litter size across the diallel, we tested for an effect of litter number (birth parity number, for a given mating pair) on the number of pups weaned in each litter. previous research suggests that the first litter can be significantly smaller than subsequent litters due to various biological factors (de la fuente and san primitivo ) . this effect was consistent in the diallel: we observed that there was significant reduction in litter size in the first litter (p , . ) compared to the overall linear effect that parity has on reducing litter size ( figure s ). litter size is moderately heritable, and maternal effects account for the majority of explained variation to estimate founder strain effects on litter size, we used a bayesian regression model that decomposes the phenotypic variation in the diallel into genetic and parent-of-origin contributions (lenarcic et al. ). using this model, the percentage of the variance in litter size explained by diallel effects was . %, with additive effects explaining . % (varp[additive]; this gca-like measure being related to narrow sense heritability), parent-of-origin effects (varp[parental.sex]) accounting for . %, the fact of being inbred (varp[inbred.overall]) at . %, and strain-by-strain interactions (varp[epistatic.symmetric] + varp[asymmetric.epistatic]) at . % (figure a) . in more detail, we present estimates for all modeled diallel effects as posterior means and highest posterior density (hpd) intervals ( figure b ). parameters are divided into two groups: general effects and strain pair-specific effects. general effects comprise strain-specific additive effects (additive), strain-specific and overall inbred (inbred), and strain-specific parent-of-origin (parental sex) effects. strain pair-specific (epistatic) effects are the effects that arise specifically in crosses of two heterologous strains, with 'v' referring to symmetric epistatic and 'w' referring to asymmetric epistatic effects (pairwise parent-of-origin effects). under the general effects, we see significant positive additive effects on average litter size from b , nod, and nzo and significant negative additive effects on litter size from cast, pwk, and wsb strain dosages. a similar pattern is seen in the parental sex effects, where b and nod dosages have a significant positive effect on average litter size, whereas cast, pwk, and wsb have significant negative effects. the overall "inbred" effect is negative, indicating that inbred status decreases figure diallel crossing scheme and weaned pup distribution. the number of litters observed per cross is given by the integers, with the largest sample sizes, along the diagonal, corresponding to the production of inbred parental strains. column and row sums are given along the bottom row and rightmost column, respectively. a total of , litters were evaluated for this analysis, resulting in a total of , weaned pups. the shading within each box corresponds to the average number of weaned pups per litter in each cross, with averages ranging from . to . pups per litter. litters for which no pups survived until weaning were not included in our analysis. the symbol "·" is used to indicate incompatible crosses that do not produce any litters. average litter size, regardless of parental strain. each strain also has an individual inbred effect in addition to the overall inbred effect. when the individual inbred effects are taken into account with the overall "inbred" effect, inbred litters are on average slightly smaller than their heterozygous counterparts. for strain pair-specific epistatic effects, there are a few marginally noteworthy effects, with the most prominent being a negative asymmetric epistatic effect for pwk·nod. to more clearly differentiate the contributions of the mother vs. the father strain, we reparameterized the bayesdiallel model to capture effects specific to dam.strain and sire.strain (figure ). b and nod dams increase litter sizes by more than . fold, by an average of . and . pups, respectively, regardless of sire. cast, pwk, and wsb dams tend to decrease average litter size by . , . , and . pups. the sire effect is similar, with nod and nzo sires having larger litters and cast sires producing smaller litters. as expected, we see that the dam.strain has a much larger influence on the variation of litter size compared with the sire.strain ( . % vs. . %). we examined genetic and non-genetic effects on the average sex ratio per litter and found no evidence that sex ratio was skewed ( figure s ; figure s ). the overall mean for sex bias, quantified as number of male pups weaned divided by the total number of weaned pups, was . , and did not significantly differ from our expectation of a : sex ratio (binominal test, two-tailed p = . ). for the eight inbred founders, s is the only strain that departed significantly from expectation, with slight reduction of males . : . (binominal test, two-tailed p = . ). however, correction for multiple testing shows no significant sex ratio bias of any inbred strain. the outbred crosses have substantially fewer litters and offspring than the inbred matings, leading to less balanced sex ratios; however, when multiple testing is accounted for, they also show no significant deviations from expected sex ratios. we have investigated factors influencing litter size in the eight cc founders and their f hybrids using a new extension of the bayesdiallel model. we note that litter size is a component of reproductive performance, but distinct from total strain productivity; a study on total strain productivity would need to take into account litter size, numbers of litters, maximum reproductive age, and pup survival until breeding. our results illustrate how mammalian litter size in a full diallel design is influenced by genotypic and environmental variation. these results present new information on cc founder strains' reproductive performance, show that maternal effects and the environment play a large role in litter size variation, and provide no evidence for seasonality effects on litter size in a controlled animal facility. the results also address some of the factors that contributed to line extinction and breeding problems in generating the cc (chesler et al. ; philip et al. ; collaborative cross consortium ; shorter et al. ) . we estimated that genetic (additive, inbred, and epistatic) effects on average litter size explained . % of variation, suggesting that most of the phenotypic variation arises from unexplained environmental effects. compared with the overall average litter, we observed substantial positive effects of b and nod strains, from both additive genetic and parent-of-origin parameters, and substantial negative effects of pwk and wsb (figure ) . we also observed, as expected, that lower litter size was associated with being inbred (figure a ). these estimates are likely driven by the unique selection history of these inbred lines, and comparisons should be limited to these eight founder strains, and the cc and do populations. during the g and g out-crossing generations of the cc, mean litter size was lower for crosses involving wild-derived strains, cast, pwk and wsb (philip et al. ) . a similar pattern is observed here, but these effects are determined to be specifically through the maternal strain, with cast, pwk and wsb having negative "dam" effects on strain-specific additive, parental sex, and inbred effects, and (right) epistatic effects between each pairwise cross. for each parameter, thin and thick horizontal lines represent % and % highest posterior density (hpd) intervals of effects, respectively, and vertical break and dash give posterior median and mean, respectively. the effects are in relation to an overall mean litter size of . ( % hpd: . - . ). the gray vertical lines indicate zero. effects are shown as the log, or latent, scale effects on the mean litter size attributable to each strain or strainpair and inheritance group, where values are centered at for each random effect class. intervals that exclude zero have non-negligible effects on the mean litter size. labels with "v" or "w" refer to symmetric or asymmetric epistatic effects, respectively. colored bars indicate corresponding variance classes in (a) and (b). litter size (figure ). it is likely that selection pressure in classical lab strains is associated with larger litters compared with the wild-derived strains. additionally, two of the wild-derived strains, cast and pwk, are from a different subspecific origin than the other six cc founders. this likely contributes to decreased productivity through subspecific incompatibilities (shorter et al. ) . we identify and report environmental factors that may influence litter size. the breeding of this diallel was performed across two different vivariums over the course of years, and we see a significant effect from housing facility. the hillsborough facility was associated with larger litters for all strains, especially s and wsb. the two facilities have many different factors that could explain these differences. the hillsborough facility housed multiple species, including dogs and mice, had smaller rooms that held approximately mouse cages, was remotely located in a rural area, had different laboratory personnel, had cage changes once a week, and was supplied with filtered well water. the diallel breeding at the gmb facility took place in a large central room, contained only laboratory mice, is located in a basement of a large seven story research building on campus, has cage changes every other week, and is supplied with filtered city water. it is possible that one or more of these factors, independently or in combination, affected productivity. another finding was that seasonality, which has previously been shown to influence litter size and frequency of litters in mammals (drickamer ) , did not seem to significantly impact litter size in this study. this is likely due to consistent lightdark cycles and temperatures as well as a steady diet. we did observe a significant effect on litter size after the transfer of the mice to the unc gmb facility (february ), which reduced overall litter sizes from march to june . this may have been due to the use of fenbendazole during the time of the transfer. other factors, such as the sex of the laboratory personnel interacting with the animals, are generally known to influence rodent behavior and could contribute to some of the environmentally-induced variation we observed (sorge et al. ) . last, we measured sex ratio across all inbred and outbred crosses. despite some departures from equality at the nominal significance threshold (alpha = . ), no associations with founder strain dosage remain significant after correction for multiple testing. recent work has suggested a potential for bias in sex ratio driven by the male germline in mus musculus (conway et al. ; macholán et al. ; cocquet et al. ; ellis et al. ; cocquet et al. ; turner et al. ; larson et al. ) , particularly in inter-subspecific hybrids that are mismatched for copy number of x-and y-linked genes expressed in postmeiotic spermatids. although there are no previous observations that suggest a bias in sex ratio in the cc or its founders, it remains an important characteristic to measure in a study on reproductive productivity. to estimate heritable effects on liter size and sex ratio we extended the original bayesdiallel model of lenarcic et al. ( ) in two new ways. first, to better understand and distinguish the effects arising from female and male parents, we reparameterized our strain-specific additive and parental-sex effects such that we could provide estimates of maternal strain and paternal strain effects separately. in the original bayesdiallel model, maternal and paternal strain effects are split into "additive" effects, which consolidates the effects they have in common, and "parental sex", which models any remaining deviation between the two. recognizing that additive effects from the sire are essentially wiped out by the additive effects, we instead collapsed the additive and parental-sex effects into "dam.strain" and "sire.strain" effects in a postprocessing step on the posterior output. this allowed us to run the original bayesdiallel model while also viewing our data from the perspective of dam strain and sire strain contributions. second, we reimplemented the original mcmc sampler, designed for modeling a continuous outcome variable, in a general package mcmcglmm (hadfield ) in order to model count and binary responses. litter size, as measured, is most naturally distributed as poisson, with zero-truncation owing to the fact that only successful litters were recorded. sex ratio is most naturally modeled as a binomial, with an underlying (male) proportion between and . although it would be possible to obtain an approximate analysis by transformations to normality using the original bayesdiallel (and we do this for litter size for some otherwise hard-to-obtain quantities), we found such approximations to be inadequate for reliable estimation of higher order effects in the case of litter size and deeply flawed in the case of sex ratio. although there is a computational cost, and added complexity to determining variance contributions, this new implementation achieves several objectives: ) we no longer break the assumptions in the original model regarding normally distributed errors; ) we easily accommodate overdispersion in our data; and ) we can select from a large number of glmms models that more closely resemble the forms of our data observations. in addition, we believe this flexibility will be appealing to many other researchers who would like to model non-gaussian distributed phenotypes using diallel designs, and we have provided the code in an r package litterdiallel (https://doi.org/ . /zenodo. ). overall, these results have implications for other avenues of future research. future multiparental research populations should test for strain incompatibilities, reproductive phenotyping, and other health traits in a full diallel before the recombinant inbreeding begins (odet et al. ) . these future research populations should also use non-related wild-derived individuals from the same subspecific origin in order to increase genetic diversity without introducing hybrid incompatibilities. this work was supported in part by the following grants from the national institutes of health: p gm , p hg / p mh , r hd (fp-mv), t hd (jrs), t ai (plm), r gm (plm, wv), and r gm figure dam.strain and sire.strain variance contributions and estimates of effects on weaned litter size. these effects are a reparameterization of additive and parental.sex effects from the previous analysis. estimates for the maternal ("dam.strain") and paternal ("sire.strain") effects on litter size, as calculated from the additive and parental sex parameters in figure , with hpd intervals defined correspondingly. (wv). the collaborative cross project is also supported by the university cancer research funds granted to lineberger comprehensive cancer center (mcr ccri). we collected data on litter size at weaning for genetic crosses of inbred lines, across four years of breeding. we use zero-truncated poisson (ztp) regression for modeling our data. this type of regression is explicit in its framework accounting for discrete observations, flexible in its ability to use linear mixed models on the latent scale, and allows for parameterization of excess variance observed, in a way that standard poisson regression does not. we account for the zero depletion in our data by using ztp regression instead of standard poisson regression, since we exclude observations of birth cohorts where no pups survived to weaning. in figure , the distribution of the observed data ðlitter sizeÞ is displayed for the wsb·wsb inbred mating, along with simulated data from a zero-truncated poisson distribution based on the data mean. for the ztp, the first two moments (mean and variance), for values y i . , are given by: the density for the ztp, for every count x ; ; :::, is given by: the relationship between the latent, expected, and data scales of the ztp regression model are illustrated by the toy example shown in figure . ztp frequencies were calculated using the r package countreg (zeileis and kleiber ) . diallel effect estimates are obtained using an mcmcglmm (hadfield ) implementation of bayesdiallel (bayesdiallel-glmm, henceforth), with : · iterations, : · iterations of burn-in, and thinning by (saving only every / th iteration), to obtain independent samples with minimal autocorrelation, and plotted as highest posterior densities (hpds) in the results. the effects estimated from the bayesdiallel-glmm model are transformed from the latent scale to the (expected) data scale via the inverse link function, i.e. expða aj Þ, for interpretability of effects on the original data scale. to avoid the problem of interpretability in transforming variance parameter (and variance projection) estimates from the latent to the observed data scales, we instead calculate and report variance projections, as calculated using the gaussian version of bayesdiallel. in order to account for heteroscedasticity (unequal variance) of the model residuals that arises from the approximately ztpoisson nature of the data, we use a variancestabilizing transformation (vst) (yu, ) and run bayesdiallel again (gaussian) to obtain variance projections on the modeled data. the varps that are calculated from these parameter estimates are an approximation of the variance contributions that we would observe in the glmm bayesdiallel model. we model the male pup counts and female pup counts jointly, using the bayesdiallel linear model, formulated for binomial glmm regression. this model directly considers genetic effects on the imbalance in male vs. female pups by parameterizing the number of males and number of total pups (or the total, and the fraction of males in the total). the model is elaborated in the methods section of the main manuscript. the proportion of weaned pups that are male, or equivalently, the proportion of weaned pups that are female, is approximated by a binomial distribution. in our data, the mean and the variance of male ratio are . and . , respectively. to generate the upper and lower % boundaries, as shown in figure s , for the expected phenotype under the null hypothesis of male pup proportion = . , we used the qbinom function in the stats package in r. to generate the upper and lower % boundaries, as shown in figure s , for the expected phenotype under the null hypothesis of male pup proportion = . , we used the qbinom function in the stats package in r. genetic improvement of litter size in pigs genetic analysis of complex traits in the emerging collaborative cross prenatal and postnatal effects in mouse lines selected for body weight and litter size: performance of postnatal dams and growth of progeny on the fitness functions relating parental care to reproductive value mouse phenome database: an integrative database and analysis suite for curated empirical phenotype data from laboratory mice genetic architecture of skewed x inactivation in the laboratory mouse out of the bottleneck: the diversity outcross and collaborative cross mouse populations in behavioral genetics research the collaborative cross at oak ridge national laboratory: developing a powerful resource for systems genetics quadratic analyses of reciprocal crosses a genetic basis for a postmeiotic x vs. y chromosome intragenomic conflict in the mouse the multicopy gene sly represses the sex chromosomes in the male mouse germline after meiosis the genome architecture of the collaborative cross mouse genetic reference population the components of genetic variance in populations of biparental progenies and their use in estimating the average degree of dominance estimation of average dominance of genes y /b: a candidate multiple-copy spermiogenesis gene on the mouse y chromosome genetics of adverse reactions to haloperidol in a mouse diallel: a drug-placebo experiment and bayesian causal analysis analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance selection for large and small litter size of the first three litters in mice discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias seasonal variation in fertility, fecundity and litter sex ratio in laboratory and wild stocks of house mice (mus domesticus) association of sly with sex-linked gene amplification during mouse evolution: a side effect of genomic conflict in spermatids? the genetics of litter size in mice introduction to quantitative genetics modeling host genetic regulation of influenza pathogenesis in the collaborative cross estimation of genetic response to selection in litter size of rabbits using a cryopreserved control population genome wide identification of sars-cov susceptibility loci using the collaborative cross allelic variation in the toll-like receptor adaptor protein ticam contributes to sars-coronavirus pathogenesis in mice. g : genes, genomes a generalized treatment of the use of diallel crosses in quantitative inheritance genetic parameters for canalisation analysis of litter size and litter weight traits at birth in mice mcmc methods for multi-response generalized linear mixed models: the mcmcglmm r package the genetic basis of family conflict resolution in mice the analysis of variance of diallel tables mating success and litter size variation within and between inbred and hybrid generations of rats diallel analysis of litter size and body weight in rats responses in ovulation rate, embryonal survival, and litter traits in swine to generations of selection to increase litter size genetic analysis of hematological parameters in incipient lines of the collaborative cross epigenetic mechanisms of mouse interstrain variability in genotoxicity of the environmental toxicant , -butadiene the composite regulatory basis of the large x-effect in mouse speciation a general bayesian approach to analyzing diallel crosses of inbred strains genetics and analysis of quantitative traits genetic conflict outweighs heterogametic incompatibility in the mouse hybrid zone? architecture of energy balance traits in emerging lines of the collaborative cross bayesian diallel analysis reveals mx -dependent and mx -independent effects on response to influenza a virus in mice genetic architecture of fitness and nonfitness traits: empirical patterns and development of ideas the mouse universal genotyping array: from substrains to subspecies natural selection and the heritability of fitness components the founder strains of the collaborative cross express a complex combination of advantageous and deleterious traits for male reproduction inbred strain variant database (isvdb): a repository for probabilistically informed sequence differences among the collaborative cross strains and their founders genetics of murine craniofacial morphology: diallel analysis of the eight founders of the collaborative cross epistasis affecting litter size in mice genetic analysis in the collaborative cross breeding population using the emerging collaborative cross to probe the immune system on the low heritability of life-history traits host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance the effects on litter size of crossing lines of mice inbred without selection the collaborative cross as a resource for modeling human disease: cc /unc, a new mouse model for spontaneous colitis perinatal nutrition interacts with genetic background to alter behavior in a parent-of-origin-dependent manner in adult collaborative cross mice male infertility is responsible for nearly half of the extinction observed in the mouse collaborative cross the optimal balance between size and number of offspring olfactory exposure to males, including men, causes stress and related analgesia in rodents general vs. specific combining ability in single crosses of corn genomes of the mouse collaborative cross selection, structure and the heritability of behaviour diallel analysis involving maternal and maternal interaction effects parent-offspring conflict reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone joint analysis of binomial and continuous traits with a recursive model: a case study using mortality and litter size of pigs variance stabilizing transformations of poisson, binomial and negative binomial distributions countreg: count data regression mixed model approaches for diallel analysis based on a bio-model key: cord- -dn t qa authors: salehi, bahare; sener, bilge; kilic, mehtap; sharifi-rad, javad; naz, rabia; yousaf, zubaida; mudau, fhatuwani nixwell; fokou, patrick valere tsouh; ezzat, shahira m.; el bishbishy, mahitab h.; taheri, yasaman; lucariello, giuseppe; durazzo, alessandra; lucarini, massimo; suleria, hafiz ansar rasul; santini, antonello title: dioscorea plants: a genus rich in vital nutra-pharmaceuticals-a review date: journal: iran j pharm res doi: . /ijpr. . . sha: doc_id: cord_uid: dn t qa dioscorea species, known as “yams,” belong to family dioscoreaceae. this genus consists of more than species distributed from africa, asia, the caribbean’s south america, and the south pacific islands. their organoleptic properties make them the most widely used carbohydrate food and dietary supplements. the underground and/or aerial tubers represent valuable sources of proteins, fats, and vitamins for millions of people in west africa. this review gives a shot of secondary metabolites of dioscorea plants, including steroids, clerodane diterpenes, quinones, cyanidins, phenolics, diarylheptanoids, and nitrogen-containing compounds. this review collected the evidence on biological properties of description dioscorea, including in-vitro and in-vivo studies. dioscorea species contain promising bioactive molecules i.e. diosgenin that support their different biological properties, including antioxidant, hypoglycaemic, hypolipidemic, anti- antimicrobial, inflammatory, antiproliferative, androgenic, estrogenic, and contraceptive drugs. indeed, besides its nutrient values, dioscorea is a potential source of bioactive substances of interest in the prevention/treatment of several diseases, and thus represents a great challenge in developing countries. however, ethnomedicinal potential should be validated and further researches on pharmacological properties and phytochemical composition should be carried out. particularly, doing some studies to convert the preclinical results to clinical efficacy should be guaranteed. dioscorea, food plant, traditional use, phytochemistry, pharmacological activities dioscorea belongs to the family dioscoreaceae, sub-family dioscoreoideae, consisting of species. dioscorea species are native to the old-world including high temperatures tropics and subtropic regions of the world. the major part of species occur in west africa, southern asia, and tropical america: they are herbaceous climbers with rhizome or tubers dioscorea species and, being usedin the formulation of pharmaceutical products, have a high medicinal, industrial and commercial importance ( ) . indeed, dioscorea species account for the most important dietary supplements ingredients used in cosmetics and pharmaceutical industries. it has also been commonly used by local people, mostly those who are engaged in the trade of medical plants worldwide. indeed, tubers of different dioscorea species are used as a cure for different diseases and ailments (cough, cold, stomach ache, leprosy, burns, fungal infections, dysentery, skin diseases, rheumatism, arthritis, etc.) in several formulations, and even for birth control ( ) . it is well known the potential of this plant is related due to the different phytochemical compounds found in dioscorea. tubers and roots contain steroidal sapogenins, mostly diosgenin as well as volatile compounds ( , ) . chemical substances like diosgenin found in dioscorea are commercially used in the pharmaceutical industry. the high demand for the medicinal use of this plant in different parts of the world suggests a strong need for conservation strategies. however, the conservation might not be easy or simple as it must involve plant protection and wellcontrolled access to its resources. there still a huge need for more research and information on the food, nutraceutical, and medicinal value worldwide of this plant species. therefore, this review tends to fill this gap by summarizing data on the current medicinal importance of dioscorea species. researches have been brought new knowledge about chemical compounds in tubers of dioscorea species and possible medicinal usage. the most common secondary metabolites are saponins, and more than steroidal saponins (based on aglycon part as stigmastanol, furostanol, spirostanol, cholestanol, ergostanol, and pregnanol glycosides (figure )) were isolated from various dioscorea species. besides these steroids, clerodane diterpenes, phenolics, cyanidins, quinones, diarylheptanoids, and nitrogen-containing compounds in the tubers of dioscorea species were quantified ( ) . steroidal saponins mainly consist of furostane, a pentacyclic ring system with a sixth open ring; spirostane, a hexacyclic ring system; and pregnane, a tetracyclic ring system. the sugar part is mainly composed of glucose and rhamnose in various proportions and linkages. steroidal saponins are glycosides consisting of an aglycone (diosgenin) and several glycosyl moieties. the most common sugars encountered in saponins are pentoses (arabinose, xylose, etc.), hexoses (glucose, galactose, etc.) and -deoxyhexose (rhamnose, etc.). the saponins of this plant species are both water-soluble and water-insoluble steroid saponins. dioscorea species are well known for containing steroidal saponins, which were used as marker compounds for quality control of the botanical products. as reported by jesus et al., , diosgenin ( -β-hydroxy- spirostene) is the primary furostanol saponin found in several plants, including dioscorea species, and is described as a promising bioactive compound with several medicinal properties, i.e. hypolipidemic, antioxidant, anti-inflammatory, hypoglycaemic, and antiproliferative activities ( ) . diosgenin obtained by hydrolysis of yam saponin were the main raw material for the industrial production of steroid drug i.e. anti-inflammatory, androgenic, estrogenic, and contraceptive drugs, by underlying its potential in the prevention/treatment of several diseases. dioscorea villosa l. roots and rhizomes, also are known as "wild yam", is a rich source of diosgenin. today, dietary supplements containing wild yam extracts are popular among women for the alleviation of menopausal symptoms and are widely used as alternatives to hormone replacement therapy ( ) . dioscorea alata l. dioscorea alata l. is one of the most popular varieties of yams. dioscorea alata (d. alata) were implicated in the promotion of the health of postmenopausal women. wild mexican yam was marketed for the treatment of irritability, hot flashes, insomnia, and depression. cheng et al., , reported the isolation and identification of two new compounds, hydro-q chromene, and γ-tocopherol- ; together with four known compounds (rrr-α-tocopherol, coenzyme q , cycloartane, and -feruloylglycerol): these compounds had phenolic hydroxyl group in common with the known phytoestrogens and have also antioxidant activity. moreover, their estrogenic activity was evaluated based on the ligand-dependent transcription activation assay ( ) . dioscorea antaly jum. and h. perrier is originated from madagascar, diffused in the west and north-west regions. the study of rakatobe et al., reported two clerodane diterpenoids, antadiosbulbins a and b and two furanoid -norclerodane diterpenes, -epidiosbulbins e and g along with the known diterpenoid diosbulbin e as well as nine known phenolics including five phenanthrenes and four flavonoids in the ethyl acetate soluble part of the methanolic extract of the tubers ( ) . dioscorea bulbifera l. dioscorea bulbifera l. (d. bulbifera l.), commonly known as air potato, is originated from africa and southern asia. the rhizome of d. bulbifera l. is called as "huang yao zi" in traditional chinese medicine and used for the treatment of thyroid diseases and cancers. in the northern districts of bangladesh, this herb is used for the treatment of cancers and leprosy. this plant has two types of storage organs, namely bulbils in the leaf axils of the stem and tubers. bulbils have been used as a folk remedy to treat diarrhea, conjunctivitis, and the extracts of the plant exhibit anti-tumor activity. the mannose-binding lectin from bulbils of d. bulbifera was studied for its clinical potential in hiv and cancer research ( ) . phytochemical studies reported as main bioactive compounds of this plant: flavonoids, clerodane diterpenoids, and steroidal saponins and phenolic compounds. as instance, liu et al. reported the isolation and identification of two new compounds, bibenzyl type- , , ′, ′-tetrahydroxy- ′methoxybibenzyl and diarylheptanone containing a dihydrophenanthrene moiety named as diobulbinone a along with sixteen known compounds ( ) . nine norclerodane diterpenoids were isolated from the tubers of d. bulbifera l. including two new compounds (diosbulbin n and diosbulbin p), and a naturally occurring compound (diosbulbin o) along with six known diosbulbins a-d, f, and g ( ) . another study also showed the molecular changes of liver dysfunction and reveal overall metabolic and physiological mechanisms of the subchronic toxic effect of dioscorea bulbifera rhizome ( ) . lam.-holl known as "yellow yam" is native to tropical west africa and its rhizomes are used as food and as a remedy for treating burns and fevers. a new furostanol glycoside namely -o-β -d-glucopyranosyl- β, -dihydroxy- , -seco- (r)-furost- en- , -dione- -o-α-l-rhamnopyranosyl-( → )-α -l-rhamnopyranosyl-( → )-[ α -l -r h a m n o p y r a n o s y l -( → ) ] -β -dglucopyranoside was isolated from the methanolic extract of the rhizome of dioscorea cayenensis growing in cameroon, together with the known spirostanol saponins described as methyl protodioscin, asperoside and prosapogenin a of dioscin ( ) . prosapogenin a of dioscin having a spirostan skeleton showed antifungal activity against candida species with mic values between . and mg/ml ( ) . two new fatty acidspirostan steroid glycoside esters, progenin iii palmitate, and progenin iii linoleate, were isolated from the methanol extract of the rhizomes of dioscorea cayenensis ( ) . it is worth mentioning the recent studies on the nutrient and antinutrient composition of yellow yam (dioscorea cayenensis) and their products; as instance raw dioscorea cayenensis tubers contained . g moisture, . g crude protein, . g lipid, . g fiber, . g ash, . g carbohydrates, . mg potassium, . mg magnesium, . mg iron, . mg zinc, and yielded . kcal energy with insignificant vitamin content/ g edible portion ( ) . dioscorea esculenta (lour.) burk. dioscorea esculenta also called as a "lesser yam" of dioscorea species, is widely distributed southern asia and the pacific. the tubers of d. esculenta were used traditionally in the treatment of various diseases. moreover, fiteen steroidal saponins were isolated from its tuber powder, and from the leaf of dioscorea esculenta (d. esculenta); four of them belonged to furostanol-type saponin,eleven as spirostane-type saponins ( ) . the rhizome of dioscorea japonica thunb., known as "san yak" in korea, is used as food and medicine. the plant was used in traditional chinese medicine to control/eliminate diarrhea, dilute sputum, improve anorexia, and moisturize skin ( ) . phytochemical studies on dioscorea japonica (d. japonica) showed the presence of active hypoglycemic compounds (dioscorans a-f), sesquiterpene, and acetophenone along with the steroidal constituents, including spirostane, furostane, and cholestane types ( ) . the rhizomes of dioscorea membrancea (d. membranacea) pierre have been used in thai traditional medicine. as instance, thongdeeying et al. isolated from the cytotoxic chloroform fraction of the rhizomes a new steroid (epi-panthogenin b), a known steroid (panthogenin b), two napthofuranoxepins dioscorealide a and dioscorealide b, phenanthraquinone (dioscoreanone) and three phenanthrene; the same authors reported that also, three steroids (β-sitosterol, stigmasterol, and β-dsitosterolglucoside) and two steroidal saponins [(diosgenin- -o-α-l-rhamnopyranosyl ( → )-β-d-glucopyranoside and diosgenin- -o -β -d -g l u c o p y r a n o s y l ( - ) -β -dglucopyranoside)] were obtained from d. membranacea ( ) . makino is bitter-sweet in taste and warm in nature according to the traditional chinese medicine. the rhizomes of d. nipponica were traditionally used in china as herbal medicine and food supplement for more than four thousand years; particularly, these plant species mainly act on the liver, kidney, and lungs, displaying anti-rheumatic, analgesic, blood circulation-stimulating, lung channeldredging, digestive, anti-diuretic, anti-tussive, panting-calming, and phlegm-dispelling activities. medicinally, it is commonly used for the treatment of rheumatoid arthritis, pain in the legs and lumbar area, kashin-beck disease, bruises, sprains, chronic bronchitis, cough and asthma and communication, and adherence to therapy is one of the main concerns ( , and ) . recently, it has been used as an important industrial raw material for the synthesis of steroidal drugs. concerning the phytochemical profile, twelve cyclic diarylheptanoids were isolated from the rhizomes of dioscorea nipponica ( ) , among which two new cyclic diarylheptanoids, diosniponol a and b; moreover, as reported by the same authors, these compounds were evaluated for their effects on nitric oxide production without cell toxicity in lipopolysaccharide-activated bv- cells. therefore, diarylheptanoid derivatives from d. nipponica may be potential candidates for the treatment of various neurodegenerative diseases associated with neuroinflammation ( ) . another study described the watersoluble non-saponin components [benzyl -o-β-d-glucopyranoside, leonuriside a, icariside d , pyrocatechol- -o-β-dglucopyranoside, (+) syringaresinol- -oβ-d-glucopyranoside, cyclo-(leu-tyr), and adenosine], and phenolic acid (piscidic acid) from the rhizomes of d. nipponica ( ) . on the other hand, -oxodioscin a new spirostan-type steroid along with known dioseptemloside g, ( r)-dracaenoside g, orbiculatoside b, dioscin, progenin iii, gracillin were determined. the presence of ( β, α, r)- - were also confirmed as furastan-type steroids ( ) . it is worth mentioning the study of that explore the chemical basis of the rhizomes and aerial parts of dioscorea nipponica makino by a combination of cheminformatics and bioinformatics, particularly their potential therapeutic and toxicity targets were screened through the drugbank's or t db's chemquery structure search: the compounds in the rhizomes possessed potential therapeutic targets and potential toxicity targets ( ) . dioscorea opposita thunb. dioscorea opposita thunb. has been cultivated in china, japan, and korea as a food, and widely used as traditional medicine, for a very long time. there are about yam cultivars that are originated in china, including the so-called "trueborn" chinese yam. the important ingredient of the yam is sugar, which provides energy and sweetness and adds to the general flavor of foods. also, the chinese yam assists in food digestion and is beneficial in cases of stomach disorders ( ) . phytochemical investigations of dioscorea opposite (d. opposita) have revealed many chemical components such as purine derivatives, phenanthrenes, stilbenes, sapogenins, and saponins. phytochemical studies of the chloroform soluble fraction of d. opposita thunb. have resulted in the isolation of four new compounds, two dihydrostilbenes: , -dihydroxy- -methoxybibenzyl, , ′, trihydroxy- ′-methoxybibenzyl; and dibenzoxepins , -dihydro-dibenz[b,f]oxepin- , diol and , -dihydro- -methoxy-dibenz[b,f] oxepin- -ol, together with an additional fifteen known compounds. all the nineteen isolated compounds were tested in the dpph, superoxide anion radical scavenging assays and cyclooxygenases (coxs) inhibition assay. among them, , ′, -trihydroxy- ′methoxybibenzyl showed the most potent cox- inhibitory activity ( ) . bioassayguided fractionation of d. opposita extracts led to the isolation and identification of phenolic compounds. of them, compounds reduced porcine pancreatic lipase activity at ic values of less than mm and from them , ′, -trihydroxy- ′-methoxybibenzyl and ( e, e)- , -bis( -hydroxyphenyl)- , -heptadien- -one showed the highest inhibition with an ic value of . mm and dose-dependently in the concentration range - mm ( ) . these findings provide that phenolic compounds with stilbenoids are considered to play important roles in the lipase inhibition of the d. opposita extract. especially, the stilbenoids possessing , -dihydroxybibenzyl moiety showed higher inhibitory potencies than the others. the lipase inhibitory activities of stilbenoids depend on the presence of the hydroxyl group in the c- position. the structural difference also influences the inhibitory effect of diarylheptanoids on pancreatic lipase. dihydrostilbene phenanthrene and diarylheptanoid structures were deemed to be responsible for lipase inhibition activity of this species ( ) . d. opposita thunb. is also rich in starch, water-soluble polysaccharides, and mucilage defined as a polysaccharide with unique viscosity characteristics are widely used in the pharmaceutical and food industries as a thickening agent and emulsion stabilizer. these agents are important in the food industry as they improve the sensory quality, flavor, texture, palatability, mouthfeel, and general appearance of the final products. therefore, the mucilage of this plant species is a potential candidate for food emulsifier resource of a natural emulsifier, especially under alkaline conditions obtained from industrial processing waste such as gum arabic, yellow mustard, and chia (salvia hispanica l.) ( ) . dioscorea preussii pax d. preussii pax collected in bambui, cameroon, was reported to have in-vitro cytotoxic, antileishmanial and antifungal activities. three new steroidal saponins, named as diospreussinosides a, b, c, along with two known compounds were determined as ( r)- α-hydroxyspirost- - r h a m n o p y r a n o s y l -( → )α -l -r h a m n o p y r a n o s y l -( → ) ] -β -dglucopyranoside were isolated from rhizomes of d. preussii ( ) . dioscorea pyrifolia kunth d. pyrifolia kunth is called also as "akar kemeyan paya" in malaysia. these plants grow wild and are diffused particularly in peninsular malaysia. sharlina et al. reported that the starch extracted from d. pyrifolia was characterized by the high amylose content ( . ± . %); the same authors marked how d. pyrifolia kunth represents a very important source of starch for food and non-food applications, such as crispy food, coating materials, hydrogels, films, bio-membranes, and adhesives ( ) . cholestane glycosides, dioseptemlosides a and b, together with six spirostane glycosides, dioseptemlosides c-h, were isolated from the rhizomes of d. septemloba. among these, spirostane aglycones containing hydroxyl group at c- were reported in the family dioscoreaceae. spirostane-type glycosides showed strong activity on nitric oxide production ( ) . on the other hand, three new diarylheptanoids, dioscorol a, dioscorosides e , e ; two new stilbenes, dioscorosides f and f were isolated from the rhizomes of dioscorea septemloba. besides, , -bis( -hydroxyphenyl)hepta- e, e-dien- -one, , -bis( -hydroxyphenyl)- , , -heptatrien- -one, , -dihydroxy- , -bis( -hydroxyphenyl)heptane, ( r, r)- , -dihydroxy- , -bis( -hydroxyphenyl)heptane -o-β-d-glucopyranoside, ( r, r)- , -dihydroxy- , -bis( -hydroxy- were also obtained as known compounds. they have also shown a significant increase in the glucose consumption in differentiated l myotubes and displayed triglyceride inhibitory effects in hepg cells . eight new compounds namely, dioscorosides g, h , h , dioscorol b, dioscorosides i, j, k , and k , together with twelve known ones were isolated from the rhizomes of dioscorea septemloba. moreover, the authors showed that some of these compounds were found to display significant inhibition of nitrite production evaluated in-vitro anti-inflammatory potential using lps-stimulated raw . murine macrophages ( ) . currently, he et al. have identified and elucidated the structure of a new phenanthropyran, dioscorone b, and a new phenanthrene, together with seven known compounds, is from the % ethanol extract of dioscorea septemloba rhizomes. moreover, the authors reported that new isolated phenanthropyran, tested for antioxidant activity, showed excellent activities with ic values of . ± . μm and . ± . μm, respectively ( ). dioscorea tokoro makino d. tokoro is one of those wild yams, and its rhizome was generally used for daily food.; in particular in the northern part of japan, the rhizome was used as health-promoting food. d. tokoro was characterized by steroidal saponins, such as dioscin and gracillin, but no data were found about its health-promoting effect. the acetonitrile extract of d. tokoro rhizome fractionation led to protodioscin as an antiproliferative compound to hl- leukemic cells ( ) . the most well-known species of this genus is dioscorea villosa, also called "wild yam." two furanostane type saponins, namely methyl parvifloside, and protodeltonin, were isolated from d. villosa as well as two spirostane types deltonin and glucosidodeltonin (zingiberensis i), whereas minor saponins were methylprotodioscin, disoscin, and prosapogenin ( ) . the same authors reported that two fla-van- -ol glycosides were also isolated from d. villosa and fifteen steroidal compounds, including two new metabolites, were isolated from the tubers of d. villosa; these compounds were characterized as cholestane type steroidal glycosides named as dioscoreavillosides a and b ( ). dong et al., described how the rhizomes were also afforded diarylheptanoids, five of which were new compounds containing a tetrahydropyran ring in the heptane portion of the molecule ( ) . in another study, three furostanol saponins, parvifloside, methyl protodeltonin, and trigofoenoside a- were isolated from the n-butanol soluble extract of d. villosa by using hsccc; moreover the authors reported that subsquent normal-phase hsccc separation also led to the identification of four spirostanol saponins identified as zingiberensis saponin i, deltonin, dioscin, and prosapogenin a of dioscin ( ) . on the other hand, two series of lipidated steroid saponins were isolated from d. villosa ( ) . the series a was identified as a mixture of five lipidated steroid saponins: the series b was characterized as a mixture of the following five compounds: -en-clionaster- these findings revealed two classes as new biomarkers: the diarylheptanoids and the lipidated steroid saponins. the rhizome of dioscorea zingiberensis c.h. wright is known as ''huang jiang" and represents an important source of diosgenin. d. zingiberensis were used for the treatment of lung heat, pyretic stranguria, anthracia, coronary heart disease, and swelling; in particular, the rhizome of dioscorea zingiberensis is extensively used for the extraction of diosgenin sapogenin and its glycoside dioscin, used for the synthesis of sex hormones and corticosteroids. it is worth mentioning the current study of zhang et al., that give comprehensive overview of the traditional usage and phytochemistry of the dioscorea zingiberensis c.h. wright: -more than compounds have been identified; -several of these have been tested in preclinical assays and clinical trials; -a wide spectrum of biological effects including cardiovascular, anti-thrombosis, hyperlipidemia, neuroprotection, anti-inflammatory, and anthelmintic effect has been verified ( ) . wang et al. reported how spirostanol based aglycon containing steroidal saponins are the representative steroidal saponins, and their quantity is higher than that of furostanol steroidal saponins in d. zingiberensis ( ) . apart from the steroidal saponins, other kinds of constituents were also identified from d. zingiberensis. until now, four steroids have been isolated from this plant including β-sitosterol, sterol, zingiberenin f, and ( r)- β-hydroxyspirost-Δ , ( ) in addition to the common steroidal saponins, other constituents classified as alkaloid, phenyl-glycoside, and benzoic acid derivatives have also been determined. -aminomethyl-phenol, -o-β-d-glucopyranosyl- -hydroxybenzoic acid and another nonsteroidal saponin called -o-β-d-glucopyranosyl- -methoxybenzoic acid, were isolated and identified from the n-buthanol extracts of fresh rhizomes. following the regular phytochemical research procedure, five compounds were obtained from d. zingiberensis ( ) . they consisted of two new phenanthrene derivatives, namely , , -trimethoxy- , -dione- , -dihydrophenanthrene and , , -trihydroxy- , -dimethoxy- , -dihydrophenanthrene; a new anthracenedione, i.e., , , -trimethoxy- , -dione-anthracene; and two known , -dihydrophenanthrenes, called , -dihydroxy- , -dimethoxy- , -dihydrophenanthrene and , -dihydroxy- , , -trimethoxy- , -dihydrophenanthrene. one new bibenzyl ( , -dihydroxy- , -dimethoxybibenzyl.) and one new phenanthrene ( , -dihydroxy- , -dimethoxy- , -dihydrophenanthrene.), together with two known bibenzyls ( , -dihydroxy- -methoxybibenzyl , ′,-dihydroxy- ′, -dimethoxybibenzyl and four known diarylheptanoids (( r, r)- , -dihydroxy- , -bis( -hydroxyphenyl)heptane, ( r, r)- , -dihydroxy- -( , -dihydroxyphenyl)- -( -hydroxyphenyl)heptane, ( r, r)- , -dihydroxy- -( -hydroxy- -methoxyphenyl)- -( -hydroxyphenyl)-heptane and ( r, r)- , -dihydroxy- , -bis( -hydroxy- -methoxyphenyl)-heptane were isolated from the dichloromethane soluble fraction of the rhizomes of d. zingiberensis ( ) . all these phenolic compounds were evaluated for their anti-pancreatitic activities on sodium taurocholate-induced pancreatic acinar necrosis as inhibition of necrotic cell death pathway activation. among them, ( r, r)- , -dihydroxy- -( , -dihydroxyphenyl)- -( -hydroxyphenyl) heptane which bears a trihydroxy-diarylheptane skeleton was shown to protect against pancreatic acinar cell injury through mediating novel potential therapy for pancreatic necrosis ( ) . the tubers of different dioscorea species present a wide variation of bioactive compounds, responsible for their pharmacological activities. generally, the evaluation of bioactive components and the assessments of their interactions could be viewed as the first step for the determination of potential of a plant ( ) ( ) ( ) ( ) ( ) ( ) ( ) . also, their ethnopharmacological importance promotes further investigations of dioscorea metabolites as a source of potential therapeutic agents to ameliorate different diseases (table ) . cytotoxic activity d. bulbifera ethanolic crude extract and different fractions (hexane, ethyl acetate, and water) at concentrations of , , and μg/ml were found to possess a potent cytotoxic activity against human colorectal carcinoma (hct ), human colorectal adenocarcinoma (ht- ), human lung carcinoma (a ), human breast carcinoma (mcf- ), human tuber immune-modulating activity ( ) . anti-inflammatory activity ( ) anti-severe acute respiratory syndrome associated coronavirus ( ) . gastroprotective activity ( ) enhance murine splenocyte proliferation ex-vivo and improve regeneration of bone marrow cells ( ) immunomodulatory activity ( ) anti-inflammatory activity ( ) . neuroprotective activity ( ) antioxidant activity ( ) mediated synthesis of gold and silver nanoparticles with catalytic activity ( ) neuroprotective activity ( ) antidiabetic activity ( ) cell shrinkage, and dna fragmentation as shown by flow cytometry. the authors also revealed that dbeaf induces apoptosis effects on hct cells through externalization of phosphatidylserine and by promoting the loss of mitochondrial membrane potential (mmp), dysregulation of the bcl- family proteins and the downregulation of the expression of procaspase - , - , - and - and parp protein expression. furthermore, their results suggested that the inhibitory effect of dbeaf on erk / and the activation of jnk were closely associated with the apoptotic cell death induction in human colorectal carcinoma ( ). rhizome antioxidant activity ( ) antitumor activity ( ) anti-hypercholesterolemic effect ( ) anti-platelet aggregation activity ( ) antifungal activity ( ) antihyperlipidemic and antioxidative activity ( ) dioscorea pentaphylla l. antioxidant activity and antibacterial activity d. collettii var. hypoglauca and its active metabolite protoneodioscin, a furostanol saponin, was also evaluated for its cytotoxic activity against cell lines including human leukemia, melanoma, and cancers of lung, colon, brain, ovary, renal, breast, and prostate. protoneodioscin exhibited significant cytotoxicity with % growth inhibition (gi ) ranging from . to mm against all cell lines from leukemia and most solid tumors ( ) . in the following year, the same research team evaluated two other compounds, namely; methyl protoneogracillin and gracillin. the results revealed that methyl protoneogracillin exhibited significant cytotoxicity with (gi ) ≤ mm. leukemia, cns cancer, and prostate cancer were the most sensitive subpanels, while ovarian cancer was the least sensitive subpanels. on the other hand, gracillin lacked selectivity against human cancer diseases ( ). a different approach was undertaken by ashri and co-workers ( ) ( ), who evaluated the cytotoxicity of modified d. hispida starchbased hydrogels on small intestine cell line (fhs- int) to ensure the safety of their use. their results revealed that the prepared hydrogels did not show any cytotoxicity and could be employed for future pharmaceutical use. a mucopolysaccharide of yam (d. batatas decne) (ymb) was found to increase the cytotoxic activity of mouse splenocyte against leukemia cell at µg/ml concentration. however, it did not affect the viability of splenocytes. the production of ifn-γ was significantly increased in the ymp treated splenocytes. at µg/ml concentration, it increases the up taking capacity and lysosomal phosphatase activity of peritoneal macrophages. in addition, ymp ( - µg/ml) significantly increased the viability of peritoneal macrophages ( ) . dioscorin, the glycoprotein isolated from d. alata was reported to activate toll-like receptor (tlr ) and induce macrophage activation via typical tlr -signaling pathways at µg/ml concentration. it induces tlr downstream cytokine expression in bone marrow cells isolated from tlr -functional c h/hen mice but not from tlr -defective c h/hej mice. dioscorin also stimulated multiple signaling molecules (nf-jb, erk, jnk, and p ) and induced the expression of cytokines (tnf-a, il- b, and il- ) in murine raw . macrophages ( ) . different dioscorea species were used traditionally for the treatment of memoryrelated disorders and dementia, i.e. alzheimer disease and other neurodegenerative diseases ( ) . the chloroform extract from d. opposite rhizomes significantly reduced the glutamateinduced toxicity in a dose-dependent manner with a maximum neuroprotective effect at µg/ml ( ) . kim ( ) . moreover, the compounds isolated from d. nipponica rhizomes exhibited anti-neuroinflammatory and neuroprotective activities, the most active of which was , -dihydroxy- , , trimethoxy-phenanthrene. the later was the most potent nerve growth factor (ngf) inducer, with . % stimulation, and strongly reduced nitric oxide (no) levels with an ic value of . μm in lps-activated bv microglial cells. also, it significantly increased neurite outgrowth in mouse neuro a (n a) cells and therefore possessed a significant neuroprotective activity ( ) . antidiabetic activity different extraction procedures as ultrasonic-assisted extraction, cold water extraction, warm water extraction and hot water extraction significantly influenced the antidiabetic potential of the rhizomes of d. hemsleyi as determined via both alphaglucosidase and alpha-amylase inhibition assays, where the ic values of alphaglucosidase inhibition assay varied from . to . µg/ml while those of alpha-amylase inhibition assay varied from . to . mg/ ml ( ) . the antioxidant activity of the extracts of dioscorea species received much scientific attention ( , , and ) . different extracts of rhizomes of d. hemsleyi, d. hispida dennst, d. opposite thunb., d. nipponica makino, d. esculenta (lour). burkill, d. japonica thunb. var. pseudojaponica and d. pentaphylla l. were all studied for their antioxidant properties by different in-vitro assays namely; reducing power assay, ferric reducing antioxidant power assay (frap), dpph radical scavenging assay, hydroxyl radical scavenging assay, superoxide radical assay, self-oxidation of , , -phentriol assay and antioxidant activity by radical cation (abts) assay. generally, the studied extracts from different dioscorea species showed a significant antioxidant potential which may account for their involvement in the treatment of various disorders via radical scavenging mechanisms. lipopolysaccharide-stimulated raw . cells were employed to determine the antiinflammatory properties of the ethanolic extract of tubers of d. japonica thunb. var. pseudojaponica and individual steroid glycosides isolated from d. septemloba rhizomes ( , ) . d. japonica extract at doses of and µg/ml significantly inhibited lps-induced inos and cox- protein expressions, also, the nitrite relative concentration percentage ranged between . % to . % for the steroid glycosides isolated from d. septemloba rhizomes. similar to the total ethanol extract of d. membranacea presented a potent inhibitory activity, with an ic value of . µg/ml. its most potent metabolite included diosgenin- -o-alpha-lrhamnosyl ( --> )-beta-d-glucopyranoside that showed a powerful inhibitory activity with ic value as low as . µg/ml ( ) . the study of the mechanism of anti-inflammatory activity of the ethanolic extract from the bark of d. batatas decne showed that it inhibited both no and pge production with an ic of - µg/ml respectively. it was also suggested that the extract act suppressing dna-binding activity and reporter gene activity as well as translocation of the nf-jb p subunit. it also down-regulated ijb kinase (ikk), thus inhibiting lps-induced both phosphorylation and the degradation of ijba. in addition, it also inhibited the lps-induced activation of erk / ( ) . a current study of hwang et al. reported the anti-inflammatory effect of aerial bulblets of dioscorea japonica thunb extract through inhibition of nf-κb and mapk signaling pathway in raw . . antiallergic activity d. membranacea ethanolic extract and compounds showed antiallergic activities by inhibiting beta-hexosaminidase release as a marker of degranulation in rbl- h cells, with an ic value of . µg/ml. from this extract, dioscorealide b showed the highest activity with an ic value of . µg/ml ( ) . du et al. reported how several compounds were isolated from the rhizomes of d. zingiberensis i.e. ( r, r)- , -dihydroxy- -( , -dihydroxyphenyl) - -( -hydroxyphenyl) heptane at a concentration of . mm showed anti-pancreatitis activities on sodium taurocholate -induced pancreatic acinar necrosis with an inhibitory effect of . %. moreover, the same authors marked that this compound's protective effects were mainly dependent on atp inhibition and excessive ros production and thus saving the cells from mitochondrial dysfunction ( ) . another study reported that the steroidal saponins obtained from the rhizomes of d. zingiberensis showed anti-arthritic activities on the lps stimulated . macrophage cells through induction of both p and iκbα protein expressions ( ) . the methanolic extract of tubers of d. pentaphylla l. showed antibacterial activity against s. pyogenes, s. mutans, v. cholera, s. typhi, and s. flexneri. the best activity was: . mm and . mm inhibition zone diameter observed (at μg/disc and μg/ disc) against s. pyogenes using disc diffusion, agar well diffusion assays, respectively. the mic values were μg/ml for the extract against v. cholera, s. typhi and s. flexneri while it was μg/ml against s. pyogenes and s. mutans. similarly, rajendra et al. reported the antibacterial activities of % and % methanolic extract of d. deltoidea wall ex griseb rhizomes against s. aureus and e. coli, but this concentration does not show activity against s. typhi and p. aeruginosa. it could be concluded that, in general, dioscorea species do not exhibit a potent antibacterial activity ( ) . the study of cho et al. studied dioscin, isolated from wild yam d. nipponica root bark, for its potential as an antifungal agent via the propidium iodide assay and calceinleakage measurement, in addition to, its ability to disrupt the plasma membrane potential, using , ′-dipropylthiadicarbocyanine iodide and bis-( , -dibarbituric acid)-trimethine oxanol. as reported by cho et al. the results showed that dioscin exerts a considerable antifungal activity, where, the dye-stained cells showed a significant increase in fluorescent intensity after treatment with dioscin, demonstrating that dioscin possess an effect on the membrane potential. the auhors concluded that dioscin could act by disrupting the fungal membrane structure resulting in cell death ( ) . globally a large number of dioscorea plants have investigated in-vivo. data on dioscorea species potency on the animal model are summarized in table . the anti-constipation activity dioscorea opposita thunb. yam tuber is rich in starch, which has always been ignored and discarded during the isolation of bioactive compounds. huang et al., studied the anti-constipation effect of native yam starch (ns), dual enzyme-treated starch (des), and cross-linked carboxymethyl starch (ccs) in constipation model induced by loperamide in km mice. the modified starches (des and ccs) could benefit the bowel function by increasing stool frequency and defecation moisture as they have the good water-binding capacity and swelling ability, the modified starches could increase the water-holding capacity of stools to remain soft. des and ccs groups had improved small intestinal propulsion through the production of more short chain fatty acids (scfas) and decrease ph by increasing acetic acid and butyric acid concentration in the feces. the study done on mice with high-fat diet showed that both native and modified starch from yam had anti-hyperlipidemic activity through a significant decrease of both cholesterol and triglycerides and the liver index. the reduction of superoxide dismutase (sod) and increase malondialdehyde (mda) was observed in hyperlipidemic mice while they were both improved in all starches-treated mice proving their antioxidant effect (table ) ( ). four types of resistant starch (rs) including native (rs ), retrograded (rs ), crosslinked with sodium trimetaphosphate/sodium tripolyphosphate (rs ) and complexed with palmitic acid (rs ) resistant starches from winged yam (d. alata l.) showed the gastroprotective activity of in ethanol-induced gastric ulcer in mice. the increase in gastric emptying rate may help in reducing the contact time of ethanol and gastric mucosa and helps to prevent the gastric tissue damage. therefore, an increase in gastric emptying induced by rs is a possible mechanism of anti-gastric ulcer. moreover, rs and rs groups generated more short chain fatty acids which are produced through fermentation of the starch by the colon microflora. the fatty acids have many physiological and biological functions, such as increasing the immunity of the stomach. etoh-ulcer can result from the imbalance between the generated reactive oxygen species (ros) and the natural antioxidant power. thus, the free radical scavenging is one of the mechanisms for inhibition of gastric ulcer ( ). mao et al. reported the decrease of sod activity in ethanol-treated rats with an increase in mda levels in gastric mucosa indicating severe oxidation reaction in ethanol-induced gastric ulcer mice. rs ( . g/kg) and rs ( . g/kg) exhibited the best antioxidant effect. therefore, the inhibition of oxidative stress by the four types of rs is a possible mechanism that may assist in decreasing the gastric mucosal damage. generally, the high dose groups of rs and rs ( . g/kg) showed the best activity ( ) . a current study also reported the protective effects of dioscorea batatas flesh and peel extracts against an ethanol-induced gastric ulcer in mice ( ) . li et al. reported the significant antithrombotic and anticoagulation effect of the total steroidal saponin extract (tsse) from the rhizomes of d. zingiberensis c.h. wright on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. oral administration of the extract significantly inhibited adenosine diphosphateinduced platelet aggregation (pag) in-vivo, which was more effective than xue-sai-tong, a commercial product of triterpenoid saponins from panax ginseng having antithrombosis activity in china. in addition, tsse inhibited the thrombosis in a dose-dependent manner (more than % inhibition rate) which was more powerful than the standard xue-saitong at . , and . mg/kg doses of tss, and markedly reduced thrombus weight. tsse also exhibited in a dose-dependent manner prolongation of thromboplastin time (aptt) (which is an intrinsic clotting index), prothrombin time (pt) (that evaluates the extrinsic clotting pathway), and thrombin time (tt) (which is a test of fibrin formation, the addition of thrombin directly induces that). the more pronounced effect of tsse on pt and tt than aptt indicates that it inhibits the extrinsic pathway of coagulation and fibrin formation, and cut down the thrombotic risk ( ) . the ethanolic extract of the tubers of d. japonica thunb. var. pseudojaponica (eedj) showed in-vivo anti-inflammatory activity by significantly inhibiting the development of carrageenan-induced paw edema in mice after the th and th hour of the treatment at . g/kg. it also decreased the levels of tnf-α, which is a mediator of carrageenan-induced inflammatory response and induced the release of kinins and leukotrienes. carrageenaninduced paw edema causes oxidative stress with the release of peroxidation products, including malondialdehyde (mda) and nitric oxide. mda increased levels of oxygen free radicals, which attack polyunsaturated fatty acids in cell membranes and causing lipid peroxidation. eedj was able to reduce nitric oxide which was excessively released after administration of carrageenan and also could reduce the levels of mda through increasing the enzymatic antioxidant defense systems such as catalase (cat), superoxide dismutase (sod) and glutathione peroxidase (gpx) in the paw oedema which are the natural protectors against lipid peroxidation ( ) . the anti-arthritic effect of the total saponin extract from the rhizomes of d. zingiberensis c.h. wright (tsrdz) in freund's complete adjuvant (fca) induced arthritis in rats ( ) . in this study, a significant increase in arthritis scores and ankle perimeter were observed after days from intraplantar administration of fca, which reaches its maximum on day . administration of tsrdz at and mg/kg produced substantial dose suppressive significant effect in the treated groups. tsrdz at and mg/kg also caused a marked reduction of fca-induced elevated indexes of the thymus and spleen. the high dose of tsrdz caused only mild synovial infiltration with few inflammatory cells and no obvious damage in cartilage bone erosion, which appeared in histopathological study. the efficient regulation of tsrdz on the inflammatory cytokines at mg/ kg, was almost equivalent to that caused by the standard methotrexate. moreover, the mitigation of tsrdz on the prostaglandins (pge ), at mg/kg, was almost equivalent to that of standard methotrexate. furthermore, administration of tsrdz at mg/kg resulted in attenuating the production of mda and no, while elevating the sod activity compared with the control group ( ) . the total % ethanol extract of the tubercles of d. trifida l.f. as well as its three subfractions, dichloromethane, butanol, and aqueous fractions showed anti-inflammatory activity in food allergy induced by ovalbumin in balb/c mice ( ) . ova-sensitized balb/c mice received the ova solution to develop local signs of inflammation characterized by eosinophil infiltration, edema, an increase in a number of mast cells in the intestinal submucosa, mucus secretion, an increase in serum anti-ova igg and ige, and weight loss because of hyper-catabolism caused by the production of inflammatory cytokines. the crude extract of d. trifida tested at the doses of and mg/kg/day corresponding to . and . mg/kg/day of allantoin, and . and . mg of diosgenin correlated substances/kg/ day, respectively did not affect the body weight, but they inhibited ige production compared to untreated groups. the inhibition tended to be more significant in aqueous fraction and butanol fraction, which contains allantoin, than dichloromethane, which contains diosgenin. the ethanolic extract and its fractions exerted a reduction in several mastocytes and edema, which was also probably due to the presence of diosgenin and allantoin ( ) . diosgenin was also reported to reduce the production of anti-ova ige and the inflammatory infiltrate in allergic balb/c mice intestines, resulting in a reduction in edema ( ) . balb/c mice having food allergy showed an increase in mucus production by the caliciform cells in the small intestine ( ) . an important allergic response that is induced by interleukins il- and il- is hypersecretion of mucus. mucus has a protective effect on the intestinal cells by limiting the antigen absorption. the presence of eosinophil peroxidase (epo) indirectly reflects the infiltration of eosinophils into intestinal mucosa, which was reduced by administration of the ethanolic extract of d. trifida. mollica et al. also showed that d. trifida extract and fractions reduced all of the inflammatory parameters associated with food allergies in ova-sensitised animals and this activity is correlated with the presence of allantoin and diosgenin correlated substances ( ) . the animals treated with ethanolic extract showed a reduction in mucus production, and this result was also observed for the other fractions. previous studies showed that diosgenin and allantoin activities reduce mucus production in animals allergic to ova ( ) . the crude extracts of d. membranaceae and d. birmania, which contain high amounts of sapogenins, inhibited the production of nitric oxide (no) most probably through inhibiting the nitric oxide synthetase (inos) and tnf-α, with consequent reduction of intestinal edema ( , ) . a study by olayemi and ajaiyeoba also associated the antiedematogenic activity of crude extract from d. esculenta to the presence of sapogenins ( ) . in-vivo studies have demonstrated that oral administration of allantoin attenuates the production of ige, il- , and il- , has an anti-inflammatory effect and promotes healing ( ) . the methanol extract of the rhizomes of d. deltoidea wall.ex griseb. at mg/ kg inhibited the rat hind paw carrageenaninduced edema. the maximum percentage of inhibition was achieved at h after the intraperitoneal administration of the extract ( ) . in a study made by yang et al., the chloroform soluble extract of the rhizomes of d. opposita (csdo) showed cognitive enhancing effects on spatial memory and learning function of mice against scopolamineinduced amnesic deficits via morris water maze and passive avoidance tests. in morris water maze test, both acute treatment by csdo ( mg/kg body weight, p.o.) and prolongedtreatment ( days' daily administration of mg/kg body weight, p.o.) exhibited significant shorter escape latencies in daily first trial than the scopolamine-administered group during a -consecutive-day training period, which suggested that csdo improves the impaired reference memory (long-term memory) induced by scopolamine. especially, the acute treatment group showed a marked decrease in escape latencies in each daily trial compared with the prolonged treatment group. in this study, csdo also showed significant enhancing effects on spatial memory retention in the probe trial. similar results were obtained in the passive avoidance test. csdo treatment significantly increased step-through latencies of scopolamine-induced amnesic mice compared to control untreated group. the results of the two animal behavioral tests demonstrated that csdo improves spatial learning and memory function against scopolamine-induced amnesia ( ) . the study of jeon et al., investigated the neuroprotective effect of herbal mixture from euphoria longana, houttuynia cordata, and dioscorea japonica, by testing the hypothesis that administration of herbs reverses memory deficits and promotes the protein expression of brain-derived neurotrophic factor (bdnf) in the mouse brain: these herbs demonstrated an inductive effect on bdnf, cyclic-amp response element-binding protein (creb) and phospho-creb (pcreb) ( ) . the study of the antinociceptive effect of the methanol extract of the bulbs of dioscorea bulbifera l. var sativa (medb) was performed by nguelefack et al. ( ) the effects of medb persisted for days after two administrations in cfa-induced hypernociception at and mg/ kg. medb significantly inhibited acute lipopolysaccharides (lps)-induced pain at mg/kg but did not affect thermal hypernociception and capsaicin-induced spontaneous nociception. the antinociceptive effects of medb in prostaglandin-e (pge ) model was antagonized by either nitro-l-arginine methyl ester (l-name) or glibenclamide. this indicated that the plant exerted its antinociceptive effect through partial activation of the no-cgmp-atpsensitive potassium channels pathway ( ) . the intraperitoneal administration of n-butanol extract of the d. nipponica rhizomes at mg/kg regulates the blood cholesterol, triglyceride, hdl and ldl than the chloroform ( mg/kg) and water extracts ( mg/kg) ( ) . wang et al. reported the potential activity of trillin, a steroidal saponin isolated from d. nipponica rhizomes, as antihyperlipidemic and anti-oxidative agent invivo. the intraperitoneal administration of trillin ( . mg/kg dissolved in . % dmso) enhanced the blood circulation and could increase the bleeding time for more than % and coagulation in the rats fed on a high-fat diet in which the blood viscosity would be changed resulting in shorter blood coagulation time. the improvement effect of trillin in restoring the blood coagulation abnormality in high-fat diet fed rat was due to its ability to reduce the levels of blood cholesterol, triglyceride and two critical lipoproteins (hdl and ldl). the level of lipid oxidation was increased in high-fat diet rats, causing oxidative stress. trillin exerted an anti-oxidative effect through lowering lipid peroxidation, via the enhancement of superoxide dismutase (sod) level in blood. thus, the combination of trillin and lovastatin in treating hyperlipidemia could represent a more effective therapy (wang et al. ). the authors of this research marked how these findings greatly support the significant role of d. nipponica in protecting the cardiovascular system in-vivo against hyperlipidemia ( ). tang ( ); the authors reported how high serum levels of lactate dehydrogenase (ldh), aspartate aminotransferase (ast) and creatine kinase (ck) indicated cell membrane damage and were thus elevated in the iso model group. intra-gastric injection of total saponins of the three species could restore the activities of myocardial injury marker enzymes to the same extent. oxidative stress plays an essential role in the pathogenesis of mi injury. sod, catalase (cat), gpx, and total antioxidant capacity (t-aoc) levels were reduced significantly in the iso model group with the increase in mda. the activities of these enzymes were normalized by intragastric injection of the total saponins of the three species. the study revealed that the anti-mi mechanism of dioscorea saponins is related not only to the enzymatic antioxidant, such as gpx and cat but also to nonenzymatic antioxidants and its effect on myocardial histology ( ) . the current studies reported the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fat-fed hamsters ( , ) . the total saponins from rhizoma dioscoreae nipponicae could effectively reverse potassium oxonate-induced alterations in renal mouse urate transporter , glucose transporter , organic anion transporter , and organic anion transporter mrna and protein levels, resulting in enhancement of renal urate excretion in mice ( ) . moreover, the authors concluded that the total saponins from rhizoma dioscoreae nipponicae had a uricosuric effect on the regulation of renal organic ion transporters in hyperuricemic animals. in a further paper, the same authors here was the potent uricosuric effect of tdn on hyperuricemic rats by decreasing the expressions of renal rurat while increasing the expressions of renal roat and roat ( ) . a double-blind, placebo-controlled, crossover study was conducted to evaluate the effects of a wild yam cream in healthy women suffering from troublesome symptoms of the menopause. every female was treated with the active cream and a placebo for months randomly. after months treatment with topical wild yam extract in women suffering from menopausal symptoms it was found that the cream is free from side-effects, and has little effect on menopausal symptoms ( ) . dioscorea species make a significant contribution both as root crops and vegetables to the diets around the world. despite their importance as a food source, dioscorea plant parts are quite useful in the treatment of various ailments and disorders due to the presence of several bioactive compounds such as diosgenin. however, several reported ethnomedicinal potential need to be validated and detailed investigations on dioscorea pharmacological properties and phytochemical composition should be carried out to standardize the formulations used. indeed, some pharmacological properties such as hypolipidemic, hypoglycaemic, antioxidant, anti-inflammatory, antimicrobial, antiproliferative, androgenic, estrogenic, and contraceptive have been reported. however, most of the active constituents have not been characterized. thus, authentication of all the secondary metabolites (alkaloids, saponin, flavonoids, tannins, and phenols) from dioscorea should be performed thoughtfully to standardize and validate its quality and biological potentials. moreover, investigations are warranted to address the poor conversion of the preclinical results to clinical efficacity. therefore, an attempt should be made to understand their mechanism of action, pharmacokinetics/pharmacodynamics, and bioavailability and to conduct clinical trials. overall, these findings suggested that dioscorea should not be ignored and should rather be considered as a good alternative source of active molecules that can prevent or alleviate both functional and infectious disease burden, representing a current big challenge in developing countries. the knowledge gaps in this review such as insufficient data on a clinical trial to support preclinical results will help the further initiative to turn dioscorea into drug and stimulate activity to promote their production and utilization as not only valuable components of a well-balanced diet but also for disease prevention. dioscorea spp. (a wild edible tuber): a study on its ethnopharmacological potential and traditional use by the local people of similipal biosphere reserve volatile compounds in intermittent frying by gas chromatography and nuclear magnetic resonance volatile components of the rhizomes of dioscorea japonica dioscorea nipponica makino: a systematic review on its ethnobotany, phytochemical and pharmacological profiles diosgenin: recent highlights on pharmacology and analytical methodology isolation and identification of novel estrogenic compounds in yam tuber (dioscorea alata cv clerodane and -norclerodane diterpenoids from the tubers of dioscorea antaly purification, characterization and biological significance of mannose binding lectin from dioscorea bulbifera bulbils phenolic compounds from the rhizomes of dioscorea bulbifera new norclerodane diterpenoids from the tubers of dioscorea bulbifera liver-specific metabolomics characterizes the hepatotoxicity of dioscorea bulbifera rhizome in rats by integration of gc-ms and h-nmr antifungal steroid saponins from dioscorea cayenensis two spirostan steroid glycoside fatty esters from dioscorea cayenensis effects of processing methods on nutrient and antinutrient composition of yellow yam (dioscorea cayenensis) products novel steroidal saponins from dioscorea esculenta (togedokoro) neurotrophic activity of da- , a mixture extract of dioscorea japonica thunb. and dioscorea nipponica makino, in-vitro a novel steroid and cytotoxic constituents from dioscorea membranacea pierre against hepatocellular carcinoma and cholangiocarcinoma cells adherence to chronic medication in older populations: application of a common protocol among three european cohorts italian translation and cultural adaptation of the communication assessment tool in an outpatient surgical clinic antineuroinflammatory diarylheptanoids from the rhizomes of dioscorea nipponica -oxodioscin, a new spirostan steroid glycoside from the rhizomes of dioscorea nipponica combination of cheminformatics and bioinformatics to explore the chemical basis of the rhizomes and aerial parts of dioscorea nipponica makino phenolic compounds with radical scavenging and cyclooxygenase- (cox- ) inhibitory activities from dioscorea opposita phenolic compounds with pancreatic lipase inhibitory activity from korean yam (dioscorea opposita) chemical components and emulsification properties of mucilage from dioscorea opposita thunb polyhydroxylated spirostanol saponins from the tubers of dioscorea p olygonoides steroidal saponins from dioscorea preussii physicochemical properties of starch from dioscorea pyrifolia tubers anti-inflammatory steroids from the rhizomes of dioscorea septemloba thunb bioactive constituents from the rhizomes of dioscorea septemloba thunb a new phenanthropyran and a new biphenanthrene from the rhizomes of dioscorea septemloba and their antioxidant activities protodioscin, isolated from the rhizome of dioscorea tokoro collected in northern japan is the major antiproliferative compound to hl- leukemic cells cholestane steroid glycosides from the rhizomes of dioscorea villosa (wild yam) diarylheptanoids from dioscorea villosa (wild yam) application of high-speed countercurrent chromatography-evaporative light scattering detection for the separation of seven steroidal saponins from dioscorea villosa lipidated steroid saponins from dioscorea villosa (wild yam) an overview on its traditional use, phytochemistry, pharmacology, clinical applications, quality control, and toxicity anthelmintic activity of steroidal saponins from dioscorea zingiberensis ch wright against dactylogyrus intermedius (monogenea) in goldfish (carassius auratus) phenolic compounds isolated from dioscorea zingiberensis protect against pancreatic acinar cells necrosis induced by sodium taurocholate principles of pharmacological research of nutraceuticals nutraceuticals: a paradigm of proactive medicine nutraceuticals -shedding light on the grey area between pharmaceuticals and food nutraceuticals: opening the debate for a regulatory framework from pharmaceuticals to nutraceuticals: bridging disease prevention and management from plant compounds to botanicals and back: a current snapshot current shot and rethinking of antioxidant research strategy dioscorin isolated from dioscorea alata activates tlr -signaling pathways and induces cytokine expression in macrophages evaluation of anti-inflammatory activity and standardisation of hydro-methanol extract of underground tuber of dioscorea alata synthesis of silver nanoparticles through green approach using dioscorea alata and their characterization on antibacterial activities and optical limiting behavior four types of winged yam (dioscorea alata l.) resistant starches and their effects on ethanol-induced gastric injury in-vivo taiwanese and japanese yam (dioscorea spp.) extracts attenuate doxorubicin-induced cardiotoxicity in mice anti-inflammatory effects of dioscorea alata l. anthocyanins in a tnbs-induced colitis model effects of madagascar yam extracts, dioscorea antaly, on embryo-larval development of medaka fish immune cell stimulating activity of mucopolysaccharide isolated from yam (dioscorea batatas) antiinflammatory activity of bark of dioscorea batatas decne through the inhibition of inos and cox- expressions in raw . cells via nf-κb and erk / inactivation traditional chinese medicine herbal extracts of cibotium barometz, gentiana scabra, dioscorea batatas, cassia tora and taxillus chinensis inhibit sars-cov replication protective effects of dioscorea batatas flesh and peel extracts against ethanol-induced gastric ulcer in mice dioscorea phytocompounds enhance murine splenocyte proliferation ex-vivo and improve regeneration of bone marrow cells in-vivo immunomodulatory effects of phytocompounds characterized by in-vivo transgenic human gm-csf promoter activity in skin tissues dioscorea bulbifera induced apoptosis through inhibition of erk / and activation of jnk signaling pathways in hct human colorectal carcinoma cells phytochemistry and therapeutic potential of medicinal plant: dioscorea bulbifera antinociceptive activities of the methanol extract of the bulbs of dioscorea bulbifera l. var sativa in mice is dependent of no-cgmp-atp-sensitive-k+ channel activation the cytotoxicity of protoneodioscin (nsc- ), a furostanol saponin from the rhizomes of dioscorea collettii var. hypoglauca, against human cancer cells invitro the cytotoxicity of methyl protoneogracillin (nsc- ) and gracillin (nsc- ), two steroidal saponins from the rhizomes of dioscorea collettii var. hypoglauca, against human cancer cells in-vitro medicinal plants in farwest nepal: indigenous uses and pharmacological validity antimicrobial activity and phytochemical screening of some traditionally used nepalese medicinal plants in-vitro antioxidant studies of dioscorea esculenta (lour) protective effects of allantoin against ovalbumin (ova)-induced lung inflammation in a murine model of asthma comparative antimicrobial studies of dioscorea hamiltonii hook. f. tubers with azadirachta indica stem the effect of different extraction techniques on property and bioactivity of polysaccharides from dioscorea hemsleyi modified dioscorea hispida starch-based hydrogels and their in-vitro cytotoxicity study on small intestine cell line (fhs- int) analysis and identification of phenolic compounds in dioscorea hispida dennst antioxidant and anti-inflammatory properties of taiwanese yam (dioscorea japonica thunb. var. pseudojaponica (hayata) yamam.) and its reference compounds nitric oxide inhibitory substances from the rhizomes of dioscorea membranacea immunomodulatory activity of dioscorea membranacea pierre rhizomes and of its main active constituent dioscorealide b. bmc complement phenolic derivatives from the rhizomes of dioscorea nipponica and their antineuroinflammatory and neuroprotective activities anti-allergic substances from the rhizomes of dioscorea membranacea total saponins from discorea nipponica makino ameliorate urate excretion in hyperuricemic rats total saponins from discorea nipponica ameliorate urate excretion in hyperuricemic mice trillin, a steroidal saponin isolated from the rhizomes of dioscorea nipponica, exerts protective effects against hyperlipidemia and oxidative stress comparative study on the morphology, chemistry, metabolism and anti-myocardial ischemia activity of three medicinal species of dioscorea [dissertation]. hkbu institutional repository neuroprotective effects of dioscorea opposita on scopolamine-induced memory impairment in in-vivo behavioral tests and in vitro assays antioxidant and antitumor activities of the extracts from chinese yam (dioscorea opposite thunb.) flesh and peel and the effective compounds dioscorea oppositifolia mediated synthesis of gold and silver nanoparticles with catalytic activity hypoglycemic effect of chinese yam (dioscorea opposita rhizoma) polysaccharide in different structure and molecular weight antioxidant activity, antibacterial potential and characterization of active fraction of dioscorea pentaphylla l. tuber extract collected from similipal biosphere reserve, odisha, india. braz cardioprotective effect of total saponins from three medicinal species of dioscorea against isoprenaline-induced myocardial ischemia antifungal effects of saponin extract from rhizomes of dioscorea panthaica prain et burk against candida albicans effects of jamaican bitter yam (dioscorea polygonoides) and diosgenin on blood and fecal cholesterol in rats changes in some liver enzymes in streptozotocin-induced diabetic rats fed sapogenin extract from bitter yam (dioscorea polygonoides) or commercial diosgenin total saponins from dioscorea septemloba thunb reduce serum uric acid levels in rats with hyperuricemia through oatp a up-regulation effect of dioscorea tokoro makino extract on hyperuricemia in mice the use of purple yam (dioscorea trifida) as a healthpromoting ingredient in bread making anti-inflammatory activity of american yam dioscorea trifida lf in food allergy induced by ovalbumin in mice valuation of the antibacterial activity of ethanolic extract of dioscorea villosa tubers-an in-vitro study hepatoprotective effect of steroidal glycosides from dioscorea villosa on hydrogen peroxideinduced hepatotoxicity in hepg cells bioassay-guided evaluation of dioscorea villosa-an acute and subchronic toxicity, antinociceptive and anti-inflammatory approach therapeutic effects of total steroid saponin extracts from the rhizome of dioscorea zingiberensis ch wright in freund's complete adjuvant induced arthritis in rats the antifungal activity and membranedisruptive action of dioscin extracted from dioscorea nipponica preparation, physicochemical characterization and biological activities of two modified starches from yam novel role of zn (ii)-curcumin in enhancing cell proliferation and adjusting proinflammatory cytokine-mediated oxidative damage of ethanolinduced acute gastric ulcers anti-thrombotic activity and chemical characterization of steroidal saponins from dioscorea zingiberensis ch wright diosgenin attenuates allergen-induced intestinal inflammation and ige production in a murine model of food allergy a model of chronic ige-mediated food allergy in ovalbumin-sensitized mice anti-inflammatory studies of yam (dioscorea esculenta) extract on wistar rats anti-inflammatory and antimicrobial property of dioscorea deltoidea l from nepal alteration in brain-derived neurotrophic factor (bdnf) after treatment of mice with herbal mixture containing euphoria longana, houttuynia cordata and dioscorea japonica the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fat-fed hamsters the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fatfed hamsters effects of wild yam extract on menopausal symptoms, lipids and sex hormones in healthy menopausal women key: cord- -bxm yxjm authors: zeng, yawen; pu, xiaoying; du, juan; yang, xiaomeng; li, xia; mandal, md. siddikun nabi; yang, tao; yang, jiazhen title: molecular mechanism of functional ingredients in barley to combat human chronic diseases date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: bxm yxjm barley plays an important role in health and civilization of human migration from africa to asia, later to eurasia. we demonstrated the systematic mechanism of functional ingredients in barley to combat chronic diseases, based on pubmed, cnki, and isi web of science databases from to . barley and its extracts are rich in ingredients to combat more than chronic diseases, which include the similar and different chronic diseases between grains and grass, due to the major molecular mechanism of six functional ingredients of barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch). the antioxidant activity of barley grass and grain has the same and different functional components. these results support findings that barley grain and its grass are the best functional food, promoting ancient babylonian and egyptian civilizations, and further show the depending functional ingredients for diet from pliocene hominids in africa and neanderthals in europe to modern humans in the world. this review paper not only reveals the formation and action mechanism of barley diet overcoming human chronic diseases, but also provides scientific basis for the development of health products and drugs for the prevention and treatment of human chronic diseases. global cost of five chronic diseases (diabetes, cardiovascular disease, mental illness, chronic respiratory disease, and cancer) treatment to reach $ trillion from to [ ] . the intake of high sodium with low whole grains and fruits was the top most dietary risk factors for deaths and disability-adjusted life years globally and in many countries [ ] . diabetes in production regions of polished rice with high glycemic index (gi ≥ ) caused the biggest reduction in health-adjusted life expectancy at birth in regions in countries from to [ ] . the micronutrients deficiencies at the highest risk are fe, zn, and vitamins (v b , v b , v b , and v c ) [ ] . the outbreak of human chronic disease is due to taste pursuit that changes a healthy diet, i.e., the ancients switched from brown rice (gi ≤ , high k and high micronutrients) and barley (gi ≤ ) or its grass flour (k/na ≥ ) as staple foods to modern polished rice (gi ≥ ) and wheat white flour (gi ≥ ) with low and low micronutrients as staple foods [ , ] . barley grass is not only the best functional food for cell nutrition and detoxification in human body but also the most abundant bioactive ingredients for lots of health-promoting effects [ , ] . it can combat more than chronic diseases due to gaba, flavonoids, sod, k-ca, vitamins, and trypto-phan mechanism in barley grass ( figure ) [ ] . the sustaining major foods+barley grass powder can achieve the who's intake target of low sodium (< g) with high potassium (> . g) every day [ ] . more than functional ingredients in barley grass can combat over chronic diseases, and functional ingredients in barley grains may prevent chronic diseases [ ] . barley enhanced the sterols accumulation through ltp gene action that take part in the abiotic stress reaction of mediating intracellular lipid transport [ ] . barley straw ( . ~ . g/l) for phenolic acid in degradation inhibited the alga (m. aeruginosa) blooms of aquatic eutrophication by cell shrinkage of metabolic activity and chlorophyll a fluorescence decay [ , ] . therefore, barley grass powder plays an important role for solving human chronic diseases. barley grains have the highest functional value (low gi with high β-glucans and resistant starch) and antioxidant properties among cereal crops. the soluble fiber β-glucans is a group of polysaccharides found in barley, oats, mushrooms, yeasts, and seaweed [ ] . hulless barley variety zangqing has a . -gb sequence with , genes in seven chromosomes [ ] ; three hvcslf genes take part in ( , ; , )-β-glucan synthesis [ ] . qingke (hulless barley) is a major food for tibetan people and an important livestock feed in the qinghai-tibetan plateau, which has lots of gene family related to stress reactions [ ] , especially different antioxidant capacities due to some polysaccharide and phytochemical compositions [ ] . regular daily consumption of whole barley flour can prevent chronic diseases, especially diabetes, colonic cancer, hyperlipidemia, high blood pressure, and gallstones [ ] . although barley grains have played an important role in health effects of human being, health contribution and different major mechanisms from barley grass for preventive human chronic diseases and functional ingredients in barley grains are unclear. [ , , ] resistant starch (%) whole grains : ± : . ~ . [ ] [ ] [ ] arabinoxylan (%) endosperms : ± : . ~ . [ ] barley bran : ± : . ~ . [ , ] grains flour : ± : . ~ . [ , ] polyphenols (mg/ g) whole grains : ± : . ~ . [ , ] barley bran : ± : . ~ . [ ] grains flour : ± : . ~ . [ ] phenolic acids (mg/ g) whole grains : ± : . ~ . [ ] total flavones (mg/ g) whole grains : ± : . ~ . [ , , ] flavonoids (mg/ g) whole grains : ± : . ~ . [ ] catechin (mg/ g) whole grains : ± : . ~ . [ , ] quercetin (mg/ g) purple grains : ± : . ~ . [ , ] kaempferol (mg/ g) whole grains : ± : . ~ . [ , ] myricetin (mg/ g) whole grains : ± : ~ . [ ] total alkaloid (mg/ g) whole grains : ± : . ~ . [ ] total anthocyanin (mg/ g) whole grains : ± : . ~ . [ ] barley bran : ± : ~ . [ ] refined flours : ± : . ~ . [ ] proanthocyanidin (mg/ g) whole grains : ± : . ~ . [ ] total tocols (mg/ g) whole grains : ± : . ~ . [ , , , ] antioxidant activity (%) whole grains : ± : . ~ . [ ] gaba (mg/ g) whole grains : ± : . ~ . [ ] protein % whole grains : ± : . ~ . [ ] folates (mg/ g) whole grains : ± : . ~ . [ , ] phytosterols (mg/ g) whole grains : ± : . ~ . [ ] p (mg/kg) whole grains , : ± , : ~ [ ] k (mg/kg) whole grains , : ± , : ~ [ ] ca (mg/kg) whole grains : ± : . ~ . [ ] mg (mg/kg) whole grains , : ± : . ~ . [ ] fe (mg/kg) whole grains : ± : . ~ . [ ] zn (mg/kg) whole grains : ± : . ~ . [ ] cu (mg/kg) whole grains : ± : . ~ . [ ] mn (mg/kg) whole grains : ± : . ~ . [ ] na (mg/kg) whole grains : ± : . ~ . [ ] s (mg/kg) whole grains , : ± : . ~ . [ ] oxidative medicine and cellular longevity and metal chelating activity . - . %) than that of plains ( ~ m altitude), but the soluble β-glucan and arabinoxylan content ranged from . % to . % and . % to . %, respectively [ ] . . . β-glucan. β-glucan in barley is the most abundant group of polysaccharides in cell wall. the molecular weight of β-d-glucan in hulless barley grains is . kda, which composes of ( → )-and ( → )-glucopyranosyl residues, especially its trisaccharide and tetrasaccharide accounted for . % of total cellulosyl units [ ] . β-glucan content (%) in naked, malt, black, waxy-naked, and blue barley is . , . , . , . , and . , respectively, especially waxy-naked barley flour has the highest extraction rate ( . %); however, gi in vitro starch digestibility was lowered by adding β-glucan [ ] . the fatty acid derived flavouring substance (dodecanoic acid, octyl butanoate, ethyl decanoate, and decyl acetate) in beer has important role in the aggregation behavior of barley β-glucan [ ] . the β-glucan concentrations in the six hulless barley grains varied from . % to . %, among shorts ( . ~ . %)>bran ( . ~ . %)>flour ( . ~ . %) [ ] . the total β-glucan contents in the nine hulless barley are . ~ . % in bran and > . ~ . % in refined flour [ ] . barley. phenolic compounds have the antioxidant, anti-inflammatory, and antitumor potentials [ ] . there are the most abundant polyphenols in barley grains, especially p-hydroxybenzoic ( . %), pcoumaric ( . %), and ferulic acids ( . %) [ ] . the most abundant polyphenol in barley extract include free polyphenols ( : ± : mg/ g) and bound polyphenols ( : ± : mg/ g), especially ferulic acid ( . mg/ g) and >procyanidin b ( . mg/ g) [ ] . phenolic acids such as ferulic acid and p-coumaric acid were . mg/ g and . mg/ g in barley grains and . mg/ g and . mg/ g in malt, respectively [ ] . the extraction polyphenols yield of barley lactobacillus fermented solution of % ethanol concentration was . %, main components (mg/ g) including rutin ( . ), vanillic acid ( . ), ferulic acid ( . ), coumaric acid ( . ), gallic acid ( . ), protocatechuic acid ( . ), and p-coumaric acid ( . ) [ ] . compared to the raw barley extract, the protein, total phenols, and βglucan of fermented barley extract with lactobacillus plantarum dy- can significantly increase to . %, . mg/g, and . %, respectively [ ] . there were larger genetic variations in the contents of total polyphenol ( : ± : mg/ g), total flavonoid content ( : ± : mg/ g), and antioxidant activity ( : ± : %) among the barley genotypes; however, major qtls between bpb- and bpb- control phenolic compounds in tibetan wild barley, especially the udp-glycosyltransferase gene with biosynthesis of flavonoid glycosides was colocated with bpb- [ ] . barley. nacl stress increased the phenolic compounds (vanillic acid, p-coumaric acid, ferulic acid, and sinapic acid) accumulation and synthesis by upregulating the gene expression of phenylalanine ammonia lyase, cinnamic acid -hydroxylase, -coumarate coenzyme a ligase, p-coumaric acid -hdroxylase, and caffeic acid/ -hydroxyferulic acid o-methyltransferase of germinated hulless barley [ ] . the blue hulless barley grains have larger variation on phenolic compounds and antioxidant activity, such as the free, bound, and total phenolic acids varied between . ~ . , . ~ . , and . ~ . mg/ g, respectively, where the major phenolic compounds include quercetin, rutin, naringenin, hesperidin, (+)-catechin, gallic acid, benzoic acid, syringic acid, and -coumaric acid [ ] . the anthocyanin and total phenolic contents in hulless barley grains are higher for high altitude, and the contents in its refined flours were . ~ . mg/ g and . ~ . mg fae/ g and in its bran were . ~ . mg/ g and . ~ . mg fae/ g, respectively [ ] . whole grain hulless barley had high contents of total phenolic ( . mg/ g), total pentosan ( . g/ g), and orac values ( : ± : mol/ g) [ ] . the bran extract of hulless barley rich in phenolic acids on the nε-carboxymethyllysine formation during processing biscuits, which can reduce glycation and benefiting health [ ] . total polyphenols ( . mg/ g) and proanthocyanidins ( . anthocyanin for human health belongs to flavonoids, which is a secondary metabolite that plants adapt to harsh environments. ant gene ( hl) with a bhlh domain control purple grain and ant gene control red leaf sheath and pericarp in barley, r r -myb (ant )+bhlh (ant ) complex promotes the synthesis by affecting expression of the anthocyanin biosynthesis structural genes (f ′ h) and ans genes [ ] . the hl alleles from barley purple pericarp synthesis the peonidin- glucoside [ ] . the anthocyanin synthesis hvmyc gene is the major variant factor for blue aleurone of barley [ ] . barley anthocyanins take part in the amino acid biosynthesis, carbon metabolism, phenylpropanoid biosynthesis, and metabolic pathways [ ] . flavonoid ′ -hydroxylase (f ′ h) and flavonoid ′ , ′ -hydroxylase (f ′ ′ h)-coding genes take part in anthocyanin synthesis in barley [ ] . the anthocyanin bran-rich fractions of yellow ( . mg/ g, anthocyanins) and purple barley ( . mg/ g, anthocyanins) are times higher than that of the whole grain flours ( . and . mg/ g), especially cyanidin -glucoside, delphinidin -glucoside, petunidin -glucoside, delphinidin -rutinoside, and cyanidin chloride [ ] . arabinoxylan in barley is the second highest cell wall polysaccharide [ ] . arabinoxylan in barley plays an important role in quality traits of malt and beer product [ ] ; however, arabinoxylan arabinofuranohydrolase i can be used as novel enzyme products in the beer industry [ ] . the starch degradation for seedling relies on cell wall degradation, where the iminosugar , dideoxy- , -imino-l-arabinitol inhibits dextrinase and arabinoxylan arabinofuranohydrolase but permits rapid diffusion of αand β-amylase [ ] . arabinoxylan contents in barley grains range from . % to . % [ ] where it ranged from . mg/ g to . mg/ g at an average value of . mg/ g in barley endosperm in spring -row barley; its two qtls include glycosyltransferases and glycoside hydrolases [ ] . arabinoxylan accounts for % of total polysaccharide [ ] and ~ % of total monosaccharide in barley husk [ ] . arabinoxylan contents in hulless barley are in bran . %>in shorts . %>in flour . % [ ] which differ from the report of moza et al. [ ] (in bran . ~ . %>in flour . ~ . %). . . phytosterols. phytosterols in plant membrane are similar in structure to cholesterol [ ] . higher phytosterols are found in the outer layers of barley grains and ranged between . mg/ g and . mg/ g, among which β-sitosterol is : ± : mg/ g and campesterol is : ± : mg/ g. the other phytosterols include stigmasterol ( . mg/ g), brassicasterol, δ -avenasterol, stigmastanol, stigmastadienol, and other minor sterols (δ -and δ -avenasterols, δ -stigmastenol, and stigmastadienol: : ± : mg/ g) [ ] . . vitamin e is the major lipid-soluble antioxidant for human health, which has eight different stereoisomers [ , ] by three chiral centers in tocopherols from barley. spring barley has higher α-tocotrienol content in four tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, and α-tocopherol) [ ] . [ ] . a tocochromanol in barley grains ranged from . to . mg/ g, but which is much higher than in oat ( . mg/ g) and triticum ( . mg/ g) [ ] . tocochromanols content in barley is % in pericarp, > % in endosperm, and > % in germ; about % of the tocochromanols were tocotrienols, and tocopherols in germ ( %) was higher than that in pericarp ( %) [ ] . the hulless barley especially with waxy, double waxy and tercel cultivars have the highest tocols content. tocol in whole grain was . mg/ g to . mg/ g, and in pearling flour was . mg/ g to . mg/ g; however, the ratios of total tocotrienols to total tocopherols ranged from . to . [ ] . the highest content of tocols ( . ~ . mg/ g) and vitamin e concentrations ( . ~ . mg/ g) was found in the waxy barley, especially in the hulless waxy washonubet (tocols . mg/ g and α-tocotrienols isomer . mg/ g) [ ] . . . resistant starch. resistant starch (rs) can prevent dietrelated chronic diseases such as diabetes and colon cancer. rs in hulless barley grains is related with b-type granules and the amylopectin f-iii fraction; however, sequential rate of enzymatic hydrolysis in diets is waxy>normal>high amylose barley [ ] . rs of spring barley cultivars approved and popularized during the past years in oxidative medicine and cellular longevity europe, in which rs content ranged from < % to > % [ ] . rs content of unprocessed grains of high-amylose, normal, and waxy barley is : ± : %, : ± : %, and : ± : %, respectively, but slowly digestible starch in unprocessed grains of normal ( : ± : %)>high-amylose ( : ± : %)>waxy barley ( : ± : %) and rapidly digestible starch in normal ( : ± : %)<high-amylose ( : ± : %)<waxy barley ( : ± : %) [ ] . the rs content (mg/ g) of accessions barley grains was : ± : %, with a highest content up to . % [ ] . snps (i.e., th exon g( )-to-t and fifth exon c( )-to-t) in three exons play different roles on the expression of the waxy transcript, granule-bound starch synthase i (gbss i), protein, amylose, and starch properties of hulless barley [ ] . the chronology of rs contents in different barley diets is ground pearled barley ( . %)>pearled barley flakes ( . %)>whole pearled barley ( . %)>cut barley . . gaba and linoleic acid. gaba increased α-amylase gene expression by treating barley aleurone with exogenous gaba, especially α-amylase activity began to rise after about h and reached a peak at h [ ] . the gaba content (mg/ g) in accessions of barley grains is : ± : mg/ g, the highest up to : : ± : mg/ g [ ] . the gaba has a very important role in mediating nacl stress phenolic compounds accumulation in germinated hulless barley [ ] . linoleic acid content increased from . % to . % and oil from . to . %, while oleic content decreased from . % to . % and palmitic acid from . % to . % during barley malting process [ ] . . . phytases. hydrolyze phytate in barley associated the bioavailable nutrient elements (p, fe, and zn), which exists as a single gene (paphy_a) in barley, but as two or three homeologous copies in wheat [ ] . the improvement of hvpaphy_a transformed barley showed phytase activity increases up to -fold in green leaves, -fold in grains, and -fold in dry straw [ ] . barley grains for preventive chronic diseases . . . antidiabetic properties. diabetes is a chronic metabolic disease with high mortality rates; therefore, search for novel natural inhibitors has gained much attention [ ] . major antidiabetic elements in barley are β-glucan, phenolic compounds (phenolic acids and flavonoids), phytosterols, tocols, arabinoxylan, and resistant starch ( table ) . oxidative stress not only leads to insulin resistance, impaired glucose tolerance, b-cell dysfunction, ultimately diabetes but also can treat diabetes and obesity by phytochemicals (phenolic acids, flavonoids, phytosterols, and tocols) in barley [ ] . chronic consumption of foods with high β-glucans in barley can improve insulin resistance and lower the postprandial glucose response and increase satiety [ ] . the β-glucan in hulless barley reduced the insulin resistance, arterial sclerosis, serum glucose, and serum lipid in high-fat mouse [ ] . high phenolic content ( . mg/ g) and low rapidly digested starch ( . %) make barley muffin to modulate glycemic response [ ] . the hypoglycemic effect of ethanol extract polysaccharide from barley malt is better for decreased fasting plasma glucose of the diabetes mice than that of water extract [ ] . the boiled barley kernels evening meal can facilitate glucose regulation, increase the release of glucagon-like peptide- , and reduce energy intake and fasting serum free fatty acids, mediated through gut microbial fermentation of the indigestible carbohydrates [ ] . the glycemic index (gi = : ) of all-wheat bread is higher than that (gi = : ) of % wheat+ % barley flour ( . % β-glucan) [ ] . gi for barley with . % β-glucan and oat tempe are and , respectively [ ] . the hulless barley can reduce postprandial glucose and improved insulin sensitivity by amino acid and biogenic amine profiles [ ] . . . . antiobesity. major antiobesity components in barley are β-glucan, resistant starch, polyphenols, dietary fiber, arabinoxylan, tocols, and phytosterols ( table ) . β-glucan in barley significantly treats obesity that reduced low-density lipoprotein, total cholesterol, and serum p-cresyl sulfate levels and increased flow-mediated dilation [ , ] . barley β-glucan can prevent visceral fat (≥ cm ) obesity and increase faecal scores, but decreased nutrient digestibility and antiobesity [ , ] . rs and β-glucans as well as soluble arabinoxylan were utilized mainly in the caecum, especially rs shifted the utilization of other polysaccharides to more distal parts of the colon of pigs [ ] . obesity and insulin resistance associated with bile acid changes and lower dietary fiber (β-glucan) in barley diet [ ] . the aqueous extract of fermented barley has antiobesity effects due to β-glucan and phenolic acids (vanillic acid and ferulic acid) [ ] . β-glucans in black and blue hulless barley for preventive obesity were very higher than that of white one, based on its molecular weights, particle sizes, viscosities, binding capacities (fat, cholesterol, and bile acid), and inhibiting activities on pancreatic lipase [ ] . the polyphenols extracted in black hulless barley show notable decreases in total cholesterol, low-density lipoprotein cholesterol, and atherosclerosis, but significant increase in high-density lipoprotein cholesterol levels [ ] . barley β-glucan can reduce low-density lipoprotein cholesterol and non-highdensity lipoprotein cholesterol as well as alters the gut microbiota for preventing cardiovascular disease (see table ) [ ] . oxidative stress and inflammation are two important factors of atherosclerosis, and polysaccharide extracts with antioxidation and anti-inflammation of hulless barley prevent cardiovascular diseases [ ] . some other functional components of barley have been associated with oxidative medicine and cellular longevity cardiovascular health, such as polyphenols, phytosterols, lignans, tocols, and folate [ ] . major anticancer elements in barley are β-glucan, phenolics, arabinoxylan, phytosterols, lignan, and resistant starch ( table ). functional ingredients of barley with antioxidative and immunomodulatory activities are associated with anticancer effects [ ] . barley with high dietary fiber (β-glucan) has an important role for the prevention of colon cancer and cardiovascular diseases [ ] ; low molecular weight β-d-glucan can enhance antioxidant and antiproliferative activities [ ] . aqueous extract of fermented barley can induce subcutaneous transplantation tumor apoptosis that can be used for a nutrient supplement in the treatment of human colon cancer [ ] . β-glucans in hulless barley has anticancer activities in vitro, but its anti-inflammatory activities increased as their molecular weights decreased [ ] . the bound phenolics in dehulled hulless barley have excellent antioxidant and antiproliferative effects to human liver cancer cells [ ] . a water soluble polysaccharide (glucose : xylose : arabinose : rhamnose = . : . : . : . ) from hulless barley can inhibit colon cancer, which induce ht- apoptosis through ros-jnk and nf-κb-regulated caspase pathways [ ] . . . . antioxidation. antioxidants are compounds that remove reactive oxygen species from cells, which play a dual role in aggravating and preventing diseases [ ] . major antioxidants in barley are phenolic compounds (phenolic acids, flavonoids, and anthocyanin), tocols (vitamin e), polysaccharide (arabinoxylan), dietary fiber, and phytic acid ( table ) . antioxidant effects of polyphenols in barley are flavanols>flavonols (quercetin)>hydroxycinnamic acids (ferulic, caffeic, and coumaric acids) [ ] . malt has higher phenolic content (sinapinic acid and epicatechin) than its barley grains, which plays a key role on antioxidant stability of beer [ ] . the antioxidant activity for anthocyanin in barley bran was markedly higher than that of whole grains flour [ ] . five of the seven associations in barley were with markers near genes associated with the tocochromanol (vitamin e with the most powerful antioxidants) pathway [ ] . overexpression of homogentisate geranylgeranyl transferase for barley enhanced the tocotrienol levels (δ-, β-, and γ-tocotrienol - %) and antioxidant capacity (radical scavenging activity - %) in barley seeds [ ] . the antioxidant effects of dietary fiber in hulless barley bran were associated with total phenolic concentration, which had the dpph ( , -diphenyl- -picrylhydrazyl radical , -diphenyl- -( , , -trinitrophenyl)-hydrazyl) radicalscavenging activity and ferric-reducing antioxidant power oxidative medicine and cellular longevity [ ] . gaba induces the accumulation of proline and total phenolics and enhances the antioxidant system in germinated hulless barley under nacl stress [ ] . the chapatti quality score reduced by % and its phenolic concentration increased from . to . mg/ g, while biscuit spread factor reduced by % and its β-glucan concentration increased from . to . % as well as phenolic content increased from . to . mg/ g after blending of % hulless barley flour, especially markedly increased antioxidant activity [ ] . . . . anti-inflammation. major anti-inflammatory ingredients in barley are β-glucans, lignans, vanillic acid, arabinoxylan, and so on ( table ) . endothelial cell adhesion molecules were identified as an early step in inflammation and atherogenesis; barley β-glucans not only have a maximum anti-inflammatory activity at mw~ : × , especially the inhibition of tnf-α-induced expression of vascular cell adhesion molecule was stronger than that of oat β-glucans, but also have a higher ratio of -o-β-cellobiosyl-d-glucose to -o-β-cellotriosyl-d-glucose oligomers in the polymeric chains [ ] . the molecular weights of β-glucans in hulless barley increased that add inhibitory abilities on α-amylase and pancreatic lipase, but the antiinflammatory abilities decreased, especially the low intrinsic viscosity and high solubility of β-glucans acid hydrolysis for min might contribute to its higher anti-inflammatory activity, which significantly affected their bioactivities (e.g., anticancer), which was beneficial for a better understanding of their structure-function relationships [ ] . the fermented barley extracts (vanillic acid) downregulate glucose consumption and reducing proinflammatory cytokine secretion [ ] . the anti-inflammatory property of malt and wholegrain barley is due to the formation of short chain fatty acid (scfa) and changes in microbiota composition [ ] . . . . immunomodulation. major immunomodulatory substances in barley are β-glucans, arabinoxylan, and so on ( table ). the immunomodulatory activity of barley β-glucans insolubility associated with its particle size, granule conformation, and particulate homogeneity. all β-glucan fractions can induce more cytokines in bone marrowderived dendritic cells than their oat equivalents; however, the insolubility of β-glucan affects its immunomodulatory activity, which is related to its particle size, particle configuration, and particle uniformity [ ] . a water-soluble polysaccharide (bp- , molecular weight : × da) from hulless barley can improve the immune ability of immunosuppressive mice through increasing the serum levels of il- , tnfα, and ifn-γ, such as bp- ( mg/kg and mg/kg) can not only significantly increase the number of bone marrow cells and peripheral blood white blood cells, as well as enhance the production of il- , tnf-α, ifn-γ, igg, and igm in the spleen and serum levels for improving the immune function, but also promote the proliferation and phagocytosis activity of macrophages as well as repair the damage induced by ctx in the spleen cells of immunosuppressive mice [ ] . table ), which shows a % survival rate after cardiac ischemia ( min)/reperfusion ( min) injury, reduces left ventricular anion superoxide production ( %) and infarct size ( %), and increases the capillary ( %) and arteriolar density ( %) and vegf expression ( . %) of hearts in mice [ ] . the products of barley grains can reduce the cardiometabolic risk and regulate the blood glucose and appetite hormones in - h after intake; however, its mechanisms are gut fermentation of indigestible carbohydrates [ ] . . . . hypocholesterolaemic effects. cholesterol is a synthesis lipid in the body [ ] . dietary β-glucan of hulless barley reduces the plasma ldl cholesterol content (see table ) by promoting the excretion of faecal lipids and regulating the activities of -hydroxy- -methyl glutaryl-coenzyme a oxidative medicine and cellular longevity reductase and cholesterol -αhydroxylase in hypercholesterolaemic rats [ ] . barley bran % and % in diet to the hypercholesterolaemic rats improved the level of lipids, lactate dehydrogenase, liver enzymes, and creatine kinase-mb [ ] . whole grain hulless barley has hypocholesterolaemic effects by promoting bile acid synthesis and reabsorption, controlling cholesterol synthesis and accumulation in peripheral tissue, decreasing the expression of -hydroxy- methylglutaryl coenzyme a reductase, while increasing the hepatic expressions of amp-activated protein kinase α, cholesterol α-hydroxylase, ldl receptor, liver x receptor, and pparα [ ] . . . . blood pressure regulation. higher consumption of barley β-glucan is associated with lower systolic and diastolic blood pressure (see table ), i.e., diets rich in β-glucans reduce systolic blood pressure by . mmhg ( % ci . to . mmhg) and diastolic blood pressure by . mmhg ( % ci . to . mmhg) for a median difference in β-glucans of g [ ] . the consumption of high molecular weight barley β-glucan can reduce blood pressure [ ] . . . . bowel health improvement. gastrointestinal tract disease is a major global health problem. β-glucan in hulless barley has protective effects to the gastrointestinal tract (see table ) [ ] . the dietary fiber in hulless barley improved indices of bowel health compared with refined cereal foods, especially himalaya possesses high amylose and resistant starch due to lacking activity of a key enzyme responsible for starch synthesis; however, consumption of himalaya foods resulted in % higher faecal weight, a lowering of faecal ph from . to . , a % higher faecal concentration, a % higher excretion of butyrate, a % higher faecal total scfa excretion, and a % lower faecal p-cresol concentration [ ] . fermented barley extract ( mg/ g) can act as a promising laxative agent to cure spastic constipation [ ] . butyric acid for improving the colonic health is produced by degradation of barley dietary fiber by microbiota [ ] . . . . gastroprotective effects. β-glucan from hulless barley can mitigate the gastric lesions and gastric mucosal damage as well as gastric oxidative stress injury through decreasing the level of malondialdehyde [ ] . the oral administration of fermented barley extract had strong gastroprotective effects through strengthening antioxidant defense system and anti-inflammatory effects, as well as decreasing lipid peroxidation and cat activity by increasing the gsh levels and sod activity in the body, and the mg/kg dose of fermented barley extract was similar gastroprotective as the mg/kg dose of omeprazole, which indicates that this dosage can be used for patients suffering from different levels of gastric damages [ ] . . . . reduce chronic kidney disease. barley β-glucans is associated with a saccharolytic shift in the gut microbiota metabolism by a reduction of pcs toxin blood levels and an increase of scfa production at colonic site, which can reduce the microbial-derived uremic toxin and cardiovascular complications in end-stage renal disease (see table ), especially chronic kidney disease [ ] . the total cholesterol and triglycerides were reduced, and hdl cholesterol increased in % and % barley intervention in breakfast diet; however, barley in the diet of stage chronic kidney disease patients has significantly improved the nutritional status and renal functions [ ] . . . . improve metabolic syndrome. barley β-glucan can improve postprandial glucose response and cholesterol levels as well as the metabolic syndrome based on individual gut microbiota composition (see table ) [ ] . tibetan hulless barley can reduce insulin resistance, dyslipidemia, and body weight gain, which can diminish the prevalence of metabolic syndrome induced by high-fat-sucrose diets, i.e., rats fed with tibetan hulless barley can increase the assessment of insulin resistance scores (body weight, abdominal fat deposition, liver weight, liver fat deposition, triglyceride, fasting blood glucose, and serum fasting insulin) and decrease lowdensity lipoprotein cholesterol levels compared to rats fed with a basal diet [ ] . . . . hepatoprotective effect. β-glucans in barley can decrease fatty liver in diabetes with obesity (see table ) [ ] . the free phenolic extract in barley added the hepatic levels of antioxidant enzymes [ ] . whole grain hulless barley had significantly lower liver lipid levels (total phenolic and pentosan) [ ] . barley sprout extract protects liver cells under oxidative stress by activating nrf and adding glutathione synthesis, especially against alcohol-induced liver injury, as it inhibits glutathione depletion and hepatic lipid accumulation, reduces serum biochemical markers of liver injury, and inhibits inflammatory responses [ ] . . . . wound healing acceleration. nowadays, β-glucans represent effective topical agents for the treatment of chronic wounds and burns due to the activation of the immune and cutaneous cells (see table ), which increase wound repair by enhancing the infiltration of macrophages and promote tissue granulation, collagen deposition, and reepithelialization based on inducing the proliferation and migration of keratinocytes and fibroblasts through specific receptors such as dectin- , cr , or tlrs [ ] . barley β-glucan in vivo promotes the wound closure in mouse skin by promoting the migration and proliferation of human dermis fibroblasts [ ] . . . . heart failure prevention. barley β-d-glucan is a natural activator of mnsod expression, which can prevent heart failure (see table ) [ ] . the food and drug administration has made a health claim between β-glucan and reduced risk of coronary heart disease, diabetes, and heart-related problems [ ] . barley products can prevent and reduce the risk of coronary heart disease, which is associated with the constituents like β-glucan, phenolics, tocols, linoleic acid, and folate [ , ] . . . . atopic dermatitis alleviation. the allergen produced by barley and the protein expressed in insect cells induce the same amount of ifn-γ and il- in pbmc from vaccinated horses (see table ) [ ] . fermented barley extract oxidative medicine and cellular longevity p reduced skin lesions by inhibiting inflammatory cytokines [ ] , but gaba alleviated atopic dermatitis by suppressing serum immunoglobulin e and splenocyte interleukin production [ ] . barley intake reduces the risk of total stroke by significantly increasing the medium and small particle sizes of high-density lipoprotein cholesterol (see table ) [ ] . increasing the expression of antioxidant genes in the liver and the regulation of nrf played a role in the regulation of metabolic diseases in stroke-prone spontaneously hypertensive rats consuming a fermented barley extract p diet [ ] . . . . allergic rhinitis alleviation. ma-al-shaeer formulation based on barley can treat for allergic rhinitis, especially reduced the nasal congestion, post nasal drip, and headache (see table ) [ ] . fermented barley extract alleviated allergic rhinitis in ova-sensitized mice by regulating cytokines (ifn-γ or il- ) related to chronic inflammation [ ] . in addition, the common edible whole-barley flour can reduce the risk of hyperlipidemia and cholelithiasis [ ] , antiaging [ ] as well as increase body strength and increase the content of linoleic acid and linolenic acid in pigs, cattle, sheep, and geese. in a word, there are more than kinds health effects for barley grain preventive chronic diseases according to the summary of current retrieval literature. the descriptions of some literatures for barley preventive chronic diseases (antidiabetes, antiobesity, anticancer, antioxidation, anti-inflammation, hypocholesterolaemic effects, blood pressure regulation, cardioprotection, immunomodulation, improve gastrointestinal, hepatoprotection, bowel health, cardiovascular disease prevention, atopic dermatitis alleviation, wound healing acceleration, heart failure prevention, and so on) are relatively sufficient, but some literatures for barley preventive chronic diseases (antiaging, reduce cholelithiasis, and increase body strength) are relatively less. these results fully demonstrate the health contribution relationship between barley functional food and human chronic disease prevention. the efficacy of preventing chronic diseases is related to barley genotype and its location, composition, extraction, and compatibility, and with the in-depth study of barley grain in prevention and treatment of human chronic diseases, the novel mechanisms for the prevention of chronic diseases may be ascertained as well as its putative role, either at major or minor level, would be further validated. grains for preventive chronic disease . . . β-glucans mechanism. β-glucans can be used as candidates for the medication in the treatment of human chronic diseases [ ] . β-glucans have many bioactivities including antidiabetes; anticancer; antiobesity; anti-inflammation; immunomodulation; cardioprotection; lower cholesterol and lower blood pressure; improve bowel health, gastroprotection, and hepatoprotection; reduce chronic kidney disease and metabolic syndrome; prevent the risk of heart and cardiovascular diseases; and accelerate wound healing activities (figure , tables and ) [ , , , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the major mechanisms of β-glucans of barley involve in the prevention of chronic diseases are as follows: β-glucans can interact with intestinal lipids and bile salt to reduce cholesterol levels and subsequently prevent diabetes, hypertension, cardiovascular disease, and metabolic syndrome [ ] . barley β-glucans not only control appetite and improve insulin sensitivity by gut hormone secretion via microbiota produced scfa [ ] due to its high molecular weight and high viscosity but also increase their antigen ability and enhances the proinflammatory cytokines, which can be degraded by macrophages and natural killer cells mediating its cellular cytotoxicity opsonized tumor cells [ ] . β-glucans of barley are the major regulators of adipogenesis, especially markedly downregulated the target genes in the adipose tissue including adipocyte fatty acid-binding protein, lipoprotein lipase, uncoupling protein- , and glucose transporter in t -l cells [ ] and also have the effect of inhibiting the α-amylase and pancreatic lipase [ ] . barley β-glucan can reduce blood pressure and cardiovascular diseases that alters the composition of gut microbiota, decrease body mass index, waist circumference, and triglyceride levels [ ] and also reduce the systemic inflammatory profile, prevent alveolar bone loss, and improve βcell function in diabetic animals [ ] . barley β-glucans can not only regulate immune responses and connect innate and adaptive immunity [ ] but also have cardioprotective mechanism of promoted angiogenesis through endothelial upregulation of the vascular growth factor [ ] . the hypocholesterolaemic effect of β-glucan in barley is due to the increased bile acid synthesis [ ] and improved bowel health by inhibited feed intake and increased cecal fermentation [ ] . barley β-glucan not only has the gastroprotective effects by increasing the sod and cat activity, decreasing the gastric ulcer index, and increasing prostaglandin e and nitric oxide in laboratory rodents [ ] but also affects lipid metabolism and scfa production, lowering microbes in patients with metabolic syndrome [ ] and improving chronic kidney disease by reducing the microbial-derived uremic toxin. chronic consumption of barley β-glucans can decrease fatty liver by increasing small intestinal contents viscosity and improving glucose, lower glycated hemoglobin and relative kidney weights [ ] , strengthen the angiogenic ability of ros-exposed endothelial cells for preventive heart disease [ ] , and accelerate the wound closure by promoting the migration and proliferation of human dermal fibroblasts [ ] . barley polyphenols have lots of bioactivities including antidiabetes, antiobesity, anticancer, antioxidant, anti-inflammation, hepatoprotection, and prevention of cardiovascular and heart diseases ( figure , tables and ). barley lignan as natural polyphenols has anticancer, antioxidant, anti-inflammation properties, and preventive role for cardiovascular diseases. anthocyanin belongs to flavonoids, and flavonoids belongs to polyphenols. black, purple, and blue barley grains have gained much attention recently because their anthocyanins have anticancer, glycemic and body weight regulation, antioxidation, anti-inflammation, neuroprotection, hypolipidemia, retinal protection, hepatoprotection, and antiaging effects [ , ] . several mechanisms of barley polyphenols for preventing chronic diseases have been documented so far. zhang et al. [ ] has verified the molecular mechanism of insulin resistance by fermented barley extract vanillic acid through regulating mir- expression. the polyphenols with antiobesity from black hulless barley has strong superoxide radical, hydroxyl radical and , -diphenyl- -picrylhydrazyl radicalscavenging activity, ferric reducing antioxidant power, and moderate metal ion-chelating activity [ ] . an efficacious antiproliferation capacity in caco- cells of black barley malt free extract was predicted due to its phenolic constituents which have cellular antioxidant and oxygen radical absorbance as well as peroxyl radical scavenging activities, dpph, and abts radical scavenging assays [ ] . black barley sprouting stimulates the phenolic biosynthesis by upregulating proline-associated pentose phosphate pathway to support structure of sprouts with antioxidant capacity [ ] . total phenolic and pentosan in hulless barley grains have antioxidant activity by downregulated expression of heat shock protein and phosphatidylethanolamine binding protein but upregulated expression of enoyl-coenzyme a hydratase and peroxiredoxin [ ] . the total polyphenol (flavonoid) content and the , -diphenyl- -picrylhydrazyl and abts radical scavenging abilities increased as the barley added to the food mixture [ ] . lignan (-)- (s)-hydroxymatairesinol inhibited tumor necrosis factor-α stimulated endothelial inflammation by inhibiting nf-κb activation and upregulating nrf antioxidant element signaling pathway [ ] . lignans in barley have high anti-inflammatory abilities in endothelial cells by reducing nuclear factor-κb and extracellular signal as well as regulating kinase phosphorylation [ ] . the polyphenols such as (+)-catechin, protocatechuate, and quercetin in barley not only have hepatoprotective effect [ ] but also prevent coronary heart disease by reducing oxidativeinduced tissue damage through modulating intracellular signaling pathways [ ] . polyphenols play an important role in alleviating cardiovascular diseases due to their antiradical scavenging abilities [ ] . barley arabinoxylan has a lot of health benefits, which include antidiabetes, antiobesity, anticancer, lowering cholesterol, immunomodulation, antioxidant, cardiovascular diseases prevention, and so on (figure , tables and ). arabinoxylan in barley is the most abundant polysaccharide that has the capacity of lowering cholesterol and glucose as well as antioxidant activities [ ] . the major mechanisms of barley arabinoxylan for preventing chronic diseases are as follows: arabinoxylan can improve urinary metabolites associated with diabetes by improvement of carbohydrate and lipid as well as amino acid metabolism [ ] . the antioxidant and antiobesity as well as immunomodulation of arabinoxylans associated with prebiotic effects and short-chain fatty acids production by interaction of gut microbiota and arabinoxylans [ ] . arabinoxylan rice bran can increase anticancer effects in the older population by increased nk activity [ ] . the arabinoxylan in barley with the immunomodulatory activity consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and -o-methylglucuronic acid [ ] . the arabinoxylan diet led to a lower postprandial blood for glucose-dependent insulinotropic polypeptide response, especially fat oxidation has an important role in the antiobesity and in the prevention of cardiovascular diseases [ ] . barley polysaccharide prevent cardiovascular diseases by the vasodilatory effect of controlling angiotensin-converting enzyme production [ ] . tables and ). barley grains contain phytosterols that can esterify to fatty acids, phenolic acids, steryl glucosides, or acylated steryl glycosides [ ] . phytosterols significantly inhibited the ability of oxysterols to activate the liver x receptors transcription in modulating cancer cell behavior [ ] , which are thought to influence multiple processes related to cancer, such as carcinogen production, cancercell growth, angiogenesis, invasion, metasis, and cancer-cell apoptosis [ ] . for the prevention of cardiovascular diseases, barley phytosterols can compete with cholesterol for micelle formation, inhibiting cholesterol absorption in intestine and lowering cholesterol in central nervous system of the brain [ , ] . the effect of plant sterols on neurodegenerative diseases is due to its passage through the blood-brain barrier, modulating cholesterol metabolism and inflammation in the central nervous system process in the brain, which involve low-density-lipoprotein, apolipoprotein e, and scavenger receptor class b type [ ] . the lowering of cholesterol and prevention of cardiovascular disease are due to the fact that the phytosterol structure in the barley membrane is similar to the configuration of different cholesterol [ ] , but functionally similar to precursors in phytohormone synthesis, but lowering the blood concentration of cholesterol is beneficial to reduce the risk of cardiovascular disease. extremely rich in content in barley embryos. barley tocols are the best in cereals due to a high concentration and favorable distribution of eight active vitamers [ ] . barley tocols have anticancer, antiobesity, and antioxidant effects and can lower cholesterol level, reduce the risk of stroke, and prevent cardiovascular and heart diseases ( figure , tables and ) [ , ] . tocotrienols can suppress various cancers (breast, lung, ovary, prostate, liver, brain, colon, myeloma, and pancreas) by its molecular mechanisms of cellular proliferation, apoptosis, angiogenesis, metastasis, and inflammation [ ] , which are associated with human intake of whole grains, especially barley and rye. tocotrienol is a functional food of obesity and diabetes by regulating adipogenesis and increase apoptosis of adipocytes and improve glucose homeostasis through oxidative medicine and cellular longevity suppression of inflammation and oxidative stress [ ] . tocol is one of the most powerful antioxidants that has the ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species, thus protecting fatty acids from lipid peroxidation [ ] . all barley pitas had the greatest antioxidant and vitamin e levels from barley malt flour [ ] . the antioxidant properties of barley tocols due to its ability to inhibit lipid peroxidation in biological membranes induce the immune system, promote apoptosis induction, and reduce the risk factors of cardiovascular diseases and stroke by atherosclerotic blockages in the carotid artery [ ] . barley rs has many immune properties like antidiabetes, antiobesity, anticancer, and so on ( figure ,table ). ceramide can promote lipid storage, impaired glucose utilization, and inhibited enzyme dihydroceramide desaturase , which can treat hepatic steatosis and metabolic disorders [ ] . antidiabetes of rs can be increased by suppressing amylopectin synthesis through silencing of starch branching enzymes in barley [ ] . t cell development and gut iga production suppress host lipid absorption by modulating cd expression [ ] to achieve the effect of antiobesity of barley rs. gbss i is mainly responsible for amylose synthesis whereas sss i and sbe ii for amylopectin synthesis in amyloplasts [ ] . barley with high β-glucan and moderate rs may benefit hyperglycemia-impaired lipid metabolism [ ] . the blending of barley starch citrate with resistant starch iv up to % can produce noodles of acceptable quality and numerous health benefits [ ] . early hominids used fruits/vegetables and leaves rich in polyphenols and k-ca as well as vitamins as staple foods to increase the dependence of the human body on these functional ingredients (see figure ). diet played an important role in early hominids evolution [ ] , but no reports have been delivered to date for diet in coevolution of human chronic diseases. miocene ( . ~ . ma) apes had a variety of foods that included folivory, soft-fruit eating, and hard-object feeding [ ] . the diet of pliocene ( . ~ . ma) and early pleistocene ( . ~ . ma) hominids in africa was mainly fruits and leaves of c plants (trees, bushes, shrubs; . ~ . ma) which was gradually transformed into grass (c plant/tropical grasses and sedges) and hard-object (seeds and nuts) ( . ~ . ma) [ ] [ ] [ ] , which was due to low availability of fruits in dry and active glacier ( . ~ . ma) as well as migration to warm grasslands. ethiopia's pliocene lucy is one of the oldest and most complete fossils in hominid bones, her death due to fall out vertically and live on tall tree [ ] . early hominids and australopithecines inhabited forests and savannas for collinearity oxidative medicine and cellular longevity found between tasty fruits (fructose/sucrose, quinine, and tannins) and primate sensory perception, which offered evidence of the two-direction evolutionary trend determining taste sensitivity [ ] . our ape ancestors possessed a digestive dehydrogenase enzyme capable of metabolizing ethanol about ma that they began using fruits fermentation from the forest floor [ ] . there were differences in the proportion of meat and vegetables between the early hominids australopithecus and paranthropus; paranthropus ate more hard food than australopithecus [ ] . early hominid australopithecus africanus, like chimpanzees, are dominated by fruit, leaves, and carbon- -enriched foods about ma [ ] . the worldwide daily consumption of fruits and vegetables as well as tea has become the main tool for prevention of cardiovascular disease, stroke, cancer and diabetes due to their polyphenols modulate tau hyperphosphorylation and beta amyloid aggregation [ ] . the anthocyanins and polyphenols for major functional ingredients in blueberry played a key role in preventing chronic diseases [ ] . the organopolysulfides and quercetin for major functional ingredients in allium genus played a key role in preventing chronic diseases [ ] . baobab was cultivated from seeds from countries in east and west africa, its leaves had the highest vitamin b content ( : ± : mg/ g) from senegal, adult leaves provided the highest ca content ( . %) and young leaves with the highest ca and k content of nankoun in burkina faso [ ] . the diets of early hominids related with five center of crop origin (mediterranean, middle east, central asia, indo-burma, and china-korea); however, the rich food structure maintained the survival and development of early hominids who lacked survival competition and migration could not improve their intelligence. therefore, polyphenols and k-ca as well as vitamins ingredients in fruits/vegetables and leaves as well as grass/seeds (such as gramineae and its ancestor species of barley) for preventive chronic disease are the results of long-term dependence for diet from pliocene hominids in africa to modern human beings ( figure ). ingredients for neanderthals. neanderthals used mushrooms and nuts rich in polysaccharide and phytosterols as well as linoleic acid as staple foods to increase the dependence of the human body on these functional ingredients (see figure ). neanderthals as well as early homo sapiens show high dietary variability in mediterranean evergreen habitats, but less diet in high latitude steppe or coniferous forests [ ] . the steppe-like neanderthals of belgium feed on the meat of the woolly rhinoceros and wild sheep, while the neanderthals of the spanish forest feature root feed on mushrooms and pine nuts [ ] ; neanderthals in northern spain roasted vegetables and used medicinal plants about . ~ . ma [ ] . neanderthals ate meat (high chloroprostol) and plants ( β-sitosterol) as staple foods [ ] . plant foods of neanderthals from iraq and belgium had the typical modern human diets, which include palms, date, legumes, and grass seeds [ ] . medicinal functions (major polysaccharide) in medicinal mushrooms and fungi can prevent and treat more than chronic diseases [ ] ; however, β-glucans in barley preventive chronic diseases. the total polysaccharide content in morchella sp. reached up to . % of dried biomass in a mixture of : of wheat grains and potato peels [ ] . β-glucans are group of polysaccharides found in mushrooms, yeasts, seaweed, barley, and oats [ ] . macrofungal β-glucans are major β- , and β- , -glycosidic bonds, which have immunomodulatory, anticancer, and antioxidant properties; total β-glucan content varied from . % in a. bisporus to . % in t. rutilans [ ] . phytosterols are diet ingredients found in an array of nuts, seeds, and vegetables which have anticancer activities via their interactions with the plasma cell membrane [ ] . oil palm (elaeis guineensis jacq.) is one of the highest oil-yield crops in the world; however, palm oil is the largest variety of plant oil produced, consumed ( %), and internationally traded in the world, rich in linoleic acid ( %) that is associated with egfad gene [ ] . ~ % carbohydrate diets (especially whole-grain breads, vegetables, and nuts) had minimal risk of mortality [ ] . fruits, vegetables, and whole grains were indispensable among four top diets (mediterranean diet with five best ranked first, dash diet with lower blood pressure, flexitarian diet with lose weight and mind diet with brain health). the diets of neanderthals was not correlated with the complete center of crop origin, poor diet (meats, mushrooms, and pine nuts) and lack of migration lead to extinction ( figure ). these results reveal the importance of whole grain and vegetables or fruits in human health; however, lack of three categories of food suggesting the cause of the neanderthal destruction. therefore, polysaccharide (βglucan) and phytosterols as well as linoleic acid in mushrooms and nuts as well as palm oil for preventive chronic disease are the results of long-term dependence for diet from neanderthals in europe to modern human beings ( figure ). ingredients for homo sapiens. homo sapiens not only used grass and seeds rich in gaba and enzymes as well as resistant starch as staple foods to increase the unique dependence of the human body on these functional ingredients (see figure ) but also inherited the staple foods of early hominids and neanderthals. feeding and diet played key roles in human evolution, especially homo sapiens have a relative masticatory structure similar to that of other primates [ ] . homo sapiens moved from africa into the middle east about ka, according to fossils at skhul and qafzeh caves in israel [ ] . the aba-mirna- in belladonna with highly homologous to homo sapiens mirna can target and downregulate human brain-enriched transcription factor (znf- ) and gene expression in the human central nervous system [ ] . for homo sapiens, foragers have greater complexity than farmers or pastoralists; meanwhile, the old world foragers had significantly higher anisotropy values than new world foragers, but similarity between hard food foragers and hard food farmers [ ] . the meat diet abuse by a herbivorous homo sapiens can lead to atherosclerosis [ ] . the diets of african homo sapiens associated with center of crop origin in ethiopia, their migration along eight center of crop origin changed the fate of mankind [ ] . variation in cranial robusticity from geographical homo sapiens associated with cranial shape, size, climate, and neutral genetic distances; however, cranial robusticity may be an adaptation to cold and harsh environments as well as masticatory differences in diet [ ] , especially higher gaba (such as barley grass . mg/ g) grass diets in crop suiting environmental extremes improved intelligence [ ] . gaba contents in picked tea leaves under anoxic treatments at h and h are : ± : and : ± : mg/kg (fresh weight) [ ] . gaba a receptors modulate vigilance, emotions, cognition, and muscle tension, and they are the targets of anxiety-reducing and sedative-hypnotic benzodiazepines and some general anesthetics [ ] . shisa regulates gaba ar trafficking, function, and pharmacology, especially modulates benzodiazepine action in the brain [ ] . barley can be grown in four seasons (spring, summer, autumn, and winter) at , - , m in yunnan province of china which may be associated with harboring enzymes in barley grass [ ] . the diet high in sodium and low four diets (whole grains, fruits, vegetables, nuts, and seeds) were major dietary risk factors for deaths and disability-adjusted life-years globally and in many countries [ ] ; however, the whole grain is the manifestation of resistant starch type i surrounded by protein matrix and bran layer for making the starch unavailable for enzymes. the least chronic disease of ancient humans due to replacing the salt with seasoning crops, such as onions, ginger, garlic, coriander, pepper, chili, and so on [ ] . the coevolution of the preventive human chronic diseases are related to major diets of vavilov's eight crop origin centers (ethiopia, mediterranean, middle east, central asia, indo-burma, china-korea, mexico-guatemala, and peru-ecuador-bolivia) [ ] . human chronic diseases are related with six dietary structures (fruits/vegetables, young grass/barley grass, carnivorous, cereals crop, polished rice/wheat flour, and polished rice/-wheat+grass powder), but polished rice/wheat+barley grass powder is the most major healthy dietary guidelines for modern humans; therefore, it is necessary to unravel coevolutionary mechanism between preventive chronic diseases and human diet for functional foods [ ] . these results support homo sapiens used grass/seeds (rich in gaba and enzymes as well as resistant starch), fruits/vegetables and leaves (rich in polyphenols and k-ca as well as vitamins), mushrooms, and nuts (rich in polysaccharide and phytosterols as well as linoleic acid) as staple foods to increase the dependence of the human body on these functional ingredients. barley is the oldest and more important cereal crop with the utmost dietary fiber in the world; its malt, as a functional food, is not only the largest beer raw material in the world but also one of the most commonly used chinese herbal medicines [ ] . our review point out that barley grass has antidiabetic, anticancer, antidepressant, antioxidant, fatigue, anti-inflammatory, hypolipidemic, antigout, calcium supplementary, and antiacne/detoxifying effects; promotes sleep; regulates blood pressure; enhances immunity; protects liver; reduces hyperuricemia; alleviates atopic dermatitis; improves cognition, constipation, gastrointestinal function; and prevents hypoxia, cardiovascular diseases, and so on [ ] . barley grass powder is known to play a pivotal role in prevention of chronic diseases that involves six molecular mechanism of gaba, flavonoids, sod, k-ca, vitamins, and tryptophan [ ] ; however, barley grains play key roles in prevention of chronic diseases that involves six molecular mechanism of β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch. modern humans had originated in the progeniture of african homo sapiens with cognitive hominin [ ] . the staple foods of modern human are the synthesis of homo sapiens that inherited early hominids and neanderthals, which carry the neanderthal dna due to interbreeding between homo sapiens and neanderthal took place in the middle east. human flt ligand isolated from transgenic barley seeds is a glycoprotein including α( , )-fucose and α( , )xylose, which showed expression of human growth factor in barley grains with active protein [ ] . the peptide ll- is a component of the human innate immune system, it accumulated . mg/kg in the barley grains [ ] . human fgf- gene was fused with barley α-amylase signal peptide dna sequence and expressed in transgenic salvia miltiorrhiza plants driven by s promoter; however, recombinant fgf- in leaves was ng/ fresh weight [ ] . therefore, functional ingredients in barley grass and grains are essential for the health contribution of modern human (homo sapiens), neanderthals, and early hominids staple food to prevent and treat human chronic diseases. barley has health beneficial properties and was part of the modern hominid diet; thus, it has evolved in its functional ingredients and contributed to a reduced risk of diseases. it is amazing that barley grains and malt as well as grass powder can prevent or treat more than human chronic diseases. we think the major scientific basis for that is as follows: first, barley prevent over humans chronic diseases which associated with the similar origin and evolution center of barley and human beings: ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley ( figure ) [ , , ] . ethiopia, morocco, fertile crescent, and tibet of china have been proposed as centers of barley origin and the primary habitat of wild barley (figure ) [ ] . wild barley is a selfing annual grass of predominantly morocco and irano-turanian and israel-jordan in arid desert or salt environments, the cold region in tibet of china and ethiopia, which has accumulated abundant functional ingredients for drought, salt, and cold resistances. the earliest modern human originate from ethiopia and morocco are dated to~ ka and~ ka (figure ) , respectively [ ] . the earliest human occupied high-altitude habitats in the andes and the tibetan plateau, especially late pleistocene humans adapted to the severe environments of these glaciated above , -meter elevation oxidative medicine and cellular longevity in the bale mountains of ethiopia ( figure ) [ ] . neanderthals in modern-day iraq and belgium ate grasses, cooked barley grains, and others. second, barley grass powder plays a key role in the promotion of human intelligence in the early stage: the incremental evolution of globular braincase associated with diets of brain health from ethiopia and middle east as well as from mediterranean center of crop origin, especially the highest gaba in crop diets suiting environmental extremes improved intelligence. brain development is a self-reinforcing process in which brain cells proliferate, differentiate, migrate, and connect functional neural circuit, especially primate-specific features of gabaergic interneuron development [ ] on the basis of gaba content in diet. survival depends on the selection of behaviors adaptive for environment; however, stimulation of dorsal raphe gaba neurons promoted movement in negative but not positive environments to promote environment-specific adaptive behaviors of serotonin [ ] . barley is a major crop in many developed countries [ ] ; gaba in barley grass suiting environmental extremes (cold, arid, and salt) can significantly increase, which can improve cognition and prevent chronic diseases [ ] . the average content of gaba in cultivars that we bred is . mg/ g which is . fold higher than that of other barley crops around the world [ ] . third, healthy effects of functional ingredients of barley grass and grains are the sum of staple foods for early hominids and neanderthals as well as homo sapiens: early hominids used fruits/vegetables and leaves rich in polyphenols (flavonoids) and k-ca as well as vitamins (tocols); neanderthals used mushrooms and nuts rich in polysaccharide (β-glucans and arabinoxylan) and phytosterols as well as linoleic acid; homo sapiens used grass and seeds rich in gaba and enzymes (sod) as well as resistant starch; modern human used barley grass rich in gaba, flavonoids, sod, k-ca, vitamins and tryptophan; however, barley grains rich in β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, resistant starch, and so on. therefore, barley played an important role in solving the problem of depending functional ingredients of homo sapiens and hominids as well as neanderthals, especially food safety in the process of migration and evolution from ancient humans to modern people. migration. food shortages and survival struggles caused by climate change were the causes of early human evolution of suiting environmental extremes from africa to asia and later to eurasia (figure ) [ , ] . barley is not only the most widely used cereal crop with comprehensive utilization of forage, materials for intoxicating liquor, functional food, stable food, ornamental weaving, and chinese medicines but also is the crop with the strongest resistance to stress (drought, cold, and salt) for the highest content of functional components, especially the growth period of barley varies from to days, which can be grown in four seasons in the world or at , ~ , m in yunnan province of china. these excellent characteristics become the best food for human migration. interestingly, there is a striking similarity between the human migration route and barley translocation/evolution route (see figure ). in [ ] . the spread of farming peoples of eurasia from the near east ( . ka), with movements both westward and eastward, especially ancestor of modern south asians is a mixture between early holocene populations of iran and south asia; however, yamnaya in the bronze age of europe moved both westward and eastward from north of the black sea [ ] . discovery of different crushing apparatuses in mountains of iran revealed that people were grinding wheat and barley about , years ago [ ] . these results support the healthy food contribution between human migration and barley translocation/evolution, especially climate change increased functional ingredients in barley for preventive chronic diseases. civilization. this point of view reveals the evolution of human skull morphology on the basis of the hybridization between h. sapiens and other hominin species in morocco at , years ago, all of which are descendants of the african homo sapiens population [ ] . all living people in europe and asia carry the same amount of neanderthal dna due to the interbreeding between homo sapiens and neanderthal that took place in the middle east [ ] . fertile crescent is the concentrated area of wild barley [ ] and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam ( figure ). barley is one of the oldest crops used by ancient farmers, its cultivation has been optimized by modern humans in the ancient era for less shattering, higher yield and better grains; however, the sharp awn of barley has become an important guarantee for the most resistant birds trouble and largescale farming civilization in cereal crops. climate change stimulated agricultural innovation and exchange across asia between , and , years ago; sorghum and millet made their way from china to central asia; wheat and barley moved from central asia to the far east and became a staple food in the north of china at . ka, which exchanges across central and high-altitude asia coalesced to form the silk road ( . ka~ . ka) and grand canal for traffic great artery of north-south in ancient china ( . ka) [ ] . tibetan barley (qingke) is derived from eastern domesticated barley, north pakistan, india, and nepal between , and , years ago, which supports a feral or hybridization origin for tibetan weedy barley [ ] . the rise of barley against stress (drought, cold, salt, and bird) to staple food has increased functional ingredients (especially gaba) in diet to prevent chronic diseases and promoted human civilization. gaba-mediated inhibitory interneurons control memory-encoding ca neurons; nucleus incertus (ni) establishes gabaergic inhibitory synapses on interneurons; ni gabaergic cells can regulate hippocampusdependent episodic memory formation bidirectionally, and its dysfunction may contribute to anxiety-like syndromes [ ] . cities and words and metallurgy as well as complex ceremonial buildings are the four standards of world civilization; fertile crescent for one of centers of barley origin is closely related to ancient babylonian and ancient egyptian civilizations, especially ancient babylonian civilizations have the earliest human civilization (agriculture ka, cities ~ ka, metallurgy ~ ka, words . ka, calendar ka, and systematic religion ka) in the world; however, ancient egyptian civilizations has the earliest empire (mathematics . ka, geometry ka, and writing tool ka). the ancestral blocks of the domesticated barley genomes were descended from all over the fertile crescent, especially levantine (western) and zagros (eastern) clusters of the origin of agriculture for nine wild barley populations, i.e., carmel and galilee, golan heights, hula valley and galilee, judean desert and jordan valley, lower mesopotamia, negev mountains, north levant, sharon, coastal plain and judean lowlands, and upper mesopotamia [ ] . the neolithic crops facilitated the early agricultural establishment; the barley evolution followed the agricultural development in the near east [ ] . for humans from hunter gathering to agriculture about ka of the levant in near east, barley was a founder crop for converting the brittle floral axis of the wild-type into a tough and nonbrittle spike, which made a major contribution to the emergence of early agrarian societies [ ] . for ancient indian civilization, the earliest known farming cultures in south asia emerged in the hills of balochistan in pakistan about . ka; however, seminomadic peoples domesticated wheat, barley, sheep, goat, and cattle [ ] . for ancient chinese civilization, the earliest beer recipe in china included broomcorn, millet, barley, job's tears, and tubers around , y ago, which may have motivated the initial translocation of barley from the western eurasia into the central plain of china [ ] . diseases. western medicine solves the issue of human organs, and traditional chinese medicine solves the issue of human body system; however, functional food is to address the problem of human cells. barley grains and its grass not only are the best functional food that provides nutrition and eliminates toxins from cells in human beings, which are rich in ingredients to combat more than chronic diseases but also have all the nutrients needed for cell nutrition and detoxification, which is the result of the long-term coevolution of the dietary structure of ancient apes for plants and early homo sapiens with the staple food of barley. interestingly, the types of prevention and treatment of human chronic diseases by key functional components in barley grain were in order: β-glucans ( )>polyphenols ( )>arabinoxylan ( ) = tocols ( )>phytosterols ( )>resistant starch ( ), but gaba ( )>flavonoids ( )>sod ( )>k-ca ( ) = vitamins ( )>tryptophan ( ) in barley grains. the key functional components of barley grains and its grass not only play an important role in the prevention and treatment of more than chronic human diseases but also interact with other nutritional functional components to provide the possibility to solve hundreds of human diseases caused by cell undernutrition and their detoxification disorders. the unique theory and practice system of the whole regulation improve the immunity of chinese medicine to prevent and treat human diseases. with advances in science and technology, barley will also find many behavioral mechanisms that combat human diseases, such as barley malt has been used in many prescriptions for the prevention and treatment of covid- in lots of provinces in china, which includes jiangxi, guangdong, gansu, guizhou, and jiangsu province in china [ ] . dietary restriction is an activator of prolongevity molecular pathways based on an escape from costs incurred under nutrient-rich conditions [ ] . chronic disease deaths due to heredity, environment, and lifestyle are as high as million, accounting for more than % of all deaths worldwide [ ] . these results support that human has only new cell disease theory, which are made up of sixty million cells, more than , diseases are due to cell nutritional deficiencies and detoxification disorder caused by the disease [ ] . ingredients of barley were preserved in the chinese crop genebank. the detection of functional ingredients in grains and grass powder of more than , accessions barley gremplasm especially combinated with high-density snp markers can not only reveal the molecular mechanism of functional ingredients and their molecular breeding and gene editing techniques but also make great contributions for improving human health by increasing functional ingredients in barley grains and grass powder. ingredients in barley. some excellent barley varieties have very high functional ingredients both in grains and grass powder, which are the result of adaptation to high and low temperature, drought and waterlogging, long and short sunshine, and day and night changes caused by altitude, latitude, and seasonal differences. first of all is the breeding of high functional ingredients in barley grains. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (β-glucan > . %, resistant starch > %, arabinoxylan > %, or polyphenols > . %) and cultivated barley at low temperature and drought as well as high altitude, such as yunke and zangqing with high β-glucan, yungongmai , and yungongmai with high vitamin c. secondly is the breeding of high functional ingredients in barley grass. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (gaba > . %, k > . %) and cultivated barley at low temperature and drought as well as high altitude. the average content of gaba in cultivars that we bred is . mg/ g which is . fold higher than that of other barley crops around the world, especially barley grass powder of yungong brand contains times gaba ( . mg/ g) and times ca as well as times k than those of polished rice [ ] . in addition, the functional ingredients of barley grain and grass powder can also be greatly improved by gene editing technology, which needs to be further studied and enriched in the future. functional ingredients of barley. functional ingredients of barley grain and its grass powder have larger variation due to latitude, altitude, season, light, temperature, water, day, and night. all the high-altitude ( , ~ , m) hulless barley can increase higher functional ingredient content than that of plains ( ~ m altitude) [ ] . therefore, it is necessary to promote the functional components of the barley grain and its grass powder under the best ecological conditions, and we also make a useful exploration of the functional components at different altitudes and in different seasons. in addition, the functional ingredients of different parts of barley grain (see table ) and the functional ingredients of different grass cutting stages were different. barley is the oldest and the richest functional food among global cereals. its grains are rich in β-glucan; polyphenols (phenolic acids, flavonoids, and anthocyanins), polysaccharide (arabinoxylan), phytosterols (β-sitosterol, campesterol), tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, α-tocopherol), resistant starch, alkaloid, gaba, folates, linoleic acid, phytate, and so on. this review paper summarizes the obvious efficacy of barley grains that includes antidiabetes, antiobesity, anticancer, antioxidants, anti-inflammation, immunomodulation, cardioprotection, gastroprotection, and hepatoprotection properties, and also, barley grains can lower blood pressure; prevent cardiovascular diseases; optimize cholesterol; improve bowel health and metabolic syndrome; prevent heart disease; reduce chronic kidney disease; decrease stroke; alleviate allergic rhinitis and atopic dermatitis; and accelerate wound healing activities. barley grains, grass, straw, husk, bran, and fine powder are rich in ingredients and food structure to defeat chronic diseases during human migration, especially molecular mechanisms of six functional ingredients barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch) involve to combat more than chronic diseases. these results suggest that barley plays an important role in a healthy diet and in the promotion of early human intelligence. in particular, the healthy effects of functional components of barley grains and grass are the result of longterm continuous evolution of early hominids (fruits/vegetables and leaves rich in polyphenols, k-ca, and vitamins), neanderthals (mushrooms and nuts rich in polysaccharides, phytosterols, and linoleic acids), and homo sapiens (grasses and seeds rich in gaba, enzymes, and resistant starch), which associate with modern humans originating in the progenitor of african homo sapiens with cognitive hominin, especially after interbreeding between homo sapiens and neanderthals that took place in the middle east. the migration route from africa to asia and then to eurasia is basically consistent with the origin and spread of barley and its domestication path, which indirectly supports that barley against stress (drought, cold, and salt) enriched with functional ingredients prevented chronic disease from ancient humans to modern people. fertile crescent is the concentrated area of wild barley and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam. these results indirectly support this great contribution of barley for promoting world civilization. the polyphenols in fruits/leaves and polysaccharide in mushrooms/nuts as well as gaba in grass/seeds for prevention of chronic disease are associated with depending functional ingredients for diet from pliocene hominids in africa to modern humans. ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley. food shortages and survival struggles caused by climate change were the causes of early human evolution associated with gaba in the barley grass increased oxidative medicine and cellular longevity sharply under environmental extremes from africa to asia and later to eurasia, especially gaba in crop diets suiting environmental extremes improved intelligence. these results support findings that barley and its grass may be the best functional food crop, especially barley prevents over human chronic diseases based on six functional ingredients of barley grass and grains due to three aspects of the scientific basis, i.e., the similar origin and evolution center of barley and human, the promotion of human intelligence in the early stage, and the sum of staple foods for early hominids and neanderthals as well as homo sapiens. we put forward the strategy of increasing the functional ingredients of barley grain and its grass powder that is as follows: ( ) exploration of excellent germplasm with high functional ingredients in barley; ( ) breeding excellent cultivars with high functional ingredients in barley; ( ) optimization of ecological conditions for high functional ingredients of barley. in addition, the functional ingredients of different parts of barley grain and the functional ingredients of different grass cutting stages are different. although therapeutic mechanisms of functional ingredients in barley grains and grass powder for prevention of human chronic diseases seem a very complicated task, and functional food for therapeutic interventions opens up new ways, it is necessary to find further scientific evidence that demonstrates the health effects of functional ingredients of barley and their extracts from barley on the treatment of chronic diseases. barley is one of the most exciting potential natural sources for the development of functional foods and new drugs with improved efficiency and safety. although we have found some relationship of origin and migration between human and barley, especially preventive role of barley for chronic diseases of human beings, it is necessary to conduct more systemic studies to unravel coevolutionary interconnection mechanism between chronic diseases prevention and human diet for barley functional foods. unfortunately, so far there, is no evidence provided of barley evolving as part of evolutionary consumption. barley plays an important role in promoting the development of functional food and has a potential underlying molecular mechanism and formation as well as action mechanism, which is worthy of further study. this review can be used as a starting point for novel nutraceuticals and functional foods and drugs for barley to improve the prognosis of chronic diseases. the authors declare that they have no conflict of interests whatsoever to declare. the global economic burden of non-communicable diseases health effects of dietary risks in countries, - : a systematic analysis for the global burden of disease study contribution of specific diseases and injuries to changes in health adjusted life expectancy in countries from to : retrospective observational study micronutrient deficiencies in medical and surgical inpatients joint association of urinary sodium and potassium excretion with cardiovascular events and mortality: prospective cohort study preventive and therapeutic role of functional ingredients of barley grass for chronic diseases in human beings barley grass juice (hordeum vulgare l.) inhibits obesity and improves lipid profile in high fat diet-induced rat model nature's contributions to crop diet for human health nature's contributions to crop diet in environmental extremes for human cognition effects of multiple abiotic stresses on lipids and sterols profile in barley leaves (hordeum vulgare l.) response of microcystis aeruginosa bccusp to barley (hordeum vulgare l.) straw degradation extract and fractions effects of tibetan hulless barley on bloom-forming cyanobacterium (microcystis aeruginosa) measured by different physiological and morphologic parameters β-glucans as potential immunoadjuvants: a review on the adjuvanticity, structure-activity relationship and receptor recognition properties assembly and analysis of a qingke reference genome demonstrate its close genetic relation to modern cultivated barley the barley genome sequence assembly reveals three additional members of the cslf ( , ; , )-β-glucan synthase gene family the draft genome of tibetan hulless barley reveals adaptive patterns to the high stressful tibetan plateau protective role of antioxidant huskless barley extracts on tnf-α-induced endothelial dysfunction in human vascular endothelial cells bioactive phytochemicals in barley mass spectrometrybased analysis of whole-grain phytochemicals phytochemical pharmacokinetics and bioactivity of oat and barley flour: a randomized crossover trial phytochemical composition and β-glucan content of barley genotypes from two different geographic origins for human health food production effect of malt process steps on bioactive properties and fatty acid composition of barley, green malt and malt grains folate distribution in barley (hordeum vulgare l.), common wheat (triticum aestivum l.) and durum wheat (triticum turgidum durum desf.) pearled fractions identification of quantitative trait loci for mineral elements in grains and grass powder of barley starch digestibility and bioactivity of high altitude hulless barley structural characterization and rheological properties of β-d-glucan from hull-less barley (hordeum vulgare l. var. nudum hook. f.) physicochemical characteristics and in vitro bile acid binding and starch digestion of βglucans extracted from different varieties of jeju barley impact of flavouring substances on the aggregation behaviour of dissolved barley β-glucans in a model beer distribution and molecular characterization of β-glucans from hull-less barley bran, shorts and flour influence of non-starchy polysaccharides on barley milling behavior and evaluating bioactive composition of milled fractions contribution of the phenolic composition to the antioxidant, anti-inflammatory and antitumor potential of equisetum giganteum l. and tilia platyphyllos scop development of extraction method for characterization of free and bonded polyphenols in barley (hordeum vulgare l.) grown in czech republic using liquid chromatographytandem mass spectrometry antioxidant-guided isolation and mass spectrometric identification of the major polyphenols in barley (hordeum vulgare) grain genetic diversity of individual phenolic acids in barley and their correlation with barley malt quality extraction and antioxidant activity of polyphenols from barley lactobacillus fermented solution the anti-obesity effect of fermented barley extracts with lactobacillus plantarum dy- and saccharomyces cerevisiae in dietinduced obese rats association mapping for total polyphenol content, total flavonoid content and antioxidant activity in barley gaba mediates phenolic compounds accumulation and the antioxidant system enhancement in germinated hulless barley under nacl stress free and bound phenolic compound content and antioxidant activity of different cultivated blue highland barley varieties from the qinghai-tibet plateau antioxidant activity of whole grain highland hull-less barley and its effect on liver protein expression profiles in rats fed with high-fat diets effects of highland barley bran extract rich in phenolic acids on the formation of n ε -carboxymethyllysine in a biscuit model phenolic acids profile, nutritional and phytochemical compounds, antioxidant properties in colored barley grown in southern italy the therapeutic potential of apigenin genetic linkage facilitates cloning of ert-m regulating plant architecture in barley and identified a strong candidate of ant involved in anthocyanin biosynthesis regulation of the flavonoid biosynthesis pathway genes in purple and black grains of hordeum vulgare metabolic pathways and genes identified by rna-seq analysis of barley near-isogenic lines differing by allelic state of the black lemma and pericarp (blp) gene barley ant , encoding flavanone -hydroxylase (f h), is a promising target locus for attaining anthocyanin/proanthocyanidin-free plants without pleiotropic reduction of grain dormancy toward identification of black lemma and pericarp gene blp in barley combining bulked segregant analysis and specific-locus amplified fragment sequencing purple-grained barley (hordeum vulgare l.): marker-assisted development of nils for investigating peculiarities of the anthocyanin biosynthesis regulatory network inheritance analysis and mapping of quantitative trait loci (qtl) controlling individual anthocyanin compounds in purple barley (hordeum vulgare l.) grains identification and characterization of regulatory network components for anthocyanin synthesis in barley aleurone quantitative proteomics analysis reveals proteins and pathways associated with anthocyanin accumulation in barley duplicatedflavonoid '-hydroxylaseandflavonoid ', '-hydroxylasegenes in barley genome anthocyanin composition and oxygen radical scavenging capacity (orac) of milled and pearled purple, black, and common barley a genome wide association study of arabinoxylan content in -row spring barley grain pod promoted oxidative gelation of water-extractable arabinoxylan through ferulic acid dimers. evidence for its negative effect on malt filterability purification and characterisation of arabinoxylan arabinofuranohydrolase i responsible for the filterability of barley malt cell wall degradation is required for normal starch mobilisation in barley endosperm barley β-glucans and arabinoxylans: molecular structure, physicochemical properties, and uses in food products-a review isolation of hemicelluloses from barley husks isolation and characterization of physicochemical and material properties of arabinoxylans from barley husks phytosterols inhibit side-chain oxysterol mediated activation of lxr in breast cancer cells molecular mechanisms of action of tocotrienols in cancer: recent trends and advancements the role of tocotrienol in protecting against metabolic diseases tocotrienols and tocopherols in colored-grain wheat, tritordeum and barley antioxidant capacity and vitamin e in barley: effect of genotype and storage determination of tocopherol and tocotrienol content of greek barley varieties under conventional and organic cultivation techniques using validated reverse phase highperformance liquid chromatography method coularray electrochemical evaluation of tocopherol and tocotrienol isomers in barley, oat and spelt grains tocopherol and tocotrienol accumulation during development of caryopses from barley (hordeum vulgare l.) tocol composition and supercritical carbon dioxide extraction of lipids from barley pearling flour effect of cultivar, year grown, and cropping system on the content of tocopherols and tocotrienols in oxidative medicine and cellular longevity grains of hulled and hulless barley barley grain constituents, starch composition, and structure affect starch in vitro enzymatic hydrolysis genome-wide association study of resistant starch (rs) phenotypes in a barley variety collection impact of micronization on rapidly digestible, slowly digestible, and resistant starch concentrations in normal, high-amylose, and waxy barley genetic variation of functional components in grains for barley improved lines from four continents effect of wide variation of the waxy gene on starch properties in hull-less barley from qinghai-tibet plateau in china compositional analysis of whole grains, processed grains, grain co-products, and other carbohydrate sources with applicability to pet animal nutrition effects of exogenous gamma-aminobutyric acid on α-amylase activity in the aleurone of barley seeds molecular advances on phytases in barley and wheat barley hvpaphy_a as transgene provides high and stable phytase activities in mature barley straw and in grains looking at marine-derived bioactive molecules as upcoming antidiabetic agents: a special emphasis on ptp b inhibitors a double-blind randomised controlled trial testing the effect of a barley product containing varying amounts and types of fibre on the postprandial glucose response of healthy volunteers effects of beta glucan in highland barley on blood glucose and serum lipid in high fat-induced c mouse total antioxidant capacity and starch digestibility of muffins baked with rice, wheat, oat, corn and barley flour study on the hypoglycemic effect of different structures of barley polysaccharide effects of indigestible carbohydrates in barley on glucose metabolism, appetite and voluntary food intake over h in healthy adults effects of barley β-glucan-enriched flour fractions on the glycaemic index of bread whole-grain cereal products based on a high-fibre barley or oat genotype lower post-prandial glucose and insulin responses in healthy humans postprandial differences in the amino acid and biogenic amines profiles of impaired fasting glucose individuals after intake of highland barley beta-glucans supplementation associates with reduction in p-cresyl sulfate levels and improved endothelial vascular reactivity in healthy individuals study on the anti-obesity effect of barley β-glucan effects of high β-glucan barley on visceral fat obesity in japanese individuals: a randomized, double-blind study the variation in chemical composition of barley feed with or without enzyme supplementation influences nutrient digestibility and subsequently affects performance in piglets influence of a diet rich in resistant starch on the degradation of non-starch polysaccharides in the large intestine of pigs effects of barley variety, dietary fiber and β-glucan content on bile acid composition in cecum of rats fed low-and high-fat diets characterization, in vitro binding properties, and inhibitory activity on pancreatic lipase of β-glucans from different qingke (tibetan hulless barley) cultivars in vitro and in vivo antioxidant activity of polyphenols extracted from black highland barley high molecular weight barley β-glucan alters gut microbiota toward reduced cardiovascular disease risk biochemical composition and nutritional evaluation of barley rihane (hordeum vulgare l.) effect of γ-irradiation on structure and nutraceutical potential of β-d-glucan from barley (hordeum vulgare) antitumor activities and apoptosis-regulated mechanisms of fermented barley extract in the transplantation tumor model of human ht- cells in nude mice phenolics content, antioxidant and antiproliferative activities of dehulled highland barley (hordeum vulgare l.) inhibitory effect on ht- colon cancer cells of a water-soluble polysaccharide obtained from highland barley antioxidants: positive or negative actors? determination of phenolic content in different barley varieties and corresponding malts by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry quantitative trait loci associated with the tocochromanol (vitamin e) pathway in barley overexpression of hvhggt enhances tocotrienol levels and antioxidant activity in barley superfine grinding improves functional properties and antioxidant capacities of bran dietary fibre from qingke (hull-less barley) grown in qinghai-tibet plateau, china hulless barley as a promising source to improve the nutritional quality of wheat products effect of oat and barley β-glucans on inhibition of cytokine-induced adhesion molecule expression in human aortic endothelial cells: molecular structure-function relations correlations of molecular weights of β-glucans from qingke (tibetan hulless barley) to their multiple bioactivities fermented barley extracts with lactobacillus plantarum dy- rich in vanillic acid modulate glucose consumption in human hepg cells effects of two whole-grain barley varieties on caecal scfa, gut microbiota and plasma inflammatory markers in rats consuming low-and high-fat diets immunomodulatory properties of oat and barley β-glucan populations on bone marrow derived dendritic cells immunomodulatory activity of a water-soluble polysaccharide obtained from highland barley on immunosuppressive mice models long-term intake of pasta containing barley ( - )beta-d-glucan increases neovascularization-mediated cardioprotection through endothelial upregulation of vascular endothelial growth factor and parkin gut microbiota mediated benefits of barley kernel products on metabolism, gut hormones, and inflammatory markers as affected by co-ingestion of commercially available probiotics: a randomized controlled study in healthy subjects hypocholesterolaemic effect of whole-grain highland hull-less barley in rats fed a high-fat diet effects of dietary hull-less barley β-glucan on the cholesterol metabolism of hypercholesterolemic hamsters the efficiency of barley (hordeum vulgare) bran in ameliorating blood and treating fatty heart and liver of male rats effects of dietary fibre type on blood pressure: a systematic review and meta-analysis of randomized controlled trials of healthy individuals protective effects of β-glucan isolated from highland barley on ethanol-induced gastric damage in rats and its benefits to mice gut conditions wholegrain foods made from a novel high-amylose barley variety (himalaya ) improve indices of bowel health in human subjects protective effects of triple fermented barley extract (fbe) on indomethacin-induced gastric mucosal damage in rats effects of variety and steeping conditions on some barley components associated with colonic health effects of barley semolina on physiological oxidative medicine and cellular longevity and biochemical parameters in chronic kidney disease patients alterations in gut microbiota composition and metabolic parameters after dietary intervention with barley beta glucans in patients with high risk for metabolic syndrome development intake of tibetan hull-less barley is associated with a reduced risk of metabolic related syndrome in rats fed high-fat-sucrose diets consumption of a high β-glucan barley flour improves glucose control and fatty liver and increases muscle acylcarnitines in the zucker diabetic fatty rat chemical composition and hepatoprotective effect of free phenolic extract from barley during malting process antioxidant effect of barley sprout extract via enhancement of nuclear factor-erythroid related factor activity and glutathione synthesis β-glucans: multi-functional modulator of wound healing barley β-glucan accelerates wound healing by favoring migration versus proliferation of human dermal fibroblasts barley beta-glucan promotes mnsod expression and enhances angiogenesis under oxidative microenvironment utilization of barley functional foods for preventing chronic diseases in china a review of extraction and analysis of bioactives in oat and barley and scope for use of novel food processing technologies barley produced culicoides allergens are suitable for monitoring the immune response of horses immunized with e. coli expressed allergens fermented barley extract suppresses the development of atopic dermatitis-like skin lesions in nc/nga mice, probably by inhibiting inflammatory cytokines effects of single and combined administration of fermented barley extract and γ-aminobutyric acid on the development of atopic dermatitis in nc/nga mice association of lifelong intake of barley diet with healthy aging: changes in physical and cognitive functions and intestinal microbiome in senescenceaccelerated mouse-prone (samp ) fermented barley extract supplementation ameliorates metabolic state in stroke-prone spontaneously hypertensive rats effects of a novel barley-based formulation on allergic rhinitis: a randomized controlled trial β-glucans and cholesterol (review) barley β-glucan improves metabolic condition via short-chain fatty acids produced by gut microbial fermentation in high fat diet fed mice the effects of β-glucans on dendritic cells and implications for cancer therapy suppressive effects of barley β-glucans with different molecular weight on t -l adipocyte differentiation effects of βglucans ingestion on alveolar bone loss, intestinal morphology, systemic inflammatory profile, and pancreatic β-cell function in rats with periodontitis and diabetes barley β-glucan reduces blood cholesterol levels via interrupting bile acid metabolism wholegrain barley β-glucan fermentation does not improve glucose tolerance in rats fed a high-fat diet anthocyanins in cereals: composition and health effects molecular mechanism and health role of functional ingredients in blueberry for chronic disease in human beings comparative study on the phytochemical profiles and cellular antioxidant activity of phenolics extracted from barley malts processed under different roasting temperatures metabolic stimulation of phenolic biosynthesis and antioxidant enzyme response in dark germinated barley (hordeum vulgare l.) sprouts using bioprocessed elicitors oxidative medicine and cellular longevity barley mix at different cooking method and mixing ratio (s)-hydroxymatairesinol protects against tumor necrosis factor-α-mediated inflammation response in endothelial cells by blocking the mapk/nf-κb and activating nrf /ho- lignans -hydroxymatairesinol and -hydroxymatairesinol exhibit anti-inflammatory activity in human aortic endothelial cells hyperglycemia-induced oxidative stress and heart disease-cardioprotective effects of rooibos flavonoids and phenylpyruvic acid- -o-β-d-glucoside arabinoxylan attenuates type diabetes by improvement of carbohydrate, lipid, and amino acid metabolism evidence-based review of biobran /mgn- arabinoxylan compound as a complementary therapy for conventional cancer treatment structure elucidation of an immunostimulatory arabinoxylan-type polysaccharide prepared from young barley leaves (hordeum vulgare l.) dietary steamed wheat bran increases postprandial fat oxidation in association with a reduced blood glucose-dependent insulinotropic polypeptide response in mice the impact of phytosterols on the healthy and diseased brain recent advances in our understanding of tocopherol biosynthesis in plants: an overview of key genes, functions, and breeding of vitamin e improved crops targeting a ceramide double bond improves insulin resistance and hepatic steatosis concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules t cell-mediated regulation of the microbiota protects against obesity starch accumulation in hulless barley during grain filling prowashonupana barley dietary fibre reduces body fat and increases insulin sensitivity in caenorhabditis elegans model rheological behavior of wheat starch and barley resistant starch (type iv) blends and their starch noodles making potential diet and the evolution of the earliest human ancestors isotopic evidence of early hominin diets the diets of early hominins perimortem fractures in lucy suggest mortality from fall out of tall tree new perspectives on taste and primate evolution: the dichotomy in gustatory coding for perception of beneficent versus noxious substances as supported by correlations among human thresholds hominids adapted to metabolize ethanol long before human-directed fermentation early hominid diets from quantitative image analysis of dental microwear isotopic evidence for the diet of an early hominid, australopithecus africanus mediterranean diet: the role of long-chain ω- fatty acids in fish; polyphenols in fruits, vegetables, cereals, coffee, tea, cacao and wine; probiotics and vitamins in prevention of stroke, age-related cognitive decline, and alzheimer disease therapeutic role of functional components in alliums for preventive chronic disease in human being variability of vitamins b , b and minerals content in baobab (adansonia digitata) leaves in east and west africa molar macrowear reveals neanderthal ecogeographic dietary variation neanderthal behaviour, diet, and disease inferred from ancient dna in dental calculus neanderthals ate their greens the neanderthal meal: a new perspective using faecal biomarkers microfossils in calculus demonstrate consumption of plants and cooked foods in neanderthal diets (shanidar iii, iraq; spy i and medicinal mushrooms in human clinical studies. part i. anticancer, oncoimmunological, and immunomodulatory activities: a review evaluation of biomass and chitin production of morchella mushrooms grown on starch-based substrates quantitative evaluation of , , , β-d-glucan contents in wild-growing species of edible polish mushrooms exploring the biophysical properties of phytosterols in the plasma membrane for novel cancer prevention strategies identification of a Δ fatty acid desaturase from oil palm (elaeis guineensis jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in saccharomyces cerevisiae effects of a low carbohydrate diet on energy expenditure during weight loss maintenance: randomized trial taylor the masticatory apparatus of humans (homo sapiens): evolution and comparative functional morphology oldest homo sapiens genome pinpoints neandertal input atropa belladonna expresses a microrna (aba-mirna- ) highly homologous to homo sapiens mirna- (hsa-mirna- ); both mirnas target the ′-untranslated region ( ′ -utr) of the mrna encoding the neurologically relevant, zinc-finger transcription factor znf- dental microwear texture analysis of homo sapiens sapiens: foragers, farmers, and pastoralists clinical biochemistry methods in objectiva evalution of overeating food of carnivores (meat) by a phylogenetically herbivorous homo sapiens (a patient) relationship of cranial robusticity to cranial form, geography and climate in homo sapiens dual mechanisms regulating glutamate decarboxylases and accumulation of gammaaminobutyric acid in tea (camellia sinensis) leaves exposed to multiple stresses modulating anxiety and activity shisa is a gabaareceptor auxiliary subunit controlling benzodiazepine actions rewilding food ecosystems for human health human diet and health how did homo sapiens evolve? barley as a green factory for the production of functional flt ligand molecular farming in barley: development of a novel production platform to produce human antimicrobial peptide ll- ectopic expression of human acidic fibroblast growth factor in the medicinal plant, salvia miltiorrhiza, accelerates the healing of burn wounds genebank genomics highlights the diversity of a global barley collection new fossils from jebel irhoud, morocco and the pan-african origin of homo sapiens middle stone age foragers resided in high elevations of the glaciated bale mountains, ethiopia assembling human brain organoids intense threat switches dorsal raphe serotonin neurons to a paradoxical operational mode die ersten menschen co-evolution of methods and thoughts in cereal domestication studies: a tale of barley (hordeum vulgare) genomic analysis of , -year-old cultivated grain illuminates the domestication history of barley climate change stimulated agricultural innovation and exchange across asia the formation of human populations in south and central asia world history / ancient civilizationsseptember origin and evolution of qingke barley in tibet conducting memory formation targeted resequencing reveals genomic signatures of barley domestication evolution of the grain dispersal system in barley revealing a , -y-old beer recipe in china remarkable efficacy of covid- treatment by traditional chinese medicine the hidden costs of dietary restriction: implications for its evolutionary and mechanistic origins are noncommunicable diseases communicable? never be sick again: health is a choice, learn how to choose it genetic analysis of four main flavonoids in barley grain analysis of functional ingredients in barley grains from different regions between southwest china and icarda nutrition analysis and food process of barley the research and publication of this article was funded by china agriculture research system (cars- - a, cars- - b) and yunnan provincial expert grass-roots scientific research workstation. key: cord- -ar cnsa authors: rouadi, philip w.; idriss, samar a.; naclerio, robert m.; peden, david b.; ansotegui, ignacio j.; canonica, giorgio walter; gonzalez-diaz, sandra nora; rosario filho, nelson a.; ivancevich, juan carlos; hellings, peter w.; murrieta-aguttes, margarita; zaitoun, fares h.; irani, carla; karam, marilyn r.; bousquet, jean title: immunopathological features of air pollution and its impact on inflammatory airway diseases (iad) date: - - journal: world allergy organ j doi: . /j.waojou. . sha: doc_id: cord_uid: ar cnsa air pollution causes significant morbidity and mortality in patients with inflammatory airway diseases (iad) such as allergic rhinitis (ar), chronic rhinosinusitis (crs), asthma, and chronic obstructive pulmonary disease (copd). oxidative stress in patients with iad can induce eosinophilic inflammation in the airways, augment atopic allergic sensitization, and increase susceptibility to infection. we reviewed emerging data depicting the involvement of oxidative stress in iad patients. we evaluated biomarkers, outcome measures and immunopathological alterations across the airway mucosal barrier following exposure, particularly when accentuated by an infectious insult. the presence in the air of one or more natural or anthropogenic substances at a concentration, or location, for a duration, above their natural levels with the potential to cause an adverse health effect defines air pollution. indoor air pollution refers to chemical, biological, and physical exposure of air pollutants in homes, schools, and workplaces. similar to indoor air pollution, ambient (outdoor) air pollution can result from chemical substances or biologically derived contaminants modified by climate change or human activity such as bioaerosols and aeroallergens. air quality guidelines endorsed by the world health organization (who) aim to provide clean air in and around the home. air pollution reduced life expectancy in by year and months on average worldwide. who has linked . million deaths globally in to household cooking using coal, wood and biomass stoves. outdoor air pollution in the same year caused an estimated . million deaths. in inflammatory airway disease (iad) patients an estimated - % increased risk in asthma-related mortality was commensurate with a rise in ambient pollutant concentrations such as no , pm . , or ozone when computed few days prior to asthma death. similar but smaller increments in chronic obstructive pulmonary disease (copd)-related mortality were attributed to pollution and ranged from . % to . %. in the respiratory tract, air pollution can impact wellness in healthy people and patients with iad, irrespective of their atopic status. hence, the airway mucosal barrier may be disrupted by immunopathological mechanisms resulting from effects of pollution and iad. the co-occurrence of iad phenotypes (allergic rhinitis, and chronic rhinosinusitis, copd and asthma) within an individual increases the likelihood of pollutant induced exacerbation of disease or infection. we reviewed the following iads in relation to air pollution. allergic rhinitis (ar), is an ige mediated inflammatory disease generated by a spectrum of outdoor aeroallergens like pollens or indoor aeroallergens such as dust mites, cockroaches, cat allergens, or molds. crs represents multiple overlapping rhinosinusitis phenotypes with different endotypes. asthma is characterized by chronic atopic or non-atopic inflammation of the airway with superimposed episodes of acute exacerbations. the majority of exacerbations are triggered by respiratory viral infections, most commonly human rhinovirus. , other triggers include allergens and atmospheric pollutants. , copd, another chronic inflammatory airway disease, is characterized by airflow limitation and cough. acute exacerbation of copd, like in the upper airway, can be triggered by infection and inhalation of irritants. , chemical pollutants are health-damaging atmospheric aerosol and non-aerosol particles originating from a variety of natural (eg, volcanic eruptions) or anthropogenic sources (eg, biomass burning, fossil fuel combustion, or traffic related particles). primary pollutants such as particulate matter (pm) and volatile organic compounds are aerosol particles directly emitted as solid or liquid droplets in the air. in the atmosphere, natural gasto-particle conversion can culminate in secondary chemical pollutant particles like are ozone and pm. particularte matter (pm) is a mixture of solid and liquid particles suspended in indoor and outdoor air. their source, size, classification, and airway distribution patterns are well described. [ ] [ ] [ ] various human indoor activities cause resuspension and deposition of particles in indoor air, a process governed primarily by the effective size of the particle. this can range from hours for pm to several months for -mm particulate pollutants. pm . broadly represents around % of the total mass of pm and can be inhaled more deeply into the lungs, with a portion depositing in the alveoli and entering the pulmonary and systemic circulation. the submicron pm family, ultrafine particles and nanoparticles, due to their small size, have a relatively large surface area allowing a greater proportion of compounds to be displayed at the surface such as metals and organic compounds. , they cannot be taken by macrophages and can escape phagocytosis. when retained in the lungs, the ensuing inflammation can result in asthma and lung fibrosis; , yet they can allocate to distant organs through systemic circulation resulting in different toxicological phenotypes such as diabetes and heart disease. [ ] [ ] [ ] the adverse health effects of pm are not uniform since pm is not a single entity; rather its constituents and their proportion in ambient air can change from one geographical location to another depending on the type of emissions inherent to each area. volatile organic compounds (vocs) and formaldehyde vocs are primary pollutants located mainly indoors and include benzene, toluene, xylenes, terpenes, and polycyclic aromatic hydrocarbons. they produce a secondary pollutant, formaldehyde, by an indoor chemical reaction between ozone or nitrogen oxide and terpene. formaldehyde appears to be associated with a higher risk of nasopharyngeal carcinoma and leukemia. the primary domestic, - microbial, and socio-cultural sources of vocs , are well elaborated. diesel exhaust represents the most important local contributor to ambient air pollution and has been classified by who as carcinogenic to humans. it is a complex mixture of chemicals and metals stratified into fractions: a solid fraction (made of a soot of carbon core, metals, and their oxides), a gaseous fraction (made of nitrogen, oxygen, and polycyclic aromatic hydrocarbons -pahs), and a liquid fraction where pahs can adsorb into soot or water droplets. , ultrafine particles, nitrogen oxide, and pm (in the range of . mm) can be produced also by internal combustion of diesel engines. metal elements include chromium, magnesium, zinc, and lead and are associated with engine emissions and abrasion of tires and brake pads. vanadium and nickel are tracers of long-range transport from the use of heavy fuel oil. the relatively large surface area of diesel exhaust particles (deps) permits many of these chemicals and metals to attach to its core. thus, most of the deleterious effects of deps are due to chemicals that are adsorbed onto their surface. ozone and nitrogen oxide (nox) to date, ozone is considered the most damaging air pollutant in terms of adverse effects on human health, vegetation, and crops. [ ] [ ] [ ] [ ] [ ] [ ] it produces short-and long-term effects on cardiorespiratory function. recent evidence suggests there is no threshold concentration below which there are no effects on health. ground-level ozone is formed in the atmosphere by a complex reaction of its precursors, nitrogen oxide (nox), carbon monoxide, and volatile organic compounds in the presence of sunlight. background ozone concentrations are strongly correlated with the increased global nox emissions derived from human-generated fossil fuel combustion and biomass burning. tobacco smoke (tbs) tobaco smoke (tbs) emits a wide range of gases, aerosolized liquids, and fine particulate matter including voc and formaldehyde, nitrogen oxide, pm . , and nicotine. , tbs is estimated to cause approximately , excess deaths per year, and it can contribute to % of all cancer deaths. among other actions, tbs can induce dna damage, change in sputum (mucin) quality, and depressed antioxidant and antimicrobial activity in smokers and among copd patients. , household dust household dust represents a convenient means to sample respiratory exposure to pollutants. in one study, the respirable fraction of dust constituted less than % of the total weight of dust surrounding us, and on scan electron microscopy consisted of large flakes (> mm diameter) to which are adherent smaller particles. the median aerodynamic diameter of respirable dust particles allows their deposition both in the nose and lungs. the chemical composition of these flakes suggests household dust might be an important carrier vehicle of organic pollutants into the airways in addition to its intrinsic risk of oxidative stress. allergens can pollute indoor and outdoor air and exacerbate ar and asthma. indoor allergenic pollutants can be derived from skin scales of pets (eg, cats, dogs), urine of rodents (eg, mice), molds, or from fecal material of arthropods such as house dust mites and cockroaches. outdoor allergens are aeroallergens originating from grasses, trees, weeds, or molds. outdoor pollen also modulates indoor aeroallergen concentration. the concentration of aeroallergens in the indoor environment is governed by complex bioaerosol dynamics. for example, airborne cat allergen (fel d ) is mostly associated with large particles (> mm), but around / of fel d are carried on particles less than micra in diameter. thus fel d can be deposited in the alveoli but most importantly suspended for several days in the air favoring distribution of the allergen in the environment. [ ] [ ] [ ] also, the groups of mite allergens listed in the who nomenclature of allergens are composed of particles ranging in diameter from to mm. hence, they can become airborne upon disturbance and can be carried on house dust that becomes a vector for exposure. how dust mite allergen particles can induce and volume , no. , month trigger asthma in lower airways remains to be determined. the diversity and functioning of the normal microbiome are crucial for maintaining the health of the host. while the effects of pm on human health are well established, the impact of infectious particles on bacterial ecosystems has been overlooked. in vitro studies suggest black carbon, a major component of pm, is strongly implicated in predisposition to respiratory infectious diseases, , and induces structural and functional changes in the biofilms of both streptococcus pneumonia and staphylococcus aureus. this is manifested by increase in biofilm thickness and tolerance to degradation by proteolytic enzymes, thereby promoting colonization of the respiratory tract. similarly, evidence suggests indoor and outdoor dust modifies microbial growth, virulence, and biofilm formation of opportunistic pathogens. by exposing opportunistic bacteria (pseudomonas aeruginosa, escherichia coli, and enterococcus faecalis) to progressively increasing concentrations of indoor and outdoor dust, a differential growth pattern of pathogens was noted. this was commensurate with increased biofilm formation and sensitivity to oxidative stress following hydrogen peroxide challenge. consequently, the detrimental impact of particulate pollutants on human health is not only due to direct effects on the host but also may involve the effect on bacterial behavior in the host. oxidative stress is a disproportionate generation of free radicals beyond the body antioxidant capacity. it translates into a non-ige mediated th airway inflammation following exposure to a pollutant. in brief, reactive oxygen species (ros), generated naturally as by-product of cell growth and metabolism, can be produced following pollutant exposure. , ros include oxygen radicals (eg, superoxide, hydroxyl, hydroperoxyl) and certain non-radicals (eg, h o , ozone, singlet oxygen) that are easily converted into radicals. ros have a pivotal role in cell signaling in the oxidation/reduction cascades following exposure and ultimately generation of anti-oxidant mechanisms thru nrf- , activator protein , and nuclear factor-kappa b. [ ] [ ] [ ] [ ] antioxidants are scavengers of ros and can be enzymatic or non-enzymatic systems, constitutive or de novo synthesized by activated gene expression, according to ros load. the inflammatory phase of oxidative stress involves cytokines-and chemokines-mediated activation and recruitment of inflammatory cells secondary to direct effect of pollutants on airway epithelial cells. this can propagate oxidative stress further and augment the inflammatory response and tissue damage. alternatively, ros can contribute directly to cell injury and apoptosis by disrupting cellular and nuclear membranes in the epithelial barrier wall and altering the function of cellular enzymes. , a different mechanism by which environmental pollution can trigger disease in the nose is via a neurogenic mechanism. another component of oxidative pathway is the exposure-driven adjuvant effect on atopy where environmental pollution acts as an exacerbating factor for allergic airway disease by enhancement of allergic airway hypersensitivity in atopic individuals. the evidence emerges from experimental protocols involving inhalation of pollutants and allergen challenge which show pollutants can act synergistically to heighten the allergic response with increased expression of th inflammatory biomarkers. , this is in contrast to healthy individuals which express either th or a mixed th /th profile in controlled exposure studies. epidemiological studies suggest pollution modulates ar, [ ] [ ] [ ] [ ] [ ] [ ] [ ] rhinosinusitis, and asthma. , other studies suggest a positive association between exposure and prevalence of ar and asthma , [ ] [ ] [ ] [ ] in children and adults predominately in reports on short-term exposure and residential proximity studies to sources of traffic pollution. , however, other long-term exposure studies provided evidence to the contrary. [ ] [ ] [ ] this could be due to differences in study design, methods of exposure assessment, and complex nature of studied pollutants. inin-vivovivo studies in both human and animal models suggest pollutant exposure induces inflammatory changes in normal, chronically diseased and allergic nasal and sinonasal tissues ( table ). the cytokine profile of affected tissues suggests activation of the oxidative inflammatory pathways. [ ] [ ] [ ] moreover, there is compelling evidence for involvement of oxidative stress inflammatory pathways following pollutant exposure in the pathogenesis of rhinitis, crs, and asthma irrespective of atopic status. this stems from an abundance of literature on oxidative stress biomarkers studied under natural or experimental allergen exposure both in seasonal and perennial ar described in table . in fact, dust mite or ragweed allergic patients exposed to diesel exhaust particlesdeps in climate chamber expressed higher nasal symptom scores following dust mite or ragweed challenge, respectively, when compared to nonexposed but allergen-challenged patients. , also in the lower airways, short-term natural increase in ambient air ozone was associated with deteriorating lungh function tests in atopic asthmatics despite use of proper asthma controller therapy. similarly, an ozone exposure protocol revealed atopic asthmatics expressed depressed spirometry testing results compared to healthy volunteers. along with this, climate chamber studies revealed (ozone) exposure of healthy or allergic asthmatics induces a neutrophilic or a mixed neutrophilic and eosinophilic inflammatory profile in the lower airways, respectively. furthermore, gene expression profiles of sputum cells recovered from healthy volunteers and allergic asthmatic patients also confirmed significant difference in inflammatory response to ozone exposure. analysis of biomarkers activity greatly improved our understanding of cascade and signal pathways involved in atopic and non-atopic phenotypes of airway disease following exposure. although most oxidative stress biomarkers require tissue specimen collection, some studies suggest an analysis of biomarkers can be determined non-invasively in exhaled breath condensates or blood. [ ] [ ] [ ] [ ] [ ] [ ] natural allergen exposure reverses oxidative and antioxidative status compared to asymptomatic period, with a persistent oxidative state outside pollination season in allergic patients when compared to healthy controls. ar and asthma comorbidity in children does not seem to augment oxidative stress markers compared to ar alone, although adult patients with seasonal ar and asthma manifest an exaggerated stress response during natural allergen exposure compared to ar alone. clinically, oxidative stress correlates with nasal symptom scores in children with perennial ar and can predict ar severity independent of total ige. additionally, ros status does not correlate with atopic skin sensitization in children with perennial ar. furthermore, dust mite challenge in asthmatics or sensitized mice resulted in oxidative damage to nucleic acids as well as lipids and proteins and subsequently triggered dna repair pathways. further blockage of dna repair proteins resulted in increased production of dna double-strand breaks and cell apoptotic enzymes suggesting importance of dna repair in suppressing airway inflammation. endogenous antioxidant response in atopic respiratory diseases is complex and oxidative stress response to anti-inflammatory drugs isare poorly understood. antioxidant enzymes mostly studied in atopic respiratory diseases include heme oxygenase and , , nadph oxidases, catalase, , superoxide dismutase, , , dual oxidases and (in crs patients), paraoxonase, and glutathione peroxidase. , antioxidant activity can also be measured by serum thiol-sh and total antioxidant status ( table ). in this respect, evidence suggests oxidative stress decreases antioxidant enzyme activity or total antioxidant status in atopic children , , or in human in vitro controlled exposure studies, whereas other studies present evidence to the contrary. for example, heme oxygenase antioxidant (iso)enzyme- activity was preferentially increased in a human in vitro model of perennial ar, and upregulated in a human exposure model of copd aggravated by infection; ; also dual oxidase antioxidant (iso) enzymes showed preferential upregulation in different phenotypes and endotypes of crs. , contrary to this, antioxidant enzymes can be downregulated in asthma and rhinitis irrespective of atopic status, and in vitro animal exposure models challenged by an infectious insult. importantly, genetic polymorphism in antioxidant/ detoxifying genes like gstm and gstp can alter oxidative stress response in patients with copd and those with ar following exposure. , exogenous (dietary) antioxidants are scavengers of oxygen free radicals and can act on different levels of defensive antioxidation pathways. , epidemiologic, in vivo , and in vitro studies suggest a beneficial role of exogenous antioxidants in patients with iad or in controlled exposure studies of healthy sinonasal epithelial cells. however, lack of clinical trials data clearly supporting their efficacy, in addition to their potential role in skewing th /th balance towards a th -type immunity as suggested in vitro, renders their indication restricted to special situations such as over exposure to environmental pollutants, among others. nacetylcysteine maintains a potent antioxidant effect in in vitro studies or in ovalbuminsensitized rats by downregulating tumor necrosis factor-alpha in recruited inflammatory cells. along these lines, intranasal steroids can exhibit an exogenous antioxidant regulatory role in seasonal ar by decreasing exhaled breath condensates of leukotriene b and -isoprostane, although no effect was seen on exhaled carbon monoxide and nitrogen oxide. in another study involving children with ar and asthma, no effect of topical nasal steroid therapy was noted on measured lipid peroxidation oxidative stress biomarkers and antioxidant enzymes. data on potential antioxidant effect of inhaled steroids in adult asthmatics is scarce. epidemiological studies suggested prior intake of oral or inhaled steroids in adult asthmatic patients had no effect on asthma control, as measured by clinical symptoms and fev testing, with pm and ozone exposure. other similar studies noted increased consumption of asthma controller therapy (bronchodilators, inhaled corticosteroids, or both) with pm or no exposure in adults. moreover, in children inhaled steroid therapy downregulated induced expression of heme oxygenase- in non-smoking patients with bronchiectasis but had no effect on exhaled carbon monoxide. furthermore, desloratadine can exert an antioxidant effect in children with perennial ar by increasing antioxidant enzyme activities (catalase and superoxide dismutase) and decreasing lipid peroxidation marker (malonaldehyde) although no effect was seen on total antioxidant status. when compared to placebo, fexofenadine improved nasal symptom scores in ragweed ar patients following ragweed challenge and dep controlled exposure. the majority of controlled human exposure studies to ambient pollutants have been conducted in climate chambers on healthy individuals. [ ] [ ] [ ] [ ] for example, relative to clean air, mixtures of vocs increased ratings of nasal irritation, odor intensity and cognitive symptoms (memory loss, dizziness), and a two-fold increase in polymorphonuclear cells in nasal lavage immediately following exposure. similar studies using different pollutants showed no detectable effects on nasal symptom scores or markers of nasal inflammation. , additionally, healthy subjects exposed to room air, nanoparticles, or o /terpene showed no significant changes in inflammatory biomarkers in blood, sputum or nasal secretions and pulmonary function tests. however, only nanoparticles exposure increased significantly high frequency variability in heart rate, thereby indicating a shift in autonomic balance to a more parasympathetic tone. low level ozone exposure in healthy subjects resulted in increased sputum production of airway inflammatory cells such as neutrophils, monocytes, and dendritic cells, and modification of cell surface phenotypes of antigen presenting cells. using a similar protocol the reported decrement in lung spirometry testing (fev ) of healthy subjects was associated with increased neutrophilic airway inflammation following exposure; the latter likely being more pronounced in healthy individuals with gstm null genotype. more importantly, comparing healthy controls to atopic asthmatics, exposure to high levels of ultrafine particles in a climate chamber was associated with a small but significant fall in arterial oxygen saturation, a fall in forced expired volume over s (fev ) the morning after exposure, and a transient slight decrease in low frequency (sympathetic) power during quiet rest. these controversial results can be related partly to the nature and concentration of the investigated pollutant or its experimental duration of exposure keeping in mind brief exposure to a single pollutant in a climate chamber does not reflect chronic exposure to multiple pollutants in real life. controlled exposure studies in atopic patients involving allergen challenge revealed more consistent results. for example, dust mite allergic patients reported worsening nasal symptom scores following intranasal dust mite challenge and dep exposure commensurate with increased histamine levels in nasal washes, all suggestive of induced mast-cell degranulation. similarly, controlled exposure studies in ragweed allergic patients challenged with dep and ragweed outside their pollen season reported higher total nasal symptoms scores or increased levels of specific ige and expression of th inflammatory cytokines, when compared to ragweed challenged alone. taken together, controlled airway exposure studies to ambient pollutants in healthy individuals show small but significant negative health effects whereas exposure studies in allergic patients support the role of pollutants in increasing atopic airway hypersensitivity. large scale translational studies are needed to correlate the bio-cellular toxic effects of pollution with epidemiological studies. signal and cascade pathways triggered across the airway mucosal barrier at first encounter of pollutants are complex (see fig. ). airway mucosal cells can recognize pollutants through an epithelial toll-like receptors (tlr)-mediated mechanism either directly or indirectly by the intermediary of pattern recognition receptors (see below). more precisely, pollutants such as pm, cigarette smoke, and ozone can present themselves directly to subclasses of surface tlrs, namely tlr and tlr , which can serve as ligands for these pollutants. alternatively, pollutants can be bound to pattern recognition receptors, a collective conglomerate of receptors which encompasses tlrs and normally can recognize conserved molecular structures derived from microbial agents or released by damaged nonmicrobial cells. once triggered, pattern recognition receptors and tlrs attract antigen presenting cells and leukocytes to the site of inflammation resulting in priming of the airway to subsequent mucosal infectious insults. afterwards, when eventuated by an infectious challenge, alveolar macrophages mount a heightened inflammatory response aimed at containing and clearing bacteria while producing minimal collateral tissue damage. , the immunological "storm" resulting from co-exposure and infection is studied in different clinical models of respiratory cells and also in patients with iad such as copd ( table ) . another signal pathway is mediated by submucosal innate lymphoid cells (ilcs) which can differentiate into adaptive subsets. ilc s relates to immune reactions in crs without nasal polyps, copd, and some viral and bacterial infections; whereas ilc s becomes important in regulating type immunity and some helminthic and viral infections. , other immunologic and antimicrobial responses to pollutant exposure modulate expression of host defense peptides and antiviral mechanisms, impair mucus production crucial for capturing pollutants or weaken tight junctions essential for the epithelial airway defense barrier. , epidemiological studies suggest indoor and outdoor air pollution increase the risk of respiratory tract infections in both pediatric - and adult populations. , , , for example, morbidity of the recent covid- pandemic disease has been linked partly to air pollution. [ ] [ ] [ ] [ ] also, air pollution can aggravate the severity of asthma caused by respiratory viral infections. moreover, in vitro studies suggest air pollution may suppress innate and adaptive immunity and increases susceptibility to bacterial and viral respiratory infections in both human and animal clinical models, following short-or long-term exposure (see table ). for example, in the upper airways diesel exhaust exposure increased the number of human nasal epithelial cells infected by influenza a virus in vitro the proposed mechanism was enhancement of virus attachment and entry into respiratory cells mediated by radical oxygen species, despite increased antiviral interferon-dependent signaling and interferon-stimulated gene expression by dep exposure. also, in vitro rrhinovirus (rv) infectivity following nitrogen oxide and ozone exposure in human respiratory epithelial cells loss of low-level dep-exposed mdmf along their differentiation into macrophages likely due to dysfunctional (loss of mitochondrial membrane electrical potential and lysosomal function) and phenotypic (tlrmediated reduction in cd and cd surface marker expression) structural changes in mdmf of healthy exposed individuals. this can likely contribute to inflammation in copd by decreased mdmf proinflammatory cytokines (cxcl ) production. resulted in increased icam receptor expression (receptor for rv ) and pro-inflammatory il- cytokine production. in another combined human and animal model, activated nasal airway microbial proteins at the surface mucosal liquid, which include lysozyme, human cathelicidin antimicrobial peptide, and human b defensins, were attenuated following (pm) exposure and staphylococcus aureus infection. the ensuing impaired bacterial killing resulted from adsorption and electrostatic interactions between either pollutant or bacteria with activated microbial proteins leading to the depletion of the latter. the literature on the lower airways exceeds that on the upper airways. in this respect, susceptibility to infections following exposure was examined at several stages of immunological alterations triggered in host cells. starting with the epithelial barrier level, an initial in vivo pm exposure of bronchial epithelial cells in mice followed by experimental infection with pseudomonas aeruginosa resulted in decreased levels of an epithelial ciliary marker (b tubulin) and a non-ciliary epithelial (clara cells) marker, and their gene expression/transcription regulator, all suggesting airway remodeling is a contributing factor to the impaired bacterial clearance. furthermore, an initial infection with pseudomonas aeruginosa induced an epithelial antimicrobial peptide human beta defensin ; but as the model was pre-exposed to pm, induction of human beta defensin was suppressed and a cell senescence biomarker (sa-b-gal) was upregulated in an ros-dependent process. also, in an in vitro human model, a pm-enhanced susceptibility to streptococcus pneumoniae infection was heightened by increased bacterial adhesion and penetration into bronchial epithelial cells. this was mediated by a receptor for platelet-activating factor, a putative receptor for pm-stimulated pneumococcal adhesion to airway cells. on a submucosal level, macrophages and monocytes play a central role in phagocytosis. the study of immunopathological alterations in phagocytosis has shown inconsistent results. for example, in an exposure (pm)-infectious animal model, impaired streptococcus pneumoniae clearance and phagocytosis resulted from decreased macrophages internalization of bacteria, although increased binding of microbe to surface of macrophages was reported. in a similar model increased susceptibility to staphylococcus aureus infection resulted from depressed phagocytosis index and abnormal natural killer cell response. also, in another animal exposure model increased infectivity to listeria monocytogenes resulted from decreased ros-induced nitric oxide production by alveolar macrophages. in contrast, natural (chronic) pm exposure of human bronchoalveolar lavage fluid decreased macrophage cytokine (cxcl ) release and downregulated induced phagosomal oxidative burst. per contra, no impairment in macrophage redox potential, proteolysis or phagocytosis was observed likely due to the experimental chronicity of exposure. additionally, in an analogous model using high levels of the same pollutant (pm), the impaired antimicrobial defense resulted from defective macrophage activation of t cells by class ii þ major histocompatibility complex and subsequent decrease in interferon-g production, but unaltered phagocytic activity. interestingly, no increase of neutrophils and tnf-a levels was observed in bronchoalveolar lavage following exposure and infection suggesting acute exposure to relatively high level of pm does not trigger a classic or sustained inflammatory response. besides suggesting interference with innate immunity, exposure studies suggest further alterations in adaptive immunity as evidenced by immunopathological relationships between antigen presenting cell cytokines, the corresponding sensitized t cells subsets, and recruited neutrophils (see table ). as such, a listeria monocytogenes-mediated suppression of macrophages immune response upon low dose dep exposure manifested as "dysfunctional" production of macrophages-derived cytokines. this was associated with downregulation of innate protective cytokines (e.g. il- b, tumor necrosis factor-a, il- , il- and interferon-g), suppression of adaptive cd and cd t cell immune response, and upregulation of macrophage bactericidal anti-inflammatory cytokines (il- and il- ). other examples of altered cytokine release include the pro-inflammatory besides the role of cytokines in fine tuning extent of inflammation in these models, t cell subsets like t cytotoxic (cd þ ) and regulatory t cells (treg) in addition to neutrophils have been studied. dep exposure in rats increased susceptibility to listeria monocytogenes infection by attenuating t cell mediated immunity, namely cd þ t helper lymphocytes and cd þ t cytotoxic cells; pm exposure in neonatal mice resulted in depression of adaptive response to influenza virus a infection and by an increased expression in treg cells and il- in lung tissues. interestingly, the induced immunosuppressive effect was reversed by treg depletion and restored by either treg transfer or recombinant il- treatment. furthermore, airway neutrophilia, which is instrumental in bacterial clearance, has been studied in inin-vitrovitro infectious exposure model in relationship to th and th proinflammatory cytokine release. the concomitant increase in bronchoalveolar lavage fluid il- with airway neutrophilia, and their attenuation in il- "knock out" mice following exposure and infection suggested the importance of il- in inducing neutrophil-mediated airway inflammation. also, decreased induction of il- a-mediated airway neutrophilia following exposure and infection in il- r mice compared with wild-type controls also suggests il- signaling is required in il- aexacerbated neutrophilia. moreover, in an in vivo exposure-infectious animal model modulated by interferon-g priming to mimic viral infection, an impaired pm-mediated bacterial phagocytosis correlated with activation of genes encoding neutrophil-recruiting chemokines and increased histopathology suggestive of severe pneumonia. still, in an animal in vivo model, exposure followed by lps infection induced cytokine changes in the lung suggestive of a th /th imbalance and manifested by increased expression of il- among others, and a concordant decrease in ifn-g expression. the infectious-exposure model is an attractive tool to explore immunopathological alterations in copd patients or in laboratory cells exposed to secondhand smoking. in a mice model, weeks secondhand smoking pre-exposure was followed by infection with non-typeable haemophilus influenza which is a pathogen commonly implicated in acute exacerbation of copd. the model revealed increased number of immune cell infiltrates except for macrophages, and a suppressed induction of a robust adaptive immune response manifested as decreased ifn-g. also, a downregulated t cell adaptive response manifested by decreased bacterial clearance and diminished efficiency of specific antibody subclass switching, both mitigated by anti-viral vaccination. in a similar animal model examining the immunological effect of antibiotic therapy, cigarette smoke exposure followed by streptococcus pneumoniae infection resulted in recruitment of macrophages and monocytes in lung tissue and alveolar fluid reportedly to confine infection to the lung; also a decreased number of neutrophils but a differential increase in neutrophil-mediated antimicrobial peptide, myeloperoxidase. antibiotic therapy had no effect on mice survival rate but reduced lung injury and induced a differential change of cytokine levels in bronchoalveolar lavage fluid most importantly downregulation of th and th inflammatory cytokines. human in-vitro pre-exposure and infectious models are designed to mimic acute exacerbations in stable but exposed copd patients. dep exposure followed by non-typeable haemophilus influenza infection did not compromise mucosal barrier function in copd or healthy patients. however, epithelial endoplasmic reticulum activity was markedly disrupted in copd patients, manifested by depressed gene expression of the integrated stress response markers in an ros-mediated process. in another model, macrophages differentiating from locally recruited monocytes in lungs of copd patients were pre-exposed to low level dep and subsequently challenged with tlr agonists or heat killed e.coli. this resulted in structural and functional changes in innate and adaptive immune system consisting of mitochondrial and lysosomal dysfunction in macrophages, decreased expression of their surface recognition markers, loss of macrophage differentiation, and reduction in proinflammatory cytokine production (e.g.il- ). the majority of exposure-infection human and animal models have examined immunological alterations following long-term (weeks) and lowdose pre-exposure periods which best mimics real-life outdoor pollutant exposure or indoor secondhand smoking relevant to copd. nevertheless, other models which studied brief and short-term (hours to days) exposure periods have yielded mixed results. for example, one-week diesel exhaust pre-exposure of mice in vivo decreased pseudomonas aeruginosa clearance from bronchial epithelial cells, whereas in the same model a six-months pre-exposure did not. also, in an in vivo model, mice were pre-exposed to pm for day (short term) or weeks (long term), later infection with influenza virus a and survival rate was assessed over the ensuing days following contamination. short-term exposure improved mice survival rate and triggered a robust immune response whereas long-term exposure did not, reportedly mediated by macrophage cytokine gene expression regulator kdm a. to model secondhand smoking exposure or for recent initiation of active smoking, mice were exposed to brief ( h per day for days) low dose of side stream cigarette smoke or to prolonged ( . weeks) high dose cigarette smoke, respectively, and later inoculated with streptococcus pneumonia. surprisingly, brief exposure did not show significant survival benefit whereas prolonged exposure in mice did, reportedly due to diminished propagation of bacteria into the systemic circulation during chronic exposure. finally, in a mice model examining only chronic secondhand smoking exposure and its impact on non-typeable haemophilus influenza antimicrobial response, weeks secondhand smoking preexposure, theoretically mimicking mainstream smoking, compromised the ability of host t cellmediated adaptive immune system to mount an effective response against non-typeable haemophilus influenza infection. taken together, these models suggest exposure impairs innate and adaptive immunity against airway microbial infections. limitations inherent to the design of these models compel a careful interpretation of results taking into consideration the response to infectivity of animal host cells, the duration and intensity , , , of pollutant pre-exposure, and the nature of microbial agents used for contamination. we reviewed evidence for the involvement of oxidative stress pathways and their nature in healthy individuals and patients with inflammatory airway diseases following exposure to a spectrum of important chemical, allergic and infectious air contaminants. when comparing exposure clinical models in patients with ar, crs, and allergic asthma, the signal and cascade pathways can generate important oxidative and anti-oxidative markers and induce specific changes in adaptive and innate immune system. thus, exposure can amplify the inflammatory process in patients with ar, crs, and allergic asthma supporting evidence that, at least in atopic individuals, exposure can increase airway hypersensitivity. when accentuated by an infectious insult, pre-exposure clinical models in patients with inflammatory airway diseases show specific immunopathological alterations at mucosal and submucosal levels of the airway epithelial barrier and ultimately in the adaptive immune system. the resultant increased susceptibility to infection can be due to either increased infectivity of microbial agents or to a ros-mediated direct effect of pollutant on host immune defense cells. the complex nature and composition of chemical air pollutants and their aerodynamic properties is reflected in conflicting epidemiological and experimental results on exposure and its impact on health. also, the oxidative stress-mediated immunopathological changes have highlighted important antioxidant markers, which can be therapeutically bio-engineered. since there is no clear consensus on efficacy of natural or synthetic antioxidants, , , current research should search for new therapeutic modalities and define the role of currently available ones such as antihistamines, intranasal or inhaled steroids, antibiotics and anti-viral vaccination in patients with inflammatory airway diseases challenged by exposure and at times by an infectious process. volume , no. , month atmospheric chemistry and physics: from air pollution to climate change who guidelines for indoor air quality: household fuel combustion. world health organization health effects institute. state of global air million premature deaths annually linked to air pollution. world health organization short-term exposure to ambient air pollution and asthma mortality short-term effect of ambient air pollution on copd mortality in four chinese cities chronic rhinosinusitis: epidemiology and medical management allergens, viruses, and asthma exacerbations asthma exacerbations: origin, effect, and prevention particulate air pollution and hospital emergency room visits for asthma in seattle personal exposures to traffic-related air pollution and acute respiratory health among bronx school children with asthma air pollution and chronic airway diseases: what should people know and do? impact of cigarette smoke exposure on host-bacterial pathogen interactions atmospheric aerosols: composition, transformation, climate and health effects clearing the air: a review of the effects of particulate matter air pollution on human health outdoor air pollution and asthma effects on health of air pollution: a narrative review estimating the resuspension rate and residence time of indoor particles toxic potential of materials at the nanolevel a work group report on ultrafine particles (aaaai) why ambient ultrafine and engineered nanoparticles should receive special attention for possible adverse health outcomes in humans aeroparticles, composition, and lung diseases short-term exposure to air pollution and cardiac arrhythmia: a metaanalysis and systematic review air particulate matter and cardiovascular disease: the epidemiological , biomedical and clinical evidence particulate matter air pollution and cardiovascular disease an update to the scientific statement from the american heart association urban air pollution and respiratory infections microbial volatile organic compounds occupational exposure to formaldehyde and wood dust and nasopharyngeal carcinoma formaldehyde and leukemia: epidemiology, potential mechanisms and implications for risk assessment indoor pollutants emitted by office equipment: a review of reported data and information needs the carcinogenicity of outdoor air pollution characterization of particulate matter deposited in diesel particulate filters: visual and analytical approach in macro-, micro-and nano-scales polycyclic aromatic hydrocarbons in diesel emission , diesel fuel and lubricant oil physical characterization of particulate emissions from diesel engines: a review phase and size distribution of polycyclic aromatic hydrocarbons in diesel and gasoline vehicle emissions pm speciation and sources in mexico during the milagro- campaign diesel exhaust: current knowledge of adverse effects and underlying cellular mechanisms impact of ozone on mediterranean forests: a review corrosion on cultural heritage buildings in italy: a role for ozone? evidence of widespread effects of ozone on crops and (semi-) natural vegetation in europe ( - ) in relation to aot -and flux-based risk maps review of evidence on health aspects of air pollution -revihaap project air quality in europe - report global topics and novel approaches in the study of air pollution , climate change and forest ecosystems the relation between ozone , no and hydrocarbons in urban and polluted rural environments european abatement of surface ozone in a global perspective oxidative stress and obesity, diabetes, smoking, and pollution: part of a -part series hhs public access mitochondrial toxicity of tobacco smoke and air pollution mortality attributable to cigarette smoking in the united states associations between the smoking-relatedness of a cancer type, cessation attitudes and beliefs, and future abstinence among recent quitters tobacco exposure inhibits splunc -dependent antimicrobial activity human neutrophil elastase degrades splunc and impairs airway epithelial defense against bacteria isolation and characterization of a respirable particle fraction from residential house-dust indoor particle dynamics animal allergens and their presence in the environment distribution , aerodynamic characteristics , and removal of the major cat allergen fel d in british homes dog and cat allergies: current state of diagnostic approaches and challenges indoor allergens and allergic respiratory disease size distribution of particulate polycyclic aromatic hydrocarbons in fresh combustion smoke and ambient air: a review air conditioning and source-specific particles as modifiers of the effect of pm on hospital admissions for heart and lung disease air pollution alters staphylococcus aureus and streptococcus pneumoniae biofilms , antibiotic tolerance and colonisation science of the total environment the effects of indoor and outdoor dust exposure on the growth , sensitivity to oxidative-stress , and bio fi lm production of three opportunistic bacterial pathogens reactive oxygen species in metabolic and inflammatory signaling oxidative stress and air pollution exposure the role of reactive oxygen species (ros) in the biological activities of metallic nanoparticles nrf protects against airway disorders oxidative stress in airway diseases synergism between rhinovirus infection and oxidant pollutant exposure enhances airway epithelial cell cytokine production h s induces th /th imbalance with triggered nf-k b pathway to exacerbate lpsinduce chicken pneumonia response what is oxidative stress? the role of the reactive oxygen species and oxidative stress in the pathomechanism of the agerelated ocular diseases and other pathologies of the anterior and posterior eye segments in adults house dust mite -induced asthma causes oxidative damage and dna double-strand breaks in the lungs international expert consensus on the management of allergic rhinitis (ar) aggravated by air pollutants impact of air pollution on patients with ar: current knowledge and future strategies combined diesel exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-specific ige and skews cytokine production to a t helper cell -type pattern immunomodulation and t helper th /th response polarization by ceo and tio nanoparticles association of indoor dampness and molds with rhinitis risk: a systematic review and meta-analysis rhinitis symptoms and asthma among parents of preschool children in relation to the home environment in chongqing , china long-term exposure to close-proximity air pollution and asthma and allergies in urban children pm , and children's respiratory symptoms and lung function in the paty study association between environmental factors and hospital visits among allergic patients: a retrospective study science of the total environment time-series studies on air pollution and daily outpatient visits for allergic rhinitis in traffic-related air pollution and the development of asthma and allergies during the first years of life air quality influences the prevalence of hay fever and sinusitis traffic-related air pollution in relation to respiratory symptoms , allergic sensitisation and lung function in schoolchildren upper airway inflammation and respiratory symptoms in domestic waste collectors an association between fine particles and asthma emergency department visits for children in seattle asthma and air pollutants: a time series study environmental and occupational allergies residential proximity to major roadways is associated with increased prevalence of allergic respiratory symptoms in children meta-analysis of air pollution exposure association with allergic sensitization in european birth cohorts the effects of air pollution on the development of atopic disease ambient particulate pollution and the world-wide prevalence of asthma, rhinoconjunctivitis and eczema in children: phase one of the international study of asthma and allergies in childhood (isaac) expression of dual oxidases and secreted cytokines in chronic rhinosinusitis expression of heme oxygenase- in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps: modulation by cytokines airborne fine particulate matter induces oxidative stress and inflammation in human nasal epithelial cells diesel exhaust as a model xenobiotic in allergic inflammation phase , single-center, sequential and parallel group, double-blind, randomized study evaluating the efficacy and safety of fexofenadine hydrochloride mg (allegra/telfast) versus placebo in subjects suffering from allergic rhinitis with symptoms aggravated in presence of pollutants: analysis of individual symptom scores low-level ozone has both respiratory & systemic effects in african-american adolescents with asthma despite asthma controller therapy atopic asthmatic subjects but not atopic subjects without asthma have enhanced inflammatory response to ozone ozone-induced inflammation is attenuated with multiday exposure prolonged acute exposure to . ppm ozone induces eosinophilic airway inflammation in asthmatic subjects with allergies airway cells from atopic asthmatics exposed to ozone display an enhanced innate immune gene profile breath markers of oxidative stress and airway inflammation in seasonal allergic rhinitis oxidative stress status and plasma trace elements in patients with asthma or allergic rhinitis total antioxidant status and oxidative stress and their relationship to total ige levels and eosinophil counts in children with allergic rhinitis plasma paraoxonase activity and oxidative stress and their relationship to disease severity in children with allergic rhinitis a new oxidative stress marker for thiol-disulphide homeostasis in seasonal allergic rhinitis oxidative stress in the airways of children with asthma and allergic rhinitis heme oxygenase (ho)- is upregulated in the nasal mucosa with allergic rhinitis overexpression of the superoxide anion and nadph oxidase isoforms and (nox and nox ) in allergic nasal mucosa influence of desloratadine on selected oxidative stress markers in patients between and years of age with allergic perennial rhinitis diesel exhaust alters the response of cultured primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (copd) to non-typeable haemophilus influenzae genetic susceptibility to the respiratory effects of air pollution effects of glutathione stransferase gene polymorphisms and antioxidant capacity per unit albumin on the pathogenesis of chronic obstructive pulmonary disease novel antioxidant approaches to the treatment of upper airway inflammation effects of nacetylcysteine on outcomes in chronic obstructive pulmonary disease (bronchitis randomized on nac cost-utility study, broncus): a randomised placebo-controlled trial antioxidant intake and allergic disease in children effect of silymarin in the treatment of allergic rhinitis n-acetylcysteine combined with home based physical activity: effect on health related quality of life in stable copd patients-a randomised controlled trial air pollutant-mediated disruption of sinonasal epithelial cell barrier function is reversed by activation of the nrf pathway potential role of antioxidant food supplements, preservatives and colorants in the pathogenesis of allergy and asthma effects of antioxidant supplements and nutrients on patients with asthma and allergies n-acetylcysteine exerts therapeutic action in a rat model of allergic rhinitis short-term effects of low-level air pollution on respiratory health of adults suffering from moderate to severe asthma air pollution and asthma control in the epidemiological study on the genetics and environment of asthma associations of pm and airborne iron with respiratory health of adults living near a steel factory increased asthma medication use in association with ambient fine and ultrafine particles increased levels of exhaled carbon monoxide in bronchiectasis: a new marker of oxidative stress realistic indoor nanoaerosols for a human exposure facility effects on heart rate variability by artificially generated indoor nano-sized particles in a chamber study health effects of a mixture of indoor air volatile organics , their ozone oxidation products , and stress sensory and cognitive effects of acute exposure to hydrogen sulfide equivalence of sensory responses to single and mixed volatile organic compounds at equimolar concentrations exposure of humans to a volatile organic mixture . iii . inflammatory response nasal effects of a mixture of volatile organic compounds and their ozone oxidation products low-level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans lung function and inflammatory responses in healthy young adults exposed to . ppm ozone for . hours the glutathione-s-transferase null genotype and increased neutrophil response to low level ozone ( . ppm) exposures of healthy and asthmatic volunteers to concentrated ambient ultrafine particles in los angeles exposures of healthy and asthmatic volunteers to concentrated ambient ultrafine particles in los angeles effects of air pollutants on innate immunity: the role of toll-like receptors and nucleotide-binding oligomerization domain -like receptors alveolar macrophages in pulmonary host defence-the unrecognized role of apoptosis as a mechanism of intracellular bacterial killing innate immune cell suppression and the link with secondary lung bacterial pneumonia human innate lymphoid cells the role of innate lymphoid cells in airway inflammation: evolving paradigms barrier function of airway tract epithelium. tissue barriers the interplay between host immunity and respiratory viral infection in asthma exacerbation respiratory disease associated with solid biomass fuel exposure in rural women and children: systematic review and meta-analysis indoor air pollution from unprocessed solid fuel use and pneumonia risk in children aged under five years : a systematic review and meta-analysis correlation between respiratory alterations and respiratory diseases due to urban pollution air pollution and respiratory infections during early childhood: an analysis of european birth cohorts within the escape project long-term exposure to ambient air pollution and risk of hospitalization with community-acquired pneumonia in older adults links between air pollution and covid- in england the potential role of particulate matter in the spreading of covid- in northern italy: first evidence-based research hypotheses. medrxiv assessing nitrogen dioxide (no ) levels as a contributing factor to the coronavirus (covid- ) fatality rate sars-cov- rna found on particulate matter of bergamo in northern italy: first preliminary evidence. medrxiv personal exposure to nitrogen dioxide (no ) and the severity of virus-induced asthma in children diesel exhaust enhances influenza virus infections in respiratory epithelial cells effects of coal fly ash particulate matter on the antimicrobial activity of airway surface liquid inhaled diesel engine emissions reduce bacterial clearance and exacerbate lung disease to pseudomonas aeruginosa infection invivo urban particulate matter (pm) suppresses airway antibacterial defence adhesion of streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter effect of concentrated ambient particles on macrophage phagocytosis and killing of streptococcus pneumoniae exposure to particular matter increases susceptibility to respiratory staphylococcus aureus infection in rats via reducing pulmonary natural killer cells diesel exhaust particles suppress macrophage function and slow the pulmonary clearance of listeria monocytogenes in rats chronic household air pollution is associated with impaired alveolar macrophage function in malawian non-smokers adverse effects of wood smoke pm . exposure on macrophage functions suppression of cell-mediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown norway rats regulatory t cells and il suppress pulmonary host defense during early-life exposure to radical containing combustion derived ultrafine particulate matter il- a and the promotion of neutrophilia in acute exacerbation of chronic obstructive pulmonary disease air pollution particles diminish bacterial clearance in the primed lungs of mice secondhand smoke induces inflammation and impairs immunity to respiratory infections cigarette smoke exposure worsens acute lung injury in antibiotic-treated bacterial pneumonia in mice diesel exhaust particle exposure in vitro alters monocyte differentiation and function biochemical and biophysical research communications down-regulating pulmonary macrophage kdm a and mediates histones modi fi cation in il- and ifn-b promoter regions cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice the importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state a review of the epidemiological evidence for the ' antioxidant hypothesis human and animal research not applicable. not applicable.availability of data and materials not applicable. we attest that all authors contributed significantly to the creation of this manuscript.-philip rouadi and samar idriss initiated the work, contributed substantially to the conception and design of the study, the acquisition, analysis, and interpretation of data. -philip rouadi supervised the manuscript.ethics committee approval not applicable. all authors agreed to the publication of this work. the authors have nothing to declare relative to this paper. key: cord- -e cd o q authors: shah, bakht ramin; li, bin; al sabbah, haleama; xu, wei; mraz, jan title: effects of prebiotic dietary fibers and probiotics on human health: with special focus on recent advancement in their encapsulated formulations date: - - journal: trends food sci technol doi: . /j.tifs. . . sha: doc_id: cord_uid: e cd o q background: dietary fibers (dfs) are known as potential formulations in human health due to their beneficial effects in control of life-threatening chronic diseases including cardiovascular disease (cvd), diabetes mellitus, obesity and cancer. in recent decades scientists around the globe have shown tremendous interest to evaluate the interplay between dfs and gastrointestinal (git) microbiota. evidences from various epidemiological and clinical trials have revealed that dfs modulate formation and metabolic activities of the microbial communities residing in the human git which in turn play significant roles in maintaining health and well-being. furthermore, interestingly, a rapidly growing literature indicates success of dfs being prebiotics in immunomodulation, namely the stimulation of innate, cellular and humoral immune response, which could also be linked with their significant roles in modulation of the probiotics (live beneficial microorganisms). scope and approach: the main focus of the current review is to expressively highlight the importance of dfs being prebiotics in human health in association with their influence on gut microbiota. now in order to significantly achieve the promising health benefits from these prebiotics, it is aimed to develop novel formulations to enhance and scale up their efficacy. therefore, finally, herein unlike previously published articles, we highlighted different kinds of prebiotic and probiotic formulations which are being regarded as hot research topics among the scientific community now a days. conclusion: the information in this article will specifically provide a platform for the development of novel functional foods the demands for which has risen drastically in recent years. the word dietary fiber (df), denoting undigestible extremely intricate carbohydrates and lignins, was first described by eben hipsley in his article after seeing people consuming diets high in fiber-rich foods (hipsley, ) . in a more elaborated way, dfs comprises a large group of is generally believed that the bacterial flora residing in the git vary from to person to person, however, mainly it remains stable over time periods (rodríguez et al., ) . in this perspective, shorter duration of rv acute watery diarrhea (islek et al., ) . also, milk fermented with b. in light of the potential health benefits of prebiotics & probiotic given in fig. nowadays a strong and efficient inner defense mechanism (immunity) is more and more in recent years a greater attention has been attracted by different kinds of prebiotic formulations. thereby influencing the production of scfas, the balance of which is vital to maintain gut health. among all the produced scfas, butyrate may be regarded as the most beneficial one for colonic health, which could be due to is principal energy source for the colonycytes as well as its role in modulating cell turnover in the colon. this implies that ensuring an adequate supply of dfs, such that to maintain raised butyrate levels is throughout the entire length of the large intestine, key factors influencing the composition as well as metabolic activities of gut microbiota roles and potential health benefits of prebiotics & probiotics interplay between dietary fiber and colonic microbiome ) the effects of dfs in control of chronic diseases and viral infections was described ) the importance of dietary fibers being prebiotics was highlighted in detail ) encapsulation of pre and prebiotics for their protection in gut environment is crucial ) advancements in new pre & probiotics-based formulations have been focused gastrointestinal effects of low-digestible carbohydrates metagenomic systems biology of the human gut microbiome reveals topological shifts associated with obesity and inflammatory bowel disease inflammatory mechanisms in obesity. annual review of immunology definition of metabolic syndrome lipolysis and thermogenesis in adipose tissues as new potential mechanisms for metabolic benefits of dietary fiber expert consensus document: the international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic dietary "fibre" and pregnancy toxaemia agave inulin supplementation affects the fecal microbiota of healthy adults participating in a randomized, double-blind, placebo-controlled, crossover trial pyrosequencing reveals a shift in fecal microbiota of healthy adult men consuming polydextrose or soluble corn fiber comparative effects of different whole grains and brans on blood lipid: a network meta-analysis the role of bifidobacterium lactis b plus inulin in the treatment of acute infectious diarrhea in children development of lamivudine containing multiple emulsions stabilized by gum odina whole grains, bran, and germ in relation to homocysteine and markers of glycemic control, lipids, and inflammation food ingredients that inhibit cholesterol absorption. preventive nutrition and food science application of inulin in cheese as prebiotic, fat replacer and texturizer: a review predominant role of host genetics in controlling the composition of gut microbiota in vitro study of the effect of a probiotic bacterium lactobacillus rhamnosus against herpes simplex virus type effects of whole grain wheat bread on visceral fat obesity in japanese subjects: a randomized double- blind study effect of a high-fiber diet vs a fiber-supplemented diet on c-reactive protein level effect of whole-grain consumption on changes in fecal microbiota: a review of human intervention trials from dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites role of whole grains versus fruits and vegetables in reducing subclinical inflammation and promoting gastrointestinal health in individuals affected by overweight and obesity: a randomized controlled trial dietary fibers, prebiotics, and exopolysaccharides produced by lactic acid bacteria: potential health benefits with special regard to cholesterol-lowering effects formulation of protein based inulin incorporated synbiotic nanoemulsion for enhanced stability of probiotic dietary fiber intake and risk of colorectal cancer and incident and recurrent adenoma in the cancer screening trial. the american journal of clinical nutrition gut microbiota in human adults with type diabetes differs from non- diabetic adults effects of millet whole grain supplementation on the lipid profile and gut bacteria in rats fed with high-fat diet how satiating are the 'satiety'peptides: a problem of pharmacology versus physiology in the development of novel foods for regulation of food intake prebiotic and probiotic treatment of nonalcoholic fatty liver disease: a systematic review and meta-analysis prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebo-controlled trial roles for dietary fibre in the upper gi tract: the importance of viscosity the impact of dietary fiber on gut microbiota in host health and disease firmicutes/bacteroidetes ratio of the human microbiota changes with age gut microbiome composition is linked to whole grain-induced immunological improvements ferulic acid may target myd -mediated pro-inflammatory signaling-implications for the health protection afforded by whole grains, anthocyanins, and coffee the first microbial colonizers of the human gut: composition, activities, and health implications of the infant gut microbiota prebiotic potential of gum odina and its impact on gut ecology: in vitro and in vivo assessments effect of dietary fibers on cecal microbiota and gastrointestinal transit time, glucose homeostasis and metabolic health: modulation by dietary fibers glucagon-like peptide (glp- ). molecular metabolism gut microbiome production of short-chain fatty acids and obesity in children investigation of stability, consistency, and oil oxidation of emulsion filled gel prepared by inulin and rice bran oil using ultrasonic radiation fat, fibre and cancer risk in african americans and rural africans the need to reassess dietary fiber requirements in healthy and critically ill patients in vitro antiviral activity of lactobacillus casei and bifidobacterium adolescentis against rotavirus infection monitored by nsp protein production production and characterization of emulsion filled gels based on inulin and extra virgin olive oil the effect of prebiotics on the viability of encapsulated probiotic bacteria dietary fibers and their fermented short-chain fatty acids in prevention of human diseases. bioactive carbohydrates and dietary fibre use of prebiotic sources to increase probiotic viability in pectin microparticles obtained by emulsification/internal gelation followed by freeze-drying dietary fibre and whole grains in diabetes management: systematic review and meta-analyses prevention of rotavirus diarrhea in suckling rats by a specific fermented milk concentrate with prebiotic mixture whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial the composition of the gut microbiota throughout life, with an emphasis on early life. microbial ecology in health and disease high- protein, reduced-carbohydrate weight-loss diets promote metabolite profiles likely to be detrimental to colonic health health benefits of konjac glucomannan with special focus on diabetes non-thermal processing of inulin-enriched soursop whey beverage using supercritical carbon dioxide technology an essential role of ffar (gpr ) in dietary fibre-mediated promotion of healthy composition of gut microbiota and suppression of intestinal carcinogenesis fiber and prebiotics: mechanisms and health benefits prebiotic supplementation over a cold season and during antibiotic treatment specifically modulates the gut microbiota composition of - year-old children dietary fiber, atherosclerosis, and cardiovascular disease in vitro assessment of immunomodulatory and anti-campylobacter activities of probiotic lactobacilli dietary fibre intake and risk of cardiovascular disease: systematic review and meta- analysis intestinal microbiota in health and disease: role of bifidobacteria in gut homeostasis water-soluble dietary fibers enhance bioavailability of quercetin and a fiber derived from soybean is most effective after long-term feeding in rats association between grains, gluten and the risk of colorectal cancer in the cancer prevention study-ii nutrition cohort substituting whole grains for refined grains in a -wk randomized trial has a modest effect on gut microbiota and immune and inflammatory markers of healthy adults. the american journal of clinical nutrition short chain fatty acids (scfas)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases whole grain food diet slightly reduces cardiovascular risks in obese/overweight adults: a systematic review and meta-analysis. bmc cardiovascular disorders immunomodulatory mechanisms of lactobacilli weight loss induced by whole grain-rich diet is through a gut microbiota-independent mechanism structural characterization and anti- tumor effects of an inulin-type fructan from atractylodes chinensis catalytic and anti-bacterial properties of biosynthesized silver nanoparticles using native inulin whole grain diet reduces systemic inflammation: a meta-analysis of randomized trials association of whole grain, refined grain, and cereal consumption with gastric cancer risk: a meta-analysis of observational studies. food science & nutrition antiviral effects of a probiotic metabolic products against transmissible gastroenteritis coronavirus rotavirus vaccines: current status and future considerations. human vaccines & immunotherapeutics probiotics and prebiotics: present status and future perspectives on metabolic disorders akkermansia muciniphila is a promising probiotic effect of probiotic supplementation on cognitive function and metabolic status in alzheimer's disease: a randomized, double-blind and controlled trial glucose-and lipid-related biomarkers are affected in healthy obese or hyperglycemic adults consuming a whole-grain pasta enriched in prebiotics and probiotics: a -week randomized controlled trial probiotics and prebiotic fiber for constipation associated with parkinson disease: an rct probiotication of cooked sausages employing agroindustrial coproducts as prebiotic co-encapsulant in ionotropic alginate-pectin gels potential application of monoglyceride structured emulsions as delivery systems of probiotic bacteria in reduced saturated fat ice cream prebiotic carbohydrates: effect on reconstitution, storage, release, and antioxidant properties of lime essential oil microparticles improving quality parameters of functional strawberry juices: optimization of prebiotic fiber enrichment and geraniol treatment probiotics for infantile colic: a randomized, double-blind, placebo-controlled trial investigating lactobacillus reuteri dsm microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions the propre-save study: effects of probiotics and prebiotics alone or combined on necrotizing enterocolitis in very low birth weight infants effects of probiotics on nonalcoholic fatty liver disease in obese children and adolescents effect of prebiotic ingredients on the rheological properties and microstructure of reduced-sodium and low-fat meat emulsions inulin-based emulsion-filled gel as a fat replacer in prebiotic-and pufa-enriched dry fermented sausages the additional effects of a probiotic mix on abdominal adiposity and antioxidant status: a double-blind, randomized trial physicochemical changes and microbial inactivation after high-intensity ultrasound processing of prebiotic whey beverage applying different ultrasonic power levels physicochemical characterization and potential prebiotic effect of whey protein isolate/inulin nano complex a double-blind, placebo-controlled study to assess the effect of a probiotic mixture on symptoms and inflammatory markers in women with diarrhea-predominant ibs the effect of probiotic and/or prebiotic on liver function tests in patients with nonalcoholic fatty liver disease: a double blind randomized clinical trial correction: the potential role of probiotics or/and prebiotic on serum lipid profile and insulin resistance in non-alcoholic fatty liver disease: a double blind randomized clinical trial effect of probiotic and prebiotic vs placebo on psychological outcomes in patients with major depressive disorder: a randomized clinical trial starch-and carboxymethylcellulose-coated bacterial nanocellulose-pectin bionanocomposite as novel protective prebiotic matrices formulation of protein based inulin incorporated synbiotic nanoemulsion for enhanced stability of probiotic developing emulsion gels by incorporating jerusalem artichoke inulin and investigating their lipid oxidative stability characterization of prebiotic emulsions stabilized by inulin and β-lactoglobulin viability of probiotic lactobacillus rhamnosus in structured emulsions containing saturated monoglycerides the impact of probiotic soy milk consumption on oxidative stress among type diabetic kidney disease patients: a randomized controlled clinical trial prebiotics reduce body fat and alter intestinal microbiota in children who are overweight or with obesity investigation of stability, consistency, and oil oxidation of emulsion filled gel prepared by inulin and rice bran oil using ultrasonic radiation production and characterization of emulsion filled gels based on inulin and extra virgin olive oil the effect of prebiotics on the viability of encapsulated probiotic bacteria probiotic bifidobacterium longum ncc reduces depression scores and alters brain activity: a pilot study in patients with irritable bowel syndrome potential use of whey concentrate and prebiotics as carrier agents to protect bifidobacterium-bb- microencapsulated by spray drying encapsulation of lactobacillus acidophilus and different prebiotic agents by external ionic gelation followed by freeze-drying growth and survival of microencapsulated probiotics prepared by emulsion and internal gelation use of prebiotic sources to increase probiotic viability in pectin microparticles obtained by emulsification/internal gelation followed by freeze-drying the effects of sodium butyrate and inulin supplementation on angiotensin signaling pathway via promotion of akkermansia muciniphila abundance in type diabetes; a randomized, double-blind, placebo-controlled trial effects of multistrain probiotic supplementation on glycemic and inflammatory indices in patients with nonalcoholic fatty liver disease: a double-blind randomized clinical trial improvement of lactic acid bacteria viability in acid conditions employing agroindustrial co-products as prebiotic on alginate ionotropic gel matrix co-encapsulation effects of probiotics on gut microbiota in patients with inflammatory bowel disease: a double-blind, placebo-controlled clinical trial biopolymer-prebiotic carbohydrate blends and their effects on the retention of bioactive compounds and maintenance of antioxidant activity probiotic supplementation in diabetic hemodialysis patients has beneficial metabolic effects effect of probiotics on obesity-related markers per enterotype: a double-blind, placebo-controlled, randomized clinical trial effect of inulin, medium-chain triglycerides and whey protein isolate on stability and in vitro digestibility of enteral nutrition formulas a randomized controlled clinical trial investigating the effect of synbiotic administration on markers of insulin metabolism and lipid profiles in overweight type diabetic patients with coronary heart disease effects of probiotic supplementation on oxidative stress indices in women with rheumatoid arthritis: a randomized double-blind clinical trial prebiotic carbohydrates: effect on physicochemical stability and solubility of algal oil nanoparticles stability, microstructural and rheological properties of complex prebiotic emulsion stabilized by sodium caseinate with inulin and konjac glucomannan effect of administering a multi-species probiotic mixture on the changes in fecal microbiota and symptoms of irritable bowel syndrome: a randomized, double-blind, placebo-controlled trial replacing modified starch by inulin as prebiotic encapsulant matrix of lipophilic bioactive compounds clinical and metabolic response to probiotic supplementation in patients with rheumatoid arthritis: a randomized, double-blind, placebo-controlled trial the increased viability of probiotic lactobacillus salivarius nrrl b- encapsulated in emulsions with multiple lipid-protein-pectin layers s/o/w emulsions prepared with sugar beet pectin to enhance the viability of probiotic lactobacillus salivarius nrrl b- probiotic encapsulation in water-in-water emulsion via heteroprotein complex coacervation of type-a gelatin/sodium caseinate zhao, l., zhang, f., ding, x., wu, g., lam, y. y., wang key: cord- - ugc odq authors: salazar-gómez, anuar; ontiveros-rodríguez, julio c.; pablo-pérez, saudy s.; vargas-díaz, m. elena; garduño-siciliano, leticia title: the potential role of sesquiterpene lactones isolated from medicinal plants in the treatment of the metabolic syndrome – a review date: - - journal: s doi: . /j.sajb. . . sha: doc_id: cord_uid: ugc odq metabolic syndrome comprises a cluster of metabolic disorders related to the development of cardiovascular disease and type diabetes mellitus. in latter years, plant secondary metabolites have become of special interest because of their potential role in preventing and managing metabolic syndrome. sesquiterpene lactones constitute a large and diverse group of biologically active compounds widely distributed in several medicinal plants used for the treatment of metabolic disorders. the structural diversity and the broad spectrum of biological activities of these compounds drew significant interests in the pharmacological applications. this review describes selected sesquiterpene lactones that have been experimentally validated for their biological activities related to risk factors of metabolic syndrome, together with their mechanisms of action. the potential beneficial effects of sesquiterpene lactones discussed in this review demonstrate that these substances represent remarkable compounds with a diversity of molecular structure and high biological activity, providing new insights into the possible role in metabolic syndrome management. metabolic syndrome (mets) refers to a cluster of risk factors related to the development of cardiovascular disease (cvd) and type diabetes mellitus (t dm) (alberti et al., ). this pathological condition represents a public health concern in most nations due to its association with mortality caused by the cardiovascular and metabolic complications (ju et al., ) . indeed, mets is recognized as a risk factor that influences progression and prognosis of coronavirus disease , the infectious disease caused by the most recently discovered coronavirus sars-cov- (costa et al., ) . lifestyle modifications remain the primary component of metss risk factors reduction and pharmacological therapies are indicated in special situations to improving more than one factor (larsen et al., ) . in latter years, secondary plant metabolites have become of special interest in the scientific community because of their potential role in preventing and managing mets (cicero and colletti, ; francini-pesenti et al., ) . many compounds, such as sesquiterpene lactones (slns), have been isolated from medicinal plants and their hypoglycemic, anti-inflammatory, and antioxidative properties coupled with their capacity to modulate key cellular functions have been confirmed by in vitro and in vivo methods (chadwick et al., ; chaturvedi et al., ; alonso et al., ) . despite these effects are linked to the pathogenesis of mets, the role of these compounds to avoid its progression is not well documented compared to many other compounds such as polyphenols into which a great deal of research has been conducted. therefore, in order to provide relevant information regarding the potential benefits of slns in preventing abbreviations: ace, angiotensin i-converting enzyme; ampk, activated protein kinase; apoc , apolipoprotein c ; at, adipose tissue; cat, catalase; cox- , cyclooxygenase ; cvd, cardiovascular disease; ffa, free fatty acids; fn, fibronectin; g pase, glucose- -phosphatase; gk, glucokinase; gpx, glutathione peroxidase; gsh, reduced glutathione; hdl-c, high-density lipoproteins-cholesterol; ifn-g, interferon gamma; il- b, interleukin beta; il- , interleukin ; inos, inducible nitric oxide synthase; ir, insulin resistance; jnk, c-jun n-terminal kinases; ldl-c, low-density lipoprotein-cholesterol; lps, lipopolysaccharide; mapk, mitogen-activated protein kinases; mcp- , monocyte chemoattractant protein ; mets, metabolic syndrome; nf-kb, nuclear factor kappa b; no, nitric oxide; ros, reactive oxygen species; slns, sesquiterpene lactones; sod, superoxide dismutase; stat , signal transducer and activator of transcription ; stz, streptozotocin; t dm, type diabetes mellitus; tbars, thiobarbituric acid reactive substances; tc, total cholesterol; tg, triglycerides; tgf-b , transforming growth factor beta; tlrs, toll-like receptor; tnf-a, tumor necrosis factor alpha; vldl, very-low-density lipoprotein and managing mets, this review addresses plant-derived slns that are potentially responsible for the positive effect in improving risk factors associated with mets. an electronic literature search was conducted on slns with hypoglycemic and/or hypolipidemic effects and isolated from plants used in traditional medicine for the same purposes. the search was done in databases of google scholar, science direct, and scifinder until june using the keyword sesquiterpene lactones and following terms: metabolic syndrome, hypoglycemic, hypolipidemic, antidiabetic, antihypertensive, and medicinal plants. additional information was identified from references located in the retrieved articles. although many slns were screened in some of the studies, only those considered active by the authors are included in this review. the plants included in this review were selected based on their ethnomedicinal records. for each species, the currently accepted name in the online "the plant list" database has been checked and the reported name has been replaced with the current one. compound class, plant source, biological activity and important aspects related with mets are summarized in the table . mets is defined as a cluster of risk factors such as raised blood pressure, atherogenic dyslipidemia, raised fasting glucose, and central obesity, related to the development of cvd and t dm (alberti et al., ) . since the world health organization (who) reported the first formalized definition of the mets, the diagnostic criteria have been sequentially developed by several public health organizations (engin, ) . nowadays, the most recognized criterion for the clinical diagnosis of mets (alberti et al., ) is based on identifying at least three of the following risk factors: ( ) elevated waist circumference (population and country-specific definitions determined by local organizations); ( ) triglycerides (tg) mg/ dl (< . mmol/l) or on drug treatment for elevated triglycerides; ( ) high-density lipoproteins-cholesterol (hdl-c) < mg/dl (< . mmol/l) in men or < mg/dl (< . mmol/l) in women or on drug treatment for reduced hdl-c; ( ) blood pressure / mmhg or on antihypertensive medication treatment, and/or a history of hypertension; and ( ) fasting glucose > mg/dl (> . mmol/l) or on drug treatment for elevated glucose. the mechanisms underlying pathogenesis of mets have not been completely explored, but obesity-induced adipocyte dysfunction and inflammation is associated with the progression of insulin resistance and metabolic disorders (kl€ oting and bl€ uher, ) . body fat mass accumulation in obesity depends on different factors including the relationship of energy intake to energy expenditure and body energy storage (gomez-hernandez et al., ) . adipose tissue (at) plays an important role as an energy storage organ, as well as an endocrine organ produces adipokines such as leptin, adiponectin, monocyte chemoattractant protein (mcp ), tumor necrosis factor alpha (tnfa) and interleukin (il- ), which circulate and regulate systemic metabolism and inflammation. the cell type composition of at includes adipocytes, fibroblasts, macrophages, stromal cells, monocytes and preadipocytes (r afols, ) . on further at expansion, hypertrophy of adipocytes and increased secretion of macrophage chemoattractants occurs, including the secretion of mcp- , which recruits additional macrophages. these actions in turn result in local inflammatory state, enhanced basal lipolysis, increasing the leakage of free fatty acids (ffa) and a dysregulated secretion of several proinflammatory adipokines (longo et al., ; xu et al., ) . subsequently, these adverse signals reach metabolic tissues (e.g. liver, pancreatic islets, and skeletal muscle) and modify inflammatory responses as well as glucose and lipid metabolism, thereby contributing to a global metabolic effect of insulin resistance. chronic low-grade inflammation in obesity results from the activation of various inflammatory mechanisms through nuclear factor kappa b (nf-kb) and c-jun n-terminal kinases (jnk) pathways. these pathways represent important modulators of cytokine gene expression downstream of toll-like receptors (tlrs) in insulin target cells (catrysse and van loo, ; rogero and calder, ) . nf-kb is an important transcription factor involved in different processes of the immune and inflammatory responses. it is composed of p and p subunits and is found in the cytoplasm in complex with inhibitory proteins of the ikb family. cytokines and lipopolysaccharide (lps) can stimulate cell surface receptors including toll-like receptor (tlr ) to initiate a signaling cascade that converge on the activation of the inhibitor of kb kinase (ikk) complex (baker et al., ) . the ikk complex phosphorylates ikba and induces its degradation, leading to the release and nuclear translocation of nf-kb to promote transcription of target genes such as tnf-a, interleukin- beta (il- b), il- , il- , cyclooxygenase (cox- ) and inducible nitric oxide synthase (inos) (knab et al., ; ruan et al., ) . on the other hand, jnk are members of the mitogen-activated protein kinases (mapk) that mediate cellular responses to a variety of intra-and extracellular stresses (zeke et al., ) . in the context of obesity, jnk pathways can be activated by proinflammatory cytokines, ffa and reactive oxygen species (ros), and regulate several nuclear and extra-nuclear substrates, specially the transcription factor activator protein (ap ) which controls the expression of proinflammatory genes and protein synthesis (e.g. tnf-a, il- b, il- and il- ) (pal et al., ; feng et al., ) . deregulated activation of nf-kb and jnk pathways results in increased transcription of il- and tnf-a, which reduce the sensitivity of insulin target cells towards insulin. thus, chronically activated nf-kb and jnk pathways leads to the promotion of insulin resistance (yung and giacca, ) . additionally, increased flux of ffa to the liver in the insulin resistant state stimulates production tg and secretion in the form of verylow-density lipoprotein (vldl). the resulting hypertriglyceridemia leads to lower hdlÀc levels and normal or slightly elevated lowdensity lipoprotein-cholesterol (ldl-c) levels (iqbal et al., ) . this is related to the typical dyslipidemic profile in mets. consistent with a potential role in the pathogenesis of mets, slns interfering with these processes described above could be useful to prevent the onset of insulin resistance and the risk of t dm and cvd in obese individuals. slns represent a diverse group of terpenoids with more than different elucidated structures. they are particularly diversified in the compositae (asteraceae) family, but also occurring in apiaceae, illiciaceae, magnoliaceae, solanaceae, and euphorbiaceae families (padilla-gonzalez et al., ) . numerous species of these families are used in traditional medicine and slns have been described as their primary active constituents. these compounds possess several biological activities such as anti-inflammatory, antidiabetic, antimalarial, anti-proliferative, anti-parasitic and antimicrobial (merfort, ; chadwick et al., ; chaturvedi et al., ). slns are characterized by the presence of a g-lactone ring. the lactone ring can be fused to the remaining skeleton in either a cis or trans configuration, being most common the trans configuration (fischer et al., ; fischer, ; . based on their carbocyclic skeleton, slns are mainly classified in four major groups: germacranolides ( -membered ring), eudesmanolides ( À bicyclic compounds), guaianolides and pseudoguaianolides ( À blood glucose levels (#), hba c (#), plasma insulin ("), tissue glycogen (") tc, tg and ldl-c (#); hdl-c (") tbars levels (#), gsh ("), sod, cat and gpx (") in brain, liver, heart, kidney, and pancreas eliza et al., a eliza et al., , b eliza et al., , costunolide germacrolide costus speciosus alantolactone eudesmanolide inula helenium antiinflammatory-associated to glucose intolerance and ir (in vitro) antiinflamatory-obesity-induced ir (in vitro) attenuates lipid accumulation (in vitro) antiinflamatory-associated to diabetic nephropathy (in vivo) stat inhibitor. inhibition of the tlr gene expression. inhibition of tlr -jnk signaling. il- and mcp- (#). apoc expression at both mrna and protein levels (#) tnf-a and il- (#) in diabetic kidney. serum creatinine and urea nitrogen levels (#). kim et al., a kim et al., , b yang et al., ; zhu et al., tirotundin , epoxygermacranolide antidiabetic (in vitro) dual ppara/g agonists lin, tagitinin a tagitinin g anti-hyperglycemic (in vitro) glucose uptake in t -l adipocytes ("). blood glucose levels (#), serum insulin and pancreatic insulin levels (") g pase activity (#) and gk activity (") tc, tg, ldl-c and vldl (#); hdl-c (") tbars levels (#), sod, cat and gpx (") in liver, kidneys, and pancreas tnf-a levels (#). normalization of blood pressure hong et al., hong et al., , reduction (#); increment ("); thiobarbituric acid reactive substances (tbars); reduced glutathione (gsh); superoxide dismutase (sod); catalase (cat); glutathione peroxidase (gpx); insulin resistance (ir); toll-like receptor (tlr ); c-jun n-terminal kinases (jnk); interleukin (il- ); monocyte chemoattractant protein (mcp- ); apolipoprotein c (apoc ); amp-activated protein kinase (ampk); peroxisome proliferator-activated receptors a and g (ppara/g); nuclear factor kappa b (nf-kb); transforming growth factor beta (tgf-b ); fibronectin (fn); glucose- -phosphatase (g pase); glucokinase (gk); cyclooxygenase (cox- ); interferon-gamma (ifn-g); tumor necrosis factor a (tnf-a); angiotensin i-converting enzyme (ace). a. salazar-g omez et al. / south african journal of botany ( ) À bicyclic compounds) and many subtypes of slns according to their skeletal arrangement (adekenov, ; padilla-gonzalez et al., ) . in many cases, the g-lactone ring contains an exocyclic double bond conjugated to the carbonyl group (a-methylene-g-lactone) but in some cases the exocyclic methylene is reduced or the double bond can be endocyclic (martínez et al., ; padilla-gonzalez et al., ; . it has been well documented that biological activity of the most common types of slns is mainly attributed to formation of covalent union between the a,b-unsaturated group in the exo-methylene-g-lactone and nucleophilic biological targets (e.g. free cysteine sulfhydryl) resulting in alkylation through michael-type addition (schmidt, ) . this chemical reaction induces steric and chemical changes in enzymes, receptors or transcriptional factors leading to a series of cellular events that culminate in diverse biologic responses. the number of alkylating structural elements present on the molecule define differences between the activity of each sln. another structural influences on the biological effects are the molecular size, hydrophobicity, chemical environment and the presence of other functional groups (e.g., epoxy groups, hydroxyls or hydroxyls esterified with acetate, propionate, isobutyrate, angelate, epoxyangelate, and benzoate) (chaturvedi, ; ivanescu et al., ; padilla-gonzalez et al., ; . the structural diversity and the broad spectrum of biological activities drew significant interests in the pharmacological applications of slns (moujir et al., ) . many slns have proved to have a significant anti-inflammatory, hypoglycemic and hypolipidemic activity therefore making them attractive for mets therapy (chaturvedi, ; chaturvedi et al., ) . . . enhydrin from smallanthus sonchifolius (poepp.) h.rob smallanthus sonchifolius (poepp.) h.rob., commonly known as yacon, is a perennial herbaceous plant native to the andean regions of south america (caetano et al., ) . yacon is consumed as a safety dietary supplement and because of its low glucose content and high fructooligosaccharide levels putative antidiabetic effects were suggested (delgado et al., ) . indeed, this suggestion is supported by the hypoglycemic (aybar et al., ; baroni et al., ) and hypolipidemic (miura et al., ; habib et al., ) reported activities. also, a potential use in metabolic disorders could be proposed based on the anti-inflammatory and antioxidant properties (feltenstein et al., ; sousa et al., ) . besides the above mentioned evidences, enhydrin ( ), a melampolide (cis,trans-germacranolide) and the major sesquiterpene lactone component in leaves of s. sonchifolius, has proven to be effective reducing post-prandial glucose levels and a useful compound for blood glucose control by the induction of a late increase in plasma insulin in streptozotocin (stz)- induced diabetic rats (genta et al., ) . more recently, in vivo and in vitro experiments showed that enhydrin ( ) is effective in the management of post-prandial hyperglycemia through inhibition of a-glucosidase in the small intestine (serra-barcellona et al., ) . the inhibition of this enzyme has been linked to the presence of the a,b-unsaturatedg-lactone ring system (yin et al., ) , similar to the intact exo-methylene-g-lactone group in (fig. ) . thus, the antia-glucosidase activity of enhydrin could be derived from this interaction. in addition, the alkylation of nucleophilic sites of factors involved in early stages of the inflammatory cascade described above allows to relate the anti-inflammatory properties reported for with the presence of the exo-methylene-g-lactone ring system (feltenstein et al., ; ma et al., ) . regarding toxicological studies, it has been reported that a single oral administration of enhydrin ( ) at doses of . , and mg/kg body weight (b.w.) did not caused deaths or acute toxic effects within days in male and female rats (genta et al., ) . in acute study in rats, there were no deaths or signs of toxicity observed after single oral administration of at any dose level up to the highest dose tested ( . g/kg b.w.) within days. in subchronic studies, after oral administration for days at daily doses of . , . and . mg/ kg b.w., did not caused hematological, biochemical and histological alterations in rats (serra et al., ) . smallanthus macroscyphus (heliantheae, asteraceae), is a perennial herb commonly known as ''wild yacon'' native from the south american region comprising from southern bolivia to northwestern argentina. this species is closely related to s. sonchifolius and possibly with similar antidiabetic properties. polymatin a ( ) is the main sesquiterpene lactone isolated from s. macroscyphus (de pedro et al., ) . this melampolide exerts an effective inhibition of post-prandial blood glucose peak and hypoglycemic activity in stz-diabetic rats probably by the stimulation of insulin release or due to an insulin-like effect (serra-barcellona et al., ) . due to the close relationship in the polymatin a ( ) and enhydrin ( ) structures, the melampolide could be effective in the management of postprandial hyperglycemia through inhibition of a-glucosidase. however, no studies of these effects were found. in contrast to the c Àc epoxy group in enhydrin ( ), polymatin a ( ) presents a double bond (fig. ). c Àc epoxy groups in melampolides such as enhydrin have been reported to hinder the ability of these compounds to inhibit nf-kb dna binding responsible to cytokines expression (schorr et al., ) commonly observed in inflammatory response. conversely, the effect is favored by the presence of a double bond between c- Àc- in other melampolides (schorr et al., ) . hence, hopeful results could be expected in future studies to explore anti-inflammatory activity in polymatin a. acute oral toxicity of polymatin a ( ) in normal healthy rats at doses assayed ( . , . and . g dried powder/kg b.w.) do not produced deaths or acute toxic effect (changes in behavior or posture, presence of convulsions or occurrence of secretions) within days. the doses were well tolerated and did not produce adverse nutritional effect. no gastrointestinal symptom such as diarrhea or constipation were observed at the doses assayed. volume, ph and urine specific gravity were within normal ranges. no nitrites, protein or blood were detected in the urine samples of the animal groups treated (serra-barcellona et al., ) . in subchronic studies, polymatin a ( ) was orally administered to wistar rats for days at daily doses of , and mg/kg b.w. no toxicity signs or deaths were observed. there were no changes in the behavior, body or organ weights, hematological, biochemical or urine parameters of the rats. no histopathological lesions were observed in the examined organs. the results indicate that polymatin a ( ) from s. macroscyphus leaves may be considered as nontoxic substance at a wide range of doses (serra-barcellona., ) . the common sunflower, helianthus annuus linn (asteraceae), is a well-known plant with edible seeds, flower petals and tender leaf petioles. this plant was proposed to offer a variety of medical benefits (lim, ; guo et al., ) . to provide a scientific explanation for its use in nigerian traditional medicine, onoja and anaga ( ) reported a hypoglycemic effect on the fasting blood glucose level in alloxan-induced diabetic rats after a single dose of the methanolic extract of h. annuus leaves. this study led to the recent isolation of the heliangolide -dehydroeucannabinolide ( ) (fig. ) . (onoja et al., ) . heliangolide slns represent an isomeric form of germacranolides with a cyclodecadiene skeleton and double bonds at c =c and c =c , which stereochemical configurations are trans, cis respectively (s€ ulsen and martino, ). the heliangolide reduced the fasting blood glucose level at dose of . mmol/kg in alloxaninduced diabetic rats (onoja et al., ) . the authors suggested that this sesquiterpene lactone might have reducing effects in glucose absorption based on the heliangolide derivative structure, which presents an intact exo-methylene-g-lactone group. as mentioned above, a,b-unsaturated g-lactone ring system has an important relation in the inhibition of a-glucosidase activity, thus the heliangolide may be inhibiting this enzyme similar to enhydrin ( ). in addition to blood glucose regulation, it has also been demonstrated the positive effects of hydromethanolic leaf extract of h. annuus l. on other components of mets, including reduced ldl-c and tg levels as well as hepatoprotective and antilipid peroxidation activities of alloxan-induced diabetic rats (onoja et al., ) . since -dehydroeucannabinolide ( ) is one of the major components of the extract, potential activity of this molecule in managing dyslipidemia may be proposed. however, additional studies are needed to ascertain the hypolipidemic property of this compound. no in vivo toxicity studies on -dehydroeucannabinolide ( ) have been reported to date. costus speciosus (j. koenig) sm is a plant used in traditional medicine in south asia to treat diabetic patients by eating one leaf of this species per day to regulate blood glucose levels (waisundara et al., ) . also, the hypoglycemic effect of c. speciosus root crude extracts have been reported (daisy et al., ) . eremanthin ( ) (fig. ) is a guaianolide sesquiterpene lactone present in c. speciosus and other plants such as pterodon pubescens (benth.) benth., eremanthus elaeagnus (mart. ex dc.) sch.bip. and laurus nobilis l. (eliza et al., a; waisundara et al., ) . another sesquiterpene lactone reported to be present in c. speciosus is the germacrolide (a trans, trans-germacranolide) costunolide ( ) (fig. ) (eliza et al., b) , which has been also isolated from magnolia spp, laurus nobilis and saussurea costus (falc.) lipsch (koo et al., ) . studies in stz-induced diabetic rats showed that oral administration of and decreased plasma glucose level, glycosylated hemoglobin (hba c), total cholesterol (tc), tg, ldl-c and at the same time markedly increased plasma insulin, tissue glycogen and hdl-c (eliza et al., a (eliza et al., , b . the specific mechanism of eremanthin ( ) bioactivity is not completely characterized, but the experimental model used in both works allowed the authors to suggest that this molecule might exert an insulin-like effect on peripheral tissues by either promoting glucose uptake metabolism, by inhibiting hepatic gluconeogenesis or by absorption of glucose into the muscle and adipose tissues through the stimulation of a regeneration process and revitalization of the remaining b cells (eliza et al., a (eliza et al., , b ). in addition, costunolide ( ) has shown to suppress lps-induced nf-kb activation that leads to the suppression of inos and a consequent nonproduction of no. this activity was stronger than the observed for parthenolide, a germacranolide sesquiterpene lactone (koo et al., ) . thus, the anti-inflammatory activity exhibited by serve as a promising and expanding strategy for treatment of metabolic disease-associated inflammation. on the other hand, the possible inhibition of inflammatory processes has not been studied in eremanthin ( ). however, this compound has a rigid skeleton and an intact exo-methylene-g-lactone group, previously related to anti-inflammatory properties (simonsen et al., ) . additionally, the absence of free hydroxyl groups in could help to improve anti-inflammatory properties since an increasing number of free hydroxyl groups are reported to diminish nf-kb inhibition activity (simonsen et al., ) . taking together these structural characteristics, we suggest that eremathin ( ) is a candidate to be tested for inhibition of inflammatory processes related to metabolic diseases. acute oral toxicity of eremanthin ( ) and costunolide ( ) in normal healthy male wistar rats at doses assayed ( , , , and mg/kg b.w.) do not produced behavioral changes on the rats and mortality was not observed during acute toxicity test ( days) (eliza et al., ) . finally, the antioxidant activity of eremanthin ( ) and costunolide ( ) has been demonstrated by significantly decreasing of the thiobarbituric acid reactive substances (tbars) and lipid peroxidation markers levels as well as by increasing reduced glutathione (gsh) levels and enzymatic antioxidants (eliza et al., ) . this activity could indicate a protective effect of both on oxidative stress in diabetes. inula (asteraceae) is one of the most popular herbs in traditional chinese medicine. this genus comprises more than one hundred species widely distributed throughout asia, africa, and europe, and many of these have been used to treat a wide range of diseases such as bronchitis, diabetes, obesity, hypertension, and inflammation (seca et al., ) . extracts of inula spp. such as inula britannica l., inula viscosa [the accepted name is dittrichia viscosa (l.) greuter.], inula racemosa hook.f., inula helenium l., etc., have been documented for their potential effects on some components of mets, especially hypoglycemic, hypolipidemic and antioxidant activities (kobayashi et al., ; zeggwagh et al., ; shan et al., ; ajani et al., ) as well as inhibition of a-glucosidase enzyme (orhan et al., ) . regarding bioactive secondary metabolites, slns represent the largest group of compounds occurring in inula spp. many of these compounds were isolated from plants mentioned above and demonstrated to exert diverse biological activities (seca et al., ; wang et al., ) . the anti-inflammatory mechanisms of some slns have been related to their influence on directly inhibit the mapk and block the activation of nf-kb, thus achieving the prevention of pro-inflammatory signaling (han et al., ; park et al., ) . alantolactone ( ) (fig. ) is the most known eudesmanolide present in several inula species, including inula helenium l. used for hypoglycemic and antiobesity purposes (seca et al., ; wang et al., ) . this compound has been extensively studied for its antitumor, antioxidant and anti-inflammatory effects (moujir et al., ) . regarding anti-inflammatory activity, the eudesmanolide suppresses no, pge and tnf-a production in lps-stimulated raw . cells by modulating the activity of the nf-kb and mapk signaling pathways (chun et al., ) , and it also suppresses tnf-a and interferon gamma (ifn-g)-induced production of chemokines by blocking the signal transducer and activator of transcription (stat ) phosphorylation in hacat cells (lim et al., ) . as previously mentioned, it is recognized the fact that tlrs are responsible for the activation of inflammatory pathways in obesity state. in vitro trials confirmed that alantolactone ( ) prevents the increase of il- levels and regulates macrophage infiltration by reduction of mcp- concentrations via inhibition of the tlr -jnk pathway in both, lean (adipocytes) and obese (adipocyte-macrophage) states (kim et al., a (kim et al., , b . besides the effect on adipocytes, alantolactone ( ) can also act in skeletal muscle and liver cells. in this context, the compound inhibits il- -induced insulin-stimulated glucose intolerance and insulin resistance in the skeletal muscle through blockade the activation of stat and the abnormal expression of tlr (kim et al., a (kim et al., , b . in l cells, alantolactone ( ) also inhibits oxldl-induced lipid accumulation trough regulating the apolipoprotein c (apoc ) expression partly by decreasing the activation of stat . thus, may be relevant to explore further its role in managing metabolic complications on e.g. modulating hepatic il- /stat signaling in high-fat diet-induced metabolic disorder in animal models, since the physiological metabolic response to il- depends on the specific source of il- in vivo (han et al., ) concerning to in vivo experiments, no hypoglycemic effect was observed on stz-induced diabetic mice after oral administration of alantolactone. however, inflammation and renal abnormalities were suppressed via inhibition of nf-kb gene expression and the high glucose-induced overexpression of pro-inflammatory cytokines and macrophage adhesion in renal nrk- e cells were inhibited. (zhu et al., ) . these results led the authors to propose a beneficial use for the treatment of diabetic neuropathy. taking together all evidences mentioned for alantolactone ( ) activity, it is probably that this compound could reduce some components of mets by the regulation of inflammatory processes. alantolactone ( ) did not induced significant hepatotoxicity and nephrotoxicity after daily administration ( mg/kg b.w.) for weeks in mice . this compound also is a contact allergen, and in vitro is cytotoxic in cancer cells and induces apoptosis (tisserand and young, ) . . . , -epoxygermacranolides from tithonia diversifolia (hemsl.) a. gray tithonia diversifolia (hemsl) a. gray, commonly known as tree marigold or mexican sunflower (asteraceae: heliantheae), is a shrublike perennial or annual invasive plant, native from north and central america. traditionally, its leaves are widely used by indigenous people for treating a wide spectrum of diseases, including diabetes mellitus. several in vitro and in vivo studies have stated the antioxidant antidiabetic, hypolipidemic and antiobesity effects of t. diversifolia (ajao and moteetee, ; tagne et al., ) . germacranolides, eudesmanolides and guaianolides are some of the major components occurring in this plant (ajao and moteetee, ; tagne et al., ) and because of their previously described activity, they apparently are involved in the important spectrum of bioactivity reported for t. diversifolia. tirotundin ( ) and tagitinin a ( ) (fig. ) isolated from the ethyl acetate soluble fraction of t. diversifolia are related to the activation of peroxisome proliferator-activated receptors (ppars) (lin, ) . ppars are members of nuclear hormone receptors superfamily involved in the metabolic regulation of lipid and lipoprotein levels, blood glucose, and abdominal adiposity. in mammals, three isoforms of ppars have been recognized namely ppar-a, ppar-b/d and ppar-g. activation of ppar-a by hypolipidemic fibrate class of drugs decreases tg levels and raises hdl-c in dyslipidemic individuals, whereas activation of ppar-g by antidiabetic thiazolidinedione agents causes insulin sensitization to enhance glucose metabolism (botta et al., ) . currently, dual ppara/g agonists, which stimulate both ppara and ppar-g isoforms to similar extents, are gaining popularity since it is believed that they are able to ameliorate the unwanted side effects of selective ppar-a and ppar-g agonists; and may also be used to treat dyslipidemia and t dm simultaneously (balakumar et al., ) . tirotundin ( ) and tagitinin a ( ) have been suggested being dual ppara/g agonists after the evaluation of their agonistic activity against ppars employing a transient transfection reporter assay in hepg cells . on the other hand, the germacranolides tagitinin g ( ), tagitinin i ( ), b-hydroxydiversifolin- -o-methyl ether ( ) and b-hydroxytirotundin- -o-methyl ether ( ) (fig. ) isolated from the aerial parts of t. diversifolia were found to significantly elevate glucose uptake in t -l adipocytes without any toxicity (zhao et al., ) . the authors suggested that these compounds had pparg agonist activity on glucose uptake, similar to pioglitazone, a ppar-g agonist used as a control compound in the study. no in vivo acute, subchronic or chronic toxicity studies on , -epoxygermacranolides described above have been reported to date. the increasing abundance of tithonia diversifolia in conservation and agricultural areas over the past ten years in south africa has been of concern. this plant is considered as a scrub resulting in the initiation of a biological control program against its propagation (simelane et al., ) . therefore, utilization for medicinal purposes of this invasive plant could be important before major eradication takes place. the genus magnolia, previously known as michellia, reported to exert various biological effects, including anti-cancer, anti-anxiety, antidepressant, antioxidant and anti-inflammatory (lee et al., ) . in later years, magnolia species and their components have been related to ameliorate characters of obesity and diabetes, such as hyperglycemia, hyperlipidemia, and other complications . since terpenoids are one of the principal substantial compounds in magnolia species, slns may be related with its medicinal properties (sun et al., ) . anticancer activity investigation lead to the isolation of the guaianolide micheliolide ( ) (fig. ) from m. compressa (maxim.) sarg. (ogura et al., ) . this compound has also been obtained in a semi-synthetic way from a bf -mediated rearrangement of parthenolide (castañeda-acosta et al., ; zhai et al., ) , presenting enhanced stability and remarkable antiinflammatory effect by suppressing activation of the nf-kb through inhibition of ikba degradation as well as for the inhibition of p translocation to the nucleus (viennois et al., ) . the compound also influenced the mapk and pi k/akt signaling pathways in lpsstimulated bv- cells (sun et al., ) . further, an in vitro assay has revealed that attenuate the high glucose-stimulated activation of nf-kb, the degradation of ikba, and the expression of mcp- in rat mesangial cells (jia et al., ) . in addition, the dimethylamino michael adduct of known as dimethylaminomicheliolide is a pro-drug of this sesquiterpene lactone approved for clinical trials for glioblastoma treatment . this compound protects the kidneys against proteinuria, renal failure, histopathological injury, and inflammation by suppressing activation of the nf-kb signaling pathway in db/db mice . these results showed that dimethylaminomicheliolide intervention mitigated diabetic kidney disease in db/db mice without directly affecting hyperglycemia. furthermore, it has been demonstrated that micheliolide ( ) alleviates hepatic steatosis in obese db/db mice, and the molecular mechanisms driving the therapeutic effects of this compound might involve ppar-g upregulation to consequently inhibit nf-kb mediated inflammation and activate ampk/mtor-dependent autophagy (zhong et al., ) . thus, it will be interesting to future work evaluate if this effect in hepatic steatosis impacts glucose metabolism. no in vivo toxicity studies on micheliolide ( ) have been reported to date. byrsonima crassifolia (malpighiaceae), commonly known as "nanche", is a tropical tree widely distributed in mexico, central and south america. this tree is commonly harvested, both for its edible fruit and for its health benefits (b ejar and malone, ; duarte, ) . in folk medicine, the fruit or bark infusion have been used as hypoglycemic remedy (andrade-cetto and heinrich, ) and according to ethnomedicinal reports, the antihyperglycemic activity of hexane and chloroform extracts from fruits and seeds of b. crassifolia in stzinduced diabetic rats was reported (perez-gutierrez et al., ) . the phytochemical analysis of antihyperglycemic extract from the seed of b. crassifolia revealed that byrsonines a ( ) and b ( ) (fig. ) , two dimeric guaianolides, are responsible for the antioxidant, hypoglycemic and hypolipidemic activities (guti errez and ramirez, ) . in particular, it has been proposed that the antihyperglycemic activity of these compounds may involve both pancreatic and extra pancreatic mechanisms, based on the observed increase in serum insulin level and the reduction of hepatic glucose output via decreasing glucose- phosphatase (g pase) activity and increasing glucokinase activity (guti errez and ramirez, ) . although the mechanisms underlying the antihyperglycemic activity of and have not been completely explored, this effect could likely be ascribed to the activation ampactivated protein kinase (ampk). activation of the ampk pathway in the liver regulates glucose homeostasis by inhibiting hepatic glucose production and downregulating the expression of gluconeogenic genes, including g pase gene expression. targeting ampk by natural products demonstrated considerable success in lowering blood glucose levels (joshi et al., ) . nevertheless, more experimental research is needed to substantiate this claim. on the other hand, the induction of antioxidant enzymes in hepatic, renal, and pancreas tissues after the administration of and , indicates a positive effect of these molecules in improving glycemic control in stz-induced diabetic rats (guti errez and ramirez, ), since oxidative stress takes an important role in the pathogenesis and progression of diabetic tissue injury. in this sense, the reported improvement in insulin resistance by byrsonines a and b can be related to the observed increase in the hdl-c levels and the decrease in tc, tg, ldl-c, and vldl (guti errez and ramirez, ). additionally, tnf-a levels were also decreased after treatment of byrsonines a and b in stz-induced diabetic rats. the bioactivity described for and makes them one of the most active guaianolides on different components of mets reported at this time. as described above, another molecule of this family with activity on different components of mets is eremathin. however, byrsonines a and b ( , ) seems to be more effective, which could be due to their dimeric structure that provides two possible michaels acceptors. dimeric slns have been found to be more potently cytotoxic than their monomers toward human cancer cells, indicating that the antitumor potential of slns are improved by the presence of additional a-methylene-g-lactone ring (singh et al., ; ren et al., ) . this phenomenon could be studied in and for mets treatment due to their extended activity reviewed here. moreover, pharmacokinetics studies are needed to understand either byrsonines suffer biotransformation reactions and act as monomeric molecules or if they can show biological activity in their original dimeric forms. no in vivo toxicity studies on byrsonines a ( ) and b ( ) have been reported to date. various species of the genus lactuca are globally important edible plants containing valuable nutrients such as polyphenols, sterols, vitamins, minerals, and dietary fiber. l. sativa l., lactuca indica l. and l. serriola l. (syn. l. scariola l.) have been used in the treatment of diabetes mellitus, hypertension, and cardiac diseases (eddouks et al., ; subramoniam, ) . in vivo experiments of some lactuca species shown the ability to reduce several metabolic risk factors, especially hyperglycemia, tg and tc (nicolle et al., ; salih, ) . slns are commonly found in lactuca species and are represented as subgroups such as lactucin-type guaianolides and the eudesmanolide-type. the latter is commonly found in some species of lactuca such as l. sativa l. var. anagustata, l. saligna l. and l. canadensis l. (han et al., ; kisiel and gromek, ; michalska et al., ) . the dimeric guaianolide lactucain c ( ) (fig. ) , isolated from l. indica linn. showed moderate lowering of plasma glucose in stz-diabetic rats (hou et al., ) . nevertheless, there are no evidences about the possible mechanism of action and no further research has been made about this compound. as mentioned before, dimeric slns could show an important activity in components of mets derived from their double a,b-unsaturated g-lactone ring. because of its hypoglycemic properties and chemical structure, is also a candidate to further studies that analyze deeply its effect in blood glucose levels and some other metss components. no in vivo toxicity studies on luctucain c ( ) have been reported to date. . . -deoxylactucin from cichorium intybus l cichorium intybus, commonly known as chicory, was historically cultivated by the ancient egyptians as a vegetable crop, a coffee substitute, and a medicinal plant. nowadays, it is appreciated for its bitter taste and widely used in india as a traditional treatment for diabetes mellitus (al-snafi, ; pushparaj et al., ) . the antidiabetic, hypolipidemic, hepatoprotective, antioxidant and anti-inflammatory effects of c. intybus have been widely studied (chandra and sk, ) . this broad spectrum of biological activities has been attributed to its high content of phytochemical constituents, being slns the most abundant in roots and the responsible of the bitterness of leaves (al-snafi, ). -deoxylactucin ( ) (fig. ) is a guaianolide found as a major component of chicory root and it is also common in species of lactuca. it has been demonstrated that possess anti-inflammatory activity by inhibiting the nuclear transcription factor nf-kb and as a selective inhibitor of cox- (cavin et al., ). an important structural characteristic of is the presence of a-methylene-g-lactone group and a,b-unsaturated cyclopentenone. compounds that possess these structures, such as helenalin, can react with sulfhydryl group of cys in nf-kb and prevent dna binding (lyß et al., ; widen et al., ) . thus, -deoxylactucin ( ) could interact with nf-kb by a similar mechanism. no in vivo toxicity studies on -deoxylactucin ( ) have been reported to date. . . artemisinin from artemisia spp artemisinin ( ) (fig. ) is a cadinanolide endoperoxide sesquiterpene lactone originally isolated from artemisa annua linn in (tu, ) . nowadays, and their derivatives are part of the protocols for malaria treatment (bridgford et al., ) . experimental studies have also established their effectiveness as an anti-inflammatory, antioxidant, anti-tumor, and vascular protection agent jiang et al., ; efferth, ) . regarding to vascular protection, cao et al. ( ) demonstrated that oral administration of artemisinin ( ) effectively alleviated inflammatory response (mcp- , ifn-g, il- and tnf-a), elevated macrophage autophagy, suppressed foamy macrophage transformation, and thereby inhibiting atherosclerotic plaque formation in high-fat diet treated apoe À/À mice. in addition, has been discovered to be a potential therapeutic agent for ameliorating type diabetes because of its ability to promote the conversion of pancreatic glucagon-producing a cells to insulinsecreting b cells in rats . nevertheless, the widespread application of artemisinin as an anti-malarial drug could contributed to the selection of resistant strains of the etiologic agent plasmodium, thus its use to combating non-communicable human diseases would raise the risk for this selection and lead to an important drug resistance development. some authors have suggested that unless necessary, artemisinin ( ) should be replaced by other therapeutic agents for the treatment of versatile types of human diseases (dong-sheng et al., ) . it is important to note that some species of artemisia have been traditionally used in treatment of diabetes such as a. dracunulus l., a. minor jacq. ex besser, a. pallens wall. ex d.c., a. sphaerocephala krasch and a. herba-alba asso, (mohamed et al., ; subramoniam, ) . several eudesmanolides and guaianolides have been isolated from a. herba-alba, all of them having in common the absence of the a-methylene in the g-lactone ring as in artemisinin ( ) structure. indeed the ethanol, chloroform and water extracts of this plant have showed important hypoglycemic activity (mohamed et al., ) . animal experiments show considerable toxicity after the application of artemisinin family (neurotoxicity, embryotoxicity, genotoxicity, hemato-and immunotoxicity, cardiotoxicity, nephrotoxicity, and allergic reactions), but large clinical studies and meta-analyzes did not show serious human side effects, although proper monitoring of adverse effects in developing countries may not be a trivial task. there is a paucity of large-scale clinical trials adequate to detect rare but significant toxicity. the lesson learned from animal and human studies is that long-term availability rather than short-term peak concentrations of artemisinin cause toxicity (efferth and kaina, ) . thus, the observation of the toxicity of artemisinin derivatives in animals, but not in humans, is most likely due to different pharmacokinetic profiles after different routes of administrations. . . scoporanolide and estafiatin from artemisia scoparia waldst. & kit artemisia scoparia waldst. & kit. (redstem wormwood) is widely distributed in southwest asia and central europe. this species has been reported to possess anti-obesity (richard et al., ) and hypolipidemic (boudreau et al., ) properties. a. scoparia ethanolic extract reduces non-alcoholic fatty liver disease by enhancing hepatic insulin and amp-ampk signaling in diet-induced obese mice . a. scoparia hot water extract reduces blood pressure in spontaneously hypertensive rats via the inhibition of plasma angiotensin i-converting enzyme (ace) activity (cho et al., ) . the phytochemical analysis of this extract revealed that seven slns contribute to the antihypertensive effect inhibition of ace activity, highlighting the activity of scoporanolide ( ) and estafiatin ( ) (fig. ) (cho et al., ) showing significantly higher ace inhibitory activities than the other isolated slns. no in vivo toxicity studies on scoporanolide ( ) and estafiatin ( ) have been reported to date. the guaianolide cumambrin a ( ) (fig. ) , isolated from chrysanthemum boreale (makino) makino, has shown a pharmacological effect on the normalization of blood pressure in spontaneously hypertensive rats (hong et al., ) . ex vivo study demonstrate that this compound is a potent relaxant of aortic smooth muscle at a concentration of £ À m (hong et al., ) . no in vivo toxicity studies on cumambrin a ( ) have been reported to date. the potential beneficial effects of slns on metss risk factors discussed in this review clearly demonstrate that these substances represent remarkable compounds with a diversity of molecular structure and biological activity. these beneficial effects include normalization of blood glucose levels, improvement of blood lipid profiles, anti-inflammatory activity, improvement of insulin sensitivity and normalization of blood pressure. the underlying molecular targets mediating these effects have not been completely understood. regardless of that, it can be noticed that at least six molecular targets or pathways could explain the effect of slns on components of mets: furthermore, the evidence from published literature demonstrates that some slns belonging to guaianolides like eremanthin ( ) and germacranolides such as costunolide ( ) and guaianolide dimers byrsonines a and b ( , ), show multifunctional benefits due to their action on many metss risk factors. these observations suggest that structural specificity may be a key for understanding the mechanisms of action of slns. looking forward, the effects of slns on mets should be explained based on the molecular targets and should be related to biochemical mechanisms. mets has become a major public health problem worldwide and represents a common clinical condition in countries with a high incidence of obesity and western dietary patterns. natural products, such as slns, are attractive drug candidates and more attention must be paid to their potential use in the treatment and prevention of mets. the authors declare no conflict of interest. sesquiterpene lactones with unusual structure. their biogenesis and biological activity evaluation of antidiabetic effect of methanolic extract of inula racemosa root in rats tithonia diversifolia (hemsl) a. gray. (asteraceae: heliantheae), an invasive plant of significant ethnopharmacological importance: a review harmonizing the metabolic syndrome: a joint interim statement of the international diabetes federation task force on epidemiology and prevention; national heart, lung, and blood institute anti-inflammatory activity medical importance of cichorium intybus-a review mexican plants with hypoglycaemic effect used in the treatment of diabetes hypoglycemic effect of the water extract of smallantus sonchifolius (yacon) leaves in normal and diabetic rats nf-kb, inflammation, and metabolic disease a contemporary overview of ppara/g dual agonists for the management of diabetic dyslipidemia effect of crude extracts of leaves of smallanthus sonchifolius (yacon) on glycemia in diabetic rats pharmacological and chemical screening of byrsonima crassifolia, a medicinal tree from mexico. part i ppar agonists and metabolic syndrome: an established role? an ethanolic extract of artemisia scoparia inhibits lipolysis in vivo and has antilipolytic effects on murine adipocytes in vitro artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome yacon (smallanthus sonchifolius) as a food supplement: health-promoting benefits of fructooligosaccharides artemisinin attenuated atherosclerosis in high-fat diet-fed apoe-/-mice by promoting macrophage autophagy through the ampk/mtor/ulk pathway biomimetic transformations of parthenolide inflammation and the metabolic syndrome: the tissuespecific functions of nf-kb inhibition of the expression and activity of cyclooxygenase- by chicory extract sesquiterpenoids lactones: benefits to plants and people therapeutic potential of cichorium intybus in lifestyle disorders: a review sesquiterpene lactones: structural diversity and their biological activities recent developments on the antidiabetic sesquiterpene lactones and their semisynthetic analogues sesquiterpene lactones and scopoletins from artemisia scoparia waldst. & kit. and their angiotensin i-converting enzyme inhibitory activities antihypertensive effects of artemisia scoparia waldst in spontaneously hypertensive rats and identification of angiotensin i converting enzyme inhibitors alantolactone suppresses inducible nitric oxide synthase and cyclooxygenase- expression by down-regulating nf-kb, mapk and ap- via the myd signaling pathway in lps-activated raw . cells role of phytochemicals in the management of metabolic syndrome metabolic syndrome and covid- : an update on the associated comorbidities and proposed therapies influence of costus speciosus (koen.) sm. rhizome extracts on biochemical parameters in streptozotocin induced diabetic rats melampolides from smallanthus macroscyphus yacon (smallanthus sonchifolius): a functional food artemisinin: a panacea eligible for unrestrictive use? postharvest biology and technology of tropical and subtropical fruits. , woodhead publishing ethnopharmacological survey of medicinal plants used for the treatment of diabetes mellitus, hypertension and cardiac diseases in the south-east region of morocco (tafilalet) from ancient herb to modern drug: artemisia annua and artemisinin for cancer therapy toxicity of the antimalarial artemisinin and its dervatives antioxidant activity of costunolide and eremanthin isolated from costus speciosus (koen ex normo-glycemic and hypolipidemic effect of costunolide isolated from costus speciosus (koen ex streptozotocin-induced diabetic rats antidiabetic and antilipidemic effect of eremanthin from costus speciosus (koen.) sm., in stz-induced diabetic rats the definition and prevalence of obesity and metabolic syndrome antiinflammatory and anti-hyperalgesic effects of sesquiterpene lactones from magnolia and bear's foot the role of jnk signaling pathway in obesity-driven insulin resistance sesquiterpene lactones: biogenesis and biomimetic transformations fortschritte der chemie organischer naturstoffe / progress in the chemistry of organic natural products potential role of phytochemicals in metabolic syndrome prevention and therapy hypoglycemic activity of leaf organic extracts from smallanthus sonchifolius: constituents of the most active fractions differential role of adipose tissues in obesity and related metabolic and vascular complications dimethylaminomicheliolide (dmamcl) suppresses the proliferation of glioblastoma cells via targeting pyruvate kinase (pkm ) and rewiring aerobic glycolysis a review of phytochemistry, metabolite changes, and medicinal uses of the common sunflower seed and sprouts (helianthus annuus l.) hypoglycemic effects of sesquiterpene lactones from byrsonima crassifolia hypolipidemic effect of smallanthus sonchifolius (yacon) roots on diabetic rats: biochemical approach ergolide, sesquiterpene lactone from inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase- expression in raw . macrophages through the inactivation of nf-kb regulation of adipose tissue inflammation by interleukin isolation and characterisation of the sesquiterpene lactones from lactuca sativa l var. anagustata effect of cumambrin a treatment on blood pressure in spontaneously hypertensive rats effect of cumambrin a on the relaxation of rat aorta antidiabetic dimeric guianolides and a lignan glycoside from lactuca indica metabolic syndrome, dyslipidemia and regulation of lipoprotein metabolism sesquiterpene lactones from artemisia genus: biological activities and methods of analysis sesquiterpene lactones and their derivatives inhibit high glucoseinduced nf-kb activation and mcp- and tgf-b expression in rat mesangial cells artesunate attenuated progression of atherosclerosis lesion formation alone or combined with rosuvastatin through inhibition of pro-inflammatory cytokines and pro-inflammatory chemokines targeting ampk signaling pathway by natural products for treatment of diabetes mellitus and its complications association of metabolic syndrome and its components with all-cause and cardiovascular mortality in the elderly: a meta-analysis of prospective cohort studies alantolactone induces apoptosis in glioblastoma cells via gsh depletion, ros generation, and mitochondrial dysfunction anti-inflammatory, antioxidant and antimicrobial effects of artemisinin extracts from artemisia annua l alantolactone improves palmitate-induced glucose intolerance and inflammation in both lean and obese states in vitro: adipocyte and adipocyte-macrophage co-culture system alantolactone improves prolonged exposure of interleukin- -induced skeletal muscle inflammation associated glucose intolerance and insulin resistance sesquiterpene lactones from lactuca saligna adipocyte dysfunction, inflammation and metabolic syndrome involvement of eicosanoids in the pathogenesis of pancreatic cancer: the roles of cyclooxygenase- and -lipoxygenase preventative effects of the flowers of inula britannica on autoimmune diabetes in c bl/ksj mice induced by multiple low doses of streptozotocin a sesquiterpene lactone, costunolide, from magnolia grandiflora inhibits nf-kb by targeting ikb phosphorylation the pharmacological management of metabolic syndrome therapeutic applications of compounds in the magnolia family artemisinins target gaba a receptor signaling and impair a cell identity alantolactone from saussurea lappa exerts antiinflammatory effects by inhibiting chemokine production and stat phosphorylation in tnfÀa and ifnÀgÀinduced in hacat cells helianthus annuus sesquiterpene lactones from tithonia diversifolia act as peroxisome proliferator-activated receptor agonists micheliolide ameliorates diabetic kidney disease by inhibiting mtdh-mediated renal inflammation in type diabetic db/db mice adipose tissue dysfunction as determinant of obesity-associated metabolic complications the anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor nf-kb by directly targeting p inhibition of nf-kb-mediated transcription and induction of apoptosis by melampolides and repandolides the artemisia l. genus: a review of bioactive sesquiterpene lactones perspectives on sesquiterpene lactones in inflammation and cancer sesquiterpene lactones from lactuca canadensis and their chemotaxonomic significance hypoglycemic and hypolipidemic activity of the leaf of samallanthus sonchifolius in genetically type diabetic mice chemical constituents and biological activities of artemisia herba-alba applications of sesquiterpene lactones: a review of some potential success cases health effect of vegetable-based diet: lettuce consumption improves cholesterol metabolism and antioxidant status in the rat anticancer sesquiterpene lactones of michelia compressa (magnoliaceae) evaluation of the antidiabetic and antioxidant potentials of methanolic leaf extract of helianthus annuus l. on alloxan-induced hyperglycemic rats potential antidiabetic and antioxidant activities of a heliangolide sesquiterpene lactone isolated from helianthus annuus l. leaves hypoglycemic, antidyslipidemic, hepatoprotective and antilipid peroxidation activities of hydromethanol leaf extract of helianthus annuus in alloxan-induced diabetic rats antioxidant potential and carbohydrate digestive enzyme inhibitory effects of five inula species and their major compounds sesquiterpene lactones: more than protective plant compounds with high toxicity the roles of cÀjun nh Àterminal kinases (jnks) in obesity and insulin resistance britanin suppresses lps-induced nitric oxide, pge and cytokine production via nf-kb and mapk inactivation in raw . cells antihyperglycemic, antihyperlipidemic and antiglycation effects of byrsonima crassifolia fruit and seed in normal and streptozotocin-induced diabetic rats anti-diabetic effects of cichorium intybus in streptozotocin-induced diabetic rats adipose tissue: cell heterogeneity and functional diversity. endocrinología y nutrici on development of anticancer agents from plantderived sesquiterpene lactones artemisia scoparia enhances adipocyte development and endocrine function in vitro and enhances insulin action in vivo obesity, inflammation, toll-like receptor and fatty acids inducible cox- dominates over cox- in prostacyclin biosynthesis: mechanisms of cox- inhibitor risk to heart disease effect of lactuca serriola on b-cell dysfunction and glucose tolerance induced by high sucrose fed in albino rats structure-activity relationships of sesquiterpene lactones a novel dimeric melampolide and further terpenoids from smallanthus sonchifolius (asteraceae) and the inhibition of the transcription factor nf-kb the genus inula and their metabolites: from ethnopharmacological to medicinal uses safety assessment of aqueous extract from leaf smallanthus sonchifolius and its main active lactone, enhydrin smallanthus macroscyphus: a new source of antidiabetic compounds enhydrin regulates postprandial hyperglycemia in diabetic rats by inhibition of a-glucosidase activity polymatin a from smallanthus macroscyphus leaves: a safe and promising antidiabetic compound anti-diabetic and hypolipidemic effects of aqueousextract from the flower of inula japonica in alloxan-induced diabetic mice prospective agents for the biological control of tithonia rotundifolia (mill.) s.f. blake and tithonia diversifolia (hemsl.) a. gray (asteraceae) in south africa guaianolide sesquiterpenoids: pharmacology and biosynthesis effects of artemisinin dimers on rat breast cancer cells in vitro and in vivo antioxidant properties of sterilized yacon (smallanthus sonchifolius) tuber flour plants with anti-diabetes mellitus properties analytical procedures overview magnolia officinalis extract contains potent inhibitors against ptp b and attenuates hyperglycemia in db/db mice micheliolide suppresses lps-induced neuroinflammatory responses tithonia diversifolia (hemsl.) a. gray as a medicinal plant: a comprehensive review of its ethnopharmacology, phytochemistry, pharmacotoxicology and clinical relevance essential oil safety. a guide for health care professionals, second ed artemisinin-a gift from traditional chinese medicine to the world (nobel lecture) micheliolide, a new sesquiterpene lactone that inhibits intestinal inflammation and colitis-associated cancer costus speciosus and coccinia grandis: traditional medicinal remedies for diabetes inula sesquiterpenoids: structural diversity, cytotoxicity and anti-tumor activity artemisia scoparia extract attenuates nonalcoholic fatty liver disease in diet-induced obesity mice by enhancing hepatic insulin and ampk signaling independently of fgf pathway targeting nf-kb p with a helenalin inspired bis-electrophile etiology of metabolic syndrome and dietary intervention alantolactone suppresses apoc expression and alters lipid homeostasis in l liver cells role of c-jun n-terminal kinase (jnk) in obesity and type diabetes study of hypoglycaemic and hypolipidemic effects of inula viscosa l. aqueous extract in normal and diabetic rats jnk signaling: regulation and functions based on complex protein-protein partnerships biomimetic semisynthesis of arglabin from parthenolide three new sesquiterpenes from tithonia diversifolia and their anti-hyperglycemic activity extracts of magnolia species-induced prevention of diabetic complications: a brief review micheliolide alleviates hepatic steatosis in db/db mice by inhibiting inflammation and promoting autophagy via ppar-g-mediated nf-kb and ampk/mtor signaling alantolactone mitigates renal injury induced by diabetes via inhibition of high glucose-mediated inflammatory response and macrophage infiltration this work was partially supported by the secretaría de investigaci on y posgrado, instituto polit ecnico nacional. asg was cona-cyt ( ) and ipn-beifi fellow. julio c. ontiveros thanks conacyt for funding conacyt project no. "c atedras cona-cyt". key: cord- -s anw authors: xu, jing; pan, li-jia; jia, chun-sheng title: exploration on the feasibility of moxibustion in prevention and treatment of covid- from the perspective of modern medical mechanism date: - - journal: world j acupunct moxibustion doi: . /j.wjam. . . sha: doc_id: cord_uid: s anw novel coronavirus pneumonia (covid- ) is rampant in many countries and regions and there is no time to delay the exploration of the scheme for its prevention and control. the pathogenic characteristics of novel coronavirus and the effect of moxibustion for warming up yang and strengthening the antipathogenic qi were analyzed in this paper. from the perspective of modern medical mechanism, during the prevention and treatment of novel coronaviral infection, moxibustion may be able to prevent and treat covid- by improving the body's immunity so as to conquer virus, by anti-inflammation to alleviate the inflammatory response of covid- and by improving lung function to inhibit pulmonary fibrosis. and treatment of covid- . the disease is clinically manifested as fever, dry cough and fatigue, accompanied with nasal obstruction, runny nose, headache, sore throat, myalgia and diarrhea in a few cases. its incubation period is day to days in generally. the patients with viral infection and asymptomatic carries may be the source of infection and the population is generally susceptible. according to the analysis of jibai xiong, the master of chinese medicine, the infectious characteristics of this disease are in line with the description in chapter of sùwèn ( 《素问》basic questions). "where the epidemic disease arrives, it is easy to infect each other, no matter young or old, with similar symptoms". hence, in tcm, this disease is in the category of pestilence [ ] . it is said by youke wu in ming dynasty, in wēnyìlùn (《温疫论》treatise on warm-heat pestilence) that pestilence results from epidemic pathogen and the people will suffer from it whenever being contagious, no matter young or old, strong or weak. regarding the nature of pathogen, the academician, xiaolin tong, from guang'anmen hospital of china academy of chinese medical sciences, believes that this pathogen pertains to cold because the disease started at the winter solstice, just around "the st nine-day period in winter" according to chinese lunar calendar. the climate in wuhan is extremely humid and the continuous rainy days aggravate the damp pathogen during the epidemic. it is shown that the nature of this disease refers to yin disorder, caused by pathogenic cold and damp, dominated by yang damage [ ] . yin pathogen easily damages yang qi. consumption of yang qi or loss of yang qi threatens the life. novel coronavirus impairs human body and depletes yang qi. "when the people are of the old age, yang qi is declining, hence, the hands and feet are not warm anymore and the primary yang is exhausted". this may be the reason why elderly patients are more seriously ill and once they are at the serious stage, the fatality rate will be greatly raised. in view of this situation, the principle of treatment should be warming and tonifying the primary yang. it is said in biănquè xīnshū (《扁鹊 心书》the teachings of bian que) that "when the genuine yang and the primary qi are deficiency, the people will be ill, when the genuine yang and the primary qi are collapse, the people will be dead, moxibustion is the first option to save the life." moxibustion acts on warming meridians, expelling cold, rescuing yang from collapse, removing stasis and resolving masses, preventing diseases and keeping healthy. academician, xiaolin tong also advocates that with moxibustion combined for warming yang and eliminating cold and damp while the chinese herbal medication is used based on syndrome/pattern differentiation, the body immunity may be improved. moxibustion works not only for strengthening the antipathogenic qi, but also for eliminating pathogens. it motivates the antipathogenic qi in the human body and enhances body resistance. moxibustion may also prevent from diseases and benefit health care. for the pathogenic cold and damp in the body, moxibustion may expel cold pathogen and promote circulation in meridian and collateral by its warming and heat effect. regarding the heat transformed by the long-term accumulation of cold and damp, moxibustion may open the sweat pore of skin, keep the pores open so as to ensure the elimination of the heat [ ] . potential modern medical mechanism of moxibusion in prevention and treatment of covid- lymphocytes can be divided into t lymphocytes, b lymphocytes and natural killer (nk) cells, which are the main executor for immune system function regulation. during the immune response, when the number and the function of each lymphocyte subset are abnormal, a series of pathological changes may occur. therefore, the imbalance of lymphocyte subsets is an important indicator of abnormal immune response [ ] . in the patients with covid- , the levels of white blood cell (wbc), l (%), red blood cell (rbc), hemoglobin (hgb), cd + , cd + , cd + , blood urea nitrogen (bun) and uric acid (ua) are lower than those in healthy people respectively [ ] . the reason for the decrease of lymphocytes in the patients may be related to the fact that covid- directly or indirectly kills lymphocytes or inhibits lymphocyte generation, which will lead to the low immune function of patients [ ] . modern research shows that moxibustion improves the body immunity by regulating various immune cells and immune factors. ginger-isolated mild moxibustion significantly regulates the immune function of children with cough variant asthma and the level of cd + and the ratio cd + /cd + are higher than those in the western medication group and the level of cd + is lower obviously than the western medication group [ ] . another study have shown that moxibustion with grain-size cone at zúsānlĭ (足三里 st ) has a positive regulatory effect on the cellular immune function of elderly patients in bed. compared with the group without moxibusiton of grain-size cone, the levels of cd + and cd + and the ratio of cd + /cd + are increased after treatment in the group of moxibustion with grain-size cone and the level of cd + decreased obviously [ ] . complement system is an important part of non-specific immunity, which is an important part of fighting against pathogen infection and participates in the specific immune response of the body. the active substances synthesized after complement activation have the functions of regulating and mediating inflammation, eliminating immune complex, reducing immune pathological injury, etc. moxibustion on governor vessel effectively improves the biased state of constitution of people with yang deficiency constitution and significantly increases the levels of c and c in serum complement. hence, c and c may be the targets of moxibustion on governor vessel in regulating human immune level [ ] . for the patients with digestive malignant tumor and receiving chemotherapy, herbal moxibustion at shénquè (神阙 cv ) significantly increases the levels of nk cells, cd + cells, cd + cells and cd + cells and improves the immune function and the quality of life [ ] . moxibustion also improves the survival status in the tumor bearing mice of gastric cancer [ ] . moxibustion based on the solar term effectively improves the sub-health state of yang deficiency constitution and increases the levels of immunoglobulins, such as immunoglobulin m (igm), immunoglobulin a (iga) and immunoglobulin g (igg) [ ] . from the perspective of pathophysiological mechanism of modern medicine, the specific mechanism is unknown on inflammatory storm caused by novel coronaviral infection. however, a previous study has shown that viral infection may trigger a large amount of cytokine secretion through activating transcription factors such as nuclear factor kappa-b (nf-κb), activator protein- (ap ) and activating transcription factor (atf ) [ ] . it is speculated that after novel coronaviral infection, immune cells are activated, tumor necrosis factor-α (tnf-α), interleukin (il)- , interferon and chemokines are released, a large number of mediated immune cells are aggregated and infiltrated to the lung tissue. simultaneously, the intracellular signal transduction pathway is activated, the waterfall inflammatory cascade reaction starts and a large number of cytokines are released. additionally, much more inflammatory cells are activated constantly and a vicious cycle is formed. eventually, cytokine storm results [ ] . in one research, the expressions of inflammatory immune response cytokines are detected in the plasma of patients with covid- . it is shown that the plasma concentrations of il b, il ra, il , il , il , il , basic fibroblast growth factor (basic fgf), granulocyte colony stimulating factor (gcsf), granulocyte-macrophage colony-stimulating factor (gmcsf), interferon gamma (ifnγ), interferon-inducible protein (ip ), monocyte chemoattractant protein (mcp ), macrophage inflammatory protein- a ( mip a), macrophage inflammatory protein- b (mip b), platelet derived growth factor (pdgf), tnf-α and vascular endothelial growth factor (vegf) in the patients are all significantly higher than those in healthy adults. for the patients in intensive care unit (icu), the plasma concentrations of il , il , il , gcsf, ip , mcp , mip a and tnf-α are higher significantly than those in the patients not in icu. it is suggested that inflammatory storm is closely related to disease severity [ ] . moxibustion plays an anti-inflammatory role by regulating the level of inflammation-related cytokines. for example, in the model mice with viral pneumonia, moxibustion at "fèishū(肺俞 bl )" can effectively control the inflammatory edema in the lungs and reduces the pulmonary indexes by regulating the relevant inflammatory factors, tnf-α and il [ ] . mild moxibustion with moxa stick can significantly decrease the mortality of mice with staphylococcus aureus infection, reduce bacterial infection and alleviate inflammatory damage, displaying its protective role. moxibustion exerted after bacteria devour by macrophages, can significantly improve the bactericidal activity of macrophages, with significant anti-bacterial infection and anti-inflammatory effects [ ] . by reducing the level of peripheral and central pro-inflammatory factor, il- and increasing the level of anti-inflammatory factor, il- and the ratio of il- /il- , moxibustion alleviates the peripheral and central inflammatory responses and relieves the secretion imbalance of pro-inflammatory factors and anti-inflammatory factors so as to reduce peripheral and central inflammatory responses in the rats with exercise fatigue [ ] . in treatment of rheumatoid arthritis, with moxibustion combined, the anti-inflammatory effect of western medication is obviously improved. treated with moxibustion in combination, the levels of hypoxia inducible factor- α (hif- α), vegf, nik and nf-κb in serum are reduced significantly in the patients and the concentration of transforming growth factor-β (tgf-β ) is increased significantly. additionally, visual analogue scale (vas) score, tenderness index, swelling index, erythrocyte sedimentation rate (esr), c-reactive protein (crp) and il- β are better improved as compared with the control group with western medication [ ] [ ] . in the treatment with moxibustion and infrared irradiation for community-acquired pneumonia, the results of purulent sputum disappearance time, fever relief time, rale absorption time, inflammation absorption time in chest x-ray test, the time length of hospital stay and the changes in serum inflammatory indexes, as well as immune function indexes are all better than those in the control group with western medication, indicating a better therapeutic effect and the improvement of immune functions in the patients [ ] . with the increasing cases of cured patients with covid- , for some patients in the recovery stage, the results of viral nucleic acid test have turned negative, but the patients still have fatigue, cough, poor mental state, etc. in particular, the changes in chest ct scanning are not coincident with the clinical symptoms, meaning that there is still unabsorbed inflammation in the lungs when the patients are discharged from the hospital [ ] . however, compared with coronaviral infectious atypical pneumonia, the incidence of diffuse interstitial pulmonary fibrosis is relatively high in the sequelae after treatment [ ] . although it has not been clinically verified whether covid- will lead to the similar sequelae as atypical pneumonia, pulmonary fibrosis may be a high-risk sequelae due to the lesions involved in the lungs [ ] . cess of pulmonary fibrosis. moxibustion at "bl " and "gāohuāngshū (膏肓俞 bl ) " can inhibit the pulmonary fibrosis process in the rats of pulmonary fibrosis induced by bleomycin a (blma ) and its mechanism may be related to the increase of the expression of e-cad gene in pulmonary epithelial cells, the decrease of the levels of α-smooth muscle actin (α-sma) and vimentin and the decrease of alveolitis degree [ ] [ ] . in terms of lung function improvement, the isolated moxibustion at cv combined with qiangli zhikening capsule effectively improves the lung function and the quality of life in the patients with chronic bronchitis [ ] . yang-supplementing fire moxibustion improves the lung function, delays the progressive decline of lung function, obviously improves the body constitution in the chronic obstructive pulmonary diseases (copd) patients in stable phase with yang deficiency, increases the quality of life and effectively improves the comprehensive effect of treatment in the patients with chronic obstructive pulmonary disease in stable stage [ ] . in history, moxibustion has been applied for many times to prevent and treat infectious diseases. epidemic cholera was introduced into the lingnan region of china in . and plague was introduced in . both of these two diseases caused high fatality rates before . the medical masters of the lingnan region believe that epidemic cholera is a yin and cold disease and the ginger-isolated moxibustion is applicable. plague refers to "heat and toxin in blood and obstruction due to blood stagnation", hence, the garlic-isolated moxibustion is applicable [ ] . at the end of , without hindering the management with western medicine (wm), meisheng zhou adopted moxibustion and fire needling therapy at the tender points on the back, dàzhuī (大椎 gv ), sānyīnjiāo (三阴交 sp ), jùquē (巨阙 cv ), zhìyáng (至阳 gv ), etc. in patients with epidemic hemorrhagic fever, with the effective rate of . % [ ] . the studies have further found that moxibustion shortens the duration of scanty urine and urinary retention, promotes the conversion of urine protein to be negative, reduces the content of urea nitrogen and protects renal function to some extent in the patients with renal insufficiency of epidemic hemorrhagic fever [ ] . a study has reported that the lamp-fire moxibustion is used in the treatment of infectious condyloma acuminatum by the direct cauterization at the foci. the total effective rate is % [ ] . this therapy is firstly found in wŭshíèr bìngfāng (《五十二病方》formulas for fifty-two diseases), unearthed from the han tomb in mawangdui, changsha. besides, this moxibustion method is often used in treatment of mumps. in a retrospective analysis on the medical cases of hepatitis b treated by xiliang xie, the contemporary master of moxibustion, with moxibustion of grain-size cone during -year clinical practice, moxibustion of grain-size cone is exerted at gānshū (肝俞 bl ) and píshū (脾俞 bl ), with shēnzhù (身柱 gv ) combined in children and zúsānlĭ (足三里 st ) in adults. the improvement rate of clinical symptoms and physical signs is %. hepatomegaly, splenomegaly, liver cirrhosis and ascites are relieved definitely. the negative conversion rate of hepatitis b surface antigen is . % and the conversion rates of e antigen and core antibody are . % and . % respectively [ ] . for pulmonary infectious diseases, the garlic-isolated moxibustion is used in treatment of pulmonary tuberculosis and the effective rate is up to %. besides, this therapy can rectify the impaired cellular immune function [ ] . the latest research results show that the genetic sequences of covid- and severe acute respiratory syndrome-cov (sars-cov) are similar by . % [ ] . in the process of diagnosis and treatment of sars, patients with sars in recovery stage had been treated in guang'anmen hospital, china academy of chinese medical sciences. in treatment, moxibustion is applied to gv , bl and st , combined with the medication of wm and tcm. after treatment, the symptoms are all relieved, such as low fever, chest oppression, fatigue, headache and general soreness, distending pain in the chest and the abdomen, poor appetite and constipation. moreover, the percentage of cd + is increased as compared with that before treatment, suggesting that moxibustion can enhance the partial immune function in sars patients [ ] . in the fight against covid- , patients with covid- of common type were treated in the affiliated hospital of jiangxi university of traditional chinese medicine, fusheng branch (jiangxi heat-sensitive moxibustion hospital) and its assisted qichun county people's hospital, hubei province. the heat-sensitive moxibustion is applied at cv and tiānshū (天枢 st ). each moxibustion lasts for min to min till the heat sensation penetrates to the deep and distal areas, as well as the patient feels feverish sensation in the body and sweating on the forehead. the treatment is given once daily. this therapy effectively alleviates the negative emotions of patients and relieves the symptoms such as chest oppression, poor appetite, etc. [ ] . according to the transmission and pathogenic characteristics of covid- , professor chen wang, the academician of chinese academy of engineering, once proposed that it is possible that this virus may be transferred to be chronic and existed for a long term. it means that the prevention, control, diagnosis and treatment of covid- will likely be sustainable. acupuncture-moxibustion plays a regulatory role in the respiratory system and systemic immune inflammatory response [ ] . hence, it is quite necessary for us to fully display the effect of acupuncture-moxibustion in improving the body immunity, in which, moxibustion is undoubtedly a non-invasive, convenient and effective approach. on tcm diagnosis and treatment program of coronavirus disease in hunan province by national tcm master xiong jibo discussion on traditional chinese medicine prevention and treatment strategies of coronavirus disease (covid- ) from the perspective of "cold-dampness pestilence the role of immunological testing and intervention in reproductive medicine: a fertile collaboration? analysis of novel coronavirus pneumonia related blood test indexes in cases clinical characteristics and laboratory results of patients with fever and cough therapeutic effect of ginger warming and moxibustion on children's cough variant asthma and its influence to on immune function regulation effect of moxibustion with small moxa-cone about the size of a wheat grain at "zusanli" point on cellular immune function in elderly bedridden patients clinical study on the effect of du-moxibustion therapy on the symptom and levels of serum supplement c and c in yang-deficiency subjects the effect of moxibustion shenque point on immune function of digestive tract cancer patients undergoing chemotherapy effect of moxibustion on survival status and nutritional metabolic factor in tumor-bearing rats with gastric cancer effect of solar-term moxibustion on hemorheology and immune function in patients with subhealth status of yang deficiency molecular pathways in virus-induced cytokine production therapeutic strategies for covid- based on its pathophysiological mechanisms clinical features of patients infected with novel coronavirus in wuhan, china effect of electroacupuncture stimulation of "feishu" (bl ) on lung index, serum and lung il- and and tnf-α levels in mice with viral pneumonia effect of mild moxibustion on bacterial infection and inflammation in mice and related autophagy mechanism effect of moxibustion on body weight and peripheral and cerebral cortical il- and il- levels in fatigue rats mechanism study on nik/nf-κb/vegf pathway, anti-inflammation and analgesia in ra treated with moxibustion effect of moxibustion on tgf-β , hif- α and vegf levels in patients with rheumatoid arthritis (ra) effects of moxa-moxibustion plus infrared illumination on community acquired pneumonia and immune function thinking of the effect of integrated chinese and western medicine on covid- dynamic changes of serum sars-coronavirus igg, pulmonary function and radiography in patients recovering from sars after hospital discharge feasibility of yiqi xuanbi decoction in treating pulmonary fibrosis after new crown pneumonia effects of moxibustion of feishu (bl ) and gaohuangshu (bl ) on expression of e-cad gene in lung epithelial cells in blma -induced pulmonary fibrosis rats regulation of e-cad, α-sma and vimentin through moxibustion at feishu and gaohuangshu of pulmonary fibrosis rats combined with qiangli zhike capsule (强力止咳宁胶囊) on chronic bronchitis clinical observation of yang-supplementing fire moxibustion for chronic obstructive pulmonary disease in stable stage contribution of physicians in the south of the five ridges in acupuncture and moxibustion treatment of plague and cholera clinical observation of moxibustion in the treatment of cases of epidemic hemorrhagic fever observation on the effect of moxibustion on renal function of epidemic hemorrhagic fever cases of condyloma acuminatum treated with moxibustion retrospective analysis of mr. xie xiliang's medical records accumulated in years on direct moxibustion for treating hepatitis b clinical observation of treating refractory pulmonary tuberculosis with garlic moxibustion a pneumonia outbreak associated with a new coronavirus of probable bat origin nine cases of the chronic stage of sars treated by moxibustion clinical observation of heat-sensitive moxibustion treatment for coronavirus disease discussion on the effect pathways of preventing and treating coronavirus disease by acupuncture and moxibustion from the regulation of immune inflammatory response key: cord- - q n t authors: nan title: cumulative pharmacological activity index volumes - date: - - journal: studies in natural products chemistry doi: . /s - ( ) - sha: doc_id: cord_uid: q n t publisher summary this chapter lists the important subjects on pharmacological activity that are discussed in the publication studies in natural products chemistry, volumes – , such as abdominal constriction test, acanthoic acid, acetaminophen, parkinson's disease, photodynamic activity, prostaglandins, and oleanolic acid. the terms are mentioned along with the page numbers in which they are discussed in the publication. use of psycotria colorata in : ( --+ )-abeo- c~, (s)-epoxylabda- ( ), -diene : activity in ebv bioassay system : -nor-abieta- ( ), , , -tetraen- one : activity in ebv assay system : abieta- , , -trien- -one : activity in ebv assay system : -nor- , a, triol : activity in ebv assay system : , , activity in ebv assay system : abieta- , , -triene- o~,l , -triol acetate : activity in ebv assay system : abietic acid : activity in tpa assay system : activity in crg (+)- -epi-acetomycin : antitumor activity of : (x-acetoxy- -hydroxy- -iso-valeroyloxy- -oxo-costunolide : activity in nfkb assay system : activity in tnf(x assay system : cx-acetoxy- ~, -epoxymiller- ( )zenolide : activity in nfkb assay system : ~-acetoxy- -isobutyryloxy-reynosin : activity in mam- assay system : activity in dif assay system : -acetoxy- ~-hydroxy- [ -methacryloyloxy- -oxo-acanthospermolide : activity in nfkb assay system : -acetoxy- cx-methacryloyloxy- ~hydroxy- -oxo-acantho-spermolide : activity in nfkb assay system : -acetoxycostunolide : activity in nfkb assay system : -acetoxy-eremantholide b : activity in nfkb assay system : activity in tnf~ assay system : ~-acetoxy-miller- ( ) : , , development of : , use of : analgesic effects : , , , , , ; : , : , ; : , ; : ; : ; : ; : , ; : , , , , , , , , , , , , , , , ; : , , , , , , , , , , , , ; : , , ; : , ; : , ; : : ; : ; : ; : , , , , , ; : ; : - : , , , , , , , , , , , - , , , , , , , , , , - ; : ; : , , , , , , , , , , ; : , , , ; : ; , , , ; : , , against candida albicans : , from hypericum roeperanum : of (e)- ( ), - abdadiene- , -dial : of (e)- b, -epoxylabd- -ene- , -deol : of (e)- [ , -epoxylabol- -ene- , - : , ; : , , , , , , ; : , , , , ; : , ; : , , : ; : , ; : ; : , , , , , , , , , , ; : , , , , ; : , , , , ; : ; : , , , , , , , , , , , , , , , , , ; : , ; : , , ; , ; : , , , , , , : , ; : , , , , , , , , , , , , , , -microbial activity : ; : ; : - ; : - ; : , , , , , , , , , , ; : ; : , , , , , , , , , , , ; : , , , , - , , , , , , , , , , , , , ; : , , , , ; : ; : , , , ; : : , , ; : , , , , , , ; : ; : , , , , , , , , , ; : , , , , , , , , , ; : , , , , , , : , - - ; : ; : , , , , , , , ; : ; : , , , , , , , , , ; : - ; : , , , ; : , , , , - ; : , , ; : ; : , , , , , ; : , , , , , , , , , , , , , , , : , ; : , , , ; : ; : , , , , , , , , , ; : , ; : , , , , , , , , ; : ; : , , , , , , , ; : ; : , ; : , - , , , , , - , , - , , , , , , , , , , , , , , , , , , , , , , , , , ; : , , , , , , , - , , , , , , , - , - , , , - ; ; : , , ; : , , , , , , , , ; : - ; : - , , ; : , , , , ; : ; : , , , , , , , , , , , , , , , , , , , , , , , , acaricidal enzymatic activity : - tx-cembra- , , -triene- , -diol : activity in ebv assay system : activity in pkc assay system : activity in skin- assay system : activity in odc assay system : ~-cembra- , , -triene- , -diol : activity in ebv assay system : activity in skin- assay system : activity in odc assay system : of (-)-deoxypodophyllotoxin : of (-)-hemone : of (-)-nymphone : of (-)-yatein : of (+)-corytuberine : of (+)-epiaschantin : of (+)-epimagnolin : of (+)-epiyangambin : of (+)-hernovine : of (+)-laurotetanine : of (+)-magnoflorine : of (+)-malekulatine : of (+)-n-formyldehydroovigerine : of (+)-n-formylnornantenine : of (+)-n-formylovigerine : of (+)-n-hydroxyhemangerine : of (+)-n-methylhemangerine : of (+)-ovigerine : of (+)-ovihernangerine : of (+)-vateamine- ' [ -n- , , , , , , , , , , , , , , , , , , , , , , , , , , ; : , , , , , , , , , , , , , , , , , , ; : , , , ; : - , as anti-fungal agents : [ , -epoxy- , -dihydroxy- c~ange lo y-lo xy- [ -is obutylo xygermacran- ot, -olide : activity in inos assay system : activity in nfkb assay system : a, c~-ep oxy- or-hydro xyabieta- ( ), -dien- -one : activity in ebv assay system : , or-epoxy- , -seco-abieta- -o-phenyl- -propionyl-neu ac-cx me : -t-butyl-dimethyl-silyl ether : -azido- -deamino-neu ac-c~ me : -n-benzyloxycarbonyl-neu-cx me : -n-propionyl-neu-c~ me : -deoxy-neu ac-c~ me : , -amino- -deoxy-neu ac : , -azido- -deoxy-neu ac : , -tosyl-neu acme ester : , -amino- -deoxy-neu ac : , -amino- -deoxy-neu ac-c~ me : -azido- -deoxy-neu ac : , -azido- -deoxy-neu ac-c~ me : -o-acetyl-neu ac-( , activity of (-)-deoxypodophyllotoxin in : activity of (-)-hemone in : activity of (-)-nymphone in : activity of (-)-yatein in : activity of (+)-corytuberine in : activity of (+)-epiaschantin in : activity of (+)-epimagnolin in : activity of (+)-epiyangambin in : activity of (+)-hernovine in : activity of (+)-laurotetanine in : activity of (+)-magnoflorine in : activity of (+)-malekulatine in : activity of (+)-n-activity of hydroxyhernangerine in : activity of (+)-n-formylnornantenine in : activity of (+)-n-formyldehydroovigerine in : activity of (+)-n-formylovigerine in : activity of (+)-ovigerine in : activity of (+)-ovihernangerine in : activity of (+)-vateamine- ' -noxide in : activity of -formyldehydrohernangerine in : activity of -formyldehydronornantenine in : activity of -formyldehydroovigerine in : activity of -hydroxy- -methoxy- -hydroxy - , -seco-abieta- , , trien- , -dial : activity in ebv assay system : -hydroxy- -methoxy- -methyliso- : , , , , , , , , - , , achyranthes aspera l , , , , , ; : , ; : - , , ; : , - , - , - ; : , , , ; : , , , , chemo-differentiation therapy : of acute monocytic leukemia chemopreventive activity : , , in mouse mammary organ culture model : in vitro : in vitro bioassays for : of chlorella vulgaris : ofisoquinoline alkaloids : of lignans : chemopreventive agent : genkwanin as chemopreventive efficacy : of limonene carcinogenesis : chemoprophylaxis : chemosensitizers : antimalarial drugs as : chemotaxonomic markers : chemotherapeutic agent : , , atovaquone : for colon cancer : for multi-drug resistant cancers : for ovarian cancer for taxol-resistant breast cancer chemotherapeutic intervention : targets for chemotherapy drug : antitumor activity of : chest pain : use of ginseng root in : childhood disintegrative disorder (cdd) gram-negative bacteria : maytenus species against gram-positive bacteria : ; : , actamycin against : antimicrobial activity of : inhibitor maytenus species against grandiflorolic acid : activity in ebv bioassay system grandiflorolic acid angelate : activity in ebv bioassay system grandisin : trypanosomicidal activity of grandmal seizures granulocyte phagocytosis : , ofjenisseensosides c : , ofjenisseensosides d green tea : , biological activity of effect of general analogues on pgf na-induced contraction griseofulvin : as anti-fungal agents grob-type fragmentation : , growth : of tumor cells : growth inhibition : growth inhibitor activity : guaiazulene : , as an anti-inflammatory agent guidimacrin : antitumor mechanism neurodegeneration : reperfusion injuries : respiratory disorders : role of free radicals in : human growth hormone human hek cell line : biological response of human hl- leukemia cells : differentiation of : inhibition of : proliferation of human leukemia cells : human leukocyte elastase : inhibitor human leukocyte elastase (hle) : , , homologous sequences of : plasminogen activator (u-pa) : human lung carcinoma : ; : discodermolide against human lung carcinoma) : phenolic triterpenes against : human medulloblastoma : human melanoma : human mesangial cell proliferation : in vitro human monoblastic leukemia cells : effect of hydroxyurea (hu) : growth inhibition of : human monoblastic leukemia u cells : growth inhibition of human neuroblastoma nbla-n- cells tamoxifen activity against : human neuroblastoma sh-sy- y cells : human neutrophils : human neutropil protein kinase c : human onchocerciasis : ivermectin for human papilloma virus : pathogenesis of : cause of hepatocellular carcinoma human papilloma virus (hpv) : human papilloma virus type : microbicidal compound against human papilloma virus type : microbicidal compound against : human platelets : study of protein kinase c in : by staurosporine : human rotavirus (hrv) : , cause of dehydrating gastroenteritis : member of : reoviridae type of human serotonin transporter gene (slc a ) : regulation of neuronal activity human spermatozoa motility : human synovial pla : inhibitor anti-glycemic effects : - anti-hyperglycemic effects of -o-methyl- -hydroxyecdysone activity in : pinnatasterone activity in : polypodine b -o-cinnamate activity in muscarinic achr antagonists : muscarinic antagonist : muscarinic receptor : muscarinic receptor antagonists : muscarinic-l, -receptor : muscle pain : aconitine for relieving activation effects of : medicinal uses of : protein kinase c activation effects : muscular p- (murine lymphocytic leukemia) : activity of (-)-deoxypodophyllotoxin against : activity of (-)-yatein against p- lymphocytic leukemia cell : p mouse leukemia : staurosperine activity against p- murine leukemia cells : pachyman : , antitumor activity of : pachyrrhizus erosus : , - anti-feeding activity of : pachyrrhizus palmatilobis : biological activity of : paclitaxel (+)-acetoxypinoresinol dimethyl ether as : arctigenin as : arctigenin methyl ether as : chamigrenal as : cinnamophilin as : dehydroschisandrol a as : denudatin as fptase inhibition by : paw oederma : inhibition of : -pectenotoxin : cytoxic activity of : pectinasic pectolinaringenin : , antispasmodic activity of : pedunculariside : activity in cox assay system : activity in epp assay system pelargonidin (anthocyanidin) : effects on lela : effects on penates sp. : as ~-glucosidase inhibitor : penicillium turbatum : antibiotic a b by : penstemide : activity against p- lymphocytic leukemia penstemon rosseus : anti-fungal activity pentacyclic triterpenes : protein kinases inhibition by in lipid autoxidation , , -p enta-o-gallo yl-~ -d-g luc o s e (galloyl ester) : effects on biological activity of : -prenyleriodictyol : biological activity of : prenylflavones : anti-human immunodeficiency virus (hiv) activity of : prenylfiavonoid : antinocicieptive effect of antitumor promoting activities of : as cytomegalovirus protease inhibitors : gastro-intestinal problems : use in hodgkins disease : use in infections : use in lupus : use in osteomyelitis : use in parkinson's disease : use in psoriasis : use in respiratory problems : use in skin ulcerations : use in syphilis : -prenylnaringenin prevention by chitosan : of myelotoxicity : primary biliary cirrhosis : primary granulosa cells : apoptosis in : primary solid-tumor growth : effect of triterpenoids on : pristimarin : cytotoxic activity of prl & prl proproteins : effects on cysteine protease probes : activity of : procurcumenol : activity in tnfct assay system procyanidin b (dimeric flavan- -ol) : effects on ace procyanidin b (dimeric flavan- -ol) : effects on ace procyanidin b- , '-di-o-gallate : effects on ace procyanidin c (trimeric flavan- -ol) : effects on ace : procyanidin polymer (flavan- -ol polymer) : effects on ace prodrug monotherapy : natural anthracyclines for : prodrugs : of natural anthracyclines pro-inflammatory cytokines : pro-inflammatory substances : in chronic painful inflammatory diseases : proliferation : , effect of serotonergic neurotransmission on : serotonin action on : prolysine : prophylactic effects pro-nociceptive prostaglandins : in inflammation pro-nociceptive transmitter : release inhibition of : prooxidant activities : in thiobarbituric acid assays : of chemiluminescence : of tert-butyl hydroperoxide : prooxidant property : of carotenoids : prophylactic drugs prostaglandin e -dependent flavonoids cytoprotection effect pgh ) : prostaglandin inhibitors : clinical use of : use in rheumatism : use in osteoarthritis : use in headache : use in dental surgery anticancer clavulones : as antiviral compound : - as pain mediator : in mast cells : nociceptive effect of prostate cancer prevention : role of lycopene protease inhibitor proteins from plants : effects on metallo protease inhibitors : , cereal bifunctionals as : chymotrypsin as : destructive potential of : in alzheimer's disease : in angiogenesis : in cancer inflammatory disease : in protozoal infection : in viral infection : kunitz as : mustard family of : of metallocarboxypeptidase : ofserine protease : pepsin as : phytocystatins as : potato type : proteins as : serpin as : trypsin as : proteases : ; : enzymatic activity : in apoptosis : in blood clotting : in cell division : in digestion : in extracellular matrix digestion : in inflammatory responses : protein kinase a (pka) in regulation of ionotropic receptors protein kinase activity : ofxestoquinolide protein kinase c (pkc) activator : protein kinase c inhibition : ; : , by xestocylamine : protein kinase inhibition : by pentacyclic triterpenes protein phosphatase inhibitors : protein phosphatases : inhibitor of : protein synthesis : inhibitor of : protein transduction domain (ptd) proteinaceous receptor : proteinase activity : protein-coupled receptor : g-proteine kinase c activity : proteins protein-tyrosine kinase activity : offlavonoid aglycones : of glycosides : ofkoelreuteria henryi protein-tyrosine kinase inhibitory activities proteolytic activity : proteolytic systemin inactivation protium kleinii : anti-inflammatory effect of : antinociceptive effect of : protocatechuic acid ( , -dihydroxybenzoic acid) : effects on proton pump inhibitors (ppis) : proton-translocating nadh:q oxidoreductase : high-affinity inhibitors protozoocidal activity : , , against leishmania : against plasmodium : against tryponosoma : genus baccharis : in vitro : of neo-clerodane diterpenoids : ofquinone derivative : protozoocidal compounds : use of proxyelocytic leukaemia (hl- ) prunella vulgaris : antiviral activity of psammaplin a (bisprasin) : effects on bacillus subtilis psammaplysilla purpurea : antimicrobial activity of : as tyrosine kinase inhibitor pseudobersana mossambicensis : bioactive steroids from pseudoguaiane a, -dihydrohelenalin acetate : activity in cro assay system against cytomegalo virus : against human immunodeficiency virus- : against influenza virus : as virucidal agents : retroviral activity of : pseudolaric acid b : pseudomonas aeruginosa : antibacterial activity against : antifungal activity against pseudorabies virus : in vitro replication of : role ofheparin : psii inhibitors : , psk in mitosis : psk-receptor : psk-a activity : psoriasis : use of lc~, (oh) d : psychiatric disorders : anorexia : bipolar disorder : bulimia : effect of circadian activity on : obsessive compulsive disorder : panic disorder : schizophrenia : seasonal effective disorder : unipolar depression : psychodelic drugs : cocaine as : morphine as : semisynthetic lsd as : psychovegetative disorders : , psycotria colorata : to relieve abdominal pain puberulin a : , as paf-induced inhibitor pulegone : activity in am assay system : pulmericin : antifungal activity of : antileukemic activity of : cytotoxic activity : pulmonary vascular injury : role of serine protease in : role of thrombin pumiliotoxin alkaloids : , alio-ptx b alkaloid : , biological activity of punta toro virus : treatment of : purifying blood : herniaria hirsuta in : purine receptors putative receptor proteins : putative satiety factor : effect of cholecystokinin : pyranocoumarin : biological activity pyranoid carbasugars : as cellular messengers : as insulin release mediators : biological activities of : effects on glycosidase enzyme : effects on glycosyltransferase enzyme : galactosyltrans ferase inhibition by pyridoacridine alkaloids : cytotoxic effects on kb cells glycosidase inhibitors of : pyrrolomycin antibiotics : pytressin-induced coronary spasm antileukaemic activity of '-quercetagetin ( -hydroxy quercetin hexahydroxyflavone) (flavonol) : effects on '-pentahydroxyflavone) : ; : , analgesic effect of quercetin -o-c~-l-arabinopyranoside : anti-oxidant activity quercetin -o-c~-l-rhamnopyranoside : , anti-oxidant activity of quercetin -o-[ -d-galactopyranoside : anti-oxidant activity o-galloyl)-glucoside : effects on ace quercetin- -o-ot-l-rhamnopyranoside : anti-oxidant activity quercitrin (quercetin- -rhamnoside) : effects on ace quillaia : of adjuvant activity : quinidine : quinine : antitumor activity of : inhibitor of dna quinoline drugs : quinoline drug family quinoline-based antimalarials : quinoline-ring antimalarial drugs : quinolines : quinolizidine derivatives : , testing against mycobacterium tuberculosis - as antiviral quinone methides : antiplasmodial activity of : quinones rabidaea platyphylla : for epilepsy : rabies virus : inhibitor of : rabies virus infection : in chicken-embryo-related cells : rabies virus replication : in nerve fibers rachitic bone healing potencies of vitamin d : of vitamin d : of vitamin d : of vitamin d : of vitamin d : of vitamin d radical scavenging activity : of coumarin : radioligands : from brain membrane synaptosomes ranitidine : , ranp-triggered signal transduction raphe nuclei : role in cognitive functions ras oncogenes : rat brain synaptosomes : p-affinity rat mammary carcinogenesis model : cancer chemopreventive activity rat serum vitamin d-binding protein (dbp) : binding assay for rebaudioside a : activity in tpa bioassay system tpa bioassay system -receptor : ~-receptor : -receptor affinity : binding properties of : ~t-receptor affinity : binding properties of : of glycopeptide : -receptor agonists k-receptor agonists : , pharmacological activity of -receptor agonists : pharmacological activity of : -receptor antagonists : pharmacological activity of k-receptor antagonists : pharmacological activity of : ~t-receptor antagonists : pharmacological activity of : receptor binding : receptor binding assay : in vitro : mouse vas deferens (mvd) activity in : of guinea-pig ileum (gpi) : receptor binding properties : of tyr-d-ala-phe-[[ -d-glc(oac) ]tyr-pro-ser-nh : of tyr-d-ala-phe-asp-val-val-gly-nh (deltorphin c) : of oac) ]-gly-nh : : of tyr-d-ala-phe-gly-tyr-pro-ser-nh (deltorphin c) : of tyr-d-ala-phe-gly-tyr-pro-thr([ -d-glc)-gly-nh : of tyr-d-ala-phe-gly-tyr-pro-thr c )-val-vai-gly-nh receptor tyrosine kinase (rtk) : receptors : ; : and cell-cell adhesion : and cell-cell communication : antigenic determinants of : for bacteria : ofchiral proteins : with enzymatic activity : without enzymatic recombinant prourokinase (pro-uk) : as fibrinolytic enzymes recombinant tissue-type plasminogen activator : as fibrinolytic enzymes : red wine : biological activity of : redox enzymes redox inhibitors : of - ipoxygenase repandusic acid (hydrolysable tannin) : effects on hiv- protease : repellent properties : of amblyomma variegatum : reperfusion injuries : role of free radicals rescorcinolic lipids : - effects on enzymatic activity : - interaction with proteins reserpine : as immunosuppressive drug resistance-modifiers : antimalarial drugs as resistant plasmodium species : in chloroquine sensitivity resorcinolic lipids : , , , - as antifungal fluids : as growth reulators : - as hair restoration-lotion : used in gingival infections respiratory ailments : respiratory depression : due to analgesic alkaloid : respiratory disorder : role of free radicals in : respiratory infection : respiratory syncytial virus : respiratory toxins : , respiratory tract infections : herniaria hirsuta in : response decay mechanism : response modifiers resveratrol : biological activity of : resveratrol : effects in llc-bearing mice : effects on tumor treatment of : use of hernandia moerenhoutiana in : use of hernandia nymphaeifolia antimalarial activity of : antiplasmodial activity of : as platelet aggregation inhibitor : effect on antiplasmodial activity : retinal : ; : , activity in am bioassay system : in visual signal transduction : retinoic acid : activity in am, ebv, skin- , odc bioassay systems : retinoic acid : , , in cell differentiation : in development : in growth regulation : retinoid retinol : activity in am retinol acetate : activity in am bioassay system retinol palmitate : activity in am bioassay system : retroviral enzymes : inhibitor of causative agent of immunodeficiency syndrome : inhibitor of rett's disorder (rtt) : , , by mutations in mecp gene : genetic basis of : reutericyclin : as antibiotic reverse transcriptase : , , role in hiv replication cycle : role in viral replication rhazinilams : as microtubule poisons rheedia gardneriana : antinocicieptive effect of : rheumatism : herniaria hirsuta in : rheumatism : , use of non-steroidal antiinflammatory drugs (nsaids) : use of prostaglandin inhibitors : rheumatoid arteriosclerosis : rheumatoid arthritis : ; : ; : , ; : , , in treatment of cph : in rats : reagent-induced : treatment of : use of kalopanax pictus rhoifolin ( , '-dihydroxy - -rhamnosyl-glucosyl-flavone) : effects on hiv- protease : rhombenone : fptase inhibition by ricinoleic acid : analgesic action of : rickettsial pathogens : cowdria ruminantium as rifamycin-type macrolides : , antiviral microbial-derived compound rift valley fever infection : treatment of rimantidine : for treatment of influenza in infections activity of deltorphin b in : of glycopeptide : rna virus : arenavirus type of : bunyavirus type of : coronavirus : influenza virus : measles virus : parainfluenza virus : picornavirus type of : rabies virus : reovirus type of : rotavirus type of rna-dependent rna polymerase activity : virion-associated rna-directed dna-polymerases : inhibition by sf- cell line : activity in mam- assay system : antileukemic activity : sarcoptes mites : cause of human scabies : symptoms sarcoptes scabiei : cause of mange mites : cause of scabies sargasm horneri : , anti-cmv activity of : polysaccharide (ps) from : sarkomycin : as antitumor agent : satratoxins : antiviral activity of scavenging effects : of tannic acid : sch : fptase inhibition by : schisandra : display platelet activating factor antagonist activity : effect on cardiovascular system : effect on heart rate : for treatment of hepatitis schisandrol a : as paf-induced inhibitor schistosomiasis (bilharzia) : schistosomicidal activity : schizonticidal drug : schizophrenia activity in crg bioassay system : from scoparia dulcis scropolioside a : activity in tpa assay system : scrovalentinoside : activity in tpa assay system scutellaria baicalensis : , , , , in allergric inflammatory disease sdb : inhibitory effect of : sdc : inhibitory effect of : sdz- - : antinociceptive activity of : anti-hyperalgesic activity of : seasonal affective disorder : effect of circadian activity on : seaweeds : anti-cmv activity of activity in ebv assay system : , -secoemetine derivatives : amoebicidal activity of : secoiridoids : antimicrobial activity of : second messenger : secondary metabolites : , , acaricidal activity of : biological activity of : secondary metastatic tumor growth : effect of triterpenoids on arnica as : artocarpus heterophyllus as basilicum polystachyon for : sedative activity : sedative in convulsions : crocus sativus l. for : sedative in epilepsy : withania somnifera l. as : g-selective agonist : dermorphin : -selective antagonist tyr-tic-phe-nh selective antitumor activity : selective cytotoxicity : -selective opioid agonist : deltorphin b : g-selective opioid peptide : -selective opioid peptide of ici : ofk opioids : ,g-selectivity : in vivo : of glyco analogues semilicoisoflavone b : biological activity of : semiochemicals semisynthetic lsd : as psychodelic drug -dihydro xy- -methoxy-naphtho sendai virus infection sensory neurons : hyperpolarisation of : er inhibitors serine : activitiy of serine : c~ -macroglobulin inhibition by serine protease inhibitor proteins : arrowhead pis api-a & api-b as cp-thionin as : effects on barley malt cysteine endoproteinases : effects on chymotrypsin : effects on subtilisin : effects on subtilisin eleusine double-headed try-~x-amylase inhibitor i- as : from brassica nigra : from brassica napus : from cassia fistula : from eleusine coracana : from hordeum vulgare : from phaseolus angularis from sagittaria sagittifolia : from sinapis arvensis : from vigna unguiculata : hordeum lipid transfer proteins as blood clotting factors as : cathepsin g as : chymase as : chymotrypsin as : granzymes as : in angiogenesis : in blood clotting : in cytosolic proteolysis : in digestion : in inflammation : in proprotein processing : in tissue remodelling : kallikrein as : plasmin as : prolyl endopeptidases as : role in blood coagulation : role in platelet activation : role in pulmonary vascular injury serine proteases (subtilases) : cysteine proteinase inhibitor : poly phenol oxidase : serine proteinase inhibitor serine proteinase inhibitor i : as defense proteins serine-threonine-specific receptor protein kinases serotonergic innervations : autoradiographic imaging : in cerebral cortex : in neonatal rats by immunohistochemical techniques : physiological role of serotonergic neurons : , in raphe nuclei : role in :regulation of neurogenesis as hyperforin inhibitor : as neurotransmitter : effect on synaptogenesis : in apoptosis : in cell proliferation : in presynaptic neuron : morphogenic properties of : neurochemical connection of : receptor activity of -ht) receptors : , , , in brain : in limbic brain regions : pharmacological aspects of serotonin ( -ht) re-uptake inhibitors : mechanism of serotonin ( -ht) synthesis : , , in autistic children serotonin ( -ht) ar receptor antagonists : in posmatal treatment -ht)sa receptor : in brain development : in phenotypic behavioral irregularities : in purkinje cells : mrna expression of : serotonin action : on apoptosis : on maturation : on neuronal functioning : on proliferation serotonin agonists : anticomplementary activity of : role of cyclic amp-dependent protein kinase : delayed-type allergy suppressant activity of serotonin synthesis inhibitor : parachlorophenylalanine as serum tc : biological activity of : serum transaminase : , , biological activity of : serum transaminase activity : of curcumia longa l serum triglyceride : biological activity sesquiterpene lactone : sesquiterpene quinones : antimicrobial activity of : cytotoxic activity of sesquiterpenoids drimane-type synthesis : avian myeloblastosis virus (amv) inhibition by by jauch : sex pheromone sexual potency : ofbroussoflavonol g : sgot : activities of : sgpt : activities of : shikimic acid derivatives : antitumoral activity of : shinpterocarpin : biological activity of : sialic acids : biological function of : model for sialyltransferase activity : side effect : , of analgesic alkaloid : of opioids sigmoidin a : biological activity sigmoidin b : biological activity of : signal reception : in autistic children : signal transduction mechanisms : signal transduction pathways signal transductors/activators of transcription (stats) : silandrin : as antihepatotoxic agent : silchristin : as antihepatotoxic agent : sildenafil : used for erectile dysfunction silymonin : as antihepatotoxic agent : simocyclinon : antibiotic activity of : cytostatic effects of : sindbis virus : sinomenine : , hepatoprotective effect glycoside) : effects on pep : skin cancer : skin diseases : skin inflammation : anthrones used for : skk moth : - juvenile hormone from slaframine : biological activity slaframine alkaloids : , as muscarinic agonist : sleeping sickness smenoquinone : , antimicrobial activity of smooth muscle relaxation : - as biological action : of kampo medicines snake bite : treatment of : use of crossopetalum gaumeri : sobrerol : activity in mam- assay system : activity in gst assay system : activity in ras assay system : social phobia sodium salt of caffeic acid tetramer : , anti-hiv activity of : soilborne fungi : bioactive metabolites from soilborne phytopathogens : biological control of : solandelactones : fptase inhibition by solanidane-induced teratogenicity : solid tumors neovasularization : somatostain : as a~ adenosine agonists somatostatin receptor : ; : ; : type of g-protein-linked receptor : sonodione : from hernandia sonora : sores : tricyclic acylphloroglucinols sorivudine : , as anti-viral agent sorocein f : biological activity of : sos chromotest : soyabean saponins : , hypocholesterdemic effects soyasaponins : anti-obesity action of : biological activity of : soybean trypsin inhibitor (sbti) spasmolytic activity : ; : , ; : , , of hernandia moerenhoutiana : ofhernandia voyronii : of imperatorin : , of plant polyphenols : , of thymus satureioides : on guinea pig ileum : on smooth muscles specionin : antifeedant activity of : spermicidal activity : of gypsophila paniculata sphingolipid receptor ca + channels : sphingolipid receptors : sphinxolide : ; : antitumor activity of : spider mite : hatching inhibitor spiroalkyl analogs : biological activity spirulina platensis : , , anti-influenza activity of : biological activity of : calcium spirulan from : polysaccharide (ps) from spleen/adipose tissue weight : , , effect of fucoidan on : , effect of oleic acid on : spontaneous apoptosis : spontaneous metastasis squarroside a : , immuno-modulatory effect of : from vaccaria segetalis squash family serine protease inhibitors : bd-ti-ii as : cmti-i as : cmti-iii as : cmti-iv as : cpgti-i as : cpti-ii as factor xiia, kallikrein, trypsin : effects on lysyl endopeptidase : effects on trypsin : effects on trypsin : effects on trypsin : effects on xa, xiia, kallikrein, plasmin, trypsin : elti-i as : from bryonia dioica : from citrullus vulgaris : from cucumis melo : from cucumis melo : from cucumis sativus : from cucurbita maxima : from cucurbita maxima : from cucurbita pepo : from cucurbita pepo : from ecballium elaterium : from echinocystis lobata : from lagenaria leucantha : from luffa acutangula : from luffa cylindrica cucurbitaceae) : from tricosanthes : hmti-i as : lati as : lati-ii as : lldti-i as : lldti-ii as : mcei-i as stachenone : activity in ebv bioassay system : standishinal : activity in ebv assay system : standishinal diacetate : activity in ebv assay system staphyllococcus aureus : minimal inhibitory concentration (mic) of staphyllococcus epidermidis : minimal inhibitory concentration (mic) of staphyllococcus saprophyticus : minimal inhibitory concentration (mic) of staphyllococcus warnieri : minimal inhibitory concentration (mic) of staphylococcus aureus : , antibacterial activity against : antibacterial properties of : antifungal activity against starfish fertilisation inhibitor : callyspongin b as : stavudin : , , as anti-viral agent : steganes : , cytotoxicity of : , tubulin assembly inhibition by : antitubulin activity of : , microtubules assembly inhibition by sterculia urens roxb. : antiviral activity of : steroid hormones : steroid saponins : teratogenic metabolites of : - toxic cardiac active steroids sterol biosynthesis : inhibitor of : a-sterol sulfate : cytotoxicity against sterol sulfates haplosamate a : as hiv- integrase inhibitor : sterols : antitumor activity of : antiinflammatory activity of : stevia stevia extract : , , for diabetics stevia leaves : immunological activity stevia plant : allergenic activity steviol : , , , acute toxicity tests of : effect on fertility : mutagenicity tests with tpa bioassaysystem : activity in skin- bioassay system : acute toxicity tests of : allergenicity problems of : chronic toxicity studies of : , , effect on fertility : , for phenylketonuria : mutagenicity tests for stigmasterol : , , , analgesic effect of stilbene derivatives : , ; : biological activity of : , in llc-bearing mice : tumor growth inhibition by : stimulatory : biological activity : stimulant agent : ofteucrium sp stimulus-response mechanisms : streptococci : pathogenicity of : serological groups streptokinase/streptodomase : streptolygidin : antibiotic activity of : as bacterial rna polymerase :inhibitor as terminal dna transferase striga asiatica : infection with ofbioactive metabolites : of scopadulan-type diterpenoids structure activity relationship studies (sar) : , , of anandamide : of aspirin : ofopioid analogues : of vitamin d analogues : structure dereplication : bioactive natural product database in : of melanin inhibition strychnine : as immunosuppressive drug : strychnopentamine : anticancer activity strychnos bisindole : anticancer activity strychnos monoindole : anticancer/protozoal subacute toxicity : from animals : of stevioside effects on furin : sudhl- - ymphoma : sugar cane cystatins : effects on cysteine proteases : suicide inhibition : of '-hydroxylase : suicide inhibitors sulfatobastadin : as endothelin a receptor inhibitor sulfide-containing pyrroloiminoquinones : as antileukaemic agents sulfur-containing cyclic peptides : biological activities of : cytotoxicity of sulphated polysaccharide (ps) : , , anti-cmv activity of : anti-hiv activity of : antiviral activity against hiv : effect on viral replication : from marine sources : inhibitory effect of : mechanisms of action sulphated polysaccharide (ps) extracts : inhibitory activity of sulphonylurea drugs : sunflower cystatin phytocystatin : effects on cathepsin h ficin : effects on papain : sunflower multicystatin : effects on papain supercritical fluid extraction : superoxide dismutase superoxide dismutase activity : suppressing activity : on cells proliferation zizyphus saponin i as : zizyphus saponin ii sweroside : , serum alt activity of symbiotic marine microorganisms : bioactive metabolites : sympathetic ganglia : blockade of : sympathetic nerve activity : sympathomimetic amines : synapic potential mediation : by non-nmda receptors : synaptic actions : of serotonin synaptic dopamine transporters : synaptic glycine transporters : synaptic serotonin transporters : synaptogenesis : , effect of serotonergic neurotransmission on : role in rodents : role of serotonin in : syndrome type-la : adipose tissue distribution as : cerebellar dysfunction as : liver insufficiency as : peripheral neuropathy as : psychomotor retardation as synergistic activity : synergistic effect : , ; : , ofhervelines b : ofhervelines c : of retinoids : of vitamin d derivatives : synergistic interaction : of acyclovir(acv) : ofganciclovir (gcv) : with ganciclovir synergistic purgative actions : of rheinanthrone : of aloe-emodin-anthrone : synthetic pharmacological agents : antioxidant activity of : synthetic podolactones : allelopathic activity synthetic psychotropic agents : syringaresinol : anti-platelet aggregation activity of systemic wound response protein : systemin activity : assay for ]-val-val-gly-nh : receptor binding properties of receptor binding properties tyr-pro-ser-nh (deltorphin c) : receptor binding properties of -d-glc)-gly-nh : receptor binding properties of oac) ]-gly-nh : receptor binding properties of -d-glc)-tyr-pro-ser-nh : receptor binding properties of -d-g lc )-v a -v al-gly-nh : receptor binding properties of tyrosinase inhibitory activity : tyrosine kinase tyrosine-specific kinase : inhibitor of tyr-tic-nh : activity in functional bioassay tyr-tic-phe-nh : as selective antagonist : u myeloma cell : ucn- -ucn- : antitumor activity of : protein kinase inhibitor ulapualides a : antifungal activity unsaturated carbapyranoses : as enzyme inhibitors : as herbicidal : biological properties of : unsaturated ketonucleosides : tumor inhibition by u-plasminogen activator (u-pa) : urdamycin a : antifungal activity of : antitumor activity ureterolithiasis-drug-therapy : urinary tract infections : herniaria hirsuta in : urine retention : as opioid's side effect : uroterpenol : activity in mam- assay system : activity in ras assay system ursane triterpene : effect on chy : ursolic acid : , ; : effects on hiv- protease : effects on lela : inhibition of hiv- protease dimerization by ursolic acid methyl ester (ursene triterpene) : effects on hiv- protease : usambarensine : anticancer activity of : uterine tumor : effect of pironetin derivatives in : uusambarine : anticancer activity uvaol (triterpene) : effects on urs- -ene- , -diol) (ursene triterpene) : effects on vaccaria segetalis : imunomodulatory effect of vaccination : for respiratory infection as neuraminidase inhibitors : as c~-amylase inhibitor : c~-glucosidase inhibitors of : ; : yeast a-glucosidase inhibitor of : valinuoctins : fptase inhibition by valiolamine : , yeast c~-glucosidase inhibitor valiolamines : as glucosidase inhibitor : biological potential of : in clinical trials : in treatment of diabetes : valproic acid : vancomycin : vancoresmycin : activity against gram-positive bacteria vanilloid receptor agonist : olvanil as : capsaicin as : structure of vanilloid receptor antagonist : , capsazepine as varicella-zoster virus : cause of varroajacobsoni : toxicity of varroa mites : vascular physiological action : , of natural products vasodilating activity : vasodilator : scoparone as vasodilator effect : of acetylcholine vasodilatory activity : ofsatujera obovata vasoprotective agent : vasoprotective effects : of green tea vateamine- ' [ -n-oxide : , , activity in human cancer cell line : activity in murine cancer cell line : as platelet aggregation inhibitor : cytotoxic effect of : veinotonic activities veraguensin : trypanosomicidal activity verapamil : , as caz+-channel blocker : vermisporin : antimicrobial activity vernonia amygdalina : tick toxicity of : verrucarins : arenavirus junin : herpes simplex virus type ii (hsv- ) verrucosin : ofantiviral activity : vertebrate toxicity : vesicant activity vesicular glutamate transporters : vesicular monoamine transporter type (vmat ) inhibitor vesicular stomatitis virus (vsv) : , replication of : vesititol : anti-microbial activity of : vestitol : anti-helicobacter pylori activity of : veterinary medicine : for ectoparasites control : vetiver grass : for controlling ticks viagra (sildenafil) : vicolides : activity in ctn bioassay system anticancer activity of : viral diseases : viral dna : integration of : ; : , , , , , , , , , , , , , , , , ; : , , ; : ; : ; : against bone resorption : , against h+,k+-atpase : against staphyllococcus aureus : mechanism action of : of (-)-( , a-di-epi-swainsonine : of (-)-swainsonine : o f (+) cyasterone activity in : -deoxy- -hyroxyecdysone activity in : -deoxymuristerone a activity in : [ , [ -dihydro xy- -chole st- en- -one activity in : [ , a-dihydroxy- [ -cholest- en- -one activity in : ecdysone activity in : -hydro xy- [ -cho le st- -one activity in : -hydroxyecdysone activity in : inokosterone activity in : kc cell bioassay as : makisterone a activity in : muristerone a activity in : podecdysone a activity in : ponasterones activity in : , , , a-tetrahydro x ycholest- -en- -one activity in : [ , ~ , -tridihydroxy- cholest- -one activity in : viticosterone e activity in : insect control : insect juvenile hormone analogues : - from thujone : - insect pheromones : [ ] [ ] [ ] via diisopropylethanediol esters : - insect repellant : insecticidal activity : , ; : , , , , , , , , , , , , ; : ; , , , ; : , , , ; : , , , , , ; : ; : - activity of (-)-deoxypodo phyllotoxin in : activity of (-)-hernone in : activity of (-)-nymphone in : activity of (-)-yatein in : activity of (+)-corytuberine in : activity of (+)-epiaschantin in : activity of (+)-epimagnolin in : activity of (+)-epiyangambin in : activity of (+)-hernovine in : activity of (+)-laurotetanine in : activity of (+)-magnoflorine in : activity of (+)-malekulatine in : activity of (+)-n-hydroxyhemangerine in : activity of (+)-n-formylnornantenine in : activity of (+)-n-formyldehydroovigerine in : activity of (+)-n-formylovigerine in : activity of (+)-ovigerine in : activity of (+)-ovihernangerine in : activity of (+)-vateamine- ' [ -noxide in : activity of -formyldehydrohernangerine in : activity of -formyldehydronornantenine in : activity of -formyldehydroovigerine in : activity of -hydroxy- -methoxy- : , ; : , , , ; : ; : , , - ; : , ; : ; : , activity in ebv assay system : activity in skin assay system : activity in crg assay system : activity in cro assay system : activity in aa assay system : activity in brk assay system : activity in cps assay system : activity in htm assay system : activity in paf assay system : , , , , , , , , , , ; : anti-inflammatory activity of : antiviral activity of : saponins : ; ; : , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ; : ajugasterone c activity in : amarasterone a activity in : amarasterone b activity in : cyasterone activity in : ponasterone a p-glucoside activity in : pterosterone activity in : rubrosterone activity in : sengosterone activity in : stachysterone c activity in : capitasterone activity in : sarcophytol a : activity in ebv assay system : activity in colon- assay system : activity in liver- assay system : activity in lung- assay system : activity in mam- assay system : activity in pancr assay system : activity in skin- assay system : sarcophytol b : ; : activity in skin- assay system : use of platycodon grandiflorum key: cord- -psnqrdo authors: paez, antonio; lopez, fernando a.; menezes, tatiane; cavalcanti, renata; pitta, maira galdino da rocha title: a spatio‐temporal analysis of the environmental correlates of covid‐ incidence in spain date: - - journal: geogr anal doi: . /gean. sha: doc_id: cord_uid: psnqrdo the novel sars‐cov has disrupted health systems and the economy, and public health interventions to slow its spread have been costly. how and when to ease restrictions to movement hinges in part on whether sars‐cov will display seasonality due to variations in temperature, humidity, and hours of sunshine. here, we address this question by means of a spatio‐temporal analysis in spain of the incidence of covid‐ , the disease caused by the virus. use of spatial seemingly unrelated regressions (sur) allows us to model the incidence of reported cases of the disease per , population as an interregional contagion process, in addition to a function of temperature, humidity, and sunshine. in the analysis we also control for gdp per capita, percentage of older adults in the population, population density, and presence of mass transit systems. the results support the hypothesis that incidence of the disease is lower at higher temperatures and higher levels of humidity. sunshine, in contrast, displays a positive association with incidence of the disease. our control variables also yield interesting insights. higher incidence is associated with higher gdp per capita and presence of mass transit systems in the province; in contrast, population density and percentage of older adults display negative associations with incidence of covid‐ . from a small outbreak linked to a live animal market at the end of to a global pandemic in a matter of weeks, the sars-cov virus and covid- , the disease it causes, have threatened to overrun health systems around the world. in efforts to contain the spread of the disease, numerous governments in many regions and nations have either recommended or mandated social distancing measures, and as of this writing, millions of people in five continents shelter in place. there are encouraging signs that these measures have mitigated the spread of the virus (e.g., lancastle ; lewnard and lo ; wilder-smith and freedman ) . even so, this has come at a high cost, and the consequences for all spheres of economic, social, and cultural life have been dire (e.g., fernandes ; luo and tsang ) . as a result, there is a sense of urgency to anticipate the progression of the pandemic, in order to plan for progressive lifting of restrictions to movement and social contact (e.g., kissler et al. ) . needless to say, this has become a delicate, and politically charged, balancing act between public health and the economy (gong et al. ) . a salient question in the debate about how and when to ease social distancing measures is whether the virus will display seasonal variations. existing research on similar pathogens suggests that the virus could be more stable and potentially easier to transmit in conditions of low temperature and low humidity. while this is encouraging, it is important to keep in mind that "not all seasonal respiratory viruses experience the same spatiotemporal patterns" (de Ángel solá et al. , sec. ) . this urges caution when extrapolating from known viruses. the evidence in this respect is as yet inconclusive, and although easing restrictions as the weather warms may appear tempting, doing so prematurely could well undo weeks or possibly months of costly measures. it is not surprising, given the stakes involved, that this issue has already triggered a lively debate. the current state of knowledge was well-summarized by the national academy of sciences, engineering, and medicine in the u.s. in a recent report (see national academies of sciences, engineering and medicine ). engaged by the office of the executive for guidance on this matter, this organization concluded that: "[some] limited data support a potential waning of cases in warmer and more humid seasons, yet none are without major limitations… additional studies as the sars-cov pandemic unfolds could shed more light on the effects of climate on transmission" (p. ). to further complicate matters, much of the relevant work has yet to be peer-reviewed (see for instance the challenge of harbert, cunningham, and tessler ; to araujo and naimi ) . with the above considerations in mind, our objective with this paper is to investigate the influence of environmental factors, concretely temperature, humidity, and sunshine, on the progression of the pandemic. we adopt a population health approach, and report results from a spatio-temporal model of the incidence of covid- in the coterminous provinces in spain, one of the countries hardest hit by the pandemic. we combine data on reported cases of the disease with metereological information, to create a spatio-temporal dataset covering a period of days. we then join this dataset with provincial-level economic and demographic information to act as controls to our key environmental variables. these data are analyzed using a spatial seemingly unrelated regressions (sur) approach, which allows us to model incidence of covid- as a contagion process. the results of this research provide evidence of the effect of temperature, humidity, and sunshine on the incidence of covid- . the clearest result with respect to these variables is a lower incidence of covid- at higher temperatures and levels of humidity, while the opposite happens with respect to hours of sunshine. our control variables also provide some intriguing insights. higher incidence is associated with higher gdp per capita and presence of mass transit systems in the province; in contrast, population density and percentage of older adults display negative associations with incidence of covid- . the results of this analysis provide support to the hypothesis of seasonality of the novel sars-cov , and should be of interest to public health officials and policy makers grappling with the question of the trajectory of the pandemic. please note that this paper is prepared as a reproducible research document. the source r markdown document, as well as all data and code needed to reproduce/review/extend the analysis can be obtained from a public repository. the global emergence of infectious diseases is mostly driven by environmental, ecological, and socio-economic factors (jones et al. ). in the case of sars-cov , the ecological factors include the interaction between humans and wildlife. once transmission of a disease begins to happen between humans, socio-economic and environmental factors become increasingly important. as noted in the introduction, the focus of the paper is on environmental variables, concretely three related to meteorological conditions: temperature, humidity, and sunshine. much of what is known about the potential seasonality of sars-cov is a result of research on other pathogens. earlier, diverse studies have shown the effect of temperature and humidity on the incidence of influenza (e.g., mäkainen et al. ; jaakkola et al. ; kudo et al. ) . jaakkola et al. ( ) , for example, found that a decrease of temperature and absolute humidity precedes the onset of symptoms of influenza a and b viruses by days in places where the temperature is low. after the - outbreak of sars, researchers also began to investigate the relationship between these factors and sars-cov (casanova et al. ; chan et al. ). casanova et al. ( , for instance, used surrogates to find that virus inactivation was likely more rapid at higher temperatures; in terms of humidity, these researchers reported that survival of the virus was lower at moderate relative humidity levels. chan et al. ( ) also found that viability of the virus that causes sars is also lost at higher temperatures (> • c) and relative humidity superior to %. whether results from laboratory experiments will hold when the virus circulates in the community remains uncertain. at a global scale, de Ángel solá et al. ( ) see less risk from sars-cov in the caribean basin; on the other hand, coelho et al. ( ) warn that at least during the exponential phase, expansion of the virus is not driven by climate. similarly, whereas araujo and naimi ( ) argue that spread of sars-cov will likely be constrained by climate, harbert, cunningham, and tessler ( ) remain unconvinced that spatial modelling can currently discriminate the distribution of the disease on the basis of climate, at least in the united states. yao et al. ( ) , examined data from china and came to the conclusion that neither temperature nor ultraviolet indices had an association with transmission of covid- . this is despite previous research that has linked less exposure to uvb radiation to higher prevalence and severity of acute respiratory tract infections (zittermann et al. ; dabrowska-leonik et al. ; dinlen et al. ; mathyssen et al. ; esposito and lelii ; jat ; moriyama, hugentobler, and iwasaki ) . in addition to the environmental variables above, from a population health perspective it is also important to account for potential socio-economic and demographic confounders. to account for population-level factors, the first variable that we consider is gdp per capita. much has been written about globalization and the spread of infectious disease . the growth in global connections has presented a challenge to spatial approaches in the initial stages of disease management, when the cause of a disease may still be unclear but the plane has already departed (zhou and coleman ) . in reference to the earlier outbreak of sars, van wagner ( ) chronicles how toronto's status as a global city turned out to be a vulnerability in this respect. in our case, we think of gdp per capita as a marker of a region's relative position in a network of global cities, and its potential to be further ahead in the trajectory of the pandemic. furthermore, wealthier regions also tend to concentrate more activities that produce non-traded goods, including building and construction (hallet ) . therefore, it is possible that wealthier regions remain relatively more active even during a lockdown. on the other hand, we cannot discount the possibility that less wealthy regions have a higher proportion of workers in manual occupations who cannot telework, and therefore have more difficulties complying with shelterin-place orders. secondly, we consider percentage of older adults (over ) in a region. early evidence regarding covid- suggests that the case rate mortality is higher at older ages (e.g., the novel coronavirus pneumonia emergency response epidemiology team ). however, it is not clear that a relatively large population of older adults necessarily translates into higher transmission rates of the infection. the tool of choice in containing the spread of the disease has been social distancing. in this respect, the evidence from the field of transportation is that older adults tend to travel less frequently, for shorter distances, and have higher rates of immobility than most everyone, except the youngest members of the public (e.g., roorda et al. ; morency et al. ; sikder and pinjari ) . in other words, many older adults are, whether by preference or otherwise, already in a form of social isolation. social distancing during the pandemic may actually reinforce that condition for them, as suggested by the analysis of age-structured social contact in india, china, and italy of singh and adhikari ( ). since the age-structured matrix of social contact in spain is similar to italy (see prem, cook, and jit ) , our expectation is that populations with higher percentages of older adults will tend to have lower levels of social contact and hence of incidence. population density is also relevant since it directly affects the contact patterns and contact rates between individuals in a population (hu, nigmatulina, and eckhoff ) . the evidence available suggests a positive relationship between the transmission of covid- and population density (e.g., cumulative incidence in urban areas like nyc). for this reason, we anticipate a positive relationship between population density and the incidence of the disease. the last variable that we consider as a control is the presence of mass transit systems in a province. every province in spain offers some form of public transportation, but only five provinces have higher order systems of mass mobility (e.g., metro or subway), namely barcelona, madrid, sevilla, valencia, and bizkaia. public transportation has been hypothesized to relate to the spread of contagious disease by some researchers using agent-based approaches and simulation (e.g., perez and dragicevic ; wang et al. ) , and while we find scant evidence of a link in the literature, the idea is intuitively appealing. after all, unlike the isolation that a car offers to travellers, most mass transit system are cauldrons of social contact. the first reported case of covid- in spain was on january st, , when a german tourist in the canary islands tested positive for the virus. after this case, it was still a few weeks before the first domestic case was reported, on february th in sevilla province (andalusia). in a short period of time, as testing started to ramp up, it became clear that an outbreak was flaring. by march th the world health organization (who) declared covid- officially a pandemic. this declaration marked a turning point for the public in spain too. as of march th, the number of cases of covid- reported in spain was , , with a majority of cases ( , ) concentrated in madrid: these numbers were at the time the worst outbreak in europe after italy. in response to the situation, on march th the spanish national government declared a state of emergency, to go into effect on saturday march th. as part of the state of emergency restrictions to most activities were imposed, with the exception of essential services (e.g., food, health) and some economic subsectors of industry and construction. a few days later, on march th, spain closed its lands borders to allow entry only to returnee nationals and permanent residents. the lockdown was further hardened between march th and april th (including the easter weekend of april th- th) and during this period only essential activities were allowed. during this period, there was a dramatic reduction in overall mobility, both within provinces as between. our dataset includes information about the daily number of cases of covid- reported in spain at the provincial level (nuts in eurostat terminology) for the period between march th and april th, inclusive. for our purposes, we consider positive cases reported, but exclude symptomatic cases diagnosed by a doctor without a polymerase chain reaction (pcr) test. the spanish national government publishes periodic updates at the regional level (nuts ) and the information is also released at the provincial level as part of a collaborative project by geovoluntarios.com, providencialdata , and esri españa (see datadista, ) . this information is compiled from several sources, mainly the official web pages of the spanish regional goverments, as documented in centro de datos covid- . we consider two sets of explanatory variables. the first one, and the focus of this research, are the three environmental variables, collected from official sources (i.g., aemet, the state meteorology agency, and mapa, the ministry of agriculture, fisheries, and food). the second set provides some relevant controls as discussed above, and are also collected from official sources, namely ine, the national statistics institute. table shows the descriptive statistics and the provenance of the data used in this research. the spatial and temporal coverage of the data is as follows. our dataset begins on march , which is the first date when every province had reported at least one case of covid- , and continues until april , for a period of days. the unit of analysis is the province. provinces in spain are the equivalent of states, and are embedded in autonomous communities. as an example, cataluña is an autonomous community and consists of four provinces, namely barcelona, gerona, lerida, and tarragona. the size of the provinces is relatively large, as seen in table . the smallest province is , . km (this is smaller than rhode island in the u.s.) and the largest province is , . km (slightly smaller than new jersey in the u.s.). while this is a relatively large degree of spatial aggregation, reporting on covid- is not yet consistent at smaller geographies, or cases are not reported at that level at all. an important aspect of working with environmental data such as temperature, humidity, and hours of sunshine, is the incubation period of the disease. lauer et al. ( ) report for the case of covid- a median incubation period of . days (with a confidence interval between . to . days). the vast majority of cases ( %) develop symptoms between . days (ci, . to . days) and . days (ci, . to . days). for this reason, we judge it best to use lagged values of the environmental variables. we test different time lags as follows. we consider a lagged -day average, from date-minus- to date-minus- days (hereafter lag ). secondly, we consider a lagged -day average, from date-minus- to date-minus- days (hereafter lag ). finally, to account for the likely duration of incubation, we consider a lagged -day weighted average, from date-minus- to date-minus- days (hereafter lag w). the weights for this variable are calculated using the parameters of the log-normal distribution reported by lauer et al. ( ) , that is, a log-mean of . and a log-standard deviation of . . with these weights, the environmental variables at date-minus- and date-minus- days are weighted as . and . respectively, whereas the environmental variables at date-minus- days are weighted as . . these weights have the effect of changing the contribution of daily values to the lagged moving average. for instance, the temperature at date-minus- -days is weighted more heavily than the temperature at date-minus- -days, as a closer approximation of the conditions at the most likely time of contagion before the disease became manifest. the seemingly unrelated regression equations model (sur hereafter) is a multivariate econometric model used in different fields when the structure of the data consists of cross-sections for different time periods. the basis of this approach is well-known since the initial works of zellner ( ) , and has become a popular methodology included in several econometrics textbook (e.g., greene ) . to our knowledge, anselin ( ) was the first author to discuss sur from a spatial perspective, in the context of spatio-temporal analysis. in his landmark text, anselin discussed a model made of "an equation for each time period, which is estimated for a cross section of spatial units" (p. ). from this milestone, a large body of research has developed to extend the classical sur into a spatial framework (e.g., rey and montouri ; lauridsen et al. ; le gallo and dall'erba ; lópez, martínez-ortiz, and cegarra-navarro ) . the classical sur model without spatial effects (from here, sur-sim) is a stack of equations as follows: where y t = (y t , … , y nt ) is a n × vector, and in our case y st is the incidence ratio in the province s (s = , …, n) the day t (t = , …, t); x t is a n × k t matrix of the k t independent variables, with associated vector of coefficients β t ,; β t = (β t , … , β nt ) is a vector of coefficients and ϵ t = (ϵ t , … , ϵ nt ) is the vector of residuals. a key feature of the sur model is the temporal dependence structure among the vectors of residuals, namely: note that this specification is very flexible, in that it allows changes in the coefficients β it in order to modulate the effect of the independent variables on y t . this flexibility can be reduced and it is posible to impose restrictions considering some β coefficients as being constant over time. in this case, we can reformulate the coefficients expression β t = (β , … , β r− , β r , β r+ , … , β nt ) to restrict the first r coefficients to be constant across equations. this is equivalent to specifying some effects to be invariant over time. equation ( ) can be rewriten in compact form: where y is now a vector of dimension nt × , x is a block-diagonal matrix nt × k (with k = ∑ t k t ) and ε is an nt × vector. using the kronecker product notation (⊗), the error matrix structure is expressed concisely as: as is the case with cross-sectional data, it is possible to test the residuals of model ( ) for spatial autocorrelation, and several tests have been developed to test the null hypothesis of spatial independence (see lópez, mur, and angulo ) . when the null hypothesis is rejected, several alternative specifications have been proposed to include spatial effects (anselin ) . in this paper we consider a spatial sur model that incorporates a spatial lag of the dependent variable as an explanatory factor. spatial analytical approaches have been used to understand contagion-difussion processes in the case of infectious disease in general (e.g., cliff, haggett, and smallman-raynor ) and the - sars outbreak in particular (e.g., meng et al. ; cao et al. ). while we are mindful of the same caveat that the novel sars-cov may not follow the patterns of previous diseases, there is still evidence from the united states that covid- displays spatial patterns that are consistent with a diffusion process (desjardins, hohl, and delmelle, ) . for this reason, the spatial lag model is appropriate to model incidence of covid- geographically, since it accounts for potential spatial patterns that result from a process of contagion, as explained next. the stack expresion for the sur model with a spatially lagged dependent variable (sur-slm) is as follows: this specification assumes that outcome (y st ) at location s and time t is partially determined by the weighted average (wy st ) of the outcome in neighboring provinces, with neighborhood defined by matrix w of spatial weights. in other words, the spatially lagged dependent variable represents a process of contagion, where the disease in neighboring provinces can spillover in a spatial way. the coefficients of the spatially lagged variable are estimated for each time period ρ t and identify the intensity and the sign of the contagion effect. it is possible test the null hypothesis of identical levels of spatial dependence (ρ i = ρ j , ∀i, j). the correspond wald test is available in the r package spsur. the sur-slm model can be estimated using maximum likelihood (lópez, mur, and angulo ) or instrumental variables (mínguez, lópez, and mur ) . a note regarding the interpretation of the model is in order. it is well-known that coefficients in linear regression models are partial derivatives of the dependent variable with respect to the independent variables, and therefore directly give the marginal effects or rates of change. spatially lagged models, however, are no longer linear. the intuition behind the non-linearity is that the spatial lag expands the information set to include information from neighbouring regions: in other words, the value of an explanatory variable in a spatial unit can have influence in other spatial units via the spatial lag. this makes interpretation of the coefficients less straightforward but also richer (golgher and voss ) . the results of lesage and pace ( ) for cross-sectional spatial lag models can be extended to the spatial sur framework. note that, according to elhorst ( ) , the partial derivatives have the following interpretation: if an explanatory variable (x k ) in a particular province changes, not only the incidence rate in that province changes, also incidence rates in other provinces change via the contagion effect. therefore, a change in x k in a particular province has a direct effect on that province, but also an indirect effect on neighbouring provinces. in this way, the ith diagonal element of the matrix of partial derivatives represents the ( ) direct effect on the ith province, whereas the non-diagonal elements of ith row of the matrix of partial derivatives represent the indirect effects on other provinces. in order to obtain a global indicator, the direct effect is calculated as the mean of the diagonal elements and captures the average change in incidence ratio caused by the change in x k . likewise, a global indicator of the indirect effects is calculated as the mean of the non-diagonal elements. the total effect is the sum of direct and indirect effects. finally, note that if ρ k = , the indirect effects are and the direct effects are equal to β k t. fig. shows the geographical variation in the incidence of covid- in spain, as well as the temporal progression of the disease in weekly averages. our analysis begins on march . albeit still low, the highest incidence at this early date was in the provice of Álava, in the north of spain. Álava is not the most populous province, with a population of only , , but it has the highest gdp per capita of all provinces. vitoria, its main city, is the capital of the basque country and has been the focus of efforts, along with san sebastian and bilbao, to develop a "global basque city" (meijers, hoekstra, and aguado ) . the other early focus of the pandemic in spain was madrid, which is the most populous province in the country and has the second highest gdp per capita after Álava. the early outbreaks in these two provinces can be traced throughout the progression of the pandemic over time, although by the end of the period under study, other provinces had matched and even surpased their incidence rates, including segovia and soria to the north of madrid, and ciudad real and albacete to the south. fig. shows the distribution of the environmental variables in spain. for ease of visualization we aggregate the provinces by autonomous community. each box-and-whisker in the figure represents the distribution of the variable for a community over the -day period. in the plot, the communities have been sorted by latitude, so that principado de asturias is the northernmost community, and andalucia the southernmost. as seen in the figure, there is a relatively wide range of values both within and between provinces over the -day period examined. the top panel of the figure shows the distribution of mean temperatures. the lowest mean temperature for a community on any given day was approximately • c, and the highest about • c, for a range of approximately • . likewise, there is a great deal of variability in humidity, as seen in the middle panel of the figure, where the lowest mean humidity for any community is approximately % and the highest is close to %. finally, the bottom panel displays mean daily hours of sunshine in the community. this variable displays the most variability within communities over time, but remains relatively constant across communities. it is important to note that these are summaries by community, and the actual values of these variables for the provinces display somewhat more variability. fig. includes three maps that display the spatial variation of our control variables, namely gdp per capita, percentage of older adults in province, population density, and presence of mass transit systems. as seen there, gdp per capita is higher in madrid and the northeast part of the country, mainly in pais vasco and cataluña. percentage of older adults is somewhat more checkered, with high values in madrid and other provinces in the center-west part of the country, but also in some provinces in the north. outside of provinces with major cities (e.g., madrid; bizkaia and gipuzkoa in pais vasco; pontevedra in galicia), population density tends to be higher in provinces along the mediterranean coast. the final panel in the figure shows the five provinces in the country that have higher order mass transit systems (e.g., metro). fig. shows the distribution of daily simple correlations of incidence of covid- with the independent variables (with the exception of transit, which is a categorical variable). these correlations are calculated after log-transforming all variables. as previously discussed, the environmental variables have a temporal lag and were calculated using different time windows. it can be seen in the figure that temperature (in its three forms) has the highest simple correlation with incidence of covid- . after temperature, gdp per capita has the highest positive correlation with the dependent variable. the distribution of these correlations is also quite tight over the -day period of the study. hours of sunshine tends to have a moderately high correlation with incidence of covid- , but the distribution of these correlations is more spread, and in some cases strays into negative values. a similar thing happens with humidity, which also tends to display more day to day variation in the correlation with the dependent variable. the percentage of older adults shows a relatively tight distribution of day-to-day correlations, and autonomous communities in spain (sorted from north to south) value is negative. population density, in contrast, tends to be negative, but is relatively spread, and on some days, the simple correlation between density and incidence of covid- is weakly positive. overall for the period under examination, the pairwise correlations between these variables and incidence are significant at p < . , with the exception of the three sunshine_hours variables. correlation analysis in the preceding section provides some insights about the potential associations between incidence of covid- and the various environmental and control variables. in this section we estimate three spatial sur models to test the differences between the various temporal lags and weighting schemes for the environmental variables. accordingly, we define three models: model , which is estimated using the lagged -day averages of the environmental variables (lag ); model , which is estimated using the lagged -day averages of the environmental variables (lag ); and finally, model , which is estimated using the lagged -day weighted averages of the environmental variables (lag w). to implement the sur approach, we must define a matrix of spatial weights w. in this case, we consider neighborhoods based on adjacency, based on the well-known queen criterion (two provinces are adjacent if they share a boundary or touch at a vertex). the analysis is of the coterminous provinces. for estimation, we log-transform the dependent and quantitative independent variables. the only variable that is not transformed is the categorical variable for transit systems. a log-log transformation is appropriate to capture non-linear relationships between variables and provides a straightforward interpretation of the coefficients as percentage change. furthermore, we introduce restrictions so that the coefficients of two of our control variables are constant over time, namely gdp per capita and percentage of older adults. we do not see an a priori reason to let those two variables vary across equations, and the correlation analysis in fig. also suggest little temporal variation. in contrast, we let the spatial autocorrelation parameter, as well as the parameters of the rest of the independent variables (including the constant) to vary over time. this will be useful to detect whether there are behavioral adaptations at the population level over the course of the period examined. as an example of behavioral adaptations, the effect of density might weaken over time, in the measure that the effects of the lockdown are felt: at full compliance with the lockdown, with people practicing social avoidance, density might matter less than other factors. after estimation, we compare the goodness of fit of the three sur models. fig. shows the equation-level coefficient of determination r , one for each time period/day. as well, the overall coefficient of determination for the system is reported for each model pooled-r . the general trend is identical for the three models, with the goodness-of-fit improving over time and plateuing around a value of r of . . model (lag ) performs somewhat better in the first few equations/days, when the goodness-of-fit is relatively poor, and then again in the last few equations/ variable correlation days. model (lag w), in contrast, does not perform well towards the end of the study period. the most balanced model in terms of equation-level goodness-of-fit appears to be model (lag ), and this impression is further supported by a slightly higher value of the pooled-r . the analysis using a lagged moving average of the environmental variables is generally in line with the incubation period reported by lauer et al. ( ) , although the results do not support the use of a weighted average. for the remainder of the paper, we will adopt model (lag ) as our best model. in the following section we discuss the results of the analysis in more depth. table presents a summary of the results of model (lag ). recall that two coefficients were constrained and are estimated only for the first equation of the system, and thus are assumed to be constant over time. these are the coefficients corresponding to gdp per capita (p ≤ . ) and percentage of older adults (p ≤ . ). the sign of the coefficient for gdp per capita is positive, which indicates that wealthier regions tend to have a higher incidence of covid- . this is in line with the idea that the epidemic started earlier in wealthier places due to their connections to a globalized world. the sign of the coefficient for percentage of older adults, on the other hand, is negative. as previously discussed, the level of social contact of older adults even under normal circumstances tends to be lower than for younger people. as a consequence, places with larger populations of older adults appear to have a natural level of social distancing in place. it is important to note that this does not detract from evidence that older adults are more vulnerable individually and in institutional settings, where their case mortality rates are perhaps the highest of all age groups. instead, this result indicates that their presence in the community at large tends to depress transmission of the virus. of the two other control variables, the coefficient of population density is significant at p ≤ . in , at p ≤ . in equations, and not significant in . the coefficient for transit notes: significance: this is the number of coefficients with p-values as indicated. non-constrained: coefficient was allowed to vary across equations. constrained: coefficient as constant across equations. is significant at p ≤ . in equations, and of those, significant at p ≤ . in equations. the next four variables are environmental factors. the coefficient for humidity is significant at p ≤ . in equations, and of those, significant at p ≤ . in equations. of the environmental variables, temperature is the only variable that has significant coefficients in every equation at p ≤ . . finally, sunshine has significant coefficients at p ≤ . in equations. to better understand the results, we proceed to plot the coefficients in their temporal sequence. at this point it is worth recalling that the state of emergency went into effect on march . in the following figures, the periods of time indicated in yellow starting on march correspond to the state of emergency, with only essential travel and selected industrial activities allowed; the period of time in orange was the stricter lockdown when only essential travel was allowed. we begin our discussion with the evolution of the spatial autocorrelation coefficient (ρ) in fig. (left panel) . we notice that the magnitude of the spatial autocorrelation coefficient ρ declines over the period under analysis, and is not significant for some days. this is an interesting result: immediately prior to the declaration of the state of emergency, there appears to have been a strong inter-provincial contagion effect. keeping in mind that the incubation period ranges between and days with a median of , it is reasonable to expect that the effect of the lockdown will be observed with some delay. indeed, as seen in the figure, the autocorrelation coefficient remains high for several days, then begins to decline around march , and continues to weaken over time. at the end of the period under examination, the strength of this effect is much diminished and we would expect that under full compliance with strict lockdown conditions (meaning no inter-provincial mobility) the spatial contagion effect would be zero-as seems to be the case. the intercept (right panel in fig. ) is indicative of the variation of the incidence of covid- , other things being equal. here we see that at the incidence declines somewhat immediately after the state of emergency, only to begin increasing again over time. then, the incidence declines again after the stricter lockdown and rebounds to a higher level by april . figure . temporal evolution of the spatial autocorrelation coefficient (rho) and the intercept of the model; dots are the point estimates and vertical lines are % confidence intervals. in yellow is the period after the declaration of the state of emergency, and in orange is the period when only essential activities were allowed. fig. shows the temporal evolution of the coefficients for the two control variables that were not fixed over time, that is, log (density) (left panel) and transit (right panel). in section "background" we had anticipated a positive sign for the coefficient of density, and indeed, at the beginning of the period the coefficient is positive, albeit not significant, and then remains mostly non-significant for the earlier part of the lockdown. we are somewhat surprised by the way this coefficient turns significant and negative in the later part of the lockdown, after april st. this effect, we surmise, is the result of risk compensation, a situation where people adapt their behavior according to the perceived level of risk, becoming more careful when the perceived risk is higher and viceversa (e.g., noland ; phillips, fyhri, and sagberg ; richens, imrie, and copas ) . consequently, residents in high density regions may perceive the risk of infection as being higher, and adapt their behavior accordingly-while the opposite may be true of residents in low density regions. the coefficient for transit is positive, as expected, but with very wide confidence intervals, and in fact not significant in the earlier part of the period. the evolution of the coefficients for the three environmental variables is shown in fig. . despite a mostly positive simple correlation with incidence (see fig. ) once that we control for other factors, humidity has a negative association with incidence of . this is in line with the literature that describes the lower viability and transmission of different viruses at higher levels of humidity. the coefficients for temperature (topright panel) are consistently negative and this variable is, besides the intercept, the only one with significant coefficients in all equations. the range of variation of this coefficient during the period examined is approximately between − and − , although it is important to recall that these values should not be interpreted directly as effects; more on this below. finally the plot for the coefficients associated with hours of sunshine (bottom panel) is more ambiguous: prior to the lockdown, the coefficient was negative, but not significant. however, five days into the lockdown, the coefficient becomes significant and positive. this result stands in contrast to previous findings regarding influenza, where more hours of sunlight reduced the strength and duration of epidemic durations (yu et al. ) . a difference with previous studies is the temporal scale of the analysis: where yu et al. ( ) use monthly averages, we use daily data for a much shorter period of time. the positive sign of sunshine may well be another instance of behavioral adaptations, whereby compliance with lockdown orders weakens on sunny days. the preceding discussion helps to establish the inferential contributions of the analysis, and indicate which variables display significant statistical associations with incidence of covid- . the remaining question is, what are the implications. as discussed in section "methods: the spatial sur model" the effect of a variable is not clear from its coefficient alone, since a change to a variable in a province influences, via the contagion effect, its neighbors. for this reason, the appropriate way to estimate the effects is to calculate both the own effect and the effect due to contagion, or in other words the direct and indirect effects, respectively. the total effect is the sum of the two. a summary of the effects appears in table . all effects in the table are interpreted as percentage change in the incidence of covid- as a consequence of a one percent change in the variable. the exception to this is figure . temporal evolution of coefficient for the environmental variables; dots are the point estimates and vertical lines are % confidence intervals. in yellow is the period after the declaration of the state of emergency, and in orange is the period when only essential activities were allowed. transit (which was not log-transformed). this variable instead represents the percentage change in incidence between provinces without and with mass transit systems. two variables had temporally constrained coefficients. the estimated effect of gdp per capita is to increase the incidence of covid- by . % for each percent increase of this variable (in € , s). in our view, this is a measure of inertia, as provinces with higher gdp per capita where among the first to see exponential growth in the pandemic. percentage of older adults has a negative effect, and each percent increase in this variable is associated with a relatively small reduction of the incidence of approximately . %. the temporal variation of the effects for the rest of the variables is shown in fig. . the largest positive direct effect is transit, and the largest direct negative effects are temperature and humidity. the direct effects of these variables are as follows: for each percent point increase in temperature, there is between a % and % reduction in the incidence of the disease. this effect is compounded via contagion, as seen in the central panel in the figure, and the indirect effect can further reduce the incidence by up to . %. the effect of humidity is also to reduce the incidence: each percent point of increase in humidity is associated with a reduction of up to % in incidence. with the addition of the indirect effect, the total effect of a % increase in humidity is to reduce incidence by up to %. as seen in the figure, the indirect (i.e., contagion) effects are stronger at the before and at the beginning of the lockdown period. nonetheless, by the end of the period under study, the indirect effects have weakened considerably. what do these effects mean? under a situation of lockdown, inter-regional contagion is reduced, as expected, and the total effects of the variables tend towards their direct effects. in the first few days covered by our analysis the total effect of all variables is greater due to the spatial contagion effect. contagion makes analysis and intervention more complex: the contagion effect essentially acts like a multiplier, whereby developments in each province spill over to their neighbors. once the contagion effect has been tamed, each province can be "treated" independently from its neighbors. it is important, before concluding our discussion, to highlight some limitations of this study. first, the analysis was conducted mostly under a situation of lockdown, and therefore, besides first days of the period examined, one must exercise caution when trying to extrapolate the findings to a situations without lockdown. secondly, it is well known that there is in many countries substantial underreporting of cases of covid- due to limited testing. in this case we do not suspect systematic provincial bias in reporting, and as long as the underreporting is consistent across units of analysis, we do not expect biased results; it is still important, however, to remain aware that the number of true cases is likely higher. finally, we defined neighborhoods based on adjacency. it would be interesting to compare adjacency to other connectivity criteria, for instance based on domestic transportation instrastructure and services. we flag this as a matter for future research. in this paper we presented a spatio-temporal analysis of incidence of covid- in spain. the analysis covers a -day period that begins immediately before the state of emergency was declared in the country. the focus of the research has been on the environmental correlates of incidence of the disease. there is a need for more empirical evidence, as policy makers, public health practitioners, and the public begin planning for the months ahead at this early stage of the pandemic. our results offer strong support for the hypothesis that incidence of covid- at the population level is lower at higher temperatures and levels of humidity: the estimated effect is a reduction in the neighborhood of % in incidence per each % increase in temperature, and a % reduction in incidence per % increase in humidity under conditions of contagion. these reductions are lower when contagion has ceased (i.e., due to lockdown conditions). the question here seems to be whether these environmental variables can yield a bigger reduction of more cases, or a smaller reduction of fewer cases. our control variables also offer some interesting insights. in particular, there is evidence of behavioral adaptations during lockdown in the form of risk compensation (density) and compliance with the lockdown (sunshine). these results offer a cautionary tale with regards to the effectiveness of the lockdown in more dense areas, and also the implications for compliance with stay-at-home orders as the northern hemisphere moves towards summer and more hours of sunshine during the day. if risk compensation is a factor, then efforts should be made to reduce or eliminate risk compensation in less densely populated regions. a key aspect of the analysis using spatial sur models is that we were able to model incidence of covid- as an interregional contagion process. here, we find that the strength of the contagion effect was dramatically reduced by the lockdown. needless to say, the analysis presented here is at the level of population health. for this reason, the analysis does not make any claims with respect to the effect of ultraviolet light on the virus, but rather about transmission of the virus in the population. for example, the analysis does not imply that the virus moves less effectively in places where more people live in close proximity to each other, but rather that humans are more contagious when they feel safe in less dense regions. similarly, more sunshine does not mean that the virus thrives, but rather that humans are more contagious to each other when their behavior adapts to this environmental condition. some directions for future research include investigating other modelling frameworks, such as geographically and temporally weighted regression and/or space-time conditional autoregressive models. in addition, the environmental variables examined here relate to meteorological conditions only, and did not include other environmental factors that may incide in the transmission of the virus, such as pollution. these other factors should be incorporated in future studies. (bivand, pebesma, and gomez-rubio ) , spsur (angulo et al. ) tidyverse (wickham et al. ) , units (pebesma, mailund, and hiebert ) . notes https://github.com/paezh a/covid -envir onmen tal-corre lates. as the globe and mail, canada's paper of record, put it in relation to the sars outbreak in : "globalization means that if someone in china sneezes, someone in toronto may one day catch a cold" (editorial, march , , p. a ) https://www.mitma.gob.es/minis terio/ covid- /evolu cion-movil idad-big-data/movil idad-provi ncial. https://www.geovo lunta rios.org/. https://www.datos covid.es/pages/ provi denci aldata . https://www.datos covid.es/pages/ sobre-la-inici ativa. as a check for robustness, we also tested the rook criterion (two provinces are adjacent if they share a boundary, but not if they only touch at a vertex), and included the three islands in the sample. in this case we made an allowance for adjacency between the two islands in the autonomous community of canarias in the pacific (las palmas and santa cruz de tenerife), and assumed that islas baleares in the mediterranean are adjacent to three provinces in pais catalans (i.e., barcelona, tarragona, and castello) the results (which can be consulted in the source r markdown document) are robust to the specification of w. we conducted sensitivity analysis letting all parameters vary over time, and while the results are qualitatively similar, the resulting models are less parsimonious. these results are available in the source r markdown document. rticles: article formats for r markdown. software manual spsur: spatial seemingly unrelated regression models. software manual spatial econometrics: methods and models, studies in operational regional science spread of sars-cov- coronavirus likely to be constrained by climate ggthemes: extra themes, scales and geoms for 'ggplot '. software manual gridextra: miscellaneous functions for "grid weathering the pandemic: how the caribbean basin can use viral and environmental patterns to predict, prepare and respond to covid- applied spatial data analysis with r spatio-temporal evolution of beijing sars epidemic effects of air temperature and relative humidity on coronavirus survival on surfaces the effects of temperature and relative humidity on the viability of the sars coronavirus detecting space-time patterns in geocoded disease data. cholera in london, measles in the united states, - exponential phase of covid expansion is not driven by climate at global scale vitamin d deficiency in children with recurrent respiratory infections, with or without immunoglobulin deficiency rapid surveillance of covid- in the united states using a prospective space-time scan statistic: detecting and evaluating emerging clusters association of vitamin d deficiency with acute lower respiratory tract infections in newborns spatial econometrics: from cross-sectional data to spatial panels vitamin d and respiratory tract infections in childhood economic effects of coronavirus outbreak (covid- ) on the world economy. available at ssrn how to interpret the coefficients of spatial models: spillovers, direct and indirect effects a balance act: minimizing economic loss while controlling novel coronavirus pneumonia econometric analysis dates and times made easy with lubridate regional specialisation and concentration in the eu spatial modeling cannot currently differentiate sars-cov- coronavirus and human distributions on the basis of climate in the united states the scaling of contact rates with population density for the infectious disease models decline in temperature and humidity increases the occurrence of influenza in cold climate vitamin d deficiency and lower respiratory tract infections in children: a systematic review and meta-analysis of observational studies global trends in emerging infectious diseases projecting the transmission dynamics of sars-cov- through the postpandemic period low ambient humidity impairs barrier function and innate resistance against influenza infection is the impact of social distancing on coronavirus growth rates effective across different settings? a non-parametric and local regression approach to test and compare the growth rate the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application a spatiotemporal analysis of public pharmaceutical expenditure evaluating the temporal and spatial heterogeneity of the european convergence process, - introduction to spatial econometrics scientific and ethical basis for social-distancing interventions against covid- spatial spillovers in public expenditure on a municipal level in spain spatial model selection strategies in a sur framework. the case of regional productivity in eu how much of china and world gdp has the coronavirus reduced? available at ssrn janssens wim ( ) vitamin d supplementation in respiratory diseases -evidence from rct. polish archives of internal medicine cold temperature and low humidity are associated with increased occurrence of respiratory tract infections strategic planning for city networks: the emergence of a basque global city? understanding the spatial diffusion process of severe acute respiratory syndrome in beijing robust standard error estimators for panel models: a unifying approach ml versus iv estimates of spatial sur models: evidence from the case of airbnb in madrid urban area distance traveled in three canadian cities: spatial analysis from the perspective of vulnerable population segments rapid expert consultation on sars-cov- survival in relation to temperature and humidity and potential for seasonality for the covid- pandemic perceived risk and modal choice-risk compensation in transportation system simple features for r: standardized support for spatial vector data an agent-based approach for modeling dynamics of contagious disease spread risk compensation and bicycle helmets projecting social contact matrices in countries using contact surveys and demographic data us regional income convergence: a spatial econometric perspective condoms and seat belts: the parallels and the lessons aemet: obtain climatic and meteorological data from spanish meteorological agency (aemet). software manual trip generation of vulnerable populations in three canadian cities: a spatial ordered probit approach immobility levels and mobility preferences of the elderly in the united states evidence from national household travel survey the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- )-china, toward a dialectical understanding of networked disease in the global city: vulnerability, connectivity, topologies use of gis and agent-based modeling to simulate the spread of influenza welcome to the tidyverse isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak knitr: a comprehensive tool for reproducible research in r dynamic documents with r and knitr no association of covid- transmission with temperature or uv radiation in chinese cities characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data an efficient method of estimating seemingly unrelated regressions and tests for aggregation bias accelerated contagion and response: understanding the relationships among globalization, time, and disease kableextra: construct complex table with 'kable' and pipe syntax. software manual märz winfried ( ) vitamin d and airway infections: a european perspective key: cord- -re v qt authors: le, thanh ninh; chiu, chiu-hsia; hsieh, pao-chuan title: bioactive compounds and bioactivities of brassica oleracea l. var. italica sprouts and microgreens: an updated overview from a nutraceutical perspective date: - - journal: plants (basel) doi: . /plants sha: doc_id: cord_uid: re v qt sprouts and microgreens, the edible seedlings of vegetables and herbs, have received increasing attention in recent years and are considered as functional foods or superfoods owing to their valuable health-promoting properties. in particular, the seedlings of broccoli (brassica oleracea l. var. italica) have been highly prized for their substantial amount of bioactive constituents, including glucosinolates, phenolic compounds, vitamins, and essential minerals. these secondary metabolites are positively associated with potential health benefits. numerous in vitro and in vivo studies demonstrated that broccoli seedlings possess various biological properties, including antioxidant, anticancer, anticancer, antimicrobial, anti-inflammatory, anti-obesity and antidiabetic activities. the present review summarizes the updated knowledge about bioactive compounds and bioactivities of these broccoli products and discusses the relevant mechanisms of action. this review will serve as a potential reference for food selections of consumers and applications in functional food and nutraceutical industries. over the past twenty years, the heavy dependence of human nutrition on the sustainability of agricultural production has been highlighted owing to rapid population growth and environmental degradation [ ] . increasing food and agricultural productivity has simultaneously imposed external expenses and consequences upon the financial resource, ecological system, and human health by excessive water use, soil occupation, fertilizers, pesticides, herbicides, and food waste treatment [ ] . thus, to meet the united nations' sustainable development goals, the agriculture and food sectors have been confronted with a major challenge: to provide adequate nutrition for global food demand while minimizing the negative impacts on the environment [ ] . furthermore, in recent years, improving public awareness about the healthy lifestyle framework has prompted the search for novel food sources, which are rich in essential nutrients and have positive effects on human health [ ] . as a result, functional foods and nutraceuticals are gaining significant attention since these foods could provide both health benefits to reduce the risk of chronic diseases and basic nutrition [ , ] . besides, many scientific projects and research groups are focusing on the recovery of food wastes and upgrading them into high-value byproducts to serve as functional ingredients in new product development [ ] [ ] [ ] [ ] . particularly, the issues of the food systems, including food safety, food security, and food sustainability, should be significantly addressed in the era of the coronavirus (covid- ) pandemic crisis. the availability of bioactive constituent of food and functional foods may become bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing healthpromoting benefits [ ] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [ ] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure and tables - ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [ ] [ ] [ ] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [ , ] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables - ). it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure ). bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing health-promoting benefits [ ] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [ ] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure and tables - ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [ ] [ ] [ ] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [ , ] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables - ) . it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure ). summary of the bioactive compounds analyzed in the last ten years of broccoli sprouts and microgreens. glucosinolates (glss), nitrogen−sulfur compounds (β-d-thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [ ] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [ ] . in broccoli sprouts and microgreens, compounds of glss were identified (table ) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [ ] . in particular, glucoraphanin accounted for over % of the total glss content, which was quantified in the range of to mg per g of fresh weight (fw) [ ] [ ] [ ] . glucosinolates (glss), nitrogen−sulfur compounds (β-d -thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [ ] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [ ] . in broccoli sprouts and microgreens, compounds of glss were identified (table ) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [ ] . in particular, glucoraphanin accounted for over % of the total glss content, which was quantified in the range of to mg per g of fresh weight (fw) [ ] [ ] [ ] . notably, glss are not the functional components in cruciferous vegetables, rather their hydrolysis products are the putative bioactive compounds [ ] . when plant tissues are mechanically damaged, glss could be hydrolyzed by the enzyme myrosinase into a variety of degradation products, including isothiocyanates (itcs), thiocyanates, nitriles, epithionitriles, and oxazolidines. in particular, itcs have been proven to present strong anticarcinogenic activities [ ] . as mentioned above, predominant glss in broccoli seedlings are glucoraphanin, glucoerucin, and glucobrassicin, which are enzymatically converted into sulforaphane (sfn), erucin (ern), and iberin, respectively [ ] . sfn is a naturally occurring inducer of phase ii enzymes in human and animal bodies to detoxify cancer-causing chemicals. thus, it can reduce the risk of different cancers, especially those of the bladder, colon, and lung [ ] . to date, sfn was analyzed as a major compound among the total of itcs identified from broccoli sprouts and microgreens ( table ). the total itc content of broccoli seedlings was reported to be around mg per g of fw. additionally, such seedlings were said to contain % sfn [ , ] . uv/vis, ultraviolet-visible spectrophotometry; gc-ms, gas chromatography combined with mass spectrometry; gc−fid, gc with a flame ionization detector. besides glss and itcs, another important group of bioactive constituents present in cruciferous vegetables is the phenolic compounds. they are secondary metabolites produced in plants through the phenylpropanoid and shikimate pathways [ ] . based on their structure, which comprises one or more aromatic rings with attached hydroxyl substituents, phenolic compounds can be categorized into various subgroups, such as phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. these compounds have been mainly reported for antioxidant activity. moreover, they are also associated with other health-promoting effects such as anticarcinogenic, antimicrobial, anti-inflammatory, and anti-aging properties [ ] . hydroxycinnamic acids and flavonoid glycosides are among the main phenolic compounds found in broccoli seedlings [ , ] . using quantitative and qualitative analysis, phenolic compounds were characterized in broccoli sprouts and microgreens, including hydroxycinnamic acids and derivatives, and flavonoids and derivatives. their phenolic profile composed mostly of sinapic acid, gallic acid, flavonoids (quercetin and kaempferol), and other hydroxycinnamic acids (chlorogenic, caffeic acid, and ferulic acids) ( table ). in general, total phenolic and flavonoid contents were determined to be in the ranges of - mg per g of fw and - mg per g of fw, respectively [ , , ] . similar to other sprouted seeds, broccoli sprouts and microgreens are known for their high concentration of essential nutrient composition [ ] . essential nutrients are compounds that must be supplied from foods, such as vitamins, minerals, fatty acids, and amino acids. they are required for normal body functions, including dna synthesis, energy production, and biosynthetic pathways [ ] . nonetheless, the number of studies, which determined the nutritional composition of broccoli seedlings and the variations occurring during germination, is significantly smaller than that of glucosinolates and phenolic compounds. these studies indicated that broccoli sprouts are expressly rich in vitamins (a, c, k, and folic acid), minerals (potassium, calcium, magnesium, and selenium), pigments (carotenoids and chlorophylls) and some other important nutrients (amino acids, fatty acids, and dietary fiber) [ ] [ ] [ ] , , , ] . in general, the biochemical composition of broccoli sprouts and microgreens depends mainly on the germination time, and -day-old sprouts displayed the highest nutrient concentration [ ] . among these compounds, carotenoids and chlorophylls were determined in several studies of broccoli sprouts [ , , , , ] . carotenoids are a group of isoprenoid molecules synthesized as secondary metabolites by all photosynthetic plants, including broccoli [ ] . they have lipophilic antioxidant and immunomodulatory activities owing to the conjugated double bonds of the long polyene chain, which are capable of inhibiting reactive oxygen species and reducing oxidative damage [ ] . thus, carotenoids might prevent degenerative diseases, such as cardiovascular diseases, skin damage, diabetes, and several types of cancer [ ] . the major carotenoids found in broccoli are β-carotene, α-xanthophyll (lutein), and β-xanthophylls (zeaxanthin, violaxanthin, and neoxanthin) [ ] . in particular, β-carotene is the most typically studied carotenoids in broccoli sprouts and microgreens, due to its importance in medical science [ ] . it supplies the human diet a substantial amount of vitamin a that is essential for organogenesis, tissue differentiation, immune function, and vision [ ] . the total carotenoid (β-carotene) concentration of broccoli sprouts was reported in several studies with a range of to mg per g of dry weight (dw) [ , ] . chlorophylls are another group of light-absorbing pigments regularly present in broccoli sprout extract. they are primary metabolites that have a porphyrin structure [ ] . similar to carotenoids, they also have antioxidant, anti-mutagenic, and anti-inflammatory potential [ ] . the content of chlorophyll in broccoli sprouts was commonly determined along with total carotenoid content in previous studies, ranging from to mg per g dw [ , ] . additionally, a study showed individual concentrations of carotenoids (lutein and neoxanthin) and chlorophyll (chlorophyll a and b) in broccoli sprouts [ ] . the human consumption of foods or nutraceuticals is not limited to bioactive compounds. in the human body, bioavailability is determined as the fraction of the dose administered that reaches the circulatory system and then distributes into target tissues so that the bioactive compounds are biologically available for exerting health-promoting benefits [ ] . many factors could affect the bioavailability of the dietary phytochemicals, including plant cell wall compositions, chemical structures, processing conditions, environmental stress, as well as an individual's gastrointestinal system [ ] . the effectiveness of a high consumption of broccoli sprouts in reducing the risk of cancer has been attributed mainly to their high content of glss. the daily intake of glss is difficult to estimate owing to the high variability of the constituent in cruciferous vegetables, particularly broccoli. a report for glucosinolate intake was indicated for european people ranging from . - mg/day [ ] . glss are broken down by both the plant enzyme myrosinase in the small intestine and the microbiota in the gastrointestinal tract into itcs, which are reported to have the prominent anticancer effect [ ] . after intake - h, the itcs are absorbed from the small bowel and colon and metabolized by the mercapturic acid pathway. the metabolites are detectable in human urine and blood [ ] . in broccoli sprouts, two of the most abundant glss are glucoraphanin and glucoerucin, and myrosinase hydrolysis of these glss form sfn and ern, respectively. sfn, distinguished itcs in broccoli sprouts, is one of the most potent naturally occurring inducers of phase detoxification enzymes, boost antioxidant status, and protect animals against chemically induced cancer [ , ] . its modes of action are involved with the repressor protein kelch-like ech associated protein (keap ), nuclear factor erythroid p -related factor (nrf ), and genes, which contain an antioxidant responsive element (are) [ ] . thus, it is important for the inclusion of potential cancer chemopreventive agents in dairy foods. moreover, it has shown an inhibitory effect for urease from helicobacter pylori, a human pathogen [ ] . the formation of other breakdown products from glss by intestinal microbiota is not well documented. for example, bifidobacterium strains belonging to the human intestinal microbiota can in vitro metabolize the glss to nitriles. it is also known that several microorganisms are able to convert nitriles into ammonia and organic acids [ ] . phenolic compounds are substantial micronutrients in the human diet and the health benefits of polyphenols depend on the amount consumed and on their bioavailability [ ] . the dietary intake of phenolic compounds has been associated with health-promoting effects, such as antioxidant, anti-aging, antiproliferative, and anti-inflammatory activities [ ] . it has been reported that the average dietary intake of phenolic compounds for humans is about - mg/day, including % of hydroxycinnamic acids, - % of flavonoids, and % of anthocyanins [ ] . bioavailability differs significantly among phenolic compounds so that the most abundant compounds in the human diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues [ ] . there are two directions for the digestion of dietary phenolics, comprising digestion along the gastrointestinal tract and digestion inside the enterocytes. these digestions are based on hydrolase enzymes, which are available in the intestinal lumen, brush border, and enterocyte [ ] . many studies showed that polyphenols are absorbed in both the small intestine and the large intestine by intestinal enzymes after microbial digestions [ ] . in the human small intestine and stomach, gallic acid and caffeic acid are the most well-absorbed compounds ( %), followed by catechins ( %). the least well-absorbed polyphenols are proanthocyanidins and the anthocyanins. they are ph-sensitive, which could be broken down in the stomach and readily absorbable. flavonoid group is one of the group molecules with molecular weights > da so that it has a low bioavailability level ( %) because molecules are improbable to be transported through passive diffusion pathways [ , , ] . the dietary intake of carotenoids, particularly vitamin a, has been linked to the protection of dna, proteins, and lipids from oxidative damage [ ] . digested carotenoids decrease oxidative dna damage so that they could protect colon cells against the stress of reactive oxygen species [ ] . the dietary chlorophylls from fresh fruits and vegetables, which compose of chlorophyll a and b, showed distinct biological potentials, including wound healing, the control of calcium oxalate crystals, the modulation of xenobiotic metabolism, and the induction of apoptosis [ ] . broccoli seedlings and their bioactive components exhibit many potential health-promoting roles. in the last decade, the beneficial effects, including antioxidant, anticarcinogenic, antimicrobial, anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [ , , ] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables and ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure ). anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [ , , ] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables and ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure ) . the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [ ] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p , lipoxygenases, and nitric oxide syntheses [ ] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [ ] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [ ] . previous studies the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [ ] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p , lipoxygenases, and nitric oxide syntheses [ ] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [ ] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [ ] . previous studies on broccoli sprouts and microgreens have demonstrated that they are recognized for their variety of naturally occurring antioxidants, comprising both nutritional antioxidants like vitamins (especially, ascorbic acid and α-tocopherol), and non-nutritional antioxidants such as carotenoids and phenolic compounds [ ] . it was reported that these compounds are responsible for − % of the total antioxidant capacity in broccoli sprouts [ ] . the antioxidant activity of broccoli sprouts and microgreens has been determined in numerous studies using various methods. the capacity of broccoli sprouts and microgreens for chelating fe + and scavenging free radicals such as dpph ( , -diphenyl- -picrylhydrazyl) and abts ( , -azino-bis- -ethylbenzothiazoline- -sulphonic acid) was demonstrated in numerous studies (table ) . among the possible methods, dpph radical scavenging assay, abts radical cation decolonization assay, and ferric reducing antioxidant power (frap) assay were commonly employed in previous studies ( table ). the dpph method is speedy, simple, and low cost in comparison to other test models. dpph, a dark crystalline molecule, is a stable chromogen radical formed by the delocalization of the spare electron over the molecule. the dpph scavenging assay is based on the electron donation of antioxidants to neutralize dpph radical. the reaction occurs with the loss of the violet color of dpph that is measured at nm, and the discoloration acts as an indicator of the antioxidant effectiveness [ ] . the abts decolonization assay is appropriate for both hydrophilic and lipophilic antioxidants. it uses a spectrophotometer to measure the ability of antioxidants to scavenge the stable radical cation abts + , a blue-green chromophore molecule with absorption at nm. antioxidants can neutralize and decolorize the radical cation abts + by electron or hydrogen atom donations [ ] . the frap assay was originally employed to measure reducing power in plasma, but it has been expanded for other biological fluids and plant extracts. thus, it is applicable for both in vitro and in vivo experiments. the frap mechanism is based on electron transfer rather than hydrogen atom transfer. the assay demonstrates the ability of antioxidants to reduce ferric iron. it measures the reduction of the ferric ion (fe + )-ligand complex to the blue-colored ferrous (fe + ) form at low ph by antioxidants (absorption at nm) [ ] . moreover, another mechanism of antioxidant activity of broccoli sprouts and microgreens have been confirmed in several in vitro and in vivo examinations by inhibiting the activity of prooxidant enzymes such as lipoxygenase (lox), and xanthine oxidase (xo), and activating antioxidant enzymes such as peroxidase (pod), catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), nad(p)h-quinone acceptor oxidoreductase (nqo ), and heme oxygenase- (ho- ) ( table ) . prooxidant enzymes are considered as the main biological source of superoxide radicals, whereas antioxidant enzymes could eliminate the excess of reactive oxygen species and reduce oxidative damage during senescence [ , ] . for example, enzyme activities of cytosolic glutathione peroxidase (gpx ), thioredoxin reductase(tr) in the thyroid, plasma glutathione peroxidase (gpx ), and ferric reducing ability of plasma (frap) significantly increased in response to broccoli sprouts ingestion in -week-old wistar rats [ ] . in summary, previous studies showed that broccoli sprout extracts rich in vitamins, carotenoids, and phenolic compounds showed very high antioxidant activity in both in vitro and in vivo tests (table ) . thus, they could be applied for antioxidant therapeutics to reduce the risk of chronic diseases caused by ros. interestingly, although the antioxidant capacity of broccoli seedlings has been reported comprehensively, it is still attracting considerable attention from investigators and researchers at present [ , , ] . abts, , -azino-bis- -ethylbenzothiazoline- -sulphonic acid decolonization activity; dpph, , -diphenyl - -picrylhydrazy radical scavenging activity; frap, ferric reducing-antioxidant power; teac, trolox equivalent antioxidant capacity; orac, oxygen radical absorbance capacity; pod, peroxidase activity; cat, catalase activity; sod, superoxide dismutase activity; gpx, glutathione peroxidase activity; chel, metal chelating activity; lpo, inhibition of lipid peroxidation; loxi, inhibition of lipoxygenase; xoi, inhibition of xanthine oxidase; nqo , nad(p)h-quinone acceptor oxidoreductase ; ho- , heme oxygenase- ; ma/gc, malonaldehyde/gas chromatography. in the last decade, many studies and projects have supported the protective effects of natural products in cancer prevention. the protective role against cancer has been mostly attributed to the high content of glss, typically found in cruciferous vegetables [ ] . broccoli sprouts and microgreens have been valued as a rich source of glss and their hydrolysis products (icts, particularly sfn), which are a well-known class of cancer chemotherapeutic agents that work by inducing apoptosis and arresting cell cycle progression (tables and ) . the potential mechanisms of action mainly involve the inhibition of proliferation and the induction of apoptosis in cancer (table ) . hence, to demonstrate the anticancer activity of broccoli sprouts and microgreens, many in vitro tests have been carried out to determine the antiproliferative activity. their results indicated that broccoli seedlings exert strong cytotoxicity against different types of cancer cell lines, including hepatocellular carcinoma cells (hepg ), prostate carcinoma cells (pc- , at- , and sum ), lung carcinoma cells (a ), and colorectal adenocarcinoma cells (caco- , and ht- ) ( table ). in these studies, the highly effective ic values were found to be from to µg/ml. on the another hand, the selectivity of broccoli seedlings was reported on normal skin fibroblasts (bj), normal colon fibroblasts (ccd -co), and normal liver cells (fl b) by displaying no toxic effects on their viability after the treatment of broccoli samples (table ) . cancer is essentially a disease of uncontrolled cell division. it is caused by an imbalance between cell proliferation and apoptosis [ ] . its development and progression are generally associated with the disorder of cell cycle regulators' activity. defects in the programmed cell death mechanism also make a key contribution to tumor pathogenesis [ ] . thus, most chemotherapeutic agents exert their cytotoxic activity against cancer cells, since they cause dna damage and activate a complex signaling network resulting in cell cycle arrest and apoptosis induction [ ] . targeting the cell cycle phase and the checkpoint signaling pathway, which leads to the arrests at g /s or g /m phases, would provide a promising opportunity for cancer treatment. besides, triggering cell apoptosis could be an effective strategy for potential chemotherapeutic agents [ , , , ] . to confirm the antiproliferative activity of broccoli sprouts and microgreens, several in vitro studies revealed the mechanism of cell death based on inducing cell cycle arrest and apoptosis (table ) . their results showed cell cycle arrests (obviously at g /g and s phases) and significant increases in cell percentage with subg dna content, which is considered as a marker of apoptosis. besides, mitochondrial changes might activate the intrinsic apoptotic pathway. the loss of mitochondrial membrane potential (mmp) leads to the activation of several proteins linked to apoptosis, such as caspase- and cytochrome c [ ] . hence, a few studies indicated the notable decrease in mmp levels of cancer cells after treatment by broccoli seedlings to prove the mechanism of programmed cell death [ , ] . furthermore, some in vivo carcinogenesis models were employed to demonstrate the anticancer effects and related molecular mechanisms of broccoli sprouts and microgreens, such as c bl/ mice, balb/c mice, skh- hairless mice, and sprague-dawley rats (table ). sfn extracted from broccoli sprouts showed the inhibition of breast cancer stem cells and downregulate the wnt/β-catenin self-renewal pathway in a nonobese diabetic/severe combined immunodeficient xenograft model [ ] . sfn isolated from broccoli seeds and sprouts also exhibited anticancer effects in lung cancer cell lines and nude balb/c mice with lung cancer xenograft by inhibiting the pi k-akt signaling pathway [ ] . the transgenic adenocarcinoma of the mouse prostate model with broccoli sprout intake demonstrated a significant decline in prostate cancer occurrence and hdac protein expression in the epithelial cells of the prostate. hdac is one of the histone deacetylase enzymes that turn off tumor suppressor genes and promote the expression of oncogenes [ ] . the broccoli sprout diet administered to adult her /neu mice showed both preventive and suppressive effects on mammary cancer. these protective effects were associated with tumor-and epigenetic-related gene expression, and changed histone acetylation, dna methylation, and dna hydroxymethylation levels [ ] . the dietary administration of broccoli sprout extract to the rat model inhibited bladder cancer development by inducing glutathione s-transferase and nad(p)h-quinone oxidoreductase in the bladder, which are important enzymes against oxidants and carcinogens [ ] . in vitro ns a , h , h , hcc , h , h cells inhibiting cell proliferation; inducing apoptosis [ ] in vitro ns caco- , ccd -co cells inducing cell cycle arrest and apoptosis; decreasing mmp level; increasing ros generation [ ] in vivo ns female nod/scid mice eliminating breast cscs in vivo; downregulating wnt/β-catenin pathway [ ] in vivo ns female nude balb/c mice inhibiting the pi k-akt signaling pathway [ ] in vivo days female skh- hairless mice stabilizing p ; inducing phase enzyme; inhibiting inos upregulation [ ] in vivo ns male tramp mice in c bl/ background decreasing hdac protein expression [ ] in vivo ns sv and her /neu mice modulating epigenetic pathways; regulating epigenetic-controlled gene expression [ ] in vivo ns female her /neu mice regulating tumor-and epigenetic-related gene expression; increasing tumor suppressor gene expression [ ] in vivo days female sprague-dawley rats inducing gst and nqo [ ] hepg the antimicrobial capacity of broccoli has been reported in several publications; however, most of these studies focused on broccoli florets. there are a few reports that displayed the detrimental effect of broccoli sprouts on pathogenic bacteria. a study investigated broccoli sprouts with high levels of gallic acid, esculetin, ferulic acid, and myricetin have the antibacterial activity against foodborne pathogens, including both gram-positive bacteria (staphylococcus aureus and bacillus subtilis) and gram-negative bacteria (salmonella typhimurium and escherichia coli), with minimum inhibition concentration (mic) values from to µg/ml [ ] . this study revealed that the gram-positive bacteria were more sensitive to broccoli sprout extract than the gram-negative bacteria, similar to the previously published results of other plant extracts [ , ] . the possible reason might be the structural differences in the cell wall between these two classes of bacteria. gram-negative bacteria are surrounded by an additional outer membrane, which is a hydrophilic layer to prevent the access of many substances including natural compounds [ , ] . thus, broccoli sprout extract was demonstrated to be more active on gram-positive bacteria. another in vitro study reported the powerful bactericidal activity of broccoli sprouts against helicobacter pylori by the presence of sulforaphane as the major anti-helicobacter active compound, with highly active inhibition zones (> cm) [ ] . many reports showed that plant-derived bioactive compounds, such as phenolics, flavonoids, and glucosinolates, can inhibit the growth and activity of various microorganisms [ ] . with the diversity in the molecular structure and chemical composition, these compounds can perform distinct antimicrobial effects, such as destabilization of the plasma membrane or inhibition of extracellular enzymes [ ] . the antimicrobial activity against pathogenic bacteria, yeast, and phytopathogenic fungi was also proven by the presence of many antimicrobial peptides in broccoli floret extract [ ] . moreover, the daily intake of sulforaphane-rich broccoli sprouts for months was demonstrated to reduce gastric bacterial colonization and attenuate gastritis in helicobacter pylori-infected mice and patients [ ] . in another clinical trial, the high-sulforaphane broccoli sprouts powder also showed a considerable effect on h. pylori eradication in type diabetic patients with positive h. pylori [ ] . collectively, these findings indicate that broccoli sprout extracts might serve as a potential source of antimicrobial agents in the food and pharmaceutical industry. several studies proved that broccoli sprouts and their bioactive components possess anti-inflammatory activity. hence, the application of broccoli sprouts and microgreens has potential in the prevention and treatment of inflammatory bowel diseases, such as ulcerative colitis and crohn's disease [ ] . the anti-inflammatory mechanisms of broccoli sprouts are probably associated with the nuclear factor-kappa b (nf-κb) and nuclear factor erythroid -related factor (nrf ) signaling pathways [ ] . both in vitro and in vivo experiments, as well as clinical trials, mainly exhibit anti-inflammatory effects of broccoli sprouts, which showed high concentrations of sulforaphane and a group of phenolic compounds, including anthocyanins, isoquercetin, chlorogenic and cinnamic acids, via inhibiting inflammatory mediators such as a nitric oxide (no), decreasing the levels of proinflammatory cytokines such as tumor necrosis factor α (tnf-α), interleukin- (il- ), and il- β, and increasing the levels of anti-inflammatory cytokines such as il- and il- [ , , , , , ] . for example, a study showed that sulforaphane-enriched broccoli sprouts inhibited activation of the nf-κb signaling pathway and the secretions of inflammatory proteins (inducible nitric oxide synthase (inos), cyclooxygenase (cox- ), tnf-α, il- , il- β, and prostaglandin e (pge )) in microglial cells (bv ) and male icr (institute of cancer research) mice. in this study, broccoli samples also upregulated the expression of nrf and heme oxygenase- (ho- ) in normal bv cells and against scopolamine-induced amnesia in mouse brain tissue samples [ ] . to analyze immunological parameters in the wistar rats with iodine deficiency, the significant decrease in il- level, along with no significant differences in il- concentration, were observed after adding broccoli sprouts to the diet [ ] . high-sulforaphane broccoli sprouts also indicated favorable effects on inflammatory markers by reducing concentrations of serum high-sensitive c reactive protein (hs-crp), il- , and tnf-α in type diabetic patients [ ] . broccoli is one of the few vegetables that is considered as a supplementary treatment for type diabetes and for the prevention of its long-term complications [ ] . a few studies investigated the potential benefits of broccoli sprouts and microgreens for patients with diabetes. a study determined the effects of broccoli sprout powder on insulin resistance in type diabetic patients as a new approach by the use of its bioactive constituents. the results showed that broccoli sprout powder containing a high concentration of sulforaphane may significantly decrease in serum insulin concentration and lessen complications of diabetes [ ] . in another report, the potential efficacy of sulforaphane extracted from young broccoli sprouts has been confirmed as an effective option for supplementary treatment in type diabetes. it could induce some peroxisome proliferators-activated receptors, which contribute to glucose homeostasis in hyperglycemia and oxidative conditions [ ] . obesity has become a worldwide health problem and leads to adverse metabolic disorders, such as cardiovascular disease and type diabetes. a couple of studies documented positive actions of broccoli sprouts on obesity by regulating lipid metabolism. in a double-blind clinical trial, broccoli sprout powder as supplementary treatment in type diabetic patients could have beneficial effects on lipid profiles and oxidized low-density lipoprotein ratio (ox-ldl/ldl), as risk factors for obesity and cardiovascular disease [ ] . another mechanism to regulate lipid metabolism of broccoli sprouts may be associated with sulforaphane capacity to induce the nrf pathway [ ] . a recent study revealed that glucoraphanin extracted from broccoli sprouts can decrease the lipid accumulation and increase the nrf activation. it leads to the downregulation of the expression of multiple genes involved in gluconeogenesis and lipogenesis, thereby alleviating obesity and diabetes [ ] . several other health-promoting effects of broccoli sprouts and microgreens have also been explored by in vitro, in vivo, and clinical research. a couple of studies indicated that broccoli sprouts are a good source of bioactive compounds that act as xanthine oxidase (xo) inhibitors [ , , ] . xo is the enzyme that catalyzes the metabolism of hypoxanthine and xanthine into uric acid, so inhibiting xo activity may be potentially useful in the treatment of gout or other xo-induced diseases [ ] . broccoli sprout supplementation during pregnancy and the early newborn period could reduce brain injury following placental insufficiency in the newborn rats. the findings provide a new approach for the prevention of cerebral palsy and disabilities related to placental insufficiency [ ] . the analgesic and antinociceptive effects of broccoli sprout extract were proven by the opioid mechanism using two in vivo experimental models of nociception. this study suggests the potential activity of broccoli sprouts in pain therapy [ ] . in a double-blind study, the short-term ingestion of broccoli sprout homogenates showed the beneficial effects on nasal responses to the influenza virus in smokers by significantly decreasing virus-induced markers of inflammation and reducing the virus quantity. it may be a promising strategy for preventing influenza risk in smokers and other people exposed to airborne pollutants [ ] . in recent years, broccoli sprouts and microgreens are one of the most consumed vegetable products. they have gained recognition as functional foods or nutraceutical foods by the increasing interest of consumers for diets that support health and longevity. consequently, the health-promoting compounds of broccoli seedlings have been extracted and isolated to integrate into food and pharmaceutical product formulations. over the past ten years, the extraction, isolation, and characterization of the functional properties of these broccoli products have been demonstrated by numerous scientific publications. in this review, we summarized for the first time the research findings of the last decade into bioactive constituents, bioactivities, and molecular mechanisms of broccoli sprouts and microgreens. they have been proven to present an abundant source of important natural compounds, including glucosinolates, phenolics, flavonoids, vitamins, minerals, and pigments. in particular, glucosinolates and their hydrolysis products are the main components analyzed, followed by phenolic compounds with a detailed profile. moreover, the previous studies have focused on several biological activities of broccoli seedlings, such as antioxidant, anticancer, antimicrobial, and anti-inflammatory, as well as the potentially beneficial effects for patients with cancers, diabetes, and obesity. in general, they are non-toxic or have low toxicity. hopefully, this updated review, in addition to being a reference for consumers' food selections, might attract more attention to broccoli sprouts and microgreens and their further applications in the food and nutraceutical industries, or even in clinical studies as cancer chemopreventive agents. in the future, more bioactive compounds in broccoli sprouts and microgreens will be isolated, identified, and evaluated. further investigations are required for exploring unrecognized biological functions and their underlying mode-of-action and molecular mechanisms in both in vitro and in vivo models, such as cardiovascular protective, hepatoprotective, neuroprotective, or enzyme inhibitory potentials. besides, well-designed clinical trials should be carried out to confirm these health-promoting benefits of broccoli seedlings on humans. food security and sustainable intensification broccoli microgreens: a mineral-rich crop that can diversify food systems how circular will you eat? the sustainability challenge in food and consumer reaction to either waste-to-value or yet underused novel ingredients in food small-seeded legumes as a novel food source. variation of nutritional, mineral and phytochemical profiles in the chain: raw seeds-sprouted seeds-microgreens relationships between bioactive food components and their health benefits the role of functional foods, nutraceuticals, and food supplements in intestinal health polyphenolic profile and varied bioactivities of processed taiwanese grown broccoli: a comparative study of edible and non-edible parts implementation of phenols recovered from olive mill wastewater as uv booster in cosmetics a facile water-induced complexation of lycopene and pectin from pink guava byproduct: extraction, characterization and kinetic studies phenols recovered from olive mill wastewater as additives in meat products the food systems in the era of the coronavirus (covid- ) pandemic crisis. foods recovery of high added-value components from food wastes: conventional, emerging technologies and commercialized applications separation of functional macromolecules and micromolecules: from ultrafiltration to the border of nanofiltration selecting sprouts of brassicaceae for optimum phytochemical composition microgreens: production, shelf life, and bioactive components micro-scale vegetable production and the rise of microgreens microgreen nutrition, food safety, and shelf life: a review bioactive compounds and bioactivities of germinated edible seeds and sprouts: an updated review broccoli and radish sprouts are safe and rich in bioactive phytochemicals evaluation of the bioaccessibility of antioxidant bioactive compounds and minerals of four genotypes of brassicaceae microgreens alone or combined, redirect the biosynthesis of glucosinolates, phenolics, carotenoids, and chlorophylls in broccoli sprouts biochemical composition of broccoli seeds and sprouts at different stages of seedling development dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in helicobacter pylori-infected mice and humans brassica oleracea l. var. italica) sprouts as the potential food source for bioactive properties: a comprehensive study on in vitro disease models the natural compound sulforaphene, as a novel anticancer reagent, targeting pi k-akt signaling pathway in lung cancer analysis and anti-helicobacter activity of sulforaphane and related compounds present in broccoli (brassica oleracea l.) sprouts sulforaphane-enriched broccoli sprouts pretreated by pulsed electric fields reduces neuroinflammation and ameliorates scopolamine-induced amnesia in mouse brain through its antioxidant ability via nrf -ho- activation effects of broccoli sprout with high sulforaphane concentration on inflammatory markers in type diabetic patients: a randomized double-blind placebo-controlled clinical trial effect of broccoli sprouts on insulin resistance in type diabetic patients: a randomized double-blind clinical trial broccoli sprouts powder could improve serum triglyceride and oxidized ldl/ldl-cholesterol ratio in type diabetic patients: a randomized double-blind placebo-controlled clinical trial extraction, chemical characterization and biological activity determination of broccoli health promoting compounds health benefits and possible risks of broccoli-an overview physiological effects of broccoli consumption methods for determining bioavailability and bioaccessibility of bioactive compounds and nutrients pressurized hot water extraction (phwe) for the green recovery of bioactive compounds and steviol glycosides from stevia rebaudiana bertoni leaves evaluation of microwave-assisted extraction technology for separation of bioactive components of saffron (crocus sativus l.). ind. crops prod. , , potential use of pulsed electric technologies and ultrasounds to improve the recovery of high-added value compounds from blackberries the effects of conventional and non-conventional processing on glucosinolates and its derived forms, isothiocyanates: extraction, degradation, and applications emerging technologies for the production of nutraceuticals from agricultural by-products: a viewpoint of opportunities and challenges glucosinolate metabolism, functionality and breeding for the improvement of brassicaceae vegetables glucosinolates in broccoli sprouts (brassica oleracea var. italica) as conditioned by sulphate supply during germination glucoraphanin, sulforaphane and myrosinase activity in germinating broccoli sprouts as affected by growth temperature and plant organs influence of light on health-promoting phytochemicals of broccoli sprouts effects of cacl on the metabolism of glucosinolates and the formation of isothiocyanates as well as the antioxidant capacity of broccoli sprouts glucosinolates fortification of cruciferous sprouts by sulphur supplementation during cultivation to enhance anti-cancer activity antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion establishing the occurrence of major and minor glucosinolates in brassicaceae by lc-esi-hybrid linear ion-trap and fourier-transform ion cyclotron resonance mass spectrometry calcium sulfate treatment enhances bioactive compounds and antioxidant capacity in broccoli sprouts during growth and storage uvb light doses and harvesting time differentially tailor glucosinolate and phenolic profiles in broccoli sprouts uv-b irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors photosynthetic pigments, phenolic, glucosinolates content and antioxidant capacity of broccoli sprouts in response to nanoselenium particles supply assessment of the anticancer compounds se-methylselenocysteine and glucosinolates in se-biofortified broccoli (brassica oleracea l. var. italica) sprouts and florets bioavailability of isothiocyanates from broccoli sprouts in protein, lipid, and fiber gels genotypic effects on the phytochemical quality of seeds and sprouts from commercial broccoli cultivars effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design effect of sucrose and mannitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts sucrose enhances the accumulation of anthocyanins and glucosinolates in broccoli sprouts metabolism and antiproliferative effects of sulforaphane and broccoli sprouts in human intestinal (caco- ) and hepatic (hepg ) cells characterization of products from the reaction of glucosinolate-derived isothiocyanates with cysteine and lysine derivatives formed in either model systems or broccoli sprouts physiological and biochemical metabolism of germinating broccoli seeds and sprouts sulforaphane bioavailability from glucoraphanin-rich broccoli: control by active endogenous myrosinase calcium mitigates the stress caused by znso as a sulphur fertilizer and enhances the sulforaphane formation of broccoli sprouts effect of se treatment on glucosinolate metabolism and health-promoting compounds in the broccoli sprouts of three cultivars increasing antioxidant content of broccoli sprouts using essential oils during cold storage. agriculture (polnohospodárstvo) response surface optimization and identification of isothiocyanates produced from broccoli sprouts free radical scavenging, antiproliferative activities and profiling of variations in the level of phytochemicals in different parts of broccoli (brassica oleracea italica) absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract optimisation of enzymatic production of sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days comparative study of predominant phytochemical compounds and proapoptotic potential of broccoli sprouts and florets selenium enrichment of broccoli sprout extract increases chemosensitivity and apoptosis of lncap prostate cancer cells effect of broccoli sprouts on thyroid function, haematological, biochemical, and immunological parameters in rats with thyroid imbalance antiproliferative effect of bioaccessible fractions of four brassicaceae microgreens on human colon cancer cells linked to their phytochemical composition sorting out the value of cruciferous sprouts as sources of bioactive compounds for nutrition and health phenolic acids: natural versatile molecules with promising therapeutic applications effect of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts influence of sodium and maturity stage on the antioxidant properties of cauliflower and broccoli sprouts phenolic profile and antioxidant activity in selected seeds and sprouts energy regulated nutritive and antioxidant properties during the germination and sprouting of broccoli sprouts (brassica oleracea var. italica) morphometric characteristics, polyphenols and ascorbic acid variation in brassica oleracea l. novel foods: sprouts, microgreens and baby leaves effect of sprouting and light cycle on antioxidant activity of brassica oleracea varieties antioxidant and antiproliferative activities in different maturation stages of broccoli (brassica oleracea italica) biofortified with selenium anticancer and antioxidant activity of bread enriched with broccoli sprouts health-affecting compounds in brassicaceae daily intake of broccoli sprouts normalizes bowel habits in human healthy subjects acylated anthocyanins in broccoli sprouts phytochemicals in daucus carota and their health benefits bioavailability of phytochemicals. in phytochemicals-a global perspective of their role in nutrition and health bioavailability of glucosinolates and their breakdown products: impact of processing bioavailability of sulforaphane following ingestion of glucoraphanin-rich broccoli sprout and seed extracts with active myrosinase: a pilot study of the effects of proton pump inhibitor administration chemical and biological characterisation of nutraceutical compounds of broccoli potential efficacy of broccoli sprouts as a unique supplement for management of type diabetes and its complications oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options reactive oxygen species: the dual role in physiological and pathological conditions of the human body reactive oxygen species in health and disease analysis and antioxidant activity of extracts from broccoli (brassica oleracea l.) sprouts measurement of antioxidant activity review on in vivo and in vitro methods evaluation of antioxidant activity enhancement of antioxidant abilities and the lipoxygenase and xanthine oxidase inhibitory activity of broccoli sprouts by biotic elicitors induction of the phase response in mouse and human skin by sulforaphane-containing broccoli sprout extracts pharmacological targeting of cell cycle, apoptotic and cell adhesion signaling pathways implicated in chemoresistance of cancer cells sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells broccoli sprouts delay prostate cancer formation and decrease prostate cancer severity with a concurrent decrease in hdac protein expression in transgenic adenocarcinoma of the mouse prostate (tramp) mice maternal epigenetic regulation contributes to prevention of estrogen receptor-negative mammary cancer with broccoli sprout consumption inhibition of urinary bladder carcinogenesis by broccoli sprouts protection against uv-light-induced skin carcinogenesis in skh- high-risk mice by sulforaphane-containing broccoli sprout extracts temporal efficacy of a sulforaphane-based broccoli sprout diet in prevention of breast cancer through modulation of epigenetic mechanisms antimicrobial activity and phytochemical analysis of organic extracts from cleome spinosa which approach is more effective in the selection of plants with antimicrobial activity? evid plant phenolics and phenolic-enriched extracts as antimicrobial agents against food-contaminating microorganisms antimicrobial activity of broccoli (brassica oleracea var. italica) cultivar avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast complementary and alternative medicinal effects of broccoli sprouts powder on helicobacter pylori eradication rate in type diabetic patients: a randomized clinical trial nutraceutical improvement increases the protective activity of broccoli sprout juice in a aqueous extract of glucoraphanin-rich broccoli sprouts inhibits formation of advanced glycation end products and attenuates inflammatory reactions in endothelial cells glucoraphanin: a broccoli sprout extract that ameliorates obesity-induced inflammation and insulin resistance broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency broccoli sprouts in analgesia-preclinical in vivo studies effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -ef l authors: potter, gregory d. m.; wood, thomas r. title: the future of shift work: circadian biology meets personalised medicine and behavioural science date: - - journal: front nutr doi: . /fnut. . sha: doc_id: cord_uid: ef l shift work is commonplace in modern societies, and shift workers are predisposed to the development of numerous chronic diseases. disruptions to the circadian systems of shift workers are considered important contributors to the biological dysfunction these people frequently experience. because of this, understanding how to alter shift work and zeitgeber (time cue) schedules to enhance circadian system function is likely to be key to improving the health of shift workers. while light exposure is the most important zeitgeber for the central clock in the circadian system, diet and exercise are plausible zeitgebers for circadian clocks in many tissues. we know little about how different zeitgebers interact and how to tailor zeitgeber schedules to the needs of individuals; however, in this review we share some guidelines to help shift workers adapt to their work schedules based on our current understanding of circadian biology. we focus in particular on the importance of diet timing and composition. going forward, developments in phenotyping and “envirotyping” methods may be important to understanding how to optimise shift work. non-invasive, multimodal, comprehensive phenotyping using multiple sources of time-stamped data may yield insights that are critical to the care of shift workers. finally, the impact of these advances will be reduced without modifications to work environments to make it easier for shift workers to engage in behaviours conducive to their health. integrating findings from behavioural science and ergonomics may help shift workers make healthier choices, thereby amplifying the beneficial effects of improved lifestyle prescriptions for these people. simplistically, our species once lived by two "clocks." one of these clocks is the environmental clock, which generates roughly -h changes in the light/dark (ld) cycle. the other clock is the endogenous biological clock, which among other rhythms generates roughly -h (circadian) rhythms in biological outputs such as the sleep/wake cycle. prior to the introduction of artificial light at night, these two clocks were probably tightly synchronised ( , ) . following industrialisation, however, people can more easily work outside of conventional daytime hours, and - % of the working population now work shifts ( ) . the burden of shift work is striking: shift workers are not only at increased risk of accidents ( ) , they are also disposed to developing numerous diseases, including certain cancers, coronary heart disease, stroke, and type-two diabetes ( ) . few studies have explored whether shift work makes individuals prone to neurodegenerative diseases ( , ) , but shift work frequently disrupts biological rhythms and sleep, and such disturbances propagate a slew of pathobiological changes that contribute to neurodegeneration ( ) . while at the time of writing little is known about the effects of the novel coronavirus disease (covid- ) pandemic on the lives of many shift workers, we would be remiss not to mention that many healthcare professionals at the frontlines of the outbreak are currently working long shifts in conditions that dispose them to developing covid- ( ) . many of the chronic conditions associated with shift work are also associated with greater risk of poor outcomes in those with covid- as well as other coronavirus and influenza infections ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . as shift workers often work jobs considered essential during the covid- pandemic, improving the health of shift workers should become a key part of current and future pandemic preparedness. importantly, however, at present there is no strong evidence that people fully adapt to shift work ( ) . and considering that the unconventional schedules of shift workers also interrupt the lives of cohabiting non-shift workers, the burden of shift work is greater still. the purpose of this manuscript is therefore to summarise some ways by which we might be able to reduce this burden. optimising shift work schedules is fundamental to the health and productivity of shift workers. in general, it appears that most shift workers tolerate rapid, forward (clockwise) rotation schedules best ( ) . to support worker wellbeing, these shifts should each last no longer than h, have at least h of recovery between them, and amount to no more than h of work per week ( ) . to hone shift work schedules for individual workers, workers may benefit from having some control of their schedules. this autonomy helps account for differences between people in nonwork responsibilities, tolerance to shift work, and commuting to and from work. chronotype is another tailoring variable that is particularly germane to optimising shift work schedules. chronotype is defined as interindividual differences in the phenotypic expression of behavioural outputs regulated by the circadian system ( ) , the most conspicuous of which is the timing of the sleep/wake cycle, and in industrialised societies there exist large differences between individuals in their chronotypes ( ) . chronotype appears to modify the association between shift work schedules and risk of health problems ( ) , such that the health of early chronotypes may be especially negatively affected by working night shifts ( ) , whereas late chronotypes find working morning shifts particularly problematic ( ) . while it is not clear precisely why this interaction exists, shift workers who have closer alignment between their chronotypes and their work schedules appear to have more robust melatonin rhythms than their fellow shift workers, suggesting that they have better circadian system function ( ) . shift workers who have chronotypes that are better matched to their work schedules may also sleep better ( ) , and it is increasingly clear that circadian system and sleep health are essential to perhaps all facets of human health ( , ) . the period of each individual's circadian system is one determinant of his or her chronotype. in the absence of time cues (zeitgebers), the free-running period of the human circadian system is slightly longer than h, on average ( ) . the circadian system therefore needs to be synchronised (entrained) each day with the -h day, and shift work complicates this process. retinal light exposure is generally regarded as the most important stimulus in entraining the human circadian system ( ) , and changes in patterns of light exposure can rapidly and substantially shift circadian system timing (phase). this is especially true of short-wavelength light, which most potently suppresses melatonin synthesis ( ) . exposure to such light in the biological morning tends to advance circadian phase, whereas exposure to such light in the late biological evening tends to delay circadian phase. the implication of this is that it is possible to bolster how well-shift workers adapt to work schedules through timely use of means to increase exposure to high-intensity, shortwavelength light at specific times of day (e.g., by using lighttherapy lamps) and means to reduce exposure to such light at specific times of day (e.g., "blue-blocking" glasses and blue-light filtering apps on electronic devices). while not all studies that have used interventions to modify exposure to light in shift workers have proven beneficial, this inconsistency likely reflects marked heterogeneity in the methods used by researchers ( ), as well as large variation between people in how they respond to light ( ) . during darkness, retinal photoreceptors no longer register exposure to light, relaying this to the central clock in the circadian system (the suprachiasmatic nucleus), which in turn signals the pineal gland to synthesise melatonin. melatonin therefore acts as an endogenous marker of darkness, agonising its receptors in cells in numerous tissues to signal them to fulfil time-ofday-specific functions. simplistically, when the concentration of melatonin in the blood surpasses a certain threshold in humans who are melatonin-proficient, it is the biological night-time. conversely, when the concentration of melatonin is below this threshold, it is the biological daytime. melatonin supplementation can shift the phase of the circadian system ( ) . melatonin ingestion in the late biological afternoon tends to advance circadian phase, while ingestion in the early biological morning tends to delay it. melatonin is therefore a chronobiotic -an agent that can modify circadian phase. when timed appropriately, light exposure and melatonin ingestion additively shift circadian phase ( ) . a growing body of evidence also shows that exercise can shift circadian phase. early research demonstrated that min of cycling exercise each hour of night shifts helped workers adjust their circadian systems to a -h delay in bedtime ( ) . more recent work has begun to clarify the precise nature of the relationship between exercise and circadian phase, showing that treadmill exercise done in the early biological morning or early biological afternoon advances circadian phase, whereas the same exercise done in the biological evening delays it ( ) . this relationship is therefore similar to how timing of exposure to light affects circadian phase, and timely exposure to both light and exercise can also additively shift circadian phase ( ) . the influence of timing of food availability on patterns of activity in rats was documented as early as a century ago ( ) , and numerous studies of such "food anticipatory activity" have since implicated nutrition as an influence on circadian system timing. whereas the ld cycle is the primary zeitgeber for the suprachiasmatic nucleus, some scientists have hypothesised that the eating/fasting cycle may be the primary time cue for some peripheral clocks in the circadian system. we now know that changing the timing of food consumption rapidly alters the timing of gene transcription in peripheral clocks in mice, for example ( ) . recent work has shown that this may be true of humans too, for changing meal timing independently shifts the expression of some genes in peripheral tissues as well as the timing of the blood glucose rhythm, without changing the phase of the melatonin rhythm ( ) . we acknowledge, however, that lack of control of variables such as ld cycles in most studies of the effects of nutrition on the human circadian system mean that this is arguably the only study of people that fulfils at least one of the criteria for diet to be classified as a zeitgeber ( ) . it could be that entrainment to ld cycles largely nullifies any zeitgeber effects of nutrition ( ) . summarising the above, it is plausible that carefully timed exposure to light, melatonin ingestion, and exercise may result in additive shifts in the phase of the suprachiasmatic nucleus. as eating/fasting cycles appear to affect the phases of some peripheral circadian clocks, we anticipate that coordinated changes in all of these variables could be used to expedite adaptation to new shift work schedules. if one could estimate shift workers' circadian phases in real time and model how subsequent changes in zeitgeber schedules would influence their circadian systems, one could develop tools that use this information to expedite adaptation to shift schedule changes by providing personalised guidance and perhaps even individual-level changes in exposure to light. this may be a particularly fruitful topic for further study. while it is plausible that one could change nutrient timing to accelerate adaptation to new shift work schedules, in many instances shift workers do not seek to fully adjust to their new shifts. this raises the question of whether workers undergoing transient changes in work schedules should adjust their diets accordingly. however, it is also crucial to consider contextual factors that influence when shift workers eat and drink. work schedules, time constraints, timing of breaks within shifts, family commitments, and prioritising behaviours such as sleep over meals all influence diet timing in shift workers, leading to erratic diet timing patterns in these people ( ) . diet timing irregularities are also affected by cultural factors (e.g., ramadan) and the nature of some jobs (e.g., many on-call workers have especially unpredictable work schedules). temporarily putting these complexities to one side, controlled experiments have begun to explore the effects of diet timing during pre-clinical and clinical simulations of shift work. table summarises our dietary and supplementation suggestions for shift workers based on our interpretation of the current literature. beginning with preclinical research, studies of mice have shown that restricting food access to the dark period (the active phase for these nocturnal animals) may protect against the obesogenic effects of repeated -h advances in the ld cycle ( ) . in addition, restricting food access to the active phase may also accelerate adaptation of circadian rhythms in core body temperature and locomotor activity to repeated -h changes in ld cycles ( ) . these findings imply that people would better cope with rotating shift work if they fixed their eating to the daytime, which is somewhat counterintuitive given that fixing eating time during shifting ld cycles might be expected to uncouple circadian rhythms between the suprachiasmatic nucleus and peripheral clocks. it is, however, intuitive that restricting food access to the active phase may be preferable to restricting it to the rest phase, and findings from initial research on humans support this contention. among healthy young men undergoing simulated night shift work for days, those who confined their consumption of calorie-containing foods and drinks (i.e., the caloric period) to between breakfast at : and dinner at : had superior postbreakfast glucose tolerance after the intervention compared to men who had dinner at : , a meal at : , and breakfast at : ( ) . the group that restricted food intake to the daytime also had superior overnight cognitive function ( ) . this is especially salient given that many shift workers redistribute their energy intakes into the night when working shifts ( ) . additional studies using larger sample sizes and investigating the effects of diet composition on a range of round-the-clock postprandial responses will be instructive. studies of adults undergoing time-restricted eating (tre) also indicate that optimising nutrient timing is likely to be important to cardiometabolic health, although the participants in these studies have generally not been shift workers. we arbitrarily define tre as consumption of all calorie-containing items within a period of h or less each day. conversely, we define workers should restrict consumption of all items containing > calories to a -to -h period each day, when possible. they should keep the timing of this period as regular as is feasible from day to day. workers should self-select the timing of this period, and the ideal time for this period may be relatively early in each worker's biological daytime. we therefore recommend that workers select a caloric period that finishes at least h before their most common bedtime. workers who have poor cardiometabolic health should aim to consume at least half of daily energy intake in the first half of the caloric period (e.g., by increasing the size of breakfast and reducing the size of dinner). this is less relevant to people who exercise in the second half of their caloric period. workers should also aim to evenly divide their protein intakes between dietary events. as a starting point, we recommend that workers aim to consume ∼ . g protein per kg bodyweight at each of to evenly-spaced dietary events each day ( ) . workers who have poor glycaemic control should consume carbohydrate-rich foods last at dietary events, when practical (e.g., consuming fibre-and protein-rich salads before meals or eating meat and vegetable foods before carbohydrate-rich foods). when workers feel it would be beneficial to snack outside of the caloric period (e.g., to abate hunger and/or support alertness), they may benefit from consuming relatively small (i.e., ∼ - % daily caloric intake), minimally processed, micronutrient-dense, satiating, easy to digest, convenient snacks. we hypothesise that relatively high-protein, low carbohydrate snacks are ideal at these times (e.g., snack items may include boiled eggs, dairy products, minimally-processed fish jerky or meat jerky, high-protein drinks, nuts, whole vegetables, and/or low-sugar whole fruits such as berries). if their goal is to support cognitive function during shifts, workers may benefit from individual doses of - mg caffeine per kg bodyweight, favouring the upper end of this range if short on sleep ( ) . repeated doses of caffeine every h or so may maximally support cognitive function during extended wakefulness ( ) . as consuming caffeine as gum leads to faster absorption than consuming caffeine as capsules ( ), caffeinated gum may be particularly helpful if the goal is to affect cognition as quickly as possible. since mistimed caffeine intake impairs sleep, workers should also stop consuming caffeine at least h before the main sleep period, if possible ( ) . individuals differ remarkably in their responses to caffeine ingestion, so they should moderate their intakes according to their individual responses. as a starting point, we recommend consuming no more than mg caffeine per kg bodyweight per h. creatine monohydrate consumption may help shift workers cope with sleep loss. during periods of insufficient sleep, we tentatively recommend that shift workers consume . g creatine monohydrate per kg bodyweight per day. because of its potential alertness-boosting properties, we speculate that the ideal time to consume creatine is with the first meal of each day. well-timed melatonin use may help some shift workers adapt to new work schedules and sleep better during these transitions. we tentatively recommend that workers consume a dose of . - mg melatonin at these times, beginning with a dose at the low end of this range and adjusting the dose according to responses. because of its potent chronobiotic properties, the optimal timing of melatonin ingestion depends on variables such as the individual's circadian phenotype and work schedule. we therefore do not offer guidance related to melatonin ingestion timing. intermittent fasting as periodic abstinence from consumption of any calories for at least h. skipping breakfast is one way to implement tre, and doing so leads to late tre. while breakfast-skipping is a controversial topic, epidemiologic studies have tended to associate breakfast consumption with lower risk of developing cardiometabolic diseases such as heart disease and type-two diabetes ( , ) . however, controlled studies have not shown large effects of skipping breakfast on cardiometabolic health ( ) . for example, lean adults who skipped breakfast for weeks inadvertently decreased their daily energy intakes, but this change was compensated by reductions in physical activity energy expenditure, resulting in no changes in energy balance or body composition ( ) . skipping breakfast did not affect most measures of cardiometabolic health -the only noteworthy difference between groups was that afternoon glycaemic variability was higher in adults who skipped breakfast. a subsequent study implemented the same intervention but only included obese adults ( ) . in this study, participants in the breakfast-skipping group expended less energy in the morning, but they did not burn fewer calories over the entire day. daily energy intake was similar in breakfast-skippers and breakfast eaters, and both groups gained weight during the study. people who skipped breakfast did have higher insulinaemic responses to an oral glucose tolerance test, however. these two rigorous studies show that skipping breakfast minimally affects energy balance but may negatively affect glycaemic regulation and some of its determinants. as sleep timing did not differ between groups, breakfast skipping led to a form of late tre, so these studies imply that late tre may not be optimal for some aspects of cardiometabolic health. skipping breakfast imposes a relatively late caloric period, and an alternative is to shorten the caloric period by way of skipping dinner or having an early dinner. several recent carefully controlled experiments have shown that such early tre may exert numerous positive effects on health. the first of these experiments reported that compared with a ∼ -h daily caloric period for weeks, weeks of early tre (∼ -h daily caloric period, finished by : ) improved insulin sensitivity, blood pressure, appetite regulation, and a marker of oxidative stress in men who have prediabetes ( ) . the same group of scientists recently reported that in overweight adults, just days of early tre reduced mean -h blood glucose levels and improved metabolic flexibility, among other benefits ( , ) . these experiments did not compare early tre to later tre while keeping the caloric period fixed, however, and to our knowledge, only one study has done this to date ( ) . the study in question showed that days of both early ( : to : ) and late ( : to : ) tre improved oral glucose tolerance in men at high risk of developing type-two diabetes, although only early tre lowered fasting glucose, suggest a small advantage of early tre ( ) . while this hypothesis needs careful testing, we believe that early tre may also enhance diet composition by reducing intakes of foods and drinks commonly consumed in the evening, such as processed snacks and alcohol. together, these studies support the superiority of relatively early tre in adults who have poor cardiometabolic health. however, non-self-selected tre schedules may interfere with some social activities and be difficult to adhere to in the context of work schedules and family commitments ( , ) . letting people self-select their tre periods helps mitigate these undesirable consequences. indeed, weeks of self-selected tre minimised these issues in adults with metabolic syndrome, also reducing daily energy intake and potently improving numerous aspects of cardiometabolic health including bodyweight, waist circumference, and blood pressure ( ) . moreover, tre led to more regular diet timing, which may independently be beneficial for cardiometabolic health ( ) . interestingly, tre also improved sleep timing regularity and increased how often participants selfreported restorative sleep. however, this study was an unblinded, single-arm study with only participants included in the data analysis ( ) . based on existing studies, tre appears to be a safe strategy that is likely to reduce energy intake, which would be especially beneficial for people who have unavoidably sedentary lifestyles. we hypothesise that fixing the timing of each worker's caloric period within regular hours each day supports metabolic health, and it is plausible that this may be especially important in workers who are subject to unpredictable changes in zeitgebers such as ld cycles (e.g., emergency service workers). we further speculate that each worker's biological daytime is the optimal time at which to fix the individual's caloric period, but self-selection of tre schedules will help people adhere to tre and avoid undesirable effects on social and family life. this said, scheduling tre as early as is practical may maximise the beneficial cardiometabolic effects of tre. while a detailed discussion of this subject is beyond the scope of this review, several recent controlled studies have shown that when daily energy intake is fixed, the distributions of energy and macronutrient intakes within the caloric period strongly influence cardiometabolic health. for example, one study divided overweight and obese women into two groups that consumed isocaloric weight loss diets for weeks ( ). one group consumed half of their daily energy intakes at breakfast, the other group consumed half at dinner. the group that consumed half at breakfast lost more than twice as much bodyweight, more than twice as many centimetres off their waists, and had greater improvements in oral glucose tolerance. subsequent work by the same scientists demonstrated that when energy intake is controlled, concentrating energy and carbohydrate intakes early in the day leads to enhanced appetite regulation, weight loss, and dramatic improvements in glycaemic control in adults with type- diabetes ( ) . this builds on research demonstrating that having carbohydrate-rich meals early in the day reduces -h glycaemia in adults with impaired fasting glucose and/or impaired glucose tolerance ( ) . while these studies highlight the advantages of concentrating energy and carbohydrate intakes relatively early in the caloric period, we note that that intelligent inclusion of physical activity leads to acute improvements in postprandial responses to dietary events such that relatively high energy and carbohydrate intakes late in the biological day may not be so problematic if they bookend exercise ( ) . and staying on the subject of exercise, there is tentative evidence that distribution of daily protein intake affects skeletal muscle protein synthetic responses to resistance training ( ) . as muscle protein synthesis is the main determinant of muscle protein balance, it is reasonable to assume that evenly dividing and spacing protein intakes between and daily dietary events may help maximise fat-free mass, a key determinant of cardiometabolic health ( ) . we would be negligent to not mention that the sequence of macronutrient intakes within dietary events may also meaningfully affect postprandial responses. several studies by one research group have shown that consuming carbohydrate last at a given dietary event (e.g., a full meal) dramatically reduces postprandial glycaemia and insulinaemia in adults who have prediabetes or type-two diabetes ( ) ( ) ( ) . shift workers who have poor glycaemic control may hence benefit from consuming carbohydrate-rich foods last at dietary events, when practical. most shift workers snack during night shifts. the problem is that night shifts often occur during the workers' biological nighttimes, and digestive and metabolic responses to dietary events are impaired during the biological night ( ) . as highlighted earlier, eating and/or drinking during the biological night-time may disrupt peripheral clocks. if workers snack during night shifts, it is therefore important to minimise energy intake and select dietary choices that lead to favourable postprandial responses. these snacks should also be convenient, minimally processed, micronutrient-dense, satiating, easy to digest, and minimally perishable, when applicable. preliminary research has shown that when -h energy and macronutrient intakes are controlled during simulated night shifts, a small snack (containing % of daily energy intake) may support cognitive function and performance in simulated driving compared with no snacking or a larger meal containing % of daily energy intake ( ) . in this instance, the small snack also reduced hunger to a comparable extent to the meal, without leading to significant digestive discomfort ( ) . compared to large night-time snacks, small night-time snacks may also be better for metabolic health. glycaemic control is relatively easy to measure and predictive of many health outcomes, and some researchers have therefore focused on the effects of nocturnal snacking on glycaemic control. compared with a small midnight snack (∼ calories), a large midnight snack (∼ calories) impaired postprandial glycaemic responses at a subsequent breakfast at : during simulated shift work ( ) . research such as this is informative, but we again need additional studies of workers in which the effects of dietary changes on metabolic parameters are measured around the clock. shift workers are not only apt to consume foods and drinks at suboptimal circadian phases, the quality of shift workers' diets is often worse than that of day workers too. many shift workers report consuming few fruits and vegetables while also consuming a variety of processed foods at work, such as biscuits, cakes, chocolates, pastries, sandwiches, and fried foods ( ) . as diet composition affects metabolic health and cognitive function, it is important to help these people make better dietary choices. one way by which diet composition influences health is via effects on the circadian clockwork, and the ketogenic diet (kd) exemplifies this. there has been a resurgence in interest in the kd of late, and while some believe that the restrictive nature of the kd is a barrier to its widespread implementation, certain properties of the kd make it an appealing option for some shift workers who are able to adhere to it. studies of mice have shown that the kd has chronobiotic actions on the clocks in multiple peripheral tissues, including the brain, gut, and liver ( ) ( ) ( ) . interestingly, tognini and colleagues found that a kd induced distinct changes in the liver and gut clocks in mice. compared to a control diet, consumption of a kd produced greater amplitudes of clock gene transcription and their downstream products in the liver, as well as inducing -h oscillations in the transcription of many genes in the gut ( ) . as disruption of the gut clock is associated with increased intestinal inflammation and permeability, as well as endotoxaemia ( , ) , if translatable to humans these results suggest that shift workers who follow a kd may protect themselves against some of the adverse consequences of consuming calories at suboptimal circadian phases. more generally, both the kd and less severe carbohydrate restriction may reduce some negative effects of shift work on metabolic health. shift workers are at an increased risk of impaired glucose tolerance and type-two diabetes, and restricting carbohydrate intake is likely to reduce fasting and postprandial glycaemia, both of which are precursory to numerous chronic diseases (e.g., some cardiovascular diseases, certain cancers, and dementia) ( ) ( ) ( ) ( ) ( ) ( ) . preliminary evidence has shown that a multicomponent lifestyle intervention centred on the kd may also improve subjective sleep quality in adults who have poor glycaemic control ( ) , suggesting that sleep enhancement may mediate some of the reported benefits of the kd. in preclinical studies, ketone bodies themselves have been found to have pleiotropic beneficial physiological effects, including modulation of inflammation, tissue-specific suppression of mtor signalling, and increased production of brain-derived neurotrophic factor ( ) ( ) ( ) . if translatable to humans, these systemic effects of ketone bodies imply that long-term consumption of a kd could reduce risk of certain cancers and neurodegenerative diseases such as alzheimer's in shift workers, particularly those that are already at increased risk ( , ) . increased production of ketone bodies may also account for some benefits of fasting and tre. for example, early tre led to greater morning beta-hydroxybutyrate levels compared to a -h caloric period ( ) . however, there have not yet been any clinical trials of the kd in shift workers, and it will be interesting to explore how the combination of the kd and tre and/or intermittent fasting interact to affect ketosis, metabolic regulation, and circadian biology in these people. in addition to effects of dietary patterns on the circadian system, specific dietary compounds have chronobiotic actions. a multitude of dietary compounds affects the circadian system and sleep ( , ) , and it is beyond the scope of this article to discuss them all. we therefore focus on some of those that we anticipate may be practical and beneficial for shift workers. in the future, screens for novel chronobiotics and hypnotics may yield compounds that support the health and performance of these workers ( ) . identifying agents that counter decrements in health and cognitive function incited by sleep disruption would also benefit shift workers. largely by antagonising adenosine receptors, consumption of caffeine can improve alertness, attention, reaction time, and mood, as well as physical performance in tests of endurance, strength, and power ( ) . studies of caffeine consumption by shift workers have consistently shown beneficial effects on multiple aspects of cognitive function, although whether this results in improved safety is not clear ( ) . the trade-off is that caffeine consumption tends to prolong sleep latency, reduce slow-wave activity during sleep (which is important to numerous restorative processes), shorten sleep duration, fragment sleep, and worsen subjective sleep quality ( ) . consumed late in the day as coffee, caffeine also delays circadian phase ( ) . thus it is clear that while judicious caffeine intake can be used to help shift workers perform at work -especially when sleepy -mistimed caffeine intake may strongly degrade sleep, which is noteworthy given that many of the adverse consequences of shift work appear to relate to its detrimental effects on sleep ( ) . it therefore seems prudent to recommend that shift workers generally stop consuming caffeine several hours before their main sleep period (more specific guidance on caffeine intake is provided in table ). antagonising adenosine receptors is one way to reduce the accumulation of pressure to sleep (sleep homeostasis), but another is to bolster the phosphorylation of adenosine. creatine (creatine monohydrate, specifically), a safe and inexpensive dietary supplement that increases brain phosphocreatine stores, countering the accumulation of extracellular adenosine in the brain during extended wakefulness. a study of rats showed that adding creatine to the rats' chow for weeks reduced the duration and slow-wave activity of the rats' sleep ( ). we do not currently know the effects of creatine supplementation on sleep in humans, however. notably, while shorter sleep would generally be expected to impair health and performance, creatine supplementation has repeatedly been shown to enhance these variables in humans. creatine supplementation routinely improves performance in -and adaptations to -many exercise tasks, and creatine has a number of therapeutic actions, including neuroprotective properties ( ) . interestingly, creatine supplementation may also acutely help protect against the deleterious consequences of sleep loss. after sleep loss, creatine supplementation seems to offset deterioration in executive function, mood, reaction time, balance, and other motor skills ( ) ( ) ( ) . although we expect creatine supplementation to be a useful strategy to help but this people cope with shift work, we are not aware of any research on this topic. we also note that there is some evidence that concurrent consumption of caffeine may reduce some of the ergogenic effects of creatine on physical performance ( ) , and additional studies are needed to better identify how the two compounds interact. several dietary amino acids may influence circadian rhythms and sleep. for instance, l-tryptophan is a precursor to melatonin that researchers have studied with respect to circadian rhythms and sleep. as an example, there appears to be a temporal relationship between consumption of l-tryptophan in breast milk and infant urinary excretion of -sulfatoxymelatonin, the primary metabolite of melatonin ( ) . furthermore, infants fed l-tryptophan-enriched night-time formula seem to experience more consolidated sleep/wake patterns ( ) . many studies of adults have also shown that ∼ g l-tryptophan each day enhances some sleep parameters, although it is not a potent hypnotic ( ) . to our knowledge, there are no rigorously controlled studies demonstrating that l-tryptophan affects circadian phase, however. overall, there has been little research on whether amino acids affect circadian system parameters. in a screen of whether amino acids affect light-induced shifts in the phase of wheel running activity in mice, l-serine increased the magnitude of phase shifts by %. this effect seems to translate to humans, as adults who consumed l-serine before bedtime experienced a greater advance in circadian phase in response to bright light exposure ( ) . another study reported that week of lornithine supplementation delayed the plasma melatonin rhythm by min ( ) . however, ld cycles and meal timing were not fully controlled in these studies. interestingly, there is also preliminary evidence that regular l-ornithine supplementation ( mg per day) may enhance sleep quality during stressful periods ( , ) . l-glycine may too affect sleep. consuming g l-glycine an hour before bedtime appears to shorten sleep latency, increase sleep efficiency, and reduce daytime sleepiness in healthy adults, effects that appear to be mediated via the suprachiasmatic nucleus ( , ) . such supplementation also seems to diminish daytime fatigue and boost vigilance during sleep restriction ( ) , implying that l-glycine may both enhance sleep and the ability to cope with sleep loss. given that l-glycine is safe, inexpensive, and may confer other health benefits ( ) , night shift workers could gain from supplementing with this amino acid. at present, however, there has been little research on effects of this amino acid on sleep. in summary, it is plausible that supplementing with certain amino acids may help shift workers adapt more quickly to changes in their shifts and/or sleep better, but this is based on few studies that did not control zeitgeber cycles or explore whether the circadian timing of amino acid ingestion interacts with the circadian timing of light exposure. going forward, it will be important to address these limitations. it will also be interesting to see whether concurrent consumption of different chronobiotic agents additively boosts circadian phase shifts. we have mentioned several ideas for future studies, and we will end by focusing on additional research avenues that may be worth exploring with respect to improving the health of shift workers. rapid recent advances in the development and uptake of digital technologies such as smartphones, apps, wearables, and artificial intelligence provide scientists with an unprecedented ability to comprehensively assess people's behaviours and health in freeliving contexts. the mycircadianclock app is a salient example of such technology. this app has already been used in multiple studies to monitor the circadian phenotypes of study participants, unveiling interesting insights into the effects of interventions such as tre on human health ( , ) . as data collected from digital devices are time-stamped, it is easier than ever to temporally map behavioural patterns and their biological sequelae, which could provide novel insights into the causes of changes in the health trajectories of shift workers. one could identify the hours of the day in which it is most frequently a shift worker's biological daytime by longitudinally assessing the timing of the individual's biological clock. as it is not currently practical to assess an individual's melatonin rhythm on a daily basis, the integration of data from surrogate markers of circadian phase such as body temperature and sleep/wake cycles could be used to approximate the timing of the biological daytime. these cycles could be monitored ambiently using data from devices such as smartphones, and the data from the devices could then be used to inform individual shift workers about how to best implement tre. where feasible, this process could be refined figure | using digital devices to optimise the health and performance of shift workers by providing personalised guidance, work schedules, and zeitgeber schedules. ( ) digital devices can be used to monitor behaviours and their biological responses, including (i) exposures to variables such as light (e.g., via head-worn wearables), (ii) dietary behaviours (e.g., via food photography and continuous glucose monitoring), and (iii) physical activity (e.g., via smart watches). digital phenotyping using patterns of smartphone use can be used to enrich this analysis. ( ) these data and their interactions can then be analysed in real time and used to ( ) personalise guidance, shift schedules, and zeitgeber schedules of individual workers. guidance can be delivered digitally and in-person, and innovative technologies may eventually allow the automation of adjustments to individual workers' zeitgeber schedules. with the addition of round-the-clock measures of metabolic regulation, such as continuous glucose monitoring. at a small scale, the feasibility of this type of approach has already been shown ( ) . ultimately, implementing such methods at a large scale and including both shift workers and non-shift-working controls may help develop models that forecast transitions in the health of shift workers, as well as how to alter these trajectories. however, the data collection process will need to be relatively frictionless (for participants, at least) to achieve this. this will be facilitated by close collaboration between scientists and workers in the technology sector. with accurate monitoring in place, digital tools could then be implemented to improve the health and productivity of shift workers by optimising variables such as zeitgeber schedules in real time (figure ) . innovative technologies could also provide novel means of generating insightful data while minimising participant burden. for example, sensors commonly built into smartphones can now be used to monitor blood parameters such as haemoglobin that once required invasive testing ( ) . smartphones can also be used to monitor some exposures that are particularly relevant to shift workers, such as patterns of locomotion and exposure to light. one problem, however, is that it would be especially useful to assess exposure to light at the level of the eye. this requires new wearable devices, for smartphones are not suited to this, and many existing wearables that measure light exposure are frequently obstructed by clothing, confounding their data. it is possible to make smart eyewear to estimate retinal light exposure, and such eyewear may be especially useful for another purpose. the utility of all of these monitoring technologies may be enhanced by the addition of the ability to digitally "envirotype" individuals, ambiently tracking information about their environments to better understand the interaction between environment and phenotype ( ) . building camera technology into eyewear is one way to accomplish this. meanwhile, digital phenotyping -assessing changes in people's phenotypes using data from digital devices -has already been used to identify patients' disease trajectories in neurological disorders such as schizophrenia ( ) . such phenotyping can proceed without active user engagement, and it can also be used to assess behaviours such as sleep ( ) . ultimately, use of multimodal novel sensors that analyse biofluids including interstitial fluid (e.g., continuous glucose monitoring), saliva, sweat, and tears may prove particularly useful in monitoring variables such as dietary intakes and associated changes in metabolites ( ) . however, the development of these sensors poses substantial challenges related to biofouling, accuracy, power, usability, calibration, and data security. these tools are promising approaches to forecasting changes in behaviours and health, and we hope they will help healthcare professionals intervene before individuals succumb to disease. we foresee that using sophisticated computational methods such as deep learning to concurrently analyse individuals' behavioural, health, and environmental data from multimodal sources will eventually enhance personalisation of guidance for individual shift workers ( ) . even if shift workers understand precisely which behaviours they should enact to improve their health, they are prone to a variety of factors that impair decision making, such as circadian system misalignment and sleep loss ( , ) . furthermore, knowledge alone is rarely sufficient to support lasting health behaviour change ( ) . it is therefore imperative to support the ability of these people to make smart decisions, and this requires applying principles from behavioural science, particularly at the level of the organisations that employ shift workers. significantly, many new technologies are strikingly habitforming, and this exemplifies the power of applying behavioural science principles to shape behaviour. if scientists and technologists can collaborate to effectively use behavioural science to create engaging, scalable products that deliver tailored health guidance to shift workers, all would benefit. we believe that technologies that deliver adaptive interventions to both help people avoid poor health decisions during states of vulnerability and support good health decisions during states of opportunity will be particularly advantageous ( ) . the built environment also affects health in numerous ways ( ) , and given that shift workers are prone to health problems, it is particularly critical to pay attention to optimising the workplaces of these people. as shift workers commonly experience circadian system disruption and do not gain tolerance to such disruption ( ) , it may be valuable to create workplaces that allow close control over exposure to light, and intelligent use of "smart" lighting systems may benefit these individuals. we also anticipate the development of closed-loop devices that will personalise light exposure at the level of the individual. the built environment influences physical activity. to support job performance and health, workplaces should have designated exercise spaces to encourage physical activity. environmental design is relevant to nutrition too. dietary choices depend strongly on where foods and drinks are sourced from. in work settings such as airplanes, food is provided for shift workers. however, most shift workers are left to source their own food, and when short on time many shift workers buy foods and drinks from vending machines ( ) . it is encouraging that many workers do select healthier dietary choices when they are available in vending machines, providing an opportunity for organisations to positively affect their employees' health decisions ( ) . furthermore, workplace interventions to promote healthier diets, such as offering free fruit and labelling meals, have sometimes been shown to facilitate healthy dietary choices ( , ) . simple changes in the placement of food in eating areas affect food selection too ( ) , and these changes can be leveraged to support the health of shift workers. similarly, if workers are using products such as melatonin supplements and blue-light-blocking glasses to shift the phases of their circadian systems, it makes sense to help them acquire efficacious products. it is also clear that social life is a strong influence on many shift workers' health behaviours, including their diets. the dietary attitudes and preferences of co-workers affect some workers' dietary choices ( ) , so group commitment to healthier dietary choices may aid the adoption of more nutritious diets. as stress strongly affects dietary choices in many people and shift workers often report high stress and abnormal dietary behaviours ( ) , interventions to nurture the resilience of shift workers and to improve workers' self-regulation skills may support their dietary choices. such interventions include mindfulnessbased approaches ( ) . shift workers could also benefit from other types of social support, including provision of additional childcare, as well as groups and events designed to minimise conflicts between their work and non-work activities. educating shift workers about how to sleep better is likely to be pivotal to their well-being, and shift work workplaces should have spaces for sleepy workers to nap. it is of course important to identify workers who have sleep disorders too, and simple screening tools such as brief questionnaires can be used for this ( ) . it may too be useful to screen for people who are simply not suited to certain shift schedules, for people differ substantially in how they tolerate shift work. certain characteristics associate with better shift work tolerance, including robust general health; young age; male sex; not having children; low languidity and neuroticism; high extraversion, flexibility in sleeping habits, and internal locus of control; and a chronotype that is neither very early nor very late ( ) . promisingly, personalising shift work schedules by removing night shifts for early chronotypes and excluding morning shifts for late chronotypes has been shown to prolong selfreported sleep, improve subjective sleep quality, and enhance worker well-being ( ) . to estimate chronotype, a study by vetter and colleagues used a shift work-specific version of the munich chronotype questionnaire ( ) , and this approach may be useful to help personalise work schedules for shift workers. nonetheless, it would be useful to develop additional questionnaires designed specifically to identify appropriate shift schedules, as well as to track how workers respond to these schedules. finally, it is worth noting that many workplace wellness programmes that have been tested have not yielded impressive results ( ) . assessing the effects of workplace interventions is difficult for numerous reasons, not the least of which are enforcing blinding and randomisation of participants. to date, marked heterogeneity between studies has made it challenging to assess the utility of workplace interventions for shift workers ( ) . and as is so often the case, the participants included in many of these studies did not comprise a diversity of ages and races, nor did the scientists attempt to define determinants of which workers responded positively to the interventions. none of this means that it is not possible to implement effective programmes, however, and we hope that lacklustre results to date do not stymie continued efforts to improve on workplace interventions by better incorporating principles from behavioural science. to assess the efficacy of interventions to improve shift-worker health, it may make sense to use alternatives to many of the hitherto-used study designs. recently, studies applying "big data" approaches have contributed to some advances in efforts to personalise medicine. however, it may be advantageous to concurrently carry out studies that use a "small data" paradigmfor example, using n-of- approaches to more rapidly assess how individual workers are responding to a given intervention and to forecast which of them are at risk of health trajectory transitions towards disease ( ). a large proportion of the workforce works shifts, and these individuals are integral to sustaining functional societies. however, the study of how to support the long-term health and well-being of these people has been somewhat neglected, and a relatively small proportion of relevant studies has included shift workers as participants. while the type of personalised interventions to support shift workers that we have discussed in this article are bound to produce logistical headaches for employers, the onus should be on supporting the long-term the health and performance of their employees. the acute difficulties arising from implementing customised shift schedule systems and suchlike may be more than made up for by the lasting benefits of these systems on health, safety, and productivity. we note also that as shift work increases the likelihood of adverse pregnancy outcomes and may lead to epigenetic modifications in parents that could plausibly affect the epigenetics and hence health of their children, supporting the health of shift workers could one day have critical effects on the well-being of future generations ( , ) . scientists now have an unprecedented ability to identify ways of helping shift workers. we hope that this ability is realised in the near future. entrainment of the human circadian clock to the natural light-dark cycle circadian entrainment to the natural light-dark cycle across seasons and the weekend working group on the evaluation of carcinogenic risk to humans. painting, firefighting, and shiftwork shift and night work and long working hoursa systematic review of safety implications health consequences of shift work and insufficient sleep occupational history of night shift work and parkinson's disease in denmark shift work and overall and cause-specific mortality in the danish nurse cohort. scand j work environ health mechanisms linking circadian clocks, sleep, and neurodegeneration when health professionals look death in the eye: the mental health of professionals who deal daily with the coronavirus outbreak presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area ons. deaths involving covid- , england and wales: deaths occurring in middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) severe acute respiratory syndrome: clinical and laboratory manifestations a novel risk factor for a novel virus: obesity and pandemic influenza a (h n ) h n influenza a in a hospital in norway working time society consensus statements: individual differences in shift work tolerance and recommendations for research and practice circadian disruption: what do we actually mean? epidemiology of the human circadian clock mismatch of sleep and work timing and risk of type diabetes shift work patterns, chronotype, and epithelial ovarian cancer risk sleep and need for recovery in shift workers: do chronotype and age matter shift work, chronotype, and melatonin rhythm in nurses the circadian clock and human health sleep, health, and society stability, precision, and near- -hour period of the human circadian pacemaker physiology of circadian entrainment effect of light wavelength on suppression and phase delay of the melatonin rhythm health-related interventions among night shift workers: a critical review of the literature high sensitivity and interindividual variability in the response of the human circadian system to evening light physiology and pharmacology of melatonin in relation to biological rhythms evening melatonin and bright light administration induce additive phase shifts in dim light melatonin onset phase-shifting human circadian rhythms with exercise during the night shift human circadian phase-response curves for exercise circadian phase-shifting effects of bright light, exercise, and bright light + exercise a behavioristic study of the activity of the rat restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus meal timing regulates the human circadian system food as a circadian time cue -evidence from human studies phase-shifting human circadian rhythms: influence of sleep timing, social contact and light exposure the factors influencing the eating behaviour of shiftworkers: what, when, where and why protein "requirements" beyond the rda: implications for optimizing health a review of caffeine's effects on cognitive, physical and occupational performance multiple dose pharmacokinetics of caffeine administered in chewing gum to normal healthy volunteers the rate of absorption and relative bioavailability of caffeine administered in chewing gum versus capsules to normal healthy volunteers caffeine effects on sleep taken , , or hours before going to bed time-fixed feeding prevents obesity induced by chronic advances of light/dark cycles in mouse models of jet-lag/shift work time-restricted feeding improves adaptation to chronically alternating light-dark cycles timing of food intake during simulated night shift impacts glucose metabolism: a controlled study the impact of meal timing on performance, sleepiness, gastric upset, and hunger during simulated night shift. ind health dietary patterns of nurses on rotational shifts are marked by redistribution of energy into the nightshift meta-analysis of relation of skipping breakfast with heart disease breakfast skipping is associated with increased risk of type diabetes among adults: a systematic review and meta-analysis of prospective cohort studies is breakfast the most important meal of the day? the causal role of breakfast in energy balance and health: a randomized controlled trial in lean adults the causal role of breakfast in energy balance and health: a randomized controlled trial in obese adults early time-restricted feeding improves insulin sensitivity, blood pressure, and oxidative stress even without weight loss in men with prediabetes early time-restricted feeding improves -hour glucose levels and affects markers of the circadian clock, aging, and autophagy in humans early timerestricted feeding reduces appetite and increases fat oxidation but does not affect energy expenditure in humans time-restricted feeding improves glucose tolerance in men at risk for type diabetes: a randomized crossover trial a pilot feasibility study exploring the effects of a moderate time-restricted feeding intervention on energy intake, adiposity and metabolic physiology in free-living human subjects a delayed morning and earlier evening time-restricted feeding protocol for improving glycemic control and dietary adherence in men with overweight/obesity: a randomized controlled trial ten-hour time-restricted eating reduces weight, blood pressure, and atherogenic lipids in patients with metabolic syndrome irregular meal-pattern effects on energy expenditure, metabolism, and appetite regulation: a randomized controlled trial in healthy normal-weight women high caloric intake at breakfast vs. dinner differentially influences weight loss of overweight and obese women reduction in glycated hemoglobin and daily insulin dose alongside circadian clock upregulation in patients with type diabetes consuming a three-meal diet: a randomized clinical trial the effect of diurnal distribution of carbohydrates and fat on glycaemic control in humans: a randomized controlled trial. sci rep exercise-stimulated glucose uptake -regulation and implications for glycaemic control timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis the impact of food order on postprandial glycaemic excursions in prediabetes food order has a significant impact on postprandial glucose and insulin levels carbohydrate-last meal pattern lowers postprandial glucose and insulin excursions in type diabetes circadian physiology of metabolism altering meal timing to improve cognitive performance during simulated nightshifts subjective hunger, gastric upset, and sleepiness in response to altered meal timing during simulated shiftwork eating on nightshift: a big vs small snack impairs glucose response to breakfast distinct circadian signatures in liver and gut clocks revealed by ketogenic diet ketogenic diet delays the phase of circadian rhythms and does not affect amp-activated protein kinase (ampk) in mouse liver ketogenic diet and fasting induce the expression of cold-inducible rna-binding protein with timedependent hypothermia in the mouse liver disruption of the circadian clock in mice increases intestinal permeability and promotes alcohol-induced hepatic pathology and inflammation systematic review and meta-analysis of different dietary approaches to the management of type diabetes effects of the mean amplitude of glycemic excursions and vascular endothelial dysfunction on cardiovascular events in nondiabetic patients with coronary artery disease prediabetes and associated disorders associations of prediabetes with all-cause and cardiovascular mortality: a meta-analysis prediabetes and the risk of cancer: a meta-analysis glucose peaks and the risk of dementia and -year cognitive decline. diabetes care improvement in patient-reported sleep in type diabetes and prediabetes participants receiving a continuous care intervention with nutritional ketosis β-hydroxybutyrate: a signaling metabolite a ketogenic diet extends longevity and healthspan in adult mice ketogenic diet improves the spatial memory impairment caused by exposure to hypobaric hypoxia through increased acetylation of histones in rats shift work and risk of incident dementia: a study of two population-based cohorts night shift work, long working hours and dementia: a longitudinal study of the danish work environment cohort study interdependence of nutrient metabolism and the circadian clock system: importance for metabolic health effects of diet on sleep quality regulation of circadian behaviour and metabolism by synthetic rev-erb agonists caffeine for the prevention of injuries and errors in shift workers coffee, caffeine, and sleep: a systematic review of epidemiological studies and randomized controlled trials effects of caffeine on the human circadian clock in vivo and in vitro creatine supplementation reduces sleep need and homeostatic sleep pressure in rats international society of sports nutrition position stand: safety and efficacy of creatine supplementation in exercise, sport, and medicine creatine supplementation, sleep deprivation, cortisol, melatonin and behavior effect of creatine supplementation and sleep deprivation, with mild exercise, on cognitive and psychomotor performance, mood state, and plasma concentrations of catecholamines and cortisol skill execution and sleep deprivation: effects of acute caffeine or creatine supplementation -a randomized placebo-controlled trial creatine and caffeine: considerations for concurrent supplementation the circadian rhythm of tryptophan in breast milk affects the rhythms of -sulfatoxymelatonin and sleep in newborn improved circadian sleep-wake cycle in infants fed a day/night dissociated formula milk effects of tryptophan loading on human cognition, mood, and sleep l-serine enhances light-induced circadian phase resetting in mice and humans a randomized, double-blind and placebo-controlled crossover trial on the effect of l-ornithine ingestion on the human circadian clock ornithine ingestion improved sleep disturbances but was not associated with correction of blood tryptophan ratio in japanese antarctica expedition members during summer randomised controlled trial of the effects of l-ornithine on stress markers and sleep quality in healthy workers glycine ingestion improves subjective sleep quality in human volunteers, correlating with polysomnographic changes the sleep-promoting and hypothermic effects of glycine are mediated by nmda receptors in the suprachiasmatic nucleus the effects of glycine on subjective daytime performance in partially sleep-restricted healthy volunteers glycine metabolism in animals and humans: implications for nutrition and health a smartphone app reveals erratic diurnal eating patterns in humans that can be modulated for health benefits a pilot characterization of the human chronobiome noninvasive hemoglobin measurement using unmodified smartphone camera and white flash digital envirotyping: quantifying environmental determinants of health and behavior relapse prediction in schizophrenia through digital phenotyping: a pilot study a comparison of passive and active estimates of sleep in a cohort with schizophrenia wearable biosensors for healthcare monitoring high-performance medicine: the convergence of human and artificial intelligence effects of circadian misalignment on cognition in chronic shift workers the sleep-deprived human brain why is changing health-related behaviour so difficult? public health just-in-time adaptive interventions (jitais) in mobile health: key components and design principles for ongoing health behavior support health and the built environment: years after effects of the internal circadian system and circadian misalignment on glucose tolerance in chronic shift workers vending assessment and program implementation in four iowa worksites a workplace feasibility study of the effect of a minimal fruit intervention on fruit intake effectiveness of offering healthy labelled meals in improving the nutritional quality of lunch meals eaten in a worksite canteen nudging consumers towards healthier choices: a systematic review of positional influences on food choice influences on dietary choices during day versus night shift in shift workers: a mixed methods study the association between shift duty and abnormal eating behavior among nurses working in a major hospital: a cross-sectional study an on-the-job mindfulnessbased intervention for pediatric icu nurses: a pilot questionnaires that screen for multiple sleep disorders aligning work and circadian time in shift workers improves sleep and reduces circadian disruption the munich chronotype questionnaire for shift-workers (mctqshift) effect of a workplace wellness program on employee health and economic outcomes: a randomized clinical trial the impact of occupational shift work and working hours during pregnancy on health outcomes: a systematic review and meta-analysis night shift work before and during pregnancy in relation to depression and anxiety in adolescent and young adult offspring gp conceived the idea and drafted the manuscript. tw edited the manuscript and produced the final draft. all authors approved the final version and performed the literature review. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © potter and wood. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -g vefajw authors: spisni, enzo; petrocelli, giovannamaria; imbesi, veronica; spigarelli, renato; azzinnari, demetrio; donati sarti, marco; campieri, massimo; valerii, maria chiara title: antioxidant, anti-inflammatory, and microbial-modulating activities of essential oils: implications in colonic pathophysiology date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: g vefajw essential oils (eos) are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants, most of them commonly used in the human diet. in recent years, many studies have analyzed their antimicrobial, antioxidant, anti-inflammatory, immunomodulatory and anticancer properties in vitro and on experimentally induced animal models of colitis and colorectal cancer. however, there are still few clinical studies aimed to understand their role in the modulation of the intestinal pathophysiology. many eos and some of their molecules have demonstrated their efficacy in inhibiting bacterial, fungi and virus replication and in modulating the inflammatory and oxidative processes that take place in experimental colitis. in addition to this, their antitumor activity against colorectal cancer models makes them extremely interesting compounds for the modulation of the pathophysiology of the large bowel. the characterization of these eos is made difficult by their complexity and by the different compositions present in the same oil having different geographical origins. this review tries to shift the focus from the eos to their individual compounds, to expand their possible applications in modulating colon pathophysiology. essential oils (eos) are a complex mixture of volatile compounds that are produced by aromatic plants as secondary metabolites. they are present in all plant organs and their main role is to defend plants from aggressions by bacteria, fungi and viruses, but also by insects. there are huge amounts of eos obtained from different plants around the world, and most of them have been at least partially characterized for their antimicrobial activity against gram-positive and gram-negative bacteria, but also against other microorganisms such as fungi and virus. the composition of the eos was selected by nature during an evolutionary process lasting millions of years. their activity is the result of the competitive selection process acting on their antibacterial, antifungal and antiviral activities in a continuous evolutionary conflict between the survival of plants and the microbial aggressions. the antimicrobial strategies of eos have been to develop multi-target mechanisms of action which make it very difficult for microorganisms to become resistant to these compounds [ ] . the enormous potentials for the possible use of eos as therapeutic agents against human pathogen the excessive amount of reactive oxygen species (ros) is at the basis of degenerative processes such as lipid peroxidation, protein glycation/oxidation and nitration, enzyme inactivation and dna damages that occur in many non-communicable diseases, including colitis and neurodegenerative diseases [ , ] . some eo single molecules, such as geraniol, easily cross the blood-brain barrier and then reach all the organs in which they can exert their antioxidant activities [ ] . ros exert multiple modulating effects on inflammation and play a key role in the regulation of immune responses [ ] . eos and their molecules are capable of modulating different signaling pathways that are overactivated or downregulated during acute or chronic inflammation responses [ ] . the recent discovery of the complexity of the human intestinal microbiota, composed of bacteria, fungi and viruses, and its intricate pathophysiological relationships with the immune system and the enteric nervous system, makes eos truly interesting for their antimicrobial activities, often selective for the different microbial components. from this point of view, eos could be considered potential powerful modulators of the intestinal microbiota. unfortunately, most eo molecules are quickly assimilated into the small intestine and do not reach the colon, where most of the intestinal microbiota is known to reside. the release of eos into the colon, therefore, becomes a fundamental point to allow these compounds to effectively modulate the pathophysiology of the colon. from this point of view, our research group is one of the first in the world to have used a single eo molecule in human clinical trials, to modulate the microbiota and manage the symptoms associated to a functional bowel disorder such as the irritable bowel syndrome (ibs) [ ] . this review explores, in a broad and modern way, the knowledge that can lead to the effective use of eos and/or their single molecules in the modulation of the physiology and pathology of the large bowel. the major components of the most common essential oils are shown in table , together with their chemical structure and major biological activity. several studies have been conducted to evaluate the effects of eos in colon inflammation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in particular, several preclinical studies have been conducted on validated models of colitis induced in rats or mice by administration of dextran sulphate sodium (dss), trinitrobenzenesulfonic acid (tnbs) or acetic acid. while dss and acetic acid induces chemical damage to the epithelial cells, with consequent disruption of mucosal barrier and activation of immune cells in the lamina propria by the intestinal microbiota, tnbs acts by directly haptenizing colonic autologous/microbial proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . from this point of view, dss and acetic acid are models used in a translational way for ulcerative colitis studies, while tnbs is a model with some characteristics that bring it closer to crohn's disease. these models allow researchers to evaluate the effects of different eos on clinical, histological, serological and molecular markers of colitis. clinical features of the experimental colitis are mainly represented by weight loss, changes in stool consistency and rectal bleeding, usually scored to obtain a disease activity index (dai). histological analyses are performed to evaluate the extent of ulcerated areas, alterations of mucosal architecture and inflammatory cell infiltrations. the molecular markers that are more often evaluated comprise some circulating pro-inflammatory cytokines and the colonic expression of a plethora of enzymes and factors involved in inflammation. they include cyclooxygenase- (cox- ), nitric oxid synthase (inos), peroxisome proliferator-activated receptor-gamma (pparγ), nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kβ) or myeloperoxydase (mpo) [ ] . the anti-inflammatory effects of eos on colitis models are summarized in table . with regard to the mechanisms of action of eos and their single molecules at the cellular and molecular levels, we underline that it is almost impossible to understand which molecule may exert anti-inflammatory effects when eos are used. even in studies carried out on single eo molecules, it is not easy to define a main molecular target, because these compounds act in a multitarget manner ( figure ). for example, the anti-inflammatory effect of these molecules could be linked to direct interaction with pro-inflammatory proteins, such as nf-kβ, pparγ cox- and inos, but this direct interaction still remains to be demonstrated for the majority of these molecules. a specific interaction between borneol, camphene and eucalyptol, major components of thyme eo, and pro-inflammatory enzymes such as inos and cox- has been only indirectly demonstrated in vitro [ ] . the multitarget effect of geraniol has been clearly demonstrated in vitro, since it is capable of regulating wnt/β-catenin, p mapk, nfκb, pparγ and cox- signaling pathways. the transcription factor nf-kβ seems to be a common target for many different eo molecules, such as geraniol, eucalyptol, α-pinene, and camphor [ ] , even if a definitive demonstration of the binding between these molecules and their supposed target is still lacking and should be provided by using molecular modeling studies. conceptually, however, these studies on colitis should be divided between those that delivered eo as it is, without carrier or controlled release and those in which the eo was delivered with retarded release systems, in order to really reach the colon. only by using these controlled release systems can the effects of these eos be analyzed at the colon level, where the experimentally-induced inflammation mainly occurs. basing on available studies, we can conclude that eos and their constituents could be very effective against the inflammatory component of experimental colitis. unfortunately, eo complexity makes it difficult to identify all the possible molecular targets responsible for these strong anti-inflammatory effects. the majority of studies on the antioxidant effects of eos, available in the scientific literature, are based on in vitro approaches. a limiting factor of these studies is that eos can themselves be oxidized within the intestinal lumen or into the stomach, thus losing some of their antioxidant properties even before reaching the small intestine. geraniol showed good antioxidant proprieties in vitro: palmrose and citronella eos, mainly composed of geraniol, have demonstrated to have an effective antioxidant activity in vitro on human lymphocyte cells. in this model, geraniol-containing eos, at a relatively low concentration ( ppm), protected lymphocytes from dna methylation damages induced by methyl methanesulfonate [ ] . these serum doses of geraniol are easy to achieve with a diet rich in aromatic plants or with food supplements [ ] . recent studies have shown that geraniol administration reduced the intestinal inflammation induced by dss [ ] , but these anti-inflammatory effects could be also linked to its antioxidant activity, since its administration resulted in a decreased inos activity and a decreased lipid peroxidation, in a rat model of colitis [ ] . geraniol seems to exert its antioxidant activity also indirectly, by increasing the synthesis of liver antioxidant enzymes after oral administration at mg/kg, in mice [ ] . ginger eo administration at , and mg/kg, significantly reduced the intestinal lipid peroxidation, increased the expression of intestinal antioxidant enzymes and serum glutathione level in a model of colitis induced by acetic acid in rats. in this model, ginger eo was able to induce free radicals neutralization and to protect the cell membrane lipids from oxidation, in a dose-dependent manner [ ] . a recent study has evaluated antioxidant proprieties of carvacrol ( -isopropyl- -methylphenol) which is a major monoterpenic component of origan (oreganum vulgarea), a plant widely used in mediterranean cuisine. carvacrol was used in a model of experimental induction of colorectal carcinoma, by using , dimethylhydrazine and dss, in rats. carvacrol was orally administrated before and after tumor induction at a dosage of mg/kg. results of this and other studies showed that carvacrol was an excellent antioxidant agent and reduced colonocyte damages caused by ros [ , ] . thymol is a natural phenol monoterpene isomer of carvacrol. thymol is one of the major constituents ( - %) of thyme (thymus vulgaris) eo. an in vitro study on colon epithelial cells showed that thymol, at low doses ( . ppm), was a protective compound against oxidative dna damage [ ] . twelve aromatic molecules from basil and thyme eos have been analyzed for their antioxidant activities, and in particular eugenol, -allylphenol, thymol and carvacrol ( µg/ml) have shown greater antioxidant activities, measured in vitro on by using the aldehyde/carboxylic acid assay [ ] . basil eo, orally-administered, has shown beneficial effects in a model of colitis induced by acetic acid in rats, at two different doses of mg/kg and mg/kg die. these effects were also linked to the reduction of mpo activity in the colon wall, an enzyme clearly involved in the oxidative damages induced by colitis [ ] . despite this, further confirmations should be provided by human clinical trials. basing on available data we can confirm that eos and their constituents have interesting antioxidant properties that could justify their use as therapeutic agents against chronic intestinal oxidative damages ( figure ). . antioxidant effect of essential olis (eos) in the gut. the chronic low-grade inflammation or dysbiosis that very soon occurs into the gut and in the gut wall increases the level of reactive oxygen species (ros). their increased levels are effectively counteracted by eos that are able to reduce the activity and expression of enzymes, such as myeloperoxydase (mpo), cyclooxygenase- (cox- ) or inducible nitric oxide synthase(inos), which are the ones most responsible for ros production and for the oxidative damages related to them. there are approximately trillion cells in the human body, and more than % of them are microbes. they make up the human microbiota, consisting of bacteria, fungi and even viruses, mainly located in the intestine where they form the intestinal microbiome. microbiota can be considered a complex human organ which closely interacts with the gut-associated linfoid tissue (galt) and with the enteric nervous system. it is involved in many digestive functions, but it is also able to modulate the physiology of the immune system both locally and in the whole body. quantitative and qualitative microbiota alterations, known as dysbiosis, may be involved in the development or in the chronicization of several diseases [ ] . the analysis of the bacterial component of the intestinal microbiota, thought their s rrna sequences, allowed to identify major phyla, firmicutes ( %), bacteroidetes ( %), actinobacteria ( %) and proteobacteria ( %). at a lower taxonomic level, the most represented bacterial genera were found to be faecalibacterium, ruminococcus, eubacterium, dorea, bacteroides, alistipes and bifidobacterium [ ] . the microbiota normally represents an ecologically stable environment, but pathogenic bacterial strands or xenobiotics can interfere in this equilibrium and give rise to dysbiosis and/or colitis. the use of broad-spectrum antibiotics to counteract infectious diseases is often associated with the onset of antibiotic resistance phenomena, other than cause a transient dysbiosis in the gastrointestinal tract. in the last few years, a great effort has been made to find new strategies to overcome the rising issue of antibiotic-resistance. in this scenario, eos may have a consistent role thanks to their capacity to control and modulate bacterial growth, acting both as bacteriostatic or bactericidal agents [ ] . in fact, due to their lipophilic properties, eos can penetrate membranes, and damage bacterial cell structure, making their membranes more unstable and permeable. membrane disruption may also lead to bacterial death caused by the significant leak of ions and other essential cytosolic components. these eo effects are generally more pronounced on gram-positive bacteria with respect to gram-negative ones [ ] . however, it has been demonstrated that eos can also affect the bacterial cell-wall and restore antibiotic susceptibility in drug-resistant gram-negative bacterial strains [ ] . several studies have explored the antibacterial properties of eo single molecules. among them, the most studied was certainly geraniol, for its interesting antimicrobial potential. geraniol antibacterial activity seems to be linked to his property to destabilize bacterial cell wall and damage transmembrane efflux pumps, thus restoring drug-sensitivity in different bacterial antibiotic-resistant strains, such as enterobacter aerogenes, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii [ ] . despite being absorbed very quickly and in an active manner by the small intestine mucosa, geraniol is reported to positively modulate the colitis-associated dysbiosis when administered by oral route by using a controlled delivery system based on microencapsulation [ ] . in mice but also in humans, geraniol has demonstrated to act as an excellent modulator of intestinal microbiota, capable to boost populations of butyrate-producer bacteria such as collinsella and faecalibacterium, normally reduced in the dysbiotic human intestinal flora of ibs patients [ ] . it is interesting to note how geraniol antibacterial activities were selective for pathogenic bacteria and do not involved commensal species [ ] . for these reasons, geraniol can be considered as an efficient positive modulator of the intestinal microbiota. another interesting eo molecule with antibacterial activities is eugenol ( -methoxy- -(prop- en- -yl)-phenol), the major compound present in clove oil, but also found in many other eos. eugenol has demonstrated antimicrobial activities based on a non-specific permeabilization of the bacterial membrane with depletion of cytosolic molecules such as adenosin triphosphate (atp), necessary for bacterial metabolism and survival [ ] . this effect has been observed against e. coli, listeria monocytogenes and lactobacillus sakei using the relatively low concentration of mm [ ] . eugenol antibacterial effects against the pathogen l. monocytogenes have been analyzed in-depth and the principal mechanism of action identified was the alteration of the respiratory bacterial chain associated with dna damages [ ] . in mice, orally administrated eugenol improved the secretion of the intestinal mucus, creating a thicker intestinal layer associated with positive changes of the mucosal microbiota ecology. in particular, it has been shown that eugenol inhibited the intestinal adherence of citrobacter rodentium, a mice pathogen that shares several biochemical features with clostridium difficile in humans [ ] . another interesting antimicrobial effect of eugenol was observed in p. aeruginosa and e. coli, where this compound was able to inhibit their chemical communication system, also known as quorum sensing [ ] . cinnamaldehyde ( e- -phenylprop- -enal) is a phenylpropanoid naturally present in the plant of the genus cinnamon. cinnamaldehyde is one of the most studied eo molecules and it has been already approved as antimicrobial food preservative [ ] . antibacterial effects of cinnamaldehyde have been demonstrated by using many different bacterial models, but only a few studies evaluated its impact on the whole intestinal microbiota. in vitro, cinnamaldehyde was capable to inhibit the growth of potentially pathogenic bacteria such as staphylococcus aureus, enterobacter cloacae, a. baumannii and l. monocytogenes [ ] and it was able to kill a pathogenic strand of e. coli at a very low concentration ( . % v/v) [ ] . one of the proposed antibacterial mechanisms of cinnamaldehyde inhibition of e. coli growth was the inactivation of its acetyl-coa carboxylase enzyme [ ] . other studies showed that cinnamaldehyde antimicrobial activity has a broad spectrum of action, being effective also against enterococcus faecalis, enterococcus faecium, e. aerogenes salmonella enterica and clostridium perfringens [ ] . finally, with a concentration of µg/ml, cinnamaldehyde was also capable to improve the bactericidal efficacy of the antibiotic clindamycin on c. difficile, significantly decreasing its minimum inhibitory concentration (mic) from . to . µg/ml [ ] . in vivo, only a few studies have been conducted on cinnamaldehyde, perhaps because of its strong aggressiveness towards the mucosal epithelia. nevertheless, in animal experimental colitis, the oral administration of cinnamon eo (approx. % in cinnamaldehyde) at mg/kg or mg/kg lead to an improvement of the ecological biodiversity of the intestinal microbiota. short-chain fatty acids (scfa)-producing bacteria family, such as bacteroidaceae, were increased while intestinal helicobacter and bacteroides were reduced [ ] . in broiler and duck farming, the supplementation of food with a mixture of thymol and cinnamaldehyde improved animal growth performances and positively modulated their intestinal microbiota composition, boosting healthy bacteria and reducing anaerobic coliforms and lactose-negative enterobacteria [ , ] . thymol is effective at extremely low concentrations (as low as ppm) against the growth of many species of pathogenic bacteria that colonize the human intestine, such as c. difficile, c. perfringens, propionibacterium shermanii, propionibacterium freudenreichii and bacteroides thetaiotaomicron. the negative aspect of its antimicrobial activity is that thymol was not selective, and could also have a negative impact on commensal bacteria [ ] . on the other hand, eos in which thymol is a major component have clearly shown to have a wide-spectrum bactericide effects on different pathogenic species such as l. monocytogenes, e. coli, s. enterica, s. aureus, clostridium botulinum, c. perfringens, shigella sonnei, sarcina lutea, mariniluteicoccus flavus, brochothrix thermosphacta, listeria innocua, l. monocytogenes, pseudomonas putida and shewanella putrefaciens [ ] . moreover, thymol seems to be effective also against the bacterial biofilm formed by β-lactamase-producing enteric bacteria [ ] . it is still debated if thymol could be or not degraded by the intestinal microbiome since it was found to be not effective against fecal fermentation reactions [ ] . nevertheless, in weaning piglets, thymol associated with carvacrol was capable of positively modulating their microbiota by increasing populations of the lactobacillus genus and by reducing populations of enterococcus and escherichia genera [ ] . in vitro, carvacrol was showed to inhibit bacterial adhesion, invasion and biofilm development in cultured intestinal cells [ ] . in the farmed broiler, treatment with carvacrol-rich eos was tested to control the pathogenic bacteria spreading inside the farms. the results of these studies demonstrated that carvacrol reduced the microbial counts of e. coli and different salmonella species in the small intestine of farmed chicken [ ] . moreover, carvacrol administration to broiler chickens was capable of eliminating the intestinal presence of campylobacter spp. after days of daily oral administration at - mg/kg. this effect was probably linked to the enhanced growth of bacteria of the lactobacillus genus, that were found to be increased in chicken microbiota, after carvacrol administration. e. coli growth in the cecum of chickens was found to be significantly reduced by carvacrol. for these reasons, this molecule is today the most used in organic breeding to positively modulate gut microbiota and improve the growth performance of farmed chickens [ ] . in intensive breeding practices carvacrol is often associated with thymol, since there is strong evidence that the combination of the two was more effective in decreasing intestinal pathogens and increasing the growth performance of chickens [ , ] . limonene ( -methyl- -(prop- -en- -yl)-cyclohex- -ene) is a cyclic monoterpene present in a high amount in eo of citrus fruit peels and, in smaller concentrations, in many other aromatic plant eos. limonene has widely demonstrated antimicrobial and anti-inflammatory effects in vivo. in mice, daily oral administration of mg ( mg/kg) of orange oil, rich in limonene, modulates the mice microbiota by enhancing the relative abundance of the lactobacillus genus and reducing the presence of scfa-producing bacteria [ ] . its particular ability to reduce the scfa synthesis has been exploited in a mouse model of obesity to reduce weight gain. mice fed with a high-fat diet (hfd) were treated daily with microencapsulated sweet orange oil for days ( ml of suspension of microcapsules containing g/l of sweet orange eo rich in limonene). the result of this study showed a reduced body weight in treated mice, associated with a modulation of the intestinal microbiota. specifically, the bifidobacterium population was enhanced with an overall reduction of the intestinal chronic inflammation induced by the hfd, in treated mice [ ] . despite the low toxicity of limonene, which would not rise concerns, it should be noted that these effects on the microbiota were obtained only with high dosages of this compound. eucalyptol ( , , -trimethyl- -oxabicyclo[ . . ]octane) is a cyclic ether and a monoterpenoid. it is the major compound in eucalyptus eo, but it can be also found in many other officinal plants eos. a recent review indicates that eucalyptus eo has extraordinary antimicrobial activities. eucalyptus eo has shown to be effective against a plethora of bacteria species and among them s. aureus, e. coli, bacillus subtilis, klebsiella pneumonia, salmonella enteritidis and p. aeruginosa [ ] . nevertheless, eucalyptus eo also contains high amounts of other antimicrobial components besides eucalyptol, therefore not all of eucalyptus eo antibacterial activity can be ascribed to the presence of eucalyptol. however, literature data regarding eucalyptol antimicrobial activity are very limited, and new studies focused on this interesting compound are needed. menthol ( -methyl- -(propan- -yl)cyclohexan- -ol) is a chiral alcohol and the main molecule present in cornmint and peppermint eos. it has been well known for its use in foods as a cooling and minty-smell aroma. many in vitro studies, reviewed by kamatou and coworkers [ ] focused on its antibacterial activities. interestingly, in vitro, menthol was capable to drastically decrease c. difficile viability at . mg/ml, with a dose-dependent effect. its mechanism of action seems to be due to the significant leakage of cellular atp induced by menthol in this pathogenic bacterium [ ] . fungi were reported to represent about . % of all the microorganisms present in the gastrointestinal tracts. maybe also for this reason, despite the presence of fungi in the intestine has been known for many years, in depth studies of the human mycobiome were only recently performed [ ] . humans are hosts to a wide number of fungi species that coexist with the other microorganisms into the gut in a complex ecological relationship of interdependence [ ] . together with bacteria, fungi contribute to the modulation of the intestinal immune system [ ] . many of them have a clear pathogenic potential even if, physiologically, they are commensals in our bodies. only in some specific conditions their overgrowth can lead to well-known mycosis. the best known fungal pathogen of humans is certainly candida albicans, which is a normal component of the gut mycobiota but may causes candidiasis in case of its intestinal and vaginal overgrowth [ ] . an altered intestinal mycobiota has also been observed in other human pathological conditions, such as ibs [ ] , inflammatory bowel disease (ibd) [ ] and also autism-spectrum disorders and rett syndrome [ ] . since human mycobiome is altered in some diseases, perhaps contributing to their pathogenesis, the therapeutic manipulation of the mycobiome could be a useful approach to treat and/or prevent these diseases [ ] . eos antimycotic activities are characterized by a broad spectrum of actions [ ] . c. albicans is responsible for the majority of fungal infections in humans and is certainly the most studied mycobiota pathogen. the overgrowth of c. albicans is usually controlled by the immune system of the host; however, in particular conditions such as in immunocompromised patients, this microorganism may cause severe infections [ ] . for this reason, c. albicans is one of the main target for studies focusing the antifungal effect of eos and their single molecules. the antifungal activities of eo obtained from thymus vulgaris, citrus limonum, pelargonium graveolens, cinnamomum cassia, ocimum basilicum, and eugenia caryophyllus have been evaluated against clinical isolates of c. albicans and candida glabrata. all of these eos exhibited both fungistatic and fungicidal activity toward these two candida species, but cinnamon oil demonstrated the highest activity [ ] . since the most represented active compounds of cinnamomum eo is cinnamaldehyde, many studies have been addressed to analyse in depth its activity against c. albicans. the mic at which cinnamaldehyde has been shown to inhibit c. albicans growht ranged from to µg/ml [ ] . limonene has shown to possess strong antifungal properties [ ] and in particular an excellent anti-candida activity. a recent study analyzed the efficacy of this compound against the growth of c. albicans isolates, including fuconazole-resistant strains. in this study, the in vitro growth of clinical isolates of c. albicans were completely inhibited at doses ranging between mm and mm of limonene. furthermore, limonene inhibited the adhesion, development and maturation of the c. albicans biofilm with % of inhibition occurred at doses between . and . mm. at the dose of mm, limonene was also capable to degrade % of mature biofilm [ ] . mentha eos obtained from m. piperita, m. spicata, m. longifolia, m. pulegium, m. cervina, and m. suaveolens, have demonstrated good antimycotic activities against different fungi genera, including candida [ ] . menthol and (+)-carvone are the major components of peppermint eos and both exhibited strong antifungal activity in vitro. these activities were evident only at a relatively high doses: peppermint oil inhibited the in vitro growth of c. albicans at mg/ml in agar dishes, whereas caraway oil showed inhibitory effects at lower doses, in the range - mg/ml [ ] . a patented peppermint and caraway oils formulation, called menthacarin ® , was tested in an ibs animal model, showing to be effective in alleviating abdominal pain in rats via mycobiome modulation, suggesting a possible role of mycobiota dysbiosis in the etiopathogenesis of ibs [ ] . thymus vulgaris eo has also shown to be effective against fungi pathobionts capable to infect humans. a study on dermatophyte, fungi that can cause superficial infections of the skin, and on aspergillus, fungus genera that can cause respiratory infections, reported mic values for thymus vulgaris eo ranging from . to . µl/ml. higher mic values, between . and . µl/ml, were reported for candida spp. the antifungal activity of this eo has been attributed to its two major components: thymol and carvacrol, that accounted respectively % and % of thymus vulgaris eo [ ] . both these phenolic compounds seem to act by disrupting the fungal cell membranes [ ] . an interesting study evaluated the antifungal activity of thymol in comparison with miconazole, a classical antifungal medication, against c. albicans growth and biofilms formation. the results of this study demonstrated that these two molecules were equally effective against c. albicans with no statistically difference between the two treatments, confirming an extraordinary antimycotic effect of thymol. however, relatively higher concentration was necessary for thymol, with a mic that corresponded to µl/ml vs. . µl/ml for miconazole [ ] . thymol antifungal activity was tested against other candida species, and it showed to be effective also against c. tropicalis. [ ] . clove eo has been traditionally used in dentistry for its anesthetic and antimicrobial activities [ ] . its anti-fungal action has been attributed to eugenol, the major clove oil molecule. a recent study indicated that clove eo, at concentrations that ranged between . % and . % (v/v), inhibited the biofilm formation in many candida species, grown on different substrates [ ] . for what the mechanism of action concerns, eugenol was able to cause permanent injury to the cell membranes of c. albicans and morphological alterations to its cell wall [ , ] . the activity of eugenol against c. albicans was multitarget and also targeted enzymatic pathways, such as the h + -atpase in mitochondria [ ] . eugenol treatment also induced an overall oxidative damage to the fungal cell (lipid peroxidation), and these multiple damages may finally lead to cell death [ ] . the antiviral activity of eos has been established for different viruses, and it can be directed against the viral particle or against their intracellular replication process [ ] . several studies demonstrated that the herpes simplex viruses, type and (hsv- and hsv- ), influenza a virus and coronavirus may be sensitive to eos and to their single molecules [ ] [ ] [ ] . it is interesting to observe that many hsv strains, resistant to synthetic antiviral drugs, are instead sensible to eos and to their components. that is probably because they have a multitarget mechanism of action compared to specific drugs that usually target single virus component or single metabolic pathways. for this reason, it is reasonable to expect that antiviral drugs and eos could act in synergy [ ] . bovine viral diarrhea virus (bvdv) is considered a good animal model for the human hepatitis c virus. this virus has been successfully treated with basil eo and single monoterpenes that are major components of this eo: camphor, thymol and eucalyptol. to perform the tests, basil eo and monoterpenes were used at a concentration of mg/ml. results obtained in this in vitro study showed that while the basil eo did not demonstrated to have significant antiviral activities, its single monoterpenes significantly decreased bvdv infectivity on bovine kidney cells, acting directly on the viral particle [ ] . these results are interesting because they suggest that for specific antimicrobial applications, it is much more useful to use individual components of eos rather than eos as such. since the antimicrobial and antiviral activity of single eo molecules are multitarget activities, it is very important to design new study focused on single eo constituents and not on blends of different eos, that are very difficult to analyze and to replicate. antiviral properties of carvacrol have been demonstrated on nonenveloped murine norovirus (mnv), a good model for the human norovirus. carvacrol . % and . % (v/v) reduced virus propagation on a murine monocyte cell line, demonstrating its ability to inactivate mnv acting firstly on the viral capsid and afterwards directly on its rna. this underlines the efficacy of carvacrol as a natural surface disinfectant and as a food preservative to control human norovirus, which are the most frequent cause of food-borne viral diseases in humans, causing non-bacterial gastroenteritis. [ ] . to date, no study has been performed to understand the impact of eos on the intestinal virome. the main physiological viral component of the gastrointestinal tract is represented by prophages or phages [ ] . the bacteriophage component is mainly composed by temperate virus of the caudovirales order, but most of the detected viral sequences in human gut virome could not be attributed to known viruses [ ] and to date it is estimated that the number of viruses in human stools is up to per gram [ ] . despite this, it is clear that eos may impact the intestinal virome composition by modulating all the microbiota components, it could be really difficult to understand the direct impact of eos on the intestinal viruses and the consequences of this modulation on the intestinal ecology. to date, there is some evidence that eos may be effective against different virus replication, but there are still not enough data to predict what could be the impact of eos on viral infectious diseases of the gastrointestinal tract. the presence of a very complex microenvironment makes it difficult to understand if eos can really act against the pathogen virus particles or if they reach mucosa cells only after the infection. since eos are potentially able to act both on bacteria and viruses, in pathological conditions it could be difficult to understand the real targets of these compounds. for example, it has been demonstrated that norovirus persistent infection can be sustained by gut bacteria dysbiosis, and the use of antibiotics in infected mice is able to counteract the viral replication [ , ] . nowadays, colorectal cancer (crc) is one of the most common tumours worldwide. it represents the second cause of cancer death in europe, even if mortality is decreasing due to the new screening programs and improvement in therapies [ ] . the aetiology of crc is not only related to genetic and environmental factors but also to gut microbiota and chronic colonic inflammation. genetic factors include mutations in genes regulating enterocyte cell growth, proliferation, differentiation or cell cycle control or polymorphisms of several proteins involved in dna repair and transcription [ ] [ ] [ ] [ ] . among the environmental factors, the most important seems to be the high consumption of red meat, smoking and drinking alcohol in huge amounts [ , ] . the involvement of the gut microbiota in crc is related to the production of noxious metabolites by bacteria, such as secondary bile acids, polyamines or genotoxins [ ] . these metabolites may cause to colonocytes oxidative stress, direct dna damage or induce inflammation [ ] . chronic inflammation condition, such as those linked to ibd, are also recognised as a risk factors for crc development [ ] . crc manifestation can be sporadic or have a familial predisposition, that's the case of familial adenomatous polyposis (fap) and other syndromes like peutz-jeghers, serrated polyposis and lynch [ , ] . classical crc therapies include surgical treatments, radiotherapy or chemotherapy [ ] , which are associated with important side effects and with the development of drug resistance [ , ] . recently new pharmacological approaches have been successfully developed for crc, including biological drugs, aimed at the treatment of previously diagnosed cancers [ ] . despite the presence of different therapeutic strategies for crc, we must not forget the potential of natural anticancer substances, especially in the prevention of a neoplasia which requires long times to transform from a benign dysplasia to a malignant adenocarcinoma. several eos and their single components have been tested in vitro and/or in vivo by using crc models and have been proved to be valid antitumoral molecules for this cancer. carvacrol was tested on different crc cell lines (htc- and lovo), where it was determined the reduction of proliferation and cell cycle arrest in g /m phase, associated with the reduction of cellular invasion and migration proprieties [ ] . geraniol has shown a strong cytotoxic effect on colo- cell line [ ] but it was not able to induce apoptosis on caco- cell line, where it only showed a cytostatic effect, by arresting their cell cycle in s phase [ ] . this demonstrate how the same compound may not have the same effect on different cellular crc models. geraniol also demonstrated its anticancer property against the regulation of polyamine metabolism, that is another target of cancer therapies [ , ] . in different crc cell lines, geraniol has been shown to downregulate the ornithine decarboxylase (odc) and to upregulate s-adenosylmethionine decarboxylase (adometdc), two enzymes involved in polyamine catabolism and elimination [ ] . thymol has shown cytotoxic effect against htc- cell line by inducing ros production and dna damages. it also induced cell-death by affecting cancer cell mitochondrial pathways [ ] . on the other hand, thymol, geraniol, nerolidol, and methyleugenol, at low doses, have demonstrated to have genoprotective effects against oxidative and dna methylation damages in ht- cell line [ ] . these data underline the importance of the effective dosage of these substances that reaches the colon, since all of them are subject to intestinal absorption. cinnamaldehyde have been tested on several crc cell lines and, like others eo components, it caused the inhibition of proliferation and the induction of apoptosis (by pi k/akt inhibition and by increasing bax/bcl- ratio) associated with a reduction of invasion and migration capability of sw- , hct- and lovo cells (by increasing e-cadherin levels and downregulating mmp- and mmp- enzymes) [ ] . for these reasons, cinnamaldehyde has been used for the development of an aspirin-like drug for the prevention of crc [ ] . cinnamaldehyde has also been associated with camptothecin, a hydrophobic anticancer drug, and then incorporated in polymeric micelles to obtain a controlled release system based on ph-gradients. these micelles have shown to induce apoptosis and generate intracellular ros with synergistic anticancer effects between cinnamaldehyde and camptothecin both on in vitro and in vivo models of sw- human colon tumour cell or bearing mice [ ] . so, the anticancer activities of eos compounds can reduce drug resistance and sensitize cancer cells to traditional chemotherapeutic agents, by acting synergistically with them. geraniol has been reported to sensitize caco- cell line to -fluorouracil ( -fu), by altering cell membrane potentials and facilitating the uptake of -fu [ , ] . the synergistic effect of geraniol and -fu has been investigated both in vitro and in vivo. geraniol potentiates -fu growth inhibition activity on sw- and caco- cell lines by downregulating the thymidylate synthase and thymidine kinase, two enzymes related to -fu cytotoxicity. these data have been confirmed in vivo on nude mice grafted with the human colorectal cells tc- [ ] . in a similar manner thymoquinone, a compound of nigella sativa eo, demonstrated-in association with doxorubicin-an improvement of drug-antineoplastic activities on ht- cell line [ ] . thymoquinone also sensitized colo- and htc- cancer cells to cisplatin and increased cancer cell death by suppressing nf-kβ [ ] . finally, it has been shown that thymoquinone was capable to sensitize resistant lovo colon cancer cells to the drug irinotecan by affecting erk / pathway and increasing the membrane permeability and the autophagy process [ , ] . β-caryophyllene is a natural bicyclic sesquiterpene found in many eos, particularly in clove oil. it has been studied in association with paclitaxel and doxorubicin on dld- and caco- cell lines, respectively. it facilitated the passage of paclitaxel through the plasma membrane, increasing its anticancer activity [ ] . moreover, β-caryophyllene treatment induced an intracellular accumulation of doxorubicin that increased its anticancer activity [ ] . β-caryophyllene has also been involved in the regulation of glucose homeostasis in crc cells, regulating genes involved in glycolysis and cell growth and finally leading to cell growth suppression and apoptosis [ ] . taken together, these results suggest that there is a real wide possibility of using eos or their individual compounds in crc prevention, but also in reducing the doses of classic chemotherapy drugs adopted to treat crc patients. this would consequently reduce the side effects of chemotherapy with a real benefit for patients during anticancer therapy but without decreasing its therapeutic efficacy. considering that the development of crc can be determined by the presence of colonic chronic inflammation for a long time, the efficacy of these natural compounds is certainly maximum in terms of crc prevention, in all subjects at high risk of developing this cancer, both for familiar history or for pre-disposing pathologies, such as ibd or fap. in this context and within long-term therapeutic preventive strategies, it has to be stressed eos multi-target properties, that makes the development of resistant tumour cells very difficult. eos, but especially their single molecules, are effective multitarget modulators of the intestinal physiology and pathology. their use to date has been limited due to several factors that include testing complex mixtures that did not allow identifying the individual active components, the difficulty of releasing these compounds in the affected intestinal tract and their toxicity and aggression on the mucous membranes which complicates their in vivo administration. most eos have very strong, sometimes unpleasant flavors, and in several cases they are also aggressive towards the oral, esophageal and gastric mucosa. administration in controlled release formulations therefore often becomes a necessity. by using normal pharmacological forms such as tablets, capsules or soft gels it is possible to obtain gastro-resistant pharmaceutical forms. once reached the intestine, the chemical compounds contained in the oes are assimilated more or less quickly, with different bioavailability, depending on their chemical structure and pharmacokinetics. geraniol rapidly crosses the enterocytes and, once reaching the bloodstream, has shown a half-life of . min with a pseudo first-order kinetics [ ] . on the contrary, eugenol is rapidly absorbed, but has shown a half-life of . h in blood and could accumulate after repeated daily administrations [ ] . cinnamaldehyde has shown a half-life of . h. however, the % of cinnamaldehyde is oxidized to cinnamic acid rapidly [ ] . carvacrol is only partially absorbed in the intestine and has shown to reach low concentration in plasma with a peak after h of oral administration [ ] . d-limonene is only partially absorbed by enterocytes and has shown a half-life of . h in plasma [ , ] . similar behavior has been found for β-caryophyllene, that has shown a half-life of . h in plasma [ ] . thymol is absorbed quickly in the gut, and it is present in plasma only as thymol sulfate, that reach a considerable concentration after min, with an half-life of . h [ ] . the current scientific knowledges regarding their activities against oxidative stress and their ability to modulate the microbiota and the intestinal inflammation are more than enough to plan clinical trials on humans and provide evidences for their use as therapeutic agents. the strong multitarget antitumor activity of some molecules present in eo make them potentially very effective therapeutic strategies against crc, also in combination with chemotherapies already in use. however, the maximum potential of these compounds seems to be expressed in the prevention of crc, inflammation and intestinal dysbiosis. table summarize the main effects of oes divided according to their main activity in the intestine and with the indication of the individual compounds mainly responsible for the action. increasing antineoplastic effect of doxorubicin, increasing cell death by suppressing nf-kβ in association with cisplatin, induction of autophagy in association with irinotecan [ ] [ ] [ ] [ ] n.a. β-caryophyllene antitumoral effect in association with chemotherapeutic agents, regulation of glucose homeostatis (in vitro) increase of anticancer activity of paclitaxel and doxorubicin regulation of genes involved in glycolysis and cell growth, induction of apoptosis [ ] [ ] [ ] a limit to their use is the need of controlled release systems that target these molecules in the intestine area where their action is required. in fact, all these compounds are absorbed in the small intestine, some of them very quickly, such as geraniol, while others with less efficient transport mechanisms, such as limonene [ , ] . nevertheless, possibilities for a controlled release of these molecules already exist, and it has already been proven that they are effective in humans to obtain a controlled release [ ] . further in vivo and clinical studies are necessary to understand their bioavailability, pharmacokinetics and mechanism of actions. unfortunately, the low cost that these eos have on average, and their non-patentability, make them little or not at all interesting for pharmaceutical industries, making missing the sponsors for the clinical studies necessary to finally validate their therapeutic efficacy in many different intestinal pathologies of the large bowel, such as inflammation, colitis, dysbiosis and crc. metabolic engineering strategies for enhancing the production of bio-active compounds from medicinal plants antimicrobial, antioxidant, and immunomodulatory properties of essential oils: a systematic review oxidant signals and oxidative stress metal dyshomeostasis and their pathological role in prion and prion-like diseases: the basis for a nutritional approach geraniol pharmacokinetics, bioavailability and its multiple effects on the liver antioxidant and xenobiotic-metabolizing enzymes nitric oxide and superoxide in inflammation and immune regulation dietary geraniol by oral or enema administration strongly reduces dysbiosis and systemic inflammation in dextran sulfate sodium-treated mice dietary geraniol ameliorates intestinal dysbiosis and relieves symptoms in irritable bowel syndrome patients: a pilot study longitudinal analysis of inflammation and microbiota dynamics in a model of mild chronic dextran sulfate sodium-induced colitis in mice in vivo study of the efficacy of the essential oil of zanthoxylum bungeanum pericarp in dextran sulfate sodium-induced murine experimental colitis protective effect of the essential oil of zanthoxylum myriacanthum var. pubescens against dextran sulfate sodium-induced intestinal inflammation in mice foeniculum vulgare essential oil ameliorates acetic acid-induced colitis in rats through the inhibition of nf-kb pathway essential oils in ginger, hops, cloves, and pepper flavored beverages-a review protective effect of ginger volatile oil against acetic acid-induced colitis in rats: a light microscopic evaluation protective effect of ocimum basilicum essential oil against acetic acid-induced colitis in rats examination of the anti-inflammatory, antioxidant, and xenobiotic-inducing potential of broccoli extract and various essential oils during a mild dss-induced colitis in rats essential turmeric oils enhance anti-inflammatory efficacy of curcumin in dextran sulfate sodium-induced colitis new insights on the anti-inflammatory potential and safety profile of thymus carnosus and thymus camphoratus essential oils and their main compounds rosmarinus officinalis essential oil: a review of its phytochemistry, anti-inflammatory activity, and mechanisms of action involved antigenotoxic and antioxidant activities of palmarosa and citronella essential oils genoprotective effects of essential oil compounds against oxidative and methylated dna damage in human colon cancer cells pharmacological properties of geraniol identification of volatile components in basil (ocimum basilicum l.) and thyme leaves (thymus vulgaris l.) and their antioxidant properties role of the gut microbiota in immunity and inflammatory disease towards the human intestinal microbiota phylogenetic core antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review a comprehensive review of the antibacterial, antifungal and antiviral potential of essential oils and their chemical constituents against drug-resistant microbial pathogens essential oils from aromatic herbs as antimicrobial agents geraniol restores antibiotic activities against multidrug-resistant isolates from gram-negative species sensitivity of pathogenic and commensal bacteria from the human colon to essential oils disruption of escherichia coli, listeria monocytogenes and lactobacillus sakei cellular membranes by plant oil aromatics antimicrobial mechanism of clove oil on listeria monocytogenes phytonutrient diet supplementation promotes beneficial clostridia species and intestinal mucus secretion resulting in protection against enteric infection eugenol inhibits quorum sensing at sub-inhibitory concentrations antibacterial activities of the crude ethanol extracts of medicinal plants against listeria monocytogenes and some other pathogenic strains inactivation of escherichia coli o :h by essential oil from cinnamomum zeylanicum constituents of cinnamon inhibit bacterial acetyl coa carboxylase composition, antimicrobial activity and in vitro cytotoxicity of essential oil from cinnamomum zeylanicum blume (lauraceae) trans-cinnamaldehyde from cinnamomum zeylanicum bark essential oil reduces the clindamycin resistance of clostridium difficile in vitro effect of cinnamon essential oil on gut microbiota in the mouse model of dextran sodium sulfate-induced colitis nutritional impacts of dietary oregano and enviva essential oils on the performance, gut microbiota and blood biochemicals of growing ducks the effect of feeding essential oils on broiler performance and gut microbiota bioactive extracts of carum copticum and thymol inhibit biofilm development by multidrug-resistant extended spectrum β-lactamase producing enteric bacteria the role of gut microbiota for the activity of medicinal plants traditionally used in the european union for gastrointestinal disorders a carvacrol-thymol blend decreased intestinal oxidative stress and influenced selected microbes without changing the messenger rna levels of tight junction proteins in jejunal mucosa of weaning piglets effects of oral administration of different dosages of carvacrol essential oils on intestinal barrier function in broilers the in vitro and in vivo effect of carvacrol in preventing campylobacter infection, colonization and in improving productivity of chicken broilers effects of thymol and carvacrol, alone or in combination, on fermentation and microbial diversity during in vitro culture of bovine rumen microbes effect of origanum chemotypes on broiler intestinal bacteria effects of orange essential oil on intestinal microflora in mice microcapsule of sweet orange essential oil changes gut microbiota in diet-induced obese rats biological, medicinal and toxicological significance of eucalyptus leaf essential oil: a review menthol: a simple monoterpene with remarkable biological properties natural products show diverse mechanisms of action against clostridium difficile human gut microbial gene catalogue established by metagenomic sequencing the fungal mycobiome and its interaction with gut bacteria in the host interactions between commensal fungi and the c-type lectin receptor dectin- influence colitis candida albicans infection and intestinal immunity intestinal fungal dysbiosis associates with visceral hypersensitivity in patients with irritable bowel syndrome and rats nion-larmurier, i.; et al. fungal microbiota dysbiosis in ibd fungal dysbiosis: immunity and interactions at mucosal barriers commensal fungi in health and disease sensitivity of candida albicans to essential oils: are they an alternative to antifungal agents? investigation of the antifungal activity and mode of action of thymus vulgaris, citrus limonum, pelargonium graveolens, cinnamomum cassia, ocimum basilicum, and eugenia caryophyllus essential oils cinnamic aldehydes affect hydrolytic enzyme secretion and morphogenesis in oral candida isolates mechanism of bacterial inactivation by (+)-limonene and its potential use in food preservation combined processes limonene inhibits candida albicans growth by inducing apoptosis antioxidant, antifungal, antibiofilm, and cytotoxic activities of mentha spp. essential oils reversal of visceral hypersensitivity in rat by menthacarin ® , a proprietary combination of essential oils from peppermint and caraway, coincides with mycobiome modulation antifungal activity of the essential oil of thymus pulegioides on candida, aspergillus and dermatophyte species cell viability of candida albicans against the antifungal activity of thymol in vitro anti-biofilm activities of citral and thymol against candida tropicalis antimicrobial activity of eugenol and essential oils containing eugenol: a mechanistic viewpoint effect of clove and thyme essential oils on candida biofilm formation and the oil distribution in yeast cells eugenol and thymol, alone or in combination, induce morphological alterations in the envelope of candida albicans proton translocating atpase mediated fungicidal activity of eugenol and thymol induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids essential oils for the treatment of herpes simplex virus infections phytochemical analysis and in vitro antiviral activities of the essential oils of seven lebanon species antiviral activity of monoterpenes beta-pinene and limonene against herpes simplex virus in vitro chemical compositions and anti-influenza activities of essential oils from mosla dianthera in vitro inhibition of the bovine viral diarrhoea virus by the essential oil of ocimum basilicum (basil) and monoterpenes antiviral efficacy and mechanisms of action of oregano essential oil and its primary component carvacrol against murine norovirus virome-host interactions in intestinal health and disease viruses in the faecal microbiota of monozygotic twins and their mothers diversity and abundance of single-stranded dna viruses in human feces enteric bacteria promote human and mouse norovirus infection of b cells commensal microbes and interferon-λ determine persistence of enteric murine norovirus infection european cancer mortality predictions for the year with focus on colorectal cancer adenomas-genetic factors in colorectal cancer prevention effectors of epidermal growth factor receptor pathway: the genetic profiling of kras, braf, pik ca, nras mutations in colorectal cancer characteristics and personalized medicine integrated analysis of molecular and clinical prognostic factors in stage ii/iii colon cancer colon cancer family registry and the genetics and epidemiology of colorectal cancer consortium. identification of genetic susceptibility loci for colorectal tumors in a genome-wide meta-analysis red meat and colon cancer: a review of mechanistic evidence for heme in the context of risk assessment methodology alcohol, smoking and the risk of premalignant and malignant colorectal neoplasms the gut microbiota, bacterial metabolites and colorectal cancer colorectal cancer in inflammatory bowel disease: the risk, pathogenesis, prevention and diagnosis molecular genetics of colorectal cancer clinical management of hereditary colorectal cancer syndromes balancing the efficacy and toxicity of chemotherapy in colorectal cancer pharmacologic resistance in colorectal cancer: a review carvacrol inhibits proliferation and induces apoptosis in human colon cancer cells. anti-cancer drugs geraniol and geranyl acetate induce potent anticancer effects in colon cancer colo- cells by inducing apoptosis, dna damage and cell cycle arrest geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells polyamine metabolism and gene methylation in conjunction with one-carbon metabolism thymol elicits hct- colorectal carcinoma cell death through induction of oxidative stress cinnamaldehyde affects the biological behaviour of human colorectal cancer cells and induces apoptosis via inhibition of the pi k/akt signalling pathway novel cinnamaldehyde-based aspirin derivatives for the treatment of colorectal cancer dual acid-responsive micelle-forming anticancer polymers as new anticancer therapeutics perturbation by geraniol of cell membrane permeability and signal transduction pathways in human colon cancer cells geraniol, a component of plant essential oils, sensitizes human colonic cancer cells to -fluorouracil treatment geraniol, a component of plant essential oils, modulates dna synthesis and potentiates -fluorouracil efficacy on human colon tumor xenografts combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin thymoquinone chemosensitizes colon cancer cells through inhibition of nf-κb thymoquinone induces caspase-independent, autophagic cell death in cpt- -resistant lovo colon cancer via mitochondrial dysfunction and activation of jnk and p inhibition of nf-κb and metastasis in irinotecan (cpt- )-resistant lovo colon cancer cells by thymoquinone via jnk and p potentiating effect of β-caryophyllene on anticancer activity of α-humulene, isocaryophyllene and paclitaxel the influence of sesquiterpenes from myrica rubra on the antiproliferative and pro-oxidative effects of doxorubicin and its accumulation in cancer cells effects of beta-caryophyllene on arginine adp-ribosyltransferase -mediated regulation of glycolysis in colorectal cancer under high-glucose conditions pharmacokinetics of eugenol and its effects on thermal hypersensitivity in rats pharmacokinetic study of cinnamaldehyde in rats by gc-ms after oral and intravenous administration pharmacokinetic and antimicrobial activity of a new carvacrol-based product against a human pathogen, campylobacter jejuni determination of d-limonene in mice plasma and tissues by a new gc-ms/ms method: comparison of the pharmacokinetics and tissue distribution by oral and inhalation administration in mice self-microemulsifying drug delivery system for improved oral delivery of limonene: preparation, characterization, in vitro and in vivo evaluation physicochemical characterization and pharmacokinetics evaluation of β-caryophyllene/β-cyclodextrin inclusion complex systemic availability and pharmacokinetics of thymol in humans chemopreventive effect of carvacrol on , -dimethylhydrazine induced experimental colon carcinogenesis carvacrol exhibits anti-oxidant and anti-inflammatory effects against , dimethyl hydrazine plus dextran sodium sulfate induced inflammation associated carcinogenicity in the colon of fischer rats this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank alberto sardo for illuminating us on the infinite potential of essential oils. the authors declare no conflict of interest. key: cord- -vu qbcy authors: zhang, xiao-rui; li, ting-na; ren, yuan-yuan; zeng, yi-jia; lv, hong-yang; wang, jin; huang, qin-wan title: the important role of volatile components from a traditional chinese medicine dayuan-yin against the covid- pandemic date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: vu qbcy aromatic chinese herbs have been used to prevent plagues since ancient times. traditional chinese medicine has unique advantages in the prevention and treatment of epidemic diseases. according to the traditional chinese medicine treatment plan in the national covid- diagnosis and treatment plan (trial seventh edition) of the national health commission, chinese patent medicines or prescriptions rich in aromatic chinese herbs are selected for prevention and treatment during the period of medical observation, clinical treatment, and recovery of confirmed covid- patients. some local health committees or traditional chinese medicine administrations recommend a variety of other ways of using traditional aromatic chinese herbs to prevent and cure covid- . these involve external fumigation, use of moxibustion, and wearing of sachet. the efficacy of aromatic chinese herbs plays a decisive role in the prevention and treatment of covid- . the unique properties, chemical composition, and mechanism of action of aromatic chinese herbs are worthy of extensive and in-depth experimental and clinical research. the findings are expected to provide a reference for follow-up treatment of novel coronavirus and the development of corresponding drugs. in , dayuan-yin produced excellent results in the treatment of the sars virus. individually, confirmed cases were administered this drug between january and april , and more than . % of the patients showed noticeable mitigation of the symptoms, as well as recovery. dayuan-yin also was selected as one of the nationally recommended prescriptions for the covid- . based on the national recommendation of dayuan-yin prescription, this review discusses the role of volatile components in the prevention and treatment of covid- , and speculates the possible mechanism of action, so as to provide a basis for the prevention and treatment of covid- . most of the volatile components of dayuan-yin have been elucidated, and their structures are well established (table ) . however, there are no data on volatile components extracted from areca catechu l. based on clinical evidence of therapeutic results with dayuan-yin, we summarized its potential bioactive volatile components in the treatment of covid- . the biological benefits of dayuan-yin seem to involve anti-inflammatory, antiviral, antibacterial, and immunomodulatory effects ( table ) . in autopsy studies and animal models, covid- manifests mainly as acute viral pneumonia leading to respiratory failure (chan et al., ; yao et al., ) . antiviral drugs have been used to treat common cold, fever and influenza viruses by destroying the viral surface structure and inhibiting its entry (hsieh et al., ) , suggesting that antiviral drugs can be used for covid- . unfortunately, no specific antiviral treatment has been recommended for covid- treatment because of insufficient evidence from randomized trials (hung et al., ) . it has been shown that many re-purposed drugs have effects against close relatives of sars-cov- , such as bcoronavirus, in vitro. furthermore, lopinavir and many interferons, especially interferon beta, have moderate effects against sars-cov in vitro and can be used in combination with ribavirin (chen et al., ; chan et al., ) . administration of antiviral drugs soon after symptoms appear reduces the release of virus in respiratory secretions of patients with covid- , thereby decreasing their infectivity to others. targeted preventive treatment for contacts reduces their risk of infection (welliver et al., ; oriol and bonaventura, ) . patchouli oil is extracted from pogostemon cablin (blanco) benth. some studies in vitro have shown that patchouli oil exerted anti-viral effects against coxsackie virus (ic = . mg/ ml, ti . ), adenovirus(ic = . mg/ml, ti . ), influenza a virus (ic = . mg/ml, ti . ), and respiratory syncytial virus (ic = . mg/ml, ti . ) (wei et al., ) . evaluation of the antiviral properties of six chemical compositions of atractylodes lancea (thunb.) d dpph.c. revealed that atractylodin produced the most significant effect at doses of - mg/kg for five days, and attenuated iav−induced pulmonary injury via regulation of the tlr signaling pathway (cheng et al., ) . moreover, , -cineole, the major constituent of the essential oil of lanxangia tsao-ko (crevost & lemarie) m.f.newman & skornick., is commonly applied for treating inflammatory diseases of the respiratory tract caused by viruses since it potentiates the antiviral effect of irf , in addition to its inhibitory effect on proinflammatory nf-kb signaling (müller et al., ) . the levels of proinflammatory factors i.e. il- , il- , il- , gcsf, ip , mcp , mip a, and tnf -a in the plasma of critically-ill patients were higher than those in plasma of patients who were not in intensive care, suggesting that "cytokine storm" is closely related to the severity of covid- . cytokine storm is a very prominent pathophysiological feature of covid- infection (lin et al., ) . extensive endothelial barrier disruption and uncontrolled cytokine storm promote uncontrolled inflammatory response which is the basis of the core mechanism underlying acute respiratory distress syndrome (ards) (huang et al., ) , although this phenotype varies among individuals. experimental models of acute lung injury (ali) and human genome-wide association studies of ards indicate that cytokine storm plays an essential role in the pathophysiology of ards ( biondi et al., ; huang et al., ) . moreover, the most common and severe complication of covid- is ards (huang et al., ) . therefore, an understanding of the cytokine storm that aggravates ards in covid- may lead to early and effective intervention in critically-ill covid- patients. it seems necessary to directly inhibit inflammatory response in the lungs because cytokine storm can be alleviated with inflammatory therapy (cao p. et al., ) . previous studies have shown the benefits of antiinflammatory drugs in lung disease: they slow down impairment of lung function, reverse the inflammatory parameters nearly back to normal values, and improve patients' survival (konstan et al., ; lubamba et al., ) . ibuprofen, a popular antiinflammatory drug, is recommended for airway inflammation in cystic fibrotic lung disease (flume et al., ) . studies have shown pogostemon cablin (blanco) benth. volatile oils patchoulene: cyclin e↓, cyclin b↓, cdk ↓; the subsequent s-phase arrest, ifn-gamma↓, il- ↓ pogostemon cablin (blanco) benth. volatile oils: tnf-a↓, il- ↓, il- ↑, np-sh↑ (chen et al., ) patchoulene: regulates on the balance between nrf and nf-kb p signaling pathways (yang et al., ) patchoulene: vitro neutrophil fmlp chemotaxis↓, phagocytic activity↑; ear edema↑, myeloperoxidase (mpo) activity↑ that long-term prediagnostic use of nonaspirin nsaids (e.g., ibuprofen) is associated with a significant reduction in lung cancer survival (brasky et al., ) . in addition, barictinib, fedratinib, and ruxolitinib are active and selective jak inhibitors which have been approved for rheumatoid arthritis and myelofibrosis. all three drugs are effective anti-inflammatory agents, and as jak-stat signaling inhibitors, they may be effective against the consequences of elevated levels of cytokines (including interferon-g) usually observed in patients with covid- (stebbing et al., ) . the uk is currently conducting a randomized evaluation of covid- treatment (recovery) trial, based on the announcement on june , , that dexamethasone had been shown to significantly improve the prognosis of covid- patients receiving respiratory support (recovery, randomised evaluation of covid- therapy trial, ). dexamethasone is a glucocorticoid which can be used as a synthetic form of the natural hormone cortisol (cain and cidlowski, ) . it has the same anti-inflammatory effect as cortisol. it inhibits the release of inflammatory chemokines by immune cells, thereby improving the prognosis of patients by reducing the severity of ards (lester et al., ) . in european patients, low-dose dexamethasone reduces mortality by % in critically patients requiring invasive ventilation (lim et al., ) . however, the implementation of appropriate dexamethasone use in low-and-middle-income countries has been a challenge. for example, corticosteroids may cause sepsis in some prevalent parasitic infections in africa (nutman, ) . therefore, the use of dexamethasone in african patients who have not been b-eudesmol has two-way regulation of gastrointestinal motility, which may be anticholinergic or directly acting on gastrointestinal smooth muscle. (wang et al., ) hinesol: h + , k + -atpase activity↓ (kanako et al., (guo et al., ) falcarindiol inhibited dc maturation by blocking the canonical pathway of nuclear factor-kappab and phosphorylated p . (mitsui et al., ) falcarindiol inhibit pseudomonas aeruginosa by repressing virulencerelated genes, including the t ss; quorum sensing synthase genes lasir and rhlir; lasb; motility-related genes flic and flig; and phenazine synthesis genes phza and phza . diagnosed with covid- may lead to unexpected consequences (brotherton et al., ) . during the treatment of covid- , dayuan-yin also reduces the severity of ards by inhibiting the release of inflammatory chemokines from immune cells. as a classic prescription in ancient china, dayuan-yin can play a safe and effective role in the treatment of respiratory infections in a more adverse environment. thus, it can avoid such problems in a large extent. anti-inflammatory property is widespread in various sources of volatile components. several data have found that , -cineole significantly improved lung function and health conditions, and reduced dyspnea in patients with asthma, acute bronchus, and chronic obstructive pulmonary disease (copd). moreover, it significantly reduced the frequency of cough in patients with acute bronchitis, and alleviated frequent exacerbations in patients with copd and frequent exacerbations, notably (worth et al., ; worth and dethlefsen, ; fischer and dethlefsen, ; vogelmeier et al., ) . in a mouse model of lps-induced acute pulmonary inflammation, , -cineole upregulated il- in lung tissues, and decreased the expressions of tnf-a, il- b, nf-kb's subunit p and tlr (zhao et al., ) . moreover, , -cineole was shown to inhibit ltb and pge (pathways of aa-metabolism in human blood monocytes) in bronchial asthma in vitro (juergens et al., b) . in addition, , -cineole decreased levels of tnfa, il- b, leukotriene b , and thromboxane b in human blood monocytes in vitro (juergens et al., a) . pogostone, a bioactive component extracted from pogostemon cablin (blanco) benth., reduced the total population of t cells under cona stimulation by blocking t cell proliferation via downregulation of cyclin e, cyclin b, and cdk . subsequent s-phase arrest inhibited the production of ifn-gamma and il- . simultaneously, pogostone pretreatment mitigated ethanolinduced gastric ulcer in rats by downregulation of il- and tnfalpha, and upregulation of il- and non-protein-sulfhydryl (np-sh) groups in the gastric mucosa (chen et al., ) . in lung disease, pogostone exerted potent protective effects against lipopolysaccharide-induced acute lung injury in mice by decreasing tnf-a-induced cell injury in a cells through modulation of the balance between nrf and nf-kb-p signaling pathways (yang et al., ) . pogostone significantly inhibited the protein and mrna expressions of proinflammatory mediators such as tnf-a, il- , il- b, no, and pge . pogostone also significantly reduced lps-induced mortality in mice, suppressed the production of proinflammatory mediators in serum. and it attenuated liver and lung injury via downregulation of the mrna expressions of inflammatory mediators in multiple organs due to inhibition of activation of nf-kb and phosphorylation of p mapk (li et al., ) . pre-treatment with pogostone markedly mitigated lps-induced acute lung injury in mice, improved survival, attenuated histological alterations in the lungs, reduced mpo and mda levels, decreased the wet/dry weight ratio of lungs, and down-regulated proinflammatory mediators, such as tnf-a, il- beta and il- . furthermore, pretreatment with pogostone enhanced the nrf dependent genes nqo- , gclc, and ho- , but suppressed the nf-kappa b regulated genes tnf-alpha, il- beta, and il- . the mechanism involved in the protective effect of pogostone was correlated with its regulation of the balance between keap -nrf and nf-kappa b signaling pathways . moreover, volatile oils from pogostemon cablin contain a bioactive component named b-patchoulene which has been shown to significantly decrease mortality and lung wet/dry weight ratio of mice, and mitigate pathological changes in lungs, when compared to model group. it suppressed lps-induced activation of nf-kappa b, and markedly upregulated nrf and mir- a (chen et al., ) . oxidative stress and inflammation form a positive feedback cycle (mittal et al., ) . in lung disease, excessive inflammation and oxidative stress lead to adverse outcomes. for instance, patients with copd are usually affected by other diseases (rabe and watz, ) . several mechanisms in lung inflammation and oxidative stress destroy dna and lead to an imbalance between tissue repair and cell proliferation, which seems to promote the link between copd and lung cancer (wilson et al., ; houghton, ; durham and adcock, ) . under normal conditions, the production and elimination of ros maintain a crucial balance between oxidation and antioxidation (cao et al., ) . in such a balance, the signal pathways are regulated, and cell proliferation can be guaranteed. when inflammatory factors destroy this balance, oxidative stress enhances the maturation of proinflammatory factors, leading to oxidative damage to cells and multisystem diseases (sies, ; kruk et al., ) . antioxidant drugs have been used in lung diseases. for example, antioxidants have been recommended for reduction of mortality or prevention of organ damage in animal models of acute lung injury induced by lipophilic acids (hsu et al., ; zhu et al., ) . vitamin c has also been shown to reduce the incidence of pneumonia in several controlled trials for human subjects (hemila, ) . the essential oil of magnolia officinalis rehder & e.h. wilson exerts antioxidant effect by scavenging , -diphenyl- picrylhydrazyl (dpph) radical and superoxide anion radical. the essential oil contains b-eucalyptol with a hydroxyl group which can provide hydrogen atom for scavenging dpph radical. moreover, with increase in volatile oil concentration, the antioxidant capacity gradually increased (guo, ) . the essential oil from atractylodes lancea (thunb.) dc. showed a strong antioxidant effect in vitro and indicated by dpph-radical scavenging property, with an ic of . mg/ml. moreover, it inhibited lipid peroxidation, and affected total antioxidant capacity (t-aoc) in the serum and organ tissues of mice . in short-term cigarette smoke (cs)induced acute lung inflammation, , -cineole decreased oxidative stress involving reactive oxygen species, by increasing superoxide dismutase and catalase, while reducing levels of malondialdehyde, inflammation, and the nf-kappa b p subunit (kennedy-feitosa et al., ) . the major components of pogostemon cablin (blanco) benth. are carvacrol ( . %) and p-cymene ( . %). it completely inhibits the growth of e. coli at a level of . % (lin et al., ) . the essential oil of atractylodes lancea (thunb.) dc. exhibited antibacterial effects against gram-positive and gram-negative bacteria due to the cell membrane . in chronic zhang et al. dayuan-yin against the covid- pandemic frontiers in pharmacology | www.frontiersin.org september | volume | article rhinosinusitis, , -cineole suppressed the growth of s. aureus, escherichia coli, moraxella catarrhalis due to downregulation of significant and critical players in biofilm generation (agra, sara, and s b ) (schürmann et al., ) . on the other hand, the major constituent of the essential oil of atractylodes lancea (thunb.) dc. is b-eudesmol. in terms of intestinal flora, b-eudesmol has two-way regulation for gastrointestinal motility: anticholinergic pathway and direct effect on gastrointestinal smooth muscle. antibacterial effect is an essential pharmacological property of volatile compounds (houdkova et al., ; riad et al., ) . secondary bacterial co-infection is common in patients with covid- infection, and it leads to adverse prognosis (macintyre et al., ) . at present, many antibiotics have been used in the treatment of covid- . for example, shufeng jiedu capsule (sfjd) prevents acute upper respiratory tract infection and positively affects fever, cough, and headache. studies have shown that sfjd significantly reduced the levels of serum pge , il- b, and tnf-a in rats with acute pharyngitis (qian, ) . being a popular antiviral and antibacterial drug, sfjd is one of the drugs for covid- treatment in china (pan x. et al., ) . in addition, shuanghuanglian (shl) is a popular anti-bacterial drug. it has various pharmacological potential such as antibacterial, antiviral, and immune-enhancing properties which can be exploited in the treatment of acute upper respiratory tract infection (zhang et al., ) . preliminary studies in vitro showed that shl oral liquid inhibited sars-cov- . indeed, shl has been used to carry out clinical research on covid- in shanghai public health clinical center and tongji hospital affiliated to huazhong university of science and technology (pan x. et al., ) . the antibacterial effects of volatile components of dayuan-yin are not limited to upper respiratory tract infections: these volatile components also regulate intestinal flora, treat gastric ulcers, and improve gastrointestinal symptoms. human gut microbes are the "second genome" of the human body (backhed et al., ; gill et al., ; cani and delzenne, ) . the composition of intestinal flora is closely related to human health status, and it plays an essential role in maintaining physiological balance (schuijt et al., ) . it has been confirmed that intestinal flora reduces ventilatorassociated pneumonia and enteritis by enhancing the function of primary alveolar macrophages (bradley et al., ) . patients with covid- showed intestinal microbial malnutrition and decreased microbial flora levels of some probiotics such as lactobacillus and bifidobacterium. the latest version of novel coronavirus pneumonia diagnosis and treatment plan released by the people's republic of china national health council suggests that intestinal microbiota should be used in severe and critical cases to maintain intestinal micro ecological balance (general office of the national., health commission of the people's republic of china. et al., ) . apart from the biological effects of the volatile components mentioned above, other non-volatile components of dayuan-yin also have abundant pharmacological properties. (zhang t. t. et al., ) (mitsui et al., ) . falcarindiol inhibited the growth of pseudomonas aeruginosa by repressing virulencerelated genes, including the t ss; quorum sensing synthase genes lasir and rhlir; lasb; motility-related genes flic and flig; and phenazine synthesis genes phza and phza . it is obvious that dayuan-yin exerts extensive biological properties such as antiviral, anti-inflammatory, antioxidative, and antibacterial effects. it can be inferred that dayuan-yin may play an essential role in preventing covid- pandemic. at present, there are no conventional drugs that can cure covid- (cao b. et al., ) . however, according to data collected by the national health commission of the people's republic of china, clinical practice in chinese hospitals have reported that traditional chinese medicine has a definite therapeutic effect in the early stages of covid- infection . as a significant part of medical practice, chinese medicine has been used to treat human diseases for more than , years (li and kan, ) . in recent decades, volatile compounds extracted from medicinal plants have attracted more and more attention due to their important biological effects such as antiviral, anti-inflammatory, and antibacterial properties. besides, they are non-toxic and have few side effects, making them suitable for use as drugs. this review discussed the potential role of traditional chinese medicine in terms of volatile components. the antiinflammatory, antiviral, antibacterial and immunomodulatory effects of these volatiles seem to play the most critical roles in treating patients infected with covid- . however, there are still lack of clinical trials on dayuan-yin. these need to be done in future. in china, the situation of covid- pandemic prevention and control has improved. the national pandemic situation has been controlled. however, with the resumption of factory work, re-opening of shopping malls, and resumption of transportation, the cross-flow of personnel has increased significantly, and the probability of close contact between people has increased tremendously too. in particular, with likelihood of increase in imported cases from abroad, the epidemic prevention and control should not be relaxed. it is essential to improve the ability of the human body to withstand infection. in addition to frequent washing of hands, wearing masks, social distancing and other measures, the "chinese medicine sachet" can be used as an essential means of prevention. this stems from a very important theory of traditional chinese medicine, namely "treating predisease". this idea in traditional chinese medicine originated in the yin and shang dynasties, took shape in zhouyi, and formed in huangdi neijing . chinese doctors in the past dynasties attached great importance to the prevention and treatment of diseases. they emphasized the prevention of diseases first, especially infectious diseases (lian et al., ) . wearing chinese medicine sachet is another special treatment of "treating pre-disease" . chinese medicine sachet has been used to prevent disease since ancient times. in this method, aromatic chinese medicine is put into a unique bag and worn on the chest to prevent respiratory diseases. this is known as "xiangpei therapy" (zhang q. et al., ) . from the perspective of modern medicine, the medicinal fragrance (i.e., volatile oil components) of chinese medicine sachet stimulates the nasal mucosa, promotes the secretion of immunoglobulins, and kills all kinds of viruses at the same time, thereby playing multiple roles in regulating immune function, and exerting antibacterial and anti-viral effects (lvy and bai, ) . interestingly, early intervention with aromatic chinese medicine blocks the course of diseases and relieves symptoms in clinical practice through oral administration, external fumigation, and moxibustion (lun and chen, ; chen et al., ) . aromatic chinese medicine dispels exterior pathogenic factors, regulates qi, activates blood circulation, breaks blood stasis, and disperses nodules. the application of aromatic chinese medicine embodies the theory of "internal disease and external treatment" of traditional chinese medicine (hu et al., ) . since the outbreak of covid- , fumigation has been used for air disinfection to prevent the spread of the virus. in the clinical treatment period, the application of moxibustion plays the role of anti-inflammatory agent, regulates immune function, and prevents deterioration of the patients (zhang, ; liu k. et al., ) . some local health committees or chinese medicine administration bureaus are actively involved in promoting aromatic traditional chinese medicine as an anti-epidemic, as well as the use of fumigation or chinese medicine sachet to prevent and control covid- . there is no doubt that the pharmacological effects of volatile components of traditional chinese medicine are beneficial in the global fight against covid- . however, each tcm prescription has multiple goals and links in the treatment of diseases, making it difficult to clearly and thoroughly explain its mechanism in a short period. more research should be carried out on volatile components of traditional chinese medicine to elucidate the associated regulatory mechanism, evaluate possible side effects, and conduct standard clinical trials. the insights provided in this review may help ease the covid- pandemic worldwide. q-wh and jw are the corresponding authors on the study. x-rz and t-nl are first authors and responsible for collecting materials and writing the paper. y-yr, y-jz, and h-yl helped in organizing the information and edited the article pictures. all authors contributed to the article and approved the submitted version. this work is financially supported by the xinglin scholar talent promotion plan of chengdu university of traditional chinese medicine (qnxz , xsgg ). we are indebted to our alma mater, chengdu university of traditional chinese medicine for provided convenience in the collection of documents. thanks for all the help from everyone in our lab. research progress on the clinical effects of chinese herbal sachets in preventing colds hypolipidemic and antioxidant activities of thymoquinone and limonene in atherogenic suspension fed rats host-bacterial mutualism in the human intestine the inflammation: lipoprotein cycle anti-rheumatoid arthritic effect of volatile components in notopterygium incisum in rats via antiinflammatory and anti-angiogenic activities the acute respiratory distress syndrome tonic interferon signals in lung stromal cells protect from influenza virus infection pressssdiagnostic nonsteroidal anti-inflammatory drug use and lung cancer survival in the vital study dexamethasone for covid- : data needed from randomised clinical dayuan-yin against the covid- pandemic frontiers in pharmacology | www immune regulation by glucocorticoids gut microflora as a target for energy and metabolic homeostasis chemical composition and antiinflammatory effects of essential oil from houpo (magnolia officinalis bark) the preventative effects of procyanidin on binge ethanol-induced lipid accumulation and ros overproduction via the promotion of hepatic autophagy a trial of lopinavir-ritonavir in adults hospitalized with severe covid- the important role of polysaccharides from a traditional chinese medicine-lung cleansing and detoxifying decoction against the covid- pandemic citrus reticulata peel oil inhibits non-small cell lung cancer cell proliferation in culture and implanted in nude mice citrus reticulata peel oil as an antiatherogenic agent: hypolipogenic effect in hepatic cells, lipid storage decrease in foam cells, and prevention of ldl oxidation broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus simulation of the clinical and pathological manifestations of coronavirus disease (covid- ) in golden syrian hamster model: implications for disease pathogenesis and transmissibility the quality of chinese medicine and its rational application in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds protective effects of pogostone from pogostemonis herba against ethanol-induced gastric ulcer in rats b-patchoulene from patchouli oil protects against lps-induced acute lung injury via suppressing nf-kb and activating nrf pathways discussion on role of aromatic chinese herbs in prevention and treatment of covid- based on theory of traditional chinese medicine antiviral activities of atractylon from atractylodis rhizoma insecticidal compounds from the essential oil of chinese medicinal herb atractylodes chinensis anti-infectious efficacy of essential oil from caoguo (fructus tsaoko) anti-inflammatory effects of essential oils of amomum aromaticum fruits in lipopolysaccharide-stimulated raw . cells chemical composition and in vitro inhibitory effects of essential oils from fruit peel of three citrus species and limonene on mycelial growth of sclerotinia sclerotiorum the relationship between copd and lung cancer a comparative study on chemical composition and antioxidant activity of ginger (zingiber officinale) and cumin (cuminum cyminum) efficacy of cineole in patients suffering from acute bronchitis: a placebo-controlled double-blind trial cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health metagenomic analysis of the human distal gut microbiome impact of air pollution on the burden of chronic respiratory diseases in china: time for urgent action the anti-viral studies of notopterygium incisum headspace solid-phase microextraction-gas chromatography-mass spectrometry for the analysis of volatile compounds from a.lancea research about effective ingredients of magnolia officinalis based on their antioxidant activity inhibitory effects of atractylone on mast cell-mediated allergic reactions antioxidant and antibacterial activities of essential oil from atractylodes lancea rhizomes vitamin c and infections evaluation of antibacterial potential and toxicity of plant volatile compounds using new broth microdilution volatilization method and modified mtt assay mechanistic links between copd and lung cancer mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus attenuation of endotoxin-induced oxidative stress and multiple organ injury by , -methylenedioxyphenol in rats application of aromatic herb in external therapy experimental lung injury reduces krüppel-like factor to increase endothelial permeability via regulation of rapgef -rac signaling clinical features of patients infected with novel coronavirus in wuhan triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial dayuan-yin against the covid- pandemic frontiers in pharmacology | www metabolite profiles of essential oils in citrus peels and their taxonomic implications covid- dashboard by the inhibition of cytokine production and arachidonic acid metabolism by eucalyptol ( . -cineole) in human blood monocytes in vitro antiinflammatory effects of euclyptol ( . -cineole) in bronchial asthma: inhibition of arachidonic acid metabolism in human blood monocytes ex vivo inhibition of h+,k+-atpase by hinesol, a major component of so-jutsu, by interaction with enzyme in the e state eucalyptol attenuates cigarette smoke-induced acute lung inflammation and oxidative stress in the mouse exhibits anti-inflammatory activity through the suppression of mapks and the nf-kb pathway in mouse peritoneal macrophages atractylone, an active constituent of kmp , attenuates allergic inflammation on allergic rhinitis in vitro and in vivo models clinical use of dornase alpha is associated with a slower rate of fev decline in cystic fibrosis oxidative stress in biological systems and its relation with pathophysiological functions: the effect of physical activity on cellular redox homeostasis analysis of tetramethylpyrazine in ephedrae herba by gas chromatography-mass spectrometry and highperformance liquid chromatography the use of dexamethasone in the treatment of covid- traditional chinese medicine for pulmonary fibrosis therapy: progress and future prospects pogostone suppresses proinflammatory mediator production and protects against endotoxic shock in mice , -cineol protect against influenza-virus-induced pneumonia in mice two cases of covid- treated effectively with modified da yuan drinking review of classical famous prescription "dayuanyin" used for the treatment of covid- and analysis of pulmonary classical famous prescriptions discussion on tcm theories of "qingwen pihui san" to prevent new coronavirus pneumonia effect of dexamethasone in hospitalized patients with covid- -preliminary report essential oils from taiwan: chemical composition and antibacterial activity against escherichia coli cytokine storms, hyper-inflammatory phenotypes, and acute respiratory distress syndrome. genes dis attach importance to research and development of chinese materia medica based on prevention and control needs of sars-cov- infection feasibility analysis of moxibustion whole process intervention in the prevention and treatment of covid- vardenafil as anti-inflammatory drug in the treatment of cystic fibrosis lung disease prevention and treatment of aromatic chinese medicine study on the mechanism of aromatic traditional chinese medicine compound on prevention and treatment of new coronavirus pneumonia discussion on the effect of air disinfection for aromatic smell of traditional chinese medicine sachet the role of pneumonia and secondary bacterial infection in fatal and serious outcomes of pandemic influenza a(h n )pdm retinal findings in patients with covid- supercritical co extracts and essential oil of ginger (zingiber officinale r.): chemical composition and antibacterial activity the herbal medicine compound falcarindiol from notopterygii rhizoma suppresses dendritic cell maturation reactive oxygen species in inflammation and tissue injury , -cineole potentiates irf -mediated antiviral response in human stem cells and in an ex vivo model of rhinosinusitis effect of some volatile oils on the affinity of intact and oxidized lowdensity lipoproteins for adrenal cell surface receptors general office of china., health commission of the people's republic of and medicine human infection with strongyloides stercoralis and other related strongyloides species use of antiviral drugs to reduce covid- transmission qualitative study on volatile components of different reticulatae pericarpium products based on gc-ms potential drugs for the treatment of the novel coronavirus pneumonia (covid- ) in china antiinflammatory effects of ginger and some of its components in human bronchial epithelial (beas- b) cells dayuan-yin against the covid- pandemic frontiers in pharmacology | www effect of shufeng jiedu capsule on serum pge , il- b and tnf-a in rats with acute pharyngitis and clinical efficacy observation chronic obstructive pulmonary disease traditional chinese medicine for covid- treatment chemical screening and antibacterial activity of essential oil and volatile fraction of dictyopteris polypodioides mechanism of dayuanyin in the treatment of coronavirus disease based on network pharmacology and molecular docking the gut microbiota plays a protective role in the host defence against pneumococcal pneumonia the therapeutic effect of , -cineol on pathogenic bacteria species present in chronic rhinosinusitis oxidative stress: a concept in redox biology and medicine effect of patchouli (pogostemon cablin) essential oil on in vitro and in vivo leukocytes behavior in acute inflammatory response covid- : combining antiviral and anti-inflammatory treatments immunosuppressive activity of pogostone on t cells: blocking proliferation via s phase arrest , -cineol reduces mucus-production in a novel human ex vivo model of late rhinosinusitis modern enlightenment on aromatic drugs in the prevention and treatment of epidemic diseases in ming and qing dynasties protective effects of pogostone against lps-induced acute lung injury in mice via regulation of keap -nrf /nf-kb signaling pathways atractylodin attenuates lipopolysaccharide-induced acute lung injury by inhibiting nlrp inflammasome and tlr pathways comparative study on chemical composition and antibacterial activity of essential oils from patchouli and magnolia officinalis alone and combination low-cost dexamethasone reduces death by up to one third in hospitalised patients with severe respiratory complications of covid- limonene: aroma of innovation in health and disease guideline for the diagnosis and treatment of copd patients -issued by the german respiratory society and the german atemwegsliga experience in treatment of covid- by elimination of pathogens through purgation and diuresis effects of beudesmol, an active constituent from rhizoma atractylodis on small intestine movement in rats study on the molecular mechanism of pogotone against staphylococcus aureus a case of dayuan yin and liujunzi decoction treating with covid- study on the effect of anti-respiratory viruses of patchouli oil in vitro effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial the pittsburgh lung screening study (pluss): outcomes within years of a first computed tomography scan patients with asthma benefit from concomitant therapy with cineole: a placebo-controlled, double-blind trial concomitant therapy with cineole (eucalyptole) reduces exacerbations in copd: a placebo-controlled double-blind trial new guaiane sesquiterpenes and furanocoumarins from notopterygium incisum a new coronavirus associated with human respiratory disease in china orthogonal array design for the optimization of supercritical co extraction of volatile oil from amomum tsaoko fruits theoretical origin and practical thinking of the theory of chinese medicine preventive treatment of disease analysis of volatile components of ephedra from different places based on gc-ms cytotoxic, apoptotic and antioxidant activity of the essential oil of amomum tsao-ko pogostone attenuates tnf-a-induced injury in a cells via inhibiting nf-kb and activating nrf pathways gc-ms analysis of volatile oil from atractylodes chinensis a pathological report of three covid- cases by minimal invasive autopsies. zhonghua bing li xue za zhi = chin limonene suppresses lipopolysaccharide-induced production of nitric oxide, prostaglandin e , and pro-inflammatory cytokines in raw . macrophages chinese medicine injection shuanghuanglian for treatment of acute upper respiratory tract infection: a systematic review of randomized controlled trials effect of miao medicine fanggan sachet on expression of nkp in peripheral blood of mice bioactivity evaluation of ingredients identified from the fruits of amomum tsaoko crevost et lemaire, a chinese spice dayuan-yin against the covid- pandemic frontiers in pharmacology | www pseudomonas aeruginosavirulence-inhibiting herbal compound falcarindiol significantly reduced mortality in mice infected with the antibacterial effect of cangzhu and its application in the disinfection of hospital environment , -cineol attenuates lps-induced acute pulmonary inflammation in mice effect of alpha-pinene on nuclear translocation of nf-kappa b in thp- cells effect of patchouli oil on acute bacterial upper respiratory infections model mice alphalinolenic acid protects against lipopolysaccharide-induced acute lung injury through anti-inflammatory and anti-oxidative pathways the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zhang, li, ren, zeng, lv, wang and huang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - bvoy authors: zhang, fan; nan, xuemei; wang, hui; guo, yuming; xiong, benhai title: research on the applications of calcium propionate in dairy cows: a review date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: bvoy simple summary: in modern dairy cattle production systems, the mycotoxins in feed and metabolic disease, such as ketosis and milk fever, seriously affect the health and milk production of dairy cows. calcium propionate is a safe and reliable food and additive that is widely used. it can be employed in silage and total mixed rations (tmr) against mycotoxin production. in the perinatal period, many cows cannot adjust to the tremendous metabolic, endocrine, and physiological changes, resulting in ketosis and fatty liver due to a negative energy balance or milk fever induced by hypocalcemia, which damages their health and reduces the production performance. studies have revealed that calcium propionate can play an active role in solving these problems. it can also regulate rumen development in calves. this paper reviews the recent research progress regarding the application of calcium propionate in dairy cows and dairy calves. the key findings and mechanisms are summarized and potential further studies are suggested. abstract: calcium propionate is a safe and reliable food and feed additive. it can be metabolized and absorbed by humans and animals as a precursor for glucose synthesis. in addition, calcium propionate provides essential calcium to mammals. in the perinatal period of dairy cows, many cows cannot adjust to the tremendous metabolic, endocrine, and physiological changes, resulting in ketosis and fatty liver due to a negative energy balance (neb) or milk fever induced by hypocalcemia. on hot weather days, cow feed (tmr or silage) is susceptible to mildew, which produces mycotoxins. these two issues are closely related to dairy health and performance. perinatal period metabolic disease significantly reduces cow production and increases the elimination rate because it causes major glucose and calcium deficiencies. feeding a diet contaminated with mycotoxin leads to rumen metabolic disorders, a reduced reproductive rate (increased abortion rate), an increased number of milk somatic cells, and decreased milk production, as well as an increased occurrence of mastitis and hoof disease. propionic acid is the primary gluconeogenic precursor in dairy cows and one of the safest mold inhibitors. therefore, calcium propionate, which can be hydrolyzed into propionic acid and ca( +) in the rumen, may be a good feed additive for alleviating neb and milk fever in the perinatal period of dairy cows. it can also be used to inhibit tmr or silage deterioration in hot weather and regulate rumen development in calves. this paper reviews the application of calcium propionate in dairy cows. there are many important challenges in dairy production, including reducing the feed intake and metabolic diseases caused by a negative energy balance (neb) [ ] and milk fever [ ] during the perinatal period and mycotoxin pollution [ ] of feed induced by environmental and climatic conditions, which have negative effects on milk production and quality and pose a potential threat to human health. in particular, ketosis and hypocalcemia represent two potentially devastating insults to the lactating dairy cow [ ] . appropriate dosages of calcium propionate as a feed additive can effectively alleviate these difficulties and serve the dairy industry. since calcium propionate does not inhibit yeast growth, it is one of the most useful antimicrobial preservatives in the fermented foods industry, especially in bread and fermented dairy products; in aqueous solution, it can dissociate to propionic acid (the active antifungal ingredient) and calcium ions [ ] . it can be used as a feed preservative, growth promoter, intestinal microbiota enhancer, or appetite suppressant in animal nutrition [ ] . in the dairy cow industry, calcium propionate can also be applied in many cases to inhibit mycotoxin production and as a metabolite precursor additive. propionic acid and ca + are basic components in the rumen fluid [ ] , which means that calcium propionate is safe to add to the feed of dairy cows. it is approved by the world health organization (who) and the united nations food and agriculture organization (fao) for use in food or feed additives. therefore, it is used as a safe and valuable additive in the dairy cow industry. to update our knowledge on calcium propionate application for dairy cow performance and metabolism, we reviewed the effects of calcium propionate supplementation on decreasing feed mycotoxins, alleviating dairy cow neb and milk fever, and promoting rumen development in dairy calves. calcium propionate is an organic salt formed by the reaction between calcium hydroxide and propionic acid [ ] and has the molecular formula (ch ch coo) ca. the compound exists in either crystalline or powder form. calcium propionate is soluble in water and can be hydrolyzed into ca + and propionic acid. the solution is alkaline, but exerts bacteriostatic effects in acidic media. it is mainly synthesized through the reaction between a calcium source (caco , cao, ca(oh) , egg, or oyster shell) and propionic acid. through the reaction, the generated calcium propionate is concentrated, dried, and dehydrated to obtain qualified products. calcium propionate is a strong preservative with little or no flavor with normal use that can be effective against mold and bacteria and is widely used in foods, feeds, and pharmaceuticals [ ] . it has the ability to inhibit the growth of molds and other microorganisms without an obvious inhibition of yeasts [ ] . the antimicrobial properties of calcium propionate are dependent upon the corresponding undissociated acids in solution and involve the uncoupling of microbial substrate transport and oxidative phosphorylation from the electron transport system [ ] . when exhibiting the same bacteriostatic effect, the effective dose of calcium propionate has been shown to be lower than that of sodium propionate. calcium propionate has no teratogenic activity or reproductive toxicity, and propionic acid can be excreted in urine; thus, there is no risk of accumulation in the human body, even at large doses [ ] . propionic acid can interfere with the electrochemical gradients in the cell membrane, disrupt transport processes, and inhibit the uptake of substrate molecules, such as phosphate and amino acids [ ] . the high-affinity potassium transporter in the cell membrane of yeasts enables the maintenance of ph homeostasis and stabilization of membrane potential by potassium uptake and accumulation, so the yeasts can develop tolerance to propionic acid [ ] . however, the molds are susceptible to propionic acid. the antimicrobial activity of calcium propionate is due to the neutral undissociated propionic acid form, which is lipophilic and readily soluble in fungal cell membranes [ ] . therefore, the antimicrobial effect of calcium propionate depends on the ph value of the product because the undissociated acid has a better antimicrobial effect than the dissociated acid [ ] . compared to % undissociated propionic acid at ph . , only % of the propionic acid can be undissociated at ph . an enhanced inhibitory effect of calcium propionate at a lower ph is expected, and the maximum ph at which calcium propionate exerts measurable antimicrobial activity is approximately . - . [ ] . therefore, a lower ph is beneficial to the improvement of the antibacterial properties of calcium propionate under appropriate conditions. many antibacterial actions of calcium propionate have been found, and it can reduce the count of aspergillus flavus [ ] , escherichia coli o :h , salmonella enterica serovar typhimurium [ ] , clostridia [ ] , and so on. because of its antimicrobial properties, calcium propionate can also reduce mold and act as a preservative in many industries. in the feed industry, a warm and humid climate and a long postharvest period favor mold growth and the production of mycotoxins [ ] . molds can cause economic losses and health problems due to the production of mycotoxins. aflatoxins, which are potent mycotoxins mainly produced by aspergillus flavus and aspergillus parasiticus, have carcinogenic, mutagenic, teratogenic, and growth inhibiting effects on animals and humans [ ] . calcium propionate is a well-established chemical mold inhibitor that can be used in the feed industry to inhibit mold growth and reduce the incidence of aflatoxicosis in animals. moreover, because of less feed spoilage, the heat production in feed is also reduced, preventing energy loss and poor palatability of the feed, which the cattle may refuse to eat. bintvihok and kositcharoenkul [ ] indicated that aflatoxin b -calcium propionate-supplemented diet groups showed increased body weight gain, feed consumption, and feed conversion, but decreased residual levels of aflatoxin b and aflatoxin m in muscle and liver tissues compared with those of aflatoxin b -supplemented groups. therefore, calcium propionate is a reliable additive for silage and tmr in dairy cows. in other fields, calcium propionate has been used for paper preservation [ ] , increasing the fruit shelf life [ ] , and inhibiting bread spoilage [ ] , due to its antibacterial properties, and can also be used as an additive for silage and tmr in dairy cows. as described above, calcium propionate can be hydrolyzed into ca + and propionic acid. calcium and propionic acid in solution are indispensable sources of nutrients for ruminants. it is well-known that calcium is essential for the formation of skeletal tissue, transmission of nervous tissue, muscle contractility, and essential minerals for blood and milk. in contrast, the role of calcium with respect to the immune function and intermediary metabolism explains the contribution of subclinical hypocalcemia to the development of several diseases observed in early lactation and underlines its importance in high-performing dairy cows [ ] . oral calcium supplementation with calcium propionate, which is a calcium source in solution, can be greatly absorbed by the rumen and increase the ionized calcium concentration in the blood. an increase in digestible energy intake has been shown to increase the posthepatic glucose supply [ ] . propionate, which is produced from the ruminal fermentation of starch and other organic matter, is the primary glucose precursor for ruminants [ ] . propionic acid, which is hydrolyzed from calcium propionate and formed under acidic conditions in the rumen, is absorbed by the rumen epithelium, passes to the liver through the portal vein, and synthesizes glucose. in fed cows, propionate is the major precursor of glucose, and the liver removal of propionate contributes to up to % of glucose hepatic release [ ] . propionate is an obligatory anaplerotic metabolite for the tricarboxylic acid (tca) cycle [ ] . it enters the tca cycle through succinate, thus providing carbons that can either remain within the tca cycle or be extracted from the cycle for gluconeogenesis [ ] . therefore, calcium propionate can be used as a good additive in ruminants for gluconeogenesis, and this property has been found to improve lamb performance [ ] , act as an energy source for finishing lambs [ ] , and improve beef quality [ ] . therfore, it is a favorable nutrient additive for dairy cows. as mentioned above, calcium propionate can be metabolized and absorbed by animals, providing them with essential calcium and glucose precursors, which are advantages that are not offered by other anti-mildew agents. furthermore, it is generally regarded as safe (gras) in the united states, where upper limits only exist for its use in specific human food items [ ] . therefore, it is widely used in dairy cows as an antimicrobial agent, glucose precursor, and calcium provider. silage is one of the most common ingredients in the diets of dairy cows and is an important source of nutrients. however, poorly made or contaminated silage can also be a source of pathogenic bacteria that may decrease the dairy cow performance, reduce the safety and quality of dairy products, and compromise animal and human health [ ] . molds identified in fermented feeds include aspergillus sp., cladosporium sp., fusarium sp., mucor sp., and penicillium sp., and their adverse effects may occur through either their deleterious effects on the nutrient quality or their production of mycotoxins [ ] . several mycotoxins have been detected in corn silage, including aflatoxin b , citrinin, deoxynivalenol, gliotoxin, and zearalenone [ ] . to enhance the quality of silage, fermentation and the aerobic stability can be improved by adding silage additives. propionic acid-based products, which are compatible with microbial inoculants, can be used as a silage additive. the combined use of propionic acid-based products and microbial inoculants can result in improvements in silage fermentation and the aerobic stability [ ] . propionic acid has excellent antifungal activity and has little impact on the activity of lactic acid bacteria. the application of propionic acid presents some problems due to its corrosive and hazardous nature, but its salt-calcium propionate-also has antimicrobial effects; additionally, it is safe and easy to handle [ ] . calcium propionate is an effective tool for suppressing the germination, growth rate, and aflatoxin production of aspergillus flavus (a- ) in different substrates [ ] . therefore, calcium propionate has the potential, as an additive in silage, to inhibit the growth of molds and decrease the mycotoxin contents in silage. alfalfa is a protein-rich forage that is widely cultivated and has become a major protein source of diets for dairy cows. fresh alfalfa silage is prone to clostridia spoilage because of its low dry matter, low sugar contents, and high buffering capacity [ ] . dong et al. [ ] demonstrated that the amount of enterobacteria, molds, and clostridia decreased linearly with an increasing calcium propionate proportion in alfalfa silage, while lactic acid bacteria counts quadratically increased; calcium propionate can improve the fermentation quality and aerobic stability of proteolysis alfalfa silage, and the recommended additive level is g/kg fresh weight. wen et al. [ ] evaluated the potential of calcium propionate as an alfalfa silage additive and found that calcium propionate decreased the butyric acid content and dry matter loss and increased the water-soluble carbohydrate content. however, the ph decreased slowly at the start of ensilage, possibly because of the alkaline properties of calcium propionate. after days of storage, calcium propionate increased the concentrations of lactic, acetic, propionic, and total organic acids and the microbial populations of lactic acid bacteria, but decreased the enterobacteria, mold, and clostridia populations [ ] . the lower population of molds and clostridia may be related to the antimicrobial effects of calcium propionate. clostridium is a kind of undesirable microorganism that is harmful to animal health. it not only destroys lactic acid, but also leads to an increase in the ph value and a decrease in the nutritional value of silage [ ] . in conclusion, calcium propionate is a good additive for silage, which can act as a significant inhibitor for the growth of molds and clostridia. warm and humid conditions are favorable for mold growth and can result in increased mycotoxin production. the spoilage of tmr in summer is an important factor affecting the production efficiency. to reduce the influence of tmr mold growth and its metabolites on the production performance, health, and milk quality of cows, appropriate methods, including chemical additives, water content control, increasing the number of fresh feed deliveries per day, and the timely cleaning of leftovers, must be used. as mentioned above, calcium propionate is a safe and effective inhibitor of mold, and can improve the aerobic stability of feed. mold growth can be prevented in coarse texture feeds and other high moisture feeds by the addition of calcium propionate [ ] . the addition of calcium propionate to tmr inhibits feed spoilage. therefore, the proper addition of calcium propionate in tmr feed may have the function of preventing feed corruption. however, research on the recommended amount of calcium propionate added to tmr to prevent feed spoilage is needs to be further explored. the perinatal period from late pregnancy to early lactation is a critical period in a dairy cow's life due to the rapidly increased drain of nutrients from the mother towards the fetus and into the colostrum and milk [ ] . after calving, with a high yield of milk, the nutrient intake of dairy cows is large under the need to supply the output of milk, resulting in a negative nutrient balance that requires the mobilization of body reserves. the metabolic diseases fatty liver and ketosis are due to the extent of glucose deficit that induces the excessive mobilization of body fat. in addition, during the perinatal period, the dry matter intake of dairy cows is reduced due to the diminution in rumen volume induced by the growth of the fetus and other hormonal changes [ ] . the high energy demands of lactation, coupled with a reduction in the dry matter intake around calving, means that the majority of dairy cows enter the state of neb in early lactation [ ] . cows showing excessive neb utilize their body fat as a source of energy to maintain the rapidly increasing milk yield, which leads to excessive body fat mobilization, ketosis, and fatty liver syndrome. metabolic or infectious diseases, including fatty liver syndrome and ketosis, affect dairy cow production during the perinatal period [ ] and further impact the welfare, productive lifespan, and economic outcomes of dairy cows [ ] . strategies for supplying energy are one way of mitigating neb. based on a large amount of data on cattle and other species, glucose is known to reduce the fatty acids mobilized from adipose tissue [ ] . the failure of cows to meet their glucose demands for lactation leads to an impaired immune response and an increased risk of disease that may affect milk production and profitability [ ] . for cow rearing, the dietary energy can be improved through fat or concentrate supplementation to alleviate neb, but excess fat supplementation inhibits rumen microbial growth, decreases the rumen ph value, and increases the rate of subclinical ruminal acidosis [ ] . glucose precursors, such as propylene glycol and calcium propionate, have been used in dairy cattle to correct metabolic problems [ ] . propionate can directly regulate its own metabolism in isolated bovine hepatocytes through upregulation of the mrna expression of cytosolic phosphoenolpyruvate carboxykinase (pck ), mitochondrial phosphoenolpyruvate carboxykinase (pck ), and pyruvate carboxylase (pc), which are the key enzymes required for the stimulation of gluconeogenesis from propionate in ruminants [ ] . propionate is the major glucose precursor in ruminants that has a positive energy balance and anti-ketogenic effects [ ] . it is used as a readily available energy source to correct metabolic problems in dairy cattle [ ] . propionate, whose liver uptake is preferential and highly efficient, can inhibit hepatic lipid oxidation and the production of ketones [ ] . during the perinatal period, calcium propionate is a good available energy source for preventing metabolic disorders in dairy cows, so it can be incorporated into the diet and increase the rumen concentration of propionate, which is the main precursor for glucose synthesis in the liver [ ] . abdel-latif et al. [ ] found that the supplementation of calcium propionate in primiparous egyptian buffalo cows during late gestation and early lactation significantly improved the body weight, reproductive parameters such as first estrus postpartum, days open, and number of services per conception; it also significantly decreased the blood metabolites of nonesterified fatty acids (nefas) and increased the glucose and insulin concentrations. the beta-hydroxybutyrate (bhba) can be considered an indicator of a negative energetic balance due to its correlation with the energetic demand and energy reserves [ ] . in the study of martins et al. [ ] , the calcium propionate-supplemented group had a lower amount of blood bhba because the propionate is the principal source of gluconeogenesis in peripartum cows. maintaining or increasing the dm intake is also crucial for alleviating neb in the perinatal period. the effects of calcium propionate on the dm intake in dairy cows are controversial; some studies have reported no difference [ ] , and some have reported a higher dm intake with calcium propionate [ ] . mcnamara and valdez [ ] showed that cows fed . kg/d calcium propionate increased their dm intake by % and % in the prepartum and postpartum groups, respectively, compared with that of the control group, and showed reduced net lipolysis, but increased adipose tissue lipogenesis, at postpartum. martins et al. [ ] confirmed that, during early lactation, g/d calcium propionate provided a better energetic supply for dairy cows, which can also increase the milk yield and protein, lactose, fat, and total solids contents in milk, despite the reduced dry matter intake. propionate is converted to glucose in the liver, supporting lactose synthesis in the mammary gland [ ] . liu et al. [ ] observed that increasing the supplementation of calcium propionate improved the energy status, as indicated by the higher blood glucose, lower blood bhba and nefa, and lower urine ketones. in ruminants, an increase in glucose precursors, such as propionate, could optimize nutrient use and improve milk production [ ] . the concentration of oxaloacetate determines whether acetyl-coa enters the tca-cycle or ketogenesis occurs. the antiketogenic effect of calcium propionate is also related to the increase of the oxaloacetate content in the mitochondria of the liver. calcium propionate can be incorporated into the diet or fed per cow per day for cows in early lactation. the optimum dose was approximately g per cow per day in the experimental conditions of liu et al. [ ] . in conclusion, calcium propionate can act as a good glucose precursor to alleviate neb in dairy cows. however, the maximum dose available in cows still needs to be studied because calcium propionate can depress appetite [ ] . milk fever is a metabolic disease characterized by clinical symptoms due to a reduction in the blood calcium concentration (hypocalcemia) during peripartum, which affects high-yielding multiparous cows [ ] . it is one of the most common periparturient abnormalities afflicting dairy cows [ ] . milk fever can decrease the dry matter intake, milk production, and reproductive performance and increase the risk of secondary diseases, such as ketosis, a retained placenta, displaced abomasum, mastitis, and the incidence of dystocia and uterine disorders [ , ] . when the concentration of blood calcium falls below a critical threshold, it results in clinical and subclinical milk fever [ , ] . serum calcium levels of and . mmol/l have been proposed as thresholds of subclinical and clinical hypocalcemia, respectively, but the external signs may not be displayed in dairy cows [ ] . improving the mobilization of calcium from bone and the absorption of calcium from the diet are two major processes that prevent the decrease in blood calcium in dairy cows. the mobilization of calcium from bone can be accomplished by feeding a calcium-deficient diet or negative dietary cation-anion difference in the pre-calving period [ ] . in addition, the infusion of -hydroxytryptophan can also improve blood calcium concentrations around parturition [ ] . however, after calving, it is important to improve the available calcium in the diet for absorption. it is well-accepted that calcium can be absorbed across the rumen wall of sheep and goats if the soluble calcium concentration is high [ , ] . schroder et al. [ ] proved that calcium can be absorbed across the cattle rumen epithelium in vitro. calcium absorption by the rumen appears to be a key factor in calcium homeostasis at the onset of lactation, and its failure causes uncontrolled hypocalcemia, leading to parturient paresis [ ] . calcium sources that are soluble at a slightly acidic ph may result in more absorption from the rumen, intestine, or both, than insoluble calcium sources [ ] . to increase the calcium absorption, an effective method is to increase the concentration of ionized calcium within the rumen by the given supplement [ ] . providing a highly soluble source of oral calcium induces high concentrations of ionized calcium in the lumen of the gastrointestinal tract. the high concentrations of ionized calcium in the rumen lumen induce a chemical gradient that passively transports ionized calcium from the mucosa through the tight junctions towards the extracellular space on the serosa side, increasing the concentrations of ionized calcium in the blood [ ] . the administration of a ruminal calcium bolus (approximately g of calcium) has been used to restore blood calcium concentrations [ ] by improving the amount of calcium that can be absorbed from feed. calcium chloride solutions and gel products offer good calcium sources that are very soluble, very concentrated ( % calcium), and rapidly absorbed, making them generally effective in reducing the incidence of milk fever [ ] . large amounts of propionic acid are produced in the rumen by carbohydrate metabolism, and there are no obvious adverse effects, so calcium propionate might also be a satisfactory source of calcium [ ] . although its effects on blood calcium are not as rapid as those of calcium chloride, calcium propionate can be used in the form of calcium propionate paste and given at calving or after calving in dairy cows to prevent milk fever. pehrson et al. [ ] showed that the incidence of milk fever in a calcium propionate-treated ( g of calcium in total) group of cows that experienced milk fever during previous calving was . %, which was lower than that in untreated cows ( . %), but similar to that in the calcium chloride-treated group ( . %). therefore, calcium propionate is considered a satisfactory alternative to calcium chloride for the prevention of milk fever [ ] . goff et al. [ ] also demonstrated that calcium propionate paste treatment was beneficial in reducing subclinical hypocalcemia and could reduce the incidence of milk fever from % in control cows to % in treated cows. calcium propionate is less soluble in water than calcium chloride, but its solubility is adequate and more soluble than that of calcium lactate, calcium sulfate, and calcium carbonate. calcium propionate is neutral in taste and has no erosive effect on the digestive tract mucosa [ ] . to prevent milk fever, calcium propionate is usually administered orally around the time of the calving of cows. kara et al. [ ] found that giving cows two drenches (each drench contained . kg calcium propionate) at calving and h after calving was beneficial in treating milk fever. in summary, calcium propionate can act as a source of calcium to prevent milk fever in dairy cows during the perinatal period. the rumen is a vital digestive organ that plays a key role in the growth, production performance, and health of ruminants. therefore, promoting rumen development has always been a key target of calf nutrition [ ] . the papilla length of the rumen is the most important factor for the evaluation of rumen development [ ] . rumen epithelium development plays a very important role in the absorption, metabolism, and transportation of volatile fatty acids (vfas). vfas, such as propionic and butyric, provide the main chemical stimuli for the proliferation of the rumen epithelium if the amount is sufficient [ ] , indicating that additives of propionate may be used in calf feed as rumen growth promoters. as one kind of propionate, the additive of calcium propionate may also stimulate the epithelium development of calves. g protein-coupled receptors (gprs) are integral membrane proteins which are activated by an external signal in the form of a ligand or other signal mediator [ ] . zhang et al. [ ] found that calves supplemented with % calcium propionate (mixed in milk replacer and starter ration) in the diet had a greater rumen papillae length and improved mrna expression of g protein-coupled receptor (gpr ), gpr , and cyclin d after feeding for days, which indicated that propionate acted as a signaling molecule to improve the rumen epithelium. propionate can be converted into glucose in the liver, and higher glucose concentrations mean that high energy can be used to increase the body weight of calves. zhang et al. [ ] pointed out that there were no differences in dmi with the different feeding levels of calcium propionate, but the addition of calcium propionate improved the growth performance and gastrointestinal tract traits of jersey calves; thus, adding % calcium propionate to the feed before days and % for to days was beneficial for calves. cao et al. [ ] also verified that calcium propionate supplementation ( % dry matter) can improve body weight gain and rumen growth both pre-and postweaning. monensin is an ion carrier that can change the number of rumen microorganisms, reduce the amount of methane production, increase propionate in the rumen, decrease the intake of dry matter, and improve the efficiency of milk production and weight gain of dairy cows [ ] . however, as an antibiotic, the use of monensin in animal feed as a growth promoter may enhance the risk of antibiotic-resistant strains, so it is important to seek alternatives to this compound [ ] . ferrerra and bittar [ ] revealed that employing calcium propionate as an additive in starter feeds of calves resulted in an equal animal performance before and after weaning in comparison to that of sodium monensin, which suggests that sodium monensin may be replaced by calcium propionate. therefore, calcium propionate can be used as a good additive to promote the rumen development and growth of dairy calves. however, the use of calcium propionate in dairy cows should be controlled at appropriate doses because an overdose has a hypophagic effect in ruminants [ ] and may decrease the dmi of dairy cows. it has also been reported that calcium propionate induces a negative causation state while reducing the feed intake in broiler breeders [ ] , rats [ ] , and steers [ ] at high doses. the metabolic feedback theory contends that when the absorption of nutrients, principally energy and protein, exceeds the requirements, negative metabolic feedback impacts dmi. calcium propionate is an important energy provider when working as an additive to alleviate neb in dairy cows. therefore, propionic acid is the fuel most likely to stimulate satiety and reduce the feed intake in dairy cows [ ] because it has a high energy concentration. propionic acid can stimulate the oxidation of acetyl coa in the liver [ ] . according to the oxidation theory, the oxidation of fuels in the liver can stimulate satiety by transmitting signals via hepatic vagal afferents to feeding centers in the brain [ ] . oba and allen [ ] confirmed that a propionate infusion linearly decreased the dmi of dairy cows at higher doses. when feeding calcium propionate at a high level, the tca cycle intermediates increase, stimulating the oxidation of acetyl coa, likely affecting the feeding behavior and satisfaction. however, propionate had a smaller hypophagic effect at low plasma glucose concentrations and had a greater hypophagic effect at elevated plasma glucose concentrations [ ] . therefore, when appetite reduction occurs in cows, the supply of calcium propionate suppresses the requirement for gluconeogenesis. however, the maximum dose available in cows remains to be determined. calcium propionate is a safe and reliable food and feed additive. the calcium ions and propionic acid generated by calcium propionate hydrolysis are the basic components in the rumen of dairy cows. the application of calcium propionate in dairy cows is summarized in figure . calcium propionate can be used as a silage additive to inhibit the growth of mildew, reduce mycotoxins, and improve the aerobic stability. adding calcium propionate to tmr feed can inhibit the putrefaction of the feed in hot weather; thus, calcium propionate can also be used as a preservative in feed. propionic acid generated by the hydrolysis of calcium propionate is the main glucose precursor in dairy cows. adding calcium propionate to the diet during the perinatal period can effectively alleviate the nutritional metabolic disease caused by neb in dairy cows, and the calcium ions generated by hydrolysis can serve as a calcium source to effectively alleviate paralysis caused by milk fever. calcium propionate can also promote dairy calves' rumen epithelium development to promote calf growth through propionic acid. proper feeding is beneficial to the health of dairy cows, but excessive feeding may inhibit the appetite and limit the intake. in summary, the applications of calcium propionate in dairy cows mainly include the inhibition of feed mildew, alleviation of neb, prevention of milk fever, and promotion of the rumen epithelial development of dairy calves. figure . summary of the calcium propionate applications in dairy cows. the effects of supplementation with calcium propionate in dairy cows can be divided into the functions of hydrolyzed ca + and propionic acid. ca + , as a water-soluble calcium source, can reduce the incidence of milk fever by increasing the calcium content in the blood of perinatal dairy cows. propionic acid is mainly used to inhibit the growth of mildew in silage or total mixed rations (tmr) or as a glucose precursor to inhibit a negative energy balance (neb). propionate can also promote the development of the rumen epithelium of calves. however, at very high doses, calcium propionate may lead to a decrease in appetite. the following are some of the limitations of the current research and areas to be explored: ( ) currently, research on calcium propionate in dairy cows has been mainly carried out by oral feeding alone, which is not convenient for application in practical production. therefore, to improve the application effectiveness of calcium propionate in dairy cows, more studies are needed to determine the optimal feeding level when calcium propionate is mixed with tmr. when calcium propionate is used to prevent neb and milk fever, the optimal feeding ratio should be revealed according to a cows' milk production level and body condition; ( ) excessive calcium propionate feeding has been shown to inhibit the appetite and limit the intake. to avoid its adverse effects, the maximum feeding level, influencing factor, and adverse impact of calcium propionate in dairy cows need to be further studied; ( ) calcium propionate can be used as an anti-mildew additive in silage and can also be directly added to feed to prevent several metabolic diseases of dairy cows during the perinatal period. however, few studies on the effects of silage with calcium propionate on perinatal dairy cows have been conducted. therefore, research in this field is worth exploring. the effects of supplementation with calcium propionate in dairy cows can be divided into the functions of hydrolyzed ca + and propionic acid. ca + , as a water-soluble calcium source, can reduce the incidence of milk fever by increasing the calcium content in the blood of perinatal dairy cows. propionic acid is mainly used to inhibit the growth of mildew in silage or total mixed rations (tmr) or as a glucose precursor to inhibit a negative energy balance (neb). propionate can also promote the development of the rumen epithelium of calves. however, at very high doses, calcium propionate may lead to a decrease in appetite. the following are some of the limitations of the current research and areas to be explored: ( ) currently, research on calcium propionate in dairy cows has been mainly carried out by oral feeding alone, which is not convenient for application in practical production. therefore, to improve the application effectiveness of calcium propionate in dairy cows, more studies are needed to determine the optimal feeding level when calcium propionate is mixed with tmr. when calcium propionate is used to prevent neb and milk fever, the optimal feeding ratio should be revealed according to a cows' milk production level and body condition; ( ) excessive calcium propionate feeding has been shown to inhibit the appetite and limit the intake. to avoid its adverse effects, the maximum feeding level, influencing factor, and adverse impact of calcium propionate in dairy cows need to be further studied; ( ) calcium propionate can be used as an anti-mildew additive in silage and can also be directly added to feed to prevent several metabolic diseases of dairy cows during the perinatal period. however, few studies on the effects of silage with calcium propionate on perinatal dairy cows have been conducted. therefore, research in this field is worth exploring. effect of diet-induced negative energy balance on the feeding behavior of dairy cows effect of subclinical and clinical hypocalcemia and dietary cation-anion difference on rumination activity in periparturient dairy cows mycotoxins occurrence and fungal populations in different types of silages for dairy cows in spain elevating serotonin pre-partum alters the holstein dairy cow hepatic adaptation to lactation antifungal treatment of paper with calcium propionate and parabens: short-term and long-term effects conditioned place avoidance using encapsulated calcium propionate as an appetite suppressant for broiler breeders calcium propionate supplementation improves development of rumen epithelium in calves via stimulating g protein-coupled receptors effects of del a- ter brca frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis influence of calcium propionate, water activity and storage time on mold incidence and aflatoxins production in broiler starter feed sanitary dips with calcium propionate, calcium chloride, or a calcium amino acid chelate maintain quality and shelf stability of fresh-cut honeydew chunks a study of the decomposition of calcium propionate, using simultaneous tg-dta evolutionary engineering in saccharomyces cerevisiae reveals a trk -dependent potassium influx mechanism for propionic acid tolerance effect of weak acid preservatives on growth of bakery product spoilage fungi at different water activities and ph values effect of dietary calcium propionate on performance, hepatic enzyme activities and aflatoxin residues in broilers fed a diet containing low levels of aflatoxin b response surface methodology-based optimization of decontamination conditions for escherichia coli o :h and salmonella typhimurium on fresh-cut celery using thermoultrasound and calcium propionate effects of four short-chain fatty acids or salts on dynamics of fermentation and microbial characteristics of alfalfa silage incorporation of lactobacillus plantarum and zeolites in poultry feed can reduce aflatoxin b levels effect of calcium propionate on the microstructure and pectin methy-lesterase activity in the parenchyma of fresh-cut fuji apples the use of sourdough fermented by antifungal lab to reduce the amount of calcium propionate in bread symposium review: transition cow calcium homeostasis-health effects of hypocalcemia and strategies for prevention effects of glucose, propionic acid, and nonessential amino acids on glucose metabolism and milk yield in holstein dairy cows whole-body glucose metabolism and mammary energetic nutrient metabolism in lactating dairy cows receiving digestive infusions of casein and propionic acid whole-body propionate and glucose metabolism of multiparous dairy cows receiving folic acid and vitamin b supplements temporal effects of ruminal infusion of propionic acid on hepatic metabolism in cows in the postpartum period the role of tca cycle anaplerosis in ketosis and fatty liver in periparturient dairy cows addition of calcium propionate to finishing lamb diets effect of calcium propionate and sorghum level on lamb performance the effect of calcium propionate supplementation on performance, meat quality, and mrna expression of finishing steers fed a high-concentrate diet silage review: foodborne pathogens in silage and their mitigation by silage additives patulin-producing molds in corn silage and high moisture corn and effects of patulin on fermentation by ruminal microbes in continuous culture airborne molds and mycotoxins associated with handling of corn silage and oilseed cakes in agricultural environment the effects of buffered propionic acid-based additives alone or combined with microbial inoculation on the fermentation of high moisture corn and whole-crop barley effects of calcium propionate on the fermentation quality and aerobic stability of alfalfa silage effect of calcium propionate and water activity on growth and aflatoxins production by aspergillus flavus effects of four short-chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage proteomics and metabolomics characterizing the pathophysiology of adaptive reactions to the metabolic challenges during the transition from late pregnancy to early lactation in dairy cows integration of metabolism and intake regulation: a review focusing on periparturient animals prevalence of excessive negative energy balance in commercial united kingdom dairy herds impact of supplementing propylene glycol and calcium propionate to primiparous buffalo cows during the late gestation and early lactation period on reproductive performance and metabolic parameters adipose tissue metabolism and production responses to calcium propionate and chromium propionate nutrition, immune function and health of dairy cattle effects of supplemental dietary energy source on feed intake, lactation performance, and serum indices of early-lactating holstein cows in a positive energy balance effects of dietary calcium propionate on growth performance and carcass characteristics of finishing lambs propionate induces mrna expression of gluconeogenic genes in bovine calf hepatocytes biology of dairy cows during the transition period: the final frontier? effects of calcium propionate supplementation on lactation performance, energy balance and blood metabolites in early lactation dairy cows effects of dietary glucogenic precursors and fat on feed intake and carbohydrate status of transition dairy cows blood parameters of lactating cows fed calcium salts as energetic source calcium propionate increased milk parameters in holstein cows influence of calcium propionate on in vitro fermentation of sorghum-based diets additive genetic and heterosis effects for milk fever in a population of jersey, holstein × jersey, and holstein cattle under grazing conditions epidemiology of subclinical hypocalcemia in early-lactation holstein dairy cows: the temporal associations of plasma calcium concentration in the first days in milk with disease and milk production a herd health approach to dairy cow nutrition and production diseases of the transition cow the monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows association of parturient hypocalcemia with eight periparturient disorders in holstein cows calcium and magnesium physiology and nutrition in relation to the prevention of milk fever and tetany (dietary management of macrominerals in preventing disease) review: endocrine pathways to regulate calcium homeostasis around parturition and the prevention of hypocalcemia in periparturient dairy cows endocrine and metabolic changes in transition dairy cows are affected by prepartum infusions of a serotonin precursor gastrointestinal calcium absorption in sheep is mostly insensitive to an alimentary induced challenge of calcium homeostasis in contrast to sheep, goats adapt to dietary calcium restriction by increasing intestinal absorption of calcium calcium transport in bovine rumen epithelium as affected by luminal ca concentrations and ca sources in vivo measurement of strontium absorption from the rumen of dairy cows as an index of calcium absorption capacity effects of high dietary calcium propionate and dietary cation-anion balance on calcium metabolism and longissimus muscle tenderness in finishing steers effects of oral calcium supplementation on mineral and acid-base status, energy metabolites, and health of postpartum dairy cows field trials of an oral calcium propionate paste as an aid to prevent milk fever in periparturient dairy cows a comparative study of the effectiveness of calcium propionate and calcium chloride for the prevention of parturient paresis in dairy cows effects of calcium propionate by different numbers of applications in first week postpartum of dairy cows on hypocalcemia, milk production and reproductive disorders review of strategies to promote rumen development in calves. animals (basel) effect of various dietaries on the anatomical development of the stomach in the calf growth performance and development of internal organ, and gastrointestinal tract of calf supplementation with calcium propionate at various stages of growth period calcium propionate supplementation alters the ruminal bacterial and archaeal communities in pre-and postweaning calves performance, rumen fermentation and blood metabolites of dairy calves fed starter mixtures supplemented with herbal plants, essential oils or monensin the effects of increasing garlic powder and monensin supplementation on feed intake, nutrient digestibility, growth performance and blood parameters of growing calves performance and plasma metabolites of dairy calves fed starter containing sodium butyrate, calcium propionate or sodium monensin systemic treatment with the enteric bacterial fermentation product, propionic acid, produces both conditioned taste avoidance and conditioned place avoidance in rats effects of diet on short-term regulation of feed intake by lactating dairy cattle hepatic metabolism of propionate relative to meals for cows in the postpartum period dose-response effects of intrauminal infusion of propionate on feeding behavior of lactating cows in early or midlactation extent of hypophagia caused by propionate infusion is related to plasma glucose concentration in lactating dairy cows we thank the national agricultural information system, chinese academy of agricultural sciences (caas), beijing , china for providing access to the literature during the period of the covid- pandemic. the authors declare no conflicts of interest. acknowledgments: we thank the national agricultural information system, chinese academy of agricultural sciences (caas), beijing , china for providing access to the literature during the period of the covid- pandemic. the authors declare no conflict of interest. key: cord- -xaxxewb authors: trump, cary e.; herrod, jessica l.; ayres, kevin m.; ringdahl, joel e.; best, lauren title: behavior momentum theory and humans: a review of the literature date: - - journal: psychol rec doi: . /s - - - sha: doc_id: cord_uid: xaxxewb behavioral momentum theory (bmt) is often described as analogous to newton’s ( ) laws of motion. that is to say, similar to an object in motion continuing in motion unless acted upon by a force, responses occurring in a static environment will continue to occur at the same rate, unless presented with a disruptor (nevin, tota, torquato, & shull, journal of the experimental analysis of behavior, , – , ). when evaluating response rates through a behavioral momentum framework, responding continuing after a change in reinforcer conditions is said to persist. previous research conducted with nonhuman animals indicates greater response persistence following conditions with either higher reinforcer rates or higher reinforcer magnitudes (nevin, journal of the experimental analysis of behavior, ( ), – , ; nevin et al., journal of the experimental analysis of behavior, , – , ). although bmt’s implications extend across human and nonhuman species, this literature review attempts to provide practitioners and researchers information regarding response persistence across various conditions with human participants. much of the behavior momentum theory (bmt) literature compares newton's first and second laws of motion (newton, ) to the behavior of organisms ). newton's first law of motion states that an object in motion remains in motion unless acted upon by an external force. the second law states an object's acceleration, or deceleration, is proportional to the relation between the object's mass and the force applied. regarding newton's first law, bmt suggests the object in motion described in newton's laws is analogous to the response rate of a particular behavior, and the external force equates to the presentation of a disruptor (e.g., satiation or extinction). bmt also compares newton's second law to increased or decreased response rates inversely related to the magnitude of obtained reinforcement and the disruptor. thus, greater magnitudes of reinforcement (i.e., history of reinforcement) result in greater resistance to change (i.e., responses persistence) following the presentation of a disruptor (nevin & shahan, ) . demonstrated that pavlovian contingencies affect response persistence. through stimulus-reinforcer pairings, the context associated with reinforcer delivery becomes conditioned, and its eliciting effect on the target behavior varies as a function of the conditioning history. however, others assert this relation between conditioning history and persistence to exist within an operant, rather than pavlovian, conditioning contingency (troisi & mauro, ) . in particular, proponents advocating an operant conditioning explanation for response persistence suggest the context associated with reinforcer delivery is not a respondent conditioned stimulus but instead serves as a discriminative stimulus signaling reinforcement availability and setting the occasion for responding. regardless of whether behavioral persistence is attributed to aspects of pavlovian or operant conditioning, responses occurring in the presence of contextual variables associated with greater rates of reinforcement often result in greater resistance following disruption (e.g., podlesnik & shahan, ) . variables shown to affect response strength (also referred to as persistence, and resistance to change) following the presentation of a disruptor involve the response's reinforcement history prior to disruption (nevin, ) . newton's ( ) second law of motion described an object's acceleration or deceleration as directly proportional, and inversely related, to an object's mass and applied force. when discussing variables related to response strength from a bmt perspective, nevin and grace ( ) described behavioral mass as the behavior's history of reinforcement. in consequence, responses associated with relatively denser reinforcement histories exhibit greater resistance to change than responses associated with leaner reinforcement histories. for example, responding associated with different stimuli (or different contexts upon which the response occurs) are paired with the respective reinforcement schedules occurring within each context. in this example, responses in context a contact reinforcement on a variable interval (vi) -s schedule of reinforcement, whereas responses in context b contact reinforcement on a vi -s schedule of reinforcement. upon contacting a disruptor (e.g., extinction), bmt predicts the behavior associated with the richer reinforcement history (i.e., greater behavioral mass; context b, in this example), will occur at proportionally higher response rates compared to baseline than the behavior associated with the leaner reinforcement history (e.g., sweeney and shahan, ) . although widely researched within the framework of an experimental analysis of behavior with nonhuman animals (e.g., igaki & sakagami, ) , the predictions made by bmt extends beyond the basic laboratory into translational and applied studies with human participants. for example, parry-cruwys et al. ( ) evaluated response persistence exhibited by students with autism in a special education classroom when simultaneously presented with distracting stimuli. in this experiment, researchers evaluated task persistence, such as writing responses, bead stringing, or puzzle building, on rich (vi -s) compared to lean (vi -s) reinforcement schedules. five of the six participants exhibited behavior that was more resistant to extinction during the task associated with the richer reinforcement schedule, suggesting bmt can have clinical applications. furthermore, romani et al. ( ) evaluated the effect of different negative reinforcement schedules on the persistence of task completion exhibited by three participants who engaged in escape maintained problem behavior. in their study, all three participants demonstrated greater response persistence in the context associated richer reinforcement schedule when reinforcement was disrupted. both of these examples provided researchers and practitioners with valuable information regarding bmt in applied settings. although research suggests bmt's predictions apply to humans as well as nonhuman animals, a comprehensive synthesis of the literature is needed to provide practitioners and researchers with information regarding persistence across various conditions. this synthesis could address several questions, such as what is represented in the literature regarding the percentage of participants exhibiting greater response persistence following disruption in rich versus lean conditions? we analyzed the literature regarding response persistence as it pertains to ( ) factors that influence initial interventions (e.g., problem behavior contacting a disruptor following a rich compared to a lean schedule of reinforcement) as well as ( ) factors that might influence persistence of trained responses (e.g., functional communicative responses and task completion). therefore, the purpose of this review was to provide a comprehensive review of studies evaluating bmt using human participants by synthesizing ( ) participant and setting characteristics, ( ) experimental characteristics, and ( ) experimental outcomes. response persistence/resistance to change can be characterized in two ways: ( ) resistance to change of a reinforced response when a disruptor is presented (often extinction), or ( ) the reemergence of a previously extinguished response under various conditions (i.e., relapse). although both are certainly important and hold practical implications, the current review focused on the first aspect of persistence. the authors conducted a literature search following guidelines recommended by moher, liberati, tetzlaff, altman, and the prisma group ( psycarticles, and socindex with full text using the term "behavior* momentum theory." studies included in the review were not limited by publication year, but were included based on the following conditions: ( ) peer-reviewed; ( ) english-language; and ( ) academic journals. after removing duplicates, the initial search provided studies in which the author conducted additional reviews using the following three additional inclusion conditions: ( ) included human participants across basic (conducted in laboratory settings to evaluate fundamental principles of behavior; cooper, heron, & heward, ) , applied (evaluating socially significant behaviors; cooper et al., ) , or translational (extending laboratory findings to clinical populations and problems; lerman, ) ; studies ( ) comparing two different conditions, such as rich versus lean schedules; and ( ) evaluated the effect of a disruptor on behavior within the context of bmt. articles were excluded if their primary purpose involved evaluating behavioral relapse phenomena (e.g., reinstatement, renewal, resurgence), as behavioral relapse occurs with a previously extinguished behavior (podlesnik & shahan, ). an initial abstract and title review resulted in potentially eligible articles. the full-text screening excluded articles with a remaining articles meeting the criteria. from these articles, the first author conducted an ancestral hand search using the same inclusion criteria listed above. the ancestral hand search involved reviewing every reference cited in the original articles to determine whether cited studies also met the inclusion criteria. the final ancestral search resulted in an additional articles for a total of articles included in the review (see fig. ). the first author coded descriptive participant and experiment characteristics ( variables) of all included experiments within each study. in addition to descriptive characteristics, reviewers coded nine variables related to outcome variables. participant characteristics included variables such as gender, age, and diagnosis. experiment characteristics included variables such as experimental setting(s), implementer(s), target behavior(s), reinforcers, and disruptor(s). experimental outcomes included coding persistence comparisons between independent variables across each dependent variable. participant and setting characteristics participant and setting characteristics included gender, age, diagnosis, experimental setting, interventionists, and functional analysis results. when coding participant's ages, reviewers coded whether the participant fell between to years old, to years old, to years old, or if they were older than years. participant diagnosis codes included autism or pervasive developmental disorders (pdd), intellectual disability (id), developmental delays (dd), multiple diagnoses, other diagnoses, or no diagnosis (i.e., participants were typically developing). experimental settings included hospital rooms, therapy or laboratory rooms, classrooms, work facilities, or living rooms, kitchens, or bedrooms, which were also coded as additional settings. interventionist codes included therapist/experimenter, teacher/paraeducator, or parent/caregiver. functional analyses results included tangible, automatic, escape, attention, and multiply maintained. experimental characteristics experimental characteristics included dependent variables, reinforcers, signaled or unsignaled stimuli through the incorporation of a discriminative stimulus, and disruptors. dependent variables included functional communicative responses (fcr), task completion, and problem behavior (e.g., aggression, self-injurious behavior, disruption). the fcrs included various mand modalities such as various augmentative alternative communicative devices (e.g., picture cards, tablets, microswitches), manual sign, and vocal requests. reinforcer codes included escape, attention, tangible, tokens, food, food compared to tokens and an "other" category. when coding whether researchers included stimuli associated with different experimental conditions, the reviewers noted whether different color task materials, different therapists, or different backgrounds were associated with different experimental conditions. researchers coded "no" under the discriminative stimulus section if these components were not present in the experiment. disruptor codes included alternative stimuli, distractors, extinction, extinction and distraction, prefeeding, noncontingent reinforcement (ncr), or other distractors not falling into any of the six listed categories. reviewers coded for extinction as the disruptor if the experiment incorporated a condition where a previously reinforced response no longer contacted reinforcement. noncontingent reinforcement was coded when reinforcers were delivered independent of participant responding. alternative stimuli were defined as stimuli signaling the availability of an alternative concurrent schedule of reinforcement. distractors included the presence of items or activities such as preferred toys, movies playing, or the presence of an additional therapist. prefeeding involved participant presession reinforcer consumption. experimental outcomes reviewers coded each comparison conducted within each experiment. for example, if an experiment compared the effect of rich versus lean schedules (e.g. vi -s versus vi -s) on fcrs and task completion, the authors coded results related to rich versus lean schedules on fcrs in addition to coding results for the effect on task responding. independent variable comparisons included studies evaluating persistence of high versus low preferred mand modalities. for example, evaluating the effect of disruption on a more preferred mand modality such as a picture exchange card compared to a less preferred mand modality such as vocalizations. the review also coded rich versus lean schedules of reinforcement. for example, evaluating the effect of a rich (vi -s) versus lean (vi -s) schedule on response persistence, or evaluating the effect of a combined vi plus fixed time (ft) schedule compared to a vi schedule alone. furthermore, experimental outcome codes also included coding results by disruptors (e.g., extinction, distractors, ncr). a second graduate student conducted an identical literature search using the aforementioned search terms, inclusion criteria, and multidatabase search engine. a third graduate student coded . % (n = ) of included articles, and . % of total experiments (n = ), using identical participant, experiment, and outcome coding templates described above. each experiment included descriptive variables and nine results variables for each comparison. interobserver agreement was calculated by dividing agreements by the number of agreements plus disagreements and multiplying by . the literature search, conducted by the second graduate student, resulted in % agreement. the second graduate student initially discovered original articles meeting the inclusion/ exclusion criteria. following conversations discussing inclusion/exclusion criteria, the first author and graduate student agreed on articles meeting criteria. coding, conducted by the third graduate student, resulted in . % overall interobserver agreement for participant, experiment, and outcome codes. the review comprised experimental evaluations (see table ). reviewers coded participant characteristics every time the participant engaged in an experimental evaluation. for example, vargo and ringdahl ( ) included multiple experiments with four to five participants. some participants were included in more than one experiment. in these instances, their participant codes were included for each experiment. likewise, lionello-denolf, dube, and mcilvane ( ) evaluated the effect of three different disruptors (alternative stimulus, prefeeding, distractor) with six participants and an additional distractor with five participants. when evaluating lionello-denolf et al.'s ( ) study, reviewers included participant demographic and setting codes for each disruptor evaluation for a total of participant evaluations. participant ages ranged from to years old with more than half of the participants under the age of ( . %). of the participants, . % (n = ) were male, . % (n = ) were female, and . % (n = ) did not have a specified gender reported. the review included participants diagnosed with intellectual disability (n = ), autism or pervasive developmental disability (n = ), developmental delays (n = ), or multiple disabilities (n = ). in addition, the experiments also included participants without reported disabilities, which primarily participated in basic or translational experiments. for example, vargo and ringdahl ( ) evaluated resistance to change with unconditioned and conditioned reinforcers with typically developing children. the review yielded five different settings in which the experiments were conducted. one hundred twenty-three experiments were conducted in a therapy or experimental room ( . %), experiments were conducted in a living or bedroom ( . %), and experiments were conducted in a classroom setting ( . %). furthermore, therapists or experimenters conducted the majority of the included experiments (n = , . %), whereas a teacher/paraprofessional conducted one experiment ( . %). the review included results of functional analyses when reported. a tangible function was reported for . % (n = ) of participants, an escape function was reported for . % (n = ) of participants, and an attention function was reported for . % (n = ) of participants. in addition, % of participants exhibited automatic or multiply maintained problem behavior; . % (n = ) of participants did not have a functional analysis conducted. as previously described, dependent variables included fcrs, problem behavior, or task completion (see table ). task completion included experiments examining participant responses involving computer navigation such as dube, thompson, silveira, and nevin's ( ) study involving a computer game written in python requiring participants to move icons using keys on a modified keyboard, or studies such as vargo and ringdahl's ( ) evaluating tasks such as number or letter tracing, and stringing beads. over % of the included experiments evaluated task completion (n = ), whereas the remaining experiments evaluated problem behavior (n = ), fcrs (n = ), or a combination of problem behavior and fcr (n = ). the type of reinforcers in each experiment are also noted in table , with almost half of the experiments ( . %) using tangible items (n = ) and . % using tokens (n = ). experiments included disruptors such as extinction (n = , . %). thirty-one ( . %) experiments evaluated the effect of distractors such as videos (e.g., mace et al., ) , and experiments ( . %) evaluated the effect of prefeeding. for example, during vargo and ringdahl's ( ) prefeeding disruptor phase, participants consumed food prior to beginning experimental sessions in addition to food consumption during intercomponent intervals. note. id = intellectual disability, pdd = pervasive developmental disorder furthermore, . % of the disruptors were coded as alternative stimuli. for example, lionello-denolf and alternative stimulus test involved an additional stimulus associated with a vi -s presented concurrently with either the rich or lean components. of the evaluated experiments, . % (n = ) involved a discriminative stimulus and . % (n = ) did not. for example, dube and mcilvane ( ) used different colored backgrounds to signal different conditions (i.e., a white background for task a and a black background for task b). likewise, lionello-denolf and dube ( ) used different computer icons associated with either the rich or lean conditions (e.g., balloon and gift). experimental outcomes are summarized in tables and . table displays the number of comparisons and the percent of comparisons displaying greater persistence across dependent variables. the independent variable comparisons are indicated with superscript numerals in the first column. the first independent variable comparison evaluates the effect of rich versus lean schedules on experiments evaluating problem behavior (n = ) and task completion (n = ). for example, mace et al. ( ) compared the effect of a rich (vi -s) versus lean (vi -s) schedule on response persistence, but other researchers, such as lieving, deleon, carreau-webster, triggs, and frank-crawford ( ) , evaluated the combination of a vi plus fixed time (ft) schedule compared to a vi schedule alone. overall, the review discovered % of the included experiments indicated problem behavior responses persisted greater in rich compared to lean schedules. likewise, % of task completion responses persisted in rich compared to lean schedules. in particular, comparisons involving dra associated with rich versus dra lean reinforcement schedules display greater persistence in % of included comparisons. however, when comparing the effect of different disruptor magnitudes on response persistence, task completion responses persisted more in conditions associated with lower magnitudes of reinforcement. for example, in carr, bailey, ecott, lucker, and weil's ( ) study, task completion was disrupted with noncontingent reinforcement of low, medium, or high reinforcer magnitudes. in these comparisons, the participant's task completion persisted at a greater rate when disrupted with the lower reinforcer magnitude. independent variable comparisons also included experiments evaluating persistence of high versus low preferred mand modalities. for example, ringdahl et al. ( ) evaluated persistence of fcrs associated with high preferred versus low preferred mands. in this example, reviewers coded the number of participants evaluated in each comparison as well as the number of participants exhibiting greater response persistence with high preferred versus low preferred mands ( %). table displays experimental outcomes by studies and also includes dependent variables, disruptors, the number of participant evaluations, independent variable comparisons, as well as comparison results. in the second column, the author identifies independent variable comparisons. for example, carr et al. ( ) evaluated the effect of a noncontingent reinforcement schedule (ncr) on low verses medium, low versus high, and medium versus high magnitude reinforcers. the third column indicates the dependent variable, which the author coded as task completion in the carr et al. ( ) experiment. finally, the last five columns indicate the number of participants (n), and the percent of participants with greater persistence in the first comparison condition. note. fcr = functional communicative response. the current review included peer-reviewed articles evaluating bmt with human participants. many of the experiments evaluated effects of rich versus lean schedules of reinforcement on response persistence. in addition, a large number of experiments evaluated response persistence when extinction functioned as a disruptor. most of the reviewed studies took place in experimental (e.g., human operant laboratory) or therapeutic (e.g., in-patient unit) settings. few experiments took place in more naturalistic settings, such as classrooms, homes, or work settings." the review indicated greater response persistence following rich (i.e., high rate and/or high magnitude) compared to lean (i.e., low rate and/or low magnitude) reinforcement schedules. the aforementioned results align with results reported in the basic, nonhuman research literature. for example, nevin ( ) showed greater persistence following relatively high rates or relatively high magnitude reinforcement schedules. the current review discovered experiments evaluating response persistence of problem behavior. it is interesting that of these experiments evaluated the effect of extinction as a disruptor whereas the remaining two evaluated the effect of motivating operation (mo) manipulation. for example, berg et al. ( ) evaluated the effect of presession attention on responding during attention session of a functional analysis for behavior that was determined to be maintained by attention. eleven of the experiments evaluating response persistence of problem behavior in rich compared to lean schedules resulted in greater persistence in the richer schedule of reinforcement. as a result of this finding, practitioners might consider the implications of implementing an extinction component in contexts in which problem behaviors contacts rich schedules of reinforcement. for example, practitioners should be aware that implementing an extinction-based procedure following a rich reinforcement schedule might require a longer time to be effective when compared to extinction-based procedures implemented following lean reinforcement schedules. on the other hand, the review also discovered % (n = ) of studies demonstrated response persistence of fcr or task completion responses in rich compared to lean schedules. therefore, when programing alternative responses (e.g., fcr), practitioners might consider programing rich schedules of reinforcement for alternative behaviors. for example, if teaching a learner to mand, the literature suggests the mand is more likely to persist in the face of disruption, if the mand contacted a richer schedule of reinforcement prior to disruption. furthermore, because it is likely a newly acquired skill such as manding might contact disruptors outside of intervention contexts, programing rich schedules of reinforcement might create robust alternative behaviors, which are paramount for the learner's success. schieltz, wacker, ringdahl, and berg ( ) described assessment and treatment decisions based on bmt. the authors focused on discrepancies between two ways behavior analytic maintenance effects are measured: ( ) treatment effects probed over long periods of time under prevailing treatment conditions (durand & carr, ) ; and ( ) evaluating behavioral persistence following the presentation of a disruptor (e.g., extinction; nevin & wacker, ) . schieltz et al. ( ) noted the former measurement system does not require an analytical evaluation directly tied to specific behavioral processes. however, the latter definition provides practitioners with a format to evaluate and program durable maintenance effects during treatment rather than conducting posthoc treatment evaluations. although dra interventions demonstrate decreased levels of problem behavior and increased rates of appropriate behavior, maintenance of these effects are seldom reported (schieltz et al., ) . moreover, when maintenance effects are reported, they are often reported as effects over time rather than a systematic analysis providing information regarding the circumstances in which maintenance is likely or unlikely to occur (e.g., nevin & wacker, ; schieltz et al., ) . evaluating maintenance based on bmt (i.e., evaluating persistence following disruption) provides a thorough assessment and treatment framework based on underlying behavioral processes (schieltz et al., ) . the second maintenance measurement method described by schieltz et al. ( ) , requires evaluating the effect of systematically manipulating the target response's history of reinforcement (i.e., magnitude and rate) and the presentation of different disruptors. therefore, evaluating the effect of different reinforcement and disruptor variables might prove instrumental in programming high levels of treatment durability when designing and monitoring intervention plans. for instance, ringdahl et al. ( ) indicated relatively preferred mand modalities (as demonstrated through a mand modality preference assessment) resulted in greater persistence when placed on extinction. perhaps practitioners can use similar evaluations to determine responses more likely to exhibit greater persistence when challenged. evaluating variables linked to bmt such as behavioral mass and various disruptors provide valuable information regarding treatment decisions. for example, assessing maintenance through a bmt framework might allow practitioners to program high levels of treatment durability. furthermore, after evaluating persistence and determining a behavior to be resistant to disruptors, this analysis might also provide a framework to systematically discontinue services. when evaluating behavioral mass, the current review indicated researchers often evaluate the effect rich versus lean reinforcement rates on responding during disruption. few ( ) manipulated three different reinforcer dimensions (quality, duration, delay) in a concurrent schedule arrangement. the authors discovered response allocation favoring the more advantageous schedule (e.g., higher quality reinforcers, longer duration exposed to reinforcers, or shorter delays to reinforcement) rather than behaviors associated with weaker reinforcer dimensions (e.g., lower quality, shorter duration, or longer delays to reinforcement). the results reported by athens and vollmer align with predictions made by matching law, a quantitative model of behavior that predicts response allocation under concurrent reinforcement schedule arrangements (baum, ; herrnstein, ) , and also align with trump, ayres, quinland, and zabala ( ) literature review, which discovered effective treatment packages with concurrent schedules resulting in greater response allocation associated with the more favorable schedule. given that they demonstrated that reinforcement schedule dimensions such as magnitude and delay to reinforcement affected response allocation, it may be interesting to see if these same dimension affect responding under schedule arrangements relevant to bmt (i.e., multiple schedule arrangements). similar to evaluating different reinforcer dimensions, few experiments evaluated different disruptor dimensions. when considering newton's second law of motion, bmt states response strength is directly proportional and inversely related to reinforcement history and the magnitude of a disruptor (e.g., nevin & shahan, ) . as previously discussed, bmt provides a framework for evaluating and predicting responding based on the target behavior's history of reinforcement and the presentation of a disruptor. numerous experiments evaluated different factors related to behavioral mass, but little or minimal variation exists when examining the effect of different disruptors. only . % (n = ) of the included experiments evaluated mo manipulation as a primary disruptor (e.g., prefeeding). moreover, few researchers evaluated different disruptor magnitudes (e.g., different distractor volumes) or disruptor combinations, and only . % (n = ) evaluated the effect of distractors. therefore, future investigations might consider quantifying and evaluating the effect of different disruptor magnitudes (e.g., quiet compared to loud disruptors), distractors (e.g., preferred compared to nonpreferred videos), and presession conditions (e.g., evaluating the effect of satiation and deprivation). for example, in applied settings, teachers might play music during independent work time; however, the effect of type of music or the music's volume has not been evaluated within the context of bmt. in addition, this review discovered experiments evaluating the effect of disruptors on task completion. it is interesting that all experiments involving typically developing individuals (n = ) evaluated response persistence of task completion. of these experiments, only % evaluated the effect of computer tasks. although task completion certainly involved experiments evaluating the effect of response persistence on computer related tasks (e.g., kuroda, cancado, & podlesnik, ) , additional tasks included spelling, math, stringing beads, and completing puzzles (parry-cruwys et al., ) , as well as number and letter tracing (vargo & ringdahl, ) . therefore, future investigations might want to evaluate the effect of different disruptor variables on different types of tasks (e.g., academic tasks compared to computer tasks). for instance, does the presentation of a distractor (e.g., music or videos) affect academic tasks (e.g., vargo & ringdahl, ) differently than computer tasks (e.g., dube et al., ) ? in particular, future investigations might evaluate the effect of disruption on multistep or complex tasks (i.e., tasks requiring the individual to engage in covert verbal behavior) compared to one-step computer tasks requiring little to no covert verbal behavior. furthermore, schieltz et al. ( ) described the impact of the number of instances target behavior contacts extinction. the authors stated that response extinction pairings correlated negatively with resurgence. therefore, schieltz et al. suggested future research evaluating pre-and posttreatment exposures to extinction and reinforcement to determine whether or not reinforcement to extinction ratios influence treatment durability. likewise, researchers evaluating response persistence might consider evaluating the effect of number of exposures to disruptors on resistance to change. although a majority of the included studies involved different stimuli associated with different conditions (i.e., blue card associated with vi -s and red card associated with vi -s), only two included articles (dube & mcilvane, ; saini & fisher, ) specifically evaluated the effect of these stimuli or stimulus variations. for example, dube, mcilvane, mazzitelli, and ncnamara ( ) evaluated the effect of stimuli with lower and high discrimination when challenged by a change in reinforcement schedules. likewise, saini and fisher ( ) evaluated the effect of different stimuli salience on response persistence. saini and fisher stated bmt, "predicts that increasing the discriminability of the change from variable-interval to variable-time reinforcement should lead to faster reductions in responding" (p. ). the current review only discovered the two aforementioned studies evaluating stimuli salience. moreover, none of the studies evaluated the effect of verbal statements such as instructions on responding when challenged, despite experiments demonstrating response differences in the presence of verbal stimuli (günther & dougher, ) . furthermore, although the purpose of this review was to synthesize the literature regarding changes in reinforcer conditions (i.e., response persistence), it should be noted that relapse paradigms, evaluating changes in treatment conditions (e.g., extinction; podlesnik & shahan, ) are also crucial to the field of applied behavior analysis. furthermore, treatment relapse involves the reemergence of a response following intervention conditions previously resulting in extinguished behaviors (pritchard, hoerger, & mace, a) . in particular, relapse involves at least three paradigms: reinstatement, resurgence, and renewal (pritchard et al., a) . reinstatement involves the delivery of the reinforcer maintaining rates of target behavior during baseline following extinction, which subsequently results in resumed rates of the target behavior (pritchard et al., a, b; podlesnik & shahan, ). resurgence involves the reappearance of a previously extinguished behavior, extinguished through extinction and dra, when the alternative behavior contacts extinction procedures (doughty & oken, ; pritchard et al., a) . the third relapse paradigm, renewal, involves extinguishing a target behavior in a context different from the baseline context, and the target behavior reappears upon returning to the baseline context (pritchard et al., a) . evaluating these three relapse paradigms through a framework of bmt might assist behavior analytic professionals in determining possible solutions to mitigate issues related to treatment relapse. therefore, the authors hope to see future reviews evaluating experiments involving behavioral relapse paradigms (e.g., reinstatement, renewal, and resurgence) such as pritchard, hoerger, mace, penney, and harris's, ( b) study evaluating the effect of high compared to low rates of reinforcement during treatment on reinstatement. pritchard et al. ( b) discovered that although both rates of reinforcement produced similar results during treatment, responding was . times as high during reinstatement conditions in the context associated with higher rates of reinforcement. this finding is similar to the results identified in the current review indicating greater response persistence in contexts associated with richer schedules of reinforcement. as with any systematic literature review, publication bias might limit search results (e.g., sham & smith, ; tincani & travers, ) . sham and smith ( ) indicated publication bias occurs when journals reject publications indicating null effects. for example, experiments indicating little to no difference in persistence following different reinforcement histories might not survive the peer-review process if the reason for the lack of difference was not explored. furthermore, publication bias occurs when researchers do not submit datasets that do not report large differences across conditions (tincani & travers, ) . in addition to publication bias, search terms might not result in a comprehensive list of experiments related to the current review. therefore, the current review might not fully represent all of the bmt studies conducted with human participants. as an additional limitation, the current review did not code the effect of repeated exposures to disruptors. for example, although not included in the review, wacker et al.'s ( ) experiment evaluated persistence of treatment effects during long-term treatment. in this experiment, wacker et al. also conceptualized maintenance as treatment durability during treatment challenges. for example, evaluating whether problem behavior relapses when presented with a disruptor such as a newly acquired fcr contacting extinction. the authors discovered decreased resistance to change over extended treatment periods, which implies longer treatment durations or repeated exposures to extinction might lead to more durable treatment outcomes. the current review did not code number of exposures to disruptors; therefore, this review does not provide information regarding the effect of continued exposure to disruptors on response persistence. in summary, this review discovered several important factors regarding response persistence within the context of bmt as well as considerations for applied settings and future research. for instance, future research could focus investigations on the phenomena described in the preceding pages, including, but not limited to, variables affecting appropriate and inappropriate behavior and how those variables might affect generalization and maintenance of responding resulting in improved quality of life for individuals exhibiting behavioral concerns. moreover, in light of recent events (i.e., the covid- pandemic), perhaps researchers can evaluate variables influencing response persistence of behaviors related to public health behaviors such as mask wearing. after all, behavior analysis is only restricted by its principles and methods (lerman, iwata, & hanley, ) . compliance with ethical standards on behalf of all authors, the corresponding author states that there is no conflict of interest. furthermore, as this is a literature review, this manuscript did not require informed consent. availability of data and materials data sharing is not applicable to this article as no new data were created or analyzed. an investigation of differential reinforcement of alternative behavior without extinction choice in free-ranging wild pigeons the effects of presession exposure to attention on the results of assessments of attention as a reinforcer on the effects of noncontingent delivery of differing magnitudes of reinforcement applied behavior analysis extinction-induced response resurgence: a selective review behavioral momentum in computer-presented discriminations in individuals with severe mental retardation reinforcer rate and stimulus control in discrimination reversal learning reinforcer rate effects and behavioral momentum in individuals with developmental disabilities the role of contingencies and stimuli in a human laboratory model of treatment of problem behavior assessing behavioral momentum in humans with mental retardation and unstable baselines reinforcer rate effects and behavioral momentum in individuals with developmental disabilities functional communication training to reduce challenging behavior: maintenance and application in new settings applied behavior analysis relative and absolute strength of response as a function of frequency of reinforcement resistance to change in goldfish resistance to change and resurgence in humans engaging in a computer task effects of noncontingent reinforcement on the persistence and resurgence of mild aggression from the laboratory to community application: translational research in behavior analysis applied behavior analysis responding maintained by intermittent reinforcement: implications for the use of extinction with problem behavior in clinical settings additional free reinforcers increase persistence of problem behavior in a clinical context: a partial replication of laboratory findings contextual influences on resistance to disruption in children with intellectual disabilities evaluation of resistance to change under different disrupter conditions in children with autism and severe intellectual disability persistence during extinction: examining the effects of continuous and intermittent reinforcement on problem behavior the momentum of human behavior in a natural setting effects of reinforcer quality on behavioral momentum: coordinated applied and basic research some effects of magnitude of reinforcement on persistence of responding preferred reporting items for systematic reviews and meta-analyses: the prisma statement response strength in multiple schedules resistance to extinction and behavioral momentum alternative reinforcement increases resistance to change: pavlovian or operant contingencies? quantitative models of persistence and relapse from the perspective of behavioral momentum theory: fits and misfits behavioral momentum and the law of effect behavioral momentum theory equations and applications alternative reinforcement increases resistance to change: pavlovian or operant contingencies? from behavioral research to clinical therapy philosophiae naturalis principia mathematica resistance to disruption in a classroom setting treatment relapse and behavioral momentum theory clinical translation of animal models of treatment relapse behavioral momentum and relapse of extinguished operant responding effects of response preference on resistance to change relations between rate of negative reinforcement and the persistence of task completion evaluating the effects of discriminability on behavioral persistence during and following time-based reinforcement basing assessment and treatment of problem behavior on behavioral momentum theory: analyses of behavioral persistence publication bias in studies of applied behavior-analytic intervention: an initial analysis modeling the effects of sensory reinforcers on behavioral persistence with alternative reinforcement behavioral momentum and resurgence: effects of time in extinction and repeated resurgence tests greater reinforcement rate during training increases spontaneous recovery publishing single-case research design studies that do not demonstrate experimental control do pavlovian processes really mediate behavioral momentum? some conflicting issues differential reinforcement without extinction: a review of the literature an evaluation of resistance to change with unconditioned and conditioned reinforcers an evaluation of persistence of treatment effects during long-term treatment of destructive behavior publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations a portion of this research was supported by the eunice kennedy shriver national institutes of child health & human development of the national institutes of health under award r hd . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord- -atbjwpo authors: nan title: poster sessions date: - - journal: febs j doi: . /febs. sha: doc_id: cord_uid: atbjwpo nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v epitope tag into the n-terminal of klf gene. insertion into the target side of klf gene via crispr-cas system provided an opportunity to overwhelm the above mentioned obstacles. v epitope tag would not interfere with the function of the klf and also enable us to recognize endogenous klf via anti-v antibody in the mouse myoblast cell lines (c c ). we confirmed the targeted insertion into the exon of the klf gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc , but not ctf is detached from chromatin. our data also shows that meanwhile, rad checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc and ctf are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc has a more specific role in the process of adaptation. our data demonstrates that mrc is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost drosophila proteins with c h zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw , pita and zipic) were found to interact with cp and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw , pita, zipic and cg . first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw , pita, zipic and cg revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project № - - ). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t and t concentrations in adult male wistar rats. fifty rats aged between and weeks ( ae g) were divided into five groups (n = in each group), and drugs were administered each day intraperitoneally (ip) for days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol ( . mg/kg, n = and mg/kg, n = ) and clozapine ( . mg/kg, n = and mg/kg, n = ). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture hours after the last injection. the t and t plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v . . kruskal-wallis and bonferroni tests were used. t plasma concentration levels significantly differ between sham (median= . mg/kg) and haloperidol ( mg/kg) (median= . mg/kg), haloperidol ( . mg/kg) (me-dian= . mg/kg) and clozapine ( mg/kg) (median= . mg/ kg), haloperidol ( mg/kg) (median= . mg/kg) and clozapine ( mg/kg) (median= . mg/kg) groups (p < . ). however, no significant differences between the groups regarding to t plasma levels were observed. in conclusion, haloperidol and clozapine increased the t plasma concentrations, but didn't have any significant effect on t plasma concentrations. p- . . - isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a . lm filter and incubated on lb agar medium for hours. based on differencies of colony morphologies, isolates were selected and purified for identification. s lipase activity assay was carried out by rhodamine b. all of the strains exhibited lipase activity. for determining the substrat specificity of isolates, different substrates were used; nitrophenyl-butyrate, -nitrophenyl-caprylate, -nitrophenyl-laurate, -nitrophenyl-myristate, -nitrophenyl-palmitate. results were measured spectrophotometrically at nm. all of the strains hydrolyzed -nitrophenyl-butirat, while there was no activity with -nitrophenyl-palmitate. bacillus sp. l was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l will be cloned and expressed for more analyses and industrial applications. p- . . - some quantitative aspects of hair follicle layers differentiation e. vsevolodov , , v. golichenkov , a. mussayeva , , i. latypov llc "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with mcm hair diameter for those with the hair diameter mcm the ratio of hair medulla diameter to hair diameter increases from . to . . the ratio of hair diameter to the diameter of inner root sheath increased from . to . . it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes % of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from to u/g pulp basis and the consistecy of the pulp was between and %. enzyme at the desired concentration was added to the pulp and the mixture was incubated at °c for hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as % pulp consistecy, u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p- . . - biochemical characterization of lipase from bacillus subtilis strain a from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec . . . ) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a is partially purified and characterized. bacillus subtilis strain a is isolated from olive factory from soke (aydin, turkey) and identified with s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with % olive oil (vol/vol) for hour at °c. after incubation, cells were harvested by centrifugation at , rpm for minutes, resuspended in mm tris-hcl (ph . ) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at °c. the enzyme was active and stable in the broad ph range of . - . and temperature range of - °c. bacillus subtilis strain a have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p- . . - investigation of pin as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a (pin ) as a potential nfib interactor. pin is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin implicating this domain in nfi-pin interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin proteins in hek t cells showed that pin precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin . we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. - - . p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during - period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy , open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were to years old and their average age was . ae . . ofthem ( . %) were under years of age whereas of them ( . %) were above years of age. number of pregnancies were scaling between to with an average of . ae . . of mothers ( . %) were not undergone amniocentesis, whereas babies with chromosomal abnormalities were detected among mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. ( . %) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with . , . and % concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses ( . , . % and %) in both species with percentages %, % and % in r. ridibunda and %, % and % in b. viridis, respectively, while in the control group it was about %. furthermore, delayed larval development was detected. thus, the larvae from the control and . % oil group reached gosner stage (gs) , tadpoles from . % and % groups were at gs- and gs- , respectively, by the th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p- . . - effect of catechin loaded plga nanoparticles on glioma cell line histone h t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h subtype and hyperacetylation of histone h tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p- . . - serum dickkopf- levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf- (dkk- ) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk- levels in obese and non-obese children and adolescents. materials and methods: the study included obese children and adolescents ( males and females) aged from to years and healthy normal-weight controls ( males and females) aged from to years. serum dkk- levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = . ). however, there was no significant difference between dkk- levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk- levels were not changed in obese children and adolescents. p- . . - transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib À/À precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia À/À hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the kb human cdo promoter region identified five nfi binding sites: one cluster in the first kb region, another in the . kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to % in hek t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd /ankk gene variants in eos development. in this study, eos patients and healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd /ankk taq a (rs ) and bdnf val met (rs ) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd /ankk rs polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = . , or = . ; % ci: . - . ). however, no significant relationship was observed in the genotype frequencies of bdnf val met polymorphism between eos patients and controls (p = . ). these results indicate that, drd /ankk rs genotypes may affect eos development. however, bdnf val met polymorphism may not be associated with eos. lack of association of bdnf val met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black ) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black ) was determined between and nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black ) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm , prm and their relationship with defective spermatogenesis. materials and methods: this study has been performed on infertile and fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm and prm . results: distinct levels of spermatozoal prm and prm mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm was reduced in (% ), and the mrna levels of prm was reduced in (% ) out of infertile patients.in the current study,we found statistical significant association between the prm expression and infertility (p < . ).although prm gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > . ). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm and prm themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf , myogenin) in white muscle were studied in salmon parr of age groups +, +, + in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr ( +) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings ( +). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age +. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age + and + then in +, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to +. there was no differences in expression level of myod, myf and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. - - . p- . . - lmp and lmp gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu , f. ozbas gerceker sanko university, gaziantep, turkey, gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide (lmp ) and low molecular weight polypeptide (lmp ) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome . these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp and lmp gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp and lmp genes were determined. results: a deviation from the hardy-weinberg equilibrium (v = . ,p < . ) was found for the genotype distribution of lmp gene polymorphism, while the lmp genotypes found to be distributed (v = . ,p > . ). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp and lmp genes should be increased to understand their effects as a genetic marker. p- . . - investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have - nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found . ; . ; . nm respectively. the morphological features were examined by sem images. antioxidant activities of q , q ve q nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: - - -gep ". p- . . the effect of environmental enrichment on spatial memory and certain nmdars, and ht a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and ht a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into groups as enviromental enrichment (ee, n = ) and standard cage control (scc,n = ) groups. eeg housed in an enriched environment and sccg were kept in standard cages for weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr a, nr b, ht a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the training days showed that eeg located the hidden platform significantly earlier than sccg on days , (p = . , p < . ). also eeg significantly spent lower time in the outer zone of the maze on days , which was the sign of low anxiety level (p = . , p = . ). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < . ), in fst comparison of groups showed no difference (p > . ). the levels of nr b and ht a were significantly increased as compared to sccg as well (p < . , p = . ). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr b and ht a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p- . . - effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, ( weeks old) were divided into groups as control (cg, n = ; + , male+female) , experiment (msg-low dose, e g, n = ; + , male+female) and experiment (msg-high dose, e g, n = ; + ) groups. msg was administered at mg/kg/d to e g, . g/kg/d to e g for weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au autoanalyser. level of total protein was significantly increased in e g and e g groups when compared to cg (p < . ). level of alb€ umine was also increased in both egs but significant difference was seen in e g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < . ). although the glucose levels in both egs were increased, the increase in e g was statistically significant (p < . ). the alt levels of in egs were also increased but the significant increase was seen in e g (p < . ). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. ( mg/kg) + tartrazine ( mg/kg) + brilliant blue fcf ( mg/kg) + ponceau r ( mg/kg) + azorubine ( mg/kg) + indigotine ( mg/kg) + erythrosine ( mg/kg). artificial food color mixture were administered to g and g and drinking water was applied simultaneously to g by oral gavage per day for weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > . ). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g were higher than g and g in serum and kidney and brain homogenate (p < . ). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p- . . - er quality control protein network in cf to modulate f del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo and gst-pi enzymes. for this purpose, mrna and protein expression of nqo and gst-pi enzymes were analyzed in ht and sw (colon); hepg and huh (liver); pnt a and pc (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo protein was expressed in all cell lines and relative protein expression is highest in the hepg ( %) and pnt a ( %) while huh ( . %) showed relatively low expression of nqo . in addition, nqo mrna expression was relatively high in ht ( . fold) and pnt a ( . fold) when compared with liver cell line hepg ( . fold). gst-pi protein expression was found very high in huh ( %) while there was no expression in hepg . gst-pi mrna expression was relatively higher in pnt a ( . fold) and ht ( . fold) when compared with huh ( . fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo and gst-pi are ht and huh , respectively. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p- . . - tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about out of every cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. mrna studies and mirna study, which were designed for different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ . , fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf transcription factor and hsa-mir- . in the literature hsa-mir- and klf are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb ) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd , cd , cd and cd as positive markers and cd as a negative marker. gene expression profilling was investigated using affymetrix hg-u -plus arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < . , fold change≥ ). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of genes were identified that differentially expressed in msc line versus primer mscs ( upregulated and down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] association between loss of q , gain of q . and progression in sporadic colorectal cancer n. belder , b. savas , m. a. kuzu , i. pak , h. s€ umer c ß elebi , a. ensari , h. € ozdag biotechnology institute, ankara university, ankara, turkey, school of medicine, ankara university, ankara, turkey, ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix . genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from stage i, stage ii and stage iii samples. paired cnv analyses were performed using partek genomic suite . and genomic segmentation algorithm was performed using a minimum of markers per segment, a signal-to-noise ratio of . and the cut-off value for the gain and loss was set of ae . . the adjusted p-value ≤ . were considered to be significant. whole genome cnv analysis revealed that amplification of q . with genes was found the most frequent ( . %) in stage ii tumors. the most frequent ( %) amplifications were q . and p . in stage iii tumors. while deletion of chromosome q . in stage iii with a frequency of % was found the most frequent loss, deletion of q . was seen the most frequent ( . %) in stage ii tumors. two tumor suppressor genes smad and smad which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of q . might have a significant role in the progression of cancer. loss of q comprising two tumor suppressor genes is also another important finding. q loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no: s . p- . . - meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin , cin and cin according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin , cin and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin , cin and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin , cin and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with common genes showed that these targets were significantly enriched (p < . ) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group ), dia-betic+glibenclamide (group ), diabetic+plant extract (group ), untreated control (group ) and control+plant extract (group ) groups (n = for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract ( mg), and the reference drug glibenclamide ( mg/kg) by gastric gavage every day for weeks. in group , blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group , serum glucose, alt, ast, triglyseride and hba c levels decreased compared to group while cholesterol and ldl levels were high. in group , serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba c were higher compared to group . body weights increased except group and hdl levels were not altered. histologically degenerative changes observed on pancreas of group were decreased in groups and . there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p- . . - on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p- . . - qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan , c. neuschl , r. alberts , g. a. brockmann school of medicine, istanbul kemerburgaz university, istanbul, turkey, department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) , , , and . most likely candidate genes within or near the regions with the highest significance levels were identified. the genes f rik, gbe , a n , and four genes cenpc , stap , uba , gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr for fat mass, on chr for fat mass gain and on chr for lean weight, and chr for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to healthy women aged - years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than . . . % of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = . ) and protein (p = . ) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = . ), isoleucine (p = . ), leucine (p = . ), lysine (p = . ), methionine (p = . ), cysteine (p = . ), tryptophan (p = . ), and glutamic acid (p = . ) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = . ). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at nm, but in contrast to pvx, did not have additional peak at nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant - - ). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k , k , k and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a- dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full -utr and -utr sequences of m satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p- . . - physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga ) and with encapsulated antigen (plga ) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga particles (plga - . %, plga - . %). the flow cytometry results demonstrated that % of plga particles conjugated with egfp-sbb, and only . % of plga particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga particles size was . nm and plga - . nm. nta data also showed that mean plga particles size a little smaller than plga ( . nm and . nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p- . . - a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt , a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at °c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt cells with the isogenic cho-k cells as a control. cells after transient transfection were incubated at °c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt and the wild type control cell lines are performed to validate the reported model system. p- . . - host-pathogen interactions: is there a relationship between tlr polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than million individuals die from tb and each year , cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher ( . / , ) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even % of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study tb patients and healthy controls were included. tlr genotyping for rs , rs was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be . . results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p- . . - lytic action of bacteriophages as a tool for the obtaining of images p. boltovets , r. radutny , t. shevchenko institute of semiconductor physics nas of ukraine, kyiv, ukraine, scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the . % agar with the nutrient medium and the . % agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t were applied. samples were incubated during the twenty-four hours at + °c. after that stencils were removed and the samples were stained by coomassie blue r- or fuchsine (with further fixation by the % acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type (st ) complexes with ctx-m gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli - / -g and attc e. coli-reference strain and (ii) to compare the proteomes of strain at two conditions: with and without antibiotics. strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein , cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p- . . - molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu , i. gheorghe , , m. popa , , a. ion , m. mihai , v. lazar , , m. c. chifiriuc , carol davila" university of medicine and pharmacy, bucharest, romania, research institute of the university of bucharest-icub, bucharest, romania, faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of s. aureus clinical strains isolated in from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of . %, meca in . %, sscmec type ivd element in . %, ccrb in . % and sccmec types i, iii, iv in . % of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa ( . % of the isolates), followed by clfb ( . %), fib ( . %), hlg ( . %) and bbp ( . %). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p- . . - analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin ( %), vancomycin ( %) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than antibiotics. it was determined that among the investigated strains % contained meca, % -mecc, % contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in . % of cases, sed - %, seg - %, sei - %. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in % of strains, beta peptide toxin genes in %, delta toxin gene in %. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p- . . - impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] host-specificity determinants of bacteriophage vb_ecom_fv considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of water buffalo milk and dairy products ( water buffalo cream and water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso - and isolates were confirmed for the presence of the s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in of water buffalo milk ( %), of water buffalo cream ( %), and of water buffalo cheese ( %). a total of isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of isolates, ( %) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in out of ( %) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea ( / ; . %), two isolates produced sec ( / ; . %), one isolate produced ( / ; . %) sed, one isolate produced ( / ; . %) see and three isolates produced sec+sed ( / ; %) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet - . . ) and this article was part of a phd thesis. p- . . - identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il- receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp ; was cloned in fusion with an n terminal cleavable his-tag. the recombinant protein, his- was purified using nickel affinity chromatography. was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of was investigated by in vitro pull-down and size-exclusion chromatography. , a amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant his- was produced with a yield of mg/l culture. a structurally stable kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that also form dimers, supporting that it is a btp. current research is focused on solving the structure of and investigating its interaction with myd . might play a direct role in reduced immune response against h.pylori by binding to myd analogous to other btps. further characterization of will provide the first solid evidence of presence of a tir domain protein in h.pylori. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il production by bone marrow-derived macrophages k. korneev , , e. sviriaeva , lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor (tlr ) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il by bone marrow-derived macrophages from c bl/ mice, using tlr -deficient macrophages to control for specificity of tlr signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk / activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p- . . - apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz , l. monturiol-gross , m. j. pineda padilla , c. araya-castillo , a. alape-gir on bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc ( - ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with . lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of . triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor (her ) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for % of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb (mdr- ) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb- was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with , and nm of the drug respectively along with lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr- gene expression with the treatment of dox. especially after nm of dox treatment, mdr- was overexpressed to be fold when compared to control. in conclusion, it was demonstrated that mda-mb- cells have shown to display elevated resistance to higher doses of dox. p- . . - targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa , t. kondo toyama prefectural university, imizu-shi, japan, university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from °c to °c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-( -aminopurin- -yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached - %). nucleosides, such as ribosides, -deoxyribosides, and arabinosides were obtained in high yields ( - %). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el- (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant - - ). p- . . - dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim , n. demir , m. yildiz health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s -). also, cytotoxicity of the compounds against hepg cell lines was assayed by the mtt ( -( , -dimethylthyazolyl- )- , -diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s . also, schiff base complex had cytotoxic activity towards hepg cell line, that it was found to be more potent than the control cisplatin. p- . . - single particle electron tomography of rnap elongation complex, stalled at position + genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering or bp into the nucleosome; ec+ or ec+ , respectively). hear we produced two complexes, both stalled in the + position, one without break in the dna, and the other with introduced ssb at position + of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae °in a jeol microscope at kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+ complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant # - - . colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to who report new incidence rate of crc in turkey is . % among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh À g>a (rs ) promotor substitution, mlh g>c (rs ) exonic substitution, mthfr c t (rs ) and apc t>a (rs ) were investigated in this study. these snps "rs , rs , rs , rs " are located on p . , q , p respectively. colonoscopic investigations were performed on both cancer and control group. the snps were genotyped using kompetitive allele specific pcr technology in cases and healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these of the snps in mlh , mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh À aa genotype in group aged ≥ was found to be . % in cancer versus % in control group. moreover apc a, mlh c alleles were detected only and allele respectively. previously, apc a allele was determined in . % of a turkish cohort. however in the present study apc a allele was detected on allele only. studies showed mlh À promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c t and mlh g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p- . . - excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil and neil , have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either -oxoguanine ( -oxog) or , -dihydrouracil (dhu) by e. coli fpg and nei and human neil and neil from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases ( to ), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil and neil efficiently excised dhu located inside a bubble. fpg and neil was generally more active than neil in excision of -oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil also have shown activity in d-loops with rna. the presence of an additional unpaired -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf ( - - ). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas ( bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+ ), fab-dc/nflu(À )) or unrepairable (nflu/nflu (+ ), nflu/nflu(À ), nant/nflu(+ ), nant/nflu(À )) structures. besides, it has been shown that model dnas bearing bulky lesions in opposite positions (fab-dc/nflu( ), nflu/nflu( )) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(À ), nflu/nflu (+ ), and nant/nflu(+ )), whereas the level of specific excision from nflu/nflu(À ), nflu/nflu(À ) and nant/nflu(À ), nant/nflu(À ) was approximately % of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex ( bp) with fab-dc has decreased structurally dependent affinity for xpc-hr b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+ ), fab-dc/nflu (À ), fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(- )) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized - [ -( morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. - [ -( -morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p- . . - target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil and neil , and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg + . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil and neil showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf ( - - ). p- . . - does causes mhz magnetic field application kras and p mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group. mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for weeks as minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < . ).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for weeks minute/day exposure to mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p mutations in colon tissue. p- . . - synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g motifs) with the potential to form a spatial structure, gquadruplexes is known. g motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed samples of pancreatic ductal adenocarcinoma and samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. - - of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from healthy controls and patients with mm. the mean age for controls and patients were . ae . and . ae . years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a lung adenocarcinoma cells. a lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for a cell line. compounds , and which were including -chlorophenyl, -nitrophenyl on pyridine ring; -fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic values lower than . ae . lg/ml. compound showed the highest cytotoxic activity with a ic value of . ae . lg/ml, whereas cisplatin ic values were . ae . lg/ml lg/ml against a cells. cytotoxic activity of compound and with a ic value were . ae . and . ae . lg/ml, respectively. also, compound showed the highest population of early apoptotic cells ( . %) of the tested compounds which was . -fold higher than for cisplatin. compound produced a comparable population of apoptotic cells with a percentage of . %, respectively according to cisplatin's percentage of . %. it was determined that synthesized compounds , and had considerable anticancer activity against a cell lines compared to cisplatin. compound including -florophenyl on pyrimidine ring was the most cytotoxic compound against the a cell line. our study results demonstrated that compound , also induced apopototic pathway on a cells. p- . . in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl- cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ , , -trimethoxyphenyl = -methoxyphenyl > dillapiol > -methoxyphenyl> , dimethoxyphenyl > , , , -tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic values ranging from . (a cells) to . lm (mda-mb- cells). compounds, containing , , -trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to lm. anti-proliferative effects of isoflavones observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones demonstrated cytotoxicity in human pbmcs, up to lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing , , -trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag- is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl- , hsp /hsc family, growth hormone receptors, raf- , ubiquitin machinery and dna to regulate cell survival. for this reason, bag- is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag- are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag- silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag- enhanced cisplatin or paclitaxel-induced apoptosis in mcf- cells by down-regulating antiapoptotic and upregulating proapoptotic bcl- family proteins, changes on cell cycle, upregulation on subg phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag- has a suppressive role in pi k and akt signaling pathway in mcf- breast cancer cells through inhibition of akt phosphorylation and downregulation on pi k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag- silencing. bag- silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc and rapamycin-sensitive mtorc complexes, respectively. knockdown strategies of bag- is important to enlighten the network interactions of bag- and clarify its interaction partners in the cells. therefore utilization of bag- targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p- . . - biological activity evaluation of new , , trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately % of all female and % of all male cancer deaths in . the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. -[ -( , -bis( -substituted phenyl)- , , -triazin- -yl)piperazin- -yl]- -[benzimidazole/benzoxazole/benzothiazole- -yl)thio]ethanone ( - ) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and -chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt ( -( , -dimethylthiazol- -yl )- , -diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic values between - lg/ml. the early and late apoptotic cell percentage was determined as . for compound by flow cytometric analysis. the lox inhibition activity was found . ae . for compound . compound bearing -chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic / , ic and ic x . p- . . - single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal -drp group in oligonucleotide dna substrates in the presence or absence for me + ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants - - -a and - - -mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u mg) were seeded into the well plates and cultured with dmem supplemented with % fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in / (v/v) dilution ratio was given to first group, lm etoposide was given to second group, / (v/v) diluted rosemary and lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly % , etoposide reduced the viability by nearly % and rosemary with the etoposide reduced the viability by nearly % . the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing -( -aryl- -oxoethyl)- -substituted benzimidazole moiety on c glioma. c glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard ( -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for c cell line. compounds , , , , and , which were including hydrogen, -methyl, -methoxy, -chloro and -floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic values lower than lg/ml. compound showed the highest cytotoxic activity with a ic value of lg/ml, whereas cisplatin ic values were lg/ml against c cells. cytotoxic activity of compound , , , and with a ic value were , , and lg/ml, respectively. compound , and showed the highest population of early apoptotic cells as . , . , and . % respectively compared to cisplatin ( . %). also, compounds caused dna synthesis inhibition depend on their ic values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c cell lines. however, compound , and including -methyl, -chloro and -floro substituents were the most active compounds against the c cell line. also our study results showed that compound , , induced apoptosis in c glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc- and du prostate cancer cells were treated with rutin ( - um) and/or trail ( ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc- and du- prostate cancer cells were treated with rutin for and hours incubation and ic doses for hours incubation were determined um and um respectively. treatment with rutin, pc- cells is more sensitive than du cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc- and du prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc- and du prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p- . . - determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst- ) methods; and antigenotoxic effects against ccl on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration ( and lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations ( , , . lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst- , ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k and aml cell line hl- were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl , bax and caspase ), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p ) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k cell line but not in hl- cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k and hl- cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl and increasing bax and caspase levels. these effects of bor were observed to correlated with increasing the p expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p- . . - analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with - kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome p . , p . , q , q , p . , q . , q . , q . , q . , p , q . , q . , q . , q . , q . , q . , and q . , and also common gains were detected in chromosome p . , q , q . , q . and xq . in colon cancer dna samples, common losses were detected in chromosome p . , q , p . , p . , q . , q . , q . , q . , p . , p . , q . , and q . , and also common gains were detected.in chromosome q . , xp . , xp . , xp . , xp . and xq . both in gastric and colon cancer dna samples, common losses were detected in chromosome q . , q . , and p . , and common gains were detected in xq . discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase (parp ) recruitment which is a sensor for single strand breaks in dna. parp catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for to seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes and . bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt and a benzoxazole derivative xt b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k s) and imatinib-resistant (k r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl- , bad, bim, bcl-xl and mcl by real-time quantitative pcr. results: treatment of k cells with xt increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt b was not found to have similar effects on k r and k s cells. combined application of xt increased cell death in the mtt assay. mtt assay demonstrated that ic for xt treated cells in k r with imatinib (ic = . ) is lower than k r without imatinib (ic = . ). discussion and conclusion: our results showed that combining xt with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh and msh . alteration in expression or a defect in mlh or msh can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k s (sensitive) and k r (resistance) were grown in rpmi- . k r cells were maintained in rpmi- medium supplemented with lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh in k r cells is dramatically lower than equal amount of imatinib treated k s cells, whereas msh expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh genes leads to imatinib resistance. p- . . - characterization of interaction between rad inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france -diisothiocyanostilbene- , -disulfonic acid (dids) has been largely used during the last years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad -mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad recombinase protein. p- . . - mechanism of tap beta-mdm autoregulation p is a transcription factor which is the member of a p family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p , p is rarely mutated in tumors and elevated p expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm is known as the key player in regulation stability and activity of p . in addition, p induces mdm transcriptional activity, and caspase- , activations which cleave mdm n-terminal at asp . cleaved form of mdm binds p and promotes its stabilization. mdm suggested as a candidate to modulate p activity and stability too. however, an interaction between p and mdm has not defined well. in this study, we aimed to analyze the role of mdm in p stability. to define this relationship, firstly, we overexpressed the tap beta isoform using trex system in hep b. tap beta and mdm protein levels were determined by western blot. to examine whether mdm mediate tap beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg for hours prior to analysis. previous studies showed that p -induced caspase- and caspase- activation cleaves mdm . considering this, we firstly examined caspase- activation by western blot in hep b tap beta cells. then we analyzed expression of cleaved mdm and tap beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap beta-induced full-length mdm- expression. furthermore, tap beta enhanced cleavage of mdm via increased caspase- activation. in addition, inhibition of caspase- activation caused a decrease in cleaved-mdm levels in parallel with tap beta expression repression. our results suggested positive regulation between mdm -tap beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p is a transcription factor which is the member of p family. it has two promoters; while p promoter expresses apoptotic ta isoforms, p promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p . it has been shown that both tap and dnp isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap and dnp affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap isoforms via western blot in all hcc cell lines. then we overexpressed the tap beta isoform using trex system in hep b and snu cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; . only tap alpha isoform is expressed in a few hcc cell lines. . there is no correlation between basal expression of p isoforms and drug responses in hcc cell lines. . there is no change in expression of p isoforms after treatment of drugs. . we showed that the ectopic expression of tap beta in hep b arrested the cell cycle in g / s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p- . . - biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease (ape ). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape and archaeal mth revealed conserved amino acid residues in the nir-specific ap endonucleases ape , mth and exoa that are absent in xth. based on these data, we constructed four ape point mutants y h, n q, g s and t d and examined their dna substrate specificities. results obtained from biochemical characterization of ape mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll ( - °) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between and °. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p- . . - ercc /xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group (ercc ) is one of the important dna repair genes.ercc codon and polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated ml blood for biomarker testing. ercc genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v . ). there was significant difference between the frequencies of ercc polymorphism in cancer cases and controls (p < . ). the frequencies of ercc gln allele were . % in controls and . % in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or= . , %=ci . À . ). the above findings indicate that the genetic polymorphism in the ercc codon is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv .x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv . -kcsa-kv . chimeras was developed by us. this approach rested on high-level expression of kcsa-kv .x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv . -channel blockers that combines bl (de ) cells expressing kcsa-kv . and rhodamine-labelled agitoxin (rh-agtx ) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv . was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv . . for system validation, wellknown kv channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx , osk , and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv . channel binding using a number of agtx mutants constructed by us, whose affinities to kcsakv . were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv . channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv . -blockers for scientific and medical purposes. the work was supported by the grant - - from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp- ). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp- by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of : achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp- cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp- cells were was exhibited to possess the highest values of around % at ar concentration of lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis- inhibition of ire signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire (inositol requiring enzyme ), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u glioma cells. it was shown that inhibition of ire leads to up-regulation of the expression of krt , cd , mest, cenpu, myl , ing , ing , mybl , and mybl genes at the mrna level in u glioma cells, with more profound changes for mest, mybl , and cd genes. hypoxia leads to up-regulation of the expression of cd , ing , and ing genes and to down-regulationof krt gene in glioma cells. at the same time, inhibition of ire modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing gene, eliminates hypoxic regulation of krt , cd , and ing genes in glioma cells. the present study demonstrates that inhibition of ire enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase (pol ) of thermus scotoductus k isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol gene of t. scotoductus strain k recently isolated from an armenian geothermal spring. the draft genome sequence of strain k was deposited under accession number ljjr . . genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~ . kb) were ligated into the pet- b(+) vector (novagen) and transformed into chemically competent top escherichia coli. inserts were sequenced with t prom and t term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol gene from k shares % and % similarity with pol of t. scotoductus sa- ( . kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide ( l- fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of l- fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf- breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf- human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst- method. the most effective extract in inhibition of mcf- cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic ( ) values of lg/ml and . lg/ml at h. and h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf- cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf- cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf- cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, . ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, -aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than %. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than %. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for hours daily for one ( ximo), two ( ximo) or ten ( ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a -adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc a and ppargc b, significantly decreased in adrenal cortex of ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp , the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc a, ppargc b, nrf and nrf a). nonselective b-adrsblocker attenuated this effect. the selective a -adrs antagonist did not change adrenaline-induced stimulation of ppargc a, ppargc b, nrf and nrf a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a -adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis- generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for hours daily for one ( ximo), two ( ximo) or ten ( ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (Δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between Δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in ximo group. the mitochondrial mass in leydig cells from ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst- assay was used to evaluate the effects of metformin and tq in dhl (b cell lymphoma) and k (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci< ), additive (ci= ) or antagonistic effects (ci> ). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl and k cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl and k cell lines, ci values were . and . , respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with -hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with -hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after weeks of adipogenic induction in the presence of the chromium complex with -hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with -hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh and gfh . while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g a (pt g a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total participants were included in this study, patients with dvt and healthy blood donors. in order to identify fvl, pt g a, mthfr c t and mthfr a c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in patients fvl was present in ( . %) patients while in controls fvl was present in only ( . %). frequency of fvl was significantly higher in cases as compared to controls (p < . ). pt g a mutation was present in patients ( . %) and in healthy participants ( . %). mthfr c t and mthfr a c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c t/a c was found in patients ( . %) and in healthy controls ( . %), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c t and mthfr a c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c t/a c with fvl may be associated with increased risk of dvt. p-mis- self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t , tx ) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. - - r_center_a ( no. - - r_center_a ( - no. - - r_center_a ( ) and - - mol_a_ved ( no. - - r_center_a ( - . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk / or btk- inhibitors. the phosphorylation of erk / and btk- was determined by western blot analysis. proteomic analysis was performed by ultimate rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef ( v/cm). comparative analysis of bc and lc cells proteomes revealed about differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk- is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk- phosphorylation in lc than bc cells. in contrast erk / activity was not necessary for electrotaxis of lc cells and ef did not induce erk / phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre / /b/nz / , poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce , n. y. saral , f. b. aksungar , , a. coskun , , m. serteser , , i. unsal , acibadem labmed, istanbul, turkey, acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between and were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be lu/ml for cut off point and lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of lu/ml in ( . %), between and lu/ml in ( . %) and above the level of lu/ml in ( . %) babies, respectively. recall rate was . %. ch cases of neonatal tsh levels greater than lu/ml were . the incidence of ch of this group was : . there were no significant differences in the number of congenital hypothyroidism between males and females (p > . ). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou , m. kazou , v. alexandraki , b. pot , e. tsakalidou agricultural university of athens, athens, greece, institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of , , bp with a g+c content of . %. the genome contains , protein-coding genes on the chromosome covering up to . % of the genome sequence, trna and rrna. according to the subsystem-based annotation, , protein-coding genes were assigned to metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = , . % of total protein-coding genes) and protein metabolism (n = , . % of total protein-coding genes). furthermore, three prophage regions were detected; one intact ( . kb), one incomplete ( . kb) and one questionable ( . kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type diabetes mellitus (t dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after month. also, a healthy control group (n = ) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = . ) between the irisin levels of the patients at the beginning and after month treatment. a similar result was found between the control and the treated groups (p = . ), while a significant difference (p = . ) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in % of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. - - of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant - - ). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn in ammonia-exposed mouse cortical astrocytes (bisi; lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn and sn , second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r a and orexin ox heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin a and b (ghs-r a , ghs-r b ) for ghrelin, and orexin and (ox , ox ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r a and ox receptors to form heterocomplexes. formation of ghs-r a -ox receptor heteromers in transfected hek t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r a and ox receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r a -ox receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = ). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than . nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than . . our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva , , b. bekmanov , , a. amirgalieva , k. dosybaev , , z. orazymbetova , , r. zhapbasov , a. zhomartov , n. zumadillaev , n. zumadillaev llp "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than . ) there were found rarely met fragments (frequency lower than . ). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) breeds ( populations) were characterized. informative value and resolving capacity of the sum of str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. informative markers were chosen for both two breeds, markers being used for both breeds, while other markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele of bm was met in birlik population of edilbay breed as often as in % of animals while in two other populations there were no this allele. in kumtekey population one can meet % animals having particular locus (dyms ), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of preeclamptic mothers and mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk ( vs u/ml, p = . ). while tos levels were significantly higher in the preeclampsia group ( . vs . lmol/l, p = . ), the tas levels were significantly higher in the control breast milks ( . vs . mmol/l, p = . ). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis- investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included subjects ( female and male with age range of - years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at nd, th and th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = . , p = . ), tg (r = . , p = . ), tc (r = . , p = . ), ldl-c (r = . , p = . ) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis- eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than , compounds, incl. confirmatory and counter screening, ic/ec determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in , but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate -hydroxy-nicotine ( hnic) produced by a. nicotinovorans pao was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate hnic by using a genetically engineered a. nicotinovorans strain. the growth and the hnic accumulation were compared for two strains: . a. nicotinovorans pao wild type strain and . a genetically engineered a. nicotinovorans pao strain (part ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of hnic were monitored by hplc using a mn nucleodur - c ec column and . m sulfuric acid at a flow rate of ml/minute. the growth curve of the part ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of hnic are observed. although the sds-page analysis of the total protein extracts from the part ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a fold accumulation of hnic in the growth medium. the first attempts to purify ndh from the part ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part ndh strain for hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeÀinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of . - lm, and ache in the range . - lm. five cinchonidines displayed a - times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda ( lm) for h increased the expression of kir . mrna and decreased that aqp -and gs mrna. the expression of kir . was decreased by h exposure to glu ( mm) and tnfa ( ng/ml). at h incubation, nmda induced a decrease of kir . expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr subunit, and decreased that of nr b mrna. glu decreased the expression of out of subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir . mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to pediatric nurses and adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x . the average age of participants was ae years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was % and after the training it was %, which showed an increase of %. the preanalytic error rates which were . % in february were decreased to . % in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was nm and had a sufficient stability over a period of months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g ) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g dna is considerably more susceptible to lightinduced modification than duplex dna; -terminal tetrads of the g dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. - - from the russian science foundation. resistin levels in denervated obese rats n. saglam , t. ahmedi rendi , c. kahraman , a. alver department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study experimental groups were formed each consisted of rats. during weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < . ). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est gene is regulated at the translation level by + programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf p (ampk) and gcn p (eif kinase) on the prf rate in est gene. prf rate of est gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf , or gcn genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn p, and snf p, respectively. prf rate of est gene was measured as % in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than -fold, giving less than % prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by -fold in the wild type cells and prf rate increased to %. when the prf rate was analyzed in gcn and snf mutants, frame shift rate of est was - % in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn p and gcn p, positive regulator of gcn p, is also essential for the regulation of prf in est in response to stress conditions. it is clear that the basal level expression of est is highly dependent on the gcn p kinase complex. gcn p is also associates with ribosomes, indicating that gcn p might have a significant function in connecting the stress signals to biosynthesis of the full length est peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf p and gcn p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = ; nonerosive-nerd, n = ; healthy controls-hc, n = ) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee ( . ae . ng/mg.prt) versus hc ( . ae . ng/mg.prt, p = . ). mpo tissue activities in ee group were significantly lower ( . ae . u/mg.prt) than hc ( . ae . u/mg.prt, p = . ) while mpo serum levels were higher in ee ( . ae . ul) versus hc ( . ae . ul, p = . ). tissue chit levels were three fold increased in ee versus hc (p = . ). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=À . , p = . ) in tissue. nfkb and ecad levels had a positive correlation in serum (r = . , p < . ). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from , patients (mean age ae sd, . ae . ) were used for the correlation (excel analyze-it v . . ) and linear regression (pasw statistics v . ) analysis between the measured parameters. there was a significant (p < . ) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp* . ) + ( . *age) + ( . *wbc) + ( . *neut%)À . . correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of . . our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c -dox ratio ( : and : ) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp ad molt- ) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h o -induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to , tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < . ) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < . ). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil ( -fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of -fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of -fu synthesized via "click reaction", namely f -[{ -( ″, ″, ″, ″-tetra-o-acetyl-b-dglycopyronosyl)- h- , , -triazole- -yl}methyl] -fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in ml of solvent for h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta , ta (for the detection of frameshift mutations) ta and ta (for detection of base pair substitutions) according to the oecd guideline . the drug was solubilized ( lg/ml) with no precipitation in lutrol Ò -f /ethanol/water ( . : . : . , wt/wt) micelles ( . ae . nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the healthy goats were divided into groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t? (r ), at t = . s (r ), t = s (r ) and t?∞ (r ). this work was supported by the russian scientific foundation ( - - ). all animals had - % fat content. the contents of total protein ( . %), albumin ( . %) and urea ( . %) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol ( . %) and creatinine ( . %) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r (r = À . ), r (r = À . ), r (r = À . ); total protein or albumin levels with r (r = À . ), r (r = À . ), r (r = À . ); aminotransferase activity with r (r = À . ), r (r = À . ). the correlations were found between the total protein and albumin levels with k (r = . ), k (r = . ); glucose levels and r (r = . ), r (r = . ). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of > were classified as the high gs group (hgg), and those with a gs less than were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of . lmol h o equiv./ l for serum tos levels predicted high gs with a sensitivity of % and a specificity of %. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was . (sensitivity % and specificity %). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci , c. velioglu , e. recber , c. unsal , g. gulsoy , b. sariyar akbulut marmara university, istanbul, turkey, istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis . the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis cells were subjected to lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for hours but then cells resumed their regular growth. upon addition of lg/ml (-)-roemerine to the bacillus subtilis cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono- , -lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf- breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf- breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase (hplscr ) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr . this study aims to identify the region of hplscr that interacts with topo iia. to identify the topo iia interacting sites in hplscr , nterminal deletion constructs of hplscr viz Δ -hplscr , Δ -hplscr , Δ -hplscr , Δ -hplscr and Δ -hplscr were generated by pcr, cloned, overexpressed and purified to homogeneity using ni + -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex p- and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr and the gst-ctd-topo iia. the binding region in hplscr will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr . Δ -hplscr did not show any enhancement of the decatenation activity compared to full length hplscr . hence, the binding region could be in the - region of hplscr . further deletions were done in the - region of hplscr as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr that binds to topo iia. we conclude that hplscr interacts with and enhances the activity of topo iia and the - region of hplscr is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr and the physiological relevance of this interaction in the cell. a. ugur kurtoglu , v. aslan , e. kurtoglu department of biochemistry, antalya education and research hospital, antalya, turkey, department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs . is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs . , ivs . , ivs . and ivs . . our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis- investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen , h. kara karadeniz technical university medical biochemistry department, trabzon, turkey, balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba ),non-lysosomal b-glucosidase (gba ), cytosolic b-glucosidase (gba ),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose b-ltyrosine- -naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba .inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba was purified at . -fold with . % yield.gba was illustrated and kda at sds-page.ic value of ampicillin/sulbactam antibiotic for gba was found . mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba was illustrated and kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba .ampicillin/sulbactam antibiotic inhibited to purfied gba at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu , p. b. sermikli , a. abbasi taghidizaj , e. yilmaz ankara university, institute of science, ankara, turkey, ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, t -l cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type- diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft , ft , vitamin b and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b , folate. the cases were - years old, male individuals who admitted to our hospital between april -march for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: ) were divided into four subgroups; group (g) : - . lg/dl, g : - . , g : - . , g : ≥ . from these, the number of cases whose th levels were measured (n: ) given respectively; g : , g : , g : , g : cases. also the number of cases whose vit b and folate levels were measured (n: ) given respectively; g : , g : , g : ,g : cases. levels of pb were determined by icp-ms. th, vit b , folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t , tsh and vitamin b (p > . ). on the other hand there was statistically significant difference between t and group , , (p < . ) but there was no difference between group (p > . ). the average folate belongs to the first group was about % higher than the other groups, and found that the difference was statistically significant (p < . ). there are many publications which have various results between pb levels and t ,t , tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b , folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b . but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged ( ). since that time this method became associated with a number of major breakthroughs in the field ( ) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors ( ) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf - - . ( ) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. , , À . ( ) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. , , - . ( ) katritch v., cherezov v., stevens r.c. ( ) . annu rev pharmacol toxicol , - . p-mis- stamp is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp knockdown. interestingly, in the crpc cell line rv , where-stamp knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of d thin layer chromatograms using d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional ( d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional ( d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for d plates, and cannot be adapted to quantitative evaluations of d plates. in this study, the applicability of the gel documentation systems and d analysis software for the analysis of d tlc plates were examined. material and method: d tlc of lipids: st dimension: methyl acetate/n-propanol/chloroform/methanol/ . % kcl ( / / / / v/v); nd dimension: chloroform/methanol/acetic acid/ water ( / / / v/v); detection: charring. d tlc of aminoacids: st dimension: . % (v/v) formic acid; nd dimension: toluene/glacial acetic acid ( : v/v); detection: uv. phospholipid and aminoacid standards, each include different classes were developed by d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest d gel analysis software. for the method validationa) plates containing same lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample b) plates containing same lipid classes were developed in different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates results: volume of each phospholipid and aminoacid had less than % intra and inter-assay cv. conclusion: gel documentation system with d gel analysis software can be used for the quantitative analysis of the d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, adult male wistar albino rats were used. rats were separated into four groups as control rats (g ), mm nac (g ), mm nac (g ) and mm nac (g ). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding mm kcl to the organ bath. then, in the first minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at . . results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < . ). similarly, nak atpase activity is also decreased in a dose dependent manner (p < . ). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk , which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of . - . . the optimum temperature for keratinase activity was investigated by varying the incubation temperature between °c and °c. optimum keratinolytic activity was observed at °c and ph . . the enzyme was stable at °c. the activity was investigated in the presence of some chemicals, including sds, tween , dmso, triton x- , edta, nacl, zncl , cacl , glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel ( %) using standard molecular weight marker and estimated about kda by sds-page. the keratinase isolated from bacillus uk could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of bthalassemia and a-(including two of . kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler Ò instrument. the a/b-globin mrna ratio of each sample was calculated based on the Àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis- self-assembling peptides mimic supramolecular biochirality r. garifullin , , m. o. guler bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis- pon activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon- activity in both total and hdl subgroups, and evaluation of performance characteristics of pon- activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed obese, overweight and normal subjects. fasting morning blood samples were taken from all study groups. hdl subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl -c concentrations were calculated with the subtraction of hdl -c from total hdl-c. hdl -c and total pon- activity were determined by using eckerson method. non-hdl pon- activitiy was calculated with subtraction of hdl pon- activity from total pon- activity. results: total hdl-c, hdl -c and hdl -c concentrations and the activity of total pon- and hdl pon- were found lower in obesity according to overweight and normal subjects (p < . ). negative correlations were found between body mass index and hdl -c, total pon- and hdl pon- (r = À . , p < . ; r = À . , p < . ; r = À . , p < . , respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl -c measurements, pon activity, measurement of total and hdl -c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis- hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > . was accepted as significant value. results: in a study of blood donors ( male, female), the overall prevelance of anti-hev igg antibodies were . %. the blood samples were collected different areas. the prevelance of anti-hev igm antibodies was . % and he was years and acting a butcher during years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were %, the animal husbandry were % and the veterians and the butcher were % were found. discussion: the prevelance of anti-hev in the north cyprus ( %) was found low such as the prevelance of the turkey ( %). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january and january . clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that blood samples were sent to our laboratory in one-year period. . % of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause ( %). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be %, %, %, %, % and % respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of patients ( female, male) aging . ae . years ( - ) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of g sugar ( hour), -hour and -hour. these parameters were statistically analyzed in comparison to simultaneous hba c results. glucose measurements at hour had better distinction power (p < . ) between these individual groups than initial and -hour glucose measurements. the average hba c (%) levels for healthy, pre-diabetic and diabetic individuals were . ae . , . ae . and . ae . , respectively. roc curve analysis showed . % sensitivity and . % specificity for the clinically accepted hba c cut-off value of . %. hba c cut-off value of . % had a higher sensitivity of . % and comparable specificity of . %. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at -hour after sugar administration in ogtt test as opposed -hours generally used for diagnosis. low sensitivity was observed for the clinically adapted . % cut-off value of hba c. the cut-off value of . % for hba c was found to be more sensitive with comparable specificity than the . % cut-off values for diabetes screening in our population. our results suggest that . % for hba c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin ( mg/kg, po) every hours for a total of doses prior to an apap challenge ( mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp e , cyp a , or cyp a in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf -dependent pathway. we found that stamp fused to cyan fluorescent protein (cfp-stamp ) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp with various fluorescent protein markers revealed that cfp-stamp puncta corresponded to lipid rafts that were labelled with caveolin- -rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp and caveolin- -rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp internalization. notably, stamp was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c ), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with -hydroxy- -nitrobenzyl bromide (hnbb) that covalently modifies the trp residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity ( %) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states -phosphofructo- -kinase/fructose- , -bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose- -phosphate (f p) to fructose- , -bisphosphate (f , bp) and vice versa, and f , bp is an allosteric activator of phosphofructokinase- (pfk ), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb - ), pfkfb is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb , variant- and variant- , coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g d)induced oncogenic transformation on pfkfb expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb mrna and protein expressions (mrna; ipde: ae . ; ipde+kras: . ae . and protein; ipde: ipde+kras: . ). we then, checked individual expressions of splice variants and observed that while pfkfb splice variant- (p -v ) expression was reduced by k-ras induction (ipde: ; ipde+kras: . ), pfkfb splice variant- (p -v ) expression was increased (ipde: ; ipde+kras: . ). then, we checked effects of p -v and p -v on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb variants increased f , bp concentration (p -v : . ; p -v : . fold; compared to empty vec), glucose uptake (p -v : %; p -v : %) and glycolysis (p -v : %; p -v : %) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb isoforms and observed that both pfkfb isozymes localize to the nucleus, with more prominent nuclear localization of p -v compared to p -v . also, nuclear localization ratio of p -v increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p map kinase inhibitors on mda-mb- cell line introduction: p mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p mapk sb and sb on mda-mb- cells to further elucidate the controversial role of p mapk on cell proliferation or cell migration. materials and methods: mda-mb- cancer line was cultured in rpmi- supplemented with % fbs. the cytotoxic and cell migration effects of sb and sb inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb- cells were determined by icelligence system. results: it was found that sb p map kinase inhibitor was more effective than sb . however, no significant effects of low doses of lm and lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to hours as determined by icelligence system. on the other hand, both sb and sb significantly prevented cell proliferation at a concentration of lm. both sb and sb significantly reduced cell migration in a time-dependent manner at a concentration of lm. then, we tested whether each p mapk inhibitors have any effect on cell adhesion during a treatment period of hours using icelligence system. only lm concentration of sb reduced cell adhesion for about . hour (p < . ). conclusion: p mapk inhibitors sb and sb differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no - . mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto , f. zilifdar , s. yilmaz , t. sarac ßbasi , i. yalc ßin , n. diril hacettepe university, ankara, turkey, ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of , -benzoxazine- -one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta and ta strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s ) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a d-common features pharmacophore model with hiphop method. according to the results, only bs , bs , bs and bs exhibited strong mutagenic effects on both strains in the presence and absence of s . additionally bs , bs , bs and bs (in the absence of the s ), bs , bs and bs (in the presence of the s ) showed weak mutagenic effects on ta . hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r and hydrophobic groups at the position of r of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since .the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = ) were evaluated in dried blood samples which sent to our laboratory for the study between august and august . electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms ,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in ( . %) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in ,organic acidemia in ,fatty acid oxidation defects in patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain- are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr /nr b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr /nr b subunits, calpain- , hsp and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain- are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp that reduces the affinity for ca + of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain- . our data suggest a physiological role of calpain- at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca + influx, the activated protease regulated by hsp , promotes the removal of nmdar from the plasma membranes, decreasing ca + entrance through this receptor-channel and protecting cells from ca + overloading. tissue transglutaminase (tg ) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg mutant constructs. renca cells were transduced by lentiviral particles encoding wttg , transaminase-defective tg -c s form with low gtpbinding affinity, gtp-binding deficient form tg -r a, and transaminase-inactive tg -w a. in order to investigate the role of tg transamidating and gtpase activity in cell migration, scattering assay was used where colonies for each mutant clone was followed for a time interval of hours. our results showed that non-transduced control and tg -c s mutant renca cells demonstrated a similar migration pattern with a % of scatter activity. on the other hand, % colonies formed by renca cells overexpressing wttg and tg -w a mutant scattered away from each other. a small insignificant increase in scattering was seen in % of the total number of colonies for renca cells overexpressing tg-r a construct. data from this study supports that gtp-binding activity of tg is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = ), subclinical hypothyroid (n = ), subclinical hyperthyroid (n = ). serum tsh, free t (ft ) and free t (ft ) levels were determined by chemiluminescence immunoassay and serum -hydroxy (oh) vitamin d (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, . to . miu/l), ft (range, . to . ng/dl) and ft (range, . to . ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t and t levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t and free t levels. results: subclinical hyperthyroid group had significantly higher (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < . ). (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co , no x and so x (ghorbani et al., ) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., ) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr , ts and ts were used as ethanologenic microorganisms. expression of vhb in ts is lower than in ts strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts strain by up to . x cfu per ml of fructose ( %, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s ( ) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology : - . ghorbani, f., younesi, h., sari, a. e., najafpour, g. ( ) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. ) . in this work, e. coli strains fbr , ts (low level vhb expressing) and ts (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for hours at °c and rpm in a ml erlenmayer flask. the ethanol production was improved over % by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as . , . and . for fbr , ts and ts strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. ( ) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., ; geckil et al., ] . in this study, ethanologic e. coli strains fbr , its derivative strains ts (vgb+) and ts (vgb+) were used. ts was constructed in such that it could express more vhb than ts . bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations ( . - mm) were used as inhibitors from lignocellulose hydrolysate. . mm acetic acid was used as an inhibitor. the growth of vhb expressing ts and ts strains was inhibited about % after hours fermentation time. strain fbr was inhibited as high as % by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd , an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd on adipogenesis in t -l preadipocytes. notably, azd treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of t -l preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription- (stat- ) during adipocyte differentiation. furthermore, azd largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd inhibits adipogenesis in t -l preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat- . effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = ) and food colouring (fcg, n = ) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than . were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment ( . ae . ) and control ( . ae . ) groups showed no statistical difference (p = . ). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr c ) polymorphisms and unipolar or bipolar depression. the nr c gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr c (bcl and rs ) in recurrent depressive disorder (rdd) patients. methods: our study included volunteers with recurrent depressive disorder and healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr c gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs ) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl- (g/c) were as follows: cc % and %, gc % and %, gg % and %, respectively. there was not a significant difference when the frequency of the allele (p = . ) and genotype frequency (p = . ) were compared between the patients and the control. genotype distribution in the rs region (a/t) of the patients and controls were tt % and %, ta % and %, aa % and %, respectively. allele frequency (p = . ) and the genotype frequencies (p = . ) were not significantly different among the groups. conclusion: numerous nr c gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr c polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy y human neuroblastoma cell line. sh-sy y cell line treated with - lm tq by solving medium for , and h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm , p , p , akt, pten, cdk , cyclin d , caspase- , - , - , - , bcl- , bax, parp, bcl-xl, bid, dr , dr , puma, noxa, mmp- , - , timp- , - and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt profiles array data analysis by using student's t test. ic value of tq in sh-sy y cells was detected as lm at th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm , akt, cdk , cyclin d , bcl- and mmp- . it is also observed that tq caused a significant increase in the expression of p , pten, caspase- , - , bid, dr , puma, noxa and timp- . it was also found that tq in sh-sy y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. - - . the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm wild type (wt) and its isogenic Δmacab mutant strains. we also used h o survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab- xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h o . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after hours of growth in lb-broth there is at least -fold increase in expression in response to the presence of h o . on the protein level macab can be detected starting from hours of growth in lb-broth and it reaches maximum expression on hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h o properties. antiproliferative and apoptotic effects of noscapine on mcf- and mda-mb- human breast cancer cell lines approximately - % of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor . these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf- and mda-mb- human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf- and mda-mb- cell lines were analyzed by roche xcelligence system. the cells were cultured in % fetal bovine serum containing dulbecco's modified eagle medium at °c in a humidified atmosphere containing % co . h after seeding, the cells were treated with different doses of docetaxel ( . to nm), tamoxifen ( . to lm), and noscapine ( . to lm). cultured cells were harvested, fixed with % formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at lm thickness and stained with h&e, ki- , bcl- , cyclin-d , and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf- cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf- cells, hovewer docetaxel and noscapine were more effective on mda-mb- cells. tamoxifen has more apoptotic and antiproliferative effects on mcf- cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb- cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a -ml centrifuge tube by a neodymium magnet through a -cm cushion of % sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was -fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. - - . epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl sandwich elisa and rt-pcr. the mechanism of action of zeb / on ccl promoter has been studied by luciferace assay and chip. ccl coding region was cloned into pcdna . and stably transfected into dld- cells. ccl deficient ct cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl and sip expression was studied using ihc or flow cytometry. emt status catagorised crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl in induced dld-sip cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl in the induced dld-sip cells as well as mesenchymal crc cells as compared to epithelial ones (p = . ). promoter studies showed that zeb / bind to ccl promoter and and activate ccl gene expression. no metastasis was observed for dld- cells overexpressing ccl but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl is up-regulated by emt inducing transcription factor sip , and mesenchymal (metastatic) crc cells secrete significantly more ccl compared to epithelial (non-metastatic) ones. ccl did not induce emt per se but abundant secretion of ccl by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for - minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of . to . lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the lg/plate concentration s. typhimurium ta . at all the doses antimutagenic response was significant at (p < . ) against both the strains with a percent mutagenicity decrease from to for ta followed by ta with percent antimutagenicity from to . the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [( , , , )-tetrathia[ . ] ( , )- , , -thiadiazolophane; ( , )dioxo- , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane; ( , , , )-tetraoxo- ( , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane and ( , , , , , )-hexaoxo- ( , , , )-tetrathia [ . ] ( , )- , , -thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for hours. at the end of the each hour, cell vitality was assessed by measuring the absorbance ( nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of : of . mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using , -diphenyl- -picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than ng/ml or greater than ng/ml were accepted as inappropriate initial testing. fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of patients who referred to the hospital phlebotomy unit were collected using holder into the different tubes. first tube was ml glass tube and with no additive, second was ml tube with gel separator, third was ml tube with gel separator. serum was separated and immediadiately analysed for biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft , ft ) which are the most common requested laboratory tests. thyroid function tests of patients are analyzed in ankara numune education and research hospital in october -may . five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below % of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for -month study period was . %. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis- development of polyclonal and monoclonal antibodies against fatty acid binding protein (fabp /ap ) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp ) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp protein made by recombinant dna technology. recombinant his-fabp was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp , as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb- , mcf- and cd + /cd -cells and ic values of thz were found to be lm, . lm and lm respectively, at hours. invasion potency of mcf- , cd + /cd -and mda-mb- cells were determined as %, %, . % when compared to relevant treatment controls. migration potency of mcf- , cd + /cd -and mda-mb- cells was determined as . %, . %, % respectively. among the three cell lines mda-mb- cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin- , b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb- , mcf- and cd + /cd cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. healthy - year children's ( girls, boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = . , p = . ) and a negative correlation with humidity (r= À . , p = . ). mean urine density in spring was higher than that of autumn (p = . ) and winter (p = . ). mean value of summer was higher than autumn (p = . ) and winter (p = . ). - months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis- implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of pharmacies located in north cyprus were involved in the study with %stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two ( . %),responded positively to this demand. it has also been identified in the study that . % of the daily sale of antibiotics in the first half of the year was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with . % and upper respiratory tract with . %. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be ( . %),compared with the studies conducted in greece,italy,malta and spain % and egypt . %that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic ) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a cells including with ultrastructural and morphological effects. ic value of aa on a cells for h was detected with mtt ( -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a cells in dose-dependent manner and ic on a cells was found to be . mm for hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on immunoassays studied by the beckman coulter unicell dxi immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of tests were measured by the beckman coulter unicell dxi . d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin ( . , . , . , . , . and . g/l hemoglobin) and intralipid ( . , . , . , and g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid ( %, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au . after spiking the pools, the tests were measured again in duplicate on beckman-coulter dxi analyzer. a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b , testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca . , ca , ca . , insulin, pth and e , and of lipemia for progesterone, ca . , vitamin b and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb andiso plus tak- . treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase- (pon- ) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p mapk and nuclear factor erythroid -related factor (nrf ) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p mapk, phosphorylated c-jun n-terminal protein kinase (pjnk / ) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk / ) and nrf expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb treatment. p mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p mitogen-activated protein kinases, sb , tak- . silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were males in each groups which contained patients with silicosis (group ), individuals having similar symptoms with silicosis from same occupational area (group ) and healthy subjects (group ). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev , fev %, fev /fvc, fev / fvc%, fef - and fef - %) were compared. the average age of group , and are . ae . , . ae and ae . years, respectively. there was a significant difference between group and in terms of the ada level (p < . ). there was a negative correlation between ada activity and fev , fev %, fev /fvc, fev /fvc%, fef - values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. - - . the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar , m. tekin , s. civelek firat university, elazig, turkey, cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by genus on the world and by genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega . program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, ll of internal standard (d - deoxycortizol) in methanol was added to ll standart or serum and centrifuged at . rpm for minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at • c and dissolved in mobile phase. ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. k ) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value , , intercept value À . and r² value . . the regressione quation was elisa= À . + . lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste polycomb repressive complex expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u , h. s€ urer , g. bilgin , d. y€ ucel ankara training and research hospital, medical biochemistry department, ankara, turkey, ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. patients with copd, patients with asthma and healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha- antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < . ). there was no significant difference for ferritine and alpha- antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova , , v. timofeev , , n. zhukhlistova , y. abramchik , t. muravieva , r. esipov shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, national research centre "kurchatov institute", moscow, russia, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at k from single crystal grown in space (sp. group p ) was collected on the spring- synchrotron-radiation facility (japan) and the structure was solved at . a resolution, using the molecular replacement method (pdb id i c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id da ) and human pnp (pdb id pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h ) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h . ara h aptamer was used to recognize any residual peanut major allergen protein ara h in food samples. recombinant ara h protein was produced and puirifed to be used as a target. ara h aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between and . kg/m . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of women. students with high bmi (> ) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (< ) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (< ) student group. waist circumference, percentage of body fat was higher in high bmi (> ) group than low bmi (< ) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (> ) and bmi (< )). students were grouped depend on their waist circumference. healty individuals who had lower than cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum ( , , aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions ( % ethanol and polyethylene glycol -peg %) and different method (ultrasonic treatment w/ o c/ minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at o c/ hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %), followed by liquid samples treated by ultrasonic method prior to transformation ( . %). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: patients, having delivery beyond weeks, and who did not have any other medical problems were included in the study. the patients had normal g. oral glucose tolerance test results. they were divided as groups, based on their newborns' birthweight as " g. and g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low ( . ng/l, . ng/ml and . ng/ml respectively), normal ( . ng/l, ng/ml, . ng/l respectively) and high ( ng/l, ng/ml, g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin ( . , . , . , , . and . g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access analyzer. the hemolysis indices were measured on beckman coulter au . a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations ( . ng/ml), and a negative interference for myoglobin at low concentrations ( . ng/ml) and high concentrations ( ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥ . g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a- , glycosidase enzyme with an incidence of : , - : , . as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a- , -glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a- , glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of patients's examined, of them were diagnosed with pompe disease depending on clinical findings & low a- , glycosidase activity. enzyme activities of pompe patients were . nmol/ml/hour as lsd suspected patients'activities had a mean of . nmol/ml/hour (p = . ).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher ( vs - u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group ( vs u/l, p = . ). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = . r=À . ) was observed between a- , -glycosidase and ck activities (n: ). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of ( h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of ( h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f ) on biofilms formed by s. aureus atcc and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic ( mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of mg/l of f the mic of kanamycin was decreased -fold, and the mics of both erythromycin and ampicillin were lowered -fold. at the concentration of mg/l f also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto and propidium iodide. the addition of f to preformed h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f at concentration of mg/l led to death of up to % of biofilm-embedded cells. this fact suggests that f efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project № - - and the german academic exchange service (№ ). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged and over is - times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin- secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed young and elderly patients with pneumonia and their respective aged matched controls (n = , n = ) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml and . ae . mg/l, . ae . ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < . and p < . ). elderly patients with pneumonia have significantly higher crp levels than their controls (p < . ). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin ( . g/dl) and normal calcium ( . mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of ml were aliquoted. purified albumin, . g, was added to one of these pools and albumin concentration was determined as . g/dl. the low and high albumin pools were mixed at different ratios and pools with . , . , and . g/dl albumin concentrations. total calcium and albumin concentrations of these pools were measured at a beckman-coulter au analyzer and ionized calcium was measured at a radiometer abl blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of . g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: . %, . %, . %, and . %, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: . %, . %, . %, and . %, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at > g/dl albumin concentrations. ionized calcium is significantly affected by . g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba c and fasting glucose concentration was investigated. lp(a), hba c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july to july . there were patients with these test results at the same time. the patients were grouped according to hba c values: group i < . % (n = ), group ii . - . % (n = ), and group iii > . % (n = ). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba c (r = . ; p < . ), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < . . there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < . ). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep -p is written in kit inserts. target values ( %) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from to mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate . % from the target when the degrees of hb are mg/dl. when the degree of hemoglobin is mg/dl, the test strays about . %. potassium levels increase ( %) at mg/dl hb while this increase reaches to . % at mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases %, at the mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. d-structure of the hu protein from mycoplasma gallisepticum have been determined with a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund ( - - ). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox . sox is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with -ethyl- -( -dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range ( . - pg/ml) with a detection limit as low as . ng/ml sox . furthermore, the developed sox immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox antigen and anti-sox antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: z ). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: . women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh ( - ) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= . -( . age)À( . fsh) + ( . lh)À( . estradiol) is detected and the model's r = . is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld- ) and endometrium (ecc- ) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic ) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at and lg/ml concentrations by hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld- and ecc- . however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca - against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca - is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+ %psa. tissues were seeded on mm petri dishes containing dmem/high glucose supplemented with % (v/v) fcs and incubated %co in air at % relative humidity and at °c. after seeding, the medium was unchanged for days and then it was changed in every days for days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over %) in culture when compared with the negative control ( %). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g /g phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g /g phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g /g phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, g ). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe o ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used - -trimethyl- -phenyl , , hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between and °c simple nanoparticles decreased the membrane fluidity. at physiological temperatures ( - °c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at °c, and at °c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. -isoprostane is considered as an indicator of oxidative stress. mao activity and -isoprostane levels were measured in some diseases. however, there are no information on -isoprostane levels and mao activity in newly diagnosed patients with stage hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and -isoprostane in newly diagnosed patients with stage hypertension. the study included newly diagnosed stage hypertensive patients with no other systemic disease. patients were selected as randomized ( women, men; range of age - years) and healthy individuals as control ( women, men; range of age - years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and -isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > . ). compared to the control group, the activity of mao and -isoprostane levels were found significantly higher in patients (p < . ). conclusion: increased -isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, -isoprostane p-mis- determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c -a guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from , males, , females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma and sigma subunits did not affect rna elongation. we showed that the sigma -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp /dis , dis l and dis l proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the s subunit at °c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the s ribosomal subunit, concomitantly with the increase of s and s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of -thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of -thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip- / modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov- g and tov- d. for this purpose, tov- g and tov- d cells were treated with limnio grape skin extracts at a range of concentrations, at °c, for , and hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov- g and tov- d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl , bclx, bax, bak and bcl l , along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. % of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse , gse and gse . we analyzed two datasets of er(+) breast cancer patient samples (n = , n = ) compared to normal breast tissue (n = ) using apadetect and sam. a total of utr shortening and utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb- cells that are otherwise of triple negative nature. our results suggested that most of the genes are utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between - bp also detecting the impurities in dna solution in %, % and % ratios to the dna of interest. the developed method was validated using the in-house dna fragments of , and bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, . lm, . mm id . cm and tskgel dna-npr, . lm, . mm id . cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for bp in the range of . ng to ng dna with the regression coefficient of r = . . lod and loq for the bp dna was found to be . ng and . ng, respectively. the recovery values for the %, % and % impurity ratios were found . . . and . , respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = . ). the length distribution of small rnas revealed nt-peak corresponding to trna-derived fragments, amount of which was increased by . -fold under dtt treatment. the trna isotypes that gave rise to almost % and % of all nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of nt fragments produced from these trnas are precisely phased halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor (rnh ). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun , , , j. kim , , j. y. hong dankook university, cheonan, south korea, institute of tissue regeneration engineering (itren), cheonan, south korea, the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; hour, day, week, month and months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage ( hour to week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at hour or day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p- . . - utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco- cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco- cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day ) caco- cells and tested sample apa events by rt-qpcr. utr isoforms were identified by using race pcr. we identified genes ( % of all apa events) to undergo utr shortening in differentiated cells compared to proliferating cells. on the contrary genes ( % of all apa events) went through utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine- of rna polymerase ii c-terminal domain by end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y -s -p -t -s -p -s at the c-terminal domain (ctd) of its largest subunit, rpb . phosphorylation of serines (s , s , and s ) and tyrosine- orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine- site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine- phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine- ctd phosphomarks using integrative structural biology. one of them, a factor involved in the -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine- that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t g human gbm cell line were used to evaluate the effects of ind ( lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p < . ), cell viability (p < . ), cell rate at s phase (p < . ) and g + m phase (p < . ), camp levels (p < . ), the levels of pdgfr-a (p < . ), mrp- (p < . ), nf-jb (p < . ) and cox- (p < . ) in comparison to control group. ind mildly increased apoptosis (p < . ) and caspase- levels (p < . ). interestingly, ind increased htert levels ( %, p < . ; %, p < . ). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox- and other tested factors. p- . . - interaction between fibrinogen and insulin-like growth factor binding protein- under physiologic conditions and influence of diabetes mellitus type on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp- , is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp- . besides glucose regulation, igfbp- stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp- , their change in dm type (dm ) patients and involvement in fibrin clot. samples from adult healthy persons and dm patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp- /fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp- . in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp- /fibrinogen complexes can be differentiated from igfbp- oligomers and igfbp- /alpha- macroglobulin complexes. the amount of igfbp- /fibrinogen complexes was lower in patients with dm . complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp- monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm was additionally able to bind exogenous igfbp- . since igfbp- stimulates wound healing, directly and by delivering igfs, igfbp- /fibrinogen complexes may be seen as igfbp- storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k q (q substitution for k in position ) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated (single mutant) and (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk- cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst- reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk- cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p- . . - his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova , m. tileva , k. maskos , i. ivanov , g. nacheva institute of molecular biology "roumen tsanev", sofia, bulgaria, proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five Ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p- . . - development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected ( - in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec . . . ) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta- , -d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall -fold purification, a . % yield and specific activity uÁmg À protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled kda. it was found that the enzyme displays pi . , prefers ortho-nitrophenyl-beta-galactoside as substrate (km mm) and shows maximum activity at °c and at ph . - . . the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km . . it was established that the gene carries an open reading frame consisting of bp ( amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family (cazy database). p- . . - different splice-forms of tdrd protein mutated in cataract's and glaucoma's interacts with s k / o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s kinases (s k) are important players in cellular pi k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s k -tdrd (trap). tdrd is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s k-tdrd interaplay and study its role in cells. bioinformatical analysis of tdrd sequence revealed the presence of potential phosphorylation sites of s k . using in vitro kinase assay, we have demonstrated that recombinant s k phosphorylate from fragments of tdrd . formation of s k -tdrd complexes in vivo was further confirmed by coimmunoprecipitation using anti-s k and anti-tdrd antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd co-localize with s k in hepg cells, predominantly in perinuclear region, enreached for one of the tdrd isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s k with methylated arg interfere with tdrd from hepg lysates. the physiological characteristics of s k -tdrd interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~ kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x- in high concentrations, but efficiently dissociates in the presence of m urea and mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of to kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a and other proteins. serum albumin, transferrin and protein with molecular weight , kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b fusion protein. we have shown that cytochrome b enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at nm in oxidized environment. cytochrome b change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b fusion tags multiples recombinant antibody yield (from to times) and doesn't affect antigen-binding properties. the bb - site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i- c (monab i- c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i- c in bb - of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb - , bb - and both their scrambled version, and bb - peptide. monab i- c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i- c was investigated by spr method using plasmon- device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb - , bb - , bb - , and the scrambled versions of bb - and bb - peptides on a binding to monab i- c and on the fibrin polymerization process. in spr analysis was showed that bb - and bb - peptides, but not their scrambled version, binds to monab i- c, immobilized to a chip. turbidity data showed that only bb - and bb - peptides caused the -fold decrease of the rate of the lateral association of protofibryls at the concentration . À m and . À m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb - site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp- ) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is - ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= , female= ) was . years (sd . years). among them ( %) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while ( %) as a hf-ref (hf with reduced ejection fraction). mean bnp values was . pg/ml (sd . pg/ml) and mean lvef was . % (sd . %). mean urine ngal was . ng/ml (sd . ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = . , p = . , wilcoxon signed rank test z = À . p < . ). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p subunit of splicing factor sf ;(iii) voltage dependent anionic channel- ;(iv) dynamin- ; (v) heterogeneous nuclear ribonucleoprotein a /b . obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near uridine derivatives were determined. the combined kinetic (nnna, , ) and structural data (acta crystallogr., d , , ) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as - kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp (forkhead box p ) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp on tumor cell migration. our results showed that foxp has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase (pde ) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde was increased in cell lines cancers thus pde inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde gene expression and cgmp signaling in the mcf- er + and mda-mb- er À . methods and materials: the ed of the hawe on both cell lines were examined by using mtt viability test then the expression of pde and cgmp concentration were measured in timedependent manner (in the ed ) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, lg/ml is ed for both cell lines and the hawe lead to reduction in pde mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf- and mda-mb- , cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having -stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to da in neutral ph but the pore is closed by going through a conformational change under ph . . as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg- s is obtained by adding amino acids to the primary chain in order to have a -stranded beta barrel porin. ompg- sl is formed by further adding amino acids to loop l and by replacing lysines with arginines. ompg- s and ompg- sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd . / . and infrared spectra are recorded. further, temperature profiling are recorded for the range between to °c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ °c, although this is less than the wt transition at $ °c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase (parp ) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt . further characterization of nudt will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group ( mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid ( mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups . % peanut oil was added throughout the experiment. the all rats were sacrificed at the end of weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in . % of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp- levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if , if and if . the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim p possesses a function of mtif . however, the mitochondrial translation has not been stopped in the yeast strain without aim p which is surprising taking into account the fact that if is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim p is almost indispensable for cox p synthesis, and that it affects the translation of cox mrna through its -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim gene than in the wild-type. taken together, our results point on the multiple role of aim p in mitochondrial translation: in addition to its function as mitochondrial if , it specifically regulates the amount of complex iv subunits and its activity. p- . . - the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of patients with pcos and healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < . ). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < . ). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). we found that bche hydrolyses this molecule at ph rather better than at ph . . at ph values, purified human bche has a km value that was times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately % that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p- . . - determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi auto analyzer. the epo levels of samples was . ae . mul/ml (ranged between . and . ) mul/ml. when we performed ae sd for the studies population we determined normal serum epo levels were as . - . mul/ml. the upper limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml and the lower limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included patients diagnosed with subclinical hypothyroidism and healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at À •c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were ng/l, control group ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > . ). apelin levels didn't show significant correlation with bmi (p > . ). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv /mia import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv is a sulfhydryl oxidase that partners with the import receptor mia to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv mutant strain (erv - ) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv s role in these processes. we discovered that the erv - strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv and mia mutants tested were similar to the wt control. in addition, while all the erv and mia mutants tested exhibit temperature-dependent defects in mia oxidation, only the erv - strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv - strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh ) that compromises gsh protein folding and/or stability. to address whether gsh deficiency in the erv - mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv and mia mutant strains. only the erv - strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv - are due to gsh depletion and neither erv nor mia play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are . - . ng/ml and - ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak Ò hplc and zivak Ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast l aaa column ( mm i.d). the mobile phase was composed of, a: mm ammonium formate (af) in water, b: mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c column ( mm i.d., mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p- . . - heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp expression and induced caspase-dependent apoptosis in c rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at °c for h in c cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase- activation in c glioma cells. pretreatment of the cells with non-toxic dose ( lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase- activation in c glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp , matrix metalloproteases ; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from mhz to mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain - amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp- and hnp- encoded by defa , defa and defa genes are firstly expressed in neutrophils. human hdp and hdp encoded by defa and defa are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd strain was cloned, expressed, characterized in escherichia coli blr(de ) strain, and then val- and phe- amino acids, supposed to be responsible for substrate preference, were substituted for met- and ile- amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe ile enzyme are %, %, %, %, % and % of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val met mrbsh enzyme are %, %, %, %, % and % of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d , t , and d ) but according to alignment is closer to chr even the last one motif is e , t , n . it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf - - . evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un , s. sahin , f. duygu university of gaziosmanpasa, department of biochemistry, tokat, turkey, oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (% - ). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha (ta ), thymuline and thymosin beta (tb ) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < . ) and plt and wbc levels were significantly lower in the cchf group (p < . ). levels of tf, ta and tb were found to be significantly higher in cchf (p < . ). there was no mortality during the study period. duration of hospitalization was . ae . days. levels of tb were significantly correlated with duration of hospitalization (r= À . , p = . ). alt levels were significantly correlated with tf levels (r = . , p = . ). patients received ffp and apheresis for the supportive treatment, while patients received only ffp and patients got only apheresis. patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > . ). we report survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic values were determined as and mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh of carbamate group of methocarbamol to the carboxyl group of asp side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser and ser residues of the pseudoenzyme. p- . . - dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc ) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than mm. moreover, primary cell cycle analysis has indicated arrestment at g /m phase in hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g /g and g / m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor alpha (hif a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor . it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif -a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of - % oxygen and - % nitrogen for week. rats in adm groups were injected intraperitoneally in a dose of . nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from - animals and adm, hif -a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif -a level. adm levels and hif -a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif -a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif -a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif -a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p- . . - covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, ( ) their circulatory-lives and stability (in different ph and temperature values) enhance ( ) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. postmortem skeletal muscle were analyzed hour periods in first -hour after death. nd and rd -hour periods were statistically significant (p < . ). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of , were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr b and akr b in hct- colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr b ; aldo-keto reductase family , member b , and akr b ; aldo-keto reductase family , member b . both are -kda cytosolic reductases that are similar in both amino acid sequence identity ( %) and tertiary structure with the (a/b) barrel topology. while hct- , a colorectal cancer cell line, cells expresses akr b robustly, there is no expression of akr b . in this study, we have stably knocked down akr b through shrna technology and overexpressed akr b in hct- cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr b , we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr b overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr b and akr b have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m medium (ph ) boosted nisin production at °c, as the addition of . lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p- . . - coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between and , the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to , , and amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf isoform in hct- colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf . we show that vegf undergoes n-linked glycosylation in hct- cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p- . . - novel methods for modulating the activity of bcl family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl- family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl- family, including the pro-survival members bcl- , bcl xl and mcl , and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl- family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl- family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl- family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora , v. castro-fern andez , c. a. ramirez-sarmiento , e. a. komives , v. guixe facultad de ciencias, universidad de chile, santiago, chile, department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt . elucidating the domain swapping mechanism of the forkhead domain of human foxp fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp follows a threestate n « i « u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp , showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt , ). p- . . - the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of . % per week for up to weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the -week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full -week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < . ). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = . , p = . , p = . , respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p- . . - pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck kinases on ents activity. methods: adenosine uptake ( lm adenosine, s, °c) was assayed in hk cells preincubated with mm or mm d-glucose for h and exposed to lm -br camp, lmh or lm tbb for h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent and ent contents were quantified by western blot. results: high d-glucose in hk cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h ) through an increased ent uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent or ent under normal d-glucose ( mm) or high d-glucose conditions ( mm). also, we saw that tbb (ck inhibitor) decreased the activity of ent and ent under normal glucose conditions, decreasing the localization of ent at cell surface, while the membrane localization of ent decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent . this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was . ; body mass index was . ; serum lipo (a) level was . and plasma fibrinogen level was . . there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = . ; p = . ), significant positive correlation between the serum lipo (a) and rdw-sd (r = . ; p = . ) and significant negative correlation between lipo (a) and mpv (r= À . ; p = . ). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is . kda as found. optimum temperature was °c and half-life times were , , and . min at , , and °c, respectively. optimum ph value was . . however, it shows significant ph stability at ph values . and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d lipase. therefore, the effect of immobilization on kinetic parameters of d lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c q tumor necrosis factor-related protein (ctrp- ), c q tumor necrosis factor-related protein (ctrp- ) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp- , ctrp- and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included patients with knee oa and healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp- , ctrp- and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp- in the patient group were significantly higher than in the control group (p = . ), serum ctrp- and kallistatin levels were not statistically different (in order of p = . , p = . ). in the patient group, there was not a significant difference between serum ctrp- , ctrp- and kallistatin levels and radiologic stages (respectively p = . , p = . , p = . ). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = . , r = . ; p = . , r = . ; p = . , r = . ; p = . , r = ). discussion and conclusion: serum ctrp- levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp- and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp- , ctrp- and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as - and - °c. in consequence of the salt test, the test isolates were grown at - % nacl. of thermophilic isolates were selected by rep-pcr and according to s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of - %. enzyme tests showed that, some of the isolates were able to produce protease (f , f , f , f , f and f ), amylase (f , f , f , f and f ), cellulase (f , f , f , f , f and f ), xylanase (f , f , f , f and f ) and lipase (f , f , f , f and f ). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p- . . - warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt a, du , hela, hep b, hek t, shsy y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter (glut ) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp- , - ) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco and casp- was higher in hek t compared to other cell lines. casp- was upregulated in cancer cell lines, mostly in hep b. glut- levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p has a repressive effect on glut , it also can lead us to study about p levels. the effect of inhibition of pi k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her , her ) play important roles in breast cancer pathogenesis. her /her interaction is the most potent heterodimer and it causes oncogenic pi k/akt activation. inhibitors of pi k/akt pathway (akt inhibitor and pi k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi k/akt inhibitor pi- on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb- cells (which has egfr overexpression and pten deficiency) and skbr- cells (which has her overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi k/akt inhibitor pi- for different time periods ( - h) . egfr, her her total rtks expression and pi k/akt pathway inhibition (p-akt and p-p s k expression) were evaluated by western blot. in mda-mb- cells, there were significant decrease in p-akt and p-p s k proteins' expression during the first h. this inhibition was followed by reactivation of the signaling pathway after h. in skbr- cells, p-akt and p-p s k proteins' expression were significantly decreased during the first h. the pi k/ akt signaling pathway in these cells were reactivated after h. basal expression of egfr and her in mda-mb- cells and basal expression of her and her in skbr- cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her and her expression levels. these results suggest that dual pi k/mtor inhibiton by pi- may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid -formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a cell line, respectively. for the whole cell transcriptome analysis a cells were treated with lg/ml of binase for h followed by mrna extraction and library preparation. sequencing was performed on solid xl wildfire next-generation sequencer. we found that binase internalized into a cells after h of incubation. the binase at lg/ml was absolutely non-cytotoxic towards a cell line and was active in the cell culture medium during h incubation. the analysis of rna-seq data showed that among thousands of protein coding transcripts transcripts were up and down regulated by binase, among them transcripts were induced and repressed. binase repressed the production of s a and tnxb which act cancer biomarkers, scn a and drd which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp , rasgrp and alpk transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. . ) is a specific group of phosphatases capable of hydrolyzing myo-inositol , , , , , -hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: -phytase, / -phytase, and -phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain . . showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. . . phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was °c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet a expression vector. multicopy plasmid pet a/agpp contained the sequence of the phytase gene of pantoea sp. . . under t promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag- (bcl- associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag- exists as three major isoforms (bag- s, bag- m and bag- l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp /hsc molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl- , raf- kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag- expression levels affect cell survival in mda-mb- (er, pr and her /neu negative) breast cancer cell lines. we first cloned bag- l gene to a cloning vector, later we transfected mda-mb- cells for overexpression of bag- . we also used bag- sirna to silence bag- gene. western blot analysis was applied to demonstrate relative expression levels of bag- , its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag- overexpressed cells to checkbag- 's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag- overexpression. in addition, our study revealed that once bag- forms a complex with c-raf/b-raf/hsp /akt/bcl- , modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag- and its isoforms are found, the role of each bag- isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type diabetes because of different physiological effects. in circulation, glp is cleaved by ddp in a very short time. if glp can be protected from cleaving, the effective time of glp would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp was performed by sds-page. the purity of fusion protein was found to be %, as confirmed by glp elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at °c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp . ) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology ( %) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with % homology was identified in the genome of clinical isolate of m. morganii . rt-pcr analysis showed that this gene had the maximum expression at h of growth. in addition, the cellular extract of m. morganii had small actinolytic activity. cloning of the gene into e. coli dh a cells led to the synthesis of the kda protein. it is known that the highest expression of serratia proteases is observed at h of growth, and the molecular weight of the mature proteins is kda. it was shown that metalloprotease gene of m. morganii expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight ( kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova , d. zhuravleva , a. laykov , k. forchhammer , a. kayumov kazan federal university, kazan, russia, eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only - % homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = lm) over atp (kd = lm) suggesting that they compete for binding to lbrglnk. neither -oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. - - a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k ) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k -fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c. . . . ) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in . u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a antibody, its kappa and lambda variants, in order to understand the difference on the active site between a and a which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a antibody kappa and lambda variants have been already determined, there was no structural information though about the a antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are s À and s À for a kappa and a lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi x . translation elongation factor ba (eef ba) is a component of a heavy form of translation elongation factor (eef h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor a (eef a) restoring its active conformation appropriate for aminoacylated trna binding. eef ba forms a tight complex with translation elongation factor bc (eef bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef ba. this suggests that the eef ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef a, eef a and eef a . we showed that recombinant eef ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef a comparing to full-length eef ba. similar effect on the catalytic activity of eef ba was observed after its interaction with eef bc. in contrast, the effect of full-length eef ba and its truncated forms on the rate of guanine nucleotide exchange in eef a was similar but relatively modest compared to eef a . this can be explained by higher rate of spontaneous gdp dissociation from eef a comparing to eef a and lower affinity of eef a to eef ba. thus, we propose that the n-terminal domain of eef ba via flexible linker region may interfere with eef a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef bc and eef ba n-terminal domains abolishes this inhibitory effect. p- . . - assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below %, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof + mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of proteins across all samples with variation across lc-ms replicates was % on average, and the second approach led to quantitation of proteins in total, of them across % of samples, all with the variation about % on average. p- . . - interaction of plasminogen fragments k - and k with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k - and k with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k - and k reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by - % in the presence of k - and k . fragments k - and k have high affinity to fibrin fragment dd (dissociation constant is . lm for k - and . lm for k ). analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k - interacts with complex of fragment dd-immobilized k as well as k with complex of fragment dd-immobilized k - . the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa - region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k - and k , which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p- . . - implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf ) cells expressing human dopamine d receptors using [ h]sch- and bodipy-fl-skf- as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf- itself were rapid with an apparent half-life of t / = . ae . s for association ( nm) and t / = . ae . s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas levels in gi cancer patients. female and male patients were included in the study (n = ): colorectal, gastric, pancreatic, liver, ampullary, gall bladder and esophageal. from all gi cancer patients, ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at g for min, and plasma samples were carefully removed and stored in À °c prior to use. the level of plasma gas was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas levels in cancer patients were determined as follows: . ae . ng/ml in colorectal; . ae . ng/ml in gastric; . ae . ng/ml in pancreatic; . ae . ng/ml in liver; . ae . ng/ml in ampullary; . ae . ng/ml in gall bladder and . ae . ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas levels. taken together, these results suggest that gas may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas would be an attractive therapeutic target for slow down the progression of gi cancer. monday september : - : computational biology p- . . - computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p- . . - computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g -dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g -dendrimer to mannose receptor (mr ) were performed using hex . . software. taa lumo atoms were conjugated to homo atoms of pg-g dendrimer using maestro software. results showed that pg-g -dendrimer displays carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g -conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g -dendrimer binds to the core of the mr receptor. increasing of the number of mannosamine conjugated to pg-g -dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, -deoxy-d-xylulose- phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the d structure of tpdxr by phyre . energy minimisation and root mean square deviation (rmsd) was performed by drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket . server a reliable d model for dxr from t. parva was modeled by using au as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be . and . a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p- . . - molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme (bace ) as a drug candidate for alzheimer's diseases g. yalcin , i. yildiz biotechnology institute, ankara university, ankara, turkey, department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme (bace ) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly , thr , gln , phe , tyr , lys , thr , arg , thr residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace . the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version . . . dst was determined by bpa - p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley . this work was supported by the russian scientific foundation (grant - - ). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r , r , r , r , k , k ): r = . + . * p À . * f r = . + . * p À . * f r = . + . * p À . * f r = . + . * p + . * f k = . + . * p + . * f k = . À . * p À . * f these formulas show that the maximum total effect of fat and protein contents influences at r and r . a significant coefficient (> ) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p- . . - molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv . channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv . with peptide blockers agitoxin and its mutant forms. d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code a h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv .x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. - - . simulations were performed using the supercomputing center of lomonosov moscow state university. p- . . - homology modeling and molecular docking study of the paraoxonase- and its polymorphic variants q/r and m/l for non-statin lipid lowering drugs paraxonase- (pon ) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon and its polymorphic structures pon q/r and m/l . fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon and its polymorphic variants pon q/r and m/l was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon (pdb code: sre). ns molecular dynamic simulation analysis was performed using gromacs . . . affinity of lipid lowering drugs to pon and its polymorphic variants was predicted by molecular docking approach using autodock . suite. unlike other lipid lowering drugs that they have negative Δg values for affinity, probucol, orlistat and betasterol was calculated by positive Δg values ( . , . and . kcal/mol). these values suggest that they may have no affinity to pon q/r polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon -m/l and sibutramine to pon q/r were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon and m/l polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon structures may reduce the activity of antioxidant pon enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over distinct functions in mammalians. the mammalian prlr gene consists of - exons and several and regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_ . , nc_ . and rn_celera, ac_ . ) were used and primary analysis showed that both sequences contain several gaps (varying from . to kbp), corresponding to about . % ( - kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_ . prlr gene has two exon- (one is about kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from . % to . % (from kbp to kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon- and - , similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the utr (exon- and - ). in rats, there are exon- variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share common non-protein coding exon- variants. in conclusion, it is found that the rnor_ . version of the prlr gene has the highest number of unidentified base pairs (corresponding to . % of the gene) and the second exon- is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase- enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon (pdb id: sre) from protein data bank. modeller software was used for homology modeling of pon and its polymorphic variants that's called as pon q/ r and m/l . amino acid sequence of human serum pon (uniprot: p ) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs . . software. molecular docking calculations on each of the polymorphic structure of the pon was performed with auto dock . . suite. for each substrate, y residue showed open conformation in pon and m/l polymorphic structures while q/r polymorphic structure showed closed conformation. in comparison between structures of pon variants, in most cases statins had lower affinity to q/r polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon . by considering that the high affinity drugs can have reducing effect of pon activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p- . . - self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar , d. alpay , a. r. atilgan , c. atilgan sabanci university, istanbul, turkey, northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever- m' and tensiometer 'bpa- p'. three groups of animals were formed from clinically healthy holstein cows at the age of - years according to the fat content in the milk sample. group i - cows (milk fat content . ae . %), group ii - cows (milk fat content . ae . %), group iii - cows (milk fat content . ae . %). this work was supported by the russian scientific foundation (grant - - ). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from . % to . %, lactose and mineral content varies from . % to . %, respectively. the dst parameters (r , r and k ) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r , r and k . obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p- . . - exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p- . . - effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas , , l. buimaga-iarinca , c. floare , l. janosi faculty of physics, babes-bolyai university, cluj-napoca, romania, national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl À pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~ % of all cancers and up to % in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c : -phosphatidyl-choline: di- : -phosphatidyl-choline: cholesterol : : ) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region - and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id n ) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant - - . serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a -ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at nm uv light. antibodies of rhd antigen were detected using differential pulse method at between . and . v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in rhdnegative pregnant women ( - th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon and of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p- . . - investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega and bioedit version . . . and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega and paup . b with bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui . using gtrgamma model with bootstrap replicates. beast v . . was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total reads (with an average read length of . bp). there were contigs having > bp sequence length whereas contigs with > bp sequence length. for further analysis we have taken contigs with > bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of À . ≥ % of total contigs were mapped to the reference with ≥ % contig match coverage. the community analysis revealed that domain bacteria is predominantly present ( . %) in the water sample, followed by eukaryota ( . %), viruses ( . %) and other sequences ( . %). most abundant phyla was proteobacteria ( . %) and the most dominant family was enterobacteriaceae ( %) followed by xanthomonadaceae ( %), vibrionaceae ( . %), pasteurellaceae ( . %), shewanellaceae ( . %). we performed functional analysis of all contigs using rapid annotation using subsystems technology (rast) which detected coding sequences and rnas in subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with % accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~ % accuracy by this single cleavage, with optimal combination of - cleavages improving the accuracy tõ %. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants - - , - - and - - ) and ras presidium program 'molecular and cellular biology'. p- . . - mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain - which was derived from soil-isolated b. pumilus p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to lg/ml) and ability to produce extracellular enzymes in quantities almost -fold higher than the parent strain. these features make the - strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus p and - strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. . % of total cdss differ in - strain in comparison to p one, % of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k n) leading to str resistance is identified in s ribosomal protein s p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain - gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided x ( p) and x ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) overall genome coverage. assembled genome sequences of p and - strains included and scaffolds > bp with a calculated genome size of bp and bp, respectively. the gc content was %. both draft genomes have been deposited at genbank (jojx . for p and jhud . for [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b and safr- ) revealed the max. ani value ( . % and . %, respectively). b. pumilus sh-b strain has been used as a reference for snp calling in strains p and - . snps for the p strain and for the - strain were classified as nonsynonymous variants. radical nucleotide substitutions from the - genome were not found in p genome. among them, the mutation in the codon of rpsl gene (coding s ribosomal protein s ) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a , that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a antibody is only m À min À , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a . the hybrid qm/mm method allows to study the reaction mechanism of interaction of a with paraoxon. it was shown that the reaction proceeds via the classical s n mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr- to paraoxon. qm/mm approach determines position for mutagenesis -leu- in light chain. for one of the mutant in this position -leu argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu arg exhibits a -fold increase in k /k d compared to a ( m À min À vs. . m À min À ). double mutant leu arg/ser ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu arg still binds paraoxon three times better, that may be due to the fact that the serine in position increase the nucleophilicity of tyr . thus, our results are in line with our computed predictions. this work was supported by rfmefi x . due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is x pixels and field of view (fov) is lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on % raw meat and % offal tissue. calculations proofed with imagejÒ. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday september : - : mechanisms of pro-inflammatory diseases p- . . - the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als mg/kg (oral) group, tor+als mg/kg (oral) group, tor/det+als mg/kg (oral) group, tor/det+als mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated °clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il- b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als and tor/det+als groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak project, coded s . ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type diabetes was induced in male c bl/j mice by i.p. injection of high-dose stz ( mg/kg b.w.). after weeks of stz-induced diabetes animals were treated with/without d ( iu/mouse per os) for weeks. blood serum ohd was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum ohd level, indicative of d deficiency. significant alterations in d -endocrine system were found as is evident from reduced expression of cyp a , cyp r , vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of ohd content and partial normalization of liver tissue structure were achieved after d treatment. d administration partially normalized expression of cytochromes involved in d metabolism and hepatic pro-inflammatory factors. d treatment prevented overexpression of rel-a and phosphorylated p /rel-a translocation to hepatocellular nuclei that is most likely mediated through , (oh) d and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d against diabetes-induced liver injury. p- . . - lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c c muscle cells s. savranoglu , h. ipek , s. arslan , h. delig€ oz , a. r. t€ ufekc ßi , i. demirtas , t. boyunegmez t€ umer graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c c myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt- (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine ( ) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c c cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser )/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the -deoxyglucose uptake assay. results: eae at and lg/ml significantly increased the glucose uptake and % compared to insulin resistant control cells. metformin at mm increased this parameter up to %. eae increased pakt ser /akt level - % and lpl expression - % for and lg/ml, in insulin resistant myotubes, respectively (p < . ). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt molecule through ser phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: t ) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, : n À ) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of . mg/kg ( mm/kg) daily during weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day . we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. - - ), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. - - mol_a. p- . . - circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd +/ cd + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd +/ cd + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd +/ cd + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥ . pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < . ) as well as high levels of aopps-alb, apoptotic emps (cd + /cd + /an-v + ), and nt-probnp (p = . , p = . , p = . respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd + /cd + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: patients who were diagnosed with obstructive sleep apnea and healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = . , p = . , respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = . ). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p- . . - decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type (dmt ) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt patients. the cross-sectional study included dmt patients, treated at the primary health care centre zenica from december to april , distributed into groups according to glycated hemoglobin (hba c) values: a (n = , hba c ≤ . %) and b (n = , hba c > . %). complete blood cell count, fasting blood glucose (fbg) and hba c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss . . p values fasting blood glucose and hba c were significantly higher in the group b compared to the group a (p < . ). there was no significant difference of nlr, plr and rpr between the groups (p = . ; p = . ; p = . , respectively). significant correlation of inflammation-based scores with fbg and hba c was found only between plr and hba c in the group a of dmt patients (r = . , p = . ). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type diabetes mellitus (t dm) and determine whether or not these are reliable markers for diagnosis. we evalueted people's results who were carried out oral glucose tolerance test (ogtt). acording to -h values of plasma glucose in the ogtt; . group (normal glucose tolerance: ngt): under mg/dl (n = ), . group (prediabetic: impared glucose tolerance (igt)): ranging from mg/dl to mg/dl (n = ), . group (firstly diagnosed diabetic by ogtt): above mg/dl (n = ). . group is clear diabetic without complication (taking treatment) group (n = ). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = . ) and plr (p = . ) values. nlr values are significantly higher in prediabetic ( . it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type diabetes mellitus (t d). this study aimed to analyze the long-term impact of altered metabolism in female t d patients at the level of mediators of inflammatory response. this study included femalet d patients and control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il- , were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = . ), crp (p = . ), il- (p = . ), leukocytes (p = . ) and sedimentation rate (p = . ) in female t d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba c (p = . ), il- and glucose ( . ), il- and bmi ( . ). in our study, female t d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il , crp and sedimentation. other studies conducted in female population associated elevated levels of il- and crp with t d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure , (oh) d levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included renal transplant recipients ( female, male; mean age: . ae . ) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month . serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, (oh)d and , (oh) d levels were measured. gfr values were estimated by ckd-epi. plasma , (oh) d levels were determined in a lcms- triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss . software was used for statistical analysis. results: although plasma , (oh) d levels significantly increased (p = . ), we did not find any significant differences for serum (oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = . , p = . for alp), we observed significantly higher calcium and gfr values (p = . ). vitamin d insufficiency was present . %, deficiency . %, severe deficiency % before transplantation, insufficiency was also seen . %, deficiency %, severe deficiency . % after transplantation at month . conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type diabetes (t d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type diabetic subjects. this study involved newly diagnosed type diabetics and healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. ra patients, as patients and healthy volunteers with mean age of - were included in the study. serum levels of vegf, angiopoetin- and tie- were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang- and no levels were significantly higher in the as group ml, p < . ; p < . ; p < . ). no differences were found between as and ra for tie- (p > . ). vegf, ang- , tie- and no levels were positively correlated in both as and ra patients (p < . ), but no correlation was detected between clinically activation index das- , basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > . ). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang- in as patients was sufficient (auc ( % cl): . , p < . ). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang- measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately - h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with patients and healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at g for min at °c. the serum samples were stored at À °c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p- . . - neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of patients with polycystic ovary syndrome and healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count ( . ae . vs. . ae . , p < . ) and platelet count ( . ae . vs. . ae . , p < . ) were higher in patients with pcos compared to the control group. lymphocyte count was . ae . in pcos patient and . ae . in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls ( . ae . , . ae . p < . , . ae . , . ae . p < . , respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b , caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b were measured in c bl mice with alf induced by single i.p. injection of aom ( mg/kg of b.w.) and after neutralization of tgf-b induced by single i.p. injection of ab-tgf-b ( mg/kg) h before aom injection. in alf mice, blood tgf-b was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s ) fraction by~ % and %, respectively, but was slightly depressed in the membrane (p ) fraction by~ % and~ %. aom induced an increase of postsynaptic proteins: psd- and nnos by~ % in p fraction. tgf-b neutralization resulted in a reduction in the expression of presynaptic proteins by~ % in s fraction and~ % in p fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd- and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b neutralization appear to be confined to the presynapse. % of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox- and cox- ) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox- and cox- , they have serious side effects on gastrointestinal system. therefore, new highly selective cox- inhibitors having fewer side effects are needed. in the present study, selective cox- inhibitory activities and cytotoxic effects of new series of -benzoxazolinone and thiazolo [ , -b ]- , , -triazole derivatives previously synthesized as specific cox- inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco- cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il- b, il- , il- and il- levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl- and tgf-b levels of patients with aa. patients with aa and healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl- and tgf-b levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b levels in the patient group were significantly lower compared to the control group whereas serum ykl- levels were significantly higher in patient group. tgf-b levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl- level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl- levels in aa patients suggests that ykl- plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin (il- ), and soluble intercellular adhesion molecule- (sicam- ) in comparison with control patients. cimt values are similar in psa ( , ae . *) and psc ( , ae . ) patients. however, both of them were significant increase compared with control ( . ae , ). regarding inflammatory markers il- serum levels in patients with aps was higher than pcs ( ae . ) and healthy controls ( , ae . ) but the difference did not achieve statistical significance (*p > . ). on other hand mean of sicam- , value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > . ). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < . ) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam- crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p- . . - protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr and increase in dcr expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type diabetes (t d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il- beta (il- b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il- b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit- mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il- b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at and h by mtt assay. trail ligand and receptor expression profiles on nit- cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit- beta cells at h, while increasing cell viability following il- /ifn/trail and il- /tnf/trail combined applications. substantial levels of death receptor (dr ) expression were detected on nit- cells before applications, yet it displayed decreased levels at h of trail treatment. lower levels of decoy receptor (dcr ) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il- b increased cell viability in nit- beta cell lines along with reduction in dr death receptor expression and an increase in the decoy receptor dcr expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u plus . microarrays, peripheral blood cell mrna profiles of bs patients (b) and matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of and for the chosen fold changes of . and . respectively (p ≤ . for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin- and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin- and homocysteine, we also investigated whether there is a correlation between these parameters. obese and healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin- and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c -esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c _ inh deficiency. materials and methods: people with c _ inh and people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss . was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = . ), looh (p = . ) and sh (p = . ). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: + . ) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, % fructose administered group, cape group, % fructose + cape administered group and ethanol group. after weeks, liver oxidant and antioxidant status, and blood tnf alpha, il- , and il- , tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il- , il- levels were found to be increased in fructose group compared with the control group (p < . ). in liver tissue of % fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul , n. uzun , e. c ß inar tanriverdi , h. € un agri ibrahim cecen university, agri, turkey, nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of pregnant women were included in this study. pregnant volunteers were divided into two groups ( healthy pregnant women and pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin- (il- ) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il- levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il- levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty -day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents hrs after lps injection. the rats were sacrificed at hrs after lps or normal saline injection (n = ). hepatic tissue tnf-a, il- , icam- and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il- , icam- and pdgf levels were statistically significantly higher than in controls (p < . ). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il- , and pdgf when compared to sepsis group (p < . ). there were no significant differences in the icam- levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il- , icam- and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of mg/kg during weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival ( %) compared to rats without treatment ( %). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel ( / nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red ( nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic doses of dtx, cis and deg. on the a and h nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v. . . ) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than . were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic values). these effects were remarkable in p -mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the % of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic doses of dtx, cddp and selenium on the a and h (p mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic concentrations at lower doses in both nsclc cells. however, cddprelated ic concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor (her ) is overexpressed in % of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her overexpression. in breast cancer cell line mcf , her overexpression (mcf-her ) results in mitochondrial dysfunction with low mitochondrial membrane potential (Δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf -her cells than vector control (mcf -vec). additionally, mcf -her cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf -her and decreases in knockdown of her in hcc cells that is her positive breast tumor cell line. based on these results, we conclude that there is a link between her overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed microrna genes deregulated by methylation and new potential target genes of mir- and mir- - p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with references (rnu , rnu ) and paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir- b- p, - - p, - - p, - a- p, - b- p, - - p, - - p, and - - p (p ≤ . , fisher's exact test) in bc. we observed mirna genes to be hypermethylated and mir- hypomethylated. hypermethylation for of these mirna genes was shown for the first time: mir- , - , and - ( - % of bc cases). a significant correlation between methylation and expression alterations was revealed for mirnas with downregulation: mir- b- p, - - p, - - p, - a- p, and - b- p (spearman's correlation coefficient (rs) was in the range À . to À . , p ≤ . ), and for mirnas with both scene (down-and upregulation) as well: mir- a- p, - a, and - (rs = À . to À . , p ≤ . ). comparative analysis of the data on expression alterations of mirna genes and protein-coding genes, which were predicted as targets by mirwalk . , revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir- /rhoa, mir- /rassf (a), mir- - p/dapk (rs = À to À . , p ≤ . ). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant - - from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: -year survival is about %. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of paired (tumor/ normal) ccrcc samples. we also used post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and paired ccrcc samples. we observed mirna genes (mir- a- /- /- , - - , - - , - b/c, - - , - a, - ) to be hypermethylated, (p ≤ . , fisher's exact test), mirna genes to be hypomethylated and mir- a with both scene (hyper-and hypomethylation was detected). methylation of mirna genes (mir- a- /- , - b/c, - - , - , - a, - a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir- - , mir- a, and mir- significantly correlated with metastasis presence (p < . , fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir- - and up-regulation of p protein-coding genes: rarb( ), rhoa, nkiras , and chl , which were predicted as targets by mirwalk . (spearman's correlation coefficients (r s ) was in the range . - . , p ≤ . ). in conclusion, novel supposed interactions of mir- - with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir- - , mir- a, and mir- could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm and eno genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset ( k illumina microarrays) we supposed that cpg methylation of pgm and eno promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm :~ bp and bp upstream tss; eno :~ bp downstream tss) that are frequently methylated in hc samples (up to %, / ) with down-regulated pgm and eno expression. thus, we demonstrated frequent and significant pgm and eno down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm and eno gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mК- . . from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of sprague dawley rats were randomly divided into six groups. rats were administered with mnng ( lg/ml) by oral gavage on days , and to initiate stomach cancer. during the experiment, rats died and those surviving were sacrificed in the rd, th, th, th and th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains rat which are given only food and water for months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows . , . , . , . and . for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < . ) and of groups iii, iv and v (p < . ) and also were lower than in the control group (p < . ). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: / ). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep- ), by considering its anticancer features. water extract of turkish propolis (wep, - mg/ml) and ethanolic extract of turkish propolis (eep, . - mg/ml) were prepared and incubated with hep- cell lines ( , , and h). mmp was investigated with a flourometric method by using dioc ( , -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep- cell series according to control ( concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf (nuclear factor ) family. there are few data suggesting that the nf factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf -b isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < . . these data suggest the involvement of the nf proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal ( ; ) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than % of chronic myeloid leukemia (cml) cases. epigallocathecin- -gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k- cells. the cytotoxic effect of egcg on k- cells was determined in time and dose dependent manner by wst- analysis. total rna was isolated from k- cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac , hdac , hdac gene that supports cell proliferation was down-regulated . , . , . folds in k- cells treated with ic dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. patients with cll and healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = . ) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = . ). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = . , p = . ). cut-off value of . absu for anti-ca i was associated with % sensitivity and % specificity and a cut-off value of . absu for anti-ca ii was associated with % sensitivity and % specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained noti-clones associated with chromosome genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, noti-sites with high ( - % of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh l , vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn , foxp , prickle , etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras , itga , lrrc b, and ctdspl genes. several genes with high hm/d frequency (aldh l , ephb , itga , and ropn ) were tested for expression alterations using qpcr. frequent ( - % of cases) and significant (> -fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh l geneon the average -fold mrna level decrease in % of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh l , ephb , itga , and ropn genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant - - from the russian science foundation. functional hypermethylation of a number of chromosome genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained noti-clones associated with chromosome genes, were used for comparative (tumor/normal) hybridization of dna from paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, noti-sites with % and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga ), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn , nbeal , and ube e ). the highest hm/d frequency was observed for ankrd , nkiras /rpl , itga , cmtm , and gor-asp /ttc a genes - - %. expression alterations were evaluated for genes with high hm/d frequency (plcl , prickle , and ppp r a) and significant mrna level decrease (> -fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl , prickle , and ppp r a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from % to % of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost %. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons - , which corresponds to a control group consisting of people living in the city of balkhash. on the whole, molecular genetic studies have shown that examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of individuals including patients and healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than . ). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than . ). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than . ). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately % of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy y cells was investigated by crystal violet staining. ec was found to be . %(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p mrna levels. on the other hand, bcl- , bcl l , erk- , jnk, caspase and mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p and pten remained unchanged. these results indicate that another effector caspase such as caspase or may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor (irf ) is upregulated in melanomas, and genome wide association studies linked variation at irf locus with skin cancers. irf was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf in various hematopoietic cancer types. the aim of this study is to investigate the role of irf in melanoma cell lines. lentiviral vectors were used to reduce irf levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf knockdown (gfp positive cells) over melanoma cells with normal irf levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf levels. cell cycle profiling showed increased g /g and decreased g /m levels in irf knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf levels. taken together, these results indicate that irf knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf . these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. , -dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, adult wistar male rats weighing - g were randomly selected and divided into eight groups. mg/kg body weight (b.wt.) morin and . mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with . mg/kg b.wt. dmba three times during the administration period ( days). cytochrome p a (cyp a) associated -ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were ae , ae , ae , ae , ae , ae , ae and ae pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were ae , ae , ae , ae , ae , ae , ae and ae nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad and smad by the type i receptor and formation of smad / / heteromeric complexes that enter the nucleus to regulate transcription. -deazaneplanocin a is an inhibitor of the histone methyltransferase ezh . we aimed to reveal the effect of -deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg cell line. hepg , a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with % fbs. the cells were seeded the day before -deazaneplanocin a administration and then the cells were treated with lm -deazaneplanocin a for days. expression levels of genes were analyzed by roche lightcyclerÒ . gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < . was accepted as statistically significant. -deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads - in hepg cells (p < . ). we also found that -deazaneplanocin induces apoptosis in treated cell line (p < . ). as a result, -deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf- compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > . /l. differential leukocyte counts were obtained by bc (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < . ). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = À . ; p = . ). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy y cells. ec was found to be . % (w/w) essential oil for sh-sy y cells. neuroblastoma cells were incubated at °c for h. after h, cells were harvested for further studies. bax and p mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl- , bcl l , casp- , casp- , erk- and jnk expression were significantly downregulated. unlike these proteins, p and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp- and casp- . additionally, erk and p mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy y cells. p- . . - the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment ( mg/kg b.w., i.p.), ng and ng-ox ( mg/kg b.w., i.p daily for days) alone treatment; cis + ng ( or mg/kg b.w., i.p daily for days) and cis+ng-ox ( or mg/kg b.w., i.p daily for days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over . million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at À . v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c. . . . ) with glutaraldehyde could be achieved over a wide range of relative mole ratios in mm phosphate buffer at ph . , glutaraldehyde concentration of % . . discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u cell line. changes of apoptosis and cell cycle in u cell line exposured to ic dose of astaxanthin ( . nm- lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u cell line to which astaxanthin was treated or not. ic dose of astaxanthin was calculated as . lm at h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of % in u cells treated by ic dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u cell line and astaxanthin caused an increase of g /m phase arrest ( . fold) and s phase arrest ( . fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g /m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor (irf ) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf expression levels are elevated in several lymphoid cancers, and irf is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf -linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf expression levels. however, there are no published studies about irf target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il- , tnf-a) in non-small cell lung cancer (nsclc). material and methods: patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi ; ifn-c, il- , tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c . post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = . , p = . , %ci; . - . ). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = . , %ci; . - . , p = . ). after crt, mean alt level was determined as . . there was survival difference in nsclc patients with high post-crt alt (hr = . , %ci; . - . , p = . ). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l ) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic concentrations for h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. -aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < . ) through both cells treated with c- and c- . only c- decreased methylation level on l but not on hela. consequently, c- and c- caused demethylation on hela cells similarly with -aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c- and c- might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il- b and il- , causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il- b signaling increases activity of nf-kb pathway. at the same time, il- , triggers jak-stat signaling pathway, which effector is stat . nf-kb and stat activity facilitates hyperexpression of mir-nas mir- , mir- and mir- as well as down-regulates expression of mirnas mir- / , mir- and let- . this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac / / / and sirt / genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir- , mir- or mir- . also, these mirnas can silence ezh , mll, mll , nsd , setd / / , smyd , suv h genes encoding histone methyltransferases. mirna mir- suppresses gene encoding de novo dna methyltransferase dnmt b. at the same time, down-regulation of mirna mir- / can allow hyperexpression of gene encoding acetyltransferase elp . these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd and bd ). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd -selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp , and ctdsp , plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp and ctdsp dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir- - - microrna cluster in co-regulation of ctdspl/ / genes in nsclc was predicted. the significant ( -fold on the average) and simultaneous decrease of mrna levels of ctdspl/ / genes was revealed in the majority of nsclc samples ( %, / ). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = . - . , p ≤ . ) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir- - - family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples ( %, / ) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir- - - micrornas in the regulation of rb protein activity via inactivation of ctdspl/ / in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for were reviewed. for , a total of patients and tumor marker requests were included. carbohydrate antigen - , cancer antigen , cancer antigen - , prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of , both positive tumor markertest resultsratio and the positive patient ratio were %. in the patients group with increased marker levels, % of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, % patients with diagnosed cancer history in remission. the ratios of positive tumor markers were % forca - , % for ca , . % for psa, %for ca - , . %for afp, and . % for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct colon carcinoma cells. ic doses of resveratrol were determined by wst- assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd , fra , ppard, egfr, birc , pcna, mcl , stat , fos, jun, p , atf , trail, puma, gadd a, rb , faslg, tnf, socs , stat gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol ( lm) causes decrease in cell viability and increase in apoptotic cell death in hct p (+/+) and hct p (À/À) cells significantly (p < . ). the fold changes of the gene expressions have shown that resveratrol has significant (p < . ) and different effects on the expressions of the genes related with the existence of p in hct cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group and group , respectively. hypermethylation was detected in atm, cdkn a, and gata genes. mortality rate was ( . %) in group and group (p > . ). mean -years survival rate in group was ae months and mean -years survival in group was ae months (p > . ). positive median lymph node count was ae for group and ae for group and the difference was statistically significant (p < . ). frequencies of perineural invasion and lymphovascular invasion rate in two groups were % (p > . ). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for % of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p mutation, which is mutated % of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p (+/+) and p (À/À) hct colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate ( mm) and disodium pentaborate decahydrate ( mm) significantly causes nearly % reduction of cell viability at h (p < . ). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi k/akt, map kinases and many epigenetic regulators. satb is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb expression in nsclc cells. we used a and h cells as a model to evaluate the effects of the crosstalk between p and akt on emt of nsclc cells. therefore, cell culture, inhibition of p activation via sb , transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb , wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb , p-p , p , p-akt and akt was examined in a and h cells by western blot analysis. we observed that e-cadherin and satb are downregulated in a cells (highly active p , lowly active akt) compared to h cells (lowly active p , highly active akt), suggesting that e-cadherin and satb are associated with the crosstalk between p and akt pathways. our results demonstrated that p inhibition in a cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p inhibition upregulated satb expression, and reversed emt in a cells. furthermore, alone satb knockdown is sufficient to induce emt, and prevented the effects of p inhibition on emt. all these results strongly indicate that the crosstalk between p and akt pathways might determine satb expression and epithelial characters of nsclc cells, and satb is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p and akt signalling pathways could regulate satb expression. this work was supported by tubitak ( s , z ). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron vntr and exon -g t (glu asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. ) and bioenergetics (pallotta ) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d -pyrroline- -carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put p and put p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta ) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put p/put p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group ), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dΨ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. (pallotta et al. , (pallotta et al. , pallotta ; di martino pallotta ) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : patients who were diagnosed with colorectal cancer between january and january ; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was . . cancer-specific survival was not significantly different between the high-and low-lmr groups (p = . ). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: . ; % confidence interval: . - . ; p = . ). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment and treatment . . ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment groups, and the treatment group were given mg/kg and mg/kg dose of luteolin, intraperitoneally, for a days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < . ) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < . ). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around - % of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu- , snu- and snu- than the well-differentiated hcc cell lines such as huh- , hepg and plc/prf/ . besides, expression of txnip was examined in non-hcc and hcc tissue samples by immunohistochemical staining. txnip positivity was observed in % of well and % of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk- b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak kinase (ag- ) on myocardial infarct size and the jak /stat signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia ( % o , weeks, h/day) and part of them recieved ag- ( mg/kg) min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak /stat signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak inhibitor ag- caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak , stat and some other proteins involved in cardioprotection (akt, erk / , gsk b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p- . . - detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection ( mg/kg, twice a week) following the unilateral nephrectomy in male -week-old wistar albino rats for weeks. blood pressure was measured by using tail-cuff method. gw ( mg/kg/day, i.p.) was administered last week. expression of various markers (grp , perk, p-perk, ikb-a, nf-kb p , tnf-a, bax, bcl- , mmp- ) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp and p-perk were increased by hypertension and gw treatment reversed them. hypertension-induced increase in nuclear nf-kb p expression and decrease in ikb-a expression were reversed by gw treatment. while bcl- expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp- expression was augmented and gw treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw treatment. these results suggest that lxr activation by gw restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph ( . mg/kg/day or mg/ kg/day, per os) from p -p . chronic mph treatment ( mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin- and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin- and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin- , with no alterations in claudin- . overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs directly interacts with and interferes in opioid receptor (or) signaling. rgs is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs can regulate transcriptional responses mediated by the stat b transcription factor. isolated neural stem cells from rgs À/À mice were immunostained for the mitosis marker ph and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro a cells stably expressing rgs . the functional assays of stat b transcriptional activation were performed in hek expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs blocks stat b phosphorylation and transcriptional activation by interfering in stat b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs results in increased mrna levels of stat b target genes such as the members of the bcl anti-apoptotic family bcl- , bcl- and bcl-xl. this upregulation of stat b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs in stat b mediated transcriptional responses and a novel selective role of rgs in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph - molten globule structures was formed while the pre-molten form took place under ph . in our tests at ph . when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph . in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [ , ] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel- (vdac- ), mitochondrial-plasma membrane cofilin and caspase- activity was observed in the hippocampus of ka treated rats. administration of mi h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca and ca subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references . r. . neuroscience letters, vol. , no. , pp. - . . r. solomonia, et al; . cell. mol. neurobiol. vol. , no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic values of . lm and . lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab / . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin- (ps ) with - mm azure b or vehicle for h. to determine the effect of azure b treatment on ab / levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab and ab levels with a dose range treatment of azure b in ps cells. ab levels were reduced by . % in lm and . % in lm azure b-treated cells when compared to control. additionally, ab levels were decreased by % in lm and . % in lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy y) used as a tumoral model or differentiated to a neuronal phenotype. pink role in this process was analyzed using genetically engineered pink deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p- . . - intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment ( - ) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage ( - ) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal - dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included female day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during weeks. the rats in the exercise group swam for weeks. after weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function ( ) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain ( ) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad ( ). we initiated a study to screen their acetylcholinesterase (ache, ec . . . ) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic values in the range of . - . nm for ache. this study was financed by turkish research council-tubi-tak (kbag z ). p- . . - f -a h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut , a. sen , d. doyduk , y. yildirir department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, gazi university, ankara, turkey fty , a sphingosine -phosphate (s p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty , namely f -a h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f -a h along with fty using pgl . luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty treatment at non-toxic doses. it was found that f -a h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty does. furthermore, f -a h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f -a h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab - ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a nicotinic acetylcholine receptor (a - nachr) domain resulted in astrogliosis, decrease of a nachr density, accumulation of ab - in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c bl/ mice were injected with lps, immunized with recombinant a - or a - endoglycosidase treated to remove carbohydrates. two immunizations with week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a - -specific antibodies and brain preparations were studied for a nachr, ab - and il- levels. the original a - ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a - immunization with 'deglyc' a - did not stimulate il- elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a resulted in similar a nachrs decrease and ab - accumulation in brain and significant episodic memory decline comparable to those after lps injections. a nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a nachr density caused by a - -specific antibody is critical for ab - accumulation and episodic memory impairment while pro-inflammatory capacity of a - -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using , -diphenyl- -picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl , the cxcr ligand. we aimed to determine whether snp rs located in the promoter of cxcr gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr promoter activity. mef c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr promoters containing different rs alleles were estimated using luciferase reporter assay. we determined that minor rs allele creates functional mef c-binding site within one of the regions required for the basal activity of the cxcr promoter. cxcr promoter containing minor rs variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef c as the main regulator of rs -dependent modulation of cxcr promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d receptors (d r) and histamine h receptors (h r) form heteromers through which h r ligands can inhibit d r function. d rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d r and h r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d r and h r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk / phosphorylation and cell death in cortical slices. endogenous d r-h r heteromers were detected in rat and mouse cortical slices, where h r ligands decreased d r signaling (erk / pathway) and were also able to block the cell death induced by overstimulation of either d r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d r and h r form heteromers with nmdar subunit a in the presence of subunit b. d r-h r-nmdar heteromers were detected by bret with bifc. endogenous d r-h r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d r, h r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d r-h r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h r ligands act as a 'molecular brake' for d r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with weeks old total male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for s to co and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at À °c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi- and cd . changes in tight junction (tj) proteins (occluding and zone occludin- ) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi- staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec . . . ) is a zinc metalloenzyme that catalyzes the reversible reactions of co and water. carbonic anhydrases (cas, ec . . . ) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose- b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained . m tris-so ph . , r-nitro phenol in ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: rats were used. for sit, animals were kept individually for month and for pst, rats were exposed to dirty cat litter for min at the first day of month stress. flu was given ( mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < . were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < . ). flu restored anxiety scores in both sexes (p < . ) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < . ). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < . ) and pst (p < . ) in both sexes. female rats had % lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < . ). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein (hapln ) is one of the major elements of pnns. hapln interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln were observed. however, the role of hapln in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln during differentiation using pc cell line as a neural differentation model, derived from rat pheochromocytoma. after pc cells were stimulated to differentiate into neurons by nerve growth factor on days , and ; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln and survival motor protein (smn ) network analysis were performed in-silico. as a result, we analyzed fold increase in hapln mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln level suggests that, hapln is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln and smn through fibulin (fbln ) was detected. fbln was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln and fbln in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf . the risk of malignancy is increased in nf , wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf , we considered the mesenchymal stem/stromal cells (msc) carrying the nf mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf -mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen . mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of g) chronically intoxicated with hepatotrophic toxine of taa. every weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by % in the experimental group. in the experimental hepatic x-amidase activity decreased by % compared to those in the control. the average x-amidase activity in the blood serum ( . nmols/ mg/min) and in the brain ( . nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about % higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached %. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi x ). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp b gene. atp b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t i, p.v i and p.r g-fsx in yeast lacking the ccc gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t i mutation mainly affects the liver and the nervous system, p.v i mutation affects the nervous system, and p.r g-fsx mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp b, we cloned human atp b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp b genes were transformed into yeast cells lacking the homologous ccc gene for functional comparison. first, we analyzed the expression of atp b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp b gene in ccc d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t i, p.v i and p.r g-fsx mutations cause functional deficiency in atp b functions and suggest that these residues are important for normal atp b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: z ). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen , , e. erem faculty of medicine, ege university, izmir, turkey, center for continuing education, ege university, izmir, turkey, recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. students (in rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was . / , that increased to . / in the posttest evaluation. % of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was % in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates ( , , lg/well) and lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by % sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < . was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv . voltage-gated potassium channel kv . belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv - subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s -s linker at the inner mouth of the pore. here we present an improved d structure of the truncated human kv . channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv . channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag channel, full-length shaker potassium channel kv . , c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a -year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of graduation. currently, there are students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube : z (becton dickinson and company, franklin lakes, nj, usa); tube : ayset (lot , turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au ) and randomised samples stored at - °c for and h. th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at , and h were statistically no significant (p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' ( / / - / / ) (search # ). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search # ) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search # revealed articles; of these were clinical trials, were observational studies. human studies were while animal studies were . overall, there were review articles and meta-analysis in the field. search # revealed articles of which were clinical trials, were review articles, were meta-analysis. human studies were and other animal studies were . the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi kinase, pi-dependent kinase and akt. then, we found the d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopÒ software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of ptb ( s, msc, phd theses) . % was in natural sciences while . % was in health sciences. the theses output-growth measured by the compund annual growth rate was % over the -years. the top clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants ( ), endocrine-metabolism ( ) and enzymology ( ) were the top research areas in ptb, followed by genetics ( ) and cancer ( ). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february , organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for days was realized with participants in total, of which from universities; representatives of their companies; from chamber-institute-public establishments and of which from public hospitals. of the participants were from izmir, of them were coming from out of izmir. conclusion: at the end of the symposium, % of the participants gave feedback. among the feedback selected; . % of the participants evaluated the symposium overall, as successful. . % of them found the symposium successful with regard to its scientific content. their feedback were . % positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and . % of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a item online survey with sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the -day period, ms students (in the thesis phase) answered the questionnaire (female: . %, male: . %). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work ( %), visits with their mentors ( %) and theoretical lessons ( %). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only % and % sufficiency, respectively). communication skills ( %) and team work ( %) were rated as the highest professional competencies followed by literature search and research planning (both %). it was striking that ms perceived themselves as quite insufficient in scientific writing ( %), data analysis ( %) and project writing ( %) while proficiency in english ( %) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority ( %) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu- phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached phd graduates who had graduated from deu graduate school of health sciences between and from different departments via e-mail. the survey was included questions, which were prepared in the light of the existing literature. among the phd graduates, ( %) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (% . ) are well satisficed from the education they had taken. beside this, interestingly we found that % . of graduates prefers staying in the academic positions and % . of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn and itsn are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using web servers we analyzed utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir- , mir- , mir- , mir- / , mir- , mir- and mir- in utr of itsn -s, predicted by servers, mir- predicted by servers for itsn -l, and mir- , mir- / , mir- , mir- and mir- predicted by - servers for itsn -l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir- , mir- , mir- / , mir- , mir- and mir- were highly enriched for all analyzed pathways. using regrna . and scan for motifs services we predicted types of different regulatory elements in utr of itsn and itsn : k-box and brd-box, musashi binding element for rbps musashi and musashi , gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn -s regulation by micrornas we cloned utr of itsn -s into luciferase reporter vector and transfect hek cells by this construction and mir- a, mir- a and mir- a. for mir- transfected cells, we found - % decrease of expression of itsn -s utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir- target site in utr of itsn -s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. % of stroked patients lose their life in year, and remaining / of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir- a, - , - , b, - , , - a and let- f of ischemic stroke patients and to investigate role in predicting prognosis methods: patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and healthy individual were included in the study. after stroke patients' blood samples were taken periodically in st day, st week, and rd month, and at the same time nihss and mrs scores were determined. set mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke st day peripheric blood levels of mir- a,- ,- a and let- f were significantly low; when st week and st day serum records were compared there was a significant increase in mir- level; and when st week and rd month records were compared we noted that there was a significant increase in mir- a,- and let- f levels. from prognosis point of view; after ischemic stroke measurements showed that mir- b in the st day, mir- a and mir- in the st week showed positive significant correlation with rd month mrs scores (p = . , p = . , p = . , respectively) conclusion: according to the outcomes of this study, after stroke st day mir- b, st week mir- a and st week mir- levels can be stipulated to use in predicting patients' rd month prognosis p- . . - inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [ ] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing -nucleotides of a random region. after rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that -fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [ ] hunsicker, et al. ( ) an rna aptamer that induces transcription. chem. biol., : - . p- . . - microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyÒ mini-kit. the expression profiles of mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir- - p was expressed only in aorta. mir- - p and were expressed both aorta and whole blood. mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > ). mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir- - p, mir- - p and might be a potential for cardiovascular disease development. also mir- - p and might serve as novel non-invasive biomarkers for cad p- . . - mir b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc- and miapaca- cells transfected with mir b mimic. our data revealed that mir b led to decrease the cell viability depending on enhanced mir b doses, which are , , and nm, as the ratio of % , , and , respectively, in miapaca- cells and as the ratio of % , , and , respectively, in panc- cells compared with control condition of each cell. this inhibition mediated by mir b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir b was more effective than gemcitabine. based on our findings, it is clearly shown that mir b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant s . breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her + breast cancers, which has role in approximately % of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her + breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt and skbr cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that out of the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t( ; ) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k cells were treated with lm (ic dose) res during h and cytotoxicity was evaluated by using wst- assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir- - p level . fold and significantly downregulated hdac gene expression (p = . ) and upregulated p / sqsmt gene expression (p = . ), according to the control cells.p /sqstm interacts with lc and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac and p /sqsmt gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac dependent induction of gene associated with autophagy like p /sqsmt and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir- , hsa-let- a, hsa-mir- b and hsa-mir- . thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let + , let + b, let + , + b, + , + let + b, + let + b. the cell viability was detected by wst- kit in a well plate elisa reader. cells were seeded as per well, mirnas incubated with cells for h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however + b and + combination increased the cell viability dramatically. on the other hand let + b and only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein (sip ), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip cernas listed at competive endogenous mrna database (cerdb), transcripts (pten, zeb , ptch , creb , acvr b, enah, robo , erbb , e f , foxo , rictor, klf , ets , cdk ) sharing at least common mre sites with sip were selected and their expression in hcc cell lines were determined by qrt-pcr. ets was found to be highly correlated with sip in hcc. furthermore, repressing sip expression by shrna in hcc cells resulted in decreased expression of ets , pten and zeb . our results suggest a possible bidirectional and post-transcriptional regulation of sip and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar , , n. belder, h. ozdag biotechnology institute, ankara university, ankara, turkey, comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of crc tumors against normal colorectal mucosa samples, using raw data from e-mtab- and e-geod- (affymetrix microarrays), and gse and e-mtab- (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ . , fold change ≥ . and stringent false discovery rates. mirtarbase and mirwalk . databases were explored to identify validated targets. we found thirty (including mir- and mir- ) and thirteen (mir- , mir- , mir- , etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol- kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan , h. g€ urdal , b. g€ ur dedeoglu biotechnology institute, ankara university, ankara, turkey, department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip ( , , ) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip is generated by hydrolysis of pip ( , ) bisphosphate, which is synthesized by pip k . any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt- was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt- cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip k c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk- -beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl- , crl- , crl- , crl- and htb- ) were evaluated with non-malignant (normal) brain cell line (hcn- ) . determinations of expression level of five mirnas (mir- , mir- , mir- , mir- , and mir- ) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir- , mir- , mir- , and mir- ) were significantly decreased while the expression level of mir- was increased in gbm cell lines according to hcn- cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v . , mir disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn b-as, locus cdkn a/b (ink /arf), chromosome p ) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan . , we predicted target sites for hsa-mir- -p/ -p/ -p/ -p/ -p/ -p and hsa-mir- - p/ in anril utr. then, we used hmdd v . and mir disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir- is implicated to alzheimer disease and mir- to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir- and mir- targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril utr. conclusions: we predicted sites for mir- and mir- in utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different ( . - mg/ml) concentrations for h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir- b- p (- , fold, p = . ), hsamir- - p (- , fold, p = . ), hsa-mir- a- p (- , fold p = . ), hsa-mir- a- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- b- p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- a- p (- , fold, p = . ) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir- b- p plays a critical role in the matrine response. ericd (e f -regulated inhibitor of cell death) is a newly found lncrna. it is located at q . . it has two exons and its transcript size is bp. ericd is regulated by e f (transcription factor ) and modulates the cellular response to dna damage. arid a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, different cancer cell lines, osteoblast cell line and different types of normal human tissues rnas were selected for expression analysis of ericd and arid a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid a had lower expression in u- os (osteosarcoma), a- (glioblastoma) and a (lung cancer). at the same time, ericd expression was lower and arid a had high expression in hfob . (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid a are cell cycle regulated and are commonly regulated by e f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif a and anti-eif c. mir- - p, mir- a, mir- b, mir- - p, mir- , mir- a*, mir- , mir- a, mir- a, mir- b, mir- a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif c immunoreactivitiy was negative in all groups. mir- , mir- a* and mir- a were highly expressed in proliferation phase of fertile group, mir- a and mir- b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over . million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately nucleotide length which regulates the expression of genes by binding to the -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed unique small rna sequences obtained from raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified putative mature microrna sequences belonging to distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. of precursor sequences showing % homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as , a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as . materials and method: for the tissue expressions analysis, human total rna master panel ii (containing different human tissue samples) was used. total number cancer cell lines and normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as were found to be tissue specific. particularly, while vim-as was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as was also found to be differentially expressed in cancer cell lines. vim-as expression was found to be lost in cal , pc , and hct and highly diminished a cancer cells. also, it is found to be highly expressed in bcpap, panc and beas b cells. discussion: results of the current study suggest that vim-as may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as and vim axis can be involved in the formation of lung tumors because vim-as was highly expressed in normal lung tissues and beas b cells and expressed very low levels in a lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately % patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h and pc cells treated by tnf-a ( ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir- a- p, mir- as- p, mir- , mir- , mir- - p, mir- q mimics can induce cellular invasion on h , mir- v, mir- h- p, mir- - p, mir- a- p, mir- as- p, mir- mimics can induce cellular invasion on pc . we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir- q, mir- a- p, mir- as- p, mir- , mir- in h , mir- - p, mir- a- p, mir- as- p, mir- in pc can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir- a- p, mir- as- p and mir- . (this study is supported by tub _ itak grand number s ). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a cells. when we inhibit nf-kb activity by p targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir- as and pten untranslated region has recognition site for mir- as. therefore; we transfected a cells by mir- as. mir- as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir- as. (this study is supported by tubitak grand nummber s ). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a (autophagy and v-atpase inhibitor, . pg/ . ml/ovary) on day of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc -i was not significantly altered throughout pregnancy, that of autophagosome-associated lc -ii increased significantly from day , showed a peak on day , and decreased on day (day of normal parturition). lc -ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day . in vivo treatment with bafilomycin a resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc -ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in . the value of low dose bpa was received as < lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i- therapy, have been examined in patients ( m/ f) with ptc and ( m/ f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at days after i- administration ( . gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp- , mmp- , timp- and timp- were measured by elisa. there were no significant changes in serum concentrations of mmp- , timp- and mmp- /timp- ratio after i- in the two groups. in ptc patients, i- administration resulted in an increase with % in timp- level (p = . ) and a reduction with % in mmp- /timp- ratio (p = . ). in ptc+ht patients it has been observed an increase with % in tgab level (p = . ), % in mmp- /timp- ratio (p = . ) and unchanged timp- serum concentration. tgab titers were positively correlated with mmp- /timp- ratio (r = . , p < . ). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp- /timp- for anti-invasion and anti-migration by augmenting timp- . in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts , and and mmp , and to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, cases have been included in this study, divided into groups according to their pmis: ' - h', ' - h' and ' - h'. from these tissues, western blotting technique is used to analyse the expression of adamts , and and mmp , and . it is determined that when pmi increases; adamts- and amounts decrease. on the other hand adamts- amounts are examined to increased related to the interval., especially on the ' - h' dataset. additionally, considering metalloprotease levels, mmp- and amounts decrease as pmi increases. unlike mmp- and ; mmp- levels increase proportional to the interval, especially on the ' - h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts- and mmp- , proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases and in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp and mmp (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp - c>t and - c>t polymorphisms cause loss of gene function, and mmp - c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp - c>t and - c>t polymorphisms, and mmp - c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from healthy subjects. the risk of abortion was . -fold higher in patients with heterozygous - c>t polymorphism (p = . ). combined genotype analysis showed that the risk of abortion was . -fold higher for patients with normal - c>t polymorphism, and heterozygous - c>t polymorphism (p = . ). functional polymorphisms of mmp and mmp may have a role in etiology of abortion. p- . . - changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses ( . and mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, min and min ischemia-reperfusion. after months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts- and - genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam , - , and - . in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = ), oligozoospermia (os; n = ), azoospermia (as; n = ) groups. adam , - and - protein levels in infertility indviduals were analysed by western blot. adam protein level was . fold lower in the os and as groups than in the ns group. adam protein level was . fold higher in the as group than in the ns group. adam protein level was fold lower in the as group accourding to ns group. we observed no change between protein level of adam and adam of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin- on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck- , timp- and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created patients with diagnosis of cchf. healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc- and timp- levels of patients and a control group were analyzed using the human elisa kits. serum timp- tck- and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp- , tck- and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p- . . - activation of calpain and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase (mmp ) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca + homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine in gene of cystic fibrosis transmembrane conductance regulator (f del-cftr), could be involved in the abnormal presence of mmp in the extracellular fluids of cf patients. pbmcs were isolated from healthy donors and cf patients homozygous for f del-cftr. mmp levels were evaluated following h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp ; conversely, in pbmcs from healthy donors, expression and secretion of mmp are undetectable but both events can be evoked, after h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and h-cultured control pbmcs mmp secretion is prevented by chelation of intracellular ca + and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases and (erk / ). moreover, the rescue of active f del-cftr reduces the extent of mmp secretion, correlating the channel defect to the [ca + ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca + concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp . these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp in fluids of cf patients. p- . . - aebp /aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir , , u. akpulat , i. onbasilar , c ß . kocaefe department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, department of stem cell, institute of health, hacettepe university, ankara, turkey, laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp /aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp is the short isoform that acts as a transcriptional repressor by targeting the ap promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp /aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp /aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp /aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated folds upon the induction of differentiation in c c cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp /aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp /aclp expression peaks up to folds in the th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp /aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp /aclp expression is upregulated up to folds in early time-point samples. these results depict a novel role for aebp /aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus - encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus - extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average % compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a -fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo a remained at the level with expression in the strain with the complete spo a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo b, spo f, spo k, spo j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p- . . - paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for min followed by h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp- and - were performed after h of iri. the effects of paricalcitol on activities and expressions of mmp- and mmp- levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp- and mmp- during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp- and - . p- . . - the changes of matrix metalloproteinase , activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko , o. shaulska , y. kot , g. ushakova , a. shevtsova dnipropetrovsk medical academy, dnipropetrovsk, ukraine, kharkiv national university, kharkiv, ukraine, dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp and mmp activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the wister rats divided to groups were used for the experiment. cdcl x . h o in doses . lg/kg and lg/kg was given to rats intragastrically in drinking water during days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp and mmp , total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium ( . lg/ kg) lead to reduced activity of pro-and active forms of mmp in myocardium ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly) and in serum ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly), but pro-mmp activity in heart was increased ( . ae . au/mgtp compare to the . ae . au/mgtp in the control rats); level of ha was decreased in both tissues ( . ae . lg/ml and . ae . lg/ml compare to the . ae . lg/ml and . ae . lg/ml in the control rats accordingly). high doses of cadmium ( lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp increased from . ae . au/mgtp to . ae . au/mgtp, prommp to . ae . au/mgtp, mmp to . ae . au/mgtp. ha level was increased in serum ( . ae . lg/ml) and decreased in heart ( . ae . lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin- repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin- (il- ) is an il- cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il- implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts in u proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of lg/ml while trypsin and chimotrypsin are used as - mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no - - ). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from to lmol resv and opposite effect from to lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn- . since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in . m kcl (ph . ) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments - of amyloid peptide, fragment - of [gly ]-amyloid peptide and - of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs ( - - ) and sp- . . . ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti , c. bernacchioni , f. scaletti , p. turano cerm (magnetic resonance centre), florence, italy, department of biochemical sciences, university of florence, florence, italy, chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort , t. ozben , a. sansone , s. barata-vallejo , c. chatgilialoglu , c. ferreri sanko university, gaziantep, turkey, akdeniz university, antalya, turkey, cnr, bologna, italy, universidad de buenos aires, buenos aires, argentina, national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at °c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr in glucose homeostasis. drugs targeting gpr may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr inhibition. in addition, gpr takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon and an additional trans double bond between carbons and . dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical -carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from to mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p- . . - trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b -arrestin or gb subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with À m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p- . . - l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev , , v. polovinkin , , , v. shevchenko , , v. gordeliy , , moscow institute of physics and technology, dolgoprudniy, russia, research centre j€ ulich, j€ ulich, germany, institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr has an absorption maximum at nm at ph . and to create more redshifted variants we analyzed available structures of the kr (pdb codes: xtl, xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m , g and s ). the mutants of kr under investigation were: m a, g v, m a/g v, s a, s f, s g, s l, s m, s n, s t, s v, s y. the protein mutants were expressed in escherichia coli c strain, expression was induced by the addition of mm isopropyl b-d- -thiogalactopyranoside. the cells were then washed trice with unbuffered mm nacl or kcl solution. finally, the ph changes in cell suspensions (od = . ) were monitored. ph changes upon the addition of lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s f, s l, s m, s n, s v. the remaining mutants have shown sodium pumping activity and s a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph . ( mm na/na-pi, mm nacl). m a mutant absorption maximum is blue-shifted to nm. g v and m a/g vblue-shift to nm. s a, a potassium pumping variant,red-shift to nm. s g, s yred-shift to nm. s tno change in absorption maximum position. based on structural analysis of kr we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to nm and red-shift up to nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than % drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins - , which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. - - ). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv- ). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in biopsies ( %). c iv deficiency was most common ( %), followed by c i ( %) and c ii-iii ( %). multiple complex deficiency was present in % and isolated deficiency was present in % of the biopsies ( % c i, % c ii-iii, % c iv). cs activity was elevated in % of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in %, % and %, respectively. comparing with histological data, biochemical analysis revealed additional findings in % of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) , resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and groups were included in the study. rats were fed with chows that were given either normal diet for control group ( % carbohydrate, % protein and % fat), high fructose ( % carbohydrate ( % fructose and % starch), % protein and % fat), or high sucrose ( % carbohydrate ( % sucrose and % starch), % protein and % fat). rats were fed with chows for weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba c, glucose, resistin and adiponectin were studied. glut and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < . ). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < . ). there was no significant differance level of hba c, glucose, resistin and glut in rats fed sucrose or hfcs (p > . ). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p- . . - nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy , n. eruygur , e. s€ uleymanoglu biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg +) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg +-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group ). secondly mice were randomly divided in four groups as: group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days, group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days. ozone/oxygen mixture and oxygen were given at a dose of mg/ kg groups ( ) ( ) ( ) ( ) . after treatment animals were sacrificed, and muscle samples were taken and stored in À °c for until the analyses. muscle tissues were homogenized in . m tris-hci and . m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). sdh activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). there was no statistically significant difference among all groups in terms of cs (p > . ) and sdh activities (p > . ). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than . %, so they were scored as . there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of -monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of -monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, -( -{-[ -(octyloxy) phenyl] diazenyl} phenoxy) propane- , -diol ( % mol), was incorporated into the -monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn m phase with lattice parameter . angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at nm we observed trans-cis-isomerization. in the same time the cubic-pn m phase was not changed but the lattice parameter reduced to . angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin , t. cora cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt that is a hnscc tumor cell line and hek- that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf- r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of women ( %), and men ( %). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant . times increase of gpx activity levels; and . times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by . times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than . times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity ( mm) and potential protective effect of different doses of boric acid (ba) ( , and mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+ mm ba, ethanol+ mm ba, ethanol+ mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < . ). however the mda level in the ethanol+ boric acid ( mm) group was shown to be significantly decreased compared with that in the ethanol group (p < . ). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba ( mm, mm) groups were close compared with control groups (p < . ). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid ( mm) groups were increased (p < . ). these results demonstrate that ethanol ( mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co /bicarbonate between metabolizing tissues and lungs, ph and co homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = . ), (p = . ), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = . , p = . ). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = . , p = . ), (r = . , p = . ), respectively. at an anti-ca i cut-off point of . absu, sensitivity was % and specificity %. at an anti-ca ii cut-off point of . absu, sensitivity was % and specificity %. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from healthy control and patients with behc ßet's disease. the mean age for controls and patients were ae . and ae , respectively (p = . ). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v . results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ). lymphocyte counts were lower in patients with behc ßet's disease compared to control group ( . ae . vs. . ae . ) (p = . ). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of neuroblastoma patients ( male, female, median age: months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of healthy children. patients had stage , , or s disease, patients had stage disease. different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir ds (p = . ), and higher expression of inhibitory kir gene dl (p = . ). additionally kir ds genes were more common in patients with early stages (stage , , or s) (p = . ) and kir dl genes were more common in patients with stage (p = . ). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir dl gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir ds can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p- . . - neuroprotective and immunomodulatory effects of urtica urens s. arslan , g. terzioglu , b. kabalay , a. r. tufekci , a. sen , i. demirtas department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco- cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine (cxcl ), and (cxcl ) were determined. whole genome transcriptome analysis was performed by using human ht- v beadchip. extract treatment caused % and % increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly ( % and %, respectively). similarly, cxcl and cxcl mrna levels decreased % and %. transcriptome analysis showed that probes were significantly changed (p < . ). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak t . p- . . - linear low molecular weight a- , -glucan from bifidobacterium bifidum bim b- d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, , ( ) : - ) that bifidobacterium bifidum bim b- d contains low molecular mass ( - kda) a- , glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b- d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, h and c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as - kda linear a- , -glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b- d cells contain linear low molecular mass a- , -glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a- , -glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba c, uric acid, creatinine, ggt, -hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > . ) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < . ). on the other hand, rdw are determined to be significantly lower (p < . ) in the non-diabetic obese group. no difference was detected in -hydroxyvitamin d levels between the control and the study groups (p > . ). non-diabetic obese patients had significantly (p < . ) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < . ). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco , which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco is also known to be present on autophagosomes, and overexpression of fyco can induce the formation of enlarged lc -containing autophagocytic structures. here we demonstrate that fyco knock-down leads to defects in autophagic mb degradation, and that fyco functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p- . . - proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d and b ) and cell cycle controller genes (p and p ). sf cells were treated with different doses ( nm- lm) of hypericine for h and h. a significant cell proliferation was observed in moderate concentrations ( . - lm; % -% ), but at high concentrations ( - lm) cytotoxic effects emerged in sf cells (ic = . m, r = . ). qrt-pcr results revealed that the most proliferative dose of hypericine ( lm) stimulates cyclin d . the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b mrna, whereas it induced expression of p and p genes, and thus apoptosis was observed by dna laddering at the same dose ( lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately months, increasing to months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant, the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f -targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p- . . - leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t( ; )(q ;q ) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k subclones (k -ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k -ir cells are not only resistant to imatinib but also to nd, rd generation tyrosine kinase inhibitors. we demonstrated that k -ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k -ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k -ir cells forms intact tumor spheroids in d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k -ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd + cscs isolated from human osteosarcoma cell line saos- using macs magnetic separation technique were characterized, and their stemness properties under hypoxic ( % o ) and normoxic ( % o ) conditions were compared in two and three dimensional culture conditions. two different d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct / , nanog and sox compared to normoxic conditions in d cultures. although similar effects of hypoxia were observed in d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on d compared to d culture systems. this study is seen as an introduction to develop a more relevant d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd + cscs were isolated from human pca pc cells using a magnetic-activated cell sorting system. pc and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for h. apigenin dose dependently inhibited cscs and pc cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p and p (kip ). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases- , - and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf- in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase- while caspase- , tnf-a and bcl- levels remained unchanged in pc cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp- and - exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi k/akt and nf-kb p / p were significantly decreased after h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. s ). (pi k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi k activity contribute to phenotypic variability. our aim is to characterise pik ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi k activation on downstream output specificity in the cell. we are currently employing crispr/cas mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p a, allowing precise spatiotemporal control over pi k signaling to unravel the extent to which pi kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi k signalling and potentially aid the development of targeted therapies for human diseases of pi k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf as transcription factors, egfr and cdk as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥ ), prostate (≥ ), and colorectal (≥ ) cancer based on assessment of . ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. . ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck , ck ) and the leukocyte-specific marker as anti-cd . all samples were analyzed by bd facs aria iii flow cytometry. in total, patients and healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but - ctcs were detected with ck , , , -based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at °c with % co and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf- and mda-mb- cells. this effect was observed more prominent on mcf- . considering prfe therapy this effect is much more pronounced especially for mda-mb- comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut , glut , glut proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut , glut and glut proteins was detected in hamscs by immunofluorescence. glut , glut , glut protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd , cd , cd , cd (mesenchymal stem cell markers) and hematopoietic markers cd , cd b, cd , cd and hla-dr were negative. the presence of glut , glut , glut proteins and genes were identified in hamscs. in this study, for the first time in literature, glut , glut and glut gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) cl ] on mammospheres obtained from mcf- human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase / activity, annexin-v-fitc and bcl- activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc- staining. mammospheres exhibited increased oct- and sox (stemness markers) expressions compared to parental mcf- cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion ( . - lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase / activity, annexin-v-fitc positivity and bcl- inactivation. apoptosis was also confirmed with increased tnfrsf a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [ ] . in the development of primary cancers, the head and neck cancer is accounting for approximately . new cases annually around the world [ ] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [ ] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than % of deaths in cancer patients are attributed to metastasis [ ] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [ ] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [ ] . in our study, . ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck , ck for positive selection and cd for negative selection [ ] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level . one of the general methods in ctc detection is flow cytometry . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy , . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which . ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck , ck for positive selection and cd for negative selection, were analyzed by bd facs aria iii flow cytometry . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p- . . - determination of effect cytotoxic, apoptotic, caspace- activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations ( . , . , . , . , , , lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase- activity in breast cancer cells (mda-mb- , mcf , bt- , sk-br ) and normal cell (mcf a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp- , bcl- , bax, birc- , gapdh, caspase- , caspase- , caspase- , caspase- ) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < . ) effect cytotoxic, apoptotic, caspase- activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl- ) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd + gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the gbm cell lines (u , u , u and t ). cd + and cd À cells were seperated by macs method via anti-cd (ac ) antibody from cultured cells and cell lines. rna isolation from cd + and (À) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd + cells express approximately fold tnfrsf and fold tnfsf when they are compared with control cells. tnfsf binds to cd that is expressed on the surface of tcells. cd does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf (dr ) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf and tnfrsf both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf and tnfrsf , these molecules may be essential markers to target cd + gbm stem cells. the effect of docetaxel on p , sin a and mdm gene expression in mcf- breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g /m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf- breast cancer cells line. in this study, mcf- breast cell line was applied different doses docetaxel ( nm, nm, lm, lm, lm) as h and h. mtt analysis was performed to the mcf- breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p , sin a and mdm gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < . ) in h as mtt analysis. the lowest cell viabilty was determinated in group applied lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < . ). the highest p , si a and mdm expressions were apperared in nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g ) and acetyl-terminated (g ) nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g and g dendrimer derivates labeled with dox were absorbed by the cells at °c with different efficiency. g -dox derivate characterized much slower internalization rate than nonacetylated g -dox. only g -dox shown partial colocalization with lysosomal marker lamp after h of incubation. internalization of afprbd-g -dox and afprbd-g -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp already after min of incubation. cytotoxicity results revealed that ic levels of g -dox and afprbd-g -dox coincided and demonstrated a bit higher activity against sensitive to dox skov and resistant skvlb cells than afprbd-g -dox conjugate after h of incubation. at the same time, after h of incubation afprbd-g -dox and afprbd-g -dox were much more than g -dox and g -dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. - - . ) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m -pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m -pk in breast cancer, and high m -pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~ . and . v potentials vs. ag/agci. m -pk activty was detected by using differential pulse method at between . and À . v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m -pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m -pk activity determination at low concentrations is possible with this method. p- . . - tie /tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie /tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie /tek in both cd + gscs and cd À gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with % fetal calf serum and % penicillin-streptomycin at °c in % co -humidified incubator. we isolated cd + and cd À cells from gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd À and cd + cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version . . tie /tek gene expression was determined as . fold higher in cd + gscs than normal brain tissue (p < . ). morever it was determined . fold higher compared to normal brain tissue in cd À (p < . ). according to our results tie /tek expression was higher in gscs, indicating that tie / tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no: s ). adenosine inhibited the breast cancer stemlike cell population through erk / pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk / protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk / pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd + /cd + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd + /cd + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for and h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd , cd , cd , cd and negative with hematopoietic markers cd , cd b, cd , cd and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for and h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in , high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the -phosphofructo- -kinase/fructose- , -bisphosphatase- (pfkfb ) regulates glycolysis by producing fructose , -bisphosphate (f , bp). given that pfkfb is induced by several established emt-inducers in tumor cells, e.g. hif- a and ras, we hypothesized that pfkfb may be involved in regulation of the emt in tumor cells. silencing of pfkfb in pancreatic adenocarcinoma cell lines panc and s vp was achieved using specific sirna molecules. mrna and protein expressions of the cdh gene (encoding e-cadherin, an established epithelial marker), as well as zeb and snai genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb was achived by transfecting cells with a plasmid carrying zeb cdna. cells that were depleted of pfkfb exhibited markedly increased cdh mrna and e-cadherin protein expressions and reduced snai and zeb mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb caused approximately % reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb depletion does not appear to be associated with reduced zeb expression, as ectopic expression of zeb did not reverse the effect of pfkfb silencing on invasion. taken together, these data suggest that pfkfb may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia hematology system and abbott cell dyn and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled . ae . x , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to . x , with a maximum value of x . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = . ), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia ( %), and chronic myeloid leukemia had % of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p- . . - effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type . the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst- tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at h time point. however, at h time point, hescs isolated with the non-enzymatic and collagenase type method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type isolation method at h, h and h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd and lower cd profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd , cd , and cd surface expression levels. only hescs isolated with collagenase type did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type , while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd + subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd . epcam+/cd + cells were sorted by magnetic separation from huh- cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd + subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd + cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd + subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su , in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd + subpopulation of huh- . in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd + hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg ) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst- assay and tg activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp- and - were investigated with zymography and rt-pcr, respectively. although tg activity was found to be % less in emscs isolated from endometriotic tissue, these cells showed times higher tg protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have . times higher tg and . fold higher itgb mrna levels when compared to the cells of healthy group. similar results were observed in sdc- gene expression with a . fold increase. endometriotic emscs demonstrated an average of . -fold increase in the mmp- activity while a onefold increase was evident in mmp- activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within h. the fact that emscs from the control tissue showed lower tg protein levels with a high enzyme activity suggested that tg might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg along with itgb and sdc was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp- and - . recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f with % fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd , cd , cd and cd (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd , cd for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd were decreased to - %. differentiation induced the expression of cd , cd for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~ % and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca + entry (soce) activated by depletion of intracellular ca + stores has been shown to control intracellular ca + homeostasis in many physiological and pathological events. stromal interactive protein, stim , as endoplasmic reticulum (er) ca + sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim and orai on soce in differentiation of huh- hccs expressing epcam and cd surface adhesion molecules (epcam + cd + ). epcam + cd + subpopulations in huh- cells were separated via flow cytometry and transfected with stim and orai- over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca + concentration were monitored via dual wavelength spectrofluorimeter in fura -loaded cells. in epcam + cd + cells, er ca + release increased without any change in soce compared to that of epcam À cd À cells. similar results were observed in stim -oe epcam + cd + cells. on the other hand, increase in orai has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost - % of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd , cd , nanog, oct / , hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd , cd , nanog and oct / significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf- release from human mesenchymal stem cells s. k€ ose , f. aerts kaya , d. uc ßkan c ß etinkaya , p. korkusuz stem cell research and application center, hacettepe university, ankara, turkey, department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor (sdf- /cxcl ) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf- release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage mscs were induced with ng/ml lipopolysaccaride (lps) for h. antagonists for cb (am ) and cb (am ) receptors were added to cultures for days. after incubation with antagonists msc culture supernatants collected and processed with human sdf- beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf- beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf- release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta- , igfr , vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n: ), media (m, n: ), stem cell in media (m+bmsc, n: ). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton ll saline, ll culture media and cell/ ll culture media. the pregnant female rats on the day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta- , anti-igfr , anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta- , igfr , vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol -kinase (pi k)/akt pathways. the effects of h s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h s agents on the in vitro cell growth and differentiation of human lin À sca + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h s-releasing agents, such as na s, gyy and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional d-systems for cardiac tissue repair, that take into account the relevant biological role of h s in the cardiovascular system. p- . . - investigation of the protective effect of boric acid and omega- fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into groups with rats in each: control, mi, mi+boric acid, mi+omega- and mi+boric acid+omega- groups. control rats were treated with ml/day saline, boric acid-treated rats received mg/kg/day boric acid and omega- -treated rats received mg/kg/day for days by oral gavage. for the experimental mi model, mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a -h interval in the last days of the boric acid and/or omega- treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp , biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < . , p < . and p < . respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < . and p < . , respectively) and pon levels were decreased (p < . ). according to the ecg recordings, st wave and heart rate were found to be decreased (p < . and p < . , respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega- after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega- after induction of mi. results of the present study suggest that omega- and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included patients diagnosed with dka, patients diagnosed with dk and voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values ( . ae . , . ae . . ae . ; p = . ). no statistically significant differences were found among groups with respect to ck-mb values ( . ae . , . ae . , . ae . ; p = . ). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group ( . ae . ; . ae . . ae . ; p = . ). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour after acidosis correction ( . ae . ; . ae . ; p = . ) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p- . . - genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac -hcms in dmem/f with %fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber ( %o , %co , %n ) for , , , h and the control group was incubated in normal conditions ( %co , %o ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u -plus- arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as h. we identified genes ( down-regulated and up-regulated) (p < . , fold change ≥ . ) were differentially expressed in hypoxic-ac vs. ac . degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif- signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number s . autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd and cd markers. purified ips cells were differentiated into flk + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days . to . . flk + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd and cd gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk expressing cell number reached to a peak level ( %) on day . followed by a progressive decline subsequently. in the second step, cd and cd positive cells were generated and enriched during day of induction. we showed optimal time for harvesting flk + cells is day . of initial differentiation. following isolation of flk + progenitor cells they were further matured into functional epcs by vegf within days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p- . . - electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups ( mg, mg, mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < . ). amplitude of p wave and qrs complex did not change (p > . ). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < . ). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t- rna polymerase, possess toxicity to leukemia cells hl- and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in - nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents ( - times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkÒ hydrogel photopolymerized in a d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd + stem cell subpopulation. cd + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkÒ hydrogel. printing was done using a lm diameter needle, under bar pressure, and mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd + cells viability is maintained after days of cultured in bioinkÒ hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd + cells expressed endothelial markers such as cd , cd , cd , cd and vegfr . in conclusion d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe o ) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe o were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at and days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe o functionalized with carvone and limonene significantly stimulated the production of il- , il- and il- , while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe o nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p- . . - new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between . and . mm and epinephrine concentrations between . and . mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be . and . mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has À . v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < . ). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than % for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, %); a positively charged phospholipid, dotap ( %) and cholesterol ( %) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll- is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll- . the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll- immobilized was determined by elisa method. . % of ll- peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll- immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using t mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll- immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio nanotube-modified ti surfaces (ti/tio ) on the cross-talk between raw . macrophages (mf) and mc t -e preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw . and mc t -e cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw . cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il- and mcp- release was significantly increased at h, while after days only il- exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for h and days, whereas the presence of the mf for weeks significantly inhibited mineralization. on ti/tio surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p- . . - synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan , , b. tural , s. tural mardin artuklu university, mardin, turkey, dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b and due to high collagen synthesis. on the other hand the other isoform tgf-b , suppresses the dense collagen production caused by tgf-b and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b was studied with elisa. hepg cell line was used on mtt cell proliferation assay for both tgf-b loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be . %. particle size, zeta index and polydispersity index for this formulation were determined as . ae . nm, . mw and . , respectively. thickness of the prepared films were ae . nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand s from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a -well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (% , % . , % wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (% , % . and % wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p- . . - evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz , s. dogan , s. celikler kasimogullari department of molecular biology and genetics, balikesir university, balikesir, turkey, department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations ( , . and %) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki , , c. dupont-gillain universit e catholique de louvain, louvain-la-neuve, belgium, j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of . kda, isoelectric point ip at ph . ) and lysozyme (lys, molecular weight of . kda, ip~ ) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly ( -(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph . (pdmaema is neutral in such conditions) and were swollen at ph . (pdmaema is positively charged). protein adsorption was performed at different ph values ( . - . ) and salt concentrations ( . - . m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [ ] , [ ] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, -amino pyridine and , -diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly( -hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe t (dsm ) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa ), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl- -pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu + -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p- . . - bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonÒ, we employed argon plasma treatment with the powers of and w for - s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst- reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p- . . - graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations ( . - wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from to days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a d composite hydrogel scaffold ( d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin À sca + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of d-chs for tissue regeneration using the emerging bioprinting technologies p- . . - gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformÒ, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite % and tricalcium phosphate %, with - % pore volume and - lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformÒ. in order to improve cell adherence, ceraformÒ was covered with fibronectin. the cells were cultivated for -day period by osteogenic induction medium. days , , , and were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor (runx ), bone morphogenetic protein- (bmp- ), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day th, continued to increase in day . bmp- gene expression was increased in , , day compared to day. on the other hand, runx gene expression was increased only on days and . these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p- . . - on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu , i. negut , f. sima , e. axente , l. e. sima national institute for lasers, plasma and radiation physics, magurele, romania, institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom ( . %) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to show negatively charged particles. when n/p was , the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than . the migration of polyplexes with different n/p ratios ( - ) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was %. tuesday september : - : aging p- . . - novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd - and vd - - ) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc values to be lm for vd - , lm for vd - - and lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc doses of vd - and vd - - showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd - - exhibited - nm ic for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited -fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd - - needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was -fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic of nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap- and on the activity of nfat and ap- transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß is more prone to aggregation to form plaques and more toxic to neurons than aß . in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß / in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin , namely ps , were treated with a dose range of tbo ( - lm) or vehicle control for h. after treatment, aß / levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß and aß secreted into the cell culture media. significant reduction for both aß species was evident at lm (p < . ), lm (p < . ), and lm (p < . ) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g and g cell cycle checkpoints by p and p mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > genes, including nine genes encoding transporters. p mapk and p were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p- . . - smad specific e ubiquitin protein ligase (smurf ) and its potential effects on inhibitory transmission in aging adams , , , interdisciplinary program in neuroscience, bilkent university, ankara, turkey, national nanotechnology research center (unam), bilkent university, ankara, turkey, department of molecular biology and genetics, bilkent university, ankara, turkey, molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, department of psychology, bilkent university, ankara, turkey smad specific e ubiquitin protein ligase (smurf ) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf , via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf transcript is significantly higher in old zebrafish brains. thus, smurf may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb /+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf and gep levels remain stable in the aging brain (p = . , p = . ), and in the ache mutants gep levels are increased compared to the wildtype controls (p = . ). further analysis of the relationships between smurf and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we evaluated patients with ihd and healthy subjects aged years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr rs and tlr rs were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr (arg gln) and tlr (asp gly) were not significantly different between the groups (p ≥ . ). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over years old (p ≥ . ). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we plan to include other types of polymorphisms in tlr and tlr genes and increase the volume of patient cohort in our future studies. p- . . - evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up % and hemorrhagic strokes % of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke patients, with acute ischemic stroke and healthy controls we reenrolled in the study. blood samples were taken within st and th days, and after rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: . ) and thiol (p: . ) levels were significantly higher in first h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the rd month values of tos and nihss score (r = . , p = . ). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p- . . - amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from infertile women with pcos and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p promoter of xp bacteriophage in presence of p protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp , in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p protein, except of bacteriophage p promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p protein in the transcription initiation and identification of the role of the individual elements of p promoter of xp bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p protein. we have designed a set of promoter probes representing the combination of sequences of p -resistant p promoter and p sensitive t n promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream À position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing À promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region , a sigma-factor domain responsible for interaction with À promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p protein. promoter probe with up-element of p promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p promoter of bacteriophage xp can escape inhibition by p protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p- . . - distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study gerbils brains were used and divided into groups (n = ): : newborn animals ( day), - : , and days respectively, : animals aged years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within days (in cerebellumamounted to . ae . lg/ mg tissue; to - days increased to . ae . lg/ mg tissue, and began to grow again in older individuals aged years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at days p.d. ( . - . lg/ mg tissue), and unchanged for days. these results revealed that the most intensive development of astrocytes in the cerebellum to p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs . in order to investigate the impact of whirly on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly (hvwhy kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs at onset of drought-induced senescence in wild type (wt) and hvwhy kd lines, stress-responsive loading with histone modifications at gene regions of hvs ( regions in the promoter, one region around translation start site and regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy kd indicating that whirly protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h k ac all over the analyzed gene regions, correlating with a massive induction of hvs expression, while drought stress caused no substantial increase of h k ac in why kd plants. the results suggest that drought induced expression of hvs is under epigenetic control, and furthermore that why is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after week of inoculated with erlich cancernome ( cells/animal, i. p.) in months male balb/c mice treated at once with or without plant extract with bbv ( mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on % and % respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold ( . % versus . % in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at lg/ml concentration against escherichia coli vkpm-m , pseudomonas aeruginosa grp , bacillus subtilis wt-a , salmonella typhimurium mdc and staphylococcus aureus mdc , candida albicans wt- and candida guilliermondii hp- . statistical analysis was done using graphpad prism . . crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p- . . - effects of aluminum stress on photosystem-i apoprotein a gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl in different doses ( . , . , and mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ . ) decreased after hours treatment with mm and mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with . and . mm al for hours increased the gene expression level of psab by . % and . % respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, mm al exposure for hours could damage the electron transport in photosystem i. p- . . - nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + mg/kg ns; iii = sham + mg/kg (n-acetyl cysteine) nac; iv = g/ kg paracetamol; v = g/kg paracetamol + mg/kg nac; vi, vii and viii = g/kg paracetamol + , and mg/kg ns, respectively. paracetamol administration (oral) was carried out h after ns and nac administrations (oral), and all animals were sacrificed h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p- . . - the study of problems of preservation of the birches e. shadenova , , e. zhumabekov , m. sembekov , m. burchaeva institute of general genetic and cytology, almaty, laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p- . . - fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht- , t and upci-scc- cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a ( . m/z) and -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at th and th hours compared to control group. this study shows that holothurin a, dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support ( z ). p- . . - alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified genes encoding sr proteins in chinese cabbage (brassica rapa chiifu- ) from brassica database and analyzed their phylogenetic relationship. b. rapa has types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage ( and days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra and bra were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p- . . - characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of u mg(- ) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to . . the optimum ph and temperature was estimated as . and °c, respectively. the purified enzyme was stable from ph . - . and able to thermal stability up to °c. the k(m) and v(max) values were . mm and . micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf- , substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd + and hg +. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, , , -trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in , but the uniprot database contains less than reviewed sequences, which is approximately -fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above , are easily detectable in the total protein extracts. p- . . - transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in transcripts, database search with trinotate tool revealed transcripts with aox domains characterized in known alternative oxidases. blast analysis of these sequences with known aox proteins revealed three distinct aox genes (nalba-aox , nalba-aox and nalba-aox ). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox , nalba-aox are predicted as mitochondrial while nalba-aox is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr rk ). p- . . - comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak , v. koukalov a , z. medvedov a , c. martin , j. hradilov a , l. sp ıchal , b. brzobohat y mendel university in brno, brno, palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde ( -chloro- -( -methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of -day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p- . . - the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as °c and , respectively. the enzyme was stable at °c, but thermal stability was dramatically decreased at higher temperatures up to °c. kinetic parameters were also measured; vmax was . u/ml min and km was . mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp , lira and lines g- / _o, g- / _k were treated for and hours with lm alcl solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about million raw reads were obtained and conserved mirnas from families were identified. significant expression alterations in flax plants under al treatment were shown for mir and mir . expression level of mir was varied in similar way in resistant and susceptible to al genotypes: mir was up-regulated after hours of alcl exposure and down-regulated after hours. mir expression was increased after hours of alcl exposure and decreased after hours in susceptible to al flax genotypes (lira, g- / _o), while in resistant genotypes (tmp , g- / _k) mir level was decreased after both and hours of al treatment. in other plant species, mir and mir were identified as al-responsive. mir targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir targets mrna of tas protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir and mir in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir in susceptible and resistant to al genotypes. this work was financially supported by grant - - from the russian science foundation. p- . . - association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from year old poplar trees ( clones x replicates x ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as . ae . lg/ml, ae . lg/ml, and ae . lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p- . . - proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum ( min), radio-frequency ef ( - min) and cp ( - min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in days in comparison to days after seed treatment. the d dige analysis revealed differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin- were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p- . . - suppression of lipopolysaccharide-induced inflammatory responses in raw . macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw . macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il- concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw. . cell were found as . - lg/ml. studied cyclamen taxa inhibited tnf-a and il- release on lps stimulated-raw. . in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p- . . - in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < . ). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < . ). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p- . . - cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase- expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp- mrna expression of human sw colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp- mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag- s . data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with . lm ic value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at lm concentration when compared to control and lm (p < . ). cub alone showed inhibitory effects on mmp- mrna expression at lm and higher doses significantly (p < . ). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp- expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp- synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using % aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between mg/ml and . mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to % sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc is gram-positive cocci (mic: mg/ml). the mic values of other gram-positive bacteria s. aureus atcc , e. faecalis atcc , l. monocytogenes f and m. smegmatis cmm were detected as , , and mg/ml respectively. mic values of gram-negative bacilli were detected as > mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc , s. aureus atcc and e. faecalis atcc ). the strongest effect was observed on s. aureus atcc strain. in contrast, extract showed less effect on gram-negative bacilli. p- . . - selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a ) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p- . . - dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu , f. zihnioglu denizli vocational school of technical sciences, pamukkale university, denizli, department of biochemistry, faculty of science, aegean university, izmir, turkey type diabetes mellitus (t dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide- (glp- ) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec . . . ) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic values were also determined by nonlinear regression curve fit using graph pad prism . with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic . lg/ml while the standard (positive control) diprotin a displayed the ic value of . lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p- . . - dna recognition determinants of arabidopsis thaliana b transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b tfs constitute a large, plant-specific protein family (approx. % of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. amino acids) b dna binding domains. currently the b tfs are divided into four groups (lec -abi /val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b domain (arf , arf family) is available, thus the mechanism of site-specific dna recognition by the lec -abi /val and rav b domains remains poorly understood. based on the arf -dna structure (pdb ldx) we have built homology models of dna-bound b domains from a. thaliana abi (lec -abi /val family) and nga (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi / nga b domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b domains and in the future may help us predict the dna binding properties of currently uncharacterized b domains. p- . . - immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili , a. zeytunluoglu denizli health services vocational high school, pamukkale university, denizli, denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp- and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group : control, group : stzinduced diabetic, group : o. hypericifolium injected, group : stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups and by a single intraperitoneal injection of stz at a dose of mg/kg body weight. in groups and , rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of ml/kg body weight for consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine (k ) of tubulin. k is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk , e. marusich , r. chuprov-netochin , j. neskorodov , y. mishutkina , s. leonov life sciences center, moscow institute of physics and technology, dolgoprudny, center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature ( °c) and light conditions ( : hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point- . od) and hours later. to validate our system we used known cytokinins and auxins ( mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in - times range, compared with only times increase in control group. eight new chemicals ( lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to %. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an % yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl , pl , pl , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total sequence tagged site (sts) sequences from ncbi were aligned for pl , pl , pl , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p- . . - investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg cells line s. altundag , pamukkale university, denizli, istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg - cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut- ,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol Ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut- ,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut- ,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg - cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut- ) analyzed the expression levels . teucrum polium plant extract is applied in hepg - cells the glycolysis pathway genes (pk m glut- ) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg - cells showed antidiabetic effect. p- . . - purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara , s. sinan , z. ekmekci , y. turan university of balikesir faculty of veterinary, balikesir, university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec . . ) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with % ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose b-ltyrosine- -naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph - and thermal optima was determined using - °c temperature range. inhibitory effects was determined with mm substances. results: the enzyme was . -fold purified with yield of %. purified b-glucosidase from apricot seed was visualized about kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of . mm and . eu, respectively. the optimum ph and temperature were determined . and °c respectively. effects of cacl , kcl, nacl, mgcl , k so , na so , cuso , fecl , pb(ii) acetate, agno , zncl and glucose on purified enzyme activity were investigated. kcl, na so , pb(ii) acetat and cuso reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p- . . - a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un , , o. arslan balikesir university, science and technology application and research center, balikesir, department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec . . . ), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose- b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose- b-aniline p-amino benzoic acid affinity chromatography gel. % . yield and . fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of kda. the v max and k m of the purified enzyme were determined , u/ml min and . mm, respectively. p- . . - phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken , b. kocat€ urk , s. dogan , h. tuner balikesir university, balikesir, kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have , , kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of s promotor region in fragmented dnas was proved by pcr. escherichia coli dh a strain was transformed by fragmented dna samples according to cacl method. s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to - bp for bacterial transformation. fragmented dnas confirmed for carrying intact s promotor sequence. none of bacterial genomic dnas were found to be s-positive. according to the transformation results, absence of s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p- . . - preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker (tm- ) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p- . . - cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in , minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima - , tm- and ps- were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in % high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a °c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p- . . - association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of - nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm- (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p- . . - pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of chls, a homozygous cotton line tm- and a double haploid line pima - were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm- and pima - (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p- . . - pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during - . chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy ( n = - ), polyploidy ( n = - ) and the occurrence of additional b-chromosomes ( n = + - b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of s rdna and s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to . %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p- . . - angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu , i. arslan biomedical equipment technology, pamukkale university, denizli, turkey, denizli, biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the kda < peptides < kda in the ultrafiltration fractions displayed highest ace inhibition ( . ae . % at lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p- . . - modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg cell line d. irtem kartal , a. altay yuzuncuyil university, van, erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than different ailments ranging from aches to epilepsy. there are around species of salvia, of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm and catalase in hepg cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg cells for hours and hours incubation and methanol extract decrease catalase mrna expression for only hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing gigabases of bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcÀesiÀqtofÀms) to identify drought-related proteins. totally proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p- . . - isolation and characterization of a hexose converter from olive s. altunok , e. d€ undar department of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer , finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was kda with a pi of . . the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number o . key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p- . . - antioxidant potentials of propolis and its bioactive components, and their effects on cyp e gene expression in ht- adenocarcinoma cell line a. altay , d. irtem kartal erzincan € university, erzincan, y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p enzymes are involved in phase i xenobiotic and drug metabolism. cyp e is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with % ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht- human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp e in ht- cells were investigated bu using q-rt-pcr technique. ic values for dpph radicals scavenging activities of the extract was calculated as . mg/ml. tpc and tfc were determined as . mg gae/g extract and . mg qe/g extract, respectively. caffeic acid content of the extract was measured as . lg/g extract. ic values for xtt assay in hours incubation with the extract and caffeic acid were evaluated as . mg/ ml and . mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp e mrna expression with . and . fold, respectively in ht- human colorectal adenocarcinoma cells for hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp e expression and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - effects of histone h lysine inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h at lysine residue by specific methyltransferases suv h in humans and kyp/suvh in arabidopsis generates a'code' for the recruitment of hp proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv h has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h k methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf- breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt ( , -bis( -metoksi- -nitro- -sulfofenil)- h-tetrazolyum) test. in order to determine the dose of ic , plant extracts were applied as time and dose dependent in the range of - ug. in nd hour, the ic value is determined as ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase- , caspase- , caspase- , caspase- , bax, bcl- , fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while . and . -fold respectively increase in the gene expressions of caspase- and caspase- of dose group cells, . -fold decrease in the gene expressions of bcl- . no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p- . . - the effect of ferulic acid and -fluorouracil combination on apoptosis in pc- human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. -fluorouracil ( -fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; -hydroxy- -methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and -fu on apoptosis in pc- human prostate cancer cell line. the effects of -fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp , casp , casp , casp , bcl , bax, fas and cycs were investigated in four groups by qpcr. the ic doses of fa and -fu were found to be lm and lm for hours in pc- cells, respectively. in order to determine combination effect, pc- cells were treated with <ic doses ( lm fa and lm -fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl were observed in the fa group, compared with the control group cells. after the treatment with -fu, the expression of casp was significantly increased compared with the control group. furthermore, combination of fa and -fu significantly increased expression of casp , casp , fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and -fu affected expression of different genes in apoptosis in pc- cell line. p- . . - combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc- human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc- human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp , casp , casp , casp , bcl , bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic doses of fa and gemcitabine were found to be lm and lm for hours in pc- cells, respectively. for determination of combination effect, pc- cells were treated with <ic doses ( lm fa and lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp , casp , casp , fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc- human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf- , hela, a , and ht- cancer cell lines and nih- t cells. the viability of cells in lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic = . ae . mg/ml) and abts radical cation scavenging activity ( . ae . mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc ( . ae . mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species ( - mm / < . - mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain ( - mm/> mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p- . . - antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention ( ) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms ( ) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: ), aureus e. coli (atcc: ), and salmonella enterica (atcc: ) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with and milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be milligrams and milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with and milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p- . . - molecular analysis of serine/arginine rich sc splicing factor from olive b. bas , e. d€ undar depertmant of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc splicing factor (oesarsc sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc sf was a protein consisting of amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was kda with an isoelectric point (pi) of . . the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number o . keywords: olea europaea l., oesarsc sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p- . . - application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli , g. arabaci kto karatay university faculty of medicine, konya, faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h o and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with % (w/v) ammonium sulfate ((nh ) so ) and added t-butanol with the ratio of : . (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh ) so ( , , , , %) and tbutanol ratios ( : . , : , : . , : ) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were . , . % and . , . . therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of : . , the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh ) so ( , , , %) were . , . , . , % and . , . , . , . . the interfacial phase was not dissolved in % (nh ) so . at constant % (nh ) so , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio ( : . , : , : . , : ) were . , . , . , . % and . , . , . , . . finally, at optimum conditions (% (nh ) so , t-butanol : . ) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were . % and . . the results of gel electrophoresis showed that the molecular weight of enzyme was between - kda. the applications of tpp gave the maximum recovery of . % and . -fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for , and days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of distinct spots in brachypodium protein repertoire. a total of differentially expressed proteins (deps), with at least -fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p- . . - immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m e epitope from matrix m protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m e sequence. accumulation level of m e-ala recombinant coat protein was significantly higher than m e-cys/ ser (ratio : ). tmv-m e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves ( d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm ae . cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw . macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase- (cox- ), tumor necrosis factor-a (tnf-a) and interleukin- (il- ) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox- and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il- in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p- . . - the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf- breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf- breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase , bax and p and antiapoptotic genes; bcl- and pi expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf- cell was only observed in and lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf- cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf- breast cancer cell lines. p- . . - reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are , bp (mon- kalr) and , bp (mon- volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and ribosomal rna genes. and trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf , ycf , accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant - - and rfbr grant - - (mon- volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), -methyl-z-tetradecen- -ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, -octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p- . . - role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut , deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature ( k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p- . . - the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature ( / °c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor , -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for days at optimal conditions. d was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p- . . - reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m ) was investigated. three weeks old tomato grown at optimal conditions ( / °c day/ night temperature and light intensity lmol/m /s) plants were exposed for and days to elevated temperature at / °c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf- cells. mcf- cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively , and ng/ml) . then different doses of silymarin ( , , , lm), curcumin ( , , , lm) and propolis ( . , . , . , . mg/ml) were added. after , , and hours incubation periods different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf- cells for , , and hours (biovision cell proliferation kit) all experiments were carried out different days, at least times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < . between and hours. it was found that there were not seen much cells at and hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was . mg/ml, silymarin and curcumin were lm. also, we evaluated the effects of on proliferation rate the mcf- cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf- cells in hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p- . . - investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; -hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, h-nmr and melting point analyses. the theoretical uv-vis spectra and d conformations of the isolated compounds were determined by using the gaussian software with hf theory at the b lyp/ - g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [ , ] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than hydroxyphysodic acid, and the toxic activities were found sufficient as much as % at the highest doses of the substances; lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately % by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [ ] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. ( ) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research : - . [ ] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. ( ) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian software with hf theory at the b lyp/ - g level was also performed to determine the theoretical uv-vis spectrum and d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [ , ] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as % at the highest dose of the substance; lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as , and -lm, and % of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size ( - nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n is %, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score or higher, p < . ) peptide mass fingerprint of cwr -de electrophoresis revealed differences in protein composition between cwr and n . information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p- . . - mir and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir s and their target by q-rt-pcr. we mined mir genes sharing sequence similarity and grf targets. the expression analyses of mir s and grf are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir s and grf in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate , and to detect expression through real-time pcr were designed using primer . amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as . . the molecular weight of the protein was calculated as kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p- . . - cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht- , t and upci-scc- cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for hours. the cell index data were compared with the control group. ic values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a ( . m/z), -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). the fraction d was the most effective on all cell lines with ic value of . ae . mg/l, . ae . mg/l and . ae . mg/l for ht- , upci-scc- and t , respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p- . . - effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol ( %) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose ( mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically % (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp- , mmp- by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of rd, th, and th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the rd and th days. the malondialdehyde levels were significantly lower in the treatment group. mmp gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the th day while it was reduced in th day. mmp gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in th, and th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp and mmp regulation. p- . . - isolation and molecular molecular characterization of vps /vam from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps /vam promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps /vam , however, have been reported. the aim of this study is to report information of olive vps /vam including expression pattern and biochemical characterization. vps /vam was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega . cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn d. the blast and bioedit analyses revealed vps /vam had base pairs coding amino acids with a molecular weight of . kda, and pi of . . the at/gc ratio was very high ( . ) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the d structure of the protein was similar to its previously reported homologs. p- . . - isolation and characterization of the ribosomal l protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l protein is larger than the lsu of ribosome and contains domains as -layered alpha/beta domain and -layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l (rl ) gene has been studied in many plants, reports on olive rl (oerl ) are very rare. this study presents molecular characterization of rl gene from olive. oerl was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional ( d) structure analysis, and phylogenetic analysis were conducted using signalp . , thhmm, i-tasser/cn d and mega , respectively. oel was found to have an open reading frame of base pairs coding amino acids that constitutes a molecular weight of . kda and a high pi of . . lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a . at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl . likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl with respect to expression pattern and biochemical function continues. p- . . - isolation and characterization of an olive splicing factor b subunit m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor b subunit (sf b ) functions in the regulation of translation and gene expression. sf b forms u small nuclear ribonucleoprotein complex (u snrnp). splicing factors a and b binds pre-mrna at the site of the intron branching point. this binding joins u snrnp to pre-mrna. although sf b from various plants have been widely studied, no studies on olive sf b (oesf b ) have been reported. this study reports information on various aspects of oesf b . oesf b was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn d were used to generate the calculated d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb . the orf contained nucleotides coding amino acids that produce a . kda peptide with a pi of . . alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf b is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf b could be synthesizing in mitochondria. the predicted d structure of oesf b was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p- . . - kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of - %. they are grouped into a ( - kda), b ( kda), c ( kda) and d ( kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of % naci concentration, and second, kafirins were obtained using tertiary butanol ( %) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as , , , and %. of the total proteins, % was identified as a, % b, % c, % d, and % non-prolamines. non-prolamin group of proteins were visualised as different bands ranging from . to kda. c and b group of proteins were only viewed when treated with reducing agents as -me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for . , . , . , . and . kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p- . . - untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu , university of agricultural sciences and veterinary medicine, cluj-napoca, center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol ( ) and chloroform/methanol ( ) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction ( ) , ranging from . to . mg/ g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. / , pncdi . glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys ko) and liver glycogen synthase (gys ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos , nos (genes encoded no producers) and pde a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos which lost rhythm and increased and delayed amplitude of nos expression. further, pinealectomy initiated cyclic transcription of gucy b and noncyclic transcription of gucy a (genes encoded cgmp producers) and increased mesor and amplitude of pde transcription. the transcription of prkg , the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi or other investigated genes (coxi , nrf , nrf a, pgc a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s fraction mix was used. cells were treated with . , and lg/ml oseltamivir, the tamiflu capsule ingradient, for or hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ . ) in the absence of s mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: . - mm of glucose, . - mm of urea, and . - mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p- . . - ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide ( amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with mm dimensions. ovary adenocarcinoma cells (efo- and onco-dg- ) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak- s ). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide ( n) contains biotin on '-and '-ends, and sterptavidin in molar ratio : . this complex represents a structure with equivalent electrophoretic mobility of bp dna and preferred "valency" of streptavidin is . this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved - times highter sensitivity than elisa. to reduce the matrix effect we used - fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only mkl of serum sample). antibodies to the disaccharide galb - glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ . ) for health donor sera (n = ; . ae . pg/ml) compared with sera from patients with breast cancer (n = ; . ae . pg/ml). sensitivity of igm anti-le c antibodies detection was pg/sample ( mkl) ie . molecules. thus for the immuno-pcr detection of antibodies the - tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (# - - ) and by russian federation president scholarships donated to d. yu. riazantsev (# sp . . bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma , as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = , the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge ( uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = , when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle- cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, u of penicillinstreptomycin and maintained in a humidified incubator at °c and a % co atmosphere. cell viability was measured using the colorimetric -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in well plates and incubated with ll/ml dmso, - lg/ml tm, . - mm tudca, or lg/ml tm + . - mm tudca for - hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for hours at °c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or mm tudca was cytotoxic. at and hours incubations mm tudca and lg/ml tm + mm tudca were significantly cytotoxic compared to control, dmso and mm tudca groups. treatment of cells with . mm tudca hours before administrating ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at mm concentration when treated with tm. therefore . mm of tudca, administered hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; s ). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a (lp-pla ) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla mass, activity, index) related genetic variations (pla g ) increase the risk for developing future cardiovascular disease in women with pe. a group of preeclamptic patients and normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla mass level. lp-pla activity were determined by kinetic method. plag snp genotyping performed by using the sequenom massarray iplex. the rs tt genotype had a higher lp-pla index (p = . ) for early onset preeclampsia, cc genotype had a higher lp-pla mass and lp-pla index for late onset preeclampsia. no difference were found for control. the rs gg genotype had higher lp-pla mass and index for late onset preeclampsia (p = . , p = . respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs and rs snps in early, late onset preeclampsia and control group. only lp-pla mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla mass, index and rs , rs snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi- mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding -hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi- mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found ae , ae , and ae hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi- mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi- mutation. it could accurately discriminate between normal and ivsi- mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il- b and il- and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in and hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio . to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il- b, il- and tnf-a genes were increased by the exposure to nanoparticles of , and lg/ml for - hours. tio nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio . oxidative stress was elevated by the treatment with tio nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il- b, il- . background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor- (vegfr ) and a v b -integrin on d and d cultures (spheroids) of ea.hy . methods: the ea.hy cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf ( ng/ml) or vegf a ( ng/ml) for hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr and a v b -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b -integrin and vegfr- increased on the d culture compared to d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b -integrin in the d culture of the ea.hy cell line activated with tnf. also increased expression of vegfr in the d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b -integrin and vegfr on the surface of d and d cultures as well as antibodies with fluorescent label. conclusions: d cultures of the ea.hy cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the d structure of tnf and ii) design of more potent and less toxic spd analogues. b. drug synthesis. a series of spd analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded %. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd derivatives have better dissociation constants than spd . the effect of exercises on serum bmp- levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp- is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp- to sustain the molecular approaches for treatments. a total of patients were included in this study. the groups are formed as follows: group , oa patients before the exercise; group , oa patients after the exercise; group , oa patients before the isokinetic exercise; group , oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after weeks of the exercise programme. self reported severity of pain was measured using the mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp- levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp- levels were significantly decreased in group compared to group ( . ae . ; . ae . respectively, p < . ) and in group compared to group ( . ae . ; . ae . respectively, p < . ). there is not any statistically differences between group and group (p > . ). as a conclusion, the decreased serum levels of bmp- may be suggested as a biochemical marker for oa patients during exercise programmes. p- . . - tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase- ), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < . ). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase- ) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < . ). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < . ). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p- . . - short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin ( mg/kg, i.p.). rats were divided into group i: control, ii: control/ mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/ mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il- b, il- (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il- b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p- . . - cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb- and mcf- breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb- and mcf- breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb- wt and mda-mb- gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as lm (for mcf- ) and lm (for mda-mb- ) decreased cell proliferation and induced apoptosis through causing jak dephoshorylation, stat , , dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for hours. lm curcumin-induced apoptotic cell death via dephosphorylating jak at tyr / residues and decreased phospho-stat , level in both breast cancer cell lines. although curcumin dephosphorylated stat in both mda-mb- and mcf- wt cells, no significant effect has been observed in mda-mb- gh+ and mcf- gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf- and mda-mb- breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat , a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb- breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb- breast cancer cell lines. we found that lm curcumin-decreased in apoptotic cell death through inactivity at jak which led to dimerization of stat , stat , stat . concomitantly, curcumin affected stat regulating socs proteins in mda-mb- breast cancer cell line. in addition, we demonstrated that lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb- breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il and il cytokines as main mediators of inflammation progression, correlated in keratinocytes with il a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam- and vcam- adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il and il in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam- over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into groups (n = ). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + mg/kg salvia l.group; group iv: i/r + mg/kg salvia l. group; group v: i/r + mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for days was given single dose as a gavage. minutes ischemia, minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)- and interleukin ib (il- b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il- and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il- , and il- b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group mg/kg salvia l. and mg/kg rosmarinic acid especially mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but mg/kg salvia l. extract were more effective than mg/kg salvia l. extract and used as synthetic mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a (spla ) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla , lpl and evaluate changes in circulating protein levels of angiopoietin-like protein (angptl ), angptl , cyclooxygenase (cox) and prostaglandin e (pge ). serum from control subjects and acne vulgaris patients with moderate and severe disease was evaluated for levels of spla , cox, pge , lpl, angptl and angptl . disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au clinical chemistry and unicel dxi immunoassay systems). serum levels of spla and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge , angptl and angptl levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla , lpl, angptl and angptl levels in acne vulgaris. future studies are aimed to understand the regulation of spla and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; # s ). p- . . - -ohdg and hogg levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. -hydroxy- -deoxyguanosine ( -ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human -oxoguanine dna glycosylase (hogg ) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after months of isotretinoin treatment by measuring -ohdg and hogg levels. the current study includes acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. -ohdg and hogg levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg and -ohdg, respectively.both -ohdg (p as a conclusion, isotretinoin increases dna damage and high serum -ohdg and hogg levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor (spi- ) in the stat -mediated manner in pinealocytes. however, the precise function of stat in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi- in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi- were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat transfection. the successful transfection and subsequent decline in stat expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi- were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat on those immunomodulators is important from the clinical point of view because inhibitors of stat are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles ( nm) using human lung mrc- fibroblast as model. cells were exposed to . lg/ml silica nanoparticles for , and hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il- b, il- , il- , tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp- , mmp- , mmp- ) and tissue inhibitor of metalloproteinase- (timp- ) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il- , il- , il- and tnf-a as well as of mmp- and timp- , was up-regulated whereas those of mmp- , mmp- was down-regulated after and hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase- in mrc-cells after and hours. p- . . - association of fto gene variant (rs ) with markers of t dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type diabetes (t d) encodes a -oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included patients with t d and prediabetes and healthy controls both sexes, aged from up to years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs with increased risk of t d (or = . , % ci . - . , p = . ). a allele was significantly associated with higher values of hba c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb ), glial fibrillary acidic protein (gfap), c-x-c motif chemokine and (cxcl , cxcl ), matrix metalloproteinase (mmp ), chemokine (c-c) motif (ccl ) and tyrosine-protein phosphatase non-receptor type (ptpn ) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak s and pamukkale university paubap fbe . p- . . - apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek , s. savranoglu , a. r. t€ ufekc ßi , f. g€ ul , i. demirtas , t. boyunegmez t€ umer graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw . macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into subfractions by column chromatography. e was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw . cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e , and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at lm decreased no production and %-ic : and lm-by inhibiting inos gene expression and % as well as protein expression and %, respectively (p < . ). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id: t ) support this work. the role of tip in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il- (interleukin ) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip conditional knockout mice, so we thought that tip may have a direct or indirect role on inflammation mechanism. tip (tat interacting protein, kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il- were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il- was increased in lps-induced inflammation group (p < . , p < . ) and liver-specific tip conditional knockout group mouse liver (p < . , p < . ). our initial data suggest that tip may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: z ). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose -phosphate dehydrogenase (g pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il ), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il was significantly increased in lps treated group. while the level of mda and h o was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: z ). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < . was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = . ) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of patients which have been diagnosed with cchf. matched for gender, healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p- . . - inhibitors of mdm ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm is an e ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p / mdm interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm and p might be playing a substantial role in autoimmune processes. we used a small molecule p /mdm inhibitor nutlin- a to test the effect of p activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p /mdm interaction by nutlin- a alters the p levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p /mdm interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant - - mol_a_dk. can ykl- be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc . and netphos . program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into groups: deletion/deletion (dd) genotype (group , n = ), insertion/deletion (id), insertion/insertion (ii) genotypes (group , n = ). blood pressure and pulse measurements were performed in all patients within minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes ( . ae . and, . ae . , p < . ). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone ( mg/kg b.w.) with and without iu of d (for days). the levels of rank, rankl, opg, a-hydroxylase (cyp b ) in bm were determined by western blotting. vitamin d receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. ohd content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp b , which is responsible for , (oh) d synthesis, in bm and ohd content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d and rank/ rankl/opg pathway. vitamin d co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d treatment. p- . . - heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor encoded by the hsf gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa a promoter (hsp promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp inhibitor, mg as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks and tks , the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks binding partners including adaptor proteins itsn , itsn , crk and grb , kinase src, amph , bin , plcg and also another member of the tks family -tks . it may indicate the possible role of tks in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph and bin , as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks isoform -tks -beta. we suggested that an active state of tks is regulated via intramolecular interactions between its proline-rich motifs and own sh -domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr and wire interact with sh domains of itsns in complex with actin. p- . . - correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in % methanol. after sample preparation obtained solutions were analyzed by hplc-agilent with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp within insulators still remains to be elucidated. recently we described an interaction between cp btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate aa fragment within ctcf c-terminal domain sufficient for interaction with cp btb, but deletions of flanking regions also lead to the loss of interaction with cp in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp insulator protein at . a resolution. overall structure is similar to other btb-domains. cp btb-domain has peptide-binding groove similar to that previously found in bcl btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project № - - ). p- . . - comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f hybrids of romanov sheep with . % of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by . % lower ( . ae . %, p < . ), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by . % and . % higher ( . ae . and . ae . (p < . ), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from . % to . %) and for oleic, linoleic, arachidonic acids (increased from . %, . %, . % to . %, . %, . %, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was . ae . , . ae . and . ae . %, that was . % lower (p < . ), and . % and . % higher (p < . ) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. - - . foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd (nbd ) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature > °c). the genome of nbd is $ kb long, and has a total of probable protein-encoding genes as well as gene for trna ser . the genome analysis revealed that nbd orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, are similar to proteins from phages that infect different members of enterobacteriaceae, while nbd orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, nbd orfs were given a putative functional annotation, including genes coding for morphogenesis-related proteins, as well as associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit- / ) from the research council of lithuania. p- . . - the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas , j. williamson , v. schw€ ammle , s. douthwaite universidad de cundinamarca, cundinamarca, colombia, university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as , as -rlma::aph and b) were grown aerobically in nutrient rich lb media at °c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be > -fold higher the wild-type b-strain of e. coli than for the hypersusceptible as strains ( lg/ml). whole genome comparison of the b and as strains were characterized by an increase in proteasome components ( proteins), chaperones ( ), error-prone dna polymerase components ( ), ribosomal hibernation factors ( ), heat shock response ( ), electron transport coupled proton transport ( ), pentose phosphate pathway ( ), flagellar assembly ( ), oxidative phosphorylation ( ) and tca cycle ( ). whereas ribosomal proteins ( ), aminoacyl-trna synthetases ( ), rnases ( ), abc transporters ( ), mismatch repair ( ) and sec secretion pathway ( ) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak /stat signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e g), asparagine to aspartic acid (n d), threonine to alanin (t- a) missense mutations, alanine to serine (a s) substitution, tryptophan to stop codon (w- x), gaaa insertion in intron of gh-n gene and both intron (+ c) and deletion of . amino acid of gh protein phenylalanine (f del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc . vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w- x, e g, f del, a s and n d mutations suppresses gh signaling via acting on either jak dephosphorylation or stat downregulation, t- a, gaaa insertion and deletion of + c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek cell lines. as a result, w- x, e g, f del, a s and n d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no tyr)-containing proteins were immunoprecipitated from serum with a no tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no tyr antibody. among the numerous immunoreactive bands observed in disease patients, the kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha- (iii) chain (co a _hu-man) with mascot algorithm analysis giving a score of (p < . ). collagen alpha- (iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no tyr modifications in co a relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs in gckr gene and rs in galnt gene with parameters of t d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved patients with t d and healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt polymorphism (rs ), while polymorphism in gckr (rs ) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs variant with waist circumference (p = . ) and fasting glucose levels (p = . ) in the control group. no such association was demonstrated for rs galnt gene. in the group of diabetic patients, significant association of gckr rs variant with levels of bilirubin (p = . ) and rs galnt variant with hba c (p = . ) and triglyceride levels (p = . ) was also demonstrated. our results suggest an association of variations of gckr and galnt genes with specific markers of t d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp binding site of the collagen type a (col a ) gene is associated with low bmd. we examined the distribution of col a gene polymorphism in young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤ . sd and controls was healthy women ( - years). mean age ( . ae . ) and ( . ae . ) respectively. the bmd, as g/cm , was measured in the hip and the lumbar spine (l -l ) with (dexa). dna was isolated from blood. col a gene was analysed with genomica clinical array system. the x test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was À . ae . . mean bmd (as g/cm ) was . ae . , and ( . ae . ) genotype distribution were ( %) ss, (% )ss, (% )ss for patients, and ( )ss, ( )ss, ( )ss for control . patients had (% )s allele, ( %) s allele, controls had ( %)s allele, ( %)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > . . genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(% ) than controls (ss % ) p < . . this study shows that high prevalences of the ss genotype at the col a locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col a and col a mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col a gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly ( - %) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep b at - and hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep b cells in three different concentration, namely - and . lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was lm. proapoptotic genes, bax and bad, and autophagy genes, beclin- and lc , were upregulated in hep b cells after hours treatment in contrast, antiapoptotic gene, bcl- , expression level was decreased for hep b cells after hours. p- . . - association of irs genetic variation with type diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate- (irs ) encodes the irs protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs single nucleotide polymorphisms (snps) were associated with type diabetes mellitus (t d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs (g>a), rs (t>c) and rs (a>t) with t d risk and related traits. our study involved t d patients and healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss . our results demonstrated a significant difference in frequency of rs (p < . ) and rs (p = . ) snps between t d patients and control subjects. interestingly, here we showed a significant association of irs rs risk t allele with increased insulin levels (p < . ) and homa-ir (p < . ) in t d patients. similarly, rs variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = . ) and homa-ir (p = . ). no such association was demonstrated for rs . however, this irs variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = . ) and decreased hdl levels. our results suggest that irs variants are associated with t d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t d biomarkers. the adra a gene encodes alpha- a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra a polymorphisms (rs a>g and rs g>t) with type diabetes (t d) and its related traits. in this study we have included t d patients and healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics software. our data showed that frequencies of both, rs and rs , variants were not significantly different between t d and control subjects. however, rs risk a allele appear to increase insulin levels (p = . ) and homa-ir index (p = . ). furthermore, this variant also seems to affect vldl levels (p = . ) and waist circumference (p = . ) in diabetic patients. the genotype analysis of rs variant demonstrated that risk g allele decreased hdl (p = . ) and increased ldl levels (p = . ), as well as affected the waist circumference (p = . ) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs a / rs g with higher homa-ir index. here we demonstrated that although both, rs and rs , adra a polymorphisms were not associated with t d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein (sip ), also known as zeb is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip mediate its binding to ebox sequences in the genome. however, there are only limited data about sip target genes. by using a home-made anti-sip monoclonal antibody, clone e , we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu , plc/prf/ and sk-hep- and found receptor tyrosine kinase-like orphan receptor (ror ) as one of the targets. sip dnabinding consensus motif cacctg was found at + kb, + kb and + kb from ror transcription start site. chip experiments validated sip binding to all consensus motifs in the ror gene region. interestingly, the strongest enrichment was at + kb suggesting that long-range interactions play an important role in the regulation of ror by sip . sip knockdown by shrna in high-sip expressing snu cells resulted in the repression of ror expression. ror is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror -positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip and ror act in similar pathophysiological processes. our finding that ror is regulated by sip at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein (sip ), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip in hcc are yet to be identified. we conducted a chip-seq study in high-sip expressing hcc cell line snu by using a home-made anti-sip antibody, clone e . among annotated genes, we selected six for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip dna-binding motif cacctg was found at - kb from transcription start site of six gene. chip qpcr experiment validated sip binding to this region with , fold enrichment. compared to healthy liver, six transcripts were upregulated in of hcc cell lines included in this study. knockdown of sip by shrna in snu cells caused upregulation of six . immunohistochemistry studies in hcc tissue arrays showed increased expression of six in tumors and inverse association with sip expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip and six in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by -hydroxy vitamin d ( (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were healthy women in age - years. the bmd was measured as a t score in the lumbar spine (l -l ) with dexa. all subjects had normal t score between (- . to . ) sd. vitd was measured by lc- -at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was ( . ae . ) lg/l, mean plasma ipth level was ( . ae . ) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = À . ). genotype distribution and allele frequency of subjects were as follows: ( %) ff), ( %) ff, ( %) ff genotype in vdrf -fok gene, ( %) bb, ( %) bb, ( %) bb in vdrb-bsmi gene. allele frequencies were f: %, f: %; b: %, b: %. when fok and bsmi were combined, %(ff-bb) and % (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > . ). but foki was not (p = ). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women ( %). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p- . . - determination of hb a c values in beta thalassemia f. g€ uzelg€ ul , g. s. seydel , a. e. yalin , e. s€ onmez , k. aksoy c ß ukurova university, adana, nigde university, nigde, mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected ml of whole blood k edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a c values were measured using the agilent hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs - /ivs - , one ivs - /ivs - , one ivs - /ivs - , two ivs - /ivs - , two ivs - /ivs - , one fsc /fsc , one fsc /fsc , two - /- , two cd /cd , one cd - /cd - , one cd - /cd - , two cd /cd -g/ cd /cd -g, two ivs - / ivs - , one ivs - / ivs - , one ivs - /fsc , one ivs - /cd , one ivs - /cd , one ivs - /cd - , one ivs - /ivs - , one ivs - / ivs - , one ivs - / ivs - , one ivs - /cd and one fsc /cd . cases were classified as diabetic ( ), prediabetic ( ) and non-diabetic ( ) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in samples from . we observed positive correlation between expression of aurc and braf (rs = . , p = . ). discussion and conclusion: previous investigation showed, that for normal prostate tissue % of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to . . based on its genome sequence, metabolic pathways for proteases, amylases, cellulases, lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of . to . makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, . and ph . . for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p- . . - biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u mg and u as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk . mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc -a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk levels in u mg were reduced by %. due to mts results, ≤ mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk level in u mg and u cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately % calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d ; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p- . . - apj receptor a c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than mm to allow rapid penetration of the fixative put in % buffered formalin for hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = ) and sinus rhythm (n = ). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu- plus platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among . transcripts representing . well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ . ) with a p-value ≤ . . results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr - and environmental pigment-producing s. marcescens strain sm . bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm . cluster has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only % of identity to xantholipin biosynthetic gene cluster. clusters and of nrps genes of s. marcescens sm did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr - contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p- . . - the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to - months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: ) active animals ) hibernated animals and ) animals immobilized for weeks in laboratory, were examined. rna libraries have been sequenced using hiseq illumina platform. coupled with genome dna sequencing provided x coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e -ubiquitin ligases murf and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. - - . in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain , and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis wild-type strain forms biofilm with its maximum on th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of . - . . the temperature optimum is in the range from °c to °c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average % lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain - , - and °c - °c respectively. the crataegus genus which is a member of rosaceae family, has approximately species worldwide and species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: , diphenyl- -picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities ( . ae . % and . ae . %, respectively), at lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values ( . , . , . ) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por gene expression, encoding vdac should increase in the stress conditions. the promoter of the por gene was used to construct some new expression systems based on yl w . a new genetic construct bearing a reporter bgalactosidase gene under control of the por promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h o and methyl viologen treated cells b-galactosidase activity increased . - . -fold reaching its maximum in the cells, grown at ph of . . thus, we demonstrated high inducibility of the por promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no - - mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine- -carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn , respectively. gene deletion was accomplished through double crossover. a cassette containing the~ bp ' and ' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn d strains isolated the achad ngn d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn d, shows azc toxicity, suggesting that ngn is a true orthologue of mpr , able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c h zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at . a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant ( . . . ). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t polymerase primer for cdna synthesis. microarray represents probes for orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps , which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane- -monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose- -phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example , -dienoyl-coa reductase, -ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of patients; healthy pregnant women (control group) and pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba c levels and homa-ir were significantly higher in gdm group compared to the control group (p = . , p = . , p = . , p = . , respectively). serum adiponectin levels were significantly lower (p < . ); whereas serum resistin levels were significantly higher (p = . ) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic , ic , amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p- . . - functional polymorphisms of ephx in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec . . . ) is encoded by ephx and catalyses the degradation of endogenous fatty acid epoxides generated by cyp epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx gene that affect the seh activity may alter the circulating levels of eets. k r and r q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k r and r q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k r polymorphism were . % wild type (aa) and . % polymorphic genotype (ag+gg) and for r q polymorphism . % wild type and . % polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a g, mtrr a g and mthfr c t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from pregnant women. the main group consisted of women (n = ) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = ) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was . ae . years compared with control of the age . ae . . the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a g (or = . , ci % = . - . ; v = . , p < , ). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr rk project number). p- . . - a study on the association between rs polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is . % above the age in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs g/c single nucleotide polymorphism (snp) is found at position À , in promoter region. the presence of a c allele for rs is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs polymorphism of the ctgf gene. study population consisted of patients with pes and controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs polymorphic allele g was . in patients, and . in controls ( . , p = . ). distribution of genotypes was gg: . %, gc: . % and cc: . % among patients, while gg: %, gc: . % and cc: . % (or = . , p = . ) in controls. statistical analysis showed that there is no significant relationship between ctgf rs snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs snp and pes. this work did not point out a role for ctgf rs in the risk for pes. a significant relationship might be found when the study population is enlarged. p- . . - evaluation of rs single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs snp of clu gene have a role in the development of pes. study population consisted of patients with pes and controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was . and that of controls was . ( . , p = . ). the distribution of genotypes was cc: . %, tc: . % and tt: . % among patients while cc: . %, tc: . % and tt: . % ( . , p = . ) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp and to evaluate the difference at mrna and protein expression levels of the tp target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp mutation. to show the tp mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of hpv negative hnscc patients. employing different databases we constructed the tp regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh was significantly associated with tp mutational state. ezh is a member of the polycomb group protein enhancer zeste homolog which is known to be directly repressed by tp and indirectly by the activation of mir- a and mir- b. we found a significant up-regulation of ezh that depend from tp mutation. it is important to understand the difference in mrna and protein expression of tp regulatory network that could depend from its mutational state. this finding suggest that ezh might be a potential therapeutic target for hnscc. p- . . - next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of all patients. for this, we used sanger sequencing of the products of multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after days of treatment. for this purpose, we performed ngs of libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to per , and , cells) for a calibration curve generation. using this approach, we analyzed all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found leukemic cell per , analyzed cells. znf is considered as a transcriptional target for p and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs , rs ) of znf were investigated in kazakh population in this study. the present case-control study was carried out with participation of kazakh females with bc and cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/À), progesterone receptor (pr+/À) and human epidermal growth factor receptor (her +/À) status were estimated. pearson p-value, odds ratio, % confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs locus in znf between the patients and control groups (p = . for allele; p = . for genotype). moreover, significant association with bc was revealed for rs after dividing patients according to er+/ À, pr+/À and her +/À status of the tumor. the g allele was associated with er+ (p = . , or = . , %ci: . - . ), pr+ (p = . , or = . , %ci: . - . ) and gg genotype with her -bc carriers (p = . , or = . , %ci: . - . ). also, g allele can be considered as a risk factors in er+/ pr+/her -luminal type of tumor (p = . , or = . , % ci: . - . ). our findings correlate with the data of several gwas where the association of the rs polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs as a marker of bc risk with predictive value, restricted to er, pr and her status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr (rs ), tox (rs ), map k (rs ), lsp (rs ) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included bc patients and healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with % confidence intervals ( % ci) were applied to data analysis. according to obtained results combinations of allele c of tox rs and a of map k rs (p = . , or = . ), also allele c of tox rs and c of lsp rs (p = . , or = . ) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox rs contribute significantly to bc risk with p-value = . , or = . . on the contrary, tt genotype of tox rs with p = . , or = . and its combination with allele t of lsp rs with p = . , or = . determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp rs and a of map k rs was found in kazakh population (p = . , or = . ). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf- ) and mouse fibroblast (l ) cell lines. cells were studied four different concentrations ( , ; ; ; lm). xtt ( , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide) protocol was applied after and hours. in our study % fetal bovine serum (fbs), % l-glutamine, iu/ml penicillin and mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in % humidity and % co incubator at °c. anti-cancer activity of synthesized complexes were determined on mcf- and l . in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control ( -fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on gem/cistreated and pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm rs , ercc rs , ercc rs , and xrcc rs . values ranged between and . ; cutoff value of . had sensitivity of . and specificity of . . patients with higher score had shorter progression-free and overall survival (p < . ). in the validation group, positive predictive value was . and negative predictive value was . . model predicting outcome of pmx/cis chemotherapy included crp level, mthfd rs , and abcc rs with scores ranging between and . . cutoff value of . had sensitivity of . and specificity of . . patients with higher score had lower probability of good response and shorter progression-free survival (p < . ). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p- . . - evaluation of anti-diabetic potential of circiliol and circilineol using caco cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt (sodium-glucose transporter- ) and glut - and glucose transport. neither ag activity nor sglt activity was increased with either circiliol or circilineol treatment in caco cells compared to positive control. similarly, neither the activity nor the expression level of glut *- was increased in caco cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak z and paubap fbe the purpose of this study was to characterize and assess the impact of a novel magnetite (fe o ) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe o nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe o @e) were characterized by ir, sem, tga and hr tem. one laboratory and p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics ( , , and hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges - nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe o @e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p- . . - specimen processing and handling for plasma ammonia measurement b. sarac ßligil , , s. abus ßo glu , f. aky€ urek , b. € ozt€ urk selc ßuk medical school, konya, biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at °c) within minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at °c) within minutes and analyse refrigerated - °c hours (a , b ) and hours (a , b ). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results ( . versus . lg/dl, p < . ). in all protocols including a , a , b , b also cause an elevation in plasma ammonia results ( . , . , . and . ; p = . , p < . , p < . , p < . , respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb (gene encoding b-catenin; mostly mutations in exon ) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with ll of extraction buffer at °c over night. the tubes were boiled for min to inactivate the proteinase k. ctnnb exon was amplified by pcr. sscp is used to observe any difference between the groups. genomic dna was isolated as described above. ll of each pcr products mixed with ll denaturating buffer and were denatured by heating at °c for minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on % acrylamide/ bis gel in . tbe buffer for . hours at v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between and % in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb exon between the tumor group and normal groups. p- . . - theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol , b. uc ßar koc ß university, istanbul, american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe , tyr -octreotate, tate) has been labelled with the positron emitter ga and the beta and gama emitter lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ ga] dota -tyr -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to mm. from mm. in d.-after fifth operation). due to its high sstr selectivity, [ lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p- . . - cardiovascular diseases can be treated by using 'tetr-odd-vp ' and 'hre' hypoxia inducible systems a. celik , t. kaya , s. cigdem , m. g€ und€ uz department of medical genetic, turgut ozal university, ankara, faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif- a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif- a protein levels increase under hypoxic conditions as a result of decreased o -dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif a between and in front of tetr dna binding domain and vp transactivation domain, so that tetr-odd-vp or odd-tetr-vp could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp and odd-tetr-vp constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p- . . - osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a d cell/ scaffold bioconstruct to a continuous magnetic field during weeks of osteogenic induction. in this view, mc t -e murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc t -e cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc t -e cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p- . . - impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = , . ae . yr, . ae . kg) and untrained (n = , . ae . yr, . ae . kg) subjects performed three soccer matches ( min) in field ( m versus m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < . was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < . ). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < . ) in trained ( . ae . u/l versus . ae . u/l, % (m) . ae . u/l versus ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n) and also untrained groups ( . ae . u/l versus . ae . u/l, % (m), . ae . u/l versus . ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p- . . - regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in subjects ( males and females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between and , were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef . %, gk . %, m . %, r . %, s-l . %, s . %, ehsani . % and sirdah . %. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, out of , out of and out of patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i- (g>a), ivsii- (g>a) and heterozygous codon deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p- . . - novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev , l. gera , r. hodges institute of molecular biology and genetics, kyiv, ukraine, university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm- appeared to be the most effective, with ic values of lm and . lm in rat glioma c and human glioblastoma u cell lines, respectively. bkm- suppressed erk / and akt phosphorylation in u cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm- and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi l inhibited the viability of u cells more effectively than tmz. furthermore, the combination of chi l and bkm- resulted in an additive cytotoxic effect. chi l -mediated decrease of cell viability was associated with a g /s transition arrest. chi l provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d expression, as well as a substantial increase in p level. in addition to the accumulation of p , we observed the upregulation of cdk inhibitor p . therefore, g /s arrest in chi l -treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p . harmful hereditary mutations in brca and brca are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca and brca genes in breast cancer-diagnosed patients, that one of them is male, and person yet healthy whose brca was sequenced by sanger sequencing. the data were analyzed by using seqscape software . and detected variations were compared with literature. in brca , we determined different benign genetic variations and variation with unknown significance and variation which has not in literature. in brca gene of patient and healthy person, benign variations, variations with unknown significance, variations which has not in literature and mutation were determined. this mutation is c. - delgtca and is located in occr. c. - a>c variation in brca gene, was determined in only male patient.c. t>a variation in exon of brca , was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p- . . - inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi x . non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi x . introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included adult male rats, divided into groups. each group included rats. i.group: control (regular feed), ii.group: cpe ( . mg/kg/day), iii.group: rw ( mg/kg/day) and iv.group: cpe ( . mg/kg/day) + rw ( mg/kg/day). following days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn + pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm À ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h dcf-da. gsh level was estimated using lowry method. an intracellular labile zn + pool was assessed using fluozin- -am. investigations were performed on the specord m , hplc system lc- prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn + in erythrocytes of patients with ah + dm + compare with ah + dm À and ah À dm À was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn + level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm À , ah À dm À ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, , : - ; lipid insights, , - ] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., , : - ] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde - -hexadecanal. it was found, that -hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal (suppl. ), abstracts: mem. biol. s , lipid signaling & dynamics, p. .] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the - - proteindependent inhibition of apoptosis signalregulating kinase protein kinase ask (apoptosis signal-regulating kinase ) is a member of the mitogen-activated protein kinase kinase kinase (map k) family that plays a crucial role in immune and stress responses. the activity of ask is regulated through homo-oligomerization and interaction with other proteins including the - - protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask is dephosphorylated at ser and the - - protein dissociates. this dissociation is then one of the factors that lead to the activation of ask . we performed low-resolution structural analysis of the kinase domain of ask (ask -cd) bound to - - using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask -cd binds to the - - protein in two to two stoichiometry through a small binding interface involving surface of - - outside its central channel and several regions from the c-lobe of ask -cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask -cd to - - modulates conformation of ask 's activation segment. these results suggest that the - - binding suppresses the catalytic activity of ask through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask and - - and offers a plausible structural explanation for the - - protein-dependent inhibition of ask kinase activity. introduction: thymoquinone ( -methyl- -isopropyl- , -benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c rat glioma cells functioning was studied. results: it has been shown that the cultivation of c cells with tq in concentrations of - lm during hours strongly inhibits cell proliferation and induces cell death with id of lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c cell death by %. we analyzed mapk pathways involvement in c cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd and jnk pathway by sp does not influence on tq-induced effects. on the contrary, the specific phosphoinositide- -kinase (pi k) inhibitor (ly ) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = ) and patients with co-infection (hiv and hepatitis b and / or c) (n = ). as a control we used the data of healthy women (n = ). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control ( . times; p = . ) and group with hivmonoinfection ( . -fold; p = . ). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control ( . times (p = . ) and . -fold (p < . ), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > , ). total antioxidant activity parameter decreased . fold (p = . ) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = . , compared to the control and p = . , compared with the group with hiv-monoinfection) and the level of a-tocopherol ( . -fold to control and . fold to hiv-monoinfection) was detected in the group with hiv-coinfection. . -fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p- . . - acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose groups were formed. rats in group served as control group animals which were only given corn oil. group , group and group were administered , , mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group , group and group were inhibition percentage of %, %, and % respectively, comparison of group 's che activity. the liver alt, ast and ldh increased in group and group compare to group and group (p < . ). we also observed that there was vacuolar and hydropic degeneration in liver of group . according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised patients with ami and healthy subjects. patients that had an acute myocardial infarction (ami) less than hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon dhc were significantly higher in group with ami (p < . , respectively p < . ). elevated levels of mda (p < . ) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < . ) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p- . . - dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet , which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft , the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet gene. however, we found that a strain lacking fet is more sensitive to cu surplus conditions than a strain carrying the aft gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet gene expression. we next evaluated whether ace could be the alternative regulator of fet under cu excess. to test this hypothesis we first constructed the double mutant aft ace and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft or ace mutants. we next monitored the expression of fet gene in cells lacking ace , using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet expression is dependent on ace when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet by ace in an aft independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon- derived from human long bones (atcc Ò crl- tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with %, the superoxide anion is reduced with % compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = ) and age matched control group (n = ) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h o -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss software package. results: dna strand breaks and h o -induced dna damage were found to be higher (p = . for all), the ratio of gsh/gssg was found to be lower (p = . ) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h o -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = . ). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = . ). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p- . . - validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product iso-prostaglandin f a in urine m. kant , m. akıs ß , h. _ is ßlekel , department of medical biochemistry, school of medicine, dokuz eylul university, izmir, department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey -iso-prostaglandin f a ( -iso-pgf a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of -iso-pgf a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version . were used for data analyses. mrm transitions used were m/z? / for -iso-pgf a and m/z? / for -iso-pgf a-d . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for -iso-pgf a was determined as ng/ml by using standards ranging from . - ng/ml. analytical sensitivity of the method was determined by lod with s/n of and loq with s/n of . lod and loq for iso-pgf a were . À and À ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = ). the intra-and interday cvs for -iso-pgf a were . % and . %, respectively. sid-mrm lc-ms/ms method for absolute quantification of -iso-pgf a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p- . . - synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase / activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g /g peak, an increase in the annexin v(+) cells and the cleavage of caspase- and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl- ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto- h cells, cis/res was less efficient in killing h- cells. h- cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin- and lc a. inhibition of autophagy by bafilomycin a rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h- cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. adult male mice ( - weeks) were randomly divided into groups: control group received normal saline ( . ml/day, ip), cp group received cp ( mg/kg/week,¬ ip), the cp+ep group received ep ( mg/kg/day, ip) along with cp, were treated for days. mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < . ). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < . ). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < . ). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < . ). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < . ). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of mg/kg (group ), mg/kg (group ) and mg/kg (group ). only plain corn oil was given to control group (group ). the rats were sacrificed hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group compare to group , besides the insulin levels of group and group were significantly higher than group (p < . ). pancreas amylase and lipase activities significantly decreased in group and group compare to group and group (p < . ). however, there was no significant change in pancreas mda and tos levels (p > . ). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < . ). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < . ). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb ( mg l- and . mg l- ) for hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. day). ml physiological saline (% . nacl solutions), mg/kg/day paracetamol, mg/kg/day paracetamol and mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p- . . - lipid rafts are involved in modulation of ca + responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim Ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan Ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca + concentration ([ca + ] i ) increase due to ca + mobilization from thapsigargin-sensitive ca + stores and subsequent store-dependent ca + entry in rat peritoneal macrophages. it is known that key proteins involved in ca + signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca + ] i in macrophages. [ca + ] i measurements were performed with fura- am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca + ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with mm mbcd for hours before molixan addition causes significant inhibition of both ca + mobilization (on average, by . ae . %) and subsequent ca + entry (on average, by . ae . %), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca + responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca + ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range ( to lm) against phenotypically different mcf- , mda-mb- and sk-br- breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p , p , p and/or p . apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of lm, whereas at the concentration of lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut- and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h ax phosphorylation and bp foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, / /d/nz / . nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn that is accompanied by the relocation of nucleolar proteins and p -based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf- , mda-mb- and sk-br- ). we found that low dose nutraceutical treatment resulted in p -mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt , dnmt a and dnmt b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, / /d/nz / . p- . . - rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < . ), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < . ). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < . ), while malondialdehyde (mda) levels increased notably (p < . ). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type diabetes mellitus (t dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t dm imply an increased enzymatic activity of pa ( . %), which correlates with increased levels of ea ( . %) and glycated hemoglobin (hba c) ( . %), compared with control group. the variables hba c, pa and ea are correlated: the identified pearson correlation coefficients r = . and r = . respectively, have an associated probability of p < . and p < . a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi , e. demir , k. cinar gaziantep university, school of medicine, gaziantep, haran university, sanliurfa, department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group (ir) received gy of gamma irradiation as a single dose to total cranium plus ml saline daily. group received daily plain dmso. group received daily plain saline. at the end of the day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p- . . - role of leptin and adiponectin in obesityassociated oxidative stress e. becer , a. c ß irakoglu near east university, nicosia, cyprus, istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included obese and non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < . ) and adiponectin (p = . ) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < . ) and malondialdehyde (p < . ). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = . ) and malondialdehyde (p = . ) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around %. an extract from aronia showed greater than % efficiency, and a net ethanol extract from all the berries showed a % effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega / / and / / . p- . . - attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins -e / pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins- e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene ( lm, hours) application have been made after resveratrol ( lm, hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins- , ins- and sirt- were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo- gene was increased while pdx- was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, adult sprague-dawley male rats were divided into groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for minutes followed by a hour of reperfusion. in the treatment group, days before ischemia mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as % in the control group, whereas it was % in the i/r group. this value was decreased to % in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost % efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak ( %) at a concentration of mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to %. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega / / and / / . the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t d wt and gh+ cells. although forced gh expression induced cell proliferation, lm curcumin inhibited cell invasion. curcumin ( lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t d cancer cells. although curcumin decreased az expression in t d wt cancer cell, increased az expression was determined in t d gh cancer cell. pao and ssat expressions were upregulated in t d gh+ cells. concomitantly, putrescine levels were increased in t d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak research project (project no: z ). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. - weeks old female sprague-dawley rats (n = ) included in current study were divided into groups: control group (n = ) with healthy rats; breast cancer group (n = ); breast cancer group (n = ) treated with mg/kg paclitaxel injection (once a week for weeks); breast cancer group (n = ) treated with mg/kg royal jelly (by oral gavage for days); and finally breast cancer group (n = ) treated mg/kg royal jelly in addition to mg/kg paclitaxel injection. rats with breast carcinoma was obtained at th days after a single dose injection of mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < . ). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > . ). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase (pon ) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese ( g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = ). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups ( rats in each group). group : sham + saline; group : anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group : anp + gallic acid ( mg/kg/day, intraperitoneal). at the end of th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin- (il- ), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il- , plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < . ). however; tas and nitrite + nitrate levels did not show any significant difference (p > . ). on the other hand, while serum amylase, il- and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > . ). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p- . . - evaluation of oxidant/antioxidant system parameters, il- and il- levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder , s. bahsi , t. bahsi , v. topc ßu , a. bakir ankara universty faculty of medicinel, ankara, zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il and il levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from pregnancies affected by down sydrome and normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il and il levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < . ). besides this, in study group, cat and il- levels are found singnificantly lower than control group (p < . ). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il- levels (p > . ). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g cells. cytotoxicity of linalool was assayed by celltiter-blue Ò cell viability assay. malondialdehyde levels result in membrane damage in hep g cells were assayed by using fluorometric method. hep g cells were incubated with linalool at , and hours. the viability of hep g cells decreased in a manner dependent upon concentration and incubation time. the ic values were calculated . lg/ml ( hours), . lg/ml ( hours) and . lg/ml ( hours). but, cell viability of hep g cells increased when the cells preincubated with linalool at lower concentrations (˂ic ) against h o cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h o was decreased when the cells preincubated with linalool before h o incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included subjects ( female and male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and nd, th and th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = . and . , respectively). thiol levels decreased significantly in both gender at postprandial nd, th and th hours as compared with fasting condition (p = . ). while tbars levels increased at postprandial nd hour, that was only significant for male individuals (p = . ). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p- . . - ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in patients with prediabetes and controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p- . . - the effects of s _ io nanoparticles of rat uterine smooth muscle specially used in textile field sio nanoparticles on uterus smooth muscle was aimed to be researched. in this study wistar albino female rats were used. rats were separated in groups as control, dose ( lg/ml), dose ( lg/ml) and dose ( lg/ml). nanoparticle's size was chosen as nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio nanoparticles, in nm size and in , and lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx )] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine -epi-prostaglandin f a ( -epi-pgf a)] were determined. the results were compared with those of controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher ( % for cuzn-sod, p = . ; % for cat, p . ) and the levels of lipid peroxidation biomarkers were lower (% for mda, p = . and % for -epi-pgf a, p > . ) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = - . , p = . ). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p- . . - investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, patients with acute limb ischemia (li group; mean age years) and healthy individuals (control group; mean age years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < . ). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase activity (pon ) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on women consisted of patients with pcos and healthy ones as control. serum pon activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at nm. molar absorptivity coefficients were used in the calculation of pon and are activities as nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group ( . ae . - . ae . u/ml p < . , . ae . - . ae . u/ml p < . , recpectively). the pon activities are higher in the patient group ( . ae . u/ml) compared to the control group ( . ae . u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were . ae . absu, ima levels of control subjects were . ae . absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < . ). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < . ). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p- . . - the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard ( % protein, % ( % sucrose) carbohydrate, % lipid) and sucrose ( % protein, % ( % sucrose) carbohydrate, % lipid) diet fed bdnf heterozygous mice liver tissues. the male c bl strain wild type (+/+) and bdnf heterozygous (+/À) mice ( weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for weeks. first group involved in c bl wild type mice and fed by standard diet. second group contained c bl bdnf heterozygous mice and fed by standard diet. third group consisted c bl wild type mice and fed by sucrose diet. fourth group involved in c bl heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c bl/ mice inbred strain wild type and bdnf heterozygous (+/À) mice. animals were divided to two groups: wild type (n = ) and bdnf heterozygote mice (n = ). the animals were fed ad libitum by high-fat diet during month. weight gain was recorded every th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p- . . - disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p- . . - determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p- . . - effects of alpha- antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha- receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha- antagonists on oxidative system of rat heart tissue. male wistar albino rats were divided into groups randomly. ) tamsulosin ( mg/kd/day), ) terazosin ( mg/kg/day), ) doksazosin ( mg/kg/day), ) alfuzosin ( mg/kg/day), ) control. all drugs were administered every other day single doze via oral. rats were sacrificed after days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = . ). in addition, tamsulosin group and terazosin group were also significantly different (p = . ). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. wistar albino rats were divided into groups randomly. control (i.p. physiological saline), sbt (i.p. mg/kg/ hours sbt), stress (i.p. physiological saline; hours cold + immobilization stress) and sbt + stress (i.p. mg/kg/ hours sbt; hours cold + immobilization stress) groups were formed. nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = . ), total glutathione (p = . ) levels and glutathione peroxidase activities (p = . ) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = . ) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker -nitrotyrosine level was found to be significantly higher when compared with control group (p = . ) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. children and adolescents with obesity (age: - ); and age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics ). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity ( . ae . lmol/l; . ae . nmol/ml) than the controls ( . ae . lmol/l; . ae . nmol/ml) (< . ). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p- . . - relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats ( - g) received three i.p. injections of taa ( mg/kg) at hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca + chelation had almost the same effect as pharmacological nos inhibition suggesting that ca + -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh ) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries ( , and mg/kg) by oral gavage during days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < . ). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < . ). the maximum hypoglycemic and antioxidant effects were observed at mg/kg dose of mc. background: human serum paraoxonase (pon ) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon prevents ldl oxidation by hydrolyzing lipid peroxides. pon is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon q r isoenzymes. methods: pon q r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with mm edta for minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of mm edta to partially purified hdl-pon q r isoenzymes inhibited % of htlase and arylesterase activities. inactivation of pon for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca + -dependent inhibition of pon q r arylesterase/htlase by using the metal chelator edta, did not alter pon 's ability to inhibit ldl oxidation. pon 's ability to protect ldl from oxidation may not require calcium. p- . . - evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad , college of applied medical sciences, aljouf university, aljouf, saudi arabia, faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type diabetic patients. a total of type diabetic patients and healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx- ). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type diabetic patients than the control group (p < . ). one of the boron compounds is sodium perborate tetrahydrate (nabo . h o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose- -phosphate dehydrogenase (g pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < . ) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan , e. aky€ uz , g. baysal furtana , s. s. ellialtioglu , r. tipirdamaz nezahat g€ okyigit botanic garden, istanbul, department of biology, faculty of sciences, gazi university, ankara, department of horticulture, faculty of agriculture, ankara university, ankara, department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h o ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including mg/l , -d and . mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing and lm h o for hours and then transplanted on the mediums including mm nacl for hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h o -only and h o pre-treated tissues. pre-treatment with h o decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h o pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. the result showed pre-treatment of lm h o induced acclimation of the plants to salinity. in addition, lm h o pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type (cb ) receptor agonist acea, the antagonist am- and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study male wistar albino rats were used weighing - g. each group was consisted of rats. study groups: : control, :penicilin( iu), :penicillin( iu) + ghrelin( lg), :penicillin( iu) + am- ( . lg), :penicilin ( iu) + acea( . lg), :penicillin( iu) + am- ( . lg) + ghrelin ( lg), :penicillin( iu) + acea( . lg) + ghrelin( lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule- (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of patients with a diagnosis of as according to newyork ctriteria and control subjects were included in our study. serum endocan, interleukin- (il- ), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il- , tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < . ). whereas, serum il- , tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il- , tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = . , p = . ; r = . , p = . ). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p- . . - investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged or hours according to parasitemia state ( - %). biochemical analyses were performed in erythrocyte culture in which parasitemia between % and %. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed times with physiological saline, erythrocyte suspensions were stored at- oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < . ). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g pdh, gshpx, gshrx p- . . - in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut , g. kanbak istanbul bilim university vocational school of health services, istanbul, eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol ( mm, mm ve mm) alone and together with lg/ml aspirin, and possible protective role of mm betaine on rat brain synaptosomes. male sprague dawley rat forebrains were divided into equal pieces and pooled to form study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < . ) was observed in mda level of mm ethanol group compared to control group. different doses of ethanol ( mm, mm ve mm) + aspirin exposure significantly increased (p < . ) mda levels compared to controls, whereas betaine administration significantly decreased (p < . ) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p- . . - analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif- a in hepatocellular carcinoma cell line, hep b under hypoxia. method: hep b cells were subjected to normoxia ( % o ) and hypoxia ( % o ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif- a, pgc- a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at lm. bbr treatment has shown significant reduction in hif- a and sirt which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt expression. bbr induces the overexpression of sirt and its deacetylated-pgc- a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif- a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with lm h o after minutes lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h o . also our data suggest that ziziphus jujuba is effective in preventing h o induced oxidative stress and nitric oxide levels. discussion: some research showed that h o was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h o . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the -hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at + . v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p- . . - impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller , h. budak , m. sisecioglu , m. c ß iftci department of chemistry, faculty of science, atat€ urk university, erzurum, department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose- -phosphate dehydrogenase (g pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: / ). p- . . - protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur ( , and mg/kg) were administered orally for days. on the sixth day, chlorpyrifos ( mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < . - . ). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < . ). cur administration reduced all of these changes (p < . - . ). similarly, cur at the doses of mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < . ). additionally, the histopathological damage scores induced by chlorpyrifos (p < . - . ) were decreased by the administration of cur (p < . - . ). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after g ogtt which was applied in pregnant women for screening. methods: the serum specimens of pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = ) and prediabetic pregnant women (n = ) in the th and th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the th and th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between th and th minutes regarding the xo enzyme activity in normal pregnant women (p = . ). in normal pregnant women, the median xo enzyme activity in the th minute was . ( . - . ) u/l and it was . ( . - . ) u/l in the th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in th and th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased . times under red and blue lights and . times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was . times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p- . . - effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c glioma and mouse n a neuroblastoma cells and incubated for hours. at sub-lethal concentrations ( , , lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein (gap ) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c cells, and of nefh and biii tubulin in n a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at g for minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h o absence and presence of curcumin ( - lg/ ml) for hours at °c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase , and activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h o ) on hepg liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg cells's - - hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg cells were determined with flow cytometry method with the help of annexin v-apc and aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: hours ic > lg/ml, hours ic . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; hours ic = . lg/ml, hours ic = . lg/ml, hours ic = . lg/ml. increasing concentrations of ehta extracts were determined to direct hepg cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of mg in g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg was cytostatic at low doses, and cytotoxic at high doses. p- . . - relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of patients ( subject as scf and subjects as ncf) were analyzed. ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group ( ae versus ae ng/ml, p = . ). also there was a significant correlation between serum cp levels and tfcs (r = À . , p = . ). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t ) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n ), and experimental (n ) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t ( mg/ g) in postoperative day. rats were sacrificed on postoperative day . serum thyroid stimulating hormone (tsh), free t (ft ), and free thyroxine (ft ) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: . ) and centrifuged at rpm for minutes ( °c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t hormone had not decline in ft levels compared with the control groups. a significant decrease in ft levels was found in t given rats on postoperative day . whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft level. although tos levels and osi values were similar in both groups, t supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip (tat interacting protein, kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to . hour of lschemia and hour of reperfusion, group iii of rats that received intraperitonealy curcumin ( mg/kg) . hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase , and were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase- and ieveis, and increased the uterine tissue levels of tac but, caspase activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group ( . ae . %) and decreased after treatment with curcumin ( . ae . %). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p - on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, -day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were , , and days old. their weight and tumor diameters were measured throughout the months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p- . . - effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e ) in food preservation for approximately years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf- , colon cancer cell line, ht- , lung adenocarcinoma cell line, a , and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with % heatinactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ ml, ug/ml, ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over hours. according to our results, ha induced a decrease in ht- , a and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf- cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf- cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p- . . - chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using male wistar albino rats weighing - g. rats were divided into five groups (n = / group). group i comprised the control group, group ii was exposed with saline + immobilization stress ( minutes daily immobilization stress for days and . ml saline was administered perorally minutes before immobilization), group iii was exposed amisulpride ( mg/kg/day) + immobilization stress, group iv was exposed fluoxetine ( mg/kg/day) + immobilization stress and v. group was exposed amisulpride ( mg/kg/ day) + fluoxetine ( mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < . ). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < . ). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < . ). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > . ). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p- . . - protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against -methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin , g. oto, f. g€ ok, y. karakus, d. yildiz y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st -methylcholanthrene ( -mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % . nacl, group ii: the b(a)p were administered mg/kg via ip. four times. group iii: the -mc-treated animals were administered mg/kg via ip. four times, group iv: ba was given mg/l/day with water. group v: bx was given mg/l/day with water. group vi: b(a)p mg/kg via ip four times + ba mg/l/day dosage with water. group vii: -mc mg/kg via ip four times + ba mg/l/day with water. group viii: b(a)p mg/kg via ip four times + bx mg/l/ day dosage with water. group ix: -mc mg/kg via ip four times + bx mg/l/day with water. the experimental period was continued for days. statistical analysis showed that the -mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < . , p < . ,p p- . . - effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed - g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to mg/kg body weight alcl in drinking water for and weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., tacal, Ö., take, g., takic, m.m., taldykbayev, z., talim, b., tamashevski, a., tamer, f., tamer, l., , , taneva, s., taniyan, g., , tanrisev, m., tanriverdi, e.c., tanriverdi, k., tarhan, m., tarhan, t., tartar, s., tas, a., , taskin, a., taskin, t., taskiran, e., taskiran, b., taskoparan, b., taspinar, r., , tastan, Ö., tatli, Ö., tauraite, d., tay, t., tayman, c., taysi, s., , teker, h.t., tekes, s., tekin neijman, s., tekin, m.h., , tekin, g., , tekin, m., tekin, n., tekin, g., telci, d., , telefoncu, a., temel, h.e., , , temelie, m., temizgül, r., temlyakova, e., teplova, v., terashima, r., tercan avci, s., terekhov, s., tereshenko, o., terzi gulel, g., terzi, e., , terzi, e., terzioglu, g., terzioglu, o., testoni, g., tetik vardarli, a., tevdoradze, t., tevzadze, l., tezcan, Ö., tezcanli kaymaz, b., thielens, n., thomaidou, d., thomas, a., thornton, j.d., ticea, a.c., tikhonova, a., tileva, m., , timofeev, v., , timofeev, v., timofeeva, e., tipirdamaz, r., tiryaki, m., , tok, m., tokay, e., toker, a., , tokgun, o., toksoy Öner, e., tokyol, Ç., toman, r., tomasi, a., tombul, n., , tooke, c., topaloglu, h., topbas, m., topcu, b., topcu, c., topcu, g., , , topçu, v., topcu, c., topçuoglu, c., , topel, h., , toprak, b., toprak, m.s., torac, e., tosner, z., tosun, m., , toy, h., toymentseva, a., tozkoparan, b., trabulus, d.c., trantirek, l., trantirkova, s., trchounian, a., , trizna, e., , troshagina, d., tro t, m., truncaite, l., , tsakalidou, e., tsarkov, d., tsarkova, m., , tsigara, e.g., tsigara, m.g., tsverava, l., tsvetkova, e., tsyba, l., tsyganov, d., tsymbal, d., tüfekçi, a.r., , tufekci, a.r., tufenkci, h., tuglu, m.i., , tuglu, i., tuli, a., , , , tuli, a., tulubas, f., tunali, g., tunca gedik, s., , tunca gedik, s., tunçdemir, m., tuncel, h., tuncel, h., tunçer, s., tuncer, e., , tuncer, z.s., tuncer, b., tuncer, e., tuncez akyurek, f., tunçez akyürek, f., tuner, h., tüney kizilkaya, i., tural, b., tural, s., turan, i., turan, m., , turan, v., turan, y., turan, c., turano, p., , türel, s., , turhan, t., , turhan, y., , turk, s., turkan, a., türkcan kayhan, c., turkekul, k., türkel, s., turkeri, o.n., turkeri, n., turkkan, b., turkmen, s., türkmen, n., turkon, h., tutar, y., tüten, a., tutkun, e., , tüylü küçükkilinç, t., tuz, m.u., twardovska, m., tyapkina, o., tzartos, s., photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf- bucharest, national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, microelements modifying oxidative stress status were determined by icp-ms/ms in the hno diluted serum samples collected from chl-positive adult females (n = ) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging - g) were randomly divided into groups, as each composed of rats. after hippophae rhamnoides l. leaf ether extracts of mg/kg, mg/kg and mg/kg doses and mg/kg doses of famotidin orally administered, mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental ( mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; hours torsion/ hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape ( lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi k, akt and mtorc. intensities were evaluated as mild ( ), moderate ( ) or strong ( ). serum ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c ( . ae . lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p- . . - binding of fas and cu(ii) ions to hsa changes its cys thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion ( . mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys -sh decreased for % after cu(ii) ion binding, and during hours incubation at °c, it was further decreased for % (stearic acid, mixfas) and % (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg ( mol/ mol hsa), lead to decrease in cys -sh content depending on fa present: %- % for myristic and stearic acid, % for oleic acid and mixfas and % for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases pirto ek p- . . - anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf- and mda-mb- background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl- is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl- levels. methods: our study group includes subjects with vitamin d deficiency (group ) and age and sex-matched healthy subjects with normal serum levels of vitamin d (group ). plasma (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl- analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > . ), plasma ykl- levels were significantly higher in group than group (p < . ). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl- levels in group . vitamin d deficiency may be related to increased ykl- levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl- , inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on unrelated cad patients and healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as . %, . % and . %, respectively. in the control group, frequencies of genotypes were found as . % for aa, . % for ac and . % for cc. we did not observe difference in apj receptor a c polymorphism between cad patients and healthy controls (v = . ; df = ; p = . ). the a allele was encountered in % ( ) of the cad and . % ( ) of the controls. the c allele was seen in % ( ) of the cad and . % ( ) of the controls. allele frequencies were not significantly different between groups (v = . ; df = ; p = . ). the frequencies of apj receptor a c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ . . in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p- . . - comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups ( rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at hours intervals, intraperitoneally (i.p.). cerulein group received cerulein ( lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide ( %), two times, at hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin ( mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < . , p < . , respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < . ). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < . ). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < . , p < . , respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < . ).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p- . . - identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx- and rtx- ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of mg/ml was prepared, cleared by membrane filter of . lm and freezed at À °c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. mg of dry extract was reacted with ll pyridin + ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including % trimethylchlorosilane (tmcs) and incubated for minutes at °c. all analyses were performed with shimadzu gcms-qp ultra by using a flame ionisation detector (fid). rtx- and rtx- ms capillary columns and helium for carrier gas at a flow time of ml/minute were used. injection was applied on split mode at °c. derivatized propolis sample was injected as ll, initial column temperature was adjusted as °c, then increased to °c with increments of °c. total analysis time was determined as minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx- column were much more than those of rtx- ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx- column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after and exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to . lg/ml of cd (cdcl .h o) and zn (zncl ), and their mixture, for , and days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph . ). homogenates were centrifuged at , g ( min, + °c). supernatants were stored at À °c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < . ). results: gst and gsh changes were recorded as decreases after all metal exposures at day . although day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor- (lox- ) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox- expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor- (lox- ) in animal model of type diabetes mellitus. methods: we compared groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz ( mg/kg). animals were kept in standard condition. in day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox- levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox- levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox- levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type diabetes. p- . . - investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur , i. ince , i. abidin , a. alver objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain animals. the rats in ns treated groups were given ns ( . ml/kg) once a day orally for weeks starting days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b ) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of , , and mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with mg/kg body wt. doses ( %). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: patients with prostate cancer and healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were . ae . lmol/l in the control group, . ae . lmol/l in the patients before rp, and . ae . lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < . ). native thiol, total thiol and tas levels were higher months after rp compared to before rp in patients, but these changes were not significant statictically (p values . , . and . respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p- . . - is there any relation between g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to pregnant women aged - years and at - weeks of gestation. plasma glucose concentrations were measured initial, hour and in addition to test hours after ingestion of g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and hour serum total antioxidant status (tas) levels (r = . ) and the oxidative stress index (osi) (r = . ) was found. there was a positive weak correlation with initial and hours total oxidant status (tos) levels (r = . ) but statistically significance difference was not found (p > . ).in this study after ingestion of g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their , -diphenyl- -picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid ( . - . -lg/g), p-coumaric ( . - . -lg/g), and total phenolic content (ranged . - . -lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at - c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after hours following cpb were significantly higher than the before surgery (p < . , p < . , p serum ngal levels in after hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = . , p < . ; r = . , p < . ; r = . , p < . ; r = . , p < . respectively). ngal levels were positively correlated with total perfusion time after cbp (r = . , p < . ).the results of our study show that, increased ngal levels hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of wistrar male rats were used. groups are named as healthy, ind control, famotidine ( mg/kg) and three different doses of tq ( . , and mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. min later, mg/kg ind administrated to each rat. animals were sacrified about hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq ( . , and mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and , diphenyl- -picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + mg/kg zn sulfate (low dose; group ), al diet + mg/kg zn sulfate (middle dose; group ) and al diet + mg/kg zn sulfate (high dose; group ). zn sulfate solutions were administered . ml/day orally for days and in day rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group and ; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group . in kidney; aopp level was decreased only in group and sod level was only decreased in group as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group ). gst level was decreased in only group conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group was composed of patients with type dm and cataract and group was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of patients, totally patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < . ). tos serum levels in dm group was statistically higher than the control group (p < . ). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > . ). group , divided into two subgroups according to their hba c levels, there was no statistically significant difference between the subgroups when hba c levels were compared with the relationship between serum and ha's tac and tos levels (p > . ). there was not an association between the gender, age and the levels of tac-tos in both groups (p > . ). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik , g. erbil , z. c ß avdar , c. ural department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to hours of ischemia followed by hours of reperfusion. ferulic acid was administered ( mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase- , inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase- and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < . ). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < . ). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < . ). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p- . . - across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [ ] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane- , p-phenol- , -p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. %) charged titanium phthalocyanine ( . , . , . , and lm) were determined on a human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc( ), tritc-phalloidin and dapi staining protocols. waltmann pdt l was used as the non-toxic light source at j/cm fluence and mw/ cm fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: rvo patients and controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > . for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of osa patients were included in the study ( controls, with mild, with moderate, and with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi< served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was . ae . years and . % ( / ) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > . ). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < . ). there was a negative correlation between ahi and serum total thiol levels (r = À . , p < . ) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera -ohdg, mda and il- were significantly higher in diabetic group than control group (p < . ). although there was a notable positive correlation between mda and -ohdg, there was no a relationship between -ohdg or mda with il- . discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il- , induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose- -phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g- -p-d) enzyme activity and the effects of quercetin on erythrocyte g- -p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = ) received physiological saline. the quercetin group (n = ) received quercetin ( mg/kg/ day) via i.g. route the alcohol group (n = ) received ethanol ( % v/v, ml/day) via i.g. route. the alcohol + quercetin group (n = ) received ml of ethanol ( % v/v) hours after quercetin treatment ( mg/ kg/day). experimental procedures were peformed for days. erythrocyte g- -p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < . ). in the alcohol group, the erythrocyte g- -p-d activity was found to be significantly decreased than those in the control group (p < . ). statistically significant differences were observed in erythrocyte g- -p-d activity between the alcohol group and the alcohol + quercetin group (p < . ).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g pd, oxidative stress, quercetin p- . . - effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = ), wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of minutes. four hours later, except control, sham-a, sham-c groups, mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca , ca , and ca region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il- b, il- , malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il- b, il- , mpo, no, and tnfa levels. it could increased il- b levels in chronic stage but not affect to il- , mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il- , and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: ( ) nitrite formation from nitrate by nar, and ( ) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-( -naphtyl)-ethylenediamine, which form red color measured at nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment ( °c, min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p- . . - the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation co) on the radiotherapeutic equipment teragam in a dose of gy and received phytomedicine eminium regelii in a dose of . mg/kg orally within days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in times in spleen, in times in thymus, in times in adrenal glands, in liver in times, in lymph nodes of small intestine it in times. mda decreased in liver up to and times, in spleen in . times, in thymus in times, in liver in times, in adrenal glands in time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p- . . - evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal , c. topc ßuoglu , m. cingi clinic of biochemistry, ankara polatli duatepe public hospital, ankara, clinic of biochemistry, ankara numune training and research hospital, ankara, clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm- ) criteria for bipolar disorder i. healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p- . . - xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in fmf patients and healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc- , sjsa as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with % heat-inactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ml, ug/ ml, ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc- and sjsa cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc- cells with an ic value of . lg/ml. contrary to this, ha induced proliferation of sjsa cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa cells with an ic value of > lg/ml. on the other hand, cell migration was reduced in pc- cell line and interestingly, migration was accelerated in sjsa cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal (suppl. ) ( )